Isolation And Identification Of Bacillus Thuringiensis And Cloning Of Insecticidal Genes

Bacillus thuringiensis (Bt) is a ubiquitous gram-positive bacterium.Its activity is attributed largely to the insecticidal crystal proteins (Insecticidal Crystal Proteins, ICPs) which is also called as delta-endotoxin. Isolation and identification of Bacillus thuringiensis and cloning of the insecticidal genes were performed in this work.41 Bt isolates were obtained from 71 soil samples collected from orchards in Hebei province using sodium acetate with antibiotics method. These Bt isolates were characterized based on their insecticidal activity, crystal protein shape,sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) result, and PCR analysis using novel general and specific primers for cry genes. Six types of cry gene, cry1, cry7, cry8, cry19, cry26-28 and cry32, were found in 39 Bt isolates. The cry genes in other two Bt strains were not identified. Most isolates showed a major protein band of 130 kDa in SDS–PAGE gel. The spore-crystal mixtures of XPZ-6, XPZ-8, XPZ-9, XPZ-39 and XPZ-42 strains all showed high toxicity against the larvae of Plutella xylostella, Helicoverpa armigera, Ostrinia furnacalis and Hyphantria cunea, and the insecticidal activities of XPZ-39 strain were highest (>97% mortalities). XPZ-28 strain showed insecticidal activities to larvae of Plagiodera versicolora and Gastrophysa atrocyanea though the cry gene was not identified.The 12 Bt strains containing cry8 genes were analysed using three methods by PCR-RFLP, plasmid profiles and protein molecular weight. The 11 strains contained the same gene of Bt crystal protein which was different to that in XPZ-46 strain. Therefore two novel cry8 gene were cloned and sequenced from XPZ-5 and XPZ-46 strain. The result of BLAST in NCBI showed XPZ-5 had cry8Ea1-like gene and XPZ-46 had cry8X-like gene, with 99% similarity to cry8Ea1 and cry8X, respectively. Two cloned genes are the fourth level of novel cry genes. The cry8Ea1and cry8X genes were expressed by pEB, a high-level prokaryotic expression vector developed by the Institute of Plant Protection of CAAS. The two genes were registered in GenBank with accession number HM021727 and HM021728. The bioassay results showed that the Cry8Ea1 and cry8X had no activity on the the leaf beetle Phaedon brassicae.vip1/vip2 and sip primers were designed by using the AlignX program. And two pairs of primer(vip1/vip2 and sip) were desigened acording the conserved block. By the primer(vip1/vip2 ), 9 Bt strains were containing vip1/vip2 gene were screened from 72 standard strains.6 Bt strains were containing vip1/vip2 gene were screened from 95 strains which have activity to Coleoptera.Their genes were cloned and sequenced. The result of BLAST in NCBI showed that these vip1/vip2-like genes had a identity of 78%-95% to the reported vip1/vip2 gene, respectively. Those genes are predicted the three-level novel vip genes. By the primer(sip)2 Bt strains were containing sip gene were screened from 167 strains.By ammonium sulfate precipitation, the Vegetative Insecticidal Proteins of 8 strains which have cry8 gene were prepaired and evaluate the toxicity to leaf beetle Phaedon brassicae, the results indicated that it have no activity against the Phaedon brassicae.