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  本文關(guān)鍵詞： 少孢節(jié)叢孢 milRNA milR289 QDE-2 捕器 HITS-CLIP RIP　出處：《云南大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：非編碼RNA廣泛存在于原核生物和各種真核生物中,在生物體的生長發(fā)育衰老疾病等生物學(xué)機(jī)制中都發(fā)揮重要的調(diào)控作用。MiRNA是非編碼RNA中研究得最為清楚的一類small RN A,大小在21 nt左右,在動植物中的功能和調(diào)控機(jī)制都有比較系統(tǒng)的報道,并已應(yīng)用到各物種的生物學(xué)功能機(jī)制研究和人類疾病治療等方面。目前真菌中已經(jīng)報道了microRNA-like RNA,但其生物學(xué)功能還不清楚。本文在捕食線蟲真菌少孢節(jié)叢孢(簡稱少孢)中發(fā)現(xiàn)milRNA,并且證明milR289能夠以qde-2等基因為靶基因。Qde-2等基因在少孢生活史轉(zhuǎn)化中起到重要作用,即在少孢形成捕器三維菌網(wǎng)的過程中發(fā)揮作用。我們通過small RNA組測序和northernblot證明少孢中存在small RNA,根據(jù)其序列信息和northern blot雜交結(jié)果,發(fā)現(xiàn)這些small RNA與miRNA結(jié)構(gòu)類似,將其命名為microRNA-like RNA (milRNA)。RIP實驗表明milR289綁定在Argonaute蛋白QDE-2上發(fā)揮作用。QDE-2是形成RISC復(fù)合體的核心成分,在RNAi的過程中起到剪切或抑制靶基因的功能。我們通過生物信息學(xué)預(yù)測、HITS-CLIP和RIP-RT-PCR發(fā)現(xiàn)了nilR289的三個可能的靶基因,有意思的是,qde-2 mRNA自身就綁定在RISC復(fù)合體成分QDE-2上。進(jìn)一步通過煙草瞬時表達(dá)系統(tǒng)體外驗證了QDE-2是mi1R289的靶基因。并且QDE-2的敲除實驗發(fā)現(xiàn)其在urea和線蟲提取液、活線蟲誘導(dǎo)的捕器形成過程中都具有重要作用。綜上,我們首次在捕食線蟲真菌中發(fā)現(xiàn)milRNA,首次在絲狀真菌領(lǐng)域證明milRNA的靶基因并闡述其功能及機(jī)制,并且該機(jī)制在捕器形成過程中發(fā)揮重要作用。功能機(jī)制研究發(fā)現(xiàn),milR289能夠以RISC核心蛋白QDE-2為靶基因,從而在RNAi過程中形成一個,milRNA與QDE-2 (RISC)作用調(diào)控的環(huán)路,即milR289綁定QDE-2形成RISC又作用于qde-2基因來抑制RNAi作用。這條功能機(jī)制在植物中有過類似報道,表明真菌中milRNA和植物中miRNA調(diào)控機(jī)制上可能存在保守性。
[Abstract]:Non-coding RNA exists widely in prokaryotes and various eukaryotes. It plays an important regulatory role in biological mechanisms, such as growth, development, senescence and other biological mechanisms. MiRNAs are the most clearly studied class of small RNA in non-coding RNA, the size of which is about 21 NT. The functions and regulatory mechanisms in plants and animals have been systematically reported. MicroRNA-like RNAs have been reported in fungi, but their biological functions are not clear. In this paper, the predatory nematode fungus oligosporium (abbreviated as oligosporium) has been used to study the biological function of RNAs. MilRNAs were found in oligospora, and it was proved that milR289 can play an important role in the transformation of oligosporal life cycle by using qde-2 isobars as the target gene. Qde-2 and other genes play an important role in the transformation of oligosporal life cycle. We proved that small RNAs existed in oligospora by small RNA sequencing and northernblot. According to the sequence information and northernblot hybridization results, we found that these small RNA were similar to miRNA structure. It was named microRNA-like RNA millet RNA.RIP experiment showed that milR289 binding on Argonaute protein QDE-2. QDE-2 is the core component of RISC complex. We found three possible target genes of nilR289 by bioinformatics prediction, HITS-CLIP and RIP-RT-PCR. It is interesting to note that the Qde-2 mRNA itself is bound to the RISC complex component QDE-2. Further, the transient expression system of tobacco proved that QDE-2 is the target gene of mi1R289 in vitro, and QDE-2 knockout test found that it was found in the urea and nematode extracts. All of them play an important role in the process of trap formation induced by living nematodes. In summary, milRNAs were first found in nematode predatory fungi, and the target genes of milRNA were demonstrated in the field of filamentous fungi for the first time, and its function and mechanism were explained. This mechanism plays an important role in the process of trap formation. It was found that Mil R289 was able to target the RISC core protein QDE-2 in the process of RNAi, thus forming a loop regulating the interaction between QDE-2 and RISC during the process of RNAi. That is, milR289 binds QDE-2 to form RISC and acts on qde-2 gene to inhibit RNAi. This mechanism has been reported in plants, suggesting that milRNA and miRNA in plants may be conserved.
【學(xué)位授予單位】：云南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q933

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1 李恒;捕食線蟲真菌少孢節(jié)叢孢中milRNA的發(fā)現(xiàn)及功能機(jī)制研究[D];云南大學(xué);2016年

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本文編號：1529821