CAJ | 학술논문

在已报道的UGPase的植物cDNA序列基础上,从膜荚黄芪( Astragalus membranaceus (Fisch.) Bunge)毛状根中分离了此酶的cDNA.此cDNA全长为1 831 bp,推测编码分子量为51.5 kD、等电点为6.01的由471个氨基酸残基组成的多肽.将此cDNA的开放阅读框载入质粒pET28(a)+并转入大肠杆菌( Escherichia coli ) BL21.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40%.酶活分析表明,转化菌中UGPase 的活性比非转化菌高0.50～3.27倍,证明此cDNA可以在原核生物中获得表达.Northern blot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.
재이보도적UGPase적식물cDNA서렬기출상,종막협황기( Astragalus membranaceus (Fisch.) Bunge)모상근중분리료차매적cDNA.차cDNA전장위1 831 bp,추측편마분자량위51.5 kD、등전점위6.01적유471개안기산잔기조성적다태.장차cDNA적개방열독광재입질립pET28(a)+병전입대장간균( Escherichia coli ) BL21.SDS-PAGE표명차매이경재 E.coli 중획득대량표체,표체량약위총세균단백적40%.매활분석표명,전화균중UGPase 적활성비비전화균고0.50～3.27배,증명차cDNA가이재원핵생물중획득표체.Northern blot표명UGPase재황기적근、경、협급모상근중균유표체,재근급모상근중표체량교고,증명료차매주요분포우식물저장조직적보도.
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 level in root and hairy root.