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 MyD88-3 from the Pacific oyster（Crassostrea gigas）, the expression pattern of CgMyD88-3 gene was analyzed in different 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 CgMyD88-3 gene was cloned. The bioinformatics analysis was analyzed by ProtScale, SWISS-MODEL and InterPro. Realtime 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 phosphorylation 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 sequence of Crassostrea virginica, which was 100%. The result of the phylogenetic tree based on MyD88 amino acid sequence 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 CgMyD88-3was 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 expressed in all tissues of healthy C. virginica, the highest relative expression is found in the mantle. Stimulation by V. alginolyticus obviously induces the up-regulation of CgMyD88-3 expression, indicating that the CgMyD88-3 may play an important role in the defense of C. virginica against external microbial and pathogen infections.