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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/88115完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 林詩舜 | zh_TW |
| dc.contributor.advisor | Shih-Shun Lin | en |
| dc.contributor.author | 洪瑄斐 | zh_TW |
| dc.contributor.author | Syuan-Fei Hong | en |
| dc.date.accessioned | 2023-08-08T16:21:59Z | - |
| dc.date.available | 2023-11-10 | - |
| dc.date.copyright | 2023-08-08 | - |
| dc.date.issued | 2023 | - |
| dc.date.submitted | 2023-06-07 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/88115 | - |
| dc.description.abstract | 植物基因靜默系統利用小分子 RNA 和 ARGONAUTE (AGO) 蛋白質，可調節發育過程以及面對生物和非生物逆境時的基因表現。本論文使用專一性的抗體進行 AGO1 免疫沉澱 (immunoprecipitation; IP) 並建立試管外生物活性測試系統，以分析阿拉伯芥和地錢 Marchantia polymorpha 中 RNA 誘導緘默化複合體 (RNA-induced silencing complex; RISC) 的剪切效率。第二章的研究揭示部分馬鈴薯 Y 病毒屬病毒 (Potyvirus) 的基因靜默抑制子 HC-Pro 抑制 AGO1 功能，達到抵抗基因靜默途徑目的。將 HC-Pro 轉基因植物中 AGO1 處理免疫沉澱後分析生化活性，顯示蕪菁嵌紋病毒的 HC-Pro 顯著抑制 RISC 切割，以利病毒入侵寄主植物。論文第三章提出 MpAGO1 在小分子 RNA 結合和 miRNA 剪切中的特性。此外，MIR11707 基因於高溫環境下負向控制 MpAGO1 平衡。結果顯示，地錢在較高環境溫度生長時加速 miR11707*降解，激活 miR11707 負調控 MpAGO1 表現。此外地錢適應溫度升高時雖降低 miRNA 生合成量，依然可穩定與 AGO1 結合。本論文凸顯 HC-Pro 通過抑制 RISC 活性干擾 AGO1 的功能，也同時提 出地錢適應環境溫度升高時，MpAGO1 調節和表型可塑性之間的聯繫。 | zh_TW |
| dc.description.abstract | Plant gene silencing system comprising microRNAs (miRNA) and ARGONAUTE proteins (AGOs) regulates multiple gene in developmental processes, and responses to biotic and abiotic stress. In this thesis, we established an in vitro assay system using specific AGO1 antibodies to analyze the cleavage efficiency of RNA-induced silencing complex (RISC) through AGO1 immunoprecipitation (IP) in different Arabidopsis and Marchantia polymorpha plants. The investigation in Chapter II uncovers a possibility that HC-Pros from different potyviruses evolve distinct suppression mechanisms to invade host plants. The suppression of RISC activity was confirmed through AGO1-IP of three P1/HC-Pro transgenic plants. Our results indicated that only the P1/HC-Pro of turnip mosaic virus harbored a unique ability to repress RISC slicing. In Chapter III, the intrinsic features of MpAGO1 in small RNA sorting and miRNA cleavage were confirmed. The activation of miR11707 through the acceleration of miR11707* turnover negatively regulated MpAGO1 at high ambient temperature. Besides, the reduction of miRNA biogenesis is not correlated to its AGO1 binding efficiency in response to elevated temperature acclimation. Our study highlights a conserved AGO1 regulatory pathway in bryophyte and the homeostasis of MpAGO1 is control by a novel MIR11707 gene. Taken together, this thesis provides the evidence to support HC-Pro mediated AGO1 suppression pathway via the inhibition of RISC activity. The study implies the link between AGO1 regulation and phenotype plasticity in acclimation to high ambient temperature in M. Polymorpha. | en |
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| dc.description.provenance | Made available in DSpace on 2023-08-08T16:21:59Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 論文審訂書……………………I
致謝……………………II 摘要……………………IV Abstract……………………V Content……………………VI Chapter 1 Literature review……………………1 1.1 Introduction……………………2 1.2 RNA silencing machinery……………………2 1.2.1 Small RNA biogenesis……………………2 1.2.2 RISC assembly……………………3 1.2.3 AGO1 slicer activity……………………6 1.2.4 AGO1 feedback regulatory……………………7 1.3. RNA silencing in antiviral defense……………………8 1.3.1 Viral-derived siRNAs as a key factor in activation of antiviral immunity……………………8 1.3.2 Inhibition of RNA silencing by viral suppressor……………………9 1.3.3 TuMV, ZyMV, and TEV in Potyvirus……………………11 1.3.4 The common functions of HC-Pro in potyviruses……………………13 1.4 Gene silencing in Marchantia polymorpha……………………14 1.4.1 General description of M. polymorpha……………………14 1.4.2 Recent study of RNA silencing in M. polymorpha……………………14 1.5 Aims of this thesis……………………16 1.6 Reference……………………17 Chapter 2 Suppression of host RISC activity by a potyvirus RNA silencing suppressor, HC-Pro……………………36 2.1 Abstract……………………37 2.2 Introduction……………………38 2.3 Materials and methods……………………40 2.3.1 Plant materials and virus inoculum……………………40 2.3.2 AGO1-IP and in vitro RISC assay……………………40 2.3.3 Immunoblotting……………………42 2.3.4 RNA extraction and bioinformatic analysis……………………42 2.4 Results……………………43 2.4.1 Establishment of the in vitro RISC activity assay……………………43 2.4.2 Unpairing nucleotides on the target site are essential but not sufficient for RISC cleavage……………………44 2.4.3 Suppression of RISC activity in P1/HC-Pro plants……………………45 2.4.4 Suppression of AGO1 activity in TuMV-infected Arabidopsis……………………47 2.4.5 Evaluation of AGO1-IP targeting viral RNA cleavage activity……………………48 2.5 Discussion……………………48 2.5.1 HC-Pro triggering AGO1 suppression……………………49 2.5.2 HC-Pro suppressing RISC cleavage……………………50 2.6 References……………………53 2.7 Figures and Legends……………………58 Figure 1. Establishment of an in vitro RISC assay……………………58 Figure 2. In vitro RISC assay for various miR166-target substrates……………………59 Figure 3. In vitro RISC assay for various miR159/ miR319-target substrates……………………61 Figure 4. Evaluation of HC-Pro suppressing AGO1 activities……………………63 Figure 5. AGO1 abundance and evaluation of miRNA-mediated target cleavage in TuMV-infected plants……………………65 Figure 6. in vitro RISC activity using TuMV-infected plants……………………66 Figure 7. in vitro RISC assay for viral RNA cleavage……………………67 2.8 Supplementary Table: The primer list for this study……………………68 Chapter 3 Features of MpAGO1 and its regulator miR11707 in high temperature acclimation……………………70 3.1. Abstract……………………71 3.2. Introduction……………………72 3.3. Materials & Methods……………………74 3.3.1 Plant materials and growth conditions……………………74 3.3.2 Vector Construction……………………74 3.3.3 Sporeling transformation……………………75 3.3.4 Structural modeling for AtAGO1 and MpAGO1……………………75 3.3.5 Antibody production……………………75 3.3.6 Immunoprecipitation (IP)……………………76 3.3.7 in vitro RISC assay……………………76 3.3.8 Immunoblotting……………………77 3.3.9 RNA folding……………………78 3.3.10 RNA extraction……………………78 3.3.11 RNA sequencing……………………78 3.3.12 Real-time RT-PCR……………………79 3.3.13 Microscopy and image processing……………………79 3.4. Results……………………80 3.4.1 Characterization of small RNA associated with MpAGO1……………………80 3.4.2 Small RNA selection strategy for MpAGO1……………………80 3.4.3 The flexible 5' nucleotide selectivity in MpAGO1……………………81 3.4.4 MpAGO1 stability maintained by miR11707 at elevated temperature……………………82 3.4.5 Disruption of miRNA biogenesis and AGO1 assembly at ambient warm temperature……………………84 3.4.6 mir11707 deficient plants presenting asymmetric growth and ectopic thalli in the early transition stage……………………86 3.4.7 MIR11707 responses at high ambient temperature……………………86 3.4.8 MiR11707 and MpAGO1 expression under the ambient temperature……………………88 3.5. Discussion……………………88 3.6. References……………………94 3.7. Figures and Legends……………………101 Figure 1. AGO1-immunoprecipitation and in vitro RISC activity assay……………………101 Figure 2. Characteristics of plant small RNAs associated with AGO1……………………102 Figure 3. Regulation of MpAGO1 by miR11707 at high ambient temperature….103 Figure 4. The alteration of miRNA biogenesis and RISC maturation in response to high temperature……………………105 Figure 5. Characterization of mir11707ge mutants……………………106 Figure 6. Mutation of MIR11707 interrupting thallus development at high temperature……………………107 Figure 7. Expression of MpAGO1 and MpC3HDZ1 in mir11707ge mutants……………………109 Figure 8. A working model for the regulation of MpAGO1 by miR11707 at different temperature……………………111 3.8. Supplementary table: DNA oligos used in Chapter 3……………………112 3.9. Supplementary figures……………………114 Supplementary figure 1. Characteristics of total small RNAs and the proportion in M. polymorpha and Arabidopsis……………………114 Supplementary figure 2. Phenotypic plasticity of mir11707ge mutant line F5 adapting to high ambient temperature……………………115 Appendix……………………116 | - |
| dc.language.iso | en | - |
| dc.title | 地錢與阿拉伯芥 RNA 誘導緘默化複合體之功能性研究 | zh_TW |
| dc.title | The functional investigation of RNA-induced silencing complex in Marchantia polymorpha and Arabidopsis | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 111-2 | - |
| dc.description.degree | 博士 | - |
| dc.contributor.oralexamcommittee | 楊俊逸;邱子珍;陳荷明;西浜竜一;小松愛乃 | zh_TW |
| dc.contributor.oralexamcommittee | Jun-Yi Yang;Tzyy-Jen Chiou;Ho-Ming Chen;Ryuichi Nishihama;Aino Komatsu | en |
| dc.subject.keyword | 馬鈴薯 Y 病毒屬病毒,基因靜默抑制子,P1/HC-Pro,ARGONAUTE 1,RNA 誘導緘默化複合體,地錢,表型可塑性, | zh_TW |
| dc.subject.keyword | Potyvirus,viral suppressor of RNA silencing,P1/HC-Pro,ARGONAUTE 1,RISC cleavage,Marchantia Polymorpha,phenotype plasticity, | en |
| dc.relation.page | 129 | - |
| dc.identifier.doi | 10.6342/NTU202300883 | - |
| dc.rights.note | 未授權 | - |
| dc.date.accepted | 2023-06-07 | - |
| dc.contributor.author-college | 生物資源暨農學院 | - |
| dc.contributor.author-dept | 生物科技研究所 | - |
| 顯示於系所單位： | 生物科技研究所 | |
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