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研究生:葛孟杰
研究生(外文):Ger, Mang-Jye
論文名稱:轉殖菸草中表現hrap基因增加其對致敏因子harpinPss的敏感度與細菌性病原的抗性
論文名稱(外文):Constitutive expression of hrap gene in transgenic tobacco plant enhances sensitivity to harpinPss and resistance against bacterial pathogens
指導教授:馮騰永
指導教授(外文):Feng, Teng-Yung
學位類別:博士
校院名稱:國防醫學院
系所名稱:生命科學研究所
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:2002
畢業學年度:90
語文別:中文
論文頁數:112
中文關鍵詞:過敏反應轉殖菸草植物抗病
外文關鍵詞:hypersensitive responsetransgenic tobaccoplant defense
相關次數:
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  • 下載下載:34
  • 收藏至我的研究室書目清單書目收藏:1
中文摘要
HRAP (hypersensitive response assisting protein)是一種來自甜椒的雙極性蛋白,可促進致敏因子harpinPss在甜椒上所引起的過敏反應(hypersensitive response)。從hrap cDNA比對上發現此基因不與任何已知序列相似,而且可在非親合性交互作用下快速反應。為了了解HRAP在植物防禦系統中所扮演的角色,吾人利用CaMV 35S啟動子在轉殖菸草中持續表現甜椒hrap基因並測試其抗病能力。致敏因子harpinPss注射測試中發現,等量的harpinPss可在轉殖菸草葉片上引發比在野生型菸草上更強的過敏反應。相較於野生型菸草,非親合性細菌性病原Pseudomonase syringae pv. syringae與Xanthomonas campestris pv. vesicatoria在轉殖菸草上引發過敏反應也較為強烈。此結果顯示轉殖菸草對harpinPss與非親合性細菌性病原有較高的敏感度。吾人亦分析轉殖菸草對親合性病原Pseudomonase syringae pv. tabaci與Erwinia carotovora subsp. carotovora的抗性。在此兩種病原接種測試中,兩者皆無法在轉殖菸草上引發病徵。進一步分析病原族群數後顯示,此兩種病原在轉殖菸草中的增殖明顯受到抑制。在未受病原感染情況下,水楊酸與過敏反應標示基因hsr203J的表現量在轉殖與野生型菸草間並無明顯差異。此結果顯示轉殖菸草對親合性細菌性病原的抗性並非來自持續表現過敏反應。而在蛋白毒性測試中也顯示HRAP重組蛋白對病原並無直接毒害作用。然而在親合性病原接種轉殖菸草後,hsr203J基因被快速誘導表現。經由Trypan blue染色法亦觀察到在轉殖菸草葉片接種區中有快速的細胞死亡現象。綜合上述結果,吾人推論hrap轉殖菸草的抗性來自被親合性病原所誘發的過敏反應。

Hypersensitive response assisting protein (HRAP) is previously reported as an amphipathic protein isolated from sweet pepper, which intensifies the harpinPss-mediated hypersensitive response (HR). The hrap gene has no appreciable similarity to any other known sequences and its activity can be rapidly induced by incompatible pathogen infection. To assess the function of the hrap gene in plant disease resistance, the CaMV 35S promoter was used to express sweet pepper hrap in transgenic tobacco. After avirulence pathogens infection (Pseudomonase syringae pv. syringae and Xanthomonas campestris pv. vesicatoria), transgenic tobacco exhibits stronger HR necrosis than wild type. Compared with wild type tobacco, transgenic tobacco plants also exhibit more sensitivity to harpinPss and show resistance to virulent pathogens (Pseudomonas syringae pv. tabaci and Erwinia carotovora subsp. carotovora). This disease resistance of transgenic tobacco does not originate from a constitutive HR because endogenous level of salicylic acid and hsr203J mRNA showed similarities in transgenic and wild type tobacco under non-infected conditions. In the protein antibacterial activity analysis, HRAP shows no directly toxicity against pathogens. However, following a virulent pathogen infection in hrap transgenic tobacco, hsr203J was rapidly induced and a micro-HR necrosis was visualized by trypan blue staining in the infiltration area. Consequently, we suggest that the disease resistance of transgenic plants may result from the induction of an HR by a virulent pathogen infection.

目錄

圖表目錄…………………………………………………………………………IV
附錄目錄……..……………………………………………………………………V
中文摘要…………….……………………………………………………………VI
英文摘要…………………………………………………………………………VII
正文目錄
第一章、 緒言……………………………………………………………………...…1
第一節、植物與病原的交互作用………………………………………..…..1
第二節、植物的防禦機制……………………………………………………..2
壹、過敏反應(hypersensitive response)……………………………….…2
貳、系統性後發抗病反應(systemic acquired resistance)……………..…5
參、防禦相關蛋白(defense-related proteins)………………………….…6
肆、植物的抗性基因(resistance gene)…………………………….….….7
第三節、細菌性病原的致病機制……………………………………….…..10
壹、Hrp蛋白質分泌系統…………………………………….…….…...10
貳、非毒力基因(avirulence gene)…………………………….………....12
參、致敏因子harpin…………………………………………………..…13
第四節、植物抗病反應的分子研究……………………………….…….….14
壹、抗性基因與非毒力基因產物間的交互作用…………………….…14
貳、抗病反應的訊號傳遞路徑………………………………….…….…15
參、利用突變株研究植物抗病反應的訊號傳遞……………….…….…17
肆、利用基因工程提高植物抗病能力………………………….…….…20
第五節、論文研究目標……………………………………………….……....22
第二章、材料與方法…………………………………………………….………24
第一節、實驗材料………………………………………………….………..24
壹、植物材料…………………………………………………..………..24
貳、菌種材料…………………………………………………..………..24
參、harpinPss的備製……………………………………………..………24
肆、HRAP重組蛋白之純化……………………………………..……...25
伍、農桿菌pS-hrap載體的構築………………………………………..26
陸、轉殖植物的建立…………………………………………….……....26
第二節、實驗方法……………………………………………………..….….27
壹、過敏反應試驗………………………………………………..….…..27
貳、蛋白質分析…………………………………………………………..27
參、植物基因組DNA分析………………………………………..……31
肆、植物總量RNA分析……………………………………………..…32
伍、HRAP重組蛋白毒性測試……………………………………….....35
陸、轉殖植物抗性分析…………………………………….…………....36
柒、水楊酸的萃取與定量……………………..………………….……..36
捌、Trypan blue染色法………………………..………………………...37
玖、HRAP與harpinPss蛋白間的交互作用測試…..……………………37
拾、電子顯微鏡……….……………………………..…………………..39
拾壹、雜項…………………………………………..…………………...39
第三章、結果
第一節、大腸桿菌所表現之HRAP重組蛋白可促進harpinPss引起的過敏反應………………………………………………………………….41
第二節、於大腸桿菌中表現HRAP重組蛋白會延遲細菌本身的增殖…..42
第三節、HRAP在甜椒組織中的分布………………………………………42
第四節、利用農桿菌轉殖系統構築hrap轉殖菸草………………………..43
第五節、hrap轉殖基因在轉殖菸草中的表現………………………………44
第六節、hrap轉殖基因可促進轉殖菸草對harpinPss的敏感度……………45
第七節、非親和性細菌性病原可在hrap轉殖菸草引起較強過敏反應…..46
第八節、hrap轉殖基因可增強轉殖菸草對親和性病原Pseudomonas syringae pv. tabaci的抗性…………………………………..…………..….47
第九節、hrap轉殖基因可增強轉殖菸草對親和性病原Erwinia carotovora subsp. carotovora的抗性………….………..……………………….48
第十節、HRAP重組蛋白對病原菌沒有直接毒害作用……………………..49
第十一節、外加HRAP重組蛋白可抑制病原菌在菸草葉片的繁殖…..….49
第十二節、hrap轉殖基因不會影響水楊酸在轉殖菸草中的含量………….50
第十三節、轉殖胺草對親和性病原也可產生過敏反應……………..……..51
第十四節、HRAP蛋白與harpinPss有直接交互作用………………….……52
第十五節、HRAP蛋白位於植物細胞表面…………………………………54
第四章、討論……………………………………………………………….…….55
第一節、HRAP蛋白特性……………………………………………………55
第二節、hrap轉殖菸草的抗性………………………………………………57
第三節、hrap轉殖菸草的抗性來自於過敏反應的誘發……………………58
第四節、hrap轉殖菸草的抗病機制…………………………………………60
第五章、結論……………………………………………………………………..63
第六章、參考資料………………………………………………………………..65
圖表目錄

圖1……………………………………………………………………………….83
圖2…………………………………………………..…..……………………….84
圖3…………………………………………………..………..………………….85
圖4…………………………………………………..……………..…………….86
圖5…………………………………………………..…………………..……….87
圖6…………………………………………………..………………………..….88
圖7…………………………………………………..……………..…………….89
圖8…………………………………………………..…………..……………….90
圖9…………………………………………………..…………………..……….91
圖10………………………………………………………………………………92
圖11………………………………………………………………………………93
圖12………………………………………………………………………………94
圖13………………………………………………………………………………95
圖14………………………………………………………………………………96
圖15………………………………………………….……………………………97
圖16………………………………………………….……………………………98
圖17……………………………………………………………………………….99
圖18……………………………………………………………………………...100
圖19……………………………………………………………………………...101
圖20……………………………………………………………………………...102
圖21………………………………………………….…………………………..103
圖22……………………………………………………………………………..104
圖23……………………………………………………………………………..105
圖24……………………………………………………………………………...106
圖25………………………………………………….…………………………..107
圖26………………………………………………….…………………………..108
圖27………………………………………………….…………………………..109
圖28……………………………………………………………………………...110
圖29……………………………………………….……………………………..111
表1……………………………………………….………………………………112
附錄目錄
中英文對照表……………………………………………………………….附錄一
縮寫表……………………………………………………………………….附錄二
已發表論文………………………………………………………………….附錄三

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