Molecular Cloning And Biochemical Characteristic Of Two Phi Glutathione S-transferase From Populus Tomentosa

Glutathione S-transferases (GSTs, EC2.5.1.18) are a supergene family of a multifunctional enzymes which play important roles in stress tolerance and detoxification metabolism. Two Phi GST genes (PtoGSTF1and PtoGSTF2) were cloned from Populus tomentosa. The gene expression pattern, protein structure and function of the two genes were studied in detail.1. The PtoGSTF1and PtoGSTF2genes encode a protein of215and218amino acid residues, with a calculated molecular mass of24.32and24.57kDa, respectively. The genomic sequence analysis showed PtoGSTF1and PtoGSTF2both contain two introns. The predicted protein three-dimensional structure showed, PtoGSTF1and PtoGSTF2have a similar structure.2. Reverse transcription PCR revealed that both PtoGSTF1and PtoGSTF2were expressed in the root, stem, leaf, phloem and bud tissues of P. tomentosa, and indicating these two genes were constitutive expression genes in P. tomentosa.3. The recombinant PtoGSTF1and PtoGSTF2proteins were overexpressed in E. coli, and were purified by Ni-affinity chromatography. The PtoGSTF1and PtoGSTF2proteins showed enzymatic activities towards the substrates CDNB、NBD-Cl、NBC and Cum-OOH. The PtoGSTF2protein showed2.5-fold higher affinity to substrate NBD-Cl than PtoGSTF1protein. But, on the other hand, the PtoGSTF1showed56.85-fold higher catalytic efficiency to substrate NBD-Cl than PtoGSTF2. The PtoGSTF1protein revealed higher thermal stability than PtoGSTF2protein.These results indicated functional divergence between the PtoGSTFl and PtoGSTF2genes.