Construction of an engineering strain expressing cry7Ab7 gene cloned from Bacillus thuringiensis

Abstract

The genotype of Bacillus thuringiensis (Bt)strain GW6 isolated in China was identified, and a novel cry7Ab gene was found based on the results of PCRs using 40 pairs of cry universal primers. The cry7Ab gene was cloned by PCR and named as cry7Ab7 by the Bt Delta Endotoxin Nomenclature Committee (GenBank Accession No. FJ940776). The construction of a novel three-dimensional structure of Cry7Ab7 protein via homology modeling methods indicated that the Cry7Ab7 protein was different from other Cry7Ab proteins within the domain structures. The cry7Ab7 gene was inserted into the BamHI/SalI sites of E. coli expression vector pET21b and the Bt-E. coli shuttle vector pSXY422b to construct the recombinant plasmids pET21b-7Ab7 and pSXY422b-7Ab7, which were then transformed into E. coli BL21 and Bt HD73cry in order to obtain the E. coli transformant EC7Ab7 and the engineering strain Bt HD7AB, respectively. The bioassay results showed that Cry7Ab7 protein inclusion bodies in EC7Ab7 and the crystal proteins in HD7AB were highly toxic to the second-instar larvae of Henosepilachna vigintioctomaculata with the LC₅₀ values of 1.167 μg·μL^−1 and 0.779 μg·μL^−1, respectively. Our results clearly indicated that cry7Ab7 gene can offer important benefits to research on Coccinellidae insect pest control.