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谷胱甘肽转移酶(glutathione S-transferase,GST)在植物的抗逆反应、细胞信号转导和抗病等方面发挥着重要作用。从毛白杨(Populustomentosa)中克隆到2个Phi类GST基因(PtoGSTF1和PtoGSTF2),它们分别编码215和218个氨基酸残基的蛋白质。DNA序列分析显示这2个基因均含有2个内含子。组织表达模式分析表明,这2个基因在毛白杨的根、茎、叶、韧皮部和顶芽组织中均表达,是组成型表达基因。在大肠杆菌中表达这2个基因并纯化重组蛋白。酶活性分析显示PtoGSTF1和PtoGSTF2蛋白对底物CDNB、NBD-Cl、NBC和Cum-OOH都具有酶学活性,但活性差异较大。动力学分析显示,PtoGSTF2对NBD-Cl的亲和力是PtoGSTF1的2.5倍,但PtoGSTF1的催化效率是PtoGSTF2的56.8倍。热力学稳定性分析显示,PtoGSTF1比PtoGSTF2具有更高的热力学稳定性。因此,酶学性质的差异预示着这2个Phi类GST基因可能存在功能上的分化。
Glutathione S-transferase (GST) plays an important role in plant anti-retrogradation, cell signal transduction and disease resistance. Two Phi-like GST genes (PtoGSTF1 and PtoGSTF2) were cloned from Populustomentosa and encoded 215 and 218 amino acid residues, respectively. DNA sequence analysis showed that both of these two genes contained two introns. Tissue expression pattern analysis showed that these two genes were expressed in roots, stems, leaves, phloem and apical bud tissue of Populus tomentosa, which were constitutively expressed genes. The two genes were expressed in E. coli and the recombinant protein was purified. Enzyme activity analysis showed that the PtoGSTF1 and PtoGSTF2 proteins were enzymatically active on the substrates CDNB, NBD-Cl, NBC and Cum-OOH, but the activity varied greatly. Kinetic analysis showed that the affinity of PtoGSTF2 for NBD-Cl was 2.5 times that of PtoGSTF1, but the catalytic efficiency of PtoGSTF1 was 56.8 times that of PtoGSTF2. Thermodynamic stability analysis showed that PtoGSTF1 has higher thermodynamic stability than PtoGSTF2. Therefore, the difference in enzymatic properties indicates that there may be functional differentiation of these two Phi GST genes.