Cloning and Activity Analysis of Transcription Factor FoxO1 Promoter in Dairy Goat (Capra hircus)
HE Qiu-Ya, LUO Jun*, GAO Liang-Jia-Hui, WU Jiao, YAO Wei-Wei
College of Animal Science and Technology/Shaanxi Provincial Key Laboratory of Molecular Biology for Agriculture, Northwest A& F University, Yangling 712100, China
Abstract：Forkhead box protein O1 (FoxO1) is a key transcription factor regulating fatty acid metabolism. Although FoxO1 promoter has been shown to be regulated by upstream transcription factors in non-ruminants, the mechanism of FoxO1 regulation during lactation in dairy goats (Capra hircus) remains unclear. This study designed primers based on the genome sequence of Yunnan black goat and cloned the 5' flanking sequence of FoxO1 gene. Bioinformatic analysis of FoxO1 promoter sequence was performed using online software to predict the binding sites of transcription factors. Double luciferase reporter vectors containing different lengths of FoxO1 promoter fragments were constructed by deletion mutation method, and double luciferase reporter vectors containing mutations of cis-acting elements were constructed by overlapping extension PCR method. The recombinant constructs and reference vectors were co-transfected into goat mammary epithelial cells (GMEC), and the promoter activity was detected by dual luciferase system. The results showed that 1 500 bp FoxO1 promoter sequence was cloned from the blood genome of dairy goats. Bioinformatics analysis revealed 2 binding sites of FoxO1 on the FoxO1 promoter. Deletion mutation results showed that region from -95 to -24 bp was necessary for basal transcription activity of FoxO1 promoter. Overexpression of FoxO1 significantly increased the activity of FoxO1 promoter. Mutation of the forkhead transcription factor 1 (FKH1) (-359 bp) site showed a significant decrease in FoxO1 promoter activity compared to wild type, but unchanged after mutation of FKH1 and FKH2 sites. Above results suggest that FoxO1 might promote FoxO1 transcription by binding to the FKH elements located at the promoter. This study provides theoretical reference for the functional study of the FoxO1 gene promoter and basic data for constructing the fatty acid metabolism network in mammary gland of dairy goats.
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