J Integr Plant Biol. ›› 2002, Vol. 44 ›› Issue (6): 689-693.

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UGPase of Astragalus membranaceus: cDNA Cloning, Analyzing and Expressing in Escherichia coli

WU Xiao Jun, DU Min, WENG Ying Qi, LIU Di, HU Zhi Bi   

Abstract:

On the basis of sequences of UGPase from plants, a cDNA encoding the enzyme was isolated from the hairy root of Astragalus membranaceus (Fisch.) Bunge. The cDNA consisted of 1 831 bp and encoded a polypeptide of 471 amino acid residues with a calculated molecular weight of 51.5 kD and a deduced isoelectric point of 6.01. Then the open read frame of the cDNA was ligated into pET28(a)+ vector and expressed in E. coli BL21. SDS PAGE showed that the expressed protein was ca. 40% in the total bacterial protein. Enzyme activity assay demonstrated that the UGPase activity in the transformed bacteria was 0.50-3.27 times higher than that of the control. Northern blotting revealed that ugp was expressed in the leaf, stem, root and hairy root of A. membranaceus, with a higher levelin root and hairy root.

黄UGPase cDNA 克隆、分析及其在大肠杆菌中的表达
吴晓俊 杜 旻 翁颖琦 刘 涤 胡之璧
(上海中医药研究院中药研究所,上海200032)

摘要: 在已报道的UGPase的植物cDNA序列基础上 ,从膜荚黄芪 (Astragalusmembranaceus (Fisch .)Bunge)毛状根中分离了此酶的cDNA。此cDNA全长为 1 831bp ,推测编码分子量为 5 1 .5kD、等电点为 6 .0 1的由 4 71个氨基酸残基组成的多肽。将此cDNA的开放阅读框载入质粒pET2 8(a) +并转入大肠杆菌 (Escherichiacoli)BL2 1。SDS_PAGE表明此酶已经在E .coli中获得大量表达 ,表达量约为总细菌蛋白的 4 0 %。酶活分析表明 ,转化菌中UGPase的活性比非转化菌高 0 .5 0~ 3.2 7倍 ,证明此cDNA可以在原核生物中获得表达。Northernblot表明UGPase在黄芪的根、茎、叶及毛状根中均有表达 ,在根及毛状根中表达量较高 ,证明了此酶主要分布于植物贮藏组织的报道。

关键词: 原核表cDNA克隆;UGPase;毛状根;膜荚黄芪

Key words: prokaryotic expression, cDNA cloning, UGPase, hairy root, Astragalus membranaceus

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