Functional study of the AfRAP2 gene in Amorpha fruticosa L. tolerance to saline-alkali and drought stress.
BACKGROUND: Amorpha fruticosa L. is a leguminous shrub with high tolerance to drought, poor soil, and saline-alkali stress conditions. As a member of the family of transcription factors in higher plants, the ethylene response factor AP2/ERF plays a crucial role in both plant adaptation to abiotic stress and in growth and development. In this study, based on genes identified from the transcriptomic sequencing of Amorpha fruticosa L. under drought stress, the upregulated gene AfRAP2 was isolated from its seedlings, with the aim of elucidating its stress-response function using molecular biological techniques. RESULTS: In this study, the AfRAP2 gene was cloned from the leaves of Amorpha fruticosa L. using RT-PCR. Bioinformatics analysis revealed that AfRAP2 contains an AP2 domain and belongs to the DREB subfamily of the AP2/ERF transcription factor family, showing close phylogenetic relationships with LaEREBP from Lathyrus albus. Real-time quantitative PCR (RT-qPCR) results indicate that AfRAP2 is expressed in various tissues of Amorpha fruticosa L., with the highest expression in leaves and the lowest in stems, furthermore, its expression is significantly upregulated in roots and leaves upon induction by NaHCO3 and PEG6000. Subcellular localization experiments confirmed that the AfRAP2 protein is localized to the nucleus, and GUS histochemical staining assay revealed that its promoter drives GUS expression in anthers. Resistance analysis of overexpressing yeast strains showed that yeast transformed with the AfRAP2 gene exhibited significantly better growth under sorbitol, mannitol, and NaHCO3 stress conditions compared to the control, indicating that this gene enhances yeast tolerance to drought and saline-alkali stress. We screened transgenic tobacco and Populus davidiana × P. alba var. Pyramidalis. The results showed that under natural drought and saline-alkali stress treatments, the transgenic lines exhibited significantly improved growth and higher activities of the physiological indicators of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) compared with wild-type plants, indicating that the overexpression of the AfRAP2 gene plays a key role in the response to saline-alkali stress and drought stress. CONCLUSION: In summary, AfRAP2 contains an AP2 domain and belongs to the DREB subfamily of transcription factors, under abiotic stress induced by NaHCO₃ and mannitol, it can induce the expression of the AfRAP2 gene in tobacco and Populus davidiana×P. alba var. pyramidalis. AfRAP2 plays a vital role in the plant response to saline-alkali stress and drought stress and is a promising candidate gene for stress-tolerant breeding.