Cloning And Expression Of A Novel Gene Cry9Ea7 From Bacillus Thuringiensis

Insecticidal Crystal Proteins (ICPs) from Bacillus thuringiensis (Bt) are great resources for biocontrol of insect pests. Identification of cry genes encoding ICPs is of great significance to the discovery of novel genes with specific bioactivity. In this study, a cry9-type gene from Bt strain Bt4 was identified by PCR-RFLP method, and subsequently a novel gene was cloned from Bt strain 4 by PCR technology. The results of sequencing and structural analysis revealed that the gene, named cry9Ea7 by International delta-endotoxin nomenclature committee of delta-endotoxin (GenBank Accession number: FJ380927),was 3453bp and Its open reading frame encoded a 130kDa protein with 1150 amino acids,and the gene is similarity of 98%-99% to compare with other cry9Ea genes.An engineering strain E.coli BL21 (cry9Ea7) was constructed by transformation of a recombinant expression plasmid pETcry9Ea into E.coli BL21 (DE3). By IPTG induction, analysis of SDS-PAGE showed that the cry9Ea7 gene was expressed in the engineering strain. Furthermore, another engineering strain, namely BioHD9Ea7, was constructed by insection of the cry9Ea7 gene into a shuttle vector pSXY422b, and by electrol transforntion of the recombinant plasmid into a crystalliferous mutant Bt HD73-(cry-). Finally, the results of SDS-PAGE and Western blot analyses showed that the cry9Ea7 gene was expressed in BioHD9Ea7 strain, too. We didn't find that there was significant difference in comparison with the growth rate between the engineering strain and the host strain. Bioassay indicated that the Cry9Ea7 toxic protein appeared high insecticidal activity against Trichoplusia ni neonates, and the LC50 value was 0.044μg/mL. However, the toxic protein showed no activity against Spodoptera exigua and Helicoverpa armigera neonates.