奶牛产奶性状候选功能基因VPS28的功能验证

发布时间：2018-02-08 12:42

本文关键词： VPS28 乳脂功能验证泛素化 ESCRTs 蛋白酶体　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

【摘要】：本课题组前期的GWAS及目标捕获测序研究发现VPS28基因与奶牛乳脂率和乳脂量显著相关,且其5’UTR区(具体位置用不用写?)有一个C/T突变与乳脂率极强关联(P=7.32E-60)。VPS28基因是内体蛋白分选转运装置ESCRTs的一个亚单位,与ESCRTs的功能和稳定性有关。ESCRTs特异性识别泛素化膜蛋白并将其运输至溶酶体降解,此外它还参与蛋白酶体对胞质中泛素化蛋白质的降解。因此,本研究预测VPS28通过影响ESCRTs或蛋白酶体的功能而影响乳脂的生成,并在细胞水平上分别从DNA、转录组及蛋白质组三种水平上分析该基因突变位点的作用方式及其调控乳脂的机制,试图探索VPS28对乳脂的作用通路。首先,本研究通过对VPS28基因进行启动子活性分析发现其5'UTR的C/T突变可导致基因的表达量下降；然后对该突变进行转录因子结合预测,发现其可导致转录因子由ADR1变为NRF2,两个转录因子的DNA结合域分别是二锌指结构和亮氨酸拉链,且前者与DNA结合的位点数及结合强度均大于后者。因此,本研究认为该突变通过改变转录因子与DNA的结合方式使VPS28基因表达下调。然后,本研究利用RNA干扰技术将BMEC中的VPS28进行干扰,并在转录水平分析其对乳脂的调控机制。通过对比分析相关基因的表达水平,发现VPS28对乳脂的影响可能主要通过影响ESCRTs及蛋白酶体的功能影响了脂肪酸的吸收和从头合成。为进一步验证,本研究又在BMEC中检测了相关蛋白等相关的生化指标的表达水平,发现将VPS28干扰后,泛素化的脂肪酸转运蛋白CD36、脂肪分化相关蛋白ADFP及甘油三酯的表达水平表达水平均上升(P0.05),蛋白酶体的活性显著下降(P0.05),并在电子显微镜下观察到干扰VPS28的细胞中存在数量更多体积更大的脂肪滴。该结果表明VPS28受抑制可通过阻碍ESCRTs对泛素化膜蛋白的降解及蛋白酶体对胞质中泛素化蛋白质的降解,以增加脂肪酸吸收和从头合成,最终提高甘油三酯合成增加乳脂生成。最后,本研究又将干扰前后的BMEC进行iTRAQ蛋白质组学分析,以全面揭示VPS28对乳脂合成与分泌的调控机制和作用通路。通过对筛选到的203个差异表达蛋白进行富集分析发现VPS28受到抑制后主要对脂类生成和转运造成影响,然后将这些差异表达蛋白进行通路图的构建,得到了以VPS28为中心且较为清晰的通路图。结合第二部分结果,本研究发现VPS28被抑制后主要通过两种方式调控乳脂生成：1：VPS28低表达阻碍ESCRT功能,蓄积泛素化膜蛋白CD36提高BMEC脂肪酸吸收；2；VPS28低表达降低蛋白酶体活性,蓄积胞质中泛素化蛋白蓄积脂肪酸合成酶FASN及乙酰辅酶A羧化酶ACACA增加脂肪酸的从头合成,最终增加乳脂合成。
[Abstract]:The results of GWAS and target capture sequencing showed that the VPS28 gene was significantly correlated with milk fat rate and milk fat content in dairy cows. ) there is a C / T mutation strongly associated with milk fat rate. VPS28 gene is a subunit of ESCRTs, which is related to the function and stability of ESCRTs. EscRTs specifically recognizes the ubiquitin membrane protein and transports it to lysosomal degradation. It is also involved in the degradation of ubiquitin proteins in the cytoplasm by proteasome. Therefore, this study predicted that VPS28 affects the production of milk fat by affecting the function of ESCRTs or proteasome. At the cellular level, we analyzed the action mode of the mutation site and the mechanism of regulating milk fat from the three levels of DNA, transcriptional and proteome, respectively, in order to explore the mechanism of VPS28 acting on milk fat. First of all, we tried to explore the mechanism of the effect of VPS28 on milk fat. In this study, we analyzed the promoter activity of VPS28 gene and found that the C / T mutation of 5 UTR could lead to the decrease of gene expression, and then the transcription factor binding prediction of the mutation was carried out. It was found that the transcription factor changed from ADR1 to NRF2. The DNA binding domain of the two transcription factors was dizinc finger structure and leucine zipper respectively, and the site number and binding strength of the former to DNA were higher than that of the latter. This study suggests that the mutation can down-regulate the expression of VPS28 gene by changing the binding of transcription factors to DNA. Then, RNA interference technique is used to interfere the VPS28 in BMEC. The regulation mechanism of milk fat was analyzed at the transcriptional level. It was found that the effect of VPS28 on milk fat may affect the absorption and ab initio synthesis of fatty acids by affecting the functions of ESCRTs and proteasome. In order to further verify, the expression level of related biochemical indexes, such as related proteins, was detected in BMEC. After finding that the VPS28 is interfered with, The expression levels of ubiquitin fatty acid transporter CD36, adipose differentiation related protein ADFP and triglyceride increased, while the activity of proteasome decreased significantly, and the presence of interfering VPS28 cells was observed under electron microscope. The results show that the inhibition of VPS28 can be achieved by blocking the degradation of ubiquitin membrane protein by ESCRTs and the degradation of ubiquitin protein in cytoplasm by proteasome. In order to increase fatty acid absorption and ab initio synthesis, triglyceride synthesis and milk fat production were increased. Finally, the BMEC before and after interference was analyzed by iTRAQ proteomics. In order to reveal the regulation mechanism and pathway of VPS28 on milk fat synthesis and secretion, the enrichment analysis of 203 differentially expressed proteins showed that the inhibition of VPS28 mainly affected the formation and transport of lipids. Then we construct the pathway map of these differentially expressed proteins and obtain a clear path map centered on VPS28. In this study, it was found that the low expression of VPS28 inhibited by VPS28 was inhibited by two ways, and the accumulation of Ubiquitin membrane protein CD36 increased the low expression of VPS28 in BMEC fatty acids and decreased the activity of proteasome. Ubiquitin fatty acid synthase (FASN) and acetyl-coenzyme A carboxylase (ACACA) in the accumulative cytoplasm increased the ab initio synthesis of fatty acids and ultimately increased the synthesis of milk fat.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S823

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