CN109112137B

CN109112137B - Gene SNG1 for controlling size and weight of rice grains and application thereof

Info

Publication number: CN109112137B
Application number: CN201711155158.6A
Authority: CN
Inventors: 何予卿; 云鹏; 李一博
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2017-11-20
Filing date: 2017-11-20
Publication date: 2021-07-09
Anticipated expiration: 2037-11-20
Also published as: CN109112137A

Abstract

The present invention belongs to the field of plant gene engineering technology. In particular to a gene SNG1 for controlling the size and the weight of rice grains and application thereof. The invention discloses a related gene SNG1 for controlling rice grain length, grain width and grain weight, wherein the SNG1 gene controls the size of chaff by influencing cell elongation and regulates the filling rate so as to influence the yield of rice. The invention obtains the rice plant with over-expression SNG1 by using the transgenic method, and the experiment shows that the transgenic rice plant is obviously improved in the relative characters of grain length, grain width and grain weight compared with the contrast. The cloned gene of the invention provides new gene resources and new application of the gene for breeding the yield and quality of rice.

Description

Gene SNG1 for controlling size and weight of rice grains and application thereof

Technical Field

The invention relates to the technical field of plant genetic engineering. In particular to a gene SNG1 for controlling the character of rice grain, especially controlling the character of length, width and weight of the rice grain and application thereof. The SNG1 gene is located at the tail end of the first chromosome long arm of the rice and controls the related characters of the length, the width and the weight of rice grains.

Background

The size of rice grains is controlled by three sub-characters of grain length, grain width and grain thickness, the grain weight is directly determined, and the grain weight is one of three constitutive factors (the effective ear number of a single plant, the solid grain number of each ear and the thousand grain weight) of the single plant yield, so the grain size is an important factor influencing the yield; the grain size of rice is also an important appearance quality character, and in the case of indica rice, most Chinese consumers prefer to have a slender grain shape, and in the national standard (high-quality rice GB/T17891-1999), the length-width ratio of the high-quality rice is required to be more than 2.8.

Rice grain size is a complex trait controlled by multiple quantitative trait loci. In recent years, with the development of genetic mapping, genome sequencing and functional genomic research, Quantitative Trait Loci (QTLs) that largely control the size of rice grains have been mapped, among which a number have been cloned: GS3(Fan et al, 2006), GW2(Song et al, 2007), GW5/qSW5(Shomura et al, 2008; Weng et al, 2008), GS5(Li et al, 2011), GW8(Wang et al, 2012), qGL3/qGL3.1(Qi et al, 2012; Zhang et al, 2012), TGW6(Ishimaru et al, 2013), GS2/GL2(Che et al, 2015; Duan et al, 2015; Hu et al, 2015), GL7/GW7(Wang et al, 2015 a; Wang et al, 2015b), GW6a (Song et al, 2015) and OsSPL13(Si al, 2016). These genes regulate rice seed size by a variety of molecular mechanisms, such as G protein signaling, proteasome degradation pathways, plant hormones, and transcriptional regulation, among others (Li and Li, 2016; Zuo and Li, 2014). We also need to clone more grain shape genes, study the interrelation of the grain shape genes, and construct the molecular regulation network of the grain shape, so as to better understand the biological basis of rice grain development and guide the genetic improvement of rice yield and appearance quality.

The applicant of the invention separates and identifies a rice granule mutant SNG1(short and narrow grain 1), clones a new gene SNG1 for positively regulating the size of seeds by using a map-based cloning method, verifies the function of the gene by using a genetic transformation method, enriches a granular molecular regulation network, and provides a new gene resource for genetic improvement of rice grain shape.

Disclosure of Invention

The invention aims to overcome the defects of the prior art, and utilizes a small-grain mutant SNG1 to isolate and clone a related gene which is positioned on a first chromosome and controls grain length, grain width and grain weight, and the gene is named as SNG1 gene by the applicant.

The invention separates and identifies a small-grain mutant sng1 produced by tissue culture, and the background is japonica rice variety Hwayoung (abbreviated as HW, the same below, conventional source variety, which is planted in Korea in large area). Compared with wild rice, the mutant seed has significantly reduced grain length, grain width and thousand kernel weight (see figure 1), the grain length, grain width and thousand kernel weight of F2 generation segregation population constructed by the HW gene and the sng1 gene are distributed in a bimodal mode, and the segregation ratio of large grain materials to small grain materials accords with the segregation ratio of 3:1 in classical genetics (see figure 1), which indicates that the mutant seed is reduced by unit points.

The applicant clones the variant sites by using a map-based cloning method, and hybridizes sng1 with indica rice variety Zhenshan 97B (ZS97 for short, large-area application in China) with larger seeds to construct an F2 generation segregation population for primary positioning. An isogenic line with Zhenshan 97B (ZS97B) as background is constructed by a backcross method, the source materials of the cloned gene are rice mutant seeds sng1 and Oryza sativa L.sng1, and the obtained seeds are delivered to the China center for type culture collection of the university of Wuhan, Wuhan in 2017 at 11 month and 17 days, wherein the collection number is CCTCC NO: p201723 (see figure 2 for a technical route for constructing a near isogenic line), and the effect of SNG1 is verified by separating a population by using the near isogenic line, which shows that the particle length, the particle width and the weight average of thousand particles of the population are in bimodal distribution (figure 3). Using the methods of genetic large population and map-based cloning of SNG1 near isogenic lines, SNG1 was finely mapped to a 5.5kb chromosomal segment (FIG. 4). The target segment contained two incomplete candidate genes, which were sequenced and aligned, and compared to the HW wild-type, the sng1 mutant had only a single nucleotide mutation (i.e., a T-a mutation) in the entire segment, which was located in the last mutexon of the candidate gene (accession number LOC _ Os01g71310), and which changed the codon encoding arginine (AGA) to the stop codon (TGA), and the candidate gene of sng1 was predicted to encode a truncated protein lacking the C-terminal 103 amino acids (fig. 4). Bioinformatics analysis indicated that LOC _ Os01g71310 comprises 9 exons, encoding 500 amino acids in total, which encodes a hexokinase (OsHXK 3). Through agrobacterium-mediated genetic transformation, over-expression (OE) SNG1 is performed in Hwayoung, transgenic T0 generation positive single plants show larger seeds (figure 5), T1 generation family co-segregation detection shows that grain length, grain width and thousand seed weight are co-segregated with genotype (figure 6), and the plant morphology, heading period and other properties of T2 generation transgenic homozygous families are not greatly different from wild type (figure 7), which indicates that the over-expression of SNG1 has certain application prospect.

The invention has the advantages that:

(1) the invention clones the gene SNG1 for positively regulating the characteristics of the rice grain length, the grain width and the grain weight in the rice, and provides a new gene resource for high-yield and high-quality breeding of the rice;

(2) the cloned gene has strong evolutionary conservation, and the invention can also provide reference for genetic improvement of other crops.

Drawings

SEQ ID NO. 1 of the sequence Listing is the nucleotide sequence of SNG1 allele of wild type rice variety Hwayoung (HW) isolated and cloned according to the present invention.

SEQ ID NO. 2 of the sequence Listing is the protein sequence encoded by the SNG1 allele of wild type Hwayoung (HW).

SEQ ID NO 3 of the sequence Listing is the nucleotide sequence of the sng1 allele of the mutant from which the present invention was isolated and cloned.

SEQ ID NO. 4 of the sequence Listing is the protein sequence encoded by the sng1 allele of the mutant of the present invention.

FIG. 1: sng1/HW F₂Separating the distribution of the grain length, the grain width and the thousand grain weight of the colony. Description of reference numerals: FIG. 1A is a graph showing the frequency distribution of particle lengths; FIG. 1B is a graph showing a frequency distribution of grain widths; the graph C in fig. 1 is a frequency distribution graph of thousand kernel weights.

Fig. 2: is the process chart of the construction of the near isogenic line of the invention (Zhenshan 97 is used as female parent in backcross).

FIG. 3: BC₃F₂The frequency distribution of grain length, width and thousand kernel weight in random populations. Description of reference numerals: FIG. 3 is a graph A showing a frequency distribution of grain lengths; FIG. 3B is a graph showing a frequency distribution of grain widths; the graph C in fig. 3 is a frequency distribution graph of thousand kernel weights. Wherein, black, striped and white bars respectively represent SNG1 site mutant SNG1 homozygous genotype (A), heterozygous genotype (H) and Zhenshan 97 homozygous genotype (B), and three genotypes of SNG1 are detected by molecular markers.

FIG. 4: map-based cloning of the SNG1 gene of the invention. Description of reference numerals: panel A in FIG. 4 is the result of preliminary mapping, with the SNG1 gene mapped into a 480kb segment; the B panel in FIG. 4 is the result of fine localization, with the SNG1 gene being localized to a 5.5kb segment; panel C in figure 4 is a graph showing the genotype and progeny of the key recombinant individual segment of interest. The numbers between the markers in FIG. 4 represent the number of recombinations that occurred between each marker and the SNG1 gene site.

FIG. 5: overexpression of the SNG1 Gene of the inventionT₀And (3) performing expression and phenotype statistical analysis on grain shape and grain weight of transgenic individuals. Description of reference numerals: panel A in FIG. 5 is the transgenic plant grain shape, on the left of which is the transgenic negative material (OE (-)) and on the right is the transgenic positive material (OE (+); panel B, panel C and panel D in fig. 5 show the results of grain length, grain width and thousand kernel weight statistics for transgenic negative and transgenic positive plants, respectively, by the two-tailed T test, with P values shown above, for a total of 12 transgenic negative plants (n-12) and 20 transgenic positive plants (n-20).

FIG. 6: overexpression of T in SNG1 Gene of the invention₁And (4) the phenotype and the genotype of the generation transgenic pedigree are subjected to cosegregation analysis. Description of reference numerals: FIG. 6A is a graph showing the results of coseparation of grain length and genotype; FIG. 6B is a graph showing the results of coseparation of grain width and genotype; panel C in FIG. 6 shows the results of cosegregation of thousand kernel weight and genotype. The white open column and the blue solid column represent the transgenic negative and transgenic positive plants, respectively, and the genotype was obtained by detecting the reporter gene GUS by the PCR method.

FIG. 7: overexpression of T in SNG1 Gene of the invention₂And (3) detecting the phenotype of the transgenic pedigree in the filling stage and the expression level of the SNG1 gene. Description of drawings: panel A in FIG. 7 is wild type (HW) and two SNG1 gene overexpression T₂The phenotype of plants in the grain filling stage of the generation transgenic homozygous family lines (OE-4 and OE-35); FIG. 7B is a graph showing the relative expression level of SNG1 gene in the heading stage flag leaves measured by Real-time PCR.

FIG. 8: map of the overexpression vector PU1301 used in the invention.

Detailed Description

This example identifies a rice mutant with smaller seeds from Korean T-DNA insertion mutant library (Jeon et al, 2000) under the code PFG _1C-12303, and the transformation receptor is japonica rice variety Hwayoung (HW for short). Compared with wild HW, the seed length and the seed width of the mutant are obviously reduced, the thousand seed weight is obviously reduced (figure 1), and other properties such as plant shape, plant height, heading period and the like are not greatly changed. The applicants named the mutant sng1 (the source material of the cloned gene of the present invention is rice mutantThe variant seed sng1, Oryza sativa L.sng1, is delivered to China at 11 months and 17 days in 2017, the China center for type culture Collection of Wuhan university, with the collection number of CCTCC NO: p201723). HW and mutant construction of F₂The grain length, the grain width and the thousand grain weight of the generation segregation population are in bimodal distribution, the grain shape of large-grain materials is close to HW, the grain shape of small-grain materials is close to that of mutants, and the segregation ratio of the small-grain materials accords with the 3:1 segregation ratio of classical genetics (figure 1), which indicates that the mutant seed reduction is caused by single-site recessive mutation. However, the applicants could not isolate the T-DNA flanking sequences of the mutant, and thought that the mutation may be caused by tissue culture, the site of the mutation was cloned by using the map-based cloning method.

Example 2: mapping and map-based cloning of the SNG1 Gene

1. Backcrossing and selection

The invention utilizes a map-based cloning method to clone the variation sites, and obtains F through hybridizing sng1 and indica rice variety Zhenshan 97B (from agricultural academy of sciences in Jiangxi province) with wider seeds₂The generation segregating population (FIG. 2), primary localization was performed using segregating population grouping Analysis (BSA). The QTL at the end of the 1 st chromosome is found to control the grain length and the grain width, the Zhenshan 97 genotype increases the phenotypic value, and the QTL is named as SNG 1.

From F₂Selecting small-particle single plants from the generation segregation population, hybridizing with female parent Zhenshan 97 once, combining molecular Marker Assisted Selection (MAS) on the basis of primary positioning, taking Zhenshan 97 as recurrent parent, carrying out continuous backcross for 3 times, and then selfing for 1 time to obtain a near-isogenic line of SNG1 for fine positioning of SNG1 (figure 2). Using 192 strains of BC₃F₂Segregating the population confirmed the effect of SNG1, indicating that the population had a bimodal distribution of particle length, particle width and thousand weight average (fig. 3). Is shown here BC₃F₂In the population, grain size is controlled by a major gene.

2. Method for genotyping and phenotyping

The genotype determination adopts an SSR method: see, e.g., sambrook et al, 2002, molecular cloning, a guide, third edition, anser geese et al (translation), methods introduced by scientific publishers, for standard procedures for PCR. PCR A20. mu.l reaction was used, comprising: 20-50ng DNA template, 10mM Tris-HCl, 50mM KCl, 0.1% Triton X-100,1.8mM MgCl2,0.1mM dNTP, 0.2. mu.M primer and 1U Taq DNA polymerase. The conditions for PCR amplification were: pre-denaturation at 94 ℃ for 4 min; 1min at 94 ℃, 1min at 55 ℃, 1min at 72 ℃ and 34 cycles; extension at 72 ℃ for 10 min. The PCR products were silver stained after separation on a 6% polyacrylamide gel (Bassam et al, 1991, anal. biochem.196: 80-83).

The SSR marker information used in the invention is all from a Gramene website database (http:// www.gramene.org/) In addition, the genome sequence of the japonica rice variety Nipponbare published on the web (http://rgp.dna.affrc.go.jp) And the genomic sequence of indica rice variety 93-11 (http:// rise. genetics. org. cn /) an Indel (Insert/Deletion) marker with polymorphism between Zhenshan 97B and SNG1 was designed for fine localization analysis of SNG 1. The sequence information of these markers is shown in Table 1.

Phenotype investigation method:

measurement of grain length, grain width and thousand grain weight: the seeds are harvested, dried in the sun and placed at room temperature for at least 3 months to ensure that the seeds are dry and the water content of the seeds among various strains is relatively consistent. Randomly selecting 30 full seeds from each individual plant, arranging 10 seeds into a group, arranging the seeds in a row in a side-by-side, non-overlapping and non-gap mode in the same direction, reading the width by using a vernier caliper, and repeatedly taking the average value for 3 times to obtain the width of the seeds; the 10 seeds are arranged end to end, the length is read by a vernier caliper, and the average value is taken repeatedly for 3 times, namely the seed length. The thousand kernel weight was calculated by weighing the randomly picked 200 full weights. Fine localization and candidate Gene determination of the SNG1 Gene

On the basis of effect verification, the SNG1 gene is finely positioned by taking the grain width as a target trait. 192 strain BC for verifying SNG1 gene effect₃F₂In the segregating population, 13 recombinant individuals exist between Indel markers Y11 and Y91 at the two ends of the segment where the SNG1 gene is located (figure 4), the 13 recombinant individuals are subjected to a Progeny test (Progeney testing), and the genotype of the recombinant individual SNG1 is deduced according to the grain width phenotype of 24 Progeny: recombining the single plants if the progeny have no segregation in grain width and are all large grainsThe SNG1 of the current generation of the strain is Zhenshan 97 homozygote; if the progeny grain width is not separated and all the progeny grain widths are small, the SNG1 of the current generation of the recombinant single plant is homozygous for SNG 1; if the progeny has segregation in grain width and seed size, the genotype of SNG1 of the current generation of the recombinant single plant is heterozygote. Molecular markers were encrypted in the target segment and the markers were genotyped for linkage analysis, fine-positioning of SNG1 into the 480kb segment between markers Y6 and Y54 (FIG. 4).

Based on this, the applicant planted ZS97/sng1BC₃F₃Isolate 7680 strains of the population, screen 125 recombinant individuals with markers Y6 and Y54, perform a progeny test on the recombinant individuals, infer the genotype of the recombinant individual SNG1, encrypt the markers further, and finally map SNG1 to a 5.5kb segment between Indel markers Y1 and Y7, co-segregate with the marker SNP3 (FIG. 4).

With reference to the japonica rice "Nipponbare" genome (http:// rice. plant biology. msu. edu/index. shtml), the 5.5kb segment between markers Y1 and Y7 contains two incomplete ORFs, and the two ORFs are arranged in a tail-to-tail fashion. By taking a Nipponbare genome as a reference, designing primer pairs (shown in table 1) and sequencing 5.5kb positioning segments of HW, sng1 and ZS97, and comparing the sequence, only one SNP (T-A) is found in the whole segment of the sng1 mutant compared with a HW wild type rice variety, the SNP is positioned on the last mutexon of a candidate gene LOC _ Os01g71310, and the mutation enables a codon (AGA) for coding arginine to be changed into a stop codon (TGA), thereby predicting that the candidate gene of the sng1 encodes a truncated protein which lacks 103C-terminal amino acids; ZS97 the SNP genotype is T (same as HW), and there are 14 SNP variations between ZS97 and sng1 in a 5.5kb segment, 12 of which are located in non-coding regions and 2 are located in exons and are synonymous mutations. LOC _ Os01g71310 was therefore identified as a candidate gene for SNG1, and SNG1QTL mapped using the map-based cloning method was generated by mutation of SNG1 (possibly from tissue culture), rather than natural variation between HW and ZS 97.

TABLE 1 primer sequences for the map-based cloning and Gene function analysis of the present invention

Example 3: transgenic complementation assay of SNG1

Construction of SNG1 overexpression vector PU1301

Based on the SNG1 full-length cDNA sequence of Nipponbare (accession number AY884171, DQ116385), primers were designed to amplify the coding region of wild type HW SNG 1. Wherein the OEF primer and the OER primer respectively have KpnI restriction endonuclease recognition sites and BamHI restriction endonuclease recognition sites, and the PCR reaction system is as follows: HW first strand cDNA 4. mu.l +2 XKOD FX PCR buffer 25. mu.l +2mM dNTPs 10. mu.l + OEF/OER 1. mu.l + KOD FX (1.0U/. mu.l) 1. mu.l + dH₂O7 mu l; the PCR amplification conditions were: 94 ℃ for 4min, 98 ℃ for 10sec, 68 ℃ for 2min, 32cycles, 72 ℃ for 10min, 25 ℃ for 1 min; the amplification length was approximately 1.6 kb. The amplification product is firstly subjected to a 3' end dA adding reaction (the high fidelity KOD enzyme amplification product is a blunt end and cannot be directly used for TA cloning), and then is connected to a pGEM-T (purchased from Promega corporation, figure 8) cloning vector after being recovered and purified by gel digging, and the connection reaction system is as follows: mu.l of the recovered amplification product + pGEM-T vector (50 ng/. mu.l) 1. mu.l +2 XT 4Ligase Buffer 5. mu.l + T4Ligase (400U/. mu.l) 1. mu.l. Reacting at 16 ℃ overnight, transforming escherichia coli, selecting a monoclonal shake bacteria to extract plasmid, carrying out double enzyme digestion with KpnI and BamHI and sequencing verification to select a positive clone without mutation, carrying out double enzyme digestion on the positive clone with KpnI and BamHI, carrying out gel excavation, recycling and purifying, and connecting to a binary over-expression vector pU1301 (figure 8), wherein the connecting reaction system is as follows: mu.l of the recovered pU1301 vector was digested simultaneously with KpnI and BamHI in 4. mu.l + 10 XT 4Ligase Buffer 1. mu.l + T4Ligase (400U/. mu.l) in 1. mu.l. Reacting at 16 ℃ overnight, transforming escherichia coli, selecting single clone, shaking bacteria, extracting plasmid, performing double enzyme digestion verification on KpnI and BamHI, and obtaining the correct clone which is the constructed SNG1 overexpression vector PU1301(SNG1 OE). Transformation of SNG1 overexpression vector PU1301

The genetic transformation method of this example refers to the following documents:

(1) patent nos. ZL 2004100133457; a method for in vitro culture of indica rice, Huazhong university of agriculture; or

(2) The patent No. ZL 2013104541628, university of Huazhong agriculture, application of histone methyltransferase SDG723 in regulating the heading stage of rice and a reported method.

The invention relates to a specific main step of genetic transformation, a culture medium and a preparation method thereof, wherein the specific main step comprises the following steps:

1) reagent and solution abbreviations

The abbreviations for the phytohormones used in the culture media in this experiment are as follows: 6-BA (6-BenzylaminoPurine, 6-benzyladenine); CN (Carbenicillin ); KT (Kinetin ); NAA (Napthalene acetic acid, naphthylacetic acid); IAA (Indole-3-acetic acid, indoleacetic acid); 2,4-D (2, 4-dichlorphenoxyacetic acid, 2,4-Dichlorophenoxyacetic acid); AS (acetosyringone); CH (Casein enzymic Hydrolysate, hydrolyzed Casein); HN (Hygromycin B, Hygromycin); DMSO (Dimethyl S μ lfoxide, Dimethyl sulfoxide); n is a radical of_6max(N6 macronutrient component solution); n is a radical of_6mix(N6 microelement component solution); MS (Mass Spectrometry)_max(MS macronutrient component solution); MS (Mass Spectrometry)_mix(MS microelement composition solution)

2) Main solution formulation

a)N_6maxCulture medium macroelement mother liquor (prepared according to 10 times of concentrated solution):

dissolving the above reagents one by one, then using distilled water to fix the volume to 1000ml at room temperature, and storing at room temperature.

b)N_6minCulture medium microelement mother liquor (prepared according to 100 times of concentrated solution):

the above reagents were dissolved at room temperature and made up to 1000ml with distilled water and stored at room temperature.

c) Iron salt (Fe)²⁺EDTA) stock solution (prepared as 100X concentrate):

3.73g of disodium ethylene diamine tetraacetate (Na)₂EDTA·2H₂O) and 2.78g FeSO₄·7H₂Dissolving O respectively, mixing, diluting with distilled water to 1000ml, warm bathing at 70 deg.C for 2 hr, and storing at 4 deg.C.

d) Vitamin stock solution (prepared as 100X concentrate):

nicotinic acid (niacin) 0.1g

Vitamin B₁(Thiamine HCl) 0.1g

Vitamin B₆(Pyridoxine HCl) 0.1g

Glycine (Glycine) 0.2g

Inositol (Inositol) 10g

Adding distilled water to a constant volume of 1000ml, and storing at 4 ℃ for later use.

e) MS culture medium macroelement mother liquor (MS)_maxMother liquor) (prepared as 10X concentrate):

ammonium Nitrate (NH)₄NO₃) 16.5g

Potassium nitrate (KNO)₃) 19.0g

Potassium dihydrogen phosphate (KH)₂PO₄) 1.7g

Magnesium sulfate (MgSO)₄·7H₂O) 3.7g

Calcium chloride (CaCl)₂·2H₂O) 4.4g

The above reagents were dissolved at room temperature, and the volume was adjusted to 1000ml with distilled water, and stored at room temperature.

f) MS culture medium microelement mother liquor (MS)_minMother liquor) (prepared as 100X concentrate):

g) Preparation of 2,4-D stock solution (1 mg/ml):

weighing 2, 4-D100 mg, dissolving with 1ml of 1N potassium hydroxide for 5 minutes, adding 10ml of distilled water to dissolve completely, fixing the volume to 100ml, and storing at room temperature.

h) Preparation of 6-BA stock solution (1 mg/ml):

weighing 6-BA 100mg, dissolving with 1ml1N potassium hydroxide for 5 minutes, adding 10ml distilled water to dissolve completely, fixing the volume to 100ml, and storing at room temperature.

i) Preparation of stock solution (1mg/ml) of Naphthylacetic acid (NAA):

weighing NAA 100mg, dissolving with 1ml1N potassium hydroxide for 5 min, adding 10ml distilled water to dissolve completely, diluting to 100ml, and storing at 4 deg.C in dark place.

j) Preparation of Indoleacetic acid (IAA) stock solution (1 mg/ml):

weighing 100mg of IAA, dissolving with 1ml of 1N potassium hydroxide for 5 minutes, adding 10ml of distilled water to dissolve completely, fixing the volume to 100ml, and storing at 4 ℃ in a dark place.

k) Preparation of glucose stock solution (0.5 g/ml):

weighing 125g of glucose, dissolving with distilled water to a constant volume of 250ml, sterilizing, and storing at 4 ℃ for later use.

l) preparation of AS stock solutions:

weighing 0.392g of AS, adding 10ml of DMSO to dissolve, subpackaging into 1.5ml of centrifuge tubes, and storing at-20 ℃ for later use.

m) preparation of 1N potassium hydroxide stock solution:

weighing 5.6g of potassium hydroxide, dissolving with distilled water to a constant volume of 100ml, and storing at room temperature for later use.

3) Culture medium formula for japonica rice genetic transformation

a) Induction medium

Adding distilled water to 900ml, adjusting pH to 5.9 with 1N potassium hydroxide, boiling to 1000ml, packaging into 50ml triangular flask (30 ml/bottle), sealing, and sterilizing by conventional method (for example, 121 deg.C for 15 min, the following method for sterilizing culture medium is the same as that for the present culture medium).

b) Subculture medium:

adding distilled water to 900ml, adjusting pH to 5.9 with 1N potassium hydroxide, boiling, diluting to 1000ml, packaging into 50ml triangular flask (30 ml/bottle), sealing, and sterilizing.

c) Suspension culture medium:

adding distilled water to 100ml, adjusting pH to 5.4, packaging into two 100ml triangular bottles, sealing, and sterilizing according to the above method.

1ml sterile glucose stock solution and 100. mu. lAS stock solution were added before use.

d) Co-culture medium:

adding distilled water to 250ml, adjusting pH to 5.6 with 1N potassium hydroxide, sealing, and sterilizing as above.

The medium was dissolved by heating and 5ml of glucose stock solution and 250. mu. lAS stock solution were added before use and dispensed into petri dishes (25ml per dish).

e) Screening a culture medium:

adding distilled water to 250ml, adjusting pH to 6.0, sealing, and sterilizing by the above method.

The medium was dissolved before use and 250. mu. lHN (50mg/ml) and 400. mu. lCN (10g CN/36ml water) were added and dispensed into petri dishes (25 ml/dish). (Note: the concentration of carbenicillin in the first selection medium was 400 mg/liter, and the concentration of carbenicillin in the second and subsequent selection media was 250 mg/liter).

f) Differentiation medium:

distilled water was added thereto to 900ml, and 1N potassium hydroxide was added to adjust the pH to 6.0.

Boiling, adding distilled water to 1000ml, packaging into 100ml triangular flask (50 ml/bottle), sealing, and sterilizing.

g) Rooting culture medium

Distilled water was added to 900ml, and the pH was adjusted to 5.8 with 1N potassium hydroxide.

Boiling, adding distilled water to 1000ml, packaging into raw tube (25 ml/tube), sealing, and sterilizing.

4) Agrobacterium-mediated genetic transformation procedure

a) Callus induction

Mature rice seeds were dehulled and then treated sequentially with 70% ethanol for 1 minute with 0.15% mercuric chloride (HgCl)₂) Seed surface disinfection15-25 minutes; washing the seeds with sterilized water for 4-5 times; placing 8-10 seeds on an induction culture medium; placing the inoculated culture medium in a dark place for culturing for 4-5 weeks at the temperature of 26 +/-1 ℃.

b) Callus subculture:

the bright yellow, compact and relatively dry embryogenic calli were selected and placed on subculture medium for 2 weeks in the dark at 25 + -1 deg.C.

c) Pre-culturing:

compact and relatively dry embryogenic calli were selected and placed on pre-culture medium for 2 weeks in the dark at 26 + -1 deg.C.

d) And (3) agrobacterium culture:

two days at 28 ℃ for preculture of Agrobacterium EHA105 (the strain is from an Agrobacterium strain publicly used by CAMBIA Inc.) in LA medium with corresponding resistance selection (see: preparation of LA medium J. SammBruke et al, molecular cloning instructions, third edition, King Dong Yan et al (translation), scientific Press, 2002, Beijing); the Agrobacterium is transferred to a suspension medium and cultured on a shaker at 28 ℃ for 2-3 hours.

e) Infection of agrobacterium:

transferring the pre-cultured callus to a sterilized bottle; adjusting the suspension of Agrobacterium to OD 6000.8-1.0; soaking the callus in agrobacterium tumefaciens suspension for 30 minutes; transferring the callus to sterilized filter paper and sucking to dry; then placed on a co-culture medium to be cultured for 3 days at a temperature of 19-20 ℃.

f) Callus washing and selective culture:

washing the callus with sterilized water until no agrobacterium is visible; soaking in sterilized water containing 400mg/L Carbenicillin (CN) and shaking for 30 min; transferring the callus to sterilized filter paper and sucking to dry; transferring the callus to a selective culture medium for selective culture for 2-3 times, 2 weeks each time until good resistant callus grows out.

g) Differentiation:

transferring the resistant callus to a pre-differentiation culture medium and culturing for 5-7 days in a dark place; transferring the pre-differentiated cultured callus to a differentiation medium, evenly distributing three independent calluses in each bottle, culturing for 5-6 weeks under illumination until large seedlings grow at 26 ℃.

h) Rooting and seedling exercising:

cutting off old roots generated during differentiation; then transferring the seedlings to a rooting culture medium, culturing for 2-3 weeks under illumination until large seedlings grow out, removing a sealing film, adding part of tap water, hardening seedlings for one week, and transplanting at the temperature of 26 ℃.

i) Transplanting:

washing off residual culture medium on the roots, transferring the seedlings with good root systems into a greenhouse, keeping moisture wet in the first few days, transplanting the seedlings to a field after the seedlings are strong in equal length.

2. Trait survey of transgenic plants

The invention obtains 32 independent SNG1 Overexpression (OE) T₀And (3) generating transgenic plants, and determining the genotypes of the transgenic plants by amplifying a reporter gene GUS through PCR, wherein 20 plants are transgenic positive individuals, and 12 plants are transgenic negative individuals. The expression level of SNG1 was detected by Real-time PCR, and the expression level of SNG1 of the transgenic positive individual was found to be increased to different degrees. Examining the grain length, grain width and thousand grain weight of the 32 transgenic single plant seeds, the grain length, grain width and thousand grain weight of the over-expressed positive single plant are found to be significantly larger than those of the negative plant (P)<0.01) (fig. 5).

To further validate the results of the superscalar transformation, from 20T₀Randomly selecting 3 single plants from the generation positive plants, and planting T₁The generation carries out coseparation detection on the genotype and the phenotype, each generation is 36 plants, and the genotype of each single plant is determined by using the PCR amplification result of a GUS reporter gene (a conventional marker gene). The results show that the genotype is completely co-segregating with grain length, grain width and thousand kernel weight (fig. 6), further demonstrating that the SNG1 gene is a candidate gene that is controlling grain length, grain width and thousand kernel weight.

In addition, the transgenic material of the over-expression SNG1 has little influence on the agronomic traits such as plant morphology, heading stage and the like (figure 7), which shows that the over-expression of SNG1 may have a certain application prospect, and the rice variety can be improved through the gene transformation.

Reference to the literature

1.Che RH,Tong HN,Shi BH,Liu YQ,Fang SR,Liu DP,Xiao YH,Hu B,Liu LC,Wang HR,Zhao MF,Chu CC.Control of grain size and rice yield by GL2-mediated brassinosteroid responses.Nature Plants,2015,2: 15195

2.Duan PG,Ni S,Wang JM,Zhang BL,Xu R,Wang YX,Chen HQ,Zhu XD,Li YH.Regulation of OsGRF4 by OsmiR396 controls grain size and yield in rice.Nature Plants,2015,2:15203

3.Fan C,Xing Y,Mao H,Lu T,Han B,Xu C,Li X,Zhang Q.GS3,a major QTL for grain length and weight and minor QTL for grain width and thickness in rice,encodes a putative transmembrane protein.Theoretical and Applied Genetics,2006,112:1164-1171

4.Hu J,Wang Y,Fang Y,Zeng L,Xu J,Yu H,Shi Z,Pan J,Zhang D,Kang S,Zhu L,Dong G,Guo L,Zeng D, Zhang G,Xie L,Xiong G,Li J,Qian Q.A rare allele of GS2 enhances grain size and grain yield in rice. Molecular Plant,2015,8:1455-1465

5.Ishimaru K,Hirotsu N,Madoka Y,Murakami N,Hara N,Onodera H,Kashiwagi T,Ujiie K,Shimizu B, Onishi A,Miyagawa H,Katoh E.Loss of function of the IAA-glucose hydrolase gene TGW6 enhances rice grain weight and increases yield.Nature Genetics,2013,45:707-711

6.Jeon JS,Lee S,Jung KH,Jun SH,Jeong DH,Lee J,Kim C,Jang S,Yang K,Nam J,An K,Han MJ,Sung RJ, Choi HS,Yu JH,Choi JH,Cho SY,Cha SS,Kim SI,An G.T-DNA insertional mutagenesis for functional genomics in rice.Plant J,2000,22:561-570

7.Li,N.,and Li,Y.Signaling pathways of seed size control in plants.Current Opinion in Plant Biology,2016, 33:23-32

8.Li Y,Fan C,Xing Y,Jiang Y,Luo L,Sun L,Shao D,Xu C,Li X,Xiao J,He Y,Zhang Q.Natural variation in GS5 plays an important role in regulating grain size and yield in rice.Nature Genetics,2011c,43:1266-1269

9.Qi P,Lin YS,Song XJ,Shen JB,Huang W,Shan JX,Zhu MZ,Jiang L,Gao JP,Lin HX.The novel quantitative trait locus GL3.1 controls rice grain size and yield by regulating Cyclin-T1；3.Cell Research,2012,22:1666-1680

10.Shomura A,Izawa T,Ebana K,Ebitani T,Kanegae H,Konishi S,Yano M.Deletion in a gene associated with grain size increased yields during rice domestication.Nature Genetics,2008,40:1023-1028

11.Si L,Chen J,Huang X,Gong H,Luo J,Hou Q,Zhou T,Lu T,Zhu J,Shangguan Y,Chen E,Gong C,Zhao Q, Jing Y,Zhao Y,Li Y,Cui L,Fan D,Lu Y,Weng Q,Wang Y,Zhan Q,Liu K,Wei X,An K,An G,Han B.OsSPL13 controls grain size in cultivated rice.Nature Genetics,2016,48:447-456

12.Song XJ,Huang W,Shi M,Zhu MZ,Lin HX.A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase.Nature Genetics,2007,39:623-630

13.Song XJ,Kuroha T,Ayano M,Furuta T,Nagai K,Komeda N,Segami S,Miura K,Ogawa D,Kamura T, Suzuki T,Higashiyama T,Yamasaki M,Mori H,Inukai Y,Wu J,Kitano H,Sakakibara H,Jacobsen SE, Ashikari M.Rare allele of a previously unidentified histone H4 acetyltransferase enhances grain weight, yield,and plant biomass in rice.Proc Natl Acad Sci USA,2015,112:76-81

14.Wang S,Li S,Liu Q,Wu K,Zhang J,Wang S,Wang Y,Chen X,Zhang Y,Gao C,Wang F,Huang H,Fu X. The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality.Nature Genetics,2015a,47:949-954.

15.Wang S,Wu K,Yuan Q,Liu X,Liu Z,Lin X,Zeng R,Zhu H,Dong G,Qian Q,Zhang G,Fu X.Control of grain size,shape and quality by OsSPL16 in rice.Nature Genetics,2012,44:950-954

16.Wang Y,Xiong G,Hu J,Jiang L,Yu H,Xu J,Fang Y,Zeng L,Xu E,Xu J,Ye W,Meng X,Liu R,Chen H, Jing Y,Wang Y,Zhu X,Li J,Qian Q.Copy number variation at the GL7 locus contributes to grain size diversity in rice.Nature Genetics,2015b,47:944-948

17.Weng J,Gu S,Wan X,Gao H,Guo T,Su N,Lei C,Zhang X,Cheng Z,Guo X,Wang J,Jiang L,Zhai H,Wan J.Isolation and initial characterization of GW5,a major QTL associated with rice grain width and weight. Cell Research,2008,18:1199-1209

18.Zhang X,Wang J,Huang J,Lan H,Wang C,Yin C,Wu Y,Tang H,Qian Q,Li J,Zhang H.Rare allele of OsPPKL1 associated with grain length causes extra-large grain and a significant yield increase in rice.Proc Natl Acad Sci USA,2012,109:21534-21539

19.Zuo,J.,and Li,J.Molecular genetic dissection of quantitative trait loci regulating rice grain size.Annual Review of Genetics.2014,48:99-118。

Sequence listing

<110> university of agriculture in Huazhong

<120> gene SNG1 for controlling size and weight of rice grains and application thereof

<141> 2017-11-20

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1503

<212> DNA

<213> Rice (Oryza sativa)

<220>

<221> gene

<222> (1)..(1503)

<220>

<221> CDS

<222> (1)..(1503)

<400> 1

atg ggg agg gtg ggg ctc ggc gtg gcg gtg ggg tgc gcg gcg gtg acc 48

Met Gly Arg Val Gly Leu Gly Val Ala Val Gly Cys Ala Ala Val Thr

1 5 10 15

tgc gcg atc gcc gcg gcg ctc gtg gcg cgc agg gcg tcg gcg cgg gcg 96

Cys Ala Ile Ala Ala Ala Leu Val Ala Arg Arg Ala Ser Ala Arg Ala

20 25 30

cgg tgg cgg cgg gcg gtg gcg ctg ctg cgg gag ttc gag gag ggg tgt 144

Arg Trp Arg Arg Ala Val Ala Leu Leu Arg Glu Phe Glu Glu Gly Cys

35 40 45

gcc acg ccg ccc gcg agg ctg cgc cag gtc gtg gac gcc atg gtc gtc 192

Ala Thr Pro Pro Ala Arg Leu Arg Gln Val Val Asp Ala Met Val Val

50 55 60

gag atg cac gcc ggc ctc gcg tcc gat ggc ggc agc aag ctc aag atg 240

Glu Met His Ala Gly Leu Ala Ser Asp Gly Gly Ser Lys Leu Lys Met

65 70 75 80

ctg ctc acc ttc gtc gac gcg ctc ccc agc ggg agt gaa gaa ggt gta 288

Leu Leu Thr Phe Val Asp Ala Leu Pro Ser Gly Ser Glu Glu Gly Val

85 90 95

tat tat tct att gat ctt gga gga aca aac ttc aga gtc ttg agg gta 336

Tyr Tyr Ser Ile Asp Leu Gly Gly Thr Asn Phe Arg Val Leu Arg Val

100 105 110

caa gtt ggt gcg gga tct gtg atc gtc aac caa aag gtt gaa cag caa 384

Gln Val Gly Ala Gly Ser Val Ile Val Asn Gln Lys Val Glu Gln Gln

115 120 125

ccc atc cct gag gaa ctg acc aaa ggc act act gag ggt tta ttc aac 432

Pro Ile Pro Glu Glu Leu Thr Lys Gly Thr Thr Glu Gly Leu Phe Asn

130 135 140

ttt gtt gcc ctg gca cta aag aat ttt ctt gaa gga gaa gat gac caa 480

Phe Val Ala Leu Ala Leu Lys Asn Phe Leu Glu Gly Glu Asp Asp Gln

145 150 155 160

gat gga aaa atg gca ctt ggt ttt aca ttt tct ttc cct gtt aga caa 528

Asp Gly Lys Met Ala Leu Gly Phe Thr Phe Ser Phe Pro Val Arg Gln

165 170 175

att tca gtg tct tca ggg tca tta att agg tgg aca aaa gga ttt tcc 576

Ile Ser Val Ser Ser Gly Ser Leu Ile Arg Trp Thr Lys Gly Phe Ser

180 185 190

atc aga gac acg gtt ggc aga gat gtt gct cag tgc tta aat gaa gcg 624

Ile Arg Asp Thr Val Gly Arg Asp Val Ala Gln Cys Leu Asn Glu Ala

195 200 205

ctt gcc aat tgt ggg cta aat gtg cgg gtc act gca ttg gtg aat gat 672

Leu Ala Asn Cys Gly Leu Asn Val Arg Val Thr Ala Leu Val Asn Asp

210 215 220

aca gtg ggg aca tta gct cta ggg cat tac tat gat gaa gac aca gtg 720

Thr Val Gly Thr Leu Ala Leu Gly His Tyr Tyr Asp Glu Asp Thr Val

225 230 235 240

gct gct gtg ata att ggg tct ggc acc aac gct tgc tac att gaa cgc 768

Ala Ala Val Ile Ile Gly Ser Gly Thr Asn Ala Cys Tyr Ile Glu Arg

245 250 255

act gat gca att atc aag tgc cag ggt ctt cta acg aac tct gga ggc 816

Thr Asp Ala Ile Ile Lys Cys Gln Gly Leu Leu Thr Asn Ser Gly Gly

260 265 270

atg gta gta aac atg gag tgg ggg aat ttc tgg tca tca cat ttg cca 864

Met Val Val Asn Met Glu Trp Gly Asn Phe Trp Ser Ser His Leu Pro

275 280 285

agg acg cca tat gac atc ttg ctg gat gat gaa aca cac aat cgc aat 912

Arg Thr Pro Tyr Asp Ile Leu Leu Asp Asp Glu Thr His Asn Arg Asn

290 295 300

gat cag ggc ttt gag aaa atg ata tca gga atg tat ctt ggg gaa att 960

Asp Gln Gly Phe Glu Lys Met Ile Ser Gly Met Tyr Leu Gly Glu Ile

305 310 315 320

gca aga ttg gta ttt cat aga atg gcc cag gaa tca gat gtt ttt ggt 1008

Ala Arg Leu Val Phe His Arg Met Ala Gln Glu Ser Asp Val Phe Gly

325 330 335

gat gct gct gat agt cta tcc aac cct ttc att ttg agc aca ccg ttt 1056

Asp Ala Ala Asp Ser Leu Ser Asn Pro Phe Ile Leu Ser Thr Pro Phe

340 345 350

ctg gcc gca att cgc gag gac gat tca cca gat ctg agc gaa gtc aga 1104

Leu Ala Ala Ile Arg Glu Asp Asp Ser Pro Asp Leu Ser Glu Val Arg

355 360 365

agg ata ctt cga gaa cat ctg aag att ccc gat gct cct ctg aaa act 1152

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

370 375 380

cga cgg ctg gtc gtg aaa gtc tgc gac att gtg act cgc aga gcc gcc 1200

Arg Arg Leu Val Val Lys Val Cys Asp Ile Val Thr Arg Arg Ala Ala

385 390 395 400

cgt cta gcc gct gca ggc atc gtg ggg ata ctg aag aag ctg ggg agg 1248

Arg Leu Ala Ala Ala Gly Ile Val Gly Ile Leu Lys Lys Leu Gly Arg

405 410 415

gat ggg agc ggc gcg gcg tcg agc ggg aga ggt aga ggg cag ccg agg 1296

Asp Gly Ser Gly Ala Ala Ser Ser Gly Arg Gly Arg Gly Gln Pro Arg

420 425 430

agg acg gtg gtg gcg atc gag ggc ggg ctg tac cag ggt tac ccg gtg 1344

Arg Thr Val Val Ala Ile Glu Gly Gly Leu Tyr Gln Gly Tyr Pro Val

435 440 445

ttc agg gag tac ctg gac gag gcc ctg gtg gag atc ctg ggg gag gag 1392

Phe Arg Glu Tyr Leu Asp Glu Ala Leu Val Glu Ile Leu Gly Glu Glu

450 455 460

gtg gcg cgg aac gtg acg ctg agg gtg acg gag gat ggg tcg ggg gtc 1440

Val Ala Arg Asn Val Thr Leu Arg Val Thr Glu Asp Gly Ser Gly Val

465 470 475 480

ggg gct gcc ctc ctc gcc gcc gta cat tcg tcg aat aga cag caa caa 1488

Gly Ala Ala Leu Leu Ala Ala Val His Ser Ser Asn Arg Gln Gln Gln

485 490 495

gga ggt ccc ata tag 1503

Gly Gly Pro Ile

500

<210> 2

<211> 500

<212> PRT

<213> Rice (Oryza sativa)

<400> 2

Met Gly Arg Val Gly Leu Gly Val Ala Val Gly Cys Ala Ala Val Thr

1 5 10 15

Cys Ala Ile Ala Ala Ala Leu Val Ala Arg Arg Ala Ser Ala Arg Ala

20 25 30

Arg Trp Arg Arg Ala Val Ala Leu Leu Arg Glu Phe Glu Glu Gly Cys

35 40 45

Ala Thr Pro Pro Ala Arg Leu Arg Gln Val Val Asp Ala Met Val Val

50 55 60

Glu Met His Ala Gly Leu Ala Ser Asp Gly Gly Ser Lys Leu Lys Met

65 70 75 80

Leu Leu Thr Phe Val Asp Ala Leu Pro Ser Gly Ser Glu Glu Gly Val

85 90 95

Tyr Tyr Ser Ile Asp Leu Gly Gly Thr Asn Phe Arg Val Leu Arg Val

100 105 110

Gln Val Gly Ala Gly Ser Val Ile Val Asn Gln Lys Val Glu Gln Gln

115 120 125

Pro Ile Pro Glu Glu Leu Thr Lys Gly Thr Thr Glu Gly Leu Phe Asn

130 135 140

Phe Val Ala Leu Ala Leu Lys Asn Phe Leu Glu Gly Glu Asp Asp Gln

145 150 155 160

Asp Gly Lys Met Ala Leu Gly Phe Thr Phe Ser Phe Pro Val Arg Gln

165 170 175

Ile Ser Val Ser Ser Gly Ser Leu Ile Arg Trp Thr Lys Gly Phe Ser

180 185 190

Ile Arg Asp Thr Val Gly Arg Asp Val Ala Gln Cys Leu Asn Glu Ala

195 200 205

Leu Ala Asn Cys Gly Leu Asn Val Arg Val Thr Ala Leu Val Asn Asp

210 215 220

Thr Val Gly Thr Leu Ala Leu Gly His Tyr Tyr Asp Glu Asp Thr Val

225 230 235 240

Ala Ala Val Ile Ile Gly Ser Gly Thr Asn Ala Cys Tyr Ile Glu Arg

245 250 255

Thr Asp Ala Ile Ile Lys Cys Gln Gly Leu Leu Thr Asn Ser Gly Gly

260 265 270

Met Val Val Asn Met Glu Trp Gly Asn Phe Trp Ser Ser His Leu Pro

275 280 285

Arg Thr Pro Tyr Asp Ile Leu Leu Asp Asp Glu Thr His Asn Arg Asn

290 295 300

Asp Gln Gly Phe Glu Lys Met Ile Ser Gly Met Tyr Leu Gly Glu Ile

305 310 315 320

Ala Arg Leu Val Phe His Arg Met Ala Gln Glu Ser Asp Val Phe Gly

325 330 335

Asp Ala Ala Asp Ser Leu Ser Asn Pro Phe Ile Leu Ser Thr Pro Phe

340 345 350

Leu Ala Ala Ile Arg Glu Asp Asp Ser Pro Asp Leu Ser Glu Val Arg

355 360 365

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

370 375 380

Arg Arg Leu Val Val Lys Val Cys Asp Ile Val Thr Arg Arg Ala Ala

385 390 395 400

Arg Leu Ala Ala Ala Gly Ile Val Gly Ile Leu Lys Lys Leu Gly Arg

405 410 415

Asp Gly Ser Gly Ala Ala Ser Ser Gly Arg Gly Arg Gly Gln Pro Arg

420 425 430

Arg Thr Val Val Ala Ile Glu Gly Gly Leu Tyr Gln Gly Tyr Pro Val

435 440 445

Phe Arg Glu Tyr Leu Asp Glu Ala Leu Val Glu Ile Leu Gly Glu Glu

450 455 460

Val Ala Arg Asn Val Thr Leu Arg Val Thr Glu Asp Gly Ser Gly Val

465 470 475 480

Gly Ala Ala Leu Leu Ala Ala Val His Ser Ser Asn Arg Gln Gln Gln

485 490 495

Gly Gly Pro Ile

500

<210> 3

<211> 1503

<212> DNA

<213> Rice (Oryza sativa)

<220>

<221> gene

<222> (1)..(1503)

<220>

<221> CDS

<222> (1)..(1503)

<400> 3

atg ggg agg gtg ggg ctc ggc gtg gcg gtg ggg tgc gcg gcg gtg acc 48

Met Gly Arg Val Gly Leu Gly Val Ala Val Gly Cys Ala Ala Val Thr

1 5 10 15

tgc gcg atc gcc gcg gcg ctc gtg gcg cgc agg gcg tcg gcg cgg gcg 96

Cys Ala Ile Ala Ala Ala Leu Val Ala Arg Arg Ala Ser Ala Arg Ala

20 25 30

cgg tgg cgg cgg gcg gtg gcg ctg ctg cgg gag ttc gag gag ggg tgt 144

Arg Trp Arg Arg Ala Val Ala Leu Leu Arg Glu Phe Glu Glu Gly Cys

35 40 45

gcc acg ccg ccc gcg agg ctg cgc cag gtc gtg gac gcc atg gtc gtc 192

Ala Thr Pro Pro Ala Arg Leu Arg Gln Val Val Asp Ala Met Val Val

50 55 60

gag atg cac gcc ggc ctc gcg tcc gat ggc ggc agc aag ctc aag atg 240

Glu Met His Ala Gly Leu Ala Ser Asp Gly Gly Ser Lys Leu Lys Met

65 70 75 80

ctg ctc acc ttc gtc gac gcg ctc ccc agc ggg agt gaa gaa ggt gta 288

Leu Leu Thr Phe Val Asp Ala Leu Pro Ser Gly Ser Glu Glu Gly Val

85 90 95

tat tat tct att gat ctt gga gga aca aac ttc aga gtc ttg agg gta 336

Tyr Tyr Ser Ile Asp Leu Gly Gly Thr Asn Phe Arg Val Leu Arg Val

100 105 110

caa gtt ggt gcg gga tct gtg atc gtc aac caa aag gtt gaa cag caa 384

Gln Val Gly Ala Gly Ser Val Ile Val Asn Gln Lys Val Glu Gln Gln

115 120 125

ccc atc cct gag gaa ctg acc aaa ggc act act gag ggt tta ttc aac 432

Pro Ile Pro Glu Glu Leu Thr Lys Gly Thr Thr Glu Gly Leu Phe Asn

130 135 140

ttt gtt gcc ctg gca cta aag aat ttt ctt gaa gga gaa gat gac caa 480

Phe Val Ala Leu Ala Leu Lys Asn Phe Leu Glu Gly Glu Asp Asp Gln

145 150 155 160

gat gga aaa atg gca ctt ggt ttt aca ttt tct ttc cct gtt aga caa 528

Asp Gly Lys Met Ala Leu Gly Phe Thr Phe Ser Phe Pro Val Arg Gln

165 170 175

att tca gtg tct tca ggg tca tta att agg tgg aca aaa gga ttt tcc 576

Ile Ser Val Ser Ser Gly Ser Leu Ile Arg Trp Thr Lys Gly Phe Ser

180 185 190

atc aga gac acg gtt ggc aga gat gtt gct cag tgc tta aat gaa gcg 624

Ile Arg Asp Thr Val Gly Arg Asp Val Ala Gln Cys Leu Asn Glu Ala

195 200 205

ctt gcc aat tgt ggg cta aat gtg cgg gtc act gca ttg gtg aat gat 672

Leu Ala Asn Cys Gly Leu Asn Val Arg Val Thr Ala Leu Val Asn Asp

210 215 220

aca gtg ggg aca tta gct cta ggg cat tac tat gat gaa gac aca gtg 720

Thr Val Gly Thr Leu Ala Leu Gly His Tyr Tyr Asp Glu Asp Thr Val

225 230 235 240

gct gct gtg ata att ggg tct ggc acc aac gct tgc tac att gaa cgc 768

Ala Ala Val Ile Ile Gly Ser Gly Thr Asn Ala Cys Tyr Ile Glu Arg

245 250 255

act gat gca att atc aag tgc cag ggt ctt cta acg aac tct gga ggc 816

Thr Asp Ala Ile Ile Lys Cys Gln Gly Leu Leu Thr Asn Ser Gly Gly

260 265 270

atg gta gta aac atg gag tgg ggg aat ttc tgg tca tca cat ttg cca 864

Met Val Val Asn Met Glu Trp Gly Asn Phe Trp Ser Ser His Leu Pro

275 280 285

agg acg cca tat gac atc ttg ctg gat gat gaa aca cac aat cgc aat 912

Arg Thr Pro Tyr Asp Ile Leu Leu Asp Asp Glu Thr His Asn Arg Asn

290 295 300

gat cag ggc ttt gag aaa atg ata tca gga atg tat ctt ggg gaa att 960

Asp Gln Gly Phe Glu Lys Met Ile Ser Gly Met Tyr Leu Gly Glu Ile

305 310 315 320

gca aga ttg gta ttt cat aga atg gcc cag gaa tca gat gtt ttt ggt 1008

Ala Arg Leu Val Phe His Arg Met Ala Gln Glu Ser Asp Val Phe Gly

325 330 335

gat gct gct gat agt cta tcc aac cct ttc att ttg agc aca ccg ttt 1056

Asp Ala Ala Asp Ser Leu Ser Asn Pro Phe Ile Leu Ser Thr Pro Phe

340 345 350

ctg gcc gca att cgc gag gac gat tca cca gat ctg agc gaa gtc aga 1104

Leu Ala Ala Ile Arg Glu Asp Asp Ser Pro Asp Leu Ser Glu Val Arg

355 360 365

agg ata ctt cga gaa cat ctg aag att ccc gat gct cct ctg aaa act 1152

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

370 375 380

cga cgg ctg gtc gtg aaa gtc tgc gac att gtg act cgc aga gcc gcc 1200

Arg Arg Leu Val Val Lys Val Cys Asp Ile Val Thr Arg Arg Ala Ala

385 390 395 400

cgt cta gcc gct gca ggc atc gtg ggg ata ctg aag aag ctg ggg agg 1248

Arg Leu Ala Ala Ala Gly Ile Val Gly Ile Leu Lys Lys Leu Gly Arg

405 410 415

gat ggg agc ggc gcg gcg tcg agc ggg aga ggt aga ggg cag ccg agg 1296

Asp Gly Ser Gly Ala Ala Ser Ser Gly Arg Gly Arg Gly Gln Pro Arg

420 425 430

agg acg gtg gtg gcg atc gag ggc ggg ctg tac cag ggt tac ccg gtg 1344

Arg Thr Val Val Ala Ile Glu Gly Gly Leu Tyr Gln Gly Tyr Pro Val

435 440 445

ttc agg gag tac ctg gac gag gcc ctg gtg gag atc ctg ggg gag gag 1392

Phe Arg Glu Tyr Leu Asp Glu Ala Leu Val Glu Ile Leu Gly Glu Glu

450 455 460

gtg gcg cgg aac gtg acg ctg agg gtg acg gag gat ggg tcg ggg gtc 1440

Val Ala Arg Asn Val Thr Leu Arg Val Thr Glu Asp Gly Ser Gly Val

465 470 475 480

ggg gct gcc ctc ctc gcc gcc gta cat tcg tcg aat aga cag caa caa 1488

Gly Ala Ala Leu Leu Ala Ala Val His Ser Ser Asn Arg Gln Gln Gln

485 490 495

gga ggt ccc ata tag 1503

Gly Gly Pro Ile

500

<210> 4

<211> 500

<212> PRT

<213> Rice (Oryza sativa)

<400> 4

Met Gly Arg Val Gly Leu Gly Val Ala Val Gly Cys Ala Ala Val Thr

1 5 10 15

Cys Ala Ile Ala Ala Ala Leu Val Ala Arg Arg Ala Ser Ala Arg Ala

20 25 30

Arg Trp Arg Arg Ala Val Ala Leu Leu Arg Glu Phe Glu Glu Gly Cys

35 40 45

Ala Thr Pro Pro Ala Arg Leu Arg Gln Val Val Asp Ala Met Val Val

50 55 60

Glu Met His Ala Gly Leu Ala Ser Asp Gly Gly Ser Lys Leu Lys Met

65 70 75 80

Leu Leu Thr Phe Val Asp Ala Leu Pro Ser Gly Ser Glu Glu Gly Val

85 90 95

Tyr Tyr Ser Ile Asp Leu Gly Gly Thr Asn Phe Arg Val Leu Arg Val

100 105 110

Gln Val Gly Ala Gly Ser Val Ile Val Asn Gln Lys Val Glu Gln Gln

115 120 125

Pro Ile Pro Glu Glu Leu Thr Lys Gly Thr Thr Glu Gly Leu Phe Asn

130 135 140

Phe Val Ala Leu Ala Leu Lys Asn Phe Leu Glu Gly Glu Asp Asp Gln

145 150 155 160

Asp Gly Lys Met Ala Leu Gly Phe Thr Phe Ser Phe Pro Val Arg Gln

165 170 175

Ile Ser Val Ser Ser Gly Ser Leu Ile Arg Trp Thr Lys Gly Phe Ser

180 185 190

Ile Arg Asp Thr Val Gly Arg Asp Val Ala Gln Cys Leu Asn Glu Ala

195 200 205

Leu Ala Asn Cys Gly Leu Asn Val Arg Val Thr Ala Leu Val Asn Asp

210 215 220

Thr Val Gly Thr Leu Ala Leu Gly His Tyr Tyr Asp Glu Asp Thr Val

225 230 235 240

Ala Ala Val Ile Ile Gly Ser Gly Thr Asn Ala Cys Tyr Ile Glu Arg

245 250 255

Thr Asp Ala Ile Ile Lys Cys Gln Gly Leu Leu Thr Asn Ser Gly Gly

260 265 270

Met Val Val Asn Met Glu Trp Gly Asn Phe Trp Ser Ser His Leu Pro

275 280 285

Arg Thr Pro Tyr Asp Ile Leu Leu Asp Asp Glu Thr His Asn Arg Asn

290 295 300

Asp Gln Gly Phe Glu Lys Met Ile Ser Gly Met Tyr Leu Gly Glu Ile

305 310 315 320

Ala Arg Leu Val Phe His Arg Met Ala Gln Glu Ser Asp Val Phe Gly

325 330 335

Asp Ala Ala Asp Ser Leu Ser Asn Pro Phe Ile Leu Ser Thr Pro Phe

340 345 350

Leu Ala Ala Ile Arg Glu Asp Asp Ser Pro Asp Leu Ser Glu Val Arg

355 360 365

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

370 375 380

Arg Arg Leu Val Val Lys Val Cys Asp Ile Val Thr Arg Arg Ala Ala

385 390 395 400

Arg Leu Ala Ala Ala Gly Ile Val Gly Ile Leu Lys Lys Leu Gly Arg

405 410 415

Asp Gly Ser Gly Ala Ala Ser Ser Gly Arg Gly Arg Gly Gln Pro Arg

420 425 430

Arg Thr Val Val Ala Ile Glu Gly Gly Leu Tyr Gln Gly Tyr Pro Val

435 440 445

Phe Arg Glu Tyr Leu Asp Glu Ala Leu Val Glu Ile Leu Gly Glu Glu

450 455 460

Val Ala Arg Asn Val Thr Leu Arg Val Thr Glu Asp Gly Ser Gly Val

465 470 475 480

Gly Ala Ala Leu Leu Ala Ala Val His Ser Ser Asn Arg Gln Gln Gln

485 490 495

Gly Gly Pro Ile

500

Claims

The application of SNG1 gene in rice grain width improvement through overexpression, wherein the nucleotide sequence of the gene is shown as SEQ ID NO:1 is shown.