虹鳟TANK基因的克隆及其在传染性造血器官坏死病毒(IHNV)感染下的表达分析  

Cloning of TANK Gene and Its Expression Analysis in Rainbow Trout(Oncorhynchus mykiss)Under Infectious hematopoietic necrosis virus(IHNV)Infection

作　　者：孙同振 黄进强[1] 吴深基 赵璐 潘玉财 雷明荃 SUN Tong-Zhen;HUANG Jin-Qiang;WU Shen-Ji;ZHAO Lu;PAN Yu-Cai;LEI Ming-Quan(College of Animal Science and Technology,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

出　　处：《农业生物技术学报》2022年第11期2174-2186,共13页Journal of Agricultural Biotechnology

基　　金：甘肃农业大学科技创新基金-青年导师扶持基金(GAU-QDFC-2018-09);甘肃省农牧厅特种养殖专项基金(GARSTSYZ-1-2);甘肃省现代丝路寒旱农业科技支撑计划(GSLK-2021-16)。

摘　　要：TRAF家族成员关联的NF-κB激活剂(TRAF family member-associated NF-κB activator,TANK)参与维甲酸诱导基因Ⅰ(retinoic acid-inducible gene I,RIG-Ⅰ)样受体介导的信号通路,在脊椎动物的先天免疫中发挥重要作用。为了解TANK基因的生物学特性及其在虹鳟(Oncorhynchus mykiss)抗病毒免疫应答中的作用,本研究通过cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术获得虹鳟TANK的全长cDNA序列(GenBank No.OK605587),并进行了生物信息学分析,同时采用q PCR技术检测该基因在健康虹鳟头肾、皮肤、眼、鳃、脑、肠、肝脏、脾脏、心脏和肌肉10个组织中的表达模式,以及感染传染性造血器官坏死病毒(Infectious hematopoietic necrosis virus,IHNV)后脾脏、肝脏、头肾、皮肤、鳃和肠6个重要免疫组织在不同时间点(0,6,12,24,48,72,96,120和144 h)的表达变化。结果显示,TANK基因cDNA全长1618 bp,包含1107 bp的开放阅读框,编码368个氨基酸。生物信息学分析表明,TANK蛋白的分子量为41.41 kD,理论等电点为6.03,不稳定系数为70.60,亲水性平均值为-0.748,脂肪系数为65.54,属于不稳定亲水蛋白。对TANK蛋白的结构分析发现,该蛋白无跨膜结构域,含有保守的TBK1/IKKi结合结构域(TBK1/IKKi-binding domain,TBD)和coiled-coil区域。同源性比对及系统进化分析显示,虹鳟与大马哈鱼(Oncorhynchus keta)的同源性最高(95.92%)且进化关系最近。组织表达分析显示,TANK基因在健康虹鳟头肾、皮肤、眼、鳃、脑、肠、肝脏、脾脏、心脏、肌肉组织中均有表达,在肝脏中表达最高,脾脏和心脏次之,皮肤中最低;经IHNV感染后,TANK基因在脾脏、肝脏、头肾、皮肤、鳃、肠中的表达均极显著上调(P<0.01),以黏膜组织的皮肤和肠中变化最为显著,分别在48和72 h达到峰值,为对照组的3.14和3.16倍;除了脾脏组织的表达量整体表现为先升后降的趋势外,肝脏、头肾、皮肤、鳃和肠均呈现先降�TRAF family member-associated NF-κB activator(TANK)is involved in retinoic acid-inducible gene I(RIG-I)-like receptor mediated signaling pathway and plays an important role in the innate immunity of vertebrate.To explore the biological properties of TANK gene and its regulatory role in the antiviral immune response of rainbow trout(Oncorhynchus mykiss),the full length cDNA sequence of TANK gene(GenBank No.OK605587)was obtained by rapid amplification of cDNA ends(RACE)technique,and bioinformatic analysis was also performed.qPCR was used to detect the gene expression pattern in tissues of healthy fish and the expression changes in 6 important immune tissues(spleen,liver,head kidney,skin,gill and intestine)at different time points(0,6,12,24,48,72,96,120 and 144 h)in rainbow trout after infection with Infectious hematopoietic necrosis virus(IHNV).The results showed that the full length cDNA of TANK gene was 1618 bp,containing 194 bp 5’untranslated region(UTR),317 bp 3’UTR and 1107 bp ORF.The ORF of TANK encoded368 amino acid residues.Bioinformatics analysis showed that molecular weight of TANK protein was 41.41kD,theoretical isoelectric point was 6.03,instability coefficient was 70.60,average hydrophily was-0.748,and fat coefficient was 65.54,which indicated that TANK protein was an unstable hydrophilic protein.Structural analysis of the TANK protein revealed that it contained a conserved TBK1/IKKi-binding domain(TBD)and a coiled-coil region without transmembrane domain.Homology matching and phylogenetic analysis showed that rainbow trout and salmon(Oncorhynchus keta)had the highest homology(95.92%)and the closest evolutionary relationship.In addition,rainbow trout and salmon had the highest amino acid sequence similarity in the TBD structural domain of the TANK protein,showing that the TBD domain had similar functions in salmon trout species.Tissue expression analysis revealed that TANK gene was expressed at different levels in all tissues of healthy fish(head kidney,skin,eye,gill,brain,intestine,liver,spleen,h

关 键 词：虹鳟 免疫 基因克隆 TRAF家族成员关联的NF-κB激活剂(TANK) 表达分析 

分 类 号：S917.4[农业科学—水产科学]