三相电机变频调速系统

学位：理学硕士院系专业：计算机与信息工程学院电子工程系_无线电物理学号：1990350

水稻高附加價值基因轉殖之研究

1.Selectable markers enable transgenic plant or cells to be identified after transformation. In this project, we construct a plasmid vector carried novel marker gene, racemase gene, which mediate the racemization of selective agents and can be applied as an alternative selection marker instead of antibiotic resistance gene in screening system of transgenic plant. This antibiotic-free screening system will reduce the impaction of antibiotic gene of the environment and increase the safety of food. 2.The aim of this study is to develop the daao gene from T. variabilis as a selectable marker in transgenic plants. The demand of both research and commercial crop production eads to explore new biosafety selectable markers and develop strategies for generate marker-free plants become an area that is growing rapidly. Our preliminary results indicated that the ability of E. coli JM109 to grow on M9 medium containing Dasparagine as sole nitrogen source required DAAO expression. The potential of using T. variabilis daao as a selectable marker in transformed bacteria will be evaluated in the ongoing project. In our previous study, two plant expression plasmids, designated as pCaPDAO and pRPDAO, each has T. variabilis daao under the control of CaMV35S and rbcS promoter, respectively, were constructed. The constructed pCaPDAO and pRPDAO plasmids had been transferred into callus of rice (Oryza sativa L. cv. Tinung 67) via Agrobacterium-mediated transformation. Several regenerated plants were then selected by antibiotic G418. The presence of daao in the transgenic plants was confirmed by PCR in our preliminary studies. Moreover, the toxicity of D-amino acid on rice germination was evaluated by plating on half-strength MS medium containing different concentration of D-alanine or D-serine. Growth inhibition was observed for medium containing 10 ppm of D-alanine. To successfully establish daao as selectable marker in transgenic rice, expression vectors with both of the daao and gus or gfp 3.Neisseria meningitidis is a capsulated Gram-negative bacterium causing meningitis and septicemia, the life-threatening invasive infections, and is therefore a potential bioweapon. To meet emergent needs in cases sufficient stock of the vaccines including antisera for passive immunization is necessary. Since the findings by E. A. von Behring showing that specific antisera was effective in curing diphtheria, antibodies have been the solving to the poisoning from bites by snakes or spiders and acteremia or septicemia caused by gram-negative bacteria. One of the bottleneck in application of antibodies in medicine is the high costs in production. As a major crop in Taiwan, rice has the potential to be used as a bioreactor for antibody production so as to decrease the cost of production and increase the value of this crop. The final goal of this project is to construct transgenic rice plants for the production of antibodies and vaccines against N. meningitidis, with production of antibody 4-7-3 and the cognate antigen Ag473 being the first goal. Development of bioindustry in Taiwan has been lagged and is moving slowly, and therefore the intellectual property rights has been relatively narrow. The hybridomas and Ag473 used in this research are all original and has no problem in violating the intellectual property rights. In contrast, our invention can file patents in different countries. In addition, our research can consolidate our agriculture and medical biotech industries. The antibodies obtained from our work can be used, in place of antibiotics, to treat N. meningitidis, as well as to prevent the invasion of this organism. They can also be used to develop diagnostic kits and for strain classification. Success in t1.建立一個以racemase基因為篩選標記的表現載體，應用於異源性蛋白表現系統開發新的水稻基因轉殖篩選系統，此系統是利用racemase的生物轉換特性對篩選劑進行消旋作用以達到植物選殖目的，並降低對生態環境的衝擊及增加基改食物的安全性。 2.本研究的目的是希望能建立由T. variabilis 菌株來源的daao基因，作為水稻或其它作物的篩選標誌。因為發展安全性篩選標誌基因或無篩選標誌基因的轉殖植物，是目前研究及應用上的趨勢。我們在上一年度的研究中，先以大腸菌菌株進行測試，發現在添加有0.08%及0.12% D-Asparagine 作為氮源的M9 基礎培養基中，僅經轉型帶有T. variabilis daao基因質體的大腸菌株才會生長，說明了daao基 因具有作為大腸菌株篩選標誌基因的潛力。為便利轉殖水稻的分析及篩選工作，在上一年度工作中，我們亦已完成了兩個可以在水稻表現載體pCaPDAO 及 pRPDAO的構築，它們分別具有可受CaMV35S 及rbcS 啟動子調控之T. variabilis daao 基因。此兩個植物表現載體也初步藉由農桿菌轉殖法，轉殖到水稻癒傷組織，並誘導再生。目前已初步完成G418抗生素的篩選及PCR分析工作。此外，將水稻種子播種於添加有不同濃度D-alanine或D-serine 的1/2 MS培養基中，發現10 ppm以上濃度的D-alanine 即可對水稻種子的發芽產生抑制作用。在這一年度的工作中，我們將先完成在同一載體上，同時具有可受CaMV35S 及rbcS 啟動子調控之daao基因及gus或gfp 報導基因表現載體的構築，以便利水稻轉殖株的分析及篩選工作。並將繼續利用D-alanine或D-serine對帶有daao基因的水稻轉殖株進行測試，最後利用 3.奈瑟氏腦膜炎雙球菌(NM)為具有頰膜的格蘭氏陰性菌，會引起腦脊髓膜炎、菌血症及敗血病，是一個潛在的生物性武器。為了因應不時之需，應儲備足夠的疫苗(包括作為被動疫苗之抗體)。自Emil A. von Behring證明抗白喉血清可用來治療白喉以來，抗體是治療毒蛇或毒蜘蛛咬傷及格蘭氏陰性菌所造成之菌血症及敗血症的救命解藥。抗體在臨床醫學應用上最大的瓶頸在於生產成本過高，水稻為台灣 主要的農產作物，若能以水稻作為抗體生產之生物反應器，不但能降低抗體之生產成本，更能提昇水稻之經濟價值。本計畫的最終目標是建立轉殖水稻生產抗奈瑟氏腦膜炎雙球菌抗體以及疫苗；其中以生產抗體4-7-3及其抗原Ag473為首要目標。 台灣生技產業起步較晚，速度較慢，而因智慧財產權的限制，使台灣的研發空間更形狹小。本研究所使用的融合瘤細胞及抗原均為自行研發之成果，無侵犯智慧財產權之虞，因此可以申請全世界各國專利。另外，藉由此研究結果，結合台灣農業與醫學生物科技，使水稻生產抗體及疫苗成為台灣生技產業特色之一。本研究所得到的抗體可替代抗生素作為治療奈瑟氏腦膜炎雙球菌所造成之菌血症、預防奈瑟氏腦膜炎雙球菌由鼻腔進入血液循環系統入侵腦組織造成腦膜炎、以及 用來研發診斷試劑或流行感染病學之菌種分類；而Ag473具有成為腦膜炎雙球菌疫苗成份，本計劃成果將提升台灣在抗體與疫苗生技產業的競爭力。 4.轉穀氨醯胺酵素(Transglutaminase, TGA)廣泛分布於大部份的動物組織和體液，並且與許多生命現象有關，如血液凝固、傷口癒合、表皮角質化和紅血球細胞膜變硬等。除了動物之外，轉穀氨醯胺酵素也存在於植物、魚類及微生物中。轉穀氨醯胺酵素會使蛋白濃縮液形成膠體化，因此在食品加工上有相當高的應用價值及潛力；例如：在漢堡、肉丸、魚漿、豆腐、植物蛋白粉末等可改善彈性、質地、口感、風味，並可增加儲存壽命。目前轉穀氨醯胺酵素大多自動物肝臟中分離純化取得，由於來源取得不易且分離純化過程複雜，使得轉穀氨醯胺酵素的價格相當高，一單位約八十美元，因此限制了其在食品加工上之應用。目前轉穀氨醯胺酵素在日本之年銷售顯達二十億日圓以上。L-homophenylalanine (L-HPA)為廣泛被使用在降血壓藥物的合成，以往多是以化學方法合成，除了費時，價格昂貴外，也會造成環境污染等問題，近年來有開發以酵素生產L-HPA。N-acylamino acid racemase (消旋酵素, NAAAR)在生產L-HPA上扮演提高原料轉換率、大幅降低成本的關鍵性角色。中興大學分子生物研究所楊明德博士及許文輝博士的實驗室已篩選出tga及naaar基因。本研究乃嘗試將tga及naaar基因黏接到水稻種子獨特之球蛋白