Kong Lingxin, Chen Ziao, Peng Yeshao, Deng Qiong, Jia Zhen, Zhu Peng, Xu Youhou, She Zhicai
Guangxi Key Laboratory of Beibu Gulf Marine Biodiversity Conservation, Pinglu Canal and Beibu Gulf Coastal Ecosystem Observation and Research Station of Guangxi, College of Marine Sciences, Beibu Gulf University, Qinzhou, 535011, China.
Guangxi Key Laboratory of Beibu Gulf Marine Biodiversity Conservation, Pinglu Canal and Beibu Gulf Coastal Ecosystem Observation and Research Station of Guangxi, College of Marine Sciences, Beibu Gulf University, Qinzhou, 535011, China.
Dev Comp Immunol. 2025 Aug;169:105397. doi: 10.1016/j.dci.2025.105397. Epub 2025 May 27.
Na/K-ATPase is a carrier protein embedded in the cell membrane. The main function of this protein is to maintain osmotic pressure balance inside and outside the cell. To investigate the role of the Na/K-ATPase gene under high-salt stress in the Hong Kong oyster (Crassostrea hongkongensis), full-length cloning and expression analysis of Na/K-ATPase β subunit (ChNa/K-ATPase-β) were performed. Additionally, the gene function was verified using RNA interference (RNAi) technology. In this study, we obtained a 1230 bp sequence of the ChNa/K-ATPase-β gene, 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 revealed the expression of ChNa/K-ATPase-β in all tissues of 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 labial palps. Under hypersalinity stress (30 PSU), the expression levels of ChNa/K-ATPase-β in the gills significantly upregulated at 8 h, downregulated at 24 h, and showed no significant change between 48 and 120 h. RNAi-mediated knockdown of ChNa/K-ATPase-β led to a concomitant downregulation of ChNa/K-ATPase-α expression. After interfering with ChNa/K-ATPase-β for 48, 72, 96, and 120 h, the Na/K-ATPase activity in the gills significantly decreased. The survival rate of the RNAi group (75 %) was lower than that of the blank control and negative control groups, which proved that Na/K-ATPase dysfunction would seriously affect the resistance of oysters to high salt stress. These results indicate that ChNa/K-ATPase-β plays an important role in enhancing the salt stress resistance in the Hong Kong oyster.
钠钾-ATP酶是一种嵌入细胞膜的载体蛋白。该蛋白的主要功能是维持细胞内外的渗透压平衡。为了研究钠钾-ATP酶基因在香港牡蛎（Crassostrea hongkongensis）高盐胁迫下的作用，对钠钾-ATP酶β亚基（ChNa/K-ATPase-β）进行了全长克隆和表达分析。此外，利用RNA干扰（RNAi）技术验证了该基因的功能。在本研究中，我们获得了ChNa/K-ATPase-β基因1230 bp的序列，包括25 bp的5'非翻译区（UTR）；263 bp的3'UTR；以及942 bp的开放阅读框，编码一个313个氨基酸的蛋白质，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显示ChNa/K-ATPase-β在香港牡蛎的所有组织中均有表达，在血窦中的相对表达水平最高，其次是鳃，在唇瓣中的表达水平最低。在高盐胁迫（30 PSU）下，鳃中ChNa/K-ATPase-β的表达水平在第8小时显著上调，在第24小时下调，在48至120小时之间无显著变化。RNAi介导的ChNa/K-ATPase-β敲低导致ChNa/K-ATPase-α表达随之下调。在干扰ChNa/K-ATPase-β 48、72、96和120小时后，鳃中的钠钾-ATP酶活性显著降低。RNAi组的存活率（75%）低于空白对照组和阴性对照组，这证明钠钾-ATP酶功能障碍会严重影响牡蛎对高盐胁迫的抗性。这些结果表明，ChNa/K-ATPase-β在增强香港牡蛎的盐胁迫抗性中起重要作用。
