| Nitrogen is one of macronutrients in plants,which plays an essential role in growth and development of plants,it is also one of major factors limiting the growth of plant.The absorption and utilization of nitrogen directly affects growth and development of plants.Studies suggests that members of Dof transcription factor family are involved in regulating a series of key genes related to absorption and utilization of nitrogen,and enhanced plant growth index.In order to select the members of Dof transcription factor family which may possible promote nitrogen assimilation;the following experiments were designed in this study:1.We identified 44 members of Dof family in Populus trichocarpa based on V3.0 database published in 2012.In this study,the members of the Dof family in Populus trichocarpa were divided into 4 subfamilies according to the results of the phylogenetic tree;Each Dof member had a complete Dof domain except PtDof29;Except for PtDof29 protein was predictably located in mitochondria,the other Dof members were located in the nucleus;Most of the Dof members were expressed in the leaves and roots.2.In order to identify the members of Dof genes which could be induced by nitrogen,this study designed two independent experiments:one group was resupplied with nitrogen for 48h after nitrogen deficiency 3 days,and then deprived the nitrogen again for 48h.The effects of nitrogen deficiency and resupplement for expression level of Populus simonii × P.nigra Dof genes.Another group was constantly treated by three concentrations of ammonium nitrogen and nitrate nitrogen for exploring the changes of Dof genes expression under different concentration and different kinds of nitrogen treatments.Through these two groups of experiments,6 nitrogen responsing Dof genes,PnDof1O,PnDof12,PnDof19,PnDof20,PnDof30,and PnDof32,were screened out.Then 3 genes were selected for subsequent experiments:PnDof19,PnDof20,PnDof30.3.Primers were designed based on V3.0 database of Populus trichocarpa which has been finished whole-genome sequencing,and then cloned the three homologous genes from Populus simonii X P.nigra:PnDofl9,PnDof20,PnDof3O.Sequence analysis found that they all had Dof domain and nuclear location signals;phylogenetic analysis showed that PnDof20 and PnDof30 were clustered together with Arabidopsis thaliana AtDofl and Coastal pine PpDof5 that had been verified to regulate nitrogen metabolism,suggesting that these two genes might be involved in the regulation of nitrogen metabolism.4.The subcellular localization of these three Dof transcriptional factor were observed through transient transformation method mediated by gene gun.The study found that these three genes were all located in nuclei,which indicated that PnDofl9,PnDof20 and PnDof3O have nuclear location signal.These results were conformed to the general characteristics of the members of the transcription factor.5.PnDof30 were transformed into Arabidopsis thaliana,and the transgenic plants were used for nitrogen treatments.Under the low nitrogen level,the overexpressed lines had better growth phenotypes;the content of soluble protein and chlorophyll was significantly increased,and the content of soluble sugar decreased significantly;among the members of AMT,NRT and GS genes,partly were up-regulated,and partly were down regulated,and the expression of PEPC and PK genes increased significantly;the activities of PEPC,PK,GS and NR enzyme increased significantly.These results indicate that overexpression of PnDof3O in Arabidopsis thaliana can increase growth and C/N metabolism at low nitrogen level. |
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