Trypsin Inhibitor 轉移於花椰菜之研究

Abstract

本實驗主要目的在台灣栽培種花椰菜基因轉移系統的建立，並將選殖 自台灣栽培種甘薯的Trypsin Inhibitors基因轉入花椰菜中，觀察轉移植 物是否具有抗蟲的效應。轉移的方法利用農桿菌媒介轉移法，研究液體共 培養時菌的濃度、溫度、固體共培養時間及感染後轉移植物篩選的時機是 否對農桿菌轉移效率有所影響。首先在建立花椰菜組織培養再生系統的過 程中瞭解：花椰菜下胚軸的分化能力較強，若將下胚軸分化以2.4-D 前處 理3 天，並置於含銀離子29.4mM的分化培養基中，下胚軸分化成不定芽的 能力均可達到95% 以上，其中以雪姬的分化能力較強。在以農桿菌媒介轉 移法建立花椰菜基因轉移系統的過程中發現：感染時液體共培養的溫度 在20至25℃之間有較好的轉移率。感染時菌的濃度與固體共培養時間以菌 液稀釋100 倍及共培養72小時的轉移效率最高。感染後轉移植物的篩選時 機以感染後培殖體置於分化培養基後一週開始加入15mg/l的G418篩選， 第3 週加入30mg/l的G418，直至抗G418的再生苗出現為止。在分生檢測方 面先以TI assay來測定抗G418的再生苗TI表現的程度。選取表現量較高的 再生苗，以PCR 及南方氏核酸雜交分析，證明TI基因已移入再生苗中，再 以免疫轉印分析亦證明轉移植物具有TI蛋白的表現。最後進行抗蟲實驗的 結果證明轉移TI的花椰菜對咀嚼式害蟲中之白粉蝶、小菜蛾及斜紋夜盜幼 蟲有不錯的表現。

The aim of this research project is to establish a gene transfer system for the Taiwan cauliflower cultivars. Agrobacterium mediated transformation was used to introduce a trypsin inhibitor (TI) gene into cauliflower hypocotyl explants. This target gene was isolated from a strain of Taiwan cultivated sweet potato. Our regeneration studies indicated that, among various tested explant types, the 3-day-old seedling hypocotyl segments had the highest regeneration capacity. Five-mm long hypocotyl explant were first pretreatedwith 1 mg/l 2,4-D for three days. They were then transferred onto a silver ion (29.4mM) containing shoot formation medium. More than 95% of the explantsregenerated adventitious shoots using this protocol. The following optimumtransformation conditions were determined. The 2,4-D pretreated explants wereinoculated with an 1:100 dilution of an 18-h Agrobacterium culture harboring TI and NPTII gene containing plasmid. Co-cultivation was carried out at 20-25 ℃ for 72hs. G418 selection was initiated one-week after co-cultivation at a concentration of 15 mg/l. G418 concentration was doubled three-weeks afterward.Confirmation of the transgenic ststus of the resultant G418 resistant R0 plantswas carried out with Southern and Western hybridization, PCR analysis assay. From 18 tested plants, 14 expressed positive reactions to all the above four molecular assays. All the transgenic plants demonstrated resistance to the chewing insects Plulella xyloshella and Spodophera litura to which the nontransformed control plants were vulnerable.