Cloning,Expression Patterns And Promoter Region Study Of Porcine Muscle Related PGM1 And UGP2 Genes

The genes function of phosphoglucomutase 1 (Phosphoglucomutase 1, PGMl) and UDP-glucose pyrophosphorylase 2 gene (UDP-glucose pyrophosphorylase 2, UGP2) are the frist study in pigs. It is not clearly the specific function and expression in pigs that the gene of porcine PGM1 and UGP2. Clustering analysis reveal that some genes had influence on porcine carcass characteristics and meat quality, including porcine PGM1 and UGP2. Moreover, the porcine PGM1 gene in protein expression screening has obvious difference. The protein of UGP2 is involved in glucose metabolism of an enzyme. There are not functional reports about them so far.The objective of this study is to identify PGM1 and UGP2 gene functions in pigs, in the hope of finding new candidate genes, which relate to meat quality improvement. The DNA samples are extracted from Yorkshire and Meishan pigs, which is distributed and expressed the genes. The gene of porcine PGM1 and UGP2 is detected in different developmental stage of skeletal muscle, and the promoter region of PGMl and UGP2 is separated and identified. It implies that the gene is important in the development of muscle. The results are as follows:1. By the bioinformatics method, the gene of porcine PGM1 is 2481 bp of mRNA sequence, and predicts 1689 bp of the ORF. There are 11 exons and 10 introns, and intron shearing mode accords with the "GT-AG" rule. The protein is encoded by 562 amino acids, about 62 KDa, and pI is 6.07. The protein, which is located in cell membrane, is a hydrophilic protein as the stability of proteins typical.The mRNA sequence of porcine UGP2 is 2056 bp, and the ORF length is 1527 bp. There are 10 exons and 9 introns, and intron shearing mode accords with the "GT-AG" rule. The protein is encoded by 508 amino acids. The molecular weight of protein is about 57 KDa and pI is 7.69. The protein, which is located in cells, is a hydrophilic protein as the stability of proteins typical.2. By phylogenetic analysis, porcine PGM1 and UGP2 is the nearest with Bos taurus, following by Pongo abelii, Homo sapiens, Macaca mulatta, and Oryctolagus cuniculus. Two genes are highly conserved in evolution.3. In the present study, we cloned both the complete genomic DNA sequence and 5’-flanking promoter sequence of porcine PGM1 and UGP2, and determined the genomic structure. It is 2311 bp and 2077 bp for the 5’-flanking promoter sequence, respectively. Following the 5’-flanking delete sequence method, the 5’-flanking promoter sequence is deleted different lengths from transcriptional site (ATG).The 5’-flanking promoter sequence of PGM1 is deleted 6 fragments, and upstream 1343 bp sequence from transcriptional site (ATG) has the greatest activity. The 5’-flanking promoter sequence of UGP2 is deleted 7 fragments, and upstream 588 bp sequence from transcriptional site (ATG) had the greatest activity.4. Real-time RT-PCR revealed that PGM1 and UGP2 are highly expressed in skeletal muscle. At the same time, the two genes are higher expression in the liver. It implies that the gene is important in carbohydrate metabolism.5. Among different types of muscle fibers, the PGM1 presents highest expression both in Longissimus dorsi and Biceps Femoris. PGM1 show a similar trend of expression that it concentrated expression of the pre-formed in the muscle fibers at different growth stages of Meishan and Yorkshire pigs. And the UGP2 presents the highest expression both in soleus muscle and Longissimus dorsi. UGP2 show a trend of expression that it concentrated expression of the pre-formed in the muscle fibers at different growth stages of Meishan and Large White pigs.6. At the Large White and Meishan pigs of different developmental stages of skeletal muscle, the expression of porcine PGM1 gene is similar and more important in pig early postnatal, and with the growth shows a downward trend.The porcine UGP2 gene expression trend is not consistent at different developmental stages of skeletal muscle. In Large White pigs, UGP2 is high expression at the early stage of development, but it is on the contrary in Meishan pigs. UGP2 has low expression level in the embryonic and early development, and it is gradually increase with the growth.