Genetic Varition And Analysis Of Their Effects And Tissue Expression Regular Pattern Of MEF2C Gene In Yellow Cattle

Muscle growth is a complex biological process regulated by many factors, and themyocyte enhancer factor2C (MEF2C) is such a transcription factor that plays a pivotal role inmuscle development. MEF2C can not only activate muscle gene expression by bindingto anA/T-rich DNA sequence as homo-and heterodimers in the control regions ofmuscle-specificgenes, but also interact with other types of factor directly via its respective DNA-bindingdomains to control the skeletal muscle development.Although MEF2C geneisso important to the muscle development, the research onMEF2C gene is currentlyfocusing on the mechanism of human skeletal and cardiac muscle ofmice, and studies in livestock and poultryare rare.In this study, bovine MEF2C gene wasexploredto analyze and study the structure, genetic variation and tissue expression, throughthe bioinformatics, sequencing and molecular biology techniques. The objects were topreliminarily explore the role of bovine MEF2C gene in the musclesgeneration, to revealmolecular markers with significant effects on important growth traits of cattle, and to providetheoretical basis for further improvement of economic traits of cattle breeds.The results were as follows:1.Genetic variation of MEF2C gene in Yellow cattleIn this study, we aimed to detect and identifyvariations of the MEF2C gene in1095individuals representing five Chinese cattle breeds and their hybrids groups by pooled DNAsamples sequencing (Pool-Seq) and forced polymerase chain reaction-restriction fragmentlength polymorphism (Forced PCR-RLP) method.A total of four novel SNPs were identifiedand they were all located innon-coding region, namely SNP1(g.T96732C), SNP2(g.C76904T), SNP3(g.G76992T) and SNP4(g.A18286C). Analysis of genetic parametersshowed that, at the SNP1,2and3loci, the Qinchuan, JiaxianandXiajia cattle populationsbelonged to intermediate genetic diversity (0.25<PIC <0.50),whereasat the SNP4locus, allpopulations belonged to low genetic diversity(PIC<0.25) and genotypic frequencies of all four loci in Qinchuan, Jiaxian, De′nanand Xiajia cattle populations were in line withHardy-Weinberg Equilibrium (P>0.05);the analysis of linkage disequilibrium revealedthatSNP2and SNP3showed a strong linkage (D’>0.7，r2>0.3); the analysis ofhaplotypeshowed that, Hap5(-TCGA-) had the highest haplotype frequency in Qinchuan andDe′nan(35.10%and34.2%), Hap1(-CCGA-) had the highest haplotype frequency in Jiaxianand Nanyang(39.70%and37.30％), and the Hap7was unique in Qinchuan cattle breed.2.The association analysis between polymorphic and growth traits of MEF2C gene inYellow cattleCorrelation analysis showed that,the four SNPsand combinedhaplotypeshad significantassociation with body weight，body length, body height,chest girth,chestdepth, rump length,hucklebone width and height at hip cross in Qinchuan, Jiaxian, Nanyang and De′nanpopulations (P<0.05); genotypesSNP1-TT/CC, SNP2-TT/CC, SNP3-GG/GT andSNP4-AC/AAcan significantly improve the corresponding traits (P<0.05). This indicates thatthese SNPsmay be used as markerfor cattle growth early marker-assisted selection andestablishthe foundation for transgenetic and functional studies.3.Tissue expression pattern analysis of MEF2C gene in Qinchuan cattleMEF2C gene was expressed in different periods (fetal bovines, calves and adult cattle)and different tissues of Qinchuan cattle, and the relative expression quantity was highest inmuscle and spleenin different stages.During the development from fetal bovines toadult cattle,the relative expression quantity of MEF2C was significantly reduced in muscle(P<0.05), butthe relative expression quantity of MEF2C was significantly increasedin spleen(P<0.05).This showed that MEF2C expression was tissue-specific and time-specific.MEF2C genehas a major role in the formation and development of skeletal muscle, controls genetranscription in the process of muscle cell differentiation, and promotes the maturation ofspleen.4.Effect of different levels of nutrition on MEF2C gene expression of Chinese HolsteincattleIn Chinese Holstein bull calves of three nutrition groups (at the age of eight months),therelative expression quantity of MEF2C gene increased firstly and then decreased in spleenand muscle. Compared with the high-energy diet group A and low-energy diet group C,theintermediate-energy dietgroup B hadsignificantlyhigher level of MEF2C geneexpression inmuscle and spleen.This showed that too high or too low energy level would inhibit the expression ofMEF2C gene, and group B is a suitable energy level.5. Cloning, identification and bioinformatics analysis of MEF2C gene in Yellow cattle The coding sequences of MEF2C gene in Qinchuan cattle wereamplified andconnectedinto pGEM-Teasy vector by PCR methods. After enzyme digestion, the fragment size of thepositive clone was consistent with the expected. Sequencing found24bp continuous fragmentwas missed and it was seventh exon.Bioinformatics methods and softwares were carried outto analyze and predict the structure and propertiesof the coding sequence of MEF2C gene andits protein. Full-length of coding region is1326bp, which encodes a protein containing441amino acids. The MEF2C protein is unstable and hydrophilic, contains phosphorylation sitesand transmembrane regions, without signal peptide, and it is also a non-secreted protein.There are MADS-box domains, α helixes and random coilsinthe secondary structure.