Single nucleotide polymorphism of myocyte enhancer factor 2C (MEF2C) gene and correlation analysis with meat traits in Chinese Simmental
Keywords Chinese Simmental Myocyte enhancer factor 2C Polymorphism Carcass traits
A family of transcription factors, the myocyte enhancer factor-2(MEF2) family, has been shown to play a pivotal role in morphogenesis and myogenesis of skeletal, cardiac, and smooth muscle cells. In vertebrates, there are four MEF2 genes, referred to as MEF2A,- B ,- C and -D that are located on different chromosomes. It is shown that the gene product of the MEF2 family had the special and unique domain, MADS-like region, which ...view middle of the document...
Despite, more and more data provide evidence that MEF2C is an essential factor for vertebrate skeletal muscle development and differentiation, it is little known about how this gene is regulated at the transcriptional and translational level, even the specific structure or functional region, especially in bovine.
Because of the economic importance of the bovine species to the livestock industry, it appears clearly essential to clarify some of the factors related to MEF2C genes expression. The present study was conducted to identify new polymorphisms in the promoter region of the bovine MEF2C gene in Chinese Simmental, and to determine the relationship between the MEF2C gene and the productive traits.
Materials and methods
Animals and DNA samples
Blood from 137 Chinese Simmental (Bos taurus) were collected and snap frozen using liquid nitrogen and then stored at -80°C pending analyses. The animals had grazed freely on nature pasture and had received a supplement of salt per 2–4 week interval at the IAS-CAAS, city of Beijing, China. Genomic DNA was extracted from blood using AxyPrep Blood Genomic DNA Midi/Maxi prep Kits(Axygen Inc. ,HangZhou, China) and stored in the eppendorf tubes contains ddH2O at -80°C.
Polymerase chain reaction (PCR) and sequencing
Basing on the sequences of the bovine chromosome 7 genomic contig (GenBank accession number: NC_007305) available in the GenBank database, 8 pairs of primers were designed using the Primer3 (v. 0.4.0)(http://frodo.wi.mit.edu/) (Table 1). They encompassed most of exons and the 5’UTR encompassed site of the promoter region to examine any potential SNP.(The MEF2C gene contains large number of poly-N as poly-G,C,A,T, that make the proper primer of few exons fail to be designed.)
The PCR was carried out in a reaction volume of 25μL, containing 1.0μL DNA (approximately 120ng/μL), 2μL 10mM/mL dNTP Mixture(2.5 mM/mL each dNTP), 2.5μL EX Taq buffer (Mg2+ Plus),0.5μL 10 μM/μL forward primer, 0.5μL 10μM/μL reverse primer, 0.5 μL EX Taq DNA polymerase (5U/μL, TAKARA BIO Inc, JAPAN.) and 16.0 μL ddH2O. Thermal cycling parameters were as follows: 5 min at 94 °C, 35 cycles of amplification (30 s at 94 °C, 30 s at individual Tm, 60 s at 72 °C), and finally 7 min at 72 °C. The 132 purified PCR products (using AxyPrep DNA Gel Extraction Kit,Axygen Inc.,china )were selected and sequenced on both strands using an ABI373X DNA Analyzer (Applied Biosystems Inc.) at the Sango Biotechnology Company (Shanghai, China)
Table 1 Forward (F) and reverse (R) primers for amplification of the MEF2C gene
Locus | Primer sequences | Mutationa | Region | Positionb | Length | Tm |
P1 | F: ACCTGATTAGAAAGTTCATGGC | | Promoter | -1389 to -1125 | 264 | 56 |
| R: TAACCACTTCATTCTTCCACTG | | | | | |
P2 | F: GAGTCAACGGTTTGGGATT | | Promoter | -591 to -226 | 305 | 57.3 |
| R: GTAACCAGACTCGTCCAGAA | | | | | |
| R: CGTTCATCCATAATCCTCGT | | | | | |
E2 | F: TGGCTTGCTATTAAAGTCGT | | Exon 2 | 53908-54138 |...