紫花苜蓿DREB1基因的原核表达与蛋白纯化

Prokaryotic expression of Ms DREB1 gene and protein purification

构建MsDREB1基因的原核表达载体,对表达产物进行鉴定和纯化,为研究DREB1基因在植株中功能奠定基础。用冷酚法从紫花苜蓿提取RNA,并转化成cDNA,然后将其克隆至pET32a原核表达载体,构建重组载体pET32aDREB1。用重组质粒转化大肠杆菌BL21,IPTG诱导表达后进行SDS-PAGE分析,用Ni-NTA亲和层析柱对重组蛋白进行纯化。结果表明,原核表达载体构建正确;SDS-PAGE分析,出现了与DNAMAN预测的43.2 kd大小一致的蛋白条带;经分析重组蛋白纯化率达到90%以上。

MsDREB1 prokaryotic expression vector was constructed and the expression products were identified and purified, as a basis for study DREB1 gene function in Alfalfa. We extracted RNA using cold phenol method from Alfalfa, and transformed into cDNA, then cloned into prokaryotic expression vector pET32 a to construct recombinant plasmid p ET32a-DREB1. Recombinant plasmid was transformed into E. coli BL21, and IPTG induced its expression; the protein was SDS-PAGE analyzed, recombinant protein was purified by Ni-NTA affinity chromatography. The results showed that the prokaryotic expression vector was constructed correctly, and by SDS-PAGE analysis, there was a 43.2 kd protein band consistent with the DNAMAN forecast. The recombinant protein purification rate was 90% or more.