《植物生理学报》 2014, 50(2): 185-191

拟南芥abi5基因的分子克隆及其在原核细胞中的表达和纯化

马燕林, 马建忠*, 王永刚

兰州理工大学生命科学与工程学院, 兰州730050

通信作者：马建忠；E-mail: majz@lut.cn；Tel: 0931-2976707

摘　要：

拟南芥abi5基因编码了一个碱性亮氨酸拉链类转录因子, 它在ABA信号转导过程中发挥着关键调控作用。本文以拟南芥为材料, 通过RT-PCR扩增、克隆了包含abi5基因编码区的片段。核苷酸序列分析表明, 所克隆的基因与NCBI数据库收录的abi5基因(GenBank登录号NM_129185.3)有99.0%的一致性; 氨基酸序列存在4个残基差异。所克隆的abi5基因被进一步亚克隆至pET-32a表达载体。序列测定核实构建正确的重组质粒(pET32a-ABI5)转化入大肠杆菌BL21 Star (DE3)中诱导表达。表达产物经Ni-NTA亲和层析柱分离纯化、SDS-PAGE分析和质谱鉴定。结果表明, 重组abi5基因在大肠杆菌表达的较适宜条件为: 异丙基-β-D-硫代半乳糖苷(IPTG)终浓度为0.3 mmol·L^-1、30 ℃下诱导4 h, 可达到细菌裂解液上清蛋白的29.1%。经Ni-NTA亲和层析柱纯化后的ABI5融合蛋白在SDS-PAGE电泳分析时呈现一条蛋白带。该条带经串联质谱分析证明为重组ABI5融合蛋白。

关键词：拟南芥; abi5; 分子克隆; 原核表达; 纯化; 质谱

收稿：2013-10-16   修定：2013-12-31

资助：国家基金委地区科学基金(31060041)和甘肃省自然科学基金B类(1212RJYA008)

Molecular Cloning, Prokaryotic Expression and Purification of abi5 Gene from Arabidopsis thaliana L.

MA Yan-Lin, MA Jian-Zhong*, WANG Yong-Gang

School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, China

Corresponding author: MA Jian-Zhong; E-mail: majz@lut.cn; Tel: 0931-2976707

Abstract:

The abi5 gene from Arabidopsis encodes a basic leucine zipper transcription factor, which plays a key regulatory role in ABA signal transduction. In this paper, the coding region of the abi5 gene of Arabidopsis thaliana Columbia was amplified by RT-PCR and then cloned into a T-vector, pMD^®19-T. The nucleotide sequencing showed that the cloned gene had a 99.0% identity with the abi5 gene sequence (GenBank No. NM_129185.3). The amino acid sequence of the cloned fragment exists four different residues. The abi5 gene was further subcloned into an expression vector, pET-32a. The recombinant plasmid construct (pET32a-ABI5) was verified by nucleotide sequencing, and transformed into Escherichia coli BL21 Star (DE3). The expression of the ABI5-fused protein was induced with 0.3 mmol·L^-1 IPTG, at 30 ℃ for 4 h. Under this condition, the content of the fusion protein could reach 29.1% of the total supernatant proteins of bacteria lysate. After Ni-NTA affinity chromatography purification, a single band was observed in the SDS-PAGE gel, with an 86.1% purity. The band was excised for MS/MS assay. The results of tandem mass spectrometry proved that the band contained the ABI5-fused recombinant protein.

Key words: Arabidopsis thaliana; abi5; molecular cloning; prokaryotic expression; purification; mass