苏云金芽胞杆菌rocE基因的转录调控

Transcription and regulation of rocE in Bacillus thuringiensis

摘要:

[目的] rocE基因编码精氨酸降解途径中的精氨酸通透酶，通过分析苏云金芽胞杆菌（Bacillus thuringiensis，Bt）rocE基因的转录活性，明确rocE基因的转录调控机制。[方法] 通过RT-PCR确定rocE基因所在基因簇的转录单元；β-半乳糖苷酶活性测定分析rocE基因启动子（ProcE）的转录活性；采用同源重组技术敲除Bt HD73菌株的rocE基因；通过融合His标签的方法在大肠杆菌中表达纯化RocR蛋白的HTH结构域；通过凝胶阻滞实验明确RocR与rocE基因启动子的结合作用。[结果] 在M9培养基中，精氨酸可诱导ProcE的转录活性；在SSM培养基和精氨酸诱导培养基中，与出发菌株HD73相比，ProcE在sigL（编码Sigma54因子）突变体和rocR突变体中的转录活性显著下降。RocR-HTH蛋白与ProcE有结合作用。rocE基因的缺失对菌体生长和Cry1Ac蛋白产量无显著影响。rocE缺失突变体的芽胞形成率为65.5%，HD73出发菌株为85.7%，显著性分析结果表明差异显著（P<0.05）。[结论] rocE基因的转录活性受Sigma54的控制，并受RocR正调控。rocE基因的缺失影响菌株的芽胞形成率。

Abstract:

[Objective] rocE encodes arginine permease in arginine degradation pathway. To determine the mechanism of transcriptional regulation of rocE, we analyzed the transcriptional activity of rocE in Bacillus thuringiensis (Bt). [Methods] Transcriptional units of rocE gene cluster were analyzed by RT-PCR. Transcriptional activity of rocE promoter (ProcE) was analyzed by β-galactosidase assay. rocE mutant of Bt HD73 strain was constructed by homologous recombination. The HTH domain of RocR with His fusion protein was purified by HiTrap chelating column. The binding of rocE promoter with RocR-HTH protein was verified by electrophoresis mobility shift assays. [Results] Transcriptional activity of ProcE was induced by arginine in M9 medium. The transcriptional activity of ProcE was sharply decreased in sigL (encodes Sigma 54) and rocR mutants compared with that in HD73 wild-type in Schaeffer's sporulation medium (SSM) and arginine induced medium. ProcE could bind to RocR-HTH protein. Mutation of rocE had no significant differences on growth and Cry1Ac protein production. However, the sporulation efficiency of the rocE mutant was 65.5%, and that of the HD73 strain was 85.7%. The results of significance analysis show that the difference was significant (P<0.05). [Conclusion] Transcriptional activation of rocE is controlled by Sigma 54, and positive regulated by RocR. rocE gene is related to sporulation efficiency.