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

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 ofc-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.%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12h后表达量达到0h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.