连城白鸭MITF基因的cDNA克隆、组织表达与分析
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摘要
对连城白鸭刺参小眼畸形相关转录因子(microphthalmia-associtated transcription factor,MITF)的序列进行克隆及序列分析,预测MITF蛋白的二、三级结构,并利用RT-PCR技术检测MITF基因在连城白鸭不同组织中的表达差异,为深入研究连城白鸭体内黑色素沉积的内在调控机制奠定理论基础。结果显示,连城白鸭MITF基因长度为1 323bp (登录号:MG516570),包括一个完整的CDS区(34～1 323bp),编码429个氨基酸;与绿头鸭氨基酸同源性100%,与原鸡等鸟类的同源性达95%以上,而与绵羊等哺乳动物的亲缘关系较远。对MITF蛋白的二级结构预测显示,无规则卷曲及α螺旋是形成其二级结构的主要结构元件;连城白鸭MITF蛋白三级结构为一个环状结构间隔开两个螺旋,从而形成MITF蛋白的基础螺旋-环-螺旋-拉链结构。荧光定量PCR分析显示,连城白鸭皮肤组织中的MITF基因mRNA表达量最高,极显著(P<0.01)高于其他各组织;各组织表达量依次为:皮肤>肾脏>肌胃>肝脏>肌肉。综上所述,MITF基因在连城白鸭组织中的表达具有组织特异性,为深入探究MITF基因能否作为调控连城白鸭黑色素沉积的主效候选基因及连城白鸭黑色素性状的辅助选择提供理论依据。
The cloning,tissue expression and structural analysis of Microphthalmia-associated transcription factor(MITF)gene were conducted in Liancheng white duck,which would provide a theoretical basis for the further research of melanin deposition.The MITF gene from the skin of female ducks was cloned through nested-PCR,and the mRNA expression detected in tissues by qRT-PCR.It was shown that the MITF cDNA was 1 323 bp in length(GenBank No.MG516570)with a complete CDS region(34-1 323bp)encoding 429 amino acids.The amino acid sequence of MITF was 100% homologous to that of mallard and more than 95% to that of Gallus gallus,but farther from sheep and other mammals.The predicted secondary structure of the protein was mainly random coil andα-helix,while the circular tertiary structure contained two separate helices to form the helix-loop-helix-chain structure.The qRT-PCR results showed that the mRNA expression of MITF was significantly higher in the skin than the other organs(P<0.01)with the relative expressions in the order of skin>kidney>gizzard>liver>muscle.It appeared that the expression of MITF gene was tissue-specific in Liancheng white ducks.The information would aid in the utilization of MITFgene for studies on the melanin deposition and trait selection of the duck.
The cloning,tissue expression and structural analysis of Microphthalmia-associated transcription factor(MITF)gene were conducted in Liancheng white duck,which would provide a theoretical basis for the further research of melanin deposition.The MITF gene from the skin of female ducks was cloned through nested-PCR,and the mRNA expression detected in tissues by qRT-PCR.It was shown that the MITF cDNA was 1 323 bp in length(GenBank No.MG516570)with a complete CDS region(34-1 323bp)encoding 429 amino acids.The amino acid sequence of MITF was 100% homologous to that of mallard and more than 95% to that of Gallus gallus,but farther from sheep and other mammals.The predicted secondary structure of the protein was mainly random coil andα-helix,while the circular tertiary structure contained two separate helices to form the helix-loop-helix-chain structure.The qRT-PCR results showed that the mRNA expression of MITF was significantly higher in the skin than the other organs(P<0.01)with the relative expressions in the order of skin>kidney>gizzard>liver>muscle.It appeared that the expression of MITF gene was tissue-specific in Liancheng white ducks.The information would aid in the utilization of MITFgene for studies on the melanin deposition and trait selection of the duck.
引文
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[15]LOERCHER A E,TANK E M,DELSTON R B,et al.MITF links differentiation with cell cycle arrest in melanocytes by transcriptional activation of INK4A[J].Cell Biology,2005,168(1):35-40.
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[18]SHI Y,LI X,JU D,et al.A novel mutation of the MITF gene in a family with Waardenburg syndrome type 2:A case report[J]. Experimental and Therapeutic Medicine,2016,11(4):1516-1518.
[19]TAKEDA K,HOZUMI H,NAKAI K,et al.Insertion of long interspersed element-1in the MITF gene is associated with altered neurobehavior of the black-eyed white(mi-bw)mouse[J].Genes Cells,2014,19(2):126-140.
[20]HAUSWIRTH R, HAASE B, BLATTER M, et al.Mutations in MITF and PAX3cause"Splashed White"and Other White Spotting Phenotypes in Horses[J].Plos Genetics,2012,8(4):1-9.
[21]MINVIELLE F,BED'HOM B,COVILLE J L,et al.The"silver"Japanese quail and the MITF gene:causal mutation,associated traits and homology with the"blue"chicken plumage[J].BMC Genetics,2010,11(15):1-7.
[22]WANG Y,LI S M,HUANG J,et al.Mutation of TYR and MITF genes with are associated with plumage color phenotypess in geese[J].Asian-Australasian Journal of Animal-Sciences,2014,27(6):778-783.
[23]SULTANA H,SEO D,CHOI N R,et al.Identification of polymorphisms in MITF and DCT genes and their associations with plumage colors in Asian duck breeds[J].AsianAustralasian Journal of Animal-Sciences,2018,31(2),180-188.
[24]PFAFFL M W. A new mathematical model for relative quantification in realtime RT-PCR[J]. Nucleic Acids Research,2001,29(9):2004-2007.
[25]HODGKIMSON C A,MOORE K J,NAKAYAMA A,et al.Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helixzipper protein[J].Cell,1993,74:395-404.
[26]朱芷葳,贺俊平,于秀菊,等.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[J].中国农业科学,2012,45(4):794-800.
[27]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):13298-13300,13308.
[28]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.
[29]安键,汪明,樊宝良,等.柔嫩艾美耳球虫马杜拉霉素抗药株与敏感株的mRNA差异显示[J].畜牧兽医学报,2006,37(5):480-484.
[30]ZHU Z,HE J,JIA X,et al. MicroRNA-25functions in regulation of pigmentation by targeting the transcription factor MITF in alpaca(Lama pacos)skin melanocytes[J].Domestic Animal Endocrinology,2010,38(3):200-209.
[31]KINGO K,AUNIN E M,RATSEP R,et al.Expressional changes in the intracellular melanogenesis pathways and their possible role in the pathogenesis of vitiligo[J].Journal of Dermatological Science,2008,52(1):39-46.
[2]ITO S, NAKANISHI Y, VALENZUELA R K,et al.Usefulness of alkaline hydrogen peroxide oxidation to analyze eumelanin and pheomelanin in various tissue samples:application to chemical analysis of human hair melanins[J].Pigment Cell Melanoma Res,2011,24,605-613.
[3]LIN J Y,FISHER.Melanocyte biology and skin pigmentation[J].Nature,2007,445(7130):843-850.
[4]DORAZIO J A,NOBUHISA T,CUI R,et al.Topical drug rescue strategy and skin protection based on the role of Mc1rin UV induced tanning[J]. Nature,2006,443(7109):340-344.
[5]WICKELGREN I.Why I have red hair,need to avoid the sun,and shouldn't commit a crime[J].Science,2007,315(5816):1215.
[6]WAN P,HU Y Q,HE L.Regulation of melanocyte pivotal transcription factor MITF by some other transcription factors[J].Molecular and Cellular Biochemistry,2011,354(1-2):241-246.
[7]LEE HD,LEE WH,ROH E,et al.Manassantin A inhibits cAMP induced melanin production by down-regulating the gene expressions of MITF and tyrosinase in melanocytes[J].Experimental Dermatology,2011,20(9):761-763.
[8]VACHTENHEIM J, BOROVANSKY J.''Transcription physiology'of pigment formation in melanocytes:central role of MITF[J].Experimental Dermatology,2010,19:617-627.
[9]GODING C R.A picture of MITF in melanoma immortality[J].Oncogene,2011,30(20):2304-2306.
[10]LEVY C,FISHER D E.Dual roles of lineage restricted transcription factors:The case of MITF in melanocytes[J].Transcription,2011,2(1):19-22.
[11]BAUER G L,PRAETORIUS C,BERGSTEINSDóTTIR K,et al.The role of MITF phosphorylation sites during coat color and eye development in mice analyzed by bacterial artificial chromosome transgene rescue[J].Genetics,2009,183(2):581-594.
[12]OTREBA M,ROK J,BUSZMAN E,et al.Regulation of melanogenesis:the role of cAMP and MITF[J].Postepy Higieny i Medycyny Doswiadczalnej,2012,66(855199):33-40.
[13]LEVY C,KHALED M,FISHER D E.Review:MITF:master regulator of melanocyte development and melanoma oncogene[J].Trends in Molecular Medicine,2006,12(9):406-414.
[14]NISHIMURA E K, GRANTER S R, FISHER D E.Mechanisms of hair graying:incomplete melanocyte stem cell maintenance in the niche[J].Science,2005,307(5710):720-724.
[15]LOERCHER A E,TANK E M,DELSTON R B,et al.MITF links differentiation with cell cycle arrest in melanocytes by transcriptional activation of INK4A[J].Cell Biology,2005,168(1):35-40.
[16]YAN X,ZHANG T,WANG Z,et al.A novel mutation in the MITF may be digenic with GJB2 mutations in a large Chinese family of Waardenburg syndrome typeⅡ[J].Journal of Genetic and Genomics, 2011,38(12):585-591.
[17]NI C,ZHANG D,BEYER LA,et al.Hearing dysfunction in heterozygous MITF(Mi-wh)/+mice, a model for Waardenburg syndrome type 2 and Tietz syndrome[J].Pigment Cell Melanoma Res,2013,26(1):78-87.
[18]SHI Y,LI X,JU D,et al.A novel mutation of the MITF gene in a family with Waardenburg syndrome type 2:A case report[J]. Experimental and Therapeutic Medicine,2016,11(4):1516-1518.
[19]TAKEDA K,HOZUMI H,NAKAI K,et al.Insertion of long interspersed element-1in the MITF gene is associated with altered neurobehavior of the black-eyed white(mi-bw)mouse[J].Genes Cells,2014,19(2):126-140.
[20]HAUSWIRTH R, HAASE B, BLATTER M, et al.Mutations in MITF and PAX3cause"Splashed White"and Other White Spotting Phenotypes in Horses[J].Plos Genetics,2012,8(4):1-9.
[21]MINVIELLE F,BED'HOM B,COVILLE J L,et al.The"silver"Japanese quail and the MITF gene:causal mutation,associated traits and homology with the"blue"chicken plumage[J].BMC Genetics,2010,11(15):1-7.
[22]WANG Y,LI S M,HUANG J,et al.Mutation of TYR and MITF genes with are associated with plumage color phenotypess in geese[J].Asian-Australasian Journal of Animal-Sciences,2014,27(6):778-783.
[23]SULTANA H,SEO D,CHOI N R,et al.Identification of polymorphisms in MITF and DCT genes and their associations with plumage colors in Asian duck breeds[J].AsianAustralasian Journal of Animal-Sciences,2018,31(2),180-188.
[24]PFAFFL M W. A new mathematical model for relative quantification in realtime RT-PCR[J]. Nucleic Acids Research,2001,29(9):2004-2007.
[25]HODGKIMSON C A,MOORE K J,NAKAYAMA A,et al.Mutations at the mouse microphthalmia locus are associated with defects in a gene encoding a novel basic-helix-loop-helixzipper protein[J].Cell,1993,74:395-404.
[26]朱芷葳,贺俊平,于秀菊,等.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[J].中国农业科学,2012,45(4):794-800.
[27]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):13298-13300,13308.
[28]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.
[29]安键,汪明,樊宝良,等.柔嫩艾美耳球虫马杜拉霉素抗药株与敏感株的mRNA差异显示[J].畜牧兽医学报,2006,37(5):480-484.
[30]ZHU Z,HE J,JIA X,et al. MicroRNA-25functions in regulation of pigmentation by targeting the transcription factor MITF in alpaca(Lama pacos)skin melanocytes[J].Domestic Animal Endocrinology,2010,38(3):200-209.
[31]KINGO K,AUNIN E M,RATSEP R,et al.Expressional changes in the intracellular melanogenesis pathways and their possible role in the pathogenesis of vitiligo[J].Journal of Dermatological Science,2008,52(1):39-46.