Study Of Interaction Mechanism Of MSTN Promoter And Foxo6 Transcription Factor

FoxO6 gene belongs to FoxO proteins transcription factor family(including FoxO1,Fox O3,FoxO4 and Fox O6).In several cells types,Fox O transcription factors play an importantrole in cell cycle arrest,oxidative stress resistance,cell survival,energy metabolism,andcell death.And study had shown:FoxO6 transcription and PGC-1? promoter formed a regulatory loop for setting oxidative metabolism level in the skeletal muscle.Myostatin(Myostatin,MSTN)is one of the major genes that plays an important negative regulator in the animal muscle's growth and development,meanwhile the impact of the tough of tendons and ligaments.Based on our laboratory preliminary experimental results:MSTN promoter has FoxOs transcription factorbinding site,we cloned CDS region of Fox O6 gene ? MSTN promoter region and constructed eukaryotic expression vector ?prokaryotic expression vector of FoxO6,eukaryotic expression vector of MSTN promoter to study FoxO6 transcription factors interact with MSTN promoter,in order to parse FoxO6 transcription factor regulation into muscle cells and adipose cell proliferation and differentiation,participate in the mechanism of skeletal muscle metabolism.In this study,Guanling Cattle is as an experimental subject.According to GenBank datebase,we designed PCR primers of CDS region of Fox O6 and MSTN promoter region?Through PCR method,we obtained CDS region of FoxO6 and MSTN promoter region,and constrcted pET32a(+)-FoxO6 prokaryotic expression vector,pcDNA3.1(+)-FoxO6 eukaryotic expression vector and PGL3-Basic-MSTN eukaryotic expression vector.Then we transformed pET32a(+)-FoxO6 prokaryotic expression vector into prokaryotic expression strain BL21(DE3),and Preliminary explored to express conditions.The pcDNA3.1(+)-FoxO6 eukaryotic expression vector and PGL3-Basic-MSTN eukaryotic expression vector were transfected into mouse C2C12 cell line.We detected the dual luciferase activity and we verified the interaction of transcription factors MyoD and MSTN promoter within cells after 24 h.The results are as follows:(1)The experiment successfully cloned CDS region of Guanling Cattle FoxO6 gene and we successfully constructed recombinant vector pcDNA3.1(+)-FoxO6 and pET32a(+)-FOxO6,transformed the pET32a(+)-FOxO6 into prokaryotic expression strain BL21(DE3)successfully.(2)The experiment successfully cloned MSTN promoter,and constructed recombinant vector PGL3-Basic-MSTN.(3)The expreession condition of FoxO6 protein is 37 ?,0.4 m M IPTG,6%glycine,induction time 4h.(4)dual luciferase assay shown that in C2C12 cell,FoxO6 transcription factor inhibit the activity of MSTN promoter.