| Over the years of selection, one of the most important goals of pig breeding is to increase the growth rate and lean rate, and get some genetic peogress on pig species. Lean hog breeding leads the proportion of glycolytic fibers increased in pigs, and also muscle fiber diameter increases. Pork tenderness, juiciness, flavor, flesh, intramuscular fat and other meat quality factors such as pH value were severely affected.Previous studies have found there is a positive correlation in type I fibers, intramuscular fat content and muscle tenderness, while the fiber diameter, pH45MIN, drip loss was negatively correlated with type Ⅰ fibers. Type ⅡB fiber has larger cross-sectional area, leads the reduction of pork hydraulic system and shear force, Type ⅡB fibers contain a lot of glycolysis enzymes, at 45 minutes after slaughter, muscle glycogen and lactate increased, decreased muscle pH, flesh-colored gray, and low water holding capacity, drip loss also showed a positive correlation with type ⅡB fibers.PGC-la is a key regulator of cellular energy metabolism, involved in the regulation of mitochondrial generation, regulation of mitochondrial fusion and fission. Experiments in mice showed that over-expressed PGC-lain skeletal muscle stimulates mitochondria generate and promote the skeletal muscle fiber type ⅡB swiching to type I, increasing the proportion of the oxidized fibers.Therefore, PGC-la, as important gene in muscle fiber change, was constructed in a muscle-specific eukaryotic expression vector. Make the transgenic model, and analyse how the PGC-la work on nergy metabolism of porcine muscle tissue, muscle changes, and the impact of pork meat quality. The main results are as follows:1. Based on the eukaryotic expression vector pEGFP-N1, construct eukaryotic expression vector p-sMCK-sPGC-la.7kb MCK promoter that obtained by homologous recombination of pig BAC library, promote PGC-la code sequence.2. By pronuclear microinjection of plasmid p-sMCK-sPGC-la, we obtained four positive transgenic mice and use it to proliferation offspring. On RNA and protein levels, we analyzed the sPGC-la’s influences on mice muscle tissue, and found that porcine PGC-la can promote mouse mitochondrial generation and stimulate formation of murine red muscle fibers, increase muscle oxidative, addition sPGC-la also regulates the expression of PDK4, thus affecting glucose metabolism and fat metabolism in mice.3. Use 30-day-old porcine embryo fibroblasts cell as host, transfect with p-sMCK-sPGC-la plasmid, after screen we got monoclonal cell which stablly express the p-sMCK-sPGC-1α.4. By somatic cell nuclear transfer, positive transgenic pigs were prepared. Analysis of the vector integration sites and copy numbers in the original transgenic pigs, we found the integration sites of the vector are mostly located on the X chromosome, and has varies copy numbers, the average copy number is greater than 20. From morphology of pig muscle, RNA expression levels, ATP activity, we analyzed the impact of muscles sPGC-la over expression, result showed that PGC-1α promote porcine muscle mitochondria generated and stimulate swith white muscle fibers into red muscle with increased muscle oxidation. PGC-la was also regulated PDK4 gene expression, thus affecting porcine muscle glucose metabolism and fat metabolism. Additional, by measuring meat quality we found that a number of genetically related indicators have significantly improved the quality of pork, such as marbling, drip loss, water loss rate, water holding capacity, intramuscular fat, tenderness, etc., illustrate the PGC-la influence pork meat by regulating a type of fiber changes. It laid a theoretical foundation for cultivate high-quality pig muscle fibers. |
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