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研究生: 熊御全
Hsiung, Yu-Chyuan
論文名稱: 植物相剋化合物月桂烯對於植物體抗氧化酵素活性與逆境反應基因之研究
Modulation of antioxidant responses and defense-response gene expression by allelopathic volatile, myrcene, in plants
指導教授: 黃浩仁
Huang, Hao-Jen
共同指導教授: 李瑞花
Lee, Ruey-Hua
學位類別: 碩士
Master
系所名稱: 生物科學與科技學院 - 熱帶植物科學研究所
Institute of Tropical Plant Sciences
論文出版年: 2012
畢業學年度: 100
語文別: 英文
論文頁數: 80
中文關鍵詞: 植物相剋作用揮發性相剋化合物活性氧簇訊息傳遞途徑月桂烯菸草水稻抗氧化酵素絲裂原活化蛋白激酶
外文關鍵詞: Myrcene, allelochemical, MAP kinase, ROS, signaling pathway, Cyclin, antioxidative enzyme
相關次數: 點閱:173下載:3
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  • 植物相剋作用在生態系統中扮演非常重要的角色,而植物相剋作用的涵義一般是描述兩個植物間之有機化學交互作用。在植物體內,植物相剋化合物可存在於根及根的分泌物、莖、葉、樹皮、花及果實。而植物相剋作用的定義則是指供體植物透過淋溶作用、來自植株 (根、莖、葉) 揮發作用、根部的泌濾作用及土壤中植物殘體分解作用所釋出至環境的植物相剋化合物,將造成受體植物生長及發育嚴重的影響,由此可知植物相剋化合物在生物多樣性中扮演重要的角色。近年研究指出,植物為了對抗環境逆境,可調節自身釋放出各種不同之生物性有機揮發物質。有機揮發物質能對鄰近植物引起反應,構成植物與植物與植物之間交互作用的平台,然而在植物揮發性相剋化合物的分子作用機制,目前所知有限。本文研究旨在深入探討植物揮發性相剋化合物月桂烯,對於水稻根部與菸草葉片之抗氧化活性酵素與逆境訊息傳遞基因之表現。此篇研究中我們發現植物相剋化合物月桂烯會抑制水稻根部的生長情形,並誘發活性氧簇的產生;在菸草葉片的實驗中亦會誘發活性氧簇的生成。為了更進一步了解植物體遭遇月桂烯逆境下的抗氧化酵素改變,我們接著偵測水稻根部與菸草葉片過氧化氫酶、過氧化酶、與過氧化物歧化酶的活性分析。結果發現在水稻根部中,過氧化氫酶、過氧化酶、過氧化物歧化酶的活性皆會被誘導而上升,而驗菸草葉片的酵素活性分析中亦發現過氧化酶活性會隨著月桂烯濃度上升而上升。而就轉錄層級而言,我們希望明瞭揮發性植物相剋化合物的訊息傳遞途徑。實驗結果指出水稻的絲裂原活化蛋白激酶三號活性會被月桂烯專一性誘導而上升;菸草植物中的WIPK與NTF6亦有被月桂烯誘導而上升的趨勢,隨後探究絲裂原活化蛋白激酶其下游訊號傳遞途徑,我們發現水稻中WRKY71似乎為絲裂原活化蛋白激酶三號的下游基因。另外我們由細胞週期相關基因(Cyclin)與脂氧化酶的反轉錄聚合酶鏈鎖反應的實驗結果推測出月桂烯造成的生長抑制情形應該是經由與產生植物逆境荷爾蒙-茉莉花酸並抑制細胞分裂週期所導致。而在後續研究中我們利用乙烯抑制劑硫代硫酸銀前處理菸草葉片後處理月桂烯,發現活性氧簇的生成情形明顯被抑制,故我們推測月桂烯所造成的活性氧簇產生途徑,須經由乙烯當做其中的訊號傳遞分子。至此,本研究已初步了解植物在月桂烯逆境下訊息傳遞途徑的分子機轉,期許能有效幫助了解揮發性植物相剋化合物的作用模式。

    Myrcene (7-Methyl-3-methylene-1,6-octadiene) is known as one of volatile allelochemicals, but its molecular mode of action is not well understood. We found that Myrcene inhibited root growth and induced reactive oxygen species (ROS) production both in rice root and tobacco leaves. To gain more insight into these cellular responses and antioxidative responses, we performed isoenzyme analysis of rice root and tobacco leaves during Myrcene stress. We found 3 kinds of antioxidative enzyme , CAT, POD and SOD all can be induced when exposure to Myrcene stress in rice roots, and only POD activity can be detected and induced in tobacco leaves.
    Myrcene also triggered changes in transcript levels of genes related to signal transduction, transcription factor, cell growth response and phytohormone.. Gene expression profiling of Myrcene-treated rice roots and tobacco leaves revealed upregulated of Mitogen-activated protein kinase (MAPK3 in rice., WIPK, NTF6 in tobacco ), WRKY71, Lipoxygenase and biosynthesis of jasmonic acid. In addition, Myrcene downregulated cyclinA1, B1-1 and D1, indicated that may induce stress response and reduce cycle cycle ratio when expose on Myrcene. We also found ethylene receptor inhibitor, STS-treated tobacco leaves may reduce ROS production, supposed that Myrcene-induced ROS production should through ethylene pathway. Further characterization of these Myrcene-responsive genes may be helpful for better understanding the mechanisms of allelochemical tolerance in plants.

    Table of Contents Abstract in Chinese 3 Abstract in English 5 Acknowledgement 6 Abbreviations 12 Chapter1 Introduction 1.1 Allelopathy 14 1.2 Biogenic volatile organic compounds as Allelochemicals 15 1.3 ROS in plant signal transduction 16 1.4 ROS vs. antioxident enzyme 18 1.5 MAP Kinase gene in plant signal transduction 19 1.6 Ethylene and ROS production 21 1.7 Aim of this study 22 Chapter2 Material and methods 2.1 Growth conditions of plant materials and root length determination 25 2.2 Determination of ROS production in rice roots 25 2.3 Histochemical detection of hydrogen peroxide in N. benthamiana leaves 25 2.4 Purification of total RNA 26 2.5 Semi-quantitive RT-PCR 26 2.6 Antioxidative enzyme analysis 26 Chapter3 Identification of antioxidant responses and defense response gene expression by Myrcene in plants 3.1 Abstract 28 3.2 Introduction 29 3.3 Result 3.3.1 Effect of Myrcene stress on root growth and viability of rice seedlings 32 3.3.2 Myrcene rapidly induced ROS accumulation in rice roots 32 3.3.3 Time course expression profiles of antioxidative enzyme by Myrcene stress 32 3.3.4 Dose-expression profiles of antioxidative enzyme by Myrcene stress 32 3.3.5 Effects of Myrcene on lipid peroxidation 33 3.3.6 Expression profiles of genes associated with signaling in rice root 33 3.3.7 Expression profiles of JA- and cell cycle-related genes in Myrcene stress 33 3.3.8 Myrcene induced ROS accumulation in tobacco leaves 34 3.3.9 Dose-depend expression profiles of antioxidative enzyme by Myrcene stress 34 3.3.10 Expression profiles of genes associated with signaling in tobacco leaves 34 3.3.11 The effect of Myrcene on ROS accumulation of STS treated tobacco leaves 34 3.4 Discussion35 Chapter4 Conclusion and References Conclusion 39 Reference 40 Figures Figure 1. Effects of Myrcene stress on root growth in O. sativa 60 Figure 2. Reactive Oxygen species (ROS) production with Myrcene treatment in rice roots 61 Figure 3. Patterns of POD isoenzyme in roots of O. sativa 62 Figure 4. Patterns of SOD isoenzyme in roots of O. sativa 63 Figure 5. Patterns of CAT isoenzyme in roots of O. sativa 64 Figure 6. Patterns of POD isoenzyme in roots of O. sativa 65 Figure 7. Patterns of SOD isoenzyme in roots of O. sativa 66 Figure 8. Patterns of CAT isoenzyme in roots of O. sativa 67 Figure 9. Patterns of POD isoenzyme in roots of O. sativa 68 Figure 10. Patterns of SOD isoenzyme in leaves of O.sativa 69 Figure 11. Patterns of CAT isoenzyme in leaves of O. sativa 70 Figure 12. The effect of Myrcene on Lipoxygenase activity in O. sativa roots 71 Figure 13. The effect of Myrcene on Lipoxygenase expression pattern in O. sativa 72 Figure 14. The effect of Myrcene on JA pathway-related gene in O. sativa 73 Figure 15. Analysis of MAPK family gene expression by RT-PCR in O. sativa roots 74 Figure 16. Analysis of cell cycle-related gene expression by RT-PCR in O. sativa roots 75 Figure17 A putative pathway of linking Myrcene stress and signal transduction in rice roots 76 Figure18 Reactive Oxygen species (ROS) production with Myrcene treatment in N. benthamiana leaves 77 Figure19. Patterns of POD isoenzyme in leaves of N. benthamiana 78 Figure 20. The effect of Myrcene on MAPK families’ gene expression pattern in N. benthamiana 79 Figure 21. Reactive Oxygen species (ROS) production with Myrcene treatment in N. benthamiana leaves 80

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