苹果MxIrt1基因的克隆与原核表达

Cloning and Prokaryotic Expression of Apple MxIrt1 Gene

根据植物IRT(Iron Regulated Transporter)家族的功能保守区设计引物,通过RACE法从缺铁胁迫处理的小金海棠根系cDNA文库中克隆得到了Fe2+转运蛋白基因cDNA全长,将其命名为MxIrt1(Malus xiaojinensis Iron regulated transporter 1)。将MxIrt1 cDNA片段与pET30a构建原核表达载体pEIrt,转化大肠杆菌BL21。SDS-PAGE电泳检测结果表明,以30℃、0.5mmol·L-1 IPTG诱导该基因表达效果最好,诱导产物为一个40kD的蛋白。为进一步纯化和鉴定目的蛋白提供了试验基础。

A Full length cDNA of Mxlrtl gene from the iron-stressed root cDNA library of Malus xiaojinensis Chent et Jiang was cloned using the RACE method with primers designed based on the conserved domain sequences of plant IRT gene families. The recombinant prokaryotic expression vector pEIrt was constructed using vector pET30a. Restriction endonuclease analysis, PCR amplifying and sequencing confirmed that the construction was correct and had no base mutant. The prokaryotic expression analysis was carried out through transformation of E. coli （ BL21 ）. The SDS - PAGE electrophoresis analysis showed that the best expression was induced by 30℃ and 0. 5 mmol · L^-1 IPTG, under which a 40 kD recombinant protein was produced. These results will provide a foundation for further purifying and identifying objective protein.