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.

References 44

[1]	WANG H. Circadian biology and its recent progresses[J]. Chinese Bulletin of Life Sciences, 2015, 27(11): 1313-1319. (in Chinese)王晗. 生物钟生物学及其研究进展[J]. 生命科学, 2015, 27(11): 1313-1319.
[2]	XING C, SONG L. Regulation system for generation and maintenance of circadian rhythms[J]. Military Medical Sciences, 2017, 41(8): 698-702. (in Chinese)邢陈, 宋伦. 昼夜节律产生和维持的调控系统[J]. 军事医学, 2017, 41(8): 698-702.
[3]	MARCHEVA B, RAMSEY K M, BUHR E D, et al. Disruption of the clock components CLOCK and BMAL1 leads to hypoinsulinaemia and diabetes[J]. Nature, 2010, 466(7306): 627-631.
[4]	ZHANG R, LAHENS N F, BALLANCE H I, et al. A circadian gene expression atlas in mammals: implications for biology and medicine[J]. Proc Natl Acad Sci U S A, 2014, 111(45): 16219-16224.
[5]	ASTIZ M, HEYDE I, OSTER H. Mechanisms of communication in the mammalian circadian timing system[J]. Int J Mol Sci, 2019, 20(2): 343.
[6]	ROSENWASSER A M, TUREK F W. Neurobiology of circadian rhythm regulation[J]. Sleep Med Clin, 2015, 10(4): 403-412.
[7]	ABBOTT S M, ZEE P C. Circadian rhythms: implications for health and disease[J]. Neurol Clin, 2019, 37(3): 601-613.
[8]	GIGUÈRE V, TINI M, FLOCK G, et al. Isoform-specific amino-terminal domains dictate DNA-binding properties of RORα, a novel family of orphan hormone nuclear receptors[J]. Genes Dev, 1994, 8(5): 538-553.
[9]	MA H Z, KANG J, FAN W G, et al. ROR: nuclear receptor for melatonin or not?[J]. Molecules, 2021, 26(9): 2693.
[10]	PILORZ V, ASTIZ M, HEINEN K O, et al. The concept of coupling in the mammalian circadian clock network[J]. J Mol Biol, 2020, 432(12): 3618-3638.
[11]	URIZ-HUARTE A, DATE A, ANG H, et al. The transcriptional repressor REV-ERB as a novel target for disease[J]. Bioorg Med Chem Lett, 2020, 30(17): 127395.
[12]	STRATMANN M, STADLER F, TAMANINI F, et al. Flexible phase adjustment of circadian albumin D site-binding protein (Dbp) gene expression by CRYPTOCHROME1[J]. Genes Dev, 2010, 24(12): 1317-1328.
[13]	UEDA H R, HAYASHI S, CHEN W B, et al. System-level identification of transcriptional circuits underlying mammalian circadian clocks[J]. Nat Genet, 2005, 37(2): 187-192.
[14]	RIPPERGER J A, SCHIBLER U. Rhythmic CLOCK-BMAL1 binding to multiple E-box motifs drives circadian Dbp transcription and chromatin transitions[J]. Nat Genet, 2006, 38(3): 369-374.
[15]	POURCET B, DUEZ H. Circadian Control of inflammasome pathways: implications for circadian medicine[J]. Front Immunol, 2020, 11: 1630.
[16]	JETTEN A M. Retinoid-related orphan receptors (RORs): critical roles in development, immunity, circadian rhythm, and cellular metabolism[J]. Nucl Recept Signal, 2009, 7: e003.
[17]	FAN J S, LV Z L, YANG G H, et al. Retinoic acid receptor-related orphan receptors: critical roles in tumorigenesis[J]. Front Immunol, 2018, 9: 1187.
[18]	LI Z Q, ZHAO J, LIU H Y, et al. Melatonin inhibits apoptosis in mouse Leydig cells via the retinoic acid-related orphan nuclear receptor α/p53 pathway[J]. Life Sci, 2020, 246: 117431.
[19]	JIA L L, JIN F, FU S Y, et al. Comparation of expression pattern of Clock genes in cashmere goat skin[J]. China Animal Husbandry & Veterinary Medicine, 2015, 42(2): 251-257. (in Chinese)贾丽丽, 金凤, 付绍印, 等. 生物钟基因在绒山羊皮肤中表达模式的比较[J]. 中国畜牧兽医, 2015, 42(2): 251-257.
[20]	ZHAO Y H, LIU Z H, WANG L, et al. Expression of the RORα gene in Inner Mongolian cashmere goat hair follicles[J]. Genet Mol Res, 2015, 14(1): 380-388.
[21]	LIU Z L, CHEN T, YANG D D, et al. Molecular cloning, bioinformatics analysis and transcriptional activity of promoter of nuclear factor erythroid 2-related factor(Nrf2) gene in pig[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(7): 1328-1339. (in Chinese)刘宗立, 陈涛, 杨丹丹, 等. 猪Nrf2基因克隆、生物信息学分析及启动子区转录活性分析[J]. 畜牧兽医学报, 2019, 50(7): 1328-1339.
[22]	DU P F, CHEN B, GAO L G, et al. Cloning and expression analysis of chicken HMIT gene[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(8): 1811-1822. (in Chinese)杜鹏飞, 陈博, 高林歌, 等. 鸡HMIT基因的克隆与表达分析[J]. 畜牧兽医学报, 2020, 51(8): 1811-1822.
[23]	WANG X J, XIANG H, ZHANG H R, et al. Cloning, sequence analysis and function prediction of HABP4 gene in goat[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(12): 3426-3438. (in Chinese)王宪军, 向华, 张焕容, 等. 山羊HABP4基因的克隆、序列分析及功能预测[J]. 畜牧兽医学报, 2021, 52(12): 3426-3438.
[24]	MAURY E. Off the Clock: from circadian disruption to metabolic disease[J]. Int J Mol Sci, 2019, 20(7): 1597.
[25]	YU E A, WEAVER D R. Disrupting the circadian clock: gene-specific effects on aging, cancer, and other phenotypes[J]. Aging (Albany NY), 2011, 3(5): 479-493.
[26]	FAN J F, HE Y, WANG W Z, et al. Research progress of small molecular compounds regulating biological clock[J]. China Pharmacy, 2021, 32(7): 890-896. (in Chinese)凡杰夫, 何颖, 王伟忠, 等. 调控生物钟的小分子化合物的研究进展[J]. 中国药房, 2021, 32(7): 890-896.
[27]	XIAO Y Y, ZHAO L J, LI W D, et al. Circadian clock gene BMAL1 controls testosterone production by regulating steroidogenesis-related gene transcription in goat Leydig cells[J]. J Cell Physiol, 2021, 236(9): 6706-6725.
[28]	ZHAO H C, GAO D K, JIANG H Z, et al. Construction of eukaryotic expression vector and bioinformatics analysis of circadian CLOCK gene in goats[J]. China Animal Husbandry & Veterinary Medicine, 2021, 48(12): 4327-4338. (in Chinese)赵泓淙, 高登科, 江海圳, 等. 山羊生物钟基因CLOCK真核表达载体的构建和生物信息学分析[J]. 中国畜牧兽医, 2021, 48(12): 4327-4338.
[29]	WANG Y Q, GAO D K, ZHAO H C, et al. Construction of a eukaryotic expression vector in goat NR1D1 gene and its bioinformatics analysis[J]. Chinese Journal of Animal Science, doi:10.19556/j.0258-7033.20210825-06.(in Chinese)王逸群, 高登科, 赵泓淙, 等. 山羊NR1D1基因真核表达载体的构建及生物信息学分析[J]. 中国畜牧杂志, doi:10.19556/j.0258-7033.20210825-06.
[30]	ZHAO Y H, SUN Y M, LIU Z H, et al. Cloning and sequence analysis of RORα gene in inner Mongolian cashmere goat[J]. Journal of Inner Mongolia Agricultural University, 2010, 31(3): 1-4. (in Chinese)赵艳红, 孙永明, 刘志红, 等. 内蒙古绒山羊孤核受体RORα部分cDNA克隆及序列分析[J]. 内蒙古农业大学学报, 2010, 31(3): 1-4.
[31]	CHEN C T, SCHULTZ J A, HAVEN S E, et al. Loss of RAR-related orphan receptor alpha (RORα) selectively lowers docosahexaenoic acid in developing cerebellum[J]. Prostaglandins Leukot Essent Fatty Acids, 2020, 152: 102036.
[32]	YASUI H, MATSUZAKI Y, KONNO A, et al. Global knockdown of retinoid-related orphan Receptor α in mature Purkinje cells reveals aberrant cerebellar phenotypes of Spinocerebellar ataxia[J]. Neuroscience, 2021, 462: 328-336.
[33]	DZHAGALOV I, GIGUÈRE V, HE Y W. Lymphocyte development and function in the absence of retinoic acid-related orphan receptor α[J]. J Immunol, 2004, 173(5): 2952-2959.
[34]	COOK D N, KANG H S, JETTEN A M. Retinoic acid-related orphan receptors (RORs): regulatory functions in Immunity, development, circadian rhythm, and metabolism[J]. Nucl Receptor Res, 2015, 2: 101185.
[35]	FUJIEDA H, BREMNER R, MEARS A J, et al. Retinoic acid receptor-related orphan receptor α regulates a subset of cone genes during mouse retinal development[J]. J Neurochem, 2009, 108(1): 91-101.
[36]	CAI Z M, ISHIBASHI T, KOZAI M, et al. ROR agonist hampers the proliferation and survival of postactivated CD8+ T cells through the downregulation of cholesterol synthesis-related genes[J]. Immunol Cell Biol, 2021, 99(3): 288-298.
[37]	SAYED R K A, MOKHTAR D M, FERNÁNDEZ-ORTIZ M, et al. Retinoid-related orphan nuclear receptor alpha (RORα)-deficient mice display morphological testicular defects[J]. Lab Invest, 2019, 99(12): 1835-1849.
[38]	SAYED R K A, MOKHTAR D M, FERNÁNDEZ-ORTIZ M, et al. Lack of retinoid acid receptor-related orphan receptor alpha accelerates and melatonin supplementation prevents testicular aging[J]. Aging (Albany NY), 2020, 12(13): 12648-12668.
[39]	YANG M H, GUAN S Y, TAO J L, et al. Melatonin promotes male reproductive performance and increases testosterone synthesis in mammalian Leydig cells[J]. Biol Reprod, 2021, 104(6): 1322-1336.
[40]	FANG Y, ZHANG J L, LI Y H, et al. Melatonin-induced demethylation of antioxidant genes increases antioxidant capacity through RORα in cumulus cells of prepubertal lambs[J]. Free Radic Biol Med, 2019, 131: 173-183.
[41]	DENG S L, ZHANG Y, YU K, et al. Melatonin up-regulates the expression of the GATA-4 transcription factor and increases testosterone secretion from Leydig cells through RORα signaling in an in vitro goat spermatogonial stem cell differentiation culture system[J]. Oncotarget, 2017, 8(66): 110592-110605.
[42]	XU Q, HUANG M, WANG X M, et al. TF promotes circadian phase advancement by affecting expressions of liver circadian clock genes[J]. Military Medical Sciences, 2021, 45(1): 1-6. (in Chinese)徐晴, 黄鸣, 王晓明, 等. TF通过影响肝脏生物钟基因表达促进昼夜节律相位前移[J]. 军事医学, 2021, 45(1): 1-6.
[43]	PANDA S. Circadian physiology of metabolism[J]. Science, 2016, 354(6315): 1008-1015.
[44]	YANG X Y, DOWNES M, YU R T, et al. Nuclear receptor expression links the circadian clock to metabolism[J]. Cell, 2006, 126(4): 801-810.

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

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