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研究生:王俊淇
研究生(外文):CHUN-CHI WANG
論文名稱:二次過氧化氫前處理誘導綠豆植株獲致比擬於冷馴化之低溫耐受性
論文名稱(外文):Double hydrogen peroxide treatment induces a chilling tolerance comparable to cold acclimation in mung bean
指導教授:游志文
指導教授(外文):CHIH-WEN YU
學位類別:碩士
校院名稱:大葉大學
系所名稱:分子生物科技學系碩士班
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:44
中文關鍵詞:過氧化氫麩胱甘肽低溫耐受性鈣離子冷馴化
外文關鍵詞:hydrogen peroxide (H2O2)glutathionecold acclimationcalciumchilling tolerance
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以綠豆植株 (Vigna radiata L. cv. TN5) 處理200 mM過氧化氫再經4 oC,36小時的低溫測試以評估植株之低溫耐受性。發現重複處理過氧化氫得到較1次處理過氧化氫佳之低溫抗性,且以間隔3小時重複處理過氧化氫所得到的電解滲漏率最低,相近於經10oC, 48小時冷馴化處理的植株。以2次過氧化氫前處理隨即誘導麩胱甘肽累積,且累積量高於控制組、1次過氧化氫,甚至是冷馴化處理的植株。經過氧化氫處理所誘導綠豆植株之麩胱甘肽累積量不受光照所影響;然而經光照之冷馴化處理植株則明顯降低麩胱甘肽含量。於過氧化氫處理前,以鈣離子螫合劑EGTA或glutathione (GSH) 生合成抑制劑BSO處理綠豆植株,均導致電解滲漏率提高,逆轉其抗寒能力,但無顯著影響麩胱甘肽累積。以細胞內鈣離子抑制劑ruthenium red處理對電解滲漏率麩胱甘肽累積並無顯著影響。經實驗結果暗示,過氧化氫處理綠豆植株所生成的訊號回應中,細胞外鈣離子與麩胱甘肽於其中扮演著重要角色。以冷馴化處理所誘導植株低溫抗寒訊息傳遞中,可能經光依賴(light-dependent)及光不依賴(light-independent)之訊息傳遞調節。
Mung bean seedlings (Vigna radiata L. cv. TN5, a chilling-sensitive cultivar) pretreated twice with 200 mM hydrogen peroxide (H2O2) by a 3-h interval apart followed by chilled at 4oC for 36 h showed a similar electrolyte leakage levels to those of seedlings cold-acclimated at 10oC for 48 h. Double H2O2 treated seedlings showed higher glutathione levels than those of control, single-treated, and even cold-acclimated plants. This H2O2 induced glutathione accumulation in seedlings did not inference by illumination; however, the glutathione levels of cold-acclimated plants diminished significantly. Seedlings treated with buthionine sulfoximine (BSO), a specific inhibitor of glutathione synthesis, prior to H2O2 application reversed the H2O2-induced tolerances and thus showed a higher electrolyte leakage. Combination of distinct treatments with ethylene glycol-bis (β-aminothyl ether)-N,N,Nˊ,Nˊ-tetraacetic acid (EGTA), a specific calcium chelator, although had no significantly influence on the glutathione accumulation. However, the tolerances of cold acclimated and H2O2 treated seedlings do decrease by EGTA. Notably, ruthenium red, inhibitor of Ca2+ flux from intracellular stores, had no obvious influence on both glutathione and electrolyte leakage levels. These observations indicated that extracellular calcium and glutathione accumulation play important roles in the response elicited by H2O2 pretreatment in mung bean seedlings. Also, cold acclimation induced chilling tolerance response could separately mediate via both light-dependent and light-independent pathways.
目錄
封面內頁
簽名頁
授權書1................................................................................................ iii
授權書2.................................................................................................iv
中文摘要................................................................................................v
英文摘要...............................................................................................vi
誌謝...................................................................................................... vii
目錄.....................................................................................................viii
圖目錄...................................................................................................xi
表目錄..................................................................................................xii
附錄圖目錄.........................................................................................xiii

第一章 前言......................................................................................1
第一節 環境逆境與游離基的關係......................................1
第二節 抗氧化防禦系統(antioxidant defense
system)與低溫防禦之關係........................1
第三節 過氧化氫與逆境防禦的關係..................................3
第四節 過氧化氫、鈣離子與低溫防禦之關係................... 4
第二章 材料與方法
第一節 實驗藥品..................................................................6
一、化學藥品及試劑...................................................... 6
第二節 植物材料與生長條件..............................................6
第三節 植物處理方法..........................................................7
第四節 電解質滲漏率測試(electrolyte leakage).................8
第五節 蛋白質測定..............................................................8
第六節 綠豆麩胱甘肽總量分析(total glutathione
assay, TG).................................................................8
第三章 結果
第一節 重複過氧化氫前處理使植物提高低溫耐
受性........................................................................10
第二節 重複過氧化氫前處理使植物累積高量麩
胱甘肽....................................................................12
第三節 以麩胱甘肽生合成抑制劑抑制麩胱甘肽
累積使綠豆植株降低低溫耐受性.........................13
第四節 以EGTA前處理綠豆植株使綠豆植株提
高電解滲漏率但經 ruthenium red 前處理
的植株影響則較小.................................................14
第五節 外生性鈣離子(exogenous calcium)亦誘發
植物生成低溫抗性................................................15
第四章 結論
第一節 重複過氧化氫前處理誘導綠豆植株獲致
同等於冷馴化之低溫耐受性................................17
第二節 重複過氧化氫前處理誘使麩胱甘肽累積,
進而顯著提升植物低溫耐受性............................18
第三節 重複過氧化氫前處理以鈣離子做為二次
訊號傳遞者(second messenger)誘使綠
豆植株生成低溫耐受性........................................20
第四節 總結........................................................................21
參考文獻..............................................................................................23
圖表......................................................................................................31
附錄......................................................................................................42






















圖目錄

圖1. 多重過氧化氫前處理對於綠豆植株低溫耐受性的影響
..................................................................................................31
(A) 2次過氧化氫前處理的時間間隔對於綠豆葉面之
電解滲率的影響................................................................31
(B) 多重過氧化氫前處理所誘導的低溫耐受性.....................32
(C) 過氧化氫誘導綠豆植株低溫耐受性之去冷馴化
(deacclimation)分析.........................................................33
圖2. 綠豆植株於4oC 光照 (圖 2A) 或黑暗下(圖 2B)麩胱
甘肽之含量變化.........................................................................34
圖3. BSO前處理對於綠豆葉子其麩胱甘肽含量 (圖3A and
3C) 及電解滲漏率(圖3B and 3D) 的影響..............................35
圖4. EGTA(圖4C及圖4D)及ruthenium red(圖4A及圖
4B)對綠豆植株電解滲漏率與麩胱甘肽含量之影響..............36
圖5. 氯化鈣前處理對電解滲漏率(圖5A)與麩胱甘肽的累
積量(圖5B)之影響...................................................................37
圖6. 以外生性鈣離子前處理可能誘發的訊號傳遞路徑.................38
圖7. 由2次過氧化氫前處理可能誘發的訊號傳遞路徑..................39
圖8. 外生性過氧化氫與冷馴化處理所引發可能的訊號傳遞
路徑............................................................................................40


表目錄

表1. 比較各前處理所生成的逆境記憶、4 oC低溫耐受
時間、麩胱甘肽累積量及是否依賴鈣離子的訊息
傳遞路徑....................................................................................41




附錄圖目錄

附錄1. 由過氧化氫所誘導的可能訊號傳遞路徑.............................41
附錄2. 將含有轉殖Aequorin之菸草轉殖株以2次過氧化
氫處理後分析其所造成的冷光強度.....................................42
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