Cloning, Polymorphisms Detection Of MYF5 And PAX7 Genes And The Analysis Of Genetic Effects On Cattle

Satellite cells are resident myogenic progenitors in postnatal skeletal muscle involved in muscle development, regeneration and also self-renew. Paired box 7 (Pax7) is the identification gene of myogenic satellite cells. It can regulate the proliferation and differentiation of satellite cells by triggering the expression of myogenic determination factor (MyoD) and myogenic factor 5 (Myf5). Therefore, Pax7 is essential for the postnatal muscle growth and development.Myf5 and Pax7 were chosen as candidate genes in this study. Firstly, the coding sequences (CDS) of Myf5 were cloned by the method of exon-Over-lap and DNA sequencing, and the protein expression in prokaryotic/ eukaryotic was detected, which will provide some basic materials for the transgenic research in cattle. Secondly, the single nucleotide polymorphisms (SNPs) of Pax7 gene were detected in 1226 cattle from five Chinese indigenous breeds (NY, JX, QC, LX, and CY) by DNA pool, sequencing and PCR-RFLP. The association between SNPs and growth traits were analyzed by using general linear model, as well as population genetic structure. These will benefit for the application of DNA maker related to the growth traits on marker-assisted selection (MAS), improvement and promotion of beef cattle. The main results were shown below:1. Cloning and expression of bovine Pax7 geneThe coding sequences (CDS) of Myf5 gene were cloned from genomic DNA of QC breed by exon-Over-lap method and constructed prokaryotic expression vector pET-28a-Myf5. The prokaryotic expression vector was transformed into E. coli BL21 (DE3) pLysS, and then the expression of fusion protein 6His-Myf5 was induced on the condition of 1mmol/L IPTG and 37℃. Demonstrated by SDS-PAGE, the fusion protein had large quantities when induced for 5 hours. In addition, the plasmid pEGFP-C1-Myf5 was constructed and transfected into PK15 cell by liposome and expressed transiently, after 24 hours the efficiency of transfection was observed by fluorescence microscope. Processed by Western blot, Myf5 gene was verified to be expressed successfully in PK15 cell.2. Polymorphisms detection and population genetic analysis of bovine Pax7 gene Genetic variations of Pax7 gene within all coding sequences and partial introns (P1-P9) were detected in five Chinese indigenous cattle breeds (NY, JX, QC, LX, and CY) by DNA pool, sequencing and PCR-RFLP. Five novel SNPs (NC₀07300: g.332 ins/del G, g.3531G>A, g.56626G>A, g.66117C>T and g.66251A>G) were identified, which were located at intron 1, exon3, exon 5, exon 7, and intron 7, respectively. Among the above mentioned five SNPs, three mutations in exons revealed synonymous mutations. Interestingly, different allele patterns of all these five loci were directly detected by specified restriction enzymes, suggesting that they could be genotyped by the methods of EcoRII,HinfI,BspT104I,PstI and ApaI-RFLP, respectively.By population genetics analysis, there were two genotypes (AG and GG) in g.332 ins/del G locus. The genotype GG was dominant in five cattle breeds than AG, and the frequencies varied from 0.652⁰.983. Three genotypes (AA,AG and GG) were identified in g.3531G>A locus, and the GG genotype was predominant in NY, JX, QC and LX breed, the frequencies varied from 0.554⁰.724. In g.56626G>A locus, genotype AA,AG and GG were detected and GG genotype was predominant in the above four breeds, the frequencies varied from 0.387⁰.492. In g.66117C>T locus, three genotypes (TT,TC and CC) were found and CC genotype was superior in NY, JX, QC and CY breed, the frequencies varied from 0.401⁰.991. Three genotypes (AA,AG and GG) were detected in g.66251A>G and GG genotype was predominant in the above four breeds, the frequencies varied from 0.590⁰.676. The g.332 ins/del G locus possessed low genetic diversity, and other four loci possessed middle genetic diversity except for the g.3531G>A locus in JX. Moreover, the genotypic frequencies of the five SNPs between CY and other four breeds (P<0.01 for NY, JX, QC and LX, respectively) appeared different based onχ2-test. The linkage disequilibrium analysis demonstrated that g.66117C>T and g.66251A>G were strongly linked in CY breed.3. Association of polymorphisms in Pax7 gene and growth traits in cattleThe association between five SNPs of Pax7 gene and growth traits in QC, JX and NY (6, 12, 18 and 24 months) cattle was analyzed by GLM model. The results showed that g.332 ins/del G locus was significantly associated with body weight in QC breed, body length in JX breed and average daily gain in NY breed aged 12 months, and the individuals with GG genotype had greater than AG (P<0.05). The different genotypes of g.3531G>A locus were significantly affected several growth traits in QC and NY breed (6 and 12 months) (P<0.05 or P<0.01). The g.56626G>A locus was only significantly associated with chest depth in QC breed, and had no association with growth traits in JX and NY breeds. The different genotypes of g.66117C>T locus were significantly affected body height and height at hip cross in QC breed and heart girth in NY breed aged 6 and 12 months (P<0.05). In g.66251A>G locus, different genotypes can not only affect the body weight, chest depth and hucklebone width in QC breed, but also affect the heart girth, hip width and rump length in JX breed and several traits in NY breed aged 6 and 12 months (P<0.05 or P<0.01).