赤眼鳟Mx1基因的cDNA克隆及表达特性

MOLECULAR CLONING AND EXPRESSION CHARACTERISTICS OF MX1 GENE IN SQUALIOBARBUS CURRICULUS

  • 摘要: 为探究赤眼鳟(Squaliobarbus curriculus)是否存在Mx1 (Myxovirus resistance)基因及参与抗病毒免疫反应, 研究利用RACE技术获得了赤眼鳟Mx1基因(ScMx1)的cDNA全长序列, 并对其进行了生物信息学分析; 采用荧光定量PCR技术, 检测了ScMx1在赤眼鳟健康组织中的表达情况以及感染GCRV后ScMx1和ScIFN-Ⅰ的表达特征。结果表明, ScMx1的cDNA全长为3000 bp, 包含5′非编码区124 bp, 开放阅读框1893 bp, 3′非编码区983 bp, 共编码630个氨基酸。预测的ScMx1蛋白包含GTP酶结合区域、中央核心结构域和GTP酶效应结构域。ScMx1与青鱼Mx1的相似性最高(97%), 与ScMx的相似性仅为50%。ScMx1在所检测的10种组织中均有表达, 其中在脾脏中表达量最高。经GCRV感染开始至168h, ScMx1和ScIFN-Ⅰ在肝脏和体肾中的表达量持续上调; 在脾脏和头肾中于感染后72h达到峰值。相关性分析显示脾脏中ScMx1和ScIFN-Ⅰ的表达水平呈显著相关(r=0.94, P=0.018)。研究发现赤眼鳟存在Mx1基因, 且可能参与了抗GCRV免疫应答反应。

     

    Abstract: In order to investigate whether the Myxovirus resistance isoform (Mx1) exists in Squaliobarbus curriculus and gets involved in antiviral immune response, the full-length cDNA of ScMx1 was cloned and characterized by using RACE-PCRs technology. The expression levels of ScMx1 and ScIFN-I were detected after injection with grass carp reovirus (GCRV) with qPCR. The full-length cDNA of ScMx1 was 3000 bp including a 5′-terminal untranslated region of 124 bp, a 3′-terminal untranslated region of 893 bp and an open reading frame of 1893 bp encoding a polypeptide of 630 amino acid residues. The ScMx1 protein consisted of Dynamin GTPase domain, Dynamin central domain and Dynamin GTPase effector domain. The BLAST analysis showed that ScMx1 shared high identity (97%) with Mylopharyngodon piceus Mx1, and low identity (50%) with ScMx. The expression of ScMx1 could be detected in all tested tissues, with the highest expression in spleen. After injection with GCRV, the ScMx1 and ScIFN-I expression levels were up-regulated to 168h in liver and trunk kidney, and peaked at 72h in spleen and head kidney. There was a significant correlation between ScMx1 and ScIFN-I expressions in spleen (r=0.94, P=0.018). These results indicated that there were ScMx1 gene existed in Squaliobarbus curriculus, and it may play an important role in the immune response of against GCRV.

     

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