SNPs Analysis And Prokaryotic Expression Of MEF2C And MEF2D Genes In Xingyi Duck

Myocyte enhancer factor 2C(MEF2C)and Myocyte enhancer factor 2D(MEF2D)are members of the myocyte enhancer factor 2(MEF2),a supergene family,and thought to be related to the development and regeneration of skeletal muscle.To investigate the effects of polymorphism of MEF2 C and MEF2 D gene on slaughter traits of Xingyi ducks,this study used PCR and DNA sequencing methods to find SNP loci and analyzed their correlation with slaughter traits.Meanwhile,we constructed the prokaryotic expression vectors of these two genes to obtain the MEF2 C and MEF2 D proteins.The results of the study are as follows:1.In this trial population,we do NOT find SNP locus in exons of MEF2 C gene.The results showed 6 SNPs in exons of MEF2 D gene,which are respectively C3894 T,G3957A,T16236 C,A16305C,G16359 C and A21071 G.All of the six sites were synonymous mutation,and those did not cause any change of amino acid encoding.By the population genetics analysis,the results showed that 6 loci were at moderately polymorphic status(0.25<PIC<0.5).The χ2 test showed that the loci were all in Hardy-Weinberg equilibrium status(P>0.05).Analysis of matching chain disequilibrium and haplotype analysis showed that there was a strong chain of balance among the six loci.Analysis of association of polymorphism with slaughter traits at all mutation loci showed that T16236 C locus and A16305 C locus had significant effect on live weight,carcass weight,semi-eviscerated weight,eviscerated weight,and breast muscle weight(P < 0.05 or P < 0.01);G16359C locus had significant effect on semi-eviscerated weight and eviscerated weight(P<0.05);A21071G locus had significant effect on carcass weight,eviscerated weight,leg muscle weight,dressing percentage and eviscerated weight rate(P < 0.05 or P < 0.01).Seven haplotype combinations were found in fifty-two samples of Xingyi duck,the CGCCCG haplotype had significant effect on semi-eviscerated weight and semi-eviscerated weight rate(P<0.05 or P<0.01);TACCCA(TATAGA)haplotype had significant effect on leg muscle weight(eviscerated weight rate)(P<0.05).2.We successfully cloned the CDS region of MEF2 C and MEF2 D genes of Xingyi duck,and constructed prokaryotic expression vector: p ET32a(+)-MEF2C-1/MEF2C-2 and p ET32a(+)-MEF2 D.We found two spliceosome of MEF2 C gene,their coding sequences are 1398 bp and 1302 bp,encoded 465 and 433 aa respectively.We successfully cloned the CDS region of MEF2 D gene,the length was 1557 bp,encoded 518 aa.3.Western-blot verificated MEF2 C and MEF2 D recombinant protein from strain BL21(DE3)was expressed successfully.After transfecting the recombinant plasmid of p ET32a(+)-MEF2C-1/MEF2C-2 and p ET32a(+)-MEF2 D into strain BL21(DE3),we used IPTG(0.1 m M,37 ℃ 4 h)to conduct the inducible expression of recombinant proteins and verified the expression by Western-blot method,and successfully obtained the MEF2 C and MEF2 D prokaryotic proteins of Xingyi duck.The results suggested that four SNPs and three haplotype combinations might havepotential effect on slaughter traits in the duck populations mentioned above and could be used for marker-assisted selection.This study provides a theoretical basis for the following analysis of structure and function of MEF2 C and MEF2 D gene in ducks,and further enrich the research of MEF2 C and MEF2 D gene.