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系統識別號 U0026-2508202011154400
論文名稱(中文) 微生物揮發性氣味對阿拉伯芥植株生長及缺氧相關基因表現的影響
論文名稱(英文) Effect of microbial volatile compounds on growth and expression of hypoxia-related genes in Arabidopsis seedings
校院名稱 成功大學
系所名稱(中) 熱帶植物與微生物科學研究所
系所名稱(英) Institute of Tropical Plant Sciences and Microbiology
學年度 108
學期 2
出版年 109
研究生(中文) 王武貴
研究生(英文) Wu-Guei Wang
學號 Z36064026
學位類別 碩士
語文別 中文
論文頁數 83頁
口試委員 指導教授-黃浩仁
共同指導教授-邱啟洲
口試委員-蔣鎮宇
口試委員-黃兆立
口試委員-李勇毅
中文關鍵字 產氣腸桿菌(Klebsiella aerogenes)  微生物揮發性氣味  自噬作用  先天性免疫反應  缺氧反應 
英文關鍵字 Klebsiella aerogenes  microbial volatile compounds  autophagy  innate immunity  hypoxia 
學科別分類
中文摘要 在自然界中植物與環境中多樣的生物皆有複雜的互動,過去植物與環境微生物交互作用的研究大多局限於植物與微生物的直接接觸或微生物分泌之可溶性化合物對植物之影響。越來越多的證據顯示,微生物,尤其是細菌會釋放多種揮發性氣味,這些揮發性氣味同樣對植物及其它病原體具有重要的生物活性,但植物對微生物揮發性氣味的感知及回應機制仍有許多待瞭解之處。本研究以產氣腸桿菌 (Klebsiella aerogenes) 作為微生物揮發性氣味的來源,探討阿拉伯芥感知到微生物揮發性氣味後,產生的生理生化及基因轉錄層次的變化。野生型阿拉伯芥與 K. aerogenes 揮發性氣味共培養下,阿拉伯芥的生長發育明顯受到抑制,共培養的早期伴隨誘發根部大量 ROS 累積、 NO 減少、生長素減少、葉片及根冠內澱粉形成失衡,到共培養晚期,可以發現葉綠素的減少而使葉片黃白化發生,細胞壁有胼胝質沉積及木質素的形成,並觀察到植株有細胞死亡的發生。透過蛋白質磷酸化分析發現 K. aerogenes 揮發性氣味能增加阿拉伯芥 MPK3 及 MPK6 的磷酸化程度。根據 qPCR 分析阿拉伯芥免疫相關途徑之基因表現發現,在 K. aerogenes 揮發性氣味影響下,植物賀爾蒙茉莉酸/乙烯及水楊酸、camalexin 及 indole glucosinolates 的生合成途徑相關基因轉錄量會受到正向調控。另一方面, K. aerogenes 揮發性氣味會誘發阿拉伯芥缺氧反應相關基因的活化,同時 K. aerogenes 揮發性氣味也會誘導阿拉伯芥根部自噬體的形成及部分自噬作用基因的轉錄表現,在探討自噬作用在阿拉伯芥回應微生物揮發性氣味的角色時發現, atg7 突變株會降低 K. aerogenes 揮發性氣味誘導的阿拉伯芥缺氧反應相關基因的轉錄量,因此知道自噬作用能參與調控阿拉伯芥對於 K. aerogenes 揮發性氣味的反應過程。
英文摘要 Mounting evidence suggests that microbes, especially bacteria, can emit diverse volatile compounds with biological activities on plants and their pathogens. However, it is still largely unknown about how plants perceive and respond to microbial volatile compounds (VCs). In the study, Arabidopsis co-cultured with microbial VCs from Klebsiella aerogenes. Physiological analysis and molecular biotechnology were used to explore the changes in Arabidopsis. Exposing to K.a.VCs, the growth of Arabidopsis was significantly inhibited. In the beginning, it caused high intracellular levels of ROS, generation of autophagosomes, reduction of NO, reduction of auxin accumulation in the roots, imbalance of starch formation in the leaves, and root caps. In the end, it caused a reduction of chlorophyll content that led to chlorosis, deposition of callose and lignin that thickened cell wall of Arabidopsis, and cell death in the plant. Results showed that K.a.VCs could activate Arabidopsis innate immunity by elevating the phosphorylation levels of MPK3 and MPK6. The downstream immune-related transcription factors were activated, including the expression of jasmonic acid/ethylene, salicylic acid, camalexin, indole glucosinolates biosynthetic pathway. K.a.VCs also induced the expression level of some autophagy genes of Arabidopsis. On the other hand, K.a.VCs induced the activation of hypoxia-related genes, and atg7 reduced the expression of hypoxia-related genes in Arabidopsis induced by K.a.VCs. In conclusion, autophagy regulates the response of Arabidopsis to K.a.VCs.
論文目次 摘要 I
Abstract II
英文延伸摘要 III
致謝 VIII
目錄 IX
表目錄 XII
圖目錄 XIII
縮寫對照表 XV
壹、前言 1
一、植物先天性免疫 1
二、自噬作用 2
三、微生物揮發性氣味 3
四、產氣腸桿菌 Klebsiella aerogenes 4
五、研究目的 4
貳、材料方法 6
一、植株材料與培養方法 6
二、菌種保存與培養 6
2.1保菌 6
2.2 Klebsiella aerogenes 培養 7
三、植物與細菌揮發性氣味共培養 7
四、植物於細菌揮發性氣味影響下之生理生化分析 7
4.1細胞死亡染色 7
4.2葉綠素含量測定 8
4.3澱粉染色 8
4.4 DR5::GFP 及 PIN::PIN-GFP 轉殖株根部 GFP 螢光表現 8
4.5偵測根部 ROS 螢光表現 8
4.6偵測根部 O2- 及 H2O2 表現 9
4.7偵測根部 NO 螢光表現 9
4.8 偵測根部自噬體 (autophagosome) 表現 (MDC staining) 9
4.9偵測根部胼胝質沉積 (callose deposition) 9
4.10偵測植株木質素累積 (phloroglucinol-HCl staining) 10
4.11 MAPK磷酸化程度檢測 (MAPK activation assay) 10
五、植物基因表現分析 12
5.1 RNA 萃取 (RNA extraction) 12
5.2 RNA濃度定量 (Quantitative Analysis of RNA) 13
5.3 瓊脂膠體電泳 (Gel Electrophoresis) 14
5.4反轉錄作用 (Reverse Transcription, RT) 14
5.5即時聚合酶連鎖反應 (Real-time Polymerase Chain Reaction) 14
六、結果分析與統計 15
參、結果 16
一、Klebsiella aerogenes 揮發性氣味對阿拉伯芥的生理生化影響 16
1.1 Klebsiella aerogenes 揮發性氣味對阿拉伯芥外表型的影響 16
1.2 Klebsiella aerogenes 揮發性氣味影響阿拉伯芥葉片澱粉及根冠澱粉粒恆定性 17
1.3 Klebsiella aerogenes 揮發性氣味減少阿拉伯芥根部 auxin 累積及 PIN3、PIN4 18
1.4 Klebsiella aerogenes 揮發性氣味影響阿拉伯芥根部 ROS 表現 18
1.5 Klebsiella aerogenes 揮發性氣味活化阿拉伯芥 MAPK cascades 19
1.6 Klebsiella aerogenes 揮發性氣味抑制阿拉伯芥根部 NO 表現 20
1.7 Klebsiella aerogenes 揮發性氣味誘導阿拉伯芥根部自噬體的形成 20
1.8 Klebsiella aerogenes 揮發性氣味影響阿拉伯芥細胞壁組成(cell wall integrity) 21
二、Klebsiella aerogenes 揮發性氣味對阿拉伯芥基因表現影響 21
2.1 Klebsiella aerogenes 揮發性氣味誘導阿拉伯芥免疫反應基因表現 22
2.2 Klebsiella aerogenes 揮發性氣味誘導阿拉伯芥乙烯及茉莉酸生合成基因表現 22
2.3 Klebsiella aerogenes 揮發性氣味誘導阿拉伯芥乙烯及茉莉酸下游基因表現 23
2.4 Klebsiella aerogenes 揮發性氣味誘導阿拉伯芥 camalexin、indole glucosinolates 轉錄因子及生合成基因表現 23
2.5 Klebsiella aerogenes 揮發性氣味誘導阿拉伯芥水楊酸轉錄因子及生合成基因表現 24
2.6 Klebsiella aerogenes 揮發性氣味誘導阿拉伯芥自噬作用相關基因表現 24
2.7 Klebsiella aerogenes 揮發性氣味誘導阿拉伯芥 RLCKs 及 MAPK cascades 訊息傳遞途徑相關基因表現 25
2.8 Klebsiella aerogenes 揮發性氣味誘導阿拉伯 PRRs 相關基因表現 25
三、阿拉伯芥 ATG7 在 Klebsiella aerogenes 揮發性氣味下之角色 26
3.1阿拉伯芥 ATG7 調控 Klebsiella aerogenes 揮發性氣味誘導之缺氧反應 27
肆、討論 29
一、Klebsiella aerogenes 揮發性氣味影響阿拉伯芥的生長 30
二、Klebsiella aerogenes 揮發性氣味誘發阿拉伯芥的免疫反應 31
三、阿拉伯芥 ATG7 在 Klebsiella aerogenes 揮發性氣味下之角色 34
四、結論 36
參考文獻 37
結果圖表 54
附錄 77
附件一、實驗中所使用細菌之菌種鑑定結果。 77
附件二、細菌揮發性氣味與植株共培養之示意圖。 78
附件三、乙烯 (ethylene) 生合成途徑。 79
附件四、茉莉酸 (jasmonic acid) 生合成途徑。 79
附件五、Camalexin 及 indole glucosinolates 生合成途徑。 80
附件六、水楊酸 (salicylic acid) 生合成途徑。 80
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