水貂DCT基因启动子的克隆及转录调控元件分析
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摘要
为了找到水貂多巴色素异构酶(DCT)基因启动子活性区域及转录因子结合位点,试验采用PCR扩增与克隆,构建双荧光素酶报告基因重组质粒,分别转染到293T细胞和A375细胞,测定其活性,并利用在线软件对序列进行生物信息学分析,预测水貂DCT基因核心启动子区域的转录因子结合位点。结果表明:得到的6个不同长度的启动子片段均具有明显的启动子活性,且-1 292～+113 bp区域活性最高,提示其为水貂DCT基因核心启动子区域;成功筛选出337 bp水貂DCT基因活性较高的启动子片段,发现转录因子特异性蛋白1(Sp1)可能是调控启动子活性的重要转录因子。
In order to find the active region and transcription factor binding site of the promoter of the mink dopachrome tautomerase(DCT) gene, the author constructed dual luciferase reporter gene recombinant plasmids by using PCR amplification and cloning, which were transfected into 293 T cells and A375 respectively to determine its activity.Bioinformatics analysis of the sequences was performed using online software to predict transcription factor binding sites in the core promoter region of the mink DCT gene. The results show that the six different lengths of the promoter fragments have significant promoter activity, and the activity of the region-1 292/+113 bp is the highest, suggesting that the it is core promoter region of the mink DCT gene. The promoter fragment of 337 bp with higher activity of DCT gene in mink is successfully screened. It indicates that transcription factor specific protein 1(Sp1) may be an important transcription factor to regulate promoter activity.
In order to find the active region and transcription factor binding site of the promoter of the mink dopachrome tautomerase(DCT) gene, the author constructed dual luciferase reporter gene recombinant plasmids by using PCR amplification and cloning, which were transfected into 293 T cells and A375 respectively to determine its activity.Bioinformatics analysis of the sequences was performed using online software to predict transcription factor binding sites in the core promoter region of the mink DCT gene. The results show that the six different lengths of the promoter fragments have significant promoter activity, and the activity of the region-1 292/+113 bp is the highest, suggesting that the it is core promoter region of the mink DCT gene. The promoter fragment of 337 bp with higher activity of DCT gene in mink is successfully screened. It indicates that transcription factor specific protein 1(Sp1) may be an important transcription factor to regulate promoter activity.
引文
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[15] 李丽莎,李兰会,李祥龙,等.水貂PMEL基因启动子活性及转录调控元件分析[J].农业生物技术学报,2017,25(6):911-920.
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[18] 汤方,涂慧珍.真核启动子研究进展[J].林业科技开发,2015,29(2):7-12.
[19] 刘春杨,张乐超,王麒,等.山羊DCT基因启动子活性区及其转录因子调控探究[J].畜牧兽医学报,2018,49(1):55-64.
[20] JIAO Z,MOLLAAGHABABA R,PAVAN W J,et al.Direct interaction of Sox10 with the promoter of murine Dopachrome Tautomerase (Dct) and synergistic activation of Dct expression with Mitf[J].Pigment Cell Res,2004,17(4):352-362.
[21] 刘栋,朱文元.MITF与黑素细胞的发育、分化和功能调节[J].中国细胞生物学学报,2002,24(6):346-351.
[22] 黎钊,王平,洪为松,等.正常人黑素细胞MITF对酪氨酸酶相关蛋白的转录调控研究[J].医学研究杂志,2013,42(3):58-62.
[23] LUDWIG A,REHBERG S,WEGNER M.Melanocyte-specific expression of dopachrome tautomerase is dependent on synergistic gene activation by the Sox10 and Mitf transcription factors[J].FEBS Lett,2004,556(1/2/3):236-244.
[24] SACHITA K,YU H J,YUN J W,et al.YM155 induces apoptosis through downregulation of specificity protein 1 and myeloid cell leukemia-1 in human oral cancer cell lines[J].J Oral Pathol Med,2015,44(10):785-791.
[25] CHOI E S,NAM J S,JUNG J Y,et al.Modulation of specificity protein 1 by mithramycin A as a novel therapeutic strategy for cervical cancer[J].Sci Rep,2014,24(4):7162.
[26] 张晓,罗军,李建华,等.西农萨能奶山羊脂肪酸合酶基因启动子的克隆及活性测定[J].中国农业科学,2010,43(3):640-647.
[27] SAMSON S L,WONG N C.Role of Sp1 in insulin regulation of gene expression[J].J Mol Endocrinol,2002,29(5):265-279.
[28] 柳云霞,张娟娟,刘晓彤,等.转录因子Sp1调控小鼠前成骨细胞中MEPE基因表达的研究[J].基因组学与应用生物学,2017,36(7):2613-2618.
[29] 田宏攀.乳腺癌中DNA甲基化对Sp1调控FOXF2转录及其功能的影响[D].天津:天津医科大学,2015.
[2] 舒文,毛华明.黑色素的研究进展[J].国外畜牧学—猪与禽,2003,23(2):31-34.
[3] 白春雨,高玉花,庞全海.影响动物毛色的基因[J].国外畜牧学—猪与禽,2008,28(5):71-73.
[4] 刘伟兰,李祥龙,周荣艳,等.不同物种TYRP2基因完整编码区生物信息学分析[J].河南农业科学,2011,40(10):144-148.
[5] 李圣彦,郎志宏,黄大昉.真核生物启动子研究概述[J].生物技术进展,2014,4(3):158-164.
[6] GUYONNEAU L,MURISIER F,ROSSIER A,et al.Melanocytes and pigmentation are affected in dopachrome tautomerase knockout mice[J].Mol Cell Biol,2004,24:3396-3403.
[7] OKUMURA N,HAYASHI T,SEKIKAWA H,et al.Sequences and mapping of genes encoding porcine tyrosinase (TYR) and tyrosinase-related proteins (TYRP1 and TYRP2)[J].Anim Genet,2005,36(6):513-516.
[8] COSTIN G,VALENCIA J C,WAKAMATSU K,et al.Mutations in dopachrome tautomerase (DCT) affect eumelanin/pheomelanin synthesis,but do not affect intracellular trafficking of the mutant protein[J].Biochem J,2005,391(Pt 2):249-259.
[9] MCEVOY B,BELEZA S,SHRIVER M D.The genetic architecture of normal variation in human pigmentation:an evolutionary perspective and model [J].Hum Mol Genet,2006,15(Suppl2):R176-R181.
[10] 高莉,董常生,赫晓燕,等.羊驼酪氨酸酶基因家族在不同毛色个体中的基因表达水平[J].畜牧兽医学报,2008,39(7):895-899.
[11] 宋兴超.水貂被毛色素沉积机理及基于高通量RNA-seq皮肤转录组注释研究[D].长春:中国农业科学院,2016.
[12] 陈明帅.黑色突变貉生产性能测定及毛色基因TYR、DCT单核苷酸多态性分析[D].长春:中国农业科学院,2017.
[13] PENG Y D,LIU X H,GENG L Y,et al.Illumina-sequencing based transcriptome study of coat color phenotypes in domestic goats[J].Genes Genomics,2017,39(8):817-830.
[14] SAMBROOK J,RUSSELL D W.分子克隆实验指南[M].3版.黄培堂,王嘉玺,朱厚础,等,译.北京:科学出版社,2002.
[15] 李丽莎,李兰会,李祥龙,等.水貂PMEL基因启动子活性及转录调控元件分析[J].农业生物技术学报,2017,25(6):911-920.
[16] HEARING V J.Biochemical control of melanogenesis and melanosomal organization [J].J Investig Dermatol Symp Proc,1999,4(1):24-28.
[17] 王涛.猪CART和AgRP基因启动子的克隆与活性分析[D].广州:华南理工大学,2011.
[18] 汤方,涂慧珍.真核启动子研究进展[J].林业科技开发,2015,29(2):7-12.
[19] 刘春杨,张乐超,王麒,等.山羊DCT基因启动子活性区及其转录因子调控探究[J].畜牧兽医学报,2018,49(1):55-64.
[20] JIAO Z,MOLLAAGHABABA R,PAVAN W J,et al.Direct interaction of Sox10 with the promoter of murine Dopachrome Tautomerase (Dct) and synergistic activation of Dct expression with Mitf[J].Pigment Cell Res,2004,17(4):352-362.
[21] 刘栋,朱文元.MITF与黑素细胞的发育、分化和功能调节[J].中国细胞生物学学报,2002,24(6):346-351.
[22] 黎钊,王平,洪为松,等.正常人黑素细胞MITF对酪氨酸酶相关蛋白的转录调控研究[J].医学研究杂志,2013,42(3):58-62.
[23] LUDWIG A,REHBERG S,WEGNER M.Melanocyte-specific expression of dopachrome tautomerase is dependent on synergistic gene activation by the Sox10 and Mitf transcription factors[J].FEBS Lett,2004,556(1/2/3):236-244.
[24] SACHITA K,YU H J,YUN J W,et al.YM155 induces apoptosis through downregulation of specificity protein 1 and myeloid cell leukemia-1 in human oral cancer cell lines[J].J Oral Pathol Med,2015,44(10):785-791.
[25] CHOI E S,NAM J S,JUNG J Y,et al.Modulation of specificity protein 1 by mithramycin A as a novel therapeutic strategy for cervical cancer[J].Sci Rep,2014,24(4):7162.
[26] 张晓,罗军,李建华,等.西农萨能奶山羊脂肪酸合酶基因启动子的克隆及活性测定[J].中国农业科学,2010,43(3):640-647.
[27] SAMSON S L,WONG N C.Role of Sp1 in insulin regulation of gene expression[J].J Mol Endocrinol,2002,29(5):265-279.
[28] 柳云霞,张娟娟,刘晓彤,等.转录因子Sp1调控小鼠前成骨细胞中MEPE基因表达的研究[J].基因组学与应用生物学,2017,36(7):2613-2618.
[29] 田宏攀.乳腺癌中DNA甲基化对Sp1调控FOXF2转录及其功能的影响[D].天津:天津医科大学,2015.