鳜鱼MDA5基因的克隆与组织表达分析  

Molecular Cloning and Expression Pattern of MDA5 cDNA in Siniperca chuatsi

作　　者：陈博雯[1] 顾天天 路璐[1] 王婧雯 田立立 黄敏 徐琪[1] 陈国宏[1] CHEN Bo-Wen;GU Tian-Tian;LU Lu;WANG Jing-Wen;TIAN Li-Li;HUANG Min;XU Qi;CHEN Guo-Hong(School of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China)

机构地区：[1]扬州大学动物科学与技术学院,江苏扬州225009

出　　处：《生物技术通讯》2017年第6期807-811,共5页Letters in Biotechnology

基　　金：江苏省高等学校大学生创新创业训练计划(201511117038z);江苏高校优势学科建设工程资助项目(苏政办发[2011]137号)

摘　　要：目的:克隆鳜鱼MDA5基因cDNA序列,研究聚肌苷酸胞苷酸[Poly(I∶C)]感染过程中MDA5的表达规律。方法:利用RT-PCR和RACE技术克隆鳜鱼MDA5全长序列,构建Poly(I∶C)感染模型,分析MDA5在鳜鱼脾脏和鳃组织中的表达规律。结果:鳜鱼MDA5 cDNA全长3556 bp,包括142 bp 5′UTR、438 bp 3′UTR和2976 bp开放读框,共编码991个氨基酸残基。MDA5蛋白具有RLR家族的典型特征,即N端含有2个CARD结构域,C端含有DEXDc、解旋酶和RD结构域,序列分析发现鳜鱼的MDA5与横斑石鲷、大黄鱼的同源性较高,分别达85.4%、79.8%。组织表达谱分析显示,鳜鱼MDA5基因在不同组织中普遍表达,用Poly(I∶C)抗原刺激后,MDA5 mRNA表达显著上调,其中脾脏组织在诱导8 h后达到峰值,而在鳃组织中表达持续显著上调。结论:MDA5在鳜鱼免疫应答中起重要作用。Objective:To clone the full-length cDNA of MDA5gene of Siniperca chuatsi,and detect the temporal spatial expression of MDA5in tissues when the fish were challenged with Poly(I∶C).Methods:The cDNA sequences of MDA5were obtained by RT-PCR and RACE technology.The expression of MDA5in spleen and gill was detected by RT-PCR or RT-qPCR.Results:The full-length cDNA of MDA5gene was3556bp,including142bp of5′UTR,438bp of3′UTR,and2976bp of open reading frame,which encodings991amino acids.Bioinformatics analysis indicated that MDA5protein had typical characteristics of the family of RLR,which contained two CARD domain structures of N side,and DEXDc,helicase and RD domain structure of C side.MDA5of S.chuatsi shared high similarities with Oplegnathus fasciatus(85.4%)and Larimichthys crocea(79.8%).RT-PCR analysis revealed that MDA5mRNA was widely expressed in healthy tissues.MDA5mRNA was significantly up-regulated following injection with Poly(I∶C),reaching peak at8h post-infection in the spleen,and increasing consistently in gill.Conclusion:MDA5contributes to the innate immune response against viral infection.

关 键 词：鳜鱼 MDA5基因 克隆 诱导 

分 类 号：Q78[生物学—分子生物学] Q811.4