北极狐TYRP1基因启动子活性及转录调控区域分析
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摘要
通过分析调控北极狐毛色基因TYRP1启动子核心区域及转录因子,为探究该基因的表达调控机制提供理论依据,并为狐狸毛皮品质分子育种和彩色毛皮新材料的创制提供思路。通过基因组测序技术获得了北极狐TYRP1基因启动子序列,并利用生物信息学方法对北极狐TYRP1基因核心启动子区域和转录因子结合位点进行预测;以北极狐基因组DNA为模板,PCR扩增北极狐TYRP1基因不同长度的启动子缺失片段克隆至pGL3-Basic载体,将重组质粒瞬时转染到A375和293T细胞,利用双荧光素酶基因检测仪进行活性验证。结果表明,成功构建了9个含有不同长度启动子片段的重组质粒,经双荧光素酶活性检测发现北极狐TYRP1基因-699/+35区域为核心启动子区域,-699/-93区域存在着TYRP1基因正调控元件。生物信息学预测分析发现该区域存在4个转录因子结合位点;利用重叠延伸PCR技术成功构建了4个突变载体,经双荧光素酶活性检测发现4个突变载体活性均显著下降(P<0.05),表明这4个转录因子是北极狐TYRP1基因转录调控的正调控元件。本研究确定了北极狐TYRP1基因启动子核心区域-699/+35,Sp1(-656/-646)、CREB(-598/-589)、Sp1(-539/-530)和Sp1(-163/-154)为北极狐TYRP1基因转录的正调控元件。
The research was designed to investigate the activity region and transcription factors of fox TYRP1 gene,and to provide a theoretical reference for exploring the breeding of fox and creating the new fur materials.The gene promoter sequence of fox TYRP1 gene was obtained through transcriptome sequencing technology,and the method of bioinformatics was used to predict the core promoter region of TYRP1 gene and the transcription factor binding site.The fragment in a 5′flanking region was amplified and cloned into the vector pGL3-Basic.The positive colonies were identified and sequenced.The recombinant plasmid was transfected to A375 and 293 Tcells,and the activity was verified by the dual-luciferase assays system.The results showed that 9 fragments with different lengths of promoter regions were amplified and cloned into the vector pGL3-Basic.The region-699/+35 of fox TYRP1 gene was detected as the core promoter region by dual-luciferase assays system.There were some positive regulatory elements in the region from-699/-93.The bioinformatics prediction analysis revealed that there were 4 transcription factor binding sites in the region.Using the overlap extension PCR technology successfully constructed 4 mutation vectors.Their activity were significantly decreased by dual luciferase assay.It is suggests that these 4 transcription factors were the positive regulatory elements in fox TYRP1 gene transcription.In this study,the identified core regions of TYRP1 gene promoter is-699/+35.Sp1(-656/-646),CREB(-598/-589),Sp1(-539/-530)and Sp1(-163/-154)binding sites are the positive regulatory elements of fox TYRP1 gene.
The research was designed to investigate the activity region and transcription factors of fox TYRP1 gene,and to provide a theoretical reference for exploring the breeding of fox and creating the new fur materials.The gene promoter sequence of fox TYRP1 gene was obtained through transcriptome sequencing technology,and the method of bioinformatics was used to predict the core promoter region of TYRP1 gene and the transcription factor binding site.The fragment in a 5′flanking region was amplified and cloned into the vector pGL3-Basic.The positive colonies were identified and sequenced.The recombinant plasmid was transfected to A375 and 293 Tcells,and the activity was verified by the dual-luciferase assays system.The results showed that 9 fragments with different lengths of promoter regions were amplified and cloned into the vector pGL3-Basic.The region-699/+35 of fox TYRP1 gene was detected as the core promoter region by dual-luciferase assays system.There were some positive regulatory elements in the region from-699/-93.The bioinformatics prediction analysis revealed that there were 4 transcription factor binding sites in the region.Using the overlap extension PCR technology successfully constructed 4 mutation vectors.Their activity were significantly decreased by dual luciferase assay.It is suggests that these 4 transcription factors were the positive regulatory elements in fox TYRP1 gene transcription.In this study,the identified core regions of TYRP1 gene promoter is-699/+35.Sp1(-656/-646),CREB(-598/-589),Sp1(-539/-530)and Sp1(-163/-154)binding sites are the positive regulatory elements of fox TYRP1 gene.
引文
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[20]叶丽萍,顾彬彬,方从诚,等.Sp1调控PIWIL1基因启动子转录活性研究[J].医学研究杂志,2016,45(6):133-136.
[21]MONTMINY M R,BILEZIKJIAN L M.Binding of a nuclear protein to the cyclic-AMP response element of thesomatostatin gene[J].Nature,1987,328(6126):175-178.
[22]COVEN E,NI Y,WIDNELL K L,et al.Cell type-specific regulation of CREB gene expression:Mutational analysis of CREB promoter activity[J].J Neurochem,1998,71(5):1865-1874.
[23]唐海斌,马越云,苏明权,等.CREB及p-CREB在前列腺癌细胞增殖中的作用[J].中国肿瘤生物治疗杂志,2016,23(2):223-229.
[2]REISSMANN M,LUDWIG A.Pleiotropic effects of coatcolour-associated mutations in humans,mice and other mammals[J].Semin Cell Dev Biol,2013,24(6/7):576-86.
[3]徐桂利,刘铮铸,巩元芳,等.毛皮动物毛色调控基因的研究进展[J].黑龙江畜牧兽医,2015(13):52-54.
[4]SULEM P,GUDBJARTSSON D F,STACEY S N,et al.Genetic determinants of hair,eye and skin pigmentation in Europeans[J].Nat Genet,2007,39(12):1443-1452.
[5]朱亮,蔡月琴,屠珏.实验兔酪氨酸酶基因家族表达水平与毛色和虹膜颜色性状的关系研究[J].中国比较医学杂志,2013,23(10):13-16.
[6]于秀菊,范阔海.毛囊的黑色素沉着[J].畜牧兽医科技信息,2009(4):16-18.
[7]FANG D,TSUJI Y,SETALURI V.Selective downregulation oftyrosinase family gene TYRP1by inhibition of the activity of melanocyte transcription factor,MITF[J].Nucleic Acids Res,2002,30(14):3096-3106.
[8]DELL′ANGELICA E C.Melanosome biogenesis:shedding light on the origin of an obscure organelle[J].Trends Cell Biol,2003,13(10):503-506.
[9]BOISSY R E,ZHAO H,OETTING W S,et al.Mutation in and lack of expression oftyrosinase-related protein-1(TRP-1)in melanocytes from an individual with brown oculocutaneous albinism:a new subtype of albinism classified as"OCA3"[J].Am J Hum Genet,1996,58(6):1145-1156.
[10]MURISIER F,GUICHARD S,BEERMANN F.Aconserved transcriptional enhancer that specifies Tyrp1expression to melanocytes[J].Dev Biol,2006,298(2):644-655.
[11]STELL A J,DOBSON J M,SCASE T J,et al.Evaluation of variants of melanoma-associated antigen genes and mRNA transcripts in melanomas of dogs[J].Am J Vet Res,2009,70(12):1512-1520.
[12]赵彦斌,孙兆增,白杰英,等.黑线仓鼠及其白化突变系TYR、TYRP1的基因表达水平比较分析[J].中国比较医学杂志,2012,22(7):1-4.
[13]LI B,HE X L,ZHAO Y P,et al.Tyrosinase-related protein 1(TYRP1)gene polymorphism and skin differential expression related to coat color in Mongolian horse[J].Livest Sci,2014,167:58-64.
[14]KWON B S.Pigmentation genes:thetyrosinase gene family and the TYRP117gene family[J].J Invest Dermatol,1993,100(2Suppl):134-140.
[15]DYNAN W S,TJIAN R.The promoter-specific transcription factor Sp1binds to upstream sequences in the SV40early promoter[J].Cell,1983,35(1):79-87.
[16]HAGEN G,MLLER S,BEATO M,et al.Cloning by recognition site screening of two novel GT box binding proteins:a family of Sp1related genes[J].Nucleic Acids Res,1992,20(21):5519-5525.
[17]WOLFE S A,NEKLUDOVA L,PABO C O.DNArecognition by Cys2His2zinc finger proteins[J].Annu Rev Biophys Biomol Struct,2000,29:183-212.
[18]PIETRZAK M,PUZIANOWSKA-KUZNICKA M.p53-dependent repression of the human MCL-1gene encoding an anti-apoptotic member of the BCL-2family:the role of Sp1and of basic transcription factor binding sites in the MCL-1promoter[J].Biol Chem,2008,389(4):383-393.
[19]AMINA E G.Otosclerosis:association of COLIA1Sp1binding site polymorphism in Alexandria,Egypt[J].Asian Biomed,2012,10:747-750.
[20]叶丽萍,顾彬彬,方从诚,等.Sp1调控PIWIL1基因启动子转录活性研究[J].医学研究杂志,2016,45(6):133-136.
[21]MONTMINY M R,BILEZIKJIAN L M.Binding of a nuclear protein to the cyclic-AMP response element of thesomatostatin gene[J].Nature,1987,328(6126):175-178.
[22]COVEN E,NI Y,WIDNELL K L,et al.Cell type-specific regulation of CREB gene expression:Mutational analysis of CREB promoter activity[J].J Neurochem,1998,71(5):1865-1874.
[23]唐海斌,马越云,苏明权,等.CREB及p-CREB在前列腺癌细胞增殖中的作用[J].中国肿瘤生物治疗杂志,2016,23(2):223-229.