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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張雅君(Ya-Chun Chang) | |
| dc.contributor.author | Khong-Sam Chia | en |
| dc.contributor.author | 謝光森 | zh_TW |
| dc.date.accessioned | 2021-06-15T16:22:45Z | - |
| dc.date.available | 2020-08-28 | |
| dc.date.copyright | 2015-08-28 | |
| dc.date.issued | 2015 | |
| dc.date.submitted | 2015-08-15 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52673 | - |
| dc.description.abstract | 複製是病毒感染過程中首要且關鍵的步驟，擁有RNA基因體階段的病毒需要調節寄主內膜系統完成複製過程。前人研究中顯示PAP85 基因在菸草嵌紋病毒 (Tobacco mosaic virus，TMV) 侵染阿拉伯芥原生質體時會被誘導，且參與調節寄主內膜系統。但單獨表現TMV主要複製酶P126時不會誘導PAP85。另外，在阿拉伯芥原生質體中表現 PAP85 和P126會使得細胞內質網結構產生變化。利用Membrane-Based Yeast-Two Hybrid系統和雙螢光分子互補系統 (Bimolecular fluorescence complementation) 探討PAP85和P126之交互作用，目前在這兩種方法中並沒有觀察到交互作用的訊號。為了研究PAP85基因是由病毒何者蛋白/基因所誘導，建立以35S為啟動子的二元載體 (binary vector)來表現TMV (p35S::U1)之感染性克隆 (infectious clone)。另外，也修改p35S::U1得到P126突變株 (p35S::Rep*)和MP/CP突變株 (p35S::MP*/CP*)。結果顯示p35S::U1和p35S::MP*/CP*可以在農桿菌注入後1天誘導PAP85基因的表現，顯示TMV replicase (P126/P128) PAP85 之誘導有關。本研究也利用PAP85啟動子區域連結β-葡糖醛酸糖酶報導基因 (GUS)的轉基因阿拉伯芥探討PAP85啟動子區域調節的情況。結果顯示PAP85基因的RNA合成起始點上游107-1227 bp為主要調控區域，而1227-2000 bp為次重要的調控區域。此外，在阿拉伯芥上轉殖一個可利用雌二醇 (Estradiol)誘導的起動子來表現PAP85反向重複序列(921-1050 bp)。此轉殖植株可以在雌二醇施加後表現髮夾 (hairpin)RNA，結果顯示在病毒接種前或後施加雌二醇可有效抑制病毒的累積。 | zh_TW |
| dc.description.abstract | Replication is the first infection process during virus infection, and viruses with RNA genome stage need to modify the host intracellular membrane for replication. Previous study indicated, Arabidopsis PAP85 is induced by Tobacco mosaic virus (TMV) infection in protoplasts and involved in modification of endoplasmic reticulum (ER) for TMV replication. Overexpression of P126 (TMV main replicase) cannot induce the expression of PAP85, and co-expression of PAP85 and TMV-P126 but not either protein expressed alone induced ER modification in protoplasts. Membrane-Based Yeast-Two Hybrid (Y2H) system and Bimolecular fluorescence complementation (BiFC) systems were used for analyze the interaction between PAP85 and TMV-P126; however, no interaction were observed between PAP85 and P126 in these two approaches. In order to identify the viral open reading frame responsible for the induction of PAP85, TMV infectious clones (p35S::U1) was constructed to a binary vector (pCAMBIA-1301) under 35S promoter for agroinoculation and two other clones with mutation on TMV P126 (p35S::Rep*) and CP/MP (p35S::MP*/CP*) were used. Result indicated that PAP85 expression is induced by p35S::U1 and p35S::MP*/CP*. Besides, transgenic Arabidopsis with different length of PAP85 promoter fused with GUS (β-glucuronidase) gene were generated. Inoculation of TMV to the transgenic Arabidopsis allowed us to identify the important region responsible for induction of PAP85. The results showed that the most important regulating region located at 107-1227 bp upstream of transcription start site and the minor regulation located at 1227-2000 bp upstream of transcription start site. Arabidoipsis with Estradiol-induced promoter fused with PAP85 directed repeat DNA segment (921-1050 bp) was constructed to express hairpin RNA of PAP85. Result showed that transgenic arabidopsis sprayed with Estradiol before or after TMV inoculation showed reduction of TMV accumulation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T16:22:45Z (GMT). No. of bitstreams: 1 ntu-104-R02633024-1.pdf: 1576769 bytes, checksum: 097a63cb3f38f785285be402ce6caf3c (MD5) Previous issue date: 2015 | en |
| dc.description.tableofcontents | 中文摘要 i Abstract ii Table of Content iv Chapter I 1 Introduction 1 Chapter II 10 Material and Methods 10 Plant Growth Condition 10 Clone construction 10 In Vitro Transcription 12 Agrobacterium-mediated transient expression assays 13 Real-time RT-PCR 14 Transgenic plants preparation 14 Histochemical GUS staining 15 Yeast two-hybrid assay 15 Protoplast isolation and PEG transfection 16 Bimolecular fluorescence complementation 17 Confocal microscopy 17 Estradiol Treatment 18 Chapter III 19 Result 19 PAP85 showed no interaction with P126 as assayed by Membrane Yeast Two-Hybrid (MYTH) and Bimolecular fluorescence complementation（BiFC） 19 TMV 126/183 replicase induce the expression of PAP85 20 In silico promoter analysis of PAP85 21 Promoter region of PAP85 response to TMV 22 Transgenic plant express PAP85 hairpin RNA regulated by Estradiol 23 Transgenic plants transformed with pK2GW7-PAP85-GFP 24 Chapter IV 25 Discussion 25 Chapter V 30 Reference 30 Table 36 Table 1. The possible transcription factors binding site (TFBS) on PAP85 analysis by AtPAN database. 36 Table 2. Primer used in this study. 44 Figure 46 Figure 1(a). PAP85 and P126 protein interaction evaluated by the yeast two-hybrid assay 46 Figure 1(b). PAP85 and P126 protein interaction evaluated by the yeast two-hybrid assay. 47 Figure 2(a). BiFC analysis of PAP85 and P126 protein. PAP85 and P126 fused with N-terminal or C-terminal tagged half protein fragment of yellow fluorescent protein (YFP), different combination of two protein were tested. 48 Figure 2(b). BiFC analysis of PAP85 and P126 protein. 49 Figure 2(c). BiFC analysis of PAP85 and P126 protein. 50 Figure 3. Schematic representation of Tobacco mosaic virus (TMV) and derived construct.. 51 Figure 4. Relative expression of PAP85 after 1,2 and 3 day post infection by agro-infiltration with pCAMBIA 1301(vector only) and p35S::U1. 52 Figure 5. Relative expression of PAP85 after 1 day post infection by agro-infiltration with pCMABIA 1301(vector only), p35S::Rep*, p35S::MP*/CP* and p35S::U1. 53 Figure 7. Schematic representation of different length of PAP85 promoter region fused with GUS gene. 55 Figure 8(a). Histochemical detection of GUS in transgenic Arabidopsis plants. 56 Figure 8(b). Histochemical detection of GUS in transgenic Arabidopsis plants 56 Figure 8(c). Histochemical detection of GUS in transgenic Arabidopsis plants. 57 Figure 9. GUS activity of 4-week-old transgenic Arabidopsis plants 1 day after agro-infiltration with p35S::U1. 58 Figure 10. Schematic diagram of pER8-GW-PAP85i vector. 59 Figure 11. Relative expression of PAP85 after 1, 2, 3 and 4 days post treatment with 20μM Estradiol. 60 Figure 12. Virus accumulation of transgenic plant (pER8-GW-PAP85i) with Estradiol treatment after TMV inoculation. 61 Figure 13. Virus accumulation of transgenic plant (pER8-GW-PAP85i) with Estradiol treatment before TMV inoculation. 62 Figure 14. Subcellular localization of PAP85-GFP overexpression transgenic plant. 63 | |
| dc.language.iso | en | |
| dc.subject | 抗病毒 | zh_TW |
| dc.subject | 阿拉伯芥 | zh_TW |
| dc.subject | P126蛋白 | zh_TW |
| dc.subject | 菸草嵌紋病毒 | zh_TW |
| dc.subject | PAP85 | zh_TW |
| dc.subject | Arabidopsis | en |
| dc.subject | Antivirus | en |
| dc.subject | TMV-P126 | en |
| dc.subject | TMV replication | en |
| dc.subject | PAP85 | en |
| dc.title | 探討阿拉伯芥PAP85與菸草嵌紋病毒P126之交互作用及其在抗病毒之應用 | zh_TW |
| dc.title | Investigation of the interaction between Arabidopsis PAP85 and Tobacco mosaic virus (TMV) P126 and application of PAP85 in antiviral strategy | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 103-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 葉信宏(Hsin-Hung Yeh) | |
| dc.contributor.oralexamcommittee | 劉瑞芬(Ruey-Fen Liou),陳仁治(Jen-Chih Chen) | |
| dc.subject.keyword | PAP85,菸草嵌紋病毒,P126蛋白,阿拉伯芥,抗病毒, | zh_TW |
| dc.subject.keyword | PAP85,TMV replication,TMV-P126,Arabidopsis,Antivirus, | en |
| dc.relation.page | 63 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2015-08-15 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 植物病理與微生物學研究所 | zh_TW |
| 顯示於系所單位： | 植物病理與微生物學系 | |
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