溶藻弧菌菌落相变关键小RNA的鉴定和调控机制研究

Vibrio alginolyticus is one of the predominant pathogens of marine aquaculture in South China Sea. Previous study has found that the pathogen underwent colony phase variation between opaque and translucent. The two variants showed significant difference in the basic biochemical characteristics such as biofilm formation, motility and flagella biosynthesis, utilization of nitrogen and carbon resources, resistance to antibiotics and stress response as well. Genetic analysis has also revealed that complex signal transduction and genetic regulatory network including global regulators ValR, Rpos and ToxR are all involved in the phenomenon. However, the non-coding RNA (ncRNA) Hfq and associated small RNAs have been indicated to be the key factor in determination of the colony morphology. Deletion of hfq led to the change of the colony morphology, attenuation of the virulence, reduction of biofilm formation and stress responsibility, indicating critical roles of small RNAs in colony phase variation and associated cellular processes. To understand the mechanism, this project will first identify the small RNAs of V. alginolyticus and expression profiles by sequencing and comparing the transcriptoms of the wild type strains and the hfq mutants. The selected small RNAs will be confirmed and characterized by experimental approach as gfp-fusion expression and deletion. The essential small RNAs involving in conoly phase variation will be selected and their regulatory targets will be discolsed and confirmed by EMSA. This project will promote the study on the adaptation of V. alginolyticus and may shed lights on the function of small RNAs in the vibios.

溶藻弧菌是南方海域养殖动物弧菌病的主要病原之一，前期研究中我们发现菌落相变是决定其环境和宿主适应性的重要机制，不同菌落形态的菌株间生物膜形成能力、鞭毛合成与运动能力、碳源和氮源利用效率、对抗生素和胁迫条件反应能力等呈现显著差异，遗传分析结果表明菌落相变涉及复杂的信号传导和基因调控网络，其中非编码RNA（ncRNA）分子伴侣hfq及相关小RNA可能是控制此现象的决定因子,hfq基因缺失突变后改变了菌落形态，导致致病力消失，影响生物膜形成、物质转运与利用、抗胁迫能力等关键生理生化途径，显示小RNA对溶藻弧菌基本生命过程，尤其是对环境适应性和致病力等方面的重要调控作用。本项目拟在此基础上，通过转录组测序和生物信息分析鉴定溶藻弧菌小RNA及其表达特征；通过基因敲除和回补筛选出调控菌落相变的关键小RNA；通过gfp融合表达、凝胶阻滞等技术阐明其功能和调控方式，从而揭示小RNA在海洋病原弧菌环境适应性

溶藻弧菌(Vibrio alginolyticus)是海水起源的常见致病菌，感染宿主十分广泛，包括鱼类、贝类、甲壳类等几乎所有养殖动物，对人的临床感染也屡见报道，是我国南方海水养殖产业和海鲜食品安全行业威胁最严重的病原菌之一；非编码RNA（ncRNA）或小RNA（sRNA）是指一类位于基因间区或反义链上不编码蛋白但具有在翻译水平上调控基因表达功能的小分子RNA，越来越多的研究表明小RNA对细菌遗传变异、生长繁殖、致病机制等具有重要的调控作用，但是弧菌小RNA的鉴定、功能及其调控机制鲜有报道。本研究分析了两个不同菌落形态的溶藻弧菌（ZJT和ZJO）背景中RNA分子伴侣Hfq突变菌株与野生株的表型与全基因表达差异；通过转录组分析初步筛选出小RNA组；通过Northern Blotting，RACE，融合表达和引物延伸等实验手段验证了多个关键小RNA；通过突变，生理生化表型分析和转录组分析等技术结合，阐明了小RNA ZGL-3083在Hfq的协助下调控丙氨酸脱氢酶的表达，从而参与丙氨酸的代谢；而另外一个小RNAmisc_RNA2157则调控下游支链氨基酸合成相关基因的表达，从而参与支链氨基酸的合成，而且缺失突变后还影响了菌落形态变化。同时我们分析了两个菌相菌株的全基因组，鉴定了4个与菌落变化相关的基因位点，并分析了2个多糖相关位点的表型功能；对两个关键小RNA的调控机制进行了详细分析，初步阐明其调控的目标基因。项目共发表论文10篇，申请专利8项，培养研究生6名。通过项目研究，在国内建立了比较成熟的小RNA研究平台和技术，为阐明溶藻弧菌的环境适应机制打下了基础，也为深入研究弧菌病害的暴发的分子机制开拓了一个新的领域。