小麦NAC转录因子TaNAC14的原核表达和分离纯化

Prokaryotic expression and purification of the transcription factor TaNAC14 in wheat(Triticum aestivum)

植物NAC转录因子参与调控多种生物学过程,在植物生长发育和逆境适应性方面发挥重要作用。前期研究发现小麦(Triticum aestivum)NAC家族成员TaNAC14正向调控幼苗根生长发育并增强其耐旱性。本研究对小麦TaNAC14蛋白的理化性质和结构进行了分析预测;对TaNAC14的亚细胞定位和转录激活活性进行了验证;构建了TaNAC14重组蛋白原核表达载体pET21a-HMT-TaNAC14,转化大肠杆菌(Escherichia coli)表达菌株BL21 CodonPlus(DE3)-RIPL,对TaNAC14重组蛋白的诱导表达条件进行了优化,对重组蛋白的可溶性进行了分析,并利用镍柱对重组蛋白进行了分离纯化。结果表明,TaNAC14具有NAM家族保守的结构域,定位于细胞核,具有转录激活活性。重组蛋白HMT-TaNAC14在大肠杆菌中的最佳诱导表达条件为0.2mmol/L的IPTG诱导4 h;该重组蛋白主要以可溶性形式存在;利用镍柱对其进行了分离纯化,获得了较高纯度的目的蛋白。本研究为后续进行TaNAC14调控靶基因的鉴定奠定了基础。

The transcription factors(TFs)in the NAC family are involved in regulating multiple biological processes,playing an important role in plant growth,development,and stress adaptation.Our previous studies have demonstrated that TaNAC14,a member of the NAC family in wheat(Triticum aestivum L.),positively regulates root growth and development and enhances the drought tolerance of wheat seedlings.In this study,we analyzed the physicochemical properties and structure and verified the subcellular localization and transcriptional activation activity of TaNAC14.The prokaryotic expression vector pET21a-HMT-TaNAC14 was constructed and transformed into Escherichia coli BL21 CodonPlus(DE3)-RIPL.The conditions for inducing the expression of the recombinant protein HMT-TaNAC14 were optimized.The solubility of the recombinant protein was analyzed,and the protein was purified by affinity chromatography on a Ni-nitrilotriacetic acid column.The results indicated that TaNAC14 had a conserved domain of the NAM family.It was located in the nucleus and had transcriptional activation activity.The optimal conditions for expression of the recombinant protein in E.coli were induction with 0.2 mmol/L IPTG for 4 h.The recombinant protein mainly existed in the soluble form,and the target protein was obtained after purification.This study lays a foundation for the identification of target genes regulated by TaNAC14.