橘色双冠丽鱼体色相关基因mc1r的组织表达分析
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摘要
黑素皮质素受体1基因(mc1r)是动物体色形成的关键调控因子,为探讨mc1r基因在橘色双冠丽鱼(Amphilophus citrinellus)体色褪黑过程中的调控作用,本研究利用cDNA末端快速克隆(RACE)技术首次获得橘色双冠丽鱼mc1r的cDNA全序列,并利用荧光定量PCR分析了橘色双冠丽鱼体色变化不同时期和不同组织中mc1r基因的表达差异。获得cDNA全长序列1 699 bp,共编码氨基酸325个,开放阅读框(ORF)为978 bp,5′非编码区(UTR)497 bp,3′非编码区(UTR)224 bp。氨基酸序列和系统发育分析表明,橘色双冠丽鱼与人(Homo sapiens)、斑马鱼(Danion rerio)、大黄鱼(Larimichthys crocea)和尼罗罗非鱼(Oreochromis niloticus)相似度分别为55.1%、77.1%、90.15%和96.92%。荧光定量PCR结果表明,mc1r在橘色双冠丽鱼胚胎发育9个时期均有不同程度的表达,且随着胚胎发育表达量逐渐减少,推测在胚胎发育的早期阶段该基因的基础表达水平即可启动参与体色细胞形成的腺苷酸循环。在"黑色-灰白色-黄色"三个体色蜕变期,mc1r在尾鳍、鳞片、皮肤的表达均呈先降低再稍升高的变化趋势,均在灰白色过渡期呈现最低值,推测这与mc1r和Agouti存在竞争性结合,及mc1r、tyr、tyr1三者是否处于适当、平衡状态有关。橘色双冠丽鱼长至近成鱼期,非正常褪黑鱼(即未完全褪黑)各组织mc1r表达量均比正常褪黑鱼(完全褪黑)高,其中皮肤组织的相对表达量显著高于正常褪黑鱼(P <0.05),可见鱼体体表褪黑程度与mc1r表达量呈负相关。在成熟鱼体11个组织中mc1r均有不同程度的表达,其中,鳞片显著高于其他组织(P <0.05),说明橘色双冠丽鱼mc1r的主要表达部位是鳞片。本研究通过了解体色变异相关基因表达特征,为鱼类体色遗传和体色改良研究积累资料。
Melanocortin receptor 1 gene(mc1r) is a crucial regulatory factor for body color formation in animals. In order to explore the role of mc1 r gene during body color variation in Amphilophus citrinellus, mc1rfull-length cDNA was obtained for the first time by rapid-amplification of cDNA ends(RACE) technique. The total length of mc1 r was 1 699 bp containing 497 bp 5′ untranslated region(UTR), 224 bp 3′-UTR, and 978 bp open reading frame(ORF) encoding 325 amino acids(Fig. 1). Sequence analysis of amino acids and phylogenetic analysis indicated that the similarity of mc1 r encoded protein sequence of A. citrinellus showed 55.1%, 77.1%, 90.15% and 96.92% similarity with that of Homo sapiens, Danion rerio, Larimichthys croceaand Oreochromis niloticus, respectively(Fig. 4). Analysis revealed that mc1 r was expressed at different levels in all 9 embryonic periods. With the development of embryo, the expression level of mc1 r decreased gradually(Fig. 7). The results suggested that the basic expression level of mc1 r initiated adenylate cycle in early stage of embryonic development, then involved in the formation of pigment cells. In three periods of body color changes(black to hoar to yellow), the expression of mc1 r was decreased first and then increased slightly in caudal fin, scales and skin and its expression was the lowest in the gray white transition stage(Fig. 8). It was presumed that there might be a competitive combination between mc1 r and Agouti, and that the color might be related to appropriate balance among three genes, mc1 r, tyr, and tyr1 in the process of body color changes of A. citrinellus. When the fish grew to near maturity, individuals with incomplete black-fading showed higher mc1 r expression compared to those with complete black-fading(P < 0.05, Fig. 9), and surface melatonin was negatively correlated with the mc1 r expression. mc1 r gene was expressed in all 11 tissues, and its expression in scale was significantly higher than in other tissues(P < 0.05, Fig. 10). The present study revelaed expression features of genes related to color variation, and the data are important for further studying fish body color heredity and body color improvement.
Melanocortin receptor 1 gene(mc1r) is a crucial regulatory factor for body color formation in animals. In order to explore the role of mc1 r gene during body color variation in Amphilophus citrinellus, mc1rfull-length cDNA was obtained for the first time by rapid-amplification of cDNA ends(RACE) technique. The total length of mc1 r was 1 699 bp containing 497 bp 5′ untranslated region(UTR), 224 bp 3′-UTR, and 978 bp open reading frame(ORF) encoding 325 amino acids(Fig. 1). Sequence analysis of amino acids and phylogenetic analysis indicated that the similarity of mc1 r encoded protein sequence of A. citrinellus showed 55.1%, 77.1%, 90.15% and 96.92% similarity with that of Homo sapiens, Danion rerio, Larimichthys croceaand Oreochromis niloticus, respectively(Fig. 4). Analysis revealed that mc1 r was expressed at different levels in all 9 embryonic periods. With the development of embryo, the expression level of mc1 r decreased gradually(Fig. 7). The results suggested that the basic expression level of mc1 r initiated adenylate cycle in early stage of embryonic development, then involved in the formation of pigment cells. In three periods of body color changes(black to hoar to yellow), the expression of mc1 r was decreased first and then increased slightly in caudal fin, scales and skin and its expression was the lowest in the gray white transition stage(Fig. 8). It was presumed that there might be a competitive combination between mc1 r and Agouti, and that the color might be related to appropriate balance among three genes, mc1 r, tyr, and tyr1 in the process of body color changes of A. citrinellus. When the fish grew to near maturity, individuals with incomplete black-fading showed higher mc1 r expression compared to those with complete black-fading(P < 0.05, Fig. 9), and surface melatonin was negatively correlated with the mc1 r expression. mc1 r gene was expressed in all 11 tissues, and its expression in scale was significantly higher than in other tissues(P < 0.05, Fig. 10). The present study revelaed expression features of genes related to color variation, and the data are important for further studying fish body color heredity and body color improvement.
引文
Adalsteinsson S,Hersteinsson P,Gunnarsson E.1987.Fox colors in relation to colors in mice and sheep.Journal of Heredity,78(4):235-237.
Dickman M C,Schliwa M,Barlow G W.1988.Melanophore death and disappearance produces color metamorphosis in the polychromatic Midas cichlid(Cichlasoma citrinellum).Cell&Tissue Research,253(1):9-14.
Djurdjevi?I,Kreft M E,Su?nik B S.2015.Comparison of pigment cell ultrastructure and organisation in the dermis of marble trout and brown trout,and first description of erythrophore ultrastructure in salmonids.Journal of Anatomy,227(5):583-595.
Fitzgerald L,Fryer J,Dwyer T,et al.2006.Effect of melanotan1,[Nle4,D-Phe7]-a-MSH,on melanin synthesis in humans with MC1R variant alleles.Peptides,27(2):388-394.
Hoekstra H E.2006.Genetics,development and evolution of adaptive pigmentation in vertebrates.Heredity,97(3):222-234.
Kim K S,Mendez E A,Marklund S,et al.2000.Rapid communication:linkage mapping of the porcine Agouti gene.Journal of Animal Science,78(5):1395.
Klovins J,Haitina T,Fridmanis D,et al.2004.The melanocortin system in fugu:determination of pomc/agrp/mcr gene repertoire and synteny,as well as pharmacology and anatomical distribution of the MCRs.Molecular Biology&Evolution,21(3):563-579.
Robbins l S.2007.Melanocyte biology and skin pigmentation.Nature,445(7130):843.
Selz Y,Braasch I,Hoffmann C,et al.2007.Evolution of melanocortin receptors in teleost fish:The melanocortin type 1 receptor.Gene,401(1/2):114-122.
郭忠宝,仇雪梅,白杨,等.2009.大菱鲆MC1R基因克隆与序列分析.生物技术通报,(7):109-112.
胡建尊,李康乐,项松平,等.2013.瓯江彩鲤体色调控相关因子MC1R的克隆与表达分析.上海海洋大学学报,22(4):518-523.
蒋燕玲.2016.橘色双冠丽鱼体色发育变化及体色相关基因TYR的克隆与表达研究.上海:上海海洋大学硕士学位论文.
蒋燕玲,宋红梅,刘奕,等.2016.橘色双冠丽鱼TYR基因的克隆及其发育时序和组织表达分析.农业生物技术学报,24(5):697-707.
林永丰.2010.血鹦鹉色素生成及调控机制之研究.基隆:台湾海洋大学硕士学位论文.
牟春艳,郑曙明,任胜杰,等.2015.不同体色血鹦鹉鱼的色素细胞种类、数量及色素含量.水产科学,34(8):497-501.
聂庆华,刘清神,方梅霞,等.2008.犬MC1R基因的分子进化分析.遗传,30(4):469-474.
任玉红,杨刚,范瑞文,等.2012.黑素皮质素受体1(MC1R)在不同毛色羊驼皮肤组织中表达与定位研究.畜牧兽医学报,43(7):1049-1055.
孙静.2012.红褐色突变貉毛色基因MC1R的序列分析及其表达水平的研究.青岛:青岛农业大学硕士学位论文.
韦敏侠,宋红梅,祁宝伦,等.2015.橘色双冠丽鱼胚后色素细胞发育与体色变化.上海海洋大学学报,24(1):28-35.
杨永升,李宁,邓学梅,等.2004.黑素皮质素受体1哺乳动物黑色素形成中的关键基因.遗传,26(4):544-550.
于云柱,何奕多,刘丽,等.2010.黑素皮质素受体1(MC1R)基因的研究进展.中国畜牧兽医,37(6):232-233.
Dickman M C,Schliwa M,Barlow G W.1988.Melanophore death and disappearance produces color metamorphosis in the polychromatic Midas cichlid(Cichlasoma citrinellum).Cell&Tissue Research,253(1):9-14.
Djurdjevi?I,Kreft M E,Su?nik B S.2015.Comparison of pigment cell ultrastructure and organisation in the dermis of marble trout and brown trout,and first description of erythrophore ultrastructure in salmonids.Journal of Anatomy,227(5):583-595.
Fitzgerald L,Fryer J,Dwyer T,et al.2006.Effect of melanotan1,[Nle4,D-Phe7]-a-MSH,on melanin synthesis in humans with MC1R variant alleles.Peptides,27(2):388-394.
Hoekstra H E.2006.Genetics,development and evolution of adaptive pigmentation in vertebrates.Heredity,97(3):222-234.
Kim K S,Mendez E A,Marklund S,et al.2000.Rapid communication:linkage mapping of the porcine Agouti gene.Journal of Animal Science,78(5):1395.
Klovins J,Haitina T,Fridmanis D,et al.2004.The melanocortin system in fugu:determination of pomc/agrp/mcr gene repertoire and synteny,as well as pharmacology and anatomical distribution of the MCRs.Molecular Biology&Evolution,21(3):563-579.
Robbins l S.2007.Melanocyte biology and skin pigmentation.Nature,445(7130):843.
Selz Y,Braasch I,Hoffmann C,et al.2007.Evolution of melanocortin receptors in teleost fish:The melanocortin type 1 receptor.Gene,401(1/2):114-122.
郭忠宝,仇雪梅,白杨,等.2009.大菱鲆MC1R基因克隆与序列分析.生物技术通报,(7):109-112.
胡建尊,李康乐,项松平,等.2013.瓯江彩鲤体色调控相关因子MC1R的克隆与表达分析.上海海洋大学学报,22(4):518-523.
蒋燕玲.2016.橘色双冠丽鱼体色发育变化及体色相关基因TYR的克隆与表达研究.上海:上海海洋大学硕士学位论文.
蒋燕玲,宋红梅,刘奕,等.2016.橘色双冠丽鱼TYR基因的克隆及其发育时序和组织表达分析.农业生物技术学报,24(5):697-707.
林永丰.2010.血鹦鹉色素生成及调控机制之研究.基隆:台湾海洋大学硕士学位论文.
牟春艳,郑曙明,任胜杰,等.2015.不同体色血鹦鹉鱼的色素细胞种类、数量及色素含量.水产科学,34(8):497-501.
聂庆华,刘清神,方梅霞,等.2008.犬MC1R基因的分子进化分析.遗传,30(4):469-474.
任玉红,杨刚,范瑞文,等.2012.黑素皮质素受体1(MC1R)在不同毛色羊驼皮肤组织中表达与定位研究.畜牧兽医学报,43(7):1049-1055.
孙静.2012.红褐色突变貉毛色基因MC1R的序列分析及其表达水平的研究.青岛:青岛农业大学硕士学位论文.
韦敏侠,宋红梅,祁宝伦,等.2015.橘色双冠丽鱼胚后色素细胞发育与体色变化.上海海洋大学学报,24(1):28-35.
杨永升,李宁,邓学梅,等.2004.黑素皮质素受体1哺乳动物黑色素形成中的关键基因.遗传,26(4):544-550.
于云柱,何奕多,刘丽,等.2010.黑素皮质素受体1(MC1R)基因的研究进展.中国畜牧兽医,37(6):232-233.