日本鳗鲡TRAF3基因的克隆、表达及亚细胞定位分析  

Molecular Cloning,Expression Analysis in vivo and in vitro,and Subcellular Localization of TRAF3 from Japanese Eel(Anguilla japonica)

作　　者：陈鹏云 林鹏 王艺磊[1,2,3] 王添禹 张炎[1,2,3] 冯建军 CHEN Pengyun;LIN Peng;WANG Yilei;WANG Tianyu;ZHANG Yan;FENG Jianjun(Key Laboratory of Healthy Mariculture for the East China Sea,Ministry of Agriculture,Xiamen 361021 China;Engineering Research Centre of Eel Modern Technical Industry,Ministry of Education,Xiamen 361021,China;Fisheries College,Jimei University,Xiamen 361021,China)

机构地区：[1]农业农村部东海海水健康养殖重点实验室,福建厦门361021 [2]鳗鲡现代产业技术教育部工程研究中心,福建厦门361021 [3]集美大学水产学院,福建厦门361021

出　　处：《集美大学学报（自然科学版）》2023年第3期193-204,共12页Journal of Jimei University：Natural Science

基　　金：福建省自然科学基金项目(2020J01671,2021J01830);鳗鲡现代产业技术教育部工程研究中心开放基金项目(RE202208,RE202307);农业农村部东海海水健康养殖重点实验室开放基金项目(2020ESHML02)。

摘　　要：肿瘤坏死因子受体(TNFR)相关因子3(TRAF3)是激活NF-κB、I型IFN信号通路的重要调节因子。克隆了日本鳗鲡TRAF 3基因全长cDNA序列,命名为AjTRAF 3,可编码568个氨基酸,具有保守的TRAF结构域(MATH结构域)、锌指(ZF)结构域和环指结构域(RING domain)。系统发育树分析表明AjTRAF3与其他鱼类TRAF3聚为一支,而哺乳类、鸟类、两栖类分别聚为一支。实时荧光定量PCR(qRT-PCR)结果显示:天然状态下日本鳗鲡各组织均检测到有AjTRAF 3基因的表达,其中肝脏表达量最高;LPS、Poly I:C、嗜水气单胞菌免疫注射日本鳗鲡后,能引起肝脏、脾脏和肾脏中AjTRAF 3基因的表达水平显著提高;日本鳗鲡肝脏细胞经不同病原相关分子模式LPS、Poly I:C、CpG、PGN和不同浓度嗜水气单胞菌感染刺激后,其AjTRAF 3基因的表达水平均显著升高。亚细胞定位研究显示天然状态下Aj TRAF3蛋白在细胞质中聚集呈散点状分布,经LPS和Poly I:C刺激后,Aj TRAF3蛋白分子可进入细胞核且呈散点状分布。以上结果表明AjTRAF 3在日本鳗鲡抗病毒、细菌免疫应答反应中发挥重要作用。Tumor necrosis factor receptor-associated factor 3(TRAF3)plays an important role in the activation of NF-κB and type I IFN signaling pathway.In the present study,the full-length cDNA of a TRAF 3 homologue,AjTRAF 3,was cloned from Japanese eel.AjTRAF 3 encoded a polypeptide of 586 amino acids,which had the conserved TRAF domain(MATH),zinc finger(ZF)domain,and ring domain.AjTRAF 3 was clustered together with other fish families in the phylogenetic tree,whereas mammals,birds,and amphibians were grouped into different separated branches.Quantitative real-time PCR(qRT-PCR)analysis revealed a broad expression for AjTRAF3 in a wide range of tissues,and was highly expressed in the liver.In vivo,the AjTRAF 3 expressions in the liver and the kidney of A.japonica were significantly increased following injection with the bacterial mimic LPS,the viral mimic Poly I:C and Aeramonas hydrophila.The expression level of AjTRAF 3 gene in eel liver cells was significantly increased after being stimulated by different pathogen related molecular models LPS,Poly I:C,CpG,PGN and different concentrations of A.hydrophila infection.Subcellular localization studies showed that Aj TRAF3 was scattered in the cytoplasm of cells under natural state.Aj TRAF3 was found to aggregate into spots partly in the nucleus after the stimulation of LPS and Poly I:C.These results collectively suggested that AjTRAF 3 was an important factor possibly involved in Japanese eel defense against viral and bacterial infection.

关 键 词：日本鳗鲡 TRAF 3基因 表达分析 亚细胞定位 

分 类 号：S968.43[农业科学—水产养殖]