Function Of Novel Recognition Molecules Containing DM9 Domain In Oyster Crassostrea Gigas

Crassostrea gigas is one of the most economical shellfish in the world and one of the most important mariculture species in China.In recent years,fulminant diseases of oyster have occurred frequently,which seriously restricted the healthy development of marine culture in China,so it is urgent to analyze the immune defense mechanism of oyster.As a new type of pattern recognition receptor,DM9 domain proteins have been found in more and more species and have been proved to be involved in the immune response of organisms.By means of bioinformatics,molecular immunology and cell biology,1.Gene sequence Analysis of DM9 Domain protein in Oyster Crassostrea gigasSeven proteins,CgDM9CP-1-7,containing DM9 domain were selected from the genome of Oyster gigas.Among them,CgDM9CP-1-6 contains two DM9 domains.CgDM9CP-7,which contain two DM9 domains and one Aldeh domain.In this paper,the CgDM9CP-2,CgDM9CP-4and CgDM9CP-5 containing two DM9 domains are studied.CgDM9CP-2,CgDM9CP-4 and CgDM9CP-5 genes were cloned by PCR technique,encoding 143,145 amino acids and 145amino acids,respectively.The similarity of CgDM9CP-2,CgDM9CP-4 and CgDM9CP-5 with CgDM9CP-1 was 60.8%,52.4%and 60.7%,respectively.The sequence of CgDM9CP-2,CgDM9CP-4 and CgDM9CP-5 was conserved in evolution.2.Characteristics of gene expression of DM9 domain protein in oysterThe expression characteristics and pathogen response patterns of CgDM9CP-2,CgDM9CP-4 and CgDM9CP-5 genes were detected by fluorescence quantitative PCR.The results showed that CgDM9CP-2,CgDM9CP-4 and CgDM9CP-5 were mainly expressed in gill.After stimulated by mannose,the mRNA expression level of three CgDM9CP was significantly increased?p<0.05?,while the mRNA expression of three CgDM9CP decreased after stimulated by Vibrio splendidus,indicating that CgDM9CP-2,CgDM9CP-4 and CgDM9CP-5 responded to Mannose stimulation at mRNA level.3.Study on the function of Recombinant protein containing DM9 Domain protein in Oyster Oyster in VitroThe recombinant and prokaryotic expression of rCgDM9CP-2,rCgDM9CP-4 and rCgDM9CP-5 protein were carried out in vitro,and the immunological recognition activity of rCgDM9CP-4 and rCgDM9CP-4 was studied.Enzyme linked immunosorbent assay?ELISA?was used to detect the binding activity of recombinant protein to PAMPs.The results showed that rCgDM9CP-2,rCgDM9CP-4 and rCgDM9CP-5 could bind to Mannose,lipopolysaccharide?LPS?and peptidoglycan?PGN?.In vitro,bacterial binding tests showed that rCgDM9CP-2could bind to fungi Yarrowia lipolytica and Pichia pastoris,Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus,Gram-negative bacteria Vibrio anguillarum and V.splendidus.Moreover,rCgDM9CP-4 and rCgDM9CP-5 could also bind to fungi Y.lipolytica and P.pastoris,Gram-positive bacteria S.aureus and M.luteus,but not to two Gram-negative bacteria.rCgDM9CP-4 had the strongest binding ability with two fungi and Gram-positive bacteria.The binding activity of bacteria showed Ca²⁺dependence,while rCgDM9CP-5 had the strongest binding ability with Gram-positive bacteria S.aureus,but it was not Ca²⁺dependent.In vitro coagulation experiments showed that rCgDM9CP-2 could agglutinate fungi P.pastoris and Y.lipolytica,while rCgDM9CP-4 could agglutinate fungi P.pastoris and Y.lipolytica and Gram-negative bacteria V.splendidus.its agglutination activity was Ca²⁺dependent.rCgDM9CP-5 was able to agglutinate the fungus P.pastoris,Y.lipolytica,but its agglutination activity was not dependent on Ca²⁺.In vitro bacteriostatic experiments showed that rCgDM9CP-2 and rCgDM9CP-4 could inhibit the growth of fungi Ppastoris,Gram-positive bacteria and Gram-negative bacteria V.splendidus,but rCgDM9CP-5 did not exhibit bacteriostatic activity.rCgDM9CPs was incubated with oyster hemocytes,and the phagocytic effect of rCgDM9CPs on blood cells of oyster was detected by means of fluorescence microscope.The results showed that the phagocytosis rate of rCgDM9CP-5 to fluorescent microspheres was significantly higher than that of the control group?31.1±2.1%?,but the phagocytosis rate of the long oyster blood cells treated with r CgDM9CP-2 and rCgDM9CP-4 was not significantly different from that of the control group.These results suggest that oyster has a new type of multifunctional pattern recognition receptor,which can respond to Mannose stimulation and possess the activity of PAMPs binding to fungi.The combination of CgDM9CP-2 with Gram-negative bacteria and Gram-positive bacteria CgDM9CP-4 had the highest phagocytic activity with Ca²⁺dependent CgDM9CP-5.This study provides data support for further revealing the mechanism of immune response mediated by DM9 domain protein,and will also promote the analysis of the immune defense system of oyster and marine invertebrates.