Cloning Of Bovine Myf6 Gene And Expression Of The Gene In Myoblasts

With the changing diet and growing concern on health,people are more and more paying attention to high grade beef. Myogenic regulatory factors(MRFs)gene family controls the whole muscle development process:from pre-muscle cell's tying,propagation and conform of muscle fiber to the maturation and the function-completing of postnatal indivldual,and affect the ability of meat production and quality. Myf6 is a mumber of myogenic regulatory factors gene family.By studying myf6 gene regulation mechanism of muscle development and controling myf6 expression ,the muscle fibers'characters can be changed the beef quality can be improved.In this article, genes of the myf6 was cloned and eukaryotic expression vector pIRES2-EGFP-myf6 was constructed.Liposome techinigue was used to transfect Luxi cattle fibroblasts and myoblasts , by G418 selecting ,a stable transfected cell line was gained. The expression changes of myf6 gene,muscle creatine kinase gene and myosin light chain gene were analyzed by western blot,real-time PCR.In this article, Liposome techinigue was established. The stable transfected cells were screened by G418 with 400μg/ml and 600μg/ml. Compared with the control group,the results showed that,after the fibroblasts transfected by plasmid, protein and mRNA expression levels of the myf6 were both increased(p<0.01), muscle creatine kinase gene and myosin light chain gene mRNA expression were both increased (p<0.01). Morphology observations indicated that fibroblasts did not fuse to myotubes.The results suggested that myf6 gene was expressed in fibroblasts.Compared with the control group,the results showed that,after myoblasts transfected by plasmid, protein and mRNA expression levels of myf6 were both increased(p<0.01), muscle creatine kinase gene and myosin light chain gene mRNA expression were both increased(p<0.01). Morphology observations indicated that myoblast had fused to myotubes. Eukaryotic expression vector pIRES2-EGFP-myf6 could highly be expressed in myoblasts .The results suggest that myf6 gene can promote myoblasts differentiation.Eukaryotic expression vectors—pIRES2-EGFP-myf6 was successfully constructed.The recombinant plasmids were transfected into fibroblast cells and myoblast cells. Myf6 gene tranfer technigues have been established. The stable transfected cells provide important materials for transgenic cattle in future.