NL2028064B1 - Gene for controlling small grain and semi-dwarf of oryza sativa and application thereof - Google Patents

Abstract

The present invention relates to an Oryza sativa small grain and semi-dwarf related gene 5 and an application thereof. Specifically, a genetic group is constructed by a large-grain Oryza sativa variety Hui 12-29 and a small-grain variety FH212 by the inventors; and a gene TGW5 that controls small grain and semi-dwarf of Oryza sativa is disclosed. The gene codes tripolymer G protein a subunit. Compared with the large-grain parent variety Hui 12-29, in the small-grain parent FH212, an intron splicing manner is changed due to change of single bases (A?T) at the 10 fourth intron terminal, 3 transcripts different from the Hui 12-29 are produced and a predicted three-dimensional structure of the TGW5 protein is changed. The present invention further relates to a mutant protein of TGW5 with protein function deletion or deficiency, a coding sequence thereof, and an application in hybrid vigour improvement.

Description

GENE FOR CONTROLLING SMALL GRAIN AND SEMI-DWARF OF ORYZA SATIVA AND

APPLICATION THEREOF Technical Field The present invention relates to the technical field of plant biology, and particularly relates to a gene for controlling small grain and semi-dwarf of Oryza sativa and an application thereof. Background Grain size of Oryza sativa is a typical quantitative trait and an important economic trait.

Formation of the trait is controlled and affected by various genetic signals and regulating ways, including parental origin effects, plant hormone signalling pathways, G proteins and the like. An Oryza sativa seed is mainly composed of embryo, endosperm, pericarp, seed coat and glume. The inside endosperm provides a part that is eaten by humans; the outside glume provides protection for the seed and is considered as a container limiting grain growth so as to achieve an important effect of controlling the grain shape. The size of the glume depends on glume cells and is cooperatively controlled in two manners such as cell division and cell enlargement. During early development of the glume, the quantity of the glume cells is increased by virtue of extensive cell division; later, the cell division slows down, and the cells are enlarged by cell expansion; and finally, phenotypes of grain length, grain width and grain thickness are determined by the quantity and size of the cells in the glume of the seed in different dimensions.

Grain weight and grain shape of Oryza sativa are key factors for forming Oryza sativa yield, and are generally measured by grain shape traits such as grain length, grain width, grain thickness and length-width ratio. The grain weight/grain shape of Oryza sativa is a typical quantitative trait, controlled by multiple genes, and often subjected to genetic analysis by a QTL (quantitative trait locus) research method. At present, more than four hundred QTLs of the grain weight/grain shape of Oryza sativa have been identified, and distributed on any chromosome of the Oryza sativa genome (www.gramene.com). The grain shape traits have extremely high heritability, and stable performance of the grain shape traits in different genetic backgrounds and environments has important significances for genetic improvement of varieties. In the last dozen years, important progress has been made on cloning and functional study of genes/QTLs that control the grain shape traits of the Oryza sativa. At present, 16 QTLs that control the grain shape and grain weight of the Oryza sativa have been subjected to molecular cloning and functional analysis. However, except for a small amount of the QTLs/genes, most of the QTLs that control the grain shape and grain weight of the Oryza sativa are still in a preliminary study phase.

Therefore, cloning of the QTLs that control the traits and functional analysis of candidate genes of the QTLs urgently need to be enhanced. In the present invention, a gene TGWS that controls the small grain and semi-dwarf of the Oryza sativa is separated and cloned by a map-based cloning method, thereby providing important gene resources for improvement breeding of the

Oryza sativa. Summary A purpose of the present invention is to provide a gene for controlling grain shape and plant height of crops and an application thereof.

According to a first aspect of the present invention, a separated polypeptide for controlling small grain and semi-dwarf of Oryza sativa is provided. The protein is selected from the following groups: (1) a polypeptide having an amino acid sequence of SEQ ID NO:2; or (2) a polypeptide formed from the amino acid sequence of SEQ ID NO:2 by substitution, deletion or addition of 1-10 amino acid residues and derived from (1).

According to a second aspect of the present invention, a separated polypeptide for controlling small grain and semi-dwarf of Oryza sativa is provided. The protein is selected from the following groups: (1) a polypeptide having an amino acid sequence of SEQ ID NO:4; or (2) a polypeptide formed from the amino acid sequence of SEQ ID NO:4 by substitution, deletion or addition of 1-10 amino acid residues and derived from (1).

According to a third aspect of the present invention, a separated polypeptide for controlling small grain and semi-dwarf of Oryza sativa is provided. The protein is selected from the following groups: (1) a polypeptide having an amino acid sequence of SEQ ID NO:6; or (2) a polypeptide formed from the amino acid sequence of SEQ ID NO:6 by substitution, deletion or addition of 1 - 10 amino acid residues and derived from (1).

According to a fourth aspect of the present invention, a separated polynucleotide is provided.

The polynucleotide is selected from the following groups: (a) a polynucleotide coding the polypeptide in the first aspect of the present invention; (b) a polynucleotide having a sequence shown as SEQ ID NO:1.

According to a fifth aspect of the present invention, a separated polynucleotide is provided. The polynucleotide is selected from the following groups: (a) a polynucleotide coding the polypeptide in the second aspect of the present invention; (b) a polynucleotide having a sequence shown as SEQ ID NO:3.

According to a sixth aspect of the present invention, a separated polynucleotide is provided. The polynucleotide is selected from the following groups: (a) a polynucleotide coding the polypeptide in the third aspect of the present invention; (b) a polynucleotide having a sequence shown as SEQ ID NO:5.

According to a seventh aspect of the present invention, a separated polynucleotide is provided. The polynucleotide is selected from the following groups:

(a) a polynucleotide transcribing the polypeptide in the fourth, fifth and sixth aspects of the present invention; (b) a polynucleotide having a sequence shown as SEQ ID NO:7.

According to an eighth aspect of the present invention, a method for improving crops (more preferably, breeding small-grain and semi-dwarf Oryza sativa varieties) is provided. The method includes: (Iy improving grain traits of Oryza sativa; (Il) regulating plant heights of the Oryza sativa.

Description of Drawings Drawings below are used for describing specific embodiments of the present invention, rather than limiting the scope of the present invention defined by claims.

Fig. 1 shows plant height and grain shape comparison of complementary transgenic plants of Oryza sativa TGW5 and a receptor variety “FH212”; Fig. 2 shows plant height and grain shape comparison of TGW5 mutant plants obtained by a crispr technology and a receptor variety “C815S”; and Fig. 3 shows plant height and grain shape comparison between improved plants with TGW5 genes introduced by utilizing molecular marker-assisted selection and a receptor variety “Wuxiang S”.

Detailed Description By virtue of extensive in-depth study, the inventors first discover a novel gene for controlling grain shape and grain weight of crops. Function decrease or deficiency of the gene may reduce the grain shape and decrease the plant height. The study proves that, due to single-base mutation and gene knockout of the TGW5 gene, the grain shape may be significantly reduced, and the plant height may be decreased, which indicates that the TGW5 gene has wide application prospects in selection breeding of small-grain crop varieties.

Terms The “crops” in the present invention include but not limited to Oryza sativa, wheat, maize, sorghum, soybean and the like.

The “separated” in the present invention means that substances are separated from a primitive environment. For example, polynucleotides and polypeptides in natural states in living cells are not separated or purified. However, if separated from other substances existing in natural states, the polynucleotides or polypeptides are separated and purified.

The “separated TGWS5 protein or polypeptide” in the present invention means that the TGW5 protein basically contains no other natural proteins related to the TGWS5 protein.

The polypeptide in the present invention may be a recombinant polypeptide, a natural polypeptide and a synthetic polypeptide. The polypeptide in the present invention includes a naturally purified product or a chemosynthetic product or is produced from prokaryotic or eukaryotic hosts by a recombinant technology.

The “long grain” and “large grain” in the present invention may be exchanged.

The “TGWS5 protein” in the present invention refers to a polypeptide with sequence of SEQ ID NO:2 having TGWS5 protein activity.

The present invention further provides a polynucleotide sequence coding the TGWS protein. The polynucleotide in the present invention may be in a DNA form or an RNA form. The DNA form includes DNA, genome DNA or synthetic DNA. The DNA may be a coding strand or a non-coding strand. A coding region for coding mature polypeptides may have the same sequence as the coding regions shown as SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7 or is a degenerated variant.

The present invention further relates to an application of a molecular marker-assisted selection technology of TGWS genes in small-grain sterile line breeding of crops.

In the present invention, a genetic population is constructed by hybridization of Hui 12-29 (a large-grain variety) and FH212 (a small-grain variety); a gene TGWS5 located on Chromosome 5 and used to control grain length and grain weight of Oryza sativa is localized by a QTL-seq location technology; and the gene is cloned by a map-based cloning technology.

The present invention has major advantages as follows: (1) A novel small-grain Oryza sativa gene is first separated; and by changing the transcription and translation of the gene or knocking out the gene, the grains of the crops (such as Oryza sativa) can be narrowed, and the plants are dwarfed.

(2) The small-grain Oryza sativa gene TGWS in the present invention may serve as a gene for controlling the grain size and plant height of the crops, and is applied to improving the crop varieties.

Embodiment 1 Obtainment of small-grain and semi-dwarf Oryza sativa gene TGW5 Hui 12-29 is a large-grain Oryza sativa variety, while FH212 is a small-grain Oryza sativa variety. A genetic population is constructed through hybridization of Hui 12-29 and FH212 by the inventors; a novel gene (or QTL) TGWS5 that controls grain shape and plant height of Oryza sativa is localized by a QTL-seq analysis method; and the gene is located on Chromosome 5. Further, the gene is cloned by the map-based cloning technology. Sequences of the gene are shown as SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7. Sequences of a coded protein are shown as SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. Embodiment 2 Transgenic experiment of TGWS Oryza sativa Through genetic transformation, it is verified that, grain shape and plant height of the Oryza sativa are controlled by TGWS5. In the present embodiment, a vector derived from a plant expression vector pPCAMBIA3301 serves as a transgenic vector of Oryza sativa. The vector codes termination signal sequences of a bacterium replication origin (ori), a kanamycin resistant gene (Kan), a hygromycin resistance gene (Hyg'), a B-glucuronidase gene (GUS), a dual-CaMV35S promoter and an NOS gene, and a restriction enzyme multiple cloning site (MCS). The DNA sequence of the TGW5 can be forwards or reversely inserted at the restriction enzyme multiple cloning site to construct the transgenic vector.

1. Construction of complementary transgenic plasmid of TGW5 5 In the present embodiment, genome DNA derived from a large-grain variety Hui 12-29 served as a template; a TGWS5 gene sequence in the Hui 12 - 29 was amplified with a high-fidelity enzyme KOD FX Neo(TOYOBO); amplified fragments were subjected to gel recovery after 1% of agar gel electrophoresis; the gel recovery fragments were cloned onto an intermediate vector pEASY- Blunt3 in a blunt end connection manner; and multiple recons were sequenced so as to verify accuracy of the sequence. Sequences of the PCR amplification primers are as follows: The sequence of 5’-end oligonucleotide primer is: 5-CCGGAATTCCAAACCCCGTTAAAGCC -3 The sequence of the 3’-end oligonucleotide primer sequence is: 5-ATAGGGTACAGACCTGAACAGC -3'.

The vectors TGW5-pEASY-Blunt3 and pCAMBIA1301 were digested with restriction enzymes of EcoRI and Hindlll; and a target fragment digested by the TGW5-pEASY-Blunt3 was connected to the EcoRI and Hindlll restriction enzyme cutting sites of the final vector pCAMBIA1301. The connection product was transformed into an Escherichia coli strain T1; a transformant was screened on an LB medium containing Kan (50 ug/ml); a single colony was selected for extracting the plasmid; and the clone was sequenced by an M13 universal primer to detect whether the sequence of the target fragment was accurate. Thus, the complementary transgenic plasmid of TGW5 was successfully constructed.

2. TGWS5 transformed Oryza sativa The complementary transgenic plasmid of TGW5 was introduced into agrobacterium strains EHA105 by a freeze-thaw method; every 200 ul of EHA105 competent cells were uniformly mixed with 0.5-1 ug (about 10ul) of plasmid DNA; the mixture was placed on ice, in liquid nitrogen and in a water bath of 37°C in sequence for 5 minutes respectively; the mixture was diluted to 1 ml with a fresh YEB fluid medium and subjected to shaking culture at 28°C for 2 - 4 hours; and 200 pl of the mixture was applied onto a YEB plate containing an antibiotic Kan (50 ug/ml) and then cultured at 28°C for 2 - 3 days. Single bacteria were streaked from growing bacterial colonies on the antibiotic-containing YEB plate for 3 consecutive times; agrobacterium single colonies were picked from the YEB plate and then inoculated into 3 ml of AB fluid medium containing antibiotics; the agrobacterium single colonies were continuously subjected to shaking culture at 200 rpm until ODsos was about 0.6-0.8; the fresh agrobacterium solution was centrifuged at 500 rpm and 4°C for 5 minutes, collected and resuspended in an AAM fluid medium of 1/3 volume; and then the product can be used for transforming various receptor materials of Oryza sativa.

In the present embodiment, embryogenic callus of FH212 was transformed by the conventional agrobacterium-mediated transformation method. Immature seeds of FH212 within

12 - 15 days after pollination were soaked in 70% of ethanol for 1 minute, disinfected in a NaCIO solution (mixed with water according to a ratio of 1:3, added with 2-3 drops of Tween-200) for more than 90 minutes, and then flushed with sterile water for 4-5 times; then young embryos were taken out by a scalpel and tweezers and inoculated onto an N6D2 medium for inducing the callus; the callus was cultivated at 26 + 1°C in a dark place and can be used for transformation 4 days later; the young embryo callus was soaked in the fresh AAM agrobacterium solution and shaken at intervals; the Oryza sativa material was moved out after 20 minutes; and the excessive bacterium solution was sucked on sterile paper and then immediately transferred onto the N6D:C medium for co-culture at 26°C for 3 days. During co-culture, acetosyringone was added into the co-culture medium to serve as an agrobacterium Vir gene activator with an application concentration of 100 umol/L. 3 days later, the callus was taken out from the co-culture medium, the germ was cut off, and the callus was transferred into a selective medium N6D2S51 (Hyg 25 mg/l) for performing selective culture. 7-12 days later, the resistant callus was transferred onto the selective medium N6D:S2 (Hyg 50 mg/l} and continuously screened. 10-12 days later, vigorously growing resistant callus was transferred onto a pre-differentiation medium, cultured for about one week and then transferred onto a differential medium for differentiation (lighting for 12 hours per day). Reproduced plantlets grew roots and sound seedlings on a 1/2 MS; medium and then transferred into phytotron potting soil for cultivation.

Embodiment 3 Grain size and plant height comparison of TGW5 complementary transgenic plants and wild-type plants of Oryza sativa The TGWS complementary transgenic plants of Oryza sativa were obtained by the method in embodiment 2; phenotypes of grain shapes and plant heights of the wild-type FH212 and the transgenic Oryza sativa were observed; and the influence of the TGWS gene on the grain size and plant height was analysed. The result was shown in Fig. 1; and compared with the control FH212, the TGW5 complementary transgenic plants (C5) of Oryza sativa have significantly enlarged grains and significantly increased plant heights.

Embodiment 4 Obtainment of TGWS mutant by utilizing crispr technology

1. The sequence of the TGW5 gene was analysed; sgRNA target sites were designed by utilizing a CRISPR-P tool (http://crispr.hzau.edu.cn/CRISPR2/); and a target sequence was as follows: target sequence: 5'-aaagaggtggagaggtatatagg -3'.

2. gRNA fragments were obtained by primer degeneration and annealing, digested and connected to obtain recombinant plasmids; the plasmids were transformed into Escherichia coli and placed in a thermostatic incubator at 37°C and then cultured overnight; single clones were selected; and the plasmids were extracted for identification.

3. The extracted plasmids were transferred to 2 single clones for sequencing identification.

4. The accurately identified plasmids were digested and connected to an expression vector and transformed into Escherichia coli competent cells DH5a; the competent cells were applied onto an LB solid medium for overnight culture; single clones were picked; and plasmids were extracted for identification.

5. The accurately identified plasmid vector infected the callus of C815S by an agrobacterium (EHA105) infection method to obtain a transgenic plant.

6. Tender leaves were picked from TO-generation transgenic plant; and plant tissue DNA was extracted by a CTAB method.

7. Primers containing target sites were designed by primer5 software; TGWS5 fragments were obtained by a PCR technology; and primer sequences were as follows: 5’-end nucleotide primer sequence TGW5-341(+): 5-CCATTTCCCGTCATTTCTTA -3’; 3'-end nucleotide primer sequence TGW5-417(-): 5-TTCCTTGGTCTCCATCATTC -3’.

8. 1% of agarose gel electrophoresis was conducted; and a target band was selected for gel cutting and recovery and then sequenced in a company.

9. Sequencing results were analysed by a website DSDECODE (nttp://dsdecode.scgene.com/home/).

10. The results showed that two TGW5 mutants C65 and C70 were obtained. Target sequences of the mutants C65 and C70 and the wild type (Control) were compared as follows: Wild type: CCTCCTACATCATACAACCTATATACCTCTCCACCTCTTT J22: CCTCCTACATCATACAACCT———TACCTCTCCACCTCTTT J42: CCTCCTACATCATACAACCTATA——CCTCTCCACCTCTTT Embodiment 5 Grain size comparison of transgenic plants of TGW5 mutants of Oryza sativa and wild-type plants TGWS5 gene knockout plants of Oryza sativa were obtained by the method in embodiment 4; phenotypes of grain shapes and plant heights of the Oryza sativa were observed; and the influence of the TGW5 gene on the grain size and the plant height was analysed. The result was shown in Fig. 2; and the grains of the TGW5 gene knockout transgenic plants (J22) of Oryza sativa were obviously smaller than those of C8158 of the receptor variety of Oryza sativa, and the plant heights were also significantly decreased. Embodiment 6 Molecular marker-assisted selection breeding of TGW5 gene In the present embodiment, PCR oligonucleotide primers (shown as SEQ ID NO:8 and SEQ ID NO:9) were designed in the TGWS5 gene; PCR amplification was conducted on DNA of a large- grain variety Wuxiang S and a small-grain variety FH212 with a Tag enzyme; the amplified fragments were digested with a restriction enzyme Alu I; DNA polymorphism (difference) existing between the large-grain variety and the small-grain variety was detected by 2.5% of agarose gel electrophoresis; the large-grain variety had a molecular weight of 286 bp; and the small-grain variety had a molecular weight of 314 bp. Therefore, the pair of primers developed into molecular markers that can specifically identify the large-grain TGWS5 gene and the small- grain TGW5 gene. The results were shown in Fig. 3. Individuals carrying the small-grain gene can be rapidly picked out of a filial generation group of the large-grain variety Wuxiang S and the small-grain variety FH212 at a seedling stage, and thus a new strain (XS1) of the small-grain Oryza sativa was cultivated.

Primer sequences of the specific molecular markers of the TGW5 gene are as follows: The sequence of the 5-end oligonucleotide primer is: 5-AGAGTTCTTTTGCTCCTTCATATAGTAGC-3' (SEQ ID NO:8); The sequence of the 3’-end oligonucleotide primer sequence is: 5-TCCGTACGCCGCTAGTTAGTC-3’ (SEQ ID NO:9).

SEQUENCE LISTING <110> China National Rice Research Institute <120> GENE FOR CONTROLLING SMALL GRAIN AND SEMI-DWARF OF ORYZA SATIVA AND

APPLICATION THEREOF <130> BJS-Small Rice Gene NL <140> NL2028064 <141> 2021-04-24 <160> 17 <170> PatentIn version 3.3 <210> 1 <211> 1179 <212> DNA <213> Oryza sativa <400> 1 atgtccgtgc ttacctgtgt gcttgataac atgggctcat cctgtagcag atctcattct 60 ttaagtgagg ctgaaacaac caaaaatgca aaatctgcag acattgacag gcgaattttg 120 caagagacaa aagcagagca acacatccac aagctcttac ttcttggtgc gggagaatca 180 gggaagtcta cgatatttaa acagattaag ctccttttcc aaactggctt tgatgaggca 240 gaacttagga gctacacatc agttatccat gcaaacgtct atcagacaat taaagtatgc 300 aatactggaa agggagcaaa agaactctca caagtggaat cagattcctc aaaatatgtt 360 atatccccag ataaccagga aattggagaa aaactatcag atattgatgg caggttggat 420 tatccactgc tgaacaaaga acttgtactc gatgtaaaaa ggttatggca agacccagcc 480 attcaggaaa cttacttacg tggaagtatt ctgcaacttc ctgattgtgc acaatacttc 540 atggaaaatt tggatcgatt agctgaagca ggttatgtgc caacaaagga ggatgtgctt 600 tatgcaagag tacggacaaa tggtgttgta caaatacaat ttagtcctgt tggagaaaac 660 aaaagaggtg gagaggtata taggttgtat gatgtaggag gccagaggaa tgagaggaga 720 aagtggattc atctttttga aggtgttaat gcggtaatct tttgtgctgc cattagcgaa 780 tatgatcaga tgctatttga agatgagaca aaaaacagaa tgatggagac caaggaactc 840 tttgactggg ttttaaagca aagatgtttt gagaaaacat cattcattct gtttctcaac 900 aaatttgata tattcgagaa gaaaatacaa aaggttcctt taagtgtgtg cgagtggttt 960 aaagactacc agcctattgc acctgggaaa caggaggttg aacatgcata tgagtttgtc 1020 aagaagaagt ttgaagagct ctacttccag agcagcaagc ctgaccgtgt ggaccgcgtc 1080 ttcaaaatct acagaactac ggccctagac cagaaacttg taaagaagac attcaagttg 1140 attgatgaga gcatgagacg ctccagggaa ggaacttga 1179

<210> 2 <211> 392 <212> PRT <213> Oryza sativa <400> 2 Met Ser Val Leu Thr Cys Val Leu Asp Asn Met Gly Ser Ser Cys Ser 1 5 10 15 Arg Ser His Ser Leu Ser Glu Ala Glu Thr Thr Lys Asn Ala Lys Ser

Ala Asp Ile Asp Arg Arg Ile Leu Gln Glu Thr Lys Ala Glu Gln His 40 45 Ile His Lys Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr 50 55 60 Ile Phe Lys Gln Ile Lys Leu Leu Phe Gln Thr Gly Phe Asp Glu Ala 65 70 75 80 Glu Leu Arg Ser Tyr Thr Ser Val Ile His Ala Asn Val Tyr Gln Thr 85 90 95 Ile Lys Val Cys Asn Thr Gly Lys Gly Ala Lys Glu Leu Ser Gln Val 100 105 110 Glu Ser Asp Ser Ser Lys Tyr Val Ile Ser Pro Asp Asn Gln Glu Ile 115 120 125 Gly Glu Lys Leu Ser Asp Ile Asp Gly Arg Leu Asp Tyr Pro Leu Leu 130 135 140 Asn Lys Glu Leu Val Leu Asp Val Lys Arg Leu Trp Gln Asp Pro Ala 145 150 155 160 Ile Gln Glu Thr Tyr Leu Arg Gly Ser Ile Leu Gln Leu Pro Asp Cys 165 170 175 Ala Gln Tyr Phe Met Glu Asn Leu Asp Arg Leu Ala Glu Ala Gly Tyr 180 185 190 Val Pro Thr Lys Glu Asp Val Leu Tyr Ala Arg Val Arg Thr Asn Gly 195 200 205 Val Val Gln Ile Gln Phe Ser Pro Val Gly Glu Asn Lys Arg Gly Gly 210 215 220

Glu Val Tyr Arg Leu Tyr Asp Val Gly Gly Gln Arg Asn Glu Arg Arg 225 230 235 240 Lys Trp Ile His Leu Phe Glu Gly Val Asn Ala Val Ile Phe Cys Ala 245 250 255 Ala Ile Ser Glu Tyr Asp Gln Met Leu Phe Glu Asp Glu Thr Lys Asn 260 265 270 Arg Met Met Glu Thr Lys Glu Leu Phe Asp Trp Val Leu Lys Gln Arg 275 280 285 Cys Phe Glu Lys Thr Ser Phe Ile Leu Phe Leu Asn Lys Phe Asp Ile 290 295 300 Phe Glu Lys Lys Ile Gln Lys Val Pro Leu Ser Val Cys Glu Trp Phe 305 310 315 320 Lys Asp Tyr Gln Pro Ile Ala Pro Gly Lys Gln Glu Val Glu His Ala 325 330 335 Tyr Glu Phe Val Lys Lys Lys Phe Glu Glu Leu Tyr Phe Gln Ser Ser 340 345 350 Lys Pro Asp Arg Val Asp Arg Val Phe Lys Ile Tyr Arg Thr Thr Ala 355 360 365 Leu Asp Gln Lys Leu Val Lys Lys Thr Phe Lys Leu Ile Asp Glu Ser 370 375 380 Met Arg Arg Ser Arg Glu Gly Thr 385 390 <210> 3 <211> 1160 <212> DNA <213> Oryza sativa <400> 3 atgtccgtgc ttacctgtgt gcttgataac atgggctcat cctgtagcag atctcattct 60 ttaagtgagg ctgaaacaac caaaaatgca aaatctgcag acattgacag gcgaattttg 120 caagagacaa aagcagagca acacatccac aagctcttac ttcttggtgc gggagaatca 180 gggaagtcta cgatatttaa acagattaag ctccttttcc aaactggctt tgatgaggca 240 gaacttagga gctacacatc agttatccat gcaaacgtct atcagacaat taaagagcaa 300 aagaactctc acaagtggaa tcagattcct caaaatatgt tatatcccca gataaccagg 360 aaattggaga aaaactatca gatattgatg gcaggttgga ttatccactg ctgaacaaag 420 aacttgtact cgatgtaaaa aggttatggc aagacccagc cattcaggaa acttacttac 480 gtggaagtat tctgcaactt cctgattgtg cacaatactt catggaaaat ttggatcgat 540 tagctgaagc aggttatgtg ccaacaaagg aggatgtgct ttatgcaaga gtacggacaa 600 atggtgttgt acaaatacaa tttagtcctg ttggagaaaa caaaagaggt ggagaggtat 660 ataggttgta tgatgtagga ggccagagga atgagaggag aaagtggatt catctttttg 720 aaggtgttaa tgcggtaatc ttttgtgctg ccattagcga atatgatcag atgctatttg 780 aagatgagac aaaaaacaga atgatggaga ccaaggaact ctttgactgg gttttaaagc 840 aaagatgttt tgagaaaaca tcattcattc tgtttctcaa caaatttgat atattcgaga 900 agaaaataca aaaggttcct ttaagtgtgt gcgagtggtt taaagactac cagcctattg 960 cacctgggaa acaggaggtt gaacatgcat atgagtttgt caagaagaag tttgaagagc 1020 tctacttcca gagcagcaag cctgaccgtg tggaccgcgt cttcaaaatc tacagaacta 1080 cggccctaga ccagaaactt gtaaagaaga cattcaagtt gattgatgag agcatgagac 1140 gctccaggga aggaacttga 1160 <210> 4 <211> 137 <212> PRT <213> Oryza sativa <400> 4 Met Ser Val Leu Thr Cys Val Leu Asp Asn Met Gly Ser Ser Cys Ser 1 5 10 15 Arg Ser His Ser Leu Ser Glu Ala Glu Thr Thr Lys Asn Ala Lys Ser

Ala Asp Ile Asp Arg Arg Ile Leu Gln Glu Thr Lys Ala Glu Gln His 40 45 Ile His Lys Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr 50 55 60 Ile Phe Lys Gln Ile Lys Leu Leu Phe Gln Thr Gly Phe Asp Glu Ala 65 70 75 80 Glu Leu Arg Ser Tyr Thr Ser Val Ile His Ala Asn Val Tyr Gln Thr 85 90 95

Ile Lys Glu Gln Lys Asn Ser His Lys Trp Asn Gln Ile Pro Gln Asn 100 105 110 Met Leu Tyr Pro Gln Ile Thr Arg Lys Leu Glu Lys Asn Tyr Gln Ile 115 120 125 Leu Met Ala Gly Trp Ile Ile His Cys 130 135 <210> 5 <211> 1321 <212> DNA <213> Oryza sativa <400> 5 atgtccgtgc ttacctgtgt gcttgataac atgggctcat cctgtagcag atctcattct 60 ttaagtgagg ctgaaacaac caaaaatgca aaatctgcag acattgacag gcgaattttg 120 caagagacaa aagcagagca acacatccac aagctcttac ttcttggtgc gggagaatca 180 gggaagtcta cgatatttaa acagattaag ctccttttcc aaactggctt tgatgaggca 240 gaacttagga gctacacatc agttatccat gcaaacgtct atcagacaat taaagtatgc 300 aatactggaa agggtgtgtc ttttttttct tattgcaaag tggggattat gtaggagatt 360 cgactaggga tttgtattct gttcataagg aaatgcgttc atacttttcc tttttgtcga 420 gtaatgtgtt aaatgttaac tgatactata tgaaggagca aaagaactct cacaagtgga 480 atcagattcc tcaaaatatg ttatatcccc agataaccag gaaattggag aaaaactatc 540 agatattgat ggcaggttgg attatccact gctgaacaaa gaacttgtac tcgatgtaaa 600 aaggttatgg caagacccag ccattcagga aacttactta cgtggaagta ttctgcaact 660 tcctgattgt gcacaatact tcatggaaaa tttggatcga ttagctgaag caggttatgt 720 gccaacaaag gaggatgtgc tttatgcaag agtacggaca aatggtgttg tacaaataca 780 atttagtcct gttggagaaa acaaaagagg tggagaggta tataggttgt atgatgtagg 840 aggccagagg aatgagagga gaaagtggat tcatcttttt gaaggtgtta atgcggtaat 900 cttttgtgct gccattagcg aatatgatca gatgctattt gaagatgaga caaaaaacag 960 aatgatggag accaaggaac tctttgactg ggttttaaag caaagatgtt ttgagaaaac 1020 atcattcatt ctgtttctca acaaatttga tatattcgag aagaaaatac aaaaggttcc 1080 tttaagtgtg tgcgagtggt ttaaagacta ccagcctatt gcacctggga aacaggaggt 1140 tgaacatgca tatgagtttg tcaagaagaa gtttgaagag ctctacttcc agagcagcaa 1200 gcctgaccgt gtggaccgcg tcttcaaaat ctacagaact acggccctag accagaaact 1260 tgtaaagaag acattcaagt tgattgatga gagcatgaga cgctccaggg aaggaacttg 1320 a 1321 <210> © <211> 117 <212> PRT <213> Oryza sativa <400> © Met Ser Val Leu Thr Cys Val Leu Asp Asn Met Gly Ser Ser Cys Ser 1 5 10 15 Arg Ser His Ser Leu Ser Glu Ala Glu Thr Thr Lys Asn Ala Lys Ser

Ala Asp Ile Asp Arg Arg Ile Leu Gln Glu Thr Lys Ala Glu Gln His

40 45 Ile His Lys Leu Leu Leu Leu Gly Ala Gly Glu Ser Gly Lys Ser Thr

50 55 60 Ile Phe Lys Gln Ile Lys Leu Leu Phe Gln Thr Gly Phe Asp Glu Ala 65 70 75 80 Glu Leu Arg Ser Tyr Thr Ser Val Ile His Ala Asn Val Tyr Gln Thr 85 90 95 Ile Lys Val Cys Asn Thr Gly Lys Gly Val Ser Phe Phe Ser Tyr Cys 100 105 110

Lys Val Gly Ile Met

115 <210> 7 <211> 3670 <212> DNA <213> Oryza sativa <400> 7 atgtccgtgc ttacctgtgt gcttgataac atgggctcat cctgtagcag atctcattct 60 ttaagtgagg ctgaaacaac caaaaatgca aaagtaagtt agcactcgga cttactgaac 120 aagtaaatgc taactcaatt cttgatttga gagttgccac atttggtttc ttctaattca 180 gctggtaaca gtctgcagac attgacaggc gaattttgca agagacaaaa gcagagcaac 240 acatccacaa gctcttactt cttggtattg ctaactttcc caaatttaag tggtcatttt 300 ccttgtcaca attatctgcg ctacctttag tatctattgg ttcagaaaat taattgtttc 360 tgttgttcct atttacctct ataaaaaaaa cctttctcat gttatttcca aaaaaaagaa 420 gataaataaa tgtatcctag aaatttttag tttgaacttg ttctcaatgt ggatccatcc 480 ttctttectct ctctcaattg cttctgtttt aaggtgcggg agaatcaggg aagtctacga 540 tatttaaaca ggtgatgaat gttatattcc atggagaatc ataatccgta cgccgctagt 600 tagtctgatg tattcttact gttcacctgc agattaagct ccttttccaa actggctttg 660 atgaggcaga acttaggagc tacacatcag ttatccatgc aaacgtctat cagacaatta 720 aagtatgcaa tactggaaag ggtgtgtctt ttttttctta ttgcaaagtg gggattatgt 780 aggagattcg actagggatt tgtattctgt tcataaggaa atgcgttcat acttttcctt 840 tttgtcgagt aatgtgttaa atgttaactg atactatatg aaggagcaaa agaactctca 900 caagtggaat cagattcctc aaaatatgtt atatccccag ataaccaggt ttgtgcttac 960 tctttactca acagttaaag ctaaatctgt gcatatgaac atgtcttgtt aaatctggga 1020 atacaaacat tttgatttgc aacatttctg ttgtagtcaa gctgctcggc tctatgtttt 1080 aacctgttaa gaccttgtag actgtgctcg gctctattgt agtcttatat tttacacggt 1140 cattctataa tgaaaacttg aaaaagatat ctattgaacc gtacaatgta ctgaacaaag 1200 tagaaaagaa caatgagatt atgtaacatt tattcttcct tgtttatttg attgcttcag 1260 acaattgttg atatgctaaa aataacttgg tatcaaatgt gggtgttata agattcaatt 1320 tttttctcaa ccaggttaaa aaaagtatac ctttgtgcat ttaccttgtt ccgttgcttt 1380 ggaactttaa aggaaaactg acttttctta ggcattgaaa gacaaatatc accagtttca 1440 cactgtacac cttaccaacc aattttgttt cttagatgtc atttactttg tcatatcatc 1500 aggaaattgg agaaaaacta tcagatattg atggcaggtt ggattatcca ctgctgaaca 1560 aagaacttgt actcgatgta aaaaggttat ggcaagaccc agccattcag gtgaaaacaa 1620 atagccattc aaatcttttg aagttatata gttttcctgg ccaggtgtgc tgaagcaatg 1680 ctctatactg taggaaactt acttacgtgg aagtattctg caacttcctg attgtgcaca 1740 atacttcatg gaaaatttgg atcgattagc tgaagcaggt tatgtgccaa caaaggtgtg 1800 ctgtccatgt tcatagacaa ttatttacat attctcagat atttgtgctg acaccatttc 1860 atgttgattt ttagtctact tagtcagagg ttgtcaaatg gttaactatg tgtactgagt 1920 cagaggttgc caaatagttt taaaagatgg gcatatgttt atccttatct tttaaataat 1980 attggaggct atcctttaaa attcaatatt agggaggaga aactattatt ctaccgttat 2040 tacgcagtct acataacgaa ggtaaaaaat gtccctgtga aacatagggt gcaaaactgc 2100 tgtgaataaa actctactta tctaagcacc ttgagctttt gagttcccac atattaatct 2160 tatgacacta gcatatattt tttttgttca gttccttcaa taagttgcaa accacaaata 2220 tgatcactgt accatccact tttgcaacca tttcccgtca tttcttaagc atagaaaatt 2280 gtttgtcact tgtttaagtc cacactgcat caaaattcca attaactttg tgtgtgctaa 2340 gtgaagatat gactccatat ttctgcattt agcagtctga tggataattt atgattgtac 2400 cttgtctaat ggttcgtttg aaaggctggt agttgatctt ccatacttaa gaatgcttgc 2460 agtattatag ttgtcaatat tatgagtcat tttgcaggag gatgtgcttt atgcaagagt 2520 acggacaaat ggtgttgtac aaatacaatt taggtaatct gctgacacta ttttttgcac 2580 atttttttgc tggttgctct actatgtaca gaacgacaag ttgaagtcct ttttttctcc 2640 cttttcactt ctaagatatg acctgagagg ttctgaatgt agctgttata agatgagttg 2700 aatcatctag ttaactgggt ttctttctgc agtcctgttg gagaaaacaa aagaggtgga 2760 gaggtatata ggttgtatga tgtaggaggc cagaggaatg agaggagaaa gtggattcat 2820 ctttttgaag gtgttaatgc ggtaatcttt tgtgctgcca ttagcgagta agtacaattt 2880 ttttgattgt tgaacttatc ctaatctgct aagttcttct cataggcttc ttgttcattt 2940 cagatatgat cagatgctat ttgaagatga gacaaaaaac agaatgatgg agaccaagga 3000 actctttgac tgggttttaa agcaaagatg ttttgaggtc tgcatgcatc catttctgca 3060 acctttgtgc tcatgctttt tttctcattt tgaaactaat tacggtgcta tattgaccat 3120 cagaaaacat cattcattct gtttctcaac aaatttgata tattcgagaa gaaaatacaa 3180 aaggtaaggc ctgctctttg taccaatgca tagtttagta ctaaatgtta ccaacattta 3240 tgtttacgct ggttacgtag gttcctttaa gtgtgtgcga gtggtttaaa gactaccagc 3300 ctattgcacc tgggaaacag gaggttgaac atgcatatga gtgagtgcac tactcgccct 3360 ctcagatgaa catgggcatt tggccatttg taatgttgct gcatggtgca cttatatgcc 3420 ttgataagtt tttccattct aatgttatat agtatcaaac gttcatcatt actgtggctt 3480 atggtctgga gtgacgtttt acaggtttgt caagaagaag tttgaagagc tctacttcca 3540 gagcagcaag cctgaccgtg tggaccgcgt cttcaaaatc tacagaacta cggccctaga 3600 ccagaaactt gtaaagaaga cattcaagtt gattgatgag agcatgagac gctccaggga 3660 aggaacttga 3670 <210> 8

<211> 29

<212> DNA

<213> Oryza sativa

<400> 8 agagttcttt tgctccttca tatagtagc 29 <210> 9

<211> 21

<212> DNA

<213> Oryza sativa

<400> 9 tccgtacgcc gctagttagt c 21 <210> 10

<211> 26

<212> DNA

<213> Artificial Sequence

<220>

<223> 5'-TGW5 primer

<400> 10 ccggaattcc aaaccccgtt aaagcc 26 <210> 11

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> 3'-TGW5 primer

<400> 11 atagggtaca gacctgaaca gc 22 <210> 12

<211> 23

<212> DNA

<213> Artificial Sequence

<220>

<223> sgRNA target site probe

<400> 12 aaagaggtgg agaggtatat agg 23 <210> 13

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> 5'-TGW5-341 primer

<400> 13 ccatttcccg tcatttctta 20 <210> 14

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> 3'-TGW5-417 primer

<400> 14 ttccttggtc tccatcattc 20 <210> 15

<211> 40

<212> DNA

<213> Artificial Sequence

<220>

<223> TGW5 wt probe

<400> 15 cctcctacat catacaacct atatacctct ccacctcttt 40 <210> 16

<211> 37

<212> DNA

<213> Artificial Sequence

<220>

<223> TGW5 J22 probe

<400> 16 cctcctacat catacaacct tacctctcca cctcttt 37 <210> 17

<211> 38

<212> DNA

<213> Artificial Sequence

<220>

<223> TGW5 J42 probe

<400> 17 cctcctacat catacaacct atacctctcc acctcttt 38

Claims (7)

CONCLUSIONS An isolated protein selected from the following groups: (1) a polypeptide having an amino acid sequence as shown as SEQ ID NO:2; or (2) a polypeptide formed from the amino acid sequence shown as SEQ ID NO:2 by substitution, deletion or addition of 1 to 10 amino acid residues and derived from (1). An isolated protein selected from the following groups: (1) a polypeptide having an amino acid sequence as shown as SEQ ID NO:4; or (2) a polypeptide formed from the amino acid sequence shown as SEQ ID NO:4 by substitution, deletion or addition of 1 to 10 amino acid residues and derived from (1). An isolated protein selected from the following groups: (1) a polypeptide having an amino acid sequence as shown as SEQ ID NO:6; or (2) a polypeptide formed from the amino acid sequence shown as SEQ ID NO:6 by substitution, deletion or addition of 1 to 10 amino acid residues and derived from (1). An isolated polynucleotide selected from the following groups: (a) a polynucleotide encoding the protein of any one of claims 1, 2 and 3; (b) a polynucleotide comprising any of the sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7; {c) a polynucleotide having a nucleotide sequence having a homology of 95% or more (preferably of 98% or more) to any of the sequences of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and SEQ ID NO:7; (d) a polynucleotide which is complementary to any of the polynucleotides of (a), (b) and (c). Use of the protein according to any one of claims 1 to 3 or a polynucleotide according to claim 4 for controlling the seed grain size of crops. Use of the protein according to any one of claims 1 to 3 or a polynucleotide according to claim 4 for regulating the plant height of crops. Use of the protein of any one of claims 1 to 3 or a polynucleotide of claim 4 for regulating crop seed weight and crop yield.