牛MED28基因编码区克隆、生物信息学分析及组织表达谱研究

Cloning,Bioinformatics Analysis and Tissue Expression Profile of Coding Region of Bovine MED 28 Gene

[目的]克隆牛介体复合物亚单位28(mediator complex subunit 28,MED28)基因CDS区,预测MED28蛋白的结构功能,并分析MED 28基因在牛各组织中的表达水平,为后续研究MED 28基因的功能奠定基础。[方法]以牛成肌细胞RNA反转录的cDNA为模板,利用PCR方法扩增并克隆牛MED 28基因CDS区。运用MegAlign、Mega 11.0软件进行MED28蛋白的相似性比对和系统进化树构建,利用ProtParam、ProtScale、SOPMA、SWISS-MODEL、MEME、STRING等在线软件分析MED28蛋白的理化性质及结构功能,并通过实时荧光定量PCR检测MED 28基因在牛心脏、肝脏、脾脏、肺脏、肾脏、小肠、肌肉组织中的表达量。[结果]试验成功克隆MED 28基因CDS区,长度为537 bp,编码178个氨基酸。相似性比对结果表明,牛MED28蛋白与山羊、绵羊的相似性最高;系统进化树显示,牛与山羊、绵羊的遗传距离最近,与鸡的遗传距离最远。MED28蛋白属于不稳定的亲水性蛋白,无跨膜区和信号肽,主要在细胞外发挥作用,含有2个N-糖基化位点和21个磷酸化位点。MED28蛋白二级结构由α-螺旋、β-转角、延伸链和无规则卷曲组成,其中α-螺旋占比最大,蛋白结构稳定,与三级结构预测结果一致。蛋白互作网络分析表明,MED28蛋白与其家族的多种蛋白之间存在互作,且主要在转录途径和甲状腺信号通路中发挥作用。MEME预测结果表明,MED28蛋白的保守基序在牛、人、猪、小鼠、马、兔、山羊、绵羊之间是相同的;MED28蛋白结构域有低复杂度和卷曲螺旋序列。实时荧光定量PCR结果表明,MED 28基因在牛肌肉组织中的表达量显著高于其他组织(P<0.05)。[结论]牛和羊的亲缘关系最近、蛋白相似性高。MED28蛋白亲水且结构稳定,存在N-糖基化位点和磷酸化位点,无跨膜区和信号肽,主要分布在细胞外,并参与转录途径和甲状腺信号通路。MED 28基因在牛肌肉组织中高表达。以上结果可为进一步研究牛MED 28基因的功能提供参考。

[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 MED 28 gene in various tissues of dairy cows,so as to provide a foundation for further exploration of the function of MED 28 gene.[Method]The CDS region of MED 28 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 MegAlign 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 MED 28 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 MED 28 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 MED 28 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 MED 28 gene was the highest in muscle of dairy cows.The results could provide reference for further study on the function of bovine MED 28 gene.