Cloning And Expression Of Cry7Ab8 Gene And Construction Of An Genetic Engineering Strain From Bacillus Thuringiensis

Bacillus thuringiensis is one of the most widely used insecticidal microbe. The toxicity resulted mainly from the insecticidal crystal protein formed during sporulation process. Cloning of insecticidal novel cry genes to pest from Bacillus thuringiensis isolates will be beneficial for appilication of pesticidal gene resources and for constrction of genetically engineered strain and transgenic plants, and will lessen the gap between domestic and abroad.Using the temperature screening method, 7 Bacillus thuringiensis isolates were obtained from 208 soil samples collected from some locations of Hebei Province. The microscope examination and SDS-PAGE results indicated that most of them expressed 130kDa and 60kDa insecticidal crystal proteins. PCR method was used to determine genotype of 7 Bacillus Thuringienses strains. The GWS7 strain showed the cry7-type gene. The gene cry7 used as a research target.A cry7-type gene from Bt GWS7 strain was identified by full-length PCR method. The cry7Ab8 gene was amplified and cloned into pET28b. The recombinant plasmid pETcry7Ab8 was transformed into E.coli BL21(DE3), resulting BL21(pETcry7Ab8). The SDS-PAGE results showed that the 130kDa Cry7Ab8 protein was expressed. An engineering strain BioHD7Ab8 was construced by transforming the shuttle vector pSXY422b containing cry7Ab8 into Bt acrystalliferous mutant HD73-(cry-). The bioassay results indicated that the Cry7Ab8 protein was highly toxic against Henosepilachna vigintioctomaculata second-instar larvae, and the LC50 value was 548μg/mL.The part cry7Ab8 gene(Δcry7Ab8) was amplified by PCR with a pair of primersΔF7Ab/ΔR7Ab.TheΔcry7Ab8 gene was amplified and cloned into pET28b. The recombinant plasmid pETΔcry7Ab8 was transformed into E.coli BL21(DE3), resulting BL21(pETΔcry7Ab8). The SDS-PAGE results showed that the 56kDaΔCry7Ab8 protein was expressed. An engineering strain BioHDΔ7Ab8 was construced by transforming the shuttle vector pSXY422b containingΔcry7Ab8 into Bt acrystalliferous mutant HD73-(cry-). The crystal shape do not form from BioHDΔ7Ab8. The bioassay results indicated that theΔCry7Ab8 protein was no toxic against Henosepilachna vigintioctomaculata second-instar larvae. We reported that the domainⅢand C-end from Cry7Ab8 have palyed an important role in the activity of toxins and the crystal shape .