Isolation Of Bacillus Thuringiensis Strains From Qian Moutain And Cloning Of Cry Genes

Bacillus thuringiensis(Bt) is mieroorganism pestieides with high speeifieity and safety for the environment. It had been extensively researched and applied in the past 30 years. Primarily insecticidal activity and its carried coding ICPs (Insecticidal Crystal Proteins, ICPs) of Bacillus thuringiensis cry genes closely related, therefore, in recent years cry genetic engineering research has became a hot research. The international community has 446 species of rodent proteincloning of genes, but with Bt applications have been expanding and Bt ICPs genes to varying degrees, by the resistance of insects, finding a new strain of high-drug efforts and genes is one of the effective ways of resolving insect resistance.In this study,a method was developed to isolate Bacillus thuringiensis from the soil samples colleeted from the three differedt places in qianshan of Liaoning province.Twenty-two Bt strains were isolated from 225 samples and the proortionwas 9.8%,there were bipyramidal and spheroealerystals. Twenty-six pairs of universal primers were used to identify cry gene-tapes of 22 Bacillus thuringiensis isolates use the method of PCR.Six isolates harbored the cry1 gene,produeed 130kDa; Two isolates harbored the cry7 gene,produeed 130kD.We found a strain QZG121 may contain cry7 genes. Cry7 have been reported that have active on Coleoptera,Lepidoptera and Orthoptera pests. Through PMD18-T vector cloning, nucleotide sequencing revealed and NCBI BLAST that the strains contains cry7Ab gene. Cry7Ab full-length gene sequences based on the design of specific primers, successfully cloned the gene. Sequence analysis results showed that the gene encoding 3417 bp, software translation 1138 amino acid residues, molecular weight of 130kD, isoelectric point pI value of 5.03 for the weak acid protein. The gene was registered in GenBank with accession number FJ716102 and was designated as a novel gene, cry7Ab9 by International Nomenclature Committee of Bt.Imultaneously,according to the published sequence of cry1Ac gene, a pair of special primers Q5UNX/QU3UNX was designed for full ength DNA cloning of cry1Ac gene in Q12 strain. The gene was designated as a novel gene,cry1Ac28,by International Nomenclature Committee of Bt. Sequence analysis results showed that the open reading frame of the cry1Ac gene was 3537 bp which coded 1178 amino acids and its molecular weight was 134kD.The cloning of new cry gene,not only has added a new member for cry genetic family,but also offered more genetic materials for constructing the new transgenic project fungus and transgenic plant.In order to examine the expression of the gene in E.coli,cry7Ab9 and cry1Ac28 was inserted into the expression vector pEB respectively.The reeombinant Plasmid,respectively designated pEB-cry7Ab and pEB-cry1Ac,was transferred into E. coli BL21(DE3). SDS-PAGE analysis showed that both cry7Ab9 and cry1Ac28 gene were expressed successfully.