Abstract：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 1 618 bp, containing 194 bp 5' untranslated region (UTR), 317 bp 3'UTR and 1 107 bp ORF. The ORF of TANK encoded 368 amino acid residues. Bioinformatics analysis showed that molecular weight of TANK protein was 41.41 kD, 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, heart and muscle), with the highest expression level in liver, followed by spleen and heart, and the lowest expression level in skin, which implied that TANK gene may play a major role in the innate immune response of rainbow trout. After IHNV infection, the expression level of TANK gene in important immune tissues (spleen, liver, head kidney, skin, intestine and gill) was extremely significantly up-regulated (P<0.01). The significant expression changes were appeared in skin and intestine of mucosal tissues, peaked at 48 and 72 h, which were 3.14 and 3.16 fold of control group, and the expression in liver, head kidney, spleen and gill peaked at 48, 48, 72 and 96 h, respectively. In the spleen tissue, the expression level increased firstly and then decreased, while the expression level of the liver, head kidney, skin, intestine and gill all decreased firstly, then rose and fell again. Meanwhile, the liver, head kidney, skin and intestine showed different degrees of rebound at 96 and 120 h. These results indicated that TANK gene may be involved in the innate immune response and played an important role in rainbow trout against IHNV infection. The results of this study provide basic data for further investigating the antiviral immune regulatory mechanism of TANK gene in teleost fish.
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