Preliminary Study On The Immunomodulatory Mechanism Of Vitellogenin In Crassostrea Gigas

Vitellogenin(Vg)is the main precursor of egg-yolk proteins,which is present in almost all egg-laying non-mammals.Vg is usually synthesized in extraovarian tissues such as the liver in vertebrates,the fat body in insects,and the hepatopancreas in crustaceans.Vg enters oocytes through receptor-mediated endocytosis to serve as a nutrient for the early development of embryos and larvae.Besides,the Vg is also involved in many biological processes,such as hormones regulation,antioxidant stress response,carrier function and host immune defense.In the present study,a Vg homologue(CgVg)was identified and characterized in oyster Crassostrea gigas,and the distribution and expression characteristics of CgVg were analyzed,and reveal the immune function of functional domains DUF1943 and VWD of CgVg,and to explore the immune defense regulation mechanism of CgVg The results obtained are as follows:A vitellinogen homologous was identified from C.gigas.The full length of CgVg is 7879 bp,and the open reading frame(ORF)was of 7539 bp which encoded 2512 amino acids residues.There are three domains in the CgVg protein,including a Vitellogenin＿N domain,a DUF1943 domain and a VWD domain.The mRNA transcripts of CgVg were detected in all tested tissues(haemocytes,gill,hepatopancreas,gonad,adductor muscle,labial palp and mantle)with high expression in the gonad,hepatopancreas and haemocytes,which was 466.29-,117.15-and 57.49-fold(p < 0.01)of that in adductor muscle,respectively.The mRNA of CgVg was specifically highly expressed in the fertilized Eggs,and then decreased rapidly,reaching the lowest level in the Early-Trochophore.After Vibrio splendidus stimulation,the mRNA expression level of CgVg in haemocytes increased significantly at 6,12 and 24 h,which was 1.97-,3.58-and 1.3-fold(p <0.01)of that in the control group,respectively.After lipopolysaccharides(LPS)stimulation,the mRNA expression level of CgVg in haemocytes also increased significantly at 3,6 and 12 h,which was 2.77-,7.9-,and 6.66-fold(p < 0.01)of that in the control group,respectively.The immunofluorescence assay showed that positive signals of CgVg protein were mainly located at the cytoplasm of haemocytes,and no distributed in the nucleus.After V.splendidus stimulation,the fluorescence signal of CgVg in the cytoplasm was enhanced,and a small amount of expression was detected in the nucleus.CgVg is involved in immune response by regulating the expression of inflammatory cytokines and antimicrobial peptides(AMPs).The mRNA expression of CgVg in haemocytes of C.gigas was significantly decreased by RNA interference(0.04-fold of that in control group,p <0.01).At the same time,the mRNA expression of inflammatory cytokines Cg IL17-1,Cg IL17-5,Cg TNF and Cg IL17-2 in haemocytes of C.gigas were significantly decreased,which were 0.17-,0.19-,0.62-fold(p < 0.01)and 0.42-fold(p < 0.05)of that in the control group,respectively.In addition,the expression levels of AMPs related genes Cg Defh2,Cg Bigdefh and Cg Defh1 were also significantly decreased,which were 0.018-,0.0012-fold(p < 0.01)and 0.53-fold(p < 0.05)of that in the control group,respectively.The recombinant protein DUF1943(rDUF1943)and recombinant protein VWD(rVWD)were obtained by prokaryotic expression.The rDUF1943 and rVWD was able to bind lipopolysaccharide(LPS),mannose(MAN),peptidoglycan(PGN)and poly(I:C),as well as Gram-positive bacteria(Staphylococcus aureus and Micrococcus luteus),Gram-negative bacteria(Escherichia coli and V.splendidus)and fungi(Pichia pastoris).rDUF1943 and rVWD also showed agglutination and antimicrobial activity against pathogenic microorganisms.rDUF1943 exhibited stronger agglutination activity towards S.aureus,M.luteus,E.coli,V.splendidus and P.pastoris,while agglutination was only observed in the rVWD group towards P.pastoris.The rVWD inhibited the growth of E.coli,S.aureus and V.splendidus,while no antibacterial activity was detected in rDUF1943 group.In conclusion,CgVg can participate in the regulation of immune defense,not only responding to the stimulation of pathogenic bacteria,but also regulating the expression of inflammatory factors and antimicrobial peptides.CgVg functional domains DUF1943 and VWD have different immune functions,among which DUF1943 and VWD can be used as(Pattern recognition receptors)PRRs to recognize and bind a variety of pathogenic microorganisms and PAMPs.The DUF1943 domain showed strong agglutination activity against pathogenic microorganisms,while the VWD domain significantly inhibited microorganism growth.The results of this study indicate that CgVg has immune defense regulation mechanism,which enrich is the innate immune mechanism of marine invertebrates and provides theoretical knowledge for immunology of marine invertebrates.