胎衣不下奶牛母体胎盘组织差异表达microRNA的筛选及验证

发布时间：2018-06-22 18:15

本文选题：胎衣不下 + 奶牛　；参考：《黑龙江八一农垦大学》2015年硕士论文

【摘要】：本实验主要目的是采用Solexa高通量测序技术和生物信息学分析方法，构建sRNA文库和分析奶牛胎衣不下和胎衣正常排出时母体胎盘组织间差异表达的microRNAs（miRNAs），影响该病发生发展的关键miRNAs，并对部分miRNA进行验证，以期从分子层面揭示该病的发病机制、为临床防治效果提供理论依据。实验方法：选取年龄、胎次、泌乳量、体重都相近，，且无其它疾病影响的胎衣正常排出奶牛母体胎盘（normal maternal placenta，NC）作为对照组，胎衣不下奶牛母体胎盘（retained maternal placenta，RC）作为实验组，每组三头奶牛。采用Trizol裂解法提取组织总RNA，构建NC和RC的cDNA文库，并采用Solexa高通量测序方法对文库进行测序，筛选两文库中差异表达的miRNAs。随机挑选8个差异表达的miRNAs，应用实时荧光定量PCR的方法对测序结果进行验证。利用生物信息学软件预测差异表达miRNAs的靶基因，最后进行GO富集分析和KEGG Pathway通路分析。实验结果：成功构建了小RNA文库，高通量测序在NC和RC文库中获得的高质量序列分别为5962381与5446898条，两个文库中高质量序列占原始小RNA序列比率均在99%以上（99.37%，99.43%），两个文库中sRNA大小主要集中在19nt-24nt，22nt大小的片断是两个文库中最多的，NC和RC文库分别占据53.76%和56.21%；69个已知miRNAs在奶牛胎衣正常排出组和胎衣不下组母体胎盘组织中差异显著表达，与胎衣正常排出组母体胎盘相比，其中36个显著下调，33个显著上调（P＜0.05）；在NC和RC文库中分别发现36种和33种新miRNA候选者；实时荧光定量PCR验证与测序结果一致，实验组与对照组相比bta-miR-423-5p，bta-miR-181a，bta-miR-185显著下调（P＜0.05）；bta-miR-411a，bta-miR-31， bta-miR-424-5p显著上调（P＜0.05）；bta-miR-1839和bta-miR-1上调但不显著（P＞0.05）；对差异表达的miRNAs GO分析结果显示，与蛋白结合，杂环化合物结合，金属离子集合和催化活性等功能相关基因出现频率最高，但无显著富集（P＞0.05）；KEGG Pathway分析结果显示，癌症通路，肌动蛋白细胞骨架调节通路和MAPK信号通路被显著富集（P＜0.05）。实验结果说明奶牛在发生胎衣不下时，母体胎盘中部分miRNA的表达发生明显变化，这些表达变化miRNA可通过调节相关基因的表达，特别是与细胞粘附作用和肌肉收缩作用相关蛋白mRNA的表达，通过复杂的调控网络，在一定程度上影响胎衣不下的发生。
[Abstract]:The main purpose of this experiment is to use Solexa high-throughput sequencing technique and bioinformatics analysis method. To construct a siRNA library and analyze the differential expression of miRNAs in placenta tissues of dairy cows during placenta normal excretion, the key to the development of the disease was miRNAs. and some miRNAs were verified in order to reveal the pathogenesis of the disease at the molecular level. To provide theoretical basis for clinical prevention and treatment. Methods: the age, birth order, lactation volume and body weight were all similar, and the placenta (normal maternal placenta NC was normally excreted from the placenta of dairy cows without other diseases. The control group was used as the control group, and the placenta (retained maternal placenta RC) was used as the experimental group. There are three cows in each group. The cDNA libraries of NC and RC were constructed by using Trizol cleavage method to extract tissue total RNAs. Solexa high-throughput sequencing method was used to sequence the libraries and to screen the differentially expressed miRNAs in the two libraries. Eight differentially expressed miRNAss were randomly selected and sequenced by real-time fluorescent quantitative PCR. The target genes of differentially expressed miRNAs were predicted by bioinformatics software. Finally, go enrichment analysis and KEGG Pathway pathway analysis were performed. Results: the small RNA library was successfully constructed. The high quality sequences obtained by high throughput sequencing in NC and RC libraries were 5962381 and 5446898, respectively. The ratio of the high quality sequence to the original small RNA sequence in the two libraries was over 99% (99. 37% or 99. 43%). The size of the sRNA in the two libraries mainly concentrated in the 19nt-24ntnt22nt size fragment, which occupied 53.76% and 56.21% respectively. There were significant differences in the expression of 69 known miRNAs in maternal placenta of cow placenta with normal excretion of fetal coat and normal placenta, 36 of them were significantly down-regulated and 33 were up-regulated (P < 0.05). 36 and 33 new miRNA candidates were found in NC and RC libraries respectively, and the results of real-time fluorescence quantitative PCR were consistent with those of sequencing. Compared with the control group, bta-miR-423-5pta-miR-181abta-miR-185 was significantly down-regulated (P < 0.05) and bta-miR-411abta-miR-31, bta-miR-424-5p was significantly up-regulated (P < 0.05). Bta-miR-1839 and bta-miR-1 were up-regulated but not significantly (P > 0.05). The results of miRNAs go analysis showed that the genes related to protein binding, heterocyclic compounds binding, metal ion set and catalytic activity were the most frequent, but not significantly enriched (P > 0.05). KEGG Pathway analysis showed that the cancer pathway, actin cytoskeleton regulatory pathway and MAPK signaling pathway were significantly enriched (P < 0.05). The results showed that the expression of partial miRNA in the placenta of the mother changed obviously when the placenta occurred. These changes of miRNA expression could regulate the expression of related genes. In particular, the expression of protein mRNA associated with cell adhesion and muscle contraction affects the occurrence of fetal coat to some extent through complex regulatory networks.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.23

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