Effects of CO2 and Pb on Rapeseed Transcriptional Metabolism

Transcription: 1. RNA Extraction and Detection: Total RNA quality assessment: Concentration and purity were detected using nanodrop <Thermo Scientific NanoDrop 2000 (Thermo Scientific, Waltham, Massachusetts, USA)>, and integrity was detected using RNA-specific agarose gel electrophoresis or 2100 <Agilent 2100 Bioanalyzer RNA 6000 Nano kit 5067-1511 (Agilent Technologies Inc, California, USA)>.2. Library Construction and Quality ControlSelect total RNA >= 1 μg. Using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs Inc; Ipswich, Massachusetts, USA), mRNA with polyA tails was enriched using Oligo(dT) magnetic beads. The mRNA was then randomly fragmented using divalent cations. Using the fragmented mRNA as a template and random oligonucleotides as primers, cDNA was synthesized. The double-stranded cDNA was purified, followed by end repair and the introduction of an "A" base at the 3' end, and ligation of sequencing adapters. cDNA samples of approximately 400-500 bp were screened using AMPure XP beads, amplified by PCR, and the PCR products were purified again using AMPure XP beads to obtain the final library. Library quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies Inc, California, USA) and an Agilent High Sensitivity DNA Kit (Agilent Technologies Inc, California, USA, 5067-4626). Total library concentration was determined using Pico Green (Quantifluor-ST, fluorometer, Promega, Madison, Wisconsin, USA, E6090; QuantiT PicoGreen dsDNA Assay Kit, Invitrogen, California, USA, P7589). Effective library concentration was quantitatively determined by qPCR (StepOnePlus Real-Time PCR Systems, Thermo Scientific, Waltham, Massachusetts, USA).Multiplexed DNA libraries were homogenized and mixed in equal volumes. The mixed libraries were then stepwise diluted and quantified before sequencing in PE150 mode on an Illumina sequencer.Metabolism:4.1 Metabolite Extraction1. Weigh 40 mg of ground plant sample (10 mg for dried, lyophilized, or lyophilized powdered plant sample) into a 2 mL centrifuge tube;2. Add 300 μL of pre-cooled methanol:acetonitrile:water (2:2:1, v/v/v, containing 5 ppm 2-chlorophenylalanine), add 2 steel beads, and vortex for 30 s;3. Place in a high-throughput tissue homogenizer, grind at 55 Hz for 60 s, repeat this step once;4. Sonicate in an ultrasonic cleaner for 10 min;5. Freeze at -20℃ for 30 min;6. Centrifuge at 12,000 rpm, 4℃ for 10 min, collect the supernatant and filter through a 0.22 μm filter membrane, add the filtrate to the detection bottle;7. Take the sample filtrate... Mix 10-20 μL to form a QC sample. No QC is performed on 5 or fewer samples (including 5). This is used to evaluate instrument stability and data reliability.4.2 Chromatographic MethodAn ACQUITY UPLC HSS T3 column (100 Å, 1.8 µm, 2.1 mm × 100 mm) was used. The flow rate was 0.4 mL/min, column temperature was 40℃, autosampler temperature was 8℃, and injection volume was 2 μL.Positive and negative mobile phases: Mobile phase A was 0.1% formic acid in water, and mobile phase B was acetonitrile (containing 0.1% formic acid). The elution gradient was as follows:4.3 Mass Spectrometry MethodsDDA mass spectrometry data were acquired in both positive and negative ion modes using a Thermo Orbitrap Exploris 120 mass spectrometer under the control of Xcalibur software (version 4.7, Thermo). A HESI source was used, with a spray voltage of 3.5 kV/-3.0 kV, sheath gas of 40 arb, auxiliary gas of 10 arb, capillary temperature of 320℃, auxiliary gas temperature of 300℃, first-order resolution of 60,000, scan range of 70-1000 m/z, AGC Target Standard, Max IT 100 ms. The top 4 ions in the response were screened for secondary fragmentation, with a dynamic exclusion time of 4 s, a second-order resolution of 15,000, HCD collision energy of 30%, AGC Target Standard, and Max IT Auto.All official samples and QC samples were processed using the chromatographic and mass spectrometric methods described above. Before the official sample injection, 2-4 injections of QC samples were used for system equilibration. During the injection process, one injection of a QC sample was performed for every 5-10 samples. For samples with 5 or fewer samples, QC was not performed; these QC samples were used for subsequent data evaluation and quality control.Metabolite Library SearchThe raw format data from the samples was imported into MS-DIAL software (version 4.9.221218). Peak extraction, alignment, filtering, and metabolite identification were performed using this software. Peaks not detected in more than 50% of the QC samples were filtered. Missing values ​​for undetected peaks were filled using the software's gap filling algorithm and then normalized. Metabolite identification was performed using the PerSonalbio Next-Generation Metabolomics Database (PSNGM), which includes a self-built standard database, the mz Cloud database (https://www.mzcloud.org/), LIPIDMAPS (https://www.lipidmaps.org/), HMDB (https://hmdb.ca/), Mona (https://mona.fiehnlab.ucdavis.edu/), NIST_2020_MSMS, and an AI-predicted MSMS map database. The main search parameters were as follows: MS1 tolerance for identification 0.01, MS2 tolerance for identification 0.05, smoothing level 3, minimum peak height 10000, minimum peak width 5, mass slice width 0.05, and identification score cut-off 70.