盐度胁迫对缢蛏渗透压、游离氨基酸及肌肽合成酶基因的影响

Effects of salinity stress on osmotic pressure, free amino acid levels, and the carnosine synthetase gene of Sinonovacula constricta

中文关键词: 缢蛏  盐度  渗透压  游离氨基酸  肌肽合成酶

英文关键词: Sinonovacula constricta  Salinity  Osmotic pressure  Free amino acids  Carnosine synthase

中文摘要:

      缢蛏(Sinonovacula constricta)为广盐性贝类，自身存在抵抗外界盐度胁迫的渗透调节机制。为探究缢蛏在适应盐度胁迫过程中发挥渗透调节作用的主要游离氨基酸(free amino acids, FAA)及其降解途径，本研究测定了不同盐度(5、20和35)胁迫下缢蛏组织的渗透压、FAA含量，并结合其肌肽合成酶基因(carnosine synthase, Sc-CARNS)的表达及相关生理指标的变化研究了Sc-CARNS基因的功能。结果显示，缢蛏鳃、足和血淋巴中渗透压和总游离氨基酸含量随着盐度的升高而升高，且各组织渗透压都在盐度胁迫24 h后达到稳态。此时，缢蛏鳃、足和血淋巴中随盐度变化含量变化最明显的FAA分别为Ala、Gly、Glu、Pro，Ala、Gly、Arg、Tua和Ala、Ser、Thr、Gly。前期研究结果显示，在缢蛏盐度胁迫转录组中，Sc-CARNS表达上调，与盐度调节相关。Sc-CARNS在缢蛏肌肉型组织中表达量最高。低盐胁迫下，Sc-CARNS mRNA表达量和肌肽含量显著升高(P<0.05)，丙氨酸(Ala)含量变化趋势与肌肽相反。受RNA干扰后，正常盐度和低盐胁迫下，Sc-CARNS基因在足中mRNA表达量和肌肽含量先下降后升高，丙氨酸含量变化则相反，48 h干扰效率和含量变化最高，且肌肽与丙氨酸是1∶1的转化。结果表明，缢蛏属于渗透压随变者，体内FAA中的丙氨酸在渗透调节方面的贡献率最大，肌肽合成酶(CARNS)是丙氨酸代谢为肌肽的关键酶，参与渗透压调节过程。本研究揭示了低盐胁迫下缢蛏渗透压调节的关键机理，为揭示竹蛏科(Solenida)贝类独特的盐度耐受性机制提供了依据。

英文摘要:

      Sinonovacula constricta is one of the four traditional cultured shellfish in China. The salinity in the aquaculture water body is easily affected by tides, seasonal rainfall, and high temperatures, and often fluctuates to different degrees. This affects the physiological activities of S. constricta and causes a series of changes in the structure of the osmoregulation organs, osmotic pressure, ion transport, and free amino acid (FAA) content in the body to adapt to the changes in environmental salinity. Aquatic animals can regulate cell volume and maintain osmotic pressure balance through FAAs. This mechanism has been proven in aquatic animals, such as Meretrix lusoria, Crassostrea gigas, Haliotis discus hannai, Penaeus vannamei, and Portunus trituberculatus. The common FAAs that regulate osmotic pressure in bivalves mainly include Ala, Gly, Pro, and Tau. Whether FAAs play an osmoregulation role in S. constricta, whether their involvement in osmoregulation is similar to that in other shellfish, and what the metabolic pathway is of main FAAs deserve further study. This study explored the changes of osmotic pressure and FAAs in the gill, foot, and hemolymph of S. constricta after salinity stress and analyzed the sequence characteristics, tissue expression, and mRNA expression characteristics after salinity stress and RNA interference (RNAi) of the Sc-CARNS gene, and the changes of alanine and carnosine contents. The osmotic pressure and FAA contents in the gills, foot, and hemolymph of S. constricta under different salinities (5, 20, and 35) were measured by freezing point osmometer and automatic amino acid analyzer. At the same time, the expression of the Sc-CARNS gene in the foot under different salinities (5, 20, and 35) was analyzed by RT-qPCR and RNAi technology. The content of alanine was determined with the shellfish alanine ELISA kit. The content of carnosine was determined by phthalaldehyde colorimetry. The results showed that the osmotic pressure in the gills, foot, and hemolymph of S. constricta significantly decreased within 1–72 h under low salt stress (P < 0.05), while under high salt stress, the osmotic pressure in the gills, foot, and hemolymph reached a steady state within 24 h, which was consistent with the osmotic pressure of external seawater. Compared with the control group, the osmotic pressure in the gills, foot, and hemolymph decreased by 66.7%, 69.7%, and 71.6%, respectively, when the salinity was low. The wet weight of tissues was then increased by 68.3%, 67.5%, and 70.2%, respectively. At the same stress time, the osmotic pressure of each group of S. constricta was salinity 35 > salinity 20 > salinity 5. Under normal salinity, the FAAs with the highest content in the gills, foot, and hemolymph of S. constricta were Gly, Arg, and Gly, respectively. After salinity stress, the content of total free amino acids in all tissues increased significantly with the increase of salinity. The main FAAs with the largest content change in the gills, foot, and hemolymph, respectively, were Ala, Gly, Glu, and Pro; Ala, Gly, Arg, and Tua; and Ala, Ser, Thr, and Gly. Ala was the most variable FAA in all tissues. According to the transcriptome results of S. constricta after salinity stress, it was speculated that Sc-CARNS is related to osmotic regulation. The expression of Sc-CARNS was the highest in the muscle type tissues of S. constricta, followed by the gills, and was the lowest in the hepatopancreas. After low salt stress, the expression of the Sc-CARNS gene mRNA in S. constricta increased at first and then decreased with the stress time, and was significantly higher than that in the control group after 4 h of stress (P < 0.01), and reached the peak at 24 h. The content of alanine decreased significantly with time, and the content of carnosine increased significantly with time (P < 0.05). After high salt stress, the expression of Sc-CARNS decreased, and there was no significant change compared with the control group. The content of alanine first increased, then decreased, and then increased after 8 h, and was not lower than that of the control group. The content of carnosine showed a decreasing trend after 24 h. After RNAi, the expression level of Sc-CARNS mRNA in the interference group decreased first and then increased with the interference time under normal salinity. At 24 and 48 h, the expression level of Sc-CARNS mRNA was significantly lower than that of negative control (P < 0.05), and the interference efficiency was 24% and 69%, respectively. The interference efficiency at 48 h was the highest. In the interference group, the content of alanine first increased and then decreased, and the content of carnosine first decreased and then increased, reaching the maximum at 48 h. Under low salt stress, the expression of Sc-CARNS mRNA increased, the content of alanine decreased, and the content of carnosine increased after interference for 0–24 h. After interference for 24 h and 96 h, the mRNA expression of Sc-CARNS and the contents of alanine and carnosine did not change significantly in control group and diethyl pyrocarbonate treated water. The expression level of Sc-CARNS mRNA in the interference group first decreased and then increased with the interference time, the content of alanine first increased and then decreased with time, and the content of carnosine first decreased and then increased. At 48 h, the expression of Sc-CARNS mRNA and the content of carnosine were significantly decreased, and the content of alanine was significantly increased. The results showed that S. constricta has a variable osmotic pressure. Ala as a FAA in vivo contributed the most to osmotic regulation. Moreover, carnosine synthetase was the key enzyme for converting alanine to carnosine. This study revealed the key mechanism of osmotic pressure regulation under low salt stress and provided a basis for revealing the unique salinity tolerance mechanism of Solenida shellfish.