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研究生:蔡孟吟
論文名稱:Deinococcusradiodurance海藻糖合成酶基因於水稻之轉殖、表現及其重組蛋白質生化分析
論文名稱(外文):Transgenic Rice with Deinococcus radiodurance Ttrhalose Synthase Gene and Characterization of the Recombinant Protein
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:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
中文關鍵詞:海藻糖合成酶水稻轉殖
外文關鍵詞:Ttrhalose SynthaseTransgenic rice
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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。而水稻穀粒正為當作生物反應器之理想植物器官,因水稻穀粒內只含少量之蛋白質水解酵素,因此外源蛋白質可長期且安全的儲存於種子中。加上,研究指出生物體中海藻糖的少量堆積,可幫助生物渡過逆境(如:高溫、低溫、乾燥和輻射等)增加其生存優勢;推測提高水稻中海藻糖的含量可改變其對逆境的耐受性。
基於以上的理由,本研究以水稻為轉殖作物,將Deinococcus radiodurance之海藻糖合成酶基因TreS ( DRTS )建構於含玉米之ubiquitin基因或水稻之glutelin基因之啟動子的載體上,構築成兩種不同之轉殖載體,以作為轉殖水稻所需,之後利用農桿菌轉殖法送入水稻中進行大量表現,並對獲得之轉殖植物的榖粒及葉子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,並利用南方墨點轉印法及西方墨點轉印法檢測證實,已成功的將DRTS基因轉殖到水稻中,且讓基因具有功能性表現能力。經由西方墨點轉印法之結果證明栽植於土壤的水稻轉殖植株之榖粒和葉子均可偵測到海藻糖合成酶的存在;又將水稻粗抽蛋白質加入以麥芽糖為受質進行反應,利用HPLC的醣類分離檢測方法分析反應後之產物,證實轉殖植株之榖粒及葉子皆有具活性的海藻糖合成酶表現。在生化特性方面,轉殖水稻所產生之重組海藻糖合成酶的最適作用pH值為7.0;最適反應溫度為37℃;於熱穩定性的分析上在經37、40、50 ℃加熱十分鐘後,此重組蛋白質尚有95 ﹪以上的活性,55 ℃加熱十分鐘後其海藻糖合成酶活性只剩下50 ﹪。而於60 ℃加熱十分鐘後則海藻糖合成酶活性完全消失。再者,我們意外地發現水稻穀粒及葉片中均含有海藻糖水解酶活性,但僅有穀粒中含有一新發現酵素可將海藻糖轉化為假定之麥芽三糖。
There is a tendency towards using genetically modified crops to replace microorganism for the production of high-value proteins and enzymes. The grain of rice is a desirable plant organ as a bioreactor. It has less protease in grain, so extraneous protein can be more steadily reservd in grain. Moreover, trehalose accumulation in cells can help organism to resistance abiotic stresses such as heat, chilling, drought and oxygen radicals. We anticipated that increasing trehalose content in rice plants may confer tolerance to different abiotic stresses.
In this study, the Deinococcus radiodurance trehalose synthase (DRTS)was fused with the maize ubiquitin or rice glutelin promoter, and inserted into binary vectors. These plasmids were transformed to Agrobaterium tumefaciens EHA101, for transformation of rice callus. Molecular analyses showed that the transgenic rice plant expressed active recombinant DRTS.
Results from Southern blotting and Western blotting demonstrated that we have obtained several independent transgenic rice lines expressing functional DRTS. The Western blotting showed that, both of grain and leaf from transgenic expressed DRTS protein. Using maltose as substrate, the transgenic rice crude extract from grain or leaf can convert it into trehalose, which confirmed active recombinant DRTS enzymes were expressed in transgenic rice. After reaction, the samples were quantitated by HPLC analysis. Biochemical analyses showed that the optimal pH value of the recombinant trehalose synthase from transgenic rice is 7.0 and the optimal temperature is 37oC. The enzyme retained 95% relative activity after 10 minutes of incubation at 37, 40, 50℃ respectively. Besides, after 10 minutes of incubation at 55℃, the enzyme still retained 50% relative activity. However after 10 minutes of incubation at 60℃, the recombinant trehalose synthase was inactived.
Accidentally, we discovered that both rice grain and leaf contain trehalase which can hydrolyze trehalose into glucose but only rice grain contains a novel enzyme which can convert trehalose into putative maltotriose.
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、海藻糖之特性 1
三、海藻糖合成酶(Trehalose Synthase)的生化特性 4
四、玉米的Ubiquitin及其基因啟動子之研究 6
五、水稻的儲藏性蛋白質Glutelin之生理特性及其調控因子 7
六、植物基因轉殖的方法 9
七、研究動機與方向 12
材料與方法 14
一、微生物、植物及質體材料 14
二、Deinococcus Radiodurance TreS海藻糖合成酶表現載體DNA(expression
vector)之構築 16
三、質體DNA的製備 17
四、DNA定序分析 20
五、農桿菌轉殖水稻的方法 20
六、水稻轉殖植株之鑑定 23
七、酵素生化特性之測定 35
結果 37
一、農桿菌轉殖法之水稻基因轉殖 37
二、水稻轉殖植株之鑑定 37
三、海藻糖合成酶基因在水稻轉殖植株之表現 38
四、重組蛋白質生化特性之分析 39
五、水稻各組織萃取液中所含特定酵素對海藻糖之作用 41
討論 42
一、Ubiquitin及Glutelin基因啟動子於水稻轉殖殖株中之啟動調控情形 42
二、農桿菌轉殖法之Deinococcus Radiodurance TreS基因轉殖水稻研究 43
三、重組海藻糖酶生化特性之分析 44
四、轉殖水稻之相關應用暨市場前景 47
參考文獻 49
圖表暨說明 56
附錄 83
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8. 活性部位苯丙胺酸殘基突變後對於海藻糖苷糊精生成酶之轉糖基與水解作用的影響
9. 利用SulfolobussolfataricusATCC35092之原生型與突變型麥芽寡糖?海藻糖生成?及麥芽寡糖?海藻糖水解?由澱粉生產海藻糖之研究
10. 基質次結合部位殘基突變後對於麥芽糖寡苷海藻糖水解酶之活性與基質選擇性的影響
11. 增加基質結合部位殘基與基質間氫鍵對於SulfolobussolfataricusATCC35092麥芽寡糖苷海藻糖生成酶轉糖苷及水解作用之影響
12. 麥芽寡糖苷海藻糖水解酶的基因選殖、特性探討與基因重組
13. 新型基因重組Picrophilustorridus海藻糖合成酶之蛋白質工程及其固定化酵素之最適化反應條件研究
14. 活性部位殘基突變後對於海藻糖生成酶之活性與基質選擇性的影響
15. Deinococcusradiodurans海藻糖合成酶與Thermoanaerobacteriumthermosulfurigenesβ-澱粉酶重組融合蛋白之生化特性分析
 
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