苏云金芽胞杆菌bkd基因簇的转录调控

Transcriptional regulation of bkd gene cluster in Bacillus thuringiensis

摘要:

摘要:【目的】通过分析苏云金芽胞杆菌bkd 基因簇的转录调控和bkdR突变体的表型特征，明确bkdR所在基因簇的转录调控机制和对Cry蛋白产量的影响。【方法】通过生物信息学方法分析bkdR所在基因簇的结构，RT-PCR分析基因簇的转录单元，采用同源重组技术敲除苏云金芽胞杆菌HD73菌株的bkdR基因，利用启动子融合lacZ的方法分析启动子的转录活性。利用总蛋白定量确定Cry1Ac蛋白产量。【结果】bkd基因簇由8个基因组成，其中ptb-bkdB7个基因组成1个转录单元。ptb基因的启动子转录活性在sigL和bkdR突 变体中均明显降低。bkdR基因的缺失对菌体生长、芽胞形成率和Cry1Ac蛋白产量无影响，但使运动能力减弱。【结论】bkd操纵子受Sigma 54控制，并由BkdR激活，bkdR基因的缺失对Cry蛋白产量无影响，对菌株的运动能力有影响。

Abstract:

Abstract:［Objective］In order to determine the effect of bkdR deletion on Cry protein production．We analyzed the transcriptional regulation of bkd gene cluster and the phenotype of bkdR mutant．［Methods］Sequence of bkd gene cluster in Bacillus thuringiensis was analyzed by sequence alignment．RT-PCRwas used to reveal the transcriptional units of the bkd gene cluster．bkdR insertion mutant was constructed by homologous recombination．Transcriptional activity was analyzed by promoter fusions with lacZ gene．Comparison of the Cry1Ac protein production was determined by protein quantitation．［Results］The bkd gene cluster was composed of eight genes．The ptb-bkdB formed one transcriptional unit．The transcriptional activity of ptb sharply decreased in sigL and bkdR mutants．Deletion of bkdR decreased the motility of cells，but no effect on growth，sporulation efficiency and Cry protein production．The bkd gene cluster is controlled by Sigma 54 and activated by BkdR． Deletion of bkdR has no effect on Cry protein production，but decreased the motility of the cells．［Conclusion］The bkd gene cluster is controlled by Sigma 54 and activated by BkdR． Deletion of bkdＲ has no effect on Cry protein production，but decreased the motility of the cells．It suggested that deletion of bkdR do not affect the Cry protein production the same as sigL mutant．It means decreasing of Cry protein productioninsigL mutant was not caused by only one EBP mutation，but might be multiple roles．