Study On Constructing Engineered Bacillus Thuringiensis Of Efficiently Expressing Tobacco Chitinase Gene TchiB And Insecticidal Activity

Bacillus thuringinsis is a kind of environment friendly microbiology agriculture pestcide, which widely used for biological control of farming and forestry pests. The research about Bt engineered strains in domestic and overseas all focused on getting optimized strains which have more better charactetities such as expanding insecticidal spectrum and high toxicity. It was reported that chitinase could increase the insecticidal activity of insecticidal crystal protein markedly by destorying the peritriphic menbrane.We employed Red/ET homologous recombination technology, putting the tobacco Chitinase gene between the cry2Aa promoter gene and cryl Ac terminator gene, to obtain mosaic gene pcry2Aa-tchiB-1Act. Then cloning pcry2Aa-tchiB-1Act gene into shuttle vecter pHT315, constructed recombination plasmid pHTtchiB. Electricing tansform pHTtchiB into Bt subsp.kurstaki HD73strain, got engineered strain HDtchiB. By means of SDS analysis and detection, we found hetero-logous gene tchiB could efficiently expressed under the control of cry2Aa strong promoter, and the protein were further identified by mass spectrometry. The insecticidal activity of the engineered strain shows higher toxicity to Helicoverpa armigera larvae compared with that of original strain HD73.Heterologous gene always is unstabitily or even losed in Bt eng in-eered strains. In order to keep it stability, it is a effective way to integrate heterologous gene into the chrosome of B. thuringinsis. The Chitinase gene located on the chromosome of B. thuringinsis was choosed as the integration site for heterologous gene, then an integrative plasmid pKCHIUN was constructed by cloning the homologous arms into a temperature-sensitive E.coli-Bt shutter vector pKSV7. Employing Red/ET homologous recombination technology constructed a integrative vector pKTCHIUN contained mosaic gene pcry2Aa-tchiB-1Act.Via Red/ET homologous recombination technology constructed the shuttle expression vector pHTtchiB and intergrative vector pKTCHIUN successfully, and then achieved to construct the B.thuringinsis engineered strain HDtchiB that could efficiently express the heterologous gene, which lay an important foundation for studying the exogeneous insecticidal genes that have synergistic effect into B.thuringinsis, and the constructing of engineered strains with superior quality.