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研究生:王嘉宏
研究生(外文):Jia-Hung Wang
論文名稱:假絲酵母(Candidarugosa)脂肪酶基因之馬鈴薯轉殖及其重組蛋白質之生化分析
論文名稱(外文):Transgenic Potato with Candida rugosa Lipase Gene and Characterization of the Recombinant Lipase
指導教授:蕭介夫蕭介夫引用關係
指導教授(外文):Jei-Fu Shaw
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:水產生物技術研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:97
中文關鍵詞:脂肪酶假絲酵母
外文關鍵詞:LipaseCandida rugosa
相關次數:
  • 被引用被引用:1
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  • 下載下載:40
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中 文 摘 要
利用基因工程方法,使基因轉殖作物取代微生物或動物細胞,來生產工業用蛋白質以降低生產成本,已成為新的趨勢。而馬鈴薯塊莖正為當作生物反應器之理想植物器官;馬鈴薯的塊莖含有數種儲存性蛋白質,其中以佔可溶性蛋白質30 ~ 40 % 之Patatin為主。由於Patatin可進行脂質醯基水解反應,針對甘油酯類的水解反應而言,Patatin無法水解三酸甘油酯,但對單甘油酯可進行水解反應;而此物質正為Lipase進行三酸甘油酯水解反應時的中間產物。因此推論兩酵素間可能具有協同作用影響(synergistic effects)現象。即Patatin可能具有幫助Lipase水解三酸甘油酯成脂肪酸的效果。
基於上述理由,本研究以馬鈴薯為轉殖作物,將假絲酵母脂肪酶基因LIP4構築於甘薯之sporamin或馬鈴薯之蔗糖合成酶(Sus4)基因之啟動子,並接上含各種可控制蛋白質定位表達的核酸序列,其中定位表達序列包括:阿拉伯芥rbcS基因轉譯transit peptide(TP)之序列或甘薯sporamin基因轉譯sequence-specific vacuolar sorting domain(spVSD)之序列;再以不同組合構築各式LIP4基因,以作為轉殖馬鈴薯之轉殖載體,之後利用農桿菌轉殖法送入馬鈴薯中進行大量表現,並對獲得之轉殖植物的塊莖萃取物質進行蛋白質生化分析。
目前每一個不同的表現載體已獲得數個細胞系,並利用PCR、南方墨點轉印法、RT-PCR及GUS組織活性染色的結果,證實已經成功的將LIP4基因轉殖到馬鈴薯中且讓基因具有功能性表現能力。藉由RT-PCR之結果發現培養於MT/KT培養基的微塊莖及CM/KT培養基的根、莖和葉之轉殖植株器官均可發現LIP4 mRNA的存在;又經滴定測定法(pH-stat)分析脂肪酶活性,也發現轉殖植株之微塊莖、根、莖和葉皆有具活性的脂肪酶表現,唯表現量以微塊莖較佳。轉殖馬鈴薯所產生之重組脂肪酶的最適作用pH值為7.0;最適反應溫度為40℃;在37、40、50、60、70、80、90及100℃加熱十分鐘後,此重組脂肪酶尚各保有100、98、72、66、57、34、11及4 ﹪之活性;酵素催化水解反應上,則對中碳鏈脂肪酸之三酸甘油酯有較佳的水解能力。
There is a tendency towards using genetically modified crops to replace microorganism for the production of high-value proteins and enzymes. The tuber of potato is a desirable plant organ as a bioreactor. Patatin is the most abundant soluble potato tuber protein, an estimated 30 ~ 40 % of total soluble protein. Patatin has been shown to exhibit lipid acyl hydrolase activity which can hydrolyze mono-acylglycerol but not di-acylglycerol or tri-acylglycerol. We predicted a potential synergistic effect on the hydrolysis of fat or oils by the combination of lipase and patatin.
In this study, the Candida rugosa LIP4 gene encoding high lipase activity have been isolated and fused with the sporamin or Sus4 promoter, with or without targeting peptide such as transit peptide (TP) from rbcS gene and sequence-specific vacuolar sorting domain (spVSD) from sporamin gene, and inserted into binary vectors. These plasmids were transferred to Agrobaterium tumefaciens LBA4404, for transformation of potato. Molecular analyses showed that the transgenic plants expressed active recombinant LIP4.
Results from the PCR, Southern blotting, RT-PCR analyses, and GUS activity staining demonstrated that we have obtained several independent transgenic potato lines expressing lipase genes. The RT-PCR assay showed that the LIP4 mRNA was accumulated in microtuber, root, stem, and leaf. The pH-stat assay also detected lipase activity in microtuber, root, stem, and leaf. The microtuber was the best tissue for the expression of active lipase. Biochemical analyses showed that the optimal pH value of the recombinant lipase from transgenic potato is 7.0 and the optimal temperature is 40oC. The enzyme retained 100, 98, 72, 66, 57, 34, 11 and 4 % relative activity after 10 minutes of incubation at 37, 40, 50, 60, 70, 80, 90 and 100 oC respectively. For the lipase substrate specificity, the recombinant lipase showed highest activity towards medium-chain triglycerides.
中文摘要 I
英文摘要 III
目錄 V
圖表目次 VII
附錄目次 IX
縮寫 X
緒論 1
一、植物作為生物反應器 1
二、脂肪酶的生化特性及應用 1
三、Candida rugosa脂肪酶之相關研究 5
四、馬鈴薯的儲藏性蛋白質Patatin之研究 9
五、甘薯的儲藏性蛋白質sporamin基因啟動子之研究 10
六、馬鈴薯的蔗糖合成酶Sus4基因啟動子之研究 11
七、植物基因轉殖的方法 13
八、研究動機與方向 15
材料與方法 17
一、植物材料 17
二、Patatin的純化 17
三、蛋白質電泳分析 19
四、Patatin與Lipase間之協同作用影響分析 22
五、Candida rugosa LIP4脂肪酶表現質體DNA之構築 23
六、質體DNA的製備 24
七、DNA定序分析 27
八、農桿菌轉殖馬鈴薯的方法 28
九、馬鈴薯轉殖植株之鑑定 29
十、酵素生化特性之測定 36
結果 39
一、Patatin與Lipase間之協同作用影響分析 39
二、農桿菌轉殖法之馬鈴薯基因轉殖 43
三、馬鈴薯轉殖植株之鑑定 43
四、脂肪酶基因在馬鈴薯轉殖植株之表現 44
五、重組蛋白質生化特性之分析 45
討論 47
一、Patatin與Lipase間協同作用影響之研究 47
二、Sporamin及Sus4基因啟動子於馬鈴薯轉殖植株中之啟動調控情形 48
三、農桿菌轉殖法之Candida rugosa Lip4基因轉殖馬鈴薯研究 50
四、重組蛋白質生化特性之分析 51
五、馬鈴薯轉殖植株中Patatin與重組LIP4間協同作用影響之研究 52
六、轉殖馬鈴薯之相關應用暨市場前景 53
參考文獻 55
圖表暨說明 64
附錄 88
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