2008-04-092024-05-15https://scholars.lib.ntu.edu.tw/handle/123456789/664828摘要：為提升蝴蝶蘭植株抗軟腐病能力，本計劃將蝴蝶蘭軟腐病原之果膠分解&#37238;（pectate lyase）基因PelE-1和PelZ，以農桿菌為媒介轉殖入蝴蝶蘭癒合組織，已經南方氏雜交分析確認轉殖株，並證實轉殖株可表現目標蛋白。另為避免排斥，以及避免篩選標誌基因對轉殖細胞產生副作用等課題，因此選殖非抗生素耐受篩選標誌 EPSP 合成&#37238;基因及標誌基因去除之 Cre/loxP 序列，搭配目標基因，構築成無標誌通用載體，可應用於文心蘭、蝴蝶蘭等蘭科植物。本年度之工作項目包括: (1) 蝴蝶蘭癒傷組織軟腐病抗性基因pelZ轉殖株之西方轉漬分析。(2) 進行軟腐病抗性基因pelE-1及pelZ基因之蝴蝶蘭癒合組織雙重轉殖，經抗生素篩選，並再生成殖株，將進行GUS活性組織化學染色、聚合&#37238;連鎖反應及南方氏雜交分析。(3) 為降低蝴蝶蘭對乙烯之敏感度，於乙烯訊息傳導受體下游之負向調控因子CTR1為對象，進行過量表現之蝴蝶蘭基因轉殖。首先進行轉殖質體之構築，構築完成後將進行蝴蝶蘭癒傷組織之農桿菌法基因轉殖。(4) 進行蝴蝶蘭細胞對於Glyphosate殺草劑之敏感度測試。(5) 將無標誌通用載體轉殖至蝴蝶蘭癒傷組織。<br> Abstract: During cultivation, bacterial soft rot disease always causes Phalaenopsis severe symptom and reduce the profit. Rresistant gene pectate lyase gene pelE-1 and pelZ isolated from Erwinia chrysanthemi were transformed into Phalaenosis via Agrobacterium mediated transformation. PelE transgenic callus lines confirmed by molecular analysis were further transformed with pelZ construct containing hpt and gus genes. Therefore, survival transformed calli after antibiotic selection will be confirmed by β-glucuronidase (GUS) activity, polymerase chain reaction PCR), and Southern blot analysis. pelZ transgenic plants will be analyzed by Western analysis for protein expression. On the other hand, ethylene signal molecule CTR1 gene will be constructed and transformed into Phalaenopsis for reduction of ethylene sensitivity. Furthermore, to develop antibiotic-free selectable markers or the ability to excise selectable marker genes becomes important for plant genenetic transformation. The elimination system of selectable marker via Cre/loxP recombinase system will be established in orchid by this project. The nonantibiotic selection marker EPSP synthase gene was cloned, mutated, and constructed into vector for transformation. This year Phalaenopsis calli will be tested for tolerance to herbicide glyphosate and transformed.蝴蝶蘭果膠分解&#37238Phalaenopsispectate lyase