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作 者:王宁[1] 陈静[1,2] 高飞[1] 李华云[1] 隋欣[1] 周宜君[1]
机构地区:[1]中央民族大学生命与环境科学学院,北京100081 [2]公安部物证鉴定中心,北京100741
出 处:《生物技术通报》2016年第4期110-115,共6页Biotechnology Bulletin
基 金:国家自然科学基金项目(31370356;31070361);中央民族大学一流大学一流学科项目(YLDX01013;2015MDTD08C);中央民族大学研究生科研创新项目(2015)
摘 要:以盐芥(Thellungiella salsuginea)为材料,获得TsGPX3的cDNA全长序列,其开放阅读框(ORF)序列长591bp,编码196个氨基酸,具有GPXs家族的3个保守的特征结构域。系统进化分析显示,TsGPX3与同属于十字花科的萝卜RsGPX3、油菜BnGPX3和花椰菜BoGPX3等具有较高的同源性。构建植物表达载体pRTL2-TsGPX3-GFP,利用PEG转化拟南芥原生质体细胞进行瞬时表达,发现TsGPX3蛋白主要定位在细胞膜上。构建原核表达载体pET-TsGPX3,采用不同浓度IPTG对转入E.coliBL21(DE3)的pET-TsGPX3进行诱导表达,获得了分子量约为27kD的蛋白,该蛋白在37℃、诱导培养5h时可获得较高的表达量。A cDNA of TsGPX3 ( Thellungiella salsuginea GPX3 ) was isolated from T. salsuginea, and it contained a complete ORF with 591 bp encoding t96 amino acid residues. The three conservative domains of TsGPX3 protein in GPXs fatuity was predicted by bioinformatics analysis. Phylogenetic analysis discovered that TsGPX3 was in high homology with RsGPX3 of Raphanus sativus, BnGPX3 of Brassica napus, and BoGPX3 of B. oleracea etc., all belonging to Brassicaceae. The plant-expressed vector pRTL2-TsGPX3-GFP was transiently expressed by transforming the protoplasts of Arabidopsis thaliana via PEG method, and it was discovered that the TsGPX3 protein was mainly located in the plasma membrane. The constructed prokaryotic vector pET-TsGPX3 was transferred into Escherichia coli strain BL21 ( DE3 ) for induced expression under different concentration of IPTG, and a 27 kD recombinant protein was highly expressed after induced for 5 h at 37℃.
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