| Pig serves as an important source of protein for human consumption as one of the major meat animals, thus pork industries have been pursuing higher lean growth rate in pigs for many years. However, with the development of life level, better meat quality is becoming more and more important in pig breeding in order to meet the pork market demands. The Tongcheng pigs originate from Tongcheng County, Hubei Province, and is one of the famous formal authorized Chinese indigenous pig breeds for its perfect characteristics, such as highly intramuscular fat content, low managing requirement, delicious flavor and so on. It was reported that lean meat percentage and meat quality of pig were largely influenced by the content and structure of skeletal muscle. The number of muscle fibers is determined by the prenatal myofibrils hyperplasia, while the fiber sizes are mainly determined in a postnatal hypertrophy. Large Numbers of genes have been reported to take part in the network regulation of muscular development, but the mechanism is still unclear. Nowadays, with the rapid development of molecular technologies and the further research in pig structural genomics and functional genomics, it is possible to scan genes related to muscle development with transcriptome analysis. The objective of this thesis was to obtain the embryonic skeletal muscle transcriptome of Tongcheng pigs, and monitor the gene activities in different developmental stages. The main results were as follows:1. Two LongSAGE libraries were constructed with the total mRNA from 33 and 65 days old fetal skeletal muscle. 50,450 and 53,927 total SAGE tags were obtained, respectively, which represented 44,558 unitags combined. The tag abundance distribution of the two libraries was similar: 80% tags were expressed in one copy, and only 0.1% tags are expressed more than 100 copies. Gene mapping results showed that 70% tags were matched to Unigene sequences, and the matched tags were composed of 64% singletons and 36% multiple matched tags.2. The transcriptome analysis showed that the genes in T33 and T65 were similar to each other, and mainly involved in the following biological processes: protein biosynthesis, signal transduction, transcription regulation, cell proliferation, metabolism, immune response and so on. In addition, a number of transcription factors with low abundance were detected in the two libraries.3. The comparative transcriptome results displayed that the genes related to metabolism of fat and fatty acid and cell adhering in T33 were more than that in T65, but, the situation for genes involved in transcription regulation was reverse. 4. Expression analysis revealed great numbers of genes differentially expressed at T33 and T65. 506 tags were up regulated, including 71 annotated genes in pig, and other 170 ESTs and unmatched tags. Only 352 tags showed down regulation pattern, which included 55 annotated genes and 118 EST or unmatched tags. 117 and 167 tags were restricted there expression to T33 and T65, respectively. The up regulated genes with difference folds higher than 2 were mainly muscular structural genes (e.g. TNNT2 and TNNI2), collagens, cytochrome c oxidase, NADH dehydrogenase and ATP synthase. Ribosomal protein (RP) factors were found differentially expressed between T33 and T65. 18 RP factors, such as RPL11 (60S ribosomal protein L11), RPL12 (60S ribosomal protein L12), RPL35 (60S ribosomal protein L35), RPS15 (Rig-analog DNA-binding protein) were up regulated, and 26 RP factors were down regulated, such as RPL21, RPL3, RPL38, RPS21, RPS26 and RPS28. Several genes showed more than 10 difference folds were also detected in this research, such as CKM (Muscle creatine kinase), VPS33B (vacuolar protein sorting 33B (yeast homolog)) and SLN (Sarcolipin) were up regulated with 24, 13.7 and 16.7 difference folds, respectively.5. The partial cDNA sequences of three up regulated genes BIN1, TNNT2 and TNNI2 were obtained with RT-PCR methods and alterative splicing transcripts for BIN1 and TNNI2 were found. The expression profile revealed that all the three genes showed highest expression in pig skeletal muscle tissue.6. The INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel was employed to determine the precise location of the three genes. The results showed that BIN1 was located to SSC15, closed to SW1118 with LOD score threshold 7.14 and TNNT2 gene was localized to SSC10, closed to SWC19 with LOD score threshold 5.45.All the above results may be of benefit to the better understanding of pig fetal muscle development, especially for Chinese pig breeds, and promote molecular breeding technologies application in pig breeding. |
