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

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

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