| In invertebrates,innate immunity is an important defense system against pathogenic microorganisms.In recent years,Toll or Toll-like receptor（TLRs）signaling pathways have been widely studied in crustaceans.Among the components of Toll or Toll like receptors（TLRs）signal pathway,tumor necrosis factor receptor-associated factor 6（TRAF6）is an important cytoplasmic junction molecule,which is common in Drosophila and humans.In this study,a new traf6 like gene,named Pc-traf6 like gene,was cloned from the hepatopancreas of Procambarus clarkii.The full length c DNA sequence of Pc-traf6 like gene was analyzed by BLAST and translated into amino acid sequence by Ex PASY.The results showed that the full length of crayfish Pc-traf6 like gene was 1746 bp,the open reading frame was 999 bp,which could translate 332 amino acids,and the first 22 amino acid residues formed the signal peptide.In order to study the amino acid sequence similarity between Pc-TRAF6 like and other TRAF6 molecules,the amino acid sequences were compared.The results showed that the sequence similarity between the target protein and other TRAF or TRAF molecules was 47.35%.In order to further study the evolutionary relationship between Pc-TRAF6 like molecules and other selected invertebrate TRAF6 or TRAF6 like molecules,phylogenetic analysis was carried out.The results showed that the evolutionary tree was obviously divided into two branches.Penaeus vannamei TRAF6,P.vannamei TRAF6 like and Pc-TRAF6 like molecules of P.vannamei are located in the same branch.Pc-TRAF6 like was closely related to P.vannamei TRAF6 and P.vannamei TRAF6 like.The Pc-TRAF6 like protein was analyzed by bioinformatics software.The results showed that the molecular formula of Pc-TRAF6 like protein was C1662H2563N483O513S17,the relative molecular weight was 38.06 k Da,and the theoretical isoelectric point was 6.13,which belonged to hydrophobic protein.Using the techniques of gene cloning and protein purification,the r Pc-TRAF6 like protein was obtained.In order to study the application of Pc-traf6 like gene in regulating the crayfish antibacterial innate immune response,the expression patterns of the target gene were detected by q RT-PCR.The results showed that Pc-traf6 like gene was highly expressed in hemocytes,hepatopancreas,and gills of normal crayfish.The expression profiles of Pc-traf6 like gene after Staphylococcus aureus or Edwardsiella ictaluri stimulation were studied.The results showed that the m RNA transcription level of Pc-traf6 like gene was significantly up-regulated in hemocytes,hepatopancreas,gills and intestine of crayfish.After the knockdown of Pc-traf6 like gene,the expression levels of transcription factors（Pc-dorsal）and some key immune effector genes（Pc-ALF3,Pc-lys1,Pc-lectin1 and Pc-crustin2）in TLRs signaling pathway were significantly inhibited.At the same time,in the RNAi experiment,the survival rate of crayfish stimulated with S.aureus or E.ictaluri decreased significantly.In the word,this study found a new Pc-traf6 like gene which played an important role in the antimicrobial innate immune response of P.clarkii.This gene played an important regulatory role in the expression of middle and downstream effectors of TLRs signal pathway in P.clarkii.All these results provided a theoretical basis for further study of the defense mechanism of antibacterial innate immunity of P.clarkii.And it also provided the theoretical support for the prevention and control of bacterial diseases of P.clarkii in aquaculture. |
