Research Of Cattle CMYA1 Gene For Core Muscle-specific Promoter Screening

Our laboratory has authenticated cattle CMYA1 gene is muscle tissue specific expression.This study cloned cattle CMYA1 gene promoter,then verified CMYA1 promoter activity and muscle specific expression.With deletion of CMYA1 promoter,eventually we obtained the core promoter sequences of CMYA1 gene.This study is the basis for CMYA1 gene research work,including CMYA1 gene transcription regulation mechanism research.For the study of transgenic cattle,an efficient muscle specific expression promoter is particularly important,at the same time,we should consider the safety for transgenic animal research,natural muscle cell-specific promoter should be selected such as CMYA1 gene promoter,rather than artificial muscle specific promoter.Muscle specific expression promoter,has significance for clinical research,and lay the foundation for the research of transgenic animals.The lab has verified cattle CMYA1 gene specific expressed in muscle tissue by RT-PCR method.This experiment cloned cattle CMYA1 gene promoter sequence 1155 bp,upstream primer in 5 'end was added enzyme locus XhoI,(CTCGAG),protection bases is CCG and downstream primers in 5 'end was added enzyme locus HindIII,(AAGCTT),protection bases is CCC.Then we constructed pEGFP-1-CMYA1 promoter,respectively transfected mice C2C12 cells and cattle fibroblasts,The result for CMYA1 promoter is that both with promoter activity and muscle specific expression.By deletion CMYA1 promoter in 5 'end,we designed8 pairs primers for CMYA1 promoter,respectively named P1,P2,P3,P4,P5,P6,P7 and P8.upstream primer in 5' end was added enzyme loci XhoI and downstream primers in5 'end was added enzyme locus HindIII respectively.Construction pGL3-basic-(P1-P8),together with the internal plasmid pRL-TK transfected C2C12 cells,tested with the luciferase report gene test kits,we finally obtained CMYA1 core promoter P7(477bp).After that we constructed vector pEGFP-1-P7,transfected C2C12 cells and cattle fibroblasts,at last,we tested CMYA1 gene core promoter sequences with the promoter activity and muscle specific expression.The experimental results showed that: 1.1155 bp cattle CMYA1 promoter sequence,with the promoter activity,and has the characteristics of muscle cell specific expression.2.Cattle CMYA1 gene core promoter sequences of 477 bp,has high promoter activity,also has the characteristics of the specific expression in muscle cell.Conclusion: obtained cattle CMYA1 gene core promoter sequence is 477 bp,has high promoter activity,also has the feature of muscle cell specific expression.At the same time,muscle specific expression promoter,has significance for clinical research and lay the foundation for transgenic animals research.