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標題: Identify the Novel Innate Immunity Genes against Virus in Plants
作者: 蔡慶修
關鍵字: 生物技術, 植物保護類;基礎研究
Identifying the novel innate immunity genes in plants is one of the important research topics in studying the interaction between pathogens and plants. These genes may play potential roles for plants in defending the invading pathogens. Identifying these potential and novel innate immunity genes in plant will be very important not only for basic research but also for the application in the future. Previously, we have used the cDNA-amplified fragment length polymorphism (cDNA-AFLP) in identifying 90 differentially expressed cDNA fragments with 8 selective primers on Bamboo mosaic virus (BaMV)-infected N. benthamiana plants. In this project we are going to focus on 31 of those differential expressed genes whose identifies are related to cell rescue, death, defense, ageing, and energy in plants. In preliminary experiments, we had knocked down the expression levels by virus-induced gene silencing (VIGS) for some of these genes and showed the characters as the potential immunity genes that enhances the viral accumulation in the knockdown plants. These results indicted that they may suppress the virus accumulation when they are normally expressed. To identify these potential immunity genes, we are planning to screen these genes by knocking down the expression levels with VIGS, and following by virus inoculation to see if virus accumulation levels are increased. Besides BaMV inoculation in the experiment, we are planning to inoculate Potato virus X and Cucumber mosaic virus to see whether the effect is specific on certain type of virus such as BaMV, or common to all plant viruses. Once we get the potential innate immunity gene candidates, we will clone the full-length of these genes by 3- and 5-RACE. Cloning the full-length of these genes will be the most important step in this project that we can use them not only in studying the basic research such as how do these genes defend the invading pathogens, but also in improving the plant defense capability by over-expressing their products in transgenic plants. Another part of this project is related to the known innate immunity genes in the pathway of autophagy. One of the early genes in the autophagy pathway, Beclin-1, was shown upregulated during BaMV infection in our preliminary experiment. We are wondering whether the autophagy used to be reported involving in hypersensitive response (HR) of pathogen invading is also involved in none-HR pathogen such as BaMV infection in N. benthamiana. We are going to inspect a few genes in the autophagy pathway to make sure this pathway is truly involved. Following up the confirmation of the genes in the pathway do involve in defending BaMV, we are going to inspect the autophagosome formation by using confocal microscopy and study how the autophagosome to remove the viral products.

尋找植物先天性免疫基因是目前研究病原與寄主交互作用的重要課題之一,如能找到這些具有潛在性的抗病基因,不僅在基礎研究的工作上能夠深耕,在未來更能夠培育出抗病的品種。我們利用cDNA-AFLP的技術,以8對選擇性引子,找到了90個在竹嵌紋病毒(BaMV)感染菸草後有差異性表現的基因,在這個計畫中我們將鎖定其中31個與植物存活及能量相關的基因進行探討。我們將先進行基因靜默,然後再以病毒感染,當基因靜默後,會使得病毒大量累積,那該基因就有可能是一個具有潛力的先天性免疫基因。除了感染BaMV外,我們還將感染相同族群的PVX與不相關的CMV,實驗結果可以得知這個基因對於不同病毒是否同樣具有抗性。經由初步篩選獲得了候選者後,我們將進行5-與3-RACE來得到全長的基因,一旦得到這些基因,我們可以進行基礎研究例如將ferredoxin-NADP(+) reductase進行靜默後,出現了與病毒感染的病癥,因此認為此基因可能與植物感染病毒的病癥有關,我們將進一步證實這個相關連,並探討此病癥引發之機制。其次,我們也發現自噬作用的相關基因會在BaMV感染後有被誘導表現,因此認為自噬作用有可能參與BaMV的感染,我們也會將自噬作用相關的部分基因進行靜默,然後觀察對BaMV複製的影響,同時利用共軛焦顯微鏡來觀察自噬體的形成。當我們確認了最有可能的先天性抗病基因後,我們亦將這些基因,進行轉殖到菸草中,看是否可以強化植物對病毒入侵的抵抗。
其他識別: NSC99-2321-B005-003-MY3
Appears in Collections:生物科技學研究所

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