A method for screening marker metabolites related to somatic embryo germination of Picea twigs based on nmr technology
A technique for spruce spruce and metabolites is applied in the field of evaluating the effect of drying on the germination of spruce spruce somatic embryos by NMR technology, which can solve the problems of lack of reliable methods for data analysis and screening of labeled metabolites, and achieve rapid reproducibility. , the effect of good application prospects
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Embodiment 1
[0035] The collection of different somatic embryo tissues of embodiment 1
[0036] (1) Drying treatment of spruce somatic embryos
[0037] Use tweezers to select mature and plump spruce somatic embryos and place them flat in a drying container, and carry out drying treatment according to the "filter paper method", specifically: spread the mature somatic embryos on two layers of filter paper, Put the filter paper with somatic embryos into a small petri dish (35mm) again, place 30 embryos scatteredly in each small petri dish, then place the small petri dish in a larger petri dish (90mm), Add 10mL of sterile water to the large Petri dish, and finally cover and seal it. 2 Drying process under the light intensity of s for 14 days, obtain the somatic embryo after drying ( figure 2 ).
[0038] (2) Collection and preservation of different tissues of spruce somatic embryos before and after drying
[0039] Under a stereo microscope, use a sterile scalpel to cut out the cotyledon an...
Embodiment 2
[0040] Example 2 Pretreatment of Metabolome Samples of Picea twigs Embryo Material Based on NMR
[0041] (1) Extraction of metabolites from somatic embryo material of Picea twigs
[0042] After cryogenic grinding, add 1000 μL extractant (50% methanol solution, v / v) to the sample tube, vortex for 1 minute; conduct ultrasonic extraction at 93 μm amplitude in ice-water bath, 4 seconds ultrasonic, 3 seconds intermittent, total Cycle 16 times; centrifuge at 4°C and 13,000 rpm for 15 minutes; take 600 μL of the clear supernatant in a centrifuge tube and dry the sample with nitrogen.
[0043] (2) Freeze-drying and reconstitution of metabolites
[0044] Freeze the sample in a -80°C refrigerator for 1 hour, then place the sample in a lyophilizer for 1 hour; add 450 μL of pure water and vortex for 1 minute; take 50 μL of ACDSS reagent (internal standard, 4.088 mM) Put in the above centrifuge tube, and vortex for 10 seconds (to fully mix the filtrate and ACDSS reagent); centrifuge at 4...
Embodiment 3
[0045] Example 3 Detection of Metabolome Samples of Picea twigs Somatic Embryos Based on NMR
[0046] The NMR spectrometer used for NMR detection is an Agilent DD2 600MHz triple resonance spectrometer. The specific detection conditions are shown in Table 1:
[0047] Table 1 NMR data acquisition parameters
[0048]
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