Method for breeding soybean mosaic virus (SMV) resistant soybeans
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A soybean mosaic virus and soybean technology, applied in the biological field, can solve problems such as improvement of resistance to soybean mosaic virus disease that are not involved
Inactive Publication Date: 2020-03-20
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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However, none of the above studies involved the improvement of soybean mosaic virus disease resistance
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Embodiment 1
[0047] Embodiment 1, SMV genome multiple nucleotide sequence alignment and determination of gRNA target
[0048] 1. Search for the full genomes of 13 SMV epidemic strains at home and abroad (the names and corresponding Genebank accession numbers of the 13 SMV epidemic strains are shown in Table 2) on the website of the National Center for Biotechnology Information (NCBI) sequence, and then utilize ClustalX 1.83 software to carry out multiple nucleotide sequence homology comparisons to the genomes of the SMV sense strand and the negative sense strand, and determine that the conserved interval of the SMVHC-Pro gene is 2039-2309nt (see figure 1 ) and 8373-8530nt (see figure 2 ), and then searched for the PAM site and determined the gRNA(+) target sequence with a similarity of 100% (see figure 1 ) and gRNA(-) target sequence (see figure 2 ).
[0049] gRNA (+) target sequence: 5'-AGGATGTACATAGCTAAAGA-3' (sequence 1 in the sequence listing).
[0050] gRNA (-) target sequence: ...
Embodiment 2
[0054] Example 2, T 1 Obtaining transgenic soybean and identification of SMV resistance at phenotype level, RNA level and protein level
[0055] 1. Obtaining recombinant Agrobacterium
[0056] 1. Take Microtube, add 5 μL HCss(+)-F aqueous solution (concentration is 10 μM), 5 μL HCss(+)-R aqueous solution (concentration is 10 μM) and 15 μL Autoclaved ddH 2 O, a mixed solution was obtained.
[0057] 2. After completing step 1, place the Microtube in a metal bath for annealing to obtain a reaction solution. The reaction liquid contains oligo dimer.
[0058] Annealing procedure: 95°C for 3 minutes, slowly cool at 95°C to 25°C (decrease about 3°C per minute), and 16°C for 5 minutes.
[0059] 3. After completing step 2, take another new Microtube, add 1 μL of the reaction solution obtained in step (2), 1 μL of Cas9 / gRNA Vector, 1 μL of Solution 1, 1 μL of Solution 2 and 6 μL of Autoclaved ddH 2 O, mix well.
[0060] 4. After step 3 is completed, the Microtube is placed in a ...
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Abstract
The invention discloses a method for breeding soybean mosaic virus (SMV) resistant soybeans. According to the method, an SMV HC-Pro gene serves as a target gene, domestic and international SMV popularplant lines are subjected to multiple nucleotide sequence comparison, a gRNA target sequence is determined in a conservative interval, a fixed-point edited vector of a CRISPR / Cas9 genome is constructed, a batch of positive transgenic materials are obtained through mediating a genetic transformation system of soybean cotyledonary nodes by further applying Agrobacterium, disease-resistant conditions of T1 soybean plants to SMV are researched separately from a phenotype level, an RNA level and a protein level through SMV disease-resistant identification, qRT-PCR and DAS-ELISA serological experiments, and then, a batch of resistant soybean materials are screened out. The method has an important theoretical significance and a practical value in researches on SMV resistant mechanisms of soybeans and disease-resistant breeding. The method has an important application value.
Description
technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for cultivating soybeans resistant to soybean mosaic virus. Background technique [0002] Soybean mosaic virus disease (SMV) caused by soybean mosaic virus (SMV) is a global soybean disease, which is widely distributed in soybean production areas in the world and seriously affects the yield and quality of soybean. SMV belongs to the Potyviridae family, the Potyvirus genus, and the soybean mosaic virus species. After soybeans are infected with SMV, symptoms such as mosaic and necrosis will occur, resulting in reduced photosynthetic area and photosynthetic capacity of soybeans, dwarfing of plants, decreased growth, brown spots on grains, resulting in a loss of about 35%-50% of soybean yield, or even a failure to harvest. . [0003] CRISPR / Cas9 technology is an efficient, precise and specific genome-targeted modification technology that has emerged in recent years...
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