Cry1 Ac基因载体构建及其在枯草芽孢杆菌中的杀虫活性表达

Construction of recombined plasmid carrying Cry1Ac gene and expression of insecticidal activities in Bacillus subtilis

根据苏云金芽孢杆菌BacillusthuringiensisHD_73基因Cry1Ac和枯草芽孢杆菌Bacillussubtilis木糖诱导型启动子PxylR序列,分别设计2对特异引物Cry1AcFR和PxyFR,扩增获得了完整的启动子PxylR和Cry1Ac基因序列,进一步以上述产物混合物为模板,以PxyFCry1AcR作引物进行重迭PCR,获得了载体PxylR_Cry1Ac,经SphⅠ和BamHⅠ完全酶切后,将PxylR_Cry1Ac插入大肠杆菌_苏云金芽孢杆菌穿梭载体pHT315,重组表达质粒pCry1Ac315转化枯草芽孢杆菌感受态细胞。工程菌株质粒酶切电泳分析、SDS_PAGE电泳分析和杀虫生物活性测定结果证实了Cry1Ac基因的导入及其在枯草芽孢杆菌JAAS01D中的有效表达。

The full length sequence of the promoter and Cry1Ac gene were obtained by PCR with two pairs of unique primers Cry1AcF/R and PxyF/R respectively, which were designed according to the Cry1Ac gene and promoter sequence of xylase operon from Bacillus subtilis 168. Then, the fused translational expression vector PxylR_Cry1Ac was constructed using overlapping PCR technique with the primers pair PxyF/Cry1AcR and the mixture of above PCR production. After being digested by SphⅠ and BamHⅠ, PxylR_Cry1Ac expression vector was inserted into E. coli_B. thuringiensis shuttle vector pHT315, and the resulted recombinant plasmids were named as pCry1Ac315. The recombinant plasmids were transferred into B. subtilis laboratory strain JAAS01D. Efficient expression of the Cry1Ac gene in the engineered JAAS01D_1Ac was proved with restriction enzyme analysis, SDS_PAGE electrophoresis analysis and insecticidal activity assay.