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系統識別號 U0026-2507201401491200
論文名稱(中文) 轉錄圖譜與生理分析探討菸草的Exo70基因在產氣桿菌(Enterobacter aerogene)氣味影響下之功能與角色
論文名稱(英文) Transcriptome Profiling and Physiological Studies Reveal a Major Role for Exo70 in response to volatile organic compounds produced by Enterobacter aerogenes in tobacco.
校院名稱 成功大學
系所名稱(中) 生命科學系
系所名稱(英) Department of Life Sciences
學年度 102
學期 2
出版年 103
研究生(中文) 鄭楷騰
研究生(英文) Kai-Teng Cheng
學號 L56014073
學位類別 碩士
語文別 中文
論文頁數 113頁
口試委員 指導教授-黃浩仁
口試委員-蔣鎮宇
口試委員-張文粲
口試委員-朱信
口試委員-林財富
中文關鍵字 胞吐複合體  次世代定序  轉錄圖譜  基因靜默  過氧化物質 
英文關鍵字 Exo70  Next Generation Sequencing  Transcriptome  gene silencing  ROS 
學科別分類
中文摘要 過去植物病理學研究,著重在病原菌接觸植物之條件下,探討植物的病徵、反應與抵抗機制,但對於病原菌產生的氣味對植物之影響則較少見。我們從野外土壤中純化出產氣桿菌(Enterobacter aerogenes),其氣味能有效抑制菸草生長、降低葉綠素含量、促使細胞死亡並誘導過氧化物質累積。利用次世代定序技術分析菸草在Enterobacter aerogenes氣味下之轉錄圖譜,發現有1320條轉錄產物(transcripts)會受菌味處理而誘導表現,並參與在WRKY轉錄調控、粒線體電子傳遞鏈、Cytochrome P450解毒機制與鈣離子訊息傳遞等相關路徑。NbExo70為菸草中的胞吐複合體成員,其同樣會受Enterobacter aerogenes氣味處理而表現上提,若利用基因靜默技術壓低NbExo70表現量,將會降低菸草對Enterobacter aerogenes氣味的耐受性,並減緩菌味誘導的氧化物質累積現象。經由比較野生型與NbExo70靜默型菸草在Enterobacter aerogenes氣味下的轉錄圖譜,發現有162條NbExo70依賴型轉錄產物(transcripts),其只在野生型菸草中會表現上提,過去研究得知粒線體電子傳遞鏈複合體1號的運作,能誘導植物累積過氧化物質,分析結果顯示,NbExo70依賴型轉錄產物(transcripts)有17條屬於粒線體電子傳遞鏈複合體1號中的成員。本研究結果可證明NbExo70參與Enterobacter aerogenes氣味下的反應機制,並透過直接或間接影響粒線體電子傳遞鏈複合體1的基因表現,來誘導過氧化物質累積。
英文摘要 Previous studies of plant pathology focusing on plant and pathogen direct surface-to-surface contact. However, the effects of volatile chemicals on plants are rare. In this stufy, we found volatiles of Enterobacter aerogenes significantly inhibited the growth, and decreased the content of chlorophyll in Nicotiana benthamiana. This volatiles also induced the cell death and H2O2 production. Transcriptome profiling revealed 1320 transcripts were significantly responsed to 12 hours exposure to E. aerogenes volatiles. These transcripts primary involved in WRKY transcriptional regulation, mitochondrial electron transport chain, cytochrome P450 in detoxification mechanism and calcium signaling pathway. NbExo70 is a member of exocytosis complex in N. benthamiana. This gene also was responsed to E. aerogenes volatiles. Using the gene silencing technique to knock down NbExo70 would reduce the tolerance and H2O2 production under E. aerogenes volatiles. We also compared the transcriptome profiles of wild-type and NbExo70 silencing tobacco under the E. aerogenes volatiles. We found 162 NbExo70-dependent transcripts were specifically induced in the wild-type tobacco plants. Seventeen out of 162 transcripts involved in mitochondrial electron transport chain complex I that could cause the accumulation of H2O2. In this study, we proposed that NbExo70 were involved in response to E. aerogenes volatiles. Moreover, NbExo70 might directly or indirectly affect gene expression of members in mitochondrial electron transport chain complex I to induce H2O2 production under the E. aerogenes volatiles.
論文目次 摘要1
Abstract2
英文延伸摘要3
誌謝6
縮寫對照表14
第一章 前言15
一、細菌氣味對植物之影響15
二、植物的胞吐複合體16
三、次世代定序17
四、基因靜默技術18
五、研究目的19
第二章材料與方法20
一、植物材料製備20
(一)菸草(Nicotiana benthamiana)組織培養20
(二)菸草(Nicotiana benthamiana)土壤培養20
二、Enterobacter aerogenes的純化與鑑定20
(一)土壤採集20
(二)菌種培養21
(三)菌種純化與保菌21
(四)菌種篩選21
(五)菌種鑑定22
三、Enterobacter aerogenes氣味對植物之處理23
(一)菌盤製備23
(二)菌味對植物之處理23
四、植物的生理與生化分析24
(一)葉綠素含量測定24
(二)過氧化物質之植物組織染色25
(三)細胞死亡測定25
五、植物之基因表現測定25
(一)RNA萃取25
(二)反轉錄聚合酶連鎖反應(reverse transcription-PCR;RT-PCR)26
(三)聚合酶連鎖反應(Polymerase chain reaction;PCR)26
(四)即時聚合酶連鎖反應( Real time polymerase chain reaction ; Q-PCR)27
六、利用β-glucuronidase(Gus)方法進行啟動子活性測定27
(一)pHGWFS7-NbExo70 promoter載體建構27
(二)利用電穿孔法置備pHGWFS7-NbExo70 promoter之轉型農桿28
(三)NbExo70promoter-β-glucuronidase之轉殖菸草製作29
(四)NbExo70之啟動子活性測定31
七、NbExo70之基因靜默轉殖株製作31
(一)pH2WG7-NbExo70載體建構31
(二)利用電穿孔法置備pH2WG7-NbExo70之轉型農桿菌33
(三)pH2WG7-NbExo70之轉殖菸草製作34
八、用次世代定序探討Enterobacter aerogenes氣味下菸草之基因表現35
(一)RNA資歷料庫(Library)製備與次世代定序流程35
(二)次世代定序結果組裝與分析流程35
第三章 結果37
一、Enterobacter aerogenes氣味對菸草的影響與生理反應37
(一)Enterobacter aerogenes篩選與鑑定結果37
1.1 細菌篩選與鑑定結果37
(二)Enterobacter aerogenes氣味對菸草生理之影響37
2.1 Enterobacter aerogenes氣味能有效抑制菸草生長37
2.2 Enterobacter aerogenes氣味能誘導菸草葉片黃化39
2.3 Enterobacter aerogenes氣味能誘導菸草累積過氧化物質39
二、利用次世代定序(Next Generation Sequencing , NGS)技術探討菸草在Enterobacter aerogenes氣味下的分子機制41
(一)次世代定序組裝結果與基本分析41
1.1 次世代定序組裝結果與功能預測41
(二)菸草在Enterobacter aerogenes氣味下之分子機制42
2.1 概觀分析結果42
2.2 荷爾蒙相關基因42
2.3 鈣離子訊息傳遞相關基因43
2.4 解毒相關基因44
2.5 過氧化物質相關基因44
2.6 轉錄因子基因45
三、菸草的NbExo70在Enterobacter aerogenes氣味下之功能與角色46
(一)NbExo70在Enterobacter aerogenes氣味的表現趨勢與位置46
1.1 Enterobacter aerogenes氣味能誘發NbExo70表現上升46
1.2 Enterobacter aerogenes氣味能誘導NbExo70的啟動子活化46
(二)利用NbExo70靜默型菸草探討NbExo70在Enterobacter aerogenes氣味的下之功能與角色47
2.1 NbExo70的靜默會降低菸草對Enterobacter aerogene氣味的耐受性47
2.2 NbExo70的靜默會增強Enterobacter aerogene氣味誘導的細胞死亡48
2.3 NbExo70靜默會減少Enterobacter aerogene氣味誘導的過氧化氫累積49
四、利用次世代定序(Next Generation Sequencing , NGS)技術探討野生型(Wt)與NbExo70靜默型(C6)菸草在Enterobacter aerogenes氣味下的基因表現差異50
(一)比較野生型(Wt)與NbExo70靜默型(C6)菸草在Enterobacter aerogenes氣味下之分子機制差異50
1.1 概觀分析50
1.2 粒線體電子傳遞鏈相關基因51
1.3 解毒相關基因51
1.4 轉錄因子相關基因51
第四章 討論53
一、Enterobacter aerogenes氣味對菸草生長之影響 53
二、乙烯與茉莉酸之合成路徑會因Enterobacter aerogenes氣味處理而誘導上升54
三、Enterobacter aerogenes氣味誘導菸草累積過氧化物質55
四、Enterobacter aerogenes氣味能活化鈣離子訊息傳遞路徑57
五、菸草的Cytochrome P450基因會受Enterobacter aerogenes氣味處理而表現上升58
六、Enterobacter aerogenes氣味下表現上升的轉錄因子59
七、NbExo70在Enterobacter aerogenes氣味下的功能與角色62
八、NbExo70依賴型基因之分析63
參考文獻65
結果圖表74
附錄112
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