Construction Of Engineering Bt Strain HD8Ea2 And Impact Of Cry8Ea2 On Midgut Microorganisms Of Holotrichia Oblita

Having damage to agriculture, forestry, pastured and other at the stage of larvae, Holotrichia oblita belongs to coleopteran of Scarabaeoidae. Holotrichia oblita is of widely distribution, large population and serious harm, and is a important plant pest in China.As a Gram-positive bacterium Bacillus thuringiensis can produce a variety of insecticidal crystal proteins in its life cycle, and is one of the most effective and widely used microbial insecticides at present. In this study, a wild-type strain named GW11 haboring cry8 gene was screened from soil samples collected from diffent areas of China. Insecticidal activity of GW11 to H. oblita was tested, and the LC₅₀ is 6.43×10⁶ cell/mL. Bt Cry8Ea2 protein is a new type of insecticidal crystal protein. cry8Ea2 was cloned into E. coli-Bt shuttle vector pSXY422b, resulting recombinant pScry8Ea2, and pScry8Ea2 was transformed into the Bt acrystalliferous mutant, generating engineered Bt strain HD8Ea2. Cry8Ea2 protein was successfully expressed in HD8Ea2 strain. By molecular sieve chromatography, 130 kDa of toxin protein Cry8Ea2 was purified. Using improved bioassay method, activity of the Cry8Ea2 toxin was evaluated, and the LC₅₀ value to first instars larvae of H. oblita was 0.2679μg/mL.H. oblita was normally feed in control group, and was feeded Cry8Ea2 protein in treatment group. Midgut microorganisms was analyzed by 16S rDNA gene amplification and PCR-RFLP analysis, 300 clones harboring 16S rDNA of bacteria from each group larvae were got, and 24 clones were randomly selected to perform PCR-RFLP analysis. 17 PCR-RFLP polymorphic bands appeared in control group, and 5 PCR-RFLP polymorphic bands in treatment group.