Characterization and functional analysis of pax3 in body color transition of polychromatic Midas cichlids (Amphilophus citrinellus).

As the representative genetic and economic trait of ornamental fish, skin color has a strong impact on speciation and adaptation. However, the genetic basis of skin color pigmentation, differentiation and change is still not understood. The Midas cichlid fish with three typical body color transition stages of "black-gray‑gold" is an ideal model system for investigating the formation and change of fish body color. In this study, to investigate the regulatory role of the pair box 3 (pax3) gene in the early body color fading process of Midas cichlids, the complete cDNA sequence (3513 bp) of pax3 was successfully isolated from Midas cichlids (Amphilophus Citrinellus), and found to encode polypeptides of 491 amino acids. Expression patterns of the pax3 gene in tissues of Midas cichlids during different periods, including embryonic development and body color fading stages were detected by quantitative real-time PCR. The qRT-PCR analysis showed that pax3 was expressed in all tissues of adult fish, with a higher expression level in muscle and skin. The highest expression level in muscle tissue was significantly higher than that in other tissues (P < 0.05). During embryonic development, the expression tendency of pax3 was first increased and then decreased. In the three typical stages of early skin color fading from black to gold, pax3 expression in skin, caudal fin and scales all showed a downward trend. The expression level in the black stage was significantly higher than that in other stages (P < 0.05). Positive signal of pax3 protein was detected in the three typical skin color conversion stages, and the highest positive signal intensity was detected in the black stage, which was consistent with qRT-PCR results. After pax3 RNA interference, pax3 and the downstream genes mitf and tyr all decreased, while dct mRNA expression increased in the skin of fish. Western blotting also showed a decrease in pax3 protein concentration. Those results suggest that pax3 plays an important role in skin color formation, distribution and change in Midas cichlids through the melanogenesis pathway.
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