Cry1Ac基因载体构建及其在枯草芽孢杆菌中的杀虫活性表达

根据苏云金芽孢杆菌Bacillus thuringiensis HD-73基因Cry1Ac和枯草芽孢杆菌Bacillus subtilis木糖诱导型启动子PxylR序列, 分别设计2对特异引物Cry1Ac F/R和Pxy F/R,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Pxy F/Cry1Ac R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。

Construction of recombined plasmid carrying Cry 1Ac gene and expression of insecticidal activities in Bacillus subtilis

 The full length sequence of the promoter and Cry1Ac gene were obtained by PCR with two pairs of unique primers Cry1Ac F/R and Pxy F/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.