Cloning and RNA interference indicate that the Na/K-ATPase gene helps Hong Kong oysters (Crassostrea hongkongensis) resist short-term hypersalinity stress

Na/K-ATPase is a carrier protein embedded in the cell membrane and its main function is to maintain the osmotic pressure balance inside and outside the cell. To investigate the role of Na/K-ATPase under high-salt stress in the Hong Kong oyster (Crassostrea hongkongensis), full-length cloning and expression analysis of Na/K-ATPase were performed, and the function of the gene was verified using RNA interference (RNAi) technology. The full-length sequence was 1,230 bp, including a 5' untranslated region (UTR) of 25 bp; a 3’ UTR of 263 bp; and an open reading frame of 942 bp, encoding a protein of 313 amino acids, with a molecular weight 35.452 KDa and a theoretical isoelectric point 5.55. Reverse transcription-quantitative polymerase chain reaction results showed that Na/K-ATPase is expressed in all tissues of the Hong Kong oyster, with the highest relative expression level in the blood sinus, followed by the gills, and the lowest level of expression in the labellum. Under conditions of acute high-salt stress, the expression level of Na/K-ATPase in the gills increased and then decreased. Using RNAi technology, it was found that Na/K-ATPase enzyme activity in the gills decreased significantly after 72, 96, and 120 h of high-salinity stress, and the survival rate of the RNAi group was lower than that of the blank control group and the negative control group with only 75%. These results indicated that Na/K-ATPase is closely related to osmoregulation in Hong Kong oysters and plays an important role in osmotic pressure regulation and resistance to salinity stress.

Na+/K+-ATP酶是一种嵌于细胞膜上的载体蛋白，其主要功能在于维持细胞内外渗透压的平衡。为探究香港牡蛎（Crassostrea hongkongensis）在高盐胁迫下Na+/K+-ATP酶的作用，本研究对其进行了全长克隆及表达分析，并通过RNA干扰（RNAi）技术验证了该基因的功能。全长序列为1,230碱基对，包含25碱基对的5'非编码区（UTR）、263碱基对的3'非编码区以及942碱基对的开放阅读框，编码由313个氨基酸组成的蛋白质，分子量为35.452 KDa，理论等电点为5.55。逆转录-定量聚合酶链反应结果显示，Na+/K+-ATP酶在香港牡蛎的所有组织中均有表达，其相对表达水平最高者为血液窦，次之为鳃，最低者为唇瓣。在急性高盐胁迫条件下，鳃中Na+/K+-ATP酶的表达水平先升高后降低。通过RNAi技术发现，在高盐胁迫下72小时、96小时和120小时后，鳃中Na+/K+-ATP酶的酶活性显著下降，RNAi组的存活率低于空白对照组及仅含阴性对照组的75%。这些结果表明Na+/K+-ATP酶与香港牡蛎的渗透调节密切相关，并在渗透压调节及耐盐胁迫中发挥着重要作用。