Supplementary file 3_MAPK pathway-mediated regulation of body coloration in the leopard coral grouper (Plectropomus leopardus): functional roles of jnk1 and p38.docx

Plectropomus leopardus is a marine fish species with high economic value, and its market price is related to its body coloration. During artificial aquaculture, as the farming scale expands, environmental fluctuations often induce stress responses in P. leopardus, leading to gradual darkening or blackening of body coloration. This discoloration reduces the species’ commercial value and has become a major factor limiting the sustainable and rapid development of the P. leopardus aquaculture industry. This study used molecular cloning, RNA interference (RNAi), yeast two-hybrid assays, and co-immunoprecipitation experiments to validate the effects of jnk1 and p38 genes on body coloration. The dsRNA was successfully synthesized, and the optimal injection dosage and interference duration were determined, providing technical support for subsequent functional studies of the jnk1 and p38 genes. Yeast two-hybrid assays revealed that the jnk1 and mitf proteins could be co-expressed in the yeast system, whereas no expression was detected between p38 and mitf in yeast. Furthermore, co-immunoprecipitation (Co-IP) analysis confirmed the interaction between jnk1 and mitf proteins, as Western blot detection following immunoprecipitation revealed clear signals for both jnk1 and mitf in the IP group. Following RNAi of jnk1 and p38, the expression levels of melanogenesis-related genes, including microphthalmia-associated transcription factor (mitf), dopachrome tautomerase (dct), melanocortin 1 receptor (mc1r), and tyrosinase (tyr), were significantly downregulated. Among these, dct showed the most pronounced decrease, with an 80% reduction following jnk1-dsRNA injection and a 50% reduction after p38-dsRNA injection. Both yeast two-hybrid and co-immunoprecipitation results showed that jnk1 interacted with mitf, suggesting a direct involvement of jnk1 in the regulation of body coloration in P. leopardus. A direct interaction was identified between jnk1 and mitf, and the binding of jnk1 to mitf was found to modulate mitf activity, thereby regulating the transcription of downstream melanogenesis-related genes and influencing the melanin biosynthesis process in P. leopardus. This study not only enriches the theoretical understanding of body color variation in P. leopardus but also provides a valuable reference for further investigating the functional roles and potential applications of jnk1 and p38 in the regulation of melanogenesis.