建立表現耐熱澱粉普魯南糖?基因之轉植甘藷; Development of transgenic sweet potato overexpressing amylopullulanase

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Title:	建立表現耐熱澱粉普魯南糖?基因之轉植甘藷;Development of transgenic sweet potato overexpressing amylopullulanase
Authors:	林惠文;Huey-Wen Lin
Contributors:	生命科學研究所
Keywords:	普魯南糖酶;農桿菌轉殖;甘藷;amylopullulanase;Agrobacterium-mediated transformation;sweet potato
Date:	2009-07-15
Issue Date:	2009-09-22 10:21:07 (UTC+8)
Publisher:	國立中央大學圖書館
Abstract:	甘藷是世界上一個很重要的糧食作物，特化的塊根提供人類良好的營養來源，適合生長在氣候溫暖的地區，特別是在熱帶以及亞熱帶國家。塊根當中含有大量的澱粉，除了供給食物來源外，在工業上也被廣泛的應用於加工食品，利用基因工程的方式促進甘薯澱粉品質的改良對於飲食以及工業上加工的應用都有良好的發展潛力。在這個研究中我們試圖發展甘薯的轉殖系統並大量表現外源蛋白，以Ubi::GFP和Ubi::GMCSF作為發展甘藷轉殖系統的對照組，另一方面也將 SPO::APU表達在甘藷當中。我們主要表達一個熱穩定的澱粉普魯南糖酶(同時具有普魯南糖酶以及α-澱粉酶的雙重酵素活性)，利用在一個載體當中放入兩段T-DNA的方式，得到不帶有抗抗生素基因的轉殖株。我們得到八棵獨立的轉殖株，並經由基因組PCR和南方墨點雜交的技術確認這些轉殖株當中含有澱粉普魯南糖酶的重組基因，我們預期普魯南糖酶會加速澱粉的分解，改變甘藷中澱粉的特性。我們利用RT-PCR分析所有的轉殖甘藷，結果顯示所有的外源基因皆可在轉殖甘藷中表現。證實我們這套農桿菌轉殖系統成功地在台農31品系中得到轉殖甘藷，未來也可以將各種重要的基因放入甘藷中，除了增加品種的優勢外，也可用來研究有興趣的基因之功能。 Sweet potato (Ipomoea batatas (L.) Lam.) is an important food crop in the world, and its tuberous roots are sources of human nutrition in many countries, especially in tropical and subtropical areas. In sweet potato tuberous roots, starch is a major constituent and it is not only applied for food production but also for a wide variety of industrial applications. Therefore, genetic engineering of starch composition and quality in sweet potato will has a great potential for new dietary and industrial product. In this study, we tried to make transgenic sweet potato which over-express foreign recombination genes – Ubi::GFP、Ubi::mGMCSF and SPO::APU. Amylopullulanase, a thermostable and bifunctional starch hydrolase, by two borders set technology to create a selection marker free transgenic plants. We obtained eight independent transgenic plants. Genomic PCR and southern blot hybridization confirmed that putative transformants contains the recombinant amylopullulanase gene. We are analyzing the expression level of amylopullulanase in these independent transgenic sweet potatos. In the further, we will examine the amylopullulanase activity in these independent transgenic plants. We expect amylopullulanase alter starch properties of sweet potato tuberous roots in order to accelerate bioprocessing of starch and produce less starch and high maltose roots. Expression of the transgenes (GFP、GMCSF and APU) in transgenic plants was confirmed by RT-PCR. Therefore, we report a successful and reliable Agrobacterium mediated transformation for sweet potato cultivar TNG31. Also, the transformation system has potential to develop new varieties of sweet potato with several important genes for value-add traits.
Appears in Collections:	[Graduate Institute of Life Science] Electronic Thesis & Dissertation

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