水产学报2023，Vol.47Issue(12)：157-173,17.DOI:10.11964/jfc.20210612931
日本鳗鲡TBK1基因的克隆与免疫功能分析
Molecular cloning and immune functional analysis of TBK1 gene in Japanese eel(Anguilla japonica)
摘要
Abstract
As an important serine/threonine kinase in the IKK family,TANK Binding Kinase 1(TBK1)plays a critical role in innate immune response by activating NF-κB and type I IFN signaling pathways in mammals.Although several studies have reported that fish TBK1 was involved in the regulation of type I IFN production,information on TBK1's close association with antimicrobial immune responses in the regulation of NF-κB and MAPK signaling pathways is still limited in teleost fish.In order to elucidate the regulation of fish TBK1 in NF-κB,I IFN,and MAPK immune response signaling pathways,the full-length cDNA of a TBK1 homologue,AjTBK1,was cloned by SMART RACE from Japanese eel,and its characteristics of expression in response to vari-ous PAMPs and Aeromonas hydrophila infection were investigated both in vivo and in vitro by quantitative real-time polymerase chain reaction(qRT-PCR).In addition,the subcellular localization of AjTBK1 GFP fusion pro-tein and the induction of AjTBK1 overexpression in the activation of NF-κB,AP1 and type I IFN performed by Dual-Glo luciferase assay system were also detected.Amino acid sequence analysis indicated that AjTBK1 encodes a polypeptide of 731 amino acids,which has the conserved N-terminal kinase domain(KD),a ubiquitin-like domain(ULD),a scaffold dimerization domain(SDD),and a C-terminal domain(CTD).The predicted three-dimensional structure of AjTBK1 is similar to that of human TBK1,and AjTBK1 is clustered with other fish famil-ies in the phylogenetic tree.Quantitative real-time PCR(qRT-PCR)analysis revealed that AjTBK1 is broadly expressed in a wide range of tissues,with high expression in liver and intestine.In vivo,the expression of AjTBK1 in the liver of Japanese eel was significantly increased by 1.8-fold at 6h,1.8-fold at 6 h,and 1.9-fold at 48 h after injection with LPS,viral mimic poly I:C and A.hydrophila infection,respectively.The AjTBK1 expression in kid-ney was found to increase at 12 h and 24 h with 1.9-and 1.6-fold after A.hydrophila infection,but decreased fol-lowing the stimulation of LPS and poly I:C.In vitro,the AjTBK1 transcripts of Japanese eel liver cells were signi-ficantly enhanced to its peak by the treatment of LPS,poly I:C,PGN,or the 106 CFU/mL A.hydrophila,being up to the 15.3-,5.8-,9.3-and 4.7-fold,respectively.Subcellular localization studies showed that AjTBK1 was evenly distributed in the cytoplasm of HEK293 cells in natural state.AjTBK1 was found to aggregate into spots in the cytoplasm upon the stimulation of LPS or poly I:C in HEK293 cells.Additionally,luciferase assays demonstrated that the AjTBK1 overexpression significantly enhanced the activation of NF-κB,AP1,and IFNβ-responsive pro-moters in HEK293 cells,and robustly up-regulated the activation of NF-κB,AP1,and IFNβ-responsive promoter,which were 1.8-,1.2-,and 1.92-fold induced at 24 h,12 h,and 24 h after cotransfection,respectively.These res-ults collectively suggest that AjTBK1 may function as important positive regulation in innate immunity of host against antibacterial and antiviral infection likely via the activation of NF-κB,AP1,and type I IFN signaling path-ways.关键词
日本鳗鲡/TANK结合激酶1(TBK1)/信号通路/亚细胞定位/双荧光素酶活性Key words
Anguilla japonica/TANK binding kinase 1(TBK1)/signal pathway/subcellular localization/dual-luciferase reporter gene assay分类
生物科学引用本文复制引用
徐元凯,彭欣慰,林鹏,王艺磊,冯建军..日本鳗鲡TBK1基因的克隆与免疫功能分析[J].水产学报,2023,47(12):157-173,17.基金项目
福建省自然科学基金(2020J01671) （2020J01671）
鳗鲡现代产业技术教育部工程研究中心开放基金(RE202110) （RE202110）
农业农村部东海海水健康养殖重点实验室开放基金(2020ESHML02) Nature Science Foundation of Fujian Province(2020J01671) （2020ESHML02）
Engineering Research Center of the Modern Technology for Eel Industry,Ministry of Education,P.R.China(RE202110) （RE202110）
Key Laboratory and Healthy Mariculture for the East China Sea,Ministry of Agriculture and Rural Affairs,P.R.China(2020ESHML02) （2020ESHML02）
