The Cloning, Expression And The Core Area Promoter Positioning Of Min Pig Myog Gene

There are very complex mechanisms in both Growth and differentiation of skeletal muscle.The number of muscle fibers that the piglet born with will determine the individual’s potential of maximumlean growth.Per unit area muscle fibers’straight has negatively correlated with the succulenttaste,and positively correlated with the meat production.The growth and development of muscle fibers have a direct link with meat.Myogenin can regulate the formation of the initial fusion of muscle cells and myofibroblasts.It plays an important role in muscle differentiation.Min pig,one of our excellent local species,with the adventages of high fecundity,fleshy,and strong resistance to roughage only distributes in the Northeast region of China,and concentrates in Heilongjiang Province, Compared with the imported species, however, Min pig has a low rate of lean meat and the research of it is still limited.In this study,we studied MyoG gene of Min pigs by using molecular biology methods in order to provide the theoretical basis for Min pig skeletal muscle differentiation mechanism.Firstly,we cloned the full-length coding sequence of Min pig MyoG gene by PCR. Secondly, we predicted and analysised the secondary and tertiary structure of MyoG gene by using biology software and construct a eukaryotic expression vector by means of double digestion and connection method.Then,the expression of MyoG gene has been analysisd in the30-day-old Min pig organization by using Realtime-PCR method.Finally,we use the dual luciferase reporter system detected and analysised the core sequence of promoter region in MyoG gene.The main findings are as follows:1) Accessed the675bp full-length coding sequence of Min pig MyoG gene,compared with other breeds MyoG gene sequence given by NCBI found a mutation sites:391Tâ†'C,which caused a change in amino acid sequence131Câ†'R.2) Constructed pcDNA3.1(+)-MyoG eukaryotic expression vector successfully.3) Accessed the MyoG gene tissue results,and revealed that MyoG gene expression only in skeletal muscle tissue.It does not express in the tissues of heart,liver,spleen,lung,kidney,large intestine,small intestine or the stomach.4) Accessed2203bp sequence of Min pig MyoG gene, including5’flank and part of the coding region.The result was99%match with the pig MyoG sequence on the NCBI.5) Constructed the vitro culture system of pig skeletal muscle satellite cells and detected MyoG mRNA in cellular RNA,which means MyoG gene expressed in pig skeletal muscle satellite cells.6) Constructed the9missing different DNA fragments luciferase vector.And the results of dual-luciferase reporter assay showed that in the MyoG gene promoter region, the interval of-1385bp～-1184bp may have possible negative regulatory element or silencer,and the interval of-199bp～-31bp may have the basic activity sequence of the promoter region, the results were consistent in the skeletal muscle satellite cells.7) For the promoter region-199bp～-31bp re-constructed4missing different DNA fragments luciferase vector.After the dual-luciferase detection reporting system analysis and bioinformatics web site analysis revealed that the promoter region-137bp～-101bp contains CdxA and MZF1transcription factor which may play an important role in regulating the expression of MyoG gene.