虹鳟TRIM25基因克隆及其在IHNV感染过程中的表达分析  

Cloning of TRIM25 Gene in Rainbow Trout(Oncorhynchus mykiss)and Its Expression Analysis

作　　者：雷明荃 黄进强[1] 李永娟[1,2] 吴深基 赵璐 潘玉财 孙同振 LEI Ming-Quan;HUANG Jin-Qiang;LI Yong-Juan;WU Shen-Ji;ZHAO Lu;PAN Yu-Cai;SUN Tong-Zhen(College of Animal Science and Technology,Gansu Agricultural University,Lanzhou 730070,China;College of Science,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物科学技术学院,兰州730070 [2]甘肃农业大学理学院,兰州730070

出　　处：《农业生物技术学报》2023年第8期1684-1695,共12页Journal of Agricultural Biotechnology

基　　金：甘肃省现代丝路寒旱农业科技支撑计划(GSLK-2021-16);甘肃农业大学“伏羲青年英才培育计划”项目(Gaufx-02Y08)。

摘　　要：三基序蛋白25(tripartite motif containing 25,TRIM25)是宿主抗病毒免疫应答中发挥重要作用的TRIM蛋白家族成员之一。为了解虹鳟(Oncorhynchus mykiss)TRIM25基因分子特征及其在感染传染性造血器官坏死病毒(Infectious hematopoietic necrosis virus,IHNV)过程中的表达变化,本研究采用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术获得虹鳟TRIM25基因cDNA全长序列(GenBank No.OL692831),并对其进行生物信息学分析,同时利用qPCR技术检测TRIM25基因在健康虹鳟不同组织及感染IHNV后不同时间点(0,6,12,24,48,72,96,120和144 h)肠道、肝脏、脾脏、皮肤、头肾和鳃中的表达变化。结果显示,虹鳟TRIM25基因cDNA全长序列4745 bp,其开放阅读框2028 bp,编码675个氨基酸。生物信息学分析表明,TRIM25蛋白分子量为76.04 kD,理论等电点为8.79,不稳定系数为49.2,平均亲水系数为-0.591,属于不稳定亲水蛋白。对TRIM25蛋白结构域分析发现,该蛋白包含RING、B-boxes、B-Box C-terminal domain和PRY-SPRY结构域。同源比对及系统进化树分析表明,虹鳟与大马哈鱼(Oncorhynchus keta)同源性最高(94.96%)且亲缘关系最近,与哺乳动物和两栖动物同源性较低。qPCR检测发现,TRIM25基因在健康虹鳟各组织中均有表达,其在肝脏中的表达量最高,头肾和脑中的表达量次之,皮肤中的表达量最低。感染IHNV后,TRIM25基因在肝脏、头肾、脾脏、肠道和皮肤的表达量均在48 h达到峰值,鳃在96 h表达量达到峰值,各组织在峰值时的表达量与对照组相比差异极显著(P<0.01),其中以头肾和脾脏表达变化较显著,分别为对照组的5.72和4.33倍。上述结果表明,TRIM25基因可能在虹鳟抗IHNV感染的免疫应答中发挥着重要作用。本研究为深入研究TRIM25基因在鱼类抗病毒免疫调控中的作用提供了基础资料。Tripartite motif containing 25(TRIM25),a member of the TRIM family,plays an important role in the host antiviral immune response.To understand the molecular characteristics of TRIM25 in rainbow trout(Oncorhynchus mikiss)and its expression changes during infection with Infectious hematopoietic necrosis virus(IHNV),in this study,the full-length cDNA sequence of TRIM25(GenBank No.OL692831)was obtained by rapid amplification of cDNA ends(RACE)technique and bioinformatically analyzed.At the same time,the qPCR technique was used to detect changes in TRIM25 expression in different tissues of healthy rainbow trout and at different time points(0,6,12,24,48,72,96,120 and 144 h)after infection with IHNV in the intestine,liver,spleen,skin,head kidney and gill.The results showed that the full-length sequence of the rainbow trout TRIM25 cDNA,4745 bp,had an ORF of 2028 bp and encoded 675 amino acids.Bioinformatics analysis showed that the TRIM25 protein had a molecular weight of 76.04 kD,a theoretical pI of 8.79,an instability coefficient of 49.2 and an average hydrophilic coefficient of-0.591,indicating TRIM25 was an unstable hydrophilic protein.Analysis of the structural domains of TRIM25 protein revealed that the protein included RING,B-boxes,B-Box C-terminal domain and PRY-SPRY structural domains.The homology and phylogenetic tree analysis showed that rainbow trout had the highest homology(94.96%)with salmon(Oncorhynchus keta)and was closely related,but had low homology with mammals and amphibians.qPCR showed that TRIM25 was widely expressed in all tissues of healthy rainbow trout,with its highest expression in liver,followed by that in head,kidney and brain,and the lowest expression in skin.After infection with IHNV,the expression of TRIM25 gene in liver,head kidney,spleen,intestine and skin peaked at 48 h,and gill reached the peak at 96 h.Expression of each tissue at the peak was extremely significant compared to the control(P<0.01),with the most significant changes in expression in head and kidney and spleen,which were 5

关 键 词：虹鳟 三基序蛋白25(TRIM25) 传染性造血器官坏死病毒(IHNV) 基因克隆 表达分析 

分 类 号：S917.4[农业科学—水产科学]