蓝狐MITF-M基因序列扩增及生物信息学分析
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摘要
试验旨在研究蓝狐MITF-M基因核心启动子活性区、转录因子结合位点、基因编码蛋白结构及功能,为探究该基因的表达调控机制提供理论依据。从成年蓝狐背部皮肤组织中采集1cm×1cm样品,采用RT-PCR扩增及测序技术首次获得MITF-M基因序列3 880bp(包括5′侧翼区2 262bp、CDS区1 260bp、3′侧翼区358bp),编码419个氨基酸残基。生物信息学分析结果显示,蓝狐MITF-M基因5′侧翼区存在潜在的启动子区域(-86~-336bp),且发现TATA框、CAAT框、GC框和CRE等调控元件及CREB、LSF和Sp1等多种转录因子。MITFM基因编码的蛋白是定位于细胞核和细胞质的不稳定、可溶性亲水蛋白质。亚细胞定位结果提示该蛋白在细胞核、细胞质和线粒体的概率较高,分别为65.2%、17.4%和8.7%。DNAMAN软件对MITF-M基因编码蛋白的二级结构预测发现,该蛋白由α螺旋(33.66%)、β折叠(16.66%)和无规卷曲(49.68%)组成,且不存在跨膜区域和信号肽。蓝狐与其他物种MITF-M基因编码的氨基酸序列同源性对比和系统进化树分析均提示蓝狐与犬的遗传亲缘关系最近,与哺乳动物和禽类均具有较高的同源性(>89.1%),说明MITF-M基因编码区在物种进化过程中较为保守。本研究为深入研究MITF-M基因调控狐狸被毛颜色的分子遗传机制奠定了理论基础。
In this study,to investigate the core promoter activity region,transcription factor binding site,the structure and function of encoding protein and the expression and regulation mechanism of Blue fox MITF-M gene,the skin tissue samples(1 cm ×1 cm)was collected from adult Blue fox,the partial sequence of MITF-M gene was obtained by RT-PCR and sequencing technique for the first time.The length of the Blue fox MITF-M gene was 3 880 bp(including2 262 bp 5′flanking region,1 260 bp CDS region and 358 bp 3′flanking region),and encoding419 amino acid residues.The bioinformatics analysis showed that there were potential promoter regions at-86 to-336 bp in the 5′flanking region of MITF-M gene,and some regulatory elements(TATA box,CAAT box,GC box and CRE)and transcription factors(CREB,LSF and Sp1)were found.The protein encoded by MITF-M gene was unstable and hydrophilic which mainly located in nucleus and cytoplasm.Subcellular localization results showed that the proteinhad a higher probability in nucleus(65.2%),cytoplasm(17.4%)and mitochondrion(8.7%),the results of secondary structure prediction by DNAMAN software found that the protein was consist ofαhelix(33.66%),βsheet(16.66%)and random coil(49.68%),and there was no transmembrane region and signal peptide.Multiple sequence alignment and phylogenetic tree analysis showed that Blue fox had the closet relationship with dog and a high homology with mammal and poultry(>89.1%)indicated that the MITF-M gene coding region was conservative in the course of evolution.This study laid the theoretical foundation for the in-depth study of the molecular genetic mechanism of the MITF-M gene regulating the hair color of the fox.
In this study,to investigate the core promoter activity region,transcription factor binding site,the structure and function of encoding protein and the expression and regulation mechanism of Blue fox MITF-M gene,the skin tissue samples(1 cm ×1 cm)was collected from adult Blue fox,the partial sequence of MITF-M gene was obtained by RT-PCR and sequencing technique for the first time.The length of the Blue fox MITF-M gene was 3 880 bp(including2 262 bp 5′flanking region,1 260 bp CDS region and 358 bp 3′flanking region),and encoding419 amino acid residues.The bioinformatics analysis showed that there were potential promoter regions at-86 to-336 bp in the 5′flanking region of MITF-M gene,and some regulatory elements(TATA box,CAAT box,GC box and CRE)and transcription factors(CREB,LSF and Sp1)were found.The protein encoded by MITF-M gene was unstable and hydrophilic which mainly located in nucleus and cytoplasm.Subcellular localization results showed that the proteinhad a higher probability in nucleus(65.2%),cytoplasm(17.4%)and mitochondrion(8.7%),the results of secondary structure prediction by DNAMAN software found that the protein was consist ofαhelix(33.66%),βsheet(16.66%)and random coil(49.68%),and there was no transmembrane region and signal peptide.Multiple sequence alignment and phylogenetic tree analysis showed that Blue fox had the closet relationship with dog and a high homology with mammal and poultry(>89.1%)indicated that the MITF-M gene coding region was conservative in the course of evolution.This study laid the theoretical foundation for the in-depth study of the molecular genetic mechanism of the MITF-M gene regulating the hair color of the fox.
引文
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[3]LEVY C,KHALED M,FISHER D E.MITF:Master regulator of melanocyte development and melanoma oncogene[J].Trends in Molecular Medicine,2006,12(9):406-414.
[4]FUSE N,YASUMOTO K,SUZUKI H,et al.Identification of a melanocyte-type promoter of the microphthalmia-associated transcription factor gene[J].Biochemical and Biophysical Research Communications,1996,219(3):702-707.
[5]郑嫩珠,辛清武,朱志明,等.白绒乌鸡MITF基因的cDNA克隆、表达及其对黑色素沉积的影响[J].中国农业科学,2015,48(18):3711-3718.ZHENG N Z,XIN Q W,ZHU Z M,et al.cDNA cloning and expression of mitf gene and its effect on melanin deposition in silky fowl[J].Scientia Agricultura Sinica,2015,48(18):3711-3718.(in Chinese)
[6]张建一,梁春年,裴杰,等.牦牛MITF-M基因序列分析及其在皮肤组织中表达水平[J].华北农学报,2015,30(3):1-7.ZHANG J Y,LIANG C N,PEI J,et al.Sequence analysis and expression level detection of MITF-M gene in different color skin tissues of yak[J].Acta Agriculturae Boreali-Sinica,2015,30(3):1-7.(in Chinese)
[7]杨玉静,张丹瑾,聂瑞强,等.绵羊MITF-M在黑素细胞中过表达后的功能分析[J].中国农业科学,2016,49(2):4214-4221.YANG Y J,ZHANG D J,NIE R Q,et al.The function analysis of over-expression of oar MITF-M in melanocytes[J].Scientia Agricultura Sinica,2016,49(2):4214-4221.(in Chinese)
[8]韩吉龙,岳耀敬,裴杰,等.藏羊刺鼠信号蛋白基因(Agouti)的多态性及不同颜色被毛皮肤组织中Agouti与小眼畸形相关转录因子基因(MITF)表达的定量分析[J].农业生物技术学报,2013,21(3):338-347.HAN J L,YUE Y J,PEI J,et al.The polymorphism of agouti signaling protein gene(Agouti)and the expression of Agouti and microphthalmia-associated transcription factor gene MITFin different coat color skin tissues of Tibetan sheep(Ovis aries)[J].Journal of Agricultural Biotechnology,2013,21(3):338-347.(in Chinese)
[9]SAMBROOK J,FRITSCH E F,MANIATIS T.Molecular Cloning[M].New York:Cold Spring Habor Laboratory Press,1989.
[10]耿荣庆,白丁平,袁超,等.从羊皮肤组织中提取总RNA方法的改进[J].中国畜牧兽医,2012,39(5):244-247.GENG R Q,BAI D P,YUAN C,et al.The improvement on the method of total RNA extraction from goat and sheep skin tissues[J].China Animal Husbandry&Veterinary Medicine,2012,39(5):244-247.(in Chinese)
[11]KOZAK M.Structural features in eukaryotic mRNA that modulate the initiation of translate[J].Biological Chemistry,1991,266(30):19867-19870.
[12]白瑞,张美萍,乔德瑞,等.MITF基因与动物毛色的研究进展[J].上海畜牧兽医通讯,2006,4:2-3.BAI R,ZHANG M P,QIAO D R,et al.Research progress of MITFgene and its effect on animal coat color[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2006,4:2-3.(in Chinese)
[13]ARNHEITER H.The discovery of the microphthalmia locus and its gene,Mitf[J].Pigment Cell&Melanoma Research,2010,23(6):729-735.
[14]WEILBAECHER K N,HERSHEY C L,TAKEMOTO C M,et al.Age-resolving osteopetrosis:A rat model implicating microphthalmia and the related transcription factor TFE3[J].Journal of Experimental Medicine,1998,187(5):775-785.
[15]POINDEXTER K M,MATTHEW S,ARONCHIK I,et al.Cooperative antiproliferative signaling by aspirin and indole-3-carbinol targets microphthalmia-associated transcription factor gene expression and promoter activity in human melanoma cells[J].Cell Biology and Toxicology,2016,32(2):103-119.
[16]刘秉胜,吴宁华,沈珝琲.CRE和AP-1元件参与人hsp90β基因的表达[J].基础医学与临床,2000,20(3):28-31.LIU B S,WU N H,SHEN Y F.CRE and AP-1elements in the expression of human hsp90βgene[J].Basic&Clinical Medicine,2000,20(3):28-31.(in Chinese)
[17]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.ZHANG X,LUO J,LI J H,et al.Cloning and activity determination of fatty acid synthase(FAS)gene promoter of Xinong Saanen dairy goat[J].Scientia Agricultura Sinica,2010,43(3):640-647.(in Chinese)
[18]HARTMAN M L,CZYZ M.MITF in melanoma:Mechanisms behind its expression and activity[J].Cellular and Molecular Life Sciences,2015,72(7):1249-1260.
[19]BERTOLOTTO C,ABBE P,HEMESATH T J,et al.Microphthalmia gene product as a signal transducer in cAMP-induced differentiation of melanocytes[J].Journal of Cell Biology,1998,142(3):827-835.
[20]DONG L H,WON H L,ROHEUNMIR I,et al.A inhibits cAMP-induced melanin production by downregulating the gene expressions of MITFand tyrosinase in melanocytes[J].Experimental Dermatology,2011,20(9):761-763.
[21]张丽英,李祥龙,周荣艳,等.不同物种MITF基因编码区生物信息学分析[J].安徽农业科学,2011,39(22):13298-13300.ZHANG L Y,LI X L,ZHOU R Y,et al.Bioinformatics analysis on the coding region of MITF gene among different species[J].Journal of Anhui Agricultural Sciences,2011,39(22):13298-13300.(in Chinese)
[22]THORNTON J M.From genome to function[J].Science,2001,292(5524):2095-2097.
[23]连云涓,熊惠霖.蛋白质二级结构预测的多核学习方法[J].计算机应用,2013,33(S1):43-45.LIAN Y J,XIONG H L.Multi-kernel learning method for predicting protein secondary structures[J].Journal of Computer Applications,2013,33(S1):43-45.(in Chinese)
[2]MARTINA J A,DIAB H I,LI H,et al.Novel roles for the MITF/TFE family of transcription factors in organelle biogenesis,nutrient sensing,and energy homeostasis[J].Cellular and Molecular Life Sciences,2014,71(13):2483-2497.
[3]LEVY C,KHALED M,FISHER D E.MITF:Master regulator of melanocyte development and melanoma oncogene[J].Trends in Molecular Medicine,2006,12(9):406-414.
[4]FUSE N,YASUMOTO K,SUZUKI H,et al.Identification of a melanocyte-type promoter of the microphthalmia-associated transcription factor gene[J].Biochemical and Biophysical Research Communications,1996,219(3):702-707.
[5]郑嫩珠,辛清武,朱志明,等.白绒乌鸡MITF基因的cDNA克隆、表达及其对黑色素沉积的影响[J].中国农业科学,2015,48(18):3711-3718.ZHENG N Z,XIN Q W,ZHU Z M,et al.cDNA cloning and expression of mitf gene and its effect on melanin deposition in silky fowl[J].Scientia Agricultura Sinica,2015,48(18):3711-3718.(in Chinese)
[6]张建一,梁春年,裴杰,等.牦牛MITF-M基因序列分析及其在皮肤组织中表达水平[J].华北农学报,2015,30(3):1-7.ZHANG J Y,LIANG C N,PEI J,et al.Sequence analysis and expression level detection of MITF-M gene in different color skin tissues of yak[J].Acta Agriculturae Boreali-Sinica,2015,30(3):1-7.(in Chinese)
[7]杨玉静,张丹瑾,聂瑞强,等.绵羊MITF-M在黑素细胞中过表达后的功能分析[J].中国农业科学,2016,49(2):4214-4221.YANG Y J,ZHANG D J,NIE R Q,et al.The function analysis of over-expression of oar MITF-M in melanocytes[J].Scientia Agricultura Sinica,2016,49(2):4214-4221.(in Chinese)
[8]韩吉龙,岳耀敬,裴杰,等.藏羊刺鼠信号蛋白基因(Agouti)的多态性及不同颜色被毛皮肤组织中Agouti与小眼畸形相关转录因子基因(MITF)表达的定量分析[J].农业生物技术学报,2013,21(3):338-347.HAN J L,YUE Y J,PEI J,et al.The polymorphism of agouti signaling protein gene(Agouti)and the expression of Agouti and microphthalmia-associated transcription factor gene MITFin different coat color skin tissues of Tibetan sheep(Ovis aries)[J].Journal of Agricultural Biotechnology,2013,21(3):338-347.(in Chinese)
[9]SAMBROOK J,FRITSCH E F,MANIATIS T.Molecular Cloning[M].New York:Cold Spring Habor Laboratory Press,1989.
[10]耿荣庆,白丁平,袁超,等.从羊皮肤组织中提取总RNA方法的改进[J].中国畜牧兽医,2012,39(5):244-247.GENG R Q,BAI D P,YUAN C,et al.The improvement on the method of total RNA extraction from goat and sheep skin tissues[J].China Animal Husbandry&Veterinary Medicine,2012,39(5):244-247.(in Chinese)
[11]KOZAK M.Structural features in eukaryotic mRNA that modulate the initiation of translate[J].Biological Chemistry,1991,266(30):19867-19870.
[12]白瑞,张美萍,乔德瑞,等.MITF基因与动物毛色的研究进展[J].上海畜牧兽医通讯,2006,4:2-3.BAI R,ZHANG M P,QIAO D R,et al.Research progress of MITFgene and its effect on animal coat color[J].Shanghai Journal of Animal Husbandry and Veterinary Medicine,2006,4:2-3.(in Chinese)
[13]ARNHEITER H.The discovery of the microphthalmia locus and its gene,Mitf[J].Pigment Cell&Melanoma Research,2010,23(6):729-735.
[14]WEILBAECHER K N,HERSHEY C L,TAKEMOTO C M,et al.Age-resolving osteopetrosis:A rat model implicating microphthalmia and the related transcription factor TFE3[J].Journal of Experimental Medicine,1998,187(5):775-785.
[15]POINDEXTER K M,MATTHEW S,ARONCHIK I,et al.Cooperative antiproliferative signaling by aspirin and indole-3-carbinol targets microphthalmia-associated transcription factor gene expression and promoter activity in human melanoma cells[J].Cell Biology and Toxicology,2016,32(2):103-119.
[16]刘秉胜,吴宁华,沈珝琲.CRE和AP-1元件参与人hsp90β基因的表达[J].基础医学与临床,2000,20(3):28-31.LIU B S,WU N H,SHEN Y F.CRE and AP-1elements in the expression of human hsp90βgene[J].Basic&Clinical Medicine,2000,20(3):28-31.(in Chinese)
[17]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.ZHANG X,LUO J,LI J H,et al.Cloning and activity determination of fatty acid synthase(FAS)gene promoter of Xinong Saanen dairy goat[J].Scientia Agricultura Sinica,2010,43(3):640-647.(in Chinese)
[18]HARTMAN M L,CZYZ M.MITF in melanoma:Mechanisms behind its expression and activity[J].Cellular and Molecular Life Sciences,2015,72(7):1249-1260.
[19]BERTOLOTTO C,ABBE P,HEMESATH T J,et al.Microphthalmia gene product as a signal transducer in cAMP-induced differentiation of melanocytes[J].Journal of Cell Biology,1998,142(3):827-835.
[20]DONG L H,WON H L,ROHEUNMIR I,et al.A inhibits cAMP-induced melanin production by downregulating the gene expressions of MITFand tyrosinase in melanocytes[J].Experimental Dermatology,2011,20(9):761-763.
[21]张丽英,李祥龙,周荣艳,等.不同物种MITF基因编码区生物信息学分析[J].安徽农业科学,2011,39(22):13298-13300.ZHANG L Y,LI X L,ZHOU R Y,et al.Bioinformatics analysis on the coding region of MITF gene among different species[J].Journal of Anhui Agricultural Sciences,2011,39(22):13298-13300.(in Chinese)
[22]THORNTON J M.From genome to function[J].Science,2001,292(5524):2095-2097.
[23]连云涓,熊惠霖.蛋白质二级结构预测的多核学习方法[J].计算机应用,2013,33(S1):43-45.LIAN Y J,XIONG H L.Multi-kernel learning method for predicting protein secondary structures[J].Journal of Computer Applications,2013,33(S1):43-45.(in Chinese)