博碩士論文 91224016 詳細資訊




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姓名 郭如玉(Ju-Yu Kuo)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 AtNPR1轉殖番茄之性狀分析及抗病機制研究
(Characterization of AtNPR1 transgenic tomato and study of defense mechanism)
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摘要(中) 植物常會遭受不同的生物及非生物性逆境的危害,然而當植物遇到環境逆境時,無法立即移動脫逃,需要仰賴體內啟動一連串的防禦機制,來達到自我保護的功能。而屬生物性逆境中的植物病蟲害,一直是造成全球農作物產量及品質受損之重要因素;近年來,利用植物遺傳工程以培育抗逆境新作物品種,已成為常被應用且有效的病害管理方法。近幾年科學家利用篩選突變株發現NPR1 (nonexpresser of PR genes),為一在植物啟動系統性誘導抗病機制時重要的調控蛋白質。本實驗室先前培育出大量表現阿拉伯芥NPR1 (AtNPR1 ) 之番茄轉殖品系 (CL5915),並將其對八種重要之番茄病害的抗病性作測試,結果證實,這些轉殖品系具有廣效抗病性,特別對維管束病害及葉部病害,然而其抗病機制至今仍不清楚。本論文針對特定AtNPR1轉殖品系,以青枯病菌為研究對象,分析其分子抗病機制,並評估其園藝性狀。實驗結果發現,在轉殖品系中,葉部組織之特定PR基因表現量確實較未轉殖品系高,推測此為轉殖品系對葉部病害有產生抗性的原因之一;但根部組織之特定PR基因表現量和未轉殖品系無顯著差異,無法解釋AtNPR1轉殖品系對於維管束病害之抗性。所以進一步利用cDNA微陣列技術分析大量基因,以探討AtNPR1轉殖品系可能誘導之抗病機制。進一步分析及比對微陣列結果發現,AtNPR1轉殖品系有部分基因會共同受青枯病菌與過量銅誘導表現,推測這兩個防禦訊息間有相互交集。而本論文也進一步將番茄植物處理園藝上常見的非生物性逆境,證實未轉殖及轉殖品系對非生物性逆境的反應耐受性一致,並沒有因為基因轉殖而造成性狀上的缺失。由於現有的轉殖品系 (CL5915) 對於番茄嚴重的病毒病害的抗性沒有顯著提高,所以本論文也將AtNPR1轉殖入一個對番茄黃葉捲曲病毒有中度抗性的番茄品種 (CLN2116B),並已成功培育具抗病性的新番茄品系,預期將來可進一步應用於栽培品種,當作抗病育種之抗病性狀來源,以傳統育種雜交法增加其抗病範圍,有效達成作物病害管理防治目的。
摘要(英) Abstract
In nature, crop plants constantly encounter various biotic and abiotic stresses, which can severely affect agricultural productivity. Pathogens, particularly, are limiting factors reducing crops quality and quantity seriously. Genetic engineering of disease-resistance through transferal of plant defense-related genes into crop is a valuable disease-control approach. Among the defense genes used to genetically engineer systemic acquired resistance in plants, Arabidopsis NPR1 (nonexpresser of PR genes) is of particular interest for its being a central regulator of plant defense responses. The exploration of empolying Arabidopsis NPR1 (AtNPR1) gene for genetics engineering disease-resistance in tomato plants have led to the production of transgenic tomato lines conferring broad-spectrum disease-resistance, especially to vascular and leaf pathogens. In this work, the possible resistance mechanism employed by a selected AtNPR1 transgenic line and its interactions with Ralstonia solanacerarum were further studied. The results showed that some PR (pathogenesis-related) genes were constitutively expressed at a high level in the leaf tissues of the transgenic plants and thus may account for the enhanced resistances to the leaf pathogens. By further employing cDNA microarray approach, using customized tomato cDNA microarray, a group of putative AtNPR1 overexpression-induced genes were identified and a model for defense mechanism is proposed. By cross-referencing these data with the microarray data obtained from other abiotic stress studies, possible cross-links between biotic stress and heavy metal stress response were revealed. Evaluation of the horticulture traits of the transgenic tomato showed that the transgenic plants responded similarity, as did the wild type plants under drought and salinity stress. Furthermore, because none of the transgenic lines generated in the genetic background of CL5915 tomato cultivar conferred enhanced resistance to virus diseases, a tomato cultivars, CLN2116B, which carries the resistance trait to tomato yellow leaf curl virus was used as the background cultivar for transformation with AtNPR1. New CLN2116B transgenic plants were selected and characterized. Molecular analyzes revealed that expression of some PR genes was constitutively enhanced in these transgenic plants, suggesting a potential enhanced of disease-resistance in these transgenic plants.
關鍵字(中) ★ cDNA 微陣列
★ AtNPR1轉殖番茄
★ 抗病機制
關鍵字(英) ★ defense resistance mechanism
★ cDNA microarray
★ AtNPR1 transgenic tomato
論文目次 目錄
中文摘要…………………………………………………………………
英文摘要…………………………………………………………………
目錄………………………………………………………………………
圖目錄……………………………………………………………………
表目錄……………………………………………………………………
附錄………………………………………………………………………
縮寫與全名對照表………………………………………………………
第一章 緒論……………………………………………………………..
一、番茄簡介……………………………………………………………
二、番茄病害……………………………………………………………12
三、植物抗病機制………………………………………………………
四、植物基因轉殖於抗病育種上之應用………………………………
五、NPR1在植物系統性誘導抗病機制中所扮演之角色…………….
六、DNA微陣列應用於植物病害研究………………………………..
七、研究動機……………………………………………………………
第二章 材料與方法……………………………………………………..
一、實驗材料……………………………………………………………
(一) 植物材料………………………………………………………
(二) 番茄的種植及逆境條件的處理………………….……………
1. 生物性逆境的處理條件……………………………………..
2. 非生物性逆境的處理條件…………………………………..
二、實驗方法……………………………………………………………
(一) DIG標記探針之製作……………………………………….…
(二) 總核醣核酸之純化及電泳分析……………………………….
(三) 北方點墨法…………………………………………………….
(四) 反轉錄聚合酶連鎖反應………………………………….……
(五) cDNA微陣列技術……………………………………….…….
1. cDNA探針之製備………………………………………..….
2. 雙色螢光基因微陣列雜交反應…………………………….
3. 資料分析…………………………………………………….
(六) 基因組去氧核醣核酸之純化及電泳分析…………….………
(七) 南方墨點法…………………………………………….………
(八) 番茄CLN2116B品系之基因轉殖…………………….………
第三章 結果…………………………………………………………
一、AtNPR1轉殖番茄品系抗病機制之探討…………………….…….
(一) 轉殖基因及防禦相關基因在轉殖品系之表現………….……
(二) 轉殖品系中可能受AtNPR1或青枯病菌誘導之基因….…….
二、AtNPR1轉殖番茄品系對非生物性逆境之耐受性比較…………..
三、番茄CLN2116B抗病品系之培育………………………………...
第四章 討論…………………………………………………………..….
第五章 建議………………………………………………………………
參考文獻…………………………………………………………………
圖目錄
圖一、 以北方點墨法分析 AtNPR1基因在轉殖植物中的表現…….
圖二、 以北方點墨法分析 PR1b1基因在轉殖植物中的表現………
圖三、 以北方點墨法分析 PR1p6基因在轉殖植物中的表現………
圖四、 以北方點墨法分析β-1, 3-glucanase基因在轉殖植物中
的表現…………………………………………………………..
圖五、 以北方點墨法分析acidic chitinase 9基因在轉殖植物中
的表現…………………………………………………………..
圖六、 在AtNPR1轉殖品系可能受誘導表現之基因……………..
圖七、 以青枯病菌處理AtNPR1轉殖品系後可能受誘導表現之基因……………………………………………………………
圖八、 轉殖品系可能受AtNPR1誘導表現之基因來源分配圖……….
圖九、 維恩圖表示可能共同受生物性及非生物性逆境誘導之
基因……………………………………………………………..
圖十、 確認微陣列實驗所得基因之表現……………………………..
圖十一、未轉殖與轉殖品系對非生物性逆境處理之耐受性比較…….
圖十二、番茄CLN2116B轉殖品系之篩選…………………………….
圖十三、以分子生物技術分析CLN2116B轉殖品系基因表現……….
圖十四、以北方點墨法分析在CLN2116B轉殖品系中防禦基因
之表現…………………………………………………………..
圖十五、AtNPR1轉殖品系可能被誘導之防禦機制……………………
表目錄
表一、番茄病害防禦微陣列中可能受AtNPR1誘導之基因……….….
表二、IBS-Tom微陣列中處理青枯病菌後受AtNPR1誘導之
基因………………………………………………………………
表三、AtNPR1轉殖品系中共同受青枯病菌與其他非生物性
逆境誘導之基因………………………………………………….
附錄
附錄一、pNPR1之建構圖……………………………………………….
附錄二、常用之細菌培養基配方……………………………………….
附錄三、實驗使用之引子序列………………………………………….
附錄四、改良式 Hoagland水耕培養液……………………………….
附錄五、番茄基因轉殖所需培養基配方………………………………..
附錄六、AtNPR1轉殖品系之抗病測試結果……………………………
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Zhou, J.M., Trifa, Y., Silva, H., Pontier, D., Lam, E., Shah, J., and Klessig, D.F. (2000). NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid. Mol Plant Microbe Interact 13, 191-202.
指導教授 詹明才、黃雪莉
(Ming-Tsair Chan、Shir-Ly Huang)
審核日期 2004-10-5
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