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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 葉信宏(Hsin-Hung Yeh) | |
| dc.contributor.author | Tze-Yu Huang | en |
| dc.contributor.author | 黃子毓 | zh_TW |
| dc.date.accessioned | 2021-06-07T17:49:38Z | - |
| dc.date.copyright | 2013-03-15 | |
| dc.date.issued | 2013 | |
| dc.date.submitted | 2013-01-30 | |
| dc.identifier.citation | Arditti J. (1992). Fundamentals of orchid biology, John Wiley & Sons.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15663 | - |
| dc.description.abstract | 蘭花為台灣重要外銷花卉，根據農委會部統計，去年出口值已突破7,200萬美元，其中蝴蝶蘭 (Phalaenopsis sp.)是台灣最具競爭力之蘭花產業然而在學術方面，因蘭花為非模式物種，生長緩慢，且蘭花轉基因植株之建構困難，造成研究上的障礙。經前人在煙草上研究之證實，在啟動子上因甲基化所產生的基因靜默之性狀是可以被遺傳的，因此本實驗擬使用雙股RNA誘發蝴蝶蘭啟動子產生甲基化，並探討其發生甲基化的情形對轉錄之影響，提供建立基因剔除轉基因植株系統的新方法之基礎。本實驗將蝴蝶蘭系統性抗病途徑中之指標基因pathogen-related protein 1 (PR1) 之啟動子分為12個片段作為標的，利用實驗室已建構之東亞蘭嵌紋病病毒載體系統，將PR1片段藉農桿菌打入蘭花葉片，在病毒載體複製過程中，會產生PR1啟動子的雙股RNA片段，以引發甲基化 (RNA-directed DNA methylation, RdDM) 導致基因靜默(Transcriptional gene silencing, TGS)。接種後一個月觀察PR1表現量，發現其中五個處理組PR1表現量有明顯下降之趨勢。由於所使用之病毒載體只會被侷限在接種區域中，但在系統葉中仍可發現基因靜默現象，推測基因靜默的訊號是可系統性移動的。進而萃取蘭花genomic DNA，利用亞硫酸氫鹽定序分析其序列，得知在 PR1啟動子區域確實產生甲基化，且甲基化程度和基因靜默程度具有相關性。過去利用可轉錄區域構築之基因靜默病毒載體，在蘭花上所造成的基因靜默效應僅能維持兩個月，但在此實驗中，持續追蹤至第四個月皆能維持基因靜默之效果，將進一步驗證此等基因靜默是否可藉遺傳將此性狀傳下去下一代。 | zh_TW |
| dc.description.abstract | Phalaenopsis orchids have successfully gained a significant market share in Taiwan. Exports of Phalaenopsis orchids earned Taiwan $72 million in 2012. Though Phalaenopsis sp. are important agricultural products, they are non-model organisms with difficulties for studies because of their large genome size, low transformation efficiency, and long regeneration time. Its long life cycle makes functional analysis hard to achieve through stable transgenic orchids. Previously, virus-induced gene silencing (VIGS) using Cymbidium mosaic virus (CymMV) recovers after 8 weeks of inoculation in flower stalks, and therefore leads to difficulty in gene functional analysis during vegetative phase. In this study, the same VIGS vector was used to conduct a silencing system with Phalaenopsis pathogen-related gene 1 (PR1) promoter. PR1 promoter was divided into 12 fragments and cloned into the VIGS vector separately. In the process of virus replication, dsRNA of PR1 promoter fragments were generated then induced RNA-directed DNA methylation (RdDM) to cause transcriptional gene silencing (TGS). After 1 month of inoculation by Agrobacteria, 5 of the 12 treatments showed repression of PR1 gene. Although the virus vector was restricted in inoculation area, the silencing signal can move systemically and cause reduction on PR1 gene expression. Further analyzing the bisulfite sequencing of Phalaenopsis genomic DNA, the homologous PR1 promoter region was discovered methylated. The degree of DNA methylation is related to the gene silencing level. And the silencing efficiency can last 4 months long in inoculation leaf. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-07T17:49:38Z (GMT). No. of bitstreams: 1 ntu-102-R99633025-1.pdf: 3741753 bytes, checksum: fe10ec6418872e6f3bb017019a3e356d (MD5) Previous issue date: 2013 | en |
| dc.description.tableofcontents | 口試委員審定書 i
誌謝 ii 摘要 iii Abstract iv Content 1 Chapter 1 Introduction 5 1.1 Orchids, the adorable agricultural products. 5 1.2 The gene functional analysis system that was established in orchid. 5 1.3 Gene silencing induced by small double-stranded RNA 6 1.3.1 Post transcriptional gene silencing (PTGS) 7 1.3.2 Transcriptional gene silencing (TGS) 7 1.4 Silencing signal transduction 10 1.5 Endogenous gene and transgene as silencing target. 11 1.6 Silencing vector used to induce gene silencing 11 1.7 The aim of this study 12 Chapter 2 Materials and methods 13 2.1 Plant materials 13 2.2 Cloning of Phalaenopsis PR1 promoter 13 2.3 Construction of PR1 promoter VIGS silencing vector 13 2.3.1 VIGS vector 13 2.3.2 Transformation 14 2.4 Culture condition and Agrobacterium infiltration 14 2.4.1 Electroporation 14 2.4.2 Agroinfiltration 15 2.5 PR1 gene expression analysis 16 2.5.1 Sampling position of the Phalaenopsis leaf 16 2.5.2 RNA extraction method 16 2.5.3 DNase treatment 17 2.5.4 Gene expression analysis by semi-RT PCR 17 2.6 Bisulfite analysis 18 2.6.1 Genomic DNA extraction of Phalaenopsis 18 2.6.2 Genomic DNA extraction of Nicotiana tabaccum 19 2.6.3 Enzyme digestion of genomic DNA 19 2.6.4 Bisulfite treatment 19 2.6.5 Bisulfite primer design 20 2.6.6 PCR condition 21 2.7 TA cloning 21 2.7.1 Isolation and purification of DNA fragment 21 2.7.2 Ligation 22 2.7.3 Transformation and blue/white screening 22 2.7.4 Plasmid extraction by commercial kit 23 2.7.5 Digestion check by restriction enzyme digestion 24 2.8 Sequencing 24 2.8.1 Sequencing 24 2.8.2 Analysis of Methylation 24 Chapter 3 Results 25 3.1 Cloning of Phalaenopsis aphrodite subsp. formosana endogenous PhaPR1 25 promoter fragments. 25 3.2 Inoculation and PhaPR1 silencing analysis 26 3.2.1 PhaPR1 promoter silencing vector can hardly move systemically 26 3.2.2 PhaPR1 promoter silencing signal can move systemically. 27 3.3 The PhaPR1 promoter-induced gene silencing has position effects of different segments of PhaPR1 promoter. 27 3.4 Gene silencing induced by promoter segments can have stronger silencing effect than by coding region. 28 3.5 Methylation status of the promoter sequence is correlated to transcriptional efficiency 29 Chapter 4 Discussion 32 4.1 The first case of inducing TGS by virus vector in monocots 32 4.2 TGS-driven gene expression knockdown is correlated to RNA-derived DNA methylation 33 4.3 The advantages of using Cymbidium mosaic virus vector to induce TGS 33 4.4 Methylation of Phalaenopsis orchids 34 4.5 DNA methylation level and gene silencing degree 35 4.6 Future aim 35 List of figure 37 Figure 1. pCymMV-Gateway system and the PhaPR1 promoter predicted cis-elements distribution. 39 Figure 2. The pCymMV-Gateway empty vector inoculation can induce the 40 Figure 3. PhaPR1 gene silencing rate of inoculation leaf after inoculation of PhaPR1pro-3, PhaPR1pro-10, and PhaPR1pro-11. 41 Figure 4. PhaPR1 gene silencing rate of side leaf and systemic leaf after inoculation of PhaPR1pro-3, PhaPR1pro-10 and PhaPR1pro-11. 43 Figure 5. The PhaPR1 gene silencing status tested in 60 dpi, 90 dpi, and 120 dpi of inoculation leaf. 45 Figure 6. Bisulfite sequencing result shows the methylation status of inoculation leaf of Phaleanopsis orchids after pCymMV-PhaPR1pro-3 treatment. 47 Figure 7. Bisulfite sequencing result shows the methylation status of inoculation leaf of Phaleanopsis orchids after pCymMV-PhaPR1pro-10 treatment. 49 Figure 8. Bisulfite sequencing result shows the methylation status of inoculation leaf of Phaleanopsis orchids after pCymMV-PhaPR1pro-11 treatment. 51 Figure 9. The bisulfite sequencing result of the inoculation leaf at 4 mpi. 52 List of tables 53 Table 1. Primers used in this study 54 Table 2. PhaPR1 gene silencing status of PhaPR1pro-1-12 inoculated Phalaenopsis orchids. 55 Table 3. Maintenance of silencing effect of 1 mpi, 2 mpi, 3 mpi, and 4 mpi. 56 Table 4. Cytosine contents in each PhaPR1 promoter segments. 57 Reference 58 Appendix 67 Appendix figure 1. Agrobacteria inoculation site and sample collection. 68 Appendix figure 2. The methylation patterns of the bottom strand of 5S rDNA in Nicotiana tabacum 69 Appendix table 1. The PhaPR1 promoter functional analysis and the cis-element prediction 72 | |
| dc.language.iso | en | |
| dc.title | 探討雙股RNA對蘭花啟動子之甲基化及對轉錄之影響 | zh_TW |
| dc.title | Investigation of dsRNA in induction of promoter methylation and its effects on transcription in orchids. | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 101-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 陳虹樺,陳仁治 | |
| dc.subject.keyword | 甲基化,轉錄前基因靜默,雙股RNA,蝴蝶蘭, | zh_TW |
| dc.subject.keyword | RNA-directed DNA methylation (RdDM),transcriptional gene silencing (TGS),double-strand RNA,Phalaenopsis, | en |
| dc.relation.page | 72 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2013-01-30 | |
| 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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