日本鳗鲡TRAF3基因的克隆及功能研究  

Identification and functional characterization of TRAF3 in Japanese eel(Anguilla japonica)

作　　者：凌露露 梁英[1,2] 黄文树 聂品[4] 黄贝 LING Lulu;LIANG Ying;HUANG Wenshu;NIE Pin;HUANG Bei(College of Fisheries,Jimei University,Xiamen 361021,China;Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture,Zhanjiang 524088,China;Engineering Research Center of the Modern Industry Technology for Eel,Ministry of Education,Xiamen 361021,China;School of Marine Science and Engineering,Qingdao Agricultural University,Qingdao 266237,China)

机构地区：[1]集美大学水产学院,福建厦门361021 [2]广东省水产动物病害防控与健康养殖重点实验室,广东湛江524088 [3]鳗鲡现代产业技术教育部工程研究中心,福建厦门361021 [4]青岛农业大学海洋科学与工程学院,山东青岛266237

出　　处：《水产学报》2023年第8期143-154,共12页Journal of Fisheries of China

基　　金：国家自然科学基金(32073011);厦门市自然科学基金(3502Z20206022);福建省自然科学基金(2020J01666);广东省水产动物病害防控与健康养殖重点实验室开放基金(PBEA2021ZD05)。

摘　　要：为探究鱼类TRAF3在鱼类抗病毒免疫应答中的功能及作用机制,实验利用逆转录PCR克隆获得了日本鳗鲡TRAF3转录本(AjTRAF3),利用生物信息学软件分析了AjTRAF3的结构特征,利用实时荧光定量PCR(qPCR)、双荧光素酶报告系统以及免疫共沉淀等方法对其表达规律、功能及作用机理进行了初步分析。AjTRAF3的开放阅读框长度为1707 bp,编码568个氨基酸。序列结构分析结果显示,AjTRAF3由N端的环结构域2个锌指结构域以及1个螺旋结构域和C端高度保守的TRAF-C(MATH)结构域组成。qPCR结果显示,AjTRAF3在日本鳗鲡各组织中均有表达,脑组织中表达量最高,其次为头肾,心脏中的表达量最低。Poly I:C刺激6 h后,日本鳗鲡脾脏组织中AjTRAF3上调倍数最高,为对照组的15.83倍。迟缓爱德华氏菌感染24 h后,日本鳗鲡脾脏组织中AjTRAF3上调倍数最高,为对照组的31.47倍。此外,本研究构建了AjTRAF3真核表达质粒,发现过表达AjTRAF3能显著上调炎症及抗病毒相关基因的表达,可显著增强AjIFN2、AjIFN4和NF-κB启动子荧光素酶活性。并能显著上调由AjRIG-IN、AjMAVS、AjIRF3诱导的AjIFN2、AjIFN4和NF-κB启动子活性。免疫荧光结果显示,AjTRAF3主要定位于细胞质中,且与AjMAVS存在共定位。免疫共沉淀结果显示,AjTRAF3通过MATH结构域与AjMAVS相互结合,缺失该结构域后,其与AjMAVS的相互作用消失,推测AjTRAF3可通过介导RIG-I/MAVS信号转导途径调控鱼类的抗病毒免疫应答。本研究结果为进一步揭示鱼类TRAF3的生物学功能奠定了基础。The aim of this study was to explore the function and regulatory mechanism of tumor necrosis factor receptor-associated factor 3(TRAF3)in fish antiviral immune response.In the present study,the transcript encoding TRAF3(AjTRAF3)was obtained from Japanese eel,Anguilla japonica,by using reverse transcription PCR.The structural characteristics of AjTRAF3 were analyzed using bioinformatics softwares and its expression profile and functional mechanism were investigated by qPCR,luciferase reporter and co-immunoprecipitation assay.The open reading frame of AjTRAF3 was 1707 bp long,encoding a polypeptide consisting of an N-terminal ring domain,two Zn Finger domains,a helical domain,and a C-terminal TRAF-C(MATH)domain.qPCR analysis revealed a wide tissue distribution of AjTRAF3,with the highest expression in brain,followed by head kidney,and the lowest in heart.The highest induction of AjTRAF3 in response to Poly I:C stimulation was observed at 24 hour post injection(hpi)in spleen,being 15.83 fold higher than control.The highest up-regulation of AjTRAF3 after E.tarda infection was observed in spleen at 24 hpi,being 31.47 fold higher than control.Furthermore,the eukaryotic expression plasmid was constructed to study the function of AjTRAF3 in vitro.Our results revealed increased expression of antiviral related genes and increased luciferase transactivation of the AjIFN2,AjIFN4 and NF-κB promoter in cells overexpressed with AjTRAF3.Further,AjRIG-IN,AjMAVS-or AjIRF3-induced AjIFN2,AjIFN4 and NF-κB promoter activation was significantly enhanced in cells co-transfected with AjTRAF3.In addition,subcellular localization analysis revealed the cytoplasmic distribution of AjTRAF3 and the co-localization of AjTRAF3 with MAVS on mitochondria.Lastly,co-immunoprecipitation assays showed that AjTRAF3 interacts with AjMAVS via the MATH domain,whereas a mutant lacking this domain lost the ability to interact with AjMAVS.Collectively,these data suggested that AjTRAF3 can induce antiviral responses by regulating RIGI/MAVS-mediated signali

关 键 词：日本鳗鲡 TRAF3 MAVS 共定位 启动子 

分 类 号：Q785[生物学—分子生物学] S942[农业科学—水产养殖]