ANK1 gene promoter in Guangxi Bama miniature pig:Cloning and activity analysis

ObjectiveIn this study, ANK1 gene promoter of Guangxi Bama miniature pig was cloned, and its core ac-tivity areas were detected, so as to lay a foundation for researching correlation between ANK1 gene promoter and meat qual-ity traits, and establishing animal disease model. MethodTranscription factor binding sites of ANK1 promoter were pre-dicted with online software, ANK1 promoter fragments of different lengths were amplified by specific primers based on tran-scription factor binding sites. To determine the activity of different fragments, fluorescence values of different promoter re-gions were tested using dual luciferase kits. ResultOne transcription starting sites (TSS), two CpG islands and multiple transcription factor binding sites were found in ANK1 promoter. The results served as basis for segmentation of ANK1 gene promoter and it was devided into eight target fragments of different length, namely P638,P791,P1113,P1163,P1648, P1694,P1796 and P2074. The eight segments was cloned. Through KpnⅠand HindⅢdouble enzyme digestion, T4 eukaryotic expression vector linkage and cell transfection, eight dual luciferase kit recombination reporter genes were established. Based on dual luciferase detection, the activity of ANK1 gene promoter was the strongest in P1796 fragment, and the difference with others fragments was significant (P<0.05). ConclusionIn this study, eight fragments of the ANK1 gene promoter from Guangxi Bama miniature pig are successfully cloned and the core region of the ANK1 gene promoter is identified as P1796 fragment by dual luciferase kits.