Cloning Of Porcine FoxO1 Gene And Its Regulation During Preadipocyte And Myoblast Differentiation

Following the improvement of living standard, consumers proposes higher request for meat quality. They not only require high lean meat but also proper fat proportion. At present, the quantity of meat product has been satisfied for people, but the quality is becoming poor. Therefore, study on meat formation and molecular mechanism has been hotspot for animal scientist. There are many factors caused decrease of meat quality, mainly is the position change of fat deposition, such as decrease of intramuscular fat content, too much deposition of subcutaneous fat and abdomen fat, pale of muscle fiber, dysregulation of type and proportion of muscle fiber and PH diminish. Adipocyte and myocyte is the major component unit of animal adipose and muscle, respectively differentiated from preadipocyte and myoblast. The state of proliferation and differentiation is essential for fat and muscle formation. The studies in recent years showed that Foxo1 might be the key regulatory factor during process of preadipocyte and myoblast differentiation in mouse cell lines. It may influence cell proliferation, differentiation, apoptosis, and cell cycle through multiple ways, hinting that procine Foxo1 plays an important modulatory role during formation of adipocyte and myocyte.Fat deposition is the process that TG accumulation, lipid droplets de novo and from many small droplets change into single droplet, which result from interplay of preadipocyte proliferation, differentiation and hypertrophy. Muscle formation is the process that myoblast confluent into myotube and further become muscle fiber with multinuclear. The primary culture system of preadipocyte and myoblast provides an effective model for studying cell differentiation. It was supposed that FoxO1 interacted with other transcription factors to regulate fat and muscle formation. In this study, FoxO1 complete cDNA was firstly cloned with RACE method and performed sequence analysis and predict of protein structure and function. Then, the refined chromosome location of FoxO1 was operated by IMpRH7000-rad. The tissue expression of pig FoxO1 was detected by Realtime PCR and Western blotting. The effects of resveratrol and IGF-1 on proliferation and differentiation of pig preadipocyte and myoblast were analyzed using cell culture technique, which possible mechanism was primarily probed. Finally, the modulation of FoxO1 on time-spatial expression of genes concerned pig fat and muscle formation was checked by liposome and RNAi. The conclusion was as follows:1. cDNA cloning, sequencing analysis and predict of protein structure and function of pig FoxO1. After clone sequence of 5' and 3'RACE, removing vector, primer and partial repeat known sequence and, 2335bp of complete cDNA was obtained through sequence junction, among which containing initiation codon ATG, termination codon TGA,and polyA sequence. The Genebank number is EF453379. Predicting by bioinformation, the AA sequence length of pig FoxO1 was 662AA; molecular weight was 69.93kD; isoelectric point pH was 6.295. It had Forkhead DNA structural domain(AA165-255) and transmembrane structure(AA 90-113), but no signal peptide. The prediction of phosphorylation site indicated that there were 51 Ser, 8 Thr and 4 Tyr phosphorylation sites. The protein secondary structure speculated that there were 22% helix, 19% sheet, 24% turn and 35% coil conformation. The free energy of FoxO1 mRNA secondary structure in pig was -671.5 kcal/mol.2. The chromosome location and tissue expression analysis of procine FoxO1 gene. Using RH radiation clone plate found that procine FoxO1 was closely linked to marker SW1632. The genetic distance of FoxO1 with SW1632 was 0.32cR and LOD was 12.17, so procine FoxO1 was located in 11p13. The analysis of tissue expression indicated that FoxO1 was higher expressed in subcutaneous adipose, visceral adipose, liver and skeletal muscle in piglets and 180-day-old pigs, which piglets was higher than that of 180-day-old pigs. The tissue expression of procine FoxO1 indicated that FoxO1 might play an important physiological role in these four tissues.3. Resveratrol and IGF-1 could respectively repress and promote procine preadipocyte proliferation and differentiation in dose and time dependent manner. Among them, there were significantly difference in 100μmol/L RES and 100 ng/mL IGF-1 groups. This might be resulted from up-regulate or down-regulate of FoxO1 mRNA causing change of PPARγmRNA expression, which Sirt1 may play assisted modulatory role.4. Resveratrol and IGF-1 respectively repressed and promoted procine myoblast proliferation and differentiation in dose and time dependent manner. Among them, there were significantly difference in 160μmol/L RES and 45 ng/mL IGF-1 groups. It might be resulted from up-regulate or down-regulate of FoxO1 mRNA expression causing change of MyoD mRNA expression. 5. Construction of procine FoxO1 RNAi expression vector. The study of procine primary preadiocyte and myoblast identified that the transfection efficiency of liposome was higher than method of calcium phosphate precipitation. It had been successfully constructed 3 FoxO1 siRNA expression vectors, pBS/U6-FoxO1-952, 1303 and 1748, respectively, then transfected into pig preadipocyte and myoblast by liposome. It was found that pBS/U6-FoxO1-1748 could effectively repress endogenous FoxO1 by RT-PCR and immunofluorescence.6. Inhibition of endogenous FoxO1 expression promoted the ability of pig preadipocyte and myoblast to form fat and muscle. The reason might be related to expression up-regulation or down-regulation of the key adipocyte genes PPARγ, A-FABP, H-FABP, Sirt1, LPL, GADD45αand muscle genes MyoD,MEF2C,CaMKII,Sirt1,NFAT,MyoG,IGFBP5 and GADD45α.