Functional Study On Drought And Salt-alkali Tolerance Of WRKY42 Gene In Amorpha Fruticosa

Due to global climate change,plants can’t move during planting,so they must endure abiotic stresses such as drought,salinity,and temperature extremes.These stressors greatly limit plant development and hinder growth,thereby reducing productivity and causing severe damage.Transcription factors have been extensively studied in many crops,as well as play an important role in plant growth and defense.WRKY transcription factors（TFs）play an important role in plant responses to abiotic stresses.However,there are still many aspects of WRKY TFs involved in stress tolerance in Amorpha fruticose L.that are unknown.In this study,the Af WRKY42 gene was isolated and characterized from data analyzed by transcriptome sequencing data under drought stress in Amorpha fruticose,and was genetically transformed and integrated into the tobacco genome for heterologous overexpression to study salinity resistance and assess the expression pattern of Af WRKY42 in response to salinity stress.1.In this study,three-month-old seedlings of Amorpha fruticose were cultured as experimental material and the Af WRKY42 gene was cloned by RT-PCR.Bioinformatic analysis revealed that it contains a WRKY structural domain,two low complexity regions and a helix region.Phylogenetic tree analysis revealed that Af WRKY42 is most closely related to WRKY47 of Cajanus cajan and WRKY42 of Cicer arietinum.And it was named as Af WRKY42.2.The Af WRKY42 gene was expressed in different tissues of the plant,with the highest in flowers and the lowest in roots.Its expression was detected in roots and leaves treated with Na Cl,Na HCO₃,and PEG,and the data showed an overall upward trend by its induction.Stress treatment,induced an increase in Af WRKY42 gene expression;it is speculated that Af WRKY42gene is associated with the regulation of stress by adversity.3.In this study,the Af WRKY42-GFP recombinant expression vector was successfully constructed,and the fluorescence expression of Af WRKY42-GFP fusion protein was detected using the transient gene expression system of Arabidopsis thaliana leaf protoplasts,showing that it was localized in the nucleus,providing a basis for studying the protein expression and the mechanism of its function.4.In this study,overexpressing T3 generation of tobacco strains driven by 35S promoter transducing Af WRKY42 gene were obtained and tobacco chlorophyll,conductivity and malondialdehyde were analyzed by salt（Na Cl）alkali（Na HCO₃）stress and drought stress treatments.Af WRKY42 was found to play an important role in response to drought,salt and alkaline stresses.5.In order to further test the functional properties of Af WRKY42 gene and to understand the basic role in the organism,the tobacco transformed with Af WRKY42 gene RNAi interfered with the homologous gene,and the tobacco transformed to silence the homologous gene showed significant differences in growth from WT,with plant clumping,smooth leaf epidermis,no leaf hairs and also changed leaf color,indicating that Af WRKY42 gene is involved in plant growth and development and has an important role,which laid the foundation for future studies on the function of Af WRKY42 gene.6.In this study,we used Agrobacterium tumefaciens to infest the cotyledon nodes of Amorpha fruticose to induce healing tissues and establish an efficient and stable genetic transformation system.Amorpha fruticose stained with the GUS gene showed the expression activity and pattern in various parts of the plant at various times,which laid a solid foundation for functional gene research in Amorpha fruticose and the development of new strains.Our results show that overexpression of Af WRKY42 improves drought and salinity tolerance in plants,indicating that it not only plays a positive role in abiotic stress response but also provides a WRKY transcription factor candidate gene that can improve stress tolerance in Amorpha fruticose and other plants.