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題名 | Expression of Trigonopsis Variabilis D-amino Acid Oxidase in Transgenic Rice for Cephalosporin Production=在轉殖水稻中表現三角酵母菌D-型胺基酸氧化酶以供應頭孢菌素生產 |
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作者 | 林詩芸; 王俊達; 林正宏; Lin, Shih Yun; Wang, Jiun Da; Lin, Jenq Horng; |
期刊 | Botanical Studies |
出版日期 | 20090400 |
卷期 | 50:2 2009.04[民98.04] |
頁次 | 頁181-192 |
分類號 | 434.114 |
語文 | eng |
關鍵詞 | 三角酵母菌; D-型胺基酸氧化酶; 頭孢菌素; 轉殖水稻植株; 梗稻栽培種-臺梗9號; Cephalosporin; D-amino acid oxidase; Japonica rice cultivar Taiken 9; Transgenic rice plant; Trigonopsis variabilis; |
中文摘要 | 轉殖植物已成為製造重組蛋白質的有利系統,而且已有許多轉殖植物生產具功能性蛋白質的成功例子。在此篇研究中,利用農桿菌基因轉殖技術表現三角酵母菌(Trigonopsis variabilis) 的D-型胺基酸氧化酶(DAAO) 於梗稻栽培種― 台梗9 號(Taiken 9) 中。DAAO 為一種黃素酵素(flavoenzyme),可催化使頭孢菌素C (cephalosporin C) 經氧化作用成為頭孢菌素類抗生素之前驅物glutaryl-7-aminocephalosporin acid (GL-7-ACA)。其中三角酵母菌的DAAO 對於頭孢菌素C 則有最好的氧化催化能力。在水稻中,分別以來自水稻本身的啟動子―actin 1 (Act1) 和玉米的啟動子―phosphoenolpyruvate carboxylase (PEPC) 調控三角酵母菌的daao 基因表現。並以南方墨點分析證實三角酵母菌的daao 基因皆有插入水稻染色體,且進一步以北方墨點和西方墨點分析,不論使用Act1 或PEPC 啟動子調控daao 基因,證實相較於未轉殖株,在轉殖株的不同組織中皆可偵測其基因正常表現,並有其蛋白之累積。偵測DAAO 活性分別於兩種調控啟動子的水稻轉殖株,發現以Act1 為啟動子的轉殖植物的葉子有最大比活性 (65.5 ± 7.4 U mg protein-1 min-1)。利用水稻Act1 啟動子的轉殖水稻具有的DAAO 活性高於使用玉米PEPC 啟動子的好幾倍,且在葉子和莖桿部位分別提高了5.3-3.7 倍。具PEPC 啟動子的轉殖水稻的穀粒則偵測不到DAAO 活性。由上述結果皆可證實,三角酵母菌的daao 基因可以穩定存在於轉殖水稻染色體中,且此基因可行正常的轉譯以及轉錄成具功能性的蛋白質。 |
英文摘要 | Transgenic plants have become an effective system to produce recombinant proteins, and there are many examples of transgenic plants that successfully produce functional proteins. In this study, the japonica rice cultivar Taiken 9 was transformed through an Agrobacterium-mediated method to express D-amino acid oxidase (DAAO) from Trigonopsis variabilis. DAAO is a flavoenzyme that catalyzes the oxidation of cephalosporin C to produce the precursor of the cephalosporin antibiotic glutaryl-7aminocephalosporin acid (Gl-7-ACA). DAAO derived from T. variabilis has the highest catalytic activity for cephalosporin C oxidation of DAAO enzymes that have been characterized. Trigonopsis daao was expressed in rice under the control of either the rice actin 1 (Act1) or maize phosphoenolpyruvate carboxylase (PEPC) promoter. Southern blot analysis demonstrated the integration of Trigonopsis daao gene into the rice genome. Furthermore, northern blot and western blot analysis demonstrated production of the daao transcript and accumulation of its protein in various tissues of transgenic rice plants using either the Act1 or PEPC promoter as compared with the wild type. DAAO activity was detected in both transgenic rice lines with a maximum specific activity of 65.5 ± 7.4 U mg protein-1 min-1 detected in the leaves of transgenic plants containing the rice Act1 promoter. The transgenic rice plant with the rice Act1 promoter exhibited several fold higher DAAO activity than the plant with the maize PEPC promoter: 5.3- and 3.7-fold higher in the leaves and sheaths, respectively. No DAAO activity was detected in the grains of transgenic rice containing the PEPC promoter. Taken together, these results demonstrate that Trigonopsis daao is stably integrated into the transgenic rice genome, transcribed efficiently, and translated into a functional protein. |
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