臺灣博碩士論文加值系統

本實驗目的擬以目前台灣佔有率最高之基因改造大豆Roundup Ready® soybean (RRS) 品系為目標，建立市售大豆產品中RRS成分之快速鑑定系統。實驗中比較緩衝液萃取法及傳統的CTAB沈澱法對於DNA之回收效率，結果顯示利用兩種方法對於完整大豆萃取皆可得高純度之genomic DNA，但在各類市售樣品之DNA萃取上，利用緩衝液對加工樣品中之DNA萃取可較傳統CTAB沈澱法更為有效，同時對於高分子量DNA回收效率亦較佳，且品質仍可供為PCR反應之模板使用。另外根據RRS插入表現序列之調節基因CaMV 35S promoter設計GM-f/GM-r引子對，作為是否含RRS之鑑定依據，同時以大豆中轉譯大豆凝集素之Le1基因為目標序列，設計Le-f/Le-r引子對，以分辨是否含大豆成分，配合multiplex PCR建立可快速檢測RRS之分析方法。利用非大豆成分得知設計引子僅對RRS及大豆成分具專一性，同時最低偵測極限為0.1%RRS，PCR反應產物亦經純化定序與原設計相符。在實驗中所收集之九類樣品中，對於A、B、D、E、F類等低加工層次之大豆原料及加工製品，可利用multiplex PCR快速鑑定是否含有基因改造大豆成分，C、G、H、I類等成分複雜或加工程度高之市售樣品，則需以單套引子PCR系統檢測，因此本系統在因應不同型態之大豆原料及其市售產品之檢測上，可適用於我國目前所訂立的5% 基因改造成分標示基準。

The objective of this study was to develop a suitable method for detection of Roundup Ready® soybean (RRS) ingredient in foodstuffs. Both buffer extraction and CTAB precipitate methods could isolate high quality DNA of whole soybean. For the different types of commercial products, buffer extraction showed higher recovery of DNA compared to CTAB method. The GM-f/GM-r primer pair that targeted to foreign gene CaMV 35S promoter and the Le-f/Le-r primer complementary to soybean Le1 sequence were constructed. Application of individual or both primer pairs in PCR or multiplex PCR could identify the presence of RRS and soybean ingredient in soy-derived foods. The detection limit of RRS was 0.1% RRS by multiplex PCR with 2 primers. The sequence of PCR product was identical to the original sequence. Using multiplex PCR could correctly and rapidly distinguish RRS content in sample A, B, D, E and F. Highly processed sample, such as C, G, H and I, could be identified the RRS ingredient by single primer PCR. Therefore, the developed multiplex PCR method could be applied for the regulation of 5% GMOs content labeling in Taiwan.

中文摘要 I
英文摘要 III
誌謝 IV
目錄 VI
圖索引 X
表索引 XII
第一章 前言 1
第二章 文獻回顧 4
2.1基因改造生物(genetically modified organisms, GMOs) 4
2.1.1基因改造生物的目的 6
2.1.2基因轉殖的方法 12
2.1.2.1粒子槍法 12
2.1.2.2農桿菌轉殖法 15
2.1.2.3電穿孔 15
2.2大豆 (Glycine max (L.) Merrill) 16
2.2.1大豆的介紹 16
2.2.2大豆凝集素 18
2.3基因改造大豆Roundup Ready® soybean 18
2.3.1除草劑的作用機制 20
2.3.2 RRS的抗除草劑機制 20
2.3.3 RRS之轉殖基因 23
2.3.3.1 CaMV 35S promoter 23
2.3.3.2 Petunia EPSPS CTP 25
2.3.3.3 CP4 EPSPS基因 25
2.3.3.4 NOS 3’ terminator 25
2.3.3.5重組基因之構築及傳送 26
2.4基因改造生物所引發的疑慮 26
2.4.1標誌基因的評估 26
2.4.2過敏反應 28
2.4.3微生物的病原性 29
2.5實質等同的概念 29
2.6各國對GMOs之規範 32
2.7台灣的消費態度及標示規範 36
2.8 GMOs之檢測方法 37
2.8.1 GMOs檢測的限制 38
2.8.2聚合酵素連鎖反應 (polymerase chain reaction) 42
2.8.3複合式聚合酵素連鎖反應 (multiplex PCR) 42
第三章 材料與方法 44
3.1儀器設備 44
3.2藥品試劑 44
3.3樣品 45
3.4樣品前處理 46
3.5樣品中DNA萃取及純化 46
3.5.1緩衝液萃取方法 46
3.5.2 CTAB沈澱法 47
3.6 DNA之定量及品質確認 48
3.7引子之設計 49
3.8聚合酵素連鎖反應 49
3.8.1 multiplex PCR 49
3.8.2 GM-f /GM-r PCR 50
3.8.3 Le-f /Le-r PCR 50
3.9瓊膠電泳分析及影像處理 51
3.10 PCR產物純化 51
3.11 PCR產物之核苷酸定序分析及序列比對 52
第四章 結果與討論 53
4.1樣品之DNA製備 53
4.2引子之設計 61
4.3 multiplex PCR系統之建立 62
4.4 multiplex PCR之系統特異性 65
4.5 multiplex PCR之系統靈敏度 67
4.6市售樣品之檢測 69
4.6.1 A-食品加工原物料之檢測 69
4.6.2 B-低加工層次大豆製品之檢測 72
4.6.3 C-市售嗜好性食品之檢測 75
4.6.4 D-各類豆腐之檢測 78
4.6.5 E-市售豆花之檢測 80
4.6.6 F-市售豆漿之檢測 80
4.6.7 G-市售大豆重組製品及仿素肉之檢測 80
4.6.8 H-大豆添加物之檢測 86
4.6.9 I -市售醱酵食品之檢測 86
4.7 PCR產物之定序確認 91
第五章 結論 92
第六章 參考文獻 93
第七章 附錄 113
附錄一「基因改造黃豆及玉米之查驗與登記」細節 113
附錄二「基因改造黃豆及玉米之食品標示」細節 114
附錄三 實驗樣品市售之品名及成份 116
圖索引
圖一、大豆加工食品之種類 17
圖二、芳香族胺基酸生合成途徑 21
圖三、glyphosate之作用機制 22
圖四、GTS line 40-3-2插入的基因序列構成圖 24
圖五、質體PV-GMGT04及構築之重組基因 27
圖六、GMOs食品中潛在過敏原的評估方法 30
圖七、目前GMOs之PCR方法檢測流程 39
圖八、Roundup Ready® soybean、Non-GM soybean及A-I九類不同型態
之市售樣品萃取純化之DNA分佈 56
圖九、GM-f /GM-r及Le-f/Le-r引子對之設計 63
圖十、以multiplex PCR系統檢測RRS及非基因改造大豆 64
圖十一、multiplex PCR之系統特異性 66
圖十二、multiplex PCR對RRS檢測系統之靈敏度 68
圖十三、以2%瓊膠電泳分析A類樣品以multiplex PCR檢測RRS成分
之產物 70
圖十四、以2%瓊膠電泳分析A-4、A-9~12以GM-f/GM-r PCR及Le-f
/Le-r PCR檢測RRS成分之產物 73
圖十五、以2%瓊膠電泳分析B類樣品以multiplex PCR檢測RRS成分
之產物 74
圖十六、以2%瓊膠電泳分析C類樣品以multiplex PCR檢測RRS成分
之產物 76
圖十七、以2%瓊膠電泳分析C類樣品以GM-f/GM-r PCR及Le-f/Le-r
PCR檢測RRS成分之產物 77
圖十八、以2%瓊膠電泳分析D類樣品以multiplex PCR檢測RRS成分
之產物 79
圖十九、以2%瓊膠電泳分析E類樣品以multiplex PCR檢測RRS成分
之產物 81
圖二十、以2%瓊膠電泳分析F類樣品以multiplex PCR檢測RRS成分
之產物 82
圖二十一、以2%瓊膠電泳分析G類樣品以multiplex PCR檢測RRS成分
之產物 83
圖二十二、以2%瓊膠電泳分析G類樣品以GM-f/GM-r PCR及Le-f
/Le-r PCR檢測RRS成分之產物 85
圖二十三、以2%瓊膠電泳分析H類樣品以multiplex PCR檢測RRS成分
之產物 87
圖二十四、以2%瓊膠電泳分析H類樣品以GM-f/GM-r PCR及Le-f
/Le-r PCR檢測RRS成分之產物 88
圖二十五、以2%瓊膠電泳分析I類樣品以multiplex PCR檢測RRS成分
之產物 89
圖二十六、以2%瓊膠電泳分析I 類樣品以GM-f/GM-r PCR及Le-f
/Le-r PCR檢測RRS成分之產物 90
表索引
表一、基因改造生物之相關名詞 5
表二、2000年全球五大基因改造作物及其栽種面積 7
表三、經FDA核准於全球已商品化之基因改造作物 8
表四、主要基因轉殖作物種類及其轉殖方法 13
表五、粒子槍於植物基因轉殖之應用 14
表六、植物中所具有的天然毒素或抗營養成分 31
表七、不同基因改造大豆中之營養抑制劑含量 33
表八、各國對基因改造食品標示之基準 35
表九、GMOs檢測所針對之目標序列及其靈敏度比較 41
表十、比較緩衝液萃取法及CTAB沈澱法對大豆粉末及不同種類
大豆加工食品DNA抽取之差異 54

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