Cloning And Salt Tolerance Analysis Of Dof Genes From Tamarix Hispida

Dof(DNA binding with one finger)proteins are one of the largest groups of plant-specific transcriptional factors,and they play diverse roles in various plant development processes.In previous study,many DOFCOREZM motifs were found in the promoters of ThbZIP and V-ATPase c subunit gene from Tamarix hispida,which were identified as stress regulation related genes and suggested that Dof transcriptional factors may involve stress tolerance by regulating stress response for the expression of T.hispida bZIP and V-ATPase c subunit.Therefore,the current study was to analyze the abiotic strss tolerance of Dof from T.hispida.A total 20 ThDof genes with full ORF were obtained by searching T.hispida seven transcriptomes cDNA libraries.The sequence of these 20 ThDofs were analyzed and their expression patterns in roots,stems and leaves under NaCl,PEG and ABA treatment were compared by real-time RT-PCR.The results showed that the expression of these ThDof genes displayed an obviously tissue-specific expression pattern.The expression levels of ThDof 2,ThDof3,ThDof 6 and ThDof 14 under stresses were highly upregulated,indicating that they may be stress response regulation related genes of Tamarix hispida.And the ThDof6 was selected for further study.A 1874 bp promoter of ThDof6 was cloned by genomic walking.To investigate the spatial and temporal expression of ThDof6,the 35S promoter of pCAMBIA1301 was replaced with the ThDof6 promoter to activate the expression of GUS gene.The transgenic Arabidopsis plants ProThDof6::GUS of T3 generation were stained with GUS assay solution.The results showed that the ThDof6 gene is tissue and developmental stage-specific which mainly expressed in leaf and root,and the expression leves increased obviously under NaCl,PEG stress and exogenous ABA treatment.The ThDof6 gene was constructed into plant expression vector and transformed into Arabidopsis by floral dip method.The root length,fresh weight and seed germination were compared between the ThDof6 overexpression Arabidopsis plants and wild type(WT)under NaCl,PEG and ABA treatment.The results indicated that the transgenic plants significantly improved salt and drought tolerance,also displayed increased sensitivity to ABA,compared to WT.Physiological indicators such as MDA content,Chlorophyll content,SOD activity,POD activity,relative conductivity and histochemical staining assays were detected in transgenic and WT plants.The results showed that the intracellular O2,H2O2 and cell damage levels of transgenic Arabidopsis were lower than those of WT Arabidopsis under salt stress condition.These results indicate that over expression of ThDof6 can reduce the injury of plant cell.The expression profiles of ThDof6 overexpression T3 transgenic Arabidopsis OE8-5 and WT Arabidopsis under salt stress were compared using Agilent Arabidopsis gene chips.A total of 2346 differentially expression genes with p-value<0.05 and fold change>2 were obtained.Among them,1173 genes were upregulated,of which 1090 are function-known genes and 83 are function-unknown.Chip data analysis suggests that ThDof6 gene can regulate downstream gene expression during salt-promoted stress response and may change plant salt tolerance.