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HAN Xiaolin, LIU Ping, GAO Baoquan, WANG Haofeng, DUAN Yafei, XU Wenfei, CHEN Ping. Na+/K+-ATPase α-subunit in swimming crab Portunus trituberculatus : molecular cloning, characterization, and expression under low salinity stress[J]. Journal of Oceanology and Limnology, 2015, 33(4): 828-837 |
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Na+/K+-ATPase α-subunit in swimming crab Portunus trituberculatus : molecular cloning, characterization, and expression under low salinity stress |
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| HAN Xiaolin1,2, LIU Ping1, GAO Baoquan1, WANG Haofeng1, DUAN Yafei1, XU Wenfei1,2, CHEN Ping1 |
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1 Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China;
2 Dalian Ocean University, Dalian 116023, China |
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| Abstract: |
| 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 cDNA was cloned from gill tissue of the swimming crab Portunus trituberculatus by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end methods. Analysis of the nucleotide sequence revealed that the cDNA 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 amino acid sequences showed that the P. trituberculatus 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. |
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| Key words:
cloning|expression|Na+/K+-ATPase|α-subunit|Portunus trituberculatus|salinity
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| Received: 2014-02-20 Revised: 2014-11-24 |
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