臺灣博碩士論文加值系統

在澱粉合成路徑中，澱粉合成?(Starch synthase, SS,alpha-1,4-glucan-glucosyltransferase ; EC 2.4.1.21) 是將 ADP-Glucose 以alpha-1,4 glucosidic bond 的方式轉移 glucose至直鏈 glucan chain的非還原端上延長鏈長度，在SS異構形中，顆粒結合性澱粉合成? (GBSS, granule-bound starch synthase) 與直鏈澱粉 (amylose) 生合成有關，因此本實驗目的為選殖出 GBSS 之 cDNA，未來可更進一步探討 GBSS 酵素的特性與功能。先前利用胰蛋白?水解與MALDI-TOF方法完成綠豆58-kDa GBSS的鑑定，本研究目的是進一步選殖出綠豆GBSS的cDNA，未來探討重組GBSS酵素之特性與功能。根據綠豆GBSS已知內部胺基酸序列及各物種GBSS的保守區間 (conserved region) 設計基因特異性引子 (gene specific primer, GSP)，以發育中的「台南五號」(cv. Tainan No. 5) 綠豆種子為材料，抽取其 total RNA 後進行反轉錄-聚合?鏈鎖反應 (reverse transcriptase-polymerase chain reaction, RT-PCR)；首先獲得中間 1025 bp片段，接著以豇豆(cowpea, Vigna unguiculata) 之GBSS (accession No. 145202752)核?酸5’ 及 3’序列往內部設計引子再進行RT-PCR，分別獲得含 ATG 起始碼的 5’ 序列 254 bp，及 3’ 包含終止密碼 519 bp 序列，因而成功的獲得全長之綠豆 GBSS cDNA 序列為 1584 bp，命名為 Vrgbss。其包含起始到終止密碼子的完整 open reading frame (ORF)，推定出編碼 527 個胺基酸，約 58 kDa 分子大小蛋白質，pI為 5.49。

總 目 錄
頁次

總目錄 I
圖目錄 IV
表目錄 V
中文摘要 VI
英文摘要 VII
第一章 研究目的 1
第二章 文獻回顧 3
第一節、澱粉及其生合成途徑 3
1.1 澱粉的組成與結構 3
1.2 澱粉的生合成與參與酵素 4
第二節、澱粉合成?之研究 7
2.1 可溶性澱粉合成?之研究 7
2.2 顆粒結合性澱粉合成?之分類 10
第三節、顆粒結合性澱粉合成?之研究 10
3.1 顆粒結合性澱粉合成?之酵素性質 10
3.2 顆粒性澱粉合成?於分子層次之研究 13
3.3 綠豆顆粒結合性澱粉58 kDa 的發現 15
第四節、綠豆 15
4.1 簡介 15
4.2 成分分析 16
4.3 物理特性 16
4.4 生理功能 17
4.5 加工性質方面 17
第三章 材料與方法 22
第一節、材料 22
1.1 樣品 22
1.2 藥品 22
1.3 儀器設備 23
第二節、Total RNA 之萃取 25
2.1 Total RNA 之抽取 25
2.2 RNA 甲醛洋菜膠體電泳分析法 27
第三節、引子的設計與合成 29
3.1 設計引子 29
3.2 合成引子 29
第四節、DNA 片段的增幅與純化 30
4.1 反轉錄?作用 (RT) 30
4.2 聚合?鏈鎖反應 (PCR) 31
4.3 快速放大 cDNA 末端序列 (RACE) 32
4.4 DNA 洋菜膠體電泳分析法 34
4.5 DNA 片段之純化方法 34
第五節、cDNA 的基因選殖 35
5.1 E. coli JM109 及勝任細胞之製備 (電轉勝任細胞
之製備) 35
5.2 TA cloning 36
5.3 質體DNA的抽取 40
5.4 DNA之限制? (restriction enzyme)的切割作用 40
5.5 全長DNA 之選殖 41
第六節、DNA 定序與序列特性分析 41
6.1 DNA定序 41
6.2 DNA 序列比對分析 41
第四章 結果與討論 43
第一節 RNA的萃取與品質分析 43
第二節 顆粒結合性澱粉合成?之基因選殖 43
2.1 以��-actin 基因作為基因表現的控制組 44
2.2 選殖Vrgbss cDNA 44
2.3 Vrgbss 全長cDNA序列確認 46
第三節 綠豆GBSS Cdna序列之特性分析 46
3.1 分析 Vrgbss與各物種植物GBSS cDNA及胺基酸
序列比較 46
3.2 分析綠豆與各物種GBSS之演化關係 47
第四節 TA-cloning 47
第五章 結論 48
5.1 論文總結 48
5.2 未來研究方向 48
結果圖與表 51
參考文獻 70
附錄一 79

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