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

Abstract

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

CLC Number:

S827.2

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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The Cloning, Expression Vector Construction and Function Analysis of Goat RORα Gene

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