内蒙古农业大学学报（自然科学版）2026，Vol.47Issue(4)：1-8,8.DOI:10.16853/j.cnki.1009-3575.2026.04.001
苜蓿源UDP-鼠李糖合成酶MtNRS/ER的克隆、表达与酶学特性研究
Cloning,Expression,and Enzymatic Characterization of UDP-Rhamnose Synthase MtNRS/ER from Medicago Truncatula
摘要
Abstract
Rhamnose is an important constituent unit of plant cell wall pectin,polysaccharides,and various secondary metabolites,playing a crucial role in the activity of natural products and the structure of plant cell walls.In this study,the bifunctional UDP-rhamnose synthase gene MtNRS/ER was cloned from Medicago truncatula,and an Escherichia coli expression system was constructed using the pET30 vector.By optimizing the induction conditions(0.2 mmol/L IPTG,16℃,14 h),large amounts of soluble recom-binant protein were obtained and purified through a two-step process involving Ni-NTA affinity chromatography and HiTrap Q HP an-ion exchange chromatography,achieving a final purity>98%and concentration of 10 mg/mL.Enzymatic experiments demonstrated that MtNRS/ER could catalyze the conversion of UDP-4-keto-6-deoxyglucose to UDP-rhamnose,with the reaction products confirmed by the liquid chromatography-mass spectrometry.Kinetic analysis yielded a Km of(373.0±7.2)μmol/L and a Vmax of(143.77±11.41)μmol/min,demonstrating good substrate affinity and catalytic efficiency.This study revealed,for the first time,the synergistic cata-lytic mechanism of the plant bifunctional MtNRS/ER enzyme,providing experimental evidence for enzymatic studies of the UDP-rhamnose biosynthetic pathway,and laying the foundation for the biosynthesis and metabolic engineering applications of rare deoxy sugars and their derived natural products.关键词
UDP-鼠李糖合成酶/重组蛋白表达/酶动力学/液相质谱/蒺藜苜蓿Key words
UDP-rhamnose synthase/Recombinant protein expression/Enzyme kinetics/Liquid chromatography-mass spectrometry/Medicago truncatula分类
农业科技引用本文复制引用
韩晓东,任雪源,罗述鹏,周佳欣,张梦妍,王桂花..苜蓿源UDP-鼠李糖合成酶MtNRS/ER的克隆、表达与酶学特性研究[J].内蒙古农业大学学报（自然科学版）,2026,47(4):1-8,8.基金项目
2025年内蒙古盐碱地饲草生产关键技术集成与推广应用项目(RZ2500002136) （RZ2500002136）
2021年自治区级大学生创新创业训练计划项目(202110129028) （202110129028）
