| In recent years our understanding of the molecular processes underlying skeletalmyogenesis has improved considerably. Overt myogenesis is preceded by a number ofsteps leading to the specification of muscle precursor cells. During this period, myogenicprecursors express mRNAs for Muscle Regulatory Factors (MRFs) of the bHLH familyof transcription factors. MRFs factors are specific for developing skeletal muscle andtheir identification belongs to the great achievements in muscle research. Othertranscriptional regulators involved in myogenesis are the myocyte enhancer factors(MEF2). In contrast to the MRFs, they are expressed in developing cardiac, skeletal andsmooth muscle. MEF2 factors can increase the efficiency of myogenic conversion ofnon-muscle cells in combination with MRFs. Although no skeletal muscle phenotype hasbeen described in MEF2 mutants, specific interactions have been observed, especiallybetween MEF2C and Myogenin, in the sense of a positive feedback loop. So thetranscription factors of MRFs and MEF2 family were cloned and identified and thepromoter regulation and genetics effect were analyzed. The main results are as follows:1. Isolation, sequencing and sequence analysis of the cDNA of porcine MEF2A,MEF2C and Myf6 genes: Using in silico cloning and rapid amplification of cDNAends (RACE) technology, we obtained 2 full-length cDNA: MEF2A gene, GenBankaccession number EF194143, full-length cDNA 2818 bp, ORF 1416 bp; MEF2C gene,full-length cDNA 4365 bp, ORF 1392 bp; Using RACE technology, we obtained3'UTR of Myf6 gene, GenBank accession number DQ139774, 3'UTR 626bp.2. Using CLUSTAL W and some related software, we analyzed the gene structure,protein structure, conserved motifs and phylogenetic relation of genes MEF2A andMEF2C. In addition, the corresponding phylogenetic trees were constructed.3. Identification of the porcine MEF2C-1/MEF2C-2 transcripts: There are two transcriptsMEF2C-1/MEF2C-2 on porcine MEF2C gene. Primers were used for amplifyingfragments of porcine cDNA from muscle tissue. The sequences of RT-PCR productswere analyzed, and the results suggested that different band of RT-PCR productsamplified. Using CLUSTAL W and some related software, we analyzed the genestructure, protein structure, conserved motifs and phylogenetic relation ofMEF2C-1/MEF2C-2 transcripts. In addition, the corresponding phylogenetic treeswere constructed. 4. MEF2A and MEF2C genes' partial genomic sequences were obtained according to thecDNA sequence, and analyzed their polymorphisms in different pig breeds. MEF2A,1169bp (containing partial exon 6 and exon 7, complete intron 6), we identified 5SNPs. Of 2 are transversion mutations, 3 are transition mutations, as well as anA157G substitution in intron 6 MEF2A of which makes the region be recognized byMspI. MEF2C, 3312 bp (containing partial exon 7 and exon 9, complete intron 7, 8and exon 8), we identified 5 SNPs. Of 3 are transition mutations, 2 areinsertion/deletion mutation. The locations of splice donor/acceptor sites in all intronsfollow the "GT/AG" rule.5. Screening of SNPs in porcine Myf5,MyoD and Myf6 gene: (1) Myf5 gene, 26 SNPswere obtained. Of 6 are transversion mutations, 11 are transition mutations, 9 areinsertion/deletion mutation. (2) MyoD gene, 8 SNPs were obtained. Of 4 aretransversion mutations, 4 are transition mutations. (3) Myf6 gene, 8 SNPs wereobtained. Of 4 are transversion mutations, 4 are transition mutations.6. Genome PCR walking technique was used to clone the proximal promoter region ofprocine MEF2C and Myf5 gene and comparative genomics was used to clone theproximal promoter region of procine Myf6 gene. The length of obtained fragment are2427bp,1970bp and 1089bp, respectively. Bioinformatics approaches were adopted toanalyze the proximal promoter region of procine MEF2C, Myf5 and Myf6 genes, andthe putative binding sites of transcription factors in the regulatory region of the threegenes were analyze by online software. In addition, 13 SNPs were obtained. Of 3 aretransversion mutations, 9 are transition mutations, 1 is insertion/deletion mutation.7. Using PCR-RFLP, we detected 7 SNPs in different pig populations and observedassociations with traits in Large White x Meishan F2 population. The results showed:Myf5- BcnI- RFLP is significant association with dressing percentage (p＜0.05) andlean meat percentage (p＜0.05). Myf5- Hsp92Ⅱ- RFLP is significant association withfat percentage (p＜0.05), shoulder backfat thickness (p＜0.05), thorax-waist fatthickness (p＜0.05), average backfat thickness (p＜0.05), carcass length to 1st rib(p＜0.05), intramuscular fat (p＜0.05) and water moisture (p＜0.01). Myf5-HinfI- RFLPis significant association with pH (LD) (p＜0.10). Myf5- MspI- RFLP is significantassociation with 6- 7 rib fat thicknesses (p＜0.05), thorax- waist fat thickness (p＜0.05),carcass length to 1st rib (p＜0.05) and pH (LD) (p＜0.05). MyoD- DdeI- RFLP issignificant association with carcass length to 1st rib (p＜0.01), 6- 7 rib fat thicknesses (p＜0.05). Myf6- TasI- RFLP is significant association with pH (LD) (p＜0.05),longissimus doris marbling (p＜0.05), biceps femoris marbling (p＜0.01) and lean meatpercentage (p＜0.10). MEF2A- MspI- RFLP is significant association with lean meatpercentage (p＜0.05), buttock backfat thickness (p＜0.05), pH (LD) (p＜0.05), pH (BF)(p＜0.05), water holding capacity (p＜0.05), drip water rate (p＜0.05) and internal fatRate (p＜0.01).
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