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。
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.NM129185.3）.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-1IPTG,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.
出处
《植物生理学报》
CAS
CSCD
北大核心
2014年第2期185-191,共7页
Plant Physiology Journal
基金
国家基金委地区科学基金(31060041)
甘肃省自然科学基金B类(1212RJYA008)
关键词
拟南芥
abi5
分子克隆
原核表达
纯化
质谱
Arabidopsis thaliana
abi5
molecular cloning
prokaryotic expression
purification
mass
