本文選題：胎衣不下 + 奶牛��； 參考：《黑龍江八一農(nóng)墾大學》2015年碩士論文

【摘要】：本實驗主要目的是采用Solexa高通量測序技術和生物信息學分析方法，構建sRNA文庫和分析奶牛胎衣不下和胎衣正常排出時母體胎盤組織間差異表達的microRNAs（miRNAs），影響該病發(fā)生發(fā)展的關鍵miRNAs，并對部分miRNA進行驗證，以期從分子層面揭示該病的發(fā)病機制、為臨床防治效果提供理論依據(jù)。 實驗方法：選取年齡、胎次、泌乳量、體重都相近，，且無其它疾病影響的胎衣正常排出奶牛母體胎盤（normal maternal placenta，NC）作為對照組，胎衣不下奶牛母體胎盤（retained maternal placenta，RC）作為實驗組，每組三頭奶牛。采用Trizol裂解法提取組織總RNA，構建NC和RC的cDNA文庫，并采用Solexa高通量測序方法對文庫進行測序，篩選兩文庫中差異表達的miRNAs。隨機挑選8個差異表達的miRNAs，應用實時熒光定量PCR的方法對測序結果進行驗證。利用生物信息學軟件預測差異表達miRNAs的靶基因，最后進行GO富集分析和KEGG Pathway通路分析。 實驗結果：成功構建了小RNA文庫，高通量測序在NC和RC文庫中獲得的高質量序列分別為5962381與5446898條，兩個文庫中高質量序列占原始小RNA序列比率均在99%以上（99.37%，99.43%），兩個文庫中sRNA大小主要集中在19nt-24nt，22nt大小的片斷是兩個文庫中最多的，NC和RC文庫分別占據(jù)53.76%和56.21%；69個已知miRNAs在奶牛胎衣正常排出組和胎衣不下組母體胎盤組織中差異顯著表達，與胎衣正常排出組母體胎盤相比，其中36個顯著下調(diào)，33個顯著上調(diào)（P＜0.05）；在NC和RC文庫中分別發(fā)現(xiàn)36種和33種新miRNA候選者；實時熒光定量PCR驗證與測序結果一致，實驗組與對照組相比bta-miR-423-5p，bta-miR-181a，bta-miR-185顯著下調(diào)（P＜0.05）；bta-miR-411a，bta-miR-31， bta-miR-424-5p顯著上調(diào)（P＜0.05）；bta-miR-1839和bta-miR-1上調(diào)但不顯著（P＞0.05）；對差異表達的miRNAs GO分析結果顯示，與蛋白結合，雜環(huán)化合物結合，金屬離子集合和催化活性等功能相關基因出現(xiàn)頻率最高，但無顯著富集（P＞0.05）；KEGG Pathway分析結果顯示，癌癥通路，肌動蛋白細胞骨架調(diào)節(jié)通路和MAPK信號通路被顯著富集（P＜0.05）。 實驗結果說明奶牛在發(fā)生胎衣不下時，母體胎盤中部分miRNA的表達發(fā)生明顯變化，這些表達變化miRNA可通過調(diào)節(jié)相關基因的表達，特別是與細胞粘附作用和肌肉收縮作用相關蛋白mRNA的表達，通過復雜的調(diào)控網(wǎng)絡，在一定程度上影響胎衣不下的發(fā)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.
【學位授予單位】：黑龍江八一農(nóng)墾大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.23

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