中华绒螯蟹C/EBPβ基因的克隆及表达OA

We investigated the molecular regulatory role of CCAAT/enhancer-binding protein β(C/EBPβ),an important bZIP transcription factor,in Eriocheir sinensis lipid metabolism.The full-length cDNA of C/EBPβ was obtained by reverse transcription PCR combined with rapid amplification of cDNA ends using hepatopancreas cDNA as the template.Bioinformatic analyses were performed to characterize sequence features and predict structural and physicochemical properties of the protein.The tissue expression profile of C/EBPβ was examined using quantitative PCR.Furthermore,functional verification was conducted via injections of the C/EBPβ agonist Rosiglitazone,inhibitor Withaferin A,and siRNA-mediated gene silencing to evaluate regulatory effects of C/EBPβ on the expression of lipid metabolism–related genes,including Fad6,Fad9,Elovl4,Elovl6,and Elovl7.The full-length cDNA of C/EBPβ was 1637 bp,containing an 879-bp open reading frame encoding a 293-amino-acid protein,a 227-bp 5′-UTR,and a 531-bp 3′-UTR.Sequence analysis revealed a conserved bZIP domain,confirming its identity as a member of the C/EBP family.Homology analysis revealed high similarity of E.sinensis C/EBPβ with Portunus trituberculatus(78.23%)and Chionoecetes opilio(79.93%).Phylogenetic analysis revealed that E.sinensis C/EBPβ clustered closely with that of other crab species.Tissue distribution analysis revealed significantly higher C/EBPβ expression in the hepatopancreas and gills than in other tissues(P<0.05).Functional assays demonstrated that Rosiglitazone injection significantly upregulated Fad6,Fad9,Elovl6,and Elovl7 expression,whereas Withaferin A and siRNA interference markedly downregulated their transcription levels(P<0.05).Elovl4 expression exhibited no significant response to any treatment.In this study,we cloned the full-length C/EBPβ gene from E.sinensis and confirmed that C/EBPβ could promote LC-PUFA biosynthesis by regulating transcription of desaturase and elongase genes in the hepatopancreas.These results provide new insights into the biological functions of C/EBPβ in crustaceans and offer a theoretical basis for elucidating the molecular mechanisms underlying lipid metabolism regulation in E.sinensis.