Abstract:

The "king of forage," alfalfa (Medicago sativa L.), is not only a high-quality forage crop but also addresses challenges arising in agricultural production and environmental governance. However, its production and application are severely impacted by drought. Physiological phenotype analysis has revealed that exogenous nitric oxide (NO) can enhance the drought tolerance of alfalfa. This study utilizes "Sandeli" alfalfa as the material to investigate the function of exogenous nitric oxide (NO)-mediated miR166 in regulating drought tolerance in alfalfa through the application of AmiRNA (Artificial microRNA) technology and STTM (Short Tandem Target Mimic) technology. The regulation of drought resistance in miR166-transformed alfalfa by exogenous nitric oxide (NO) indicates that silencing miR166e-5p leads to the serration of alfalfa leaf margins and delayed wilting in detached leaves. The content of proline, an osmoregulatory substance, increases to 1.58 times that of the wild type. The activities of antioxidant enzymes increase to 1.39 to 1.59 times those of the wild type, while the contents of H₂O₂ (Hydrogen Peroxide) and MDA (Malondialdehyde) decrease to 61 % and 72 % of the wild type, respectively. Additionally, the expression of target genes and NO synthase genes is upregulated, with the NR2 gene showing a 2.27-fold upregulation. Overexpression of miR166e-5p exacerbates the damage to the antioxidant system and reduces osmoregulatory substances. In summary, silencing miR166e-5p enhances the drought tolerance of alfalfa. The application of exogenous NO causes the physiological indicators and gene expression levels in the overexpressing lines to trend towards those of the wild type, effectively reducing the drought sensitivity caused by miR166e-5p overexpression. This study screens out miR166-5p related to drought tolerance, laying a theoretical foundation for the rapid breeding of drought-tolerant alfalfa.