苏云金芽胞杆菌3-羟基丁酮代谢基因簇的转录调控

Transcriptional regulation of aco gene cluster in Bacillus thuringiensis

【目的】通过分析苏云金芽胞杆菌(Bacillus thuringiensis)3-羟基丁酮代谢基因簇aco的转录调控和acoR突变体的表型特征,明确aco基因簇的转录调控机制和对芽胞产量及Cry蛋白产量的影响。【方法】通过生物信息学方法分析aco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acoR基因,利用启动子融合lac Z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】aco基因簇由aco ABCL 4个基因组成,形成一个转录单元。aco基因簇的启动子Paco A转录活性在sig L(编码Sigma 54因子)和acoR突变体中均明显降低。acoR基因的缺失对菌体生长和Cry1Ac蛋白产量无显著影响,但使菌体运动能力减弱,使芽胞产量略有下降,并且不能利用3-羟基丁酮。【结论】aco操纵子受Sigma 54控制,并由AcoR激活,aocR基因的缺失影响菌体对3-羟基丁酮的利用,但对Cry蛋白产量无显著影响。

[ Objective] We analyzed the transcriptional regulation of aco gene cluster and the phenotype of acoR mutant, to determine the effect of acoR deletion on sporulation efficiency and Cry protein production. [ Methods] Sequence of aco gene cluster in Bacillus thuringiensis was analyzed by sequence alignment. RT-PCR was carried out to reveal the transcriptional units of the aco gene cluster, acoR insertion mutant was constructed by homologous recombination. Transcriptional activity was analyzed by promoter fusions with lacZ gene. Comparison of the Cryl Ac protein production was determined by protein quantitation. [ Results] The aco gene cluster was composed of four genes. The acoABCL formed one transcriptional unit. The transcriptional activity of acoA promoter sharply decreased in sigL and acoR mutants, respectively. Deletion of acoR had no effect on growth and Cry protein production, but decreased the motility of cells and sporulation efficiency. [ Conclusion ] The aco gene cluster is controlled by Sigma 54 and activated by AcoR. Deletion of acoR has no effect on Cry protein production, but decreased the motility of the cells.