Identification And Cloning Of Cry Genes On 20kb DNA In Crystalline Inclusion From Bacillus Thuringiensis Strain 4.0718

Bacillus thuringiensis strain 4.0718 produces two different kinds of crystalline inclusions with forms of bipyramidal and cuboidal. They are composed of 130kD Cryl and 65kD Cry2 protoxin respectively. On the basis of PCR-RFLP strategy which has proven to be a powerful tool for identification of the specific insecticidal genes carried by different Bt strains, the err-type genes on the 20kb DNA fragment were determined. Subsequently, an PCR method was applied to amplify coding sequence of crylAa and crylAc genes on the 20kb DNA fragment. Then 3.5kb PCR product encoding CrylAa protoxin was cloned into pUCm-T for further studies.1. Selective solubiIization of crystalline inclusions from Bt strain 4.0718 and extraction of 20kb DNACrystalline inclusions from Bt strain 4.0718 were solubilized selectively under different conditions. Then the obtained protoxin-DNA complex was monitered by agarose gel electrophoresis and SDS-PAGE. Experimental result showed that cryl and Cry2 protoxin were associated with 20kb DNA. The condition of solubilization required by bipyramidal crystalline inclusions was more gentle compared with that of cuboidal crystalline inclusion. Consequently, 20kb DNA in association with Cryl protoxin remained intact while DNA fragment associated with Cry2 protoxin was partially broken down by being boiled. The extraction of DNA from Cryl protoxin was achieved by addition of phenol/chloroform at 65 癈 previously equilibrated to pHlO.5. The target 20kb DNA was recovered effectively from agarose gel by electroelution.2. Identification of cry-type genesTwo pairs of universal oligonucleotide primers to probe the most conserved regions of all known cryl and cry2 genes were chosen to analyse the nucleotide sequence of 20kb DNA preliminarily. The DNA fragment was positive for cryl and cry2 genes and copies of cryl gene were lower than that of crj2gene. Then the positive DNA fragment was further probed with a set of specific primers and subject to PCR-RFLP analysis which was known to be a facile method to detect both known and novel cry genes. The tested DNA template produced the predicted sizes of PCR fragment with the sizes of 1. 6kb for cryl genes and 1. 2kb for cry2 genes. The quantity of PCR product of 1. 6kb was more than that of PCR product of 1.2kb. The restriction fragment length polymorphism patterns of the PCR-amplified fragments revealed that 20kb DNA contains crylAa crylAc cry2Aa and cry2Ab genes. DNA sequencing results of 1. 6kb PCR product were consistent with the results of RFLP pattern analysis.3. Cloning of crylAa gene from 20kb DNAAfter the sequence of N-terminal region and C-terminal region from crylAa and crylAc were amplified successfully, another pair of primers was designed to amplify the coding sequence of crylAa and crylAc gene. The 3. 5kb PCR product was cloned into pUCm-T vector, and then transformed into E. coli. DH5 a . PCR-RFLP analysis and enzyme digestion results demonstrated that the selected positive transformant clone contained 3.5kb PCR product for crylAa gene.