【摘要】：植物體內(nèi)有一系列基因抵抗病原侵染,以保證植物的正常生長。RNA干擾(RNAinterference,RNAi)是植物針對病毒侵染的一個重要的應(yīng)答反應(yīng)。人工微RNA(artificial microRNA,amiRNA)是一種高效沉默基因表達(dá)的第二代RNAi技術(shù),是利用天然miRNA的生成和作用原理設(shè)計的,以調(diào)控生物體內(nèi)某一個或多個基因的表達(dá)的一項新的基因沉默技術(shù)。病毒誘導(dǎo)的基因沉默(VIGS)也是沉默植物基因的有力手段。使用amiRNA的VIGS(MIR VIGS)是一種新開發(fā)的植物反向遺傳學(xué)研究技術(shù),其兼具amiRNA和VIGS技術(shù)的優(yōu)勢。本氏煙草(Nicotiana benthamiana)對超過500種不同的病毒敏感,廣泛應(yīng)用于植物與病原微生物相互作用以及高等植物基因功能的研究中,并且可以作為VIGS的良好宿主。植物的RNA沉默途徑中的幾乎所有的基因都已在擬南芥中發(fā)現(xiàn)和功能驗證。但是在本氏煙草中RNA沉默途徑相關(guān)基因的研究鮮有報道。本研究對本氏煙草中RNA沉默途徑相關(guān)的11條基因(AGO1a、AGO1b、AG02、DCL2、DCL3、DCL4、DRB4、HEN1、RDR1、RDR6、SGS3)進(jìn)行干擾研究。載體構(gòu)建成功后通過瞬時表達(dá)實驗分析,得到了如下結(jié)論:1)利用MIR VIGS技術(shù)在植物病毒載體pCVA中構(gòu)建了32個干擾載體,沉默了其中9條基因,沉默效率均在50%以上;2)利用amiRNA技術(shù)在植物雙元表達(dá)載體pBI121(Kpn Ⅰ)中構(gòu)建了 8個干擾載體,沉默了其中7條基因,沉默效率均在45%以上,其沉默效率整體上不如病毒載體pCVA的沉默效率高;3)農(nóng)桿菌介導(dǎo)法侵染本氏煙草,病毒載體pCVA和病毒共侵染存在強(qiáng)烈的競爭關(guān)系,而植物雙元載體pBI121和病毒在侵染液濃度較低(如OD600=0.1)時共侵染則互不影響;4)利用植物雙元載體pBI121瞬時表達(dá),在侵染液濃度為OD600=0.1的條件下,NbAG02基因的下調(diào)使本氏煙草抗煙草花葉病毒(pJL24)的能力顯著減弱,而NbAGO1a、AGO1b、NbRDR1、NbDCL2基因的下調(diào)與煙草花葉病毒則沒有關(guān)系。
[Abstract]:RNA interference (RNAi) is an important plant response to viral infection. Artificial microRNA (amiRNA) is a second-generation RNAi technology that silences gene expression efficiently and utilizes natural microRNA production and production. Virus-induced gene silencing (VIGS) is also a powerful tool for silencing plant genes. VIGS using amiRNA (MIR VIGS) is a newly developed technique for plant reverse genetics, which combines amiRNA and VIGS techniques. Advantages. Nicotiana benthamiana is susceptible to more than 500 different viruses. It is widely used in the study of plant-pathogenic microorganism interactions and gene functions in higher plants. It can also serve as a good host for VIGS. Almost all genes in plant RNA silencing pathways have been found and functioned in Arabidopsis. Verification. However, few studies have been reported on RNA silencing pathway related genes in Ben's tobacco. In this study, 11 genes (AGO1a, AGO1b, AG02, DCL2, DCL3, DCL4, DRB4, HEN1, RDR1, RDR6, SGS3) related to RNA silencing pathway in Ben's tobacco were interfered with. After successful construction of the vector, the following conclusions were obtained: 1) 32 interfering vectors were constructed in plant virus vector pCVA by MIR VIGS technology, 9 of them were silenced, and the silencing efficiency was above 50%. 2) Eight interfering vectors were constructed in plant binary expression vector pBI121 (Kpn I) by amiRNA technology. Seven of them were silenced, and the silencing efficiency was above 45%. For example, the silencing efficiency of virus vector pCVA is high; 3) Agrobacterium tumefaciens-mediated infection of tobacco, virus vector pCVA and virus co-infection have a strong competitive relationship, while the co-infection of plant binary vector pBI121 and virus at low concentration (e.g. OD600 = 0.1) does not affect each other; 4) the use of plant binary vector pBI121 transient expression in the infection solution; Under the condition of OD600=0.1, the down-regulation of NbAG02 gene significantly weakened the resistance of tobacco to tobacco mosaic virus (pJL24), but the down-regulation of NbAGO1a, AGO1b, NbRDR1, NbDCL2 gene had no relationship with tobacco mosaic virus.
【學(xué)位授予單位】：內(nèi)蒙古大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2

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