新型非编码RNA Vvrr1参与溶藻弧菌毒力调控的机制研究

Because of the huge economic losses caused by Vibrio alginolyticus, the molecular mechanism underlying pathogenesis of V. alginolyticus has raised increasing attention. Resent research found that non-coding RNAs (ncRNAs) are important post-transcriptional regulators in bacteria and have been demonstrated to participate in most of the cellular processes. We previously found a new ncRNA, which was named as Vvrr1 (Vibrio virulence regulatory RNA 1). Vvrr1 was predicted to regulate several virulence genes. Over-expression of Vvrr1 could significantly reduce the pathogenecity of V. alginolyticus. The present study will investigate the molecular mechanism underlying regulation of V. alginolyticus virulence by Vvrr1. Firstly, iTRAQ will be performed to compare the expression of proteins in the wild type, △Vvrr1 and Vvrr1+, in order to predict the target genes of Vvrr1. Then, QPCR and Northern blot will be used to verify the target genes. After that, stable gene silencing will be performed to determine the effect of these target genes on V. alginolyticus virulence. Finally, EMSA and GFP reporter gene analysis will be performed to investigate the molecular mechanism underlying regulation of the target genes by Vvrr1. Our results would help to further understand the role of ncRNA in regulating the virulence of bacteria and the mechanism underling V. alginolyticus infection.

溶藻弧菌是海水养殖中最常见的致病菌之一，给我国的海水养殖业带来巨大危害。近来研究发现非编码RNA（non-coding RNA，ncRNA）广泛存在于细菌，并具有多种调控功能，对其研究越来越受到关注。本课题组前期在溶藻弧菌中发现一种可能调控多种毒力基因的新型ncRNA——Vvrr1，过表达Vvrr1可以显著降低溶藻弧菌的毒力。本项目将在此基础上，围绕Vvrr1调控溶藻弧菌毒力的机理开展研究。首先采用比较蛋白组学的方法筛选Vvrr1调控毒力的靶基因，并通过QPCR和Northern blot验证Vvrr1对靶基因的调控；然后检测这些靶基因的稳定沉默株对溶藻弧菌毒力的影响；最后通过GFP报告基因分析技术、EMSA等方法分析Vvrr1调控靶基因的机制，达到阐明Vvrr1调控溶藻弧菌毒力的机理的目的。本项目将增进对ncRNA在细菌毒力中的作用的认识，加深对溶藻弧菌致病机理的了解。

溶藻弧菌是海洋养殖环境中最常见的条件致病菌之一，给我国水产养殖业带来了巨大的经济损失。本课题组早期通过转录组测序分析，发现了一种具有毒力调控作用的溶藻弧菌非编码RNA Vvrr1。本研究通过构建Vvrr1过表达菌株并进行蛋白质组学分析，发现多种与毒力相关的Vvrr1潜在靶基因，其中pykF及leuO在Vvrr1参与的溶藻弧菌毒力调控机制中发挥着重要的枢纽作用。此外，我们不仅利用GFP报告基因分析技术明确了Vvrr1与pykF的作用方式，还利用转录组测序及Chip-seq预测、筛选了LeuO靶基因及其靶标ncRNA，为Vvrr1、pykF、leuO参与调控溶藻弧菌毒力的机制提供理论基础。通过以上研究，我们在溶藻弧菌中总结出两种Vvrr1参与毒力调控的可能模式：（1） 以“Vvrr1-pykF”为主轴的与粘附相关的毒力调控模式。其中Vvrr1通过直接调控pykF，间接调控pykF相关基因（eno、sdhB、glpF、cysH、ndk）来参与调控溶藻弧菌粘附的毒力机制。（2） 以“Vvrr1-leuO-LeuO靶基因/靶标ncRNA-BF通路基因”为主轴的与生物成膜相关的毒力调控模式。其中Vvrr1通过直接负调控leuO，进而间接调控LeuO靶基因或靶标ncRNA及BF通路基因的表达水平来参与调控溶藻弧菌生物成膜通路中的多种代谢及信号传导过程。本研究有助于进一步了解溶藻弧菌中由ncRNA和基因构成的毒力调控网络，为该菌病害的防治提供参考。受本课题经费资助，目前本课题发表SCI学术论文4篇，其中在Emerging Microbes & Infections上发表论文1篇。