Abstract
Bacillus thuringiensis (Bt) is the most widely applied type of microbial pesticide due to its high specificity and environmental safety. The activity of Bt is largely attributed to the insecticidal crystal protein encoded by the cry genes. Different insecticidal crystal proteins of Bt have different bioactivity against distinct agricultural insect pests, and combination of these proteins not only increases insecticidal activity, but also overcomes and delays development of resistance. A Bt strain, S185, was isolated from a soil sample collected in Songfeng Shan district, Heilongjiang Province, China. Bt S185 has highly specific insecticidal activity against Coleoptera, and was determined to contain cry8 -type genes by peptide mess fingerprint (PMF) analysis. Application of polymerase chain reaction-restricted fragment length polymorphisms (PCR-RFLP) analysis further determined the genotype due to the high homology of cry8Ea1 and cry8Fa1 genes. Through the full-length primers design, two insecticidal crystal protein genes cry8Ca and cry8Ea were obtained. Using prokaryotic cloning vectors, the recombinant plasmids pEB-cry8Ca and pEB-cry8Ea were transferred into expression host strain Escherichia coli Rosetta, thus the two genes were successfully expressed in heterologous bacteria. Keywords: Bacillus thuringiensis , peptide mess fingerprint, identification, clone, insecticidal crystal protein. African Journal of Biotechnology , Vol 13(25) 2484-2490
Highlights
Bacillus thuringiensis (Bt) is a gram-positive soil bacterium characterized by its ability to produce crystalline inclusions named cry or cyt during sporulation (Höfte and Whiteley, 1989)
The present study describes the identification, cloning and expression of cry8Ca and cry8Ea from a new indigenous isolate of Bt S185 strain, which has high-virulence against Holotrichia parallela
The Bt strain S185 used in this study was isolated from soil obtained from Songfeng Shan district, Heilongjiang Province, China
Summary
Northeast Agricultural University, HarBin 150030, People’s Republic of China. State Key Laboratory of Biology for Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094, People’s Republic of China. The activity of Bt is largely attributed to the insecticidal crystal protein encoded by the cry genes. Different insecticidal crystal proteins of Bt have different bioactivity against distinct agricultural insect pests, and combination of these proteins increases insecticidal activity, and overcomes and delays development of resistance. Bt S185 has highly specific insecticidal activity against Coleoptera, and was determined to contain cry8-type genes by peptide mess fingerprint (PMF) analysis. Application of polymerase chain reaction-restricted fragment length polymorphisms (PCR-RFLP) analysis further determined the genotype due to the high homology of cry8Ea1 and cry8Fa1 genes. Through the full-length primers design, two insecticidal crystal protein genes cry8Ca and cry8Ea were obtained.
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