苏云金芽胞杆菌CcpA蛋白对几丁质酶基因chiA和chiB的表达调控作用

Regulation effect of CcpA on chitinase gene chiA and chiB in Bacillus thuringiensis

摘要:

【目的】研究苏云金芽胞杆菌Bti75中糖代谢蛋白CcpA对两种几丁质酶基因chiA和chiB的表达调控。【方法】利用PREDetector软件分析Bti75 chiA和chiB的基因上游区序列，EMSA方法在体外验证CcpA是否能与chiA和chiB基因的启动子区域片段特异性结合。构建ccpA基因敲除载体以获得敲除突变株ΔccpA，运用实时荧光定量PCR技术和western blot技术比较有无葡萄糖存在的情况下，CcpA对chiA和chiB基因表达的影响。【结果】计算机分析显示，chiB上游启动子区存在一个潜在的CcpA结合位点crechiB，而chiA上游启动子区未发现类似序列。体外实验表明，CcpA蛋白在共阻遏蛋白Hpr-Ser45-P的参与下可与chiB基因启动子区特异性结合，而与chiA基因启动子区没有特异性结合；实时荧光定量PCR和western blot结果均显示，Bti75中ccpA基因敲除后，同样在葡萄糖存在下chiB的表达量提高而chiA的表达量变化不明显。【结论】在葡萄糖存在的情况下，CcpA蛋白能抑制苏云金芽胞杆菌中几丁质酶chiB的表达，而chiA的表达不受CcpA调控。

Abstract:

[Objective] Purpose of this work was to research the regulation effect of catabolite control protein A (CcpA) on chitinase gene chiA and chiB in Bacillus thuringiensis subsp. israelensis 75 (Bti75). [Methods] We used the PREDetector software program to analyze the upstream regulatory region of chiA and chiB in Bti75, in addition, specific binding of CcpA protein to the promoter region of chiA and chiB was determined by electrophoretic mobility shift assay (EMSA). In order to acquire ccpA deletion mutant ΔccpA, we constructed a ccpA knockout vector. Then, the influence of CcpA to chiA and chiB in Bti75 strains with and without glucose was detected by quantitative real-time reverse transcription PCR (qRT-PCR) and Western blot. [Results] PREDetector software program analysis showed that there is a potential CcpA binding site crechiB in the promoter region of the chiB, while the similar site is not found in the chiA promoter region. In vitro, CcpA could bind specifically to the chiB promoter with the assist of Hpr-Ser45-P, but the binding of CcpA to chiA promoter is non-specific. The results of qRT-PCR and western blot revealed that ccpA deletion led to a significantly increased expression of chiB, however, the change of chiA expression was not obvious. [Conclusion] The expression of chiB not chiA in Bti75 was negative controlled by CcpA in the presence of glucose.