臺灣博碩士論文加值系統

DHT 能夠水解 D,L-p-hydroxyphenylhydantoin(D-p-HPH) 生成 D,L-p-hydroxyphenylglycine(D-p-HPG)。D-p-HPG 可與 6-aminopenicillanic acid 合成Amoxicillin，Amoxicillin 和 ß-lactamase inhibitor混合使用後，抗菌效果大為提昇。因此市場上對前驅物(D-p-HPG)需求擴大，每年成長12% 以上。國內D-p-HPG年需求量約60公噸，全部仰賴進口，因此國內存在生產D-p-HPG的空間，然而欲發展D-p-HPG 工業必須在技術上有所突破。 目前在工業上希望能以二段酵素法來生產D-p-HPG ，二段酵素法是利用DHT將D-p-HPH轉換成N-carbamoyl-D-HPG，再由N-carbamoylase 將其轉換成D-p-HPG。然而，目前遭遇的困難是D-p-HPH 的溶解度不佳，為解決此一問題，就必須提高反應溫度到50 oC 增加溶解度，但目前的DHT在此溫度下都已失去或降低活性。為克服此一問題，已從微生物選殖出熱穩定DHT，是由四個次單元組成，每個次單元分子量為51 kD。DHT酵素分析，發現於50℃恆溫中，半衰期可長達35天，能夠突破D-p-HPH受質難溶問題。然而微生物所生產的DHT容易生成 inclusion body，不具酵素活性，因此需要適合的DHT表現系統。
本研究目的在於建立水稻生產DHT蛋白之系統。首先構築含dht 的表現載體，使用Ose721、Ose705、Actin和CaMV35S四種啟動子來啟動dht 基因，利用農桿菌轉殖法送入水稻基因組內，並觀察dht在不同組織之表現。轉殖水稻經由聚合酶鏈鎖反應及南方墨點分析，證明轉殖基因的存在，其嵌入位置及數目也不盡相同。西方墨點分析，可以偵測到DHT 蛋白的表現。Ose721、Ose705啟動子所啟動的轉殖株，可在種子中偵測到DHT 蛋白的存在，而葉片卻沒有。Actin啟動子所啟動的轉殖株，則在種子和葉片中皆能偵測到DHT 蛋白；至於含CaMV35S啟動子的轉殖株，只在葉片偵測到有DHT 蛋白的表現，在種子中卻沒有。以上各種轉殖株所萃取的DHT 蛋白皆具有生物活性。

Abstract
D-hydantoinase (DHT) catalyzes a reversible ring-opening hydrolysis of D, L-p-hydroxyphenyl-hydantoins (D, L-p-HPH). DHT has been used as biocatalyst for the stereospecific production of optically active non-proteinogenic amino acids, which are valuable synthons for the synthesis of antibiotics. DHT has been successfully exploited in different enzymatic systems in the production of pharmaceuticals. However there is a problem in the application of DHT in these operations, since the solubility of the substrate (D, L-p-HPH) requires a temperature over 50 ºC. At the temperature of 50 ºC, activity of the regular DHT is significantly decreased. Therefore, it is important to isolate a thermal stable DHT for this process. Although DHT can be easily expressed in microorganisms, mostly forms inclusion body, and usually does not retain enzyme activity. In this study, the possibility to produce DHT in transgenic rice plants was evaluated.
Two full-length of DHT genes (including regular and thermal stable DHTs) were isolated and cloned in plant expression vectors under the control of constitutive (CaMV35S and rice actin) or seed specific (Ose721 and Ose705) promoters, respectively. These expression vectors were introduced into rice plants through agrobacterium-mediated transformation method. The existence of DHT gene in antibiotic resistant plants was confirmed by southern blot assays. Expression of DHT gene in various tissues was analyzed through western analyses. For gene driven by constitutive promoters, DHT was detected in both leaves and seeds, while gene driven by Ose721 and Ose705 promoters, DHT was observed only in seeds. Our preliminary results indicated that the DHTs produced in transgenic rice retain their enzymatic activities.

目 錄
目 錄………………………………………………………………………..Ⅰ
英文摘要……………….……………………………………..………..….Ⅲ
中文摘要………………….……………………………………..….Ⅳ
前言………………………….………………………..………..1
前人研究…………………….…………………………..……..2
一、生物反應器……..…………...….……………………………….2
二、DHT之研究…………………………………………….3
三、水稻胚發育的研究………….…………………………………..5
四、Ose705基因及起動子…………………………………………..6
五、Ose721基因及起動子…………………………………………..7
六、水稻肌動蛋白基因及其起動子…………..…………………….7
七、農桿菌基因轉殖技術…………………………………………...7
材料與方法……………………………………………………..………9
一、材料………………………………………………………..……9
二、方法………………………………………………………..9
1.少量質體DNA之萃取………………………..………….……9
2.DNA片段之純化回收…………………………..……….…….10
3.DNA片段之黏接作用…………………………………..…….…….10
4.大腸桿菌勝任細胞的製備………………………..………..…..11
5.質體轉形作用…………………………………..………….…...11
6.農桿菌之確認…………………………………..………….…...12
7.水稻基因組DNA之萃取…………………..……..…………....12
8.聚合酶鏈鎖反應……………………………………..….……...13
9.耐熱性dht 基因在E.coli 的表現……………….….………....13
10.DHT 的純化………………………………………...14
11.蛋白質濃度測定…………………………………………….….…15
12.DHT在E.coli的活性分析…………..……….……..15
13.南方墨點分析…………………………………………….….…16
14.水稻總蛋白之萃取……………………………………..………17
15. 蛋白質膠體電泳分析……………….……………………………..18
16. 西方墨點分析……………………………….………………….….18
17. 水稻所生產DHT 的活性分析……………………...…………….19
結果……………………………………………………………21
一、一般性dht 基因轉殖…...…………………....……….………..…..21
1. 植物表現載體之構築……………….…………………….21
2. 水稻轉殖………………………….……..………………...22
3. 轉殖水稻之分析…...……………….………………..……22
二、耐熱性 dht 基因之轉殖..…………….………………….…..…….23
1. E.coli 表現載體之構築………………………………..…23
2. tdht在E.coli的大量表現….……………………...……24
3. tDHT 的純化……………………………………….…….24
4. tDHT活性分析…………………………………….….…..25
5. tdht基因DNA定序………………………………….…...25
6. 水稻表現載體之構築………………………………………26
7. 轉殖水稻之南方墨點分析………………………….……...…27
8. 轉殖水稻之西方墨點分析…………………………….………29
9. 轉殖水稻之tDHT活性分析……….….….……………………….31
討論……………………………………………………….…….…32
一、 一般性dht基因轉殖水稻…….….……………..…...32
1. 水稻之確認與分析………………..…………….….………32
2. 轉殖水稻生產DHT………….…………………………..….32
二、 耐熱性dht基因轉殖水稻….………………………….33
1. 聚合酶鏈鎖反應分離tdht基因……………………………………33
2. DNA定序………..……..………………………………..…..33
3. 耐熱性DHT………….………………………………..…..34
4. 表現卡匣嵌入水稻基因組…………………………….…...34
5. 轉殖水稻生產tDHT………………… …………………….35
6. 活性測試……………………………………………….….37
結論……………………………………………………………………….37
參考文獻…………………………………………………………………….38圖表…………………………………………………………………………42

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