【作者】 庞卫军；

【导师】 杨公社；

【作者基本信息】 西北农林科技大学 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 随着人们生活水平的提高,消费者对肉类产品的质量提出了更高的要求,不仅要求肉品瘦肉率高、脂肪分布适中,而且要求肉品优质。目前,肉产品的数量已能满足人们的消费需求,但其品质却越来越差。因此,研究肉质形成及其分子机理成为目前家畜育种科研工作者关注的焦点。导致肉质变差的因素很多,主要有脂肪的沉积部位发生改变,如肌内脂肪含量下降、皮下脂肪和腹脂沉积过多以及肌纤维颜色变苍白、肌纤维类型比例失调、PH值下降等。脂肪细胞和肌细胞是动物脂肪组织和肌肉组织主要组成单位,分别由前体脂肪细胞和成肌细胞分化而来,两类细胞的增殖与分化状况对脂肪形成和肌形成起关键作用。近年来研究表明,在啮齿动物鼠及其细胞系中,叉头转录因子O亚家族1(forkhead transcription factor group o1,Foxo1)可能是前体脂肪细胞和成肌细胞分化过程的重要调控因子,通过多条通路影响细胞增殖、分化、凋亡和细胞周期以及葡萄糖代谢和氧化应激等,暗示小鼠Foxo1在脂肪细胞和肌细胞的形成过程中发挥重要的调控作用。体脂沉积是脂肪细胞内甘油三脂聚积、脂滴从无到有、从多室变为单室的过程,实质是前体脂肪细胞增殖、分化及肥大共同作用的结果;而肌形成则是成肌细胞融合为肌管,进而成为多核的肌纤维的过程。前体脂肪细胞和成肌细胞原代培养体系为研究细胞分化及调控提供了一个可操作模型。推测猪FoxO1很可能与其它转录因子组成网络共同调控成脂和成肌过程。本实验首先通过RACE技术克隆了猪FoxO1 cDNA全长,并对其进行了序列分析和蛋白结构与功能的预测;其次,利用猪×仓鼠体细胞辐射杂种克隆板(IMpRH7000-rad)对FoxO1基因进行染色体精细定位,并运用Realtime PCR和Western blotting技术分析了猪FoxO1基因组织表达规律;再次,运用细胞培养技术研究了不同浓度的百藜芦醇和IGF-1对猪前体脂肪细胞和成肌细胞增殖与分化的影响,并初步探讨了可能的作用机理;最后,运用脂质体转染和RNAi技术研究了FoxO1对猪成肌和成脂关键基因时序表达的调控作用,获得如下结论:1.猪FoxO1基因cDNA克隆、序列分析及其蛋白结构与功能预测。5’和3’RACE克隆测序后,去掉载体序列、引物序列和重复的部分已知序列,经拼结得到cDNA全长序列2335bp,其中含有起始密码子ATG和终止密码子TGA,polyA序列,Genebank登陆号EF453379。运用生物信息学预测,猪FoxO1基因氨基酸序列长度为662AA,蛋白质分子量为69.93kD,等电点pH为6.295;具有Forkhead DNA结构域(AA 165-255),跨膜结构(AA90-113),但无信号肽;磷酸化位点预测结果显示含有51个Ser、8个Thr和4Tyr磷酸化位点;蛋白二级结构推测含有螺旋构象(helix)22%,折叠构象(sheet)19%,转角构象(turn)24%,无规则卷曲(coil)35%;预测了猪FoxO1 mRNA的二级结构,其结构的自由能为-671.5 kcal/mol。2.猪FoxO1基因染色体精细定位及其组织表达分析。运用RH辐射克隆板方法研究发现,猪FoxO1基因与标记SW1632紧密连锁,与SW1632的遗传距离为0.32cR,两点分析结果LOD值为12.17,故将猪FoxO1基因定位于11p13。组织表达分析发现,初生和180日龄时猪皮下脂肪、腹腔脂肪和肝脏组织FoxO1基因表达均相对较高,骨骼肌组织FoxO1基因表达则相对较低;初生与180日龄猪相比,相应组织中FoxO1表达量较高(P<0.05),提示猪FoxO1在这4种组织中可能发挥重要生理作用。3.百藜芦醇和IGF-1分别以剂量和时间依赖的方式抑制和促进猪前体脂肪细胞增殖与分化,以100μmol/L RES组和100 ng/mL IGF-1组作用最为明显,其机理可能与FoxO1 mRNA的上调或下调而导致PPARγmRNA表达变化有关,其中Sirt1可能起到辅助调节的作用。4.百藜芦醇和IGF-1分别以剂量和时间依赖的方式抑制和促进猪成肌细胞增殖与分化,以160μmol/L RES组和45 ng/mL IGF-1组作用最为明显,其机理可能与FoxO1 mRNA的上调或下调而导致MyoD mRNA表达变化有关。5.成功构建了猪FoxO1干扰表达载体。以原代猪前体脂肪细胞和成肌细胞为模型的研究中,证实脂质体的转染效率明显高于磷酸钙沉淀法。构建了3个猪FoxO1 siRNA表达载体,分别为pBS/U6-FoxO1-952、1303和1748,并运用脂质体转染猪前体脂肪细胞和成肌细胞,经RT-PCR和免疫荧光检测发现pBS/U6-FoxO1-1748对细胞内源FoxO1的抑制效果最明显。6.内源FoxO1表达的抑制可导致猪前体脂肪细胞和成肌细胞增殖与分化能力增强,原因很可能与关键成脂基因PPARγ、A-FABP、H-FABP、Sirt1、LPL和GADD45α及成肌基因MyoD、MEF2C、CaMKII、Sirt1、NFAT、MyoG、IGFBP5和GADD45α表达上调或下调有关。更多还原

【Abstract】 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.更多还原