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

    摘要
  • 摘要: 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。

     

    Abstract: 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 TRAF3 proteins. 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.

     

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