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dc.contributorChing-Hsiu Tsaien_US
dc.contributor.authorHao-Yun Yangen_US
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dc.description.abstract竹嵌紋病毒(Bamboo mosaic virus, BaMV)為單一核酸的正股RNA病毒,隸屬於Alphaflexiviridae科 Potexvirus屬。為了了解竹嵌紋病毒在感染的過程中與宿主蛋白之間的競合關係,我們實驗室利用cDNA-AFLP技術在病毒感染後的菸草(Nicotiana benthamiana)分析出具有差異性表現的基因片段。而這些基因可能扮演著協助病毒複製或是協助植物對抗病毒的功能。其中被命名為ACCT7-1的基因對BaMV複製具有正調控的功能。在前人的研究中,利用病毒誘導基因靜默技術(virus-induced gene silencing, VIGS)使ACCT7-1表達量下降,並且分別在葉片及原生質體接種病毒,發現病毒的外鞘蛋白(coat protein, CP)的累積量與對照組相比皆有明顯的下降,據此推斷ACCT7-1可能扮演協助病毒複製的角色。為了更進一步的研究,前人以RACE技術延長ACCT7-1之上下游基因序列,並透過National Center for Biology Information (NCBI)資料庫比對,推測ACCT7-1為DELLA蛋白家族的成員且與菸草(N. sylvestris)的GA-INSENSITIVE (GAI)具有高度的相似性,因此將ACCT7-1正名為NbGAI。將NbGAI過量表現在菸草上後感染病毒發現BaMV的外鞘蛋白累積量有顯著的上升,由此更確定NbGAI會協助BaMV在菸草細胞內的複製。DELLA蛋白家族的特徵之一是具有核定位訊號(nuclear localization signal, NLS), 可以將蛋白帶入細胞核後調節植物的賀爾蒙路徑。使用共軛焦顯微鏡已經確認NbGAI融合螢光蛋白的表現位置位於細胞核中。更進一步利用網路軟體預測NbGAI的核定位訊號的位置,刪除NbGAI的NLS並過量表現在菸草上後,發現BaMV的CP也有相較於控制組顯著的上升,這個結果表示NbGAI幫助病毒的複製並不需要進核,然而根據文獻BaMV的複製位置是位於葉綠體中,在已刪除NLS的NbGAI接上chloroplast transit peptide (TP)。過量表現於菸草後,病毒複製效率獲得更顯著的提升。因此我們推論NbGAI很有可能是被BaMV脅持共同進入葉綠體以利於病毒在葉綠體內複製,探討NbGAI是如何進入葉綠體以及NbGAI如何幫助病毒的複製將會是未來要探討的重要課題。zh_TW
dc.description.abstractBamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus which belongs to Potexvirus genus of Alphaflexiviridae. To understand the infection mechanism of BaMV as well as the involved host factors, our laboratory conducted cDNA-AFLP technique to screen the differentially expressed genes of BaMV-infected Nicotiana benthamiana. These genes might play roles either assisting the virus infection or defending against virus. One of the upregulated genes designated as ACCT7-1 was extensively studied. The accumulation of BaMV in ACCT7-1 knockdown leaves and protoplasts was revealed lower than those in the controls. These results suggested that ACCT7-1 could assist the replication of BaMV. Thereafter, the full-length gene of ACCT7-1 was cloned by RACE technique; and the sequence was compared in the data bases of National Center for Biology Information (NCBI). The result indicated that ACCT7-1 is a member of DELLA protein family with a high homology to GA-INSENSITIVE protein of N. slvestris. Thus, ACCT7-1 is then designated as NbGAI. While NbGAI was overexpressed in N. benthamiana leaves, the accumulation of BaMV coat protein was significantly increased compared to that of the control leaves. The results confirmed the hypothesis that NbGAI could play a role in assisting BaMV replication. DELLA protein family is a group of proteins containing a nuclear localization signal (NLS). We have confirmed the nucleus-localization of NbGAI by confocal microscopy. Furthermore, a mutant was created to remove NLS and expressed in plants by transient expression. The coat protein accumulation of BaMV was also significantly increased in those plants. The results suggest that the nucleus-localization play no role in assisting BaMV accumulation. Based on previous study that BaMV was demonstrated to replicate in chloroplast, NbGAI might also get into the chloroplast. Therefore, I fused a chloroplast transit peptide (TP) to the N-terminus of NbGAI/NLS and transiently expressed in N. benthamiana, BaMV replication efficiency was enhanced significantly. Overall of these results suggested that NbGAI could be hijacked from the cytoplasm and co-entry with BaMV together to the chloroplast. To understand how NbGAI is entering to the chloroplast and how does it assist BaMV replication are important issues to focus on in the future.en_US
dc.description.tableofcontents中文摘要 i Abstract iii 目錄 v 前言 1 竹嵌紋病毒 (Bamboo mosaic virus, BaMV) 1 NbGAI與BaMV複製的關係 2 Gibberellic acid insensitive(GAI)與gibberellin(GAs)訊息途徑 2 目標與宗旨 4 材料與方法 5 菸草種植與病毒純化 5 目標蛋白質萃取 5 建構刪除核定位訊號的NbGAI及其過量表現 6 在NbGAI/ΔNLS上建構含有PGK transit peptide的突變株及其過量表現 6 SDS-polyacrylamide gel electrophoresis與西方墨點法 7 結果 9 BaMV在NbGAI-knockdown的植株及原生質體中的累積量下降 9 NbGAI參與了BaMV的複製週期 9 NbGAI/ΔNLS-OFP在本氏菸草原生質體中的訊號定位 10 刪除NbGAI的入核訊號對於BaMV複製有正向的影響 10 PGKtp-NbGAI/ΔNLS-OFP在本氏菸草原生質體中的訊號定位 10 PGKtp-NbGAI/ΔNLS-OFP對於BaMV複製亦有正向的影響 11 討論 12 Figures 14 Figure 1. The localization of NbGAI in NbGAI/∆NLS -OFP transiently expressed N. benthamiana by fluorescent confocal microscope. 14 Figure 2. The accumulation level of BaMV coat protein in NbGAI/ΔNLS-OFP transiently expressed plants. 15 Figure 3. The localization of NbGAI protein in PGKtp-NbGAI/∆NLS –OFP transiently expressed N. benthamiana by fluorescent confocal microscope. 16 Figure 4. The accumulation level of BaMV coat protein in PGKtp-NbGAI/∆NLS –OFP transiently expressed plants. 17 Appendix Figures 18 Appendix Figure 1. NbGAI-knockdown Nicotiana benthamiana showed no significant change on phenotypes. 18 Appendix Figure 2. (A) NbGAI (ACCT7-1)-knockdown efficiency in Nicotiana benthamiana. (B) The accumulation of BaMV coat protein is significantly decreased in NGAI (ACCT7-1)-knockdown plants. 19 Appendix Figure 3. (A) The expression of ACCT7-1 in Luc- and NbGAI-knockdown protoplasts quantified by real-time RT-PCR. (B) The accumulation of BaMV coat protein in NbGAI-knockdown protoplasts at 24 and 48 hpi. 20 Appendix Figure 4. The relative accumulation of BaMV RNA in NbGAI-knockdown protoplasts at 24 hpi. 21 References 22zh_TW
dc.titleThe study of NbGAI from Nicotiana benthamiana involved in the infection cycle of Bamboo mosaic virusen_US
dc.typethesis and dissertationen_US
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