TWI332508B - Plant myb proteins - Google Patents

Description

1332508 发明, DESCRIPTION OF INVENTION: TECHNICAL FIELD The present invention relates to a MYB protein and its isolated nucleic acid, and a transformed cell or a genetically transformed plant containing the same. [Prior Art] The MYB protein is a group of transcription factors. The myb of the cell is an oncogene (c-myb) involved in the proliferation and/or differentiation of hematopoietic cells (Graf (1992) cun> Op. Gen. Devel. 2, 249-255). Common to all MYB gene products is the DNA-binding domain, which is highly retained at the N-terminus. In animals, the DNA binding region consists of three incomplete repetitive units, each unit having about 51 to 52 amino acids (designated R2 and R3) having 18 or 19 amino acids regularly. Separation of the remaining tryptophan residues at three degrees (Weston (1999) Curr. Op. Gene. Devel. 8, 76-81) » In plants and yeast, the main MYB protein has two repeats 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 repetitive unit or containing three repetitive 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 Cell 9, 491-507; Braun and Grotewold (1999) Plant Physiol. 121, 21-24) 〇【Contents of the Invention】

O:\85\859SI.DOC 1332508 The basis of the present invention is three novel rice genes encoding MYB proteins, which are designated OsMYBSl, OsMYBS2 and OsMYBS3, respectively. These proteins bind to a promoter containing one or more sets of TATCC A sequences and modulate promoter activity. The full-length OsMYBS1, OsMYBS2 and OsMYBS3 cDNAs (designated SEQ ID NOs: 1 to 3, respectively) are shown below, wherein the start codon and the stop codon are underlined.

OsMYBSl cDNA ( SEQ ID NO : 1):

GTGCGAGATCCACCACCCGATGACCTCCCAGGCGGCGACGACGACGACCACGGCGGCGGCGGCGGCGGCGT

GGACCAGGGAGGACGACAAGGCGTTCGAGAACGCGCTCGCGGCTTGCGCGGCGCCGCCGCCCGCGGACGGA

GGCGCGCCCGACGACGACTGGTTCGCCGCGCTCGCCGCGAGCGTGCCCGGGGCGAGGTCGGCGGAGGAGGT

GCGGAGGCACTACGAGGCGCTGGTGGAGGACGTCGCGGCCATCGACGCGGGCCGCGTCCCGCTCCCGCGCT

ACGCCGGGGAGGAGTCCGCGGCGCCGCCCGACGGAGCCGGAGCCGCCGCCGCCGCGTCCAAGGACGGCGGA

CACCGGCGCGACGAGCGCAAGGGCGGCGGCGGCGGGTACGACGGCGGCAAGAGCTGCTCCAAGGCGGAGCA

GGAGAGGCGCAAGGGCATCCCATGGACGGAGGAAGAGCACAGGCTGTTCTTGCTGGGGCTGGACAAGTTCG

GCAAGGGGGACTGGCGGAGCATCTCGCGCAACTTCGTCATCTCGCGGACGCCAACGCAGGTGGCGAGCCAC

GCGCAGAAGTACTTCATCCGCCTCAACTCCATGAACCGCGACCGCCGCCGCTCCAGCATCCACGACATCAC

CAGCGTCACCGCCGGCGATCAGGTCGCCGCGCAGCAGGGCGCCCCGATCACCGGCCACCAGGCCACGGGCA

ACCCCGCGGCGGCGGCGCTGGGCCCGCCGGGCATGAAGCACCACCACCACCACCACCCGGGCGGCGCGCCG

CCGCCCATGCCCATGTACAGCGCCGCGCCCATGGGCCACCCCGTCGCCGGCCACATGGTGCCCGCCGCCGT

CGGCACGCCGGTGGTGTTCCCGCCGGGCCACGCGCCGTACGTCGTGCCCGTCGGCTACCCGGCGCCTCCGG

CCAAGATGCACCAATGACGCGCCATGGACGGACATGAGCAGCATTTCTTCCTCCTCCTTTCTTGATGTCAA

TCTTGATTTGTTCTTTGTGTAGTCGCCGGCTCATCGTCCCTGATCATCTTGTTCTTCTCACAATCTCACTA

ATGTAAACATACATAGATCAGATGCCAAGAGTGCAGGGATTGGGGATTAAAGGCGAATAAGTAAAGTATTT

TGCTGACTGTTTGCAAGTGATCATCACGTACACCCGGTGA^AGCTTAGCTCCAAATGTGGATGTAATTAGC

AGCGC-CCTTCCGTACGTGGTGGCGCCGATCGATGATCTTGCAGGGGTTGCAATTAGGGATTGATTTCCATT

TTGCTGATGTAAATTTGCCAACTGTCTCATTGGACCAAftAAAAAAAAAAAAA O:\8S\8S981.DOC -6- 1332508

OsMYBS2 cDNA (SEQ ID NO: 2):

3CAAACGGCGCGCC ^AGAAAGGGGTACC

3AACTTTG

CGAGGTCCGCGGCGGCGGCGGCGGAGTTGACGAGGAGGAGTACGAGGAGGAGGAGGTGGAGGGTGGATTGTTCATCA AGAAGAGCTCCAGTATGCCCAACCTCACCTCCATCGACCCGCTGCCGGTGCCGGCCGACGGCGGC TCCGACGACTCCGAGCTCGCCTCCGGCCAGCAGAAGCGCCGCCGCCGCAAGGTGCAGGAGAGGAAG ATGGACTGAGGAGGAGCACAAGAAATTCCTGGAAGGGCTGAGGCAGCTGGGGAAAGGGGACTGGAGAGGCATCTCCA rGTGACCAGCAGGACGGCGACTCAGGTGGCCAGCCACGCCCAGAAGTACTTCCTCCGGCAGACCAACCCT GGCAAAAAGAAGCGCCGGGCCAGCCTCTTTGATGTTGTTGCTGAGTGCAGTGATGATCAACTTCCAAGTCCTCAGAG TGTTGGAACTAAGCCTCCTACCCAGGATATAATTCATACAGATCGCGGCGATGTCCCGATACTAAGCTATCCAGTTG CTAGAGGCTTTAGAGGCGATAGCGTGCAGGTTGATGAACTAACTGAATATGTGAAGAGATTAAAGGCCGCCGAGGAC ATGTCGCTCTCCATGATCTCTGGACTGGAAATGGCATCATCATCCATCAGCAGTCTAGAGCTCAGTATCGCGCCCTC TCATTTGCGGATCGACGGGGCCATCAAGGGGCTGGGATCCAAACCCAATTTTCCCCCGAAGGAArrrGGATCGGCTT CAGCTACTGTTTTTTGTCCCCCCTGTTGTTGTTTGTTGTTGTTGTrrTTTTTTTTTTTTriTGCGGGGGTTGITrG ^ TGTTGTTGTTGTTGTAGTTGTCATGCTAACTTTGTATTTGGGTCATGTGGGGTTTCTTTCACCAGTTTTATATAATA CAGAGAGAATGTCAGTCCCTTCCGAGACATGTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

OsMYBS3 cDNA (SEQ ID NO: 3):

ATCGATCGATCGATCTCCATAGGTGGGGGAAGGGAAGCTTTGGAAGGTGGAGGGACGGAGGGGGGGATGACGAGGCG

GTGCTCGCACTGCAGCCACAACGGGCACAACTCGCGGACGTGCCCCAACCGCGGGGTCAAGATCTTCGGGGTGCGCC

TCACCGATGGCTCCATCCGCAAGAGCGCCAGCATGGGGAACCTCTCCCTCCTCTCCTCCGCCGCCGGATCCACCAGC

GGCGGCGCCTCCCCCGCCGACGGCCCCGACGCCGCCCCCACCGCCGCCGACGGCTACGCCTCCGACGACTTCGTCCA

GGGCTTCTCCTCCGCCACCCGCGACCGCAAGAAGGGTGTTCCTTGGACTGAAGAAGAACACCGGAGGTTTTTGCTTG

GATTGCAAAAGCTTGGCAAAGGTGATTGGCGAGGAATCTCTCGTAATTTCGTGGTCTCAAGAACACCTACTCAAGTA

GCCAGTCATGCTCAGAAATATTTTATACGCCAATCCAATATGACCAGAAGGAAAAGAAGGTCTAGCCTTTTTGACAT

GGTGCCAGATGAGTCTATGGACCrrCCACCACTTCCTGGAGGTCAAGAACCAGAGACCCAA.GTArTAAATCAACCAG

CACTACCTCCACCGAAGGAGGAAGAGGAGGTAGATTCTATGGAGTCAGATACTTCTGCCGTTGCAGAGAGCTCrrCC

GCTTCTGCTATCATGCCAGATAATTrGCAGTCGACCTATCCAGTGATTGTTCCAGCrrATTrCTCGCCCTTTTTCCA

ATTCTCGGTTCCTTTCTGGCAAAATCAGAAAGATGAAGATGGTCCTGTGCAAGAAACACATGAGATTGTCAAGCCTG

TTCCAGTTCATTCAAAGAGCCCAATCAACGTTGATGAGCTTGTTGGCATGTCGAAGCTCAGCATAGGAGAGTCCAAT

CAAGAGACAGAGTCTACTrCTCTTTCATTAAATCTGGTAGGAGGTCAAAATAGACAATCAGCTTTCCATGCAAATCC

ACCAACAAGGGCACAGGCATGATCTGGTTGTGCACACAACTGCATTTAGATGAATCCCAGGCAAAATAAGCTTTGCC

TCCTTGTTTTTTrGnTTTATTTTAAGATTAACCGTTCTCCGTAGTCrGTATCATGTGCTGTAAGTTATGCTATGTA

TGAATGTATCTGTTGTTTGTCTGGCACACATGATAAATCACTCfATGTTAACAAAATCAGTAATGGTAGTGCTGATC

TTCGTGGTTGTACTGTTGTAAACTCTrTTATAAGAAAAAAAAATATTAGTTAGTC

O:\8S\8S9S1.DOC 1332508 The nucleic acid sequences encoding the OsMYBS1, OsMYBS2 and OsMYBS3 proteins (ie, the ATG start codon in SEQ ID NOS: 1 to 3 to a codon before the stop codon) are designated as SEQ ID, respectively. NOS: 4 to 6. The OsMYBS1, OsMYBS2 and OsMYBS3 proteins (designated SEQ ID NOS: 7 to 9 respectively) encoded by the above cDNAs are as follows:

OsMYBS1 protein (SEQ ID NO: 7):

MTSQAATTTTTAAAAAAWTREDDKAFENALAACAAPPPADGGAPDDDWFAALAASVPGARSAZEVRRHYEALVEDVA

AIDAGRVPLPRYAGE^SAAPPDGAGAAAAASkDGGHRRDERKGGGGGYDGGK^CSKAEQERRKGIPWXEEZHRLFLL

• 23LDKFG: PAAAALGPPGM

ICGDWRSISHiiTV GPPGMKHHHHHH

ISRTPTQVASHAQKYFIRLNSMNRDRRilSSIHIMTSVTAGDQVAAQQGAPlTGHQATGN

PGGAPPPMPMYSAAPMGHPVAGHMVPAAVGTPWFPPGHAPYWPVGYPAPPAXMHQ

OsMYBS2 protein (SEQ ID NO 8):

MPNLTSIDPLPVPADGGKRRAS SRTATQVASHAQKYFL'RQTNPG

DDSELAS KKKRRAS

GQQKRRRRKVQERKKGVPWTEEEHKKFLEGLRCiLGKGDWRG SKNFVT LFDWASCSDDQLPSPQSVGTKPPTQDIIHTDRGDVPILSYPVARGFR

GDSVQVDEI/TSYVKRLKAAZDMSLSMISGLEMASSSISSLELSIAPSHLRIDGAIKG^iGSKPNFPPKEFGSASATVF

CPPCCCLLLLFFFFFFAGWCCCCCCSCHANFVFGSCGVSFTSFI

OsMYBS3 protein (SEQ ID NO 9): ΜΤΚΙ^5Η<:5ΗΝσΗΝ5ΙΙΤζ:ΡΝΗσνΚΙΓσνΚΙίΤ〇σ5ΙΗΚ5Α3ΜσΝΙ^Ι^55ΑΑσ5Τ5〇σΑ3ΡΑΌΟΡ0ΑΑΡΤΑΑΒσΥΑ50

DFVQGFSSATRDRKKGVPWTESEHRRFLLGLQKLGKGDWRGISRNFWSRTPTQVAiSHAQKyFIRQSNMTRRKRRSS

LFDMVPDESMDLPPLPGGQEPETQVLNQPALPPPKEESEVDSMESDTSAVAESSSASAIMPDNLQSTYPVIVPAYFS

PFLQFSVPFWQNQKDEDGPVQETH2IVKPV?VKSKSPINVDELVGMSKLSIGESNQETESTSLSLNLVGGQNHQSAF

HANPPTRAQA Accordingly, the invention features a pure polypeptide comprising at least 70% (e.g., at least 75, 80, 85, 90 or 95, or 100%) identical to SEQ ID NO: 7, 8, or 9. Amino acid sequence. When expressed in a cell, the polypeptide binds to a promoter and modulates the transcription under the control of the promoter. The cell may be a plant cell, especially a monocot plant cell (e.g., a terpene plant cell, such as a rice cell or a barley cell). The promoter may contain one

O:\8S\8S98I.DOC 1332508 Set or sets of TATCCA sequences or variants thereof. Furthermore, HvMYBGa proteins can be expressed together in cells. HvMYBGa interacts with OsMYBs proteins and cooperates to regulate gene expression. The polypeptide of the present invention can be used to produce an OsMYBS antibody (either monoclonal antibody or polyclonal antibody). These antibodies can ultimately be used to detect the presence and distribution of OsMYBS proteins in tissues and cell compartments. For example, such antibodies can be used to demonstrate the performance of OsMYBS proteins in genetically transgenic plants.

"Pure polypeptide" refers to a polypeptide that is substantially free of naturally associated molecules, i.e., at least 75% pure (e.g., at least 80, 85, 90, 95, or 100 deg. pure) on a dry weight basis. Purity can be determined by any suitable standard method, for example, by column chromatography, polypropylene gel electrophoresis or HPLC. The "percent identity of the two-stage amino acid sequence" was determined by the algorithm of Karlin and Altschul ((1990) Proc. Natl. Acad. Sci. USA. 87, 2264-2268), which was followed by Karlin and Altschul (( 1993) Proc. Natl. Acad. Sci. USA 90, 5873-5877) Modifications. The algorithm was incorporated

Altschul et al., XBLAST program ((1990) J. Mol. Biol. 215, 403-410). The BLAST protein search was performed using the XBLAST program, with 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 employed. When using the BLAST and Gapped BLAST programs, the default parameter 0 for using an individual program (for example, XBLAST) is available at www.ncbi.nlm.nih.gov. The invention includes isolated nucleic acids (e.g., vectors) encoding a polypeptide of the invention and cells comprising the nucleic acid of the invention (in culture or in gene transfer)

O:\85\85981.DOC in the plant). The cells may be plant cells, especially monocot plant cells (e. g. 'shell plant cells, such as rice cells or barley cells). Examples of the nucleic acid in the present invention include a nucleic acid characterized by being hybridizable to SEQ m Ν〇 1, 2 or 3 or its complementary sequence under severe conditions. The nucleic acid can be at least 15 (e.g., at least 3, 5, 1, 2, 5 or 1) nucleotides in length. Such nucleic acids and cells can be used to produce a genetically transformed plant or to produce a polypeptide of the invention. For example, the nucleic acid of the present invention can be used to determine whether mRNA of OsMYBS is expressed in tissues or cells. The nucleic acid can be used as a primer in a PCR detection method or as a labeled probe in a nucleic acid dot collapse method (e.g., northern blotting). An isolated nucleic acid is a nucleic acid that differs in structure from any naturally occurring nucleic acid structure or any fragment of a naturally occurring genomic nucleic acid. The term encompasses, for example, (a) a NA sequence having a partial sequence of a naturally occurring genomic DNA molecule, both of which are flanked by coding sequences flanking the portion of the molecule in the genome of the naturally occurring product; (b) incorporated A vector or nucleic acid in the genomic DNA of a prokaryote or eukaryote, and incorporated in such a way that the resulting molecule is not identical to any naturally occurring vector or genomic DNA; (c) individual knives, such as cDNA, genomic fragments a fragment or restriction fragment produced by a polymerase chain reaction (pCR), and (d) a recombinant nucleotide sequence of a portion of a hybrid gene (i.e., a gene encoding a fusion protein). Heterozygous under severe conditions means heterozygous at 65 °C, 0.5 X SSC, followed by 0.1 X SSC at 45 °C. The present invention <plant cells can be cultured to produce a genetically transformed plant. The genetically transgenic plant may be a monocotyledonous plant, for example, a mossy plant such as rice.

O:\8S\8S98I.OOC -10· 1332508 or barley. The genome of the genetically transgenic plant contains a transgenic gene comprising a nucleic acid characterized by being hybridizable to SEQ ID NO: 1, 2 or 3 or its complement under severe conditions. The transgene, when expressed in a cell, modulates the activity of a promoter (e.g., one or more sets of TATCCA sequences or variants thereof). Further, the present invention includes a method of expressing a transcript in a cell (e.g., a plant cell). The method comprises introducing a vector comprising a nucleic acid encoding a transcript into a cell, and rendering the transcript in the cell. This transcript is characterized by its ability to hybridize to SEQ ID NO: 1, 2 or 3 or its complement under stringent conditions. The transcript can encode a polypeptide of the invention that modulates the activity of an promoter (e.g., one or more sets of TATCCA sequences or variants thereof). Alternatively, the transcript can be an antisense RNA that inhibits the expression of the endogenous OsMYBS gene' thereby disabling the activity of a promoter (e.g., containing one or more sets of TATCCA sequences or variants thereof). The present invention provides a system for the highly regulated expression of endogenous or exogenous genes in plant cells or gene transfer plants. Some specific embodiments of the invention are described in the following description. The remaining features, objects, and advantages of the invention will be apparent from the description and appended claims. [Embodiment] The expression of the 'α-amylase hydrolase gene in the shell is rusted by Gibsin (Ga) and sugar starvation. All of the _ mobilization hydrolyzing enzyme genes isolated from the steroids contain TATCCA units or variants thereof at positions about 9 〇 to 15 bp upstream of the transcription start position. TATCCA unit is

O:\85\a5981.DOC 1332508 The GA reaction complex (GARC) of the α-starch hydrolase gene promoter and the important component of the sugar reaction complex (SRC).

Three cDNA lines encoding the novel MYB protein and having a single DNA binding region have been isolated from the rice suspension cell cDNA library and designated OsMYBS, OsMYBS2 and OsMYBS3. Unexpectedly, the three OsMYBS proteins specifically bind to the TATCCA unit in vitro. In vivo, OsMYBS1 and OsMYBS2 bind to the TATCCA unit, and when sugar is provided, it activates the promoter containing the TATCCA unit, while OsMYBS3 suppresses transcription of the promoter when the sugar is deficient. Furthermore, the three OsMYBS proteins cooperate with a GA-regulated transcriptional factor GAMyb (HvMYBGa) to activate a low pi barley powder hydrolyzing enzyme gene promoter in the absence of GA.

In one aspect, the invention relates to a pure OsMYBS polypeptide (eg, SEQ ID NS: 7 to 9) comprising a functional OsMYBS polypeptide. "Functional polypeptide" refers to a polypeptide 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 invention relates to an isolated OsMYBS nucleic acid (i.e., DNA, cDNA, and RNA) characterized by being hybridized to SEQ ID NO: 1, 2, or 3, or a complement thereof, under stringent conditions. The nucleic acids of the invention include degenerate sequences derived from the genetic code table. The nucleic acids of the invention can be expressed in vitro by transferring the DNA into a suitable host cell by methods known in the art. For example, the nucleic acid can be inserted into a recombinant expression vector. The 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 that have been transformed with phage DNA, plastid DNA, or viscous DNA expression vectors by weight O:\85\8598l.DOC • 12-1332508; recombinant yeast Bacteria expressing a transgenic yeast; and a plant cell transformed with a recombinant viral expression vector (eg, broccoli mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed by a recombinant plastid expression vector (eg, Ti plastid) system. The isolation and purification of the recombinant polypeptide or fragment thereof provided by the present invention can be carried out by a conventional method, including column chromatography for preparation and immunoseparation involving single or multiple antibodies.

The invention also provides an antibody against an OsMYBS polypeptide comprising a monoclonal antibody and a plurality of antibodies. The term "antibody" includes intact molecules and fragments thereof, such as Fab, F(ab')2 and Fv which bind to the epitopes present in the OsMYBS polypeptide. Methods of making single and multiple antibodies and fragments thereof are known in the art. See, for example, Harlow and Lane (1 988)

Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York °

Also included within the scope of the invention are gene-transgenic plants whose genome contains a transgenic gene comprising a nucleic acid which hybridizes under stringent conditions to SEQ ID NO: 1, 2 or 3 or its complement. The transgene, when expressed in a cell, modulates the activity of a promoter (e.g., one or more sets of TATCCA sequences or variants thereof). As used herein, the term "plant" refers to a whole plant, plant part, plant cell or a group of plant cells, such as plant tissue. Young plants are also included in the meaning of "plants". Plants encompassed by the present invention are any plants that can be modified by transformation techniques, including angiosperms, gymnosperms, monocots, and dicots. Examples of monocots include, but are not limited to, O:\85\8598I.DOC -13- 1332508 Asparagus, field corn and sweet 'corn, barley, wheat, rice, sorghum, scallions, pearl oysters, rye and oats. Examples of dicots include, but are not limited to, potatoes, tomatoes, tobacco, cotton, canola, farm beans, soybeans, peppers, lettuce, peas, alfalfa, alfalfa, mustard crops, or kale (eg, kale) Vegetables, broccoli, broccoli, Brussels sprouts, radishes, carrots, beets, glutinous rice, tired vegetables, forked melons, pumpkins, melons, cantaloupes, radish, and various decorative plants. Woody plants include poplar, pine, sequoia, cedar and oak. The genetically transformed plants can be made by methods known in the art. See, for example, Weissbach and Weissbach (1988) Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; Grierson and Corey (1988) Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9; Horsch, et al. (1985) Science 227, 1229

Furthermore, the invention provides a method of expressing a transcript in a cell (e.g., a plant cell). The method comprises introducing a vector comprising a nucleic acid encoding a transcript into a cell' and rendering the transcript in a cell. The transcript is characterized by being heterozygous for SEQ ID NO: 1, 2 or 3 or its complementary sequences under stringent conditions. The transcript can interfere with another RNA in the plant cell

Functions (eg, RNA that prevents mRNA from being translated into a polypeptide, or that triggers specific RNA degradation to facilitate the cessation of activity of the target post-transcriptional gene) (Mol et al. (1990) FEBS Lett 268(2), 427-30; and Fire et al (1998)

Nature 391, 806-811). Alternatively, the transcript can encode a polypeptide such that the polypeptide overexpresses in a transgenic plant cell or a genetically transgenic plant "at O:\8S\8S98I.DOC • 14-1332508

The altered tone of OsMYBS expression results in a change in the performance of the OsMYBS protein-regulated gene (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 invention and are in no way intended to limit the remainder of the disclosure. Those skilled in the art will be able to make full use of the present invention based on the teachings herein without undue effort. Quoted in this article

All publications are incorporated herein by reference in their entirety. Materials and Methods 1. Rice cell cultures as previously described (Yu, et al. (1991) J. Biol. Chem. 266, 2113 1- 21137) Establish a suspension cell culture of rice (Oryza sativa cv. Tainan 5). 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 30/〇 sucrose. Subculture is carried out. Set the cells to i2〇 rpm

Incubate on a reciprocating shaker and incubate at 26 ° C in the dark. 2. Plastoplasts cn Amy8-C carries a 1.4 kb rice alpha-housefly hydrolysate cDNA insert in τρ* pBluescript KS + (Stratagene) (Yu, et al. (1992) Gene 122, 247-253). The plastid pcRAcl.3 contains the 1.4 kb rice actin gene (Actl) in pBluescdptll-KS

cDNA insert (McElroy, et al. (1990) Plant Mol. Biol. 15, 257-268). The plastid pRY18 carries a 3_8 kb DNA fragment containing the rice genome rDNA plexus. The plexus includes 1 gs rRNA in puc 13 O:\S5U5981.DOC -15- 1332508

The 3' side half of the gene, the complete 5.8S rRNA gene and the 5' side half of the 25S rRNA gene (Sano and Sano (1990) Genome 33, 209-218). The plastid pGAMyb contains the HvMYBGa cDNA fused between the maize pantoxin (Ubi) promoter and the nopaline synthase (Nos) terminator (Cerc6s, et al. (1999) Plant J. 19, 107-118). The plastid pAHC 18 contains a luciferase (Luc) cDNA fused between the Ubi promoter and the Nos terminator (Bruce, et al. (1989) Proc. Natl. Acad. Sci. USA 86, 9692-9696). The plastid pAmy32b-GUS contains a 331-bp promoter region, a complete 5' untranslated sequence, the first intron of Amy32b 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. Southern-Western Screening of cDNA Library Rice suspension cells were cultured for 5 days in sucrose-containing medium and then transferred to a medium containing sucrose (+S) or sucrose-free (-S) for 4 hours. The cells were collected and the entire RNA was purified. Poly(A)+ RNA was further purified (5 Prime-3 Prime) using an oligo(dT) cellulose spin column. A cDNA library isolated from the AGE cells was constructed using poly(A)+RNA isolated from _s cells. The Escherichia coli Y1090 strain (Stratagene) was used as a bacterial host. For screening and plaque purification, a 416-bp DNA fragment containing 8 vertical repeat units of the _33 to -82 region of the amy3 promoter was excised from p3Luc.44 with pstI and xhol (Lu, et al (1998) J. Bi〇L chem. 273, 10120-10131) and used as a probe for cdNA library screening. The eDNA library was screened according to the method of Singh et al. (Singh et al, (1988) Cell 52, O: \85\8598l. DOC -16 - 1332508 4 15-423). Positive phage plaques were isolated and the phage plaques were purified for further identification of the features. 4. plastid construction

OsMYBSl and OsMYBS2 cDNAs were excised from λ GEM-2 with Sail and Notl and inserted into the same portion of pBluescript (Stratagene) to generate pBS-Sl and pBS-S2, respectively. Has one in the OsMYBS3 cDNA

Sail sites, therefore, OsMYBS3 cDNA with EcoRI and Notl

Cut out from the long GEM-2 and insert it into the same part of the pBluescrip to produce pBS-S3. pAHC 13 was cut with BamHI to remove the Luc cDNA insert and then blunt-ended. OsMYBSl and OsMYBS2 were excised from pBS-Sl and pBS-S2 by Sail and Notl, respectively, and blunt-ended. OsMYBS3 was excised from pBS_S3 with EcoRI and Notl and blunt-ended. The OsMYBS cDNAs were then ligated to the truncated pAHC18 to generate the Ubi-OsMYBS-Nos fusion gene. 5. Genomic DNA gel point collapse analysis Genomic DNA was isolated from rice callus according to the method of Sheu, et al. (1996) J. Biol. Chem. 27JU 26998-27004. Ten milligrams of genomic DNA was cleaved with restriction enzymes, separated on a 0.8% agarose gel, and then transferred to a nylon membrane (MSI). The 32P random primer-labeled OsMYBS gene-specific DNA was used as a probe and hybridization was carried out at 42 °C. pBS-Sl is cleaved with SacII and contains a 220-bp coding region and

The 613-bp DNA fragment of the 3 93-bp 3' untranslated region of OsMYBS1 cDNA was used as OsMYBS1-specific DNA. pBS-S2 was digested with SacI and O:\85\8598I.DOC -17- 1332508 was used as a 241-bp coding region and a 334-bp DNA fragment of the 93-bp 3' untranslated region of OsMYBS2 cDNA as OsMYBS2 - specific DNA. pBS-S3 was digested with SacI, and a 599-bp DNA fragment containing a 332-bp coding region and a 267 bp 3' untranslated region of OsMYBS3 was used as OsMYBS3-specific DNA. 6. RNA gel point collapse analysis Using TRIZOL reagent (GIBCO-BRL) to completely mobilize all RNA from rice

Purified in the cells. An α-32P-labeled DNA probe was prepared, and then subjected to RNA gel dot collapse analysis as described in the literature (Sheu, et al. (1996) J. Biol. Chem. 271, 26998-27004). When the spot on the membrane collapses with various probes

At the time, the membrane was removed and hybridized as described in the literature (Sheu, et al., (1994) Plant J. 5, 655-664). The plastid DNA of a Amy8-C and pcRAcl .3 was cleaved with EcoRI. a Amy3 and a Amy8 gene-specific DNA were prepared as described in the literature (Sheu, et al., (1996) J. Biol. Chem. 271, 26998-27004). These insert DNAs were individually isolated, labeled with a-32P and used as probes. A DNA fragment containing 25S, 18S and 5.8S rDNAs was excised from pRY18 with BamHI, labeled with α-32P and used as a probe to equalize RNA loading. 7. Expression and purification of recombinant protein OsMYBS was digested with Sail and Notl and ligated into the same portion of pET28b(+) (Novagen) to generate pET-S 1, pET-S2 and pET-S3. Each of the three plastids was transferred to Escherichia coli BL21 (DE3) strain and then expressed as OsMYBS, OsMYBS2 and OsMYBS3. Purification of OsMYBs protein was performed according to instructions provided by Novagen. Protein Concentration: \85\8 Outer 81.DOC -18· 1332508 Degrees were determined using Bradford reagent (6io-Rad). 8. Gel Mob丨丨ity Shift Assay Gel mobility shift analysis was performed as previously described (Lu, et al. (1998) j. Biol. Chem. 273, 10120-1013 1 ), but the DNA-protein binding reaction is achieved by 0.02 ng of 32P-labeled oligonucleotide: y: acid with 0.2 μg of recombinant OsMYBS purified from E. coli and 1 μg of poly(dl-dC) in total volume It was carried out in a reaction of 20 μl of the solution. The DNA fragment containing the TATA box was prepared as previously described (Lu, et al. (1998) J. Biol. Chem. 273, 10120-10131" 9. The construction of the reporter reporter strain (Reporter Strains) will be performed with EcoRI and Xhol A DNA fragment containing 8 column repeating units at -133 to -82 of the a Amy3 promoter was excised from p3Luc.44 (Lu, et al. (1998) J. Biol. Chem. 273, 10120-10131) Then, it is blunt-ended and inserted into the Smal site of pLacZi (Clontech) to generate the 8x (TATCCA+F)-Cyc 1 minimal promoter-LacZ fusion gene. The second-segment complementary oligonucleotide 5'-AATTCTATCCATATCCATATCCATATCC ATATCCATATCCAC- 3 and 5,-GTGGATATGGATATGGATATG GATATGGATATGGATAG-3' was ligated and inserted into the EcoRI and Smal sites of pLacZi to generate the 6xTATCCA-Cycl minimal promoter-LacZ fusion gene. According to the MATCHMAKER Single-Hybrid System method (Clontech) ), these plastids are linearized with Ncol and introduced into the genome of yeast YM4271 to produce a yeast reporter strain. 10. A yeast single-hybrid system for OsMYBS binding activity analysis in order to construct a GAD-OsMYBS fusion gene, Will be all OsMYBS cDNAs 1332508 were amplified by PCR using pBS-Sl, pBS-S2 and pBS-S3 as DNA templates, and OsMYBSl-5' (5'-AAACTCGAGAATGACCT CCCAGGCGGCGA-3', Xhol site underlined )and

OsMYBSl-3, (5'-ATCGAATTCTCATTGGTGCATCTTGGCCGGA-3', EcoRI part underlined) as an introduction to OsMYBSl, used

OsMYBS2-5, (5,-AAACTCGAGAATGCCCAACCTCACCTCCA-3, Xhol part is underlined) and OsMYBS2-3' (5’-

AGCGAATTCTTATATAAAACTGGTGAA-3 ', the EcoRI part is underlined) as the primer for OsMYBS2, and OsMYBS3-5' (5'-AAA CTCGAGTATGACGAGGCGGTGCTCGCA-3 ', Xhol part is below

Square line) and OsMYBS3-3' (5,-ATCGAATTCTCATGCCTGTGCC

CTTGT-3 ', the EcoRI part is underlined) as an introduction to OsMYBS3. The GAD sequence was amplified by PCR using pGAD-424 (Clontech) as a DNA template, and using an oligonucleotide: y: acid GAD-5H5, -CCAGAATTCTGCAAAGATGGATAAA-3, > EcoRI site underlined) and GAD-3' (5'-CCACTCGAGCTCTCTTT TTTTGGGT-3 ', Xho I site is underlined) as a primer. All PCR products were cut with Xhol and EcoRI. The OsMYBS cDNAs were each ligated to the GAD sequence in the Xhol site and inserted into the EcoRI site of pBluescript to generate pGAD-OsMYBS. The pGAD-424 was cleaved with Mlul and EcoRI to remove the 3' portion of the GAD region. The GAD3' partial-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:\8S\8S981 containing the ADH1 promoter-GAD-OsMYBS fusion gene. DOC -20· 1332508 pGAD-OsMYBS-424. Each of these plastids was introduced into a yeast reporter strain according to the MATCHMAKER single hybrid system method (Clontech), which has an 8x (TATCCA+F)-Cycl minimal promoter-LacZ fusion gene or a 6xTATCCA-Cycl minimal promoter- LacZ fusion gene. The enzyme activity of the /3-galactose site in the yeast was analyzed. 11. Yeast single-hybrid system for OsMYBS transactivation analysis In order to construct the GBD-OsMYBS fusion gene, the GBD sequence was amplified by PCR using pGBT9 (Clontech) as a DNA template and oligonucleotide nucleoside GBD-5' (5'-CCAGAATTCAGATGAAGCTACTG ^ TCT-3', EcoRI site is underlined) and GBD-3' (5'-CCA CTCGAGTTCGATACAGTCAACTGT-35, Xho I site underlined) as an introduction. Since there is a Xhol cleavage site in the GBD sequence, the PCR product was first cut with EcoRI and then cut with Xhol. The resulting DNA fragment was ligated to the OsMYBS cDNA and inserted into the EcoRI site of pBluescript to generate pGBD-OsMYBS. will

pGAD-424 was cleaved with Hindlll to remove the GAD sequence and blunt it. The GBD-OsMYBS fusion gene was excised from pGBD-OsMYBS with EcoRI and Hindlll, sharpened and ligated with truncated pGAD-424 to generate pGBD-OsMYBS containing the ADH1 promoter-GBD-OsMYBS fusion gene. 424 » These plastids were introduced into yeast strain CG-1945, which has a 3xUAS-Cycl minimal promoter-LacZ and a GAL1 promoter-HIS3 indicator construct (Clontech). 3 xUAS is a three-column repeating unit of the synthetic UASG17-mer universal sequence that can be recognized by GBD. Analysis of the /5-galactosidase activity in yeast or the growth of yeast on a selective culture of 0:\SS\8S9ei.00C • 21· 1332508. 12./3 galactose chymase activity and yeast cell growth fraction in the transfected yeast cells without galactose chymase activity by using ONPG (o-nitrophenyl cold-galactopyranoside) as Quantitative and quantitative. The transformed cells were screened in a medium containing 5 mM 3-aminotriazole but no histidine according to the yeast method manual (Clontech). Temporary performance analysis of I3·barley aleurone tissue Particle bombardment of barley tissue of Hordeum vulgare and GUS transient analysis of large systems such as Lanahan, et al. (1992) Plant Cell 4, 203-211 and Cerc0s, et al. (1999) Plant J. 19, 107-118. The bombarded barley embryo-free half-seeded seeds were cultured for 20 hours in a buffer containing or lacking glucose or GA3 (CaCl2 and sodium succinate, 20 mM each, pH 5.0), and assayed for luciferase or GUS activity. All Mori hits at least 4 times. Results 1. Three OsMYBS genes encoding a MYB protein with one DNA-binding repeater unit. In order to obtain cDNAs encoding proteins capable of specifically binding to the TATCCA unit, screening from poly(A)+ mRNA was performed using a Southern blotting method. The resulting cDNA library 'where the poly(A)+ mRNA line was prepared from rice suspension cells depleted of sucrose for 4 hours. A 416-bp probe consisting of 8 column repeat units of the -33 to 82 DNA fragment of the Amy3 promoter was used (Lu, et al. (1998) J. Biol. Chem. 273, 10120-10131) , which contains the TATCCA unit, separates the cdna O:\ftSSS5991.DOC • 22- 1332508 that specifically interacts with the probe and analyzes its sequence. Three different genes were identified and named OsMYBS, OsMYBS2 and OsMYBS3. The open coding regions of OsMYBS, 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, Drosophila 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 are less correlated with AtMYBL2, PcMYBl and CCA1 (AtMYBCCAl). StMYBl will activate the cauliflower mosaic virus 335RNA gene (CaMV35S) promoter, which is expressed in various organs of the potato.

(Baranowskij, et al. (1994) EMBO J 13,5383-5392). AtMYBL2 is expressed in the leaves of Arabidopsis, but its function has not yet been identified (Kirik and BSumlein, (1996) Gene 183, 109-113). PcMYB1 interacts with light-regulatory promoter units in vivo and is expressed in cell cultures and seedlings of Dutch criaww (Feldbriigge, et al. (1997) Plant J. 11, 1079-1093) « AtMYBCCAl Binding to Arabidopsis light-collecting the promoter of the chloroplast a/b binding protein gene (Lhcb), and the light-sensitive element of the Lhcb

Modulation of (phytochrome) (Wang, et al. (1997) Plant Cell 9, 491-507). The 1R sequence of OsMYBS3 is most similar to StMYBl (92°/〇 identical) and is similar to OsMYBS 1 (87% identical) and OsMYBS2 (85% identical), while the 1R sequences of OsMYBS1 and OsMYBS2 are the most similar to each other. (77% identical). All MYB O:\SS\8S98I.DOC -23- 1332508 proteins with 1R DNA_binding region are very similar to each other in the N- and C-terminal regions outside the 1R region, but OsMYBS3 and StMYBl are at the N-terminal The 90-amino acid region has 71% identity and is 62% identical in the 70-amino acid region of the C-terminus. Comparison of the 1R region of the three OsMYBS proteins with the amino acid residues between the R2 and R3 repeating units of the animal and plant MYB proteins revealed that the 1R region contains retained tryptophanic acid (W), chitosan at the same position ( E), glycine (G) and arginine (R). A tryptophan residue that plays an important role in the DNA binding region of the MYB protein of the stable animal (Ogata, et al. (1992) Proc. Natl. Acad. Sci. USA 89, 6428-6432) in most IR intervals, The first and second positions are reserved, but not in the third position. The putative base-contact residue in the third helix of the animal MYB protein is 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% identity), which is used as a gene-specific DNA probe for rice genomic DNA gel break analysis. The three gene-specific DNA probes are each only heterozygous for a single band. This result shows that each of the three OsMYBS gene lines in the rice MYB gene family is a single set of genes. 2. OsMYBS gene expression in various rice tissues and barley aleurone The performance pattern of OsMYBS in rice was analyzed. All RNA was purified from various rice tissues, and OsMYBS-specific DNA was used as a probe for gel point collapse analysis. OsMY.BS 1 is in the middle of the organization; it is now - and has the highest degree of performance in the leaves. -0sMYBS2 main · be expressed in roots, leaves and senescent leaves, and the degree of expression is the same, while OsMYBS3 is expressed in all tissues and has the highest performance in aged leaves. O:\8S\SS981.DOC -24- 1332508 In order to determine whether the OsMYBS gene is regulated by sugar, all RNA is isolated from rice suspension cells cultured for 48 hours in the presence or absence of sucrose, and OsMYBS-specific Sex DNAs were used as probes for gel point collapse analysis. OsMYBS 1 and OsMYBS3 mRNA were maintained in a low amount in the presence of sucrose, but increased in the absence of the recommended sugar, which was consistent with the mRNA content of a Amy3 and another rice Q:-starch 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 carried out in the following experiment in which half of the barley seeds were used as a system for studying the functions of sugar and hormone regulation of the OsMYBS protein. To determine whether the OsMYBS gene was expressed in barley aleurone cells, all RNA was isolated from half of the barley seeds and then gel-pointed using OsMYBS-specific DNAs as probes. All of these OsMYBS genes are in barley aleurone cells in the absence of glucose

which performed. The performance of OsMYBSl and OsMYBS3 is in the presence of glucose or GA

It is suppressed, but remains unchanged in the presence of sputum. The performance of OsMYBS2 increased in the presence of glucose, but was slightly inhibited by sputum treatment. 3. Specific interaction of OsMYBS protein with TATCCA in vitro In order to determine whether the three OsMYBS proteins specifically bind to the TATCCA unit, they are expressed in E. coli. The interaction between the affinity-purified recombinant OsMYBS protein and the five DNA fragments covering the -171 to -82 region of a Amy3 SRS was analyzed by gel shift. The five fragments (designated F1 to F5) were previously explored for their gel migration shift analysis with the interaction of nuclear protein extracts from rice suspension cells. O:\85\8S981.DOC •25· 1332508 (Lu, et al. (1998) J. Biol Chem. 273, 10120-10131). F2 was used as a probe for gel shift analysis, which contained 2 TATCCA units and some flanking sequences in the SRS. Three kinds of reorganization

The OsMYBS protein binds to probe F2 to form a low-shift complex. The interaction between F2 and OsMYBSl or 〇sMYBS2, only the DNA fragments of F2 and 6 vertical repeat units containing 6_bp TATCCA units can compete with it's but the other DNA fragments of 500-fold excess cannot compete with it. . The interaction between F2 and 0sMYBS3, and only the F2, F5 (including TATCCA units) and the DNA fragments of the 6 column repeat units containing the 6-bp TATCCA unit can compete with it. A 17-bp DNA fragment containing the TATA box sequence (taTATA) (only two cores are different from the TATCCA unit: y: acid) The method is combined with the competition. To further demonstrate the specificity of the association between the OsMYBS protein and the TATCCA unit, a series of site-directed mutagenesis spanning the -119 to -102 region of the TATCCA column repeat unit was constructed. The wild type (Wt) sequence was used as a probe for gel shift analysis. The three recombinant OSMYbs proteins bind to the wt# knowing needle to form a low migration complex. Furthermore, the combination of these individual sMYBS proteins can be completely competitive with unlabeled Wt sequences. The DNA sequence (Ml), which has mutations in both repetitive units, is incapable of competing, and in the two early years, the displaced DNA sequences (M2 and M3) compete with lower efficiency. Three of the six bases of the two overlapping units (M4, M5, and M6) competed only slightly. These results suggest that all of these 〇sMYBS proteins can specifically bind to two sets of TATCCA units. However, if only one set of TATCCA exists, the binding affinity will decrease by O:\85\8598I.DOC •26- 1332508. 4. Interaction between OsMYBS protein and TATCCA unit in vivo

In vivo, the binding activity of the OsMYBS protein to the TATCCA unit was analyzed using a yeast single hybrid system. The full-length OsMYBS sequences were each fused at the C-terminus to the activation region of the yeast GAL4 transcriptional activator (GAD). The GAD-OsMYBS chimeric gene was then fused to the downstream region of the yeast ADH1 promoter to serve as an effector construct. The yeast strain containing the Cycl minimal promoter-LacZ fusion reporter construct was then transformed with the construct of the construct. The presence of GAD alone or in combination with the GAD-OsMYBS fusion protein does not result in the performance of LacZ. The 8 vertical column repeating units of the A Amy3-133 to -82 DNA sequence (designated 8 X (TATCCA+F) are fused to the upstream region of the Cycl minimal promoter-LacZ coding region to serve as a reporter construct. First, the reporter construct is transported into the yeast, and then the yeast is transformed into a construct containing the ADH1-GAD-OsMybS fusion gene. The GAD alone stores % - which increases the LacZ performance. 5 times, this means that the TATCCA unit with the flanking sequence will slightly increase the Cycl minimum promoter activity even in the absence of OsMYBS. Compared with the LacZ performance in the absence of OsMYBS, the presence of GAD-OsMYBSl and GAD-OsMYBS2 will be Increasing the LacZ performance by 3 and 5 times. In contrast, the presence of OsMYBS3 reduces the LacZ performance by a factor of 2. To test whether the flanking sequence of the TATCCA unit will affect,

The binding affinity of OsMYBS 1 and OsMYBS2, 6-bp TATCCA single O:\8S\8S98I.DOC -27· 1332508 yuan 6 column repeat unit (named 6xTATCCA) and Cycl minimal promoter-LacZ coding region The upstream area is fused to serve as a reporting sub-structure. The reporter construct was transferred into a yeast, and the yeast was transformed into a construct containing an ADPU-GAD-OsMYBS fusion gene. The LacZ performance is increased only in the presence of GAD-OsMYBS2. 5.0sMYBSl and OsMYBS2 are transcriptional activators

To determine whether OsMYBS is a transcriptional activator, the full-length OsMYBS sequence was fused to the C-terminus of the DNA-binding region of GAL4 (GBD). This chimeric gene was then fused to the downstream region of the yeast ADH1 promoter to serve as an effector construct. The yeast strain containing the 3xUAS-Cycl minimal promoter-LacZ fusion reporter construct was transformed with the construct of the activator. The presence of the GBD-OsMybSl fusion protein increased the LacZ performance by 8 times, and the GBD-OsMYBS2 was only slightly Increasing LacZ performance, as well as GBD-OsMYBS3 does not increase LacZ performance.

The yeast strain containing the GAL1 promoter-HIS3 fusion reporter construct was also transformed into a construct construct containing the ADH1-GBD-OsMYBS fusion gene. The presence of the GBD-OsMYBS1 fusion protein allows the yeast cells to grow well in the absence of histidine and in the presence of 3-AT. The presence of the GBD-OsMYBS2 fusion protein allows the yeast cells to grow slowly, but the presence of the GBD-OsMYBS3 fusion protein does not allow the yeast cells to grow on selective media. The above results suggest that in yeast, OsMYBS1 is a strong transcriptional activator, OsMYBS2 is a weak transcriptional activator, and OsMYBS3 is a non-transcriptional activating factor. OA85\8S98I.DOC -28- 1332508 6, OsMYBSl and OsMYBS2 for SRS-containing promoters

In order to determine the function of OsMYBS in sugar regulation of α-starch hydrolase gene expression in plant cells, it is analyzed in barley aleurone cells (which can be more consistent than in rice protoplasts), OsMYBS for SRS - The transactivation ability of the CaMV35S chimeric promoter, wherein the SRS_CaMV35S chimeric promoter is fused by SRS with the CaMV3 5S minimal promoter (the -46 bp upstream region of the transcription initiation site). Three OsMYBS cDNAs were fused to the maize pantoxin gene (Ubi) promoter to form a Ubi-OsMYBS chimeric gene and used as an effector. The coding region of the luciferin gene (Luc) was fused to the downstream region of the SRS-CaMV35S chimeric promoter to serve as a reporter. Half of the barley seeds were subjected to particle bombardment together with the effector and the reporter plastid. The bombarded half of the seeds were divided into two halves and then each incubated with 0.3 M glucose or 0.3 M mannitol for 24 hours and assayed for luciferase activity. Luciferase activity when the half of the seed was bombarded with the reporter plastid and plastid pRS426 (unrelated yeast plastids with molecular weight similar to the action plastid, which was used as a negative control) 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, 10120-10131). When half seed is bombarded with the reporter plastid and the proton plastid, OsMYBS1 significantly increases the luciferase activity in the presence of glucose to a similar extent as in the absence of glucose. OsMYBS1 can also be present in the absence of glucose. Increased luciferase O:\8S\8S981.DOC •29· 1332508 activity, but with a small increase. OsMYBS2 increases luciferase activity in the presence of glucose, but does not affect activity in the absence of glucose. OsMYBS3 does not significantly increase luciferase activity in the presence of glucose, but luciferase activity in the absence of glucose. This result shows that for the transcription of the promoter containing SRS, OsMYBSl and OsMYBS2 are strong and weak activating factors, respectively, and 0sMYBS3 is a suppressing factor. 7. OsMYBSl for the initiation of TATCCA-containing units and adjacent flanking sequences

Suppression of the sugar-suppressed transcription of the sub-segment. In order to determine whether OsMYBS will activate the promoter containing only the TATCCA unit and the adjacent flanking sequences, the DNA of the 6-column repeating unit containing the SRS-133 to -82 sequence will be used. The fragment (designated 6x (TATCCA+F)) was fused to the CaMV3 5S minimal promoter. The plastid containing the Ubi-OsMYBS construct was used as a reporter, and the Luc cDNA fused to the downstream region of the 6x (TATCCA+F)-CaMV35S promoter was used as a reporter. Half of the barley seeds were subjected to particle bombardment together with the action and the reporter plastids. The bombarded half of the seeds were divided into two halves and then each incubated with 0.3 M glucose or 0.3 M mannitol for 24 hours and assayed for luciferase activity. OsMYBS 1 significantly increases luciferase activity in the presence of glucose to a level similar to that in the absence of glucose. Although OsMYBS2 and OsMYBS3 appear to slightly increase luciferase performance in the presence of glucose, luciferase activity is significantly suppressed in the absence of glucose. This result indicates that OsMYBS 1 activates transcription of a promoter containing only the TATCCA unit and adjacent flanking sequences, and

OsMYBS2 and OsMYBS3 suppress their transcription. 8. OsMYBS protein and HvMYBGa co-activated barley Amy32b O:\8S\8S98t.DOC -30· 1332508 Promoter GARC in the α-starch hydrolase 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 vitro and to activate transcription of barley high-pi α-amylase and cysteine proteinase gene promoter in the absence of GA in vivo (Gubler, et al (1995) Plant Cell 7, 1879-1891; Cercos, et al. (1999) Plant J. 19, 107-118). Since the TATCCA unit is required for the high transcription of the a-starch hydrolase gene promoter by GA activation (Gubler and Jacobsen (1992) Plant Cell 4, 1435-1441; Lanahan, et al. (1992) Plant Cell 4, 203- 211), therefore, whether the OsMYBS protein is tested together with HvMYBGa to promote transcription of the starch hydrolase gene promoter. The barley semi-seed seeds were subjected to particle bombardment with the action of the Ubi-OsMYBSGa construct, the other proton plastid containing the Ubi-OsMYBS construct, and the reporter plastid containing the Amy32b-GUS construct. When no HvMYBGa is present, GUS only appears in the presence of GA, and in the absence of GA, the excessive performance of HvMYBGa significantly increases GUS activity. This result is consistent with other results reported by the literature (Gubler, et al. (1995) Plant Cell 7, 1879-1891; Cerc0s, et al. (1999) Plant J. 19, 107-118). Interestingly, HvMYBGa and OsMYBS OsMYBS2 or OsMYBS3 together can further increase GUS activity, which is about 2-3 times that of HvMYBGa alone and in the absence of GA. These results imply the three O:\85\85981.DOC -31 · 1332508

The OsMYBS protein will work with HvMYBGa to activate the barley alpha-starch hydrolase gene promoter. 9. Formation of homodimers in OsMYBSl in barley aleurone cells

In c-Myb, R2 and R3 together bind to a specific DNA sequence. To test the possibility of OsMYBS binding to DNA in the form of dimers, the homozygous system was used to analyze the homologous and heterologous interactions between OsMYBS proteins in barley aleurone cells. The full-length OsMYBS sequences were each associated with GAD.

Or C-terminal fusion of GBD. The GAD-OsMYBS and GBD-OsMYBS chimeric genes were fused to the downstream region of the Ubi promoter as a constructor. A DNA fragment containing five column repeating units of UAS (5xUAS) was fused to the upstream region of the CaMV35S minimal promoter-Luc chimeric gene, and as a reporter construct. The barley half-seed seeds were subjected to particle bombardment together with the two action plastids (each containing the GAD-OsMYBS and GBD-OsMYBS fusion genes) and the reporter plastids. The bombarded half seeds were incubated in a glucose-free buffer for 24 hours and assayed for luciferase activity. The common performance of GBD-OsMYBSl and GAD-OsMYBSl significantly increased luciferase performance compared with GBD-OsMYBSl alone. However, the common performance of GBD-OsMYBSl and GAD-OsMYBS2 or GAD-OsMYBS3, compared with GBD-OsMYBSl alone, does not further increase luciferase activity. Similar experiments with OsMYBS2 and OsMYBS3 show the two Proteins do not interact between isoforms or isoforms. In order to confirm that only OsMYBS1 will interact with each other in the same type, and the three OsMYBS proteins do not interact with each other, the constructor and the reporter plastid are further constructed for expression in the yeast. Except for O:\SS\SS981.DOC •32- 1332508 in the presence of GBD-OsMYBSl alone, the background concentration of the reporter gene expression is too high to detect the interaction between GBD-OsMYBSl and GAD-OsMYBS proteins. In this experiment, results similar to those of the yeast two-hybrid experiment were obtained. These results suggest that only OsMYBSI forms homodimers under these experimental conditions. Other Embodiments All of these features disclosed in this specification can be combined in any combination. Features disclosed in this specification can be replaced by another feature for the same, equivalent or similar purpose. Therefore, unless expressly stated, the features disclosed are merely examples of a series of equivalent or similar features. From the above description, those skilled in the art can readily recognize the subject matter of the present invention and various changes and modifications of the present invention to adapt to various uses and conditions without departing from the spirit and scope of the invention. It is also within the scope of the following patent application.

OrN85\85981.DOC 33- 1332508 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 (Sakamoto cultivation population) <220><221> CDS <222> (20)...(937) <400> 1 gtgcgagatc caccacccg atg ace 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 aag gcg ttc gag Ala Ala Ala Ala Ala Ala Trp Thr Arg Glu Asp Asp Lys Ala Phe Glu 15 20 aac gcg etc gcg get tgc gcg gcg ccg 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 Trp Phe Ala Ala 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 lie 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 95 100 l〇5 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

52 100 148 196 244 292 340

388 2 1332508 110 115 120 ggg tac gac ggc ggc This ugly g age tgc tcc aag gcg g pressure g caig gag aigg cgc 436

Gly Tyr Asp Gly Gly Lys Ser Cys Ser Lys Ala Glu Gin Glu Arg Arg 125 130 135 aag ggc ate 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 ege tee age ate 628

Phe lie 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 lie 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 cct 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 X147 tgcaagtgat catcacgtac acccggtgaa agcttagctc caaatgtgga tgtaattage 1207 agcggccttc cgtacgtggt ggcgccgatc gatgatettg caggggttgc aattagggat 1267 tgatttccat tttgctgatg taaatttgcc aactgtctca ttggaccaaa aaaaaaaaaa 1327 aaa 1330 1332508

<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 gggtggattg 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 10 15 20 gac tee gag etc gee tee ggc cag cag aag ege ege ege ege aag 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 1332508

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 688

Leu Glu Met Ala Ser Ser Ser lie Ser Ser Leu Glu Leu Ser lie Ala 185 190 195 ccc tet cat ttg egg ate gac ggg gee ate aag ggg ctg gga tec aaa 735

Pro Ser His Leu Arg lie Asp Gly Ala lie Lys Gly Leu Gly Ser Lys 200 205 210 215 ccc aat ttt ccc ccg aag gaa ttt gga teg get tea get act gtt ttt 784

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 832

Cys Pro Pro Cys Cys Cys Leu Leu Leu Leu Phe Phe Phe Phe Phe Phe 235 240 245 SCS SSS 9tt: gtt tgt tgt tgt tgt tgt tgt agt tgt cat get aac ttt 880

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 929

Val Phe Gly Ser Cys Gly Val Ser Phe Thr Ser Phe lie 265 270 275 agaatgtcag tcccttccga gacatgttta aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 989 aaaaaaaaaa aaaaaaaaaa aaa 1012

<210> 3 <211> 1287 <212> DNA <213> Rice (Japanese cultivated population) <220>

<221> CDS

<222> (67) ... (1020) <400> 3 ategategat cgatctccat aggtggggga agggaagett tggaaggtgg agggaeggag 60 999999 ac9 agg egg tgc teg cac tgc age cac aac ggg cac ugly c 108

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

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

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 252

Leu Ser Ser Ala Ala Gly Ser Thr Ser Gly Gly Ala Ser Pro Ala Asp 50 55 60 1332508 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 He 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 lie 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 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 lie 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 lie Val Lys Pro Val Pro Val His Ser Lys Ser Pro lie 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 1020 6 1020 61332508

Gin Glu Thr Glu Ser Thr Ser Leu Ser Leu Asn Leu Val Gly Gly Gin 290 295 300 aat aga caa tea get ttc cat gca aat cca cca aca agg gca cag gca Asn Arg Gin Ser Ala Phe His Ala Asn Pro Pro Thr Arg Ala Gin ala 305 310 315 tgatctggtt gtgcacacaa ctgcatttag atgaatccca ggcaaaataa gctttgcctc 1080 cttgtttttt tgtttttatt ttaagattaa ccgttctccg tagtctgtat catgtgctgt 1140 aagttatgct atgtatgaat gtatctgttg tttgtctggc acacatgata aatcactcta 1200 tgttaacaaa atcagtaatg gtagtgctga tcttcgtggt tgtactgttg taaactcttt 1260 tataagaaaa aaaaatatta gttagtc 1287

≪ 210 > 4 < 211 > 918 < 212 > DNA < 213 > rice (japonica cultivar population) < 400 > 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 aaccccgcgg cggcggcgct gggcccgccg 720 ggeatgaage accaccacca ccaccacccg ggcggcgcc cgccgcccat gccc Atgtac 780 agcgccgcgc ccatgggcca ccccgtcgcc ggccacatgg tgcccgccgc cgtcggcacg 840 ccggtggtgt tcccgccggg ccacgcgccg tacgtcgtgc ccgtcggcta cccggcgcct 900 ccggccaaga tgcaccaa 918

≪ 210 > 5 < 211 > 828 < 212 > DNA < 213 > rice (japonica cultivar population) < 400 > 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 cctgttgttg tttgttgttg 720 ttgttttttt tttttttttt tgcgggggtt gtttgttgtt gttgttgttg tagttg Tcat 780 gctaactttg tatttgggtc atgtggggtt tctttcacca gttttata 828 1332508

≪ 210 > 6 < 211 > 954 < 212 > DNA < 213 > rice (japonica cultivar population) < 400 > 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 agatggtcct gtgcaagaaa cacatgagat tgtcaagcct 780 gttccagttc attcaaagag cccaatcaac gttgatgagc ttgttggcat gtcgaagctc 840 agcataggag agtccaatca agagacagag tctacttctc tttcattaaa tctggtagga 900 ggtcaaaata gacaatcagc tttccatgca aatccaccaa caagggcaca ggca 954

<210> 7 <211> 306 <212> PRT <213> Rice (Japanese cultivated population) <400>

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 /

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 lie 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 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 1332508

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

<210> Θ <211> 276 <212> PRT

<213> Rice (Japanese cultivated population) <400> 8

Met Pro Asn Leu Thr Ser lie 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 lie Ala Pro Ser His Leu Arg lie Asp Gly Ala 195 200 205 lie 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 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 <210> 9 9 1332508

<211> 318 <212> PRT <213 > Rice (Japanese cultivated population) <400>

Met Thr Arg Arg Cys Ser His Cys 1 5

Thr Cys Pro Asn Arg Gly Val Lys 20

Gly Ser lie Arg Lys Ser Ala Ser 35 40

Ser Ala Ala Gly Ser Thr Ser Gly 50 55

Asp Ala Ala Pro Thr Ala Ala Asp 65 70

Gin Gly Phe Ser Ser Ala Thr Arg 85

Thr Glu Glu Glu His Arg Arg Phe 100

Lys Gly Asp Trp Arg Gly lie Ser 115 120

Pro Thr Gin Val Ala Ser His Ala 130 135

Asn Met Thr Arg Arg Lys Arg Arg 145 150

Asp Glu Ser Met Asp Leu Pro Pro 165

Thr Gin Val Leu Asn Gin Pro Ala 180

Glu Val Asp Ser Met Glu Ser Asp 195 200

Ser Ala Ser Ala lie Met Pro Asp 210 215 lie Val Pro Ala Tyr Phe Ser Pro 225 230

Trp Gin Asn Gin Lys Asp Glu Asp 245 work le Val Lys Pro Val Pro Val His 260

Glu Leu Val Gly Met Ser Lys Leu 275 280

Thr Glu Ser Thr Ser Leu Ser Leu 290 295

Gin Ser Ala Phe His Ala Asn Pro 305 310 <210> 10 <211> 42 <212> DNA <2;L3> Artificial sequence <220><223> Synthesis of generated oligonucleotide

Ser His Asn Gly His Asn Ser Arg 10 15 lie Phe Gly Val Arg Leu Thr Asp 25 30

Met Gly Asn Leu Ser Leu Leu Ser 45

Gly Ala Ser Pro Ala Asp Gly Pro 60

Gly Tyr Ala Ser Asp Asp Phe Val 75 80

Asp Arg Lys Lys Gly Val Pro Trp 90 95

Leu Leu Gly Leu Gin Lys Leu Gly 105 110

Arg Asn Phe Val Val Ser Arg Thr 125

Gin Lys Tyr Phe lie Arg Gin Ser 140

Ser Ser Leu Phe Asp Met Val Pro 155 160

Leu Pro Gly Gly Gin Glu Pro Glu 170 175

Leu Pro Pro Pro Lys Glu Glu Glu 185 190

Thr Ser Ala Val Ala Glu Ser Ser 205

Asn Leu Gin Ser Thr Tyr Pro Val 220

Phe Leu Gin Phe Ser Val Pro Phe 235 240

Gly Pro Val Gin Glu Thr His Glu 250 255

Ser Lys Ser Pro lie Asn Val Asp 265 270

Ser lie Gly Glu Ser Asn Gin Glu 285

Asn Leu Val Gly Gly Gin Asn Arg 300

Pro Thr Arg Ala Gin Ala 315

<400> 10 aattctatcc atatccatat ccatatccat atccatatcc ac 42 101332508 <210> 11 <211> 38 <212> DNA <213> Artificial sequence <220><223> Synthesis of generated oligonucleotide <400> 11 gtggatatgg atatggatat <210> 12 <211> 29 <2X2> DNA <213>Artificial sequence<220><223>Introduction<400> 12 aaactcgaga atgacctccc <210> 13 <211&gt 31 <212> DNA <213>Artificial sequence<220><223>Introduction<400> 13 atcgaattct cattggtgca <210> 14 <211> 29 <212> DNA <213> Artificial sequence <220><223>Introduction<400> 14 aaactcgaga atgcccaacc ggatatggat atggatag aggcggcga tcttggccgg a tcacctcca 38 29 31 29 >>>> 0 12 3 1 1 1 2 2 2 2 << &lt < 15 27 DNA artificial sequence <220><223> primer <400> 15 agcgaattct tatataaaac tggtgaa <210> 16 <211> 30 27 1332508 <212><213> DNA artificial sequence <220><223>primer<400> 16 Aaactcgagt atgacgaggc ggtgctcgca <210><2X1><212><213> 17 27 DNA artificial sequence <220><223> Introduction <400> 17 atcgaattct catgcctgtg cccttgt <210><211><;212><213> 18 25 DNA artificial sequence <220><223><400> 18 ccagaattct gcaaagatgg ataaa <210><211><212><213> 19 25 DNA artificial Sequence <220><223> primer <400> 19 ccactcgagc tctctttttt tgggt <210><211><212><213> 20 26 DNA artificial sequence <220><223>primer<400> 20 ccagaattca gatgaagcta ctgtct <210><211><212><213> 21 27 DNA artificial sequence 1332508 12 <220><223>Introduction<400> 21 ccactcgagt tcgatacagt caactgt 27

Claims (1)

1332508 Patent Application No. 092121002 This patent application is hereby incorporated by reference in its entirety in its entirety in its entirety in the the the the the the the the the the the the the the the the the the the the the the A sequence which will hybridize to the sequence under severe conditions and which encodes a polypeptide having the activity of the SMYBS 1 polypeptide. 2. The nucleic acid of claim i, wherein the sequence is seq ID NO : 1 〇 3. The nucleic acid of claim 1 wherein the nucleic acid encodes an amino group comprising at least 95% identical to SEQ ID NO: 7. A polypeptide of an acid sequence, and the polypeptide has OsMYBS 1 polypeptide activity. 4. The nucleic acid of claim 3, wherein the nucleic acid encodes a polypeptide comprising SEQ ID NO: 7. A cell comprising a nucleic acid according to any one of claims 1 to 4 wherein the nucleic acid is expressed. 6. The cell of claim 5, which comprises the nucleic acid of claim 4, in the scope of the patent application. A method for producing a genetically transgenic plant, which comprises the step of introducing a plant cell genome of (4) which is to be referred to in the first to fourth paragraphs of the patent application. 8. The method of producing a genetically transgenic plant according to claim 7, wherein the plant is a monocot. 9. The method of preparing a gene-transformed plant according to claim 8 of the patent application, wherein the plant is a moss plant. W. The method for preparing a genetically transformed plant according to Item 9 of the application (4), wherein the plant is rice. A method of producing a genetically transgenic plant according to claim 9 wherein the plant is barley. 12. A method for expressing a transcript in a cell, the method comprising: introducing a vector comprising a nucleic acid encoding the transcript into a cell, and expressing the transcript in the cell; wherein the transcript is characterized It will hybridize with seq ID NO: i or its complementary sequence under highly stringent conditions, and the transcript encodes a polypeptide having OsMYBS 1 polypeptide activity. 13. The method of claim 12, wherein the nucleic acid encodes a polypeptide comprising an amino acid sequence at least 95% identical to SEQ ID NO: 7, and the polypeptide has OsMYBS 1 polypeptide activity. 14. The method of claim 13, wherein the sequence of the polypeptide consists of SEQ ID NO: 7. 15. The method of claim 13, wherein the polypeptide binds to DNA comprising one or more TATCCA sequences. 16. The method of claim 15, wherein the sequence of the polypeptide consists of SEQ ID NO: 7. 17. An isolated nucleic acid which encodes a polypeptide comprising an amino acid sequence which is at least 95% identical to SEQ ID NO: 7, and which polypeptide has 〇sMYBS 1 polypeptide activity 'where the polypeptide encoded contains one or DNA binding of multiple portions of the TATCCA sequence. 18. The isolated nucleic acid of claim 17, wherein the amino acid sequence of the encoded polypeptide consists of SEQ ID NO: 7. 85981-990820.doc -2-