Cotton gene editing method based on gene gun mediation and application
A gene editing and gene gun technology, applied in the field of plant genetic engineering, can solve the problems of cell reproduction and development hazards, troublesome suspension culture operation, labor and time consumption, etc., so as to shorten the genetic transformation cycle, improve the efficiency of gene editing, and save time. and workload effects
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Embodiment 1
[0055] Embodiment 1: optimize the induction system of embryogenic callus;
[0056] Cut the hypocotyls of sterile seedlings cultured in the seedling medium for 7 days into 1.0-1.5cm long stem segments, place the stem segments on the embryogenic callus induction medium for embryogenic callus induction culture, and subculture for 4 weeks during the period Once, replace with new medium until embryogenic callus appears. Pick the newly induced embryogenic callus and subculture on the new medium. After about 2 weeks, when the embryogenic callus turns into a beige rice-grained callus, select the embryogenic callus with the same callus state as the bombardment of the gene gun The explants of 0.2g embryogenic callus were bombarded once;
[0057] The tissue culture conditions for sterile seedlings and embryogenic callus culture are as follows: temperature is 28±2°C, photoperiod is day / night 16h / 8h, air humidity is 40%-60%, light intensity is 12000-15000Lx.
Embodiment 2
[0058] Embodiment 2: Construction of sgRNA-pKSE401 carrier;
[0059] Log in http: / / crispr.hzau.edu.cn / CRISPR / Website, enter the target gene sequence into the corresponding sequence box, search for high-scoring targets and evaluate off-target situations; after selecting the target sequence, design primers according to the following rules:
[0060] oligo-F: 5'-ATTGNNNNNNNNNNNNNNNNNNNN-3'; (N is the first 19nt sequence of the PAM sequence)
[0061] oligo-R: 5'-AAACNNNNNNNNNNNNNNNNNNNN-3'; (N is the reverse complementary sequence of the first 19nt sequence of the PAM sequence)
[0062] Mix the above two oligonucleotide strands (100 μmol / L each), heat at 95°C for 5 minutes, then cool to room temperature to form a double-stranded insert, and construct the vector according to the following system (see Table 2); the reaction conditions are : 5h at 37°C, 5min at 50°C, 10min at 80°C; transform 5 μL of the above mixture into competent E. coli, and select positive clones on an LB plat...
Embodiment 3
[0072] Embodiment 3: establish gene gun transformation method system;
[0073] 1) Preparation of plasmid DNA: Streak and activate the preserved sgRNA-pKSE401 positive Escherichia coli liquid, pick a small clone, then absorb a small amount of bacterial liquid to expand the culture, and use the plasmid mini-extraction kit (Magen, Guangzhou) to extract the plasmid .
[0074] 2) Biolistic bombardment conditions: the embryogenic callus was subcultured on a hyperosmotic medium for 4 hours, and used for gene bombardment transformation. Embedding steps of gene editing plasmid DNA: prepare 60 mg / mL gold powder; draw 25 μL of thoroughly vortexed gold powder suspension, draw 10 μL sterile ultrapure water, add 6 μl (3 μg) gene editing plasmid DNA (500ng / uL ), and mix by pipetting. Add 50 μL of 2.5M calcium chloride dihydrate, then add 20 μL of 3mg / mL protamine, quickly mix and vortex for 3 minutes, centrifuge for 30 seconds, remove the supernatant, add 200 μL of absolute ethanol, vortex...
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