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標題: Application of marker gene-free technology in chloroplast transformation
作者: 曾夢蛟
關鍵字: 技術發展
selectable marker gene
chloroplast gene transformation
D-amino acid oxidase
摘要: Resistance to antibiotics and herbicides mediated by selectable marker genes remains a powerful selection tool for transgenic event production. However, the presence of antibiotic and herbicide resistance genes in genetically engineered crops has generated a number of environmental and consumer's concerns for long time. Expression of foreign genes <I>via</I> chloroplast genomes offers several unique advantages, including high protein levels, expressing multiple genes in operons, transgene containment by maternal inheritance, as well as lack of gene silencing, position and pleiotropic effects. The most widely used selectable marker for chloroplast transformation is a gene encoding aminoglycoside 3'-adenylyltransferase (<I>aada</I>). Transplastomic plantlets are then selected by spectinomycin and/or streptomycin resistance. However, there are concerns associated with antibiotic resistance marker genes including the metabolic burden imposed by high-level expression of chloroplast and the potential for development of resistant strains of bacteria. D-amino acid racemase (alaR) can mediate the racemization of D-amino acids. D-amino acid oxidase (DAAO) catalyzes the dehydrogenation of D-amino acids to yield α-keto acid and accompany with NH<SUB>3</SUB> and H<SUB>2</SUB>O<SUB>2 </SUB>production. Our previous studies has successfully employed biolistic bombardment to obtain a stable and highly efficient transformation platform for transplastomic cabbageand rice by using the <I>alaR </I>or<I> daao </I>gene as a selectable marker gene, conferring resistance to the D-alanine. Moreover, we had demonstrated the elimination of marker gene by the mechanism of transient cointegration of the marker gene. By using the system of <I>daao</I> gene as marker genes and followed by elimination of marker gene, we propose to engineering the phage <I>lysozyme </I>(<I>lys</I>) and <I>holin</I> (<I>hol</I>) genes into the chloroplast of cabbage, pai-tsai and rice. The objective of this study is to engineer transplastomic cabbage, pai-tsai and rice with a high level of resistance against plant disease using the approach of the marker gene-free technology.
隨著基因改造作物的廣泛種植,其對人類建康及環境生態的衝擊,也廣受到社會大眾關著。目前被廣泛應用於轉殖作物篩選上的耐抗生素及抗除草劑標誌基因,將面臨減少或無法使用。植物葉綠體基因轉殖具有:增加轉殖基因大量表現,不會造成基因污染、基因靜寂及插入位置效應,與較細胞核基因轉移穩定等優點;因此開發葉綠體基因轉殖技術為近代生物技術的主力研發工作。由於葉綠體基因的大量表現轉殖之基因,因此對非耐抗生素基因或無篩選標誌基因之轉殖系統的需求,更具必要性及急迫性。D型胺基酸氧化酵素(DAAO)及D-丙胺酸消旋酵素(alaR)可以代謝D型胺基酸。 本研究之先期試驗已建立以<I>alaR</I>或<I>daao</I>基因作為篩選標誌基因,用D-alanine篩選甘藍、小白菜及水稻之葉綠體基因轉殖系統,並證實剔除篩選標誌基因之無篩選標誌基因的葉綠體基因轉殖系統是可行的。本研究乃結合所建立的<I>daao</I>基因作為葉綠體篩選標誌基因系統,及剔除篩選標誌基因之無篩選標誌基因的葉綠體基因轉殖系統,應用於植物抗病基因工程的研發。本研究計畫擬將分離與構築之<I>lysozyme </I>(<I>lys</I>)及<I>holin</I> (<I>hol</I>)基因轉移至甘藍及水稻之葉綠體中,探討利用無篩選標誌基因之葉綠體基因轉殖系統,培育出抗病甘藍、小白菜及水稻之可行性。此外,利用<I>lysozyme</I>及<I>holin</I>基因表現抗病的特性,當篩選標誌基因剔除後,方便篩選基因轉殖植株後裔,健全無篩選標誌基因之葉綠體基因轉殖系之建立
其他識別: 101農科-9.1.1-糧-Z1(5)
Appears in Collections:園藝學系



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