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研究生:沈翰祖
研究生(外文):Han-Tzu Shen
論文名稱:農桿菌法共同轉移基因到甘藍之研究
論文名稱(外文):Studies on Co-transfer Genes into Cabbage (Brassica oleracea L. var. capitata L.) via Agrobacterium-Mediated Transformation
指導教授:曾夢蛟
指導教授(外文):Menq-Jiau Tseng
學位類別:碩士
校院名稱:國立中興大學
系所名稱:園藝學系
學門:農業科學學門
學類:園藝學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
論文頁數:130
中文關鍵詞:熱休克蛋白基因蘇力菌殺蟲晶體蛋白基因超氧化歧化酵素基因過氧化氫酵素基因農桿菌基因轉移法共同轉移基因導引訊息胜肽
外文關鍵詞:Heat shock proteinBacillus thuringiensissuperoxide dismutasecatalaseAgrobacterium-mediated transformationCo-transformtransit peptide
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隨著地球人口增加、科技進步,人類對環境的破壞不斷加速且擴大影響範圍,溫室效應、酸雨、臭氧層破洞等對氣候的影響逐漸佈及全球,而位於亞熱帶的台灣自然無法自外於地球氣候變遷之影響,且臺灣氣候夏季本就高溫多濕,加速了病蟲害的孳生,因此近年常出現高溫、酸雨、淹水、乾旱等環境與病蟲害等逆境的發生,因此培育出抗病蟲害、且耐環境逆境的蔬菜品種,是育種工作的重要方向,而利用基因轉移技術,改善植物對病蟲害與環境逆境的耐受性,將是最有效率的途徑之ㄧ。
熱休克蛋白 (heat shock proteins cognate, HSC)與植物耐高溫逆境有關;蘇力菌殺蟲晶體蛋白基因 (Bt) 具殺蟲的效果;而利用超氧化歧化酵素 (superoxide dismutase, SOD)與過氧化氫酵素 (catalase, CAT) 的抗氧化酵素系統來紓解氧自由基的危害,可提高其抗逆境的能力。本實驗是利用農桿菌基因共同轉移法,研究同時轉移番茄的熱休克蛋白、蘇力菌殺蟲晶體蛋白、玉米的超氧化歧化酵素及過氧化氫酵素等四種基因至甘藍之可行性;其目的:(一)建立農桿菌基因共同轉移系統與培殖體再生模式,(二)利用生物科技,探討開發增加植物抗蟲且抗環境逆境之可行性,(三)比較大豆葉綠體transit peptide (TP) 對甘藍基因表現之影響。
以南方墨點分析經PCR篩選出之轉殖植株結果顯示,帶與未帶TP序列之四種基因均已轉移到轉殖植株染色體;北方墨點之分析結果顯示轉殖植株大多有很強的基因表現;西方墨點與SOD及CAT酵素活性分析結果顯示,轉殖帶有TP之轉殖植株的HSP蛋白及SOD與CAT酵素的表現優於未帶有TP之轉殖植株,且在葉綠體的HSP蛋白及SOD與CAT酵素活性的分析結果也顯示,轉殖帶有TP之植株表現優於未帶有TP之轉殖植株。生物檢定結果顯示轉殖植株較對照組耐SO2 (800ppb, 3天) 及高溫 (42℃, 2天),且有殺小菜蛾之特性,帶有TP之轉殖植株較未帶有TP之轉殖植株之殺蟲及耐逆境之效果更佳。
The results of increasing human population and scientific technology bring about the ruining of environment as well as enlarging the injured area. For example, greenhouse effect, acid rain and ozonosphere spoil etc. have resulted in a worldwide hurt for the nature, including Taiwan, where can’t be separated from the global weather change. In addition that Taiwan’s summer is extremely hot and humid, torrential rain, and drought stresses are speeding up the propagating the disease and destructive insects for crops. Therefore, breeding the vegetables of tolerates stress is an important direction. And using the transgenic technique is an effective way to improve the ability of tolerating the stress. The heat shock proteins cognate (HSC) is relation with tolerating high temperature, Bacillus thuringiensis (Bt) has the effect to kill insects, and the superoxide dismutase (SOD) and catalase (CAT) has the ability to decrease the free-radicle and tolerating the stress. In this study, hsc, bt, sod, and cat genes were co-transferred into cabbage (Brassica oleracea L. var. capitata L.) via Agrobacterium-mediated transformation.
The purposes of this study are to establish a co-transformation system of genes via Agrobacterium-mediated transformation and an explants regeneration model, to study the possibility of increase in the resistance to insects and environmental stress by gene transformation, and to explore the role of chloroplast transit peptide (TP) in delivering transformed proteins in the chloroplast.
The results of PCR, Southern blot, and Northern blot hybridization indicated that hsc70-2, bt, sod, and cat genes had been transformed into the chromosome of transformed cabbage and expressed the mRNA. The results of western analysis confirmed that the significant amounts of HSC70 were presented in the co-transformed plants than those of non-transformed plants. Moreover, the results of Western blot and SOD and CAT activity analysis showed that the expression of four transformed genes was higher in the co-transformed plants harbored with the TP than those of without TP. The results of bioassay showed increases in the resistance of heat, SO2, and insect (Plutella xylostella) in co-transformed cabbage. A better performance in the resistance to insect and environmental stresses were found in co-transformed plants harbored with the TP than those of without TP.
中文摘要
前言
前人研究
一、基因共同轉移的研究
二、利用基因轉移提高作物對環境逆境耐受性之研究
(一)、轉殖熱休克蛋白基因之研究
(二)、轉殖超氧化歧化酵素基因之研究
(三)、轉殖過氧化氫酵素基因之研究
三、利用基因轉移提高作物抗蟲能力之研究
材料與方法
結果
一、含有大豆葉綠體transit peptide之 bt 基因轉殖載體的構築
二、共同轉移熱休克蛋白、蘇力菌殺蟲晶體蛋白、超氧化歧化酵素、過氧化氫酵素等基因
(一)、轉殖植株再生情形
(二)、轉殖植株篩選與分析
討論
一、pKrtBn 的構築
二、共同轉移熱休克蛋白、蘇力菌殺蟲晶體蛋白、超氧化歧化酵素、過氧化氫酵素等基因到甘藍
(一)、轉殖植株再生情形
(二)、轉殖植株檢定與分析
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