基于CRISPRi系统的IncF型质粒接合转移调控元件和抑制技术研究

细菌获得耐药性已成为危及食品安全和人畜健康的棘手问题，其耐药获得机制及抑制技术的研究对保障人畜安全具有重大意义。本研究以肠杆菌科中普遍存在的IncF型多重耐药质粒为研究对象，探讨其转移调控机制和抑制方法。前期转录组学数据分析发现，核糖转运和趋化受体蛋白RbsB的低水平表达下IncF型质粒接合转移效率显著降低。本研究拟建立野生型IncF型耐药质粒模型，构建高效特异的CRISPRi基因敲降系统和利用抗生素诱导，分析rbsB基因的不同转录水平与接合转移效率的关系，运用EMSA、DNaseI足迹、SPRi、氨基酸定点突变和细菌双杂交系统等确定RbsB蛋白与关键调控元件TraJ蛋白和tra操纵子启动子相互作用关系，结合能力和关键结合位点，解析RbsB蛋白对质粒接合转移调控机制。本课题将将拓宽对质粒接合转移调控元件及其相互作用的认识，为建立干预基因水平转移的耐药细菌控制技术提供理论依据和技术基础。

The acquisition of drug resistance by bacteria has become a thorny problem that endangers food safety, human and animal health. In this study, we will study the transfer regulation mechanism and inhibition technology of IncF type multidrug-resistant plasmids, which are most commonly found in Enterobacteriaceae. On the basis of previous studies on transcriptomics, at the low expression level of RbsB, the conjugation efficiency of IncF plasmid was significantly reduced. In this study, we will use wild type IncF resistant plasmid as the model, analyze the relationship of different transcription level of rbsB gene with the efficiency of conjugation by constructing CRISPRi gene knock down system and utilizing antibiotics for induction. Electrophoretic mobility shift assay, DNaseI footprint, surface plasmon resonance analysis imaging system, amino acid fixed-point mutation and bacterial double hybrid system are used to illustrate the interaction, binding ability and key binding sites of RbsB with the key modulation elements, TraJ and promoter of tra operons, and the regulatory mechanism of RbsB on conjugation. This study will broaden the understanding of transfer regulatory elements of plasmid conjugation and their interactions, and provide theoretical and technical basis for establishing the control technology of drug-resistant bacteria by intervention gene horizontal transfer.