臺灣博碩士論文加值系統

白胺酸N端切位酶（Leucine aminopeptidase；LAP）可水解大分子量食物蛋白質，特異性（specific）切除胜肽兩端的疏水性胺基酸，除去苦味，使食物呈現正面的風味。本研究以食品發酵工業常使用的菌種Aspergillus oryzae的LAP基因，構築於pMON530表現載體上，並轉型至番茄植株以建立含Aspergillus oryzae LAP基因的轉基因株系。爲此，藉內含LAP基因之質體pQE30-lap為模板，設計引子進行聚合酶連鎖反應（PCR），以擴增含LAP基因的DNA片段。所增殖之LAP基因5’-端另包含6個histidine殘基，以方便LAP蛋白純化分析。將PCR擴增之DNA片段接合於pMON530載體，利用質體上之CaMV35S promoter為啟動子，建構成pMON530-LAP質體。再藉農桿菌轉殖方式，將Aspergillus oryzae LAP基因轉型至番茄植株中，並藉由genomic PCR鑑定LAP基因確已嵌插至轉殖植株之染色體中。經馴化後各轉基因株系隨即出瓶轉移至溫室培養。

Aspergillus oryzae leucine aminopeptidase (LAP) specifically and effectively catalyzes the hydrolytic digestion of the hydrophobic amino acid residues in the N-terminus of the peptides of different origin. For the purpose of reducing the production cost of LAP, the LAP gene was amplified by polymerase chain reaction (PCR), and cloned into plant expression vector pMON530. A hexa-histidine affinity tag was fused to the N-terminus of the LAP gene for protein purification by affinity chromatography. With leaf-disc method, the recombinant plasmid was introduced into tomato cells via Agrobacterium tumefaciens-mediated transformation to overexpress LAP gene in plants. LAP gene was already transformation in tomato plant, and determine by genomic PCR. The transgenic tomato of Aspergillus oryzae Leucine Aminopeptidase also domestication and growing in green house.

封面內頁
簽名頁
授權書1...........................................................................................iii
授權書2............................................................................................iv
中文摘要...........................................................................................v
英文摘要..........................................................................................vi
誌謝.................................................................................................vii
目錄................................................................................................viii
圖目錄.............................................................................................xii
表目錄............................................................................................xiii

第一章 前言.....................................................................................1
1.1 食品與胜肽之關係………………...…......................1
1.2 胜肽與風味之關係…………………………………..2
1.3 食品風味之改善……………………………………..2
1.4 Leucine aminopeptidase與胜肽苦味………………..4
1.5 LAP於食品工業之重要性…………………………..6
1.6 植物與外源性蛋白之生產…………………………..7
1.7 研究目的及展望........................................................8
第二章 材料與方法..........................................................................9
2.1 實驗材料....................................................................9
2.1.1 轉殖材料........................................................9
2.1.2 菌株及質體....................................................9
2.1.3 培養基..........................................................10
2.1.3.1 菌類培養基....................................10
2.1.3.2 植物培養基（番茄植株）.................10
2.1.4 植物荷爾蒙..................................................11
2.1.5 其他緩衝液及試劑......................................11
2.2 實驗方法..................................................................12
2.2.1 質體（plasmid）DNA之小量製備.............12
2.2.2 以試劑組萃取小量質體DNA......................13
2.2.3 以試劑組萃取大量質體DNA......................14
2.2.4 引子(primer)設計.........................................15
2.2.4.1 LAP基因特異性引子.....................15
2.2.4.2 NPTⅡ基因特異性引子..................15
2.2.5 聚合酶鏈鎖反應（polymerase chain
reaction；PCR）...........................................15
2.2.6 瓊脂凝膠電泳(agarose gel electrophoresis).17
2.2.7 DNA 片段的回收及純化............................17
2.2.8 限制酵素剪切（enzyme digestion）...........18
2.2.9 DNA 的黏接反應（ligation）....................18
2.2.10 勝任細胞(competent cell)之製備.................19
2.2.10.1 大腸桿菌化學法勝任細胞之製備.19
2.2.10.2 大腸桿菌電勝任細胞之製備.........20
2.2.10.3 農桿菌電勝任細胞之製備.............20
2.2.11 轉形作用（transformation）...........................21
2.2.11.1 化學法轉型作用...........................21
2.2.11.2 大腸桿菌電穿孔法
（Electroporation）轉型作用.........21
2.2.11.3 農桿菌電穿孔法轉型作用............22
2.2.12 Colony PCR..................................................22
2.2.13 番茄基因轉殖..............................................23
2.2.13.1 植物材料........................................23
2.2.13.2 農桿菌培養....................................24
2.2.13.3 農桿菌基因轉殖............................24
2.2.13.4 再生植株誘導................................25
2.2.13.5 馴化處理........................................25
2.2.14 轉基因株系之分子分析..............................26
2.2.14.1 植物Genomic DNA抽取法...........26
2.2.14.2 聚合酶鏈鎖反應鑑定轉基因植株.27
第三章 結果與討論........................................................................29
3.1 構築植物表現（轉殖）載體pMON530-LAP................29
3.2 LAP蛋白3D結構的預測...........................................30
3.3 pMON530-LAP轉型於農桿菌...................................31
3.4 番茄轉殖LAP基因....................................................31
3.5 番茄轉基因株系馴化及健化.....................................33
3.6 番茄轉基因株系之分子分析.....................................34
3.7 未來展望....................................................................34
參考文獻.........................................................................................54
附錄一 A. oryzae lap基因之核甘酸及胺基酸序列.......................62

圖目錄

圖一、LAP基因片段與引子設計..................................................36
圖二、PCR產物之電泳分析圖......................................................37
圖三、LAP基因選殖策略圖..........................................................38
圖四、pMON530-LAP表現構築鑑定……..…................................39
圖五、利用PCR鑑定構築的pMON530-LAP表現載體..............40
圖六、LAP蛋白3D結構預測.......................................................41
圖七、LAP蛋白3D結構預測…………….……………………....42
圖八、利用PCR判定pMON530-LAP有無轉型至農桿菌當中…..43
圖九、農桿菌轉殖法......................................................................44
圖十、成功誘導番茄培殖體芽體再生之健康轉殖植株...............45
圖十一、切下健康轉殖植株移至誘導發根培養基.........................46
圖十二、誘導番茄再生的芽體抽長及發根...................................47
圖十三、生長至蘭花瓶頂可增生的番茄植株...............................48
圖十四、切下番茄帶有生長點的間節培養增生...........................49
圖十五、轉基因植物之發根及馴化處理.......................................50
圖十六、抽取擬轉基因番茄genomic DNA以專一性引子進行
PCR偵測NPTⅡ基因.....................................................51

表目錄

表一、菌種特性及來源..................................................................52
表二、質體特性及來源..................................................................53

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