Patent application title: INCREASING PLANT GROWTH BY MODULATING OMEGA-AMIDASE EXPRESSION IN PLANTS
Inventors:
Pat J. Unkefer (Los Alamos, NM, US)
Pat J. Unkefer (Los Alamos, NM, US)
Penelope S. Anderson (Los Alamos, NM, US)
Penelope S. Anderson (Los Alamos, NM, US)
Thomas J. Knight (Raymond, ME, US)
Thomas J. Knight (Raymond, ME, US)
IPC8 Class: AC12N1582FI
USPC Class:
800287
Class name: Multicellular living organisms and unmodified parts thereof and related processes method of introducing a polynucleotide molecule into or rearrangement of genetic material within a plant or plant part the polynucleotide contains a tissue, organ, or cell specific promoter
Publication date: 2016-03-10
Patent application number: 20160068854
Abstract:
The present disclosure relates to compositions and methods for increasing
the leaf-to-root ratio of the signal metabolite 2-oxoglutaramate and
related proline molecules in plants by modulating levels of
Ω-amidase to increase nitrogen use efficiency, resulting in
enhanced growth, faster growth rates, greater seed and fruit/pod yields,
earlier and more productive flowering, increased tolerance to high salt
conditions, and increased biomass yields.Claims:
1-41. (canceled)
42. A method for generating transgenic plants having increased leaf-to-root ratio of 2-oxo-glutaramate production to thereby improve growth characteristics of the transgenic plants, comprising: (a) introducing an Ω-amidase transgene into a plurality of plant cells, wherein the Ω-amidase transgene is operably linked to a heterologous promoter; (b) generating a transgenic plant from the plurality of plant cells; and (c) expressing the Ω-amidase transgene in root tissue of the transgenic plant or the progeny of the transgenic plant, wherein said transgenic plant has an increased leaf-to-root ratio of 2-oxoglutaramate production relative to an analogous wild type or untransformed plant.
43. The method of claim 42, wherein the Ω-amidase transgene codes for a polypeptide having Ω-amidase catalytic activity and an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, and (b) an amino acid sequence that has at least 90% sequence identity to any one of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38.
44. The method according to claim 42, wherein the Ω-amidase transgene is incorporated into the genome of the plant.
45. The method according to claim 42, wherein the root-preferred promoter is selected from the group consisting of RolD promoter, RolD-2 promoter, glycine rich protein promoter, GRP promoter, ADH promoter, maize ADH1 promoter, PHT promoter, Pht1 gene family promoter, metal uptake protein promoter, maize metallothionein protein promoter, 35S CaMV domain A promoter, pDJ3S promoter, SIREO promoter, pMe1 promoter, Sad1 promoter, Sad2 promoter, TobRB7 promoter, RCc3 promoter, FaRB7 promoter, SPmads promoter, IDS2 promoter, pyk10 promoter, Lbc3 leghemoglobin promoter, PEPC promoter, Gns1 glucanase root promoter, 35S2promoter, GI4 promoter, GI5 promoter, and GRP promoter.
46. The method according to claim 42, wherein endogenous Ω-amidase expression in leaf tissue is inhibited.
47. The method according to claim 46, wherein the endogenous Ω-amidase expression in leaf tissue is inhibited by recessive gene disruption, dominant gene silencing, or a chemical inhibitor.
48. The method according to claim 46, wherein the endogenous Ω-amidase expression in leaf tissue is inhibited by a recessive gene disruption selected from the group consisting of a mutant Ω-amidase gene that eliminates endogenous Ω-amidase expression, an endogenous Ω-amidase knockout mutant, and an endogenous Ω-amidase knockdown mutant.
49. The method according to claim 46, wherein the endogenous Ω-amidase expression in leaf tissue is inhibited by an RNAi antisense oligonucleotide that is specific for an endogenous Ω-amidase gene.
50. The method according to claim 46, wherein the endogenous Ω-amidase expression in leaf tissue is inhibited by a chemical inhibitor selected from the group consisting of 6-diazo-5-oxonor-leucine, p-hydroxymercuribenzoate, diisopropyl fluorophosphates, sodium cyanide, phenylmercuriacetate, Iodoacetate, silver nitrate, chloromercuricphenylsulfonic acid, and copper sulfate.
51. The method according to claim 42, wherein the transgenic plant has an increased leaf-to-root ratio of GS activity in comparison to an analogous wild type plant or untransformed plant.
52. The method according to claim 42, wherein the transgenic plant further comprises a GPT transgene.
53. The method according to claim 52, wherein the GPT transgene is a GPT/F:L mutant encoded by SEQ ID NO:1.
54. The method according to claim 42, wherein the transgenic plant further comprises a GPT transgene and a GS transgene.
55. The method according to claim 54, wherein the GPT transgene and GS transgene are each operably linked to a leaf-preferred promoter.
56. The method according to claim 42, wherein endogenous GPT expression in the transgenic plant is increased by gene activation.
57. The method according to claim 42, wherein endogenous GS expression in the transgenic plant is increased by gene activation.
58. The method according to claim 42, wherein the Ω-amidase transgene is codon optimized for expression in the plant.
59-72. (canceled)
73. The method according to claim 42, wherein said transgenic plant or said progeny of said transgenic plant has at least one increased growth characteristic selected from the group consisting of increased biomass yield, increased NO3 uptake, increased chlorophyll per unit weight, increased CO2 fixation, and increased seed yield.
74. The method according to claim 42, further comprising selecting a progeny said transgenic plant having increased growth characteristic relative to an analogous wild type or untransformed plant, wherein the growth characteristic is selected from the group consisting of increased biomass yield, increased NO3 uptake, increased chlorophyll per unit weight, increased CO2 fixation, and increased seed yield.
75. The method according to claim 42, wherein the plant is a monocotyledonous plant.
76. The method according to claim 42, wherein the plant is a dicotyledonous plant.
77. The method according to claim 42, wherein the plurality of plant cells are taken from a plant selected from the group consisting of wheat, oats, rice, corn, bean, soybean, tobacco, alfalfa, Arabidopsis, grasses, fruits, vegetables, flowering plants, and trees.
78. The method according to claim 42, wherein the promoter is a root-preferred promoter.
79. The method according to claim 42, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence according to SEQ ID NO: 3.
80. The method according to claim 42, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 3.
81. The method according to claim 42, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 3.
82. A progeny of any generation of the transgenic plant generated according to claim 42, wherein the progeny comprises the Ω-amidase transgene.
83. A seed of any generation of the transgenic plant generated according to claim 42, wherein the progeny comprises the Ω-amidase transgene.
84. A method for generating and selecting transgenic plants having increased leaf-to-root ratio of 2-oxo-glutaramate production and improved growth characteristics of the transgenic plants, comprising: (a) introducing an Ω-amidase transgene into a plurality of plant cells, wherein said Ω-amidase transgene includes a heterologous plant promoter; (b) generating a plurality of transgenic plants from the plant cells; (c) expressing the Ω-amidase transgene in root tissue of the plant or the progeny of the plant; and (d) selecting a transgenic plant having an increased leaf-to-root ratio of 2-oxo-glutaramate production relative to an analogous wild type or untransformed plant.
85. The method according to claim 84, wherein said transgenic plant or said progeny of the transgenic plant has at least one increased growth characteristic selected from the group consisting of increased biomass yield, increased NO3 uptake, increased chlorophyll per unit weight, increased CO2 fixation, and increased seed yield.
86. The method according to claim 84, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence according to SEQ ID NO: 3.
87. The method according to claim 84, wherein the heterologous plant promoter is a root-preferred promoter.
88. The method according to claim 87, wherein the root-preferred promoter is selected from the group consisting of RolD promoter, RolD-2 promoter, glycine rich protein promoter, GRP promoter, ADH promoter, maize ADH1 promoter, PHT promoter, Pht1 gene family promoter, metal uptake protein promoter, maize metallothionein protein promoter, 35S CaMV domain A promoter, pDJ3S promoter, SIREO promoter, pMe1 promoter, Sad1 promoter, Sad2 promoter, TobRB7 promoter, RCc3 promoter, FaRB7 promoter, SPmads promoter, IDS2 promoter, pyk10 promoter, Lbc3 leghemoglobin promoter, PEPC promoter, Gns1 glucanase root promoter, 35S2promoter, GI4 promoter, GI5 promoter, and GRP promoter.
89. The method according to claim 84, wherein the Ω-amidase transgene encodes a polypeptide having Ω-amidase catalytic activity and an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, and (b) an amino acid sequence that has at least 90% sequence identity to any one of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38.
90. The method according to claim 84, wherein the Ω-amidase transgene is incorporated into the genome of the plant.
91. The method according to claim 84, wherein the plant is a monocotyledonous plant.
92. The method according to claim 84, wherein the plant is a dicotyledonous plant.
93. The method according to claim 84, wherein the plurality of plant cells are taken from a plant selected from the group consisting of wheat, oats, rice, corn, bean, soybean, tobacco, alfalfa, Arabidopsis, grasses, fruits, vegetables, flowering plants, and trees.
94. The method according to claim 84, wherein said transgenic plant produces more 2-oxo-glutaramate relative to an analogous wild type or untransformed plant.
95. The method according to claim 84, wherein the transgenic plant has an increased leaf-to-root ratio of GS activity in comparison to an analogous wild type or untransformed plant.
96. The method according to claim 84, wherein the transgenic plant has an increased leaf-to-root ratio of GPT activity in comparison to an analogous wild type or untransformed plant.
97. The method according to claim 84, wherein the plant further comprises a GPT transgene.
98. The method according to claim 97, wherein the GPT transgene is a GPT/F:L mutant encoded by SEQ ID NO:1.
99. The method according to claim 84, wherein the plant further comprises a GPT transgene and a GS transgene.
100. The method according to claim 99, wherein the GPT transgene and GS transgene are each operably linked to a leaf-preferred promoter.
101. The method according to claim 84, wherein endogenous GPT expression in the plant is increased by gene activation.
102. The method according to claim 84, wherein endogenous GS expression in the plant is increased by gene activation.
103. The method according to claim 84, wherein the transgene is codon optimized for expression in the plant.
104. The method according to claim 84, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 3.
105. The method according to claim 84, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 3.
106. A method for generating and selecting transgenic plants having increased leaf-to-root ratio of 2-oxo-glutaramate production and increased growth characteristics of the transgenic plants, comprising: (a) introducing an Ω-amidase transgene into a plurality of plant cells, wherein said Ω-amidase transgene includes a heterologous plant promoter; (b) generating a plurality of transgenic plants from the plant cells; and (c) selecting from said plurality of transgenic plants a transgenic plant having an increased biomass yield relative to an analogous wild type or untransformed plant, wherein a polypeptide encoded by said Ω-amidase transgene catalyzes the synthesis of 2-oxo-glutaramate.
107. The method according to claim 106, wherein said transgenic plant or said progeny of the transgenic plant has at least one additional increased growth characteristic selected from the group consisting of increased NO3 uptake, increased chlorophyll per unit weight, increased CO2 fixation, and increased seed yield.
108. The method according to claim 106, wherein said polypeptide has an amino acid sequence according to SEQ ID NO: 3.
109. The method according to claim 106, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 90% sequence identity to SEQ ID NO: 3.
110. The method according to claim 106, wherein the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 3.
111. The method according to claim 106, wherein the heterologous plant promoter is a root-preferred promoter.
112. The method according to claim 111, wherein the root-preferred promoter is selected from the group consisting of RolD promoter, RolD-2 promoter, glycine rich protein promoter, GRP promoter, ADH promoter, maize ADH1 promoter, PHT promoter, Pht1 gene family promoter, metal uptake protein promoter, maize metallothionein protein promoter, 35S CaMV domain A promoter, pDJ3S promoter, SIREO promoter, pMe1 promoter, Sad1 promoter, Sad2 promoter, TobRB7 promoter, RCc3 promoter, FaRB7 promoter, SPmads promoter, IDS2 promoter, pyk10 promoter, Lbc3 leghemoglobin promoter, PEPC promoter, Gns1 glucanase root promoter, 35S2promoter, GI4 promoter, GI5 promoter, and GRP promoter.
113. The method according to claim 106, wherein said polypeptide has an amino acid sequence selected from the group consisting of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, and (b) an amino acid sequence that has at least 90% sequence identity to any one of (a) SEQ ID NO: 3; SEQ ID NO: 4; SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO 8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38.
114. The method according to claim 106, wherein the Ω-amidase transgene is incorporated into the genome of the transgenic plant.
115. The method according to claim 106, wherein the transgenic plant is a monocotyledonous plant.
116. The method according to claim 106, wherein the transgenic plant is a dicotyledonous plant.
117. The method according to claim 106, wherein the transgenic plant is selected from the group consisting of wheat, oats, rice, corn, bean, soybean, tobacco, alfalfa, Arabidopsis, grasses, fruits, vegetables, flowering plants, and trees.
118. The method according to claim 106, wherein said transgenic plant produces more 2-oxo-glutaramate relative to an analogous wild type or untransformed plant.
119. The method according to claim 106, wherein the transgenic plant has an increased leaf-to-root ratio of GS activity in comparison to an analogous wild type or untransformed plant.
120. The method according to claim 106, wherein the transgenic plant has an increased leaf-to-root ratio of GPT activity in comparison to an analogous wild type or untransformed plant.
121. The method according to claim 106, wherein the transgenic plant further comprises a GPT transgene.
122. The method according to claim 121, wherein the GPT transgene is a GPT/F:L mutant encoded by SEQ ID NO:1.
123. The method according to claim 106, wherein the transgenic plant further comprises a GPT transgene and a GS transgene.
124. The method according to claim 123, wherein the GPT transgene and GS transgene are each operably linked to a leaf-preferred promoter.
125. The method according to claim 106, wherein endogenous GPT expression in the transgenic plant is increased by gene activation.
126. The method according to claim 106, wherein endogenous GS expression in the transgenic plant is increased by gene activation.
127. The method according to claim 106, wherein the Ω-amidase transgene is codon optimized for expression in the plant.
Description:
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. patent application Ser. No. 13/037,307, filed Feb. 28, 2011, and which claims the benefit under 35 USC 119(e) of U.S. Provisional Patent Application No. 61/308,971, filed Feb. 28, 2010, the disclosure of which is hereby incorporated by reference in its entirety.
REFERENCE TO SEQUENCE LISTING
[0003] This application includes a Sequence Listing as a text file named "87485-946184-SEQLIST.txt" created Jun. 4, 2015, and containing 183,340 bytes. The material contained in this text file is incorporated by reference in its entirety for all purposes.
BACKGROUND OF THE INVENTION
[0004] As the human population increases worldwide, and available farmland continues to be destroyed or otherwise compromised, the need for more effective and sustainable agriculture systems is of paramount interest to the human race. Improving crop yields, protein content, and plant growth rates represent major objectives in the development of agriculture systems that can more effectively respond to the challenges presented.
[0005] In recent years, the importance of improved crop production technologies has only increased as yields for many well-developed crops have tended to plateau. Many agricultural activities are time sensitive, with costs and returns being dependent upon rapid turnover of crops or upon time to market. Therefore, rapid plant growth is an economically important goal for many agricultural businesses that involve high-value crops such as grains, vegetables, berries and other fruits.
[0006] Genetic engineering has and continues to play an increasingly important yet controversial role in the development of sustainable agriculture technologies. A large number of genetically modified plants and related technologies have been developed in recent years, many of which are in widespread use today (Factsheet: Genetically Modified Crops in the United States, Pew Initiative on Food and Biotechnology, August 2004). The adoption of transgenic plant varieties is now very substantial and is on the rise, with approximately 250 million acres planted with transgenic plants in 2006.
[0007] While acceptance of transgenic plant technologies may be gradually increasing, particularly in the United States, Canada and Australia, many regions of the World remain slow to adopt genetically modified plants in agriculture, notably Europe. Therefore, consonant with pursuing the objectives of responsible and sustainable agriculture, there is a strong interest in the development of genetically engineered plants that do not introduce toxins or other potentially problematic substances into plants and/or the environment. There is also a strong interest in minimizing the cost of achieving objectives such as improving herbicide tolerance, pest and disease resistance, and overall crop yields. Accordingly, there remains a need for transgenic plants that can meet these objectives.
[0008] The goal of rapid plant growth has been pursued through numerous studies of various plant regulatory systems, many of which remain incompletely understood. In particular, the plant regulatory mechanisms that coordinate carbon and nitrogen metabolism are not fully elucidated.
[0009] These regulatory mechanisms are presumed to have a fundamental impact on plant growth and development.
[0010] The metabolism of carbon and nitrogen in photosynthetic organisms must be regulated in a coordinated manner to assure efficient use of plant resources and energy. Current understanding of carbon and nitrogen metabolism includes details of certain steps and metabolic pathways which are subsystems of larger systems. In photosynthetic organisms, carbon metabolism begins with CO2 fixation, which proceeds via two major processes, termed C-3 and C-4 metabolism. In plants with C-3 metabolism, the enzyme ribulose bisphosphate carboxylase (RuBisCo) catalyzes the combination of CO2 with ribulose bisphosphate to produce 3-phosphoglycerate, a three carbon compound (C-3) that the plant uses to synthesize carbon-containing compounds. In plants with C-4 metabolism, CO2 is combined with phosphoenol pyruvate to form acids containing four carbons (C-4), in a reaction catalyzed by the enzyme phosphoenol pyruvate carboxylase. The acids are transferred to bundle sheath cells, where they are decarboxylated to release CO2, which is then combined with ribulose bisphosphate in the same reaction employed by C-3 plants.
[0011] Numerous studies have found that various metabolites are important in plant regulation of nitrogen metabolism. These compounds include the organic acid malate and the amino acids glutamate and glutamine. Nitrogen is assimilated by photosynthetic organisms via the action of the enzyme glutamine synthetase (GS) which catalyzes the combination of ammonia with glutamate to form glutamine. GS plays a key role in the assimilation of nitrogen in plants by catalyzing the addition of ammonium to glutamate to form glutamine in an ATP-dependent reaction (Miflin and Habash, 2002, Journal of Experimental Botany, Vol. 53, No. 370, pp. 979-987). GS also reassimilates ammonia released as a result of photorespiration and the breakdown of proteins and nitrogen transport compounds. GS enzymes may be divided into two general classes, one representing the cytoplasmic form (GS1) and the other representing the plastidic (i.e., chloroplastic) form (GS2).
[0012] Previous work has demonstrated that increased expression levels of GS1 result in increased levels of GS activity and plant growth, although reports are inconsistent. For example, Fuentes et al. reported that CaMV S35 promoter-driven overexpression of Alfalfa GS1 (cytoplasmic form) in tobacco resulted in increased levels of GS expression and GS activity in leaf tissue, increased growth under nitrogen starvation, but no effect on growth under optimal nitrogen fertilization conditions (Fuentes et al., 2001, J. Exp. Botany 52: 1071-81). Temple et al. reported that transgenic tobacco plants overexpressing the full length Alfalfa GS1 coding sequence contained greatly elevated levels of GS transcript, and GS polypeptide which assembled into active enzyme, but did not report phenotypic effects on growth (Temple et al., 1993, Molecular and General Genetics 236: 315-325). Corruzi et al. have reported that transgenic tobacco overexpressing a pea cytosolic GS1 transgene under the control of the CaMV S35 promoter show increased GS activity, increased cytosolic GS protein, and improved growth characteristics (U.S. Pat. No. 6,107,547). Unkefer et al. have more recently reported that transgenic tobacco plants overexpressing the Alfalfa GS1 in foliar tissues, which had been screened for increased leaf-to-root GS activity following genetic segregation by selfing to achieve increased GS1 transgene copy number, were found to produce increased 2-hydroxy-5-oxoproline levels in their foliar portions, which was found to lead to markedly increased growth rates over wild type tobacco plants (see, U.S. Pat. Nos. 6,555,500; 6,593,275; and 6,831,040).
[0013] Unkefer et al. have further described the use of 2-hydroxy-5-oxoproline (also known as 2-oxoglutaramate) to improve plant growth (U.S. Pat. Nos. 6,555,500; 6,593,275; 6,831,040). In particular, Unkefer et al. disclose that increased concentrations of 2-hydroxy-5-oxoproline in foliar tissues (relative to root tissues) trigger a cascade of events that result in increased plant growth characteristics. Unkefer et al. describe methods by which the foliar concentration of 2-hydroxy-5-oxoproline may be increased in order to trigger increased plant growth characteristics, specifically, by applying a solution of 2-hydroxy-5-oxoproline directly to the foliar portions of the plant and over-expressing glutamine synthetase preferentially in leaf tissues.
SUMMARY OF THE INVENTION
[0014] The present disclosure is based on the surprising discovery that increasing Ω-amidase (omega-amidase) expression in the root tissues of a plant results in an increase in the plant's leaf-to-root ratio of the signal metabolite 2-oxoglutaramate and related proline molecules. Additionally, increasing Ω-amidase expression in the root tissues of a plant results in decreased Ω-amidase expression in leaf tissue. Advantageously, modulating the expression of Ω-amidase in plants results in plants with increased nitrogen use efficiency, which in turn results in enhanced growth and other agronomic characteristics, including faster growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, increased tolerance to high salt conditions, and increased biomass yields.
[0015] Accordingly, one aspect of the present disclosure relates to a transgenic plant containing an Ω-amidase transgene, where the Ω-amidase transgene is operably linked to a root-preferred promoter.
[0016] Another aspect of the present disclosure provides a transgenic plant having inhibited expression of endogenous Ω-amidase in leaf tissue.
[0017] Still another aspect of the present disclosure relates to a method for increasing nitrogen use efficiency of a plant relative to a wild type or untransformed plant of the same species, by: (a) introducing an Ω-amidase transgene into the plant, where the Ω-amidase transgene is operably linked to a root-preferred promoter; (b) expressing the Ω-amidase transgene in root tissue of the plant or the progeny of the plant; and (c) selecting a plant having an increased leaf-to-root ratio of 2-oxoglutaramate relative to a plant of the same species that does not contain an Ω-amidase transgene, where the increased leaf-to-root ratio of 2-oxoglutaramate results in increased nitrogen use efficiency.
[0018] Yet another aspect of the present disclosure relates to a method for increasing nitrogen use efficiency of a plant relative to a wild type or untransformed plant of the same species, by: (a) inhibiting endogenous Ω-amidase expression in leaf tissue of the plant; and (b) selecting a plant having an increased leaf-to-root ratio of 2-oxoglutaramate relative to a plant of the same species that does not have inhibited endogenous Ω-amidase expression in leaf tissue, where the increased leaf-to-root ratio of 2-oxoglutaramate results in increased nitrogen use efficiency
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 depicts a schematic of a metabolic pathway of nitrogen assimilation and 2-oxoglutaramate biosynthesis.
[0020] FIG. 2A-C depict amino acid sequence alignments of an Arabidopsis thaliana Ω-amidase with other putative plant Ω-amidases.
[0021] FIG. 3A-B depict amino acid sequence alignments of an Arabidopsis thaliana Ω-amidase with other putative animal Ω-amidases.
[0022] FIG. 4 graphically depicts a plot of plant fresh weight values plotted against 2-oxoglutaramate concentration.
[0023] FIG. 5 depicts a photograph comparing Ω-amidase transgenic and control alfalfa plants.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0024] Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodology by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons, Inc. 2001; Transgenic Plants: Methods and Protocols (Leandro Pena, ed., Humana Press, 1st edition, 2004); and, Agrobacterium Protocols (Wan, ed., Humana Press, 2nd edition, 2006). As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.
[0025] Each document, reference, patent application or patent cited in this text is expressly incorporated herein in its entirety by reference, and each should be read and considered as part of this specification. That the document, reference, patent application or patent cited in this specification is not repeated herein is merely for conciseness.
[0026] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof ("polynucleotides") in either single- or double-stranded form.
[0027] Unless specifically limited, the term "polynucleotide" encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g. degenerate codon substitutions) and complementary sequences and as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19: 5081; Ohtsuka et al., 1985 J. Biol. Chem. 260: 2605-2608; and Cassol et al., 1992; Rossolini et al., 1994, Mol. Cell. Probes 8: 91-98). The term nucleic acid is used interchangeably with gene, cDNA, and mRNA encoded by a gene.
[0028] The term "promoter" refers to a nucleic acid control sequence or sequences that direct transcription of an operably linked nucleic acid. As used herein, a "plant promoter" is a promoter that functions in plants. Promoters include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. As used herein, a promoter may include the full nucleotide sequence, or may only include the core domain or sequence that directs transcription of the operably linked nucleic acid. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
[0029] The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers.
[0030] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
[0031] Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
[0032] The term "plant" includes whole plants, plant organs (e.g., leaves, stems, flowers, roots, reproductive organs, embryos and parts thereof, etc.), seedlings, seeds and plant cells and progeny thereof. The class of plants which can be used in the method of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), as well as gymnosperms. It includes plants of a variety of ploidy levels, including polyploid, diploid, haploid and hemizygous.
[0033] The terms "GPT polynucleotide" and "GPT nucleic acid" are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a polypeptide involved in catalyzing the synthesis of 2-oxoglutaramate, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof. The term "GPT coding sequence" refers to the part of the gene which is transcribed and encodes a GPT protein. The term "targeting sequence" and "transit peptide" are used interchangeably and refer to the amino terminal part of a protein which directs the protein into a subcellular compartment of a cell, such as a chloroplast in a plant cell. GPT polynucleotides are further defined by their ability to hybridize under defined conditions to the GPT polynucleotides specifically disclosed herein, or to PCR products derived therefrom.
[0034] A "GPT transgene" is a nucleic acid molecule comprising a GPT polynucleotide which is exogenous to transgenic plant, or plant embryo, organ or seed, harboring the nucleic acid molecule, or which is exogenous to an ancestor plant, or plant embryo, organ or seed thereof, of a transgenic plant harboring the GPT polynucleotide. A "GPT transgene" may encompass a polynucleotide that encodes either a full length GPT protein or a truncated GPT protein, including but not limited to a GPT protein lacking a chloroplast transit peptide. More particularly, the exogenous GPT transgene will be heterogeneous with any GPT polynucleotide sequence present in wild-type plant, or plant embryo, organ or seed into which the GPT transgene is inserted. To this extent the scope of the heterogeneity required need only be a single nucleotide difference. However, preferably the heterogeneity will be in the order of an identity between sequences selected from the following identities: 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, and 20%.
[0035] The terms "GS polynucleotide" and "GS nucleic acid" are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a glutamine synthetase protein, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof. The term "GS coding sequence" refers to the part of the gene which is transcribed and encodes a GS protein. The terms "GS1 polynucleotide" and "GS1 nucleic acid" are used interchangeably herein, and refer to a full length or partial length polynucleotide sequence of a gene which encodes a glutamine synthetase isoform 1 protein, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof. The term "GS1 coding sequence" refers to the part of the gene which is transcribed and encodes a GS1 protein.
[0036] A "GS transgene" is a nucleic acid molecule comprising a GS polynucleotide which is exogenous to transgenic plant, or plant embryo, organ or seed, harboring the nucleic acid molecule, or which is exogenous to an ancestor plant, or plant embryo, organ or seed thereof, of a transgenic plant harboring the GS polynucleotide. A "GS transgene" may encompass a polynucleotide that encodes either a full length GS protein or a truncated GS protein, including but not limited to a GS protein lacking a transit peptide. A "GS1 transgene" is a nucleic acid molecule comprising a GS1 polynucleotide which is exogenous to transgenic plant, or plant embryo, organ or seed, harboring the nucleic acid molecule, or which is exogenous to an ancestor plant, or plant embryo, organ or seed thereof, of a transgenic plant harboring the GS1 polynucleotide. A "GS1 transgene" may encompass a polynucleotide that encodes either a full length GS protein or a truncated GS1 protein, including but not limited to a GS1 protein lacking a transit peptide. More particularly, the exogenous GS or GS1 transgene will be heterogeneous with any GS or GS1 polynucleotide sequence present in wild-type plant, or plant embryo, organ or seed into which the GS or GS1 transgene is inserted. To this extent the scope of the heterogeneity required need only be a single nucleotide difference. However, preferably the heterogeneity will be in the order of an identity between sequences selected from the following identities: 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, and 20%.
[0037] The terms "Ω-amidase (omega-amidase) polynucleotide" and "Ω-amidase nucleic acid" are used interchangeably herein, and refer to a polynucleotide sequence of a gene which encodes a polypeptide involved in the enzymatic breakdown of 2-oxoglutaramate, and includes polynucleotides containing both translated (coding) and un-translated sequences, as well as the complements thereof. The term "Ω-amidase coding sequence" refers to the part of the gene which is transcribed and encodes an Ω-amidase protein. The Ω-amidase polynucleotides are further defined by their ability to hybridize under defined conditions to the Ω-amidase polynucleotide specifically disclosed herein, or to PCR products derived therefrom.
[0038] An "Ω-amidase transgene" is a nucleic acid molecule comprising an Ω-amidase polynucleotide which is exogenous to transgenic plant, or plant embryo, organ or seed, harboring the nucleic acid molecule, or which is exogenous to an ancestor plant, or plant embryo, organ or seed thereof, of a transgenic plant harboring the Ω-amidase polynucleotide. An "Ω-amidase" may encompass a polynucleotide that encodes either a full length Ω-amidase protein or a truncated Ω-amidase protein, including but not limited to an Ω-amidase protein lacking a chloroplast transit peptide. More particularly, the exogenous Ω-amidase transgene will be heterogeneous with any Ω-amidase polynucleotide sequence present in wild-type plant, or plant embryo, organ or seed into which the Ω-amidase transgene is inserted. To this extent the scope of the heterogeneity required need only be a single nucleotide difference. However, preferably the heterogeneity will be in the order of an identity between sequences selected from the following identities: 0.01%, 0.05%, 0.1%, 0.5%, 1%, 5%, 10%, 15%, and 20%.
[0039] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
[0040] As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
[0041] The following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
[0042] Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al., Molecular Biology of the Cell (3rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: The Conformation of Biological Macromolecules (1980). "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 25 to approximately 500 amino acids long. Typical domains are made up of sections of lesser organization such as stretches of β-sheet and α-helices. "Tertiary structure" refers to the complete three dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three dimensional structure formed by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
[0043] The term "isolated" refers to material which is substantially or essentially free from components which normally accompany the material as it is found in its native or natural state. However, the term "isolated" is not intended refer to the components present in an electrophoretic gel or other separation medium. An isolated component is free from such separation media and in a form ready for use in another application or already in use in the new application/milieu. An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using Coomassie blue or, preferably, silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.
[0044] The term "heterologous" when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, a nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a nucleic acid encoding a protein from one source and a nucleic acid encoding a peptide sequence from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
[0045] The terms "identical" or percent "identity," in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, or 95% identity) over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a sequence comparison algorithms, or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence. This definition also refers to the complement of a test sequence, which has substantial sequence or subsequence complementarity when the test sequence has substantial identity to a reference sequence.
[0046] When percentage of sequence identity is used in reference to polypeptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, where amino acids residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the polypeptide. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.
[0047] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
[0048] A "comparison window", as used herein, includes reference to a segment of any one of the number of contiguous positions selected from about 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
[0049] A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1977, Nuc. Acids Res. 25:3389-3402 and Altschul et al., 1990, J. Mol. Biol. 215:403-410, respectively. BLAST and BLAST 2.0 are used, typically with the default parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, 1993, Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
[0050] The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, highly stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. Low stringency conditions are generally selected to be about 15-30° C. below the Tm. Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0M sodium ion, typically about 0.01 to 1.0M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization.
[0051] Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cased, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
[0052] Genomic DNA or cDNA comprising GPT polynucleotides may be identified in standard Southern blots under stringent conditions using the GPT polynucleotide sequences disclosed here. For this purpose, suitable stringent conditions for such hybridizations are those which include a hybridization in a buffer of 40% formamide, 1M NaCl, 1% SDS at 37° C., and at least one wash in 0.2×SSC at a temperature of at least about 50° C., usually about 55° C. to about 60° C., for 20 minutes, or equivalent conditions. A positive hybridization is at least twice background. Those of ordinary skill will readily recognize that alternative hybridization and wash conditions may be utilized to provide conditions of similar stringency.
[0053] A further indication that two polynucleotides are substantially identical is if the reference sequence, amplified by a pair of oligonucleotide primers, can then be used as a probe under stringent hybridization conditions to isolate the test sequence from a cDNA or genomic library, or to identify the test sequence in, e.g., a northern or Southern blot.
[0054] Transgenic Plants with Altered Levels of Ω-Amidase Expression:
[0055] The present disclosure provides transgenic plants containing higher levels of the signal metabolite 2-oxoglutaramate and its analogs in foliar tissues versus root or below-ground tissues and methods for increasing nitrogen use efficiency of a plant generating plants. 2-oxoglutaramate is a metabolite which is an extremely potent effector of gene expression, metabolism and plant growth (U.S. Pat. No. 6,555,500), and which may play a pivotal role in the coordination of the carbon and nitrogen metabolism systems (Lancien et al., 2000, Enzyme Redundancy and the Importance of 2-Oxoglutarate in Higher Plants Ammonium Assimilation, Plant Physiol. 123: 817-824).
[0056] The levels of 2-oxoglutaramate in leaf tissue may be increased by increasing the biosynthesis of 2-oxoglutaramate. The biosynthesis of 2-oxoglutaramte in leaf tissue may be preferentially increased to a level sufficient to exceed the breakdown rate by, for example, by decreasing the activity of the enzyme catalyzing the breakdown of 2-oxoglutaramate (FIG. 1). Additionally, the levels of 2-oxoglutaramate in root tissue may be decreased by increasing the breakdown of 2-oxoglutaramate. The breakdown rate of 2-oxoglutaramte in root tissue may be preferentially increased by, for example, increasing the activity of the enzyme catalyzing the breakdown of 2-oxoglutaramate (FIG. 1).
[0057] The methods disclosed herein are used to generate transgenic plants exhibiting higher leaf-to-root ratios of 2-oxoglutaramate when compared to wild type or progenitor plants. In the practice of the disclosed methods, 2-oxoglutaramate concentration in the leaf and root tissues may be modulated by any one of or a combination of several approaches disclosed herein. For example, the leaf-to-root ratio of 2 may be increased by increasing the activity of Ω-amidases in root tissues or by inhibiting the activity of Ω-amidases in leaf tissues.
[0058] Modulation of the Ω-Amidase Pathway in Plants:
[0059] The present disclosure is based on the surprising discovery that the leaf-to-root ratio of 2-oxoglutaramate may be increased in plants by modulating Ω-amidase expression in the root and/or leaf tissue of plants. Moreover, increasing the leaf-to-root ratio of 2-oxoglutaramate results in increased nitrogen use efficiency.
[0060] Applicants have identified an Ω-amidase pathway that may be modulated to achieve the objective of increasing the relative leaf to root concentration of 2-oxoglutaramate thereby triggering higher Nitrogen assimilation and carbon metabolism dynamics resulting in enhanced growth rates and agronomic characteristics. The only intermediate in this pathway, 2-oxoglutaramate, ostensibly functions as a signal metabolite that reflects the flux of assimilated nitrogen. Increased levels of 2-oxoglutaramate trigger a striking increase in resource acquisition rates, carbon and nitrogen metabolism, and overall growth. Plants treated with 2-oxoglutaramate or engineered to produce increased 2-oxoglutaramate levels in leaf show greater leaf nitrogen and media nitrogen use efficiency. The resulting increase in overall growth metabolism is accompanied by increased leaf-to-root ratios in 2-oxoglutaramate pools, which are in turn controlled by glutamine synthetase, glutamine phenylpyruvate transaminase and Ω-amidase activities, as well as by the availability of nitrogen, in a complex, interrelated and tissue-specific manner. Moreover, increasing the leaf-to-root ratio of 2-oxoglutaramate results in increased nitrogen use efficiency. As used herein, "increased nitrogen use efficiency" and "increasing nitrogen use efficiency" refers to plants that have enhanced growth and better agronomic characteristics. Agronomic characteristics include, without limitation, faster growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, increased tolerance to high salt conditions, and/or increased biomass yields resulting from increased nitrogen utilization mediated by an increased leaf-to-root ratio of 2-oxoglutaramate.
[0061] As described in Example 1, infra, transgenic plants engineered to over-express GS and/or GPT show lower Ω-amidase activities in the leaves and greater Ω-amidase activity in the roots. GPT and GS+GPT transgenic plants showed the largest increases in root Ω-amidase activity. These plants responded to expression of the transgenes by altering their Ω-amidase activities such that they tend to increase the leaf 2-oxoglutaramate pool and maintain the root 2-oxoglutaramate pool. These responses combined in the GS+GPT over-expressing plants to generate the highest leaf and lowest root 2-oxoglutaramate pools and the highest leaf and lowest GS and GPT activities.
[0062] Without wishing to be bound by theory, it is believed that the signal metabolite, 2-oxoglutaramate, provides two different messages, depending upon the tissue. Increased 2-oxoglutaramate in the leaves is stimulatory (Tables 2-4) and appears to effectively convey the message that nitrogen is abundant and carbon must be fixed to take advantage of the increased nitrogen. The increased nitrogen supply-driven faster growth is accompanied by an increase in leaf-to-root 2-oxoglutaramate pools. The increase in the leaf 2-oxoglutaramate pool is apparently key to the stimulation. Without wishing to be bound by theory, it is believed that the plant's strategy to maintain near normal or diminished 2-oxoglutaramate pool in the roots is a mechanism they may be using to overcome the apparent contradiction of how plants grow faster when fertilized with nitrogen and yet display the long-observed inhibition of nitrate uptake and assimilation in roots by a "N metabolite downstream of NH3" (Foyer et al., 2002, Kluwer Academic Publishers, The Netherlands). If the inhibitory nitrogen metabolite is 2-oxoglutaramate, then the mechanism could be to manage its concentration downward when nitrogen is abundant to let the plant prosper and, conversely keep 2-oxoglutaramate higher when nitrogen is scarce and the plant tries to conserve a limiting resource to survive to reproduce.
[0063] Without wishing to be bound by theory, it is believed that the results disclosed herein indicate that modulating Ω-amidase activity in plants is useful in driving increased levels of 2-oxoglutaramate in leaves relative to roots. Two approaches are hereinafter described: (1) promoting the breakdown of 2-oxoglutaramate by upregulating Ω-amidase activity in root tissues, and (2) impairing the breakdown of 2-oxoglutaramate in leaf tissues by impairing, downregulating, or deactivating leaf Ω-amidase activity.
[0064] Promoting the Breakdown of 2-Oxoglutaramate by Upregulating Ω-Amidase Activity in Root Tissues:
[0065] Certain aspects of the present disclosure relate to transgenic plants containing an Ω-amidase transgene, where the Ω-amidase transgene is operably linked to a root-preferred promoter.
[0066] It has been previously shown that an Ω-amidase-like pathway in plants that is possibly involved in 2-oxoglutaramate breakdown (Von Gusgtav Schwab 1936, Planta Archiv fur wissenschaftliche botanik. 25.Band, 4. Heft. p 579-606. [German publication]; Olenicheva, LS1955, Biokhimiia 20(2):165-172. [Russian publication]; and Yamamoto, Y. 1955, Journal of Biochemistry 42:763-774). However, none of these studies identified an Ω-amidase protein.
[0067] Accordingly, it is believed that that an Ω-amidase pathway is involved in the breakdown of 2-oxoglutaramate and its analogs in plants (FIG. 1). Additionally, an Ω-amidase enzyme has been shown to be capable of catalyzing the breakdown of 2-oxoglutaramate in animal cells by opening the ring of 2-oxoglutaramate and removing the nitrogen to yield a keto acid (Cooper and Meister, 1977, CRC Critical Reviews in Biochemistry, pages 281-303; Meister, 1952, J. Biochem. 197: 304).
[0068] Applicants have identified a putative plant Ω-amidase gene and protein (Arabidopsis thaliana gene AT5g12040, F14F18 210 mRNA). The identification of the putative plant Ω-amidase was based on sequence homology analysis to Ω-amidase gene sequences from other organisms including human and rat. The nucleotide coding and translated amino acid sequences thereof are shown below.
[0069] Arabidopsis ω-omega.-amidase nucleotide coding sequence (Genbank accessions AY075592.1 with GI 19715573 corresponding to protein NP445766 (full-length protein AAL91613.1=SEQ ID NO:46)) [SEQ ID NO: 2]:
TABLE-US-00001 AAAGTGAAATGAAGTCAGCAATTTCATCGTCACTCTTCTTCAATTCGAAG AATCTTTTAAACCCTAATCCTCTTTCTCGCTTCATTTCTCTCAAATCTA ACTTCCTCCCTAAATTATCTCCGAGATCGATCACTAGTCACACCTTGAA GCTCCCATCTTCGTCAACCTCAGCTTTAAGATCCATTTCCTCTTCCATG GCTTCTTCTTTCAACCCTGAACAAGCTAGAGTTCCCTCTGCTCTTCCTC TCCCAGCTCCTCCGTTGACCAAATTCAACATCGGATTGTGTCAGCTATC TGTTACATCTGACAAAAAGAGAAACATCTCTCATGCTAAAAAAGCCATT GAAGAAGCTGCTTCTAAAGGAGCTAAGCTTGTTCTCTTACCCGAAATTT GGAACAGTCCGTATTCCAATGATAGTTTTCCAGTTTATGCGGAGGAGAT TGATGCAGGTGGTGATGCTTCTCCTTCAACGGCAATGCTTTCTGAAGTT TCCAAACGTCTCAAGATTACAATCATTGGTGGATCTATACCAGAAAGAG TTGGAGATCGTTTGTATAACACTTGCTGTGTCTTTGGTTCCGATGGAGA GCTAAAAGCTAAGCATCGGAAGATACATTTATTTGATATAGACATTCCC GGGAAGATTACTTTTATGGAATCCAAAACTCTTACTGCTGGAGAGACAC CAACAATCGTTGACACAGATGTAGGGCGTATTGGAATAGGCATCTGTTA TGATATCAGGTTCCAGGAGTTAGCTATGATATATGCTGCAAGAGGGGCT CATTTGCTGTGCTACCCGGGAGCCTTTAACATGACAACTGGACCATTGC ATTGGGAATTACTACAAAGGGCCAGGGCTACGGATAATCAGTTATATGT GGCGACATGCTCACCTGCCAGAGATTCAGGAGCTGGCTACACTGCTTGG GGGCACTCAACACTCGTTGGGCCTTTTGGAGAAGTACTAGCAACGACTG AGCATGAGGAGGCCATTATCATAGCAGAGATTGATTACTCTATCCTTGA ACAACGAAGGACTAGCCTTCCATTGAATAGGCAGCGGCGGGGAGATCTT TACCAGCTTGTAGACGTACAGCGCTTAGACTCTAAATGAACGCAGCAGTA ACTGTATATCTGAGAGATATTGCGAGTTGAGCACGATTTGGTTACTTACA ACTTCATGCATGATCAGTCATTTCTCCACAACTTTGCTGAGATATGTAAA AGAATAAAAATCAAACTTTTGAGTTAAAATCGAACAAAGGCAAGTAAATT CTGCTTAGATAATGTGAACTCCACCCACTTGCCATGTGTTTGTTGTTTAT AAACTTCAATGCATTCTGATAACG
[0070] Mature Arabidopsis ω-amidase amino acid sequence; and derived from the translation product of SEQ ID NO: 2, above (Genbank accession AAL91613.1) [SEQ ID NO: 3]:
TABLE-US-00002 MASSFNPEQARVPSALPLPAPPLTKFNIGLCQLSVTSDKKRNISHAKKA IEEAASKGAKLVLLPEIWNSPYSNDSFPVYAEEIDAGGDASPSTAMLSE VSKRLKITIIGGSIPERVGDRLYNTCCVFGSDGELKAKHRKIHLFDIDI PGKITFMESKTLTAGETPTIVDTDVGRIGIGICYDIRFQELAMIYAARG AHLLCYPGAFNMTTGPLHWELLQRARATDNQLYVATCSPARDSGAGYTA WGHSTLVGPFGEVLATTEHEEAIIIAEIDYSILEQRRTSLPLNRQRRGD LYQLVDVQRLDSK
[0071] Based on initial BLAST analysis in Genbank, the Arabidopsis Ω-amidase has homologs in other plant species, as well as in bacteria, fungi, frogs, fish, and mammal. None of the identified homologs were annotated as Ω-amidases. However, without wishing to be bound by theory, it is believed that these sequences also encode Ω-amidases. The amino acid sequences of the identified putative Ω-amidases are shown below.
[0072] Vitis vinifera amino acid sequence (Genbank accession XP 002279687.1) [SEQ ID NO: 4]:
TABLE-US-00003 MKSAALSALLSSTLSYASPPHLNLLRPATAVLCRSLLPTSTPNPFHTQL RTAKISASMSSSFKPEQARVPPAIPPPTPPLSKFKIGLCQLSVTADKER NIAHARKAIEEAVEKGAQLVLLPEIWNSPYSNDSFPVYAEDIDAGSDAS PSTAMLSEVSHALKITIVGGSIPERCGDQLYNTCCVFGSDGKLKAKHRK IHLFDINIPGKITFMESKTLTAGGSPTIVDTEVGRIGIGICYDIRFSEL AMLYAARGAHLICYPGAFNMTTGPLHWELLQRARAADNQLYVATCSPAR DAGAGYVAWGHSTLVGPFGEVLATTEHEEAIIISEIDYSLIELRRTNLP LLNQRRGDLYQLVDVQRLDSQ
[0073] Zea mays amino acid sequence (Genbank accession ACN30911.1) [SEQ ID NO: 5]:
TABLE-US-00004 MVAAAAAAAAATATAAALLAPGLKLCAGRARVSSPSGLPLRRVTAMASA PNSSFRPEEARSPPALELPIPPLSKFKVALCQLSVTADKSRNIAHARAA IEKAASDGAKLVVLPEIWNGPYSNDSFPEYAEDIEAGGDAAPSFSMLSE VARSLQITLVGGSIAERSGNNLYNTCCVFGSDGQLKGKHRKIHLFDIDI PGKITFKESKTLTAGQSPTVVDTDVGRIGIGICYDIRFQELAMLYAARG AHLLCYPGAFNMTTGPLHWELLQRARAADNQLFVATCAPARDTSAGYVA WGHSTLVGPFGEVIATTEHEEATIIADIDYSLIEQRRQFLPLQHQRRGD LYQLVDVQRLGSQ
[0074] Populus trichocarpa amino acid sequence (Genbank accession XP 002309478.1) [SEQ ID NO: 6]:
TABLE-US-00005 MKSAISSTTTLLSSKNLSLKLHLNHSPLSRLPSSLFRSKSNTHFPSLLP RNNSTHNQKSQIHTPIMASSFMPEQARAPPALPLPVPPFKIGLCQLSVT ADKERNIAHARKAIEEAAAKGAKLVMLPEIWNSPYSNDCFPVYAEDIDA GGEASPSTAMLSEAAGLLKVTIVGGSIPERSGDRLYNTCCVFDSDGKLK AKHRKIHLFDIDIPGKITFIESKTLTAGETPTIVDTEVGRIGIGICYDI RFQELAIIYAARGAHLICYPGAFNMTTGPLHWELLQRARAADNQLYVAT CSPARDVAAGYVAWGHSTLVGPFGEVLATTEHEEDIIIAEIDYSLLEVR RTNLPLTKQRRGDLYQLVDVQRLKSDS
[0075] Picea sitchensis amino acid sequence (Genbank accession ABK22312.1) [SEQ ID NO: 7]:
TABLE-US-00006 MTPLLSYSLRVVASALRPKSSIASAVGRLSATPKRFPANRLRISYRNYN AAMAKPEDARSPPALPLPSAPNGGKFKIALCQLSVTENKERNIAHARDA IEAAADNGAQLVVLPEIWNGPYSNASFPVYAEDIDAGGSASPSTSMLSE VARSKGITIVGGSISERSGDHLYNTCCIFGKDGELKAKHRKIHLFDIDI PGKISFMESKTLTAGNTPTIVDTDVGRIGIGICYDIRFQELAMLYAARG AHLICYPGAFNMTTGPLHWELLQRARAIDNQLYVATCSPARDINAGYVA WGHSTLVAPFGEIVATTEHEEATVIADIDYSRIEERRMNMPLEKQRHGD LYQLVDVSRLDTAKH
[0076] Oryza sativa amino acid sequence (Genbank accession NP--001049134.1) [SEQ ID NO: 8]:
TABLE-US-00007 MATAASFRPEAARSPPAVQPPAPPLSKFKVALCQLSVTADKARNIARAR EAIEAAAAGGAKLVLLPEIWNGPYSNDSFPEYAEDIEAGGDAAPSFSMM SEVARSLQITLVGGSISERSGNKLYNTCCVFGSDGELKGKHRKIHLFDI DIPGKITFKESKTLTAGQDLTVVDTDVGRIGIGICYDIRFQELAMLYAA RGAHLLCYPGAFNMTTGPLHWELLQRARAADNQLFVATCAPARDTSAGY IAWGHSTLVGPFGEVIATAEHEETTIMAEIDYSLIDQRRQFLPLQYQRR GDLYQLVDVQRSGSDE
[0077] Sorghum bicolor amino acid sequence (Genbank accession XP--002468410.1) [SEQ ID NO: 9]:
TABLE-US-00008 MRAAAAAAATSTAAALLAPGLKLCAGRARVSSCRLPLRRVAAMASAPNS SFRPEEARSPPALELPTPPLSKFKVALCQLSVTADKSRNIAHARAAIEK AASDGAKLVLLPEIWNGPYSNDSFPEYAEDIEAGGDAAPSFSMMSEVAR SLQITLVDGQLKGKHRKIHLFDIDIPGKITFKESKTLTAGQSPTVVDTD VGRIGIGICYDIRFQELAMLYAARGAHLLCYPGAFNMTTGPLHWELLQR ARQPAVCCNVRSSSRYQCRLCCLGTLHACWTFWRGDCNN
[0078] Ricinus communis amino acid sequence (Genbank accession XP--002516116.1) [SEQ ID NO: 10]:
TABLE-US-00009 MSASFNPEQARSPPALPLPTPPLTKAQFLLTSYLTILIYMIFKIGLCQL LVTPDKAKNIAHARKAIEEAAAKGAKLVLLPEIWNSPYSNDSFPVYAED IDAGHVASPSTAMLSQLARLLNITIVGGSIPERSGDRLYNTCCVFDTQG NLIAKHRKIHLFDIDIPGKITFIESKTLTAGETPNIVDTEVGRIGIGIC YDIRFQELAVLYAARGAHLICYPGAFNMTTGPLHWELLQRARAADNQLY VATCSPARDVGAGYVAWGHSTLVGPFGEILATTEHEQDIIIAEIDYSLI ELRSQLSTTHLPLPTPTTTRDSTIEEEDDLVYIYI
[0079] Physcomitrella patens subsp. patens amino acid sequence (Genbank accession XP--001766085.1) [SEQ ID NO: 11]:
TABLE-US-00010 MASDFQPHMARQPPSESLPNAPNGGKYKLAVCQLSVTSDKAANIAHARQ KIEAAADSGAQLIVLPEMWNCPYSNDSFPTYAEDIDAGLEASPSSHMLS EVARKKKVTIVGGSIPERNDGKLYNTCCVFDKNGELKAKFRKIHLFDID IPGKITFKESDTLTPGEGLCVVDTDVGRIAVGICYDIRFPEMAMLYSAR GAHIICYPGAFNMTTGPLHWELLQKARAVDNQIFVVTCSPARDTEAGYI AWGHSTVVGPFGEILATTEHEEATIFADIDYSQLDTRRQNMPLESQRR GDLYHLIDVTRKDTVKSS
[0080] Selaginella moellendorffii amino acid sequence (Genbank accession XP--002969787.1) [SEQ ID NO: 12]:
TABLE-US-00011 MPSSRYFWFLWQFKLAVCQLSICADKEQNIRHAREAIQTAADGGSKLVL LPEMWNCPYSNASFPIYAEDIDAGDSPSSKMLSDMAKSKEVTIIGGSIP ERSGNHLYNTCCIYGKDGSLKGKHRKVHLFDIDIPGKIQFKESDTLTPG DKYTVVDTDVGRIGVGICYDIRFPEMAMTYAARGVHMICYPGAFNMTTG PAHWELLQKARAVDNQLFVATCSPARNPSAGYVAWGHSSVIGPFGEILA STGREEAIFYADIDYAQIKERRMNMPLDHQRRGDLYQLVDLTFTT
[0081] Medicago truncatula amino acid sequence (Genbank accession ACJ85250.1) [SEQ ID NO: 13]:
TABLE-US-00012 MAASSINSELARSPPAIPLPTPPLTNFKIGLCQLSVTSDKDKNIAHART AIQDAAAKGAKLILLPEIWNSPYSNDSFPVYAEDIDAGGDASPSTAMLS ELSSLLKITIVGGSIPERSGDRLYNTCCVFGTDGKLKAKHRKIHLFDID IPGKITFIESLTLTAGDTPTIVDTEVGRIGIGICYDIRFPELAMIYAAR GAHLLCYPGAFNMTTGPLHWELLQRARATDNQLYVATCSPARDTTGWLC GLGVTPLLLVLLEKFWLLQNARRQPL
[0082] Chlorella variabilis amino acid sequence (Genbank accession EFN54567.1) [SEQ ID NO: 14]:
TABLE-US-00013 MQALAKGMALVGVAGLSAAAGRRAACLRPLSSYTSATADVIDPPPPQKV PPPLPCCRCRHCCHRLASNQQLARPLLAGPSAQIKVALCQLAVGADKQA NLTTARSAIEEAATAGADLVVLPEMWNCPYSNDSFPTYAEDVEAGDSPS TSMLSAAAAANRVVLVGGSIPERANGGRLYNTCFVYGRDGRLLGRHRKV HLFDIDIPGKITFKESLTLTPGEGLTVVGRLGIGICYDIRFPELALLYA ARGVQLIVYPGAFNMTTGPVHWELLQRARAVDGQLFVATCSPARSEGTG YIAWGHSTAVGPFAEVLATTDEKAGIVYCHMDFAQLGERRANMPLRHQK RADLYSLLDLTRPNSLSNAGLHNGPVQRTLAGSSGIVGSGITRQLLMEG AKVVALLRKVDQKAGLLRDCQGAPIENLYPAVVEDVSKEEQCAAFVHEV VEQHGAIDHAVSCFGAWWQGGLLTEQSYAEFSRVLANFAGSHFTFVKYI LPAMRQSHTSSMLFVTGGVGKRVLSADSGLATVGGAALYGIVRAAQAQY QGRPPRINELRIFALVTRHGEMPRSHSSIVEGLRAHSNRKVGNLAAEAL AAAADDELLEVTSERLDGVMLMVGD
[0083] Volvox carteri f. nagariensis amino acid sequence (Genbank accession XP--002948137.1) [SEQ ID NO: 15]:
TABLE-US-00014 MHVTADKAQNLQTAKRAIEDAAAQGAKLVVLPEMWNCPYSNDSFPTYAE DIEGGASGSVAMLSAAAAAACVTLVAGSIPERCGDRLYNTCCVFNSRGE LLAKHRKVHLFDIDIPGKITFKESLTLSPGPGPTVVDTEAGRLGIGICY DIRFPELAQLYAARGCQVLIYPGAFNMTTGPVHWELLARARAVDNQIFV ITCSPARNPSSSYQAWGHSTVVGPFAEILATTDHQPGTIYTELDYSQLA ERRANMPLRQQKRHDLYVLLDKTA
[0084] Chlamydomonas reinhardtii amino acid sequence (Genbank accession XP--001690839.1) [SEQ ID NO: 19]:
TABLE-US-00015 KVALCQLHVTADKEQNLRTARKAIEDAAAAGAKLVVLPEMFNCPYSNDS FPTYAEDIEGGASGSVAALSAAAAAARVTLVAGSIPERCQGKLYNTCCV FDSSGKLLAKHRKVHLFDIDIPGKITFKESLTLSPGPGPTVVDTEAGRL GIGICYDIRFPELAQIYAARGCQVLIYPGAFNMTTGPVHWELLAKARAV DNQVFVLTCSPARNPDSSYQAWGHSTALGPFAEVLATTEHSPATVFAEL DYAQLDERRAAMPLRQQKRHDLYLLLDKTA
[0085] Micromonas pusilla CCMP1545 amino acid sequence (Genbank accession XP--003064056.1) [SEQ ID NO: 20]:
TABLE-US-00016 MRATKTTAAAAAAAAASSSGAGAPVPFARVPAPWSASGASASDAATPTP TPAPRVVKVALCOLACPTADKVANIARAREAIRNAAEGGAALVVLPEMW NCPYANESFPAHAETIGANDPTPSVTMLSEAAAAHDIVLVGGSIPERGV GVGGGGGADEEDVLYNACCVFDGKRGLIARHRKTHLFDVDIPGEISFRE SDTLTEGEGLTVVDTAVGRVGVGICFDVRFGEMAAAMANRGADVLIYPG AFNTVTGPHHWELLQRARAVDNQARSIHWSPYDRCFVLTCSPARNTTGE GYQAWGHSTAVGPFAEVLATTDERPGIVFADLDLGEVTRRRRNMPLATQ RRGDLYALHDLGAVRGDA
[0086] Ectocarpus siliculosus amino acid sequence (Genbank accession CBJ25483) [SEQ ID NO: 21]:
TABLE-US-00017 MFLAAARRASPILLSLAVKTSTTAAFCSPRLANARTNTAAGATRTAYAA CSISRNISLLSRPLSSMSASGASEGATAGAGSRRFVVAACQILCGSDKL ANIATAESAVRDAAAAGAQVVVLPECWNGPYDTASFPVYAEPVPDPQGD ETAADMPSAEQSPSAAMLCRAAAENKVWLVGGSVPEAGKDGGVYNTCIV VGPSGRIVAKHRKVHLFDIDVPGGITFKESDTLSPGDSITTVETPFGTI GVGICYDMRFPELSMAMRAAGSVLLCFPGAFNMTTGPAHWELLQRARAL MDNQCFVVTASPARNPDSKYQAWGHSSIVDPWGTVVATTEHEEALVAEV DVGRVAEVRTSIPVSLQKRPDLYRLELP
[0087] Phaeodactylum tricornutum CCAP 1 055/1 amino acid sequence (Genbank accession XP--002183613.1) [SEQ ID NO: 22]:
TABLE-US-00018 MSASRQNDDDDDDDPSVLRVALCQLPVTNDKAQNHQTAREYLNRAANQG ARLVVLPEIWNSPYATAAFPEYAEQLPDVLAQDGDGHTGVYESPSADLL RESAKEHKLWIVGGSIPERDDDDKIYNTSLVFDPQGNLVAKHRKMHLFD IDVPGGITFFESDTLSPGNTVSHFATPWGNIGLGICYDIRFPEYAMLLA KEHDCGILIYPGAFNLTTGPAHWELLQRGRAVDNQCFVLTASPARTEPP SKAGLYPHYTAWGHSTAVSPWGEVIATTNEKAGIVFADLDLSKVTEMRT SIPIGKQKRTDLYQLVGKS
[0088] Schizosaccharomyces pombe 972h amino acid sequence (Genbank accession NP--594154.1) [SEQ ID NO: 23]:
TABLE-US-00019 MNSKFFGLVQKGTRSFFPSLNFCYTRNIMSVSASSLVPKDFRAFRIGLV QLANTKDKSENLQLARLKVLEAAKNGSNVIVLPEIFNSPYGTGYFNQYA EPIEESSPSYQALSSMAKDTKTYLFGGSIPERKDGKLYNTAMVFDPSGK LIAVHRKIHLFDIDIPGGVSFRESDSLSPGDAMTMVDTEYGKFGLGICY DIRFPELAMIAARNGCSVMIYPGAFNLSTGPLHWELLARARAVDNEMFV ACCAPARDMNADYHSWGHSTVVDPFGKVIATTDEKPSIVYADIDPSVMS TARNSVPIYTQRRFDVYSEVLPALKKEE
[0089] Aspergillus oryzae RIB40 amino acid sequence (Genbank accession XP--001819629.1) [SEQ ID NO: 24]:
TABLE-US-00020 MAALLKQPLKLALVQLASGADKAVNLAHARTKVLEAAQAGAKLIVLPEC FNSPYGTQYFPKYAETLLPSPPTEDQSPSYHALSAIAAEAKAYLVGGSI PELEPTTKKYYNTSLVFSPTGSLIGTHRKTHLFDIDIPGKITFKESEVL SPGNQLTIVDLPDYGKIGLAICYDIRFPEAAMIAARKGAFALIYPGAFN MTTGPMHWSLLARARAVDNQLYVGLCSPARDMEATYHAWGHSLIANPAA EVLVEAEDKETIVYADLDNDTIQSTRKGIPVYTQRRFDLYPDVSAEK
[0090] Neurospora crassa OR74A amino acid sequence (Genbank accession XP--960906.1) [SEQ ID NO: 25]:
TABLE-US-00021 MASSTKHPILLKKPVKLACIQLASGADKSANLSHAADKVREAASGGANI VVLPECFNSPYGCDFFPSYAEQLLPSPPTVEQSPSFHALSAMARDNGIY LVGGSIPELAIEEGTEDKKTYYNTSLVFGPDGKLLASHRKVHLFDIDIP GKIKFKESDVLSPGNSVTLVDLPDYGRIAVAICYDIRFPELAMIAARKG CFALVYPGAFNTTTGPLHWRLQGQARAMDNQIYVALCSPARDISASYHA YGHSLIVDPMARVLVEAEESETIVSAELDGTKIEEARSGIPLRDQRRF DIYPDVSQAKPFF
[0091] Rhizobium leguminosarum by. viciae 3841 amino acid sequence (Genbank accession YP--769862.1) [SEQ ID NO: 26]:
TABLE-US-00022 MSFKAAAVQMCSGVDPVRNAAAMARLVREAAGQGAIYVQTPEMTGMLQR DRAAARAVLADEAHDIIVKTGSDLARELGIHMHVGSTAIALADGKIANR GFLFGPDGRILNRYDKIHMFDVDLDNGESWRESAAYTAGSEARVLSLPF AEMGFAICYDVRFPALFRAQAMAGAEVMTVPAAFTKQTGEAHWEILLRA RAIENGVFVIAAAQAGRHEDGRESFGHSMIIDPWGTVLASAGATGEAVI VAEIDPSAVKAAHDKIPNLRNGREFSVEKIAGAIAGGVAA
[0092] Rhizobium etli CFN 42 amino acid sequence (Genbank accession YP--471237.1) [SEQ ID NO: 27]:
TABLE-US-00023 MSFKAAAIQMCSGVDPVKNAASMARLVREAAAQGATYVQTPEMTGMLQR DRAAARAVLADEAHDIIVKTGSELARELGIHVHVGSTAIALSDGKIANR GFLFGPDGRILNRYDKIHMFDVDLDNGESWRESAAYTAGSEARVLSLPF AEMGFAICYDVRFPALFRAQAVAGAEVMTVPSSFSRQTGEAHWEILLRA RAIENGVFVIAAAQAGRHEDGRETFGHSIIIDPWGTVLASAGATGEAVI LAEIDPGAVKAAHDKIPNLRDGREFSVEKIAGAVAGGVAA
[0093] Rhizobium leguminosarum by. trifolii WSM1325 amino acid sequence (Genbank accession YP--002977603.1) [SEQ ID NO: 28]:
TABLE-US-00024 MSFKAAAVQMCSGVDPVKNAAAMARLVREAAGQGATYVQTPEMTGMLOR DRTAARAVLADEAHDIIVKTGSELAIELGIHMHVGSTAIALADGKIANR GFLFGPDGRVLNRYDKIHMFDVDLDNGESWRESAAYTAGSEARVLSLPF AEMGFAICYDVRFPALFCAQAVAGAEVMTVPAAFTKQTGEAHWEILLRA RAIENGVFVIAAAQAGRHEDGRETFGHSMIIDPWGTVLASAGATGEAVI VAEIDPAAVKAAHDKIPNLRNGREFSVEKIAGAIAGGVAA
[0094] Bradyrhizobium sp. ORS278 amino acid sequence (Genbank accession YP--001202760.1) [SEQ ID NO: 29]:
TABLE-US-00025 MSNDRSFTAAMVQMRTALLPEPSLEQGTRLIREAVAQGAQYVQTPEVSN MMQLNRTALFEQLKSEEEDPSLKAYRALAKELNIHLHIGSLALRFSAEK AVNRSFLIGPDGQVLASYDKIHMFDIDLPGGESYRESANYQPGETAVIS DLPWGRLGLTICYDVRFPALYRALAESGASFISVPSAFTRKTGEAHWHT LLRARAIETGCFVFAAAQCGLHENKRETFGHSLIIDPWGEILAEGGVEP GVILARIDPSRVESVRQTIPSLQHGRRFGIADPKGGPDYLHLVRGSA
[0095] Sinorhizobium meliloti BL225C amino acid sequence (Genbank accession ZP--07592670.1) [SEQ ID NO: 30]:
TABLE-US-00026 MPSSRYFWFLWQFKLAVCQLSICADKEQNIRHAREAIQTAADGGSKLVL LPEMWNCPYSNASFPIYAEDIDAGDSPSSKMLSDMAKSKEVTIIGGSIP ERSGNHLYNTCCIYGKDGSLKGKHRKVHLFDIDIPGKIQFKESDTLTPG DKYTVVDTDVGRIGVGICYDIRFPEMAMTYAARGVHMICYPGAFNMTTG PAHWELLQKARAVDNQLFVATCSPARNPSAGYVAWGHSSVIGPFGEILA STGREEAIFYADIDYAQIKERRMNMPLDHQRRGDLYQLVDLTFTT
[0096] Sinorhizobium meliloti 1021 amino acid sequence (Genbank accession NP--386723.1) [SEQ ID NO: 31]:
TABLE-US-00027 MTFKAAAVQICSGVDPAGNAETMAKLVREAASRGATYVQTPEMTGAVQR DRTGLRSVLKDGENDVVVREASRLARELGIYLHVGSTPIARADGKIANR GFLFGPDGAKICDYDKIHMFDVDLENGESWRESAAYHPGNTARTADLPF GKLGFSICYDVRFPELFRQQAVAGAEIMSVPAAFTRQTGEAHWEILLRA RAIENGLFVIAAAQAGTHEDGRETFGHSMIVDPWGRVLAEAGATGEEII VAEIDVAAVHAARAKIPNLRNARSFVLDEVVPVGK GGAAA
[0097] Phytophthora infestans T30-4 amino acid sequence (Genbank accession XP--002999170.1) [SEQ ID NO: 32]:
TABLE-US-00028 MLGRTIRSQARHLRSPFLRLSSPMSTTAPKFKLALCQIAVGDDKQKNIA TATAAVTEAAQNAAQVVSLPECWNSPYATTSFPQYAEEIPEKKAALNEK EHPSTFALSQLAAKLQIFLVGGSIPEKDATGKVYNTSVIFSPEGEILGK HRKVHLFDIDVPGKITFKESDTLSPGNSMTLFDTPYGKMGVGICYDIRF PELSMLMKKQGAKVLLFPGAFNLTTGPAHWELLQRARAVDNQLYVAATS PARGPEGGYQAWGHSTVISPWGEVVATCGHGESIVYAEVDLEKVEEMRR NIPTTNQTRSDLYELVQK
[0098] Homo sapiens amino acid sequence (Genbank accession NP--064587.1) [SEQ ID NO: 33]:
TABLE-US-00029 MTSFRLALIQLQISSIKSDNVTRACSFIREAATQGAKIVSLPECFNSPY GAKYFPEYAEKIPGESTQKLSEVAKECSIYLIGGSIPEEDAGKLYNTCA VFGPDGTLLAKYRKIHLFDIDVPGKITFQESKTLSPGDSFSTFDTPYCR VGLGICYDMRFAELAQIYAQRGCQLLVYPGAFNLTTGPAHWELLQRSRA VDNQVYVATASPARDDKASYVAWGHSTVVNPWGEVLAKAGTEEAIVYSD IDLKKLAEIRQQIPVFRQKRSDLYAVEMKKP
[0099] Equus caballus amino acid sequence (Genbank accession XP--001502234.1) [SEQ ID NO: 34]:
TABLE-US-00030 MAAHSILDLSGLDRESQIDLQRPLKARPGKAKDLSSGSACTFRLALIQL QVSSVKSDNLTRACGLVREAAAQGAKIVCLPECFNSPYGTNYFPQYAEK IPGESTQKLSEVAKECSIYLIGGSIPEEDAGKLYNTCAVFGPDGALLVK HRKLHLFDIDVPGKITFQESKTLSPGDSFSTFDTPYCRVGLGICYDLRF AELAQIYAQRGCQLLVYPGAFNLTTGPAHWELLQRGRAVDNQVYVATAS PARDDKASYVAWGHSTVVTPWGEVLATAGTEEMIV YSDIDLKKLAEIR QQIPIFSQKRLDLYAVEAKKP
[0100] Xenopus (Silurana) tropicalis amino acid sequence (Genbank accession NP--001016633.1) [SEQ ID NO: 35]:
TABLE-US-00031 MAKFRLSLVQFLVSPVKSENLNRACKLIKEAAQKGAQIVALPECFNSPY GTKYFPEYAEKIPGESTERLSQVAKECGIYLIGGSIPEEDSGKLYNTCA VFGPDGTLLVKHRKIHLFDIDVPGKIRFQESETLSPGDSFSVFETPYCK VGVGICYDIRFAELAQLYSKKGCQLLVYPGAFNMTTGPAHWELLQRARA LDNQVYVATASPARDEKASYVAWGHSTIVSPWGEVIAKAGSEETVISAD IDLEYLAEIREQIPIRRQRRHDLYSVEEKKN
[0101] Danio rerio amino acid sequence (Genbank accession AAQ97821.1) [SEQ ID NO: 36]:
TABLE-US-00032 MSKFRLAVVQLHVSKIKADNLGRAQTLVTEAAGQGAKVVVLPECFNSPY GTGFFKEYAEKIPGESTQVLSETAKKCGIYLVGGSIPEEDGGKLYNTCS VFGPDGTLLVTHRKIHLFDIDVPGKIRFQESETLSPGKSLSMFETPYCK VGVGICYDIRFAELAQIYAKKGCQLLVYPGAFNMTTGPAHWELLQRGRA VDNQVYVATASPARDETASYVAWGHSSVINPWGEVISKAGSEESVVYAD IDLQYLADVRQQIPITKQRRNDLYSVNSVQEG
[0102] Nematostella vectensis amino acid sequence (Genbank accession XP--001622809.1) [SEQ ID NO: 37]:
TABLE-US-00033 MAVPILVFRIGLVQLAVTANKLQNLQRAREKIKEAVAAGAKIVALPECF NSPYGTQYFKDYAEEIPGESSNMLAEVAKETGAYIVGGSIPERASNGKL YNTSLSYDPSGNLMGKHRKIHLFDIDVPGKIRFQESEVLSPGENLTILD TEYCKIGIGICYDMRFPELAQLYAKKGCHLLLYPGAFNMTTGPAHWELL TRARALDNQLYVATISPARDDNATYIAWGHSTVVNPWGKIVSKADHTEQ ILYAEIDLKYLNEVRSQIPVQFQKRDDVYELQVK
[0103] Mus musculus amino acid sequence (Genbank accession NP--075664.1) [SEQ ID NO: 38]:
TABLE-US-00034 MSTFRLALIQLQVSSIKSDNLTRACSLVREAAKQGANIVSLPECFNSPY GTTYFPDYAEKIPGESTQKLSEVAKESSIYLIGGSIPEEDAGKLYNTCS VFGPDGSLLVKHRKIHLFDIDVPGKITFQESKTLSPGDSFSTFDTPYCK VGLGICYDMRFAELAQIYAQRGCQLLVYPGAFNLTTGPAHWELLQRARA VDNQVYVATASPARDDKASYVAWGHSTVVDPWGQVLTKAGTEETILYS DIDLKKLAEIRQQIPILKQKRADLYTVESKKP
[0104] The Arabidopsis thaliana ω-amidase amino acid sequence was aligned with the amino acid sequence of other putative plant Ω-amidases and with other putative animal Ω-amidases to identify conserved regions. The results of the sequence alignments are shown in FIG. 2 and FIG. 3. Regions of homology are depicted in color shading and in the consensus sequences (SEQ ID NO: 44 for FIG. 2, and SEQ ID NO: 45 for FIG. 3).
[0105] Additional homologs of the Arabidopsis thaliana Ω-amidase are listed in Table 1 below.
TABLE-US-00035 TABLE 1 Genbank Accession No. Source Organism AAL91613.1 Arabidopsis thaliana NP_196765.2 Arabidopsis thaliana XP_002871509.1 Arabidopsis lyrata subsp. lyrata NP_974769.1 Arabidopsis thaliana XP_002309478.1 Populus trichocarpa XP_002279687.1 Vitis vinifera NP_001146676.1 Zea mays NP_001146295.1 Zea mays NP_001049134.1 Oryza sativa Japonica Group XP_002516116.1 Ricinus communis XP_001766085.1 Physcomitrella patens subsp. patens XP_001756522.1 Physcomitrella patens subsp. patens XP_002969787.1 Selaginella moellendorffii XP_002985119.1 Selaginella moellendorffii XP_002948137.1 Volvox carteri f. nagariensis XP_001690839.1 Chlamydomonas reinhardtii NP_001057093.1 Oryza sativa Japonica Group XP_002468410.1 Sorghum bicolor NP_064587.1 Homo sapiens XP_001089575.2 Macaca mulatta XP_001502234.1 Equus caballus XP_002502298.1 Micromonas sp. RCC299 XP_526254.2 Pan troglodytes XP_535718.2 Canis familiaris XP_002716659.1 Oryctolagus cuniculus NP_001033222.1 Bos taurus NP_001029298.1 Rattus norvegicus NP_001016633.1 Xenopus (Silurana) tropicalis NP_001085409.1 Xenopus laevis XP_002758928.1 Callithrix jacchus XP_003064056.1 Micromonas pusilla CCMP1545 NP_001135127.1 Salmo salar XP_001622809.1 Nematostella vectensis NP_991174.2 Danio rerio XP_002594716.1 Branchiostoma floridae NP_075664.1 Mus musculus XP_001370849.1 Monodelphis domestica NP_001090454.1 Xenopus laevis XP_002999170.1 Phytophthora infestans T30-4 XP_002917137.1 Ailuropoda melanoleuca XP_002741281.1 S accoglossus kowalevskii XP_002131764.1 Ciona intestinalis NP_594154.1 Schizosaccharomyces pombe 972h- XP_001742101.1 Monosiga brevicollis MX1 XP_416604.2 Gallus gallus XP_002194275.1 Taeniopygia guttata XP_001599587.1 Nasonia vitripennis XP_002410555.1 Ixodes scapularis XP_003035898.1 Schizophyllum commune H4-8 XP_002183613.1 Phaeodactylum tricornutum CCAP 1055/1 XP_001875493.1 Laccaria bicolor S238N-H82 XP_002112209.1 Trichoplax adhaerens XP_636983.1 Dictyostelium discoideum AX4 XP_002158547.1 Hydra magnipapillata XP_002839272.1 Tuber melanosporum Mel28 XP_307722.3 Anopheles gambiae str. PEST XP_001819629.1 Aspergillus oryzae RIB40 Aspergillus flavus NRRL3357 XP_001268376.1 Aspergillus clavatus NRRL 1 ZP_08115581.1 Desulfotomaculum nigrificans DSM 574 YP_001320997.1 Alkaliphilus metalliredigens QYMF XP_369268.1 Magnaporthe oryzae 70-15 XP_002626458.1 Ajellomyces dermatitidis SLH14081 XP_751200.1 Aspergillus fumigatus Af293 XP_001657673.1 Aedes aegypti XP_002173486.1 Schizosaccharomyces japonicus yFS275 XP_001212538.1 Aspergillus terreus NIH2624 XP_001258462.1 Neosartorya fischeri NRRL 181 XP_002434512.1 Ixodes scapularis XP_960906.1 Neurospora crassa OR74A XP_002847679.1 Arthroderma otae CBS 113480 XP_967861.1 Tribolium castaneum XP_002426154.1 Pediculus humanus corporis XP_003176259.1 Arthroderma gypseum CBS 118893 XP_500602.1 Yarrowia lipolytica XP_001428419.1 Paramecium tetraurelia strain d4-2 XP_003014235.1 Arthroderma benhamiae CBS 112371 XP_001393123.1 Aspergillus niger CBS 513.88 ZP_03608460.1 Methanobrevibacter smithii DSM 2375 XP_002147261.1 Penicillium marneffei ATCC 18224 ZP_03293831.1 Clostridium hiranonis DSM 13275 XP_002290043.1 Thalassiosira pseudonana CCMP1335 XP_003065597.1 Coccidioides posadasii C735 delta SOWgp XP_001588734.1 Sclerotinia sclerotiorum 1980 YP_001273073.1 Methanobrevibacter smithii ATCC 35061 > Methanobrevibacter smithii DSM 2374 XP_001552800.1 Botryotinia fuckeliana B05.10 XP_446414.1 Candida glabrata CBS 138 XP_002792830.1 Paracoccidioides brasiliensis Pb01 XP_001998501.1 Drosophila mojavensis YP_003780301.1 Clostridium ljungdahlii DSM 13528 NP_013455.1 Saccharomyces cerevisiae S288c XP_002404736.1 Ixodes scapularis YP_001086961.1 Clostridium difficile 630 ZP_05328587.1 Clostridium difficile QCD-63q42 ZP_05399936.1 Clostridium difficile QCD-23m63 Clostridium difficile NAP08 Clostridium difficile NAP07 YP_001113615.1 Desulfotomaculum reducens MI-1 XP_001247884.1 Coccidioides immitis RS XP_390426.1 Gibberella zeae PH-1 XP_003025334.1 Trichophyton verrucosum HKI 0517 XP_002052999.1 Drosophila virilis ZP_07325748.1 Acetivibrio cellulolyticus CD2 ZP_05349666.1 Clostridium difficile ATCC 43255
[0106] Accordingly, in certain embodiments, the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that is at least 75%, at least 80%, at least 85%, at last 90%, at last 91%, at last 92%, at last 93%, at last 94%, at last 95%, at last 96%, at last 97%, at last 98%, at last 99%, or 100% identical to an amino acid sequence encoded by a polypeptide selected from AAL91613.1, ACN30911.1, ABK22312.1, ACJ85250.1, AAQ97821.1, CBJ25483.1, EFN54567.1, NP--196765.2, XP--002871509.1, NP--974769.1, XP--002309478.1, XP--002279687.1, NP--001146676.1, NP--001146295.1, NP--001049134.1, XP--002516116.1,
[0107] XP--001766085.1, XP--001756522.1, XP--002969787.1, XP--002985119.1, XP--002948137.1, XP--001690839.1, NP--001057093.1, XP--002468410.1, NP--064587.1, XP--001089575.2, XP--001502234.1, XP--002502298.1, XP--526254.2, XP--535718.2, XP--002716659.1, NP--001033222.1, NP--001029298.1, NP--001016633.1, NP--001085409.1, XP--002758928.1, XP--003064056.1, NP--001135127.1, XP--001622809.1, NP--991174.2, XP--002594716.1,
[0108] NP--075664.1, XP--001370849.1, NP--001090454.1, XP--002999170.1, XP--002917137.1, XP--002741281.1, XP--002131764.1, NP--594154.1, XP--001742101.1, XP--416604.2, XP--002194275.1, XP--001599587.1, XP--002410555.1, XP--003035898.1, XP--002183613.1, XP--001875493.1, XP--002112209.1, XP--636983.1, XP--002158547.1, XP--002839272.1, XP--307722.3, XP--001819629.1, XP--001268376.1, ZP--08115581.1, YP--001320997.1, XP--369268.1, XP--002626458.1, XP--751200.1, XP--001657673.1, XP--002173486.1, XP--001212538.1, XP--001258462.1, XP--002434512.1, XP--960906.1, XP--002847679.1, XP--967861.1, XP--002426154.1, XP--003176259.1, XP--500602.1, XP--001428419.1, XP--003014235.1, XP--001393123.1, ZP--03608460.1, XP--002147261.1, ZP--03293831.1, XP--002290043.1, XP--003065597.1, XP--001588734.1, YP--001273073.1, XP--001552800.1,
[0109] XP--446414.1, XP--002792830.1, XP--001998501.1, YP--003780301.1, NP--013455.1, XP--002404736.1, YP--001086961.1, ZP--05328587.1, ZP--05399936.1, YP--001113615.1, XP--001247884.1, XP--390426.1, XP--003025334.1, XP--002052999.1, YP--769862.1, ZP--07325748.1, ZP--05349666.1, YP--471237.1, YP--002977603.1, YP--001202760.1, ZP--07592670.1, and NP--386723.1. In other embodiments, the Ω-amidase transgene is incorporated into the genome of the transgenic plants.
[0110] Certain aspects of the present disclosure relate to an Ω-amidase transgene that is operably linked to a root-preferred promoter. As used herein, a "root-preferred promoter" refers to expression driven by a promoter that is selectively enhanced in root cells or tissues, in comparison to one or more non-root cells or tissues. For example, a root-preferred promoter may preferentially drive high levels of expression of a gene in root cells or tissue but may still drive low levels of expression of the gene in other non-root cells or tissues, such as leaves. Root tissues include but are not limited to at least one of root cap, apical meristem, protoderm, ground meristem, procambium, endodermis, cortex, vascular cortex, epidermis, and the like.
[0111] In certain other embodiments, 2-oxoglutaramate levels in root tissues are decreased in order to increase the leaf-to-root ratio thereof by increasing the natural breakdown of 2-oxoglutaramate in root tissue. For example, the breakdown of 2-oxoglutaramate in root tissue may be increased by upregulating Ω-amidase activity in the roots. Thus, in one embodiment, an Ω-amidase transgene, including without limitation any of the Ω-amidase genes and coding sequences disclosed herein, is introduced into the plant under the control of a root-preferred promoter, such as the rolD promoter of Agrobacterium rhizogenes (Kamo and Bowers, 1999, Plant Cell Reports 18: 809-815 and references cited therein). The rolD promoter controls the expression of rolD, which functions to promote root elongation. GUS protein expression under control of the rolD-promoter has been shown to yield mainly root-preferred GUS expression (Leach and Aoyagi, 1991, Plant Sci. 79, 69-76). Root-preferred promoters may be either constitutive or inducible.
[0112] Additional constitutive and/or inducible root-preferred promoters include, without limitation, the RolD-2 promoter; glycine rich promoters (GRP); ADH promoters, including the maize ADH1 promoter (Kyozuka, J et al., 1994, The Plant Cell 6:799-810); PHT promoters, including the Pht1 gene family promoters (Schunmann et al., 2004, J. Experimental Botany 55:855-865); metal uptake protein promoters, including the maize metallothionein promoter (Diehn, S, 2006 Maize, U.S. Pat. Application Publication US 2006/0005275); the 35S CaMV domain A promoter (Elmayan, T and M. Tepfer. 1995, Transgenic Research 4:388-396); the pDJ3S, SIREO, and pMe1 promoters (Arango et al., Plant Cell Rep. 2010 June; 29 (6):651-9. Epub 2010 Apr. 6.); the Sad1 and Sad2 promoters (U.S. Patent Application Publication US 2008/0244791); the TobRB7 promoter (Yamamoto et al., 1991, Plant Cell 3:371-3); the RCc3 promoter (Xu et al., 1995, Plant Mol. Biol. 27:237-248); the FaRB7 promoter (Vaugn et al., Exp. Bot. (2006) 57 (14): 3901-3910); the SPmads promoter (Noh et al., American Society of Plant Biologists, Plant Biology 2005 Conference, Abstract #1097); the IDS2 promoter (Kobayashi et al., The Plant Journal, Vol. 36(6): 780-793, December 2003); the pyk10 promoter (Nitz et al., Plant Sci. 2001 July; 161(2):337-346); the Pt2L4 promoter (De Souza et al., Genet. Mol. Res. 8 (1): 334-344 (2009)); the Lbc3 leghemoglobin promoter (Bak, K, et al., 1993, The Plant Journal 4(3) 577-580); the PEPC promoter (Kawamura et al. 1990, J. Biochem 107: 165-168); the Gns1 Glucanase root promoter (Simmons, C et al., 1992, Plant Molecular Biology 18: 33-45), the 35S2promoter (Elmayan, T and M. Tepfer. 1995, Transgenic Research 4:388-396); the GI4 and GI5 promoters (European Patent EP 1 862 473 B1; and the GRP promoter. Additionally, any of the disclosed root-preferred promoters may be in a truncated form that contains only the core domain or functional domain of the promoter sufficient to drive expression in root tissue. Moreover, root-preferred promoters also include isoforms of any of the root-preferred promoters disclosed herein. For example, the RolD-2 promoter is one of several truncated isoforms of the RolD promoter described in Leach and Aoyagi, 1991, Plant Sci. 79, 69-76. Moreover, Leach and Aoyagi describe the RolD2 promoter as being a highly root-preferred promoter. Accordingly, a root-preferred promoter also includes any of the RolD isomers described by Leach and Aoyagi.
[0113] Thus, in certain embodiments, the root-preferred promoter is selected from RolD promoter, RolD-2 promoter, glycine rich protein promoter, GRP promoter, ADH promoter, maize ADH1 promoter, PHT promoter, Pht1 gene family promoter, metal uptake protein promoter, maize metallothionein protein promoter, 35S CaMV domain A promoter. pDJ3S promoter, SIREO promoter, pMe1 promoter, Sad1 promoter, Sad2 promoter, TobRB7 promoter, RCc3 promoter, FaRB7 promoter, SPmads promoter, IDS2 promoter, pyk10 promoter, Lbc3 leghemoglobin promoter, PEPC promoter, Gns1 glucanase root promoter, 35S2promoter, GI4 promoter, GI5 promoter, and GRP promoter.
[0114] In stable transformation embodiments, one or more copies of the Ω-amidase transgene become integrated into the genome of the transgenic plant, thereby providing increased Ω-amidase enzyme capacity into the root tissue of the plant, which serves to mediate synthesis of 2-oxoglutaramate, which in turn signals metabolic gene expression, resulting in increased nitrogen use efficiency, which in turn results in increased plant growth and the enhancement of other agronomic characteristics.
[0115] In other embodiments, root-preferred expression of the Ω-amidase transgene results in an increased leaf-to-root ratio of 2-oxoglutaramte relative to a plant of the same species that does not contain an Ω-amidase transgene. In certain preferred embodiments, the leaf-to-root ratio of 2-oxoglutaramate is at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, or more higher than that of a plant of the same species that does not contain an Ω-amidase transgene.
[0116] In further embodiments, the transgenic plant has increased nitrogen use efficiency. The disclosed transgenic plants with increased nitrogen use efficiency may further contain other transgenes known to increase nitrogen utilization efficiency, including without limitation those described in U.S. Pat. No. 7,560,626.
[0117] Impairing the Breakdown of 2-Oxoglutaramate in Leaf Tissues by Inhibiting Ω-Amidase Activity:
[0118] Other aspects of the present disclosure relate to transgenic plants having inhibited expression of endogenous Ω-amidase in leaf tissue.
[0119] Accordingly, in certain embodiments, the breakdown of 2-oxoglutaramate or its analogs may be impaired by decreasing Ω-amidase activity in order allow accumulation of 2-oxoglutaramate in the leaves, thereby increasing the leaf-to-root ratio. More specifically, the following approaches to impeding the 2-oxoglutaramate breakdown pathway may be used.
[0120] In one specific embodiment, the normal metabolic breakdown of 2-oxogluataramate catalyzed by an Ω-amidase enzyme, including but not limited to any of the Ω-amidase enzymes disclosed herein, is inhibited in leaf tissue of any of the disclosed transgenic plants by the application of a chemical inhibitor, including without limitation 6-diazo-5-oxo-nor-leucine, p-hydroxymercuribenzoate, diisopropyl fluorophosphates, sodium cyanide, phenylmercuriacetate, Iodoacetate, silver nitrate, chloromercuricphenylsulfonic acid, and copper sulfate. Accordingly, in certain embodiments, endogenous Ω-amidase expression in leaf tissue of any of the disclosed transgenic plants is inhibited by a chemical inhibitor selected from 6-diazo-5-oxo-nor-leucine, p-hydroxymercuribenzoate, diisopropyl fluorophosphates, sodium cyanide, phenylmercuriacetate, Iodoacetate, silver nitrate, chloromercuricphenylsulfonic acid, and copper sulfate.
[0121] In another embodiment, Ω-amidase function may be inhibited in leaf tissue of any of the disclosed transgenic plants by genetically impeding the transcription and/or translation of an Ω-amidase gene, including but not limited to the Ω-amidase genes and coding sequences disclosed herein. Methods for impeding Ω-amidase expression and function include, without limitation, recessive gene disruption and dominant gene silencing.
[0122] As used herein, "recessive gene disruption" refers to mutating a target Ω-amidase sequence to eliminate either expression or function. Methods for mutating a target sequence are known in the art, and include, without limitation, the generation of mutations via chemical or radiation damage followed by isolation of the mutant. In addition, known molecular biology approaches for decreasing the expression of a functional phenotype may be used, and include without limitation, various knockout or knockdown methods. These methods capitalize upon knowledge of sequence either in the gene of interest or in the DNA sequence flanking the gene. Such sequences are then examined to find suitable sequences that can be targeted to accomplish either excision of the target gene or fragments of the gene. Thus, in certain embodiments, the endogenous Ω-amidase expression in leaf tissue of any of the disclosed transgenic plants is inhibited by a recessive gene disruption selected from a mutant Ω-amidase gene that eliminates endogenous Ω-amidase expression, an endogenous Ω-amidase knockout mutant, and an endogenous Ω-amidase knockdown mutant.
[0123] As used herein, "dominant gene silencing" refers to inducing or destroying/inhibiting the mRNA transcript of the gene, a means which provides the benefit of being done in a spatial or temporal manner by the selection of specific promoters. Of the dominant gene silencing approaches, dsRNA-triggered RNAi is one of the most powerful and the most efficient at gene silencing, and allows one to enhance or capitalize upon a natural regulatory mechanism which destroys intact mRNA by providing an antisense oligonucleotide that is specific for an endogenous Ω-amidase gene (For review, see, Behlke, 2006, Molecular Therapy 13(4): 644-670; see also, Tang and Galili, 2004, Trends Biotechnology 22:463-469; Rajewsky and Socci, 2004, Developmental Biology 267:529-535; Hamilton et al., 2002, EMBO J. 21:4671-46794 In one embodiment, a construct comprising a suitable RNAi sequence under the control of a leaf specific promoter such as the RuBisCo small subunit promoter is introduced into the plant in order to silence Ω-amidase protein expression. Accordingly, in certain embodiments, the endogenous Ω-amidase expression in leaf tissue of any of the disclosed transgenic plants is inhibited by an RNAi antisense oligonucleotide that is specific for an endogenous Ω-amidase gene.
[0124] In certain embodiments, inhibition of endogenous Ω-amidase expression in leaf tissue results in an increased leaf-to-root ratio of 2-oxoglutaramte relative to a plant of the same species that does not comprise inhibited endogenous Ω-amidase expression in leaf tissue. In certain preferred embodiments, the leaf-to-root ratio of 2-oxoglutaramate is at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, or more higher than that of a plant of the same species that does not comprise inhibited endogenous Ω-amidase expression in leaf tissue. In further embodiments, the transgenic plant has increased nitrogen use efficiency.
[0125] Similarly, the expression of the substrate GS and/or the catalytic protein GPT may be impaired in root tissue using any of the approaches disclosed herein.
[0126] Embodiments Relating to Transgenic Plants with Increased Ω-Amidase Activity in Root Tissues and Inhibited Ω-Amidase Activity in Leaf Tissue:
[0127] Certain aspects of the present disclosure relate to transgenic plants with increased Ω-amidase expression in root tissue and inhibited endogenous Ω-amidase expression in leaf tissue, which results in an increased leaf-to-root ratio of 2-oxoglutaramte.
[0128] Accordingly, in certain embodiments, transgenic plants containing an Ω-amidase transgene that is operably linked to a root-preferred promoter, further have inhibited endogenous Ω-amidase expression in leaf tissue. Exemplary transgenic plants Ω-amidase transgenes, and root-preferred promoters are as described in previous sections. In other embodiments, the endogenous Ω-amidase expression in leaf tissue is inhibited by recessive gene disruption, dominant gene silencing, or a chemical inhibitor. In still other embodiments, the endogenous Ω-amidase expression in leaf tissue is inhibited by a recessive gene disruption selected a mutant Ω-amidase gene that eliminates endogenous Ω-amidase expression, an endogenous Ω-amidase knockout mutant, and an endogenous Ω-amidase knockdown mutant. In yet other embodiments, the endogenous Ω-amidase expression in leaf tissue is inhibited by an RNAi antisense oligonucleotide that is specific for an endogenous Ω-amidase gene. In further embodiments, the endogenous Ω-amidase expression in leaf tissue is inhibited by a chemical inhibitor selected from 6-diazo-5-oxo-nor-leucine, p-hydroxymercuribenzoate, diisopropyl fluorophosphates, sodium cyanide, phenylmercuriacetate, Iodoacetate, silver nitrate, chloromercuricphenylsulfonic acid, and copper sulfate. In further embodiments, the transgenic plant has an increased leaf-to-root ratio of 2-oxoglutaramte. In certain preferred embodiments, the leaf-to-root ratio of 2-oxoglutaramate is at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, or more higher than that of an unmodified plant of the same species. In still further embodiments, the transgenic plant has increased nitrogen use efficiency.
[0129] In certain embodiments, transgenic plants having inhibited expression of endogenous Ω-amidase in leaf tissue relative to a plant of the same species that does not comprise inhibited expression of endogenous Ω-amidase in leaf tissue, where the endogenous Ω-amidase expression in leaf tissue is inhibited by recessive gene disruption or dominant gene silencing of at least one endogenous Ω-amidase gene, further contain an Ω-amidase transgene, where the Ω-amidase transgene is operably linked to a root-preferred promoter. Exemplary transgenic plants, recessive gene disruption, dominant gene silencing, Ω-amidase transgenes, and root-preferred promoters are as described in previous sections. In further embodiments, the transgenic plant has an increased leaf-to-root ratio of 2-oxoglutaramte. In certain preferred embodiments, the leaf-to-root ratio of 2-oxoglutaramate is at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, or more higher than that of an unmodified plant of the same species. In still further embodiments, the transgenic plant has increased nitrogen use efficiency.
[0130] Expression of Glutamine Phenylpyruvate Transaminase and Glutamine Synthetase:
[0131] Other aspects of the present disclosure relate to transgenic plants with increased Ω-amidase expression in root tissue or inhibited endogenous Ω-amidase expression in leaf tissue that further contain increased expression of glutamine phenylpyruvate transaminase (GPT) and/or glutamine synthetase (GS).
[0132] In a particular embodiment, any of the transgenic plants disclosed herein further over-express the GPT protein, which is directly involved in the synthesis of 2-oxoglutaramate, which results in higher leaf-to-root ratios of the 2-oxoglutaramate compound. In a related embodiment, any of the transgenic plants disclosed herein further over-express the GPT protein and the GS protein. These transgenic plants further containing GPT and GS have an even higher leaf-to-root ratios of 2-oxoglutaramate, resulting in a further increase in nitrogen use efficiency. This increase in nitrogen use efficiency also results in plants that grow faster, produce greater seed and fruit/pod yields, display earlier and more productive flowering, demonstrate increased tolerance to high salt conditions, and produce superior biomass yields (See, co-owned, co-pending U.S. patent application Ser. No. 12/551,271).
[0133] More particularly, applicants have determined that the over-expression of GPT and GS in transgenic plants results in a disproportionate increase in the relative concentrations of 2-oxoglutaramate in the foliar and below ground tissues. Moreover, the ratio of the concentration of 2-oxoglutaramate in above ground tissue to below ground tissue (leaf-to-root ratio) is positively correlated with plant biomass. Faster growing, larger genetically-engineered plants have a greater ratio of the concentrations of 2-oxoglutaramate in leaf tissue versus root tissue when compared to wild type plants. In one particular embodiment, transgenic tobacco plants carrying GPT and GS1 transgenes under the control of robust constitutive promoters showed substantially greater leaf-to-root ratios of 2-oxoglutaramate and demonstrated high growth phenotypes when compared to wild type tobacco plants. In particular, two transgenic tobacco lines over-expressing GPT and GS1 transgenes were found to have: (1) well over two-times the fresh weight of wild type plants, (2) two-times the 2-oxoglutaramate foliar concentration compared to the wild type plants, and (3) between two- and three-times the leaf-to-root ratio seen in wild type plants.
[0134] In a wild type or engineered plant, the ratio can be expected to reflect the relative concentrations of 2-oxoglutaramate, as well as glutamine, the substrate from which it is made, in the leaves versus the roots of the plant. The actual ratios would be expected to differ from species to species. This is due to the fact that leaves and roots house differing fractions of the nitrogen assimilation machinery and activity, as a function of plant species (Pate, 1980, Ann. Rev. Plant Physiol. 31: 313-340). In fact, some plant species assimilate most of their nitrogen in their roots, and thus have high amino acid concentrations in their roots and xylem sap, and lower concentrations in their leaves. Other plants assimilate most of their nitrogen in their leaves, and thus have high amino acid concentrations in their leaves, and lower concentrations in their roots. Plant species distribute themselves along a continuum of this distribution of labor and amino acid concentrations between leaves and roots.
[0135] In addition to the over-expression of natural GPT proteins in transgenic plant systems, genetically-engineered, enhanced GPT enzymes may be developed and used to improve 2-oxoglutaramate synthesis kinetics, thereby increasing the rate of 2-oxoglutaramate accumulation in leaves. The GPT enzyme may be broadly classed as being a member of aspartate amino transferase type enzymes, based on sequence homology with known well characterized aspartate amino transferase enzymes. The major gene sequence databases include this classification of the transferase enzymes as a part of their sequence analysis (Gen Bank for example). Characteristically these are vitamin B6-dependent enzymes which catalyze transamination reactions between an amino acid and a ketoacid. The kinetic properties of these many (1000) transaminases differ in such properties as substrate specificities, binding constants, maximal velocity (Vmax) and unimolecular turnover rates (Kcat). The specific arginine residues involved directly in the hydrogen-bonding of the substrate dicarboxlyic acid substrates have been highly conserved (Fotheringham et al., 1986, Biochem J. 234:593-604; Seville et al., 1988, Biochemistry 27:8344-8349: Jager et al., 1992, FEBS Lett. 306:234-238) and thus the changes in specificities and kinetic properties are often conferred by changes in other amino acid residues. The enzyme's performance has proven to be very sensitive to subtle changes in the structure of the residue, for example the addition of a single CH2 group in a residue not in direct contact with either substrate or co-factor (Jansonius and Vincent, 1987; Seville et al., 1988, supra). Various studies have shown that it is possible to change an aspartate amino transferase enzyme's properties with directed mutation of the wild type protein (Kohler et al., 1994, Biochemistry 33:90-97; Jager et al., 1994, Protein Engineering. 7:605-612).
[0136] Within the plant GPT sequences, the region NLGQGFP (SEQ ID NO: 18) is highly conserved (and completely conserved among soybean, grape, rice hordeum, and Arabidopsis sequences (See, U.S. patent application Ser. No. 12/551,271 and U.S. patent application Ser. No. 12/551,193). Applicants have used such a directed mutation approach to generate a mutant GPT from the natural Arabidopsis GPT, by substituting V (valine) for the F (phenylalanine) residue in the wild type sequence. The resulting GPT/F:V mutant was expressed in E. coli, using the common PET vector system, and showed improved maximal velocity and unimolecular turnover. Maximal velocity was determined using the formula: Vmax=Kcat[E] tot. The apparent unimolecular rate constant Kcat is also called turnover number and denotes the maximum number of enzymatic reactions catalyzed per second.
[0137] Vmax for the mutant increased to 6.04, a 20% increase over the wild type Vmax value of 5.07. Care was taken to assure that the same amount of protein was used in these experiments and thus the relationship of Vmax=Kcat[E]tot can be applied to show that the mutant's unimolecular turnover rate has increased. The glutamine Km for the mutant is 0.75 millimolar, a slight increase from the 0.30 mM Km measured for the wild type enzyme. This mutant GPT enzyme may be expected to produce more product, the 2-oxoglutaramate, per unit time than the wild type GPT when the concentration of the substrate glutamine is present in millimolar or greater quantities, thus assuring that the mutant is saturating. A survey of the plant and agricultural literature shows that well nourished plants contain millimolar glutamine concentrations (Dzuibany et al., 1998, Plants 206:515-522; Knight and Weissman, 1982, Plant Physiol 70:1683-1688; Sivasankar and Oaks, 1995, Plant Physiol. 107: 1225-1231; Yanagisawa et al., 2004, Proc. Natl. Acad. Sci. USA 101:7833-7838; Udy and Dennison, 1997, Australia J Experimental Marine Biology and Ecology 217:253-277).
[0138] The amino acid sequence of the GPT/F:V mutant protein is as follows (V substitution shown in bold) [SEQ ID NO: 1]:
TABLE-US-00036 MYLDINGVMIKQFSFKASLLPFSSNFRQSSAKIHRPIGATMTTVSTQNES TQKPVQVAKRLEKFKTTIFTQMSILAVKHGAINLGQGVPNFDGPDFVKE AAIQAIKDGKNQYARGYGIPQLNSAIAARFREDTGLVVDPEKEVTVTSG CTEAIAAAMLGLINPGDEVILFAPFYDSYEATLSMAGAKVKGITLRPPDF SIPLEELKAAVTNKTRAILMNTPHNPTGKMFTREELETIASLCIENDVLV FSDEVYDKLAFEMDHISIASLPGMYERTVTMNSLGKTFSLTGWKIGWA IAPPHLTWGVRQAHSYLTFATSTPAQWAAVAALKAPESYFKELKRDY NVKKETLVKGLKEVGFTVFPSSGTYFVVADHTPFGMENDVAFCEYL IEEVGVVAIPTSVFYLNPEEGKNLVRFAFCKDEETLRGAIERMKQKLK R KV
[0139] Two other substitution mutants were made at this residue, and one was also expressed in E. coli and analyzed kinetically (F:L mutation), but showed a higher (less desirable) Km value of 1.98 mM and a decreased Vmax value of 4.0.
[0140] In another approach to this aspect of the present disclosure, GPT and/or GS transgenes may be designed to utilize the codon usage preferred by the target plant species. Codon usage in plants is well established to vary particularly between monocots and dicots; in general, monocots seem to have higher GC usage overall with a very pronounced GC preference at the third base position (Kawabe and Miyashita, 2003, Genes & Genetic Systems 78(5): 343-52). Codon usage bias has been correlated with protein expression levels (Hiroaka et al., 2009). Thus one skilled in the art can refer to such sources as the Codon Usage Database or the work of Kawabe and Miyashita comparing several monocots and dicots or other genome sequence information and use or deduce the preferred codon usage for the target plant and simply design and synthesize the optimized gene sequence.
[0141] In yet a further approach to this aspect of the present disclosure, consensus engineering of the GPT and/or GS structures is used to generate consensus variants showing significant increases in protein stability in order to improve the amount of GPT activity in the plant. In this approach the native sequence is modified to more closely resemble a consensus sequence derived from the alignment of numerous proteins of a particular family (Schiller et al., 1994, J Mol. Biol. 240:188-192).
[0142] Accordingly, in certain embodiments, any of the transgenic plants with modulated Ω-amidase expression disclosed herein further contain a GPT transgene. In certain embodiments, the GPT transgene is a GPT/F:V mutant given by SEQ ID NO:1. In other embodiments, any of the transgenic plants disclosed herein further contain a GPT transgene and a GS transgene. In yet other embodiments, the GPT transgene and GS transgene are each operably linked to a leaf-preferred promoter. As used herein, a "leaf-preferred promoter" refers to expression driven by a promoter that is selectively enhanced in leaf cells or tissues, in comparison to one or more non-leaf cells or tissues. For example, a leaf-preferred promoter may preferentially drive high levels of expression of a gene in leaf cells or tissue but may also drive low levels of expression of the gene in other non-leaf cells or tissues, such as roots.
[0143] In other embodiments, any of the disclosed transgenes are codon optimized for expression in the plant. In further embodiments, the transgenic plant has an increased leaf-to-root ratio of 2-oxoglutaramte. In certain preferred embodiments, the leaf-to-root ratio of 2-oxoglutaramate is at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, or more higher than that of an unmodified plant of the same species. In still further embodiments, the transgenic plant has increased nitrogen use efficiency.
[0144] Increasing 2-Oxoglutaramate Biosynthesis in Leaf Tissues by Gene Activation:
[0145] As yet another approach, genes encoding proteins involved in the metabolic pathway which produces 2-oxoglutaramate, which genes may be "silent" and not expressed in particular cell type in a plant, may be activated, or turned-on, via gene activation methodologies, such as the homologous recombination methods developed by Transkaryotic Therapies, Inc. (see, for example U.S. Pat. No. 6,187,305). In these methods, the endogenous regulatory region of a gene is replaced with a regulatory sequence from a different gene or a novel regulatory whose presence in the cell results in expression of the gene. Such regulatory sequences may be comprised of promoters, enhancers, scaffold-attachment regions, negative regulatory elements, transcriptional initiation sites, regulatory protein binding sites or combinations of these sequences. As a result, an endogenous copy of a gene encoding a desired gene product is turned on and expressed, and an exogenous copy of the gene need not be introduced.
[0146] In a related embodiment, transcription factor upregulation or over-expression may be used to increase the transcription of genes which promote higher leaf-to-root ratios of 2-oxoglutaramate and/or its analogs. In one embodiment, the Dof-1 transcription factor is introduced as a transgene in order to induce the up-regulation of genes encoding enzymes for carbon skeleton production, a marked increase in amino acid content and a reduction in the glucose level, as previously reported in transgenic Arabidopsis. Over-expression of the Dof-1 transcription factor has been shown to improved nitrogen assimilation and growth under low-nitrogen conditions (Yanagisawa et al., 2004, PNAS 101:7833-7838). In this report, the transcription factor was expressed constitutively in the plant. Over expression of the Dof-1 transcription factor, alone, or in combination with other measures such as the over-expression of GS and GPT (or functionally-improved mutants thereof) in above-ground plant tissues can be expected to increase the leaf to root ratio of 2-oxoglutaramate.
[0147] Accordingly, in certain embodiments, any of the transgenic plants with modulated Ω-amidase expression disclosed herein also contain increased endogenous GPT expression, where the endogenous GPT expression is increased by gene activation. In still further embodiments, any of the transgenic plants disclosed herein contain increased endogenous GS expression, where the endogenous GS expression is increased by gene activation. In further embodiments, the transgenic plant has an increased leaf-to-root ratio of 2-oxoglutaramte. In certain preferred embodiments, the leaf-to-root ratio of 2-oxoglutaramate is at least two times, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, or more higher than that of an unmodified plant of the same species. In still further embodiments, the transgenic plant has increased nitrogen use efficiency.
[0148] Suitable Transgenic Plants:
[0149] Certain aspects of the present disclosure relate to transgenic plants. The transgenic plants disclosed herein may be any vascular plant of the phylum Tracheophyta, including angiosperms and gymnosperms. Angiosperms may be a monocotyledonous (monocot) or a dicotyledonous (dicot) plant. Important monocots include those of the grass families, such as the family Poaceae and Gramineae, including plants of the genus Avena (Avena sativa, oats), genus Hordeum (i.e., Hordeum vulgare, Barley), genus Oryza (i.e., Oryza sativa, rice, cultivated rice varieties), genus Panicum (Panicum spp., Panicum virgatum, Switchgrass), genus Phleum (Phleum pratense, Timothy-grass), genus Saccharum (i.e., Saccharum officinarum, Saccharum spontaneum, hybrids thereof, Sugarcane), genus Secale (i.e., Secale cereale, Rye), genus Sorghum (Sorghum vulgare, Sorghum), genus Triticum (wheat, various classes, including T. aestivum and T. durum), genus Fagopyrum (buckwheat, including F. esculentum), genus Triticosecale (Triticale, various hybrids of wheat and rye), genus Chenopodium (quinoa, including C. quinoa), genus Zea (i.e., Zea mays, numerous varieties) as well as millets (i.e., Pennisetum glaucum) including the genus Digitaria (D. exilis).
[0150] Important dicots include those of the family Solanaceae, such as plants of the genus Lycopersicon (Lycopersicon esculentum, tomato), genus Capiscum (Capsicum annuum, peppers), genus Solanum (Solanum tuberosum, potato, S. lycopersicum, tomato); genus Manihot (cassava, M. esculenta), genus Ipomoea (sweet potato, I. batatas), genus Olea (olives, including O. europaea); plants of the Gossypium family (i.e., Gossypium spp., G. hirsutum, G. herbaceum, cotton); the Legumes (family Fabaceae), such as peas (Pisum spp, P. sativum), beans (Glycine spp., Glycine max(soybean); Phaseolus vulgaris, common beans, Vigna radiata, mung bean), chickpeas (Cicer arietinum)), lentils (Lens culinaris), peanuts (Arachis hypogaea); coconuts (Cocos nucifera) as well as various other important crops such as camelina (Camelina sativa, family Brassicaceae), citrus (Citrus spp, family Rutaceae), coffee (Coffea spp, family Rubiaceae), melon (Cucumis spp, family Cucurbitaceae), squash (Cucurbita spp, family Cucurbitaceae), roses (Rosa spp, family Rosaceae), sunflower (Helianthus annuus, family Asteraceae), sugar beets (Beta spp, family Amaranthaceae), including sugarbeet, B. vulgaris), genus Daucus (carrots, including D. carota), genus Pastinaca (parsnip, including P. sativa), genus Raphanus (radish, including R. sativus), genus Dioscorea (yams, including D. rotundata and D. cayenensis), genus Armoracia (horseradish, including A. rusticana), genus Elaeis (Oil palm, including E. guineensis), genus Linum (flax, including L. usitatissimum), genus Carthamus (safflower, including C. tinctorius L.), genus Sesamum (sesame, including S. indicum), genus Vitis (grape, including Vitis vinifera), and plants of the genus Brassica (family Brassicaceae, i.e., broccoli, brussel sprouts, cabbage, swede, turnip, rapeseed B. napus, and cauliflower).
[0151] Other specific plants which may be transformed to generate the transgenic plants of the present disclosure include various other fruits and vegetables, such as apples, asparagus, avocado, banana, blackberry, blueberry, brussel sprout, cabbage, cotton, canola, carrots, radish, cucumbers, cherries, cranberries, cantaloupes, eggplant, grapefruit, lemons, limes, nectarines, oranges, peaches, pineapples, pears, plums, tangelos, tangerines, papaya, mango, strawberry, raspberry, lettuce, onion, grape, kiwi fruit, okra, parsnips, pumpkins, and spinach. In addition various flowering plants, trees and ornamental plants may be used to generate transgenic varietals, including without limitation lily, carnation, chrysanthemum, petunia, geranium, violet, gladioli, lupine, orchid and lilac.
[0152] In certain embodiments, the transgenic plant is selected from wheat, oats, rice, corn, bean, soybean, tobacco, alfalfa, Arabidopsis, grasses, fruits, vegetables, flowering plants, and trees.
[0153] Other aspects of the present disclosure also relate to a progeny of any generation of any of the transgenic plants disclosed herein. A further aspect relates to a seed of any generation of the transgenic plants disclosed herein.
[0154] Production of Transgenic Plants:
[0155] Certain aspects of the present disclosure relate to methods for generating transgenic plants with increased nitrogen use efficiency. Exemplary methods for the production of transgenic plants are described below. Further examples are described in co-owned, co-pending U.S. patent application Ser. Nos. 12/551,271, and 12/660,501, both of which are incorporated in their entireties by reference herein.
[0156] Transgene Constructs/Expression Vectors:
[0157] In order to generate the transgenic plants of the present disclosure, the gene coding sequence for the desired transgene(s) must be incorporated into a nucleic acid construct (also interchangeably referred to herein as a/an (transgene) expression vector, expression cassette, expression construct or expressible genetic construct), which can direct the expression of the transgene sequence in transformed plant cells. Such nucleic acid constructs carrying the transgene(s) of interest may be introduced into a plant cell or cells using a number of methods known in the art, including but not limited to electroporation, DNA bombardment or biolistic approaches, microinjection, and via the use of various DNA-based vectors such as Agrobacterium tumefaciens and Agrobacterium rhizogenes vectors. Once introduced into the transformed plant cell, the nucleic acid construct may direct the expression of the incorporated transgene(s) (i.e., Ω-amidase), either in a transient or stable fashion. Stable expression is preferred, and is achieved by utilizing plant transformation vectors which are able to direct the chromosomal integration of the transgene construct. Once a plant cell has been successfully transformed, it may be cultivated to regenerate a transgenic plant.
[0158] A large number of expression vectors suitable for driving the constitutive or induced expression of inserted genes in transformed plants are known. In addition, various transient expression vectors and systems are known. To a large extent, appropriate expression vectors are selected for use in a particular method of gene transformation (see, infra). Broadly speaking, a typical plant expression vector for generating transgenic plants will comprise the transgene of interest under the expression regulatory control of a promoter, a selectable marker for assisting in the selection of transformants, and a transcriptional terminator sequence.
[0159] More specifically, the basic elements of a nucleic acid construct for use in generating the transgenic plants of the present disclosure are: a suitable promoter, such as a root-preferred promoter, capable of directing the functional expression of the transgene(s) in a transformed plant cell, the transgene (s) (i.e., Ω-amidase coding sequence) operably linked to the promoter, preferably a suitable transcription termination sequence (i.e., nopaline synthetic enzyme gene terminator) operably linked to the transgene, and sometimes other elements useful for controlling the expression of the transgene, as well as one or more selectable marker genes suitable for selecting the desired transgenic product (i.e., antibiotic resistance genes).
[0160] As Agrobacterium tumefaciens is the primary transformation system used to generate transgenic plants, there are numerous vectors designed for Agrobacterium transformation. For stable transformation, Agrobacterium systems utilize "binary" vectors that permit plasmid manipulation in both E. coli and Agrobacterium, and typically contain one or more selectable markers to recover transformed plants (Hellens et al., 2000, Technical focus: A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446-451). Binary vectors for use in Agrobacterium transformation systems typically comprise the borders of T-DNA, multiple cloning sites, replication functions for Escherichia coli and A. tumefaciens, and selectable marker and reporter genes.
[0161] So-called "super-binary" vectors provide higher transformation efficiencies, and generally comprise additional virulence genes from a Ti (Komari et al., 2006, Methods Mol. Biol. 343: 15-41). Super binary vectors are typically used in plants which exhibit lower transformation efficiencies, such as cereals. Such additional virulence genes include without limitation virB, virE, and virG (Vain et al., 2004, The effect of additional virulence genes on transformation efficiency, transgene integration and expression in rice plants using the pGreen/pSoup dual binary vector system. Transgenic Res. 13: 593-603; Srivatanakul et al., 2000, Additional virulence genes influence transgene expression: transgene copy number, integration pattern and expression. J. Plant Physiol. 157, 685-690; Park et al., 2000, Shorter T-DNA or additional virulence genes improve Agrobacterium-mediated transformation. Theor. Appl. Genet. 101, 1015-1020; Jin et al., 1987, Genes responsible for the supervirulence phenotype of Agrobacterium tumefaciens A281. J. Bacteriol. 169: 4417-4425).
[0162] Plant Promoters:
[0163] In order to generate the transgenic plants of the present disclosure, the gene coding sequence for the desired transgene(s) is are operably linked to a promoter in order to drive expression of the transgene. A large number of promoters which are functional in plants are known in the art. In constructing Ω-amidase, GPT, or GS transgene constructs and Ω-amidase RNAi constructs, the selected promoter(s) may be constitutive, non-specific promoters such as the Cauliflower Mosaic Virus 35S ribosomal promoter (CaMV 35S promoter), which is widely employed for the expression of transgenes in plants. Examples of other strong constitutive promoters include without limitation the rice actin 1 promoter, the CaMV 19S promoter, the Ti plasmid nopaline synthase promoter, the alcohol dehydrogenase promoter and the sucrose synthase promoter.
[0164] Alternatively, in some embodiments, it may be desirable to select a promoter based upon the desired plant cells to be transformed by the transgene construct, the desired expression level of the transgene, the desired tissue or subcellular compartment for transgene expression, the developmental stage targeted, and the like. For example, a root-preferred promoter may include, without limitation, a RolD promoter, a RolD-2 promoter, a glycine rich protein promoter, a GRP promoter, an ADH promoter, a maize ADH1 promoter, a PHT promoter, a Pht1 gene family promoter, a metal uptake protein promoter, a maize metallothionein protein promoter, a 35S CaMV domain A promoter, a pDJ3S promoter, an SIREO promoter, a pMe1 promoter, an Sad1 promoter, an Sad2 promoter, a TobRB7 promoter, an RCc3 promoter, an FaRB7 promoter, an SPmads promoter, an IDS2 promoter, a pyk10 promoter, an Lbc3 leghemoglobin promoter, a PEPC promoter, a Gns1 glucanase root promoter, a 35S2promoter, a GI4 promoter, a GI5 promoter, and a GRP promoter
[0165] In addition to constitutive promoters, various inducible promoter sequences may be employed in cases where it is desirable to regulate transgene expression as the transgenic plant regenerates, matures, flowers, etc. Examples of such inducible promoters include promoters of heat shock genes, protection responding genes (i.e., phenylalanine ammonia lyase; see, for example Bevan et al., 1989, EMBO J. 8(7): 899-906), wound responding genes (i.e., cell wall protein genes), chemically inducible genes (i.e., nitrate reductase, chitinase) and dark inducible genes (i.e., asparagine synthetase; see, for example U.S. Pat. No. 5,256,558). Also, a number of plant nuclear genes are activated by light, including gene families encoding the major chlorophyll a/b binding proteins (cab) as well as the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) (see, for example, Tobin and Silverthorne, 1985, Annu Rev. Plant Physiol. 36: 569-593; Dean et al., 1989, Annu Rev. Plant Physiol. 40: 415-439.).
[0166] Other inducible promoters include ABA- and turgor-inducible promoters, the auxin-binding protein gene promoter (Schwob et al., 1993, Plant J. 4(3): 423-432), the UDP glucose flavonoid glycosyl-transferase gene promoter (Ralston et al., 1988, Genetics 119(1): 185-197); the MPI proteinase inhibitor promoter (Cordero et al., 1994, Plant J. 6(2): 141-150), the glyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al., 1995, Plant Mol. Biol. 29(6): 1293-1298; Quigley et al., 1989, J. Mol. Evol. 29(5): 412-421; Martinez et al., 1989, J. Mol. Biol. 208(4): 551-565) and light inducible plastid glutamine synthetase gene from pea (U.S. Pat. No. 5,391,725; Edwards et al., 1990, supra).
[0167] For a review of plant promoters used in plant transgenic plant technology, see Potenza et al., 2004, In Vitro Cell. Devel. Biol--Plant, 40(1): 1-22. For a review of synthetic plant promoter engineering, see, for example, Venter, M., 2007, Trends Plant Sci., 12(3): 118-124.
[0168] In certain embodiments, a 3' transcription termination sequence is also incorporated downstream of the transgene in order to direct the termination of transcription and permit correct polyadenylation of the mRNA transcript. Suitable transcription terminators are those which are known to function in plants, including without limitation, the nopaline synthase (NOS) and octopine synthase (OCS) genes of Agrobacterium tumefaciens, the T7 transcript from the octopine synthase gene, the 3' end of the protease inhibitor I or II genes from potato or tomato, the CaMV 35S terminator, the tml terminator and the pea rbcS E9 terminator. In addition, a gene's native transcription terminator may be used. In specific embodiments, described by way of the Examples, infra, the nopaline synthase transcription terminator is employed.
[0169] Selectable Markers:
[0170] Selectable markers are typically included in transgene expression vectors in order to provide a means for selecting plant transformants. While various types of markers are available, various negative selection markers are typically utilized, including those which confer resistance to a selection agent that inhibits or kills untransformed cells, such as genes which impart resistance to an antibiotic (such as kanamycin, gentamycin, anamycin, hygromycin and hygromycinB) or resistance to a herbicide (such as sulfonylurea, gulfosinate, phosphinothricin and glyphosate). Screenable markers include, for example, genes encoding β-glucuronidase (Jefferson, 1987, Plant Mol. Biol. Rep 5: 387-405), genes encoding luciferase (Ow et al., 1986, Science 234: 856-859) and various genes encoding proteins involved in the production or control of anthocyanin pigments (See, for example, U.S. Pat. No. 6,573,432). The E. coli glucuronidase gene (gus, gusA or uidA) has become a widely used selection marker in plant transgenics, largely because of the glucuronidase enzyme's stability, high sensitivity and ease of detection (e.g., fluorometric, spectrophotometric, various histochemical methods). Moreover, there is essentially no detectable glucuronidase in most higher plant species.
[0171] Transformation Methodologies and Systems:
[0172] Various methods for introducing the transgene expression vector constructs of the present disclosure into a plant or plant cell are well known to those skilled in the art, and any capable of transforming the target plant or plant cell may be utilized.
[0173] Agrobacterium-mediated transformation is perhaps the most common method utilized in plant transgenics, and protocols for Agrobacterium-mediated transformation of a large number of plants are extensively described in the literature (see, for example, Agrobacterium Protocols, Wan, ed., Humana Press, 2nd edition, 2006). Agrobacterium tumefaciens is a Gram negative soil bacteria that causes tumors (Crown Gall disease) in a great many dicot species, via the insertion of a small segment of tumor-inducing DNA ("T-DNA", `transfer DNA`) into the plant cell, which is incorporated at a semi-random location into the plant genome, and which eventually may become stably incorporated there. Directly repeated DNA sequences, called T-DNA borders, define the left and the right ends of the T-DNA. The T-DNA can be physically separated from the remainder of the Ti-plasmid, creating a `binary vector` system.
[0174] Agrobacterium transformation may be used for stably transforming dicots, monocots, and cells thereof (Rogers et al., 1986, Methods Enzymol., 118: 627-641; Hernalsteen et al., 1984, EMBO J., 3: 3039-3041; Hoykass-Van Slogteren et al., 1984, Nature, 311: 763-764; Grimsley et al., 1987, Nature 325: 167-1679; Boulton et al., 1989, Plant Mol. Biol. 12: 31-40; Gould et al., 1991, Plant Physiol. 95: 426-434). Various methods for introducing DNA into Agrobacteria are known, including electroporation, freeze/thaw methods, and triparental mating. The most efficient method of placing foreign DNA into Agrobacterium is via electroporation (Wise et al., 2006, Three Methods for the Introduction of Foreign DNA into Agrobacterium, Methods in Molecular Biology, vol. 343: Agrobacterium Protocols, 2/e, volume 1; Ed., Wang, Humana Press Inc., Totowa, N.J., pp. 43-53). In addition, given that a large percentage of T-DNAs do not integrate, Agrobacterium-mediated transformation may be used to obtain transient expression of a transgene via the transcriptional competency of unincorporated transgene construct molecules (Helens et al., 2005, Plant Methods 1:13).
[0175] A large number of Agrobacterium transformation vectors and methods have been described (Karimi et al., 2002, Trends Plant Sci. 7(5): 193-5), and many such vectors may be obtained commercially (for example, Invitrogen, Carlsbad, Calif.). In addition, a growing number of "open-source" Agrobacterium transformation vectors are available (for example, pCambia vectors; Cambia, Can berra, Australia). See, also, subsection herein on TRANSGENE CONSTRUCTS, supra. In a specific embodiment described further in the Examples, a pMON316-based vector was used in the leaf disc transformation system of Horsch et. al. (Horsch et al., 1995, Science 227:1229-1231) to generate growth enhanced transgenic tobacco and tomato plants.
[0176] Other commonly used transformation methods that may be employed in generating the transgenic plants of the present disclosure include, without limitation microprojectile bombardment, or biolistic transformation methods, protoplast transformation of naked DNA by calcium, polyethylene glycol (PEG) or electroporation (Paszkowski et al., 1984, EMBO J. 3: 2727-2722; Potrykus et al., 1985, Mol. Gen. Genet. 199: 169-177; Fromm et al., 1985, Proc. Nat. Acad. Sci. USA 82: 5824-5828; Shimamoto et al., 1989, Nature, 338: 274-276.
[0177] Biolistic transformation involves injecting millions of DNA-coated metal particles into target cells or tissues using a biolistic device (or "gene gun"), several kinds of which are available commercially. Once inside the cell, the DNA elutes off the particles and a portion may be stably incorporated into one or more of the cell's chromosomes (for review, see Kikkert et al., 2005, Stable Transformation of Plant Cells by Particle Bombardment/Biolistics, in: Methods in Molecular Biology, vol. 286: Transgenic Plants: Methods and Protocols, Ed. L. Pena, Humana Press Inc., Totowa, N.J.).
[0178] Electroporation is a technique that utilizes short, high-intensity electric fields to permeabilize reversibly the lipid bilayers of cell membranes (see, for example, Fisk and Dandekar, 2005, Introduction and Expression of Transgenes in Plant Protoplasts, in: Methods in Molecular Biology, vol. 286: Transgenic Plants: Methods and Protocols, Ed. L. Pena, Humana Press Inc., Totowa, N.J., pp. 79-90; Fromm et al., 1987, Electroporation of DNA and RNA into plant protoplasts, in Methods in Enzymology, Vol. 153, Wu and Grossman, eds., Academic Press, London, UK, pp. 351-366; Joersbo and Brunstedt, 1991, Electroporation: mechanism and transient expression, stable transformation and biological effects in plant protoplasts. Physiol. Plant. 81, 256-264; Bates, 1994, Genetic transformation of plants by protoplast electroporation. Mol. Biotech. 2: 135-145; Dillen et al., 1998, Electroporation-mediated DNA transfer to plant protoplasts and intact plant tissues for transient gene expression assays, in Cell Biology, Vol. 4, ed., Celis, Academic Press, London, UK, pp. 92-99). The technique operates by creating aqueous pores in the cell membrane, which are of sufficiently large size to allow DNA molecules (and other macromolecules) to enter the cell, where the transgene expression construct (as T-DNA) may be stably incorporated into plant genomic DNA, leading to the generation of transformed cells that can subsequently be regenerated into transgenic plants.
[0179] Newer transformation methods include so-called "floral dip" methods, which offer the promise of simplicity, without requiring plant tissue culture, as is the case with all other commonly used transformation methodologies (Bent et al., 2006, Arabidopsis thaliana Floral Dip Transformation Method, Methods Mol Biol, vol. 343: Agrobacterium Protocols, 2/e, volume 1; Ed., Wang, Humana Press Inc., Totowa, N.J., pp. 87-103; Clough and Bent, 1998, Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana, Plant J. 16: 735-743). However, with the exception of Arabidopsis, these methods have not been widely used across a broad spectrum of different plant species. Briefly, floral dip transformation is accomplished by dipping or spraying flowering plants in with an appropriate strain of Agrobacterium tumefaciens. Seeds collected from these T0 plants are then germinated under selection to identify transgenic T1 individuals. Example 16 demonstrated floral dip inoculation of Arabidopsis to generate transgenic Arabidopsis plants.
[0180] Other transformation methods include those in which the developing seeds or seedlings of plants are transformed using vectors such as Agrobacterium vectors. For example, such vectors may be used to transform developing seeds by injecting a suspension or mixture of the vector (i.e., Agrobacteria) directly into the seed cavity of developing pods (i.e., pepper pods, bean pods, pea pods and the like). Still other transformation methods include those in which the flower structure is targeted for vector inoculation.
[0181] In addition, although transgenes are most commonly inserted into the nuclear DNA of plant cells, transgenes may also be inserted into plastidic DNA (i.e., into the plastome of the chloroplast). In most flowering plants, plastids do not occur in the pollen cells, and therefore transgenic DNA incorporated within a plastome will not be passed on through propagation, thereby restricting the trait from migrating to wild type plants. Plastid transformation, however, is more complex than cell nucleus transformation, due to the presence of many thousands of plastomes per cell (as high as 10,000).
[0182] Transplastomic lines are genetically stable only if all plastid copies are modified in the same way, i.e. uniformly. This is typically achieved through repeated regeneration under certain selection conditions to eliminate untransformed plastids, by segregating transplastomic and untransformed plastids, resulting in the selection of homoplasmic cells carrying the transgene construct and the selectable marker stably integrated therein. Plastid transformation has been successfully performed in various plant species, including tobacco, potatoes, oilseed rape, rice, and Arabidopsis.
[0183] To generate transplastomic lines carrying an Ω-amidase transgene, the transgene expression cassette is inserted into flanking sequences from the plastome. Using homologous recombination, the transgene expression cassette becomes integrated into the plastome via a natural recombination process. The basic DNA delivery techniques for plastid transformation include particle bombardment of leaves or polyethylene glycol-mediated DNA transformation of protoplasts. Transplastomic plants carrying transgenes in the plastome may be expressed at very high levels, due to the fact that many plastids (i.e., chloroplasts) per cell, each carrying many copies of the plastome. This is particularly the case in foliar tissue, where a single mature leaf cell may contain over 10,000 copies of the plastome. Following a successful transformation of the plastome, the transplastomic events carry the transgene on every copy of the plastid genetic material. This can result in the transgene expression levels representing as much as half of the total protein produced in the cell.
[0184] Plastid transformation methods and vector systems are described, for example, in recent U.S. Pat. Nos. 7,528,292; 7,371,923; 7,235,711; and, 7,193,131. See also U.S. Pat. Nos. 6,680,426 and 6,642,053.
[0185] The foregoing plant transformation methodologies may be used to introduce at least one transgene into a number of different plant cells and tissues, including without limitation, whole plants, tissue and organ explants including chloroplasts, flowering tissues and cells, protoplasts, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells, tissue cultured cells of any of the foregoing, any other cells from which a fertile regenerated transgenic plant may be generated. Callus is initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation.
[0186] Methods of regenerating individual plants from transformed plant cells, tissues or organs are known and are described for numerous plant species.
[0187] As an illustration, transformed plantlets (derived from transformed cells or tissues) are cultured in a root-permissive growth medium supplemented with the selective agent used in the transformation strategy. Once rooted, transformed plantlets are then transferred to soil and allowed to grow to maturity. Upon flowering, the mature plants are preferably selfed (self-fertilized), and the resultant seeds harvested and used to grow subsequent generations.
[0188] T0 transgenic plants may be used to generate subsequent generations (e.g., T1, T2, etc.) by selfing of primary or secondary transformants, or by sexual crossing of primary or secondary transformants with other plants (transformed or untransformed). During the mature plant growth stage, the plants are typically examined for growth phenotype, nitrogen use efficiency, CO2 fixation rate, etc. (see following subsection).
[0189] Selection of Transgenic Plants with Increased Nitrogen Use Efficiency:
[0190] Transgenic plants may be selected, screened and characterized using standard methodologies. The preferred transgenic plants of the present disclosure will exhibit one or more phenotypic characteristics indicative of increased nitrogen use efficiency, including without limitation, faster growth rates, greater seed and fruit/pod yields, earlier and more productive flowering, increased tolerance to high salt conditions, and increased biomass yields. Transgenic plants are typically regenerated under selective pressure in order to select transformants prior to creating subsequent transgenic plant generations. In addition, the selective pressure used may be employed beyond T0 generations in order to ensure the presence of the desired transgene expression construct or cassette.
[0191] T0 transformed plant cells, calli, tissues or plants may be identified and isolated by selecting or screening for the genetic composition of and/or the phenotypic characteristics encoded by marker genes contained in the transgene expression construct used for the transformation. For example, selection may be conducted by growing potentially-transformed plants, tissues or cells in a growth medium containing a repressive amount of antibiotic or herbicide to which the transforming genetic construct can impart resistance. Further, the transformed plant cells, tissues and plants can be identified by screening for the activity of marker genes (i.e., β-glucuronidase) which may be present in the transgene expression construct.
[0192] Various physical and biochemical methods may be employed for identifying plants containing the desired transgene expression construct, as is well known. Examples of such methods include Southern blot analysis or various nucleic acid amplification methods (i.e., PCR) for identifying the transgene, transgene expression construct or elements thereof, Northern blotting, 51 RNase protection, reverse transcriptase PCR (RT-PCR) amplification for detecting and determining the RNA transcription products, and protein gel electrophoresis, Western blotting, immunoprecipitation, enzyme immunoassay, and the like may be used for identifying the protein encoded and expressed by the transgene.
[0193] In another approach, expression levels of genes, proteins and/or metabolic compounds that are know to be modulated by transgene expression in the target plant may be used to identify transformants. In one embodiment of the present disclosure, increased levels of the signal metabolite 2-oxoglutaramate in leaf tissue, or decreased levels in the root tissue, or a higher leaf-to-root ratio of 2-oxoglutaramate may be used to screen for desirable transformants.
[0194] Ultimately, the transformed plants of the present disclosure may be screened for increased nitrogen use efficiency. Nitrogen use efficiency may be expressed as plant yield per given amount of nitrogen. Indeed, some degree of phenotypic screening is generally desirable in order to identify transformed lines with the fastest growth rates, the highest seed yields, etc., particularly when identifying plants for subsequent selfing, cross-breeding and back-crossing.
[0195] Various parameters may be used for this purpose, including without limitation, growth rates, total fresh weights, dry weights, seed and fruit yields (number, weight), seed and/or seed pod sizes, seed pod yields (e.g., number, weight), leaf sizes, plant sizes, increased flowering, time to flowering, overall protein content (in seeds, fruits, plant tissues), specific protein content (i.e., Ω-amidase), nitrogen content, free amino acid, and specific metabolic compound levels (i.e., 2-oxoglutaramate). Generally, these phenotypic measurements are compared with those obtained from a parental identical or analogous plant line, an untransformed identical or analogous plant, or an identical or analogous wild-type plant (i.e., a normal or parental plant). Preferably, and at least initially, the measurement of the chosen phenotypic characteristic(s) in the target transgenic plant is done in parallel with measurement of the same characteristic(s) in a normal or parental plant. Typically, multiple plants are used to establish the phenotypic desirability and/or superiority of the transgenic plant in respect of any particular phenotypic characteristic.
[0196] Preferably, initial transformants are selected and then used to generate T1 and subsequent generations by selfing (self-fertilization), until the transgene genotype breeds true (i.e., the plant is homozygous for the transgene). In practice, this is accomplished by screening at each generation for the desired traits and selfing those individuals, often repeatedly (i.e., 3 or 4 generations). As exemplified herein, transgenic plant lines propagated through at least one sexual generation (Tobacco, Arabidopsis, Tomato) demonstrated higher transgene product activities compared to lines that did not have the benefit of sexual reproduction and the concomitant increase in transgene copy number.
[0197] Stable transgenic lines may be crossed and back-crossed to create varieties with any number of desired traits, including those with stacked transgenes, multiple copies of a transgene, etc. Various common breeding methods are well known to those skilled in the art (see, e.g., Breeding Methods for Cultivar Development, Wilcox J. ed., American Society of Agronomy, Madison Wis. (1987)). Additionally, stable transgenic plants may be further modified genetically, by transforming such plants with further transgenes or additional copies of the parental transgene. Also contemplated are transgenic plants created by single transformation events which introduce multiple copies of a given transgene or multiple transgenes.
[0198] Nitrogen use efficiency may be expressed as plant yield per given amount of nitrogen.
[0199] Methods for Increasing Nitrogen Use Efficiency:
[0200] Certain aspects of the present disclosure relate to methods for increasing nitrogen use efficiency of a plant.
[0201] One particular aspect relates to a method for increasing nitrogen use efficiency of a plant relative to a wild type or untransformed plant of the same species, by: (a) introducing an Ω-amidase transgene into the plant, where the Ω-amidase transgene is operably linked to a root-preferred promoter; (b) expressing the Ω-amidase transgene in root tissue of the plant or the progeny of the plant; and (c) selecting a plant having an increased leaf-to-root ratio of 2-oxoglutaramate relative to a plant of the same species that does not contain an Ω-amidase transgene, where the increased leaf-to-root ratio of 2-oxoglutaramate results in increased nitrogen use efficiency.
[0202] In certain embodiments, the Ω-amidase transgene encodes a polypeptide having an amino acid sequence that is at least 90% identical to an amino acid sequence encoded by a polypeptide selected from AAL91613.1, ACN30911.1, ABK22312.1, ACJ85250.1, AAQ97821.1, CBJ25483.1, EFN54567.1, NP--196765.2, XP--002871509.1, NP--974769.1, XP--002309478.1, XP--002279687.1, NP--001146676.1, NP--001146295.1, NP--001049134.1, XP--002516116.1, XP--001766085.1, XP--001756522.1, XP--002969787.1, XP--002985119.1, XP--002948137.1, XP--001690839.1, NP--001057093.1, XP--002468410.1, NP--064587.1, XP--001089575.2, XP--001502234.1, XP--002502298.1, XP--526254.2, XP--535718.2, XP--002716659.1, NP--001033222.1, NP--001029298.1, NP--001016633.1, NP--001085409.1, XP--002758928.1, XP--003064056.1, NP--001135127.1, XP--001622809.1, NP--991174.2, XP--002594716.1, NP--075664.1, XP--001370849.1, NP--001090454.1, XP--002999170.1, XP--002917137.1, XP--002741281.1, XP--002131764.1, NP--594154.1, XP--001742101.1, XP--416604.2, XP--002194275.1, XP--001599587.1, XP--002410555.1, XP--003035898.1, XP--002183613.1, XP--001875493.1, XP--002112209.1, XP--636983.1, XP--002158547.1, XP--002839272.1, XP--307722.3, XP--001819629.1, XP--001268376.1, ZP--08115581.1, YP--001320997.1, XP--369268.1, XP--002626458.1, XP--751200.1, XP--001657673.1, XP--002173486.1, XP--001212538.1, XP--001258462.1, XP--002434512.1, XP--960906.1, XP--002847679.1, XP--967861.1, XP--002426154.1, XP--003176259.1, XP--500602.1, XP--001428419.1, XP--003014235.1, XP--001393123.1, ZP--03608460.1, XP--002147261.1, ZP--03293831.1, XP--002290043.1, XP--003065597.1, XP--001588734.1, YP--001273073.1, XP--001552800.1, XP--446414.1, XP--002792830.1, XP--001998501.1, YP--003780301.1, NP--013455.1, XP--002404736.1, YP--001086961.1, ZP--05328587.1, ZP--05399936.1, YP--001113615.1, XP--001247884.1, XP--390426.1, XP--003025334.1, XP--002052999.1, YP--769862.1, ZP--07325748.1, ZP--05349666.1, YP--471237.1, YP--002977603.1, YP--001202760.1, ZP--07592670.1, and NP--386723.1. In other embodiments, the Ω-amidase transgene is incorporated into the genome of the plant. In still other embodiments, the root-preferred promoter is selected from RolD promoter, RolD-2 promoter, glycine rich protein promoter, GRP promoter, ADH promoter, maize ADH1 promoter, PHT promoter, Pht1 gene family promoter, metal uptake protein promoter, maize metallothionein protein promoter, 35S CaMV domain A promoter. pDJ3S promoter, SIREO promoter, pMe1 promoter, Sad1 promoter, Sad2 promoter, TobRB7 promoter, RCc3 promoter, FaRB7 promoter, SPmads promoter, IDS2 promoter, pyk10 promoter, Lbc3 leghemoglobin promoter, PEPC promoter, Gns1 glucanase root promoter, 35S2promoter, GI4 promoter, GI5 promoter, and GRP promoter.
[0203] In other embodiments, endogenous Ω-amidase expression in leaf tissue is inhibited. In still other embodiments, the endogenous Ω-amidase expression in leaf tissue is inhibited by recessive gene disruption, dominant gene silencing, or a chemical inhibitor. In yet other embodiments, the endogenous Ω-amidase expression in leaf tissue is inhibited by a recessive gene disruption selected from a mutant Ω-amidase gene that eliminates endogenous Ω-amidase expression, an endogenous Ω-amidase knockout mutant, and an endogenous Ω-amidase knockdown mutant. In further embodiments, the endogenous Ω-amidase expression in leaf tissue is inhibited by an RNAi antisense oligonucleotide that is specific for an endogenous Ω-amidase gene. In still further embodiments, the endogenous Ω-amidase expression in leaf tissue is inhibited by a chemical inhibitor selected from 6-diazo-5-oxo-nor-leucine, p-hydroxymercuribenzoate, diisopropyl fluorophosphates, sodium cyanide, phenylmercuriacetate, Iodoacetate, silver nitrate, chloromercuricphenylsulfonic acid, and copper sulfate.
[0204] In other embodiments, the leaf-to-root ratio of 2-oxoglutaramate is at least two times higher than that of a progenitor or wild type plant of the same species. In still other embodiments, the plant further contains a GPT transgene. In yet other embodiments, the GPT transgene is a GPT/F:V mutant given by SEQ ID NO:1. In further embodiments, the plant further comprises a GPT transgene and a GS transgene. In other embodiments, the GPT transgene and GS transgene are each operably linked to a leaf-preferred promoter. In still further embodiments, endogenous GPT expression in the plant is increased by gene activation. In yet other embodiments, endogenous GS expression in the plant is increased by gene activation. In other embodiments, each transgene is codon optimized for expression in the plant.
[0205] Another aspect relates to a method for increasing nitrogen use efficiency of a plant relative to a wild type or untransformed plant of the same species, by: (a) inhibiting endogenous Ω-amidase expression in leaf tissue of the plant; and (b) selecting a plant having an increased leaf-to-root ratio of 2-oxoglutaramate relative to a plant of the same species that does not have inhibited endogenous Ω-amidase expression in leaf tissue, where the increased leaf-to-root ratio of 2-oxoglutaramate results in increased nitrogen use efficiency.
[0206] In certain embodiments, the endogenous Ω-amidase expression in leaf tissue is inhibited by recessive gene disruption, dominant gene silencing, or a chemical inhibitor. In other embodiments, the endogenous Ω-amidase expression in leaf tissue is inhibited by a recessive gene disruption selected from a mutant Ω-amidase gene that eliminates endogenous Ω-amidase expression, an endogenous Ω-amidase knockout mutant, and an endogenous Ω-amidase knockdown mutant. In still other embodiments, the endogenous Ω-amidase expression in leaf tissue is inhibited by an RNAi antisense oligonucleotide that is specific for an endogenous Ω-amidase gene. In yet other embodiments, the chemical inhibitor selected from 6-diazo-5-oxo-nor-leucine, p-hydroxymercuribenzoate, diisopropyl fluorophosphates, sodium cyanide, phenylmercuriacetate, Iodoacetate, silver nitrate, chloromercuricphenylsulfonic acid, and copper sulfate.
[0207] In other embodiments, the plant further contains an Ω-amidase transgene, wherein the Ω-amidase transgene is operably linked to a root-preferred promoter. In still other embodiments, the leaf-to-root ratio of 2-oxoglutaramate is at least two times higher than that of a progenitor or wild type plant of the same species. In yet other embodiments, the plant further contains a GPT transgene. In further embodiments, the GPT transgene is a GPT/F:V mutant given by SEQ ID NO:1. In still further embodiments, the plant further contains a GPT transgene and a GS transgene. In other embodiments, the GPT transgene and GS transgene are each operably linked to a leaf-preferred promoter. In yet further embodiments, endogenous GPT expression in the plant is increased by gene activation. In other embodiments, endogenous GS expression in the plant is increased by gene activation. In still other embodiments, each transgene is codon optimized for expression in the plant.
[0208] In further embodiments of the methods for increasing nitrogen use efficiency of a plant, the plant may contain other transgenes known to increase nitrogen utilization efficiency, including without limitation those described in U.S. Pat. No. 7,560,626.
[0209] In the Examples provided herein, the transgene and control plants all received the same nutrient solutions in the same amounts. The transgenic plants were consistently characterized by higher yields, and thus have higher nitrogen use efficiencies.
EXAMPLES
Example 1
Effects of Increasing Expression Levels of GS and GPT on OMEGA-Amidase Pathway
[0210] Materials and Methods:
[0211] Generation of Transgenic Plants: Plants were genetically engineered to over-produce 2-oxoglutaramate by over expressing GS and GPT transgenes, as described in U.S. patent application Ser. No. 12/551,271. The resulting phenotypic effects were evaluated. Three sets of transgenic tobacco lines were generated: one set over-expressing GPT to increase GRMT catalytic capacity; a second set over-expressing GS to increase, in the leaves only, the catalytic capacity to make the glutamine substrate of GPT; and a third set over expressing GS and GPT, produced by sexually crossing fast-growing progeny of the single transgene lines.
[0212] Growth of Engineered Tobacco and Arabidopsis: Wild type and engineered tobacco seeds were surface sterilized and germinated in phytotrays containing M&S medium. The medium for the engineered plants contained kanamycin (10 ug/ml). The vigorously growing seedlings were transferred at 17 d to a sand culture, covered to control humidity for 4 d to aid adaptation to ambient conditions; plants were continuously provided a nutrient solution (Knight and Langston-Unkefer, 1988, Science 241: 951) containing 10 mM KNO3. The growth conditions were as described previously (Knight and Langston-Unkefer, supra). Tissue was harvested between 32-35 d after the transplant unless the plants were being grown for seed production. Wild type and engineered Arabidopsis seeds were surface sterilized and germinated in phytotrays containing M&S medium. For the engineered plants the medium contained kanamycin (10 ug/ml).
[0213] The seedlings were transferred to the ArabiSystem using the Promix (Lehle Seeds) growth medium and grown at 24° C. with 16 h light and 8 h dark periods. Plants were grown to maturity.
[0214] Results:
[0215] The over-expression of GS in tobacco generated two classes of progeny; those that grew faster and over-expressed GS only in their leaves, thus increasing their leaf-to ratio of GS, and a second class that grew at normal rates and over-expressed GS in both leaves and roots, thereby maintaining a normal leaf-to-root ratio. Regulation of GS and GPT expression in these plants appears complex and expression of each gene appears to influence the other (see Tables 2 and 3); over-expression of only GPT in leaves and roots was accompanied by increased GS activity in the leaf and only normal GS activity in the root. Increased GS expression only in the leaf was accompanied by increased GPT activity in the leaf and lower GPT activity in the root. These responses were evident in the GS+GPT transgenic plants as well. Over-expression of either GS or GPT was accompanied by lower Ω-amidase activity in the leaves and greater Ω-amidase activity in the roots. GPT and GS+GPT transgenic plants showed the largest increases in root Ω-amidase activity. These plants responded to expression of the transgenes by altering their Ω-amidase activities such that they tend to increase the leaf root 2-oxoglutaramate pool, and maintain the root 2-oxoglutaramate pool. These responses combined in the GS+GPT over-expressing plants to generate the highest leaf and lowest root 2-oxoglutaramate pools and the highest leaf and lowest GS and GPT activities.
[0216] Tables 2, 3, and 4 below depict the effects of engineering for greater 2-oxoglutaramate (2-0GM) biosynthesis. Tables 2 and 3 depict results from tobacco plants, and Table 4 depicts results from Arabidopsis thaliana plants.
TABLE-US-00037 TABLE 2 Effects of engineering greater 2-oxoglutaramate biosynthesis capability Leaf Root 2-OGM Leaf Leaf Leaf Amidase Root GS Root Root Amidase 2-OGM nmol/ GS GPT nmol/ umol/ GPT nmol/ Tobacco nmol/ gfwt umol/ nmol/ gfwt/h gfwt/m nmol/ gfwt/h Genotype gfwt (Leaf/Root) gfwt/m gfwt/h (GPT/Amidase) (Leaf/Root) gfwt/h (GPT/Amidase) Wild type 191 116 (1.6) 7.8 100 191 (0.5) 2.1 (3.7) 236 252 (0.9) +GPT 384 143 (2.7) 10.5 196 118 (1.7) 1.9 (5.5) 566 440 (1.3) +GS 502 131 (3.8) 11.6 288 112 (2.6) 1.7 (6.8) 136 372 (0.4) +GS + GPT 701 80 (8.7) 16.3 731 149 (4.9) 1.8 (9.1) 117 292 (0.4)
[0217] TABLE-US-00005 TABLE 2 Effects of engineering greater 2-oxoglutaramate biosynthesis capability Root Leaf Amidase Root GS 2-OGM Leaf nmol/umol/Root Root Amidase Leaf nmol/gfwt Leaf GS GPT gfwt/h gfwt/m GPT nmol/gfwt/h Tobacco 2-OGM (Leaf/umol/nmol/(GPT/(Leaf/nmol/(GPT/Genotype nmol/gfwt Root) gfwt/m gfwt/h Amidase) Root) gfwt/h Amidase) Wild type 191 116 (1.6) 7.8 100 191 (0.5) 2.1 (3.7) 236 252 (0.9)+GPT 384 143 (2.7) 10.5 196 118 (1.7) 1.9 (5.5) 566 440 (1.3)+GS 502 131 (3.8) 11.6 288 112 (2.6) 1.7 (6.8) 136 372 (0.4)+GS+GPT 701 80 (8.7) 16.3 731 149 (4.9) 1.8 (9.1) 117 292 (0.4)
[0218] In Table 2 above, "gfwt" refers to grams fresh weight and "nmol/gfwt/h" refers to nano moles per grams fresh weight per hour.
TABLE-US-00038 TABLE 3 Effects of engineering greater 2-oxoglutaramate biosynthesis capability NO3 CO2 Seed Whole Tobacco uptake rate Leaf NO3 Root NO3 Leaf Protein Chlorophyll Fixed Rate RGR yield Plant Genotype mm/gfwth μmol/gfwt μmol/gfwt Mg/gfwt μg/gfwt mm/m2/s Mg/g/d g/plt gfwt Wild type 4.3 ± 2.8 69.3 ± 4.9 29.9 ± 2.2 4.3 818 ± 10 7.7 226 1.0 21.0 (100%) (100%) +GPT 10.9 ± 2.4 77.6 ± 8.2 57.5 ± 6.0 5.2 1044 ± 3 12.9 ND NM 31.sup. (120%) (147%) +GS 11.3 ± 1.9 22.8 ± 2.7 12.1 ± 3.2 6.9 1109 ± 6 13.5 269 NM 35.6 (160%) (169%) +GS + GPT 19.5 ± 3.1 51.1 ± 1.8 30.1 ± 3.5 7.3 1199 ± 11 20.6 346 2.87 71.9 (170%) (342%)
TABLE-US-00039 TABLE 4 Arabidopsis engineered to over-produce 2-oxoglutaramate Whole Leaf Leaf Leaf Leaf Plant GS GPT 2-OGM Protein Fresh Arabidopsis Activity Activity nmol mg/gfwt Wt. g Genotype μmol/gfwt. m nmol/gfwt/h gfwt/h (% wt) (% wt) Wild type 6.89 184 184 6.06* 0.246 +GS + GPT 18.7 1077 395.5 7.46* 1.106 (123%) (449%)
Example 2
Plant Expression Vector Modulating Root Ω-Amidase Expression Levels
[0219] OMEGA-amidase expression levels are increased in root tissues by generating transgenic plants transformed with expression constructs containing an Ω-amidase coding sequence, including but not limited to any of the Ω-amidase coding sequences disclosed herein, under the control of a root-preferred promoter. It is believed that increased levels of Ω-amidase in root tissues result in increased breakdown of the signal metabolite 2-oxoglutaramate.
[0220] A construct for transforming plants includes an expression cassette encoding a suitable root-preferred promoter, a sequence encoding a plant Ω-amidase, and a terminator sequence. In this example, the expression cassette contains the glycine-rich protein (GRP) promoter (Goddemeier et al., 1998, Plant Mol. Biol. 36(5): 799-802), the Arabidopsis thaliana Ω-amidase coding sequence, and the NOS terminator. The GRP promoter sequence is shown below [SEQ ID NO: 16]:
TABLE-US-00040 GAAATTAAACCCAGGGTCGACAGCGCCCACTATAGAGAAAAAATTG AAATGTTTTGAGAATCGGATGATTTTTTTTAACTATTAGGTCTAGTTTG AAAACCCTATTTTCTAACAAAGGGATTTTCATTTTTATAAGAGAAAAT AAACTAACTTTTCTTGAGAAAATAAAATTCTTTGGAAAAATGGATTTC TCAAACTAGCTCTTACGGCTAGTTTGGAAACCCCAATTTCACACGG GATTCTCATTTTCCCAAGGGAAAAATGAACTAATTTCCCTTAGAAAA ATGAGAATCCCGTGGGAAATTGGGATTTTCAAAGTAGCCCTTATAGT GGAAATAAGTTATGGTGTCTCGCTCGTATGGTTATGTAGGGCCGCGC GTGTATTCCAGCGCCGGCCGCATGGATACCCTATCGATTCTGACTT CTCTGTCTCAGGAAAATAATACAGCCACGATTAACGGAACCTGCTG GCTGGATCCATGATTACTCACTTGACTTCACATCGATCCAAATTATC TAGCTTGCACGTTCATGGGTCGCCTCGCTCGCCCGATCGATATTAC GTACACCATAGATTAGTACTATATGGAGTGGAGTGTTGAATGGATGC TCTTTATTATTCTAGCCAAGTTATCAAGCCGGGCACTTGCATCGGAAG GAGTACCAGTGTACGCATCAGATCAGACGATAATCGATCAAGATGG GTACGAGATTTGCCGCTTGCTTCCTGTTCTTGATGGGCAATCTTTTC GGGCCTTGAACGTCGGAGAATCGACTATACGAAATCCTAGGTCAAC TATACATTGGTTGATGCTTCCGTGTAGTTTTACCAGTTCATCGGTCTC TAGCTTGTTGTTTGCGACGACTTCACGTGGCCACGCGTTTACTGCGC TCTGCTCAAAGAAATTGCCTACAGTGCCTGGCGTCAGCTGCAGGCG TTGAATCCGAGGTCGCGCGCCGCAGAATAAGTACGAGTCAAAGGCT GAGCTGCATGCCGTACCGGCCTTTATTAATAGCTGAGCTCTACTCGC TACGTCAGTATAGTATAGCACGGTCATATATATACTATAGCTATAGCT GTGGGGTACCGTGTCCGTATCGTGAATCTGAAGTCGAACAGTGATAT GGCGTACTATCTAATAATGTCCCGTGCAGTAATATCACTGTTGCCGAC GATGGGAATCTCTAGTTTTGACAGAAACCAAAGCAACTGCTAGCTAAT TAATTCCAGAGAGATCGATTTCTACAGTGCTGCAACAATCAATGCAAT TGGCATCAGACGATATATGCTAATGGTTTCTTTATCGATACGTGGTCAA CAGAGCTCTCTCGCCCGCCCTGATCAGATCTCATCGCACATGGACAC CCATCTGCCAACCCAACACGGGCGGGGGAACCACCGTGAAACATCG CGTTCATGCACGACCCCCCCGCAGGCCGCAGCTATAAATACCCATG CAATGCAATGCAGCGGGTCATCATCGACTCCACCTGGACTCGCTCAC TGGCAATGGCTACCACCAGC
[0221] Alternatively, an expression cassette containing the rolD promoter and the Arabidopsis thaliana Ω-amidase coding sequence may be used. The sequence of this construct is shown below: (ATG start codon of the Ω-amidase gene shown in bold) [SEQ ID NO: 17]:
TABLE-US-00041 GACGTCGGTACCGAATTTGTTCGTGAACTATTAGTTGCGGGCCTTGG CATCCGACTACCTCTGCGGCAATATTATATTCCCTGGGCCCACCGT GAACCCAATTTCGCCTATTTATTCATTACCCCCATTAACATTGAAGTA GTCATGATGGGCCTGCAGCACGTTGGTGAGGCTGGCACAACTCATC CATATACTTTCTGACCGGATCGGCACATTATTGTAGAAAACGCGGAC CCACAGCGCACTTTCCAAAGCGGTGCCGCGTCAGAATGCGCTGGC AGAAAAAAATTAATCCAAAAGTACCCTCCAAGCAGCCCATATAAAC GCGTTTACAAATCCGCTAACCTCAACAATTTGAGCAGAGAAAATTCG CACCTACAAGGCAGATGGCATCATCATTCAATCCAGAGCAGGCAAG AGTTCCTTCAGCATTACCTTTACCAGCACCACCACTTACCAAATTCA ACATCGGACTTTGTCAATTGAGTGTTACTTCTGATAAGAAAAGAAACA TTTCACATGCTAAGAAAGCAATCGAAGAGGCTGCTAGTAAGGGAGC TAAACTCGTTCTTTTGCCTGAAATATGGAACTCACCATACAGTAACG ATTCTTTTCCTGTGTACGCAGAAGAGATCGATGCTGGAGGTGATGC ATCTCCATCAACTGCTATGCTCTCAGAAGTTAGTAAGAGACTCAAGA TTACAATTATCGGAGGTTCAATTCCTGAGAGAGTTGGAGATAGGTTG TATAACACATGTTGCGTGTTCGGATCTGATGGAGAGCTCAAGGCTAA GCATAGGAAGATTCACCTCTTCGATATAGATATTCCTGGAAAGATCA CCTTCATGGAATCAAAAACACTTACCGCTGGAGAGACTCCAACAAT TGTTGATACAGATGTGGGTAGAATCGGAATAGGTATATGTTAC GATATCAGGTTCCAAGAATTGGCTATGATATATGCTGCAAGAGGAGC ACATCTCTTATGCTACCCTGGAGCTTTCAATATGACTACAGGTCCATT GCACTGGGAGCTTTTGCAAAGAGCTAGGGCAACAGATAACCAGCTC TATGTTGCTACCTGCTCTCCTGCAAGAGATTCAGGAGCTGGTTACAC CGCATGGGGTCATTCTACTCTTGTTGGACCATTTGGTGAAGTGTTGGC TACCACTGAGCACGAAGAGGCTATTATAATCGCAGAAATCGATTACA GTATACTTGAGCAGAGAAGGACTTCTCTCCCATTAAATAGGCAGAGG AGGGGTGATTTATACCAGTTAGTTGATGTTCAGAGATTAGATAGTAAGT GACACGTGTGAATTACAGGTGACCAGCTCGAATTTCCCCGATCGTTC AAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTT GCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATAA TTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAG AGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCG CGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGA TCGGGGGTACCGACGTC
[0222] For transformation of plants, the expression cassette, above, is cloned into a suitable vector. For Agrobacterium mediated transformation, the above construct is cloned into the TF101.1 vector, which carries the spectinomycin resistance selectable marker gene.
[0223] All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
[0224] The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any which are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.
Example 3
Increased Growth of Transgenic Alfalfa Plants Carrying Root-Preferred OMEGA-Amidase Transgene
[0225] In this example, alfalfa plant growth was increased by introducing an Ω-amidase transgene under the control of a highly root-preferred promoter. The resulting transgenic alfalfa plants showed decreased 2-oxoglutaramate concentration in roots, increased leaf-to-root ratio of 2-oxoglutaramate, and enhanced growth relative to wild type alfalfa plants. Alfalfa plants (Medicago sativa, var Ladak) were transformed with the Arabidopsis Ω-amidase coding sequence truncated to remove the chloroplast transit peptide, under the control of a truncated Agrobacterium rhizogenes RolD promoter within the expression vector pTF101.1.
[0226] Materials and Methods:
[0227] Agrobacterium Vectors: The expression vector pTF101.1 was engineered to carry the Ω-amidase transgene expression cassette of SEQ ID NO: 39 (RolD promoter+Ω-amidase+NOS terminator) and was transferred to Agrobacterium tumefaciens strain LBA4404 cultures using a standard electroporation method (McCormac et al., 1998, Molecular Biotechnology 9:155-159). The truncated Agrobacterium rhizogenes RolD promoter utilized is the pD-02 isoform (RolD2) described in Leach and Aoyagi, 1991, Plant Sci. 79, 69-76. Leach and Aoyagi describe the RolD2 promoter as being a highly root-preferred promoter that drove high levels of expression in root tissue. Transformed Agrobacterium were selected on media containing 50 μg/ml of chloroamphenicol. Transformed Agrobacterium cells were grown in LB culture media containing 25 μg/ml of antibiotic for 36 hours. At the end of the 36 hr growth period cells were collected by centrifugation and cells from each transformation were resuspended in 100 ml LB broth without antibiotic.
[0228] The nucleotide sequence of the pTF101.1 vector+rolD-02promoter+Arabidopsis Ω-amidase (codon optimized for Arabidopsis)+nos terminator (SEQ ID NO: 39) is set forth below.
[0229] Underlined nucleotides=rolD-02 promoter, bold nucleotides=Ω-amidase coding region, italicized nucleotides include the nos terminator region (and some Cambia vector sequence), and other nucleotides=pTF101.1 vector.
TABLE-US-00042 AGTACTTTAAAGTACTTTAAAGTACTTTAAAGTACTTTGATCCAACCCCT CCGCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCT GAAAACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCC TGCCCTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTA GAATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCC GCCGCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACT TGACCAACCAACGGGCCGAACTGCACGCGGCCGGCTGCACCAAGCTGTT TTCCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGG ATGCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAG ACCGCCTGGCCCGCAGCACCCGCGACCTACTGGACATTGCCGAGCGCAT CCAGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGAC ACCACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTG CCGAGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGA GGCCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACC CCGGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCA CCGTGAAAGAGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTA CCGCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCGG CGCGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGG CCGCCGAGAATGAACGCCAAGAGGAACAAGCATGAAACCGCACCAGGAC GGCCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGA TCGCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCG GCTGCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGG CCGGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGT GATGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATAT GATGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTA TCGCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAAC CCATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTC GATTCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAG ATCAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGA CGTGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCC CAGGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGC TGATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCT GGTGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAA GCGGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTG AGGTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCG TATCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACC GTTCTTGAATCAGAACCCGAGGGCGACGCTGCCCGCGAGGTCCAGGCGC TGGCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAG AAAATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCAC GCAGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCAT GAAGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAG ATGTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAAT ACATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGAT GAATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAACCAGG CACCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCA GGCGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCC CCAAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCCGGTAC AAATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGC GCAGGCCGCCCAGCGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAA TCGTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGC CGGCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCA ACCAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGT CGCAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGAC GAGCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGT TTCCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTA CTGATGGCGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAG GGAAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGT ACTCAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTG GTAGAAACCTGCATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTA CGAAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGC CTTGATTAGCCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAG TACATCGAGATCGAGCTAGCTGATTGGATGTACCGCGAGATCACAGAAG GCAAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGA TCCCGGCATCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGC AAGGCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCA GCGCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGG GTCAAATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCT GGCCCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGGCGAAGCAT CCGCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGC AGGGGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATT GGGAACCCAAAGCCGTACATTGGGAACCGGAACCCGTACATTGGGAACC CAAAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAA AGAGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAA ACTCTTAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCCAGCGCACAG CCGAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACG CCCCGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATG GCTGGCCTACGGCCAGGCAATCTACCAGGGCGCGGACAAGCCGCGCCGT CGCCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTT TCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGT CACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGC GCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCAC GTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAG ATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCG TAAGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGA CTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCA AAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGA ACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGC GTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAA AATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGAT ACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGAC CCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG GCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCG TTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCG CTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACAC GACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGA GGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGG CTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTT ACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCG CTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAA AAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCT CAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATGATATAT CTCCCAATTTGTGTAGGGCTTATTATGCACGCTTAAAAATAATAAAAGC AGACTTGACCTGATAGTTTGGCTGTGAGCAATTATGTGCTTAGTGCATC TAATCGCTTGAGTTAACGCCGGCGAAGCGGCGTCGGCTTGAACGAATTT CTAGCTAGACATTATTTGCCGACTACCTTGGTGATCTCGCCTTTCACGT AGTGGACAAATTCTTCCAACTGATCTGCGCGCGAGGCCAAGCGATCTTC TTCTTGTCCAAGATAAGCCTGTCTAGCTTCAAGTATGACGGGCTGATAC TGGGCCGGCAGGCGCTCCATTGCCCAGTCGGCAGCGACATCCTTCGGCG CGATTTTGCCGGTTACTGCGCTGTACCAAATGCGGGACAACGTAAGCAC TACATTTCGCTCATCGCCAGCCCAGTCGGGCGGCGAGTTCCATAGCGTT AAGGTTTCATTTAGCGCCTCAAATAGATCCTGTTCAGGAACCGGATCAA AGAGTTCCTCCGCCGCTGGACCTACCAAGGCAACGCTATGTTCTCTTGC TTTTGTCAGCAAGATAGCCAGATCAATGTCGATCGTGGCTGGCTCGAAG ATACCTGCAAGAATGTCATTGCGCTGCCATTCTCCAAATTGCAGTTCGC GCTTAGCTGGATAACGCCACGGAATGATGTCGTCGTGCACAACAATGGT GACTTCTACAGCGCGGAGAATCTCGCTCTCTCCAGGGGAAGCCGAAGTT TCCAAAAGGTCGTTGATCAAAGCTCGCCGCGTTGTTTCATCAAGCCTTA CGGTCACCGTAACCAGCAAATCAATATCACTGTGTGGCTTCAGGCCGCC ATCCACTGCGGAGCCGTACAAATGTACGGCCAGCAACGTCGGTTCGAGA TGGCGCTCGATGACGCCAACTACCTCTGATAGTTGAGTCGATACTTCGG CGATCACCGCTTCCCCCATGATGTTTAACTTTGTTTTAGGGCGACTGCC CTGCTGCGTAACATCGTTGCTGCTCCATAACATCAAACATCGACCCACG GCGTAACGCGCTTGCTGCTTGGATGCCCGAGGCATAGACTGTACCCCAA AAAAACATGTCATAACAAGAAGCCATGAAAACCGCCACTGCGCCGTTAC CACCGCTGCGTTCGGTCAAGGTTCTGGACCAGTTGCGTGACGGCAGTTA
CGCTACTTGCATTACAGCTTACGAACCGAACGAGGCTTATGTCCACTGG GTTCGTGCCCGAATTGATCACAGGCAGCAACGCTCTGTCATCGTTACAA TCAACATGCTACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCT TAGTTGCCGTTCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCG CCTTACAACGGCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCT GTATCGAGTGGTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGC TGGCTGGTGGCAGGATATATTGTGGTGTAAACAAATTGACGCTTAGACA ACTTAATAACACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAA TTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGAT CGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGC TGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGT TGTAAAACGACGGCCAGTGCCAAGCTAATTCTTCAAGACGTGCTCAAAT CACTATTTCCACACCCCTATATTTCTATTGCACTCCCTTTTAACTGTTT TTTATTACAAAAATGCCCTGGAAAATGCACTCCCTTTTTGTGTTTGTTT TTTTGTGAAACGATGTTGTCAGGTAATTTATTTGTCAGTCTACTATGGT GGCCCATTATATTAATAGCAACTGTCGGTCCAATAGACGACGTCGATTT TCTGCATTTGTTTAACCACGTGGATTTTATGACATTTTATATTAGTTAA TTTGTAAAACCTACCCAATTAAAGACCTCATATGTTCTAAAGACTAATA CTTAATGATAACAATTTTCTTTTAGTGAAGAAAGGGATAATTAGTAAAT ATGGAACAAGGGCAGAAGATTTATTAAAGCCGCGTAAGAGACAACAAGT AGGTACGTGGAGTGTCTTAGGTGACTTACCCACATAACATAAAGTGACA TTAACAAACATAGCTAATGCTCCTATTTGAATAGTGCATATCAGCATAC CTTATTACATATAGATAGGAGCAAACTCTAGCTAGATTGTTGAGAGCAG ATCTCGGTGACGGGCAGGACCGGACGGGGCGGTACCGGCAGGCTGAAGT CCAGCTGCCAGAAACCCACGTCATGCCAGTTCCCGTGCTTGAAGCCGGC CGCCCGCAGCATGCCGCGGGGGGCATATCCGAGCGCCTCGTGCATGCGC ACGCTCGGGTCGTTGGGCAGCCCGATGACAGCGACCACGCTCTTGAAGC CCTGTGCCTCCAGGGACTTCAGCAGGTGGGTGTAGAGCGTGGAGCCCAG TCCCGTCCGCTGGTGGCGGGGGGAGACGTACACGGTCGACTCGGCCGTC CAGTCGTAGGCGTTGCGTGCCTTCCAGGGGCCCGCGTAGGCGATGCCGG CGACCTCGCCGTCCACCTCGGCGACGAGCCAGGGATAGCGCTCCCGCAG ACGGACGAGGTCGTCCGTCCACTCCTGCGGTTCCTGCGGCTCGGTACGG AAGTTGACCGTGCTTGTCTCGATGTAGTGGTTGACGATGGTGCAGACCG CCGGCATGTCCGCCTCGGTGGCACGGCGGATGTCGGCCGGGCGTCGTTC TGGGCTCATGGTAGATCCCCCGTTCGTAAATGGTGAAAATTTTCAGAAA ATTGCTTTTGCTTTAAAAGAAATGATTTAAATTGCTGCAATAGAAGTAG AATGCTTGATTGCTTGAGATTCGTTTGTTTTGTATATGTTGTGTTGAGA ATTAATTCTCGAGGTCCTCTCCAAATGAAATGAACTTCCTTATATAGAG GAAGGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACGTCAG TGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTC TTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCA CTGTCGGTAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGAT GGCATTTGTAGGAGCCACCTTCCTTTTCCACTATCTTCACAATAAAGTG ACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCGGATATTACCCTT TGTTGAAAAGTCTCAATTGCCCTTTGGTCTTCTGAGACTGTATCTTTGA TATTTTTGGAGTAGACAAGTGTGTCGTGCTCCACCATGTTATCACATCA ATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCACGATG CTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAGGCAT CTTCAACGATGGCCTTTCCTTTATCGCAATGATGGCATTTGTAGGAGCC ACCTTCCTTTTCCACTATCTTCACAATAAAGTGACAGATAGCTGGGCAA TGGAATCCGAGGAGGTTTCCGGATATTACCCTTTGTTGAAAAGTCTCAA TTGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTTTTGGAGTAGAC AAGTGTGTCGTGCTCCACCATGTTGACCTGCAGGCATGCAAGCTTGCAT GCCTGCAGGTCGACTCTAGAGGATCCCCGTCGGTACCGAATTTGTTCGT GAACTATTAGTTGCGGGCCTTGGCATCCGACTACCTCTGCGGCAATATT ATATTCCCTGGGCCCACCGTGAACCCAATTTCGCCTATTTATTCATTAC CCCCATTAACATTGAAGTAGTCATGATGGGCCTGCAGCACGTTGGTGAG GCTGGCACAACTCATCCATATACTTTCTGACCGGATCGGCACATTATTG TAGAAAACGCGGACCCACAGCGCACTTTCCAAAGCGGTGCCGCGTCAGA ATGCGCTGGCAGAAAAAAATTAATCCAAAAGTACCCTCCAAGCAGCCCA TATAAACGCGTTTACAAATCCGCTAACCTCAACAATTTGAGCAGAGAAA ATTCGCACCTACAAGGCAGATGGCATCATCATTCAATCCAGAGCAGGCA AGAGTTCCTTCAGCATTACCTTTACCAGCACCACCACTTACCAAATTCAA CATCGGACTTTGTCAATTGAGTGTTACTTCTGATAAGAAAAGAAACATTT CACATGCTAAGAAAGCAATCGAAGAGGCTGCTAGTAAGGGAGCTAAACTC GTTCTTTTGCCTGAAATATGGAACTCACCATACAGTAACGATTCTTTTCC TGTGTACGCAGAAGAGATCGATGCTGGAGGTGATGCATCTCCATCAACTG CTATGCTCTCAGAAGTTAGTAAGAGACTCAAGATTACAATTATCGGAGGT TCAATTCCTGAGAGAGTTGGAGATAGGTTGTATAACACATGTTGCGTGTT CGGATCTGATGGAGAGCTCAAGGCTAAGCATAGGAAGATTCACCTCTTCG ATATAGATATTCCTGGAAAGATCACCTTCATGGAATCAAAAACACTTACC GCTGGAGAGACTCCAACAATTGTTGATACAGATGTGGGTAGAATCGGAAT AGGTATATGTTACGATATCAGGTTCCAAGAATTGGCTATGATATATGCTG CAAGAGGAGCACATCTCTTATGCTACCCTGGAGCTTTCAATATGACTACA GGTCCATTGCACTGGGAGCTTTTGCAAAGAGCTAGGGCAACAGATAACCA GCTCTATGTTGCTACCTGCTCTCCTGCAAGAGATTCAGGAGCTGGTTACA CCGCATGGGGTCATTCTACTCTTGTTGGACCATTTGGTGAAGTGTTGGCT ACCACTGAGCACGAAGAGGCTATTATAATCGCAGAAATCGATTACAGTAT ACTTGAGCAGAGAAGGACTTCTCTCCCATTAAATAGGCAGAGGAGGGGTG ATTTATACCAGTTAGTTGATGTTCAGAGATTAGATAGTAAGTGACACGTG TGAATTACAGGTGACCAGCTCGAATTTCCCCGATCGTTCAAACATTTGGC AATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATC ATATAATTTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATG CATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTAT ACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAA TTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGGGTACCGACGGGT ACCGAGCTCGAATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATT GTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGT AAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGC GCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTA ATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGAGCTTGA GCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAACTATCAGTGTT TGACAGGATATATTGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAG AATAACGGATATTTAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATT TGTATGTGCATGCCAACCACAGGGTTCCCCTCGGGATCAA
[0230] Seedling Inoculations: Alfalfa seedlings were grown to less than about 1/2 inch tall, and were then soaked in paper toweling that had been flooded with the Agrobacteria containing the TF101.1 vector carrying the Ω-amidase transgene expression cassette. The seedlings were left in the paper toweling for two to three days, removed and then planted in potting soil. Resulting TO and control plants were then grown for 27 days in a growth chamber, harvested and analyzed for biochemical and physical characteristics.
[0231] Biochemical Characterization: HPLC Assay for 2-oxoglutaramate: HPLC was used to assay 2-oxoglutaramate, following a modification of Calderon et al., 1985, J Bacteriol 161(2): 807-809. Briefly, 2-oxoglutaramate was extracted from plant tissue in distilled de-ionized water acidified to less than pH 2.0 with HCl using a weight to volume ratio of 2:1. 2-Oxoglutaramate was detected and quantified by HPLC, using an ION-300 7.8 mm ID×30 cm L column, with a mobile phase in 0.01NH2SO4, a flow rate of approximately 0.2 ml/min, at 40° C. Injection volume is approximately 20 and retention time between about 38 and 39 minutes. Detection is achieved with 210 nm UV light. Authentic 2-oxoglutaramate was used to calibrate the assay.
[0232] HPLC Assays for GPT and GS Activities: GPT was extracted from fresh plant tissue after grinding in cold 100 mM Tris-HCl, pH 7.6, containing 1 mm ethylenediaminetetraacetic, 200 mM pyridoxal phosphate and 6 mM mercaptoethanol in a ratio of 3 ml per gram of tissue. The extract was clarified by centrifugation and used in the assay. GS activity was extracted from fresh plant tissue after grinding in cold 50 mM Imidazole, pH 7.5 containing 10 mM MgCl2, and 12.5 mM mercaptoethanol in a ratio of 3 ml per gram of tissue. The extract was clarified by centrifugation and used in the assay. GPT activity was assayed as described in Calderon and Mora, 1985, Journal Bacteriology 161:807-809. GS activity was measured as described in Shapiro and Stadtmann, 1970, Methods in Enzymology 17A: 910-922. Both assays involve an incubation with substrates and cofactor at the proper pH. Detection was by HPLC.
[0233] HPLC Assay for Ω-Amidase Activity: Ω-amidase activity was determined using the 96-well plate assay as described in Krasnikov et al., 2009, Analytical Biochemistry 391: 144-150.
[0234] Results:
[0235] Plant fresh weight and leaf and root 2-oxoglutaramate concentrations and ratios in wild type and transgenic alfalfa plants were measured and are shown in Table 5 below. A comparison of the GS and GPT activities in the best performing transgenic alfalfa with a wild type control plant average values is shown in Table 6. Plant fresh weight values are plotted against 2-oxoglutaramate concentrations in FIG. 4. A photograph comparing transgenic and control alfalfa plants is shown in FIG. 5.
[0236] Transgenic alfalfa plants carrying the Ω-amidase directed for root expression showed significantly reduced root 2-oxoglutaramate concentrations, presumably as a result of the added Ω-amidase activity introduced by the transgene (Table 5). Activity levels for both GS and GPT remain constant in root tissues but are very significantly elevated in transgenic alfalfa (Table 6).
[0237] The transgenic plants also showed faster growth, yielding substantially increased biomass (Table 5), which correlated in near-linear fashion with the level of reduced 2-oxoglutaramate in these plants (FIG. 4). The fastest growing transgenic alfalfa line exhibited a 274% increase in biomass relative to the average biomass of the controls. This line also showed the most reduction in 2-oxoglutaramate root concentration.
TABLE-US-00043 TABLE 5 FRESH ROOT LEAF LEAF/ ALFALFA WEIGHT 2-OGM 2-OGM ROOT GENOTYPE (g) (nmol/g) (nmol/g) 2-OGM Control 1 1.43 269.7 910.7 3.5 Control 2 1.75 286 1536.5 5.4 Control 3 1.16 383.4 1826.6 4.8 Transgene 4 2.31 332 2144.6 6.5 Transgene 6 2.80 189 1479.4 7.8 Transgene 13 3.16 241.2 3038.5 12.8 Transgene 5 5.42 125.14 2729.9 21.8 TG = Trangenic
TABLE-US-00044 TABLE 6 GS Activity GPT Activity ALFALFA micromoles/gfwt/min nmoles/gfw/hr GENOTYPE Leaf Root Leaf Root Control. avg 4.3 3.3 339.1 66.3 Transgene 5 15.9 3.5 551.5 63.6
Example 4
Increased Growth of Transgenic Arabidopsis Plants Carrying Root-Preferred OMEGA-Amidase Transgene
[0238] In this example, Arabidopsis plant growth was increased by introducing an Ω-amidase transgene under the control of a highly root-preferred promoter. The resulting transgenic Arabidopsis plants showed markedly decreased 2-oxoglutaramate concentration in roots, very significantly increased leaf-to-root ratios of 2-oxoglutaramate, highly elevated GS and GPT levels in leaf, greatly reduced Ω-amidase levels in leaves, and astounding enhanced growth relative to wild type Arabidopsis plants.
[0239] Materials and Methods:
[0240] Agrobacterium Vectors: The Ω-amidase transgene expression vector and Agrobacteria preparation were generated as described in Example 3, supra.
[0241] Transformation: Transformation of Arabidopsis was achieved using Agrobacterium-mediated "floral dip" transfer as described (Harrison et al., 2006, Plant Methods 2:19-23; Clough and Bent, 1998, Plant J. 16:735-743). Agrobacteria transformed with the TF101.1 vector carrying the 52-amidase transgene expression cassette were grown under antibiotic selection, collected by centrifugation resuspended in LB broth with antibiotic and used to floral dip Arabidopsis inflorescence. Floral dipped Arabidopsis plants were taken to maturity and self-fertilized and seeds were collected.
[0242] Germination and Selection: Seeds from plants transformed with the TF101.1 vector carrying the Ω-amidase transgene expression cassette were germinated on a media containing 15 mg/L of BASTA or an equivalent amount of phosphinothricin. For the additional constructs and combinations described in Examples 5-7: Seeds derived from plants transformed with glutamine synthetase construct were germinated on a media contain 20 micrograms hygromycin and regular selection procedures were followed to obtain the surviving seedlings. Seeds derived from plants transformed with glutamine phenylpyruvate transaminase were geminated on a media containing 20 ug/ml of kanamycin and regular selection procedures were followed to obtain the surviving seedlings. For seedlings containing more than one of these genes the seedlings were transferred to media containing the next selection chemical and surviving seedlings were obtained. The surviving seedlings were examined.
[0243] Biochemical Characterization: Assays for 2-oxoglutaramate, Ω-amidase, GS and GPT were conducted as described in Example 3, supra.
[0244] Results:
[0245] GPT activity and GS activity of wild type and transgenic Arabidopsis plants were measured and are shown in Table 7, below. Ω-amidase activities and 2-oxoglutaramate concentrations in wild type and transgenic Arabidopsis plants were measured in both leaf and root tissues, and are shown in Table 8, below.
TABLE-US-00045 TABLE 7 GS Activity GPT Activity Arabidopsis FWt mg umoles/gfwt/min nmoles/gfwt/hr Genotype Whole plant Root Leaf L/R Root Leaf L/R Wild type* 77 1.5 3.4 2.3 167 132 0.8 TG Amidase** 479 1.4 5.8 4.1 192 486 2.5 TG = Transgenic *Average values of 9 plants **Average values of 6 plants
TABLE-US-00046 TABLE 8 2-Oxoglutaramate Ω-amidase Concentration nmoles/gfwt/hr nmoles/gfwt Arabidopsis FWt mg L/R L/R Genotype Whole plant Root Leaf Ratio Root Leaf Ratio Wild type* 77 86 1090 12.7 410 163 0.4 TG 479 243 127 0.5 98 305 3.1 ω-amidase** TG = Transgenic *Average values of 9 plants **Average values of 6 plants
[0246] Compared to wild type control Arabidopsis plants, the transgenic Arabidopsis plants carrying the Ω-amidase directed for root expression showed dramatic biochemical changes within the Ω-amidase pathway, including increased root Ω-amidase activity (186% increase), reduced levels of root 2-oxoglutaramate (76% reduction) and increased leaf-to-root 2-oxoglutaramate ratios. Moreover, these transgenic plants also show great reductions in leaf Ω-amidase activity levels (88% reduction), a near-doubling of leaf 2-oxoglutaramate levels, and higher leaf GS and leaf GPT activities (70% and 533%, respectively). The resulting impact on growth was astounding, with the transgenic plants weighing more than six times the weight of the wild type plants, on average.
Example 5
Increased Growth of Transgenic Arabidopsis Plants Carrying Root-Preferred Ω-Amidase Transgene and Leaf-Directed GPT
[0247] In this example, Arabidopsis plant growth was increased by introducing an Ω-amidase transgene under the control of a highly root-preferred promoter and a GPT transgene under the control of a leaf-directing promoter. The resulting transgenic Arabidopsis plants show astounding increases in growth, as well as various biochemical changes, relative to wild type Arabidopsis plants.
[0248] Materials and Methods:
[0249] Agrobacterium Vectors: The Ω-amidase transgene expression vector and Agrobacterium preparation were generated as described in Example 3, supra. For the leaf-directed GPT transgene, an expression cassette comprising the tomato rubisco small subunit promoter and an Arabidopsis codon-optimized GPT truncated to delete the first 45 codons of the full length GPT (eliminating the chloroplast transit peptide) was constructed and cloned into the Cambia 2201 expression vector. The resulting GPT transgene expression vector construct (6c) is shown below, and was used to transform Agrobacteria as described in Example 3.
[0250] The nucleotide sequence of the Cambia 2201 with tomato rubisco SSU promoter+(-45) truncated, optimized for Arabidopsis GPT+nos terminator is set forth below as SEQ ID NO: 40. Underlined nucleotides=tomato rubisco promoter, bold nucleotides=GPT coding region (codon optimized for Arabidopsis), italicized nucleotides=nos terminator region (and some Cambia vector sequence), and other nucleotides=Cambia 2201 vector and additional cloning sites.
TABLE-US-00047 CCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGA CTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACT CATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGT GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCA TGATTACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGATCTAGAGAAT TCATCGATGTTTGAATCCTCCTTAAAGTTTTTCTCTGGAGAAACTGTAGT AATTTTACTTTGTTGTGTTCCCTTCATCTTTTGAATTAATGGCATTTGTT TTAATACTAATCTGCTTCTGAAACTTGTAATGTATGTATATCAGTTTCTT ATAATTTATCCAAGTAATATCTTCCATTCTCTATGCAATTGCCTGCATAA GCTCGACAAAAGAGTACATCAACCCCTCCTCCTCTGGACTACTCTAGCTA AACTTGAATTTCCCCTTAAGATTATGAAATTGATATATCCTTAACAAACG ACTCCTTCTGTTGGAAAATGTAGTACTTGTCTTTCTTCTTTTGGGTATAT ATAGTTTATATACACCATACTATGTACAACATCCAAGTAGAGTGAAATGG ATACATGTACAAGACTTATTTGATTGATTGATGACTTGAGTTGCCTTAGG AGTAACAAATTCTTAGGTCAATAAATCGTTGATTTGAAATTAATCTCTCT GTCTTAGACAGATAGGAATTATGACTTCCAATGGTCCAGAAAGCAAAGTT CGCACTGAGGGTATACTTGGAATTGAGACTTGCACAGGTCCAGAAACCAA AGTTCCCATCGAGCTCTAAAATCACATCTTTGGAATGAAATTCAATTAGA GATAAGTTGCTTCATAGCATAGGTAAAATGGAAGATGTGAAGTAACCTGC AATAATCAGTGAAATGACATTAATACACTAAATACTTCATATGTAATTAT CCTTTCCAGGTTAACAATACTCTATAAAGTAAGAATTATCAGAAATGGGC TCATCAAACTTTTGTACTATGTATTTCATATAAGGAAGTATAACTATACA TAAGTGTATACACAACTTTATTCCTATTTTGTAAAGGTGGAGAGACTGTT TTCGATGGATCTAAAGCAATATGTCTATAAAATGCATTGATATAATAATT ATCTGAGAAAATCCAGAATTGGCGTTGGATTATTTCAGCCAAATAGAAGT TTGTACCATACTTGTTGATTCCTTCTAAGTTAAGGTGAAGTATCATTCAT AAACAGTTTTCCCCAAAGTACTACTCACCAAGTTTCCCTTTGTAGAATTA ACAGTTCAAATATATGGCGCAGAAATTACTCTATGCCCAAAACCAAACGA GAAAGAAACAAAATACAGGGGTTGCAGACTTTATTTTCGTGTTAGGGTGT GTTTTTTCATGTAATTAATCAAAAAATATTATGACAAAAACATTTATACA TATTTTTACTCAACACTCTGGGTATCAGGGTGGGTTGTGTTCGACAATCA ATATGGAAAGGAAGTATTTTCCTTATTTTTTTAGTTAATATTTTCAGTTA TACCAAACATACCTTGTGATATTATTTTTAAAAATGAAAAACTCGTCAGA AAGAAAAAGCAAAAGCAACAAAAAAATTGCAAGTATTTTTTAAAAAAGAA AAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGGACGA GTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCAC AAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTT GTCCGTTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAA CCTCTTAGTAACCAATTATTTCAGCAACTAGTATGGCGACTCAAAATGAG TCAACACAAAAGCCTGTTCAGGTGGCTAAGAGACTTGAGAAGTTTAAAAC TACAATTTTCACTCAAATGTCTATCCTCGCAGTTAAGCACGGAGCTATTA ATCTTGGACAGGGTTTTCCTAACTTCGATGGTCCAGATTTCGTGAAAGAA GCTGCAATTCAAGCAATCAAGGATGGAAAAAATCAGTATGCTAGAGGATA CGGTATTCCTCAGTTGAACTCTGCTATCGCTGCAAGATTCAGAGAAGATA CAGGACTTGTTGTGGATCCAGAAAAAGAGGTTACTGTGACATCAGGTTGT ACTGAGGCTATTGCTGCAGCTATGCTCGGACTTATTAACCCTGGAGATGA AGTTATCCTTTTTGCACCATTCTATGATTCTTACGAGGCTACATTGTCAA TGGCAGGAGCTAAGGTGAAAGGTATTACTCTCAGACCTCCAGATTTCTCT ATCCCTTTGGAAGAGCTCAAGGCAGCTGTTACTAATAAGACAAGAGCTAT CTTGATGAATACTCCTCATAACCCAACAGGAAAGATGTTTACTAGAGAAG AGCTCGAAACTATTGCTTCTCTTTGCATCGAGAACGATGTTTTGGTGTTC TCAGATGAAGTGTATGATAAACTCGCATTTGAGATGGATCACATTTCTAT CGCTTCACTTCCAGGAATGTACGAAAGAACTGTTACTATGAATTCTTTGG GAAAGACTTTTTCTCTCACAGGATGGAAAATTGGTTGGGCAATCGCTCCT CCACATCTCACATGGGGTGTTAGACAAGCACACTCTTATCTTACTTTCGC AACTTCAACACCTGCTCAGTGGGCAGCTGTGGCAGCTCTTAAGGCTCCAG AATCTTACTTCAAGGAGTTGAAGAGAGATTACAACGTTAAGAAAGAAACA CTTGTGAAGGGATTGAAAGAGGTTGGTTTTACAGTGTTCCCTTCTTCAGG AACTTACTTTGTTGTGGCAGATCATACTCCATTCGGTATGGAAAACGATG TTGCTTTTTGTGAGTATCTTATTGAAGAGGTTGGAGTTGTGGCTATCCCT ACATCTGTGTTTTACCTTAATCCAGAAGAGGGAAAGAATCTTGTTAGATT TGCATTCTGCAAAGATGAAGAGACTTTGAGAGGTGCTATTGAGAGGATGA AGCAAAAACTCAAGAGAAAAGTTTGACACGTGTGAATTACAGGTGACCAG CTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGAT TGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAAT TACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAG ATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAG AAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCA TCTATGTTACTAGATCGGGAATTAAACTATCAGTGTTTGACAGGATATAT TGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATT TAAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGC CAACCACAGGGTTCCCCTCGGGATCAAAGTACTTTGATCCAACCCCTCC GCTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGA AAACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTG CCCTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTAGA ATACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGC CGCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTG ACCAACCAACGGGCCGAACTGCACGCGGCCGGCTGCACCAAGCTGTTTT CCGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGAT GCTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGAC CGCCTGGCCCGCAGCACCCGCGACCTACTGGACATTGCCGAGCGCATCC AGGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACAC CACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCC GAGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGAGG CCGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACCCC GGCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACC GTGAAAGAGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTACC GCGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCGGCG CGGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCC GCCGAGAATGAACGCCAAGAGGAACAAGCATGAAACCGCACCAGGACGG CCAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATC GCGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGC TGCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGGCC GGCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGTGA TGTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGA TGCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTATC GCTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCC ATCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGA TTCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAGAT CAACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACG TGAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCA GGCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGCTG ATTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGG TGGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGC GGCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAG GTTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGTA TCACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGT TCTTGAATCAGAACCCGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTG GCCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAA AATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGC AGCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCATGA AGCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGAT GTACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATAC ATCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGA ATTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAACCAGGCA CCGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGG CGTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCC AAGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCCGGTACAA ATCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGC AGGCCGCCCAGCGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATC GTGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCG GCAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAAC CAGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCG CAGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGA GCTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTT
CCGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACT GATGGCGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGG AAGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTAC TCAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGT AGAAACCTGCATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTACG AAGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCT TGATTAGCCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTA CATCGAGATCGAGCTAGCTGATTGGATGTACCGCGAGATCACAGAAGGC AAGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATC CCGGCATCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAA GGCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCAGC GCCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGT CAAATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGG CCCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGGCGAAGCATCC GCCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAG GGGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGG GAACCCAAAGCCGTACATTGGGAACCGGAACCCGTACATTGGGAACCCA AAGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAG AGAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAAC TCTTAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCCAGCGCACAGCC GAAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCC CCGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGC TGGCCTACGGCCAGGCAATCTACCAGGGCGCGGACAAGCCGCGCCGTCG CCACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTC GGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCA CAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGC GTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGT AGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGAT TGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTA AGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACT CGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA GGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAAC ATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGT TGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAA TCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATAC CAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCC TGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTT CGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGA CTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGG TATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCT ACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTAC CTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCT GGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAA AAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCA GTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATGATATATCT CCCAATTTGTGTAGGGCTTATTATGCACGCTTAAAAATAATAAAAGCAG ACTTGACCTGATAGTTTGGCTGTGAGCAATTATGTGCTTAGTGCATCTA ATCGCTTGAGTTAACGCCGGCGAAGCGGCGTCGGCTTGAACGAATTTCTA GCTAGAGGATCGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCT GCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATG GAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGC ACCTTGTCGCCTTGCGTATAATATTTGCCCATTGTGAAAACGGGGGCGA AGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCAC CCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGG AAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGT GTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAA CGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCC CATATCACCAGCTCACCGTCTTTCATTGCCATACGGAACTCCGGATGAG CATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTG CTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACG GTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTT CTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTT TTTCTCCATGATGTTTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTA ACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCG CTTGCTGCTTGGATGCCCGAGGCATAGACTGTACCCCAAAAAAACATGT CATAACAAGAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCG TTCGGTCAAGGTTCTGGACCAGTTGCGTGACGGCAGTTACGCTACTTGC ATTACAGCTTACGAACCGAACGAGGCTTATGTCCACTGGGTTCGTGCCC GAATTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCT ACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGT TCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCGCCTTACAACG GCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTG GTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGG CAGGATATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATAAC ACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTAATTCGGG GGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAATTAGAAATTTTA TTGATAGAAGTATTTTACAAATACAAATACATACTAAGGGTTTCTTATA TGCTCAACACATGAGCGAAACCCTATAGGAACCCTAATTCCCTTATCTG GGAACTACTCACACATTATTATGGAGAAACTCGAGCTTGTCGATCGACT CTAGCTAGAGGATCGATCCGAACCCCAGAGTCCCGCTCAGAAGAACTCG TCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATAC CGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGC AATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCC AGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGA TATTCGGCAAGCAGGCATCGCCATGTGTCACGACGAGATCCTCGCCGTC GGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCC TGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCC GAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCA GGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATG GATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCG GCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTC GAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGC GCTGCCTCGTCCTGGAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGA CAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATC AGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCC ACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCCCCA TGGTCGATCGACAGATCTGCGAAAGCTCGAGAGAGATAGATTTGTAGAG AGAGACTGGTGATTTCAGCGTGTCCTCTCCAAATGAAATGAACTTCCTT ATATAGAGGAAGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTT ACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTG GAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTT GGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCG CAATGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCCTTTTGA TGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATAT TACCCTTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTA TCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTAT CACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTC CACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCA GAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGT AGGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGC TGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAA GTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGG AGTAGACGAGAGTGTCGTGCTCCACCATGTTGGCAAGCTGCTCTAGCCA ATACGCAAACCGCCTCTC
[0251] Transformation and Selection: Transformation of Arabidopsis was achieved using Agrobacterium-mediated "floral dip" transfer as described in Example 4. Transformed plants were grown under selection as described in Example 4.
[0252] Biochemical Characterization: Assays for 2-oxoglutaramate, Ω-amidase, GS and GPT were conducted as described in Example 3, supra.
[0253] Results:
[0254] GPT activity and GS activity of wild type and transgenic Arabidopsis plants were measured and are shown in Table 9, below. Ω-amidase activities and 2-oxoglutaramate concentrations in wild type and transgenic Arabidopsis plants were measured in both leaf and root tissues, and are shown in Table 10, below.
TABLE-US-00048 TABLE 9 GS Activity GPT Activity Arabidopsis FWt mg umoles/gfwt/min nmoles/gfwt/hr Genotype Whole plant Root Leaf L/R Root Leaf L/R Wild type* 77 1.5 3.4 2.3 167 132 0.8 TG GPT (6c) + 513 1.8 8.25 4.6 232 389 1.7 ω-amidase** TG = Transgenic GPT (6c) = -45 truncated GPT [SEQ ID NO: 40] *Average values of 9 plants **Average values of 5 plants
TABLE-US-00049 TABLE 10 2-Oxoglutaramate Ω-amidase Concentration nmoles/gfwt/hr nmoles/gfwt Arabidopsis FWt mg L/R L/R Genotype Whole plant Root Leaf Ratio Root Leaf Ratio Wild type* 77 86 1090 12.7 410 163 0.4 TG GPT (6c) + 513 308 584 1.9 268 275 1.0 ω-amidase ** TG = Transgenic GPT (6c) = -45 truncated GPT [SEQ ID NO: 40] *Average values of 9 plants ** Average values of 5 plants
[0255] Compared to wild type control Arabidopsis plants, the transgenic Arabidopsis plants carrying the Ω-amidase directed for root expression and the truncated GPT (for cyosolic expression) directed for leaf expression showed biochemical changes within the Ω-amidase pathway similar to those observed in the transgenic plants of Example 5, supra. More specifically, transgenic GPT+Ω-amidase plants showed increased root Ω-amidase activity, reduced root 2-oxoglutaramate concentration, and increased leaf-to-root 2-oxoglutaramate ratios. Also, similar to the results seen in the transgenic plants of Example 5, the GPT+Ω-amidase transgenic plants showed significant reductions in leaf Ω-amidase activity levels, increased leaf 2-oxoglutaramate, and higher leaf GS and leaf GPT activities. The resulting impact on growth was even greater than observed for the transgenic plants of Example 5, with the transgenic plants weighing more than 6.7 times the weight of the wild type plants, on average.
Example 6
Increased Growth of Transgenic Arabidopsis Plants Carrying Root-Preferred Ω-Amidase Transgene and Leaf-Directed GPT and GS
[0256] In this example, Arabidopsis plant growth was increased by introducing an Ω-amidase transgene under the control of a highly root-preferred promoter and GPT and GS transgenes under the control of leaf-directing promoters. The resulting transgenic Arabidopsis plants showed astounding increases in growth, as well as various biochemical changes, relative to wild type Arabidopsis plants.
[0257] Materials and Methods:
[0258] Agrobacterium Vectors: The Ω-amidase transgene expression vector and Agrobacteria preparation were generated as described in Example 3, supra. For the leaf-directed GPT transgene, an expression cassette comprising the tomato rubisco small subunit promoter and the coding sequence for an Arabidopsis codon-optimized GPT truncated to delete the first 45 codons of the full length GPT (eliminating the chloroplast transit peptide), and containing an F to V mutation at amino acid residue 45, was constructed and cloned into the Cambia 2201 expression vector. The resulting GPT transgene expression vector construct (9c) is shown in SEQ ID NO: 41 below, and was used to transform Agrobacteria as described in Example 5.
[0259] The nucleotide sequence of the Cambia 2201 with tomato rubisco SSU promoter+(-45) truncated, optimized for Arabidopsis GPT F-to-V mutation+nos terminator is set forth below as SEQ ID NO: 41. Underlined nucleotides=tomato rubisco promoter, bold nucleotides=GPT coding region (codon optimized for Arabidopsis), italicized nucleotides=nos terminator region (and some Cambia vector sequence), and other nucleotides=Cambia 2201 vector and additional cloning sites.
TABLE-US-00050 CCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGA CTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACT CATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGT GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCA TGATTACGAATTCGAGCTCGGTACCCGGGGATCCTCTAGATCTAGAGAAT TCATCGATGTTTGAATCCTCCTTAAAGTTTTTCTCTGGAGAAACTGTAGT AATTTTACTTTGTTGTGTTCCCTTCATCTTTTGAATTAATGGCATTTGTT TTAATACTAATCTGCTTCTGAAACTTGTAATGTATGTATATCAGTTTCTT ATAATTTATCCAAGTAATATCTTCCATTCTCTATGCAATTGCCTGCATAA GCTCGACAAAAGAGTACATCAACCCCTCCTCCTCTGGACTACTCTAGCTA AACTTGAATTTCCCCTTAAGATTATGAAATTGATATATCCTTAACAAACG ACTCCTTCTGTTGGAAAATGTAGTACTTGTCTTTCTTCTTTTGGGTATAT ATAGTTTATATACACCATACTATGTACAACATCCAAGTAGAGTGAAATGG ATACATGTACAAGACTTATTTGATTGATTGATGACTTGAGTTGCCTTAGG AGTAACAAATTCTTAGGTCAATAAATCGTTGATTTGAAATTAATCTCTCT GTCTTAGACAGATAGGAATTATGACTTCCAATGGTCCAGAAAGCAAAGTT CGCACTGAGGGTATACTTGGAATTGAGACTTGCACAGGTCCAGAAACCAA AGTTCCCATCGAGCTCTAAAATCACATCTTTGGAATGAAATTCAATTAGA GATAAGTTGCTTCATAGCATAGGTAAAATGGAAGATGTGAAGTAACCTGC AATAATCAGTGAAATGACATTAATACACTAAATACTTCATATGTAATTAT CCTTTCCAGGTTAACAATACTCTATAAAGTAAGAATTATCAGAAATGGGC TCATCAAACTTTTGTACTATGTATTTCATATAAGGAAGTATAACTATACA TAAGTGTATACACAACTTTATTCCTATTTTGTAAAGGTGGAGAGACTGTT TTCGATGGATCTAAAGCAATATGTCTATAAAATGCATTGATATAATAATT ATCTGAGAAAATCCAGAATTGGCGTTGGATTATTTCAGCCAAATAGAAGT TTGTACCATACTTGTTGATTCCTTCTAAGTTAAGGTGAAGTATCATTCAT AAACAGTTTTCCCCAAAGTACTACTCACCAAGTTTCCCTTTGTAGAATTA ACAGTTCAAATATATGGCGCAGAAATTACTCTATGCCCAAAACCAAACGA GAAAGAAACAAAATACAGGGGTTGCAGACTTTATTTTCGTGTTAGGGTGT GTTTTTTCATGTAATTAATCAAAAAATATTATGACAAAAACATTTATACA TATTTTTACTCAACACTCTGGGTATCAGGGTGGGTTGTGTTCGACAATCA ATATGGAAAGGAAGTATTTTCCTTATTTTTTTAGTTAATATTTTCAGTTA TACCAAACATACCTTGTGATATTATTTTTAAAAATGAAAAACTCGTCAGA AAGAAAAAGCAAAAGCAACAAAAAAATTGCAAGTATTTTTTAAAAAAGAA AAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGGACGA GTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCAC AAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTT GTCCGTTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAA CCTCTTAGTAACCAATTATTTCAGCAACTAGTATGGCGACTCAAAATGAG TCAACACAAAAGCCTGTTCAGGTGGCTAAGAGACTTGAGAAGTTTAAAAC TACAATTTTCACTCAAATGTCTATCCTCGCAGTTAAGCACGGAGCTATTA ATCTTGGACAGGGTGTTCCTAACTTCGATGGTCCAGATTTCGTGAAAGAA GCTGCAATTCAAGCAATCAAGGATGGAAAAAATCAGTATGCTAGAGGATA CGGTATTCCTCAGTTGAACTCTGCTATCGCTGCAAGATTCAGAGAAGATA CAGGACTTGTTGTGGATCCAGAAAAAGAGGTTACTGTGACATCAGGTTGT ACTGAGGCTATTGCTGCAGCTATGCTCGGACTTATTAACCCTGGAGATGA AGTTATCCTTTTTGCACCATTCTATGATTCTTACGAGGCTACATTGTCAA TGGCAGGAGCTAAGGTGAAAGGTATTACTCTCAGACCTCCAGATTTCTCT ATCCCTTTGGAAGAGCTCAAGGCAGCTGTTACTAATAAGACAAGAGCTAT CTTGATGAATACTCCTCATAACCCAACAGGAAAGATGTTTACTAGAGAAG AGCTCGAAACTATTGCTTCTCTTTGCATCGAGAACGATGTTTTGGTGTTC TCAGATGAAGTGTATGATAAACTCGCATTTGAGATGGATCACATTTCTAT CGCTTCACTTCCAGGAATGTACGAAAGAACTGTTACTATGAATTCTTTGG GAAAGACTTTTTCTCTCACAGGATGGAAAATTGGTTGGGCAATCGCTCCT CCACATCTCACATGGGGTGTTAGACAAGCACACTCTTATCTTACTTTCGC AACTTCAACACCTGCTCAGTGGGCAGCTGTGGCAGCTCTTAAGGCTCCAG AATCTTACTTCAAGGAGTTGAAGAGAGATTACAACGTTAAGAAAGAAACA CTTGTGAAGGGATTGAAAGAGGTTGGTTTTACAGTGTTCCCTTCTTCAGG AACTTACTTTGTTGTGGCAGATCATACTCCATTCGGTATGGAAAACGATG TTGCTTTTTGTGAGTATCTTATTGAAGAGGTTGGAGTTGTGGCTATCCCT ACATCTGTGTTTTACCTTAATCCAGAAGAGGGAAAGAATCTTGTTAGATT TGCATTCTGCAAAGATGAAGAGACTTTGAGAGGTGCTATTGAGAGGATGA AGCAAAAACTCAAGAGAAAAGTTTGACACGTGTGAATTACAGGTGACCAG CTCGAATTTCCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGAT TGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCTGTTGAAT TACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAG ATGGGTTTTTATGATTAGAGTCCCGCAATTATACATTTAATACGCGATAG AAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCA TCTATGTTACTAGATCGGGAATTAAACTATCAGTGTTTGACAGGATATAT TGGCGGGTAAACCTAAGAGAAAAGAGCGTTTATTAGAATAACGGATATTT AAAAGGGCGTGAAAAGGTTTATCCGTTCGTCCATTTGTATGTGCATGCC AACCACAGGGTTCCCCTCGGGATCAAAGTACTTTGATCCAACCCCTCCG CTGCTATAGTGCAGTCGGCTTCTGACGTTCAGTGCAGCCGTCTTCTGAA AACGACATGTCGCACAAGTCCTAAGTTACGCGACAGGCTGCCGCCCTGC CCTTTTCCTGGCGTTTTCTTGTCGCGTGTTTTAGTCGCATAAAGTAGAA TACTTGCGACTAGAACCGGAGACATTACGCCATGAACAAGAGCGCCGCC GCTGGCCTGCTGGGCTATGCCCGCGTCAGCACCGACGACCAGGACTTGA CCAACCAACGGGCCGAACTGCACGCGGCCGGCTGCACCAAGCTGTTTTC CGAGAAGATCACCGGCACCAGGCGCGACCGCCCGGAGCTGGCCAGGATG CTTGACCACCTACGCCCTGGCGACGTTGTGACAGTGACCAGGCTAGACC GCCTGGCCCGCAGCACCCGCGACCTACTGGACATTGCCGAGCGCATCCA GGAGGCCGGCGCGGGCCTGCGTAGCCTGGCAGAGCCGTGGGCCGACACC ACCACGCCGGCCGGCCGCATGGTGTTGACCGTGTTCGCCGGCATTGCCG AGTTCGAGCGTTCCCTAATCATCGACCGCACCCGGAGCGGGCGCGAGGC CGCCAAGGCCCGAGGCGTGAAGTTTGGCCCCCGCCCTACCCTCACCCCG GCACAGATCGCGCACGCCCGCGAGCTGATCGACCAGGAAGGCCGCACCG TGAAAGAGGCGGCTGCACTGCTTGGCGTGCATCGCTCGACCCTGTACCG CGCACTTGAGCGCAGCGAGGAAGTGACGCCCACCGAGGCCAGGCGGCGC GGTGCCTTCCGTGAGGACGCATTGACCGAGGCCGACGCCCTGGCGGCCG CCGAGAATGAACGCCAAGAGGAACAAGCATGAAACCGCACCAGGACGGC CAGGACGAACCGTTTTTCATTACCGAAGAGATCGAGGCGGAGATGATCG CGGCCGGGTACGTGTTCGAGCCGCCCGCGCACGTCTCAACCGTGCGGCT GCATGAAATCCTGGCCGGTTTGTCTGATGCCAAGCTGGCGGCCTGGCCG GCCAGCTTGGCCGCTGAAGAAACCGAGCGCCGCCGTCTAAAAAGGTGAT GTGTATTTGAGTAAAACAGCTTGCGTCATGCGGTCGCTGCGTATATGAT GCGATGAGTAAATAAACAAATACGCAAGGGGAACGCATGAAGGTTATCG CTGTACTTAACCAGAAAGGCGGGTCAGGCAAGACGACCATCGCAACCCA TCTAGCCCGCGCCCTGCAACTCGCCGGGGCCGATGTTCTGTTAGTCGAT TCCGATCCCCAGGGCAGTGCCCGCGATTGGGCGGCCGTGCGGGAAGATC AACCGCTAACCGTTGTCGGCATCGACCGCCCGACGATTGACCGCGACGT GAAGGCCATCGGCCGGCGCGACTTCGTAGTGATCGACGGAGCGCCCCAG GCGGCGGACTTGGCTGTGTCCGCGATCAAGGCAGCCGACTTCGTGCTGA TTCCGGTGCAGCCAAGCCCTTACGACATATGGGCCACCGCCGACCTGGT GGAGCTGGTTAAGCAGCGCATTGAGGTCACGGATGGAAGGCTACAAGCG GCCTTTGTCGTGTCGCGGGCGATCAAAGGCACGCGCATCGGCGGTGAGG TTGCCGAGGCGCTGGCCGGGTACGAGCTGCCCATTCTTGAGTCCCGTAT CACGCAGCGCGTGAGCTACCCAGGCACTGCCGCCGCCGGCACAACCGTT CTTGAATCAGAACCCGAGGGCGACGCTGCCCGCGAGGTCCAGGCGCTGG CCGCTGAAATTAAATCAAAACTCATTTGAGTTAATGAGGTAAAGAGAAA ATGAGCAAAAGCACAAACACGCTAAGTGCCGGCCGTCCGAGCGCACGCA GCAGCAAGGCTGCAACGTTGGCCAGCCTGGCAGACACGCCAGCCATGAA GCGGGTCAACTTTCAGTTGCCGGCGGAGGATCACACCAAGCTGAAGATG TACGCGGTACGCCAAGGCAAGACCATTACCGAGCTGCTATCTGAATACA TCGCGCAGCTACCAGAGTAAATGAGCAAATGAATAAATGAGTAGATGAA TTTTAGCGGCTAAAGGAGGCGGCATGGAAAATCAAGAACAACCAGGCAC CGACGCCGTGGAATGCCCCATGTGTGGAGGAACGGGCGGTTGGCCAGGC GTAAGCGGCTGGGTTGTCTGCCGGCCCTGCAATGGCACTGGAACCCCCA AGCCCGAGGAATCGGCGTGACGGTCGCAAACCATCCGGCCCGGTACAAA TCGGCGCGGCGCTGGGTGATGACCTGGTGGAGAAGTTGAAGGCCGCGCA GGCCGCCCAGCGGCAACGCATCGAGGCAGAAGCACGCCCCGGTGAATCG TGGCAAGCGGCCGCTGATCGAATCCGCAAAGAATCCCGGCAACCGCCGG CAGCCGGTGCGCCGTCGATTAGGAAGCCGCCCAAGGGCGACGAGCAACC AGATTTTTTCGTTCCGATGCTCTATGACGTGGGCACCCGCGATAGTCGC AGCATCATGGACGTGGCCGTTTTCCGTCTGTCGAAGCGTGACCGACGAG CTGGCGAGGTGATCCGCTACGAGCTTCCAGACGGGCACGTAGAGGTTTC
CGCAGGGCCGGCCGGCATGGCCAGTGTGTGGGATTACGACCTGGTACTG ATGGCGGTTTCCCATCTAACCGAATCCATGAACCGATACCGGGAAGGGA AGGGAGACAAGCCCGGCCGCGTGTTCCGTCCACACGTTGCGGACGTACT CAAGTTCTGCCGGCGAGCCGATGGCGGAAAGCAGAAAGACGACCTGGTA GAAACCTGCATTCGGTTAAACACCACGCACGTTGCCATGCAGCGTACGA AGAAGGCCAAGAACGGCCGCCTGGTGACGGTATCCGAGGGTGAAGCCTT GATTAGCCGCTACAAGATCGTAAAGAGCGAAACCGGGCGGCCGGAGTAC ATCGAGATCGAGCTAGCTGATTGGATGTACCGCGAGATCACAGAAGGCA AGAACCCGGACGTGCTGACGGTTCACCCCGATTACTTTTTGATCGATCC CGGCATCGGCCGTTTTCTCTACCGCCTGGCACGCCGCGCCGCAGGCAAG GCAGAAGCCAGATGGTTGTTCAAGACGATCTACGAACGCAGTGGCAGCG CCGGAGAGTTCAAGAAGTTCTGTTTCACCGTGCGCAAGCTGATCGGGTC AAATGACCTGCCGGAGTACGATTTGAAGGAGGAGGCGGGGCAGGCTGGC CCGATCCTAGTCATGCGCTACCGCAACCTGATCGAGGGCGAAGCATCCG CCGGTTCCTAATGTACGGAGCAGATGCTAGGGCAAATTGCCCTAGCAGG GGAAAAAGGTCGAAAAGGTCTCTTTCCTGTGGATAGCACGTACATTGGG AACCCAAAGCCGTACATTGGGAACCGGAACCCGTACATTGGGAACCCAA AGCCGTACATTGGGAACCGGTCACACATGTAAGTGACTGATATAAAAGA GAAAAAAGGCGATTTTTCCGCCTAAAACTCTTTAAAACTTATTAAAACT CTTAAAACCCGCCTGGCCTGTGCATAACTGTCTGGCCAGCGCACAGCCG AAGAGCTGCAAAAAGCGCCTACCCTTCGGTCGCTGCGCTCCCTACGCCC CGCCGCTTCGCGTCGGCCTATCGCGGCCGCTGGCCGCTCAAAAATGGCT GGCCTACGGCCAGGCAATCTACCAGGGCGCGGACAAGCCGCGCCGTCGC CACTCGACCGCCGGCGCCCACATCAAGGCACCCTGCCTCGCGCGTTTCG GTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCAC AGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCG TCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTA GCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATT GTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAA GGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTC GCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAG GCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTT GCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAAT CGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACC AGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCT GCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCG CTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTC GCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTG CGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGAC TTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGT ATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTA CACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACC TTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG GTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAA AGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAG TGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGCATGATATATCTC CCAATTTGTGTAGGGCTTATTATGCACGCTTAAAAATAATAAAAGCAGA CTTGACCTGATAGTTTGGCTGTGAGCAATTATGTGCTTAGTGCATCTAA TCGCTTGAGTTAACGCCGGCGAAGCGGCGTCGGCTTGAACGAATTTCTA GCTAGAGGATCGCACCAATAACTGCCTTAAAAAAATTACGCCCCGCCCT GCCACTCATCGCAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATG GAAGCCATCACAAACGGCATGATGAACCTGAATCGCCAGCGGCATCAGC ACCTTGTCGCCTTGCGTATAATATTTGCCCATTGTGAAAACGGGGGCGA AGAAGTTGTCCATATTGGCCACGTTTAAATCAAAACTGGTGAAACTCAC CCAGGGATTGGCTGAGACGAAAAACATATTCTCAATAAACCCTTTAGGG AAATAGGCCAGGTTTTCACCGTAACACGCCACATCTTGCGAATATATGT GTAGAAACTGCCGGAAATCGTCGTGGTATTCACTCCAGAGCGATGAAAA CGTTTCAGTTTGCTCATGGAAAACGGTGTAACAAGGGTGAACACTATCC CATATCACCAGCTCACCGTCTTTCATTGCCATACGGAACTCCGGATGAG CATTCATCAGGCGGGCAAGAATGTGAATAAAGGCCGGATAAAACTTGTG CTTATTTTTCTTTACGGTCTTTAAAAAGGCCGTAATATCCAGCTGAACG GTCTGGTTATAGGTACATTGAGCAACTGACTGAAATGCCTCAAAATGTT CTTTACGATGCCATTGGGATATATCAACGGTGGTATATCCAGTGATTTT TTTCTCCATGATGTTTAACTTTGTTTTAGGGCGACTGCCCTGCTGCGTA ACATCGTTGCTGCTCCATAACATCAAACATCGACCCACGGCGTAACGCG CTTGCTGCTTGGATGCCCGAGGCATAGACTGTACCCCAAAAAAACATGT CATAACAAGAAGCCATGAAAACCGCCACTGCGCCGTTACCACCGCTGCG TTCGGTCAAGGTTCTGGACCAGTTGCGTGACGGCAGTTACGCTACTTGC ATTACAGCTTACGAACCGAACGAGGCTTATGTCCACTGGGTTCGTGCCC GAATTGATCACAGGCAGCAACGCTCTGTCATCGTTACAATCAACATGCT ACCCTCCGCGAGATCATCCGTGTTTCAAACCCGGCAGCTTAGTTGCCGT TCTTCCGAATAGCATCGGTAACATGAGCAAAGTCTGCCGCCTTACAACG GCTCTCCCGCTGACGCCGTCCCGGACTGATGGGCTGCCTGTATCGAGTG GTGATTTTGTGCCGAGCTGCCGGTCGGGGAGCTGTTGGCTGGCTGGTGG CAGGATATATTGTGGTGTAAACAAATTGACGCTTAGACAACTTAATAAC ACATTGCGGACGTTTTTAATGTACTGAATTAACGCCGAATTAATTCGGG GGATCTGGATTTTAGTACTGGATTTTGGTTTTAGGAATTAGAAATTTTA TTGATAGAAGTATTTTACAAATACAAATACATACTAAGGGTTTCTTATA TGCTCAACACATGAGCGAAACCCTATAGGAACCCTAATTCCCTTATCTG GGAACTACTCACACATTATTATGGAGAAACTCGAGCTTGTCGATCGACT CTAGCTAGAGGATCGATCCGAACCCCAGAGTCCCGCTCAGAAGAACTCG TCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATAC CGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGC AATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCC CAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACATGA TATTCGGCAAGCAGGCATCGCCATGTGTCACGACGAGATCCTCGCCGTC GGGCATGCGCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCC TGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCC GAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCA GGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATG TGATACTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCG GCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTC GAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGC GCTGCCTCGTCCTGGAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGA CAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATC AGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCC ACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCCCCA TGGTCGATCGACAGATCTGCGAAAGCTCGAGAGAGATAGATTTGTAGAG AGAGACTGGTGATTTCAGCGTGTCCTCTCCAAATGAAATGAACTTCCTT ATATAGAGGAAGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTT ACGTCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTG GAACGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTT GGGACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCG CAATGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCCTTTTGA TGAAGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATAT TACCCTTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTA TCTTTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTAT CACATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTC CACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCA GAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGT AGGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGC TGGGCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAA GTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGG AGTAGACGAGAGTGTCGTGCTCCACCATGTTGGCAAGCTGCTCTAGCCA ATACGCAAACCGCCTCTC
[0260] For the leaf-directed GS transgene, an expression cassette comprising the tomato rubisco small subunit promoter and an Arabidopsis GS1 coding sequence was constructed and cloned into the Cambia 1305.1 expression vector with the tomato rubisco small subunit promoter (rbcS3C). The resulting GS transgene expression vector construct (4c) is set forth as SEQ ID NO: 43, and was used to transform Agrobacteria as described in Example 5.
[0261] The nucleotide sequence of the Cambia 1305.1 with rubisco small subunit promoter (rbcS3C)+ARGS (Arabidopsis GS1) is set forth below as SEQ ID NO: 43. Underlined nucleotides=tomato rubisco promoter, double underlined nucleotides=catI intron in the Cambia 1305.1 vector first 10 amino acids are from GUSplus enzyme and cloning sites in 1305.1 vector, bold nucleotides=Arabidopsis GS1 coding region (cloned into SpeI to pmII sites), italicized nucleotides=nos terminator region (and some Cambia vector sequence), and other nucleotides=Cambia 2201 vector and additional cloning sites.
TABLE-US-00051 GGTACCGTTTGAATCCTCCTTAAAGTTTTTCTCTGGAGAAACTGTAGTAA TTTTACTTTGTTGTGTTCCCTTCATCTTTTGAATTAATGGCATTTGTTTT AATACTAATCTGCTTCTGAAACTTGTAATGTATGTATATCAGTTTCTTAT AATTTATCCAAGTAATATCTTCCATTCTCTATGCAATTGCCTGCATAAGC TCGACAAAAGAGTACATCAACCCCTCCTCCTCTGGACTACTCTAGCTAAA CTTGAATTTCCCCTTAAGATTATGAAATTGATATATCCTTAACAAACGAC TCCTTCTGTTGGAAAATGTAGTACTTGTCTTTCTTCTTTTGGGTATATAT AGTTTATATACACCATACTATGTACAACATCCAAGTAGAGTGAAATGGAT ACATGTACAAGACTTATTTGATTGATTGATGACTTGAGTTGCCTTAGGAG TAACAAATTCTTAGGTCAATAAATCGTTGATTTGAAATTAATCTCTCTGT CTTAGACAGATAGGAATTATGACTTCCAATGGTCCAGAAAGCAAAGTTCG CACTGAGGGTATACTTGGAATTGAGACTTGCACAGGTCCAGAAACCAAAG TTCCCATCGAGCTCTAAAATCACATCTTTGGAATGAAATTCAATTAGAGA TAAGTTGCTTCATAGCATAGGTAAAATGGAAGATGTGAAGTAACCTGCAA TAATCAGTGAAATGACATTAATACACTAAATACTTCATATGTAATTATCC TTTCCAGGTTAACAATACTCTATAAAGTAAGAATTATCAGAAATGGGCTC ATCAAACTTTTGTACTATGTATTTCATATAAGGAAGTATAACTATACATA AGTGTATACACAACTTTATTCCTATTTTGTAAAGGTGGAGAGACTGTTTT CGATGGATCTAAAGCAATATGTCTATAAAATGCATTGATATAATAATTAT CTGAGAAAATCCAGAATTGGCGTTGGATTATTTCAGCCAAATAGAAGTTT GTACCATACTTGTTGATTCCTTCTAAGTTAAGGTGAAGTATCATTCATAA ACAGTTTTCCCCAAAGTACTACTCACCAAGTTTCCCTTTGTAGAATTAAC AGTTCAAATATATGGCGCAGAAATTACTCTATGCCCAAAACCAAACGAGA AAGAAACAAAATACAGGGGTTGCAGACTTTATTTTCGTGTTAGGGTGTGT TTTTTCATGTAATTAATCAAAAAATATTATGACAAAAACATTTATACATA TTTTTACTCAACACTCTGGGTATCAGGGTGGGTTGTGTTCGACAATCAAT ATGGAAAGGAAGTATTTTCCTTATTTTTTTAGTTAATATTTTCAGTTATA CCAAACATACCTTGTGATATTATTTTTAAAAATGAAAAACTCGTCAGAAA GAAAAAGCAAAAGCAACAAAAAAATTGCAAGTATTTTTTAAAAAAGAAAA AAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGATAAGGACGAGT GAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGCCAAGAACCACAA AATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGCTAACTCTTTTTGT CCGTTAGATAGGAAGCCTTATCACTATATATACAAGGCGTCCTAATAACC TCTTAGTAACCAATTATTTCAGCACCATGGTAGATCTGAGGGTAAATTTC TAGTTTTTCTCCTTCATTTTCTTGGTTAGGACCCTTTTCTCTTTTTATTT TTTTGAGCTTTGATCTTTCTTTAAACTGATCTATTTTTTAATTGATTGGT TATGGTGTAAATATTACATAGCTTTAACTGATAATCTGATTACTTTATTT CGTGTGTCTATGATGATGATGATAGTTACAGAACCGACGAACTAGTATGT CTCTGCTCTCAGATCTCGTTAACCTCAACCTCACCGATGCCACCGGGAAA ATCATCGCCGAATACATATGGATCGGTGGATCTGGAATGGATATCAGAAG CAAAGCCAGGACACTACCAGGACCAGTGACTGATCCATCAAAGCTTCCCA AGTGGAACTACGACGGATCCAGCACCGGTCAGGCTGCTGGAGAAGACAGT GAAGTCATTCTATACCCTCAGGCAATATTCAAGGATCCCTTCAGGAAAGG CAACAACATCCTGGTGATGTGTGATGCTTACACACCAGCTGGTGATCCTA TTCCAACCAACAAGAGGCACAACGCTGCTAAGATCTTCAGCCACCCCGAC GTTGCCAAGGAGGAGCCTTGGTATGGGATTGAGCAAGAATACACTTTGAT GCAAAAGGATGTGAACTGGCCAATTGGTTGGCCTGTTGGTGGCTACCCTG GCCCTCAGGGACCTTACTACTGTGGTGTGGGAGCTGACAAAGCCATTGGT CGTGACATTGTGGATGCTCACTACAAGGCCTGTCTTTACGCCGGTATTGG TATTTCTGGTATCAATGGAGAAGTCATGCCAGGCCAGTGGGAGTTCCAAG TCGGCCCTGTTGAGGGTATTAGTTCTGGTGATCAAGTCTGGGTTGCTCGA TACCTTCTCGAGAGGATCACTGAGATCTCTGGTGTAATTGTCAGCTTCGA CCCGAAACCAGTCCCGGGTGACTGGAATGGAGCTGGAGCTCACTGCAACT ACAGCACTAAGACAATGAGAAACGATGGAGGATTAGAAGTGATCAAGAAA GCGATAGGGAAGCTTCAGCTGAAACACAAAGAACACATTGCTGCTTACGG TGAAGGAAACGAGCGTCGTCTCACTGGAAAGCACGAAACCGCAGACATCA ACACATTCTCTTGGGGAGTCGCGAACCGTGGAGCGTCAGTGAGAGTGGGA CGTGACACAGAGAAGGAAGGTAAAGGGTACTTCGAAGACAGAAGGCCAGC TTCTAACATGGATCCTTACGTTGTCACCTCCATGATCGCTGAGACGACCA TACTCGGTTGACACGTGTGAATTGGTGACCAGCTCGAATTTCCCCGATCG TTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTC TTGCGATGATTATCATATAATTTCTGTTGAATTACGTTAAGCATGTAATA ATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAG AGTCCCGCAATTATACATTTAATACGCGATAGAAAACAAAATATAGCGCG CAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGG GAATTAAACTATCAGTGTTTGACAGGATATATTGGCGGGTAAACCTAAGA GAAAAGAGCGTTTATTAGAATAACGGATATTTAAAAGGGCGTGAAAAGG TTTATCCGTTCGTCCATTTGTATGTGCATGCCAACCACAGGGTTCCCCT CGGGATCAAAGTACTTTGATCCAACCCCTCCGCTGCTATAGTGCAGTCG GCTTCTGACGTTCAGTGCAGCCGTCTTCTGAAAACGACATGTCGCACAA GTCCTAAGTTACGCGACAGGCTGCCGCCCTGCCCTTTTCCTGGCGTTTT CTTGTCGCGTGTTTTAGTCGCATAAAGTAGAATACTTGCGACTAGAACC GGAGACATTACGCCATGAACAAGAGCGCCGCCGCTGGCCTGCTGGGCTA TGCCCGCGTCAGCACCGACGACCAGGACTTGACCAACCAACGGGCCGAA CTGCACGCGGCCGGCTGCACCAAGCTGTTTTCCGAGAAGATCACCGGCA CCAGGCGCGACCGCCCGGAGCTGGCCAGGATGCTTGACCACCTACGCCC TGGCGACGTTGTGACAGTGACCAGGCTAGACCGCCTGGCCCGCAGCACC CGCGACCTACTGGACATTGCCGAGCGCATCCAGGAGGCCGGCGCGGGCC TGCGTAGCCTGGCAGAGCCGTGGGCCGACACCACCACGCCGGCCGGCCG CATGGTGTTGACCGTGTTCGCCGGCATTGCCGAGTTCGAGCGTTCCCTA ATCATCGACCGCACCCGGAGCGGGCGCGAGGCCGCCAAGGCCCGAGGCG TGAAGTTTGGCCCCCGCCCTACCCTCACCCCGGCACAGATCGCGCACGC CCGCGAGCTGATCGACCAGGAAGGCCGCACCGTGAAAGAGGCGGCTGCA CTGCTTGGCGTGCATCGCTCGACCCTGTACCGCGCACTTGAGCGCAGCG AGGAAGTGACGCCCACCGAGGCCAGGCGGCGCGGTGCCTTCCGTGAGGA CGCATTGACCGAGGCCGACGCCCTGGCGGCCGCCGAGAATGAACGCCAA GAGGAACAAGCATGAAACCGCACCAGGACGGCCAGGACGAACCGTTTTT CATTACCGAAGAGATCGAGGCGGAGATGATCGCGGCCGGGTACGTGTTC GAGCCGCCCGCGCACGTCTCAACCGTGCGGCTGCATGAAATCCTGGCCG GTTTGTCTGATGCCAAGCTGGCGGCCTGGCCGGCCAGCTTGGCCGCTGA AGAAACCGAGCGCCGCCGTCTAAAAAGGTGATGTGTATTTGAGTAAAAC AGCTTGCGTCATGCGGTCGCTGCGTATATGATGCGATGAGTAAATAAAC AAATACGCAAGGGGAACGCATGAAGGTTATCGCTGTACTTAACCAGAAA GGCGGGTCAGGCAAGACGACCATCGCAACCCATCTAGCCCGCGCCCTGC AACTCGCCGGGGCCGATGTTCTGTTAGTCGATTCCGATCCCCAGGGCAG TGCCCGCGATTGGGCGGCCGTGCGGGAAGATCAACCGCTAACCGTTGTC GGCATCGACCGCCCGACGATTGACCGCGACGTGAAGGCCATCGGCCGGC GCGACTTCGTAGTGATCGACGGAGCGCCCCAGGCGGCGGACTTGGCTGT GTCCGCGATCAAGGCAGCCGACTTCGTGCTGATTCCGGTGCAGCCAAGC CCTTACGACATATGGGCCACCGCCGACCTGGTGGAGCTGGTTAAGCAGC GCATTGAGGTCACGGATGGAAGGCTACAAGCGGCCTTTGTCGTGTCGCG GGCGATCAAAGGCACGCGCATCGGCGGTGAGGTTGCCGAGGCGCTGGCC GGGTACGAGCTGCCCATTCTTGAGTCCCGTATCACGCAGCGCGTGAGCT ACCCAGGCACTGCCGCCGCCGGCACAACCGTTCTTGAATCAGAACCCGA GGGCGACGCTGCCCGCGAGGTCCAGGCGCTGGCCGCTGAAATTAAATCA AAACTCATTTGAGTTAATGAGGTAAAGAGAAAATGAGCAAAAGCACAAA CACGCTAAGTGCCGGCCGTCCGAGCGCACGCAGCAGCAAGGCTGCAACG TTGGCCAGCCTGGCAGACACGCCAGCCATGAAGCGGGTCAACTTTCAGT TGCCGGCGGAGGATCACACCAAGCTGAAGATGTACGCGGTACGCCAAGG CAAGACCATTACCGAGCTGCTATCTGAATACATCGCGCAGCTACCAGAG TAAATGAGCAAATGAATAAATGAGTAGATGAATTTTAGCGGCTAAAGGA GGCGGCATGGAAAATCAAGAACAACCAGGCACCGACGCCGTGGAATGCC CCATGTGTGGAGGAACGGGCGGTTGGCCAGGCGTAAGCGGCTGGGTTGT CTGCCGGCCCTGCAATGGCACTGGAACCCCCAAGCCCGAGGAATCGGCG TGACGGTCGCAAACCATCCGGCCCGGTACAAATCGGCGCGGCGCTGGGT GATGACCTGGTGGAGAAGTTGAAGGCCGCGCAGGCCGCCCAGCGGCAAC GCATCGAGGCAGAAGCACGCCCCGGTGAATCGTGGCAAGCGGCCGCTGA TCGAATCCGCAAAGAATCCCGGCAACCGCCGGCAGCCGGTGCGCCGTCG ATTAGGAAGCCGCCCAAGGGCGACGAGCAACCAGATTTTTTCGTTCCGA TGCTCTATGACGTGGGCACCCGCGATAGTCGCAGCATCATGGACGTGGC CGTTTTCCGTCTGTCGAAGCGTGACCGACGAGCTGGCGAGGTGATCCGC TACGAGCTTCCAGACGGGCACGTAGAGGTTTCCGCAGGGCCGGCCGGCA TGGCCAGTGTGTGGGATTACGACCTGGTACTGATGGCGGTTTCCCATCT AACCGAATCCATGAACCGATACCGGGAAGGGAAGGGAGACAAGCCCGGC CGCGTGTTCCGTCCACACGTTGCGGACGTACTCAAGTTCTGCCGGCGAG
CCGATGGCGGAAAGCAGAAAGACGACCTGGTAGAAACCTGCATTCGGTT AAACACCACGCACGTTGCCATGCAGCGTACGAAGAAGGCCAAGAACGGC CGCCTGGTGACGGTATCCGAGGGTGAAGCCTTGATTAGCCGCTACAAGA TCGTAAAGAGCGAAACCGGGCGGCCGGAGTACATCGAGATCGAGCTAGC TGATTGGATGTACCGCGAGATCACAGAAGGCAAGAACCCGGACGTGCTG ACGGTTCACCCCGATTACTTTTTGATCGATCCCGGCATCGGCCGTTTTC TCTACCGCCTGGCACGCCGCGCCGCAGGCAAGGCAGAAGCCAGATGGTT GTTCAAGACGATCTACGAACGCAGTGGCAGCGCCGGAGAGTTCAAGAAG TTCTGTTTCACCGTGCGCAAGCTGATCGGGTCAAATGACCTGCCGGAGT ACGATTTGAAGGAGGAGGCGGGGCAGGCTGGCCCGATCCTAGTCATGCG CTACCGCAACCTGATCGAGGGCGAAGCATCCGCCGGTTCCTAATGTACG GAGCAGATGCTAGGGCAAATTGCCCTAGCAGGGGAAAAAGGTCGAAAAG GTCTCTTTCCTGTGGATAGCACGTACATTGGGAACCCAAAGCCGTACAT TGGGAACCGGAACCCGTACATTGGGAACCCAAAGCCGTACATTGGGAAC CGGTCACACATGTAAGTGACTGATATAAAAGAGAAAAAAGGCGATTTTT CCGCCTAAAACTCTTTAAAACTTATTAAAACTCTTAAAACCCGCCTGGC CTGTGCATAACTGTCTGGCCAGCGCACAGCCGAAGAGCTGCAAAAAGCG CCTACCCTTCGGTCGCTGCGCTCCCTACGCCCCGCCGCTTCGCGTCGGC CTATCGCGGCCGCTGGCCGCTCAAAAATGGCTGGCCTACGGCCAGGCAA TCTACCAGGGCGCGGACAAGCCGCGCCGTCGCCACTCGACCGCCGGCGC CCACATCAAGGCACCCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAAC CTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGG ATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGG GTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTAT ACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCA TATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATC AGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTC GGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATC CACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAG CAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATA GGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAG GTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACC TGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACG CTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACT ATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGC AGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACA GAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTAT TTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGG TAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTT GTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATC CTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACG TTAAGGGATTTTGGTCATGCATTCTAGGTACTAAAACAATTCATCCAGT AAAATATAATATTTTATTTTCTCCCAATCAGGCTTGATCCCCAGTAAGT CAAAAAATAGCTCGACATACTGTTCTTCCCCGATATCCTCCCTGATCGA CCGGACGCAGAAGGCAATGTCATACCACTTGTCCGCCCTGCCGCTTCTC CCAAGATCAATAAAGCCACTTACTTTGCCATCTTTCACAAAGATGTTGC TGTCTCCCAGGTCGCCGTGGGAAAAGACAAGTTCCTCTTCGGGCTTTTC CGTCTTTAAAAAATCATACAGCTCGCGCGGATCTTTAAATGGAGTGTCT TCTTCCCAGTTTTCGCAATCCACATCGGCCAGATCGTTATTCAGTAAGTA ATCCAATTCGGCTAAGCGGCTGTCTAAGCTATTCGTATAGGGACAATCC GATATGTCGATGGAGTGAAAGAGCCTGATGCACTCCGCATACAGCTCGA TAATCTTTTCAGGGCTTTGTTCATCTTCATACTCTTCCGAGCAAAGGAC GCCATCGGCCTCACTCATGAGCAGATTGCTCCAGCCATCATGCCGTTCA AAGTGCAGGACCTTTGGAACAGGCAGCTTTCCTTCCAGCCATAGCATCA TGTCCTTTTCCCGTTCCACATCATAGGTGGTCCCTTTATACCGGCTGTC CGTCATTTTTAAATATAGGTTTTCATTTTCTCCCACCAGCTTATATACC TTAGCAGGAGACATTCCTTCCGTATCTTTTACGCAGCGGTATTTTTCGA TCAGTTTTTTCAATTCCGGTGATATTCTCATTTTAGCCATTTATTATTT CCTTCCTCTTTTCTACAGTATTTAAAGATACCCCAAGAAGCTAATTATA ACAAGACGAACTCCAATTCACTGTTCCTTGCATTCTAAAACCTTAAATA CCAGAAAACAGCTTTTTCAAAGTTGTTTTCAAAGTTGGCGTATAACATA GTATCGACGGAGCCGATTTTGAAACCGCGGTGATCACAGGCAGCAACGC TCTGTCATCGTTACAATCAACATGCTACCCTCCGCGAGATCATCCGTGT TTCAAACCCGGCAGCTTAGTTGCCGTTCTTCCGAATAGCATCGGTAACA TGAGCAAAGTCTGCCGCCTTACAACGGCTCTCCCGCTGACGCCGTCCCG GACTGATGGGCTGCCTGTATCGAGTGGTGATTTTGTGCCGAGCTGCCGG TCGGGGAGCTGTTGGCTGGCTGGTGGCAGGATATATTGTGGTGTAAACA AATTGACGCTTAGACAACTTAATAACACATTGCGGACGTTTTTAATGTA CTGAATTAACGCCGAATTAATTCGGGGGATCTGGATTTTAGTACTGGAT TTTGGTTTTAGGAATTAGAAATTTTATTGATAGAAGTATTTTACAAATA CAAATACATACTAAGGGTTTCTTATATGCTCAACACATGAGCGAAACCC TATAGGAACCCTAATTCCCTTATCTGGGAACTACTCACACATTATTATG GAGAAACTCGAGCTTGTCGATCGACAGATCCGGTCGGCATCTACTCTAT TTCTTTGCCCTCGGACGAGTGCTGGGGCGTCGGTTTCCACTATCGGCGA GTACTTCTACACAGCCATCGGTCCAGACGGCCGCGCTTCTGCGGGCGAT TTGTGTACGCCCGACAGTCCCGGCTCCGGATCGGACGATTGCGTCGCAT CGACCCTGCGCCCAAGCTGCATCATCGAAATTGCCGTCAACCAAGCTCT GATAGAGTTGGTCAAGACCAATGCGGAGCATATACGCCCGGAGTCGTGG CGATCCTGCAAGCTCCGGATGCCTCCGCTCGAAGTAGCGCGTCTGCTGC TCCATACAAGCCAACCACGGCCTCCAGAAGAAGATGTTGGCGACCTCGT ATTGGGAATCCCCGAACATCGCCTCGCTCCAGTCAATGACCGCTGTTAT GCGGCCATTGTCCGTCAGGACATTGTTGGAGCCGAAATCCGCGTGCACG AGGTGCCGGACTTCGGGGCAGTCCTCGGCCCAAAGCATCAGCTCATCGA GAGCCTGCGCGACGGACGCACTGACGGTGTCGTCCATCACAGTTTGCCA GTGATACACATGGGGATCAGCAATCGCGCATATGAAATCACGCCATGTA GTGTATTGACCGATTCCTTGCGGTCCGAATGGGCCGAACCCGCTCGTCT GGCTAAGATCGGCCGCAGCGATCGCATCCATAGCCTCCGCGACCGGTTG TAGAACAGCGGGCAGTTCGGTTTCAGGCAGGTCTTGCAACGTGACACCC TGTGCACGGCGGGAGATGCAATAGGTCAGGCTCTCGCTAAACTCCCCAA TGTCAAGCACTTCCGGAATCGGGAGCGCGGCCGATGCAAAGTGCCGATA AACATAACGATCTTTGTAGAAACCATCGGCGCAGCTATTTACCCGCAGG ACATATCCACGCCCTCCTACATCGAAGCTGAAAGCACGAGATTCTTCGC CCTCCGAGAGCTGCATCAGGTCGGAGACGCTGTCGAACTTTTCGATCAG AAACTTCTCGACAGACGTCGCGGTGAGTTCAGGCTTTTTCATATCTCAT TGCCCCCCGGGATCTGCGAAAGCTCGAGAGAGATAGATTTGTAGAGAGA GACTGGTGATTTCAGCGTGTCCTCTCCAAATGAAATGAACTTCCTTATA TAGAGGAAGGTCTTGCGAAGGATAGTGGGATTGTGCGTCATCCCTTACG TCAGTGGAGATATCACATCAATCCACTTGCTTTGAAGACGTGGTTGGAA CGTCTTCTTTTTCCACGATGCTCCTCGTGGGTGGGGGTCCATCTTTGGG ACCACTGTCGGCAGAGGCATCTTGAACGATAGCCTTTCCTTTATCGCAA TGATGGCATTTGTAGGTGCCACCTTCCTTTTCTACTGTCCTTTTGATGA AGTGACAGATAGCTGGGCAATGGAATCCGAGGAGGTTTCCCGATATTAC CCTTTGTTGAAAAGTCTCAATAGCCCTTTGGTCTTCTGAGACTGTATCT TTGATATTCTTGGAGTAGACGAGAGTGTCGTGCTCCACCATGTTATCAC ATCAATCCACTTGCTTTGAAGACGTGGTTGGAACGTCTTCTTTTTCCAC GATGCTCCTCGTGGGTGGGGGTCCATCTTTGGGACCACTGTCGGCAGAG GCATCTTGAACGATAGCCTTTCCTTTATCGCAATGATGGCATTTGTAGG TGCCACCTTCCTTTTCTACTGTCCTTTTGATGAAGTGACAGATAGCTGG GCAATGGAATCCGAGGAGGTTTCCCGATATTACCCTTTGTTGAAAAGTC TCAATAGCCCTTTGGTCTTCTGAGACTGTATCTTTGATATTCTTGGAGT AGACGAGAGTGTCGTGCTCCACCATGTTGGCAAGCTGCTCTAGCCAATA CGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGC ACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAA TGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTT CCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAG GAAACAGCTATGACCATGATTACGAATTCGAGCTC
[0262] Transformation and Selection: Transformation of Arabidopsis was achieved using
[0263] Agrobacterium-mediated "floral dip" transfer as described in Example 4. Transformed plants were grown under selection as described in Example 4.
[0264] Biochemical Characterization:
[0265] Assays for 2-oxoglutaramate, Ω-amidase, GS and GPT were conducted as described in Example 3, supra.
[0266] Results:
[0267] GPT activity and GS activity of wild type and transgenic Arabidopsis plants were measured and are shown in Table 11, below. Ω-amidase activities and 2-oxoglutaramate concentrations in wild type and transgenic Arabidopsis plants were measured in both leaf and root tissues, and are shown in Table 12, below.
TABLE-US-00052 TABLE 11 GS Activity GPT Activity Arabidopsis FWt mg umoles/gfwt/min nmoles/gfwt/hr Genotype Whole plant Root Leaf L/R Root Leaf L/R Wild type* 77 1.5 3.4 2.3 167 132 0.8 TG GPT (9c) + 709 1.6 9.4 5.9 182 414 2.3 GS (4c) + ω-amidase** TG = Transgenic GPT (9c) = -45 truncated GPT variant [SEQ ID NO: 41] GS 4c = SEQ ID NO: 43 *Average values of 9 plants **Average values of 5 plants
TABLE-US-00053 TABLE 12 2-Oxoglutaramate Ω-amidase Concentration nmoles/gfwt/hr nmoles/gfwt Arabidopsis FWt mg L/R L/R Genotype Whole plant Root Leaf Ratio Root Leaf Ratio Wild type* 77 86 1090 12.7 410 163 0.4 TG GPT (9c) + 709 195 344 1.8 113 223 2.0 GS (4c) + ω-amidase ** TG = Transgenic GPT (9c) = -45 truncated GPT variant [SEQ ID NO: 41] *Average values of 9 plants ** Average values of 5 plants
[0268] Compared to wild type control Arabidopsis plants, the transgenic Arabidopsis plants carrying the Ω-amidase directed for root expression, and truncated GPT (for cyosolic expression) and GS1 directed for leaf expression, showed biochemical changes within the Ω-amidase pathway similar to those observed in the transgenic plants of Examples 4 and 5, supra. More specifically, transgenic GPT+Ω-amidase plants showed increased root Ω-amidase activity, reduced root 2-oxoglutaramate concentration, and increased leaf-to-root 2-oxoglutaramate ratios. Also, similar to the results seen in the transgenic plants of Examples 5 and 6, the GPT+GS+Ω-amidase transgenic plants showed significant reductions in leaf Ω-amidase activity levels, increased leaf 2-oxoglutaramate, and higher leaf GS and leaf GPT activities. The resulting impact on growth was greater than observed for the transgenic plants of either Example 4 or Example 5, with the transgenic plants weighing more than nine times the weight of the wild type plants, on average.
Example 7
Comparison of Transgenic Arabidopsis Genotypes Carrying Root-Preferred Ω-Amidase Transgene
[0269] The data generated from the studies of Examples 4, 5, and 6, which were conducted in parallel, are presented together for comparison in Tables 13 and 14 below.
TABLE-US-00054 TABLE 13 GS Activity GPT Activity FWt mg umoles/gfwt/min nmoles/gfwt/hr Genotype Whole plant Root Leaf L/R Root Leaf L/R Wild type* 77 1.5 3.4 2.3 167 132 0.8 WT + 479 1.4 5.8 4.1 192 486 2.5 ω-amidase** GPT (6c) + 513 1.8 8.25 4.6 232 389 1.7 ω-amidase GPT (9c) + 709 1.6 9.4 5.9 182 414 2.3 GS + ω-amidase *** *Average values of 9 plants **Average values of 6 plants *** Average values of 5 plants
TABLE-US-00055 TABLE 14 2-Oxoglutaramate Ω-amidase Concentration FWt mg nmoles/gfwt/hr nmoles/gfwt Genotype Whole plant Root Leaf L/R Root Leaf L/R Wild type* 77 86 1090 12.7 410 163 0.4 WT + 479 243 127 0.5 98 305 3.1 ω-amidase ** GPT (6c) + 513 308 584 1.9 268 275 1.0 ω-amidase *** GPT (9c) + 709 195 344 1.8 113 223 2.0 GS + ω-amidase *** *Average values of 9 plants ** Average values of 6 plants *** Average values of 5 plants
Example 8
Chemical Inhibition of Ω-Amidase Activity in Leaf and Root Tissues and Modulation of Leaf-to-Root Ratio of 2-Oxoglutaramate
[0270] This example demonstrates how the concentration of 2-oxoglutaramate changes in response to foliar and root treatment with 6-diazo-5-oxo-nor-leucine (DON), an inhibitor of the Ω-amidase enzyme which breaks-down 2-oxoglutaramate (Duran and Calderon, 1995, Role of the glutamine transaminase-Ω-amidase pathway and glutaminase in glutamine degradation in Rhizobium etli. Microbiology 141:589-595).
[0271] Materials and Methods:
[0272] Nutrient Solution:
[0273] Columbia nutrient solution was utilized (Knight and Weissman, 1982, Plant Physiol. 70: 1683).
[0274] Leaf Treatments:
[0275] Plants were grown in sand at 24° C. using a 16 hour/day light and 8 hour/day dark photoperiod. Seeds were germinated in the sand and allowed to grow for 9-14 days after seedling emergence. Seedlings (1 per 3 inch pot) were provided nutrients daily. The top of each pot was covered with Saran plastic film, with a slit cut to allow the seedlings room to emerge, in order to prevent treatment solution from reaching the soil. Leaves were treated by spraying DON treatment/nutrient solution twice daily. The DON treatment/nutrient solution contained 1 microgram/ml DON, 50 micoliters/liter SILWET L77 (a surfactant) and 0.02 vol/vol % glycerol (a humectant) at pH 6.3, dissolved in nutrient solution. Control plants were sprayed daily with the same solution without DON. An airbrush was used to apply the solutions until drip. Plants were sacrificed and analyzed for fresh weight, Ω-amidase activity, and 2-oxoglutaramate concentrations in leaf and root tissue 14 days after initiation of treatment.
[0276] Root Treatments:
[0277] Plants were grown hydroponically at 24° C. using a 16 hour/day light and 8 hour/day dark photoperiod. Seeds were first germinated and seedlings were grown for 9 days, at which time the individual seedlings were suspended with their roots in the nutrient solution (600 ml of nutrient, pH 6.3) in an 800 ml beaker covered with aluminum foil, with a slit for the seedlings, to prevent algal growth. The beakers were aerated with air provided by a small pump and delivered into the solution through a glass Pasteur pipette. For the treatments, DON was added to the nutrient solution to a final concentration of 1 microgram per ml. Thus DON was supplied continuously to the treated seedlings. The controls were grown in the same nutrient solution without DON. The solutions were refreshed every third day. Plants were sacrificed and analyzed for fresh weight, Ω-amidase activity, and 2-oxoglutaramate concentrations in leaf and root tissue 14 days after initiation of treatment.
[0278] Results:
[0279] Treatment of Roots with Ω-Amidase Inhibitor:
[0280] Sweet corn and pole bean plant roots were treated with DON as described above. The results of the root treatments are shown in Tables 15 and 16 below. None of the DON treated plants had detectable levels of Ω-amidase activity, whereas the control wild type plants maintained normal levels of Ω-amidase activity. Indeed, all corn and bean plants subjected to continuous DON treatment of roots showed severely stunted growth, in contrast to vigorous growth of untreated control plants. Control plants increased their fresh weighs throughout the experimental period up to nearly eight-fold.
[0281] Dramatic reductions in the 2-oxoglutaramate leaf-to-root ratio were observed in both corn and bean plants treated with the Ω-amidase inhibitor. Treated plants accumulated very large amounts of 2-oxoglutaramate in their roots (over 30-fold increase in corn; 15-fold increase in beans) while maintaining normal levels in their leaves.
TABLE-US-00056 TABLE 15 INHIBITION OF Ω-AMIDASE IN CORN ROOTS % INCREASE 2-OGM 2-OGM LEAF/ IN FWT LEAF ROOT ROOT CORN (initial wt) nmoles/gfwt nmoles/gfwt RATIO CONTROL 370% 275 65 4.25 (1.26 g) TREATED 174% 300 2286 0.13 (0.96 g) 2-OGM = 2-oxoglutaramate
TABLE-US-00057 TABLE 16 INHIBITION OF Ω-AMIDASE IN BEAN ROOTS % INCREASE 2-OGM 2-OGM LEAF/ IN FWT LEAF ROOT ROOT BEAN (initial wt) nmoles/gfwt nmoles/gfwt RATIO CONTROL 839% 311.2 101.4 3.07 (1.134 g) TREATED 198% 279 1586 0.18 (1.104 g) 2-OGM = 2-oxoglutaramate
[0282] Treatment of Leaves with Ω-Amidase Inhibitor:
[0283] Corn plant leaves were treated with DON as described above. The results are shown in Tables 17 and 18, below. Ω-amidase activity in leaf was effectively suppressed by DON, with the treated plants showing only about 50% the Ω-amidase activity observed in untreated plants (Table 18). A dramatic increase in leaf 2-oxoglutaramate and the leaf-to-root 2-oxoglutaramate ratio was observed in the treated plants (Tables 17 and 18). Specifically, treated plants accumulated very large amounts of 2-oxoglutaramate in their leaves (more than 7-fold increase over untreated plants). Moreover, inhibition of Ω-amidase activity in leaves also resulted in a near doubling of corn plant fresh weights (Table 18).
TABLE-US-00058 TABLE 17 INHIBITION OF Ω-AMIDASE IN CORN LEAVES RESULTS IN INCREASED LEAF-TO-ROOT RATIO 2-OXOGLUTARAMATE 2-OGM LEAF 2-OGM ROOT LEAF/ROOT CORN nmoles/gfwt nmoles/gfwt RATIO CONTROL 101.3 344.1 0.29 TREATED 753.9 126.8 5.0 2-OGM = 2-oxoglutaramate gfwt = grams fresh weight
TABLE-US-00059 TABLE 18 INHIBITION OF Ω-AMIDASE IN CORN LEAVES RESULTS IN INCREASED GROWTH 2-Oxoglutaramate Whole Plant Fresh Amidase activity Leaf, Concentration Wt, g μmole/gfw/hr nmoles/gfwt CORN Control Treated Control Treated Control Treated 9.3 20.9 12.4 19.2 11.3 24.3 Average 11.0 21.4 0.261 0.135 101.3 838.8 gfwt = grams fresh weight
Example 9
Comparison of GPT Isoforms in Combination with Root-Preferred Ω-Amidase
[0284] This Example compares the growth-enhancing performance of three different GPT transgene isoforms in combination with root-preferred expression of an Ω-amidase transgene on Arabidopsis plant growth. The Ω-amidase expression construct used in all three combinations is as described in Example 3 [SEQ ID NO: 39]. The three GPT transgene isoforms were: (1) GPT 5c, full length Arabidopsis GPT codon optimized [SEQ ID NO: 42]; (2) GPT 6c, truncated -45 GPT (deleted chloroplast targeting sequence), codon optimized [SEQ ID NO: 40]; and, (3) GPT 9c, truncated -45 GPT (deleted chloroplast targeting sequence), codon optimized, mutation F to V at amino acid residue 45 [SEQ ID NO: 41].
[0285] The nucleotide sequence of the Cambia 1305.1 with rbcS3C promoter+catI intron with Arabidopsis GPT gene is set forth below as SEQ ID NO: 42. Underlined ATG is start site, parentheses are the catI intron and the underlined actagt is the speI cloning site used to splice in the Arabidopsis gene.
TABLE-US-00060 AAAAAAGAAAAAAAAAACATATCTTGTTTGTCAGTATGGGAAGTTTGAGA TAAGGACGAGTGAGGGGTTAAAATTCAGTGGCCATTGATTTTGTAATGC CAAGAACCACAAAATCCAATGGTTACCATTCCTGTAAGATGAGGTTTGC TAACTCTTTTTGTCCGTTAGATAGGAAGCCTTATCACTATATATACAAG GCGTCCTAATAACCTCTTAGTAACCAATTATTTCAGCA TA GATCTGAGG(GTAAATTTCTAGTTTTTCTCCTTCATTTTCTTGGTTAGG ACCCTTTTCTCTTTTTATTTTTTTGAGCTTTGATCTTTCTTTAAACTGAT CTATTTTTTAATTGATTGGTTATGGTGTAAATATTACATAGCTTTAACTG ATAATCTGATTACTTTATTTCGTGTGTCTATGATGATGATGATAGTTACA G)AACCGACGA ATGTACCTGGACATAAATGGTGTGATGATC AAACAGTTTAGCTTCAAAGCCTCTCTTCTCCCATTCTCTTCTAATTTCCG ACAAAGCTCCGCCAAAATCCATCGTCCTATCGGAGCCACCATGACCACAG TTTCGACTCAGAACGAGTCTACTCAAAAACCCGTCCAGGTGGCGAAGAGA TTAGAGAAGTTCAAGACTACTATTTTCACTCAAATGAGCATATTGGCAGT TAAACATGGAGCGATCAATTTAGGCCAAGGCTTTCCCAATTTCGACGGTC CTGATTTTGTTAAAGAAGCTGCGATCCAAGCTATTAAAGATGGTAAAAAC CAGTATGCTCGTGGATACGGCATTCCTCAGCTCAACTCTGCTATAGCTGC GCGGTTTCGTGAAGATACGGGTCTTGTTGTTGATCCTGAGAAAGAAGTTA CTGTTACATCTGGTTGCACAGAAGCCATAGCTGCAGCTATGTTGGGTTTA ATAAACCCTGGTGATGAAGTCATTCTCTTTGCACCGTTTTATGATTCCTA TGAAGCAACACTCTCTATGGCTGGTGCTAAAGTAAAAGGAATCACTTTAC GTCCACCGGACTTCTCCATCCCTTTGGAAGAGCTTAAAGCTGCGGTAACT AACAAGACTCGAGCCATCCTTATGAACACTCCGCACAACCCGACCGGGAA GATGTTCACTAGGGAGGAGCTTGAAACCATTGCATCTCTCTGCATTGAAA ACGATGTGCTTGTGTTCTCGGATGAAGTATACGATAAGCTTGCGTTTGAA ATGGATCACATTTCTATAGCTTCTCTTCCCGGTATGTATGAAAGAACTG TGACCATGAATTCCCTGGGAAAGACTTTCTCTTTAACCGGATGGAAGAT CGGCTGGGCGATTGCGCCGCCTCATCTGACTTGGGGAGTTCGACAAGCA CACTCTTACCTCACATTCGCCACATCAACACCAGCACAATGGGCAGCCG TTGCAGCTCTCAAGGCACCAGAGTCTTACTTCAAAGAGCTGAAAAGAGA TTACAATGTGAAAAAGGAGACTCTGGTTAAGGGTTTGAAGGAAGTCGGA TTTACAGTGTTCCCATCGAGCGGGACTTACTTTGTGGTTGCTGATCACA CTCCATTTGGAATGGAGAACGATGTTGCTTTCTGTGAGTATCTTATTGA AGAAGTTGGGGTCGTTGCGATCCCAACGAGCGTCTTTTATCTGAATCCA GAAGAAGGGAAGAATTTGGTTAGGTTTGCGTTCTGTAAAGACGAAGAGA CGTTGCGTGGTGCAATTGAGAGGATGAAGCAGAAGCTTAAGAGAAAAGT CTGA
[0286] The results are shown in Table 19 below. Both of the GPT isoforms in which the chloroplast transit peptide was deleted outperform the wild type and full length GPT isoform plants.
TABLE-US-00061 TABLE 19 TRUNCATED GPT ISOFORMS OUTPERFORM NATIVE GPT Fresh Mean % Genotype Wt, mg weight, mg SD+/- Increase Wild type (average of 77 27 0 9 plants 5c GFT + ω-amidase 357 456 459 198 371 149 482 6cGPT + ω-amidase 525 438 560 501 552 513 56 666 9c GPT + ω-amidase 390 359 405 387 574 431 99 560
Sequence CWU
1
1
461440PRTArtificial Sequencesynthetic Arabidopsis glutamine phenylpyruvate
transaminase (GPT) mutant GPT/FV with V substitution in conserved
region at position 88 1Met Tyr Leu Asp Ile Asn Gly Val Met Ile Lys Gln
Phe Ser Phe Lys 1 5 10 15
Ala Ser Leu Leu Pro Phe Ser Ser Asn Phe Arg Gln Ser Ser Ala Lys
20 25 30 Ile His Arg Pro
Ile Gly Ala Thr Met Thr Thr Val Ser Thr Gln Asn 35
40 45 Glu Ser Thr Gln Lys Pro Val Gln Val
Ala Lys Arg Leu Glu Lys Phe 50 55 60
Lys Thr Thr Ile Phe Thr Gln Met Ser Ile Leu Ala Val Lys
His Gly65 70 75 80
Ala Ile Asn Leu Gly Gln Gly Val Pro Asn Phe Asp Gly Pro Asp Phe
85 90 95 Val Lys Glu Ala Ala
Ile Gln Ala Ile Lys Asp Gly Lys Asn Gln Tyr 100
105 110 Ala Arg Gly Tyr Gly Ile Pro Gln Leu Asn
Ser Ala Ile Ala Ala Arg 115 120
125 Phe Arg Glu Asp Thr Gly Leu Val Val Asp Pro Glu Lys Glu
Val Thr 130 135 140
Val Thr Ser Gly Cys Thr Glu Ala Ile Ala Ala Ala Met Leu Gly Leu145
150 155 160 Ile Asn Pro Gly Asp
Glu Val Ile Leu Phe Ala Pro Phe Tyr Asp Ser 165
170 175 Tyr Glu Ala Thr Leu Ser Met Ala Gly Ala
Lys Val Lys Gly Ile Thr 180 185
190 Leu Arg Pro Pro Asp Phe Ser Ile Pro Leu Glu Glu Leu Lys Ala
Ala 195 200 205 Val
Thr Asn Lys Thr Arg Ala Ile Leu Met Asn Thr Pro His Asn Pro 210
215 220 Thr Gly Lys Met Phe Thr
Arg Glu Glu Leu Glu Thr Ile Ala Ser Leu225 230
235 240 Cys Ile Glu Asn Asp Val Leu Val Phe Ser Asp
Glu Val Tyr Asp Lys 245 250
255 Leu Ala Phe Glu Met Asp His Ile Ser Ile Ala Ser Leu Pro Gly Met
260 265 270 Tyr Glu Arg
Thr Val Thr Met Asn Ser Leu Gly Lys Thr Phe Ser Leu 275
280 285 Thr Gly Trp Lys Ile Gly Trp Ala
Ile Ala Pro Pro His Leu Thr Trp 290 295
300 Gly Val Arg Gln Ala His Ser Tyr Leu Thr Phe Ala Thr
Ser Thr Pro305 310 315
320 Ala Gln Trp Ala Ala Val Ala Ala Leu Lys Ala Pro Glu Ser Tyr Phe
325 330 335 Lys Glu Leu Lys
Arg Asp Tyr Asn Val Lys Lys Glu Thr Leu Val Lys 340
345 350 Gly Leu Lys Glu Val Gly Phe Thr Val
Phe Pro Ser Ser Gly Thr Tyr 355 360
365 Phe Val Val Ala Asp His Thr Pro Phe Gly Met Glu Asn Asp
Val Ala 370 375 380
Phe Cys Glu Tyr Leu Ile Glu Glu Val Gly Val Val Ala Ile Pro Thr385
390 395 400 Ser Val Phe Tyr Leu
Asn Pro Glu Glu Gly Lys Asn Leu Val Arg Phe 405
410 415 Ala Phe Cys Lys Asp Glu Glu Thr Leu Arg
Gly Ala Ile Glu Arg Met 420 425
430 Lys Gln Lys Leu Lys Arg Lys Val 435
440 21353DNAArabidopsis thalianaArabidopsis omega-amidase,
AT5g12040/F14F18_210 cDNA 2aaagtgaaat gaagtcagca atttcatcgt
cactcttctt caattcgaag aatcttttaa 60accctaatcc tctttctcgc ttcatttctc
tcaaatctaa cttcctccct aaattatctc 120cgagatcgat cactagtcac accttgaagc
tcccatcttc gtcaacctca gctttaagat 180ccatttcctc ttccatggct tcttctttca
accctgaaca agctagagtt ccctctgctc 240ttcctctccc agctcctccg ttgaccaaat
tcaacatcgg attgtgtcag ctatctgtta 300catctgacaa aaagagaaac atctctcatg
ctaaaaaagc cattgaagaa gctgcttcta 360aaggagctaa gcttgttctc ttacccgaaa
tttggaacag tccgtattcc aatgatagtt 420ttccagttta tgcggaggag attgatgcag
gtggtgatgc ttctccttca acggcaatgc 480tttctgaagt ttccaaacgt ctcaagatta
caatcattgg tggatctata ccagaaagag 540ttggagatcg tttgtataac acttgctgtg
tctttggttc cgatggagag ctaaaagcta 600agcatcggaa gatacattta tttgatatag
acattcccgg gaagattact tttatggaat 660ccaaaactct tactgctgga gagacaccaa
caatcgttga cacagatgta gggcgtattg 720gaataggcat ctgttatgat atcaggttcc
aggagttagc tatgatatat gctgcaagag 780gggctcattt gctgtgctac ccgggagcct
ttaacatgac aactggacca ttgcattggg 840aattactaca aagggccagg gctacggata
atcagttata tgtggcgaca tgctcacctg 900ccagagattc aggagctggc tacactgctt
gggggcactc aacactcgtt gggccttttg 960gagaagtact agcaacgact gagcatgagg
aggccattat catagcagag attgattact 1020ctatccttga acaacgaagg actagccttc
cattgaatag gcagcggcgg ggagatcttt 1080accagcttgt agacgtacag cgcttagact
ctaaatgaac gcagcagtaa ctgtatatct 1140gagagatatt gcgagttgag cacgatttgg
ttacttacaa cttcatgcat gatcagtcat 1200ttctccacaa ctttgctgag atatgtaaaa
gaataaaaat caaacttttg agttaaaatc 1260gaacaaaggc aagtaaattc tgcttagata
atgtgaactc cacccacttg ccatgtgttt 1320gttgtttata aacttcaatg cattctgata
acg 13533307PRTArabidopsis
thalianaArabidopsis mature omega-amidase, AT5g12040/F14F18_210 3Met
Ala Ser Ser Phe Asn Pro Glu Gln Ala Arg Val Pro Ser Ala Leu 1
5 10 15 Pro Leu Pro Ala Pro Pro
Leu Thr Lys Phe Asn Ile Gly Leu Cys Gln 20 25
30 Leu Ser Val Thr Ser Asp Lys Lys Arg Asn Ile
Ser His Ala Lys Lys 35 40 45
Ala Ile Glu Glu Ala Ala Ser Lys Gly Ala Lys Leu Val Leu Leu Pro
50 55 60 Glu Ile Trp
Asn Ser Pro Tyr Ser Asn Asp Ser Phe Pro Val Tyr Ala65 70
75 80 Glu Glu Ile Asp Ala Gly Gly Asp
Ala Ser Pro Ser Thr Ala Met Leu 85 90
95 Ser Glu Val Ser Lys Arg Leu Lys Ile Thr Ile Ile Gly
Gly Ser Ile 100 105 110
Pro Glu Arg Val Gly Asp Arg Leu Tyr Asn Thr Cys Cys Val Phe Gly
115 120 125 Ser Asp Gly Glu
Leu Lys Ala Lys His Arg Lys Ile His Leu Phe Asp 130
135 140 Ile Asp Ile Pro Gly Lys Ile Thr
Phe Met Glu Ser Lys Thr Leu Thr145 150
155 160 Ala Gly Glu Thr Pro Thr Ile Val Asp Thr Asp Val
Gly Arg Ile Gly 165 170
175 Ile Gly Ile Cys Tyr Asp Ile Arg Phe Gln Glu Leu Ala Met Ile Tyr
180 185 190 Ala Ala Arg
Gly Ala His Leu Leu Cys Tyr Pro Gly Ala Phe Asn Met 195
200 205 Thr Thr Gly Pro Leu His Trp Glu
Leu Leu Gln Arg Ala Arg Ala Thr 210 215
220 Asp Asn Gln Leu Tyr Val Ala Thr Cys Ser Pro Ala Arg
Asp Ser Gly225 230 235
240 Ala Gly Tyr Thr Ala Trp Gly His Ser Thr Leu Val Gly Pro Phe Gly
245 250 255 Glu Val Leu Ala
Thr Thr Glu His Glu Glu Ala Ile Ile Ile Ala Glu 260
265 270 Ile Asp Tyr Ser Ile Leu Glu Gln Arg
Arg Thr Ser Leu Pro Leu Asn 275 280
285 Arg Gln Arg Arg Gly Asp Leu Tyr Gln Leu Val Asp Val Gln
Arg Leu 290 295 300
Asp Ser Lys305 4364PRTVitis viniferawine grape cultivar PN40024
omega-amidase NIT2-like, LOC100266241 4Met Lys Ser Ala Ala Leu Ser
Ala Leu Leu Ser Ser Thr Leu Ser Tyr 1 5 10
15 Ala Ser Pro Pro His Leu Asn Leu Leu Arg Pro Ala
Thr Ala Val Leu 20 25 30
Cys Arg Ser Leu Leu Pro Thr Ser Thr Pro Asn Pro Phe His Thr Gln
35 40 45 Leu Arg Thr Ala
Lys Ile Ser Ala Ser Met Ser Ser Ser Phe Lys Pro 50 55
60 Glu Gln Ala Arg Val Pro Pro Ala Ile
Pro Pro Pro Thr Pro Pro Leu65 70 75
80 Ser Lys Phe Lys Ile Gly Leu Cys Gln Leu Ser Val Thr Ala
Asp Lys 85 90 95
Glu Arg Asn Ile Ala His Ala Arg Lys Ala Ile Glu Glu Ala Val Glu
100 105 110 Lys Gly Ala Gln Leu
Val Leu Leu Pro Glu Ile Trp Asn Ser Pro Tyr 115
120 125 Ser Asn Asp Ser Phe Pro Val Tyr Ala
Glu Asp Ile Asp Ala Gly Ser 130 135
140 Asp Ala Ser Pro Ser Thr Ala Met Leu Ser Glu Val Ser
His Ala Leu145 150 155
160 Lys Ile Thr Ile Val Gly Gly Ser Ile Pro Glu Arg Cys Gly Asp Gln
165 170 175 Leu Tyr Asn Thr
Cys Cys Val Phe Gly Ser Asp Gly Lys Leu Lys Ala 180
185 190 Lys His Arg Lys Ile His Leu Phe Asp
Ile Asn Ile Pro Gly Lys Ile 195 200
205 Thr Phe Met Glu Ser Lys Thr Leu Thr Ala Gly Gly Ser Pro
Thr Ile 210 215 220
Val Asp Thr Glu Val Gly Arg Ile Gly Ile Gly Ile Cys Tyr Asp Ile225
230 235 240 Arg Phe Ser Glu Leu
Ala Met Leu Tyr Ala Ala Arg Gly Ala His Leu 245
250 255 Ile Cys Tyr Pro Gly Ala Phe Asn Met Thr
Thr Gly Pro Leu His Trp 260 265
270 Glu Leu Leu Gln Arg Ala Arg Ala Ala Asp Asn Gln Leu Tyr Val
Ala 275 280 285 Thr
Cys Ser Pro Ala Arg Asp Ala Gly Ala Gly Tyr Val Ala Trp Gly 290
295 300 His Ser Thr Leu Val Gly
Pro Phe Gly Glu Val Leu Ala Thr Thr Glu305 310
315 320 His Glu Glu Ala Ile Ile Ile Ser Glu Ile Asp
Tyr Ser Leu Ile Glu 325 330
335 Leu Arg Arg Thr Asn Leu Pro Leu Leu Asn Gln Arg Arg Gly Asp Leu
340 345 350 Tyr Gln Leu
Val Asp Val Gln Arg Leu Asp Ser Gln 355 360
5356PRTZea maysmaize strain B73 unknown protein 5Met Val Ala Ala
Ala Ala Ala Ala Ala Ala Ala Thr Ala Thr Ala Ala 1 5
10 15 Ala Leu Leu Ala Pro Gly Leu Lys Leu
Cys Ala Gly Arg Ala Arg Val 20 25
30 Ser Ser Pro Ser Gly Leu Pro Leu Arg Arg Val Thr Ala Met
Ala Ser 35 40 45
Ala Pro Asn Ser Ser Phe Arg Pro Glu Glu Ala Arg Ser Pro Pro Ala 50
55 60 Leu Glu Leu Pro Ile
Pro Pro Leu Ser Lys Phe Lys Val Ala Leu Cys65 70
75 80 Gln Leu Ser Val Thr Ala Asp Lys Ser Arg
Asn Ile Ala His Ala Arg 85 90
95 Ala Ala Ile Glu Lys Ala Ala Ser Asp Gly Ala Lys Leu Val Val
Leu 100 105 110 Pro
Glu Ile Trp Asn Gly Pro Tyr Ser Asn Asp Ser Phe Pro Glu Tyr 115
120 125 Ala Glu Asp Ile Glu Ala
Gly Gly Asp Ala Ala Pro Ser Phe Ser Met 130 135
140 Leu Ser Glu Val Ala Arg Ser Leu Gln Ile Thr
Leu Val Gly Gly Ser145 150 155
160 Ile Ala Glu Arg Ser Gly Asn Asn Leu Tyr Asn Thr Cys Cys Val Phe
165 170 175 Gly Ser Asp
Gly Gln Leu Lys Gly Lys His Arg Lys Ile His Leu Phe 180
185 190 Asp Ile Asp Ile Pro Gly Lys Ile
Thr Phe Lys Glu Ser Lys Thr Leu 195 200
205 Thr Ala Gly Gln Ser Pro Thr Val Val Asp Thr Asp Val
Gly Arg Ile 210 215 220
Gly Ile Gly Ile Cys Tyr Asp Ile Arg Phe Gln Glu Leu Ala Met Leu225
230 235 240 Tyr Ala Ala Arg Gly
Ala His Leu Leu Cys Tyr Pro Gly Ala Phe Asn 245
250 255 Met Thr Thr Gly Pro Leu His Trp Glu Leu
Leu Gln Arg Ala Arg Ala 260 265
270 Ala Asp Asn Gln Leu Phe Val Ala Thr Cys Ala Pro Ala Arg Asp
Thr 275 280 285 Ser
Ala Gly Tyr Val Ala Trp Gly His Ser Thr Leu Val Gly Pro Phe 290
295 300 Gly Glu Val Ile Ala Thr
Thr Glu His Glu Glu Ala Thr Ile Ile Ala305 310
315 320 Asp Ile Asp Tyr Ser Leu Ile Glu Gln Arg Arg
Gln Phe Leu Pro Leu 325 330
335 Gln His Gln Arg Arg Gly Asp Leu Tyr Gln Leu Val Asp Val Gln Arg
340 345 350 Leu Gly Ser
Gln 355 6370PRTPopulus trichocarpawestern balsam poplar (black
cottonwood) hypothetical protein, locus POPTRDRAFT_819468 6Met Lys
Ser Ala Ile Ser Ser Thr Thr Thr Leu Leu Ser Ser Lys Asn 1 5
10 15 Leu Ser Leu Lys Leu His Leu
Asn His Ser Pro Leu Ser Arg Leu Pro 20 25
30 Ser Ser Leu Phe Arg Ser Lys Ser Asn Thr His Phe
Pro Ser Leu Leu 35 40 45
Pro Arg Asn Asn Ser Thr His Asn Gln Lys Ser Gln Ile His Thr Pro
50 55 60 Ile Met Ala
Ser Ser Phe Met Pro Glu Gln Ala Arg Ala Pro Pro Ala65 70
75 80 Leu Pro Leu Pro Val Pro Pro Phe
Lys Ile Gly Leu Cys Gln Leu Ser 85 90
95 Val Thr Ala Asp Lys Glu Arg Asn Ile Ala His Ala Arg
Lys Ala Ile 100 105 110
Glu Glu Ala Ala Ala Lys Gly Ala Lys Leu Val Met Leu Pro Glu Ile
115 120 125 Trp Asn Ser Pro
Tyr Ser Asn Asp Cys Phe Pro Val Tyr Ala Glu Asp 130
135 140 Ile Asp Ala Gly Gly Glu Ala Ser
Pro Ser Thr Ala Met Leu Ser Glu145 150
155 160 Ala Ala Gly Leu Leu Lys Val Thr Ile Val Gly Gly
Ser Ile Pro Glu 165 170
175 Arg Ser Gly Asp Arg Leu Tyr Asn Thr Cys Cys Val Phe Asp Ser Asp
180 185 190 Gly Lys Leu
Lys Ala Lys His Arg Lys Ile His Leu Phe Asp Ile Asp 195
200 205 Ile Pro Gly Lys Ile Thr Phe Ile
Glu Ser Lys Thr Leu Thr Ala Gly 210 215
220 Glu Thr Pro Thr Ile Val Asp Thr Glu Val Gly Arg Ile
Gly Ile Gly225 230 235
240 Ile Cys Tyr Asp Ile Arg Phe Gln Glu Leu Ala Ile Ile Tyr Ala Ala
245 250 255 Arg Gly Ala His
Leu Ile Cys Tyr Pro Gly Ala Phe Asn Met Thr Thr 260
265 270 Gly Pro Leu His Trp Glu Leu Leu Gln
Arg Ala Arg Ala Ala Asp Asn 275 280
285 Gln Leu Tyr Val Ala Thr Cys Ser Pro Ala Arg Asp Val Ala
Ala Gly 290 295 300
Tyr Val Ala Trp Gly His Ser Thr Leu Val Gly Pro Phe Gly Glu Val305
310 315 320 Leu Ala Thr Thr Glu
His Glu Glu Asp Ile Ile Ile Ala Glu Ile Asp 325
330 335 Tyr Ser Leu Leu Glu Val Arg Arg Thr Asn
Leu Pro Leu Thr Lys Gln 340 345
350 Arg Arg Gly Asp Leu Tyr Gln Leu Val Asp Val Gln Arg Leu Lys
Ser 355 360 365 Asp
Ser 370 7358PRTPicea sitchensisSitka spruce cultivar FB3-425 unknown
protein, similar to Arabidopsis At5g12040 7Met Thr Pro Leu Leu Ser
Tyr Ser Leu Arg Val Val Ala Ser Ala Leu 1 5
10 15 Arg Pro Lys Ser Ser Ile Ala Ser Ala Val Gly
Arg Leu Ser Ala Thr 20 25 30
Pro Lys Arg Phe Pro Ala Asn Arg Leu Arg Ile Ser Tyr Arg Asn Tyr
35 40 45 Asn Ala Ala
Met Ala Lys Pro Glu Asp Ala Arg Ser Pro Pro Ala Leu 50
55 60 Pro Leu Pro Ser Ala Pro Asn Gly
Gly Lys Phe Lys Ile Ala Leu Cys65 70 75
80 Gln Leu Ser Val Thr Glu Asn Lys Glu Arg Asn Ile Ala
His Ala Arg 85 90 95
Asp Ala Ile Glu Ala Ala Ala Asp Asn Gly Ala Gln Leu Val Val Leu
100 105 110 Pro Glu Ile Trp Asn
Gly Pro Tyr Ser Asn Ala Ser Phe Pro Val Tyr 115
120 125 Ala Glu Asp Ile Asp Ala Gly Gly Ser
Ala Ser Pro Ser Thr Ser Met 130 135
140 Leu Ser Glu Val Ala Arg Ser Lys Gly Ile Thr Ile Val
Gly Gly Ser145 150 155
160 Ile Ser Glu Arg Ser Gly Asp His Leu Tyr Asn Thr Cys Cys Ile Phe
165 170 175 Gly Lys Asp Gly
Glu Leu Lys Ala Lys His Arg Lys Ile His Leu Phe 180
185 190 Asp Ile Asp Ile Pro Gly Lys Ile Ser
Phe Met Glu Ser Lys Thr Leu 195 200
205 Thr Ala Gly Asn Thr Pro Thr Ile Val Asp Thr Asp Val Gly
Arg Ile 210 215 220
Gly Ile Gly Ile Cys Tyr Asp Ile Arg Phe Gln Glu Leu Ala Met Leu225
230 235 240 Tyr Ala Ala Arg Gly
Ala His Leu Ile Cys Tyr Pro Gly Ala Phe Asn 245
250 255 Met Thr Thr Gly Pro Leu His Trp Glu Leu
Leu Gln Arg Ala Arg Ala 260 265
270 Ile Asp Asn Gln Leu Tyr Val Ala Thr Cys Ser Pro Ala Arg Asp
Ile 275 280 285 Asn
Ala Gly Tyr Val Ala Trp Gly His Ser Thr Leu Val Ala Pro Phe 290
295 300 Gly Glu Ile Val Ala Thr
Thr Glu His Glu Glu Ala Thr Val Ile Ala305 310
315 320 Asp Ile Asp Tyr Ser Arg Ile Glu Glu Arg Arg
Met Asn Met Pro Leu 325 330
335 Glu Lys Gln Arg His Gly Asp Leu Tyr Gln Leu Val Asp Val Ser Arg
340 345 350 Leu Asp Thr
Ala Lys His 355 8310PRTOryza sativarice Japonica group
cultivar Nipponbare hypothetical protein Os03g0175600, similar to
Nit protein 2 (CUA002) 8Met Ala Thr Ala Ala Ser Phe Arg Pro Glu Ala
Ala Arg Ser Pro Pro 1 5 10
15 Ala Val Gln Pro Pro Ala Pro Pro Leu Ser Lys Phe Lys Val Ala Leu
20 25 30 Cys Gln Leu
Ser Val Thr Ala Asp Lys Ala Arg Asn Ile Ala Arg Ala 35
40 45 Arg Glu Ala Ile Glu Ala Ala Ala
Ala Gly Gly Ala Lys Leu Val Leu 50 55
60 Leu Pro Glu Ile Trp Asn Gly Pro Tyr Ser Asn Asp Ser
Phe Pro Glu65 70 75 80
Tyr Ala Glu Asp Ile Glu Ala Gly Gly Asp Ala Ala Pro Ser Phe Ser
85 90 95 Met Met Ser Glu Val
Ala Arg Ser Leu Gln Ile Thr Leu Val Gly Gly 100
105 110 Ser Ile Ser Glu Arg Ser Gly Asn Lys Leu
Tyr Asn Thr Cys Cys Val 115 120
125 Phe Gly Ser Asp Gly Glu Leu Lys Gly Lys His Arg Lys Ile
His Leu 130 135 140
Phe Asp Ile Asp Ile Pro Gly Lys Ile Thr Phe Lys Glu Ser Lys Thr145
150 155 160 Leu Thr Ala Gly Gln
Asp Leu Thr Val Val Asp Thr Asp Val Gly Arg 165
170 175 Ile Gly Ile Gly Ile Cys Tyr Asp Ile Arg
Phe Gln Glu Leu Ala Met 180 185
190 Leu Tyr Ala Ala Arg Gly Ala His Leu Leu Cys Tyr Pro Gly Ala
Phe 195 200 205 Asn
Met Thr Thr Gly Pro Leu His Trp Glu Leu Leu Gln Arg Ala Arg 210
215 220 Ala Ala Asp Asn Gln Leu
Phe Val Ala Thr Cys Ala Pro Ala Arg Asp225 230
235 240 Thr Ser Ala Gly Tyr Ile Ala Trp Gly His Ser
Thr Leu Val Gly Pro 245 250
255 Phe Gly Glu Val Ile Ala Thr Ala Glu His Glu Glu Thr Thr Ile Met
260 265 270 Ala Glu Ile
Asp Tyr Ser Leu Ile Asp Gln Arg Arg Gln Phe Leu Pro 275
280 285 Leu Gln Tyr Gln Arg Arg Gly Asp
Leu Tyr Gln Leu Val Asp Val Gln 290 295
300 Arg Ser Gly Ser Asp Glu305 310
9284PRTSorghum bicolorsorghum cultivar BTx623 hypothetical protein
SORBIDRAFT_01g045480, similar to hydrolase, carbon-nitrogen family
protein, expressed 9Met Arg Ala Ala Ala Ala Ala Ala Ala Thr Ser Thr Ala
Ala Ala Leu 1 5 10 15
Leu Ala Pro Gly Leu Lys Leu Cys Ala Gly Arg Ala Arg Val Ser Ser
20 25 30 Cys Arg Leu Pro Leu
Arg Arg Val Ala Ala Met Ala Ser Ala Pro Asn 35 40
45 Ser Ser Phe Arg Pro Glu Glu Ala Arg Ser
Pro Pro Ala Leu Glu Leu 50 55 60
Pro Thr Pro Pro Leu Ser Lys Phe Lys Val Ala Leu Cys Gln Leu
Ser65 70 75 80 Val
Thr Ala Asp Lys Ser Arg Asn Ile Ala His Ala Arg Ala Ala Ile
85 90 95 Glu Lys Ala Ala Ser Asp
Gly Ala Lys Leu Val Leu Leu Pro Glu Ile 100
105 110 Trp Asn Gly Pro Tyr Ser Asn Asp Ser Phe
Pro Glu Tyr Ala Glu Asp 115 120
125 Ile Glu Ala Gly Gly Asp Ala Ala Pro Ser Phe Ser Met Met
Ser Glu 130 135 140
Val Ala Arg Ser Leu Gln Ile Thr Leu Val Asp Gly Gln Leu Lys Gly145
150 155 160 Lys His Arg Lys Ile
His Leu Phe Asp Ile Asp Ile Pro Gly Lys Ile 165
170 175 Thr Phe Lys Glu Ser Lys Thr Leu Thr Ala
Gly Gln Ser Pro Thr Val 180 185
190 Val Asp Thr Asp Val Gly Arg Ile Gly Ile Gly Ile Cys Tyr Asp
Ile 195 200 205 Arg
Phe Gln Glu Leu Ala Met Leu Tyr Ala Ala Arg Gly Ala His Leu 210
215 220 Leu Cys Tyr Pro Gly Ala
Phe Asn Met Thr Thr Gly Pro Leu His Trp225 230
235 240 Glu Leu Leu Gln Arg Ala Arg Gln Pro Ala Val
Cys Cys Asn Val Arg 245 250
255 Ser Ser Ser Arg Tyr Gln Cys Arg Leu Cys Cys Leu Gly Thr Leu His
260 265 270 Ala Cys Trp
Thr Phe Trp Arg Gly Asp Cys Asn Asn 275 280
10329PRTRicinus communiscastor bean cultivar Hale nitrilase and
fragile histidine triad fusion protein, putative, locus
RCOM_0706590A transcript 10Met Ser Ala Ser Phe Asn Pro Glu Gln Ala
Arg Ser Pro Pro Ala Leu 1 5 10
15 Pro Leu Pro Thr Pro Pro Leu Thr Lys Ala Gln Phe Leu Leu Thr
Ser 20 25 30 Tyr
Leu Thr Ile Leu Ile Tyr Met Ile Phe Lys Ile Gly Leu Cys Gln 35
40 45 Leu Leu Val Thr Pro Asp
Lys Ala Lys Asn Ile Ala His Ala Arg Lys 50 55
60 Ala Ile Glu Glu Ala Ala Ala Lys Gly Ala Lys
Leu Val Leu Leu Pro65 70 75
80 Glu Ile Trp Asn Ser Pro Tyr Ser Asn Asp Ser Phe Pro Val Tyr Ala
85 90 95 Glu Asp Ile
Asp Ala Gly His Val Ala Ser Pro Ser Thr Ala Met Leu 100
105 110 Ser Gln Leu Ala Arg Leu Leu Asn
Ile Thr Ile Val Gly Gly Ser Ile 115 120
125 Pro Glu Arg Ser Gly Asp Arg Leu Tyr Asn Thr Cys Cys
Val Phe Asp 130 135 140
Thr Gln Gly Asn Leu Ile Ala Lys His Arg Lys Ile His Leu Phe Asp145
150 155 160 Ile Asp Ile Pro Gly
Lys Ile Thr Phe Ile Glu Ser Lys Thr Leu Thr 165
170 175 Ala Gly Glu Thr Pro Asn Ile Val Asp Thr
Glu Val Gly Arg Ile Gly 180 185
190 Ile Gly Ile Cys Tyr Asp Ile Arg Phe Gln Glu Leu Ala Val Leu
Tyr 195 200 205 Ala
Ala Arg Gly Ala His Leu Ile Cys Tyr Pro Gly Ala Phe Asn Met 210
215 220 Thr Thr Gly Pro Leu His
Trp Glu Leu Leu Gln Arg Ala Arg Ala Ala225 230
235 240 Asp Asn Gln Leu Tyr Val Ala Thr Cys Ser Pro
Ala Arg Asp Val Gly 245 250
255 Ala Gly Tyr Val Ala Trp Gly His Ser Thr Leu Val Gly Pro Phe Gly
260 265 270 Glu Ile Leu
Ala Thr Thr Glu His Glu Gln Asp Ile Ile Ile Ala Glu 275
280 285 Ile Asp Tyr Ser Leu Ile Glu Leu
Arg Ser Gln Leu Ser Thr Thr His 290 295
300 Leu Pro Leu Pro Thr Pro Thr Thr Thr Arg Asp Ser Thr
Ile Glu Glu305 310 315
320 Glu Asp Asp Leu Val Tyr Ile Tyr Ile 325
11311PRTPhyscomitrella patensPhyscomitrella patens subspecies patens
ecotype Gransden 2004 moss hypothetical protein, locus
PHYPADRAFT_130338 11Met Ala Ser Asp Phe Gln Pro His Met Ala Arg Gln Pro
Pro Ser Glu 1 5 10 15
Ser Leu Pro Asn Ala Pro Asn Gly Gly Lys Tyr Lys Leu Ala Val Cys
20 25 30 Gln Leu Ser Val Thr
Ser Asp Lys Ala Ala Asn Ile Ala His Ala Arg 35 40
45 Gln Lys Ile Glu Ala Ala Ala Asp Ser Gly
Ala Gln Leu Ile Val Leu 50 55 60
Pro Glu Met Trp Asn Cys Pro Tyr Ser Asn Asp Ser Phe Pro Thr
Tyr65 70 75 80 Ala
Glu Asp Ile Asp Ala Gly Leu Glu Ala Ser Pro Ser Ser His Met
85 90 95 Leu Ser Glu Val Ala Arg
Lys Lys Lys Val Thr Ile Val Gly Gly Ser 100
105 110 Ile Pro Glu Arg Asn Asp Gly Lys Leu Tyr
Asn Thr Cys Cys Val Phe 115 120
125 Asp Lys Asn Gly Glu Leu Lys Ala Lys Phe Arg Lys Ile His
Leu Phe 130 135 140
Asp Ile Asp Ile Pro Gly Lys Ile Thr Phe Lys Glu Ser Asp Thr Leu145
150 155 160 Thr Pro Gly Glu Gly
Leu Cys Val Val Asp Thr Asp Val Gly Arg Ile 165
170 175 Ala Val Gly Ile Cys Tyr Asp Ile Arg Phe
Pro Glu Met Ala Met Leu 180 185
190 Tyr Ser Ala Arg Gly Ala His Ile Ile Cys Tyr Pro Gly Ala Phe
Asn 195 200 205 Met
Thr Thr Gly Pro Leu His Trp Glu Leu Leu Gln Lys Ala Arg Ala 210
215 220 Val Asp Asn Gln Ile Phe
Val Val Thr Cys Ser Pro Ala Arg Asp Thr225 230
235 240 Glu Ala Gly Tyr Ile Ala Trp Gly His Ser Thr
Val Val Gly Pro Phe 245 250
255 Gly Glu Ile Leu Ala Thr Thr Glu His Glu Glu Ala Thr Ile Phe Ala
260 265 270 Asp Ile Asp
Tyr Ser Gln Leu Asp Thr Arg Arg Gln Asn Met Pro Leu 275
280 285 Glu Ser Gln Arg Arg Gly Asp Leu
Tyr His Leu Ile Asp Val Thr Arg 290 295
300 Lys Asp Thr Val Lys Ser Ser305 310
12290PRTSelaginella moellendorffiiclub moss hypothetical protein, locus
SELMODRAFT_92478 12Met Pro Ser Ser Arg Tyr Phe Trp Phe Leu Trp Gln
Phe Lys Leu Ala 1 5 10 15
Val Cys Gln Leu Ser Ile Cys Ala Asp Lys Glu Gln Asn Ile Arg His
20 25 30 Ala Arg Glu Ala
Ile Gln Thr Ala Ala Asp Gly Gly Ser Lys Leu Val 35
40 45 Leu Leu Pro Glu Met Trp Asn Cys Pro
Tyr Ser Asn Ala Ser Phe Pro 50 55 60
Ile Tyr Ala Glu Asp Ile Asp Ala Gly Asp Ser Pro Ser Ser
Lys Met65 70 75 80
Leu Ser Asp Met Ala Lys Ser Lys Glu Val Thr Ile Ile Gly Gly Ser
85 90 95 Ile Pro Glu Arg Ser
Gly Asn His Leu Tyr Asn Thr Cys Cys Ile Tyr 100
105 110 Gly Lys Asp Gly Ser Leu Lys Gly Lys His
Arg Lys Val His Leu Phe 115 120
125 Asp Ile Asp Ile Pro Gly Lys Ile Gln Phe Lys Glu Ser Asp
Thr Leu 130 135 140
Thr Pro Gly Asp Lys Tyr Thr Val Val Asp Thr Asp Val Gly Arg Ile145
150 155 160 Gly Val Gly Ile Cys
Tyr Asp Ile Arg Phe Pro Glu Met Ala Met Thr 165
170 175 Tyr Ala Ala Arg Gly Val His Met Ile Cys
Tyr Pro Gly Ala Phe Asn 180 185
190 Met Thr Thr Gly Pro Ala His Trp Glu Leu Leu Gln Lys Ala Arg
Ala 195 200 205 Val
Asp Asn Gln Leu Phe Val Ala Thr Cys Ser Pro Ala Arg Asn Pro 210
215 220 Ser Ala Gly Tyr Val Ala
Trp Gly His Ser Ser Val Ile Gly Pro Phe225 230
235 240 Gly Glu Ile Leu Ala Ser Thr Gly Arg Glu Glu
Ala Ile Phe Tyr Ala 245 250
255 Asp Ile Asp Tyr Ala Gln Ile Lys Glu Arg Arg Met Asn Met Pro Leu
260 265 270 Asp His Gln
Arg Arg Gly Asp Leu Tyr Gln Leu Val Asp Leu Thr Phe 275
280 285 Thr Thr 290 13271PRTMedicago
truncatulabarrel medic unknown protein 13Met Ala Ala Ser Ser Ile Asn Ser
Glu Leu Ala Arg Ser Pro Pro Ala 1 5 10
15 Ile Pro Leu Pro Thr Pro Pro Leu Thr Asn Phe Lys Ile
Gly Leu Cys 20 25 30
Gln Leu Ser Val Thr Ser Asp Lys Asp Lys Asn Ile Ala His Ala Arg
35 40 45 Thr Ala Ile Gln
Asp Ala Ala Ala Lys Gly Ala Lys Leu Ile Leu Leu 50 55
60 Pro Glu Ile Trp Asn Ser Pro Tyr Ser
Asn Asp Ser Phe Pro Val Tyr65 70 75
80 Ala Glu Asp Ile Asp Ala Gly Gly Asp Ala Ser Pro Ser Thr
Ala Met 85 90 95
Leu Ser Glu Leu Ser Ser Leu Leu Lys Ile Thr Ile Val Gly Gly Ser
100 105 110 Ile Pro Glu Arg Ser
Gly Asp Arg Leu Tyr Asn Thr Cys Cys Val Phe 115
120 125 Gly Thr Asp Gly Lys Leu Lys Ala Lys
His Arg Lys Ile His Leu Phe 130 135
140 Asp Ile Asp Ile Pro Gly Lys Ile Thr Phe Ile Glu Ser
Leu Thr Leu145 150 155
160 Thr Ala Gly Asp Thr Pro Thr Ile Val Asp Thr Glu Val Gly Arg Ile
165 170 175 Gly Ile Gly Ile
Cys Tyr Asp Ile Arg Phe Pro Glu Leu Ala Met Ile 180
185 190 Tyr Ala Ala Arg Gly Ala His Leu Leu
Cys Tyr Pro Gly Ala Phe Asn 195 200
205 Met Thr Thr Gly Pro Leu His Trp Glu Leu Leu Gln Arg Ala
Arg Ala 210 215 220
Thr Asp Asn Gln Leu Tyr Val Ala Thr Cys Ser Pro Ala Arg Asp Thr225
230 235 240 Thr Gly Trp Leu Cys
Gly Leu Gly Val Thr Pro Leu Leu Leu Val Leu 245
250 255 Leu Glu Lys Phe Trp Leu Leu Gln Asn Ala
Arg Arg Gln Pro Leu 260 265
270 14613PRTChlorella variabilisgreen algae strain NC64A hypothetical
protein CHLNCDRAFT_58195, similar to hydrolase, carbon-nitrogen
family protein, expressed 14Met Gln Ala Leu Ala Lys Gly Met Ala Leu Val
Gly Val Ala Gly Leu 1 5 10
15 Ser Ala Ala Ala Gly Arg Arg Ala Ala Cys Leu Arg Pro Leu Ser Ser
20 25 30 Tyr Thr Ser
Ala Thr Ala Asp Val Ile Asp Pro Pro Pro Pro Gln Lys 35
40 45 Val Pro Pro Pro Leu Pro Cys Cys
Arg Cys Arg His Cys Cys His Arg 50 55
60 Leu Ala Ser Asn Gln Gln Leu Ala Arg Pro Leu Leu Ala
Gly Pro Ser65 70 75 80
Ala Gln Ile Lys Val Ala Leu Cys Gln Leu Ala Val Gly Ala Asp Lys
85 90 95 Gln Ala Asn Leu Thr
Thr Ala Arg Ser Ala Ile Glu Glu Ala Ala Thr 100
105 110 Ala Gly Ala Asp Leu Val Val Leu Pro Glu
Met Trp Asn Cys Pro Tyr 115 120
125 Ser Asn Asp Ser Phe Pro Thr Tyr Ala Glu Asp Val Glu Ala
Gly Asp 130 135 140
Ser Pro Ser Thr Ser Met Leu Ser Ala Ala Ala Ala Ala Asn Arg Val145
150 155 160 Val Leu Val Gly Gly
Ser Ile Pro Glu Arg Ala Asn Gly Gly Arg Leu 165
170 175 Tyr Asn Thr Cys Phe Val Tyr Gly Arg Asp
Gly Arg Leu Leu Gly Arg 180 185
190 His Arg Lys Val His Leu Phe Asp Ile Asp Ile Pro Gly Lys Ile
Thr 195 200 205 Phe
Lys Glu Ser Leu Thr Leu Thr Pro Gly Glu Gly Leu Thr Val Val 210
215 220 Gly Arg Leu Gly Ile Gly
Ile Cys Tyr Asp Ile Arg Phe Pro Glu Leu225 230
235 240 Ala Leu Leu Tyr Ala Ala Arg Gly Val Gln Leu
Ile Val Tyr Pro Gly 245 250
255 Ala Phe Asn Met Thr Thr Gly Pro Val His Trp Glu Leu Leu Gln Arg
260 265 270 Ala Arg Ala
Val Asp Gly Gln Leu Phe Val Ala Thr Cys Ser Pro Ala 275
280 285 Arg Ser Glu Gly Thr Gly Tyr Ile
Ala Trp Gly His Ser Thr Ala Val 290 295
300 Gly Pro Phe Ala Glu Val Leu Ala Thr Thr Asp Glu Lys
Ala Gly Ile305 310 315
320 Val Tyr Cys His Met Asp Phe Ala Gln Leu Gly Glu Arg Arg Ala Asn
325 330 335 Met Pro Leu Arg
His Gln Lys Arg Ala Asp Leu Tyr Ser Leu Leu Asp 340
345 350 Leu Thr Arg Pro Asn Ser Leu Ser Asn
Ala Gly Leu His Asn Gly Pro 355 360
365 Val Gln Arg Thr Leu Ala Gly Ser Ser Gly Ile Val Gly Ser
Gly Ile 370 375 380
Thr Arg Gln Leu Leu Met Glu Gly Ala Lys Val Val Ala Leu Leu Arg385
390 395 400 Lys Val Asp Gln Lys
Ala Gly Leu Leu Arg Asp Cys Gln Gly Ala Pro 405
410 415 Ile Glu Asn Leu Tyr Pro Ala Val Val Glu
Asp Val Ser Lys Glu Glu 420 425
430 Gln Cys Ala Ala Phe Val His Glu Val Val Glu Gln His Gly Ala
Ile 435 440 445 Asp
His Ala Val Ser Cys Phe Gly Ala Trp Trp Gln Gly Gly Leu Leu 450
455 460 Thr Glu Gln Ser Tyr Ala
Glu Phe Ser Arg Val Leu Ala Asn Phe Ala465 470
475 480 Gly Ser His Phe Thr Phe Val Lys Tyr Ile Leu
Pro Ala Met Arg Gln 485 490
495 Ser His Thr Ser Ser Met Leu Phe Val Thr Gly Gly Val Gly Lys Arg
500 505 510 Val Leu Ser
Ala Asp Ser Gly Leu Ala Thr Val Gly Gly Ala Ala Leu 515
520 525 Tyr Gly Ile Val Arg Ala Ala Gln
Ala Gln Tyr Gln Gly Arg Pro Pro 530 535
540 Arg Ile Asn Glu Leu Arg Ile Phe Ala Leu Val Thr Arg
His Gly Glu545 550 555
560 Met Pro Arg Ser His Ser Ser Ile Val Glu Gly Leu Arg Ala His Ser
565 570 575 Asn Arg Lys Val
Gly Asn Leu Ala Ala Glu Ala Leu Ala Ala Ala Ala 580
585 590 Asp Asp Glu Leu Leu Glu Val Thr Ser
Glu Arg Leu Asp Gly Val Met 595 600
605 Leu Met Val Gly Asp 610 15269PRTVolvox
carteriVolvox carteri forma nagariensis hypothetical protein
VOLCADRAFT_73623 15Met His Val Thr Ala Asp Lys Ala Gln Asn Leu Gln Thr
Ala Lys Arg 1 5 10 15
Ala Ile Glu Asp Ala Ala Ala Gln Gly Ala Lys Leu Val Val Leu Pro
20 25 30 Glu Met Trp Asn Cys
Pro Tyr Ser Asn Asp Ser Phe Pro Thr Tyr Ala 35 40
45 Glu Asp Ile Glu Gly Gly Ala Ser Gly Ser
Val Ala Met Leu Ser Ala 50 55 60
Ala Ala Ala Ala Ala Cys Val Thr Leu Val Ala Gly Ser Ile Pro
Glu65 70 75 80 Arg
Cys Gly Asp Arg Leu Tyr Asn Thr Cys Cys Val Phe Asn Ser Arg
85 90 95 Gly Glu Leu Leu Ala Lys
His Arg Lys Val His Leu Phe Asp Ile Asp 100
105 110 Ile Pro Gly Lys Ile Thr Phe Lys Glu Ser
Leu Thr Leu Ser Pro Gly 115 120
125 Pro Gly Pro Thr Val Val Asp Thr Glu Ala Gly Arg Leu Gly
Ile Gly 130 135 140
Ile Cys Tyr Asp Ile Arg Phe Pro Glu Leu Ala Gln Leu Tyr Ala Ala145
150 155 160 Arg Gly Cys Gln Val
Leu Ile Tyr Pro Gly Ala Phe Asn Met Thr Thr 165
170 175 Gly Pro Val His Trp Glu Leu Leu Ala Arg
Ala Arg Ala Val Asp Asn 180 185
190 Gln Ile Phe Val Ile Thr Cys Ser Pro Ala Arg Asn Pro Ser Ser
Ser 195 200 205 Tyr
Gln Ala Trp Gly His Ser Thr Val Val Gly Pro Phe Ala Glu Ile 210
215 220 Leu Ala Thr Thr Asp His
Gln Pro Gly Thr Ile Tyr Thr Glu Leu Asp225 230
235 240 Tyr Ser Gln Leu Ala Glu Arg Arg Ala Asn Met
Pro Leu Arg Gln Gln 245 250
255 Lys Arg His Asp Leu Tyr Val Leu Leu Asp Lys Thr Ala
260 265 161525DNAArtificial
Sequencesynthetic glycine-rich protein (GRP) promoter 16gaaattaaac
ccagggtcga cagcgcccac tatagagaaa aaattgaaat gttttgagaa 60tcggatgatt
ttttttaact attaggtcta gtttgaaaac cctattttct aacaaaggga 120ttttcatttt
tataagagaa aataaactaa cttttcttga gaaaataaaa ttctttggaa 180aaatggattt
ctcaaactag ctcttacggc tagtttggaa accccaattt cacacgggat 240tctcattttc
ccaagggaaa aatgaactaa tttcccttag aaaaatgaga atcccgtggg 300aaattgggat
tttcaaagta gcccttatag tggaaataag ttatggtgtc tcgctcgtat 360ggttatgtag
ggccgcgcgt gtattccagc gccggccgca tggataccct atcgattctg 420acttctctgt
ctcaggaaaa taatacagcc acgattaacg gaacctgctg gctggatcca 480tgattactca
cttgacttca catcgatcca aattatctag cttgcacgtt catgggtcgc 540ctcgctcgcc
cgatcgatat tacgtacacc atagattagt actatatgga gtggagtgtt 600gaatggatgc
tctttattat tctagccaag ttatcaagcc gggcacttgc atcggaagga 660gtaccagtgt
acgcatcaga tcagacgata atcgatcaag atgggtacga gatttgccgc 720ttgcttcctg
ttcttgatgg gcaatctttt cgggccttga acgtcggaga atcgactata 780cgaaatccta
ggtcaactat acattggttg atgcttccgt gtagttttac cagttcatcg 840gtctctagct
tgttgtttgc gacgacttca cgtggccacg cgtttactgc gctctgctca 900aagaaattgc
ctacagtgcc tggcgtcagc tgcaggcgtt gaatccgagg tcgcgcgccg 960cagaataagt
acgagtcaaa ggctgagctg catgccgtac cggcctttat taatagctga 1020gctctactcg
ctacgtcagt atagtatagc acggtcatat atatactata gctatagctg 1080tggggtaccg
tgtccgtatc gtgaatctga agtcgaacag tgatatggcg tactatctaa 1140taatgtcccg
tgcagtaata tcactgttgc cgacgatggg aatctctagt tttgacagaa 1200accaaagcaa
ctgctagcta attaattcca gagagatcga tttctacagt gctgcaacaa 1260tcaatgcaat
tggcatcaga cgatatatgc taatggtttc tttatcgata cgtggtcaac 1320agagctctct
cgcccgccct gatcagatct catcgcacat ggacacccat ctgccaaccc 1380aacacgggcg
ggggaaccac cgtgaaacat cgcgttcatg cacgaccccc ccgcaggccg 1440cagctataaa
tacccatgca atgcaatgca gcgggtcatc atcgactcca cctggactcg 1500ctcactggca
atggctacca ccagc
1525171615DNAArtificial Sequencesynthetic Agrobacterium rhizogenes roID
promoter with Arabidopsis thaliana omega-amidase coding sequence
construct 17gacgtcggta ccgaatttgt tcgtgaacta ttagttgcgg gccttggcat
ccgactacct 60ctgcggcaat attatattcc ctgggcccac cgtgaaccca atttcgccta
tttattcatt 120acccccatta acattgaagt agtcatgatg ggcctgcagc acgttggtga
ggctggcaca 180actcatccat atactttctg accggatcgg cacattattg tagaaaacgc
ggacccacag 240cgcactttcc aaagcggtgc cgcgtcagaa tgcgctggca gaaaaaaatt
aatccaaaag 300taccctccaa gcagcccata taaacgcgtt tacaaatccg ctaacctcaa
caatttgagc 360agagaaaatt cgcacctaca aggcagatgg catcatcatt caatccagag
caggcaagag 420ttccttcagc attaccttta ccagcaccac cacttaccaa attcaacatc
ggactttgtc 480aattgagtgt tacttctgat aagaaaagaa acatttcaca tgctaagaaa
gcaatcgaag 540aggctgctag taagggagct aaactcgttc ttttgcctga aatatggaac
tcaccataca 600gtaacgattc ttttcctgtg tacgcagaag agatcgatgc tggaggtgat
gcatctccat 660caactgctat gctctcagaa gttagtaaga gactcaagat tacaattatc
ggaggttcaa 720ttcctgagag agttggagat aggttgtata acacatgttg cgtgttcgga
tctgatggag 780agctcaaggc taagcatagg aagattcacc tcttcgatat agatattcct
ggaaagatca 840ccttcatgga atcaaaaaca cttaccgctg gagagactcc aacaattgtt
gatacagatg 900tgggtagaat cggaataggt atatgttacg atatcaggtt ccaagaattg
gctatgatat 960atgctgcaag aggagcacat ctcttatgct accctggagc tttcaatatg
actacaggtc 1020cattgcactg ggagcttttg caaagagcta gggcaacaga taaccagctc
tatgttgcta 1080cctgctctcc tgcaagagat tcaggagctg gttacaccgc atggggtcat
tctactcttg 1140ttggaccatt tggtgaagtg ttggctacca ctgagcacga agaggctatt
ataatcgcag 1200aaatcgatta cagtatactt gagcagagaa ggacttctct cccattaaat
aggcagagga 1260ggggtgattt ataccagtta gttgatgttc agagattaga tagtaagtga
cacgtgtgaa 1320ttacaggtga ccagctcgaa tttccccgat cgttcaaaca tttggcaata
aagtttctta 1380agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt
gaattacgtt 1440aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt
ttttatgatt 1500agagtcccgc aattatacat ttaatacgcg atagaaaaca aaatatagcg
cgcaaactag 1560gataaattat cgcgcgcggt gtcatctatg ttactagatc gggggtaccg
acgtc 1615187PRTArtificial Sequencesynthetic conserved glutamine
phenylpyruvate transaminase (GPT) region 18Asn Leu Gly Gln Gly Phe
Pro1 5 19275PRTChlamydomonas
reinhardtiiChlamydomonas reinhardtii strain CC-503 cw92 mt+
predicted protein, partial, locus CHLREDRAFT_114854 19Lys Val Ala Leu Cys
Gln Leu His Val Thr Ala Asp Lys Glu Gln Asn 1 5
10 15 Leu Arg Thr Ala Arg Lys Ala Ile Glu Asp
Ala Ala Ala Ala Gly Ala 20 25
30 Lys Leu Val Val Leu Pro Glu Met Phe Asn Cys Pro Tyr Ser Asn
Asp 35 40 45 Ser
Phe Pro Thr Tyr Ala Glu Asp Ile Glu Gly Gly Ala Ser Gly Ser 50
55 60 Val Ala Ala Leu Ser Ala
Ala Ala Ala Ala Ala Arg Val Thr Leu Val65 70
75 80 Ala Gly Ser Ile Pro Glu Arg Cys Gln Gly Lys
Leu Tyr Asn Thr Cys 85 90
95 Cys Val Phe Asp Ser Ser Gly Lys Leu Leu Ala Lys His Arg Lys Val
100 105 110 His Leu Phe
Asp Ile Asp Ile Pro Gly Lys Ile Thr Phe Lys Glu Ser 115
120 125 Leu Thr Leu Ser Pro Gly Pro Gly
Pro Thr Val Val Asp Thr Glu Ala 130 135
140 Gly Arg Leu Gly Ile Gly Ile Cys Tyr Asp Ile Arg Phe
Pro Glu Leu145 150 155
160 Ala Gln Ile Tyr Ala Ala Arg Gly Cys Gln Val Leu Ile Tyr Pro Gly
165 170 175 Ala Phe Asn Met
Thr Thr Gly Pro Val His Trp Glu Leu Leu Ala Lys 180
185 190 Ala Arg Ala Val Asp Asn Gln Val Phe
Val Leu Thr Cys Ser Pro Ala 195 200
205 Arg Asn Pro Asp Ser Ser Tyr Gln Ala Trp Gly His Ser Thr
Ala Leu 210 215 220
Gly Pro Phe Ala Glu Val Leu Ala Thr Thr Glu His Ser Pro Ala Thr225
230 235 240 Val Phe Ala Glu Leu
Asp Tyr Ala Gln Leu Asp Glu Arg Arg Ala Ala 245
250 255 Met Pro Leu Arg Gln Gln Lys Arg His Asp
Leu Tyr Leu Leu Leu Asp 260 265
270 Lys Thr Ala 275 20361PRTMicromonas pusillagreen algae
Micromonas pusilla strain CCMP1545 hypothetical protein, locus
MICPUCDRAFT_23156 20Met Arg Ala Thr Lys Thr Thr Ala Ala Ala Ala Ala Ala
Ala Ala Ala 1 5 10 15
Ser Ser Ser Gly Ala Gly Ala Pro Val Pro Phe Ala Arg Val Pro Ala
20 25 30 Pro Trp Ser Ala Ser
Gly Ala Ser Ala Ser Asp Ala Ala Thr Pro Thr 35 40
45 Pro Thr Pro Ala Pro Arg Val Val Lys Val
Ala Leu Cys Gln Leu Ala 50 55 60
Cys Pro Thr Ala Asp Lys Val Ala Asn Ile Ala Arg Ala Arg Glu
Ala65 70 75 80 Ile
Arg Asn Ala Ala Glu Gly Gly Ala Ala Leu Val Val Leu Pro Glu
85 90 95 Met Trp Asn Cys Pro Tyr
Ala Asn Glu Ser Phe Pro Ala His Ala Glu 100
105 110 Thr Ile Gly Ala Asn Asp Pro Thr Pro Ser
Val Thr Met Leu Ser Glu 115 120
125 Ala Ala Ala Ala His Asp Ile Val Leu Val Gly Gly Ser Ile
Pro Glu 130 135 140
Arg Gly Val Gly Val Gly Gly Gly Gly Gly Ala Asp Glu Glu Asp Val145
150 155 160 Leu Tyr Asn Ala Cys
Cys Val Phe Asp Gly Lys Arg Gly Leu Ile Ala 165
170 175 Arg His Arg Lys Thr His Leu Phe Asp Val
Asp Ile Pro Gly Glu Ile 180 185
190 Ser Phe Arg Glu Ser Asp Thr Leu Thr Glu Gly Glu Gly Leu Thr
Val 195 200 205 Val
Asp Thr Ala Val Gly Arg Val Gly Val Gly Ile Cys Phe Asp Val 210
215 220 Arg Phe Gly Glu Met Ala
Ala Ala Met Ala Asn Arg Gly Ala Asp Val225 230
235 240 Leu Ile Tyr Pro Gly Ala Phe Asn Thr Val Thr
Gly Pro His His Trp 245 250
255 Glu Leu Leu Gln Arg Ala Arg Ala Val Asp Asn Gln Ala Arg Ser Ile
260 265 270 His Trp Ser
Pro Tyr Asp Arg Cys Phe Val Leu Thr Cys Ser Pro Ala 275
280 285 Arg Asn Thr Thr Gly Glu Gly Tyr
Gln Ala Trp Gly His Ser Thr Ala 290 295
300 Val Gly Pro Phe Ala Glu Val Leu Ala Thr Thr Asp Glu
Arg Pro Gly305 310 315
320 Ile Val Phe Ala Asp Leu Asp Leu Gly Glu Val Thr Arg Arg Arg Arg
325 330 335 Asn Met Pro Leu
Ala Thr Gln Arg Arg Gly Asp Leu Tyr Ala Leu His 340
345 350 Asp Leu Gly Ala Val Arg Gly Asp Ala
355 360 21371PRTEctocarpus siliculosusbrown
algae Ectocarpus siliculosus strain Ec 32 hydrolase, carbon-nitrogen
family protein, locus Esi_0003_0068 21Met Phe Leu Ala Ala Ala Arg Arg Ala
Ser Pro Ile Leu Leu Ser Leu 1 5 10
15 Ala Val Lys Thr Ser Thr Thr Ala Ala Phe Cys Ser Pro Arg
Leu Ala 20 25 30
Asn Ala Arg Thr Asn Thr Ala Ala Gly Ala Thr Arg Thr Ala Tyr Ala 35
40 45 Ala Cys Ser Ile Ser
Arg Asn Ile Ser Leu Leu Ser Arg Pro Leu Ser 50 55
60 Ser Met Ser Ala Ser Gly Ala Ser Glu Gly
Ala Thr Ala Gly Ala Gly65 70 75
80 Ser Arg Arg Phe Val Val Ala Ala Cys Gln Ile Leu Cys Gly Ser
Asp 85 90 95 Lys
Leu Ala Asn Ile Ala Thr Ala Glu Ser Ala Val Arg Asp Ala Ala
100 105 110 Ala Ala Gly Ala Gln
Val Val Val Leu Pro Glu Cys Trp Asn Gly Pro 115
120 125 Tyr Asp Thr Ala Ser Phe Pro Val Tyr
Ala Glu Pro Val Pro Asp Pro 130 135
140 Gln Gly Asp Glu Thr Ala Ala Asp Met Pro Ser Ala Glu
Gln Ser Pro145 150 155
160 Ser Ala Ala Met Leu Cys Arg Ala Ala Ala Glu Asn Lys Val Trp Leu
165 170 175 Val Gly Gly Ser
Val Pro Glu Ala Gly Lys Asp Gly Gly Val Tyr Asn 180
185 190 Thr Cys Ile Val Val Gly Pro Ser Gly
Arg Ile Val Ala Lys His Arg 195 200
205 Lys Val His Leu Phe Asp Ile Asp Val Pro Gly Gly Ile Thr
Phe Lys 210 215 220
Glu Ser Asp Thr Leu Ser Pro Gly Asp Ser Ile Thr Thr Val Glu Thr225
230 235 240 Pro Phe Gly Thr Ile
Gly Val Gly Ile Cys Tyr Asp Met Arg Phe Pro 245
250 255 Glu Leu Ser Met Ala Met Arg Ala Ala Gly
Ser Val Leu Leu Cys Phe 260 265
270 Pro Gly Ala Phe Asn Met Thr Thr Gly Pro Ala His Trp Glu Leu
Leu 275 280 285 Gln
Arg Ala Arg Ala Leu Asp Asn Gln Cys Phe Val Val Thr Ala Ser 290
295 300 Pro Ala Arg Asn Pro Asp
Ser Lys Tyr Gln Ala Trp Gly His Ser Ser305 310
315 320 Ile Val Asp Pro Trp Gly Thr Val Val Ala Thr
Thr Glu His Glu Glu 325 330
335 Ala Met Leu Val Ala Glu Val Asp Val Gly Arg Val Ala Glu Val Arg
340 345 350 Thr Ser Ile
Pro Val Ser Leu Gln Lys Arg Pro Asp Leu Tyr Arg Leu 355
360 365 Glu Leu Pro 370
22313PRTPhaeodactylum tricornutumdiatom Phaeodactylum tricornutum strain
CCAP 1055/1 hypothetical protein, locus PHATRDRAFT_15536 22Met Ser
Ala Ser Arg Gln Asn Asp Asp Asp Asp Asp Asp Asp Pro Ser 1 5
10 15 Val Leu Arg Val Ala Leu Cys
Gln Leu Pro Val Thr Asn Asp Lys Ala 20 25
30 Gln Asn His Gln Thr Ala Arg Glu Tyr Leu Asn Arg
Ala Ala Asn Gln 35 40 45
Gly Ala Arg Leu Val Val Leu Pro Glu Ile Trp Asn Ser Pro Tyr Ala
50 55 60 Thr Ala Ala
Phe Pro Glu Tyr Ala Glu Gln Leu Pro Asp Val Leu Ala65 70
75 80 Gln Asp Gly Asp Gly His Thr Gly
Val Tyr Glu Ser Pro Ser Ala Asp 85 90
95 Leu Leu Arg Glu Ser Ala Lys Glu His Lys Leu Trp Ile
Val Gly Gly 100 105 110
Ser Ile Pro Glu Arg Asp Asp Asp Asp Lys Ile Tyr Asn Thr Ser Leu
115 120 125 Val Phe Asp Pro
Gln Gly Asn Leu Val Ala Lys His Arg Lys Met His 130
135 140 Leu Phe Asp Ile Asp Val Pro Gly
Gly Ile Thr Phe Phe Glu Ser Asp145 150
155 160 Thr Leu Ser Pro Gly Asn Thr Val Ser His Phe Ala
Thr Pro Trp Gly 165 170
175 Asn Ile Gly Leu Gly Ile Cys Tyr Asp Ile Arg Phe Pro Glu Tyr Ala
180 185 190 Met Leu Leu
Ala Lys Glu His Asp Cys Gly Ile Leu Ile Tyr Pro Gly 195
200 205 Ala Phe Asn Leu Thr Thr Gly Pro
Ala His Trp Glu Leu Leu Gln Arg 210 215
220 Gly Arg Ala Val Asp Asn Gln Cys Phe Val Leu Thr Ala
Ser Pro Ala225 230 235
240 Arg Thr Glu Pro Pro Ser Lys Ala Gly Leu Tyr Pro His Tyr Thr Ala
245 250 255 Trp Gly His Ser
Thr Ala Val Ser Pro Trp Gly Glu Val Ile Ala Thr 260
265 270 Thr Asn Glu Lys Ala Gly Ile Val Phe
Ala Asp Leu Asp Leu Ser Lys 275 280
285 Val Thr Glu Met Arg Thr Ser Ile Pro Ile Gly Lys Gln Lys
Arg Thr 290 295 300
Asp Leu Tyr Gln Leu Val Gly Lys Ser305 310
23322PRTSchizosaccharomyces pombefission yeast Schizosaccharomyces pombe
strain 972h- amidohydrolase, locus SPAC26A3.11 23Met Asn Ser Lys Phe
Phe Gly Leu Val Gln Lys Gly Thr Arg Ser Phe 1 5
10 15 Phe Pro Ser Leu Asn Phe Cys Tyr Thr Arg
Asn Ile Met Ser Val Ser 20 25
30 Ala Ser Ser Leu Val Pro Lys Asp Phe Arg Ala Phe Arg Ile Gly
Leu 35 40 45 Val
Gln Leu Ala Asn Thr Lys Asp Lys Ser Glu Asn Leu Gln Leu Ala 50
55 60 Arg Leu Lys Val Leu Glu
Ala Ala Lys Asn Gly Ser Asn Val Ile Val65 70
75 80 Leu Pro Glu Ile Phe Asn Ser Pro Tyr Gly Thr
Gly Tyr Phe Asn Gln 85 90
95 Tyr Ala Glu Pro Ile Glu Glu Ser Ser Pro Ser Tyr Gln Ala Leu Ser
100 105 110 Ser Met Ala
Lys Asp Thr Lys Thr Tyr Leu Phe Gly Gly Ser Ile Pro 115
120 125 Glu Arg Lys Asp Gly Lys Leu Tyr
Asn Thr Ala Met Val Phe Asp Pro 130 135
140 Ser Gly Lys Leu Ile Ala Val His Arg Lys Ile His Leu
Phe Asp Ile145 150 155
160 Asp Ile Pro Gly Gly Val Ser Phe Arg Glu Ser Asp Ser Leu Ser Pro
165 170 175 Gly Asp Ala Met
Thr Met Val Asp Thr Glu Tyr Gly Lys Phe Gly Leu 180
185 190 Gly Ile Cys Tyr Asp Ile Arg Phe Pro
Glu Leu Ala Met Ile Ala Ala 195 200
205 Arg Asn Gly Cys Ser Val Met Ile Tyr Pro Gly Ala Phe Asn
Leu Ser 210 215 220
Thr Gly Pro Leu His Trp Glu Leu Leu Ala Arg Ala Arg Ala Val Asp225
230 235 240 Asn Glu Met Phe Val
Ala Cys Cys Ala Pro Ala Arg Asp Met Asn Ala 245
250 255 Asp Tyr His Ser Trp Gly His Ser Thr Val
Val Asp Pro Phe Gly Lys 260 265
270 Val Ile Ala Thr Thr Asp Glu Lys Pro Ser Ile Val Tyr Ala Asp
Ile 275 280 285 Asp
Pro Ser Val Met Ser Thr Ala Arg Asn Ser Val Pro Ile Tyr Thr 290
295 300 Gln Arg Arg Phe Asp Val
Tyr Ser Glu Val Leu Pro Ala Leu Lys Lys305 310
315 320 Glu Glu24292PRTAspergillus oryzaeyellow koji
mold Aspergillus oryzae strain RIB40 hypothetical protein, locus
AOR_1_782154 24Met Ala Ala Leu Leu Lys Gln Pro Leu Lys Leu Ala Leu Val
Gln Leu 1 5 10 15
Ala Ser Gly Ala Asp Lys Ala Val Asn Leu Ala His Ala Arg Thr Lys
20 25 30 Val Leu Glu Ala Ala
Gln Ala Gly Ala Lys Leu Ile Val Leu Pro Glu 35 40
45 Cys Phe Asn Ser Pro Tyr Gly Thr Gln Tyr
Phe Pro Lys Tyr Ala Glu 50 55 60
Thr Leu Leu Pro Ser Pro Pro Thr Glu Asp Gln Ser Pro Ser Tyr
His65 70 75 80 Ala
Leu Ser Ala Ile Ala Ala Glu Ala Lys Ala Tyr Leu Val Gly Gly
85 90 95 Ser Ile Pro Glu Leu Glu
Pro Thr Thr Lys Lys Tyr Tyr Asn Thr Ser 100
105 110 Leu Val Phe Ser Pro Thr Gly Ser Leu Ile
Gly Thr His Arg Lys Thr 115 120
125 His Leu Phe Asp Ile Asp Ile Pro Gly Lys Ile Thr Phe Lys
Glu Ser 130 135 140
Glu Val Leu Ser Pro Gly Asn Gln Leu Thr Ile Val Asp Leu Pro Asp145
150 155 160 Tyr Gly Lys Ile Gly
Leu Ala Ile Cys Tyr Asp Ile Arg Phe Pro Glu 165
170 175 Ala Ala Met Ile Ala Ala Arg Lys Gly Ala
Phe Ala Leu Ile Tyr Pro 180 185
190 Gly Ala Phe Asn Met Thr Thr Gly Pro Met His Trp Ser Leu Leu
Ala 195 200 205 Arg
Ala Arg Ala Val Asp Asn Gln Leu Tyr Val Gly Leu Cys Ser Pro 210
215 220 Ala Arg Asp Met Glu Ala
Thr Tyr His Ala Trp Gly His Ser Leu Ile225 230
235 240 Ala Asn Pro Ala Ala Glu Val Leu Val Glu Ala
Glu Asp Lys Glu Thr 245 250
255 Ile Val Tyr Ala Asp Leu Asp Asn Asp Thr Ile Gln Ser Thr Arg Lys
260 265 270 Gly Ile Pro
Val Tyr Thr Gln Arg Arg Phe Asp Leu Tyr Pro Asp Val 275
280 285 Ser Ala Glu Lys 290
25306PRTNeurospora crassared bread mold Neurospora crassa strain OR74A
hypothetical protein, locus NCU06726.1, similar to nit protein 2 25Met
Ala Ser Ser Thr Lys His Pro Ile Leu Leu Lys Lys Pro Val Lys 1
5 10 15 Leu Ala Cys Ile Gln Leu
Ala Ser Gly Ala Asp Lys Ser Ala Asn Leu 20 25
30 Ser His Ala Ala Asp Lys Val Arg Glu Ala Ala
Ser Gly Gly Ala Asn 35 40 45
Ile Val Val Leu Pro Glu Cys Phe Asn Ser Pro Tyr Gly Cys Asp Phe
50 55 60 Phe Pro Ser
Tyr Ala Glu Gln Leu Leu Pro Ser Pro Pro Thr Val Glu65 70
75 80 Gln Ser Pro Ser Phe His Ala Leu
Ser Ala Met Ala Arg Asp Asn Gly 85 90
95 Ile Tyr Leu Val Gly Gly Ser Ile Pro Glu Leu Ala Ile
Glu Glu Gly 100 105 110
Thr Glu Asp Lys Lys Thr Tyr Tyr Asn Thr Ser Leu Val Phe Gly Pro
115 120 125 Asp Gly Lys Leu
Leu Ala Ser His Arg Lys Val His Leu Phe Asp Ile 130
135 140 Asp Ile Pro Gly Lys Ile Lys Phe
Lys Glu Ser Asp Val Leu Ser Pro145 150
155 160 Gly Asn Ser Val Thr Leu Val Asp Leu Pro Asp Tyr
Gly Arg Ile Ala 165 170
175 Val Ala Ile Cys Tyr Asp Ile Arg Phe Pro Glu Leu Ala Met Ile Ala
180 185 190 Ala Arg Lys
Gly Cys Phe Ala Leu Val Tyr Pro Gly Ala Phe Asn Thr 195
200 205 Thr Thr Gly Pro Leu His Trp Arg
Leu Gln Gly Gln Ala Arg Ala Met 210 215
220 Asp Asn Gln Ile Tyr Val Ala Leu Cys Ser Pro Ala Arg
Asp Ile Ser225 230 235
240 Ala Ser Tyr His Ala Tyr Gly His Ser Leu Ile Val Asp Pro Met Ala
245 250 255 Arg Val Leu Val
Glu Ala Glu Glu Ser Glu Thr Ile Val Ser Ala Glu 260
265 270 Leu Asp Gly Thr Lys Ile Glu Glu Ala
Arg Ser Gly Ile Pro Leu Arg 275 280
285 Asp Gln Arg Arg Phe Asp Ile Tyr Pro Asp Val Ser Gln Ala
Lys Pro 290 295 300
Phe Phe305 26285PRTRhizobium leguminosarumnitrogen-fixing plant
microsymbiont bacteria of tribe Viciae Rhizobium leguminosarum
biovar viciae strain 3841 aminohydrolase, locus RL4288 26Met Ser Phe
Lys Ala Ala Ala Val Gln Met Cys Ser Gly Val Asp Pro 1 5
10 15 Val Arg Asn Ala Ala Ala Met Ala
Arg Leu Val Arg Glu Ala Ala Gly 20 25
30 Gln Gly Ala Ile Tyr Val Gln Thr Pro Glu Met Thr Gly
Met Leu Gln 35 40 45
Arg Asp Arg Ala Ala Ala Arg Ala Val Leu Ala Asp Glu Ala His Asp 50
55 60 Ile Ile Val Lys Thr
Gly Ser Asp Leu Ala Arg Glu Leu Gly Ile His65 70
75 80 Met His Val Gly Ser Thr Ala Ile Ala Leu
Ala Asp Gly Lys Ile Ala 85 90
95 Asn Arg Gly Phe Leu Phe Gly Pro Asp Gly Arg Ile Leu Asn Arg
Tyr 100 105 110 Asp
Lys Ile His Met Phe Asp Val Asp Leu Asp Asn Gly Glu Ser Trp 115
120 125 Arg Glu Ser Ala Ala Tyr
Thr Ala Gly Ser Glu Ala Arg Val Leu Ser 130 135
140 Leu Pro Phe Ala Glu Met Gly Phe Ala Ile Cys
Tyr Asp Val Arg Phe145 150 155
160 Pro Ala Leu Phe Arg Ala Gln Ala Met Ala Gly Ala Glu Val Met Thr
165 170 175 Val Pro Ala
Ala Phe Thr Lys Gln Thr Gly Glu Ala His Trp Glu Ile 180
185 190 Leu Leu Arg Ala Arg Ala Ile Glu
Asn Gly Val Phe Val Ile Ala Ala 195 200
205 Ala Gln Ala Gly Arg His Glu Asp Gly Arg Glu Ser Phe
Gly His Ser 210 215 220
Met Ile Ile Asp Pro Trp Gly Thr Val Leu Ala Ser Ala Gly Ala Thr225
230 235 240 Gly Glu Ala Val Ile
Val Ala Glu Ile Asp Pro Ser Ala Val Lys Ala 245
250 255 Ala His Asp Lys Ile Pro Asn Leu Arg Asn
Gly Arg Glu Phe Ser Val 260 265
270 Glu Lys Ile Ala Gly Ala Ile Ala Gly Gly Val Ala Ala
275 280 285 27285PRTRhizobium
etlinitrogen-fixing plant microsymbiont bacteria Rhizobium etli
strain CFN 42 aminohydrolase, locus RHE_CH03761 27Met Ser Phe Lys Ala Ala
Ala Ile Gln Met Cys Ser Gly Val Asp Pro 1 5
10 15 Val Lys Asn Ala Ala Ser Met Ala Arg Leu Val
Arg Glu Ala Ala Ala 20 25 30
Gln Gly Ala Thr Tyr Val Gln Thr Pro Glu Met Thr Gly Met Leu Gln
35 40 45 Arg Asp Arg
Ala Ala Ala Arg Ala Val Leu Ala Asp Glu Ala His Asp 50
55 60 Ile Ile Val Lys Thr Gly Ser Glu
Leu Ala Arg Glu Leu Gly Ile His65 70 75
80 Val His Val Gly Ser Thr Ala Ile Ala Leu Ser Asp Gly
Lys Ile Ala 85 90 95
Asn Arg Gly Phe Leu Phe Gly Pro Asp Gly Arg Ile Leu Asn Arg Tyr
100 105 110 Asp Lys Ile His Met
Phe Asp Val Asp Leu Asp Asn Gly Glu Ser Trp 115
120 125 Arg Glu Ser Ala Ala Tyr Thr Ala Gly
Ser Glu Ala Arg Val Leu Ser 130 135
140 Leu Pro Phe Ala Glu Met Gly Phe Ala Ile Cys Tyr Asp
Val Arg Phe145 150 155
160 Pro Ala Leu Phe Arg Ala Gln Ala Val Ala Gly Ala Glu Val Met Thr
165 170 175 Val Pro Ser Ser
Phe Ser Arg Gln Thr Gly Glu Ala His Trp Glu Ile 180
185 190 Leu Leu Arg Ala Arg Ala Ile Glu Asn
Gly Val Phe Val Ile Ala Ala 195 200
205 Ala Gln Ala Gly Arg His Glu Asp Gly Arg Glu Thr Phe Gly
His Ser 210 215 220
Ile Ile Ile Asp Pro Trp Gly Thr Val Leu Ala Ser Ala Gly Ala Thr225
230 235 240 Gly Glu Ala Val Ile
Leu Ala Glu Ile Asp Pro Gly Ala Val Lys Ala 245
250 255 Ala His Asp Lys Ile Pro Asn Leu Arg Asp
Gly Arg Glu Phe Ser Val 260 265
270 Glu Lys Ile Ala Gly Ala Val Ala Gly Gly Val Ala Ala
275 280 285 28285PRTRhizobium
leguminosarumnitrogen-fixing plant microsymbiont bacteria of South
American clover Trifolium polymorphum Rhizobium leguminosarum biovar
trifolii strain WSM1325 nitrilase/cyanide hydratase and
apolipoprotein N-acyltransferase, locus Rleg_3821 28Met Ser Phe Lys Ala
Ala Ala Val Gln Met Cys Ser Gly Val Asp Pro 1 5
10 15 Val Lys Asn Ala Ala Ala Met Ala Arg Leu
Val Arg Glu Ala Ala Gly 20 25
30 Gln Gly Ala Thr Tyr Val Gln Thr Pro Glu Met Thr Gly Met Leu
Gln 35 40 45 Arg
Asp Arg Thr Ala Ala Arg Ala Val Leu Ala Asp Glu Ala His Asp 50
55 60 Ile Ile Val Lys Thr Gly
Ser Glu Leu Ala Ile Glu Leu Gly Ile His65 70
75 80 Met His Val Gly Ser Thr Ala Ile Ala Leu Ala
Asp Gly Lys Ile Ala 85 90
95 Asn Arg Gly Phe Leu Phe Gly Pro Asp Gly Arg Val Leu Asn Arg Tyr
100 105 110 Asp Lys Ile
His Met Phe Asp Val Asp Leu Asp Asn Gly Glu Ser Trp 115
120 125 Arg Glu Ser Ala Ala Tyr Thr Ala
Gly Ser Glu Ala Arg Val Leu Ser 130 135
140 Leu Pro Phe Ala Glu Met Gly Phe Ala Ile Cys Tyr Asp
Val Arg Phe145 150 155
160 Pro Ala Leu Phe Cys Ala Gln Ala Val Ala Gly Ala Glu Val Met Thr
165 170 175 Val Pro Ala Ala
Phe Thr Lys Gln Thr Gly Glu Ala His Trp Glu Ile 180
185 190 Leu Leu Arg Ala Arg Ala Ile Glu Asn
Gly Val Phe Val Ile Ala Ala 195 200
205 Ala Gln Ala Gly Arg His Glu Asp Gly Arg Glu Thr Phe Gly
His Ser 210 215 220
Met Ile Ile Asp Pro Trp Gly Thr Val Leu Ala Ser Ala Gly Ala Thr225
230 235 240 Gly Glu Ala Val Ile
Val Ala Glu Ile Asp Pro Ala Ala Val Lys Ala 245
250 255 Ala His Asp Lys Ile Pro Asn Leu Arg Asn
Gly Arg Glu Phe Ser Val 260 265
270 Glu Lys Ile Ala Gly Ala Ile Ala Gly Gly Val Ala Ala
275 280 285 29292PRTBradyrhizobium
sp.photosynthetic bacteria Bradyrhizobium sp. strain ORS278
nitrilase, locus BRADO0581 29Met Ser Asn Asp Arg Ser Phe Thr Ala Ala Met
Val Gln Met Arg Thr 1 5 10
15 Ala Leu Leu Pro Glu Pro Ser Leu Glu Gln Gly Thr Arg Leu Ile Arg
20 25 30 Glu Ala Val
Ala Gln Gly Ala Gln Tyr Val Gln Thr Pro Glu Val Ser 35
40 45 Asn Met Met Gln Leu Asn Arg Thr
Ala Leu Phe Glu Gln Leu Lys Ser 50 55
60 Glu Glu Glu Asp Pro Ser Leu Lys Ala Tyr Arg Ala Leu
Ala Lys Glu65 70 75 80
Leu Asn Ile His Leu His Ile Gly Ser Leu Ala Leu Arg Phe Ser Ala
85 90 95 Glu Lys Ala Val Asn
Arg Ser Phe Leu Ile Gly Pro Asp Gly Gln Val 100
105 110 Leu Ala Ser Tyr Asp Lys Ile His Met Phe
Asp Ile Asp Leu Pro Gly 115 120
125 Gly Glu Ser Tyr Arg Glu Ser Ala Asn Tyr Gln Pro Gly Glu
Thr Ala 130 135 140
Val Ile Ser Asp Leu Pro Trp Gly Arg Leu Gly Leu Thr Ile Cys Tyr145
150 155 160 Asp Val Arg Phe Pro
Ala Leu Tyr Arg Ala Leu Ala Glu Ser Gly Ala 165
170 175 Ser Phe Ile Ser Val Pro Ser Ala Phe Thr
Arg Lys Thr Gly Glu Ala 180 185
190 His Trp His Thr Leu Leu Arg Ala Arg Ala Ile Glu Thr Gly Cys
Phe 195 200 205 Val
Phe Ala Ala Ala Gln Cys Gly Leu His Glu Asn Lys Arg Glu Thr 210
215 220 Phe Gly His Ser Leu Ile
Ile Asp Pro Trp Gly Glu Ile Leu Ala Glu225 230
235 240 Gly Gly Val Glu Pro Gly Val Ile Leu Ala Arg
Ile Asp Pro Ser Arg 245 250
255 Val Glu Ser Val Arg Gln Thr Ile Pro Ser Leu Gln His Gly Arg Arg
260 265 270 Phe Gly Ile
Ala Asp Pro Lys Gly Gly Pro Asp Tyr Leu His Leu Val 275
280 285 Arg Gly Ser Ala 290
30290PRTSinorhizobium melilotinitrogen-fixing plant microsymbiont
bacteria Sinorhizobium meliloti strain BL225C nitrilase/cyanide
hydratase and apolipoprotein N-acyltransferase, locus
SinmeBDRAFT_5722 30Met Pro Ser Ser Arg Tyr Phe Trp Phe Leu Trp Gln Phe
Lys Leu Ala 1 5 10 15
Val Cys Gln Leu Ser Ile Cys Ala Asp Lys Glu Gln Asn Ile Arg His
20 25 30 Ala Arg Glu Ala Ile
Gln Thr Ala Ala Asp Gly Gly Ser Lys Leu Val 35 40
45 Leu Leu Pro Glu Met Trp Asn Cys Pro Tyr
Ser Asn Ala Ser Phe Pro 50 55 60
Ile Tyr Ala Glu Asp Ile Asp Ala Gly Asp Ser Pro Ser Ser Lys
Met65 70 75 80 Leu
Ser Asp Met Ala Lys Ser Lys Glu Val Thr Ile Ile Gly Gly Ser
85 90 95 Ile Pro Glu Arg Ser Gly
Asn His Leu Tyr Asn Thr Cys Cys Ile Tyr 100
105 110 Gly Lys Asp Gly Ser Leu Lys Gly Lys His
Arg Lys Val His Leu Phe 115 120
125 Asp Ile Asp Ile Pro Gly Lys Ile Gln Phe Lys Glu Ser Asp
Thr Leu 130 135 140
Thr Pro Gly Asp Lys Tyr Thr Val Val Asp Thr Asp Val Gly Arg Ile145
150 155 160 Gly Val Gly Ile Cys
Tyr Asp Ile Arg Phe Pro Glu Met Ala Met Thr 165
170 175 Tyr Ala Ala Arg Gly Val His Met Ile Cys
Tyr Pro Gly Ala Phe Asn 180 185
190 Met Thr Thr Gly Pro Ala His Trp Glu Leu Leu Gln Lys Ala Arg
Ala 195 200 205 Val
Asp Asn Gln Leu Phe Val Ala Thr Cys Ser Pro Ala Arg Asn Pro 210
215 220 Ser Ala Gly Tyr Val Ala
Trp Gly His Ser Ser Val Ile Gly Pro Phe225 230
235 240 Gly Glu Ile Leu Ala Ser Thr Gly Arg Glu Glu
Ala Ile Phe Tyr Ala 245 250
255 Asp Ile Asp Tyr Ala Gln Ile Lys Glu Arg Arg Met Asn Met Pro Leu
260 265 270 Asp His Gln
Arg Arg Gly Asp Leu Tyr Gln Leu Val Asp Leu Thr Phe 275
280 285 Thr Thr 290
31285PRTSinorhizobium melilotinitrogen-fixing plant microsymbiont
bacteria Sinorhizobium meliloti strain 1021 hydrolase, locus
SMe02442 31Met Thr Phe Lys Ala Ala Ala Val Gln Ile Cys Ser Gly Val Asp
Pro 1 5 10 15 Ala
Gly Asn Ala Glu Thr Met Ala Lys Leu Val Arg Glu Ala Ala Ser 20
25 30 Arg Gly Ala Thr Tyr Val
Gln Thr Pro Glu Met Thr Gly Ala Val Gln 35 40
45 Arg Asp Arg Thr Gly Leu Arg Ser Val Leu Lys
Asp Gly Glu Asn Asp 50 55 60
Val Val Val Arg Glu Ala Ser Arg Leu Ala Arg Glu Leu Gly Ile
Tyr65 70 75 80 Leu
His Val Gly Ser Thr Pro Ile Ala Arg Ala Asp Gly Lys Ile Ala
85 90 95 Asn Arg Gly Phe Leu Phe
Gly Pro Asp Gly Ala Lys Ile Cys Asp Tyr 100
105 110 Asp Lys Ile His Met Phe Asp Val Asp Leu
Glu Asn Gly Glu Ser Trp 115 120
125 Arg Glu Ser Ala Ala Tyr His Pro Gly Asn Thr Ala Arg Thr
Ala Asp 130 135 140
Leu Pro Phe Gly Lys Leu Gly Phe Ser Ile Cys Tyr Asp Val Arg Phe145
150 155 160 Pro Glu Leu Phe Arg
Gln Gln Ala Val Ala Gly Ala Glu Ile Met Ser 165
170 175 Val Pro Ala Ala Phe Thr Arg Gln Thr Gly
Glu Ala His Trp Glu Ile 180 185
190 Leu Leu Arg Ala Arg Ala Ile Glu Asn Gly Leu Phe Val Ile Ala
Ala 195 200 205 Ala
Gln Ala Gly Thr His Glu Asp Gly Arg Glu Thr Phe Gly His Ser 210
215 220 Met Ile Val Asp Pro Trp
Gly Arg Val Leu Ala Glu Ala Gly Ala Thr225 230
235 240 Gly Glu Glu Ile Ile Val Ala Glu Ile Asp Val
Ala Ala Val His Ala 245 250
255 Ala Arg Ala Lys Ile Pro Asn Leu Arg Asn Ala Arg Ser Phe Val Leu
260 265 270 Asp Glu Val
Val Pro Val Gly Lys Gly Gly Ala Ala Ala 275 280
285 32312PRTPhytophthora infestanspotato blight oomycete
Phytophthora infestans strain T30-4 carbon-nitrogen hydrolase,
putative, locus PITG_05539 32Met Leu Gly Arg Thr Ile Arg Ser Gln Ala Arg
His Leu Arg Ser Pro 1 5 10
15 Phe Leu Arg Leu Ser Ser Pro Met Ser Thr Thr Ala Pro Lys Phe Lys
20 25 30 Leu Ala Leu
Cys Gln Ile Ala Val Gly Asp Asp Lys Gln Lys Asn Ile 35
40 45 Ala Thr Ala Thr Ala Ala Val Thr
Glu Ala Ala Gln Asn Ala Ala Gln 50 55
60 Val Val Ser Leu Pro Glu Cys Trp Asn Ser Pro Tyr Ala
Thr Thr Ser65 70 75 80
Phe Pro Gln Tyr Ala Glu Glu Ile Pro Glu Lys Lys Ala Ala Leu Asn
85 90 95 Glu Lys Glu His Pro
Ser Thr Phe Ala Leu Ser Gln Leu Ala Ala Lys 100
105 110 Leu Gln Ile Phe Leu Val Gly Gly Ser Ile
Pro Glu Lys Asp Ala Thr 115 120
125 Gly Lys Val Tyr Asn Thr Ser Val Ile Phe Ser Pro Glu Gly
Glu Ile 130 135 140
Leu Gly Lys His Arg Lys Val His Leu Phe Asp Ile Asp Val Pro Gly145
150 155 160 Lys Ile Thr Phe Lys
Glu Ser Asp Thr Leu Ser Pro Gly Asn Ser Met 165
170 175 Thr Leu Phe Asp Thr Pro Tyr Gly Lys Met
Gly Val Gly Ile Cys Tyr 180 185
190 Asp Ile Arg Phe Pro Glu Leu Ser Met Leu Met Lys Lys Gln Gly
Ala 195 200 205 Lys
Val Leu Leu Phe Pro Gly Ala Phe Asn Leu Thr Thr Gly Pro Ala 210
215 220 His Trp Glu Leu Leu Gln
Arg Ala Arg Ala Val Asp Asn Gln Leu Tyr225 230
235 240 Val Ala Ala Thr Ser Pro Ala Arg Gly Pro Glu
Gly Gly Tyr Gln Ala 245 250
255 Trp Gly His Ser Thr Val Ile Ser Pro Trp Gly Glu Val Val Ala Thr
260 265 270 Cys Gly His
Gly Glu Ser Ile Val Tyr Ala Glu Val Asp Leu Glu Lys 275
280 285 Val Glu Glu Met Arg Arg Asn Ile
Pro Thr Thr Asn Gln Thr Arg Ser 290 295
300 Asp Leu Tyr Glu Leu Val Gln Lys305
310 33276PRTHomo sapienshuman omega-amidase NIT2, Nit protein 2,
nitrilase homolog 2, nitrilase family, member 2 33Met Thr Ser Phe Arg
Leu Ala Leu Ile Gln Leu Gln Ile Ser Ser Ile 1 5
10 15 Lys Ser Asp Asn Val Thr Arg Ala Cys Ser
Phe Ile Arg Glu Ala Ala 20 25
30 Thr Gln Gly Ala Lys Ile Val Ser Leu Pro Glu Cys Phe Asn Ser
Pro 35 40 45 Tyr
Gly Ala Lys Tyr Phe Pro Glu Tyr Ala Glu Lys Ile Pro Gly Glu 50
55 60 Ser Thr Gln Lys Leu Ser
Glu Val Ala Lys Glu Cys Ser Ile Tyr Leu65 70
75 80 Ile Gly Gly Ser Ile Pro Glu Glu Asp Ala Gly
Lys Leu Tyr Asn Thr 85 90
95 Cys Ala Val Phe Gly Pro Asp Gly Thr Leu Leu Ala Lys Tyr Arg Lys
100 105 110 Ile His Leu
Phe Asp Ile Asp Val Pro Gly Lys Ile Thr Phe Gln Glu 115
120 125 Ser Lys Thr Leu Ser Pro Gly Asp
Ser Phe Ser Thr Phe Asp Thr Pro 130 135
140 Tyr Cys Arg Val Gly Leu Gly Ile Cys Tyr Asp Met Arg
Phe Ala Glu145 150 155
160 Leu Ala Gln Ile Tyr Ala Gln Arg Gly Cys Gln Leu Leu Val Tyr Pro
165 170 175 Gly Ala Phe Asn
Leu Thr Thr Gly Pro Ala His Trp Glu Leu Leu Gln 180
185 190 Arg Ser Arg Ala Val Asp Asn Gln Val
Tyr Val Ala Thr Ala Ser Pro 195 200
205 Ala Arg Asp Asp Lys Ala Ser Tyr Val Ala Trp Gly His Ser
Thr Val 210 215 220
Val Asn Pro Trp Gly Glu Val Leu Ala Lys Ala Gly Thr Glu Glu Ala225
230 235 240 Ile Val Tyr Ser Asp
Ile Asp Leu Lys Lys Leu Ala Glu Ile Arg Gln 245
250 255 Gln Ile Pro Val Phe Arg Gln Lys Arg Ser
Asp Leu Tyr Ala Val Glu 260 265
270 Met Lys Lys Pro 275 34314PRTEquus
caballusthoroughbred horse predicted omega-amidase NIT2-like,
LOC100072286 34Met Ala Ala His Ser Ile Leu Asp Leu Ser Gly Leu Asp Arg
Glu Ser 1 5 10 15
Gln Ile Asp Leu Gln Arg Pro Leu Lys Ala Arg Pro Gly Lys Ala Lys
20 25 30 Asp Leu Ser Ser Gly
Ser Ala Cys Thr Phe Arg Leu Ala Leu Ile Gln 35 40
45 Leu Gln Val Ser Ser Val Lys Ser Asp Asn
Leu Thr Arg Ala Cys Gly 50 55 60
Leu Val Arg Glu Ala Ala Ala Gln Gly Ala Lys Ile Val Cys Leu
Pro65 70 75 80 Glu
Cys Phe Asn Ser Pro Tyr Gly Thr Asn Tyr Phe Pro Gln Tyr Ala
85 90 95 Glu Lys Ile Pro Gly Glu
Ser Thr Gln Lys Leu Ser Glu Val Ala Lys 100
105 110 Glu Cys Ser Ile Tyr Leu Ile Gly Gly Ser
Ile Pro Glu Glu Asp Ala 115 120
125 Gly Lys Leu Tyr Asn Thr Cys Ala Val Phe Gly Pro Asp Gly
Ala Leu 130 135 140
Leu Val Lys His Arg Lys Leu His Leu Phe Asp Ile Asp Val Pro Gly145
150 155 160 Lys Ile Thr Phe Gln
Glu Ser Lys Thr Leu Ser Pro Gly Asp Ser Phe 165
170 175 Ser Thr Phe Asp Thr Pro Tyr Cys Arg Val
Gly Leu Gly Ile Cys Tyr 180 185
190 Asp Leu Arg Phe Ala Glu Leu Ala Gln Ile Tyr Ala Gln Arg Gly
Cys 195 200 205 Gln
Leu Leu Val Tyr Pro Gly Ala Phe Asn Leu Thr Thr Gly Pro Ala 210
215 220 His Trp Glu Leu Leu Gln
Arg Gly Arg Ala Val Asp Asn Gln Val Tyr225 230
235 240 Val Ala Thr Ala Ser Pro Ala Arg Asp Asp Lys
Ala Ser Tyr Val Ala 245 250
255 Trp Gly His Ser Thr Val Val Thr Pro Trp Gly Glu Val Leu Ala Thr
260 265 270 Ala Gly Thr
Glu Glu Met Ile Val Tyr Ser Asp Ile Asp Leu Lys Lys 275
280 285 Leu Ala Glu Ile Arg Gln Gln Ile
Pro Ile Phe Ser Gln Lys Arg Leu 290 295
300 Asp Leu Tyr Ala Val Glu Ala Lys Lys Pro305
310 35276PRTXenopus tropicaliswestern clawed frog
omega-amilase NIT2, nitrilase homolog 2, nitrilase family, member 2
35Met Ala Lys Phe Arg Leu Ser Leu Val Gln Phe Leu Val Ser Pro Val 1
5 10 15 Lys Ser Glu Asn
Leu Asn Arg Ala Cys Lys Leu Ile Lys Glu Ala Ala 20
25 30 Gln Lys Gly Ala Gln Ile Val Ala Leu
Pro Glu Cys Phe Asn Ser Pro 35 40
45 Tyr Gly Thr Lys Tyr Phe Pro Glu Tyr Ala Glu Lys Ile Pro
Gly Glu 50 55 60
Ser Thr Glu Arg Leu Ser Gln Val Ala Lys Glu Cys Gly Ile Tyr Leu65
70 75 80 Ile Gly Gly Ser Ile
Pro Glu Glu Asp Ser Gly Lys Leu Tyr Asn Thr 85
90 95 Cys Ala Val Phe Gly Pro Asp Gly Thr Leu
Leu Val Lys His Arg Lys 100 105
110 Ile His Leu Phe Asp Ile Asp Val Pro Gly Lys Ile Arg Phe Gln
Glu 115 120 125 Ser
Glu Thr Leu Ser Pro Gly Asp Ser Phe Ser Val Phe Glu Thr Pro 130
135 140 Tyr Cys Lys Val Gly Val
Gly Ile Cys Tyr Asp Ile Arg Phe Ala Glu145 150
155 160 Leu Ala Gln Leu Tyr Ser Lys Lys Gly Cys Gln
Leu Leu Val Tyr Pro 165 170
175 Gly Ala Phe Asn Met Thr Thr Gly Pro Ala His Trp Glu Leu Leu Gln
180 185 190 Arg Ala Arg
Ala Leu Asp Asn Gln Val Tyr Val Ala Thr Ala Ser Pro 195
200 205 Ala Arg Asp Glu Lys Ala Ser Tyr
Val Ala Trp Gly His Ser Thr Ile 210 215
220 Val Ser Pro Trp Gly Glu Val Ile Ala Lys Ala Gly Ser
Glu Glu Thr225 230 235
240 Val Ile Ser Ala Asp Ile Asp Leu Glu Tyr Leu Ala Glu Ile Arg Glu
245 250 255 Gln Ile Pro Ile
Arg Arg Gln Arg Arg His Asp Leu Tyr Ser Val Glu 260
265 270 Glu Lys Lys Asn 275
36277PRTDanio reriozebrafish Nit protein 2, NIT2 36Met Ser Lys Phe Arg
Leu Ala Val Val Gln Leu His Val Ser Lys Ile 1 5
10 15 Lys Ala Asp Asn Leu Gly Arg Ala Gln Thr
Leu Val Thr Glu Ala Ala 20 25
30 Gly Gln Gly Ala Lys Val Val Val Leu Pro Glu Cys Phe Asn Ser
Pro 35 40 45 Tyr
Gly Thr Gly Phe Phe Lys Glu Tyr Ala Glu Lys Ile Pro Gly Glu 50
55 60 Ser Thr Gln Val Leu Ser
Glu Thr Ala Lys Lys Cys Gly Ile Tyr Leu65 70
75 80 Val Gly Gly Ser Ile Pro Glu Glu Asp Gly Gly
Lys Leu Tyr Asn Thr 85 90
95 Cys Ser Val Phe Gly Pro Asp Gly Thr Leu Leu Val Thr His Arg Lys
100 105 110 Ile His Leu
Phe Asp Ile Asp Val Pro Gly Lys Ile Arg Phe Gln Glu 115
120 125 Ser Glu Thr Leu Ser Pro Gly Lys
Ser Leu Ser Met Phe Glu Thr Pro 130 135
140 Tyr Cys Lys Val Gly Val Gly Ile Cys Tyr Asp Ile Arg
Phe Ala Glu145 150 155
160 Leu Ala Gln Ile Tyr Ala Lys Lys Gly Cys Gln Leu Leu Val Tyr Pro
165 170 175 Gly Ala Phe Asn
Met Thr Thr Gly Pro Ala His Trp Glu Leu Leu Gln 180
185 190 Arg Gly Arg Ala Val Asp Asn Gln Val
Tyr Val Ala Thr Ala Ser Pro 195 200
205 Ala Arg Asp Glu Thr Ala Ser Tyr Val Ala Trp Gly His Ser
Ser Val 210 215 220
Ile Asn Pro Trp Gly Glu Val Ile Ser Lys Ala Gly Ser Glu Glu Ser225
230 235 240 Val Val Tyr Ala Asp
Ile Asp Leu Gln Tyr Leu Ala Asp Val Arg Gln 245
250 255 Gln Ile Pro Ile Thr Lys Gln Arg Arg Asn
Asp Leu Tyr Ser Val Asn 260 265
270 Ser Val Gln Glu Gly 275 37279PRTNematostella
vectensisstarlet sea anemone strain CH2 x CH6 predicted protein,
locus NEMVEDRAFT_v1g139747 37Met Ala Val Pro Ile Leu Val Phe Arg Ile Gly
Leu Val Gln Leu Ala 1 5 10
15 Val Thr Ala Asn Lys Leu Gln Asn Leu Gln Arg Ala Arg Glu Lys Ile
20 25 30 Lys Glu Ala
Val Ala Ala Gly Ala Lys Ile Val Ala Leu Pro Glu Cys 35
40 45 Phe Asn Ser Pro Tyr Gly Thr Gln
Tyr Phe Lys Asp Tyr Ala Glu Glu 50 55
60 Ile Pro Gly Glu Ser Ser Asn Met Leu Ala Glu Val Ala
Lys Glu Thr65 70 75 80
Gly Ala Tyr Ile Val Gly Gly Ser Ile Pro Glu Arg Ala Ser Asn Gly
85 90 95 Lys Leu Tyr Asn Thr
Ser Leu Ser Tyr Asp Pro Ser Gly Asn Leu Met 100
105 110 Gly Lys His Arg Lys Ile His Leu Phe Asp
Ile Asp Val Pro Gly Lys 115 120
125 Ile Arg Phe Gln Glu Ser Glu Val Leu Ser Pro Gly Glu Asn
Leu Thr 130 135 140
Ile Leu Asp Thr Glu Tyr Cys Lys Ile Gly Ile Gly Ile Cys Tyr Asp145
150 155 160 Met Arg Phe Pro Glu
Leu Ala Gln Leu Tyr Ala Lys Lys Gly Cys His 165
170 175 Leu Leu Leu Tyr Pro Gly Ala Phe Asn Met
Thr Thr Gly Pro Ala His 180 185
190 Trp Glu Leu Leu Thr Arg Ala Arg Ala Leu Asp Asn Gln Leu Tyr
Val 195 200 205 Ala
Thr Ile Ser Pro Ala Arg Asp Asp Asn Ala Thr Tyr Ile Ala Trp 210
215 220 Gly His Ser Thr Val Val
Asn Pro Trp Gly Lys Ile Val Ser Lys Ala225 230
235 240 Asp His Thr Glu Gln Ile Leu Tyr Ala Glu Ile
Asp Leu Lys Tyr Leu 245 250
255 Asn Glu Val Arg Ser Gln Ile Pro Val Gln Phe Gln Lys Arg Asp Asp
260 265 270 Val Tyr Glu
Leu Gln Val Lys 275 38276PRTMus musculushouse
mouse omega-amidase NIT2, 1190017B19Rik, D16Ertd502e 38Met Ser Thr
Phe Arg Leu Ala Leu Ile Gln Leu Gln Val Ser Ser Ile 1 5
10 15 Lys Ser Asp Asn Leu Thr Arg Ala
Cys Ser Leu Val Arg Glu Ala Ala 20 25
30 Lys Gln Gly Ala Asn Ile Val Ser Leu Pro Glu Cys Phe
Asn Ser Pro 35 40 45
Tyr Gly Thr Thr Tyr Phe Pro Asp Tyr Ala Glu Lys Ile Pro Gly Glu 50
55 60 Ser Thr Gln Lys Leu
Ser Glu Val Ala Lys Glu Ser Ser Ile Tyr Leu65 70
75 80 Ile Gly Gly Ser Ile Pro Glu Glu Asp Ala
Gly Lys Leu Tyr Asn Thr 85 90
95 Cys Ser Val Phe Gly Pro Asp Gly Ser Leu Leu Val Lys His Arg
Lys 100 105 110 Ile
His Leu Phe Asp Ile Asp Val Pro Gly Lys Ile Thr Phe Gln Glu 115
120 125 Ser Lys Thr Leu Ser Pro
Gly Asp Ser Phe Ser Thr Phe Asp Thr Pro 130 135
140 Tyr Cys Lys Val Gly Leu Gly Ile Cys Tyr Asp
Met Arg Phe Ala Glu145 150 155
160 Leu Ala Gln Ile Tyr Ala Gln Arg Gly Cys Gln Leu Leu Val Tyr Pro
165 170 175 Gly Ala Phe
Asn Leu Thr Thr Gly Pro Ala His Trp Glu Leu Leu Gln 180
185 190 Arg Ala Arg Ala Val Asp Asn Gln
Val Tyr Val Ala Thr Ala Ser Pro 195 200
205 Ala Arg Asp Asp Lys Ala Ser Tyr Val Ala Trp Gly His
Ser Thr Val 210 215 220
Val Asp Pro Trp Gly Gln Val Leu Thr Lys Ala Gly Thr Glu Glu Thr225
230 235 240 Ile Leu Tyr Ser Asp
Ile Asp Leu Lys Lys Leu Ala Glu Ile Arg Gln 245
250 255 Gln Ile Pro Ile Leu Lys Gln Lys Arg Ala
Asp Leu Tyr Thr Val Glu 260 265
270 Ser Lys Lys Pro 275 3910798DNAArtificial
Sequencesynthetic pTF101.1 vector + roID-02 promoter + Arabidopsis
omega-amidase (codon optimized) + nos terminator region construct
39agtactttaa agtactttaa agtactttaa agtactttga tccaacccct ccgctgctat
60agtgcagtcg gcttctgacg ttcagtgcag ccgtcttctg aaaacgacat gtcgcacaag
120tcctaagtta cgcgacaggc tgccgccctg cccttttcct ggcgttttct tgtcgcgtgt
180tttagtcgca taaagtagaa tacttgcgac tagaaccgga gacattacgc catgaacaag
240agcgccgccg ctggcctgct gggctatgcc cgcgtcagca ccgacgacca ggacttgacc
300aaccaacggg ccgaactgca cgcggccggc tgcaccaagc tgttttccga gaagatcacc
360ggcaccaggc gcgaccgccc ggagctggcc aggatgcttg accacctacg ccctggcgac
420gttgtgacag tgaccaggct agaccgcctg gcccgcagca cccgcgacct actggacatt
480gccgagcgca tccaggaggc cggcgcgggc ctgcgtagcc tggcagagcc gtgggccgac
540accaccacgc cggccggccg catggtgttg accgtgttcg ccggcattgc cgagttcgag
600cgttccctaa tcatcgaccg cacccggagc gggcgcgagg ccgccaaggc ccgaggcgtg
660aagtttggcc cccgccctac cctcaccccg gcacagatcg cgcacgcccg cgagctgatc
720gaccaggaag gccgcaccgt gaaagaggcg gctgcactgc ttggcgtgca tcgctcgacc
780ctgtaccgcg cacttgagcg cagcgaggaa gtgacgccca ccgaggccag gcggcgcggt
840gccttccgtg aggacgcatt gaccgaggcc gacgccctgg cggccgccga gaatgaacgc
900caagaggaac aagcatgaaa ccgcaccagg acggccagga cgaaccgttt ttcattaccg
960aagagatcga ggcggagatg atcgcggccg ggtacgtgtt cgagccgccc gcgcacgtct
1020caaccgtgcg gctgcatgaa atcctggccg gtttgtctga tgccaagctg gcggcctggc
1080cggccagctt ggccgctgaa gaaaccgagc gccgccgtct aaaaaggtga tgtgtatttg
1140agtaaaacag cttgcgtcat gcggtcgctg cgtatatgat gcgatgagta aataaacaaa
1200tacgcaaggg gaacgcatga aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa
1260gacgaccatc gcaacccatc tagcccgcgc cctgcaactc gccggggccg atgttctgtt
1320agtcgattcc gatccccagg gcagtgcccg cgattgggcg gccgtgcggg aagatcaacc
1380gctaaccgtt gtcggcatcg accgcccgac gattgaccgc gacgtgaagg ccatcggccg
1440gcgcgacttc gtagtgatcg acggagcgcc ccaggcggcg gacttggctg tgtccgcgat
1500caaggcagcc gacttcgtgc tgattccggt gcagccaagc ccttacgaca tatgggccac
1560cgccgacctg gtggagctgg ttaagcagcg cattgaggtc acggatggaa ggctacaagc
1620ggcctttgtc gtgtcgcggg cgatcaaagg cacgcgcatc ggcggtgagg ttgccgaggc
1680gctggccggg tacgagctgc ccattcttga gtcccgtatc acgcagcgcg tgagctaccc
1740aggcactgcc gccgccggca caaccgttct tgaatcagaa cccgagggcg acgctgcccg
1800cgaggtccag gcgctggccg ctgaaattaa atcaaaactc atttgagtta atgaggtaaa
1860gagaaaatga gcaaaagcac aaacacgcta agtgccggcc gtccgagcgc acgcagcagc
1920aaggctgcaa cgttggccag cctggcagac acgccagcca tgaagcgggt caactttcag
1980ttgccggcgg aggatcacac caagctgaag atgtacgcgg tacgccaagg caagaccatt
2040accgagctgc tatctgaata catcgcgcag ctaccagagt aaatgagcaa atgaataaat
2100gagtagatga attttagcgg ctaaaggagg cggcatggaa aatcaagaac aaccaggcac
2160cgacgccgtg gaatgcccca tgtgtggagg aacgggcggt tggccaggcg taagcggctg
2220ggttgtctgc cggccctgca atggcactgg aacccccaag cccgaggaat cggcgtgacg
2280gtcgcaaacc atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga
2340agttgaaggc cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg
2400aatcgtggca agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg
2460gtgcgccgtc gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga
2520tgctctatga cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc
2580tgtcgaagcg tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg
2640tagaggtttc cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga
2700tggcggtttc ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc
2760ccggccgcgt gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg
2820gcggaaagca gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg
2880ccatgcagcg tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag
2940ccttgattag ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga
3000tcgagctagc tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga
3060cggttcaccc cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg
3120cacgccgcgc cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca
3180gtggcagcgc cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa
3240atgacctgcc ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca
3300tgcgctaccg caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga
3360tgctagggca aattgcccta gcaggggaaa aaggtcgaaa aggtctcttt cctgtggata
3420gcacgtacat tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc
3480caaagccgta cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag
3540gcgatttttc cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct
3600gtgcataact gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt
3660cgctgcgctc cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa
3720aaatggctgg cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac
3780tcgaccgccg gcgcccacat caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa
3840aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
3900agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg
3960acccagtcac gtagcgatag cggagtgtat actggcttaa ctatgcggca tcagagcaga
4020ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta aggagaaaat
4080accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
4140tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
4200ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
4260ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
4320gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
4380gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
4440ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
4500tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
4560gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
4620tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
4680tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc
4740tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
4800ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
4860ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
4920gttaagggat tttggtcatg catgatatat ctcccaattt gtgtagggct tattatgcac
4980gcttaaaaat aataaaagca gacttgacct gatagtttgg ctgtgagcaa ttatgtgctt
5040agtgcatcta atcgcttgag ttaacgccgg cgaagcggcg tcggcttgaa cgaatttcta
5100gctagacatt atttgccgac taccttggtg atctcgcctt tcacgtagtg gacaaattct
5160tccaactgat ctgcgcgcga ggccaagcga tcttcttctt gtccaagata agcctgtcta
5220gcttcaagta tgacgggctg atactgggcc ggcaggcgct ccattgccca gtcggcagcg
5280acatccttcg gcgcgatttt gccggttact gcgctgtacc aaatgcggga caacgtaagc
5340actacatttc gctcatcgcc agcccagtcg ggcggcgagt tccatagcgt taaggtttca
5400tttagcgcct caaatagatc ctgttcagga accggatcaa agagttcctc cgccgctgga
5460cctaccaagg caacgctatg ttctcttgct tttgtcagca agatagccag atcaatgtcg
5520atcgtggctg gctcgaagat acctgcaaga atgtcattgc gctgccattc tccaaattgc
5580agttcgcgct tagctggata acgccacgga atgatgtcgt cgtgcacaac aatggtgact
5640tctacagcgc ggagaatctc gctctctcca ggggaagccg aagtttccaa aaggtcgttg
5700atcaaagctc gccgcgttgt ttcatcaagc cttacggtca ccgtaaccag caaatcaata
5760tcactgtgtg gcttcaggcc gccatccact gcggagccgt acaaatgtac ggccagcaac
5820gtcggttcga gatggcgctc gatgacgcca actacctctg atagttgagt cgatacttcg
5880gcgatcaccg cttcccccat gatgtttaac tttgttttag ggcgactgcc ctgctgcgta
5940acatcgttgc tgctccataa catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg
6000gatgcccgag gcatagactg taccccaaaa aaacatgtca taacaagaag ccatgaaaac
6060cgccactgcg ccgttaccac cgctgcgttc ggtcaaggtt ctggaccagt tgcgtgacgg
6120cagttacgct acttgcatta cagcttacga accgaacgag gcttatgtcc actgggttcg
6180tgcccgaatt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca tgctaccctc
6240cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg aatagcatcg
6300gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg tcccggactg
6360atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg agctgttggc
6420tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa cttaataaca
6480cattgcggac gtttttaatg tactgaatta acgccgaatt gctctagcat tcgccattca
6540ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg
6600cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac
6660gacgttgtaa aacgacggcc agtgccaagc taattcttca agacgtgctc aaatcactat
6720ttccacaccc ctatatttct attgcactcc cttttaactg ttttttatta caaaaatgcc
6780ctggaaaatg cactcccttt ttgtgtttgt ttttttgtga aacgatgttg tcaggtaatt
6840tatttgtcag tctactatgg tggcccatta tattaatagc aactgtcggt ccaatagacg
6900acgtcgattt tctgcatttg tttaaccacg tggattttat gacattttat attagttaat
6960ttgtaaaacc tacccaatta aagacctcat atgttctaaa gactaatact taatgataac
7020aattttcttt tagtgaagaa agggataatt agtaaatatg gaacaagggc agaagattta
7080ttaaagccgc gtaagagaca acaagtaggt acgtggagtg tcttaggtga cttacccaca
7140taacataaag tgacattaac aaacatagct aatgctccta tttgaatagt gcatatcagc
7200ataccttatt acatatagat aggagcaaac tctagctaga ttgttgagag cagatctcgg
7260tgacgggcag gaccggacgg ggcggtaccg gcaggctgaa gtccagctgc cagaaaccca
7320cgtcatgcca gttcccgtgc ttgaagccgg ccgcccgcag catgccgcgg ggggcatatc
7380cgagcgcctc gtgcatgcgc acgctcgggt cgttgggcag cccgatgaca gcgaccacgc
7440tcttgaagcc ctgtgcctcc agggacttca gcaggtgggt gtagagcgtg gagcccagtc
7500ccgtccgctg gtggcggggg gagacgtaca cggtcgactc ggccgtccag tcgtaggcgt
7560tgcgtgcctt ccaggggccc gcgtaggcga tgccggcgac ctcgccgtcc acctcggcga
7620cgagccaggg atagcgctcc cgcagacgga cgaggtcgtc cgtccactcc tgcggttcct
7680gcggctcggt acggaagttg accgtgcttg tctcgatgta gtggttgacg atggtgcaga
7740ccgccggcat gtccgcctcg gtggcacggc ggatgtcggc cgggcgtcgt tctgggctca
7800tggtagatcc cccgttcgta aatggtgaaa attttcagaa aattgctttt gctttaaaag
7860aaatgattta aattgctgca atagaagtag aatgcttgat tgcttgagat tcgtttgttt
7920tgtatatgtt gtgttgagaa ttaattctcg aggtcctctc caaatgaaat gaacttcctt
7980atatagagga agggtcttgc gaaggatagt gggattgtgc gtcatccctt acgtcagtgg
8040agatatcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga
8100tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggtagaggca tcttgaacga
8160tagcctttcc tttatcgcaa tgatggcatt tgtaggagcc accttccttt tccactatct
8220tcacaataaa gtgacagata gctgggcaat ggaatccgag gaggtttccg gatattaccc
8280tttgttgaaa agtctcaatt gccctttggt cttctgagac tgtatctttg atatttttgg
8340agtagacaag tgtgtcgtgc tccaccatgt tatcacatca atccacttgc tttgaagacg
8400tggttggaac gtcttctttt tccacgatgc tcctcgtggg tgggggtcca tctttgggac
8460cactgtcggc agaggcatct tcaacgatgg cctttccttt atcgcaatga tggcatttgt
8520aggagccacc ttccttttcc actatcttca caataaagtg acagatagct gggcaatgga
8580atccgaggag gtttccggat attacccttt gttgaaaagt ctcaattgcc ctttggtctt
8640ctgagactgt atctttgata tttttggagt agacaagtgt gtcgtgctcc accatgttga
8700cctgcaggca tgcaagcttg catgcctgca ggtcgactct agaggatccc cgtcggtacc
8760gaatttgttc gtgaactatt agttgcgggc cttggcatcc gactacctct gcggcaatat
8820tatattccct gggcccaccg tgaacccaat ttcgcctatt tattcattac ccccattaac
8880attgaagtag tcatgatggg cctgcagcac gttggtgagg ctggcacaac tcatccatat
8940actttctgac cggatcggca cattattgta gaaaacgcgg acccacagcg cactttccaa
9000agcggtgccg cgtcagaatg cgctggcaga aaaaaattaa tccaaaagta ccctccaagc
9060agcccatata aacgcgttta caaatccgct aacctcaaca atttgagcag agaaaattcg
9120cacctacaag gcagatggca tcatcattca atccagagca ggcaagagtt ccttcagcat
9180tacctttacc agcaccacca cttaccaaat tcaacatcgg actttgtcaa ttgagtgtta
9240cttctgataa gaaaagaaac atttcacatg ctaagaaagc aatcgaagag gctgctagta
9300agggagctaa actcgttctt ttgcctgaaa tatggaactc accatacagt aacgattctt
9360ttcctgtgta cgcagaagag atcgatgctg gaggtgatgc atctccatca actgctatgc
9420tctcagaagt tagtaagaga ctcaagatta caattatcgg aggttcaatt cctgagagag
9480ttggagatag gttgtataac acatgttgcg tgttcggatc tgatggagag ctcaaggcta
9540agcataggaa gattcacctc ttcgatatag atattcctgg aaagatcacc ttcatggaat
9600caaaaacact taccgctgga gagactccaa caattgttga tacagatgtg ggtagaatcg
9660gaataggtat atgttacgat atcaggttcc aagaattggc tatgatatat gctgcaagag
9720gagcacatct cttatgctac cctggagctt tcaatatgac tacaggtcca ttgcactggg
9780agcttttgca aagagctagg gcaacagata accagctcta tgttgctacc tgctctcctg
9840caagagattc aggagctggt tacaccgcat ggggtcattc tactcttgtt ggaccatttg
9900gtgaagtgtt ggctaccact gagcacgaag aggctattat aatcgcagaa atcgattaca
9960gtatacttga gcagagaagg acttctctcc cattaaatag gcagaggagg ggtgatttat
10020accagttagt tgatgttcag agattagata gtaagtgaca cgtgtgaatt acaggtgacc
10080agctcgaatt tccccgatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct
10140gttgccggtc ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata
10200attaacatgt aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa
10260ttatacattt aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg
10320cgcgcggtgt catctatgtt actagatcgg gggtaccgac gggtaccgag ctcgaattcg
10380taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
10440atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
10500ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
10560taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttggagcttg agcttggatc
10620agattgtcgt ttcccgcctt cagtttaaac tatcagtgtt tgacaggata tattggcggg
10680taaacctaag agaaaagagc gtttattaga ataacggata tttaaaaggg cgtgaaaagg
10740tttatccgtt cgtccatttg tatgtgcatg ccaaccacag ggttcccctc gggatcaa
107984011845DNAArtificial Sequencesynthetic GPT 6c construct; Cambia 2201
with tomato rubisco SSU promoter + GPT (-45) truncated (deleted
chloroplast targeting sequence), codon optimized for Arabidopsis +
nos terminator 40cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
ctggaaagcg 60ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
ccaggcttta 120cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
atttcacaca 180ggaaacagct atgaccatga ttacgaattc gagctcggta cccggggatc
ctctagatct 240agagaattca tcgatgtttg aatcctcctt aaagtttttc tctggagaaa
ctgtagtaat 300tttactttgt tgtgttccct tcatcttttg aattaatggc atttgtttta
atactaatct 360gcttctgaaa cttgtaatgt atgtatatca gtttcttata atttatccaa
gtaatatctt 420ccattctcta tgcaattgcc tgcataagct cgacaaaaga gtacatcaac
ccctcctcct 480ctggactact ctagctaaac ttgaatttcc ccttaagatt atgaaattga
tatatcctta 540acaaacgact ccttctgttg gaaaatgtag tacttgtctt tcttcttttg
ggtatatata 600gtttatatac accatactat gtacaacatc caagtagagt gaaatggata
catgtacaag 660acttatttga ttgattgatg acttgagttg ccttaggagt aacaaattct
taggtcaata 720aatcgttgat ttgaaattaa tctctctgtc ttagacagat aggaattatg
acttccaatg 780gtccagaaag caaagttcgc actgagggta tacttggaat tgagacttgc
acaggtccag 840aaaccaaagt tcccatcgag ctctaaaatc acatctttgg aatgaaattc
aattagagat 900aagttgcttc atagcatagg taaaatggaa gatgtgaagt aacctgcaat
aatcagtgaa 960atgacattaa tacactaaat acttcatatg taattatcct ttccaggtta
acaatactct 1020ataaagtaag aattatcaga aatgggctca tcaaactttt gtactatgta
tttcatataa 1080ggaagtataa ctatacataa gtgtatacac aactttattc ctattttgta
aaggtggaga 1140gactgttttc gatggatcta aagcaatatg tctataaaat gcattgatat
aataattatc 1200tgagaaaatc cagaattggc gttggattat ttcagccaaa tagaagtttg
taccatactt 1260gttgattcct tctaagttaa ggtgaagtat cattcataaa cagttttccc
caaagtacta 1320ctcaccaagt ttccctttgt agaattaaca gttcaaatat atggcgcaga
aattactcta 1380tgcccaaaac caaacgagaa agaaacaaaa tacaggggtt gcagacttta
ttttcgtgtt 1440agggtgtgtt ttttcatgta attaatcaaa aaatattatg acaaaaacat
ttatacatat 1500ttttactcaa cactctgggt atcagggtgg gttgtgttcg acaatcaata
tggaaaggaa 1560gtattttcct tattttttta gttaatattt tcagttatac caaacatacc
ttgtgatatt 1620atttttaaaa atgaaaaact cgtcagaaag aaaaagcaaa agcaacaaaa
aaattgcaag 1680tattttttaa aaaagaaaaa aaaaacatat cttgtttgtc agtatgggaa
gtttgagata 1740aggacgagtg aggggttaaa attcagtggc cattgatttt gtaatgccaa
gaaccacaaa 1800atccaatggt taccattcct gtaagatgag gtttgctaac tctttttgtc
cgttagatag 1860gaagccttat cactatatat acaaggcgtc ctaataacct cttagtaacc
aattatttca 1920gcaactagta tggcgactca aaatgagtca acacaaaagc ctgttcaggt
ggctaagaga 1980cttgagaagt ttaaaactac aattttcact caaatgtcta tcctcgcagt
taagcacgga 2040gctattaatc ttggacaggg ttttcctaac ttcgatggtc cagatttcgt
gaaagaagct 2100gcaattcaag caatcaagga tggaaaaaat cagtatgcta gaggatacgg
tattcctcag 2160ttgaactctg ctatcgctgc aagattcaga gaagatacag gacttgttgt
ggatccagaa 2220aaagaggtta ctgtgacatc aggttgtact gaggctattg ctgcagctat
gctcggactt 2280attaaccctg gagatgaagt tatccttttt gcaccattct atgattctta
cgaggctaca 2340ttgtcaatgg caggagctaa ggtgaaaggt attactctca gacctccaga
tttctctatc 2400cctttggaag agctcaaggc agctgttact aataagacaa gagctatctt
gatgaatact 2460cctcataacc caacaggaaa gatgtttact agagaagagc tcgaaactat
tgcttctctt 2520tgcatcgaga acgatgtttt ggtgttctca gatgaagtgt atgataaact
cgcatttgag 2580atggatcaca tttctatcgc ttcacttcca ggaatgtacg aaagaactgt
tactatgaat 2640tctttgggaa agactttttc tctcacagga tggaaaattg gttgggcaat
cgctcctcca 2700catctcacat ggggtgttag acaagcacac tcttatctta ctttcgcaac
ttcaacacct 2760gctcagtggg cagctgtggc agctcttaag gctccagaat cttacttcaa
ggagttgaag 2820agagattaca acgttaagaa agaaacactt gtgaagggat tgaaagaggt
tggttttaca 2880gtgttccctt cttcaggaac ttactttgtt gtggcagatc atactccatt
cggtatggaa 2940aacgatgttg ctttttgtga gtatcttatt gaagaggttg gagttgtggc
tatccctaca 3000tctgtgtttt accttaatcc agaagaggga aagaatcttg ttagatttgc
attctgcaaa 3060gatgaagaga ctttgagagg tgctattgag aggatgaagc aaaaactcaa
gagaaaagtt 3120tgacacgtgt gaattacagg tgaccagctc gaatttcccc gatcgttcaa
acatttggca 3180ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca
tataatttct 3240gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat
ttatgagatg 3300ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa
acaaaatata 3360gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag
atcgggaatt 3420aaactatcag tgtttgacag gatatattgg cgggtaaacc taagagaaaa
gagcgtttat 3480tagaataacg gatatttaaa agggcgtgaa aaggtttatc cgttcgtcca
tttgtatgtg 3540catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc
ctccgctgct 3600atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac
atgtcgcaca 3660agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt
cttgtcgcgt 3720gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac
gccatgaaca 3780agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac
caggacttga 3840ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc
gagaagatca 3900ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta
cgccctggcg 3960acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac
ctactggaca 4020ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag
ccgtgggccg 4080acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt
gccgagttcg 4140agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag
gcccgaggcg 4200tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc
cgcgagctga 4260tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg
catcgctcga 4320ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc
aggcggcgcg 4380gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc
gagaatgaac 4440gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt
ttttcattac 4500cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc
ccgcgcacgt 4560ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc
tggcggcctg 4620gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt
gatgtgtatt 4680tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag
taaataaaca 4740aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg
cgggtcaggc 4800aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc
cgatgttctg 4860ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg
ggaagatcaa 4920ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa
ggccatcggc 4980cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc
tgtgtccgcg 5040atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga
catatgggcc 5100accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg
aaggctacaa 5160gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga
ggttgccgag 5220gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg
cgtgagctac 5280ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg
cgacgctgcc 5340cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt
taatgaggta 5400aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc
gcacgcagca 5460gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg
gtcaactttc 5520agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa
ggcaagacca 5580ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc
aaatgaataa 5640atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga
acaaccaggc 5700accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg
cgtaagcggc 5760tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga
atcggcgtga 5820cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg
acctggtgga 5880gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag
cacgccccgg 5940tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac
cgccggcagc 6000cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt
ttttcgttcc 6060gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg
ccgttttccg 6120tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc
cagacgggca 6180cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg
acctggtact 6240gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga
agggagacaa 6300gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc
ggcgagccga 6360tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca
ccacgcacgt 6420tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat
ccgagggtga 6480agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg
agtacatcga 6540gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc
cggacgtgct 6600gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc
tctaccgcct 6660ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga
tctacgaacg 6720cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc
tgatcgggtc 6780aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc
cgatcctagt 6840catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat
gtacggagca 6900gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct
ttcctgtgga 6960tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt
acattgggaa 7020cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa
aagagaaaaa 7080aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa
cccgcctggc 7140ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc
ctacccttcg 7200gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg
ctggccgctc 7260aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg
cgccgtcgcc 7320actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt
gatgacggtg 7380aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
gcggatgccg 7440ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
ggcgcagcca 7500tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg
catcagagca 7560gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
taaggagaaa 7620ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
cggtcgttcg 7680gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
cagaatcagg 7740ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
accgtaaaaa 7800ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
acaaaaatcg 7860acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
cgtttccccc 7920tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
acctgtccgc 7980ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
atctcagttc 8040ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
agcccgaccg 8100ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
acttatcgcc 8160actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
gtgctacaga 8220gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
gtatctgcgc 8280tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
gcaaacaaac 8340caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
gaaaaaaagg 8400atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
acgaaaactc 8460acgttaaggg attttggtca tgcatgatat atctcccaat ttgtgtaggg
cttattatgc 8520acgcttaaaa ataataaaag cagacttgac ctgatagttt ggctgtgagc
aattatgtgc 8580ttagtgcatc taatcgcttg agttaacgcc ggcgaagcgg cgtcggcttg
aacgaatttc 8640tagctagagg atcgcaccaa taactgcctt aaaaaaatta cgccccgccc
tgccactcat 8700cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca
caaacggcat 8760gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa
tatttgccca 8820ttgtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca
aaactggtga 8880aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct
ttagggaaat 8940aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga
aactgccgga 9000aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca
tggaaaacgg 9060tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt
gccatacgga 9120actccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga
taaaacttgt 9180gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg
gtctggttat 9240aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc
cattgggata 9300tatcaacggt ggtatatcca gtgatttttt tctccatgat gtttaacttt
gttttagggc 9360gactgccctg ctgcgtaaca tcgttgctgc tccataacat caaacatcga
cccacggcgt 9420aacgcgcttg ctgcttggat gcccgaggca tagactgtac cccaaaaaaa
catgtcataa 9480caagaagcca tgaaaaccgc cactgcgccg ttaccaccgc tgcgttcggt
caaggttctg 9540gaccagttgc gtgacggcag ttacgctact tgcattacag cttacgaacc
gaacgaggct 9600tatgtccact gggttcgtgc ccgaattgat cacaggcagc aacgctctgt
catcgttaca 9660atcaacatgc taccctccgc gagatcatcc gtgtttcaaa cccggcagct
tagttgccgt 9720tcttccgaat agcatcggta acatgagcaa agtctgccgc cttacaacgg
ctctcccgct 9780gacgccgtcc cggactgatg ggctgcctgt atcgagtggt gattttgtgc
cgagctgccg 9840gtcggggagc tgttggctgg ctggtggcag gatatattgt ggtgtaaaca
aattgacgct 9900tagacaactt aataacacat tgcggacgtt tttaatgtac tgaattaacg
ccgaattaat 9960tcgggggatc tggattttag tactggattt tggttttagg aattagaaat
tttattgata 10020gaagtatttt acaaatacaa atacatacta agggtttctt atatgctcaa
cacatgagcg 10080aaaccctata ggaaccctaa ttcccttatc tgggaactac tcacacatta
ttatggagaa 10140actcgagctt gtcgatcgac tctagctaga ggatcgatcc gaaccccaga
gtcccgctca 10200gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag
cggcgatacc 10260gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa
tatcacgggt 10320agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt
cgatgaatcc 10380agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat
gtgtcacgac 10440gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg
ctggcgcgag 10500cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca
tccgagtacg 10560tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg
gatcaagcgt 10620atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag
caaggtgaga 10680tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc
ccgcttcagt 10740gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg
atagccgcgc 10800tgcctcgtcc tggagttcat tcagggcacc ggacaggtcg gtcttgacaa
aaagaaccgg 10860gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg
tctgttgtgc 10920ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt
gcaatccatc 10980ttgttcaatc cccatggtcg atcgacagat ctgcgaaagc tcgagagaga
tagatttgta 11040gagagagact ggtgatttca gcgtgtcctc tccaaatgaa atgaacttcc
ttatatagag 11100gaaggtcttg cgaaggatag tgggattgtg cgtcatccct tacgtcagtg
gagatatcac 11160atcaatccac ttgctttgaa gacgtggttg gaacgtcttc tttttccacg
atgctcctcg 11220tgggtggggg tccatctttg ggaccactgt cggcagaggc atcttgaacg
atagcctttc 11280ctttatcgca atgatggcat ttgtaggtgc caccttcctt ttctactgtc
cttttgatga 11340agtgacagat agctgggcaa tggaatccga ggaggtttcc cgatattacc
ctttgttgaa 11400aagtctcaat agccctttgg tcttctgaga ctgtatcttt gatattcttg
gagtagacga 11460gagtgtcgtg ctccaccatg ttatcacatc aatccacttg ctttgaagac
gtggttggaa 11520cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc atctttggga
ccactgtcgg 11580cagaggcatc ttgaacgata gcctttcctt tatcgcaatg atggcatttg
taggtgccac 11640cttccttttc tactgtcctt ttgatgaagt gacagatagc tgggcaatgg
aatccgagga 11700ggtttcccga tattaccctt tgttgaaaag tctcaatagc cctttggtct
tctgagactg 11760tatctttgat attcttggag tagacgagag tgtcgtgctc caccatgttg
gcaagctgct 11820ctagccaata cgcaaaccgc ctctc
118454111845DNAArtificial Sequencesynthetic GPT 9c construct;
Cambia 2201 + tomato rubisco SSU promoter + GPT (-45) truncated
(deleted chloroplast targeting sequence) FV mutation codon optimized
for Arabidopsis + nos terminator; GPT transgene expression vector
construct (9c) 41cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga
ctggaaagcg 60ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
ccaggcttta 120cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
atttcacaca 180ggaaacagct atgaccatga ttacgaattc gagctcggta cccggggatc
ctctagatct 240agagaattca tcgatgtttg aatcctcctt aaagtttttc tctggagaaa
ctgtagtaat 300tttactttgt tgtgttccct tcatcttttg aattaatggc atttgtttta
atactaatct 360gcttctgaaa cttgtaatgt atgtatatca gtttcttata atttatccaa
gtaatatctt 420ccattctcta tgcaattgcc tgcataagct cgacaaaaga gtacatcaac
ccctcctcct 480ctggactact ctagctaaac ttgaatttcc ccttaagatt atgaaattga
tatatcctta 540acaaacgact ccttctgttg gaaaatgtag tacttgtctt tcttcttttg
ggtatatata 600gtttatatac accatactat gtacaacatc caagtagagt gaaatggata
catgtacaag 660acttatttga ttgattgatg acttgagttg ccttaggagt aacaaattct
taggtcaata 720aatcgttgat ttgaaattaa tctctctgtc ttagacagat aggaattatg
acttccaatg 780gtccagaaag caaagttcgc actgagggta tacttggaat tgagacttgc
acaggtccag 840aaaccaaagt tcccatcgag ctctaaaatc acatctttgg aatgaaattc
aattagagat 900aagttgcttc atagcatagg taaaatggaa gatgtgaagt aacctgcaat
aatcagtgaa 960atgacattaa tacactaaat acttcatatg taattatcct ttccaggtta
acaatactct 1020ataaagtaag aattatcaga aatgggctca tcaaactttt gtactatgta
tttcatataa 1080ggaagtataa ctatacataa gtgtatacac aactttattc ctattttgta
aaggtggaga 1140gactgttttc gatggatcta aagcaatatg tctataaaat gcattgatat
aataattatc 1200tgagaaaatc cagaattggc gttggattat ttcagccaaa tagaagtttg
taccatactt 1260gttgattcct tctaagttaa ggtgaagtat cattcataaa cagttttccc
caaagtacta 1320ctcaccaagt ttccctttgt agaattaaca gttcaaatat atggcgcaga
aattactcta 1380tgcccaaaac caaacgagaa agaaacaaaa tacaggggtt gcagacttta
ttttcgtgtt 1440agggtgtgtt ttttcatgta attaatcaaa aaatattatg acaaaaacat
ttatacatat 1500ttttactcaa cactctgggt atcagggtgg gttgtgttcg acaatcaata
tggaaaggaa 1560gtattttcct tattttttta gttaatattt tcagttatac caaacatacc
ttgtgatatt 1620atttttaaaa atgaaaaact cgtcagaaag aaaaagcaaa agcaacaaaa
aaattgcaag 1680tattttttaa aaaagaaaaa aaaaacatat cttgtttgtc agtatgggaa
gtttgagata 1740aggacgagtg aggggttaaa attcagtggc cattgatttt gtaatgccaa
gaaccacaaa 1800atccaatggt taccattcct gtaagatgag gtttgctaac tctttttgtc
cgttagatag 1860gaagccttat cactatatat acaaggcgtc ctaataacct cttagtaacc
aattatttca 1920gcaactagta tggcgactca aaatgagtca acacaaaagc ctgttcaggt
ggctaagaga 1980cttgagaagt ttaaaactac aattttcact caaatgtcta tcctcgcagt
taagcacgga 2040gctattaatc ttggacaggg tgttcctaac ttcgatggtc cagatttcgt
gaaagaagct 2100gcaattcaag caatcaagga tggaaaaaat cagtatgcta gaggatacgg
tattcctcag 2160ttgaactctg ctatcgctgc aagattcaga gaagatacag gacttgttgt
ggatccagaa 2220aaagaggtta ctgtgacatc aggttgtact gaggctattg ctgcagctat
gctcggactt 2280attaaccctg gagatgaagt tatccttttt gcaccattct atgattctta
cgaggctaca 2340ttgtcaatgg caggagctaa ggtgaaaggt attactctca gacctccaga
tttctctatc 2400cctttggaag agctcaaggc agctgttact aataagacaa gagctatctt
gatgaatact 2460cctcataacc caacaggaaa gatgtttact agagaagagc tcgaaactat
tgcttctctt 2520tgcatcgaga acgatgtttt ggtgttctca gatgaagtgt atgataaact
cgcatttgag 2580atggatcaca tttctatcgc ttcacttcca ggaatgtacg aaagaactgt
tactatgaat 2640tctttgggaa agactttttc tctcacagga tggaaaattg gttgggcaat
cgctcctcca 2700catctcacat ggggtgttag acaagcacac tcttatctta ctttcgcaac
ttcaacacct 2760gctcagtggg cagctgtggc agctcttaag gctccagaat cttacttcaa
ggagttgaag 2820agagattaca acgttaagaa agaaacactt gtgaagggat tgaaagaggt
tggttttaca 2880gtgttccctt cttcaggaac ttactttgtt gtggcagatc atactccatt
cggtatggaa 2940aacgatgttg ctttttgtga gtatcttatt gaagaggttg gagttgtggc
tatccctaca 3000tctgtgtttt accttaatcc agaagaggga aagaatcttg ttagatttgc
attctgcaaa 3060gatgaagaga ctttgagagg tgctattgag aggatgaagc aaaaactcaa
gagaaaagtt 3120tgacacgtgt gaattacagg tgaccagctc gaatttcccc gatcgttcaa
acatttggca 3180ataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca
tataatttct 3240gttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat
ttatgagatg 3300ggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa
acaaaatata 3360gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag
atcgggaatt 3420aaactatcag tgtttgacag gatatattgg cgggtaaacc taagagaaaa
gagcgtttat 3480tagaataacg gatatttaaa agggcgtgaa aaggtttatc cgttcgtcca
tttgtatgtg 3540catgccaacc acagggttcc cctcgggatc aaagtacttt gatccaaccc
ctccgctgct 3600atagtgcagt cggcttctga cgttcagtgc agccgtcttc tgaaaacgac
atgtcgcaca 3660agtcctaagt tacgcgacag gctgccgccc tgcccttttc ctggcgtttt
cttgtcgcgt 3720gttttagtcg cataaagtag aatacttgcg actagaaccg gagacattac
gccatgaaca 3780agagcgccgc cgctggcctg ctgggctatg cccgcgtcag caccgacgac
caggacttga 3840ccaaccaacg ggccgaactg cacgcggccg gctgcaccaa gctgttttcc
gagaagatca 3900ccggcaccag gcgcgaccgc ccggagctgg ccaggatgct tgaccaccta
cgccctggcg 3960acgttgtgac agtgaccagg ctagaccgcc tggcccgcag cacccgcgac
ctactggaca 4020ttgccgagcg catccaggag gccggcgcgg gcctgcgtag cctggcagag
ccgtgggccg 4080acaccaccac gccggccggc cgcatggtgt tgaccgtgtt cgccggcatt
gccgagttcg 4140agcgttccct aatcatcgac cgcacccgga gcgggcgcga ggccgccaag
gcccgaggcg 4200tgaagtttgg cccccgccct accctcaccc cggcacagat cgcgcacgcc
cgcgagctga 4260tcgaccagga aggccgcacc gtgaaagagg cggctgcact gcttggcgtg
catcgctcga 4320ccctgtaccg cgcacttgag cgcagcgagg aagtgacgcc caccgaggcc
aggcggcgcg 4380gtgccttccg tgaggacgca ttgaccgagg ccgacgccct ggcggccgcc
gagaatgaac 4440gccaagagga acaagcatga aaccgcacca ggacggccag gacgaaccgt
ttttcattac 4500cgaagagatc gaggcggaga tgatcgcggc cgggtacgtg ttcgagccgc
ccgcgcacgt 4560ctcaaccgtg cggctgcatg aaatcctggc cggtttgtct gatgccaagc
tggcggcctg 4620gccggccagc ttggccgctg aagaaaccga gcgccgccgt ctaaaaaggt
gatgtgtatt 4680tgagtaaaac agcttgcgtc atgcggtcgc tgcgtatatg atgcgatgag
taaataaaca 4740aatacgcaag gggaacgcat gaaggttatc gctgtactta accagaaagg
cgggtcaggc 4800aagacgacca tcgcaaccca tctagcccgc gccctgcaac tcgccggggc
cgatgttctg 4860ttagtcgatt ccgatcccca gggcagtgcc cgcgattggg cggccgtgcg
ggaagatcaa 4920ccgctaaccg ttgtcggcat cgaccgcccg acgattgacc gcgacgtgaa
ggccatcggc 4980cggcgcgact tcgtagtgat cgacggagcg ccccaggcgg cggacttggc
tgtgtccgcg 5040atcaaggcag ccgacttcgt gctgattccg gtgcagccaa gcccttacga
catatgggcc 5100accgccgacc tggtggagct ggttaagcag cgcattgagg tcacggatgg
aaggctacaa 5160gcggcctttg tcgtgtcgcg ggcgatcaaa ggcacgcgca tcggcggtga
ggttgccgag 5220gcgctggccg ggtacgagct gcccattctt gagtcccgta tcacgcagcg
cgtgagctac 5280ccaggcactg ccgccgccgg cacaaccgtt cttgaatcag aacccgaggg
cgacgctgcc 5340cgcgaggtcc aggcgctggc cgctgaaatt aaatcaaaac tcatttgagt
taatgaggta 5400aagagaaaat gagcaaaagc acaaacacgc taagtgccgg ccgtccgagc
gcacgcagca 5460gcaaggctgc aacgttggcc agcctggcag acacgccagc catgaagcgg
gtcaactttc 5520agttgccggc ggaggatcac accaagctga agatgtacgc ggtacgccaa
ggcaagacca 5580ttaccgagct gctatctgaa tacatcgcgc agctaccaga gtaaatgagc
aaatgaataa 5640atgagtagat gaattttagc ggctaaagga ggcggcatgg aaaatcaaga
acaaccaggc 5700accgacgccg tggaatgccc catgtgtgga ggaacgggcg gttggccagg
cgtaagcggc 5760tgggttgtct gccggccctg caatggcact ggaaccccca agcccgagga
atcggcgtga 5820cggtcgcaaa ccatccggcc cggtacaaat cggcgcggcg ctgggtgatg
acctggtgga 5880gaagttgaag gccgcgcagg ccgcccagcg gcaacgcatc gaggcagaag
cacgccccgg 5940tgaatcgtgg caagcggccg ctgatcgaat ccgcaaagaa tcccggcaac
cgccggcagc 6000cggtgcgccg tcgattagga agccgcccaa gggcgacgag caaccagatt
ttttcgttcc 6060gatgctctat gacgtgggca cccgcgatag tcgcagcatc atggacgtgg
ccgttttccg 6120tctgtcgaag cgtgaccgac gagctggcga ggtgatccgc tacgagcttc
cagacgggca 6180cgtagaggtt tccgcagggc cggccggcat ggccagtgtg tgggattacg
acctggtact 6240gatggcggtt tcccatctaa ccgaatccat gaaccgatac cgggaaggga
agggagacaa 6300gcccggccgc gtgttccgtc cacacgttgc ggacgtactc aagttctgcc
ggcgagccga 6360tggcggaaag cagaaagacg acctggtaga aacctgcatt cggttaaaca
ccacgcacgt 6420tgccatgcag cgtacgaaga aggccaagaa cggccgcctg gtgacggtat
ccgagggtga 6480agccttgatt agccgctaca agatcgtaaa gagcgaaacc gggcggccgg
agtacatcga 6540gatcgagcta gctgattgga tgtaccgcga gatcacagaa ggcaagaacc
cggacgtgct 6600gacggttcac cccgattact ttttgatcga tcccggcatc ggccgttttc
tctaccgcct 6660ggcacgccgc gccgcaggca aggcagaagc cagatggttg ttcaagacga
tctacgaacg 6720cagtggcagc gccggagagt tcaagaagtt ctgtttcacc gtgcgcaagc
tgatcgggtc 6780aaatgacctg ccggagtacg atttgaagga ggaggcgggg caggctggcc
cgatcctagt 6840catgcgctac cgcaacctga tcgagggcga agcatccgcc ggttcctaat
gtacggagca 6900gatgctaggg caaattgccc tagcagggga aaaaggtcga aaaggtctct
ttcctgtgga 6960tagcacgtac attgggaacc caaagccgta cattgggaac cggaacccgt
acattgggaa 7020cccaaagccg tacattggga accggtcaca catgtaagtg actgatataa
aagagaaaaa 7080aggcgatttt tccgcctaaa actctttaaa acttattaaa actcttaaaa
cccgcctggc 7140ctgtgcataa ctgtctggcc agcgcacagc cgaagagctg caaaaagcgc
ctacccttcg 7200gtcgctgcgc tccctacgcc ccgccgcttc gcgtcggcct atcgcggccg
ctggccgctc 7260aaaaatggct ggcctacggc caggcaatct accagggcgc ggacaagccg
cgccgtcgcc 7320actcgaccgc cggcgcccac atcaaggcac cctgcctcgc gcgtttcggt
gatgacggtg 7380aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa
gcggatgccg 7440ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg
ggcgcagcca 7500tgacccagtc acgtagcgat agcggagtgt atactggctt aactatgcgg
catcagagca 7560gattgtactg agagtgcacc atatgcggtg tgaaataccg cacagatgcg
taaggagaaa 7620ataccgcatc aggcgctctt ccgcttcctc gctcactgac tcgctgcgct
cggtcgttcg 7680gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
cagaatcagg 7740ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
accgtaaaaa 7800ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
acaaaaatcg 7860acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
cgtttccccc 7920tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
acctgtccgc 7980ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
atctcagttc 8040ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
agcccgaccg 8100ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
acttatcgcc 8160actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
gtgctacaga 8220gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg
gtatctgcgc 8280tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
gcaaacaaac 8340caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
gaaaaaaagg 8400atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
acgaaaactc 8460acgttaaggg attttggtca tgcatgatat atctcccaat ttgtgtaggg
cttattatgc 8520acgcttaaaa ataataaaag cagacttgac ctgatagttt ggctgtgagc
aattatgtgc 8580ttagtgcatc taatcgcttg agttaacgcc ggcgaagcgg cgtcggcttg
aacgaatttc 8640tagctagagg atcgcaccaa taactgcctt aaaaaaatta cgccccgccc
tgccactcat 8700cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca
caaacggcat 8760gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa
tatttgccca 8820ttgtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca
aaactggtga 8880aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct
ttagggaaat 8940aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga
aactgccgga 9000aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca
tggaaaacgg 9060tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt
gccatacgga 9120actccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga
taaaacttgt 9180gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg
gtctggttat 9240aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc
cattgggata 9300tatcaacggt ggtatatcca gtgatttttt tctccatgat gtttaacttt
gttttagggc 9360gactgccctg ctgcgtaaca tcgttgctgc tccataacat caaacatcga
cccacggcgt 9420aacgcgcttg ctgcttggat gcccgaggca tagactgtac cccaaaaaaa
catgtcataa 9480caagaagcca tgaaaaccgc cactgcgccg ttaccaccgc tgcgttcggt
caaggttctg 9540gaccagttgc gtgacggcag ttacgctact tgcattacag cttacgaacc
gaacgaggct 9600tatgtccact gggttcgtgc ccgaattgat cacaggcagc aacgctctgt
catcgttaca 9660atcaacatgc taccctccgc gagatcatcc gtgtttcaaa cccggcagct
tagttgccgt 9720tcttccgaat agcatcggta acatgagcaa agtctgccgc cttacaacgg
ctctcccgct 9780gacgccgtcc cggactgatg ggctgcctgt atcgagtggt gattttgtgc
cgagctgccg 9840gtcggggagc tgttggctgg ctggtggcag gatatattgt ggtgtaaaca
aattgacgct 9900tagacaactt aataacacat tgcggacgtt tttaatgtac tgaattaacg
ccgaattaat 9960tcgggggatc tggattttag tactggattt tggttttagg aattagaaat
tttattgata 10020gaagtatttt acaaatacaa atacatacta agggtttctt atatgctcaa
cacatgagcg 10080aaaccctata ggaaccctaa ttcccttatc tgggaactac tcacacatta
ttatggagaa 10140actcgagctt gtcgatcgac tctagctaga ggatcgatcc gaaccccaga
gtcccgctca 10200gaagaactcg tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag
cggcgatacc 10260gtaaagcacg aggaagcggt cagcccattc gccgccaagc tcttcagcaa
tatcacgggt 10320agccaacgct atgtcctgat agcggtccgc cacacccagc cggccacagt
cgatgaatcc 10380agaaaagcgg ccattttcca ccatgatatt cggcaagcag gcatcgccat
gtgtcacgac 10440gagatcctcg ccgtcgggca tgcgcgcctt gagcctggcg aacagttcgg
ctggcgcgag 10500cccctgatgc tcttcgtcca gatcatcctg atcgacaaga ccggcttcca
tccgagtacg 10560tgctcgctcg atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg
gatcaagcgt 10620atgcagccgc cgcattgcat cagccatgat ggatactttc tcggcaggag
caaggtgaga 10680tgacaggaga tcctgccccg gcacttcgcc caatagcagc cagtcccttc
ccgcttcagt 10740gacaacgtcg agcacagctg cgcaaggaac gcccgtcgtg gccagccacg
atagccgcgc 10800tgcctcgtcc tggagttcat tcagggcacc ggacaggtcg gtcttgacaa
aaagaaccgg 10860gcgcccctgc gctgacagcc ggaacacggc ggcatcagag cagccgattg
tctgttgtgc 10920ccagtcatag ccgaatagcc tctccaccca agcggccgga gaacctgcgt
gcaatccatc 10980ttgttcaatc cccatggtcg atcgacagat ctgcgaaagc tcgagagaga
tagatttgta 11040gagagagact ggtgatttca gcgtgtcctc tccaaatgaa atgaacttcc
ttatatagag 11100gaaggtcttg cgaaggatag tgggattgtg cgtcatccct tacgtcagtg
gagatatcac 11160atcaatccac ttgctttgaa gacgtggttg gaacgtcttc tttttccacg
atgctcctcg 11220tgggtggggg tccatctttg ggaccactgt cggcagaggc atcttgaacg
atagcctttc 11280ctttatcgca atgatggcat ttgtaggtgc caccttcctt ttctactgtc
cttttgatga 11340agtgacagat agctgggcaa tggaatccga ggaggtttcc cgatattacc
ctttgttgaa 11400aagtctcaat agccctttgg tcttctgaga ctgtatcttt gatattcttg
gagtagacga 11460gagtgtcgtg ctccaccatg ttatcacatc aatccacttg ctttgaagac
gtggttggaa 11520cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc atctttggga
ccactgtcgg 11580cagaggcatc ttgaacgata gcctttcctt tatcgcaatg atggcatttg
taggtgccac 11640cttccttttc tactgtcctt ttgatgaagt gacagatagc tgggcaatgg
aatccgagga 11700ggtttcccga tattaccctt tgttgaaaag tctcaatagc cctttggtct
tctgagactg 11760tatctttgat attcttggag tagacgagag tgtcgtgctc caccatgttg
gcaagctgct 11820ctagccaata cgcaaaccgc ctctc
11845421780DNAArtificial Sequencesynthetic GPT 5c construct;
Cambia 1305.1 with rbcS3C promoter + catI intron with full-length
codon optimized Arabidopsis GPT gene 42aaaaaagaaa aaaaaaacat
atcttgtttg tcagtatggg aagtttgaga taaggacgag 60tgaggggtta aaattcagtg
gccattgatt ttgtaatgcc aagaaccaca aaatccaatg 120gttaccattc ctgtaagatg
aggtttgcta actctttttg tccgttagat aggaagcctt 180atcactatat atacaaggcg
tcctaataac ctcttagtaa ccaattattt cagcaccatg 240gtagatctga gggtaaattt
ctagtttttc tccttcattt tcttggttag gacccttttc 300tctttttatt tttttgagct
ttgatctttc tttaaactga tctatttttt aattgattgg 360ttatggtgta aatattacat
agctttaact gataatctga ttactttatt tcgtgtgtct 420atgatgatga tgatagttac
agaaccgacg aactagtatg tacctggaca taaatggtgt 480gatgatcaaa cagtttagct
tcaaagcctc tcttctccca ttctcttcta atttccgaca 540aagctccgcc aaaatccatc
gtcctatcgg agccaccatg accacagttt cgactcagaa 600cgagtctact caaaaacccg
tccaggtggc gaagagatta gagaagttca agactactat 660tttcactcaa atgagcatat
tggcagttaa acatggagcg atcaatttag gccaaggctt 720tcccaatttc gacggtcctg
attttgttaa agaagctgcg atccaagcta ttaaagatgg 780taaaaaccag tatgctcgtg
gatacggcat tcctcagctc aactctgcta tagctgcgcg 840gtttcgtgaa gatacgggtc
ttgttgttga tcctgagaaa gaagttactg ttacatctgg 900ttgcacagaa gccatagctg
cagctatgtt gggtttaata aaccctggtg atgaagtcat 960tctctttgca ccgttttatg
attcctatga agcaacactc tctatggctg gtgctaaagt 1020aaaaggaatc actttacgtc
caccggactt ctccatccct ttggaagagc ttaaagctgc 1080ggtaactaac aagactcgag
ccatccttat gaacactccg cacaacccga ccgggaagat 1140gttcactagg gaggagcttg
aaaccattgc atctctctgc attgaaaacg atgtgcttgt 1200gttctcggat gaagtatacg
ataagcttgc gtttgaaatg gatcacattt ctatagcttc 1260tcttcccggt atgtatgaaa
gaactgtgac catgaattcc ctgggaaaga ctttctcttt 1320aaccggatgg aagatcggct
gggcgattgc gccgcctcat ctgacttggg gagttcgaca 1380agcacactct tacctcacat
tcgccacatc aacaccagca caatgggcag ccgttgcagc 1440tctcaaggca ccagagtctt
acttcaaaga gctgaaaaga gattacaatg tgaaaaagga 1500gactctggtt aagggtttga
aggaagtcgg atttacagtg ttcccatcga gcgggactta 1560ctttgtggtt gctgatcaca
ctccatttgg aatggagaac gatgttgctt tctgtgagta 1620tcttattgaa gaagttgggg
tcgttgcgat cccaacgagc gtcttttatc tgaatccaga 1680agaagggaag aatttggtta
ggtttgcgtt ctgtaaagac gaagagacgt tgcgtggtgc 1740aattgagagg atgaagcaga
agcttaagag aaaagtctga 17804311960DNAArtificial
Sequencesynthetic GS 4c construct; Cambia 1305.1 with rbcS3C tomato
rubisco small subunit promoter + catI intron +Arabidopsis GS1 coding
region + nos terminator 43ggtaccgttt gaatcctcct taaagttttt ctctggagaa
actgtagtaa ttttactttg 60ttgtgttccc ttcatctttt gaattaatgg catttgtttt
aatactaatc tgcttctgaa 120acttgtaatg tatgtatatc agtttcttat aatttatcca
agtaatatct tccattctct 180atgcaattgc ctgcataagc tcgacaaaag agtacatcaa
cccctcctcc tctggactac 240tctagctaaa cttgaatttc cccttaagat tatgaaattg
atatatcctt aacaaacgac 300tccttctgtt ggaaaatgta gtacttgtct ttcttctttt
gggtatatat agtttatata 360caccatacta tgtacaacat ccaagtagag tgaaatggat
acatgtacaa gacttatttg 420attgattgat gacttgagtt gccttaggag taacaaattc
ttaggtcaat aaatcgttga 480tttgaaatta atctctctgt cttagacaga taggaattat
gacttccaat ggtccagaaa 540gcaaagttcg cactgagggt atacttggaa ttgagacttg
cacaggtcca gaaaccaaag 600ttcccatcga gctctaaaat cacatctttg gaatgaaatt
caattagaga taagttgctt 660catagcatag gtaaaatgga agatgtgaag taacctgcaa
taatcagtga aatgacatta 720atacactaaa tacttcatat gtaattatcc tttccaggtt
aacaatactc tataaagtaa 780gaattatcag aaatgggctc atcaaacttt tgtactatgt
atttcatata aggaagtata 840actatacata agtgtataca caactttatt cctattttgt
aaaggtggag agactgtttt 900cgatggatct aaagcaatat gtctataaaa tgcattgata
taataattat ctgagaaaat 960ccagaattgg cgttggatta tttcagccaa atagaagttt
gtaccatact tgttgattcc 1020ttctaagtta aggtgaagta tcattcataa acagttttcc
ccaaagtact actcaccaag 1080tttccctttg tagaattaac agttcaaata tatggcgcag
aaattactct atgcccaaaa 1140ccaaacgaga aagaaacaaa atacaggggt tgcagacttt
attttcgtgt tagggtgtgt 1200tttttcatgt aattaatcaa aaaatattat gacaaaaaca
tttatacata tttttactca 1260acactctggg tatcagggtg ggttgtgttc gacaatcaat
atggaaagga agtattttcc 1320ttattttttt agttaatatt ttcagttata ccaaacatac
cttgtgatat tatttttaaa 1380aatgaaaaac tcgtcagaaa gaaaaagcaa aagcaacaaa
aaaattgcaa gtatttttta 1440aaaaagaaaa aaaaaacata tcttgtttgt cagtatggga
agtttgagat aaggacgagt 1500gaggggttaa aattcagtgg ccattgattt tgtaatgcca
agaaccacaa aatccaatgg 1560ttaccattcc tgtaagatga ggtttgctaa ctctttttgt
ccgttagata ggaagcctta 1620tcactatata tacaaggcgt cctaataacc tcttagtaac
caattatttc agcaccatgg 1680tagatctgag ggtaaatttc tagtttttct ccttcatttt
cttggttagg acccttttct 1740ctttttattt ttttgagctt tgatctttct ttaaactgat
ctatttttta attgattggt 1800tatggtgtaa atattacata gctttaactg ataatctgat
tactttattt cgtgtgtcta 1860tgatgatgat gatagttaca gaaccgacga actagtatgt
ctctgctctc agatctcgtt 1920aacctcaacc tcaccgatgc caccgggaaa atcatcgccg
aatacatatg gatcggtgga 1980tctggaatgg atatcagaag caaagccagg acactaccag
gaccagtgac tgatccatca 2040aagcttccca agtggaacta cgacggatcc agcaccggtc
aggctgctgg agaagacagt 2100gaagtcattc tataccctca ggcaatattc aaggatccct
tcaggaaagg caacaacatc 2160ctggtgatgt gtgatgctta cacaccagct ggtgatccta
ttccaaccaa caagaggcac 2220aacgctgcta agatcttcag ccaccccgac gttgccaagg
aggagccttg gtatgggatt 2280gagcaagaat acactttgat gcaaaaggat gtgaactggc
caattggttg gcctgttggt 2340ggctaccctg gccctcaggg accttactac tgtggtgtgg
gagctgacaa agccattggt 2400cgtgacattg tggatgctca ctacaaggcc tgtctttacg
ccggtattgg tatttctggt 2460atcaatggag aagtcatgcc aggccagtgg gagttccaag
tcggccctgt tgagggtatt 2520agttctggtg atcaagtctg ggttgctcga taccttctcg
agaggatcac tgagatctct 2580ggtgtaattg tcagcttcga cccgaaacca gtcccgggtg
actggaatgg agctggagct 2640cactgcaact acagcactaa gacaatgaga aacgatggag
gattagaagt gatcaagaaa 2700gcgataggga agcttcagct gaaacacaaa gaacacattg
ctgcttacgg tgaaggaaac 2760gagcgtcgtc tcactggaaa gcacgaaacc gcagacatca
acacattctc ttggggagtc 2820gcgaaccgtg gagcgtcagt gagagtggga cgtgacacag
agaaggaagg taaagggtac 2880ttcgaagaca gaaggccagc ttctaacatg gatccttacg
ttgtcacctc catgatcgct 2940gagacgacca tactcggttg acacgtgtga attggtgacc
agctcgaatt tccccgatcg 3000ttcaaacatt tggcaataaa gtttcttaag attgaatcct
gttgccggtc ttgcgatgat 3060tatcatataa tttctgttga attacgttaa gcatgtaata
attaacatgt aatgcatgac 3120gttatttatg agatgggttt ttatgattag agtcccgcaa
ttatacattt aatacgcgat 3180agaaaacaaa atatagcgcg caaactagga taaattatcg
cgcgcggtgt catctatgtt 3240actagatcgg gaattaaact atcagtgttt gacaggatat
attggcgggt aaacctaaga 3300gaaaagagcg tttattagaa taacggatat ttaaaagggc
gtgaaaaggt ttatccgttc 3360gtccatttgt atgtgcatgc caaccacagg gttcccctcg
ggatcaaagt actttgatcc 3420aacccctccg ctgctatagt gcagtcggct tctgacgttc
agtgcagccg tcttctgaaa 3480acgacatgtc gcacaagtcc taagttacgc gacaggctgc
cgccctgccc ttttcctggc 3540gttttcttgt cgcgtgtttt agtcgcataa agtagaatac
ttgcgactag aaccggagac 3600attacgccat gaacaagagc gccgccgctg gcctgctggg
ctatgcccgc gtcagcaccg 3660acgaccagga cttgaccaac caacgggccg aactgcacgc
ggccggctgc accaagctgt 3720tttccgagaa gatcaccggc accaggcgcg accgcccgga
gctggccagg atgcttgacc 3780acctacgccc tggcgacgtt gtgacagtga ccaggctaga
ccgcctggcc cgcagcaccc 3840gcgacctact ggacattgcc gagcgcatcc aggaggccgg
cgcgggcctg cgtagcctgg 3900cagagccgtg ggccgacacc accacgccgg ccggccgcat
ggtgttgacc gtgttcgccg 3960gcattgccga gttcgagcgt tccctaatca tcgaccgcac
ccggagcggg cgcgaggccg 4020ccaaggcccg aggcgtgaag tttggccccc gccctaccct
caccccggca cagatcgcgc 4080acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa
agaggcggct gcactgcttg 4140gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcag
cgaggaagtg acgcccaccg 4200aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac
cgaggccgac gccctggcgg 4260ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg
caccaggacg gccaggacga 4320accgtttttc attaccgaag agatcgaggc ggagatgatc
gcggccgggt acgtgttcga 4380gccgcccgcg cacgtctcaa ccgtgcggct gcatgaaatc
ctggccggtt tgtctgatgc 4440caagctggcg gcctggccgg ccagcttggc cgctgaagaa
accgagcgcc gccgtctaaa 4500aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg
gtcgctgcgt atatgatgcg 4560atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg
ttatcgctgt acttaaccag 4620aaaggcgggt caggcaagac gaccatcgca acccatctag
cccgcgccct gcaactcgcc 4680ggggccgatg ttctgttagt cgattccgat ccccagggca
gtgcccgcga ttgggcggcc 4740gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc
gcccgacgat tgaccgcgac 4800gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg
gagcgcccca ggcggcggac 4860ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga
ttccggtgca gccaagccct 4920tacgacatat gggccaccgc cgacctggtg gagctggtta
agcagcgcat tgaggtcacg 4980gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga
tcaaaggcac gcgcatcggc 5040ggtgaggttg ccgaggcgct ggccgggtac gagctgccca
ttcttgagtc ccgtatcacg 5100cagcgcgtga gctacccagg cactgccgcc gccggcacaa
ccgttcttga atcagaaccc 5160gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg
aaattaaatc aaaactcatt 5220tgagttaatg aggtaaagag aaaatgagca aaagcacaaa
cacgctaagt gccggccgtc 5280cgagcgcacg cagcagcaag gctgcaacgt tggccagcct
ggcagacacg ccagccatga 5340agcgggtcaa ctttcagttg ccggcggagg atcacaccaa
gctgaagatg tacgcggtac 5400gccaaggcaa gaccattacc gagctgctat ctgaatacat
cgcgcagcta ccagagtaaa 5460tgagcaaatg aataaatgag tagatgaatt ttagcggcta
aaggaggcgg catggaaaat 5520caagaacaac caggcaccga cgccgtggaa tgccccatgt
gtggaggaac gggcggttgg 5580ccaggcgtaa gcggctgggt tgtctgccgg ccctgcaatg
gcactggaac ccccaagccc 5640gaggaatcgg cgtgacggtc gcaaaccatc cggcccggta
caaatcggcg cggcgctggg 5700tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc
cagcggcaac gcatcgaggc 5760agaagcacgc cccggtgaat cgtggcaagc ggccgctgat
cgaatccgca aagaatcccg 5820gcaaccgccg gcagccggtg cgccgtcgat taggaagccg
cccaagggcg acgagcaacc 5880agattttttc gttccgatgc tctatgacgt gggcacccgc
gatagtcgca gcatcatgga 5940cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct
ggcgaggtga tccgctacga 6000gcttccagac gggcacgtag aggtttccgc agggccggcc
ggcatggcca gtgtgtggga 6060ttacgacctg gtactgatgg cggtttccca tctaaccgaa
tccatgaacc gataccggga 6120agggaaggga gacaagcccg gccgcgtgtt ccgtccacac
gttgcggacg tactcaagtt 6180ctgccggcga gccgatggcg gaaagcagaa agacgacctg
gtagaaacct gcattcggtt 6240aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc
aagaacggcc gcctggtgac 6300ggtatccgag ggtgaagcct tgattagccg ctacaagatc
gtaaagagcg aaaccgggcg 6360gccggagtac atcgagatcg agctagctga ttggatgtac
cgcgagatca cagaaggcaa 6420gaacccggac gtgctgacgg ttcaccccga ttactttttg
atcgatcccg gcatcggccg 6480ttttctctac cgcctggcac gccgcgccgc aggcaaggca
gaagccagat ggttgttcaa 6540gacgatctac gaacgcagtg gcagcgccgg agagttcaag
aagttctgtt tcaccgtgcg 6600caagctgatc gggtcaaatg acctgccgga gtacgatttg
aaggaggagg cggggcaggc 6660tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag
ggcgaagcat ccgccggttc 6720ctaatgtacg gagcagatgc tagggcaaat tgccctagca
ggggaaaaag gtcgaaaagg 6780tctctttcct gtggatagca cgtacattgg gaacccaaag
ccgtacattg ggaaccggaa 6840cccgtacatt gggaacccaa agccgtacat tgggaaccgg
tcacacatgt aagtgactga 6900tataaaagag aaaaaaggcg atttttccgc ctaaaactct
ttaaaactta ttaaaactct 6960taaaacccgc ctggcctgtg cataactgtc tggccagcgc
acagccgaag agctgcaaaa 7020agcgcctacc cttcggtcgc tgcgctccct acgccccgcc
gcttcgcgtc ggcctatcgc 7080ggccgctggc cgctcaaaaa tggctggcct acggccaggc
aatctaccag ggcgcggaca 7140agccgcgccg tcgccactcg accgccggcg cccacatcaa
ggcaccctgc ctcgcgcgtt 7200tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
ggagacggtc acagcttgtc 7260tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
gtcagcgggt gttggcgggt 7320gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
agtgtatact ggcttaacta 7380tgcggcatca gagcagattg tactgagagt gcaccatatg
cggtgtgaaa taccgcacag 7440atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct
tcctcgctca ctgactcgct 7500gcgctcggtc gttcggctgc ggcgagcggt atcagctcac
tcaaaggcgg taatacggtt 7560atccacagaa tcaggggata acgcaggaaa gaacatgtga
gcaaaaggcc agcaaaaggc 7620caggaaccgt aaaaaggccg cgttgctggc gtttttccat
aggctccgcc cccctgacga 7680gcatcacaaa aatcgacgct caagtcagag gtggcgaaac
ccgacaggac tataaagata 7740ccaggcgttt ccccctggaa gctccctcgt gcgctctcct
gttccgaccc tgccgcttac 7800cggatacctg tccgcctttc tcccttcggg aagcgtggcg
ctttctcata gctcacgctg 7860taggtatctc agttcggtgt aggtcgttcg ctccaagctg
ggctgtgtgc acgaaccccc 7920cgttcagccc gaccgctgcg ccttatccgg taactatcgt
cttgagtcca acccggtaag 7980acacgactta tcgccactgg cagcagccac tggtaacagg
attagcagag cgaggtatgt 8040aggcggtgct acagagttct tgaagtggtg gcctaactac
ggctacacta gaaggacagt 8100atttggtatc tgcgctctgc tgaagccagt taccttcgga
aaaagagttg gtagctcttg 8160atccggcaaa caaaccaccg ctggtagcgg tggttttttt
gtttgcaagc agcagattac 8220gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt
tctacggggt ctgacgctca 8280gtggaacgaa aactcacgtt aagggatttt ggtcatgcat
tctaggtact aaaacaattc 8340atccagtaaa atataatatt ttattttctc ccaatcaggc
ttgatcccca gtaagtcaaa 8400aaatagctcg acatactgtt cttccccgat atcctccctg
atcgaccgga cgcagaaggc 8460aatgtcatac cacttgtccg ccctgccgct tctcccaaga
tcaataaagc cacttacttt 8520gccatctttc acaaagatgt tgctgtctcc caggtcgccg
tgggaaaaga caagttcctc 8580ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc
ggatctttaa atggagtgtc 8640ttcttcccag ttttcgcaat ccacatcggc cagatcgtta
ttcagtaagt aatccaattc 8700ggctaagcgg ctgtctaagc tattcgtata gggacaatcc
gatatgtcga tggagtgaaa 8760gagcctgatg cactccgcat acagctcgat aatcttttca
gggctttgtt catcttcata 8820ctcttccgag caaaggacgc catcggcctc actcatgagc
agattgctcc agccatcatg 8880ccgttcaaag tgcaggacct ttggaacagg cagctttcct
tccagccata gcatcatgtc 8940cttttcccgt tccacatcat aggtggtccc tttataccgg
ctgtccgtca tttttaaata 9000taggttttca ttttctccca ccagcttata taccttagca
ggagacattc cttccgtatc 9060ttttacgcag cggtattttt cgatcagttt tttcaattcc
ggtgatattc tcattttagc 9120catttattat ttccttcctc ttttctacag tatttaaaga
taccccaaga agctaattat 9180aacaagacga actccaattc actgttcctt gcattctaaa
accttaaata ccagaaaaca 9240gctttttcaa agttgttttc aaagttggcg tataacatag
tatcgacgga gccgattttg 9300aaaccgcggt gatcacaggc agcaacgctc tgtcatcgtt
acaatcaaca tgctaccctc 9360cgcgagatca tccgtgtttc aaacccggca gcttagttgc
cgttcttccg aatagcatcg 9420gtaacatgag caaagtctgc cgccttacaa cggctctccc
gctgacgccg tcccggactg 9480atgggctgcc tgtatcgagt ggtgattttg tgccgagctg
ccggtcgggg agctgttggc 9540tggctggtgg caggatatat tgtggtgtaa acaaattgac
gcttagacaa cttaataaca 9600cattgcggac gtttttaatg tactgaatta acgccgaatt
aattcggggg atctggattt 9660tagtactgga ttttggtttt aggaattaga aattttattg
atagaagtat tttacaaata 9720caaatacata ctaagggttt cttatatgct caacacatga
gcgaaaccct ataggaaccc 9780taattccctt atctgggaac tactcacaca ttattatgga
gaaactcgag cttgtcgatc 9840gacagatccg gtcggcatct actctatttc tttgccctcg
gacgagtgct ggggcgtcgg 9900tttccactat cggcgagtac ttctacacag ccatcggtcc
agacggccgc gcttctgcgg 9960gcgatttgtg tacgcccgac agtcccggct ccggatcgga
cgattgcgtc gcatcgaccc 10020tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc
tctgatagag ttggtcaaga 10080ccaatgcgga gcatatacgc ccggagtcgt ggcgatcctg
caagctccgg atgcctccgc 10140tcgaagtagc gcgtctgctg ctccatacaa gccaaccacg
gcctccagaa gaagatgttg 10200gcgacctcgt attgggaatc cccgaacatc gcctcgctcc
agtcaatgac cgctgttatg 10260cggccattgt ccgtcaggac attgttggag ccgaaatccg
cgtgcacgag gtgccggact 10320tcggggcagt cctcggccca aagcatcagc tcatcgagag
cctgcgcgac ggacgcactg 10380acggtgtcgt ccatcacagt ttgccagtga tacacatggg
gatcagcaat cgcgcatatg 10440aaatcacgcc atgtagtgta ttgaccgatt ccttgcggtc
cgaatgggcc gaacccgctc 10500gtctggctaa gatcggccgc agcgatcgca tccatagcct
ccgcgaccgg ttgtagaaca 10560gcgggcagtt cggtttcagg caggtcttgc aacgtgacac
cctgtgcacg gcgggagatg 10620caataggtca ggctctcgct aaactcccca atgtcaagca
cttccggaat cgggagcgcg 10680gccgatgcaa agtgccgata aacataacga tctttgtaga
aaccatcggc gcagctattt 10740acccgcagga catatccacg ccctcctaca tcgaagctga
aagcacgaga ttcttcgccc 10800tccgagagct gcatcaggtc ggagacgctg tcgaactttt
cgatcagaaa cttctcgaca 10860gacgtcgcgg tgagttcagg ctttttcata tctcattgcc
ccccgggatc tgcgaaagct 10920cgagagagat agatttgtag agagagactg gtgatttcag
cgtgtcctct ccaaatgaaa 10980tgaacttcct tatatagagg aaggtcttgc gaaggatagt
gggattgtgc gtcatccctt 11040acgtcagtgg agatatcaca tcaatccact tgctttgaag
acgtggttgg aacgtcttct 11100ttttccacga tgctcctcgt gggtgggggt ccatctttgg
gaccactgtc ggcagaggca 11160tcttgaacga tagcctttcc tttatcgcaa tgatggcatt
tgtaggtgcc accttccttt 11220tctactgtcc ttttgatgaa gtgacagata gctgggcaat
ggaatccgag gaggtttccc 11280gatattaccc tttgttgaaa agtctcaata gccctttggt
cttctgagac tgtatctttg 11340atattcttgg agtagacgag agtgtcgtgc tccaccatgt
tatcacatca atccacttgc 11400tttgaagacg tggttggaac gtcttctttt tccacgatgc
tcctcgtggg tgggggtcca 11460tctttgggac cactgtcggc agaggcatct tgaacgatag
cctttccttt atcgcaatga 11520tggcatttgt aggtgccacc ttccttttct actgtccttt
tgatgaagtg acagatagct 11580gggcaatgga atccgaggag gtttcccgat attacccttt
gttgaaaagt ctcaatagcc 11640ctttggtctt ctgagactgt atctttgata ttcttggagt
agacgagagt gtcgtgctcc 11700accatgttgg caagctgctc tagccaatac gcaaaccgcc
tctccccgcg cgttggccga 11760ttcattaatg cagctggcac gacaggtttc ccgactggaa
agcgggcagt gagcgcaacg 11820caattaatgt gagttagctc actcattagg caccccaggc
tttacacttt atgcttccgg 11880ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
cacaggaaac agctatgacc 11940atgattacga attcgagctc
1196044394PRTArtificial Sequencesynthetic plant
omega-amidase consensus sequence 44Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Leu Xaa
Xaa Xaa Xaa 20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
35 40 45 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Xaa 50 55
60 Xaa Xaa Ala Ser Ser Phe Xaa Pro Glu
Gln Ala Arg Ser Pro Pro Ala65 70 75
80 Leu Pro Leu Pro Thr Pro Pro Leu Thr Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 85 90 95
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Lys Ile Gly Leu Cys
100 105 110 Gln Leu Ser Val Thr
Ala Asp Lys Asp Arg Asn Ile Ala His Ala Arg 115
120 125 Lys Ala Ile Glu Glu Ala Ala Ala Lys
Gly Ala Lys Leu Val Leu Leu 130 135
140 Pro Glu Ile Trp Asn Ser Pro Tyr Ser Asn Asp Ser Phe
Pro Val Tyr145 150 155
160 Ala Glu Asp Ile Asp Ala Gly Gly Asp Ala Ser Pro Ser Thr Ala Met
165 170 175 Leu Ser Glu Val
Ala Arg Xaa Leu Lys Ile Thr Ile Val Gly Gly Ser 180
185 190 Ile Pro Glu Arg Ser Gly Asp Arg Leu
Tyr Asn Thr Cys Cys Val Phe 195 200
205 Gly Ser Asp Gly Xaa Leu Lys Ala Lys His Arg Lys Ile His
Leu Phe 210 215 220
Asp Ile Asp Ile Pro Gly Lys Ile Thr Phe Ile Glu Ser Lys Thr Leu225
230 235 240 Thr Ala Gly Asp Thr
Pro Thr Ile Val Asp Thr Glu Val Gly Arg Ile 245
250 255 Gly Ile Gly Ile Cys Tyr Asp Ile Arg Phe
Gln Glu Leu Ala Met Leu 260 265
270 Tyr Ala Ala Arg Gly Ala His Leu Leu Cys Tyr Pro Gly Ala Phe
Asn 275 280 285 Met
Thr Thr Gly Pro Leu His Trp Glu Leu Leu Gln Arg Ala Arg Ala 290
295 300 Ala Asp Asn Gln Leu Tyr
Val Ala Thr Cys Ser Pro Ala Arg Asp Thr305 310
315 320 Gly Ala Gly Tyr Val Ala Trp Gly His Ser Thr
Leu Val Gly Pro Phe 325 330
335 Gly Glu Val Leu Ala Thr Thr Glu His Glu Glu Ala Ile Ile Ile Ala
340 345 350 Glu Ile Asp
Tyr Ser Leu Ile Glu Xaa Arg Arg Gln Xaa Xaa Xaa Xaa 355
360 365 Xaa Leu Pro Leu Xaa Xaa Gln Arg
Arg Gly Asp Leu Tyr Gln Leu Val 370 375
380 Asp Val Gln Arg Leu Xaa Ser Xaa Xaa Xaa385
390 45318PRTArtificial Sequencesynthetic animal
omega-amidase consensus sequence 45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 20 25 30
Xaa Xaa Xaa Xaa Xaa Xaa Met Ser Lys Phe Arg Leu Ala Leu Ile Gln
35 40 45 Leu Gln Val Ser
Ser Ile Lys Ser Asp Asn Leu Arg Ala Cys Ser Leu 50 55
60 Val Arg Glu Ala Ala Xaa Gln Gly Ala
Lys Ile Val Xaa Leu Pro Glu65 70 75
80 Cys Phe Asn Ser Pro Tyr Gly Thr Xaa Tyr Phe Pro Glu Tyr
Ala Glu 85 90 95
Lys Ile Pro Gly Xaa Xaa Xaa Xaa Xaa Glu Ser Thr Gln Lys Leu Ser
100 105 110 Glu Val Ala Lys Glu
Cys Ile Tyr Leu Ile Gly Gly Ser Ile Pro Glu 115
120 125 Glu Asp Ala Gly Lys Leu Tyr Asn Thr
Cys Ala Val Phe Gly Pro Asp 130 135
140 Gly Thr Leu Leu Val Lys His Arg Lys Ile His Leu Phe
Asp Ile Asp145 150 155
160 Val Pro Gly Lys Ile Thr Phe Gln Glu Ser Lys Thr Leu Ser Pro Gly
165 170 175 Asp Phe Ser Thr
Phe Asp Thr Pro Tyr Cys Lys Val Gly Leu Gly Ile 180
185 190 Cys Tyr Asp Ile Arg Phe Ala Glu Leu
Ala Gln Ile Tyr Ala Gln Arg 195 200
205 Gly Cys Gln Leu Leu Val Tyr Pro Gly Ala Phe Asn Leu Thr
Thr Gly 210 215 220
Pro Ala His Trp Glu Leu Leu Gln Arg Ala Arg Ala Asp Asn Gln Val225
230 235 240 Tyr Val Ala Thr Ala
Ser Pro Ala Arg Asp Asp Lys Ala Ser Tyr Val 245
250 255 Ala Trp Gly His Ser Thr Val Val Xaa Pro
Trp Gly Glu Val Leu Ala 260 265
270 Lys Ala Gly Thr Glu Glu Thr Ile Leu Tyr Ala Asp Ile Asp Leu
Xaa 275 280 285 Xaa
Leu Ala Glu Ile Arg Gln Ile Pro Ile Xaa Lys Gln Arg Arg Xaa 290
295 300 Asp Leu Tyr Thr Val Glu
Xaa Lys Lys Xaa Xaa Xaa Xaa Xaa305 310
315 46369PRTArabidopsis thalianaArabidopsis full-length
omega-amidase, AT5g12040/F14F18_210 46Met Lys Ser Ala Ile Ser Ser
Ser Leu Phe Phe Asn Ser Lys Asn Leu 1 5 10
15 Leu Asn Pro Asn Pro Leu Ser Arg Phe Ile Ser Leu
Lys Ser Asn Phe 20 25 30
Leu Pro Lys Leu Ser Pro Arg Ser Ile Thr Ser His Thr Leu Lys Leu
35 40 45 Pro Ser Ser Ser
Thr Ser Ala Leu Arg Ser Ile Ser Ser Ser Met Ala 50 55
60 Ser Ser Phe Asn Pro Glu Gln Ala Arg
Val Pro Ser Ala Leu Pro Leu65 70 75
80 Pro Ala Pro Pro Leu Thr Lys Phe Asn Ile Gly Leu Cys Gln
Leu Ser 85 90 95
Val Thr Ser Asp Lys Lys Arg Asn Ile Ser His Ala Lys Lys Ala Ile
100 105 110 Glu Glu Ala Ala Ser
Lys Gly Ala Lys Leu Val Leu Leu Pro Glu Ile 115
120 125 Trp Asn Ser Pro Tyr Ser Asn Asp Ser
Phe Pro Val Tyr Ala Glu Glu 130 135
140 Ile Asp Ala Gly Gly Asp Ala Ser Pro Ser Thr Ala Met
Leu Ser Glu145 150 155
160 Val Ser Lys Arg Leu Lys Ile Thr Ile Ile Gly Gly Ser Ile Pro Glu
165 170 175 Arg Val Gly Asp
Arg Leu Tyr Asn Thr Cys Cys Val Phe Gly Ser Asp 180
185 190 Gly Glu Leu Lys Ala Lys His Arg Lys
Ile His Leu Phe Asp Ile Asp 195 200
205 Ile Pro Gly Lys Ile Thr Phe Met Glu Ser Lys Thr Leu Thr
Ala Gly 210 215 220
Glu Thr Pro Thr Ile Val Asp Thr Asp Val Gly Arg Ile Gly Ile Gly225
230 235 240 Ile Cys Tyr Asp Ile
Arg Phe Gln Glu Leu Ala Met Ile Tyr Ala Ala 245
250 255 Arg Gly Ala His Leu Leu Cys Tyr Pro Gly
Ala Phe Asn Met Thr Thr 260 265
270 Gly Pro Leu His Trp Glu Leu Leu Gln Arg Ala Arg Ala Thr Asp
Asn 275 280 285 Gln
Leu Tyr Val Ala Thr Cys Ser Pro Ala Arg Asp Ser Gly Ala Gly 290
295 300 Tyr Thr Ala Trp Gly His
Ser Thr Leu Val Gly Pro Phe Gly Glu Val305 310
315 320 Leu Ala Thr Thr Glu His Glu Glu Ala Ile Ile
Ile Ala Glu Ile Asp 325 330
335 Tyr Ser Ile Leu Glu Gln Arg Arg Thr Ser Leu Pro Leu Asn Arg Gln
340 345 350 Arg Arg Gly
Asp Leu Tyr Gln Leu Val Asp Val Gln Arg Leu Asp Ser 355
360 365 Lys
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