Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/20632
標題: Trigonopsis variabilis D型胺基酸氧化脢基因之水稻轉殖
Transformation of Trigonopsis variabilis D-amino acid oxidase gene into rice
作者: 李姬緩
Li, Chi-Huan
關鍵字: Trigonopsis variabilis
D-型胺基酸氧化酶
D-amino acid oxidase
transformation of rice
基因
水稻轉殖
出版社: 植物學系
摘要: 中文摘要 D-型胺基酸氧化酶(DAO)為轉換頭孢菌素C,生產頭孢菌類抗生素之前驅物7-ACA的重要酵素,而三角酵母菌Trigonopsis variabilis的DAO酶是截至目前為止,轉換頭孢菌素C能力較強者。本實驗之轉殖基因材料是以T. variabilis所分離到的D-型胺基酸氧化酶cDNA選殖體為模版,利用PCR分離得到D-型胺基酸氧化酶基因dao之全長cDNA片段,其全長為1068 bp,再將此dao基因分別構築於CaMV35S及水稻油體膜蛋白18 kDa基因(Ose721)啟動子的後方,形成融合轉殖質體pCAM721dao及pCAM35Sdao,利用農桿菌轉殖水稻技術,將轉殖基因轉入水稻台梗九號品系中,探討此二種啟動子表現D-型胺基酸氧化酶基因的差異。結果顯示,水稻的癒傷組織經由農桿菌感染,培養在含hygromycin抗生素培養基中篩選生長,兩種轉殖質體皆有轉殖水稻癒傷組織之生成,其中以含pCAM721dao轉殖質體的癒傷組織生成較多。以PCR分析,證實轉殖癒傷組織,含有外來轉殖基因dao,進一步經由南方墨點分析,顯示轉殖基因的 確成功地嵌入水稻基因組內。
Abstract D-amino acid oxidase(DAO) is a flavoenzyme that catalyzes the oxidation of cephalosporin C to produce the precursor of cephalosporin antibiotic glutaryl-7-aminocephalosporin acid(GL-7-ACA). Up to date, the D-amino-acid oxidase derived from Trigonopsis variabilis has been shown to have higher catalytic activity of oxidation of cephalosporin C. A full-length D-amino acid oxidase cDNA fragment was isolated by PCR using plasmid pGEM-dao as template. This cDNA was cloned into plant transformation vectors under the control of CaMV35S or rice oleosin promoter to form recombinant plasmid pCAM721dao or pCAM35Sdao, respetively. These recombinant plasmid were then introduced into rice calli through Agrobacterium-mediated transformation method. After hygromycin selection, cells that were resistant antibiotic were able to continue proliferation. The growth of transgenic calli pCAM721dao were better than that of pCAM35Sdao, and there were enough tissues to assay. The existence of D-amino acid oxidase gene in antibiotic resistant rice calli was confirmed by PCR and Southern blot assays. The result has demonstrated that the transgenic calli contain the D-amino acid oxidase gene, and transgene was successfully integrated into rice genome.
URI: http://hdl.handle.net/11455/20632
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