种子2025，Vol.44Issue(9)：64-74,11.DOI:10.16590/j.cnki.1001-4705.2025.09.064
紫花苜蓿G6PD蛋白的原核表达及蛋白纯化研究
Prokaryotic Expression and Protein Purification of Medicago sativa G6PD Protein
摘要
Abstract
To explore the biological functions of glucose-6-phosphate dehydrogenase(G6PD)in Medi-cago sativa,the soluble expression and efficient purification of this protein were achieved through a prokaryotic expression system.The coding sequence of the MsG6PD gene in Medicago sativa was amplified by PCR technology.They were respectively cloned into prokaryotic expression vectors pCold-SUMO,pGEX-4T-1,and pMAL-c2X to construct recombination plasmids pCold-SUMO-MsG6PD9,pGEX-4T-1-MsG6PD9,and pMAL-c2XMsG6PD9.The recombinant plasmid was trans-formed into Escherichia coli BL21(DE3)competent cells,and the expression was induced by IPTG.By optimizing the induction conditions(temperature,IPTG concentration and induction time),it was found that the expression induction effect of pMAL-c2X-MsG6PD9 was better.SDS-PAGE analysis showed that the soluble expression level of approximately 66 kDa of MsG6PD protein was the highest under the condition of 28℃ and 1.0 mmol/L IPTG induction for 9 hours.The target protein was fur-ther purified by affinity chromatography of MBP tag cross-linked purification resin,and high-purity recombinant MBP-MSG6PD protein was obtained after elution with maltose buffer.The prokaryotic expression and purification system of G6PD protein in Medicago sativa was successfully established in the research,providing a reference for the subsequent analysis of its molecular mechanism and enzy-matic characteristics in the stress resistance physiology of Medicago sativa.关键词
紫花苜蓿/G6PD蛋白/原核表达/蛋白纯化Key words
Medicago sativa L./G6PDH protein/prokaryotic expression/protein purification分类
农业科技引用本文复制引用
马兰民,李幸儿,赵爽,梁佳林,李含笑,梁佳,李萍..紫花苜蓿G6PD蛋白的原核表达及蛋白纯化研究[J].种子,2025,44(9):64-74,11.基金项目
青海省科学技术厅基础研究项目(2022-ZJ-714) （2022-ZJ-714）
国家林业草原高寒草地适应性管理工程技术研究中心自主基金项目(2025GJLCGCZX002) （2025GJLCGCZX002）
青海大学一流专业建设项目(YLZY2025-3) （YLZY2025-3）
青海大学大学生创新训练项目(2024-QX-62) （2024-QX-62）
三江源生态一流学科硕士研究生科技创新项目(2024-stxy-Y11) （2024-stxy-Y11）
青海大学大学生科研训练计划项目(SRT202590) （SRT202590）
