2009-02-272024-05-17https://scholars.lib.ntu.edu.tw/handle/123456789/684277摘要：細菌性軟腐病為蝴蝶蘭常見且嚴重之病害之一，為了培育具抗病能力之蝴蝶蘭品種，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，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。本年度將進行PelE-1/PelZ 基因蝴蝶蘭癒傷組織雙重轉殖，轉殖之擬原球體及細胞系將經抗生素hygromycin 篩選後，進行再生、GUS 活性組織化學染色、聚合&#37238;連鎖反應及南方氏雜交分析，以驗證外源基因確實整合入蝴蝶蘭染色體中。再者，蝴蝶蘭對乙烯相當敏感，故選殖乙烯訊息傳導路徑相關基因CTR1，進行蝴蝶蘭基因轉殖，期望降低蝴蝶蘭對乙烯的敏感度，以維持貯運品質。本年度將以抗生素 G418 持續篩選轉殖過量表現抗老化基因 CTR1 之蝴蝶蘭癒傷組織，再生後進行分子驗證，包括 GUS 活性組織化學染色分析、聚合&#37238;連鎖反應及南方氏雜交分析，以確認轉殖植株。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。本年度之工作內容包括無篩選標誌通用載體之構築，採用之去除篩選標誌基因的策略包括Cre/loxP 及轉位子 (transposon) 兩種，以點突變之EPSP 合成&#37238;基因或 cDNA 作為篩選標誌，使用誘導性啟動子驅動重組&#37238;或轉位&#37238;基因。接著將上述通用載體以農桿菌法轉殖至蝴蝶蘭癒傷組織，以殺草劑嘉磷塞進行篩選，待得到轉殖細胞系後，誘發重組或轉位以去除篩 選標誌基因。<br> Abstract: Bacterial soft rot disease always causes Phalaenopsis severe symptom during cultivation and reduces the profit. Pectate lyase gene pelE-1 and pelZ isolated from Erwinia chrysanthemi were transformed into Phalaenopsis via Agrobacterium-mediated transformation. PelE transgenic callus lines confirmed by molecular analysis were further transformed with pelZ construct containing hpt and gus genes. On the other hand, ethylene signal molecule CTR1 gene had been introduced into Phalaenopsis for reduction of ethylene sensitivity. All transformed calli after antibiotic selection will be confirmed by β-glucuronidase (GUS) activity, polymerase chain reaction PCR), and Southern blot analysis. Furthermore, to develop antibiotic-free selectable markers or the ability to excise selectable marker genes becomes important for plant genetic transformation. The nonantibiotic selection marker EPSP synthase gene was cloned, mutated, and constructed into vector for transformation. The elimination system of selectable marker via Cre/loxP recombinase system or transposition will be established in orchid by this project this year. Transformed Phalaenopsis calli will be selected by herbicide glyphosate.蝴蝶蘭果膠分解&#37238無篩選標誌系統Phalaenopsispectate lyasemarker-free system