奶牛NR1D1基因的真核表达载体构建、表达谱及其在卵巢组织的定位

doi:10.11843/j.issn.0366-6964.2023.01.013

Abstract

Abstract: This study aimed to clone the coding sequence (CDS) of the nuclear receptor subfamily 1 group D member 1 gene (NR1D1) in dairy cows, construct its eukaryotic expression vector, predict and analyze the biological function of the NR1D1 gene, detect the tissue expression profiles of the gene and its expression distribution in ovary. In this study, 5 healthy female dairy cows (24 months old) were used as experimental animals. The full-length fragment of the CDS region in dairy cow NR1D1 gene with homologous arms was amplified by PCR, and the target fragment was ligated to the linearized pcDNA3.1-Puro-N-3HA vector by homologous recombination to construct a recombinant plasmid. Recombinant plasmids were identified by enzyme digestion, PCR and sequencing techniques. Combined with the results of plasmid sequencing, bioinformatics softwares were used to predict and analyze the biological function of NR1D1. The correctly identified plasmid was transfected into HEK293T cells, and the expression of NR1D1 gene at mRNA and protein levels was detected by real-time quantitative PCR (qPCR) and Western blotting. In addition, qPCR was used to detect the expression profile of dairy cow NR1D1 gene in different tissues, and immunohistochemistry was used to focus on its expression distribution in ovary. The full-length fragment of the CDS (1 884 bp) region of NR1D1 gene in dairy cow with homology arms was obtained by PCR amplification. The results of restriction enzyme digestion, PCR identification and sequencing showed that the eukaryotic expression vector pcDNA3.1-3HA-cNR1D1 was successfully constructed. The results of bioinformatics analysis showed that the NR1D1 protein had no transmembrane structure and was a nucleoprotein, its nuclear localization sequence existed near the 200th amino acid, and there were 86 potential phosphorylation sites in NR1D1 protein. The tertiary structure of cow and mouse NR1D1 protein was similar and the NR1D1 protein may play an important role in the physiological process of reproduction, metabolism and immunity in dairy cow. The pcDNA3.1-3HA-cNR1D1was transfected into HEK293T cells, and the cow NR1D1 gene was successfully overexpressed at both mRNA and protein levels. The qPCR results showed that the expression of NR1D1 gene in dairy cow’s rumen, colon and liver was significantly higher than other tissues. Immunohistochemical results showed that NR1D1 was mainly expressed in granulosa cells of follicles in different developmental stages. This study successfully constructed dairy cow NR1D1 gene’s eukaryotic expression vector, and successfully overexpressed NR1D1 in HEK293T cells. The tissue expression profile of NR1D1 and its expression distribution in the ovary of dairy cows were preliminarily predicted and analyzed. This study provided the preliminary basis and key materials for in-depth exploration of NR1D1’s biological function.

Key words: ruminants, circadian clock, nuclear receptor, reproduction, expression profiles

CLC Number:

S823.91

WANG Yiqun, LIU Zupei, LI Yating, ZHANG Haisen, LI Dan, JIN Yaping, CHEN Huatao. The Dairy Cow NR1D1 Gene’s Eukaryotic Expression Vector Construction, Expression Profile and Its Ovarian Localization[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 133-145.

References 41

[1]	PATKE A,YOUNG M W,AXELROD S.Molecular mechanisms and physiological importance of circadian rhythms[J].Nat Rev Mol Cell Biol,2020,21(2):67-84.
[2]	DUNLAP J C.Molecular bases for circadian clocks[J].Cell,1999,96(2):271-290.
[3]	MOHAWK J A,GREEN C B,TAKAHASHI J S.Central and peripheral circadian clocks in mammals[J].Annu Rev Neurosci,2012,35:445-462.
[4]	COX K H,TAKAHASHI J S.Circadian clock genes and the transcriptional architecture of the clock mechanism[J].J Mol Endocrinol,2019,63(4):R93-R102.
[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]	SCHIBLER U,GOTIC I,SAINI C,et al.Clock-talk:interactions between central and peripheral circadian oscillators in mammals[J]. Cold Spring Harb Symp Quant Biol,2015,80:223-232.
[7]	PARTCH C L,GREEN C B,TAKAHASHI J S.Molecular architecture of the mammalian circadian clock[J].Trends Cell Biol,2014, 24(2):90-99.
[8]	EVERETT L J,LAZAR M A.Nuclear receptor Rev-erbα:up,down,and all around[J].Trends Endocrinol Metab,2014,25(11):586-592.
[9]	LAZAR M A,HODIN R A,DARLING D S,et al.A novel member of the thyroid/steroid hormone receptor family is encoded by the opposite strand of the rat c-erbA alpha transcriptional unit[J].Mol Cell Biol,1989,9(3):1128-1136.
[10]	SUN Z,FENG D,EVERETT L J,et al.Circadian epigenomic remodeling and hepatic lipogenesis:lessons from HDAC3[J].Cold Spring Harb Symp Quant Biol,2011,76:49-55.
[11]	HUNTER A L,PELEKANOU C E,ADAMSON A,et al.Nuclear receptor REVERBα is a state-dependent regulator of liver energy metabolism[J].Proc Natl Acad Sci U S A,2020,117(41):25869-25879.
[12]	WANG S,LI F,LIN Y K,et al.Targeting REV-ERBα for therapeutic purposes:promises and challenges[J]. Theranostics, 2020, 10(9):4168-4182.
[13]	WANG S,LIN Y K,YUAN X,et al.REV-ERBα integrates colon clock with experimental colitis through regulation of NF-κB/NLRP3 axis[J].Nat Commun,2018,9(1):4246.
[14]	张芹,董珍,吴曦,等.生物钟基因Rev-erb-α缺失导致雌鼠生理周期延长[J].生理学报,2013,65(4):395-401.ZHANG Q,DONG Z,WU X,et al.Circadian clock gene Rev-erb-α deficiency prolongs female estrous cyclicity[J].Acta Physiologica Sinica,2013,65(4):395-401.(in Chinese)
[15]	GUAN D Y,XIONG Y,TRINH T M,et al.The hepatocyte clock and feeding control chronophysiology of multiple liver cell types[J].Science,2020,369(6509):1388-1394.
[16]	LI H,LI K Q,ZHANG K X,et al.The circadian physiology:implications in livestock health[J].Int J Mol Sci,2021,22(4):2111.
[17]	PETERS R R,CHAPIN L T,LEINING K B,et al.Supplemental lighting stimulates growth and lactation in cattle[J]. Science,1978,199(4331):911-912.
[18]	THUN R,EGGENBERGER E,ZEROBIN K,et al.Twenty-four-hour secretory pattern of cortisol in the bull:evidence of episodic secretion and circadian rhythm[J].Endocrinology,1981,109(6):2208-2212.
[19]	EVANS N M,HACKER R R,HOOVER J.Effect of chronobiological alteration of the circadian rhythm of prolactin and somatotropin release in the dairy cow[J].J Dairy Sci,1991,74(6):1821-1829.
[20]	JING Y J,CHEN Y F,WANG S,et al.Circadian gene PER2 silencing downregulates PPARG and SREBF1 and suppresses lipid synthesis in bovine mammary epithelial cells[J].Biology (Basel),2021,10(12):1226.
[21]	NIU M,HARVATINE K J.Short communication:the effects of morning compared with evening feed delivery in lactating dairy cows during the summer[J].J Dairy Sci,2018,101(1):396-400.
[22]	罗玉茹.EGF和EGFR在牛发情周期卵巢组织的定位和表达研究[D].杨凌:西北农林科技大学,2019.LUO Y R.Research of the localization and expression of EGF and EGFR in bovine estrus cycle[D].Yangling:Northwest A&F University,2019.(in Chinese)
[23]	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.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)
[24]	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,2022,58(10):215-222.WANG Y Q,GAO D K,ZHAO H C,et al.Construction of a eukaryotic expression vector of goat NR1D1 gene and its bioinformatics analysis[J].Chinese Journal of Animal Science,2022,58(10):215-222.(in Chinese)
[25]	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.
[26]	高登科,赵泓淙,董浩,等.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 D K,ZHAO H C,DONG H,et al.The cloning,expression vector construction and function analysis of goat RORα gene[J].Acta Veterinaria et Zootechnica Sinica,2022,53(6):1779-1794.(in Chinese)
[27]	GAO D K,ZHAO H C,DONG H,et al.Transcriptional feedback loops in the caprine circadian clock system[J].Front Vet Sci,2022,9:814562.
[28]	GRIFFETT K,BEDIA-DIAZ G,ELGENDY B,et al.REV-ERB agonism improves liver pathology in a mouse model of NASH[J].PLoS One,2020,15(10):e0236000.
[29]	ISAYAMA K,ZHAO L J,CHEN H T,et al.Removal of Rev-erbα inhibition contributes to the prostaglandin G/H synthase 2 expression in rat endometrial stromal cells[J].Am J Physiol Endocrinol Metab,2015,308(8):E650-E661.
[30]	AMIR M,CHAUDHARI S,WANG R,et al.REV-ERBα regulates TH17 cell development and autoimmunity[J].Cell Rep,2018, 25(13):3733-3749.e8.
[31]	SAITO S,CAO D Y,VICTOR A R,et al.RASAL3 is a putative RasGAP modulating inflammatory response by neutrophils[J]. Front Immunol,2021,12:744300.
[32]	ZHANG H H,XIE T,SHUI Y J,et al.Knockdown of PLCB2 expression reduces melanoma cell viability and promotes melanoma cell apoptosis by altering Ras/Raf/MAPK signals[J].Mol Med Rep,2020,21(1):420-428.
[33]	WANG G H,YING Z K,JIN X J,et al.Essential requirement for both hsf1 and hsf2 transcriptional activity in spermatogenesis and male fertility[J].Genesis,2004,38(2):66-80.
[34]	王雯.HSF2调控肠上皮细胞线粒体途径凋亡在溃疡性结肠炎中的作用机制研究[D].昆明:昆明医科大学,2021.WANG W.The mechanism of HSF2 regulating intestinal epithelial cell apoptosis through the mitochondrial pathway in ulcerative colitis[D].Kunming:Kunming Medical University,2021.(in Chinese)
[35]	DAI L J,ZHANG Q W,SHI J,et al.The distribution,expression patterns and functional analysis of NR1D1 and NR4A2 in the reproductive axis tissues of the male Tianzhu White Yak[J].Animals (Basel),2021,11(11):3117.
[36]	李文茜.丁酸梭菌对犊牛及育成牛生长性能、血液指标和瘤胃发酵的影响[D].哈尔滨:东北农业大学,2019.LI W X.Effects of Clostridium butyricum on growth performance,blood index and rumen fermentation in calves and bred cattle[D].Harbin:Northeast Agricultural University,2019.(in Chinese)
[37]	GAO J,XU Q Y,WANG M Z,et al.Ruminal epithelial cell proliferation and short-chain fatty acid transporters in vitro are associated with abundance of period circadian regulator 2(PER2)[J].J Dairy Sci,2020,103(12):12091-12103.
[38]	王强龙,潘阳阳,闫翠霞,等.雌性牦牛主要生殖器官中DBI的表达与定位分析[J].畜牧兽医学报,2022,53(1):169-178.WANG Q L,PAN Y Y,YAN C X,et al.Expression and localization of DBI in main reproductive organs of female yaks[J].Acta Veterinaria et Zootechnica Sinica,2022,53(1):169-178.(in Chinese)
[39]	CHEN H T,CHU G Y,ZHAO L J,et al.Rev-erbα regulates circadian rhythms and StAR expression in rat granulosa cells as identified by the agonist GSK4112[J].Biochem Biophys Res Commun,2012,420(2):374-379.
[40]	SEN A,HOFFMANN H M.Role of core circadian clock genes in hormone release and target tissue sensitivity in the reproductive axis[J].Mol Cell Endocrinol,2020,501:110655.
[41]	WANG L G,LI J J,ZHANG L T,et al.NR1D1 targeting CYP19A1 inhibits estrogen synthesis in ovarian granulosa cells[J].Theriogenology,2022,180:17-29.

The Dairy Cow NR1D1 Gene’s Eukaryotic Expression Vector Construction, Expression Profile and Its Ovarian Localization

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