Allene oxide synthase (AOS) is a key enzyme involved in the jasmonic acid (JA) synthesis pathway in plants. To explore its function on the regulatory mechanism of JA synthesis, we screened and identified two genes and in qingke. Both HvnAOS1 and HvnAOS2 contained conserved heme-binding motif, which is most closely related to AtsAOS2, indicating controlled dehydration of fatty acid hydroperoxides to allene oxides. Molecular docking simulations identified the key amino acid sites that were important for heme binding and interaction with 13()-HPOT, respectively. The expression pattern also indicated that and were highly induced by JA, abscisic acid, and salicylic acid. Subcellular localization of and using transient expression of showed the green fluorescent protein signal in the cell cytoplasm of the . leaves. Overexpression of and in mutant restored male fertility and plant resistance to , indicating that and can restore the functions of in mutant.