尼罗罗非鱼TRIF基因克隆及功能初步分析  

Gene Cloning and Primary Functional Analysis of TRIF in Nile Tilapia(Oreochromis niloticus)

作　　者：刘洁 高风英[2] 陈刚[1] 卢迈新[2] 曹建萌[2] 刘志刚[2] 王淼[2] 韩雪晴 LIU Jie;GAO Feng-Ying;CHEN Gang;LU Mai-Xin;CAO Jian-Meng;LIU Zhi-Gang;WANG Miao;HAN Xue-Qing(College of Fisheries,Guangdong Ocean University,Zhanjiang 524025,China;Pearl River Fisheries Research Institute/Key Laboratory of Tropical&Subtropical Fishery Resource Application&Cultivation,Ministry of Agriculture,Chinese Academy of Fishery Science,Guangzhou 510380,China;College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]广东海洋大学水产学院,湛江524025 [2]中国水产科学研究院珠江水产研究所/农业部热带亚热带水产资源利用与养殖重点实验室,广州510380 [3]上海海洋大学水产学院,上海201306

出　　处：《农业生物技术学报》2020年第6期1039-1051,共13页Journal of Agricultural Biotechnology

基　　金：现代农业产业技术体系专项资金(CARS-46);国家重点研发计划(2018YFD0900302)。

摘　　要：干扰素诱导型含TIR结构域的接头分子(TIR domain-containing adaptor inducing interferon-β,TRIF)介导Toll样受体(Toll-like receptor,TLR)信号传导途径中的下游信号传导,在宿主固有免疫应答中具有重要作用。为探究TRIF是否参与尼罗罗非鱼(Oreochromis niloticus)抗病免疫反应,本研究通过反转录PCR(reverse transcription PCR,RT-PCR)和分段扩增获得TRIF基因的cDNA(GenBank No.MT199561),并对其进行生物信息学分析;利用实时荧光定量PCR(real time quantitative PCR,q RT-PCR)技术检测该基因在健康个体及无乳链球菌(Streptococcus agalactiae)感染、脂多糖(lipopolysaccharides,LPS)和聚肌胞苷酸(polyinosinic polycytidylic acid,Poly I:C)刺激后的组织表达;进一步构建重组真核表达载体,分析TRIF在人(Homo sapiens)胚肾293T细胞的定位及对核因子κB(nuclear factor-κB,NF-κB)的激活作用。结果显示,尼罗罗非鱼TRIF cDNA全长3135 bp,其ORF为1653 bp,编码550个氨基酸。其蛋白质分子包含保守的Toll/IL-1受体(Toll/interleukin-1 receptor,TIR)结构域。系统进化树中,尼罗罗非鱼TRIF与其他鱼类成簇后再与斑马鱼(Danio rerio)和哺乳动物聚成一支。组织表达分析表明,TRIF在尼罗罗非鱼11个组织/器官中均有表达,在肌肉和血液中表达量最高。人工感染无乳链球菌可引起TRIF基因在脾脏和肾脏中的上调表达,均在感染后1 d达到最大值;LPS刺激后,TRIF在肝脏和脾脏中的表达呈上调趋势,在肠和肾脏中呈下调趋势;Poly I:C刺激后,TRIF在肠和肝脏中上调表达,在肾脏中下调表达。亚细胞定位和双荧光素酶报告分析显示,TRIF在293T细胞中定位于细胞质,可增强NF-κB活性。上述结果表明,TRIF基因在尼罗罗非鱼的免疫应答中可能具有重要作用,本研究可为进一步探讨硬骨鱼类TRIF基因在免疫进化中的功能提供参考依据。As an adaptor in Toll-like receptor(TLR)signaling pathway,TIR domain-containing adaptor inducing interferon-βreceptor(TRIF)mediates downstream signaling cascades and plays an important role in host innate immune responses.In order to investigate the potential role of TRIF in anti-disease immune response of Nile tilapia(Oreochromis niloticus),the cDNA sequence(GenBank No.MT199561)of TRIF gene was obtained by reverse transcription PCR(RT-PCR)and fragment amplification,and bioinformatic analysis was also performed.Real time quantitative PCR(qRT-PCR)was used to detect TRIF expression in healthy individuals and those with intraperitoneal injection of Streptococcus agalactiae,lipopolysaccharides(LPS)and polyinosinic polycytidylic acid(Poly I:C).The recombinant eukaryotic expression vector was constructed to analyze the subcellular localization of TRIF in 293T human(Homo sapiens)embryonic kidney cells and the activation of nuclear factorκB(NF-κB).The results showed that TRIF cDNA was 3135 bp in length which contained an ORF of 1653 bp encoding a polypeptide with 550 amino acid residues.The deduced amino acid sequence contained Toll/IL-1 receptor(TIR)domain.In the phylogenetic tree,Nile tilapia was clustered with other fish and then clustered with zebrafish(Danio rerio)and mammals.The TRIF expression were widespread in all the tested tissues and organs(liver,skin,heart,kindey,stomach,gill,brain,spleen,intestine,blood and muscle),with the highest expression in the muscle and blood.After challenged with pathogenic bacteria S.agalactiae,the TRIF gene expression increased in the spleen and kidney,and then reached the peak at 24 h post infecton(hpi).After infection with LPS,TRIF was up-regulated in the liver and spleen,while down-regulated in the intestine and kidney.TRIF was up-regulated in the intestine and liver,and down-regulated in the kidney after infection with Poly I:C.The result of subcellular localization showed that TRIF protein distributed in the cytoplasm and could significantly increase NF-кB activity.Taken

关 键 词：尼罗罗非鱼 β干扰素诱导型含TIR结构域的接头分子基因(TRIF) 基因克隆 表达分析 亚细胞定位 

分 类 号：S917.4[农业科学—水产科学]