延緩花瓣老化及抗病毒蘭花基因轉殖之研究

Abstract

嘉德麗雅蘭 (Cattleya spp.) 因對乙烯敏感，以致於花期不長。本研究欲利用生物技術抑制乙烯訊息傳導，以延長嘉德麗雅蘭之花期。以嘉德麗雅蘭葉片為材料，利用農桿菌媒介法，將過量表現香蕉之constitutive triple response 1 (Musa spp. Constitutive Triple Response 1, MhCTR1) 基因之質體轉殖至嘉德麗雅蘭中，測試條件包含預培養、農桿菌液濃度、超音波震盪時間及共培養天數。為驗證策略之可行性，將過量表現MhCTR1質體轉殖至阿拉伯芥。過量表現MhCTR1之T1代阿拉伯芥，與未轉殖株相比，有較大之花朵及較長之花期。過量表現MhCTR1之T3代阿拉伯芥種子，經ACC處理後，與未轉殖株相比並無明顯之乙烯三相反應，顯示此延緩老化之策略可有效抑制乙烯反應。嘉德麗雅蘭葉片經15秒超音波震盪及暗培養2週之處理，為最適預培養條件；以低農桿菌液濃度進行轉殖，有較高之芽體再生率及較低之農桿菌復發率。抗病基因轉殖方面，係利用核糖核酸干擾技術，將同時含蕙蘭嵌紋病毒 (Cymbidium mosaic virus, CymMV) 及齒舌蘭輪點病毒 (Odontoglossum ringspot virus, ORSV) 鞘蛋白基因之默化載體，轉殖至蝴蝶蘭中，期望獲得抗病之蝴蝶蘭。部分經抗生素篩選存活之蝴蝶蘭擬原球體已再生為植株，葉片經GUS活性組織化學染色分析呈藍色反應。擬轉殖株DNA經聚合酶連鎖反應可合成預期片段909 bp，進一步以南方氏雜交分析，確定得到7棵轉殖蝴蝶蘭。

To prolong the longevity of Cattleya by application of biotechnology, inhibition of ethylene signal transduction is used as the strategy. To overexpress negative regulator of ethylene signal transduction MhCTR1 (Musa spp. Constitutive Triple Response 1), driven by CaMV 35S promoter was transformed into Cattleya via Agrobacterium-mediated transformation. Pre-culture condition of explant, concentration of Agrobacterium, time of sonication assistant Agrobacterium-mediated transformation (SAAT), and co-culture days were tested to establish the transformation system of Cattleya. To prove of concept, MhCTR1 was also expressed in Arabidopsis. The T1 transgenic Arabidopsis produced larger flowers with longer flowering period than wild-type. T3 seeds of transgenic Arabidopsis, shown no obvious triple response under ACC treatment, indicated that this strategy inhibit ethylene response. For pre-culture test, the leaves of Cattleya were treated with sonication 15 sec. and cultured in dark for 2 weeks had greater expanded rate. Lower concentration of Agrobacterium used in transformation resulted higher regeneration rate of shoot and lower relapse rate of Agrobacterium. On the other hand, to achieve virus-resistant transgenic Phalaenopsis, the construct express hairpin small RNA of both Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) coat protein genes based on RNA interference (RNAi) technology was transformed into Phalaenopsis previously. After transgenic plantlet regenerated from antibiotic-resistant Phalaenopsis PLBs had been confirmed by GUS staining and polymerase chain reaction in which expected 909 bp fragment was synthesized. Moreover, 7 transgenic Phalaenopsis have been confirmed by Southern hybridization analysis.