橘色双冠丽鱼体色相关基因mitf的结构及表达调控特性

Structure and Expression Analysis of Body Color-Related mitf Gene in Amphilophus citrinellus

中文关键词: 橘色双冠丽鱼  小眼畸形相关转录因子(MITF)  qRT-PCR  黑色素细胞

英文关键词: Amphilophus citrinellus  mitf  qRT-PCR  Melanocyte

中文摘要:

      为了解小眼畸形相关转录因子mitf基因在鱼类早期体色褪黑过程中的调控作用，本研究采用cDNA末端快速扩增(RACE)技术获得橘色双冠丽鱼(Amphilophus citrinellus) mitf基因cDNA序列全长，利用实时荧光定量PCR (qRT-PCR)技术检测其在橘色双冠丽鱼胚胎不同发育时期、体色褪黑不同时期和各个组织中的表达规律。获得mitf基因2个亚型，其中，mitf1的cDNA全长为1816 bp，包括5′非编码区(UTR) 158 bp、3′UTR 428 bp、开放阅读框(ORF) 1230 bp，共编码409个氨基酸；mitf2的cDNA全长为1638 bp，包括5´UTR 160 bp、3´UTR 428 bp和ORF 1050 bp，共编码349个氨基酸。同源性和系统进化分析显示，mitf1和mitf2聚在一小支，与慈鲷科(Cichlidae)鱼类同源性最高，与哺乳类动物同源性较低。qRT-PCR结果显示，在成鱼各个组织中，mitf1和mitf2均有不同程度表达，其中，眼部表达量最高且显著高于其他组织(P<0.05)，肌肉、脑和肾脏也有较高表达；mitf1和mitf2在胚胎各个发育时期均有表达，在受精卵时期表达量最高，显著高于其他胚胎期；随着橘色双冠丽鱼体色由黑色过渡到橘黄色，mitf1和mitf2在鱼皮肤、鳞片、尾鳍中的表达均呈逐渐下降的趋势，表明mitf基因表达与鱼体色由黑到黄转变的表型间存在关联性，推测与鱼体色发育阶段色素细胞的分化和分布比例的动态变化相关。本研究通过了解鱼类体色发育和变异的分子基础，可为鱼类色素细胞发育和体色人工改良积累资料。

英文摘要:

      Microphthalmia-associated transcription factor (MITF) is a transcription factor with a typical basic helix-loop-helix-leucine zipper structure. It is a key transcription factor in the melanin synthesis pathway in animals by activating downstream genes to initiate melanin synthesis. In order to explore the regulatory action of mitf during body color variation in Amphilophus citrinellus, the full-length cDNA of the mitf gene was cloned using the rapid amplification of cDNA ends technique, and the difference in gene expression in different developmental stages of embryos, different stages of body melatonin, and various tissues in A. citrinellus were analyzed through qRT-PCR. Two mutant sequences of mitf were obtained. The full-length of mitf1 cDNA was 1816 bp, including a 158 bp 5′UTR, a 428 bp 3′UTR, and a 1230 bp ORF coding a polypeptide of 409 amino acids. The full-length of mitf2 cDNA was 1638 bp, including a 160 bp 5′ UTR, a 428 bp 3′UTR, and a 1050 bp ORF coding a polypeptide of 349 amino acids. The analysis of the homology and phylogenetic tree results showed that mitf1 and mitf2 clustered into a small branch in the phylogenetic tree, which had the highest homology with Cichlidae fish and low homology with mammals. The results of the qRT-PCR showed that mitf1 and mitf2 were expressed to varying degrees in various tissues of adult fish, among which the expression in the eyes was the highest and was significantly higher than in other tissues (P<0.05), and the expression in muscle, brain, and kidney tissue was also higher. The mitf1 and mitf2 genes were expressed in all stages of embryonic development, and the expression level was highest in the cleavage stage, which was significantly higher than the other embryonic stages. With the body color transition from black to orange, the expression of mitf1 and mitf2 genes decreased gradually in the fish skin, scales, and tail fins. It showed that the expression of mitf was positively correlated with changes in body color from black to yellow in A. citrinellus, which may be related to the dynamic change in the differentiation and distribution ratio of pigment cells in the body color development stage of fish. Through understanding the molecular basis of fish body color development and variation, this study accumulates data for fish pigment cell development and the artificial improvement of fish body color.