臺灣博碩士論文加值系統

植物生長常會遭受生物性與非生物性逆境的影響，其中由 Ralstonia solanacearum 引起之青枯病 (bacterial wilt, BW) 與缺水逆境 (water deficit, WD) 更是造成全球作物生產重大損失的重要因子。本研究針對番茄 SlZFP 基因 (C3HC4-RING zinc finger type) 與茄科抗菌蛋白合成之關鍵酵素 5-Epi-aristolochene synthase (EAS) 在病害與非生物逆境反應之功能進行探討。首先，先前短暫靜默番茄 SlZFP 基因詴驗發現此基因參與番茄抗青枯病機制中，本研究進一步發現在正常生長條件下，SlZFP 蛋白主要表現在花及成熟果實，蛋白質則坐落於細胞骨架；在抗/感品系處理青枯病菌及各式病原相關物質後，SlZFP 在轉錄層次之表現皆受到抑制，但不受乾旱逆境或荷爾蒙調控。此外，SlZFP 基因被靜默後導致番茄較不耐乾旱，而過量表現 SlZFP 可使轉基因菸草植株更抗青枯病菌、缺水、氧化逆境和鹽害，且在正常生長條件下其 SA 路徑相關防禦基因之表現已明顯提升。綜合以上結果證實 SlZFP 基因在青枯病和多種非生物逆境防禦反應中皆扮演正面的重要角色。另一方面，當 SlZFP 之阿拉伯芥同源基因 At5g24870 基因缺失時，其種子萌芽率在鹽害、模擬乾旱、糖分、滲透壓及 ABA 等逆境下皆較低，但並不影響對青枯病與軟腐病菌之反應，顯示 At5g27870 基因在植物對抗多數非生物逆境上亦具正面功能。第二部分的研究係探討茄科 EAS 基因在逆境反應之功能，先前研究短暫靜默番茄 SlEAS 基因詴驗發現此基因參與番茄抗青枯病機制中，本研究進一步發現過量表現煙草 NtEAS 之轉基因菸草可更抗青枯病菌、氧化逆境和鹽害，且其 SA 路徑相關防禦基因之表現亦明顯提升。此外，先前研究顯示在不同煙草品種中，以 Nicotiana glutinosa EAS (NgEAS) 基因受青枯病菌及氧化逆境誘導表現最為顯著，經進一步釣取啟動子序列並分析後發現，NgEAS 啟動子可受多種重要植物病原菌與病原相關物質誘導，並經由維管束傳導達成系統性的表現，而 GT-1 box 為誘發訊號傳遞之必需 cis-element。綜合本研究顯示 SlZFP 與 EAS 基因在青枯病、乾旱、氧化逆境和鹽害逆境防禦反應皆具正面功能，除在學術上提供更多關於植物抗逆機制的重要資訊外，未來也可將之應用在培育抗病/逆新品種的研發工作上。

Plant constantly encounters environmental stresses, including biotic and abiotic factors. Bacterial wilt (BW, caused by Ralstonia solanacearum or Rs) and water deficit (WD) are very important factors limiting crop production worldwide. The aim of this work was to study function of a tomato zinc-finger protein SlZFP (a C3HC4-RING finger protein) and a Solanaceae gene encoding 5-epi-aristolochene synthase (EAS) in biotic and abiotic stresses. Previously, SlZFP was suggested to involve in tomato BW defense mechanism by virus-induced gene silencing (VIGS). This study further revealed that SlZFP was abundantly expressed in tomato flower and mature fruit, and that GFP::SlZFP recombinant protein localized on cytoskeleton in Arabidopsis protoplasts. SlZFP expression at transcriptional level was suppressed by Rs and various pathogen-associated molecular patterns (PAMPs), but not significantly changed under drought and phytohormone treatments. In addition, VIGS of SlZFP in tomato resulted in increased sensitivity to drought stress, and the 35S::SlZFP transgenic tobacco displayed enhanced tolerance to BW, drought, oxidative stress and salinity. Furthermore, expression of SA-dependent pathway-related genes was enhanced in 35S::SlZFP transgenic tobacco under normal growth condition. These results together evidences an important and positive role of SlZFP in defense response to multiple stresses. On the other hand, null mutants of the Arabidopsis orthologous gene At5g24870 of SlZFP exhibited significantly increased susceptibility to various abiotic stresses during seed germination stage, but their response to Rs and Pectobacterium chrysanthemi infection was not obviously affected, suggesting that At5g24870 is involved in plant defense to various abiotic stresses. Secondly, tomato SlEAS was suggested to involve in tomato BW defense mechanism by VIGS. Current study further showed that 35S::NtEAS transgenic tobacco conferred enhanced tolerance to BW, oxidative and salinity, and that expression of SA-dependent pathway-related genes in these transgenic plants was enhanced under normal growth condition. In addition, our previous study identified and isolated a Nicotiana glutinosa EAS (NgEAS) whose expresion could be significantly induced by Rs and oxidative stress. This study further revealed that NgEAS promoter can be systemically induced by a group of economically important phytopathogenic bacteria and fungi, as well as PAMP in vascular tissues, and the GT-1 box is an essential cis-element for systemic activation. These results consistently demonstrate that Solanaceae EAS positively regulate defense response to microbial pathogens and various abiotic streses. Collectively, current study paves the way not only for elucidating mechanisms and determinants involved in plant stress defense responses, but also for potentially the establishing useful means of breeding crops with enhanced tolerance to diseases and abiotic stresses.

口試委員會審定書 i
謝誌 ii
中文摘要 .iii
英文摘要 iv
縮寫與全名對照表 vi
目錄 viii
表目錄 xiii
圖目錄 xiv
附錄目錄 xvi

第一章 前言 1
1. 番茄簡介 1
2. 青枯病 (Bacterial wilt) 1
3. 植物與病原菌交互作用 2
4. 植物荷爾蒙在抗逆境之功能扮演 3
5. 植物對於青枯病害之相關研究 4
6. 植物非生物逆境之研究 8
7. 植物細胞骨架 9
8. 鋅指蛋白 (Zinc finger proteins, ZFP) 10
9. 病毒誘導性基因靜默 (Virus-induced gene silencing, VIGS) 11
10. 研究動機 12

第二章 材料與方法 13
1. 植物材料簡介 13
2. 供試質體及菌體培養條件 13
3. 常用實驗方法 14
4. 植物 RNA 萃取及 CDNA 之製備 17
5. 反轉錄聚合酶連鎖反應 (Reverse transcription PCR, RT-PCR) 19
6. 半定量 RT-PCR (Semi-quantitative RT-PCR, sqRT-PCR) 19
7. 核酸即時定量 PCR (Real-time PCR) 20
8. 番茄短暫性病毒誘導性基因靜默 20
9. 番茄澆灌青枯病菌 21
10. 番茄乾旱逆境測試 22
11. 番茄不同組織之 RNA 樣品製備 22
12. 番茄處理各式逆境及植物荷爾蒙 RNA 樣品製備 22
13. 螢光重組蛋白質之定位分析 (sub-cellular localization) 24
14. 菸草基因轉殖 (農桿菌轉殖法) 25
15. 阿拉伯芥花序浸潤轉殖法 25
16. 植物 DNA 萃取 26
17. 菸草轉殖株各式逆境測試 27
18. 阿拉伯芥各式逆境測試 29
19. 菸草轉殖株 Nicotiana tabacum W38 接種茄科病害後 NgEAS 啟動子之活性 分析 30

第三章 結果 33
I. SlZFP 基因之功能性分析 33
1. 番茄 SlZFP 基因之全長選殖、結構組成及其序列比對及分析 33
2. SlZFP 蛋白質在細胞之表現位置 34
3. SlZFP 基因在轉錄層次之調控 34
3.1 SlZFP 基因在番茄植株的內在分佈表現情形 34
3.2 SlZFP 基因於番茄接種青枯病菌後之調控情形 34
3.3 SlZFP 基因於番茄接種各式 PAMPs 後之調控情形 35
3.4 SlZFP 基因於乾旱逆境下之調控情形 35
3.5 SlZFP 基因於番茄植株處理植物荷爾蒙後之調控情形 35
4. 短暫靜默番茄 SlZFP 基因進行乾旱耐受性測試 36
5. 檢測番茄 SlZFP 基因靜默植株其防禦路徑相關基因表現情形 36
6. 表現番茄 SlZFP 之轉殖 N. benthamiana 植物分子檢驗及逆境分析 36
6.1 過量表現番茄 SlZFP 之轉基因植物分析 36
6.2 青枯病菌抗性測試 37
6.3 菸草幼苗於非生物逆境下之耐受性測試 37
6.4 乾旱耐受性測試 38
6.5 氧化及鹽害逆境耐受性測試 38
7. 過量表現 SlZFP 之菸草中參與生物與非生物逆境反應之基因表現情形 38
8. 阿拉伯芥同源基因之功能分析 39
8.1 同源基因之尋找與表現預測 39
8.2同源基因突變體之分子檢驗 39
8.3 各式非生物逆境下之萌芽率測試 39
8.4 接種青枯病菌及軟腐病菌抗病性測試 40
II. 茄科作物抗菌蛋白合成酵素 (5-Epi-aristolochene synthase, EAS) 之功能性分析 40
1. 表現菸草 NtEAS 之轉殖 N. benthamiana 植物分子檢驗及逆境分析 40
1.1過量表現番茄 NtEAS 之轉基因植物分析 40
1.2 青枯病菌抗性測試 41
1.3 氧化及鹽害逆境耐受性測試 41
2. 過量表現 NtEAS 之菸草中病害防禦相關基因表現情形 41
3. 分離及分析新的逆境誘導性茄科基因啟動子 42
3.1各啟動子片段受青枯病菌之誘發活性測試 42
3.2 真菌與細菌病原菌之誘發表現情形 42
3.3 不同病原型式之誘發表現 43
3.4 在植物不同組織中之表現 43

第四章 討論 44
I. SlZFP 基因之功能探討 44
1. SlZFP 主要表現於細胞骨架與植物的花與成熟果實 44
2. 番茄 SlZFP 基因正調控青枯病防禦反應 45
3. 番茄 SlZFP 基因正調控乾旱、氧化逆境及鹽害之防禦反應 46
4. 阿拉伯芥同源基因 at5g24870 突變體對多數非生物逆境耐受性降低 48
5. At5g24870 基因在病害防禦之功能探討 48
6. SlZFP 基因功能分析結語 49
II. 茄科 EAS 基因之功能探討 49
1. 表現菸草 NtEAS 基因之轉殖菸草增加對青枯病菌及非生物逆境抗性 49
2. NgEAS 基因啟動子可受多種病菌誘導，且可能具未知受病原菌誘導 cis-elements 50
3. NgEAS 基因啟動子專一表現在維管束組織 51
4. GT-1 box 為 NgEAS 啟動子誘發訊號接收之必須cis-element 52
5. 茄科 EAS 基因功能分析結語 53
III. 本論文總結 53

第五章 未來展望 54
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