Department of Microbiology, College of Life Sciences, Nankai University, Tianjin 300071, People's Republic of China.
Curr Microbiol. 2012 May;64(5):492-500. doi: 10.1007/s00284-012-0100-0. Epub 2012 Feb 25.
The expression and application of Bacillus thuringiensis (Bt) chitinase genes have been extensively investigated. However, little information is available regarding the regulation of chitinase gene expression in Bt. In this study, a shuttle promoter-probe vector was constructed incorporating the thermostable β-galactosidase gene bgaB of B. stearothermophilus as the reporter for the study of Bt promoters. Using this plasmid, the activity of the chiA gene promoter in Bt was investigated. Deletion analysis of the putative chiA promoter region revealed that the sequence located ~75 bp DNA from positions -116 to -42, with respect to the translation start site, is the core promoter of chiA gene. Furthermore, a site for chitin induction was identified near position -36. This site for negative regulation was indicated downstream of the RNA polymerase binding sites of the promoter of chiA. The expression of chiA started in cell grown for about 6 h and reached the maximum after 60 h of incubation. Induction of chiA expression by chitin was demonstrated by an increase in β-galactosidase activity of ~2.5-fold.
苏云金芽孢杆菌（Bt）几丁质酶基因的表达和应用已经得到了广泛的研究。然而，关于 Bt 中几丁质酶基因表达的调控，信息却很少。在本研究中，构建了一个穿梭启动子-探针载体，其中包含嗜热脂肪芽孢杆菌的热稳定β-半乳糖苷酶基因 bgaB 作为报告基因，用于研究 Bt 启动子。利用该质粒，研究了 Bt 中 chiA 基因启动子的活性。chiA 启动子区的缺失分析表明，与翻译起始位点相对应的-116 至-42 位置的约 75 bp DNA 序列是 chiA 基因的核心启动子。此外，还在-36 位置附近鉴定到了一个几丁质诱导位点。该负调控位点位于 chiA 启动子的 RNA 聚合酶结合位点的下游。chiA 的表达在细胞生长约 6 小时后开始，并在孵育 60 小时后达到最大值。几丁质诱导 chiA 表达的结果表现为β-半乳糖苷酶活性增加约 2.5 倍。
