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本研究旨在克隆奶牛(Bos taurus)维甲酸相关孤儿受体α (retinoic acid-related orphan receptor alpha,RORα)的蛋白编码序列(coding sequence, CDS),预测分析其结构与功能特征并验证其真核表达载体在HEK293T细胞中的过表达效果，进一步检测RORα基因在奶牛不同组织的表达谱。本研究提取了奶牛肝脏组织的总RNA,反转录得到cDNA,经过PCR扩增后获得奶牛RORα基因的CDS区片段，随后将其与pcDNA3.1-Puro-N-3HA线性化载体进行同源重组连接。转化感受态细胞DH5α后，对初步鉴定为阳性克隆的重组质粒进行测序。结合测序结果，利用ExPASy、 Protscale和DNAstar等生物信息学软件，对奶牛RORα蛋白的理化性质和功能特性进行预测分析。将对照组空质粒pcDNA3.1-Puro-N-3HA和试验组重组质粒pcDNA-3.1-3HA-RORα分别转染至HEK293T细胞，借助实时定量PCR和蛋白质印迹法检测奶牛RORα基因的过表达效果。借助半定量RT-PCR技术检测奶牛RORα基因在肝脏、脾脏和肌肉等7个组织的相对表达量。PCR结果显示，成功克隆了奶牛RORα基因的CDS区片段；酶切及测序结果表明重组质粒pcDNA3.1-3HA-RORα构建成功；生物信息学软件预测分析结果表明，奶牛RORα蛋白三级结构与山羊(Capra hircus)、小鼠(Mus musculus)的高度相似；不同物种间同源性比对显示，奶牛RORα基因与山羊的相似性最高；实时定量PCR和蛋白印迹法结果表明，与对照组相比，试验组HEK293T细胞中奶牛RORα基因的mRNA和蛋白表达水平均显著升高；半定量RT-PCR结果表明，在所检测的7个组织中，奶牛RORα基因在肝脏表达量最高，在脾脏表达量最低。本研究成功克隆了奶牛RORα基因的CDS区片段，在HEK293T细胞中验证了其真核表达载体在mRNA和蛋白水平的表达效果，并分析了其结构与功能特征，检测了其在不同组织的表达谱，为深入探究其在奶牛生殖与代谢调控中的作用机制提供了前期基础。
Abstract：This study aimed to clone the protein coding sequence(CDS) of retinoic acid-related orphan receptor alpha(RORα) in Bos taurus. The structural and functional characteristics of the gene were predicted and analyzed, the overexpression effect of the eukaryotic expression vector in HEK293T cells was verified, and the expression profile of the gene in different tissues of dairy cows was further detected. In this study, total RNA from liver tissue of Bos taurus and cDNA was obtained by reverse transcription. After PCR amplification, the CDS region fragment of Bos taurus RORα gene was obtained. It was ligated with the linearized vector pcDNA3.1-Puro-N-3HA by homologous recombination. After transforming competent cells DH5α, the recombinant plasmids initially identified as positive clones were sequenced. Combined with the sequencing results, bioinformatics softwares such as ExPASy, Protscale and DNAstar were used to predict and analyze the physicochemical properties and functional properties of Bos taurus RORα protein. Then, the control group empty plasmid pcDNA3.1-Puro-N-3HA and the experimental group recombinant plasmid pcDNA-3.1-3HA-RORα were respectively transfected to HEK293T cells, the overexpression effect of Bos taurus RORα gene was detected by real time quantitative PCR and western blotting techniques. Finally, the relative expression of Bos taurus RORα gene in seven tissues including liver, spleen, and muscle tissue was detected by semi-quantitative RT-PCR technology. The PCR results showed that the CDS region fragment of the Bos taurus RORα gene was successfully cloned. The results of enzyme digestion and sequencing showed that the recombinant plasmid pcDNA3.1-3HA-RORα was successfully constructed. The results of bioinformatics software prediction analysis showed that the tertiary structure of Bos taurus RORα protein was highly similar to that of Capra hircus and Mus musculus. The comparison of homology among different species showed that Bos taurus RORα gene was highly similar to that of Capra hircus. The results of real time quantitative PCR and western blotting showed that compared with the control group, the mRNA and protein expression levels of Bos taurus RORα gene in HEK293T cells in the experimental group were significantly increased. The results of semi-quantitative RT-PCR showed that the expression level of Bos taurus RORα gene was the highest in liver and the lowest in spleen among the seven tissues tested. In this study, the CDS region of Bos taurus circadian clock gene RORα was successfully cloned, and the expression effect of its eukaryotic expression vector at mRNA and protein levels was verified in HEK293T cells. The structural and functional characteristics of Bos taurus RORα gene were analyzed, and its expression profile in different tissues was detected. This study provided a preliminary basis for further exploring its mechanism of action in Bos taurus reproductive and metabolic regulation.
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基本信息:
DOI：10.13417/j.gab.042.000013
中图分类号:S823.91
引用信息:
[1]刘薇,王博,王逢博,等.奶牛RORα基因的生物信息学分析与组织表达谱检测[J].基因组学与应用生物学,2023,42(01):13-24.DOI:10.13417/j.gab.042.000013.
基金信息:
陕西省农业农村厅农业专项基金项目[NYKJ-2021-YL(XN)10]; 国家自然科学基金项目(31771301,31602125); 中国博士后科学基金项目(2018T111112)共同资助
2022-11-17
2022-11-17
2022-11-17