A method for obtaining high oleic acid cotton by applying gene editing technology
A gene editing, high oleic acid technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problems of insufficient genetic stability, incomplete gene silencing, and easy weakening of the effect of offspring silencing, so as to increase the oleic acid content and reduce the The effect of linoleic acid content
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Embodiment 1
[0025] Example 1: Construction of CRISPR / Cas9 gene editing vector
[0026] 1. gRNA target selection
[0027] The cotton FAD2 genes (Gh_A13G1850 and Gh_D13G2238) were used as target genes, and the targets screened by the online software CRISPR-Pv2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / ) were selected, and the target sites sgRNA1 and sgRNA2 were selected. Cotton FAD2 gene contains two conserved domains, DUF3474 (PF11960) and FA_desaturase (PF00487), the target sgRNA1 is located in the DUF3474 domain, and its PAM sequence is CCA, while the target sgRNA2 is located in the FA_desaturase domain, and its PAM sequence is CCG ;
[0028] The sgRNA1 sequence is: 5'-CCATTCCGCCCCACTGTTTTCGC-3';
[0029] The sgRNA2 sequence is: 5'-CCGTCACCACTCGAACACCGGTT-3'.
[0030]2. Construction of dual-target CRISPR / Cas9 gene editing vector
[0031] Adapter primers for sgRNA1 and sgRNA2: Synthesize and construct vector-related primers according to the designed target sites, see Table 1:
[0032] Tab...
Embodiment 2
[0038] Example 2: Acquisition of gene-edited cotton plants
[0039] 1. Preparation of sterile receptor material
[0040] The seeds of the upland cotton variety Huamian No. 1 (HM1) were used as materials, and the seed shells were peeled off after delinting with sulfuric acid, sterilized with 70% ethanol solution for 1 minute, and then soaked in 0.1% mercuric chloride (HgCl). 2 ) sterilized for 15 minutes, rinsed with sterile water for 5 times, inoculated on the seed germination medium, and incubated in the dark at 22-28°C for 3-5 days before use.
[0041] The seed germination medium was: 1 / 2 MS medium macroelements + 15 g / L glucose + 2.5 g / L Phytagel, pH 5.8-6.0.
[0042] 2. Culture of donor Agrobacterium GV3101
[0043] Agrobacterium tumefaciens GV3101 carrying dual-target sgRNA1 and sgRNA2 was inoculated on LB solid medium plates and cultured in the dark at 28°C for 36-48 hours. Use a sterile toothpick to pick a single colony with good growth, inoculate it into LB liquid m...
Embodiment 3
[0056] Example 3: PCR detection of transgenic cotton plants
[0057] 1. DNA extraction from transgenic cotton plants
[0058] The "Polysaccharide and Polyphenol Plant Genome DNA Extraction Kit (spin column type)" of Tiangen Biochemical Technology (Beijing) Co., Ltd. was used to extract T. 0 DNA in the leaves of transgenic cotton. Specifically include the following steps:
[0059] (1) Cut 80-100 mg of fresh young leaves of each transgenic cotton plant and wild-type cotton (HM1) respectively, immediately add liquid nitrogen to fully grind, and transfer the ground powder to a numbered 1.5ml centrifuge tube;
[0060] (2) Quickly add 400 μl of buffer GPS and 10 μl of RNase A (10 mg / ml), vortex and mix quickly, place the centrifuge tube in a 65°C water bath for 15 minutes, invert the centrifuge tube 5 times during the water bath to better mix the sample ;
[0061] (3) Add 100 μl of buffer GPA, vortex for 1 min, centrifuge at 12,000 rpm for 5 min, transfer the supernatant to the ...
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