牛MED28基因编码区克隆、生物信息学分析及组织表达谱研究OA北大核心CSTPCD

[Objective]This study was aimed to clone the coding(CDS)region of mediator complex subunit 28(MED28)gene,predict the structure and function of MED28 protein,and analyze the expression of MED28 gene in various tissues of dairy cows,so as to provide a foundation for further exploration of the function of MED28 gene.[Method]The CDS region of MED28 gene was amplified using PCR and cloned with cDNA template derived from myoblast RNA reverse transcription.Similarity alignment and phylogenetic tree construction of MED28 protein were performed using Meg Align and Mega 11.0 softwares,respectively.Additionally,various online platforms,including ProtParam,ProtScale,SOPMA,SWISS-MODEL,MEME and STRING,were employed to analyze the physicochemical properties and structural functions.The expression of MED28 gene in heart,liver,spleen,lung kidney,small intestine and muscle of dairy cows were detected using Real-time quantitative PCR.[Result]The CDS region of MED28 gene was successfully amplified,the length was 537 bp which encoded 178 amino acids.Similarity alignment results showed that MED28 protein of dairy cows shared the highest similarity with Capra hircus and Ovis aries.Phylogenetic tree showed that there were the closest genetic relationship between dairy cows and Capra hircus and Ovis aries,and there was the farthest genetic relationship between dairy cows and Gallus gallus.The MED28 protein,containing 2 N-glycosylation sites and 21 phosphorylation sites,was characterized as an unstable,hydrophilic protein lacking transmembrane regions and signal peptides,predominantly functioning extracellularly.The secondary structure of MED28 protein was composed of alpha helix,beta turn,extended chain,and random coil,with alpha helix being predominant,suggesting a stable protein structure that was consistent with predicted tertiary structure.Protein-protein interaction network analysis demonstrated that MED28 protein interacted with many proteins in its family,and they mainly played a role in transcriptional pathways and thyroid signaling pathways.MEME prediction indicated that the conserved motifs of MED28 protein were the same among dairy cows,Homo sapiens,Sus scrofa,Mus musculus,Equus caballus,Oryctolagus cuniculus,Capra hircus and Ovis aries.MED28 protein domain was composed of low complexity and coiled coil sequences.Real-time quantitative PCR results showed that the expression of MED28 gene in muscle of dairy cows was significantly higher than that in other tissues(P＜0.05).[Conclusion]The genetic relationship was the closest between dairy cows and Capra hircus and Ovis aries,and the protein similarity was high.MED28 protein exhibited a hydrophilic and structurally stable nature,featuring N-glycosylation and phosphorylation sites,while lacking a transmembrane region and signal peptide.MED28 protein functioned mainly in extracellular and was involved in transcriptional pathways and thyroid signaling pathways.The expression of MED28 gene was the highest in muscle of dairy cows.The results could provide reference for further study on the function of bovine MED28 gene.