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| 研究生： |
李岳軒 Li, Yue-Xuan |
|---|---|
| 論文名稱： |
三個來自液化澱粉芽孢桿菌的糖解作用基因對其生長速度的影響 The effect of three glycolysis genes on the growth rate of Bacillus amyloliquefaciens |
| 指導教授： |
陳又嘉
Chen, Yo-Chia |
| 學位類別： |
碩士 Master |
| 系所名稱： |
農學院 - 生物科技系 Department of Biological Science and Technology |
| 畢業學年度： | 107 |
| 語文別： | 中文 |
| 論文頁數： | 52 |
| 中文關鍵詞： | 液化澱粉芽孢桿菌 、葡萄糖消耗 、甘油醛三磷酸去氫酶 、生長速率 、糖解作用 、丙酮酸激酶 、磷酸果糖激酶 |
| 外文關鍵詞： | Bacillus amyloliquefaciens, Glucose consumption, Glyceraldehyde 3 phosphate dehydrogenase, Growth rate, Glycolysis, Pyruvate kinase, Phosphofructokinase |
| DOI URL： | http://doi.org/10.6346/THE.NPUST.BST.009.2019.D01 |
| 相關次數： | 點閱：26 下載：0 |
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糖解作用是生物重要的代謝路徑，能夠使生物經由代謝醣類獲得能量，已經有研究指出pyk、pfk及gap為糖解作用關鍵基因。本研究的目的是探討過度表現三個糖解作用基因對於菌株生長的影響。在E.coli表現系統中探討導入外源的糖解作用基因對其生長的影響，結果顯示在生長速度上與對照組相比沒有明顯的成長，但在葡萄糖代謝方面，與對照組相比發現pfk2、gap1及gap2轉型株分別增加了230%、1530%及150%的葡萄糖代謝，但pyk及pfk1的導入卻使菌體的葡萄糖代謝分別下降40%及30%。為了瞭解葡萄糖濃度與細胞生長的關係，增加了葡萄糖的濃度，然而各基因生長趨勢相較於增加前的轉型株無提升，在葡萄糖消耗方面，除gap2轉型株外，各基因轉型株的葡萄糖消耗量消較於控制組減少，pyk轉型株、pfk1轉型株、pfk2轉型株及gap1轉型株分別減少了65%、42%、63%及48%的葡萄糖代謝。在液化澱粉芽孢桿菌方面，我們表現了三個內生性糖解作用基因，根據生長曲線圖結果顯示，在各轉型株的生長速度皆有增加，其中在pyk轉型株及gap1轉型株的生長提升效果最好，分別增加了115%及116%，而pyk轉型株的葡萄糖代謝量降低了22%，但gap1增加了10%。由結果可以說明三個糖解作用基因可以藉由調節體內蛋白改變生長速度及葡萄糖的代謝。
Glycolysis is an important pathway in organism. Energy was obtained via carbohydrate degradation. pyk, pfk, gap were indicated as key enzymes of glycolysis. The aim of this study was to investigate the influence of glycolysis gene overexpression on the growth rate of Escherichia coli and B.amyloliquefaciens. Three exogenous glycolysis genes were expressed in E.coli systems. we expressed three exogenous glycolysis genes. The growth curve results showed that the growth rates of transformants were increased insignificantly. In glucose metabolism, pfk2 transformant, gap1 transformant and gap2 transformant increased 230%, 1530% and 150% of glucose consumption, respectively after IPTG induction, the glucose utilizations of transformants expressing pyk gene and pfk1 gene were decreased to 60% and 70%, respectively. In order to understand the relationship between cell growth and glucose consumption, the concentration of glucose was increased. However, the growth rate didn’t increase significantly. But every transformant decreased glucose consumption except gap2 transformant. In pyk transformant, pfk1 transformant, pfk2 transformant and gap1 transformant were decreased 126%, 65%, 42%, 63% and 48% respectively after IPTG induction. In B.amyloliquefaciens system, we also expressed three endogenous glycolysis genes. The results of growth curve showed that the growth rate of all transformants was increased. The pyk gene and gap1 gene transformed strains were obviously increased 115% abd 116% during exponential phase. The level of glucose consumption was decreased 22% in pyk transformant but increased 10% in gap1 transformant. It represneted the pyk transformant have ability to increase the growth rate without high glucose consumption.
中文摘要 I
Abstract II
謝誌 IV
目錄 V
圖目錄 VIII
表目錄 IX
第一章 前言 1
第二章 文獻回顧 2
2.1 糖解作用 2
2.1.1 糖解作用之介紹 2
2.1.2 糖解作用之步驟 2
2.1.3 Pyruvate kinase介紹暨應用 3
2.1.4 Phosphofructokinase介紹暨應用 4
2.1.5 Glyceraldehyde 3 phosphate dehydrogenase介紹暨應用 4
2.2 液化澱粉芽孢桿菌 5
2.2.1 液化澱粉芽孢桿菌之介紹 5
2.2.2 液化澱粉芽孢桿菌之表觀特徵 5
2.2.3 液化澱粉芽孢桿菌之應用 6
第三章 材料與方法 7
3.1 實驗架構 7
3.2 yT&A載體的建構 8
3.2.1 菌種活化 8
3.2.2 Genomic DNA extraction 8
3.2.3 糖解作用基因的Polymerase Chain Reaction( PCR)反應 9
3.2.4 PCR產物純化 10
3.2.5 yT&A vector建構 11
3.2.6 DH5α勝任細胞製備 12
3.2.7 yT&A載體轉型/藍白篩 13
3.2.8 Colony PCR篩選轉型株 13
3.2.9 質體抽取法 14
3.2.10 限制酶確認 14
3.2.11 菌體及質體的保存 15
3.3 表現菌株構築 15
3.3.1 限制酶作用 15
3.3.2 切膠純化 15
3.3.3 載體去磷酸化反應 16
3.3.4 表現載體的接合反應 16
3.3.5 表現菌株勝任細胞製備 17
3.3.6 表現載體轉型 18
3.4 蛋白質分析 19
3.4.1 蛋白質最適誘導時間 19
3.4.2 蛋白質濃度定量 19
3.4.3 總蛋白的SDS-PAGE膠體分析 19
3.4.4 二維電泳蛋白質定量分析 21
3.4.5 一維蛋白質等電點電泳 21
3.4.6 一維strip平衡 22
3.4.7 二維蛋白質電泳分析 22
3.5 微生物生長評估 23
3.5.1 微生物生長曲線 23
3.5.2 培養基葡萄糖分析 25
3.5.3 葡萄糖檢量線製作 25
第四章 結果 26
4.1. 糖解基因選殖 26
4.1.1 B.amyloliquefaciens DNA抽取 26
4.1.2 糖解基因的PCR擴增 26
4.1.3 TA載體選殖確認 27
4.2. 表現載體選殖 30
4.2.1 大腸桿菌 BL21(DE3)選殖 30
4.2.2 液化澱粉芽孢桿菌選殖 31
4.3. E.coli BL21(DE3)的蛋白質表現確認 31
4.3.1 最適蛋白質誘導時間 31
4.4. 生長曲線暨葡萄糖觀測 33
4.4.1 Xylanase轉型株生長觀測 33
4.4.2 PYK轉型株生長觀測 35
4.4.3 PFK1轉型株生長觀測 36
4.4.4 PFK2轉型株生長觀測 37
4.4.5 GAP1轉型株生長觀測 39
4.4.6 GAP2轉型株生長觀測 40
4.5. 蛋白質體檢測 42
第五章 討論 47
第六章 結論 48
第七章 參考文獻 49
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