Na^＋/K^＋-ATPase α-subunit in swimming crab Portunus trituberculatus： molecular cloning, characterization, and expression under low salinity stress

• Published in: Chinese journal of oceanology and limnology Vol. 33; no. 4; pp. 828 - 837
• Main Author: 韩晓琳 刘萍 高保全 王好锋 段亚飞 徐文斐 陈萍
• Format: Journal Article
• Language: English
• Published: Heidelberg Science Press 01.07.2015; Springer Nature B.V
• Subjects: 3' Untranslated regions; 5' Untranslated Regions; Active transport; Adenosine triphosphate; Amino acid sequence; Amino acid sequences; Amino acids; Analysis; Aquatic crustaceans; ATP; ATP酶; Binding sites; Biology; Cell membranes; Cladistics; Cloning; Complementary DNA; Crabs; Crustaceans; Earth and Environmental Science; Earth Sciences; Enzymes; Hepatopancreas; ions; Marine biology; Marine crustaceans; Membranes; molecular cloning; Molecular weight; muscles; Na+/K+-exchanging ATPase; Nucleotide sequence; Nucleotides; Oceanography; open reading frames; PCR; Phosphorylation; Phylogeny; Polymerase chain reaction; Portunus trituberculatus; potassium; rapid amplification of cDNA ends; reverse transcriptase polymerase chain reaction; RT-PCR分析; Salinity; Salinity effects; salt stress; Sequencing; sodium; Swimming; Transcription; Transmembrane domains; 三疣梭子蟹; 亚单位; 分子克隆; 核苷酸序列分析; 盐胁迫; 逆转录聚合酶链反应; salinity; expression; Na; ATPase; α-subunit; K; cloning
• ISSN: 0254-4059; 2096-5508; 1993-5005; 2523-3521
• DOI: 10.1007/s00343-015-4018-9

Na^＋/K^＋-ATPases are membrane-associated enzymes responsible for the active transport of Na^＋ and K^＋ ions across cell membranes, generating chemical and electrical gradients. These enzymes＇ α-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na^＋/K^＋-ATPase α-subunit eDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction （RT-PCR） and rapid amplification of eDNA end methods. Analysis of the nucleotide sequence revealed that the eDNA had a full-length of 3 833 base pairs （bp）, with an open reading frame of 3 120 bp, 5＇ untranslated region （UTR） of 317 bp, and 3＇ UTR of 396 bp. The sequence encoded a 1 039 amino acid protein with a predicted molecular weight of 115.57 kDa and with estimated pI of 5.21. It was predicted here to possess all expected features of Na^＋/K^＋-ATPase members, including eight transmembrane domains, putative ATP-binding site, and phosphorylation site. Comparison of arnino acid sequences showed that the P. tritubereulatus α-subunit possessed an overall identity of 75%-99% to that of other organisms. Phylogenetic analysis revealed that this α-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this α-subunit＇s transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that α-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, α-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab＇s Na^＋/K^＋-ATPase α-subunit was potentially involved in the observed acute response to low salinity stress.