Screening Of Bacillus Thuringiensis Strains With High Toxicity And Cloning, Expression And Insecticidal Activity Of Cry2Ac10 Gene

Bacillus thuringiensis (Bt) is microorganism pesticides with high specificity and safety for the environment. The cry genes of Bt strains also is most widely and potentially used against pest insects. This study mainly includes isolation of Bt stains from soil of Shandong Province, identification of cry gene-type of Bt strains and cloning, expression and insecticidal activity of cry gene from Bt strain QCL-1. The main contents are as follows:57 Bt strains were isolated from 750 soil samples from Shandong Province by selective medium (NGKG) method. Crystal shapes could be observed, such as bipyramidal, spherical and so on.The bioassay results showed that the corrected mortality of strains QCL-1, QCY-3, QJN-1, WRC-1 and WZ-1 were over 50% for Mythimna separata, while the corrected mortality of strains QCL-1, WRC-1 and HJY-1 were over 50% for Spodoptera exigua. Toxicity comparison results showed that strain QCL-1 was more toxicity than strain HD-1 to S. exigua, and as same as toxicity to M. separata and Trichoplusia ni.The SDS-PAGE analysis showed that proteins of 130 kDa and 70 kDa were expressed in Bt strain QCL-1. Results of cry-type gene identification indicated that the strain QCL-1 contained the cry1Cb, cry1Fa, cry1Fb, cry2Ab and unknown cry2A genes.The cry2A gene was amplified by designing special primers, FY2A5 and FY2A3, based on the plasmid of Bt strain QCL-1. The gene cry2Ac10 was cloned and identified as GenBank accession number No. EF405952. Alignment results showed that the cry2Ac was composed of 1872 base pairs, encoding 623 amino acids, which were homolog of 97.4～99.7% compared with published Cry2Ac.The recombinant vector, pET21b-cry2Ac, was constructed by control of T7 promotor in E.coli BL21(DE3). The cry2Ac10 gene was expressed as 70kDa protein induced by IPTG The insecticidal protein form transformant possessed high toxicity to the larvae of Helicoverpa armigera, M. separata. LC₅₀ of the protein was 30.0μg/g to H. armigera and 16.7μg/g to M. separata respectively.