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.