本試驗將分離自三角酵母的 D-胺基酸氧化酵素基因 (DAO)，構築到 攜帶有 Cauliflower Mosaic Virus 35S (CaMV 35S) 及 rubisco small subunit (rbcS) 啟動子的轉殖載體，並同時以蘇力菌殺蟲晶體蛋白基因 (Bt)，比目魚抗凍蛋白基因 (AF)為材料，利用農桿菌將其轉移到甘藍 (新豐、初秋)、結球白菜 (濱綠、新 38 號)的子葉或下胚軸。本實驗之 目的在建立甘藍及結球白菜基因轉移及植株再生系統，研究不同啟動子對 表現此三種基因的影響，並探討培育成抗菌、抗蟲及抗凍之蔬菜的可行性 。 轉移的三種基因，皆有轉殖植株再生，結果顯示，以農桿菌轉殖兩 個品種的甘藍再生率在 1.4% ~ 4.8% 之間，轉殖兩個品種結球白菜的再 生率在 0.24 ~ 0.7% 之間。再生植株經PCR反應作初步篩選後，以南方墨 點雜交法分析檢驗，可在轉殖植株的DNA上偵測到雜交訊號。北方墨點雜 交分析的結果顯示，存在轉殖植株內的D-胺基酸氧化酵素基因，蘇力菌殺 蟲晶體蛋白基因及抗凍蛋白基因皆可轉錄出RNA。轉殖攜帶 rbcS 啟動子 質體的轉殖後再生植株，其 RNA 表現量與轉殖攜帶 CaMV 35S 啟動子質 體的植株，均相似。經 Bt 基因轉移之植株的生物檢定結果顯示，以 rbcS 啟動子為轉殖質體的植株，殺小菜蛾效果較以 CaMV 35S 啟動子為 轉殖質體的植株高。初步分析經AF基因轉移之再生植株的抗凍性，顯示轉 殖植株較對照組可耐較低的溫度。 This research focuses on the use of cruciferous vegetables as a model system to establish the gene transfer technology, and to study the possibility for improvement of cruciferous vegetable with bacteria, insect and frost resistance, through the art of genetic engineering. The results are summarized as following: We had constructed the D-amino acid oxidase gene (DAO) isolated from Trigonopsis variabilis with the promoter of CaMV 35S, rubisco small subunit (rbcS). The constructed plasmids included δ-toxin gene of Bacillus thuringiensis (Bt) and antifreezing protein gene (AF) were transfer into hypocotyl and cotyledon of cabbage (New top, K-Y cross) and Chinese cabbage (Tropical pride, New No. 38) via Agrobacterium mediated transformation. Regenerated plants of two Brassica vegetables were obtained after transformation with six kinds of plasmids. The regeneration rate of transformation in Cabbage was 1.4% to 4.8% , in Chinese cabbage was 0.24% to 0.7%. The transformed plants were examined by PCR, southern and northern blot hybridization. The results indicated that the expression of constructed genes was little higher in transgenic plants transferred with rbcS as promoter than CaMV 35S promoter and did not obvious. High biological activity in killing Plutella xylostellac was show in Bt-transformed plants.