酪氨酸酶(TYR)、小眼畸形相关转录因子(MITF)和刺鼠信号蛋白(ASIP)基因对白绒乌骨鸡黑色素沉积的遗传效应
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摘要
为研究白绒乌骨鸡(Gallus gallus domesticus Brisson)黑色素沉积分子机制,本研究采用实时荧光定量PCR(quantitative Real-time PCR,q RT-PCR)的2-ΔΔCt检测酪氨酸酶(tyrosinase,TYR)、刺鼠信号蛋白(agouti signaling protein,ASIP)和小眼畸形相关转录因子(microphthalmia-associated transcription factor,MITF)基因在乌骨鸡皮肤、肌肉、肝脏、肾脏和肌胃的表达情况,采用紫外分光光度法测定不同组织黑色素含量,探索3个基因在不同组织中m RNA表达量与黑色素沉积之间的关系。结果表明,TYR、MITF和ASIP在所有组织中均有表达,且各基因m RNA相对表达量在各组织间差异极显著(P<0.01),其中MITF和TYR m RNA表达量依次为皮肤>肾脏>肌胃>肝脏>肌肉,与乌骨鸡各组织黑色素沉积规律基本一致;ASIP基因表达量则与其相反,表现为肌肉>肝脏>肌胃>肾脏>皮肤。相关性分析表明,TYR和MITF基因在各个组织中的表达量与其黑色素含量呈显著正相关(P<0.05),ASIP基因表达量与其呈显著负相关(P<0.05)。由此提示,TYR、MITF和ASIP与白绒乌骨鸡黑色素沉积均具有一定的相关性,其中TYR和MITF基因的高表达可能促进乌骨鸡黑色素沉积,ASIP则相反。本研究为进一步阐明黑色素沉积的遗传调控机制提供了理论基础。
In order to study the molecular mechanism of melanin deposition in White Silky Fowl(Gallus gallus domesticus Brisson), the quantitative Real-time PCR(q RT-PCR) was used to detect the expression level of tyrosinase(TYR), agouti signaling protein(ASIP) and microphthalmia- associated transcription factor(MITF) genes in the skin, muscle, liver, kidney and gizzard of White Silky Fowl and the ultraviolet spectrophotometry was adopted to measure the melanin content in tissues for exploring the relationship between m RNA expression of the 3 genes and melanin deposition in tissues of White Silky Fowl. Results showed that the m RNA of TYR, MITF and ASIP expressed very significantly differently in all test tissues of White Silky Fowl(P<0.01) with an order of skin>kidney>gizzard>liver>muscle of MITF and TYR, which were consistent with the law of melanin deposition in White Silky Fowl. However, the m RNA expression of ASIP in tissues showed a contrary trend of muscle>liver>gizzard>kidney>skin. Correlation analysis indicated that the m RNA expression of TYR and MITF were significently positively correlated with the melanin content in tissues of White Silky Fowl(P<0.05), while that of ASIP showed significently negative correlation(P<0.05). It was speculated that the expression of TYR, MITF and ASIP had certain correlation with the melanin deposition in White Silky Fowl, respectively. High expression of TYR and MITF might promote melanin deposition while ASIP might inhibit melanin deposition in White Silky Fowl. The results would provide a theoretical basis for furtherly elucidating the genetic regulation mechanism of melanin deposition.
In order to study the molecular mechanism of melanin deposition in White Silky Fowl(Gallus gallus domesticus Brisson), the quantitative Real-time PCR(q RT-PCR) was used to detect the expression level of tyrosinase(TYR), agouti signaling protein(ASIP) and microphthalmia- associated transcription factor(MITF) genes in the skin, muscle, liver, kidney and gizzard of White Silky Fowl and the ultraviolet spectrophotometry was adopted to measure the melanin content in tissues for exploring the relationship between m RNA expression of the 3 genes and melanin deposition in tissues of White Silky Fowl. Results showed that the m RNA of TYR, MITF and ASIP expressed very significantly differently in all test tissues of White Silky Fowl(P<0.01) with an order of skin>kidney>gizzard>liver>muscle of MITF and TYR, which were consistent with the law of melanin deposition in White Silky Fowl. However, the m RNA expression of ASIP in tissues showed a contrary trend of muscle>liver>gizzard>kidney>skin. Correlation analysis indicated that the m RNA expression of TYR and MITF were significently positively correlated with the melanin content in tissues of White Silky Fowl(P<0.05), while that of ASIP showed significently negative correlation(P<0.05). It was speculated that the expression of TYR, MITF and ASIP had certain correlation with the melanin deposition in White Silky Fowl, respectively. High expression of TYR and MITF might promote melanin deposition while ASIP might inhibit melanin deposition in White Silky Fowl. The results would provide a theoretical basis for furtherly elucidating the genetic regulation mechanism of melanin deposition.
引文
安健,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m RNA差异显示[J].畜牧兽医学报,37(5):480-484.(An J,Wang M,Fan B L,et al.2006.m RNA differential display PCR of maduramisin sensitive and resistant eimeria tenella strain[J].Acta Veterinaria et Zootechnica Sinica,37(5):480-484.)
白春雨,高玉花,庞全海.2008.影响动物毛色的基因[J].国外畜牧学(猪与禽),28(5):71-73.(Bai C Y,Gao Y H,Pang Q H.2008.Gene of effecting animal coat color[J].Animal Husbandry Abroad(Pigs and Poultry),28(5):71-73.)
高莉,董常生,赫晓燕,等.2008.羊驼酪氨酸酶基因家族在不同毛色个体中的基因表达水平[J].畜牧兽医学报,39 (7):895-899.(Gao L,Dong C S,He X Y,et al.2008.Gene expression levels of alpaca tyrosinase gene family in individuals of different colors[J].Acta Veterinaria et Zootechnica Sinica,39(7):895-899.)
韩吉龙,岳耀敬,裴杰,等.2013.藏羊刺鼠信号蛋白基N(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N(MITF)表达的定量分析[J].农业生物技术学报,21(3):338-347.(Han J L,YueY J,Pei J,et al.2013.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)[J].Journal of Agricultural Biotechnology,21(3):338-347.)
姜俊兵,赵志军,贺俊平,等.2005.Agouti在不同颜色被毛羊驼皮肤组织中的表达[J].畜牧与兽医,39(4):38-41.(Jiang J B,Zhao Z J,He J P,et al.2005.Expression of Agouti in skin stissues of Alpaca(Lama pacos)with different coat colors[J].Animal Husbandry&Veterinary Medicine,39(4):38-41.)
黎观红,徐海燕,许兰姣,等.2011.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[J].中国农业科学,44(13):2777-2786.(Li G H,Xu H Y,Xu L J,et al.2011.Effects of dietary selenium supplementation on growth performance and melanin content in tissues of Taihe Silky Fowls[J].Scientia Agricultura Sinica,44(13):2777-2786.)
李亮,赵小玲,李琴,等.2010.利用m RNA差异显示技术筛选丝羽乌骨鸡肝脏差异表达序列标签[J].畜牧兽医学报,41(4):387-391.(Li L,Zhao X L,Li Q,et al.2010.Identifying ESTs differential expressed in liver organ of Black-Bone Silky Fowl by using differential displayed PCR[J].Acta Veterinaria et Zootechnica Sinica,41(4):387-391.)
李瑞成,尚永彪,管俊峰.2010.乌骨鸡黑色素及其研究进展[J].肉类研究,(10):54-59.(Li R C,Shang Y B,Guan J F.2010.Research on melanin from Black-bone Silky Fowl[J].Meat Research,(10):54-59.)
刘辉.2008.乌骨鸡黑色素的形成与杂交应用[J].新疆农垦科技,6:32-33.(Liu H.2008.The formation of Silky Fowl melanin and hybrid application[J].Xinjiang Farmland Reclamation Science&Technology,6:32-33.)
伍革民,柳小春,娄义洲,等.2005.差异显示筛选武定乌骨鸡“乌色"性状相关基因[J].遗传学报,32(1):30-37.(Wu G M,Liu X C,Lou Y Z,et al.2005.Screening genes related to‘Black’character in Wu-Ding Chicken by delta diferential display[J].Acta Genetica Sinica,32(1):30-37.)
原昊,张红艳,田勇,等.2011.SYBR GreenⅠ荧光定量PCR法检测朗德鹅填饲前后黑素皮质素受体-4基因(MC4R)的表达[J].农业生物技术学报,19(4):692-697.(Yuan H,Zhang H Y,Tian Y,et al.2011.The melanocortin-4 receptor gene(MC4R)expression detected by SYBR greenⅠReal-time quantitative PCR in Landes geese before and after overfeeding[J].Journal of Agricultural Biotechnology,19(4):692-697.)
郑嫩珠,辛清武,缪中纬,等.2013.黑色素基因在半番鸭及番鸭不同羽色中的差异表达研究[J].福建农业学报,28 (5):427-431.(Zheng N Z,Xing Q W,Miao Z W,et a1.2013.Differential expressions of melanin genes in the Mule and Muscovy duck with different plumage colors[J].Fujian Journal of Agricultural Sciences,28(5):427-431.)
Chang C M,Coville J L,Coquerelle G,et al.2006.Complete association between a retroviral insertion in the tyrosi nase gene and the recessive white mutation in chicken[J].BMC Genomics,7:19.
Commo S,Gaillard O,Thibau T S,et al.2004.Absence of TRP-2 in melanogenic melanocytes of human hair[J].Pigment Cell Research,17(5):488-497.
Fontanesi L,Forestier L,Allain D,et al.2010.Characterization of the rabbit agouti signaling protein(ASIP)gene:Transcripts and phylogenetic analyses and identification of the causative mutation of the nonagouti black coat colour[J].Genomics,95(3):166-175.
Goding C R.2011.A picture of Mitf in melanoma immortality[J].Oncogene,30(20):2304-2306.
Gratten J,Pilkington J G,Brown E A,et al.2010.The genetic basis of recessive self-colour pattem in a wild sheep population[J].Heredity(Edinb),104(2):206-214.
Gutierrez-Gil B,Wiener P,Williams J L.2007.Genetic effects on coat colour in cattle:Dilution of eumelanin and phaeomelanin pigments in an F2-backcross Charolais x Holstein population[J].BMC Genetics,8:56-67.
Hida T,Wakamatsu K,Sviderskaya E V,et al.2009.Agouti protein,mahogunin,and attractin in pheomelanogenesis and melanoblast-like alteration of melanocytes:A c AMP-independent pathway[J].Pigment Cell Melanoma Research,22(5):623-634.
Ito S.2006.Encapsulation of a reactive core in neuromelanin[J].Proceedings of the National Academy of Sciences of the USA,103:14647-14648.
Ito S,Wakamatsu K,Ozeki H.2000.Chemical analysis of melanins and its application to the study of the regulation of melanogenesis[J].Pigment Cell Research,13(s8):103-109.
Jiri V,Jan B.2010.‘Transcription physiology’of pigment formation in melanocytes:Central role of MITF[J].Experimental Dermatology,19(7):617-627.
Kerns J A,Newton J,Berryere T G,et al.2004.Characterization of the dog Agouti gene and a nonagouti mutation in German Shepherd dogs[J].Mammalian Genome,15(10):798-808.
Lamoreux M L,Wakamatsu K,Ito S.2011.Interaction of major coat color gene functions in mice as studied by chemical analysis of eumelanin and pheomelanin[J].Pigment Cell Research,14(1):23-31.
Li S J,Wang C,Yu W H,et al.2012.Identification of genes related to white and black plumage formation by RNASeq from White and Black Feather Bulbs in ducks[J].PLo S ONE,7(5):e36592.
Norris B J,Whan V A.2008.A gene duplication affecting expression of the ovine ASIP gene is responsible for white and black sheep[J].Genome Research,18(8):1282-1293.
Otreba M,Rok J,Buszman E,et al.2012.Regulation of melanogenesis:The role of c AMP and MITF[J].Postepy Hig Med Dosw(Online),66:33-40.
Ozeki H,Ito S,Wakamatsu K,et al.1996.Spectrophotometric characterization of eumelan in and pheomelanin in hair[J].Pigment Cell Research,9(5):265-270.
Sobajima S,Shimer A L,Chadderdon R C,et al.2005.Quantitative analysis of gene expression in a rabbit model of intervertebral disc degeneration by Real-time polymerase chain reaction[J].The Spine Journal,5(1):14-23.
Steingrimsson E,Copeland N G,Jenkins N A.2004.Melancoytes and microphthalmia transcription factor network[J].Annual Review of Genetics,38:365-411.
Tu Y G,Sun Y Z,Tian Y G,et al.2009.Physicochemical characterization and antioxidant activity of melanin from the muscles of Taihe Black-Bone Silky Fowl(Gallus gallus domesticus Brisson)[J].Food Chemistry,114:1345-135.
Voisey J,Carroll L,van Daal A.2003.Melanocortins and their receptors and antagonists[J].Current Drug Targets,4(7):586-597.
Wang Y,Li S M,Huang J,et al.2014.Mutation of TYR and MITF genes are associated with plumage color phenotypess in geese[J].Asian-Australasian Journal of Animal-Sciences(AJAS),27(6):778-783.
Xu Y,Zhang X H,Pang Y Z.2013.Association of tyrosinase(TYR)and Tyrosinase-related Protein 1(TYRP1)with melanic plumage color in Korean Quails(Coturnix coturnix)[J].Asian Australasian Journal of Animal Sciences,26:1518-1522.
Zhu Z,He J,Jia X,et al.2010.Micro RNA-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.
白春雨,高玉花,庞全海.2008.影响动物毛色的基因[J].国外畜牧学(猪与禽),28(5):71-73.(Bai C Y,Gao Y H,Pang Q H.2008.Gene of effecting animal coat color[J].Animal Husbandry Abroad(Pigs and Poultry),28(5):71-73.)
高莉,董常生,赫晓燕,等.2008.羊驼酪氨酸酶基因家族在不同毛色个体中的基因表达水平[J].畜牧兽医学报,39 (7):895-899.(Gao L,Dong C S,He X Y,et al.2008.Gene expression levels of alpaca tyrosinase gene family in individuals of different colors[J].Acta Veterinaria et Zootechnica Sinica,39(7):895-899.)
韩吉龙,岳耀敬,裴杰,等.2013.藏羊刺鼠信号蛋白基N(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N(MITF)表达的定量分析[J].农业生物技术学报,21(3):338-347.(Han J L,YueY J,Pei J,et al.2013.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)[J].Journal of Agricultural Biotechnology,21(3):338-347.)
姜俊兵,赵志军,贺俊平,等.2005.Agouti在不同颜色被毛羊驼皮肤组织中的表达[J].畜牧与兽医,39(4):38-41.(Jiang J B,Zhao Z J,He J P,et al.2005.Expression of Agouti in skin stissues of Alpaca(Lama pacos)with different coat colors[J].Animal Husbandry&Veterinary Medicine,39(4):38-41.)
黎观红,徐海燕,许兰姣,等.2011.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[J].中国农业科学,44(13):2777-2786.(Li G H,Xu H Y,Xu L J,et al.2011.Effects of dietary selenium supplementation on growth performance and melanin content in tissues of Taihe Silky Fowls[J].Scientia Agricultura Sinica,44(13):2777-2786.)
李亮,赵小玲,李琴,等.2010.利用m RNA差异显示技术筛选丝羽乌骨鸡肝脏差异表达序列标签[J].畜牧兽医学报,41(4):387-391.(Li L,Zhao X L,Li Q,et al.2010.Identifying ESTs differential expressed in liver organ of Black-Bone Silky Fowl by using differential displayed PCR[J].Acta Veterinaria et Zootechnica Sinica,41(4):387-391.)
李瑞成,尚永彪,管俊峰.2010.乌骨鸡黑色素及其研究进展[J].肉类研究,(10):54-59.(Li R C,Shang Y B,Guan J F.2010.Research on melanin from Black-bone Silky Fowl[J].Meat Research,(10):54-59.)
刘辉.2008.乌骨鸡黑色素的形成与杂交应用[J].新疆农垦科技,6:32-33.(Liu H.2008.The formation of Silky Fowl melanin and hybrid application[J].Xinjiang Farmland Reclamation Science&Technology,6:32-33.)
伍革民,柳小春,娄义洲,等.2005.差异显示筛选武定乌骨鸡“乌色"性状相关基因[J].遗传学报,32(1):30-37.(Wu G M,Liu X C,Lou Y Z,et al.2005.Screening genes related to‘Black’character in Wu-Ding Chicken by delta diferential display[J].Acta Genetica Sinica,32(1):30-37.)
原昊,张红艳,田勇,等.2011.SYBR GreenⅠ荧光定量PCR法检测朗德鹅填饲前后黑素皮质素受体-4基因(MC4R)的表达[J].农业生物技术学报,19(4):692-697.(Yuan H,Zhang H Y,Tian Y,et al.2011.The melanocortin-4 receptor gene(MC4R)expression detected by SYBR greenⅠReal-time quantitative PCR in Landes geese before and after overfeeding[J].Journal of Agricultural Biotechnology,19(4):692-697.)
郑嫩珠,辛清武,缪中纬,等.2013.黑色素基因在半番鸭及番鸭不同羽色中的差异表达研究[J].福建农业学报,28 (5):427-431.(Zheng N Z,Xing Q W,Miao Z W,et a1.2013.Differential expressions of melanin genes in the Mule and Muscovy duck with different plumage colors[J].Fujian Journal of Agricultural Sciences,28(5):427-431.)
Chang C M,Coville J L,Coquerelle G,et al.2006.Complete association between a retroviral insertion in the tyrosi nase gene and the recessive white mutation in chicken[J].BMC Genomics,7:19.
Commo S,Gaillard O,Thibau T S,et al.2004.Absence of TRP-2 in melanogenic melanocytes of human hair[J].Pigment Cell Research,17(5):488-497.
Fontanesi L,Forestier L,Allain D,et al.2010.Characterization of the rabbit agouti signaling protein(ASIP)gene:Transcripts and phylogenetic analyses and identification of the causative mutation of the nonagouti black coat colour[J].Genomics,95(3):166-175.
Goding C R.2011.A picture of Mitf in melanoma immortality[J].Oncogene,30(20):2304-2306.
Gratten J,Pilkington J G,Brown E A,et al.2010.The genetic basis of recessive self-colour pattem in a wild sheep population[J].Heredity(Edinb),104(2):206-214.
Gutierrez-Gil B,Wiener P,Williams J L.2007.Genetic effects on coat colour in cattle:Dilution of eumelanin and phaeomelanin pigments in an F2-backcross Charolais x Holstein population[J].BMC Genetics,8:56-67.
Hida T,Wakamatsu K,Sviderskaya E V,et al.2009.Agouti protein,mahogunin,and attractin in pheomelanogenesis and melanoblast-like alteration of melanocytes:A c AMP-independent pathway[J].Pigment Cell Melanoma Research,22(5):623-634.
Ito S.2006.Encapsulation of a reactive core in neuromelanin[J].Proceedings of the National Academy of Sciences of the USA,103:14647-14648.
Ito S,Wakamatsu K,Ozeki H.2000.Chemical analysis of melanins and its application to the study of the regulation of melanogenesis[J].Pigment Cell Research,13(s8):103-109.
Jiri V,Jan B.2010.‘Transcription physiology’of pigment formation in melanocytes:Central role of MITF[J].Experimental Dermatology,19(7):617-627.
Kerns J A,Newton J,Berryere T G,et al.2004.Characterization of the dog Agouti gene and a nonagouti mutation in German Shepherd dogs[J].Mammalian Genome,15(10):798-808.
Lamoreux M L,Wakamatsu K,Ito S.2011.Interaction of major coat color gene functions in mice as studied by chemical analysis of eumelanin and pheomelanin[J].Pigment Cell Research,14(1):23-31.
Li S J,Wang C,Yu W H,et al.2012.Identification of genes related to white and black plumage formation by RNASeq from White and Black Feather Bulbs in ducks[J].PLo S ONE,7(5):e36592.
Norris B J,Whan V A.2008.A gene duplication affecting expression of the ovine ASIP gene is responsible for white and black sheep[J].Genome Research,18(8):1282-1293.
Otreba M,Rok J,Buszman E,et al.2012.Regulation of melanogenesis:The role of c AMP and MITF[J].Postepy Hig Med Dosw(Online),66:33-40.
Ozeki H,Ito S,Wakamatsu K,et al.1996.Spectrophotometric characterization of eumelan in and pheomelanin in hair[J].Pigment Cell Research,9(5):265-270.
Sobajima S,Shimer A L,Chadderdon R C,et al.2005.Quantitative analysis of gene expression in a rabbit model of intervertebral disc degeneration by Real-time polymerase chain reaction[J].The Spine Journal,5(1):14-23.
Steingrimsson E,Copeland N G,Jenkins N A.2004.Melancoytes and microphthalmia transcription factor network[J].Annual Review of Genetics,38:365-411.
Tu Y G,Sun Y Z,Tian Y G,et al.2009.Physicochemical characterization and antioxidant activity of melanin from the muscles of Taihe Black-Bone Silky Fowl(Gallus gallus domesticus Brisson)[J].Food Chemistry,114:1345-135.
Voisey J,Carroll L,van Daal A.2003.Melanocortins and their receptors and antagonists[J].Current Drug Targets,4(7):586-597.
Wang Y,Li S M,Huang J,et al.2014.Mutation of TYR and MITF genes are associated with plumage color phenotypess in geese[J].Asian-Australasian Journal of Animal-Sciences(AJAS),27(6):778-783.
Xu Y,Zhang X H,Pang Y Z.2013.Association of tyrosinase(TYR)and Tyrosinase-related Protein 1(TYRP1)with melanic plumage color in Korean Quails(Coturnix coturnix)[J].Asian Australasian Journal of Animal Sciences,26:1518-1522.
Zhu Z,He J,Jia X,et al.2010.Micro RNA-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.