| Sinonovacula constricta is an important economic aquaculture shellfish,mainly distributed in the intertidal zone and estuarine area where salinity changes greatly.High temperature and thermal stress in summer accelerated water evaporation,which increased the salinity of aquaculture water,affecting their growth.Proline is a kind of free amino acid in vivo,and also an important organic osmotic regulator,which affects the body’s ability of osmotic regulation of salinity.Therefore,this study took proline as the entry point and S.constricta as the object to explore the effect of proline on its high salt tolerance by adding exogenous amino acids under high salt stress,providing theoretical reference for further analysis of its regulatory mechanism.1.Taking the salinity of 20 as the control group,high salt(32)stress was applied to S.constricta.The contents of total free amino acid,isoleucine,proline,taurine and alanine in serum and the osmotic pressure of seawater of razor clam were detected.The results showed that the contents of total free amino acid,isoleucine,proline,taurine and alanine in branchial tissue and serum osmotic pressure increased significantly under high salt condition.Among them,the accumulation of proline was the most vigorous,the accumulation amount reached more than 10 μg/m L,and reached the peak at 3 h,the accumulation amount was about 1.5 times of the normal condition.Alanine and isoleucine were the next,taurine accumulation was the least,and the proliferation was between 1.1 and 1.2 times.The osmolality test results showed that the serum osmolality reached a balance with the external seawater osmolality at 24 h,and the difference between the two increased significantly from the late stage of stress to 72 h.2.Add three kinds of exogenous amino acids(proline,taurine,alanine)solid powder on the basis of high salt(32),with the added concentration of 5 μg/m L.Amino acid content,osmotic pressure and relative cell volume of each group were analyzed under different conditions.The results showed that the contents of proline,taurine and alanine increased continuously after the addition of exogenous amino acids,and the synthesis of proline was still in a high level.The serum osmolality of S.constricta continued to increase after the addition of exogenous amino acids.Add three groups of exogenous amino acid after the osmotic adjustment of the comparative analysis found that proline respectively after adding serum osmotic pressure water osmotic pressure in 6 h with the outside world tends to equilibrium state and the taurine group in 12 h into balance,alanine group of more than 6 h.Flow cytometry was used to analyze the fluorescence channels of forward Angle scattered light(FSC)produced by S.constricta gill cells.It was found that the average fluorescence channels of FSC treated with proline,taurine and alanine were significantly higher than those under high salt stress.Under the three amino acid addition conditions,the difference of the average fluorescence intensity channel number was significantly reduced,and the relative volume of gill cells was the same.3.On the basis of high salt(32),proline solid powder(5 μg/m L)was added to detect the contents of soluble protein,ATP,ammonia expulsion rate and the expression of proline transporter gene in the gill tissue of S.constricta under different conditions.The results showed that the content of soluble protein in branchial tissue decreased under high salt stress.ATP content increased;Ammonia discharge rate increased;The expressions of proline transporter SLC6A7-1 and SLC6A7-2 were significantly increased.After the addition of exogenous proline,the soluble protein content began to increase significantly,and the ammonia expulsion rate and ATP content decreased significantly,both of which showed a trend of first increasing and then decreasing(P<0.05).The expression of proline transporter SLC6A7-1 increased first and then decreased,while SLC6A7-2 remained in a high expression state and reached a peak expression level at 24 h. |
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