本文關(guān)鍵詞： 擬南芥 十字花科黑腐病菌 RNA高通量測序 miRNA差異表達(dá)　出處：《廣西大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：miRNA(microRNA)是存在于真核生物細(xì)胞中長度～22nt(nucleotide)的內(nèi)源非編碼RNA。miRNA參與生長發(fā)育、組織分化、腫瘤發(fā)生、細(xì)胞凋亡等一系列重要的生命過程。此外,miRNA在植物對(duì)抗病原菌入侵的防衛(wèi)反應(yīng)中起重要作用。擬南芥是研究寄主植物與病原菌互作分子機(jī)理的模式植物。目前人們已經(jīng)在擬南芥中發(fā)現(xiàn)了多個(gè)參與對(duì)抗丁香假單胞菌(Pseudomonas syringaepv.Syringae,Pst)入侵的miRNA。十字花科黑腐病菌(Xanthomonas campestrispv.Campestris,c)是引起十字花科植物黑腐病的病原菌,同時(shí)也可以侵染擬南芥。但是,參與對(duì)抗Xcc侵染的擬南芥miRNA至今未見報(bào)道。研究表明,在對(duì)抗病原菌入侵的防衛(wèi)反應(yīng)中起重要作用的miRNA的表達(dá)往往會(huì)受到病原菌的誘導(dǎo)或者抑制。本研究的目的是鑒定擬南芥中受Xcc誘導(dǎo)或者抑制的miRNA,為鑒定在對(duì)抗Xcc侵染過程中起重要作用的miRNA打下基礎(chǔ)。為此,我們首先采用高通量RNA測序(RNA-seq)技術(shù),測定了擬南芥在沒有感染以及在感染Xcc后的不同時(shí)期的miRNA表達(dá)譜,并通過差異表達(dá)分析確定受Xcc誘導(dǎo)或抑制的候選的miRNA;然后采用定量Northern雜交方法對(duì)這些候選miRNA進(jìn)行驗(yàn)證。在進(jìn)行高通量RNA測序前,我們首先建立了一種適合于測定擬南芥與病原菌互作過程中寄主和病原菌轉(zhuǎn)錄組變化的接種方法——真空抽濾法。隨后,我們測定了哥倫比亞0生態(tài)型(Col-O)擬南芥在沒有感染以及在感染Xcc和hrcV基因突變體(△hrcV)后2、6、12和24小時(shí)的miRNA表達(dá)譜;通過表達(dá)差異分析,確定了 10個(gè)受Xcc誘導(dǎo)的候選miRNA。定量Northern雜交驗(yàn)證結(jié)果顯示,miR166和miR393在接種Xcc后2小時(shí)時(shí)的達(dá)量顯著升高,而miR160和miR156在接種后24小時(shí)時(shí)的表達(dá)量顯著升高,說明這些miRNA的表達(dá)受Xcc誘導(dǎo)。此外,通過Northern雜交,我們還發(fā)現(xiàn)了 3個(gè)新的擬南芥miRNA。本研究采用RNA-Seq技術(shù)對(duì)擬南芥中受Xcc誘導(dǎo)或抑制的miRNA進(jìn)行了系統(tǒng)鑒定,發(fā)現(xiàn)miR156、miR160、miR166和miR393受Xcc誘導(dǎo)。這是首次在擬南芥中發(fā)現(xiàn)受Xcc誘導(dǎo)或抑制的miRNA,為進(jìn)一步研究miRNA在擬南芥—Xcc互作過程的作用的打下了基礎(chǔ)。
[Abstract]:MiRNA-microRNAs are endogenous non-coding RNA.miRNA that exist in eukaryotic cells with a length of 22 NT nucleotide. they are involved in the growth and development of Eukaryotic cells. Tissue differentiation, tumorigenesis, apoptosis and other important life processes. MiRNA plays an important role in plant defense against pathogen invasion. Arabidopsis thaliana is a model plant to study the molecular mechanism of host plant and pathogen interaction. Several ginseng have been found in Arabidopsis thaliana. Against Pseudomonas clove. Pseudomonas syringaepv.Syringae. Xanthomonas campestrispv.Campestris. C) is the pathogen causing black rot of cruciferous plants and can also infect Arabidopsis thaliana. However, the miRNA involved in the resistance to Xcc infection has not been reported. The expression of miRNA, which plays an important role in defense response to pathogen invasion, is often induced or inhibited by pathogens. The purpose of this study was to identify the Xcc induced or inhibited mi in Arabidopsis thaliana. RNA. In order to lay a foundation for the identification of miRNA which plays an important role in the process of combating Xcc infection, we first adopted the technique of high-throughput RNA sequencing RNA-seq. The miRNA expression profiles of Arabidopsis thaliana at different stages of infection and after infection with Xcc were determined, and the candidate miRNAs induced or inhibited by Xcc were identified by differential expression analysis. Then the candidate miRNA was verified by quantitative Northern hybridization before high-throughput RNA sequencing. We first established a inoculation method, vacuum filtration method, which was suitable for the determination of transcriptional changes in host and pathogen during the interaction between Arabidopsis thaliana and pathogenic bacteria. We measured the level of Xcc and hrcV gene mutants (hrcV6) in Arabidopsis thaliana without infection and after infection with Xcc and hrcV gene mutants (HRCV). MiRNA expression profiles were observed at 12 and 24 hours. Ten candidate miRNAs induced by Xcc were identified by differential expression analysis. The results of quantitative Northern hybridization showed that 10 candidate miRNAs were induced by Xcc. The levels of miR166 and miR393 increased significantly at 2 hours after Xcc inoculation, while the expression of miR160 and miR156 increased significantly at 24 hours after inoculation. These results indicated that the expression of miRNA was induced by Xcc. In addition, the expression of these miRNA was induced by Northern hybridization. We also found three new Arabidopsis miRNAs. MiRNA induced or inhibited by Xcc in Arabidopsis thaliana was systematically identified by RNA-Seq technique. It was found that miR156, miR160, miR166 and miR393 were induced by Xcc. This is the first time that miRNA induced or inhibited by Xcc was found in Arabidopsis thaliana. The results provide a basis for further study on the role of miRNA in Arabidopsis-Xcc interaction.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S432.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Gang Wu;;Plant MicroRNAs and Development[J];Journal of Genetics and Genomics;2013年05期

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本文編號(hào)：1461513