Comparative Transcriptional Profile Of Embryonic Skeletal Muscle At Different Development Stages Between Chinese And Exotic Pig Breeds By LongSAGE Technique

Chinese indigenous pigs have great differences comparing to exotic pig breeds in meat production traits, such as growth, carcass and meat quality. Phenotype variations between breeds coming from different genetic backgrounds were directly determined by gene expression during skeletal muscle development. The muscle growth and development is complex processing involved in thousands of genes. Gene expression was subtly regulated in network mechanism. The production and meat quality of pork were largely detemined by the total number and cross sectionarea of skeletal muscle fiber. However, total number of skeletal muscle fibers was fixed at prenatal stage. In this study, transcriptome of skeletal muscle from pig embryo of Tongcheng (T) and Landrace (L) pigs at different stages (at 33,65 and 90days of gestation) were analyzed using long serial analysis of gene expression technique (LongSAGE), and cell ultrastructure of skeletal muscle was also observed through electronic transmission microscope. The novel LongSAGE tags were further extended using GLGI (generation of longer cDNA fragments from SAGE tags for gene identification) technique. Four novel swine genes that were represented by LongSAGE unique tags differentially expressed in libraries were cloned, mapped, and the temporal and spatial expression of these genes was detected. The results of this study were presented as follows:1. Transcrptional profiling of six Long SAGE libraries from T33 (50,450 tags), T65 (53,927 tags), T90 (53,761 tags), L33 (53,104 tags), L65 (54,483 tags), and L90 (51,188 tags) were generated and compared. A total of 317,115 tags, corresponding to 98,437 unique tags were obtained. A total of 14,683 unique tags with more than one copy at least in one library were detected. On average, 30.87% of unique tags could not be assigned to UniGene entries in NCBI database.2. The clustering results of the LongSAGE libraries showed that T65 was distinctive from other transcriptional profiles. L65 and L90 were merged firstly into a single cluster, and then T33 and L33 formed another group. Finally, T90, L65 and L90 formed a big group. The expression pattern of Tongcheng pigs changed more smoothly than those of Landrace. And the expression pattern changed more sharply at early stages (33-65) than later stages (65-90) during skeletal muscle development in Landrace. 3. The comparative analysis of the transcriptomes at the three different gestation stages identified 1,400 and 1,201 differentially expressed unique tags in Tongcheng and Landrace pigs, respectively. Of these differentially expressed unique tags, 832 (p＜0.05) were exclusively differentially expressed in Tongcheng pigs and 683 (p＜0.05) were in Landrace, respectively. The expression patterns of differentially expressed unique tags could be grouped into eight expression modes.4. The main biological process in which these differently expressed genes participate in myogenesis of Tongcheng pigs generally similar to Landrace. These processes mainly included cellular physiological process, metabolism, localization, cell communication, response to stimulus and development. More biological processes were found to be represented in Tongchenhg pigs. Furthermore, more differentially expressed genes be concerned with organic acid metabolism (6.70%vs.0.79%, p=0.017), amine metabolism (4.47%vs.0.79%, p=0.08), as well as amino acid and derivative metabolism (4.47%vs.0.79%, p=0.08) in Tongcheng pigs than Landrace. In contrary, more genes were involved in alcohol metabolism (5.56%vs.0.56%, p=0.009), nucleic acid metabolism (18.99%vs.29.37%, p=0.039) and positive regulation of metabolism (2.38%vs.0.00%, p=0.069) in Landrace than Tongcheng pigs.5. Signal pathway analysis indicated that there were 25 primary pathways involved in genes differentially expressed, and ribosome pathway was the leading pathway during skeletal muscle development in both Tongcheng pigs and Landrace. A number of genes associated with calcium signalling pathway, regulation of actin cytoskeleton and carbon fixation pathway were found in Landrace, while a number of genes associated with focal adhesion, ECM receptor interaction and apoptosis pathway were in Tongcheng pigs.6. A total of 653, 532, and 459 unqiue tags were differentially expressed at 65, 33, and 90 days of gestation between Landrace and Tongcheng pigs, respectively. Most unique tags were differentially expressed at 65 day of gestation between Tongcheng pigs and Landrace.7. A total of 113 ESTs were obtained from 67 novel LongSAGE tags using GLGI method. BLAST analysis showed that 100 ESTs did not assigne to any sequence, and which indicated that these ESTs may potentially deriv from novel genes or transcripts.8. The cDNA sequence of TOB1,FSCN1,OLFML3 and NPM genes were isolated and cloned together with the second intron of OLFML3 and fourth intron of NPM. TOB1, FSCN1, OLFML3 and NPMgene were assigned to SSC12, SSC3, SSC14 and SSC16, and linked to SS04H11 (LOD=5.52), SSC10E12 (LED=8.03), IGLV (LED=5.26) and SW977 (LOD=9.16) by IMpRH panel, respectively. Furthermore, NPM gene was mapped to SSC16q21 using SCHP panel.9. The temporal and spatial expression of TOB1,FSCN1 and OLFML3 genes were examined using quantitative real time PCR (qPCR) method. Expression level of gene TOB1 is highest at 65 day of gestation in Tongcheng pigs. Secondly, it was highly expressed in adult pig. Spatial expression profile of TOB1 showed that it was expressed in many tissues from Wuzhishan pigs, while it had abundant expression in skeletal muscle. Expression of FSCN1 peaked in prenatal skeletal muscle at 65 days of Landrace and 90 days of Tongcheng pigs, respectively. And FSCNI was most highly expressed in brain from Wuzhishan pigs. OLFML3 gene was down-regulated during skeletal muscle development, and hardly was examined in postnatal skeletal muscle. This gene was abundantly expressed in stomach, liver and fat tissues, and rarely expressed in skeletal muscle from adult Wuzhishan pigs.10. The ultrastructure of embroynic skeletal muscle via electronic transmission microscope suggested that Landrace had longer sarcomere and more obvious structure of myofiber than Tongcheng pigs. However, compared with Toncheng pigs, more typical characteristic were observed in Landrace, such as sarcomere with A and I region, and Z-lines. Muscle cell had more mature structure at 65 days than 33 days of gestation, and more abundant hepatin granules were distributed in Landrace than Tongcheng pigs. The assignment of myofibrils in Landrace was more compact than in Tongcheng pigs at 90 days of gestation.