Study On The Anti-bacterial Immune Mechanism Mediated By Mannose Receptor Of Red Swamp Crayfish,procambarus Clarkii And LGBP Of Mud Crab,Scylla Paramamosain

Invertebrates lack adaptive immunity and only rely on the innate immune system to resist the invasion of pathogenic microorganisms.Invertebrates use pattern recognition receptors(PPRs)to recognize the invasion pathogens,by recognizing the pathogen associated molecular patterns(PAMPs)found on the surface of microorganisms,and further trigger immune response to defend themselves.In recent years,with the continuous expansion of shrimp and crab artificial cultivation scale,the outbreak of a variety of bacteria,fungal and viral diseases has caused great economic losses to the crustacean aquaculture.Therefore,further understanding of the relationship between hosts and their pathogens,especially immune-related genes,is of far-reaching significance for the prevention and treatment of crustacean diseases,and also contributes to enriching the theoretical knowledge of crustacean innate immunity.In this study,we used red swamp crayfish,Procambarus clarkii and mud crab,Scylla paramamosain as experiment materials to characterize the roles of a mannose receptor(MR)gene and a Lipopolysaccharide and ?-1,3-glucan-binding protein(LGBP)gene in immunity.1.Identification and molecular characterization of Pc MR from red swamp crayfish,Procambarus clarkiiMannose receptor(MR),a member of pattern-recognition receptors(PRRs),is the first MR family member to be discovered that plays a critical role in immunity.The function of MRs has been reported in mammals and teleosts while none in invertebrates.In the present study,we identified a MR-like gene(designated as Pc MR)from red swamp crayfish,Procambarus clarkii.The Pc MR c DNA is 6848 bp long with a 6288 bp open reading frame that encodes a polypeptide with 2095 amino acid residues,and also includes a putative signal peptide with 31 aa.Pc MR transcripts were mainly detected in hepatopancreas and hemocytes,and upregulated by Vibrio anguillarum challenge.The Pc MR protein contained 14 C-type lectin domains(CTLDs)and they were divided into four fragments(CTLD 1–3,CTLD 4–6,CTLD 7–10,CTLD 11–14).The four recombinant proteins encoded by the four fragments were all expressed and purified.Microorganism-binding and sugar-binding assay showed that CTLD 1–3,CTLD 4–6,CTLD 7–10,CTLD 11–14 could bind to a variety of bacteria,as well as glycoconjugates on the bacterial surface.Moreover,they agglutinated bacteria in a calcium-dependent manner.Bacteria clearance experiment manifested that the mixed proteins facilitated the clearance of injected bacteria in crayfish.Pc MR silencing by si RNA interference impaired the bacterial clearance ability.These results suggest Pc MR is involved in the antibacterial defense of crayfish,and this study can help us better understand the functions of invertebrate MRs.2.Identification and molecular characterization of Sp LGBP from mud crab,Scylla paramamosainAccording to transcriptome sequencing results,we identified LGBP gene(designated as Sp LGBP)from mud crab,Scylla paramamosain.The ORF sequences of Sp LGBP was obtained with specific primers.The Sp LGBP c DNA is 1217 bp long with a 1195 bp open reading frame that encodes a polypeptide with364 amino acid residues.The predicted p I and molecular weight of Sp LGBP are 4.53 and 39.9 k Da,respectively.Quantitative RT-PCR was used to detect the tissue distribution of Sp LGBP,including hemocytes,hepatopancreas,gills,intestines,stomach,connective tissue and muscles.The results showed that Sp LGBP gene was expressed in all tested tissues,with the highest expression in hepatopancreas.We used Vibrio parahaemolyticus and Staphylococcus aureus to stimulate Scylla paramamosain and found that Sp LGBP was up-regulated after 6 h,12 h of Vibrio parahemolyticus and Staphylococcus aureus challenge.Sp LGBP was overexpressed and purified,and the recombinant protein Sp LGBP was used for bacterial binding experiment,microbial polysaccharide binding experiment,agglutination experiment RNAi,detection of the expression of antimicrobial peptides to study its immune function.In addition,we also explored whether r Sp LGBP had an effect on the activity of phenol oxidase,and the results showed that r Sp LGBP may combine with LPS to improve the activity of phenol oxidase.