TW200413403A - Plant MYB proteins - Google Patents

Abstract

A pure polypeptide containing an amino acid sequence at least 70% identical to SEQ ID NO: 7, 8, or 9. The polypeptide regulates expression of a gene in a cell. Also disclosed is an isolated nucleic acid characterized in that it hybridizes under stringent conditions to SEQ ID NO: 1, 2, or 3, or a complementary sequence thereof. Also within the scope of the invention are a transformed cell or a transgenic plant containing such a nucleic acid.

Description

200413403 (1) Description of the invention: [Technical Field] The present invention relates to a MYB protein and its isolated nucleic acid, and a transformed cell or a gene-transplanted plant containing the nucleic acid. [Prior art] MYB protein is a group of transcription factors. Cell myb is an oncogene (c-myb) involved in the proliferation and / or differentiation of hematopoietic cells (0 ^ € (1992) (^ 111 ·! ·· Op. Gen. Devel. 2, 249-255). All MYB genes The common feature of the products is the highly-reserved DNA-binding domain at the N-terminus. In animals, the DNA-binding domain is composed of three incomplete repeating units, each unit having about 51 to 52 Amino acids (named R1, R2, and! 13), which have three highly retained tryptophan residues separated by 18 or 19 amino-acids (Weston (1999) Curr. Op. Gene DeveL 8, 76-81). In plants and yeast, the main MYB protein has two repeating units (R2 and R3) (Martin and Paz-Ares (1997) Trends Genet. 13, 67-73; Jin and Martin (1999) Plant Mol. Biol. 41, 577-585. However, MYB proteins containing only one repeat unit or three repeat units have also been identified in plants (Baranowskij, et al. (1994 ) EMBO J 13, 5383-5392; Kirik and Baumlein (1996) Gene 183, 109-1 13; Feldbriigge, et al. (1997) Plant J. 11? 1079-1093; Wang, et al. (1997) Plant Ce ll 9,491-507; Braun and Grotewold (1999) Plant Physiol. 121, 21-24) 〇 [Content of the invention]

O: \ 85 \ 85981.DOC 200413403 The present invention is based on three novel rice genes encoding MYB proteins, which are named OsMYBS1, OsMYBS2, and OsMYBS3, respectively. These proteins bind to promoters containing one or more sets of TACCC A sequences and can regulate promoter activity. The full-length OsMYBSl, OsMYBS2 and OsMYBS3 cDNAs (named SEQ ID NOs: 1 to 3 respectively) are shown below, where the start codon

And the stop codon is underlined. OsMYBSl cDNA (SEQ ID NO: 1):

GTGCGAGATCCACCACCCGATGACCTCCCAGGCGGCGACGACGACGACCACGGCGGCGGCGGCGGCGGCGT

GGACCAGGGAGGACGACAAGGCGTTCGAGAACGCGCTCGCGGCTTGCGCGGCGCCGCCGCCCGCGGAGAGAGA

GGCGCGCCCGACGACGACTGGTTCGCCGCGCTCGCCGCGAGCGTGCCCGGGGCGAGGTCGGCGGAGGAGGT

GCGGAGGCACTACGAGGCGCTGGTGGAGGACGTCGCGGCCATCGACGCGGGCCGCGTCCCGCTCCCGCGCT

ACGCCGGGGAGGAGTCCGCGGCGCCGCCCGACGGAGCCGGAGCCGCCGCCGCCGCGTCCAAGGACGGCGGA

CACCGGCGCGACGAGCGCAAGGGCGGCGGCGGCGGGTACGACGGCGGCAAGAGCTGCTCCAAGGCGGAGCA

GGAGAGGCGCAAGGGCATCCCATGGACGGAGGAAGAGCACAGGCTGTTCTTGCTGGGGCTGGACAAGTTCG

GCAAGGGGGACTGGCGGAGCATCTCGCGCAACTTCGTCATCTCGiCGGACGCCAACGCAGGTGGCGAGCCAC

GCGCAGAAGTACTTCATCCGCCTCAACTCCATGAACCGCGACCGCCGCCGCTCCAGCATCCACGACATCAC

CAGCGTCACCGCCGGCGATCAGGTCGCCGCGCAGCAGGGCGCCCCGATCACCGGCCACCAGGCCACGGGCA

ACCCCGCGGCGGCGGCGCTGGGCCCGCCGGGCATGAAGCACCACCACCACCACCACCCGGGCGGCGCGCCG

CCGCCCATGCCCATGTACAGCGCCGCGCCCATGGGCCACCCCGTCGCCGGCCACATGGTGCCCGCCGCCGT

CGGCACGCCGGTGGTGTTCCCGCCGGGCCACGCGCCGTACGTCGTGCCCGTCGGCTACCCGGCGCCTCCGG

CCAAGATGCACCA ^ .TGACGCGCCATGGACGGACATGAGCAGCATTTCTTCCTCCTCCTTTCTTGATGTCAA

TCTTGATTTGTTCTTTGTGTAGTCGCCGGCTCATCGTCCCTGATCATCTTGTTCTTCACAATCTCACTA

ATGTAAACATACATAGATCAGATGCCAAGAGTGCAGGGATTGGGGATTAAAGGCGAATAAGTAAAGTATTT

TGCTGACTGTTTGC ^ GTGATCATCACGTACACCCGGTGA ^ AGCTTAGCTCCAAATGTGGATGTAATTAGC

AGCGC-CCTTCCGTACGTGGTGGCGCCGATCGATGATCTTGCAGGGGTTGCAATTAGGGATTGATTTCCATT

TTGCTGATGTAAATTTGCCAACTGTCTCATTGGACCAAAAAAAAAAAAAAAA O: \ 85 \ 85981.DOC -6- 200413403

OsMYBS2 cDNA (SEQ ID NO: 2):

CGAGGTCCGCGGCGGCGGCGGCGGAGTTGACGAGGAGGAGTACGAGGAGGAGGAGGTGGAGGGTGGATTGTTCATCA

AGAAGAGCTCCAGTATGCCCAACCTCACCTCCATCGACCCGCTGCCGGTGCCGGCCGACGGCGGCAAACGGCGCGCC

TCCGACGACTCCGAGCTCGCCTCCGGCCAGCAGAAGCGCCGCCGCCGCAAGGTGCAGGAGAGGAAGAAAGGGGTACC

ATGGACTGAGGAGGAGCACAAGAAATTCCTGGAAGGGCTGAGGCAGCTGGGGAAAGGGGACTGGAGGCATCTCCA

AGAACTTTGTGACCAGCAGGACGGCGACTCAGGTGGCCAGCCACGCCCAGAAGTACTTCCTCCGGCAGACCAACCCT

GGCAAAAAGAAGCGCCGGGCCAGCCTCTTTGATGTTGTTGCTGAGTGCAGTGATGATCAACTTCCAAGTCCTCAGAG

TGTTGGAACTAAGCCTCCTACCCAGGATATAATTCATACAGATCGCGGCGATGTCCCGATACTAAGCTATCCAGTTG

CTAGAGGCTTTAGAGGCGATAGCGTGCAGGTTGATGAACTAACTGAATATGTGAAGAGATTAAAGGCCGCCGAGGAC

ATGTCGCTCTCCATCATTCTCTGGACTGGAAATGGCATCATCATCCATCAGCAGTCTAGAGCTCAGTATCGCGCCCTC

TCATTTGCGGATCGACGGGGCCATCAAGGGGCTGGGATCCAAACCCAATTTTCCCCCGAAGGAATTTGGATCGGCTT CAGCTACTGTTTTTTGTCCCCTGTTGTTGTTTTTGTTGTTGTTGTTTTTTTTTTTTTTTTTTTTGCGGGGGTTGTTTG ^

TGTTGTTGTTGTTGTAGTTGTCATGCTAACTTTGTATTTGGGTCATGTGGGGTTTCTTTCACCAGTTTTATATAATA

CAGAGAGAATGTCAGTCCCTTCCGAGACATGTTTAAAAAAAAAAAAAAAWU ^ AAAAAAAAAAAAAAAAAAAAAAAAA

OsMYBS3 cDNA (SEQ ID NO ... 3) ...

ATCGATCGATCGATCTCCATAGGTGGGGGAAGGGAAGCTTTGGAAGGTGGAGGGACGGAGGGGGGGATGACGAGGCG

GTGCTCGCACTGCAGCCACAACGGGCACAACTCGCGGACGTGCCCCAACCGCGGGGTCAAGATCTTCGGGGTGCGCC

TCACCGATGGCTCCATCCGCAAGAGCGCCAGCATGGGGAACCTCTCCCTGCTCTCCTCCGCCGCCGGATCCACCAGC

GGCGGCGCCTCCCCCCCGCCGACGGCCCCGACGCCGCCCCCACCGCCGCCGACGGCTACGCCTCCGACGACTTCGTCCA

GGGCTTCTCCTCCGCCACCCGCGACCGCAAGAAGGGTGTTCCTTGGACTGAAGAAGAACACCGGAGGTTTTTGCTTG

GATTGCAAAAGCTTGGCAAAGGTGATTGGCGAGGAATCTCTCGTAATTTCGTGGTCTCAAGAACACCTACTCAAGTA

GCCAGTCATGCTCAGAAATATTTTATACGCCAATCCAATATGACCAGAAGGAAAAGAAGGTCTAGCCTTTTTGACAT

GGTGCCAGATGAGTCTATGGACCTTCCACCACTTCCTGGAGGTCAAGAACCAGAGACCCAAGTATTAAATCAACCAG

CACTACCTCCACCGAAGGAGGAAGAGGAGGTAGATTCTATGGAGTCAGATACTTCTGCCGTTGCAGAGAGCTCTTCC

GCTTCTGCTATCATGCCAGATAATTTGCAGTCGACCTATCCAGTGATTGTTCCAGCTTATTTCTCGCCCTTTTTGCA attctcggttcctttctggcaaaatcagaaagatgaagatggtcctgtgcaagaaacacatgagattgtcaagcctg

TTCCAGTTCATTCAAAGAGCCCAATCAACGTTGATGAGCTTGTTGGCATGTCGAAGCTCAGCATAGGAGAGTCCAAT

CAAGAGACAGAGTCTACTTCTCTTTCATTAAATCTGGTAGGAGGTCAAAATAGACAATCAGCTTTCCATGCAAATCC

ACCAACAAGGGCACAGGCATGATCTGGTTGTGCACACAACTGCATTTAGATGAATCCCAGGCAAAATAAGCTTTGCC

TCCTTGTTTTTTTTTTTTTATTTTAAGATTAACCGTTCTCCGTAGTCTGTATCATGTGCTGTAAGTTATGCTATGTA

TGAATGTATCTGTTGTTTGTCTGGCACACATGATAAATCACTCTATGTTAACAAAATCAGTAATGGTAGTGCTGATC

TTCGTGGTTGTACTGTTGTAAACTCTTTTATAAGAAAAAAAAATATTAGTTAGTC

O: \ 8S \ 85981.DOC 200413403 Nucleic acid sequences encoding OsMYBSl, OsMYBS2 and OsMYBS3 proteins (ie, the codons from the ATG start codon to the stop codon in SEQ ID NOS: 1 to 3) are named as SEQ ID NOS: 4 to 6. The OsMYBS1, OsMYBS2 and OsMYBS3 proteins encoded by the above cDNAs (separately designated as SEQ ID NOS: 7 to 9) are as follows:

OsMYBSl protein (SEQ ID NO ·· 7):

MTS QAATTTTTAAAAAAWTREDDKAFENAIJUVCAAP P P ADGGAPDDDWFAALAAS VPGARS AEEVRRHYEAL VED VA AID AGRVPLPRYAGE three S AAP PDGAGAAAAAS kDGGHRRDERKGGGGGYDGGK ^ CS KAEQERRKGIPWTEEEHRL FLL · • ISLDKFGICGDWRSISRliFVISRTPTQVASHAQKYFIRLNSMNRDRRRSSIHDITSVTAGDQVAAQQGAPITGHQATGN PAAAALGPPGMKHHHHHHPGGAPPPMPMYSAAPMGHPVAGHMVPAAVGTPWFPPGHAPYWPVGYPAPPAXMHQ

OsMYBS2 protein (SEQ ID NO 8):

MPNLTSIDPLPVPADGGKRRASDDSELASGQQKRRRRKVQERKKGVPWTEEEHKKFLEGLRQLGKGDWRGISKNFVT

SRTATQVASHAQKYFL'RQTNPGKKKRRASLFDWAECSDDQLPSPQSVGTKPPTQDIIHTDRGDVPILSYPVARGFR

GDSVQVDELTEYVKRLKAAEDMSLSMISGLEMASSSISSLELSIAPSHLRIDGAIKGI ^ GSKPNFPPKEFGSASATVF

CPPCCCLLLLFFFFFFAGWCCCCCCSCHANFVFGSCGVSFTSFI

OsMYBS3 protein (SEQ ID NO 9):

MTRRCSHCSHNGHNSRTCPNRGVKIFGVRLTDGS IRKS ASMGNLS LLS S AAGSTSGGAS PADGPDAAPTAADGYASD DFVQGFSSATRDRKKGVPWTEEEHRRFLLGLQKLGKGDWRGISRNFWSRTPTQVASHAQKYFIRQSNMTRRKRRSS LFDMVPDESMDLPPLPGGQEPETQVLNQPALPPPKEEEEVDSMESDTSAVASSSSASAIMPDNLQSTYPVIVPAYFS PFLQFSVPFWQNQKDEDGPVQETHEIVKPV VKSKSPINVDELVGMSKLSIGESNQETESTSLSIiNLVGGQ_SAF HANPPTRAQA Accordingly, the present invention is characterized in that a pure polypeptide comprising the SEQ ID NO:? 7,8 9 or at least 70% (e.g., at least 75,80,85, 90 or 95, or 100%) amino acid sequences. When expressed in a cell, the polypeptide binds to a promoter and regulates transcription controlled by the promoter. The cell may be a plant cell, especially a monocotyledonous plant cell (e.g., a cereal plant cell, such as a rice cell or a barley cell). The promoter may contain a

O: \ 8S \ 85981.DOC 200413403 One or more sets of TATCCA sequences or variants thereof. Furthermore, HvMYBGa protein can be co-expressed in cells. HvMYBGa interacts with OsMYBs proteins and cooperates to regulate gene performance. The polypeptides of the present invention can be used to make OsMYBS antibodies (single or multiple antibodies). These antibodies can eventually be used to detect the presence and distribution of OsMYBS proteins in tissues and cell compartments. For example, these antibodies can be used to demonstrate the performance of OsMYBS protein in transgenic plants. A "pure polypeptide" means a polypeptide that is substantially free of naturally-bound molecules, that is, on a dry weight basis, that is at least 75% pure (eg, at least 80, 85, 90, 95, or 100% pure). Purity can be determined by any suitable standard method, for example, by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. The "percent identity" of the two amino acid sequences was determined using the algorithm of Karlin and Altschul ((1990) Proc. Natl. Acad. Sci. USA. 87, 2264-2268), which was tested by Karlin and Altschul (( 1993) Proc. Natl. Acad. Sci. USA 90, 5873_5 877). This algorithm was incorporated into the XBLAST program of Altschul et al. ((1990) J. Mol. Biol. 215, 403-4 10). The BLAST protein search was performed using the XBLAS Ding program, where score = 50 and wordlength = 3. When there is a gap between the two sequences, Gapped BLAST as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402 is used. When BLAST and Gapped BLAST programs are used, individual programs (for example, XBLAST ). See www.ncbi.nlm.nih.gov. The invention includes isolated nucleic acids (e.g., vectors) encoding a polypeptide of the invention, as well as cells (in culture or genetically transformed) containing a nucleic acid of the invention

O: \ 85 \ 85981.DOC 200413403 plant). The cell may be a plant cell, especially a monocotyledonous plant cell (e.g., a '榖 plant cell, such as a rice cell or a barley cell). Examples of nucleic acids within the present invention include nucleic acids characterized by being able to hybridize to SEQ m NO: 1, 2 or 3 or its complementary sequence under severe conditions. The nucleic acid can be at least 15 (e.g., at least 30, 50, 100, 2000, 5000, or 10,000) nucleotides in length. These nucleic acids and cells can be used to produce transgenic plants or to produce polypeptides of the invention. For example, the nucleic acid of the present invention can be used to determine whether the mRNA of OsMYBS is expressed in tissues or cells. This nucleic acid can be used as a primer in a PCR detection method, or as a labeled probe in a nucleic acid spotting method (for example, the northern spotting method). "Isolated nucleic acid" is a nucleic acid having a structure different from that of any naturally occurring nucleic acid or the structure of any fragment of a naturally occurring genomic nucleic acid. This term covers, for example, (a) DNA with a partial sequence of a naturally occurring dn A molecule, but not on both sides of which is a coding sequence located on both sides of the molecule in the naturally occurring genome; (b) by Nucleic acid incorporated into a vector or genomic DNA of a prokaryote or eukaryote, and incorporated in such a way that the resulting molecule is not the same as any naturally occurring vector or genomic DNA; (c) an individual knife, i.e., cDNA, genomic fragments, fragments or restriction fragments produced by polymerase chain reaction (pcR), and (d) the recombinant nucleus of a part of a hybrid gene (ie, a gene encoding a fusion protein): y: acid sequence. Under "stringent conditions" heterozygous means heterozygous at 65 ° C, 0.5 X SSC, and then washed with 0.1 X SSC at 45 ° C. The plant cells of the invention can be cultured to produce transgenic plants. The gene transgenic plant may be a monocotyledonous plant, for example, a plant of the tribe type, such as rice O: \ 85 \ 8598l.DOC -10-200413403 or barley. The genome of the gene-transplanted plant contains a transgene, which includes a nucleic acid characterized by being capable of hybridizing to SEQ ID NO ·· 1, 2 or 3 or a complementary sequence thereof under severe conditions. This transgene, when expressed in a cell, will activate the activity of a promoter (for example, one containing one or more sets of TA but ccA sequences or variants thereof). In addition, the invention includes a method for expressing a transcript in a cell (e.g., a plant cell). The method includes introducing a vector containing a nucleic acid encoding a transcript into a cell, and causing the transcript to be expressed in the cell. The transcript is specifically intended to hybridize to SEq ID No. 1, 2, or 3 or its complementary sequence under severe conditions. The transcript may encode a polypeptide of the present invention, and its promoter (for example, one containing one or more sets of TATCCA sequences or variants thereof) is inactive. On the other hand, the transcript may be antisense RNA, which can suppress the expression of the endogenous OsMYBS gene, thereby deactivating the activity of the promoter (for example, one containing one or more sets of TATCCA sequences or variants thereof). The present invention provides a system for highly regulated expression of endogenous or exogenous genes in plant cells or transgenic plants. Some specific embodiments of the present invention are described in the accompanying description below. The remaining features, objects and advantages of the present invention will become clearer through detailed description and patent application scope. [Embodiment] In tadpoles, the expression of starch hydrolase gene is induced by gabein (ga) and sugar starvation. All j _ amylolytic enzyme genes isolated from tadpoles contain TATCCA units or their variants at positions approximately 90 to 150 b ρ upstream of the transcription initiation position. tatcca unit is

O: \ 85 \ 85981.DOC 200413403 The GA-response complex (GARC) and sugar response complex (SRC) are important components of the α-amylase enzyme promoter. Three pure cDNA lines encoding a novel MYB protein and having a single DNA binding region have been isolated from the rice suspension cell cDNA library and named OsMYBS1, OsMYBS2, and OsMYBS3. Unexpectedly, three OsMYBS proteins specifically bind to the TATCCA unit in vitro. In vivo, OsMYBS1 and OsMYBS2 are combined with TATCCA units, and when sugar is provided, they can activate a promoter containing TATCCA units, while OsMYBS3 suppresses the transcription of the promoter when sugar is deficient. Furthermore, the three OsMYBS proteins cooperate with a GA-regulated transcription factor GAMyb (HvMYBGa) to activate the low-pi barley α-amylase enzyme promoter in the absence of GA. In one aspect, the invention relates to a pure OsMYBS polypeptide (eg, SEQ ID NS: 7 to 9), which includes a functional OsMYBS polypeptide. A "functional peptide" refers to a peptide having the same biological activity as the wild-type OsMYB protein, for example, a fragment of the wild-type OsMYBS protein. In another aspect, the present invention relates to a single isolated OsMYBS nucleic acid (ie, DNA, cDNA and RNA), which is characterized in that it can hybridize with SEQ ID NO: 1, 2 or 3 or its complementary sequence under severe conditions. Nucleic acids of the invention include degenerate sequences derived from the codebook. The nucleic acid of the present invention can be expressed in vitro by transferring the DNA into an appropriate host cell by a method known in the art. For example, the nucleic acid can be inserted into a recombinant expression vector. Nucleic acids of the invention can be expressed using a variety of host-expression vector systems. These include (but are not limited to) microorganisms, such as bacteria transformed with the expression vectors of phage-, plastid- or plastid-type DNA; O: \ 85 \ 85981.DOC -12- 200413403; recombinant yeast Yeast transformed with a fungal expression vector; and plant cells infected with a recombinant viral expression vector (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with a recombinant plastid expression vector (eg, Ti plastid) system. Isolation and purification of the recombinant polypeptide or fragment thereof provided by the present invention can be performed by conventional methods, including column chromatography for preparation and immunoseparation methods involving single or multiple antibodies. The invention also provides an antibody against OsMYBS polypeptide, which includes a monoclonal antibody and a multiple antibody. The term "antibody" includes intact molecules and fragments thereof, such as Fab, F (ab ') 2, and Fv capable of binding to epitopes present in the OsMYBS polypeptide. Methods for making single and multiple antibodies and fragments thereof are known in the art. See, for example, Harlow and Lane (1988)

Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Within the scope of the present invention are also gene transgenic plants, the genome of which contains transgenic genes, and the transgenic genes include SEQ ID NOs under severe conditions: Nucleic acids hybridized with 1, 2, or 3 or their complementary sequences. The transgenic gene, when expressed in a cell, can regulate the activity of a promoter (for example, one containing one or more sets of TATCCA sequence 歹 ij or a variant thereof). As used herein, the term "plant" refers to a whole plant, plant part, plant cell, or group of plant cells, such as plant tissue. Young plants are also included in the meaning of "plants". The plants included in the present invention are any plants that can be modified by transformation techniques, and include angiosperms, gymnosperms, monocots and dicots. Examples of monocotyledons include (but are not limited to) O: \ 85 \ 85981.DOC -13- 200413403 Asparagus, field corn and sweet corn, barley, wheat, rice, sorghum, spring onion, pearl cormorant, rye and oats . Examples of dicotyledons include, but are not limited to, potatoes, tomatoes, tobacco, cotton, rapeseed, beans, soybeans, peppers, lettuce, peas, alfalfa, alfalfa, mustard crops or kale vegetables (eg, cabbage, Broccoli, broccoli, brussels sprouts), radishes, carrots, beets, eggplant, spinach, cucumber, pumpkin, melon, cantaloupe, sunflower and various decorative plants. Woody plants include poplar, pine, North American red (sequoia), red pine (cedar), and oak. Transgenic plants can be produced by methods known in the art. See, for example, Weissbach and Weissbach (1988) Methods plant Molecular Biology, Academic Press, NY, Section Vin 5 PP 421-463; Grierson and Corey (1988) Plant Molecular Biology 2d Ed., Blackie, London, Ch. 7-9; Horsch, et aL (1985)

Science 227, 1229 In addition, the present invention provides a method for expressing a transcript in a cell (e.g., a plant cell). The method includes introducing a vector containing a nucleic acid encoding a transcript into a cell, and causing the transcript to be expressed in the cell. The transcript is specifically intended to be hybridizable to SEQ ID NO: 1, 2 or 3 or its complementary sequence under severe conditions. The transcript can be an RNA that can interfere with the function of another RNA in a plant cell (for example, to prevent mRNA from being translated into a polypeptide, or to stimulate the degradation of a specific RNA in order to stop the target gene from being post-transcribed) iRNA (Moletal (1990) FEBS Leu 268 (2), 427-30; and Fire et al. (1998) Nature 391, 806-81 1). In addition, the transcript may encode a polypeptide such that the polypeptide is overexpressed in a transformed plant cell or a transgenic plant. in

O: \ 85 \ 85981.DOC -14- 200413403

Changes in the performance of OsMYBS result in changes in the performance of genes that are regulated by the 0sMYBS protein (e.g., 'by a promoter containing one or more sets of TATCCA sequences or variants thereof). The following specific examples are merely illustrative of the present invention and are not intended to limit the rest of the disclosure in any way. Those skilled in the art can make full use of the present invention without extra effort based on the description herein. All publications cited herein are incorporated herein by reference in their entirety. Materials and methods 1. Rice cell culture A suspension cell culture of rice (Oryza sativa cv. Tainan 5) was established as previously described (Yu, et al (1991) J. Biol. Chem. 266, 21131-21137). Approximately 0.5 ml of cells were transferred every 7 days into 25 ml of fresh liquid Murashige and Skoog medium (Murashige and Skoog (1962) Physiol · Plant 15, 473-497) in a 125 ml culture flask containing 3% sucrose, For subculture. Cells were cultured on a reciprocating shaker set at 120 rpm and incubated at 26 ° C in the dark. 2. Plastid plastid a Amy8-C carries a 1.4 kb rice alpha-amylase cdNA insert from pBluescript KS + (Stratagene) (Yu, et al. (1992) Gene 122, 247-253). Plastid pcRAcl.3 contained in

1-4 kb rice actin gene (Actl) cDNA insert in pBluescriptll-KS (McElroy, et al. (1990) Plant Mol. Biol. 15, 257-268). The plastid PRY18 carries a 3.8 kb DNA fragment containing the rice genomic rDNA cluster. The cluster includes the 18S rRNA O: \ 85 \ 85981.DOC in PUC 13 and the third half of the 200413403 gene, a complete 5.8S. The 5 'half of the rRNA gene and 25S rRNA gene (Sano and Sano (1 990) Genome 3 3, 209-218). The plastid pGAMyb contains the HvMYBGa cDNA (Cei * c0s, et al. (1999) Plant J. 19, 107-118) fused between the maize ubiquitin (Ubi) promoter and the nopaline synthase (Nos) terminator. Plastid pAHC 18 contains luciferase fused between Ubi promoter and Nos terminator

(Luc) cDNA (Bruce, et al. (1989) Proc. Natl. Acad. Sci. USA 86, 9692-9696). Plastid pAmy32b-GUS contains a 331-bp promoter region, a complete 5 'untranslated sequence, the first intron of Amy3 2b fused to the GUS coding sequence, and the 3' untranslated region of Amy32b (Gomez- Gadenas, et al. (1999) Proc. Natl. Acad. Sci. USA 96, 1767-1772). 3.South-West Screening of cDNA Library

The rice suspension cells were cultured in a medium containing sucrose for 5 days, and then transferred into a medium containing sucrose (+ S) or non-sucrose (-S) for 4 hours. Cells were collected and all RNA was purified. Poly (A) + RNA was further purified using an oligo (dT) cellulose spin column (5 Prime- > 3 Prime). Poly (A) + RNA isolated from -S cells was used to construct a cDNA library in the AGEM-2 vector (Promega). E. coli Y1090 strain (Stratagene) was used as the bacterial host. For screening and plaque purification, a 416-bp DNA fragment containing 8 tandem repeat units from the -133 to -82 region of the a Amy3 promoter was excised from p3Luc.44 with PstI and Xhol (Lu, et al. (1998) J. Biol. Chem. 273, 10120-10131), and used as a probe for cDNA library screening. The cDNA library was screened according to the method of Singh et al. (Singh et al, (1988) Cell 52, O: \ 85 \ 85981.DOC -16- 200413403 4 1 5-423). The positive phage plaque was isolated and purified for further characterization. 4. Construction of plastids

OsMYBSl and OsMYBS2 cDNAs were excised from λ GEM-2 using Sail and Notl and inserted into the same site in pBluescript (Stratagene) to generate pBS-Sl and pBS-S2, respectively. There is a Sail site in the OsMYBS3 cDNA. Therefore, the OsMYBS3 cDNA was cut out from λ GEM-2 with EcoRI and Notl, and inserted into the same site in pB lues crip to generate pBS-S3. PAHC13 was cut with BamHI to remove the Luc cDNA insert, and then it was end-blunted. Use Sail and Notl to separate OsMYBSl and OsMYBS2 from pBS-Sl and pBS-S2 and blunt-end them. OsMYBS3 was excised from pBS-S3 with EcoRI and Notl and purely terminated. OsMYBS cDNAs were then each ligated to the truncated pAHC18 to generate Ubi-OsMYBS-Nos fusion genes.

5. Genomic DNA gel spot analysis According to the method of Sheu, et al. (1996) J. Biol. Chem. 271, 26998-27004, genomic DNA was isolated from rice callus. 10 mg of genomic DNA was cut with a restriction enzyme, separated in a 0.8% agarose gel, and then transferred to a nylon membrane (MSI). 32P random primer-labeled OsMYBS gene-specific DNA was used as a probe, and hybridization was performed at 42 ° C. PBS-Sl was cut with SacII, and it contained a 220-bp coding region and

The 3 93-bp 3 'untranslated region 613-bp DNA fragment of the OsMYBSl cDNA was used as OsMYBSl-specific DNA. PBS-S2 was cut with SacI, and O: \ 85 \ 85981.DOC-17 · 200413403 93-bp 3 containing 241-bp coding region and OsMYBS2 cDNA, and 334-bp DNA fragment of untranslated region was used as OsMYBS2- Specific DNA. PBS-S3 was cut with SacI, and a 599-bp DNA fragment containing a 332-bp coding region and a 267bp 3 'untranslated region of OsMYBS3 was used as OsMYBS3-specific DNA. 6 · RNA gel spotting analysis

All RNA was purified from rice suspension cells using TRIZOL reagent (GIBCO-BRL). Α-32P-labeled DNA probes were prepared and then described in the literature (Sheu, et al. (1996) J. Biol. Chem. 271, 26998-27004).

Method for RN A gel spot rupture analysis. When the dots on the membrane are hybridized with various probes, the membrane is removed and rehybridized as described in the literature (Sheu, et al., (1994) Plant J. 5, 6 5 5-664). 〇; 八 11 ^ 8- <: and? 〇11 八 (: 1.3 plastid DNA was cut with EcoRI. A Amy3 and a Amy8 gene-specific DNA was prepared as described in the literature (Sheu, et al., (1996) J. Biol. Chem. 271, 26998-27004 ). Separate these insert DNAs separately, use a -32P

Label and use it as a probe. DNA fragments containing 25S, 18S and 5.8S rDNAs were cut out from pRY18 with BamHI, labeled with α-32P and used as probes to equalize the RNA load. 7. Expression and purification of recombinant protein OsMYBS was cut with Sail and Notl, and then connected to the same site in pET28b (+) (Novagen) to produce pET-Sl, pET-S2 and pET-S3. These three plastids were each transferred into E. coli BL21 (DE3) strains, and then expressed OsMYBS, OsMYBS2 and OsMYBS3. OsMYBs protein was purified according to the instructions provided by Novagen. Protein concentration O: \ 85 \ 85981.DOC -18- 200413403 Degree was determined using Bradford reagent (Bio-Rad). 8. Gel Mobility Shift Assay. Gel Mobility Shift Assay was performed as described previously (Lu, et al · (1998) J. Biol. Chem. 273, 10120-1013 1), but The DNA-protein binding reaction was performed by combining 0.02 nanograms of 32P-labeled oligonucleotide with 0.2 micrograms of recombinant OsMYBS purified from E. coli and 1 microgram of poly (dl-dC) in a total volume of 20 microliters. The reaction proceeds in solution. The DNA fragment containing the TATA box was prepared as described previously (Lu, et al. (1998) J. Biol. Chem. 273, 10120-10131). 9. Construction of Yeast Reporter Strains

A DNA fragment containing 8 tandem repeat units at _133 to -82 of a amy3 promoter was excised from p3Luc.44 using EcoRI and Xhol (Lu, et al. (1998) J. Biol. Chem. 273 10120-10131), and then blunt-ended and inserted into the Smal site of pLacZi (Clontech) to generate 8x (TATCCA + F) -Cycl minimal promoter-LacZ fusion gene. The two complementary oligonucleotides 5, -AATTCTATCCATATCCATATCCATATCC ATATCCATATCCAC-3 and 5, -GTGGATATGGATATGGATATG and Smal sites were generated to generate the 6xTATCCA-Cycl minimal promoter-LacZ fusion gene. According to the MATCHMAKER One-Hybrid System method (Clontech), these plastids were linearized with Ncol and introduced into the genome of yeast YM4271 to generate a yeast reporter strain. 10. Yeast single hybrid system for OsMYBS binding activity analysis To construct GAD-OsMYBS fusion gene, the full-length OsMYBS cDNAs O: \ 85 \ 8598l.DOC -19- 200413403 were amplified by PCR, using pBS-Sl, pBS -S2 and pBS-S3 as DNA templates, and OsMYBSl-5, (5, -AAACTCGAGAATGACCT CCCAGGCGGCGA-3 ', Xhol is underlined) and OsMYBSl-3, (5, -ATCGAATTCTCATTGGTGCATCTTGGCCGGA-3', EcoRI The underlined part is used as the primer of OsMYBS1. OsMYBS2-5, (5, -AAACTCGAGAATGCCCAACCTCTCACCTCCA-3, and the Xhol part is underlined) and OsMYBS2-3 '(5'-

AGCGAATTCTTATATAAAACTGGTGAA-3, with EcoRI as underlined) as primer of 0sMYBS2, and OsMYBS3-5 '(5'-AAA CTCGAGTATGACGAGGCGGTGCTCGCA-3, with Xhol as underlined) and OsMYBS3-3, (5 , -ATCGAATTCTCATGCCTGTGCC CTTGT-3, (EcoRI is underlined) as the primer of OsMYBS3. The GAD sequence was amplified by PCR using pGAD-424 (Clontech) as the DNA template and oligo: y: acid GAD-5, (5, -CCAGAATTCTGCAAAGATGGATAAA-3 ', EcoRI is underlined ) And GAD-3, (5, -CCACTCGAGCTCTCTTT TTTTGGGT-3, and Xho I is underlined) as primers. All PCR products were cut with Xhol and EcoRI. OsMYBS cDNAs were individually ligated to GAD sequences in the Xhol site and inserted into the EcoRI site of pBluescript to generate pGAD-OsMYBS. PGAD-424 was cut with Mlul and EcoRI to remove the 3 'portion of the GAD region. The GAD3'-OsMYBS fusion gene was excised from pGAD-OsMYBS using Mlul and EcoRI and inserted into the Mlul and EcoRI sites of pGAD-424 to generate O: \ 85 \ 85981 containing the ADH1 promoter-GAD-OsMYBS fusion gene. DOC -20- 200413403 pGAD-OsMYBS-424. According to the MATCHMAKER single-hybrid system method (Clontech), each of these plastids was introduced into a yeast reporter strain having 8x (TATCCA + F) -Cycl minimal promoter-LacZ fusion gene or 6xTATCCA-Cycl minimal promoter- LacZ fusion gene. Analysis of the dot-galactosidase activity in the yeast. 11 · Yeast Monohybrid System for OsMYBS Transactivation Analysis

In order to construct the GBD-OsMYBS fusion gene, the GBD sequence was amplified by PCR, in which pGBT9 (Clontech) was used as a DNA template and oligonucleotide # acid GBD-5, (5, -CCAGAATTCAGATGAArTrTACTG TCT-3 ', with the EcoRI site below Underlined) and GBD-3 '(5'-CCA CTCGAGTTCGATACAGTCAACTGT-3, Xho I is underlined) as primers. Since there is an Xhol cleavage site in the GBD sequence, the PCR product is cut first with EcoRI and then with Xhol. Ligation and insertion of the generated DNA fragment with OsMYBS cDNA

The EcoRI site of pBluescript to generate pGBD-OsMYBS. PGAD-424 was cut with Hindlll to remove the GAD sequence 歹 ij and blunt end. The GBD-OsMYBS fusion gene was excised from pGBD-OsMYBS using EcoRI and Hindlll, blunt-ended and linked to the truncated pGAD-424 to generate pGBD-OsMYBS- containing the ADH1 promoter-GBD-OsMYBS fusion gene 424. This plastid was introduced into the yeast strain CG-1945, which has a 3xUAS-Cycl minimal promoter-LacZ and a GAL1 promoter-HIS3 indicator construct (Clontech). 3 xUAS is the 3 vertical repeating unit of the universal UASG17-mer universal sequence recognized by GBD. Analyze the / 3 galactosidase activity in yeast or the growth of yeast on selective culture O: \ 85 \ 85981.DOC -21- 200413403. 12. Analysis of galactosidase activity and yeast cell growth in galactosidases in transformed yeast cells was performed by using 0NPG Qualitative and quantitative. According to the Yeast Methodology Manual (Clontech), the transformed cells were incubated with 5 mM 3-aminotris. Screened in a medium that does not contain histidine. 13. Analysis of temporary performance of barley aleurone tissue

Particle bombardment of Hordeum vulgare aleurone tissue and large systems for temporary analysis of GUS such as Lanahan, et al_ (1992) Plant Cell 4, 203-21 1 and Cerc0s, et al · (1999) Plant J · 19, 107 -118 as described. The bombarded barley embryo-free half-seed seeds were cultured in a buffer containing or lacking glucose or GA3 (CaCl2 and sodium succinate 20mM each, pH 5.0) for 20 hours, and the luciferase or GUS activity was measured. All bombardments are repeated at least 4 times. Results 1. Three OsMYBS genes encoding three MYB proteins with one DNA-binding repeat unit

In order to obtain cDNAs encoding proteins that can specifically bind to the TATCCA unit, a cDNA library constructed from poly (A) + mRNA was screened using the Southern Point Method, where the poly (A) + mRNA was depleted from sucrose over 4 hours. Preparation of rice suspension cells. Using a 416-bp probe consisting of 8 tandem repeat units of the -133 to -82 DNA fragment of the a Amy3 promoter (Lu, et al. (1998) J. Biol. Chem. 273, 10120-10131), It contains TATCCA units. Isolate the cDNA O: \ 85 \ 85981.DOC -22- 200413403 which can specifically interact with this probe and analyze its sequence. Three different genes were identified and named OsMYBS, OsMYBS2 and OsMYBS3. The open coding regions of OsMYBS and OsMYBS2 and OsMYBS3 encode polypeptides of 306, 276, and 318 amino acid residues, respectively. These three OsMYBS proteins contain a highly retained single DNA-binding region that is highly similar to the DNA-binding regions of other mammalian, fruit fly, and plant MYB proteins.

Compared with other MYB proteins containing 1R in the DNA-binding region, the 1R residues of the three OsMYBS proteins are most closely related to MybStl (StMYBl), and less related to AtMYBL2, PcMYBl, and CCA1 (AtMYBCCAl). StMYBl will activate the cauliflower mosaic virus 335RNA gene (CaMV35S) promoter and it is expressed in various organs of potato (Baranowskij5 et al. (1994) EMBO J 13, 5383-5392). AtMYBL2 in Arabidopsis leaves Performance, but its function has not been identified (Kirik and Baumlein, (1996) Gene 183, 109-113). PcMYBl interacts with the light-regulatory promoter unit fork in vivo 'and is expressed in cell cultures and seedlings of the Dutch bud crbpwm) (Feldbriigge, et al. (1997) Plant J. 11, 1079-1093). AtMYBCCAl binds to the promoter of Arabidopsis thaliana light-collecting chloroplast a / b binding protein gene (Lhcb) and regulates the regulation of phytochrome of Lhcb (Wang, et al. (1997) Plant Cell 9, 491-507 ). The 1R sequence of OsMYBS3 is most similar to StMYBl (92% identity) and somewhat similar to OsMYBS 1 (87% identity) and 〇smYBS2 (85% identity), while the 1R sequences of OsMYBSl and 〇SMYBS2 are the least similar to each other. (77% identical). All MYB η · \ α < \ β < 〇βΐ -23-200413403 proteins with 1R DNA-binding regions are extremely similar to each other in the N- and C-terminal regions outside the 1R region, but OsMYBS3 and StMYBl are in the N region. -The terminal 90-amino acid region has 71% identity and the C-terminal 70-amino acid region has 62% identity.

Comparison of the amino acid residues between the 1R regions of the three OsMYBS proteins and the repeating units of R2 and R3 of animal and plant MYB proteins shows that the 1R region contains the retained tryptophan (W), amidine ( E), Glycine (G) and Arginine (R). Tryptophan residues (Ogata, et al. (1 992) Pro c. Natl. Acad · Sci. USA 89, 6428-6432) which play an important role in the DNA-binding region of stable animal MYB protein The interval is reserved in the first and second positions, but not in the third position. The putative base-contact residues in the third helix of the animal MYB protein are retained in the plant R2R3 MYB protein, but not in the plant 1R MYB protein.

The 3 ′ untranslated regions of the three OsMYBS genes share a very low similarity (33 to 35% of the same degree), and they are used as gene-specific DNA probes for the analysis of rice genome DNA gel spots. Each of these three gene-specific DNA probes is hybridized to a single band only. This result shows that the three OsMYBS gene lines in the rice MYB gene family are each a single set of genes. 2.0sMYBS gene expression in various rice tissues and barley aleurone Analysis of the expression pattern of OsMYBS in rice. All RNA was purified from various rice tissues, and OsMYBS-specific DNA was used as a probe for gel spot analysis. OsMYBS 1 is expressed in the ground tissues and the highest in leaves. OsMYBS2 is mainly expressed in roots, leaves, and senescent leaves with the same degree of expression, while OsMYBS3 is expressed in all tissues and has the highest degree of expression in aging leaves. O: \ 85 \ 85981.DOC -24- 200413403 In order to determine whether OsMYBS gene expression is regulated by sugar, all RNA was isolated from rice suspension cells cultured in the presence or absence of sucrose for 48 hours, and OsMYBS-specific Sex DNAs were used as probes for gel spot analysis. OsMYBS 1 and OsMYBS3 mRNA remained low in the presence of sucrose, but increased in the absence of stored sugar, which is consistent with the mRNA content of a Amy3 and another rice α-dianfan hydrolase gene (a Amy8). In contrast, the concentration of OsMYBS2 is higher in the presence of sucrose than in the absence of sucrose.

A temporary performance analysis was performed in the following experiment, in which half-seed seeds of barley were used as a system to study the sugar and hormone regulation function of OsMYBS protein. In order to determine whether the OsMYBS gene was expressed in barley aleurone cells, all RNA was isolated from half of the seeds of barley, and then gel spotting analysis was performed using OsMYBS-specific DNAs as probes. All of these OsMYBS genes are expressed in barley aleurone cells in the absence of glucose. The expression of OsMYBSl and OsMYBS3 was suppressed in the presence of glucose or GA, but remained unchanged in the presence of ΑΑΑ. OsMYBS2 showed an increase in the presence of glucose, but was slightly inhibited by ABA treatment. 3. Specific interaction between OsMYBS protein and TATCCA unit in vitro In order to determine whether three OsMYBS proteins specifically bind to TATCCA unit, they are expressed in E. coli. The interactions between the affinity-purified recombinant OsMYBS protein and five DNA fragments covering the -171 to -82 region of a Amy3 SRS were analyzed by gel electrophoresis shift. The five fragments (named F1 to F5) were previously used in gel electrophoretic shift analysis to investigate their interaction with nuclear protein extracts from rice suspension cells. O: \ 85 \ 85981.DOC -25- 200413403 (Lu, et al. (1998) J. Biol Chem. 273, 10120-1013 1). F 2 was used as a probe for gel electrophoresis shift analysis. The ρ 2 contains 2 TATCCA units and some flanking sequences in the SRS. Three kinds of reorganization

OsMYBS protein binds to probe F2 to form a low-shift complex. The interaction between F2 and OsMYBS1 or 0sMYBS2, only the DNA fragments of F2 and 6 mediastinal ij repeat units containing 6-bp TATCCA units can compete with it, but other DNA fragments with a 500-fold excess cannot Competition knot

Together. For the interaction between F2 and 0sMYBS3, only the DNA fragments of F2, F5 (including TATCCA units) and 6 longitudinal 歹 J repeat units containing 6-bp TATCCA units can compete with it. The 17-bp DNA fragment containing the TATA box sequence (TATATA) (only 2 nucleotides different from the TATCCA unit) cannot compete with it. In order to further demonstrate the specificity of the binding between the OsMYBS protein and the TATCCA unit, a series of site-directed mutations spanning the -119 to -102 region containing TATCCA tandem repeat units were constructed. A wild-type (Wt) sequence was used as a probe for gel electrophoresis shift analysis. These three recombinant OsMYBS proteins bind to the Wt% probe to form a low-mobility complex. Furthermore, the binding of each of these OsMYBS proteins can compete completely via unlabeled Wt sequences. DNA sequences (M1) that have mutations in both repeating units cannot compete, and DNA sequences (M2 and M3) that have been replaced in either of the two units compete at a lower efficiency. Mutants (M4, M5, and M6) with mutations in 3 of the 6 bases of the two repeating units competed only slightly. These results suggest that all of these OsMYBS proteins can specifically bind to the two sets of TATCCA units. However, if only one set of TATCCA is present, the binding affinity will be reduced O: \ 85 \ 8598l.DOC -26- 200413403. 4. Interaction between OsMYBS protein and TATCCA unit in vivo In vivo, the binding activity of OsMYBS protein and TATCCA unit was analyzed by yeast single hybrid system. The full-length 0sMYbs sequences were fused at the c-terminus to the activation region of yeast GAL4 transcription activating factor (Gad), respectively. The GAD-OsMYBS chimeric gene was then fused to the downstream region of the yeast ADm promoter as an effector construct. The yeast strain containing the Cycl minimal promoter-LacZ fusion reporter (reporter ·) construct was then transformed with the offending construct. GAD, either alone or with GAD-OsMYBS fusion protein, does not cause LacZ performance. Fusion of the 8 tandem repeat units (named 8 X (TATCCA + F)) of the A1333 -133 to -82 DNA sequence with the upstream region of the Cycl minimal promoter-LacZ coding region as a reporter construct First, the reporter construct was transported into yeast, and then the yeast was transformed with the acting construct containing the ADH and GAD-OsMybS fusion gene. GAD alone could increase the performance of LacZ by 5 times, which means that The DATCCA unit with flanking sequences slightly increases Cyel's minimum promoter activity even in the absence of OsMYBS. Compared to the performance of LacZ in the absence of OsMYBS, the presence of GAD-OsMYBS 1 and GAD-OsMYBS2 will increase the performance of LacZ, respectively. 3 times and 5 times. In contrast, the presence of OsMYBS3 will reduce LacZ performance by 2 times. In order to test whether the flanking sequence of the TATCCA unit will affect the binding affinity of OsMYBSl and 0sMYBS2, the 6-bp TATCCA single O: \ 85 \ 85981 .DOC -27- 200413403 yuan 6 vertical repeating units (named 6xTATCCA) are fused with the upstream region of the Cycl minimal promoter-LacZ coding region as the reporter construct. The reporter construct is transported into In yeast, After the effect of sub-containing ADHl-GAD-OsMYBS fusion gene construct Transformation The yeast to be only at the presence of GAD-OsMYBS2 increase LacZ manifestations. 5, OsMYBSl transcription activator and OsMYBS2

To determine whether OsMYBS is a transcription activator, the full-length OsMYBS sequence 歹 ij was fused to the C-terminus of the DNA-binding region of GAL4 (GBD). Then, the chimeric gene was fused to a region downstream of the yeast ADH1 promoter to serve as an acting construct. With this acting construct, the yeast strain containing the 3xUAS-Cycl minimal promoter-LacZ fusion reporter construct was transformed. The presence of GBD-OsMybSl fusion protein will increase LacZ performance by 8 times, GBD-OsMYBS2 will only slightly increase LacZ performance, and GBD-OsMYBS3 will not increase LacZ performance.

Yeast strains containing the GAL1 promoter-HIS3 fusion reporter construct were also transformed with the effector construct containing the ADH1-GBD-OsMYBS fusion gene. The presence of the GBD-OsMYBSl fusion protein allows yeast cells to grow well on media lacking histidine and in the presence of 3-AT. (} BD-OsMYBS2 fusion protein makes yeast cells grow slowly, but GBD-OsMYBS3 fusion protein does not allow yeast cells to grow on selective media. The above results suggest that in yeast, OsMYBSl is a strong transcriptional activation Factor, OsMYBS2 is a weak transcriptional activating factor, while OsMYBS3 is a non-transcriptional activating factor. O: \ 85 \ 85981.DOC -28-200413403 6.0sMYBSl and OsMYBS2 depressor effect on sugar-repressed transcription of SRS-containing promoters

In order to determine the function of OsMYBS in the regulation of sugar in the expression of the α-amylase enzyme gene in plant cells, we analyzed here in barley aleurone cells (which can obtain more consistent results than in rice protoplasts). OsMYBS for SRS -The transactivation capacity of the CaMV35S chimeric promoter, wherein the SRS_CaMV3 5S chimeric promoter is a fusion of the SRS and the smallest promoter of CaMV3 5S (the -46 bp upstream region of the transcription initiation site). The three OsMYBS cDNAs were fused with the maize ubiquitin gene (Ubi) promoter downstream to form a Ubi-OsMYBS chimeric gene, which was used as an operator. The coding region of the luciferin gene (Luc) was fused to the downstream region of the SRS-CaMV35S chimeric promoter as a reporter. Half the barley seeds were bombarded with particles and traits and reporter protoplasts. The bombarded half of the seeds were divided into two halves, and then each was cultured with 0.3 M glucose or 0.3 M mannitol for 24 hours and luciferase activity was measured. Luciferase activity when the half seed was bombarded with the reporter protoplasts and plastid pRS426 (—irrelevant yeast plastids, the molecular weight of which is similar to the active protoplasts, which is used as a negative control group) Higher in the absence of glucose. This result is consistent with previous findings that "in rice protoplasts, the SRS-CaMV35S chimeric promoter is activated in the absence of glucose and is suppressed in the presence of glucose" (Lu, et al. (1998) J · Biol. Chem. 273, 10120-1013 1). When half-seeds were bombarded with reporter protoplasts and acting protoplasts, OsMYBSl significantly increased luciferase activity in the presence of glucose to a level similar to that in the absence of glucose, and OsMYBSl was also available in the absence of glucose. Increasing the activity of luciferase O: \ 85 \ 8598l.DOC • 29- 200413403, but to a lesser extent. 0sMYBS2 increases luciferase activity in the presence of glucose, but does not affect activity in the absence of glucose. 0sMYBS3 does not significantly increase luciferase activity in the presence of glucose, but suppresses luciferase activity in the absence of glucose. This result shows that for the transcriptional effect of the SRS-containing promoter, OsMYBS1 and 0smYBS2 are strong and weak activating factors, respectively, and OsMYBS3 is a repressor. 7.0sMYBSl Hostility to TATCCA-containing Units and Adjacent Flanking Sequences

Unsuppressed effect of glycosyl-repressed transcription in order to determine whether OsMYBS will activate the promoter containing only TATCCA units and adjacent flanking sequences, DNA containing 6 repeating units of the -133 to -82 sequence of SRS The fragment (named 6x (TATCCA + F)) was fused to the CaMV3 5S minimal promoter. A plastid containing the Ubi-OsMYBS construct was used as a reporter, and a Luc cDNA fused to the downstream region of the 6x (TATCCA + F) -CaMV35S promoter was used as a reporter. Half the barley seeds were bombarded with particles and interactors and reporter protoplasts. The bombarded half of the seed was divided into two halves, and then each was cultured with 0.3M glucose or 0.3M mannitol for 24 hours and the luciferase activity was measured. OsMYBSl significantly increased luciferase activity to a similar degree in the absence of glucose in the presence of glucose. Although 0sMYBS2 and OsMYBS3 appear to slightly increase luciferase performance in the presence of glucose, they significantly suppress luciferase activity in the absence of glucose. The results show that OsMYBS1 activates transcription of the promoter containing only the TATCCA unit and adjacent flanking sequences, while 0sMYBS2 and 0sMYBS3 beta4 suppress its transcription. 8-OsMYBS protein and HvMYBGa together activate barley Amy32b O: \ 85 \ 85981.DOC • 30- 200413403 The GARC of the promoter in the α-amylase gene promoter includes GARE and TATCCA units (Gubler and Jacobsen (1992 ) Plant Cell 4, 1453-1441; Lanahan, et al. (1992) Plant Cell 4, 203-211). HvMYBGa has been shown to specifically bind to GARE in test tubes and to activate transcription of the barley high-pi α-amylase and cysteine protease gene promoters in the absence of GA in vivo (Gubler, et al (1995) Plant Cell 7, 1879-1891; Cercos, et al. (1999) Meal | Plant J. 19, 107-118). Since the TATCCA unit is necessary for the high transcription of the a-amylase enzyme promoter by GA activation (Gubler and Jacobsen (1992) Plant Cell 4, 1435-1441; Lanahan, et al. (1992) Plant Cell 4, 203- 211), so it was tested whether the OsMYBS protein and HvMYBGa together promoted the transcription of the α-dianfan hydrolase gene promoter. Barley half-grain seeds were bombarded with particles of Ubi-OsMYBSGa-containing constructs, protons containing Ubi-OsMYBS constructs, and reporter protoplasts containing Amy32b-GUS constructs. When% HvMYBGa is absent, GUS only manifests in the presence of GA, and when GA is absent, excessive expression of HvMYBGa will significantly increase GUS activity. This result is consistent with other results reported in the literature (Gubler, et al. (1995) Plant Cell 7, 1879-1891; Cercos, et al. (1999) Plant J. 19, 107-118). It is interesting that the combined performance of HvMYBGa and OsMYBS and OsMYBS2 or OsMYBS3 can further enhance the GUS activity, which is about 2-3 times that of GUS activity when HvMYBGa is alone and without GA. These results suggest that the three O: \ 85 \ 85981.DOC -31-200413403

OsMYBS protein and HvMYBGa will co-activate the barley α-starch hydrolysate gene promoter. 9 · OsMYBSl forms homodimer in barley aleurone cells

In c-Myb, R2 and R3 together bind to specific DNA sequences. To test the possibility of OsMYBS binding to DNA as a dimer, a two-hybrid system was used in barley aleurone cells to analyze the allogeneic and heterogeneous interactions between OsMYBS proteins. The full-length OsMYBS sequences were each fused to the C-terminal of GAD or GBD. The GAD-OsMYBS and GBD-OsMYBS chimeric genes were fused to the downstream region of the Ubi promoter as the acting construct. A DNA fragment containing 5 tandem repeat units of UAS (5xUAS) was fused to the upstream region of the CaMV35S minimal promoter-Luc chimeric gene and used as a reporter construct. Half-grain barley seeds were bombarded with two acting protoplasts (each containing GAD-OsMYBS and GBD-OsMYBS fusion genes) and the reporter protoplasts. The bombarded half-seeds were incubated in a glucose-free buffer for 24 hours, and the luciferase activity was measured. The common performance of GBD-OsMYBSl and GAD-〇sMYBSl is inconsistent with that of GBD-OsMYBS 1 alone, which significantly enhances the performance of leukoenzyme. However, the common performance of GBD-OsMYBSl and GAD-OsMYBS2 or GAD-OsMYBS3 does not further increase the luciferase activity compared to the GBD-OsMYBSl alone. Similar experiments with OsMYBS2 and OsMYBS3 have shown that the two proteins do not interact between isotypes or isotypes. In order to confirm that only OsMYBSl will interact with each other among the isotypes, and that the three OsMYBS proteins will not interact with each other, further construct the effector and reporter protoplasts for expression in yeast. Except that O: \ 85 \ 8598I.DOC -32- 200413403 in the presence of GBD-OsMYBSl alone, the background concentration of the reporter gene expression is too high to detect the interaction between the GBD-OsMYBS 1 and GAD-QsMYBS proteins In this experiment, similar results were obtained with the yeast two-hybrid experiment. These results suggest that only 0sMYBS 1 forms homodimers under these experimental conditions. Other Specific Embodiments All of these features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification may be replaced by another feature serving the same, equivalent, or similar purpose. Therefore, unless expressly stated, each feature disclosed is only an example of a series of equal or similar features.

From the above description, those skilled in the art can easily confirm the main features of the invention, and make various changes and modifications to the invention to adapt to various uses and conditions without departing from the spirit and scope of the invention. Therefore, other specific embodiments are also within the scope of the following patent application parks. A

O: \ 85 \ 85981.DOC 33 · 200413403 Sequence Listing < 110 > Yu Shumei < 120 > Plant MYB Gene < 130 > 08919-088001 < 140 > 092121002 < 141 > 2003-07-31 < 150 > US 60 / 399,999 < 151 > 2002-07-31 < 160 > 21 < 170 > FastSEQ for Windows Version 4.0 < 210 > 1 < 211 > 1330 < 212 > DNA < 213 > Rice (Japan Cultivated population) < 220 > < 221 > CDS < 222 > (20) ... (937) < 400 > 1 gtgcgagatc caccacccg atg acc tcc cag gcg gcg acg acg acg acc acg Met Thr Ser Gin Ala Ala Thr Thr Thr Thr Thr 15 10 gcg gcg gcg gcg gcg gcg tgg acc agg gag gac gac aac gag gcg ttc gag Ala Ala Ala Ala Ala Ala Ala Trp Thr Arg Glu Asp Asp Lys Ala Phe Glu 15 20 25 aac gcg etc gcg g gg ccg ccc gcg gac gga ggc gcg Asn Ala Leu Ala Ala Cys Ala Ala Pro Pro Pro Ala Asp Gly Gly Ala 30 35 40 ccc gac gac gac tgg ttc gcc gcg etc gee gcg age gtg ccc ggg gcg Pro Asp Asp Asp Arp Trp Phe Leu Ala Ala Ser Val Pro Gly Ala 45 50 55 agg teg gcg gag gag gtg egg agg cac tac gag gcg ctg gtg gag gac Arg Ser Ala Glu Glu Val Arg Arg His Tyr Glu Ala Leu Val Glu Asp 60 65 70 75 gtc gcg gcc ate gac gcg ggc ege gtc ccg etc ccg ege tac gcc ggg Val Ala Ala workers Asp Ala Gly Arg Val Pro Leu Pro Arg Tyr Ala Gly 80 85 90 gag gag tec gcg gcg ccg ccc gac gga gcc gga gcc gcc gcc gcc gcg Glu Glu Ser Ala Ala Pro Pro Asp Gly Ala Gly Ala Ala Ala Ala Ala Ala 95 100 105 52 100 148 196 244 292 340 tec aag gac ggc gga cac egg ege gac gag ege aag ggc ggc ggc ggc Ser Lys Asp Gly Gly His Arg Arg Asp Glu Arg Lys Gly Gly Gly Gly Gly 388 2 2200413403 110 115 120 ggg tac gac ggc ggc aag age tgc gag cag gag agg ege 436

Gly Tyr Asp Gly Gly Lys Ser Cys Ser Lys Ala Glu Gin Glu Arg Arg 125 130 135 aa9 ggc atc cca tgg aeg gag gaa gag cac agg ctg ttc ttg ctg ggg 484

Lys Gly lie Pro Trp Thr Glu Glu Glu His Arg Leu Phe Leu Leu Gly 140 145 150 155 ctg gac aag ttc ggc aag ggg gac tgg egg age ate teg ege aac ttc 532

Leu Asp Lys Phe Gly Lys Gly Asp Trp Arg Ser lie Ser Arg Asn Phe 160 165 170 gtc ate teg egg aeg cca aeg cag gtg geg age cac geg cag aag tac 580

Val lie Ser Arg Thr Pro Thr Gin Val Ala Ser His Ala Gin Lys Tyr 175 180 185 ttc ate ege etc aac tee atg aac ege gac ege ege tee age ate 628

Phe Engineering Arg Leu Asn Ser Met Asn Arg Asp Arg Arg Arg Ser Ser lie 190 195 200 cac gac ate acc age gtc acc gee ggc gat cag gtc gee geg cag cag 676

His Asp Worker Thr Ser Val Thr Ala Gly Asp Gin Val Ala Ala Gin Gin 205 210 215 ggc gee ccg ate acc ggc cac cag gee aeg ggc aac ccc geg geg geg 724

Gly Ala Pro lie Thr Gly His Gin Ala Thr Gly Asn Pro Ala Ala Ala 220 225 230 235 geg ctg ggc ccg ccg ggc atg aag cac cac cac cac cac cac ccg ggc 772

Ala Leu Gly Pro Pro Gly Met Lys His His His His His His Pro Gly 240 245 250 ggc geg ccg ccg ccc atg ccc atg tac age gee geg ccc atg ggc cac 820

Gly Ala Pro Pro Pro Met Pro Met Tyr Ser Ala Ala Pro Met Gly His 255 260 265 ccc gtc gee ggc cac atg gtg ccc gee gee gtc ggc aeg ccg gtg gtg 868

Pro Val Ala Gly His Met Val Pro Ala Ala Val Gly Thr Pro Val Val 270 275 280 ttc ccg ccg ggc cac geg ccg tac gtc gtg ccc gtc ggc tac ccg geg 916

Phe Pro Pro Gly His Ala Pro Tyr Val Val Pro Val Gly Tyr Pro Ala 285 290 295 ect ccg gee aag atg cac caa tgacgcgcca tggacggaca tgageageat 967

Pro Pro Ala Lys Met His Gin 300 305 ttcttcctcc tcctttcttg atgtcaatct tgatttgttc tttgtgtagt cgccggctca 1027 tcgtccctga tcatcttgtt cttctcacaa tctcactaat gtaaacatac atagatcaga 1087 tgccaagagt gcagggattg gggattaaag gegaataagt aaagtatttt gctgactgtt 1147 tgcaagtgat catcacgtac acccggtgaa agcttagctc caaatgtgga tgtaattage 1207 agcggccttc cgtacgtggt ggcgccgatc gatgatettg caggggttgc aattagggat 1267 tgatttccat tttgctgatg taaatttgcc aactgtctca ttggaccaaa aaaaaaaaaa 1327 aaa 1330 200413403

< 210 > 2 < 211 > 1012 < 212 > DNA < 213 > Rice (Japanese cultivated population) < 220 >

< 221 > CDS < 222 > (92) ... (919) < 400 > 2 cgaggtccgc ggcggcggcg gcggagttga cgaggaggag tacgaggagg aggaggtgga 60 ttcatcaaga agagctccag t atg ccc aac etc acc tee ate 112

Met Pro Asn Leu Thr Ser lie 1 5

gac ccg ctg ccg gtg ccg gee gac ggc ggc aaa egg ege gee tee gac 160

Asp Pro Leu Pro Val Pro Ala Asp Gly Gly Lys Arg Arg Ala Ser Asp l0 15 20 gac tee gag etc gee tee ggc cag cag aag ege ege ege ag gtg 208

Asp Ser Glu Leu Ala Ser Gly Gin Gin Lys Arg Arg Arg Arg Lys Val 25 30 35 cag gag agg aag aaa ggg gta cca tgg act gag gag gag cac aag aaa 256

Gin Glu Arg Lys Lys Gly Val Pro Trp Thr Glu Glu Glu His Lys Lys 40 45 50 55 ttc ctg gaa ggg ctg agg cag ctg ggg aaa ggg gac tgg aga ggc ate 304

Phe Leu Glu Gly Leu Arg Gin Leu Gly Lys Gly Asp Trp Arg Gly lie 60 65 70 tee aag aac ttt gtg acc age agg aeg geg act cag gtg gee age cac 352

Ser Lys Asn Phe Val Thr Ser Arg Thr Ala Thr Gin Val Ala Ser His 75 80 85

gee cag aag tac ttc etc egg cag acc aac cct ggc aaa aag aag ege 400

Ala Gin Lys Tyr Phe Leu Arg Gin Thr Asn Pro Gly Lys Lys Lys Arg 90 95 100 egg gee age etc ttt gat gtt gtt get gag tgc agt gat gat caa ett 448

Arg Ala Ser Leu Phe Asp Val Val Ala Glu Cys Ser Asp Asp Gin Leu 105 110 115 cca agt cct cag agt gtt gga act aag cct cct acc cag gat ata att 496

Pro Ser Pro Gin Ser Val Gly Thr Lys Pro Pro Thr Gin Asp lie lie 120 125 130 135 cat aca gat ege ggc gat gtc ccg ata eta age tat cca gtt get aga 544

His Thr Asp Arg Gly Asp Val Pro lie Leu Ser Tyr Pro Val Ala Arg 140 145 150 ggc ttt aga ggc gat age gtg cag gtt gat gaa eta act gaa tat gtg 592

Gly Phe Arg Gly Asp Ser Val Gin Val Asp Glu Leu Thr Glu Tyr Val 155 160 165 aag aga tta aag gee gee gag gac atg teg etc tee atg ate tet gga 640 4 200413403

Lys Arg Leu Lys Ala Ala Glu Asp Met Ser Leu Ser Met lie Ser Gly 170 175 180 ctg gaa atg gca tea tea tcc ate age agt eta gag etc agt ate geg Leu Glu Met Ala Ser Ser Ser lie Ser Ser Leu Glu Leu Ser lie Ala 185 190 195 ecc tet cat ttg egg ate gac ggg gee ate aag ggg ctg gga tec aaa Pro Ser His Leu Arg lie Asp Gly Ala lie Lys Gly Leu Gly Ser Lys 200 205 210 215 ecc aat ttt ccc ccg aag gaa ttt gga teg get tea get act gtt ttt Pro Asn Phe Pro Pro Lys Glu Phe Gly Ser Ala Ser Ala Thr Val Phe 220 225 230 tgt ccc ccc tgt tgt tgt ttg ttg ttg ttg ttt ttt ttt ttt ttt ttt ttt Cys Pro Pro Cys Cys Cys Leu Leu Leu Leu Phe Phe Phe Phe Phe Phe 235 240 245 9C <3 999 gtt gtt tgt tgt tgt tgt tgt tgt agt tgt cat get aac ttt Ala Gly Val Val Cys Cys Cys Cys Cys Cys Ser Cys His Ala Asn Phe 250 255 260 gta ttt ggg tea tgt ggg gtt tet ttc acc agt ttt ata taatacagag Val Phe Gly Ser Cys Gly Val Ser Phe Thr Ser Phe lie 265 270 275 agaatgtcag tcccttccga gacatgttta aaaaaaaaaa aaaaaa aaaaaaa aa &lt; 210 &gt; 3 &lt; 211 &gt; 1287 &lt; 212 &gt; DNA &lt; 213 &gt; Rice (Japanese cultivated population) 688 736 784 832 880 929 989 1012 &lt; 220 &gt;

&lt; 221 &gt; CDS &lt; 222 &gt; (67)… (1020) &lt; 400 &gt; 3 60 108 ategategat cgatctccat aggtggggga agggaagett tggaaggtgg agggaeggag gggggg aeg agg egg tgc teg cac tgc age cac aac gac aac gac

Met Thr Arg Arg Cys Ser His Cys Ser His Asn Gly His Asn 15 10 156 teg egg aeg tgc ccc aac ege ggg gtc aag ate ttc ggg gtg ege etc

Ser Arg Thr Cys Pro Asn Arg Gly Val Lys lie Phe Gly Val Arg Leu 15 20 25 30 204 acc gat ggc tec ate ege aag age gee age atg ggg aac etc tec etc

Thr Asp Gly Ser lie Arg Lys Ser Ala Ser Met Gly Asn Leu Ser Leu 35 40 45 etc tec tec gee gee gga tec acc age ggc ggc gee tec ccc gee gac

Leu Ser Ser Ala Ala Gly Ser Thr Ser Gly Gly Ala Ser Pro Ala Asp 50 55 60 252 5 5200413403 ggc ccc gac gcc gcc ccc acc gcc gcc gac ggc tac gcc tcc gac gac 300

Gly Pro Asp Ala Ala Pro Thr Ala Ala Asp Gly Tyr Ala Ser Asp Asp 65 70 75 ttc gtc cag ggc ttc tcc tcc gcc acc cgc gac cgc aag aag ggt gtt 348

Phe Val Gin Gly Phe Ser Ser Ala Thr Arg Asp Arg Lys Lys Gly Val 80 85 90 cct tgg act gaa gaa gaa cac egg agg ttt ttg ett gga ttg caa aag 396

Pro Trp Thr Glu Glu Glu His Arg Arg Phe Leu Leu Gly Leu Gin Lys 95 100 105 110 ett ggc aaa ggt gat tgg ega gga ate tet cgt aat ttc gtg gtc tea 444

Leu Gly Lys Gly Asp Trp Arg Gly lie Ser Arg Asn Phe Val Val Ser 115 120 125 aga aca cct act caa gta gcc agt cat get cag aaa tat ttt ata cgc 492

Arg Thr Pro Thr Gin Val Ala Ser His Ala Gin Lys Tyr Phe Engineering Arg 130 135 140 caa tcc aat atg acc aga agg aaa aga agg tet age ett ttt gac atg 540

Gin Ser Asn Met Thr Arg Arg Lys Arg Arg Ser Ser Leu Phe Asp Met 145 150 155 gtg cca gat gag tet atg gac ett cca cca ett cct gga ggt caa gaa 588

Val Pro Asp Glu Ser Met Asp Leu Pro Pro Leu Pro Gly Gly Gin Glu 160 165 170 cca gag acc caa gta tta aat caa cca gca eta cct cca ccg aag gag 636

Pro Glu Thr Gin Val Leu Asn Gin Pro Ala Leu Pro Pro Lys Glu 175 180 185 190 gaa gag gag gta gat tet atg gag tea gat act tet gcc gtt gca gag 684

Glu Glu Glu Val Asp Ser Met Glu Ser Asp Thr Ser Ala Val Ala Glu 195 200 205 age tet tcc get tet get ate atg cca gat aat ttg cag teg acc tat 732

Ser Ser Ser Ala Ser Ala lele Met Pro Asp Asn Leu Gin Ser Thr Tyr 210 215 220 cca gtg att gtt cca get tat ttc teg ccc ttt ttg caa ttc teg gtt 780

Pro Val lie Val Pro Ala Tyr Phe Ser Pro Phe Leu Gin Phe Ser Val 225 230 235 cct ttc tgg caa aat cag aaa gat gaa gat ggt cct gtg caa gaa aca 828

Pro Phe Trp Gin Asn Gin Lys Asp Glu Asp Gly Pro Val Gin Glu Thr 240 245 250 cat gag att gtc aag cct gtt cca gtt cat tea aag age cca ate aac 876

His Glu Le Val Lys Pro Val Pro Val His Ser Lys Ser Pro Le Asn 255 260 265 270 gtt gat gag ett gtt ggc atg teg aag etc age ata gga gag tcc aat 924

Val Asp Glu Leu Val Gly Met Ser Lys Leu Ser lie Gly Glu Ser Asn 275 280 285 caa gag aca gag tet act tet ett tea tta aat ctg gta gga ggt caa 972 6200413403

Gin Glu Thr Glu 290

Ser Thr Ser Leu Ser Leu Asn Leu Val 295

Gly Gly Gin 300 1020 aat aga caa tea get ttc Asn Arg Gin Ser Ala Phe 305 cat gca aat cca cca aca agg gca cag gca His Ala Asn Pro Pro Thr Arg Ala Gin Ala 310 315 tgatctggtt gtgcacaa ctgcatttag ctatttatgatccattatattatatatgatcc agtatttatatatatgatc agtattt ccgttctccg tagtctgtat catgtgctgt 1140 aagttatgct atgtatgaat gtatctgttg tttgtctggc acacatgata aatcactcta 1200 tgttaacaaa atc ^ gtaatg gtagtgctga tcttcgtgtttgttgtgttg taaactcta gata 1a tata

&Lt; 210 &gt; 4 &lt; 211 &gt; 918 &lt; 212 &gt; DNA air) &lt; 213 &gt; rice (japonica cultivar population) &lt; 400 &gt; 4 atgacctccc aggeggegae gacgacgacc aeggeggegg cggcggcggc gtggaccagg 60 gaggacgaca aggegttega gaacgcgctc geggettgeg cggcgccgcc gcccgcggac 120 ggaggegege ccgacgacga ctggttcgcc gcgctcgccg cgagcgtgcc cggggcgagg 180 teggeggagg aggtgcggag gcactacgag gcgctggtgg aggaegtege ggccatcgac 240 gcgggccgcg tcccgctccc gcgctacgcc ggggaggagt ccgcggcgcc gcccgacgga 300 gccggagccg ccgccgccgc gtccaaggac ggcggacacc ggegegaega gcgcaagggc 360 ggcggcggcg ggtaegaegg cggcaagagc tgctccaagg eggageagga gaggcgcaag 420 ggcatcccat ggacggagga agagcacagg ctgttcttgc tggggctgga caagttcggc 480 aagggggact ggeggageat ctcgcgcaac ttegteatet cgcggacgcc aacgcaggtg 540 gcgagccacg egeagaagta cttcatccgc ctcaactcca tgaaccgcga ccgccgccgc 600 tccagcatcc acgacatcac cagcgtcacc gccggcgatc aggtcgccgc gcagcagggc 660 gccccgatca ccggccacca ggccacgggc aaccccgcgggg ggcgcggggcc accgggcc gcccatgtac 780 agcgccgcgc ccatgggcca ccccgtcgcc ggccacatgg tgcccggccgc cgtcggcacg 840 ccggtggtgt tcccgccgcggg ccacgcgccg tacgtcgtgc ccgtcggcta cccggcgccaga 900

&Lt; 210 &gt; 5 &lt; 211 &gt; 828 &lt; 212 &gt; DNA &lt; 213 &gt; rice (japonica cultivar population) &lt; 400 &gt; 5 atgcccaacc tcacctccat cgacccgctg ccggtgccgg ccgacggcgg caaacggcgc 60 gcctccgacg actccgagct cgcctccggc cagcagaagc gccgccgccg caaggtgcag 120 gagaggaaga aaggggtacc atggactgag gaggagcaca agaaattcct ggaagggctg 180 aggcagctgg ggaaagggga ctggagaggc atctccaaga actttgtgac cagcaggacg 240 gcgactcagg tggccagcca cgcccagaag tacttcctcc ggcagaccaa ccctggcaaa 300 aagaagegee gggccagcct ctttgatgtt gttgctgagt gcagtgatga tcaacttcca 360 agtcctcaga gtgttggaac taagcctcct acccaggata taattcatac agategegge 420 gatgtcccga tactaagcta tccagttgct agaggettta gaggegatag cgtgcaggtt 480 gatgaactaa ctgaatatgt gaagagatta aaggccgccg aggacatgtc gctctccatg 540 atctctggac tggaaatggc atcatcatcc atcagcagtc tagagctcag tatcgcgccc 600 tctcatttgc ggategaegg ggccatcaag gggctgggat ccaaacccaa ttttcccccg 660 aaggaatttg gateggette agctactgtt ttttgtcccc cctgttgtttg tttgttgtttgg 720 ttgttttttt tttttttttt tgcgggggtgtt ggt gtcat 780 gctaactttg tatttgggtc atgtggggtt tctttcacca gttttata 828 200413403

&Lt; 210 &gt; 6 &lt; 211 &gt; 954 &lt; 212 &gt; DNA &lt; 213 &gt; rice (japonica cultivar population) &lt; 400 &gt; 6 atgacgaggc ggtgctcgca ctgcagccac aacgggcaca actcgcggac gtgccccaac 60 cgcggggtca agatcttcgg ggtgcgcctc accgatggct ccatccgcaa gagcgccagc 120 atggggaacc tctccctcct ctcctccgcc gccggatcca ccagcggcgg cgcctccccc 180 gccgacggcc ccgacgccgc ccccaccgcc gccgacggct acgcctccga cgacttcgtc 240 cagggcttct cctccgccac ccgcgaccgc aagaagggtg ttccttggac tgaagaagaa 300 caccggaggt ttttgcttgg attgcaaaag cttggcaaag gtgattggcg aggaatctct 360 cgtaatttcg tggtctcaag aacacctact caagtagcca gtcatgctca gaaatatttt 420 atacgccaat ccaatatgac cagaaggaaa agaaggtcta gcctttttga catggtgcca 480 gatgagtcta tggaccttcc accacttcct ggaggtcaag aaccagagac ccaagtatta 540 aatcaaccag cactacctcc accgaaggag gaagaggagg tagattctat ggagtcagat 600 acttctgccg ttgcagagag ctcttccgct tctgctatca tgccagataa tttgcagtcg 660 acctatccag tgattgttcc agcttatttc tcgccctttt tgcaattctc ggttcctttc 720 tggcaaaatc agaaagatga taga gt aagcct 780 gttccagttc attcaaagag cccaatcaac gttgatgagc ttgttggcat gtcgaagctc 840 agcataggag agtccaatca agagacagag tctacttctc tttcattaaa tctggtagtag 900 ggtcaaaata gacaatcagc tcaccagca tcaccagca

&lt; 210 &gt; 7 &lt; 211 &gt; 306 &lt; 212 &gt; PRT &lt; 213 &gt; Rice (Japanese cultivated population) &lt; 400 &gt; 7

Met Thr Ser Gin Ala Ala Thr Thr Thr Thr Thr Ala Ala Ala Ala Ala 1 5 10 15

Ala Trp Thr Arg Glu Asp Asp Lys Ala Phe Glu Asn Ala Leu Ala Ala 20 25 30 〆

Cys Ala Ala Pro Pro Pro Ala Asp Gly Gly Ala Pro Asp Asp Asp Trp 35 40 45

Phe Ala Ala Leu Ala Ala Ser Val Pro Gly Ala Arg Ser Ala Glu Glu 50 55 60

Val Arg Arg His Tyr Glu Ala Leu Val Glu Asp Val Ala Ala Engineer Asp 65 70 75 80

Ala Gly Arg Val Pro Leu Pro Arg Tyr Ala Gly Glu Glu Ser Ala Ala 85 90 95

Pro Pro Asp Gly Ala Gly Ala Ala Ala Ala Ala Ser Lys Asp Gly Gly 100 105 110

His Arg Arg Asp Glu Arg Lys Gly Gly Gly Gly Gly Gly Gly Tyr Asp Gly Gly 115 120 125

Lys Ser Cys Ser Lys Ala Glu Gin Glu Arg Arg Lys Gly lie Pro Trp 130 135 140

Thr Glu Glu Glu His Arg Leu Phe Leu Leu Gly Leu Asp Lys Phe Gly 145 150 155 160

Lys Gly Asp Trp Arg Ser lie Ser Arg Asn Phe Val lie Ser Arg Thr 165 170 175

Pro Thr Gin Val Ala Ser His Ala Gin Lys Tyr Phe lie Arg Leu Asn 180 185 190

Ser Met Asn Arg Asp Arg Arg Arg Ser Ser lie His Asp lie Thr Ser 195 200 205

Val Thr Ala Gly Asp Gin Val Ala Ala Gin Gin Gly Ala Pro lie Thr 210 215 220 8 8200413403

Gly His Gin Ala Thr Gly Asn Pro Ala Ala Ala Ala Leu Gly Pro Pro 225 230 235 240

Gly Met Lys His His His His His His Pro Gly Gly Ala Pro Pro Pro 245 250 255

Met Pro Met Tyr Ser Ala Ala Pro Met Gly His Pro Val Ala Gly His 260 265 270

Met Val Pro Ala Ala Val Gly Thr Pro Val Val Phe Pro Pro Gly His 275 280 285

Ala Pro Tyr Val Val Pro Val Gly Tyr Pro Ala Pro Pro Ala Lys Met 290 295 300

His Gin 305

&lt; 210 &gt; 8 &lt; 211 &gt; 276 &lt; 212 &gt; PRT &lt; 213 &gt; Rice (Japanese cultivated population) &lt; 400 &gt; 8

Met Pro Asn Leu Thr Ser Gong Asp Pro Leu Pro Val Pro Ala Asp Gly 15 10 15

Gly Lys Arg Arg Ala Ser Asp Asp Ser Glu Leu Ala Ser Gly Gin Gin 20 25 30

Lys Arg Arg Arg Arg Lys Val Gin Glu Arg Lys Lys Gly Val Pro Trp 35 40 45

Thr Glu Glu Glu His Lys Lys Phe Leu Glu Gly Leu Arg Gin Leu Gly 50 55 60

Lys Gly Asp Trp Arg Gly lie Ser Lys Asn Phe Val Thr Ser Arg Thr 65 70 75 80

Ala Thr Gin Val Ala Ser His Ala Gin Lys Tyr Phe Leu Arg Gin Thr 85 90 95

Asn Pro Gly Lys Lys Lys Arg Arg Ala Ser Leu Phe Asp Val Val Ala 100 105 110

Glu Cys Ser Asp Asp Gin Leu Pro Ser Pro Gin Ser Val Gly Thr Lys 115 120 125

Pro Pro Thr Gin Asp lie lie His Thr Asp Arg Gly Asp Val Pro lie 130 135 140

Leu Ser Tyr Pro Val Ala Arg Gly Phe Arg Gly Asp Ser Val Gin Val 145 150 155 160

Asp Glu Leu Thr Glu Tyr Val Lys Arg Leu Lys Ala Ala Glu Asp Met 165 170 175

Ser Leu Ser Met lie Ser Gly Leu Glu Met Ala Ser Ser Ser lie Ser 180 185 190

Ser Leu Glu Leu Ser Gle Ala Pro Ser His Leu Arg Gle Asp Gly Ala 195 200 205

He Lys Gly Leu Gly Ser Lys Pro Asn Phe Pro Pro Lys Glu Phe Gly 210 215 220

Ser Ala Ser Ala Thr Val Phe Cys Pro Pro Cys Cys Cys Leu Leu Leu 225 230 235 240

Leu Phe Phe Phe Phe Phe Phe Phe Ala Gly Val Val Cys Cys Cys Cys Cys 245 250 255

Cys Ser Cys His Ala Asn Phe Val Phe Gly Ser Cys Gly Val Ser Phe 260 265 270

Thr Ser Phe lie 275 &lt; 210 &gt; 9 9 9200413403

&lt; 211 &gt; 318 &lt; 212 &gt; PRT &lt; 213 &gt; Rice (Japanese cultivated population) &lt; 400 &gt; 9

Met Thr Arg Arg Cys Ser His Cys Ser His Asn Gly His Asn Ser Arg 15 10 15

Thr Cys Pro Asn Arg Gly Val Lys lie Phe Gly Val Arg Leu Thr Asp 20 25 30

Gly Ser Worker Arg Lys Ser Ala Ser Met Gly Asn Leu Ser Leu Leu Ser 35 40 45

Ser Ala Ala Gly Ser Thr Ser Gly Gly Ala Ser Pro Ala Asp Gly Pro 50 55 60

Asp Ala Ala Pro Thr Ala Ala Asp Gly Tyr Ala Ser Asp Asp Phe Val 65 70 75 80

Gin Gly Phe Ser Ser Ala Thr Arg Asp Arg Lys Lys Gly Val Pro Trp 85 90 95

Thr Glu Glu Glu His Arg Arg Phe Leu Leu Gly Leu Gin Lys Leu Gly 100 105 110

Lys Gly Asp Trp Arg Gly lie Ser Arg Asn Phe Val Val Ser Arg Thr 115 120 125

Pro Thr Gin Val Ala Ser His Ala Gin Lys Tyr Phe lie Arg Gin Ser 130 135 140

Asn Met Thr Arg Arg Lys Arg Arg Ser Ser Leu Phe Asp Met Val Pro 145 150 155 160

Asp Glu Ser Met Asp Leu Pro Pro Leu Pro Gly Gly Gin Glu Pro Glu 165 170 175

Thr Gin Val Leu Asn Gin Pro Ala Leu Pro Pro Pro Lys Glu Glu Glu 180 185 190

Glu Val Asp Ser Met Glu Ser Asp Thr Ser Ala Val Ala Glu Ser Ser 195 200 205

Ser Ala Ser Ala lie Met Pro Asp Asn Leu Gin Ser Thr Tyr Pro Val 210 215 220 Industrial Val Pro Ala Tyr Phe Ser Pro Phe Leu Gin Phe Ser Val Pro Phe 225 230 235 240

Trp Gin Asn Gin Lys Asp Glu Asp Gly Pro Val Gin Glu Thr His Glu 245 250 255 Le Val Lys Pro Val Pro Val His Ser Lys Ser Pro lie Asn Val Asp 260 265 270

Glu Leu Val Gly Met Ser Lys Leu Ser lie Gly Glu Ser Asn Gin Glu 275 280 285

Thr Glu Ser Thr Ser Leu Ser Leu Asn Leu Val Gly Gly Gin Asn Arg 290 295 300

Gin Ser Ala Phe His Ala Asn Pro Pro Thr Arg Ala Gin Ala 305 310 315 &lt; 210 &gt; 10 &lt; 211 &gt; 42 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &lt; 223 &gt; Nucleic acid &lt; 400 &gt; 10 aattctatcc atatccatat ccatatccat atccatatcc ac 42 200413403 ίο &lt; 210 &gt; 11 &lt; 211 &gt; 38 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence &lt; 220 &gt; &lt; 223 &gt; Ru acid &lt; 400 &gt; 11 gtggatatgg atatggatat ggatatggat atggatag &lt; 210 &gt; 12 &lt; 211 &gt; 29 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223 &gt; Primer &lt; 400 &gt; 12 aaccgag agg ; 210 &gt; 13 &lt; 211 &gt; 31 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223 &gt; Primer &lt; 400 &gt; 13 atcgaattct cattggtgca tcttggccgg a &lt; 210 &gt; 14 &lt; 211 &gt; 29 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223 &gt; Primer &lt; 400 &gt; 14 aaactcgaga atgcccaacc tcacctcca &lt; 210 &gt; 15 &lt; 211 &gt; 27 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223 &gt; Primer &lt; 400 &gt; 15 agcgaattct tatataaaac tggtgaa &lt; 210 &gt; 16 &lt; 211 &gt; 30 38 29 31 29

27 11200413403 &lt; 212 &gt; DMA &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 223 &gt; primer &lt; 400 &gt; 16 aaactcgagt atgacgaggc ggtgctcgca 30 &lt; 210 &gt; 17 &lt; 211 &gt; 27 &lt; 212 &gt; DNA &lt;213; Artificial Sequence &lt; 220 &gt; &lt; 223 &gt; Primer &lt; 400 &gt; 17 atcgaattct catgcctgtg cccttgt 27 &lt; 210 &gt; 18 &lt; 211 &gt; 25 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 220 &gt; &lt; 223 &gt; Primers &lt; 400 &gt; 18 ccagaattct gcaaagatgg ataaa 25 &lt; 210 &gt; 19 &lt; 211 &gt; 25 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 223 &gt; primer &lt; 400 &gt; 19 ccactcgagc tctctttt &lt; 210 &gt; 20 &lt; 211 &gt; 26 &lt; 212 &gt; DNA &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 223 &gt; primer &lt; 400 &gt; 20 ccagaattca gatgaagcta ctgtct 26 &lt; 210 &gt; 21 &lt; 211 &gt; 27 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial sequence 200413403 &lt; 220 &gt; &lt; 223 &gt; Primer &lt; 400 &gt; 21 ccactcgagt tcgatacag t caactgt

Claims (1)

The scope of patent application ... L A pure multi-month-old, which contains at least the same amino acid sequence as SEQ ID NO: 7, 8, or 9, wherein the polypeptide can regulate the expression in the cell. 1 is as long as one month as the scope of the patent application, wherein the amino acid sequence is at least 80% identical to SEQ ID NO: 7, 8 or 9. The polypeptide of item 2 of the patent claim, wherein the amino acid sequence and π. ID NO: 7, 8 or 9 is at least 90% identical. 4. The polypeptide as described in claim 3 of the patent scope, wherein the amino acid sequence is at least 95% identical to SEQ ID NO: 7, 8 or 9. 5. The polypeptide according to item 4 of the patent application, wherein the amino acid sequence is SEQ ID NO: 7, 8 or 9. 6. The polypeptide according to claim 1, wherein the cell is a plant cell. 7. The polypeptide according to item 6 of the application, wherein the cell is a monocotyledonous plant cell. 8. The polypeptide according to item 7 in the scope of the patent application, wherein the cell is a plant cell of pupae. 9. The polypeptide according to item 8 of the application, wherein the cell is a rice cell. 10. The polypeptide of claim 8 in which the cell is a barley cell. 11. The polypeptide according to item 1 of the patent application scope, wherein the expression of the gene is driven by a promoter containing one or more sets of TATCCA sequences. 12. The polypeptide according to item n of the application, wherein the cell is a plant cell. 13. A polypeptide as claimed in claim 1, wherein the cell expresses a HvMYBGa protein. O: \ 85 \ 85981.DOC 200413403 14. The polypeptide according to item 13 of the patent application scope, wherein the cell is a plant cell. 15 · The polypeptide according to item 13 of the patent application, wherein the expression of the gene is driven by a promoter containing one or more sets of TATCCA sequences. 16 · The polypeptide according to item 15 of the application, wherein the cell is a plant cell. 17. An isolated nucleic acid 'is characterized in that it will hybridize to g E Q ID NO: 1, 2 or 3 or its complementary sequence under severe conditions. 18. The nucleic acid according to item 17 of the patent application, wherein the nucleic acid is a complementary sequence of SEQ m NO: 1, 2 or 3 ω. 19. The nucleic acid according to item 7 of the scope of patent application, wherein the nucleic acid encodes the polypeptide of item 1 of the scope of patent application. 20. The nucleic acid according to claim 19, wherein the nucleic acid encodes the polypeptide according to claim 5. 21. · An antibody against the polypeptide of SEQ ID NO: 7, 8 or 9. 22. Cells containing a nucleic acid according to claim 17 in which the nucleic acid is expressed. 2 3 If the cell of claim 22 is claimed, the cell contains the nucleic acid of claim 20 of the patent. 24. A method for producing a transgenic plant, comprising the step of culturing a plant cell comprising a nucleic acid according to claim 17 of the patent application, wherein the nucleic acid is expressed in the plant cell. 25. The transgenic plant according to item 24 of the application, wherein the plant is a monocotyledon. 6. The transgenic plant according to item 25 of the Shen Qiao patent, wherein the plant is a pheasant. 0: \ 85 \ 8598l.D〇C -2-^ 00413403 27. 28. 29. 30. 31. 32. 33. 34. 35. The transgenic plant of item 26 in the scope of patent application, such as the plant For rice. The female claims that the transgenic plant of the patent No. 26, wherein the plant is barley. For example, the genetically-transplanted plant with the scope of the patent claim No. 24, wherein the transfer gene contains the nucleic acid with the scope of the patent application no. ^ The genetically modified plant of claim 29, wherein the plant is a monocotyledonous plant. # 专 ^ gItem 3g is a gene transgenic plant, wherein the plant is a tadpole. For example, the transgenic plant in the scope of application for item J1, wherein the plant is rice. For example, the gene transgenic plant of item 3 丨 alfalfa L +, wherein the plant is a method of expressing a transcript in a cell: the method includes: The transcript is introduced into a cell, and the transcript is expressed in the cell; wherein the transcript has a special NO:!, Which is hybridized with the complementary sequence of SEQ I] 5 under severe conditions. The method of applying #patent scope No. 34, and the scope of patent application No. 5, Xixixi, wherein the nucleic acid encoding application ^ v &lt; multi-month too. O: \ 85 \ 85981.DOC 200413403 柒. Designated representative map: (1) The designated representative map in this case is: (none) map. (2) Brief description of the component representative symbols in this representative figure: (No component representative symbols) 捌. If there is a chemical formula in this case, please disclose the chemical formula that can best show the characteristics of the invention: (none) O: \ 85 \ 85981.DOC