Abstract：Abstract The premelanosome protein gene (PMEL) can directly initiate the formation of fibers in the melanin small body and promote the melanosome synthesis, which is one of the main candidate genes to regulate animal coat color. At present, there are relatively few researches on the differentially expressed genes of the coat color of the Vulpes lagopus. This research was designed to study the activity region and transcription factors of PMEL gene in Vulpes lagopus. The gene promoter sequence of Vulpes lagopus PMEL gene was obtained through transcriptome sequencing technology, and the method of bioinformatics was used to predict the core promoter region of PMEL gene and the transcription factor binding site. Using Vulpes lagopus DNA as template, promoter deletion fragments of different lengths of the gene were amplified and ligated to pGL3-Basic vector. The recombinant plasmid was transiently transfected into A375 and 293T cells, and the activity was verified by the dual-luciferase assays system. The results showed that 8 fragments with different lengths of promoter regions were amplified and cloned into the vector pGL3-Basic.The region -1 162/+8 of PMEL gene in Vulpes lagopus was detected by dual-luciferase assays system as the core promoter region. There were some positive regulatory elements in the region from -1 162/-655. The bioinformatics prediction analysis revealed that there were 5 transcription factor binding sites in the region. Using the overlap extension PCR technology successfully constructed 5 mutation vectors. Their activity was significantly decreased by dual luciferase assay. It was suggested that these 5 transcription factors were the positive regulatory elements in Vulpes lagopus PMEL gene transcription. In this study, the core region of PMEL gene promoter were identified that 1 162/+8. Sp1 (-966/-952), Sp1 (-912/-900), Sp1 (-750/-736), NF-1 (-712/-699) and NF-1 (-682/-673) binding sites were the positive regulatory elements of Vulpes lagopus PMEL gene.This study provides a scientific basis for exploring the gene expression regulation mechanism, and provides ideas for Vulpes lagopus fur quality breeding and creating new materials.
