應用轉位子去除篩選標誌系統於蝴蝶蘭基因轉殖

Abstract

基因轉殖作物中抗生素抗性基因與目標基因同時存在的情況下，可能造成人類對生態和食品安全的疑慮，可選擇非抗生素抗性基因作為篩選標誌，並於轉殖後去除轉殖植株內的篩選標誌來消除疑慮。突變的烯醇丙酮莽草酸磷酸合成酶 (5-enolpyruvylshikimate 3-phosphate synthase; EPSPS) 基因產物對殺草劑嘉磷塞 (Glyphosate) 具抗性，由於嘉磷塞對於環境不具影響且對動物是低毒性的，故本研究以聚合酶連鎖反應 (Polymerase chain reaction)，從轉殖水稻中選殖出突變之 EPSPS 基因，連接 CaMV 35S作為啟動子，構築為轉殖質體。以蝴蝶蘭癒傷組織作為材料進行轉殖，建立以 EPSPS 基因為篩選標誌的蝴蝶蘭轉殖系統。轉殖前測試蝴蝶蘭癒傷組織對嘉磷塞天然耐受性，結果顯示 1 mM 為合適篩選濃度。試驗中以嘉磷塞篩選蝴蝶蘭轉殖細胞，得到存活的細胞在 GUS組織化學活性分下，均具有活性表現，顯示利用嘉磷塞作為蝴蝶蘭癒傷組織的篩選試劑是可行的，且篩選效率較 G418 高約 4 倍。另外，本研究採取轉位子 (transpostion system) 系統去除篩選標誌之策略，轉殖細胞或植株經水楊酸誘導後，將可移除篩選標誌基因。經由聚合酶連鎖反應分析，已確認蝴蝶蘭轉殖細胞及菸草轉殖株。再以 5 mM 水楊酸進行轉位誘導後；觀察到轉位作用的發生。

The presence of antibiotic resistant genes in genetically engineered crops together with the target gene has generated a number of environmental and consumer concerns. use of the non-antibiotic selectable marker, or elimination of marker gene from transgenic plants are suitable strategies to reduce the concerns. Plants with mutated 5-enolpyruvyl shikimate 3-phosphate synthase (EPSPS) gene are resisted to the herbicide glyphosate, which has low toxicity and non-impact on environment and animals. In order to establish a non-antibiotic selection system for Phalaenopsis transformation in this research, the modified EPSPS gene was cloned by polymerase chain reaction (PCR) from transgenic rice, ligated with CaMV 35S promoter, constructed into vector. and transformed into Phalaenopsis calli. The nature tolerance of Phalaenopsis calli against glyphosate is at the concentration of 1 mM. After transformation, the Phalaenopsis calli were selected by glyphosate and the putative transgenic calli got positive results in GUS histochemical analysis. The selection efficiency was four folds higher than G418. It suggested that glyphosate is efficient to be a selective reagent for transformed Phalaenopsis calli. Meanwhile, transposon was used as a strategy to remove selection marker. Transgenesis of Phalaenops cells and tobacco plants were performed by PCR. Transposition was confirmed after transgenic calli treated with 5 mM salicylic acid for elimination of the transposon, via PCR with suitable primers.