本文關(guān)鍵詞：亞麻籽油與紅花油對(duì)奶牛乳腺ncRNA的影響及牛奶miRNA表達(dá)譜研究　出處：《西北農(nóng)林科技大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 奶牛 乳脂合成 脂肪酸 牛奶 miRNA 長(zhǎng)鏈非編碼RNA

【摘要】：大量研究已經(jīng)證實(shí),在日糧中添加富含不飽和脂肪酸的植物油(大豆油、亞麻籽油、紅花油和魚(yú)油等)可以產(chǎn)生乳脂抑制現(xiàn)象,即:乳脂生成特別是從頭合成途徑生成的短中鏈脂肪酸的含量會(huì)受到顯著抑制,而不飽和脂肪酸的含量會(huì)相對(duì)升高,這樣乳脂中脂肪酸成分會(huì)發(fā)生明顯變化,更符合人類(lèi)健康的需求。這種乳脂抑制現(xiàn)象也為研究乳脂合成和代謝調(diào)控機(jī)理提供了一個(gè)理想的試驗(yàn)?zāi)Ｐ�。牛奶的乳脂生成涉及到一個(gè)復(fù)雜的代謝調(diào)控網(wǎng)絡(luò),需要大量影響脂肪酸合成的關(guān)鍵酶和轉(zhuǎn)錄因子的參與。非編碼RNA(nc RNA,包括mi RNA和長(zhǎng)鏈非編碼RNA)在轉(zhuǎn)錄水平對(duì)基因的表達(dá)具有重要的調(diào)控作用,已有研究發(fā)現(xiàn)mi RNA和長(zhǎng)鏈非編碼RNA(lnc RNA)參與了脂肪生成和肌肉分化的調(diào)控,還有研究提示mi RNA也在反芻動(dòng)物的乳腺發(fā)育、泌乳和泌乳周期調(diào)控上可能發(fā)揮重要作用,但非編碼RNA是否在乳脂合成過(guò)程中發(fā)揮作用仍不清楚。本研究以處于泌乳中期的加拿大荷斯坦奶牛為對(duì)象,分析了日糧中分別添加5%亞麻籽油(亞麻籽油處理組)或5%紅花油(紅花油處理組)對(duì)奶牛乳脂生成和乳脂組成成分的影響,并利用高通量測(cè)序技術(shù)(小RNA測(cè)序和RNA測(cè)序)研究乳腺乳脂生成變化過(guò)程中乳腺組織mi RNA和長(zhǎng)鏈非編碼RNA表達(dá)譜的變化,并利用生物信息學(xué)方法研究相關(guān)關(guān)鍵非編碼RNA在乳脂合成中的生物學(xué)功能。近年來(lái)研究發(fā)現(xiàn),牛奶中也存在RNA,因此,探索牛奶中mi RNA的種類(lèi)將為研究乳腺的活體采樣替代方法和牛奶mi RNA功能提供重要參考。本研究取得了以下創(chuàng)新性結(jié)果:1、奶牛日糧中分別添加5%亞麻籽油或5%紅花油對(duì)乳脂合成和脂肪酸成分的影響在亞麻籽油處理組和紅花油組的處理后期Day+28(相對(duì)于對(duì)照組Day-14),兩種處理組均導(dǎo)致乳脂率顯著降低,分別降低了34.2%和29.9%。在兩個(gè)處理中,飽和脂肪酸含量受到的影響一致,6個(gè)飽和脂肪酸(C4:0、C6:0、C8:0、C14:0、C16:0和C17:0)的含量均顯著降低,C18:0在處理前期(Day+7)顯著升高,隨后降低到對(duì)照組水平。不飽和脂肪酸中,有4個(gè)(C18:1n11t、C20:3n3、C20:5n3和CLA:10t12c)的含量在兩種處理中均顯著升高;而C14:1的含量受兩種處理的影響均顯著下降。少量脂肪酸僅在一種處理中受到顯著調(diào)控。2、奶牛日糧中分別添加5%亞麻籽油或5%紅花油對(duì)乳腺mi RNA轉(zhuǎn)錄組的影響本研究構(gòu)建了小RNA測(cè)序的分析流程,分析了乳腺mi RNA轉(zhuǎn)錄組的特點(diǎn),并分別研究了日糧中添加5%亞麻籽油或5%紅花油對(duì)乳腺轉(zhuǎn)錄組的影響,發(fā)現(xiàn)在處理后期(Day+28)共有27個(gè)mi RNA的表達(dá)受到顯著調(diào)控(P0.05)。在乳腺中,有321個(gè)已知mi RNA表達(dá)并鑒定了176個(gè)高可信度的新mi RNA。表達(dá)最高的10個(gè)mi RNA占總mi RNA表達(dá)量的70.48%。在亞麻籽油處理組和紅花油處理組,分別有14個(gè)和22個(gè)mi RNA的表達(dá)發(fā)生顯著變化;其中7個(gè)mi RNA在兩個(gè)處理組中均受到顯著調(diào)控,被定義為核心差異表達(dá)mi RNA。IPA功能富集分析表明,核心差異表達(dá)mi RNA與乳脂合成相關(guān)。3、奶牛日糧中添加5%亞麻籽油或5%紅花油對(duì)乳腺長(zhǎng)鏈非編碼RNA(lnc RNA)轉(zhuǎn)錄組的影響本研究構(gòu)建了完整嚴(yán)格的lnc RNA鑒定流程,分析了乳腺lnc RNA轉(zhuǎn)錄組的特點(diǎn),并分別研究了日糧中添加5%亞麻籽油或5%紅花油對(duì)乳腺轉(zhuǎn)錄組的影響。研究發(fā)現(xiàn),在乳腺組織共鑒定了4113個(gè)lnc RNA及其編碼的6497個(gè)轉(zhuǎn)錄本。在亞麻籽油處理組和紅花油處理組,乳腺組織分別有140和85個(gè)lnc RNA差異表達(dá)。差異表達(dá)的lnc RNA不僅出現(xiàn)在處理后期(Day+28),也出現(xiàn)在處理前期(Day+7)。4、牛奶mi RNA轉(zhuǎn)錄組與乳腺mi RNA轉(zhuǎn)錄組的比較分析本研究建立并優(yōu)化了牛奶三種組分(乳脂、乳清和體細(xì)胞)中總RNA的提取方法,并成功構(gòu)建了小RNA測(cè)序文庫(kù),以用于小RNA測(cè)序分析。通過(guò)對(duì)乳脂、乳清和體細(xì)胞中的小RNA測(cè)序分析,分別鑒定了210、200和249個(gè)已知mi RNA,以及33、31和36個(gè)新mi RNA。通過(guò)對(duì)比乳脂、乳清、體細(xì)胞和乳腺組織的mi RNA表達(dá)譜,發(fā)現(xiàn)乳脂mi RNA表達(dá)譜與乳腺mi RNA表達(dá)譜最為接近而且相關(guān)系數(shù)很高,因此,本研究認(rèn)為,乳脂是替代乳腺活體采樣以研究乳腺mi RNA轉(zhuǎn)錄組的最佳替代方法。乳脂、乳清和體細(xì)胞各自表達(dá)最高的20個(gè)mi RNA中有14個(gè)是共有的;乳清、體細(xì)胞和乳腺組織也存在各自特異高表達(dá)的mi RNA。牛奶中高表達(dá)的14個(gè)mi RNA與乳腺泌乳以及個(gè)體發(fā)育等相關(guān)。
[Abstract]:A number of studies have confirmed that adding rich in unsaturated fatty acids in the diets of vegetable oils (soybean oil, linseed oil, safflower oil and fish oil) can produce milk fat suppression phenomenon, namely: fat generation especially content of short chain fatty acids generated de novo pathway will be inhibited, and the content of unsaturated fatty acid will be relatively increased, so milk fatty acid composition will change significantly, more in line with the needs of human health. The milk fat suppression also provides an ideal experimental model for the study of the mechanism of lipid synthesis and metabolic regulation. Milk fat formation involves a complex metabolic regulation network that requires the participation of a large number of key enzymes and transcription factors that affect fatty acid synthesis. Non encoding RNA (NC RNA, including mi RNA and long chain non encoding RNA) on gene expression at the transcriptional level has an important role in the regulation of MI, RNA and RNA long chain non encoding has been found (LNC RNA) is involved in the regulation of adipogenesis and muscle differentiation, and the results suggest that Mi RNA in ruminant animal the mammary gland development, lactation and lactation cycle regulation may play an important role, but non encoding whether RNA play a role in fat synthesis process is still not clear. In this study, in mid lactation Holstein cows in Canada as the object, analyzed 5% linseed oil were added in diets (treatment group or 5% safflower oil flax seed oil) (safflower oil treatment group) effect on milk production and milk composition, and the use of high-throughput sequencing technologies (small RNA sequencing and RNA sequencing) generation the change process of breast milk in the breast tissue mi RNA and long chain non expression of RNA encoding, and studying related key non biological function of encoding RNA in milk fat synthesis in the use of biological information. In recent years, studies have found that there is also RNA in milk. Therefore, exploring the types of MI RNA in milk will provide important references for studying the methods of breast body in vivo sampling and milk mi RNA function. This study has obtained the following innovative results: 1, dairy cows were added 5% linseed oil or 5% safflower oil on milk fat synthesis and fatty acid composition of Day+28 in the treatment of late treatment group and safflower oil group (flax seed oil compared with the control group Day-14), two groups were in milk fat percentage decreased significantly, respectively. 34.2% and 29.9%. In the two treatments, the contents of saturated fatty acids were the same. The contents of 6 saturated fatty acids (C4:0, C6:0, C8:0, C14:0, C16:0 and C17:0) were significantly decreased, C18:0 increased significantly in the early stage (Day+7), and then decreased to the control level. The contents of 4 unsaturated fatty acids (C18:1n11t, C20:3n3, C20:5n3 and CLA:10t12c) increased significantly in two treatments, while the contents of C14:1 were significantly decreased by two treatments. A small amount of fatty acids can be significantly regulated in one treatment. 2, dairy cows were added 5% linseed oil or safflower oil on 5% breast mi RNA transcription group was constructed based on process analysis of small RNA sequencing, analysis of the characteristics of the breast mi RNA transcriptome, and studied the effect of adding 5% linseed oil or 5% safflower oil diets. The group of breast found in the later stages of processing, (Day+28) a total of 27 mi RNA expression was significant regulation (P0.05). In the breast, 321 known mi RNA were expressed and 176 new mi RNA with high reliability were identified. The highest expression of 10 mi RNA accounted for 70.48% of the total mi RNA expression. In the treatment group and the treatment group of safflower oil flax seed oil, respectively 14 and 22 mi RNA significantly changed; one of the 7 mi RNA in the two treatment groups were significantly affected by the regulation, is defined as the core of the differential expression of MI RNA. IPA functional enrichment analysis showed that the core differential expression of MI RNA was related to milk fat synthesis. 3, cow diets supplemented with 5% linseed oil or 5% safflower oil on the breast of long chain non encoding RNA (LNC RNA) of the transcriptome of the establishment of LNC RNA identification process complete and rigorous, analyzes the characteristics of the breast LNC RNA transcriptome, and investigated 5% flax seed oil or safflower oil diet 5% in the impact of breast transcriptome. The study found that 4113 LNC RNA and 6497 transcriptional transcripts were identified in the breast tissue. In the treatment group and the treatment group of safflower oil flax seed oil, breast tissue respectively 140 and 85 LNC RNA difference. The differential expression of LNC RNA appeared not only in the late stage of treatment (Day+28), but also in the early stage of treatment (Day+7). 4, a comparative analysis of milk mi RNA transcriptome and MI RNA transcriptome. This study established and optimized the extraction method of total RNA in three components of milk (cream, whey and somatic cells), and successfully constructed a small RNA sequencing library for small RNA sequencing analysis. 210, 200 and 249 known mi RNA, as well as 33, 31 and 36 new mi RNA, were identified by small RNA sequencing in milk fat, whey and somatic cells. By contrast, MI RNA milk whey, somatic cells and breast tissue expression, and the expression of RNA in breast Mi found spectral spectrum closest and the correlation coefficient is very high, the expression of milk mi RNA therefore, this study suggests that fat is the best alternative to replace breast biopsy sampling method of breast Mi transcription of RNA group. 14 of the 20 highest mi RNA expressed in milk fat, whey and somatic cells were common; whey, somatic cells and mammary tissues also had their own highly expressed mi RNA. The high expression of 14 mi RNA in milk is associated with mammary lactation and ontogenesis.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S823
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本文編號(hào)：1339188