Identification of a tyrosinase gene and its functional analysis in melanin synthesis of Pteria penguin.

Tyrosinase is a key rate-limiting enzyme in melanin synthesis. In this study, a new tyrosinase gene (Tyr) was identified from Pteria penguin and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of PpTyr was 1728 bp long, containing a 5'untranslated region (UTR) of 11 bp, a 3'UTR of 295 bp, and an open reading fragment of 1422 bp encoding 473 amino acids. Amino acid alignment showed PpTyr had the highest (50%) identity to tyrosinase-like protein 1 from Pinctada fucata. Phylogenetic tree analysis classified PpTyr into α-subclass of type-3 copper protein. Tissue expression analysis indicated that PpTyr was highly expressed in mantle, a nacre formation related tissue. After PpTyr RNA interference, PpTyr mRNA was significantly inhibited by 71.0% (P < 0.05). For other melanin-related genes, PpCreb2 and PpPax3 expression showed no significant change, but PpBcl2 was obviously increased. By liquid chromatograph-tandem mass spectrometer (LC-MS/MS) analysis, the total content of PDCA (pyrrole-2, 3-dicarboxylic acid) and PTCA (pyrrole-2,3,5-tricarboxylic acid), as main markers for eumelanin, was sharply decreased by 66.6% after PpTyr RNAi (P < 0.05). The percentage of PDCA was also obviously decreased from 20.1% to 13.9%. This indicated that tyrosinase played a key role in melanin synthesis and color formation of P. penguin.