The Effect Of Bovine Myostatin Propeptide Mutations On Muscle Proliferation

Currently, beef has become one of the most important meat products for people. People havepaid more attention to food safety and meat quality with the improvement of living standards inrecent years. Therefore, improving beef quality is an important task which we are facing. Previousstudies have indicated that myostatin was expression in the skeletal muscle of mice, the weight ofskeletal muscle will increase dramatically after MSTN gene was deleted. The growth of musclefibers can be negatively regulated by bovine myostatin. The first two exons of myostatin genecode for the N-propeptide and its third exon codes for the C-polypeptide. Mutations of bovineMSTN mature peptide coding region could lead to inactivation of its function. When the structureof the leader peptide is altered via mutations resulting in more tight binding with the maturepeptide, MSTN function is inhibited. In this study, bioinformatics methods were taken for acomparative analysis of physical and chemical properties, structure and function between mutanttype and normal type, assuming that bovine MSTN gene coding sequence with E1-224-A>Cmutation site. Meanwhile, the A224C and G938A mutations were introduced in the coding regionof bMSTN gene and the pEF1a-IRES-DsRed-Express2-bMSTN-mut eukaryotic expression vectorwas successfully constructed. The expression of bMSTN-mut and the P21and CDK2which wereassociated with cell cycle were determined after the transfection of bovine myoblasts. Finally, thechanges of cell cycle were detected. The effects of bMSTN-mut gene immunization on muscleproliferation and reproductive capacity of mice were investigated. The results were as following.(1) The bMSTN gene coding region was amplified by nested PCR and bioinformaticsmethods were taken for a comparative analysis of physical and chemical properties, structure andfunction between mutant type and normal type, assuming that bMSTN gene coding sequence withE1-224-A>C mutation site. ProtParam analysis results showed that the atoms, atomic number,molecular weight, isoelectric point, the number of acidic amino acids, hydrophobic amino acidsand negatively charged residues were changed. The EBcutter V2.0, SingnalP, TMpred andTMHMM, TargetP1.1, WOLFPSORT and ProtCompV9.0analysis results indicated that the restricted enzyme digested maps of bMSTN gene, transmembrane structure and subcellularlocalization of encoded protein were not changed. SOPMA analysis revealed that the positiondistribution of each element was changed. The α helix of mutant protein was increased by1.87%,stretch was increased by0.54%, β angle was decreased by0.8%and random coil was reduced by1.6%. Phyre2analysis showed that the spatial configuration of mutant bMSTN protein was stable.The mutant bMSTN leader peptide and the mature peptide would bind with each other moreclosely. The ramachandran plot confirmed the reliability of the results.(2) The A224C and G938A mutations were introduced in the coding region of bMSTN geneby fusion PCR and the pEF1a-IRES-DsRed-Express2-bMSTN-mut eukaryotic expression vectorwas successfully constructed. The expression of bMSTN-mut and the P21and CDK2which wereassociated with cell cycle were determined by qPCR and Western blot after the transfection ofbovine myoblasts. The results showed that the P21expression level was down-regulated andCDK2expression level was up-regulated by mutant bMSTN in bovine myoblasts. Ultimatelypromoting cell proliferation.(3) The method of electrical pulses mediated was taken for recombinant plasmidimmunization of mice and the injection site was quadriceps of right hind leg. ELISA detectionresults in different periods showed that GH and TG levels of mice in positive group showed anincreasing trend, and the final concentration was higher than control group and PBS group. MSTNantibody level in positive group was decreased. However, the concentration in different periodswas higher than that of control group and PBS group. The analysis results of mice body weight atdifferent phases showed that the body weight increased significantly at the age of5weeks and9weeks in positive group (P>0.05), the differences were not significant among the groups in the restof the periods. The growth rate of the positive group at the age of5weeks and9weeks wassignificantly increased (P<0.05), which was significantly reduced at the age of6weeks and7weeks(P＜0.05). The differences among the groups were not significant in the rest of the period.The differences among length, wide, weight and quadriceps weight of left and right hind legs inpositive group, control group and PBS group were not significant. Further comparative analysisconcluded that the comprehensive growth trends of right leg was higher than left leg in positivegroup and the growth level was higher than that of the control group and PBS group. The numberof quadriceps muscle fiber were increased the muscle fiber were thickening. After bMSTN-mut gene immunization, the litter size, average birth weight and weaning weight of mice were detected.The results showed that the litter size of the mice in positive group was lower than that of controlgroup and PBS group, but the difference was not significant (P>0.05); The average birth weight inpositive group was higher compared with control group and PBS group, but the difference was notsignificant (P>0.05); The rats weaning weight in positive group was significantly higher than thatin control group and PBS group (P<0.05).In conclusion, bMSTN-mut gene could be expressed in bovine myoblasts and the body of miceto promote muscle proliferation. The above results laid a foundation for further studies of themechanism of bMSTN in regulating muscle growth. Meanwhile, the research has an importantsignificance in improving meat quality and production performance.