Molecular Cloning And Expression Analysis Of Adaptor Protein Syntenin And ADP Ribosylation Factor 2 Genes In Macrobrachium Nipponense

Macrobrachium nipponinse,an important economic shrimp in freshwater aquaculture in China.With intensive farming,environmental stress has become a key issue in the breeding process of M.nipponense,and ammonia nitrogen is one of the most important environmental stress factors in aquaculture,which can seriously affect the growth and physiological health of aquatic animals.Therefore,it is particularly important to study the molecular mechanism of resistance to ammonia nitrogen stress in M.nipponense.The adaptor protein Syntenin(MnSyn)is an intracellular adaptor protein commonly found in eukaryotes and prokaryotes,and plays an important role in cellular activities such as cellular immunity and signal transduction.ADP ribosylation factor 2(MnArf2),a member of the Ras gene superfamily,regulates phospholipase D activity and plays an important role in material transport and signal transduction.In this experiment,RACE-PCR,Real Time PCR,Western blot and enzyme activity assays were used to analyze the clonal identification and gene expression and function detection of MnSyn and MnArf2 genes.The changes of antioxidant enzymes in M.nipponense under high ammonia nitrogen stress were also studied.These results are shown as follows:First,under the laboratory conditions,the LD50(48h)was 96.8mg/L by the ammonia nitrogen stress test of M.nipponense.The enzyme activity assay showed that there was a significant upward trend in the hepatopancreas,gills and muscle tissue T-AOC and SOD of M.nipponense in the early stage of ammonia nitrogen stress.At the late stage of stress,T-AOC and SOD were significantly down-regulated,and the body's antioxidant enzyme activity was significantly inhibited.The MDA did not change significantly at the early stage of stress.The level of MDA was significantly higher than that of the control group after 24 hours of stress,and it showed a gradual increase trend.This indicates that the tissue cells of M.nipponense are severely damaged.The complete cDNA sequence of MnSyn gene with a length of 2432 bp was obtained by molecular cloning and RACE technology.It contains a 62 bp 5'UTR,a 1475 bp 3'UTR and a957 bp CDS region.The open reading frame encodes a protein of 318 amino acids.Themolecular weight of the protein is predicted to be 34.26 KDa.The protein encoded by this gene has two PDZ domains that exist in tandem.The homology analysis found that MnSyn was similar to the Syntenin gene of Penaeus vannamei,P.monodon and P.serrata,with 78%,77% and 75%,and was conservative during evolution.The MnArf2 gene of M.nipponense.is 949 bp in length and contains a 264 bp 5'UTR,a 148 bp 3'UTR and a 537 bp CDS region.The 537 bp open reading frame encodes a protein consisting of 178 amino acids,which is predicted to have a molecular weight of 19.98 KDa.Sequence alignment analysis showed that MnArf2 was 70% similar to Penaeus japonicus Arf,and 69% similar to Penaeus vannamei Arf-like.MnArf2 contains a typical Switch,Interswitch and Ploop regions.The Switch and Interswitch regions are involved in regulating the interconversion of Arf molecules between GDP/GTP.These structures can reflect that MnArf2 is highly conserved in evolution.Quantitative Real-time PCR(qRT-PCR)showed that the expression levels of MnSyn gene and MnArf2 gene in M.nipponense were significantly changed at different time points after ammonia-nitrogen stress.The results of real-time PCR showed that the expression of MnSyn gene began to increase after 6 h of ammonia nitrogen stress,and reached the peak at24 h,then began to decrease.After 72 hours,there was still a significant difference from the control group.The expression of MnArf2 was significantly up-regulated at 6 h after ammonia nitrogen stress,peaked at 12 h,then decreased after 72 h,and gradually returned to the initial level after 72 h.Western Blot results showed that MnSyn protein was expressed in hepatopancreas,gill,muscle,heart,stomach and eye stalk of M.nipponense.The expression level was highest in hepatopancreas and gill tissues,and the expression level in eye stalk and stomach tissue was low,and the difference was significant.The results showed that MnSyn and MnArf2 were involved in the immune response induced by ammonia nitrogen stress in the organism of M.nipponense,and played a certain role in the physiological process of ammonia nitrogen stress.The results indicated that the MnSyn and MnArf2 genes were involved in the immune response induced by ammonia nitrogen stress in M.nipponense,and provide a scientific basis for the healthy breeding of M.nipponense.