Studies On Vip3A Genes From Bacillus Thuringiensis And Construction Of Genetically Engineered Strains Against Insect Pests And Plant Pathogens

The novel vegetative insecticidal protein (VIPs) are secreted outside the cell during Bacillus thuringiensis (Bt) mid-logarithmic growth. VIP3A shows activity against many lepidopteran insect larvae. It exerts insecticidal properties on susceptible insect cells by triggering an apoptotic-type of programmed cell death(PCD).This mechanism is different from that of insecticidal crystal proteins(ICPs).Bt strains kept in the lab were screened for the \ip3A genes using PCR amplification and 30 strains were found to contain the vip3A genes. The vip3A gene amplified from wild-type Bt strains WB5 with high level of activity against lepidopteran bisect larvae was cloned into pMD18-T vector. The Vip3A-WB5 deduced amino acid sequence showed more than 90% high degree of identity with other Vip3A of Bt. Moreover, it showed 98% identity with CBM-4-9, a carbohydrate binding domain. With the signal peptide analysis software of the TMHMM2.0(http://genome.cbs.dtu.dk/services /TMHMM-2.0), the preceding peptide of Vip3A-WB5 was a transmembrane peptide. With PredictProtein software online from Colombia University for structure analysis, it showed that 28.96% helix, 25.69% sheet, and 45.35% loop in secondary structure of Vip3A-WB5. The whole Vip3A-WB5 protein appeared as compact, as a globular domain in tertiary structure.The expression plasmid pETvip-WB5 was constructed by insertion of vip3A gene (vip3A-WB5) into the expression vector pET29a(+) , and then was transformed into E. coli BL21. E. coli BL21 cells harboring the pETvip-WB5 plasmid was induced with Immol/L IPTG to express 89kDa protein. Bioassay showed that the Vip3A protein had high toxicity against 2nd instar larvae of S. litura. Experiments showed that Vip3A-WB5 proteins were almost soluble and didn't formed inclusion bodies which could be observed by transmission electronmicroscopy(TEM). The target protein was purified under the native condition and the polyclonal was prepared by immunizing rabbits.vip3A -WB5 gene was cloned into Bacillus subtilis secretive expression vector pUS186. The hybrid plasmid was transformed into endophytic bacterium Bacillus subtilis BS2, endophytic biocontrol bacterium. The results showed that BS2 and engineered strain had in vitro Inhibitory activity to phytopathogen of fungi. Inoculated by dipping seeds, BS2 and engineered strain were proved to be able to colonize in the cabbage plant. The growth of cabbage was significantly promoted by WB5 . BS2 and engineered strain. Compare with the water control, the fresh weight of the 15d old cabbage was increased 155.89%. 158.54% and 145.27% respectively tested in the pot.