Screening And Identification Of The Target Gene Of TDR1 Transcription Factor In Arabidopsis Thaliana Under Salt Stress

Plants preserved lifestyles make it more susceptible to various biological and abiotic stresses which is different from animals.Among them,soil salinization is one of the major abiotic stresses that seriously affect plant growth and threaten agricultural production safety.For a long time,researchers have been working on the mechanism of salt tolerance in plants and have made great progress.It was found that the SOS(Salt Overly Sensitive)signal pathway is the main signaling pathway for regulating salt tolerance of plants.Among them,the Na+/ H+ reverse transporter encoded by the SOS1 gene is involved in the tolerance of plants to high concentrations of Na+ or low concentrations of K+,SOS2-SOS3 complex react with SOS1,phosphorylating and activating SOS1 protein that participate in the salt discharge process,which together to involve in plant salt control process.At present,although people have a certain understanding of the molecular mechanism of plant salt tolerance.It is still necessary to further analyze the molecular mechanism of plant salt tolerance by identifying some new salt-tolerant regulators.So as to provide theoretical guidance for the use of biotechnology to improve plant salt tolerance and cultivate new varieties of salt-tolerant plants.Studies have shown that plants in the seed germination and seedling development is more sensitive to salt stress,which directly determines the survival of plants.Therefore,it is of great significance to identify the key factors of regulating plant tolerance during seed germination and seedling development.It is known that the TESTA DEVELOPMENT REGULATION 1(TDR1)gene regulates the synthesis of flavonoids and seed coat color,which regulates seed germination and seedling growth and development,but it is unclear whether it is involved in plant salt tolerance progress.In order to identify the key factors involved in plant salt tolerance regulation during early seedling development.In this study,the TDR1 T-DNA insertionmutant was used as the research object,and its phenotype of salt treatment was analyzed.The results showed that under the condition of Na Cl treatment,the growth of mutant seedlings of tdr1 was significantly different from that of wild type.The mutants showed that the cotyledons were yellow and the plants were weak.The results of tissue expression analysis showed that TDR1 was expressed in the whole Arabidopsis thaliana,and its expression in roots was significantly higher than that in other parts.And TDR1 was upregulated under Na Cl treatment,indicating that TDR1,a regulator of seed development,was involved in regulating plant tolerance to salt stress.In this study,we first analyzed the salt stress sensitivity of tdr1 and found that tdr1 showed a growth-inhibited phenotype at low concentrations of Na Cl.In addition,we found that it was sensitive to salt stress in the early stages of seedling growth and development.On the basis of this,we analyzed the transcript groups of wild type and tdr1 of Arabidopsis thaliana under Na Cl treatment condition,selected a number of genes that changed at expression level,and selected some genes that we are interested in,carried out experiment of RT-PCR.Among them,the expression of LIPID TRANSFER PROTEIN 4(LTP4),PHOSPHATE TRANSPORTER 3;2(PHT3;2),AT2G38905 and AT5G38940 in the tdr1 mutant was significantly up-regulated under Na Cl treatment,indicating that it could act on the downstream of TDR1,mediated TDR1 regulation to plant salt tolerance.Furthermore,by means of promoter analysis,we found that the LTP4,PHT3;2 promoter region-1114 bp and-650 bp,respectively,containing CACATG binding elements of TDR1,suggesting that it may be the direct downstream target gene of TDR1.We have demonstrated that the TDR1 transcription factor can bind to the promoter region of LTP4 and PHT3;2 to initiate downstream gene expression by performing yeast single hybridization experiments under in vitro conditions.At the same time,combined with the chromatin immunoprecipitation test(Ch IP),we confirmed that TDR1 transcription factor in vivo conditions can be combined with CACATG components,and thenregulate LTP4 and PHT3;2 gene expression.In order to further verify the above conclusions,we constructed the overexpression vector of the TDR1 downstream target gene and carried out the Arabidopsis thaliana genetic transformation,in order to be able to verify the function of the target gene.In conclusion,we found that TDR1 was involved in the regulation of salt tolerance during early plant development.At the same time,we screened the downstream target gene of TDR1,and PHT3;2,LTP4 may be the direct target gene of TDR1 mediated TDR1 regulation of plant salt tolerance.