Method for cultivating soybean resisting soybean mosaic virus
阅读说明：本技术 一种培育抗大豆花叶病毒的大豆的方法 (Method for cultivating soybean resisting soybean mosaic virus ) 是由 高乐 侯文胜 韩天富 孙�石 吴存祥 蒋炳军 陈莉 于 2018-08-23 设计创作，主要内容包括：本发明公开了一种培育抗大豆花叶病毒的大豆的方法。本发明以SMV HC-Pro基因作为靶标基因,对国内外SMV流行株系进行多重核苷酸序列比对,在保守区间内确定了gRNA靶点序列,构建了CRISPR/Cas9基因组定点编辑载体,进一步应用农杆菌介导大豆子叶节的遗传转化体系,获得一批阳性转基因材料,通过SMV抗病鉴定、qRT-PCR、DAS-ELISA血清学实验,分别从表型水平、RNA水平和蛋白水平研究了T<Sub>1</Sub>代大豆植株对SMV的抗病情况,筛选出一批抗性大豆材料。本发明在大豆SMV抗性机制的研究和抗病育种中具有重要的理论意义和实用价值。本发明具有重要的应用价值。(The invention discloses a method for cultivating soybean resisting soybean mosaic virus. The invention takes SMV HC-Pro gene as target gene, multiple nucleotide sequence comparison is carried out on SMV epidemic strains at home and abroad, gRNA target sequence is determined in a conservative interval, CRISPR/Cas9 genome fixed-point editing vector is constructed, agrobacterium tumefaciens is further applied to mediate a genetic transformation system of soybean cotyledonary node to obtain a batch of positive transgenic material, and T is researched from phenotype level, RNA level and protein level respectively through SMV disease-resistant identification, qRT-PCR and DAS-ELISA serological experiments 1 And (5) screening a batch of resistant soybean materials according to the disease resistance of the soybean plant to the SMV. The invention has important theoretical significance and practical value in the research of the soybean SMV resistance mechanism and the breeding for disease resistance. The invention has important application value.)
1. A method for breeding soybean with resistance to soybean mosaic virus comprises the steps of enabling the soybean to contain a CRISPR-Cas9 system; the CRISPR-Cas9 system contains sgRNA;
the target recognized by the sgRNA is HC-Pro gene of soybean mosaic virus.
2. The method of claim 1, wherein: the target site recognized by the sgRNA is a DNA molecule shown as a sequence 1 in a sequence table or a sequence 2 in the sequence table.
3. The method of claim 1 or 2, wherein: the CRISPR-Cas9 system also contains the gene encoding Cas9 protein.
4. A method according to claims 1 to 3, characterized by: the strain of the soybean mosaic virus is at least one of a1) to a 13); a1) strain SMV SC 3; a2) strain SMV G2; a3) strain SMV G3; a4) strain SMV G4; a5) strain SMV G6; a6) strain SMV G7; a7) SMV N strain; a8) strain SMVG 1; a9) strain SMV SC 6; a10) strain SMV SC15 (6067-1); a11) strain SMV SC21 (6202-2); a12) strain SMV HH 5; a13) SMV HZ strain.
5. A CRISPR-Cas9 system containing sgrnas; the target recognized by the sgRNA is HC-Pro gene of soybean mosaic virus.
6. The CRISPR-Cas9 system of claim 5, wherein: the target site recognized by the sgRNA is a DNA molecule shown as a sequence 1 in a sequence table or a sequence 2 in the sequence table.
7. The CRISPR-Cas9 system of claim 5 or 6, wherein: the CRISPR-Cas9 system also contains the gene encoding Cas9 protein.
8. Use of the CRISPR-Cas9 system of any of claims 5 to 7 for breeding soybean resistant to soybean mosaic virus.
9. Use of the CRISPR-Cas9 system of any of claims 5 to 7 to increase resistance of soybean to soybean mosaic virus.
10. Use of the method of claims 1 to 4, or the CRISPR-Cas9 system of any of claims 5 to 7, in soybean breeding.
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for cultivating soybean resisting soybean mosaic virus.
Background
Soybean Mosaic Virus (SMV) caused Soybean mosaic virus is a global Soybean disease, widely distributed in Soybean production areas of the world, and seriously affects the yield and quality of soybeans. SMV belongs to Potyviridae (Potyviridae), Potyvirus (Potyvirus), soybean mosaic virus species. After the soybeans are infected with the SMV, symptoms such as flower leaves and necrosis can be generated, the photosynthetic area and photosynthetic capacity of the soybeans are reduced, plants are dwarfed, the growth amount is reduced, brown spots appear on grains, the yield loss of the soybeans is about 35% -50%, and even the soybeans are completely harvested.
The CRISPR/Cas9 technology is an efficient, accurate and specific genome targeted modification technology which is newly developed in recent years, can realize targeted cutting and insertion, deletion, mutation or replacement of specific sites of a genome by using sequence-specific nuclease, and provides a new effective means for identification of gene functions and targeted improvement of crop traits. In recent years, scientists have successfully applied CRISPR/Cas9 technology to soybean research, including knockout of exogenous green fluorescent protein (gfp) and herbicide resistance (bar), knockout of endogenous functional genes (such as GmFEI2, GmSHR, GmFT2a, GmPDS11 or GmPDS18), development of online network tools for rapidly recognizing target sites and restriction enzyme sites in soybean genome, and the like. None of the above studies have been directed to the improvement of resistance to soybean mosaic virus.
Disclosure of Invention
The invention aims to improve the resistance of soybeans to soybean mosaic virus.
The invention firstly protects a method for cultivating soybean resisting soybean mosaic virus, which can lead the soybean to contain a CRISPR-Cas9 system; the CRISPR-Cas9 system contains sgRNA;
the target recognized by the sgRNA can be HC-Pro gene of soybean mosaic virus.
In the method, the soybean containing the CRISPR-Cas9 system can be realized by introducing a vector containing the CRISPR-Cas9 system into the soybean.
In the method, the soybean can be a soybean mosaic virus infected variety.
In the above method, the soybean may be specifically soybean variety Jack.
In the method, the target recognized by the sgRNA may be a conserved region of HC-Pro gene of soybean mosaic virus. The conserved region of HC-Pro gene of soybean mosaic virus can be 2039-2309 (shown in figure 1) or 8373-8530 (shown in figure 2) from the 5' end of HC-Pro gene of soybean mosaic virus.
In the above method, the target site recognized by the sgRNA may be a DNA molecule represented by
In the method, the CRISPR-Cas9 system can also contain a coding gene of a Cas9 protein.
The invention also protects a CRISPR-Cas9 system containing sgRNA; the target recognized by the sgRNA can be HC-Pro gene of soybean mosaic virus.
In the CRISPR-Cas9 system, the target recognized by the sgRNA may be a conserved region of HC-Pro gene of soybean mosaic virus. The conserved region of HC-Pro gene of soybean mosaic virus can be 2039-2309 (shown in figure 1) or 8373-8530 (shown in figure 2) from the 5' end of HC-Pro gene of soybean mosaic virus.
In the CRISPR-Cas9 system, the target site recognized by the sgRNA may be a DNA molecule represented by
In the CRISPR-Cas9 system, the CRISPR-Cas9 system also can contain a coding gene of a Cas9 protein.
The CRISPR-Cas9 system may specifically be a vector containing the CRISPR-Cas9 system.
Hereinbefore, the strain of soybean mosaic virus may be at least one of a1) to a 13); a1) strain SMV SC3 (Genebank accession number JF 833013); a2) strain SMV G2 (Genebank accession number S42280); a3) strain SMV G3 (Genebank accession number FJ 640978); a4) strain SMV G4 (Genebank accession number FJ 640979); a5) strain SMVG6 (Genebank accession number FJ 640980); a6) strain SMV G7 (Genebank accession number AF 241739); a7) SMV N strain (Genebank accession number D00507); a8) strain SMVG1 (Genebank accession number FJ 640977); a9) strain SMV SC6 (Genebank accession number HM 590054); a10) SMV SC15(6067-1) strain (Genebank accession number JF 833015); a11) SMV SC21(6202-2) strain (Genebank accession number JF 833014); a12) strain SMV HH5 (Genebank accession number AJ 310200); a13) SMV HZ strain (Genebank accession No. AJ 312439).
The vector containing the CRISPR-Cas9 system can be specifically the recombinant plasmid Cas9-HCss (+) mentioned in the examples. The recombinant plasmid Cas9-HCss (+) can be obtained by homologous recombination of oligo dimer methyl and Cas9/gRNA Vector. Cas9/gRNA Vector can be a component in a plant Cas9/gRNA plasmid construction kit. The plant Cas9/gRNA plasmid construction kit can be specifically a product of Beijing Weishanglide biotechnology and technology Limited, and the product number is VK 005-15. oligo dimer formazan can be synthesized from HCss (+) -F: 5'-TTGTCTTTAGCTATGTACATCCT-3' and HCss (+) -R: 5'-AACAGGATGTACATAGCTAAAGA-3' are annealed.
The vector containing CRISPR-Cas9 system can be specifically the recombinant plasmid Cas9-HCss (-) mentioned in the examples. The recombinant plasmid Cas9-HCss (-) can be specifically obtained by homologous recombination of oligo dimer B and the
The HC-Pro gene of soybean mosaic virus may specifically be at least one of D1) to D13); D1) HC-Pro gene of
The invention also protects the application of any CRISPR-Cas9 system in breeding soybean resisting soybean mosaic virus.
The invention also protects the application of any CRISPR-Cas9 system in improving the resistance of soybean to soybean mosaic virus.
The application of any of the above methods or any of the above CRISPR-Cas9 systems in soybean breeding also belongs to the protection scope of the present invention.
The invention takes SMV HC-Pro gene as target gene, multiple nucleotide sequence comparison is carried out on SMV epidemic strains at home and abroad, gRNA target sequence is determined in a conservative interval, CRISPR/Cas9 genome fixed-point editing vector is constructed, agrobacterium tumefaciens is further applied to mediate a genetic transformation system of soybean cotyledonary node to obtain a batch of positive transgenic material, and T is researched from phenotype level, RNA level and protein level respectively through SMV disease-resistant identification, qRT-PCR and DAS-ELISA serological experiments1And (5) screening a batch of resistant soybean materials according to the disease resistance of the soybean plant to the SMV. The invention has important theoretical significance and practical value in the research of the soybean SMV resistance mechanism and the breeding for disease resistance. The invention has important application value.
Drawings
Figure 1 is a multiple nucleotide sequence homology alignment of SMV sense strand genomes.
FIG. 2 is a multiple nucleotide sequence homology alignment of SMV minus-strand genomes.
FIG. 3 shows PCR amplification of E.coli.
FIG. 4 is T0And (3) herbicide application identification of the transgenic soybean.
FIG. 5 is T0PCR detection of transgenic soybean.
FIG. 6 is T0And (3) carrying out PAT/bar transgenic detection test paper identification on the transgenic soybean.
FIG. 7 is T1Development of SMV leaf disease symptoms in transgenic soybean.
FIG. 8 is T1The development symptom of the SMV strain of the transgenic soybean is generated.
FIG. 9 is T1qRT-PCR partitioning of transgenic Soybean generationsAnd (6) analyzing.
FIG. 10 is T1DAS-ELISA analysis of transgenic soybean.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
2 × Taq MasterMix is a product of Beijing Tiangen Biochemical technology Ltd. The plant Cas9/gRNA plasmid construction kit is a product of Beijing Weishanglide biotechnology limited, and the product number is VK 005-15. Cas9/gRNA Vector,
LB liquid medium: adding 10g of tryptone, 5g of yeast extract and 30g of sodium chloride into a proper amount of distilled water, fully dissolving, adjusting the pH to 8.0, and then adding distilled water to a constant volume of 1L; autoclaving at 121 deg.C for 15 min.
LB solid medium: adding 10g of tryptone, 5g of yeast extract and 30g of sodium chloride into a proper amount of distilled water, fully dissolving, adjusting the pH to 8.0, then adding 15g of agar, and fixing the volume to 1L by using distilled water; autoclaving at 121 deg.C for 15 min.
LB resistant plates: adding kanamycin to the LB solid culture medium with the concentration of 50mg/L in the system when the temperature of the LB solid culture medium is about 55 ℃, uniformly mixing, pouring into a culture dish, and naturally cooling.
The soybean variety Jack is a product of a national crop germplasm resource bank and is numbered WDD 01579. The soybean variety Jack is an SMV susceptible variety.
The soybean varieties Nannong 1138-2 and SMV SC3 are provided by the national soybean improvement center of Nanjing agriculture university.
The nucleotide sequences of the primers referred to in the examples below are shown in Table 1.
TABLE 1
Primer name
Primer sequence (5'→ 3')
dpCas9-F
TTGGGGCTCACACCAAACTT
dpCas9-R
CGATCGCCTTCTTTTGCTCG
bar-F
CGAGACAAGCACGGTCAACTT
bar-R
AAACCCACGTCATGCCAGTTC
Cas9-TY
CGTTGACTCCCCCGTAGGTT
HCss(+)-F
TTGTCTTTAGCTATGTACATCCT
HCss(+)-R
AACAGGATGTACATAGCTAAAGA
HCss(-)-F
TTGCATGTGGAGAATCATGAATG
HCss(-)-R
AACCATTCATGATTCTCCACATG
Note: specific recombination sites are underlined.
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