| Crassostrea hongkongensis is the main species of mariculture along the coast of South China due to its high nutritional and economic values.With the rapid development of coastal economy,the water environment in aquaculture area is increasingly polluted and the deterioration of water quality provides a favorable environment for pathogenic bacteria.In recent years,mass mortality of oysters due to environmental factors and diseases has often occurred.Currently,it is widely believed that oysters do not have specific immunity in their bodies and can only rely on the innate immune system to resist invading pathogens,so it is of great practical importance to study the molecular mechanism of innate immunity in Crassostrea hongkongensis.The cation-dependent mannose-6-phosphate receptor(CD-M6PR)is a P-type lectin that plays a crucial role in lysosomal enzyme transport,bacterial resistance,and viral entry.In this study,we cloned and analyzed the ORF of the CD-M6 PR gene from Crassostrea hongkongensis and named it Ch CD-M6 PR using molecular cloning and raw letter site.Its tissue expression pattern and immune response to Vibrio alginolyticus also was analyzed by using real-time fluorescence quantitative PCR.We also constructed a prokaryotic expression vector for the Ch CD-M6 PR extracellular region and analyzed its expression by SDS-PAGE and Western Blot.The results showed that the ORF of Ch CD-M6 PR was 801 bp long and encoded a protein of 266 amino acids with a signal peptide at the N-terminus,as well as Man-6-P＿recep,ATG27 and transmembrane structural domains.Phylogenetic analysis indicated that Crassostrea hongkongensis shared the highest similarity with Crassostrea gigas in the terms of CD-M6 PR.The Ch CD-M6 PR gene was found to be expressed in various tissues,with the highest expression observed in the hepatopancreas and the lowest in the hemocytes by the fluorescence quantitative PCR.Furthermore,Ch CD-M6 PR gene was significantly upregulated in the hemocytes for a short time after Vibrio alginolyticus stimulation,and was upregulated in hepatopancreas for 24 h.After Ch CD-M6 PR gene knocking out,the cumulative mortality rate of Crassostrea hongkongensis with Vibrio alginolyticus challenge significantly increased at 96 hours,reaching 73%.In addition,the prokaryotic expression system successfully produced the Ch CD-M6 PR extracellular region protein,which had a molecular weight size of 20 k Da as confirmed by SDSPAGE and Western Blot analysis.Overall,the findings suggests that Ch CD-M6 PR plays a crucial role in the immune response of Crassostrea hongkongensis to Vibrio alginolyticus infection. |
