發(fā)布時(shí)間：2018-04-12 06:49

  本文選題：脂尾型綿羊 + 尾脂　； 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2016年博士論文

【摘要】：脂尾型綿羊適應(yīng)性進(jìn)化而來(lái)的尾部大量脂肪富集已經(jīng)不符合產(chǎn)業(yè)化生產(chǎn)需求,育種實(shí)踐中亟需對(duì)脂尾羊進(jìn)行瘦尾化改良,但國(guó)內(nèi)外對(duì)于脂尾羊和瘦尾羊尾部脂肪組織發(fā)育的形態(tài)發(fā)生及其脂肪富集的分子機(jī)理研究較少,尤其是對(duì)尾脂發(fā)育起決定性作用的生長(zhǎng)早期尾部脂肪組織的差異蛋白組學(xué)研究仍為空白,本試驗(yàn)通過(guò)組織冰凍切片比較五種尾型綿羊的脂肪組織形態(tài)發(fā)育規(guī)律,脂肪酸定性和定量分析來(lái)比較五種尾型幼年羊生長(zhǎng)早期脂肪組織脂肪酸組成;對(duì)五種尾型幼年羊生長(zhǎng)早期尾脂全蛋白的抽提,液內(nèi)酶切(磷酸化肽段IMAC富集)后質(zhì)譜鑒定和數(shù)據(jù)定性和定量以及生物信息學(xué)分析;對(duì)重要蛋白進(jìn)行Realtime PCR、Western Blot、免疫組化和免疫熒光驗(yàn)證,從差異蛋白表達(dá)和磷酸化修飾來(lái)闡述不同尾型綿羊生長(zhǎng)早期尾脂的發(fā)育、形成相關(guān)的生物學(xué)機(jī)理,為脂肪組織發(fā)育及局部富集的深入研究奠定基礎(chǔ)。1.通過(guò)對(duì)五種尾型綿羊不同生長(zhǎng)階段三種脂肪組織的脂肪細(xì)胞大小及形態(tài)結(jié)構(gòu)觀察分析,發(fā)現(xiàn)幼年脂尾羊生長(zhǎng)早期脂肪細(xì)胞均顯著大于瘦尾羊,且隨著月齡增加脂肪細(xì)胞大小都有增大的趨勢(shì);幼年脂尾羊生長(zhǎng)早期其尾脂細(xì)胞較大而同時(shí)期瘦尾羊脂肪細(xì)胞較小,揭示該階段尾脂發(fā)育主要以細(xì)胞數(shù)量增多為主且伴隨細(xì)胞逐步變大,表明脂尾羊尾脂發(fā)育較早且幼年脂尾羊生長(zhǎng)早期尾部沉積脂肪效率更高,說(shuō)明幼年脂尾羊生長(zhǎng)早期尾部脂肪組織快速發(fā)育對(duì)于脂尾羊生長(zhǎng)和成年后尾部脂肪富集具有重要意義。2.五種尾型幼年羊生長(zhǎng)早期脂肪組織脂肪酸定量和定性分析,發(fā)現(xiàn)該階段其不同脂肪組織都以C18 cis-9:1、C16:0和C18:0三種脂肪酸含量最高,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長(zhǎng)早期相比同時(shí)期瘦尾羊尾部脂肪的主要脂肪酸含量有較大差異,可能由于組織特異性、脂尾與瘦尾羊品種遺傳差異所致。3.比較不同尾型幼年羊生長(zhǎng)早期尾脂差異蛋白組學(xué),五組尾脂中共鑒定到3400個(gè)蛋白,定量分析有804個(gè)差異表達(dá)蛋白,且涉及細(xì)胞分化以及脂肪酸合成的蛋白主要在脂尾羊中高表達(dá),說(shuō)明這有利于幼年脂尾羊尾脂的發(fā)育;參與脂肪代謝和脂解相關(guān)的重要蛋白也在脂尾羊中高表達(dá),表明幼年脂尾羊生長(zhǎng)早期尾脂不是主要儲(chǔ)存脂肪酸階段而可能以細(xì)胞分化數(shù)量增加為主為生長(zhǎng)后期及成年能夠儲(chǔ)存大量脂肪做準(zhǔn)備;對(duì)部分差異蛋白相應(yīng)基因進(jìn)行Realtime PCR及Western blot驗(yàn)證,其表達(dá)趨勢(shì)與蛋白組學(xué)基本一致;蛋白表達(dá)定位發(fā)現(xiàn)FABP4在脂尾羊尾脂的毛細(xì)血管壁內(nèi)外膜高表達(dá),可能介導(dǎo)前脂肪細(xì)胞分化和脂肪酸的轉(zhuǎn)運(yùn)功能;ERK2在脂尾羊尾脂脂肪細(xì)胞和毛細(xì)血管壁高表達(dá),揭示其可能介導(dǎo)幼年羊生長(zhǎng)早期脂肪組織中脂肪前體細(xì)胞分化增殖,且ACSL1在脂尾羊尾脂中高表達(dá)以參與脂肪細(xì)胞脂滴中甘油三酯的合成和儲(chǔ)存;表明以FABP4、ERK2為主的蛋白對(duì)于脂尾羊尾脂的發(fā)育具有重要意義。4.對(duì)不同尾型幼年羊生長(zhǎng)早期尾脂中蛋白磷酸化修飾分析,在五種尾型綿羊尾部脂肪組織中鑒定到1493個(gè)磷酸化位點(diǎn)分布在804個(gè)磷酸化蛋白,其主要參與翻譯、基因表達(dá)、蛋白代謝、細(xì)胞形態(tài)和細(xì)胞骨架、蛋白降解、細(xì)胞凋亡及蛋白修飾,說(shuō)明脂肪組織發(fā)育需要以上這些途徑來(lái)參與細(xì)胞代謝分化和脂肪沉積等重要生物學(xué)功能;定量發(fā)現(xiàn)435個(gè)磷酸化差異表達(dá)蛋白,涉及細(xì)胞骨架、蛋白翻譯后修飾的正調(diào)控、細(xì)胞凋亡信號(hào)通路的調(diào)控、蛋白質(zhì)分解代謝的調(diào)控、翻譯起始等15個(gè)生物學(xué)進(jìn)程,包括28個(gè)參與蛋白磷酸化修飾作用蛋白,揭示不同尾型綿羊尾脂的發(fā)育可能與差異磷酸化修飾相關(guān),基序分析發(fā)現(xiàn)以ERK1/2為主的激酶可能介導(dǎo)幼年脂尾羊生長(zhǎng)早期尾脂中相關(guān)蛋白發(fā)生磷酸化修飾,進(jìn)而對(duì)尾脂中脂肪前體細(xì)胞的分化具有正向調(diào)控作用。本試驗(yàn)為脂尾羊尾脂大量富集的生物學(xué)機(jī)制研究奠定基礎(chǔ),對(duì)幼年脂尾羊生長(zhǎng)早期參與脂肪細(xì)胞分化和脂肪沉積相關(guān)蛋白進(jìn)行表達(dá)干預(yù)實(shí)現(xiàn)阻止其尾脂快速發(fā)育,將對(duì)育種實(shí)踐中脂尾羊瘦尾化具有重要意義。
[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.

【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S826

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