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

Abstract

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 48-120 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.