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

文献类型： 外文期刊

第一作者： Han, Xiaolin

作者： Han, Xiaolin;Liu, Ping;Gao, Baoquan;Wang, Haofeng;Duan, Yafei;Xu, Wenfei;Chen, Ping;Han, Xiaolin

作者机构：

关键词： cloning;expression;Na+/K+-ATPase;alpha-subunit;Portunus trituberculatus;salinity

期刊名称：CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY （ 影响因子：1.068； 五年影响因子：0.983 ）

ISSN： 0254-4059

年卷期： 2015 年 33 卷 4 期

页码：

收录情况： SCI

摘要： 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' alpha-subunit provides catalytic function, binding and hydrolyzing ATP, and itself becoming phosphorylated during the transport cycle. In this study, Na+/K+-ATPase alpha-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 alpha-subunit possessed an overall identity of 75%-99% to that of other organisms. Phylogenetic analysis revealed that this alpha-subunit was in the same category as those of crustaceans. Quantitative real-time RT-PCR analysis indicated that this alpha-subunit's transcript were most highly expressed in gill and lowest in muscle. RT-PCR analysis also revealed that alpha-subunit expression in crab gill decreased after 2 and 6 h, but increased after 12, 24, 48, and 72 h. In addition, alpha-subunit expression in hepatopancreas of crab decreased after 2-72 h. These facts indicated that the crab's Na+/K+-ATPase alpha-subunit was potentially involved in the observed acute response to low salinity stress.

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