Study On The Function Of AfRAP2 Gene In Amorpha Fruticosa L. For Drought Resistance And Alkaline Salt Tolerance

As a widespread legume in China,Amorpha fruticosa L.is highly adaptable and can grow normally on drought,infertile and mildly saline soils.It is feasible to mine stress-related genes from Sophora fruticosa as it inevitably suffers from adverse environments throughout its growth life.The AP2/ERF family is a plant-specific family of transcription factors involved in the regulation of plant responses to biotic and abiotic stresses,as well as the regulation of target gene expression for various physiological programs.In this study,the differentially expressed gene AfRAP2 gene was selected from the PEG6000 simulated drought stress transcriptome sequencing in Sophora japonica and integrated into tobacco and Sambucus niloticus for heterologous overexpression by genetic transformation method to study the role of drought and salinity tolerance.1、AfRAP2 has an AP2 structural domain and belongs to the DREB subfamily of the AP2/ERF transcription factor family by bioinformatics analysis.Evolutionary tree analysis showed a close kinship with La EREBP of white lupine.2、AfRAP2 was expressed in roots,stems,leaves,flowers,spikes,bark and seedlings of Sophora japonica,with the highest expression in leaves and the lowest expression in stems.Nine stress treatments were also applied to the roots and leaves of Sophora japonica,in which NaHCO₃ and PEG6000 stresses showed significant increases in both roots and leaves.3、AfRAP2 is a protein localized in the nucleus,where it exerts its function.4、AfRAP2 was found to have self-activating activity by the test of yeast self-activation.5、The pYES2-AfRAP2 recombinant plasmid was successfully transferred into yeast INVSc1 for stress experiments to analyze the growth under drought and alkaline salt stress.Under sorbitol,mannitol and NaHCO3 stress conditions,it could be seen that the transgenic yeast grew significantly better than the control group,indicating that the transfer of AfRAP2gene into yeast improved the tolerance of the yeast strain to alkaline salt and drought stress.6、In this study,we successfully heterologously expressed AfRAP2 gene in Ben’s tobacco and obtained pure AfRAP2 overexpressing T3 generation transgenic tobacco strains,which grew significantly better than wild-type plants through natural drought and alkaline salt stress treatment,indicating that AfRAP2 plays an important role in response to alkaline salt and drought stress.7、The transgenic poplar strain was transformed by Agrobacterium-mediated infestation,and after molecular testing,the transgenic poplar strain was subjected to NaHCO₃ stress treatment,which showed that the transgenic poplar was significantly better than t he wild type according to the degree of leaf damage,again demonstrating that the AfRAP2 gene has good alkaline salt tolerance.