白绒乌鸡MITF基因的cDNA克隆、表达及其对黑色素沉积的影响
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摘要
【目的】克隆白绒乌鸡小眼畸形相关转录因子(microphthalmia-associaated transcription factor,MITF)基因序列,研究MITF基因在白绒乌鸡各组织中的表达,探索其对白绒乌鸡体内黑色素沉积的影响,为深入研究乌鸡体内黑色素沉积的内在调控机制奠定理论基础。【方法】以150日龄健康的白绒乌鸡为研究对象,采用简并引物和巢式PCR方法从白绒乌鸡皮肤组织中克隆MITF基因c DNA序列,通过DNAStar、Clustal X、Bio Edit和MEGA4.0等软件对获得的序列进行生物信息学分析;利用实时荧光定量PCR法和紫外分光光度法分别研究白绒乌鸡各组织MITF基因的表达特性和黑色素的沉积规律,并应用线性回归方法分析白绒乌鸡各组织MITF基因表达量与黑色素沉积之间的相关性。【结果】经测序和拼接,最终获得了长度为1 431 bp的白绒乌鸡MITF基因的c DNA序列(登录号:KC879241),该序列包含1个完整的CDS区(25—1 431 bp),编码468个氨基酸,预测蛋白分子量为52.42 k D,等电点为6.47;结构域分析发现该基因编码的氨基酸具有1个保守的基础-螺旋-环-螺旋-亮氨酸拉链(b HLH-zip)结构。同源比对结果表明,该基因CDS区氨基酸序列与原鸡的同源性高达100%;与其他鸟类同源性达98%以上,与哺乳动物同源性为90%左右;氨基酸系统进化树显示,白绒乌鸡MITF与原鸡的亲缘关系最近,与猪和人的亲缘关系最远。荧光定量PCR结果表明MITF基因表达量在白绒乌鸡各组织间差异达极显著水平(P<0.01),其中以皮肤表达量最高,大约为肾的2.9倍、肌胃的4.4倍、肝的11.4倍、肌肉的86.2倍;紫外分光光度计检测结果显示各组织黑色素的沉积规律与各组织MITF基因的表达规律大体相似,总体趋势表现为皮肤>肾>肌胃>肝>肌肉。相关分析表明,白绒乌鸡组织中MITF基因的表达量与黑色素含量呈显著的正相关关系(P<0.05)。【结论】MITF基因在动物进化中高度保守,该基因表达具有组织特异性,对白绒乌鸡体内黑色素沉积起正向调控作用,其高表达可能有助于乌鸡体内黑色素的形成。
【Objective】The aim of this paper was to clone microphthalmia-associaated transcription factor(MITF), explore its expression and relationship with melanin deposition in different tissues of Silky Fowl, for further studying molecular mechanism of melanin deposition among Silky Fowl.【Method】A total of 150 day-old healthy Silky Fowls were selected as the research materials in this experiments. Degenerate primers and Nested PCR were used to isolate MITF gene from the skin of Silky Fowl, and DNAStar, Clustal X, Bio Edit and MEGA4.0 software were adopted to analyze biological information of the obtained sequence. Quantitative real-time PCR(q RT-PCR) and ultraviolet spectrophotometry(US) were employed, respectively, to study MITF expression characteristics and melanin deposition in tissues of Silky Fowl, and linear regression estimation was applied to identify the correlation between MITF gene expression and melanin deposition in the tissues of Silky Fowl.【Result】Results showed that the 1 431 bp c DNA sequence of MITF gene was obtained by sequencing and splicing, which contained a complete CDS(25-1 431bp). The CDS domain was composed by 468 amino acids(Gen Bank accession No. KC879241) with a predicted molecular weight of 52.42 k D, an isoelectric point of 6.47, and a conserved basic-helix-loop-helix leucine-zipper(b HLH-zip) protein structure.The homology comparison indicated that the Silky MITF gene shared 100% amino acid homology to Gallus gallus, above 98% to other birds,and about 90% to mammal in CDS domaim. The amino acid phylogenetic tree analysis demonstrated that Silky Fowl MITF had the closest genetic relationship to Gallus gallus, but the farthest to pig and human. The q RT-PCR confirmed that different expressions of MITF gene were detected among tissues of Silky Fowl(P<0.01), with the highest expression in the skin which was about 2.9 times that of the kidney, 4.4 times that of the gizzard, 11.4 times that of the liver, and 86.2 times that of the muscle. The ultraviolet spectrophotmeter examination showed that the law of melanin deposition was consistent with that of MITF expression in tissues, with an overall trend: skin>kidney>gizzard >liver>muscular. Correlation analysis diplayed that there was a positive correlation between MITF expression and melanin deposition(P<0.05).【Conclusion】 MITF gene is highly conservative in the evolution of animal species, the gene expression is specific in tissues, and plays a positive role in the regulation of melanin deposition on Silky Fowl, whose high expression may contribute to the formation of melanin.
【Objective】The aim of this paper was to clone microphthalmia-associaated transcription factor(MITF), explore its expression and relationship with melanin deposition in different tissues of Silky Fowl, for further studying molecular mechanism of melanin deposition among Silky Fowl.【Method】A total of 150 day-old healthy Silky Fowls were selected as the research materials in this experiments. Degenerate primers and Nested PCR were used to isolate MITF gene from the skin of Silky Fowl, and DNAStar, Clustal X, Bio Edit and MEGA4.0 software were adopted to analyze biological information of the obtained sequence. Quantitative real-time PCR(q RT-PCR) and ultraviolet spectrophotometry(US) were employed, respectively, to study MITF expression characteristics and melanin deposition in tissues of Silky Fowl, and linear regression estimation was applied to identify the correlation between MITF gene expression and melanin deposition in the tissues of Silky Fowl.【Result】Results showed that the 1 431 bp c DNA sequence of MITF gene was obtained by sequencing and splicing, which contained a complete CDS(25-1 431bp). The CDS domain was composed by 468 amino acids(Gen Bank accession No. KC879241) with a predicted molecular weight of 52.42 k D, an isoelectric point of 6.47, and a conserved basic-helix-loop-helix leucine-zipper(b HLH-zip) protein structure.The homology comparison indicated that the Silky MITF gene shared 100% amino acid homology to Gallus gallus, above 98% to other birds,and about 90% to mammal in CDS domaim. The amino acid phylogenetic tree analysis demonstrated that Silky Fowl MITF had the closest genetic relationship to Gallus gallus, but the farthest to pig and human. The q RT-PCR confirmed that different expressions of MITF gene were detected among tissues of Silky Fowl(P<0.01), with the highest expression in the skin which was about 2.9 times that of the kidney, 4.4 times that of the gizzard, 11.4 times that of the liver, and 86.2 times that of the muscle. The ultraviolet spectrophotmeter examination showed that the law of melanin deposition was consistent with that of MITF expression in tissues, with an overall trend: skin>kidney>gizzard >liver>muscular. Correlation analysis diplayed that there was a positive correlation between MITF expression and melanin deposition(P<0.05).【Conclusion】 MITF gene is highly conservative in the evolution of animal species, the gene expression is specific in tissues, and plays a positive role in the regulation of melanin deposition on Silky Fowl, whose high expression may contribute to the formation of melanin.
引文
[1]Steingrimsson E,Copeland N G,Jenkins N A.Melancoytes and microphthalmia transcription factor network.Annual Review of Genetics,2004,38:365-411.
[2]Vachtenheim J,Borovansky J.‘‘Transcription physiology’’of pigment formation in melanocytes:central role of MITF.Experimental Dermatology,2010,19:617-627.
[3]Goding C R.A picture of Mitf in melanoma immortality.Oncogene,2011,30:2304-2306.
[4]Levy C,Fisher D E.Dual roles of lineage restricted transcription factors:The case of MITF in melanocytes.Transcription,2011,2(1):19-22.
[5]Bauer G L,Praetorius C,Bergsteinsdottir K,Hallsson J H,Gisladottir B K,Schepsky A,Swing D A,Sullivan T N,Arnheiter H,Bismuth K,Debbache J,Fletcher C,Warming S,Copeland N G,Jenkins N A,Steingr?′msson E.The role of MITF phosphorylation site during coat color and eye development in mice analyzed by bacterial artificial chromosome transgene rescue.Genetics,2009,183:581-594.
[6]Yan X K,Zhang T Y,Wang H Y,Jiang Y,Chen Y,Wang H Y,Ma D,Wang L,Li H W.A novel mutation in the MITF may be digenic with GJBZ mutations in a large Chinese family of Waardenburg syndrome typeⅡ.Journal of Genetic and Genomies,2011,38:585-591.
[7]Watanabe A,Takeda K,Ploplis B,Tachibana M.Epistatic relationship between Waardenburg syndrome genes MITF and PAX3.Nature Genetics,1998,18:283-286.
[8]Smith S D,Kelley P M,Kenyon J B,Hoover D.Tietz syndrome(hypopigmentation/deafness)caused by mutation of MITF.Journal of Medicine Genetics,2000,37:446-448.
[9]Kitamura R,Tsukamoto K,Harada K,Shimizu A,Shimada S,Kobayashi T,Imokawa G.Mechanisms underlying the dysfunction of melanocytes in vitiligo epidermis:Role of SCF/KIT protein interactions and the downstream efector,MITF-M.Journal of Pathology,2004,202:463-475.
[10]Hayes B J,Pryce J,Chamberlain A J,Bowman P J,Goddard M E.Genetic architecture of complex traits and accuracy of genomic prediction:coat colour,milk-fat percentage,and type in Holstein cattle as contrasting model traits.PLo S Genetics,2010,6(9):e1001139.
[11]Schmutz S M,Berryere T G,Dreger D L.MITF and white spotting in dogs:a population study.Journal of Heredity,2009,100(Suppl.):66-74.
[12]Zhu Z,He J,Jia X,Jiang J,Bai R,Yu X,Lv L,Fan R,He X,Geng J,You R,Dong Y,Qiao D,Lee K B,Smith G W,Dong C.Micro RNA-25 functions in regulation of pigmentation by targeting the transcription factor MITF in Alpaca(Lama pacos)skin mel-anocytes.Domestic Animal Endocrinology,2010,38:200-209.
[13]Mochii M,Mazaki Y,Mizuno N,Hayashi H,Eguchi G:Role of Mitf in differentiation and transdifferentiation of chicken pigmented epitheli cell.Developmental Biology,1998,193:47-62.
[14]Minvielle F,Bedhom B,Coville J L,Ito S,Inoue-Murayama M,Gourichon D.The“silver”Japanese quail and the MITF gene:causal mutation,associated traits and homology with the“blue”chicken plumage.BMC Genetics,2010,11:15.
[15]Wang Y,Li S M,Huang J,Chen S Y,Liu Y P.Mutation of TYR and MITF genes with are associated with plumage color phenotypess in geese.Asian-Australasian Journal of Animal-Sciences(AJAS),2014,27(6):778-783.
[16]Ozeki H,Ito S,Wakamatsu K,Thody A J.Spectrophotometric characterization of eumelanin and pheomelanin in hair.Pigment Cell Research,1996,9:265-270.
[17]黎观红,徐海燕,许兰姣,瞿明仁,游金明,易中华,潘珂.日粮硒添加水平对泰和乌骨鸡生产性能及组织黑色素含量的影响.中国农业科学,2011,44(13):2777-2786.Li G H,Xu H Y,Xu L J,Qu M R,You J M,Yi Z H,Pan K.Effects of dietary selenium supplementation on growth performance and melanin content in tissues of Taihe Silky Fowls.Scientia Agricultura Sinica,2011,44(13):2777-2786.(in Chinese)
[18]Pfaffl M W.A new mathematical model for relative quantification in realtime RT-PCR.Nucleic Acids Research,2001,29(9):2004-2007.
[19]Hodgkimson C A,Moore K J,Nakayama A,Steingrfmsson E,Copeland N G,Jenkins N A,Arnheiter H.Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein.Cell,1993,74:395-404.
[20]朱芷葳,贺俊平,于秀菊,程志学,董常生.Mitf-M在羊驼皮肤组织的表达与序列分析及免疫组织化学定位.中国农业科学,2012,45(4):794-800.Zhu Z W,He J P,Yu X J,Cheng Z X,Dong C S.Expression,sequence analysis and immunohistochemical localization of Mitf-M transcription factor in Alpaca skin.Scientia Agricultura Sinica,2012,45(4):794-800.(in Chinese)
[21]Otreba M,Rok J,Buszman E,Wrzesniok D.Regulation of melanogenesis:the role of c AMP and MITF.Postepy Higieny I Medycyny Doswiadczalnej,2012,66:33-40.
[22]安健,汪明,樊宝良,赵兴波,李宁.柔嫩艾美耳球虫马杜拉霉素抗药株与敏感株的m RNA差异显示.畜牧兽医学报,2006,37(5):480-484.An J,Wang M,Fan B L,Zhao X B,Li N.m RNA differential display PCR of maduramisin sensitive and resistant eimeria tenella strain,Acta Veterinaria et Zootechnica Sinica,2006,37(5):480-484.(in Chinese)
[23]Li S J,Wang C,Yu W H,Zhao S H,Gong Y Z.Identification of genes related to white and black plumage formation by RNA-Seq from white and black feather bulbs in ducks.PLo S ONE,2012,7(5):e36592.
[24]韩吉龙,岳耀敬,裴杰,郭婷婷,刘建斌,郭健,孙晓萍,冯瑞林,杨博辉.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.农业生物技术学报,2013,21(3):338-347.Han J L,Yue Y J,Pei J,Guo T T,Liu J B,Guo J,Sun X P,Feng R L,Yang B H.The polymorphism of agouti signaling protein gene mgouti and the expression of agouti and microphthalmia-associated transcrip Ition factor gene MITF in different coat color skin tissues of Tibetan sheep(Ovis aries).Journal of Agricultural Biotechnology,2013,21(3):338-347.(in Chinese)
[25]Kingo K,Aunin E,Karelson M,Ratsep R,Silm H,Vasar E,Koks S.Expressional changes in theintracellular melanogenesis pathways and their possible role in the pathogenesis of vitiligo.Journal of Dermatological Science,2008,52:39-46.
[2]Vachtenheim J,Borovansky J.‘‘Transcription physiology’’of pigment formation in melanocytes:central role of MITF.Experimental Dermatology,2010,19:617-627.
[3]Goding C R.A picture of Mitf in melanoma immortality.Oncogene,2011,30:2304-2306.
[4]Levy C,Fisher D E.Dual roles of lineage restricted transcription factors:The case of MITF in melanocytes.Transcription,2011,2(1):19-22.
[5]Bauer G L,Praetorius C,Bergsteinsdottir K,Hallsson J H,Gisladottir B K,Schepsky A,Swing D A,Sullivan T N,Arnheiter H,Bismuth K,Debbache J,Fletcher C,Warming S,Copeland N G,Jenkins N A,Steingr?′msson E.The role of MITF phosphorylation site during coat color and eye development in mice analyzed by bacterial artificial chromosome transgene rescue.Genetics,2009,183:581-594.
[6]Yan X K,Zhang T Y,Wang H Y,Jiang Y,Chen Y,Wang H Y,Ma D,Wang L,Li H W.A novel mutation in the MITF may be digenic with GJBZ mutations in a large Chinese family of Waardenburg syndrome typeⅡ.Journal of Genetic and Genomies,2011,38:585-591.
[7]Watanabe A,Takeda K,Ploplis B,Tachibana M.Epistatic relationship between Waardenburg syndrome genes MITF and PAX3.Nature Genetics,1998,18:283-286.
[8]Smith S D,Kelley P M,Kenyon J B,Hoover D.Tietz syndrome(hypopigmentation/deafness)caused by mutation of MITF.Journal of Medicine Genetics,2000,37:446-448.
[9]Kitamura R,Tsukamoto K,Harada K,Shimizu A,Shimada S,Kobayashi T,Imokawa G.Mechanisms underlying the dysfunction of melanocytes in vitiligo epidermis:Role of SCF/KIT protein interactions and the downstream efector,MITF-M.Journal of Pathology,2004,202:463-475.
[10]Hayes B J,Pryce J,Chamberlain A J,Bowman P J,Goddard M E.Genetic architecture of complex traits and accuracy of genomic prediction:coat colour,milk-fat percentage,and type in Holstein cattle as contrasting model traits.PLo S Genetics,2010,6(9):e1001139.
[11]Schmutz S M,Berryere T G,Dreger D L.MITF and white spotting in dogs:a population study.Journal of Heredity,2009,100(Suppl.):66-74.
[12]Zhu Z,He J,Jia X,Jiang J,Bai R,Yu X,Lv L,Fan R,He X,Geng J,You R,Dong Y,Qiao D,Lee K B,Smith G W,Dong C.Micro RNA-25 functions in regulation of pigmentation by targeting the transcription factor MITF in Alpaca(Lama pacos)skin mel-anocytes.Domestic Animal Endocrinology,2010,38:200-209.
[13]Mochii M,Mazaki Y,Mizuno N,Hayashi H,Eguchi G:Role of Mitf in differentiation and transdifferentiation of chicken pigmented epitheli cell.Developmental Biology,1998,193:47-62.
[14]Minvielle F,Bedhom B,Coville J L,Ito S,Inoue-Murayama M,Gourichon D.The“silver”Japanese quail and the MITF gene:causal mutation,associated traits and homology with the“blue”chicken plumage.BMC Genetics,2010,11:15.
[15]Wang Y,Li S M,Huang J,Chen S Y,Liu Y P.Mutation of TYR and MITF genes with are associated with plumage color phenotypess in geese.Asian-Australasian Journal of Animal-Sciences(AJAS),2014,27(6):778-783.
[16]Ozeki H,Ito S,Wakamatsu K,Thody A J.Spectrophotometric characterization of eumelanin and pheomelanin in hair.Pigment Cell Research,1996,9:265-270.
[17]黎观红,徐海燕,许兰姣,瞿明仁,游金明,易中华,潘珂.日粮硒添加水平对泰和乌骨鸡生产性能及组织黑色素含量的影响.中国农业科学,2011,44(13):2777-2786.Li G H,Xu H Y,Xu L J,Qu M R,You J M,Yi Z H,Pan K.Effects of dietary selenium supplementation on growth performance and melanin content in tissues of Taihe Silky Fowls.Scientia Agricultura Sinica,2011,44(13):2777-2786.(in Chinese)
[18]Pfaffl M W.A new mathematical model for relative quantification in realtime RT-PCR.Nucleic Acids Research,2001,29(9):2004-2007.
[19]Hodgkimson C A,Moore K J,Nakayama A,Steingrfmsson E,Copeland N G,Jenkins N A,Arnheiter H.Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helix-zipper protein.Cell,1993,74:395-404.
[20]朱芷葳,贺俊平,于秀菊,程志学,董常生.Mitf-M在羊驼皮肤组织的表达与序列分析及免疫组织化学定位.中国农业科学,2012,45(4):794-800.Zhu Z W,He J P,Yu X J,Cheng Z X,Dong C S.Expression,sequence analysis and immunohistochemical localization of Mitf-M transcription factor in Alpaca skin.Scientia Agricultura Sinica,2012,45(4):794-800.(in Chinese)
[21]Otreba M,Rok J,Buszman E,Wrzesniok D.Regulation of melanogenesis:the role of c AMP and MITF.Postepy Higieny I Medycyny Doswiadczalnej,2012,66:33-40.
[22]安健,汪明,樊宝良,赵兴波,李宁.柔嫩艾美耳球虫马杜拉霉素抗药株与敏感株的m RNA差异显示.畜牧兽医学报,2006,37(5):480-484.An J,Wang M,Fan B L,Zhao X B,Li N.m RNA differential display PCR of maduramisin sensitive and resistant eimeria tenella strain,Acta Veterinaria et Zootechnica Sinica,2006,37(5):480-484.(in Chinese)
[23]Li S J,Wang C,Yu W H,Zhao S H,Gong Y Z.Identification of genes related to white and black plumage formation by RNA-Seq from white and black feather bulbs in ducks.PLo S ONE,2012,7(5):e36592.
[24]韩吉龙,岳耀敬,裴杰,郭婷婷,刘建斌,郭健,孙晓萍,冯瑞林,杨博辉.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.农业生物技术学报,2013,21(3):338-347.Han J L,Yue Y J,Pei J,Guo T T,Liu J B,Guo J,Sun X P,Feng R L,Yang B H.The polymorphism of agouti signaling protein gene mgouti and the expression of agouti and microphthalmia-associated transcrip Ition factor gene MITF in different coat color skin tissues of Tibetan sheep(Ovis aries).Journal of Agricultural Biotechnology,2013,21(3):338-347.(in Chinese)
[25]Kingo K,Aunin E,Karelson M,Ratsep R,Silm H,Vasar E,Koks S.Expressional changes in theintracellular melanogenesis pathways and their possible role in the pathogenesis of vitiligo.Journal of Dermatological Science,2008,52:39-46.