Patent application title: Methods for Increasing the Resistance of Plants to Hypoxic Conditions
Inventors:
Michael Metzlaff (Tervuren, BE)
Marc De Block (Merelbeke, BE)
Marc De Block (Merelbeke, BE)
Assignees:
Bayer CropScience N.V.
IPC8 Class: AC12N1582FI
USPC Class:
504253
Class name: Additional hetero ring containing the additional hetero ring is five-membered including nitrogen plural ring nitrogens in the additional hetero ring
Publication date: 2013-03-14
Patent application number: 20130065761
Abstract:
Methods are provided for increasing the resistance of plants to hypoxic
or anoxic conditions. Such methods may be applied to increase the
penetrance of plant roots in the growth medium or into soil. The methods
according to the invention may include providing plants with a stress
tolerance gene. Similar effects can be obtained by applying chemical
compounds, including neonicotinoid compounds, to the plants.Claims:
1. A method for increasing the tolerance of a plant cell or plant to
hypoxic or anoxic conditions, comprising the step of: a) providing a
stress tolerance enhancing transgene to said plant cell or cells of said
plant; wherein said stress tolerance enhancing transgene is i. a stress
tolerance enhancing transgene capable of reducing the expression of plant
endogenous PARP genes; ii. a stress tolerance enhancing transgene capable
of reducing the expression of plant endogenous PARG genes; iii. a stress
tolerance enhancing transgene coding for a plant-functional enzyme of the
nicotinamide adenine dinucleotide salvage synthesis pathway selected from
nicotinamidase, nicotinate phosphoribosyltransferase, nicotinic acid
mononucleotide adenyl transferase or nicotinamide adenine dinucleotide
synthetase.
2. A method for increasing the penetrance of roots of a plant into a growth medium, comprising the step of: a) providing a stress tolerance enhancing transgene to said plant cell or cells of said plant; wherein said stress tolerance enhancing transgene is i. a stress tolerance enhancing transgene capable of reducing the expression of plant endogenous PARP genes; ii. a stress tolerance enhancing transgene capable of reducing the expression of plant endogenous PARG genes; or iii. a stress tolerance enhancing transgene coding for a plant-functional enzyme of the nicotinamide adenine dinucleotide salvage synthesis pathway selected from nicotinamidase, nicotinate phosphoribosyltransferase, nicotinic acid mononucleotide adenyl transferase or nicotinamide adenine dinucleotide synthetase.
3. The method according to claim 1, wherein said stress tolerance enhancing transgene codes for a PARP inhibitory RNA molecule.
4. The method according to claim 3, wherein said transgene comprises the following operably linked DNA fragments: a) a plant expressible promoter; b) a DNA region coding for a PARP inhibitory RNA molecule comprising at least 19 out of 20 consecutive nucleotides from the nucleotide sequence of SEQ ID No 1, the nucleotide sequence of SEQ ID No 2, the nucleotide sequence of SEQ ID No 3, the nucleotide sequence of SEQ ID No 4, the nucleotide sequence of SEQ ID No 5, or the nucleotide sequence of SEQ ID No 6; and c) a transcription termination and polyadenylation DNA region.
5. The method according to claim 3, wherein said transgene comprises the following operably linked DNA fragments: a) a plant expressible promoter; b) a DNA region coding for a PARP inhibitory RNA molecule comprising at least 19 out of 20 consecutive nucleotides from the complement of the nucleotide sequence of SEQ ID No 1, the nucleotide sequence of SEQ ID No 2, the nucleotide sequence of SEQ ID No 3, the nucleotide sequence of SEQ ID No 4, the nucleotide sequence of SEQ ID No 5, or the nucleotide sequence of SEQ ID No 6; and c) a transcription termination and polyadenylation DNA region.
6. The method according to claim 3, wherein said transgene comprises the following operably linked DNA fragments: a) a plant expressible promoter; b) a DNA region coding for a PARP inhibitory RNA molecule, said RNA molecule comprising: i. a sense nucleotide sequence comprising at least 19 out of 20 consecutive nucleotides from the nucleotide sequence of SEQ ID No 1, the nucleotide sequence of SEQ ID No 2, the nucleotide sequence of SEQ ID No 3, the nucleotide sequence of SEQ ID No 4, the nucleotide sequence of SEQ ID No 5, or the nucleotide sequence of SEQ ID No 6; and ii. an antisense nucleotide sequence comprising a nucleotide sequence complementary to said at least 20 consecutive nucleotides in said sense nucleotide sequence wherein said sense and antisense nucleotide sequence are capable of forming a double stranded RNA region; and c) a transcription termination and polyadenylation DNA region.
7. The method according to claim 6, wherein said antisense nucleotide sequence has about 95% sequence identity or is identical to said sense nucleotide sequence.
8. The method according to claim 1, wherein said transgene codes for a ParG inhibitory RNA molecule.
9. The method according to claim 8, wherein said transgene comprises the following operably linked DNA fragments: a) a plant expressible promoter; b) a DNA region coding for a PARG inhibitory RNA molecule comprising at least 19 out of 20 consecutive nucleotides from the nucleotide sequence of SEQ ID No 7, the nucleotide sequence of SEQ ID No 8, the nucleotide sequence of SEQ ID No 9 or the nucleotide sequence of SEQ ID No 10; and c) a transcription termination and polyadenylation DNA region.
10. The method according to claim 8, wherein said transgene comprises the following operably linked DNA fragments: a) a plant expressible promoter; b) a DNA region coding for a PARG inhibitory RNA molecule comprising at least 19 out of 20 consecutive nucleotides from the complement of the nucleotide sequence of SEQ ID No 7, the nucleotide sequence of SEQ ID No 8, the nucleotide sequence of SEQ ID No 9, or the nucleotide sequence of SEQ ID No 10; and c) a transcription termination and polyadenylation DNA region.
11. The method according to claim 8, wherein said transgene comprises the following operably linked DNA fragments: a) a plant expressible promoter; b) a DNA region coding for a PARG inhibitory RNA molecule, said RNA molecule comprising: i. a sense nucleotide sequence comprising at least 19 out of 20 consecutive nucleotides from the nucleotide sequence of SEQ ID No 7, the nucleotide sequence of SEQ ID No 8, the nucleotide sequence of SEQ ID No 9 or the nucleotide sequence of SEQ ID No 10; and ii. an antisense nucleotide sequence comprising a nucleotide sequence complementary to said at least 20 consecutive nucleotides in said sense nucleotide sequence wherein said sense and antisense nucleotide sequence are capable of forming a double stranded RNA region; and c) a transcription termination and polyadenylation DNA region.
12. The method according to claim 11, wherein said antisense nucleotide sequence has about 95% sequence identity or is identical to said sense nucleotide sequence.
13. The method according to claim 1, wherein said transgene codes for a plant-functional enzyme of the nicotinamide adenine dinucleotide salvage synthesis pathway selected from nicotinamidase, nicotinate phosphoribosyltransferase, nicotinic acid mononucleotide adenyl transferase or nicotinamide adenine dinucleotide synthetase.
14. The method according to claim 13, wherein said transgene comprises a the nucleotide sequence of SEQ ID No. 11, the nucleotide sequence of SEQ ID No. 12, the nucleotide sequence of SEQ ID No. 13, the nucleotide sequence of SEQ ID No. 14, the nucleotide sequence of SEQ ID No. 15, the nucleotide sequence of SEQ ID No. 16, the nucleotide sequence of SEQ ID No. 17, the nucleotide sequence of SEQ ID No. 18, the nucleotide sequence of SEQ ID No. 19, the nucleotide sequence of SEQ ID No. 20, the nucleotide sequence of SEQ ID No. 21 or the nucleotide sequence of SEQ ID No. 22.
15. The method according to claim 1, comprising the further step of applying an effective amount of a compound of formula (I) ##STR00011## wherein Het represents a heterocycle which is either mono- or polysubstituted by fluorine, chlorine, methyl or ethyl, wherein said heterocycle is pyrid-3-yl, pyrid-5-yl, 3-pyridinio, 1-oxido-5-pyridinio, 1-oxido-5-pyridinio, tetra-hydrofuran-3-yl, or thiazol-5-yl, A represents C1-C6-alkyl, --N(R1)(R2) or S(R2), in which R1 represents hydrogen, C1-C6-alkyl, phenyl-C1-C4-alkyl, C3-C6-cycloalkyl, C2-C6-alkenyl or C2-C6-alkynyl, and R2 represents C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, --C(═O)--CH3 or benzyl, R represents hydrogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, --C(═O)--CH3 or benzyl or together with R2 represents the groups below: --CH2--CH2--, --CH2--CH2--CH2--, --CH2--O--CH2--, --CH2--S--CH2--, --CH2--NH--CH2--, or --CH2--N(CH1)--CH2--, and X represents N--NO2, N--CN or CH--NO2 on said plant or on its locus, or on seeds of said plant.
16. The method according to claim 15, wherein said heterocycle represented by Het of said compound of formula (I) is a pyrid-3-yl heterocycle substituted by chlorine.
17. The method of claim 16, wherein said compound of formula (I) is imidacloprid or thiacloprid.
18-29. (canceled)
30. A method for increasing the protrusion of the roots of a plant into a growth medium comprising a) transforming said plant with a foreign DNA comprising a stress tolerance enhancing transgene or a variant of an endogenous gene corresponding to such stress tolerance enhancing transgene, and/or b) applying an effective amount of 6-chloronicotinic acid or a compound of formula (I) ##STR00012## wherein Het represents a heterocycle which is either mono- or polysubstituted by fluorine, chlorine, methyl or ethyl, wherein said heterocycle is pyrid-3-yl, pyrid-5-yl, 3-pyridinio, 1-oxido-5-pyridinio, 1-oxido-5-pyridinio, tetra-hydrofuran-3-yl, or thiazol-5-yl, A represents C1-C6-alkyl, --N(R1)(R2) or S(R2), in which R1 represents hydrogen, C1-C6-alkyl, phenyl-C1-C4-alkyl, C3-C6-cycloalkyl, C2-C6-alkenyl or C2-C6-alkynyl, and R2 represents C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, --C(═O)--CH3 or benzyl, R represents hydrogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, --C(═O)--CH3 or benzyl or together with R2 represents the groups below: --CH2--CH2--, --CH2--CH2--CH2--, --CH2--O--CH2--, --CH2--S--CH2--, --CH2--NH--CH2--, or --CH2--N(CH3)--CH2--, and X represents N--NO2, N--CN or CH--NO.sub.2.
31. A method for increasing the tolerance of a plant to hypoxic or anoxic conditions comprising a) transforming a plant with a foreign DNA comprising a stress tolerance enhancing transgene or a variant of an endogenous gene corresponding to such stress tolerance enhancing transgene, and/or b) applying an effective amount of 6-chloronicotinic acid or a compound of formula (I) ##STR00013## wherein Het represents a heterocycle which is either mono- or polysubstituted by fluorine, chlorine, methyl or ethyl, wherein said heterocycle is pyrid-3-yl, pyrid-5-yl, 3-pyridinio, 1-oxido-5-pyridinio, 1-oxido-5-pyridinio, tetra-hydrofuran-3-yl, or thiazol-5-yl, A represents C1-C6-alkyl, --N(R1)(R2) or S(R2), in which R1 represents hydrogen, C1-C6-alkyl, phenyl-C1-C4-alkyl, C3-C6-cycloalkyl, C2-C6-alkenyl or C2-C6-alkynyl, and R2 represents C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, --C(═O)--CH3 or benzyl, R represents hydrogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, --C(═O)--CH3 or benzyl or together with R2 represents the groups below: --CH2--CH2--, --CH2--CH2--CH2--, --CH2--O--CH2--, --CH2--S--CH2--, --CH2--NH--CH2--, or --CH2--N(CH1)--CH2--, and X represents N--NO2, N--CN or CH--NO.sub.2.
32. The method of claim 31, wherein said hypoxic or anoxic conditions are brought on said plant by exposure to waterlogging, submergence, or flooding.
Description:
[0001] Methods are provided for increasing the resistance of plants to
hypoxic or anoxic conditions. Such methods may be applied to increase the
penetrance of plant roots in the growth medium or into soil. The methods
according to the invention may include modification of the genome of the
plants by providing such plants with an exogenous stress tolerance gene
or with a stress tolerant variant of an endogenous gene corresponding to
such an exogenous gene. The methods according to the invention may also
include applying neonicotinoid compounds, such as but not limited to
imidacloprid, nitenpyram, acetamiprid, thiacloprid, thiamethoxam,
clothianidin, and dinotefuran, to plants or their habitats, or to cells
or seeds thereof. Particularly effective neonicotinoid compounds are
neonicotinoid compounds which comprise a chloropyridine side chain, such
as imidacloprid, nitenpyram, acetamiprid, and thiacloprid, particularly
those during the degradation of which in plants 6-chloronicotininc acid
(6-CNA) can be set free, like, e.g., imidacloprid and thiacloprid. The
plants or their habitats can also be treated directly with 6-CNA.
BACKGROUND ART
[0002] Plants engineered to be stress tolerant are known in the art. Stress tolerance in plant cells and plants can, e.g., be achieved by reducing the activity or the level of the endogenous poly-ADP-ribose polymerases (PARP) or poly(ADP-ribose) glycohydrolases (PARG) as described in WO00/04173 and WO04/090140, respectively.
[0003] European patent application No. 04077624.7 describes that stress tolerance in plants and plant cells is achieved using nucleotide sequences encoding enzymes involved in the NAD salvage synthesis pathway and/or the NAD de novo synthesis pathway e.g. for overexpression in plants.
[0004] However, none of these documents disclose the possibility to use the stress tolerance genes mentioned therein for obtaining tolerance to hypoxic or anoxic conditions in plant cells and plants. Neither do they disclose the use of the stress tolerance genes described therein for the purpose of allowing the root system of the plant to penetrate deeper into the growth medium or the soil.
[0005] The application of compounds of the class of neonicotinoids on plants for purposes other than insect control is also known from the art (WO 01/26468, WO 03/096811).
[0006] WO 01/26468 discloses a method of improving the growth of plants comprising applying to the plants or the locus thereof at least one compound selected from the class of the neonicotinoids.
[0007] WO03/096811 describes that the yield and/or the vigor of an agronomic plant can be increased or improved in locations where the level of insect infestation below that indicating the need for the use of an insecticide for insect control purposes by treating a seed of the plant with a neonicotinoid compound. The method is deemed useful for non-transgenic plants and for plants having a foreign gene that encodes for the production of a modified Bacillus thuringiensis delta-endotoxin protein.
[0008] None of these documents however describe the use of compounds of the class of the neonicotinoids on plants for the purpose of increasing the tolerance of plant cells or plants to hypoxic or anoxic conditions or to allow the root system of the plant to penetrate deeper into the growth medium or the soil.
[0009] Thus, the art remains silent on methods to increase the depth of penetration of a root system or roots of a plant into the growth medium or soil, or to increase tolerance of plant cells or plants to hypoxic or anoxic stress conditions using stress tolerance genes or by application of a chemical compound of the neonicotinoid class to plants, or cells thereof as described hereinafter in the different embodiments and claims.
SUMMARY OF THE INVENTION
[0010] In one embodiment of the invention, a novel method of increasing the tolerance of plant cells or plants to hypoxic or anoxic conditions is provided comprising, providing the plant cells or plants with a stress tolerance enhancing transgene, wherein the stress tolerance enhancing transgene is selected from:
[0011] a stress tolerance enhancing transgene capable of reducing the expression of plant endogenous PARP genes, particularly wherein the transgene codes for a PARP inhibitory RNA molecule
[0012] a stress tolerance enhancing transgene capable of reducing the expression of plant endogenous PARG genes, particularly wherein the transgene codes for a PARG inhibitory RNA molecule; or
[0013] a stress tolerance enhancing transgene coding for a plant-functional enzyme of the nicotinamide adenine dinucleotide salvage synthesis pathway selected from nicotinamidase, nicotinate phosphoribosyltransferase, nicotinic acid mononucleotide adenyl transferase or nicotinamide adenine dinucleotide synthetase.
[0014] In another embodiment, the invention relates to the use of such stress tolerance enhancing transgenes to increase the penetrance of plant roots in growth medium, including soil.
[0015] In yet another embodiment of the invention, a method for increasing the tolerance of plant cells or plants to hypoxic or anoxic conditions is provided comprising applying to the plant cell, plant or seed from which such plant is grown, or to the habitat thereof, an effective amount of a neonicotinoid compound.
[0016] In still another embodiment, the invention relates to the use of such compounds to increase the penetrance of plant roots in growth medium, including soil.
[0017] The invention further relates to a method for increasing the tolerance of plant cells or plants to hypoxic or anoxic conditions comprising the step of providing cells of said plant with an effective amount of 6-chloronicotinic acid.
[0018] The invention also relates to the use of 6-CNA for increasing the penetrance of plant roots into the growth medium.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1: Schematic representation of the assay to measure root depth of a plant growing in a agar solution. A container (1) with a seal (2) is filled with a transparent or translucent growth medium (3), such as 0.4% agar-water or 0.7% agar-water, to which additional test-compounds may be added. One pregerminated seed is added to the tube and allowed to grow for three weeks. After three weeks of growth in vertical position, the root depth (5) of the plant (4) is measured from the top of the medium to the lowest point of the roots.
[0020] FIG. 2: Boxplot representation of the root depth (mm) of Arabidopsis thaliana cv. Col-0 plants comprising a transgene encoding a dsRNA molecule capable of reducing the expression of endogenous PARP2 genes compared to non-transgenic Arabidopsis thaliana plants (Col-0).
[0021] The following populations were analysed:
[0022] Col-0: data points for wild-type Arabidopsis line
[0023] 427-16: data points for A. thaliana transgenic line comprising anti-PARP2 gene with a weak tolerance to high light stress conditions
[0024] 427-20: data points for A. thaliana transgenic line comprising anti-PARP2 gene with a weak tolerance to high light stress conditions
[0025] 427-20: data points for A. thaliana transgenic line comprising anti-PARP2 gene with a moderate tolerance to high light stress conditions
[0026] The graph represents a typical boxplot (or box and whisker plot) on the left of each of the groups of values, summarizing the following statistical measures:
[0027] median
[0028] upper and lower quartiles
[0029] minimum and maximum data values.
[0030] In addition, on the right hand for each of the groups of values, the mean and the standard error of the mean for those values is indicated.
[0031] The boxplot is interpreted as follows:
[0032] the box itself contains the middle 50% of the data. The upper edge (hinge) of the box indicates the 75th percentile of the data set, and the lower hinge indicates the 25th percentile. The range of the middle two quartiles is known as the inter-quartile range.
[0033] The line in the box indicates the median value of the data.
[0034] The ends of the whiskers indicate the minimum and maximum data values, unless outliers are present in which case the whiskers extend to the nearest value points within a range of 1.5 times the interquartile range.
[0035] FIG. 3: Boxplot representation and standard error of the mean for the measured values for root depth (mm) of Arabidopsis thaliana cv. Col-plants comprising a transgene encoding a dsRNA molecule capable of reducing the expression of endogenous PARP genes.
[0036] The following populations were analyzed:
[0037] Col-0: data points for wild-type Arabidopsis line
[0038] 427-22: data points for A. thaliana transgenic line comprising anti-PARP2 gene with a high tolerance to high light stress conditions
[0039] 427-24: data points for A. thaliana transgenic line comprising anti-PARP2 gene with a low tolerance to high light stress conditions
[0040] FIG. 4: Boxplot representation and standard error of the mean for the measured values for root depth (mm) of Arabidopsis thaliana cv. C24-plants comprising a transgene encoding a dsRNA molecule capable of reducing the expression of endogenous PARP genes.
[0041] The following populations were analyzed:
[0042] C24: data points for wild-type Arabidopsis line
[0043] 1599: data points for A. thaliana transgenic line comprising anti-PARP2 gene with a high tolerance to high light stress conditions
[0044] 1463: data points for A. thaliana transgenic line comprising anti-PARP2 gene with a moderate tolerance to high light stress conditions
[0045] 1681: data points for A. thaliana transgenic line comprising anti-PARP1 gene with a moderate tolerance to high light stress conditions
[0046] 1690: data points for A. thaliana transgenic line comprising anti-PARP1 gene with a moderate tolerance to high light stress conditions
[0047] FIG. 5: Boxplot representation and standard error of the mean for the measured values for root depth (mm) measured on an A. thaliana Col-0 population segregating for the anti-PARP2 transgene.
[0048] The following populations were analyzed:
[0049] Azygous: data points for A. thaliana plants from the population which do not contain an anti-PARP2 gene
[0050] Transgenic: data points for A. thaliana plants from the population which contain an anti-PARP2 gene
[0051] FIG. 6: Boxplot representation and standard error of the mean for the measured values for root depth (mm) of Arabidopsis thaliana cv. C24 plants treated with various concentrations of imidacloprid as compared to untreated Arabidopsis thaliana cv. C24 plants.
[0052] The following populations were analyzed:
[0053] 0: untreated A. thaliana C24 plants
[0054] 50: A. thaliana C24 plants treated with 50 mg/L imidacloprid
[0055] 100: A. thaliana C24 plants treated with 100 mg/L imidacloprid
[0056] FIG. 7: Boxplot representation and standard error of the mean for the measured values for root depth (mm) of Arabidopsis thaliana cv. C24 plants treated with various concentrations of 6-chloronicotinic acid as compared to untreated Arabidopsis thaliana cv. C24 plants.
[0057] The following populations were analyzed:
[0058] 0: untreated A. thaliana C24 plants
[0059] 1: A. thaliana C24 plants treated with 1 mg/L 6-chloronicotinic acid
[0060] 5: A. thaliana C24 plants treated with 5 mg/L 6-chloronicotinic acid
DETAILED DESCRIPTION
[0061] The current invention is based on the realization that plants comprising stress tolerance genes, such as the chimeric genes encoding dsRNA targeted for silencing the expression of parp1 or parp2 genes of plants, developed a root system with roots that protruded deeper into the growth medium than the roots of control plants. The deeper penetrance of the roots has remained unnoticed with the plants comprising stress tolerance genes as described in the prior art and required the development of a particular assay, as described herein, to analyze statistically the root penetrance into the medium.
[0062] Although not intending to limit the invention to a particular mode of action, it is tought that the stress tolerance genes increase the tolerance of plant cells, including the plant cells of roots to hypoxic or anoxic conditions, thereby allowing the roots comprising such a stress tolerance gene to grow in less favourable oxygen conditions, as can be found in the deeper areas of a growth medium or the deeper soil layers, where the oxygen tension is lower. The increased penetrance of the root system of plants into the deeper layers of the soil, provides a partial explanation for the increased drought resistance under field conditions observed for plants comprising the stress tolerance genes as described herein, such as the dsRNA encoding genes silencing the expression of the endogeneous parp1 or parp2 genes.
[0063] A similar effect on the root protrusion could be observed in described assay after addition of compound of the neonicotinoid class or 6-chloronicotinic acid. The effect of the application of neonicotinoids on root growth depth is independent of the presence of insects which are the targets of the above-mentioned neonicotinoids. Accordingly, the effect is also connected with the biochemical improvement of stress tolerance, particularly hypoxia- or anoxia-related stress tolerance, of a plant or plant cell or the seed from which it is grown.
[0064] Accordingly, in a first embodiment, the invention is directed towards the use of a stress tolerance enhancing transgene to increase the tolerance of a plant cell, plant or seed to hypoxic or anoxic conditions.
[0065] As used herein, "hypoxic or anoxic conditions" refer to conditions to which plant cells, plants or parts of such plants are exposed wherein the availability of oxygen is low to very low. Anoxic conditions refer to conditions where there is almost no oxygen available. Typically, conditions wherein the dissolved oxygen concentration is below about 2 mg/L, are indicated as hypoxic (0.1 mg/L to 2 mg/L), conditions wherein the dissolved oxygen is below 0.1 mg/L, particularly below 0.05 mg/L are indicated as anoxic. Normal dissolved oxygen concentration in water is about 8 mg/L. Hypoxic conditions in soil refer to those conditions where the oxygen tension is low, particularly where oxygen drops below 5% in the soil atmosphere.
[0066] Hypoxic conditions may occur e.g. upon flooding of the plants or parts of the plants. Hypoxic conditions may also occur where oxygen consumption is high, such as in soil layers comprising a lot of organic debris in the process of metabolization by microorganisms. Furhtermore, hypoxic conditions occur in the deeper layers of a growth medium where diffusion of oxygen occurs from the surface. Hypoxic conditions also occur in the deeper layers of the soil as oxygen diffusion and consequently oxygen tension decreases from the surfaces. The rate of decrease in oxygen depends on the compactness of the soil (whereby the more compact the soil, the less soil atmosphere is present), the presence of decomposing organic material, water content etc.
[0067] As used herein, "a stress tolerance enhancing transgene" refers to a transgene which when introduced or expressed in a plant cell or plant, provides the cell or the plant with a better tolerance to stress which is brought on a plant, e.g., by the application of chemical compounds (e.g., herbicides, fungicides, insecticides, plant growth regulators, adjuvants, fertilizers), exposure to abiotic stress (e.g., drought, waterlogging, submergence, high light conditions, high UV radiation, increased hydrogen peroxide levels, extreme (high or low) temperatures, ozone and other atmospheric pollutants, soil salinity or heavy metals, hypoxia, anoxia, etc.) or biotic stress (e.g., pathogen or pest infection including infection by fungi, viruses, bacteria, insects, nematodes, mycoplasms and mycoplasma like organisms, etc.).
[0068] Such a stress tolerance enhancing transgene may be a transgene capable of reducing the expression and/or the activity of poly(ADP-ribose)polymerase (PARP) gene in the plant cells or plants as described in WO 00/04173 or EP 04077984.5 (herein incorporated by reference).
[0069] Poly(ADP-ribose) polymerase (PARP), also known as poly(ADP-ribose) transferase (ADPRT) (EC 2.4.2.30), is a nuclear enzyme found in most eukaryotes, including vertebrates, arthropods, molluscs, slime moulds, dinoflagellates, fungi and other low eukaryotes with the exception of yeast. The enzymatic activity has also been demonstrated in a number of plants (Payne et al., 1976; Willmitzer and Wagner, 1982; Chen et al., 1994; O'Farrell, 1995).
[0070] PARP catalyzes the transfer of an ADP-ribose moiety derived from NAD+, mainly to the carboxyl group of a glutamic acid residue in the target protein, and subsequent ADP-ribose polymerization. The major target protein is PARP itself, but also histones, high mobility group chromosomal proteins, topoisomerase, endonucleases and DNA polymerases have been shown to be subject to this modification.
[0071] As a particular embodiment, the stress tolerance enhancing transgene may comprise the following operably linked DNA fragments:
[0072] a) a plant-expressible promoter;
[0073] b) a DNA region which when transcribed results in an RNA molecule capable of reducing the expression of the endogenous PARP encoding genes of a plant (a PARP inhibitory RNA molecule);
[0074] c) a DNA region involved in transcription termination and polyadenylation.
[0075] The mentioned DNA region may result upon transcription in a so-called antisense RNA molecule reducing in a transcriptional or post-transcriptional manner the expression of a PARP encoding gene in the target plant or plant cell, comprising at least 20 or 21 consecutive nucleotides having at least 95% to 100% sequence identity to the complement of the nucleotide sequence of a PARP encoding gene present in the plant cell or plant.
[0076] The mentioned DNA region may also result in a so-called sense RNA molecule comprising reducing in a transcriptional or post-transcriptional manner the expression of a PARP encoding gene in the target plant or plant cell, comprising at least 20 or 21 consecutive nucleotides having at least 95% to 100% sequence identity to the nucleotide sequence of a PARP encoding gene present in the plant cell or plant.
[0077] However, the minimum nucleotide sequence of the antisense or sense RNA region of about 20 nt of the PARP coding region may be comprised within a larger RNA molecule, varying in size from 20 nt to a length equal to the size of the target gene. The mentioned antisense or sense nucleotide regions may thus be about from about 21 nt to about 5000 nt long, such as 21 nt, 40 nt, 50 nt, 100 nt, 200 nt, 300 nt, 500 nt, 1000 nt, 2000 nt or even about 5000 nt or larger in length. Moreover, it is not required for the purpose of the invention that the nucleotide sequence of the used inhibitory PARP RNA molecule or the encoding region of the transgene, is completely identical or complementary to the endogenous PARP gene the expression of which is targeted to be reduced in the plant cell. The longer the sequence, the less stringent the requirement for the overall sequence identity is. Thus, the sense or antisense regions may have an overall sequence identity of about 40% or 50% or 60% or 70% or 80% or 90% or 100% to the nucleotide sequence of the endogenous PARP gene or the complement thereof. However, as mentioned antisense or sense regions should comprise a nucleotide sequence of 20 consecutive nucleotides having about 100% sequence identity to the nucleotide sequence of the endogenous PARP gene. Preferably the stretch of about 100% sequence identity should be about 50, 75 or 100 nt.
[0078] For the purpose of this invention, the "sequence identity" of two related nucleotide sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (×100) divided by the number of positions compared. A gap, i.e. a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues. The alignment of the two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch 1970) Computer-assisted sequence alignment, can be conveniently performed using standard software program such as GAP which is part of the Wisconsin Package Version 10.1 (Genetics Computer Group, Madison, Wis., USA) using the default scoring matrix with a gap creation penalty of 50 and a gap extension penalty of 3.
[0079] It will be clear that whenever nucleotide sequences of RNA molecules are defined by reference to nucleotide sequence of corresponding DNA molecules, the thymine (T) in the nucleotide sequence should be replaced by uracil (U). Whether reference is made to RNA or DNA molecules will be clear from the context of the application.
[0080] The efficiency of the above mentioned transgenes in reducing the expression of the endogenous PARP gene may be further enhanced by inclusion of DNA elements which result in the expression of aberrant, unpolyadenylated PARP inhibitory RNA molecules. One such DNA element suitable for that purpose is a DNA region encoding a self-splicing ribozyme, as described in WO 00/01133 A1.
[0081] The efficiency of the above mentioned transgenes in reducing the expression of the endogenous PARP gene of a plant cell may also be further enhanced by including into one plant cell simultaneously a transgene as herein described encoding a antisense PARP inhibitory RNA molecule and a transgene as herein described encoding a sense PARP inhibitory RNA molecule, wherein said antisense and sense PARP inhibitory RNA molecules are capable of forming a double stranded RNA region by base pairing between the mentioned at least 20 consecutive nucleotides, as described in WO 99/53050 A1.
[0082] As further described in WO 99/53050 A1, the sense and antisense PARP inhibitory RNA regions, capable of forming a double stranded RNA region may be present in one RNA molecule, preferably separated by a spacer region. The spacer region may comprise an intron sequence. Such a transgene may be conveniently constructed by operably linking a DNA fragment comprising at least 20 nucleotides from the isolated or identified endogenous PARP gene, the expression of which is targeted to be reduced, in an inverted repeat, to a plant expressible promoter and 3' end formation region involved in transcription termination and polyadenylation. To achieve the construction of such a transgene, use can be made of the vectors described in WO 02/059294 A1.
[0083] Current nomenclature refers to the classical Zn-finger-containing polymerases as PARP1 proteins (and corresponding parp1 genes) whereas the structurally non-classical PARP proteins are currently referred to as PARP2 (and corresponding parp2 genes) and "PARP encoding genes" as used herein, may refer to either type.
[0084] The following database entries (herein incorporated by reference) identifying experimentally demonstrated and putative poly ADP-ribose polymerase protein sequences, parts thereof or homologous sequences, could be used according to the current invention: BAD53855 (Oryza sativa); BAD52929 (Oryza sativa); XP--477671 (Oryza sativa); BAC84104 (Oryza sativa); AAT25850 (Zea mays); AAT25849 (Zea mays); NP--197639 (Arabidopsis thaliana); NP--850165 (Arabidopsis thaliana); NP--188107 (Arabidopsis thaliana); NP--850586 (Arabidopsis thaliana); BAB09119 (Arabidopsis thaliana); AAD20677 (Arabidopsis thaliana); Q11207 (Arabidopsis thaliana); C84719 (Arabidopsis thaliana); T51353 (Arabidopsis thaliana); TO1311 (Arabidopsis thaliana); AAN12901 (Arabidopsis thaliana); AAM13882 (Arabidopsis thaliana); CAB80732 (Arabidopsis thaliana); CAA10482 (Arabidopsis thaliana); AAC79704 (Zea mays): AAC19283 (Arabidopsis thaliana); CAA10888 (Zea mays); CAA10889 (Zea mays); CAA88288 (Arabidopsis thaliana).
[0085] As a particular embodiment of the invention, the PARP gene expression reducing gene may comprise the following operably linked DNA fragments:
[0086] a) a plant expressible promoter
[0087] b) a DNA region which when transcribed yields an RNA molecule, the RNA molecule comprising:
[0088] a. An antisense nucleotide sequence comprising at least about 20 consecutive nucleotides having about 96% sequence identity to a nucleotide sequence of about 20 consecutive nucleotides selected from the nucleotide sequences of SEQ ID 1 (Arabidopsis parp 1 coding region) SEQ ID 2 (Arabidopsis parp 2 coding region) SEQ ID 3 (Zea mays parp1 coding region), SEQ ID 4 (another Zea mays parp1 coding region), SEQ ID 5 (Zea mays parp2 coding region) or SEQ ID 6 (cotton parp2 partial cDNA) or from nucleotide sequences encoding proteins with similar or identical amino acid sequences as encoded by the mentioned nucleotide sequences.
[0089] b. A sense nucleotide sequence comprising at least about 20 nucleotides which are complementary to the antisense nucleotide sequence. The sense nucleotide sequence may thus comprise a sequence of at least about 20 consecutive nucleotides having about 96% sequence identity to a nucleotide sequence of about 20 consecutive nucleotides selected from the nucleotide sequences of SEQ ID 1 (Arabidopsis parp1 coding region) SEQ ID 2 (Arabidopsis parp2 coding region) SEQ ID 3 (Zea mays parp1 coding region), SEQ ID 4 (another Zea mays parp1 coding region), SEQ ID 5 (Zea mays parp2 coding region) or SEQ ID 6 (cotton parp2 partial cDNA) of from nucleotide sequences encoding proteins with similar or identical amino acid sequences as encoded by the mentioned nucleotide sequences;
[0090] whereby the sense and antisense nucleotide sequence are capable of forming a double stranded RNA molecule (dsRNA);
[0091] c) A DNA region for transcription termination and polyadenylation.
[0092] However, it will be clear that other PARP gene expression reducing genes as described in WO00/04173 or EP 04077984.5 may be used.
[0093] In another embodiment of the invention, the stress tolerance enhancing transgene may be a transgene capable of reducing the expression and/or the activity of the PARG encoding genes of the plants or plants cells, as described e.g. in WO 2004/090140 (herein incorporated by reference).
[0094] PARG (poly (ADP-ribose) glycohydrolase; E.C.3.2.1.143) converts poly (ADP-ribose) polymers to free ADP-ribose by its exoglycosidase and endoglycosidase activity (PARG).
[0095] In plants, a poly(ADP-ribose) glycohydrolase has been identified by map-based cloning of the wild-type gene inactivated in a mutant affected in clock-controlled transcription of genes in Arabidopsis and in photoperiod dependent transition from vegetative growth to flowering (tej). The nucleotide sequence of the gene can be obtained from nucleotide databases under the accession number AF394690 (Panda et al., 2002 Dev. Cell. 3, 51-61; SEQ ID No 7)
[0096] Nucleotide sequences of other plant PARG encoding genes from plants can be found in WO 2004/090140 A2, such as the PARG gene from Solanum tuberosum (SEQ ID No 8); Oryza sativa (SEQ ID No 9) or Zea mays (SEQ ID No 10) as well as methods to isolate additional PARG encoding genes and variants thereof from other plants.
[0097] Thus, in one embodiment, the plants or plant cells engineered to be stress resistant may comprise the following operably linked DNA fragments:
[0098] a) a plant expressible promoter
[0099] b) a DNA region, which when transcribed yields an inhibitory RNA molecule, the RNA molecule comprising
[0100] i. a antisense nucleotide region comprising at least 20 consecutive nucleotides having at least 96% sequence identity to a nucleotide sequence of about 20 nucleotides selected from the complement of a nucleotide sequence encoding a plant PARG protein, such as the nucleotide sequences of SEQ ID 7, SEQ ID 8, SEQ ID 9 or SEQ ID 10 or nucleotide sequences encoding proteins with similar or identical amino acid sequences as the nucleotide sequences mentioned; or
[0101] ii. a sense nucleotide region comprising at least 20 consecutive nucleotides selected from a nucleotide sequence encoding a plant PARG protein, such as the nucleotide sequences of SEQ ID 7, SEQ ID 8, SEQ ID 9 or SEQ ID 10 or nucleotide sequences encoding proteins with similar or identical amino acid sequences as the nucleotide sequences mentioned; or
[0102] iii. a antisense and sense nucleotide sequences as mentioned sub i) or ii) whereby said antisense and sense nucleotide sequence are capable of forming a double stranded RNA molecule;
[0103] c) A DNA region involved in transcription termination and polyadenylation.
[0104] It will be immediately clear to the skilled artisan that additional parameters of length of sense and antisense nucleotide sequences or dsRNA molecules, and sequence identity for the ParG inhibitory RNA molecules can be used as mentioned above for the PARP inhibitory RNA molecules.
[0105] In yet another embodiment of the invention, the stress tolerance enhancing transgene may a transgene coding for a plant-function enzyme of the nicotineamide adenine dinucleotide salvage synthesis pathway. Accordinly, the stress tolerance enhancing gene may comprise the following operably linked DNA molecules:
[0106] a) a plant-expressible promoter;
[0107] b) a DNA region coding for a plant-functional enzyme of the nicotinamide adenine dinucleotide salvage synthesis pathway selected from nicotinamidase, nicotinate phosphoribosyltransferase, nicotinic acid mononucleotide adenyl transferase or nicotinamide adenine dinucleotide synthetase; and
[0108] c) a 3' end region involved in transcription termination and polyadenylation, as described in EP 04077624.7 (herein incorporated by reference).
[0109] As used herein, "a plant-functional enzyme of the nicotinamide adenine dinucleotide salvage synthesis pathway" is an enzyme which when introduced into plants, linked to appropriate control elements such as plant expressible promoter and terminator region, can be transcribed and translated to yield a enzyme of the NAD salvage synthesis pathway functional in plant cells. Included are the enzymes (and encoding genes) from the NAD salvage synthesis, which are obtained from a plant source, but also the enzymes obtained from yeast (Saccharomyces cereviseae) or from other yeasts or fungi. It is thought that the latter proteins may be even more suitable for the methods according to the invention, since these are less likely to be subject to the enzymatic feedback regulation etc. to which similar plant-derived enzymes may be subject.
[0110] Enzymes involved in the NAD salvage synthesis pathway comprise the following
[0111] Nicotinamidase (EC 3.5.1.19) catalyzing the hydrolysis of the amide group of nicotinamide, thereby releasing nicotinate and NH3. The enzyme is also known as nicotinamide deaminase, nicotinamide amidase, YNDase or nicotinamide amidohydrolase
[0112] Nicotinate phosphoribosyltransferase (EC 2.4.2.11) also known as niacin ribonucleotidase, nicotinic acid mononucleotide glycohydrolase; nicotinic acid mononucleotide pyrophosphorylase; nicotinic acid phosphoribosyltransferase catalyzing the following reaction
[0112] Nicotinate-D-ribonucleotide+diphosphate=nicotinate+5-phospho-.alp- ha.-D ribose 1-diphosphate
[0113] Nicotinate-nucleotide adenylyltransferase, (EC 2.7.7.18) also known as deamido-NAD+ pyrophosphorylase; nicotinate mononucleotide adenylyltransferase; deamindonicotinamide adenine dinucleotide pyrophsophorylase; NaMT-ATase; nicotinic acid mononucleotide adenylyltransferase catalyzing the following reaction
[0113] ATP+nicotinate ribonucleotide=diphosphate+deamido-NAD+
[0114] NAD-synthase (EC 6.3.1.5) also known as NAD synthetase; NAD+synthase; nicotinamide adenine dinucleotide synthetase; diphosphopyridine nucleotide synthetase, catalyzing the following reaction
[0114] Deamido-NAD++ATP+NH3=AMP+diphosphate+NAD+
[0115] In one embodiment of the invention, the DNA regions coding for a plant functional enzyme of the NAD salvage pathway may comprise a nucleotide sequence from SEQ ID Nos 11, 12, 13, 14 or 15 or a nucleotide sequence encoding a protein with similar or identical amino acid sequences as the proteins encoded by the above mentioned nucleotide sequences.
[0116] As described by Hunt et al., 2004, plant homologues of these enzymes have been identified and these DNA sequences may be used to similar effect (Hunt et al., 2004, New Phytologist163(1): 31-44). The identified DNA sequences have the following Accession numbers: for nicotinamidase: At5g23220 (SEQ ID No 16); At5g23230 (SEQ ID No 17) and At3g16190 (SEQ ID No 18); for nicotinate phosphoribosyltransferase: At4g36940 (SEQ ID No 19), At2g23420 (SEQ ID No 20), for nicotinic acid mononucleotide adenyltransferase: At5g55810 (SEQ ID No 21) and for NAD synthetase: At1g55090 (SEQ ID No 22).
[0117] However, it will be clear that the plants engineered to be stress resistant may also comprise variants of these nucleotide sequences, including insertions, deletions and substitutions thereof. Equally, homologues to the mentioned nucleotide sequences from species different from Saccharomyces cerevisea can be used. These include but are not limited to nucleotide sequences from plants, and nucleotide sequences encoding proteins with the same amino acid sequences, as well as variants of such nucleotide sequences.
[0118] Variants of the described nucleotide sequence will have a sequence identity which is preferably at least about 80%, or 85 or 90% or 95% with identified nucleotide sequences encoding enzymes from the NAD salvage pathway, such as the ones identified in the sequence listing. Preferably, these variants will encode functional proteins with the same enzymatic activity as the enzymes from the NAD salvage pathway.
[0119] Having read the above description of the use according to the invention of stress tolerance enhancing transgenes to increase tolerance of plant cells, plants or seeds to hypoxic or anoxic conditions, the skilled person will immediately realize that similar effects can be obtained using variants of an endogenous gene corresponding to such a stress tolerance enhancing transgene, which variant results in higher stress tolerance of the plant cells or plants harbouring such a variant. By way of example, variants of an endogenous parp2 gene of a plant, having a low expression level and providing the harbouring plant with increased stress tolerance could be used in a similar way as a transgene reducing the expression of the endogenous parp2 gene. Such variants gene can be introduced into plant cells or plants by breeding techniques.
[0120] A person skilled in the art will also be aware that expression of the different stress tolerance enhancing genes or transgenes may lead to a population of different events, which exhibit a distribution of effects ranging from almost no effect to a very pronounced effect. However, a person skilled in the art will clearly be able to distinguish, identify or isolate those representatives of a population that best suit the needs.
[0121] In another embodiment, the invention provides a method for increasing the penetrance of the roots of a plant into growth medium or soil comprising the step of providing the plant with a stress tolerance enhancing transgene, or with a endogenous variant of such stress tolerance enhancing transgene, as herein described in its different embodiments.
[0122] As used herein, "protrusion of plant roots" or "the penetrance of the roots of a plant into growth medium or soil" refers to the depth of the growth of roots in solid growth medium, including soil, as measured from the surface of the growth medium to the lowest point of the roots (see also FIG. 1).
[0123] As a rule, an "increase in the protrusion or penetrance of plant roots" means at least a statistically significant increase in the depth of the growth of roots in growth medium as measured from the surface of the medium to the lowest point of root growth, which can be measured either as a difference in a comparison of the root depth of wild-type reference plants versus the root depth of plants engineered to be stress tolerant, or as a difference in a comparison of the root depth of plants treated with particular chemical compounds versus the root depth of untreated plants.
[0124] For a correct understanding of the invention, it is important to realize that deeper penetration of a root system of a plant into the growth medium or soil, achieved by the methods according to the invention, is not to be equalled with an increase of the root system in volume or dry or fresh weight. Indeed, the volume of a root system may be increased significantly, while the roots all remain quite superficial below the surface of the growth medium or soil. By contrast, roots of plants treated according to the invention may be equal in size, volume, weight or even length, yet protrude much deeper below the surface of the growth medium or the soil.
[0125] As used herein, "growth medium" is intended to refer to any medium suitable for plant growth including soil. Such media may include solidified or gellified liquids, such as water-agar, peat, turf, different types of soil etc.
[0126] In another embodiment, the invention is directed towards the use of a compound of the neonicotinoid class to increase the tolerance of a plant cell, plant or seed to hypoxic or anoxic conditions. Thus, a method is provided to increase the tolerance of a plant cell, plant or seed to hypoxic or anoxic conditions comprising the step of applying an effective amound of a neonicotinoid compound of the formula (I) to the plant cells, plants or seeds or to the habitat of the plants, or to the growth medium.
##STR00001##
wherein
[0127] Het represents a heterocycle which is in each case optionally mono- or polysubstituted by fluorine, chlorine, methyl or ethyl, which heterocycle is selected from the following group of heterocycles:
[0128] pyrid-3-yl, pyrid-5-yl, 3-pyridinio, 1-oxido-5-pyridinio, 1-oxido-5-pyridinio, tetra-hydrofuran-3-yl, thiazol-5-yl,
[0129] A represents C1-C6-alkyl, --N(R1)(R2) or S(R2),
[0130] in which
[0131] R1 represents hydrogen, C1-C6-alkyl, phenyl-C1-C4-alkyl, C3-C6-cycloalkyl, C2-C6-alkenyl or C2-C6-alkynyl, and
[0132] R2 represents C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, --C(═O)--CH3 or benzyl,
[0133] R represents hydrogen, C1-C6-alkyl, C2-C6-alkenyl, C2-C6-alkynyl, --C(═O)--CH3 or benzyl or together with R2 represents the groups below:
[0134] --CH2--CH2--, --CH2--CH2--CH2--, --CH2--O--CH2--, --CH2--S--CH2--, --CH2--NH--C1-12--, --CH2--N(CH3)--CH2--, and
[0135] X represents N--NO2, N--CN or CH--NO2.
[0136] Saturated or unsaturated hydrocarbon radicals, such as alkyl or alkenyl, can in each case be straight-chain or branched as far as this is possible, including in combination with heteroatoms, such as, for example, in alkoxy.
[0137] These compounds are known to have insecticidal activity (see, for example, EP-A1-192 606, EP-A2-580 533, EP-A2-376 279, EP-A2-235 725).
[0138] Compounds of the formula (I) which may be mentioned are the neonicotinoids listed in "The Pesticide Manual", 13th Edition, 2003 (British Crop Protection Council).
[0139] One compound is imidacloprid of the formula
##STR00002##
known, for example, from EP A1 0 192 060.
[0140] Another compound is nitenpyram of the formula
##STR00003##
known, for example, from EP A2 0 302 389.
[0141] Another compound is acetamiprid of the formula
##STR00004##
known, for example, from WO A1 91/04965.
[0142] Another compound is thiacloprid of the formula
##STR00005##
known, for example, from EP A2 0 235 725.
[0143] Another compound is thiamethoxam of the formula
##STR00006##
known, for example, from EP A2 0 580 553.
[0144] Another compound is clothianidin of the formula
##STR00007##
know, for example, from EP A2 0 376 279.
[0145] Another compound is dinotefuran of the formula
##STR00008##
known, for example, from EP A 1 0 649 845.
[0146] Particularly suited for the current inventions are compounds of the formula (I) wherein the substituent "Het" represents chloropyridyl
##STR00009##
such as imidacloprid, nitenpyram, acetamiprid, and thiacloprid.
[0147] Particularly preferred compounds are imidacloprid and thiacloprid.
[0148] 6-Chloronicotinic acid can be set free during the degradation of the above mentioned neonicotinoids which carry this group, such as imidacloprid, nitenpyram, and thiacloprid. For example, imidacloprid is degraded stepwise to the primary metabolite 6-chloronicotinic acid, which eventually breaks down into carbon dioxide. It was found that this metabolite also increases the stress tolerance and health of a plant or plant cell or seed from which such plant is grown and which is engineered to be stress tolerant and can be also be used according to the methods of the current invention.
[0149] One way of determining whether 6-CNA is set free during the degradation of the above mentioned neonicotinoids in plants or in particular plants is described by Placke and Weber (Pflanzenschutz-Nachrichten Bayer 46/1993, 2 109-182).
[0150] Thus, in another embodiment of the invention, a method is described which is useful to increase the tolerance of plants cells or plants or parts thereof to hypoxic or anoxic conditions comprising the step of providing to said plant, to a plant cell or to seed from which said plants are grown an effective amount of 6-chloronicotinic acid (niacin, CAS NO: 5326-23-8) of the formula (3)
##STR00010##
[0151] The effective amound of 6-chloronicotinic acid may be provided to the plant cell, plant or seed by applying directly the compound of the formula (3) to the plant cell, plant, seed and/or the habitat thereof. However, the 6-CNA may also be provided to the plant by providing a compound which can be metabolized by the plant to yield 6-CNA as a metabolite, such as the compounds mentioned above.
[0152] It will be immediately clear that the above described compounds can also be used to increase the penetrance of the roots of a plant into growth medium or soil comprising the step of providing an effective amount of a neonicotinoid compound of the formula (I), such as a neonicotinoid compound of the formula (I) comprising a chloropyridine side chain, particularly those neonicotinoids of the formula I comprising a chloropyridine side chain which can be metabolized in plants to yield 6-CNA, including imidacloprid or thiacloprid or providing 6-CNA to the plant cells, plants or seeds or to the habitat of the plants or to the growth medium.
[0153] One of the advantages of the present invention is that the systemic properties of the compounds according to the invention and compositions comprising said compounds mean that treatment of the seed of plants with these compositions is sufficient to increase the protrusion of the roots of the germinating plant and the resulting plant after emergence.
[0154] In another embodiment of the invention, a method is described which is useful to increase the protrusion of the roots of a plant, comprising applying to said plant and/or its habitat, to a plant cell or to seed from which said plants are grown an effective amount of a composition comprising the compounds of the formula (I).
[0155] Accordingly, the invention also relates to compositions comprising the compounds of the formula (I) for the use of such compositions according to the invention.
[0156] The compounds of formula (I) can be used also in a mixture with other active compounds, for example, insecticides, bactericides, miticides, fungicides, etc. in the form of their commercially useful formulations or in the application forms prepared from such formulations. This can be done to obtain compositions which in addition to increasing the protrusion of plant roots according to the invention also to combat pests which may be present. Insecticides which can be used are, for example, organophosphorous agents, carbamate agents, carboxylate type chemicals, chlorinated hydrocarbon type chemicals, insecticidal substances produced by microbes, etc.
[0157] In many cases, this results in synergistic effects, i.e. the activity of the mixture exceeds the activity of the individual components. Such formulations and application forms are commercially and ecologically especially useful as generally lower amounts of active ingredients can be used. A synergist, however, must not necessarily be active itself, as long as it enhances the action of the active compound.
[0158] A mixture with other known active compounds, such as herbicides, or with safeners, fertilizers and growth regulators is also possible.
[0159] Treatment according to the invention of the plants and plant parts with the active compounds is carried out directly or by allowing the compounds to act on their surroundings, environment or storage space by the customary treatment methods, for example by immersion, spraying, evaporation, fogging, scattering, painting on and, in the case of propagation material, in particular in the case of seed, also by applying one or more coats.
[0160] The active compounds can be converted into the customary formulations, such as solutions, emulsions, wettable powders, suspensions, powders, dusts, pastes, soluble powders, granules, suspension-emulsion concentrates, natural and synthetic materials impregnated with active compound, and microencapsulations in polymeric substances.
[0161] The content of the active compounds of the present invention in a commercially useful formulation or application form can be varied in a wide range. The active-compound content of the use forms prepared from the commercial formulations can vary within wide limits.
[0162] These formulations are produced in a known manner, for example by mixing the active compounds with extenders, that is liquid solvents and/or solid carriers, optionally with the use of surfactants, that is emulsifiers and/or dispersants, and/or foam-formers.
[0163] If the extender used is water, it is also possible to employ for example organic solvents as auxiliary solvents. Essentially, suitable liquid solvents are: aromatics such as xylene, toluene or alkylnaphthalenes, chlorinated aromatics or chlorinated aliphatic hydrocarbons such as chlorobenzenes, chloroethylenes or methylene chloride, aliphatic hydrocarbons such as cyclohexane or paraffins, for example petroleum fractions, mineral and vegetable oils, alcohols such as butanol or glycol and also their ethers and esters, ketones such as acetone, methyl ethyl ketone, methyl isobutyl ketone or cyclohexanone, strongly polar solvents such as dimethylformamide and dimethyl sulphoxide, and also water.
[0164] As solid carriers there are suitable: for example ammonium salts and ground natural minerals such as kaolins, clays, talc, chalk, quartz, attapulgite, montmorillonite or diatomaceous earth, and ground synthetic minerals, such as highly disperse silica, alumina and silicates; as solid carriers for granules there are suitable: for example crushed and fractionated natural rocks such as calcite, marble, pumice, sepiolite and dolomite, and also synthetic granules of inorganic and organic meals, and granules of organic material such as sawdust, coconut shells, maize cobs and tobacco stalks; as emulsifiers and/or foam-formers there are suitable: for example nonionic and anionic emulsifiers, such as polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, for example alkylaryl polyglycol ethers, alkylsulphonates, alkyl sulphates, arylsulphonates and also protein hydrolysates; as dispersants there are suitable: for example lignin-sulphite waste liquors and methylcellulose.
[0165] Tackifiers such as carboxymethylcellulose and natural and synthetic polymers in the form of powders, granules or latices, such as gum arabic, polyvinyl alcohol and polyvinyl acetate, as well as natural phospholipids such as cephalins and lecithins, and synthetic phospholipids, can be used in the formulations. Other additives can be mineral and vegetable oils.
[0166] It is possible to use colorants such as inorganic pigments, for example iron oxide, titanium oxide and Prussian Blue, and organic dyes, such as alizarin dyes, azo dyes and metal phthalocyanine dyes, and trace nutrients such as salts of iron, manganese, boron, copper, cobalt, molybdenum and zinc.
[0167] The formulations generally comprise between 0.1 and 98% by weight of active compound, preferably between 0.1 and 90% and particularly preferably between 0.5 and 70% by weight of active compound.
[0168] The effect of the neonicotinoid compounds and 6-CNA on root growth depth is particularly strongly pronounced at certain application rates. However, the application rates of the active compounds can be varied within relatively wide ranges. In general, the rates of applications are from 1 g to 1600 g of the active compound per hectare, preferably from 10 g to 800 g of the active compound per hectare, and particularly preferably from 10 g to 600 g of the active compound per hectare
[0169] As mentioned before, the invention relates to methods which are useful to increase the protrusion of the roots of a plant into the growth medium or to increase the tolerance to hypoxia conditions, comprising applying to the plant propagation material including seed from which the plant is grown an effective amount of a composition comprising the compounds of the formula (I). The plant propagation material may be treated before planting, for example seed may be dressed before sowing. The compounds according to the invention may also be applied to seed grains either by impregnating the grains with a liquid formulation or by coating them with a solid formulation. The composition may also be applied to the planting site when the propagation material is being planted, e.g. during sowing.
[0170] In connection with the treatment of plant propagation material such as seeds, favourable rates of application are in general 0.1 to 1000 g, in particular 1 to 800 g, preferably 10 to 500 g of one of the neonicotinoid compounds or 6-CNA per 100 kg of material to be treated.
[0171] All plants and plant parts can be treated in accordance with the invention. Plant parts are to be understood to mean all above-ground and underground parts and organs of plants, such as shoot, leaf, flower and root, examples which may be mentioned being leaves, needles, stalks, stems, flowers, fruit bodies, fruits, seeds, roots, tubers and rhizomes. The plant parts also include harvested material, and vegetative and generative propagation material, for example cuttings, tubers, rhizomes, offsets and seeds. They also include plant cells, such as may be used or result from the transformation of a plant cell in accordance with the invention. It is also possible to apply the aforementioned compounds onto or into the soil, e.g. before planting or sowing to achieve the effect described, e.g. to enhance the stress tolerance of the plants after planting and the emerging plant which grows from a seed which has been sown into treated soil.
[0172] It will also be immediately clear that the methods of the invention comprising the use of stress tolerance enhancing transgenes or stress tolerance enhancing endogenous variants can be combined with the methods of the invention comprising the use of a neonicotinoid compound or 6-CNA, to yield additive and synergistic effects in increasing the tolerance to hypoxic or anoxic conditions or in increasing the root depth of a plant in a growth medium or soil.
[0173] The method of the current invention may be suitable for any plant, both dicotyledonous and monocotyledonous plants including but not limited to cotton, Brassica vegetables, oilseed rape, wheat, corn or maize, barley, sunflowers, rice, oats, sugarcane, soybean, vegetables (including chicory, lettuce, tomato), tobacco, potato, sugarbeet, papaya, pineapple, mango, Arabidopsis thaliana, but also plants used in horticulture, floriculture or forestry, cereal plants including wheat, oat, barley, rye, rice, turfgrass, sorghum, millet or sugarcane plants. The methods of the invention can also be applied to any plant including but not limited to cotton, tobacco, canola, oilseed rape, soybean, vegetables, potatoes, Lemna spp., Nicotiana spp., sweet potatoes, Arabidopsis, alfalfa, barley, bean, corn, cotton, flax, pea, rape, rice, rye, safflower, sorghum, soybean, sunflower, tobacco, wheat, asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery, cucumber, eggplant, lettuce, onion, oilseed rape, pepper, potato, pumpkin, radish, spinach, squash, tomato, zucchini, almond, apple, apricot, banana, blackberry, blueberry, cacao, cherry, coconut, cranberry, date, grape, grapefruit, guava, kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passion fruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry, strawberry, tangerine, walnut and watermelon.
[0174] As used herein "comprising" is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein. A transgene comprising a DNA region which is functionally or structurally defined, may comprise additional DNA regions etc.
[0175] Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK. Other references for standard molecular biology techniques include Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY, Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK). Standard materials and methods for polymerase chain reactions can be found in Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and in McPherson at al. (2000) PCR--Basics: From Background to Bench, First Edition, Springer Verlag, Germany.
[0176] Throughout the description and Examples, reference is made to the following sequences:
[0177] SEQ ID No. 1: parp1 coding region from Arabidopsis thaliana.
[0178] SEQ ID No. 2: parp2 coding region from Arabidopsis thaliana.
[0179] SEQ ID No. 3: parp1 coding region 1 from Zea mays.
[0180] SEQ ID No. 4: parp1 coding region 2 from Zea mays.
[0181] SEQ ID No. 5: parp2 coding region from Zea mays.
[0182] SEQ ID No. 6: parp2 partial coding region from cotton.
[0183] SEQ ID No.7: parG coding region from Arabidopsis thaliana.
[0184] SEQ ID No. 8: parG coding region from Solanum tuberosum.
[0185] SEQ ID No. 9: parG coding region from Oryza sativa.
[0186] SEQ ID No. 10: parG coding region from Zea mays.
[0187] SEQ ID No. 11: nucleotide sequence of the nicotinamidase from Saccharomyces cereviseae (PNC1).
[0188] SEQ ID No. 12: nucleotide sequence of the nicotinate phosphoribosyltransferase from Saccharomyces cereviseae (NPT1) (complement)
[0189] SEQ ID No. 13: nucleotide sequence of the nicotinic acid mononucleotide adenyl transferase 1 (NMA 1) from Saccharomyces cereviseae.
[0190] SEQ ID No. 14: nucleotide sequence of the nicotinic acid mononucleotide adenyl transferase 2 (NMA2) from Saccharomyces cereviseae.
[0191] SEQ ID No. 15: nucleotide sequence of the NAD synthetase (QNS1) from Saccharomyces cereviseae.
[0192] SEQ ID No. 16: nucleotide sequence of the nicotinamidase from Arabidopsis thaliana (isoform 1).
[0193] SEQ ID No. 17: nucleotide sequence of the nicotinamidase from Arabidopsis thaliana (isoform 2)
[0194] SEQ ID No. 18: nucleotide sequence of the nicotinamidase from Arabidopsis thaliana (isoform 3)
[0195] SEQ ID No. 19: nucleotide sequence of the nicotinate phosphoribosyltransferase from Arabidopsis thaliana (isoform 1).
[0196] SEQ ID No. 20: nucleotide sequence of the nicotinate phosphoribosyltransferase from Arabidopsis thaliana (isoform 2).
[0197] SEQ ID No. 21: nucleotide sequence of the nicotinic acid mononucleotide adenyl transferase from Arabidopsis thaliana.
[0198] SEQ ID No. 22: nucleotide sequence of the NAD synthetase from Arabidopsis thaliana.
EXAMPLES
Example 1
Protocol for Measurement of Depth of Arabidopsis Root Growth in Growth Medium
Media
[0199] Gemination medium: Half concentrated Murashige and Skoog salts; B5 vitamins; 1.5% sucrose; pH 5.8; 0.4% Difco agar.
Arabidopsis Plants
[0200] Sterilization of Arabidopsis seeds: 2 min. 70% ethanol; 10 min. bleach (6% active chlorine)+1 drop Tween 20 for 20 ml solution; wash 5 times with sterile tap water; sterilization is done in 2 ml eppendorf tubes. Arabidopsis seeds sink to the bottom of the tube, allowing removal of the liquids by means of a 1 ml pipetman.
[0201] Pregermination of seeds: In 9 cm Optilux Petridishes (Falcon) containing 10 ml sterile tap water. Low light overnight to 24 hours.
[0202] Growing of Arabidopsis plants: Seeds are sown in 25×150 mm glass tubes (Sigma C5916) with natural (transparant) colored closure (Sigma C5791) containing ±34 ml germination medium: 1 seed/tube. The tubes are put in the two outer rows of tube holders for 40 tubes (V WR nalg5970-0025) wrapped in aluminium foil so that the roots can grow in the dark. Plants are grown at 23° C. 30-50 μEinstein S-1m-2. 12 hours light--12 hours dark. (See FIG. 1)
Measuring Root Depth
[0203] After three weeks, the root depth is measured from the surface of the medium to the lowest point of root growth. (See FIG. 1).
Example 2
Analysis of Depth of Root Growth of Arabidopsis Plants Comprising a Transgene which Enhances Stress Tolerance
[0204] Arabidopsis thaliana plants comprising a transgene encoding a dsRNA molecule which is capable of reducing the expression of endogenous PARP1 or PARP2 genes, as described in WO 00/04173 A1, (e.g. in Example 8 thereof) were grown as described in Example 1. After three weeks, the depth of the roots of the transgenic plants was measured and compared to the depth of the roots of non-transgenic control plants or to non-transgenic isogenic plants grown in a similar manner.
[0205] In a first experiment, various populations of Arabidopsis thaliana cv. Col-0 plants comprising a transgene encoding a dsRNA molecule which is capable of reducing the expression of endogenous PARP2 genes (with lines 427-16 and 427-20 showing weak tolerance to high light stress and line 427-19 showing a moderate tolerance to high light stress) were compared with a population of non-transgenic Arabidopsis thaliana cv. Col-0 plants.
[0206] The results of the measurements were subjected to statistical analysis, summarized in Table 1, which also represents the mean, standard deviation and confidence intervals.
[0207] The roots of transgenic Arabidopsis thaliana plants with the highest tolerance to high light stress conditions (line 427-19) protruded statistically significant deeper (at 99% confidence level) into the growth medium than non-transgenic Arabidopsis thaliana cv. Col-0 control plants (see FIG. 2 and Table 1)
TABLE-US-00001 TABLE 1 Root depth (mm) of Arabidopsis thaliana cv. Col-0 plants comprising a transgene encoding a dsRNA molecule which is capable of reducing the expression of endogenous PARP2 genes as compared to non-transgenic Arabidopsis thaliana cv. Col-0 plants Col-0 427-16 427-19 427-20 mean 25.173611 26.388889 27.42 24.942029 Standard dev. 3.64166 3.422548 3.361185 3.69174 Standard error 0.429174 0.46575 0.388116 0.444433 95% Confidence 0.858348 0.94128 0.776233 0.888866 99% Confidence 1.141602 1.259388 1.032389* 1.182192 *p < 0.01
[0208] In a further experiment, a population of transgenic Arabidopsis thaliana cv. Col-0 plants comprising a transgene encoding a dsRNA molecule which is capable of reducing the expression of endogenous PARP2 genes and which which are tolerant to high light stress (line 427-22) were compared to a transgenic line containing a similar transgene but which was sensitive to high light stress (line 427-24), as well as to non-transgenic Arabidopsis thaliana cv. Col-0 control plants.
[0209] The results of the measurements were subjected to statistical analysis, summarized in Table 2, which also represents the mean, standard deviation and confidence intervals.
[0210] The roots of transgenic Arabidopsis thaliana cv. Col-0 plants of the stress tolerant transgenic line (line 427-22) protruded deeper into the growth medium (comprising 0.7% Difco agar instead of 0.4%) than the roots Arabidopsis thaliana cv. Col-0 control plants and than the roots of stress-sensitve transgenic Arabidopsis thaliana cv. Col-0 plant line (line 427-24) (see FIG. 3 and Table 2).
TABLE-US-00002 TABLE 2 Root depth (mm) of Arabidopsis thaliana cv. Col-0 plants comprising a transgene encoding a dsRNA molecule which is capable of reducing the expression of endogenous PARP2 genes as compared to non-transgenic Arabidopsis thaliana cv. Col-0 plants Col-0 427-22 427-24 Mean 18.291339 21.146154 17.384058 Standard dev. 2.928677 5.368981 2.740149 Standard error 0.259878 0.66594 0.329875 95% Confidence 0.514558 1.33188 0.65975 99% Confidence 0.680101 1.771401* 0.877468 *p < 0.01
[0211] In another experiment, the following populations were analyzed:
[0212] C24: wild-type Arabidopsis line; line 1599: A. thaliana transgenic line comprising anti-PARP2 transgene with a high tolerance to high light stress conditions; line 1463: A. thaliana transgenic line comprising anti-PARP2 transgene with a moderate tolerance to high light stress conditions; line 1681: A. thaliana transgenic line comprising anti-PARP1 gene with a moderate tolerance to high light stress conditions; and line 1690: A. thaliana transgenic line comprising anti-PARP1 gene with a moderate tolerance to high light stress conditions. The stress tolerance of line 1599 is very high, the stress tolerance of lines 1463, 1681 and 1690 varies from moderate to high.
[0213] The results of the measurements were subjected to statistical analysis, summarized in Table 3, which represents the mean, standard deviation and confidence intervals.
[0214] The roots of transgenic Arabidopsis thaliana cv. C24 plants comprising a transgene encoding a dsRNA molecule which is capable of reducing the expression of endogenous PARP1 genes (lines 1681 and 1690) or PARP2 genes (lines 1599 and 1463) protruded deeper in the growth medium than the non-transgenic Arabidopsis thaliana cv. C24 control plants (see FIG. 4 and Table 3)
TABLE-US-00003 TABLE 3 Root depth (mm) of Arabidopsis thaliana cv. C24 plants comprising a transgene encoding a dsRNA molecule which is capable of reducing the expression of endogenous PARP1 or PARP2 genes as compared to non- transgenic Arabidopsis thaliana cv. C24 plants C24 1599 1463 1681 1690 mean 16.18 18.125 17.405063 17.433333 17.897727 Standard 2.192181 2.992481 2.16036 2.112246 1.885007 dev. Standard 0.219218 0.451133 0.243059 0.27269 0.284175 error 95% 0.438436 0.911741 0.486119 0.551106 0.574319 Confidence 99% 0.58312 1.219865* 0.646538* 0.737353* 0.76841* Confidence *p < 0.01
[0215] In a further experiment, a 1:1 segregating population of transgenic Arabidopsis thaliana cv. Col-0 line comprising a transgene encoding a dsRNA molecule which is capable of reducing the expression of endogenous PARP2 genes and with high tolerance to high light stress, was analyzed. The presence of the transgene was verified by PCR analysis.
[0216] Plants comprising the transgene had roots which protruded deeper into the growth medium than the azygous Arabidopsis thaliana cv. Col-0 plants derived from line 427-19 (see FIG. 5 and Table 4)
TABLE-US-00004 TABLE 4 Root depth (mm) of Arabidopsis thaliana cv. Col-0 plants comprising a transgene encoding a dsRNA molecule which is capable of reducing the expression of endogenous PARP2 genes as compared to the azygous Arabidopsis thaliana cv. Col-0 plants Azygous Transgenic 427-19 427-19 Mean 25.296512 27.38172 Standard dev. 3.573907 3.244894 Standard error 0.385384 0.33648 95% Confidence 0.770769 0.67296 99% Confidence 1.025122 0.895036* *p < 0.01
Example 3
Analysis of Depth of Root Growth of Arabidopsis Plants after Application of Imidacloprid
[0217] Arabidopsis thaliana cv. C24 plants were grown as described in Example 1 on germination medium (with 0.7% Difco agar in stead of 0.4%) comprising various concentrations of imidacloprid (0, 50, and 100 mg/l). After three weeks, the depth of the roots of the plants treated with 50 and 100 mg/l imidacloprid was measured and compared to the depth of the roots of untreated plants grown in a similar manner.
[0218] The roots of the treated Arabidopsis plants protruded deeper in the growth medium than the roots of the non-treated Arabidopsis plants (see FIG. 6 and Table 5)
TABLE-US-00005 TABLE 5 Root depth (mm) of Arabidopsis thaliana cv. C24 plants treated with 50 and 100 mg/l imidacloprid as compared to Arabidopsis thaliana cv. C24 plants not treated with imidacloprid 0 mg/L 50 mg/L 100 mg/L Mean 1.9475 2.1875 2.2725 Standard dev. 0.289714 0.353508 0.456072 Standard error 0.037402 0.045638 0.058879 95% Confidence 0.075589 0.092234 0.118994 99% Confidence 0.101135 0.123404* 0.159208* *p < 0.01
Example 4
Analysis of Depth of Root Growth of Arabidopsis Plants after Application of 6-chloronicotinic Acid (6-CNA)
[0219] Arabidopsis thaliana cv. C24 plants were grown as described in Example 1 on germination medium comprising various concentrations of 6-CNA (0, 1, and 5 mg/l). After three weeks, the depth of the roots of the plants treated with 1 and 5 mg/l 6-CNA was measured and compared to the depth of the roots of the plants, not treated with 6-CNA grown in a similar manner.
[0220] The roots of the treated Arabidopsis plants protruded deeper in the growth medium than the roots of the non-treated Arabidopsis plants (see FIG. 7 and Table 6)
TABLE-US-00006 TABLE 6 Root depth (mm) of Arabidopsis thaliana cv. C24 plants treated with 1 and 5 mg/l 6-CNA as compared to Arabidopsis thaliana cv. Col-0 plants not treated with 6-CNA 0 mg/L 1 mg/L 5 mg/L Mean 2.031034 2.179661 2.118367 Standard dev. 0.189373 0.186418 0.237762 Standard error 0.024866 0.02427 0.033966 95% Confidence 0.050254 0.049049 0.068645** 99% Confidence 0.067237 0.065625* 0.091844 *p < 0.01 **p < 0.05
Sequence CWU
1
1
2213187DNAArtificial Sequenceparp1 coding region from Arabidopsis thaliana
1taccggagaa atggcaagcc cacataagcc gtggagggcg gagtatgcaa agtcgtcgag
60gtcttcatgt aaaacttgca agtccgtcat taacaaggag aactttcgtc ttggaaagtt
120ggttcaatct actcacttcg atggcatcat gcccatgtgg aaccatgctt cttgtatact
180gaagaagacg aagcagataa aatcagttga tgatgttgaa ggcatagaat cacttcgttg
240ggaagatcag caaaagatta gaaaatatgt cgaatctgga gcagggagta acacaagcac
300aagcacaggc acaagcacga gcagtaccgc taataatgcc aaactagaat atgggattga
360agtgtcacaa acttcccgtg ccggttgcag aaagtgtagc gaaaagatct tgaaaggaga
420ggtacgtata ttctccaagc ctgaaggccc gggtaacaaa ggtttgatgt ggcatcacgc
480taaatgtttc cttgaaatgt cttcctctac tgaactggaa agtttgtctg gatggagaag
540tataccagac tcagaccaag aagctcttct tcccttagtg aagaaagctc tgccggcagc
600caaaactgag acagcagaag cacgtcaaac aaattcaaga gcaggcacaa aacgaaaaaa
660tgattctgtt gataacgaga agtcgaaact agcaaaaagt agttttgaca tgtctacaag
720tggtgcttta caaccttgta gcaaagaaaa ggaaatggag gcccaaacta aggaattgtg
780ggacctgaag gatgatctga aaaaatatgt aacatcagct gagttgcggg aaatgcttga
840agtaaatgaa caaagtacaa gaggatctga acttgatctg cgtgataaat gtgctgatgg
900catgatgttt ggcccactcg ctctctgccc aatgtgctct gggcatcttt ctttctccgg
960aggactttac cgatgccatg gatacatctc agaatggagc aaatgttctc attccacttt
1020ggatccagac cgcatcaaag ggaagtggaa aatccctgac gaaacagaaa atcaattcct
1080tctgaagtgg aataagtctc aaaagagtgt gaagccaaaa cgtattctgc gtcctgtatt
1140gtctggcgag acatctcagg gtcaaggttc taaagatgca actgactcct caaggagtga
1200aaggctagca gatcttaaag tttcaattgc tggaaatact aaggaaaggc aaccatggaa
1260gaagagaatt gaggaagctg gtgcagagtt tcatgctaat gttaaaaaag gtacaagctg
1320tttggttgtt tgtggactga cagatatcag agacgctgaa atgagaaagg caaggaggat
1380gaaagtggca atcgtgagag aggattattt ggttgattgt tttaaaaaac agaggaaact
1440tccatttgac aagtacaaaa ttgaagacac tagtgagagc cttgtcactg ttaaagtaaa
1500aggacgaagc gctgtgcatg aagcgtctgg cctccaagag cactgtcaca ttcttgaaga
1560tgggaacagt atctataaca caactctgag catgtctgat ctctctaccg gtatcaatag
1620ttattacata ctccagataa tccaagaaga taaaggttca gattgttacg tatttcgtaa
1680atggggccga gttggaaatg aaaagattgg tggtaacaaa gtggaggaaa tgtcaaagtc
1740tgatgcggtt cacgaattca aacgtctatt tcttgaaaaa accggaaaca catgggaatc
1800ttgggaacaa aaaacgaatt tccagaaaca acctggaaaa tttctcccgt tggacattga
1860ttatggagtt aataagcaag tagccaaaaa agagccattt cagaccagta gcaaccttgc
1920tccatcatta atagaattga tgaagatgct ttttgatgtg gaaacttaca gatctgcaat
1980gatggagttc gagataaata tgtcagagat gccacttggg aagctcagca aacataatat
2040acagaagggt tttgaggcat tgacggagat acagaggcta ttgactgaaa gcgaccccca
2100gcctactatg aaagaaagct tgcttgttga tgctagtaac agatttttta ccatgatccc
2160ttctattcat cctcatatta tccgagatga agatgacttt aagtcaaagg tgaaaatgct
2220cgaggctctg caggatatcg aaatagcttc aagaatagtt ggctttgatg ttgatagcac
2280cgaatctctt gatgataagt ataagaagct gcattgcgat atctcaccac ttcctcatga
2340tagcgaagat tatcgattaa tcgagaagta tcttaacaca actcatgccc caacgcatac
2400agagtggagt cttgagctag aggaagtttt tgctcttgaa agagaaggag agtttgataa
2460atatgctccc cacagagaaa aacttggcaa taagatgctc ctatggcatg gttctcgatt
2520aacgaatttt gttggaatat tgaaccaagg actgagaatt gcacctccag aagctcctgc
2580tactggttac atgtttggaa aagggatata ctttgctgac cttgtcagta aaagtgctca
2640gtactgctac acttgtaaga aaaatccggt gggtctaatg cttctgagtg aagttgcatt
2700gggagaaata catgagctaa caaaagctaa gtatatggat aaacctccga gagggaaaca
2760ctcgaccaaa gggctcggca agaaagtgcc tcaagattcc gagtttgcca agtggagagg
2820tgatgtgact gttccctgtg gaaaacctgt ttcatcaaag gtcaaggctt ctgagcttat
2880gtacaatgag tatatcgtct acgatacagc ccaggtgaag ttgcagttct tgttgaaagt
2940aaggtttaag cacaagagat gagcctgaac caaacaagaa gacgtcactt ctgttaacta
3000aatgtttttt tgggaaatcg aatccaacac gaagacttaa cttttgtaac taaattgctt
3060ttgataaatt gaattcaaca tgtagtcaca gatttaactc tctggcgttg tagatgtttc
3120tggttttaaa agagcgtact ctacattttg ttatgctttt tctcagtaat gacacttctt
3180aagactt
318722147DNAArtificial Sequenceparp2 coding region from Arabidopsis
thaliana 2attgatgaag aagaaaacga agaagaagac tcttcaaatg ctcgcgcgaa
ctcacttctg 60acgaaaacca tacttcctca gtctcattcc ctttccgacg aactattctc
ctgaagaaga 120agacgaaaat ggcgaacaag ctcaaagtcg acgaactccg tttaaaactc
gccgagcgtg 180gactcagtac tactggagtc aaagccgttc tggtggagag gcttgaagag
gctatcgcag 240aagacactaa gaaggaagaa tcaaagagca agaggaaaag aaattcttct
aatgatactt 300atgaatcgaa caaattgatt gcaattggcg aatttcgtgg gatgattgtg
aaggaattgc 360gtgaggaagc tattaagaga ggcttagata caacaggaac caaaaaggat
cttcttgaga 420ggctttgcaa tgatgctaat aacgtttcca atgcaccagt caaatccagt
aatgggacag 480atgaagctga agatgacaac aatggctttg aagaagaaaa gaaagaagag
aaaatcgtaa 540ccgcgacaaa gaagggtgca gcggtgctag atcagtggat tcctgatgag
ataaagagtc 600agtaccatgt tctacaaagg ggtgatgatg tttatgatgc tatcttaaat
cagacaaatg 660tcagggataa taataacaag ttctttgtcc tacaagtcct agagtcggat
agtaaaaaga 720catacatggt ttacactaga tggggaagag ttggtgtgaa aggacaaagt
aagctagatg 780ggccttatga ctcatgggat cgtgcgatag agatatttac caataagttc
aatgacaaga 840caaagaatta ttggtctgac agaaaggagt ttatcccaca tcccaagtcc
tatacatggc 900tcgaaatgga ttacggaaaa gaggaaaatg attcaccggt caataatgat
attccgagtt 960catcttccga agttaaacct gaacaatcaa aactagatac tcgggttgcc
aagttcatct 1020ctcttatatg taatgtcagc atgatggcac agcatatgat ggaaatagga
tataacgcta 1080acaaattgcc actcggcaag ataagcaagt ccacaatttc aaagggttat
gaagtgctga 1140agagaatatc ggaggtgatt gaccggtatg atagaacgag gcttgaggaa
ctgagtggag 1200agttctacac agtgatacct catgattttg gttttaagaa aatgagtcag
tttgttatag 1260acactcctca aaagttgaaa cagaaaattg aaatggttga agcattaggt
gaaattgaac 1320tcgcaacaaa gttgttgtcc gtcgacccgg gattgcagga tgatccttta
tattatcact 1380accagcaact taattgtggt ttgacgccag taggaaatga ttcagaggag
ttctctatgg 1440ttgctaatta catggagaac actcatgcaa agacgcattc gggatatacg
gttgagattg 1500cccaactatt tagagcttcg agagctgttg aagctgatcg attccaacag
ttttcaagtt 1560cgaagaacag gatgctactc tggcacggtt cacgtctcac taactgggct
ggtattttat 1620ctcaaggtct gcgaatagct cctcctgaag cgcctgtaac tggttacatg
tttggaaaag 1680gggtttactt tgcggatatg ttctccaaga gtgcgaacta ttgctatgcc
aacactggcg 1740ctaatgatgg cgttctgctc ctctgcgagg ttgctttggg agacatgaat
gaacttctgt 1800attcagatta taacgcggat aatctacccc cgggaaagct aagcacaaaa
ggtgtgggga 1860aaacagcacc aaacccatca gaggctcaaa cactagaaga cggtgttgtt
gttccacttg 1920gcaaaccagt ggaacgttca tgctccaagg ggatgttgtt gtacaacgaa
tatatagtct 1980acaatgtgga acaaatcaag atgcgttatg tgatccaagt caaattcaac
tacaagcact 2040aaaacttatg tatattagct tttgaacatc aactaattat ccaaaaatca
gcgttttatt 2100gtatttcttt caaactcctt catctctgat tttgcacggt tcactcg
214733211DNAArtificial Sequenceparp1 coding region 1 from Zea
mays 3acctacctga atacgtcatc cctaagtgtt ccgcttcctc tgtcgtccgg cctccaactc
60catcgaaggg gctagggaga ggagggaacc cgaaccacag caggccggcg caatggcggc
120gccgccaaag gcgtggaagg cggagtatgc caagtctggg cgggcctcgt gcaagtcatg
180ccggtcccct atcgccaagg accagctccg tcttggcaag atggttcagg cgtcacagtt
240cgacggcttc atgccgatgt ggaaccatgc cagcgttgac gatgttgaag ggatagatgc
300acttagatgg gatgatcaag agaagatacg aaactacgtt gggagtgcct cagctggtac
360aagttctaca gctgctcctc ctgagaaatg tacaattgag attgctccat ctgcccgtac
420ttcatgtaga cgatgcagtg aaaagattac aaaaggatcg gtccgtcttt cagctaagct
480tgagagtgaa ggtcccaagg gtataccatg gtatcatgcc aactgtttct ttgaggtatc
540cccgtctgca actgttgaga agttctcagg ctgggatact ttgtccgatg aggataagag
600aaccatgctc gatcttgtta aaaaagatgt tggcaacaat gaacaaaata agggttccaa
660gcgcaagaaa agtgaaaatg atattgatag ctacaaatcc gccaggttag atgaaagtac
720atctgaaggt acagtgcgaa acaaagggca acttgtagac ccacgtggtt ccaatactag
780ttcagctgat atccaactaa agcttaagga gcaaagtgac acactttgga agttaaagga
840tggacttaag actcatgtat cggctgctga attaagggat atgcttgagg ctaatgggca
900ggatacatca ggaccagaaa ggcacctatt ggatcgctgt gcggatggaa tgatatttgg
960agcgctgggt ccttgcccag tctgtgctaa tggcatgtac tattataatg gtcagtacca
1020atgcagtggt aatgtgtcag agtggtccaa gtgtacatac tctgccacag aacctgtccg
1080cgttaagaag aagtggcaaa ttccacatgg aacaaagaat gattacctta tgaagtggtt
1140caaatctcaa aaggttaaga aaccagagag ggttcttcca ccaatgtcac ctgagaaatc
1200tggaagtaaa gcaactcaga gaacatcatt gctgtcttct aaagggttgg ataaattaag
1260gttttctgtt gtaggacaat caaaagaagc agcaaatgag tggattgaga agctcaaact
1320tgctggtgcc aacttctatg ccagggttgt caaagatatt gattgtttaa ttgcatgtgg
1380tgagctcgac aatgaaaatg ctgaagtcag gaaagcaagg aggctgaaga taccaattgt
1440aagggagggt tacattggag aatgtgttaa aaagaacaaa atgctgccat ttgatttgta
1500taaactagag aatgccttag agtcctcaaa aggcagtact gtcactgtta aagttaaggg
1560ccgaagtgct gttcatgagt cctctggttt gcaagatact gctcacattc ttgaagatgg
1620gaaaagcata tacaatgcaa ccttaaacat gtctgacctg gcactaggtg tgaacagcta
1680ctatgtactc cagatcattg aacaggatga tgggtctgag tgctacgtat ttcgtaagtg
1740gggacgggtt gggagtgaga aaattggagg gcaaaaactg gaggagatgt caaaaactga
1800ggcaatcaag gaattcaaaa gattatttct tgagaagact ggaaactcat gggaagcttg
1860ggaatgtaaa accaattttc ggaagcagcc tgggagattt tacccacttg atgttgatta
1920tggtgttaag aaagcaccaa aacggaaaga tatcagtgaa atgaaaagtt ctcttgctcc
1980tcaattgcta gaactcatga agatgctttt caatgtggag acatatagag ctgctatgat
2040ggaatttgaa attaatatgt cagaaatgcc tcttgggaag ctaagcaagg aaaatattga
2100gaaaggattt gaagcattaa ctgagataca gaatttattg aaggacaccg ctgatcaagc
2160actggctgtt agagaaagct taattgttgc tgcgagcaat cgctttttca ctcttatccc
2220ttctattcat cctcatatta tacgggatga ggatgatttg atgatcaaag cgaaaatgct
2280tgaagctctg caggatattg aaattgcttc aaagatagtt ggcttcgata gcgacagtga
2340tgaatctctt gatgataaat atatgaaact tcactgtgac atcaccccgc tggctcacga
2400tagtgaagat tacaagttaa ttgagcagta tctcctcaac acacatgctc ctactcacaa
2460ggactggtcg ctggaactgg aggaagtttt ttcacttgat cgagatggag aacttaataa
2520gtactcaaga tataaaaata atctgcataa caagatgcta ttatggcacg gttcaaggtt
2580gacgaatttt gtgggaattc ttagtcaagg gctaagaatt gcacctcctg aggcacctgt
2640tactggctat atgttcggca aaggcctcta ctttgcagat ctagtaagca agagcgcaca
2700atactgttat gtggatagga ataatcctgt aggtttgatg cttctttctg aggttgcttt
2760aggagacatg tatgaactaa agaaagccac gtccatggac aaacctccaa gagggaagca
2820ttcgaccaag ggattaggca aaaccgtgcc actggagtca gagtttgtga agtggaggga
2880tgatgtcgta gttccctgcg gcaagccggt gccatcatca attaggagct ctgaactcat
2940gtacaatgag tacatcgtct acaacacatc ccaggtgaag atgcagttct tgctgaaggt
3000gcgtttccat cacaagaggt agctgggaga ctaggcaagt agagttggaa ggtagagaag
3060cagagttagg cgatgcctct tttggtatta ttagtaagcc tggcatgtat ttatgggtgc
3120tcgcgcttga tccattttgg taagtgttgc ttgggcatca gcgcgaatag caccaatcac
3180acacttttac ctaatgacgt tttactgtat a
321143212DNAArtificial Sequenceparp1 coding region 2 from Zea mays
4gcttcctctg tcgtccggcc tccaactcca tcgaaggggc tagggagagg agggaacccg
60aaccacagca ggccggcgca atggcggcgc cgccaaaggc gtggaaggcg gagtatgcca
120agtctgggcg ggcctcgtgc aagtcatgcc ggtcccctat cgccaaggac cagctccgtc
180ttggcaagat ggttcaggcg tcacagttcg acggcttcat gccgatgtgg aaccatgcca
240ggtgcatctt cagcaagaag aaccagataa aatccgttga cgatgttgaa gggatagatg
300cacttagatg ggatgatcaa gagaagatac gaaactacgt tgggagtgcc tcagctggta
360caagttctac agctgctcct cctgagaaat gtacaattga gattgctcca tctgcccgta
420cttcatgtag acgatgcagt gaaaagatta caaaaggatc ggtccgtctt tcagctaagc
480ttgagagtga aggtcccaag ggtataccat ggtatcatgc caactgtttc tttgaggtat
540ccccgtctgc aactgttgag aagttctcag gctgggatac tttgtccgat gaggataaga
600gaaccatgct cgatcttgtt aaaaaagatg ttggcaacaa tgaacaaaat aagggttcca
660agcgcaagaa aagtgaaaat gatattgata gctacaaatc cgccaggtta gatgaaagta
720catctgaagg tacagtgcga aacaaagggc aacttgtaga cccacgtggt tccaatacta
780gttcagctga tatccaacta aagcttaagg agcaaagtga cacactttgg aagttaaagg
840atggacttaa gactcatgta tcggctgctg aattaaggga tatgcttgag gctaatgggc
900aggatacatc aggaccagaa aggcacctat tggatcgctg tgcggatgga atgatatttg
960gagcgctggg tccttgccca gtctgtgcta atggcatgta ctattataat ggtcagtacc
1020aatgcagtgg taatgtgtca gagtggtcca agtgtacata ctctgccaca gaacctgtcc
1080gcgttaagaa gaagtggcaa attccacatg gaacaaagaa tgattacctt atgaagtggt
1140tcaaatctca aaaggttaag aaaccagaga gggttcttcc accaatgtca cctgagaaat
1200ctggaagtaa agcaactcag agaacatcat tgctgtcttc taaagggttg gataaattaa
1260ggttttctgt tgtaggacaa tcaaaagaag cagcaaatga gtggattgag aagctcaaac
1320ttgctggtgc caacttctat gccagggttg tcaaagatat tgattgttta attgcatgtg
1380gtgagctcga caatgaaaat gctgaagtca ggaaagcaag gaggctgaag ataccaattg
1440taagggaggg ttacattgga gaatgtgtta aaaagaacaa aatgctgcca tttgatttgt
1500ataaactaga gaatgcctta gagtcctcaa aaggcagtac tgtcactgtt aaagttaagg
1560gccgaagtgc tgttcatgag tcctctggtt tgcaagatac tgctcacatt cttgaagatg
1620ggaaaagcat atacaatgca accttaaaca tgtctgacct ggcactaggt gtgaacagct
1680actatgtact ccagatcatt gaacaggatg atgggtctga gtgctacgta tttcgtaagt
1740ggggacgggt tgggagtgag aaaattggag ggcaaaaact ggaggagatg tcaaaaactg
1800aggcaatcaa ggaattcaaa agattatttc ttgagaagac tggaaactca tgggaagctt
1860gggaatgtaa aaccaatttt cggaagcagc ctgggagatt ttacccactt gatgttgatt
1920atggtgttaa gaaagcacca aaacggaaag atatcagtga aatgaaaagt tctcttgctc
1980ctcaattgct agaactcatg aagatgcttt tcaatgtgga gacatataga gctgctatga
2040tggaatttga aattaatatg tcagaaatgc ctcttgggaa gctaagcaag gaaaatattg
2100agaaaggatt tgaagcatta actgagatac agaatttatt gaaggacacc gctgatcaag
2160cactggctgt tagagaaagc ttaattgttg ctgcgagcaa tcgctttttc actcttatcc
2220cttctattca tcctcatatt atacgggatg aggatgattt gatgatcaaa gcgaaaatgc
2280ttgaagctct gcaggatatt gaaattgctt caaagatagt tggcttcgat agcgacagtg
2340atgaatctct tgatgataaa tatatgaaac ttcactgtga catcaccccg ctggctcacg
2400atagtgaaga ttacaagtta attgagcagt atctcctcaa cacacatgct cctactcaca
2460aggactggtc gctggaactg gaggaagttt tttcacttga tcgagatgga gaacttaata
2520agtactcaag atataaaaat aatctgcata acaagatgct attatggcac ggttcaaggt
2580tgacgaattt tgtgggaatt cttagtcaag ggctaagaat tgcacctcct gaggcacctg
2640ttactggcta tatgttcggc aaaggcctct actttgcaga tctagtaagc aagagcgcac
2700aatactgtta tgtggatagg aataatcctg taggtttgat gcttctttct gaggttgctt
2760taggagacat gtatgaacta aagaaagcca cgtccatgga caaacctcca agagggaagc
2820attcgaccaa gggattaggc aaaaccgtgc cactggagtc agagtttgtg aagtggaggg
2880atgatgtcgt agttccctgc ggcaagccgg tgccatcatc aattaggagc tctgaactca
2940tgtacaatga gtacatcgtc tacaacacat cccaggtgaa gatgcagttc ttgctgaagg
3000tgcgtttcca tcacaagagg tagctgggag actaggcaag tagagttgga aggtagagaa
3060gcagagttag gcgatgcctc ttttggtatt attagtaagc ctggcatgta tttatgggtg
3120ctcgcgcttg atccattttg gtaagtgttg cttgggcatc agcgcgaata gcaccaatca
3180cacactttta cctaatgacg ttttactgta ta
321252295DNAArtificial Sequenceparp2 coding region fro Zea mays
5tgacctgttc catcccgcca gcccttccgc tcccacgacc caaccccact gcccggagcc
60cccgagcctt ctcgaatctt gcgagaaccc caggggcgag gagcagatgt cggcgaggct
120acgggtggcg gacgtccgcg cggagcttca gcgccgcggc ctcgatgtat ccggcaccaa
180gcctgctctc gtgcggaggc tggacgccgc aatttgcgag gcggagaagg ccgtggtggc
240tgctgcgcca accagtgtgg caaatgggta tgacgtagcc gtagatggca aaaggaactg
300cgggaataat aagaggaaaa ggtccgggga tgggggtgaa gagggaaacg gcgatacgtg
360tacagatgtg acaaaactag agggcatgag ctatcgtgag ctgcagggat tggccaaggc
420acgtggagtt gcggcaaatg ggggcaagaa agatgttatc cagaggttgc tctcggcgac
480tgctggtcct gctgcagttg cagatggtgg tcctctgggc gccaaggaag tcataaaagg
540tggtgatgag gaggttgagg tgaaaaagga gaagatggtt actgccacga agaagggagc
600tgcagtgctg gatcagcaca ttcccgatca cataaaagtg aactatcatg tcttgcaagt
660gggcgatgaa atctatgatg ccaccttgaa ccagactaat gttggagaca acaacaataa
720gttctatatc attcaagttt tagaatctga tgctggtgga agctttatgg tttacaatag
780atggggaaga gttggggtac gaggtcaaga taaactacat ggtccctccc caacacgaga
840ccaagcaata tatgaatttg aggggaagtt ccacaacaaa accaataatc attggtctga
900tcgcaagaac ttcaaatgtt atgcaaagaa atacacttgg cttgaaatgg attatggtga
960aactgagaaa gaaatagaga aaggttccat tactgatcag ataaaagaga caaaacttga
1020aactagaatt gcgcagttca tatccctgat ctgcaatatt agcatgatga agcaaagaat
1080ggtggaaata ggttataatg ctgaaaagct tccccttgga aagctaagga aagctacaat
1140acttaagggt tatcatgttt tgaaaaggat atccgatgtt atttcaaagg cggacaggag
1200acatcttgag caattgactg gggaattcta caccgtgatt cctcatgact ttggtttcag
1260aaagatgcgt gaatttatta tcgatactcc tcagaaacta aaagctaagc tggagatggt
1320tgaagccctt ggtgagattg aaattgcaac taaacttttg gaggatgatt caagtgacca
1380ggatgatccg ttgtatgctc gatacaagca acttcattgt gatttcacac ctcttgaagc
1440tgattcagat gagtactcta tgataaaatc atatttgaga aatacacatg gaaaaacaca
1500ctctggttat acggtggaca tagtgcaaat atttaaggtt tcaaggcatg gtgaaacaga
1560gcgatttcaa aaatttgcta gtacaagaaa taggatgctt ttgtggcatg gttctcggtt
1620gagcaactgg gctgggatcc tttctcaggg tctgcgaatc gctcctcctg aagcacctgt
1680tactggttac atgtttggca agggtgttta ctttgctgac atgttttcaa agagtgcaaa
1740ctattgctac gcctctgaag catgtagatc tggagtactg cttttatgtg aggttgcatt
1800gggcgatatg aatgagctac tgaatgcaga ttacgatgct aataacctgc ccaaaggaaa
1860attaagatcc aagggagttg gtcaaacagc acctaacatg gtcgagtcta aggtcgctga
1920cgatggtgtt gttgttcccc ttggcgaacc caaacaggaa ccttccaaaa ggggtggctt
1980gctttataat gagtacatag tgtacaacgt agaccagata agaatgcggt atgtcttaca
2040tgttaacttc aatttcaaga gacggtagat gttgcaaaga gctgaaactg ttgctgagat
2100cttagcagaa catatgtgga cttatagcac caggtgccct cagcctcatt ttctgagcaa
2160atttggtagc ctttgcattt cgattttggt ttcagcttct agccccattg atgattgata
2220ctgagtgtat atatgaacca ttgatatcca ccttccatgt acttaagttt ttttaacatg
2280tcccatgcat aataa
229561384DNAArtificial Sequenceparp2 partial coding region from cotton
6gagaagatbg ttacagcgac gaggaagggg tggctgttct ggatcaaggg atcccagatg
60acataaaggc tcattatcat gttctacaaa agggtgatga tatctatgat gccatgttaa
120atcagacgaa tgttgggcaa aacaataaca aattctttgt gatccagctt ctagaatctg
180atgactcgaa gacatacatg gttcataaca gatggggtag agttggtgtg aagggtcaaa
240ttaagttaca tggccccttt acttcacgac aagccgcaat tgatgagttt caaaccaaat
300tctttaacaa gaccaaaaac tattggtaca acagaaaaga ctttgtttgt cacccaaagt
360gctacacctt gctggagatg gactatgatg aaaaagaaaa ggaatctgat gtcaaaagaa
420aggctaactc ttccattggt gctcaattgc gggagacaaa gcttgaacaa cgtgttgcta
480agtttatctc tattatatgc aatatcagca tgatgaagca acaaatgatg gaaataggat
540acaatgctga caagttgcct cttggtaagc taagcaaatc cacaatttta aaggggtatg
600atgtcttaaa gaaaattgct gatgtgattg accagtcaaa caggagcaag cttgagcaat
660taagttcgga attttacacc gtgattccac atgattttgg atttagaaaa atgcgtgatt
720ttgtcatcga cacacctcag aagttgaaaa agaagttgga aatggttgaa gccctgggag
780aaatagaggt cgcatcaaaa ttattaatgg atgacattac gatggaggaa gatcctttat
840attatcggta ccaacagctt cactgtgaac tgtttcctct tgacaatgat actgaggagt
900tcgctttgat tgtaaagtat attcagaata ctcatgctca gacacattca aattatacag
960ttgatgttgt tcaaatattc aaggtgacaa gagacggtga aagtgaacgc tttaaaaagt
1020tttctggaac aaaaaataga atgctgttgt ggcatggttc tcggcttact aactggactg
1080gcattctgtc ccaaggtttg cgcattgctc cacctgaagc gcctgccacg ggttatatgt
1140ttgggaaggg ggtttacttt gctgatatgt tctccaaaag tgcaaattat tgctatacta
1200attctgcctt cacaacaggg gtgttgcttc tatgtgaggt tgccctgggt gacatggctg
1260agcttctaca agctaaaagc gatgctgata agctgccgga tgggaagttg agcacaaaag
1320gtgttggtgc aactgcaccg gatccttctg aagcccagtc acttgatgat ggtgttgttg
1380ttcc
138471647DNAArabidopsis thaliana 7atggagaatc gcgaagatct taactcaatt
cttccgtacc ttccacttgt aattcgttcg 60tcgtcgctgt attggccgcc gcgtgtggtg
gaggcgttaa aggcaatgtc tgaaggacca 120tctcacagcc aagttgactc aggagaggtt
ctacggcaag ctattttcga tatgagacga 180tccttatctt tctctactct cgagccatct
gcttctaatg gctacgcatt tctctttgac 240gaattgattg atgagaaaga atcaaagaga
tggttcgatg agattatccc agcattggcg 300agcttacttc tacagtttcc atctctgtta
gaagtgcatt tccaaaatgc tgataatatt 360gttagtggaa tcaaaaccgg tcttcgtttg
ttaaattccc aacaagctgg cattgttttc 420ctcagccagg agttgattgg agctcttctt
gcatgctctt tcttttgttt gtttccggat 480gataatagag gtgcaaaaca ccttccagtc
atcaactttg atcatttgtt tgcaagcctt 540tatataagtt atagtcaaag tcaagaaagc
aagataagat gtattatgca ttactttgaa 600aggttttgct cctgcgtgcc tattggtatt
gtttcatttg aacgcaagat taccgctgct 660cctgatgctg atttctggag caagtctgac
gtttctcttt gtgcatttaa ggttcactct 720tttgggttaa ttgaagatca acctgacaat
gctctcgaag tggactttgc aaacaagtat 780ctcggaggtg gttccctaag tagagggtgc
gtgcaggaag agatacgctt catgattaac 840cctgaattaa tcgctggcat gcttttcttg
cctcggatgg atgacaatga agctatagaa 900atagttggtg cggaaagatt ttcatgttac
acagggtatg catcttcgtt tcggtttgct 960ggtgagtaca ttgacaaaaa ggcaatggat
cctttcaaaa ggcgaagaac cagaattgtt 1020gcaattgatg cattatgtac accgaagatg
agacacttta aagatatatg tcttttaagg 1080gaaattaata aggcactatg tggcttttta
aattgtagca aggcttggga gcaccagaat 1140atattcatgg atgaaggaga taatgaaatt
cagcttgtcc gaaacggcag agattctggt 1200cttctgcgta cagaaactac tgcgtcacac
cgaactccac taaatgatgt tgagatgaat 1260agagaaaagc ctgctaacaa tcttatcaga
gatttttatg tggaaggagt tgataacgag 1320gatcatgaag atgatggtgt cgcgacaggg
aattggggat gtggtgtttt tggaggagac 1380ccagagctaa aggctacgat acaatggctt
gctgcttccc agactcgaag accatttata 1440tcatattaca cctttggagt agaggcactc
cgaaacctag atcaggtgac gaagtggatt 1500ctttcccata aatggactgt tggagatctg
tggaacatga tgttagaata ttctgctcaa 1560aggctctaca agcaaaccag tgttggcttc
ttttcttggc tacttccatc tctagctacc 1620accaacaaag ctatccagcc gccttga
16478598DNASolanum tuberosum 8gcaatggaga
atagagaaga cgtgaagtca atccttccct ttttgccggt gtgtctccga 60tcatcttctc
ttttctggcc gccgctagtt gttgaagcac tgaaagccct ctctgaaggc 120cctcattaca
gcaatgttaa ctccggccaa gtcctcttcc tcgcaatctc cgacattcgg 180aattcccttt
cactacctga ttcttcaatt tcctcttctg cttcagacgg attttctctc 240ttatttgatg
atttaattcc tagggatgaa gctgttaaat ggttcaaaga agtggtgccg 300aaaatggcgg
atttgctatt gcggttgcct tccttattgg aggctcacta tgagaaggct 360gatggtggaa
ttgttaaagg agtcaacact ggtcttcgct tattggaatc acaacagcct 420ggcattgttt
tcctcagtca ggaattagtc ggtgctcttc ttgcatgttc cttcttttgc 480tattccctac
caatgataga ggtatctgta tgatcagtat gacgagaaat ttgaaaataa 540attgaagtgc
attcttcact attttgagag gattggctca ttgatacctg cgggctac
59891530DNAOryza sativa 9atggaggcgc gcggcgacct gcgctcgatc ctgccctacc
tccccgtcgt gctccgcggc 60ggcgcgctct tctggccgcc ggcggcgcag gaggcgctca
aggcgctggc gctgggcccc 120gacgtgagcc gcgtctcctc cggcgacgtc ctcgccgacg
ccctcaccga cctccgcctc 180gcgctcaacc tcgacccact cccgcgccgc gccgccgagg
gcttcgcgct cttcttcgac 240gacctcctgt cgcgggcgca ggcgcgggac tggttcgacc
acgtcgcccc ctccctcgcc 300cgcctcctcc tccgcctccc cacgctgctc gagggccact
accgcgccgc cggcgacgag 360gctcgcgggc tccgcatcct gagctcgcag gatgccgggc
tcgtgctcct cagccaggag 420ctcgccgccg cgctgctcgc ctgcgcgctc ttctgcctgt
tccccaccgc cgatagggcc 480gaggcgtgcc tcccggcgat caatttcgat agcctatttg
cggcactgtg ttataattcg 540aggcaaagcc aggagcagaa ggtgaggtgc cttgttcact
attttgacag ggtgaccgct 600tctacaccta ctggttccgt ttcgtttgag cgtaaggttc
ttcctcgccg tcctgaatct 660gatggcatta cgtaccctga catggatact tggatgaaat
ctggtgttcc cctttgcaca 720ttccgggtat tttcctcagg cttgatagaa gatgaggaac
aagaagccct tgaagttgac 780tttgcaaata gatatttggg aggtggcgca ctttccagag
gctgcgtgca ggaagaaatc 840cggttcatga taaacccaga attgatcgtg ggcatgctct
tcatggtttc aatggaagat 900aatgaagcta tagaaattgt tggtgcagaa aggttctcac
agtacatggg gtatggttcc 960tcattccgtt ttactggtga ctacttagat agcaaaccct
ttgatgcgat gggtagacgg 1020aaaactagga tagtggcaat tgatgctttg gactgtccaa
ctaggttaca gtttgaatct 1080agtggtcttc taagggaagt gaacaaggct ttttgtggat
ttttggatca atcaaatcat 1140cagctctgtg caaagcttgt ccaggattta aatacaaagg
ataactgtcc aagtgtcatt 1200cctgatgaat gcataggagt ttcaactgga aactggggtt
gcggggcttt tggtggaaac 1260cctgaaatca agagcatgat tcaatggatt gctgcatcac
aggcactccg atcttttatt 1320aactactaca cttttgagtc cgaatcactg aaaagattag
aagaggtgac ccagtggata 1380ttgcgccata ggtggacggt tggcgagttg tgggacatgc
ttgtggagta ttcatcccag 1440aggctaagag gagacaccaa tgagggcttt ttaacatggc
tacttcccaa ggacatcccc 1500aatggtgatg tagattacat gtgtgaatag
153010603DNAZea mays 10tagggctgtg tgcaggagga
aatccgcttc atgataaacc ccgaattgat tgtgggtatg 60ctattcttgt cttgtatgga
agataacgag gctatagaaa tctttggtgc agaacggttc 120tcacagtata tgggttatgg
ttcctccttt cgctttgttg gtgactattt agataccaaa 180ccctttgatt cgatgggcag
acggagaact aggattgtgg ctatcgatgc tttggactgt 240ccagctaggt tacactatga
atctggctgt ctcctaaggg aagtgaacaa ggcattttgt 300ggatttttcg atcaatcgaa
acaccatctc tatgcgaagc ttttccagga tttgcacaac 360aaggatgact tttcaagcat
caattccagt gagtacgtag gagtttcaac aggaaactgg 420ggttgtggtg cttttggtgg
aaaccctgaa atcaagagca tgattcagtg gattgctgca 480tcacaggctc ttcgcccttt
tgttaattac tacacttttg agaacgtgtc tctgcaaaga 540ttagaggagg tgatccagtg
gatacggctt catggctgga ctgtcggcga gctgtggaac 600ata
60311651DNASaccharomyces
cerevisiae 11atgaagactt taattgttgt tgatatgcaa aatgatttta tttcaccttt
aggttccttg 60actgttccaa aaggtgagga attaatcaat cctatctcgg atttgatgca
agatgctgat 120agagactggc acaggattgt ggtcaccaga gattggcacc cttccagaca
tatttcgttc 180gcaaagaacc ataaagataa agaaccctat tcaacataca cctaccactc
tccaaggcca 240ggcgatgatt ccacgcaaga gggtattttg tggcccgtac actgtgtgaa
aaacacctgg 300ggtagtcaat tggttgacca aataatggac caagtggtca ctaagcatat
taagattgtc 360gacaagggtt tcttgactga ccgtgaatac tactccgcct tccacgacat
ctggaacttc 420cataagaccg acatgaacaa gtacttagaa aagcatcata cagacgaggt
ttacattgtc 480ggtgtagctt tggagtattg tgtcaaagcc accgccattt ccgctgcaga
actaggttat 540aagaccactg tcctgctgga ttacacaaga cccatcagcg atgatcccga
agtcatcaat 600aaggttaagg aagagttgaa ggcccacaac atcaatgtcg tggataaata a
651121290DNASaccharomyces cerevisiae 12ttaggtccat ctgtgcgctt
cgttatcacc actccaactt cgttcagtat atcccaattc 60ctctttcact ctcttcacag
tggcaggatc tcccatattt ttacctaagt tatcagaaat 120tttgatagcg tgattaccat
ttacttctaa tagtttgata acgatgttta acggctcact 180tttaacctgg ggttctgact
tcttacgaaa atcattagta aagtttgtgc caataccgaa 240tgtggctagc attccattct
ctttagctgc atgggagtaa gttattgcct tttcgacgtt 300caaagaatcg gaataacaga
taatcttcga gaatttaggc aatttcaaca cgtcatggta 360atggtgggaa atctttttgg
tatactcaac tgggtctcca gaatcttgtc taacaccgac 420gtaagcatca gaatatggtg
gacggaatga ttttaaaaag tcatcagttc caaaagtatc 480cgttaatgct aaaccagcat
tttttgcacc aaaagtattg atccaacaat ccattgcatt 540tttattggca tgcaaataat
cttcactaat agaagcgact cccataaccc actcgtgagc 600cacagtaccg attggcttga
ctccatattt cttggcaaat aaaatatttg atgtgcctaa 660taatagcgat ttgtttctgt
ctgggttacc gttcacagct ttcatgattc cttgcataat 720tagatcttga gccttcagag
atctacgacg tcttgtacca aattcactga atctaatacc 780attatcaaac aaagtttccg
ccttcttctc agcttgttct aattggtttt cgtagtccca 840gtcgatgtca acaaatttaa
aatacgcttc tgatattagg gacagtaagg ggatctcata 900aaggatagta tccttccaac
taccactgac taaaattttc aatttgtagt gggtgggctt 960gccctcgatt tcttctgaag
tgaaggaaat ctgctcttca gggtgtagtt tgtaattaga 1020actgctaata tacttaatat
atgccgatgg caaatatggg atttcctgtt ttaagtattc 1080aatttcctct tctgtgaacc
tcaaatttcc caaatacgaa aattgctctt tcaaccaatt 1140aatggcttcc ttattgaagg
tcaattggga cgacctgttg gtatatttat aagtaactgt 1200aacatctgga aaattagtga
agacagcagc atgcatcgta atcttgtaca tgtctgtgtc 1260caaaagagac tttatcactg
gttctgacat 1290131206DNASaccharomyces
cerevisiae 13atggatccca caagagctcc ggatttcaaa ccgccatctg cagacgagga
attgattcct 60ccacccgacc cggaatctaa aattcccaaa tctattccaa ttattccata
cgtcttagcc 120gatgcgaatt cctctataga tgcacctttt aatattaaga ggaagaaaaa
gcatcctaag 180catcatcatc accatcatca cagtcgtaaa gaaggcaatg ataaaaaaca
tcagcatatt 240ccattgaacc aagacgactt tcaaccactt tccgcagaag tgtcttccga
agatgatgac 300gcggatttta gatccaagga gagatacggt tcagattcaa ccacagaatc
agaaactaga 360ggtgttcaga aatatcagat tgctgattta gaagaagttc cacatggaat
cgttcgtcaa 420gcaagaacct tggaagacta cgaattcccc tcacacagat tatcgaaaaa
attactggat 480ccaaataaac tgccgttagt aatagtagca tgtgggtctt tttcaccaat
cacctacttg 540catctaagaa tgtttgaaat ggctttagat gcaatctctg aacaaacaag
gtttgaagtc 600ataggtggat attactcccc tgttagtgat aactatcaaa agcaaggctt
ggccccatcc 660taccatagag tacgtatgtg tgaattggcc tgcgaaagaa cctcatcttg
gttgatggtg 720gatgcatggg agtcattgca accttcatac acaagaactg ccaaggtctt
ggatcatttc 780aatcacgaaa tcaatattaa gagaggtggt gtagctactg ttactggaga
aaaaattggt 840gtgaaaataa tgttgctggc tggtggtgac ctaatagagt caatgggtga
accaaacgtt 900tgggcggacg ccgatttaca tcacattctc ggtaattacg gttgtttgat
tgtcgaacgt 960actggttctg atgtaaggtc ttttttgtta tcccatgata ttatgtatga
acatagaagg 1020aatattctta tcatcaagca actcatctat aatgatattt cttccacgaa
agttcgtcta 1080tttatcagac gcgccatgtc tgtacaatat ttgttaccta attcggtcat
caggtatatc 1140caagaacata gactatatgt ggaccaaacc gaacctgtta agcaagttct
tggaaacaaa 1200gaatga
1206141188DNASaccharomyces cerevisiae 14atggatccca ccaaagcacc
cgattttaaa ccgccacagc caaatgaaga actacaacca 60ccgccagatc caacacatac
gataccaaaa tctggaccca tagttccata tgttttagct 120gattataatt cttcgatcga
tgctcctttc aatctcgaca tttacaaaac cctgtcgtca 180aggaaaaaaa acgccaactc
aagcaaccga atggaccata ttccattaaa tactagtgac 240ttccagccac tatctcggga
tgtatcatcg gaggaggaaa gtgaagggca atcgaatgga 300attgacgcta ctctacagga
tgttacgatg actgggaatt tgggggtact gaagagccaa 360attgctgatt tggaagaagt
tcctcacaca attgtaagac aagccagaac tattgaagat 420tacgaatttc ctgtacacag
attgacgaaa aagttacaag atcctgaaaa actgcctctg 480atcatcgttg cttgtggatc
attttctccc ataacatacc tacatttgag aatgtttgaa 540atggctttag atgatatcaa
tgagcaaacg cgttttgaag tggttggtgg ttatttttct 600ccagtaagtg ataactatca
aaagcgaggg ttagccccag cttatcatcg tgtccgcatg 660tgcgaattag catgcgagcg
gacatcatct tggttaatgg ttgatgcctg ggaatcttta 720caatcaagtt atacaaggac
agcaaaagtc ttggaccatt tcaatcatga aataaatatc 780aagagaggtg gaatcatgac
tgtagatggt gaaaaaatgg gcgtaaaaat catgttattg 840gcaggcggtg atcttatcga
atccatgggc gagcctcatg tgtgggctga ttcagacctg 900caccatattt tgggtaatta
tggatgtttg atcgtggaaa ggactggttc tgatgttagg 960tccttcttgc tttcccatga
tatcatgtat gaacacagaa gaaatatcct tattatcaaa 1020caacttattt acaatgatat
ttcctctacg aaagtgcggc ttttcatcag acgtggaatg 1080tcagttcaat atcttcttcc
aaactctgtc atccgttaca tccaagagta taatctatac 1140attaatcaaa gtgaaccggt
caagcaggtc ttggatagca aagagtga 1188152145DNASaccharomyces
cerevisiae 15atgtcacatc ttatcacttt agctacatgc aacttgaatc aatgggccct
agattttgaa 60ggtaatagag accgtatcct acagtccatt aagattgcca aagagagggg
tgccaggtta 120cgtgtcggcc cagaactgga aataactggc tacggatgtt tagatcattt
tttagaaaat 180gacgtttgcc ttcattcatg ggaaatgtat gctcaaatca ttaagaataa
agaaacccat 240ggattaatac ttgacattgg tatgcccgtt ctacacaaga atgttcgtta
taattgtcgt 300ttgttatcct tggatggtga gatattgttc ataagaccta agatttggtt
agctaatgat 360ggtaactata gggaaatgag atttttcaca ccttggatga aacctggcgt
ggtggaggac 420tttatccttc cacctgagat tcagaaagtt accggccaga gacttgtgcc
atttggggac 480gctgtgataa attcattgga tacatgcatt ggtacagaaa cttgtgaaga
attgtttaca 540cctcaatccc cccacatcgc catgtcttta gatggtgtgg aaatcatgac
aaactcatct 600ggttctcatc atgaactgcg taagttaaat aaaaggttag acctaatttt
aaatgccact 660aaacgttgtg gtggtgttta cttgtatgca aatcaaagag gttgtgatgg
tgacagatta 720tattatgatg gctgtgcact aattgccatc aatggtacaa ttgtagccca
aggttcacaa 780ttttcgctag atgatgtgga agtagttact gctactgtgg acctagaaga
ggtgaggagt 840tatcgtgcag ctgtcatgtc tcgtggccta caagcctcct tggcagaaat
aaagttcaag 900cgtattgata ttcctgtaga attggcttta atgacctcca gatttgatcc
tacagtgtgt 960ccaacaaaag tccgcgagcc tttctatcac tctcctgagg aagaaattgc
actgggacct 1020gcttgctgga tgtgggatta tttaagacgt tgtaacggaa cagggttttt
ccttccctta 1080tctgggggca ttgactcttg tgcaactgca atgattgtcc actctatgtg
ccgtttagtg 1140accgacgctg ctcaaaatgg aaatgagcaa gttatcaaag acgttcgtaa
gataacacgt 1200agcggcgatg attggattcc agacagtcca caggatctag cctcaaaaat
atttcactcc 1260tgtttcatgg gtacggaaaa ttcatccaag gagacaagaa acagagcaaa
ggacctttcc 1320aatgcaattg gatcttacca cgtggattta aagatggact cattggtatc
cagtgtggtg 1380tccttattcg aagtagccac tggcaaaaaa ccaatataca aaatatttgg
gggatctcaa 1440atcgagaact tggctttaca aaacatccag gcgcgtctaa gaatggttct
ttcttatctt 1500tttgcgcaac tgttgccgtg ggttcgtggt atcccaaact cgggtggatt
gttagtactt 1560ggtagcgcaa atgttgatga gtgcttacgt gggtatctaa caaaatatga
ctgctcctcc 1620gcagatatca accctattgg gggtatttca aaaactgact tgaaaagatt
cattgcctac 1680gcatcaaaac aatataacat gccaatcttg aatgactttt taaacgctac
accaactgca 1740gaattagaac ctatgactaa agattacgtt caatcggatg agatagatat
ggggatgacg 1800tatgaagaat tgggcgtgtt tggttaccta agaaaggttg aaaaatgtgg
tccttattct 1860atgttcttaa aacttcttca tcaatggtcc ccaaagttaa cacctcgtca
aatatctgaa 1920aaggtgaaaa gatttttctt cttctatgcc atcaacagac acaagcaaac
tgttttaact 1980cctagttatc atgctgaaca gtattcacca gaagacaaca gatttgactt
acgtcctttc 2040ttaatcaacc caagatttcc atgggcttca agaaaaattg atgaagttgt
cgagcagtgt 2100gaagcacata aaggctcaac gcttgacatt atgtctattg attag
214516597DNAArabidopsis thaliana 16atggcttcct catcaacgag
aaagtacgag acacgaaagc gagatccaaa ctctaaaatc 60gcagctcttc tcgttatcga
catgcagaat cacttctcct ccatggccaa acccatcctc 120aacaacgttc tcaccaccat
cgacatctgc cgacgcgcct cagtccccgt attctttacg 180cgtcacaacc acaaatcccc
gaccgaccac ggcatgctcg gcgagtggtg taacggcgat 240gtaatccttg acggaaccac
cgattctgaa atcatccagg agatacaagg ccaagtaacc 300ggaccagacg agatggtgga
gaagaacacg tacagtgcgt ttaacaaaac ccgcctccag 360gaaaacctgg aaaagatcgg
agtaaaggag gtgatcgtga tcggagtgat gacgaacttg 420tgctgtgaga caacggcgcg
tgaagcgttt attaagggtt ttagggtttt tttctcgacg 480gacgcgactg cgacgtttaa
tgaggagctt cacgaggcta cgctaatgaa tctcgctttt 540ggcttcgctt atctcgtcga
ttgcgataaa ctccggcgaa gtctactcgg taactaa 59717597DNAArabidopsis
thaliana 17atggcttctt catcatcgag aacgtacgag acacgaaagc gagagccaaa
tcctaaaatc 60gcagctcttc tcgtcatcga tatgcagaat cacttctact ctatggctga
accaatcctc 120caaaacgctc tcaccaccat cgacatctgc cgacgcgctt caatccccgt
attcttcacg 180cgccacaacc acaaatcccc aaccgaccac ggcatgctcg gagagtggtg
gaacggcgat 240ctaatcctcg acggaaccac tgattccgaa atcatcccgg aaatcaatcg
ccaggtcacc 300ggaccagacg aaatcgtgga gaagagcacg tacagtgcgt ttaacaacac
gcaccttcag 360gagaagctgg acaagatcgg agtgaaggag gtgatcgtta tcggagtgat
gacgaaccta 420tgctgtgaga cgacggcgcg tgaagcgttt gtaaaggggt ttagggtttt
tttctcgacg 480gacgcgactg cgacggttaa tgaagagctt cacgaggcta ctctaatgaa
tctcgcgtat 540ggctttgctt atctcgtcga ttgcgataga ctccggcgag gtctactcag
tagttaa 59718591DNAArabidopsis thaliana 18atggccgaga gatggaggaa
cacggctcta ctcgtcatcg acatgcagaa cgatttcata 60gaggaaggtg ctgtgacgca
agtgaaagga ggaaaatcta tagttcctaa tgttatcaga 120gtcgtcgaac tcgcgaggca
gcgtggtatt ctcgtaattt gggttgttcg agaacatgat 180cgtcaaggaa gagatgttga
attattcagg cgccataact acagttctga gaaagtcggg 240ccagttatta aaggcaccgt
aggagcagaa ttggttgatg gattgatgat caacgaagaa 300gatgactata agattgtgaa
aactcgtttc agtgctttct ttagtaccaa tcttcattcc 360ttcttgcaaa cttcaggggt
taccaagtta gtgattgctg gtgtgcaaac gccgaactgt 420atccggcaaa cggtgtttga
tgcagtggcg ctggattatc ccaatgtgac tgttattaca 480gatgccacag ctgctgcaac
accagagatc catactgcga atattcttga catgaagaat 540attggagtca agactcctac
attacacgag tggtccgaag aacttgcttg a 591191680DNAArabidopsis
thaliana 19atggagaaga aagaaaatgg tctcgatgga aagcaatcgg gtcgggtcat
taacggaccc 60actaacccga tggtcacacc tctgctcaac gatctttacc aattcaccat
ggcttatgct 120tattggaaag ctggcaaaca atctgagcga tctgtgtttg atctgtattt
tcgtaagaat 180ccttttggtg gagaatacac tatctttgct ggtttagaag aatgcatcaa
atttctcgct 240aatttcaatt tgactgatga agagatcgat ttcgttcgtg attcgttacc
tggatgtgag 300gaagctttct gtgattatct tcgagggctt gattgttctg acattgaagt
gtatgccatt 360tcggaaggat cagttgtttt tcctaaagtt cctttactca gaatcgaagg
tcctgttgct 420gtggtgcaat tgttggaaac tccattcctc aatctcatca attacgcatc
tttggttgct 480acaaatgcag caagacatcg gtttgttgca ggaaaatcta agcttctgct
tgagtttggt 540gctagaagag ctcagggacc cgatggtgca ataagcgcat caaagtattg
ctaccttgga 600ggttttgatg caacaagtaa tgttgcagcg ggaaaactgt ttgggatacc
cctccgtggt 660actcattccc atgcttttgt tagctcattc atgagccttg atgaaattgt
tgacaaagtg 720cttcgaagtt ctgatgggaa aagcacttgt aaggatttta tatgtttggt
ccaaacttgc 780ctaacaaaga ttcagaattc atcttcatta caaggaattt tttccgagac
aaatcaaagc 840gagcttgcag cgttcatttc atatgcactg gcattcccaa actcctttct
cgctcttgta 900gacacttatg atgtgatgaa gagtggtatt ccaaacttct gtgctgttgc
tctagcactt 960aatgaattgg gatacaaagc agtaggcatt agactggatt caggtgactt
agcctatctt 1020tctactgagg tcaggaaatt cttttgtgcc atagagagag acctcaaagt
tcctgatttc 1080gggaagatga tcgtcactgc tagtaacgat ctaaacgaag agacagtcga
tgctctaaat 1140aaacagggtc atgaagtaga tgcatttgga attggaacca acttagtgac
ttgctatgcg 1200caagctgcgt taggttgtgt tttcaaactt gtggaaataa acaatcagcc
tcggatcaaa 1260ctttctgaag atgttactaa ggtatcgatt ccatgtaaaa agcgtactta
cagattgttc 1320ggaaaagagg gttaccctct tgttgatata atgactggag agaacgaacc
acctccaaag 1380gtcggtgaaa ggttactttg ccgtcatcca ttcaatgaat caaaaagggc
ttatgtggtt 1440ccacaacgcg ttgaagagct tctgaaatgt tattggcgtg gcaatgcaga
tgaagctagg 1500gaagagctag agccattgaa agagctaaga aatcgttgca tcaaacagct
cgaaaatatg 1560cgacccgatc atatgagaag attaaaccct actccttata aggttagtgt
cagcgccaag 1620ttgtatgact tcatccactt cctctggctc aacgaagctc ctgtcggtga
actgcattga 1680201674DNAArabidopsis thaliana 20atggagccga aagagaacgg
ctcagaattg ggtcagaaga tcattgacgg accaacgaat 60ccaatggtca cacctttact
caatgatctt tatcaattca ccatggctta tgcttattgg 120aaagctggca aacacaacga
acgatccgtt ttcgatctgt attttcgtaa gaacccattt 180ggtggtgagt acactgtgtt
tgctggatta gaagagtgtg ttaagttctt agccaatttc 240aaattgactg atgaagaaat
cgatttcgtt caagagtgtt tgcctggatc tgaggaagct 300ttttgtgatt atcttagagg
gcttgattgt tctgatgttg aagtttatgc aattccggaa 360ggatcagttg tttttcctaa
agtacctctc atgagagttg aaggacctgt tggtgttgtt 420caattgttgg aaactccatt
cctcaatctt gtcaattttg catctttggt agctactaac 480gcagctaggc atcgctttgt
tgccggaaaa tctaagagtc tactcgagtt tggtgctcga 540agggctcagg gtccggatgg
tgcaataagc gcatcaaaat attgctacct tggaggtttt 600gatgcaacaa gtaatgtagc
agctggaaaa ctttttggga ttcctcttcg tggaacacac 660tctcatgctt atgttagctc
attcatgagt actgatgaga ttgttgacaa agtacttcgt 720agtgctgatg ggaaaaccac
gtgcgaggat tttgttagtc atgttcagac atggttaaaa 780aagattcagt attcaccatc
tctaagtggc attttctctg agacaaatca aagcgagcta 840gcagctttca cctcatatgc
actggcattc cccaaaactt ttcttgccct cgtagataca 900tacgatgtga tgaagagtgg
aatccctaac ttctgtgcag ttgctttagc actcaatgac 960tttggatata aagcattagg
tattagactg gattcaggtg atttagctta tctatctaga 1020gaggccagaa atttcttctg
cacggtagag agagaactaa aagtgcctgg ttttgggaag 1080atggtcgtca ctgctagtaa
tgatctaaat gaagagacga ttgacgcttt aaataaacag 1140ggacatgagg tggatgcttt
tggcatcggg acctacttgg tcacttgcta ttcacaagcg 1200gccttaggtt gcgttttcaa
acttgtggag ataaacaatc agcctcggat taaactttct 1260gaagatgtta caaaggtatc
aataccgtgt aaaaagcgaa gttacagatt atacggcaaa 1320gaaggttacc ctctggtaga
tataatgact ggagagaacg aaccacctcc aaaggttggt 1380gagcgtttac tttgtcgtca
cccattcaac gaatccaaaa gagcatatgt agtgccacaa 1440cgtgtcgaag agctcctcaa
atgttattgg cgtggaagtg cagatgaagc aagagaagta 1500ttaccgcctt tgaaagagat
aagagaccgt tgcatcaaac agctcgaaaa catgcgacct 1560gatcatatga ggagattaaa
cccaactcct tataaggtta gtgtaagcgc aaagctgtac 1620gatttcatcc acttcttatg
gctaaacgaa gcacctgttg gtgaattgca gtga 167421717DNAArabidopsis
thaliana 21atggatgtcc cgttaccagt cgagaaatta tcttatggat caaacactga
ggacaaaact 60tgtgtagtgc ttgtggcaac tgggagtttc aatcctccta ctttcatgca
tttacgcatg 120tttgagctgg cgagagatga attacgctca aaaggatttc atgttcttgg
aggatatatg 180tctcctgtta atgatgcata taagaagaag ggccttttat ctgcagaaca
tcgtttagag 240atgtgtaatg tatcatgtca aagctctgac tttgtaatgg ttgatccgtg
ggaggcatct 300caaagcaact accaacgaac tttgacggtt ttatcaaggg tcaagacttt
cttaacaaca 360aatcgacatg tacccgagga atctctcaaa gtcatgctac tatgtggctc
ggatttactg 420ctatctttct gcactcccgg tgtttggatc cctgaacagt taagaactat
ttgcaaagat 480tatggcattg tgtgcatccg tagagaagga caagatgttg aaaatatgat
ctctggtgac 540gaaatcttaa acgaaaactg tgctaacgtc aaaatcgttg acaatactgt
tcctaatcaa 600atcagttcga gtagattaag gcaatgcatt tcgcgagggt tatcggttaa
atacttgact 660gaagatggag taatagatta tatcagacaa catcaactat acactgagct
cacatga 717222178DNAArabidopsis thaliana 22atgaggctgt tgaaggttgc
tacgtgtaac ttgaaccaat gggccatgga tttcgagagc 60aacatgaaga acatcaaggc
ttcgatcgct gaggcaaagg ctgctggtgc tgttatcagg 120cttggacccg agctcgaggt
cactggctat ggttgcgagg atcacttctt ggaactcgac 180actgtcactc atgcgtggga
gtgtttgaag gaattgctgc ttggtgattg gacggatgat 240attttgtgca gcataggaat
gcctgtgatt aaaggagcag agcgttataa ctgccaggtt 300ctctgtatga acagaagaat
catcatgatt cgaccgaaaa tgtggctcgc aaacgatgga 360aactataggg agctacggtg
gttcacagct tggaagcaga gagaagagct agaggaattt 420cagctcccca ttgaaatttc
agaggctttg gagcagaaat cagtcccttt tggttatggt 480tacatccagt ttatcgacac
ggctgttgca gctgaagtct gtgaggaact gtttagtcca 540cttcctcctc atgccgagct
cgcattgaat ggtgttgaag tatttatgaa tgcaagtggg 600agtcatcacc aacttaggaa
actagatatt cgtctgaatg cttttatggg ggctactcat 660gctcgtggtg gggtgtatat
gtacagtaat caacaaggat gcgatggtag ccgcttatac 720tacgatggat gtgcatgtat
tgttgtaaac gggaatgttg ttgctcaagg ctcacaattc 780tcgttgagag acgttgaggt
catcatttca caagtggatc ttgatgcggt tgctagcctt 840cgtggatcta taagtagctt
tcaggaacaa gcaagctgca aggttaaagt atcttcagta 900gctgtgccct gtagacttac
acagtccttc aacctgaaaa tgacactaag cagtccgaag 960aagatcattt accactctcc
acaagaagaa atagcctttg gtcccgcttg ctggatgtgg 1020gactatttga gaagaagtgg
cgcttcagga tttttgcttc ctctttctgg cggagcagac 1080agctcctccg tggcagctat
tgttggctgc atgtgccaac ttgttgttaa agagattgca 1140aagggagatg agcaagtaaa
agctgatgcg aaccgaattg ggaattatgc taatgggcag 1200tttcctactg atagcaaaga
gtttgccaaa cgaatatttt acactgtctt tatgggttct 1260gaaaacagtt ctgaggagac
aaaaaggcgt tcaaagcagc tggcagacga gattggtgct 1320tggcatcttg atgtttgcat
agatggtgtt gtctctgcag ttttatcatt atttcaaaca 1380gttacaggca agcgaccaag
gtataaggtt gatggaggat caaatgctga gaaccttggg 1440ttgcagaaca ttcaagcccg
gatgagaatg gtgttagcat ttatgttagc gtctctcttg 1500ccttgggttc atagcaaacc
aggcttttac cttgttctag gcagctccaa cgttgatgaa 1560ggacttcgtg gttacctgac
aaagtatgat tgcagctcag cagacataaa tcctatagga 1620agtatcagta aaatggattt
gaggttgttc ttaaaatggg ctgcaacgaa tctcggatat 1680ccatccttgg cagagataga
agctgctcca ccaacagctg agcttgagcc cattcgttct 1740gactattctc agctcgatga
agtcgacatg ggaatgacat atgaagagct ttcagtctat 1800ggaaggatga ggaagatatt
ccgttgtgga ccagtatcta tgttcaagaa tctatgttac 1860aagtggggaa caaagctaag
cccagcagaa gtagctgaga aagtgaagta tttcttcaaa 1920tattattcga tcaatcgaca
caaaatgact gtcctcacac cgtcttatca cgctgagagt 1980tactccccag aggacaacag
attcgatctg aggcagtttc tgtacaacag caagtggcca 2040taccagttta agaagattga
cgagattgtt gacagcttaa atggtgactc agttgctttc 2100ccggaagaag aagcaaactc
caacaaagaa attggagttg tagcagcaaa ctccggagac 2160ccaagtgcgg gtctctga
2178
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