赤眼鳟Mx基因全长cDNA克隆及其经GCRV攻毒后的组织表达分析

MOLECULAR CLONING AND TISSUE EXPRESSION ANALYSIS OF Mx GENE IN SQUALIOBARBUS CURRICULUS

为研究赤眼鳟(Squaliobarbus curriculus)Mx蛋白(Myxovirus resistance protein)的功能,采用简并PCR和SMART RACE方法从赤眼鳟脾脏中克隆得到Mx基因全长cDNA,并通过生物信息学方法分析其同源性,再利用实时荧光定量PCR(RT-qPCR)检测其在脾、肝、肠、肾等9个组织中的表达,以及感染草鱼呼肠孤病毒(Reovirus of Grass carp)GCRV-104后不同时间点赤眼鳟Mx的时空表达规律。结果表明:赤眼鳟Mx基因cDNA序列(ScMx)全长2325 bp,包含5′-UTR 40 bp,3′-UTR 371 bp和ORF 1884 bp,共编码627个氨基酸,其编码的Mx蛋白分子量约为70.9 kD,理论等电点pI为8.25,具有脊椎动物Mx蛋白共有的结构特征;赤眼鳟Mx与鲫鱼Mx3同源性最高;Mx在赤眼鳟脾、肝、肠、肾等9个组织中均有表达,其中肝脏中的相对表达量最高,脾脏次之,肠组织中的表达量最低;经GCRV-104病毒感染刺激后,ScMx在肝和脾组织中的表达量显著上调,均在48h到达峰值,分别为对照组的10倍(肝)和5倍(脾),且在这两个组织中的表达模式相似,均表现为先升高后下降的波动型变化趋势。研究表明ScMx参与了赤眼鳟抗GCRV-104病毒的免疫反应。

Mx protein, a GTP-enzyme active protein induced by interferon, exhibits broad-spectrum antiviral activity. As member of economically fresh water fish with superior quality, Squaliobarbus curriculus has the highest fitness and disease resistance;however, studies on its immunity are limited. In the current study, we amplified a full-length cDNA of Mx (ScMx) from the spleen by using degenerate PCR and SMART RACE, and conducted homology analysis and tis-sue expression analysis of ScMx mRNA in different tissues including liver, spleen, intestine, gill, kidney, head kidney, heart, brain and muscle. Furthermore, we investigated the temporal expression levels of ScMx in the liver and spleen after GCRV-104 infection. The results indicated that the full-length of ScMx gene was 2325 bp consisted of 40 bp 5′-UTR, 371 bp 3′-UTR and 1884 bp ORF encoding 627 amino acids. The theoretical molecular weight and isoelectric point (PI) of ScMx were 70.9 kD and 8.25, respectively. ScMx shared the most homology with Carassius aruatus Mx3. ScMx was broadly expressed in all tested tissues. After infected with GCRV-104, the ScMx expression levels in the liver and spleen showed a tendency of fluctuation:the relative expression of ScMx increased reached the peak at 48h after the viral infection, and then decreased. The infection significantly induced the expression of ScMx (P<0.05). These results suggested that ScMx may play an important role in the immune response of Squaliobarbus curriculus against GCRV-104 infection.