Identifying And Clonging The Cry30Ga2 Gene From B. Thuringiensis Isolate S2160-1

In this study, a novel cry30 gene was identified by the technique of PCR restriction fragment length polymorphism (PCR-RFLP) from B. thuringiensis S2160-1. Constructing the plasmid libraris of B. thuringiensis strain S2160-1 and the single-oligonucleotide nested-PCR (SON-PCR) method was employed for cloning the new insecticidal genes.We constructed three plasmid libraries of B. thuringiensis strain S2160-1. One of them which was the least library contains at least 1,800 clones. We extracted plasmid DNA of strain S2160-1. The plasmid was digested with BamH1 and Bglâ…¡. The fragments were inserted in pBU4 vector in vitro digested with BamH1 and then transformed into E.coli JM110. The recombinant plasmids were screened using blue and white, and detected using PCR-RFLP. We obtained a positive clone and digested with BamH1 and Sall. It failed to sequence.The single-oligonucleotide nested-PCR (SON-PCR) method has been employed for cloning the new insecticidal genes from the B. thuringiensis S2160-1. PCR was performed to produce a 1.4kb fragment by using plasmid DNA from Bt S2160-1as the template and using cry30 gene universal primers. The amplified fragments were cloned and sequenced. According to obtaining gene sequences of 5'-terminal and 3'-terminal, a pair of primers for full-length cry30 gene was designed. SON-PCR was performed by using plasmid DNA form Bt S2160-1 as the template. The production was cloned and sequenced. These sequences were assembled by the DNAstar. We obtained the cry30 full-length sequence. Blastx on the NCBI site ananlysis showed that the deduced amino acid sequence of PCR fragment had the highest identities with Cry30Ga1(>95%). The ORF was submitted to GenBank and assigned accession number:HQ638217; The cry gene was designated cry30Ga2 by the Bacillus thuringiensis Pesticide Crystal Protein Nomenclature Committee on the basis of the data obtained in this study. According to the open reading frames (ORF) of cry30Ga2, a pair of primers GF and GR was designed to obtain the full-length of cry30Ga2. Furthermore, the cry30Ga2 gene was expressed in Escherichia coli BL21 (DE3). SDS-PAGE analyzed the recombinant did not expressed the 75kDa protein. It was testify that the mutation of the insertion fragment did not occur by sequencing the E.coli. BL21(DE3)-30Ga2. Multiple sequence alignment showed that the Cry30Ga2 protein exhibited 95% similarity with Cry30Ga2 protein which protein larvicidal activity against Aedes aegypt. It was deduced that the Cry30Ga2 protein was toxic to A. aegyptii(Diptera). It was benefit of development of biological pesticides. A new cry gene was identified and cloned to provide a wealth of genenetic resources for the transgenic plants.