Plant folate biosynthetic genes

This application claims the benefit of U.S. Provisional Application No. 60/112,735, filed Dec. 18, 1998.

This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding folate biosynthetic enzymes in plants and seeds.

Tetrahydrofolic acid and its derivatives N 5 ,N 10 -methylenetetrahydrofolate, N 5 ,N 10 -methenyltetrahydrofolate, N 10 -formyltetrahydrofolate and N 5 -methyl-tetrahydrofolate are biologically active forms of folic acid. The tetrahydrofolates are coenzymes that function in a variety of enzyme catalyzed reactions as specialized cosubstrates for one-carbon metabolism. For example, tetrahydrofolate plays an important role in nucleic acid biosynthesis by serving as the immediate source of one-carbon units in purine and pyrimidine biosynthesis. The cellular tetrahydrofolate coenzyme pool must be maintained at specific levels to assure one-carbon metabolism operates efficiently. Thus, one of the most important reactions of the cell is the reduction of dihydrofolate to tetrahydrofolate by dihydrofolate reductase. The importance of this reaction in mammalian cells can be shown by the fact that methorexate, a very effective chemotherapy drug, is a potent inhibitor of dihydrofolate reductase (Zubay, G. (1983) Biochemistry , Addison-Wesley Publishing Co. Reading, Mass.). Other enzymes involved in the folic acid biosynthetic pathway to maintain the tetrahydrofolate coenzyme pool are tetrahydrofolypolyglutamate synthase, dihydropteroate synthase and dihydroneopterin aldolase.

There is a great deal of interest in identifying the genes that encode proteins required for tetrahydrofolate biosynthesis in plants. These genes may be used in plant cells to alter the tetrahydrofolate coenzyme pool concentration and modulate one-carbon metabolism. Accordingly, the availability of nucleic acid sequences encoding all or a portion of the tetrahydrofolypolyglutamate synthase, dihydropteroate synthase and dihydroneopterin aldolase enzymes would facilitate studies to better understand one-carbon metabolism in plants, provide genetic tools to one-carbon metabolism. The tetrahydrofolate biosynthetic enzymes may also provide targets to facilitate design and/or identification of inhibitors of cell cycle that may be useful as herbicides.

The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 131 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn dihydroneopterin aldolase polypeptide of SEQ ID NO:2, a soybean dihydroneopterin aldolase polypeptide of SEQ ID NO:4 and a wheat dihydroneopterin aldolase polypeptide of SEQ ID NO:6. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.

The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 75 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn dihydropteroate synthase/dihydropteroate pyrophosphorylase polypeptide of SEQ ID NO:8, a rice dihydropteroate synthase/dihydropteroate pyrophosphorylase polypeptide of SEQ ID NO:10 and a soybean dihydropteroate synthase/dihydropteroate pyrophosphorylase polypeptide of SEQ ID NO:12. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.

The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 553 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a corn tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide of SEQ ID NO:14. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.

The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 133 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a corn tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide of SEQ ID NO:16 and a soybean tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide of SEQ ID NO:18. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.

It is preferred that the isolated polynucleotides of the claimed invention consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and the complement of such nucleotide sequences.

The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.

The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.

The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.

The present invention relates to a dihydroneopterin aldolase polypeptide of at least 131 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4 and 6.

The present invention relates to a dihydropteroate synthase/dihydropteroate pyrophosphorylase polypeptide of at least 75 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:8, 10 and 12.

The present invention relates to a tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide of at least 553 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide of SEQ ID NO:14.

The present invention relates to a tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide of at least 133 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs: 16 and 18.

The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level a dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide in the host cell containing the isolated polynucleotide; and (d) comparing the level of a dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide in the host cell containing the isolated polynucleotide with the level of a dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide in the host cell that does not contain the isolated polynucleotide.

The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide gene, preferably a plant dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutarnate synthase polypeptide gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase amino acid sequence.

The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.

A further embodiment of the instant invention is a method for evaluating at least one compound for its ability to inhibit the activity of a dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase, the method comprising the steps of: (a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase, operably linked to suitable regulatory sequences; (b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase in the transformed host cell; (c) optionally purifying the dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase expressed by the transformed host cell; (d) treating the dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase with a compound to be tested; and (e) comparing the activity of the dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase that has been treated with a test compound to the activity of an untreated dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase, thereby selecting compounds with potential for inhibitory activity.

The present invention relates to a composition, such as a hybridization mixture, comprising an isolated polynucleotide or polypeptide of the present invention.

The present invention relates to an isolated polynucleotide of the present invention comprising at least one of 30 contiguous nucleotides derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29 and 31.

The present invention relates to an expression cassette comprising an isolated polynucleotide of the present invention operably linked to a promoter.

The present invention relates to a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably plant cell, such as a monocot or a dicot, under conditions which allow expression of the dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase polynucleotide in an amount sufficient to complement a null mutant and folic acid biosynthesis auxotroph to provide a positive selection means.

The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing which form a part of this application.

Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. Table 1 also identifies the cDNA clones as individual ESTs (“EST”), the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”), contigs assembled from two or more ESTs (“Contig”), contigs assembled from an FIS and one or more ESTs (“Contig*”), or sequences encoding the entire protein derived from an FIS, a contig, or an FIS and PCR (“CGS”). Nucleotide sequences, SEQ ID NOs:1, 3, 5, 7, 9, 13 and 17 and amino acid sequences SEQ ID NOs:2, 4, 6, 8, 10, 14, 16 and 18 were determined by further sequence analysis of cDNA clones encoding the amino acid sequences set forth in SEQ ID NOs:20, 22, 24, 26, 28, 30 and 32. Nucleotide SEQ ID NOs:19, 21, 23, 25, 27, 29 and 31 and amino acid SEQ ID NOs:20, 22, 24, 26, 28, 30 and 32 were presented in a U.S. Provisional Application No. 60/112,735, filed Dec. 18, 1998.

The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.

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 Res . 13:3021-3030 (1985) and in the Biochemical J . 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

In the context of this disclosure, a number of terms shall be utilized. As used herein, a “polynucleotide” is a nucleotide sequence such as a nucleic acid fragment. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolated polynucleotide of the present invention may include at least one of 60 contiguous nucleotides, preferably at least one of 40 contiguous nucleotides, most preferably one of at least 30 contiguous nucleotides derived from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 or the complement of such sequences.

As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co-suppression technology. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-à-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof.

Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications of the nucleic acid fragments of the instant invention, wherein one or more nucleotides are substituted, deleted and/or inserted, for their ability to affect the level of the polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell. For example, a substantially similar nucleic acid fragment representing at least one of 30 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell. The level of the polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level of the polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.

For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Consequently, an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of a polypeptide (dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase) in a host cell. A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a host cell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial, or viral) may comprise the steps of: constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level a polypeptide in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide in the host cell containing the isolated polynucleotide with the level of a polypeptide in a host cell that does not contain the isolated polynucleotide.

Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6×SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2×SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2×SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2×SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1×SSC, 0.1% SDS at 65° C.

Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, preferably at least about 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least about 50 amino acids, preferably at least about 100 amino acids, more preferably at least about 150 amino acids, still more preferably at least about 200 amino acids, and most preferably at least about 250 amino acids. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

A “substantial portion” of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol . 215:403-410. In general, a sequence of ten or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed, sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

“Synthetic nucleic acid fragments” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. “Chemically synthesized”, as related to nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the nucleic acid fragments can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

“Coding sequence” refers to a nucleotide sequence that codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 15:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.

The “translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Mol. Biotechnol . 3:225-236).

The “3′ non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.

“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into polypeptide by the cell. “cDNA” refers to a double-stranded DNA that is complementary to and derived from mRNA. “Sense” RNA refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (see U.S. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. “Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.

The term “operably linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. “Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated herein by reference).

“Altered levels” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

“Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. “Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. “Chloroplast transit sequence” refers to a nucleotide sequence that encodes a chloroplast transit peptide. A “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol . 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys . 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. Examples of methods of plant transformation include Agrobacterium -mediated transformation (De Blaere et al. (1987) Meth. Enzymol . 143:277) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature ( London ) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual ; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of several folate biosynthetic enzymes have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad Sci. USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220). Products generated by the 3′ and 5′ RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165). Consequently, a polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably one of at least 40, most preferably one of at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide. The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a polypeptide of a gene (such as dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase) preferably a substantial portion of a plant polypeptide of a gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a polypeptide (dihydroneopterin aldolase, dihydropteroate synthase/dihydropteroate pyrophosphorylase or tetrahydrofolypolyglutamate synthase/folylpolyglutamate synthase).

Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lemer (1984) Adv. Immunol . 36:1-34; Maniatis).

The nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed polypeptides are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of folic acid in those cells.

Overexpression of the proteins of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. The chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3′ Non-coding sequences encoding transcription termination signals may also be provided. The instant chimeric gene may also comprise one or more introns in order to facilitate gene expression.

Plasmid vectors comprising the isolated polynucleotide (or chimeric gene) may be constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al. (1985) EMBO J . 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

For some applications it may be useful to direct the instant polypeptides to different cellular compartments, or to facilitate its secretion from the cell. It is thus envisioned that the chimeric gene described above may be further supplemented by directing the coding sequence to encode the instant polypeptides with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 56:247-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol . 42:21-53), or nuclear localization signals (Raikhel (1992) Plant Phys . 100:1627-1632) with or without removing targeting sequences that are already present. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of use may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genes encoding the instant polypeptides in plants for some applications. In order to accomplish this, a chimeric gene designed for co-suppression of the instant polypeptide can be constructed by linking a gene or gene fragment encoding that polypeptide to plant promoter sequences. Alternatively, a chimeric gene designed to express antisense RNA for all or part of the instant nucleic acid fragment can be constructed by linking the gene or gene fragment in reverse orientation to plant promoter sequences. Either the co-suppression or antisense chimeric genes could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated.

Molecular genetic solutions to the generation of plants with altered gene expression have a decided advantage over more traditional plant breeding approaches. Changes in plant phenotypes can be produced by specifically inhibiting expression of one or more genes by antisense inhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression construct would act as a dominant negative regulator of gene activity. While conventional mutations can yield negative regulation of gene activity these effects are most likely recessive. The dominant negative regulation available with a transgenic approach may be advantageous from a breeding perspective. In addition, the ability to restrict the expression of specific phenotype to the reproductive tissues of the plant by the use of tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.

The person skilled in the art will know that special considerations are associated with the use of antisense or cosuppression technologies in order to reduce expression of particular genes. For example, the proper level of expression of sense or antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan. Once transgenic plants are obtained by one of the methods described above, it will be necessary to screen individual transgenics for those that most effectively display the desired phenotype. Accordingly, the skilled artisan will develop methods for screening large numbers of transformants. The nature of these screens will generally be chosen on practical grounds. For example, one can screen by looking for changes in gene expression by using antibodies specific for the protein encoded by the gene being suppressed, or one could establish assays that specifically measure enzyme activity. A preferred method will be one which allows large numbers of samples to be processed rapidly, since it will be expected that a large number of transformants will be negative for the desired phenotype.

The instant polypeptides (or portions thereof) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the these proteins by methods well known to those skilled in the art. The antibodies are useful for detecting the polypeptides of the instant invention in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production of the instant polypeptides are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a chimeric gene for production of the instant polypeptides. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded folate biosynthetic enzyme. An example of a vector for high level expression of the instant polypeptides in a bacterial host is provided (Example 8).

Additionally, the instant polypeptides can be used as a targets to facilitate design and/or identification of inhibitors of those enzymes that may be useful as herbicides. This is desirable because the polypeptides described herein catalyze various steps in folic acid biosynthesis. Accordingly, inhibition of the activity of one or more of the enzymes described herein could lead to inhibition of plant growth. Thus, the instant polypeptides could be appropriate for new herbicide discovery and design.

All or a substantial portion of the nucleic acid fragments of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet . 32:314-331).

The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4:37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide , Academic press 1996, pp. 319-346, and references cited therein).

In another embodiment, nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet . 7:149-154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan et al. (1995) Genome Res . 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med . 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1 990) Nucleic Acid Res . 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet . 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res . 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.

Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter approach may be accomplished in two ways. First, short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra). The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding the instant polypeptides. Alternatively, the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor. With either method, a plant containing a mutation in the endogenous gene encoding the instant polypeptides can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function of the instant polypeptides disclosed herein.

The present invention is further defined in the following Examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Zea mays

1

gcacgagggc ggcggctacg tggggtggcg acgacaagct cattctgcgc ggccttcagt 60

tccatggctt ccacggtgtc ctgcaggagg agaagacgtt gggacagaag ttcgtggttg 120

acatcgacgc ctggatagac ctcgccgctg ccggcgagtc cgactgcatt gctgacaccg 180

tcagctacac cgatatctac agcattgcaa aggatgttgt cgagggcacg ccacgcaacc 240

tcttggagtc ggtagctcac tcgatcgcag aggccacgct gctcaagttc cctcagatct 300

ccgcagtccg agtgaaggtt ggcaagcctc acgtcgcggt gcgaggcgtt ctggactacc 360

tgggcgtgga gataacgagg cacagaaaga aagaatgaga tgctgtacac atgtggtgat 420

ggggagccag ttcaatgctg atggcactgc ggccataacc ataatccacg cacgcttgtt 480

gcttgttggc aactaggcat atccctttca cctctgaact gttggaatat cgggaatctg 540

ttcccctagt tgctttatta cgaattcaga tcatatctgg ctagtaagat caaccttctt 600

ctggtctgta acaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 658

2

131

Zea mays

2

Thr Arg Ala Ala Ala Thr Trp Gly Gly Asp Asp Lys Leu Ile Leu Arg

1 5 10 15

Gly Leu Gln Phe His Gly Phe His Gly Val Leu Gln Glu Glu Lys Thr

20 25 30

Leu Gly Gln Lys Phe Val Val Asp Ile Asp Ala Trp Ile Asp Leu Ala

35 40 45

Ala Ala Gly Glu Ser Asp Cys Ile Ala Asp Thr Val Ser Tyr Thr Asp

50 55 60

Ile Tyr Ser Ile Ala Lys Asp Val Val Glu Gly Thr Pro Arg Asn Leu

65 70 75 80

Leu Glu Ser Val Ala His Ser Ile Ala Glu Ala Thr Leu Leu Lys Phe

85 90 95

Pro Gln Ile Ser Ala Val Arg Val Lys Val Gly Lys Pro His Val Ala

100 105 110

Val Arg Gly Val Leu Asp Tyr Leu Gly Val Glu Ile Thr Arg His Arg

115 120 125

Lys Lys Glu

130

3

705

Glycine max

3

gcacgagcgg agaggcgagg gagtgaggga ctagcacaga aagatattgt ttggtgtacg 60

gtggtgagtg tcgacgctgc cactctcgcc tgtgtctgtg ataaatggaa tctgatgcac 120

cgacatgggg agacaaactc atgttgaggg gattgtcatt ccatggtttt catggagcaa 180

agcctgaaga aaggacactg ggccagaagt tcttcataga tatagatgct tggatggatc 240

tcaaagcagc tggcaaatct gatcacttat cagattctgt tagttacaca gaaatatatg 300

atatagctaa ggatgttctt gaagggtcac ctcacaatct tctggagtca gtggcccaaa 360

aaattgcaat cactactctt acaaatcata aagaaatatc tgctgtccga gtgaaggttg 420

gaaagcctca tgtggcagtt cggggtccag ttgattactt aggcgttgag attcttagac 480

gcagaagcga cttgtcaggc tagaaatttc atatttattg ctgcacaatt tttatatttt 540

cacattccac ttgatacaaa agtaatgtaa ctctttcctt catgccccat tagtcttttc 600

tctcttaagc aatcttgcta atgaaattaa aagatcaaag ttaggcatat taaaggaact 660

atacaattaa tttggattct ccaaaacaaa aaaaaaaaaa aaaaa 705

4

132

Glycine max

4

Met Glu Ser Asp Ala Pro Thr Trp Gly Asp Lys Leu Met Leu Arg Gly

1 5 10 15

Leu Ser Phe His Gly Phe His Gly Ala Lys Pro Glu Glu Arg Thr Leu

20 25 30

Gly Gln Lys Phe Phe Ile Asp Ile Asp Ala Trp Met Asp Leu Lys Ala

35 40 45

Ala Gly Lys Ser Asp His Leu Ser Asp Ser Val Ser Tyr Thr Glu Ile

50 55 60

Tyr Asp Ile Ala Lys Asp Val Leu Glu Gly Ser Pro His Asn Leu Leu

65 70 75 80

Glu Ser Val Ala Gln Lys Ile Ala Ile Thr Thr Leu Thr Asn His Lys

85 90 95

Glu Ile Ser Ala Val Arg Val Lys Val Gly Lys Pro His Val Ala Val

100 105 110

Arg Gly Pro Val Asp Tyr Leu Gly Val Glu Ile Leu Arg Arg Arg Ser

115 120 125

Asp Leu Ser Gly

130

5

759

Triticum aestivum

5

gcacgagcca ggttccactc cacccaccca cctgcgccgc cagctctaaa ggaggcggcg 60

tcggccggcg ggcgagcgca cgcccaggcc caatcgatcg atcccagctc tagaggggag 120

ggagcaacca tggcggggga cggggaggac gaggtgccgg cgatgggcgg agacaagctg 180

atcctgcggg ggctgcagtt ccacggcttc cacggcgtga agcaggagga gaagaagctg 240

ggccagaagt tcgtggtcga cgtggacgcc tggatggacc tcgccgccgc cggggactcc 300

gacgacatcg cccacaccgt cagctacacc gacatctaca ggatagccaa gggcgtggtg 360

gaaggcccgt cgcggaacct cctggagtcg gtggcgcagt cgatcgccgg caccacgctg 420

ctcgagtttc cccagatctc cgccgtccgg gtgaaggtcg ggaagcccca cgtcgcggtg 480

cagggcgtcg tcgactacct cggggtggag atactgagga ggcgcagaga ggcatgagca 540

caagaaccgg agtacctcat atgagaagcc tgaacagagt tgatctcagt tgagcccatc 600

gatccctgtg tcttatatct atcaatctat gtatgtatgg acatgatgtt tgtctgcgct 660

caataatttc tgaattggga atcatgttct tgccaaaaaa aaaaaaaaaa aaaaaaaaaa 720

aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 759

6

178

Triticum aestivum

6

Ala Arg Ala Arg Phe His Ser Thr His Pro Pro Ala Pro Pro Ala Leu

1 5 10 15

Lys Glu Ala Ala Ser Ala Gly Gly Arg Ala His Ala Gln Ala Gln Ser

20 25 30

Ile Asp Pro Ser Ser Arg Gly Glu Gly Ala Thr Met Ala Gly Asp Gly

35 40 45

Glu Asp Glu Val Pro Ala Met Gly Gly Asp Lys Leu Ile Leu Arg Gly

50 55 60

Leu Gln Phe His Gly Phe His Gly Val Lys Gln Glu Glu Lys Lys Leu

65 70 75 80

Gly Gln Lys Phe Val Val Asp Val Asp Ala Trp Met Asp Leu Ala Ala

85 90 95

Ala Gly Asp Ser Asp Asp Ile Ala His Thr Val Ser Tyr Thr Asp Ile

100 105 110

Tyr Arg Ile Ala Lys Gly Val Val Glu Gly Pro Ser Arg Asn Leu Leu

115 120 125

Glu Ser Val Ala Gln Ser Ile Ala Gly Thr Thr Leu Leu Glu Phe Pro

130 135 140

Gln Ile Ser Ala Val Arg Val Lys Val Gly Lys Pro His Val Ala Val

145 150 155 160

Gln Gly Val Val Asp Tyr Leu Gly Val Glu Ile Leu Arg Arg Arg Arg

165 170 175

Glu Ala

7

1824

Zea mays

7

gcacgagcct cgaacgaggg ccgtacctag cgcctctgtc cttcgtcggc cgtcgcactg 60

tgctcccgtc cgcctccggc ctccgccaac ccgcgtccgc ccacgactag gcggctctgg 120

gcaggtcctt ccacaaagat gtgaaggatt aaagctcatg tgaaagattc taagactaca 180

attggtatca agcggttgct ttcttatttc tcatacgctc aaccatgctc ctgcatgcta 240

aggattcagt taggaagatg cattcagttg ctaagaacta ctttgtgtct gatcttactc 300

atcctccaag atccttgaac agagcttcca gacatgttgt tccattcaag acccgtttct 360

ttacgcattg ctcacttgag agccgttcag ttgaccaaga gattgtgatt gctatgggaa 420

gcaatgtagg cgatagagtc agtacattca acagggcatt gcagctgatg aaaagctctg 480

acgtgaacat cactaggcat gcctgtctct atgagaccgc ccctgcttat ttgactgatc 540

agccgcggtt tcttaactct gccattcggg gcacaactag gctcaggcca catgagcttc 600

ttaaactgct aaaggaaatt gagaaggata ttggccgcac tggcggaata aggtgcatct 660

agtgacaacg gtatcgaaac aagttggcac tctctctcaa agtgtagtgg aggtttcttt 720

gagttatgga ataaccttgg gggtgaatct ataattggaa cagaaagcat taaaagggta 780

ttacctgttg gggatcgttt gttggattgg tgtgagagga ctcttgtcat gggggtcctt 840

aatttgacac cagacagctt tagtgatgga ggtaagtttc tagaagtggg agctgccatt 900

tctcaggcta agtcattaat ctcagaaggt gcagatatca ttgatattgg tgctcaatct 960

accaggccct ttgcaaaaag attatctcca aatgaggagc ttgagaggtt ggttcctgtt 1020

ctggatgaga ttacaaaaat ccctgagatg gagggcaagt tactctcagt ggatacattc 1080

tatgcagaag ttgccagtga agctgtgaaa agaggagctc acatgatcaa cgatgtatcc 1140

agtggacagc ttgatccaat aattcttaaa gtggcagctg aacttggagt tccatatgtt 1200

gcaatgcaca tgaggggaga tccgtcaact atgcaaagcg aacaaaatgt tcactatgat 1260

aatgtctgca aggaagttgc tttggagcta tacacacagg tgagagaagc agagttatct 1320

gggattccat tgtggaggct ggttcttgat cctggcattg gcttctccaa gaaatctgaa 1380

cataaccttg aagtaattat gggattggaa tccattagga gggagatggg taaaatgagt 1440

ataggtgctt cacatgtgcc aatattactg ggaccttcaa ggaaaagctt tttgggtgaa 1500

atatgcaatc gtgccaatcc agttgagaga gatgttgcta ctgttgcagc cgtgacagct 1560

gggattttga atggtgctaa cattgtaaga gtccataatg ctggatatgg tgtagacgcc 1620

gcaaaggttt gtgatgcatt gcgtaagcgt aagggaagtt gcagaaactg aactatcgct 1680

ccagttttat acaagaaaaa agtgatgtcg aaaaatgtga tttgtgaagt atcgttgttg 1740

taatgaacca gagataatgc tttttcttgt gtcaccaagg aataaagtca agaagctgct 1800

actcaaaaaa aaaaaaaaaa aaaa 1824

8

481

Zea mays

8

Met Leu Leu His Ala Lys Asp Ser Val Arg Lys Met His Ser Val Ala

1 5 10 15

Lys Asn Tyr Phe Val Ser Asp Leu Thr His Pro Pro Arg Ser Leu Asn

20 25 30

Arg Ala Ser Arg His Val Val Pro Phe Lys Thr Arg Phe Phe Thr His

35 40 45

Cys Ser Leu Glu Ser Arg Ser Val Asp Gln Glu Ile Val Ile Ala Met

50 55 60

Gly Ser Asn Val Gly Asp Arg Val Ser Thr Phe Asn Arg Ala Leu Gln

65 70 75 80

Leu Met Lys Ser Ser Asp Val Asn Ile Thr Arg His Ala Cys Leu Tyr

85 90 95

Glu Thr Ala Pro Ala Tyr Leu Thr Asp Gln Pro Arg Phe Leu Asn Ser

100 105 110

Ala Ile Arg Gly Thr Thr Arg Leu Arg Pro His Glu Leu Leu Lys Leu

115 120 125

Leu Lys Glu Ile Glu Lys Asp Ile Gly Arg Thr Gly Gly Ile Arg Cys

130 135 140

Thr Ser Asp Asn Gly Ile Glu Thr Ser Trp His Ser Leu Ser Lys Cys

145 150 155 160

Ser Gly Gly Phe Phe Glu Leu Trp Asn Asn Leu Gly Gly Glu Ser Ile

165 170 175

Ile Gly Thr Glu Ser Ile Lys Arg Val Leu Pro Val Gly Asp Arg Leu

180 185 190

Leu Asp Trp Cys Glu Arg Thr Leu Val Met Gly Val Leu Asn Leu Thr

195 200 205

Pro Asp Ser Phe Ser Asp Gly Gly Lys Phe Leu Glu Val Gly Ala Ala

210 215 220

Ile Ser Gln Ala Lys Ser Leu Ile Ser Glu Gly Ala Asp Ile Ile Asp

225 230 235 240

Ile Gly Ala Gln Ser Thr Arg Pro Phe Ala Lys Arg Leu Ser Pro Asn

245 250 255

Glu Glu Leu Glu Arg Leu Val Pro Val Leu Asp Glu Ile Thr Lys Ile

260 265 270

Pro Glu Met Glu Gly Lys Leu Leu Ser Val Asp Thr Phe Tyr Ala Glu

275 280 285

Val Ala Ser Glu Ala Val Lys Arg Gly Ala His Met Ile Asn Asp Val

290 295 300

Ser Ser Gly Gln Leu Asp Pro Ile Ile Leu Lys Val Ala Ala Glu Leu

305 310 315 320

Gly Val Pro Tyr Val Ala Met His Met Arg Gly Asp Pro Ser Thr Met

325 330 335

Gln Ser Glu Gln Asn Val His Tyr Asp Asn Val Cys Lys Glu Val Ala

340 345 350

Leu Glu Leu Tyr Thr Gln Val Arg Glu Ala Glu Leu Ser Gly Ile Pro

355 360 365

Leu Trp Arg Leu Val Leu Asp Pro Gly Ile Gly Phe Ser Lys Lys Ser

370 375 380

Glu His Asn Leu Glu Val Ile Met Gly Leu Glu Ser Ile Arg Arg Glu

385 390 395 400

Met Gly Lys Met Ser Ile Gly Ala Ser His Val Pro Ile Leu Leu Gly

405 410 415

Pro Ser Arg Lys Ser Phe Leu Gly Glu Ile Cys Asn Arg Ala Asn Pro

420 425 430

Val Glu Arg Asp Val Ala Thr Val Ala Ala Val Thr Ala Gly Ile Leu

435 440 445

Asn Gly Ala Asn Ile Val Arg Val His Asn Ala Gly Tyr Gly Val Asp

450 455 460

Ala Ala Lys Val Cys Asp Ala Leu Arg Lys Arg Lys Gly Ser Cys Arg

465 470 475 480

Asn

9

589

Oryza sativa

9

gcacgagctt acagtttagg tgcttcacat gtgccaattt tacttggacc ctcaaggaaa 60

agatttttag gtgaaatatg caatcgtgtc aatcccactg agagagatgc tgctaccatg 120

gtcgttgcta ctgctgggat attgaatggt gctaatatag taagggtgca taatgttaaa 180

tatggcgtgg atactgcaaa ggtctctgat gcattgagca aaggcagaag atgattatac 240

caccttcgga aaatagatca tactccagtt ttgtactaga aaataatgat caataatagt 300

aactcggcca taatgttggc ttctcagata ataccatagg gcgagtatca tcatagaaag 360

catgtgcaca caactgttat gtgagcttga gatggaattt ttctttttgt cacatcattt 420

caataatctt ctgaggtaac ggttatacag atctctagag ttttgacctt tcaggattca 480

caaattttct acaggtctga tttgtttgga actttgggcc ataacttgaa gttattctcc 540

atgtaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 589

10

75

Oryza sativa

10

Ala Arg Ala Tyr Ser Leu Gly Ala Ser His Val Pro Ile Leu Leu Gly

1 5 10 15

Pro Ser Arg Lys Arg Phe Leu Gly Glu Ile Cys Asn Arg Val Asn Pro

20 25 30

Thr Glu Arg Asp Ala Ala Thr Met Val Val Ala Thr Ala Gly Ile Leu

35 40 45

Asn Gly Ala Asn Ile Val Arg Val His Asn Val Lys Tyr Gly Val Asp

50 55 60

Thr Ala Lys Val Ser Asp Ala Leu Ser Lys Gly

65 70 75

11

cttgactggt cgcggagaac ttccgtcatg gggatcctta atgtgactcc agatagtttt 60

agtgatgggg gaaatttcaa gtctgtggag tctgctgttt atcaggttcg gttaatgatt 120

tcagaaggag cagatatgat tgatatcggg gctcagtcta ctcggccaac ggcctctagg 180

atctctgctg cagaagaatt aggtagatta atccctgtcc tggaagctgt agtgtcaatg 240

cctgaggtag aaggaaagct catttctgtg gatactttct actctgaagt tgcttcacaa 300

gcagtgagta aaggggctca tcttataaat gatgtatcct gcctggacag ttggatagta 360

acatgtttaa agtccnnggg ctggatcttg atgttcttaa tgttgcaaat ggcacaatga 420

nggggggaac catccttaca nnngcaagaa taagtngnaa antctgnaaa tannggnaat 480

tgttntgtta naa 493

12

Leu Asp Trp Ser Arg Arg Thr Ser Val Met Gly Ile Leu Asn Val Thr

1 5 10 15

Pro Asp Ser Phe Ser Asp Gly Gly Asn Phe Lys Ser Val Glu Ser Ala

20 25 30

Val Tyr Gln Val Arg Leu Met Ile Ser Glu Gly Ala Asp Met Ile Asp

35 40 45

Ile Gly Ala Gln Ser Thr Arg Pro Thr Ala Ser Arg Ile Ser Ala Ala

50 55 60

Glu Glu Leu Gly Arg Leu Ile Pro Val Leu Glu Ala Val Val Ser Met

65 70 75 80

Pro Glu Val Glu Gly Lys Leu Ile Ser Val Asp Thr Phe Tyr Ser Glu

85 90 95

Val Ala Ser Gln Ala Val Ser Lys Gly Ala His Leu Ile Asn Asp Val

100 105 110

Ser Cys Leu Asp Ser Trp Ile Val Thr Cys Leu Lys Ser Xaa Gly Trp

115 120 125

Ile Leu Met Phe Leu Met Leu Gln Met Ala Gln

130 135

13

2041

Zea mays

13

gcacgagccc tcgcctgctc cacgacgctt atgcgctcgc gtcccgctct cgccgcccac 60

ctccggcgcc tgctcctcct ctctccctcc gcccacctca tcatcatccg ccgcgccatg 120

gcatccgccg ccgccgcgca ggcgcagcca ggtggcgccc cgccggcgac cgcggagtac 180

gaggaggtgc tggggcggct ctcctcgctc atcacgcaga aggtgcgcgc gaacagcgcc 240

aaccgcggca accagtggga cctcatggag cactacgtca agattctgga gctggaggag 300

tcgatcgcgc ggatgaaagt gattcacgtc gcggggacca aggggaaggg ttccacatgc 360

acattcaccg agtcaatcct gcgatcgtgt ggcttccata ctgggctgtt cacctcacca 420

catttgatgg atgttagaga gcgatttcag ctagatgggg ttaatatttc tgaagagaaa 480

tttttgaagt acttctggtg gtgctggaac aagttgaagg agaagactga tgatgatatt 540

cccatgccag cctatttcag gttcctcgcg ttgctcgcat tcaagatatt ttctgctgag 600

caggtagatg ttgctgttct cgaggttggc ctaggaggga agtttgatgc aactaatgtg 660

gttaaagcac ctgtagtttg tggcatatct tcccttggat atgatcatat ggaaattctt 720

gggaatacac ttggagaaat cgcaggagag aaggctggga ttttcaagaa aggagttccg 780

gcctatactg ctccacaacc agaagaggca atgactgctc tcaaacaaag agcttcggaa 840

ttgggtatct ctctccaagt cgttgatcct ttggagcccc atcacctaaa agatcagcat 900

cttgggctgc atggagaaca tcaatatata aatgctggcc ttgcagttgc tttggctagt 960

acgtggcttg agaagcaggg acataaagat acgttaccac tcaatcgtac tgatccctta 1020

ccagatcatt ttattagagg gctatcaagt gcttctttgc aaggccgagc acagattgtt 1080

ccagattcac aagtgaattc agaagagaag gacaaaaatt gttctcttgt tttttatttg 1140

gatggagcgc acagtcctga aagcatggaa gtatgtggca agtggttttc ccatgtcaca 1200

aaggatgata caaggctacc atcttctgtg gagcagtctc atacatctat gtctcaaaag 1260

atccttctgt tcaactgcat gtctgtgaga gatccgatga gattgcttcc ttgtctcctg 1320

gatgcatcaa ctcaaaatgg agtccacttt gacctggccc tatttgtgcc gaaccaatca 1380

caacacacga agcttggttc taacacttca gcaccagcgg agcctgagca aatcgatttg 1440

tcatggcagc tgtcacttca aacagtgtgg gagaagttac ttcaggataa aggtataaat 1500

actacaaaat ccagtgatac tagtaaagtt tttgattcgc ttccaatcgc aatcgagtgg 1560

ctaaggagaa atgcccgaga aaaccaatct acttctttcc aggtgctggt tactggctcc 1620

ctgcatctcg ttggggatgt cttgagaata atcaagaagt gatacgccgc ctcgaaatcc 1680

aaactggaac tggactatga tctatggtct ctcccaaggc taacatgatt agcaagggga 1740

gacatttgaa cggtgcttgc ttattggtgc caaccaagct gcgagcttct tgtgtttttt 1800

ttgtgtggcc acggtcgcct gcctaccact cgggaaaccg ccgcgcccgt tcttgtgaag 1860

gcatgaaata ggatgatcgt gccaccatag aacataactg ggaaatgaat tcgacatgga 1920

actgggacag tctgtatact cacaaaataa gatcgcatgg ggttttcttg ttcaagtgca 1980

aagaaaccag tcaattctta tccagagtag caagaattca ttcaaaaaaa aaaaaaaaaa 2040

a 2041

14

553

Zea mays

14

Ala Arg Ala Leu Ala Cys Ser Thr Thr Leu Met Arg Ser Arg Pro Ala

1 5 10 15

Leu Ala Ala His Leu Arg Arg Leu Leu Leu Leu Ser Pro Ser Ala His

20 25 30

Leu Ile Ile Ile Arg Arg Ala Met Ala Ser Ala Ala Ala Ala Gln Ala

35 40 45

Gln Pro Gly Gly Ala Pro Pro Ala Thr Ala Glu Tyr Glu Glu Val Leu

50 55 60

Gly Arg Leu Ser Ser Leu Ile Thr Gln Lys Val Arg Ala Asn Ser Ala

65 70 75 80

Asn Arg Gly Asn Gln Trp Asp Leu Met Glu His Tyr Val Lys Ile Leu

85 90 95

Glu Leu Glu Glu Ser Ile Ala Arg Met Lys Val Ile His Val Ala Gly

100 105 110

Thr Lys Gly Lys Gly Ser Thr Cys Thr Phe Thr Glu Ser Ile Leu Arg

115 120 125

Ser Cys Gly Phe His Thr Gly Leu Phe Thr Ser Pro His Leu Met Asp

130 135 140

Val Arg Glu Arg Phe Gln Leu Asp Gly Val Asn Ile Ser Glu Glu Lys

145 150 155 160

Phe Leu Lys Tyr Phe Trp Trp Cys Trp Asn Lys Leu Lys Glu Lys Thr

165 170 175

Asp Asp Asp Ile Pro Met Pro Ala Tyr Phe Arg Phe Leu Ala Leu Leu

180 185 190

Ala Phe Lys Ile Phe Ser Ala Glu Gln Val Asp Val Ala Val Leu Glu

195 200 205

Val Gly Leu Gly Gly Lys Phe Asp Ala Thr Asn Val Val Lys Ala Pro

210 215 220

Val Val Cys Gly Ile Ser Ser Leu Gly Tyr Asp His Met Glu Ile Leu

225 230 235 240

Gly Asn Thr Leu Gly Glu Ile Ala Gly Glu Lys Ala Gly Ile Phe Lys

245 250 255

Lys Gly Val Pro Ala Tyr Thr Ala Pro Gln Pro Glu Glu Ala Met Thr

260 265 270

Ala Leu Lys Gln Arg Ala Ser Glu Leu Gly Ile Ser Leu Gln Val Val

275 280 285

Asp Pro Leu Glu Pro His His Leu Lys Asp Gln His Leu Gly Leu His

290 295 300

Gly Glu His Gln Tyr Ile Asn Ala Gly Leu Ala Val Ala Leu Ala Ser

305 310 315 320

Thr Trp Leu Glu Lys Gln Gly His Lys Asp Thr Leu Pro Leu Asn Arg

325 330 335

Thr Asp Pro Leu Pro Asp His Phe Ile Arg Gly Leu Ser Ser Ala Ser

340 345 350

Leu Gln Gly Arg Ala Gln Ile Val Pro Asp Ser Gln Val Asn Ser Glu

355 360 365

Glu Lys Asp Lys Asn Cys Ser Leu Val Phe Tyr Leu Asp Gly Ala His

370 375 380

Ser Pro Glu Ser Met Glu Val Cys Gly Lys Trp Phe Ser His Val Thr

385 390 395 400

Lys Asp Asp Thr Arg Leu Pro Ser Ser Val Glu Gln Ser His Thr Ser

405 410 415

Met Ser Gln Lys Ile Leu Leu Phe Asn Cys Met Ser Val Arg Asp Pro

420 425 430

Met Arg Leu Leu Pro Cys Leu Leu Asp Ala Ser Thr Gln Asn Gly Val

435 440 445

His Phe Asp Leu Ala Leu Phe Val Pro Asn Gln Ser Gln His Thr Lys

450 455 460

Leu Gly Ser Asn Thr Ser Ala Pro Ala Glu Pro Glu Gln Ile Asp Leu

465 470 475 480

Ser Trp Gln Leu Ser Leu Gln Thr Val Trp Glu Lys Leu Leu Gln Asp

485 490 495

Lys Gly Ile Asn Thr Thr Lys Ser Ser Asp Thr Ser Lys Val Phe Asp

500 505 510

Ser Leu Pro Ile Ala Ile Glu Trp Leu Arg Arg Asn Ala Arg Glu Asn

515 520 525

Gln Ser Thr Ser Phe Gln Val Leu Val Thr Gly Ser Leu His Leu Val

530 535 540

Gly Asp Val Leu Arg Ile Ile Lys Lys

545 550

15

catggctncc aaannttcgg cactanacgt actgagaaga gctcgcgtnc ccgctactcg 60

ccggcccacc tccgggcgcc tgctcctcct ctctccctcc gcccacctca tcatcatccg 120

ccgcgccatg gcctccgccg ccgccgcgca ggcgcagcag gtggcgcccc accggcgacc 180

gnggagtang aggaggtgct ggggcggctc tcctcgctca tcacgcagaa ggtgcgcgcg 240

aacagcgcca accgcggcaa ccagtgggac ntcatggagc actangtcaa gattctggag 300

ctggaggagt cgatcgcgcg nnatgaaagt gattcacgtc gnagggacca aggggaaggg 360

ttccacatgc acattcaccg agtcaatcct gcgatcgtgt ggcttccata atnggctgtt 420

taactcacca acatttgatt ggatgttaga gagcgaattc agctagattg gggttaataa 480

tttctgaaga gaaatttttn aagtactctn gntgtgntgg aacaagttna agga 534

16

Met Ala Xaa Lys Xaa Ser Ala Leu Xaa Val Leu Arg Arg Ala Arg Val

1 5 10 15

Pro Ala Thr Arg Arg Pro Thr Ser Gly Arg Leu Leu Leu Leu Ser Pro

20 25 30

Ser Ala His Leu Ile Ile Ile Arg Arg Ala Met Ala Ser Ala Ala Ala

35 40 45

Ala Gln Ala Gln Gln Val Ala Pro His Arg Arg Pro Xaa Ser Xaa Arg

50 55 60

Arg Cys Trp Gly Gly Ser Pro Arg Ser Ser Arg Arg Arg Cys Ala Arg

65 70 75 80

Thr Ala Pro Thr Ala Ala Thr Ser Gly Thr Ser Trp Ser Thr Xaa Ser

85 90 95

Arg Phe Trp Ser Trp Arg Ser Arg Ser Arg Xaa Met Lys Val Ile His

100 105 110

Val Xaa Gly Thr Lys Gly Lys Gly Ser Thr Cys Thr Phe Thr Glu Ser

115 120 125

Ile Leu Arg Ser Cys Gly Phe His Asn Xaa Leu Phe Asn Ser Pro Thr

130 135 140

Phe Asp Trp Met Leu Glu Ser Glu Phe Ser

145 150

17

ggcttctcag ttaaatgtac ctcttcaagt ggtaacccca ttagatgcca aattgctaaa 60

tggttcaaga ctagcgcttg gaggtgaaca ccaatatata aatgctggtc ttgctattgc 120

attatgctct acgtggctga aaatgaatgg gcatcttgaa gactcgtact tgaaacatat 180

acaacacact ttaccagaga agttcataaa agggttaaca actgcaagtt tgcaaggaag 240

ggctcagatt gttcctgatc agttcatcaa tgatgaaata ccaaatgaac ttgtcttctt 300

tttagatggg gctcatagtc ctgaaagcat ggaagcatgt gccaggtggt tttctcttgc 360

tattaaagat caagaccaga ttttgtttca tcaagaaact tgataattct aacttctcaa 420

accaagtagt gaagatgcac aatggtgaaa ctgtacagaa gaaatccaca cagattttgc 480

tgttcaattg tatgtctgag cgaaaccctc aattgcttcn tccccacttg atgaaaacat 540

gtgctgatna agg 553

18

133

Glycine max

18

Ala Ser Gln Leu Asn Val Pro Leu Gln Val Val Thr Pro Leu Asp Ala

1 5 10 15

Lys Leu Leu Asn Gly Ser Arg Leu Ala Leu Gly Gly Glu His Gln Tyr

20 25 30

Ile Asn Ala Gly Leu Ala Ile Ala Leu Cys Ser Thr Trp Leu Lys Met

35 40 45

Asn Gly His Leu Glu Asp Ser Tyr Leu Lys His Ile Gln His Thr Leu

50 55 60

Pro Glu Lys Phe Ile Lys Gly Leu Thr Thr Ala Ser Leu Gln Gly Arg

65 70 75 80

Ala Gln Ile Val Pro Asp Gln Phe Ile Asn Asp Glu Ile Pro Asn Glu

85 90 95

Leu Val Phe Phe Leu Asp Gly Ala His Ser Pro Glu Ser Met Glu Ala

100 105 110

Cys Ala Arg Trp Phe Ser Leu Ala Ile Lys Asp Gln Asp Gln Ile Leu

115 120 125

Phe His Gln Glu Thr

130

19

ggcggcggct acgtggggtg gcgacgacaa gctcattctg cgcggccttc agttccatgg 60

cttccacggt gtcctgcagg aggagaagac gttgggacag aagttcgtgg ttgacatcga 120

cgcctggata gacctcgccg ctgccggcga agtccgactg cattgctgac accgtcagct 180

acaccgatat ctacagcatt gcaaaggatg ttgtcgaggg cacgccacgc aacctcttgg 240

agtcggtagc tcactcnatc gcagaggcca cgctgctcaa gttccctcaa atctccgcag 300

tccgagtgaa ggttggcaag cctcacctcg cggtgcgagg cgttctggac taactgggcg 360

tggggataac naggcacaaa aagaaagaat tgagattctg tncacatgtg gtgatggggg 420

aaccagttca atnctgatgg nactgcnggc aanaccataa tccacccncc ccntgttgcn 480

tgntgggaac taagcnantn cctttcacct ctgaactgnt gggaatatcg ggnaatctng 540

ttcccctaaa ttgctttatt acna 564

20

116

Zea mays

20

Asp Lys Leu Ile Leu Arg Gly Leu Gln Phe His Gly Phe His Gly Val

1 5 10 15

Leu Gln Glu Glu Lys Thr Leu Gly Gln Lys Phe Val Val Asp Ile Asp

20 25 30

Ala Trp Thr Ser Pro Leu Pro Ala Lys Ser Asp Cys Ile Ala Asp Thr

35 40 45

Val Ser Tyr Thr Asp Ile Tyr Ser Ile Ala Lys Asp Val Val Glu Gly

50 55 60

Thr Pro Arg Asn Leu Leu Glu Ser Val Ala His Ser Ile Ala Glu Ala

65 70 75 80

Thr Leu Leu Lys Phe Pro Gln Ile Ser Ala Val Arg Val Lys Val Gly

85 90 95

Lys Pro His Leu Ala Val Arg Gly Val Leu Asp Leu Gly Val Gly Ile

100 105 110

Thr Arg His Lys

115

21

cggagaggcg agggagtgag ggactagcac agaaagatat tgtttggtgt acggtggtga 60

gtgtcgacgc tgccactctc gcctgtgtct gtgataaatg gaatctgatg caccgacatg 120

gggagacaaa ctcatgttga ggggattgtc attccatggt tttcatggag caaagcctga 180

agaaaggaca ctgggccaga agttcttcat agatatagat gcttggatgg atctcaaagc 240

agctgggcaa atctgatcac ttatcaaatt ctgttagtta cacagaaata tatgatatag 300

ctaaggatgt tcttgaaggg tcacctcaca atcctctggg agtcaagtgg gccaaaaaaa 360

ttgcaatcac tactcttaca aatcaaaaag aaatatctgc tgtccnagtg aanggtggga 420

aaccccatgt ggcaattccg ggtccaattn attacttaag cgtttgagaa tcctaaacnc 480

aaaaaccaac ntnttcaagg ctaaaaaatt taanatttan tgctgcacaa attttatant 540

ttcaaaatcc accttgatac aaaaagtaaa ggtactccnt tcccntcaag gccccaatta 600

g 601

22

Asp Lys Leu Met Leu Arg Gly Leu Ser Phe His Gly Phe His Gly Ala

1 5 10 15

Lys Pro Glu Glu Arg Thr Leu Gly Gln Lys Phe Phe Ile Asp Ile Asp

20 25 30

Ala Trp Met Asp Leu Lys Ala Ala Gly Gln Xaa Xaa His Leu Ser Asn

35 40 45

Ser Val Ser Tyr Thr Glu Ile Tyr Asp Ile Ala Lys Asp Val Leu Glu

50 55 60

Gly Ser Pro

65

23

439

Triticum aestivum

23

ccaggttcca ctccacccac ccacctgcgc cgccagctct aaaggaggcg gcgtcggccg 60

gcgggcgagc gcacgcccag gcccaatcga tcgatcccag ctctagaggg gagggagcaa 120

ccatggcggg ggacggggag gacgaggtgc cggcgatggg cggagacaag ctgatcctgc 180

gggggctgca gttccacggc ttccacggcg tgaagcagga ggagaagaag ctgggccaga 240

agttcgtggt cgacgtggac gcctggatgg acctcgccgc cgccggggac tccgacgaca 300

tcgcccacac cgtcagctac accgacatct acaggatagc caagggcgtg gtggaaggcc 360

cgtcgcggaa acctcctgga gtcggtggcg cagtcgatcg ccggcaacaa cgctgctccg 420

aagtttcccc aaatctccg 439

24

65

Triticum aestivum

24

Asp Lys Leu Ile Leu Arg Gly Leu Gln Phe His Gly Phe His Gly Val

1 5 10 15

Lys Gln Glu Glu Lys Lys Leu Gly Gln Lys Phe Val Val Asp Val Asp

20 25 30

Ala Trp Met Asp Leu Ala Ala Ala Gly Asp Ser Asp Asp Ile Ala His

35 40 45

Thr Val Ser Tyr Thr Asp Ile Tyr Arg Ile Ala Lys Gly Val Val Glu

50 55 60

Gly

65

25

cctcgaacga gggccgtacc tagcgcctct gtccttcgtc ggccgtcgca ctgtgctccc 60

gtccgcctcc ggcctccgcc aacccgcgtc cgcccacgac taggcggctc tgggcaggtc 120

cttccacaaa gatgtgaagg attaaagctc atgtgaaaga ttctaagact acaattggta 180

tcaagcggtt gctttcttat ttctcatacg ctcaaccatg ctcctgcatg ctaaggattc 240

agttaggaag atgcattcag ttgctaagaa ctactttgtg tctgatctta ctcatcctcc 300

aagatccttg aacagagctt ccagacatgt tgttccattc aagacccgtt tctttacgca 360

ttgctcactt gagagccgtt cagttgacca agagattgtg attgctatgg gaagcaatgt 420

aggcgataga gtcagtacat tcaacagggc attgcagctg atgaaaagct cagacgtgaa 480

catcactagg catgcctgtc tctatgaaac cgcccctgct tatttgactg atcagccacg 540

gtttcttaac tctgccattc ggggnacaac taggctccag gccacatgag cttcttaaac 600

tggctaaagg aaattgagaa gggaattggc cgcactgggg ganataaagg tacggnccaa 660

gacctatcga ttaagna 677

26

Ser Leu Glu Ser Arg Ser Val Asp Gln Glu Ile Val Ile Ala Met Gly

1 5 10 15

Ser Asn Val Gly Asp Arg Val Ser Thr Phe Asn Arg Ala Leu Gln Leu

20 25 30

Met Lys Ser Ser Asp Val Asn Ile Thr Arg His Ala Cys Leu Tyr Glu

35 40 45

Thr Ala Pro Ala Tyr Leu Thr Asp Gln Pro Arg Phe Leu Asn Ser Ala

50 55 60

Ile Arg Gly Thr Xaa Ala Pro Gly His Met Ser Phe Leu Asn Trp Leu

65 70 75 80

Lys Glu Ile Glu Lys Gly Ile Gly Arg Thr Gly Xaa Xaa Arg Tyr Gly

85 90 95

Pro Arg Pro Ile Asp

100

27

cttacagttt aggtgcttca catgtgccaa ttttacttgg accctcaagg aaaagatttt 60

taggtgaaat atgcaatcgt gtcaatccca ctgagagaga tgctgctacc atggtcgttg 120

ctacngctgg gatattgaat ggtgctaata tagtaagggt gcataatgtt aaatanggct 180

nggatacngn aaaggtctct aatcctttgc caaaggggan angtgtt 227

28

Ser Leu Gly Ala Ser His Val Pro Ile Leu Leu Gly Pro Ser Arg Lys

1 5 10 15

Arg Phe Leu Gly Glu Ile Cys Asn Arg Val Asn Pro Thr Glu Arg Asp

20 25 30

Ala Ala Thr Met Val Val Ala Thr Ala Gly Ile Leu Asn Gly Ala Asn

35 40 45

Ile Val Arg Val His Asn Val Lys Xaa Gly Xaa Asp Thr Xaa Lys Val

50 55 60

Ser Asn Pro Leu Pro Lys

65 70

29

catggctncc aaannttcgg cactanacgt actgagaaga gctcgcgtnc ccgctactcg 60

ccggcccacc tccgggcgcc tgctcctcct ctctccctcc gcccacctca tcatcatccg 120

ccgcgccatg gcctccgccg ccgccgcgca ggcgcagcag gtggcgcccc accggcgacc 180

gnggagtang aggaggtgct ggggcggctc tcctcgctca tcacgcagaa ggtgcgcgcg 240

aacagcgcca accgcggcaa ccagtgggac ntcatggagc actangtcaa gattctggag 300

ctggaggagt cgatcgcgcg nnatgaaagt gattcacgtc gnagggacca aggggaaggg 360

ttccacatgc acattcaccg agtcaatcct gcgatcgtgt ggcttccata atnggctgtt 420

taactcacca acatttgatt ggatgttaga gagcgaattc agctagattg gggttaataa 480

tttctgaaga gaaatttttn aagtactctn gntgtgntgg aacaagttna agga 534

30

Met Lys Val Ile His Val Xaa Gly Thr Lys Gly Lys Gly Ser Thr Cys

1 5 10 15

Thr Phe Thr Glu Ser Ile Leu Arg Ser Cys Gly Phe His Asn Xaa Leu

20 25 30

Phe Asn Ser Pro

35

31

ggcttctcag ttaaatgtac ctcttcaagt ggtaacccca ttagatgcca aattgctaaa 60

tggttcaaga ctagcgcttg gaggtgaaca ccaatatata aatgctggtc ttgctattgc 120

attatgctct acgtggctga aaatgaatgg gcatcttgaa gactcgtact tgaaacatat 180

acaacacact ttaccagaga agttcataaa agggttaaca actgcaagtt tgcaaggaag 240

ggctcagatt gttcctgatc agttcatcaa tgatgaaata ccaaatgaac ttgtcttctt 300

tttagatggg gctcatagtc ctgaaagcat ggaagcatgt gccaggtggt tttctcttgc 360

tattaaagat caagaccaga ttttgtttca tcaagaaact tgataattct aacttctcaa 420

accaagtagt gaagatgcac aatggtgaaa ctgtacagaa gaaatccaca cagattttgc 480

tgttcaattg tatgtctgag cgaaaccctc aattgcttcn tccccacttg atgaaaacat 540

gtgctgatna agg 553

32

24

Glycine max

32

Leu Ala Leu Gly Gly Glu His Gln Tyr Ile Asn Ala Gly Leu Ala Ile

1 5 10 15

Ala Leu Cys Ser Thr Trp Leu Lys

20