橘色双冠丽鱼体色相关基因mc1r的组织表达分析

Expression Analysis of mc1r Gene Relating to Body Color Variation in Amphilophus citrinellus

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 mc1r gene during body color variation in Amphilophus citrinellus,mc1r full-length cDNA was obtained for the first time by rapid-amplification of cDNA ends(RACE)technique.The total length of mc1r 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 mc1r 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 crocea and Oreochromis niloticus,respectively(Fig.4).Analysis revealed that mc1r was expressed at different levels in all 9 embryonic periods.With the development of embryo,the expression level of mc1r decreased gradually(Fig.7).The results suggested that the basic expression level of mc1r 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 mc1r 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 mc1r and Agouti,and that the color might be related to appropriate balance among three genes,mc1r,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 mc1r expression compared to those with complete black-fading(P<0.05,Fig.9),and surface melatonin was negatively correlated with the mc1r expression.mc1r 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.