赤眼鳟TRAF6基因的cDNA克隆及其应对GCRV的免疫表达特性

Molecular cloning and expression characteristics response to GCRV of TRAF6 in Squaliobarbus curriculus

为研究赤眼鳟(Squaliobarbus curriculus)肿瘤坏死因子受体相关因子6基因的结构特性及其应对GCRV的免疫表达特性,采用RACE技术获得了赤眼鳟TRAF6基因的cDNA全长(共2 621 bp),其中包含5′端非编码区50 bp,开放阅读框1 629 bp,3′端非编码区942 bp;该基因编码542个氨基酸,推导的蛋白质相对分子质量为61670,理论等电点为6.01。SMART结构分析结果显示,TRAF6基因编码的蛋白包含1个RING结构域、2个锌指结构域、1个螺旋卷曲结构域和1个MATH结构域。系统进化树分析结果表明,赤眼鳟TRAF6与团头鲂TRAF6的亲缘关系最近。实时荧光定量PCR结果显示,TRAF6在赤眼鳟的12种组织中均有表达,其中在肝脏中的表达量最高,在中肠中的表达量最低;经GCRV感染后,赤眼鳟脾脏和头肾中TRAF6均呈波动上调趋势,在24 h达到峰值,表明TRAF6参与了赤眼鳟抗GCRV的免疫应答反应。

In order to investigate whether tumor necrosis factor-associated factor 6 gene involved inantiviral immune response, the full-length cDNA of TRAF6 in Squaliobarbus curriculus was cloned by using RACE–PCRs method. The full-length cDNA of TRAF6 is 2 621 bp including a 5′–terminal untranslated region of 50 bp, a 3′–terminal untranslated region of 942 bp and an open reading frame of 1 629 bp encoding a polypeptide of 542 amino acid residues. The putative isoelectric point and molecular weight of TRAF6 was 6.01 and 61 670, respectively. The protein encoded by this gene includes one ring domain, two zinc fingers, one coiled-coil region, and one MATH domain. Phylogenetic analysis showed that the TRAF6 was the most homogeneous to Megalobrama amblycephala. The expression of TRAF6 could be detected in all the 12 tested tissues by RT–PCR, which the highest expressed in the liver and the lowest expressed in midgut. After injection with grass carp reovirus（GCRV）, the TRAF6 expression level was up and down regulated in both spleen and head kidney, and reached the peak at 24 h, respectively. The results suggest that TRAF6 gene might play important roles in the immune response of Squaliobarbus curriculus against GCRV.