MAIZE MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN POLYNUCLEOTIDES AND METHODS OF USE

Abstract

Compositions and methods are provided for modulating the level of phytate in plants. More specifically, the invention relates to methods of modulating the level of phytate utilizing nucleic acids comprising multidrug resistance-associated protein (MRP) nucleotide sequences to modulate the expression of MRP(s) in a plant of interest. The compositions and methods of the invention find use in agriculture for improving the nutritional quality of food and feed by reducing the levels of phytate and/or increasing the levels of non-phytate phosphorus in food and feed. The invention also finds use in reducing the environmental impact of animal waste.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 12/371,209, filed Feb. 13, 2009, which is a continuation of U.S. application Ser. No. 11/133,075, filed May 19, 2005, which claims the benefit of U.S. Provisional Application No. 60/572,704, filed May 20, 2004, the contents of which are hereby incorporated by reference in their entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

[0002] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 411724SEQLIST.TXT, created on Nov. 11, 2011, and having a size of 199 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.

FIELD OF THE INVENTION

[0003] The present invention relates to the field of animal nutrition. Specifically, the present invention relates to the identification and use of genes encoding enzymes involved in the metabolism of phytate in plants and the use of these genes and mutants thereof to reduce the levels of phytate, and/or increase the levels of non-phytate phosphorus in food or feed.

BACKGROUND OF THE INVENTION

[0004] The role of phosphorous in animal nutrition is well recognized. Phosphorus is a critical component of the skeleton, nucleic acids, cell membranes and some vitamins. Though phosphorous is essential for the health of animals, not all phosphorous in feed is bioavailable.

[0005] Phytates are the major form of phosphorous in seeds. For example, phytate represents about 60-80% of total phosphorous in corn and soybean. When seed-based diets are fed to non-ruminants, the consumed phytic acid forms salts with several important mineral nutrients, such as potassium, calcium, and iron, and also binds proteins in the intestinal tract. These phytate complexes cannot be metabolized by monogastric animals and are excreted, effectively acting as anti-nutritional factors by reducing the bioavailability of dietary phosphorous and minerals. Phytate-bound phosphorous in animal excreta also has a negative environmental impact, contributing to surface and ground water pollution.

[0006] There have been two major approaches to reducing the negative nutritional and environmental impacts of phytate in seed. The first involves post-harvest interventions, which increase the cost and processing time of feed. Post-harvest processing technologies remove phytic acid by fermentation or by the addition of compounds, such as phytases.

[0007] The second is a genetic approach. One genetic approach involves developing crop germplasm with heritable reductions in seed phytic acid. While some variability for phytic acid was observed, there was no change in non-phytate phosphorous. Further, only 2% of the observed variation in phytic acid was heritable, whereas 98% of the variation was attributed to environmental factors. Another genetic approach involves selecting low phytate lines from a mutagenized population to produce germplasm. Most mutant lines exhibit a loss of function and are presumably blocked in the phytic acid biosynthetic pathway; therefore, low phytic acid accumulation will likely be a recessive trait. In certain cases, this approach has revealed that homozygosity for substantially reduced phytate can be lethal. Another genetic approach is transgenic technology, which has been used to increase phytase levels in plants. These transgenic plant tissues or seed have been used as dietary supplements.

[0008] The biosynthetic route leading to phytate is complex and not completely understood, and it has been proposed that the production of phytic acid occurs by one of two possible pathways. One possible pathway involves the sequential phosphorylation of Ins(3)P or myo-inositol, leading to the production of phytic acid. Another possible pathway involves hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C, followed by the phosphorylation of Ins(1,4,5)P₃ by inositol phosphate kinases. In developing plant seeds, accumulating evidence favors the sequential phosphorylation pathway. Such evidence includes studies of the Lpa2 gene, a gene encoding a maize inositol phosphate kinase which has multiple kinase activities. The Lpa2 gene has been cloned, and the lpa2 mutation has been shown to impair phytic acid synthesis. Mutant lpa2 seeds accumulate myo-inositol and inositol phosphate intermediates.

[0009] The maize low phytic acid 1 mutant (lpa1) was isolated from an EMS-mutagenized population in the early 1990s by USDA scientists. However, the original lpal-1 allele was previously known to have a phenotype of up to 15% loss of seed dry weight, which could translate into a yield drag if the lpal-1 mutant was used in product development. Since the discovery of lpa1, the gene responsible for the lpa1 mutation has been sought for two reasons: 1) the mutant has a phenotype of low phytic acid and high available phosphorus in grain which makes it useful in animal feeding and phosphorus waste management; and 2) the lpa1 mutant does not accumulate myo-inositol phosphate intermediates, indicating that mutation in this locus impairs a critical step in the phytic acid biosynthesis pathway which was previously uncharacterized.

[0010] Based on the foregoing, there exists the need to improve the nutritional content of plants, particularly corn and soybean, by increasing non-phytate phosphorous and reducing seed phytate. Accordingly, it is desirable to isolate and characterize the Lpa1 gene in order to place the expression of this gene under tight control so as to produce plants which have reduced seed phytate and increased non-phytate phosphorus.

SUMMARY OF THE INVENTION

[0011] Compositions and methods are provided for modulating the level of phytate in plants. More specifically, the invention relates to methods of modulating the level of phytate utilizing Lpa1 (ZmMRP3) nucleic acids to produce transformed plants that exhibit decreased expression of at least one multidrug resistance-associated protein (MRP). The compositions and methods of the invention find use in agriculture for improving the nutritional quality of food and feed by reducing the levels of phytate and/or increasing the levels of non-phytate phosphorus in food and feed. Thus, the invention finds use in producing food and feed products as well as in reducing the environmental impact of animal waste. Also provided are compositions and methods for producing MRP proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGS. 1A and 1B: Alignment of ZmMRP3 (SEQ ID NO: 3) with Pfam consensus sequences for ABC transporter ("ABC_tran"; SEQ ID NO: 62) and ABC transporter transmembrane ("ABC_membrane"; SEQ ID NO: 63) region.

[0013] FIG. 2: Diagram of ZmMRP3 and rice OsMRP13 gene structure.

[0014] FIG. 3: Phylogenetic comparison of maize, rice and Arabidopsis MRP genes, showing that maize ZmMRP3, rice OsMRP13 and Arabidopsis AtMRP5 are closely related.

[0015] FIG. 4A, 4B, 4C, 4D, 4E: cDNA sequence alignment of the maize Lpa1 gene (SEQ ID NO: 2) and its rice homolog OsMRP13 (SEQ ID NO: 6).

[0016] FIG. 5A, 5B, 5C: Protein Sequence alignment of maize Lpa1 (ZmMRP3; SEQ ID NO: 3) with rice and Arabidopsis homologs OsMRP13 (SEQ ID NO: 7) and AtMRP5 (SEQ ID NO: 9). Matches to the consensus are indicated by bold type; conservative changes are indicated by underlined text.

[0017] FIG. 6: Diagram of sample constructs. These sample constructs illustrate various configurations that can be used in expression cassettes for use in inhibition of expression, for example, for use in hairpin RNA interference. Sample construct 1 shows a single promoter and fully or partially complementary sequences of "region 1" and "region 2." Sample construct 2 illustrates a configuration of two sets of fully or partially complementary sequences. In this sample construct, "region 1" is fully or partially complementary to "region 2" and "region 3" is fully or partially complementary to "region 4." Sample construct 3 illustrates yet another configuration of two sets of fully or partially complementary sequences; here, too, "region 1" is fully or partially complementary to "region 2" and "region 3" is fully or partially complementary to "region 4."

DETAILED DESCRIPTION OF THE INVENTION

[0018] The invention is drawn to compositions and methods for modulating the level of phytate in plants. Compositions of the invention comprise multidrug resistance-associated proteins ("MRPs") of the invention (i.e., proteins that have multidrug resistance-associated protein activity ("MRP activity")), polynucleotides that encode them, and associated noncoding regions as well as fragments and variants of the exemplary disclosed sequences. For example, the disclosed Lpa1 polypeptides having amino acid sequences set forth in SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15 are MRPs and therefore have multidrug resistance-associated protein ("MRP") activity. In particular, the present invention provides for isolated polynucleotides comprising nucleotide sequences set forth in SEQ ID NOs: 1, 2, 4, 6, 8, 10, 12, and 14, or encoding the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15, and fragments and variants thereof. In addition, the invention provides polynucleotides comprising the complements of these nucleotide sequences. Also provided are polypeptides comprising the amino acid sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, and 15, polypeptides comprising the conserved domains set forth in SEQ ID NOs: 16, 17, 18, 19, 20, 21, 22, 23, and 24, fragments and variants thereof, and nucleotide sequences encoding these polypeptides. Compositions of the invention also include polynucleotides comprising at least a portion of the promoter sequence set forth in nucleotides 1 to 3134 of SEQ ID NO: 1 as well as polynucleotides comprising other noncoding regions

[0019] Thus, the compositions of the invention comprise isolated nucleic acids that encode MRP proteins (e.g., Lpa1), fragments and variants thereof, cassettes comprising polynucleotides of the invention, and isolated MRP proteins. The compositions also include nucleic acids comprising nucleotide sequences which are the complement, or antisense, of these MRP nucleotide sequences. The invention further provides plants and microorganisms transformed with these novel nucleic acids as well as methods involving the use of such nucleic acids, proteins, and transformed plants in producing food (including food products) and feed with reduced phytate and/or increased non-phytate phosphorus levels. In some embodiments, the transformed plants of the invention and food and feed produced therefrom have improved nutritional quality due to increased availability (bioavailability) of nutrients including, for example, zinc and iron.

[0020] In some embodiments, MRP activity is reduced or eliminated by transforming a maize plant cell with an expression cassette that expresses a polynucleotide that inhibits the expression of an MRP enzyme such as, for example, an Lpa1 polypeptide. The polynucleotide may inhibit the expression of one or more MRPs directly, by preventing translation of the MRP messenger RNA, or indirectly, by encoding a polypeptide that inhibits the transcription or translation of a maize gene encoding an MRP. Methods for inhibiting or eliminating the expression of a gene in a plant are well known in the art, and any such method may be used in the present invention to inhibit the expression of one or more maize MRPs. Because MRP activity is difficult to measure directly, a decrease in MRP activity can be measured by a decreased level of phytate in a plant or plant part. See, e.g., the working examples in the Experimental section.

[0021] In accordance with the present invention, the expression of an MRP protein is inhibited if the transcript or protein level of the MRP is statistically lower than the transcript or protein level of the same MRP in a plant that has not been genetically modified or mutagenized to inhibit the expression of that MRP. In particular embodiments of the invention, the transcript or protein level of the MRP in a modified plant according to the invention is less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of the protein level of the same MRP in a plant that is not a mutant or that has not been genetically modified to inhibit the expression of that MRP. The expression level of the MRP may be measured directly, for example, by assaying for the level of MRP expressed in the cell or plant, or indirectly, for example, by measuring the amount of phytate in the cell or plant. The activity of an MRP protein is "eliminated" according to the invention when it is not detectable by at least one assay method.

[0022] In other embodiments of the invention, the activity of one or more MRPs is reduced or eliminated by transforming a plant cell with an expression cassette comprising a polynucleotide encoding a polypeptide that inhibits the activity of one or more MRPs. The activity of an MRP is inhibited according to the present invention if the activity of that MRP in the transformed plant or cell is statistically lower than the activity of that MRP in a plant that has not been genetically modified to inhibit the activity of at least one MRP. In particular embodiments of the invention, an MRP activity of a modified plant according to the invention is less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, or less than 5% of that MRP activity in an appropriate control plant that has not been genetically modified to inhibit the expression of that MRP. Changes in MRP activity may be inferred, for example, by alterations in phytate content of a transformed plant or plant cell.

[0023] In other embodiments, the activity of an MRP may be reduced or eliminated by disrupting the gene encoding the MRP. The invention encompasses mutagenized plants that carry at least one mutation in an MRP gene, wherein the at least one mutation reduces expression of an MRP gene or inhibits the activity of an MRP.

[0024] Thus, many methods may be used to reduce or eliminate the activity of an MRP. More than one method may be used to reduce the activity of a single plant MRP. In addition, combinations of methods may be employed to reduce or eliminate the activity of two or more different MRPs. Non-limiting examples of methods of reducing or eliminating the expression of a plant MRP are given below.

[0025] In some embodiments of the present invention, a plant cell is transformed with an expression cassette that is capable of producing a polynucleotide that inhibits the expression of an MRP. The term "expression" as used herein refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product. For example, for the purposes of the present invention, an expression cassette capable of expressing a polynucleotide that inhibits the expression of at least one maize MRP is an expression cassette capable of producing an RNA molecule that inhibits the transcription and/or translation of at least one maize MRP.

[0026] "Expression" generally refers to the transcription and/or translation of a coding region of a DNA molecule, messenger RNA, or other nucleic acid molecule to produce the encoded protein or polypeptide. In other contexts, "expression" refers to the transcription of RNA from an expression cassette, such as, for example, the transcription of a hairpin construct from an expression cassette for use in hpRNA interference.

[0027] "Coding region" refers to the portion of a messenger RNA (or the corresponding portion of another nucleic acid molecule such as a DNA molecule) which encodes a protein or polypeptide. "Noncoding region" refers to all portions of a messenger RNA or other nucleic acid molecule that are not a coding region, including, for example, the promoter region, 5' untranslated region ("UTR"), and/or 3' UTR.

[0028] Some examples of polynucleotides and methods that inhibit the expression of an MRP are given below. While specific examples are given below, a variety of methods are known in the art by which it is possible to inhibit expression. While the invention is not bound by any particular theory of operation or mechanism of action, the invention provides the exemplary nucleotide and protein sequences disclosed herein and thereby provides a variety of methods by which expression can be inhibited. For example, fragments of noncoding region can be used to make constructs that inhibit expression of an MRP; such fragments can include portions of the promoter region or portions of the 3' noncoding region (i.e., the 3' UTR).

[0029] In some embodiments of the invention, inhibition of the expression of an MRP may be obtained by sense suppression or cosuppression. For cosuppression, an expression cassette is designed to express an RNA molecule corresponding to all or part of a messenger RNA encoding an MRP in the "sense" orientation. Overexpression of the RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the cosuppression expression cassette are screened to identify those that show suitable inhibition of MRP expression.

[0030] The polynucleotide used for cosuppression or other methods to inhibit expression may correspond to all or part of the sequence encoding the MRP, all or part of the 5' and/or 3' untranslated region of an MRP transcript, or all or part of both the coding region and the untranslated regions of a transcript encoding MRP. A polynucleotide used for cosuppression or other gene silencing methods may share 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 85%, 80%, or less sequence identity with the target sequence. When portions of the polynucleotides are used to disrupt the expression of the target gene, generally, sequences of at least 15, 20, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, or 900 nucleotides or 1 kb or greater may be used. In some embodiments where the polynucleotide comprises all or part of the coding region for the MRP, the expression cassette is designed to eliminate the start codon of the polynucleotide so that no protein product will be transcribed. In this manner, an expression cassette may cause permanent modification of the coding and/or noncoding region of an endogenous gene.

[0031] Thus, in some embodiments, for example, the polynucleotide used for cosuppression or another method to inhibit expression will comprise a sequence selected from a particular region of the coding and/or noncoding region. That is, the polynucleotide will comprise a sequence or the complement of a sequence selected from the region between nucleotides 1 and 5139 of the sequence set forth in SEQ ID NO: 2, or selected from the region with a first endpoint at nucleotide 1, 150, 250, 400, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, 3100, 3250, 3400, 3550, 3700, 3850, 4000, 4150, 4300, 4450, 4600, 4750, 4900, 5050, or 5139 and a second endpoint at nucleotide 244, 400, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, 3100, 3250, 3400, 3550, 3700, 3850, 4000, 4150, 4300, 4450, 4600, 4750, 4900, 5050, or 5139. As discussed elsewhere herein, fragments and/or variants of the exemplary disclosed sequences may also be used.

[0032] In some embodiments, for example, the polynucleotide will comprise a sequence or the complement of a sequence selected from the region between nucleotides 1 and 3134 of the sequence set forth in SEQ ID NO:1, or selected from the region with a first endpoint at nucleotide 1, 150, 400, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, or 3134, and a second endpoint at nucleotide 1, 150, 400, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, or 3134. Where a noncoding region is used for cosuppression or other method to inhibit expression, it may be advantageous to use a noncoding region that comprises CpG islands (see, e.g., Tariq et al. (2004) Trends Genet. 20: 244-251). As discussed elsewhere herein, variants and/or fragments of the exemplary disclosed sequences may also be used.

[0033] In some embodiments, for example, the polynucleotide will comprise a sequence or the complement of a sequence selected from the region between nucleotides 1 and 5123 of the sequence set forth in SEQ ID NO:6, or selected from the region with a first endpoint at nucleotide 1, 150, 300, 450, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, 3100, 3250, 3400, 3550, 3700, 3850, 4000, 4150, 4300, 4450, 4600, 4750, 4900, or 5123, and a second endpoint at nucleotide 1, 150, 300, 450, 550, 700, 850, 1000, 1150, 1300, 1450, 1600, 1750, 1900, 2050, 2200, 2350, 2500, 2650, 2800, 2950, 3100, 3250, 3400, 3550, 3700, 3850, 4000, 4150, 4300, 4450, 4600, 4750, 4900, or 5123. As discussed elsewhere herein, variants and/or fragments of the exemplary disclosed sequences may also be used.

[0034] In some embodiments, for example, the polynucleotide will comprise a sequence or the complement of a sequence selected from the region between nucleotides 1 and 1350 of the sequence set forth in SEQ ID NO:10, or selected from the region with a first endpoint at nucleotide 1, 150, 300, 450, 550, 700, 850, 1000, 1150, 1300, or 1350, and a second endpoint at nucleotide 1, 150, 300, 450, 550, 700, 850, 1000, 1150, 1300, or 1350. As discussed elsewhere herein, variants and/or fragments of the exemplary disclosed sequences may also be used.

[0035] In some embodiments, for example, the polynucleotide will comprise a sequence or the complement of a sequence selected from the region between nucleotides 1 and 465 of the sequence set forth in SEQ ID NO:12, or selected from the region with a first endpoint at nucleotide 1, 150, 300, 450, or 465, and a second endpoint at nucleotide 1, 150, 300, 450, or 465. As discussed elsewhere herein, variants and/or fragments of the exemplary disclosed sequences may also be used.

[0036] In some embodiments, for example, the polynucleotide will comprise a sequence or the complement of a sequence selected from the region between nucleotides 1 and 556 of the sequence set forth in SEQ ID NO:71, or selected from the region with a first endpoint at nucleotide 1, 150, 300, 450, or 556, and a second endpoint at nucleotide 1, 150, 300, 450, or 556. As discussed elsewhere herein, variants and/or fragments of the exemplary disclosed sequences may also be used.

[0037] Cosuppression may be used to inhibit the expression of plant genes to produce plants having undetectable protein levels for the proteins encoded by these genes. See, for example, Broin et al. (2002) Plant Cell 14: 1417-1432. Cosuppression may also be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Pat. No. 5,942,657. Methods for using cosuppression to inhibit the expression of endogenous genes in plants are described in Flavell et al. (1994) Proc. Natl. Acad. Sci. USA 91: 3490-3496; Jorgensen et al. (1996) Plant Mol. Biol. 31: 957-973; Johansen and Carrington (2001) Plant Physiol. 126: 930-938; Broin et al. (2002) Plant Cell 14: 1417-1432; Stoutjesdijk et at (2002) Plant Physiol. 129: 1723-1731; Yu et al. (2003) Phytochemistry 63: 753-763; and U.S. Pat. Nos. 5,034,323; 5,283,184; and 5,942,657; each of which is herein incorporated by reference. The efficiency of cosuppression may be increased by including a poly-dT region in the expression cassette at a position 3' to the sense sequence and 5' of the polyadenylation signal. See, e.g., U.S. Patent Publication No. 20020048814, herein incorporated by reference. Typically, such a nucleotide sequence has substantial sequence identity to the sequence of the transcript of the endogenous gene, for example, greater than about 65%, 80%, 85%, 90%, 95%, or more sequence identity. See, U.S. Pat. Nos. 5,283,184 and 5,034,323, herein incorporated by reference.

[0038] In some embodiments of the invention, inhibition of the expression of the MRP may be obtained by antisense suppression. For antisense suppression, the expression cassette is designed to express an RNA molecule complementary to all or part of a messenger RNA comprising a region encoding the MRP. Overexpression of the antisense RNA molecule can result in reduced expression of the native gene. Accordingly, multiple plant lines transformed with the antisense suppression expression cassette are screened to identify those that show the greatest inhibition of MRP expression.

[0039] The polynucleotide for use in antisense suppression may correspond to all or part of the complement of the sequence encoding the MRP, all or part of the complement of the 5' and/or 3' untranslated region of the MRP transcript, or all or part of the complement of both the coding sequence and the untranslated regions of a transcript encoding the MRP. In addition, the antisense polynucleotide may be fully complementary (i.e., 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target sequence. That is, an antisense polynucleotide may share 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 85%, 80%, or less sequence identity with the target sequence. Antisense suppression may be used to inhibit the expression of multiple proteins in the same plant. See, for example, U.S. Pat. No. 5,942,657. Furthermore, portions of the antisense nucleotides may be used to disrupt the expression of the target gene. Generally, sequences of at least 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 450, 500, or 550 nucleotides or greater may be used.

[0040] Methods for using antisense suppression to inhibit the expression of endogenous genes in plants are described, for example, in Liu et at (2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829 and 5,942,657, each of which is herein incorporated by reference. Efficiency of antisense suppression may be increased by including a poly-dT region in the expression cassette at a position 3' to the antisense sequence and 5' of the polyadenylation signal. See, U.S. Patent Publication No. 20020048814, herein incorporated by reference.

[0041] In some embodiments of the invention, inhibition of the expression of an MRP may be obtained by double-stranded RNA (dsRNA) interference. For dsRNA interference, a sense RNA molecule like that described above for cosuppression and an antisense RNA molecule that is fully or partially complementary to the sense RNA molecule are expressed in the same cell, resulting in inhibition of the expression of the corresponding endogenous messenger RNA.

[0042] Expression of the sense and antisense molecules can be accomplished by designing the expression cassette to comprise both a sense sequence and an antisense sequence. Alternatively, separate expression cassettes may be used for the sense and antisense sequences. Multiple plant lines transformed with the dsRNA interference expression cassette or expression cassettes are then screened to identify plant lines that show the greatest inhibition of MRP expression. Methods for using dsRNA interference to inhibit the expression of endogenous plant genes are described in Waterhouse et al. (1998) Proc. Natl. Acad. Sci. USA 95: 13959-13964, Liu et al. (2002) Plant Physiol. 129: 1732-1743, and WO 99/49029, WO 99/53050, WO 99/61631, and WO 00/49035; each of which is herein incorporated by reference.

[0043] In some embodiments of the invention, inhibition of the expression of one or more MRPs may be obtained by hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference. These methods are highly efficient at inhibiting the expression of endogenous genes. See, Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4: 29-38 and the references cited therein. These methods can make use of either coding region sequences or promoter or regulatory region sequences.

[0044] For hpRNA interference, the expression cassette is designed to express an RNA molecule that hybridizes with itself to form a hairpin structure that comprises a single-stranded loop or "spacer" region and a base-paired stem. In some embodiments, the base-paired stem region comprises a sense sequence corresponding to all or part of the endogenous messenger RNA encoding the gene whose expression is to be inhibited, and an antisense sequence that is fully or partially complementary to the sense sequence. The antisense sequence may be located "upstream" of the sense sequence (i.e., the antisense sequence may be closer to the promoter driving expression of the hairpin RNA than the sense sequence). In some embodiments, the base-paired stem region comprises a first portion of a noncoding region such as a promoter and a second portion of the noncoding region that is in inverted orientation relative to the first portion and that is fully or partially complementary to the first portion. In some embodiments, the base-paired stem region comprises a first portion and a second portion which are fully or partially complementary to each other but which comprise both coding and noncoding regions.

[0045] In some embodiments, the expression cassette comprises more than one base-paired "stem" region; that is, the expression cassette comprises sequences from different coding and/or noncoding regions which have the potential to form more than one base-paired "stem" region, for example, as diagrammed in FIG. 6 (construct 2 and construct 3). Where more than one base-paired "stem" region is present in an expression cassette, the "stem" regions may flank one another as diagrammed in FIG. 6 (construct 3) or may be in some other configuration (for example, as diagrammed in FIG. 6 (construct 2)). That is, for example, an expression cassette may comprise more than one combination of promoter and complementary sequences as shown in FIG. 6 (construct 1), and each such combination may be driven by a separate promoter. One of skill will be able to create and test a variety of configurations to determine the optimal construct for use in this or any other method for inhibition of expression.

[0046] Thus, the base-paired stem region of the molecule generally determines the specificity of the RNA interference. The sense sequence and the antisense sequence (or first and second portion of the noncoding region) are generally of similar lengths but may differ in length. Thus, these sequences may be portions or fragments of at least 10, 19, 20, 30, 50, 70, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 500, 600, 700, 800, 900 nucleotides in length, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb in length. The loop region of the expression cassette may vary in length. Thus, the loop region may be at least 100, 200, 300, 400, 500, 600, 700, 800, 900 nucleotides in length, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kb in length. In some embodiments, the loop region comprises an intron such as, for example, the Adh1 intron.

[0047] hpRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants. See, for example, Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97: 4985-4990; Stoutjesdijk et al. (2002) Plant Physiol. 129: 1723-1731; and Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4: 29-38. Methods for using hpRNA interference to inhibit or silence the expression of genes are described, for example, in Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA 97: 4985-4990; Stoutjesdijk et al. (2002) Plant Physiol. 129: 1723-1731; Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4: 29-38; Pandolfini et al. BMC Biotechnology 3: 7, and U.S. Patent Publication No. 20030175965; each of which is herein incorporated by reference. A transient assay for the efficiency of hpRNA constructs to silence gene expression in vivo has been described by Panstruga et al. (2003) Mol. Biol. Rep. 30: 135-140, herein incorporated by reference.

[0048] For ihpRNA, the interfering molecules have the same general structure as for hpRNA (including the same sizes of sense sequences and antisense sequences), but the RNA molecule additionally comprises an intron in the loop or "spacer" region that is capable of being spliced in the cell in which the ihpRNA is expressed. The use of an intron minimizes the size of the loop in the hairpin RNA molecule following splicing, and this increases the efficiency of interference. See, for example, Smith et al. (2000) Nature 407: 319-320 (which demonstrated 100% suppression of endogenous gene expression using ihpRNA-mediated interference). Methods for using ihpRNA interference to inhibit the expression of endogenous plant genes are described, for example, in Smith et al. (2000) Nature 407: 319-320; Wesley et al. (2001) Plant J. 27: 581-590; Wang and Waterhouse (2001) Curr. Opin. Plant Biol. 5: 146-150; Waterhouse and Helliwell (2003) Nat. Rev. Genet. 4: 29-38; Helliwell and Waterhouse (2003) Methods 30: 289-295, and U.S. Patent Publication No. 20030180945, each of which is herein incorporated by reference.

[0049] The expression cassette for hpRNA interference may also be designed such that the sense sequence and the antisense sequence do not correspond to an endogenous RNA. In this embodiment, the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the endogenous messenger RNA of the target gene. Thus, in this embodiment, it is the loop region that determines the specificity of the RNA interference. See, for example, WO 02/00904, herein incorporated by reference.

[0050] Transcriptional gene silencing (TGS) may be accomplished through use of hpRNA constructs wherein the inverted repeat of the hairpin shares sequence identity with the promoter region of a gene to be silenced. Processing of the hpRNA into short RNAs which can interact with the homologous promoter region may trigger degradation or methylation to result in silencing (Aufsatz et al. (2002) Proc. Nat'l. Acad. Sci. USA 99 (Suppl. 4): 16499-16506; Mette et al. (2000) EMBO J. 19(19): 5194-5201). As the invention is not bound by a particular mechanism or mode of operation, a decrease in expression may also be achieved by other mechanisms.

[0051] Amplicon expression cassettes comprise a plant virus-derived sequence that contains all or part of the target gene but generally not all of the genes of the native virus. The viral sequences present in the transcription product of the expression cassette allow the transcription product to direct its own replication. The transcripts produced by the amplicon may be either sense or antisense relative to the target sequence (i.e., the messenger RNA for MRP). Methods of using amplicons to inhibit the expression of endogenous plant genes are described, for example, in Angell and Baulcombe (1997) EMBO J. 16: 3675-3684, Angell and Baulcombe (1999) Plant J. 20: 357-362, and U.S. Pat. No. 6,646,805, each of which is herein incorporated by reference.

[0052] In some embodiments, the polynucleotide expressed by the expression cassette of the invention is catalytic RNA or has ribozyme activity specific for the messenger RNA of MRP. Thus, the polynucleotide causes the degradation of the endogenous messenger RNA, resulting in reduced expression of the MRP. This method is described, for example, in U.S. Pat. No. 4,987,071, herein incorporated by reference.

[0053] In some embodiments of the invention, inhibition of the expression of one or more MRPs may be obtained by RNA interference by expression of a gene encoding a micro RNA (miRNA). miRNAs are regulatory agents consisting of about 22 ribonucleotides. miRNAs are highly efficient at inhibiting the expression of endogenous genes. See, for example Javier et al. (2003) Nature 425: 257-263, herein incorporated by reference.

[0054] For miRNA interference, the expression cassette is designed to express an RNA molecule that is modeled on an endogenous miRNA gene. The miRNA gene encodes an RNA that forms a hairpin structure containing a 22-nucleotide sequence that is complementary to another endogenous gene (target sequence). For suppression of MRP expression, the 22-nucleotide sequence is selected from an MRP transcript sequence and contains 22 nucleotides of said MRP sequence in sense orientation and 21 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence. miRNA molecules are highly efficient at inhibiting the expression of endogenous genes, and the RNA interference they induce is inherited by subsequent generations of plants.

[0055] In one embodiment, the polynucleotide encodes a zinc finger protein that binds to a gene encoding an MRP resulting in reduced expression of the gene. In particular embodiments, the zinc finger protein binds to a regulatory region of an MRP gene. In other embodiments, the zinc finger protein binds to a messenger RNA encoding an MRP and prevents its translation. Methods of selecting sites for targeting by zinc finger proteins have been described, for example, in U.S. Pat. No. 6,453,242, and methods for using zinc finger proteins to inhibit the expression of genes in plants are described, for example, in U.S. Patent Publication No. 20030037355; each of which is herein incorporated by reference.

[0056] In some embodiments of the invention, the polynucleotide encodes an antibody that binds to at least one maize MRP and reduces the phytate level of the plant. In another embodiment, the binding of the antibody results in increased turnover of the antibody-MRP complex by cellular quality control mechanisms. The expression of antibodies in plant cells and the inhibition of molecular pathways by expression and binding of antibodies to proteins in plant cells are well known in the art. See, for example, Conrad and Sonnewald (2003) Nature Biotech. 21: 35-36, incorporated herein by reference. In other embodiments of the invention, the polynucleotide encodes a polypeptide that specifically inhibits the MRP activity of a maize MRP, i.e., an MRP inhibitor.

[0057] In some embodiments of the present invention, the activity of an MRP is reduced or eliminated by disrupting the gene encoding the MRP. The gene encoding the MRP may be disrupted by any method known in the art. For example, in one embodiment, the gene is disrupted by transposon tagging. In another embodiment, the gene is disrupted by mutagenizing maize plants using random or targeted mutagenesis, and selecting for plants that have reduced MRP activity.

[0058] In one embodiment of the invention, transposon tagging is used to reduce or eliminate the activity of one or more MRPs. Transposon tagging comprises inserting a transposon within an endogenous MRP gene to reduce or eliminate expression of the MRP. "MRP gene" is intended to mean the gene that encodes an MRP protein according to the invention.

[0059] In this embodiment, the expression of one or more MRPs is reduced or eliminated by inserting a transposon within a regulatory region or coding region of the gene encoding the MRP. A transposon that is within an exon, intron, 5' or 3' untranslated sequence, a promoter, or any other regulatory sequence of an MRP gene may be used to reduce or eliminate the expression and/or activity of the encoded MRP.

[0060] Methods for the transposon tagging of specific genes in plants are well known in the art. See, for example, Maes et al. (1999) Trends Plant Sci. 4: 90-96; Dharmapuri and Sonti (1999) FEMS Microbiol. Lett. 179: 53-59; Meissner et al. (2000) Plant J. 22: 265-274; Phogat et al. (2000) J. Biosci. 25: 57-63; Walbot (2000) Curr. Opin. Plant Biol. 2: 103-107; Gai et al. (2000) Nucleic Acids Res. 28: 94-96; Fitzmaurice et al. (1999) Genetics 153: 1919-1928. In addition, the TUSC process for selecting Mu insertions in selected genes has been described in Bensen et al. (1995) Plant Cell 7: 75-84; Mena et al. (1996) Science 274: 1537-1540; and U.S. Pat. No. 5,962,764; each of which is herein incorporated by reference.

[0061] Additional methods for decreasing or eliminating the expression of endogenous genes in plants are also known in the art and can be similarly applied to the instant invention. These methods include other forms of mutagenesis, such as ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis, and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines in which the endogenous gene has been deleted. For examples of these methods see Ohshima et al. (1998) Virology 243: 472-481; Okubara et al. (1994) Genetics 137: 867-874; and Quesada et al. (2000) Genetics 154: 421-436; each of which is herein incorporated by reference. In addition, a fast and automatable method for screening for chemically induced mutations, TILLING (Targeting Induced Local Lesions In Genomes), using denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the instant invention. See McCallum et al. (2000) Nat. Biotechnol. 18: 455-457, herein incorporated by reference.

[0062] Mutations that impact gene expression or that interfere with the function of the encoded protein are well known in the art. Insertional mutations in gene exons usually result in null-mutants. Mutations in conserved residues are particularly effective in inhibiting the MRP activity of the encoded protein. Conserved residues of plant MRPs suitable for mutagenesis with the goal to eliminate MRP activity are described herein, for example in the conserved domains set forth in SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24. Such mutants can be isolated according to well-known procedures, and mutations in different MRP loci can be stacked by genetic crossing. See, for example, Gruis et al. (2002) Plant Cell 14: 2863-2882.

[0063] In another embodiment of this invention, dominant mutants can be used to trigger RNA silencing due to gene inversion and recombination of a duplicated gene locus. See, for example, Kusaba et al. (2003) Plant Cell 15: 1455-1467.

[0064] The invention encompasses additional methods for reducing or eliminating the activity of one or more MRPs. Examples of other methods for altering or mutating a genomic nucleotide sequence in a plant are known in the art and include, but are not limited to, the use of chimeric vectors, chimeric mutational vectors, chimeric repair vectors, mixed-duplex oligonucleotides, self-complementary oligonucleotides, and recombinogenic oligonucleobases. Such vectors and methods of use are known in the art. See, for example, U.S. Pat. Nos. 5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984; each of which are herein incorporated by reference. See also, WO 98/49350, WO 99/07865, WO 99/25821, and Beetham et al. (1999) Proc. Natl. Acad. Sci. USA 96: 8774-8778; each of which is herein incorporated by reference. Other methods of suppressing expression of a gene involve promoter-based silencing. See, for example, Mette et al. (2000) EMBO J. 19: 5194-5201; Sijen et al. (2001) Curr. Biol. 11: 436-440; Jones et al. (2001) Curr. Biol. 11: 747-757.

[0065] Where polynucleotides are used to decrease or inhibit MRP activity, it is recognized that modifications of the exemplary sequences disclosed herein may be made as long as the sequences act to decrease or inhibit expression of the corresponding mRNA. Thus, for example, polynucleotides having at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the exemplary sequences disclosed herein may be used. Furthermore, portions or fragments of the exemplary sequences or portions or fragments of polynucleotides sharing a particular percent sequence identity to the exemplary sequences may be used to disrupt the expression of the target gene. Generally, fragments or sequences of at least 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 250, 260, 280, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more contiguous nucleotides, or greater may be used. It is recognized that in particular embodiments, the complementary sequence of such sequences may be used. For example, hairpin constructs comprise both a sense sequence fragment and a complementary, or antisense, sequence fragment corresponding to the gene of interest. Antisense constructs may share less than 100% sequence identity with the gene of interest, and may comprise portions or fragments of the gene of interest, so long as the object of the embodiment is achieved, i.e., so long as expression of the gene of interest is decreased.

[0066] Accordingly, the methods of the invention include methods for modulating the levels of endogenous transcription and/or gene expression by transforming plants with antisense or sense constructs to produce plants with reduced levels of phytate. In some embodiments, such modifications will alter the amino acid sequence of the proteins encoded by the genomic sequence as to reduce or eliminate the activity of a particular endogenous gene, such as MRP, in a plant or part thereof, for example, in a seed.

[0067] Furthermore, it is recognized that the methods of the invention may employ a nucleotide construct that is capable of directing, in a transformed plant, the expression of at least one protein, or the transcription of at least one RNA, such as, for example, an antisense RNA that is complementary to at least a portion of an mRNA. Typically such a nucleotide construct is comprised of a coding sequence for a protein or an RNA operably linked to 5' and 3' transcriptional regulatory regions. Alternatively, it is also recognized that the methods of the invention may employ a nucleotide construct that is not capable of directing, in a transformed plant, the expression of a protein or transcription of an RNA.

[0068] In addition, it is recognized that methods of the present invention do not depend on the incorporation of the entire nucleotide construct into the genome, only that the plant or cell thereof is altered as a result of the introduction of the nucleotide construct into a cell. In one embodiment of the invention, the genome may be altered following the introduction of the nucleotide construct into a cell. For example, the nucleotide construct, or any part thereof, may incorporate into the genome of the plant. Alterations to the genome of the present invention include, but are not limited to, additions, deletions, and substitutions of nucleotides in the genome. While the methods of the present invention do not depend on additions, deletions, or substitutions of any particular number of nucleotides, it is recognized that such additions, deletions, or substitutions comprise at least one nucleotide.

[0069] The use of the term "nucleotide constructs" herein is not intended to limit the present invention to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides, comprised of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides may also be employed in the methods disclosed herein. Thus, the nucleotide constructs of the present invention encompass all nucleotide constructs that can be employed in the methods of the present invention for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs of the invention also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

[0070] The invention encompasses isolated or substantially purified nucleic acid or protein compositions. An "isolated" or "purified" nucleic acid molecule or protein, or biologically active portion thereof, is substantially or essentially free from components that normally accompany or interact with the nucleic acid molecule or protein as found in its naturally occurring environment. Thus, an isolated or purified nucleic acid molecule or protein 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. Preferably, an "isolated" nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. When the protein of the invention or biologically active portion thereof is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

[0071] By "modulating" or "modulate" as used herein is intended that the level or amount of a product is increased or decreased in accordance with the goal of the particular embodiment. For example, if a particular embodiment were useful for producing purified MRP enzyme, it would be desirable to increase the amount of MRP protein produced. As another example, if a particular embodiment were useful for decreasing the amount of phytate in a transgenic plant, it would be desirable to decrease the amount of MRP protein expressed by the plant.

[0072] The article "a" and "an" are used herein to refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one or more element.

[0073] Throughout the specification the word "comprising," or variations such as "comprises," will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0074] Fragments and/or variants of the disclosed polynucleotides and proteins encoded thereby are also encompassed by the present invention. By "fragment" is intended a portion of the polynucleotide or a portion of the nucleotide sequence and hence protein encoded thereby, if any. Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein and hence have MRP activity. Alternatively, fragments of a nucleotide sequence that are useful as hybridization probes or in sense or antisense suppression generally do not encode fragment proteins retaining biological activity. Thus, fragments of a nucleotide sequence may range in length from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides, and up to the full-length nucleotide sequence encoding the proteins of the invention.

[0075] A fragment of an MRP nucleotide sequence that encodes a biologically active portion of an MRP protein of the invention will encode at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500 contiguous amino acids, or up to the total number of amino acids present in a full-length MRP protein of the invention (for example, 1510 amino acids for SEQ ID NO: 3). Fragments of an MRP nucleotide sequence that are useful in non-coding embodiments, for example, as PCR primers or for sense or antisense suppression, generally need not encode a biologically active portion of an MRP protein. A fragment of an MRP polypeptide of the invention will contain at least 15, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, or 1500 contiguous amino acids, or up to the total number of amino acids present in a full-length MRP protein of the invention (for example, 1510 amino acids for SEQ ID NO: 3).

[0076] Thus, a fragment of an MRP nucleotide sequence may encode a biologically active portion of an MRP protein, or it may be a fragment that can be used, for example, as a hybridization probe or in sense or antisense suppression using methods disclosed herein and known in the art. A biologically active portion of an MRP protein can be prepared by isolating a portion of one of the MRP polynucleotides of the invention, expressing the encoded portion of the MRP protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the MRP protein. Nucleic acid molecules that are fragments or portions of an MRP polynucleotide comprise at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 3,000, 4,000, or 5,000 contiguous nucleotides, or up to the number of nucleotides present in a full-length MRP polynucleotide disclosed herein (for example, 5139 nucleotides for SEQ ID NO: 2).

[0077] "Variants" is intended to mean substantially similar sequences. For polynucleotides, a variant comprises a deletion and/or addition at one or more nucleotides at one or more internal sites within the native polynucleotide and/or a substitution of one or more nucleotides at one or more sites in the native polynucleotide. As used herein, a "native" polypeptide or polynucleotide comprises a naturally occurring amino acid sequence or nucleotide sequence. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the MRP polypeptides of the invention. Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant polynucleotides also include synthetically-derived polynucleotides, such as those generated, for example, by using site-directed mutagenesis but which still encode an MRP protein of the invention. Generally, variants of a particular polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters described elsewhere herein.

[0078] Variants of a particular polynucleotide of the invention (i.e., the reference polynucleotide) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Thus, for example, an isolated polynucleotide that encodes a polypeptide with a given percent sequence identity to the polypeptide of SEQ ID NO: 3 are disclosed. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein. Where any given pair of polynucleotides of the invention is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. Sequences of the invention may be variants or fragments of an exemplary polynucleotide sequence, or they may be both a variant and a fragment of an exemplary sequence.

[0079] "Variant" protein is intended to mean a protein derived from the native protein by deletion or addition of one or more amino acids at one or more sites in the native protein and/or substitution of one or more amino acids at one or more sites in the native protein. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, MRP activity as described herein. Such variants may result from, for example, genetic polymorphism or from human manipulation. Biologically active variants of a native MRP protein of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 87%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein. A biologically active variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue. Sequences of the invention may be variants or fragments of an exemplary protein sequence, or they may be both a variant and a fragment of an exemplary sequence.

[0080] The proteins of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants and fragments of the MRP proteins can be prepared by the creation of mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA 82: 488-492; Kunkel et al. (1987) Methods in Enzymol. 154: 367-382; U.S. Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Nat'l. Biomed. Res. Found., Washington, D.C.), herein incorporated by reference. Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be made.

[0081] Thus, the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms. Likewise, the proteins of the invention encompass both naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired MRP activity. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. See, EP Patent Application Publication No. 75,444.

[0082] The deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated by the methods used in Example 1 and references cited therein as well as by other assays known in the art.

[0083] Variant polynucleotides and proteins also encompass sequences and proteins derived from a mutagenic and recombinogenic procedure such as DNA shuffling. With such a procedure, one or more different MRP coding sequences can be manipulated to create a new MRP possessing the desired properties. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising sequence regions that have substantial sequence identity and can be homologously recombined in vitro or in vivo. For example, using this approach, sequence motifs encoding a domain of interest may be shuffled between the MRP gene of the invention and other known MRP genes to obtain a new gene coding for a protein with an improved property of interest, such as an increased K_m in the case of an enzyme. Strategies for such DNA shuffling are known in the art. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA 91: 10747-10751; Stemmer (1994) Nature 370: 389-391; Crameri et al. (1997) Nature Biotech. 15: 436-438; Moore et al. (1997) J. Mol. Biol. 272: 336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA 94: 4504-4509; Crameri et al. (1998) Nature 391: 288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

[0084] The present invention further provides a method for modulating (i.e., increasing or decreasing) the concentration or composition of the polypeptides of the claimed invention in a plant or part thereof. Modulation can be effected by increasing or decreasing the concentration and/or the composition (i.e., the ratio of the polypeptides of the claimed invention) in a plant.

[0085] In some embodiments, the method comprises transforming a plant cell with a cassette comprising a polynucleotide of the invention to obtain a transformed plant cell, growing the transformed plant cell under conditions allowing expression of the polynucleotide in the plant cell in an amount sufficient to modulate concentration and/or composition of the corresponding protein in the plant cell. In some embodiments, the method comprises utilizing the polynucleotides of the invention to create a deletion or inactivation of the native gene. Thus, a deletion may constitute a functional deletion, i.e., the creation of a "null" mutant, or it may constitute removal of part or all of the coding region of the native gene. Methods for creating null mutants are well-known in the art and include, for example, chimeraplasty as discussed elsewhere herein.

[0086] In some embodiments, the content and/or composition of polypeptides of the present invention in a plant may be modulated by altering, in vivo or in vitro, the promoter of a non-isolated gene of the present invention to up- or down-regulate gene expression. In some embodiments, the coding regions of native genes of the present invention can be altered via substitution, addition, insertion, or deletion to decrease activity of the encoded enzyme. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868. One method of down-regulation of the protein involves using PEST sequences that provide a target for degradation of the protein.

[0087] In addition to sense and antisense suppression, catalytic RNA molecules or ribozymes can also be used to inhibit expression of plant genes. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al. (1988) Nature 334: 585-591.

[0088] A variety of cross-linking agents, alkylating agents and radical-generating species as pendant groups on polynucleotides of the present invention can be used to bind, label, detect, and/or cleave nucleic acids. For example, Vlassov et al. (1986) Nucl. Acids Res. 14: 4065-4076 describes covalent bonding of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to target sequences. Similar work is reported in Knorre et al. (1985) Biochimie 67: 785-789. Others have also showed sequence-specific cleavage of single-stranded DNA mediated by incorporation of a modified nucleotide which was capable of activating cleavage (Iverson and Dervan (1987) J. Am. Chem. Soc. 109: 1241-1243). Meyer et al. ((1989) J. Am. Chem. Soc. 111: 8517-8519) demonstrated covalent crosslinking to a target nucleotide using an alkylating agent complementary to the single-stranded target nucleotide sequence. Lee et al. ((1988) Biochemistry 27: 3197-3203) disclosed a photoactivated crosslinking to single-stranded oligonucleotides mediated by psoralen. Home et al. ((1990) J. Am Chem. Soc. 112: 2435-2437) used crosslinking with triple-helix-forming probes. Webb and Matteucci ((1986) J. Am. Chem. Soc. 108: 2764-2765) and Feteritz et al. ((1991) J. Am. Chem. Soc. 113: 4000) used N4, N4-ethanocytosine as an alkylating agent to crosslink to single-stranded oligonucleotides. In addition, various compounds to bind, detect, label, and/or cleave nucleic acids are known in the art. See, for example, U.S. Pat. Nos. 5,543,507; 5,672,593; 5,484,908; 5,256,648; and 5,681,941. Such embodiments are collectively referred to herein as "chemical destruction."

[0089] In some embodiments, an isolated nucleic acid (e.g., a vector) comprising a promoter sequence is transfected into a plant cell. Subsequently, a plant cell comprising the promoter operably linked to a nucleic acid or polynucleotide comprising a nucleotide sequence of the present invention is selected for by means known to those of skill in the art such as, but not limited to, Southern blot, DNA sequencing, or PCR analysis using primers specific to the promoter and to the gene and detecting amplicons produced therefrom. A plant or plant part altered or modified by the foregoing embodiments is grown under plant-forming conditions for a time sufficient to modulate the concentration and/or composition of polypeptides of the present invention in the plant. Plant-forming conditions are well known in the art.

[0090] In general, when an endogenous polypeptide is modulated using the methods of the invention, the content of the polypeptide in a plant or part or cell thereof is increased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more relative to a native control plant, plant part, or cell lacking the aforementioned cassette. Modulation in the present invention may occur during and/or subsequent to growth of the plant to the desired stage of development. Modulating nucleic acid expression temporally and/or in particular tissues can be controlled by employing the appropriate promoter operably linked to a polynucleotide of the present invention in, for example, sense or antisense orientation.

[0091] A transformed plant or transformed plant cell of the invention is one in which genetic alteration, such as transformation, has been effected as to a gene of interest, or is a plant or plant cell which is descended from a plant or cell so altered and which comprises the alteration. A "control" or "control plant" or "control plant cell" provides a reference point for measuring changes in phenotype of the subject plant or plant cell. A control plant or plant cell may comprise, for example: (a) a wild-type plant or cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e., with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or plant cell; (d) a plant or plant cell genetically identical to the subject plant or plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.

[0092] The polynucleotides of the invention can be used to isolate corresponding sequences from other organisms, particularly other plants. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences set forth herein. Sequences isolated based on their sequence identity to the entire MRP sequences set forth herein or to variants and fragments thereof are encompassed by the present invention. Such sequences include sequences that are orthologs of the disclosed sequences. "Orthologs" is intended to mean genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and/or their encoded protein sequences share at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater sequence identity. Functions of orthologs are often highly conserved among species. Thus, isolated sequences that encode an MRP protein or have Lpa1 promoter activity and which hybridize under stringent conditions to the Lpa1 sequences disclosed herein, or to variants or fragments thereof, are encompassed by the present invention.

[0093] In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers, and the like.

[0094] In hybridization techniques, all or part of a known polynucleotide is used as a probe that selectively hybridizes to other nucleic acids comprising corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as ³²P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the MRP sequences of the invention. Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0095] For example, the entire MRP sequences disclosed herein, or one or more portions thereof, may be used as probes capable of specifically hybridizing to corresponding MRP sequences and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among MRP sequences and are at least about 10, 12, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 50, 60, 70, 80, 90, or more nucleotides in length. Such probes may be used to amplify corresponding MRP sequences from a chosen plant by PCR. This technique may be used to isolate additional coding sequences from a desired plant or as a diagnostic assay to determine the presence of coding sequences in a plant. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).

[0096] Hybridization of such sequences may be carried out under stringent conditions. By "stringent conditions" or "stringent hybridization conditions" is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Generally, a probe is less than about 1000 or 500 nucleotides in length.

[0097] Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS. Duration of hybridization is generally less than about 24 hours, usually about 4, 8, or 12 hours.

[0098] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the T_m can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: T_m=81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)-500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, "% form" is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The T_m is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. T_m is reduced by about 1° C. for each 1% of mismatching; thus, T_m, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the T_m can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (T_m) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (T_m); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (T_m); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (T_m). Using the equation, hybridization and wash compositions, and desired T_m, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a T_m of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). The duration of the wash time will be at least a length of time sufficient to reach equilibrium, for example, 4 hours, 8 hours, or 12 hours.

[0099] The following terms are used to describe the sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", and (d) "percentage of sequence identity."

[0100] (a) As used herein, "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.

[0101] (b) As used herein, "comparison window" makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, or 100 nucleotides in length, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.

[0102] Methods of alignment of sequences for comparison are well known in the art. Thus, the determination of percent sequence identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4: 11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453; the search-for-local-alignment-method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85: 2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877.

[0103] Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (1988) Gene 73: 237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153; Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang et al. (1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24: 307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST programs of Altschul et at (1990) J. Mol. Biol. 215: 403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed with the BLASTX program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See http://www.ncbi.nlm.nih.gov. Alignment may also be performed manually by inspection.

[0104] Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3 and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2; and the BLOSUM62 scoring matrix or any equivalent program thereof. By "equivalent program" is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.

[0105] GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453, to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. Default gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences the default gap creation penalty is 50 while the default gap extension penalty is 3. The gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200. Thus, for example, the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

[0106] GAP presents one member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP displays four figures of merit for alignments: Quality, Ratio, Identity, and Similarity. The Quality is the metric maximized in order to align the sequences. Ratio is the quality divided by the number of bases in the shorter segment. Percent Identity is the percent of the symbols that actually match. Percent Similarity is the percent of the symbols that are similar. Symbols that are across from gaps are ignored. A similarity is scored when the scoring matrix value for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The scoring matrix used in Version 10 of the GCG Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915).

[0107] (c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).

[0108] (d) As used herein, "percentage of sequence identity" means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.

[0109] The use of the term "polynucleotide" is not intended to limit the present invention to polynucleotides 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 invention 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.

[0110] The MRP polynucleotide of the invention can be provided in expression cassettes for expression in the plant of interest. The cassette will include any necessary 5' and 3' regulatory sequences operably linked to an MRP polynucleotide of the invention. "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, "operably linked" is intended to mean 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 MRP polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes. If protein expression is desired, the cassette may be referred to as a protein expression cassette and will include in the 5'-3' direction of transcription: a transcriptional and translational initiation region (i.e., a promoter), an MRP nucleotide sequence of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in plants.

[0111] The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the MRP polynucleotide of the invention may be native/analogous to the host cell or to each other. Alternatively, the regulatory regions and/or the MRP polynucleotide of the invention may be heterologous to the host cell or to each other. 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 that from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form, or the promoter is not the native promoter for the operably linked polynucleotide.

[0112] While it may be optimal to express the sequences using heterologous promoters, the native promoter sequences (e.g., the promoter sequence set forth in SEQ ID NO: 1) may be used. Such constructs can change expression levels of MRP in the plant or plant cell. Thus, the phenotype of the plant or plant cell can be altered. The promoter sequence set forth in SEQ ID NO:1 contains a putative TATA box from nucleotides 2464 to 2470; the 5' UTR may contain an intron.

[0113] In an expression cassette, the termination region may be native with the transcriptional initiation region, may be native with the operably linked nucleotide sequence of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, the nucleotide sequence of interest, the plant host, or any combination thereof). Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15: 9627-9639.

[0114] Where appropriate, the polynucleotides may be optimized for increased expression in the transformed plant. That is, the genes can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, for example, U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17: 477-498, herein incorporated by reference.

[0115] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well-characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell, and the sequence may be modified to avoid predicted hairpin secondary mRNA structures.

[0116] The expression cassettes may additionally contain 5' leader sequences in the cassette construct. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6126-6130); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2): 233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Virology 154: 9-20), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353: 90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, N.Y.), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81: 382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968.

[0117] The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markers include phenotypic markers such as β-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85: 610-9 and Fetter et al. (2004) Plant Cell 16: 215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117: 943-54 and Kato et al. (2002) Plant Physiol 129: 913-42), and yellow florescent protein (PhiYFP® from Evrogen; see Bolte et al. (2004) J. Cell Science 117: 943-54).

[0118] See generally, Yarranton (1992) Curr. Opin. Biotech. 3: 506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89: 6314-6318; Yao et al. (1992) Cell 71: 63-72; Reznikoff (1992) Mol. Microbiol. 6: 2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al. (1987) Cell 48: 555-566; Brown et al. (1987) Cell 49: 603-612; Figge et al. (1988) Cell 52: 713-722; Deuschle et al. (1989) Proc. Natl. Acad. Aci. USA 86: 5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86: 2549-2553; Deuschle et al. (1990) Science 248: 480-483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90: 1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10: 3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89: 3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA 88: 5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19: 4647-4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidt et al. (1988) Biochemistry 27: 1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89: 5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36: 913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334: 721-724. Such disclosures are herein incorporated by reference.

[0119] The above list of selectable marker genes is not meant to be limiting. Any suitable selectable marker gene can be used in the present invention, and one of skill in the art will be able to determine which selectable marker gene is suitable for a particular application.

[0120] In preparing the 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.

[0121] A number of promoters can be used in the practice of the invention. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, or other promoters. Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al. (1985) Nature 313: 810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18: 675-689); pEMU (Last et al. (1991) Theon. Appl. Genet. 81: 581-588); MAS (Velten et al. (1984) EMBO J. 3: 2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611.

[0122] Chemical-regulated promoters can be used to modulate the transcription and/or expression of a particular nucleotide sequence in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88: 10421-10425 and McNellis et al. (1998) Plant J. 14(2): 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991) Mol. Gen. Genet. 227: 229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

[0123] Tissue-preferred promoters can be utilized to target enhanced MRP transcription and/or expression within a particular plant tissue. Tissue-preferred promoters include those described in Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2): 525-535; Canevascini et al. (1996) Plant Physiol. 112(2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20): 9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3): 495-505. Such promoters can be modified, if necessary, for weak expression.

[0124] Leaf-preferred promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kwon et al. (1994) Plant Physiol. 105: 357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Gotor et al. (1993) Plant J. 3: 509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20): 9586-9590.

[0125] Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2): 207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10): 1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3): 433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1): 11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7): 633-641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described. The promoters of these genes were linked to a β-glucuronidase reporter gene and introduced into both the nonlegume Nicotiana tabacum and the legume Lotus corniculatus, and in both instances root-specific promoter activity was preserved. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes (see Plant Science (Limerick) 79(1): 69-76). They concluded that enhancer and tissue-preferred DNA determinants are dissociated in those promoters. Teeri et al. (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene (see EMBO J. 8(2): 343-350). The TR1' gene, fused to nptII (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD-GRP3 gene promoter (Kuster et al. (1995) Plant Mol. Biol. 29(4): 759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol. 25(4): 681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.

[0126] "Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seed-germinating" promoters (those promoters active during seed germination). See Thompson et al. (1989) BioEssays 10: 108, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphate synthase); oleosin; and celA (cellulose synthase) (see WO 00/11177 and U.S. Pat. No. 6,225,529, herein incorporated by reference). Gamma-zein is a preferred endosperm-specific promoter. Globulin (Glb-1) is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, where seed-preferred promoters from end1 and end2 genes are disclosed; herein incorporated by reference.

[0127] Where low level transcription or expression is desired, weak promoters will be used. Generally, by "weak promoter" is intended a promoter that drives transcription and/or expression of a coding sequence at a low level. By low level is intended at levels of about 1/1000 transcripts to about 1/100,000 transcripts to about 1/500,000 transcripts. Alternatively, it is recognized that weak promoters also encompasses promoters that are expressed in only a few cells and not in others to give a total low level of transcription and/or expression. Where a promoter is expressed at unacceptably high levels, portions of the promoter sequence can be deleted or modified to decrease transcription and/or expression levels.

[0128] Such weak constitutive promoters include, for example, the core promoter of the Rsyn7 promoter (WO 99/43838 and U.S. Pat. No. 6,072,050), the core 35S CaMV promoter, and the like. Other constitutive promoters include, for example, U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142. See also, U.S. Pat. No. 6,177,611, herein incorporated by reference.

[0129] In one embodiment, the polynucleotides of interest are targeted to the chloroplast for expression. In this manner, where the nucleic acid of interest is not directly inserted into the chloroplast, the expression cassette will additionally contain a nucleic acid encoding a transit peptide to direct the gene product of interest to the chloroplasts. Such transit peptides are known in the art. See, for example, Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196: 1414-1421; and Shah et al. (1986) Science 233: 478-481.

[0130] Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30:769-780; Schnell et al. (1991) J. Biol. Chem. 266(5): 3335-3342); 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al. (1990) J. Bioenerg. Biomemb. 22(6): 789-810); tryptophan synthase (Zhao et al. (1995) J. Biol. Chem. 270(11): 6081-6087); plastocyanin (Lawrence et al. (1997) J. Biol. Chem. 272(33): 20357-20363); chorismate synthase (Schmidt et al. (1993) J. Biol. Chem. 268(36): 27447-27457); and the light harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al. (1988) J. Biol. Chem. 263: 14996-14999). See also Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968; Romer et al. (1993) Biochem. Biophys. Res. Commun. 196: 1414-1421; and Shah et al. (1986) Science 233: 478-481.

[0131] Methods for transformation of chloroplasts are known in the art. See, for example, Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87: 8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90: 913-917; Svab and Maliga (1993) EMBO J. 12: 601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91: 7301-7305.

[0132] The polynucleotides of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the polynucleotides of interest may be synthesized using chloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831, herein incorporated by reference.

[0133] In specific embodiments, the MRP sequences of the invention can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, the introduction of the MRP protein or variants and fragments thereof directly into the plant or the introduction of an MRP transcript into the plant. Such methods include, for example, microinjection or particle bombardment. See, for example, Crossway et al. (1986) Mol Gen. Genet. 202: 179-185; Nomura et al. (1986) Plant Sci. 44: 53-58; Hepler et al. (1994) Proc. Natl. Acad. Sci. 91: 2176-2180 and Hush et al. (1994) The Journal of Cell Science 107: 775-784, all of which are herein incorporated by reference. Alternatively, the MRP polynucleotide can be transiently transformed into the plant using techniques known in the art. Such techniques include viral vector system and the precipitation of the polynucleotide in a manner that precludes subsequent release of the DNA. Thus, the transcription from the particle-bound DNA can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use particles coated with polyethylimine (PEI; Sigma #P3143).

[0134] Thus, transgenic plants having low phytic acid content and high levels of bioavailable phosphorus can be generated by reducing or inhibiting MRP gene expression in a plant. For example, the transgenic plant can contain a transgene comprising an inverted repeat of Lpa1 that suppresses endogenous Lpa1 gene expression. In this manner, transgenic plants having the low phytic acid phenotype of lpa1 mutant plants can be generated. The transgenic plant can contain an MRP suppressor sequence alone or an MRP suppressor sequence can be "stacked" with one or more polynucleotides of interest, including, for example, one or more polynucleotides that can affect phytic acid levels or that provide another desirable phenotype to the transgenic plant. For example, such a transgene can be "stacked" with similar constructs involving one or more additional inositol phosphate kinase genes such as ITPK-5 (inositol 1,3,4-trisphosphate 5/6 kinase; e.g., SEQ ID NO: 65; see also WO 03/027243), IPPK (inositol polyphosphate kinase; e.g., SEQ ID NO: 64; see also WO 02/049324), and/or a myo-inositol-1 phosphate synthase gene (milps; see U.S. Pat. Nos. 6,197,561 and 6,291,224; e.g., milps-3 (SEQ ID NO: 25)). With such "stacked" transgenes, even greater reduction in phytic acid content of a plant can be achieved, thereby making more phosphorus bioavailable.

[0135] Thus, in certain embodiments the nucleic acid sequences of the present invention can be "stacked" with any combination of nucleic acids of interest in order to create plants with a desired phenotype. By "stacked" or "stacking" is intended that a plant of interest contains one or more nucleic acids collectively comprising multiple nucleotide sequences so that the transcription and/or expression of multiple genes are altered in the plant. For example, antisense nucleic acids of the present invention may be stacked with other nucleic acids which comprise a sense or antisense nucleotide sequence of at least one of ITPK-5 (e.g., SEQ ID NO: 65) and/or inositol polyphosphate kinase (IPPK; e.g., SEQ ID NO: 64), or other genes implicated in phytic acid metabolic pathways such as Lpa3 or myo-inositol kinase (see, e.g., copending application entitled, "Plant Myo-Inositol Kinase Polynucleotides and Methods of Use, Appl. No. 60/573,000, filed May 20, 2004; SEQ ID NO: 68); Lpa2 (see U.S. Pat. Nos. 5,689,054 and 6,111,168); myo-inositol 1-phosphate synthase (milps; e.g., SEQ ID NO: 25), myo-inositol monophosphatase (IMP) (see WO 99/05298 and U.S. application Ser. No. 10/042,465, filed Jan. 9, 2002); IP2K (e.g., SEQ ID NO: 67); and the like. The addition of such nucleic acids could enhance the reduction of phytic acid and InsP intermediates, thereby providing a plant with more bioavailable phosphate and/or reduced phytate. The nucleic acids of the present invention can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations. For example, in some embodiments, a phytase gene (e.g., SEQ ID NO: 66) is stacked with an lpa1 mutant so that phytase is expressed at high levels in the transgenic plant. Phytase genes are known in the art. See, for example, Maugenest et al. (1999) Plant Mol. Biol. 39: 503-514; Maugenest et al. (1997) Biochem. J. 322: 511-517; WO 200183763; WO200200890.

[0136] An MRP polynucleotide also can be stacked with any other polynucleotide(s) to produce plants having a variety of desired trait combinations including, for example, traits desirable for animal feed such as high oil genes (see, e.g., U.S. Pat. No. 6,232,529, which is incorporated herein by reference); balanced amino acids (e.g., hordothionins; see U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409, each of which is incorporated herein by reference); barley high lysine (Williamson et al. (1987) Eur. J. Biochem. 165: 99-106 and WO 98/20122); high methionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261: 6279; Kirihara et al. (1988) Gene 71: 359; and Musumura et al. (1989) Plant Mol. Biol. 12: 123); increased digestibility (e.g., modified storage proteins) and thioredoxins (U.S. Pat. No. 7,009,087).

[0137] An MRP polynucleotide also can be stacked with one or more polynucleotides encoding a desirable trait such as a polynucleotide that confers, for example, insect, disease or herbicide resistance (e.g., Bacillus thuringiensis toxic proteins; U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et al. (1986) Gene 48: 109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24: 825); fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence and disease resistance genes (Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432; Mindrinos et al. (1994) Cell 78: 1089); acetolactate synthase mutants that lead to herbicide resistance such as the S4 and/or Hra mutations; inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., the bar gene); and glyphosate (e.g., the EPSPS gene and the GAT gene; see, for example, U.S. Publication No. 20040082770 and WO 03/092360). Additional polynucleotides that can be stacked with a MRP polynucleotide include, for example, those encoding traits desirable for processing or process products such as modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516); modified starches (e.g., ADPG pyrophosphorylases, starch synthases, starch branching enzymes, and starch debranching enzymes); and polymers or bioplastics (e.g., U.S. Pat. No. 5.602,321). An MRP polynucleotide of the invention also can be stacked with one or more polynucleotides that provide desirable agronomic traits such as male sterility (e.g., U.S. Pat. No. 5.583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g., WO 99/61619; WO 00/17364; WO 99/25821). Other desirable traits that are known in the art include high oil content; increased digestibility; balanced amino acid content; and high energy content. Such traits may refer to properties of both seed and non-seed plant tissues, or to food or feed prepared from plants or seeds having such traits; such food or feed will have improved quality.

[0138] These stacked combinations can be created by any method including but not limited to cross breeding plants by any conventional or TopCross methodology, or genetic transformation. In this regard, it is understood that transformed plants of the invention include a plant that contains a sequence of the invention that was introduced into that plant via breeding of a transformed ancestor plant. If traits are stacked by genetically transforming the plants, the nucleic acids of interest can be combined at any time and in any order. More generally, where any method requires more than one step to be performed, it is understood that steps may be performed in any order that accomplishes the desired end result. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of cassettes suitable for transformation. For example, if two sequences will be introduced, the two sequences can be contained in separate cassettes (trans) or contained on the same transformation cassette (cis). Transcription and/or expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other cassettes to generate the desired combination of traits in the plant. Alternatively, traits may be stacked by transforming different plants to obtain those traits; the transformed plants may then be crossed together and progeny may be selected which contains all of the desired traits.

[0139] Stacking may also be performed with fragments of a particular gene or nucleic acid. In such embodiments, a plants is transformed with at least one fragment and the resulting transformed plant is crossed with another transformed plant; progeny of this cross may then be selected which contain the fragment in addition to other transgenes, including, for example, other fragments. These fragments may then be recombined or otherwise reassembled within the progeny plant, for example, using site-specific recombination systems known in the art. Such stacking techniques could be used to provide any property associated with fragments, including, for example, hairpin RNA (hpRNA) interference or intron-containing hairpin RNA (ihpRNA) interference.

[0140] It is understood that in some embodiments the nucleic acids to be stacked with MRP can also be designed to reduce or eliminate the expression of a particular protein, as described in detail herein for MRP. Thus, the methods described herein with regard to the reduction or elimination of expression of MRP are equally applicable to other nucleic acids and nucleotide sequences of interest, such as, for example, IPPK, ITPK-5, and milps, examples of which are known in the art and which are expected to exist in most varieties of plants. Accordingly, the descriptions herein of MRP fragments, variants, and other nucleic acids and nucleotide sequences apply equally to other nucleic acids and nucleotide sequences of interest such as milps (e.g., SEQ ID NO: 25), IPPK (e.g., SEQ ID NO: 64), ITPK-5 (e.g., SEQ ID NO: 65), IP2K (e.g., SEQ ID NO:67), and Lpa3 or MIK (myo-inositol kinase; e.g., SEQ ID NO: 68). For example, an antisense construct could be designed for milps comprising a nucleotide sequence that shared 90% sequence identity to the complement of SEQ ID NO: 25 or was at least a 19-nucleotide fragment of the complement of SEQ ID NO: 25.

[0141] Transformation protocols as well as protocols for introducing polypeptides or polynucleotides into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing polypeptides or polynucleotides into plant cells and subsequent insertion into the plant genome 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 (Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al., U.S. Pat. No. 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3: 2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., U.S. Pat. No. 5,879,918; Tomes et al., U.S. Pat. No. 5,886,244; Bidney et al., U.S. Pat. No. 5,932,782; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," 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 Weissinger et al. (1988) Ann. Rev. Genet. 22: 421-477; Sanford et al. (1987) Particulate Science and Technology 5: 27-37 (onion); Christou et al. (1988) Plant Physiol. 87: 671-674 (soybean); McCabe et al. (1988) Bio/Technology 6: 923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96: 319-324 (soybean); Datta et al. (1990) Biotechnology 8: 736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85: 4305-4309 (maize); Klein et al. (1988) Biotechnology 6: 559-563 (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment," in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); 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; Bowen et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9: 415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84: 560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12: 250-255 and Christou and Ford (1995) Annals of Botany 75: 407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.

[0142] 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; the resulting progeny having the desired phenotypic characteristic can then be identified. Two or more generations may be grown to ensure that the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure that stable transformants exhibiting the desired phenotypic characteristic have been achieved. In this manner, the present invention provides transformed seed (also referred to as "transgenic seed") having a nucleotide construct of the invention, for example, a cassette of the invention, stably incorporated into their genome.

[0143] As used herein, the term "plant" includes plant cells, plant protoplasts, plant cell tissue cultures from which maize plant can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, 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. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced polynucleotides.

[0144] The present invention may be used for transformation of any plant species, including, but not limited to, monocots and dicots. Examples of plant species of interest include, but are not limited to, corn (Zea mays), Brassica spp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, and conifers.

[0145] Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulcherrima), and chrysanthemum.

[0146] Conifers that may be employed in practicing the present invention include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis). In specific embodiments, plants of the present invention are crop plants (for example, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments, corn and soybean plants are optimal, and in yet other embodiments corn plants are optimal.

[0147] Other plants of interest include grain plants that provide seeds of interest, oil-seed plants, and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.

[0148] The methods of the invention involve introducing a polypeptide or polynucleotide into a plant. "Introducing" is intended to mean presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the invention do not depend on a particular method for introducing a sequence into a plant, only that the polynucleotide or polypeptides gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide or polypeptides into plants are known in the art, including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0149] "Stable transformation" is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or that a polypeptide is introduced into a plant.

[0150] Thus, it is recognized that methods of the present invention do not depend on the incorporation of an entire nucleotide construct into the genome, only that the plant or cell thereof is altered as a result of the introduction of a nucleotide construct or polypeptide into a cell. In one embodiment of the invention, the genome may be altered following the introduction of a nucleotide construct into a cell. For example, the nucleotide construct, or any part thereof, may incorporate into the genome of the plant. Alterations to the genome of the present invention include, but are not limited to, additions, deletions, and substitutions of nucleotides in the genome. While the methods of the present invention do not depend on additions, deletions, or substitutions of any particular number of nucleotides, it is recognized that such additions, deletions, or substitutions comprise at least one nucleotide.

[0151] In other embodiments, the polynucleotides of the invention may be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the invention within a viral DNA or RNA molecule. It is recognized that an MRP of the invention 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 of the invention also encompass promoters utilized for transcription by viral RNA polymerases. Methods for introducing nucleotide constructs 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.

[0152] The use of the term polynucleotides herein is not intended to limit the present invention to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides, comprised of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides may also be employed in the methods disclosed herein. Thus, the nucleotide constructs of the present invention encompass all nucleotide constructs that can be employed in the methods of the present invention for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs of the invention also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures, and the like.

[0153] The promoter nucleotide sequences and methods disclosed herein are useful in regulating expression of any heterologous nucleotide sequence in a host plant in order to vary the phenotype of a plant. Because the Lpa1 promoter provides weak constitutive expression of operably linked coding regions, the Lpa1 promoter finds particular use in altering gene expression in various tissues.

[0154] Various changes in phenotype are of interest including modifying the fatty acid composition in seeds, altering the amino acid content of seeds, altering a seed's pathogen defense mechanism, and the like. These results can be achieved by providing expression of heterologous products or increased expression of endogenous products in embryos. Alternatively, the results can be achieved by providing for a reduction of expression of one or more endogenous products, particularly enzymes or cofactors in the seed. These changes result in a change in phenotype of the transformed plant.

[0155] Genes of interest are reflective of the commercial markets and interests of those involved in the development of the crop. Crops and markets of interest change, and as developing nations open up world markets, new crops and technologies will emerge also. In addition, as our understanding of agronomic traits and characteristics such as yield and heterosis increase, the choice of genes for transformation will change accordingly. General categories of genes of interest include, for example, those genes involved in information, such as zinc fingers, those involved in communication, such as kinases, and those involved in housekeeping, such as heat shock proteins. More specific categories of transgenes, for example, include genes encoding important traits for agronomics, insect resistance, disease resistance, herbicide resistance, sterility, grain characteristics, and commercial products. Genes of interest include, generally, those involved in oil, starch, carbohydrate, or nutrient metabolism as well as those affecting kernel size, sucrose loading, and the like.

[0156] Agronomically important traits such as oil, starch, and protein content can be genetically altered by genetic engineering in addition to using traditional breeding methods. Modifications include increasing content of oleic acid, saturated and unsaturated oils, increasing levels of lysine and sulfur, providing essential amino acids, and also modification of starch. Hordothionin protein modifications are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389, herein incorporated by reference. Another example is lysine and/or sulfur rich seed protein encoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016, and the chymotrypsin inhibitor from barley, described in Williamson et al. (1987) Eur. J. Biochem. 165: 99-106, the disclosures of which are herein incorporated by reference.

[0157] Derivatives of the coding sequences can be made by site-directed mutagenesis to increase the level of preselected amino acids in the encoded polypeptide. For example, the gene encoding the barley high lysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor, U.S. application Ser. No. 08/740,682, filed Nov. 1, 1996, and WO 98/20133, the disclosures of which are herein incorporated by reference. Other proteins include methionine-rich plant proteins such as from sunflower seed (Lilley et al. (1989) Proceedings of the World Congress on Vegetable Protein Utilization in Human Foods and Animal Feedstuffs, ed. Applewhite (American Oil Chemists Society, Champaign, Ill.), pp. 497-502); corn (Pedersen et al. (1986) J. Biol. Chem. 261: 6279; Kirihara et al. (1988) Gene 71: 359); and rice (Musumura et al. (1989) Plant Mol. Biol. 12: 123). Other agronomically important genes encode latex, Floury 2, growth factors, seed storage factors, and transcription factors.

[0158] Insect resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48: 109, and the like.

[0159] Genes encoding disease resistance traits include detoxification genes, such as against fumonisin (U.S. Pat. No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432; and Mindrinos et al. (1994) Cell 78: 1089); and the like.

[0160] Herbicide resistance traits may include genes coding for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS), in particular the sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS) gene containing mutations leading to such resistance, in particular the S4 and/or Hra mutations), genes coding for resistance to herbicides that act to inhibit action of glutamine synthase, such as phosphinothricin or basta (e.g., the bar gene), or other such genes known in the art. The bar gene encodes resistance to the herbicide basta, the nptII gene encodes resistance to the antibiotics kanamycin and geneticin, and the ALS-gene mutants encode resistance to the herbicide chlorsulfuron. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development.

[0161] The quality of grain is reflected in traits such as, for example, levels and types of oils, saturated and unsaturated, quality and quantity of essential amino acids, and levels of cellulose. In corn, modified hordothionin proteins are described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802, and 5,990,389.

[0162] Commercial traits can also be encoded on a gene or genes that could increase for example, starch for ethanol production, or provide expression of proteins. Another important commercial use of transformed plants is the production of polymers and bioplastics such as described in U.S. Pat. No. 5,602,321. Genes such as β-Ketothiolase, PHBase (polyhydroxyburyrate synthase), and acetoacetyl-CoA reductase (see Schubert et al. (1988) J. Bacteriol. 170: 5837-5847) facilitate expression of polyhyroxyalkanoates (PHAs).

[0163] Exogenous products include plant enzymes and products as well as those from other sources including procaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones, and the like. The level of proteins, particularly modified proteins having improved amino acid distribution to improve the nutrient value of the plant, can be increased. This is achieved by the expression of such proteins having enhanced amino acid content.

[0164] Some chemicals can inhibit MRP protein transport activity. For example, the sulfonylurea glibenclamide can inhibit the glucuronide transport activity of Arabidopsis AtMRP5 and can affect its function in guard cells (Gaedeke et al. (2001) EMBO J. 20: 1875-1887; Lee et al. (2004) Plant Physiol. 134: 528-538). It is expected that glibenclamide would also inhibit maize MRP3 transport activity and thus would produce a low phytic acid phenotype.

[0165] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

EXPERIMENTAL

Example 1

Identification and Characterization of Maize Low Phytic Acid (Lpa) Mutant Plants

[0166] A collection of indexed mutagenized F2 families derived from several Mu active stocks (Bensen et al. (1995) Plant Cell 7: 75-84) was screened for seeds having high inorganic phosphate content using a rapid P_i assay as described below. Candidates identified as producing high-P_i seed were crossed with suitable maize and the progeny examined to confirm the mutations and to determine whether the mutations were allelic to the previously identified lpa1 mutant (referred to herein as "lpa1-1"; see U.S. Pat. No. 5,689,054; Raboy et al. (2000) Plant Physiol. 124: 355-68). Several of these lpa lines were allelic to the earlier-identified lpa1 mutant, and these Mu-insertion alleles of the lpa1 mutant were used to clone the gene responsible for the lpa1 mutation. Segregation populations were created by crossing heterozygous line PV03 57 C-05 (carrying Mu-tagged lpa1) with homozygous line GP24L3 (carrying EMS allele lpa1-1). F1 plants were self-pollinated to produce F2 seeds. The phenotype of F1 plants was determined by analyzing F2 seed Pi and phytic acid. Genomic DNA was extracted from leaves of individual F1 plants and used for PCR analysis as further described in Example 2.

Inorganic Phosphate (Pi) Assay

[0167] A rapid test was used to assay inorganic phosphate content in kernels. Individual kernels were placed in a 25-well plastic tray and crushed at 2000 psi using a hydraulic press. Two milliliters of 1N H₂SO₄ was added to each sample. The samples were incubated at room temperature for two hours, after which four milliliters of 0.42% ammonium molybdate-1N H₂SO₄:10% ascorbic acid (6:1) was added to each sample. Increased P_i content was signaled by the development of blue color within about 20 minutes. Positive controls included lpa2 mutant kernels, and negative controls included wild-type kernels.

Determination of Phytic Acid and Inorganic Phosphate Content

[0168] Dry, mature seeds were assayed for phytic acid and P_i content using modifications of the methods described by Haug and Lantzsch ((1983) J. Sci. Food Agric. 34: 1423-1426, entitled "Sensitive method for the rapid determination of phytate in cereals and cereal products") and Chen et al. ((1956) Anal. Chem. 28: 1756-1758, entitled "Microdetermination of phosphorus"). Single kernels were ground using a Geno/Grinder2000® grinder (Sepx CertiPrep®, Metuchen, N.J.). Samples of 25 to 35 mg were placed into 1.5 ml Eppendorf® tubes and 1 ml of 0.4 N HCl was added to the tubes, which were then shaken on a gyratory shaker at room temperature for 3.5 hours. The tubes were then centrifuged at 3,900 g for 15 minutes. Supernatants were transferred into fresh tubes and used for both phytic acid and P_i determinations; measurements were performed in duplicate.

[0169] For the phytic acid assay, 35 μl of each extract was placed into wells of a 96-well microtiter plate and then 35 μl of distilled H₂O and 140 μl of 0.02% ammonium iron (III) sulphate-0.2 N HCl were added to each sample. The microtiter plate was covered with a rubber lid and heated in a thermal cycler at 99° C. for 30 minutes, then cooled to 4° C. and kept on an ice water bath for 15 minutes, and then left at room temperature for 20 minutes. The plate was then sealed with sticky foil and centrifuged at 3,900 g at 24° C. for 30 minutes. Eighty μl of each supernatant was placed into wells of a fresh 96-well plate. For absorbance measurements, 120 μl of 1% 2,2'-bipyridine-1% thioglycolic acid solution (10 g 2,2'-bipyridine (Merck Art. 3098), 10 ml thioglycolic acid (Merck Art. 300) in ddw to 1 liter) was added to each well and absorbance was recorded at 519 nm using a VERSAmax® microplate reader (Molecular Devices®, Sunnyvale, Calif.). Phytic acid content is presented as phytic acid phosphorus (PAP). Authentic phytic acid (Sigma®, P-7660) served as a standard. This phytic acid assay also measured InsP₅ and InsP₄ present in the samples.

[0170] Phytic acid was also assayed according to modifications of the methods described by Latta & Eskin (1980) (J. Agric Food Chem. 28: 1313-1315) and Vaintraub & Lapteva (1988) (Analytical Biochemistry 175: 227-230). For this assay, 25 μl of extract was placed into wells of a 96-well microtiter plate; then 275 μl of a solution of 36.3 mM NaOH and 100 μl of Wade reagent (0.3% sulfosalicylic acid in 0.03% FeCl₃.6H₂O) was added to each well. The samples were mixed and centrifuged at 39,000 g at 24° C. for 10 minutes. An aliquot of supernatant (200 μl) from each well was transferred into a new 96-well plate, and absorbance was recorded at 500 nm using a VERSAmax® microplate reader (Molecular Devices®, Sunnyvale, Calif.).

[0171] To determine P_i, 200 μl of each extract was placed into wells of a 96 well microtiter plate. 100 μl of 30% aqueous trichloroacetic acid was then added to each sample and the plates were shaken and then centrifuged at 3,900 g for 10 minutes. Fifty μl of each supernatant was transferred into a fresh plate and 100 μl of 0.42% ammonium molybdate-1N H₂SO₄:10% ascorbic acid (7:1) was added to each sample. The plates were incubated at 37° C. for 30 minutes and then absorbance was measured at 800 nm. Potassium phosphate was used as a standard. P_i content was presented as inorganic phosphate phosphorus.

Determination of Seed Myo-Inositol

[0172] Myo-inositol was quantified in dry, mature seeds and excised embryos. Tissue was ground as described above and mixed thoroughly. 100 milligram samples were placed into 7 ml scintillation vials and 1 ml of 50% aqueous ethanol was added to each sample. The vials were then shaken on a gyratory shaker at room temperature for 1 hour. Extracts were decanted through a 0.45 μm nylon syringe filter attached to a 1 ml syringe barrel. Residues were re-extracted with 1 ml fresh 50% aqueous ethanol and the second extracts were filtered as before. The two filtrates were combined in a 10×75 mm glass tube and evaporated to dryness in a SpeedVac® microcentrifuge (Savant). The myo-inositol derivative was produced by redissolving the residues in 50 μl of pyridine and 50 μl of trimethylsilyl-imidazole:trimethylchlorosilane (100:1) (Tacke and Casper (1996) J. AOAC Int. 79: 472-475). Precipitate appearing at this stage indicates that the silylation reaction did not work properly. The tubes were capped and incubated at 60° C. for 15 minutes. One milliliter of 2,2,4-trimethylpentane and 0.5 milliliters of distilled water were added to each sample. The samples were then vortexed and centrifuged at 1,000 g for 5 minutes. The upper organic layers were transferred with Pasteur pipettes into 2 milliliter glass autosampler vials and crimp capped.

[0173] Myo-inositol was quantified as a hexa-trimethylsilyl ether derivative using an Agilent Technologies® model 5890 gas chromatograph coupled with an Agilent Technologies® model 5972 mass spectrometer. Measurements were performed in triplicate. One μl samples were introduced in the splitless mode onto a 30 m×0.25 mm i.d.×0.25 μm film thickness 5MS column (Agilent Technologies®). The initial oven temperature of 70° C. was held for 2 minutes, then increased at 25° C. per minute to 170° C., then increased at 5° C. per minute to 215° C., and finally increased at 25° C. per minute to 250° C. and then held for 5 minutes. The inlet and transfer line temperatures were 250° C. Helium at a constant flow of 1 ml per minute was used as the carrier gas. Electron impact mass spectra from m/z 50-560 were acquired at -70 eV after a 5-minute solvent delay. The myo-inositol derivative was well resolved from other peaks in the total ion chromatograms. Authentic myo-inositol standards in aqueous solutions were dried, derivatized, and analyzed at the same time. Regression coefficients of four-point calibration curves were typically 0.999-1.000.

Determination of Seed Inositol Phosphates

[0174] The presence of significant amounts of inositol phosphates in mature seeds was determined by HPLC according to the Dionex Application Note AN65, "Analysis of inositol phosphates" (Dionex Corporation®, Sunnyvale, Calif.). Tissue was ground and mixed as described above. 500 mg samples were placed into 20 ml scintillation vials and 5 ml of 0.4 M HCl was added to the samples. The samples were shaken on a gyratory shaker at room temperature for 2 hours and then allowed to sit at 4° C. overnight. Extracts were centrifuged at 1,000 g for 10 min and filtered through a 0.45 μm nylon syringe filter attached to a 5 ml syringe barrel. Just prior to HPLC analysis, 600 μl aliquots of each sample were clarified by passage through a 0.22 μm centrifugal filter. A Dionex Corporation® DX 500 HPLC with a Dionex Corporation® model AS3500 autosampler was used. 25 μl samples were introduced onto a Dionex Corporation® 4×250 mm OmniPac® PAX-100 column; Dionex Corporation® 4×50 mm OmniPac® PAX-100 guard and ATC-1 anion trap columns also were used. Inositol phosphates were eluted at 1 ml/min with the following mobile phase gradient: 68% A (distilled water)/30% B (200 mM NaOH) for 4.0 min; 39% A/59% B at 4.1 through 15.0 min; return to initial conditions at 15.1 min. The mobile phase contained 2% C (50% aqueous isopropanol) at all times to maintain column performance. A Dionex Corporation® conductivity detector module II was used with a Dionex Corporation® ASRS-Ultra II anion self-regenerating suppressor set up in the external water mode and operated with a current of 300 mA. Although quantitative standards were available, InsP₃, InsP₄ and InsP₅ were partially but clearly resolved from each other and InsP₆.

[0175] The results of the above assays demonstrated that the lpa1 mutant maize plants have a phenotype of reduced phytic acid and increased P_i in seeds, but lpa1 seeds do not accumulate inositol phosphate intermediates.

Example 2

Isolation and Characterization of Maize MRP3 (Lpa1) Gene

[0176] Initially, a PCR-based method was used in an effort to clone the lpa1 gene, but this effort was unsuccessful. However, a Mu-insertion site in a transcriptional activator gene was identified, and co-segregation analysis indicated that this Mu-insertion site was very closely linked to the Lpa1 locus. This marker, designated "TAP," was used for map-based cloning of the Lpa1 gene.

[0177] The PCR protocol used to identify the TAP marker is known as SAIFF: Selected Amplification of Insertion Flanking Fragments. First, genomic DNA was prepared from 5-8 plants of individual lines which were Mu.sup.+ and Mu.sup.-. The genomic DNA was digested with BfaI or MseI, neither of which cuts the Mu TIR (Terminal Inverted Repeat). The restriction ends generated by BfaI and MseI are the same and are compatible with the Mse/Bfa adaptor. [0178] 10× RL buffer: 2.5 μl [0179] BSA: 0.25 μl [0180] DNA: 0.3-0.5 μg [0181] Enzymes: 1 μl [0182] Water: bring to 25 μl

[0183] This mixture was incubated at 37° C. for 3 to 6 hours and then denatured at 65° C. for 20 minutes. Adaptors were then ligated to the digested DNA by adding 5 μl of adaptor mixture to each reaction: [0184] 100 mM rATP: 0.3 μl [0185] 10× RL buffer: 0.5 μl [0186] 40 uM Adaptor: 1 μl [0187] T4 ligase: 1 μl (3 U/μl) [0188] Water: bring to 5 μl

[0189] This mixture was then incubated at 4° C. overnight. The ligation reaction was purified with a PCR Purification Kit (Qiagen®) to remove excess adaptors, and the reaction was brought to a final volume of 50 μl in water or elution buffer.

[0190] Control PCR was performed to check the digestion and ligation. Either regular Taq enzyme or another non-hot start DNA polymerase was used for the control PCR. 1 μl of the purified ligation reaction was used as the template in a 10 μl PCR reaction. The primer used was the adaptor primer (MspExt18 or the nested MseInt18 primer). DMSO was added to the mixture to a final level of 5%. The PCR conditions were 94° C. 2 min; 35 cycles of 94° C. 30 sec, 55° C. 30 sec, and 72° C. 2 min 30 sec; and a final extension at 72° C. for 7 min. The reaction was then run on a 1% agarose gel and the amplification reaction visualized. Non-specific adaptor-to-adaptor amplification should occur, and there should be a nice smear on the gel ranging in size from 300 bp to 3 kb.

[0191] 1 μl of the purified ligation reaction was then used as the template in a 10 μl PCR reaction using Hot Start® DNA polymerase (Qiagen®). Primers MuExt22D and MspExt18 were added to a final concentration of 0.3-0.5 μM. DMSO was added to a final level of 5%. PCR conditions were 95° C. 15 min, 20 cycles of 94° C. 30 sec, 55° C. 30 sec, and 72° C. 2 min 30 sec, followed by a final extension at 72° C. for 7 min. The reaction was then diluted 1:10 with water.

[0192] Nested (2^nd round) PCR was performed with Ex Taq DNA polymerase, but any robust enzyme could be used. 1 μl of the Mu+ and Mu- pools was used as template in a 10 μl reaction. The primers were MuInt19 and Adaptor nested primers (+2 selective primers, 0.3-0.5 μM final concentration). DMSO was added to a final level of 5%. "Touchdown" PCR conditions were: 95° C. 2 min, 11 cycles of 94° C. 30 sec, (65° C.-0.8° C./cycle) for 30 sec, and 72° C. 2 min 30 sec, followed by 24 cycles of 94° C. 30 sec, 56° C. 30 sec, and 72° C. 2 min 30 sec, with a final extension at 72'C for 7 min. PCR reactions were electrophoresed on a 1.5% agarose gel and examined to identify bands which were present in the Mu+ pool but absent in the Mu- pool.

[0193] The second-round (nested) PCR was then repeated using as template first round PCR reactions from individual plants to confirm the co-segregation. DNA fragments that were present in all Mu+ individuals and absent in all Mu- individuals were isolated from the gel and purified. The purified DNA was cloned into a vector such as TOPO TA or pGEM-T Easy according to the manufacturer's instructions (Invitrogen®, Carlsbad, Calif.; Promega®, Madison, Wis.).

[0194] Clones were screened with PCR to identify correctly-cloned inserts for each fragment of interest. White colonies (8) were selected and resuspended in 40 μl water; the remainder of the colony was streaked on selective media (LB+Amp) for later recovery. 1 μl of the resuspended colonies were used as the template in a 10 μl PCR reaction. PCR conditions were the same as described above for nested PCR, and one positive clone was selected for each fragment.

[0195] Cultures of bacteria carrying the selected clone were grown in liquid selective media (LB+Amp). Plasmid minipreps were performed using a Spin Column Miniprep Kit (Qiagen®). The final volume was brought to 50 μl with elution buffer, and the minipreps were checked by digesting 2 μl of plasmid DNA with EcoRI. The DNA was then sequenced to confirm that each plasmid contained the MuTIR (53 bp including the MuInt19 site). The sequence of the fragment was then used to design a fragment-specific primer to pair with MuInt19 or MuExt22D, and co-segregation analysis was performed using PCR on DNA from all individuals in the segregation population.

[0196] BfaI and MseI share the same adaptor:

TABLE-US-00001 MseI/BfaI adaptor--lower: (SEQ ID NO: 26) 5'-TACTCAGGACTCATCGACCGT-3' MseI/BfaI adaptor--upper: (SEQ ID NO: 27) 5'-GTGAACGGTCGATGAGTCCTGAG-3'

[0197] Adaptors were made by mixing these two oligonucleotides, denaturing at 95° C. for 5 minutes, and then cooling the mixture down slowly to room temperature. The adaptor is designed in such a way that the original restriction sites are not restored after the ligation.

TABLE-US-00002 Adaptor Ext 18 primer (MspExtl8): (SEQ ID NO: 28) 5'-GTGAACGGTCGATGAGTC-3' MseI/BfaI adp Int18 primer (MseInt18): (SEQ ID NO: 29) 5'-GTCGATGAGTCCTGAGTA-3' BfaI +2 selective primers (16): (SEQ ID NO: 30) BfaIntGAA: GATGAGTCCTGAGTAGAA (SEQ ID NO: 31) BfaIntGAC: GATGAGTCCTGAGTAGAC (SEQ ID NO: 32) BfaIntGAG: GATGAGTCCTGAGTAGAG (SEQ ID NO: 33) BfaIntGAT: GATGAGTCCTGAGTAGAT (SEQ ID NO: 34) BfaIntGCA: GATGAGTCCTGAGTAGCA (SEQ ID NO: 35) BfaIntGCC: GATGAGTCCTGAGTAGCC (SEQ ID NO: 36) BfaIntGCG: GATGAGTCCTGAGTAGCG (SEQ ID NO: 37) BfaIntGCT: GATGAGTCCTGAGTAGCT (SEQ ID NO: 38) BfaIntGGA: GATGAGTCCTGAGTAGGA (SEQ ID NO: 39) BfaIntGGC: GATGAGTCCTGAGTAGGC (SEQ ID NO: 40) BfaIntGGG: GATGAGTCCTGAGTAGGG (SEQ ID NO: 41) BfaIntGGT: GATGAGTCCTGAGTAGGT (SEQ ID NO: 42) BfaIntGTA: GATGAGTCCTGAGTAGTA (SEQ ID NO: 43) BfaIntGTC: GATGAGTCCTGAGTAGTC (SEQ ID NO: 44) BfaIntGTG: GATGAGTCCTGAGTAGTG (SEQ ID NO: 45) BfaIntGTT: GATGAGTCCTGAGTAGTT MseI +2 selective primers (16): (SEQ ID NO: 46) MseIntAAA: CGATGAGTCCTGAGTAAAA (SEQ ID NO: 47) MseIntAAC: CGATGAGTCCTGAGTAAAC (SEQ ID NO: 48) MseIntAAG: CGATGAGTCCTGAGTAAAG (SEQ ID NO: 49) MseIntAAT: CGATGAGTCCTGAGTAAAT (SEQ ID NO: 50) MseIntACA: CGATGAGTCCTGAGTAACA (SEQ ID NO: 51) MseIntACC: GATGAGTCCTGAGTAACC (SEQ ID NO: 52) MseIntACG: GATGAGTCCTGAGTAACG (SEQ ID NO: 53) MseIntACT: GATGAGTCCTGAGTAACT (SEQ ID NO: 54) MseIntAGA: CGATGAGTCCTGAGTAAGA (SEQ ID NO: 55) MseIntAGC: GATGAGTCCTGAGTAAGC (SEQ ID NO: 56) MseIntAGG: GATGAGTCCTGAGTAAGG (SEQ ID NO: 57) MseIntAGT: CGATGAGTCCTGAGTAAGT (SEQ ID NO: 58) MseIntATA: CGATGAGTCCTGAGTAATA (SEQ ID NO: 59) MseIntATC: GATGAGTCCTGAGTAATC (SEQ ID NO: 60) MseIntATG: GATGAGTCCTGAGTAATG (SEQ ID NO: 61) MseIntATT: CGATGAGTCCTGAGTAATT

[0198] 10× RL buffer: [0199] 100 mM Tris-HCl, pH 7.5 [0200] 100 mM MgOAc, [0201] 500 mM KOAc, [0202] 50 mM DTT

[0203] Map-based cloning requires a high-resolution genetic map and a physical map around the locus of interest. Using the TAP marker, which was closely linked to the Lpa1 locus, the inventors identified a BAC contig containing about 120 BAC clones from a proprietary BAC library. PCR markers were developed based on BAC-end sequences and EST sequences, and the segregating populations of individuals described above were also used for genetic mapping. Individual F1 seeds were phenotyped by measuring Pi and phytic acid content. DNA was extracted from the individual F1 seeds with the Qiagen® Genomic DNA Purification Kit. Individuals were genotyped using PCR carried out according to the instructions of the Expand High Fidelity PCR system (Roche®). 792 individuals were analyzed to construct a fine map around the Lpa1 locus.

[0204] Based on the genetic map and the BAC physical map, the inventors identified two over-lapping BACs which cover the Lpa1 locus. The two BACs, b149a.i9 and b156a.m1, were sequenced. Open reading frames in each BAC were identified by using the Fgenesh computer program and BLAST searching against maize EST databases. BAC b149a.i9 is 140 kb in length and has several ORFs predicted by Fgenesh. Only two ORFs were found to have corresponding ESTs. One of the ORFs encodes an MRP ABC transporter protein. Gene-specific primers were synthesized from these two ORFs and used to search for the Mu insertion in the lpa1 mutant Mu-insertion alleles. A Mu insertion was found in the MRP ABC transporter gene in lpa1 allele PV03 56 C-05. A Mu insertion was also found in the same gene for eight other lpa1 alleles. Mu is inserted in Exon 1 at nucleotide 585 in Mu82978.17; at nucleotide 874 in PV03 57 C-3; and in Exon 11 at nucleotide 6069 in Mu82911.08. The remaining 6 alleles all have the same Mu insertion site as Mu82978.17. The MRP gene was also sequenced from four lpa1 EMS alleles. In two alleles (91286 and 94580), a stop codon was introduced in place of codons encoding R and Q at amino acids 371 and 595, respectively. In allele 91281, E was changed to L at amino acid 680, while in the original lpa1-1 allele, A was mutated to V at amino acid 1432.

[0205] The maize MRP ABC transporter gene was designated ZmMRP3 (Zea mays multidrug resistance-associated protein 3), or Lpa1 (low phytic acid). The MRP group of the ABC transporter family includes many proteins which are involved in diverse cellular responses. MRPs can transport a great range of substances. Some of the MRPs also have regulatory activity on other transporters or channel proteins. This maize MRP (ZmMRP3) is the first MRP shown to play a role in phytic acid metabolism and cellular function, and provides a new way in which phytic acid and available phosphorus content of plant seeds may be manipulated. Previously, the phytic acid biosynthesis pathway was altered by manipulating genes encoding the enzymes that convert glucose 6-P to phytic acid. In contrast, while the invention is not bound by a particular mechanism of operation, MRP is a transporter and/or transporter regulator. Thus, altering MRP expression and/or functionality in transgenic plants would be expected to have minimal effects on InsP intermediates of phytic acid biosynthesis pathway.

[0206] During the course of this study, the inventors determined that knockout lpa1 alleles are lethal when they are homozygous. Because the Lpa1 gene has now been cloned and further characterized as disclosed herein, it is now possible to make transgenic plants with Lpa1 expression constructs under tight control. An advantage of using Lpa1 is that it could be used to develop the low phytic acid trait without changing the composition of myo-inositol phosphate intermediates. In addition, a suppression of Lpa1 expression that was limited to suppression in developing embryos could produce transgenic plants having low phytic acid and high available phosphorus in seeds with minimal impact on agronomic performance.

[0207] Thus, SEQ ID NO:1 sets forth the genomic sequence of ZmMRP3 (Lpa1), SEQ ID NO:2 sets forth the deduced cDNA sequence, and SEQ ID NO: 3 sets forth the deduced amino acid sequence of the ZmMRP3 (Lpa1) protein. The Lpa1 protein contains 1510 amino acids and has a calculated molecular weight of about 166.8 kiloDaltons and a pI of about 8.44.

Zm-MRP3 Protein Structure

[0208] The Lpa1 polypeptide was identified as an ABC transporter, as it contains consensus features of the ABC transporter family of proteins. ABC transporters are a large family of proteins found in bacteria, fungi, plants and animals. In coupling to ATP hydrolysis, the ABC transporter transports a great variety of substrates across the plasma membrane and various intracellular membranes. Among the substrates known to be transported by ABC transporters are sugars, amino acids, inorganic acids, lipids, peptides, heavy metal ions, glutathione conjugates, alkloids, and secondary metabolites.

[0209] The member of the ABC superfamily can be divided into several subfamilies based on phylogenic pathways and structural features. The names used to define the subfamilies are historic and related to the function of drug resistance, although many members are not involved in drug transport. The three best characterized subfamilies are the pleiotropic drug resistance protein (PDR), multidrug resistance protein (MDR), and multidrug resistance-associated protein (MRP). The maize Lpa1 is a MRP ABC transporter. Previously, two MRP genes, ZmMRP1 and ZmMRP2, have been cloned from maize and their function is not known. The Lpa1 gene differs from those two ZmMRPs and thus was designated ZmMRP3.

[0210] FIG. 1A and FIG. 1B show a comparison of the Lpa1 polypeptide with Pfam consensus sequences for the ABC transporter ("ABC_tran"; Pfam Accession No. PF00005; SEQ ID NO: 62) and the ABC transporter transmembrane region ("ABC_membrane"; Pfam Accession No. PF00664; SEQ ID NO: 63). All ABC proteins consist of one or two copies of a modular structure which has two basic structural elements: an integral transmembrane domain (TMD) and a cytosolic ATP-binding domain (also known as nucleotide binding fold, or NBF). The NBF is involved in binding ATP and it contains a Walker A box, an ABC signature motif, and a Walker B box. The Walker A and B boxes also are found in other nucleotide-binding proteins, such as P-, F- and V-ATPase, G-proteins and adenylate kinase. The ABC signature motif, however, is unique to the NBFs of ABC transporters.

[0211] The members of the MRP subfamily of ABC transporters have two copies of the modular structure (see FIG. 1). Maize ZmMRP3 contains about 10 transmembrane spans in the first copy and 4 in the second copy. Two ATP-binding domains of ZmMRP3 are located at amino acids 631-843 and amino acids 1267-1450, respectively. Within the ATP-binding domains, a Walker A box is at amino acids 664-672 (GVIGSGKSS; SEQ ID NO: 18) and amino acids 1301-1309 (GRTGSGKST; SEQ ID NO: 19), an ABC signature motif is at amino acids 754-765 (LSGGQKQRVQLA; SEQ ID NO: 20) and amino acids 1404-1415 (WSVGQRQLIALG; SEQ ID NO: 21), and a Walker B box is at amino acids 774-779 (IYLLDD; SEQ ID NO: 22) and amino acids 1424-1428 (ILVLD; SEQ ID NO: 23). The second ATP-binding domain of ZmMRP3 is followed by a C1 domain with a motif of IAHRI (SEQ ID NO: 24) from amino acids 1458-1462.

[0212] The MRP gene was amplified from different maize lines by PCR and sequenced. This revealed a variant Lpa1 polypeptide (SEQ ID NO: 5) which differs from Lpa1 at positions 3, 17, and 61. This variant polypeptide is encoded by the cDNA set forth in SEQ ID NO: 4.

Example 3

Identification of Lpa1 Homologs in Other Plants

[0213] Database searches identified similar proteins from other plants which were not previously known to have a role in phytic acid metabolism as discussed herein. Accordingly, the invention additionally provides Lpa1 plant proteins and proteins comprising Lpa1 consensus sequences and domains as well as polynucleotides encoding them.

[0214] The maize MRP3 (Lpa1) gene is located on the short arm of chromosome 1 and consists of 11 exons and 10 introns. It is well known that there is significant conservation of gene content and gene order among the genomes of the plant family Gramineae. Previously, extensive studies have been focused on comparison of rice and maize gene linkage blocks and a comparative map established. Using the Lpa1 locus and its surrounding sequences, the inventors found the corresponding region in rice on chromosome 3 and identified an MRP gene in this region. Although twelve rice MRP genes had been annotated previously (Jasinski et al. (2003) Plant Physiol. 131: 1169-77), this annotation did not include this MRP on chromosome 3, which we designated OsMRP13 (SEQ ID NO: 6). OsMRP13 has the same number of exons and introns as the maize Lpa1 gene ZmMRP3 and encodes a protein of 1505 amino acids (SEQ ID NO: 7). The maize MzMRP3 and rice OsMRP13 genes share 83% nucleotide sequence identity and the encoded proteins share 91% amino acid sequence identity (see FIGS. 4 and 5). The two genes also share similar structures (see FIG. 2). The inventors conducted a Lynx® study to determine the expression patterns of the rice gene. Lynx® gene expression profiling technology utilizes massively parallel signature sequence (MPSS; see Brenner et al. (2000) Nature Biotechnology 18: 630-634; Brenner et al. (2000) Proc. Nat'l. Acad. Sci. USA 97: 1665-1670). MPSS generates 17-mer sequence tags of millions of cDNA molecules, which are cloned on microbeads. The technique provides an unprecedented depth and sensitivity of mRNA detection, including messages expressed at very low levels. The Lynx® database search revealed that the rice gene OsMRP13 is expressed in developing seeds but has lower levels of expression in other tissues. It is very likely that the rice OsMRP13 has the same function as the maize Lpa1 gene in phytic acid metabolism in developing seeds.

[0215] Arabidopsis has 14 known MRP genes (AtMRP15 is a pseudogene). The inventors discovered that AtMRP5 has the same exon/intron organization as the maize ZmMRP3 gene, and that the sizes of corresponding exons and introns also are similar. The maize ZmMRP3 and Arabidopsis AtMRP5 share 62% nucleotide sequence identity and 67% amino acid sequence identity. Among the 14 known Arabidopsis MRPs, AtMRP5 shares the highest level of sequence identity with ZmMRP3. A Lynx® study was performed on AtMRP5 and confirmed that AtMRP5 is expressed in Arabidopsis seeds. It remains to be determined whether Arabidopsis AtMRP5 has the same function as maize ZmMRP3 in phytic acid metabolism.

[0216] A soybean homolog of maize ZmMRP3 also was identified by searching a soybean EST database. The inventors conducted a Lynx® study to characterize the expression of the soybean gene (corresponding to the sequence set forth in SEQ ID NO: 10). The Lynx® study revealed that the soybean gene is expressed in developing seeds but has lower levels of expression in other tissues. A study of EST distribution in various plant tissues also indicated that the soybean gene expression is seed-preferred.

Example 4

Stacking Lpa1 with Other Inositol Phosphate Kinase Genes

[0217] By "stacking" (i.e., transforming a plant with) constructs designed to reduce or eliminate the expression of Lpa1 and other proteins, it is expected that the reduction of phytic acid and increase in available phosphorus will be enhanced in comparison to plants transformed with constructs designed to reduce or eliminate the expression of Lpa1 alone. Accordingly, expression cassettes are prepared making use of inverted repeat constructs known as Inverted Repeats Without Terminators, or "IRNTs." The first and second portion of such constructs self-hybridize to produce a hairpin structure which can suppress expression of the relevant endogenous gene. Each expression cassette contains an IRNT ("Lpa1 IRNT") that can suppress endogenous Lpa1 gene expression. This Lpa1 IRNT includes two portions of an Lpa1 inverted repeat surrounding the Adh1 gene intron. Other expression cassettes contain an additional IRNT that can suppress expression of IPPK, ITPK-5, myo-inositol kinase (MIK), IP2K, phytase, and MI1PS3, respectively. "Glb1" indicates the globulin 1 promoter, and "Ole" indicates the oleosin promoter. Each expression cassette is provided in a plasmid which contains additional useful features for transformation and expression in plants. Lpa1 constructs can also be stacked with constructs designed to increase the expression of other proteins, such as, for example, phytase.

[0218] The plasmids are inserted into Agrobacterium vectors and used to transform maize cells. Sample protocols for creation of Agrobacterium strains harboring a plasmid are described, for example, in Lin (1995) in Methods in Molecular Biology, ed. Nickoloff, J. A. (Humana Press, Totowa, N.J.). Successful transformation can be verified by restriction analysis of the plasmid after transformation back into E. coli DH5α cells. The Agrobacterium is used to transform a host plant such as maize, and the resulting transgenic plants are screened for transformation and for phytic acid phenotype as described in detail above.

[0219] In some embodiments, the Lpa1 gene is mutated and the mutated Lpa1 gene is over-expressed in order to generate transgenic plants with dominant phenotype of reduced Lpa1 activity. For example, the mutation found in EMS-generated allele lpa1-1 is A1432V (i.e., the alanine at position 1432 is changed to valine). This mutation can be introduced into a polynucleotide by PCR-based mutagenesis in which a primer is synthesized with an altered nucleotide corresponding to the desired change. The resulting PCR product is then ligated with other fragments to make a full-length mutated Lpa1 gene carrying the lpa1-1 mutation. A transformation construct consisting of the mutated Lpa1 gene driven by the oleosin promoter could be used to produce transgenic plants having the dominant phenotype of reduced Lpa1 activity; these plants would yield grain with reduced phytate and increased available phosphorus.

[0220] Total knockout of the Lpa1 gene (for example, in Mutator-insertion alleles) is lethal. It is believed that the lethality of an Lpa1 knockout could be rescued by overexpressing phytase in a plant lacking Lpa1 activity.

[0221] Plants with Lpa1 constructs or mutations can then be crossed with plants containing other constructs to obtain progeny containing multiple constructs. Thus, for example, a plant with an Lpa1 construct can be crossed with a plant containing an Lpa3 construct; progeny containing both the Lpa1 and the Lpa3 construct may then be obtained.

Example 5

Production of Lpa1 Transgenic Plants using Agrobacterium-Mediated Transformation

[0222] For Agrobacterium-mediated transformation of maize with an Lpa1 construct of the invention, preferably the method of Zhao is employed (U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326; the contents of which are hereby incorporated by reference). Briefly, immature embryos are isolated from maize and the embryos contacted with a suspension of Agrobacterium, where the bacteria are capable of transferring the Lpa1 construct to at least one cell of at least one of the immature embryos (step 1: the infection step). In this step the immature embryos are preferably immersed in an Agrobacterium suspension for the initiation of inoculation. The embryos are co-cultured for a time with the Agrobacterium (step 2: the co-cultivation step). Preferably the immature embryos are cultured on solid medium following the infection step. Following this co-cultivation period, an optional "resting" step is contemplated. In this resting step, the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium without the addition of a selective agent for plant transformants (step 3: resting step). Preferably the immature embryos are cultured on solid medium with antibiotic, but without a selecting agent, for elimination of Agrobacterium and for a resting phase for the infected cells. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step). Preferably, the immature embryos are cultured on solid medium with a selective agent resulting in the selective growth of transformed cells. The callus is then regenerated into plants (step 5: the regeneration step), and preferably calli grown on selective medium are cultured on solid medium to regenerate the plants.

Bombardment and Culture Media

[0223] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (Sigma® C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000× Sigma®-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-I H₂O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite® (added after bringing to volume with D-I H₂O); and 8.5 mg/l silver nitrate (added after sterilizing the medium and cooling to room temperature). Selection medium (560R) comprises 4.0 g/l N6 basal salts (Sigma® C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000× Sigma®-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose, and 2.0 mg/l 2,4-D (brought to volume with D-I H₂O following adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite® (added after bringing to volume with D-I H₂O); and 0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added after sterilizing the medium and cooling to room temperature).

[0224] Plant regeneration medium (288J) comprises 4.3 g/l MS salts (Gibco® 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H₂O) (Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume with polished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite® (added after bringing to volume with D-I H₂O); and 1.0 mg/l indoleacetic acid and 3.0 mg/l bialaphos (added after sterilizing the medium and cooling to 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (Gibco® 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and 0.40 g/l glycine brought to volume with polished D-I H₂O), 0.1 g/l myo-inositol, and 40.0 g/l sucrose (brought to volume with polished D-I H₂O after adjusting pH to 5.6); and 6 g/l Bacto-agar (added after bringing to volume with polished D-I H₂O), sterilized and cooled to 60° C.

Example 6

Production of Lpa1 Transgenic Plants using Soybean Embryo Transformation

[0225] Soybean embryos are bombarded with a plasmid containing an Lpa1 construct as follows. To induce somatic embryos, cotyledons 3-5 mm in length dissected from surface-sterilized, immature seeds of the soybean cultivar A2872 are cultured in the light or dark at 26° C. on an appropriate agar medium for six to ten weeks. Somatic embryos producing secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos that multiplied as early, globular-staged embryos, the suspensions are maintained as described below.

[0226] Soybean embryogenic suspension cultures can maintained in 35 ml liquid media on a rotary shaker at 150 rpm at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 ml of liquid medium.

[0227] Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont Biolistic PDS1000/HE instrument (helium retrofit) can be used for these transformations.

[0228] A selectable marker gene that can be used to facilitate soybean transformation is a transgene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313: 810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188), and the 3' region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The expression cassette comprising the Lpa1 construct operably linked to the CaMV 35S promoter can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.

[0229] To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (in order): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂ (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μl 70% ethanol and resuspended in 40 μl of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five microliters of the DNA-coated gold particles are then loaded on each macro carrier disk.

[0230] Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm Petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi, and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.

[0231] Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post-bombardment with fresh media containing 50 mg/ml hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post-bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.

Example 7

Production of Lpa1 Transgenic Plants using Brassica Napus Seed Transformation

[0232] Brassica napus seeds are transformed using a transformation and regeneration protocol modified from Mehra-Palta et al. (1991), "Genetic Transformation of Brassica napus and Brassica rapa," in Proc. 8^th GCIRC Congr., ed. McGregor (University Extension Press, Saskatoon, Sask., Canada), pp. 1108-1115 and Stewart et al. (1996), "Rapid DNA Extraction From Plants," in Fingerprinting Methods Based on Arbitrarily Primed PCR, Micheli and Bova, eds. (Springer, Berlin), pp. 25-28. See Cardoza and Stewart (2003) Plant Cell Rep. 21: 599-604.

[0233] Seeds are surface-sterilized for 5 minutes with 10% sodium hypochlorite with 0.1% Tween® added as a surfactant, rinsed for one minute with 95% ethanol, and then washed thoroughly with sterile distilled water. Seeds are germinated on MS basal medium (Murashige and Skoog (1962) Physiol. Plant 15: 473-497) containing 20 g/liter sucrose and 2 g/liter Gelrite®. Hypocotyls are excised from 8- to 10-day-old seedlings, cut into 1-cm pieces, and preconditioned for 72 hours on MS medium supplemented with 1 mg/liter 2,4-D (2,4-dichlorophenoxy acetic acid) and containing 30 g/liter sucrose and 2 g/liter Gelrite®.

[0234] Agrobacterium containing a plasmid comprising an Lpa1 construct of the invention is grown overnight in liquid LB medium to an OD₆₀₀ of 0.8, pelleted by centrifugation, and resuspended in liquid callus induction medium containing acetosyringone at a final concentration of 0.05 mM. Agrobacterium is then cocultivated with the preconditioned hypocotyl segments for 48 hours on MS medium with 1 mg/liter 2,4-D. After the cocultivation period, explants are transferred to MS medium containing 1 mg/liter 2,4-D, 400 mg/liter timentin, and 200 mg/liter kanamycin to select for transformed cells. After 2 weeks, in order to promote organogenesis, the explants are transferred to MS medium containing 4 mg/liter BAP (6-benzylaminopurine), 2 mg/liter zeatin, 5 mg/liter silver nitrate, antibiotics selective for the transformation construct, 30 g/liter sucrose, and 2 g/liter Gelrite®. After an additional 2 weeks, in order to promote shoot development, tissue is transferred to MS medium containing 3 mg/liter BAP, 2 mg/liter zeatin, antibiotics, 30 g/liter sucrose, and 2 g/liter Gelrite®. Shoots that develop are transferred for elongation to MS medium containing 0.05 mg/liter BAP, 30 g/liter sucrose, antibiotics, and 3 g/liter Gelrite®. Elongated shoots are then transferred to root development medium containing half-strength MS salts, 10 mg/liter sucrose, 3 g/liter Gelrite®, 5 mg/liter IBA (indole-3-butyric acid), and antibiotics. All cultures are maintained at 25° C.+/-2° C. in a 16-hour light/8-hour dark photoperiod regime with light supplied by cool white daylight fluorescent lights. The rooted shoots are transferred to soil and grown under the same photoperiod regime at 20° C. in a plant growth chamber.

[0235] Transformation of plants with the Lpa1 construct is confirmed using PCR of DNA extracted from putative transgenic plants.

Example 8

Variants of Lpa1

[0236] A. Variant Nucleotide Sequences of Lpa1 (SEQ ID NO: 2) that do not Alter the Encoded Amino Acid Sequence

[0237] The Lpa1 nucleotide sequence set forth in SEQ ID NO: 2 is used to generate variant nucleotide sequences having the nucleotide sequence of the open reading frame with about 70%, 76%, 81%, 86%, 92%, and 97% nucleotide sequence identity when compared to the starting unaltered ORF nucleotide sequence of SEQ ID NO: 2. In some embodiments, these functional variants are generated using a standard codon table. In these embodiments, while the nucleotide sequence of the variant is altered, the amino acid sequence encoded by the open reading frame does not change.

[0238] B. Variant Amino Acid Sequences of Lpa1

[0239] Variant amino acid sequences of Lpa1 are generated. In this example, one amino acid is altered. Specifically, the open reading frame set forth in SEQ ID NO: 2 is reviewed to determined the appropriate amino acid alteration. The selection of the amino acid to change is made by consulting the protein alignment (with the other homologs or orthologs and other gene family members from various species). See FIGS. 1, 4, and 5. An amino acid is selected that is deemed not to be under high selection pressure (not highly conserved) and which is rather easily substituted by an amino acid with similar chemical characteristics (i.e., similar functional side-chain). Using the alignments set forth in FIGS. 1, 4, and 5, an appropriate amino acid can be changed. Variants having about 70%, 75%, 80%, 85%, 90%, 95%, and 97% nucleic acid sequence identity to SEQ ID NO: 2 are generated using this method.

[0240] C. Additional Variant Amino Acid Sequences of Lpa1

[0241] In this example, artificial protein sequences are created having about 80%, 85%, 90%, 95%, and 97% identity relative to the reference protein sequence. This latter effort requires identifying conserved and variable regions from the alignments set forth in FIGS. 1, 4, and 5 and then the judicious application of an amino acid substitutions table. These parts will be discussed in more detail below.

[0242] Largely, the determination of which amino acid sequences are altered is made based on the conserved regions among MRPs. See FIGS. 1, 4, and 5. It is recognized that conservative substitutions can be made in the conserved regions below without altering function. In addition, one of skill will understand that functional variants of the Lpa1 sequence of the invention can have minor non-conserved amino acid alterations in the conserved domain.

[0243] Artificial protein sequences are then created that are different from the original in the intervals of 80-85%, 85-90%, 90-95%, and 95-100% identity. Midpoints of these intervals are targeted, with liberal latitude of plus or minus 1%, 2%, or 3%, for example. The amino acids substitutions will be effected by a custom Perl script. The substitution table is provided below in Table 1.

TABLE-US-00003 TABLE 1 Substitution Table Strongly Similar and Rank of Amino Optimal Order to Acid Substitution Change Comment I L, V 1 50:50 substitution L I, V 2 50:50 substitution V I, L 3 50:50 substitution A G 4 G A 5 D E 6 E D 7 W Y 8 Y W 9 S T 10 T S 11 K R 12 R K 13 N Q 14 Q N 15 F Y 16 M L 17 First methionine cannot change H Na No good substitutes C Na No good substitutes P Na No good substitutes

[0244] First, any conserved amino acids in the protein that should not be changed is identified and "marked off" for insulation from the substitution. The start methionine will of course be added to this list automatically. Next, the changes are made.

[0245] H, C, and P are not changed in any circumstance. The changes will occur with isoleucine first, sweeping N-terminal to C-terminal, then leucine, and so on down the list until the desired target of percent change is reached. Interim number substitutions can be made so as not to cause reversal of changes. The list is ordered 1-17, so start with as many isoleucine changes as needed before leucine, and so on down to methionine. Clearly, many amino acids will in this manner not need to be changed. Changes between L, I, and V will involve a 50:50 substitution of the two alternate optimal substitutions.

[0246] The variant amino acid sequences are written as output. Perl script is used to calculate the percent identities. Using this procedure, variants of Lpa1 are generated having about 80%, 85%, 90%, and 95% amino acid identity to the starting unaltered ORF nucleotide sequence of SEQ ID NO: 2.

Example 9

Pedigree Breeding

[0247] Pedigree breeding starts with the crossing of two genotypes, such as a transformed (i.e., transgenic) inbred line and one other elite inbred line having one or more desirable characteristics that is lacking or which complements the first transgenic inbred line. If the two original parents do not provide all the desired characteristics, other sources can be included in the breeding population. In the pedigree method, superior segregating plants are selfed and selected in successive filial generations. In the succeeding filial generations the heterozygous condition gives way to homogeneous lines as a result of self-pollination and selection. Typically in the pedigree method of breeding, five or more successive filial generations of selfing and selection are practiced: F1→F2; F2→F3; F3→F4; F4→F5, etc. After a sufficient amount of inbreeding, successive filial generations will serve to increase seed of the developed inbred. Preferably, the inbred line comprises homozygous alleles at about 95% or more of its loci.

[0248] In addition to being used to create a backcross conversion, backcrossing can also be used in combination with pedigree breeding to modify a transgenic inbred line and a hybrid that is made using the transgenic inbred line. Backcrossing can be used to transfer one or more specifically desirable traits from one line, the donor parent, to an inbred called the recurrent parent, which has overall good agronomic characteristics yet lacks that desirable trait or traits.

[0249] Therefore, an embodiment of this invention is a method of making a backcross conversion of a maize transgenic inbred line containing an Lpa1 construct, comprising the steps of crossing a plant of an elite maize inbred line with a donor plant comprising a mutant gene or transgene conferring a desired trait, selecting an F1 progeny plant comprising the mutant gene or transgene conferring the desired trait, and backcrossing the selected F1 progeny plant to a plant of the elite maize inbred line. This method may further comprise the step of obtaining a molecular marker profile of the elite maize inbred line and using the molecular marker profile to select for a progeny plant with the desired trait and the molecular marker profile of the maize elite inbred line. In the same manner, this method may be used to produce an F1 hybrid seed by adding a final step of crossing the desired trait conversion of the elite maize inbred line with a different maize plant to make F1 hybrid maize seed comprising a mutant gene or transgene conferring the desired trait.

Recurrent Selection and Mass Selection

[0250] Recurrent selection is a method used in a plant breeding program to improve a population of plants. The method entails individual plants cross-pollinating with each other to form progeny. The progeny are grown and superior progeny are selected by any number of selection methods, which include individual plant, half-sib progeny, full-sib progeny, selfed progeny and topeross yield evaluation. The selected progeny are cross-pollinated with each other to form progeny for another population. This population is planted and again superior plants are selected to cross-pollinate with each other. Recurrent selection is a cyclical process and therefore can be repeated as many times as desired. The objective of recurrent selection is to improve the traits of a population. The improved population can then be used as a source of breeding material to obtain inbred lines to be used in hybrids or used as parents for a synthetic cultivar. A synthetic cultivar is the resultant progeny formed by the intercrossing of several selected inbreds.

[0251] Mass selection is a useful technique when used in conjunction with molecular marker enhanced selection. In mass selection seeds from individuals are selected based on phenotype and/or genotype. These selected seeds are then bulked and used to grow the next generation. Bulk selection requires growing a population of plants in a bulk plot, allowing the plants to self-pollinate, harvesting the seed in bulk and then using a sample of the seed harvested in bulk to plant the next generation. Instead of self-pollination, directed pollination could be used as part of the breeding program.

Mutation Breeding

[0252] Mutation breeding is one of many methods that could be used to introduce new traits into a particular maize inbred line. Mutations that occur spontaneously or are artificially induced can be useful sources of variability for a plant breeder. The goal of artificial mutagenesis is to increase the rate of mutation for a desired characteristic. Mutation rates can be increased by many different means. Such means include: temperature; long-term seed storage; tissue culture conditions; radiation such as X-rays, Gamma rays (e.g., cobalt 60 or cesium 137), neutrons, (product of nuclear fission by uranium 235 in an atomic reactor), Beta radiation (emitted from radioisotopes such as phosphorus 32 or carbon 14), or ultraviolet radiation (preferably from 2500 to 2900 nm); genetic means such as transposable elements or DNA damage repair mutations; chemical mutagens (such as base analogues (5-bromo-uracil); and related compounds (8-ethoxy caffeine), antibiotics (streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, or acridines. Once a desired trait is observed through mutagenesis the trait may then be incorporated into existing germplasm by traditional breeding techniques, such as backcrossing. Details of mutation breeding can be found in Fehr (1993) "Principals of Cultivar Development" (Macmillan Publishing Company), the disclosure of which is incorporated herein by reference. In addition, mutations created in other lines may be used to produce a backcross conversion of a transgenic elite line that comprises such mutation.

Example 10

Gene Silencing with the Lpa1 Promoter

[0253] The promoter of a target gene (e.g., Lpa1) is inactivated by introducing into a plant an expression cassette comprising a promoter and an inverted repeat of fragments of the Lpa1 promoter. For example, an expression cassette may be created that comprises the Ole promoter operably linked to an inverted repeat comprising fragments of the Lpa1 promoter that are approximately 200 bp in length and that are separated by the Adh1 intron. The Lpa1 promoter fragments may be selected from a portion of the promoter which is rich in CpG islands, such as, for example, the 3' portion of the Lpa1 promoter. The sequence of the Lpa1 promoter is set forth in nucleotides 1-3134 of SEQ ID NO: 1. The expression cassette is used to transform a plant, which is then assayed for lack of expression of the Lpa1 gene. While the invention is not bound by any particular mechanism of operation, the method is thought to produce a small RNA molecule which recognizes the native promoter of the target gene and leads to methylation and inactivation (i.e., gene silencing) of the native promoter. Consequently, the gene associated with the promoter is not expressed. This trait is heritable and cosegregates with the transgenic construct.

Example 11

Construction of an Lpa1 Silencing Plasmid Driven by KT13

[0254] An expression cassette was prepared making use of an inverted repeat construct known as Inverted Repeats Without Terminators, or "IRNTs." The first and second portion of such a construct hybridize to each other to produce a hairpin structure which can suppress expression of the corresponding endogenous gene (e.g., Lpa1). In this Lpa1 IRNT, the first and second portions are separated by a "spacer" portion.

[0255] To make the spacer DNA, a polynucleotide fragment encoding part of the soybean Fad2-1 and soybean Fad2-2 proteins (Heppard et al. (1996) Plant Physiol. 110: 311-9) was produced as follows. First, a recombinant DNA fragment ("KSFad2-hybrid", set forth in SEQ ID NO: 72) was produced that contained a polynucleotide fragment of about 890 nucleotides comprising about 470 nucleotides from the soybean Fad2-2 gene and about 420 nucleotides from the soybean Fad2-1 gene. This KSFad2-hybrid recombinant DNA fragment was constructed by PCR amplification as follows. A DNA fragment of approximately 0.47 kb was obtained by PCR amplification using primers KS1 (SEQ ID NO: 73) and KS2 (SEQ ID NO: 74) from a template of genomic DNA purified from leaves of Glycine max cv. Jack. An approximately 0.42 kb DNA fragment was obtained from the same template by PCR amplification using primers KS3 (SEQ ID NO: 75) and KS4 (SEQ ID NO: 76). The 0.47 kb DNA fragment and 0.42 kb DNA fragment were gel-purified using GeneClean® (Qbiogene, Irvine Calif.), and then were mixed together and used as a template for PCR amplification with primers KS1 and KS4 to yield an approximately 890 bp fragment ("KSFad2-hybrid", set forth in SEQ ID NO: 72) that was cloned into the commercially available plasmid pGEM-T Easy (Promega, Madison, Wis.).

[0256] The KSFad2 hybrid fragment was then modified to contain additional restriction enzyme recognition sites, as follows. The KSFad2 hybrid fragment named "KSFad2-hybrid" was re-amplified by standard PCR methods using Pfu Turbo DNA polymerase (Stratagene®, La Jolla, Calif.), a plasmid containing KSFad2-hybrid as DNA template, and the following primer sets. The oligonucleotide primers (SEQ ID NO: 77 and SEQ ID NO: 78) were designed to add a BsiWI restriction endonuclease to the 5' end of the amplified fragment and to add an AvrII site to its 3' end. The resulting DNA "spacer" sequence comprising about 470 nucleotides from the soybean Fad2-2 gene and 418 nucleotides from the soybean Fad2-1 is shown in SEQ ID NO: 79.

[0257] To prepare the first and second portions of the inverted repeat constructs, a polynucleotide fragment encoding part of the soybean Lpa1 protein (Lpa1, SEQ ID NO: 10) was amplified by standard PCR methods using Pfu Turbo® DNA polymerase (Stratagene®, La Jolla, Calif.) and the following primer sets. Lpa1 oligonucleotide primers (SEQ ID NO: 69 and SEQ ID NO: 70) were designed to add NotI and SalI restriction endonuclease sites at the 5' end of the amplified fragment and BsiWI and AvrII restriction endonuclease sites at the 3' end of the amplified fragment as well as a stop codon (TAA) at its 3' end. The DNA sequence comprising the 556 bp polynucleotide from soybean Lpa1 is set forth in SEQ ID NO: 71.

Preparation of Expression Cassette

[0258] An expression cassette was constructed comprising the Lpa1 "IRNTs" operably linked to the strong seed-specific promoter KTI3 (Jofuku et al. (1989) Plant Cell 1: 1079-1093).

[0259] A plasmid derived from pKS121 was used to construct the expression cassette. Plasmid pKS121 was described in PCT Pub. No. WO 02/00904; this plasmid contains the KTI3 promoter/NotI/Kti3 3' terminator fragment. For use in the present expression cassette, the plasmid pKS121 was engineered to contain a second hygromycin phosphotransferase gene with a 35S-CaMV promoter. The plasmid was then digested with the restriction enzymes NotI and SalI and the digest was run on a 0.8% TAE-agarose gel to isolate and purify a 7350 bp DNA fragment using the Qiagen® gel extraction kit.

[0260] In order to insert the inverted repeat constructs and the spacer region into this plasmid, several polynucleotide fragments were prepared. Aliquots of the polynucleotide fragment comprising the 556 bp polynucleotide from soybean Lpa1 (SEQ ID NO: 71) were digested with two separate sets of restriction enzymes. First, an aliquot of the amplified Lpa1 fragment was digested with NotI and BsiWI and run on a 0.8% TAE-agarose gel to isolate a 566 bp DNA fragment, which was purified using the Qiagen® gel extraction kit. A separate aliquot of the amplified Lpa1 fragment was digested with SalI and AvrII and run on a 0.8% TAE-agarose gel to isolate a 579 bp DNA fragment, which was also purified using the Qiagen® gel extraction kit. Furthermore, the amplified polynucleotide comprising the DNA "spacer" sequence (SEQ ID NO: 79) was digested with BsiWI and AvrII, run on a 0.8% TAE-agarose gel and a 901 bp DNA fragment was purified using the Qiagen® gel extraction kit.

[0261] To assemble the expression cassette comprising the Lpa1 "IRNTs" operably linked to the strong seed-specific promoter KTI3, all four isolated and purified fragments described above were ligated together. The ligation mixture was transformed into E. coli and transformed colonies were selected on hygromycin. Hygromycin-resistant colonies were selected and grown overnight in LB media with appropriate antibiotic selection. Proper construction of the expression cassette was confirmed by isolating DNA from these bacterial cultures using a Qiagen® miniprep kit according to the manufacturer's protocol and then analyzing with appropriate restriction digests.

Example 12

Production of High P_i Lpa1 Transgenic Soybean Somatic Embryos

[0262] The expression cassettes comprising the Lpa1 "IRNTs" operably linked to the strong seed-specific promoter KTI3 (described in Example 11) was transformed into soybean embryogenic suspension cultures using the protocol described in Example 6. Individual immature soybean embryos were then dried down by transferring them into an empty small Petri dish that was seated on top of a 10-cm Petri dish containing some agar gel to allow slow dry down. This process is intended to mimic the last stages of soybean seed development, and dried-down embryos are capable of producing plants when transferred to soil or soil-less media. Storage products produced by embryos at this stage are similar in composition to storage products produced by zygotic embryos at a similar stage of development and most importantly the storage product profile is predictive of plants derived from a somatic embryo line (see PCT Pub. No. WO 94/11516).

Determination Inorganic Phosphate Content

[0263] Somatic soybean embryos were assayed for P_i (inorganic phosphate) content using modifications of Chen et al. ((1956) Anal. Chem. 28: 1756-1758). Single embryos were weighed and placed into 1.2 ml deep-well tubes of a 96 well rack (Corning® Incorporated). Metal balls were then added to the tubes and the samples were ground using a Geno/Grinder2000® grinder (Sepx CertiPrep®, Metuchen, N.J.). Then 150 μl water was added to each tube and the rack was shaken for 5 minutes and centrifuged at 3,000 g for 5 minutes. The pellet was resuspended and the complete slurry was transferred (without the metal balls) to a new set of into 1.2 ml deep-well tubes of a 96 well rack. The original tubes (still containing the metal balls) were washed with an additional 150 μl water and then shaken for 5 minutes and centrifuged at 2,500 g for 5 minutes. This solution was then pooled with the complete slurry in the new tubes, and 75 μl of 2N HCl was added to each tube. The tubes were incubated for 2 hours at room temperature. Thereafter, 188 μl of 30% aqueous trichloroacetic acid was added to each sample, and the samples were mixed and centrifuged at 2,500 g for 10 minutes. The supernatants were transferred into fresh tubes and used for P_i determinations; measurements were performed in duplicate.

[0264] To determine P_i, 100 μl of each supernatant was placed into a well of a 96 well microtiter plate and 100 μl of a mixture of 0.42% ammonium molybdate-1N H₂SO₄:10% ascorbic acid (7:1) was added to each sample. The plates were incubated at 37° C. for 30 minutes and absorbance was measured at 800 nm; sodium phosphate (NaH₂PO₄) was used as a standard. Table 2 shows data comparing the P_i content of transgenic soybean lines transformed with pJMS33 (described in Example 11) to wild type somatic embryos. Multiple events were generated expressing the Lpa1 IRNT described in Example 11. Ten out of twenty lines analyzed (50%) showed an increased P_i content when compared to wild-type somatic embryos, ranging from 3.5-fold higher than wild type to nearly 8-fold higher than wild type.

TABLE-US-00004 TABLE 2 P_i content of somatic soybean embryos from different transgenic events expressing the Lpa1 IRNT (as % of wild type (wt) content) Event P_i (% of wt) Wild type 100 embryo 4-3 755 3-1 464 4-2 350 7-7 432 1-2 496 7-1 520 8-2 759 7-6 381 4-1 543 8-3 478

Example 13

Transgenic Maize Seeds have Reduced Phytic Acid and Increased P_i Content

[0265] Two expression cassettes were constructed to provide cosuppression of an MRP. These expression cassettes (designated plasmids P36 and P94) were made using MRP polynucleotide fragments. Each construct contained an inverted repeat of an MRP polynucleotide such that the first and second portions self-hybridized to produce a hairpin structure that can suppress expression of the relevant endogenous gene (e.g., maize Lpa1). Between the two fragments of the inverted repeat was an intron that helped to form the loop portion in the hairpin structure. Transcription was driven by the oleosin promoter in plasmid P36 and by the Glb1 promoter in plasmid P94; neither construct had a terminator. In both plasmids P36 and P94, the intron used was the Adh1 intron (GenBank Accession No. X04050), although other introns may also be used.

[0266] The plasmids were used to produce transgenic maize using protocols described in Example 1. Transgenic T1 seeds were screened for elevated P_i content using a rapid P_i assay, and quantitative analysis of phytic acid and P_i were also performed. The results of these assays demonstrated that cosuppression of MRP expression resulted in a decrease in phytic acid content and an increase in P_i in the transgenic seeds (see Table 3).

TABLE-US-00005 TABLE 3 Maize plants transformed with MRP expression constructs produced transgenic seeds with reduced phytic acid and increased P_i content. Wt K CS K Plasmid #/ Wt K P_i CS K P_i PAP PAP PA Event (mg/g) (mg/g) (mg/g) (mg/g) reduction Plasmid 36 1 0.31 1.17 2.76 0.72 74% 2 0.18 1.05 2.75 0.73 74% 3 0.27 0.99 2.53 0.99 61% 4 0.26 1.21 2.43 0.84 66% 5 0.43 1.12 2.15 0.85 60% 6 0.31 1.20 2.41 0.79 67% 7 0.34 1.06 2.59 0.61 77% 8 0.26 1.15 2.60 0.57 78% 9 0.21 1.09 2.61 0.70 73% 10 0.31 1.26 2.55 0.82 68% 11 0.19 1.08 2.46 0.66 73% 12 0.32 1.09 2.50 0.78 69% Plasmid 94 1 0.14 1.01 3.47 2.29 34% 2 0.12 1.37 3.10 1.12 64% 3 0.16 1.44 3.09 1.00 68% 4 0.10 1.20 3.44 1.75 49% 5 0.24 1.25 3.04 1.53 50% 6 0.24 1.47 2.67 0.98 63% 7 0.21 1.46 2.98 1.11 63% 8 0.18 1.17 3.00 1.76 41% Wt K = wild-type kernels in a segregation ear; CS K = cosuppression kernels in a segregation ear; P_i = inorganic phosphate phosphorus; PAP = phytic acid phosphorus; PA = phytic acid

[0267] As indicated in the table legend, "Wt K" were kernels in a segregation ear without the MRP transgene and "CS K" were the kernels in the same segregation ear that did contain the MRP transgene. The PAP values in Table 3 were measured according to modifications of the methods described by Latta & Eskin (1980) J. Agric Food Chem. 28: 1313-1315 and Vaintraub & Lapteva (1988) Analytical Biochemistry 175: 227-230; see Example 1 for detail.

Example 14

Production of Transgenic Sorghum

[0268] The promoter construct prepared in Example 10 is used to transform sorghum according to the teachings of U.S. Pat. No. 6,369,298. Briefly, a culture of Agrobacterium is transformed with a vector comprising an expression cassette containing the promoter construct prepared in Example 10. The vector also comprises a T-DNA region into which the promoter construct is inserted. General molecular techniques used in the invention are provided, for example, by Sambrook et al. (eds.) Molecular Cloning: A Laboratory Manual, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0269] Immature sorghum embryos are obtained from the fertilized reproductive organs of a mature sorghum plant. Immature embryos are aseptically isolated from the developing kernel at about 5 days to about 12 days after pollination and held in sterile medium until use; generally, the embryos are about 0.8 to about 1.5 mm in size.

[0270] The Agrobacterium-mediated transformation process of the invention can be broken into several steps. The basic steps include: an infection step (step 1); a co-cultivation step (step 2); an optional resting step (step 3); a selection step (step 4); and a regeneration step (step 5). In the infection step, the embryos are isolated and the cells contacted with the suspension of Agrobacterium.

[0271] The concentration of Agrobacterium used in the infection step and co-cultivation step can affect the transformation frequency. Very high concentrations of Agrobacterium may damage the tissue to be transformed, such as the immature embryos, and result in a reduced callus response. The concentration of Agrobacterium used will vary depending on the Agrobacterium strain utilized, the tissue being transformed, the sorghum genotype being transformed, and the like. Generally a concentration range of about 0.5×10⁹ cfu/ml to 1×10⁹ cfu/ml will be used.

[0272] The embryos are incubated with the suspension of Agrobacterium about 5 minutes to about 8 minutes. This incubation or infection step takes place in a liquid solution that includes the major inorganic salts and vitamins of N6 medium (referred to as "N6 salts," or medium containing about 463.0 mg/l ammonium sulfate; about 1.6 mg/l boric acid; about 125 mg/l calcium chloride anhydrous; about 37.25 mg/l Na₂-EDTA; about 27.8 mg/l ferrous sulfate.7H₂O; about 90.37 mg/l magnesium sulfate; about 3.33 mg/l manganese sulfate H₂O; about 0.8 mg/l potassium iodide; about 2,830 mg/l potassium nitrate; about 400 mg/l potassium phosphate monobasic; and about 1.5 mg/l zinc sulfate.7 H₂O.

[0273] In addition, the media in the infection step generally excludes AgNO₃. AgNO₃ is generally included in the co-cultivation, resting (when used) and selection steps when N6 media is used. In the co-cultivation step, the immature embryos are co-cultivated with the Agrobacterium on a solid medium. The embryos are positioned axis-down on the solid medium and the medium can include AgNO₃ at a range of about 0.85 to 8.5 mg/l. The embryos are co-cultivated with the Agrobacterium for about 3-10 days.

[0274] Following the co-cultivation step, the transformed cells may be subjected to an optional resting step. Where no resting step is used, an extended co-cultivation step may utilized to provide a period of culture time prior to the addition of a selective agent. For the resting step, the transformed cells are transferred to a second medium containing an antibiotic capable of inhibiting the growth of Agrobacterium. This resting phase is performed in the absence of any selective pressures on the plant cells to permit preferential initiation and growth of callus from the transformed cells containing the heterologous nucleic acid. The antibiotic added to inhibit Agrobacterium growth may be any suitable antibiotic; such antibiotics are known in the art and include Cefotaxime, timetin, vancomycin, carbenicillin, and the like. Concentrations of the antibiotic will vary according to what is standard for each antibiotic, and those of ordinary skill in the art will recognize this and be able to optimize the antibiotic concentration for a particular transformation protocol without undue experimentation. The resting phase cultures are preferably allowed to rest in the dark at 28° C. for about 5 to about 8 days. Any of the media known in the art can be utilized for the resting step.

[0275] Following the co-cultivation step, or following the resting step, where it is used, the transformed plant cells are exposed to selective pressure to select for those cells that have received and are expressing polypeptide from the heterologous nucleic acid introduced by Agrobacterium. Where the cells are embryos, the embryos are transferred to plates with solid medium that includes both an antibiotic to inhibit growth of the Agrobacterium and a selection agent. The agent used to select for transformants will select for preferential growth of explants containing at least one selectable marker insert positioned within the superbinary vector and delivered by the Agrobacterium. Generally, any of the media known in the art suitable for the culture of sorghum can be used in the selection step, such as media containing N6 salts or MS salts. During selection, the embryos are cultured until callus formation is observed. Typically, calli grown on selection medium are allowed to grow to a size of about 1.5 to about 2 cm in diameter.

[0276] After the calli have reached the appropriate size, the calli are cultured on regeneration medium in the dark for several weeks to allow the somatic embryos to mature, generally about 1 to 3 weeks. Preferred regeneration media includes media containing MS salts. The calli are then cultured on rooting medium in a light/dark cycle until shoots and roots develop. Methods for plant regeneration are known in the art (see, e.g., Kamo et al. (1985) Bot. Gaz. 146(3): 327-334; West et al. (1993) Plant Cell 5:1361-1369; and Duncan et al. (1985) Planta 165: 322-332).

[0277] Small plantlets are then transferred to tubes containing rooting medium and allowed to grow and develop more roots for approximately another week. The plants are then transplanted to soil mixture in pots in the greenhouse.

[0278] All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0279] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claim(s).

Sequence CWU 1

79111347DNAZea mayspromoter(1)...(2495)exon(3135)...(5195)exon(5276)...(5596)exon(5674).- ..(5760)exon(6018)...(6692)exon(6767)...(6931)exon(7380)...(7674)exon(7818- )...(8032)exon(8238)...(8543)exon(8622)...(8685)exon(8788)...(9027)exon(92- 33)...(9698)3'UTR(9302)...(9699)terminator(9699)...(11347) 1ggtaaataac cgcggttatt tacccgttgg gtacggatat ggtgaagttt catatccgcg 60ggtacgggta tggatactat atggtatcca cgggtaataa tttcgcgggt atggatatct 120gctatccata tccgttaccc ggtggacata tgtcatgtgg acccaaacat ctaatggacg 180catcccaggt cccagcggcc agcatccttg ggcttgttca ctgcttcacg gatggagtct 240ggatcagcgc ctcaacactt cagcagcaag gccgcattgc cacaaggcca gcagacagca 300gtccaagcgg cccaagactg ctcacggacg gagtcttaga gtctcggtca gtctctctct 360cactctctct tatctcccag ttttaaccct agccgccaag gccaagctcc ctcccaaatc 420ccaatctgct cgtctgcggt gcagggctgc aggcatccac ctccaccagg cgctggccca 480gttttcgcca tccaccaggc gtccttagaa tatggtgcag ggctaccgag cagcgagcaa 540caagctgccg agcacagggc gccgaccgtc gagcagcgag caccgaggac cgccgagtag 600tcgagcaccg agcagctagc accgagcagc cgagcagcaa gcagagggct gcgaccaccg 660agcagccgag ccgcctcgtc ttctctcgtc ttctctagta aattaaacag gcttggatga 720attagttttt tgatttagag ttttggagag attatttgta cggatttgaa aagattactt 780gtatggaacg ttgaaattat tgtttggtgc taaaaaatat tattggaatg ttgttggaat 840gttaatttgt gataaatttc tgctagaatg atgctgtaat tttagtgggc gggcgggtaa 900tggatatccg ttggataacg gatacccgac gggtatgggt acggatatag atccgtaccc 960acgagcgtaa atgggtatgg gtatggattg ggttttacct cgtgggtatg gatacgcgaa 1020acatatatcc gccttctacc cgcccgattg ccatccctac tcgcctcgcc tccccgtgga 1080cgggcgggcc gggcgtgctc ggcactgacc ggtgccccac cgcgatatac catagggggc 1140cacgcgagat tcccgccccc catcccatct ggtgccgccg ccgcgctgcc gctgcgtgcg 1200gcctccccac ctgggcagcc tacgcccgca gccggcgcgc atgcatgtgc cacgtcgcgg 1260cagacctacc accacgccgc tcgatctggc gactggcggt gcggtggtcc gctgctgctg 1320cacggcgttt tccgcgtttg tttgcttgtt ccgttgtgtt gtccgcatcg cgcctctctc 1380tctctctcgc cctcgctcgc cgggacggag acggcgaggc cggggcggca tctttctgct 1440ggctttccgg catctcgtca gaccagaggg gaaaaaaggc gcctttccgg ccgtaacact 1500aaccccctgt ctttttttta tcacccgtgt gccatctgtt tctaggatgg taggacgtga 1560tcgatccaac gtttcgcaag taccaccacc accaccgtga ctgttcggcc ccagacggac 1620gcaacgccaa ccgtgcctgc gtcggctcaa caatcattgc cacgcgtacc cggtatcccg 1680gccaggcggt tccagtcagt ccgcggacgc caagaagaat ttttaggtgc acggagtcca 1740ggttctttgt tcatgacagt actcaaagcg aaaacgcaga agcattgtct cgactgaaca 1800gcgggttcag ttcagtttct gaggctgtct gaactggatt acagcacagc gccagcgcgc 1860gcccgcgtac acacagacga cacatctgtg acactgtcgt gttccctcct gcgtacgaag 1920cgatcgcttt tccccccact cgctgcacgt tatccctgtc gcctcctcct cagaacgcct 1980cccttttatt agtagtacaa agaaagatgt ccataaaaat aaagcctttt gtaataaatg 2040gtaaataaag gcggatattc tgcgacctcc gcaaatacgc gcacaacaat cttgtgctcc 2100gctcgatata agcctcaatc tcataagata atgtgtgtat gttttcagaa acaaaatcaa 2160cgttttaaaa ttttgaccaa cagttcagaa aaaaacatat atttttagtg tatgtatgtt 2220gtatacctga atttatacta aaaatacttt aacgtaatac ttgatttgtt tttattgatg 2280ttctatttca cagtaaatac tttatcaaaa gaatatacgc ctcgtgtttt atggaacgga 2340gggagtattt gttttcatgt atagacaata aaaacatatt aatcctgtat aataaaaatt 2400taatgcggca tgcacgtcat ggtagtagcc ggaaggcaaa gccggctacg aactctcctc 2460ctctataaaa accatcaatc gccgttccat ctgtttgcaa gccgaccgaa accaggcagt 2520ggtgtggagt ggacacaaca caacaggaga ggaggaaaag ggaaaacgga aattcactgc 2580tactactccg tctccgtact agcacgccca taacctctct ctctctcctg cctctcaccg 2640catcgtcttc ctctcccccc accacacccc ccacccgccg atccatcccc caaaagccga 2700agccgaaacc gaaacctccc tccgcacgcc acctgctacc acacacacac ccgcgccgcc 2760gcccccacgg ccggccgcgc ggaggtgact cgagaaggac gcaggaacca aggagagagt 2820ttggtgaggg gatcagagac gtaacccgcc cggacccggg ctgcattagc cggaatccca 2880tcccaggcga gcctctctct cccctcctcg aaccaggcgc aggcgagcgt ctctgcccgc 2940ccgcctgctg ctaccgccaa aacgcctcct ttgttgccat ccgccgatgc cgtaatccgc 3000cgcccaaagc tcttcctttt tccctctctc tcgcccgcgg ccgcactccc tgccccagtg 3060cctgccgtgg cgagcccaac cccaatgcct tttaaacccc tccccgctcc ctcactgatc 3120cccaccgcct cccaatgccg ccctccttcc cctccctccc gctcccggag gccgttgccg 3180ccaccgccca cgccgcgctg ctcgcgctcg ccgcactcct gctcctcctc cgcgccgcgc 3240gcgcgctcgc ctcccgctgc gcgtcatgcc tcaaggcgcc gcgccgccgc gggggccccg 3300ccgtcgtcgt gggcgacggc gccggcggcg ccctcgcggc ggcgactgcc ggcgcctggc 3360acagggccgt gctggcgtcc tgcgcctacg ccctgctctc gcaggtcgcc gtgctgagct 3420acgaggtggc cgtcgccggc tcgcgcgtct cggcgcgggc gctgctgctg ccggccgtgc 3480aggcggtgtc ctgggccgcg ctgctggcgc tcgcgcttca ggcccgcgcc gtcggctggg 3540ccaggttccc tgcgctggtg cggctctggt gggtggtctc cttcgcgctc tgcgttgtca 3600ttgcgtacga cgactccagg cgcctgatag gccagggcgc gcgcgctgtg gattacgcgc 3660acatggttgc caacttcgcg tccgtgccgg ccctgggctt cctgtgcttg gttggtgtca 3720tgggttccac cggtttggaa ttggagttta cggaggatgg caacggcctg catgagccgc 3780tgctgctcgg caggcagcgc agagaggcag aggaggagct cggctgtctg agggtcactc 3840cctacgctga tgctgggatc ctcagccttg caacattgtc atggcttagt ccgttgctct 3900ctgttggtgc gcagcggcca cttgagttgg ctgacatacc cttgctggcg cacaaggacc 3960gtgcaaagtc atgctataag gcgatgagcg ctcactacga gcgccagcgg ctagaatacc 4020ctggcaggga gccatcactc acatgggcaa tactcaagtc attctggcga gaggccgcgg 4080tcaatggcac atttgctgct gtcaacacga ttgtgtcgta tgttggacct tacttgatca 4140gctattttgt ggactacctc agtggcaaca ttgctttccc ccatgaaggt tacatccttg 4200cctctatatt ttttgtagca aaactgcttg agacactcac tgcccgacag tggtacttgg 4260gtgtggacat catggggatc catgtcaagt ctggcctcac tgccatggtg tataggaagg 4320gtctccgact gtcaaacgcc tcacggcaga gccacacgag tggtgagatt gtgaattaca 4380tggccgtcga tgtgcagcgt gtgggggact atgcatggta tttccatgac atctggatgc 4440ttcccctgca gatcattctt gctctcgcca tcctgtacaa gaacgtcggg atcgccatgg 4500tttcaacatt ggtagcaact gtgctatcga tcgcagcctc tgttcctgtg gcaaagctgc 4560aggagcacta ccaagataag ttaatggcat caaaagatga gcgcatgcgc aagacttcag 4620agtgcttgaa aaatatgagg attttgaagc ttcaggcatg ggaggatcgg taccggctgc 4680agttggaaga gatgaggaac gtggaatgca gatggcttcg gtgggctctg tactcacagg 4740ctgcagttac atttgttttc tggagctcgc caatctttgt cgcagtcata acttttggga 4800cttgcatatt actcggtggc cagctcactg caggaggggt tctatccgct ttagcaacgt 4860ttcggatcct ccaagagcct ctgaggaact tcccggatct catctctatg atggcacaga 4920caagggtgtc tttggaccgt ttgtctcatt ttctgcagca agaagaactg ccagatgacg 4980caactataaa tgttccacaa agtagtacag ataaggcagt cgatattaag gatggcgcat 5040tctcttggaa cccatacact ctgaccccta cactttctga tatacacctt agtgtagtga 5100gaggcatgag agtagcagtc tgtggtgtca ttggttctgg taaatcaagt ctactatcgt 5160ctatactcgg ggagataccc aaattatgtg gccatgtaag tataaatgca aaaaaaaatc 5220gacattgatt ttgcttgttc tgttacattg acctttctcc tgcctcatat tccaggtcag 5280gataagtggc acagcagcgt atgttcctca gactgcatgg atacagtctg gaaatattga 5340ggagaatatt ctgtttggca gtcaaatgga tagacaacgt tacaagagag tcattgcagc 5400ttgctgtctt aagaaagatc ttgagctgct ccagtacgga gatcagactg ttattggtga 5460tagaggcatt aatttgagtg gaggtcagaa acaaagagtt cagcttgcta gagcactcta 5520ccaagatgct gatatttatt tgcttgatga tcccttcagt gctgttgatg ctcatactgg 5580gagcgaactg tttaaggttg gtacagctgt ttgcctatta tatttgtttc taagctgttt 5640ctgttccata acacatctgc ttctgtgtta caggagtata tattgactgc actagcaacc 5700aaaacagtaa tctatgtaac acatcaagtt gaatttctac cagctgctga tctgatattg 5760gtaagcggta gacatatttt cgtattgata tgtatgctat tatgagtaat tcttatgggc 5820atgcttttct gattttcatc atcatatcga gttgttctct gtaatatcct attggttcat 5880cttttccttt tggaagctaa ccatgcatgt aacctctaaa tgagagctag ttaccttcag 5940gattgttttc atgggactat aagtgtgact agtgggcctg tattaatctc tctttgatgg 6000ttctgtcgca tttacaggtt cttaaggatg gccatatcac acaagctgga aagtatgatg 6060atcttctgca agctggaact gatttcaatg ctctggtttc tgctcataag gaagctattg 6120aaaccatgga tatatttgaa gattccgata gtgatacagt ttcttctatt cccaacaaaa 6180gattgacacc aagtatcagc aatattgata acctgaaaaa taagatgtgt gaaaatggac 6240aaccatctaa tacacgggga attaaggaaa aaaagaagaa agaagagcgt aagaagaagc 6300gtactgttca agaggaggaa agggaacgtg gaaaagtgag ctccaaagtt tatttgtcat 6360acatggggga agcttacaaa ggtacactga taccactaat tatcttggct caaaccatgt 6420tccaagttct tcagattgcg agcaactggt ggatggcatg ggcaaaccca caaacagaag 6480gagatgctcc caagacagat agtgtggtcc ttctggttgt ttatatgtcc cttgcctttg 6540gaagttcact atttgtgttc atgagaagcc ttcttgtggc tacgtttggt ttagcagctg 6600cccagaagct ttttataaaa atgcttaggt gtgtctttcg agctccaatg tcattctttg 6660acaccacacc atctggtcgg attttgaaca gagtaagtat tgctcttgcc tatgctaata 6720taagtttgta atatgtgctt tcctccttat tcattcttta tatcaggttt ctgtagatca 6780aagtgttgtg gaccttgata tagcgttcag acttggtgga tttgcatcaa cgacaattca 6840actccttgga attgttgctg tcatgagcaa agtcacatgg caagttctga ttcttatagt 6900ccccatggct gttgcatgca tgtggatgca ggtaaatgtt gtgatcacca aacattacat 6960ttcaatctat atttgaggtt taatatcaca agctgttttt tcccttaaca tttagcaaat 7020tggtatatga cagtctagat ttatttgaga acaccttttg caagatgggc catataacta 7080gagtttactt tcagctaatg atccttattc cttaaagaat gtttattagt cactcggcat 7140aggcacatca tgtattgcac tctatgttta gtaattagta tgtcattggt tcactgttga 7200tgtcttagaa attgctatgc ttgcagatgt ttattaattg agatacttct agctcaattc 7260tcttaatttt ttatattaaa ccattgtagt cataaggaat tacctgttta aaaggatatg 7320ttttctggta aatcagagtg gcatttttac taaagctcca attactgtca cctttgcaga 7380ggtattatat tgcttcatca agggaactaa ctaggatttt gagtgttcag aagtctccag 7440tgatccattt gtttagtgaa tcaattgctg gtgctgctac aataaggggt tttggtcaag 7500agaagcggtt tatgaaaagg aatctttatc ttcttgactg ttttgctcgc cctttatttt 7560ccagccttgc tgctattgaa tggctctgcc tgcgaatgga attgctttcg actttcgtct 7620ttgctttttg catggcaata cttgtgagct ttcctcctgg cacaatcgaa ccaagtatgt 7680ttatgttcca catgctgctc cagttctcta ctatgtttgg tcgctttctc caatgcctta 7740ttctgtgcag tagaaaacct gcatcttctt gtctgttaaa atttattcag catctaaatg 7800gattttcaaa ttgataggta tggctggcct cgctgtaaca tatggactta atttaaatgc 7860tcgcatgtca agatggatat tgagcttctg taaattagag aacaggataa tctctgttga 7920gcgcatttat caatattgca ggcttcctag tgaagcacca ttgattattg agaactgccg 7980tccaccatca tcatggcctc agaatggaaa cattgaactg attgatctca aggtatgctt 8040tatcattggg gggcagttaa ggatacgatt ttcattagca ttgctataga gctgattgtc 8100atttccagca tgcaaatatt atattctaac ataatctatt tacatttttc tctttactat 8160gtataattac catacatatc taatttatga tctatttagt tttggcttct gagtttgctt 8220tttcatgatt atgaaaggtc cgctacaagg acgatctacc attagttctt catggtgtaa 8280gttgtatgtt tcctggcggg aaaaagattg ggattgtagg gcgtactgga agcggtaaat 8340ctactcttat tcaggccctt ttccgcctaa ttgagcccac tggagggaag attataattg 8400acaacattga catctctgca attggccttc atgatctgcg gtcacggttg agcatcattc 8460cccaagaccc tacattgttt gagggtacta tcagaatgaa ccttgatcct cttgaggagt 8520gcactgatca agaaatttgg gaggtacatc ctggtcactt tgacgctata ctcatgttga 8580gtctgtgtga ttcttatctt aaggaacaca atctgttgca ggcactagaa aagtgtcagc 8640taggagaggt cattcgttcc aaggaagaga aacttgacag tccaggttag cctgacattt 8700tgctgccaag cctcctttga agagtgggaa tgtggtttct taatgcgtaa acttattgct 8760cctggacctt tttttttgct tttgcagtgc tagaaaacgg ggataactgg agcgtgggac 8820agcgccaact tattgcactg ggtagggcgc tgctcaagca ggcaaaaatt ttggtactcg 8880atgaggcgac agcatctgtc gacacagcaa cagacaatct tatccaaaag atcatccgca 8940gtgaattcaa ggactgcaca gtctgtacca ttgctcaccg tattcccacc gttattgaca 9000gtgaccttgt tctggtcctt agtgatggta tgagttcttt gactaaacta accacgcctc 9060ctttacctgt tcatagttag atttcctgag ctctggtcct tttccaactc gtgcatccga 9120ttcttggata aacatttaga aagtagaaac cgtagcaaac tgacagtttt tcttctgcac 9180agaatttgga aacaagcctt cgctgaactt ttctcatcgt cttgatttcc aggtaaaatc 9240gcagagttcg acacgcccca gaggctttta gaggacaagt catctatgtt catacagcta 9300gtatcggaat actccactcg gtcgagctgt atatagagag gcttagctta aaaccccgcc 9360ccaaacctgg caacagaggc tgggaggcaa atagcccgta tctgccatgc ttgcgccata 9420gaggtccctg cgaacaccgg agggcggcgt agaagacgag gtgtacatga gtgggaggaa 9480cactgggcgt tccctgacct gaataccgtg gaatcggcga gggagcgcgg ttggtattgg 9540taggcaccag gggaggagtt ggtgacacta gtacattacc cgaagctgat gcttcagtat 9600gtatgtataa caacaatgca tactgcttct ccctttgcag agtggagaac caagggaata 9660actcgtgcgt aataagagga gaaagatttg ttttttggca tcagactggt gtgtgtgcgc 9720ttttgtttgc tgtgtccatt agaccattac tgtatttctc tgccaaattt tactgtagcc 9780ggtgccagtt tctgcttcag aaattcagca tctcaaatcg ccaggtgaaa aaggttcagc 9840aaccagcagt ttgctcgatg gccgaggcta gtaactcatc ctgtgctgaa tacagagtat 9900caccacgtca ggttcactgc cctgacctga aaacaatact ccctgtggag atgacggctg 9960gattacgcag atactgtagt gtaaaataag agatttacat tgtagattat actttttaca 10020gattaaagct ttaaatagga gatgaataga ctgtgtagtg taaaataaga tatttacatt 10080gtagattata ctgtttacag attaaagcta tgaatagatt gctgttttta gagagagaga 10140ggctgtaggg taaaccctac atcataattt ttgtttagtg gaaaaaggta acaagatcaa 10200tagaaacaag agagagggag ggaggggggg gggggggggg gtgttatctt ttttctaaaa 10260aaaccaggct ctggaggaaa aagggtttag ttctcctaag ttaaatttta tctgtgcccc 10320acacctccaa tatttccaaa tttatttaga atattaaata gatttatttt gatttaaaaa 10380aattgttttg gcttttactt agattttaac aagtttaaaa acaacgcgcc ctctctagtg 10440taaaatttat tttttggcgc gcacgattaa aatggagcaa attaccccta tttattttat 10500atagccctct ttttttatct ctgtaaaata tatgagcttt atttttatag tgttaaatat 10560acaattttgt ataaggaaga agcctaaatt taattattta attattgaac ttcaaactca 10620ggtatctttg gtatcgaacc aggggcagac ctacgtgtat atgagtgggg gatcaggccc 10680cactcatttc tttgttgtaa gtagtagaac ctagattttc accatgaggg tccctgctaa 10740gcataactta gatgctcctg ctctgatatt ttggtacttg tttttggaag tgtgccctca 10800ttttgtattt tgttctggga ccacctctgt atcgaactac taaaattaga catacttggt 10860tcatcgagaa gccttcgatt gtactttctc cattttttat ttgacgtagt tgtccccccc 10920tcccaaaaaa aaaactctaa tcgcttatct tattaaaaat ttgtgtgttc tttaaaagct 10980actccatcct aaagtatagt tttgtccatc ctaaagtata gtttgaagtt caatggttaa 11040atctaaactt cttccattat agaacatgtg tttatttata tgtttaacac tatacttaac 11100aatatgaaaa tagagctctt atacttgtaa cagaatagat aaaaaataag ctatgaaatg 11160ataagggaca atttggataa tttgcttgag aggtgagaaa taaaagaatg gaaaatatag 11220atagagtgct attttctgaa tttatttgat gaaactactg ttgttcttat aattgcttga 11280aaacgagtgc ttttttttaa cacagtctca taccagtgtt gtttcgtgga ttcgaaggaa 11340ggctttg 1134725139DNAZea maysCDS(244)...(4776) 2cctctctctc ccctcctcga accaggcgca ggcgagcgtc tctgcccgcc cgcctgctgc 60taccgccaaa acgcctcctt tgttgccatc cgccgatgcc gtaatccgcc gcccaaagct 120cttccttttt ccctctctct cgcccgcggc cgcactccct gccccagtgc ctgccgtggc 180gagcccaacc ccaatgcctt ttaaacccct ccccgctccc tcactgatcc ccaccgcctc 240cca atg ccg ccc tcc ttc ccc tcc ctc ccg ctc ccg gag gcc gtt gcc 288 Met Pro Pro Ser Phe Pro Ser Leu Pro Leu Pro Glu Ala Val Ala 1 5 10 15gcc acc gcc cac gcc gcg ctg ctc gcg ctc gcc gca ctc ctg ctc ctc 336Ala Thr Ala His Ala Ala Leu Leu Ala Leu Ala Ala Leu Leu Leu Leu 20 25 30ctc cgc gcc gcg cgc gcg ctc gcc tcc cgc tgc gcg tca tgc ctc aag 384Leu Arg Ala Ala Arg Ala Leu Ala Ser Arg Cys Ala Ser Cys Leu Lys 35 40 45gcg ccg cgc cgc cgc ggg ggc ccc gcc gtc gtc gtg ggc gac ggc gcc 432Ala Pro Arg Arg Arg Gly Gly Pro Ala Val Val Val Gly Asp Gly Ala 50 55 60ggc ggc gcc ctc gcg gcg gcg act gcc ggc gcc tgg cac agg gcc gtg 480Gly Gly Ala Leu Ala Ala Ala Thr Ala Gly Ala Trp His Arg Ala Val 65 70 75ctg gcg tcc tgc gcc tac gcc ctg ctc tcg cag gtc gcc gtg ctg agc 528Leu Ala Ser Cys Ala Tyr Ala Leu Leu Ser Gln Val Ala Val Leu Ser80 85 90 95tac gag gtg gcc gtc gcc ggc tcg cgc gtc tcg gcg cgg gcg ctg ctg 576Tyr Glu Val Ala Val Ala Gly Ser Arg Val Ser Ala Arg Ala Leu Leu 100 105 110ctg ccg gcc gtg cag gcg gtg tcc tgg gcc gcg ctg ctg gcg ctc gcg 624Leu Pro Ala Val Gln Ala Val Ser Trp Ala Ala Leu Leu Ala Leu Ala 115 120 125ctt cag gcc cgc gcc gtc ggc tgg gcc agg ttc cct gcg ctg gtg cgg 672Leu Gln Ala Arg Ala Val Gly Trp Ala Arg Phe Pro Ala Leu Val Arg 130 135 140ctc tgg tgg gtg gtc tcc ttc gcg ctc tgc gtt gtc att gcg tac gac 720Leu Trp Trp Val Val Ser Phe Ala Leu Cys Val Val Ile Ala Tyr Asp 145 150 155gac tcc agg cgc ctg ata ggc cag ggc gcg cgc gct gtg gat tac gcg 768Asp Ser Arg Arg Leu Ile Gly Gln Gly Ala Arg Ala Val Asp Tyr Ala160 165 170 175cac atg gtt gcc aac ttc gcg tcc gtg ccg gcc ctg ggc ttc ctg tgc 816His Met Val Ala Asn Phe Ala Ser Val Pro Ala Leu Gly Phe Leu Cys 180 185 190ttg gtt ggt gtc atg ggt tcc acc ggt ttg gaa ttg gag ttt acg gag 864Leu Val Gly Val Met Gly Ser Thr Gly Leu Glu Leu Glu Phe Thr Glu 195 200 205gat ggc aac ggc ctg cat gag ccg ctg ctg ctc ggc agg cag cgc aga 912Asp Gly Asn Gly Leu His Glu Pro Leu Leu Leu Gly Arg Gln Arg Arg 210 215 220gag gca gag gag gag ctc ggc tgt ctg agg gtc act ccc tac gct gat 960Glu Ala Glu Glu Glu Leu Gly Cys Leu Arg Val Thr Pro Tyr Ala Asp 225 230 235gct ggg atc ctc agc ctt gca aca ttg tca tgg ctt agt ccg ttg ctc 1008Ala Gly Ile Leu Ser Leu Ala Thr Leu Ser Trp Leu Ser Pro Leu Leu240 245 250 255tct gtt ggt gcg cag cgg cca ctt gag ttg gct gac ata ccc ttg ctg 1056Ser Val Gly Ala Gln Arg Pro Leu Glu Leu Ala Asp Ile Pro Leu Leu 260 265 270gcg cac aag gac cgt gca aag tca tgc tat aag gcg atg agc gct cac 1104Ala His Lys Asp Arg Ala Lys Ser Cys Tyr Lys Ala Met Ser Ala His 275 280 285tac gag cgc cag cgg cta gaa tac cct ggc agg gag cca tca ctc aca 1152Tyr Glu Arg Gln Arg Leu Glu Tyr Pro Gly Arg Glu Pro Ser Leu Thr 290 295 300tgg gca ata ctc aag tca ttc tgg cga gag gcc gcg gtc aat ggc aca 1200Trp Ala Ile Leu Lys Ser Phe Trp Arg Glu Ala Ala Val Asn Gly Thr 305

310 315ttt gct gct gtc aac acg att gtg tcg tat gtt gga cct tac ttg atc 1248Phe Ala Ala Val Asn Thr Ile Val Ser Tyr Val Gly Pro Tyr Leu Ile320 325 330 335agc tat ttt gtg gac tac ctc agt ggc aac att gct ttc ccc cat gaa 1296Ser Tyr Phe Val Asp Tyr Leu Ser Gly Asn Ile Ala Phe Pro His Glu 340 345 350ggt tac atc ctt gcc tct ata ttt ttt gta gca aaa ctg ctt gag aca 1344Gly Tyr Ile Leu Ala Ser Ile Phe Phe Val Ala Lys Leu Leu Glu Thr 355 360 365ctc act gcc cga cag tgg tac ttg ggt gtg gac atc atg ggg atc cat 1392Leu Thr Ala Arg Gln Trp Tyr Leu Gly Val Asp Ile Met Gly Ile His 370 375 380gtc aag tct ggc ctc act gcc atg gtg tat agg aag ggt ctc cga ctg 1440Val Lys Ser Gly Leu Thr Ala Met Val Tyr Arg Lys Gly Leu Arg Leu 385 390 395tca aac gcc tca cgg cag agc cac acg agt ggt gag att gtg aat tac 1488Ser Asn Ala Ser Arg Gln Ser His Thr Ser Gly Glu Ile Val Asn Tyr400 405 410 415atg gcc gtc gat gtg cag cgt gtg ggg gac tat gca tgg tat ttc cat 1536Met Ala Val Asp Val Gln Arg Val Gly Asp Tyr Ala Trp Tyr Phe His 420 425 430gac atc tgg atg ctt ccc ctg cag atc att ctt gct ctc gcc atc ctg 1584Asp Ile Trp Met Leu Pro Leu Gln Ile Ile Leu Ala Leu Ala Ile Leu 435 440 445tac aag aac gtc ggg atc gcc atg gtt tca aca ttg gta gca act gtg 1632Tyr Lys Asn Val Gly Ile Ala Met Val Ser Thr Leu Val Ala Thr Val 450 455 460cta tcg atc gca gcc tct gtt cct gtg gca aag ctg cag gag cac tac 1680Leu Ser Ile Ala Ala Ser Val Pro Val Ala Lys Leu Gln Glu His Tyr 465 470 475caa gat aag tta atg gca tca aaa gat gag cgc atg cgc aag act tca 1728Gln Asp Lys Leu Met Ala Ser Lys Asp Glu Arg Met Arg Lys Thr Ser480 485 490 495gag tgc ttg aaa aat atg agg att ttg aag ctt cag gca tgg gag gat 1776Glu Cys Leu Lys Asn Met Arg Ile Leu Lys Leu Gln Ala Trp Glu Asp 500 505 510cgg tac cgg ctg cag ttg gaa gag atg agg aac gtg gaa tgc aga tgg 1824Arg Tyr Arg Leu Gln Leu Glu Glu Met Arg Asn Val Glu Cys Arg Trp 515 520 525ctt cgg tgg gct ctg tac tca cag gct gca gtt aca ttt gtt ttc tgg 1872Leu Arg Trp Ala Leu Tyr Ser Gln Ala Ala Val Thr Phe Val Phe Trp 530 535 540agc tcg cca atc ttt gtc gca gtc ata act ttt ggg act tgc ata tta 1920Ser Ser Pro Ile Phe Val Ala Val Ile Thr Phe Gly Thr Cys Ile Leu 545 550 555ctc ggt ggc cag ctc act gca gga ggg gtt cta tcc gct tta gca acg 1968Leu Gly Gly Gln Leu Thr Ala Gly Gly Val Leu Ser Ala Leu Ala Thr560 565 570 575ttt cgg atc ctc caa gag cct ctg agg aac ttc ccg gat ctc atc tct 2016Phe Arg Ile Leu Gln Glu Pro Leu Arg Asn Phe Pro Asp Leu Ile Ser 580 585 590atg atg gca cag aca agg gtg tct ttg gac cgt ttg tct cat ttt ctg 2064Met Met Ala Gln Thr Arg Val Ser Leu Asp Arg Leu Ser His Phe Leu 595 600 605cag caa gaa gaa ctg cca gat gac gca act ata aat gtt cca caa agt 2112Gln Gln Glu Glu Leu Pro Asp Asp Ala Thr Ile Asn Val Pro Gln Ser 610 615 620agt aca gat aag gca gtc gat att aag gat ggc gca ttc tct tgg aac 2160Ser Thr Asp Lys Ala Val Asp Ile Lys Asp Gly Ala Phe Ser Trp Asn 625 630 635cca tac act ctg acc cct aca ctt tct gat ata cac ctt agt gta gtg 2208Pro Tyr Thr Leu Thr Pro Thr Leu Ser Asp Ile His Leu Ser Val Val640 645 650 655aga ggc atg aga gta gca gtc tgt ggt gtc att ggt tct ggt aaa tca 2256Arg Gly Met Arg Val Ala Val Cys Gly Val Ile Gly Ser Gly Lys Ser 660 665 670agt cta cta tcg tct ata ctc ggg gag ata ccc aaa tta tgt ggc cat 2304Ser Leu Leu Ser Ser Ile Leu Gly Glu Ile Pro Lys Leu Cys Gly His 675 680 685gtc agg ata agt ggc aca gca gcg tat gtt cct cag act gca tgg ata 2352Val Arg Ile Ser Gly Thr Ala Ala Tyr Val Pro Gln Thr Ala Trp Ile 690 695 700cag tct gga aat att gag gag aat att ctg ttt ggc agt caa atg gat 2400Gln Ser Gly Asn Ile Glu Glu Asn Ile Leu Phe Gly Ser Gln Met Asp 705 710 715aga caa cgt tac aag aga gtc att gca gct tgc tgt ctt aag aaa gat 2448Arg Gln Arg Tyr Lys Arg Val Ile Ala Ala Cys Cys Leu Lys Lys Asp720 725 730 735ctt gag ctg ctc cag tac gga gat cag act gtt att ggt gat aga ggc 2496Leu Glu Leu Leu Gln Tyr Gly Asp Gln Thr Val Ile Gly Asp Arg Gly 740 745 750att aat ttg agt gga ggt cag aaa caa aga gtt cag ctt gct aga gca 2544Ile Asn Leu Ser Gly Gly Gln Lys Gln Arg Val Gln Leu Ala Arg Ala 755 760 765ctc tac caa gat gct gat att tat ttg ctt gat gat ccc ttc agt gct 2592Leu Tyr Gln Asp Ala Asp Ile Tyr Leu Leu Asp Asp Pro Phe Ser Ala 770 775 780gtt gat gct cat act ggg agc gaa ctg ttt aag gag tat ata ttg act 2640Val Asp Ala His Thr Gly Ser Glu Leu Phe Lys Glu Tyr Ile Leu Thr 785 790 795gca cta gca acc aaa aca gta atc tat gta aca cat caa gtt gaa ttt 2688Ala Leu Ala Thr Lys Thr Val Ile Tyr Val Thr His Gln Val Glu Phe800 805 810 815cta cca gct gct gat ctg ata ttg gtt ctt aag gat ggc cat atc aca 2736Leu Pro Ala Ala Asp Leu Ile Leu Val Leu Lys Asp Gly His Ile Thr 820 825 830caa gct gga aag tat gat gat ctt ctg caa gct gga act gat ttc aat 2784Gln Ala Gly Lys Tyr Asp Asp Leu Leu Gln Ala Gly Thr Asp Phe Asn 835 840 845gct ctg gtt tct gct cat aag gaa gct att gaa acc atg gat ata ttt 2832Ala Leu Val Ser Ala His Lys Glu Ala Ile Glu Thr Met Asp Ile Phe 850 855 860gaa gat tcc gat agt gat aca gtt tct tct att ccc aac aaa aga ttg 2880Glu Asp Ser Asp Ser Asp Thr Val Ser Ser Ile Pro Asn Lys Arg Leu 865 870 875aca cca agt atc agc aat att gat aac ctg aaa aat aag atg tgt gaa 2928Thr Pro Ser Ile Ser Asn Ile Asp Asn Leu Lys Asn Lys Met Cys Glu880 885 890 895aat gga caa cca tct aat aca cgg gga att aag gaa aaa aag aag aaa 2976Asn Gly Gln Pro Ser Asn Thr Arg Gly Ile Lys Glu Lys Lys Lys Lys 900 905 910gaa gag cgt aag aag aag cgt act gtt caa gag gag gaa agg gaa cgt 3024Glu Glu Arg Lys Lys Lys Arg Thr Val Gln Glu Glu Glu Arg Glu Arg 915 920 925gga aaa gtg agc tcc aaa gtt tat ttg tca tac atg ggg gaa gct tac 3072Gly Lys Val Ser Ser Lys Val Tyr Leu Ser Tyr Met Gly Glu Ala Tyr 930 935 940aaa ggt aca ctg ata cca cta att atc ttg gct caa acc atg ttc caa 3120Lys Gly Thr Leu Ile Pro Leu Ile Ile Leu Ala Gln Thr Met Phe Gln 945 950 955gtt ctt cag att gcg agc aac tgg tgg atg gca tgg gca aac cca caa 3168Val Leu Gln Ile Ala Ser Asn Trp Trp Met Ala Trp Ala Asn Pro Gln960 965 970 975aca gaa gga gat gct ccc aag aca gat agt gtg gtc ctt ctg gtt gtt 3216Thr Glu Gly Asp Ala Pro Lys Thr Asp Ser Val Val Leu Leu Val Val 980 985 990tat atg tcc ctt gcc ttt gga agt tca cta ttt gtg ttc atg aga agc 3264Tyr Met Ser Leu Ala Phe Gly Ser Ser Leu Phe Val Phe Met Arg Ser 995 1000 1005ctt ctt gtg gct acg ttt ggt tta gca gct gcc cag aag ctt ttt ata 3312Leu Leu Val Ala Thr Phe Gly Leu Ala Ala Ala Gln Lys Leu Phe Ile 1010 1015 1020aaa atg ctt agg tgt gtc ttt cga gct cca atg tca ttc ttt gac acc 3360Lys Met Leu Arg Cys Val Phe Arg Ala Pro Met Ser Phe Phe Asp Thr 1025 1030 1035aca cca tct ggt cgg att ttg aac aga gtt tct gta gat caa agt gtt 3408Thr Pro Ser Gly Arg Ile Leu Asn Arg Val Ser Val Asp Gln Ser Val1040 1045 1050 1055gtg gac ctt gat ata gcg ttc aga ctt ggt gga ttt gca tca acg aca 3456Val Asp Leu Asp Ile Ala Phe Arg Leu Gly Gly Phe Ala Ser Thr Thr 1060 1065 1070att caa ctc ctt gga att gtt gct gtc atg agc aaa gtc aca tgg caa 3504Ile Gln Leu Leu Gly Ile Val Ala Val Met Ser Lys Val Thr Trp Gln 1075 1080 1085gtt ctg att ctt ata gtc ccc atg gct gtt gca tgc atg tgg atg cag 3552Val Leu Ile Leu Ile Val Pro Met Ala Val Ala Cys Met Trp Met Gln 1090 1095 1100agg tat tat att gct tca tca agg gaa cta act agg att ttg agt gtt 3600Arg Tyr Tyr Ile Ala Ser Ser Arg Glu Leu Thr Arg Ile Leu Ser Val 1105 1110 1115cag aag tct cca gtg atc cat ttg ttt agt gaa tca att gct ggt gct 3648Gln Lys Ser Pro Val Ile His Leu Phe Ser Glu Ser Ile Ala Gly Ala1120 1125 1130 1135gct aca ata agg ggt ttt ggt caa gag aag cgg ttt atg aaa agg aat 3696Ala Thr Ile Arg Gly Phe Gly Gln Glu Lys Arg Phe Met Lys Arg Asn 1140 1145 1150ctt tat ctt ctt gac tgt ttt gct cgc cct tta ttt tcc agc ctt gct 3744Leu Tyr Leu Leu Asp Cys Phe Ala Arg Pro Leu Phe Ser Ser Leu Ala 1155 1160 1165gct att gaa tgg ctc tgc ctg cga atg gaa ttg ctt tcg act ttc gtc 3792Ala Ile Glu Trp Leu Cys Leu Arg Met Glu Leu Leu Ser Thr Phe Val 1170 1175 1180ttt gct ttt tgc atg gca ata ctt gtg agc ttt cct cct ggc aca atc 3840Phe Ala Phe Cys Met Ala Ile Leu Val Ser Phe Pro Pro Gly Thr Ile 1185 1190 1195gaa cca agt atg gct ggc ctc gct gta aca tat gga ctt aat tta aat 3888Glu Pro Ser Met Ala Gly Leu Ala Val Thr Tyr Gly Leu Asn Leu Asn1200 1205 1210 1215gct cgc atg tca aga tgg ata ttg agc ttc tgt aaa tta gag aac agg 3936Ala Arg Met Ser Arg Trp Ile Leu Ser Phe Cys Lys Leu Glu Asn Arg 1220 1225 1230ata atc tct gtt gag cgc att tat caa tat tgc agg ctt cct agt gaa 3984Ile Ile Ser Val Glu Arg Ile Tyr Gln Tyr Cys Arg Leu Pro Ser Glu 1235 1240 1245gca cca ttg att att gag aac tgc cgt cca cca tca tca tgg cct cag 4032Ala Pro Leu Ile Ile Glu Asn Cys Arg Pro Pro Ser Ser Trp Pro Gln 1250 1255 1260aat gga aac att gaa ctg att gat ctc aag gtc cgc tac aag gac gat 4080Asn Gly Asn Ile Glu Leu Ile Asp Leu Lys Val Arg Tyr Lys Asp Asp 1265 1270 1275cta cca tta gtt ctt cat ggt gta agt tgt atg ttt cct ggc ggg aaa 4128Leu Pro Leu Val Leu His Gly Val Ser Cys Met Phe Pro Gly Gly Lys1280 1285 1290 1295aag att ggg att gta ggg cgt act gga agc ggt aaa tct act ctt att 4176Lys Ile Gly Ile Val Gly Arg Thr Gly Ser Gly Lys Ser Thr Leu Ile 1300 1305 1310cag gcc ctt ttc cgc cta att gag ccc act gga ggg aag att ata att 4224Gln Ala Leu Phe Arg Leu Ile Glu Pro Thr Gly Gly Lys Ile Ile Ile 1315 1320 1325gac aac att gac atc tct gca att ggc ctt cat gat ctg cgg tca cgg 4272Asp Asn Ile Asp Ile Ser Ala Ile Gly Leu His Asp Leu Arg Ser Arg 1330 1335 1340ttg agc atc att ccc caa gac cct aca ttg ttt gag ggt act atc aga 4320Leu Ser Ile Ile Pro Gln Asp Pro Thr Leu Phe Glu Gly Thr Ile Arg 1345 1350 1355atg aac ctt gat cct ctt gag gag tgc act gat caa gaa att tgg gag 4368Met Asn Leu Asp Pro Leu Glu Glu Cys Thr Asp Gln Glu Ile Trp Glu1360 1365 1370 1375gca cta gaa aag tgt cag cta gga gag gtc att cgt tcc aag gaa gag 4416Ala Leu Glu Lys Cys Gln Leu Gly Glu Val Ile Arg Ser Lys Glu Glu 1380 1385 1390aaa ctt gac agt cca gtg cta gaa aac ggg gat aac tgg agc gtg gga 4464Lys Leu Asp Ser Pro Val Leu Glu Asn Gly Asp Asn Trp Ser Val Gly 1395 1400 1405cag cgc caa ctt att gca ctg ggt agg gcg ctg ctc aag cag gca aaa 4512Gln Arg Gln Leu Ile Ala Leu Gly Arg Ala Leu Leu Lys Gln Ala Lys 1410 1415 1420att ttg gta ctc gat gag gcg aca gca tct gtc gac aca gca aca gac 4560Ile Leu Val Leu Asp Glu Ala Thr Ala Ser Val Asp Thr Ala Thr Asp 1425 1430 1435aat ctt atc caa aag atc atc cgc agt gaa ttc aag gac tgc aca gtc 4608Asn Leu Ile Gln Lys Ile Ile Arg Ser Glu Phe Lys Asp Cys Thr Val1440 1445 1450 1455tgt acc att gct cac cgt att ccc acc gtt att gac agt gac ctt gtt 4656Cys Thr Ile Ala His Arg Ile Pro Thr Val Ile Asp Ser Asp Leu Val 1460 1465 1470ctg gtc ctt agt gat ggt aaa atc gca gag ttc gac acg ccc cag agg 4704Leu Val Leu Ser Asp Gly Lys Ile Ala Glu Phe Asp Thr Pro Gln Arg 1475 1480 1485ctt tta gag gac aag tca tct atg ttc ata cag cta gta tcg gaa tac 4752Leu Leu Glu Asp Lys Ser Ser Met Phe Ile Gln Leu Val Ser Glu Tyr 1490 1495 1500tcc act cgg tcg agc tgt ata tag agaggcttag cttaaaaccc cgccccaaac 4806Ser Thr Arg Ser Ser Cys Ile 1505 1510ctggcaacag aggctgggag gcaaatagcc cgtatctgcc atgcttgcgc catagaggtc 4866cctgcgaaca ccggagggcg gcgtagaaga cgaggtgtac atgagtggga ggaacactgg 4926gcgttccctg acctgaatac cgtggaatcg gcgagggagc gcggttggta ttggtaggca 4986ccaggggagg agttggtgac actagtacat tacccgaagc tgatgcttca gtatgtatgt 5046ataacaacaa tgcatactgc ttctcccttt gcagagtgga gaaccaaggg aataactcgt 5106gcgtaataag aggagaaaga tttgtttttt ggc 513931510PRTZea mays 3Met Pro Pro Ser Phe Pro Ser Leu Pro Leu Pro Glu Ala Val Ala Ala1 5 10 15Thr Ala His Ala Ala Leu Leu Ala Leu Ala Ala Leu Leu Leu Leu Leu 20 25 30Arg Ala Ala Arg Ala Leu Ala Ser Arg Cys Ala Ser Cys Leu Lys Ala 35 40 45Pro Arg Arg Arg Gly Gly Pro Ala Val Val Val Gly Asp Gly Ala Gly 50 55 60Gly Ala Leu Ala Ala Ala Thr Ala Gly Ala Trp His Arg Ala Val Leu65 70 75 80Ala Ser Cys Ala Tyr Ala Leu Leu Ser Gln Val Ala Val Leu Ser Tyr 85 90 95Glu Val Ala Val Ala Gly Ser Arg Val Ser Ala Arg Ala Leu Leu Leu 100 105 110Pro Ala Val Gln Ala Val Ser Trp Ala Ala Leu Leu Ala Leu Ala Leu 115 120 125Gln Ala Arg Ala Val Gly Trp Ala Arg Phe Pro Ala Leu Val Arg Leu 130 135 140Trp Trp Val Val Ser Phe Ala Leu Cys Val Val Ile Ala Tyr Asp Asp145 150 155 160Ser Arg Arg Leu Ile Gly Gln Gly Ala Arg Ala Val Asp Tyr Ala His 165 170 175Met Val Ala Asn Phe Ala Ser Val Pro Ala Leu Gly Phe Leu Cys Leu 180 185 190Val Gly Val Met Gly Ser Thr Gly Leu Glu Leu Glu Phe Thr Glu Asp 195 200 205Gly Asn Gly Leu His Glu Pro Leu Leu Leu Gly Arg Gln Arg Arg Glu 210 215 220Ala Glu Glu Glu Leu Gly Cys Leu Arg Val Thr Pro Tyr Ala Asp Ala225 230 235 240Gly Ile Leu Ser Leu Ala Thr Leu Ser Trp Leu Ser Pro Leu Leu Ser 245 250 255Val Gly Ala Gln Arg Pro Leu Glu Leu Ala Asp Ile Pro Leu Leu Ala 260 265 270His Lys Asp Arg Ala Lys Ser Cys Tyr Lys Ala Met Ser Ala His Tyr 275 280 285Glu Arg Gln Arg Leu Glu Tyr Pro Gly Arg Glu Pro Ser Leu Thr Trp 290 295 300Ala Ile Leu Lys Ser Phe Trp Arg Glu Ala Ala Val Asn Gly Thr Phe305 310 315 320Ala Ala Val Asn Thr Ile Val Ser Tyr Val Gly Pro Tyr Leu Ile Ser 325 330 335Tyr Phe Val Asp Tyr Leu Ser Gly Asn Ile Ala Phe Pro His Glu Gly 340 345 350Tyr Ile Leu Ala Ser Ile Phe Phe Val Ala Lys Leu Leu Glu Thr Leu 355 360 365Thr Ala Arg Gln Trp Tyr Leu Gly Val Asp Ile Met Gly Ile His Val 370 375 380Lys Ser Gly Leu Thr Ala Met Val Tyr Arg Lys Gly Leu Arg Leu Ser385 390 395 400Asn Ala Ser Arg Gln Ser His Thr Ser Gly Glu Ile Val Asn Tyr Met 405 410 415Ala Val Asp Val Gln Arg Val Gly Asp Tyr Ala Trp Tyr Phe His Asp 420 425 430Ile Trp Met Leu Pro Leu Gln Ile Ile Leu Ala Leu Ala Ile Leu Tyr 435 440 445Lys Asn Val Gly Ile Ala Met Val Ser Thr Leu Val Ala Thr Val Leu 450 455 460Ser Ile Ala Ala Ser Val Pro Val Ala Lys Leu Gln Glu His Tyr Gln465 470 475 480Asp Lys Leu Met Ala Ser Lys

Asp Glu Arg Met Arg Lys Thr Ser Glu 485 490 495Cys Leu Lys Asn Met Arg Ile Leu Lys Leu Gln Ala Trp Glu Asp Arg 500 505 510Tyr Arg Leu Gln Leu Glu Glu Met Arg Asn Val Glu Cys Arg Trp Leu 515 520 525Arg Trp Ala Leu Tyr Ser Gln Ala Ala Val Thr Phe Val Phe Trp Ser 530 535 540Ser Pro Ile Phe Val Ala Val Ile Thr Phe Gly Thr Cys Ile Leu Leu545 550 555 560Gly Gly Gln Leu Thr Ala Gly Gly Val Leu Ser Ala Leu Ala Thr Phe 565 570 575Arg Ile Leu Gln Glu Pro Leu Arg Asn Phe Pro Asp Leu Ile Ser Met 580 585 590Met Ala Gln Thr Arg Val Ser Leu Asp Arg Leu Ser His Phe Leu Gln 595 600 605Gln Glu Glu Leu Pro Asp Asp Ala Thr Ile Asn Val Pro Gln Ser Ser 610 615 620Thr Asp Lys Ala Val Asp Ile Lys Asp Gly Ala Phe Ser Trp Asn Pro625 630 635 640Tyr Thr Leu Thr Pro Thr Leu Ser Asp Ile His Leu Ser Val Val Arg 645 650 655Gly Met Arg Val Ala Val Cys Gly Val Ile Gly Ser Gly Lys Ser Ser 660 665 670Leu Leu Ser Ser Ile Leu Gly Glu Ile Pro Lys Leu Cys Gly His Val 675 680 685Arg Ile Ser Gly Thr Ala Ala Tyr Val Pro Gln Thr Ala Trp Ile Gln 690 695 700Ser Gly Asn Ile Glu Glu Asn Ile Leu Phe Gly Ser Gln Met Asp Arg705 710 715 720Gln Arg Tyr Lys Arg Val Ile Ala Ala Cys Cys Leu Lys Lys Asp Leu 725 730 735Glu Leu Leu Gln Tyr Gly Asp Gln Thr Val Ile Gly Asp Arg Gly Ile 740 745 750Asn Leu Ser Gly Gly Gln Lys Gln Arg Val Gln Leu Ala Arg Ala Leu 755 760 765Tyr Gln Asp Ala Asp Ile Tyr Leu Leu Asp Asp Pro Phe Ser Ala Val 770 775 780Asp Ala His Thr Gly Ser Glu Leu Phe Lys Glu Tyr Ile Leu Thr Ala785 790 795 800Leu Ala Thr Lys Thr Val Ile Tyr Val Thr His Gln Val Glu Phe Leu 805 810 815Pro Ala Ala Asp Leu Ile Leu Val Leu Lys Asp Gly His Ile Thr Gln 820 825 830Ala Gly Lys Tyr Asp Asp Leu Leu Gln Ala Gly Thr Asp Phe Asn Ala 835 840 845Leu Val Ser Ala His Lys Glu Ala Ile Glu Thr Met Asp Ile Phe Glu 850 855 860Asp Ser Asp Ser Asp Thr Val Ser Ser Ile Pro Asn Lys Arg Leu Thr865 870 875 880Pro Ser Ile Ser Asn Ile Asp Asn Leu Lys Asn Lys Met Cys Glu Asn 885 890 895Gly Gln Pro Ser Asn Thr Arg Gly Ile Lys Glu Lys Lys Lys Lys Glu 900 905 910Glu Arg Lys Lys Lys Arg Thr Val Gln Glu Glu Glu Arg Glu Arg Gly 915 920 925Lys Val Ser Ser Lys Val Tyr Leu Ser Tyr Met Gly Glu Ala Tyr Lys 930 935 940Gly Thr Leu Ile Pro Leu Ile Ile Leu Ala Gln Thr Met Phe Gln Val945 950 955 960Leu Gln Ile Ala Ser Asn Trp Trp Met Ala Trp Ala Asn Pro Gln Thr 965 970 975Glu Gly Asp Ala Pro Lys Thr Asp Ser Val Val Leu Leu Val Val Tyr 980 985 990Met Ser Leu Ala Phe Gly Ser Ser Leu Phe Val Phe Met Arg Ser Leu 995 1000 1005Leu Val Ala Thr Phe Gly Leu Ala Ala Ala Gln Lys Leu Phe Ile Lys 1010 1015 1020Met Leu Arg Cys Val Phe Arg Ala Pro Met Ser Phe Phe Asp Thr Thr1025 1030 1035 1040Pro Ser Gly Arg Ile Leu Asn Arg Val Ser Val Asp Gln Ser Val Val 1045 1050 1055Asp Leu Asp Ile Ala Phe Arg Leu Gly Gly Phe Ala Ser Thr Thr Ile 1060 1065 1070Gln Leu Leu Gly Ile Val Ala Val Met Ser Lys Val Thr Trp Gln Val 1075 1080 1085Leu Ile Leu Ile Val Pro Met Ala Val Ala Cys Met Trp Met Gln Arg 1090 1095 1100Tyr Tyr Ile Ala Ser Ser Arg Glu Leu Thr Arg Ile Leu Ser Val Gln1105 1110 1115 1120Lys Ser Pro Val Ile His Leu Phe Ser Glu Ser Ile Ala Gly Ala Ala 1125 1130 1135Thr Ile Arg Gly Phe Gly Gln Glu Lys Arg Phe Met Lys Arg Asn Leu 1140 1145 1150Tyr Leu Leu Asp Cys Phe Ala Arg Pro Leu Phe Ser Ser Leu Ala Ala 1155 1160 1165Ile Glu Trp Leu Cys Leu Arg Met Glu Leu Leu Ser Thr Phe Val Phe 1170 1175 1180Ala Phe Cys Met Ala Ile Leu Val Ser Phe Pro Pro Gly Thr Ile Glu1185 1190 1195 1200Pro Ser Met Ala Gly Leu Ala Val Thr Tyr Gly Leu Asn Leu Asn Ala 1205 1210 1215Arg Met Ser Arg Trp Ile Leu Ser Phe Cys Lys Leu Glu Asn Arg Ile 1220 1225 1230Ile Ser Val Glu Arg Ile Tyr Gln Tyr Cys Arg Leu Pro Ser Glu Ala 1235 1240 1245Pro Leu Ile Ile Glu Asn Cys Arg Pro Pro Ser Ser Trp Pro Gln Asn 1250 1255 1260Gly Asn Ile Glu Leu Ile Asp Leu Lys Val Arg Tyr Lys Asp Asp Leu1265 1270 1275 1280Pro Leu Val Leu His Gly Val Ser Cys Met Phe Pro Gly Gly Lys Lys 1285 1290 1295Ile Gly Ile Val Gly Arg Thr Gly Ser Gly Lys Ser Thr Leu Ile Gln 1300 1305 1310Ala Leu Phe Arg Leu Ile Glu Pro Thr Gly Gly Lys Ile Ile Ile Asp 1315 1320 1325Asn Ile Asp Ile Ser Ala Ile Gly Leu His Asp Leu Arg Ser Arg Leu 1330 1335 1340Ser Ile Ile Pro Gln Asp Pro Thr Leu Phe Glu Gly Thr Ile Arg Met1345 1350 1355 1360Asn Leu Asp Pro Leu Glu Glu Cys Thr Asp Gln Glu Ile Trp Glu Ala 1365 1370 1375Leu Glu Lys Cys Gln Leu Gly Glu Val Ile Arg Ser Lys Glu Glu Lys 1380 1385 1390Leu Asp Ser Pro Val Leu Glu Asn Gly Asp Asn Trp Ser Val Gly Gln 1395 1400 1405Arg Gln Leu Ile Ala Leu Gly Arg Ala Leu Leu Lys Gln Ala Lys Ile 1410 1415 1420Leu Val Leu Asp Glu Ala Thr Ala Ser Val Asp Thr Ala Thr Asp Asn1425 1430 1435 1440Leu Ile Gln Lys Ile Ile Arg Ser Glu Phe Lys Asp Cys Thr Val Cys 1445 1450 1455Thr Ile Ala His Arg Ile Pro Thr Val Ile Asp Ser Asp Leu Val Leu 1460 1465 1470Val Leu Ser Asp Gly Lys Ile Ala Glu Phe Asp Thr Pro Gln Arg Leu 1475 1480 1485Leu Glu Asp Lys Ser Ser Met Phe Ile Gln Leu Val Ser Glu Tyr Ser 1490 1495 1500Thr Arg Ser Ser Cys Ile1505 151045139DNAZea maysCDS(244)...(4776) 4cctctctctc ccctcctcga accaggcgca ggcgagcgtc tctgcccgcc cgcctgctgc 60taccgccaaa acgcctcctt tgttgccatc cgccgatgcc gtaatccgcc gcccaaagct 120cttccttttt ccctctctct cgcccgcggc cgcactccct gccccagtgc ctgccgtggc 180gagcccaacc ccaatgcctt ttaaacccct ccccgctccc tcactgatcc ccaccgcctc 240cca atg ccg ctc tcc ttc ccc tcc ctc ccg ctc ccg gag gcc gtt gcc 288 Met Pro Leu Ser Phe Pro Ser Leu Pro Leu Pro Glu Ala Val Ala 1 5 10 15gcc gcc gcc cac gcc gcg ctg ctc gcg ctc gcc gca ctc ctg ctc ctc 336Ala Ala Ala His Ala Ala Leu Leu Ala Leu Ala Ala Leu Leu Leu Leu 20 25 30ctc cgc gcc gcg cgc gcg ctc gcc tcc cgc tgc gcg tca tgc ctc aag 384Leu Arg Ala Ala Arg Ala Leu Ala Ser Arg Cys Ala Ser Cys Leu Lys 35 40 45gcg ccg cgc cgc cgc ggg ggc ccc gcc gtc gtc gtg ggc gcc ggc gcc 432Ala Pro Arg Arg Arg Gly Gly Pro Ala Val Val Val Gly Ala Gly Ala 50 55 60ggc ggc gcc ctc gcg gcg gcg act gcc ggc gcc tgg cac agg gcc gtg 480Gly Gly Ala Leu Ala Ala Ala Thr Ala Gly Ala Trp His Arg Ala Val 65 70 75ctg gcg tcc tgc gcc tac gcc ctg ctc tcg cag gtc gcc gtg ctg agc 528Leu Ala Ser Cys Ala Tyr Ala Leu Leu Ser Gln Val Ala Val Leu Ser80 85 90 95tac gag gtg gcc gtc gcc ggc tcg cgc gtc tcg gcg cgg gcg ctg ctg 576Tyr Glu Val Ala Val Ala Gly Ser Arg Val Ser Ala Arg Ala Leu Leu 100 105 110ctg ccg gcc gtg cag gcg gtg tcc tgg gcc gcg ctg ctg gcg ctc gcg 624Leu Pro Ala Val Gln Ala Val Ser Trp Ala Ala Leu Leu Ala Leu Ala 115 120 125ctt cag gcc cgc gcc gtc ggc tgg gcc agg ttc cct gcg ctg gtg cgg 672Leu Gln Ala Arg Ala Val Gly Trp Ala Arg Phe Pro Ala Leu Val Arg 130 135 140ctc tgg tgg gtg gtc tcc ttc gcg ctc tgc gtt gtc att gcg tac gac 720Leu Trp Trp Val Val Ser Phe Ala Leu Cys Val Val Ile Ala Tyr Asp 145 150 155gac tcc agg cgc ctg ata ggc cag ggc gcg cgc gct gtg gat tac gcg 768Asp Ser Arg Arg Leu Ile Gly Gln Gly Ala Arg Ala Val Asp Tyr Ala160 165 170 175cac atg gtt gcc aac ttc gcg tcc gtg ccg gcc ctg ggc ttc ctg tgc 816His Met Val Ala Asn Phe Ala Ser Val Pro Ala Leu Gly Phe Leu Cys 180 185 190ttg gtt ggt gtc atg ggt tcc acc ggt ttg gaa ttg gag ttt acg gag 864Leu Val Gly Val Met Gly Ser Thr Gly Leu Glu Leu Glu Phe Thr Glu 195 200 205gat ggc aac ggc ctg cat gag ccg ctg ctg ctc ggc agg cag cgc aga 912Asp Gly Asn Gly Leu His Glu Pro Leu Leu Leu Gly Arg Gln Arg Arg 210 215 220gag gca gag gag gag ctc ggc tgt ctg agg gtc act ccc tac gct gat 960Glu Ala Glu Glu Glu Leu Gly Cys Leu Arg Val Thr Pro Tyr Ala Asp 225 230 235gct ggg atc ctc agc ctt gca aca ttg tca tgg ctt agt ccg ttg ctc 1008Ala Gly Ile Leu Ser Leu Ala Thr Leu Ser Trp Leu Ser Pro Leu Leu240 245 250 255tct gtt ggt gcg cag cgg cca ctt gag ttg gct gac ata ccc ttg ctg 1056Ser Val Gly Ala Gln Arg Pro Leu Glu Leu Ala Asp Ile Pro Leu Leu 260 265 270gcg cac aag gac cgt gca aag tca tgc tat aag gcg atg agc gct cac 1104Ala His Lys Asp Arg Ala Lys Ser Cys Tyr Lys Ala Met Ser Ala His 275 280 285tac gag cgc cag cgg cta gaa tac cct ggc agg gag cca tca ctc aca 1152Tyr Glu Arg Gln Arg Leu Glu Tyr Pro Gly Arg Glu Pro Ser Leu Thr 290 295 300tgg gca ata ctc aag tca ttc tgg cga gag gcc gcg gtc aat ggc aca 1200Trp Ala Ile Leu Lys Ser Phe Trp Arg Glu Ala Ala Val Asn Gly Thr 305 310 315ttt gct gct gtc aac acg att gtg tcg tat gtt gga cct tac ttg atc 1248Phe Ala Ala Val Asn Thr Ile Val Ser Tyr Val Gly Pro Tyr Leu Ile320 325 330 335agc tat ttt gtg gac tac ctc agt ggc aac att gct ttc ccc cat gaa 1296Ser Tyr Phe Val Asp Tyr Leu Ser Gly Asn Ile Ala Phe Pro His Glu 340 345 350ggt tac atc ctt gcc tct ata ttt ttt gta gca aaa ctg ctt gag aca 1344Gly Tyr Ile Leu Ala Ser Ile Phe Phe Val Ala Lys Leu Leu Glu Thr 355 360 365ctc act gcc cga cag tgg tac ttg ggt gtg gac atc atg ggg atc cat 1392Leu Thr Ala Arg Gln Trp Tyr Leu Gly Val Asp Ile Met Gly Ile His 370 375 380gtc aag tct ggc ctc act gcc atg gtg tat agg aag ggt ctc cga ctg 1440Val Lys Ser Gly Leu Thr Ala Met Val Tyr Arg Lys Gly Leu Arg Leu 385 390 395tca aac gcc tca cgg cag agc cac acg agt ggt gag att gtg aat tac 1488Ser Asn Ala Ser Arg Gln Ser His Thr Ser Gly Glu Ile Val Asn Tyr400 405 410 415atg gcc gtc gat gtg cag cgt gtg ggg gac tat gca tgg tat ttc cat 1536Met Ala Val Asp Val Gln Arg Val Gly Asp Tyr Ala Trp Tyr Phe His 420 425 430gac atc tgg atg ctt ccc ctg cag atc att ctt gct ctc gcc atc ctg 1584Asp Ile Trp Met Leu Pro Leu Gln Ile Ile Leu Ala Leu Ala Ile Leu 435 440 445tac aag aac gtc ggg atc gcc atg gtt tca aca ttg gta gca act gtg 1632Tyr Lys Asn Val Gly Ile Ala Met Val Ser Thr Leu Val Ala Thr Val 450 455 460cta tcg atc gca gcc tct gtt cct gtg gca aag ctg cag gag cac tac 1680Leu Ser Ile Ala Ala Ser Val Pro Val Ala Lys Leu Gln Glu His Tyr 465 470 475caa gat aag tta atg gca tca aaa gat gag cgc atg cgc aag act tca 1728Gln Asp Lys Leu Met Ala Ser Lys Asp Glu Arg Met Arg Lys Thr Ser480 485 490 495gag tgc ttg aaa aat atg agg att ttg aag ctt cag gca tgg gag gat 1776Glu Cys Leu Lys Asn Met Arg Ile Leu Lys Leu Gln Ala Trp Glu Asp 500 505 510cgg tac cgg ctg cag ttg gaa gag atg agg aac gtg gaa tgc aga tgg 1824Arg Tyr Arg Leu Gln Leu Glu Glu Met Arg Asn Val Glu Cys Arg Trp 515 520 525ctt cgg tgg gct ctg tac tca cag gct gca gtt aca ttt gtt ttc tgg 1872Leu Arg Trp Ala Leu Tyr Ser Gln Ala Ala Val Thr Phe Val Phe Trp 530 535 540agc tcg cca atc ttt gtc gca gtc ata act ttt ggg act tgc ata tta 1920Ser Ser Pro Ile Phe Val Ala Val Ile Thr Phe Gly Thr Cys Ile Leu 545 550 555ctc ggt ggc cag ctc act gca gga ggg gtt cta tcc gct tta gca acg 1968Leu Gly Gly Gln Leu Thr Ala Gly Gly Val Leu Ser Ala Leu Ala Thr560 565 570 575ttt cgg atc ctc caa gag cct ctg agg aac ttc ccg gat ctc atc tct 2016Phe Arg Ile Leu Gln Glu Pro Leu Arg Asn Phe Pro Asp Leu Ile Ser 580 585 590atg atg gca cag aca agg gtg tct ttg gac cgt ttg tct cat ttt ctg 2064Met Met Ala Gln Thr Arg Val Ser Leu Asp Arg Leu Ser His Phe Leu 595 600 605cag caa gaa gaa ctg cca gat gac gca act ata aat gtt cca caa agt 2112Gln Gln Glu Glu Leu Pro Asp Asp Ala Thr Ile Asn Val Pro Gln Ser 610 615 620agt aca gat aag gca gtc gat att aag gat ggc gca ttc tct tgg aac 2160Ser Thr Asp Lys Ala Val Asp Ile Lys Asp Gly Ala Phe Ser Trp Asn 625 630 635cca tac act ctg acc cct aca ctt tct gat ata cac ctt agt gta gtg 2208Pro Tyr Thr Leu Thr Pro Thr Leu Ser Asp Ile His Leu Ser Val Val640 645 650 655aga ggc atg aga gta gca gtc tgt ggt gtc att ggt tct ggt aaa tca 2256Arg Gly Met Arg Val Ala Val Cys Gly Val Ile Gly Ser Gly Lys Ser 660 665 670agt cta cta tcg tct ata ctc ggg gag ata ccc aaa tta tgt ggc cat 2304Ser Leu Leu Ser Ser Ile Leu Gly Glu Ile Pro Lys Leu Cys Gly His 675 680 685gtc agg ata agt ggc aca gca gcg tat gtt cct cag act gca tgg ata 2352Val Arg Ile Ser Gly Thr Ala Ala Tyr Val Pro Gln Thr Ala Trp Ile 690 695 700cag tct gga aat att gag gag aat att ctg ttt ggc agt caa atg gat 2400Gln Ser Gly Asn Ile Glu Glu Asn Ile Leu Phe Gly Ser Gln Met Asp 705 710 715aga caa cgt tac aag aga gtc att gca gct tgc tgt ctt aag aaa gat 2448Arg Gln Arg Tyr Lys Arg Val Ile Ala Ala Cys Cys Leu Lys Lys Asp720 725 730 735ctt gag ctg ctc cag tac gga gat cag act gtt att ggt gat aga ggc 2496Leu Glu Leu Leu Gln Tyr Gly Asp Gln Thr Val Ile Gly Asp Arg Gly 740 745 750att aat ttg agt gga ggt cag aaa caa aga gtt cag ctt gct aga gca 2544Ile Asn Leu Ser Gly Gly Gln Lys Gln Arg Val Gln Leu Ala Arg Ala 755 760 765ctc tac caa gat gct gat att tat ttg ctt gat gat ccc ttc agt gct 2592Leu Tyr Gln Asp Ala Asp Ile Tyr Leu Leu Asp Asp Pro Phe Ser Ala 770 775 780gtt gat gct cat act ggg agc gaa ctg ttt aag gag tat ata ttg act 2640Val Asp Ala His Thr Gly Ser Glu Leu Phe Lys Glu Tyr Ile Leu Thr 785 790 795gca cta gca acc aaa aca gta atc tat gta aca cat caa gtt gaa ttt 2688Ala Leu Ala Thr Lys Thr Val Ile Tyr Val Thr His Gln Val Glu Phe800 805 810 815cta cca gct gct gat ctg ata ttg gtt ctt aag gat ggc cat atc aca 2736Leu Pro Ala Ala Asp Leu Ile Leu Val Leu Lys Asp Gly His Ile Thr 820 825 830caa gct gga aag tat gat gat ctt ctg caa gct gga act gat ttc aat 2784Gln Ala Gly Lys Tyr Asp Asp Leu Leu Gln Ala Gly Thr Asp Phe Asn 835 840 845gct ctg gtt tct gct cat aag gaa gct att gaa acc atg gat ata ttt 2832Ala Leu Val Ser Ala His Lys Glu Ala Ile Glu Thr Met Asp Ile Phe 850 855 860gaa gat tcc gat agt gat aca gtt tct tct att ccc aac aaa aga ttg 2880Glu Asp Ser Asp Ser Asp Thr

Val Ser Ser Ile Pro Asn Lys Arg Leu 865 870 875aca cca agt atc agc aat att gat aac ctg aaa aat aag atg tgt gaa 2928Thr Pro Ser Ile Ser Asn Ile Asp Asn Leu Lys Asn Lys Met Cys Glu880 885 890 895aat gga caa cca tct aat aca cgg gga att aag gaa aaa aag aag aaa 2976Asn Gly Gln Pro Ser Asn Thr Arg Gly Ile Lys Glu Lys Lys Lys Lys 900 905 910gaa gag cgt aag aag aag cgt act gtt caa gag gag gaa agg gaa cgt 3024Glu Glu Arg Lys Lys Lys Arg Thr Val Gln Glu Glu Glu Arg Glu Arg 915 920 925gga aaa gtg agc tcc aaa gtt tat ttg tca tac atg ggg gaa gct tac 3072Gly Lys Val Ser Ser Lys Val Tyr Leu Ser Tyr Met Gly Glu Ala Tyr 930 935 940aaa ggt aca ctg ata cca cta att atc ttg gct caa acc atg ttc caa 3120Lys Gly Thr Leu Ile Pro Leu Ile Ile Leu Ala Gln Thr Met Phe Gln 945 950 955gtt ctt cag att gcg agc aac tgg tgg atg gca tgg gca aac cca caa 3168Val Leu Gln Ile Ala Ser Asn Trp Trp Met Ala Trp Ala Asn Pro Gln960 965 970 975aca gaa gga gat gct ccc aag aca gat agt gtg gtc ctt ctg gtt gtt 3216Thr Glu Gly Asp Ala Pro Lys Thr Asp Ser Val Val Leu Leu Val Val 980 985 990tat atg tcc ctt gcc ttt gga agt tca cta ttt gtg ttc atg aga agc 3264Tyr Met Ser Leu Ala Phe Gly Ser Ser Leu Phe Val Phe Met Arg Ser 995 1000 1005ctt ctt gtg gct acg ttt ggt tta gca gct gcc cag aag ctt ttt ata 3312Leu Leu Val Ala Thr Phe Gly Leu Ala Ala Ala Gln Lys Leu Phe Ile 1010 1015 1020aaa atg ctt agg tgt gtc ttt cga gct cca atg tca ttc ttt gac acc 3360Lys Met Leu Arg Cys Val Phe Arg Ala Pro Met Ser Phe Phe Asp Thr 1025 1030 1035aca cca tct ggt cgg att ttg aac aga gtt tct gta gat caa agt gtt 3408Thr Pro Ser Gly Arg Ile Leu Asn Arg Val Ser Val Asp Gln Ser Val1040 1045 1050 1055gtg gac ctt gat ata gcg ttc aga ctt ggt gga ttt gca tca acg aca 3456Val Asp Leu Asp Ile Ala Phe Arg Leu Gly Gly Phe Ala Ser Thr Thr 1060 1065 1070att caa ctc ctt gga att gtt gct gtc atg agc aaa gtc aca tgg caa 3504Ile Gln Leu Leu Gly Ile Val Ala Val Met Ser Lys Val Thr Trp Gln 1075 1080 1085gtt ctg att ctt ata gtc ccc atg gct gtt gca tgc atg tgg atg cag 3552Val Leu Ile Leu Ile Val Pro Met Ala Val Ala Cys Met Trp Met Gln 1090 1095 1100agg tat tat att gct tca tca agg gaa cta act agg att ttg agt gtt 3600Arg Tyr Tyr Ile Ala Ser Ser Arg Glu Leu Thr Arg Ile Leu Ser Val 1105 1110 1115cag aag tct cca gtg atc cat ttg ttt agt gaa tca att gct ggt gct 3648Gln Lys Ser Pro Val Ile His Leu Phe Ser Glu Ser Ile Ala Gly Ala1120 1125 1130 1135gct aca ata agg ggt ttt ggt caa gag aag cgg ttt atg aaa agg aat 3696Ala Thr Ile Arg Gly Phe Gly Gln Glu Lys Arg Phe Met Lys Arg Asn 1140 1145 1150ctt tat ctt ctt gac tgt ttt gct cgc cct tta ttt tcc agc ctt gct 3744Leu Tyr Leu Leu Asp Cys Phe Ala Arg Pro Leu Phe Ser Ser Leu Ala 1155 1160 1165gct att gaa tgg ctc tgc ctg cga atg gaa ttg ctt tcg act ttc gtc 3792Ala Ile Glu Trp Leu Cys Leu Arg Met Glu Leu Leu Ser Thr Phe Val 1170 1175 1180ttt gct ttt tgc atg gca ata ctt gtg agc ttt cct cct ggc aca atc 3840Phe Ala Phe Cys Met Ala Ile Leu Val Ser Phe Pro Pro Gly Thr Ile 1185 1190 1195gaa cca agt atg gct ggc ctc gct gta aca tat gga ctt aat tta aat 3888Glu Pro Ser Met Ala Gly Leu Ala Val Thr Tyr Gly Leu Asn Leu Asn1200 1205 1210 1215gct cgc atg tca aga tgg ata ttg agc ttc tgt aaa tta gag aac agg 3936Ala Arg Met Ser Arg Trp Ile Leu Ser Phe Cys Lys Leu Glu Asn Arg 1220 1225 1230ata atc tct gtt gag cgc att tat caa tat tgc agg ctt cct agt gaa 3984Ile Ile Ser Val Glu Arg Ile Tyr Gln Tyr Cys Arg Leu Pro Ser Glu 1235 1240 1245gca cca ttg att att gag aac tgc cgt cca cca tca tca tgg cct cag 4032Ala Pro Leu Ile Ile Glu Asn Cys Arg Pro Pro Ser Ser Trp Pro Gln 1250 1255 1260aat gga aac att gaa ctg att gat ctc aag gtc cgc tac aag gac gat 4080Asn Gly Asn Ile Glu Leu Ile Asp Leu Lys Val Arg Tyr Lys Asp Asp 1265 1270 1275cta cca tta gtt ctt cat ggt gta agt tgt atg ttt cct ggc ggg aaa 4128Leu Pro Leu Val Leu His Gly Val Ser Cys Met Phe Pro Gly Gly Lys1280 1285 1290 1295aag att ggg att gta ggg cgt act gga agc ggt aaa tct act ctt att 4176Lys Ile Gly Ile Val Gly Arg Thr Gly Ser Gly Lys Ser Thr Leu Ile 1300 1305 1310cag gcc ctt ttc cgc cta att gag ccc act gga ggg aag att ata att 4224Gln Ala Leu Phe Arg Leu Ile Glu Pro Thr Gly Gly Lys Ile Ile Ile 1315 1320 1325gac aac att gac atc tct gca att ggc ctt cat gat ctg cgg tca cgg 4272Asp Asn Ile Asp Ile Ser Ala Ile Gly Leu His Asp Leu Arg Ser Arg 1330 1335 1340ttg agc atc att ccc caa gac cct aca ttg ttt gag ggt act atc aga 4320Leu Ser Ile Ile Pro Gln Asp Pro Thr Leu Phe Glu Gly Thr Ile Arg 1345 1350 1355atg aac ctt gat cct ctt gag gag tgc act gat caa gaa att tgg gag 4368Met Asn Leu Asp Pro Leu Glu Glu Cys Thr Asp Gln Glu Ile Trp Glu1360 1365 1370 1375gca cta gaa aag tgt cag cta gga gag gtc att cgt tcc aag gaa gag 4416Ala Leu Glu Lys Cys Gln Leu Gly Glu Val Ile Arg Ser Lys Glu Glu 1380 1385 1390aaa ctt gac agt cca gtg cta gaa aac ggg gat aac tgg agc gtg gga 4464Lys Leu Asp Ser Pro Val Leu Glu Asn Gly Asp Asn Trp Ser Val Gly 1395 1400 1405cag cgc caa ctt att gca ctg ggt agg gcg ctg ctc aag cag gca aaa 4512Gln Arg Gln Leu Ile Ala Leu Gly Arg Ala Leu Leu Lys Gln Ala Lys 1410 1415 1420att ttg gta ctc gat gag gcg aca gca tct gtc gac aca gca aca gac 4560Ile Leu Val Leu Asp Glu Ala Thr Ala Ser Val Asp Thr Ala Thr Asp 1425 1430 1435aat ctt atc caa aag atc atc cgc agt gaa ttc aag gac tgc aca gtc 4608Asn Leu Ile Gln Lys Ile Ile Arg Ser Glu Phe Lys Asp Cys Thr Val1440 1445 1450 1455tgt acc att gct cac cgt att ccc acc gtt att gac agt gac ctt gtt 4656Cys Thr Ile Ala His Arg Ile Pro Thr Val Ile Asp Ser Asp Leu Val 1460 1465 1470ctg gtc ctt agt gat ggt aaa atc gca gag ttc gac acg ccc cag agg 4704Leu Val Leu Ser Asp Gly Lys Ile Ala Glu Phe Asp Thr Pro Gln Arg 1475 1480 1485ctt tta gag gac aag tca tct atg ttc ata cag cta gta tcg gaa tac 4752Leu Leu Glu Asp Lys Ser Ser Met Phe Ile Gln Leu Val Ser Glu Tyr 1490 1495 1500tcc act cgg tcg agc tgt ata tag agaggcttag cttaaaaccc cgccccaaac 4806Ser Thr Arg Ser Ser Cys Ile 1505 1510ctggcaacag aggctgggag gcaaatagcc cgtatctgcc atgcttgcgc catagaggtc 4866cctgcgaaca ccggagggcg gcgtagaaga cgaggtgtac atgagtggga ggaacactgg 4926gcgttccctg acctgaatac cgtggaatcg gcgagggagc gcggttggta ttggtaggca 4986ccaggggagg agttggtgac actagtacat tacccgaagc tgatgcttca gtatgtatgt 5046ataacaacaa tgcatactgc ttctcccttt gcagagtgga gaaccaaggg aataactcgt 5106gcgtaataag aggagaaaga tttgtttttt ggc 513951510PRTZea mays 5Met Pro Leu Ser Phe Pro Ser Leu Pro Leu Pro Glu Ala Val Ala Ala1 5 10 15Ala Ala His Ala Ala Leu Leu Ala Leu Ala Ala Leu Leu Leu Leu Leu 20 25 30Arg Ala Ala Arg Ala Leu Ala Ser Arg Cys Ala Ser Cys Leu Lys Ala 35 40 45Pro Arg Arg Arg Gly Gly Pro Ala Val Val Val Gly Ala Gly Ala Gly 50 55 60Gly Ala Leu Ala Ala Ala Thr Ala Gly Ala Trp His Arg Ala Val Leu65 70 75 80Ala Ser Cys Ala Tyr Ala Leu Leu Ser Gln Val Ala Val Leu Ser Tyr 85 90 95Glu Val Ala Val Ala Gly Ser Arg Val Ser Ala Arg Ala Leu Leu Leu 100 105 110Pro Ala Val Gln Ala Val Ser Trp Ala Ala Leu Leu Ala Leu Ala Leu 115 120 125Gln Ala Arg Ala Val Gly Trp Ala Arg Phe Pro Ala Leu Val Arg Leu 130 135 140Trp Trp Val Val Ser Phe Ala Leu Cys Val Val Ile Ala Tyr Asp Asp145 150 155 160Ser Arg Arg Leu Ile Gly Gln Gly Ala Arg Ala Val Asp Tyr Ala His 165 170 175Met Val Ala Asn Phe Ala Ser Val Pro Ala Leu Gly Phe Leu Cys Leu 180 185 190Val Gly Val Met Gly Ser Thr Gly Leu Glu Leu Glu Phe Thr Glu Asp 195 200 205Gly Asn Gly Leu His Glu Pro Leu Leu Leu Gly Arg Gln Arg Arg Glu 210 215 220Ala Glu Glu Glu Leu Gly Cys Leu Arg Val Thr Pro Tyr Ala Asp Ala225 230 235 240Gly Ile Leu Ser Leu Ala Thr Leu Ser Trp Leu Ser Pro Leu Leu Ser 245 250 255Val Gly Ala Gln Arg Pro Leu Glu Leu Ala Asp Ile Pro Leu Leu Ala 260 265 270His Lys Asp Arg Ala Lys Ser Cys Tyr Lys Ala Met Ser Ala His Tyr 275 280 285Glu Arg Gln Arg Leu Glu Tyr Pro Gly Arg Glu Pro Ser Leu Thr Trp 290 295 300Ala Ile Leu Lys Ser Phe Trp Arg Glu Ala Ala Val Asn Gly Thr Phe305 310 315 320Ala Ala Val Asn Thr Ile Val Ser Tyr Val Gly Pro Tyr Leu Ile Ser 325 330 335Tyr Phe Val Asp Tyr Leu Ser Gly Asn Ile Ala Phe Pro His Glu Gly 340 345 350Tyr Ile Leu Ala Ser Ile Phe Phe Val Ala Lys Leu Leu Glu Thr Leu 355 360 365Thr Ala Arg Gln Trp Tyr Leu Gly Val Asp Ile Met Gly Ile His Val 370 375 380Lys Ser Gly Leu Thr Ala Met Val Tyr Arg Lys Gly Leu Arg Leu Ser385 390 395 400Asn Ala Ser Arg Gln Ser His Thr Ser Gly Glu Ile Val Asn Tyr Met 405 410 415Ala Val Asp Val Gln Arg Val Gly Asp Tyr Ala Trp Tyr Phe His Asp 420 425 430Ile Trp Met Leu Pro Leu Gln Ile Ile Leu Ala Leu Ala Ile Leu Tyr 435 440 445Lys Asn Val Gly Ile Ala Met Val Ser Thr Leu Val Ala Thr Val Leu 450 455 460Ser Ile Ala Ala Ser Val Pro Val Ala Lys Leu Gln Glu His Tyr Gln465 470 475 480Asp Lys Leu Met Ala Ser Lys Asp Glu Arg Met Arg Lys Thr Ser Glu 485 490 495Cys Leu Lys Asn Met Arg Ile Leu Lys Leu Gln Ala Trp Glu Asp Arg 500 505 510Tyr Arg Leu Gln Leu Glu Glu Met Arg Asn Val Glu Cys Arg Trp Leu 515 520 525Arg Trp Ala Leu Tyr Ser Gln Ala Ala Val Thr Phe Val Phe Trp Ser 530 535 540Ser Pro Ile Phe Val Ala Val Ile Thr Phe Gly Thr Cys Ile Leu Leu545 550 555 560Gly Gly Gln Leu Thr Ala Gly Gly Val Leu Ser Ala Leu Ala Thr Phe 565 570 575Arg Ile Leu Gln Glu Pro Leu Arg Asn Phe Pro Asp Leu Ile Ser Met 580 585 590Met Ala Gln Thr Arg Val Ser Leu Asp Arg Leu Ser His Phe Leu Gln 595 600 605Gln Glu Glu Leu Pro Asp Asp Ala Thr Ile Asn Val Pro Gln Ser Ser 610 615 620Thr Asp Lys Ala Val Asp Ile Lys Asp Gly Ala Phe Ser Trp Asn Pro625 630 635 640Tyr Thr Leu Thr Pro Thr Leu Ser Asp Ile His Leu Ser Val Val Arg 645 650 655Gly Met Arg Val Ala Val Cys Gly Val Ile Gly Ser Gly Lys Ser Ser 660 665 670Leu Leu Ser Ser Ile Leu Gly Glu Ile Pro Lys Leu Cys Gly His Val 675 680 685Arg Ile Ser Gly Thr Ala Ala Tyr Val Pro Gln Thr Ala Trp Ile Gln 690 695 700Ser Gly Asn Ile Glu Glu Asn Ile Leu Phe Gly Ser Gln Met Asp Arg705 710 715 720Gln Arg Tyr Lys Arg Val Ile Ala Ala Cys Cys Leu Lys Lys Asp Leu 725 730 735Glu Leu Leu Gln Tyr Gly Asp Gln Thr Val Ile Gly Asp Arg Gly Ile 740 745 750Asn Leu Ser Gly Gly Gln Lys Gln Arg Val Gln Leu Ala Arg Ala Leu 755 760 765Tyr Gln Asp Ala Asp Ile Tyr Leu Leu Asp Asp Pro Phe Ser Ala Val 770 775 780Asp Ala His Thr Gly Ser Glu Leu Phe Lys Glu Tyr Ile Leu Thr Ala785 790 795 800Leu Ala Thr Lys Thr Val Ile Tyr Val Thr His Gln Val Glu Phe Leu 805 810 815Pro Ala Ala Asp Leu Ile Leu Val Leu Lys Asp Gly His Ile Thr Gln 820 825 830Ala Gly Lys Tyr Asp Asp Leu Leu Gln Ala Gly Thr Asp Phe Asn Ala 835 840 845Leu Val Ser Ala His Lys Glu Ala Ile Glu Thr Met Asp Ile Phe Glu 850 855 860Asp Ser Asp Ser Asp Thr Val Ser Ser Ile Pro Asn Lys Arg Leu Thr865 870 875 880Pro Ser Ile Ser Asn Ile Asp Asn Leu Lys Asn Lys Met Cys Glu Asn 885 890 895Gly Gln Pro Ser Asn Thr Arg Gly Ile Lys Glu Lys Lys Lys Lys Glu 900 905 910Glu Arg Lys Lys Lys Arg Thr Val Gln Glu Glu Glu Arg Glu Arg Gly 915 920 925Lys Val Ser Ser Lys Val Tyr Leu Ser Tyr Met Gly Glu Ala Tyr Lys 930 935 940Gly Thr Leu Ile Pro Leu Ile Ile Leu Ala Gln Thr Met Phe Gln Val945 950 955 960Leu Gln Ile Ala Ser Asn Trp Trp Met Ala Trp Ala Asn Pro Gln Thr 965 970 975Glu Gly Asp Ala Pro Lys Thr Asp Ser Val Val Leu Leu Val Val Tyr 980 985 990Met Ser Leu Ala Phe Gly Ser Ser Leu Phe Val Phe Met Arg Ser Leu 995 1000 1005Leu Val Ala Thr Phe Gly Leu Ala Ala Ala Gln Lys Leu Phe Ile Lys 1010 1015 1020Met Leu Arg Cys Val Phe Arg Ala Pro Met Ser Phe Phe Asp Thr Thr1025 1030 1035 1040Pro Ser Gly Arg Ile Leu Asn Arg Val Ser Val Asp Gln Ser Val Val 1045 1050 1055Asp Leu Asp Ile Ala Phe Arg Leu Gly Gly Phe Ala Ser Thr Thr Ile 1060 1065 1070Gln Leu Leu Gly Ile Val Ala Val Met Ser Lys Val Thr Trp Gln Val 1075 1080 1085Leu Ile Leu Ile Val Pro Met Ala Val Ala Cys Met Trp Met Gln Arg 1090 1095 1100Tyr Tyr Ile Ala Ser Ser Arg Glu Leu Thr Arg Ile Leu Ser Val Gln1105 1110 1115 1120Lys Ser Pro Val Ile His Leu Phe Ser Glu Ser Ile Ala Gly Ala Ala 1125 1130 1135Thr Ile Arg Gly Phe Gly Gln Glu Lys Arg Phe Met Lys Arg Asn Leu 1140 1145 1150Tyr Leu Leu Asp Cys Phe Ala Arg Pro Leu Phe Ser Ser Leu Ala Ala 1155 1160 1165Ile Glu Trp Leu Cys Leu Arg Met Glu Leu Leu Ser Thr Phe Val Phe 1170 1175 1180Ala Phe Cys Met Ala Ile Leu Val Ser Phe Pro Pro Gly Thr Ile Glu1185 1190 1195 1200Pro Ser Met Ala Gly Leu Ala Val Thr Tyr Gly Leu Asn Leu Asn Ala 1205 1210 1215Arg Met Ser Arg Trp Ile Leu Ser Phe Cys Lys Leu Glu Asn Arg Ile 1220 1225 1230Ile Ser Val Glu Arg Ile Tyr Gln Tyr Cys Arg Leu Pro Ser Glu Ala 1235 1240 1245Pro Leu Ile Ile Glu Asn Cys Arg Pro Pro Ser Ser Trp Pro Gln Asn 1250 1255 1260Gly Asn Ile Glu Leu Ile Asp Leu Lys Val Arg Tyr Lys Asp Asp Leu1265 1270 1275 1280Pro Leu Val Leu His Gly Val Ser Cys Met Phe Pro Gly Gly Lys Lys 1285 1290 1295Ile Gly Ile Val Gly Arg Thr Gly Ser Gly Lys Ser Thr Leu Ile Gln 1300 1305 1310Ala Leu Phe Arg Leu Ile Glu Pro Thr Gly Gly Lys Ile Ile Ile Asp 1315 1320 1325Asn Ile Asp Ile Ser Ala Ile Gly Leu His Asp Leu Arg Ser Arg Leu 1330 1335 1340Ser Ile Ile Pro Gln Asp Pro Thr Leu Phe Glu Gly Thr Ile Arg Met1345 1350 1355 1360Asn Leu Asp Pro Leu Glu Glu Cys Thr Asp Gln Glu Ile Trp Glu Ala

1365 1370 1375Leu Glu Lys Cys Gln Leu Gly Glu Val Ile Arg Ser Lys Glu Glu Lys 1380 1385 1390Leu Asp Ser Pro Val Leu Glu Asn Gly Asp Asn Trp Ser Val Gly Gln 1395 1400 1405Arg Gln Leu Ile Ala Leu Gly Arg Ala Leu Leu Lys Gln Ala Lys Ile 1410 1415 1420Leu Val Leu Asp Glu Ala Thr Ala Ser Val Asp Thr Ala Thr Asp Asn1425 1430 1435 1440Leu Ile Gln Lys Ile Ile Arg Ser Glu Phe Lys Asp Cys Thr Val Cys 1445 1450 1455Thr Ile Ala His Arg Ile Pro Thr Val Ile Asp Ser Asp Leu Val Leu 1460 1465 1470Val Leu Ser Asp Gly Lys Ile Ala Glu Phe Asp Thr Pro Gln Arg Leu 1475 1480 1485Leu Glu Asp Lys Ser Ser Met Phe Ile Gln Leu Val Ser Glu Tyr Ser 1490 1495 1500Thr Arg Ser Ser Cys Ile1505 151065123DNAOryza sativaCDS(245)...(4762) 6actctttctc gctcgacgag gaggtgaggt gaggtgggag agctagcgaa caaaggcttg 60gtttggtgcc atctggcggc tccgatggcg taacccgccg ccgccctcag agctcggcct 120ttgcctgcct tgcctgcctt ctgccccgcc gccccctgcc ctctgccgtg gcgtggcgag 180gcccaatgcc ttttaaaccc cgccccgctg ccatcctgac gcccccgatc cccaccgcct 240ccca atg ccg cac ttc ccg aac ctc ccg ctc ccg gag gct gcc gcc gcc 289 Met Pro His Phe Pro Asn Leu Pro Leu Pro Glu Ala Ala Ala Ala 1 5 10 15gcc gcg cac gcc gcg ctg ctc gcc ctc gcc ctg ctc ctg ctc ctc ctc 337Ala Ala His Ala Ala Leu Leu Ala Leu Ala Leu Leu Leu Leu Leu Leu 20 25 30cgc tcc gcg cgc gcc ctc gcc tcg cgc tgc gcg tca tgc ctc aag acc 385Arg Ser Ala Arg Ala Leu Ala Ser Arg Cys Ala Ser Cys Leu Lys Thr 35 40 45gcc ccg cgc cga gcc gcg gcg gtc gac ggg ggg ctc gcc gcc gcg tcg 433Ala Pro Arg Arg Ala Ala Ala Val Asp Gly Gly Leu Ala Ala Ala Ser 50 55 60tcc gtg ggc gcg tgg tac agg gcg gcg ctg gcg tgc tgc ggc tac gcc 481Ser Val Gly Ala Trp Tyr Arg Ala Ala Leu Ala Cys Cys Gly Tyr Ala 65 70 75ctg ctg gcg cag gtc gcc gcc ctg agc tac gag gtc gcg gtg gcc ggt 529Leu Leu Ala Gln Val Ala Ala Leu Ser Tyr Glu Val Ala Val Ala Gly80 85 90 95tct cat gtc gcc gtg gag gcc ctg ctg ctg ccc gcg gtg cag gcg ctg 577Ser His Val Ala Val Glu Ala Leu Leu Leu Pro Ala Val Gln Ala Leu 100 105 110gcg tgg gcg gcg ctc ctg gcg ctc gcg atg cag gcc cgg gcc gtc ggg 625Ala Trp Ala Ala Leu Leu Ala Leu Ala Met Gln Ala Arg Ala Val Gly 115 120 125tgg ggc agg ttc ccc gta ctg gtg cgc gtc tgg tgg gtg gtc tcc ttc 673Trp Gly Arg Phe Pro Val Leu Val Arg Val Trp Trp Val Val Ser Phe 130 135 140gtg ctc tgt gtt ggc atc gcg tac gac gat acc agg cac ctc atg ggc 721Val Leu Cys Val Gly Ile Ala Tyr Asp Asp Thr Arg His Leu Met Gly 145 150 155gat gat gat gat gat gag gtg gac tac gct cac atg gtt gcc aac ttc 769Asp Asp Asp Asp Asp Glu Val Asp Tyr Ala His Met Val Ala Asn Phe160 165 170 175gcg tcg gcg ccg gcc ctc ggg ttc ctc tgc ttg gtt ggt gtc atg ggt 817Ala Ser Ala Pro Ala Leu Gly Phe Leu Cys Leu Val Gly Val Met Gly 180 185 190tcc acc ggt gtt gaa ttg gag ttc acc gac gac gac agc agt gtt cat 865Ser Thr Gly Val Glu Leu Glu Phe Thr Asp Asp Asp Ser Ser Val His 195 200 205gaa ccg ctc ttg ctc ggt ggg cag cgg aga gac gcc gac gag gag ccc 913Glu Pro Leu Leu Leu Gly Gly Gln Arg Arg Asp Ala Asp Glu Glu Pro 210 215 220ggg tgc ttg cgg gtg acg ccg tat ggc gat gct ggg att gtt agc ctt 961Gly Cys Leu Arg Val Thr Pro Tyr Gly Asp Ala Gly Ile Val Ser Leu 225 230 235gca aca tta tca tgg ctt agt ccg ctg ctg tca gtt ggt gcg cag cga 1009Ala Thr Leu Ser Trp Leu Ser Pro Leu Leu Ser Val Gly Ala Gln Arg240 245 250 255cca ctt gag ctg gct gac ata ccc ttg atg gca cac aaa gac cgt gcc 1057Pro Leu Glu Leu Ala Asp Ile Pro Leu Met Ala His Lys Asp Arg Ala 260 265 270aaa tcc tgc tac aag gcg atg agc agt cac tat gaa cgc cag cgg atg 1105Lys Ser Cys Tyr Lys Ala Met Ser Ser His Tyr Glu Arg Gln Arg Met 275 280 285gag cgc ccc ggc agc gaa cca tca ctg gca tgg gca ata ttg aag tcg 1153Glu Arg Pro Gly Ser Glu Pro Ser Leu Ala Trp Ala Ile Leu Lys Ser 290 295 300ttc tgg cgt gag gca gcg atc aat ggt gct ttc gca gcg gtg aac aca 1201Phe Trp Arg Glu Ala Ala Ile Asn Gly Ala Phe Ala Ala Val Asn Thr 305 310 315att gtc tcc tat gtt ggc cca tac ctg atc agc tac ttt gtg gac tac 1249Ile Val Ser Tyr Val Gly Pro Tyr Leu Ile Ser Tyr Phe Val Asp Tyr320 325 330 335ctc agt ggc aaa att gaa ttc ccc cat gaa ggt tac atc ctt gcc tct 1297Leu Ser Gly Lys Ile Glu Phe Pro His Glu Gly Tyr Ile Leu Ala Ser 340 345 350gta ttt ttt gta gca aag tta ctt gag acg ctc act gct cgg cag tgg 1345Val Phe Phe Val Ala Lys Leu Leu Glu Thr Leu Thr Ala Arg Gln Trp 355 360 365tac ttg ggc gtg gat gtc atg ggg atc cat gtc aag tct ggg ctg acg 1393Tyr Leu Gly Val Asp Val Met Gly Ile His Val Lys Ser Gly Leu Thr 370 375 380gcc atg gtg tac agg aag ggc ctt agg ctg tcg aat tcc tcg cgg cag 1441Ala Met Val Tyr Arg Lys Gly Leu Arg Leu Ser Asn Ser Ser Arg Gln 385 390 395agc cac acc agt ggt gag att gtg aat tac atg gcg gtt gat gta cag 1489Ser His Thr Ser Gly Glu Ile Val Asn Tyr Met Ala Val Asp Val Gln400 405 410 415cgt gtg ggg gac tat gca tgg tac ttt cat gac atc tgg atg ctt cca 1537Arg Val Gly Asp Tyr Ala Trp Tyr Phe His Asp Ile Trp Met Leu Pro 420 425 430ctg cag atc atc ctc gcc ctc gcc atc ctg tac aag aat gtt gga atc 1585Leu Gln Ile Ile Leu Ala Leu Ala Ile Leu Tyr Lys Asn Val Gly Ile 435 440 445gcc atg gtt tca aca ttg gta gct act gta tta tca att gct gcc tca 1633Ala Met Val Ser Thr Leu Val Ala Thr Val Leu Ser Ile Ala Ala Ser 450 455 460gtt cct gtg gcg aag ctg cag gag cac tac caa gat aag ctt atg gcc 1681Val Pro Val Ala Lys Leu Gln Glu His Tyr Gln Asp Lys Leu Met Ala 465 470 475tca aag gat gag cgc atg cgc aag aca tca gag tgc ctg aag aat atg 1729Ser Lys Asp Glu Arg Met Arg Lys Thr Ser Glu Cys Leu Lys Asn Met480 485 490 495agg att ttg aag ctc caa gcg tgg gag gat cga tac agg ctg aag ttg 1777Arg Ile Leu Lys Leu Gln Ala Trp Glu Asp Arg Tyr Arg Leu Lys Leu 500 505 510gaa gag atg aga aat gtg gaa tgc aag tgg ctt cgg tgg gct ctg tat 1825Glu Glu Met Arg Asn Val Glu Cys Lys Trp Leu Arg Trp Ala Leu Tyr 515 520 525tca cag gcc gca gtt aca ttt gtt ttc tgg agt tca cca atc ttt gtc 1873Ser Gln Ala Ala Val Thr Phe Val Phe Trp Ser Ser Pro Ile Phe Val 530 535 540gcc gtg ata aca ttt ggg act tgt ata ttg ctt ggt ggc gaa ctc act 1921Ala Val Ile Thr Phe Gly Thr Cys Ile Leu Leu Gly Gly Glu Leu Thr 545 550 555gct gga ggt gtt ctt tct gct tta gca aca ttt agg atc ctt caa gaa 1969Ala Gly Gly Val Leu Ser Ala Leu Ala Thr Phe Arg Ile Leu Gln Glu560 565 570 575cca ctt agg aat ttc cca gat ctt atc tct atg att gct cag acg agg 2017Pro Leu Arg Asn Phe Pro Asp Leu Ile Ser Met Ile Ala Gln Thr Arg 580 585 590gta tct ttg gac cgg ttg tct cac ttt ctt caa caa gaa gaa ttg cca 2065Val Ser Leu Asp Arg Leu Ser His Phe Leu Gln Gln Glu Glu Leu Pro 595 600 605gat gat gca act ata acg gtt cca cat ggt agt aca gat aag gca atc 2113Asp Asp Ala Thr Ile Thr Val Pro His Gly Ser Thr Asp Lys Ala Ile 610 615 620aat ata aat gat gct aca ttc tct tgg aac cca tct tct cca acc cct 2161Asn Ile Asn Asp Ala Thr Phe Ser Trp Asn Pro Ser Ser Pro Thr Pro 625 630 635aca ctt tct ggc atc aac ctt agt gtg gtg agg ggt atg cga gta gca 2209Thr Leu Ser Gly Ile Asn Leu Ser Val Val Arg Gly Met Arg Val Ala640 645 650 655gtg tgt ggt gtc att ggt tct ggc aaa tca agc ttg ttg tct tct ata 2257Val Cys Gly Val Ile Gly Ser Gly Lys Ser Ser Leu Leu Ser Ser Ile 660 665 670ctc ggc gag ata ccc aaa ttg tgt ggt caa gtg agg atc agt gga tca 2305Leu Gly Glu Ile Pro Lys Leu Cys Gly Gln Val Arg Ile Ser Gly Ser 675 680 685gca gca tat gtc cct cag act gcc tgg ata cag tcc gga aac att gag 2353Ala Ala Tyr Val Pro Gln Thr Ala Trp Ile Gln Ser Gly Asn Ile Glu 690 695 700gag aac att ctt ttt ggc agt cca atg gac aaa cag cgt tac aag aga 2401Glu Asn Ile Leu Phe Gly Ser Pro Met Asp Lys Gln Arg Tyr Lys Arg 705 710 715gtt att gag gct tgc tcc ctg aag aaa gat ctt cag ttg ctc caa tat 2449Val Ile Glu Ala Cys Ser Leu Lys Lys Asp Leu Gln Leu Leu Gln Tyr720 725 730 735gga gat cag acc atc atc ggt gat agg ggc att aat ttg agt ggg ggt 2497Gly Asp Gln Thr Ile Ile Gly Asp Arg Gly Ile Asn Leu Ser Gly Gly 740 745 750cag aaa caa aga gta cag ctt gca aga gca cta tac caa gat gct gat 2545Gln Lys Gln Arg Val Gln Leu Ala Arg Ala Leu Tyr Gln Asp Ala Asp 755 760 765att tat ttg ctc gat gat ccc ttc agt gcg gtt gat gct cat act ggg 2593Ile Tyr Leu Leu Asp Asp Pro Phe Ser Ala Val Asp Ala His Thr Gly 770 775 780agt gaa tta ttt agg gaa tat ata ttg act gca cta gca agc aag acc 2641Ser Glu Leu Phe Arg Glu Tyr Ile Leu Thr Ala Leu Ala Ser Lys Thr 785 790 795gta att tat gta acc cat caa att gag ttt cta cca gct gct gac ttg 2689Val Ile Tyr Val Thr His Gln Ile Glu Phe Leu Pro Ala Ala Asp Leu800 805 810 815ata ctg gtt ctt aag gat ggt cat atc acc caa gct gga aaa tat gat 2737Ile Leu Val Leu Lys Asp Gly His Ile Thr Gln Ala Gly Lys Tyr Asp 820 825 830gat ctt ctc caa gct ggc act gat ttc aat gct ttg gtt tgt gct cat 2785Asp Leu Leu Gln Ala Gly Thr Asp Phe Asn Ala Leu Val Cys Ala His 835 840 845aag gaa gct att gag acc atg gaa ttt tcc gaa gat tcc gat gag gat 2833Lys Glu Ala Ile Glu Thr Met Glu Phe Ser Glu Asp Ser Asp Glu Asp 850 855 860act gtc tct tct gtt cct atc aaa aga ctg acg cca agt gtt agc aat 2881Thr Val Ser Ser Val Pro Ile Lys Arg Leu Thr Pro Ser Val Ser Asn 865 870 875ata gat aat ctg aaa aac aag gtg tcc aat aat gaa aaa cca tct agt 2929Ile Asp Asn Leu Lys Asn Lys Val Ser Asn Asn Glu Lys Pro Ser Ser880 885 890 895acg cgt gga ata aaa gaa aag aag aag aag cct gaa gag cgt aag aag 2977Thr Arg Gly Ile Lys Glu Lys Lys Lys Lys Pro Glu Glu Arg Lys Lys 900 905 910aag cgg tct gtt caa gag gag gag agg gag cga gga agg gtt agc tta 3025Lys Arg Ser Val Gln Glu Glu Glu Arg Glu Arg Gly Arg Val Ser Leu 915 920 925cag gtt tac ttg tca tac atg gga gaa gca tac aaa ggt aca ctg ata 3073Gln Val Tyr Leu Ser Tyr Met Gly Glu Ala Tyr Lys Gly Thr Leu Ile 930 935 940ccc ctc att atc ctg gcc caa acc atg ttt caa gta ctt cag att gcg 3121Pro Leu Ile Ile Leu Ala Gln Thr Met Phe Gln Val Leu Gln Ile Ala 945 950 955agt aac tgg tgg atg gca tgg gca aac cca caa aca gaa gga gat gca 3169Ser Asn Trp Trp Met Ala Trp Ala Asn Pro Gln Thr Glu Gly Asp Ala960 965 970 975cct aag aca gac agt gtg gtt ctc ttg gtt gtt tat atg tcc ctt gcc 3217Pro Lys Thr Asp Ser Val Val Leu Leu Val Val Tyr Met Ser Leu Ala 980 985 990ttt ggg agt tca ttg ttt gtg ttt gtg aga agt ctt ctt gtg gct aca 3265Phe Gly Ser Ser Leu Phe Val Phe Val Arg Ser Leu Leu Val Ala Thr 995 1000 1005ttt ggt tta gca act gca cag aag ctg ttt gta aag atg cta agg tgt 3313Phe Gly Leu Ala Thr Ala Gln Lys Leu Phe Val Lys Met Leu Arg Cys 1010 1015 1020gtt ttt cga gcg cca atg tca ttc ttt gat act aca cca tct ggt cga 3361Val Phe Arg Ala Pro Met Ser Phe Phe Asp Thr Thr Pro Ser Gly Arg 1025 1030 1035att ttg aac cga gtt tct gta gat caa agt gtc gtg gac ctt gat ata 3409Ile Leu Asn Arg Val Ser Val Asp Gln Ser Val Val Asp Leu Asp Ile1040 1045 1050 1055gca ttc aga ctt ggt gga ttt gca tca aca aca att caa cta ctt gga 3457Ala Phe Arg Leu Gly Gly Phe Ala Ser Thr Thr Ile Gln Leu Leu Gly 1060 1065 1070att gtt gct gtc atg agc aaa gtc aca tgg caa gtt ttg att ctt ata 3505Ile Val Ala Val Met Ser Lys Val Thr Trp Gln Val Leu Ile Leu Ile 1075 1080 1085gtt cct atg gct gtt gca tgc atg tgg atg cag aga tat tat att gct 3553Val Pro Met Ala Val Ala Cys Met Trp Met Gln Arg Tyr Tyr Ile Ala 1090 1095 1100tca tca agg gaa ttg act agg atc tta agc gta cag aag tcg ccg gtg 3601Ser Ser Arg Glu Leu Thr Arg Ile Leu Ser Val Gln Lys Ser Pro Val 1105 1110 1115atc cat ttg ttt agt gag tca att gct ggt gct gct aca atc aga ggt 3649Ile His Leu Phe Ser Glu Ser Ile Ala Gly Ala Ala Thr Ile Arg Gly1120 1125 1130 1135ttt ggt caa gag aaa cga ttc atg aaa aga aat ctt tac ctt ctt gac 3697Phe Gly Gln Glu Lys Arg Phe Met Lys Arg Asn Leu Tyr Leu Leu Asp 1140 1145 1150tgt ttt gct cgg cct cta ttt tcc agc ctg gca gct att gaa tgg ctg 3745Cys Phe Ala Arg Pro Leu Phe Ser Ser Leu Ala Ala Ile Glu Trp Leu 1155 1160 1165tgc ctg cga atg gaa ttg ctc tcg acc ttt gtc ttc gct ttt tgc atg 3793Cys Leu Arg Met Glu Leu Leu Ser Thr Phe Val Phe Ala Phe Cys Met 1170 1175 1180gcg ata cta gtg agc ttc cct cct ggc aca att gaa cca agt atg gct 3841Ala Ile Leu Val Ser Phe Pro Pro Gly Thr Ile Glu Pro Ser Met Ala 1185 1190 1195ggg ctt gct gtc act tat gga ctt aat tta aat gct cgc atg tca agg 3889Gly Leu Ala Val Thr Tyr Gly Leu Asn Leu Asn Ala Arg Met Ser Arg1200 1205 1210 1215tgg ata ctg agc ttc tgt aaa tta gag aat aga atc atc tct gtt gaa 3937Trp Ile Leu Ser Phe Cys Lys Leu Glu Asn Arg Ile Ile Ser Val Glu 1220 1225 1230cgc att tat cag tat tgc aag ctt ccc agt gaa gca cca ctc atc att 3985Arg Ile Tyr Gln Tyr Cys Lys Leu Pro Ser Glu Ala Pro Leu Ile Ile 1235 1240 1245gag aat agc cgt ccc tca tcc tcg tgg cct gag aat gga aac att gag 4033Glu Asn Ser Arg Pro Ser Ser Ser Trp Pro Glu Asn Gly Asn Ile Glu 1250 1255 1260ctg gtc gat ctc aag gta cgg tac aaa gat gac ctg ccc tta gtt cta 4081Leu Val Asp Leu Lys Val Arg Tyr Lys Asp Asp Leu Pro Leu Val Leu 1265 1270 1275cat gga atc agt tgt ata ttt ccc ggt gga aaa aag att ggg att gtg 4129His Gly Ile Ser Cys Ile Phe Pro Gly Gly Lys Lys Ile Gly Ile Val1280 1285 1290 1295ggg cga act gga agt ggt aaa tct act ctt att cag gcc ctt ttc cgc 4177Gly Arg Thr Gly Ser Gly Lys Ser Thr Leu Ile Gln Ala Leu Phe Arg 1300 1305 1310tta att gaa cct aca gga ggg aaa gtt atc atc gat gac gtc gat att 4225Leu Ile Glu Pro Thr Gly Gly Lys Val Ile Ile Asp Asp Val Asp Ile 1315 1320 1325tct aga att ggc ctg cat gat ctg cgg tca cgg ttg agc atc att ccc 4273Ser Arg Ile Gly Leu His Asp Leu Arg Ser Arg Leu Ser Ile Ile Pro 1330 1335 1340cag gac cct acg ttg ttt gag ggt act atc aga atg aat ctt gat cct 4321Gln Asp Pro Thr Leu Phe Glu Gly Thr Ile Arg Met Asn Leu Asp Pro 1345 1350 1355ctt gaa gaa tgt act gat cag gaa att tgg gag gca cta gaa aag tgt 4369Leu Glu Glu Cys Thr Asp Gln Glu Ile Trp Glu Ala Leu Glu Lys Cys1360 1365 1370 1375cag ctc gga gag gtc att cgg tcc aag gat gaa aag ctg gac agt cca 4417Gln Leu Gly Glu Val Ile Arg Ser Lys Asp Glu Lys Leu Asp Ser Pro 1380 1385 1390gta ctg gag aat gga gat aac tgg agt gtg gga caa cgc cag ctt att 4465Val Leu Glu Asn Gly Asp Asn Trp Ser Val Gly Gln Arg Gln Leu Ile 1395 1400 1405gca ttg ggt agg gcc ctg ctg aaa cag gca aaa att ttg gtg ctt gac 4513Ala Leu Gly Arg Ala Leu Leu Lys Gln Ala Lys Ile Leu Val Leu Asp 1410 1415

1420gag gca aca gca tca gtt gac aca gct acg gac aat ctt att caa aag 4561Glu Ala Thr Ala Ser Val Asp Thr Ala Thr Asp Asn Leu Ile Gln Lys 1425 1430 1435att att cgc agt gaa ttc aag gat tgc acg gtc tgc acc att gca cac 4609Ile Ile Arg Ser Glu Phe Lys Asp Cys Thr Val Cys Thr Ile Ala His1440 1445 1450 1455cgt atc ccg acg gtt att gat agt gac cta gtc ctg gtg ctt agt gat 4657Arg Ile Pro Thr Val Ile Asp Ser Asp Leu Val Leu Val Leu Ser Asp 1460 1465 1470ggt aaa att gca gag ttt gac aca ccc cag agg ctc ttg gag gac aag 4705Gly Lys Ile Ala Glu Phe Asp Thr Pro Gln Arg Leu Leu Glu Asp Lys 1475 1480 1485tcc tcc atg ttc atg cag cta gta tct gaa tac tca act cgg tca agc 4753Ser Ser Met Phe Met Gln Leu Val Ser Glu Tyr Ser Thr Arg Ser Ser 1490 1495 1500tgt ata tag agaggcttag cttaaaatcc cccacaccaa gtaggaacag 4802Cys Ile 1505ggaggtagga tagccacatc tgccagtgga ctcacgccat agaagtacca acatcatagg 4862gcaagacaca agccgaggtg tatatgagcg gaaacaaaat gttccctgac gtgaataaac 4922catggaatcg atgagggaac gcagcgggca gcaccacggg aggagttggt gagattaccc 4982gaagctctga tgcttctgaa tgtataaaca atgcggtact acttctccct tgcatagtgg 5042aaaaagggaa ggcaatgttc atgggtaata aaggggtaac aagtttcatt ttggcaccag 5102attggagtgc tttggtctac t 512371505PRTOryza sativa 7Met Pro His Phe Pro Asn Leu Pro Leu Pro Glu Ala Ala Ala Ala Ala1 5 10 15Ala His Ala Ala Leu Leu Ala Leu Ala Leu Leu Leu Leu Leu Leu Arg 20 25 30Ser Ala Arg Ala Leu Ala Ser Arg Cys Ala Ser Cys Leu Lys Thr Ala 35 40 45Pro Arg Arg Ala Ala Ala Val Asp Gly Gly Leu Ala Ala Ala Ser Ser 50 55 60Val Gly Ala Trp Tyr Arg Ala Ala Leu Ala Cys Cys Gly Tyr Ala Leu65 70 75 80Leu Ala Gln Val Ala Ala Leu Ser Tyr Glu Val Ala Val Ala Gly Ser 85 90 95His Val Ala Val Glu Ala Leu Leu Leu Pro Ala Val Gln Ala Leu Ala 100 105 110Trp Ala Ala Leu Leu Ala Leu Ala Met Gln Ala Arg Ala Val Gly Trp 115 120 125Gly Arg Phe Pro Val Leu Val Arg Val Trp Trp Val Val Ser Phe Val 130 135 140Leu Cys Val Gly Ile Ala Tyr Asp Asp Thr Arg His Leu Met Gly Asp145 150 155 160Asp Asp Asp Asp Glu Val Asp Tyr Ala His Met Val Ala Asn Phe Ala 165 170 175Ser Ala Pro Ala Leu Gly Phe Leu Cys Leu Val Gly Val Met Gly Ser 180 185 190Thr Gly Val Glu Leu Glu Phe Thr Asp Asp Asp Ser Ser Val His Glu 195 200 205Pro Leu Leu Leu Gly Gly Gln Arg Arg Asp Ala Asp Glu Glu Pro Gly 210 215 220Cys Leu Arg Val Thr Pro Tyr Gly Asp Ala Gly Ile Val Ser Leu Ala225 230 235 240Thr Leu Ser Trp Leu Ser Pro Leu Leu Ser Val Gly Ala Gln Arg Pro 245 250 255Leu Glu Leu Ala Asp Ile Pro Leu Met Ala His Lys Asp Arg Ala Lys 260 265 270Ser Cys Tyr Lys Ala Met Ser Ser His Tyr Glu Arg Gln Arg Met Glu 275 280 285Arg Pro Gly Ser Glu Pro Ser Leu Ala Trp Ala Ile Leu Lys Ser Phe 290 295 300Trp Arg Glu Ala Ala Ile Asn Gly Ala Phe Ala Ala Val Asn Thr Ile305 310 315 320Val Ser Tyr Val Gly Pro Tyr Leu Ile Ser Tyr Phe Val Asp Tyr Leu 325 330 335Ser Gly Lys Ile Glu Phe Pro His Glu Gly Tyr Ile Leu Ala Ser Val 340 345 350Phe Phe Val Ala Lys Leu Leu Glu Thr Leu Thr Ala Arg Gln Trp Tyr 355 360 365Leu Gly Val Asp Val Met Gly Ile His Val Lys Ser Gly Leu Thr Ala 370 375 380Met Val Tyr Arg Lys Gly Leu Arg Leu Ser Asn Ser Ser Arg Gln Ser385 390 395 400His Thr Ser Gly Glu Ile Val Asn Tyr Met Ala Val Asp Val Gln Arg 405 410 415Val Gly Asp Tyr Ala Trp Tyr Phe His Asp Ile Trp Met Leu Pro Leu 420 425 430Gln Ile Ile Leu Ala Leu Ala Ile Leu Tyr Lys Asn Val Gly Ile Ala 435 440 445Met Val Ser Thr Leu Val Ala Thr Val Leu Ser Ile Ala Ala Ser Val 450 455 460Pro Val Ala Lys Leu Gln Glu His Tyr Gln Asp Lys Leu Met Ala Ser465 470 475 480Lys Asp Glu Arg Met Arg Lys Thr Ser Glu Cys Leu Lys Asn Met Arg 485 490 495Ile Leu Lys Leu Gln Ala Trp Glu Asp Arg Tyr Arg Leu Lys Leu Glu 500 505 510Glu Met Arg Asn Val Glu Cys Lys Trp Leu Arg Trp Ala Leu Tyr Ser 515 520 525Gln Ala Ala Val Thr Phe Val Phe Trp Ser Ser Pro Ile Phe Val Ala 530 535 540Val Ile Thr Phe Gly Thr Cys Ile Leu Leu Gly Gly Glu Leu Thr Ala545 550 555 560Gly Gly Val Leu Ser Ala Leu Ala Thr Phe Arg Ile Leu Gln Glu Pro 565 570 575Leu Arg Asn Phe Pro Asp Leu Ile Ser Met Ile Ala Gln Thr Arg Val 580 585 590Ser Leu Asp Arg Leu Ser His Phe Leu Gln Gln Glu Glu Leu Pro Asp 595 600 605Asp Ala Thr Ile Thr Val Pro His Gly Ser Thr Asp Lys Ala Ile Asn 610 615 620Ile Asn Asp Ala Thr Phe Ser Trp Asn Pro Ser Ser Pro Thr Pro Thr625 630 635 640Leu Ser Gly Ile Asn Leu Ser Val Val Arg Gly Met Arg Val Ala Val 645 650 655Cys Gly Val Ile Gly Ser Gly Lys Ser Ser Leu Leu Ser Ser Ile Leu 660 665 670Gly Glu Ile Pro Lys Leu Cys Gly Gln Val Arg Ile Ser Gly Ser Ala 675 680 685Ala Tyr Val Pro Gln Thr Ala Trp Ile Gln Ser Gly Asn Ile Glu Glu 690 695 700Asn Ile Leu Phe Gly Ser Pro Met Asp Lys Gln Arg Tyr Lys Arg Val705 710 715 720Ile Glu Ala Cys Ser Leu Lys Lys Asp Leu Gln Leu Leu Gln Tyr Gly 725 730 735Asp Gln Thr Ile Ile Gly Asp Arg Gly Ile Asn Leu Ser Gly Gly Gln 740 745 750Lys Gln Arg Val Gln Leu Ala Arg Ala Leu Tyr Gln Asp Ala Asp Ile 755 760 765Tyr Leu Leu Asp Asp Pro Phe Ser Ala Val Asp Ala His Thr Gly Ser 770 775 780Glu Leu Phe Arg Glu Tyr Ile Leu Thr Ala Leu Ala Ser Lys Thr Val785 790 795 800Ile Tyr Val Thr His Gln Ile Glu Phe Leu Pro Ala Ala Asp Leu Ile 805 810 815Leu Val Leu Lys Asp Gly His Ile Thr Gln Ala Gly Lys Tyr Asp Asp 820 825 830Leu Leu Gln Ala Gly Thr Asp Phe Asn Ala Leu Val Cys Ala His Lys 835 840 845Glu Ala Ile Glu Thr Met Glu Phe Ser Glu Asp Ser Asp Glu Asp Thr 850 855 860Val Ser Ser Val Pro Ile Lys Arg Leu Thr Pro Ser Val Ser Asn Ile865 870 875 880Asp Asn Leu Lys Asn Lys Val Ser Asn Asn Glu Lys Pro Ser Ser Thr 885 890 895Arg Gly Ile Lys Glu Lys Lys Lys Lys Pro Glu Glu Arg Lys Lys Lys 900 905 910Arg Ser Val Gln Glu Glu Glu Arg Glu Arg Gly Arg Val Ser Leu Gln 915 920 925Val Tyr Leu Ser Tyr Met Gly Glu Ala Tyr Lys Gly Thr Leu Ile Pro 930 935 940Leu Ile Ile Leu Ala Gln Thr Met Phe Gln Val Leu Gln Ile Ala Ser945 950 955 960Asn Trp Trp Met Ala Trp Ala Asn Pro Gln Thr Glu Gly Asp Ala Pro 965 970 975Lys Thr Asp Ser Val Val Leu Leu Val Val Tyr Met Ser Leu Ala Phe 980 985 990Gly Ser Ser Leu Phe Val Phe Val Arg Ser Leu Leu Val Ala Thr Phe 995 1000 1005Gly Leu Ala Thr Ala Gln Lys Leu Phe Val Lys Met Leu Arg Cys Val 1010 1015 1020Phe Arg Ala Pro Met Ser Phe Phe Asp Thr Thr Pro Ser Gly Arg Ile1025 1030 1035 1040Leu Asn Arg Val Ser Val Asp Gln Ser Val Val Asp Leu Asp Ile Ala 1045 1050 1055Phe Arg Leu Gly Gly Phe Ala Ser Thr Thr Ile Gln Leu Leu Gly Ile 1060 1065 1070Val Ala Val Met Ser Lys Val Thr Trp Gln Val Leu Ile Leu Ile Val 1075 1080 1085Pro Met Ala Val Ala Cys Met Trp Met Gln Arg Tyr Tyr Ile Ala Ser 1090 1095 1100Ser Arg Glu Leu Thr Arg Ile Leu Ser Val Gln Lys Ser Pro Val Ile1105 1110 1115 1120His Leu Phe Ser Glu Ser Ile Ala Gly Ala Ala Thr Ile Arg Gly Phe 1125 1130 1135Gly Gln Glu Lys Arg Phe Met Lys Arg Asn Leu Tyr Leu Leu Asp Cys 1140 1145 1150Phe Ala Arg Pro Leu Phe Ser Ser Leu Ala Ala Ile Glu Trp Leu Cys 1155 1160 1165Leu Arg Met Glu Leu Leu Ser Thr Phe Val Phe Ala Phe Cys Met Ala 1170 1175 1180Ile Leu Val Ser Phe Pro Pro Gly Thr Ile Glu Pro Ser Met Ala Gly1185 1190 1195 1200Leu Ala Val Thr Tyr Gly Leu Asn Leu Asn Ala Arg Met Ser Arg Trp 1205 1210 1215Ile Leu Ser Phe Cys Lys Leu Glu Asn Arg Ile Ile Ser Val Glu Arg 1220 1225 1230Ile Tyr Gln Tyr Cys Lys Leu Pro Ser Glu Ala Pro Leu Ile Ile Glu 1235 1240 1245Asn Ser Arg Pro Ser Ser Ser Trp Pro Glu Asn Gly Asn Ile Glu Leu 1250 1255 1260Val Asp Leu Lys Val Arg Tyr Lys Asp Asp Leu Pro Leu Val Leu His1265 1270 1275 1280Gly Ile Ser Cys Ile Phe Pro Gly Gly Lys Lys Ile Gly Ile Val Gly 1285 1290 1295Arg Thr Gly Ser Gly Lys Ser Thr Leu Ile Gln Ala Leu Phe Arg Leu 1300 1305 1310Ile Glu Pro Thr Gly Gly Lys Val Ile Ile Asp Asp Val Asp Ile Ser 1315 1320 1325Arg Ile Gly Leu His Asp Leu Arg Ser Arg Leu Ser Ile Ile Pro Gln 1330 1335 1340Asp Pro Thr Leu Phe Glu Gly Thr Ile Arg Met Asn Leu Asp Pro Leu1345 1350 1355 1360Glu Glu Cys Thr Asp Gln Glu Ile Trp Glu Ala Leu Glu Lys Cys Gln 1365 1370 1375Leu Gly Glu Val Ile Arg Ser Lys Asp Glu Lys Leu Asp Ser Pro Val 1380 1385 1390Leu Glu Asn Gly Asp Asn Trp Ser Val Gly Gln Arg Gln Leu Ile Ala 1395 1400 1405Leu Gly Arg Ala Leu Leu Lys Gln Ala Lys Ile Leu Val Leu Asp Glu 1410 1415 1420Ala Thr Ala Ser Val Asp Thr Ala Thr Asp Asn Leu Ile Gln Lys Ile1425 1430 1435 1440Ile Arg Ser Glu Phe Lys Asp Cys Thr Val Cys Thr Ile Ala His Arg 1445 1450 1455Ile Pro Thr Val Ile Asp Ser Asp Leu Val Leu Val Leu Ser Asp Gly 1460 1465 1470Lys Ile Ala Glu Phe Asp Thr Pro Gln Arg Leu Leu Glu Asp Lys Ser 1475 1480 1485Ser Met Phe Met Gln Leu Val Ser Glu Tyr Ser Thr Arg Ser Ser Cys 1490 1495 1500Ile150584992DNAArabidopsis thalianaCDS(207)...(4751) 8ctcgattgct ctcaagaacc caagtgacgt ctggtttcag ctgatttgtt tcttctcatt 60ctctatcttc ttctctggga aatatcgatt ttgatctatt aagagctgct acgagctttg 120ggatgtggtg agatgcttgt tctatctcga acaatccgcc ggttgttgat tttaaacaaa 180ctctctatca caaatctttc ccgatc atg gat ttt att gag atc tcg ttg atc 233 Met Asp Phe Ile Glu Ile Ser Leu Ile 1 5ttt cga gag cat ttg cca cta ctg gaa cta tgt tcg gtc atc atc aac 281Phe Arg Glu His Leu Pro Leu Leu Glu Leu Cys Ser Val Ile Ile Asn10 15 20 25ctc cta ctc ttt ctt gtc ttt cta ttt gct gtc tcc gcg agg cag att 329Leu Leu Leu Phe Leu Val Phe Leu Phe Ala Val Ser Ala Arg Gln Ile 30 35 40ctc gtc tgc gtg aga aga ggc aga gat agg ctc tct aag gac gat acg 377Leu Val Cys Val Arg Arg Gly Arg Asp Arg Leu Ser Lys Asp Asp Thr 45 50 55gtt tca gct tct aat ctt agc ttg gaa aga gag gtt aac cat gtt agt 425Val Ser Ala Ser Asn Leu Ser Leu Glu Arg Glu Val Asn His Val Ser 60 65 70gtt ggg ttt ggg ttt aat ctg tct ttg ctc tgt tgc tta tat gtg tta 473Val Gly Phe Gly Phe Asn Leu Ser Leu Leu Cys Cys Leu Tyr Val Leu 75 80 85ggc gtc caa gtt ttg gtg tta gta tat gat ggg gtt aag gtt aga aga 521Gly Val Gln Val Leu Val Leu Val Tyr Asp Gly Val Lys Val Arg Arg90 95 100 105gaa gtc agt gac tgg ttt gtt ctt tgc ttt cca gct tct cag agt tta 569Glu Val Ser Asp Trp Phe Val Leu Cys Phe Pro Ala Ser Gln Ser Leu 110 115 120gct tgg ttt gtc ctt agc ttc tta gtt ctt cat ttg aaa tac aag tct 617Ala Trp Phe Val Leu Ser Phe Leu Val Leu His Leu Lys Tyr Lys Ser 125 130 135tca gag aag cta ccc ttc ttg gtg agg ata tgg tgg ttc ctg gcg ttt 665Ser Glu Lys Leu Pro Phe Leu Val Arg Ile Trp Trp Phe Leu Ala Phe 140 145 150tcg att tgc ctc tgt act atg tat gtc gat gga aga agg cta gcc att 713Ser Ile Cys Leu Cys Thr Met Tyr Val Asp Gly Arg Arg Leu Ala Ile 155 160 165gaa ggt tgg agc aga tgt tct tct cat gtt gtc gcc aat tta gct gtt 761Glu Gly Trp Ser Arg Cys Ser Ser His Val Val Ala Asn Leu Ala Val170 175 180 185aca cct gct ctt ggg ttt ctc tgc ttt ctg gcc tgg aga ggc gtt tct 809Thr Pro Ala Leu Gly Phe Leu Cys Phe Leu Ala Trp Arg Gly Val Ser 190 195 200ggt att caa gtt acc aga agc tcc tct gat ctt caa gag cct ttg ctt 857Gly Ile Gln Val Thr Arg Ser Ser Ser Asp Leu Gln Glu Pro Leu Leu 205 210 215gtt gaa gaa gag gca gct tgt ctt aaa gtt act cca tat agt act gct 905Val Glu Glu Glu Ala Ala Cys Leu Lys Val Thr Pro Tyr Ser Thr Ala 220 225 230ggg cta gtt agc ctt att acg ctt tca tgg ttg gat cca ctt ctc tcg 953Gly Leu Val Ser Leu Ile Thr Leu Ser Trp Leu Asp Pro Leu Leu Ser 235 240 245gct ggt tca aaa aga ccg ctt gag ctt aag gat ata ccg ctt ctt gca 1001Ala Gly Ser Lys Arg Pro Leu Glu Leu Lys Asp Ile Pro Leu Leu Ala250 255 260 265cca aga gat aga gcc aaa tca agt tac aag gtc ttg aag tcg aat tgg 1049Pro Arg Asp Arg Ala Lys Ser Ser Tyr Lys Val Leu Lys Ser Asn Trp 270 275 280aag aga tgc aag tcc gag aat cct tca aag cct cct tct tta gct cgt 1097Lys Arg Cys Lys Ser Glu Asn Pro Ser Lys Pro Pro Ser Leu Ala Arg 285 290 295gca att atg aaa tca ttt tgg aaa gaa gct gct tgc aat gcc gta ttt 1145Ala Ile Met Lys Ser Phe Trp Lys Glu Ala Ala Cys Asn Ala Val Phe 300 305 310gct ggg ttg aat act ctt gtg tcc tat gtc ggt cct tac ttg atc agc 1193Ala Gly Leu Asn Thr Leu Val Ser Tyr Val Gly Pro Tyr Leu Ile Ser 315 320 325tac ttt gtt gat tat ctt gga ggg aag gag att ttc cct cat gaa gga 1241Tyr Phe Val Asp Tyr Leu Gly Gly Lys Glu Ile Phe Pro His Glu Gly330 335 340 345tac gta ctc gct ggg ata ttc ttt acg tcc aag ctt ata gag aca gtc 1289Tyr Val Leu Ala Gly Ile Phe Phe Thr Ser Lys Leu Ile Glu Thr Val 350 355 360acc acc cgc cag tgg tat atg ggt gtt gat atc cta ggg atg cat gtt 1337Thr Thr Arg Gln Trp Tyr Met Gly Val Asp Ile Leu Gly Met His Val 365 370 375aga tca gct ctt aca gca atg gta tac cga aaa ggt ctc aaa ctt tca 1385Arg Ser Ala Leu Thr Ala Met Val Tyr Arg Lys Gly Leu Lys Leu Ser 380 385 390agt ata gcc aag cag aac cac acg agc ggt gaa att gta aac tac atg 1433Ser Ile Ala Lys Gln Asn His Thr Ser Gly Glu Ile Val Asn Tyr Met 395 400 405gca gtc gat gtc cag cgc ata gga gat tac tca tgg tat ctt cat gat 1481Ala Val Asp Val Gln Arg Ile Gly Asp Tyr Ser Trp Tyr Leu His Asp410 415 420 425att tgg atg ctt ccg atg caa ata gtt ctt gct ctt gca atc cta tat 1529Ile Trp Met Leu Pro Met Gln Ile Val Leu Ala Leu Ala Ile Leu Tyr 430 435 440aaa agc gtg ggc ata gct gct gta gct aca

ttg gtt gct aca ata atc 1577Lys Ser Val Gly Ile Ala Ala Val Ala Thr Leu Val Ala Thr Ile Ile 445 450 455tcg att ctt gtc acg att cca ctc gct aag gtc cag gaa gac tat caa 1625Ser Ile Leu Val Thr Ile Pro Leu Ala Lys Val Gln Glu Asp Tyr Gln 460 465 470gat aag ttg atg act gcg aaa gat gaa aga atg agg aaa acc tca gag 1673Asp Lys Leu Met Thr Ala Lys Asp Glu Arg Met Arg Lys Thr Ser Glu 475 480 485tgt ctt agg aac atg aga gtt ctg aaa ttg cag gca tgg gaa gat cgt 1721Cys Leu Arg Asn Met Arg Val Leu Lys Leu Gln Ala Trp Glu Asp Arg490 495 500 505tat aga gtg aga ttg gaa gaa atg agg gaa gag gag tat ggt tgg ctt 1769Tyr Arg Val Arg Leu Glu Glu Met Arg Glu Glu Glu Tyr Gly Trp Leu 510 515 520cgc aaa gcc tta tat tct cag gct ttt gtt act ttt atc ttt tgg agt 1817Arg Lys Ala Leu Tyr Ser Gln Ala Phe Val Thr Phe Ile Phe Trp Ser 525 530 535tcc ccc att ttt gtc gcc gca gtt aca ttc gct act tcg ata ttt cta 1865Ser Pro Ile Phe Val Ala Ala Val Thr Phe Ala Thr Ser Ile Phe Leu 540 545 550ggc act caa ctt acc gct gga ggt gtt ctt tct gct ctg gcg aca ttc 1913Gly Thr Gln Leu Thr Ala Gly Gly Val Leu Ser Ala Leu Ala Thr Phe 555 560 565aga att ctt cag gag cca ctt cgg aac ttt cct gat ctg gtt tca atg 1961Arg Ile Leu Gln Glu Pro Leu Arg Asn Phe Pro Asp Leu Val Ser Met570 575 580 585atg gct cag aca aag gtt tct ctt gat agg att tct ggg ttc ttg cag 2009Met Ala Gln Thr Lys Val Ser Leu Asp Arg Ile Ser Gly Phe Leu Gln 590 595 600gag gaa gaa ctt caa gaa gat gca act gtt gtt att cca cgg gga ctt 2057Glu Glu Glu Leu Gln Glu Asp Ala Thr Val Val Ile Pro Arg Gly Leu 605 610 615tcg aat ata gcc ata gag att aaa gat ggt gtg ttt tgt tgg gac cct 2105Ser Asn Ile Ala Ile Glu Ile Lys Asp Gly Val Phe Cys Trp Asp Pro 620 625 630ttt tct tca agg ccg aca tta tct ggg att cag atg aaa gtg gag aag 2153Phe Ser Ser Arg Pro Thr Leu Ser Gly Ile Gln Met Lys Val Glu Lys 635 640 645ggt atg cgt gtg gct gtc tgt ggc aca gtt ggc tct gga aaa tca agt 2201Gly Met Arg Val Ala Val Cys Gly Thr Val Gly Ser Gly Lys Ser Ser650 655 660 665ttt atc tct tgc atc cta ggg gaa atc cca aaa atc tct ggc gaa gtt 2249Phe Ile Ser Cys Ile Leu Gly Glu Ile Pro Lys Ile Ser Gly Glu Val 670 675 680aga ata tgt ggt act act ggt tat gtg tct caa tcg gct tgg att cag 2297Arg Ile Cys Gly Thr Thr Gly Tyr Val Ser Gln Ser Ala Trp Ile Gln 685 690 695tct ggt aac att gaa gaa aac att cta ttt ggc agt cca atg gag aaa 2345Ser Gly Asn Ile Glu Glu Asn Ile Leu Phe Gly Ser Pro Met Glu Lys 700 705 710aca aag tac aag aat gtg ata caa gca tgt tcc cta aag aaa gat ata 2393Thr Lys Tyr Lys Asn Val Ile Gln Ala Cys Ser Leu Lys Lys Asp Ile 715 720 725gag ctt ttc tca cat ggg gac caa act att atc ggg gag aga ggt ata 2441Glu Leu Phe Ser His Gly Asp Gln Thr Ile Ile Gly Glu Arg Gly Ile730 735 740 745aat ctc agc gga ggt cag aaa cag cgt gta caa ctt gca agg gca tta 2489Asn Leu Ser Gly Gly Gln Lys Gln Arg Val Gln Leu Ala Arg Ala Leu 750 755 760tat caa gat gct gac att tat tta cta gac gac cct ttt agt gct ctt 2537Tyr Gln Asp Ala Asp Ile Tyr Leu Leu Asp Asp Pro Phe Ser Ala Leu 765 770 775gat gca cac act ggc tct gat ttg ttt agg gat tat att cta tct gca 2585Asp Ala His Thr Gly Ser Asp Leu Phe Arg Asp Tyr Ile Leu Ser Ala 780 785 790ttg gct gag aaa act gtg gtt ttt gta acg cat caa gtt gag ttt ctc 2633Leu Ala Glu Lys Thr Val Val Phe Val Thr His Gln Val Glu Phe Leu 795 800 805cct gca gct gat cta ata ttg gtt ctg aag gaa ggc agg att att caa 2681Pro Ala Ala Asp Leu Ile Leu Val Leu Lys Glu Gly Arg Ile Ile Gln810 815 820 825tcg ggt aaa tat gat gat ctg ctg caa gca ggt act gat ttt aag gcc 2729Ser Gly Lys Tyr Asp Asp Leu Leu Gln Ala Gly Thr Asp Phe Lys Ala 830 835 840tta gtg tct gcc cac cat gaa gca atc gag gca atg gac atc cca agt 2777Leu Val Ser Ala His His Glu Ala Ile Glu Ala Met Asp Ile Pro Ser 845 850 855ccc tcc tca gaa gac tct gat gaa aat cct att cgc gat agt ttg gtc 2825Pro Ser Ser Glu Asp Ser Asp Glu Asn Pro Ile Arg Asp Ser Leu Val 860 865 870ttg cat aat cca aag tct gat gtt ttt gaa aat gac atc gag act ttg 2873Leu His Asn Pro Lys Ser Asp Val Phe Glu Asn Asp Ile Glu Thr Leu 875 880 885gca aag gaa gta caa gag gga gga tct gct tca gat cta aag gca atc 2921Ala Lys Glu Val Gln Glu Gly Gly Ser Ala Ser Asp Leu Lys Ala Ile890 895 900 905aaa gag aag aag aag aaa gct aag cgt tcc cgc aaa aag cag ctt gtt 2969Lys Glu Lys Lys Lys Lys Ala Lys Arg Ser Arg Lys Lys Gln Leu Val 910 915 920caa gaa gag gaa cga gta aag gga aaa gtc agc atg aag gtg tac ttg 3017Gln Glu Glu Glu Arg Val Lys Gly Lys Val Ser Met Lys Val Tyr Leu 925 930 935tca tac atg ggt gct gca tat aaa ggg gct ctg att cct ctt att ata 3065Ser Tyr Met Gly Ala Ala Tyr Lys Gly Ala Leu Ile Pro Leu Ile Ile 940 945 950ctc gca caa gct gct ttc caa ttt ctt cag att gct agt aat tgg tgg 3113Leu Ala Gln Ala Ala Phe Gln Phe Leu Gln Ile Ala Ser Asn Trp Trp 955 960 965atg gct tgg gca aat cct caa act gaa ggt gac gaa tct aaa gtg gat 3161Met Ala Trp Ala Asn Pro Gln Thr Glu Gly Asp Glu Ser Lys Val Asp970 975 980 985cct act ctg ctt ctc atc gtt tat acg gct tta gct ttc ggg agc tct 3209Pro Thr Leu Leu Leu Ile Val Tyr Thr Ala Leu Ala Phe Gly Ser Ser 990 995 1000gtg ttc ata ttt gtt cga gct gct ctg gtt gca act ttt ggt ctt gca 3257Val Phe Ile Phe Val Arg Ala Ala Leu Val Ala Thr Phe Gly Leu Ala 1005 1010 1015gct gca cag aaa ctg ttc tta aat atg ctc aga agt gtg ttc cga gcg 3305Ala Ala Gln Lys Leu Phe Leu Asn Met Leu Arg Ser Val Phe Arg Ala 1020 1025 1030cca atg tca ttc ttt gat tcc act cct gca gga aga att ttg aat cgg 3353Pro Met Ser Phe Phe Asp Ser Thr Pro Ala Gly Arg Ile Leu Asn Arg 1035 1040 1045gtt tct att gat caa agt gtt gta gat ctt gac att cct ttt aga ctc 3401Val Ser Ile Asp Gln Ser Val Val Asp Leu Asp Ile Pro Phe Arg Leu1050 1055 1060 1065ggt ggg ttt gct tca aca aca ata caa ctc tgt ggc att gtc gct gta 3449Gly Gly Phe Ala Ser Thr Thr Ile Gln Leu Cys Gly Ile Val Ala Val 1070 1075 1080atg acc aat gtt acc tgg caa gtt ttc ctt ctt gtt gtt ccg gta gct 3497Met Thr Asn Val Thr Trp Gln Val Phe Leu Leu Val Val Pro Val Ala 1085 1090 1095gtt gct tgc ttt tgg atg cag aaa tat tac atg gct tct tca aga gaa 3545Val Ala Cys Phe Trp Met Gln Lys Tyr Tyr Met Ala Ser Ser Arg Glu 1100 1105 1110ttg gtt cgg ata gtt agt atc cag aag tct cca ata att cat ctt ttt 3593Leu Val Arg Ile Val Ser Ile Gln Lys Ser Pro Ile Ile His Leu Phe 1115 1120 1125gga gaa tca att gct ggt gct gcc aca ata aga gga ttt ggc cag gaa 3641Gly Glu Ser Ile Ala Gly Ala Ala Thr Ile Arg Gly Phe Gly Gln Glu1130 1135 1140 1145aaa aga ttt atc aaa agg aat ctt tat ctt cta gat tgt ttt gtt cga 3689Lys Arg Phe Ile Lys Arg Asn Leu Tyr Leu Leu Asp Cys Phe Val Arg 1150 1155 1160cct ttc ttc tgc agt atc gct gct atc gaa tgg ctt tgt tta cgc atg 3737Pro Phe Phe Cys Ser Ile Ala Ala Ile Glu Trp Leu Cys Leu Arg Met 1165 1170 1175gaa tta ctt tcc aca ctt gta ttt gct ttc tgt atg gtt tta ctc gtc 3785Glu Leu Leu Ser Thr Leu Val Phe Ala Phe Cys Met Val Leu Leu Val 1180 1185 1190agt ttt cca cat gga acc att gat cca agc atg gca ggt ctt gct gtg 3833Ser Phe Pro His Gly Thr Ile Asp Pro Ser Met Ala Gly Leu Ala Val 1195 1200 1205aca tat gga ctt aac ttg aat gga cgt cta tca cga tgg ata ctt agc 3881Thr Tyr Gly Leu Asn Leu Asn Gly Arg Leu Ser Arg Trp Ile Leu Ser1210 1215 1220 1225ttt tgt aag ctt gaa aac aaa ata atc tca atc gaa agg att tat cag 3929Phe Cys Lys Leu Glu Asn Lys Ile Ile Ser Ile Glu Arg Ile Tyr Gln 1230 1235 1240tac agt cag att gta gga gag gcc cca gca att ata gaa gat ttc cgc 3977Tyr Ser Gln Ile Val Gly Glu Ala Pro Ala Ile Ile Glu Asp Phe Arg 1245 1250 1255ccg cct tcc tcg tgg cct gca acg gga aca att gag cta gtt gat gtt 4025Pro Pro Ser Ser Trp Pro Ala Thr Gly Thr Ile Glu Leu Val Asp Val 1260 1265 1270aag gtt cgt tat gct gag aat ctt cca aca gta ctc cat ggg gtg agc 4073Lys Val Arg Tyr Ala Glu Asn Leu Pro Thr Val Leu His Gly Val Ser 1275 1280 1285tgt gtg ttt ccg ggt gga aaa aag att ggg att gtt ggg cga acg gga 4121Cys Val Phe Pro Gly Gly Lys Lys Ile Gly Ile Val Gly Arg Thr Gly1290 1295 1300 1305agc gga aag tcg act ttg att caa gct ttg ttt cga ttg att gag cca 4169Ser Gly Lys Ser Thr Leu Ile Gln Ala Leu Phe Arg Leu Ile Glu Pro 1310 1315 1320act gct gga aaa att act att gac aac att gac att tct caa att ggt 4217Thr Ala Gly Lys Ile Thr Ile Asp Asn Ile Asp Ile Ser Gln Ile Gly 1325 1330 1335ctt cat gat ctt cgt agt cgc ctt ggg att ata cct caa gat cct aca 4265Leu His Asp Leu Arg Ser Arg Leu Gly Ile Ile Pro Gln Asp Pro Thr 1340 1345 1350tta ttt gaa gga aca atc cga gca aat ctt gac cca ctt gaa gaa cat 4313Leu Phe Glu Gly Thr Ile Arg Ala Asn Leu Asp Pro Leu Glu Glu His 1355 1360 1365tca gat gat aaa atc tgg gag gcg ctt gat aaa tcc cag ctt gga gac 4361Ser Asp Asp Lys Ile Trp Glu Ala Leu Asp Lys Ser Gln Leu Gly Asp1370 1375 1380 1385gtt gtt aga gga aaa gac cta aaa ctt gac tct cca gta ctg gaa aat 4409Val Val Arg Gly Lys Asp Leu Lys Leu Asp Ser Pro Val Leu Glu Asn 1390 1395 1400gga gat aac tgg agt gta ggg cag aga cag ctt gtg tca ctt gga cga 4457Gly Asp Asn Trp Ser Val Gly Gln Arg Gln Leu Val Ser Leu Gly Arg 1405 1410 1415gca tta ctg aaa caa gcc aaa ata ctt gtt ctt gat gaa gca aca gca 4505Ala Leu Leu Lys Gln Ala Lys Ile Leu Val Leu Asp Glu Ala Thr Ala 1420 1425 1430tcg gtt gac aca gca aca gac aat ctg atc cag aag ata atc aga aca 4553Ser Val Asp Thr Ala Thr Asp Asn Leu Ile Gln Lys Ile Ile Arg Thr 1435 1440 1445gag ttt gaa gac tgc acg gtc tgc acc att gct cac cgg ata cca act 4601Glu Phe Glu Asp Cys Thr Val Cys Thr Ile Ala His Arg Ile Pro Thr1450 1455 1460 1465gtt ata gac agt gac cta gtt ttg gtt ctc agc gac ggt aga gta gca 4649Val Ile Asp Ser Asp Leu Val Leu Val Leu Ser Asp Gly Arg Val Ala 1470 1475 1480gag ttt gat act cct gca cgg cta tta gaa gac aaa tca tcg atg ttc 4697Glu Phe Asp Thr Pro Ala Arg Leu Leu Glu Asp Lys Ser Ser Met Phe 1485 1490 1495ttg aaa ttg gta aca gaa tac tcc tca aga tct act gga atc cct gaa 4745Leu Lys Leu Val Thr Glu Tyr Ser Ser Arg Ser Thr Gly Ile Pro Glu 1500 1505 1510tta tga tcctccatgt taaaaattca gtttaggggg tttcttttct caagaggata 4801Leutaaaagaact gatatgtgac aaaagcttaa ggtctaaagt aagagagagt tttccacagg 4861gtttaagaaa agaaaaagca tgaaaggatg ccaaaatctc cgcgcttaaa aaactttggg 4921tttaaatctc ttctgtcgaa cattgggaga aacttttttt gtatggaaca gttagtttct 4981ttggttttca t 499291514PRTArabidopsis thaliana 9Met Asp Phe Ile Glu Ile Ser Leu Ile Phe Arg Glu His Leu Pro Leu1 5 10 15Leu Glu Leu Cys Ser Val Ile Ile Asn Leu Leu Leu Phe Leu Val Phe 20 25 30Leu Phe Ala Val Ser Ala Arg Gln Ile Leu Val Cys Val Arg Arg Gly 35 40 45Arg Asp Arg Leu Ser Lys Asp Asp Thr Val Ser Ala Ser Asn Leu Ser 50 55 60Leu Glu Arg Glu Val Asn His Val Ser Val Gly Phe Gly Phe Asn Leu65 70 75 80Ser Leu Leu Cys Cys Leu Tyr Val Leu Gly Val Gln Val Leu Val Leu 85 90 95Val Tyr Asp Gly Val Lys Val Arg Arg Glu Val Ser Asp Trp Phe Val 100 105 110Leu Cys Phe Pro Ala Ser Gln Ser Leu Ala Trp Phe Val Leu Ser Phe 115 120 125Leu Val Leu His Leu Lys Tyr Lys Ser Ser Glu Lys Leu Pro Phe Leu 130 135 140Val Arg Ile Trp Trp Phe Leu Ala Phe Ser Ile Cys Leu Cys Thr Met145 150 155 160Tyr Val Asp Gly Arg Arg Leu Ala Ile Glu Gly Trp Ser Arg Cys Ser 165 170 175Ser His Val Val Ala Asn Leu Ala Val Thr Pro Ala Leu Gly Phe Leu 180 185 190Cys Phe Leu Ala Trp Arg Gly Val Ser Gly Ile Gln Val Thr Arg Ser 195 200 205Ser Ser Asp Leu Gln Glu Pro Leu Leu Val Glu Glu Glu Ala Ala Cys 210 215 220Leu Lys Val Thr Pro Tyr Ser Thr Ala Gly Leu Val Ser Leu Ile Thr225 230 235 240Leu Ser Trp Leu Asp Pro Leu Leu Ser Ala Gly Ser Lys Arg Pro Leu 245 250 255Glu Leu Lys Asp Ile Pro Leu Leu Ala Pro Arg Asp Arg Ala Lys Ser 260 265 270Ser Tyr Lys Val Leu Lys Ser Asn Trp Lys Arg Cys Lys Ser Glu Asn 275 280 285Pro Ser Lys Pro Pro Ser Leu Ala Arg Ala Ile Met Lys Ser Phe Trp 290 295 300Lys Glu Ala Ala Cys Asn Ala Val Phe Ala Gly Leu Asn Thr Leu Val305 310 315 320Ser Tyr Val Gly Pro Tyr Leu Ile Ser Tyr Phe Val Asp Tyr Leu Gly 325 330 335Gly Lys Glu Ile Phe Pro His Glu Gly Tyr Val Leu Ala Gly Ile Phe 340 345 350Phe Thr Ser Lys Leu Ile Glu Thr Val Thr Thr Arg Gln Trp Tyr Met 355 360 365Gly Val Asp Ile Leu Gly Met His Val Arg Ser Ala Leu Thr Ala Met 370 375 380Val Tyr Arg Lys Gly Leu Lys Leu Ser Ser Ile Ala Lys Gln Asn His385 390 395 400Thr Ser Gly Glu Ile Val Asn Tyr Met Ala Val Asp Val Gln Arg Ile 405 410 415Gly Asp Tyr Ser Trp Tyr Leu His Asp Ile Trp Met Leu Pro Met Gln 420 425 430Ile Val Leu Ala Leu Ala Ile Leu Tyr Lys Ser Val Gly Ile Ala Ala 435 440 445Val Ala Thr Leu Val Ala Thr Ile Ile Ser Ile Leu Val Thr Ile Pro 450 455 460Leu Ala Lys Val Gln Glu Asp Tyr Gln Asp Lys Leu Met Thr Ala Lys465 470 475 480Asp Glu Arg Met Arg Lys Thr Ser Glu Cys Leu Arg Asn Met Arg Val 485 490 495Leu Lys Leu Gln Ala Trp Glu Asp Arg Tyr Arg Val Arg Leu Glu Glu 500 505 510Met Arg Glu Glu Glu Tyr Gly Trp Leu Arg Lys Ala Leu Tyr Ser Gln 515 520 525Ala Phe Val Thr Phe Ile Phe Trp Ser Ser Pro Ile Phe Val Ala Ala 530 535 540Val Thr Phe Ala Thr Ser Ile Phe Leu Gly Thr Gln Leu Thr Ala Gly545 550 555 560Gly Val Leu Ser Ala Leu Ala Thr Phe Arg Ile Leu Gln Glu Pro Leu 565 570 575Arg Asn Phe Pro Asp Leu Val Ser Met Met Ala Gln Thr Lys Val Ser 580 585 590Leu Asp Arg Ile Ser Gly Phe Leu Gln Glu Glu Glu Leu Gln Glu Asp 595 600 605Ala Thr Val Val Ile Pro Arg Gly Leu Ser Asn Ile Ala Ile Glu Ile 610 615 620Lys Asp Gly Val Phe Cys Trp Asp Pro Phe Ser Ser Arg Pro Thr Leu625 630 635 640Ser Gly Ile Gln Met Lys Val Glu Lys Gly Met Arg Val Ala Val Cys 645 650 655Gly Thr Val Gly Ser Gly Lys Ser Ser Phe Ile Ser Cys Ile Leu Gly 660 665 670Glu Ile Pro Lys Ile Ser Gly Glu Val Arg Ile Cys Gly Thr Thr Gly 675 680 685Tyr Val Ser Gln Ser Ala Trp Ile Gln Ser Gly Asn Ile Glu

Glu Asn 690 695 700Ile Leu Phe Gly Ser Pro Met Glu Lys Thr Lys Tyr Lys Asn Val Ile705 710 715 720Gln Ala Cys Ser Leu Lys Lys Asp Ile Glu Leu Phe Ser His Gly Asp 725 730 735Gln Thr Ile Ile Gly Glu Arg Gly Ile Asn Leu Ser Gly Gly Gln Lys 740 745 750Gln Arg Val Gln Leu Ala Arg Ala Leu Tyr Gln Asp Ala Asp Ile Tyr 755 760 765Leu Leu Asp Asp Pro Phe Ser Ala Leu Asp Ala His Thr Gly Ser Asp 770 775 780Leu Phe Arg Asp Tyr Ile Leu Ser Ala Leu Ala Glu Lys Thr Val Val785 790 795 800Phe Val Thr His Gln Val Glu Phe Leu Pro Ala Ala Asp Leu Ile Leu 805 810 815Val Leu Lys Glu Gly Arg Ile Ile Gln Ser Gly Lys Tyr Asp Asp Leu 820 825 830Leu Gln Ala Gly Thr Asp Phe Lys Ala Leu Val Ser Ala His His Glu 835 840 845Ala Ile Glu Ala Met Asp Ile Pro Ser Pro Ser Ser Glu Asp Ser Asp 850 855 860Glu Asn Pro Ile Arg Asp Ser Leu Val Leu His Asn Pro Lys Ser Asp865 870 875 880Val Phe Glu Asn Asp Ile Glu Thr Leu Ala Lys Glu Val Gln Glu Gly 885 890 895Gly Ser Ala Ser Asp Leu Lys Ala Ile Lys Glu Lys Lys Lys Lys Ala 900 905 910Lys Arg Ser Arg Lys Lys Gln Leu Val Gln Glu Glu Glu Arg Val Lys 915 920 925Gly Lys Val Ser Met Lys Val Tyr Leu Ser Tyr Met Gly Ala Ala Tyr 930 935 940Lys Gly Ala Leu Ile Pro Leu Ile Ile Leu Ala Gln Ala Ala Phe Gln945 950 955 960Phe Leu Gln Ile Ala Ser Asn Trp Trp Met Ala Trp Ala Asn Pro Gln 965 970 975Thr Glu Gly Asp Glu Ser Lys Val Asp Pro Thr Leu Leu Leu Ile Val 980 985 990Tyr Thr Ala Leu Ala Phe Gly Ser Ser Val Phe Ile Phe Val Arg Ala 995 1000 1005Ala Leu Val Ala Thr Phe Gly Leu Ala Ala Ala Gln Lys Leu Phe Leu 1010 1015 1020Asn Met Leu Arg Ser Val Phe Arg Ala Pro Met Ser Phe Phe Asp Ser1025 1030 1035 1040Thr Pro Ala Gly Arg Ile Leu Asn Arg Val Ser Ile Asp Gln Ser Val 1045 1050 1055Val Asp Leu Asp Ile Pro Phe Arg Leu Gly Gly Phe Ala Ser Thr Thr 1060 1065 1070Ile Gln Leu Cys Gly Ile Val Ala Val Met Thr Asn Val Thr Trp Gln 1075 1080 1085Val Phe Leu Leu Val Val Pro Val Ala Val Ala Cys Phe Trp Met Gln 1090 1095 1100Lys Tyr Tyr Met Ala Ser Ser Arg Glu Leu Val Arg Ile Val Ser Ile1105 1110 1115 1120Gln Lys Ser Pro Ile Ile His Leu Phe Gly Glu Ser Ile Ala Gly Ala 1125 1130 1135Ala Thr Ile Arg Gly Phe Gly Gln Glu Lys Arg Phe Ile Lys Arg Asn 1140 1145 1150Leu Tyr Leu Leu Asp Cys Phe Val Arg Pro Phe Phe Cys Ser Ile Ala 1155 1160 1165Ala Ile Glu Trp Leu Cys Leu Arg Met Glu Leu Leu Ser Thr Leu Val 1170 1175 1180Phe Ala Phe Cys Met Val Leu Leu Val Ser Phe Pro His Gly Thr Ile1185 1190 1195 1200Asp Pro Ser Met Ala Gly Leu Ala Val Thr Tyr Gly Leu Asn Leu Asn 1205 1210 1215Gly Arg Leu Ser Arg Trp Ile Leu Ser Phe Cys Lys Leu Glu Asn Lys 1220 1225 1230Ile Ile Ser Ile Glu Arg Ile Tyr Gln Tyr Ser Gln Ile Val Gly Glu 1235 1240 1245Ala Pro Ala Ile Ile Glu Asp Phe Arg Pro Pro Ser Ser Trp Pro Ala 1250 1255 1260Thr Gly Thr Ile Glu Leu Val Asp Val Lys Val Arg Tyr Ala Glu Asn1265 1270 1275 1280Leu Pro Thr Val Leu His Gly Val Ser Cys Val Phe Pro Gly Gly Lys 1285 1290 1295Lys Ile Gly Ile Val Gly Arg Thr Gly Ser Gly Lys Ser Thr Leu Ile 1300 1305 1310Gln Ala Leu Phe Arg Leu Ile Glu Pro Thr Ala Gly Lys Ile Thr Ile 1315 1320 1325Asp Asn Ile Asp Ile Ser Gln Ile Gly Leu His Asp Leu Arg Ser Arg 1330 1335 1340Leu Gly Ile Ile Pro Gln Asp Pro Thr Leu Phe Glu Gly Thr Ile Arg1345 1350 1355 1360Ala Asn Leu Asp Pro Leu Glu Glu His Ser Asp Asp Lys Ile Trp Glu 1365 1370 1375Ala Leu Asp Lys Ser Gln Leu Gly Asp Val Val Arg Gly Lys Asp Leu 1380 1385 1390Lys Leu Asp Ser Pro Val Leu Glu Asn Gly Asp Asn Trp Ser Val Gly 1395 1400 1405Gln Arg Gln Leu Val Ser Leu Gly Arg Ala Leu Leu Lys Gln Ala Lys 1410 1415 1420Ile Leu Val Leu Asp Glu Ala Thr Ala Ser Val Asp Thr Ala Thr Asp1425 1430 1435 1440Asn Leu Ile Gln Lys Ile Ile Arg Thr Glu Phe Glu Asp Cys Thr Val 1445 1450 1455Cys Thr Ile Ala His Arg Ile Pro Thr Val Ile Asp Ser Asp Leu Val 1460 1465 1470Leu Val Leu Ser Asp Gly Arg Val Ala Glu Phe Asp Thr Pro Ala Arg 1475 1480 1485Leu Leu Glu Asp Lys Ser Ser Met Phe Leu Lys Leu Val Thr Glu Tyr 1490 1495 1500Ser Ser Arg Ser Thr Gly Ile Pro Glu Leu1505 1510101350DNAGlycine maxCDS(3)...(1350) 10gc acg agt gga ctt gct gtg aca tat ggc ctg aat tta aat gca cgt 47 Thr Ser Gly Leu Ala Val Thr Tyr Gly Leu Asn Leu Asn Ala Arg 1 5 10 15cta tca cgg tgg ata ctc agc ttt tgc aaa ctt gaa aat aaa att ata 95Leu Ser Arg Trp Ile Leu Ser Phe Cys Lys Leu Glu Asn Lys Ile Ile 20 25 30tct att gag aga att tat cag tac agc caa att cct agt gaa gca ccc 143Ser Ile Glu Arg Ile Tyr Gln Tyr Ser Gln Ile Pro Ser Glu Ala Pro 35 40 45aca gtt att gaa gat tat cgc cct cca tcc tca tgg cct gaa aat ggg 191Thr Val Ile Glu Asp Tyr Arg Pro Pro Ser Ser Trp Pro Glu Asn Gly 50 55 60aca att gaa ata att gat ttg aag att cgt tac aag gag aat ctt cct 239Thr Ile Glu Ile Ile Asp Leu Lys Ile Arg Tyr Lys Glu Asn Leu Pro 65 70 75ttg gtg ctt tat gga gta aca tgc aca ttt cct ggt gga aag aag att 287Leu Val Leu Tyr Gly Val Thr Cys Thr Phe Pro Gly Gly Lys Lys Ile80 85 90 95gga ata gta gga cgt act ggc agt gga aaa tct act tta att cag gcg 335Gly Ile Val Gly Arg Thr Gly Ser Gly Lys Ser Thr Leu Ile Gln Ala 100 105 110tta ttt cga ttg att gaa cca aca agt ggg agt atc ctt ata gac aac 383Leu Phe Arg Leu Ile Glu Pro Thr Ser Gly Ser Ile Leu Ile Asp Asn 115 120 125att aat att tca gag att ggc ctt cat gac ctt cga agc cat ctc agt 431Ile Asn Ile Ser Glu Ile Gly Leu His Asp Leu Arg Ser His Leu Ser 130 135 140atc ata cca caa gat cca acc tta ttt gaa ggt acc att cga ggc aat 479Ile Ile Pro Gln Asp Pro Thr Leu Phe Glu Gly Thr Ile Arg Gly Asn 145 150 155ctt gat cct ctg gat gag cac tca gat aaa gag att tgg gag gca ctt 527Leu Asp Pro Leu Asp Glu His Ser Asp Lys Glu Ile Trp Glu Ala Leu160 165 170 175gat aag tct cag ctt gga gag gtt atc cgt gag aaa gga caa cag ctt 575Asp Lys Ser Gln Leu Gly Glu Val Ile Arg Glu Lys Gly Gln Gln Leu 180 185 190gat acg cca gtt cta gaa aat gga gat aat tgg agt gta gga cag cga 623Asp Thr Pro Val Leu Glu Asn Gly Asp Asn Trp Ser Val Gly Gln Arg 195 200 205caa ctt gtt gct ctg ggc cga gct ctg ctg cag cag tca aga ata ctt 671Gln Leu Val Ala Leu Gly Arg Ala Leu Leu Gln Gln Ser Arg Ile Leu 210 215 220gta cta gat gaa gca aca gca tca gtt gat acc gcc aca gat aat ctt 719Val Leu Asp Glu Ala Thr Ala Ser Val Asp Thr Ala Thr Asp Asn Leu 225 230 235ata cag aag att atc cga agt gag ttc aaa gaa tgc act gtt tgc acc 767Ile Gln Lys Ile Ile Arg Ser Glu Phe Lys Glu Cys Thr Val Cys Thr240 245 250 255att gca cat cga ata cct act gtc att gac agt gat cta gtt ctt gtg 815Ile Ala His Arg Ile Pro Thr Val Ile Asp Ser Asp Leu Val Leu Val 260 265 270ctc agt gat ggt cga gtt gca gag ttc aac act cct tca aga cta tta 863Leu Ser Asp Gly Arg Val Ala Glu Phe Asn Thr Pro Ser Arg Leu Leu 275 280 285gag gat aag tca tcc atg ttt ctg aag ctg gtg act gag tac tca tca 911Glu Asp Lys Ser Ser Met Phe Leu Lys Leu Val Thr Glu Tyr Ser Ser 290 295 300cgt tca agt ggc ata cca gac ttt tag aac aaa tgg aag gtg tga atg 959Arg Ser Ser Gly Ile Pro Asp Phe Asn Lys Trp Lys Val Met 305 310 315ctt tca tag tgt ggt ggc tgg agc tta aga tag ttc aaa agt tga atc 1007Leu Ser Cys Gly Gly Trp Ser Leu Arg Phe Lys Ser Ile 320 325 330agg aag tga tgc cac cct tgc atg tca ctg ctg cat tcg ggg cat gca 1055Arg Lys Cys His Pro Cys Met Ser Leu Leu His Ser Gly His Ala 335 340 345tag aga cac gag atg gaa aca aac aaa ata aaa ggg aga ggt ttg tgc 1103Arg His Glu Met Glu Thr Asn Lys Ile Lys Gly Arg Gly Leu Cys 350 355 360ctc ctc atg aat caa gca tcc tac tgg ggg aaa ttt gtt tga tta ttc 1151Leu Leu Met Asn Gln Ala Ser Tyr Trp Gly Lys Phe Val Leu Phe 365 370 375ccc tta aag ttg aga aat tca tgc aag gtt agc atg ctt tgt aac aca 1199Pro Leu Lys Leu Arg Asn Ser Cys Lys Val Ser Met Leu Cys Asn Thr 380 385 390aaa taa gat gat ctg tga tta cag gaa agt aac gaa ata gtt tgt aga 1247Lys Asp Asp Leu Leu Gln Glu Ser Asn Glu Ile Val Cys Arg 395 400 405atg agg cac tag gat ttt gct tgg tta gaa aaa gtg tag agt tta aac 1295Met Arg His Asp Phe Ala Trp Leu Glu Lys Val Ser Leu Asn 410 415tag ttt tgt gta ttc cac aat ttt ctt gta gtg aaa gtt tag aat taa 1343Phe Cys Val Phe His Asn Phe Leu Val Val Lys Val Asn420 425 430gcc aaa a 1350Ala Lys 11434PRTGlycine max 11Thr Ser Gly Leu Ala Val Thr Tyr Gly Leu Asn Leu Asn Ala Arg Leu1 5 10 15Ser Arg Trp Ile Leu Ser Phe Cys Lys Leu Glu Asn Lys Ile Ile Ser 20 25 30Ile Glu Arg Ile Tyr Gln Tyr Ser Gln Ile Pro Ser Glu Ala Pro Thr 35 40 45Val Ile Glu Asp Tyr Arg Pro Pro Ser Ser Trp Pro Glu Asn Gly Thr 50 55 60Ile Glu Ile Ile Asp Leu Lys Ile Arg Tyr Lys Glu Asn Leu Pro Leu65 70 75 80Val Leu Tyr Gly Val Thr Cys Thr Phe Pro Gly Gly Lys Lys Ile Gly 85 90 95Ile Val Gly Arg Thr Gly Ser Gly Lys Ser Thr Leu Ile Gln Ala Leu 100 105 110Phe Arg Leu Ile Glu Pro Thr Ser Gly Ser Ile Leu Ile Asp Asn Ile 115 120 125Asn Ile Ser Glu Ile Gly Leu His Asp Leu Arg Ser His Leu Ser Ile 130 135 140Ile Pro Gln Asp Pro Thr Leu Phe Glu Gly Thr Ile Arg Gly Asn Leu145 150 155 160Asp Pro Leu Asp Glu His Ser Asp Lys Glu Ile Trp Glu Ala Leu Asp 165 170 175Lys Ser Gln Leu Gly Glu Val Ile Arg Glu Lys Gly Gln Gln Leu Asp 180 185 190Thr Pro Val Leu Glu Asn Gly Asp Asn Trp Ser Val Gly Gln Arg Gln 195 200 205Leu Val Ala Leu Gly Arg Ala Leu Leu Gln Gln Ser Arg Ile Leu Val 210 215 220Leu Asp Glu Ala Thr Ala Ser Val Asp Thr Ala Thr Asp Asn Leu Ile225 230 235 240Gln Lys Ile Ile Arg Ser Glu Phe Lys Glu Cys Thr Val Cys Thr Ile 245 250 255Ala His Arg Ile Pro Thr Val Ile Asp Ser Asp Leu Val Leu Val Leu 260 265 270Ser Asp Gly Arg Val Ala Glu Phe Asn Thr Pro Ser Arg Leu Leu Glu 275 280 285Asp Lys Ser Ser Met Phe Leu Lys Leu Val Thr Glu Tyr Ser Ser Arg 290 295 300Ser Ser Gly Ile Pro Asp Phe Asn Lys Trp Lys Val Met Leu Ser Cys305 310 315 320Gly Gly Trp Ser Leu Arg Phe Lys Ser Ile Arg Lys Cys His Pro Cys 325 330 335Met Ser Leu Leu His Ser Gly His Ala Arg His Glu Met Glu Thr Asn 340 345 350Lys Ile Lys Gly Arg Gly Leu Cys Leu Leu Met Asn Gln Ala Ser Tyr 355 360 365Trp Gly Lys Phe Val Leu Phe Pro Leu Lys Leu Arg Asn Ser Cys Lys 370 375 380Val Ser Met Leu Cys Asn Thr Lys Asp Asp Leu Leu Gln Glu Ser Asn385 390 395 400Glu Ile Val Cys Arg Met Arg His Asp Phe Ala Trp Leu Glu Lys Val 405 410 415Ser Leu Asn Phe Cys Val Phe His Asn Phe Leu Val Val Lys Val Asn 420 425 430Ala Lys 12465DNAGlycine maxCDS(1)...(465)misc_feature(0)...(0)"n" can be any nucleotide 12ngn aat tgc ttn cnt ngg tgc ana cgt ttg gtt gct nnc aac caa tnc 48Xaa Asn Cys Xaa Xaa Xaa Cys Xaa Arg Leu Val Ala Xaa Asn Gln Xaa1 5 10 15ccc att ngt tgc can ntg tnc ctt gtg gcc tag ggt cca nga aga ttt 96Pro Ile Xaa Cys Xaa Xaa Xaa Leu Val Ala Gly Pro Xaa Arg Phe 20 25 30ttc agg aca aat tgn tgc cca cct aag gan tga aag gnt gag aaa gcc 144Phe Arg Thr Asn Xaa Cys Pro Pro Lys Xaa Lys Xaa Glu Lys Ala 35 40 45act cag agt gtc tta gga ntn tga gga ttc tca agc tnc aag ctt ggg 192Thr Gln Ser Val Leu Gly Xaa Gly Phe Ser Ser Xaa Lys Leu Gly 50 55 60agg atc gat atc gat tga agt tgg agg aaa tgc gtg gag tng agt tca 240Arg Ile Asp Ile Asp Ser Trp Arg Lys Cys Val Glu Xaa Ser Ser 65 70 75agt ggc ata agg aaa nca ctc tat tct cag gct tgc ata act ttc atg 288Ser Gly Ile Arg Lys Xaa Leu Tyr Ser Gln Ala Cys Ile Thr Phe Met 80 85 90ttc tgg agc tcc cct ata ttt gtt tca gct gtt act ttt gct act tcc 336Phe Trp Ser Ser Pro Ile Phe Val Ser Ala Val Thr Phe Ala Thr Ser 95 100 105ata ttg ttg ggg ggt cag ttg aca gca ggt ggt gtt ctc tct gct cta 384Ile Leu Leu Gly Gly Gln Leu Thr Ala Gly Gly Val Leu Ser Ala Leu 110 115 120gct act ttc agg att cgc caa gan cct ntg agg aat ttt cct gac ttg 432Ala Thr Phe Arg Ile Arg Gln Xaa Pro Xaa Arg Asn Phe Pro Asp Leu125 130 135 140gta tca acc atg gct cag aca aaa gtt tct ctt 465Val Ser Thr Met Ala Gln Thr Lys Val Ser Leu 145 15013151PRTGlycine maxVARIANT(0)...(0)"Xaa" can be any amino acid 13Xaa Asn Cys Xaa Xaa Xaa Cys Xaa Arg Leu Val Ala Xaa Asn Gln Xaa1 5 10 15Pro Ile Xaa Cys Xaa Xaa Xaa Leu Val Ala Gly Pro Xaa Arg Phe Phe 20 25 30Arg Thr Asn Xaa Cys Pro Pro Lys Xaa Lys Xaa Glu Lys Ala Thr Gln 35 40 45Ser Val Leu Gly Xaa Gly Phe Ser Ser Xaa Lys Leu Gly Arg Ile Asp 50 55 60Ile Asp Ser Trp Arg Lys Cys Val Glu Xaa Ser Ser Ser Gly Ile Arg65 70 75 80Lys Xaa Leu Tyr Ser Gln Ala Cys Ile Thr Phe Met Phe Trp Ser Ser 85 90 95Pro Ile Phe Val Ser Ala Val Thr Phe Ala Thr Ser Ile Leu Leu Gly 100 105 110Gly Gln Leu Thr Ala Gly Gly Val Leu Ser Ala Leu Ala Thr Phe Arg 115 120 125Ile Arg Gln Xaa Pro Xaa Arg Asn Phe Pro Asp Leu Val Ser Thr Met 130 135 140Ala Gln Thr Lys Val Ser Leu145 150144050DNAArtificial SequenceConsensus sequence 14tggaytacgc kcacatggtt gccaacttcg cgtcsgygcc ggccctsggs ttcctstgct 60tggttggtgt catgggttcc accggtktkg aattggagtt yacsgasgay grcarcrgys 120tkcatgarcc gctsytgctc ggyrggcagc gsagagasgc mgasgaggag cycggstgyy 180tgmgggtsac kccstaygsy gatgctggga tystyagcct tgcaacattr tcatggctta 240gtccgytgct stcwgttggt gcgcagcgrc cacttgagyt ggctgacata cccttgmtgg 300crcacaarga

ccgtgcmaar tcmtgctaya aggcgatgag crstcactay garcgccagc 360ggmtrgaryr cccyggcags garccatcac tsrcatgggc aataytsaag tcrttctggc 420gwgaggcmgc grtcaatggy rcwttygcwg ckgtsaacac rattgtstcs tatgttggmc 480cwtacytgat cagctayttt gtggactacc tcagtggcaa mattgmwttc ccccatgaag 540gttacatcct tgcctctrta ttttttgtag caaarytrct tgagacrctc actgcycgrc 600agtggtactt gggygtggay rtcatgggga tccatgtcaa gtctggscts ackgccatgg 660tgtayaggaa gggyctymgr ctgtcraayk cctcrcggca gagccacacs agtggtgaga 720ttgtgaatta catggcsgty gatgtrcagc gtgtggggga ctatgcatgg tayttycatg 780acatctggat gcttccmctg cagatcatyc tygcyctcgc catcctgtac aagaaygtyg 840gratcgccat ggtttcaaca ttggtagcwa ctgtrytatc ratygcwgcc tcwgttcctg 900tggcraagct gcaggagcac taccaagata agytwatggc mtcaaargat gagcgcatgc 960gcaagacwtc agagtgcytg aaraatatga ggattttgaa gctycargcr tgggaggatc 1020grtacmggct gmagttggaa gagatgagra aygtggaatg carrtggctt cggtgggctc 1080tgtaytcaca ggcygcagtt acatttgttt tctggagytc rccaatcttt gtcgcmgtsa 1140taacwtttgg gacttgyata ttrctyggtg gcsarctcac tgcwggaggk gttctwtcyg 1200ctttagcaac rtttmggatc ctycaagarc cwctkaggaa yttcccrgat ctyatctcta 1260tgatkgcwca gacragggtr tctttggacc gkttgtctca ytttctkcar caagaagaay 1320tgccagatga ygcaactata amkgttccac awrgtagtac agataaggca rtcratatwa 1380akgatgsyrc attctcttgg aacccatmyw ctcyracccc tacactttct gryatmmacc 1440ttagtgtrgt gagrggyatg mgagtagcag tstgtggtgt cattggttct ggyaaatcaa 1500gyytrytrtc ktctatactc ggsgagatac ccaaattrtg tggycawgts aggatmagtg 1560gmwcagcagc rtatgtycct cagactgcmt ggatacagtc yggaaayatt gaggagaaya 1620ttctktttgg cagtcmaatg gayaracarc gttacaagag agtyattgmr gcttgctsyc 1680tkaagaaaga tcttsagytg ctccartayg gagatcagac yrtyatyggt gatagrggca 1740ttaatttgag tggrggtcag aaacaaagag twcagcttgc wagagcactm taccaagatg 1800ctgatattta tttgctygat gatcccttca gtgckgttga tgctcatact gggagygaay 1860trtttargga rtatatattg actgcactag caascaarac mgtaatytat gtaacmcatc 1920aarttgartt tctaccagct gctgayytga taytggttct taaggatggy catatcacmc 1980aagctggaaa rtatgatgat cttctscaag ctggmactga tttcaatgct ytggtttstg 2040ctcataagga agctattgar accatggaww twtyygaaga ttccgatrrk gatacwgtyt 2100cttctrttcc yawcaaaaga ytgacrccaa gtrtyagcaa tatwgataay ctgaaaaaya 2160agrtgtsyra waatgramaa ccatctarta crcgkggaat waargaaaar aagaagaagc 2220cwgaagagcg taagaagaag cgkwctgttc aagaggagga ragggarcgw ggaarrgtka 2280gctymmargt ttayttgtca tacatgggrg aagcwtacaa aggtacactg ataccmctma 2340ttatcytggc ycaaaccatg ttycaagtwc ttcagattgc gagyaactgg tggatggcat 2400gggcaaaccc acaaacagaa ggagatgcwc cyaagacaga yagtgtggty ctyytggttg 2460tttatatgtc ccttgccttt ggragttcay trtttgtgtt yrtgagaagy cttcttgtgg 2520ctacrtttgg tttagcarct gcmcagaagc tktttrtaaa ratgctwagg tgtgtytttc 2580gagckccaat gtcattcttt gayacyacac catctggtcg rattttgaac mgagtttctg 2640tagatcaaag tgtygtggac cttgatatag crttcagact tggtggattt gcatcaacra 2700caattcaact mcttggaatt gttgctgtca tgagcaaagt cacatggcaa gttytgattc 2760ttatagtycc yatggctgtt gcatgcatgt ggatgcagag rtattatatt gcttcatcaa 2820gggaaytrac taggatyttr agygtwcaga agtckccrgt gatccatttg tttagtgart 2880caattgctgg tgctgctaca atmagrggtt ttggtcaaga gaarcgrtty atgaaaagra 2940atctttayct tcttgactgt tttgctcgsc ctytattttc cagcctkgcw gctattgaat 3000ggctstgcct gcgaatggaa ttgctytcga cyttygtctt ygctttttgc atggcratac 3060twgtgagctt ycctcctggc acaatygaac caagtatggc tggsctygct gtmacwtatg 3120gacttaattt aaatgctcgc atgtcaagrt ggataytgag cttctgtaaa ttagagaaya 3180gratmatctc tgttgarcgc atttatcart attgcargct tccyagtgaa gcaccaytsa 3240tyattgagaa ywgccgtccm ycatcmtcrt ggcctsagaa tggaaacatt garctgrtyg 3300atctcaaggt mcgstacaar gaygayctrc cmttagttct wcatggwrtm agttgtatrt 3360ttccyggygg raaaaagatt gggattgtrg ggcgwactgg aagyggtaaa tctactctta 3420ttcaggccct tttccgcyta attgarccya cwggagggaa rrttatmaty gaymacrtyg 3480ayatytctrs aattggccts catgatctgc ggtcacggtt gagcatcatt ccccargacc 3540ctacrttgtt tgagggtact atcagaatga aycttgatcc tcttgargar tgyactgatc 3600argaaatttg ggaggcacta gaaaagtgtc agctmggaga ggtcattcgk tccaaggawg 3660araarctkga cagtccagtr ctrgaraayg grgataactg gagygtggga carcgccarc 3720ttattgcayt gggtagggcs ctgctsaarc aggcaaaaat tttggtrcty gaygaggcra 3780cagcatcwgt ygacacagcw acrgacaatc ttatycaaaa gatyatycgc agtgaattca 3840aggaytgcac rgtctgyacc attgcwcacc gtatyccsac sgttattgay agtgacctwg 3900tyctggtsct tagtgatggt aaaatygcag agttygacac rccccagagg ctyttrgagg 3960acaagtcmtc yatgttcatr cagctagtat ckgaatactc mactcggtcr agctgtatat 4020agagaggctt agcttaaaay cccscmcmmm 4050151522PRTArtificial SequenceConsensus sequence 15Xaa Xaa Xaa Ile Pro Xaa Phe Pro Xaa Leu Pro Leu Pro Glu Ala Leu1 5 10 15Ala Ala Xaa Ala His Ala Ala Leu Leu Ala Leu Ala Xaa Leu Leu Leu 20 25 30Leu Leu Arg Ala Ala Arg Ala Leu Ala Ser Arg Cys Ala Ser Cys Leu 35 40 45Lys Xaa Xaa Xaa Arg Arg Xaa Xaa Xaa Xaa Xaa Xaa Ala Ala Xaa Xaa 50 55 60Xaa Gly Gly Xaa Leu Ala Ala Ala Ser Val Gly Ala Trp His Arg Ala65 70 75 80Ala Leu Ala Cys Cys Ala Tyr Ala Leu Leu Ala Gln Val Ala Val Leu 85 90 95Ser Tyr Glu Val Ala Val Ala Gly Ser Xaa Val Ser Xaa Xaa Xaa Ala 100 105 110Leu Leu Leu Pro Ala Val Gln Ala Leu Ala Trp Ala Ala Leu Leu Ala 115 120 125Leu Ala Leu Gln Ala Arg Ala Val Gly Trp Ala Arg Phe Pro Xaa Leu 130 135 140Val Arg Ile Trp Trp Val Val Ser Phe Ala Leu Cys Val Xaa Ile Ala145 150 155 160Tyr Asp Asp Ser Arg Arg Leu Ile Gly Asp Gly Xaa Xaa Xaa Xaa Xaa 165 170 175Xaa Xaa Ala His Met Val Ala Asn Phe Ala Ser Xaa Pro Ala Leu Gly 180 185 190Phe Leu Cys Leu Val Gly Val Met Gly Ser Thr Gly Ile Glu Leu Glu 195 200 205Phe Thr Asp Asp Xaa Xaa Xaa Leu His Glu Pro Leu Leu Leu Gly Xaa 210 215 220Gln Arg Arg Asp Ala Glu Glu Glu Xaa Gly Cys Leu Arg Val Thr Pro225 230 235 240Tyr Ala Asp Ala Gly Ile Val Ser Leu Ala Thr Leu Ser Trp Leu Ser 245 250 255Pro Leu Leu Ser Val Gly Ala Gln Arg Pro Leu Glu Leu Ala Asp Ile 260 265 270Pro Leu Leu Ala His Lys Asp Arg Ala Lys Ser Cys Tyr Lys Ala Met 275 280 285Ser Ser His Tyr Glu Arg Gln Arg Leu Glu Xaa Pro Gly Lys Glu Pro 290 295 300Ser Leu Ala Trp Ala Ile Leu Lys Ser Phe Trp Arg Glu Ala Ala Ile305 310 315 320Asn Gly Xaa Phe Ala Ala Val Asn Thr Ile Val Ser Tyr Val Gly Pro 325 330 335Tyr Leu Ile Ser Tyr Phe Val Asp Tyr Leu Ser Gly Lys Ile Xaa Phe 340 345 350Pro His Glu Gly Tyr Ile Leu Ala Ser Ile Phe Phe Val Ala Lys Leu 355 360 365Leu Glu Thr Leu Thr Ala Arg Gln Trp Tyr Leu Gly Val Asp Ile Met 370 375 380Gly Ile His Val Lys Ser Gly Leu Thr Ala Met Val Tyr Arg Lys Gly385 390 395 400Leu Arg Leu Ser Asn Ala Ser Arg Gln Ser His Thr Ser Gly Glu Ile 405 410 415Val Asn Tyr Met Ala Val Asp Val Gln Arg Val Gly Asp Tyr Ala Trp 420 425 430Tyr Phe His Asp Ile Trp Met Leu Pro Leu Gln Ile Ile Leu Ala Leu 435 440 445Ala Ile Leu Tyr Lys Asn Val Gly Ile Ala Met Val Ser Thr Leu Val 450 455 460Ala Thr Val Leu Ser Ile Ala Ala Ser Val Pro Val Ala Lys Leu Gln465 470 475 480Glu His Tyr Gln Asp Lys Leu Met Ala Ser Lys Asp Glu Arg Met Arg 485 490 495Lys Thr Ser Glu Cys Leu Lys Asn Met Arg Ile Leu Lys Leu Gln Ala 500 505 510Trp Glu Asp Arg Tyr Arg Leu Lys Leu Glu Glu Met Arg Asn Val Glu 515 520 525Cys Lys Trp Leu Arg Trp Ala Leu Tyr Ser Gln Ala Ala Val Thr Phe 530 535 540Val Phe Trp Ser Ser Pro Ile Phe Val Ala Val Ile Thr Phe Gly Thr545 550 555 560Cys Ile Leu Leu Gly Gly Gln Leu Thr Ala Gly Gly Val Leu Ser Ala 565 570 575Leu Ala Thr Phe Arg Ile Leu Gln Glu Pro Leu Arg Asn Phe Pro Asp 580 585 590Leu Ile Ser Met Met Ala Gln Thr Arg Val Ser Leu Asp Arg Leu Ser 595 600 605His Phe Leu Gln Gln Glu Glu Leu Pro Asp Asp Ala Thr Ile Xaa Val 610 615 620Pro Xaa Gly Ser Thr Asp Lys Ala Ile Asp Ile Lys Asp Gly Xaa Phe625 630 635 640Ser Trp Asn Pro Phe Ser Xaa Thr Pro Thr Leu Ser Gly Ile Asn Leu 645 650 655Ser Val Val Arg Gly Met Arg Val Ala Val Cys Gly Val Ile Gly Ser 660 665 670Gly Lys Ser Ser Leu Leu Ser Ser Ile Leu Gly Glu Ile Pro Lys Leu 675 680 685Cys Gly Xaa Val Arg Ile Ser Gly Thr Ala Ala Tyr Val Pro Gln Thr 690 695 700Ala Trp Ile Gln Ser Gly Asn Ile Glu Glu Asn Ile Leu Phe Gly Ser705 710 715 720Pro Met Asp Lys Gln Arg Tyr Lys Arg Val Ile Xaa Ala Cys Ser Leu 725 730 735Lys Lys Asp Leu Glu Leu Leu Gln Tyr Gly Asp Gln Thr Ile Ile Gly 740 745 750Asp Arg Gly Ile Asn Leu Ser Gly Gly Gln Lys Gln Arg Val Gln Leu 755 760 765Ala Arg Ala Leu Tyr Gln Asp Ala Asp Ile Tyr Leu Leu Asp Asp Pro 770 775 780Phe Ser Ala Val Asp Ala His Thr Gly Ser Glu Leu Phe Arg Glu Tyr785 790 795 800Ile Leu Thr Ala Leu Ala Ser Lys Thr Val Ile Tyr Val Thr His Gln 805 810 815Val Glu Phe Leu Pro Ala Ala Asp Leu Ile Leu Val Leu Lys Asp Gly 820 825 830His Ile Thr Gln Ala Gly Lys Tyr Asp Asp Leu Leu Gln Ala Gly Thr 835 840 845Asp Phe Asn Ala Leu Val Ser Ala His Lys Glu Ala Ile Glu Thr Met 850 855 860Asp Ile Xaa Glu Asp Ser Asp Glu Asp Thr Val Ser Xaa Xaa Xaa Xaa865 870 875 880Xaa Ser Ile Xaa Xaa Xaa Asn Lys Arg Leu Thr Pro Ser Ile Ser Asn 885 890 895Ile Asp Asn Leu Lys Asn Lys Val Xaa Glu Asn Gly Xaa Pro Ser Xaa 900 905 910Thr Arg Gly Ile Lys Glu Lys Lys Lys Lys Xaa Glu Arg Xaa Lys Lys 915 920 925Lys Arg Ser Val Gln Glu Glu Glu Arg Glu Arg Gly Lys Val Ser Leu 930 935 940Lys Val Tyr Leu Ser Tyr Met Gly Glu Ala Tyr Lys Gly Thr Leu Ile945 950 955 960Pro Leu Ile Ile Leu Ala Gln Thr Met Phe Gln Val Leu Gln Ile Ala 965 970 975Ser Asn Trp Trp Met Ala Trp Ala Asn Pro Gln Thr Glu Gly Asp Ala 980 985 990Pro Lys Thr Asp Ser Val Val Leu Leu Val Val Tyr Met Ser Leu Ala 995 1000 1005Phe Gly Ser Ser Leu Phe Val Phe Val Arg Ser Leu Leu Val Ala Thr 1010 1015 1020Phe Gly Leu Ala Ala Ala Gln Lys Leu Phe Ile Lys Met Leu Arg Cys1025 1030 1035 1040Val Phe Arg Ala Pro Met Ser Phe Phe Asp Thr Thr Pro Ser Gly Arg 1045 1050 1055Ile Leu Asn Arg Val Ser Val Asp Gln Ser Val Val Asp Leu Asp Ile 1060 1065 1070Ala Phe Arg Leu Gly Gly Phe Ala Ser Thr Thr Ile Gln Leu Leu Gly 1075 1080 1085Ile Val Ala Val Met Ser Lys Val Thr Trp Gln Val Leu Ile Leu Ile 1090 1095 1100Val Pro Met Ala Val Ala Cys Met Trp Met Gln Arg Tyr Tyr Ile Ala1105 1110 1115 1120Ser Ser Arg Glu Leu Thr Arg Ile Leu Ser Val Gln Lys Ser Pro Val 1125 1130 1135Ile His Leu Phe Ser Glu Ser Ile Ala Gly Ala Ala Thr Ile Arg Gly 1140 1145 1150Phe Gly Gln Glu Lys Arg Phe Met Lys Arg Asn Leu Tyr Leu Leu Asp 1155 1160 1165Cys Phe Ala Arg Pro Leu Phe Ser Ser Leu Ala Ala Ile Glu Trp Leu 1170 1175 1180Cys Leu Arg Met Glu Leu Leu Ser Thr Phe Val Phe Ala Phe Cys Met1185 1190 1195 1200Ala Ile Leu Val Ser Phe Pro Pro Gly Thr Ile Glu Pro Ser Met Ala 1205 1210 1215Gly Leu Ala Val Thr Tyr Gly Leu Asn Leu Asn Ala Arg Met Ser Arg 1220 1225 1230Trp Ile Leu Ser Phe Cys Lys Leu Glu Asn Arg Ile Ile Ser Val Glu 1235 1240 1245Arg Ile Tyr Gln Tyr Cys Lys Leu Pro Ser Glu Ala Pro Leu Ile Ile 1250 1255 1260Glu Asn Xaa Arg Pro Pro Ser Ser Trp Pro Xaa Asn Gly Asn Ile Glu1265 1270 1275 1280Leu Val Asp Leu Lys Val Arg Tyr Lys Asp Asp Leu Pro Leu Val Leu 1285 1290 1295His Gly Val Ser Cys Ile Phe Pro Gly Gly Lys Lys Ile Gly Ile Val 1300 1305 1310Gly Arg Thr Gly Ser Gly Lys Ser Thr Leu Ile Gln Ala Leu Phe Arg 1315 1320 1325Leu Ile Glu Pro Thr Gly Gly Lys Ile Ile Ile Asp Asn Ile Asp Ile 1330 1335 1340Ser Xaa Ile Gly Leu His Asp Leu Arg Ser Arg Leu Ser Ile Ile Pro1345 1350 1355 1360Gln Asp Pro Thr Leu Phe Glu Gly Thr Ile Arg Met Asn Leu Asp Pro 1365 1370 1375Leu Glu Glu Cys Thr Asp Gln Glu Ile Trp Glu Ala Leu Glu Lys Cys 1380 1385 1390Gln Leu Gly Glu Val Ile Arg Ser Lys Asp Glu Lys Leu Asp Ser Pro 1395 1400 1405Val Leu Glu Asn Gly Asp Asn Trp Ser Val Gly Gln Arg Gln Leu Ile 1410 1415 1420Ala Leu Gly Arg Ala Leu Leu Lys Gln Ala Lys Ile Leu Val Leu Asp1425 1430 1435 1440Glu Ala Thr Ala Ser Val Asp Thr Ala Thr Asp Asn Leu Ile Gln Lys 1445 1450 1455Ile Ile Arg Ser Glu Phe Lys Asp Cys Thr Val Cys Thr Ile Ala His 1460 1465 1470Arg Ile Pro Thr Val Ile Asp Ser Asp Leu Val Leu Val Leu Ser Asp 1475 1480 1485Gly Lys Ile Ala Glu Phe Asp Thr Pro Gln Arg Leu Leu Glu Asp Lys 1490 1495 1500Ser Ser Met Phe Ile Gln Leu Val Ser Glu Tyr Ser Thr Arg Ser Ser1505 1510 1515 1520Cys Ile16213PRTZea mays 16Ile Lys Asp Gly Ala Phe Ser Trp Asn Pro Tyr Thr Leu Thr Pro Thr1 5 10 15Leu Ser Asp Ile His Leu Ser Val Val Arg Gly Met Arg Val Ala Val 20 25 30Cys Gly Val Ile Gly Ser Gly Lys Ser Ser Leu Leu Ser Ser Ile Leu 35 40 45Gly Glu Ile Pro Lys Leu Cys Gly His Val Arg Ile Ser Gly Thr Ala 50 55 60Ala Tyr Val Pro Gln Thr Ala Trp Ile Gln Ser Gly Asn Ile Glu Glu65 70 75 80Asn Ile Leu Phe Gly Ser Gln Met Asp Arg Gln Arg Tyr Lys Arg Val 85 90 95Ile Ala Ala Cys Cys Leu Lys Lys Asp Leu Glu Leu Leu Gln Tyr Gly 100 105 110Asp Gln Thr Val Ile Gly Asp Arg Gly Ile Asn Leu Ser Gly Gly Gln 115 120 125Lys Gln Arg Val Gln Leu Ala Arg Ala Leu Tyr Gln Asp Ala Asp Ile 130 135 140Tyr Leu Leu Asp Asp Pro Phe Ser Ala Val Asp Ala His Thr Gly Ser145 150 155 160Glu Leu Phe Lys Glu Tyr Ile Leu Thr Ala Leu Ala Thr Lys Thr Val 165 170 175Ile Tyr Val Thr His Gln Val Glu Phe Leu Pro Ala Ala Asp Leu Ile 180 185 190Leu Val Leu Lys Asp Gly His Ile Thr Gln Ala Gly Lys Tyr Asp Asp 195 200 205Leu Leu Gln Ala Gly 21017184PRTZea mays 17Ile Glu Leu Ile Asp Leu Lys Val Arg Tyr Lys Asp Asp Leu Pro Leu1 5 10 15Val Leu His Gly Val Ser Cys Met Phe Pro Gly Gly Lys Lys Ile Gly 20 25 30Ile Val Gly Arg Thr Gly Ser Gly Lys Ser Thr Leu Ile Gln Ala Leu 35 40 45Phe Arg Leu Ile Glu Pro Thr Gly Gly Lys Ile Ile Ile Asp Asn Ile 50 55 60Asp Ile Ser Ala Ile Gly Leu His Asp Leu Arg Ser Arg Leu Ser Ile65 70 75 80Ile Pro Gln Asp Pro Thr Leu Phe Glu Gly Thr Ile Arg Met Asn Leu 85 90 95Asp Pro Leu Glu Glu Cys Thr Asp Gln Glu Ile Trp Glu Ala Leu Glu

100 105 110Lys Cys Gln Leu Gly Glu Val Ile Arg Ser Lys Glu Glu Lys Leu Asp 115 120 125Ser Pro Val Leu Glu Asn Gly Asp Asn Trp Ser Val Gly Gln Arg Gln 130 135 140Leu Ile Ala Leu Gly Arg Ala Leu Leu Lys Gln Ala Lys Ile Leu Val145 150 155 160Leu Asp Glu Ala Thr Ala Ser Val Asp Thr Ala Thr Asp Asn Leu Ile 165 170 175Gln Lys Ile Ile Arg Ser Glu Phe 180189PRTZea mays 18Gly Val Ile Gly Ser Gly Lys Ser Ser1 5199PRTZea mays 19Gly Arg Thr Gly Ser Gly Lys Ser Thr1 52012PRTZea mays 20Leu Ser Gly Gly Gln Lys Gln Arg Val Gln Leu Ala1 5 102112PRTZea mays 21Trp Ser Val Gly Gln Arg Gln Leu Ile Ala Leu Gly1 5 10226PRTZea mays 22Ile Tyr Leu Leu Asp Asp1 5235PRTZea mays 23Ile Leu Val Leu Asp1 5245PRTZea mays 24Ile Ala His Arg Ile1 5251931DNAZea mays 25ggcacgagca gcagcctcct tcctcctctc actctcgctc gcgctgcgct cgctacctcg 60cttcgcattc cattcgaaaa gaggggagga aaggcaagat gttcatcgag agcttccgcg 120tcgagagccc ccacgtgcgg tacggcccga tggagatcga gtcggagtac cggtacgaca 180cgacggagct ggtacacgag ggcaaggacg gcgcctcacg ctgggtcgtc cgccccaagt 240ccgtcaagta caacttccgg accagaaccg ccgtccccaa gctcggggtg atgcttgtgg 300ggtggggagg caacaacggg tccacgctga cggctggggt cattgccaac agggagggga 360tctcatgggc gaccaaggac aaggtgcagc aagccaacta ctacggctcc ctcacccacg 420cctccaccat cagagtcggc agctacaacg gggaggagat ctatgcgccg ttcaagagcc 480tccttcccat agtgaaccca gacgacattg tgttcggagg ctgggacatt agcaacatga 540acctggccga ctccatgacc agggccaagg tgctggatat tgacctgcag aagcagctca 600ggccctacat ggagtccatg gtgccacttc ccggtatcta tgatccggac ttcatcgcgg 660ctaaccaggg ctctcgcgcc aacagtgtca tcaagggcac caagaaagaa caggtggagc 720agatcatcaa ggatatcagg gagtttaagg agaagaacaa agtggacaag atagttgtgt 780tgtggactgc aaacactgaa aggtatagca atgtgtgcgc tggtctcaac gacacgatgg 840agaatctact ggcatctgtg gacaagaacg aagcggaggt atcaccatca acactatatg 900ccattgcctg tgtcatggaa ggggtgccgt tcatcaatgg gagcccccag aacacctttg 960tgcctgggct gattgatctt gctataaaaa acaactgctt gattggtggt gacgacttca 1020agagtggaca gaccaagatg aaatctgtct tggtcgattt ccttgttggt gctggaataa 1080agcccacctc aatcgtgagc tacaaccact tgggaaacaa cgatggcatg aacctgtctg 1140cccctcaaac attcaggtcc aaggagatct ccaagagcaa cgtggtggat gacatggtct 1200cgagcaatgc catcctctat gagcccggcg agcatcccga tcatgtcgtt gtcatcaagt 1260atgtgccgta cgtgggagac agcaagaggg ctatggacga gtacacctca gagatcttca 1320tgggcggcaa gaacaccatc gtgctgcaca acacctgtga ggactcgctc ctcgccgcac 1380ctatcatcct tgatctggtg ctcttggctg agctcagcac caggatccag ctgaaagctg 1440agggagagga caaattccac tccttccacc cggtggccac catcttgagt tacttcacca 1500aggcacccct ggttccccct ggcacaccgg tggtgaacgc tctggccaag cagagggcga 1560tgctggagaa catcatgagg gcctgcgttg ggctggcccc agagaacaac atgatcttgg 1620agtacaagtg agccaagtgg cgtgccctgc agcgcgaggt tagctgctgg aagggaacta 1680gaaaggcgag attagctgtg ggattgtgtt gggcttgtcg tgttttcttt tgcgttcttt 1740cctagtcatt gctgttgcgc ttttgtattt gtcggacccg taactaccag ggctctgcta 1800ttagcggcac ggagcctgta attgtattgt atgataatgt gatcgagggt gctacttccc 1860ctcggcattc ctagtgttgg ttaaaagtcg ttcgacagca acttatcgac ccaaaaaaaa 1920aaaaaaaaaa a 19312621DNAArtificial SequenceAdaptor/primer 26tactcaggac tcatcgaccg t 212723DNAArtificial SequenceAdaptor/primer 27gtgaacggtc gatgagtcct gag 232818DNAArtificial SequenceAdaptor/primer 28gtgaacggtc gatgagtc 182918DNAArtificial SequenceAdaptor/primer 29gtcgatgagt cctgagta 183018DNAArtificial SequencePrimer 30gatgagtcct gagtagaa 183118DNAArtificial SequencePrimer 31gatgagtcct gagtagac 183218DNAArtificial SequencePrimer 32gatgagtcct gagtagag 183318DNAArtificial SequencePrimer 33gatgagtcct gagtagat 183418DNAArtificial SequencePrimer 34gatgagtcct gagtagca 183518DNAArtificial SequencePrimer 35gatgagtcct gagtagcc 183618DNAArtificial SequencePrimer 36gatgagtcct gagtagcg 183718DNAArtificial SequencePrimer 37gatgagtcct gagtagct 183818DNAArtificial SequencePrimer 38gatgagtcct gagtagga 183918DNAArtificial SequencePrimer 39gatgagtcct gagtaggc 184018DNAArtificial SequencePrimer 40gatgagtcct gagtaggg 184118DNAArtificial SequencePrimer 41gatgagtcct gagtaggt 184218DNAArtificial SequencePrimer 42gatgagtcct gagtagta 184318DNAArtificial SequencePrimer 43gatgagtcct gagtagtc 184418DNAArtificial SequencePrimer 44gatgagtcct gagtagtg 184518DNAArtificial SequencePrimer 45gatgagtcct gagtagtt 184619DNAArtificial SequencePrimer 46cgatgagtcc tgagtaaaa 194719DNAArtificial SequencePrimer 47cgatgagtcc tgagtaaac 194819DNAArtificial SequencePrimer 48cgatgagtcc tgagtaaag 194919DNAArtificial SequencePrimer 49cgatgagtcc tgagtaaat 195019DNAArtificial SequencePrimer 50cgatgagtcc tgagtaaca 195118DNAArtificial SequencePrimer 51gatgagtcct gagtaacc 185218DNAArtificial SequencePrimer 52gatgagtcct gagtaacg 185318DNAArtificial SequencePrimer 53gatgagtcct gagtaact 185419DNAArtificial SequencePrimer 54cgatgagtcc tgagtaaga 195518DNAArtificial SequencePrimer 55gatgagtcct gagtaagc 185618DNAArtificial SequencePrimer 56gatgagtcct gagtaagg 185719DNAArtificial SequencePrimer 57cgatgagtcc tgagtaagt 195819DNAArtificial Sequenceprimer 58cgatgagtcc tgagtaata 195918DNAArtificial SequencePrimer 59gatgagtcct gagtaatc 186018DNAArtificial SequencePrimer 60gatgagtcct gagtaatg 186119DNAArtificial SequencePrimer 61cgatgagtcc tgagtaatt 1962199PRTArtificial SequencePfam consensus sequence 62Gly Glu Val Leu Ala Leu Val Gly Pro Asn Gly Ala Gly Lys Ser Thr1 5 10 15Leu Leu Lys Leu Ile Ser Gly Leu Leu Pro Pro Thr Glu Gly Thr Ile 20 25 30Leu Leu Asp Gly Ala Arg Asp Leu Ser Asp Leu Ser Lys Leu Lys Glu 35 40 45Arg Leu Glu Leu Leu Arg Lys Asn Ile Gly Val Val Phe Gln Asp Pro 50 55 60Thr Leu Phe Pro Asn Pro Glu Leu Thr Val Arg Glu Asn Ile Ala Phe65 70 75 80Gly Leu Arg Leu Ser Leu Gly Leu Ser Lys Asp Glu Gln Asp Asp Arg 85 90 95Leu Lys Lys Ala Gly Ala Glu Glu Leu Leu Glu Arg Leu Gly Leu Gly 100 105 110Tyr Asp Asp Leu Leu Asp Arg Arg Pro Gly Thr Leu Ser Gly Gly Gln 115 120 125Lys Gln Arg Val Ala Ile Ala Arg Ala Leu Leu Thr Lys Pro Lys Leu 130 135 140Leu Leu Leu Asp Glu Pro Thr Ala Gly Leu Asp Pro Ala Ser Arg Ala145 150 155 160Gln Leu Leu Glu Leu Leu Arg Glu Leu Arg Gln Gln Gly Gly Thr Val 165 170 175Leu Leu Val Thr His Asp Leu Asp Leu Leu Asp Arg Leu Ala Asp Arg 180 185 190Ile Leu Val Leu Glu Asp Gly 19563286PRTArtificial SequencePfam consensus sequence 63Leu Leu Ile Ala Ile Leu Leu Leu Ile Leu Ala Gly Ala Thr Ala Leu1 5 10 15Val Thr Phe Pro Leu Leu Leu Gly Arg Leu Leu Asp Ser Gly Phe Pro 20 25 30Leu Ser Asp Gly Asn Asp Asp His Glu Ala Arg Ser Ser Leu Ile Ser 35 40 45Leu Ala Ile Leu Ser Leu Leu Ala Val Phe Val Leu Ile Gly Leu Leu 50 55 60Leu Gln Gly Ser Phe Tyr Leu Leu Ala Gly Glu Arg Leu Gly Gln Arg65 70 75 80Leu Arg Lys Arg Leu Phe Arg Ala Leu Leu Arg Gln Ile Leu Gly Leu 85 90 95Phe Asp Ser Phe Phe Asp Thr Asn Ser Val Gly Glu Leu Thr Ser Arg 100 105 110Leu Thr Asn Asp Val Glu Lys Ile Arg Asp Gly Leu Gly Glu Lys Leu 115 120 125Gly Leu Leu Phe Gln Ser Leu Ala Thr Val Val Gly Gly Leu Ile Val 130 135 140Met Phe Tyr Tyr Ser Trp Lys Leu Thr Leu Ile Leu Leu Ala Ile Leu145 150 155 160Pro Leu Leu Ile Leu Leu Ser Ala Val Leu Ala Lys Lys Leu Arg Lys 165 170 175Leu Ser Arg Lys Glu Gln Lys Ala Tyr Ala Lys Ala Gly Ser Val Ala 180 185 190Glu Glu Ser Leu Ser Gly Ile Arg Thr Val Lys Ala Phe Gly Arg Glu 195 200 205Glu Tyr Glu Leu Glu Arg Phe Asp Lys Ala Leu Glu Asp Ala Glu Lys 210 215 220Ala Gly Ile Lys Lys Ala Ile Ile Ala Gly Leu Leu Phe Gly Ile Thr225 230 235 240Gln Leu Ile Ser Tyr Leu Ser Tyr Ala Leu Ala Leu Trp Phe Gly Gly 245 250 255Tyr Leu Val Ala Ser Val Ile Ser Gly Gly Leu Ser Val Gly Thr Leu 260 265 270Phe Ala Phe Leu Ser Leu Gly Asn Gln Leu Ile Gly Pro Leu 275 280 285641170DNAZea mays 64gaaaatctct ttctccgctg cgctgcaaac ccaccgcttc caccatcgcc actcgtcacc 60ccttgctccc atagtcccca tacc atg ccc gac ctc cac ccg ccg gag cac 111caa gtc gcc ggt cac cgc gcc tcc gcc agc aag ctg ggc ccg ctc atc 159gac ggc tcc ggc ctc ttc tac aag ccg ctc cag gcc ggc gac cgt ggg 207gag cac gag gtc gcc ttc tat gag gcg ttc tcc gcc cac gcc gcc gtc 255ccg gcc cgc atc cga gac acc ttc ttc ccc cgg ttc cac ggc acg cga 303ctc ctc ccc acc gag gcg cag ccc ggg gag ccg cat ccg cac ctc gtc 351ctc gac gac ctc ctc gcg ggg ttt gag gcg ccc tgc gtc gca gac atc 399aag atc ggc gcc atc acg tgg cca ccg agt tcg ccg gag ccc tac atc 447gcc aag tac ctc gcc aag gac cgc ggg acc acg agc gtt ctg ctc gga 495ttc cgc gtc ttg cgt ccg agt cgt cgg ccc cga ggg cgc cgt gtg gcg 543gac gga gcg ccc gga ggt gaa ggc tat gga cac cgt cgg cgt ccg ccg 591cgt gct ccg gcg cta cgt gtc atc cgc ttg ccg acg agg gga tgg act 639gcg cgc tcg cgg cgg cgg tgt acg gag gaa aag gtg gag tct tgt cac 687agc tgc gcg agc tca agg cat ggt tgg agg agc aga ctc tgt tcc act 735tct act cgg cgt cga ttc ttc tgg gct atg atg ctg ctg cag tcg cag 783cag gcg gag gtg ggg gtg ggg taa cagtgaagct ggtggacttt gcccatgtgc 837ccgagggtga tggggtgatt gaccacaact tcctgggcga gctctgctag ctgatcaagt 897tcgtttctga cattgttcca gagactcctt agacgcagcc tttgggtcct tcttaagaga 957ggatcctgac atttttgatt tgataacaaa ggaagcactt tcagctgcaa aaaaagaaag 1017cagcagtgag gatgaagatg acagtagtga ggaaagttcg gatgatgagc caacaaaagt 1077tgaagaaaag aaggctccaa aagtatcaga aaacattgga tctgaggatg aatcttctga 1137agacgagagt gataaagaca gtgaagagcc tca 1170651388DNAZea mays 65ccacgcgtcc gcaaatttca atctccatcg atcgattcct cccgaacccg acccgatggc 60ctccgacgcc gccgccgagc cctcctccgg cgtcacccac cccccgcgct acgtcatcgg 120ttacgcgctc gcgccgaaga agcagcaaag cttcatccag ccgtcgctgg tggcccaggc 180ggcgtcgcgg ggcatggacc tcgtccccgt ggatgcgtcg cagcccctgg cagagcaagg 240gcccttccac ctcctcatcc acaagctcta cggagacgac tggcgcgccc agctcgtggc 300cttcgccgcg cgccacccgg ccgtccccat cgtcgacccg ccccacgcca tcgaccgcct 360ccacaaccgc atctccatgc tccaggtcgt ctccgagctc gaccacgccg ccgaccagga 420cagcactttc ggtatcccca gccaggtcgt cgtctacgac gctgccgcgc tcgccgactt 480cggactcctt gccgcgctcc gcttcccgct catcgccaag cccctcgtcg ccgacggcac 540cgccaagtcc cacaagatgt cgctcgtcta ccaccgcgag ggcctcggca agctccgccc 600gccgcttgtg ctccaggagt tcgtcaacca tggcggcgtc atcttcaagg tctacgtcgt 660cggcggccac gtcacttgcg tcaagcgccg tagcctgccc gacgtgtccc ccgaggatga 720cgcatcggcc cagggatccg tctccttctc ccaggtctcc aacctcccca ctgagcgcac 780ggcggaggag tactacggcg aaaagagtct cgaggacgcc gtcgtgccgc ccgccgcatt 840catcaaccag atcgcgggcg gcctccgccg cgcgctgggc ctgcaactct tcaacttcga 900catgatccgc gacgtccgcg ccggcgaccg ctatctcgtc attgacatca actacttccc 960gggctacgcc aagatgccag gatacgagac tgtcctcacg gatttcttct gggagatggt 1020ccataaggac ggcgtgggca accaacagga ggagaaaggg gccaaccatg ttgtcgtgaa 1080ataagatgat gattgatggc actggatatc tggcgaatgc tgctgattct ggatgcagaa 1140ttcgatgagg ggatttagtt ggttgtagta tctggcgaat gctgctggtt ctggatgcag 1200aatttgatga ggggatttag ttggatttca acccatagca tgccgaggac ctcctagctc 1260tttccaaacc agttgtttag gtatcttttc tgggtaagtc agcttcatct agtttagtct 1320gtctgaacaa aagagtggga catgacccaa acggaattct aatgaaaaac gagctctcta 1380tctgcaaa 1388661313DNAZea mays 66ctactactca aatccatcct tattgagctt agtgtttgat ccatggactc ggaaggagta 60gcagcaaagg tggcagatga gactactaaa ccggcaagcc aagaagacgg cgagagcaag 120gccgggatga ctgatctgct gatgctgacc gacaagtcgc agctgcaggc gctagcgatg 180ctgctgcgga acaacgagga gctcatgatg agccaagcga tcaagtcgga gacggagcgc 240gttgagtacc tcaagacggt gagcgactgc tacacgcgga caatgaagct ccttgacgac 300tccatggcgg ccaggatcac gtacgagcgt tcgggcggaa cgaggagcct cgtcgcccgg 360gacatggacg actacgtcgt ctacggcctc aacgcgtgct tgcagaacgt ccgcaactgc 420tgcgtgcgtc tggacgccat cgacaagctg cgggcgcact acgacgccct cgccgacgcc 480gtcgccgaac cggccgccaa cgtcgagggc ctcgccgcgg aggcgtccga gtacaaggcc 540gccatgtggc agtactgcta caaccagcgg agcgcctccg cgcgggcgca ctcccgcgcc 600tactcccagg cgctcaagct ggagggcatc gacttcgccg agcttgtgcg gaggcaccag 660ctccggctcg ggtacggcag caagggcgag gagttcgagg acctggacga cacccagaag 720ctggaggtgt acaacagcat catcgtcgag tcggggcggg cggggctacc ggtgcggatg 780ttctcgtcgg gccgctctgc cggtggccct aagattgcag ccacgacgtg ggcgcaggcg 840gtgagcgtct tcatcatggc ggcgggcaac ctggcgtggg acgtgttcac cacggagcac 900gaggtggagg ccatcctcaa gggcagcctc aacctcctgg cggggctagg gggcttcgcc 960gtggaggccg tcgtcggcgc ggctgtcacc aaggcggtcg caaacgtcgg cgccggcgtc 1020tttgcttgct ctctcgcggg cttcgtcgtg ggcgccatag ccgggctgat cttcatcggc 1080gtcagcggcc tcctcattaa cctcatcatc ggctccccta ggaaggtgcc tgacatgagc 1140aagctcatgt tccacaccgc cgtcatgccc gatggaatgg cccttgcgta tgcggtatct 1200cattaattac ttattatcat cgcagtgact accgatgcaa ctgcttcaga tcctactgtt 1260ggaacgcgtg tggaaataat aaaggaataa taataattat tattgtaata aaa 1313671932DNAZea mays 67gtcgacccac gcgtccgagc accagcatct cttcaggtct ccaccaagcg cagacaccgc 60agcagcggca gcggcacgat ctggtgaccc ccccgccgcg tcaagcctgc tcctccggtg 120atcgccggac tggcggggta ggaaccagcg gagcgcagcc cgcctccttc cgctgtgtct 180gacagcagca gatcctcgat ggagatggat ggggttctgc aagccgcgga tgccaaggat 240tgggtttaca agggggaagg cgccgcgaat cttatcctca gctacaccgg ctcgtcgccc 300tccatgcttg gcaaggtact gcggctcaag aagattctaa aaaacaagtc gcagcgggca 360ccaagttgta ttgtattctc aagtcatgag caactcctgt ggggccatat cccagaactg 420gttgagtcgg tcaaacaaga ttgcttggct caagcctatg cagtgcatgt tatgagccaa 480cacctgggtg ccaatcatgt cgatggtggg gtccgtgtac gtgtttctag ggattttctg 540gagcttgtcg aaaagaatgt tcttagcagc cgtcctgctg ggagagtaaa tgcaagttca 600attgataaca ctgctgatgc cgctcttctg atagcagacc actctttatt ttctggcaat 660cctaagggta gcagctgcat agctgtagag ataaaggcca aatgtgggtt tctgccatca 720tcagaatata tatcagaaga taatactatc aagaaacaag taacgagata taagatgcat 780cagcacctca aattttatca gggtgagata tcgaagacta gtgagtacaa tcctcttgat 840ctattttctg ggtcaaaaga gagaatatgc atggccatca agtccctttt ctcaactcct 900cagaacaact taaggatttt tgtcaatgga tctttagctt ttggtggcat gggaggtggt 960gcagatagtg ttcatcctgc tgacactctt aagtgtcttg aagatctcag caagattagt 1020ggcctaaaac tccctgactt cactgagctc ctgtcagaga caatttttag gtctgaggta 1080ttaggcaacc tgttggccac tcaaaagttg gatgatcatg acattgaagg ggtaattcat 1140ctgtactaca acataatttc tcagccttgt ttagtctgca aaaacctaac tgatgtagag 1200ctattgcgga agtacacttt cttgcattct cttccgttgg acaaaagcct gaagatcgtt 1260agggacttcc tcatttctgc taccgcaaag gactgtagcc tgatgatcag ctttcggcca 1320agagagaatg gtagtacaga ttctgagtat gattcagtgt ttcttgaatc agtgaagcga 1380acctatgagt acaaggcata tttccttgat ctggatgtga aacctctgga taagatggag 1440cattatttta aactggatca gaggatagtc aatttctaca caagaaatgg gggaggtctt 1500gccatctcca aagggcagta ataccaaaga cacttcgagg atttatacat ctggagaagg 1560gtgcatcagg gagtgttggt tgttgttcct gctgcttggt gctgctgttg taacttcatg 1620agtacagtcc caaggttggg aggctcgacc cttaacgcct

ggaaagggca cagggagctg 1680tgttgtccgt cagtcgctgt tgtaactcaa actagtgcat acaccgtggc ttgtcacggt 1740aatttccgaa gatgtccaac gttagttgag acaaccgaac tgcttaccgt ggcaatcact 1800cattgtaaca tcaagttgaa aatgagggct gaagtttccc tcacaggcta ccatatgtca 1860gatatgtcct ttgtaccact aataagtgcc cctggggtca tgtatgaatg tatctcaatt 1920tgctattgca aa 1932681879DNAZea mays 68caaacgtacg tcgccgcagc agctcagacg tgcgccgcta ccacgtgtcc tgccgcacgc 60cccgcgtcag cggcatctgt aaagccgctt gtcgccgccc cgacgcccac cccgccccgc 120gcttttattc cccacttcac cgcatctccc cctcgtctac gatgccgttg cgcacctctt 180ctctctcgcc gccccgagac ccccacgctt ccctctccgc ccccgaactg tggcgcctcc 240ccccgccgcc gcagcgatgc cactcgcggc agagcccgac gacgctcatg aggaaaggga 300gaatcagcag ctgctaatta cgacgaaggg agggcccggg cttgagggac tggtggtggg 360gagctactgc cacgatgtgc taatccgggg cgggcgcata gtgggggaga ctctcggcgg 420ggctgcggcc ttcgtgtcca acgtgctcga cgccgcttcg ccccaggacg cggcgctcaa 480cgagacatcc ccctttgtcg ttgtggccaa ggtgggccac gacttcatct acgcccgcgc 540gccggcgtcc gcgcggcatc cgcctctgct ctgctcgtcc ccaaccacct ccttccacgc 600ccagttctcg gagaccgccg cctcggcgca cgcccccgac cgggagctcc ggcgcgtgcg 660cgcctgcgac ccgatctacc ccgccgacct tcccgaccgc cgcttcgcct atggcctcgc 720tgtcggcgtc gcgggggagg tgctaccgga gacgctcgag cagatgatca ggctctgccg 780cacggtgctc gtggacgcgc aggcgctgat ccgggcgttc gacggtgacg gcgccgtcgg 840tcacgtggcg cttgacgata ccccgtacgc gcggcttctg ccccgagtgg cgttcgttaa 900ggcgtcgtcg gaggaagcgc catacgttgg ggtggaaacg acgaggcggc agtgctgtgt 960gatcgtcacg gaggggaggg acgggtgccg gctgtactgg gacggtgggg aggcgcacgt 1020tgcgccgttc cccgccgtcc aggtggaccc tactggcgcc ggagatagct ttctcgcggg 1080ctttgcagcc ggattgctgt gggggttgtc ggccacggac gccgcgctgc tggggaactt 1140ctttggcgcc gctgctgtat cgcaggtcgg cgtgcccacc ttccatccca agatgttgca 1200ggcagttaaa gaaatacttg aagagaagac aaggaaacga tctagtccat gtatgaacgg 1260cgctagtttt accttggaga agtcaaatat gcacaacgag ttacacgcag ctctccaaga 1320agctgcggtg ctgatgtctg aacagcagca ggctgatccg gcgaacggca gtggcggtga 1380tatttgctcg gcataggtac ctcacagtga agctgaagca gtcagacgcc aaactgaaat 1440ttgtggcaaa aataaccagc actgcagtcc tgaactcctg atctcacatt gagatctgta 1500aacacggtgc caacaagtgg aggaagtttg tacatacgct ctctccggcc tttacactac 1560tattctgctg gcaaggccgt cagggatcgt ttctaccttg ctatcgctga cgaggaaatg 1620aagacaactg aacagttgag ctgtggcgct tgcacgcacc atgttttctc cgctgaacaa 1680gtgcgcattt ttgagctttc gggcattcgt gctgttaact ttttaccatt ctatatgtcg 1740acttctacca aaaggtctag cgttttaccc tgactgaaca cagggaaatt tgtgtgactg 1800aactgagaag ggccaacaca caagttagga tgtgtttggt tggatgtaca cggagggatg 1860aaatggggcg gccataaaa 18796941DNAArtificial Sequenceprimer 69atcgtcgacg cggccgctga gagaatttat cagtacagga t 417046DNAArtificial Sequenceprimer 70atggcggccg cctaggcgta cgttactgca gcagagctcg gcccag 4671556DNAGlycine max 71tgagagaatt tatcagtaca gccaaattcc tagtgaagca cccacagtta ttgaagatta 60tcgccctcca tcctcatggc ctgaaaatgg gacaattgaa ataattgatt tgaagattcg 120ttacaaggag aatcttcctt tggtgcttta tggagtaaca tgcacatttc ctggtggaaa 180gaagattgga atagtaggac gtactggcag tggaaaatct actttaattc aggcgttatt 240tcgattgatt gaaccaacaa gtgggagtat ccttatagac aacattaata tttcagagat 300tggccttcat gaccttcgaa gccatctcag tatcatacca caagatccaa ccttatttga 360aggtaccatt cgaggcaatc ttgatcctct ggatgagcac tcagataaag agatttggga 420ggcacttgat aagtctcagc ttggagaggt tatccgtgag aaaggacaac agcttgatac 480gccagttcta gaaaatggag ataattggag tgtaggacag cgacaacttg ttgctctggg 540ccgagctctg ctgcag 55672890DNAArtificial Sequencerecombinant DNA fragment 72cggtcctctc tctttccgtg gcatggcaat ctattgggct gtccagggtt gcatccttac 60tggtgtttgg gtcattgccc atgagtgtgg tcaccatgca ttcagtgact accagctgct 120tgatgatatt gttggcctta tcctccactc cgctctccta gtcccgtact tttcatggaa 180atacagccat cgccgtcacc actccaacac tggttctctt gagcgggatg aagtatttgt 240gccaaagcag aagtcctgta tcaagtggta ctctaaatac cttaacaatc ctccaggcag 300agtcctcact cttgctgtca ccctcacact tggttggccc ttgtacttgg ctttaaatgt 360ttctggaagg ccttatgata gatttgcttg ccactatgac ccatatggtc ccatttactc 420tgatcgtgaa cgacttcaaa tatatatatc agatgcagga gtacttgcag gacttactct 480ctctaccgtg ttgcaaccct gaaagggttg gtttggctgc tatgtgttta tggggtgcct 540ttgctcattg tgaacggttt tcttgtgact atcacatatt tgcagcacac acactttgcc 600ttgcctcatt acgattcatc agaatgggac tggctgaagg gagctttggc aactatggac 660agagattatg ggattctgaa caaggtgttt catcacataa ctgatactca tgtggctcac 720catctcttct ctacaatgcc acattaccat gcaatggagg caaccaatgc aatcaagcca 780atattgggtg agtactacca atttgatgac acaccatttt acaaggcact gtggagagaa 840gcgagagagt gcctctatgt ggagccagat gaaggaacat ccgagaaggg 8907328DNAArtificial Sequenceoligonucleotide primer 73gcggccgccg gtcctctctc tttccgtg 287430DNAArtificial Sequenceprimer 74tagagagagt aagtcctgca agtactcctg 307530DNAArtificial Sequenceprimer 75caggagtact tgcaggactt actctctcta 307629DNAArtificial Sequenceprimer 76gcggccggcc ccttctcgga tgttccttc 297739DNAArtificial Sequenceprimer 77gcggccgcgt acgtgacggt cctctctctt tccgtggca 397837DNAArtificial Sequenceprimer 78gcggccgcct aggtcacttc tcggatgttc cttcatc 3779921DNAArtificial Sequencespacer sequence 79gcggccgcgt acgtgacggt cctctctctt tccgtggcat ggccaatcta ttgggctgtc 60cagggttgca tccttactgg tgtttgggtc attgcccatg agtgtggtca ccatgcattc 120agtgactacc agctgcttga tgatattgtt ggccttatcc tccactccgc tctcctagtc 180ccgtactttt catggaaata cagccatcgc cgtcaccact ccaacacagg ttctcttgag 240cgagatgaag tatttgtgcc aaagcagaag tccagtatca tgtggtactc taaatacctt 300aacaatccac caggcagagt cctcactctt gccgtcaccc tcacgcttgg ttggcccttg 360tacttggctt ttaatgtttc tggaaggcct tatgatagat ttgcttgcca ctatgaccct 420tatggtccca tttactctga ccgagaacga cttcaaatat atatatcaga tgcaggagta 480cttgcaggac ttactctctc taccgtgttg caaccctgaa agggttggtt tggctgctat 540gtgtttatgg ggtgcctttg ctcattgtga acggttttct tgtgactatc acatatttgc 600agcacacaca ctttgccttg cctcattacg attcatcaga atgggactgg ctgaagggag 660ctttggcaac tatggacaga gattatggga ttctgaacaa ggtgtttcat cacataactg 720atactcatgt ggctcaccat ctcttctcta caatgccaca ttaccatgca atggaggcaa 780ccaatgcaat caagccaata ttgggtgagt actaccaatt tgatgacaca ccattttaca 840aggcactgtg gagagaagcg agagagtgcc tctatgtgga gccagatgaa ggaacatccg 900agaagtgacc taggcggccg c 921

Claims

1. An isolated nucleic acid molecule comprising a nucleotide sequence that encodes a polypeptide that modulates the level of phytate in a plant, wherein the nucleotide sequence is selected from the group consisting of: a) a nucleotide sequence which has at least 90% sequence identity to the sequence set forth in nucleotides 244-4776 of SEQ ID NO: 2; b) a nucleotide sequence which encodes a polypeptide having an amino acid sequence that shares at least 93% sequence identity with the amino acid sequence set forth in SEQ ID NO: 3 or 5; c) a nucleotide sequence which has at least 90% sequence identity to the sequence set forth in nucleotides 245-4762 of SEQ ID NO: 6; d) a nucleotide sequence which encodes a polypeptide having an amino acid sequence that shares at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 7; e) a nucleotide sequence which has at least 90% sequence identity to the sequence set forth in nucleotides 3-1350 of SEQ ID NO: 10; f) a nucleotide sequence which encodes a polypeptide having an amino acid sequence that shares at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 11; g) a nucleotide sequence which has at least 90% sequence identity to the sequence set forth in nucleotides 1-465 of SEQ ID NO: 12; h) a nucleotide sequence which encodes a polypeptide having an amino acid sequence that shares at least 90% sequence identity with the amino acid sequence set forth in SEQ ID NO: 13; i) a nucleotide sequence which has at least 90% sequence identity to the sequence set forth in SEQ ID NO: 71; and j) a nucleotide sequence which encodes a polypeptide having an amino acid sequence that comprises the sequence set forth in SEQ ID NO: 15.

2. The nucleic acid molecule of claim 1, wherein said nucleotide sequence encodes a polypeptide comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3.

3. The nucleic acid molecule of claim 2, wherein said nucleotide sequence encodes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 3.

4. An expression cassette comprising the nucleic acid molecule of claim 1, wherein said nucleotide sequence is operably linked to a promoter that drives expression in a microorganism or in a plant cell.

5. An isolated polypeptide comprising an amino acid sequence which has at least 93% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, wherein said polypeptide modulates the level of phytate in a plant.

6. An expression cassette comprising a first nucleotide sequence selected from the group consisting of: a) a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence comprising at least 50 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; b) a nucleotide sequence comprising at least 19 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; c) a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and d) a nucleotide sequence which is the complement of a), b), or c).

7. A method for producing food or feed with a reduced level of phytate, said method comprising: a) transforming a plant with a nucleic acid molecule comprising a first nucleotide sequence selected from the group consisting of: i) a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence comprising at least 50 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; ii) a nucleotide sequence comprising at least 19 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; iii) a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and iv) a nucleotide sequence which is the complement of i), ii), or iii); b) growing said plant under conditions in which said nucleotide sequence is expressed; and c) producing food or feed from said plant, wherein said plant has a reduced level of phytate in comparison to a control plant.

8. The method of claim 7, wherein said first nucleotide sequence has at least 95% sequence identity to the nucleotide sequence set forth in nucleotides 244-4776 of SEQ ID NO: 2.

9. The method of claim 7, wherein said plant is further transformed with a nucleic acid molecule comprising a second nucleotide sequence selected from the group consisting of: a) a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence comprising at least 50 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; b) a nucleotide sequence comprising at least 19 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; c) a nucleotide sequence encoding an amino acid sequence that has at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 3, 5, 7, 9, 11, 13, or 15; and d) a nucleotide sequence which is the complement of i), ii), or iii); wherein said plant has a reduced level of phytate in comparison to a control plant.

10. The method of claim 7, wherein said plant is further transformed with a nucleic acid molecule comprising a second nucleotide sequence selected from the group consisting of: a) an mi1ps nucleotide sequence; b) an IPPK nucleotide sequence; c) an ITPK-5 nucleotide sequence; d) an IP2K nucleotide sequence; e) an MIK nucleotide sequence; f) a phytase nucleotide sequence; g) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 25, 64, 65, 67, or 68; h) a nucleotide sequence comprising at least 19 nucleotides of the sequence set forth in SEQ ID NO: 25, 64, 65, 67, or 68; i) a nucleotide sequence which is the complement of (a), (b), (c), (d), (e), (g), or (h); and j) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 66.

11. The method of claim 7, wherein said plant is further transformed with a nucleic acid molecule comprising a second nucleotide sequence conferring a trait of interest on said transformed plant.

12. The method of claim 11, wherein said trait of interest is selected from the group consisting of: a) high oil; b) increased digestibility; c) high energy; d) balanced amino acid; e) high oleic acid; f) insect resistance; g) disease resistance; h) herbicide resistance; i) drought tolerance; and j) male sterility.

13. A transformed plant comprising in its genome at least one stably incorporated nucleic acid molecule having a first nucleotide sequence selected from the group consisting of: a) a nucleotide sequence having at least 90% sequence identity to a nucleotide sequence comprising at least 50 contiguous nucleotides of the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; b) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; c) a nucleotide sequence comprising at least 19 nucleotides of the sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, 71, or 14; and d) a nucleotide sequence which is the complement of a), b), or c); wherein said plant has a reduced level of phytate compared to a control plant.

14. The transformed plant of claim 13, wherein said plant is further transformed with a nucleic acid molecule comprising a second nucleotide sequence selected from the group consisting of: a) an mi1ps nucleotide sequence; b) an IPPK nucleotide sequence; c) an ITPK-5 nucleotide sequence; d) an IP2K nucleotide sequence; e) an MIK nucleotide sequence; f) a phytase nucleotide sequence; g) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 25, 64, 65, 67, or 68; h) a nucleotide sequence comprising at least 19 nucleotides of the sequence set forth in SEQ ID NO: 25, 64, 65, 67, or 68; i) a nucleotide sequence which is the complement of (a), (b), (c), (d), (e), (g) or (h); and j) a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 66.

15. The transformed plant of claim 13, wherein said plant is further transformed with a nucleic acid molecule comprising at least one second nucleotide sequence that confers at least one trait of interest on said transformed plant.

16. The transformed plant of claim 15, wherein said trait of interest is selected from the group consisting of: a) high oil; b) increased digestibility; c) high energy; d) balanced amino acid composition; e) high oleic acid; f) insect resistance; g) disease resistance; h) herbicide resistance; i) drought tolerance; and j) male sterility.

17. Transformed seed of the plant of claim 13, wherein said seed comprises said first nucleotide sequence.

18. Food or feed comprising the plant of claim 13.

19. Food or feed comprising the transformed seed of claim 13.

20. The transformed plant of claim 13, wherein said plant is a monocot.

21. A method for producing food or feed with a reduced level of phytate, said method comprising the steps of: (a) transforming a plant cell with at least one first polynucleotide comprising at least 19 nucleotides of the sequence set forth in SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, or 71; (b) transforming a plant cell with at least one second polynucleotide having at least 94% sequence identity to the complement of the polynucleotide of step (a); (c) regenerating a transformed plant from said plant cell; and (d) producing food or feed from said transformed plant or from seed of said transformed plant; wherein the level of phytate in said plant is reduced in comparison to a control plant.

22. A plant containing an Lpa1 insertion mutation comprising a Mu element, wherein said Mu element is inserted in the Lpa1 gene in a location selected from the group consisting of: a) in exon 1; b) at nucleotide 585 in exon 1; c) at nucleotide 874 in exon 1; d) in exon 11; and e) at nucleotide 6069 in exon 11.

Images