魁蚶(Scapharca broughtonii)半乳糖凝集素(SbGal)基因cDNA的克隆及表达分析  

CLONING AND EXPRESSION ANALYSIS OF GALECTIN FROM SCAPHARCA BROUGHTONII(SbGal)

作　　者：郑利兵[1,2] 吴彪[1] 刘志鸿[1] 周丽青[1] 孙秀俊[1] 田吉腾[1] 杨爱国[1] 郑言鑫 

机构地区：[1]农业部海洋渔业可持续发展重点实验室中国水产科学研究院黄海水产研究所,青岛266071 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《海洋与湖沼》2015年第5期1061-1070,共10页Oceanologia Et Limnologia Sinica

基　　金：山东省自然科学基金项目;ZR2013CQ047号;科技基础条件平台项目"黄渤海区水生物种质资源标准化整理;整合与共享"

摘　　要：半乳糖凝集素(Galectin)作为动物凝集素家族的一员,是一种重要的免疫因子,在脊椎动物和无脊椎动物中都参与了重要的免疫防御反应。为明确魁蚶(Scapharca broughtonii)半乳糖凝集素基因的结构特征、组织分布及其对病原菌刺激的免疫反应规律,本研究从构建的魁蚶转录组文库中筛选得到半乳糖凝集素基因部分序列,通过c DNA末端快速扩增(rapid amplication of c DNA ends,RACE)技术获得该基因c DNA全长序列(命名为Sb Gal)。序列和结构分析表明,该c DNA全长3120bp,编码554个氨基酸,是一个含有4个糖识别结构域(CRD)的且在双壳贝类中较为独特的半乳糖凝集素;预测蛋白的分子量(Mw)为63.3158 k Da,理论等电点(p I)为4.99。同源性及系统分析表明,Sb Gal基因与美洲牡蛎、合浦珠母贝、海湾扇贝galectin的同源性分别为70%、65%、46%,Sb Gal基因四个CRD彼此的相似性为36%—41%。实时荧光定量PCR(q RT-PCR)结果表明:Sb Gal m RNA在魁蚶血细胞、斧足、闭壳肌、外套膜、鳃和肝胰腺中均有表达,血细胞中的表达量最高,在肝胰腺中的表达量最低;注射鳗弧菌后,相对于对照组,Sb Gal基因在所检测的每个组织中m RNA水平上的表达量都显著上调(P<0.01),而且表达量具有显著的时间依赖性和瞬时表达趋势,呈现先升高后降低的趋势。Galectins are a family of β-galactoside-binding lectins that play important roles in innate immunity of both vertebrates and invertebrates. In the present study, the c DNA of a galectin with multiple carbohydrate recognition domains（CRDs） was cloned from ark shell Scapharca broughtonii（designated Sb Gal） by partial c DNA sequence selected from the transcriptome library by rapid amplification of c DNA ends（RACE） technique. The full length c DNA of Sb Gal, another specific galectin in bivalve, was 3120 bp in length encoding a polypeptide of 554 amino acids, and contained four carbohydrate-recognition domains. The predicted molecular weight was 63.3158 k Da and the theoretical isoelectric point was 4.99. The results of multiple sequences alignment and phylogeny analysis show that the identity of Sb Gal shared with Crassostrea viginica, Pinctada fucata, and Argopecten irradians was 70%, 65%, and 46%, respectively. And the four domains of Sb Gal shared 36%—41% identities with each other. Quantitative Real-time PCR（q RT-PCR） was used to assess the m RNA distribution in all tested tissues, including hemocytes, foot, adductor muscle, mantle, gill, and hepatopancreas. The highest expression level was observed in hemocytes, while the lowest in hepatopancreas. After being challenged by Vibrio anguillarum, the relative m RNA expression level of Sb Gal was up-regulated significantly（P0.01）. The timing of maximum expression occurrence and returning to the initial level were different among the tissues. Therefore, Sb Gal expression is of obvious time dependence and instantaneous expression pattern, and it could function as a pattern recognition receptor in immune defense of ark shells.

关 键 词：魁蚶 半乳糖凝集素 基因表达 免疫反应 

分 类 号：Q75[生物学—分子生物学]