Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/29354
標題: 以農桿菌法共同轉移蘇力菌殺蟲晶體蛋白、D型胺基酸氧化酵素、轉酮醇酵素、熱休克蛋白、超氧化歧化酵素及過氧化氫酵素等基因到蕓苔屬蔬菜之研究
Studies on Co-transfer of Bt-toxin, D-Amino Acid Oxidase, Transketolase, Heat Shock Protein, Superoxide Dismutase, and Catalase Genes into Brassica Vegetables via Agrobacterium
作者: 黃群益
Huang, Chun-I
關鍵字: co-transformation
共同基因轉移
Bt-toxin
D-Amino acid oxidase
transketolase
heat shock protein
superoxide dismutase
catalase
蘇力菌殺蟲晶體蛋白基因
D型胺基酸氧化酵素
轉酮醇酵素
熱休克蛋白
超氧化歧化酵素
過氧化氫酵素
出版社: 園藝學系
摘要: 本省地處亞熱帶,夏季氣候高溫多濕,造成病蟲危害嚴重,且常有溫度逆境的發生,因此培育出抗病蟲害、且耐逆境的蔬菜品種,是育種工作的重要目標。本研究是利用農桿菌共同轉移法,研究同時轉移帶有 CaMV35S 及 rbcS 為啟動子之蘇力菌殺蟲晶體蛋白 (Bt)、D型胺基酸氧化酵素 (DAO)、轉酮醇酵素 (TKL) 及熱休克蛋白 (HSP) 等四種基因,或同時轉移蘇力菌殺蟲晶體蛋白 (Bt)、超氧化歧化酵素 (SOD)、過氧化氫酵素 (CAT) 及熱休克蛋白 (HSP) 等四種基因至同一棵甘藍 (高峰) 或青花菜 (綠王) 之可行性。其目的為建立蕓薹屬蔬菜之農桿菌共同基因轉移系統與培殖體再生模式,並開發培育成同時具有抗病、抗蟲且抗逆境之蔬菜品種之基因轉移技術。 目前已獲得共同轉移二種、三種、四種基因之轉殖植物。再生之Bt、DAO、TKL及HSP基因轉移之甘藍植株與Bt、SOD、CAT及HSP基因轉移之青花菜植株以南方墨點slot分析,篩選基因轉殖植物,再進一步以PCR及南方墨點電泳分析證實轉移基因已插入到轉殖植物染色體上。北方墨點電泳分析轉殖植株顯示,可偵測出1.35 kb的Bt RNA條帶、 1.1 kb的DAO條帶、2.2 kb 的TKL RNA條帶、2.1 kb的HSP RNA條帶、0.65 kb的SOD RNA條帶及2.3 kb的CAT RNA條帶。試驗結果顯示,共同轉移二基因之轉殖率為14 %,同時轉移三種基因之共同轉移率約為14 %,四種基因的共同轉移率為10 - 15 %。本研究証實利用農桿菌混合感染培殖體可達到多基因同時轉移到同一棵甘藍或青花菜,且能正常表現RNA。目前尚繼續探討這些多基因轉殖植物是否能抗病蟲及耐逆境。
Taiwan is located at the sub-tropic area. In summer, the high temperature and humidity cause very serious of plant disease and insect pests. Attempts had been made to co-transfer the Bt-toxin (Bt), D-amino acid oxidase (DAO), transketolase (TKL) and heat shock protein (HSP) genes into the cabbage and co-transfer the Bt-toxin (Bt), superoxide dismutase (SOD), catalase (TKL) and heat shock protein (HSP) genes into the broccoli. The objectives of this study are to establish the gene co-transfer technology and to study the possibility for improvement of Brassica vegetables with insect, disease, and stresses resistance by gene transformation. Regenerated plants were obtained after co-transformation with four genes (Bt + DAO + TKL + HSP) into cabbage and co-transformation with four genes (Bt + SOD + CAT + HSP) into broccoli and screen by Southern slot hybridization. The transgenic plants were further examined by PCR, Southern and Northern hybridization. Several transgenic plants contained four, three, and two genes in one plant were demonstrated. The results of genetic analysis indicated that the transgenes were inserted into the chromosome of transgenic plants and expressed Bt transcript with 1.35 kb RNA, DAO transcript with 1.1 kb RNA, TKL transcript with 2.2 kb RNA, HSP transcript with 2.1 kb RNA, SOD transcript with 0.65 kb RNA, and CAT transcript with 2.3 kb RNA. The co-transformation rate of two, three, and four genes was 14%, 14% and 10~15%, respectively. The study demonstrated that two, three and four foreign genes could be co-transferred into one cabbage or broccoli plant simultaneously via Agrobacterium mediated transformation.
URI: http://hdl.handle.net/11455/29354
Appears in Collections:園藝學系

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