| Pattern recognition receptors(PRRs)-mediated immune recognition is essential for the initiation of innate immune responses in crustacean organisms.Procambarus clarkii,an important crustacean in China,is susceptible to Aeromonas hydrophila and White spot syndrome virus(WSSV),which have caused significant disease onset during the culture process,and there are no effective prevention and control measures yet.Lipopolysaccharide and β-1,3-glucan binding protein(LGBP)is an important pattern recognition receptor which is widely widespread in crustaceans and plays an important role in innate immunity.In this study,a novel LGBP gene,named PcLGBP,was identified by whole genome sequence analysis of P.clarkii.In this study,the molecular characteristics,tissue distribution,expression profile and protein functions of PcLGBP were investigated to elucidate its function and mechanism in the innate immunity of P.clarkii,thus providing a theoretical basis for disease prevention and control of P.clarkii.The main results are as follows:1.Cloning,expression and bioinformatics analysis of PcLGBPThe full-length sequence of the PcLGBP was obtainde by RACE-PCR.The c DNA is 1,200 bp in length and contains a 1,086 bp open reading frame(ORF),encoding a protein with 361 amino acids.The molecular sequence of PcLGBP has a typical Glyco＿hydro＿16 domain,a sequence of signal peptides,a low-complexity region and some conserved motifs,it has high similarity to other invertebrate LGBP homologs and is conserved among the evolution.2.Functional studies of PcLGBP against A.hydrophila and WSSV infectionPcLGBP was expressed in hemocytes,gills,antennal glands,heart,hepatopancreas,stomach,intestines and muscles of P.clarkii,with the highest expression level in the hemocytes and located on the cytoplasm of hemocytes.It was also highly expressed in gills and antennal glands,but least expressed in muscle.The purified recombinant PcLGBP protein could combine polysaccharides(LPS,PGN,β-glucan and Mannan)and different kinds of pathogenic microorganisms(Gram-positive bacteria,Gram-negative bacteria and fungi),indicating that PcLGBP functions as a pattern recognition receptor with a broad-spectrum binding capacity.PcLGBP could response to the challenge of A.hydrophila and WSSV with a upregulated expression level,suggesting it could be involved in the disease resistance immune response of P.clarkii.Using r PcLGBP and RNA interference,we found that PcLGBP could improve the bacterial clearance ability of hemolymph after A.hydrophila stimulation,inhibit the WSSV proliferation,improve the survival of A.hydrophila and WSSV infected P.clarkii,possessing antibacterial and antiviral activities.3.Molecular mechanisms of PcLGBP against A.hydrophila and WSSV infectionIn vitro,the recombinant protein of PcLGBP could bind to polysaccharides and enhance phenol oxidase activity.In vivo,knockdown of PcLGBP,the PO gene expression and PO activity were inhibited in the hemolymph of P.clarkii,suggesting that PcLGBP is involved in the activation of the phenoloxidase system.Besides,knockdown of PcLGBP,the PO activity and PO gene expression,and the expression level of multiple genes involved in the NF-κB signaling pathway were inhibited in A.hydrophila and WSSV-infection crayfish,suggesting that PcLGBP playing an important role in antibacterial and antiviral through activating both NF-κB pathway and pro PO system in P.clarkii.The results of this study suggest that PcLGBP is a broad-spectrum PRR that may be involved in the resistance to A.hydrophila and WSSV infection in P.clarkii through activation of the phenol oxidase system and the NF-κB signalling pathway.The present study shows that the newly identified PcLGBP not only has antimicrobial functions like other reported LGBPs,but also plays an important role in the anti-WSSV immunity of P.clarkii.The results enriched the theoretical basis of the molecular mechanisms of antiviral and antibacterial resistance in crustaceans,and provided insights into disease control and disease resistance breeding of P.clarkii. |
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