摘要： 为了研究马铃薯WRKY57基因（PGSC transcript number PGSC0003DMT400072958）功能，通过RT-PCR克隆得到马铃薯耐旱品种PB06的StWRKY57基因cDNA序列，命名为StWRKY57-PB。利用生物信息学方法对其序列特征进行了分析；以pGEX-KG为基本骨架，构建了StWRKY57-PB蛋白的原核表达载体并在大肠杆菌中进行了表达和纯化分析。结果表明，StWRKY57-PB基因开放阅读框（ORF）长981 bp，编码蛋白属于WRKY转录因子家族Ⅱ c类，含326个氨基酸残基，含有WRKY结构域（WRKYGQK）和一个类锌指结构基序（C-X₄-C-X₂₃-H-X₁-H）。序列比对及构建系统发育树结果表明，它与马铃薯基因组数据库及NCBI公布的WRKY57（XP_006348711.1）有4个氨基酸的差异，与番茄WRKY57分子距离较近，相似性为93%，与拟南芥WRKY57亲缘关系较远，相似性为57%。二级结构预测发现，StWRKY57-PB所编码蛋白由延伸链、α螺旋、β折叠和不规则卷曲组成，四者比例分别为13.50%，16.56%，7.98%，61.96%。在0.5 mmol/L IPTG诱导下，该蛋白质在3种大肠杆菌表达菌株BL21、Rosetta和Tuner中稳定表达，利用GST亲和树脂分离纯化了GST-StWRKY57-PB融合蛋白，Western Blot验证了蛋白质的表达和纯化。对进一步确定该蛋白质的体外活性及生物学功能具有重要意义。

关键词: 马铃薯, StWRKY57, GST标签, 序列分析, 原核表达

Abstract: In order to explore the function of StWRKY57 (PGSC0003DMT400072958) in potato, a full-length cDNA sequence of the homologous StWRKY57 gene in Solanum tuberosum PB06 named StWRKY57-PB was cloned by RT-PCR and bioinformatics methods were used to analyze the sequence characteristic of StWRKY57-PB. For prokaryotic expression and purify, the prokaryotic expression vector of StWRKY57-PB was constructed based on pGEX-KG, then fusion protein was induced in Escherichia coli strains. The results showed that the StWRKY57-PB gene contained a 981 bp open reading frame(ORF), and the coding protein belonged to the Ⅱ c class of the WRKY transcription factor family, containing 326 amino acid residues with a WRKY(WRKYGQK) domain and a zinc-finger-like motif(C-X₄-C-X₂₃-H-X₁-H). StWRKY57-PB was determined structurally to be 16.56% a-helix, 13.50% extended strand, 7.98% β-turn and 61.96% random coil. Compared with StWRKY57(XP_006348711.1), there were 4 amino acid mutations in StWRKY57-PB. Multiple alignment and phylogenetic tree analyses indicated that StWRKY57-PB showed the next-closest relationship with SlWRKY57 with 93% similarity and was farthest to AtWRKY57. StWRKY57-PB protein was stably expressed in three Escherichia coli strains of Rosetta, BL21 and Tuner with 0.5 mmol/L IPTG. Fusion protein of GST-StWRKY57-PB was purified through GST SefiroseTM resin, and then Western Blot was carried to verify the successful expression and purification of the protein. It is of great significance to further determine the activity and biological function of this protein in vitro.

Key words: Potato, StWRKY57, GST tag, Sequence analysis, Prokaryotic expression

中图分类号: 

• S532.03

• Q78