日本鳗鲡IFN-γ基因的鉴定、表达模式及启动子活性分析

CHARACTERIZATION, EXPRESSION PATTERN AND PROMOTER ACTIVITY ANALYSIS OF INTERFERON-GAMMA IN JAPANESE EEL, ANGUILLA JAPONICA

  • 摘要: 为揭示鱼类IFN-γ的生物学功能, 研究从日本鳗鲡(Anguilla japonica)中克隆获得了IFN-γ基因, 命名为AjIFN-γAjIFN-γ具有脊椎动物IFN-γ的典型特征: 包括4外显子/3内含子的基因结构、C端的IFN-γ特征性氨基酸基序和1个核定位信号, 以及6个α-螺旋反向平行构成的二级结构。AjIFN-γ在日本鳗鲡所有组织中均低水平转录表达, 其中肝脏中表达量最高, 其次是皮肤和头肾。Poly I:C刺激和迟缓爱德华氏菌感染均可显著诱导AjIFN-γ在鳃、头肾、体肾和(或)脾脏中的转录表达, 表明AjIFN-γ能够参与日本鳗鲡抗菌和抗病毒的免疫过程。此外, 研究还克隆了AjIFN-γ基因的5′调控区序列共1536 bp, 并构建了一系列Aj IFN-γ 5′调控区删节突变体, 分析其启动子活性, 结果表明, 上游–240/+136区域中含有起始AjIFN-γ转录的关键启动子调控元件, –1062/–814区域存在转录的正调控元件, 而–1252/–1062区域存在转录的负调控元件。上述结果进一步丰富了鱼类IFN-γ的基础知识。

     

    Abstract: IFN-γ is a cytokine that is critical for innate and adaptive immunity against viral, some bacterial and protozoal infections. In this study, an interferon-γ gene, named AjIFN-γ, was cloned and characterized from Japanese eel, Anguilla japonica. The AjIFN-γ shared some common features with its vertebrate homologues including a 4-exon/3-intron gene structure, a typical IFN-γ characteristic motif and a predicted nuclear localization site in the predicted protein. AjIFN-γ mRNA could be detected in all the tested tissues from healthy Japanese eel with the highest in liver, followed by skin and head kidney by real-time RT-PCR. A significantly increased expression of AjIFN-γ could be found in gill, head kidney, trunk kidney and (or) spleen post intraperitoneal injection with Poly I:C or Edwardsiella tarda, which indicated a role in defense of Japanese eel against both viruses and bacteria. Furthermore, luciferase reporter assay demonstrated that the sequence from –240 bp to +136 bp in the 5′ flanking region of AjIFN-γ gene was essential for initiating the transcription of AjIFN-γ, and the sequence form –1062 bp to –814 bp may contain some positive transcriptional regulatory elements while the sequence from –1252 bp to –1062 bp may contain negative transcriptional regula-tory elements. This study provided the basis for further investigation of the expanding functions of IFN-γ molecules in immunity and other physiological processes in teleost and other animals.

     

/

返回文章
返回