The Genetic Research Of MEF2A, DLK1 And CLPG Genes, And Their Associations With Growth Traits

The genetic variations of MEF2A, DLK1 and CLPG gene in 18 loci were detected DNA sequence analysis, PCR-SSCP and F-PCR-RFLP techniques in 1223 individuals from eight breeds/species (Nanyang, Qinchuan, Jiaxian Red cattle, Jinnan, Chinese-Holstein, Angus, Yak and buffalo), and the genetic structure and genetic diversity were analyzed in this study. The association analysis was carried out to evaluate the effects of genotypes of those three genes on growth traits of three Chinese cattle breeds(Nanyang,Qinchuan,Jiaxian Red cattle). Furthermore, we obtained corresponding genetic information and explored the genetic effects on the growth traits of those three cattle populations. Through this study, we found the molecular markers have dramatic effects on the important economic characters,which provided the genetic basis for the efficient selection of Chinese cattle and the foundation of molecular marker database as well as the conservation and application of germplasm. Besides, we construted the MEF2A gene of prokaryotic expression system and realized the expressing of MEF2A Gene in E-Coli. The results were as following:1. The clone and bioinformatics prediction of cattle MEF2A geneWe cloned CDS of and 1.2 Kb of the 3'UTR of MEF2A gene.The cattle MEF2A gene,a 1494bp length coding region (CDS), coded 498 amino acid, including 11 exons. In addition part of the 3'UTR( about 1.2 Kb) was cloned. The prediction demonstrated that the molecular weight of the protein was 53.38 KDa and the isoelectric point was 7.13; MEF2A is composed of a MADS-box domain (No.1～56 amino acid), a MEF2 domain(No.57～86 amino acid) and c-terminal transactivation domain, there was no transmembrane structure in the MEF2A protein. In the 1.2 Kb of 3'UTR might containing the AU (ATTTA) motifs associated with mRNA instability. pET-MEF2A prokaryotic expression system was constructed, and MEF2A was successfully expressed in BL21 under the induction of IPTG.2. Genetic variations of cattle MEF2A gene12 pairs of primers were designed to detect 9 exons and the 3'UTR of MEF2A gene, six SNPs (C1598T, G1599A, A1602G, G1641A, C1734T and G2032C) were detected. Among them, 5 SNPs were in exon 11, and the other SNP was in the 3'UTR, leading to a missense (P420L) and five silent mutations (P420P, P421P, E434E, P465P and 3'UTR). While no mutation was found in other exons. These results proved that the N-terminal highly conserved in the same species, and mutations were high probability in C-terminal transcription activation.3. MEF2A gene polymorphisms are associated with growth traits in cattleThe polymorphism of E11-A locus was caused by C1598T mutation. The individuals with genotype 1598CT and CC had lower body length at 12 months than the individuals with genotype 1598TT (P<0.05). The polymorphism of E11-B locus was caused by G1641A mutation. In this locus, The locus could affect the daily gain of 12 month of age (P<0.05). The individuals with genotype 1641AA had better average daily gain (ADG) at 12 months than the individuals with genotype 1641AT (P<0.05). The polymorphism of E11-C locus was caused by C1734T mutation. The genotype 1734TT was better than the genotype 1734CT in BW and ADG at 6 month in cattle (P<0.05).The polymorphism of E11-D locus was caused by G1599A mutation, the locus of G1599A was associated with average daily gain at 6 month of cattle. The genotype AA was better than the genotype GA in ADG at 12 month in cattle (P<0.05).The polymorphism of E11-E locus was caused by A1601G mutation. The locus didn't affect the growth traits of cattle (P>0.05).The polymorphism of 3'UTR locus was caused by G2032C mutation. The G1599A was associated with BW, BH and BL at 6 month of cattle (P<0.05). The heterozygote AB genetype was significantly better than the genotype homozygous AA in these growth traits. The high conserved regions of 3'UTR might be indicate that there was a repressor in this region.4. Genetic variations and interspecific differences of DLK1 geneIn this study, the polymorphisms (SNPs) were detected in exon 2-5 and 3'UTR of DLK1 gene in the individuals belonging to eight breeds of Bovini, including cattle, buffalo and yak. The results showed that DLK1 gene contained a highly conserved imprinted domain, and the rate of gene mutation was very low, only one synonymous mutation (C451T) in exon5 and three base deletion (delC) in 3'UTR were detected. There was an insertion of base T which located between the 1087th and 1088th nucleotides, and a transition (T>C) at the 1089th nucleotide, and not causing the amino acid change.The polymorphism of E5 locus was caused by C451T synonymous mutation. No polymorphism was found in the orther populations. The relationship between SNPs and the cattle growth traits were analyzed. The individuals with genotype E5-A had better characters than those of the homozygous E5-B genotype in QC cattle breed, however, there was no significant difference (P>0.05).The polymorphism of 3'UTR locus was resulted by delC. In this locus, the SSCP genotype AA and AB were detected and genotype AA was predominant in NY, QC and JX populations. The homozygous AA genetype was significantly better than the genotype heterozygote AB in HG at 12 month in cattle.The cattle DLK1 gene, a 927 bp length coding region (CDS), coded 308 amino acid.The prediction demonstrated that the protein molecular weight was 33.01 KDa and the theoretical isoelectric point was 4.47. The prediction also showed that EGF domain, the transmembrane region and the EGF_CA Calcium-binding EGF-like domain belong to No.25~206 amino acid, No.230~252 amino acid and No.88~125 amino acid respectively.α-helices was dominent in terminals of the second structure andβ-strands was in middle part.5. Population genetics analysis of CLPG in bovidaeThe analysis of population genetics was involved in eight population which belonged to eight breeds/species, including cattle, buffalo and yak. A mutation of CLPG was detected in QC, but no polymorphism was identified in other populations. After the sequence analysis of the domain, the result showed four AT-GC base substitions in this region between cattle and buffulo, a base transition of A-G at 151 between yak and cattle,but there was no base substitions between cattle and Chinese-Holstein. The distance among cattle and buffalo was rather distant than that among cattle and yak. The Holstein is a kind of special breed of Bos taurus, and there is a few genetic diversities among them. At the CLPG locus, there is no genetic diversity between cow and cattle.