Expression Regulation Search Of MEF2C Gene Variable Shear Body In Xingyi Duck

MEF2C(Myocyte Enhancer Factor 2C)is a member of the Myocyte Enhancer Factor 2(MEF2)family,It is involved in the expression and regulation of various genes and is an important transcriptional regulator.The m RNA precursors of MEF2 C gene can produce different variable shear fragments through different shearing methods.Therefore,MEF2 C gene can encode a variety of proteins.At the present stage,multiple splicers body of MEF2 C gene have been identified in humans,mice,and other corresponding livestock and poultry resources.Studies on MEF2 C gene variable splicers body on ducks are rarely reported and belong to basic research,It has not been systematically discussed about it;promoter fragments were identified as the key to regulate gene transcription initiation time and expression degree,and also played a great role in gene expression regulation.And there are few researches on MEF2 C gene promoter in livestock and poultry.therefore,this study used xingyi duck as a test material,MEF2 C as a candidate gene.on the one hand,the promoter polymorphism of MEF2 C gene was studied by reverse transcription polymerase chain reaction(RT-PCR),bioinformatics analysis.on the other hand,the expression difference of MEF2 C gene-variable shears body of xingyi duck was investigated by real-time fluorescence quantitative PCR technique(qRT-PCR),rapid reverse transcription,transfection,and in vitro culture of duck myoblasts.the expression difference relationship of their variable shears body was explored from the m RNA and cell level.The main results of this study are as follows:1.The MEF2 C gene promoter fragment of Xingyi duck was successfully cloned,and 8 SNP loci in the MEF2 C gene promoter region of Xingyi duck were screening namely T-190 A,A-232 G,A-285 G,A-617 T,G-1420 A,A-1437 C,T-1504 G andA-1524 G.The bioinformatics analysis found that the key transcription factor binding sites,such as the HOXA4,GCN4,Pit-1a,IRF-1 around the T-190 A,A-232 G,G-1420 A mutation site disappeared and then produced Oct-1,ICSBP two new transcription factor binding sites.The results of correlation analysis with slaughter traits showed that there was a significant difference in the living weight of xingyi ducks in different genotypes of T-190A(P<0.05),and there was a significant difference in the leg muscle rate of xingyi ducks in different genotypes of G-1420A(P<0.05).2.Two variable shear bodies of MEF2 C genes were found and identified in different tissues of Xingyi duck,named MEF2C-a and MEF2C-b,the lengths were1398 bp and 1302 bp,encoding 465 and 433 amino acids,respectively.the difference between them was 96 bp,homology analysis found that the two transcripts had up to95% homology among different species,and were highly conserved.3.The m RNA expression of MEF2 C gene in 7 different tissues of Xingyi duck(female duck,male duck)at 30 days,60 days,90 days,120 days and 150 days was detected showed that MEF2C-a and MEF2C-b had significant tissue and sex effects.the two variable shear bodies were expressed between tissues at each age stage,and the expression difference between different tissues m RNA different genders reached significant(P<0.05)and extremely significant(P<0.01)levels.Shear body MEF2C-a、MEF2C-b maintains high levels of expression in the leg muscle,pectoral muscle,myocardium and brain(except for Individual day age)and low levels in liver,lung and kidney.4.The eukaryotic expression vectors of two variable shear bodies of xingyi duck MEF2 C gene were first constructed: pEGFP-C1-MEF2C-a and pEGFP-C1-MEF2C-b,and successfully expressed in duck myoblasts.The qRT-PCR method was used to detect the m RNA expression levels of muscle differentiation-related genes Myf5,Myo D1,p38,MYOG,MRF4,NFAT,p300,ERK5 after over-expression of MEF2 C gene variable shear bodies MEF2C-a and MEF2C-b,The results showed that: after over-expression of MEF2C-a,the expressions of Myf5,Myo D1 and MRF4 allincreased,while the expressions of p38,NFAT,p300,ERK5 and myogenin all decreased.After over-expression of MEF2C-b,the expression of Myf5 increased,and the expression levels of related muscle differentiation key genes Myo D1,p38,MRF4,NFAT,p300,ERK5,and myogenin all decreased.