文心蘭金魚草素合成酶基因功能及蝴蝶蘭ACC氧化酶基因啟動子活性之分析

Abstract

本論文分為兩部分。第一部分是建立黃花色研究平台，希望了解文心蘭金魚草素 (aureusidin) 合成酶基因功能，故將 2X CaMV 35S 及蝴蝶蘭花器專一表現基因PtACS2啟動子分別接上文心蘭金魚草素合成酶基因，轉殖至菸草，進行穩定性分析。南方氏雜交分析結果顯示，外源基因於轉殖株基因組中為單一拷貝。菸草轉殖株之外觀形態生長勢及花色與未轉殖株並無明顯差異，皆具有深粉紅色及淡粉紅色花。以高效液相層析儀分析花朵中色素的組成及含量變化，已知金魚草素之最大吸收波在405 nm，於10.98分鐘出現，2X 35Spro::OgAS1及PtACS2 pro::OgAS1轉殖株萃取物於405 nm未見明顯波峰，於370 nm則有兩個波峰，推測為Flavone或Flavonols類化合物；而PtACS2 pro::OgAS1之深色花及淺色花之第二個波峰較未轉殖株高1.7倍。 第二部分是分析不同長度的蝴蝶蘭ACC氧化酶基因PtACO1啟動子之活性，以瞭解此啟動子組織專一性或特殊誘導因子之調控區域。啟動子序列比對分析，顯示全長之PtACO1啟動子有多種保守性的反應元件 (response element)， 如G-box、Sp1及BoxΙ等光調控相關、JA、GA3、ABA、乙烯及受熱誘導之熱休克元件 ( heat shock response element) 等序列。進一步將與報導基因GUS連接之不同長度啟動子構築轉殖至菸草，以南方氏雜交分析，確認轉殖片段已嵌入菸草轉殖株基因組中。分析PtACO1啟動子活性，結果顯示PtACO1啟動子於不同株齡轉殖株中均於葉片及根表現，株齡30天時，於老葉表現較幼葉強且根部表現減弱。接著利用生長調節劑處理及非生物性誘導處理探討PtACO1啟動子缺失之活性。啟動子活性分析顯示-2521到-2296間則有影響葉部表現之乾旱轉錄增強子 (enhancer)。-2296到-1676及-1676到-987之間，也各含有一個JA、GA3、BA、ABA、高溫、低溫、黑暗、高鹽相關之轉錄增強子且同時處理低溫及黑暗之結果顯示低溫增強子之作用大於黑暗增強子。-2521到-987間各有兩個淹水逆境相關之轉錄增強子及沉寂子 (silencer)。-2296到-987間含兩個乙烯相關之轉錄增強子及一個沉寂子。

This thesis mainly divides into two parts. The goal of the first part is to establish a research platform for production of yellow flower. Therefore, the 2X CaMV 35S and the flower-specific PtACS2 promoter were fused with aureusidin synthase gene OgAS1 from Oncidium to OgAS1 overexpress in tobacco for functional analysis. After Agrobacterium-mediated transformation, transgenic tobaccos were confirmed by Southern analysis for identification of transgene integrity and copy number. No obvious changes in phenotype of transgenic plants. To analyze content whether aureusidin or other compound was affected by OgAS1, extracts from flowers were by high performance liquid chromatography separated and no aureusidin peak was detected at retention time 10.98 minutes under photodiode array at 405 nm. On the other hand, when detected at 370 nm, peakⅠand peakⅡin extracts from 2X 35S pro::OgAS1 and PtACS2 pro:: OgAS1 transgenic plants were higher than that of untransformed plants about 1.7 times. The second part of this thesis is to analyze the promoter activity of ACC oxidase gene PtACO1 from Phalaenopsis. According to sequence analysis of PtACO1 promoter, putative JA-, GA3-, ABA-, ACC-, and heat-responsive elements were found. Six deletion mutants of PtACO1 promoter constructed in stable transformation vector driving reporter gene β–glucuronidase (GUS), was transformed into tobacco. After confirmed by Southern analysis for transgenesis, transgenic plants containg different promoter deletion mutant were treated with different plant growth regulators and then stained for GUS activity. The region between -2521 to -2296 contained a drought response element and could enhance the promoter activity in leaves. JA, GA3, BA, ABA, heat, cold, cold and dark, dark and high salt related enchancers localized in the region between -2296 to -1676 and -1676 to -987. The effect of the cold enhancer is stronger than that of the dark enhancer according to the results of could treatment in the dark. The region between -2521 to -987 contained four flooding response element and could enhance and silencer the promoter activity. The region between -2296 to -987 contained three ethylene response element and could enhance and silencer the promoter activity.