三疣梭子蟹水通道蛋白 1 cDNA 及其盐度胁迫下的表达分析

采用RACE技术克隆获得总长为1 712 bp的三疣梭子蟹(Portunus trituberculatus)水通道蛋白基因全长cDNA序列,命名为PtAQP基因.该基因5'和3'非编码区分别为153 bp和788 bp,开放阅读框为771 bp,推测编码256个氨基酸,预测分子量为27.0 kD,理论等电点为8.26.生物信息学分析表明,PtAQP含有6个跨膜区和2个NPA单元,具有与MIP家族匹配的保守氨基酸序列,属于稳定蛋白;同源性和系统进化分析表明,三疣梭子蟹PtAQP氨基酸序列与可口美青蟹(Callinectes sapidus) AQP1的同源性最高(87％),与可口美青蟹紧密聚为一支;荧光定量RT-PCR分析表明,PtAQP基因在各组织中均有表达,而在胃中的相对表达量最高.通过分析PtAQP基因在盐度胁迫中的表达规律发现,盐度胁迫可显著改变PtAQP基因在三疣梭子蟹鳃和肝胰腺中的表达模式,整体呈先下降后上升最后下降到初始水平的表达趋势.该研究结果表明,PtAQP基因在三疣梭子蟹渗透压调节中发挥重要作用.

Cloning and characterization of aquaporins 1 and its expression analysis under salinity stress in Portunus trituberculatus

 The swimming crab, Portunus trituberculatus, is a commercially important fishery species and widely distributed on sandy and muddy bottoms in the coastal waters of Japan, Democeratic People’s Republic of Korea , China and Malaysia. It is a euryhaline crab species, surviving in wide-range salinity conditions, and water salinity condition influenced its artificial propagations significantly. Aquaporins (AQP) which belong to a major intrinsic proteins (MIP) family plays important roles in salinity stress response. In this study, aquaporins cDNA of Portunus trituberculatus was cloned by rapid amplification of cDNA ends (RACE) and named PtAQP. The full-length of PtAQP cDNA is 1 712 bp, including an open reading frame (ORF) of 771 bp which encodes a 256 amino acid polypeptide. Bioinformatics software analysis reavealed that PtAQP gene contains 6 transmembrane domains, 2 NPA structural units and conservative amino acid sequence which is matched to MIP family. Homologous analysis showed that PtAQP of Portunus trituberculatus has the highest homology to AQP1 of Callinectes sapidus. The quantitative real-time RT-PCR showed that PtAQP gene could be detected in all tested tissues of Portunus trituberculatus, with the highest expression level in stomach. After challenged by high and low salinity, the expression of PtAQP gene in gill and hepatopancreas of Portunus trituberculatus first fell then rase. Our results confirmed the osmotic regulation function of PtAQP gene and revealed the regulatory mechanism of PtAQP gene under salinity stress.