【摘要】 磷酸乙醇胺甲基转移酶是一种S-腺苷甲硫氨酸依赖性甲基转移酶，是催化磷酸乙醇胺三步甲基化合成磷酸胆碱的关键酶，而磷酸胆碱是植物中磷脂酰胆碱和甘氨酸甜菜碱生物合成的重要前体。木碱蓬转录组测序分析结果显示，磷酸乙醇胺甲基转移酶基因在盐胁迫下显著上调表达。本研究以800 mmol/L NaCl处理1 d的cDNA为模板扩增获得木碱蓬磷酸乙醇胺甲基转移酶基因SdNMT的编码区序列，构建原核表达载体pET28a-SdNMT并导入大肠杆菌菌株BL21。对重组蛋白进行IPTG诱导表达、表达条件筛选优化及可溶性分析，采用镍亲和层析柱纯化目标蛋白。结果显示，成功克隆的SdNMT基因编码区序列长1 485 bp,编码494个氨基酸，预测蛋白分子质量为56.56 ku,理论等电点为5.52。重组蛋白在大肠杆菌中得到可溶性表达。1.0 mmol/L IPTG,15℃培养振荡16 h是目的蛋白诱导表达的最佳条件。通过Ni NTA beads 6FF纯化获得在毫克级范围内获得纯度达85%的SdNMT蛋白。研究结果为蛋白酶活性检测提供足够的材料，为深入分析SdNMT在植物生长、发育及环境胁迫响应中的功能奠定基础。更多还原

【Abstract】 Phosphoethanolamine methyltransferase is an S-adenosylmethionine dependent methyltransferase that catalyzes the three-step methylation of phosphoethanolamine to synthesize phosphatidylcholine, an essential membrane component and a precursor for glycine betaine biosynthesis.Transcriptome sequencing analysis of S.dendroides showed that the phosphoethanolamine methyltransferase gene was significantly up-regulated under salt stress.In this study, the coding sequence of the phosphoethanolamine methyltransferase gene, SdNMT, from S.dendroides was amplified using the cDNA treated with 800 mmol/L NaCl for 1 day.The prokaryotic expression vector pET28a-SdNMT was constructed and transformed into E.coli BL21 cells.The recombinant protein was induced using IPTG.The expression conditions were screened and optimized, and the solubility was analyzed.The target protein was purified through a nickel affinity chromatography column.The results showed that the coding region sequence length of SdNMT was 1 485 bp, encoding 494 amino acids.The predicted protein molecular mass was 56.56 ku, and the theoretical isoelectric point was 5.52.The recombinant protein was successfully expressed in a soluble form in E.coli.The optimal induction of protein SdNMT in E.coli was 1.0 mmol/L IPTG for 16 hours at 15 ℃.The target protein, with a purity of 85%,was successfully obtained in the milligram range using the Ni-NTA 6FF beads purification method, providing sufficient materials for enzyme activity assays.The findings of this study will serve as a foundation for further exploration of the role of SdNMT in plant growth, development, and response to environmental stresses.更多还原