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

Description

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.

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1 Experimental animals and husbandry conditions Healthy Hong Kong oysters with an average weight of 134.54 ± 36.05 g were collected from the Maowei Sea culture area of Qinzhou City, Guangxi Province. The oysters were collected in polyvinyl chloride boxes and temporarily reared for 7 d under natural conditions of salinity 16 ‰ and a water temperature of 25.8 °C. During the temporary rearing period, the oysters were continuously oxygenated and baited, and the water was changed every day. 2 Tissue expression of ChNa/K-ATPase and expression under high-salt stress in the Hong Kong oyster The gills, adductor muscles, mantles, digestive glands, labial palps, hemolymphs, sinusoids, and gonads of 15 Hong Kong oysters maintained under normal conditions were collected and placed in liquid nitrogen for snap-freezing. Two groups of oysters maintained at salinity 16 ‰ (control group) and 30 ‰ (high-salt stress group) were established, with three replicates per group. Thirty Hong Kong oysters were placed in replicates per group, and samples were collected at 0, 8, 24, 48, 72, 96, and 120 h after high-salt stress. snap frozen in liquid nitrogen, and then stored at -80℃. Total RNA was extracted using Trizol, and was reversed into cDNA using the SweScript All in One RT SuperMix for qPCR kit produced by TransGen Biotech Co., Ltd. Using 2 × Universal Blue SYBR Green qPCR Master Mix (TransGen Biotech, China) as a fluorescent quantitative reagent to detect the mRNA expression levels of ChNa/K-ATPase gene in different tissues and under high salt stress. 3 ChNa/K-ATPase interference assay 3.1 Effect of RNA interference on ChNa/K-ATPase gene expression Two hundred and seventy oysters were divided into three groups with Ninety oysters in each group. Before the test, the oysters were immersed in 35 g/L MgCl2 and the prepared siRNA solution was injected into the adductor muscles of the oysters after they opened their shells naturally. For the ChNaK-ATPase-siRNA group, 100 μL of Na/KRNAi was injected into each oyster and for the Negative group, 100 μL of 100μl NC (Negative control) was injected into each oyster. The blank control group, 100 μL of PBS was injected into each oyster. RNA was prepared from the gill tissues of nine oysters randomly selected from each group at each time point (0, 8, 24, 48, 72, and 96 h after injection) for reverse transcription and the level of RNA interference was confirmed by RT-qPCR. 3.2 Determination of Na/K-ATPase enzyme activity after RNA interference The Hong Kong oyster gill tissue was placed in 9 volumes (W/V) of normal saline, and after grinding and centrifugation at 4℃, the supernatant was collected. Total protein content was determined using Coomassie Blue reagent. At the same time, Na/ K-ATPase activity was measured using the Na/ K-ATPase kit (Nanjing Jiancheng Bioengineering lnstitute), and the activity was expressed as U/mgprot.

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