Functional Divergence Of Phi Glutathione S-transferase Gene Family From Populus Tomentosa

Glutathione transferases (GSTs) are an ancient and diverse group of multi-functional proteins, playing significant roles in plant stress. In this thesis, we identified three phi class GST genes (PtoGSTF4, PtoGSTF5and PtoGSTFT) from Populus tomentosa, and then we studied their gene structure, gene expression pattern, protein structure and function in detail.1. The sequence analysis of the three genes showed that PtoGSTF4, PtoGSTF5and PtoGSTF7contain an open reading frame of642bp,642bp and654bp, encoding proteins consist of213,213and217amino acid residues, respectively, with a respectively calculated molecular mass of23.7,23.1and24.6kDa. The predicted protein three-dimensional structure of the three phi GSTs showed that they have a typical structure with largely conserved overall structures, except some subtle structural modifications present in partial loop regions.2. The study of gene expression pattern using reverse transcription PCR revealed that PtoGSTF4, PtoGSTF5and PtoGSTF7were expressed in all studied tissues (stem, leaf and phloem of stem) under normal growth condition, together with atrazine and H2O2stressed conditions, indicating all these three genes were constitutive expressed in Populus tomentosa.3. PtoGSTF4, PtoGSTF5and PtoGSTF7were recombined and overexpressed in E. coli, and soluble proteins were purified by Ni-affinity chromatography. In the substrates spectrum anlysis, the three proteins all showed enzymatic activities towards the substrates CDNB, NBD-C1, NBC and Cum-OOH, while no activity was detected towards the substrate DCNB, DHA and HED. And the enzymatic activities were divergent towards different substrates. The activity of PtoGSTF4towards CDNB is2.6-fold and38.7-fold to PtoGSTF5and PtoGSTF7, respectively. While The activity of PtoGSTF5towards NBD-C1is7.3-fold and150.8-fold to PtoGSTF4and PtoGSTF7, respectively.. In the dynamic anlysis, PtoGSTF5showed a12.3-flod and4.8-fold higher affinity to GSH and CDNB when compared to PtoGSTF4, respectively. Additionally, PtoGSTF4and PtoGSTF5showed divergent temperature spetrum and pH spetrum. The activity of PtoGSTF5was94%of the maximize activity at70℃, while PtoGSTF4remained just18%; the activity of PtoGSTF5was94%of the maximize activity at pH6.0, while PtoGSTF4was just18%.The three phi GSTs were systematically analysed by integrating phylogeny, expression, substrate specificity and enzyme kinetic data in Populus tomentosa, suggesting functional divergence among these three genes.