Functional Divergence of Dehydroascorbate Reductase Genes in Pinus densata, P. tabulaeformis and P. yunnanensis
高山松及其亲本种油松和云南松DHAR基因的功能分化

Plant dehydroascorbate reductase (DHAR) is a physiologically important reducing enzyme in the ascorbateglutathione recycling reaction. In this study, we cloned 6 DHAR genes from a hybrid pine species complex of Pinus densata, P. yunnanensis and P. tabulaeformis. P. densata originated by natural hybridization of P. yunnanensis and P. tabulaeformis. The 6 DHAR genes were divided into 2 types: DHAR1 and DHAR2. Phylogenetic analyses indicated that the 3 DHAR1 and 3 DHAR2 genes from the 3 Pinus species were 2 orthologous groups. DHAR1 and DHAR2 genes originated from an ancestral duplication event that occurred in the most recent common ancestor of the early land plants. P. densata contains a copy of DHAR1 similar to that of P. tabulaeformis and a copy of DHAR2 similar to that of P. yunnanensis. RT-PCR revealed that the 6 DHAR were constitutive expression genes in the 3 Pinus species. The recombinant Pinus DHAR proteins were overexpressed in E. coli and purified by Ni-affinity chromatography. P. densata and P. tabulaeformis DHAR1 proteins showed similar enzymatic activities, catalytic efficiency, thermal stabilities and optimal pH profiles towards substrate DHA but about 300-fold higher enzymatic activities than P. yunnanensis DHAR1 protein. The enzymatic activity and thermal stability of P. densata DHAR2 protein were higher than those of P. tabulaeformis DHAR2 protein. Joint analyses of sequence structure, phylogenetic relationships, expression patterns, enzymatic properties and protein 3-D structure revealed selective DHAR gene composition in the hybrid genome of P. densata. Such a combination of divergent copies of DHAR gene in P. densata may have adaptive implications for its colonization of novel habitats on the Tibetan Plateau.