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研究生:莊緒怡
研究生(外文):Shi-Yi Chuang
論文名稱:綠竹筍蔗糖磷酯合成酶在酵母菌中之表現
論文名稱(外文):Expression of Sucrose Phosphate Synthase from Shoots of Bamboo (Bambusa oldhamii)in Yeast (Pichia pastoris)
指導教授:李平篤李平篤引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:微生物與生化學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
畢業學年度:93
語文別:中文
論文頁數:87
中文關鍵詞:蔗糖磷酯合成酶綠竹筍酵母菌
外文關鍵詞:Sucrose phosphate synthaseBamboo(Bambusa oldhamii)Yeast(Pichia pastoris)
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蔗糖磷酯合成酶 (sucrose phosphate synthase,簡稱SPS) 為高等植物調控蔗糖生合成之重要酵素。本論文目的,即希望以具真核細胞轉譯後修飾作用之表現系統,表現綠竹筍蔗糖磷酯合成酶(BOSPS)。
首先,依據BOSPS cDNA 之5‘及3’ 端序列,設計含特定限制酶 (NotⅠ及ApaⅠ) 切位之引子,利用聚合酶鏈鎖反應 (PCR),即得兩端分別具 NotⅠ 及 ApaⅠ 切位之BOSPS cDNA;將此cDNA 接入T載體yT&A,轉形至大腸桿菌 (JM109) 中保存。續以限制酶NotⅠ及ApaⅠ將其自T載體切下,與經相同限制酶處理之pPICZ A表現載體接合(pPBOSPS)後,轉形至大腸桿菌 (JM109) 進行定序分析;待序列確認無誤,以限制酶 SacⅠ將此重組表現載體切成線狀,轉形至Pichia pastoris 中 (X-33)。
確定酶母菌轉形株表現型及最適表現時間點後,即可以甲醇誘導BOSPS大量表現;繼則以10~30% PEG沈澱及鎳離子親和層析管柱純化之,所得酵素則可分別進行各項生化性質分析。
Sucrose phosphate synthase (SPS) is an important regulatory enzyme of sucorse biogenic reaction in plants. The purpose of this study is to express sucrose phosphate synthase from shoots of Bamboo (BOSPS) by eukaryotic expression system with posttranslational modification.
At first, forward (with restriction site NotⅠ) and reverse (with restriction site ApaⅠ) primers were designed according to the both terminal sequences of BOSPS cDNA. Then, BOSPS cDNA containing these restriction sites was amplified by PCR and cloned into T vector (yT&A). Both of the recombinant plasmid pTBOSPS and pPICZ A vector were digested by restriction enzymes ApaⅠand NotⅠ sequentially. Therefore, BOSPS cDNA can be cloned into pPICZ A vector (pPBOSPS) by the terminal complementary cohesive end. Being confirmed the correct nucleotide sequence by sequencing, recombinant plasmid pPBOSPS could be linearized and transformed into Pichia pastoris (X-33).
Before scale-up of BOSPS expression induced by the addition of methanol, determining the Mut phenotype and optimal time post-induction to harvest. Finally, expressed BOSPS was purified by a procedure involving precipitation with 10~30% PEG and affinity chromatography, and analyzed for its characterization.
中文摘要………………………………………………………………Ⅰ
Abstract………………………………………………………………Ⅱ
縮寫表…………………………………………………………………Ⅲ
第一章 緒論……………………………………………………………1
第一節 蔗糖……………………………………………………………1
第二節 蔗糖磷酯合成酶………………………………………………2
第三節 Pichia pastoris表現系統…………………………………9
第四節 實驗緣起……………………………………………………14
第二章 材料與方法……………………………………………………16
第一節 實驗材料、藥品與儀器………………………………………16
1.1 實驗材料…………………………………………………………16
1.2 實驗藥品與儀器…………………………………………………16
第二節 一般實驗法……………………………………………… 17
2.1 瓊脂糖膠體電泳…………………………………………………17
2.2 質體DNA小量.………………………………………………… 18
2.3 瓊脂糖膠體中DNA片段之純化………………………………20
2.4 電穿孔法competent cell之製備………………………………20
2.5 綠竹筍蔗糖磷酯合成酶酵素活性測定………………………23
2.6 蛋白質定量法……………………………………………………24
2.7 蛋白質電泳檢定系統……………………………………………26
2.8 蛋白質轉印法…………………………………………………31
2.9 免疫染色法………………………………………………………31
第三節 Pichia pastoris表現系統之建立…………………………33
3.1 聚合酶鏈鎖反應 (PCR)……………………………………35
3.2 yT&A 重組載體之建構、轉形與檢定…………………………32
3.3 pPICZ A重組載體之建構、轉形與檢定……………………39
第四節 綠竹筍蔗糖磷酯合成酶之大量表現與純化…………………44
4.1 Pichia pastoris轉形株表現型之檢定………………………44
4.2 綠竹筍蔗糖磷酯合成酶最適誘導時間點之探討………………45
4.3 綠竹筍蔗糖磷酯合成酶之大量表現……………………………47
4.4 純化經表現之綠竹筍蔗糖磷酯合成酶…………………………47
第五節 經表現之綠竹筍蔗糖磷酯合成酶生化性質探討……………50
5.1 最適反應溫度……………………………………………………50
5.2 最適反應pH值…………………………………………………50
5.3 Glc 6-P對酵素活性之影響……………………………………51
5.4 Pi對酵素活性之影響………………………………………52
5.5 不同金屬離子對酵素活性之影響……………………………53
第三章 結果與討論…………………………………………………54
第一節 Pichia pastoris表現系統之建立…………………………54
1.1 聚合酶鏈鎖反應 (PCR) ………………………………………54
1.2 yT&A重組載體之建構、轉形與檢定……………………………56
1.3 pPICZ A重組載體之建構、轉形與檢定……………………58
第二節 綠竹筍蔗糖磷酯合成酶之大量表現與純化…………………59
2.1 Pichia pastoris轉形株表現型之檢定………………………59
2.2 綠竹筍蔗糖磷酯合成酶最適誘導時間點之探討……………60
2.3 綠竹筍蔗糖磷酯合成酶之大量表現與純化…………………61
第三節 經表現之綠竹筍蔗糖磷酯合成酶生化性質探討……………62
3.1 最適反應溫度.…………………………………………………62
3.2 最適反應pH值………………………………………………… 62
3.3 Glc 6-P對酵素活性之影響…………………………………62
3.4 Pi對酵素活性之影響……………………………………………63
3.5 不同金屬離子對酵素活性之影響………………………………63
第四章 結論與展望…………………………………………………73
附錄……………………………………………………………………75
參考文獻………………………………………………………………81
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