江南卷柏Phi类谷胱甘肽S-转移酶的分子特性  

Molecular characteristics of two Phi glutathione S-transferases in Selaginella moellendorffii

作　　者：张元杰[1] 杨志灵[1] 杨海灵[1] 

机构地区：[1]北京林业大学生物科学与技术学院,北京100083

出　　处：《生物工程学报》2016年第7期927-936,共10页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.31270641)资助~~

摘　　要：谷胱甘肽S-转移酶(Glutathione S-transferase,GST)在帮助植物抵抗各种胁迫中发挥重要作用。该研究从江南卷柏Selaginella moellendorffii中克隆到两个Phi类GST基因,分别命名为Sm GSTF1和Sm GSTF2,两个基因均编码215个氨基酸残基的蛋白质。表达模式分析发现,这两个基因在江南卷柏根、茎和叶中均有表达。将这两个基因在大肠杆菌中诱导表达重组蛋白并纯化,酶学性质分析表明Sm GSTF1和Sm GSTF2对CDNB、NBD-Cl和NBC等3种底物都有活性。Sm GSTF1对Fluorodifen和Cum-OOH也有活性,而Sm GSTF2对它们没有活性。酶动力学分析表明Sm GSTF1和Sm GSTF2对GSH有较高的亲和力,而对CDNB的亲和力都相对较低。在不同p H及温度条件下对Sm GSTF1和Sm GSTF2重组蛋白进行活性测定,发现这两个蛋白在p H 7-8.5,45-55℃温度范围内有较高的催化活性。研究推测,Sm GSTF1和Sm GSTF2可能在江南卷柏的抗逆生理过程中有重要的作用。Glutathione S-transferase（GST） is important in plants to resist various stresses. In this study, two Phi GSTgenes（Sm GSTF1 and Sm GSTF2） were cloned from Selaginella moellendorffii. Sm GSTF1 and Sm GSTF2 genes encode proteins of 215 amino acid residues. Gene expression analysis showed that the two genes were expressed in roots, stems and leaves. The recombinant Sm GSTF1 and Sm GSTF2 proteins were overexpressed in Escherichia coli, and purified by Ni-affinity chromatography. Sm GSTF1 and Sm GSTF2 had the catalytic activity towards 1-Chloro-2,4-Dieitrobenzene, 4-Chloro-7-nitro-1,2,3-benzoxadiazole（NBD-Cl）, and 4-Nitrobenzyl chloride substrates. Sm GSTF1 also had the activity towards Fluorodifen and Cumyl hydroperoxide（Cum-OOH）, whereas Sm GSTF2 not. The enzyme kinetics analysis showed that Sm GSTF1 and Sm GSTF2 had high affinity towards glutathione, and low affinity towards 1-Chloro-2, 4-Dieitrobenzene. The enzymatic activity of Sm GSTF1 and Sm GSTF2 had high catalytic activity between p H 7 and 8.5, and between 45 and 55 °C. Sm GSTF1 and Sm GSTF2 may have an important role in the resistance of Selaginella moellendorfii against stress.

关 键 词：江南卷柏 谷胱甘肽S-转移酶 基因表达 酶学性质 

分 类 号：Q946[生物学—植物学]