Cloning of MyD88-3 gene in the Pacific oyster(Crassostrea gigas)and expression pattern in response to Vibrio alginolyticus challenge
[Objective]The purpose of the study to clone a novel myeloid differentiation factor 88(MyD88)gene ferent tissues of C.gigas under Vibrio alginolyticus challenge,so as to provide a theoretical basis for exploring the role of MyD88 gene in the immune regulation and the healthy cultivation of C.gigas.[Method]Coding region(CDS)of Cg-MyD88-3 gene was cloned.The bioinformatics analysis was analyzed by ProtScale,SWISS-MODEL and InterPro.Real-time fluorescence quantitative PCR was used to analyze the expression changes of CgMyD88-3 gene in different tissues of C.gigas.[Result]The CDS length of CgMyD88-3 gene was 537 bp,encoding 178 amino acids residues.The theoretical molecular weight of CgMyD88-3 protein was 20.74 kD and the isoelectric point was 6.10.The sub-cellular localizations of the CgMyD88-3 protein were predicted in the cytoplasm.CgMyD88-3 was predicted to have five casein kinase II phos-phorylation sites(17TEED20,29SNLE32,76TQNE79,118TAND121,123TKED126)and three protein kinase C phosphorylation sites(26TMK28,148TAK150,169SEK171).CgMyD88-3 amino acids possessed only TIR(Toll/interleukin-1 receptor)domain but lacked Death domain.CgMyD88-3 amino acid sequence had the highest similarity with CvMyD88-like amino acid se-quence of Crassostrea virginica,which was 100%.The result of the phylogenetic tree based on MyD88 amino acid se-quence similarity showed that CgMyD88-3 firstly clustered with C.virginica CvMyD88-like,and then with CgMyD88-T1 and CgMyD88-T2 of C.gigas.The real-time fluorescence quantitative RT-PCR detection revealed that CgMyD88-3 was expressed in all six tissues of healthy C.virginica.The highest relative expression was found in the mantle,followed by gills,and in other tissues,the order was haemocytes>gonads>digestive glands,with the lowest relative expression level in the adductor.After infection by Vibrio alginolyticus,the relative expression of CgMyD88-3 was up-regulated in the adductor,digestive glands,haemocytes and gills.The expression of CgMyD88-3 reached peak at 72 h in adductor,digestive glands and haemocytes,respectively,and peaked at 6 h in gills,while the expression of CgMyD88-3 at other times was lower than 0 h after infection in the mantle.[Conclusion]The present results show that CgMyD88-3 is ex-pressed in all tissues of healthy C.virginica,the highest relative expression is found in the mantle.Stimulation by V.algi-nolyticus obviously induces the up-regulation of CgMyD88-3 expression,indicating that the CgMyD88-3 may play an im-portant role in the defense of C.virginica against external microbial and pathogen infections.
万方数据
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