Function And Expression Analysis Of GC1qR Gene From Macrobrachium Nipponense

Macrobrachium nipponense(M.nipponense),known as the oriental river prawn,is an economically important prawn species in China.However,with the deterioration of the ecological environment,various kinds of environmental stresses have proven to be the main limiting factors for the prawn culture industry,causing high and huge economic losses.Ammonia nitrogen(ammonia-N)is a common environmental stress factor and high concentrations of ammonia-N has a significant effect on the immunity and growth of M.nipponense.Therefore,a comprehensive understanding of immune mechanisms of the oriental river prawn is necessary for improving the immunity,protecting the germplasm resources and cultivating stress-resistant varieties of M.nipponense.gC1qR is a versatile,multilig and binding protein,which is vital to the innate immunity of vertebrates and invertebrates,and play a crucial role in protecting the body against the kinds of pathogens invasion and environmental stresses.It is a 33-kD single-chain protein,which could be detected in various tissues and numerous cellular compartments.Furthermore,gC1 qR has been identified on the cell surface and serves as a receptor for many structurally or functionally diverse molecules.Indeed,due to its manifold roles,gC1 qR has been called a multifunctional chaperone.In this study,the prawns are randomly separated into two groups: the ammonia-N stress(98 mg/L)group and the control group.The prawns are kept in the ammonia-N stress condition for 96 h and no feed is supplied during this period.Thereafter,serum,muscle,hearts,hepatopancreas,gills,eyestalk and stomach of the experimental prawns(n = 6)and control prawns(n = 6)are sampled at 0 h,3 h,6 h,12 h,24 h,48 h,72 h and 96 h after ammonia-N stress.All samples are stored at-80 °C for RNA extraction,enzyme activities assays(PO?LSZ?SOD?CAT),histology assays,RACE cloning,qRT-PCR,Western blot and RNAi.The main results are as follows:(1)The activities of SOD and CAT in hepatopancreas from M.nipponense are increased significantly suggests that the high concentrations of ammonia-N(98.0 mg/L)could causeoxidative stress and induce the antioxidant defense system after 24 h of exposure.Furthermore,after 48 h,the decreased of SOD and CAT activities indicates the presence of severe oxidative damage.PO and LSZ activity in serum have significant increased at 3 h and6 h,respectively.This result may be linked with the non-specific immunity response of M.nipponense is induced by ammonia-N.Furthermore,the activities of PO and LSZ decreased significantly from 12 h to 96 h suggests that long-term exposure to high concentrations of ammonia-N can damage the immune system of M.nipponense.(2)Histological analysis shows that the pancreatic ducts of the control group are well-structured,while the hepatopancreas tissues of M.nipponense are damaged more and more severely with the prolongation of stress time under high concentrations of ammonia-N.The pancreatic ducts are obviously swollen,the wall of the ducts become thinner,the gap between the ducts is decreased,and the vacuoles increased.The stellate lumen lost its original shape and tends to be round and even there are fragmented nuclei and exfoliated cell tissues.(3)The full-length cDNA sequence of MngC1 qR is cloned and identified from M.nipponense by RACE and RT-PCR.It is 1164 bp,including a 106 bp 5'-untranslated region(UTR),a 774 bp open reading frame(ORF)encoding 257 amino acids and a 284 bp 3'-UTR with a stop codon(TAG),a polyadenylation signal(AATAAA)and a Poly(A)tail.The molecular weight and theoretical isoelectric point of MngC1 qR is 28.73 kDa and 4.64,respectively.What's more,the deduced protein of MngC1 qR contained a domain of MAM33 with 174 amino acids and an RGD motif.Multiple alignments and phylogenetic tree demonstrates that MngC1 qR has a closer evolutionary relationship with crustacean gC1 qRs and high identities with M.rosenbergii and P.carinicauda.(4)The tissues distribution and expression level of MngC1 qR are analyzed at both mRNA and protein levels by qRT-PCR and Western blot,respectively.The mRNA transcripts of MngC1 qR are widely distributed in all tested tissues.The highest expression is observed in hepatopancreas,followed by gills and the eyestalk is the lowest.Compared with the control group,the MngC1 qR expression is significantly increased and reached a peak in the hepatopancreas and gills at 12 h.Thereafter,it decreases at 24 h and decreases to a minimum value at 48 h then returns to the original levels at 96 h.Similarly,at the protein level,MngC1 qR exists in all examined tissues and the hepatopancreas is the highest,followed bygills,which is consistent with the transcript levels detected by qRT-PCR.(5)The expression profiles of MngC1 qR in M.nipponense is knocked down using RNAi strategy,through injection of MngC1qR-specific dsRNA.QRT-PCR and Western blot are performed to detect the knockdown efficiency and the expression of gC1 qR gene of the silenced M.nipponense.The cumulative mortality of the silenced M.nipponense under ammonia-N stress is recorded.The results of qRT-PCR and Western blot showed that the expression of gC1 qR in M.nipponense decreases significant and reaches the lowest level at48 h after being injected with dsgC1 qR.The results demonstrate that the dsRNA is quite effective in MngC1 qR silencing.At 48 h after dsRNA injection,the knockdown of MngC1 qR results in high mortality of prawns at 24-96 h under ammonia-N stress,which reveals that the MngC1 qR may play a crucial role against ammonia-N stress.Furthermore,qRT-PCR is performed to detect the expression level of MngC1 qR in the dsgC1 qR group and dsgC1 qR +ammonia-N group,the results show that the expression level of MngC1 qR is significant increased at 12 h,which clearly indicates that the increased expression of MngC1 qR is mainly caused by the induction of ammonia-N.(6)In conclusion,the full-length cDNA sequence of MngC1 qR is cloned and identified from M.nipponense.The activities of various enzymes are investigated and show that high concentrations of ammonia-N has a significant effects on the immunity of M.nipponense.The MngC1 qR is expressed in all examined tissues,with the high expression level in the hepatopancreas and gills.The expression profile of MngC1 qR after exposed to ammonia-N stress demonstrates that MngC1 qR is inducible and may relate to prawn immune response.RNAi further indicates that its knockdown significant increased prawn mortality,and the expression level of MngC1 qR is found to be induced significantly by ammonia-N stress.All findings in this study suggest that MngC1 qR may play a crucial role in the immune defense against ammonia-N stress.