合浦珠母贝PfCLEC17A基因的克隆及表达分析  

Molecular Cloning and Expression Analysis of PfCLEC17A in Pearl Oyster Pinctada fucata

作　　者：郑玉斯 王培 郭颖 白丽蓉 喻达辉 赵森 ZHENG Yusi;WANG Pei;GUO Ying;BAI Lirong;YU Dahui;ZHAO Sen(Guangxi Key Laboratory of Beibu Gulf Marine Biodiversity Conservation,Beibu Gulf University,Qinzhou 535011,China)

机构地区：[1]北部湾大学广西北部湾海洋生物多样性养护重点实验室,广西钦州535011

出　　处：《华北农学报》2022年第5期230-238,共9页Acta Agriculturae Boreali-Sinica

基　　金：广西自然科学基金项目(2021GXNSFAA220031);国家自然科学基金项目(31860734,31873042)。

摘　　要：为了探究C型凝集素(C-type lectin)家族成员在合浦珠母贝机体内的作用及表达模式,为进一步解析合浦珠母贝PfCLEC17A的免疫应答机制,通过RACE技术克隆获得合浦珠母贝C型凝集素基因PfCLEC17A;利用生物信息学方法分析了PfCLEC17A的分子特征,并通过荧光定量PCR检测PfCLEC17A基因在合浦珠母贝的鳃、外套膜、闭壳肌、性腺、血液、足和肝胰腺等组织及溶藻弧菌感染后在肝胰腺中的表达情况。结果显示,从合浦珠母贝中克隆获得新的C型凝集素基因PfCLEC17A,cDNA全长为699 bp,其中开放阅读框(ORF)长534 bp,编码178个氨基酸残基,含有一个CTL结构域。系统进化树分析表明,合浦珠母贝与其他贝类的PfCLEC17A基因聚集在一个分支。实时荧光定量PCR结果显示,PfCLEC17A在合浦珠母贝的鳃、外套膜、闭壳肌、性腺、血液、足和肝胰腺等各个组织中均有表达,其中在肝胰腺中的相对表达量最高且显著高于在其他组织中的相对表达量。肝胰腺作为软体动物重要的免疫器官,暗示PfCLEC17A基因可能参与机体免疫过程。藻弧菌感染1 h后,合浦珠母贝PfCLEC17A基因表达水平显著升高并达到最大值,说明该基因参与弧菌等微生物刺激的早期反应。综上所述,PfCLEC17A基因参与合浦珠母贝的免疫应激反应,具有一定的免疫功能。To explore the immune function and expression pattern of C-type lectin family members in Pinctada fucata,a cDNA sequence of C-type lectin gene PfCLEC17 A in P.fucata was cloned through RACE technique.The molecular characteristics of PfCLEC17 A were determined by bioinformatics analysis and its mRNA expression level was detected in different tissues including gill,mantle,adductor muscle,gonad,blood,foot and hepatopancreas,as well as expression level in hepatopancreas after Vibrio alginolyticus challenged using the Real-time fluorescent quantitative PCR.The results showed that a new C-type lectin gene PfCLEC17 A was successfully cloned from P.fucata,the full cDNA was 699 bp in length with an open reading frame(ORF)of 534 bp,which encoded 178 amino acids.The deduced amino acid sequence was predicated containing a CTL domain.Phylogenetic tree analysis showed that the PfCLEC17 A of P.fucata and other shellfish proteins were clustered together in a branch.The Real-time fluorescent quantitative PCR results showed that PfCLEC17 A was expressed in all tissues detected in P.fucata,and the highest was found in hepatopancreas,which was significantly higher than the other tissues.The high expression level in hepatopancreas,one of the most important immune tissues in mollusc,suggested that PfCLEC17 A gene might be involved in the body′s immune reaction.The expression level of PfCLEC17 A increased significantly and reached the peak at 1 h after V.alginolyticus challenge,indicating that the gene expression was induced by microbial stimulation at early stage.Therefore,based on the above findings,it could be concluded that the PfCLEC17 A gene participates in the immune activation response of P.fucata and functions in immune system.

关 键 词：合浦珠母贝 C型凝集素 PfCLEC17A 基因克隆 基因表达 

分 类 号：Q78[生物学—分子生物学] S917[农业科学—水产科学]