臺灣大學: 植物科學研究所鄭秋萍陳雅婷Chen, Ya-TingYa-TingChen2013-03-212018-07-062013-03-212018-07-062011http://ntur.lib.ntu.edu.tw//handle/246246/248365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.6861368 bytesapplication/pdfen-US鋅指蛋白青枯病菌乾旱茄科抗菌蛋白誘導性啟動子Zinc finger proteinRalstonia solanacearumdrought5-epi-aristolocheneinducible promoterhttp://ntur.lib.ntu.edu.tw/bitstream/246246/248365/1/ntu-100-R97b42034-1.pdf