CN109554371A - BnGRF7a gene and application thereof - Google Patents

BnGRF7a gene and application thereof Download PDF

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CN109554371A
CN109554371A CN201811319445.0A CN201811319445A CN109554371A CN 109554371 A CN109554371 A CN 109554371A CN 201811319445 A CN201811319445 A CN 201811319445A CN 109554371 A CN109554371 A CN 109554371A
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gene
bngrf7a
seed
ser
snp
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谭小力
曹维
朱克明
王政
杨艳华
曹军
丁丽娜
李玉龙
李凯霞
王伟杰
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Jiangsu University
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Jiangsu University
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The present invention relates to a kind of gene, especially BnGRF7a gene and application thereof, the purposes and method being specifically related in promotion seed kernel becomes larger belong to plant genetic engineering and field of biotechnology；The invention further relates to the SNP for promoting seed kernel to become larger, the SNP is located at BnGRF7a gene promoter region；The present invention, which demonstrates the overexpression gene BnGRF7a in rape by some tests, can generate bigger seed, to improve yield；Experimental material is provided to study function and the mechanism of action of gene BnGRF7a, new big seed germ plasm resource is also can be used as, new GENE SOURCES is provided for rapeseed breeding, help to push agricultural sciences progress.

Description

BnGRF7a gene and application thereof
Technical field
The present invention relates to a kind of gene, especially BnGRF7a gene and application thereof, it is specifically related to promoting seed kernel Purposes and method in becoming larger, belong to plant genetic engineering and field of biotechnology.
Background technique
Cabbage type rape (Brassica napus L.) is the main oil crops in China, while cabbage type rape is also A kind of multipurpose industrial crops.For rape as one of five grande culture object of China, annual output is very huge, and in China, main oil plant is made Main status is occupied in object.
Rapeseed oil all occupies weight as a kind of vegetalitas edible oil in China or even worldwide edible oil market The status wanted.Currently, the vegetalitas edible oil in China is still needed from a large amount of imports in the whole world, and be most important rapeseed oil into Mouth country.Therefore, the primary Task that yield of rape is current numerous agriculturists and scientific research personnel is improved.Yield of rape Mainly by the tied silique number of every plant of rape (siliques per plant, SPP), Seed number per pod (seeds per Siliques, SPS) and the big factor of rape seed size (or mass of 1000 kernel) three determined that wherein seed size is agricultural production In one of economical character of greatest concern.Rape seed mass of 1000 kernel very little, compared to having very for silique number and Seed number per pod Big room for promotion is found the gene for adjusting control seed size and is studied its function, is most effective at present most direct Method and molecular breeding main thought and precondition.
Summary of the invention
The present invention is directed to solve one of above-mentioned technical problem at least to a certain extent.For this purpose, one object of the present invention It is to propose that one kind is related to vegetable seeds mass of 1000 kernel shape, the gene and SNP (single of plant seed increase can be effectively facilitated Nucleotide polymorphism, single nucleotide polymorphism) label.
Growth regulatory factor (growth-regulating factors, GRFs) is the distinctive transcription factor of a kind of plant Family.GRFs plant growth, develop, bloom, in the adjusting of the life processes such as the adjusting and controlling growth under adverse circumstance and breeding time It plays an important role.First conservative region of GRFs is fragrant hydrophobic amino acid QLQ (glutamic acid, leucine, paddy ammonia Acid) structural domain, this conservative region is likely to be important interactions between protein region；Second conservative region is a WRC (color ammonia Acid, arginine, cysteine) structural domain, which includes 1 functional core positioning signal and 1 DNA binding motif, that is, zinc Refer to structure, can work in DNA combination.QLQ and WRC structural domain is respectively positioned on the N-terminal of GRFs.However, the GRFs of some species In C-terminal, there is also the two conserved domains, some then lack one, or even do not have both；Contain relative to N-terminal conservative QLQ and WRC structural domain, the C-terminal of some GRFs family members also contain smaller conserved sequence, such as TQL (threonine, paddy ammonia Acid, leucine), GGPL (glycine, glycine, proline, leucine) and FFD (FRQ-FRH interacting domain) Structural domain.
The present invention carries out candidate gene association analysis to cabbage type rape (Brassica napus) GRF family and verifies it Function obtains gene BnGRF7a relevant to mass of 1000 kernel (seed size).Sequence is analysis shows the gene has 4 in code area A SNP for causing nonsynonymous mutation, 1 causes the SNP of same sense mutation；Promoter region has 8 SNP.The present invention is directed to verify The function of BnGRF7a, and functional SNP is filtered out for molecular breeding.
The present invention also further predicts the miRNA396 target spot of BnGRF7a gene and carries out point mutation (same sense mutation), It is created that the BnGRF7a gene order that can prevent miRNA396 from shearing, the segment have the nucleotide as shown in SEQ.ID.NO.2 Sequence.
Present invention firstly provides the gene BnGRF7a that one controls vegetable seeds seed size, the gene can promote to plant Object seed kernel increases, and the nucleotide sequence of the gene is selected from as follows:
(1) nucleotide sequence as shown in SEQ.ID.NO.1；
(2) nucleotides sequence of the point mutation of one or more bases is carried out to nucleotide sequence shown in SEQ.ID.NO.1 Column.
Specifically, sequence described in (2) is as shown in SEQ.ID.NO.2.
Purposes of the gene BnGRF7a of the present invention in terms of breeding is especially promoting seed kernel to increase, is improving kind Purposes in suboutput.
The present invention on this basis, provides a kind of SNP marker relevant to vegetable seeds seed size shape, Neng Gouyou Effectiveness is in plant breeding according to an embodiment of the invention, the SNP marker is located at the promoter region of BnGRF7a gene, for rape The base A or G of 566683 position of C02 chromosome.
Plant according to the present invention is not particularly limited specific floristics, according to an embodiment of the invention, described Plant is selected from rape, arabidopsis.
The present invention provides a kind of over-express vector, and the carrier includes nucleotide sequence shown in SEQ.ID.NO.2, described Over-express vector can be by obtaining above-mentioned nucleotide sequence insertion cloning vector or expression vector.
Carrier suitable for constructing over-express vector of the present invention includes but is not limited to PHZM08.Implementation according to the present invention Example, the present invention construct the recombinant vector of the BnGRF7a gene of miRNA396 target spot same sense mutation with Gateway technology PHZM08-BnGRF7a-miRNA。
The present invention also provides a kind of recombinant bacterial strain, the bacterial strain includes above-mentioned over-express vector, and the recombinant bacterial strain can lead to It crosses to convert containing over-express vector of the present invention to host cell and obtain.Suitable for constructing the host of recombinant cell of the present invention Cell includes but is not limited to agrobatcerium cell GV3101 etc..
The present invention also resides in the transformation of Arabidopsis thaliana that recombinant vector PHZM08-BnGRF7a-miRNA is obtained with flower-dipping method Strain provides material for subsequent further investigation.
Advantages of the present invention:
1. the present invention is associated analysis using the natural population with 324 rape germplasm, control mass of 1000 kernel is obtained Gene BnGRF7a, the degree of association is up to 9.365%.This method is true and reliable, has convincingness.Gene is overexpressed in rape BnGRF7a can generate bigger seed, to improve yield.
2. the present invention proves that gene BnGRF7a and its promoter region exist with 3 kinds of bioinformatic analysis methods Selection pressure is received in breeding or evolutionary process, shows that the function of studying this gene has far-reaching meaning to yield of rape is improved Justice.
3. the functional SNP that the present invention in numerous SNPs, is filtered out by genotypic categorization statistics and analysis: ChrC02_random_566683A/G, when the base in the site is A, rape can generate bigger seed, therefore the SNP is available In molecular breeding, the rapeseed plants with big seed are quickly filtered out, promote the development of rape molecular breeding.
4. the transformant obtained after the over-express vector PHZM08-BnGRF7a-miRNA arabidopsis thaliana transformation that the present invention constructs, Experimental material is provided to study function and the mechanism of action of gene BnGRF7a, new big seed germ plasm resource is also can be used as, is Rapeseed breeding provides new GENE SOURCES, helps to push agricultural sciences progress.
Detailed description of the invention
The chadogram of Fig. 1 cabbage type rape GRFs family member, institute's target is gene of the present invention in box in figure BnaC02g44780D(BnGRF7a)。
Fig. 2 carries out candidate gene association to cabbage type rape GRFs family member and analyzes resulting Manhattan figure, side in figure Institute's target is gene BnaC02g44780D (BnGRF7a) of the present invention in frame.
Tissue expression pattern map of Fig. 3 .BnGRF7a gene in cabbage type rape.Fig. 4 gene BnGRF7a CDS's The comparison result of cloned sequence and forecasting sequence；It is forecasting sequence that YBnGRF7a is corresponding in figure, and BnGRF7a is corresponding for clone Sequence.
The miRNA target spot of Fig. 5 .BnGRF7a predictive genes.
The base of same sense mutation in the miRNA target spot of Fig. 6 .BnGRF7a predictive genes, institute's target is synonymous in box in figure The base position of mutation.
The schematic diagram of Fig. 7 over-express vector PBI121-BnGRF7a-miRNA.
The arabidopsis sun for the conversion over-express vector PHZM08-BnGRF7a-miRNA that Fig. 8 is filtered out on LB culture medium Property plant；Non- positive seedling cannot grow true leaf, and cotyledon is yellow.
Fig. 9 converts the PCR qualification result of the Arabidopsis plant of over-express vector PHZM08-BnGRF7a-miRNA；1 in figure It is transformation of Arabidopsis thaliana pnca gene group for DNA standard Marker 5000,2-13,14 be positive control, that is, PHZM08-BnGRF7a-miRN Vector plasmid, 15 be negative control, that is, wildtype Arabidopsis thaliana col, is the position of purpose band in box.
The arabidopsis positive seedling for the conversion over-express vector PHZM08-BnGRF7a-miRNA that Figure 10 is identified and quasi- south The comparison diagram of mustard wild type col；1#, 2#, 3# and 11# are positive plants, and col is WT lines；Left side cream-laid is in figure Object of reference, length 2cm；Scale bar size is 0.5cm in figure.
The blade area measurement and analysis of the transformation of Arabidopsis thaliana strain of Figure 11 over-express vector PHZM08-BnGRF7a-miRNA Result figure.* *: P < 0.0001 indicates extremely significant.
The transformation of Arabidopsis thaliana strain of Figure 12 over-express vector PBI121-BnGRF7a-miRNA and the seed of wild type col.2# It is positive transformants, col is WT lines.
The seed of the transformation of Arabidopsis thaliana strain and wild type col of Figure 13 over-express vector PBI121-BnGRF7a-miRNA 100-grain weight measurement.2# is positive transformants, and col is WT lines.
Specific embodiment
According to following embodiment, the present invention can be better understood, but the embodiment described is to preferably explain this Invention, rather than limiting the invention.
The identification of embodiment 1:BnGRF7a gene
In cabbage type rape, there are 35 members (Fig. 1, Ma J Q, Jian H J, Yang B, et in GRFs family al.Genome-wide analysis and expression profiling of the GRF gene family in oilseed rape(Brassica napus L.).[J].Gene,2017,620:36-45.).GRFs major regulatory plant Growth and development and stress response.Mass of 1000 kernel (seed size) is important economical character, is that the three of decision crop seed yield are big The gene pairs breeding of one of factor, research control seed size is of great significance.The present embodiment to this 35 member gene (at Member's gene information is see http://www.genoscope.cns.fr/brassicanapus/cgi-bin/gbrowse/ Colza/ the association analysis of progress mass of 1000 kernel in the natural population with 324 rape germplasm) is collected in market.
First the group DNA of each 2500bp before and after gene and gene is extracted, also just SNP variation is extracted, is passed through Cross the filtering of maf (minimum gene frequency) 0.05.Then these data point reuses are the genotype data format of hapmap, by base Association analysis is done because type data and corresponding phenotypic data (mass of 1000 kernel data) import Tassel software.
A large amount of SNP is obtained as a result, and further the SNP to P value less than 0.0001 is analyzed, and is analyzed result (Fig. 2) Show that gene BnaC02g44780D is the most significant related to thousand grain weigth character, the significant relevant SNP that it includes has 42 It is a, it wherein SNP highest to mass of 1000 kernel contribution rate reaches 9.356%, therefore is BnGRF7a by this unnamed gene, and by this base Because of the candidate gene as control seed size.
The tissue-specific expression pattern of embodiment 2:BnGRF7a gene is analyzed
(1) it samples
It quickly takes in cabbage type rape at the same time and No. 11 double (to be purchased from the limited public affairs of oil kind industry science and technology in Wuhan Department) root, stem, stem apex, leaf, flower, bud, silique, seed and silique skin, be temporarily stored in liquid nitrogen, be placed in -70 DEG C of ultra low temperature freezers It saves.
(2) RNA extraction is synthesized with the first chain of cDNA
The extraction of the RNA of silique and seed: using vegetable seeds RNA extracts kit, (precious day cures the limited public affairs of biotechnology Department), take appropriate amount of sample in mortar with powdery is ground into after liquid nitrogen frozen, the freezing powder for quickly weighing about 100mg, which is added, to be contained In the 2mL EP pipe for there are 500 μ L Buffer RA reagents (pre-cooling), 150 μ L water-saturated phenols and 100 μ L chloroforms are added, it is then acute It is strong to rock 15 seconds.Then, at room temperature, 14,000 × g are centrifuged 5min；Upper honest and upright and thrifty 300 μ L to one of transfer later it is new remove nucleic acid In the 2mL EP pipe of enzyme, 300 μ L Buffer RC are added, are mixed with vortex.Mixed liquor is transferred in a gDNA Filter column, This pillar is attached in 2mL collecting pipe, at room temperature, 14,000 × g is centrifuged 2min.300 μ L dehydrated alcohols (room temperature) are added to upper It states in filtrate, and is inhaled up and down with liquid-transfering gun and play 5-10 mixing.By above-mentioned all mixed liquors (including any precipitating being likely to form Object) one is transferred in the HiBind RNA micro-column of new 2mL collecting pipe, room temperature, 10,000 × g is centrifuged 1min, abandons Filtrate is gone, and pillar is reinstalled in collecting pipe and (if sample volume is more than the capacity of pillar, loads by several times and repeats this step Suddenly).400 μ L RWC washing buffers are added, are centrifuged by above-mentioned condition.Discard filtrate and collecting pipe.Pillar is attached to one newly 2mL collecting pipe in, and be added 500 μ L RNA Wash Buffer II (being used again after being diluted with ethyl alcohol).By above-mentioned condition from The heart discards filtrate.Add 500 μ L RNA Wash Buffer II to wash pillar again, is centrifuged by above-mentioned condition and discards filtrate.So Collecting pipe in rear enclosure is centrifuged pillar 10,000 × g, 2min, to be completely dried column matrix.Transfer pillar clean is gone to one In the 1.5mL EP pipe of nuclease, RNA is mentioned with 30 μ L DEPC washing.Ensure that water is applied directly on column matrix.It is stored at room temperature 2min, then 10,000 × g, are centrifuged 1min, and it is primary to repeat this step.Agarose gel electrophoresis identifies total serum IgE quality, and running glue has 3 clearly band it is i.e. available, total RNA content is then measured in microplate reader.
Root, stem, stem apex, leaf, flower, bud and silique skin RNA extraction: use UNIQ-10 pillar Trizol kit (the handsome Bioisystech Co., Ltd in Shanghai) takes a small amount of sample in mortar with powdery is ground into after liquid nitrogen frozen, and addition fills In the 2mL EP pipe of 1mL TRNzol-A reagent (Tiangeng biochemical technology Co., Ltd), sufficiently oscillation after, 4 DEG C, 12,000rpm from Heart 10min；Honest and upright and thrifty 800 μ L is taken later, and chlorination is imitated 300 μ L (pre-cooling is conducive to separation), after acutely shaking 15s, is placed at room temperature for 3min；Afterwards after 4 DEG C, 12,000rpm centrifugation 10min, about 300 μ L ZCX of supernatant is moved in new 2mL EP pipe, is added and is waited bodies Product isopropanol precipitating RNA, 4 DEG C, 12000rpm is centrifuged 10min；It discards supernatant, 800 μ L, 75% ethyl alcohol is added to dissolve, 4 DEG C, 12, 000rpm is centrifuged 5min；Supernatant is abandoned, the DEPC water dissolution precipitating for adding 20 μ L to sterilize.Electroresis appraisal total serum IgE quality, is then being divided Rna content is measured on photometer.
It pressesRT SuperMix for qPCR (+gDNA wiper) kit (is purchased from Nanjing promise and only praises life Object Technology Co., Ltd.) operational manual carry out reverse transcription experiment.
A. genomic DNA removes
Mixed liquor is formulated as follows in the centrifuge tube of RNase free:
RNase free ddH2O to 8 μ L
4×gDNA wiper Mix 2μL
Template ribonucleic acid total serum IgE: 500ng
Mixing is gently blown and beaten with pipettor.42℃2min.
B. reverse transcription reaction system is prepared:
The 8 μ L of reaction solution of 5 × qRT SuperMix II, 2 μ L, step a are directly added into the reaction tube of step a, with shifting Liquid device gently blows and beats mixing.
C. reverse transcription reaction is carried out
Standardization program (cDNA that can get maximum output): 25 DEG C of reactions 10min, 50 DEG C of * react 30min, 85 DEG C of reactions 5min.Reaction product is saved backup at -20 DEG C.
* if template has complicated secondary structure or the high region GC, reaction temperature can be improved to 55 DEG C, is helped to improve Yield.
(3) qRT-PCR identifies expression quantity of the BnGRF7a gene in each tissue
Expression quantity of the present invention using fluorescent quantitative RT-PCR method analysis BnGRF7a gene in each tissue, fluorescence are fixed PCR is measured to useGreen Realtime PCR Master Mix-Plus- kit (Takara).
Reference gene is used as using B.napus TIP41 (Gene ID:AT4G34270), the primer of BnGRF7a gene is BnGRF7a-qF and BnGRF7a-qR.Fluorescent quantitation reaction system is 20 μ L, is prepared in the first external 1.5mL EP pipe of removing template Reaction solution (such as the following table 1), then be dispensed into PCR Strip Tubes.The cDNA of each sample dilutes 10 times and suction is beaten after mixing 2 μ L, three repetitions of each sample are successively added in each reaction solution.
Table 1.qPCR response procedures
Amplification condition are as follows: 95 DEG C, 30sec；Then 95 DEG C of 5s, 57 DEG C of 30s, 72 DEG C of 27s, 40 circulations；Each it is circulated in 72 DEG C of renaturation ends carry out fluorescence detection.It is first to heat to 95 DEG C after reaction, is then down to 72 DEG C, then be to slowly warm up to 95 DEG C, the variation of fluorescence signal is recorded, obtains the melting curve of amplified production.Every group of experiment is completed three biology and is repeated, often A biology repeats at least to do technology repetition three times.
First using melting curve as standard, using instrument institute band software, optimizes internal standard gene and target gene PCR reacts item It is special to be allowed to amplified production for part.This experiment measures B.napus TIP41 using rape B.napus TIP41 as reference gene respectively With the Ct value of target gene BnGRF7a, its average value is taken, with the Δ Ct for comparing Ct method and obtaining target gene to reference gene, so It is afterwards the Δ Δ Ct for obtaining other tissues respectively referring to (its value is converted into 1) with root, finally by 2-ΔΔCtEstimate purpose base The Relative Expression values of cause, and find out systematic error.
The primer sequence table of table 2.B.napus TIP41 gene and BnGRF7a gene
Primer Sequence Length Annealing temperature
B.napus TIP41-F(SEQ.ID.NO.3) 5'-TGAAGAGCAGATTGATTTGGCT-3' 22 56.3℃
B.napus TIP41-R(SEQ.ID.NO.4) 5'-ACACTCCATTGTCAGCCAGTT-3' 21 58℃
BnGRF7a-qF(SEQ.ID.NO.5) 5'-TCGTGTCTTCTTCACTTGATGA-3' 22 55.6℃
BnGRF7a-qR(SEQ.ID.NO.6) 5'-CTCAGTATCTCCGCTAATGGTC-3' 22 56.1℃
Then it is mapped with GraphPad Prism software, as shown in figure 3, being with expression quantity of the BnGRF7a gene in root The control group of significance difference analysis, the results showed that, expression quantity highest of the BnGRF7a gene in developmental seed, secondly It is stem apex.These results of study show that BnGRF7a gene may be by promoting seed cell division, to generate the kind of increase Son.
Embodiment 3: whether analysis BnGRF7a gene is in breeding or evolutionary process by selection pressure
The present embodiment is arranged the mass of 1000 kernel of 324 germplasm in the natural population of 324 rape germplasm from small to large Sequence, preceding 25% (2.83-3.55g) draft the material small for mass of 1000 kernel, and rear 25% (4.17-5.52g) drafts big for mass of 1000 kernel Material.25% small material of 25% material that has selected mass of 1000 kernel big respectively, mass of 1000 kernel is as being grouped.With three kinds of methods It is analyzed:
(1) Fst (Fixation index) is analyzed, and window 20kb, step-length 2kb, general Fst value is bigger, illustrates two Asias The big germplasm of group, i.e. mass of 1000 kernel and mass of 1000 kernel it is small germplasm materials differentiation it is more serious, then illustrate that the region is sent out in two subgroups Raw obvious differentiation, then be the character by selection region (table 3).
(2) θ Pi method, window 20kb, step-length 2kb calculate two groups of materials and calculate separately genetic diversity Pi, if two groups Some region Pi significant difference of material, then ratio then can be higher or relatively low, illustrates the region by selection (table 4).
(3) Tajima'D is analyzed, window 20kb, and without step-length, Tajima'D value illustrates that the region is to be selected not equal to 0 (table 5).Although window is 20kb, without other genes in BnGRF7a upstream region of gene 22kb and the downstream region 8kb.
These three analysis methods all show that BnGRF7a gene is selected in breeding or evolutionary process, illustrate the gene It is a protogene in breeding or evolutionary process, and the mass of 1000 kernel of this gene control rape seed, show to further investigate this The function of gene has great meaning to breeding.
Table 3.Fst analyzes result
4. θ Pi of table analyzes result
Table 5.Tajima'D analyzes result
* the position of BnGRF7a gene on chromosome is about are as follows:
ChrC02_random564860-chrC02_random566156 (originate in 564860 sites on C02 chromosome, Terminate at 566156 sites).Without other genes in BnGRF7a upstream region of gene 22kb and the downstream region 8kb.
The functional snp analysis of embodiment 4:BnGRF7a gene
It based on the conclusion that embodiment 1 obtains, analyzes SNP is obtained, analysis shows that in BnGRF7a gene and its up and down 52 SNP are shared in trip 2.5kbp sequence, wherein SNP of the P value less than 0.0001 there are 42, positioned at BnGRF7a downstream of gene Have 29, have on exon 5 (have 1 on conserved domain WRC, but cause same sense mutation, other 4 cause it is non- Same sense mutation), promoter region has 8 (tables 6).
In turn, the present embodiment analyzes the connection between these SNP and mass of 1000 kernel phenotype.By 324 in natural population The corresponding mass of 1000 kernel ascending sort of germplasm drafts the genotype that first 15 are mass of 1000 kernel minimum (2.83-3.18g), and latter 15 are The genotype of mass of 1000 kernel greatly (4.65-5.52g).It was found that SNP chrC02_random564820, have in minimum mass of 1000 kernel germplasm 11 are shown as A, and 2 are shown as G；SNP chrC02_random564666, only 3 are shown as in minimum mass of 1000 kernel germplasm A, other are T；SNP chrC02_random565578 and chrC02_random565490, in minimum mass of 1000 kernel germplasm respectively There are 11 to be shown as G and T, there are 3 to be shown as A and C respectively；SNP chrC02_random567282, in minimum mass of 1000 kernel germplasm There are 11 to be shown as G, 3 are shown as A (table 7).These are the result shows that SNP chrC02_random564820A, chrC02_ Random564666T, chrC02_random565578G, chrC02_random565490T and SNP chrC02_ Random567282G may be relevant with seedlet phenotype.And at SNP chrC02_random566683A/G, very big mass of 1000 kernel There are 11 to be shown as A in germplasm, there are 2 to be shown as G, shows that SNP chrC02_random566683A may be with big seed phenotypes It is relevant.
Further, analysis is expanded to preceding 108 germplasm and rear 108 germplasm by the present embodiment, further to verify State contacting between SNP and mass of 1000 kernel.Analysis has found that it is likely that those of the association of seedlet phenotype SNP, two kinds of bases respectively correspond Seedlet germplasm (first 108) number difference it is unobvious.For example, SNP chrC02_random565578G in seedlet germplasm 52 are accounted for, and chrC02_random565578A accounts for 48 (tables 8).However, may related SNP with big seed phenotypes ChrC02_random566683A/G (promoter region), in 108 big seed germplasm, chrC02_random566683A is accounted for 72, and chrC02_random566683G accounts for 20 (tables 9), gap is more obvious.It is thus determined that SNP:chrC02_ Random_566683A/G is functionality SNP, by adjusting the expression quantity of BnGRF7a gene to control seed size, energy Promote vegetable seeds seed to increase, improves mass of 1000 kernel.
SNP distribution in table 6.BnGRF7a gene and its region upstream and downstream 2.5kb
* what is listed in table 6 is the distribution situation of the SNP of P < 0.0001
Genotype of the several SNP of table 7. in minimum germplasm (15)
Genotype of the several SNP of table 8. in small germplasm (108)
Genotype of the table 9.SNP chrC02_random566683 in big germplasm
* minimum germplasm mass of 1000 kernel: 2.83-3.18g；Very big germplasm mass of 1000 kernel: 4.65-5.52g；Small germplasm mass of 1000 kernel: 2.83-3.65g；Big germplasm mass of 1000 kernel: 4.02-5.52g.
Embodiment 5: building is overexpressed the transgenic Arabidopsis plants after BnGRF7a gene miRNA target spot same sense mutation
(1) clone of BnGRF7a gene
According to predicted in Genoscope rape BnGRF7a gene C DS (Coding sequence, coded sequence, below It is identical) sequence (SEQ.ID.NO.7), in the primer of initiation codon and termination codon subregion design primer amplification BnGRF7a Sequence is respectively as follows:
Upstream primer BnGRF7a-F (5 '-ATGGACTTTCTCAAAGTTTCTGACAAG-3 ') (SEQ.ID.NO.8)；Under It swims primer BnGRF7a-R (5 '-CTAAGTCGACAACGGCGACGAGC-3 ') (SEQ.ID.NO.9).Double No. 11 rapes in CDNA is template, carries out PCR using KOD-Plus Neo enzyme (TOYOBO), according to following reaction condition: 94 DEG C, 2min, then 35cycles (98 DEG C, 10s, 59 DEG C, 30s, 68 DEG C, 35s), 68 DEG C of 10min amplify the overall length code sequence of BnGRF7a gene Column.Then, referring to UNIQ-10 pillar DNA plastic recovery kit (Sangon Biotech (Shanghai) Co., Ltd.) from agar Pcr amplification product is recycled in sugared gel, is then that the target gene BnGRF7a amplified is cloned into pMD18-T load by PCR product Body (precious bioengineering (Dalian) Co., Ltd), linked system are as follows: 4.5 μ L target fragments, 0.5 μ L pMD-18T carrier, 5 μ L Solution I (precious bioengineering (Dalian) Co., Ltd) is connected overnight at 16 DEG C, is transformed into competent escherichia coli cell In.It is sent to Sangon Biotech's sequencing.As shown in figure 4, the sequence of sequencing result and prediction There is the difference of 3 bases, but completely the same with the amino acid sequence of prediction (SEQ.ID.NO.10).Amplify target gene BnGRF7a complete encoding sequence as shown in SEQ.ID.NO.1, CDS translate the gene amino acid sequence (SEQ.ID.NO.11)。
(2) the miRNA target spot of same sense mutation BnGRF7a gene
According to BnGRF7a gene miRNA target spot (Fig. 5, Hoe K J, Tsukaya the H.Regulation of of prediction plant growth and development by the GROWTH-REGULATING FACTOR and GRF- INTERACTING FACTOR duo[J].Journal of Experimental Botany,2015,66(20):6093- 6107) base (Fig. 6) for needing same sense mutation is determined, then design primer, is divided into intermediary downstream primer and intermediate upstream primer. Intermediary downstream primer BnGRF7a-Rm:5 '-TTCAACGTGCTTGCGGCTGCGTGGACGGCCTCTGTGCAAGTGT-3 ' (SEQ.ID.NO.12)；Intermediate upstream primer BnGRF7a-Fm:5 '-ACGCAGCCGCAAGCACGTTGAACTTCCTTACTCTC GCCCTAGC-3'(SEQ.ID.NO.13).Then respectively with two pairs of primers: BnGRF7a-F/BnGRF7a-Rm and BnGRF7a- Fm/BnGRF7a-R carries out first round PCR to expand two small fragments before and after BnGRF7a gene miRNA target spot.After glue recycling, The second wheel PCR is carried out to splice two segments as template and primer using two obtained segments.PCR amplification system: preceding 2 μ of segment 0.2 1 μ L, 2mM dNTPs of μ L, 10 × buffer for KOD-Plus Neo of L, 2 μ L, KOD-Plus Neo of post-fragment, 1 μ L, 25mM MgSO40.6 μ L, ddH2O 3.2μL；Response procedures be 94 DEG C, 2min, then 15 circulation (98 DEG C, 10s, 40 DEG C, 1min, 68 DEG C, 1min), 68 DEG C of 10min.Then, using the PCR product of the second wheel as template, BnGRF7a-F/BnGRF7a-R is Upstream and downstream primer carries out third round PCR to expand the complete encoding sequence after mutation.Last glue recycling, connects pMD18-T carrier, Convert Escherichia coli, picking positive bacterium colony, sequence verification (Sangon Biotech (Shanghai) Co., Ltd.), sequencing result with It is expected that the mutant nucleotide sequence (SEQ.ID.NO.14) formed is consistent.
(3) over-express vector of the BnGRF7a gene after the same sense mutation of miRNA target spot is constructed
According toII One Step Cloning Kit (is purchased from Nanjing promise and only praises the limited public affairs of biotechnology Department) specification constructed.Steps are as follows:
(1) with restriction endonuclease sacI and BamHI (being purchased from precious bioengineering Co., Ltd) double digestion empty carrier PBI121 (is purchased from Wuhan Miao Ling Biotechnology Co., Ltd), obtains linearized vector.
(2) 5 ' end of design introduces the upstream and downstream primer of two terminal homologous sequence of linearized vector and restriction enzyme site respectively to expand Increase Insert Fragment,
Upstream primer PBI121-F:
5’-ACGGGGGACTCTAGAGGATCCATGGACTTTCTCAAAGTTTCTGACAAG-3’
(SEQ.ID.NO.15) (dashed part: BamHI restriction enzyme site；Before scribing line: the line adjacent with BamHI restriction enzyme site Property carrier end homologous sequence；After scribing line: gene specific upstream primer)；
Downstream primer PBI121-R:
5’-CGATCGGGGAAATTCGAGCTCCTAAGTCGACAACGGCGACGAGC-3’
(SEQ.ID.NO.16) (dashed part: sacI restriction enzyme site；Before scribing line: adjacent with sacI restriction enzyme site is linear Change carrier end homologous sequence；After scribing line: gene specific downstream primer).Then with the correct bacterium solution institute of sequencing in step (2) The plasmid mentioned is template, amplifies Insert Fragment.
(3) recombining reaction, in preparing reaction system on ice: 3 μ L (257.16ng) of linearized vector PBI121 is inserted into piece Section 1 μ L (39.36ng), 5 × CE II Buffer, 4 μ L, Exnase II 2 μ L, ddH2O 10μL.It is gently inhaled using pipettor It beats and mixes, of short duration centrifugation collects reaction solution to tube bottom；37 DEG C of reaction 30min；It is down to 4 DEG C or is immediately placed on cooled on ice.So The product of recombining reaction is converted into competent escherichia coli cell DH5 α afterwards, identifies positive bacterium colony, sequence verification (raw work biology Engineering (Shanghai) limited liability company), correct bacterium solution upgrading grain will be sequenced to get over-express vector PBI121- is arrived BnGRF7a-miRNA (Fig. 7).The over-express vector PBI121-BnGRF7a-miRNA carrier built is transformed into Agrobacterium In GV3101 (being purchased from Shanghai Shang Ya Bioisystech Co., Ltd).
(4) the transformation of Arabidopsis thaliana strain of over-express vector PBI121-BnGRF7a-miRNA is constructed
Arabidopsis thaliana transformation is flower-dipping method, and steps are as follows:
A. the Agrobacterium colonies containing over-express vector are inoculated in the 5mL LB culture medium (Kan containing final concentration of 50 μ g/mL And Rif) in, 28 DEG C, the concussion of 220rpm shaking table is overnight.
B. it is transferred in 200mmL LB culture medium with 1:50 ratio, grows into OD in the case where 28 DEG C600=1.2~ 1.6.C.4000rpm, the collection of thallus is carried out after centrifugation 15min.
D. it is resuspended in permeabilization buffer (+5% sucrose of MS solution), while as control, adjusting OD600=0.8~ 1.0.E. the arabidopsis for removing and having occurred that pollination and bearing pods is cut off with disinfection, they are upside down in equipped with permeabilization buffer Infecting for 2~3min is carried out on container, selects plastic film to cover entire pallet while reserving several stomatas at this time, in dark Under the conditions of be incubated overnight, for 24 hours after film is removed, placement continue to cultivate at room temperature.
F., each plant is carried out to 4-5 conversion, the harvest of seed is carried out after general 30d.
The screening of positive plant: obtained seed is placed in EP pipe, and discoloration silica gel then is added to the inside and is planted The drying process of son；Silica gel is changed at regular intervals, after waiting seeds sufficiently to dry.Seed is placed in vernalization 1-2 in 4 DEG C of refrigerators It or so；Seed after vernalization moves on in sterilized EP pipe, and with outwelling ethyl alcohol after 75% ethanol disinfection 20min；It is added new 75% ethyl alcohol re-disinfection 20min after outwell ethyl alcohol；Sterile water washing 4-5 times, every time about 2min；Transgenic seed is set with water In on the 1/2MS plate of the Kan containing 30mg/L, dried up under superclean bench；The seed of Kan resistance screening at 22 DEG C, 16h illumination/ The 8h photoperiod at night cultivates, and the seedling (non-positive seedling yellow and can not grow true leaf) (Fig. 8) for growing true leaf is transferred to after 2 weeks Native the inside, when growing into 3 weeks to transgenic seedlings, extracts the total DNA in blade, then carries out PCR identification.Such as Shown in Fig. 9, it is positive transformants that in 12 transgenic seedlings of identification, 1#, 2#, which have purpose band in 3# and 11#,.
(5) the blade area measurement of the transformation of Arabidopsis thaliana strain of over-express vector PBI121-BnGRF7a-miRNA
Positive transformants seedling 1#, 2#, 3# and 11# and a representative wild type are measured using software I mage J The area of maximum four leaves in seedling col (Columbia) (life science institute, Jiangsu University) (Figure 10) then will Measurement result is mapped with software GraphPad Prism, and carries out significance test of difference (t tests).Such as Figure 11 institute Show, the blade area of positive transformants seedling is noticeably greater than the blade area of col.Show to be overexpressed miRNA target spot in arabidopsis Gene BnGRF7a after mutation can be such that plant leaf area increases.
(6) the seed size measurement of the transformation of Arabidopsis thaliana strain of over-express vector PBI121-BnGRF7a-miRNA
Implement arabidopsis the positive transformants seedling 2# and wild type col in 5 step of example (5) using electronic balance weighing (Columbia) 100-grain weight of the seed (Figure 12) of (life science institute, Jiangsu University): 100 seeds are randomly selected respectively It is weighed, and repeats this operation five times.Then measurement result is mapped with software GraphPad Prism, and into Row significance test of difference (t tests) (Figure 13).As shown in Figure 12 and Figure 13, the seed of positive transformants seedling is noticeably greater than col Seed, show in arabidopsis be overexpressed miRNA target spot mutation after gene BnGRF7a seed can be made to increase, further demonstrate,prove Gene BnGRF7a is illustrated to the control action of seed size.
The sequence information that other are designed into is as follows:
SEQ.ID.NO.1
atggactttctcaaagtttctgacaagacaacaactctctatagaaataactctctatttgggttgaa tcaacaacaaatggagattcatcctcatcctcatgtcattactcctcatgcaggaaatggagttatgggatgttac tattattaccctttcacaaacgcgcaactaaaggagctcgagagacaagcgatgatctacaagtacatgatcgcat ccattcccgttccttttgatctccttgtctcatcatcctatgcacctccctgtaataataaaaacgctgtcggaga cttagagccggggagatgtcggagaacagacgggaagaaatggagatgctcgaaagaagtagtctctaatcacaaa tactgtgagagacacttgcacagaggccgtcctcgttcaagaaagcatgtggaacttccttactctcgccctagca acattggtggtggttccgagaaaaacagagttcttagctctataaaagacacaacagtcgtcgagccaaaggaggt ttcatcagccctctcaaactacagaggaatcgagatctttcctgcatcggcgtcaaacgagcaagaaaacaagtat ctgaactttatagatgtgtggtccgatggagtaagatcatctgagaaactgagtacccctgcttcttcttccccca atggcaatccttctctttactcgcttgatctctctatgggaggaaacagcttaatgggccacgatgaactgggctt aagggtaattgggccgggccgtgatgatcctcacgggtatggtccatatggcgctgtcgtgtcttcttcacttgat gagatgtcgagatggcttgctcccacttccgccacgcccgggggaccattagcggagatactgaggccgaacccga gttcagcgttttctggcgaaatggaagcgaatagcttgacggcgactcctactccgagctcgtcgccgttgtcgac ttag
SEQ.ID.NO.2
atggactttctcaaagtttctgacaagacaacaactctctatagaaataactctctatttgggttgaa tcaacaacaaatggagattcatcctcatcctcatgtcattactcctcatgcaggaaatggagttatgggatgttac tattattaccctttcacaaacgcgcaactaaaggagctcgagagacaagcgatgatctacaagtacatgatcgcat ccattcccgttccttttgatctccttgtctcatcatcctatgcacctccctgtaataataaaaacgctgtcggaga cttagagccggggagatgtcggagaacagacgggaagaaatggagatgctcgaaagaagtagtctctaatcacaaa tactgtgagagacacttgcacagaggccgtccacgcagccgcaagcacgttgaacttccttactctcgccctagca acattggtggtggttccgagaaaaacagagttcttagctctataaaagacacaacagtcgtcgagccaaaggaggt ttcatcagccctctcaaactacagaggaatcgagatctttcctgcatcggcgtcaaacgagcaagaaaacaagtat ctgaactttatagatgtgtggtccgatggagtaagatcatctgagaaactgagtacccctgcttcttcttccccca atggcaatccttctctttactcgcttgatctctctatgggaggaaacagcttaatgggccacgatgaactgggctt aagggtaattgggccgggccgtgatgatcctcacgggtatggtccatatggcgctgtcgtgtcttcttcacttgat gagatgtcgagatggcttgctcccacttccgccacgcccgggggaccattagcggagatactgaggccgaacccga gttcagcgttttctggcgaaatggaagcgaatagcttgacggcgactcctactccgagctcgtcgccgttgtcgac ttag
SEQ.ID.NO.7
atggactttctcaaagtttctgacaagacaacaactctctatagaaataactctctatttgggttgaa tcaacaacaaatggagattcatcctcatcctcatgtcattactcctcatgcaggaaatggagttatgggatgttac tattattaccctttcacaaacgcgcaactaaaggagctcgagagacaagcgatgatctacaagtacatgatcgcat ccattcccgttccttttgatctccttgtctcatcatcctatgcacctccctgtaataataaaaacgctgtcggaga cttagagccggggagatgtcggagaacagacgggaagaaatggagatgctcgaaagaagtagtctctaatcacaaa tactgtgagagacacttgcacagaggccgtcctcgttcaagaaagcatgtggaacttccttactctcgccctagca acattggtggtggttccgagaaaaacagaattcttagctctataaaagacacaacagtcgtcgagccaaaggaggt ttcatcagccctctcaaactacagaggactcgagatctttcctgcatcggcgtcaaacgagcaagaaaacaagtat ctgaactttatagatgtgtggtccgatggagtaagatcatctgagaaactgagtacccctgcttcttcttccccca atggcaatccttctctttactcgcttgatctctctatgggaggaaacagcttaatgggccacgatgaaatgggctt aagggtaattgggccgggccgtgatgatcctcacgggtatggtccatatggcgctgtcgtgtcttcttcacttgat gagatgtcgagatggcttgctcccacttccgccacgcccgggggaccattagcggagatactgaggccgaacccga gttcagcgttttctggcgaaatggaagcgaatagcttgacggcgactcctactccgagctcgtcgccgttgtcgac ttag
SEQ.ID.NO.10
MDFLKVSDKTTTLYRNNSLFGLNQQQMEIHPHPHVITPHAGNGVMGCYYYYPFTNAQLKELERQAMIYK YMIASIPVPFDLLVSSSYAPPCNNKNAVGDLEPGRCRRTDGKKWRCSKEVVSNHKYCERHLHRGRPRSRKHVELPYS RPSNIGGGSEKNRVLSSIKDTTVVEPKEVSSALSNYRGIEIFPASASNEQENKYLNFIDVWSDGVRSSEKLSTPASS SPNGNPSLYSLDLSMGGNSLMGHDELGLRVIGPGRDDPHGYGPYGAVVSSSLDEMSRWLAPTSATPGGPLAEILRPN PSSAFSGEMEANSLTATPTPSSSPLST
SEQ.ID.NO.11
MDFLKVSDKTTTLYRNNSLFGLNQQQMEIHPHPHVITPHAGNGVMGCYYYYPFTNAQLKELERQAMIYK YMIASIPVPFDLLVSSSYAPPCNNKNAVGDLEPGRCRRTDGKKWRCSKEVVSNHKYCERHLHRGRPRSRKHVELPYS RPSNIGGGSEKNRVLSSIKDTTVVEPKEVSSALSNYRGIEIFPASASNEQENKYLNFIDVWSDGVRSSEKLSTPASS SPNGNPSLYSLDLSMGGNSLMGHDELGLRVIGPGRDDPHGYGPYGAVVSSSLDEMSRWLAPTSATPGGPLAEILRPN PSSAFSGEMEANSLTATPTPSSSPLST
SEQ.ID.NO.14
atggactttctcaaagtttctgacaagacaacaactctctatagaaataactctctatttgggttgaa tcaacaacaaatggagattcatcctcatcctcatgtcattactcctcatgcaggaaatggagttatgggatgttac tattattaccctttcacaaacgcgcaactaaaggagctcgagagacaagcgatgatctacaagtacatgatcgcat ccattcccgttccttttgatctccttgtctcatcatcctatgcacctccctgtaataataaaaacgctgtcggaga cttagagccggggagatgtcggagaacagacgggaagaaatggagatgctcgaaagaagtagtctctaatcacaaa tactgtgagagacacttgcacagaggccgtccacgcagccgcaagcacgttgaacttccttactctcgccctagca acattggtggtggttccgagaaaaacagagttcttagctctataaaagacacaacagtcgtcgagccaaaggaggt ttcatcagccctctcaaactacagaggaatcgagatctttcctgcatcggcgtcaaacgagcaagaaaacaagtat ctgaactttatagatgtgtggtccgatggagtaagatcatctgagaaactgagtacccctgcttcttcttccccca atggcaatccttctctttactcgcttgatctctctatgggaggaaacagcttaatgggccacgatgaactgggctt aagggtaattgggccgggccgtgatgatcctcacgggtatggtccatatggcgctgtcgtgtcttcttcacttgat gagatgtcgagatggcttgctcccacttccgccacgcccgggggaccattagcggagatactgaggccgaacccga gttcagcgttttctggcgaaatggaagcgaatagcttgacggcgactcctactccgagctcgtcgccgttgtcgac ttag
Sequence table
<110>Jiangsu University
<120>BnGRF7a gene and application thereof
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 984
<212> DNA
<213>cabbage type rape (Brassica napus)
<400> 1
atggactttc tcaaagtttc tgacaagaca acaactctct atagaaataa ctctctattt 60
gggttgaatc aacaacaaat ggagattcat cctcatcctc atgtcattac tcctcatgca 120
ggaaatggag ttatgggatg ttactattat taccctttca caaacgcgca actaaaggag 180
ctcgagagac aagcgatgat ctacaagtac atgatcgcat ccattcccgt tccttttgat 240
ctccttgtct catcatccta tgcacctccc tgtaataata aaaacgctgt cggagactta 300
gagccgggga gatgtcggag aacagacggg aagaaatgga gatgctcgaa agaagtagtc 360
tctaatcaca aatactgtga gagacacttg cacagaggcc gtcctcgttc aagaaagcat 420
gtggaacttc cttactctcg ccctagcaac attggtggtg gttccgagaa aaacagagtt 480
cttagctcta taaaagacac aacagtcgtc gagccaaagg aggtttcatc agccctctca 540
aactacagag gaatcgagat ctttcctgca tcggcgtcaa acgagcaaga aaacaagtat 600
ctgaacttta tagatgtgtg gtccgatgga gtaagatcat ctgagaaact gagtacccct 660
gcttcttctt cccccaatgg caatccttct ctttactcgc ttgatctctc tatgggagga 720
aacagcttaa tgggccacga tgaactgggc ttaagggtaa ttgggccggg ccgtgatgat 780
cctcacgggt atggtccata tggcgctgtc gtgtcttctt cacttgatga gatgtcgaga 840
tggcttgctc ccacttccgc cacgcccggg ggaccattag cggagatact gaggccgaac 900
ccgagttcag cgttttctgg cgaaatggaa gcgaatagct tgacggcgac tcctactccg 960
agctcgtcgc cgttgtcgac ttag 984
<210> 2
<211> 984
<212> DNA
<213>cabbage type rape (Brassica napus)
<400> 2
atggactttc tcaaagtttc tgacaagaca acaactctct atagaaataa ctctctattt 60
gggttgaatc aacaacaaat ggagattcat cctcatcctc atgtcattac tcctcatgca 120
ggaaatggag ttatgggatg ttactattat taccctttca caaacgcgca actaaaggag 180
ctcgagagac aagcgatgat ctacaagtac atgatcgcat ccattcccgt tccttttgat 240
ctccttgtct catcatccta tgcacctccc tgtaataata aaaacgctgt cggagactta 300
gagccgggga gatgtcggag aacagacggg aagaaatgga gatgctcgaa agaagtagtc 360
tctaatcaca aatactgtga gagacacttg cacagaggcc gtccacgcag ccgcaagcac 420
gttgaacttc cttactctcg ccctagcaac attggtggtg gttccgagaa aaacagagtt 480
cttagctcta taaaagacac aacagtcgtc gagccaaagg aggtttcatc agccctctca 540
aactacagag gaatcgagat ctttcctgca tcggcgtcaa acgagcaaga aaacaagtat 600
ctgaacttta tagatgtgtg gtccgatgga gtaagatcat ctgagaaact gagtacccct 660
gcttcttctt cccccaatgg caatccttct ctttactcgc ttgatctctc tatgggagga 720
aacagcttaa tgggccacga tgaactgggc ttaagggtaa ttgggccggg ccgtgatgat 780
cctcacgggt atggtccata tggcgctgtc gtgtcttctt cacttgatga gatgtcgaga 840
tggcttgctc ccacttccgc cacgcccggg ggaccattag cggagatact gaggccgaac 900
ccgagttcag cgttttctgg cgaaatggaa gcgaatagct tgacggcgac tcctactccg 960
agctcgtcgc cgttgtcgac ttag 984
<210> 3
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tgaagagcag attgatttgg ct 22
<210> 4
<211> 21
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
acactccatt gtcagccagt t 21
<210> 5
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
tcgtgtcttc ttcacttgat ga 22
<210> 6
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ctcagtatct ccgctaatgg tc 22
<210> 7
<211> 984
<212> DNA
<213>cabbage type rape (Brassica napus)
<400> 7
atggactttc tcaaagtttc tgacaagaca acaactctct atagaaataa ctctctattt 60
gggttgaatc aacaacaaat ggagattcat cctcatcctc atgtcattac tcctcatgca 120
ggaaatggag ttatgggatg ttactattat taccctttca caaacgcgca actaaaggag 180
ctcgagagac aagcgatgat ctacaagtac atgatcgcat ccattcccgt tccttttgat 240
ctccttgtct catcatccta tgcacctccc tgtaataata aaaacgctgt cggagactta 300
gagccgggga gatgtcggag aacagacggg aagaaatgga gatgctcgaa agaagtagtc 360
tctaatcaca aatactgtga gagacacttg cacagaggcc gtcctcgttc aagaaagcat 420
gtggaacttc cttactctcg ccctagcaac attggtggtg gttccgagaa aaacagaatt 480
cttagctcta taaaagacac aacagtcgtc gagccaaagg aggtttcatc agccctctca 540
aactacagag gactcgagat ctttcctgca tcggcgtcaa acgagcaaga aaacaagtat 600
ctgaacttta tagatgtgtg gtccgatgga gtaagatcat ctgagaaact gagtacccct 660
gcttcttctt cccccaatgg caatccttct ctttactcgc ttgatctctc tatgggagga 720
aacagcttaa tgggccacga tgaaatgggc ttaagggtaa ttgggccggg ccgtgatgat 780
cctcacgggt atggtccata tggcgctgtc gtgtcttctt cacttgatga gatgtcgaga 840
tggcttgctc ccacttccgc cacgcccggg ggaccattag cggagatact gaggccgaac 900
ccgagttcag cgttttctgg cgaaatggaa gcgaatagct tgacggcgac tcctactccg 960
agctcgtcgc cgttgtcgac ttag 984
<210> 8
<211> 27
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
atggactttc tcaaagtttc tgacaag 27
<210> 9
<211> 23
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ctaagtcgac aacggcgacg agc 23
<210> 10
<211> 327
<212> PRT
<213>cabbage type rape (Brassica napus)
<400> 10
Met Asp Phe Leu Lys Val Ser Asp Lys Thr Thr Thr Leu Tyr Arg Asn
1 5 10 15
Asn Ser Leu Phe Gly Leu Asn Gln Gln Gln Met Glu Ile His Pro His
20 25 30
Pro His Val Ile Thr Pro His Ala Gly Asn Gly Val Met Gly Cys Tyr
35 40 45
Tyr Tyr Tyr Pro Phe Thr Asn Ala Gln Leu Lys Glu Leu Glu Arg Gln
50 55 60
Ala Met Ile Tyr Lys Tyr Met Ile Ala Ser Ile Pro Val Pro Phe Asp
65 70 75 80
Leu Leu Val Ser Ser Ser Tyr Ala Pro Pro Cys Asn Asn Lys Asn Ala
85 90 95
Val Gly Asp Leu Glu Pro Gly Arg Cys Arg Arg Thr Asp Gly Lys Lys
100 105 110
Trp Arg Cys Ser Lys Glu Val Val Ser Asn His Lys Tyr Cys Glu Arg
115 120 125
His Leu His Arg Gly Arg Pro Arg Ser Arg Lys His Val Glu Leu Pro
130 135 140
Tyr Ser Arg Pro Ser Asn Ile Gly Gly Gly Ser Glu Lys Asn Arg Val
145 150 155 160
Leu Ser Ser Ile Lys Asp Thr Thr Val Val Glu Pro Lys Glu Val Ser
165 170 175
Ser Ala Leu Ser Asn Tyr Arg Gly Ile Glu Ile Phe Pro Ala Ser Ala
180 185 190
Ser Asn Glu Gln Glu Asn Lys Tyr Leu Asn Phe Ile Asp Val Trp Ser
195 200 205
Asp Gly Val Arg Ser Ser Glu Lys Leu Ser Thr Pro Ala Ser Ser Ser
210 215 220
Pro Asn Gly Asn Pro Ser Leu Tyr Ser Leu Asp Leu Ser Met Gly Gly
225 230 235 240
Asn Ser Leu Met Gly His Asp Glu Leu Gly Leu Arg Val Ile Gly Pro
245 250 255
Gly Arg Asp Asp Pro His Gly Tyr Gly Pro Tyr Gly Ala Val Val Ser
260 265 270
Ser Ser Leu Asp Glu Met Ser Arg Trp Leu Ala Pro Thr Ser Ala Thr
275 280 285
Pro Gly Gly Pro Leu Ala Glu Ile Leu Arg Pro Asn Pro Ser Ser Ala
290 295 300
Phe Ser Gly Glu Met Glu Ala Asn Ser Leu Thr Ala Thr Pro Thr Pro
305 310 315 320
Ser Ser Ser Pro Leu Ser Thr
325
<210> 11
<211> 327
<212> PRT
<213>cabbage type rape (Brassica napus)
<400> 11
Met Asp Phe Leu Lys Val Ser Asp Lys Thr Thr Thr Leu Tyr Arg Asn
1 5 10 15
Asn Ser Leu Phe Gly Leu Asn Gln Gln Gln Met Glu Ile His Pro His
20 25 30
Pro His Val Ile Thr Pro His Ala Gly Asn Gly Val Met Gly Cys Tyr
35 40 45
Tyr Tyr Tyr Pro Phe Thr Asn Ala Gln Leu Lys Glu Leu Glu Arg Gln
50 55 60
Ala Met Ile Tyr Lys Tyr Met Ile Ala Ser Ile Pro Val Pro Phe Asp
65 70 75 80
Leu Leu Val Ser Ser Ser Tyr Ala Pro Pro Cys Asn Asn Lys Asn Ala
85 90 95
Val Gly Asp Leu Glu Pro Gly Arg Cys Arg Arg Thr Asp Gly Lys Lys
100 105 110
Trp Arg Cys Ser Lys Glu Val Val Ser Asn His Lys Tyr Cys Glu Arg
115 120 125
His Leu His Arg Gly Arg Pro Arg Ser Arg Lys His Val Glu Leu Pro
130 135 140
Tyr Ser Arg Pro Ser Asn Ile Gly Gly Gly Ser Glu Lys Asn Arg Val
145 150 155 160
Leu Ser Ser Ile Lys Asp Thr Thr Val Val Glu Pro Lys Glu Val Ser
165 170 175
Ser Ala Leu Ser Asn Tyr Arg Gly Ile Glu Ile Phe Pro Ala Ser Ala
180 185 190
Ser Asn Glu Gln Glu Asn Lys Tyr Leu Asn Phe Ile Asp Val Trp Ser
195 200 205
Asp Gly Val Arg Ser Ser Glu Lys Leu Ser Thr Pro Ala Ser Ser Ser
210 215 220
Pro Asn Gly Asn Pro Ser Leu Tyr Ser Leu Asp Leu Ser Met Gly Gly
225 230 235 240
Asn Ser Leu Met Gly His Asp Glu Leu Gly Leu Arg Val Ile Gly Pro
245 250 255
Gly Arg Asp Asp Pro His Gly Tyr Gly Pro Tyr Gly Ala Val Val Ser
260 265 270
Ser Ser Leu Asp Glu Met Ser Arg Trp Leu Ala Pro Thr Ser Ala Thr
275 280 285
Pro Gly Gly Pro Leu Ala Glu Ile Leu Arg Pro Asn Pro Ser Ser Ala
290 295 300
Phe Ser Gly Glu Met Glu Ala Asn Ser Leu Thr Ala Thr Pro Thr Pro
305 310 315 320
Ser Ser Ser Pro Leu Ser Thr
325
<210> 12
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
ttcaacgtgc ttgcggctgc gtggacggcc tctgtgcaag tgt 43
<210> 13
<211> 43
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
acgcagccgc aagcacgttg aacttcctta ctctcgccct agc 43
<210> 14
<211> 984
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
atggactttc tcaaagtttc tgacaagaca acaactctct atagaaataa ctctctattt 60
gggttgaatc aacaacaaat ggagattcat cctcatcctc atgtcattac tcctcatgca 120
ggaaatggag ttatgggatg ttactattat taccctttca caaacgcgca actaaaggag 180
ctcgagagac aagcgatgat ctacaagtac atgatcgcat ccattcccgt tccttttgat 240
ctccttgtct catcatccta tgcacctccc tgtaataata aaaacgctgt cggagactta 300
gagccgggga gatgtcggag aacagacggg aagaaatgga gatgctcgaa agaagtagtc 360
tctaatcaca aatactgtga gagacacttg cacagaggcc gtccacgcag ccgcaagcac 420
gttgaacttc cttactctcg ccctagcaac attggtggtg gttccgagaa aaacagagtt 480
cttagctcta taaaagacac aacagtcgtc gagccaaagg aggtttcatc agccctctca 540
aactacagag gaatcgagat ctttcctgca tcggcgtcaa acgagcaaga aaacaagtat 600
ctgaacttta tagatgtgtg gtccgatgga gtaagatcat ctgagaaact gagtacccct 660
gcttcttctt cccccaatgg caatccttct ctttactcgc ttgatctctc tatgggagga 720
aacagcttaa tgggccacga tgaactgggc ttaagggtaa ttgggccggg ccgtgatgat 780
cctcacgggt atggtccata tggcgctgtc gtgtcttctt cacttgatga gatgtcgaga 840
tggcttgctc ccacttccgc cacgcccggg ggaccattag cggagatact gaggccgaac 900
ccgagttcag cgttttctgg cgaaatggaa gcgaatagct tgacggcgac tcctactccg 960
agctcgtcgc cgttgtcgac ttag 984
<210> 15
<211> 47
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
acgggggact ctagaggatc catggacttt ctcaaagttt ctgacaa 47
<210> 16
<211> 44
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
cgatcgggga aattcgagct cctaagtcga caacggcgac gagc 44

Claims (9)

1. a kind of gene BnGRF7a for promoting vegetable seeds seed to increase, the gene can promote vegetable seeds seed to increase, should The nucleotide sequence of gene is selected from as follows:
(1) nucleotide sequence as shown in SEQ.ID.NO.1；
(2) nucleotide sequence of the point mutation of one or more bases is carried out to nucleotide sequence shown in SEQ.ID.NO.1.
2. gene according to claim 1, which is characterized in that the nucleotide sequence of gene described in (2) is such as Shown in SEQ.ID.NO.2.
3. gene according to claim 1, which is characterized in that the plant includes rape.
4. gene described in claim 1 is promoting the purposes in the increase of vegetable seeds seed.
5. purposes according to claim 4, which is characterized in that the purposes is by being overexpressed base described in claim 1 Because realizing.
6. a kind of SNP marker for promoting vegetable seeds seed to increase, the SNP marker are located at opening for gene described in claim 1 Sub-area is the base A or G of No. 02 566683 position of chromosome of rape C.
7. a kind of over-express vector, the carrier includes the nucleotide sequence of gene as claimed in claim 2.
8. a kind of recombinant bacterial strain, the bacterial strain includes over-express vector as claimed in claim 6.
9. a kind of method for promoting vegetable seeds seed to increase, which is characterized in that the method includes by base described in claim 1 The nucleotide sequence of cause or/and over-express vector as claimed in claim 6 are transformed into target plant, or described in claim 7 Recombinant bacterial strain infect target plant.
CN201811319445.0A 2018-11-07 2018-11-07 BnGRF7a gene and application thereof Pending CN109554371A (en)

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