ermC核糖开关调控大环内酯类抗生素耐药基因表达研究

诱导性耐药是细菌产生耐药性的主要原因之一。大环内酯类抗生素诱导大量耐药基因的表达，如红霉素诱导甲基化酶ERMC表达使细菌产生高水平耐药。有研究表明甲基化酶的表达与核糖体停靠有关，但核糖体停靠理论仍不能解释抗性基因的诱导表达机制。课 题组前期工作表明，ERMC的诱导表达与ermC基因 5'端前导序列RNA 结构变化相关，证实此RNA为核糖开关。本课题拟构建报告基因表达系统，通过平板扩散实验、lacZ 酶活性、实时荧光定量PCR，检测ermC 核糖开关在细菌体内对耐药基因表达的调控作用；通过非变性凝胶电泳、表面等离子共振SPR、SHAPE、in-line probing、DMS等技术及突变分析明确大环内酯类抗生素的结合位点，建立ermC核糖开关结构变化模型。通过研究核糖开关模型调控大环内酯类抗生素耐药基因表达，为抗生素新靶点的研发提供新的思路。

Antibiotic resistance of bacterium commonly occurs through induction by the antibiotics. The macrolide antibiotics can induce high level of expressions in a large number of resistance genes, such as erythromycin methylase ERMC. It's reported that ribosomal stalling controls the expression of ERMC. However, the induction mechanism remains unclear.Our previous work showed that induction of ERMC expression is related to the RNA structure of 5'-UTR. We therefore confirmed that this RNA could be a novel riboswitch. This study intends to construct the reporter gene expression system in vivo by disc-diffusion method,lacZ activity and real-time PCR, and verify the riboswitch biochemically by surface plasmon resonance(SPR), selective 2-hydroxyl acylation analyzed by primer extension(SHAPE), in-line probing，DMS and mutational analysis. We will show that macrolides bind to ermC riboswitch and cause a significant structural transition in the conformation of the RNA and thus induce the expression of ERMC. We will propose a model of riboswitch which regulates the expression of the macrolide resistance genes. Understanding the mechanism of inducible macrolide antibiotic resistance would provide newways for developing new antibiotic targets.