建立表現耐熱澱粉普魯南糖酶基因之轉植甘藷

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姓名

林惠文(Huey-Wen Lin) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

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

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摘要(中)

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。

摘要(英)

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.

關鍵字(中)

★ 普魯南糖酶
★ 農桿菌轉殖
★ 甘藷

關鍵字(英)

★ amylopullulanase
★ Agrobacterium-mediated transformation
★ sweet potato

論文目次

中文摘要.....................................................i
Abstract .....................................................ii
致謝...........................................................iii
縮寫表......................................................iv
本文目錄....................................................vi
圖目錄......................................................ix
表目錄......................................................x
本文目錄
壹、緒論.........................................................1
貳、實驗材料與方法...............................................5
一、植物表現載體............................................5
二、植物基因轉殖............................................5
1.農桿菌媒介基因轉殖法..................................5
1.1 製作農桿菌勝任細胞................................5
1.2 農桿菌轉型........................................6
1.3 農桿菌的生化檢測..................................7
1.4 農桿菌感染阿拉伯芥................................7
1.5 農桿菌感染甘藷和共培養............................8
1.6 轉殖甘藷的篩選和再生..............................8
三轉殖株之分析..................................8
2. GUS染色.............................................8
3. 基因組DNA PCR......................................8
3.1 基因組DNA的抽取與純化...........................8
3.2 利用PCR合成外來基因DNA片段.....................9
3.3 DNA膠體電泳分析.................................10
4. 南方墨點轉印.........................................10
4.1 基因組DNA轉印....................................10
4.2 探針的製備........................................11
4.3 純化探針..........................................12
4.4 探針雜交..........................................12
4.5 自動放射顯影......................................12
5. DEPC¬-水製備.........................................13
6. RNA的萃取與純化.....................................13
6.1 RNA電泳..........................................13
7. cDNA製備............................................14
7.1 去除RNA樣品中的DNA..............................14
7.2 cDNA的合成.......................................14
7.3 以PCR合成DNA片段................................14
8. 瓊脂膠體回收限制酵素切過的DNA.......................15
參、實驗結果......................................................17
I. 以阿拉伯芥當作模式植物測試不含篩選基因的轉殖系統..............17
1. 轉殖阿拉伯芥T1植株轉基因分析...............................17
2. 轉殖阿拉伯芥T2植株轉殖基因分析.............................18
II. 基因轉殖甘藷的建立.............................................18
1. 甘藷癒傷組織再生能力的分析..................................18
2. 基因轉殖甘藷的建立..........................................19
III. 轉殖甘薯的分析................................................19
1. 轉殖甘藷中轉基因的分析......................................19
2. 轉殖甘薯中轉基因的表現分析..................................20
2.1 GUS活性分析...........................................21
2.2 轉基因的表現............................................21
2.3 Sporamin和普魯南糖酶在轉殖甘藷中的表現..................22
肆、討論...........................................................23
I. 以阿拉伯芥當作模式植物測試不含篩選基因的轉殖系統...............23
II. 基因轉殖甘藷的建立.............................................24
III. 轉殖甘藷中轉基因的分析.........................................25
1. GUS活性分析................................................25
2. SPO::GUS在轉殖甘薯中的表現.................................25
IV. 後續工作......................................................27
伍、參考文獻........................................................41
附錄I.............................................................43

參考文獻

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指導教授

陸重安、余淑美
(Chung-An Lu、Su-May Yu)

審核日期

2009-7-17

推文