三疣梭子蟹Na+/H+-exchanger基因克隆鉴定及在盐度胁迫下的表达分析

为研究Na+/H+-exchanger基因在三疣梭子蟹（Portunus trituberculatus）盐度胁迫过程中的功能作用，克隆了三疣梭子蟹Na+/H+-exchanger基因并进行表达分析。结果显示，Na+/H+-exchanger基因（GenBank:KU519329）全长4233 bp，5和3非编码区（UTR）长分别为519和753 bp，开放阅读框（ORF）长2961 bp。编码986个氨基酸，预测蛋白质分子量和等电点分别为110.8 kD和7.42，具有信号肽和典型的Na+/H+-exchanger蛋白结构域，含12个跨膜螺旋；三疣梭子蟹Na+/H+-exchanger基因与普通滨蟹（Carcinus maenas）同源性最高，达到87.2%，系统进化分析也显示该序列与普通滨蟹聚为一支；表达分析显示，三疣梭子蟹Na+/H+-exchanger基因在鳃中表达量最高；在低盐（盐度5、10和20）胁迫过程中，Na+/H+-exchanger基因在0-12h上调表达明显，在24-168h间表达量呈下降趋势；在高盐（盐度50）胁迫初期（0-12h），该基因表达量相对稳定，之后（24-168h）显著下调表达。研究表明低盐显著诱导Na+/H+-exchanger基因的高表达，推测三疣梭子蟹Na+/H+-exchanger基因在低盐环境下发挥重要的渗透调节功能。

NA+/H+-EXCHANGER IN SWIMMING CRAB (PORTUNUS TRITUBERCULATUS):CLONING,CHARACTERIZATION AND MRNA EXPRESSION UNDER SALINITY STRESS

Na+/H+-exchanger is a membrane-associated enzyme responsible for the active transport of Na+ and H+ ions across cell membranes and generating chemical and electrical gradients. It plays an important role in salinity adaptation process of aquatic crustacean. The swimming crab (Portunus trituberculatus) distributes widely in the coastal waters of Japan, Korean and China, which is a commercially important marine crab in China. Growth, development and immunity of the crab are significantly affected by salinity variation. In order to investigate the function of Na+/H+-exchanger in P. trituberculatus under salinity stress, Na+/H+-exchanger cDNA (GenBank:KU519329) was cloned from gill tissue of P. trituberculatus by RACE (rapid-amplification of cDNA ends). The full-length of Na+/H+-exchanger cDNA was 4 233 bp (base pairs) including a 519 bp 5'-untranslated region (UTR), a 2961 bp ORF (open reading frame) and a 753 bp 3'-UTR. The ORF encoded 986 amino acids with calculated molecular weight 110.8 kD and theoretical isoelectric point 7.42. Comparison with homologous proteins showed that the deduced Na+/H+-exchanger sequence has the highest sequence identity to Carcinus maenas (87.2%), and the two sequences were clustered into one group by phylogenetic analysis. Typical domains including one signal peptide, one Na+/H+-exchanger domain and twelve transmembrane alpha helixes were found in amino acid sequence of Na+/H+-exchanger. Results of RT-qPCR showed that P. trituberculatus Na+/H+-exchanger was the highest expressed in gill. During salinity stress, the expression of Na+/H+-exchanger of gill in low salinity groups (5, 10 and 20) increased significantly during 0-12h. Subsequently, the expression was down-regulated during 24-168h. The expression of Na+/H+-exchanger in gill in high salinity group (50) was almost no change compared to control group during 0-12h, and the expression level significantly decreased compared with the control group during 24-168h. Low salinity significantly induced the expression of Na+/H+-exchanger gene, suggesting that Na+/H+-exchanger of P. trituberculatus plays a vital role mainly in low salt conditions in the process of salinity adaptation.