CN109112137A

CN109112137A - A kind of gene SNG1 and its application of control rice grain size and grain weight

Info

Publication number: CN109112137A
Application number: CN201711155158.6A
Authority: CN
Inventors: 何予卿; 云鹏; 李博; 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: 2019-01-01
Anticipated expiration: 2037-11-20
Also published as: CN109112137B

Abstract

The invention belongs to field of plant genetic project technology.More particularly to the gene SNG1 and its application of a kind of control rice grain size and grain weight.The invention discloses a kind of control rice grain length, related gene SNG1, the SNG1 genes that grain is wide and grain is heavy to control the size of husk by influencing cell elongation, and adjust filling rate, and then influence the yield of rice.The present invention obtains the rice plant of SNG1 overexpression using transgenic method, experiments have shown that, the rice plant of transgenosis is in grain length, grain is wide and grain weight correlated traits on significantly improve compared with the control.The gene that the present invention clones provides the new application of new genetic resources and gene for the yield and quality breeding of rice.

Description

A kind of gene SNG1 and its application of control rice grain size and grain weight

Technical field

The present invention relates to field of plant genetic project technology.It is especially controlled specifically related to a kind of control rice grain character Rice grain grain length processed, grain be wide and gene SNG1 and its application of grain principal characteristic shape.The SNG1 gene is located at the dye of rice first The long-armed end of colour solid, gene control rice grain grain length, grain are wide and grain weighs correlated traits.

Background technique

The size of rice grain is by grain length, grain is wide, grain thick three sub- characters control, it directly determines grain weight, and grain is again One of big Components of the three of single plant yield (panicle number per plant, Defined daily doses and mass of 1000 kernel), therefore, grain size is An important factor for influencing yield；Rice grain size is also an important Appearance Quality Traits, most of by taking long-grained nonglutinous rice as an example It is tall and thin that Chinese Consumer's prefer particle shape, in national standard (high quality paddy GB/T17891-1999), it is desirable that superior rice Length-width ratio is more than 2.8.

Rice grain size is the complex character controlled by multiple quantitative trait locus.In recent years, with genetic mapping, base Because of a group development for sequencing and functional genome research, a large amount of quantitative trait locus for controlling rice grain size (Quantitative Trait Loci, QTLs) is positioned, wherein there is multiple be 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.,2015a；Wang et al.,2015b),GW6a(Song et al., And OsSPL13 (Si et al., 2016) 2015).These gene regulation rice paddy seed sized molecules mechanism are varied, such as G Protein signaling, proteasome degradation pathway, plant hormone and transcriptional control etc. (Li and Li, 2016；Zuo and Li,2014).We also need to continue to clone more particle shape genes, and study the correlation of particle shape gene, construct particle shape Molecular regulation network is just better understood on the Basic of Biology of rice grain development in this way, and guides rice yield and appearance The genetic improvement of quality.

Applicant of the present invention's separation identifies the small particle mutant sng1 of a rice (short and narrow grain 1) the gene SNG1 of a new positive regulation seed size, has been cloned using the method for map based cloning, while utilizing genetic transformation The method validation function of the gene, enriches the molecular regulation network of particle shape, also provides for the genetic improvement of rice grain shape New genetic resources.

Summary of the invention

It is an object of the invention to overcome the deficiencies in the prior art, are obtained using particle mutant sng1 one small separation clone One is located at the related gene for controlling that grain length, grain are wide and grain is heavy on the first chromosome, this unnamed gene is SNG1 by applicant Gene.

Present invention separation identifies the small particle mutant sng1 of tissue cultures generation, and background is japonica rice variety Hwayoung (abbreviation HW, similarly hereinafter, usual sources kind is planted in South Korea's large area).Compared with wild rice, mutation Body seed grain length, grain are wide and mass of 1000 kernel significantly reduces (see Fig. 1), utilize the gene constructed F2 generation separation group of HW gene and sng1 Grain length, the grain of body are wide and bimodal distribution is presented in mass of 1000 kernel, and the segregation ratio of big grain material and granule material meets classical heredity The segregation ratio (see Fig. 1) of 3:1 is learned, showing that mutant seeds become smaller is as caused by unit point.

Applicant utilizes the method clonal vaviation site of map based cloning, by sng1 and the biggish rice variety treasure Shan of seed 97B (abbreviation ZS97, Chinese large-area applications) hybridization building F2 carries out just positioning for segregating population.And pass through the method for backcrossing The near isogenic lines with Zhenshan 97B (ZS97B) for background is constructed, the source material of clone gene of the present invention is rice mutation Body seed sng1, Oryza sativa L.sng1 delivers the China of the Chinese Wuhan Wuhan University on November 17th, 2017 Type Tissue Collection preservation, deposit number are that CCTCC NO:P201723 (be shown in by the technology path of building near isogenic lines Fig. 2), using the effect of near isogenic lines segregating population verifying SNG1, show that the grain length of group, grain are wide and mass of 1000 kernel is in double Peak is distributed (Fig. 3).Using the method for the heredity of SNG1 near isogenic lines big group and map based cloning, SNG1 finely positioning is arrived In the chromosome segment of 5.5kb (Fig. 4).Include two incomplete candidate genes in target zone, is sequenced and carries out sequence ratio To discovery, compared with HW wild type, only one single nucleotide mutation (i.e. the mutation of T-A) in the entire section of sng1 mutant, The variation is located on the last one exon of candidate gene (number of logging in LOC_Os01g71310), and the variation makes coding essence The codon (AGA) of propylhomoserin becomes terminator codon (TGA), predicts that the candidate gene of sng1 encodes one and lacks C-terminal 103 The truncated albumen (Fig. 4) of amino acid.Bioinformatic analysis shows that LOC_Os01g71310 includes 9 exons, encodes altogether 500 amino acid, the gene encode a hexokinase (OsHXK3).By Agrobacterium-mediated genetic transformation, Overexpression (OE) SNG1 in Hwayoung, transgenosis T0 generation positive single plant show bigger seed (Fig. 5), and T1 is for family System isolates wide detection discovery grain length, grain and mass of 1000 kernel and genotype isolates (Fig. 6), and T2 is for transgenic homozygous family The characters such as plant forms and heading stage and wild type difference less (Fig. 7), show overexpressing with before certain application for SNG1 Scape.

The present invention has the advantages that

(1) present invention has cloned that positive adjusting and controlling rice grain is long, grain is wide and the gene SNG1 of grain principal characteristic shape in rice, is water The high yield and high quality breeding of rice provides new genetic resources；

(2) gene cloned in the present invention has the conservative in very strong evolution, and the present invention may be other crops Genetic improvement offer reference.

Detailed description of the invention

Sequence table SEQ ID NO:1 is present invention separation clone from wild rice kind Hwayoung (HW) The nucleotide sequence of SNG1 allele.

Sequence table SEQ ID NO:2 is the protein sequence of the SNG1 allele coding of wild type Hwayoung (HW).

Sequence table SEQ ID NO:3 is the nucleotides sequence of the sng1 allele of the source mutant of present invention separation clone Column.

Sequence table SEQ ID NO:4 is the protein sequence of the sng1 allele coding of mutant of the invention.

Fig. 1: sng1/HW F₂Segregating population grain length, grain be wide and the distribution of mass of 1000 kernel.Description of symbols: the A figure in Fig. 1 It is the chart of frequency distribution of grain length；B figure in Fig. 1 is the wide chart of frequency distribution of grain；C figure in Fig. 1 is the frequency disribution of mass of 1000 kernel Figure.

Fig. 2: being near isogenic lines building flow chart of the invention (when backcrossing, using precious Shan 97 as female parent).

Fig. 3: BC₃F₂Grain grain length, grain be wide in random population and the frequency distribution of mass of 1000 kernel.Description of symbols: in Fig. 3 A figure be grain length chart of frequency distribution；B figure in Fig. 3 is the wide chart of frequency distribution of grain；C figure in Fig. 3 is the frequency of mass of 1000 kernel Number distribution map.Wherein, the stick of black, striped and white respectively indicates SNG1 site mutant sng1 homozygous genotype (A), miscellaneous Genotype (H) and precious 97 homozygous genotype of Shan (B) are closed, three kinds of genotype of SNG1 are obtained by Markers for Detection.

Fig. 4: the map based cloning of SNG1 gene of the present invention.Description of symbols: the A figure in Fig. 4 be Primary Location as a result, SNG1 gene is positioned in the section of 480kb；B figure in Fig. 4 is finely positioning as a result, SNG1 gene is positioned to In the section of 5.5kb；C figure in Fig. 4 is the result for showing crucial recombination single plant purpose section genotype and progeny test.Fig. 4 Number between middle label represents the recombination number occurred between each label and SNG1 gene loci.

Fig. 5: SNG1 gene overexpression T of the invention₀The performance of particle shape and grain weight for transgenosis single plant and phenotype system Meter analysis.Description of symbols: the A figure in Fig. 5 is transgenic plant particle shape, is transgene negative material on the left side of the figure (OE (-)), the right are transgenic positive material (OE (+))；In Fig. 5 B figure, C figure and D figure respectively indicate transgene negative with Grain length, the grain of transgenic positive plant are wide and mass of 1000 kernel statistical result, statistical method are double tail T tests, and P value is shown above, Transgene negative plant totally 12 plants (n=12), transgenic positive plant totally 20 plants (n=20).

Fig. 6: SNG1 gene overexpression T of the invention₁For the knot of transgenic lines phenotype and genotype coseparation analysis Fruit.Description of symbols: the A figure in Fig. 6 is grain length and the result that genotype isolates；B figure in Fig. 6 is that grain is wide and gene The result that type isolates；C figure in Fig. 6 is mass of 1000 kernel and the result that genotype isolates.White hollow pillar and blue are solid Pillar respectively indicates transgene negative and transgenic positive plant, and genotype is by the method examining report gene with PCR What GUS was obtained.

Fig. 7: SNG1 gene overexpression T of the invention₂For transgenic lines pustulation period plant phenotype and SNG1 gene table Up to the testing result of amount.Description of symbols: the A figure in Fig. 7 is wild type (HW) and two SNG1 gene overexpression T₂Generation The pustulation period plant phenotype of transgenic homozygous family (OE-4 and OE-35)；B figure in Fig. 7 is with Real-time PCR method The relative expression quantity of SNG1 gene in the heading stage sword-like leave of detection.

Fig. 8: the map for the overexpression carrier PU1301 that the present invention uses.

Specific embodiment

The present embodiment identifies the rice mutant that a seed becomes smaller, and comes from South Korea T-DNA insertional mutagenesis library (Jeon Et al., 2000), number PFG_1C-12303, transformation receptor is japonica rice variety Hwayoung (abbreviation HW).With wild type HW is compared, mutant seeds grain length and grain is wide obviously becomes smaller, and mass of 1000 kernel significantly reduces (Fig. 1), and plant shape, plant height, heading stage etc. The variation of other characters is little.Applicant by mutant be named as sng1 (source material of clone gene of the present invention be rice mutation Body seed sng1, Oryza sativa L.sng1 delivers the China of the Chinese Wuhan Wuhan University on November 17th, 2017 Type Tissue Collection preservation, deposit number are CCTCC NO:P201723).The F of HW and mutation construction₂Generation separation Grain length, the grain of group are wide and bimodal distribution is presented in mass of 1000 kernel, and the particle shape and HW of big grain material are close, the particle shape of granule material It is close with mutant, and its segregation ratio meets classical genetics 3:1 segregation ratio (Fig. 1), show mutant seeds become smaller be by Caused by unit point recessive mutation.But applicant separates the T-DNA flanking sequence less than mutant, it is believed that variation may be group Caused by knitting culture, the method clonal vaviation site of map based cloning is utilized.

The positioning of embodiment 2:SNG1 gene and map based cloning

1. backcrossing and selection

The present invention utilizes the method clonal vaviation site of map based cloning, by sng1 and the wider rice variety treasure Shan of seed 97B (deriving from Jiangxi Academy of Agricultural Sciences) hybridization building obtains F₂For segregating population (Fig. 2), with segregating population fractional analysis Method (Bulked Segregant Analysis, BSA) carries out just positioning.It was found that the 1st end of chromosome there are a QTL simultaneously It controls grain length and grain is wide, precious 97 genotype of Shan increases phenotypic number, which is named as SNG1.

From F₂For granule single plant is selected in segregating population, hybridize once with maternal precious Shan 97, on the basis of first positioning, knot It closes molecular marker assisted selection (MAS), is recurrent parent with precious Shan 97, continuous backcross 3 times, is selfed 1 time later, obtains SNG1 Near isogenic lines, the finely positioning (Fig. 2) for SNG1.Utilize 192 plants of BC₃F₂Segregating population verifies the effect of SNG1, table Grain length, the grain of bright group are wide and mass of 1000 kernel is in bimodal distribution (Fig. 3).Show in this BC₃F₂In group, seed size is by one A major gene resistance control.

2. genotype and phenotypic evaluation method

Genotype detection uses SSR method: PCR standardization program is referring to referring to J. Pehanorm Brooker etc., and 2002, molecular cloning Experiment guide, the third edition, golden winter wild goose etc. (translating), the method that Science Press is introduced.PCR uses the reaction system of 20 μ l, packet Contain: 20-50ng DNA profiling, 10mMTris-HCl, 50mMKCl, 0.1%Triton X-100,1.8mM MgCl2,0.1mM DNTP, 0.2 μM of primer and 1U Taq DNA polymerase.The condition of PCR amplification are as follows: 94 DEG C of initial denaturation 4min；94℃ 1min, 55 DEG C of 1min, 72 DEG C of 1min, 34 circulations；72 DEG C of extension 10min.PCR product divides on 6% polyacrylamide gel From rear progress silver staining (Bassam etc., 1991, Anal.Biochem.196:80-83).

In the present invention used SSR marker information all from Gramene site databases (http:// www.gramene.org/) is in addition, genome sequence also according to the japonica rice variety Nipponbare announced on the net (http://rgp.dna.affrc.go.jp) and rice variety 93-11 genome sequence (http: // Rise.genomics.org.cn/) design is between Zhenshan 97B and sng1 with the Indel (Insert/Deletion) of polymorphism Label, the finely positioning for SNG1 are analyzed.The sequence information of these labels is shown in Table 1.

Phenotype investigates method:

Grain length, grain be wide and the measurement of mass of 1000 kernel: drying after seed harvest, room temperature is at least placed 3 months, to guarantee seed Seed moisture content is relatively uniform between dry and each strain.30 full seeds are randomly selected from every single plant, 10 are one Group, is not overlapped shoulder to shoulder, by the same direction, is not put into a line in the way of clearance, reads width, 3 weights with vernier caliper It is averaged again wide as grain；10 seeds join end to end arrangement, read length with vernier caliper, 3 repetitions are averaged i.e. For grain length.The weight for the 200 full weight selected at random is weighed to calculate mass of 1000 kernel.3.SNG1 the finely positioning of gene and Candidate gene determines

Effect verifying on the basis of, using grain it is wide as objective trait to SNG1 gene progress finely positioning.For verifying 192 plants of BC of SNG1 gene effect₃F₂In segregating population, where SNG1 gene between section both ends Indel label Y11 and Y91 There are 13 plant weight group single plants (Fig. 4), progeny test (Progeny testing) is carried out to this 13 plant weight group single plant, after 24 plants The wide phenotype of the grain in generation infers the genotype of recombination single plant SNG1: not separating and recombinates if being big grain if offspring grain is wide The SNG1 in the single plant present age is that precious Shan 97 is homozygous；It does not separate and recombinates the single plant present age if being granule if offspring's grain is wide SNG1 is sng1 homozygous；The genotype for the SNG1 for recombinating the single plant present age if separation occurs for the wide and seed size of offspring's grain is Heterozygous.Molecular labeling is encrypted in target zone, and detects the genotype of label, linkage analysis is carried out, by SNG1 finely positioning To label Y6 and Y54 between 480kb section in (Fig. 4).

Based on this, applicant plants ZS97/sng1BC₃F₃It 7680 plants of segregating population, is screened with label Y6 and Y54 125 plant weight group single plants carry out progeny test to recombination single plant, infer the genotype of recombination single plant SNG1, further encryption mark SNG1 is finally navigated between Indel label Y1 and Y7 in the section of 5.5kb, isolates (Fig. 4) with label SNP3 by note.

With reference to japonica rice " OryzasativaLcv.Nipponbare " genome (http://rice.plantbiology.msu.edu/index.shtml), Marking the section of 5.5kb between Y1 and Y7 interior includes two incomplete ORF, and two ORF are arranged in such a way that tail is to tail. It is reference with " OryzasativaLcv.Nipponbare " genome, design primer carries out the 5.5kb location area section of (shown in part table 1) HW, sng1 and ZS97 Sequencing, sequence alignment discovery, compared with rice varieties HW wild type, only one SNP (T- in the entire section of sng1 mutant A), which is located on the last one exon of candidate gene LOC_Os01g71310, and the variation make to encode it is arginic Codon (AGA) becomes terminator codon (TGA), predicts that the candidate gene of sng1 encodes one and lacks 103 amino acid of C-terminal Truncated albumen；The ZS97 SNP genotype is T (identical as HW), and there are also 14 between ZS97 and sng1 in the section of 5.5kb A SNP variation, wherein 12 are located at noncoding region, 2 are located at exon and are same sense mutation.Therefore, by LOC_ Os01g71310 is set to the candidate gene of SNG1, and is the mutation by sng1 with the SNG1QTL that the method for map based cloning navigates to (may be from tissue cultures) generated, rather than the natural variation between HW and ZS97.

Table 1 is used for the primer sequence of map based cloning of the present invention and gene function analysis

The transgenosis complementary assay of embodiment 3:SNG1

The building of 1.SNG1 overexpression carrier PU1301

According to SNG1 " OryzasativaLcv.Nipponbare " full length cDNA sequence (number of logging in AY884171, DQ116385), design primer amplification is wild The code area of raw type HW SNG1.Wherein OEF and OER primer is respectively provided with KpnI and BamHI restriction enzyme enzyme recognition site, PCR reaction system are as follows: 4 25 μ l+2mM dNTPs of μ l+2 × KOD FX PCR buffer of the first chain of HW cDNA, 10 μ l+OEF/ OER each 1.5 μ l+KOD FX (1.0U/ μ l) 1 μ l+dH₂O 7μl；PCR amplification condition are as follows: 94 DEG C of 4min, 98 DEG C of 10sec, 68 DEG C 2min, 32cycles, 72 DEG C of 10min, 25 DEG C of 1min；Amplification length is about 1.6kb.Amplified production first carries out 3 ' ends plus dA is anti- It answers (high-fidelity KOD enzymatic amplification product is flat end, cannot be used directly to be TA clone), then is dug glue recycling and connected after purification It is connected on pGEM-T (purchased from Promega company, Fig. 8) cloning vector, coupled reaction system are as follows: the 3 μ l+ of amplified production of recycling 5 μ l+T4Ligase (400U/ μ l) of pGEM-T carrier (50ng/ μ l) 1 μ l+2 × T4Ligase Buffer, 1 μ l.16 DEG C of reactions Overnight, Escherichia coli are converted, monoclonal is chosen and shakes bacterium pumping plasmid, selected with KpnI and BamHI double digestion and sequence verification without prominent The positive colony of change, positive colony KpnI and BamHI double digestion are connected to double base overexpression after digging glue recovery purifying and carry Body pU1301 (Fig. 8), coupled reaction system are as follows: the pU1301 carrier 4 recycled after 4 μ l+KpnI and BamHI double digestion of recovery product μl+ 10×T4Ligase Buffer 1μl+T4Ligase(400U/μl)1μl.16 DEG C of reactions overnight, convert Escherichia coli, choose Monoclonal shakes bacterium and takes out plasmid, and the verifying of KpnI and BamHI double digestion, which is the SNG1 overexpression built Carrier PU1301 (SNG1OE).The conversion of 2.SNG1 overexpression carrier PU1301

The genetic transforming method of the present embodiment is with reference to following documents:

(1) patent No. ZL 2004100133457；Hua Zhong Agriculture University, a kind of in-vitro culture method of long-grained nonglutinous rice；Or

(2) patent No. ZL 2013104541628, Hua Zhong Agriculture University, histone methyltransferase SDG723 adjusting and controlling rice Application in heading stage, the method for report.

Specific key step, culture medium and its method of preparation of genetic transformation of the invention:

1) reagent and solution abbreviation

The abbreviation of plant hormone used in culture medium is expressed as follows in this experiment: 6-BA (6- BenzylaminoPurine, 6-benzyladenine)；CN (Carbenicillin, carbenicillin)；KT (Kinetin, excitement Element)；NAA (Napthalene acetic acid, methyl α-naphthyl acetate)；IAA (Indole-3-acetic acid, heteroauxin)；2, 4-D (2,4-Dichlorophenoxyacetic acid, 2,4- dichlorphenoxyacetic acids)；AS (Acetosringone, acetyl fourth Ketone musk)；CH (Casein Enzymatic Hydrolysate, caseinhydrolysate)；HN (Hygromycin B, hygromycin)； DMSO (Dimethyl S μ lfoxide, dimethyl sulfoxide)；N_6max(N6 a great number of elements ingredient solution)；N_6mix(N6 microelement Ingredient solution)；MS_max(MS a great number of elements ingredient solution)；MS_mix(MS Trace Elements solution)

2) main solution formula

a)N_6maxCulture medium a great number of elements mother liquor (is prepared) according to 10 times of concentrates:

Mentioned reagent is dissolved one by one, is then settled to 1000ml, room temperature storage with distilled water at room temperature.

b)N_6minCulture medium microelement mother liquor (is prepared) according to 100 times of concentrates:

Mentioned reagent is dissolved at room temperature and is settled to 1000ml, room temperature storage with distilled water.

C) molysite (Fe²⁺EDTA liquid (preparing according to 100X concentrate)) is stored:

By 3.73g b diammonium disodium edta (Na₂EDTA·2H₂) and 2.78g FeSO O₄·7H₂O dissolves respectively, mixing And be settled to 1000ml with distilled water, until 70 DEG C warm bath 2 hours, 4 DEG C save backup.

D) vitamins stock liquid (being prepared according to 100X concentrate):

Niacin (Nicotinic acid) 0.1g

Vitamin B₁(Thiamine HCl) 0.1g

Vitamin B₆(Pyridoxine HCl) 0.1g

Glycine (Glycine) 0.2g

Inositol (Inositol) 10g

Distilled water is added to be settled to 1000ml, 4 DEG C save backup.

E) MS culture medium a great number of elements mother liquor (MS_maxMother liquor) (being prepared according to 10X concentrate):

Ammonium nitrate (NH₄NO₃) 16.5g

Potassium nitrate (KNO₃) 19.0g

Potassium dihydrogen phosphate (KH₂PO₄) 1.7g

Magnesium sulfate (MgSO₄·7H₂O) 3.7g

Calcium chloride (CaCl₂·2H₂O) 4.4g

Mentioned reagent is dissolved at room temperature, and is settled to 1000ml, room temperature storage with distilled water.

F) MS culture medium microelement mother liquor (MS_minMother liquor) (being prepared according to 100X concentrate):

G) 2,4-D stores the preparation of liquid (1mg/ml):

2,4-D 100mg is weighed, is dissolved 5 minutes with 1ml1N potassium hydroxide, then plus after the dissolution completely of 10ml distilled water is determined Hold to 100ml, saves at room temperature.

H) 6-BA stores the preparation of liquid (1mg/ml):

6-BA 100mg is weighed, is dissolved 5 minutes with 1ml 1N potassium hydroxide, then plus after the dissolution completely of 10ml distilled water is determined Hold to 100ml, room temperature preservation.

I) preparation of methyl α-naphthyl acetate (NAA) storage liquid (1mg/ml):

NAA 100mg is weighed, is dissolved 5 minutes with 1ml 1N potassium hydroxide, then plus after the dissolution completely of 10ml distilled water is determined Hold to 100ml, 4 DEG C are kept in dark place.

J) preparation of heteroauxin (IAA) storage liquid (1mg/ml):

IAA 100mg is weighed, is dissolved 5 minutes with 1ml1N potassium hydroxide, then plus 10ml distilled water dissolves completely rear constant volume To 100ml, 4 DEG C are kept in dark place.

K) preparation of glucose storage liquid (0.5g/ml):

Glucose 125g is weighed, then 250ml is settled to distilled water dissolution, is saved backup for 4 DEG C after sterilizing.

L) AS stores the preparation of liquid:

AS 0.392g is weighed, DMSO 10ml dissolution is added, dispenses to 1.5ml centrifuge tube, -20 DEG C save backup.

M) 1N potassium hydroxide storage liquid is prepared:

Potassium hydroxide 5.6g is weighed, is settled to 100ml with distilled water dissolution, room temperature preservation is spare.

3) it is used for the culture medium prescription of japonica rice genetic transformation

A) induced medium

Add distilled water to 900ml, 1N potassium hydroxide adjusts pH value to 5.9, boils and is settled to 1000ml, be dispensed into 50ml Triangular flask (30ml/ bottles), sterilize according to a conventional method after sealing (such as sterilize 15 minutes at 121 DEG C, following medium sterilizations Method is identical as the sterilizing methods of basal culture medium).

B) subculture medium:

Add distilled water to 900ml, 1N potassium hydroxide adjusts pH value to 5.9, boils and is settled to 1000ml, be dispensed into 50ml Triangular flask (30ml/ bottles), sealing, sterilizes according to the above method.

C) suspension medium:

Add distilled water to 100ml, adjusts pH value to 5.4, be dispensed into the triangular flask of two 100ml, seal, by above-mentioned side Method sterilizing.

Liquid is stored using preceding addition 1ml sterile dextrose and 100 μ lAS store liquid.

D) base is co-cultured:

Add distilled water to 250ml, 1N potassium hydroxide adjusts pH value to 5.6, and sealing sterilizes according to the above method.

Using preceding heating for dissolving culture medium and 5ml glucose storage liquid and 250 μ lAS storage liquid is added, culture is poured into packing In ware (the every ware of 25ml/).

E) screening and culturing medium:

Add distilled water to 250ml, adjusts pH value to 6.0, sealing sterilizes according to the above method.

Using preceding dissolution culture medium, 250 μ lHN (50mg/ml) and 400 μ lCN (10g CN/36ml water) packing is added and pours into In culture dish (25ml/ ware).(note: first time Selective agar medium carbenicillin concentration is 400mg/ liter, second and after Selective agar medium carbenicillin concentration is 250mg/ liter).

F) differential medium:

Add distilled water to 900ml, 1N potassium hydroxide adjusts pH value to 6.0.

It boils and is settled to 1000ml with distilled water, be dispensed into 100ml triangular flask (50ml/ bottles), seal, according to the above method Sterilizing.

G) root media

Add distilled water to 900ml, adjusts pH value to 5.8 with 1N potassium hydroxide.

It boils and is settled to 1000ml with distilled water, be dispensed into pipe of taking root (25ml/ pipe), seal, go out according to the above method Bacterium.

4) Agrobacterium-mediated genetic transformation step

A) callus induces

By mature rice paddy seed decladding, then successively with alcohol treatment 1 minute of 70%, 0.15% mercury chloride (HgCl₂) the surface of the seed disinfection 15-25 minutes；With sterilizing washing seed 4-5 times；8-10 seeds are placed on induced medium On；Culture medium after inoculation is placed at dark and is cultivated 4-5 weeks, 26 ± 1 DEG C of temperature.

B) callus subculture:

The embryo callus subculture for selecting glassy yellow, consolidation and relatively dry is put on subculture medium dark lower culture 2 weeks, temperature 25 ± 1 DEG C of degree.

C) preculture:

The embryo callus subculture for selecting consolidation and relatively dry is put on pre-culture medium dark lower culture 2 weeks, 26 ± 1 DEG C of temperature.

D) Agrobacterium is cultivated:

In the LA culture medium with corresponding resistance selection, (preparation of LA culture medium is referring to J. Pehanorm Brooker etc., molecule gram Grand experiment guide, the third edition, golden winter wild goose etc. (translating), Science Press, 2002, Beijing) on cross preculture Agrobacterium EHA105 (bacterial strain discloses the agrobacterium strains used from CAMBIA company) two days, 28 DEG C of temperature；Agrobacterium is transferred to suspension training It supports in base, is cultivated 2-3 hours on 28 DEG C of shaking tables.

E) Agrobacterium is infected:

The callus of preculture is transferred in the bottle for bacterium of having gone out；The suspension of Agrobacterium is adjusted to OD600 0.8-1.0； Callus is impregnated 30 minutes in agrobacterium suspension；It is blotted in transfer callus to the filter paper to have sterilized；It is then placed within total training It supports and is cultivated 3 days, 19-20 DEG C of temperature on base.

F) callus washing and selection culture:

The water washing callus that sterilizes is to invisible Agrobacterium；It is immersed in the aqua sterilisa of carbenicillin containing 400mg/L (CN) It shakes 30 minutes；It is blotted in transfer callus to the filter paper to have sterilized；Selection culture 2-3 times in callus to Selective agar medium is shifted, often Secondary 2 weeks to the kanamycin-resistant callus tissue grown.

G) break up:

Kanamycin-resistant callus tissue is transferred on pre- differential medium and is cultivated 5-7 days at dark；The callus of the pre- differentiation culture of transfer To differential medium, every bottle is evenly distributed three independent callus, 5 weeks -6 weeks is cultivated under illumination to growing big young plant, temperature 26 DEG C of degree.

H) it takes root and hardening:

Cut the old root generated when differentiation；It is then transferred in root media under illumination and cultivates 2-3 weeks to growing Removing sealed membrane after big young plant adds transplant again for part of running water hardening one week, and 26 DEG C of temperature.

I) it transplants:

The remaining medium on root is washed off, the seedling with good root system is transferred to greenhouse, while protecting at initial several days Water holding point is wet, etc. growing ways are very strong is transplanted again to crop field.

2. the character observation of transgenic plant

The present invention obtains 32 plants of independent SNG1 overexpression (OE) T altogether₀For transgenic plant, base is reported by PCR amplification The genotype of transgenic plant is determined because of GUS, wherein 20 plants are transgenic positive single plant, 12 plants are transgene negative single plant. The expression quantity of SNG1 is detected by Real-time PCR, the expression quantity of discovery transgenic positive single plant SNG1 has different degrees of Rise.Examine or check above-mentioned 32 transgenosis single-strain seeds grain length, grain be wide and mass of 1000 kernel, find the grain of the positive single plant of overexpression Length, grain are wide and mass of 1000 kernel is extremely significant greater than negative plant (P < 0.01) (Fig. 5).

In order to further verify the conversion of super table as a result, from 20 T₀For 3 single plants have been randomly choosed in positive plant, plant Plant T₁In generation, isolates detection to genotype and phenotype, and each family is 36 plants, utilizes gus reporter gene (conventional labels base Cause) PCR amplification result determines the genotype of each single plant.The result shows that genotype with grain length, grain is wide and mass of 1000 kernel is completely total It separates (Fig. 6), further proof SNG1 gene is positive control grain length, grain is wide and the candidate gene of mass of 1000 kernel.

In addition, the transgenic line of overexpression SNG1 influences less (figure to economical characters such as the form of plant and heading stages 7), this shows that the overexpression of SNG1 may have certain application prospect, and rice product can be improved by this genetic transformation Kind.

Bibliography

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 table

<110>Hua Zhong Agriculture University

<120>the gene SNG1 and its application of a kind of control rice grain size and grain weight

<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

Application of the 1.SNG gene in rice grain character improvement, which is characterized in that the nucleotide sequence of the gene such as SEQ ID Shown in NO:1.
Application of the 2.SNG1 gene in rice grain character improvement, which is characterized in that the protein sequence of the gene such as SEQ Shown in ID NO:2.
3. application as described in claim 1, including in rice grain grain length, grain is wide and grain improve again in application.
4. application as claimed in claim 2, including in rice grain grain length, grain is wide and grain improve again in application.