Molecular Cloning And Functional Analysis Of A2M And LRP1 Genes In Macrobrachium Nipponense

Ammonia is a major physiological challenge for prawns in culture,it has an negative effect on the growth and development of shrimp and their physiological health.The?2-macroglobulin?A2M?gene,a non-specific protease inhibitor,it is involved in several immune responses in invertebrates such as blood clotting system,proPO activating system,plays a major role in the migration of cells and the tolerance of environmental stress.Low-density lipoprotein receptor-related protein?LRP1?gene,a member of the family of low-density lipoprotein receptor?LDLR?,plays an important role in the uptake of lipoprotein,blood vessel damage,cell growth and development,also can be used as a cellular signaling receptor of the activation of A2M,the fibrinolytic enzyme activation agent and other ligands.The research studied the histopathological,the cloning,expression and identification of A2M and LRP1 by RACE-PCR,Real Time PCR and Western blot from Macrobrachium nipponense?M.nipponense?.These results are shown as follows:We have created a controlled of ammonia nitrogen stress environment in the laboratory,the half lethal dose(LD₅₀=96.8mg/L)of 48 hours was observed in these prawns.Histological pathology investigation showed that the hepatopancreas and gills exhibited different pathological changes between ammonia-N stress and control groups and the symptoms were much related to the time after ammonia-N stress.Hepatopancreas of the M.nipponense following 0-96h?H0-6?exposure to ammonia-N stress showing the arrangement of hepatic lobule becomes loose,the appearance of hemorrhage and large numbers of vacuoles in the tubular epithelial cells and abnormal lumens,thinning of the fringes and duct atrophic in affected hepatopancreas,and have then undergone a karyolysis.The histoarchitecture of gills in the test group?G0-6?showed lesions are mainly hyperplasia and hypertrophy of the gill epithelium,gill edema and swollen,intercellular spaces dilatation and apart of chromatin was dissolved and migrated to the cell edge.,even necrotic gill lamellae,complete disorganization of lamellae with the time of ammonia-N stress,and the internal structure may not be apparent.We present full-length A2M cDNA sequences of M.nipponense,The 4805-bp cDNA contains an open reading frame?ORF?4422-bp,a 99-bp 5'-untranslated region?UTR?and a284-bp 3'UTR containing the poly A tail.The CDS encodes a polypeptide of 1473 amino acids with a predicted molecular weight of 163.45 kDa and the pI is 5.0.The M.nipponense A2M contains a transmembrane domain,a A2M_N2 structure domain,a c-terminal domain structure and a receptor binding domain.Sequence comparison showed that A2M deduced amino acids sequence of M.nipponense has an overall similarity of 92%and 80%to those of Macrobrachium rosenbergii and Exopalaemon corinicauda.The full-length cDNA of LRP1from M.nipponense was 4121-bp,it contains an open reading frame?ORF?2739-bp,a 318-bp5'-untranslated region UTR and a 1064-bp 3'UTR containing the poly A tail.The CDS encodes a polypeptide of 912 amino acids with a predicted molecular weight of 101.71 kDa and the pI is 4.61.The M.nipponense LRP1 predicted domains,repeats,motifs and features:EGF_CA domain,transmembrane domains,LDLa domains,LY domains and EGF domains.Sequence comparison showed that LRP1 deduced amino acids sequence of M.nipponense has an overall similarity of 77%and 71%to Pandalopsis Japonica and Penaeus Vannamei?PREDICTED?.The A2M and LRP1 gene expression have an obvious changes in the muscles,eyestalk,hepatopancreas,gills,stomach and heart of prawns at different time points after ammonia-N stress.Quantitative Real-time PCR?qRT-PCR?revealed that the A2M expression increased at3h,and increased gradually with the stress,then decreased after ammonia-N stress,the A2M has a significantly level in test groups at 12-48 h after the ammonia-N stress,and then no significant difference was detected at 96 h after the stress,compared to control groups during the experimental period.The LRP1 expression has a slight increase after stress,the LRP1mRNA expression increased at 6 h and the expression showed significant high level at 12 h,then decreased gradually at 24 h,and no significant difference was detected at 96 h after the stress.Western blot showed that the protein expression profiles of A2M among these tissues of the muscles,gills,hepatopancreas,eyestalk,stomach and heart from M.nipponense after ammonia-N stress.The A2M had a high expression in hepatopancreas and gills,and then were muscles and heart,and finally were stomach and eyestalk.Overall,significant up-regulation of A2M expression was observed after ammonia-N stress and there were significantly higher expression in hepatopancreas and gills.LRP1 appeared up-regulation and there were higher expression in hepatopancreas and muscles.These results suggested that as the key factors to induce the immune responses and inflammatory reactions,A2M and LRP1 play an important role in resistance to ammonia-N stress.