臺灣博碩士論文加值系統

澱粉是高等植物之醣類儲存的主要形式，在澱粉生合成路徑中，澱粉磷解酶 (starch phosphorylase，EC 2.4.1.1，SP) 是具有催化澱粉降解與合成的雙向活性。綠豆 (mungbean, Vigna radiata L.) 澱粉最特別的是直鏈澱粉 (amylose) 含量較一般澱粉的 15-30% 高，推測 SP 對合成直鏈澱粉扮演重要角色。先前利用胰蛋白酶水解、MALDI-TOF 與免疫分析方法，完成綠豆 105-kDa SP 的鑑定與活性相關性探討，本研究目的是進一步選殖出綠豆 SP 的 cDNA，未來探討重組 SP 酵素特性與功能。根據綠豆 SP 靠近 N 端及 C 端的內部序列，設計出退化性引子 (degenerate primer) 當作基因特異性引子 (gene-specific primers；GSP)，以發育中的「台南五號」(Vigna radiata cv. Tainan no.5) 綠豆種子為材料，抽取其 total RNA、純化為 mRNA 後進行反轉錄酶-聚合酶鏈鎖反應 (reverse transcriptase-polymerase chain reaction, RT-PCR)，首先獲得中間 1819 bp 序列片段，接著往內部序列設計引子，並以蠶豆、甘薯和馬鈴薯 5’ 及 3’ 核苷酸保守序列設計出之引子再進行 RT-PCR，分別獲得含 ATG 起始碼的 5’ 序列 640 bp，及 3’ 包含終止密碼 997 bp 序列，因而成功的獲得全長之綠豆 SP cDNA 序列為 2961 bp，命名為 Vrsp，其包含起始到終止密碼子的完整 open reading frame (ORF)，並編碼 987 個胺基酸，預估約 111 kDa 分子大小蛋白質，pI 為 5.38 ；序列的特性包括具有澱粉結合位、L-78、催化部位 (catalytic site) 及磷酸吡哆醛 (pyridoxal phosphate ; PLP) 結合位，由於 Vrsp 含有 L-78 插入序列，歸類於 L-型的 SP 異構酶。

Starch is the major carbohydrate reserve in higher plants. In the starch biosynthetic pathway, starch phosphorylase (EC 2.4.1.1, SP) catalyzes a reversible reaction between synthesis and degradation of starch with bidirectional activities. Mungbean (Vigna radiata L.) starch is the most special that amylose content was 15-30% higher than normal level, which implied that SP plays an important role in amylose synthesis. Previous studies have identified the tryptic fragments of mungbean 105-kDa SP by MALDI-TOF and conducted immunological analysis. The objective of this study was to further clone the full-length cDNA of SP in order to investigate the characteristics and function of recombination SP in the future. Based on the internal amino acid sequence of N- and C-terminal of mungbean SP, degenerate primers were designed as gene-specific primers (GSP). The developing mungbean seed (Vigna radiata cv. Tainan no.5) was used as the material to extract its total RNA, and mRNA was purified and used as the template in RT-PCR. First, a 1819 bp sequence of a middle fragment was obtained, and then primers were designed from its internal sequence. The 5’-nucleotide conserved region sequences from fave bean, sweet potato and potato were used to design primers which were coupled with internal GSPs for RT-PCR amplification. Sequences containing the start codon ATG of 5’ 640 bp and 3’ sequences containing the stop codon TAG of 997 bp were obtained successfully. The full-length cDNA of the mungbean SP which possessed nucleotides of 2961 bp in length, containing complete open reading frame (ORF) that covers from start codon to stop codon was designated as Vrsp. Vrsp encodes a polypeptide of 987 amino acids with predicted molecular mass of 111 kDa and pI of 5.38. Putative protein sequences possess the motifs of starch binding site, L-78, catalytic site and pyridoxal phosphate (PLP) binding site. In addition, Vrsp includes the L-78 insertion sequence, it belongs to the known L-type isoform.

總目錄 I
圖目錄 III
表目錄 IV
中文摘要 VI
英文摘要 VII
第一章 前言 1
第二章 文獻整理 3
第一節 澱粉的生合成作用 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 23
第二節 澱粉磷解酶之研究 7
2.1 澱粉磷解酶的發現 7
2.2 澱粉磷解酶的催化作用與分類 8
2.3 澱粉磷解酶異構型的生化探討 10
2.4 澱粉磷解酶異構型的表現位置探討 12
2.5 綠豆中澱粉磷解酶 105-kDa蛋白質的發現 13
2.6 澱粉磷解酶基因選殖的研究 13
2.7 肝醣磷解酶的介紹 14
第三節 綠豆 19
3.1 簡介 19
3.2 成分分析 20
3.3 物理特性方面 21
3.4 生理功用 21
3.5 加工性質方面 22
第三章 材料與方法 29
第一節 材料 29
1.1 樣品 29
1.2 藥品 29
1.3 儀器設備 30
1.4 勝任細胞商品 31
第二節 引子的設計與合成 31
2.1 設計引子 31
2.2 合成引子 32
第三節 Total RNA 之萃取 32
3.1 Total RNA 之抽取 32
3.2 RNA 甲醛洋菜膠體電泳分析法 34
3.3 mRNA 之純化 36
第四節 DNA 片段的增幅與純化 38
4.1 反轉錄酶作用 (reverse transcriptase, RT) 38
4.2 聚合酶鏈鎖反應 ( polymerase chain reaction, PCR) 39
4.3 快速放大 cDNA 末端序列 (rapid amplification of cDNA ends, RACE) 40
4.4 DNA 洋菜膠體電泳分析法 42
4.5 DNA 片段之純化方法 43
第五節 cDNA 的基因選殖 43
5.1 TA cloning 43
5.2質體DNA的抽取 47
5.3 DNA 之限制酶 (restriction enzyme) 的切割作用 48
5.4全長DNA 之選殖 48
第六節 DNA定序與序列特性分析 48
6.1 DNA定序 48
6.2 DNA 序列比對分析 49
第四章 結果與討論 50
第一節 RNA 的萃取與品質分析 50
第二節 澱粉磷解酶之基因選殖 50
2.1 以β-actin 基因作為基因表現的控制組 51
2.2 選殖 Vrsp cDNA 51
2.3 Vrsp 全長cDNA 序列確認 54
第三節 綠豆 SP cDNA 序列之特性分析 54
3.1 分析 Vrsp 與各物種植物 SP cDNA 及胺基酸序列比較 54
3.2 Vrsp 之酵素活性區域結構比較 55
3.3 L-78 區域之比較 56
3.4 分析綠豆與各物種 SP 之演化關係 56
第五章 結論 57
5.1 論文總結 57
5.2 未來研究方向 57
結果圖與表 60
參考文獻 87

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