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Method for screening germination-related marker metabolites of Picea asperata Mast. somatic embryo based on NMR technology

A technology of spruce and metabolites, which is applied in the field of NMR technology to evaluate the effect of desiccation on the germination of spruce somatic embryos. It can solve the problems of lack of reliable methods for data analysis and marker metabolite screening, and achieve rapid repeatability , the effect of good application prospects

Active Publication Date: 2018-12-07
INST OF FORESTRY CHINESE ACAD OF FORESTRY
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the metabolomics of spruce somatic embryos before and after treatment at home and abroad, especially the lack of reliable methods for sample processing, extraction, data analysis and marker metabolite screening of spruce metabolome detection based on NMR technology. method

Method used

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  • Method for screening germination-related marker metabolites of Picea asperata Mast. somatic embryo based on NMR technology
  • Method for screening germination-related marker metabolites of Picea asperata Mast. somatic embryo based on NMR technology
  • Method for screening germination-related marker metabolites of Picea asperata Mast. somatic embryo based on NMR technology

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Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to a method for screening germination marker metabolites of Picea asperata Mast. somatic embryo based on an NMR technology. The method comprises: drying somatic embryo of Picea asperata Mast. through a filter paper method, separating cotyledon and radicle of non-dry somatic embryo and cotyledon and radicle of dried somatic embryo, carrying out extraction, freeze-drying and redissolution on different somatic embryo tissue metabolites, and carrying out NMR detection, metabolome data processing and screening of labeled metabolites. Through metabolomics study on different tissue samples of the Picea asperata Mast. somatic embryo, a high-efficiency, rapid and reproducible method for pretreatment on metabolomics samples of Picea asperata Mast. somatic embryo, extraction, data processing and differential metabolite screening is built. The method can effectively reveal and evaluate the related metabolic information about the influence of drying treatment on conversion ofmorphological maturation of the Picea asperata Mast. somatic embryo into physiological maturation.

Description

technical field [0001] The invention relates to the fields of culture technology, instrument analysis technology and data analysis technology in the tissue culture link, in particular to a method for evaluating the influence of desiccation on the germination of spruce somatic embryos by NMR technology. Background technique [0002] Metabolomics is a new technology that assists gene expression and protein quantification by analyzing a large number of metabolites during biological development and studying their concentration differences under different physiological and genetic conditions. The impact of any environmental stress, pathology and other factors on the plant life system will cause changes in the concentration or metabolic flow of metabolites in the plant, and most metabolic pathways do not exist in isolation, so metabolomics is a reflection of a plant life process. The dynamic change process of metabolites at the overall level is being widely used in the research of...

Claims

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Application Information

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IPC IPC(8): G01N24/08G01N1/34G01N1/28
CPCG01N1/286G01N1/34G01N24/08G01N2001/2866
Inventor 王军辉夏燕景丹龙欧阳芳群卢楠张守攻
Owner INST OF FORESTRY CHINESE ACAD OF FORESTRY
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