Expression Of Insect-resistant Gene In Pichia And Genetic Analysis Of The Gene In The Hybrid Cotton Lines G75

Pichia pastoris, which is excellent expression system, has been used extensively and successfully to express heterologous proteins. In this study, We used site-directed mutagenesis through PCR technology to change Pstâ… restriction site in the rear of Bt CrylA gene for facilitating the construction of expression vector. Then we successfully constructed secreted recombinant gene expression vector pPIC9KVBA and pPIC9KVBCA expressing Bt and Bt-CpTI protein respectively. And we obtained the recombinant yeast GS115/pPIC9KVBA, GS115/pPIC9KVBCA by screening the expression of Bt and Bt-CpTI proteins. The detection results by Bt test strip and SDS-PAGE demonstrated the yeasts had expressed the proteins successfully. In addition, Insecticidal activities showed that Bt and Bt-CpTI proteins expressing in Pichia are toxical to the bollworms.Cotton has obvious hybrid advantages. Excellent hybrid cotton can increase the yield significantly than conventional cotton. At the same time, It can improve the quality of the fiber and resistance to the insect, so it is great significant to develop insect-resistant hybrid cotton for the realization of cotton's high-yield, stability, high efficiency. In this paper, We made molecular detection and analysis of agronomic characters in three-line hybrid cotton G75 and its parent. The results were as follows: PCR detected Bt gene of the cotton G75 in 23 lines, all the results were positive; Southern blot was a good result by optimizing conditions, which showed that the Bt Cry1A gene was 3~4 copy numbers of the hybrid cotton G75, and their parents with the same copy number; ELISA results showed that the content of Bt toxic protein in the top leaves of hybrid cotton lines G75 exhibit difference at different stages, Bt protein expression of G75 in the early and post growth stages was significantly higher than the parents, lower during bud stage and boll stage. The G75 of agronomic traits in plant height and branch number were closer to paternal Y18, performing higher plant height and more fruit branch number. Heterobeltiosis displayed in bolls per plant and lint percentage, the both parameter was related to the seed cotton yield directly. Thus seed cotton production of the G75 increased significantly to parents. Its seed cotton yield increased 13.3% than the control cotton LU-15 production. Although Heterosis of cotton G75 in fiber quality was weaker, it showed mid-parent heterosis meeting the requirements of the textile.