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| 三疣梭子蟹(Portunus trituberculatus)胞内氯离子通道蛋白基因克隆及其表达分析 |
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王渝,吕建建,刘萍,高保全,段亚飞,李健,陈萍
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中国水产科学研究院黄海水产研究所；上海海洋大学,中国水产科学研究院黄海水产研究所,中国水产科学研究院黄海水产研究所,中国水产科学研究院黄海水产研究所,中国水产科学研究院黄海水产研究所,中国水产科学研究院黄海水产研究所,中国水产科学研究院黄海水产研究所
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| 摘要: |
| 本研究克隆了三疣梭子蟹(Portunus trituberculatus)胞内氯离子通道蛋白基因, 命名为PtCLIC。该基因cDNA全长2000bp, 5’和3’非编码区(UTR)长分别为76bp和1162bp, 开放阅读框长762bp, 推测编码253个氨基酸, 预测分子量为29.2kDa, 理论等电点为5.93。生物信息学分析表明, PtCLIC基因中未发现跨膜结构域, 属于不稳定蛋白; 同源性分析表明, PtCLIC基因编码的氨基酸序列与蚤状溞(Daphnia pulex)CLIC基因的同源性高达83%; 系统进化分析表明, 三疣梭子蟹与拟穴青蟹首先聚为一支; 实时荧光定量RT-PCR分析表明, PtCLIC基因在选取的所有组织中均有表达, 在肝胰腺中的相对表达量最高, 且显著高于其它组织(P <0.05)。低盐度胁迫显著改变了PtCLIC基因在三疣梭子蟹鳃和肝胰腺中的表达模式, 整体呈先上调后下调的趋势, 并发现该基因在低盐耐受和低盐敏感家系中的表达规律差异显著。研究结果表明PtCLIC基因在三疣梭子蟹渗透压调节中发挥重要作用, 可辅助三疣梭子蟹耐低盐品系的选育。 |
| 关键词: 三疣梭子蟹 胞内氯离子通道蛋白 克隆 低盐胁迫 表达分析 |
| DOI：10.11693/hyhz20140300077 |
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| 基金项目:科技部农业科技成果转化资金项目, 2013GB23260589 号; 山东省自主创新专项, 2013CXC80202 号; 国家自然科学基金资助项目, 41306177 号; 中国博士后科学基金特别资助, 2014T70668 号 |
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| CLONING AND CHARACTERIZATION OF CHLORIDE INTRACELLULAR CHANNEL GENE AND ITS EXPRESSION UNDER LOW SALINITY STRESS IN PORTUNUS TRITUBERCULATUS |
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wangyu,lvjianjian,liuping,gaobaoquan,duanyafei,lijian and chenping
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Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences；Shanghai Ocean University,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences,Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences
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| Abstract: |
| Swimming crab Portunus trituberculatus is a commercially important species in China. It is a euryhaline species and survives a wide-range salinity. However, salinity could influence its propagation significantly in an artificial environment. Chloride channels were found on the plasma membrane of virtually all eukaryotic cells and play important roles in maintenance of water and electrolyte balance, in the regulation of cell volume, and in the control of membrane potential. Chloride intracellular channel (CLIC) gene is a type of chloride channels gene important in salinity stress response. In this study, we cloned the chloride intracellular channel cDNA of P. trituberculatus by rapid amplification of cDNA ends (RACE) and named PtCLIC. The full-length of PtCLIC cDNA is 2 000 bp, including an open reading frame (ORF) of 762 bp encoding a 253-amino-acid polypeptide. PtCLIC gene had no transmembrane domains as an instability protein as revealed by bioinformatics software. PtCLIC of P. trituberculatus shared highest homology of 83% with CLIC of Daphnia pulex as indicated in homologous analysis. Shown in phylogenetic tree, P. trituberculatus cluster to Scylla paramamosian. PtCLIC gene could be detected in all tested tissues of P. trituberculatus, with the highest expression level in hepatopancreas as depicted in quantitative real-time RT-PCR analysis. After challenged by low salinity, the expression of PtCLIC gene in gill and hepatopancreas of P. trituberculatus up-regulated first and then down-regulated; and the expression of PtCLIC gene in low salinity tolerant and susceptive family showed significant difference. Therefore, PtCLIC gene was confirmed having osmotic regulation function, which may help breed low-salinity tolerant strain of P. trituberculatus. |
| Key words: Portunus trituberculatus chloride intracellular channel gene cloning low salinity stress gene expression |