Abstract：Abstract Mink(Mustela vison) coat color, which is an important quality trait of mink, is also an extremely important factor affecting the price. Premelanosome protein gene (PMEL) is an important candidate gene that affects the variation of coat color and has been paid more and more attention in recent years. The aim of this study were to screen the mink coat color gene PMEL promoter active region and transcription factors binding sites, to provide a theoretical basis for elucidating the gene expression and regulation mechanism, and to provide ideas for color mink breeding and improvement. The specific primers were designed based on the domestic ferret (M. putorius furo) PMEL gene sequence (GenBank: NW_004569320.1), which was highly homologous to the mink. The fragment in a 5' flanking region was amplified and cloned into the vector pMD19 vector. The positive colonies were identified and sequenced. Six fragments with different lengths of promoter regions were amplified and cloned into the vector pMD19 vector. The positive colonies and vector pGL3-Basic were simultaneously digested with 2 restriction enzymes Kpn Ⅰ and Hind Ⅲ. The digested mixture were purified and ligated with T4 ligase to get the circular plasmid. The endo-free plamids were isolated after the positive colonies, which were identified by PCR, double enzyme digestion, sequencing. 293T and A375 cells were transiently transfected with lip2000 liposome. The dual-luciferase assay system was used to measure the luciferase activity. Transcription factor binding sites in core promoter region were predicted and verified. The length of the fragment in 5' flanking region of PMEL gene in mink was 1 401 bp. The predicted active region in the promoter, conserved motifs and multiple transcription factor binding sites were involved in the cloned fragment in the 5' flanking region. Six different lengths of fragments were obtained and ligated with luciferase reported vector. When the promoter 5' was truncated, luciferase transcriptional activity firstly increased and then decreased. The core promoter was involved in 5' flanking region of PMEL gene in mink and this region was from -671 bp to +87 bp. The activity of the promote decreased from -671 to -477, which indicated that there were some positive regulatory elements in the region from -671 to -477. The transcription factor binding site were predicted with bioinformatic methods and online program, which showed that there were many transcription factors binding sites in the strong active promoter. Sp1 (-516/-506), Sp1 (-505/-495) and Sp1 (-499/-489) binding sites were obtained based on the prediction of at least 2 softwares, and the 3 Sp1 binding sites were mutated, respectively. The results showed that -516/-506, -505/-495 and -499/-489 were positive transcription regulatory regions. The core promoter region from -671 bp to +87 bp was identified in mink PMEL gene, and 3 predicted Sp1 binding sites were positived regulatory regions and played critical role in regulating the activity of the promoter. The results provide important information for understanding the biological function of PMEL gene in mink and a new theoretical basis for further studying the molecular genetic mechanism of mink's coat color.
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