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研究生:曾博文
研究生(外文):Bo-Wen Tseng
論文名稱:甘藷傷害誘導基因ipomoelin的表現與性質測定
論文名稱(外文):Expression and Properties of a Wound-inducible Ipomoelin Gene from Sweet Potato
指導教授:鄭石通鄭石通引用關係
指導教授(外文):Shin-Tong Jeng
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物學研究所
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:2000
畢業學年度:88
語文別:中文
論文頁數:93
中文關鍵詞:傷害誘導基因甘藷
外文關鍵詞:wound-inducible genesweet potatoipomoelin
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本實驗主要目的在探討傷害誘導基因ipomoelin在甘藷中訊息傳遞的機轉。根據本實驗室的研究已知ipomoelin基因是能為蟲咬傷害及機械性傷害所誘導表現的基因。本實驗將進一步研究甘藷傷害的訊息傳導過程,主要研究目標有(1)傷害訊息( wounding signal )如:機械性傷害、Chitosan( CHI )及polygalacturonic acid( PGA )處理,(2)植物賀爾蒙及生長調節素( plant hormones and growth regulators )如:ABA、Ethylene、Methyl jasmonate ( MeJA )及salicyli acid( SA )處理,(3)蛋白質磷酸酉每( phosphatase )、蛋白質激酉每( kinase )之分析 (4)二級訊息( Secondary messenger )主要是鈣離子的研究,以LaCl3和GdCl3使細胞膜外的鈣離子無法進入細胞內、LiCl和neomycin會抑制住胞器內尤其是葉綠體內鈣離子的釋放、或是鈣離子的螯合劑 [ EGTA ( ethylene glyco tetraacetic acid ) ]研究之。
用鑷子傷害葉片偵測ipomoelin基因表現時間點,發現在受傷害之葉片( wounding leaf )中表現高峰在6小時,16小時後恢復原狀,系統性葉片( systemic leaf )則在6小時開始表現,於16小時的時候仍然持續表現。將葉片於葉柄處剪下,以chitosan、PGA處理,發現ipomoelin mRNA會在2小時內有大量的表現量 ; 泡在MeJA中6小時後,也會誘導基因表現;但是將葉片泡在ABA、SA及乙烯的處理中,均有抑制ipomoelin基因表現的現象,其中ABA和SA可能妨礙MeJA的合成,因而抑制了ipomoelin基因的表現。用OKA ( 蛋白質磷酸酉每的抑制劑 )及STA ( 蛋白質激酉每的抑制劑 )處理葉片,發現在ipomoelin基因被誘導的過程中,有蛋白質磷酸化及去磷酸化作用之參與。以Ca2+的作用抑制劑處理葉片2和6小時,發現2小時有明顯抑制ipomoelin基因表現而6小時則沒有抑制現象,推測Ca2+的作用主要在傷害訊息傳遞的早期就發生。
綜合以上結果,Ipomoelin基因在葉片的表現是屬於局部性且系統性的,不僅機械性傷害可誘導,並且CHI和PGA也可以促使ipomoelin基因表現。在甘藷傷害訊息的傳導可以分成兩類,首先是鈣離子在受到刺激訊息後會迅速地在2小時內誘導ipomoelin mRNA大量表現,扮演著早期的傷害訊息傳導及警訊的功能,但鈣離子的作用雖然快速卻也短暫,其後會有一受MeJA誘導、比較緩慢的傷害訊息,會在6小時乃至16小時後誘導ipomoelin基因的表現,促使植物體進一步防禦先前所造成的傷害。
The purpose of this research is to study the wounding signal transduction pathway of a wound-inducible gene, ipomoelin, from sweet potato. Previous study from our laboratory indicated that ipomoelin gene was activated by insect herbivory and mechanical damage, but the detailed activation process of ipomoelin upon wounding is still unclear. Therefore, several factors involving in signal transduction were analyzed in this study, including: (1) wounding signals, like mechanical damage, chitoasn ( CHI ), and polygalacturonic acid ( PGA ), (2) plant hormones and growth regulators, such as ABA, ethylene, methyl jasmonate( MeJA ), and salicylic acid ( SA ), (3) protein phosphatases and protein kinases, and (4) secondary messenger, Ca2+. The functions of Ca2+ were studied by plasma membrane Ca2+ channel blockers ( LaCl3 and GdCl3 ), vacuole membrane Ca2+ channel blockers ( LiCl and neomycin ), and the Ca2+ chelator [ EGTA( ethylene glyco tetraacetic acid ) ]。
After mechanical wounding of the plant leaves, the expression of ipomoelin mRNA reaches the maximum production at 6 hr followed by a continuous decline. The expression of ipomoelin mRNA in the systemic leaves began at 6 hr, and continuously expressed up to 16 hr after wounding. Leaf-petiole cuttings treated with MeJA, chtiosan, or PGA enhanced the expression of ipomoelin mRNA, however, those with ABA、SA and ethylene inhibited its expression. ABA and SA may block the biosynthesis of MeJ, and decrease the ipomoelin gene expression. Protein phosphorylation/dephosphorylation was also involved in the signal transduction of ipomoelin gene expression by the study with STA (a protein phosphatase inhibitor) and OKA (a protein kinase inhibitor). Furthermore, Ca2+ inhibitors obviously repressed the expression of ipomoelin gene at 2 hr after wounding, but not at 6 hr.
Accordingly, ipomoelin gene expression was both local and systemic in leaves. There are two signal pathways for inducing ipomoelin gene. The fast one was conducted by Ca2+ at about 2 hr after wounding, and the slow one was done through MeJA and the dephosphorylated proteins. Ca2+ plays a role of wounding signal transduction, and works quickly and transiently at the early stage. The slow signal induced by MeJA prolongs plants defending the stress of wounding.
中文摘要--------------------------------------------------------------------1
英文摘要--------------------------------------------------------------------3
第一章 前言----------------------------------------------------------------5
第二章 材料與方法-------------------------------------------------------17
Ⅰ實驗材料------------------------------------------------------------17
Ⅱ實驗方法------------------------------------------------------------17
第一節 甘藷傷害誘導基因於不同時間在不同組織之表現--17
一 甘藷總RNA的抽取---------------------------------------------17
二 CsCl梯度離心法大量純化製備質體DNA------------------20
三 北方墨點法偵測--------------------------------------------------22
第二節 訊息傳導抑制劑與誘導劑之處理-----------------------26
一 脫乙醯殼多糖及多半乳糖醛酸之處理-----------------------26
二 蛋白質磷酸酉每、激酉每的抑制劑及茉莉酸、MeJA合成
抑制劑DIECA之處理------------------------------------------27
三 訊息傳導抑制劑之處理------------------------------------------28
四 鈣離子( Ca2+ )作用抑制劑的處理------------------------------29
第三節 ipomoelin基因表現的普遍性之分析 -------------------30
第三章 結果------------------------------------------------------------------31
第一節ipomoelin mRNA被誘導表現的時間點-------------------31
第二節 訊息傳導相關之誘導劑及抑制劑的處理----------------32
一 脫乙醯殼多糖及多半乳糖醛酸之處理-------------------------32
二 蛋白質磷酸酉每、激酉每的抑制劑及茉莉酸之處理-------------33
三 茉莉酸( MeJA )與蛋白質磷酸化的作用順序-----------------34
四 茉莉酸類合成抑制劑DIECA之處理--------------------------34
五 訊息傳導抑制劑SA及ABA之處理---------------------------35
六 乙烯( ethylene )及乙烯合成抑制劑( AVG )藥劑之處理-----35
第三節ipomoelin 基因誘導表現與鈣離子相關抑制劑之關係-36
第四節 ipomoelin基因表現的普遍性之分析----------------------37
第四章 討論-------------------------------------------------------------------39
第一節ipomoelin基因之表現時間點--------------------------------39
第二節脫乙醯殼多糖及多半乳糖醛酸之處理----------------------41
第三節茉莉酸類、MeJA合成抑制劑DIECA 及蛋白質磷酸酉每
抑制劑、激酉每抑制劑之分析---------------------------------41
第四節植物荷爾蒙與生長調節素之處理分析----------------------44
第五節ipomoelin基因與鈣離子作用的分析------------------------45
第六節ipomoelin基因表現普遍性之分析---------------------------46
第七節 ipomoelin基因訊息傳導之模式------------------------------47
參考文獻-------------------------------------------------------------------------49
圖表-------------------------------------------------------------------------------66
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