2012-03-012024-05-15https://scholars.lib.ntu.edu.tw/handle/123456789/664062摘要：使用非抗生素抗性基因作為篩選標誌，或於轉殖後去除轉殖植株內的篩選標誌，來減少抗生素篩選基因使用之疑慮，本研究利用非抗生素抗性基因EPSP合成&#37238;突變基因為篩選標誌，配合Transposon系統移除篩選標誌基因，目標基因為蕙蘭嵌紋病毒(Cymbidium mosaic virus, CymMV)、齒舌蘭輪斑病毒 (Odontoglossum ringspot virus, ORSV)之RNA干擾構築，轉殖至蝴蝶蘭癒傷組織，期望育成具雙重病毒抗性且無篩選標誌之轉殖蝴蝶蘭。今年度之工作項目包括：1.完成轉位子系統於蝴蝶蘭可剔除篩選標誌之驗證。2. 進行雙抗病毒之RNA干擾構築於剔除篩選標誌通用載體之菸草轉殖和分子檢測。3. 進行雙抗病毒之RNA干擾於剔除篩選標誌通用載體構築之蝴蝶蘭基因轉殖。<br> Abstract: Use of the non-antibiotic selectable markers, or elimination of marker gene from transgenic plants are suitable strategies to reduce the concerns. In order to establish a non-antibiotic selection system for Phalaenopsis transformation, the modified 5-enolpyruvyl shikimate 3- phosphate synthase (EPSPS) gene was constructed into vector combined with transposon as marker-free system and transformed into Phalaenopsis calli via Agrobacterium -mediated transformation. The target gene is RNA interference construct for both Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) coat protein gene. Transformed calli will be analyzed by polymerase chain reaction and Southern analysis to verify transposition in Phalaenopsis. Plasmid containing RNA interference construct for double virus resistance against CymMV and ORSV will be transformed to tobacco, and then the transformed plants will be analyzed by GUS staining and polymerase chain reaction to confirmed transgenic events. Furthermore, plasmid containing RNA interference construct for double virus resistance coupled with markerfree system will be transformed into Phalaenopsis calli.剔除篩選標誌系統雙重抗病蘭科marker-free systemdouble resistanceOrchidaceae