CN110241125A

CN110241125A - Soybean 100-grain weight synergy gene and its molecular labeling and application

Info

Publication number: CN110241125A
Application number: CN201910702026.3A
Authority: CN
Inventors: 盖钧镒; 陈先连; 王吴彬; 刘成
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2019-07-31
Filing date: 2019-07-31
Publication date: 2019-09-17
Anticipated expiration: 2039-07-31
Also published as: CN110241125B

Abstract

The invention discloses a soybean 100-grain weight synergy gene and its molecular labeling and application, for the gene base sequence as shown in SEQ ID No.2, which has synergistic effect；The present invention identifies the gene GmSLK for being located at regulating and controlling soybean 100-grain weight on soybean Gm16 chromosome, and being overexpressed in arabidopsis for the gene can significantly improve its seed size.The label InDelSLK for identifying the gene is provided simultaneously.Important judgement value should be provided for soybean breeder selection labeled as identifying that its GmSLK is that synergy gene or leaky gene provide convenience in germ plasm resource and filial generation.

Description

Soybean 100-grain weight synergy gene and its molecular labeling and application

Technical field

The invention belongs to breeding fields, and in particular to the excavation of the gene GmSLK of regulating and controlling soybean seed 100-grain weight and its answer With.

Background technique

Soybean is the crop with Important Economic meaning, provides edible oil and protein etc. for human and animal.Now with The continuous improvement of human life quality, it is also increasing to the demand of soybean, so improving the yield of soybean all the time all It is the main target of domestic and international soybean breeder.However, important determinant of the 100-grain weight as soybean yields, is also arranged by breeder For one of main breeding character.But soybean 100-grain weight character is the quantitative character by controlled by multiple genes, the shadow vulnerable to environment It rings, this brings very big obstruction to researcher.

For soybean 100-grain weight character, many scholars at home and abroad are using different primary mapping populations and based on not isolabeling Identification a large amount of QTL (Quantitative trait loci).But primary mapping population can make QTL position section Excessive, lots of genes site separates simultaneously, causes Genetic Background Effects, increases the difficulty of main effect QTL finely positioning.Using secondary Grade mapping population, such as answering for chromosome segment substitution line (chromosome segment substitution line, CSSL) With having found desirable route to solve this problem.Ideally, chromosome segment substitution line genes of individuals group has donor A small amount of segment of parental set of chromosome, genetic background height is consistent, undoubtedly carries out the ideal material of QTL positioning.Theoretically, The character value of chromosome segment substitution line can be compared with the character value of recurrent parent, and Traits change is all because lead between two strains The introgressed segments for entering strain have differences QTL and caused by, therefore, the chromosome segment substitution line for utilizing this homologous carries out The accuracy of positioning can be improved in qtl analysis.

It, can be according to the positioning of chromosome segment substitution line in order to which QTL finely positioning and marker assisted selection is better achieved As a result, the substitution line with important goal character detected is purposefully marked auxiliary backcrossing, secondary separation is constructed These important sites are dispersed in different individuals by group, evaluate each candidate one by one under the consistent genetic background of height The effect in site, it can be achieved that the positioning of candidate gene and marker assisted selection and gene functional research effective means.

Although located the largely QTL about soybean 100-grain weight at present, direct regulation and control soybean 100-grain weight is determined Gene is really seldom, therefore excavates the functional study of the candidate gene and gene of regulating and controlling soybean 100-grain weight, can be soybean 100-grain weight point Sub- assistant breeding provides useful information and important breeding material.

Summary of the invention

The technical problems to be solved by the invention are as follows: the crucial candidate gene GmSLK of a regulating and controlling soybean 100-grain weight is provided And application, useful information and important breeding material are provided for soybean 100-grain weight marker assisted selection.

The technical solution of the present invention is as follows:

Soybean 100-grain weight synergy gene, base sequence is as shown in SEQ ID No.2.

The albumen of soybean 100-grain weight synergy gene coding, amino acid sequence is as shown in SEQ ID No.4.

Encode the polynucleotide passage of amino acid sequence shown in SEQ ID No.4.

Recombinant vector contains polynucleotide passage described above；Preferably, the sequence of the polynucleotide passage is such as Shown in SEQ ID No.2.

The special primer of soybean 100-grain weight synergy gene described above is expanded, the upstream primer sequence of the special primer is SEQ ID No.5, the downstream primer sequence of special primer are SEQ ID No.6.

The molecular labeling of soybean 100-grain weight synergy gene, the molecular labeling are located at No. 16 chromosome of soybean chromosome The position of Chr16:35923082-35923257bp, the primer of the molecular labeling such as SEQ ID No.7 and SEQ ID No.8 institute Show.

Whether identification soybean contains the method for soybean 100-grain weight synergy gene described above, with the gene of soybean to be identified Group DNA is template, PCR amplification is carried out with primer shown in SEQ ID No.7 and SEQ ID No.8, if the expansion of 175bp can be obtained Increase production object, then the soybean contains the soybean 100-grain weight synergy gene.

Soybean 100-grain weight synergy gene described above or albumen or recombinant vector or special primer or molecular labeling are big Application in beans breeding.

Present invention finds a regulating and controlling soybean 100-grain weight gene GmSLK, the gene is located at soybean chromosome the 16th Chromosome Chr16:35920962-35928190bp, full genome group leader 7229bp, the long 2640bp of complete encoding sequence analyze ratio It compares compared with GmSLK gene order of the discovery from wild beans N24852 and cultivates the GmSLK gene order of beans NN1138-2 the The more glutamine in the glutamic acid tandem repeat region of one exon.And the GmSLK gene pairs soybean from wild beans 100-grain weight has reduction effect, and the GmSLK gene pairs soybean 100-grain weight from cultivation beans has synergistic effect.By further fixed Position one molecular labeling InDelSLK of discovery is located at GmSLK gene in the difference section of wild beans N24852 and cultivation beans NN1138-2 Between, therefore can be synergy gene or leaky gene the GmSLK gene that judge in different soybean varieties by this label, There is important value in soybean breeder selection.

Compared with prior art, the invention has the following advantages:

The present invention identifies the gene GmSLK for being located at regulating and controlling soybean 100-grain weight on soybean Gm16 chromosome, the base The overexpression of cause can significantly improve soybean 100-grain weight character.The label InDelSLK for identifying the gene is provided simultaneously. It should be labeled as identifying that its GmSLK is synergy gene or the leaky gene side of providing in soya seeds resource and filial generation Just, important judgement value is provided for soybean breeder selection.

Detailed description of the invention

Fig. 1: informative population schematic diagram；Using wild beans N24852 as male parent, cultivation beans NN1138-2 is recurrent parent, building Chromosome segment substitution line (constructs) for Wang Wubin et al. 2013, and CSSL3068 is a family in substitution line.Secondary group Using CSSL3068 as female parent, NN1138-2 is used as male parent, and hybridization, selfing obtain 294 offsprings, single-strain planting and harvest obtains The F of 294 familys₂Group, then each family plants a line, selfing, and harvests to obtain 294 F by plant_2:3Family, similarly Obtain F_2:4And F_2:5Family.

Fig. 2: parent and target group's 100-grain weight phenotype；(A) kind for being secondary group parent NN1138-2 and CSSL3068 The big logotype of son；(B) compare (P < 0.01 * *) for the 100-grain weight of secondary group parent NN1138-2 and CSSL3068；(C) fixed Position group F_2:3In plantation Nanjing (2015NJ) in 2015, F_2:4Group in plantation in 2016 in Nanjing (2016NJ) and F_2:5Group planted Nanjing (2017NJ) and Anhui in 2017 when the number point for the 100-grain weight phenotype for applying (2017AH) respectively Butut.

Fig. 3: finely positioning process；It (A) is the primary physical map of No. 16 chromosome；It (B) is the part of No. 16 chromosome High density physical map；(C) high density of map spectrum analysis secondary group is combined using One marker analysis method (IciMapping2.0) F_2:3、F_2:4And F_2:5Phenotypic data in four environment；(D) using displacement graphing method analyze 11 recombination individual phenotypes and Genotype positional candidate gene；(E) GmSLK gene structure display.

Fig. 4: GmSLK amplified band figure；GmSLK-N24852 represents GmSLK from wild beans N24852, GmSLK- NN1138-2 represents GmSLK and represents GmSLK from substitution line from cultivation beans NN1138-2, GmSLK-CSSL3068 CSSL3068, Marker 5000bp, the band of red arrow instruction are 3000bp band.

Fig. 5: compare the seed size and fresh weight of parent's Grain Development different times.(A) compare parent NN1138-2 and CSSL3068 is in Grain Development different times seed size；(B) compare parent NN1138-2 and CSSL3068 Grain Development not Same time seed fresh weight.Each sample statistics analyze 3 biology and repeat to repeat (p < 0.01 * p < 0.05, * *) with 3 technologies.

Fig. 6: GmSLK parent's NN1138-2 and CSSL3068 Grain Development different times relative expression quantity.Internal reference base Because of UBI3 gene, each sample statistics analyze 3 biology and repeat to repeat (p < 0.01 * p < 0.05, * *) with 3 technologies.

Fig. 7: transgenic arabidopsis analyzes GmSLK-NN1138-2 (left side) and GmSLK-CSSL3068 (the right) gene function Energy.(A) relative expression quantity analysis of Transgenic wheat line, reference gene are Actin gene；(B) transgenic plant and wild 3 weeks plant forms sizes of type compare；(C) compare 1000 weights of transgenic plant and WT lines, each sample statistics 3 biology are analyzed to repeat to repeat (p < 0.001 * p < 0.05, * *) with 3 technologies.

Specific embodiment

The acquisition of 1. soybean 100-grain weight main effect site qSW16.1 of embodiment

(1), target group's building and plantation

Wang Wubin in 2013 et al. constructs a set of 151 family groups using wild beans N24852 and cultivation beans NN1138-2 At chromosome segment substitution line group, one of family CSSL3068 is had found according to the 100-grain weight phenotypic information of group's family 100-grain weight be 14.68g, the recurrent parent NN1138-2 100-grain weight (18.7g) that compares significantly becomes smaller, according to the label of group letter Breath discovery CSSL3068 contains the wild site Sat_224 an of 100-grain weight.Utilize family CSSL3068 and circulation parent within 2014 This NN1138-2 hybridization building secondary group, obtains the F of 294 strains₂Generation grade target group.2015 in Nanjing (2015NJ) is by F₂Secondary group plants 294 plants, and 3 repetitions, then every row individually harvests, and obtains the F of 294 familys_2:3 Group measures F_2:3The 100-grain weight phenotype of group's family.2016 in Nanjing (2016NJ) by F_2:3294 strains are planted by group Row, 3 repetitions, then every row individually harvests, and obtains the F of 294 familys_2:4Group measures F_2:4The 100-grain weight table of group's family Type.2017 by F_2:4294 plants of group are planted respectively in Nanjing (2017NJ) and Anhui when painting (2017AH), 3 weights Multiple, then every row individually harvests, and obtains the F of 294 familys_2:5Group measures F_2:5The 100-grain weight phenotype (Fig. 1) of group's family.

(2), the measurement and analysis of parent and target group's 100-grain weight phenotype

Plant plantation, plant harvest, then selects development in 30 DEG C of constant temperature ovens to seed constant weight, each parent and family Well, 100 full seeds weighing.

Seed size (Fig. 2A and B) first between two parents of identification and analysis, as a result, it has been found that parent NN1138-2 100-grain weight is significantly higher than the 100-grain weight (such as table 1) of parent CSSL3068 under four different environment.Also with SPSS 2.0 have statisticallyd analyze the frequency distribution (Fig. 2 C) of secondary group's 100-grain weight phenotype under four environment, as the result is shown all in company Continuous distribution, and F2:5 group 100-grain weight average value is up to 16.94g in the environment of 2017AH, the group under 2015NJ environment The minimum 13.88g of group's 100-grain weight average value (such as table 1), these results all show that soybean 100-grain weight phenotype is quantitative character, are Controlled by multiple genes.In addition, target group all has lower phenotypic variation coefficient (CV) and higher something lost in different environments Biography rate (h²), this, which illustrates that soybean 100-grain weight phenotype mainly makes a variation, is controlled by gene.

100-grain weight under 1 parent's NN1138-2 and CSSL3068 varying environment of table

CV, the coefficient of variation；h², heritability.

(3), group's genotype identification

Secondary group F is extracted using CTAB method₂294 strains soybean leaves genomic DNA, the primer is by English The synthesis of Weihe River victory base (Shanghai) trading company.PCR reaction system 10ul, wherein containing 3ulDNA (15ul), upstream and downstream primer (0.2mmol/L) each 1.5 μ L, 1.2 μ L Mgcl₂(2.5mmol/L), 0.24 μ L dNTP (10mmol/L, N=A, C, G, T), 0.12 μ L Taq enzyme (5U/ul) and 1.4 μ L ddH₂O.PCR response procedures: 94 DEG C of denaturation 5min；Then carry out 33 circulations 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C of extension 40s；Thoroughly extend 10min using 72 DEG C；Last 4 DEG C of preservations.PCR expands Increase production 8% polyacrylamide gel electrophoresis of object, the polymorphism of silver staining colour developing identification band.

(4), the building of genetic linkage maps

Two parents are analyzed using 49 SSR molecular markers on No. 16 chromosome, are had between statistical analysis parent more The hereditary form with polymorphism mark between parent is identified, then to it in totally 7, the site of state property in secondary group's F2 strain Linkage analysis is carried out, the genetic linkage maps (Fig. 3 A) of secondary group are constructed.

(5), molecular markers development

Existed using the soybean genome SSR marker information of the announcements such as Song and wild beans N24852 and cultivation beans NN1138-2 The InDel information of Gm16 chromosome devises new molecular labeling, statisticallys analyze the SSR between parent with polymorphism and marks Note 3,16 InDel mark (table 2), the hereditary form with polymorphism mark between parent are identified in secondary group F2, so Linkage analysis is carried out to it afterwards, constructs the high density map (Fig. 3 B) of secondary group.

The Molecular Marker Information of the present invention of table 2

(6), interpretation of result

The main effect of soybean 100-grain weight is shown using positioning result at the beginning of SAS9.4 combination secondary group phenotypic data and molecular data Site qSW16.1 is located between No. 16 chromosome Sat_224 and Satt431.

In conjunction with high density map and secondary group F_2:3、F_2:4And F_2:5Phenotypic data in four environment, utilizes single label Analytic approach (IciMapping2.0) carries out finely positioning, is as a result positioned at soybean 100-grain weight main effect site qSW16.1 Between InDel16_01 and BARCSOYSSR_16_1215 two labels (Fig. 3 C).

The acquisition of 2 regulating and controlling soybean 100-grain weight gene GmSLK of embodiment

The determination of gene GmSLK

Soybean 100-grain weight main effect site qSW16.1 is positioned at Indel16_01 and BARCSOYSSR_ by finely positioning Between 16_1215 two labels, find that there are two candidate bases altogether between the two labels with reference to gene order by comparing soybean Cause, in order to further determine candidate gene, from F₂11 are picked in group in Gm16 chromosome 35889420bp- There is the family of recombination in the region 35952811bp, passes through the base of this 11 recombinations familys and two parents of displacement graphing method comparative analysis Because of type and 100-grain weight phenotypic data further progress finely positioning, InDel16_03 and BARCSOYSSR_16_1215 two are found There is no recombination site between label, isolates (Fig. 3 D and E) with gene Glyma.16g198300.It is analyzed and is sent out by gene annotation Existing gene Glyma.16g198300 belongs to LIM domain, and functional annotation is transcription regulatory factor (Transcriptional Regulator, SLK2), therefore it is named as GmSLK.

Application of the 3 regulating and controlling soybean 100-grain weight gene GmSLK of embodiment in soybean breeder

The specific amplified upstream primer sequence of regulating and controlling soybean 100-grain weight character gene of the present invention is SEQ ID No.5, specific amplified downstream primer sequence are SEQ ID No.6 (table 3).

The specific primer sequences of the amplification GmSLK gene of table 3

Using primer sequence SEQ ID No.5 and SEQ ID No.6, respectively with wild beans N24852 and two parents The RNA of (NN1138-2 and CSSL3068) is code area (CDS) sequence of template amplification GmSLK.Amplification uses Takara The high fidelity enzyme (Phanta Super-Fidelity DNA Poymerase, Vazyme) of company, PCR reaction system and program Such as table 4 and table 5, amplified band size such as Fig. 4:

Table 4.PCR reaction system

Table 5.PCR response procedures

The CDS sequence for comparing the gene between two parents and wild beans N24852 by sequencing amplified production analysis is poor It is different, it is found that sequence of the gene GmSLK in parent CSSL3068 and wild beans N24852 is consistent, nucleotide sequence such as SEQ ID Shown in No.1, the amino acid sequence of coding is as shown in SEQ ID No.3.Illustrate the GmSLK of parent CSSL3068 from wild beans. The GmSLK gene nucleotide series of beans NN1138-2 are cultivated as shown in SEQ ID No.2, the amino acid sequence of coding such as SEQ ID Shown in No.4.The GmSLK gene of wild beans is in the glutamic acid tandem repeat region of first exon than cultivation beans NN1138-2 GmSLK gene it is more a glutamine (Fig. 3 E).And the GmSLK gene pairs soybean 100-grain weight from wild beans has reduction Effect, the GmSLK gene pairs soybean 100-grain weight from cultivation beans (NN1138-2) have synergistic effect.

Find that InDel16_03 is located at GmSLK gene in wild beans N24852 and cultivation beans NN1138-2 by sequence alignment Difference section, therefore Indel16_03 is named as InDelSLK.Therefore different soybean varieties can be judged by this label In GmSLK gene be synergy gene or leaky gene, there is important value in soybean breeder selection.

The verifying of 4 GmSLK gene function of embodiment

In order to the influence of Rapid identification GmSLK gene pairs seed grain weight, this experimental selection mode crop arabidopsis (Arabidopsis thaliana) Columbia-0 wild type as transgenic acceptor, is overexpressed parent respectively The GmSLK (GmSLK-CSSL3068) of CSSL3068 and GmSLK (GmSLK-NN1138-2) gene for cultivating beans NN1138-2, point Analysis is relatively more single to copy homozygotic 1000 weights, to verify the influence of GmSLK gene pairs seed grain weight.

(1) GmSLK gene expression dose is analyzed

In order to better understand the influence of GmSLK gene pairs soybean kernel developmental stage, GmSLK base is compared in this experimental analysis Because of the expression of the Grain Development different times in parent (NN1138-2 and CSSL3068).8 single plant NN1138- are taken respectively 2 and CSSL3068 Post flowering 7_DAF, 14_DAF, 21_DAF, 28_DAF, 35_DAF and 42_DAF (DAF:day after Flower seed), 3 biology of each sample repeat, and measure phenotype (Fig. 5) respectively and extract RNA and are (Real-time RT- PCR) RT-PCR analyzes (Fig. 6).

Observation compares parent NN1138-2 and CSSL3068 in the seed size of Grain Development different times, discovery The seed of NN1138-2 is all larger (Fig. 5 A) in the different developmental stage seeds CSSL3068 seed that compares, and passes through measurement seed Fresh weight, the results showed that it is aobvious that the seed of NN1138-2 in different developmental stage seed fresh weights is significantly higher than CSSL3068 seed It writes (Fig. 5 B).

Analysis compares GmSLK gene in the relative expression levels of the Grain Development different times of parent, this experiment is chosen big UBI3 (ribosomal-ubiquitin fusion protein) gene of beans is as internal reference.Interpretation of result shows GmSLK base Increase because of development of the expressed in abundance degree in parents with seed, and reaches maximum in 42_DAF period expression quantity.However, GmSLK gene is much like in parents' Grain Development early period and mid-term (14_DAF-35_DAF) expression quantity level, but in 42_DAF Period, GmSLK gene are significantly higher than the relative expression in parent CSSL3068 in the relative expression quantity level of parent NN1138-2 Amount is horizontal.

(2) GmSLK gene transgenic arabidopsis is verified

It is 3301 carrier of pCAMBIA selected by this research transformation of Arabidopsis thaliana, restriction enzyme site is (Bst EII and NcoI), GmSLK gene magnification upstream and downstream primer is SEQ ID No.3 and SEQ ID No.4 (table 6) respectively.Use clone's recombination kit (ClonExperss II) carries out recombining reaction.Then the over-express vector pCAMBIA 3301-GmSLK- that will be built NN1138-2 and pCAMBIA 3301-GmSLK-CSSL30068 be transformed into respectively in DH5a competence carry out plasmid expand it is numerous, then By clone identification, it is transferred in Agrobacterium EHA105 after extracting plasmid.By pollen soaking method, the overexpression that will be built respectively Carrier is transferred in arabidopsis Columbia-0 wild type.

The gene constructed vector primer design of table 6GmSLK

Over-express vector pCAMBIA 3301 has glufosinate-resistant, the glufosinate that positive seedling passes through 1/2MSA+10mg/l Culture medium is screened.The T1 of the Arabidopsis plant infected is harvested for seed, it is positive to screen T2 generation on resistance culture base again first Seedling, then plantation harvests T2 seed, and survival/death rate is planted and statisticallyd analyze to T2 seed on resistance culture base, passes through card Side detects and selects survival/death and meets 3:1, illustrates that the transgenic plant belongs to single copy plant, then plants and harvest T3 generation Seed, then plant in the homozygous plants of resistance culture base selection survival rate 100%, plant and harvest T4 seed and count 1000 Weight phenotype.Plant is overexpressed for GmSLK-NN1138-2 and GmSLK-CSSL30068 and selects 3 single copy homozygous strains respectively System, each strain plant 3 respectively, then take blade to carry out RT-PCR analysis, select the Actin gene of arabidopsis as internal reference Gene detects the relative expression quantity (Fig. 7 A) of the foreign gene of transgenic plant.This experiment also observation analysis transgenic plant 3 The plant size of Zhou great little finds that GmSLK-NN1138-2 with the GmSLK-CSSL30068 plant being overexpressed compares wild type Plant size does not have difference (Fig. 7 B).It harvests T5 seed and statisticallys analyze 1000 weights phenotype (Fig. 7 C), interpretation of result shows to compare Compared with wild type, the GmSLK-NN1138-2 of overexpression can significantly increase grain weight, however it is significant to be overexpressed GmSLK-CSSL30068 Reduce grain weight.Therefore it can prove that the GmSLK genotype from wild beans or parent CSSL3068 belongs to leaky gene, it can be with Seed grain weight is reduced, and the GmSLK genotype from cultivation beans NN1138-2 belongs to synergy gene, can increase seed grain weight, This provides strong evidence for the Yield Breeding of soybean.

Sequence table

<110>Agricultural University Of Nanjing

<120>soybean 100-grain weight synergy gene and its molecular labeling and application

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2640

<212> DNA

<213>soybean (Glycine max)

<400> 1

atgacacctt tgcgagtggc aggtggatta acccaatcat catcaaattc tggaattttc 60

tatcaaggag atgggcagtc acagaatgta gttaactctc acttaagctc atcttttgtt 120

aactcgtcta gcacggtttc tggagcttct cgttcaaacc tgggtccggt ttctggggat 180

atgaataatg cagttttgaa cagtgtggca aactcagcac caagtgtagg agccagctct 240

ttagttacag atgcaaattc tgcactctct ggtggcccac atttgcagag aagtgccagc 300

gttaacacag actcatactt acgattacct gcctcaccta tgtcatttac atcaaataat 360

atcagtattt ctggatcatc agtgatggat gtttcctctg tagtacaaca gagctctcat 420

caagatcaga atgttcaaca attgcagcag aatcagcagc agccacaggg tgcttcaagt 480

gctatgtctt tgtctgcatc tcaaactggc ccttctatgc tccaaatggg tgcacaaatc 540

ccaggatctt tcattcaaga tccaaataat atgtctcatc tgtcaaagaa acctagaatg 600

gatatcaaac aggaagatat gatgcagcag caggttatac aacagattct tcagagacaa 660

gattccatgc aattccaggg tcgtaatccc cagttacagg ctttccttca gcagcagcag 720

cagagactga gacaacaaca aatgtttcag caaatgccac aattacaccg agcacacttg 780

cagcagcagc aacaacaaca acaaatgcaa ttgaggcagc agcagcagca gcagcaacaa 840

caacaacaac aacaacaaca acaacaacaa caacaacaac aacaacaaca acaacaacag 900

caacagcaag tgatgcagcc ctcttctgtg gtcaagcgtc catatgacag tagtgttagt 960

ggggtatgtg cccgtcgatt gatgcagtat ctctatcatc aaaggcaacg accaaatgat 1020

aatagtattg cctattggag aaaatttgtg gctgaatatt actctcctcg tgcaaagaaa 1080

cggtggtgct tgtcattata tagtaatgtt gggcatcacg cacttggtgt ttttccccaa 1140

gcatctatgg atgcatggca ctgtgacata tgtggttcta aatctggaag gggatttgag 1200

gcaacttatg aagtattacc tagacttaat gaaatcaaat ttggcagtgg agtaattgat 1260

gaactattgt ttctggacat gccacgtgaa atgagattcg cttctggtgc gatgatgtta 1320

gaatatggaa aagcagttca agagagtgta tatgagcagc ttcgtgttgt tcgtgaaggt 1380

caacttcgta tcatattcac tcaagacttg aagatattat cttgggagtt ctgtgcaagg 1440

tgccatgagg aacttcttcc tcgaaggttg gttgcaccac aggtcaacca gttagttcag 1500

gtagctaaaa aatgtcaaag tacaattgct gaaagtggtt ctgatggggt ttctcaacaa 1560

gacatccaaa caaacagcaa catgttgttg acagctgggg gtcagcttgc gaagattttg 1620

gagatgcaat cgctaaatga gttgggcttt tctaaaagat acgtgagatg tttgcaaatt 1680

tcggaggttg tcaatagcat gaaagaccta atagatatct gtgcagatca caaaattggt 1740

gccattgaga gtttgaaaaa ttttcctcgt ctagcaacag cctcaaaggt ccagatgcag 1800

aagatgcagg aaatggaaca gctagcaaat gttcaaggtc tgccaactga tcgaaacaca 1860

ctcaataagc taatggcact gaatcctgga ttgaacaacc atataaacaa ccctcataat 1920

atggttaatc gtggtgcttt gagtgggtca gcccaagctg ctttagcact gaacaactac 1980

caaaatcttc tcatgaggca aaattcaatg aactctagcc ctggctcact tcagcgcgaa 2040

gggtcctctt tcaataattc aaaccagagt ccatcttcag ctttgcaagg agctggtcct 2100

gctttaattc caggcccaat gcagaattca tctgttagtg gtttcccaag cccccgtcta 2160

cccccacagc agcagcaaca ccacctacaa cagccctcat taagtgcaaa tgctttactg 2220

caacaaaatc attcacaggg ttcccaagga aatcaagctc tgcagcagca gatgatccat 2280

caactactgc aggagatgtc aaataacaac gggggagtgc aaccacagtc tcttggtgga 2340

cccagtgcaa atatggcaaa gaatgcactg gggtttgggg gccattatcc atccttaagt 2400

ggaggttctg ccaatgttac aggaaacaat ggacctatgt caaggaataa tagcttcaaa 2460

acaactgcaa atagtgattc ttctgctgct ggtggcaaca atggattaaa ccagagaaca 2520

tctgagatgc cacaaaatct acatttgcaa gatgtggttc aggatattgg caatgaattc 2580

acggataatc ccttccttaa cagtgatctt gatgataaca tgggttttgg ctggaaggca 2640

<210> 2

<211> 2637

<212> DNA

<213>soybean (Glycine max)

<400> 2

atgacacctt tgcgagtggc aggtggatta acccaatcat catcaaattc tggaattttc 60

tatcaaggag atgggcagtc acagaatgta gttaactctc acttaagctc atcttttgtt 120

aactcgtcta gcacggtttc tggagcttct cgttcaaacc tgggtccggt ttctggggat 180

atgaataatg cagttttgaa cagtgtggca aactcagcac caagtgtagg agccagctct 240

ttagttacag atgcaaattc tgcactctct ggtggcccac atttgcagag aagtgccagc 300

gttaacacag actcatactt acgattacct gcctcaccta tgtcatttac atcaaataat 360

atcagtattt ctggatcatc agtgatggat gtttcctctg tagtacaaca gagctctcat 420

caagatcaga atgttcaaca attgcagcag aatcagcagc agccacaggg tgcttcaagt 480

gctatgtctt tgtctgcatc tcaaactggc ccttctatgc tccaaatggg tgcacaaatc 540

ccaggatctt tcattcaaga tccaaataat atgtctcatc tgtcaaagaa acctagaatg 600

gatatcaaac aggaagatat gatgcagcag caggttatac aacagattct tcagagacaa 660

gattccatgc aattccaggg tcgtaatccc cagttacagg ctttccttca gcagcagcag 720

cagagactga gacaacaaca aatgtttcag caaatgccac aattacaccg agcacacttg 780

cagcagcagc aacaacaaca acaaatgcaa ttgaggcagc agcagcagca gcagcaacaa 840

caacaacaac aacaacaaca acaacaacaa caacaacaac aacaacaaca acaacagcaa 900

cagcaagtga tgcagccctc ttctgtggtc aagcgtccat atgacagtag tgttagtggg 960

gtatgtgccc gtcgattgat gcagtatctc tatcatcaaa ggcaacgacc aaatgataat 1020

agtattgcct attggagaaa atttgtggct gaatattact ctcctcgtgc aaagaaacgg 1080

tggtgcttgt cattatatag taatgttggg catcacgcac ttggtgtttt tccccaagca 1140

tctatggatg catggcactg tgacatatgt ggttctaaat ctggaagggg atttgaggca 1200

acttatgaag tattacctag acttaatgaa atcaaatttg gcagtggagt aattgatgaa 1260

ctattgtttc tggacatgcc acgtgaaatg agattcgctt ctggtgcgat gatgttagaa 1320

tatggaaaag cagttcaaga gagtgtatat gagcagcttc gtgttgttcg tgaaggtcaa 1380

cttcgtatca tattcactca agacttgaag atattatctt gggagttctg tgcaaggtgc 1440

catgaggaac ttcttcctcg aaggttggtt gcaccacagg tcaaccagtt agttcaggta 1500

gctaaaaaat gtcaaagtac aattgctgaa agtggttctg atggggtttc tcaacaagac 1560

atccaaacaa acagcaacat gttgttgaca gctgggggtc agcttgcgaa gattttggag 1620

atgcaatcgc taaatgagtt gggcttttct aaaagatacg tgagatgttt gcaaatttcg 1680

gaggttgtca atagcatgaa agacctaata gatatctgtg cagatcacaa aattggtgcc 1740

attgagagtt tgaaaaattt tcctcgtcta gcaacagcct caaaggtcca gatgcagaag 1800

atgcaggaaa tggaacagct agcaaatgtt caaggtctgc caactgatcg aaacacactc 1860

aataagctaa tggcactgaa tcctggattg aacaaccata taaacaaccc tcataatatg 1920

gttaatcgtg gtgctttgag tgggtcagcc caagctgctt tagcactgaa caactaccaa 1980

aatcttctca tgaggcaaaa ttcaatgaac tctagccctg gctcacttca gcgcgaaggg 2040

tcctctttca ataattcaaa ccagagtcca tcttcagctt tgcaaggagc tggtcctgct 2100

ttaattccag gcccaatgca gaattcatct gttagtggtt tcccaagccc ccgtctaccc 2160

ccacagcagc agcaacacca cctacaacag ccctcattaa gtgcaaatgc tttactgcaa 2220

caaaatcatt cacagggttc ccaaggaaat caagctctgc agcagcagat gatccatcaa 2280

ctactgcagg agatgtcaaa taacaacggg ggagtgcaac cacagtctct tggtggaccc 2340

agtgcaaata tggcaaagaa tgcactgggg tttgggggcc attatccatc cttaagtgga 2400

ggttctgcca atgttacagg aaacaatgga cctatgtcaa ggaataatag cttcaaaaca 2460

actgcaaata gtgattcttc tgctgctggt ggcaacaatg gattaaacca gagaacatct 2520

gagatgccac aaaatctaca tttgcaagat gtggttcagg atattggcaa tgaattcacg 2580

gataatccct tccttaacag tgatcttgat gataacatgg gttttggctg gaaggca 2637

<210> 3

<211> 880

<212> PRT

<213>soybean (Glycine max)

<400> 3

Met Thr Pro Leu Arg Val Ala Gly Gly Leu Thr Gln Ser Ser Ser Asn

1 5 10 15

Ser Gly Ile Phe Tyr Gln Gly Asp Gly Gln Ser Gln Asn Val Val Asn

20 25 30

Ser His Leu Ser Ser Ser Phe Val Asn Ser Ser Ser Thr Val Ser Gly

35 40 45

Ala Ser Arg Ser Asn Leu Gly Pro Val Ser Gly Asp Met Asn Asn Ala

50 55 60

Val Leu Asn Ser Val Ala Asn Ser Ala Pro Ser Val Gly Ala Ser Ser

65 70 75 80

Leu Val Thr Asp Ala Asn Ser Ala Leu Ser Gly Gly Pro His Leu Gln

85 90 95

Arg Ser Ala Ser Val Asn Thr Asp Ser Tyr Leu Arg Leu Pro Ala Ser

100 105 110

Pro Met Ser Phe Thr Ser Asn Asn Ile Ser Ile Ser Gly Ser Ser Val

115 120 125

Met Asp Val Ser Ser Val Val Gln Gln Ser Ser His Gln Asp Gln Asn

130 135 140

Val Gln Gln Leu Gln Gln Asn Gln Gln Gln Pro Gln Gly Ala Ser Ser

145 150 155 160

Ala Met Ser Leu Ser Ala Ser Gln Thr Gly Pro Ser Met Leu Gln Met

165 170 175

Gly Ala Gln Ile Pro Gly Ser Phe Ile Gln Asp Pro Asn Asn Met Ser

180 185 190

His Leu Ser Lys Lys Pro Arg Met Asp Ile Lys Gln Glu Asp Met Met

195 200 205

Gln Gln Gln Val Ile Gln Gln Ile Leu Gln Arg Gln Asp Ser Met Gln

210 215 220

Phe Gln Gly Arg Asn Pro Gln Leu Gln Ala Phe Leu Gln Gln Gln Gln

225 230 235 240

Gln Arg Leu Arg Gln Gln Gln Met Phe Gln Gln Met Pro Gln Leu His

245 250 255

Arg Ala His Leu Gln Gln Gln Gln Gln Gln Gln Gln Met Gln Leu Arg

260 265 270

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln

275 280 285

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Val

290 295 300

Met Gln Pro Ser Ser Val Val Lys Arg Pro Tyr Asp Ser Ser Val Ser

305 310 315 320

Gly Val Cys Ala Arg Arg Leu Met Gln Tyr Leu Tyr His Gln Arg Gln

325 330 335

Arg Pro Asn Asp Asn Ser Ile Ala Tyr Trp Arg Lys Phe Val Ala Glu

340 345 350

Tyr Tyr Ser Pro Arg Ala Lys Lys Arg Trp Cys Leu Ser Leu Tyr Ser

355 360 365

Asn Val Gly His His Ala Leu Gly Val Phe Pro Gln Ala Ser Met Asp

370 375 380

Ala Trp His Cys Asp Ile Cys Gly Ser Lys Ser Gly Arg Gly Phe Glu

385 390 395 400

Ala Thr Tyr Glu Val Leu Pro Arg Leu Asn Glu Ile Lys Phe Gly Ser

405 410 415

Gly Val Ile Asp Glu Leu Leu Phe Leu Asp Met Pro Arg Glu Met Arg

420 425 430

Phe Ala Ser Gly Ala Met Met Leu Glu Tyr Gly Lys Ala Val Gln Glu

435 440 445

Ser Val Tyr Glu Gln Leu Arg Val Val Arg Glu Gly Gln Leu Arg Ile

450 455 460

Ile Phe Thr Gln Asp Leu Lys Ile Leu Ser Trp Glu Phe Cys Ala Arg

465 470 475 480

Cys His Glu Glu Leu Leu Pro Arg Arg Leu Val Ala Pro Gln Val Asn

485 490 495

Gln Leu Val Gln Val Ala Lys Lys Cys Gln Ser Thr Ile Ala Glu Ser

500 505 510

Gly Ser Asp Gly Val Ser Gln Gln Asp Ile Gln Thr Asn Ser Asn Met

515 520 525

Leu Leu Thr Ala Gly Gly Gln Leu Ala Lys Ile Leu Glu Met Gln Ser

530 535 540

Leu Asn Glu Leu Gly Phe Ser Lys Arg Tyr Val Arg Cys Leu Gln Ile

545 550 555 560

Ser Glu Val Val Asn Ser Met Lys Asp Leu Ile Asp Ile Cys Ala Asp

565 570 575

His Lys Ile Gly Ala Ile Glu Ser Leu Lys Asn Phe Pro Arg Leu Ala

580 585 590

Thr Ala Ser Lys Val Gln Met Gln Lys Met Gln Glu Met Glu Gln Leu

595 600 605

Ala Asn Val Gln Gly Leu Pro Thr Asp Arg Asn Thr Leu Asn Lys Leu

610 615 620

Met Ala Leu Asn Pro Gly Leu Asn Asn His Ile Asn Asn Pro His Asn

625 630 635 640

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

645 650 655

Leu Asn Asn Tyr Gln Asn Leu Leu Met Arg Gln Asn Ser Met Asn Ser

660 665 670

Ser Pro Gly Ser Leu Gln Arg Glu Gly Ser Ser Phe Asn Asn Ser Asn

675 680 685

Gln Ser Pro Ser Ser Ala Leu Gln Gly Ala Gly Pro Ala Leu Ile Pro

690 695 700

Gly Pro Met Gln Asn Ser Ser Val Ser Gly Phe Pro Ser Pro Arg Leu

705 710 715 720

Pro Pro Gln Gln Gln Gln His His Leu Gln Gln Pro Ser Leu Ser Ala

725 730 735

Asn Ala Leu Leu Gln Gln Asn His Ser Gln Gly Ser Gln Gly Asn Gln

740 745 750

Ala Leu Gln Gln Gln Met Ile His Gln Leu Leu Gln Glu Met Ser Asn

755 760 765

Asn Asn Gly Gly Val Gln Pro Gln Ser Leu Gly Gly Pro Ser Ala Asn

770 775 780

Met Ala Lys Asn Ala Leu Gly Phe Gly Gly His Tyr Pro Ser Leu Ser

785 790 795 800

Gly Gly Ser Ala Asn Val Thr Gly Asn Asn Gly Pro Met Ser Arg Asn

805 810 815

Asn Ser Phe Lys Thr Thr Ala Asn Ser Asp Ser Ser Ala Ala Gly Gly

820 825 830

Asn Asn Gly Leu Asn Gln Arg Thr Ser Glu Met Pro Gln Asn Leu His

835 840 845

Leu Gln Asp Val Val Gln Asp Ile Gly Asn Glu Phe Thr Asp Asn Pro

850 855 860

Phe Leu Asn Ser Asp Leu Asp Asp Asn Met Gly Phe Gly Trp Lys Ala

865 870 875 880

<210> 4

<211> 879

<212> PRT

<213>soybean (Glycine max)

<400> 4

Met Thr Pro Leu Arg Val Ala Gly Gly Leu Thr Gln Ser Ser Ser Asn

1 5 10 15

Ser Gly Ile Phe Tyr Gln Gly Asp Gly Gln Ser Gln Asn Val Val Asn

20 25 30

Ser His Leu Ser Ser Ser Phe Val Asn Ser Ser Ser Thr Val Ser Gly

35 40 45

Ala Ser Arg Ser Asn Leu Gly Pro Val Ser Gly Asp Met Asn Asn Ala

50 55 60

Val Leu Asn Ser Val Ala Asn Ser Ala Pro Ser Val Gly Ala Ser Ser

65 70 75 80

Leu Val Thr Asp Ala Asn Ser Ala Leu Ser Gly Gly Pro His Leu Gln

85 90 95

Arg Ser Ala Ser Val Asn Thr Asp Ser Tyr Leu Arg Leu Pro Ala Ser

100 105 110

Pro Met Ser Phe Thr Ser Asn Asn Ile Ser Ile Ser Gly Ser Ser Val

115 120 125

Met Asp Val Ser Ser Val Val Gln Gln Ser Ser His Gln Asp Gln Asn

130 135 140

Val Gln Gln Leu Gln Gln Asn Gln Gln Gln Pro Gln Gly Ala Ser Ser

145 150 155 160

Ala Met Ser Leu Ser Ala Ser Gln Thr Gly Pro Ser Met Leu Gln Met

165 170 175

Gly Ala Gln Ile Pro Gly Ser Phe Ile Gln Asp Pro Asn Asn Met Ser

180 185 190

His Leu Ser Lys Lys Pro Arg Met Asp Ile Lys Gln Glu Asp Met Met

195 200 205

Gln Gln Gln Val Ile Gln Gln Ile Leu Gln Arg Gln Asp Ser Met Gln

210 215 220

Phe Gln Gly Arg Asn Pro Gln Leu Gln Ala Phe Leu Gln Gln Gln Gln

225 230 235 240

Gln Arg Leu Arg Gln Gln Gln Met Phe Gln Gln Met Pro Gln Leu His

245 250 255

Arg Ala His Leu Gln Gln Gln Gln Gln Gln Gln Gln Met Gln Leu Arg

260 265 270

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln

275 280 285

Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Val Met

290 295 300

Gln Pro Ser Ser Val Val Lys Arg Pro Tyr Asp Ser Ser Val Ser Gly

305 310 315 320

Val Cys Ala Arg Arg Leu Met Gln Tyr Leu Tyr His Gln Arg Gln Arg

325 330 335

Pro Asn Asp Asn Ser Ile Ala Tyr Trp Arg Lys Phe Val Ala Glu Tyr

340 345 350

Tyr Ser Pro Arg Ala Lys Lys Arg Trp Cys Leu Ser Leu Tyr Ser Asn

355 360 365

Val Gly His His Ala Leu Gly Val Phe Pro Gln Ala Ser Met Asp Ala

370 375 380

Trp His Cys Asp Ile Cys Gly Ser Lys Ser Gly Arg Gly Phe Glu Ala

385 390 395 400

Thr Tyr Glu Val Leu Pro Arg Leu Asn Glu Ile Lys Phe Gly Ser Gly

405 410 415

Val Ile Asp Glu Leu Leu Phe Leu Asp Met Pro Arg Glu Met Arg Phe

420 425 430

Ala Ser Gly Ala Met Met Leu Glu Tyr Gly Lys Ala Val Gln Glu Ser

435 440 445

Val Tyr Glu Gln Leu Arg Val Val Arg Glu Gly Gln Leu Arg Ile Ile

450 455 460

Phe Thr Gln Asp Leu Lys Ile Leu Ser Trp Glu Phe Cys Ala Arg Cys

465 470 475 480

His Glu Glu Leu Leu Pro Arg Arg Leu Val Ala Pro Gln Val Asn Gln

485 490 495

Leu Val Gln Val Ala Lys Lys Cys Gln Ser Thr Ile Ala Glu Ser Gly

500 505 510

Ser Asp Gly Val Ser Gln Gln Asp Ile Gln Thr Asn Ser Asn Met Leu

515 520 525

Leu Thr Ala Gly Gly Gln Leu Ala Lys Ile Leu Glu Met Gln Ser Leu

530 535 540

Asn Glu Leu Gly Phe Ser Lys Arg Tyr Val Arg Cys Leu Gln Ile Ser

545 550 555 560

Glu Val Val Asn Ser Met Lys Asp Leu Ile Asp Ile Cys Ala Asp His

565 570 575

Lys Ile Gly Ala Ile Glu Ser Leu Lys Asn Phe Pro Arg Leu Ala Thr

580 585 590

Ala Ser Lys Val Gln Met Gln Lys Met Gln Glu Met Glu Gln Leu Ala

595 600 605

Asn Val Gln Gly Leu Pro Thr Asp Arg Asn Thr Leu Asn Lys Leu Met

610 615 620

Ala Leu Asn Pro Gly Leu Asn Asn His Ile Asn Asn Pro His Asn Met

625 630 635 640

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

645 650 655

Asn Asn Tyr Gln Asn Leu Leu Met Arg Gln Asn Ser Met Asn Ser Ser

660 665 670

Pro Gly Ser Leu Gln Arg Glu Gly Ser Ser Phe Asn Asn Ser Asn Gln

675 680 685

Ser Pro Ser Ser Ala Leu Gln Gly Ala Gly Pro Ala Leu Ile Pro Gly

690 695 700

Pro Met Gln Asn Ser Ser Val Ser Gly Phe Pro Ser Pro Arg Leu Pro

705 710 715 720

Pro Gln Gln Gln Gln His His Leu Gln Gln Pro Ser Leu Ser Ala Asn

725 730 735

Ala Leu Leu Gln Gln Asn His Ser Gln Gly Ser Gln Gly Asn Gln Ala

740 745 750

Leu Gln Gln Gln Met Ile His Gln Leu Leu Gln Glu Met Ser Asn Asn

755 760 765

Asn Gly Gly Val Gln Pro Gln Ser Leu Gly Gly Pro Ser Ala Asn Met

770 775 780

Ala Lys Asn Ala Leu Gly Phe Gly Gly His Tyr Pro Ser Leu Ser Gly

785 790 795 800

Gly Ser Ala Asn Val Thr Gly Asn Asn Gly Pro Met Ser Arg Asn Asn

805 810 815

Ser Phe Lys Thr Thr Ala Asn Ser Asp Ser Ser Ala Ala Gly Gly Asn

820 825 830

Asn Gly Leu Asn Gln Arg Thr Ser Glu Met Pro Gln Asn Leu His Leu

835 840 845

Gln Asp Val Val Gln Asp Ile Gly Asn Glu Phe Thr Asp Asn Pro Phe

850 855 860

Leu Asn Ser Asp Leu Asp Asp Asn Met Gly Phe Gly Trp Lys Ala

865 870 875

<210> 5

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 5

atgacacctt tgcgagtggc 20

<210> 6

<211> 22

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 6

tcatgccttc cagccaaaac cc 22

<210> 7

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

acaattacac cgagcaca 18

<210> 8

<211> 18

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

cttgaccaca gaagaggg 18

Claims

1. soybean 100-grain weight synergy gene, base sequence is as shown in SEQ ID No.2.

2. the albumen of soybean 100-grain weight synergy gene coding described in claim 1, amino acid sequence such as SEQ ID No.4 institute Show.

3. encoding the polynucleotide passage of amino acid sequence shown in SEQ ID No.4.

4. recombinant vector contains polynucleotide passage as claimed in claim 3.

5. recombinant vector according to claim 4, which is characterized in that the sequence of the polynucleotide passage such as SEQ ID Shown in No.2.

6. expanding the special primer of soybean 100-grain weight synergy gene described in claim 1, the upstream primer sequence of the special primer It is classified as SEQ ID No.5, the downstream primer sequence of special primer is SEQ ID No.6.

7. the molecular labeling of soybean 100-grain weight synergy gene described in claim 1, which is located at soybean chromosome The position of No. 16 chromosome Chr16:35923082-35923257bp, the primer of the molecular labeling such as SEQ ID No.7 and SEQ Shown in ID No.8.

8. whether identification soybean contains the method for soybean 100-grain weight synergy gene described in claim 1, which is characterized in that with to The genomic DNA for identifying soybean is template, carries out PCR amplification with primer shown in SEQ ID No.7 and SEQ ID No.8, if The amplified production of 175bp can be obtained, then the soybean contains the soybean 100-grain weight synergy gene.

9. weight described in soybean 100-grain weight synergy gene or albumen as claimed in claim 2 or claim 5 described in claim 1 The application of group carrier or special primer as claimed in claim 6 or molecular labeling as claimed in claim 7 in soybean breeder.