| Homologous genes in hybrid species are usually derived from parental species,and the selective retention of parental genes in the offspring may confer new ecological niches or new biological traits to the hybrid species.Functional divergence of homologous genes between species is common and may lead to new adaptations.In the present study,the homoploid hybrid species Pinus densata and its parent species Pinus tabuliformis and Pinus yunnanensis are taken as the research objects.Natural distribution areas of trees of P.tabuliformis are ranged from the northeast to the southwest of China,and trees of P.yunnanensis mainly distribute in the Yunnan Province,while trees of P.densata are mainly distributed in the southeastern part of the Tibetan Plateau.These three species occupy different ecological niches in the presence of natural distribution areas.The large Glutathione S-transferase（GST）family genes play an important role in plant resistance and detoxification.The current study takes GST family as an example and underlies the molecular mechanism of selective retention of parental genes in homoploid hybrid species.The main results are as follows:（1）Forty-three GST genes,including 30 Tau class and six Phi class GST genes,were amplified from P.densata.Fifty-one GST genes were amplified from P.yunnanensis,including30 Tau class and 12 Phi class GST genes.By analyzing the phylogenetic relationships of Tau class and Phi class GSTs among P.densata,P.tabuliformis,and P.yunnanensis,19 GST orthologous groups were identified.Of which,P.densata retained GST genes from P.yunnanensis in 17 cases and two from P.tabuliformis.（2）Analysis of gene expression patterns revealed that among the 19 GST orthologous groups,GST genes of 12 groups were expressed in all examined tissues,while GST genes of the remaining seven groups showed divergent expression patterns.Among these seven groups,GST genes of five groups exhibited the same expression patterns between P.densata and P.yunnanensis,which differed in the expression of GST genes in P.tabuliformis.Meanwhile,GST genes in P.densata and P.yunnanensis were expressed in more examined tissues than those of P.tabuliformis.In accordance with the expression profiles,P.densata preferentially retains the GST genes of P.yunnanensis.（3）Through measurement of the enzymatic properties among the three pine GST orthologues,we found a general biochemical functional differentiation between the GST orthologues and three patterns of differentiation:（a）the three orthologues showed similar substrate profiles;（b）the P.densata and P.yunnanensis GST proteins had similar substrate profiles,these similar substrate profiles were different from that of GSTs in P.tabuliformis;（c）substrate profiles of the three orthologous proteins were partially overlapped with each other.Statistical analysis of the enzymatic activities of GST proteins revealed the similarity between P.densata and P.yunnanensis,and the enzymatic activities of GST proteins of P.densata and P.yunnanensis were higher than those of the P.tabuliformis GSTs.（4）To investigate the mechanism of functional divergence of the GST orthologous proteins in the three pines,we constructed ten mutant proteins with single-site amino acid mutations in PdeGSTU1 and Pta GSTU1.Enzymatic functional analysis revealed that amino acids at positions 38th and 111th（i.e.Ala38 and Gly111 in P.densata PdeGSTU1 and Pro38 and Ser111 in P.tabuliformis Pta GSTU1）contributed significantly to the functional divergence of the orthologous group of GSTU1 in P.densata,P.tabuliformis,and P.yunnanensis.Enzyme kinetic analysis revealed that Ala38 increased the affinity of the protein for the first substrate GSH,while Gly111 increased the affinity of the protein for the second substrate CDNB.Differential scanning calorimetry（DSC）results revealed that both Ala38 and Gly111 increased the protein thermal stability.（5）We obtained the crystal structure of the PdeGSTU1 protein complexed with GSH.From the PdeGSTU1 crystal structure,we found that Ala38 of PdeGSTU1 was located near the G-site,and when Ala38 of PdeGSTU1 was mutated to Pro38 in Pta GSTU1,it led to a change in the protein surface structure,which altered the structure of the GSH binding pocket.Gly111of PdeGSTU1 was located at the bottom of the hydrophobic pocket where the protein binded to the second substrate.When Gly111 of PdeGSTU1 was mutated to Ser111 in Pta GSTU1,the Gly side chain hydrogen atom was replaced by a hydroxyl group of the Ser pointing pocket,resulting in reduced hydrophobicity within the hydrophobic pocket,leading to the reduced affinity of the protein for the second substrate.In combination with the data on molecular evolution,gene expression,enzymatic properties,site-specific mutagenesis,and crystal structure analysis,we identify a functional mechanism that homoploid hybrid species of P.densata preferentially retain the GST genes of P.yunnanensis.These results provide a new perspective for understanding the molecular mechanisms by which homoploid hybrid species generate new adaptations. |
