| Meat quality is controlled by many factors and genetic is the mainly factor influencing on pork quality. The glycogen content in muscle affects the meat quality traits of pigs that include ultimate pH,color measures,tenderness and cooking loss.The glycogen metablism has a relationship with meat quality, So many researchers focus on the glycogen metabolism. It will be useful for further investigation on the molecule mechanism of the pork quality. In this study, we focus on the important glycogen metabolism signalling transduction pathway (IGF1-PI3K-AKT-GSK3β).The important genes in this pathway were obtained. Moreover, their potential involvement in the mechanism of action of insulin is discussed in porcine satellite cells.The main results are as follows:1.The full-length of coding sequence of porcine GYS1 and GYS2 genes were cloned. Porcine GYS1 and GYS2 showed the highest homology toward their catalytic domains in the central region. The main structural differences between GYS1 and GYS2 isoforms lie in the N and C-terminal regions, which contain the known phosphorylation sites.2.GYS1 was detected in all eleven tissues, and GYS2 was only detectable by RT-PCR in liver and fat. Fibres showing granular cytoplasmic accumulations of GYS1 are seen in porcine muscle, and porcine liver sections showed strong positive staining within the hepatocyte cytoplasm.3.Association analyses revealed that both the GYS1 Hin6I and MvaI polymorphisms had significant associations (p<0.05) with pH of M. longissimus dorsi (pHLD), pH of M.biceps femoris (pHBF) and pH of M. semipinalis capitis (pHSC) at post slaughter 45 minutes.4. The full-length of coding sequence of porcine GSK3αand GSK3βgenes were cloned. The difference in size is due to a glycine-rich extension at the N-terminus of GSK-3α. Although highly homologous within their kinase domains (98% identity), the two gene products share only 36% identity in the C-terminal residues.5.During the cloning of porcine GSK3β, sequencing of 32 clones from different tissues cDNA pool revealed five forms of GSK3βmRNA. We named GSK3β1,GSK3β2, GSK3β3,GSK3β4,GSK3β5 respectively.6.The expression level of different GSK3βisoforms in different embryo and adult tissues were detected by qRT-PCR.7.The cellular locations of each GSK3βisoform were determined by fluorescence and confocal analysis of PK15 cells transiently transfected with different GSK3β-GFP vectors respectively. GSK3β1-GFP,GSK3β2-GFP and GSK3β5-GFP were all found to localize in cytoplasm, however, GSK3β4-GFP displayed a nuclei distribution.8.Porcine myogenic satellite cell culture and identification. The pax7 and myogenin expression profile was used to for monitoring the capacity of satellite cell progeny to proliferate and differentiate.9.The effect of insulin on the expression of different porcine GSK3βtranstripts.10. The expression level of porcine GYS1 is up-regulated in the porcine satellite cells from proliferation to differentiation, but can not be induced significantly during differentiation by insulin.11.Insulin promotes phosphorylation of GSK3βand dephosphorylation of GS in differentiated porcine satellite cells.12.The promoter regions of porcine GYS1 and GYS2 genes were obtained by genomics walking. The promoter of porcine GYS1 contained several putative transcription factor binding sites (PPARs, HNF4a, NF-kappaB and CREB).13.Treatment with insulin resulted in a dramatic reduction in reporter gene activity of the deletion fragment of GYS1 promoter.14. The overexpression of GSK3P5 isoforms increases the activity of pGYS1-175/+60, whereas GSK3β1,GSK3β2 and GSK3β4 induce the activity of pGYS1-175/+60.15.To assess the validity of the microarray approach to identify differentially expressed genes (AMPD1 and ALP),qRT-PCR was performed. We also cloned and characterized (AMPD1 and ALP) from porcine muscle. Moreover, we analyze the expression patterns of them during skeletal muscle development and in four different muscles contained different muscle fibre types.
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