乙烯屬於植物賀爾蒙的一種，它調控的範圍包括植物從發芽到老化的所有生理過程，也產生於許多植物對於外在環境逆境的反應中。
本實驗係探討嘉德麗雅蘭 (Cattleya bicolor) 中參與乙烯合成途徑之ACC氧化酶 (ACC oxidase) 。利用RACE (Rapid amplification of cDNA ends) 技術放大目標基因終端片段。蘭花ACC oxidase選殖序列之分析發現，在3’端非轉錄區域 (3’-untranslated region, 3’-UTR) 之讀序結果有些微不同，推測是因ACC oxidase各基因家族成員序列近似，惟其常於3’-UTR有變異之緣故。利用軟體將cDNA序列之開放讀架 (open reading frame, ORF) 轉譯成胺基酸並經比對後，發現此胺基酸序列與其他物種所具有之序列相似；經基因組DNA之比較顯示C. bicolor的ACC oxidase基因組結構在內含子 (intron) 與外顯子 (exon) 數目上與其他物種並不相同。另外，藉由分析胺基酸序列，預測蛋白質之表現位置應是在細胞質中。
本研究進行至此，已完成嘉德麗雅蘭花 (C. bicolor) 中ACC oxidase基因選殖，並對之進行初步探討。期望將來能利用分子生物學方法與植物轉殖技術，從植物之遺傳物質著手以抑制乙烯生成，最終使花朵壽命延長，進而增加花卉於市場之競爭力。

Ethylene, a plant hormone, plays an essential role in many aspects about plant development, growth, ripening, and senescence. In addition, it also regulates several responses when plants suffer stress from drought, flood, herbivore bites, wound, etc. ACC synthase and ACC oxidase belong to two multigene families. In this study, PCR (polymerase chain reaction) and RACE (rapid amplification of cDNA ends) methods were used to amplify the ACC oxidase sequences in Cattleya bicolor orchid flower. The results show that there exists differences in the 3’-UTR (untranslated region) of orchid gene sequences. Compare the ACC oxidase sequences, including the cDNA ORF (open reading frame) sequences and the amino acid sequences, of several different species, the sequence similarity among the three Laeliinae orchids, namely C.bicolor, C. intermedia, and Laelia anceps, is the highest. The similarity of cDNA ORF sequences and amino acid sequences between orchids and the other plants, such as rice, apple and torenia, is comparatively lower. It was proposed that the protein located in cytoplasma (or in mitochondrial matrix space), agrees with the result from analysis of amino acid hydrophilicity prediction.
The ultimate goal of this study is to postpone the flower senescence by the way of plant transfection. In the present findings, it only deals with the cloning and analysis of the ACC oxidase genes in C. bicolor.

致謝…………………………………………………………………… …I
中文摘要……………………………………………………………… ..II
英文摘要………………………………………………………………..III
目錄……………………………………………………………………..IV
圖表目錄………………………………………………………………..VI
前言………………………………………………………………………1
一、乙烯………………………………………………………………………………1
二、ACC氧化酶特性與基因的研究………………………………………………..9
三、研究目的………………………………………………………………………..13
材料與方法……………………………………………………………..15
一、研究材料…………………………………………………………………………15
（一）嘉德麗雅蘭………………………………………………………………………………………15
（二）大腸桿菌 (Escherichia coli) ……………………………………………………………………15
二、研究方法…………………………………………………………………………17
（一）總量RNA萃取與cDNA合成…………………………………………………………………...17
（二）基因組DNA萃取……………………………………………………………………………….18
（三）聚合酶連鎖反應（polymerase chain reaction, PCR）………………………………………….19.
（四）瓊脂膠體電泳與回收…………………………………………………………………………...22
（五）細菌值體之利用…………………………………………………………………………………26
（六）勝任細胞製備與大腸桿菌轉形作用……………………………………………………………28
（七）質體抽取…………………………………………………………………………………………29
（八）接合酶反應……………………………………………………………………………………....30
結果與討論……………………………………………………………..31
一、ACC氧化酶cDNA序列之分析…………………….……………………….…31
（一）定序結果…………………………………………………………………………………………31
（二）相似度比對………………………………………………………………………………………32
（三）胺基酸序列預測結果……………………………………………………………………………39
二、ACC氧化酶基因組DNA序列之分析…………………………………………..45
結論……………………………………………………………………..49
參考文獻………………………………………………………………..53

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