Prokaryotic Expression And Effect Of FTO Gene On Porcine Intramuscular Preadipocyte Proliferation And Differentiation

Fat mass and obesity-related gene (FTO) is a recently discovered gene that could affect the body fat deposition. The single nucleotide polymorphism sites of FTO had significant positive correlation with pork quality traits, suggesting FTO may be a candidate gene for IMF deposition. This study aimed at revealing the role of FTO in intramuscular preadipocytes proliferation and differentiation. The major contents and findings of this study are as follows:1 Prokaryotic expression of porcine FTO geneThe total RNA was extracted from pig longissimus dorsi muscle and reverse transcription to synthesize cDNA, and then it as a template to clone the FTO gene. The porcine FTO gene was inserted to pET30a(+) vector, the obtained recombinant plasmid was named pET30a(+)-pFTO. In order to acquire the recombinant protein, the recombinant plasmid was transformed to E. coli BL21 (DE3). The recombinant porcine FTO was induced by addition of IPTG, and we also optimized the expression conditions for enhancing the production of recombinant porcine FTO. The recombinant protein was purfied by Ni2+-IDA. The purified product was identificated by SDS-PAGE and western blot. The results showed that the recombinant protein could be efficiently induced with 0.75 mmol/L IPTG at 30? for 4 h. The molecular weight of the recombinant protein was approximate 56 kDa by SDS-PAGE analysis, and the purification recombinant protein could be specificity identification by His-tag monoclonal antibody. These results suggested that we successfully obtained high purity of recombinant porcine FTO protein.2 Study on tissue expression and subcellular localization of porcine FTOPolyclonal antibodies were preparated by immunizing Kunming mice using the purified porcine FTO recombinant protein. Antibody titer and specificity were evaluated using the ELISA and western blot. In order to reveal the tissue expression profile of FTO in DLY pig, the polyclonal antibodies were used to detect the expression of FTO in DLY pig. The intramuscular preadipocytes were isolated from 3-day pig longissimus dorsi muscle by sterile dissection following collagenase digestion. In addition, the subcellular localization of FTO protein in porcine intramuscular preadipocytes was detected. The results showed that the antibody titer was approximately 1:40960. Western blot analysis revealed that the polyclonal antibodies could specially recognize recombinant porcine FTO protein, and the FTO protein was mainly present in subcutaneous adipose, but had a lower expression in liver and skeletal muscles. Real time quantitative PCR detection found that the isolated cells could express adiponectin, PPARy and C/EBPa. The oil red o staining found that the cells were able to differentiate into mature adipocytes. Finally, we found that FTO protein was located in the nucleus. These suggested that the polyclonal antibody was successfully prepared and FTO may play an important role in fat deposition.3 Effect of FTO on porcine intramuscular preadipocyte proliferation and differentiationThe effects of FTO on porcine intramuscular preadipocytes proliferation and differentiation were unknowned. To determine whether FTO affects porcine intramuscular preadipocyte proliferation and differentiation, the cells were transfected with plasmid pcDNA3.1(+)-pFTO or siRNA for 24 h in proliferation medium and for 8 d in differentiation medium. We found that overexpression of porcine FTO significantly increased the percentage of EdU-positive cells and the protein expression levels of phospho-Histone H3. Furthermore, siRNA treatment got the opposite result. Meanwhile, we found that the mRNA levels of the adipocyte differentiation transcription factors PPARy, C/EBPa and LPL were significantly increased. Western blot analysis also showed that the protein levels of PPARy and C/EBPa were significantly upregulated after transfection with recombinant plasmid pcDNA3.1(+)-pFTO, while the siRNA treatment got the opposite result. These results show that the porcine FTO could significantly promote porcine intramuscular preadipocytes proliferation and differentiation.In summary, the study prepared porcine FTO recombinant protein and its polyclonal antibody. FTO protein was mainly present in subcutaneous adipose, then in liver and skeletal muscles, and it was located in the nucleus. In addition, overexpression FTO or FTO silencing significantly affected porcine intramuscular preadipocytes proliferation and differentiation. These results suggested that FTO might be a candidate gene for IMF deposition.