蘇力菌殺蟲晶體蛋白基因轉移到青花菜、花椰菜及小白菜

Transfer of the Insecticidal Crystal Protein Gene of Bacillus thuringiensis into Broccoli, Cauliflower, and Pak-Choi

本試驗將分離自蘇力菌（Bacillus thuringiensis, Bt）殺蟲晶體蛋白基因cry 1A（b），構築到攜帶有rubisco small subunit （rbc S）及CaMV 35S啟動子的轉殖戰體，並利用農桿菌經幅子葉或下胚軸將其轉移到青花菜（綠王及翠光）、花椰菜（秋王及鳳山極早生）及小白菜（臺農一號）。結果顯示，以農桿菌轉殖供試之三種藝薹屬的五個品種蔬菜之再生率在1~2.5%之間。以南方墨點法分析PCR正反應之轉殖植株，均可偵測到轉移基因存在於轉殖植株的DNA。北方墨點法分析之結果顯示，轉移之二種啟動子的cry 1A（b）基因在轉殖植物體內均與轉綠。且以rcb S啟動子質體的轉殖再生植株，其RNA表現量較轉殖以CaMV 35S啟動子質體的植株高。經cry 1A（b）基因轉移之植株的生物檢定結果顯示，以rbc S啟動子為轉殖質體的植株，殺小菜效果較以CaMV 35S啟動子為轉殖質體的植株高約30~40%。以北方墨點法雜交分析cry 1A（b）基因轉移植株的F2，可偵測出其mRNA，且其植株轉綠之結果符合孟德爾遺傳模式，呈3:1的分離。經cry 1A（b）基因轉移植株的後裔，其植株亦具有殺蟲效果。

This research is to use cruciferous vegetables as a model system to establish the gene transfer technology and to study the possibility for improvement of cruciferous vegetables with insect resistance. We had constructed the cry 1A (b) gene of Bacillus thuringiensis (Bt) with the promoter of CaMV 35S and rubisco small subunit (rbc S) as plant transfer vectors. The constructed plasmids were transferred into hypocotyl and cotyledon of broccoli (Early Value, Green King), cauliflower (Chiu-Wang, Fengshan Extra Early), and Pak-Choi (Tainung No.1) via Agrobacterium mediated transformation.Regenerated plants of three Brassica vegetables were obtained after transformation with two kinds of plasmids. The regeneration rate of transformants was 1% to 2.5%. The regenerated plants were examined by PCR, Southern and Northern blots. The results indicated that the expression of the constructed gene was higher in transgenic plants with rbc S as promoter than CaMV 35S as promoter. Insecticidal effects on Plutella xylostellac was demonstrated in cry 1A (b)-transformed plants. Segregation of the cry 1A (b) gene in progeny analysis followed a 3:1 Mendelian ratio for a single locus insertion in a heterozygous state. The progeny of transformed plants also expressed the insecticidal effect.