The Impact of Mutant EDNRB on the Two-End Black Coat Color Phenotype in Chinese Local Pigs.

Endothelin Receptor Type B (EDNRB) is expressed in a variety of cells during embryonic stage, including melanocyte precursors cells. Our previous studies found that 11 bp deletion of EDNRB caused the two-end black (TEB) coat color in Chinese pigs. In this study, we aimed to explore the mutant EDNRB on the formation of TEB coat color in Chinese pigs. We constructed recombinant plasmid for wild and mutant EDNRB and EDN1, respectively, and transfected the recombinant plasmid into mouse B16 melanoma cells in groups. Real-time fluorescent quantitative PCR (RT-qPCR) was performed to detect expression of genes that participate in melanin pathway, including PLCγ, Raf, MITF. Comparing to the wild-type EDNRB cells, expression of the three genes in the cell line expressing mutant EDNRB cells was significantly reduced. We measured the melanin content produced by transfected recombinant granulocytes of wild and mutant EDNRB and found that the amount of melanin in mutant EDNRB cells was significantly lower than that of the wild. Wound-healing assay confirmed that the migration and mobility rate of mutant EDNRB cells were significantly lower than the wild. Co-immunoprecipitation further confirmed that mutant EDNRB could not interact with the EDN1 protein. In conclusion, this study revealed that the 11 bp deletion of EDNRB reduced the melanin production, which may be caused by inhibiting the expression of PLCγ, Raf, and MITF. The mutant EDNRB reduced melanocyte migration and could not interact with the EDN1 protein. We explored the effect of mutant EDNRB in Chinese pigs with TEB coat color, and the results provided a reference for exploring molecular mechanism of mutant EDNRB on the formation of TEB coat color pigs.