Screening, Identification And Function Analysis Of Genes Differentially Expressed In Porcine Skeletal Muscle Between Meishan And Yorkshire Pigs

Pork is a major part of human nutrition containing essential protein ingredients, and pigs could be considered a model species for human health issues. So understanding the growth and development of porcine skeletal muscle is one of the most important goals in animal and meat science and is also related to particular aspects of human medicine. In order to investigate the molecular mechanisms underlying porcine skeletal muscle growth and development and meat quality traits, some work had been done. The results are as follows:1. We investigated the expression of 10 commnly used reference genes in porcine skeletal muscle using quantitative PCR. Both geNorm and NormFinder were used to evaluate the expression stability of these 10 genes in all samples, Yorkshire and Meishan, respectively. As the results of geNorm was preferred over that of NormFinder, PPIA, HPRT and eEF-1γwere suitable internal controls for all samples, and PPIA, HPRT and SDHA were suitable for samples of Yorkshire while PPIA, eEF-1γand HPRT for samples of Meishan. GAPDH and ACTB were not suitable as reference genes. The results of our study could provide recommendations for choice of reference genes in gene expression studies concerning porcine skeletal muscle in the future.2. We measured the expression of four myosin heavy chain in porcine longissimus dorsi muscle at four developmental stages in Meishan and Yorkshire pigs. The principle that how four myosin heavy chain distribute during the development process were in the "Ⅰ<->Ⅱa<->Ⅱx<->Ⅱb" balance. The II x to II b fibre type transformation in Yorkshire pigs was later than Meishan pigs.3. We compared the transcriptional profile of porcine longissimus dorsi muscle at 65 days post coitus,3 days,60 days and 120 days afterbirth between Meishan and Yorkshire pigs using microarrays. Most genes differentially expressed at 65 days post coitus and 60 days afterbirth, while few genes at 120 days afterbirth, suggesting 65 days post coitus and 60 days afterbirth as important stages leading to differences in meat quality traits between Meishan and Yorkshire pigs.4. According to the number of genes differentially expressed during the whole processs, we found pathways differed between the two pig breeds, including focal adhesion, MAPK signaling pathway, actin cytoskeleton regulation, Jak-STAT signaling pathway signaling pathway, insulin signaling pathway, Wnt signaling pathway, TGF-beta signaling pathway, cell communication, calcium signaling pathway, fatty acid metabolism, cell cycle and other signaling pathways. And ROCK2 gene is involved in many of these pathways.5. The expression pattern of ROCK2 gene differed between Meishan and Yorkshire pigs. ROCK1 and ROCK2 have been reported to play important roles in skeletal muscle growth and development. Genes with the same expression pattern as ROCK1 and ROCK2 were involved in most of the differential pathways between two pig breeds. As a result, ROCK1 and ROCK2 were considered as the candidated genes important for porcine skeletal muscle growth and development.6. More information could be obtained comparing transcriptome data with proteome data of longissimus dorsi muscle at four developmental stages of Meishan pigs, and the transcriptional data was consistent with the proteomic data of Meishan pig.7. We cloned most of the cDNA sequence of ROCK1 and ROCK2 genes using PCR and 5’RACE, two transcripts were found for two genes. We explored the temporal and spatial expression pattern of two genes. ROCK1 and transcript ROCK2 were ubiquitously expressed in different tissues and expressed in testis when the pig became mature. Transcript ROCK2m were specifically expressed in skeletal muscle. ROCK1 was highly expressed in fetal period, while ROCK2 was highly expressed in period of rapid growth. The 5’flanking sequence were obtained and the core promoter region was confirmed to be located from-1210bp to-870bp (ATG was set as+1) using 5’-deleted fragment recombinant vector construction and the transcription activity measurement. Three SNPs in ROCK2 coding sequence were investigated with pyrosequencing and associated with carcasss and meat quality traits.