Abstract:

 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.

Key words: Bacillus thuringiensis, Bacillus subtilis, overlapping PCR, Cry1Acgene, insecticidal activity