蝴蝶兰三唇瓣突变种表现基因之分析.docVIP

蝴蝶蘭三唇瓣突變種表現基因之分析 Gene Analysis of Peloric Mutants of Phalaenopsis 研 究 生：陳雅惠 Chen,Ya-Huei 指導教授：陳福旗 Chen,Fure-Chyi 【摘要】 組織培養技術已廣泛應用於蝴蝶蘭繁殖，但是在繁殖過程往往會產生體細胞變異，造成組培苗品質的不穩定。本研究採用蝴蝶蘭正常及三唇瓣突變種花苞作為供試材料，利用cDNA-RAPD及cDNA扣除雜交法，試圖篩選與蝴蝶蘭三唇瓣花朵發育之相關基因。結果顯示在總數209個正常花苞特異性的ESTs及總數230個突變花苞特異性的ESTs中包含了許多不同功能範圍的基因，其中最為顯著是功能未知蛋白質及蘭花病毒基因、再來是代謝相關基因、訊號傳遞相關基因、轉錄相關基因、細胞生長與分裂、蛋白質合成及標的基因等，顯示了蝴蝶蘭三唇瓣的形成的影響層面不只有單一方面。以即時PCR定量分析顯示bZIP轉錄因子的表現量在三唇瓣花苞中降低、APK1 protein kinase、cyclophilin及TCP基因在突變花苞中的表現量反而提高，可能代表了訊號傳遞及轉錄作用在正常蝴蝶蘭與三唇瓣突變種間的差異，以及形成三唇瓣花朵的影響。而逆轉因子retroelement在突變種花苞的表現量也高達7倍。另外， DNA methyltransferase、histone acetyltransferase、ERECTA-like、DEAD/DEAH RNA helicase等基因在突變花苞的表現降低亦代表了DNA甲基化、染色質重塑及轉錄後基因調控機制參與三唇瓣花朵形成之基因調控的可能。 關鍵字： 【Abstract】 Tissue culture has been widely used for mass propagation of Phalaenopsis, however, the occurrence of somaclonal variation during micropropagation process is a severe problem affecting product quality. Normal and peloric flower buds of Phalaenopsis were used for cDNA-RAPD and cDNA subtraction analysis to study their differences in terms of expressed genes. A total of 209 ESTs from normal flower buds and 230 from mutants were sequenced and found to belong to several gene categories with different functions, including the most unknown proteins, orchid virus and related genes on metabolism, signal transduction, transcription, cell growth and division, protein synthesis, and protein destination. Several selected cDNA clones were analyzed for their preferential expression using real-time PCR. The result showed that bZIP transcription factor is down-regulated in mutant flower, while APK1 protein kinase, cyclophilin, and TCP-like genes are up-regulated, which probably present the effects from the changing of signal transduction and transcription. A retroelement clone was preferentially expressed in mutants. Some other ESTs involving DNA methylation, chromatin remodeling and post-transcriptional regulation were selectively expressed in normal flower buds, such as DNA methylt