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研究生:王盈茹
研究生(外文):Ying-Ju Wang
論文名稱:番茄 ERF38 與 ERF35 在逆境反應之功能研究
論文名稱(外文):Functional Study of Tomato ERF38 and ERF35 in Stress Responses
指導教授:鄭秋萍鄭秋萍引用關係
指導教授(外文):Chiu-Ping Cheng
口試委員:詹明才鄭石通葉開溫
口試委員(外文):Ming-Tsair ChanShih-Tong JengKai-Wun Yeh
口試日期:2013-07-30
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:97
中文關鍵詞:乙烯反應轉錄因子青枯病細菌性軟腐病乾旱逆境鹽害逆境
外文關鍵詞:Ethylene-response factors (ERFs)bacterial wilt (BW)Pectobacterium carotovoradroughtsalt stress
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植物在自然界會遭遇各種病害性與非生物性逆境並造成巨大的農業損失,例如青枯病與乾旱皆會引起作物致死性危害,且影響範圍極廣。植物特有的乙烯反應轉錄因子 (ethylene-response factors, ERFs) 在植物的逆境反應扮演重要角色,然而,目前其詳細機制尚未完全明朗。本研究室先前研究發現屬於VIII 群 ERF之番茄SlERF35 與 SlERF38可能參與青枯病與乾旱逆境反應,故本研究進一步培育其過量表現與基因靜默之基因轉殖植物,以利分析其確切功能與可能之調控機制。首先,本研究證實 SlERF38 蛋白具有抑制轉錄活性的功能;過量表現 SlERF38 之轉殖菸草,其花與種子數量減少、對青枯病抗性增加、對細菌性軟腐病較感病,且其乙烯生合成基因之表現增加,而茉莉酸生合成基因之表現則下降,推論 SlERF38 可能會使乙烯訊息傳導之抑制基因 (repressors) 的表現降低進而增強乙烯訊息傳導,同時也使茉莉酸訊息傳導之促進基因 (activators) 的表現降低進而抑制茉莉酸訊息傳導,進而造成上述性狀改變;另外,此基因也會受乙烯與茉莉酸回饋負調控。此外,遭遇逆境時,植物可能藉由離層酸的大量產生來抑制 SlERF38 轉錄,進而減少 SlERF38 對乾害及鹽害逆境耐受性之負面影響。在另一方面,前人研究發現乙烯、茉莉酸、水楊酸及離層酸會促進 SlERF35 轉錄,本研究結果則顯示 SlERF35 蛋白具有抑制轉錄活性的功能,過量表現 SlERF35 之轉殖菸草,其葉部會黃化,且成株對青枯病之抗性增加,推測在逆境時,SlERF35 可能參與上述逆境賀爾蒙誘導之葉綠素分解途徑與青枯病抗病反應的調控機制。未來可利用本論文已建立好的過量表現與基因靜默之基因轉殖植物,更深入探討 SlERF35 與 SlERF38 在各種逆境上之功能,期望所獲資訊將有助於累積對極重要且複雜之植物病害與非生物逆境防禦之瞭解與可用資源。

Plants constantly encounter a wide range of abiotic and biotic stresses, leading to tremendous crop losses. Bacterial wilt (BW), a very complex deadly disease caused by Ralstonia solanacearum, and water deficit are the most serious stress factors of many economically important crops worldwide. Ethylene-response factors (ERFs) are a large family of plant-specific transcription factors involved in various stress responses; however, our knowledge about their functions and the involved regulatory mechanisms is still rudimentary. Our previous study suggested the involvement of two Group VIII ERFs of tomato, namely SlERF35 and SlERF38, in BW and drought responses. In this study, transgenic plants with altered expression levels of these genes were generated and analyzed for their stress responses and the involved regulatory mechanisms. The results showed that SlERF38 was a functional transcriptional repressor, Overexpression of SlERF38 in tobacco led to reduced flower and seed production, enhanced tolerance to BW, increased susceptibility to bacterial soft rot, augmented expression of ethylene (ET) biosynthesis genes, and decreased expression of a jasmonic acid (JA) biosynthesis gene. These results together enable to propose that SlERF38 may de-repress the ET signaling by suppressing the expression of certain ET repressors, as well as repress the JA signaling by reducing the expression of certain JA activators, leading these phenotypes. In addition, it is hypothesized that, when plant encounters stresses, the induced abscisic acid (ABA) production may repress SlERF38 transcription and subsequently reduce its negative effect on stress defense signaling, leading to enhanced tolerance to drought and salinity. Furthermore, our previous study showed that SlERF35 transcription was promoted by ET, JA, salicylic acid (SA) and ABA. Current study showed that it is a functional transcriptional repressor and its overexpression led to leaf yellowing and increased tolerance to BW. These data suggest that SlERF35 may be involved in stress hormones-induced chlorophyll degradation and tolerance to BW. The transgenic plants generated in this study can be used for future analyses to gain further insights into functions and regulatory mechanisms of these proteins in stress responses.

第一章 前言 1
一、植物病害反應與防禦機制 1
二、植物病害與非生物逆境交互作用 2
三、植物防禦荷爾蒙對植物生長發育之影響 3
四、植物 AP2/EREBP 轉錄因子超級家族 4
五、青枯病 (Bacterial wilt, BW) 6
六、研究動機與目標 7
第二章 材料與方法 9
1. 植物材料簡介 9
2. 基因選殖常用實驗 9
3.植物 DNA 萃取 13
4. 植物 RNA 萃取 13
5. 反轉錄聚合酶連鎖反應 (Reverse transcription PCR, RT-PCR) 15
6. 調控轉錄活性分析法 (Transactivation assay) 17
7. 菸草與番茄基因轉殖 19
8. 菸草轉殖株之性狀觀察 22
9. 植物之病害反應檢測 22
10. 植物之非生物性逆境檢測 24
第三章 結果 26
1. 利用生物資訊分析番茄 SlERF35 與 SlERF38 基因結構組成與序列分析 26
2. SlERF35 與 SlERF38 之調控轉錄活性分析 26
3. 35S::SlERF38 轉殖菸草之性狀分析 26
4. SlERF38-RNAi 轉殖番茄性狀分析 29
5 GVG::SlERF35 轉殖菸草之性狀分析 30
6. SlERF35-RNAi 轉殖番茄性狀分析 31
第四章 討論 33
1. SlERF38 可被逆境荷爾蒙調控,且可調控 ET 與 JA 之合成基因轉錄 33
2. SlERF38 負調控花及種子發育 33
3. SlERF38 正調控青枯病抗性,但負調控細菌性軟腐病抗性 34
4. SlERF38 負調控乾旱與鹽害耐受性 36
5. SlERF35 可能參與葉綠素分解途徑 37
6. SlERF35 正調控青枯病抗性,但可能未參與細菌性軟腐病、乾旱及鹽害逆境反應 38
7. 總結 39
參考文獻 40


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