metabolomic analysis in WT and 3miR-OE#3 rice plants.

Published: 16 September 2024| Version 1 | DOI: 10.17632/3y2hfcj8ss.1
Contributor:
hong zheyuan

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The observed opposite alteration in key genes in the lignin and flavonoid synthesis pathways prompted us to analyze the differences in metabolic products between WT and 3miR-OE plants. To this end, we carried out a non-targeted metabolomic and targeted metabolomic analysis to identify metabolites derived from phenylpropanoid pathways. 01-04 named as 3miR-OE 41-44 named as miR408-OE N1-N4 named as Nip the CK

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Metabolome analysis. Rice samples were snap-frozen in liquid nitrogen and then lyophilized to preserve metabolite integrity. The freeze-dried samples were ground in an MM400 mixer mill (Retsch) at 30 Hz for 1.5 min. Approximately, 100 mg of power was extracted with 0.8 mL of 70% methanol in water and pure methanol overnight at 4 ℃, followed by centrifugation at 10,000 g for 10 min. The clear supernatant was filtered (0.22 μm, SCAA-104; ANPEL) for LC-MS analysis. For UHPLC-MS/MS, UHPLC-Orbitrap MS The Thermo Fisher Q Exactive Plus mass spectrometer equipped with HESI source and a Vanquish UHPLC system was used. A Waters CORTECS T3 column ensured separation, with the column and autosampler set to 40 ℃ and 10 ℃. With 0.4 mL/min flow in gradient mode, the mobile phases included acidified water (0.04% acetic acid, A) and acetonitrile (0.04% acetic acid, B). B was increased from 5% to 95% over 10 minutes, held for 1 minute, then reverted to 5% by 11.1 minutes for a total 15-minute run time. Samples of 2 μL were analyzed in positive ESI mode, with full MS scans (resolution 70,000, AGC 3 × 10^6, max IT 100 ms, scan range 120–1800 m/z) and data-dependent MS2 scans (resolution 35,000, AGC 1 × 10^5, max IT 50 ms, isolation window 3 m/z, with specific NCE). Xcalibur 4.1 facilitated data acquisition. UHPLC-TQ-MSThree biological replicates per sample underwent UHPLC-MS/MS on the AB Sciex 7500 system and were used for quantification. The ACQUITY HSS T3 column was employed with a gradient system consisting of 0.04% acetic acid in water and acetonitrile maintained at 40 ℃. An injection volume of 2 μL and a flow rate of 0.35 mL/min were employed. MRM mode recorded specified transitions, with CE individually ascertained to fine-tune the response. Data handling in MultiQuant 3.0.3 included peak area integration, relying on retention times and shapes to verify quantification accuracy.

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Metabolomics

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