cDNA Cloning, Sequence and Characterization of Starch Branching Enzyme from Colocasia esculenta var. esculenta

• Main Authors: Yu-Hui Yang, 楊于慧
• Other Authors: Chii-Ling Jeang
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/62668167497846993329

碩士 === 國立中興大學 === 食品科學系 === 92 === Starch is a major constituent of higher plants, and is widely utilized in foods as well as in industrial processes. Starch is composed of the two polymers, amylose and amylopectin. Although the pathway of starch biosynthesis is not completely understood, there is no doubt that it involves at least four groups of committed enzymes：ADP-glucose pyrophosphorylases (AGPs), starch synthases (SSs), starch branching enzymes (SBEs) and starch debranching enzymes (SDBEs). SBEs catalyze the formation of α-1,6 linkage. Based on their predicted primary protein sequences deduced from the respective genes, all isoforms can be separated into two classes: family A and family B. Enzymatic and biochemistry of SBE maybe import- ant factors to influence the structure of starch. We have isolated the total RNA from taro (Colocasia esculenta var. esculenta), and than have used gene specific primers of starch branching enzyme family A from plants, combined with the RT-PCR、PCR、3’-RACE and 5’-RACE technology to identify the full-length SBEA cDNA. The full-length of SBEA cDNA is 3156bp. The cDNA sequence contains one open reading frame (ORF) starting from nucleotide 76~78 with a ATG start codon and ending at nucleotide 2608~2610 with a TGA stop codon. A polypeptide of 844 amino acid residues, was deduced from the amino acid sequence ORF was compared with the SBEA from various plants, it was found that was most closely related to maize SBEIIa (74%), rice RBE4 (75%) and kidney bean PvSBE2 (72%). Partial SBEA cDNA fragment (about 2.2 kb) was subcloned into an expression vector, pET-21b(＋), and the newly constructed recommbinant plasmids was designated as pSBEA2.2. The transformant, E.coli (pSBEA2.2), was grown at 37℃ to A600 reached 0.6, then production of rSBEA2.2 was induced by 0.5 mM IPTG and continuously cultured for five hours. Then, IgY from egg yolk was raised by immuneizing chicken with rSBEA2.2. By western blot analysis, the results showed two protein signals about 86 and 93 kDa in size from samples of taro leaf and tuber. Besides, there is a protein signal about 150 kDa in leaf of taro, it maybe granule-bound starch branching enzyme. Combined 2D-PAGE electrophoresis and western blot analysis of total proteins extracted from taro tuber, there are four and five protein spots around 86 to 93 kDa at pI 5.4, respectively. It was suggested that there may be at least four different isoforms of SBEA.