脂尾型绵羊尾部脂肪富集的蛋白质组学研究

发布时间：2018-04-12 06:49

本文选题：脂尾型绵羊 + 尾脂　；参考：《中国农业科学院》2016年博士论文

【摘要】：脂尾型绵羊适应性进化而来的尾部大量脂肪富集已经不符合产业化生产需求,育种实践中亟需对脂尾羊进行瘦尾化改良,但国内外对于脂尾羊和瘦尾羊尾部脂肪组织发育的形态发生及其脂肪富集的分子机理研究较少,尤其是对尾脂发育起决定性作用的生长早期尾部脂肪组织的差异蛋白组学研究仍为空白,本试验通过组织冰冻切片比较五种尾型绵羊的脂肪组织形态发育规律,脂肪酸定性和定量分析来比较五种尾型幼年羊生长早期脂肪组织脂肪酸组成;对五种尾型幼年羊生长早期尾脂全蛋白的抽提,液内酶切(磷酸化肽段IMAC富集)后质谱鉴定和数据定性和定量以及生物信息学分析;对重要蛋白进行Realtime PCR、Western Blot、免疫组化和免疫荧光验证,从差异蛋白表达和磷酸化修饰来阐述不同尾型绵羊生长早期尾脂的发育、形成相关的生物学机理,为脂肪组织发育及局部富集的深入研究奠定基础。1.通过对五种尾型绵羊不同生长阶段三种脂肪组织的脂肪细胞大小及形态结构观察分析,发现幼年脂尾羊生长早期脂肪细胞均显著大于瘦尾羊,且随着月龄增加脂肪细胞大小都有增大的趋势;幼年脂尾羊生长早期其尾脂细胞较大而同时期瘦尾羊脂肪细胞较小,揭示该阶段尾脂发育主要以细胞数量增多为主且伴随细胞逐步变大,表明脂尾羊尾脂发育较早且幼年脂尾羊生长早期尾部沉积脂肪效率更高,说明幼年脂尾羊生长早期尾部脂肪组织快速发育对于脂尾羊生长和成年后尾部脂肪富集具有重要意义。2.五种尾型幼年羊生长早期脂肪组织脂肪酸定量和定性分析,发现该阶段其不同脂肪组织都以C18 cis-9:1、C16:0和C18:0三种脂肪酸含量最高,且占到80%以上,主要脂肪酸含量的主成分分析表明幼年脂尾羊生长早期相比同时期瘦尾羊尾部脂肪的主要脂肪酸含量有较大差异,可能由于组织特异性、脂尾与瘦尾羊品种遗传差异所致。3.比较不同尾型幼年羊生长早期尾脂差异蛋白组学,五组尾脂中共鉴定到3400个蛋白,定量分析有804个差异表达蛋白,且涉及细胞分化以及脂肪酸合成的蛋白主要在脂尾羊中高表达,说明这有利于幼年脂尾羊尾脂的发育;参与脂肪代谢和脂解相关的重要蛋白也在脂尾羊中高表达,表明幼年脂尾羊生长早期尾脂不是主要储存脂肪酸阶段而可能以细胞分化数量增加为主为生长后期及成年能够储存大量脂肪做准备;对部分差异蛋白相应基因进行Realtime PCR及Western blot验证,其表达趋势与蛋白组学基本一致;蛋白表达定位发现FABP4在脂尾羊尾脂的毛细血管壁内外膜高表达,可能介导前脂肪细胞分化和脂肪酸的转运功能;ERK2在脂尾羊尾脂脂肪细胞和毛细血管壁高表达,揭示其可能介导幼年羊生长早期脂肪组织中脂肪前体细胞分化增殖,且ACSL1在脂尾羊尾脂中高表达以参与脂肪细胞脂滴中甘油三酯的合成和储存;表明以FABP4、ERK2为主的蛋白对于脂尾羊尾脂的发育具有重要意义。4.对不同尾型幼年羊生长早期尾脂中蛋白磷酸化修饰分析,在五种尾型绵羊尾部脂肪组织中鉴定到1493个磷酸化位点分布在804个磷酸化蛋白,其主要参与翻译、基因表达、蛋白代谢、细胞形态和细胞骨架、蛋白降解、细胞凋亡及蛋白修饰,说明脂肪组织发育需要以上这些途径来参与细胞代谢分化和脂肪沉积等重要生物学功能;定量发现435个磷酸化差异表达蛋白,涉及细胞骨架、蛋白翻译后修饰的正调控、细胞凋亡信号通路的调控、蛋白质分解代谢的调控、翻译起始等15个生物学进程,包括28个参与蛋白磷酸化修饰作用蛋白,揭示不同尾型绵羊尾脂的发育可能与差异磷酸化修饰相关,基序分析发现以ERK1/2为主的激酶可能介导幼年脂尾羊生长早期尾脂中相关蛋白发生磷酸化修饰,进而对尾脂中脂肪前体细胞的分化具有正向调控作用。本试验为脂尾羊尾脂大量富集的生物学机制研究奠定基础,对幼年脂尾羊生长早期参与脂肪细胞分化和脂肪沉积相关蛋白进行表达干预实现阻止其尾脂快速发育,将对育种实践中脂尾羊瘦尾化具有重要意义。
[Abstract]:Fat tail sheep adaptability evolved tail fat enrichment has not meet the demand of production industry, the urgent need for the breeding of fat tail sheep were thin tail of improvement, but at home and abroad for the development of fat tail sheep and thin tailed sheep tail fat morphogenesis and its molecular mechanism of less fat accumulation, especially differential proteomic analysis of play a decisive role in the growth of early tail fat tail adipose tissue is still blank, the frozen tissue sections of five tail sheep adipose tissue morphology and development rule, qualitative and quantitative analysis of fatty acids to compare the five kinds of juvenile sheep tail early growth of adipose tissue fatty acid composition; the five tail sheep growth early juvenile tail fat protein extraction, liquid digestion (IMAC phosphopeptide enrichment) after mass spectrometry and qualitative and quantitative data and bioinformatics Division Realtime PCR analysis; the Western protein Blot, immunohistochemistry and immunofluorescence test, from the differences in protein expression and phosphorylation of different tail sheep tail fat of the early growth and development, the formation of biological mechanisms related to adipose tissue development, and research the local rich set of.1. lay the foundation through observation and analysis fat cell size and morphology of different growth stages of three kinds of fatty tissue of five tail sheep, found that juvenile fat tailed sheep in the early growth of fat cells were significantly higher than that of thin tail sheep, and with the increasing of age in the small fat cells have increased; juvenile fat tailed sheep tail fat cells in the early stage of the growth of large at the same time, thin tail sheep fat small cells and reveal the development mainly in the stage of tail fat cells and cells with gradually increased, showed that the fat tail sheep tail fat and fat tailed sheep developed earlier Juvenile Students Early long tail fat deposition efficiency is higher, that young fat tailed sheep tail fat growth early rapid growth for the growth of fat tail sheep tail fat and adult adipose tissue fatty acid enriched with quantitative and qualitative analysis of the early growth of important.2. five kinds of juvenile sheep tail, found the stage of different adipose tissue with C18 cis-9:1 C16:0 and C18:0, three kinds of fatty acid content is the highest, and accounted for more than 80% principal component analysis showed that the fatty acid content of juvenile fat tailed sheep growth compared to the same period the main early fat thin tail sheep tail fat acid content have great difference, may be due to tissue specificity, fat tail and thin tail sheep breeds genetic differences.3. by comparison of different early differential proteomics of tail fat tail juvenile growth sheep tail fat the five groups identified 3400 proteins, the quantitative analysis of 804 differentially expressed proteins involved in cell division and And the synthesis of fatty acid protein is mainly expressed in the fat tailed sheep, that this is conducive to the early development of fat tail sheep tail fat; in protein metabolism and lipolysis related fat is also highly expressed in fat tailed sheep, that young fat tailed sheep tail fat is stored mainly in early growth stage and fatty acid to increase the number of cells for later growth and adult can store large amounts of fat in preparation for Realtime PCR and Western blot; verification of some proteins related genes, their expression trends and proteomics are basically the same; protein expression and localization of the high expression of FABP4 in the capillary wall, inner and outer membrane of fat tail sheep tail fat, may mediated preadipocyte differentiation and fatty acid transport function; the expression of ERK2 in fat tailed sheep tail fat fat cells and capillary wall, reveal the possible mediated growth of early juvenile sheep fat in adipose tissue Precursor cell differentiation and proliferation, and ACSL1 in fat tailed sheep tail fat high expression in the synthesis and storage of triglyceride in adipocyte lipid droplets; that with FABP4, ERK2 protein plays an important role in.4. phosphorylation in the growth analysis of tail fat modification on different early juvenile sheep tail for fat tailed sheep tail fat the development in five kinds of sheep tail fat tail type identified 1493 phosphorylation sites in 804 phosphorylated proteins, which are involved in translation, gene expression, protein metabolism, cell morphology and cytoskeleton, protein degradation, cell apoptosis and protein modification that adipose tissue development need to participate in these ways an important cell differentiation and metabolism of fat deposition and other biological functions; quantitative expression of protein found 435 phosphorylation differences involved in cytoskeleton regulation, protein post-translational modification, cell apoptosis signal pathway regulation Control and regulation of protein catabolism, translation initiation of 15 biological processes, including 28 proteins involved in protein phosphorylation and protein function, development may reveal the differences of different types of fat tail sheep tail phosphorylation, motif analysis based on ERK1/2 kinase mediates the growth of fat tail sheep early childhood protein phosphorylation occurs in tail fat, and fat tail fat in the differentiation of precursor cells has positive regulation effect. This experiment lays a foundation for the research on the biological mechanisms of fat tail sheep tail fat accumulation, early in adipocyte differentiation and fat deposition related protein expression intervention to prevent the rapid development of juvenile tail fat fat tailed sheep growth, will have important significance for the breeding of fat tail sheep thin tail.

【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S826

【相似文献】