| Macrobrachium nipponense is one of the important freshwater economic breeding species with rich nutritional value.It belongs to arthropoda,and widely distributes in all the fresh water in our country.In recent years,due to the continuous expansion of the scale of breeding,the degree of concentration has increased,M.nipponense has been threatened by a variety of pathogenic microorganisms,causing huge economic losses and seriously hindering the development of prawn aquaculture.M.nipponense is an invertebrate and can only rely on innate immunity to resist bacterial infection.Therefore,it is very imporant to study the innate immune mechanism of M.nipponense.As a classical signaling pathway,Wnt signaling pathway can resist the invasion of pathogens.In this study,the imporant immune functions genes Wnt4,Wnt4-like and ?-Catenin in M.nipponense were investigated,and the following results were obtained.1.Two Wnt4 genes,named Mn-Wnt4 and Mn-Wnt4-like genes were cloned from M.nipponense.The length of Mn-Wnt4 gene was 3144 bp,the length of the open reading frame was 1074 bp,and encoded 358 amino acids.This protein had a conserved Wnt1 domain and a transmembrane domain.The expression of this gene was high in hepatopancreas,stomach and intestines,low in heart,and the least in hemocytes and gills.The expression level of Mn-Wnt4 changed after infected with Staphylococcus aureus,Vibro parahaemolyticus or WSSV.After silenced this gene,the expression of some antimicrobical peptides(ALF1,CRU4)showed a downward trend in M.nipponense infected with V.parahaemolyticus.The length of Mn-Wnt4-like gene was 2154 bp,the length of the open reading frame was 1602 bp,and encoded 519 amino acids.This protein had a conserved Wnt1 domain and a transmembrane domain.The expression of Mn-Wnt4-like gene was mainly in the stomach,the second the gills,intestines and least in hemocytes,heart,gills.Had been infected with S.aureus and V.parahaemolyticus,the expression of this gene was down-regulated to different degrees.After the Mn-Wnt4-like gene was interfered,M.nipponense was infected with V.parahaemolyticus and S.aureus respectively,the expression of some antimicrobical peptides(ALF4,CRU6)showed a downward trend in M.nipponense infected with V.parahaemolyticus.while the expression of some antimicrobical peptides(ALF3,ALF4)showed a downward trend in M.nipponense infected with S.aureus.2.A Wnt16 gene named Mn-Wnt16 was cloned in M.nipponense,and the full length of the gene was 2893 bp,the open reading frame was 1281 bp and it encoded427 amino acids,the protein contained a conserved Wnt1 domain and a low complexity domain.Mn-Wnt16 had the highest expression in stomach,and followed by gills and intestines,and low expressed in hepatopancreas and heart and least expressed in the hemocytes.The expression level of Mn-Wnt16 changed after infected with Staphylococcus aureus,Vibro parahaemolyticus or WSSV.After the Mn-Wnt16 gene was interfered,M.nipponense was infected with V.parahaemolyticus,it was found that the expression levels of some AMPs(CRU3,CRU4)decreased after the stimulation of V.parahaemolyticus in stomach.3.A Catenin gene named Mn-?-Catenin was cloned in M.nipponense,Mn-?-Catenin gene had a 2424 bp open reading frame,and it encoded 808 amino acids.SMART online analysis result showed that the protein contained a 12continuous(ARM)domain.Mn-?-Catenin mainly expressed in intestines,followed by hemocytes and hepatopancreas,the least in heart.Had been infected with S.aureus and V.parahaemolyticus,Mn-?-Catenin was down regulated to different degrees at different time points.After Mn-?-Catenin gene was interfered,The expression levels of antimicrobial peptides(ALF1,Lys1)decreased in gill tissue;antimicrobial peptides(ALF1,Lys1,Lys2)decreased in stomach tissue;antimicrobial peptides(ALF1,Lys1,Lys2)decreased in intestinal tissue.The expression levels of downstream Wnt genes(Wnt4,Wnt4-like,Wnt16)also decreased to varying degrees in intestinal tissues.In summary,Wnts and ?-Catenin play an imporant role in the process of M.nipponense against pathogen infection. |
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