Study On Promoter Activity Of Fox Fur Gene TYR,CBD103,and MITF

TYR,CBD103 and MITF gene are the major genes regulating the color of animal hair.The colourful hair not only plays an important role in protecting themselves,avoiding disadvantages,mutual recognition and courtship,but also has an important influence on the production and process of fur products and their economic value.Promoter can determine the initiation of gene transcription and regulate the intensity of gene expression in regulation of gene expression,about which had few reports in fox research.Therefore,bioinformatics,molecular cloning,cell transient transfection,double luciferase reporting system and point mutation technique were used to study transcriptional activities of the promoters in TYR,CBD103 and MITF gene of Arctic fox,which can provide a theoretical basis for the regulation of molecular genetic mechanism of the coat color in Arctic fox,and have great significance for reveal the genetic diversity of the coat color in Arctic fox.Bioinformatics and molecular cloning were used to amplify the 5? flanking region and all encoding region sequences of TYR,CBD103 and MITF gene firstly.Amplified results showed CBD103 gene 2 327 bp containing 5? flanking region 2 071 bp,CDS region 204 bp,and 3? flanking region 52 bp;MITF-M gene 3 880 bp containing 5? flanking region 2 262 bp,CDS region 1 260 bp,and 3? flanking region 358 bp);and TYR gene 4 300 bp containing 5? flanking region 2 681 bp,CDS region 1 593 bp,and 3? flanking region 26 bp.5? flanking sequences of TYR,MITF and CBD103 gene construct dual-luciferase reporter gene expression vectors of 5? deletion promoters in different length,and the plasmids were transfected into 293 T cells and A375 cells.The activity of the promoters of these three genes was detected by a dual luciferase assay system,determining the functional sequence location of each gene promoter and conducting the site-directed mutation of transcription factor binding site to explore the role of transcription factor binding site in the transcriptional regulation of these gene.The results showed that: 1.The highest activity was found in-1 604/+51 region of CBD103 gene.After these three transcription factor binding sites Sp1(-1 552/-1 564),Sp1(-1 439/-1 454)and Sp1(-329/-339)mutated in this region,the promoter activity of mutant decreased obviously compared with the wild type,which suggesting that these three sites are the positive regulatory region of CBD103 gene transcription.2.The highest activity was found in-510/+13 region of MITF-M gene,and-510/-222 was the core transcription region.After the three transcription factor binding sites CREB(-312/-319),Sp1(-295/-307)and Sp1(-249/-262)mutated in this region,the promoter activity of mutant increased significantly compared with the wild type,which speculating that these three sites may be the negative regulatory region of MITF-M gene transcription,or these three sites may be the positive regulatory region of MITF-M gene transcription due to the DNA methylation modification at this site,which resulting the binding of CREB and Sp1 is inhibited,so the transcription activation of the MITF-M gene promoter is inhibited.3.The highest activity was found in-2 033/+32 region of TYR gene.After these three transcription factor binding sites Sp1(-1 775/-1 785),NF-1(-199/-208)and Sp1(-119/-129)mutated in this region,the promoter activity of mutant decreased obviously compared with the wild type,which suggesting that these three sites are the positive regulatory region of TYR gene transcription.In this study,the role of promoter region and transcription factor binding sites of CBD103,MITF-M and TYR gene in the transcriptional regulation of arctic fox were determined.