Metabolic analysis
Tissues of rosemary leaves, stems and roots (three biological replicates) were used for RNA extraction and metabolite analyses. High performance liquid chromatography (Waters e2695) and high-performance gas chromatography (Agilent Technologies 7890B-5977B) were used for metabolite analysis of S. rosmarinus . The rosemary samples were oven-dried to a constant weight at 60°C, crushed with a grinder, and sieved through a 60-mesh screen. An aliquot of the test sample powder (0.02g) was added to 1 mL of 70% methanol, ultrasonically extracted for 45 minutes, centrifuged at 8,000 rpm/min for 10 minutes, and the supernatant was collected and passed through a 0.22 µm filter membrane to obtain the test solution, each group of extracts (3 replicates) was placed in a 4 ℃ refrigerator for later use.
A Waters e2695 high performance liquid chromatography system was used for analysis, the detector model was a Waters2998 ultraviolet light detector, the chromatographic column model was a Waters sunfire C18 reversed-phase chromatographic column, and the chromatographic acquisition software was Empoeder 2. The chromatographic conditions were as follows: flow rate 1 mL/min, column temperature 30°C, sample loading volume 20 µL. The sample running time was 96 minutes, 0.02 percent phosphoric acid water and acetonitrile, utilizing gradient elution.
Qualitative and quantitative analysis of volatile components in different parts of the rosemary plant was carried out using GC-MS. Precisely weighed 1 g of fresh sample of rosemary was placed in a 20 mL headspace bottle, sealed, and then the sample was injected directly into the headspace sample tray. The chromatographic and headspace conditions were as follows: chromatography initial temperature 35°C, held for 2 min; 3°C/min to 130°C, 25°C/min to 250°C, held for 3 minutes; cooling to 35°C. Headspace conditions: Equilibrium: 80℃ for 30 min, oven temperature: 80℃, loop temperature: 90℃, transfer line temperature: 100℃, GC cycle time: 70 min, split ratio: 1:5, flow rate: 1mL/min, M/Z: 35-600, EI: 70eV, MS ion source temperature: 230°C, detector temperature: 260°C.