半滑舌鳎(Cynoglossus semilaevis) c-Jun基因的克隆及免疫应答分析

Cloning and Expression of the c-Jun Gene in Immune Response to Pathogen Challenge in Half-Smooth Tongue Sole (Cynoglossus semilaevis)

中文关键词: 半滑舌鳎  转录因子c-Jun基因克隆  表达分析  病原感染  免疫应答

英文关键词: Half-smooth tongue sole Cynoglossus semilaevis  Transcription factor c-Jun gene cloning  Expression analysis  Pathogen challenge  Immune response

中文摘要:

      半滑舌鳎(Cynoglossus semilaevis)AP-1家族转录因子c-Jun基因(Jun proto-oncogene)及其在免疫应答中的作用尚无报道。本研究根据半滑舌鳎转录组数据库中预测的c-Jun序列，通过RACE技术和PCR扩增方法获得了半滑舌鳎c-Jun基因cDNA全长2093 bp，CDS区域共981 bp，编码326个氨基酸，5¢ UTR区域377 bp，3¢ UTR区域735 bp。SMART分析显示，C-JUN蛋白具有2个结构域：AP-1家族典型结构域Jun，以及高度保守的亮氨酸拉链结构域(BRLZ)。经蛋白多序列同源比对、系统进化树分析，发现半滑舌鳎c-Jun与虹鳟(Oncorhynchus mykiss) c-Jun亲缘关系最近。实时荧光定量PCR分析显示，c-Jun基因在半滑舌鳎不同组织中普遍表达，在卵巢中表达量最高。鳗弧菌(Vibrio anguillarum)人工感染半滑舌鳎后，c-Jun基因在肝脏、脾脏、头肾、小肠、鳃、血液中表达量都出现不同程度上调，其中，鳃中变化最明显：感染12 h后表达量达到0 h时的13.20倍。使用LPS、PGN、PolyI:C和WGP病原模拟物刺激半滑舌鳎外周血淋巴细胞，结果显示，WGP诱导c-Jun基因上调表达，而LPS与PolyI:C均下调基因表达。以上实验结果表明，c-Jun基因在半滑舌鳎的免疫防御中发挥重要作用。

英文摘要:

      The transcription factor c-Jun (jun proto-oncogene) of AP-1 family and its role in immune response is unknown in half-smooth tongue sole (Cynoglossus semilaevis) so far. Based on c-Jun genetic partial cDNA sequence of half-smooth tongue sole, the complete cDNA sequence is obtained by RACE and PCR application. The cDNA of c-Jun is 2093 bp in length, including a 377 bp 5¢-terminal untranslated region (UTR), a 735 bp 3¢-terminal UTR and a 981 bp open reading frame (ORF), encoding a polypeptide of 326 amino acids. The deduced amino acid sequence of c-Jun possesses a Jun domain which is a typical structure domain in AP-1 family and a high conservative leucine zipper structure domain (BRLZ). The protein sequence alignment and phylogenetic tree analysis suggested that the half-smooth tongue sole c-Jun gene is closely related to that of the rainbow trout’s (Oncorhynchus mykiss). Tissue expression analysis by quantitative real-time reverse transcription PCR showed that the c-Jun mRNA was constitutively expressed in all examined tissues with the predominant expression in the ovay. After infection with Vibrio anguillarum, the half-smooth tongue sole c-Jun transcript levels exhibited distinct time-dependent response patterns in the liver, spleen, head kidney, intestine, gill and blood. Among the tested tissues, changes in gill is the most obvious, and it reached the peak level 13.20-fold at 12 h post-infection. After challenge with different pathogenic (LPS, PGN, PolyI:C, WGP) in lymphocyte, the c-Jun gene in the WGP group showed up-regulation, while in the LPS group and PolyI:C group it showed down-regulation, while, there was no significant change in the PGN group. The results suggested that the c-Jun gene plays a vital role in the immune response in half-smooth tongue sole and might play an important role in half-smooth tongue sole host defense against the bacteria infection.