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研究生:陳音全
研究生(外文):Yin-Chung Chen
論文名稱:以cDNA-AFLP分析受harpin誘導之蕃茄全基因體的表達
論文名稱(外文):Analysis of tomato transcriptome in response to harpin by cDNA-AFLP
指導教授:莊慧文
指導教授(外文):Huey-wen Chuang
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:農業生物技術研究所
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:2004
畢業學年度:95
語文別:中文
中文關鍵詞:逆境生理有關訊息傳遞荷爾蒙有關
外文關鍵詞:harpin
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Harpin是一種天然的細菌蛋白質,可以誘導植物產生hypersensitive response(HR),並活化多種防禦路徑的訊息傳導途徑,以增強植物的抗病性。其中harpin所誘導的system acquired resistance(SAR)是一種有效的植物防禦反應機制,可提高植物對各種的病毒、細菌和真菌的抵抗力。基於此發現,harpin已被商業化生產,用於田間以增強栽培作物之抗病性。cDNA/AFLP是一種分析全基因體基因表達之技術,將反轉錄的cDNA以限制酶切割後,接上具序列專一性的片段,再進行DNA片段增幅,來找出具表達差異的基因。本實驗利用此分析技術比較對照組與harpin處理組蕃茄基因體基因表達之差異,來了解harpin誘導抗病性的分子機制。本實驗共完成分析約8300個cDNA片段,724個在對照組與處理組間有差異的表達,其中147在DNA dot blot 可獲得確認。序列分析這些被確認的DNA片段,這些基因包括光合作用有關,DNA複製、轉錄轉譯和修復,細胞結構,代謝有關,訊息傳遞,逆境生理有關,蛋白質的輸送,離子的輸送,及荷爾蒙有關等。以northern blot確認基因受harpin誘導的程度,結果發現harpin可誘導基因表達參與下列生理反應,如光合作用、細胞壁、生物性逆境,及蛋白質輸送等途徑。本實驗結果證明,harpin可在植物體中誘導廣泛基因的表達,以提高植物對生物性逆境的抵抗力。
目錄......................................................I
圖次.....................................................IV
表次......................................................V
中文摘要.................................................01
英文摘要.................................................03
第一章、前人研究..........................................05
第二章、材料與方法........................................17
第一節、 番茄幼苗之栽植...................................17
第二節、番茄之harpin處理及葉片取材.......................17
第三節、RNA之萃取.........................................17
第四節、蕃茄 PR (Pathogenesis-Related)基因選殖............18
4.1 引子(primer)之設計及製備.............................18
4.2 番茄PR1a(P4)及PR1b(P6)以RT-PCR方式分離..............19
4.3 PCR DNA片段之回收....................................20
4.4 TA cloning RT-PCR產物................................21
4.5 質體DNA之轉形作用(Transformation)...................22
4.6 質體(plasmid)DNA之萃取...............................22
4.7 DNA的定序............................................24
第五節、北方墨點轉漬法(Northern blot analysis) ...........25
5.1 北方墨點轉印法.......................................25
5.2 以random primer labeling方法放射線標定探針...........26
5.3 探針雜合.............................................26
第六節、執行cDNA-AFLP分離多樣性的DNA片段................27
6.1 para-magnetic beads 磁珠配置..........................27
6.2 hybridization of biotinylated polyT primer............27
6.3 cDNA之合成和切割.....................................27
6.4 adaptors的黏合和擴增.................................28
6.5 非選擇性PCR增幅.....................................29
6.6 選擇性PCR增幅.......................................31
6.7 以acrylamide sequencing gel分析PCR片段..............32
6.8 以銀染呈色DNA片段...................................32
6.9 cDNA之擴增...........................................32
第七節、DNA墨點法(DNA dot blot)分析........................33
7.1 本試驗使用BioRad, Bio-Dot製備DNA尼龍膜..............33
7.2 以RT-PCR放射線標定cDNA探針..........................34
7.3 雜合反應.............................................34
第三章、結果..............................................36
第一節、蕃茄PR基因之表現.................................36
第二節、cDNA-AFLP 分析受harpin所誘導的基因................36
第三節、Northern blot確認polymorphic TDFs之基因表達.......39
第四節、受harpin誘導之基因................................47
4.1 光合作用之相關基因...................................47
4.2 逆境生理之相關基因...................................48
4.3 細胞結構.............................................49
4.4 多元胺生合成.........................................49
4.5 分泌性蛋白質輸送.....................................49
4.6 離子的輸送...........................................50
4.7 荷爾蒙有關之基因.....................................50
第四章、討論.............................................52
第五章、參考文獻.........................................60

圖目錄
圖一、Harpin誘導PR基因表現的情況.........................37
圖二、cDNA-AFLP的電泳膠分析..............................38
圖三、DNA dot blot確認polymorphic TDFs之差異表達........40
圖四、以北方轉漬法分析蕃茄受harpin誘導之基因表達.........46

表目錄
表一、蕃茄受harpin調控的基因.............................41
表二、受harpin調控之蕃茄基因的功能分類...................45
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