Patent application title: GENERATING MAIZE PLANTS WITH ENHANCED RESISTANCE TO NORTHERN LEAF BLIGHT
Inventors:
IPC8 Class: AA01H646FI
USPC Class:
1 1
Class name:
Publication date: 2019-03-14
Patent application number: 20190075749
Abstract:
Compositions and methods for generating maize plants that exhibit
resistance to northern leaf blight are provided herein. Isolated
polynucleotides encoding a polypeptide that confers resistance to
northern leaf blight, polynucleotide constructs comprising such, and
maize plants comprising the polynucleotide constructs are provided. The
methods include expressing an isolated polynucleotide in a maize cell via
standard transformation methods and obtaining a maize plant from said
maize cell.Claims:
1. A polynucleotide construct comprising an isolated polynucleotide
selected from the group consisting of: a. the nucleotide sequence set
forth in SEQ ID NO:1 (PH4GP c-DNA), SEQ ID NO:3 (PH1W2 cDNA), or SEQ ID
NO:9 (PH4GP Genomic sequence); b. a nucleotide sequence encoding a
CC-NB-LRR polypeptide having an amino acid sequence of at least 90%
sequence identity when compared to SEQ ID NO:2 or SEQ ID NO:4, based on
the CLUSTAL W method of alignment with default parameters; c. a
nucleotide sequence encoding a CC-NB-LRR polypeptide having an amino acid
sequence of at least 90% sequence identity when compared to SEQ ID NO:2
or SEQ ID NO:4, based on the CLUSTAL W method of alignment with default
parameters, wherein said polypeptide comprises the amino acid sequence
set forth in SEQ ID NO:10; and d. a nucleotide sequence encoding a
CC-NB-LRR polypeptide having the sequence set forth in SEQ ID NO:2 or SEQ
ID NO:4, wherein said isolated polynucleotide is operably linked to a
promoter.
2. The polynucleotide construct of claim 1, wherein the polynucleotide construct further comprises one or more additional heterologous nucleic acid sequences that encode a polypeptide selected from the group consisting of: a polypeptide conferring disease resistance, a polypeptide conferring herbicide resistance, a polypeptide conferring insect resistance, a polypeptide involved in carbohydrate metabolism, a polypeptide involved in fatty acid metabolism, a polypeptide involved in amino acid metabolism, a polypeptide involved in plant development, a polypeptide involved in plant growth regulation, a polypeptide involved in yield improvement, a polypeptide involved in drought resistance, a polypeptide involved in cold resistance, a polypeptide involved in heat resistance, and/or a polypeptide involved in salt resistance, wherein each heterologous nucleic acid sequence is operably linked to a promoter.
3. The polynucleotide construct of claim 2, wherein a polypeptide conferring disease resistance is a polypeptide that confers resistance to northern leaf blight (NLB).
4. The polynucleotide construct of claim 3, wherein said polypeptide conferring resistance to northern leaf blight is a polypeptide having an amino acid sequence of at least 90% sequence identity when compared to SEQ ID NO:11 or 12, based on the CLUSTAL W method of alignment with default parameters.
5. A maize plant cell comprising the polynucleotide construct of claim 1.
6. A maize plant comprising the maize plant cell of claim 5.
7. A method for producing a maize plant that exhibits resistance to northern leaf blight (NLB) comprising, a) expressing in a regenerable maize plant cell a polynucleotide construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide is selected from the group consisting of: i. a nucleotide sequence set forth in SEQ ID NO:1 (PH4GP c-DNA), SEQ ID NO:3 (PH1W2 cDNA), or SEQ ID NO:9 (PH4GP Genomic sequence); ii. a nucleotide sequence encoding a CC-NB-LRR polypeptide having an amino acid sequence of at least 90% sequence identity when compared to SEQ ID NO:2 or SEQ ID NO:4, based on the CLUSTAL W method of alignment with default parameters; iii. a nucleotide sequence encoding a CC-NB-LRR polypeptide having an amino acid sequence of at least 90% sequence identity when compared to SEQ ID NO:2 or SEQ ID NO:4, based on the CLUSTAL W method of alignment with default parameters, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:10; iv. a nucleotide sequence encoding a CC-NB-LRR polypeptide having the sequence set forth in SEQ ID NO:2 or SEQ ID NO:4; or v. the nucleotide sequence complementary to (i), (ii), (iii) or (iv); and b) generating a maize plant that exhibits resistance to northern leaf blight, wherein said maize plant comprises in its genome the polynucleotide construct.
8. The method of claim 7, wherein said at least one regulatory sequence is a promoter.
9. The method of claim 7, wherein said at least one regulatory sequence is a terminator.
10. The method of claim 7, wherein said regulatory sequence is native to maize.
11. The method of claim 7, wherein said regulatory sequence is native to the Ht1 gene.
12. The method of claim 7, wherein said polynucleotide construct comprises one or more additional heterologous nucleic acid sequences that encode a polypeptide selected from the group consisting of: a polypeptide conferring disease resistance, a polypeptide conferring herbicide resistance, a polypeptide conferring insect resistance, a polypeptide involved in carbohydrate metabolism, a polypeptide involved in fatty acid metabolism, a polypeptide involved in amino acid metabolism, a polypeptide involved in plant development, a polypeptide involved in plant growth regulation, a polypeptide involved in yield improvement, a polypeptide involved in drought resistance, a polypeptide involved in cold resistance, a polypeptide involved in heat resistance, and/or a polypeptide involved in salt resistance, wherein each heterologous nucleic acid sequence is operably linked to a promoter.
13. The method of claim 12, wherein the polypeptide conferring disease resistance is a polypeptide that confers resistance to northern leaf blight (NLB).
14. The method of claim 13, wherein said polypeptide conferring resistance to northern leaf blight is a polypeptide having an amino acid sequence of at least 90% sequence identity when compared to SEQ ID NO:11 or 12, based on the CLUSTAL W method of alignment with default parameters.
15. A method of obtaining a maize plant that exhibits resistance to northern leaf blight (NLB), said method comprising, a) crossing a maize plant generated by the method of claim 7 with a maize plant that does not comprise in its genome the polynucleotide construct; b) obtaining a progeny plant that exhibits resistance to northern leaf blight, wherein said progeny plant comprises the polynucleotide construct in its genome.
Description:
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 62/242,691, filed Oct. 16, 2015, the entire contents of which are hereby incorporated by reference.
FIELD
[0002] The present disclosure relates to compositions and methods useful in generating maize plants with enhanced resistance to northern leaf blight.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0003] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 20160928_BB2396PCT_SequenceListing.txt created on Sep. 28, 2016 and having a size 65 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.
BACKGROUND
[0004] Northern leaf blight (NLB), induced by the fungal pathogen Exserohilum turcicum (previously called Helminthosporium turcicum), is a serious foliar wilt disease of maize in many tropical and temperate environments. Symptoms can range from cigar-shaped lesions on the lower leaves to complete destruction of the foliage, thereby reducing the amount of leaf surface area available for photosynthesis. A reduction in photosynthetic capability leads to a lack of carbohydrates needed for grain fill, which impacts grain yield. Mid-altitude regions of the tropics, about 900-1600 m above sea level, have a particularly favorable climate for northern leaf blight, as dew periods are long and temperatures moderate. However, northern leaf blight can also yield losses of 30-50% in temperate environments, such as in the United States, during wet seasons, particularly if the infection is established on the upper leaves of the plant by the silking stage.
[0005] The most effective and most preferred method of control for northern leaf blight is the planting of resistant hybrids. Several varieties or races of Exserohilum turcicum are present in nature, leaving growers with two hybrid options: partial resistant hybrids, which offer low-level, broad spectrum protection against multiple races, and race-specific resistant hybrids, which protect against a specific race. Genetic sources of resistance to Exserohilum turcicum have been described, and four Exserohilum turcicum resistance loci have been identified: Ht1, Ht2, Ht3, and Htn1. Gene Ht1 maps to the long arm of chromosome 2 where it is closely linked to umc36 (Coe, E. H. et al. (1988), Corn and Corn Improvement, 3rd edn., pp. 81-258), sgcr506 (Gupta, M. et al. (1989) Maize Genet. Coop. Newsl. 63, 112), umc150B (Bentolila, S. et al. (1991) Theor. Appl. Genet., 82:393-398), and pic18a (Collins et al. (1998) Molecular Plant-Microbe Interactions, 11:968-978), and it is closely flanked by umc22 and umc122 (Li et al. (1998) Hereditas, 129:101-106). Gene Ht2 maps to the long arm of chromosome 8 in the umc48-umc89 interval (Zaitlin et al. (1992) Maize Genet. Coop. Newsl., 66, 69-70), and gene Ht3 maps to chromosome 7 near bnIg1666 (Van Staden, D et al. (2001) Maize Genetics Conference Abstracts 43:P134). The Htn1 gene maps to chromosome 8, approximately 10 cM distal to Ht2 and 0.8 cM distal to the RFLP marker umc117 (Simcox and Bennetzen (1993) Maize Genet. Coop. Newl. 67, 118-119; Simcox and Bennetzen (1993) Phytopathology, 83:1326-1330).
[0006] The methods of controlling northern leaf blight by reducing fungal inoculum require additional time and resources on the part of the farmer, and in addition, can have detrimental effects on the environment. This makes the planting of resistant hybrids even more attractive to farmers and the general public. Thus, it is desirable to provide compositions and methods for generating maize plants with enhanced resistance to northern leaf blight.
SUMMARY
[0007] Presented herein are compositions and methods for generating maize plants exhibiting resistance to northern leaf blight, whether that resistance is newly conferred or enhanced.
[0008] Isolated polynucleotides are presented herein that can be used to generate maize plants that exhibit resistance to northern leaf blight. An isolated polynucleotide may be selected from the group consisting of: (a) the nucleotide sequence set forth in SEQ ID NO:1 (PH4GP c-DNA), SEQ ID NO:3 (PH1W2 cDNA), or SEQ ID NO:9 (PH4GP Genomic sequence); (b) a nucleotide sequence encoding a CC-NB-LRR polypeptide having an amino acid sequence of at least 90% sequence identity when compared to SEQ ID NO:2 or SEQ ID NO:4, based on the CLUSTAL W method of alignment with default parameters; (c) a nucleotide sequence encoding a CC-NB-LRR polypeptide having an amino acid sequence of at least 90% sequence identity when compared to SEQ ID NO:2 or SEQ ID NO:4, based on the CLUSTAL W method of alignment with default parameters, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:10; and (d) a nucleotide sequence encoding a CC-NB-LRR polypeptide having the sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
[0009] Polynucleotide constructs comprising the isolated polynucleotides are also provided, wherein an isolated polynucleotide is operably linked to a promoter. A polynucleotide construct may further comprise one or more heterologous nucleic acid sequences that encode a polypeptide selected from the group consisting of: a polypeptide conferring disease resistance, a polypeptide conferring herbicide resistance, a polypeptide conferring insect resistance, a polypeptide involved in carbohydrate metabolism, a polypeptide involved in fatty acid metabolism, a polypeptide involved in amino acid metabolism, a polypeptide involved in plant development, a polypeptide involved in plant growth regulation, a polypeptide involved in yield improvement, a polypeptide involved in drought resistance, a polypeptide involved in cold resistance, a polypeptide involved in heat resistance, and/or a polypeptide involved in salt resistance, wherein the one or more heterologous nucleic acid sequences are operably linked to a promoter. For example, a polypeptide conferring disease resistance may be a polypeptide that confers resistance to northern leaf blight (NLB), which may further have an amino acid sequence of at least 90% sequence identity when compared to SEQ ID NO:11 or 12, based on the CLUSTAL W method of alignment with default parameters.
[0010] Maize plant cells comprising the polynucleotide constructs and maize plants comprising the maize plant cells are also provided.
[0011] Methods for generating maize plants that exhibit resistance to northern leaf blight are provided herein, in which a polynucleotide construct comprising an isolated polynucleotide provided herein, wherein said isolated polynucleotide is operably linked to at least one regulatory sequence, is expressed in a regenerable maize plant cell, and a maize plant that exhibits resistance to northern leaf blight is generated from the maize plant cell. The maize plant generated by the method comprises in its genome the polynucleotide construct. The regulatory sequence may be a promoter and/or a terminator and may be native to maize. In some aspects, the regulatory sequence is native to the Ht1 gene. In still other aspects, the polynucleotide construct comprises one or more additional heterologous nucleic acid sequences that encode a polypeptide selected from the group consisting of: a polypeptide conferring disease resistance, a polypeptide conferring herbicide resistance, a polypeptide conferring insect resistance, a polypeptide involved in carbohydrate metabolism, a polypeptide involved in fatty acid metabolism, a polypeptide involved in amino acid metabolism, a polypeptide involved in plant development, a polypeptide involved in plant growth regulation, a polypeptide involved in yield improvement, a polypeptide involved in drought resistance, a polypeptide involved in cold resistance, a polypeptide involved in heat resistance, and/or a polypeptide involved in salt resistance, wherein each heterologous nucleic acid sequence is operably linked to a promoter. The polypeptide may be one that confers resistance to northern leaf blight (NLB), such as for example, a polypeptide having an amino acid sequence of at least 90% sequence identity when compared to SEQ ID NO:11 or 12, based on the CLUSTAL W method of alignment with default parameters. A progeny plant comprising the polynucleotide construct may also be generated by crossing the maize plant generated by the method to a second maize plant that does not comprise in its genome the polynucleotide construct.
BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTINGS
[0012] The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing which form a part of this application. The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2): 345-373 (1984), which are herein incorporated by reference in their entirety. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. .sctn. 1.822.
[0013] FIGS. 1A-1D show the alignment of the CC-NB-LRR variants from PH4GP (SEQ ID NO:2), PH1W2 (SEQ ID NO:4), and B73 (SEQ ID NOs:6 and 8). The deletion in the LRR region in the B73 alleles is boxed in FIG. 1C.
[0014] SEQ ID NO:1 is the nucleotide sequence of the Ht1 cDNA found in inbred line PH4GP.
[0015] SEQ ID NO:2 is the amino acid sequence of the polypeptide encoded by SEQ ID NO:1.
[0016] SEQ ID NO:3 is the nucleotide sequence of the Ht1 cDNA found in inbred line PH1W2.
[0017] SEQ ID NO:4 is the amino acid sequence of the polypeptide encoded by SEQ ID NO:3.
[0018] SEQ ID NO:5 is the nucleotide sequence of the Ht1 cDNA found in inbred line B73 and herein referred to as the "B73-high allele".
[0019] SEQ ID NO:6 is the amino acid sequence of the polypeptide encoded by SEQ ID NO:5.
[0020] SEQ ID NO:7 is the nucleotide sequence of the Ht1 cDNA found in inbred line B73 and herein referred to as the "B73-low allele".
[0021] SEQ ID NO:8 is the amino acid sequence of the polypeptide encoded by SEQ ID NO:7.
[0022] SEQ ID NO:9 is the nucleotide sequence of the Ht1 genomic DNA found in inbred line PH4GP.
[0023] SEQ ID NO:10 is the amino acid sequence of a region found in the Ht1 polypeptides of resistant alleles.
[0024] SEQ ID NO:11 is the amino acid sequence of NLB18 from line PH99N in patent application WO2011163590.
[0025] SEQ ID NO:12 is the amino acid sequence of NLB18 from line PH26N in patent application WO2011163590.
DETAILED DESCRIPTION
[0026] Before describing the present invention in detail, it is to be understood that this invention is not limited to particular embodiments, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, terms in the singular and the singular forms "a", "an" and "the", for example, include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "plant", "the plant" or "a plant" also includes a plurality of plants; also, depending on the context, use of the term "plant" can also include genetically similar or identical progeny of that plant; use of the term "a nucleic acid" optionally includes, as a practical matter, many copies of that nucleic acid molecule; similarly, the term "probe" optionally (and typically) encompasses many similar or identical probe molecules.
[0027] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.
I. Compositions
A. Ht1 Polynucleotides and Polypeptides
[0028] Mapping of a QTL associated with northern leaf blight resistance on chromosome 2, using a population derived from a cross between northern leaf blight resistant line PH4GP and northern leaf blight susceptible line PH5W4, was described in US2010095395. Presented herein is the cloning of the Ht1 gene in maize and identification of a putative CC-NB-LRR (coiled-coil, nucleotide-binding, leucine-rich repeat) gene as the causal gene. Ht1 cDNA sequences from PH4GP and PH1W2, the two resistant sources described in US2010095395, are represented by SEQ ID NOs:1 and 3, respectively, while the amino acid sequences of the encoded polypeptides are represented by SEQ ID NO:2 and 4. Moreover, a construct containing the genomic sequence of the PH4GP (resistant) allele (SEQ ID NO:9) was generated and transformed into a susceptible transformation line using Agrobacterium-mediated transformation, resulting in maize plants with resistance to northern leaf blight.
[0029] The Zea mays CC-NB-LRR (coiled-coil, nucleotide-binding, leucine-rich repeat; also referred to as Ht1) gene is a member of a large and complex family of disease resistance genes. The mechanism of NB-LRR protein activation and subsequent signaling in effector triggered immunity is not well understood (Eitas and Dangl. 2010. Curr Opin Plant Biol 13(4):472-477).
[0030] Thus, presented herein are polynucleotides that can be used to generate maize plants with resistance to northern leaf blight. The polynucleotide may be (a) the nucleotide sequence set forth in SEQ ID NO:1 (PH4GP c-DNA), SEQ ID NO:3 (PH1W2 cDNA), or SEQ ID NO:9 (PH4GP Genomic sequence); (b) a nucleotide sequence encoding a CC-NB-LRR polypeptide having an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity when compared to SEQ ID NO:2 or SEQ ID NO:4, based on the CLUSTAL W method of alignment with default parameters; (c) a nucleotide sequence encoding a CC-NB-LRR polypeptide having an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity when compared to SEQ ID NO:2 or SEQ ID NO:4, based on the CLUSTAL W method of alignment with default parameters, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO:10, which is the sequence of the deleted region of Ht1 in B73; and (d) a nucleotide sequence encoding a CC-NB-LRR polypeptide having the sequence set forth in SEQ ID NO:2 or SEQ ID NO:4.
[0031] The use of the term "polynucleotide" is not intended to limit a polynucleotide of the disclosure to a polynucleotide comprising DNA. Those of ordinary skill in the art will recognize that polynucleotides can comprise ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the disclosure also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.
[0032] As used herein, an "isolated" or "purified" polynucleotide or polypeptide, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the polynucleotide or polypeptide as found in its naturally occurring environment. Thus, an isolated or purified polynucleotide or polypeptide is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Optimally, an "isolated" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived. For purposes of this disclosure, "isolated" or "recombinant" when used to refer to nucleic acid molecules excludes isolated unmodified chromosomes. For example, in various embodiments, the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived. A polypeptide that is substantially free of cellular material includes preparations of polypeptides having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the polypeptide of the disclosure or a biologically active portion thereof is recombinantly produced, optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.
[0033] As used herein, a "recombinant" polynucleotide comprises a combination of two or more chemically linked nucleic acid segments which are not found directly joined in nature. By "directly joined" is intended the two nucleic acid segments are immediately adjacent and joined to one another by a chemical linkage. In specific embodiments, the recombinant polynucleotide comprises a polynucleotide of interest such that an additional chemically linked nucleic acid segment is located either 5', 3' or internal to the polynucleotide of interest. Alternatively, the chemically-linked nucleic acid segment of the recombinant polynucleotide can be formed by the deletion of a sequence. The additional chemically linked nucleic acid segment or the sequence deleted to join the linked nucleic acid segments can be of any length, including for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or greater nucleotides. Various methods for making such recombinant polynucleotides are disclosed herein, including, for example, by chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering techniques. In specific embodiments, the recombinant polynucleotide can comprise a recombinant DNA sequence or a recombinant RNA sequence.
[0034] A "recombinant polypeptide" comprises a combination of two or more chemically linked amino acid segments which are not found directly joined in nature. In specific embodiments, the recombinant polypeptide comprises an additional chemically linked amino acid segment that is located either at the N-terminal, C-terminal or internal to the recombinant polypeptide. Alternatively, the chemically-linked amino acid segment of the recombinant polypeptide can be formed by deletion of at least one amino acid. The additional chemically linked amino acid segment or the deleted chemically linked amino acid segment can be of any length, including for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or amino acids.
[0035] Sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MEGALIGN.RTM. program of the LASERGENE.RTM. bioinformatics computing suite (DNASTAR.RTM. Inc., Madison, Wis.). Unless stated otherwise, multiple alignment of the sequences provided herein were performed using the CLUSTAL V method of alignment (Higgins and Sharp, CABIOS. 5:151 153 (1989)) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the CLUSTAL V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences, using the CLUSTAL V program, it is possible to obtain "percent identity" and "divergence" values by viewing the "sequence distances" table on the same program; unless stated otherwise, percent identities and divergences provided and claimed herein were calculated in this manner.
[0036] Alternatively, the Clustal W method of alignment may be used. The Clustal W method of alignment (described by Higgins and Sharp, CABIOS. 5:151-153 (1989); Higgins, D. G. et al., Comput. Appl. Biosci. 8:189-191 (1992)) can be found in the MegAlign.TM. v6.1 program of the LASERGENE.RTM. bioinformatics computing suite (DNASTAR.RTM. Inc., Madison, Wis.). Default parameters for multiple alignment correspond to GAP PENALTY=10, GAP LENGTH PENALTY=0.2, Delay Divergent Sequences=30%, DNA Transition Weight=0.5, Protein Weight Matrix=Gonnet Series, DNA Weight Matrix=IUB. For pairwise alignments the default parameters are Alignment=Slow-Accurate, Gap Penalty=10.0, Gap Length=0.10, Protein Weight Matrix=Gonnet 250 and DNA Weight Matrix=IUB. After alignment of the sequences using the Clustal W program, it is possible to obtain "percent identity" and "divergence" values by viewing the "sequence distances" table in the same program.
B. Polynucleotide Constructs
[0037] The Ht1 polynucleotides disclosed herein can be provided in expression cassettes (such as, for example, in the form of polynucleotide constructs) for expression in the plant of interest or any organism of interest. The cassette can include 5' and 3' regulatory sequences operably linked to an Ht1 polynucleotide. "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (i.e., a promoter) is a functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous. When used to refer to the joining of two protein coding regions, by operably linked is intended that the coding regions are in the same reading frame. The cassette may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the Ht1 polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes.
[0038] The expression cassette can include in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), an Ht1 polynucleotide, and a transcriptional and translational termination region (i.e., termination region) functional in plants. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the Ht1 polynucleotide may be native/analogous to the maize plant cell or to each other. Alternatively, the regulatory regions and/or the Ht1 polynucleotide may be heterologous to the maize plant cell or to each other.
[0039] As used herein, "heterologous" in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form and/or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.
[0040] The termination region may be native with the transcriptional initiation region, may be native with a maize plant, or may be derived from another source (i.e., foreign or heterologous) with respect to the promoter, the Ht1 polynucleotide, the maize plant, or any combination thereof.
[0041] The expression cassettes may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include viral translational leader sequences.
[0042] In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.
[0043] A number of promoters can be used to express the various Ht1 sequences disclosed herein, including the native promoter of the polynucleotide sequence of interest (such as, for example, the native promoter of the Ht1 gene). The promoters can be selected based on the desired outcome. Such promoters include, for example, constitutive, inducible, tissue-preferred, or other promoters for expression in plants or in any organism of interest. Synthetic promoters can also be used to express Ht1 sequences. Synthetic promoters include for example a combination of one or more heterologous regulatory elements.
[0044] A polynucleotide construct may be a recombinant DNA construct. A "recombinant DNA construct" comprises two or more operably linked DNA segments which are not found operably linked in nature. Non-limiting examples of recombinant DNA constructs include a polynucleotide of interest operably linked to heterologous sequences which aid in the expression, autologous replication, and/or genomic insertion of the sequence of interest. Such heterologous and operably linked sequences include, for example, promoters, termination sequences, enhancers, etc, or any component of an expression cassette; a plasmid, cosmid, virus, autonomously replicating sequence, phage, or linear or circular single-stranded or double-stranded DNA or RNA nucleotide sequence; and/or sequences that encode heterologous polypeptides.
C. Maize Plant Cells and Maize Plants
[0045] "Maize" refers to a plant of the Zea mays L. ssp. mays and is also known as "corn".
[0046] Maize plants, maize plant cells, maize plant parts and seeds, and maize grain having the Ht1 sequences disclosed herein are also provided. In specific embodiments, the plants and/or plant parts have stably incorporated at least one heterologous Ht1 polypeptide disclosed herein. In addition, the plants or organism of interest can comprise multiple Ht1 polynucleotides (i.e., at least 1, 2, 3, 4, 5, 6 or more).
[0047] As used herein, the term maize plant includes maize plant cells, maize plant protoplasts, maize plant cell tissue cultures from which maize plants can be regenerated, maize plant calli, maize plant clumps, and maize plant cells that are intact in maize plants or parts of maize plants such as embryos, pollen, ovules, seeds, leaves, flowers, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Grain is intended to mean the mature seed produced by commercial growers for purposes other than growing or reproducing the species.
D. Other Traits of Interest
[0048] In some embodiments, the Ht1 polynucleotides disclosed herein may be engineered into a molecular stack. Thus, the various maize plants, maize plant cells and maize seeds disclosed herein can further comprise one or more traits of interest, and in more specific embodiments, the maize plant, maize plant part or maize plant cell is stacked with any combination of polynucleotide sequences of interest in order to create plants with a desired combination of traits.
[0049] As used herein, the term "stacked" includes having the multiple traits present in the same plant or organism of interest. In one non-limiting example, "stacked traits" comprise a molecular stack where the sequences are physically adjacent to each other. A trait, as used herein, refers to the phenotype derived from a particular sequence or groups of sequences.
[0050] A polynucleotide DNA construct described herein may also comprise one or more heterologous nucleic acid sequences that encode a polypeptide selected from the group consisting of: a polypeptide conferring disease resistance, a polypeptide conferring herbicide resistance, a polypeptide conferring insect resistance, a polypeptide involved in carbohydrate metabolism, a polypeptide involved in fatty acid metabolism, a polypeptide involved in amino acid metabolism, a polypeptide involved in plant development, a polypeptide involved in plant growth regulation, a polypeptide involved in yield improvement, a polypeptide involved in drought resistance, a polypeptide involved in cold resistance, a polypeptide involved in heat resistance, and/or a polypeptide involved in salt resistance, wherein each heterologous nucleic acid sequence is operably linked to a promoter.
[0051] A polypeptide conferring disease resistance may be another polypeptide that confers resistance to northern leaf blight (NLB). For example, a polynucleotide DNA construct may comprise a resistant allele of Ht1 and a resistant allele of NLB18 (in WO2011163590). The amino acid sequence of the NLB18 polypeptide from line PH99N is presented herein as SEQ ID NO:11; the amino acid sequence of the NLB18 polypeptide from line PH26N is presented herein as SEQ ID NO:12. Both PH99N and PH26N are maize lines showing resistance to northern leaf blight that reflect different sources of resistance with respect to the chromosome 8 QTL, as described in application WO2011163590. A resistant allele of NLB18 may encode a polypeptide having an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% sequence identity when compared to SEQ ID NO:11 or 12, based on the CLUSTAL W method of alignment with default parameters.
II. Methods of Generating Maize Plants with Northern Leaf Blight Resistance
[0052] "Exserohilum turcicum", previously referred to as Helminthosporium turcicum, is the fungal pathogen that induces northern leaf blight infection. The fungal pathogen is also referred to herein as Exserohilum or Et.
[0053] "Disease resistance" (such as, for example, northern leaf blight resistance) is a characteristic of a plant, wherein the plant avoids the disease symptoms that are the outcome of plant-pathogen interactions, such as maize-Exserohilum turcicum interactions. That is, pathogens are prevented from causing plant diseases and the associated disease symptoms, or alternatively, the disease symptoms caused by the pathogen are minimized or lessened. One of skill in the art will appreciate that the compositions and methods disclosed herein can be used with other compositions and methods available in the art for protecting plants from pathogen attack.
[0054] "Resistance" is a relative term, indicating that the infected plant produces better yield of maize than another, similarly treated, more susceptible plant. That is, the conditions cause a reduced decrease in maize survival and/or yield in a tolerant maize plant, as compared to a susceptible maize plant. One of skill will appreciate that maize plant resistance to northern leaf blight, or the pathogen causing such, can represent a spectrum of more resistant or less resistant phenotypes, and can vary depending on the severity of the infection. However, by simple observation, one of skill can determine the relative resistance or susceptibility of different plants, plant lines or plant families to northern leaf blight, and furthermore, will also recognize the phenotypic gradations of "resistant". For example, a 1 to 9 visual rating indicating the level of resistance to northern leaf blight can be used. A higher score indicates a higher resistance. Data should be collected only when sufficient selection pressure exists in the experiment measured. The terms "tolerance" and "resistance" are used interchangeably herein.
[0055] The resistance may be "newly conferred" or "enhanced". "Newly conferred" or "enhanced" resistance refers to an increased level of resistance against a particular pathogen, a wide spectrum of pathogens, or an infection caused by the pathogen(s). An increased level of resistance against a particular fungal pathogen, such as Et, for example, constitutes "enhanced" or improved fungal resistance. The embodiments of the invention will enhance or improve fungal plant pathogen resistance, such that the resistance of the plant to a fungal pathogen or pathogens will increase, which in turn, will increase resistance to the disease caused by the fungal pathogen. The term "enhance" refers to improve, increase, amplify, multiply, elevate, raise, and the like.
[0056] The maize plants generated by the methods described herein may provide durable and broad spectrum resistance to the maize plant and may assist in breeding of northern leaf blight resistant maize plants. For instance, if multiple northern leaf blight resistance genes are stacked into one unit, this reduces the number of specific loci that require trait introgression through backcrossing and minimizes linkage drag from non-elite resistant donors.
[0057] Various methods can be used to introduce a sequence of interest into a maize plant cell, maize plant or maize plant part. "Introducing" is intended to mean presenting to the maize plant cell, maize plant, or maize plant part the polynucleotide in such a manner that the sequence gains access to the interior of a cell of the maize plant. The methods of the disclosure do not depend on a particular method for introducing a sequence into an organism or a maize plant or maize plant part, only that the polynucleotide gains access to the interior of at least one cell of the maize plant. Methods for introducing polynucleotides into various organisms, including maize plants, are known in the art, including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
[0058] "Stable transformation" is intended to mean that the polynucleotide construct introduced into a maize plant integrates into the genome of the maize plant and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the maize plant and does not integrate into the genome of the maize plant.
[0059] Transformation protocols as well as protocols for introducing polynucleotide sequences into plants such as maize may vary. Suitable methods of introducing polynucleotides into maize plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320 334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602 5606, Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717 2722), and ballistic particle acceleration (see, for example, U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244; and, 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al. (1988) Biotechnology 6:923 926); and Lec1 transformation (WO 00/28058). Also see Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305 4309 (maize); Klein et al. (1988) Biotechnology 6:559 563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al. (1988) Plant Physiol. 91:440 444 (maize); Fromm et al. (1990) Biotechnology 8:833 839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); and Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.
[0060] In other embodiments, the Ht1 polynucleotide disclosed herein thereof may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a polynucleotide construct of the disclosure within a DNA or RNA molecule. It is recognized that the Ht1 sequence may be initially synthesized as part of a viral polyprotein, which later may be processed by proteolysis in vivo or in vitro to produce the desired recombinant protein. Further, it is recognized that promoters disclosed herein also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involving viral DNA or RNA molecules, are known in the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology 5:209-221; herein incorporated by reference.
[0061] Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the polynucleotide at a desired genomic location is achieved using a site-specific recombination system. See, for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, and WO99/25853, all of which are herein incorporated by reference. Briefly, the polynucleotide disclosed herein can be contained in transfer cassette flanked by two non-recombinogenic recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its genome a target site which is flanked by two non-recombinogenic recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome. Other methods to target polynucleotides are set forth in WO 2009/114321 (herein incorporated by reference), which describes "custom" meganucleases produced to modify plant genomes, in particular the genome of maize. See, also, Gao et al. (2010) Plant Journal 1:176-187. The cells that have been transformed may be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting progeny having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved. In this manner, the present disclosure provides transformed seed (also referred to as "transgenic seed") having a polynucleotide disclosed herein, for example, as part of an expression cassette, stably incorporated into their genome.
[0062] Transformed maize plant cells which are derived by plant transformation techniques, including those discussed above, can be cultured to regenerate a whole plant which possesses the transformed genotype (i.e., an Ht1 polynucleotide that encodes a polypeptide that confers resistance to northern leaf blight), and thus the desired phenotype, such as resistance to northern leaf blight, whether that resistance is newly conferred or enhanced. For transformation and regeneration of maize see, Gordon-Kamm et al., The Plant Cell, 2:603-618 (1990). Plant regeneration from cultured protoplasts is described in Evans et al. (1983) Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp 124-176, Macmillan Publishing Company, New York; and Binding (1985) Regeneration of Plants, Plant Protoplasts pp 21-73, CRC Press, Boca Raton. Regeneration can also be obtained from plant callus, explants, organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. (1987) Ann Rev of Plant Phys 38:467. See also, e.g., Payne and Gamborg.
[0063] One of skill will recognize that after the expression cassette containing the Ht1 gene is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
[0064] In some embodiments, the methods comprise introducing by way of expressing in a regenerable maize plant cell a polynucleotide construct comprising a polynucleotide operably linked to at least one regulatory sequence, wherein the polynucleotide encodes a resistant allele of the Ht1 gene presented herein, and generating a maize plant that has resistance to northern leaf blight from the maize plant cell. The maize plant generated by the method comprises in its genome the polynucleotide construct. The regulatory sequence may be a promoter and/or a terminator and may be native to maize. In some embodiments, the regulatory sequence is native to the Ht1 gene. A progeny plant comprising the polynucleotide construct may also be generated by crossing the maize plant generated by the method to a second maize plant that does not comprise in its genome the polynucleotide construct. In some embodiments, the Ht1 gene is overexpressed (either as a genomic fragment or cDNA) to impart greater resistance than the level of expression in the native state.
EXAMPLES
[0065] The following examples are offered to illustrate, but not to limit, the appended claims. It is understood that the examples and embodiments described herein are for illustrative purposes only and that persons skilled in the art will recognize various reagents or parameters that can be altered without departing from the spirit of the invention or the scope of the appended claims.
Example 1
Fine Mapping of Northern Leaf Blight Resistance Locus
[0066] A large backcross-derived population was created for fine mapping of a northern leaf blight resistance QTL located on chromosome 2. The population was created from a cross between resistant line PH4GP (score=9) and susceptible line PH5W4 (score=1), and the susceptible line was used as the recurrent parent. BC5 individuals were scored for northern leaf blight infection using the phenotyping method disclosed in U.S. Pat. No. 8,921,646. Marker recombination data from a large set of individuals from population 2 placed the gene in an 18 kb region in the B73 genome, encompassing a putative protein phosphatase 2C, a putative PHD-type zinc finger protein, and a putative disease resistance protein. There is no known EST for the putative disease resistance protein; moreover, based on the B73 predicted gene sequence, the putative disease resistance protein has low, constitutive expression.
Example 2
Identification of Candidate Gene for Ht1 and Comparison of Allelic Variants
[0067] A BAC library from resistant donor PH4GP was constructed, and a BAC clone covering the Ht1 interval was identified and sequenced. The Ht1 interval in PH4GP was less than 10 kb, with only a single annotated gene that encodes a putative CC-NB-LRR (coiled-coil, nucleotide-binding, leucine-rich repeat) gene. Ht1 cDNA sequences from PH4GP and from PH1W2 (another source of a resistant allele of Ht1; US2010095395) are represented by SEQ ID NOs:1 and 3, respectively, while the amino acid sequences of the encoded polypeptides are represented by SEQ ID NO:2 and 4. B73 has two splicing variants, and the novel variant expresses at a much higher level (referred to herein as B73-high) than the known variant (referred to herein as B73-low). SEQ ID NO:5 is the cDNA sequence of the B73-high allele, while the amino acid sequence of the encoded polypeptide is represented by SEQ ID NO:6. SEQ ID NO:7 is the cDNA sequence of the B73-low allele, while the amino acid sequence of the encoded polypeptide is represented by SEQ ID NO:8.
[0068] FIGS. 1A-1D show that the CC and NB domains are highly similar between the susceptible allele (from B73) and resistant alleles (from PH4GP and PH1W2). However, both B73 alleles have a deletion in the LRR (shown boxed in FIG. 1C). The amino acid sequence of this region in the Ht1 resistant alleles is represented by SEQ ID NO:10.
Example 3
Transgenic Validation
[0069] A construct containing the genomic sequence of the PH4GP (resistant) allele (SEQ ID NO:9) was generated and transformed into a susceptible transformation line using Agrobacterium-mediated transformation. The genomic sequence (SEQ ID NO:9) contained the native promoter, exons, intron and terminator regions. Regenerated transgenic plants were planted in a greenhouse, and a quantitative PCR analysis was done to confirm insertion of the T-DNA cassette that contains the genomic sequence of the PH4GP (resistant) allele. Many of the events had a single copy of the T-DNA insert, which was confirmed by qPCR using four flanking markers. Based on the marker data, of the 50 events, 41 were positive for the insert and 9 were negative (null).
[0070] All events were tested in the greenhouse for efficacy against the northern leaf blight pathogen (Exserohilum turcicum). First, all events were challenged with race 0 of the pathogen for which Ht1 gene is known to provide resistance; then the events were subjected to race 1, to which Ht1 does not provide resistance. All positive events, as determined by qPCR, were resistant to race 0, and all negative events were susceptible to race 0. As expected, all events were susceptible to race 1.
Example 4
Production of Northern Leaf Blight Resistant Maize Plants Expressing the Maize Ht1 Polypeptide
[0071] Northern leaf blight resistant maize plants expressing the maize Ht1 gene can be produced using recombinant DNA-based transformation, for example. Recombinant DNA based transformation methods are well known in the art, e.g. Agrobacterium tumefaciens-mediated and particle bombardment based transformations. With respect to Agrobacterium tumefaciens based plant transformation, vectors are constructed according to methods known in the art. The vectors contain a T-DNA insert having a promoter, an intron, an optional enhancer such as a 35S enhancer element, an Ht1 variant DNA that confers resistance to northern leaf blight, and a plant terminator. Maize immature embryos are excised and infected with an Agrobacterium tumefaciens vector containing the Ht1 variant of interest. After infection, embryos are transferred and cultured in co-cultivation medium. After co-cultivation, the infected immature embryos are transferred onto media to grow transgenic calli. The putative transgenic callus tissues are sampled using PCR and optionally a Western assay to confirm the presence of the Ht1 variant gene. The putative transgenic callus tissues are maintained on media for further growth and selection before plant regeneration. At regeneration, callus tissue confirmed to be transgenic are transferred onto maturation medium and cultured for somatic embryo maturation. Mature embryos are then transferred onto regeneration medium for shoot and root formation. After shoots and roots emerge, individual plantlets are transferred into tubes with rooting medium. Plantlets with established shoots and roots are transplanted into pots in the greenhouse for further growth and to produce T1 seed.
[0072] Furthermore, a DNA construct containing an Ht1 variant DNA that confers resistance to northern leaf blight may also include another gene encoding a polypeptide that confers northern leaf blight resistance, such as the gene encoding for NLB18 (in WO2011163590). Both PH99N and PH26N are maize lines showing resistance to northern leaf blight that reflect different sources of resistance with respect to the chromosome 8 QTL, as described in application WO2011163590. The amino acid sequence of the NLB18 polypeptide from line PH99N is presented herein as SEQ ID NO:11; the amino acid sequence of the NLB18 polypeptide from line PH26N is presented herein as SEQ ID NO:12. The introduction of Ht1 and NLB18 in a plant may have the effect of increasing resistance to race 0 of the Exserohilum turcicum pathogen and/or may provide resistance to an additional race or races (e.g. race 1), for which the Ht1 gene does not provide resistance.
Sequence CWU
1
1
1212658DNAZea mays 1atggagaacc cagacgcgca ggcgaaggcg tgggcggcgg agatgcgcga
gctggcctac 60gacatggagg acagcatcga tctcttcacc caccacgtcg accacgaacc
ggccgacacc 120gccaccaccg gcgtcaagag gttcttcctc cggatcatcc ggaagcttaa
gaaactccac 180taccgccaca ggtttgttca ggagatcaaa caactccacg accttgccaa
cgaatcgtac 240cggcgtagga agaggtacag gattgaggag ggcggttcaa gcctctcgca
cgcggagatc 300gatcctcggt tagaggcgct ctacgtggag gtggagaaac tcgtgggcat
ccagggccca 360agccaggaga tcattggaca gctcgtcggc gagaacgcag cggagcgacg
gagggttgtc 420gccgttgttg gatctggagg ttcaggcaag accacacttg ccaaacaggt
gtacgagaaa 480atcaggtgcc aattctcttg tgcagccttt gtgtctgtgt cgcaaaagcc
caacatgaat 540agcctcctgt gggagttgct atctcaaatc gggaaccatg gtggagattt
aggaatgatg 600gcagtaggat attgcagtga caaacaactg atcgacagac taagatcaca
tcttgaaaag 660cagaggtatc tcgttgtgat agatgatgtt tggacaaact cagcgtggga
gaccatacaa 720tgtgcgctcc ctaaaaatgc ccatgcaagt aaaataattc tgacaacacg
aatcaacagt 780gtaggccagt tctcctgcac tccagatgag ggttttatct atcagatgaa
gcctctttgc 840agaaacgatt ctgaaaatct gtttctgaaa aggacactat gtgataaaga
taagtttcct 900gctcagctgg aggggattaa aaacgagata atcgagaaat gcgatggttt
gccactggct 960attgttactc tagctagcat gttagctact aaacagagaa caagggaaga
atgggagagg 1020gcacttgatt caatccattc tatgcacaag aaagatagtg gcctggaagt
gatggacaag 1080atactgtctc tgagttacag ggatctacct cacaacatga gaaattgctt
gctgtatctc 1140agtacatttc cagaggacca cacgatttac aaagatgccc tagtatggag
atggatggct 1200gaagggttta tcgctgaaac acaaggcttt actttggagc aggttgccga
gggctacttc 1260tacgagtttg tgaacaggag tttggttcag cccataacct tgcgttcaag
atatgaaatg 1320cgtggagaag gaggttgccg agtccatgac attgtactga acttcctcat
ctctcgtgca 1380gctgaagaga actttttaac tacgctgtat ggcgcccagg gggttccatc
ttcagaccga 1440aggattcgcc ggctctctgt ctgggacagt ccagaacacg cactggcagt
ctctagagcg 1500accatgaatc tgtcccatct ccggtcagtt agaatatgca acgttggaga
ctggcccgtg 1560cctgctgttc tagacttacc tgtccttcga gtgttagatc tagagggatg
ccgtgatctg 1620aggatcgacg aacctgactg cattctaagc ttgtttcatc tgagatacct
gggtttccgc 1680agcgcaagtg gtgtcgtgct accggctcaa atcggaaatt tacaccatct
gcagaccatc 1740gatttaagcg ggactggagt gacacagctg ccagaaagca ttgtccagct
caagcgactg 1800atgcatcttg ttgggcaacg gctcatcatg ccagacgggt ttggtagcat
ggaatccctt 1860gaggagttag gtactatcga ctgctgcaag tgccccgtca gttttgggga
agacctagca 1920cttctgagca ggctgagggt gctccgagtg gctttcatcg gggtcgaaac
aagtgacatg 1980gaaaccagaa ggaaatcttt gatgtcatcc ctctgcaaac tcggaggaga
caaccttcgg 2040cgtgtcacta ttatcgacct cgctggcggt ggagattgct ttgtggagtc
gtggcaccct 2100cctcctcgtc tcctccagaa gttcatccat atcagtcagc aacagcactt
ctccaggttt 2160ccagaatgga tcagttcctg cctatgtgat ctcacccacc tggatataaa
ggccgaaaag 2220atggaaaggg agcatctaag tgttcttgaa cacctgcccg ccatccgttg
cctatacctt 2280ttcgtgaagc gagtctccga agacgggctc gccatcagcc acggcgcgtt
ccgatgtcta 2340cggcgtctcg agttctgcaa cgtagatgga cctggtttga tgtttgcagg
aggcgttcca 2400atgttggaat ggctgaggct cgggttcgac gcggatagag cgcaatcgac
atacggcggt 2460ctggaggttg gcatccagcg cctctcgtct ctcaaacatg tcgtgctcat
tgtatggatg 2520gtttctgaag gcggtgatga tccagcggag caagccgtct ggtctgccat
caatggccaa 2580gtagagatgc tccccaactc tccgacggtt gatatccggt ttcgtagacg
gagtcagctg 2640caggcaagct cagaataa
26582885PRTZea mays 2Met Glu Asn Pro Asp Ala Gln Ala Lys Ala
Trp Ala Ala Glu Met Arg 1 5 10
15 Glu Leu Ala Tyr Asp Met Glu Asp Ser Ile Asp Leu Phe Thr His
His 20 25 30 Val
Asp His Glu Pro Ala Asp Thr Ala Thr Thr Gly Val Lys Arg Phe 35
40 45 Phe Leu Arg Ile Ile Arg
Lys Leu Lys Lys Leu His Tyr Arg His Arg 50 55
60 Phe Val Gln Glu Ile Lys Gln Leu His Asp Leu
Ala Asn Glu Ser Tyr 65 70 75
80 Arg Arg Arg Lys Arg Tyr Arg Ile Glu Glu Gly Gly Ser Ser Leu Ser
85 90 95 His Ala
Glu Ile Asp Pro Arg Leu Glu Ala Leu Tyr Val Glu Val Glu 100
105 110 Lys Leu Val Gly Ile Gln Gly
Pro Ser Gln Glu Ile Ile Gly Gln Leu 115 120
125 Val Gly Glu Asn Ala Ala Glu Arg Arg Arg Val Val
Ala Val Val Gly 130 135 140
Ser Gly Gly Ser Gly Lys Thr Thr Leu Ala Lys Gln Val Tyr Glu Lys 145
150 155 160 Ile Arg Cys
Gln Phe Ser Cys Ala Ala Phe Val Ser Val Ser Gln Lys 165
170 175 Pro Asn Met Asn Ser Leu Leu Trp
Glu Leu Leu Ser Gln Ile Gly Asn 180 185
190 His Gly Gly Asp Leu Gly Met Met Ala Val Gly Tyr Cys
Ser Asp Lys 195 200 205
Gln Leu Ile Asp Arg Leu Arg Ser His Leu Glu Lys Gln Arg Tyr Leu 210
215 220 Val Val Ile Asp
Asp Val Trp Thr Asn Ser Ala Trp Glu Thr Ile Gln 225 230
235 240 Cys Ala Leu Pro Lys Asn Ala His Ala
Ser Lys Ile Ile Leu Thr Thr 245 250
255 Arg Ile Asn Ser Val Gly Gln Phe Ser Cys Thr Pro Asp Glu
Gly Phe 260 265 270
Ile Tyr Gln Met Lys Pro Leu Cys Arg Asn Asp Ser Glu Asn Leu Phe
275 280 285 Leu Lys Arg Thr
Leu Cys Asp Lys Asp Lys Phe Pro Ala Gln Leu Glu 290
295 300 Gly Ile Lys Asn Glu Ile Ile Glu
Lys Cys Asp Gly Leu Pro Leu Ala 305 310
315 320 Ile Val Thr Leu Ala Ser Met Leu Ala Thr Lys Gln
Arg Thr Arg Glu 325 330
335 Glu Trp Glu Arg Ala Leu Asp Ser Ile His Ser Met His Lys Lys Asp
340 345 350 Ser Gly Leu
Glu Val Met Asp Lys Ile Leu Ser Leu Ser Tyr Arg Asp 355
360 365 Leu Pro His Asn Met Arg Asn Cys
Leu Leu Tyr Leu Ser Thr Phe Pro 370 375
380 Glu Asp His Thr Ile Tyr Lys Asp Ala Leu Val Trp Arg
Trp Met Ala 385 390 395
400 Glu Gly Phe Ile Ala Glu Thr Gln Gly Phe Thr Leu Glu Gln Val Ala
405 410 415 Glu Gly Tyr Phe
Tyr Glu Phe Val Asn Arg Ser Leu Val Gln Pro Ile 420
425 430 Thr Leu Arg Ser Arg Tyr Glu Met Arg
Gly Glu Gly Gly Cys Arg Val 435 440
445 His Asp Ile Val Leu Asn Phe Leu Ile Ser Arg Ala Ala Glu
Glu Asn 450 455 460
Phe Leu Thr Thr Leu Tyr Gly Ala Gln Gly Val Pro Ser Ser Asp Arg 465
470 475 480 Arg Ile Arg Arg Leu
Ser Val Trp Asp Ser Pro Glu His Ala Leu Ala 485
490 495 Val Ser Arg Ala Thr Met Asn Leu Ser His
Leu Arg Ser Val Arg Ile 500 505
510 Cys Asn Val Gly Asp Trp Pro Val Pro Ala Val Leu Asp Leu Pro
Val 515 520 525 Leu
Arg Val Leu Asp Leu Glu Gly Cys Arg Asp Leu Arg Ile Asp Glu 530
535 540 Pro Asp Cys Ile Leu Ser
Leu Phe His Leu Arg Tyr Leu Gly Phe Arg 545 550
555 560 Ser Ala Ser Gly Val Val Leu Pro Ala Gln Ile
Gly Asn Leu His His 565 570
575 Leu Gln Thr Ile Asp Leu Ser Gly Thr Gly Val Thr Gln Leu Pro Glu
580 585 590 Ser Ile
Val Gln Leu Lys Arg Leu Met His Leu Val Gly Gln Arg Leu 595
600 605 Ile Met Pro Asp Gly Phe Gly
Ser Met Glu Ser Leu Glu Glu Leu Gly 610 615
620 Thr Ile Asp Cys Cys Lys Cys Pro Val Ser Phe Gly
Glu Asp Leu Ala 625 630 635
640 Leu Leu Ser Arg Leu Arg Val Leu Arg Val Ala Phe Ile Gly Val Glu
645 650 655 Thr Ser Asp
Met Glu Thr Arg Arg Lys Ser Leu Met Ser Ser Leu Cys 660
665 670 Lys Leu Gly Gly Asp Asn Leu Arg
Arg Val Thr Ile Ile Asp Leu Ala 675 680
685 Gly Gly Gly Asp Cys Phe Val Glu Ser Trp His Pro Pro
Pro Arg Leu 690 695 700
Leu Gln Lys Phe Ile His Ile Ser Gln Gln Gln His Phe Ser Arg Phe 705
710 715 720 Pro Glu Trp Ile
Ser Ser Cys Leu Cys Asp Leu Thr His Leu Asp Ile 725
730 735 Lys Ala Glu Lys Met Glu Arg Glu His
Leu Ser Val Leu Glu His Leu 740 745
750 Pro Ala Ile Arg Cys Leu Tyr Leu Phe Val Lys Arg Val Ser
Glu Asp 755 760 765
Gly Leu Ala Ile Ser His Gly Ala Phe Arg Cys Leu Arg Arg Leu Glu 770
775 780 Phe Cys Asn Val Asp
Gly Pro Gly Leu Met Phe Ala Gly Gly Val Pro 785 790
795 800 Met Leu Glu Trp Leu Arg Leu Gly Phe Asp
Ala Asp Arg Ala Gln Ser 805 810
815 Thr Tyr Gly Gly Leu Glu Val Gly Ile Gln Arg Leu Ser Ser Leu
Lys 820 825 830 His
Val Val Leu Ile Val Trp Met Val Ser Glu Gly Gly Asp Asp Pro 835
840 845 Ala Glu Gln Ala Val Trp
Ser Ala Ile Asn Gly Gln Val Glu Met Leu 850 855
860 Pro Asn Ser Pro Thr Val Asp Ile Arg Phe Arg
Arg Arg Ser Gln Leu 865 870 875
880 Gln Ala Ser Ser Glu 885 32652DNAZea mays
3atggagaacc cagacgcgca ggcgaaggcg tgggcggcgg agatgcgcga gctggcctac
60gacatggagg acagcatcga tctcttcacc caccacgtcg accacgaacc ggccgacacc
120gccaccaccg gcgtcaagag gttcttcctc cggatcatcc ggaagcttaa gaaactccac
180taccgccaca ggtttgttca ggagatcaaa caactccacg accttgccaa cgaatcgtac
240cggcgtagga agaggtacag gattgaggag ggcggttcaa gcctctcgca cgcggagatc
300gatcctcggt tagaggcgct ctacgtggag gtggagaaac tcgtgggcat ccagggccca
360agccaggaga tcattggaca gctcgtcggc gagaacgcag cggagcgacg gagggttgtc
420gccgttgttg gatctggagg ttcaggcaag accacacttg ccaaacaggt gtacgagaaa
480atcaggtgcc aattctcttg tgcagccttt gtgtctgtgt cgcaaaagcc caacatgaat
540agcctcctgt gggagttgct atctcaaatc gggaaccatg gtggagattt aggaatgatg
600gcagtaggat attgcagtga caaacaactg atcgacagac taagatcaca tcttgaaaag
660cagaggtatc tcgttgtgat agatgatgtt tggacaaact cagcgtggga gaccatacaa
720tgtgcgctcc ctaaaaatgc ccatgcaagt aaaataattc tgacaacacg aatcaacagt
780gtaggccagt tctcctgcac tccagatgag ggttttatct atcagatgaa gcctctttgc
840agaaacgatt ctgaaaatct gtttctgaaa aggacactat gtgataaaga taagtttcct
900gctcagctgg aggggattaa aaacgagata atcgagaaat gcgatggttt gccactggct
960attgttactc tagctagcat gttagctact aaacagagaa caagggaaga atgggagagg
1020gcacttgatt caatccattc tatgcacaag aaagatagtg gcctggaagt gatggacaag
1080atactgtctc tgagttacag ggatctacct cacaacatga gaaattgctt gctgtatctc
1140agtacatttc cagaggacca cacgatttac aaagatgccc tagtatggag atggatggct
1200gaagggttta tcgctgaaac acaaggcttt actttggagc aggttgccga gggctacttc
1260tacgagtttg tgaacaggag tttggttcag cccataacct tgcgttcaag atatgaaatg
1320cgtggagaag gaggttgccg agtccatgac attgtactga acttcctcat ctctcgtgca
1380gctgaagaga actttttaac tacgctgtat ggcgcccagg gggttccatc ttcagaccga
1440aggattcgcc ggctctctgt ctgggacagt ccagaacacg cactggcagt ctctagagcg
1500accatgaatc tgtcccatct ccggtcagtt agaatatgca acgttggaga ctggcccgtg
1560cctgctgttc tagacttacc tgtccttcga gtgttagatc tagagggatg ccgtgatctg
1620aggatcgacg aacctgactg cattctaagc ttgtttcatc tgagatacct gggtttccgc
1680agcgcaagtg gtgtcgtgct accggctcaa atcggaaatt tacaccatct gcagaccatc
1740gatttaagcg ggactggagt gacacagctg ccagaaagca ttgtccagct caagcgactg
1800atgcatcttg ttgggcaacg gctcatcatg ccagacgggt ttggtagcat ggaatccctt
1860gaggagttag gtactatcga ctgctgcaag tgccccgtca gttttgggga agacctagca
1920cttctgagca ggctgagggt gctccgagtg gctttcatcg gggtcgaaac aagtgacatg
1980gaaaccagaa ggaaatcttt gatgtcatcc ctctgcaaac tcggaggaga caaccttcgg
2040cgtgtcacta ttatcgacct cgctggcggt ggagattgct ttgtggagtc gtggcaccct
2100cctcctcgtc tcctccagaa gttcatccat atcagtcagc acttctccag gtttccagaa
2160tggatcagtt cctgcctatg tgatctcacc cacctggata taaaggccga aaagatggaa
2220agggagcatc taagtgttct tgaacacctg cccgccatcc gttgcctata ccttttcgtg
2280aagcgagtct ccgaagacgg gctcgtcatc agccacggcg cgttccgatg tctacggcgt
2340ctcgagttct gtaacgtaga tggacctggt ttgatgtttg caggaggcgt tccaatgttg
2400gaatggctga ggctcgggtt cgacgcggat agagcgcaat cgacatacgg cggtctggag
2460gttggcatcc agcgcctctc gtctctcaaa catgtcgtgc tcattgtatg gatggtttct
2520gaaggcggtg atgatccagc ggagcaagcc gtctggtctg ccatcaatgg ccaagtagag
2580atgctcccca actctccgac ggttgatatc cggtttcgta gacggagtca gctgcaggca
2640agctcagaat aa
26524883PRTZea mays 4Met Glu Asn Pro Asp Ala Gln Ala Lys Ala Trp Ala Ala
Glu Met Arg 1 5 10 15
Glu Leu Ala Tyr Asp Met Glu Asp Ser Ile Asp Leu Phe Thr His His
20 25 30 Val Asp His Glu
Pro Ala Asp Thr Ala Thr Thr Gly Val Lys Arg Phe 35
40 45 Phe Leu Arg Ile Ile Arg Lys Leu Lys
Lys Leu His Tyr Arg His Arg 50 55
60 Phe Val Gln Glu Ile Lys Gln Leu His Asp Leu Ala Asn
Glu Ser Tyr 65 70 75
80 Arg Arg Arg Lys Arg Tyr Arg Ile Glu Glu Gly Gly Ser Ser Leu Ser
85 90 95 His Ala Glu Ile
Asp Pro Arg Leu Glu Ala Leu Tyr Val Glu Val Glu 100
105 110 Lys Leu Val Gly Ile Gln Gly Pro Ser
Gln Glu Ile Ile Gly Gln Leu 115 120
125 Val Gly Glu Asn Ala Ala Glu Arg Arg Arg Val Val Ala Val
Val Gly 130 135 140
Ser Gly Gly Ser Gly Lys Thr Thr Leu Ala Lys Gln Val Tyr Glu Lys 145
150 155 160 Ile Arg Cys Gln Phe
Ser Cys Ala Ala Phe Val Ser Val Ser Gln Lys 165
170 175 Pro Asn Met Asn Ser Leu Leu Trp Glu Leu
Leu Ser Gln Ile Gly Asn 180 185
190 His Gly Gly Asp Leu Gly Met Met Ala Val Gly Tyr Cys Ser Asp
Lys 195 200 205 Gln
Leu Ile Asp Arg Leu Arg Ser His Leu Glu Lys Gln Arg Tyr Leu 210
215 220 Val Val Ile Asp Asp Val
Trp Thr Asn Ser Ala Trp Glu Thr Ile Gln 225 230
235 240 Cys Ala Leu Pro Lys Asn Ala His Ala Ser Lys
Ile Ile Leu Thr Thr 245 250
255 Arg Ile Asn Ser Val Gly Gln Phe Ser Cys Thr Pro Asp Glu Gly Phe
260 265 270 Ile Tyr
Gln Met Lys Pro Leu Cys Arg Asn Asp Ser Glu Asn Leu Phe 275
280 285 Leu Lys Arg Thr Leu Cys Asp
Lys Asp Lys Phe Pro Ala Gln Leu Glu 290 295
300 Gly Ile Lys Asn Glu Ile Ile Glu Lys Cys Asp Gly
Leu Pro Leu Ala 305 310 315
320 Ile Val Thr Leu Ala Ser Met Leu Ala Thr Lys Gln Arg Thr Arg Glu
325 330 335 Glu Trp Glu
Arg Ala Leu Asp Ser Ile His Ser Met His Lys Lys Asp 340
345 350 Ser Gly Leu Glu Val Met Asp Lys
Ile Leu Ser Leu Ser Tyr Arg Asp 355 360
365 Leu Pro His Asn Met Arg Asn Cys Leu Leu Tyr Leu Ser
Thr Phe Pro 370 375 380
Glu Asp His Thr Ile Tyr Lys Asp Ala Leu Val Trp Arg Trp Met Ala 385
390 395 400 Glu Gly Phe Ile
Ala Glu Thr Gln Gly Phe Thr Leu Glu Gln Val Ala 405
410 415 Glu Gly Tyr Phe Tyr Glu Phe Val Asn
Arg Ser Leu Val Gln Pro Ile 420 425
430 Thr Leu Arg Ser Arg Tyr Glu Met Arg Gly Glu Gly Gly Cys
Arg Val 435 440 445
His Asp Ile Val Leu Asn Phe Leu Ile Ser Arg Ala Ala Glu Glu Asn 450
455 460 Phe Leu Thr Thr Leu
Tyr Gly Ala Gln Gly Val Pro Ser Ser Asp Arg 465 470
475 480 Arg Ile Arg Arg Leu Ser Val Trp Asp Ser
Pro Glu His Ala Leu Ala 485 490
495 Val Ser Arg Ala Thr Met Asn Leu Ser His Leu Arg Ser Val Arg
Ile 500 505 510 Cys
Asn Val Gly Asp Trp Pro Val Pro Ala Val Leu Asp Leu Pro Val 515
520 525 Leu Arg Val Leu Asp Leu
Glu Gly Cys Arg Asp Leu Arg Ile Asp Glu 530 535
540 Pro Asp Cys Ile Leu Ser Leu Phe His Leu Arg
Tyr Leu Gly Phe Arg 545 550 555
560 Ser Ala Ser Gly Val Val Leu Pro Ala Gln Ile Gly Asn Leu His His
565 570 575 Leu Gln
Thr Ile Asp Leu Ser Gly Thr Gly Val Thr Gln Leu Pro Glu 580
585 590 Ser Ile Val Gln Leu Lys Arg
Leu Met His Leu Val Gly Gln Arg Leu 595 600
605 Ile Met Pro Asp Gly Phe Gly Ser Met Glu Ser Leu
Glu Glu Leu Gly 610 615 620
Thr Ile Asp Cys Cys Lys Cys Pro Val Ser Phe Gly Glu Asp Leu Ala 625
630 635 640 Leu Leu Ser
Arg Leu Arg Val Leu Arg Val Ala Phe Ile Gly Val Glu 645
650 655 Thr Ser Asp Met Glu Thr Arg Arg
Lys Ser Leu Met Ser Ser Leu Cys 660 665
670 Lys Leu Gly Gly Asp Asn Leu Arg Arg Val Thr Ile Ile
Asp Leu Ala 675 680 685
Gly Gly Gly Asp Cys Phe Val Glu Ser Trp His Pro Pro Pro Arg Leu 690
695 700 Leu Gln Lys Phe
Ile His Ile Ser Gln His Phe Ser Arg Phe Pro Glu 705 710
715 720 Trp Ile Ser Ser Cys Leu Cys Asp Leu
Thr His Leu Asp Ile Lys Ala 725 730
735 Glu Lys Met Glu Arg Glu His Leu Ser Val Leu Glu His Leu
Pro Ala 740 745 750
Ile Arg Cys Leu Tyr Leu Phe Val Lys Arg Val Ser Glu Asp Gly Leu
755 760 765 Val Ile Ser His
Gly Ala Phe Arg Cys Leu Arg Arg Leu Glu Phe Cys 770
775 780 Asn Val Asp Gly Pro Gly Leu Met
Phe Ala Gly Gly Val Pro Met Leu 785 790
795 800 Glu Trp Leu Arg Leu Gly Phe Asp Ala Asp Arg Ala
Gln Ser Thr Tyr 805 810
815 Gly Gly Leu Glu Val Gly Ile Gln Arg Leu Ser Ser Leu Lys His Val
820 825 830 Val Leu Ile
Val Trp Met Val Ser Glu Gly Gly Asp Asp Pro Ala Glu 835
840 845 Gln Ala Val Trp Ser Ala Ile Asn
Gly Gln Val Glu Met Leu Pro Asn 850 855
860 Ser Pro Thr Val Asp Ile Arg Phe Arg Arg Arg Ser Gln
Leu Gln Ala 865 870 875
880 Ser Ser Glu 52631DNAZea mays 5atggagaacc cagacgcgca ggcgaaggcg
tgggcggcgg agatgcgcga gctggcctac 60gacatggagg acagcatcga tctcttcacc
caccacgtcg accacgaacc ggccgacacc 120gccaccaccg gcgtcaagag gttcttcctc
cggatcatcc ggaagcttaa gaaactccac 180taccgccaca ggtttgctca ggagatcaaa
caactccacg accttgccaa cgaatcgtac 240cggcgtagga agaggtacag gattgaggag
ggcggttcaa gcctcccgca cgcggagatc 300gatcctcggt tagaggcgct ctacgtggag
gtggagaaac tcgtgggcat ccagggccca 360agccaggaga tcattggaca gctcgtcggc
gagaacgcag cggagcggcg gagggttgtc 420gccgttgttg gatctggagg ttcaggcaag
accacacttg ccaaacaggt gtacgagaaa 480atcaggtgcc aattctcttg tgcagccttt
gtgtccgtgt cgcaaaagcc caacatgaat 540agcctcctgt gggagttgtt atctcaaatc
gggaaccatg gtggagattt aggaatgatg 600gcagtaggat attgcagtga caaacaactg
atcgacagac taagatcaca tcttgaaaag 660cagaggtatc tcgttgtgat agatgatgtt
tggacaaact cagcgtggga gaccatacaa 720tgtgcgctcc ctaaaaatgc ccatgcaagt
aaaataattc tgacaacacg aatcaacagt 780gtaggccagt tctcctgcac tccagatgag
ggttttatct atcagatgaa gcctctttgc 840agaaacgatt ctgaaaatct gtttctgaaa
aggacactat gtgataaaga taagtttcct 900gctcagctgg aggggattaa aaacgagata
atcgagaaat gcgatggttt gccactggct 960attgttactc tagctagcat gttagctact
aaacagagaa caagggaaga atgggagagg 1020gcacttgatt caatccattc tacgcacaag
aaagatagta gcctggaagt gatggacaag 1080atactgtctc tgagttacag ggatctacct
cacaacatga gaaattgctt gctgtatatc 1140agtacatttc cagaggacca cacgatttac
aaagatgctc tagtatggag atggatggct 1200gaagggttta tcgctgaaac acaaggcttt
actttggagc aggttgccga gggctacttc 1260tacgagtttg tgaacaggag tttggttcag
cccataacct tgcgttcaag atatgaaatg 1320cgtggagaag gaggttgccg agtccatgac
attgtactga acttcctcat ctctcgtgca 1380gctgaagaga actttttaac tacgctgtat
ggcgcccagg gggttccatc ttcagaccga 1440aggattcgcc ggctctctgt ctgggacagt
ccagaacacg cactggcagt ctctagagcg 1500accatgaatc tgtcccatct ccggtcagtt
agaatatgca acgttggaga ctggcccgtg 1560cctgctgttc tagacttacc tgtccttcga
gtgttagatc tagagggatg ccgtgatctg 1620aggatcgtcg accctgactg cattctaagc
ttgtttcatc tgaggtacct gggtttccgc 1680agcgcaagtg gtgtcgtgct accggctcaa
ataggaaatt tacaccatct gcagaccatc 1740gatttaagcg ggactggagt gacacagctg
ccagaaagca ttgtccagct caagcgactg 1800atgcatcttg ttgggcaacg gctcatcatg
ccagacgggt ttggtagcat ggaatccctt 1860gaggagttag gtactatcga ctgctgcaag
tgccccgctg agggtgctcc gagtgaccga 1920gtggctttcg tcggggtcga aacaagtgac
atggaaacca gaaggaaatc tttgatgtca 1980tccctctgca aactcggagg agacaacctt
cggcgtgtca ctattatcga cctcgctggc 2040ggtggagatt gctttgtgga gtcgtggcac
cctcctcctc gtctcctcca gaagttcatc 2100catatcagtc agcaacagca cttctccagg
tttccagaat ggatcagttc ctgcctatgt 2160gatctcaccc acctggatat aaaggccgaa
aagatggaaa gggagcatct aagtgttctt 2220gaacacctgc ccgccatccg ttatctatac
cttttcgtga agcgagtctc cgaagacggg 2280ctcgtcatca gccacagcgc gttccgatgt
ctacggcgtc tcgagttctg taacttagat 2340ggacctggtt tgatgtttgc aggaggcgtt
ccaatgctgg aatggctgag gctcgggttc 2400gacgcggata gagcgcaatc gacatacggc
ggtctggagg ttggcatcca gcgcctctcg 2460tctctcaaac atgtcgtgct cattgtctgt
atggtttctg aaggcggtga tgatccagcg 2520gagcaagccg tctggtctgc catcaatggc
caagtagaga tgctccccaa ttctccgacg 2580gttgatatcc ggtttcgtag acggagtcag
ctgcaggcaa gctcagaata a 26316876PRTZea mays 6Met Glu Asn Pro
Asp Ala Gln Ala Lys Ala Trp Ala Ala Glu Met Arg 1 5
10 15 Glu Leu Ala Tyr Asp Met Glu Asp Ser
Ile Asp Leu Phe Thr His His 20 25
30 Val Asp His Glu Pro Ala Asp Thr Ala Thr Thr Gly Val Lys
Arg Phe 35 40 45
Phe Leu Arg Ile Ile Arg Lys Leu Lys Lys Leu His Tyr Arg His Arg 50
55 60 Phe Ala Gln Glu Ile
Lys Gln Leu His Asp Leu Ala Asn Glu Ser Tyr 65 70
75 80 Arg Arg Arg Lys Arg Tyr Arg Ile Glu Glu
Gly Gly Ser Ser Leu Pro 85 90
95 His Ala Glu Ile Asp Pro Arg Leu Glu Ala Leu Tyr Val Glu Val
Glu 100 105 110 Lys
Leu Val Gly Ile Gln Gly Pro Ser Gln Glu Ile Ile Gly Gln Leu 115
120 125 Val Gly Glu Asn Ala Ala
Glu Arg Arg Arg Val Val Ala Val Val Gly 130 135
140 Ser Gly Gly Ser Gly Lys Thr Thr Leu Ala Lys
Gln Val Tyr Glu Lys 145 150 155
160 Ile Arg Cys Gln Phe Ser Cys Ala Ala Phe Val Ser Val Ser Gln Lys
165 170 175 Pro Asn
Met Asn Ser Leu Leu Trp Glu Leu Leu Ser Gln Ile Gly Asn 180
185 190 His Gly Gly Asp Leu Gly Met
Met Ala Val Gly Tyr Cys Ser Asp Lys 195 200
205 Gln Leu Ile Asp Arg Leu Arg Ser His Leu Glu Lys
Gln Arg Tyr Leu 210 215 220
Val Val Ile Asp Asp Val Trp Thr Asn Ser Ala Trp Glu Thr Ile Gln 225
230 235 240 Cys Ala Leu
Pro Lys Asn Ala His Ala Ser Lys Ile Ile Leu Thr Thr 245
250 255 Arg Ile Asn Ser Val Gly Gln Phe
Ser Cys Thr Pro Asp Glu Gly Phe 260 265
270 Ile Tyr Gln Met Lys Pro Leu Cys Arg Asn Asp Ser Glu
Asn Leu Phe 275 280 285
Leu Lys Arg Thr Leu Cys Asp Lys Asp Lys Phe Pro Ala Gln Leu Glu 290
295 300 Gly Ile Lys Asn
Glu Ile Ile Glu Lys Cys Asp Gly Leu Pro Leu Ala 305 310
315 320 Ile Val Thr Leu Ala Ser Met Leu Ala
Thr Lys Gln Arg Thr Arg Glu 325 330
335 Glu Trp Glu Arg Ala Leu Asp Ser Ile His Ser Thr His Lys
Lys Asp 340 345 350
Ser Ser Leu Glu Val Met Asp Lys Ile Leu Ser Leu Ser Tyr Arg Asp
355 360 365 Leu Pro His Asn
Met Arg Asn Cys Leu Leu Tyr Ile Ser Thr Phe Pro 370
375 380 Glu Asp His Thr Ile Tyr Lys Asp
Ala Leu Val Trp Arg Trp Met Ala 385 390
395 400 Glu Gly Phe Ile Ala Glu Thr Gln Gly Phe Thr Leu
Glu Gln Val Ala 405 410
415 Glu Gly Tyr Phe Tyr Glu Phe Val Asn Arg Ser Leu Val Gln Pro Ile
420 425 430 Thr Leu Arg
Ser Arg Tyr Glu Met Arg Gly Glu Gly Gly Cys Arg Val 435
440 445 His Asp Ile Val Leu Asn Phe Leu
Ile Ser Arg Ala Ala Glu Glu Asn 450 455
460 Phe Leu Thr Thr Leu Tyr Gly Ala Gln Gly Val Pro Ser
Ser Asp Arg 465 470 475
480 Arg Ile Arg Arg Leu Ser Val Trp Asp Ser Pro Glu His Ala Leu Ala
485 490 495 Val Ser Arg Ala
Thr Met Asn Leu Ser His Leu Arg Ser Val Arg Ile 500
505 510 Cys Asn Val Gly Asp Trp Pro Val Pro
Ala Val Leu Asp Leu Pro Val 515 520
525 Leu Arg Val Leu Asp Leu Glu Gly Cys Arg Asp Leu Arg Ile
Val Asp 530 535 540
Pro Asp Cys Ile Leu Ser Leu Phe His Leu Arg Tyr Leu Gly Phe Arg 545
550 555 560 Ser Ala Ser Gly Val
Val Leu Pro Ala Gln Ile Gly Asn Leu His His 565
570 575 Leu Gln Thr Ile Asp Leu Ser Gly Thr Gly
Val Thr Gln Leu Pro Glu 580 585
590 Ser Ile Val Gln Leu Lys Arg Leu Met His Leu Val Gly Gln Arg
Leu 595 600 605 Ile
Met Pro Asp Gly Phe Gly Ser Met Glu Ser Leu Glu Glu Leu Gly 610
615 620 Thr Ile Asp Cys Cys Lys
Cys Pro Ala Glu Gly Ala Pro Ser Asp Arg 625 630
635 640 Val Ala Phe Val Gly Val Glu Thr Ser Asp Met
Glu Thr Arg Arg Lys 645 650
655 Ser Leu Met Ser Ser Leu Cys Lys Leu Gly Gly Asp Asn Leu Arg Arg
660 665 670 Val Thr
Ile Ile Asp Leu Ala Gly Gly Gly Asp Cys Phe Val Glu Ser 675
680 685 Trp His Pro Pro Pro Arg Leu
Leu Gln Lys Phe Ile His Ile Ser Gln 690 695
700 Gln Gln His Phe Ser Arg Phe Pro Glu Trp Ile Ser
Ser Cys Leu Cys 705 710 715
720 Asp Leu Thr His Leu Asp Ile Lys Ala Glu Lys Met Glu Arg Glu His
725 730 735 Leu Ser Val
Leu Glu His Leu Pro Ala Ile Arg Tyr Leu Tyr Leu Phe 740
745 750 Val Lys Arg Val Ser Glu Asp Gly
Leu Val Ile Ser His Ser Ala Phe 755 760
765 Arg Cys Leu Arg Arg Leu Glu Phe Cys Asn Leu Asp Gly
Pro Gly Leu 770 775 780
Met Phe Ala Gly Gly Val Pro Met Leu Glu Trp Leu Arg Leu Gly Phe 785
790 795 800 Asp Ala Asp Arg
Ala Gln Ser Thr Tyr Gly Gly Leu Glu Val Gly Ile 805
810 815 Gln Arg Leu Ser Ser Leu Lys His Val
Val Leu Ile Val Cys Met Val 820 825
830 Ser Glu Gly Gly Asp Asp Pro Ala Glu Gln Ala Val Trp Ser
Ala Ile 835 840 845
Asn Gly Gln Val Glu Met Leu Pro Asn Ser Pro Thr Val Asp Ile Arg 850
855 860 Phe Arg Arg Arg Ser
Gln Leu Gln Ala Ser Ser Glu 865 870 875
72667DNAZea mays 7atggagaacc cagacgcgca ggcgaaggcg tgggcggcgg
agatgcgcga gctggcctac 60gacatggagg acagcatcga tctcttcacc caccacgtcg
accacgaacc ggccgacacc 120gccaccaccg gcgtcaagag gttcttcctc cggatcatcc
ggaagcttaa gaaactccac 180taccgccaca ggtttgctca ggagatcaaa caactccacg
accttgccaa cgaatcgtac 240cggcgtagga agaggtacag gattgaggag ggcggttcaa
gcctcccgca cgcggagatc 300gatcctcggt tagaggcgct ctacgtggag gtggagaaac
tcgtgggcat ccagggccca 360agccaggaga tcattggaca gctcgtcggc gagaacgcag
cggagcggcg gagggttgtc 420gccgttgttg gatctggagg ttcaggcaag accacacttg
ccaaacaggt gtacgagaaa 480atcaggtgcc aattctcttg tgcagccttt gtgtccgtgt
cgcaaaagcc caacatgaat 540agcctcctgt gggagttgtt atctcaaatc gggaaccatg
gtggagattt aggaatgatg 600gcagtaggat attgcagtga caaacaactg atcgacagac
taagatcaca tcttgaaaag 660cagagaactg atttttcaac tgcttcacaa tctgctctta
ggtatctcgt tgtgatagat 720gatgtttgga caaactcagc gtgggagacc atacaatgtg
cgctccctaa aaatgcccat 780gcaagtaaaa taattctgac aacacgaatc aacagtgtag
gccagttctc ctgcactcca 840gatgagggtt ttatctatca gatgaagcct ctttgcagaa
acgattctga aaatctgttt 900ctgaaaagga cactatgtga taaagataag tttcctgctc
agctggaggg gattaaaaac 960gagataatcg agaaatgcga tggtttgcca ctggctattg
ttactctagc tagcatgtta 1020gctactaaac agagaacaag ggaagaatgg gagagggcac
ttgattcaat ccattctacg 1080cacaagaaag atagtagcct ggaagtgatg gacaagatac
tgtctctgag ttacagggat 1140ctacctcaca acatgagaaa ttgcttgctg tatatcagta
catttccaga ggaccacacg 1200atttacaaag atgctctagt atggagatgg atggctgaag
ggtttatcgc tgaaacacaa 1260ggctttactt tggagcaggt tgccgagggc tacttctacg
agtttgtgaa caggagtttg 1320gttcagccca taaccttgcg ttcaagatat gaaatgcgtg
gagaaggagg ttgccgagtc 1380catgacattg tactgaactt cctcatctct cgtgcagctg
aagagaactt tttaactacg 1440ctgtatggcg cccagggggt tccatcttca gaccgaagga
ttcgccggct ctctgtctgg 1500gacagtccag aacacgcact ggcagtctct agagcgacca
tgaatctgtc ccatctccgg 1560tcagttagaa tatgcaacgt tggagactgg cccgtgcctg
ctgttctaga cttacctgtc 1620cttcgagtgt tagatctaga gggatgccgt gatctgagga
tcgtcgaccc tgactgcatt 1680ctaagcttgt ttcatctgag gtacctgggt ttccgcagcg
caagtggtgt cgtgctaccg 1740gctcaaatag gaaatttaca ccatctgcag accatcgatt
taagcgggac tggagtgaca 1800cagctgccag aaagcattgt ccagctcaag cgactgatgc
atcttgttgg gcaacggctc 1860atcatgccag acgggtttgg tagcatggaa tcccttgagg
agttaggtac tatcgactgc 1920tgcaagtgcc ccgctgaggg tgctccgagt gaccgagtgg
ctttcgtcgg ggtcgaaaca 1980agtgacatgg aaaccagaag gaaatctttg atgtcatccc
tctgcaaact cggaggagac 2040aaccttcggc gtgtcactat tatcgacctc gctggcggtg
gagattgctt tgtggagtcg 2100tggcaccctc ctcctcgtct cctccagaag ttcatccata
tcagtcagca acagcacttc 2160tccaggtttc cagaatggat cagttcctgc ctatgtgatc
tcacccacct ggatataaag 2220gccgaaaaga tggaaaggga gcatctaagt gttcttgaac
acctgcccgc catccgttat 2280ctataccttt tcgtgaagcg agtctccgaa gacgggctcg
tcatcagcca cagcgcgttc 2340cgatgtctac ggcgtctcga gttctgtaac ttagatggac
ctggtttgat gtttgcagga 2400ggcgttccaa tgctggaatg gctgaggctc gggttcgacg
cggatagagc gcaatcgaca 2460tacggcggtc tggaggttgg catccagcgc ctctcgtctc
tcaaacatgt cgtgctcatt 2520gtctgtatgg tttctgaagg cggtgatgat ccagcggagc
aagccgtctg gtctgccatc 2580aatggccaag tagagatgct ccccaattct ccgacggttg
atatccggtt tcgtagacgg 2640agtcagctgc aggcaagctc agaataa
26678888PRTZea mays 8Met Glu Asn Pro Asp Ala Gln
Ala Lys Ala Trp Ala Ala Glu Met Arg 1 5
10 15 Glu Leu Ala Tyr Asp Met Glu Asp Ser Ile Asp
Leu Phe Thr His His 20 25
30 Val Asp His Glu Pro Ala Asp Thr Ala Thr Thr Gly Val Lys Arg
Phe 35 40 45 Phe
Leu Arg Ile Ile Arg Lys Leu Lys Lys Leu His Tyr Arg His Arg 50
55 60 Phe Ala Gln Glu Ile Lys
Gln Leu His Asp Leu Ala Asn Glu Ser Tyr 65 70
75 80 Arg Arg Arg Lys Arg Tyr Arg Ile Glu Glu Gly
Gly Ser Ser Leu Pro 85 90
95 His Ala Glu Ile Asp Pro Arg Leu Glu Ala Leu Tyr Val Glu Val Glu
100 105 110 Lys Leu
Val Gly Ile Gln Gly Pro Ser Gln Glu Ile Ile Gly Gln Leu 115
120 125 Val Gly Glu Asn Ala Ala Glu
Arg Arg Arg Val Val Ala Val Val Gly 130 135
140 Ser Gly Gly Ser Gly Lys Thr Thr Leu Ala Lys Gln
Val Tyr Glu Lys 145 150 155
160 Ile Arg Cys Gln Phe Ser Cys Ala Ala Phe Val Ser Val Ser Gln Lys
165 170 175 Pro Asn Met
Asn Ser Leu Leu Trp Glu Leu Leu Ser Gln Ile Gly Asn 180
185 190 His Gly Gly Asp Leu Gly Met Met
Ala Val Gly Tyr Cys Ser Asp Lys 195 200
205 Gln Leu Ile Asp Arg Leu Arg Ser His Leu Glu Lys Gln
Arg Thr Asp 210 215 220
Phe Ser Thr Ala Ser Gln Ser Ala Leu Arg Tyr Leu Val Val Ile Asp 225
230 235 240 Asp Val Trp Thr
Asn Ser Ala Trp Glu Thr Ile Gln Cys Ala Leu Pro 245
250 255 Lys Asn Ala His Ala Ser Lys Ile Ile
Leu Thr Thr Arg Ile Asn Ser 260 265
270 Val Gly Gln Phe Ser Cys Thr Pro Asp Glu Gly Phe Ile Tyr
Gln Met 275 280 285
Lys Pro Leu Cys Arg Asn Asp Ser Glu Asn Leu Phe Leu Lys Arg Thr 290
295 300 Leu Cys Asp Lys Asp
Lys Phe Pro Ala Gln Leu Glu Gly Ile Lys Asn 305 310
315 320 Glu Ile Ile Glu Lys Cys Asp Gly Leu Pro
Leu Ala Ile Val Thr Leu 325 330
335 Ala Ser Met Leu Ala Thr Lys Gln Arg Thr Arg Glu Glu Trp Glu
Arg 340 345 350 Ala
Leu Asp Ser Ile His Ser Thr His Lys Lys Asp Ser Ser Leu Glu 355
360 365 Val Met Asp Lys Ile Leu
Ser Leu Ser Tyr Arg Asp Leu Pro His Asn 370 375
380 Met Arg Asn Cys Leu Leu Tyr Ile Ser Thr Phe
Pro Glu Asp His Thr 385 390 395
400 Ile Tyr Lys Asp Ala Leu Val Trp Arg Trp Met Ala Glu Gly Phe Ile
405 410 415 Ala Glu
Thr Gln Gly Phe Thr Leu Glu Gln Val Ala Glu Gly Tyr Phe 420
425 430 Tyr Glu Phe Val Asn Arg Ser
Leu Val Gln Pro Ile Thr Leu Arg Ser 435 440
445 Arg Tyr Glu Met Arg Gly Glu Gly Gly Cys Arg Val
His Asp Ile Val 450 455 460
Leu Asn Phe Leu Ile Ser Arg Ala Ala Glu Glu Asn Phe Leu Thr Thr 465
470 475 480 Leu Tyr Gly
Ala Gln Gly Val Pro Ser Ser Asp Arg Arg Ile Arg Arg 485
490 495 Leu Ser Val Trp Asp Ser Pro Glu
His Ala Leu Ala Val Ser Arg Ala 500 505
510 Thr Met Asn Leu Ser His Leu Arg Ser Val Arg Ile Cys
Asn Val Gly 515 520 525
Asp Trp Pro Val Pro Ala Val Leu Asp Leu Pro Val Leu Arg Val Leu 530
535 540 Asp Leu Glu Gly
Cys Arg Asp Leu Arg Ile Val Asp Pro Asp Cys Ile 545 550
555 560 Leu Ser Leu Phe His Leu Arg Tyr Leu
Gly Phe Arg Ser Ala Ser Gly 565 570
575 Val Val Leu Pro Ala Gln Ile Gly Asn Leu His His Leu Gln
Thr Ile 580 585 590
Asp Leu Ser Gly Thr Gly Val Thr Gln Leu Pro Glu Ser Ile Val Gln
595 600 605 Leu Lys Arg Leu
Met His Leu Val Gly Gln Arg Leu Ile Met Pro Asp 610
615 620 Gly Phe Gly Ser Met Glu Ser Leu
Glu Glu Leu Gly Thr Ile Asp Cys 625 630
635 640 Cys Lys Cys Pro Ala Glu Gly Ala Pro Ser Asp Arg
Val Ala Phe Val 645 650
655 Gly Val Glu Thr Ser Asp Met Glu Thr Arg Arg Lys Ser Leu Met Ser
660 665 670 Ser Leu Cys
Lys Leu Gly Gly Asp Asn Leu Arg Arg Val Thr Ile Ile 675
680 685 Asp Leu Ala Gly Gly Gly Asp Cys
Phe Val Glu Ser Trp His Pro Pro 690 695
700 Pro Arg Leu Leu Gln Lys Phe Ile His Ile Ser Gln Gln
Gln His Phe 705 710 715
720 Ser Arg Phe Pro Glu Trp Ile Ser Ser Cys Leu Cys Asp Leu Thr His
725 730 735 Leu Asp Ile Lys
Ala Glu Lys Met Glu Arg Glu His Leu Ser Val Leu 740
745 750 Glu His Leu Pro Ala Ile Arg Tyr Leu
Tyr Leu Phe Val Lys Arg Val 755 760
765 Ser Glu Asp Gly Leu Val Ile Ser His Ser Ala Phe Arg Cys
Leu Arg 770 775 780
Arg Leu Glu Phe Cys Asn Leu Asp Gly Pro Gly Leu Met Phe Ala Gly 785
790 795 800 Gly Val Pro Met Leu
Glu Trp Leu Arg Leu Gly Phe Asp Ala Asp Arg 805
810 815 Ala Gln Ser Thr Tyr Gly Gly Leu Glu Val
Gly Ile Gln Arg Leu Ser 820 825
830 Ser Leu Lys His Val Val Leu Ile Val Cys Met Val Ser Glu Gly
Gly 835 840 845 Asp
Asp Pro Ala Glu Gln Ala Val Trp Ser Ala Ile Asn Gly Gln Val 850
855 860 Glu Met Leu Pro Asn Ser
Pro Thr Val Asp Ile Arg Phe Arg Arg Arg 865 870
875 880 Ser Gln Leu Gln Ala Ser Ser Glu
885 92930DNAZea mays 9atggagaacc cagacgcgca ggcgaaggcg
tgggcggcgg agatgcgcga gctggcctac 60gacatggagg acagcatcga tctcttcacc
caccacgtcg accacgaacc ggccgacacc 120gccaccaccg gcgtcaagag gttcttcctc
cggatcatcc ggaagcttaa gaaactccac 180taccgccaca ggtttgttca ggagatcaaa
caactccacg accttgccaa cgaatcgtac 240cggcgtagga agaggtacag gattgaggag
ggcggttcaa gcctctcgca cgcggagatc 300gatcctcggt tagaggcgct ctacgtggag
gtggagaaac tcgtgggcat ccagggccca 360agccaggaga tcattggaca gctcgtcggc
gagaacgcag cggagcgacg gagggttgtc 420gccgttgttg gatctggagg ttcaggcaag
accacacttg ccaaacaggt gtacgagaaa 480atcaggtgcc aattctcttg tgcagccttt
gtgtctgtgt cgcaaaagcc caacatgaat 540agcctcctgt gggagttgct atctcaaatc
gggaaccatg gtggagattt aggaatgatg 600gcagtaggat attgcagtga caaacaactg
atcgacagac taagatcaca tcttgaaaag 660cagaggttag tttacctttt cattccggtt
agcttaattc ggtacaccaa ctagagattt 720gtgatttgct attaattaca ccaaatttct
cctacacaac aataactggt ttagcatgat 780ggcgatccaa agtcaaaact atcttctact
actagtgtat gccatactca tatagatatt 840ttcttttcat aaactctcgt agcattttta
catgcattca tattcctatt gcctttatac 900agaactgatt tttcactgct tcacaatctg
ctcttaggta tctcgttgtg atagatgatg 960tttggacaaa ctcagcgtgg gagaccatac
aatgtgcgct ccctaaaaat gcccatgcaa 1020gtaaaataat tctgacaaca cgaatcaaca
gtgtaggcca gttctcctgc actccagatg 1080agggttttat ctatcagatg aagcctcttt
gcagaaacga ttctgaaaat ctgtttctga 1140aaaggacact atgtgataaa gataagtttc
ctgctcagct ggaggggatt aaaaacgaga 1200taatcgagaa atgcgatggt ttgccactgg
ctattgttac tctagctagc atgttagcta 1260ctaaacagag aacaagggaa gaatgggaga
gggcacttga ttcaatccat tctatgcaca 1320agaaagatag tggcctggaa gtgatggaca
agatactgtc tctgagttac agggatctac 1380ctcacaacat gagaaattgc ttgctgtatc
tcagtacatt tccagaggac cacacgattt 1440acaaagatgc cctagtatgg agatggatgg
ctgaagggtt tatcgctgaa acacaaggct 1500ttactttgga gcaggttgcc gagggctact
tctacgagtt tgtgaacagg agtttggttc 1560agcccataac cttgcgttca agatatgaaa
tgcgtggaga aggaggttgc cgagtccatg 1620acattgtact gaacttcctc atctctcgtg
cagctgaaga gaacttttta actacgctgt 1680atggcgccca gggggttcca tcttcagacc
gaaggattcg ccggctctct gtctgggaca 1740gtccagaaca cgcactggca gtctctagag
cgaccatgaa tctgtcccat ctccggtcag 1800ttagaatatg caacgttgga gactggcccg
tgcctgctgt tctagactta cctgtccttc 1860gagtgttaga tctagaggga tgccgtgatc
tgaggatcga cgaacctgac tgcattctaa 1920gcttgtttca tctgagatac ctgggtttcc
gcagcgcaag tggtgtcgtg ctaccggctc 1980aaatcggaaa tttacaccat ctgcagacca
tcgatttaag cgggactgga gtgacacagc 2040tgccagaaag cattgtccag ctcaagcgac
tgatgcatct tgttgggcaa cggctcatca 2100tgccagacgg gtttggtagc atggaatccc
ttgaggagtt aggtactatc gactgctgca 2160agtgccccgt cagttttggg gaagacctag
cacttctgag caggctgagg gtgctccgag 2220tggctttcat cggggtcgaa acaagtgaca
tggaaaccag aaggaaatct ttgatgtcat 2280ccctctgcaa actcggagga gacaaccttc
ggcgtgtcac tattatcgac ctcgctggcg 2340gtggagattg ctttgtggag tcgtggcacc
ctcctcctcg tctcctccag aagttcatcc 2400atatcagtca gcaacagcac ttctccaggt
ttccagaatg gatcagttcc tgcctatgtg 2460atctcaccca cctggatata aaggccgaaa
agatggaaag ggagcatcta agtgttcttg 2520aacacctgcc cgccatccgt tgcctatacc
ttttcgtgaa gcgagtctcc gaagacgggc 2580tcgccatcag ccacggcgcg ttccgatgtc
tacggcgtct cgagttctgc aacgtagatg 2640gacctggttt gatgtttgca ggaggcgttc
caatgttgga atggctgagg ctcgggttcg 2700acgcggatag agcgcaatcg acatacggcg
gtctggaggt tggcatccag cgcctctcgt 2760ctctcaaaca tgtcgtgctc attgtatgga
tggtttctga aggcggtgat gatccagcgg 2820agcaagccgt ctggtctgcc atcaatggcc
aagtagagat gctccccaac tctccgacgg 2880ttgatatccg gtttcgtaga cggagtcagc
tgcaggcaag ctcagaataa 29301016PRTZea mays 10Val Ser Phe Gly
Glu Asp Leu Ala Leu Leu Ser Arg Leu Arg Val Leu 1 5
10 15 11667PRTZea mays 11Met Ala Ala His
Gln Pro His Leu Ser Val Leu Leu Leu Val Leu Leu 1 5
10 15 Ala Ala His Val Val Ser Thr Ser Ala
His Gly Glu Pro Pro Leu Pro 20 25
30 Ser Pro Tyr Asn Thr Ser Ala His Gly Glu Pro Pro Leu Pro
Ser Thr 35 40 45
Tyr Asn Ala Ser Met Cys Ser Ser Phe Trp Cys Gly Gly Val Glu Ile 50
55 60 Arg Tyr Pro Phe Tyr
Leu Ala Asn Ala Ile Ala Asp Tyr Ser Gly Ser 65 70
75 80 Tyr Tyr Ser Cys Gly Tyr Thr Asp Leu Ser
Val Ser Cys Glu Leu Glu 85 90
95 Val Glu Gly Ser Pro Thr Thr Trp Thr Pro Thr Ile Arg Leu Gly
Gly 100 105 110 Gly
Asp Tyr Thr Val Lys Asn Ile Ser Tyr Leu Tyr Asp Gln Gln Thr 115
120 125 Ile Ser Leu Ala Asp Arg
Asp Val Leu Gly Gly Gly Gly Cys Pro Val 130 135
140 Val Arg His Asn Val Ser Phe Asp Glu Thr Trp
Leu His Leu His Asn 145 150 155
160 Ala Ser Ala Phe Asp Asn Leu Thr Phe Phe Phe Gly Cys His Trp Gly
165 170 175 Pro Arg
Asn Thr Pro Pro Glu Phe Ala Asp Tyr Asn Ile Ser Cys Ala 180
185 190 Gly Phe Asn Thr Pro Thr Ile
Ser Gly Gly Arg Ser Phe Val Phe Lys 195 200
205 Thr Gly Asp Leu Asp Glu Gln Glu Glu Gln Glu Leu
Ala Leu His Cys 210 215 220
Asp Glu Val Phe Ser Val Pro Val Arg Arg Asp Ala Leu Gln Ala Ile 225
230 235 240 Val Ser Asn
Phe Ser Leu Thr Arg Asp Gly Tyr Gly Glu Val Leu Arg 245
250 255 Gln Gly Phe Glu Leu Glu Trp Asn
Arg Thr Ser Glu Asp Gln Cys Gly 260 265
270 Arg Cys Glu Gly Ser Gly Ser Gly Gly Trp Cys Ala Tyr
Ser Gln Lys 275 280 285
Arg Glu Phe Leu Gly Cys Leu Cys Ser Gly Gly Lys Val Gly Ser Pro 290
295 300 Phe Cys Lys Pro
Ser Arg Ser Lys Arg Lys Glu Gly Pro Ile Val Gly 305 310
315 320 Ala Val Ala Val Ala Phe Leu Cys Leu
Val Ile Leu Thr Cys Phe Leu 325 330
335 Ala Cys Arg His Gly Ser Leu Pro Phe Lys Ser Glu Asn Lys
Pro Gly 340 345 350
Thr Arg Ile Glu Ser Phe Leu Gln Lys Asn Glu Ser Ile His Pro Lys
355 360 365 Arg Tyr Thr Tyr
Ala Asp Val Lys Arg Met Thr Lys Ser Phe Ala Val 370
375 380 Lys Leu Gly Gln Gly Gly Phe Gly
Ala Val Tyr Lys Gly Ser Leu His 385 390
395 400 Asp Gly Arg Gln Val Ala Val Lys Met Leu Lys Asp
Thr Gln Gly Asp 405 410
415 Gly Glu Glu Phe Met Asn Glu Val Ala Ser Ile Ser Arg Thr Ser His
420 425 430 Val Asn Val
Val Thr Leu Leu Gly Phe Cys Leu Gln Gly Ser Lys Arg 435
440 445 Ala Leu Ile Tyr Glu Tyr Met Pro
Asn Gly Ser Leu Glu Arg Tyr Ala 450 455
460 Phe Thr Gly Asp Met Asn Ser Glu Asn Leu Leu Thr Trp
Glu Arg Leu 465 470 475
480 Phe Asp Ile Ala Ile Gly Thr Ala Arg Gly Leu Glu Tyr Leu His Arg
485 490 495 Gly Cys Asn Thr
Arg Ile Val His Phe Asp Ile Lys Pro His Asn Ile 500
505 510 Leu Leu Asp Gln Asp Phe Cys Pro Lys
Ile Ser Asp Phe Gly Leu Ala 515 520
525 Lys Leu Cys Leu Asn Lys Glu Ser Ala Ile Ser Ile Ala Gly
Ala Arg 530 535 540
Gly Thr Ile Gly Tyr Ile Ala Pro Glu Val Tyr Ser Lys Gln Phe Gly 545
550 555 560 Ile Ile Ser Ser Lys
Ser Asp Val Tyr Ser Tyr Gly Met Met Val Leu 565
570 575 Glu Met Val Gly Ala Arg Asp Arg Asn Thr
Ser Ala Asp Ser Asp His 580 585
590 Ser Ser Gln Tyr Phe Pro Gln Trp Leu Tyr Glu His Leu Asp Asp
Tyr 595 600 605 Cys
Val Gly Ala Ser Glu Ile Asn Gly Glu Thr Thr Glu Leu Val Arg 610
615 620 Lys Met Ile Val Val Gly
Leu Trp Cys Ile Gln Val Ile Pro Thr Asp 625 630
635 640 Arg Pro Thr Met Thr Arg Val Val Glu Met Leu
Glu Gly Ser Thr Ser 645 650
655 Asn Leu Glu Leu Pro Pro Arg Val Leu Leu Ser 660
665 12666PRTZea mays 12Met Ala Ala His Leu Pro Arg
Leu Pro Val Leu Leu Leu Val Leu Leu 1 5
10 15 Ala Ala His Val Val Ser Thr Ser Ala His Ala
Glu Pro Pro Leu Pro 20 25
30 Ser Pro Tyr Ser Thr Ser Ala His Gly Glu Pro Pro Leu Pro Ser
Thr 35 40 45 Tyr
Asn Val Ser Met Cys Ser Glu Ser Phe Trp Cys Gly Gly Val Glu 50
55 60 Ile Arg Tyr Pro Phe Tyr
Leu Ala Asn Ala Thr Ala Asp Tyr Ser Gly 65 70
75 80 Ser Tyr Tyr Ser Cys Gly Tyr Thr Asp Leu Ser
Val Ser Cys Lys Leu 85 90
95 Glu Val Glu Gly Pro Thr Thr Thr Trp Thr Pro Thr Ile Arg Leu Gly
100 105 110 Gly Asp
Asn Tyr Thr Val Lys Asn Ile Leu Tyr Asp Tyr His Thr Ile 115
120 125 Ser Leu Ala Asp Ser Asp Val
Leu Gly Gly Gly Glu Cys Pro Val Val 130 135
140 His His Asn Val Ser Phe Asp Glu Thr Trp Leu His
Asn Pro Ser Ala 145 150 155
160 Phe Asp Asn Leu Thr Phe Phe Phe Gly Cys His Trp Gly Pro Arg Asp
165 170 175 Thr Leu Pro
Glu Phe Ala Gly Asn Asn Ile Ser Cys Ala Gly Phe Ser 180
185 190 Thr Pro Ala Ile Ser Gly Gly Gly
Ser Phe Val Phe Lys Pro Glu Asp 195 200
205 Leu Asp Glu His Ala Glu Gln Glu Leu Ala Ser His Cys
Asp Glu Val 210 215 220
Phe Ser Val Pro Val Arg Ser Glu Ala Leu Gln Gln Ala Ile Val Ser 225
230 235 240 Asn Leu Ser Leu
Gly Asp Gly Tyr Gly Glu Leu Leu Arg Gln Gly Ile 245
250 255 Glu Leu Glu Trp Lys Arg Thr Ser Glu
Asp Gln Cys Gly Gln Cys Glu 260 265
270 Glu Ser Gly Ser Gly Gly Arg Cys Ala Tyr Ser Gln Lys Arg
Glu Phe 275 280 285
Leu Gly Cys Leu Cys Ser Gly Gly Lys Ala Gly Asn Pro Phe Cys Lys 290
295 300 Pro Ser Arg Ser Lys
Arg Lys Glu Ala Ser Ile Val Gly Ala Val Ala 305 310
315 320 Val Ala Phe Leu Cys Leu Val Ile Leu Thr
Cys Phe Leu Ala Cys Arg 325 330
335 His Gly Ser Leu Pro Phe Lys Ser Glu Asn Lys Pro Gly Thr Arg
Ile 340 345 350 Glu
Ser Phe Leu Gln Lys Asn Glu Ser Ile His Pro Lys Arg Tyr Thr 355
360 365 Tyr Thr Asp Val Lys Arg
Met Thr Lys Ser Phe Ala Val Lys Leu Gly 370 375
380 Gln Gly Gly Phe Gly Ala Val Tyr Lys Gly Ser
Leu His Asp Gly Arg 385 390 395
400 Gln Val Ala Val Lys Met Leu Lys Asp Thr Gln Gly Asp Gly Glu Glu
405 410 415 Phe Met
Asn Glu Val Ala Ser Ile Ser Arg Thr Ser His Val Asn Val 420
425 430 Val Thr Leu Leu Gly Phe Cys
Leu Gln Gly Ser Lys Arg Ala Leu Ile 435 440
445 Tyr Glu Tyr Met Pro Asn Gly Ser Leu Glu Arg Tyr
Ala Phe Thr Gly 450 455 460
Asp Met Asn Ser Glu Asn Leu Leu Thr Trp Glu Arg Leu Phe Asp Ile 465
470 475 480 Ala Ile Gly
Thr Ala Arg Gly Leu Glu Tyr Leu His Arg Gly Cys Asn 485
490 495 Thr Arg Ile Val His Phe Asp Ile
Lys Pro His Asn Ile Leu Leu Asp 500 505
510 Gln Asp Phe Cys Pro Lys Ile Ser Asp Phe Gly Leu Ala
Lys Leu Cys 515 520 525
Leu Asn Lys Glu Ser Ala Ile Ser Ile Val Gly Ala Arg Gly Thr Ile 530
535 540 Gly Tyr Ile Ala
Pro Glu Val Tyr Ser Lys Gln Phe Gly Thr Ile Ser 545 550
555 560 Ser Lys Ser Asp Val Tyr Ser Tyr Gly
Met Met Val Leu Glu Met Val 565 570
575 Gly Ala Arg Glu Arg Asn Thr Ser Ala Ser Ala Asp Ser Asp
His Ser 580 585 590
Ser Gln Tyr Phe Pro Gln Trp Ile Tyr Glu His Leu Asp Asp Tyr Cys
595 600 605 Val Gly Ala Ser
Glu Ile Asn Gly Glu Thr Thr Glu Leu Val Arg Lys 610
615 620 Met Ile Val Val Gly Leu Trp Cys
Ile Gln Val Ile Pro Thr Asp Arg 625 630
635 640 Pro Thr Met Thr Arg Val Val Glu Met Leu Glu Gly
Ser Thr Ser Asn 645 650
655 Leu Glu Leu Pro Pro Arg Val Leu Leu Ser 660
665
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