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WRKY转录因子是一类含有高度保守的WRKY结构域的锌指蛋白,是植物所特有的转录因子家族。该研究将已克隆的SmWRKY1 cDNA构建到原核表达载体pET28a上,转化大肠杆菌BL21(DE3)进行诱导表达,SDS-PAGE结果显示该蛋白的大小约36 kDa,与预测的蛋白相对分子质量一样,说明该基因在大肠杆菌中成功表达。对影响蛋白表达的4个因素:诱导时间、诱导温度、IPTG浓度及诱导前菌液的浓度进行优化,结果表明在大肠杆菌BL21(DE3)中,当A600达到约1.0~1.5时,加入0.2 mol·L-1的IPTG,在20℃诱导培养12 h后SmWRKY1蛋白的表达量较高。原核表达产物SmWRKY1蛋白以包涵体的形式存在,采用尿素提取包涵体蛋白,用Ni2+亲和色谱法纯化表达蛋白,纯化后蛋白的质量浓度为2.454 g·L-1。经蛋白质印迹检测,His-Tag单克隆抗体可以特异性的识别SmWRKY1蛋白,进一步证实丹参SmWRKY1蛋白在大肠杆菌中成功表达。本研究为进一步开展SmWRKY1基因的表达与丹参酮类等化合物的生物合成相关研究奠定了工作基础。
WRKY transcription factor is a family of zinc finger proteins that contain a highly conserved WRKY domain and is a family of plant-specific transcription factors. In this study, the cloned SmWRKY1 cDNA was constructed into the prokaryotic expression vector pET28a and transformed into E. coli BL21 (DE3) for expression. SDS-PAGE showed that the size of the SmWRKY1 cDNA was about 36 kDa, which is the same as the predicted protein relative molecular mass The gene was successfully expressed in E. coli. Four factors influencing protein expression, such as induction time, induction temperature, IPTG concentration and concentration of pre-induction bacteria, were optimized. The results showed that when A600 reached about 1.0-1.5 in E. coli BL21 (DE3), 0.2 mol · L-1 IPTG, SmWRKY1 protein expression was higher after induction at 20 ℃ for 12 h. The prokaryotic expression product SmWRKY1 was present in the form of inclusion bodies. Inclusion body protein was extracted by urea and purified by Ni2 + affinity chromatography. The purified protein was 2.454 g · L-1. His-Tag monoclonal antibody can specifically recognize SmWRKY1 protein by Western blotting, further confirming the successful expression of SmWRKY1 protein in E. coli. This study laid the foundation of further research on the relationship between the expression of SmWRKY1 gene and the biosynthesis of tanshinones and other compounds.