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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 葉信宏 | |
| dc.contributor.author | Tan-Tung Wang | en |
| dc.contributor.author | 王丹彤 | zh_TW |
| dc.date.accessioned | 2021-06-07T17:56:32Z | - |
| dc.date.copyright | 2012-08-19 | |
| dc.date.issued | 2012 | |
| dc.date.submitted | 2012-08-14 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/15961 | - |
| dc.description.abstract | 蘭花屬於Orchidaceae科，其成員超過35000種，是被子植物中最大的科之一，然而蘭花在病原菌入侵的情形下，如何引發抗性反應還有其機制仍有許多部分有待研究。實驗室先前以能夠引發病毒(Cymbidium mosaic virus)誘導性基因靜默(VIGS)之病毒載體─pCymMV-Gateway攜帶187個預測的Phlaenodopsis aphrodite var. Formosa轉錄因子，篩選會參與在病毒誘導水楊酸相關抗性反應之基因，由此發現PhaTF13會影響P. aphrodite中pathogenesis related gene 1的同源性基因PhaPR1的表現。本實驗選殖PhaTF13基因，透過Rapid Amplification of cDNA Ends (RACE)取得其完整的序列及正確的轉錄起始、終止點。再經由序列比對、演化分析發現PhaTF13蛋白帶有兩個轉錄因子常見的鋅指型功能區塊(zinc finger domain)且和其他單子葉植物之相似蛋白歸為同一群。為了更確切分析此轉錄因子在水楊酸相關抗性反應中所扮演的角色，利用短暫表現小片段短夾型RNA (hairpin RNA, hpRNA)造成基因靜默現象以降低PhaTF13之表現量，並偵測蘭花中的水楊酸相關之抗性指標基因PhaNPR1、PhaPR1，來針對PhaTF13進行功能性分析，確認PhaTF13位於PhaPR1及PhaNPR1的上游。並且發現在健康P. aphrodite植株中，PhaTF13只有在蕊柱及葉片中有些微表現，而在CymMV病毒感染的P. aphrodite植株中，PhaTF13的表現量在全株中皆明顯上升。最後構築PhaTF13 open reading frame融合綠色螢光蛋白(green fluorescence protein, GFP) 的表現載體(PhaTF13::GFP及GFP::PhaTF13)，在蘭花的原生質體(protoplast)中觀察其表現的區域(subcellular localization)，顯示PhaTF13蛋白主要在細胞核內表現。 | zh_TW |
| dc.description.abstract | Orchidaceae consists of at least 35000 species and is among one of the largest families within angiosperm; however, how they trigger plant defense and response to pathogens remains largely unsolved. In our previous experiments using a Cymbidium mosaic virus (CymMV) induced gene silencing system to screen 187 predicted Phalaenopsis aphrodite var. Formosa transcription factors on virus induced salicylic acids (SA) related plant defense response allowed us to identify PhaTF13 involved in the expression of P. aphrodite homolog of pathogenesis related gene 1, PhaPR1. To identify the transcription start site and stop site of PhaTF13, we used Rapid Amplification of cDNA Ends (RACE) to obtain the 5’- and 3’-end sequences. Phylogenetic analysis revealed that PhaTF13 protein contains conserved A20/AN1 zinc finger domain and is grouped together with other monocotyledons homologs. To more specifically analyze the role of PhaTF13 in SA-related plant defense, we delivered two 21-nt hairpin RNA into P. aphrodite by agroinfiltration, and the data suggested that knockdown of PhaTF13 reduced the expression of PhaPR1 and PhaNPR1. The PhaTF13 only slightly expressed in column and leaf but induced to higher levels afterz CymMV infection in all P. aphrodite parts. Expression of PhaTF13::GFP or GFP::PhaTF13 in P. aphrodite protoplasts revealed that the PhaTF13 is mainly localized in nucleolus. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-07T17:56:32Z (GMT). No. of bitstreams: 1 ntu-101-R98633011-1.pdf: 1824384 bytes, checksum: 1046a451e8d0582ef653cb718a6846fd (MD5) Previous issue date: 2012 | en |
| dc.description.tableofcontents | 目錄
口試委員審定書…………………………………………………………………………i 致謝……………………………………………………………………………………ii 中文摘要………………………………………………………………………………iii 英文摘要………………………………………………………………………………iv 目錄……………………………………………………………………………………v 表目錄…………………………………………………………………………………viii 圖目錄…………………………………………………………………………………ix 第一章 前言…………………………………………………………………………1 1.1植物抗性……………………………………………………………………1 1.1-1 基本性抗性(basal resistance) ………………………………………1 1.1-2 抗性基因調控型抗性(R gene-mediated resistance)………………2 1.1-2-1 局部性抗性…………………………………………………2 1.1-2-2 誘導性系統抗性(Induced Systemic Resistance, ISR)……3 1.1-2-3 系統性獲得抗性(Systemic Acquired Resistance, SAR)…3 1.2 轉錄因子(transcription factors)…………………………………………5 1.2-1 植物中的抗性相關轉錄因子………………………………………5 1.2-2 參與在系統性獲得抗性(SAR)中的轉錄因子……………………6 1.3 蘭花之簡介………………………………………………………………7 1.4 病毒誘導性基因靜默之簡介……………………………………………8 1.5 NPR1上游的重要性………………………………………………………9 1.6 前人研究…………………………………………………………………10 第二章 材料與方法…………………………………………………………………12 2.1 植物材料以及生長環境…………………………………………………12 2.2 化學藥劑處理……………………………………………………………12 2.3 萃取植株RNA…………………………………………………………12 2.4 去除核酸中的DNA……………………………………………………13 2.5 聚合酶鏈鎖反應(Polymerace chain reaction, PCR)……………………13 2.6 Semi-quantitative RT-PCR定量分析……………………………………14 2.7 瓊脂膠體電泳分析………………………………………………………14 2.8 瓊脂膠體核酸產物的回收………………………………………………14 2.9 黏合反應(Ligation reaction)……………………………………………15 2.10 轉型作用(Transformation)……………………………………………15 2.11 質體抽取(Purification of plasmids)……………………………………16 2.12 Rapid Amplification of cDNA Ends (RACE)……………………………16 2.12-1 5’、3’-RACE-Ready cDNA 之合成……………………………16 2.12-2 RACE PCR反應………………………………………………17 2.13 建構PhaTF13-pCymMV-Gateway載體………………………………17 2.14 建構pB7GWIWG2-PhaTF13-hpRNA表現載體………………………18 2.15 構築PhaTF13融合綠色螢光蛋白(GFP)表現載體……………………18 2.16 PhaTF13 ORF及21 nt DNA選殖進入pENTR™ TOPO®載體………18 2.17 LR重組 (LR recombination) …………………………………………19 2.18 序列比對………………………………………………………………19 2.19 親緣演化分析…………………………………………………………20 2.20 農桿菌注入法(Agroinfiltration) ………………………………………20 2.21 分離蝴蝶蘭原生質體(protoplast)及聚乙二醇(PEG)轉染……………20 2.22 共軛焦顯微鏡觀察(confocal microscopy)……………………………21 第三章 結果…………………………………………………………………………22 3.1 利用病毒誘導性基因靜默之pCymMV-Gateway載體偵測PhaTF13…22 3.2 PhaTF13轉錄起始點及終止點之確立…………………………………22 3.3 PhaTF13序列分析………………………………………………………23 3.4 PhaTF13親緣演化分析(phylogenetic analysis)…………………………23 3.5 PhaTF13位於PhaNPR1 及PhaPR1的上游……………………………24 3.6 PhaTF13在蘭花各部位基因表現量……………………………………25 3.7 PhaTF13轉錄因子在細胞中表現位置實驗(Subcellular localization)…25 第四章 討論…………………………………………………………………………27 第五章 參考文獻……………………………………………………………………31 第六章 圖表集………………………………………………………………………48 表一 論文中所使用之引子對序列…………………………………………48 圖一 利用RT-PCR分析PhaTF13被靜默時PhaPR1的表現……………50 圖二 以Rapid-amplification of cDNA ends (RACE)分析PhaTF13全長基， 相關之圖示及RACE產物之電泳……………………………………51 圖三 PhaTF13蛋白之圖示及序列排比……………………………………53 圖四 PhaTF13親緣演化分析結果…………………………………………55 圖五 PhaTF13功能性分析…………………………………………………56 圖六 PhaTF13在蘭花植株中各部位的基因表現量………………………57 圖七 PhaTF13轉錄因子在細胞中表現位置………………………………59 第七章 附錄…………………………………………………………………………61 圖一 pCymMV-Gateway載體之基因結構…………………………………61 表一 PhaTF13之open reading frame (ORF)序列…………………………62 圖二 PhaTF13基因功能性分析所使用之產生hairpin RNA載體圖譜……63 圖三 建構PhaTF13融合綠色螢光蛋白(GFP)之表現載體圖譜…………64 | |
| dc.language.iso | zh-TW | |
| dc.title | 一個參與在病毒誘導水楊酸相關抗性之轉錄因子PhaTF13之分子選殖與鑑定 | zh_TW |
| dc.title | Molecular Cloning and Characterization of PhaTF13 that May Involve in Virus-induced Salicylic Acid-related Plant Defense Response | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 100-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 張孟基,洪傳揚,陳仁治 | |
| dc.subject.keyword | 轉錄因子,A20/AN1 zinc finger domain,水楊酸,NPR1,PR1, | zh_TW |
| dc.subject.keyword | transcription factor,A20/AN1 zinc finger domain,salicylic acid,NPR1,PR1, | en |
| dc.relation.page | 65 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2012-08-15 | |
| 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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