尼罗罗非鱼Crtam基因的克隆鉴定与mRNA表达分析

Identification and mRNA Expression Analysis of Crtam Gene in Nile tilapia

Crtam作为NK细胞和CD8+T细胞的活化标志物,已被证实在哺乳动物中参与多种免疫途径,但在罗非鱼中的作用未知。本研究克隆鉴定了尼罗罗非鱼(Oreochromis niloticus)Crtam cDNA全长(命名为On-Crtam),对该基因进行生物信息学分析,并运用荧光定量PCR对无乳链球菌刺激后On-Crtam mRNA在各免疫组织及器官中的表达模式进行了研究。结果显示,On-Crtam基因CDS区为1335 bp,编码444个氨基酸,理论分子质量为110.15 kD,等电点为5.01。On-Crtam与奥利亚罗非鱼(Oreochromis aureus)相似性最高(98.65%),与鱼类的同源性均在90%以上,表明Crtam在鱼类进化过程中较保守,但与其它物种同源性较低,在不同物种之间可能具有一定差异性。On-Crtam在健康尼罗罗非鱼各组织及器官中均有表达,在肝脏中表达量最高,其次是肌肉,胸腺,在脑中表达量最低。经poly I:C病毒类似物和灭活无乳链球菌刺激后,On-Crtam的表达量在肝脏、头肾和皮肤等免疫组织及器官中都表现为极显著上调。这表明On-Crtam基因可能在无乳链球菌等病原入侵宿主过程中发挥重要作用,但其作用机制尚需进一步研究阐明。

Crtam,as an activation marker of NK cells and CD8+T cells,has been shown to be involved in various immune pathways in mammals,but its role in tilapia remains unknown.In this study,the full-length Crtam cDNA of Nile tilapia(named On-Crtam)was cloned and identified,bioinformatics analysis of the gene was performed,and the expression patterns of On-Crtam mRNA in various tissue species after stimulation by Streptococcus agalactis and poly I:Cwas studied by fluorescence quantitative PCR.The results showed that the CDS region of On-Crtam gene was 1335 bp,encoding 444 amino acids,the theoretical molecular mass was 110.15 kD,and the isoelectric point was 5.01.On-Crtam has the highest similarity to Oreochromis aureus(98.65%),and the homology with other fish species was more than 90%,indicating that Crtam was more conservative in the evolution of fish,but it has low homology with other species.On-Crtam was expressed in all tested tissues of healthy Nile tilapia,with the highest expressionin the liver,followed by muscle and thymus,and the lowest expression in the brain.After the stimulation of poly I:C virus analogs and inactivated Streptococcus agalactis,the expression level of On-Crtam was significantly up-regulated in liver,head kidney,and skin,reaching the maximum at 96 h in the liver.This study shows that the On-Crtam gene may play an important role in the hostdefense against the challenges of Streptococcus agalactiae and poly I:C virus analogs,but its mechanism of action needs further exploration.