梅花鹿Zfx基因CDS区克隆、生物信息学及组织表达分析OA

By cloning the Zfx gene of sika deer and analyzing its molecular characteristics,this study aimed to provide key sequence targets and a theoretical basis for designing sika deer Zfx interference vectors.After collecting testicular tissue of sika deer,total RNA was extracted and reverse-transcribed into cDNA.Two pairs of primers were designed to amplify the CDS region of the Zfx gene segmentally,followed by cloning and sequencing to obtain its full-length se-quence.Bioinformatics tools were used to analyze its physicochemical properties,hydrophilicity/hydrophobicity,glyco-sylation/phosphorylation sites,and secondary/tertiary structures,as well as construct phylogenetic tree and conduct ho-mology analysis.In addition,RT-qPCR was employed to detect its tissue expression pattern.The results showed that the full-length CDS region of the sika deer Zfx gene was successfully cloned,with a length of 1 479 bp.This gene en-codes 492 amino acids,the molecular formula of its encoded protein is C2 322H3 675N635O791S28,which showed 54 041.18,4.5,46.84 of its relative molecular weight,theoretical isoelectric point and instability index,respectively.Therefore,Zfx protein is classified as an unstable hydrophilic protein and contains 36 phosphorylation sites 19 Ser,5 Tyr,12 Thr)and 3 glycosylation sites.Consistent with the tertiary structure prediction,the secondary structure is dominated by ran-dom coils(68.70％).Phylogenetic tree analysis revealed that sika deer emerged the closest genetic relationship with red deer(Cervidae),followed by bovidae animals,such as domestic cattle,wild yaks,goats,and sheep,these animals are all belong to the suborder Ruminantia(Artiodactyla);and it showed relatively distant relationship with non-ruminant animals such as tigers,domestic cats,and domestic dogs.Overlapping region homology analysis indicated that the Zfx gene sequence of sika deer shares 97.05％homology with that of 9 other species,among which the homology of red deer is the highest.Interestingly,GO functional enrichment analysis showed that the Zfx gene is involved in biological processes such as sex determination,while KEGG pathway enrichment analysis indicated its involvement in cancer-related pathways.Tissue expression analysis demonstrated that the expression level of Zfx gene is significantly higher in testis and liver than that of muscle tissue(P＜0.01).In summary,this study found that the Zfx gene of sika deer is highly conserved during evolution.The structural characteristics of its encoded protein suggest that it may participate in the ubiquitin-proteasome degradation pathway through phosphorylation modification to regulate spermatogenesis.