罗非鱼TRAF3基因的表达及功能研究

CHARACTERIZATION OF TUMOR NECROSIS FACTOR RECEPTOR ASSOCIATED FACTOR 3(TRAF3)IN NILE TILAPIA:EXPRESSION PROFILES AND FUNCTIONS IN NF-κB PATHWAY

肿瘤坏死因子受体(TNFR)相关因子3(TRAF3)是多种免疫途径中的关键调控因子。从尼罗罗非鱼(Oreochromis niloticus)中克隆获得了TRAF3基因,命名为OnTRAF3(GeneBank No.MN258118),该基因包含1个环指结构域、1个锌指结构域、1个卷曲螺旋和MATH结构域。多序列比对表明,OnTRAF3与其他已知的TRAF3蛋白具有高度的相似性,尤其是MATH结构域。实时荧光定量PCR(qRT-PCR)分析显示,OnTRAF3在各组织中广泛分布,且在脑、皮肤、肠和鳃中表达量较高。在无乳链球菌诱导后,多个组织中的OnTRAF3表达量出现了不同程度的上调,说明OnTRAF3参与了罗非鱼的抗菌免疫应答。亚细胞定位实验显示,OnTRAF3分布在HEK293的细胞质和细胞核中。此外,荧光素酶报告基因实验结果显示,野生型(WT)OnTRAF3可显著激活NF-κB信号,而coiled-coil和MATH结构域缺失后,依然能够显著激活NF-κB活性,而RING和Zinc缺失后,该激活作用则明显减弱,表明RING和Zinc结构域是OnTRAF3在免疫信号通路中行使功能的关键结构域。研究为探索TRAF3在罗非鱼免疫应答中的功能提供了重要的基础。

Tumor necrosis factor receptor(TNFR)-associated factor 3(TRAF3)is a highly versatile regulator in many immune pathways,including TNFR and Toll-like receptor(TLRs)/RIG-I-like receptors(RLRs)signal pathway.In this study,a TRAF3 gene was cloned from Oreochromis niloticus(named OnTRAF3),(GenBank No.MN258118),which contained a RING finger,a zinc finger,a coiled-coil and MATH domain.Multiple sequence alignment showed that OnTRAF3 shares a relatively high identity with other known TRAF3proteins.Quantitative real-time PCR(qRT-PCR)analysis showed OnTRAF3 transcripts were highly expressed in brain,skin,intestine and gill,and it could be induced by Streptococcus agalactiae.In HEK293T cells,OnTRAF3 presented both in the cytoplasm and nucleus.In addition,wide type(WT)OnTRAF3,activated NF-κB signal significantly,RING or Zinc finger was important for this activation.This study provides significant insights into the functions of TRAF3 in tilapia.