SMAD2、SMAD3和SMAD4基因在小尾寒羊卵巢活动中的功能分析OA北大核心CSTPCD

To investigate the biological functions of SMAD2,SMAD3 and SMAD4 genes in the reproductive activity of small-tailed cold sheep,which are important members of the SMAD gene family,the bioinformatics techniques were used to analyze the physicochemical properties,hydrophilicity and subcellular localisation of the SMAD2,SMAD3 and SMAD4 genes,as well as secondary structures,protein interaction prediction and enrichment analysis of gene ontology(GO)and kyoto encyclopedia of genes and genomes(KEGG).The small-tailed sheep was as material,positive expression of SMAD2,SMAD3 and SMAD4 proteins in ovarian tissue was determined by immunohistochemistry staining(IHC),and the real-time quantitative PCR(RT-qPCR)and western blot(WB)were used to determine the expression levels of SMAD2,SMAD3 and SMAD4 mRNAs and their encoded proteins in ovarian tissues and follicles of different diameters at different physiological stages.The results showed that SMAD2,SMAD3 and SMAD4 proteins were hydrophilic proteins with theoretical isoelectric points of 6.67,6.73 and 6.50.The positive expressions in ovarian tissues at different physiological stages were mainly located in oocytes,follicular membrane cells and granulosa cells,and the highest proportion of α-helices and random nematic clusters in the secondary structure,which were associated with FOXH1,ACVR1B,SMAD1,TGFβR1,TGFβR2,ZFYVE9,SKI,SKIL,TGIF1 and ENSOARP0000001869 proteins.SMAD2,SMAD3 and SMAD4 genes played biological functions in the TGF-β receptor signalling pathway,TGF-β receptor binding,TGF-β activated receptor activity and were enriched in TGF-β signalling pathway.The expressions of SMAD2 and SMAD4 proteins were significantly higher in ovarian tissues at 42 h after bolus withdrawal than those at 18 h after bolus withdrawal,and their mRNA expression showed the consistent trends with proteins.However,the expressions of SMAD3 gene mRNA and its encoded protein were not significant in ovarian tissues between different physiological stages.The expressions of SMAD3 and its encoded protein were significantly higher in medium follicles than those in large follicles,while the expressions of SMAD2 and SMAD4 were no significant differences between different follicle diameters.In conclusion,SMAD2,SMAD3 and SMAD4 genes had different biological functions in ovarian cycling of small-tailed cold sheep,i.e.SMAD2 and SMAD4 genes were involved in ovarian physiological changes from early oestrus to preovulation in small-tailed cold sheep,but not in follicle development;SMAD3 was involved in follicle development.Above results provided a theoretical reference to further explore the molecular mechanism of the SMAD gene family in ovarian activity in small-tailed cold sheep.