亚麻籽油与红花油对奶牛乳腺ncRNA的影响及牛奶miRNA表达谱研究

发布时间：2017-12-26 23:00

  本文关键词：亚麻籽油与红花油对奶牛乳腺ncRNA的影响及牛奶miRNA表达谱研究　出处：《西北农林科技大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 奶牛 乳脂合成 脂肪酸 牛奶 miRNA 长链非编码RNA

【摘要】：大量研究已经证实,在日粮中添加富含不饱和脂肪酸的植物油(大豆油、亚麻籽油、红花油和鱼油等)可以产生乳脂抑制现象,即:乳脂生成特别是从头合成途径生成的短中链脂肪酸的含量会受到显著抑制,而不饱和脂肪酸的含量会相对升高,这样乳脂中脂肪酸成分会发生明显变化,更符合人类健康的需求。这种乳脂抑制现象也为研究乳脂合成和代谢调控机理提供了一个理想的试验模型。牛奶的乳脂生成涉及到一个复杂的代谢调控网络,需要大量影响脂肪酸合成的关键酶和转录因子的参与。非编码RNA(nc RNA,包括mi RNA和长链非编码RNA)在转录水平对基因的表达具有重要的调控作用,已有研究发现mi RNA和长链非编码RNA(lnc RNA)参与了脂肪生成和肌肉分化的调控,还有研究提示mi RNA也在反刍动物的乳腺发育、泌乳和泌乳周期调控上可能发挥重要作用,但非编码RNA是否在乳脂合成过程中发挥作用仍不清楚。本研究以处于泌乳中期的加拿大荷斯坦奶牛为对象,分析了日粮中分别添加5%亚麻籽油(亚麻籽油处理组)或5%红花油(红花油处理组)对奶牛乳脂生成和乳脂组成成分的影响,并利用高通量测序技术(小RNA测序和RNA测序)研究乳腺乳脂生成变化过程中乳腺组织mi RNA和长链非编码RNA表达谱的变化,并利用生物信息学方法研究相关关键非编码RNA在乳脂合成中的生物学功能。近年来研究发现,牛奶中也存在RNA,因此,探索牛奶中mi RNA的种类将为研究乳腺的活体采样替代方法和牛奶mi RNA功能提供重要参考。本研究取得了以下创新性结果:1、奶牛日粮中分别添加5%亚麻籽油或5%红花油对乳脂合成和脂肪酸成分的影响在亚麻籽油处理组和红花油组的处理后期Day+28(相对于对照组Day-14),两种处理组均导致乳脂率显著降低,分别降低了34.2%和29.9%。在两个处理中,饱和脂肪酸含量受到的影响一致,6个饱和脂肪酸(C4:0、C6:0、C8:0、C14:0、C16:0和C17:0)的含量均显著降低,C18:0在处理前期(Day+7)显著升高,随后降低到对照组水平。不饱和脂肪酸中,有4个(C18:1n11t、C20:3n3、C20:5n3和CLA:10t12c)的含量在两种处理中均显著升高;而C14:1的含量受两种处理的影响均显著下降。少量脂肪酸仅在一种处理中受到显著调控。2、奶牛日粮中分别添加5%亚麻籽油或5%红花油对乳腺mi RNA转录组的影响本研究构建了小RNA测序的分析流程,分析了乳腺mi RNA转录组的特点,并分别研究了日粮中添加5%亚麻籽油或5%红花油对乳腺转录组的影响,发现在处理后期(Day+28)共有27个mi RNA的表达受到显著调控(P0.05)。在乳腺中,有321个已知mi RNA表达并鉴定了176个高可信度的新mi RNA。表达最高的10个mi RNA占总mi RNA表达量的70.48%。在亚麻籽油处理组和红花油处理组,分别有14个和22个mi RNA的表达发生显著变化;其中7个mi RNA在两个处理组中均受到显著调控,被定义为核心差异表达mi RNA。IPA功能富集分析表明,核心差异表达mi RNA与乳脂合成相关。3、奶牛日粮中添加5%亚麻籽油或5%红花油对乳腺长链非编码RNA(lnc RNA)转录组的影响本研究构建了完整严格的lnc RNA鉴定流程,分析了乳腺lnc RNA转录组的特点,并分别研究了日粮中添加5%亚麻籽油或5%红花油对乳腺转录组的影响。研究发现,在乳腺组织共鉴定了4113个lnc RNA及其编码的6497个转录本。在亚麻籽油处理组和红花油处理组,乳腺组织分别有140和85个lnc RNA差异表达。差异表达的lnc RNA不仅出现在处理后期(Day+28),也出现在处理前期(Day+7)。4、牛奶mi RNA转录组与乳腺mi RNA转录组的比较分析本研究建立并优化了牛奶三种组分(乳脂、乳清和体细胞)中总RNA的提取方法,并成功构建了小RNA测序文库,以用于小RNA测序分析。通过对乳脂、乳清和体细胞中的小RNA测序分析,分别鉴定了210、200和249个已知mi RNA,以及33、31和36个新mi RNA。通过对比乳脂、乳清、体细胞和乳腺组织的mi RNA表达谱,发现乳脂mi RNA表达谱与乳腺mi RNA表达谱最为接近而且相关系数很高,因此,本研究认为,乳脂是替代乳腺活体采样以研究乳腺mi RNA转录组的最佳替代方法。乳脂、乳清和体细胞各自表达最高的20个mi RNA中有14个是共有的;乳清、体细胞和乳腺组织也存在各自特异高表达的mi RNA。牛奶中高表达的14个mi RNA与乳腺泌乳以及个体发育等相关。
[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.
【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S823
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本文编号：1339188