TRIF依赖的信号通路在大黄鱼抗病免疫反应中的作用

In vertebrates, Toll-like receptors (TLRs) mainly via myeloid differentiation primary-response protein 88 (MyD88) and/or TIR domain-containing adaptor inducing IFN-β (TRIF)-dependent signalling pathway to play an important role in host pathogen recognition and immune response. To date, 20 TLR types or more have been identified in fish species, even some only exist in fish. However, the functional characterization of the fish TLRs signalling pathway is still limited, especially TRIF-dependent signalling pathway. This proposed research is thus designed to identify TRIF in large yellow croaker (Larimichthys crocea), which is an important marine commercial fish species in China, and to investigate the gene structure, subcellular localization as well as the mechanism of transcription regulation of TRIF. Then, the expression patterns of TRIF in different tissues of healthy fish and/or under different types of pathogen associated molecular patterns (PAMPs) stimulation will be determined. The effect of TRIF in host immune response will be evaluated by examining its direct antiviral roles and by examining the expression immune factors, such as antimicrobial peptides and interferon inducible proteins. It is also expected that the proteins associated with TRIF will be revealed and the mechanism of TRIF-dependent signalling pathway in fish will be further clarified. It is believed that the completion of this proposed research will for the first time provide novel information of TRIF-dependent signalling pathway in fish immune response, being thus significantly important in understanding the role of TRIF in fish as well as in vertebrates, which will also have an important role in the disease control and prevention of marine commercial fishes.

脊椎动物中，模式识别受体中的Toll样受体主要通过MyD88和TRIF依赖的信号通路，在机体对外界病原的识别和抗病免疫反应中发挥重要作用。鱼类中已发现20余种Toll样受体，且存在一些鱼类所特有的Toll样受体分子，而这些受体分子的信号转导，尤其是TRIF依赖的信号通路及其分子机制尚不明确。本项目拟对我国重要海水经济鱼类大黄鱼的TRIF基因进行克隆，研究其基因结构、亚细胞定位与转录调控机制；解析其在不同组织及不同病原相关分子模式刺激下的时空表达特征，以及在机体抗病免疫反应中的作用；阐明TRIF与模式识别受体及抗病免疫相关通路上其他关键分子的相互关系；揭示TRIF分子在鱼类中介导的细胞信号通路及其分子机制。本项目的完成将系统揭示TRIF依赖的信号通路在鱼类抗病免疫反应中的作用，丰富和完善脊椎动物模式识别受体及信号通路作用机制的研究，同时为海水经济鱼类的病害防治及抗病分子模块育种奠定理论基础。

脊椎动物模式识别受体中的Toll样受体主要通过MyD88和TRIF依赖的信号通路，在宿主对外界病原的识别和免疫激活中发挥重要作用。目前已在硬骨鱼类中发现20余种Toll样受体，且存在一些鱼类所特有的Toll样受体分子，而这些受体分子的信号转导，尤其是TRIF依赖的信号通路及其分子机制尚不明确。本项目以海水经济鱼类大黄鱼为实验对象，克隆并鉴定了大黄鱼TRIF分子，揭示了其基因结构及蛋白分子的保守结构域，比较分析了大黄鱼TRIF与脊椎动物Toll样受体5类接头蛋白TRIF、TRAM、TIRAP、MyD88和SARM的系统进化关系，通过对脊椎动物TRIF和TRAM基因的共线性分析揭示了硬骨鱼类缺失TRIF的“桥梁分子”TRAM；揭示了大黄鱼TRIF的亚细胞定位、在健康大黄鱼不同组织中的表达水平以及响应不同PAMPs和变形假单胞菌（P. plecoglossicida）感染刺激的表达特征；克隆了大黄鱼TRIF的启动子，初步解析了大黄鱼TRIF的转录调控机制；研究了大黄鱼TRIF及其不同结构域在激活NF-κB、IRF3、IRF7及IFN1信号通路中的功能，初步阐明了大黄鱼TRIF在宿主抗病免疫反应中的作用；对大黄鱼TRIF与与宿主抗病免疫相关信号通路上其他关键分子TRAF3、TRAF6、IRF3和IRF7的相互作用进行了分析，解析了大黄鱼TRIF介导的细胞信号转导通路及其分子机制。在上述研究工作的基础上，我们还拓展研究了与大黄鱼TRIF相关信号通路中的关键分子TRAF3、RIP1和RIP3的功能，对上述重要分子进行了克隆与鉴定，解析其时空表达特征，并对其与宿主免疫相关信号通路中的互作分子进行了分析与鉴定。本项目的完成系统揭示了TRIF依赖的信号通路在硬骨鱼类免疫反应中的作用，丰富和完善脊椎动物模式识别受体及信号通路作用机制的研究，同时本项目的研究成果还为海水经济鱼类制定合理的疾病防御和防治策略及抗病分子模块育种提供参考。