Screening Of Key Genes Modulating Intramuscular Fat Deposition And Functional Study Of PFLJ

Intramuscular fat content is a key index of pork quality, not only affect pork color, flavor and tenderness, but also on water-holding capacity. Jinhua pig, one of Chinese excellent local pig breeds showing excellent meat quality traits, its intramuscular fat content is higher, with redder pork when compared with European commercial pig breed such as Landrace. Therefore, Jinhua pig and Landrace pig were used as experimental animal model in this study. This study obtained different gene expression profiling in longissimus muscle of Jinhua pig and landrace pig by microarray, bioinformatics analysis on the basis of the comparison of carcass traits and meat quality between two breeds. The next, validation of microarray data using semi-quantitative or real-time quantitative PCR was conducted. Furtherly, we obtained cDNA full length of pFLJ gene, one of new genes modulating intramuscular fat deposition through the RACE technology, and then investigated the mechanisms of pFLJ regulating intramuscular fat deposition using RNAi technology and gene over expression technology at transcription level. The primary results are as follows:1. Comparative Study of carcass traits and meat quality between Jinhua pig and Landrace pigsThe overall appearance of a typical adult Jinhua pig is very different from a Landrace pig. Meat quality and carcass traits were compared between Jinhua and Landrace pigs at the same age (d30, d60, d90, d120, d150, days of age), respectively. Our results showed that from age of d30to d150, on average, Jinhua pigs gained approximately40kg of the weight, whilst Landrace gained about70kg, demonstrating that Jinhua pigs were growing apparently slower than Landrace. Analysis of the lean meat ratio (LMR) and loin meat area (LMA) showed that LMR and LMA were both significantly lower in Jinhua pigs from the age of d30to d150. In contrast, Jinhua pigs exhibited significantly higher back fat thicknesses (BFT) and fat meat ratios (FMR) than that in Landrace pigs (P<0.01). For example, at d150, BFT in Jinhua pigs is about two folds higher than that of the Landrace pigs (23.7mm in Jinhua pigs versus12.0mm in Landrace pigs). FMR in Jinhua pigs is about2.4folds higher than that of the Landrace pigs (32.4%in Jinhua pigs versus13.3%in Landrace pigs). We determined the pH45values at d30, d60, d90, d120and d150for both Jinhua and Landrace pigs, and found that pH45values, all ranged between6.0-6.5, did not show statistically significant differences between two breeds. Meat color parameters (L*, lightness; a*, redness; b*, yellowness) are together used as an index for meat quality. Analysis of the color parameters showed that there was a significant tendency for a*value in muscle Longissimus dorsi to be lower in Jinhua pigs than in landrace pigs at the age of d150whilst L*and b*are not significantly different between these two breeds. Oil red O staining showed that the fat in the longissimus dorsi muscles in Jinhua pigs exhibited a more abundant distribution pattern than that in the Landrace pigs. Measurement of the fat contents revealed that there were significant differences in intramuscular fat (IMF) contents between Jinhua and Landrace pigs, where Jinhua pigs showed a higher IMF content at all stages examined. Notably, the IMF contents showed a steady increase in the Jinhua pigs from d60(1.48%), d90(2.25%), d120(3.20%) to d150(3.38%) age stages while the IMF contents in Landrace pigs maintained relatively unchanged from d60(1.13%), d90(1.28%) to d120(1.31%) and showed slight increase at d150(1.79%). Interestingly, the IMF contents in both breeds were slightly reduced from d30to d60. Our results support the previous findings that Jinhua pigs exhibited higher back fat thickness and intramuscular fat but lower lean meat ratio and lean meat area than that of the Landrace at the same age. This experiment provided the following experiment two for comparison data of meat quality between two pig breeds and experimental animal samples.2. Global gene expression profiles of longissimus dorsi muscle in Jinhua and Landrace pigs at d30, d90, and d150Total RNAs extracted from the longissimus dorsi muscle in Jinhua or Landrace pigs at d30, d90, and d150were used in microarray hybridization.1) We compared the global gene expression profiles of Jinhua pigs at d90or d150with that of Jinua pigs at d30. The data showed that, in comparison with their expression in pigs at d30, a total of419differentially expressed genes were identified in longissinus dorsi muscles at d90, including177up-regulated genes (d90-up) and242down-regulated genes (d90-down). A total of490differentially expressed genes were identified in longissinus dorsi muscles at d150, including101d150-up genes and389d150-down genes. In longissinus dorsi muscles of Landrace pigs,106d90-up,231d90-down,93d150-up,383d150-down genes were identified, respectively, when compared with that at d30.2) The global expression profiles of longissimus doris at d30, d90, and d150in Jinhua pigs was compared with that of the Landrace pigs at the corresponding stage. Compared with the expression of longissimus dorsi muscles of Landrace pigs,176,276and525genes corresponding to the stage of d30, d90and d150were identified to be up-regulated in Jinhua pigs (Jinhua-up genes), and199,155and670genes corresponding to the stage of d30, d90and d150were down-regulated in Jinhua pigs3) We also compared the d90-up and d90-down genes in Jinhua pigs correspondingly with that of Landrace pigs. The result showed that only0.7%of d90-up and1.7%of d90-down genes were in common in these two breeds. For d150-up and d150-down genes, only3.2%of d150-up and7.9%of d150-down genes were in common in these breeds. This data clearly indicates that different transcriptomes are mobilized to govern the development of the longissinus dorsi muscle in these two breeds.Among the differentially expressed genes identified by microarray in longissimus dorsi muscles of Jinhua and Landrace pigs at age of d90, randomly selected Jinhua-up genes and Jinhua-down genes were selected for validation by qRT-PCR or semi-quantitative PCR. We noticed that, although the differential expression patterns of the examined genes were qualitatively the same between the results obtained from microarray and qRT-PCR analysis, showing the reliability of the microarray analysis.3. Screening, full-length cDNA cloning and sequence analysis of the new functional gene pFLJ According to the above differentially gene list obtained by microarray, a novel gene tagged by EST sequences with public No. BI184304, showed significantly extreme higher expression level (more than9fold changes) in longissimus dorsi muscle of Jinhua than that of landrace pig, but no significant difference in the subcutaneous fat tissue between two pig breeds at90d. Furthermore, this result was validated through real-time quantitative PCR. Moreover, tissue distribution study results showed that the novel gene transcriptional expression level is highest in muscle, to a lesser digree in brain tissue of Jinhua pigs. The developmental expression pattern of different age in longissimus dorsi muscle study results showed that its expression level began to increase at90days of age, showing significantly higher in longissimus dorsi muscle of Jinhua pig than that of Landrace, accompanied by the intramuscular fat deposition. Presumably, this novel gene tagged by EST sequences with public No. BI184304may be involved in porcine intramuscular fat regulation and be selected as a candidate gene for modulating intramuscular fat deposition.For the study of function of the candidate gene mentioned above, full-length cDNA sequence of the candidate gene was obtained for three steps by RACE technology according to the known EST sequences. Full length cDNA sequence of the candidate gene was2793bp, including283bp in5’UTR region (5’-untranslated translation of237amino acids. The BLAST analysis of the deduced amino acid sequences of the candidate gene with those of the other species indicated that the candidate gene sharing FLJ99%,84%similarity with human and rat FLJ gene. Therefore, the gene was named as porcine FLJ gene, abbreviated as pFLJ for the following experiments. This study laid the molecular basis for the study of the function of FLJ genes.5. Molecular mechanism of pFLJ regulating intramuscular fat depositionFor investigating the pFLJ gene function, porcine intramuscular fat cell culture model was established using the differential velocity adherent method. The experiments were carried out based on this cell model as follows:For preliminarily exploration of pFLJ gene function, this experiment studied lipid droplet deposition, pFLJ gene and lipolysis key gene, ATGL and HSL, gene expression pattern of porcine intramuscular adipocytes at different stages of differentiation. Results show that FLJ gene expression and lipid droplet deposition increased gradually with cell differentiation after4days of differentiation, but the key lipolysis genes, ATGL and HSL, decreased gradually. Secondly, the study administered the classic fat deposition inhibitor (SR141716) in porcine intramuscular adipocytes, the results showed that along with the concentration of SR141716increased, compared with the control group, the expression of pFLJ gene decreased significantly. Furtherly, we treated intramuscular adipocytes with CLA which can significantly improve the intramuscular fat deposition and improve meat quality, results showed that, with the concentration of CLA increasing, pFLJ gene mRNA levels increased significantly (p<0.05) compared with that of the control group, at the same time can significantly enhance fat synthesis of key enzyme gene FAS mRNA expression, while increasing of porcine intramuscular adipocyte triglyceride content. Therefore, based on results comparisons of meat quality between two pig breeds and pFLJ expression pattern results above, we speculated that FLJ gene may be a key functional gene positively modulating intramuscular fat deposition.To prove speculation mentioned above, the effective siRNA expression vector of pFLJ was screen out at first. The conditions were opotimized and the interference effect reached70.9%. After treatment of intramuscular adipocytes with pFLJ-siRNA expression vector, the fat biosynthesis genes FAS and ACC and fat lipolysis genes ATGL and HSL gene expression of intramuscular adipocytes were significant reduced. Treated with CLA and pFLJ-siRNA further proved that inhibition of pFLJ not only reduced the fat biosynthesis genes FAS and ACC but also decreased fat lipolysis genes ATGL and HSL gene expression. At the same time, we also found that inhibition of pFLJ suppressed the lipid droplet deposition of intramuscular adipocytes induced by exogenous fatty acids such as CLA. Therefore, we implied that pFLJ gene may be positively involved in deposition of lipid droplets of intramuscular adipocytes induced by exogenous fatty acid.Furthermore, we constructed pFLJ gene fluorescence over expression vector and optimized the transfection condition. Results showed that overexpression of pFLJ gene increased fat synthesis gene FAS and ACC genes and Iipolysis key gene ATGL and HSL gene expression, which are consistent with results of pFLJ interference research. CLA and pEGFP-FLJ expression of FLJ gene synergy results showed that overexpression of pFLJ gene significantly increased the fat metabolism key functional gene FAS gene expression and enhanced the lipid droplets of intramuscular adipocytes induced by exogenous fatty acid. The pFLJ overexpression results further revealed that overexpression of the pFLJ gene not only increased FAS, ACC but also enhanced the ATGL and HSL genes expression. This study implied that pFLJ gene may play a pivotal role in the positive regulation of deposition of lipid droplets induced by exogenous fatty acids in intramuscular adipocytes.In conclusion, based on comparisons of carcass traits and meat quality between Jinhua pigs and landrace pigs, gene transcriptional expression profiling in longissimus dorsi muscle of two pig breeds at age of30,90,150days were obtained using microarray. Furtherly, we screened and cloned key new functional gene pFLJ regulating intramuscular fat deposition from the data above. This study preliminary implied that pFLJ gene may improve the intramuscular fat deposition through positively regulation of fat metabolism, during this process, the FLJ gene probably play a pivotal role in the regulation of exogenous fatty acid transporting to the intramuscular adipocytes.