| 摘要: | 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. |
