| Grass carp(Ctenopharyngodon idella) is one of the most economically important freshwater fish species in China. However, this species is susceptible to grass carp reovirus(GCRV). Interferon(IFN) is an important functional cytokines, involving in the antiviral immunity, anti-tumor and immune regulation. Interferon(IFN) response is the first line of host defense against virus infection and is one of the core researches of immunology. TANK-binding kinase 1(TBK1), one member of the IκB kinase(IKK) family, plays essential roles in both antiviral and antibacterial innate immunity. TBK1 can phosphorylate interferon regulatory factors 3/7(IRF3/7) turning on the induction of IFN-I. TBK1-mediated signaling pathway is still poorly understood in teleosts, compared to the investigation in mammals.In this study, TBK1 gene was identified in C. idella(CiTBK1) by local EST database searching and RACE techniques; promoter vector was constructed and transfected into cells to explore the promoter activity; quantitative real-time RT-PCR(qRT-PCR) was performed to investigate the tissue distribution of CiTBK1 transcripts and mRNA expression profiles of CiTBK1 at different time points post viral/bacterial PAMPs stimulations; overexpression plasmid was constructed and trasfeced into cells to explore the CiTBK1-mediated immune response. The main results and conclusion were summarized as below:1. The CiTBK1 cDNA is 2840 bp in length encoding 727 amino acids. The complete genomic sequence of CiTBK1 is 10339 bp in length, containing an 1194 bp 5′-flanking region, 21 exons and 20 introns.2. Promoter activity investigation showed that dual promoters may exist for CiTBK1, one promoter upstream of exon 1 that requires the interaction with some enhancer elements and a second, stronger promoter within intron 1.3. CiTBK1 was widely expressed in all the examined tissues and expressed predominantly in trunk kidney, fllowed by foregut and spleen. After GCRV infection, expression levels of CiTBK1 mRNA were up-regulated in spleen and head kidney. In CIK cells, transcripts of CiTBK1 were elevated post GCRV infection or poly I:C, LPS, PGN stimulations.4. CiTBK1 participated in the regulation of IFN-I pathway. Post GCRV challenge, CiTBK1 over-expression inhibited viral infection by induction of CiIFN-I and CiMx1 mainly via CiIRF7 but not CiIRF3; after poly I:C stimulation, CiMx1 expression mediated by CiTBK1 was in IFN-I independent way; post LPS stimulation, CiTBK1-triggered CiIFN-I activation was in IRF3/IRF7-independent manner; CiTBK1 negatively regulated PGN-induced IFN-I immune response.5. CiTBK1 had antiviral activityand could inhibit GCRV replication.This study investigated that CiTBK1 participates in broad antiviral and antibacterial immune responses, regulating IFN-I signaling in different manners. The results will facilitate functional studies on TBK1 gene in teleosts and provided ideas for exploring effect mechanism and prevention of grass carp hemorrhagic disease as well as resistance genetic breeding. |
