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

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