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研究生:顏政宏
研究生(外文):Cheng-Hung Yen
論文名稱:植物葉片老化過程中屬於程式性死亡早期特殊表現基因的初步選殖與分析
論文名稱(外文):Towards Cloning and Characterization of Genes Specifically Expressed in The Initiation of Programmed Cell Death (PCD) During Leaf Senescence in Plants
指導教授:楊 長 賢
指導教授(外文):Chang-Hsien Yang
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:78
中文關鍵詞:自殺細胞DNA斷裂現象葉片老化程式性細胞死亡特殊染色法
外文關鍵詞:Apoptotic cellDNA fragmentationLeaf senescenceProgrammed cll deathTUNEL
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葉片老化是一個複雜的過程其中包含著細胞的大量死亡。這個機制必定是在基因的調控之下,藉由內生性的因子諸如老化因素和植物荷爾蒙之間的平衡性,及受到外在環境訊息的調節。由細胞的層次來看,老化過程總伴隨著葉片組織的脫水,核醣核酸分解酵素和蛋白分解酵素活性的上升,和葉綠體的減少,結果造成葉片產生微黃的顏色。在植物界中,葉片老化被認為是屬於程式性死亡(Programmed Cell Death或 Apoptosis)已有多年了,然而卻沒有任何分子生物學上的研究報告去證實葉片老化確實為程式性死亡。在此藉由使用不同種類的植物葉片,如鳳凰樹,膠蟲樹,羊蹄甲,蔓綠絨,黃金葛等,來觀察老化葉片組織是否有程式性死亡現象的發生。DNA的階梯現象為證實細胞死亡是否為PCD的重要指標,其結果是因Apoptotic cell核內DNA的斷裂會造成。藉由DNA電泳及南方墨漬法偵測到這五種植物老化葉片中皆有DNA階梯之現象發生,但在非老化葉中則無此現象的發生。DNA的斷裂及核的凝縮現象在進一步藉由螢光染色(TUNEL Assay)得到佐證。在老化葉片的葉肉細胞上細胞核有明顯的TUNEL螢光反應,對照之下綠色健康葉片並無TUNEL螢光染色的反應。這個結果提供了直接證據證實在植物發育過程中葉片的老化確實是PCD的現象。利用cDNA subtraction的方法已由羊蹄甲及擬南芥中鑑識出許多可能是表現在PCD初期的cDNA clones。進一步的選殖和鑑定將使我們更深入的瞭解這個機制調控的過程。

Leaf senescence is a complex process which is involved in cell death in a large-scale manner. At the cellular level, senescence is always accompanied by dehydration of the leaf tissue, increased activity of RNases and proteinases, and diminution of the chloroplasts, resulting in the yellowish color of the leaves. In plants, senescence of the leaves has been thought to be a type of programmed cell death (PCD or apoptosis) for many years, however, no studies at the nuclear level associated with PCD have been reported. By using leaf tissue from five different plant species, Philodendron hastatum, Epipremnum aureum, Bauhinia purpurea, Delonix regia, and Butea monosperma, the detection of PCD in senescent leaf tissue was observed. DNA ladders, the hallmarks of apoptosis, resulting from the cleavage of nuclear DNA into oligonucleosomal fragments in apoptotic cells, were detected by gel electrophoresis and southern hybridization only in senescent (but not in non-senescent) leaves in all five plant species. DNA fragmentation and nuclear DNA condensation were further confirmed by using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end in situ labeling (TUNEL) method. Fluorescence was clearly detected in the nuclei of mesophyll cells in senescent leaves. By contrast, there was no TUNEL staining in green healthy leaves. The results provide direct evidence to support the notion that natural senescence of the leaves is indeed an apoptotic process during plant development. A strategy by using cDNA subtraction was used to identify several cDNA clones potentially involved in the initial step of this process from B. purpurea and Arabidopsis. Further cloning and characterization of these genes are in progress and should lead to a deeper understanding of its mechanism.

目錄
壹. 中文摘要---------------------------------------------------------------------4
貳. 英文摘要----------------------------------------------------------------------5
參. 前言----------------------------------------------------------------------------6
肆. 材料與方法-------------------------------------------------------------------11
一. 植物種類-----------------------------------------------------------------11
二. Genomic DNA的純化與分析----------------------------------------11
三. 南方墨漬法 -------------------------------------------------------------12
四. 植物葉片組織的石蠟切片--------------------------------------------13
五. 植物葉片組織切片的雙重螢光染色(TUNEL-assay and DAPI-stain) -------------------------------------------------------------------------------15
六. cDNA差異性篩選實驗中植物葉片種類的選取 -----------------16
七. 植物葉片Total RNA的萃取 -----------------------------------------17
八. mRNA的純化 ----------------------------------------------------------18
九. cDNA差異性表現篩選法(cDNA Subtraction)-----------------19
十. cDNA差異性篩選法所得產物的進一步確認分析 --------------25
十一. 北方墨漬法(Northern Hybridization)-----------------------28
十二. DNA的定序(DNA sequences)------------------------------30
十三. DNA序列的電腦比對 ------------------------------------------33
十四. PCR 增殖DNA片段 -------------------------------------------33
伍. 結果----------------------------------------------------------------------------35
Ⅰ.葉片老化與程式性死亡關係之探討 -------------------------------------35
一. DNA ladders現象隨著老化過程有日趨明顯的結果 -----------35
二.DNA ladders的現象在五種不同種類的老化葉片中皆可被偵測到
------------------------------------------------------------------------------36
三.老化葉片之組織切片檢測出DNA階梯及核縮現象的發生----37
Ⅱ.葉片老化及程式性死亡相關基因之選殖 ------------------------------38
一. cDNA Subtraction Hybridization選殖的結果 --------------------38
二. LSG-clones在葉片中表現的分析(北方墨漬法的結果)-----39
三. LSG-3和LSG-10 DNA序列及電腦比對結果 ------------------39
四. LSG-3和其Homologue之探討 ------------------------------------40
陸. 討論 ---------------------------------------------------------------------------42
柒. 結論 ---------------------------------------------------------------------------45
捌. 引用文獻 ---------------------------------------------------------------------46
玖. 圖 ------------------------------------------------------------------------------53
拾. 表 ------------------------------------------------------------------------------78

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