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研究生:劉文彰
研究生(外文):Liu WenChang
論文名稱:利用大腸桿菌系統來表現純化蚜蟲內共生菌Buchneraaphidicola芳香族胺基酸生成酵素DAHPS
論文名稱(外文):Expression, purification, and characterization of the aromatic amino acid-synthesizing enzyme DAHPS of Buchnera aphidicola in an E. coli system
指導教授:賴吉永賴吉永引用關係
指導教授(外文):Chi-Yung Lai
學位類別:碩士
校院名稱:國立彰化師範大學
系所名稱:生物學系
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:2007
畢業學年度:96
語文別:中文
論文頁數:91
中文關鍵詞:蚜蟲內共生菌
外文關鍵詞:BuchneraDAHPSaroHoverlap-extension PCRE. coli
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蚜蟲是常見的農業害蟲,以吸食植物韌皮部汁液維生,體內含有內共生菌 Buchnera aphidicola,兩者為絕對的互利共生關係。Buchnera會提供宿主蚜蟲色胺酸(tryptophan)和苯丙胺酸(phenylalanine)兩種芳香族胺基酸。 3-deoxy-D-arabino- heptulosonate-7-phosphate synthase (DAHPS) 為芳香族胺基酸合成路徑所需要的酵素,aroH 為Buchnera DAHPS 唯一的編碼基因。Buchnera aroH 缺乏轉錄及轉譯層級的調控,其異位調控推測區和其他細菌沒有完全保守,且因為Buchnera基因的表現不需要隨生長環境的變化而改變,是故認為Buchnera DAHPS可能不受回饋調控。由於Buchnera 無法在蚜蟲體外進行一般細菌培養實驗,所以本研究的目的在於建立 E. coli 表現系統,來研究 Buchnera DAHPS 酵素功能及回饋調控。實驗結果得知,Buchnera DAHPS 可以補償大腸桿菌芳香族胺基酸突變株的營養缺失,測得Buchnera DAHPS對受質PEP及E4P的Km分別為176 μM及290 μM,會受到最終產物tryptophan與phenylalanine的部份抑制,I0.5分別為19.7 μM與168.4 μM,最大抑制百分比為61.7%與72.1%。進一步利用overlap-extension PCR建立帶有 T7 promoter 的表現載體,轉型到大腸桿菌 BL21 (DE3) T7 系統,雖然可以大量表現 Buchnera DAHPS,但表現蛋白出現在不可溶的部份,嘗試低溫誘導、共同表現及重新折疊,仍無法改善其錯誤折疊及穩定使其恢復活性。
Aphid is a major agricultural pest insect feeding on the phloem sap of plants. The bacterium Buchnera aphidicola is an essential endosymbiont of aphid. Their symbiotic association is a nutritionally-based absolute mutualism in which Buchnera provides aphid with essential amino acids and vitamins. The enzyme 3-deoxy-D-arabino-heptulosonate-7- phosphate synthase (DAHPS), encoded by aroH, is the key enzyme in the first committed step in the biosynthesis of aromatic amino acids. Buchnera aroH is not regulated transcriptionally, and its expression does not fluctuate with environmental changes. Because the putative allosteric control site of the enzyme is also different from other bacteria, it was hypothesized that Buchnera DAHPS lacks feedback inhibition as well. As Buchnera cannot be cultivated in vitro, I used an E. coli expression system to test this hypothesis. My data show that Buchnera DAHPS complemented an E. coli DAHPS mutant. The Michealis constants (Km) of partially purified enzyme were 176 μM for phosphoenolpyruvate and 290 μM for erythrose-4- phosphate. The enzyme activity was partial inhibited by tryptophan and phenylalanine, with half-inhibition concentrations (I0.5) of 19.7 μM and 168.4 μM and maximum inhibition at 61.7% and 72.1%, respectively. I used overlap-extension PCR to make an expression vector for the production of a HIS-tagged version of Buchnera DAHPS. This attempt failed because the product was insoluble, and I could not obtain an active enzyme preparation using the strategies of lowering the induction temperature, coexpression with the chaperonin gene groE, or denaturing and refolding of the inactive product.
中文摘要 I
英文摘要 II
前言 1
文獻回顧
一、 蚜蟲 4
二、 蚜蟲內共生菌Buchnera aphidicola 5
三、 蚜蟲及內共生菌的共生關係 9
四、 E.coli 及B. aphidicola的芳香族胺基酸調控機制 11
材料與方法 15
結果
一、 突變株生理活性測試 32
二、 DAHPS選殖載體及表現載體之建立 32
三、 B. aphidicola DAHPS功能互補測試 33
四、 B. aphidicola DAHPS spot test 33
五、 B. aphidicola DAHPS蛋白質表現 34
六、 DAHPS的初步純化 35
七、 內涵體的改善 36
八、 DAHPS Steady-State Kinetic 38
九、 不同芳香族胺基酸對DAHPS酵素活性的影響 38
討論 40
結論 48
參考文獻 49
表 60
圖 66
附錄 90
表目錄
表一、選殖細菌基因所用引子對及聚合酶鏈反應條件 60
表二、實驗所用載體一覽表 61
表三、本實驗所用引子的名稱和序列 62
表四、實驗所用引子的特性 62
表五、本實驗所使用E. coli菌株一覽表 63
表六、CB717菌株不同培養基spot test稀釋終點比較 63
表七、DAHPS的純化回收 64
表八、腸內細菌科細菌AroH 胺基酸Identity 比對 65
圖目錄
圖一、CB717生理測試 66
圖二、pWC3及pWC6質體建構圖 67
圖三、B.aphidicola aroH 定序結果 68
圖四、Buchnera aroH基因與pRSF-1b之乳糖操作子及前導序列聚合酶鏈鎖反應結果 70
圖五、Buchnera aroH不同溫度下功能互補測試 71
圖六、CB717菌株不同培養基功能spot test 73
圖七、B. aphidicola aroH選殖質體蛋白質表現 74
圖八、B. aphidicola aroH表現質體蛋白質表現 75
圖九、B. aphidicola aroH 蛋白質表現屬於非溶解性部分 76
圖十、B. aphidicola aroH 表現之重組酵素的純化 77
圖十一、B. aphidicola aroH 低溫蛋白質表現及共同表現 78
圖十二、B. aphidicola aroH 內涵體重新折疊復性比活性圖 79
圖十三、B. aphidicola DAHPS對E4P Lineweaver-Burk plot 雙倒數圖 80
圖十四、B. aphidicola DAHPS對PEP Lineweaver-Burk plot 雙倒數圖 81
圖十五、B. aphidicola DAHPS對E4P做Hill plot 圖 82
圖十六、B. aphidicola DAHPS對PEP做Hill plot圖 83
圖十七、芳香族胺基酸對B. aphidicola DAHPS活性的影響 84
圖十八、苯丙胺酸存在下,色胺酸對B. aphidicola DAHPS活性的影響 85
圖十九、色胺酸存在下,苯丙胺酸對B. aphidicola DAHPS活性的影響 86
圖二十、DAHPS(AroH)胺基酸排序 87
圖二十一、E.coli 和B. aphidicola DAHPS 活性區位比較 89
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