2013-01-012024-05-15https://scholars.lib.ntu.edu.tw/handle/123456789/664701摘要：為了減少使用抗生素篩選基因之疑慮，本研究利用非抗生素抗性基因EPSP合成&#37238;突變基因作為篩選標誌，並配合轉位子 (transposon) 系統之使用，以去除轉殖植株內的篩選標誌基因，加上蕙蘭嵌紋病毒 (Cymbidium mosaic virus, CymMV) 及齒舌蘭輪斑病毒 (Odontoglossum ringspot virus, ORSV) 外鞘蛋白基因之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。含剔除篩選標誌系統之蝴蝶蘭轉殖細胞系再生成植株後，經以 5 mM 水楊酸誘導轉位&#37238;基因表達，促使轉位作用發生，已經由聚合&#37238;連鎖反應及南方氏雜交分析，證實蝴蝶蘭轉殖株內篩選標誌已被剔除。另已取得轉殖成功之兼具雙抗病毒及剔除篩選標誌系統之菸草轉殖株。今年度之工作項目包括：1.雙抗病毒RNA干擾構築之菸草轉殖株的抗病分析。2. 雙抗病毒RNA干擾構築於剔除篩選標誌通用載體之蝴蝶蘭轉殖系的分子驗證。<br> Abstract: In order to establish a non-antibiotic selection system for Phalaenopsis transformation to reduce the biosafety concerns, the modified 5-enolpyruvyl shikimate 3-phosphate synthase (EPSPS) gene was constructed into vector combined with transposon as marker-free system and RNA interference construct for both Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) coat protein gene. The vectors were transformed into Phalaenopsis calli via Agrobacterium-mediated transformation to produce double virus resistance and marker-free transgenic Phalaenopsis. Transformed calli with marker-free system have been confirmed by polymerase chain reaction and Southern analysis to verify transposition in Phalaenopsis. Transgenic tobaccos with RNA interference construct for double virus resistance against CymMV and ORSV coupled with marker-free system have been identified by GUS staining, polymerase chain reaction, and Southern analysis. The degree of virus resistance in transgenic tobaccos will be analyzed by inoculation test this year. Furthermore, Phalaenopsis transgenic lines containing RNA interference construct for double virus resistance against CymMV and ORSV coupled with marker-free system will also be confirmed in molecular level this year.剔除篩選標誌系統雙重抗病蘭科marker-free systemdouble resistanceOrchidaceae