Functional Analysis And Genetic Variations Of PPARGC1A Gene In Qinchuan Cattle

Peroxisome proliferator-activated receptor gamma coactivator1alpha (PPARGC1A or PGC-la), encoded by PPARGC1A gene, was a transcription coactivator. PGC-1a participaits in many metabolic pathways through interacting with series of transcription factors, for example adaptive thermogenesis, mitochondria biogenesis, glucose metabolism, hepatic gluconeogenesis, fatty acid oxidation, adipocyte differentiation and muscle fiber conversion. As PGC-la involved in energy homeostasis and regulation of fat metabolism, PPARGC1A gene was studied as a candidate gene on milk traits, meat quality and growth characteristics of domestic animals in animal breeding. In this study, the gene cloning, construction of adenovirus vector, cell culture, real-time quantitative polymerase chain reaction(qPCR), luciferase reporter assay, DNA pool sequencing, PCR-SSCP and PCR-RFLP technologies were emploied to carry on the Qinchuan cattle PPARGC1A gene cloning, gene expression regulating through gene overexpress and RNA interference techniques mediated by adenovirus, functional verification on the regulation of mitochondrial oxidative respiratory chain genes and myocyte proliferation and differentiation related genes, PPARGC1A gene promoter cloning and the core promoter region analysis, genetic variation scanning and its associated with growth traits. The main results obtained were as follows:1. The Qinchuan cattle PPARGC1A gene codons region was cloned, which contains a total length of2391bp nucleotides, encoding796amino acids. The recombinant adenovirus vector overexpress bovine PPARGC1A gene was generated. The titer of the adenoviral stock was determined by TCID50method and reaches6×109PFU/mL. The optimum multiplicity of infection (MOI) for bovine myoctye was150μL. The PPARGC1A promoting the expression of genes related to mitochondrial respiratory chain oxidation and bovine myocyte proliferation in the growth medium and differentiation in medium containing2%horse serum was verified through the Ad-PPARGCIA adenovirus infection of bovine myocyte.2. Three shRNA sequences targeting different area of bovine PPARGC1A gene (shRNA-574, shRNA-749, shRNA-1013) were designed using online software and synthesized. After annealing, shRNA templates were constructed into pENTR/CMV-GFP/U6vecor. By cotransfecting HEK293A Cell, the result shows that entry vector expressing shRNA-574and shRNA-749sequences caused an obvious interference effect. Then we generated recombinant adenovirus vectors pAd-shRNA-574, pAd-shRNA-749by LR recombination and adenovirus were successfully packed in HEK293A Cell. The titers of the adenoviral stock were3×109PFU/mL and8×108PFU/mL for Ad-shRNA-574and Ad-shRNA-749, which were determined by TCID50method, respectively. The optimum multiplicity of infection (MOI) of adenovirus Ad-shRNA-574and Ad-shRNA-749were both200μL for bovine myoctye. The adenovirus Ad-shRNA-574and Ad-shRNA-749had more interference efficient in the differentiation culture medium than that in the growth culture medium, with the efficency of41%and51%, respectively. The expression of mitochondrial oxidative respiratory chain related genes were reduced and the normal proliferation and differentiation of myocyte related genes were affected after the bovine PPARGC1A gene was interfered through Ad-shRNA-574and Ad-shRNA-749adenovirus infection bovine myocyte.3. Qinchuan cattle PPARGC1A gene promoter region was cloned with a lengh of2119bp including-2072bp and+47bp region according to the initiation codon. The recombinant vectors pGL3-Basic-Promoter containing different truncated promoter region fragments were constructed. It was suggested that the gene promoter core activity region was in the-2072bp to-2001bp through dual-luciferase reporter assay combined with software prediction results.4. Twenty polymorphic loci of bovine PPARGC1A gene were found by the DNA pool sequencing.13loci were located in exons and introns region:g.64897G>A, g.72175A>G, g.72202A>G, g.84063T>C, g.84163G>A, g.85272C>T, g.85329C>T, g.85352C>T, g.85400A>G, g.85442G>A, g.85540C>T, g.85609T>C, g.85631T>G;7loci were located in the promoter region:-259T>A,-301_-298insCTTT,-915A>G,-1175T>G,-1590C>T,-1665C>T and-1690G>A. Association analysis between growth traits and SNPs was carried out and found that SNP g.64897G>A sites was associated with body weight and average daily gain in24months age within Nanyang cattle, and individuals with genotype GG were the dominant ones.