| Summary: | 碩士 === 國立陽明大學 === 生物化學研究所 === 89 === Bacillus thuringiensis (Bt) is an endospore-forming bacterium characterized by the presence of a crystal protein within the cytoplasm of sporulating cell. The proteins within this crystal are toxic to insects. Most importantly, toxicity of Cry protein are highly specific. Thus the application of Cry protein does not cause any harmful effect on human being other than target insects, which explains the extensive use of Bt as a biological insecticide.
It was known that endogenous proteases in Bt might degrade Cry protein. Stability and yield of Cry protein could be improved by deletion of specific protease from Bt. In order to identify these protease, we designed five pairs of degenerated primer for serine and neutral protease genes. Sequencing of PCR product reveals several protease gene fragments.
Disrupting these genes individually by homologous recombination, interestingly, we found that one mutant refereed to ispI (intracellular serine protease) deficient showed delay phenomenon on sporulation. Proteomics approach has been utilized to explain this observation. After IEF and SDS-PAGE separation, we compared the protein spots of the wild type with ispI mutant and differences of protein expression between them were found. Interesting protein spots were picked for further was found to be sigma H, which is a subunit of RNA polymerase holoenzyme for the transcription of several genes related to the initiation of sporulation. This data suggests that IspI may have direct or indirect processing on sigma H during the initiation of sporulation. Probably, this may explain the sporulation delay phenomenon of ispI mutant. In addition, this experimental approach implied that Proteomics is a feasible way to characterize the function of a gene.
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