Rare minnow (Gobiocypris rarus), a small cyprinid species that is highly sensitive to the grass carp reovirus (GCRV), is regarded as an ideal model to study the mechanisms of innate immunity in fish. In the present study, a TBK1 homologue from rare minnow (GrTBK1) was identified and its roles in defence against viral infection were investigated. Sequence analysis showed that GrTBK1 encoded a 727-amino acid peptide which shared 98% and 72% identity to the black carp (Mylopharyngodon piceus) and human (Homo sapiens) orthologues, respectively. The amino acid sequence analysis demonstrated that GrTBK1 contains a conserved Serine/Threonine protein kinases catalytic domain (S_TKc) at the N-terminus. Furthermore, cellular distribution proved that GrTBK1 was located in the cytoplasm region. Quantitative real-time PCR analysis revealed that GrTBK1 was ubiquitously expressed in all examined organs, but especially highly in liver. Temporal expression analysis in vivo showed that the expression levels of GrTBK1 were obviously up-regulated in response to GCRV infection. Meanwhile, qRT-PCR assay revealed that the levels of S7 RNA, an important segment of GCRV genome, were higher in the liver than in other tissues. This indicates that GrTBK1 might play a crucial role in responses to GCRV infection in fish. In addition, GrTBK1 activated several type I interferon (IFN) promoters and induced the expression of downstream type I IFN-stimulated genes (ISGs). Furthermore, GrTBK1 obviously phosphorylated the interferon regulatory factor 3 (IRF3). Furthermore, overexpression of GrTBK1 remarkably decreased the GCRV proliferation. In summary, we systematically characterized GrTBK1 and illustrated its role in the innate immune response to GCRV infections.

稀有鲦鱼（Gobiocypris rarus）是一种对草鱼呼肠孤病毒（GCRV）高度敏感的小型鲤科鱼类，被认为是研究鱼类先天免疫机制的理想模型。在本研究中，鉴定了来自稀有鲦鱼的TBK1同源物 ( GrTBK1 )，并研究了其在防御病毒感染中的作用。序列分析表明，GrTBK1编码一个 727 个氨基酸的肽段，与黑鲤（Mylopharyngodon piceus）和人类（Homo sapiens）具有 98% 和 72% 的同一性。) 直系同源物，分别。氨基酸序列分析表明 GrTBK1 在 N 端含有一个保守的丝氨酸/苏氨酸蛋白激酶催化结构域 (S_TKc)。此外，细胞分布证明GrTBK1位于细胞质区域。定量实时 PCR 分析显示GrTBK1在所有检查的器官中普遍表达，但在肝脏中尤其高。体内时间表达分析显示GrTBK1的表达水平对 GCRV 感染的反应明显上调。同时，qRT-PCR 分析显示 S7 RNA（GCRV 基因组的重要片段）在肝脏中的水平高于其他组织。这表明 GrTBK1 可能在鱼类对 GCRV 感染的反应中起关键作用。此外，GrTBK1 激活几个 I 型干扰素 ( IFN ) 启动子并诱导下游 I 型干扰素刺激基因 ( ISG ) 的表达。此外，GrTBK1 明显磷酸化干扰素调节因子 3 (IRF3)。此外，GrTBK1 的过表达显着降低了GCRV增殖。总之，我们系统地描述了GrTBK1 并说明了它在对 GCRV 感染的先天免疫反应中的作用。