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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 詹明才 | |
| dc.contributor.author | Chun-To Chao | en |
| dc.contributor.author | 趙鐸駿 | zh_TW |
| dc.date.accessioned | 2021-05-20T20:05:50Z | - |
| dc.date.available | 2012-08-19 | |
| dc.date.available | 2021-05-20T20:05:50Z | - |
| dc.date.copyright | 2009-08-19 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-08-12 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/8986 | - |
| dc.description.abstract | 植物生長會遭受非生物性與生物性逆境的影響，其中缺水逆境(water deficit, WD)與青枯病菌(Ralstaonia solanacearum)造成的細菌性萎凋(bacterial wilt, BW)是限制作物正常的重要因子。BW與WD皆會造成植物缺乏水分，而關於植物對抗這些逆境之相關機制研究尚需探討。本論文針對WD/BW microarray分析所選出之基因群，進ㄧ步用virus-induced gene silencing (VIGS)確認其中基因是否參與番茄抗青枯病機制。利用半定量PCR檢測已證實在番茄抗病品系中，目標基因經過VIGS後，其RNA含量確實下降；再進一步將經過基因靜默之番茄植株接種青枯病菌後，植株內之病菌量顯著比對照組植物高，且可造成青枯病萎凋病徵。利用半定量PCR測試也證實其中ㄧ些基因的RNA表現量確實會受青枯病菌感染而上升。為了瞭解各基因之確切功能，本研究進一步針對SlZFP進行功能性研究。結果發現SlZFP的RNA表現量會受水楊酸及乙烯所抑制，而SlZFP之綠色螢光重組蛋白在阿拉伯芥原生質體中的表現位置是位於細胞骨架(cytoskeleton)。此外，多個過量表現SlZFP或SlZFP-GFP的阿拉伯芥或菸草轉基因品系也已建立。環境逆境測試結果顯示：轉殖SlZFP可以提升阿拉伯芥對鹽害逆境之抗性，對軟腐病之反應未顯著改變，但卻提早青枯病之發病的時間。從這些結果推測，位於細胞骨架上的蛋白SlZFP的表現量可能必須維持在平衡的狀態方能維持植物最佳之抗病反應，而SlZFP在植物病害-逆境反應之相抵觸交互作用中可能具某種的功能。透過本研究所得結果，使得我們對植物對抗缺水逆境與病害機制有更多的了解，且預期可能對研擬青枯病之有效防治管理策略將有所啟發。 | zh_TW |
| dc.description.abstract | Plant constantly encounters environmental stresses, including abiotic and biotic factors. Water deficit (WD) and bacterial wilt (BW, caused by Ralstaonia solanacearum or Rs) are very important factors limiting crop production worldwide. The nature of BW shares commonness with that of WD. However, information on plant defense response to these stresses is far from sufficient. To elucidate plant stress defense mechanisms, this study aimed to study functions of a group of genes previously selected from tomato WD/BW microarray analyses. First, the genes were subjected to VIGS assays to study their roles in tomato BW defense response. The transcript accumulation of the test genes in stembases of a silenced BW-resistant tomato cultivar (Hawaii 7996, H7996) was reduced at various levels. Further BW bioassays revealed that silencing of a few genes led to a significant increase of R. solanacearum growth and BW symptom development. Additionally, The expression of a few of these genes in a H7996 was enhanced in response to R. solanacearum infection, further suggesting their involvement in tomato BW response. Moreover, functional study was performed on SlZFP. Its expression at transcriptional level was reduced by salicylic acid and ethephon treatment. Localization assay showed that SlZFP:GFP recombinant proteins colocalized on cytoskeleton with the microtubule marker protein in Arabidopsis protoplasts. Transgenic Arabidopsis and Nicotiana benthamiana plants containing 35S::SlZFP or 35S::SlZFP-GFP have been generated. Compared to the control plants, the transgenic Arabidopsis lines conferred enhanced tolerance to salinity, similar response to Erwinia carotovora subsp. carotovora, and increased BW development. These results suggest that the level of SlZFP might need to be fine-tuned in order to achieve the optimal disease defense response. This protein might play some role in the possible antagonistic interaction in plant responses to abiotic and biotic stresses. This study is expected to pave the way not only for elucidating mechanisms and determinants involved in plant stress defense responses, but also potentially benefit the establishment of useful disease control means. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-20T20:05:50Z (GMT). No. of bitstreams: 1 ntu-98-R96B42026-1.pdf: 2568860 bytes, checksum: 05df111840260c18e804c0327c1657b5 (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 目錄
口試委員會審定書 i 致謝 ii 中文摘要 iii 英文摘要 iv 常用名詞之縮寫與全名對照表 vi 目錄 vii 表目錄 x 圖目錄 xi 附錄目錄 xii 第一章 前言 1 1. 番茄簡介 1 2. 植物與環境逆境 1 3. 番茄青枯病之相關研究 2 4. 植物缺水逆境之研究 4 5. 病毒誘導性基因靜默 6 6. 鋅指蛋白 7 7. 植物細胞骨架 9 8. 研究目標……………………………………………………………………….10 第二章 材料與方法 11 1. 植物材料 11 2. 常用實驗方法 11 2.1 聚合酶連鎖反應 11 2.2 DNA凝膠電泳法 11 2.3 小量質體製備 11 2.4 TOPO® cloning (Invitrogen) 12 2.5 LR recombination (Invitrogen) 12 2.6 大腸桿菌熱震盪轉型作用 12 2.7 農桿菌電穿孔法 13 3. 植物RNA萃取 13 3.1 RNeasy Mini Kit (Invitrogen) 13 3.2 Trizol method 13 3.3 DNase treatment 14 4. 反轉錄聚合酶連鎖反應(RT-PCR) 14 5. 病毒誘導性基因靜默 14 6. 番茄接種青枯病菌之評估 15 7. 半定量RT-PCR 16 8. Real-time PCR 16 9. 番茄接種青枯病菌之樣本收取 16 10. SlZFP於植物細胞中表現位置之分析 16 10.1 阿拉伯芥原生質體之分離 16 10.2 PEG transfection 17 11. 阿拉伯芥花序浸潤轉殖法 17 12. 快速抽取植物DNA 18 13. 葉綠素含量測量 18 14. 番茄處理植物荷爾蒙 18 15. 植物逆境檢測 19 15.1 鹽害逆境 19 15.2 種子萌發率測試 19 15.3 乾旱逆境 19 15.3 青枯病菌接種 19 15.3 軟腐病菌接種 20 16. 生物資訊分析與統計分析 20 第三章 結果 21 1. VIGS 基因靜默之效率檢測 21 2. VIGS靜默特定基因後使番茄對抗青枯病菌能力下降 21 3. 番茄接種青枯病菌後之基因表現分析 22 4. SlZFP-cDNA之全長調取 22 5. 水楊酸、乙烯及茉莉酸處理番茄後之SlZFP表現分析 23 6. SlZFP可能位在植物細胞骨架上 24 7. 阿拉伯芥轉殖株之篩選與分子檢驗 24 8. 過量表現SlZFP增加阿拉伯芥青枯病菌的發病速率 25 9. 過量表現SlZFP未顯著影響阿拉伯芥對軟腐病菌的抗性 25 10. 過量表現SlZFP稍微增加阿拉伯芥之鹽害耐受性 25 12. 乾旱逆境測試顯示SlZFP未顯著影響植物的耐旱性 26 第四章 討論 27 1. 靜默番茄中特定基因後造成番茄外表型改變或降低對抗青枯病菌的能力 27 2. 青枯病菌誘導番茄特定基因之表現量上升 28 3. SlZFP表現量受到青枯病菌及水楊酸調控 29 4. SlZFP可能位於植物細胞的細胞骨架 30 5. 35S::SlZFP轉殖株生長正常 31 6. 過量表現SlZFP可能造成阿拉伯芥轉殖株對青枯病菌的抵抗能力下降 32 7. SlZFP在鹽害中可能扮演的角色 34 8. 總結.....................................................................................................................34 參考文獻 35 表目錄 表一、VIGS靜默番茄特定基因並檢測接種青枯菌後之菌量 44 表二、檢測VIGS靜默效率所使用之引子 45 表三、用來設計VIGS片段所使用之引子 46 表四、Real-time PCR及SlZFP選殖所使用之引子 47 圖目錄 圖ㄧ、以半定量RT-PCR檢測VIGS靜默特定基因效率之測定 48 圖二、利用VIGS靜默番茄特定之基因使番茄出現生長受阻 49 圖三、半定量RT-PCR檢測番茄接種青枯病菌之標示基因 50 圖四、Real-time PCR檢測番茄接種青枯病菌後一群基因的表現分析 52 圖五、SlZFP與阿拉伯芥中相似的蛋白質胺基酸序列比對 53 圖六、Real-time PCR檢測番茄處裡水楊酸後Pti4以及SlZFP的表現情況 54 圖七、Real-time PCR檢測番茄處理ethephon後Pti4以及SlZFP的表現情況…55 圖八、Real-time PCR檢測番茄處裡茉莉酸後WIPI-2以及SlZFP的表現情況…56 圖九、SlZFP之綠色螢光重組蛋白在阿拉伯芥原生質體中的表現位置分析 58 圖十、利用RT-PCR檢測35S::SlZFP轉殖阿拉伯芥之SlZFP表現量 59 圖十一、35S::SlZFP阿拉伯芥的外表型與野生株及轉殖空載體之阿拉伯芥比較無太大差異 60 圖十二、三週大阿拉伯芥接種青枯病菌RD15進行抗病性測試 61 圖十三、三週大阿拉伯芥接種軟腐病菌 62 圖十四、過量表現SlZFP之阿拉伯芥擁有較高的鹽害耐受性 63 圖十五、鹽害逆境下的阿拉伯芥種子萌發率 64 圖十六、乾旱逆境測試顯示SlZFP不影響轉殖阿拉伯芥的耐旱性 65 附錄目錄 附錄ㄧ、利用VIGS篩選DNA微陣列選出的可能防禦基因 66 附錄二、反轉錄PCR檢測SlZFP在AtNPR1轉殖番茄中的表現情況 67 附錄三、35S::SlZFP轉殖菸草已培育到R1世代 68 附錄四、35S::ZFP-GFP轉殖菸草已經培育到R1世代 69 附錄五、常用培養基及藥劑 70 附錄六、用於TA clone的pCR8®/GW/TOPO®載體……………………………….73 附錄七、阿拉伯芥及菸草轉殖所用載體………………………………………...... 74 附錄八、表現SlZFP綠色螢光崇組蛋白所使用的載體…………………………...75 附錄九、TRV-based VIGS vectors…………………………………………………..76 | |
| dc.language.iso | zh-TW | |
| dc.title | 一個番茄鋅指蛋白基因之鑑認與功能分析 | zh_TW |
| dc.title | identification and functional study of a tomato zinc finger protein gene | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 鄭秋萍 | |
| dc.contributor.oralexamcommittee | 林讚標,張孟基 | |
| dc.subject.keyword | 青枯病,缺水逆境,鋅指蛋白,細胞骨架,植物荷爾蒙, | zh_TW |
| dc.subject.keyword | bacterial wilt,water deficit,zinc finger protein,cytoskeleton,phytohormone, | en |
| dc.relation.page | 76 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2009-08-13 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 植物科學研究所 | zh_TW |
| 顯示於系所單位： | 植物科學研究所 | |
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