QsvR调控副溶血弧菌生物膜形成的分子机制研究

副溶血弧菌的生物膜形成需要一些特殊的结构（如鞭毛、胞外多糖等）和信号通路（如c-di-GMP）的参与，且是一个被紧密调控的过程。转录调控子QsvR对副溶血弧菌生物膜形成的调控作用受胞内是否表达OpaR的影响，但是机制未被阐明。本项目拟系统分析野生株、qsvR突变株、qsvR和opaR的双基因突变株，突变株的回补株等的生物膜相关表型（如结晶紫染色、菌落褶皱、透明度表型等）的差异；进而，采用RNA-seq、ChIP-seq、凝胶阻滞实验等，鉴定不同遗传背景及生物膜形成条件下，QsvR的最小调控元；最后，从最小调控元中挑选出与生物膜形成密切相关的基因或基因座位中各操纵子的首基因为研究靶标，采用LacZ报告基因融合实验、实时定量RT-PCR等分子调控实验技术，深入探究不同遗传背景下，QsvR对靶基因转录的精细分子调控机制，从而认识QsvR是如何通过调控相关基因的转录而调控副溶血弧菌生物膜的形成。

The biofilm formation of Vibrio parahaemolyticus is a complicated process involving many specific structures (such as flagella, pili and extracellular polysaccharides) and regulatory processes (such as c-di-GMP and various regulators). QsvR-dependent biofilm formation in V. parahaemolyticus has been shown to be affected by the intracellular concentration of OpaR, but lacks the detailed regulatory mechanisms. In this study, the biofilm-related phenotypes (such as the crystal violet staining, colony morphology, and opaque-translucent colony switching) will be systematically characterized for the wild-type, qsvR mutant, double-gene deletion mutants of qsvR and opaR, and complementory mutants of the pandemic V. parahaemolyticus. Further, the RNA-seq, ChIP-seq, and gel shift assays will be employed to identify the minimum QsvR regulon in different genetic backgrounds under the growth conditions of biofilm formation. The biofilm-related genes within the minimum QsvR regulon will be selected as the target genes. The classic molecular experiments such as the LacZ fusion and real-time quantitative PCR will be performed to investigate the molecular regulation mechanisms of QsvR on the target genes under different genetic backgrounds. The final combined phenotypic and molecular dataset will promote us to understand how QsvR regulate the biofilm formation through synergistically regulating the transcription of specific gene loci in V. parahaemolyticus.