Regulation Of UCP3 Gene On Differentiation Of Quail Myoblast Cells

With the improvement of the current living standards,people’s demand for livestock and poultry products has gradually increased,among which poultry and pork have always been the first choices of people.However,after the outbreak of African swine fever,pork prices have risen sharply,people’s consumption patterns have undergone certain changes,and the proportion of poultry meat consumption has gradually increased.As a kind of poultry,quail produces meat with extremely high nutritional value and certain medicinal value.Therefore,it is of great significance to understand the muscle development process of quail.Muscles can be divided into skeletal muscles attached to bones,smooth muscles on the surface of internal organs,and myocardium in the heart according to their muscle types.Among them,skeletal muscle is the main consumer.The growth and development of skeletal muscle consists of a series of complex and delicate processes,which are regulated by a variety of factors.Mitochondria are important subcellular organelles in the cytoplasm,which can generate energy to maintain cell growth and differentiation.In mature myotubes,the abundance,morphology,mt DNA copy number and functional properties of mitochondria change.Uncoupling proteins are a kind of mitochondrial membrane proteins,which can convert the potential energy generated by mitochondrial electron transfer into heat energy by mediating proton leakage to maintain a constant animal body temperature.Five uncoupling protein family members UCP1-UCP5 have been found in mammals,which play different functions in different life processes.However,in birds,it was found that there is a gene with the highest homology to UCP3 through comparison,and it was named UCP3 or av-UCP(avain Uncoupling protein).At present,the research on the UCP3 gene of birds mainly focuses on heat stress,and there is little research on muscle development.Because only quail has mature cell lines in poultry,chickens,ducks and other poultry do not.Therefore,quail and its myoblasts were selected as the research object.Knock down and overexpress UCP3 to understand the effect of UCP3 on muscle development of quail and poultry.1、Evolutionary analysis of UCP3 gene familyThe UCPs sequences of multiple species were obtained through the database,and the mouse UCPs genes were compared and analyzed with a variety of birds.Only one UCP gene was found in the avian genome,and this gene is up to 74% homologous to mouse UCP3 It has 59% similarity with UCP1,so this gene is named UCP3.The phylogenetic tree results show that the selected bird UCP3 genes are highly similar to the UCP3 genes of other species,and are clustered into one group.In order to understand the expression of bird UCP3 gene in different tissues,this experiment used quail as an experimental model to detect the expression level of UCP3 in different tissues and organs of quail.It was found that UCP3 had the highest expression in breast muscle,leg muscle and heart.Subsequently,Snger sequencing was performed on quail leg muscles,and it was found that compared with the reference sequence of quail,quail UCP3 had 7 synonymous mutations and 2 nonsynonymous mutations in the expression of quail leg muscles,and the 116 th amino acid occurred.The mutation from valine to methionine resulted in a change in the hydrophilicity of the protein,and the amino acid at this position was conserved in a variety of birds.It is speculated that the mutation of this gene played an important role in the evolution of quail.2、Establishment of the differentiation model of quail myoblast cell lineUsing low-concentration fetal bovine serum to induce differentiation of quail myoblasts,the formation of myotubes can be observed under a microscope.The q RT-PCR results showed that at the m RNA level,the expression of Myo D gradually decreased with the progress of differentiation,while the expression of Myo G,Desmin continued to increase,and was significantly higher than that of the undifferentiated period on the third day of differentiation(P< 0.05).Western blot results showed that the My HC expression level was significantly higher than the control group on the second day after the initiation of differentiation,and the immunofluorescence staining results showed that the number of My HC-specific myotubes increased with the differentiation time after QM7 cells were induced to differentiate.Increase and increase and thicken.In addition,this experiment also detected the expression of UCP3 in myoblasts.PCR results showed that the expression of UCP3 continued to increase after induced differentiation,and the expression in the D3 phase was significantly higher than that in the D0 phase.In addition,the determination of lactic acid content showed that the lactic acid content was in a high state during the proliferation phase,but only a small amount of lactic acid was produced after differentiation,and there was no significant difference(P>0.05).The results of pyruvate showed that there was no significant difference in its content within 0-2d of myoblast differentiation,and it was significantly higher than that of the control group only after 3d of differentiation(P<0.05).These results indicate that the myoblast differentiation model in this experiment can be used for follow-up experiments,and that myoblasts mainly rely on aerobic respiration for energy acquisition after differentiation is initiated,and UCP3 plays an important role in the differentiation of myoblasts.3、The effect of UCP3 on the differentiation of quail myoblastsIn order to explore the effect of UCP3 on myogenic differentiation,this study first designed three si RNAs that specifically recognize the UCP3 gene for transfection.The q RT-PCR results showed that the UCP3 results after transfection did not have the expected knockdown effect,combined with previous studies Based on the results of this experiment,it is speculated that UCP3 is expressed in muscle tissue at a very high level,but the expression level in cells is extremely low,so it cannot be effectively knocked down in myoblasts.Subsequently,this study constructed UCP3 and e GFP-UCP3 overexpression vectors and transfected them into QM7 cells respectively.Through the detection of e GFP fluorescent protein in the cells,it was found that most of the cells were successfully transferred into the fusion vector,and e GFP was only expressed in the cytoplasm and did not enter the nucleus.Fluorescence quantitative PCR results showed that compared with the control group,the m RNA expression of Pax7,Myf5,Myo D,Myo G and Desmin was significantly reduced after UCP3 transfection(P<0.05).Western blot results showed that My HC expression decreased significantly after over-expression of UCP3 and induced differentiation(P<0.05).Immunofluorescence staining showed that after overexpression of UCP3,the number and size of myotube formation were inhibited,indicating that the high expression of UCP3 affects the differentiation process of myoblasts.In addition,the results also found that after overexpression of UCP3 and induction of myogenic differentiation,the absolute content of lactic acid was significantly higher than that of the control group(P<0.05),but the content of pyruvate was significantly lower than that of the control group(P<0.05).Therefore,this experiment also studied the effect of overexpression of UCP3 on cell proliferation.The cell proliferation test was carried out by CCK8 method,and it was found that the cell proliferation ability of the overexpression group was significantly higher than that of the control group.These results indicate that after overexpression of UCP3,the intracellular uncoupling effect is increased,and the breathing mode begins to change from aerobic respiration to anaerobic respiration,which reduces the m RNA expression of Pax7,Myf5,Myo D,Myo G and Desmin,and My HC protein.Fuse horizontally with myotubes,inhibit myoblast differentiation and promote cell proliferation.Conclusion: In summary,the selected bird genome only contains the homologous sequence of UCP3.Overexpression of UCP3 gene during the differentiation of quail myoblasts can change the type of cell respiration,inhibit myoblast differentiation and promote proliferation.This study shows that UCP3 gene plays a role in the process of myoblastization,laying a theoretical foundation for the study of the function of bird UCP3 gene in the process of muscle development.