无乳链球菌sRNA筛选、鉴定及功能研究

Streptococcus agalactiae is one of the important causative agents of streptococcicosis that endangered the aquaculture of cultured tilapia in South China. The lethal ability of S. agalactiae to tilapia is regulated by sRNA (small RNA). We completed the sequences of genome from S. agalactiae strain ZQ0910, and predicted some sRNA with the bioinformatical protocol in recent study. Based on the results of the previous study, we hope to screen, identify sRNA and study the prelimary function of sRNA which can regulate the virulence in S. agalactiae by RNA deep sequence, Northern blotting, gene knockout and two-dimensional difference gel electrophoresis (2D-DIGE) technique. The research proposal is as following: 1) Screening and identifying the candidates of sRNA obtained from the RNA deep sequence; 2) Cloning the complete sequences of sRNA and analyzing the sites of transcriptional start and stop；3）Analyzing the distribution S. agalactiae-sRNA in the other bactera and the regulated target DNA sequence of the genome；4) Constructing the knockout mutant strain of the different sRNA, observing the growing status of recombinant strain and original strain in the different meduim, pH, metal ion etc, and measuring the virulence to tilpia with recombinant strain and original strain; 5) Comparing the difference of whole-cell protein with 2D-DIGE between the original strain and recombinant strain, further studying the function of sRNA. The results will be positive to further study the function of sRNA and can help exploring the proceeding of invading tiplia by S. agalactiae.

无乳链球菌是引起我国南方罗非鱼链球菌病暴发的主要病原之一,sRNA是其重要毒力调控分子。申请人在课题组已完成无乳链球菌ZQ0910株全基因组序列基础上，应用高通量RNA深度测序、Northern杂交、基因敲除及荧光差异双向电泳技术研究：1）筛选并鉴定无乳链球菌ZQ0910株的所有候选sRNA；2）克隆sRNA序列全长及分析其在基因组序列中的转录起始和终止位点；3）分析sRNA在其它种属细菌中的分布并预测sRNA在无乳链球菌基因组上的靶序列及作用方式；4）构建不同sRNA缺失突变株，并对比分析突变株和野生株不同培养条件下生长状态、毒力变化；5）对比分析突变株和野生株全菌蛋白差异，结合生物信息学预测结果进一步探讨无乳链球菌sRNA的生物学功能。研究结果有助于我们发现、识别具有重要调控功能的无乳链球菌sRNA，为进一步阐明sRNA在无乳链球菌感染过程中对毒力基因表达的调控机制奠定基础。

本项目应用高通量RNA-Seq测定无乳链球菌ZQ0910在不致病温度（25℃）和致病温度（37℃）下RNA转录组深度测序，分析受温度调控与无乳链球菌毒力相关的调控因子，通过对转录组数据分析筛选无乳链球菌毒力相关基因和预测sRNA；并通过荧光定量PCR技术对筛选得到的毒力相关基因的表达水平进行验证；利用RT-PCR和Northern Blotting对预测的部分sRNA进行验证，共鉴定了3条sRNA，对其中的2条sRNA克隆其全长；运用pSET4s自杀质粒构建SAR-1的sRNA的无乳链球菌缺失株ZQ0910△SAR-1，分析了缺失株ZQ090△SAR-1与野生株ZQ0910生长特性的异同；运用2D-DIGE技术制备了缺失株与野生株的全菌蛋白的双向电泳图谱，对比分析突变株和野生株全菌蛋白和差异。其重要结果对探讨sRNA在无乳链球菌感染宿主过程中对毒力基因表达的调控机制有重要意义，对制定罗非鱼链球菌病的防控策略奠定理论基础。