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研究生: 蘇珮瑜
Su, Pei-Yu
論文名稱: 阿拉伯芥MKK1和MKK3在微生物揮發物誘導防禦反應調控之角色
Arabidopsis MKK1 and MKK3 play different roles in microbial volatile compounds-induced defense response
指導教授: 黃浩仁
Huang, Hao-Jen
學位類別: 碩士
Master
系所名稱: 生物科學與科技學院 - 生命科學系
Department of Life Sciences
論文出版年: 2020
畢業學年度: 108
語文別: 中文
論文頁數: 59
中文關鍵詞: AtMKK1AtMKK3微生物揮發性化合物絲裂原活化蛋白激酶途徑植物防禦素產氣腸桿菌
外文關鍵詞: AtMKK1, AtMKK3, microorganism volatile organic compounds;mitogen-activated protein kinase cascades, plant defensin, Enterobacter aerogenes
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  • 植物是一種固著性的生物,因此在面臨生物與非生物逆境時,必須啟動相關機制來進行防禦反應。在病原菌誘發植物先天性免疫反應中,前人研究已知許多絲裂原活化蛋白激酶 (mitogen-activated protein kinase, MAPK) 在其扮演重要的角色。近年來已有許多研究證實微生物可以影響植物之生長與發育,但是對於微生物揮發性氣味 (microbial volatile organic compounds, mVCs) 所誘導植物免疫反應的防禦知之甚少。本研究將以植物生理分析以及分子生物技術來探討,當植物暴露於 mVCs 之下誘發的 AtMKK1 與 AtMKK3 相關免疫分子機制。在本研究中利用實驗室前人篩選出的植物生長抑制菌 Enterobacter aerogenes 與阿拉伯芥野生型 Col-0、mkk1 與 mkk3 共同培養,並觀察其生理及活化的訊息調路徑。本研究發現Enterobacter aerogenes 揮發性氣味可以抑制阿拉伯芥的生長,也會誘導過氧化物質 (reactive oxygen species, ROS) 的累積以及細胞死亡的情形,我發現不同的 MKKs 扮演不同的角色,其中 AtMKK1 可能參與短時間 E.a. mVCs 下的 ROS 生成機制,AtMKK3 可能參與 E.a. mVCs 誘導下的細胞死亡機制。此外,本研究使用即時聚合酶連鎖反應 (real time polymerase chain reaction, RT-qPCR) 了解相關基因在mVCs處理後之表現量變化,結果可得知植物能藉由偵測與辨識 E.a. mVCs 而誘發免疫防禦之反應,分析AtMKK1 突變株中的基因表現量時,發現 AtMKK1參與在防禦蛋白 PDF1.2、乙烯生合成路徑之 ACS2 以及 camalexin 生合成路徑之 CYP71A13、PAD3 的調控,促使植物產生防禦反應,並且誘發植株的先天性免疫訊息傳遞途徑,藉此抵抗 mVCs 對植物產生之逆境。

    Plants are sessile organisms. Therefore, in the face of biotic and abiotic stresses, relevant mechanisms must be activated to carry out defensive responses. Many mitogen-activated protein kinase (MAPKs) have been shown to play important roles in the innate immune response induced by pathogens in plants. In recent years, many studies have confirmed that microorganisms can affect plant growth and development, but little is known about the defense responses induced by microbial volatile organic compounds (mVCs). In this study, I used plant physiological analysis and molecular biotechnology to explore the immune molecular mechanisms induced by the volatile substance when plants are exposed to mVCs. The effects of Enterobacter aerogenes mVCs on Arabidopsis thaliana Col-0, AtMKK1 mutant and immune response were observed. This study found that Enterobacter aerogenes mVCs can inhibit Arabidopsis. growth. Accumulation of reactive oxygen species (ROS) and cell death were also induced after mVCs treatment. I found that AtMKK1 and AtMKK3 have different roles in the regulation of mVCs-induced responses. AtMKK1 may be involved in the ROS generation mechanism under short-term exposure to E.a. mVCs, and AtMKK3 may be involved in the regulation of E.a. mVCs-induced cell death, which have a certain degree of physiological impact on Arabidopsis. In addition, real-time polymerase chain reaction (Q-PCR) was used to understand the changes in mRNA abundance of E.a. mVCs regulated genes. The results show that plants can induce immune responses by detecting and identifying E.a. mVCs. Analysis of the mRNA abundance of CYP71A13, PAD3, ACS2 and PDF1.2 in AtMKK1 mutant revealed that AtMKK1 is involved in the regulation of defensin response, ethylene and camalexin biosynthesis. Leads to plant defense responses and induces the plant innate immune signaling pathways, thereby resisting the stress of mVCs to plants.

    摘要I AbstractII 英文延伸摘要III 誌謝VII 目錄VIII 表目錄XI 圖目錄XII 縮寫對照表XIII 壹、前言1 一、微生物揮發性氣味 (mVCs) 對植物之影響1 二、植物先天性免疫 (plant innate immunity)3 三、絲裂原活化蛋白激酶訊息傳遞途徑 (MAPK signaling transduction pathway)4 四、植物抗毒素 (phytoalexins)5 五、產氣腸桿菌 (Enterobacter aerogenes)6 六、研究目的7 貳、材料與方法8 一、植株材料培養8 二、菌種保存與培養8 2.1 菌種純化與保菌9 2.2 Enterobacter aerogenes細菌培養9 三、植物與微生物揮發性氣味共培養9 四、微生物揮發性氣味對植物影響之生理生化分析10 4.1 細胞死亡之染色測定10 4.2 過氧化物質之植物組織染色10 五、植物之基因表現測定10 5.1 RNA萃取10 5.2 RNA 濃度定量 (Nucleic acid quantitative analysis)12 5.3 反轉錄作用 (Reverse Transcription, RT-PCR)12 5.4 即時聚合酶連鎖反應 (Real time polymerase chain reaction, Q-PCR)12 參、結果14 一、 Enterobacter aerogenes揮發性氣味對阿拉伯芥的影響與生理反應14 1.1 Enterobacter aerogenes揮發性氣味能有效抑制阿拉伯芥野生型之根部生長14 1.2 Enterobacter aerogenes揮發性氣味能誘導阿拉伯芥 MKKs基因之表現15 1.3 Enterobacter aerogenes揮發性氣味能有效抑制阿拉伯芥 AtMKK1與AtMKK3 突變株之根部生長15 1.4 Enterobacter aerogenes揮發性氣味能有效抑制阿拉伯芥 AtMKK1 突變株之葉片生長16 1.5 Enterobacter aerogenes揮發性氣味能誘導阿拉伯芥產生過氧化物累積17 1.6 Enterobacter aerogenes揮發性氣味能誘導阿拉伯芥產生細胞死亡現象18 二、利用Q-PCR技術探討阿拉伯芥在Enterobacter aerogenes揮發性氣味共培養處理下的分子機制19 2.1 AtMKK1參與 Enterobacter aerogenes 揮發性氣味誘導植物先天性免疫反應相關基因之表現19 2.2 Enterobacter aerogenes揮發性氣味誘導阿拉伯芥在乙烯生合成相關基因之表現量19 2.3 Enterobacter aerogenes氣味誘導阿拉伯芥在水楊酸生合成相關基因之表現量20 2.4 AtMKK1 參與Enterobacter aerogenes揮發性氣味誘導 camalexin 生合成相關之基因表現21 肆、討論23 一、 Enterobacter aerogenes揮發性氣味對阿拉伯芥的影響23 1.1 Enterobacter aerogenes揮發性氣味對阿拉伯芥之生理影響23 1.2 Enterobacter aerogenes揮發性氣味能誘發MAPK 訊息傳遞途徑24 二、 AtMKK1在 Enterobacter aerogenes 揮發性氣味下之功能與角色25 2.1 Enterobacter aerogenes氣味誘導植物先天性免疫反應相關基因之表現25 2.2 Enterobacter aerogenes氣味誘導乙烯生合成相關基因之表現25 2.3 Enterobacter aerogenes氣味誘導水楊酸生合成相關基因之表現26 2.4 Enterobacter aerogenes氣味誘導camalexin生合成之基因表現26 伍、結論28 參考文獻29 圖表41 附錄58

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