應用轉位子去除篩選標誌於基因轉殖植物抗東亞蘭嵌紋病毒及齒舌蘭輪點病毒之研究

台灣蘭科植物病害主要以東亞蘭嵌紋病毒 (Cymbidium mosaic virus, CymMV) 及齒舌蘭輪點病毒 (Odontoglossum ringspot virus, ORSV) 感染為主，嚴重影響蘭花生長及降低其觀賞價值，又基因轉殖作物內含篩選標誌基因，易引起大眾對其安全性有所疑慮，本研究利用核糖核酸干擾技術 (RNA interference, RNAi)，將同時含CymMV及ORSV鞘蛋白基因 250 bp 以反義-隱子-順義方式連接之默化載體，構築於轉位子剔除篩選標誌通用載體，轉殖至蝴蝶蘭及嘉德麗雅蘭中，期望獲得抗病且無篩選標誌之轉殖蘭花，並以模式植物邊沁菸草進行默化效力檢測。於邊沁菸草部分已完成轉殖株病毒接種試驗，證實轉殖株具有抗CymMV及ORSV之能力。蝴蝶蘭癒傷組織經轉殖後，以抗生素篩選，部分擬轉殖癒傷組織經GUS活性組織化學染色分析呈藍色反應，聚合酶連鎖反應分析結果合成出預期片段，確定轉殖事件之發生；轉殖癒傷組織經誘導再生為擬原球體， GUS染色分析亦呈藍色反應，目前持續於篩選培養基誘導抽芽中。為克服因蝴蝶蘭癒傷組織轉殖之篩選及再生時間過於冗長，本研究另以蝴蝶蘭葉片為培植體誘導擬原球體產生，約六個月即可完成再生，明顯快於癒傷組織之再生途徑，並已進行初步轉殖。於嘉德麗雅蘭轉殖部分，亦以再生速率較快之葉片為培植體進行轉殖，目前培植體持續於篩選培養基篩選中。

關鍵字

核糖核酸干擾；雙股RNA模式；無篩選標誌；蘭花基因轉殖；蘭花葉片再生

並列摘要

Orchid diseases in Taiwan are caused mainly by infection of Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV). Both viruses affect normal growth of orchids seriously and reduce their marketable value. To achieve virus-resistant transgenic orchid without selectable marker genes, 25 bp DNA fragment based on RNA interference (RNAi) technology for both coat protein genes from CymMV and ORSV was constructed into vector combined with transposon as marker-free system and was transformed into Phalaenopsis and Cattleya. Nicotiana benthamiana was also transformed as a model to test silencing efficiency of RNAi plasmid. After inoculation with viruses, transgenic tobaccos have been proven resistant against CymMV and ORSV. Transformation events had been confirmed by GUS staining for Phalaenopsis survived calli and protocorm-like bodies after antibiotic selection. In order to shorten the time of antibiotic screening and regeneration for Phalaenopsis transformation, regeneration in six months was accomplished using leaf disc as explant. Both Phalaenopsis and Cattleya transformed leaf discs are on selective medium for shoot induction at this moment.