Molecular Mechanism Research Of Dopa Decarboxylase Regulating Innate Immune System Of Procambarus Clarkii

China is the largest aquaculture country in Asia.In recent years,the frequent outbreak of bacterial diseases,resulting in heavy losses of aquiculturist.How to improve the disease resistance of cultured species is an important issue in the current aquaculture industry.Crustaceans are rely mainly on the innate immune system to defend themselves against pathogens.The study on the immune mechanism of crustaceans helps to understand well the immunity response mechanisms of the cultured species.In this study,an open reading frame(ORF)sequence of dopa decarboxylase(DDC)was obtained,on the base of the transcriptome libraries of Procambarus clarkii.And the full-length cDNA of Pc-DDC gene was obtained.Bioinformatics analysis showed that Pc-DDC is conserved among different species.Pc-DDC was expressed in different tissues of P.clarkii,and the highest expression is found in hemocytes by qRT-PCR.Both Staphylococcus aureus and Edwardsiella ictaluri could induce significant up-regulation of Pc-DDC in different tissues.To clarify how PcDDC is involved in the innate immunity response,a series of studies including gene cloning,protein purification and antibody preparation was carried out.And the results showed that recombinant PcDDC could promote the capsulation activity of the P.clarkii hemocytes in vitro.Recombinant PcDDC could combine with S.aureus and E.ictaluri to some extent.After knocking down the expression of Pc-DDC,the melanization in the hemolymph was significantly reduced,leading to an increase in death rate of P.clarkii.It is speculated that Pc-DDC may be used to participate in the innate immunity response against bacteria by affecting the melanization.In order to further explore the molecular mechanism research of Pc-DDC participating in innate immunity system of P.clarkii,the full-length cDNA of Pc-DAR(dopamine receptor)gene locating in the downstream of Pc-DDC was obtained.And the expression levels of Pc-DAR in different tissues of crayfish were detected by qRT-PCR.The results showed that the expression level of Pc-DAR was the highest in hepatopancreas.After stimulated by S.aureus or E.ictaluri,the expression levels of Pc-DAR was obviously down regulated.Then the relationship between the PcDDC and Pc-DAR was detected by RNAi of Pc-DDC.And the expression levels of Pc-DAR were tested.As a result,we found that when Pc-DDC gene was knocked down,a decrease in melanization will happen.And the expression of the Pc-DAR gene in crayfish was significantly upregulated.However,after stimulated only by bacterias,the expression level of Pc-DAR in crayfish was significantly down-regulated to promote melanization.Thus,we hypothesized that P.clarkiiwill enhance their resistance to bacterias by expression high levels of Pc-DAR gene,when the melanization was weak.After Pc-DDC gene was knocked down,the expression changes of antioxidant enzyme related genes and Toll-like signaling pathway related genes were detected.The results showed that crayfish enhanced their resistance to bacterias by expressing high levels of the ROS and the Toll-like signaling pathway related genes,when the melanization was weak.In summary,this study clarified the important role of Pc-DDC gene in the innate immunity response of P.clarkii.And we found that the Pc-DDC and dopamine-mediated melanization response is closely related to the production of antimicrobial peptides and the synthesis of ROS.This is an important complement to the antibacterial innate immunity response in invertebrates,also provides a scientific basis for the prevention and control of bacterial diseases of P.clarkii.