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.