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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林詩舜(Shih-Shun Lin) | |
dc.contributor.author | Pin-Chun Lin | en |
dc.contributor.author | 林品均 | zh_TW |
dc.date.accessioned | 2021-06-17T02:24:25Z | - |
dc.date.available | 2022-08-24 | |
dc.date.copyright | 2017-08-24 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-08-19 | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68535 | - |
dc.description.abstract | 植物基因靜默系統包含了兩種微小核酸,微型核酸 (microRNA; miRNA) 以及短片段干擾核酸 (short-interfering RNA; siRNA),除了在基因表現、各式生理發育路徑扮演重要調控角色之外,也對病原菌如病毒等入侵提供了防禦系統。本篇論文中我們首先建立了 miRNA鑑定流程並且於演化地位重要的地錢 Marchantia polymorpha 中,整合並運用轉錄體,微小核酸轉錄體等大數據分析工具,並使用降解體來準確篩選,找到了包含保守種類共129種 miRNA。另外並利用病毒衍生出來抑制植物基因靜默抗性系統的植物病毒抑制子,蕪菁嵌紋病毒的P1/HC-Pro,研究基因靜默系統的分子機制,並同時找出P1/HC-Pro的作用位點。我們使模式植物Arabidopsis分別表現原型的 P1/HC-Pro (P1HCR) 以及 HC-Pro 上的 FRNK保守片段之R182K點突變HC-ProK (P1HCK),發現P1HCR會造成嚴重 miRNA抑制並表現出畸形表型但P1HCK不會,顯示後者失去了抑制基因靜默的功能。我們並發現P1HCR中,基因靜默路徑下游的重要蛋白AGO1 會受到細胞自噬 (autophagy) 的降解。此外,FRNK中的 Arg residue會直接與 HEN1甲基酶交互作用並使 HEN1無法接觸原本的受質 miRNA,而使 miRNA甲基化受到抑制;失去甲基化的 miRNA 將對後續的 AGO1 loading 或 RISC (RNA-Induced Silencing Complex) 的組裝受到干擾,此時 autophagy 被誘導並將不完全組裝或失去功能的 AGO1及RISC 等降解,造成基因靜默抑制現象。我們並賦予了 miRNA 甲基化一樣新的定義:miRNA甲基化將是決定其能否 AGO1 loading 或 RISC 組裝之關鍵,在此之前科學家普遍認為 miRNA甲基化主要控制 miRNA是否穩定不被 uridylation 或降解。最後,回到病毒本身,我們詢問弱系病毒造成交叉保護 (cross protection) 的分子機制為何? HC-Pro突變後的弱系病毒TuMVK 將作為很好的研究材料,結果發現除了已知的 protein-mediated resistance之外, RNA-mediated resistance 也有參與交叉保護,而初級免疫系統中的水楊酸 (SA) 路徑亦與交叉保護系統交互作用共同對抗並做出貢獻。 | zh_TW |
dc.description.abstract | Plant gene silencing system that comprises two major small RNAs, microRNA (miRNA) and short-interfering RNA (siRNA), play a broad role in gene regulation, various biological or developmental processes, as well as stress response such as defense against virus. In this thesis, we first established an miRNA identification and characterization pipeline using the ContigView platform to manipulate the high throughput data through integrated bioinformatics strategies and successfully got an miRNA profile of a novel organism, liverwort Marchantia polymorpha. The investigation of viral suppressor not only helps understanding the virus-plant crosstalk but also uncovering the mechanism of hidden molecular gene silencing pathway. Being the first discovered suppressor, P1/HC-Pro of Potyvirus; however, is still remained mysterious. We built a transgenic Arabidopsis model separately expressing the wild-type P1/HC-Pro (P1HCR) from Turnip mosaic virus (TuMV) and the mutant form P1HCK with the Arg182Lys mutation on the conserved motif FRNK. The data suggested the HCK almost lost all capability to interfere with miRNA pathways. A genetic model of atg mutant was then used to demonstrate that HCR destabilizes Argonaute 1 (AGO1), a core component of RNA-Induced Silencing Complex (RISC), via autophagy pathway, which is a stress response and protein down-regulation machinery. Moreover, the FRNK motif on HCR plays a significant role in the methyltransferase HEN1-binding rather than small RNA-binding to inhibit HEN1 activity on miRNA 3'-end 2'-O-methylation, resulting in unmethylated miRNA abnormal accumulation as free-form status, which might lead to incomplete RISC assembly and the recruitment of autophagy system. Therefore, the autophagy pathway might serve as a surveillance system to clean up the incompletely assembled or dysfunctional RISC. All together, we provide the evidence to link autophagy and HCR-mediated gene silencing suppression pathways. Last but not least, using the in vivo model of P1HCR plant we found the 2'-O-methylation on miRNA is the determinant to load the miRNA into AGO1 or help assemble miRNA-RISC and this is going to be the first report of a novel function of miRNA methylation other than the protection aspect that was approved for decades. Finally, the Tu-GK mutant virus that carries HCK helps to establish a mild strain platform for understanding the molecular mechanism of cross protection. This attenuated virus confers complete cross protection against infection by a severe wild type strain in Arabidopsis thaliana. In addition to protein and RNA-mediated resistance, we revealed here that salicylic acid (SA) pathway takes part in cross protection mechanism | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T02:24:25Z (GMT). No. of bitstreams: 1 ntu-106-D00642004-1.pdf: 4406367 bytes, checksum: 84e7a5ccda71c7e27469f13af411334f (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | Acknowledgement III
中文摘要 IV Abstract V Publications VII Contents VIII Chapter I Literature review 1 1. Gene silencing 2 2. Small RNA and key components of post-transcriptional gene silencing 2 3. Plant viral suppressor 5 4. HC-Pro 6 5. HC-Pro inhibits HEN1 activity 8 6. P1 enhance HC-Pro suppression ability 8 7. HC-Pro vs. cross protection 9 8. Autophagy 10 9. Autophagy vs. gene silencing 11 10. Identification of miRNA 12 11. Reference 13 Chapter II Inhibition of miRNA 2'-O-methylation and activation of AGO1 autophagic degradation by silencing suppressor P1/HC-Pro 20 Abstract 21 Introduction 22 Materials and Methods 25 Plant material and growth condition 25 Protein extraction and western blotting 25 RNA extraction, Northern blotting and real-time RT-PCR 26 Chemical treatments 26 Size exclusion chromatography and small RNA northern 27 Detection of miRNA 3'-end methylation by periodate treatment and -elimination 27 Results 29 Destabilized AGO1 protein and ago1 mutant-like phenotype in P1HCR plants 29 P1HCR caused AGO1 autophagic degradation 30 The atg mutants did not alter P1HCR-triggered miRNA and miRNA target accumulations 32 Evaluation the miRNA/miRNA* distributions 33 MiRNA methylation is the determinant to load miRNA into AGO1 or RISC 34 Autophagy serves as a surveillance system for degrading unmethylated miRNA-RISC complex 35 HCR binds HEN1 to impair the miRNA methylation 36 Overexpressing HEN1 compensates the P1HCR-triggered miRNA pathway suppression 37 Unmethylated miRNA interferes RISC activity 38 Discussion 40 P1 and HCR work together to cause miRNA pathway suppression 40 Arg of the FRNK motif of TuMV HC-Pro is sufficient and necessary for HEN1 binding 41 Autophagy is a surveillance system for gene silencing pathway 42 Working hypothesis 42 Reference 44 Figures and legends 47 Chaptor III Genetic Analyses of the FRNK Motif Function of Turnip Mosaic Virus Uncover Multiple and Potentially Interactive Pathways of Cross-Protection 58 Abstract 59 Introduction 60 Materials and methods 63 Plant materials and growth conditions 63 Infectious clones and virus inocula 63 Challenge inoculation with recombinant viruses and cross-protection assays 64 Transgenic Arabidopsis for P1/HC-ProR and P1/HC-ProK expression 64 Confocal microscopy 65 Enzyme-linked immunosorbent assay 65 Real-time RT-PCR 66 High-throughput deep sequencing of small RNAs 66 Results 68 Mutagenesis identified a TuMV strain that conferred effective cross-protection in Nicotiana benthamiana 68 Tu-GK caused mild symptoms and retained partial silencing suppression function in Arabidopsis 69 Tu-GK conferred complete cross-protection in both Col-0 and dcl2/4 plants 71 DCL2/4-dependent and -independent accumulation of vsiRNAs 71 P1/HC-ProK sustained a low level of sense-PTGS suppression activity 72 P1/HC-ProK did not cause abnormal phenotypes or interfere with miRNA pathways in Arabidopsis 73 HC-ProK had similar stability as the wild-type HC-ProR in planta 74 Evidence for crosstalk between RNA silencing and plant innate immunity 74 Discussion 76 Criteria for a suitable mild strain for cross-protection 76 DCL2/DCL4-dependent and -independent vsiRNAs generation 77 The crosstalk between PTGS and innate immunity 78 A working model for cross-protection 78 References 80 Figures and Legends 84 Chapter IV Identification of miRNAs and their targets in the liverwort Marchantia polymorpha by integrating RNA-Seq and degradome analyses 98 Abstract 99 Introduction 101 Materials and Methods 105 Plant material 105 Total RNA extraction and RNA-Seq 105 Gene and miRNA target identification in M. polymorpha 106 Statistical analysis 107 Small RNA detection and -elimination 107 Gene cloning for reporter assay 108 Transient expression by agroinfiltration 108 MpMADS1 overexpression in M. polymorpha and real-time RT-PCR 109 Results 111 Whole-transcriptome analysis and gene annotation in M. polymorpha 111 MiRNA prediction in M. polymorpha 112 Target prediction in M. polymorpha 114 Identification of gene silencing-related pathways 115 Conserved miRNAs in M. polymorpha 116 Conserved miRNAs without identified precursors 117 MiRNAs control diverse plant processes 118 Novel miRNAs regulate MpAGO1 and MpMADS genes in M. polymorpha 119 Reporter assay to confirm miRNA-mediated target down-regulation 120 Role of long miRNA precursors in miRNA biogenesis and target regulation 122 Discussion 123 An integrated approach for characterizing M. polymorpha miRNAs 123 MiRNAs regulate defense/stress responses and chromatin remodeling 124 Evolution of land plant miRNAs 125 Convergent evolution of miRNA regulation 127 Lineage-specific functions 128 PPR proteins in M. polymorpha 129 Figures and legends 146 Supplementary information 157 | |
dc.language.iso | en | |
dc.title | 微型核酸註解及 P1/HC-Pro 參與植物病毒交互保護與基因靜默抑制作用 | zh_TW |
dc.title | MicroRNA annotation and mechanisms of P1/HC-Pro-mediated cross protection and silencing suppression | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 吳素幸(Shu-Hsing Wu),邱子珍(Tzyy-Jen Chiou),陳荷明(Ho-Ming Chen),楊俊逸(Jun-Yi Yang),蔡宜芳(Yi-Fang Tsay) | |
dc.subject.keyword | 基因靜默,微型核酸,病毒基因靜默抑制子,細胞自噬,交互保護,微型核酸鑑定, | zh_TW |
dc.subject.keyword | miRNA,P1/HC-Pro,Argonaute 1 (AGO1),Autophagy,miRNA methylation,HEN1,cross protection,mild strain virus,miRNA identification, | en |
dc.relation.page | 162 | |
dc.identifier.doi | 10.6342/NTU201703839 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2017-08-19 | |
dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
dc.contributor.author-dept | 生物科技研究所 | zh_TW |
顯示於系所單位: | 生物科技研究所 |
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