牛UCP3和MYH1基因启动子在C2C12和3T3-L1细胞中的启动活性分析

doi:10.16431/j.cnki.1671-7236.2020.12.004

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (12): 3825-3832.doi: 10.16431/j.cnki.1671-7236.2020.12.004

牛UCP3和MYH1基因启动子在C2C12和3T3-L1细胞中的启动活性分析

陈伟^1,2, 许厚强^1,2, 陈祥^1,2, 覃海^1,2, 吴雨^1,2, 黄明捷^1,2, 卢贤君^1,2

1. 贵州大学动物科学学院, 高原山地动物遗传育种与繁殖教育部重点实验室, 贵阳 550025;
2. 贵州省动物遗传育种与繁殖重点实验室, 贵阳 550025

收稿日期:2020-02-23 出版日期:2020-12-20 发布日期:2020-12-18
通讯作者: 许厚强 E-mail:gzdxxhq@163.com
作者简介:陈伟(1980-),女,锡伯族,内蒙古通辽人,博士,研究方向:遗传学,E-mail:chenweigzu@163.com
基金资助:
国家自然科学基金（31401054）；贵州省农业领域重点项目（黔科合NY[2015]3002）；贵州省重大专项（黔科合重大专项字[2013]6008号）

Promoters Activity Analysis of UCP3 and MYH1 Genes of Cattle in C2C12 and 3T3-L1 Cells

CHEN Wei^1,2, XU Houqiang^1,2, CHEN Xiang^1,2, QIN Hai^1,2, WU Yu^1,2, HUANG Mingjie^1,2, LU Xianjun^1,2

1. Key Laboratory of Animal Genetics, Breeding and Reproduction in the Plateau Mountainous Region, College of Animal Science, Guizhou University, Guiyang 550025, China;
2. Key Laboratory of Animal Genetics, Breeding and Reproduction in Guizhou, Guiyang 550025, China

Received:2020-02-23 Online:2020-12-20 Published:2020-12-18

摘要/Abstract

摘要： 为阐明牛解偶联蛋白3（uncoupling protein 3，UCP3）和肌球蛋白重链1（myosin heavy chain 1，MYH1）基因启动子的核心区及其在小鼠C2C12和3T3-L1两种细胞株中的启动活性，本研究利用PCR、双荧光素酶和脂质体转染等方法进行关岭牛UCP3和MYH1基因启动子的扩增、重组载体构建和启动子活性分析。结果显示，试验成功构建了pGL3-Basic-UCP3-pro和pGL3-Basic-MYH1-pro重组真核表达载体，分别转染至小鼠C2C12和3T3-L1细胞后发现，UCP3、MYH1基因启动子的核心区分别为UCP3-P6（－385~+3 bp）和MYH1-P5（－255~+15 bp）区域；pGL3-Basic-MYH1-P2~pGL3-Basic-MYH1-P5在C2C12细胞中启动子活性均高于3T3-L1细胞，表明MYH1基因启动子活性在成肌细胞C2C12中较高。本研究阐明了UCP3和MYH1基因启动子的核心区及其启动子活性，为进一步研究UCP3和MYH1基因的转录调控机理提供了科学依据。

关键词: 牛; UCP3基因; MYH1基因; 启动子; C2C12细胞; 3T3-L1细胞

Abstract: To confirmed the core regions of uncoupling protein 3 (UCP3) and myosin heavy chain 1 (MYH1) genes promoter and the promoter activity in C2C12 and 3T3-L1 cells,PCR,double luciferase and liposome transfection methods were used to amplify of the promoter of UCP3 and MYH1 genes,construct recombinant vectors,and analyze promoter activity in this study.The results showed that pGL3-Basic-UCP3-pro and pGL3-Basic-MYH1-pro were successfully constructed and transfected into C2C12 and 3T3-L1 cells,respectively.The core promoter region of UCP3 and MYH1 genes were UCP3-P6 (－385 to +3 bp) and MYH1-P5 (－255 to +15 bp),respectively.The promoter activity of pGL3-Basic-MYH1-P2 to pGL3-Basic-MYH1-P5 in C2C12 cells were higher than that in 3T3-L1 cells,indicating that the promoter activity of MYH1 gene in C2C12 cells was higher.This study clarified the core region and promoter activity of UCP3 and MYH1 genes promters,provided a scientific basis for further study on the transcriptional regulation mechanism of UCP3 and MYH1 genes.

Key words: cattle; UCP3 gene; MYH1 gene; promoter; C2C12 cells; 3T3-L1 cells

中图分类号:

S823

陈伟, 许厚强, 陈祥, 覃海, 吴雨, 黄明捷, 卢贤君. 牛UCP3和MYH1基因启动子在C2C12和3T3-L1细胞中的启动活性分析[J]. 中国畜牧兽医, 2020, 47(12): 3825-3832.

CHEN Wei, XU Houqiang, CHEN Xiang, QIN Hai, WU Yu, HUANG Mingjie, LU Xianjun. Promoters Activity Analysis of UCP3 and MYH1 Genes of Cattle in C2C12 and 3T3-L1 Cells[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(12): 3825-3832.

参考文献

[1] HUGHES J,CRISCUOLO O F.Evolutionary history of the UCP gene family:Gene duplication and selection[J].BMC Evolutionary Biology,2008,8(1):306.
[2] NOWACKA-WOSZUK J,SZCZERBAL I,FIJAK-NOWAK H,et al.Chromosomal localization of 13 candidate genes for human obesity in the pig genome[J].Journal of Applied Genetics,2008,49(4):373.
[3] HAYASHI M,FUTAWAKA K,MATSUSHITA M,et al.GH directly stimulates UCP3 expression[J].Growth Hormone & IGF Research,2018,40:44-54.
[4] LIMA T I,GUIMARĀES D,SPONTON C H,et al.Essential role of the PGC-1α/PPARβ axis in UCP3 gene induction[J].The Journal of Physiology,2019,597(16):4277-4291.
[5] CROCCO P,MONTESANTO A,PASSARINO G,et al.A common polymorphism in the UCP3 promoter influences hand grip strength in elderly people[J].Biogerontology,2011,12(3):265-271.
[6] CHEN W,XU H Q,CHEN X,et al.Functional and activity analysis of cattle UCP3 promoter with MRFs-related factors[J].International Journal of Molecular Sciences,2016,17(5):682-697.
[7] LOPEZ-BERNARDO E,ANEDDA A,SANCHEZ-PEREZ P,et al.4-hydroxynonenal induces Nrf2-mediated UCP3 upregulation in mouse cardio-myocytes[J].Free Radical Biology and Medicine,2015,88(Pt B):427-438.
[8] LIN R,LIN W,CHEN Q,et al.Gene expression and promoter methylation of porcine uncoupling protein 3 gene[J].Asian-Australasian Journal of Animal Sciences,2019,32(2):170-175.
[9] AHN J S,KIM D H,PARK H B,et al.Ectopic overexpression of porcine MYH1 increased in slow muscle fibers and enhanced endurance exercise in transgenic mice[J].International Journal of Molecular Sciences,2018,19(10):2959.
[10] GARCIA DE LA SERRANA D,WREGGELSWORTH K,JOHNSTON I A.Duplication of a single myhz1.1 gene facilitated the ability of goldfish (Carassius auratus) to alter fast muscle contractile properties with seasonal temperature change[J].Frontiers in Physiology,2018,9:1724.
[11] TAKEDA S,NORTH D L,LAKICH M M,et al.A possible regulatory role for conserved promoter motifs in an adult-specific muscle myosin gene from mouse[J].Journal of Biological Chemistry,1992,67(24):16957-16967.
[12] BROWN D M,BRAMELD J M,PARR T.Expression of the myosin heavy chain ⅡB gene in porcine skeletal muscle:The role of the CArG-box promoter response element[J].PLoS One,2014,9(12):e114365.
[13] KINOSHITA S,CEYHUN S B,MD A,et al.Promoter analysis of the fish gene of slow/cardiac-type myosin heavy chain implicated in specification of muscle fiber types[J].Fish Physiology and Bioche-mistry,2018,44(2):679-691.
[14] SINGH A,KUKRETI S.A triple stranded G-quadruplex formation in the promoter region of human myosin beta(Myh7) gene[J].Journal of Biomolecular Structure & Dynamics,2018,36(11):2773-2786.
[15] KIM D,JITRAPAKDEE S,THOMPSON M.Differential regulation of the promoter activity of the mouse UCP2 and UCP3 genes by MyoD and myogenin[J].Journal of Biochemistry and Molecular Biology,2007,40(6):921-927.
[16] FROMME T,HOFFMANN C,NAU K,et al.An intronic single base exchange leads to a brown adipose tissue-specific loss of UCP3 expression and an altered body mass trajectory[J].Physiological Genomics,2009,38(1):54-62.
[17] GIROUSSE A,TAVERNIER G,TIRABY C,et al.Transcription of the human uncoupling protein 3 gene is governed by a complex interplay between the promoter and intronic sequences[J].Diabetologia,2009,52(8):1638.
[18] HO P W,HO J W,LIU H F,et al.Mitochondrial neuronal uncoupling proteins:A target for potential disease-modification in Parkinson's disease[J].Translational Neurodegeneration,2012,1(1):3.
[19] RIQUET F B,RODRIGUEZ M,GUIGAL N,et al.In vivo characterisation of the human UCP3 gene minimal promoter in mice tibialis anterior muscles[J].Biochemical and Biophysical Research Communications,2003,311(3):583-591.
[20] TONG B,SASAKI S,MURAMATSU Y,et al.Polymorphisms in promoter regions of MYH1 and IRS1 genes showing marbling-associated expression changes[J].Journal of Animal and Veterinary Advances,2012,11(23):4441-4445.
[21] WU K,TONG B,SASAKI S,et al.Preliminary association study of single nucleotide polymorphism in MYH1 and TRDN genes for growth-related traits in Japanese Black beef cattle[J].Journal of Animal and Veterinary Advances,2012,11(24):4540-4543.
[22] DAVID L A,JESSE N W,LAURA K S,et al.Myocyte enhancer factor-2 and serum response factor binding elements regulate fast myosin heavy chain transcription in vivo[J].The Journal of Biological Chemistry,2005,280(17):17126-17134.
[23] MIRETTI S,VOLPE M G,MARTIGNANI E,et al.Temporal correlation between differentiation factor expression and microRNAs in Holstein bovine skeletal muscle[J].Animal,2017,11(2):227-235.
[24] YU Y T,NADAL-GINARD B.Interaction of nuclear proteins with a positive cis-acting element of rat embryonic myosin heavy-chain promoter:Identifica-tion of a new transcriptional factor[J].Molecular and Cellular Biology,1989,9(5):1839-1849.
[25] WEIMER K,DIMARIO J X.Muscle fiber type specific activation of the slow myosin heavy chain 2 promoter by a non-canonical E-box[J].Biochemical and Biophysical Research Communications,2016,469(4):842-847.

牛UCP3和MYH1基因启动子在C2C12和3T3-L1细胞中的启动活性分析

Promoters Activity Analysis of UCP3 and MYH1 Genes of Cattle in C2C12 and 3T3-L1 Cells

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