WO2004018508A1

WO2004018508A1 - A protein controlling rice tiller, a gene encoding the protein and a method of manipulating plant tiller or branching using the gene

Info

Publication number: WO2004018508A1
Application number: PCT/CN2003/000429
Authority: WO
Inventors: Jiayang Li; Qian Qian; Xueyong Li; Dali Zeng; Zhiming Fu; Yonghong Wang; Guosheng Xiong; Xiaoqun Wang; Xinfang Liu
Original assignee: Institute Of Genetics And Developmental Biology, Chinese Academy Of Sciences
Priority date: 2002-08-20
Filing date: 2003-06-03
Publication date: 2004-03-04
Also published as: CN1477112A; CN1185256C; AU2003245803A1

Abstract

The present invention provides a protein controlling rice tiller or branching which has a amino acid sequence shown by SEQ ID NO: 2 or a functional analog sequence thereof obtained by substitution, deletion, or addition of one or more amino acids of SEQ ID No: 2, wherein the functional analog is still able to control rice tiller or branching. The present invention also provides a nucleotide sequence encoding the protein, a vector containing this nucleotide sequence and a method of manipulating rice tiller or branching using the vector.

Description

A PROTEIN CONTROLLING RICE TILLER, A GENE ENCODING THE PROTEIN AND A METHOD OF MANIPULATING PLANT TILLER OR BRANCHING USING THE GENE Technical Field This invention relates to a protein controlling rice tiller, a gene encoding the protein, a vector carrying the gene and a method of manipulating plant tiller or branching using the gene.

Background Art Tiller is an important morphological character of most grass family plants. Tillers are a kind of lateral branches that originate from the axillary bud formed on each internode of the stem and grow independently of the mother stem due to their own adventitious roots. Tillering patterns of grass family plants can be divided into different types according to genesic sites thereof, rhizome-like tillering, sparse tillering and dense tillering pattern. In plants with rhizome-like tillering pattern such as sugarcane, rhizomes grow horizontally under the ground and produce a vertical shoot at a long distance. In plants with sparse tillering pattern, lateral branches and adventitious roots are formed on the caudex internodes under the ground, and the lateral shoots incline upward outside the leaf sheath. In plants with dense tillering pattern such as rice, lateral branches and adventitious roots are formed on the caudex internodes near the ground, and the lateral shoots grow parallely with the mother plant in the leaf sheath. There is a meristem region in the first internode of rice stem. Cell divisions in this region make internode elongation and regulate internode site of tillering to maintain the development of tiller bud in different planting depth and keep the dense tillering pattern.

The rice tillering stage starts after the formation of the fourth complete leaf. This is a very important stage in the rice growth period during which most vegetative organs of rice plants such as leaves, tillers and roots are formed. Tiller number per plant determines panicle number which is a key determination factor of rice grain yield. Extremely high or low number of tillers will influence grain yield. Therefore, tillering ability is an important agronomic trait that determines grain yield.

The study on tiller focused on the morphology and physiology in the past. The study on physiology of rice cultivation made a better illustration about the relationship of tiller number and environmental conditions, including the regulation of tiller development by factors such as temperature, light, water, farming techniques and mineral nutrition and like, particularly, the effect on tiller by the level of nitrogen nutrition. Genetics studies on rice tiller developed later, it is generally deemed that tiller is regulated cooperatively by multiple genes. With the growth of rice plants, contribution of additive action of genes to tiller increases and becomes predominant, whereas that of nonadditive action of genes and environmental factors becomes subordinate. In addition, other genes such as dwarf genes also affect rice tillering ability, and plants with these genes show very strong tillering ability. In the case of some key genes mutate, tiller number will alter significantly. For example, Takamura and Kinoshita obtained several tillering mutants rail, 2, 3, 4, 5 (rcn, reduced culm number) with low tillering number by Y -ray radiation mutagenesis. But rcnl-rcnS mutants were only primarily mapped to certain rice chromosomes, they have not been isolated so far (Takamura I and T.Kinoshita 1987 Genie identification of the mutant genes for reduced culm-number in rice. Japan J. Breed37 (suppl): 182-183). We isolated a spontaneous tillering mutant mono culm 1 (mod) in the japonica cultivar H89025, which is a mutant with extremely low tillering number and has only one main culm without any tillers. Genetic analysis showed that the mod mutant was caused by a recessive mutation in a single nuclear locus. We have cloned the MOC1 gene through a map-based cloning approach, which is the first defined gene that controls tiller in rice and other monocots. Although several genes controlling dicot plants branching have also been isolated previously, such as tomato IS gene (Schumacher K., Schmitt T., Rossberg M., Schmitz G. and Theres K. 1999. The Lateral suppressor (Ls) gene of tomato encodes a new member of the VHIID protein family. Proc. Natl. Acad. Sci. USA 96: 290-295) and Arabidopsis AtLS gene (Greb, T.,Schafer, E.,Herrero, R., Muller, D., Tillmann, E., Schmitz, G., and Theres, K. 2001. Mutation in the Arabidopsis and tomato lateral suppressor genes suggest a common control mechanism for lateral shoot formation. Program and abstracts of 12^th international conference on arabidospis research. P.225.), However, LS gene and AtLS gene might not be utilized in manipulating tiller of monocots due to the large evolution distance between dicots and monocots. In order to improve grain yield and quality, today farmers mainly take cultivation techniques to manipulate tillering number of rice and other grass family crops. It will be more efficient and economical if tillering number of grass family crops can be manipulated through genetic engineering techniques. Therefore, the MOC1 gene obtained in this invention, which controls rice tillering number, has great application potential.

Contents of the Invention

An object of the invention is to provide a protein that controls rice tiller or branching.

Another object of the invention is to provide a nucleotide sequence that encodes the protein of the invention.

A further object of the invention is to provide a vector carrying the nucleotide sequence of the invention.

A further object of the invention is to provide a transformant containing the vector of the invention. A further object of the invention is to provide a method manipulating plant tiller and branching.

Therefore, the present invention provides a protein controlling rice tiller or branching which has a amino acid sequence shown by SEQ ID NO: 2 or a functional analog sequence thereof obtained by substitution, deletion, or addition of one or more amino acids of SEQ ID No: 2, wherein the functional analog is still able to control rice tiller or branching. The MOC1 protein obtained in this invention belongs to the GARS transcription factors family, and shares 44% and 37% identity to tomato LS protein and Arabidopsis AtLS protein respectively. Although also involved in plant branching, LS and AtLS were originated from dicot plants, the large evolution distance between rice and them makes them unsuitable for genetic manipulation of tiller number in rice and other monocots. Therefore, the creativity and novelty of this invention are not affected.

The protein of the invention preferably has the amino acid sequence shown by SEQ TO No: 2. The invention also provides a nucleotide sequence that encodes the amino acid sequence shown by SEQ ID No: 2 or a functional analog sequence thereof obtained by substitution, deletion, or addition of one or more amino acids of SEQ ID No: 2, wherein the functional analog is still able to control rice tiller or branching

The MOCl gene obtained in this invention is a novel gene that is isolated from the rice mod mutant.

The gene of the invention preferably has the sequence shown by SEQ ID No: 1.

The invention also provides a vector carrying a nucleotide sequence that encodes the amino acid sequence shown by SEQ ID No: 2 or a functional analog sequence thereof obtained by substitution, deletion, or addition of one or more amino acids of SEQ ID No: 2, wherein the functioanl analog is still able to control rice tiller or branching. This vector might be pC8247 or pC8247S.

The invention also provides a transformant containing the said vector of the invention. The said transformant includes E.coli, Agrobacterium tumefaciens or plant cells. The invention also provides a method of manipulating plant tiller or plant branching including transformation of plant cells using the said vector of the invention; and cultivation of the plants from the transformed plant cells.

Today, there is a worldwide contradiction between the large-scale decrease in farming land and the rapid increase in population. It is very pressing to greatly improve the grain yield on per unit area, wherein proper plant type and population structure are important basis for improving grain yield and quality. Isolation of the MOCl gene makes it feasible to manipulate plant type and to form proper plant population structure through genetic engineering techniques.

Description of Figures The following drawings will further illustrate the present invention, but not be interpreted to limit the present invention.

Figure 1. Phenotype of the rice monoculm 1 mutant (mod), a, wild-type rice breed H89025; b, the mod mutant. Figure 2. Primary location of the MOCl locus on the rice Chromosome 6. RM3 is a SSLP marker, and the others are RFLP markers.

Figure 3. Physical mapping of the MOCl gene. Y4149 and Y2242 are YAC clones; 18dD02, 45cD09, and 4cAll are BAC clones. Arabic numerals indicate the crossover events on 560 chromosomes detected by each RFLP marker. The MOCl gene was located between the 18dD02R and Y2242R, both of which lie on the same

BAC clone 4cAll.

Figure 4. Fine localization of the MOCl gene. 12-2, 15-1, 17-2 and 17-3 are newly developed CAPS markers. Arabic numerals indicate the crossover events in

4020 chromosomes detected by each CAPS marker. The MOCl gene was located on a 20-kb region between the marker 12-2 and 17-3. Plasmid clones P4123 and P4124 from the random library of BAC clone 4cAll cover the MOCl gene.

Figure 5. Plasmid map of the binary plant expression vectors pC8247 and pC8247S. Plasmid pC8247 carries the entire MOCl gene, whereas control plasmid pC8247S carries a truncated MOCl gene missing the coding sequence of 188 amino acids at the C-terminal.

Figure 6. Phenotype of T, transgenic rice plants in the function complementation test, a, wild-type H89025; b, pC8247S transformant; c, pC8247 transformant. Plasmid pC8247 that carries the entire MOCl gene resorted the tillering number of the mod mutant, but the control plasmid pC8247S transformant still shown monoculm phenotype.

Figure 7. Tillering numbers of each order in the MOCl transgenic rice plants. 1,

2, 3, 4 and 5 represent primary, secondary, tertiary, quaternary, and quinteraary tiller respectively. Tillering numbers were counted at the heading stage and shown as Mean

± SE (n = 10). The MOCl transgenic rice plants have much more high order tillering number (tertiary, quaternary, and quinternary tiller) than wild-type H89025. Figure 8. Plasmid map of the E. coli expression vector pGΕX-2T/MOCl. The entire MOCl gene was fused in the same open reading frame with GST and was driven by the IPTG inducible Lac promoter.

Figure 9. The inducible expression of the GST-MOCl fusion protein, lane 1, protein molecular marker; lane 2, the vector pGEX-2T before induction; lane 3, the vector pGEX-2T/MOCl before induction; lane 4-8, the vector pGEX-2T/MOCl that was induced by 0.4 mM IPTG for 1, 2, 4, 6, and 8 hours respectively, lane 9-10, the vector pGEX-2T that was induced by 0.4 mM IPTG for 2 and 6 hours respectively. The molecular weight of GST and GST-MOCl protein is 27 kDa and 72 kDa respectively. Expression of the GST-MOCl fusion protein reached the highest level after induction for 6 hours by 0.4 mM IPTG.

Figure 10. Purification of the GST-MOCl fusion protein, a, After affinity purification, there is still mixed proteins besides the 72 kDa GST-MOCl fusion protein. M, protein molecular weight marker; 1, 2 and 3, after elution for the first, second and third time with 10 mM reductive glutathione respectively, b, the GST- MOCl fusion protein recovered from the gel. M, protein molecular weight marker; 1, the GST-MOCl fusion protein after electronic elution; 2, the GST-MOCl fusion protein after dialyse.

Mode of Carrying Out the Invention

Example 1. Map-based cloning the MOCl gene controlling rice tiller

1. Isolation and genetic analysis of the rice monoculm 1 mutant (mod)

The spontaneous monoculm 1 mutant (mod) was isolated in the wild-type rice breed H89025 (Oryza sativa L. ssp. Japonica). The mod mutant has only one main culm without any tillers, as shown in Figure 1. Reciprocal cross experiment between the mod mutant and H89025 revealed that the mod mutant phenotype was caused by a recessive mutation in a single nuclear locus.

2.Mapping population and genomic DNA isolation of MOCl gene

In order to isolate the MOCl gene using a map-based cloning approach, a large F₂ mapping population with high polymorphism was obtained from crosses between the mo (Japonica ) and Minghui 63 (Oryza sativa L. ssp. indica) plants. About 2 gram of leave material was collected from each of the 2010 F₂ plants that show the monoculm phenotype in the tiller fastigium stage. Genomic DNA was isolated from rice leaves with modified CTAB method (Barczak AJ, Zhao J,Pruitt K.D. Genetics 1995,140:303-31). About 100 mg rice leaves was ground into powder in a mortar with 5 cm diameter after freezing in liquid nitrogen and transferred into 1.5 ml centrifuge tube for total DNA isolation. The DNA pellet was resuspended in 100 μl ultrapure water. 1 μl DNA sample was used in each SSLP and CAPS reaction while 30 μl for RFLP analysis. 3. Primary localization of the MOCl gene

A small mapping population of 280 F₂ plants was used for SSLP (Simple Sequence Length Polymorphism) and RFLP (Restriction Fragment Length Polymorphism) analysis in the primary localization stage. According to the previous reports (Akagi, H., Yokozeki, Y, Inagaki, A., Fujimura, T. 1996. Microsatellite DNA markers for rice chromosomes. Theor. Appl. Genet. 93:1071-1077. Chen, X., Temnykh, S., Xu, Y, Clio, Y.G. McCouch, S.R. 1997. Development of a microsatellite framework map providing genome-wide coverage in rice. Theor. Appl. Genet. 95:553-567.), 90 SSLP primer pairs were synthesized, and PCR amplification was performed according to the reported conditions, after separation by 4% agarose gel electrophoresis and dying by EB, the polymorphism of the PCR products were detected. For RFLP and other Southern analysis, the genomic DNA was completely digested by different restriction enzymes, separated by 0.8% agarose gel electrophoresis, and then transferred onto the Hybond N* nylon membrane (Amersham), and hybridized with probes labeled with α -³²P-dCTP (Prime-a-Gene labeling system, Promega, Cat. No. UllOO). The polymorphism was detected on the Phosphorlmager (Molecular Dynamics). All the RFLP probes were chosen from the Nipponbare-Kasalath high density molecular linkage map (Harushima, Y et al. 1998. A high-density rice genetic linkage map with 2275 markers using a single F₂ population. Genetics 148: 479-494.) The results of primary localization shown that the MOCl gene was primarily located between the RFLP marker S1437 and R1559 on the long arm of the rice Chromosome 6 (Figure 2).

4. Physical localization of the MOCl gene

On the reported YAC-based physical map of rice breed Nipponbare (Saji, S. et al. 2001. A physical map with yeast artificial chromosome clones covering 63% of the 12 rice chromosomes. Genome 44: 32-37), there are 11 YAC clones within the MOCl region. We isolated several YAC end DNA clones such as Y2242R, Y2242L, Y4149R and Y4149L using the iPCR (inverse Polymerase Chain Reactions) method, wherein Y2242R was developed into a RFLP probe successfully. Then, the RFLP markers flanking the MOCl gene (R1559 and Y2242R) were used as probes to screen the rice breed Nipponbare BAC library (provided by Clemson University Genomics Institute, CUGI). Positive BAC clones were arranged into a BAC contig according to the fingerprint of BAC clones provided by CUGI. Primers were designed according to the BAC end sequences produced by the CUGI STC sequencing project (http://www. genome.clemson.edu /projects /rice/rice_bac_end/), some BAC end DNA clones were isolated using PCR method, and 18dD02R and 45cD09F were developed into RFLP markers successfully. According to the linkage analysis of MOCl gene with YAC or BAC ends i.e., Y2242R, 18dD02R and 45cD09F, further confined physically the MOCl gene onto a single BAC clone 4cAll (Figure 3). The BAC clone 4cAll containing the MOCl gene was sonicated into fragments of 1.5-3.0 kb, and then cloned into plasmid vector pUC19 after filling-in. Thus a random library of 4cAll for BAC sequencing was constructed. Single clone in this library was randomly picked up, plasmid DNA was extracted and sequenced.

5. Fine localization of the MOCl gene

In order to finely localize the MOCl gene, we first developed 4 new CAPS (Cleaved Amplified Polymorphic Sequence) markers i.e. 12-2, 15-1, 17-2 and 17-3 according to the sequence of the BAC clone 4cAll, the primer sequences thereof and the restriction enzymes that were used shown in Table 1. Then, linkage analysis was carried out with these CAPS markers in a large mapping population of 2010 F₂ mutant plants to screen the individual that occurred cross-over between the MOCl gene and each CAPS marker. The MOCl gene is finally fine localized between the CAPS marker 17-3 and 12-2, the physical distance is only 20 kb (Figure 4). Table 1. New CAPS markers developed in the invention.

6. Prediction and comparison analysis of the MOCl gene Four putative open reading frames (ORF) were predicted using the GENSCAN software (http:/genes.mit.edu/GENSCAN.html) in the 20-kb genomic sequence region between the marker 17-3 and 12-2. Then, these ORFs were analyzed in the protein database of the GenBank using the BLASTX program. Among them, the protein encoded by ORF1 shared high identity with the tomato LS (44 %) and Arabidopsis AtLS (37 %), all of which belong to the GRAS transcription factors family. Since both LS and AtLS control lateral branching of dicot plants, the homologous ORF1 is very likely the MOCl gene that controls rice tiller. The genomic fragment of ORF1 was thus amplified from both wild-type H89025 and the mod mutant using primers designed according to the sequence of the BAC clone 4cAll (MOClfl: 5'- TCGTTGTAG TAGCTCTG GTG-3' and MOClr3: 5'-

CTAACTAGAGATCGAGTAGC-3'), and the PCR products were sequenced directly. Sequence analysis revealed that a 1.9-kb retrotransposon was inserted into the mod mutant, which causes a premature translation stop of the MOCl protein. Example 2. Transformation of the rice mod mutant and function analysis of the MOCl gene

1. Construction of plant expression vector

In order to confirm the identity of ORF1 as the MOCl gene that controls rice tiller, the wild- type genomic fragment of ORF1 was transformed into the mod mutant, then observed whether the tillering number of the transgenic rice restored normal. Therefore, we constructed a plant expression vector for rice transformation as follows. Plasmid clones P4123 and P4124 from the random library of the BAC clone 4cAll for sequencing the sequence of BAC clone 4cAllwere jointed together through the common Sad site, and a 3.2 kb fragment containing the entire coding region of the MOCl gene, 1.5 kb upstream sequence and 0.3 kb downstream sequence was obtained (Figure 4). In addition, a 2.4 kb fragment was obtained by digestion of the plasmid P4123 using the enzyme Sad, which contains a partial ORF of the MOCl gene missing the coding sequence of 188 amino acids at the C-terminal (Figure 4). The 3.2 kb and 2.4 kb fragments were cloned into the binary vector pCAMBIA1300 (CAMBIA, Clunies Ross St, Black Mountain/GPO Box 3200, Canberra, ACT 2601, Australia), and transformation plasmids pC8247 and pC8247S were obtained respectively (Figure 5). pC8247 and pC8247S were transformed into the Agrobacterium tumefaciens strain LBA4404 respectively through the electroporation method.

2. Transformation of the rice mod mutant

The mature seeds of the rice mod mutant were dehusked, sterilized, and inoculated onto the callus-inducing medium. After 3 weeks, the vigorously growing, light-yellow, incompact embryogenic calli derived from the scutella were used as transformations. The calli were infected by the LBA4404 strains containing binary plasmid vector pC8247 and pC8247S respectively, and then grown in darkness at 25 °C for 3 days. Antibiotic-resistant calli and transgenic plantlets were selected on the medium containing 50 mg/L hygromycin. Hygromycin-resistant plants were transplanted into paddy field after training for several days in the shade. 15 and 4 independent transgenic lines were obtained for the plasmid pC8247 and pC8247S respectively, and each line includes several to several hundreds single plantlet. Southern blot analysis of genomic DNA of the transgenic rice plants revealed that two lines have 3 copies of the MOCl transgene and the other lines have only one copy.

3. Phenotype analysis of the MOCl transgenic rice plants The tillering number restored normal in almost all the plasmid pC8247 transformants, but all the plasmid pC8247S transformants still remained the monoculm phenotype of the mod mutant (Figure 6). This indicated that the entire MOCl gene can restore the normal tillering ability, but cannot the mutated MOCl gene. T₂ plants of the pC8247 single copy transgenic lines separated for the normal and monoculm phenotype basically in a 3:1 ratio. This result strongly confirmed that ORF1 is indeed the MOCl gene controlling rice tiller.

4. Function analysis of the MOCl gene

In the MOCl transgenic rice plants, the mod mutant recovered the monoculm phenotype, and the tillering number thereof increased to 3-5 times of that of the wild type H89025 plants (Figures 6-7). Careful observation revealed several characteristics of the MOCl transgenic plants tillering pattern. First, transgenic plants sometimes form 2 or more tillers at a same node, whereas wild type plants only form one tiller. Second, most tiller buds formed on the elongated upper internodes of the transgenic plants and tiller buds of higher orders (tertiary, quaternary, and quinternary) usually grow out into tillers, but these tiller buds were usually arrested in wild-type plants. These characteristics indicate that the MOCl gene is not only required for tiller bud formation, but also promotes the subsequent development of tiller buds. This also suggested that rice tillering number can be increased by over-expression of the MOCl gene using transgenic technique and be decreased by reducing the expression level of the MOCl gene using antisense or RNAi transgenic techniques.

Example 3. Expression of the MOCl protein in E. coli 1. Construction of E. coli expression vector

The MOCl gene was amplified from wild-type genomic DNA using primers containing designed BamHI and EcoRI site (Sense: 5'-ggatccATgCTCC ggTCACTCCAC-3'; Antisense, 5'-gaattcgTCgTCTTCgTCgCCCgC-3'). PCR products were ligated with T-easy vector (Promeag), and then transformed into DH10B competent cells by electroporation method. Positive recombinants were screened and sequenced to confirm no mutations present in the MOCl coding region. The above T-easy construct and E. coli expression vector pGΕX2T (4.9 kb, Amersham Pharmacia Biotech UK Limited, Amersham Place, Little Chalfont, Buckinghamshire, HP79NA, England. 27-4801-01) were both double digested with BamHI and EcoRI. The interesting digestion products were recovered, ligated using T4 DNA ligase, and then transformed into DH10B competent cells. Positive recombinants were screened and sequenced to confirm that MOCl and GST were in the same open reading frame and can be expressed in fusion protein. The plasmid map of the E. coli expression vector pGEX-2T/MOCl was shown in Figure 8. Then, GEX- 2T/MOC1 was transformed into BL21 (DE3) competent cells for further inducible expression of the MOCl protein. 2. Inducible expression of the GST-MOCl fusion protein

A single colony is inoculated into 5 ml 2xYTA medium and cultured at 37 ^°C overnight. The overnight culture was diluted with 2 <YTA medium at a ratio of 1:100 and continues to grow at 37°C until OD₆₀₀=O.6-1.0. Then IPTG (Sigma) with a final concentration of 0.4 mM was added into the culture, and the cells were allowed to grow at 30°C for another 1, 2, 4, 6, and 8 hours. As shown in Figure 9, expression of the GST-MOCl fusion protein reached the highest level after induction for 6 hours. 3. Purification of the GST-MOCl fusion protein

Cell culture for purification of the GST-MOCl fusion protein was prepared as described above. The cells were centrifuged at 3000 rpm for 15 minutes and then washed once with I xPBS. The cell pellets were resuspended with IxPBS at a ratio of 50 ml IxPBS /L culture medium, and DTT (Sigma) and EDTA (Sigma) were added to a final concentration of 5 mM and 1 mM respectively. The cells were repeatedly frozen and thawed for 2-3 times, and then sonicated for 6 times with a mode of 15 seconds on and 20 seconds off. To the cells were added TritonxlOO (sigma) to a final concentration of 1% and agitated gently to mix well. The cells were placed on ice for 30 minutes, then centrifuged at 10000 rpm for 15 minutes, and transfered the supernatant into a new tube. A proper amount of 50% gluthione-coupled dextran beads (Pharmacia) were added to the supernatant at the ratio of 1 xbed volume per liter culture, then incubated on a rotator with a speed of 50 rpm at room temperature for 1 hour. Centrifuged to collect beads, discarded the supernatant, and washed the beads 3 times with lOxbed volumes of IxPBS (containing 1% Triton). The GST-MOCl fusion proteins were eluted from the beads using 10 mM reduced gluthione elution buffer (pH 8.0) at the ratio of 1 ml elution buffer per bed volume. Repeat the elution for 3 times. The collected eluents were run on SDS-PAGE (12%o separation gel) at 10 V/cm for 4 hours to separate the GST-MOCl fusion proteins. As shown in Figure 10a, besides the 72 KDa GST-MOCl fusion proteins, there are still other proteins mixed in the affinity-purified protein fractions. Therefore, the GST-MOCl fusion proteins were further recovered from the gel. Stain the gel in cooled 0.25 M KC1 solution, cut down the 72 KDa GST-MOCl protein band, pound it to pieces and transfered into a electronic elution tube (BioRad). The GST-MOCl fusion protein was electronically eluted from the gel at the constant currency of 10 mA for 4 to 6 hours. The eluted proteins solution was dialysed overnight in 0.1 mM NaCl and 10 mM Tris pH 7.5 buffer. The GST-MOCl fusion proteins were confirmed on 10% SDS PAGE gel after electronical elution and dialysis, and only a single band of 72 KDa can be detected (Figure 10b). The recovery rate of the GST-MOCl fusion proteins is about 20%. The purified MOCl -GST fusion proteins were stored at -70 ^°C.

Claims

1. A protein controlling rice tiller or branching, characterized by having a amino acid sequence shown by SEQ ID No: 2 or a functional analog sequence thereof obtained by substitution, deletion, or addition of one or more amino acids of SEQ

ID No: 2, wherein the functional analog is still able to control rice tiller or branching.

2. The protein according to claim 1, characterized by having the amino acid sequence shown by SEQ ID No: 2.

3. A nucleotide sequence, characterized by encoding the amino acid sequence shown by SEQ ID No: 2 or the functional analog sequence thereof obtained by substitution, deletion, or addition of one or more amino acids of SEQ ID No: 2, wherein the functional analog is still able to control rice tiller or branching.

4. The nucleotide sequence according to claim 3, characterized by having the sequence shown by SEQ ID No: 1.

5. A vector containing a nucleotide sequence, wherein the nucleotide sequence encodes the amino acid sequence shown by SEQ ID No: 2 or the functional analog sequence thereof obtained by substitution, deletion, or addition of one or more amino acids of SEQ ID No: 2, wherein the functional analog is still able to control rice tiller or branching.

6. The vector according to claim 5, which is plasmid pC8247 or pC8247S.

7. A transformant, characterized by comprising the vector of claim 5 or claim 6.

8. A method of manipulating rice tiller or branching, comprising transformation of plant cells with the vector of claim 5 or claim 6 and cultivation of the plants from the transformed plant cells.