臺灣博碩士論文加值系統

中文摘要
澱粉分支酶 (starch branching enzyme, SBE, EC 2.4.1.18) 是澱粉生合成路徑的酵素之一，作用為形成分支，在支鏈澱粉 (amylopectin)的合成上扮演相當重要的角色。本論文目的是選殖SBE cDNA全長，分析其結構，並且探討其特性。依據資料庫檢索已知SBE的保守區間所設計的基因特異性引子，對成長中之台南五號綠豆 (Vigna radiata cv. Tainan no.5) 所萃取出的mRNA進行cDNA選殖。先以反轉錄聚合酶鏈鎖反應 (RT-PCR) 得到部分cDNA片段，經GCG核酸資料庫比對，確定為SBEII與SBEI異構酶形式，再由內部序列設計引子進行快速放大cDNA末端序列 (5’與3’ RACE)方法，成功的獲得兩異構酶編碼區之全長cDNA，分別為2571 bp及2208 bp，命名為VrsbeII及VrsbeI，已在Genebank 註冊 (accession no. AY622199與AY667492 )；其包含起始到終止密碼子的完整ORF，並分別轉譯出856個與735個胺基酸，預估約 97 kDa 及84 kDa分子大小蛋白質，pI為5.47及6.35；且都具有符合α-amylase family的特徵，包括四個活化部位的保留區域-分別為HSHS/A S、GFRFDGVT、G/AEDVS和AESHDQ，及特有的催化區 (β/α)8-barrel domain。與資料庫比對後，發現序列及演化關係均和菜豆 (Phaseolus vulgaris，kidney bean) 及豌豆 (Pisum sativum，pea) 最為相似，而兩者間cDNA的相同性為59%，胺基酸同質性也只有56%，並由演化分析證實這兩種SBE異構型分別屬於不同的演化族群 (Family A及B)。因此，本論文結果可得知，在綠豆種子發育過程中，至少有兩種不同的SBE異構酶參與澱粉生合成作用。

Abstract
Starch branching enzyme (SBE, EC 2.4.1.18) is one of the enzymes in the starch biosynthetic pathway. It catalyzes the formation of branches, which plays a vital role in amylopectin synthesis. The objectives of this thesis are to clone full-length cDNA of SBE and analyze its structure and characteristics. Based on database search, the conserved regions from published SBE genes were obtained and used to design gene-specific primers for cloning. First, the mRNA from developing mungbean (Vigna radiata, cv. Tainan no.5) was extracted and used as template in RT-PCR. The cDNA sequences of the amplified RT-PCR products, after comparing with GCG nucleotide database, demonstrates that partial cDNA of two distinct mungbean SBE isoforms (SBEII and SBEI) were found. Then, primers designed from internal sequences of SBEII and SBEI were used in cloning their 5’ and 3’ cDNA ends by 5’/3’ RACE (rapid amplification of cDNA ends). The full-length cDNAs of the two SBE isoforms were obtained successfully, which possess sequences of 2571bp and 2208bp in length (designated VrsbeII and VrsbeI), respectively. VrsbeII and VrsbeI have also been registered in GenBank with accession numbers of AY622199 and AY667492. They both contain complete open reading frame (ORF) that covers from start codon to stop codon. VrsbeII encodes a polypeptide of 856 amino acids with predicted molecular mass of 97 kDa and pI of 5.47. Whereas, VrsbeI encodes a polypeptide of 735 amino acids with predicted molecular mass of 84 kDa and pI of 6.35. Besides, their putative protein sequences possess the properties of the α- amylase family, including four active conserved region- HSHS/A S, GFRFDG VT, G/AEDVS and AESHDQ, and catalytic (β/α)8-barrel domain. When compared in database, both VrsbeII and VrsbeI showed substantial similarity to the SBEs of kidney bean and pea.
Furthermore, the identities between mungbean SBEII and SBEI at nucleic acid and protein levels are 59% and 56%, respectively. The deduced amino acid sequences from VrsbeII and VrsbeI via phylogenetic analysis is evident that they fall into two distinct gene families (family A and B). In conclusion, there are at least two different SBE isoforms involved in starch biosynthesis during the development of mungbean.

總目錄
總目錄……………………………………………………………………I
圖目錄…………………………………………………………………..III
表目錄…………………………………………………………………..IV
中文摘要………………………………………………………………...V
英文摘要……………………………………………………………….VI
第一章? 緒論
第一節 澱粉生合成作用
1.1澱粉的組成及結構…………………………………………….1
1.2 參與澱粉生合成的酵素……………………………………....2
第二節 澱粉分支酵素之分子生物學研究
2.1澱粉分支酵素基因選殖研究………………………………….7
2.2基因結構的研究………………………………………………10
2.3 SBEII (family A)與SBEI (family B) 異構型間的差異……...14
第三節豆科植物之澱粉分支酵素的研究
3.1 豌豆 (Pisum sativum ,Pea)…………………………………..17
3.2 菜豆 (Phaseolus vulgaris, Kidney bean)…………………….19
第四節 綠豆之簡介………………………………………………...20
第五節 本論文之研究起源與目的...................................................24
第二章? 材料與方法
第一節 材料
1.1 樣品.........................................................................................25
1.2 藥品………………………………………………………….25
1.3 儀器設備…………………………………………………….26
第二節 引子的設計與合成
2.1設計引子..................................................................................27
2.2合成引子……………………………………………………..28
第三節 RNA之抽取與分析
3.1 Total RNA之抽取................................................................28
3.2 Poly (A) RNA之分離..........................................................30
第四節DNA片段的製備與純化
4.1 反轉錄酶-聚合酶鏈鎖反應 (RT-PCR)…………………….32
4.2 聚合酶鏈鎖反應 (PCR)…………………………………….33
4.3 快速放大cDNA末端序列 (RACE).....................................34
4.4 DNA片段之純化方法…………………………………….38
第五節 cDNA之選殖與分析
5.1 T-A cloning…………………………………………………...40
5.2抽質體DNA………………………………………………….44
第六節 核酸電泳分析法
6.1 DNA洋菜膠體電泳分析法.....................................................45
6.2 RNA甲醛洋菜膠體電泳分析法………………………….....46
第七節 DNA 序列分析
7.1定序部份……………………………………………………..48
7.2 DNA序列比對分析…………………………………………48
第三章? 結果與討論?
第一節RNA 的抽取與品質分析…………………………………50
第二節 澱粉分支酵素 cDNA之選殖
2.1選殖VrsbeII cDNA………………………………………….51
2.2選殖VrsbeI cDNA…………………………………………..54
2.3 VrsbI 與VrsbII全長cDNA序列確認……………………..56
第三節綠豆SBE全長cDNA序列之特性分析
3.1 cDNA及胺基酸序列之特性……………………………….56
3.2 VrSBEII與VrSBEI之酵素活性區域結構...........................58
3.3 比對分析綠豆與各種植物 SBE胺基酸序列間的差異….59
3.4 預測訊息生肽 (signal peptide) 之切位..............................60
3.5 分析綠豆與各物種SBE之演化關係……………………..61
第四章? 結論
4.1 論文總結…………………………………………………...63
4.2 未來研究方向……………………………………………...64
結果圖與表…………………………………………………………..67
參考文獻……………………………………………………………..91
附錄………………………………………………………………….100
圖目錄
圖一、支鏈澱粉的分支結構………………………………………….2
圖二、澱粉的生合成路徑圖………………………………………….6
圖三、SBE family A與B之基因結構................................................13
圖四、5’-RACE的反應機制…………………………………………35
圖五、3’-RACE的反應機制…………………………………………37
圖六、pGEM-T Easy 載體 DNA的結構圖 (Promega)……………42
圖七、選殖綠豆SBE cDNA的流程圖...............................................66
圖八、綠豆total RNA之甲醛洋菜膠體電泳圖.................................68
圖九、以SBEF1和SBER1為引子進行RT-PCR及PCR二次放
大後產物的洋菜膠體電泳圖…………………………………70
圖十、以 SBEF2及NPU為引子進行3’RACE所獲得之產物洋
菜膠體電泳圖…………………………………………………71
圖十一、以SBEF6及SBER5為引子進行RT-PCR及PCR二次
放大後產物的洋菜膠體電泳圖……………………………72
圖十二、以SBEF6及SBER9為引子進行RT-PCR放大後所得
全長Vrsbe II cDNA的洋菜膠體電泳圖…………………..73
圖十三、由VrsbeIIp-0.8抽出之質體DNA及以EcoRI切出接入
載體上之DNA (VrsbeII-0.8)的電泳圖…………………….74
圖十四、VrsbeII cDNA基因選殖之RT-PCR及RACE對照圖……75
圖十五、VrsbeII cDNA及其演繹出之胺基酸序列對照圖…………76
圖十六、以SBEF9和SBER13引l進行RT-PCR放大後產物的
洋菜膠體電泳圖……………………………………………78
圖十七、以SBEF10和SBER17引子進行RT-PCR放大後產物的
洋菜膠體電泳圖……………………………………………79
圖十八、以SBEF11和SBER16引子進行RT-PCR放大後產物的
洋菜膠體電泳圖……………………………………………80
圖十九、以SBEF9及SBER21為引子進行RT-PCR及PCR二次
放大後所得全長Vrsbe I cDNA的洋菜膠體電泳圖...........81
圖二十、VrsbeI cDNA基因選殖之RT-PCR及RACE對照圖…….82
圖二十一、VrsbeI cDNA及其演繹出之胺基酸序列對照圖……….83
圖二十二、VrsbeII, VrsbeI 與各種植物SBE胺基酸序列之比較....84
圖二十三、根據綠豆與不同物種之SBE cDNA演繹出之胺基酸序
列所推測出之演化樹狀圖................................................90
附錄圖二十四、VrsbeII cDNA的限制酵素切位圖譜…………..….100
附錄圖二十五、VrsbeI cDNA的限制酵素切位圖譜…………….…103
表目錄
表一、不同物種之SBEI異構型的研究………………………………8
表二、不同物種之SBEII異構型的研究……………………………...9
表三、澱粉分支酵素異構型的催化特性比較.....................................16
表四、綠豆與菜豆及豌豆之signal peptide切位的比較.....................61
表五、綠豆 total RNA 與 poly (A) RNA 之萃取純化結果..............67
表六、VrsbeII cDNA 專一性引子序列................................................69
表七、VrsbeI cDNA 專一性引子序列………………………………..77
表八、綠豆SBE與不同物種SBE胺基酸序列之同質性比較...........88
表九、根據Distance program分析 (Kimura protein-distance algorithm)
綠豆與不同物種之SBE cDNA演繹出之胺基酸序列間的演化
相關……………………………………………………………..89
附錄表十及十一、綠豆SBE cDNA 演繹之胺基酸序列在NCBI
? ? ? ? ? 資料庫的搜尋比…………………………………106

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