Screening And Verification Of Bovine Skeletal Muscle Specific Promoter

Promoter is the main component of genetic engineering vector,which is a "switch" that regulates gene expression,and has the function of opening and regulating gene expression.Tissue-specific promoters are regarded as organ-specific promoters.Exogenous gene expression generally occurs only in some organs or tissues,and often exhibits developmentally regulated tissue specificity.Skeletal muscle is a kind of animal muscle,belonging to the striated muscle,mainly distributed in the limbs.Skeletal muscle is the main muscle group of the body movement and is also a major component of livestock meat products.Screening of skeletal muscle-specific promoters is of great significance for the creation of high-yield,high-quality and safe transgenic beef cattle breeding new materials.In this study,the specific expression of bovine skeletal muscle tissue was detected by quantitative PCR method for CAPN3,MYOZ1 and MYF6 genes,and the genes with high tissue-specific expression were screened out.The promoter region of the gene was then cloned and the more active promoter was screened by the dual luciferase reporter system and verified in the cells.The results of the study are as follows:1.The expression of CAPN3,MYOZ1 and MYF6 genes in bovine skeletal muscle,myocardium,stomach and small intestine was detected by real-time fluorescent quantitative PCR,which indicated that CAPN3 gene and MYF6 gene were specifically expressed in bovine skeletal muscle.Using the bovine genome as a template,the promoter sequences of CAPN3 and MYF6 genes were cloned by PCR,and the promoter was ligated into pGL4.10,which was identified by enzyme digestion and sequencing,indicating that pGL4.10-pCAPN3,pGL4.10-pMYF6 The nuclear expression vector was successfully constructed.Subsequently,the mouse C2C12 cell line was co-transfected with pGL4.73 and the promoter expression vector,and the CAPN3 gene promoter was selected to be a highly active bovine skeletal muscle-specific promoter by the dual luciferase reporter assay.2.Bovine skeletal muscle,myocardium,and smooth muscle cells were isolated from bovine skeletal muscle,heart,and stomach,respectively,and the isolated cells were identified by immunofluorescence staining.The isolated bovine skeletal muscle cells were differentiated and cultured,and the expression level of CAPN3 gene at different degrees of differentiation was quantified.The results showed that the expression abundance of CAPN3 gene reached the highest on the 5th day of bovine skeletal muscle differentiation.3.The bovine CAPN3 promoter was gradually truncated,and the dual luciferase reporter gene expression vector was constructed with different truncated fragments.The mouse skeletal muscle C2C12 cell line was transfected,and the highly active promoter fragment pCAPN3-216/+90 was successfully screened according to the fluorescence intensity.,consistent with bioinformatics predictive promoter positions.4.The expression vector was constructed by replacing the promoter of EGFP-N1 with pCAPN3-216/+90,and transfected into bovine skeletal muscle cells,cardiomyocytes and smooth muscle cells.Immunofluorescence assay showed that it was only expressed in bovine skeletal muscle cells,which proved that the screening was performed.The CAPN3 core promoter allows the exogenous gene EGFP-N1 to be specifically expressed in bovine skeletal muscle cells.The CDS region of the fat deposition gene PPAR? was ligated into the promoter of EGFP-N1-pCAPN3-216/+90,and the vector EGFP-N1-Q7-PPAR? was constructed and transfected into bovine skeletal muscle cells by real-time fluorescent quantitative PCR and Western.Blot detected the mRNA and protein levels of PPAR? gene,and found that the gene could be expressed in bovine skeletal muscle,which proved that the bovine skeletal muscle-specific promoter screened in this study can express PPAR? gene in skeletal muscle.In summary,this study screened the CAPN3 gene and MYF6 gene into bovine skeletal muscle-specific genes by real-time fluorescent quantitative PCR,and verified that the CAPN3 promoter activity is greater than the MYF6 promoter activity,and the core promoter region was screened by gradual truncation.The CAPN3 core promoter region was verified at the cellular level to specifically express the exogenous genes EGFP-N1 and PPAR? genes in skeletal muscle.