吉林大学学报(医学版) ›› 2022, Vol. 48 ›› Issue (1): 94-103.doi: 10.13481/j.1671-587X.20220112

• 基础研究 • 上一篇    下一篇

小鼠NR1D1基因过表达载体的构建及其生物信息学分析

董浩1,2,江海圳1,2,李超1,2,高登科1,2,靳亚平1,2,陈华涛1,2()   

  1. 1.西北农林科技大学动物医学院临床兽医系,陕西 杨凌 712100
    2.西北农林科技大学 农业农村部动物生物技术重点实验室,陕西 杨凌 712100
  • 收稿日期:2021-06-11 出版日期:2022-01-28 发布日期:2022-01-17
  • 通讯作者: 陈华涛 E-mail:htchen@nwafu.edu.cn
  • 作者简介:董 浩(1998-),男,河北省唐山市人,在读硕士研究生,主要从事哺乳动物生物钟调控生殖和代谢机制方面的研究。
  • 基金资助:
    国家自然科学基金面上项目(31771301);中国博士后科学基金第11批特别资助项目(2018T111112)

Construction of mouse NR1D1 gene overexpression vector and its bioinformatics analysis

Hao DONG1,2,Haizhen JIANG1,2,Chao LI1,2,Dengke GAO1,2,Yaping JIN1,2,Huatao CHEN1,2()   

  1. 1.Department of Clinical Veterinary Medicine,College of Veterinary Medicine,Northwest A&F University,Yangling 712100,China
    2.Key Laboratory of Animal Biotechnology of Ministry of Agriculture and Rural Affairs,Northwest A&F University,Yangling 712100,China
  • Received:2021-06-11 Online:2022-01-28 Published:2022-01-17
  • Contact: Huatao CHEN E-mail:htchen@nwafu.edu.cn

摘要: 目的

构建小鼠核受体亚家族1组D成员1(NR1D1)基因的过表达载体,分析小鼠NR1D1蛋白的基本特征。

方法

从小鼠肝脏组织中提取总RNA,反转录后获得cDNA,采用PCR技术扩增得到小鼠NR1D1基因的编码区片段,经同源重组与pcDNA3.1载体相连接,将酶切鉴定和测序正确的重组质粒命名为pcDNA3.1-NR1D1。将pcDNA3.1空质粒和pcDNA3.1-NR1D1重组质粒分别转染至HEK293T细胞中,分别为对照组和pcDNA3.1-NR1D1转染组,48 h后提取总RNA和总蛋白样品,通过实时荧光定量PCR(RT-qPCR)和Western blotting法检测2组细胞中NR1D1 mRNA和蛋白表达水平。使用DNAStar和MEGA7软件对小鼠与其他物种的NR1D1基因编码区序列(CDS)进行相似性分析,并构建系统进化树;利用ExPASy、ProtScale、SingalP5.0、TMHMM-2.0、PhosphoSitePlus?、SOPMA和SWISS-MODEL等在线工具分析NR1D1蛋白的氨基酸组成、亲/疏水性、跨膜区域、信号肽、二级结构和三级结构。

结果

酶切鉴定和测序结果均表明过表达载体pcDNA3.1-NR1D1构建成功。与对照组比较,pcDNA3.1-NR1D1转染组细胞中NR1D1 mRNA和蛋白表达水平明显升高(P<0.01)。生物信息学分析,小鼠NR1D1 CDS区与人、大鼠、中国仓鼠、黑猩猩、兔、犬、猪、马、牛、绵羊、山羊、家猫和斑马鱼的相似性分别为90.0%、94.8%、86.5%、90.0%、89.6%、88.3%、89.7%、89.8%、88.8%、89.1%、89.1%、89.7%和65.1%。系统进化树分析,小鼠NR1D1基因与中国仓鼠和大鼠的遗传距离最近,与斑马鱼的遗传距离最远。小鼠NR1D1蛋白是一种疏水性碱性蛋白质,不属于分泌蛋白和跨膜蛋白,含有12个磷酸化位点和10个泛素化位点;二级结构中无规则卷曲占54.63%,α-螺旋占26.50%,延伸链占12.68%,β-转角占6.18%;三级结构预测,小鼠与人的NR1D1蛋白相似度大,差异较小。

结论

成功构建了小鼠NR1D1基因过表达载体pcDNA3.1-NR1D1,验证了其在HEK293T细胞中的过表达效果,为进一步研究NR1D1基因及其蛋白的功能提供了依据。

关键词: 小鼠,ICR, 核受体亚家族1组D成员1, 生物钟, 过表达载体, 生物信息学

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

中图分类号: 

  • Q78