哈氏弧菌感染后红鳍东方鲀非编码RNA对免疫相关基因调控作用的研究

Japanese pufferfish (Takifugu rubripes) is an economically important cultured fish species in Northern China. However, with the development of its production, disease has become a serious problem for its sustainable development due to genetical characterization decline and its way of farming. Among which, Vibrio harveyi is one of the most serious pathogen, it can cause huge loss for Japanese pufferfish production. In this proposal, the whole transcriptome analyses will be conducted for Japanese pufferfish to compare their ways of expression before and after Vibrio harveyi infection, the results can gain more insights into pathogenesis of Vibrio harveyi infection. Briefly, Japanese pufferfish were challenged with Vibrio harveyi, large scales of non-coding RNAs will be first discovered throughout the whole transcriptome, including miRNAs and lncRNAs. Furthermore, correlation analyses will be conducted on these non-coding RNAs and previously identified differentially expressed immune related genes in order to explore their ways of regulation. In addition, lncRNA-miRNA-gene regulation network will be built to give an overall explanation of the regulatory mechanism of non-coding RNA on immune related genes after Vibrio harveyi infection. This study will provide valuable reference for the vaccine development of Japanese pufferfish, offer new ways of molecular marker assistant selection, and guarantee the prosperous sustainable development of Japanese pufferfish.

红鳍东方鲀是我国北方重要的名贵海水养殖鱼类，然而由于种质退化和养殖方式的问题，病害已严重困扰红鳍东方鲀产业的健康发展。其中哈氏弧菌引起的皮肤溃烂病是最为常见的一种，给养殖业带来了巨大的损失。本研究以红鳍东方鲀为研究对象，对前期实验筛选出的哈氏弧菌感染后关键免疫基因IL8、SOCS3、C1ql4等在免疫过程中的调节机制进行探讨。研究主要通过比较哈氏弧菌感染后红鳍东方鲀全转录组数据，鉴定具有调控功能的非编码RNA-miRNA及lncRNA，筛选出调节免疫相关基因的非编码RNA，分析非编码RNA对免疫相关基因的调控机理，构建lncRNA-miRNA-基因的调控网络，综合解析哈氏弧菌感染红鳍东方鲀后非编码RNA对免疫相关基因的调控机制。本研究成果将为红鳍东方鲀相关疫苗的研制提供有价值的参考，并为红鳍东方鲀的分子育种提供新的研究思路，保证红鳍东方鲀的健康养殖。

随着我国有条件开放养殖红鳍东方鲀的经营与加工，红鳍东方鲀的养殖规模不断扩大，然而其养殖过程一直受到病害的影响，成为红鳍东方鲀养殖业发展的重大困扰。本项目以红鳍东方鲀为研究对象，聚焦于引发红鳍东方鲀皮肤溃烂病的病原菌哈氏弧菌，对红鳍东方鲀脾脏免疫应答机制展开系统研究，高通量筛选和鉴定关键免疫基因和非编码RNA，了解其表达模式及免疫调控作用。通过转录组测序测定了红鳍东方鲀感染哈氏弧菌后脾脏组织的转录表达谱，并对关键免疫基因进行了结构分析和表达测定，确定了其在哈氏弧菌感染后的免疫调节模式。通过全转录组测序完成了miRNA、lncRNA、circRNA的鉴定，共鉴定出106条已知miRNA和170条新预测miRNA、8888条lncRNA和837条circRNA。差异表达分析揭示出非编码RNA在感染哈氏弧菌后发生了显著变化，包括110条miRNA、684条lncRNA和67条circRNA。通过靶基因鉴定，功能富集分析，明确了红鳍东方鲀非编码RNA在哈氏弧菌感染过程中的差异表达模式。其中fru-miR-138被预测与关键免疫基因socs3有互作关系，体外双荧光素酶报告基因实验表明fru-miR-138通过抑制socs3基因的表达，参与到socs3基因与细胞因子转导途径的负反馈调控作用中，进而调控红鳍东方鲀的免疫应答反应。本研究成果为全面解析红鳍东方鲀免疫应答机制奠定了基础，为红鳍东方鲀的疾病防治提供理论依据。