迟缓爱德华氏菌毒力相关非编码小RNA的鉴定

Edwardsiella tarda(E.tarda) is a common and important pathogen of fish, which causes enormous economic losses to the world-wide aquaculture industry. The fundamental pathogenic mechanism of E.tarda still remains to be discovered. In recent years significant experimental and theoretical evidence suggests small non-coding RNAs (sRNAs) can coordinate virulence gene regulations and pathogen survival during E.tarda infecting the host. At the same time, sRNAs are the key of connecting environment factors with spatial and temporal expression of virulence gene. To date, little is known about the role of RNA-mediated regulation in E. tarda. And the potential roles of sRNAs during E. tarda infecting Japanese flounder have not been investigated. Here we will first systematically identify sRNAs in E. tarda genome by bioinformatics-based prediction methods. And E.tarda sRNAs will experimentally be confirmed by quantitative RT-PCR and Northern blot. And the potential targets of the sRNAs，whose expression differs significantly during E.tarda infecting Japanese flounder, will be identified by bioinformatics prediction,co-immunoprecipitation assay and gel retardation assay. The sRNAs of target genes coding virulence factors are candicates,in which we will choose 1-2 sRNAs for detailed investigation to define the underlying mechanism leading to virulence gene expression at molecular level. Examination of sRNAs function in regulating virulence at molecular level will be preliminarily performed by overexpression and inhibition of sRNAs in E. tarda LSE40. Our results will provide insight into the knowledge on pathogenesis of E. tarda and provide theoretical basis for prevention and treatment of edwardsiellosis.

迟缓爱德华氏菌（Edwardsiella tarda，E.tarda）是水产养殖鱼类的重要致病菌，给水产养殖业造成了巨大损失。目前对E.tarda致病机制的研究还不很透彻。近年来，研究人员发现，作为致病菌毒力调控网络中新的种类，非编码小RNA（small non-coding RNA,sRNA）极有可能是联系环境因素和毒力基因时空表达调控的纽带。本课题通过生物信息学从E.tarda基因组中预测sRNA基因，通过实验验证从中鉴定出E. tarda感染牙鲆过程中表达显著差异的sRNA；通过生物信息学预测并验证上述sRNA的靶基因，从中选出靶基因编码对象为毒力因子的sRNA;选定上述1-2个sRNA；通过sRNA体外过表达或抑制表达，初步探讨其调控毒力的分子机制。本研究有助于深入理解E.tarda的致病机理，为迟缓爱德华氏病的防治提供理论基础。

迟缓爱德华氏菌（Edwardsiella tarda，简写E.tarda）是水产养殖鱼类的重要致病菌，给水产养殖业造成了巨大损失。目前对E.tarda致病机制的研究还不很透彻。近年来，研究人员发现，作为致病菌毒力调控网络中新的种类，非编码小RNA（small non-coding RNA，sRNA）极有可能是联系环境因素和毒力基因时空表达调控的纽带。本课题对E.tarda sRNA的研究取得如下进展：（1）通过两次sRNA测序，结合生物信息学预测，获得了19个E.tarda S01sRNA，其中只有3个（ET_sRNA_1，ET_sRNA_2和ET_sRNA_10）是在别的致病菌中报道过的，其余都是新发现的sRNA。（2）通过RNA测序、Northern blot和quantitative PCR（qPCR）对获得的部分sRNA进行了验证。（3）通过qPCR对19个sRNA在菌体生长过程中的变化进行了跟踪，发现其中18个sRNA在对数生长期和稳定期表达，属于生长时期依赖型；而其中一个sRNA的变化趋势明显不同于其他，初步认为这个sRNA可能为管家sRNA。（4）采用TargetRNA2预测sRNA的靶标，其中大部分sRNA的靶标基因表达产物跟毒性直接或间接有关系。（5）采用无标记基因敲除方法，通过构建含缺陷型sRNA基因的自杀质粒pRE112，进行两次同源重组交换从而得到E.tarda S01 △ET_sRNA_1和E.tarda S01 △ET_sRNA_8突变株和互补株。两突变株和其各自互补株在菌落形态上没有明显区别。利用构建的两个sRNA缺失突变株进行sRNA对牙鲆致病性影响的实验正在进行中。本研究为E.tarda的致病机理的研究提供了新的思路，为迟缓爱德华氏病的防治提供理论。