核转录因子κB抑制因子α(IκBα)是NFκB/IκB信号传导通路的重要成员, 参与机体抗细菌感染等多种免疫反应, 可通过蛋白质间的相互作用结合核转录因子NFκB, 从而调控生物体多种免疫基因表达。该研究采用RACE技术从香鱼中克隆得到核转录因子κB抑制因子PaIκBα基因的cDNA全长序列(1 341 bp, GenBank Accession No.JN801027), 开放阅读框ORF为936 bp, 编码311个氨基酸, 5'非编码区为64 bp, 3'非编码区为341 bp。生物信息学分析表明, 香鱼IκBα蛋白的序列中包含5个保守的锚蛋白重复序列, N末端含有信号诱导蛋白, C末端含有PEST序列。同源性比对结果表明, 香鱼IκBα蛋白与胡瓜鱼IκBα的同源性最高, 为95%;其次是大西洋鲑、虹鳟、尼罗罗非鱼和鳜鱼等, 同源性分别为76%、75%、70%和68%。系统进化树分析表明, 香鱼IκBα蛋白与胡瓜鱼、虹鳟、尼罗罗非鱼、鳜鱼和大西洋鲑等亲缘关系最近。RT-PCR分析表明, PaIκBα基因在香鱼肝脏、肾脏、脾脏和鳃中表达水平较高, 其次是肠、脑和肌肉, 在心脏中表达极少。嗜水气单胞菌(Aeromonas hydrophil)感染香鱼后, PaIκBα基因表达增强, 感染24 h达最大值, 表明PaIκBα基因在香鱼受到嗜水气单胞菌刺激的免疫过程中可能发挥着重要作用。
The NFκB inhibitor (IκBα) is an integral part of NFκB/IκB signaling pathways, which plays roles in a variety of immune responses, such as bacterial infection resistance. By interacting with nuclear transcription factor NFκB, IκBα controls a variety of biological immune gene expressions. In this study, full-length cDNA (1341 bp) of the NFκB inhibitor IκBα (PaIκBα, GenBank Accession No. JN801027) of Plecoglossus altivelis was obtained by RACE and PCR, and included a 5' untranslated region (UTR) (64 bp), a 3' untranslated region (UTR) (341 bp) and an open reading frame (ORF) (936 bp) encoding a polypeptide of 311 amino acids. PaIκBα had high homology with other IκBαs, containing a conserved ankyrin repeat domain, which was required for interacting with NFκB, a PEST sequence in the C-terminus and a signal responsive domain in the N-terminus. The deduced amino acid sequence of PaIκBα shared 95% homology with Osmerus mordax, and 76%, 75%, 70%, and 68% homology with Salmo salar, Oncorhynchus mykiss, Nile tilapia, and Siniperca chuatsi, respectively. Phylogenetic analysis revealed that IκBα of ayu and Osmerus mordax, Salmo salar, Oncorhynchus mykiss, Nile tilapia, and Siniperca chuatsi were in the same phylogenetic tree. RT-PCR analysis showed that PaIκBα mRNA expression was highest in the liver, kidney, intestine, and gills, then followed by the spleen, brain and muscle, and was lowly expressed in the heart. Likewise, after Aeromonas hydrophila infection, the mRNA level of ayu PaIκBα in the liver was also up-regulated.