Functional Characterization Of Abiotic Stress Response Of AtDof2.3 From Arabidopsis

Dof are members of a major family of higher plant transcription factors,which exist in all higher plants.Dof transcription factor play important function in the regulation of many growth processes,such as light signal regulation,hormonal regulation,seed germination and growth,drought,salinity resistances and so on.The studies of Dof transcription factor in Arabidopsis have been more comprehensive than other plants.However,the functional characterization and mechanisms of Dof transcription factor responded to abiotic stress in Arabidopsis have not fully understood.In this study,the role of AtDof2.3 in salt stress response was analyzed.The expression patterms of AtDof2.3 in different tissues of Arabidopsis and after salt,drought,ABA stresses were monitored by using the real time RT-PCR.The results showed that AtDof2.3 expression level was the lowest in the roots.And the expression in inflorescence is the highest(47.43,folds of roots).The expression level in silique was 46.04-fold of roots.Its in stem was 24.81 fold of its in roots.The expression level in leaves is 6.11-fold of its in roots.These results indicated that AtDof2.3 gene expressions were tissue specific.Furthermore,AtDof2.3 gene expressions were upregulated under NaCI,PEG and ABA treatment.Compared to the normal growth conditions,its expression level was upregulated 14.32 folds after NaCI stress.It was induced 27.06 folds after ABA treatment.The expression level was the highest under PEG stress(69.46-folds),indicated that AtDof2.3 maybe involved in stress response in Arabidopsis.In order to understand the position of AtDof2.3 in the cell,the fusion gene AtDof2.3-GFP were constructed to detect the location of AtDof2.3.The results showed that the AtDof2.3 was expressed in the nucleus.In addition,the AtDof2.3 protein was detected no transcription activity by yeast two-hybrid.The results of yeast one-hybrid showed that AtDof2.3 can recognize an unknown element(GCAG)besides the Dof(AAAG)elements.To further study the function of AtDof2.3 under stress response,the germination rate,root lengths and fresh weights between overexpression AtDof2.3 Arabidopsis and the wild-type(WT)under NaCI,ABA and Mannitol stress were compared.Under abiotic stress conditions,the germination rate in overexpression AtDof2.3 was average be up to 3.87-folds of its in WT.The root lengths were average up to 3.12-folds.The fresh weights of WT were only 60%of transgenic lines.These results showed that AtDof2.3 gene obvious improved the abiotic stress resistance ability in transgenic Arabidopsis.Simultaneously,histochemical staining and physiological indexes were measured.And the results showed that DAB,NBT,Evans blue dyeing of transgenic lines were lower than of its in WT.The highest SOD activity of overexpression lines was up to 4.69 folds of WT.POD activity is up to 5.93 folds.Chlorophyll content is up to 9.91 folds.And the lowest MDA content of overexpression lines is only 40%of WT.The relative electric conductivity is only 33.4%of the WT.These results demonstrate that AtDof2.3 genes maybe have the ability of regulating plants to adapt the adversity environment through regulating plant physiological and biochemical reaction.For a preliminary comprehensive understanding of the network regulation of AtDof2.3 under stress,AtDof2.3 gene promoter sequences were analyzed.A variety of cis-acting elements were found,such as IBOXCORE,ARRrTA,GTGANTG10,MYBST1 and WRKY71OS,and so on.The Dof and ABRE elements were selected to analyze the possible upstream regulation gene by using yeast one-hybrid and Agrobacterium instantaneous transformation.The results showed that AtDof2.1 can specific binding to Dof element which is in the promoter sequence of AtDof2.3.And AtbZIP40 was specific bound with ABRE which is also in the promoter sequence of AtDof2.3.These two genes may be combine with Dof and ABRE elements to regulate the expression ofAtDof2.3.In summary,we have made a preliminary comprehensive understanding of the salt tolerance ability about AtDof2.3 and its regulation network regulation under stress.The signal molecules can regulate transcription factors AtDof2.1 and AtbZIP40.They will specific binding to Dof and ABRE element which is in the AtDof2.3 promoter to make the plant exhibits a series of physiological and biochemical changes to adapt to the adverse conditions.