东北七鳃鳗TRAF6基因克隆、表达分析及亚细胞定位研究

MOLECULAR CLONING, EXPRESSION ANALYSIS AND SUBCELLULAR LOCALIZATION OF TUMOR NECROSIS FACTOR RECEPTOR-ASSOCIATED FACTOR 6 IN KOREAN LAMPREY,LETHENTERON MORⅡ

为研究TRAF6在TLR信号介导的先天免疫中的调控作用,研究通过克隆技术获得了东北七鳃鳗(Lethenteron morii)TRAF6基因的cDNA全长,命名为LmTRAF6。利用实时荧光定量方法(qPCR)分析了LmTRAF6在幼鱼和成鱼中各组织的表达情况以及在铜绿假单胞菌(Pseudomonas aeruginosa)感染后脂肪体、鳃、肠和肾组织在不同时间点的表达量变化。利用双酶切技术构建pEGFP-TRAF6重组质粒并转染HEK293T细胞, 48h后进行荧光观察并拍照。结果表明, LmTRAF6的cDNA全长为2751 bp,开放阅读框(ORF)为1785 bp,编码594个氨基酸。其蛋白结构域高度保守,具有RING结构、两个锌指结构、环-环(Coiledcoil)α螺旋结构域和MATH结构域。通过系统进化树分析,发现LmTRAF6与哺乳类以及鱼类TRAF6的亲缘关系较近,与果蝇、中国对虾TRAF6的亲缘关系较远。qPCR结果显示, LmTRAF6在各组织中均有表达,在幼鱼的心脏、皮肤、鳃、肝中的表达量相对较高,而在肠中表达量相对较低。在成鱼的肾、鳃、肌肉中的表达量相对较高,而在心脏中表达量相对较低。成鱼LmTRAF6在铜绿假单胞菌感染后,鳃、肠和肾的表达量在24h达到峰值。细胞定位显示, LmTRAF6在HEK293T的细胞质和细胞核中均有表达。以上结果为进一步探究七鳃鳗中TRAF6在TLR信号通路中的作用奠定了理论基础。

In order to investigate the role of TRAF6 in Lethenteron morii with signal pathway of TLR,the full-length cDNA of LmTRAF6 was cloned.The distribution of tissues(supraneural body,gill,intestine,kidney)and related gene expression in both juvenile and adult were studied with real-time quantitative PCR.Additionally,the change of gene expression in those tissues from adult L.morii was also examined after challenged by Pseudomonas aeruginosa.A recombinant plasmid pEGFP-TRAF6 was constructed by the double digestion method,and transformed into the HEK293T cells.Fluorescence observation was conducted after 48 hours,and the results were photographed.The results showed that the length of LmTRAF6 cDNA was 2751 bp,including 1785 bp open reading frame(ORF)and 594 amino acids.The structure of LmTRAF6 was highly similar to that in other mammal and fish species,containing one RING domain,one coiled-coil region,one MATH domain and two zinc fingers.Phylogenetic tree showed that LmTRAF6 was evolutionarily closer to that in mammals and fishes,but not to that in Drosophila melanogaster and Penaeus chinensis.LmTRAF6 was expressed in all tested tissues and their developmental stages.However,higher expression was detected in heart,skin,gill,liver of juvenile,as well as in kidney,gill,and muscle of adult.When adult L.morii was challenged by Pseudomonas aeruginosa,the expression of LmTRAF6 in gill,intestine and kidney reached to the peak after 24h.Subcellular localization showed that LmTRAF6 was expressed in both cytoplasm and nucleus.