Isolation And Cloning, Expression Patterns And Functional Study Of Porcine Muscle Related Six1 And Pitx2c Genes

Meat is the main source of human nutrition, which contains many important proteins. Improvement in the quality of meat would likely increase consumer acceptance and perception of meat, and promote the consumption of meat. Thus, the quality of meat would affect the sustainable development of the pig industry directly. In the past decades, growth rate, food conversion ratio and lean meat content had been improved efficiently by traditional breeding methods, whereas the strength selection on these traits led to dramatic consequences, such as abnormal behaviors, physiological dysfunction, decreased immunity, and increase the incidence of pale, soft and exudative meat (PSE) and reduce the intramuscular fat content (IMF). Currently, most of the studies suggest that genetic factors are the main factors affecting meat quality, but the molecular mechanisms were still unclear. Skeletal muscle is the main component of carcass, the growth rate of muscle would determine the growth rate of pigs directly. Skeletal muscle is mainly composed of muscle fiber, whereas different characteristics of muscle fiber types exert different effects on meat quality involving in meat color, pH value, tenderness, intramuscular fat content, water holding capacity, flavour, etc. Therefore, the understanding of growth and development of skeletal muscle, the formation and transformation of muscle fiber types would be useful for revealing the molecular mechanism of meat quality. Previous studies have shown that Six1 gene and Pitx2c gene were closed to the formation of muscle. Hence, both of them were chosen as candidate genes that influences the growth and meat quality of pigs in present study. Starting from the isolation and cloning of both of the genes, and comprehensive analysis of the genes expression patterns were studied, further polymorphisms and association of both genes were analyzed and the mechanisms of expression and regulation of Six1 gene was investigated systematically. The present study may provide more theoretical evidences for the molecular mechanism of the formation of meat quality. Following are the main results:1. Six1 and Pitx2c genes were isolated using PCR and RACE in present study. The DNA full-length of Six1 gene was obtained containing 4248bp, while the full-length cDNA of Six1 gene contains 2201bp. The cDNA full-length of Pitx2c gene contains 2316bp, while the first intron length of Pitx2c gene was 1482bp.2. The expression patterns of both of the genes were analyzed completely, including the expression patterns in different tissues, the expression patterns at different development stages and expression patterns in different muscles. (1) The tissue distributions of Six1 and Pitx2c genes were analyzed using semi-quantitative RT-PCR. The results showed that both of the genes were highly expressed in skeletal muscle. (2) The expression patterns at different development stages were analyze using real-time PCR, the results showed that both of the genes were highly expressed at stage of 65 days post conception (65 dpc), and the expression levels of Six1 gene in Yorkshire pigs at 60 days and 120 days after birth were significantly higher than that of Meishan pigs. While the expression levels of Pitx2c gene in Yorkshire pigs at 65 days post conception, and 21 days,60 days and 120 days after birth were higher than that of Meishan pigs. (3) The expression patterns in different muscles was obtained using real-time PCR, the results showed that both of the genes were expressed higher in fast muscle (biceps femoris) than that in other tested muscles.3. Genetic diversity of several SNP sites in both of the genes in different pig breeds was analyzed, their genetic effects on economic traits in F2 resource population involving Yorkshire and Meishan cross pig were further analyzed. The analyzed polymorphic sites of Sixl gene included:(1) The A/G transition mutation at 1595bp position in intron. Association analysis showed that A/G transition mutation polymorphisms were significantly associated with the following meat traits:meat color value (MCV1) (P< 0.05) and meat marbling (MM1) (P<0.01) of longissimus dorsi, and dressing percentage (DP) (P<0.05). (2) The C/T transition mutation at 136bp position in promoter (position relative to transcription start site). Association analysis showed that C/T transition mutation polymorphisms were significantly associated with the following meat traits:meat color of biceps femoris (MCV2) (P<0.05), intramuscular fat of longissimus dorsi (IMF) (P<0.05), shoulder fat thickness (SFT) (P<0.05) and ratio of lean meat versus fat meat (RFL) (P<0.05). (3) The C/T transition mutation at 1363bp position in promoter (position relative to transcription start site), Association analysis showed that C/T transition mutation polymorphisms were significantly associated with the following meat traits:meat pH of longissimus dorsi (pH LD) (P< 0.05) and biceps femoris (pH BF) (P<0.05), drip loss rate (DLR) (P<0.05) and water holding capacity (WHC) of longissimus dorsi (P<0.05). The analyzed polymorphic sites of Pitx2c gene included:(1) Two C/T nonsense mutations at 474bp and 636bp positions in coding region, respectively (position relative to translation start site). The linkage disequilibrium analysis showed SNPs c.474C>T and c.636C>T in the coding region were in LD with each other (r2=0.85). Association analysis showed that the polymorphisms of c.474C>T (P<0.01) and c.636C>T (P<0.05) were significantly associated with meat color value (MCV1). (2) Two G/A transition mutations at 37bp and 47bp positions in 3’untranslated region (3’UTR), respectively (position relative to translation stop code). The linkage disequilibrium analysis showed SNPs c.*37G>A and c.*47G>A in 3’UTR were in high LD with each other (r2=0.95). Association analysis showed that the polymorphisms of c.*37G>A and c.*47G>A (P<0.05) were significantly associated with meat color of longissimus dorsi (MCV1) (P<0.05), drip loss rate (DLR) (P<0.05) and water holding capacity (WHC) (P<0.05) of longissimus dorsi. Association analysis between haplotype combinations and meat quality traits showed the haplotype had a significant effect on meat color of longissimus dorsi MCV1 (P<0.05) and the presence of two copies of haplotype 1-CCGG- may improve meat quality.4. The core promoter of porcine Six1 gene had been identified using nested deletions method combing the dual-luciferase reporter assay and the effect of MyoD on the promoter activity was analyzed, further the effect of methylation of Six1 gene promoter on promoter activity was also analyzed. The results indicated that the core promoter of porcine Six1 gene was presented in the region of -360bp/+170bp, which contains two common upstream promoter elements GC box and CATT box. Relative luciferase activities analysis indicated that the activities of all deleted promoter in C2C12 cells were higher than that in C3H10T1/2 and PK15 cells expect P2-530 promoter (-360/+170bp). Interestingly, the expression level and promoter relative activities of Six1 gene were down-regulated in the differentiated C2C12 cell. Furthermore, the results of co-transfection indicated that MyoD could activate the promoter activities of Six1 gene, whereas methylation of promoter could inhibit the activities of Six1 gene promoter.5. The methylation levels of promoter (Region 1) and the first exon (Region 2) of porcine Six1 gene were detected in different tissues and different muscle. The results from different tissues showed that the average methylation levels were the lowest in muscle. The results from different muscles showed that the average methylation level of two methylated sites in region 1 was the lowest in masseter, while the average methylation level of ten methylated sites in region 2 was lowest in biceps femoris.6. The subcellular localization of porcine Six1 protein different deletion fragments were analyzed, the results indicated that full-length CDS and HD domain were mainly located in the nucleus, the 3’domain-free zone (ND) and HD+ND were located bias in the nucleus, whereas SD domain was located in the cytoplasm.7. The effects of Six1 gene in the regulatory networks of myogenic were analyzed using overexpression technology. The results from transient and stable transfection experiments showed that overexpressing Six1 gene could promote the expression of myogenic determination factors (Myf5, myogenin and MyoD) and fast-type muscle genes (Atp2a1, Srl and Mylpf), but the expression levels of Pitx2c and Pax3 genes, which are the upstream regulatory genes of MRFs, were unaffected.8. The effects of Six1 gene on proliferation capacity and cell cycles were analyzed using MTT method and flow cytometry, respectively. The results of MTT showed that the capability of cell proliferation was started to be influenced after 48h culture in overexpring Six1 C2C12 cells, and showed significant inhibitoty effects at the stage of 72h to 96h. Cell cycles were further analyzed using stable and transient transfections. The results from stable transfection showed that Six1 gene could affect the cell cycles mainly by promoting the cells arrest in G2 phase, while the results from transient transfection showed that Six1 gene had an effect on cell cycles, reflecting in the reduction of the cells in S phase and the increase of the cells in G1 phase.