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標題: 鎘結合蛋白基因、抗凍蛋白基因及蘇力菌殺蟲晶體蛋白轉移至三種蕓薹屬蔬菜之研究
Studies on the Cd-binding protein, antifreeze protein and Bt- toxin genes transfer into three species of Brassica spp.
作者: 尤進欽
Yiu, Jin-Chin
關鍵字: Cd-binding protein
antifreeze protein
Bt-toxin genes
出版社: 園藝學系
摘要: 本試驗將分離自天竺鼠的鎘結合蛋白基因(MTⅡ )、比目魚抗凍蛋白基 因(AF)及蘇力菌殺蟲晶體蛋白基因( Bt),構築到攜帶有rubisco small subunit(rbcS)及 alcohol hydrogenase( adh )啟動子的轉殖 載體,並利用農桿菌將其轉移到青花菜(綠王及翠光)、花椰菜(秋王及 鳳山極早生)及小白菜(臺農一號)的子葉或下胚軸。本試驗的目的為建 立青花菜、花椰菜及小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,並探討培育成耐鎘、抗凍及抗小菜蛾之蔬菜的可 行性。本研究的結果顯示,以農桿菌轉殖供試之三種蕓薹屬的五個品種蔬 菜之再生率在 1 - 3% 之間。檢測轉殖植株之 GUS 基因,有 95% 以上呈 正反應。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,轉移之三種基因 在轉殖植物體內均有轉錄。無論 MT-Ⅱ、AF 或 Bt 基因以rbcS啟動子質 體的轉殖後再生植株,其 RNA 表現量較轉殖以 CaMV 35S 啟動子質體的 植株高。經 Bt 基因轉移之植株的生物檢定結果顯示,以 rbcS 啟動子為 轉殖質體的質株,殺小菜蛾效果較以 CaMV 35S 啟動子為轉殖質體的植株 高。目前轉殖三種基因的基因的三種轉殖植株均有再生。其中轉殖Bt及AF 基因之再生植物已完成生物檢定,轉殖MT基因已偵測有mRNA的存在,需再 做進一步生物檢定並探討轉殖植株後代之轉殖性狀的遺傳分析。
This project includes construction of MT binding protein genes from Geaniue pig (MTⅡ), antifreezing protein genes from winter floumder(AF), and insecticidal protein genes seperated from Bacillus thuringiensis (Bt) with vector carrying with rubisco small subumit (rbcS) and alcohol dehydrogenase (adh) promoter sites respectively. Constructed DNAs were transferred into hypocotyls and cotyledons of three Brassica vegetables on agrobacterium-mediated base. We have focused on establishing gene transter and regeneration systems on Broccoli, Cauliflower and Pak-choi, comparisons in gene expression led by different promoters. Also, assessments on the possibilities of vegetables that are resistant to Cadmium, freezing, and Plutella xylostella are made during discussion. Results have shown that the regeneration rate of five transgenic species among three Brassica vegetables ranges around 1-3%, more than 95% of these plants are GUS positive. Transferred genes are detected by southern assay among PCR selected plantlets; transcriptions of the three genes in transgenic plantlets are also confirmed resulting from Northern assay. Plantlets with rbcS promoter perform a higher RNA expression than those with CaMV 35S promoter, no correlations are fond with different genes and their RNA expression. Beside, results also have show that plantlets with rbcS promoter get higher mortalities of Plutella xylostella rather than those with CaMV 35S promoter which are evidenced by Bt bioassay. Regenerations of three Brassica vegetables with three distinct genes have been observed nowadays, among which the Bt transgenic plantlets have complete bioassays,the other AF and MTⅡ transgenic plants have also exhibit transgenic mRNA that need further bioassays.
Appears in Collections:園藝學系



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