Study Of Differential Expression And Polymorphisms Of Genes Associated With Skeletal Muscle Development In Pigs

The first part of this dissertation focused on the differential expression of extracellular matrix and cellular adhesion (ECMs and CAMs)genes in skeletal muscles from different types of pig breeds.Extracellular matrix and adhesion molecules are involved in the regulation of cell proliferation, differentiation, adhesion and migration, playing important roles during skeletal muscle growth and development. In this study, we used the cDNA microarray containing 96 extracellular matrix and adhesion (extracellular matrix proteins, cellular adhesion molecules, proteases and its inhibitors) genes to screen the differently expressed genes in skeletal muscles from Landrace, Tongcheng and Wuzhishan pigs at 65 days post copulation (dpc) (L65, T65 and W65). Subsequently, the analysis of clustering and enrichment of biological process and molecular function of gene ontology as well as the pathway was performed using DAVID database. In addition, 10 differently expressed genes were selected for validation of microarray results by QPCR. Subsequently, 5 differentially expressed genes (ITGA7, ITGB3, CAV1, FN1å’ŒMMP2) were selected to comparatively analyze their expression profiles in skeletal muscles during gestation and after birth in Landrace and Tongcheng pigs. Microarray results showed that a total of 35 genes were differently expressed among the three breeds, of which 18 were in L65/T65 (15 upregulated in L65), 18 were in L65/W65 (13 upregulated in L65) and 20 were in T65/W65 (14 downregulated in T65); the differential expressed genes between W65 and L65 or T65 mostly focused on genes encoding extracellular matrix proteins and cellular adhesion molecules; the larger number of upregulated genes involved in positively regulating skeletal muscle development were detected in L65, a smaller number was inT65 and W65. Cluster analysis showed that ECMs and CAMs genes had a more similar expression pattern between L65 and T65, while the enrichment analysis of differently expressed genes showed that they were mainly involved in GO biological process of cell adhesion, cell-matrix adhesion and integrin-mediated signalling pathway, and the KEGG pathways of focal adhesion, ECM-receptor interaction and regulation of actin cytoskeleton, indicating these genes closely involving skeletal muscle development in pigs. The results of validation for microarray results showed that our microarray data were reliable and differential expression patterns of ECM and CAM genes in skeletal muscles were present among the three pig breeds. The results of genes expression patterns showed that the expression levels of ITGA7, ITGB3, CAV1, FN1 and MMP2 genes were significantly higher than those detected after birth in both Landrace and Tongcheng pigs; the differential expression patterns of these five genes were mostly detected during gestation rather than after birth, and a greater range of their high expression levels were observed in Landrace than Tongcheng pigs during gestation. In summary, ECMs and CAMs genes were differentially expressed among the three pig breeds, and mainly participated in the regulation of skeletal muscle development during gestation in different pig breeds. A series of particular molecular events involving extracellular matrix and adhesion molecules were found in Wuzhishan pigs. This part of study will be beneficial to further elucidate the molecular mechanisms underlying phenotypic variation, and provide helpful resources for the identification of genes associated with meat production traits in pigs.The second part of this dissertation focused on the study of MSTN polymorphisms in pigsMyostatin (MSTN) is a key negative regulator during muscle growth and development. The mutations in MSTN gene can result in the inactivation of its functional activity, subsequently inducing the double-muscle phenotype in many animals. In this study, we selected MSTN gene as a candidate gene to sequence its complete DNA sequence containing promoter and 3'UTR in 76 pigs including Landrace, Largewhite, Duroc, Laiwu, Tongcheng and Wuzhishan pigs using PCR method. A total of 16 SNPs were identified, of which 4 were in the promoter, 5 were in the first intron and 7 were in the second intron. Two SNPs (P4 and P5) only showed wild-type and heterozygous mutant-type genotypes, respectively. Subsequently, we selected nine SNPs (P1, P3, P4, P5, P6, P7, P8, P9 and P10) to study the distribution of their genotypes in 372 pigs using MALDI-TGF-MS technology, and analyzed the association between their polymorphisms and body weight using the method of least square mean. The results showed that the polymorphisms in P1, P3, P4 and P5 were identified in the two groups, and P4 and P5 only showed wild-type and heterozygous mutant-type genotypes which were completely linked. The results of association between polymorphisms and body weight showed that pigs with heterozygous mutant-type genotype had a significantly higher birth weigh and 21 days-weaning weight than those with wild-type genotype in P4 and P5 (p<0.05), respectively. The combined analysis of P4 and P5 showed that pigs with TAGA genotype had a significantly higher birth weight and 21 days-weaning weight than those ones with TTGG genotype (p<0.05), respectively. In addition, the mutation in P4 was showed to introduce two binding sites for CdxA transcription factor using TFSEARCH tool. In conclusion, the polymorphisms in P4 and P5 of MSTN gene significantly associated with birth weight and 21 days-weaning weight, possessing potential application values for molecular marker-assisted selection in pig breeding.