小鼠NR1D1基因过表达载体的构建及其生物信息学分析

doi:10.13481/j.1671-587X.20220112

Abstract

Abstract: Objective

To construct the overexpression vector for the mouse Nuclear receptor subfamily 1 group D member 1（NR1D1） gene， and to further analyze the basic properties of NR1D1 protein.

Methods

The total RNA was extracted from the mouse liver tissue， and the cDNA was obtained by reverse transcription. PCR was utilized to amplify the coding fragment for the NR1D1 gene， and the NR1D1 CDS fragment was then ligated into the pcDNA3.1 vector by homologous recombination reactions. The recombinant plasmids were identified by restriction enzyme digestion and sequencing， and the recombinant plasmid was named as pcDNA3.1-NR1D1， and then the pcDNA3.1 plasmids and pcDNA3.1-NR1D1 recombinant plasmids were transfected respectively into the HEK293T cells and named as control group and pcDNA3.1-NR1D1 transfection group.After 48 h，the extraction of total RNA and total protein from cell samples were performed，and the expression levels of NR1D1 mRNA and protein were detected by real-time fluorescence quantitative PCR（RT-qPCR） and Western blotting methods， respectively. Meanwhile， the similarities of NR1D1 coding sequence（CDS） between the mice and other species were analyzed by DNAStar software and MEGA7 software， and the phylogenetic trees were constructed. In addition， the amino acid composition，hydrophilicity/hydrophobicity， transmembrane regions， signal peptides， secondary and tertiary structures of the NR1D1 protein were analyzed with online tools， such as ExPASy， ProtScale， SingalP5.0， TMHMM-2.0， PhosphoSitePlus^?， SOPMA and SWISS-MODEL.

Results

The restriction enzyme digestion and sequencing results showed that the overexpression vector pcDNA3.1-NR1D1 was successfully constructed. Compared with control group， the expression levels of NR1D1 mRNA and protein in the cells in pcDNA3.1-NR1D1 transfection group were significantly increased （P<0.01）. In addition， the bioinformatics analysis revealed that the similarities of the Mus musculus NR1D1 CDS region with Homo sapiens， Rattus norvegicus， Cricetulus griseus， Pan troglodytes， Oryctolagus cuniculus， Canis lupus familiaris， Sus scrofa， Equus caballus， Bos taurus， Ovis aries， Capra hircus， Felis catus and Danio rerio were 90.0%， 94.8%， 86.5%， 90%， 89.6%， 88.3%， 89.7%， 89.8%， 88.8%， 89.1%， 89.1% and 65.1%， respectively. The phylogenetic tree showed that the Mus musculus NR1D1 gene was the closest genetically to the Cricetulus griseus and Rattus norvegicus， and the most distant from Danio rerio. Mouse NR1D1 protein was a hydrophobic basic protein， not a secreted protein or a transmembrane protein， which contained 12 phosphorylation sites and 10 ubiquitination sites. The secondary structure of mouse NR1D1 protein included 54.63% random coil， 26.50% α-helix， 12.68% extended chain， and 6.18% β-turn， and NR1D1 protein had larger similarity and less variation in tertiary structure compared with the human.

Conclusion

The mouse NR1D1 gene overexpression vector pcDNA3.1-NR1D1 is successfully constructed， and its overexpression efficiency is verified in the HEK293T cells， which provide the basis for further research on the function of NR1D1 gene and protein.

Key words: Mouse，ICR, Nuclear receptor subfamily 1 group D member 1, Circadian clock, Over-expression vector, Bioinformatics

CLC Number:

Hao DONG,Haizhen JIANG,Chao LI,Dengke GAO,Yaping JIN,Huatao CHEN. Construction of mouse NR1D1 gene overexpression vector and its bioinformatics analysis[J].Journal of Jilin University(Medicine Edition), 2022, 48(1): 94-103.

Figures/Tables 13

Tab.1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Tab. 2

Fig. 6

Fig. 7

Fig. 8

Fig. 9

Fig. 10

Fig. 11

References 34

1	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.
2	MARVIN K A， REINKING J L， LEE A J， et al. Nuclear receptors homo sapiens Rev-erb beta and Drosophila melanogaster E75 are thiolate-ligated heme proteins which undergo redox-mediated ligand switching and bind CO and NO［J］. Biochemistry， 2009， 48（29）： 7056-7071.
3	YIN L， WU N， LAZAR M A. Nuclear receptor Rev-erb alpha： a heme receptor that coordinates circadian rhythm and metabolism［J］. Nucl Recept Signal， 2010， 8： e001.
4	CASTAGNÉ C， TERENZI H， ZAKIN M M， et al. Solution structure of the orphan nuclear receptor rev-erb beta response element by 1H， 31P NMR and molecular simulation［J］. Biochimie， 2000， 82（8）： 739-748.
5	RAMAKRISHNAN S N， LAU P， BURKE L J， et al. Rev-erbβ regulates the expression of genes involved in lipid absorption in skeletal muscle cells［J］. J Biol Chem， 2005， 280（10）： 8651-8659.
6	RAGHURAM S， STAYROOK K R， HUANG P， et al. Identification of heme as the ligand for the orphan nuclear receptors REV-ERBalpha and REV-ERB beta［J］. Nat Struct Mol Biol， 2007，14（12）：1207-1213.
7	BUGGE A， FENG D， EVERETT L J， et al. Rev-erb and Rev-erb coordinately protect the circadian clock and normal metabolic function［J］. Genes Dev， 2012，26（7）： 657-667.
8	PREITNER N， DAMIOLA F， LOPEZ-MOLINA L， et al. The orphan nuclear receptor REV-ERB alpha controls circadian transcription within the positive limb of the mammalian circadian oscillator［J］. Cell， 2002， 110（2）： 251-260.
9	TRIQUENEAUX G， THENOT S， KAKIZAWA T， et al. The orphan receptor Rev-erb alpha gene is a target of the circadian clock pacemaker［J］. J Mol Endocrinol， 2004， 33（3）： 585-608.
10	HARDING H P， LAZAR M A. The monomer-binding orphan receptor Rev-Erb represses transcription as a dimer on a novel direct repeat［J］. Mol Cell Biol， 1995， 15（9）： 4791-4802.
11	MAKISHIMA M. Nuclear receptors as targets for drug development： regulation of cholesterol and bile acid metabolism by nuclear receptors［J］. J Pharmacol Sci， 2005， 97（2）： 177-183.
12	GRANT D， YIN L， COLLINS J L， et al. GSK4112， a small molecule chemical probe for the cell biology of the nuclear heme receptor Rev-erbα［J］. ACS Chem Biol， 2010， 5（10）： 925-932.
13	SOLT L A， WANG Y J， BANERJEE S， et al. Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists［J］.Nature，2012，485（7396）： 62-68.
14	LI C， ZHANG L， MA T， et al. Bisphenol A attenuates testosterone production in Leydig cells via the inhibition of NR1D1 signaling［J］.Chemosphere，2021，263： 128020.
15	BONNELYE E， VANACKER J M， DESBIENS X，et al.Rev-erb beta， a new member of the nuclear receptor superfamily， is expressed in the nervous system during chicken development［J］. Cell Growth Differ， 1994， 5（12）： 1357-1365.
16	CHO H， ZHAO X， HATORI M， et al. Regulation of circadian behaviour and metabolism by REV-ERB-α and REV-ERB-Β［J］. Nature， 2012， 485（7396）： 123-127.
17	李翠梅. 双酚A通过生物钟基因Nr1d1影响睾酮合成的机制研究［D］. 杨凌：西北农林科技大学， 2019.
18	ERCOLANI L， FERRARI A， DE MEI C， et al. Circadian clock： Time for novel anticancer strategies？［J］. Pharmacol Res， 2015， 100： 288-295.
19	GEKAKIS N， STAKNIS D， NGUYEN H B， et al. Role of the CLOCK protein in the mammalian circadian mechanism［J］. Science， 1998， 280（5369）： 1564-1569.
20	YOO S H， KO C H， LOWREY P L， et al. A noncanonical E-box enhancer drives mouse Period2 circadian oscillations in vivo ［J］. PNAS， 2005， 102（7）： 2608-2613.
21	OHNO T， ONISHI Y， ISHIDA N. A novel E4BP4 element drives circadian expression of mPeriod2［J］. Nucleic Acids Res， 2007， 35（2）： 648-655.
22	CHAIX A， ZARRINPAR A， PANDA S. The circadian coordination of cell biology［J］. J Cell Biol， 2016， 215（1）： 15-25.
23	EVERETT L J， LAZAR M A. Nuclear receptor Rev-erb α： up， down， and all around［J］. Trends Endocrinol Metab， 2014， 25（11）： 586-592.
24	HUNTER A L， PELEKANOU C E， ADAMSON A， et al. Nuclear receptor REVERBα is a state-dependent regulator of liver energy metabolism［J］. PNAS， 2020， 117（41）： 25869-25879.
25	WANG J， LIU N， LIU Z， et al. The orphan nuclear receptor Rev-erb beta recruits Tip60 and HDAC1 to regulate apolipoprotein CIII promoter［J］. Biochim Biophys Acta， 2008， 1783（2）： 224-236.
26	DELEZIE J， DUMONT S， DARDENTE H， et al. The nuclear receptor REV-ERBα is required for the daily balance of carbohydrate and lipid metabolism［J］. FASEB J， 2012， 26（8）： 3321-3335.
27	LE MARTELOT G， CLAUDEL T， GATFIELD D， et al. REV-ERBalpha participates in circadian SREBP signaling and bile acid homeostasis［J］. PLoS Biol， 2009， 7（9）： e1000181.
28	WOLDT E， SEBTI Y， SOLT L A， et al. Rev-erb-α modulates skeletal muscle oxidative capacity by regulating mitochondrial biogenesis and autophagy［J］. Nat Med， 2013， 19（8）： 1039-1046.
29	ZHANG T P， ZHAO M J， LU D Y， et al. REV-ERBα regulates CYP7A1 through repression of liver receptor homolog-1［J］.Drug Metab Dispos，2018，46（3）：248-258.
30	XING C， HUANG X， ZHANG Y， et al. Sleep disturbance induces increased cholesterol level by NR1D1 mediated CYP7A1 inhibition［J］. Front Genet， 2020， 11： 610496.
31	MIGITA H， MORSER J， KAWAI K. Rev-erbα upregulates NF-κB-responsive genes in vascular smooth muscle cells［J］. FEBS Lett， 2004， 561（1-3）： 69-74.
32	MAZZOCCOLI G， SOTHERN R B， GRECO A，et al. Time-related dynamics of variation in core clock gene expression levels in tissues relevant to the immune system［J］.Int J Immunopathol Pharmacol，2011，24（4）： 869-879.
33	CHANDRA V， MAHAJAN S， SAINI A， et al. Human IL10 gene repression by Rev-erbα ameliorates Mycobacterium tuberculosis clearance［J］. J Biol Chem， 2013， 288（15）： 10692-10702.
34	ZVONIC S， PTITSYN A A， CONRAD S A， et al. Characterization of peripheral circadian clocks in adipose tissues［J］. Diabetes， 2006， 55（4）： 962-970.

Animo acid	n	Frequency(η/%)	Animo acid	n	Frequency(η/%)
Ala (A)	39	6.3	Lys (K)	20	3.3
Arg (R)	36	5.9	Met (M)	19	3.1
Asn (N)	36	5.9	Phe (F)	26	4.2
Asp (D)	20	3.3	Pro (P)	51	8.3
Cys (C)	15	2.4	Ser (S)	86	14.0
Gln (Q)	29	4.7	Thr (T)	38	6.2
Gly (G)	41	6.7	Trp (W)	3	0.5
His (H)	17	2.8	Tyr (Y)	11	1.8
Ile (I)	12	2.0	Val (V)	30	4.9
Leu (L)	57	9.3	Glu (E)	29	4.7

[1]	Xiaohui LIANG,Wenna LIU,Jianbo ZHAO. Bioinformatic analysis on PAQR3 expression in hepatocellular carcinoma and its correlations with T lymphocyte infiltration, immune checkpoint genes, and patient survival [J]. Journal of Jilin University(Medicine Edition), 2026, 52(3): 781-790.
[2]	Zhipeng PAN,Le WANG,Weiwen HUANG,Zhen YI,Yuxuan GAO. Bioinformatics analysis and experimental validation based on screening and identification of diagnostic candidate genes of bone marrow mononuclear cells of acute myeloid leukemia patients and their impact on prognosis [J]. Journal of Jilin University(Medicine Edition), 2026, 52(3): 791-805.
[3]	Ruihan GE,Chen LI,Shengpeng WANG,Yang LU,Caixia TAN,Haotian CUI,Xinmin WANG,Le ZHANG. Bioinformatic analysis on regulatory mechanism of MAPK-Mcl-1 signaling pathway and macrophage polarization during Bacillus Calmette-Guérin infection and its experimental validation [J]. Journal of Jilin University(Medicine Edition), 2026, 52(2): 440-450.
[4]	Xiao LIN,Meng ZHOU,Fan LIN,Xiujuan YAO. Bioinformatics analysis and experimental validation of fatty acid metabolism-related genes in idiopathic pulmonary fibrosis tissue [J]. Journal of Jilin University(Medicine Edition), 2026, 52(1): 93-104.
[5]	Yu LIANG,Jinyu YU,Zhonggao XU,Wanning WANG. Expression characteristics of FOSB in kidney tissue from IgA nephropathy and other common kidney diseases [J]. Journal of Jilin University(Medicine Edition), 2025, 51(5): 1281-1292.
[6]	Weigao SHEN,Yuqi LIU,Jun ZHANG,Jiayu LIN,Hang CUI,Yanbo LIU. Bioinformatics analysis on effect of interleukin-33 on occurrence and development of malignant brain glioma and its experimental validation [J]. Journal of Jilin University(Medicine Edition), 2025, 51(5): 1318-1332.
[7]	Linrui XU,Yiyu ZHANG,Jiaqi CUI,Xianzhu CONG,Shuang LI,Jiayu GE,Yujia KONG,Suzhen WANG,Fuyan SHI,Jinrong WANG. Construction of diagnostic model for Alzheimer’s disease and immune analysis based on bioinformatics and machine learning [J]. Journal of Jilin University(Medicine Edition), 2025, 51(4): 1039-1051.
[8]	Xianwei JIANG,Minghang WANG,Huiru LI,Xiaosheng DONG,Yuanyuan LIU. Mendelian randomization and GEO database identification analysis based on potential therapeutic targets for chronic obstructive pulmonary disease [J]. Journal of Jilin University(Medicine Edition), 2025, 51(4): 1072-1083.
[9]	Changyu SHI,Yong LI,Jing DENG,Chunmei PIAO,Ming JIN. Bioinformatics analysis on adjustment effect of colorectal liver metastases model in mice based on complement alternative pathway and its experimental verification [J]. Journal of Jilin University(Medicine Edition), 2025, 51(3): 703-715.
[10]	Wenchang CAI,Yuqi LIU,Han WANG,Helin WANG,Zhenjiang WANG,Zishen XIAO,Shiyuan MA,Liping AN,Yanbo LIU. Expression of protein kinase D2 in bladder cancer tissue and its effect on tumor immune microenvironment [J]. Journal of Jilin University(Medicine Edition), 2025, 51(2): 378-391.
[11]	Bo LIU,Chao SUN,Xu WANG,Kewei MA. Bioinformatics analysis on differentially expressed genes in multiple primary lung cancers based on GEO database [J]. Journal of Jilin University(Medicine Edition), 2025, 51(2): 437-446.
[12]	Xiaoyan WANG,Xuelian LI,Bin LIANG,Wenfei TIAN,Hailin MA,Zhijing MO. Analysis on relationship between CALU and prognosis of hepatocellular carcinoma patients and its mechanism based on transcriptome and single cell sequencing data [J]. Journal of Jilin University(Medicine Edition), 2025, 51(2): 447-459.
[13]	Pengli WU,Fengyu LI,Bo LIU,Yang LYU. Effect of silencing DDX39A gene on proliferation, migration and invasion of esophageal cancer TE-1 cells and its mechanism [J]. Journal of Jilin University(Medicine Edition), 2025, 51(1): 115-123.
[14]	Lyuyin SUN,Zhuping MA,Runlin LI,Yonggang LI,Xiaoli TAO. Screening of host proteins interacting with Nelson Bay orthoreovirus σNS based on yeast two-hybrid technology [J]. Journal of Jilin University(Medicine Edition), 2024, 50(5): 1313-1321.
[15]	Jinlian LI,Lanzhen HUANG,Xishi HUANG,Kangzhi LI,Jiali JIANG,Miaomiao ZHANG,Qunying WU. Bioinformatics analysis on key genes related to prognosis, diagnosis， and immune cell infiltration of hepatocellular carcinoma and their potential therapeutic drugs [J]. Journal of Jilin University(Medicine Edition), 2024, 50(4): 1062-1075.

Construction of mouse NR1D1 gene overexpression vector and its bioinformatics analysis

RichHTML

PDF (PC)

Like

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 13

References 34

Related Articles 15

Metrics

Comments

Recommended 10