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研究生:徐偉恩
研究生(外文):Wei-En Hsu
論文名稱:文心蘭切花老化相關基因之篩選
論文名稱(外文):Molecular cloning of senescence-ssociated genes in Oncidium cut flower
指導教授:鄭石通鄭石通引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:植物科學研究所
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:2004
畢業學年度:92
語文別:中文
論文頁數:72
中文關鍵詞:文心蘭老化
外文關鍵詞:senescenceOncidium
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文心蘭為蘭科文心蘭屬(Oncidium)之作物,原產於亞熱帶及熱帶美洲,因種類多且形態豐富,甚受市場歡迎,現已成為台灣重要的外銷切花之一。在夏秋盛產期,文心蘭較快老化,即使經過繁複的保鮮劑處理,瓶插壽命只能多3-4日。爲了延長其切花壽命,期望能選殖出與老化相關之基因,了解其功能,以期對延
長文心蘭切花壽命有所助益。
本實驗中,將文心蘭從全開到老化分為+10 (未老化)、-8、-6、-4四個時期,利用mRNA differential display,protein 2-D page assay,Suppression Subtractive Hybridization三種不同的篩選方法,比較+10與其他各老化時期,選殖出文心蘭因老化所誘導表現的差異性片段。結果共選殖出230個差異性片段(mRNA different display 55個;protein 2-D page assay 2個;Suppression Subtractive hybridization 173個)。而利用文心蘭未老化與老化的RNA對Arabidopsis oligo chip做micro array分析,挑選出14個在文心蘭老化時期中有增加表現量的阿拉伯芥基因,釣取其阿拉伯芥基因片段(計15個)。此外,由台灣大學王淑美實驗室提供一經比對結果為ACC synthase之文心蘭基因片段,一併進行文心蘭cDNA晶片
篩選。
由上述方式,最後選取出246個cDNA片段,經PCR放大後點至玻片上,利用文心蘭未老化與老化的cDNA做探針,對於玻片上的cDNA片段進行偵測,進行cDNA micro array。晶片結果發現,共有48個cDNA片段在文心蘭老化時期表現量為未老化時期表現量兩倍以上。而定序結果發現,在45個序列中,共有30個為已知序列,12個為病毒之序列,另外仍有6個在基因庫中找不到有任何相似之序列,以半定量RT-PCR進行已知序列之篩選,發現有8個基因在文心蘭老化時期中表現量確實有上升的情形,推測其為文心蘭老化相關之基因。而經由阿拉伯芥晶片與文心蘭晶片的雙重篩選後,有6個阿拉伯芥基因在與文心蘭探針雜合下,其基因與文心蘭老化花瓣 cDNA之雜合訊息為未老化花瓣 cDNA之雜合訊息兩倍以上。這些基因參與文心蘭花朵老化過程的位置與機制,仍有待日後進一步分析。
The cut flower of Oncidium Gower Ramsey is one of the most important export flowers in Taiwan. After Oncidium flowers are harvested, the operation of classification and package of flowers always results in the dislodgment of pollinia cap. Consequently, ethylene is produced from flowers within hours, and the senescence of flowers is stimulated. Even the application of preservatives, the vase life of Oncidium cut flowers only increases for another 3-4 days. Therefore, it is important to find senescence-associated genes to increase the vase life of Oncidium cut flowers and delay Oncidium senescence.
In this study, mRNA differential display, suppression subtractive hybridization, and protein 2D analysis were performed to clone senescence-related cDNAs. Also, Arabidopsis oligo chip was also used to isolate up-regulated genes during Oncidium senescence. From above methods, 246 senescence-related cDNAs were further spotted on gene chip. After chip screening, 48 cDNAs were up-regulated during Oncidium senescence. Using NCBI BLAST X, 30 cDNAs were predicted to be known genes, 12 cDNAs were predicted to be Cymbidium mosaic virus, and 6 cDNAs were predicted to be unknown sequences. Eight genes of them were further identified by semi-quantitative RT-PCR, indicating they were up-regulated during Oncidium senescence. Besides, there were 6 Arabidopsis genes were up-regulated through both Arabidopsis oligo chip and Oncidium cDNA chip during Oncidium senescence. Their function in Oncidium senescence will be analysis in the future.
目 錄

中文摘要
英文摘要

第一章、前言
1-1. 背景說明…………………………………………………………1
1-2. 花朵老化之生理、生化及其基因表現…………………………1
1-3. 文心蘭花朵老化與乙烯的關係…………………………………4
1-4. Microarray 技術在植物基因表現研究上的應用…………… 6
1-5. 研究目的…………………………………………………………7

第二章、材料與方法
材料…………………………………………………………………… 8
一、文心蘭花瓣RNA的萃取……………………………………………8
二、文心蘭花瓣蛋白質的萃取……………………………………… 9
三、mRNA Differential Display System ……………………… 10
四、蛋白質二維電泳分析……………………………………………14
五、文心蘭花瓣mRNA的純化…………………………………………16
六、Suppression Subtractive Hybridization …………………16
七、質體構築與挑選…………………………………………………22
八、大量PCR產物之純化…………………………………………… 24
九、文心蘭晶片與探針之製作………………………………………24
十、阿拉伯芥oligo晶片與文心蘭mRNA之雜合反應……………… 24
十一、資料分析………………………………………………………25
十二、半定量RT-PCR ……………………………………………… 25
第三章、結果
一、mRNA Differential Display System ……………………… 27
二、Protein 2-D analysis ……………………………………… 28
三、Suppression Subtractive Hybridization …………………28
四、阿拉伯芥oligo晶片與文心蘭mRNA之雜合反應……………… 29
五、文心蘭cDNA microarray ………………………………………29
六、半定量RT-PCR ………………………………………………… 32

第四章、討論
一、Differential Display system的建立與文心蘭晶片分析… 33
二、Protein 2-D analysis的建立與文心蘭晶片分析……………34
三、Suppression Subtractive Hybridization的建立
與文心蘭晶片分析………………………………………………35
四、1 -aminocyclopropane-1-carboxylate(ACC)synthase …37
五、半定量RT-PCR ………………………………………………… 38
六、阿拉伯芥基因與文心蘭不同時期之
cDNA雜合結果分析………………………………………………… 43

第五章、參考文獻……………………………………………………46

圖表與附圖……………………………………………………………56
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