中文摘要：

【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。

英文摘要：

[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.