Pax3 Gene Regulated Melanin Synthesis by Tyrosinase Pathway in Pteria penguin

Abstract

The paired-box 3 (Pax3) is a transcription factor and it plays an important part in melanin synthesis. In this study, a new Pax3 gene was identified from Pteria penguin (Röding, 1798) (P. penguin) by RACE-PCR (rapid-amplification of cDNA ends-polymerase chain reaction) and its effect on melanin synthesis was deliberated by RNA interference (RNAi). The cDNA of PpPax3 was 2250 bp long, containing an open reading fragment of 1365 bp encoding 455 amino acids. Amino acid alignment and phylogenetic tree showed PpPax3 shared the highest (69.2%) identity with Pax3 of Mizuhopecten yessoensis. Tissue expression profile showed that PpPax3 had the highest expression in mantle, a nacre-formation related tissue. The PpPax3 silencing significantly inhibited the expression of PpPax3, PpMitf, PpTyr and PpCdk2, genes involved in Tyr-mediated melanin synthesis, but had no effect on PpCreb2 and an increase effect on PpBcl2. Furthermore, the PpPax3 knockdown obviously decreased the tyrosinase activity, the total content of eumelanin and the proportion of PDCA (pyrrole-2,3-dicarboxylic acid) in eumelanin, consistent with influence of tyrosinase (Tyr) knockdown. These data indicated that PpPax3 played an important regulating role in melanin synthesis by Tyr pathway in P. penguin.

Kojic acid inhibited melanin synthesis by tyrosinase pathway in Pteria penguin

“…Tyrosinase activity assays were performed following the previous reports with minor modification (Choi, Sohn, et al, ; Szekely‐Klepser et al, ; Yu, Qu, et al, ). Briefly, 1 g mantle tissue was homogenized in 1 ml of 0.1 mol/L phosphate‐buffered saline (PBS, pH 6.8) and was centrifuged at 10,000 rpm for 10 min to obtain the supernate.…”

“…The total melanin was isolated from mantle of P. penguin and oxidized following our previous report (Yu, Pan, et al, ; Yu, Qu, et al, ). Briefly, 1 g mantle sample was finely homogenized and incubated in 15 ml PBS (pH 7.4) with 2% (m/V) papain at 55°C for 20 hr.…”

“…Analyses were performed at 40°C at a flow rate of 0.3 ml/min. MS/MS detection was performed using a Xevo TQ triple quadrupole mass spectrometer operated in positive electrospray ionization (ESI) mode similar to Yu, Qu, et al (). The source temperature and desolvation temperature were 150 and 550°C respectively.…”

“…Most pearls produced by P. penguin are bronze, black green or dark blue, which is closely connected with shell colour and nacre colour (Li et al, ; Naganuma et al, ). Because the P. penguin has purely black shell in population, melanin is considered to be the main pigment, especially the brown‐black eumelanin (Szekely‐Klepser et al, ; Yu, Pan, et al, ; Yu, Qu, et al, ). Inhibiting the synthesis of eumelanin will be a practical method to weaken shell colour and nacre colour, and to produce colourful pearl from P. penguin .…”

“…Observations in mammals indicated the anti‐melanogenic activity of kojic acid proceeded by a direct inhibition of cellular tyrosinase activity (Choi et al, ; Gupta et al, ), and only a few reports showed kojic acid could inhibit Mitf and Tyr mRNA (Choi et al, ). In bivalves, our previous studies showed that Mitf (microphthalmia‐associated transcription factor), Tyr (Tyrosinase), Yel (Yellow), Cdk2 (cyclin‐dependent kinase 2) and Bcl2 (B‐cell lymphoma 2) genes were involved in the melanin synthesis pathway, and a Mitf‐Tyr‐Yel axis was considered to be exist in melanin synthesis of P. penguin (Yu, Pan, et al, ; Yu, Qu, et al, ). However, it is doubtful whether kojic acid inhibits melanin synthesis by the Mitf‐Tyr‐Yel axis in bivalves.…”