Characterization of insecticidal Bacillus thuringiensis 1-3 strain and destruction of its sigG gene by homologous recombination

다국어 초록 (Multilingual Abstract)

A strain of Bacillus thuringiensis, named Bt 1-3, was isolated from Korean soil sample and it showed high insecticidal activity against Plutella xylostella. Bt 1-3 was deterimined to belong to ssp. aizawai (H7) by an H antiserum agglutination test and produced bipyramidal-shaped crystal proteins. PCR analysis with specific cry gene primers showed that Bt 1-3 contained cry1Aa, cry1Ab, cry1C, cry1D and cry2Ab genes. In addition, this isolate showed high uptake rate of foreign plasmid by electroporation. Based on these characteristics of Bt 1-3, we tried to construct a spore-free Bt 1-3 mutant by knock-out sigG gene, which is known as a key transcription factor during sporulation. First, we constructed a basal vector, named pDST, consisting of erythromycin resistant gene (EmR), partial polyhedrin gene and temperature sensitive origin of replication gene (Orits). Subsequently, according to the chromosomal DNA sequence of Bt subsp. konkukian 97-27, we amplifed upstream and downstream regions of Bt 1-3 sigG, and cloned into pDST (pDST-G). So far, several EmR colonies were obtained by electroporating into the wildtype Bt 1-3 and crossover by homologous recombination is going on.