Cloning, Expression And Genetics Effect Analysis Of The Transcrition Factors Related To Bovine Skeletal Muscle Growth And Lipid Metabolism

The bioinformatics, DNA sequencing, DNA sequence analysis, RT-PCR and PCR-RFLP techniques were applied to not only clone and predict the bovine Pax3, Pax7 and LXRA gene, but also scan and analyze the genetic variations of key function gene and their association with economic trais in 326 cattle, which comprised of eight breed, namely, Simmental (n=110), Angus (n=48), Luxi (n=30), Qinchuan (n=29), Jinnan (n=24), Charolais (n=30), Limousin (n=25) and Hereford (n=30). The six key function gene included MRFs family gene, LXRA gene and TG gene. The economic traits comprised of carcass and meat quality traits. The association between genetic variations and economic traits were analyzed by using general linear model (GLM) or adjusted linear model, as well as population genetic structure and distribution of genotype and allele were calculated by chi-square and quantitative genetic method. These will benefit for the application of DNA marker related to the economic traits on marker-assist-selection (MAS), improvement and promotion of beef cattle. The main results were shown below:1. The main cDNA sequences and partial UTR sequences of Pax3,Pax7 and LXRA gene, which contained complete CDS, were cloned and identified from skeletal muscle of simmental.2. The amino acids sequence deduced by ORF of Pax3 and Pax7 cDNA were analyzed while the character and protein domain were predicted by bio-informatics, the results showed bovine Pax3 and Pax7 protein were consisted of 390 and 503 amino acids. They both contained PAX domain (was consisted of 126 amino acids) and HOX domain (was consisted of 63 amino acids). The amino acids sequence in the domain showed homology with different species.3. The amino acids sequence deduced by ORF of LXRA cDNA was analyzed while the character and protein domain were predicted by bio-informatics, the results showed that bovine LXRA protein was consisted of 447 amino acids, and contained ZnF_C4 domain (was consisted of 71 amino acids) and HOLI domain (was consisted of 159 amino acids).4. Identification for mutations in the amplified fragments of MRFs family and LXRA genes were performed and the 5 SNPs were detected by PCR-RFLP. Allele and genotype frequencies of 5 SNPs that can be detected by PCR-RFLP were analyzed in the experimental population. The analysis of least square was used to employ to association between these SNPs and carcass and meat quality traits. The results showed that: (1) The MyoD SNP was significantly associated with Loin muscle area (LMA). Animals with the genotype BB and AB had larger LMA than the AA genotype (P<0.05). (2) The MyoG SNP was significantly associated with Live weight (LW),Loin muscle area (LMA) and Carcass length (CL). Animals with the genotype BB and AB had larger LW and LMA than the AA genotype (P<0.05); Animals with the genotype BB had longer CL than the AB genotype (P<0.01). (3) No significant associations were observed between the Myf5 SNP and any traits. (4) The Myf6 SNP was significantly associated with Carcass length (CL). Animals with the genotype BB had longer CL than the AA and AB genotype (P<0.05). (5) The LXRA SNP was significantly associated with Marbling score (MS). Animals with the genotype AB had higher MS than the AA genotype (P<0.05).4. Identification for mutations in the amplified fragments of TG genes were performed and the 18 SNPs were detected by DNA sequencing. Allele and genotype frequencies of 18 SNPs were analyzed in the experimental population. The analysis of least square was used to employ to association between these SNPs and carcass and meat quality traits. The results showed that: the linkage disequilibrium analysis indicated that these 12 SNPs were linked completely (r2=1). The G133C SNP was significantly associated with Marbling score (MS). Animals with the genotype CC had higher MS than the GG and GC genotype (P<0.05).5. Six candidate genes (Myf5, MyoG, Myf6, MyoD, Pax3 and Pax7) were detected in skeletal muscle of simmental, which were colleted from four different physiological phases: embryonic phase,12 months old,24 months old and 36 months old, by RT-PCR and Real-time Q-PCR, and the results showed that: The expression in the physiological phases Myf5, MyoG, Myf6, MyoD, Pax3 and Pax7 gene is higher than in other phases, there are no different in the 12 months old,24months old and 36months old.