摘要： 为构建拟南芥抗病相关 T1N6_22 基因的原核表达载体并进行高效表达,获得纯化的T1N6_22蛋白,以拟南芥Col-0的cDNA为模板,扩增获得了 T1N6_22 基因CDS,对其进行克隆、测序后与含有GST标签蛋白的pGEX4T-1载体相连,构建 T1N6_22 的原核表达载体pGEX4T-1- T1N6_22 -GST。经酶切和测序鉴定正确后,将构建好的载体及空载体转化大肠杆菌BL21。结果表明,在大肠杆菌BL21菌株中成功表达了与标签蛋白融合的T1N6_22蛋白,大小约57 kDa。SDS-PAGE分析表明,高效表达的诱导条件为0.1 mmol/L IPTG诱导培养3 h。研究结果为进一步T1N6_22互作蛋白的筛选及其调控拟南芥抗病分子机制的研究奠定了基础。

关键词: 拟南芥, T1N6_22, 原核表达, 纯化

Abstract: This study was aimed to construct prokaryotic expression vector of resistance-related gene T1N6_22 from Arabidopsis thaliana and obtain the purified protein T1N6_22 with high-efficiency expression.The CDS of T1N6_22 was amplified by RT-PCR technology using the cDNA of the Arabidopsis Col-0 and was fused into a prokaryotic expression vector pGEX4T-1.Restriction enzyme digestion and sequencing showed that the recombinant vector pGEX4T-1- T1N6_22 -GST was successfully constructed and transformed into E.coli BL21 cells.The results indicated that the pGEX4T-1- T1N6_22 -GST with the predicted molecular weight of about 57 kDa was successfully expressed in E.coli BL21 strain.SDS-PAGE indicated that the best expression quantity was induced with 0.1 mmol/L IPTG treatment for 3 h.These results would provide basis for screening interacting proteins of T1N6_22 and the regulation mechanism in Arabidopsis resistance.

Key words: Arabidopsis thaliana, T1N6_22, Prokaryotic expression, Purification

中图分类号: 

• S432.1

• Q78