Figure 8.

Electrophoretic mobility shift assay (EMSA) and the predicted model for the process of melanin biosynthesis. (A) EMSA for Vdpf (EGY18187.1) and the promoters of Ac, Pka, Mkk1, Mkh1, Gp1, Gp2 and Gp3. Ac represents the adenylate cyclase‐encoding gene, and Pka represents the cyclic adenosine monophosphate (cAMP)‐dependent protein kinase A (PKA)‐encoding gene; these genes are involved in the cAMP signalling pathway. Mkk1 and Mkh1 represent the mitogen‐activated protein (MAP) kinase kinase MKK1/SSP32 gene and the MAP kinase kinase kinase mkh1 gene, respectively, which play important roles in the MAP kinase signalling pathway. Gp1, Gp2 and Gp3 are genes that encode the G‐protein‐coupled receptor; they participate in the G‐protein‐mediated signalling pathway. The binding assays used 0, 3, 4 or 5 μL of purified Vdpf (EGY18187.1). The red arrow points to the shift bands. (B) Real‐time reverse transcription‐polymerase chain reaction (RT‐PCR) analysis of the signalling genes. The expression level was measured by Quantity One software. (C) Deducible signalling pathways in melanin synthesis. Chk1 and Mps1 are two MAP kinase genes involved in the MAP kinase signalling pathway. They have been demonstrated to be involved in melanin synthesis through the transcription factor Cmr1/Amr1. The purple arrows represent known results, and the black arrows represent results in this research study. The broken lines represent unconfirmed results. Three biological replicates (n = 3) were used for this study.