Effects Of Low-salinity Stress On Liver Metabolizing Enzyme And Osmoregulation Genes Expression Of Turbot(Scophthalmus Maximus)

Turbot(Scophthalmus maximus)is a low temperature economic fish native to the coast of Europe.The introduced turbot into China in 1992 by Yellow Sea Fisheies Research Institute,and now it has become one of the leading varieties of coastal aquaculture in northern China.In recent years,with the increase of consumption demand of turbot and the guidance of environmental protection policy,available water resources and culture region suitable for turbot aquaculture in northern China are declining.In order to meet the demand of consumption,the cultivation area of turbot gradually expanded to the beach and inland areas.Consequently,the salinity of aquaculture water has gradually become an important factor affecting the aquaculture production of turbot.In order to speed up the breeding process of new salt tolerant varieties of turbot and analyze the molecular mechanism of turbot to low-salinity stress,this paper studies and analyzes the changes of liver metabolizing enzymes and osmoregulation genes of turbot under low salt stress.It is expected to provide theoretical reference for the cultivation of turbot in ow-salinity environment and the cultivation of turbot varieties with low-salinity tolerance.1.Salinity is an important factor of environmental stress in aquaculture,and its changes affect various physiological and biochemical activities such as fish metabolism.To some extent,the changes of metabolic regulation can reflect the physiological status of fish in different environments,which can be used as an important physiological index to measure the adaptability of fish to salinity stress.In order to understand the effect of low-salinity stress on the metabolism regulation of turbot liver and explore the possibility of turbot culture in low salt environment,the changes of antioxidant enzymes and transaminase activities in turbot liver under low-salinity stress(0 ppt)were measured and analyzed.The results showed that the activities of catalase(CAT)and superoxide dismutase(SOD)decreased first and then increased with time under low-salinity stress,and they were the lowest at the 12 hour,significantly lower than those at the 0 hour and 48 hours(P<0.05);the activities of Glutamic-oxaloacetic transaminase(GOT)and Glutamic-pyruvic transaminase(GPT)decreased first and then increased with time,The activity was the lowest at 24 hours,and significantly lower than that at 0 hour and 48 hours(P<0.05).The results showed that the activities of four metabolizing enzymes decreased significantly at the initial stage of adaptation to low salt stress,but then recovered rapidly,which indicated that turbot may have a strong ability to adapt to low-salinity stress.2.Salinity stress will cause the imbalance of ion/osmotic homeostasis in fish body fluid,and then affect the physiological activities such as metabolism.Therefore,when salinity changes,fish will carry out osmotic regulation to maintain the normal water salt balance and metabolic physiological activities of fish.It is one of the important osmotic regulation ways to control the inflow and outflow of various ions and water through osmotic regulation genes.Based on a variety of osmoregulation related genes screened by transcriptome analysis in the early stage of the research group,four representative osmoregulation genes were selected,including cystic fibrosis transmembrane transfer regulator(CFTR),Na+/H+-exchanger(NHE),aquaporins(AQPs)AQP1 and AQP3,to study their osmoregulation in gill,kidney and intestine of turbot Response of organs to low salt stress.In this study,the expression of four genes in gill,kidney and intestine of turbot at different time points under 5 and 10 salinity were detected by Quantitative real-time PCR.The results showed that the expression level of AQP1 was very low in gill,but high in kidney and intestine.Under low-salinity stress,the expression of AQP1 in gill did not change significantly in 5 ppt group and 10 ppt group,but increased significantly in kidney and intestine(P<0.05).The expression level of AQP3 was very low in kidney,with high in gill and low in intestine.Under low-salinity stress,the expression level of AQP3 in kidney did not change significantly in 5 ppt group and 10 ppt group,but increased significantly in gill and intestine(P<0.05).The expression level of CFTR was very low in kidney,with high level in gill and low level in intestine.Under low-salinity stress,the expression level of CFTR in kidney did not change significantly in 5 ppt group and 10 ppt group,but decreased significantly in gill and intestine(P<0.05).NHE1 was less expressed in gill and intestine,but high in kidney.Under low-salinity stress,the expression of NHE1 in gill did not change significantly in 5 ppt group and 10 ppt group,but increased significantly in kidney and intestine(P<0.05).These results indicate that the expression levels of the four genes vary with tissue,salinity and time,reflecting the functional specificity of the four genes.Under low-salinity stress,the four genes responded positively and the expression levels changed to varying degrees,indicating that AQP1,AQP3,CFTR and NHE1 may have potential roles in the adaptation of turbot to low-salinity environment.3.The adaptation of fish to the change of environmental salinity is related to many signal transduction pathways,and 14-3-3 protein can bind to a variety of ligand proteins and play an important role in the regulation of many signal transduction pathways.Therefore,the response of these genes to low-salinity stress may be regulated by 14-3-3 protein.To further understand the osmoregulation mechanism of turbot and whether 14-3-3beta/alpha is involved in the regulation the response of NHE1,CFTR and Na+-K+-ATPase(NKA)to low-salinity stress.We cloned and analyzed the c DNA sequence of 14-3-3beta/alpha,discussed the tissue distribution of 14-3-3beta/alpha and the transcription mode of the main expression tissues under low salt stress,For the first time,we silenced 14-3-3beta/alpha of turbot by intramuscular injection of ds RNA.On this basis,we studied the regulatory effect of 14-3-3 protein on the expression of NHE1,CFTR and NKA under low-salinity stress,and provided a new and effective way to study gene function at the individual level.The results show that the 14-3-3beta/alpha c DNA has a total length of 892 bp and contains 774 bp ORF.It is speculated that the primary structure is 257 residues.The deduced amino acid sequence shares a highly conserved structure with other eukaryotes.The results of real-time quantitative PCR showed that 14-3-3beta/alpha transcripts were expressed in all tissues of turbot,the most abundant in gill(P<0.05),the least in kidney,intestine,brain and spleen,and the least in pituitary.Under low-salinity stress,the expression of 14-3-3beta/alpha in turbot first increased and then decreased with the extension of stress time,reaching the highest value at 12 h(P<0.05).The m RNA expression of 14-3-3beta/alpha decreased significantly after injection of different concentrations of ds RNA,and the interference effect was the best 12 hours after injection of 4 ?g/g ds RNA.In addition,the interference of the gene can cause changes in the expression of various ion channel proteins,such as the decrease of NKA,NHE1 and the increase of CFTR.Therefore,14-3-3beta/alpha may be an important molecular regulator of osmoregulation in gills of turbot.