Construction of ethylene-insensitive cotton, and applications of ethylene-insensitive cotton in verticillium wilt resistance
A technology for cotton verticillium wilt and ethylene, which is applied in the field of plant genetic engineering, can solve problems such as the lack of clear conclusions about the effect of ethylene, and achieve the effects of weakening sensitivity, increasing cotton yield and reducing disease incidence.
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Embodiment 1
[0042] Example 1: Construction and Transformation of Overexpression Vectors
[0043] In order to verify whether AtCTR1 has a regulatory function after being transferred into cotton, the applicant obtained an overexpression vector by constructing it, and then transformed cotton hypocotyls.
[0044] Design overexpression primers according to the ORF of AtCTR1 obtained in the "Summary of the Invention" of this specification, that is, add SacI and XbalI restriction sites at both ends of the primers respectively, and the primers are respectively named AtCTR1oe-F and AtCTR1oe-R, and then The plasmid of the positive clone was used as a template for PCR amplification, and the obtained PCR product was constructed on the pCAMBIA2300S vector by enzyme digestion and ligation reaction, and 2 μl of the reaction product was transferred into E. coli Top10 by heat shock method, and finally the recovered bacterial liquid was Apply to a solid LB dish containing k+ resistance, overnight at 37°C. ...
Embodiment 4
[0080] Example 4: Inoculation identification of Verticillium dahliae in transgenic material cotton
[0081] 1. Seedling cultivation in greenhouse
[0082] Select the seeds of the cotton line YZ1 with full grains and the seeds of transgenic cotton, peel off the cotton seed hulls, sterilize with 0.1% mercury chloride for 10 minutes, rinse with sterile water three times, and inoculate on the sterile seedling medium (formulation: MS 25ml of macronutrients + 15g of glucose + 2.5g of phytagel, supplemented with distilled water to 1L), after 24-36 hours of dark culture at 28°C, insert the radicle into the culture medium, turn to light culture, set the temperature at 28°C, and light Time 16h.
[0083] 2. Activation and cultivation of Verticillium dahliae V991 in cotton
[0084] Put Verticillium dahliae V991 on a PDA plate (recipe: take 200g potatoes, wash, peel and chop, add 1000mL of water and boil for 15min, filter with gauze, add 15g of glucose and 7g of agar, filter with gauze w...
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