【摘要】：番鴨呼腸孤病毒(Muscovy duck reovirus, MDRV)是近10年來(lái)發(fā)現(xiàn)的引起雛番鴨較高發(fā)病率和死亡率的病毒。肝臟和脾臟是MDRV感染的主要靶器官。為探討MDRV感染的分子致病機(jī)制,本研究以肝臟和脾臟為主要研究對(duì)象,應(yīng)用轉(zhuǎn)錄組學(xué)方法,對(duì)10日齡番鴨轉(zhuǎn)錄組進(jìn)行測(cè)序、組裝和注釋；篩選和分析了MDRV感染后肝臟和脾臟的差異表達(dá)基因,并對(duì)差異表達(dá)基因進(jìn)行了功能分析；最后對(duì)MDRV感染激活機(jī)體先天性免疫功能,誘導(dǎo)細(xì)胞凋亡的分子機(jī)制和導(dǎo)致肝臟脂肪變性的分子機(jī)制進(jìn)行了分析和驗(yàn)證。研究結(jié)果闡明了MDRV感染的分子致病機(jī)理,主要結(jié)果如下：1.本研究得到總長(zhǎng)度17G的轉(zhuǎn)錄組序列,組裝得到65,535個(gè)Unigenes,平均長(zhǎng)度987bp; COG注釋共獲得41,651個(gè)Unigenes的注釋,包含25個(gè)生物學(xué)功能。2. MDRV感染5日齡雛番鴨后第5天,差異表達(dá)基因篩選發(fā)現(xiàn),脾臟有1,352個(gè)基因表達(dá)量上調(diào),4,906個(gè)基因表達(dá)量下調(diào)；GO顯著性富集分析表明,這些基因共參與了48個(gè)生物學(xué)功能,KEGG顯著性富集229個(gè)Pathway。3.差異表達(dá)基因篩選發(fā)現(xiàn)肝臟有4,190個(gè)基因表達(dá)量上調(diào),1,113個(gè)基因下調(diào)。GO顯著性富集分析表明,這些基因共參與了48個(gè)生物學(xué)功能,KEGG顯著性富集237個(gè)Pathway。4. MDRV感染肝臟和脾臟差異基因的比較分析發(fā)現(xiàn),脾臟的差異表達(dá)基因以下調(diào)為主,肝臟以上調(diào)為主,兩個(gè)器官的共有差異表達(dá)基因1,350個(gè),GO功能分析表明,這些共有差異表達(dá)基因參與了42個(gè)生物學(xué)功能,KEGG功能分析表明,共有差異表達(dá)基因涉及的免疫功能的有炎癥因子,先天性免疫功能,抗原提呈過(guò)程等。共有差異表達(dá)基因的獲得將為進(jìn)一步闡明MDRV感染的致病機(jī)理提供了有力的依據(jù)。5. KEGG分析表明,MDRV感染,上調(diào)了脾臟中模式識(shí)別受體RIG-I, MDA5, TLR-1, TLR-2, TLR-4的表達(dá)來(lái)識(shí)別MDRV,活化IRF7促進(jìn)Ⅰ型IFN的分泌和調(diào)節(jié)NF-kB信號(hào)通路促進(jìn)炎癥因子IL6的分泌,激活JAK-STAT信號(hào),刺激IFN的分泌,增強(qiáng)機(jī)體先天性免疫功能。結(jié)果經(jīng)Q-PCR驗(yàn)證,證實(shí)轉(zhuǎn)錄組數(shù)據(jù)準(zhǔn)確可靠。6. TUNEL法和流式細(xì)胞術(shù)證實(shí),MDRV感染誘導(dǎo)了肝臟細(xì)胞凋亡。KEGG分析表明,MDRV感染通過(guò)激活了Fas信號(hào)通路,IL-1R信號(hào)通路,抑制了PI3K/AKT信號(hào)通路誘導(dǎo)細(xì)胞凋亡。Western blot和Q-PCR驗(yàn)證表明,轉(zhuǎn)錄組數(shù)據(jù)準(zhǔn)確可靠。7. MDRV感染肝臟中脂肪酸含量極顯著的增加(P0.01)，，從而誘導(dǎo)肝臟脂肪變性。KEGG分析表明,MDRV感染抑制了肝細(xì)胞中膽固醇外排和脂肪酸分解代謝關(guān)鍵酶蛋白基因的表達(dá),從而導(dǎo)致脂肪酸和膽固醇在肝細(xì)胞中的蓄積,結(jié)果闡明了MDRV感染肝臟發(fā)生脂肪變性的分子機(jī)制。綜上,本研究運(yùn)用轉(zhuǎn)錄組學(xué)方法,闡明了MDRV感染肝臟和脾臟的差異表達(dá)基因和肝臟、脾臟的共有差異表達(dá)基因,分析了這些差異表達(dá)基因的生物學(xué)功能,同時(shí)分析和驗(yàn)證了MDRV激活宿主先天性免疫功能,誘導(dǎo)細(xì)胞凋亡和肝臟脂肪變性的分子機(jī)制�？梢�(jiàn),宿主調(diào)動(dòng)了多個(gè)基因和多條通路共同參與調(diào)控MDRV感染過(guò)程。
[Abstract]:Muscovy duck reovirus (Muscovy duck reovirus, MDRV) is a virus found in recent 10 years to cause higher morbidity and mortality in young muscovy ducks. Liver and spleen are the main target organs of MDRV infection. In order to study the molecular pathogenesis of MDRV infection, the transcriptome of 10-day-old muscovy duck was sequenced, assembled and annotated by transcriptome method. The differentially expressed genes in liver and spleen after MDRV infection were screened and analyzed, and the function of differentially expressed genes was analyzed. Finally, the molecular mechanism of MDRV infection which activates innate immune function, induces apoptosis and leads to hepatic steatosis is analyzed and verified. The main results are as follows: 1. In this study, a total length of 17G transcriptome sequence was obtained, and 65535 Unigenes, average lengths were 987 BP, while 41651 Unigenes annotations were obtained, including 25 biological functions. 2. On the 5th day after MDRV infection, 1352 genes were up-regulated and 4906 genes were down-regulated in spleen. GO significant enrichment analysis showed that these genes were involved in 48 biological functions, and KEGG significantly enriched 229 Pathway.3.. Screening of differentially expressed genes revealed that 4190 genes were up-regulated and 113 genes down-regulated in liver. GO significant enrichment analysis showed that these genes participated in 48 biological functions, and KEGG significantly enriched 237 Pathway.4.. The comparative analysis of differentially expressed genes in liver and spleen of MDRV infection showed that the differential expression genes of spleen were mainly down-regulated and that of liver were up-regulated. There were 1350 differentially expressed genes in the two organs. GO functional analysis showed that there were 1350 differentially expressed genes in the two organs. These co-differentially expressed genes were involved in 42 biological functions. KEGG functional analysis showed that the common differentially expressed genes involved inflammatory factors, congenital immune function, antigen presentation process and so on. The acquisition of common differentially expressed genes will provide a powerful basis for further elucidating the pathogenic mechanism of MDRV infection. KEGG analysis showed that MDRV infection upregulated the expression of pattern recognition receptor (RIG-I, MDA5, TLR-1, TLR-2, TLR-4) in spleen to recognize MDRV,. Activation of IRF7 promoted the secretion of type I IFN and regulated the NF-kB signaling pathway, promoted the secretion of inflammatory factor IL6, activated JAK-STAT signal, stimulated the secretion of IFN, and enhanced the innate immune function of the body. Results the transcriptional data were confirmed to be accurate and reliable by Q-PCR. 6. 6%. TUNEL assay and flow cytometry confirmed that MDRV infection induced apoptosis of liver cells. KEGG analysis showed that MDRV infection activates Fas signaling pathway, IL-1R signal pathway. The inhibition of PI3K/AKT signaling pathway induced apoptosis by. Western blot and Q-PCR showed that the transcriptional data were accurate and reliable. The fatty acid content in the liver of MDRV infection was significantly increased (P0.01), which induced hepatic steatosis. KEGG analysis showed that MDRV infection inhibited the expression of key enzymes in cholesterol excretion and fatty acid catabolism in hepatocytes. The accumulation of fatty acids and cholesterol in liver cells may lead to the molecular mechanism of fatty degeneration in MDRV infected liver. In summary, we used transcriptome method to elucidate the differentially expressed genes in the liver and spleen of MDRV infection, and analyze the biological functions of these differentially expressed genes in the liver and spleen. At the same time, the molecular mechanism that MDRV activates innate immune function, induces apoptosis and steatosis of liver is analyzed and verified. It can be seen that the host involved in the regulation of MDRV infection by multiple genes and multiple pathways.
【學(xué)位授予單位】：福建師范大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.32

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本文編號(hào)：2340892