黄河鲤酪氨酸酶基因的克隆、表达模式及定位分析

为探究Tyrosinase(Tyr)基因在黄河鲤体色形成中的作用,利用生物显微镜对黄河鲤黑色鳞片、银色鳞片、鳍条色素细胞的组成进行观察,并利用RACE克隆黄河鲤Tyr基因全长cDNA序列,荧光定量PCR分析其在不同组织中的表达情况,Western blot印迹和免疫组织化学方法检测其蛋白的表达定位。结果显示,黄河鲤具有黑色素细胞、黄色素细胞和虹彩细胞3种色素细胞,3种色素细胞的数量、种类和分布在不同组织中存在较大差异。黄河鲤Tyr基因cDNA全长1717 bp(GenBank ID:No.KY305667),其中ORF长1605 bp,编码535个氨基酸,5′-UTR区41 bp,3′-UTR区71 bp。蛋白同源分析发现,黄河鲤Tyr与鲤科鱼类相似性最高,与哺乳类及鸟类等脊椎动物相似性最低。系统进化分析显示,黄河鲤Tyr与鲤、瓯江彩鲤、锦鲤和斑马鱼等鲤科鱼类的亲缘关系最近。实时定量PCR分析表明,Tyr基因在黄河鲤不同组织中均有表达,肌肉中的表达量最高,其次是黑色皮肤,另外在黄色皮肤、臀鳍和尾鳍等黑色素细胞存在的组织中也有较高表达。Western blot印迹结果显示,Tyr基因在黑色皮肤中表达最高,其次是臀鳍和尾鳍,银色皮肤中没有表达。免疫组织化学结果显示,Tyr基因在黑色皮肤的黑色素细胞和黏液细胞中有表达。研究表明,Tyr基因表达水平与黑色素细胞数量和分布具有一定相关性,参与黄河鲤体色的形成。

Molecular cloning, expression pattern and location of Tyrosinase gene in Yellow River carp (Cyprinus carpio haematopterus)

Tyrosinase (Tyr) is the essential rate-limiting enzyme in melanogenesis. To study the relationship between Tyr gene and the formation of body color in Cyprinus carpio haematopterus, the black scales, silvery scales and fins were observed using microscope. Besides, we also obtained the full-length cDNA sequence of Tyr by rapid-amplification of cDNA ends (RACE) and examined the mRNA and protein expression levels of Tyr in different tissues using quantitative real-time PCR (qRT-PCR), Western blotting and immunohistochemical methods. The results showed that there were 3 kinds of pigment cells including melanophores, xanthophores and iridophores in different tissues, and the quantity, combination, and density of the chromatophores are varied. The full length cDNA of Tyr was 1717 bp (Genbank ID: No. KY305667), including 41bp at 5''-UTR, 71 bp at 3''-UTR, and a 1605 bp open reading frame encoded a peptide of 535 amino acids. Sequence analysis exhibited that Tyr from Yellow River carp had the highest amino acid similarity with Cyprinidae, while it had low similarity with mammalian and bird. And phylogenetic tree analysis indicated Yellow River carp has close relationship with Cyprinidae species like Cyprinus carpio, Cyprinus carpio Koi, Cyprinus carpio color and Danio rerio. qRT-PCR results revealed that Tyr mRNA was checked in all different tissues of Yellow River carp, among which the maximum level was detected in muscle, followed by black skin. In addition, Tyr also displayed a higher expression in the yellow skin, anal fin and caudal fin with abundant melanophores. But Tyr was not detected in the silvery skin lacking melanophores. The protein expression of Tyr in black skin, anal fin, caudal fin and silvery skin exhibited similar trend with Tyr mRNA. The immunohistochemical positive signals were detected in the melanophores and mucous cells of black skin. In conclusion, the expression level of Tyr is relevant to the abundance and location of melanophores, and could be associated with the body color formation of the Yellow River carp.