Molecular Mechanism Of Gene Expression Of PPARδ And Myoglobin And Its Effect On Pork Color

Myoglobin is the important physiological basis of the color formation of meat. Our research used the Jinhua pig, which is Chinese native breed and foreign fine breed Landrace pigs as animal model and investigated the developmental changes of Myoglobin gene expression pattern and difference of breeds in M. Longissimus Dorsi muscle of Jinhua and Landrace barrows by real-time PCR and immunohistochemistry methods, and elucidated the close relationship of Myoglobin gene etc. with meat color and muscle fiber type and studied the molecular mechanism and effect of PPARδon the gene expression of Mb in vivo and in vitro, the main results are as fellows:1. The developmental expression pattern and breed differences of Mb and other related genes (PPARδetc.) in longissimus dorsi muscle of Jinhua and Landrace.We selected the Jinhua pig and Landrace pig as research objects to investigate the developmental changes of Mb and other candidate genes of meat color on the base of results of compared objective meat color measurement values with different ages of Jinhua and Landrace pigs, The results showed that:the objective meat color measurement values of Jinhua pigs basically changed in accordance with Landrace pigs and Hunter L* and b* values decreased with ages, while the a* values increased with ages. The change rules of SM concentration were similar in LD of two breeds, both were lower at birth and accumulated with ages but were significant (P< 0.05) in LD of Jinhua pigs and not significant in LD of Landrace pigs (P> 0.05). Mb gene expression was lower at birth in LD of Jinhua pigs, and increased significantly with ages (P< 0.05), and Mb gene expression was lower at birth in LD of Landrace pigs too, but increased significantly (P< 0.05) only at 120d and 150d; mRNA expression of PGC-la and PPARδwas similar with Mb, both were lower at birth in LD of Jinhua pigs; as regards breeds difference, Hunter L* values had no significance between the two breeds (P> 0.05), a* values were significantly higher in LD of Jinhua pigs except for birth (P< 0.05), b* values were significantly higher only at 120d and 150d (P< 0.05); Mb gene expression was higher in LD of Jinhua pigs than Landrace and had significant difference (P< 0.05) from the age of 60d to 120d and the changes of SM concentration were similar with Mb gene expression; the mRNA expressions of PGC-1αwere higher in LD of Jinhua pigs than in LD of Landrace pigs only at 120d (P<0.05), while the mRNA expressions of PPARδwere higher in LD of Jinhua pigs than in LD of Landrace pigs and were significantly higher from the age of 60d to 150d(P< 0.05). These results showed that intensity of meat color in LD had a tendency to increased gradually and the color was more redder in LD of Jinhua pigs than in LD of Landrace pigs, and the gene expression of Mb was at transcriptional control and the change rules of SM concentration and mRNA expression of Mb and PPARδwas similar.2. Analysis of the correlation coefficient between the investigated meat quality traits, SM, muscle fiber types and other candidate genes.This part of the experiments we also choosed the Jinhua pig and Landrace pig as research objects to investigate the developmental changes of four muscle fiber types (Ⅰ,Ⅱa,Ⅱx andⅡb) and differences of breeds. We found that the changes of mRNA expression of four MyHC with ages in LD of Jinhua pigs were consistent with Landrace pigs. The mRNA expression of MyHC I was higher at birth in LD of Jinhua pigs, and decreased significantly (P<0.05) and maintained at a stabilized level, while the changes of mRNA expression of MyHC I in LD of Landrace pigs were similar with Jinhua pigs but had no significant difference(P>0.05); The changes of mRNA expression of MyHCⅡa and MyHCⅡx in LD of two breeds were similar, both were higher at birth and decreased significantly (P<0.05) from birth to 120d but increased after that age stage and were significant (P<0.05) in LD of Jinhua pigs but not in LD of Landrace(P>0.05); The changes of mRNA expression of MyHCⅡb in LD of two breeds were also similar, both were lower ar birth and increased significantly (P<0.05) and maintained at at a stabilized level.In different breeds, the gene expression of MyHC I in LD of Jinhua pigs was significantly higher than Landrace pigs at the ages of birth,60d,90d and the other muscle fiber types didn't have any significance between two breeds. The gene expressions of MyHCⅠand MyHCⅡa in LD of Jinhua pigs were significantly higher than Landrace pigs at 120d (P<0.05), but the MyHCⅡb were inverse(P<0.05), then the gene expressions of MyHCⅠ, MyHCⅡa and MyHCⅡx were significantle higher than Landrace but the MyHCⅡb were inverse at the age of 150d. After Pearson's bivariate correlation analysis with SPSS 15.0 we found that, the absolute value of the correlation between Hunter a* values and Mb and PPARδgene expression and SM concentration were as high as 0.72,0.84 and 0.77, and the absolute value of the correlation between Mb gene expression and PPARδgene expression and SM concentration were as high as 0.78 and 0.82, these results showed that Mb had very close relationship with meat color and regulation of mRNA expression of Mb was mostly at transcriptional level and had close relationship with PPARδwhich means PPARδmay be the key regulator gene of gene expression of Mb.3. Study on the effect of PPARδon the expression of porcine moyglobin gene.On the basis of previous study, we established in vitro successsfully a culture system of porcine skelatal muscle satellite cells, using the PPARδagonist GW501516 and antagonist GSK0660 to explore the effect of PPARδon the gene expression of moyglobin. Results showed that, before induced with 2% HS, the gene expression of PPARδwas significantly increased (P <0.05) by using GW501516, and GW501516 can also inceased significantly the Mb and PGC-1a gene expression (P<0.05). PPARδinhibitor (GSK0660) suppressed significantly the PPARδmRNA level and the Mb and PGC-la mRNA level were also decreased significantly (P <0.05); Ater induced with with 2% HS, GW501516 can incease significantly the PPARδgene expression (P<0.05) and the Mb mRNA level were also inceased significantly (P< 0.05), after GSK0660 suppressed significantly the PPARδmRNA level (P<0.05), the Mb and PGC-1αmRNA level were also decreased (P<0.05). These results indicated that porcine PPARδcould affect the gene expression of Mb and PGC-la; Mb mRNA level was fluctuated with PPARδ.4. The effect and the mechanism of PPARδagonist GW501516 on the meat color, muscle fiber type and the expression of related genes (Mb etc.) in LD of C57BJ6 mice.This part of study aimed to further reveal the effect and mechanism of the PPARδagonist GW501516 on the meat color, muscle fiber types and related mRNA expression (Mb etc.) in mice.35-day-old C57B6J mice were used and randomly divided into 5 groups (10 per group, half male and half female). The treatments were as follows:normal control group, fed with normal saline solution;Ⅰgroup, gavage with 10mg/Kg·BW GW501516;Ⅱgroup, gavage with 20mg/Kg·BW GW501516;Ⅲgroup, gavage with 30mg/Kg·BW GW501516; andⅣgavage with 100mg/Kg·BW GW501516. The experiment period was 15d and the mice were weighted at day 0,6 and 15. At the end of the experiment, mice were killed by cervical dislocation for samples. The results showed that treatment with different concentrations of GW501516 for 15 days the body weight of mice significantly decreased (P<0.05); while increased the SM concentrations in skeletal muscle of mice (P<0.05). Treated mice with different dosees of GW501516 significantly increased MyHCⅠnd MyHCⅡx mRNA expression (P<0.05), while significantly reduced the MyHCⅡb mRNA expression in skeletal muscle (P<0.05), furthermore affected muscle fiber type. GW501516 treatment significantly increased (P<0.05) the mRNA and protein levels of PPARδ, as well as Mb, in the skeletal muscle of C57BJ6 mice. GW501516 treatment also increased significantly the PGC-1αmRNA expression (P<0.05). These results suggested that PPARδagonist GW501516 could regulate the gene expression of PGC-1αand Mb by activating the gene expression of PPAR8, thereby affecting the meat color and muscle fiber type.