Screening Of Fat Deposition Related Genes And Functional Identification Of The Novel Gene PFAM134B

Fat content is a key index to evaluate pork quality. On one hand, intramuscular fat (IMF) content has high correlation with pork color, flavor and tenderness, and water-holding capacity. On the other hand, subcutaneous fat affects economic effectiveness remarkably. How to increase the intramuscular fat content while reducing the subcutaneous fat is a big problem. Jinhua pig, a Chinese local pig breeds showing excellent meat quality traits such as higher intramuscular fat content, better pork color but with thicker subcutaneous fat and slow growth rate. Therefore, Jinhua pig and Landrace pigs, which owns less IMF content and thinner back fat thick, were used as experimental animal model to obtain different gene expression profiles in longissimus muscle and subcutaneous fat and differential expressing gene FAM134B was screened out by microarray and bioinformatics analysis. Then cDNA full length of pFAM134B gene was obtained by RACE technology, and the mechanisms of pFAM134B regulating fat deposition using RNAi technology and gene over expression technology at transcription level was investigated.1. Global gene expression profiles of longissimus dorsi muscle and subcutaneous fat in Jinhua pigs and Landrace pigs at d30and d90Microarray and bioinformatics analysis technique was used to analyze differential expression genes in longissimus dorsi muscle and subcutaneous fat at30d and90d between Jinhua pigs and Landrace pigs. Comparing with Landrace pigs,176higher expressed genes and199lower expressed genes1in longissimus dorsi muscle were screened out at d30in Jinhua pigs. While at the age of90d,276higher expressed genes and155lower expressed genes were screened out. Meanwhile, in subcutaneous fat of jinhua pigs, there were188genes up-regulated and147genes down-regulated at30d,235genes up-regulated and223genes down-regulated at90d compared with that of Landrace pigs. Moreover, there were14genes higher expressed and11genes lower expressed in longissimus dorsi muscle of Jinhua pigs at both30d and90d, while38genes higher expressed and18genes lower expressed in subcutaneous fat. This data clearly indicates that transcriptomes for fatty acid biosynthesis in both longissimus dorsi muscles and subcutaneous fat showed highly expression level, suggested that the jinhua pig fat deposition was much stronger than Landrace pigs.2. Screening, full-length cDNA cloning and sequence analysis of the new gene pFAM134BAccording to the trend that Jinhua pig has rich IMF content and subcutaneous fat at d90, we screened out one up-regulated gene both in IMF and subcutaneous fat at d90from differentially gene list obtained by microarray. Presumably, this gene tagged by EST sequences with public No. BF702245may be involved in porcine fat regulation and be selected as a candidate gene for modulating fat deposition. Full-length cDNA sequence of the candidate gene was cloned by RACE technology according to the known EST sequences. Full length cDNA sequence of the candidate gene was2889bp, including179bp in5’UTR region (5’-untranslated region),978bp coding region of translating325amino acids and1732bp in3’UTR region. The BLAST analysis of the deduced amino acid sequences with the other species indicated that the candidate gene sharing FAM134B84%similarity with human and rat FAM134B gene. Therefore, the gene was named as porcine FAM134B gene, abbreviated as pFAM134B for the following experiments.3. The study on the tissue developmental expression pattern and breed differences of pFAM134BTissue-specific study results showed that, pFAM134B mRNA level in the two kinds of pigs is highly expressed in liver which the most activated organ related with lipometabolism, lower in the subcutaneous fat, longissimus dorsi mucle and brain successively. The developmental expression pattern of different growth stage study results showed that, in the liver, subcutaneous fat and longissimus dorsi mucle, pFAM134B gene expression is increased with age but slightly decreased between d90and d120. The expression pattern of pFAM134B in the brain was not obviously. The breed difference study results showed that, in the subcutaneous fat and LD, pFAM134B gene expression in Landrace is higher than that in Jinhua pig before d60, but lower after d90significantly. The pFAM134B gene expression in brain of jinhua pig is significantly higher than landrace pig after d90. The above results is in accordance with the development pattern of intramuscular fat and fat content, which show that the expression of the pFAM134B may play an important role in fat deposition.4. Molecular mechanism of pFAM134B regulating intramuscular fat depositionWe culture porcine intramuscular fat cell to investigate the function of pFAM134B gene in regulating intramuscular fat deposition using RNAi technology and gene over expression technology efficiently. At first, the effective siRNA expression vector of pFAM134B was screen out. After The conditions were opotimized and the stable FAM134B-interference positive intramuscular preadipocyte clones were screened out by adding G418, the interference effect reached65.0%. After treatment of intramuscular adipocytes with pFAM134B-siRNA expression vector, expression of the fat biosynthesis genes FAS and ACC and expression of cell differentiation gene PPARy were significantly reduced. Meanwhile, the expression of fat lipolysis genes ATGL, HSL and sirt1gene were highly increased along with the decrease of lipid droplets of intramuscular adipocytes. It shows that, inhibition of the FAM134B expression can reduce the expression of the Lipogenesis functional genes and cell differentiation gene, increase the expression of the lipolysis functional genes and weaken the capacity of intramuscular fat deposition. Furthermore, we constructed pFAM134B gene over expression vector and optimized the transfection condition. Results showed that over expression of pFAM134B increased the expression of fat synthesis gene FAS and ACC genes and cell differentiation gene PPARy, decreased fat lipolysis key gene ATGL and HSL expression and enhanced the fat deposition of intramuscular adipocytes, which are consistent with results of pFAM134B interference research. This study implied that pFAM134B gene may play a role in the positive regulation of deposition of lipid droplets in intramuscular adipocytes through regulating the cell differentiation genes and fat metabolism gene.4. Molecular mechanism of pFAM134B regulating subcutaneous fat depositionThis test carried out to investigate Molecular mechanism of pFAM134B regulating subcutaneous fat deposition through porcine subcutaneous fat cell culture combining with RNAi technology and gene over expression technology efficiently. At first, the effective siRNA expression vector of pFAM134B was screen out. After the conditions were opotimized and the stable FAM134B-interference positive subcutaneous preadipocyte clones were screened out by adding G418, the interference effect reached74.5%. After treatment of subcutaneous adipocytes with pFAM134B-siRNA expression vector, expression of the fat biosynthesis genes FAS and ACC were significantly reduced but expression of cell differentiation gene PPARy was increased remarkably. Meanwhile, the expression of fat lipolysis genes ATGL, HSL and sirt1gene were highly increased along with the decrease of lipid droplets of intramuscular adipocytes. It shows that, inhibition of the FAM134B expression can reduce the capacity of subcutaneous fat deposition. Then we constructed pFAM134B gene over expression vector and optimized the transfection condition. Results showed that over expression of pFAM134B increased the expression of fat synthesis gene FAS and ACC genes, decreased cell differentiation gene PPARy and fat lipolysis key gene ATGL, HSL and Sirt1expression and enhanced the fat deposition of subcutaneous adipocytes. This study implied that pFAM134B gene may positively regulate the subcutaneous fat deposition through regulating the expression of fat metabolism gene to enhancing fat synthesis and cell differentiation did not help in this process.In conclusion, this study was Jinhua pigs and landrace pigs, gene transcriptional expression profiles of longissimus dorsi muscle and subcutaneous fat at both d30and d90were obtained using Jinhua and Landrace pigs as model. Screening out and successfully cloned a new functional gene pFAM134B regulating fat deposition. This study showed that pFAM134B gene may improve the fat deposition through positively regulation of fat metabolism during this process. Moreover pFAM134B gene can regulate IMF deposition through modulating PPARy expression and IMF adipocytes differenciation. This might be a great difference between intramuscular fat deposition and subcutaneous fat deposition, the former mainly lies in cell differenciation and fat metabolism, the latter relies on lipogenesis.