山羊RORα基因的克隆、表达载体构建及功能分析

doi:10.11843/j.issn.0366-6964.2022.06.012

摘要/Abstract

摘要： 旨在克隆山羊维甲酸相关孤儿受体α(RORα)基因并构建其真核表达载体，然后利用生物信息学工具系统分析RORα的生物学特性，最后对其在山羊生物钟系统中的作用机制进行初步探究。本研究以1只健康的12月龄雄性西农萨能奶山羊为试验动物，提取其肝组织总RNA，经逆转录PCR反转录为cDNA后，利用常规PCR扩增山羊RORα基因的编码区(coding sequence，CDS)片段，使用同源重组法将其与pcDNA3.1-Puro-N-3HA载体相连接; 重组载体经PCR、酶切和测序鉴定后，将阳性质粒命名为pcDNA3.1-3HA-gRORα; 将pcDNA3.1-Puro-N-3HA和pcDNA3.1-3HA-gRORα质粒分别转染至HEK293T细胞后，通过实时荧光定量PCR(real-time quantitative PCR，qPCR)和蛋白质免疫印迹(western blotting，WB)技术检测山羊RORα的表达; 同时利用ExPASy、ProtScale等软件对山羊RORα基因进行系统的生物信息学分析。另外，使用同源重组法分别构建含有山羊生物钟基因BMAL1和NR1D1启动子片段的pGL4.10-BMAL1-Promoter-Luc及pGL4.10-NR1D1-Promoter-Luc的荧光素酶报告质粒，并通过双荧光素酶报告试验探究山羊RORα蛋白调控BMAL1和NR1D1基因启动子转录活性的作用机制。PCR、酶切和测序鉴定结果表明，pcDNA3.1-3HA-gRORα重组质粒构建成功; qPCR和WB结果显示，pcDNA3.1-3HA-gRORα转染组RORα基因的mRNA表达水平和蛋白表达水平均显著高于pcDNA3.1-Puro-N-3HA转染组(P < 0.01)。生物信息学分析结果显示，山羊RORα基因CDS区序列与绵羊、牛和猪的相似性较高，分别为97.5%、97.1%和95.2%。山羊的RORα蛋白是一种亲水性蛋白。二级结构由α-螺旋、延伸链、β-转角和无规则卷曲组成，有信号肽的概率较小，无跨膜区; 三级结构与小鼠和人的RORα蛋白差异性极小，三者具有高度相似性。此外，PCR、酶切和测序结果表明，pGL4.10-BMAL1-Promoter-Luc和pGL4.10-NR1D1-Promoter-Luc重组质粒构建成功。双荧光素酶报告试验结果表明，山羊RORα蛋白可以显著上调山羊BMAL1和NR1D1基因启动子的转录活性。本研究成功构建了山羊RORα基因的真核表达载体，并证明了RORα蛋白可以正向调控山羊BMAL1和NR1D1基因的启动子活性，这为进一步探究山羊核受体RORα的功能及山羊生物钟的转录调控机制提供了前期基础。

Abstract: This study aimed to clone the retinoic acid-related orphan receptor alpha (RORα) gene and construct its eukaryotic expression vector, and systematically analyze the biological characteristics of RORα by bioinformatics tools. In addition, the regulatory role of RORα in goat circadian clock system was investigated. One healthy male Saanen dairy goat (12 months old) was used as experimental animal. Total RNA extracted from goat liver tissue was reverse transcribed into cDNA by reverse transcription PCR, and the coding sequence (CDS) fragment of goat RORα gene was amplified by PCR using the cDNA template. Then, the PCR product was ligated to pcDNA3.1-Puro-N-3HA vector by homologous recombination. The recombinant plasmid was identified by PCR, restriction enzyme digestion and sequencing. The positive plasmid was named as pcDNA3.1-3HA-gRORα. The pcDNA3.1-Puro-N-3HA and pcDNA3.1-3HA-gRORα plasmids were transfected into HEK293T cells, respectively. The expression of RORα was examined by real-time quantitative PCR (qPCR) and Western blotting (WB) analysis. Meanwhile, the goat RORα gene was systematically analyzed by bioinformatics softwares including ExPASy and ProtScale. In addition, the luciferase reporter plasmids of pGL4.10-BMAL1-Promoter-Luc and pGL4.10-NR1D1-Promoter-Luc containing the promoter fragments of goat circadian clock genes BMAL1 and NR1D1 were constructed by homologous recombination methods. At last, the dual luciferase reporter assay was used to explore the function of goat RORα protein in regulating BMAL1 and NR1D1 gene promoter activity. PCR, enzyme digestion and sequencing results showed that the eukaryotic expression vector of pcDNA3.1-3HA-gRORα was successfully constructed. The qPCR and WB results showed that the mRNA and protein expression of goat RORα gene in HEK293T cells with the transfection of pcDNA3.1-3HA-gRORα were significantly higher than those in the group of pcDNA3.1-Puro-N-3HA transfection(P < 0.01). Bioinformatics analysis showed that the similarities of the CDS region of goat RORα gene were 97.5% with sheep, 97.1% with cattle, and 95.2% with pig, respectively. Goat RORα protein was a hydrophilic protein, and its secondary structure mainly consisted of α-helix, extended chain, β-turn, and irregular coiled structures. It had a small probability of having a signal peptide, and it had no transmembrane region. Meanwhile, the tertiary structure of goat RORα protein was highly similar to the corresponding protein of mouse and human. PCR, enzyme digestion and sequencing results showed that pGL4.10-BMAL1-Promoter-Luc and pGL4.10-NR1D1-Promoter-Luc recombinant plasmids were successfully constructed. The results of dual luciferase reporter assay showed that goat RORα protein could significantly upregulate the transcriptional activity of goat BMAL1 and NR1D1 gene promoters. In this study, the eukaryotic expression vector of goat RORα was successfully constructed. Meanwhile, it is proved that goat RORα protein can positively regulate the activity of goat BMAL1 and NR1D1 gene promoters. These results provide a preliminary basis for further investigate the biological role of goat nuclear receptor RORα and the transcriptional regulation mechanism of goat circadian clock system.

Key words: goat, RORα, eukaryotic expression vector, bioinformatics analysis, circadian clock

中图分类号:

S827.2

高登科, 赵泓淙, 董浩, 靳亚平, 陈华涛. 山羊RORα基因的克隆、表达载体构建及功能分析[J]. 畜牧兽医学报, 2022, 53(6): 1779-1794.

GAO Dengke, ZHAO Hongcong, DONG Hao, JIN Yaping, CHEN Huatao. The Cloning, Expression Vector Construction and Function Analysis of Goat RORα Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(6): 1779-1794.

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