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Title: 綠豆cDNA Library的建構與澱粉合成酵素基因的選殖
Authors: 柯源悌
Contributors: 國立臺灣海洋大學:食品科學系
Keywords: 綠豆;澱粉合成脢;基因選殖
Mung bean (Vigna radiata L.);Starch synthase;Gene cloning;RT-PCR
Date: 2002-08
Issue Date: 2013-06-07T07:33:45Z
Publisher: 行政院國家科學委員會
Abstract: 摘要:依據參與澱粉生合成的酵素群BE、SSS、GBSS及SP保守區間所設計的基因特異性引子組,對成長中之台南五號綠豆(Vigna radiata L. cv Tainan no. 5)的PolyA mRNA進行反轉錄聚合脢鏈鎖反應(RT-PCR)的cDNA 選殖,只有SBE F1 及SBE R1引子對,成功的量化出一段794 bp 的cDNA。其內部序列經過與GCG Database 比對,發現和Phaseolus vulgaris物種的BE I(kbe1; accession no. AB029548; 3,360 bp)全長序列的重疊部分有97%的相似度,和 Ipomoea batatas (sweet potato) 的SBE II (AB071286; 3,123 bp) 全長序列的重疊部分有84%的相似度,經過Pile Up並排後,所得綠豆cDNA 序列為靠近中間部位,已將此cDNA 片段選殖到pGEM T-Eazy載體中作為株系保存,命名為pVrbeIp。為了得到全長序列,目前由所得的SBE cDNA片段內部序列已設計引子在單股cDNA library 中進行5'與3' RACE,3' RACE反應較平順,獲得之1,200 bp定序當中。
Abstract:Gene-specific primer pairs designed from cDNA conserved motifs of BE, SSS, GBSS and SP, a group of enzymes involved in starch biosynthesis, were used in RT-PCR cloning on the polyA mRNA of premature mung bean (Vigna radiata L. cv Tainan no. 5). Only SBE F1 and SBE R1 pairs were successfully amplify a partial-length of cDNA (794 bp) encoding starch branching enzyme (SBEI; The internal sequence of the amplicon was searched in the GCG database and found to have 97% similarity with Phaseolus vulgaris BE I (kbe1; accession no. AB029548; 3,360 bp) and 84% similarity with Ipomoea batatas (sweet potato) SBE II (AB071286; 3,123 bp) within the overlapped regions and thus designated VrbeIp. Besides, it was also shown to be located in the central region of the postulated full-length cDNA by PileUp alignment and was then cloned into pGEM T-Eazy vector, designated pVrbeIp for further use. In order to pursue a full-length clone, internal primers were designed from VrbeIp sequence to conduct RACE on the first strand cDNA library. 3'-RACE was performed more smoothly than 5'-RACE and a 1,200 bp fragment were generated and currently subjected to sequencing.
Relation: NSC91-2313-B039-002
URI: http://ntour.ntou.edu.tw/handle/987654321/33818
Appears in Collections:[食品科學系] 研究計畫

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