莆田黑鸭MITF基因表达特性及其与黑色素沉积的关联分析
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摘要
莆田黑鸭(Anas anas domesticus)是我国唯一的黑羽蛋鸭地方品种,为中国珍贵稀有的水禽资源。小眼畸形相关转录因子(microphthalmia-associtated transcription factor, MITF)作为黑色素细胞最重要的转录因子,可通过α-MSH (melanocyte-stimulating hormone)诱导,与cAMP应答原件结合蛋白(cAMP responsive element binding protein, CREB)结合作用于cAMP,激活MITF基因在黑色素细胞中特异表达,对于黑色素的合成具有关键作用。为探讨莆田黑鸭各组织MITF基因的表达特性,及其与真黑色素在莆田黑鸭各组织中沉积规律的相关性,本研究利用巢式PCR方法,首次从莆田黑鸭皮肤组织克隆MITF基因(GenBank No. MG516571),并进行了序列分析。进而利用实时荧光定量PCR方法检测莆田黑鸭各组织的MITF基因表达,利用紫外分光光度法测定莆田黑鸭各组织真黑色素的沉积量,应用线性相关分析方法分析莆田黑鸭各组织MITF基因表达与真黑色素沉积的相关性。结果表明,莆田黑鸭MITF基因长度为1 341bp,包含完整的CDS序列(12~1334 bp);编码440个氨基酸,与绿头鸭(Anas platyrhynchos)的氨基酸同源性为100%,与原鸡(Gallus gallus)、鹌鹑(Coturnix japonica)等鸟类的氨基酸同源性在95%以上。遗传进化分析发现,莆田黑鸭MITF与绿头鸭的亲缘关系最近,与鸿雁(Anser cygnoides)、白绒乌鸡(Silky flow)、原鸡等亲缘关系次之,与猪(Sus scrofa)、绵羊(Ovis aries)等哺乳动物的亲缘关系最远。实时荧光定量PCR分析发现,MITF基因mRNA表达量以莆田黑鸭皮肤组织最高,极显著高于其他各组织(P<0.01);各组织依次为:皮肤>肾脏>肌胃>肝脏>肌肉。紫外分光光度计检测结果显示,莆田黑鸭各组织真黑色素的沉积规律与各组织MITF基因的表达量基本一致;其中肝脏组织MITF基因表达量与真黑色素含量呈显著正相关(P<0.05)。综上所述,MITF基因在莆田黑鸭中的表达具有组织特异性,可能对真黑色素的沉积具有正向调控作用;MITF可作为黑色素沉积调控的主效候选基因。本研究为莆田黑鸭黑色素性状的选择提供了基础资料。
Putian black duck(Anas anas domesticus) is the sole native variety in black-feather laying duck,which is rare waterfowl resource in China. The microphthalmia-associated transcription factor(MITF), the most important transcription factor of melanocytes, could be induced by α-MSH(melanocyte-stimulating hormone) and bind to the c AMP responsive element binding protein(CREB) to activate MITF gene expression in melanocytes which plays a key role in the synthesis of melanin. To investigate the relationship between the expression of the MITF gene and regularity of eumelanin deposition in different tissues of Putian black duck,the MITF gene was firstly cloned and analyzed from the skin of Putian black duck with nested PCR and sequenced(GenBank No. MG516571). MITF gene expression was detected in various tissues of Putian black ducks via real-time quantitative PCR, and deposition of eumelanin in the tissues was measured by UV spectrophotometry. The results showed that the length of MITF gene from Putian black duck was 1 341 bp,which contained the complete CDS sequence(12～1 334 bp) and encoded 440 amino acids. The amino acid homology compared with Anas platyrhynchos was 100%, and it was above 95% compared with chicken(Gallus gallus) and quail(Coturnix japonica). The genetic relationship of MITF between Putian black duck and Anas platyrhynchos was the closest, followed with Anser cygnoides, Silky flow, Gallus gallus, and relationship compared with pig(Sus scrofa), sheep(Ovis aries), and other mammals was the farthest. The mRNA expression of MITF gene in Putian black duck skin tissue was the highest in the tissues(skin>kidney>gizzard>liver>muscle)(P<0.01). The deposition of eumelanin was basically consistent with the expression of MITF gene in each tissue of Putian black duck. The expression of MITF gene had significant positive correlation with the production of eumelanin in the liver tissues of Putian black duck(P<0.05). In summary, the expression of MITF gene had tissue specificity in Putian black duck, which might have positive regulation on the deposition of eumelanin and could be used as a candidate gene for the regulation of melanin deposition. The study could provide basic material for the selection of melanin trait in Putian black duck.
Putian black duck(Anas anas domesticus) is the sole native variety in black-feather laying duck,which is rare waterfowl resource in China. The microphthalmia-associated transcription factor(MITF), the most important transcription factor of melanocytes, could be induced by α-MSH(melanocyte-stimulating hormone) and bind to the c AMP responsive element binding protein(CREB) to activate MITF gene expression in melanocytes which plays a key role in the synthesis of melanin. To investigate the relationship between the expression of the MITF gene and regularity of eumelanin deposition in different tissues of Putian black duck,the MITF gene was firstly cloned and analyzed from the skin of Putian black duck with nested PCR and sequenced(GenBank No. MG516571). MITF gene expression was detected in various tissues of Putian black ducks via real-time quantitative PCR, and deposition of eumelanin in the tissues was measured by UV spectrophotometry. The results showed that the length of MITF gene from Putian black duck was 1 341 bp,which contained the complete CDS sequence(12～1 334 bp) and encoded 440 amino acids. The amino acid homology compared with Anas platyrhynchos was 100%, and it was above 95% compared with chicken(Gallus gallus) and quail(Coturnix japonica). The genetic relationship of MITF between Putian black duck and Anas platyrhynchos was the closest, followed with Anser cygnoides, Silky flow, Gallus gallus, and relationship compared with pig(Sus scrofa), sheep(Ovis aries), and other mammals was the farthest. The mRNA expression of MITF gene in Putian black duck skin tissue was the highest in the tissues(skin>kidney>gizzard>liver>muscle)(P<0.01). The deposition of eumelanin was basically consistent with the expression of MITF gene in each tissue of Putian black duck. The expression of MITF gene had significant positive correlation with the production of eumelanin in the liver tissues of Putian black duck(P<0.05). In summary, the expression of MITF gene had tissue specificity in Putian black duck, which might have positive regulation on the deposition of eumelanin and could be used as a candidate gene for the regulation of melanin deposition. The study could provide basic material for the selection of melanin trait in Putian black duck.
引文
安键,汪明,樊宝良,等.2006.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,37(5):480-484.(An J,Wang M,Fan B L,et al2006.mRNA differential display PCR of maduramisin sensitive and resistant Eimeria tenella strain[J].Acta Veterinaria et Zootechnica Sinica,37(5):480-484.)
韩吉龙,岳耀敬,裴杰,等.2013.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Agout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,21(3):338-347.(Han J LYue Y J,Pei J,et al.2013.The polymorphism of agout signaling protein gene(Agouti)and the expression of Agouti and microphthalmia-associated transcription factor gene MITF in different coat color skin tissues of Tibetan sheep(Ovis aries)[J].Journal of Agricultural Biotechnology,21(3):338-347.)
张丽英,李祥龙,周荣艳,等.2011.不同物种Mitf基因编码区生物信息学分析[J].安徽农业科学,39(22):13298-13300,13308.(Zhang L Y,Li X L,Zhou R Y,,et al.2011.Bioinformatics analysis on the coding region of Mitf gene among different species[J].Journal of Anhui Agricultural Sciences,39(22):13298-13300,13308.)
郑嫩珠,辛清武,朱志明,等.2015.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,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,48(18):3711-3718.)
张瑞娜,范瑞文,程志学,等.2011.CDK5对羊驼皮肤黑色素细胞TYR和MITF mRNA表达的调节[J].畜牧兽医学报,42(12):1712-1717.(Zhang R N,Fan R W,Cheng Z X,et al.2011.Regulation of TYR and MITF mRNAexpression by CDK5 in Alpaca melanocytes[J].Chinese Journal of Animal and Veterinary Sciences,42(12):1712-1717.)
Bauer G L,Praetorius C,Bergsteinsdottir K,et al.2009.The role of MITF phosphorylation sites during coat color and eye development in mice analyzed by bacterial artificial chromosome transgene rescue[J].Genetics,183(2):581-594.
Dorazio J A,Nobuhisa T,Cui R,et al.2006.Topical drug rescue strategy and skin protection based on the role of Mc1r in UV induced tanning[J].Nature,443(7109):340-344.
Dong C S,Wang H D,Xue L L,et al.2012.Coat color determination by miR-137 mediated down-regulation of microphthalmia-associated transcription factor in a mouse mode[J].RNA,18(9):1679-1686.
Goding C R.2011.A picture of Mitf in melanoma immortality[J].Oncogene,30(20):2304-2306.
Hauswirth R,Haase B,Blatter M,et al.2012.Mutations in MITF and PAX3 cause"Splashed White"and other white spotting phenotypes in horses[J].PLoS Genetics,8(4):1-9.
Ito S,Nakanishi Y,Valenzuela R K,et al.2011.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 Research,24(4):605-613.
Kingo K,Aunin E M,Ratsep R,et al.2008.Expressional changes in the intracellular melanogenesis pathways and their possible role in the pathogenesis of vitiligo[J]Journal of Dermatological Science,52(1):39-46.
Lin J Y,Fisher D E.2007.Melanocyte biology and skin pigmentation[J].Nature,445(7130):843-850.
Lee H D,Lee W H,Roh E,et al.2011.Manassantin A inhibits cAMP induced melanin production by down-regulating the gene expressions of MITF and tyrosinase in melanocytes[J].Experimental Dermatology,20(9):761-763.
Levy C,Fisher D E.2011.Dual roles of lineage restricted transcription factors:The case of MITF in melanocytes[J].Transcription,2(1):19-22.
Levy C,Khaled M,Fisher D E.2006.MITF:master regulator of melanocyte development and melanoma oncogene[J]Trends in Molecular Medicine,12(9):406-414.
Loercher A E,Tank E M,Delston R B,Harbour J W.2005MITF links differentiation with cell cycle arrest in melanocytes by transcriptional activation of INK4A[J].Cel Biology,168(1):35-40.
Li S J,Wang C,Yu W H,et al.2012.Identification of genes related to white and black plumage formation by RNA-Seq from white and black feather bulbs in ducks[J]PLoS One,7(5):e36592.
Minvielle F,Bedhom B,Coville J L,et al.2010.The'silver Japanese quail and the MITF gene:Causal mutation,associated traits and homology with the'blue'chicken plumage[J].BMC Genetics,11(15):1-7.
Nishimura E K,Granter S R,Fisher D E.2005.Mechanisms of hair graying:Incomplete melanocyte stem cell maintenance in the niche[J].Science,307(5710)720-724.
Ni C,Zhang D,Beyer L A,et al.2013.Hearing dysfunction in heterozygous MitfMi-wh/+mice,a model for Waardenburg syndrome type 2 and Tietz syndrome[J].Pigment Cel&Melanoma Research,26(1):78-87.
Otreba M,Rok J,Buszman E,Wrzesniok D.2012.Regulation of melanogenesis:The role of c AMP and MITF[J].Postepy HigienyⅠMedycyny Do?wiadczalnej,66(855199):33-40.
Ozeki H,Ito S,Wakamatsu K,Thody A J.1996.Spectrophotometric characterization of eumelanin and pheomelanin in hair[J].Pigment Cell Research,9(5):265-270.
Pfaffl M W.2001.A new mathematical model for relative quantification in realtime RT-PCR[J].Nucleic Acids Research,29(9):2004-2007.
Shi Y F,Li X Z,Ju D,et al.2016.A novel mutation of the MITF gene in a family with Waardenburg syndrome type 2:A case report[J].Experimental&Therapeutic Medicine,11(4):1516-1518.
Sultana H,Seo D,Choi NR,et al.2018.Identification of polymorphisms in MITF and DCT genes and their associations with plumage colors in Asian duck breeds[J].Asian-Australasian Journal of Animal-Sciences,31(2):180-188.
Takeda K,Hozumi H,Nakai K,et al.2014.Insertion of long interspersed element-1 in the Mitf gene is associated with altered neurobehavior of the black-eyed white Mitf(mi-bw)mouse[J].Genes Cells,19(2):126-140.
Vachtenheim J,Borovansky J.2010.'Transcription physiology'of pigment formation in melanocytes:Central role of MITF[J].Experimental Dermatology,19(7):617-627.
WickelgrenⅠ.2007.Why I have red hair,need to avoid the sun,and shouldn't commit a crime[J].Science,315(5816):1215.
Wan P,Hu Y Q,He L.2011.Regulation of melanocyte pivotal transcription factor MITF by some other transcription factors[J].Molecular and Cellular Biochemistry,354(1-2):241-246.
Wang Y,Li S M,Huang J,et al.2014.Mutation of TYR and MITF genes are associated with plumage color phenotypes in geese[J].Asian-Australasian Journal of AnimalSciences,27(6):778-783.
Yan X K,Zhang T Y,Wang Z M,et al.2011.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,38(12):585-591.
Zhu Z,He J,Jia X,et al.2010.MicroRNA-25 functions in regulation of pigmentation by targeting the transcription factor MITF in alpaca(Lama pacos)skin melanocytes[J].Domestic Animal Endocrinology,38(3):200-209.
韩吉龙,岳耀敬,裴杰,等.2013.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Agout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,21(3):338-347.(Han J LYue Y J,Pei J,et al.2013.The polymorphism of agout signaling protein gene(Agouti)and the expression of Agouti and microphthalmia-associated transcription factor gene MITF in different coat color skin tissues of Tibetan sheep(Ovis aries)[J].Journal of Agricultural Biotechnology,21(3):338-347.)
张丽英,李祥龙,周荣艳,等.2011.不同物种Mitf基因编码区生物信息学分析[J].安徽农业科学,39(22):13298-13300,13308.(Zhang L Y,Li X L,Zhou R Y,,et al.2011.Bioinformatics analysis on the coding region of Mitf gene among different species[J].Journal of Anhui Agricultural Sciences,39(22):13298-13300,13308.)
郑嫩珠,辛清武,朱志明,等.2015.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,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,48(18):3711-3718.)
张瑞娜,范瑞文,程志学,等.2011.CDK5对羊驼皮肤黑色素细胞TYR和MITF mRNA表达的调节[J].畜牧兽医学报,42(12):1712-1717.(Zhang R N,Fan R W,Cheng Z X,et al.2011.Regulation of TYR and MITF mRNAexpression by CDK5 in Alpaca melanocytes[J].Chinese Journal of Animal and Veterinary Sciences,42(12):1712-1717.)
Bauer G L,Praetorius C,Bergsteinsdottir K,et al.2009.The role of MITF phosphorylation sites during coat color and eye development in mice analyzed by bacterial artificial chromosome transgene rescue[J].Genetics,183(2):581-594.
Dorazio J A,Nobuhisa T,Cui R,et al.2006.Topical drug rescue strategy and skin protection based on the role of Mc1r in UV induced tanning[J].Nature,443(7109):340-344.
Dong C S,Wang H D,Xue L L,et al.2012.Coat color determination by miR-137 mediated down-regulation of microphthalmia-associated transcription factor in a mouse mode[J].RNA,18(9):1679-1686.
Goding C R.2011.A picture of Mitf in melanoma immortality[J].Oncogene,30(20):2304-2306.
Hauswirth R,Haase B,Blatter M,et al.2012.Mutations in MITF and PAX3 cause"Splashed White"and other white spotting phenotypes in horses[J].PLoS Genetics,8(4):1-9.
Ito S,Nakanishi Y,Valenzuela R K,et al.2011.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 Research,24(4):605-613.
Kingo K,Aunin E M,Ratsep R,et al.2008.Expressional changes in the intracellular melanogenesis pathways and their possible role in the pathogenesis of vitiligo[J]Journal of Dermatological Science,52(1):39-46.
Lin J Y,Fisher D E.2007.Melanocyte biology and skin pigmentation[J].Nature,445(7130):843-850.
Lee H D,Lee W H,Roh E,et al.2011.Manassantin A inhibits cAMP induced melanin production by down-regulating the gene expressions of MITF and tyrosinase in melanocytes[J].Experimental Dermatology,20(9):761-763.
Levy C,Fisher D E.2011.Dual roles of lineage restricted transcription factors:The case of MITF in melanocytes[J].Transcription,2(1):19-22.
Levy C,Khaled M,Fisher D E.2006.MITF:master regulator of melanocyte development and melanoma oncogene[J]Trends in Molecular Medicine,12(9):406-414.
Loercher A E,Tank E M,Delston R B,Harbour J W.2005MITF links differentiation with cell cycle arrest in melanocytes by transcriptional activation of INK4A[J].Cel Biology,168(1):35-40.
Li S J,Wang C,Yu W H,et al.2012.Identification of genes related to white and black plumage formation by RNA-Seq from white and black feather bulbs in ducks[J]PLoS One,7(5):e36592.
Minvielle F,Bedhom B,Coville J L,et al.2010.The'silver Japanese quail and the MITF gene:Causal mutation,associated traits and homology with the'blue'chicken plumage[J].BMC Genetics,11(15):1-7.
Nishimura E K,Granter S R,Fisher D E.2005.Mechanisms of hair graying:Incomplete melanocyte stem cell maintenance in the niche[J].Science,307(5710)720-724.
Ni C,Zhang D,Beyer L A,et al.2013.Hearing dysfunction in heterozygous MitfMi-wh/+mice,a model for Waardenburg syndrome type 2 and Tietz syndrome[J].Pigment Cel&Melanoma Research,26(1):78-87.
Otreba M,Rok J,Buszman E,Wrzesniok D.2012.Regulation of melanogenesis:The role of c AMP and MITF[J].Postepy HigienyⅠMedycyny Do?wiadczalnej,66(855199):33-40.
Ozeki H,Ito S,Wakamatsu K,Thody A J.1996.Spectrophotometric characterization of eumelanin and pheomelanin in hair[J].Pigment Cell Research,9(5):265-270.
Pfaffl M W.2001.A new mathematical model for relative quantification in realtime RT-PCR[J].Nucleic Acids Research,29(9):2004-2007.
Shi Y F,Li X Z,Ju D,et al.2016.A novel mutation of the MITF gene in a family with Waardenburg syndrome type 2:A case report[J].Experimental&Therapeutic Medicine,11(4):1516-1518.
Sultana H,Seo D,Choi NR,et al.2018.Identification of polymorphisms in MITF and DCT genes and their associations with plumage colors in Asian duck breeds[J].Asian-Australasian Journal of Animal-Sciences,31(2):180-188.
Takeda K,Hozumi H,Nakai K,et al.2014.Insertion of long interspersed element-1 in the Mitf gene is associated with altered neurobehavior of the black-eyed white Mitf(mi-bw)mouse[J].Genes Cells,19(2):126-140.
Vachtenheim J,Borovansky J.2010.'Transcription physiology'of pigment formation in melanocytes:Central role of MITF[J].Experimental Dermatology,19(7):617-627.
WickelgrenⅠ.2007.Why I have red hair,need to avoid the sun,and shouldn't commit a crime[J].Science,315(5816):1215.
Wan P,Hu Y Q,He L.2011.Regulation of melanocyte pivotal transcription factor MITF by some other transcription factors[J].Molecular and Cellular Biochemistry,354(1-2):241-246.
Wang Y,Li S M,Huang J,et al.2014.Mutation of TYR and MITF genes are associated with plumage color phenotypes in geese[J].Asian-Australasian Journal of AnimalSciences,27(6):778-783.
Yan X K,Zhang T Y,Wang Z M,et al.2011.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,38(12):585-591.
Zhu Z,He J,Jia X,et al.2010.MicroRNA-25 functions in regulation of pigmentation by targeting the transcription factor MITF in alpaca(Lama pacos)skin melanocytes[J].Domestic Animal Endocrinology,38(3):200-209.