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Co-transformation of Bt-toxin, Heat Shock Protein, Superoxide Dismutase, and Catalase Genes into Chinese Cabbage (Brassica campestris L. ssp. Pekinensis) via Agrobacterium Mediated Transformation

By 蔡元純, 吳佳春, 曾夢蛟, Yuan-Chun Yeh, Chia-Chune Wu and Menq-Jiau Tseng

Abstract

本研究利用農桿菌共同轉移法,研究同時轉移攜帶有rbcS為啟動子及大豆葉綠磯訊息 transit peptide 之蘇力菌殺蟲晶體蛋白(bt)、結球白菜之起氧化歧化酵素 (sod62)、過氧化氫酵素 (cat78) 及番茄之熱休克蛋白 (hsc70) 等四種基因至同一棵之'濱綠'結球白菜並探討轉運蛋白質到葉綠體之可行性。\ud 以PCR、南方墨點及北方墨點雜交分析所篩選之轉殖植株,顯示轉移之基因已插入到轉殖植株染色體上,且可偵測到bt、hsc70、sod62及cat78基因之mRNA條帶。電泳膠片酵素活性染色分析之結果顯示,轉殖植株之SOD及CAT條帶,均較未轉殖植株之條帶濃,其SOD及CAT酵素活性均較未轉殖之對照組顯著增加。西方墨點分析結果顯示,轉殖植株之HSC70顯著較未轉殖植株之對照組多,且能轉運到葉綠體內。\ud 轉殖結球白菜再生植株經過高溫處理 (42℃ 4天) 之後,有hsc70基因之轉殖植株明顯可以抵抗高溫。具有轉殖bt基因之結球白菜再生植株葉片經餵食小菜蛾幼蟲,證實具有抗蟲之效果。以800ppb之SO2燻蒸轉殖植株三天,具有sod及cat基因之結球白菜再生植株,對於SO2之危害較未轉殖或只轉殖單一sod或cat基因之轉殖株具有抗性。本研究結果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結球白菜,且能正常表現有功能之蛋白質,使轉殖植株具有抗蟲、耐高溫及耐逆境之特性。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 gene (bt), superoxide dismutase gene (sod62) of Chinese cabbage, catalase gene (cat78) of Chinese cabbage, and heat shock protein gene (hsc70) of tomato into the Chinese cabbage (Brassica campestrus L. ssp. Pekinensis cv. Tropical Pride). The objectives of this study are to establish the technology of gene co-transfer and targeting the foreign protein into the chloroplast by chloroplast transit peptide, and to study the possibility for improvement of Chinese cabbage with insect, disease, and stresses resistance by genes co-transformation. The results of PCR, Southern and Northern hybridization indicated that the bt, hsc70, sod62 and cat78 genes had been inserted into the genome of Chinese cabbage, and expressed. The result of isozyme profiles assay revealed that the transformed SOD62 and CAT78 were present in the transformed plants. The SOD and CAT activities in leaves and chloroplast of transformed plants were higher than those of the controls. Increase in amounts of HSC70 protein were found in the transformed plants as analyzing by western blot hybridization. Preliminary results of bioassay indicated that the increases in tolerance to high temperature (42℃, 4 days), SO2 (800 ppb, 3 days), and insect resistance (Plutella xylostella) were found in the transgenic plants. This study demonstrated that two, three and four foreign genes could be co-transferred into a Chinese cabbage plant simultaneously via Agrobacterium mediated transformation

Topics: 過氧化氫酵素, 農桿菌共同轉移法, 蘇力菌殺蟲晶體蛋白, 熱休克蛋白, 超氧化歧化酵素, Chinese cabbage, Co-transformation, Bt-toxin, Heat Shock Protein, Superoxide Dismutase, Catalase
Year: 2014
OAI identifier: oai:ir.lib.nchu.edu.tw:11455/81729
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