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(Populus tomentosa)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。

Plant glutathione S-transferases (GSTs) play important roles in stress tolerance and detoxification metabolism. We cloned two Phi GST genes (PtoGSTF1 and PtoGSTF2) from Populus tomentosa. PtoGSTF1 and PtoGSTF2 encode a protein of 215 and 218 amino acid residues, with a calculated molecular mass of 24.32 and 24.57 kDa, respectively. Genomic sequence analysis showed that PtoGSTF1 and PtoGSTF2 contained 2 introns. RT-PCR revealed PtoGSTF1 and PtoGSTF2 constitutively expressed in P. tomentosa. Recombinant PtoGSTF1 and PtoGSTF2 proteins were overexpressed in E. coli and purified by Ni-affinity chromatography. PtoGSTF1 and PtoGSTF2 showed enzymatic activities towards the substrates CDNB, NBD-Cl, NBC and Cum-OOH. The affinity to substrate NBD-Cl was 2.5-fold higher for PtoGSTF2 than PtoGSTF1, but the catalytic efficiency to NBD-Cl was 56.85-fold greater for PtoGSTF1 than PtoGSTF2. PtoGSTF1 showed greater thermal stability than PtoGSTF2. These results indicate the functional divergence between PtoGSTF1 and PtoGSTF2.