CN108956679B - Method for screening spruce embryo germination related marker metabolites based on NMR technology - Google Patents
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Abstract
The invention relates to a method for screening spruce embryo germination marker metabolites from rough branches based on an NMR technology, which comprises the following steps: drying the spruce embryo with filter paper, separating cotyledon and radicle of un-dried embryo and cotyledon and radicle of dried embryo with scalpel, extracting, freeze drying and re-dissolving metabolites of different embryo tissues, NMR detection, treating metabolic group data and screening labeled metabolites. According to the invention, a set of efficient, rapid and good-repeatability methods for pre-treating, extracting, processing data and screening differential metabolites of the metabolism group sample of the spruce embryo with the rough branch can be established through the metabolism group research of different tissue samples of the spruce embryo with the rough branch, and the related metabolic information of the spruce embryo with the rough branch, which is influenced by drying treatment, from morphological maturation to physiological maturation can be effectively disclosed and evaluated.
Description
Technical Field
The invention relates to the technical field of culture technology, instrument analysis technology and data analysis in a tissue culture link, in particular to a method for evaluating the influence of desiccation on the germination of spruce embryos on rough branches by an NMR technology.
Background
Metabolomics is a new technology for assisting gene expression and protein quantification by analyzing a large number of metabolites in the biological development process and researching the concentration difference of the metabolites under different physiological and genetic conditions. The concentration or metabolic flux of metabolites in a plant body is changed due to the influence of any environmental stress, pathology and other factors on the plant life system, and most metabolic pathways do not exist independently, so that metabolomics is widely applied to the research in the fields of plant functional genomics, molecular phenotype, molecular pathology and the like as a metabolic substance dynamic change process reflecting the whole level in the life process of a certain plant.
Picea asperata (Picea asperata mask.) is a plant of Picea of Pinaceae, and is a special tree species in China, and has wide distribution range, excellent material quality and strong adaptability. The method is not only an important material tree species, but also a top polar community and a zonal vegetation species of forest succession in high mountain areas such as Sichuan-xi plateau, south Gansu and south Shaanxi. At present, somatic embryogenesis technology of spruce with rough branches is basically established, but internal regulation mechanisms of some key links are unknown, and especially the change and accumulation of substances in somatic embryos before germination have the effect on somatic embryo germination.
Desiccation is a treatment mode before somatic embryo germination and can promote somatic embryo germination. Research shows that desiccation mainly converts the morphological maturation into physiological maturation of somatic embryos, and a higher germination rate can be obtained after the somatic embryos are transferred into a germination culture medium. At present, the molecular mechanism of desiccation to promote somatic embryo germination is not clear. Based on analysis of results of cotyledon and radicle metabolism groups before and after drying treatment of spruce with rough branches, the method has important significance for revealing the drying action mechanism and finding potential labeled metabolites. However, the research on metabonomics before and after drying treatment of spruce embryos is not reported at home and abroad at present, and particularly, a reliable method for sample treatment, extraction, data analysis and labeled metabolite screening of spruce metabolome detection based on an NMR technology is lacked.
Disclosure of Invention
The invention aims to provide a method for determining and analyzing a spruce rough branch somatic embryo metabolome based on an NMR technology and evaluating the influence of drying treatment on the germination capacity of the spruce rough branch somatic embryo.
In order to realize the purpose of the invention, the invention provides a method for screening spruce embryo germination marker metabolites from rough branches based on an NMR technology, which comprises the following steps: drying the spruce embryo with filter paper, cutting cotyledon and radicle of non-dried embryo and dried embryo, extracting, freeze drying and re-dissolving metabolites of different embryo tissues, NMR detection, analyzing metabolic group data and screening labeled metabolites.
According to the method, the drying treatment method comprises the following steps: placing somatic embryo of spruce with thick branch on filter paper, and performing 16h photoperiod at 24 + -1 deg.C and 15-18 μmol/m2S, culturing for 7-14 days under the condition of light intensity to obtain the dried somatic embryos.
Further, the drying treatment method specifically comprises the following steps: spreading the differentiated mature somatic embryos on 1-4 layers of filter paper, placing the filter paper with the somatic embryos in small culture dishes (35mm), placing 30-40 embryos in each small culture dish in a scattered mode, then placing the small culture dishes without covers in large culture dishes (90mm), finally adding 10mL of sterile water (the height of the small culture dishes is not more than two thirds) in the large culture dishes, and covering and sealing. At 24 +/-1 deg.C, 16h photoperiod and 15-18 μmol/m2S light intensity for 7-14 days.
In the drying treatment by the filter paper method, the number of filter paper layers padded in the culture container for placing the spruce somatic embryos with the thick branches is preferably 2-3, and more preferably 2; the light intensity is preferably 15 mu mol/m2S; the drying treatment time is preferably 14 days.
According to the method, the separation step comprises the steps of cutting off cotyledons and radicles of the dried somatic embryos under a stereomicroscope, and simultaneously cutting off cotyledons and radicles of non-dried somatic embryos; and placing the cut target tissue in a freezing tube, and freezing and storing.
Furthermore, in the method, the cutting position of the cotyledon is 0.1-0.3 mm below the unfolded cotyledon, the cutting position of the radicle is 1-1.5 mm above the tip of the radicle, and 4 groups of materials are obtained, wherein each group of materials is 6 in repetition. Accurately weighing a single sample after sampling, quickly freezing the single sample by using liquid nitrogen in time, and storing the single sample in a refrigerator at the temperature of 80 ℃ below zero for later use after the temperature is stable.
According to the method, the method for extracting the somatic embryo tissue metabolites comprises the following steps: adding 50% (v/v) methanol solution as extractant into the sample after low-temperature grinding, performing ultrasonic extraction under 93 μm condition, performing ultrasonic extraction for 4 seconds, intermittently for 3 seconds, and circulating for 16 times; centrifuging, taking supernatant fluid in a centrifuge tube, and drying by using nitrogen;
the freeze-drying and re-dissolving method comprises the following steps: adding pure water into the freeze-dried sample, adding the ACDSS reagent after vortex, uniformly mixing by vortex again, and centrifuging to obtain supernatant serving as an extracting solution for NMR detection.
Further, the extraction method of the somatic embryo tissue metabolites comprises the following steps: after cryogrinding, 1000. mu.L of extractant (50% methanol solution, v/v) was added directly to the sample tube and vortexed for 1 min; performing ice-water bath, performing ultrasonic extraction under the amplitude condition of 93 mu m, performing ultrasonic treatment for 4 seconds, performing intermittent treatment for 3 seconds, and circulating for 16 times; then centrifuging for 15 minutes at 4 ℃ and 13000 rpm; 600 μ L of clear and transparent supernatant was taken and dried in a centrifuge tube with nitrogen.
The freeze-drying and redissolving method comprises the following steps: freezing the extract in a refrigerator at-80 deg.C for 1 hr, and freeze drying in a freeze dryer for 1 hr; add 450. mu.L of purified water, vortex for 1 minute; 50 μ L of ACDSS reagent (internal standard, 4.088mM) was placed in the tube and vortexed for 10 seconds (the filtrate was mixed well with ACDSS reagent); centrifuging at 13000rpm at 4 deg.C for 2 min; 480. mu.L of the supernatant was placed in a nuclear magnetic tube and used as a sample for NMR detection.
NMR detection the NMR spectrometer used was an Agilent DD 2600 MHz triple resonance spectrometer. The specific detection parameter conditions are shown in the following table:
the invention also provides a data processing method of the spruce embryo material metabolome with rough branches, which comprises the following specific steps:
(1) data preprocessing: will be provided with1The H NMR Free Induction Decay (FID) signal is introduced into the Chenomx NMR sit (version 8.0, Chenomx, Edmonton, Canada) software, and fourier transform is automatically performed to adjust the phase and correct the baseline. In DSS-d6The peak (0.0ppm) was used as a standard for the chemical shift of the entire spectrum and was subjected to an inverse convolution operation to adjust the peak shape (CSI) of the spectrum. According to1Correlation information (e.g., chemical shift, peak shape, half-peak width, coupling split, etc.) of the signals in the H NMR spectrum as DSS-d6The peak concentration and the peak area are taken as standards, and the signals of 24 sample spectrograms are compared and analyzed one by combining a Chenomx self-contained database. Then, the metabolites and the corresponding absolute value concentrations are exported to an EXCEL table, and a metabolite variable matrix is obtained.
(2) Chemometric analysis: the variable matrix was normalized to the initial mass of the sample and used as source data for the subsequent Principal Component Analysis (PCA) and orthonormal partial least squares-discriminant analysis (OPLS-DA). Differential metabolic markers between samples were screened from complex data. Through analyzing the difference of metabolites of cotyledons and radicles before and after desiccation, arginine, glucose and quinic acid are screened out to be used as labeled metabolites for judging the germination capacity of spruce embryos on rough branches.
The invention also provides labeled metabolites related to the germination capacity of the spruce embryo of the rough branch, wherein the labeled metabolites comprise arginine, glucose and/or quinic acid.
The invention also provides application of the NMR technology in screening the spruce embryo germination marker metabolites, and further, the metabolites are arginine, glucose and/or quinic acid.
The invention also provides application of arginine, glucose and/or quinic acid as a marker metabolite for germination of the spruce embryo, and further, the content of the metabolite in the spruce embryo germination process is increased.
The invention also provides application of arginine, glucose and/or quinic acid as a labeled metabolite in indicating the germination strength of the spruce embryo with the thick branch.
The invention firstly separates cotyledon and radicle of the somatic embryo before and after drying the spruce with rough branches for detection and analysis.
According to the invention, a set of efficient, rapid and good-repeatability methods for pre-treating, extracting, processing data and screening differential metabolites of the spruce body embryo material metabolome sample are established through researching the metabolome of the spruce body embryo material, so that the metabolic information closely related to the spruce body embryo germination influenced by drying treatment can be effectively disclosed. Although the spruce with thick branches is taken as an example, the method provides good application prospects for the detection of the somatic embryo material metabolome of the whole needle-leaved tree species, the screening of labeled metabolites and the evaluation of the influence of drying treatment on the germination capacity of the somatic embryos and the seed embryos.
Drawings
FIG. 1 is a process flow chart of screening spruce embryo germination marker metabolites based on NMR technology.
FIG. 2 shows somatic embryos before and after the stem treatment in example 1 of the present invention. Wherein, A and C: non-dried somatic embryos; b and D: dried somatic embryos; c and D: the non-dehydrated cotyledon (NC), non-dehydrated radicle (NR), Dehydrated Cotyledon (DC) and Dehydrated Radicle (DR) sites of the samples were labeled, respectively. Scale bar: a and B, 10 mm; c and D, 500 μm.
FIG. 3 is a PCA score plot for different tissue metabolome before and after desiccation in example 4 of the present invention. A: an overall PCA score plot of four groups of samples, namely, non-desiccated cotyledons (NC), non-desiccated radicles (NR), Desiccated Cotyledons (DC) and Desiccated Radicles (DR); b: the analysis results of DC and NC; c: the results of the analysis of DR and NR; d: the analysis results of DC and NR; e: and (5) analysis results of DR and NC.
FIG. 4 is a graph of OPLS-DA scores of different tissue metabolomes before and after desiccation in example 4 of the present invention. A: the analysis results of DC and NC; b: the results of the analysis of DR and NR; c: the analysis results of DC and NR; d: and (5) analysis results of DR and NC.
FIG. 5. summary of the inter-group differential metabolites of example 5 of the present invention, wherein the content of Arginine (Arginine), Glucose (Glucose) and Quinic acid (Quinic acid) was significantly higher in the dried group than in the undried group.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
The Standard referred to in the examples was ACDSS (Anachro Certified DSS Standard solution) -4.088 mM, from Anachro; an ultrasonic crusher: sonic VCX 130; a freeze dryer: LABCONCO Freezone 2.5; miniature swirl mixer: XW-80A, purchased from Shanghai Lucy Analyzer Mill; a centrifuge: eppendorf Centrifuge 5415R and 5425R; an NMR spectrometer: agilent DD 2600 MHz dispersive instrument equaled with a triple-response cryoprobe.
The process flow chart of the invention for screening the spruce embryo germination marker metabolite based on the NMR technology is shown in figure 1.
EXAMPLE 1 Collection of different somatic embryo tissues
(1) Drying treatment of spruce embryo with thick branches
Selecting mature and plump rough branch spruce embryo with tweezers, placing the embryo into a drying container, and drying according to a filter paper method, wherein the drying method specifically comprises the following steps: spreading the mature somatic embryos on two layers of filter paper, placing the filter paper with the somatic embryos in small culture dishes (35mm) with 30 embryos dispersed in each small culture dish, placing the small culture dishes in larger culture dishes (90mm), adding 10mL of sterile water in the large culture dishes, and finally capping and sealing at 24 ℃, 16h photoperiod and 15 mu mol/m2S light intensity for 14 days to obtain dried embryos (FIG. 2).
(2) Collecting and storing different tissues of spruce embryos with thick branches before and after drying
Under a stereomicroscope, cotyledons and radicles of an undried somatic embryo and a dried somatic embryo are respectively cut by using a sterile scalpel, the cutting position of the cotyledons is about 2mm below an unfolded cotyledon, the cutting position of the radicles is about 1.2mm above the tip of the radicles, the cut cotyledons and radicles are respectively placed in a cryopreservation tube and accurately weighed, and 6 biological replicates of each group of samples are obtained. And (3) completely quick-freezing by using liquid nitrogen, and then storing in a refrigerator at the temperature of-80 ℃ for later use.
Example 2 Pre-treatment of spruce somatic embryo material metabolome samples based on NMR
(1) Extraction of metabolite of spruce somatic embryo material with thick branches
After cryomilling, 1000. mu.L of extractant (50% methanol solution, v/v) was added to the sample tube and vortexed for 1 minute; performing ice-water bath, performing ultrasonic extraction under the amplitude condition of 93 mu m, performing ultrasonic treatment for 4 seconds, performing intermittent treatment for 3 seconds, and circulating for 16 times; centrifuging for 15 minutes at 13000rpm and 4 ℃; 600. mu.L of the clear and transparent supernatant was taken and placed in a centrifuge tube and the sample was blown dry with nitrogen.
(2) Freeze-drying and reconstitution of metabolites
Freezing the sample in a refrigerator at-80 ℃ for 1 hour, and then putting the sample in a freeze dryer for continuous freeze drying for 1 hour; add 450. mu.L of purified water, vortex for 1 minute; 50 μ L of ACDSS reagent (internal standard, 4.088mM) was placed in the tube and vortexed for 10 seconds (the filtrate was mixed well with ACDSS reagent); centrifuging at 13000rpm at 4 deg.C for 2 min; 480 mu L of supernatant is taken in a nuclear magnetic tube and used as samples of different tissue metabolome of the spruce embryo with the thick branch detected by NMR.
Example 3 detection of spruce somatic embryo metabolome samples based on NMR
NMR detection the NMR spectrometer used was an Agilent DD 2600 MHz triple resonance spectrometer. Specific detection conditions are shown in table 1:
TABLE 1 Nuclear magnetic data acquisition parameters
Example 4 analysis of different tissue metabolome data of spruce embryos
(1) Data pre-processing
Will be provided with1The H NMR Free Induction Decay (FID) signal is introduced into the Chenomx NMR sit (version 8.0, Chenomx, Edmonton, Canada) software, and fourier transform is automatically performed to adjust the phase and correct the baseline. In DSS-d6The peak (0.0ppm) was used as a standard for the chemical shift of the entire spectrum and was subjected to an inverse convolution operation to adjust the peak shape (CSI) of the spectrum. Based on the related information (such as chemical shift, peak shape, half-peak width, coupling split, etc.) of the signals in the 1H NMR spectrum, the signals are processed with DSS-d6The peak concentration and the peak area are taken as standards, and the signals of 24 sample spectrograms are compared and analyzed one by combining a Chenomx self-contained database. Then, the metabolites and the corresponding absolute value concentrations are exported to an EXCEL table, and a metabolite concentration variable matrix is obtained. And finally, carrying out normalization processing on the data by using a Pareto Scaling method.
(2) Principal Component Analysis (PCA)
The normalized data from the four samples were subjected to pattern recognition multivariate analysis using OmicShare Tools and model fitting was performed on the first and second principal components of the data. The overall analysis results for the four groups of samples (NC, NR, DC and DR) and the analysis results for pairwise comparisons (DC vs NC, DR vs NR, DC vs NR and DR vs NC) were obtained (fig. 3).
(3) Chemometric analysis
The variable matrix was used as source data to perform inter-group (DC vs NC, DR vs NR, DC vs NR, and DR vs NC) orthogonal partial least squares-discriminant analysis (OPLS-DA) to obtain inter-group discrimination results (fig. 4).
Example 5 differential metabolite analysis and labeled metabolite screening of different tissues of spruce embryos on rough branches before and after desiccation
(1) Differential metabolite analysis between different tissues before and after desiccation
Differential metabolites between groups (DC vs NC, DR vs NR, DC vs NR and DR vs NC) were identified from complex data. The method adopts the VIP (variable immunity in the project) value of the first main component in the OPLS-DA model and combines the p value of Student's t test (Student's t-test) to screen differential metabolites. VIP ≧ 1, and those with p <0.05 were considered as differential metabolites.
Differential metabolite of dried cotyledons and undried cotyledons (DC vs NC)
The content of 8 metabolites in the Desiccated Cotyledons (DC) was significantly changed compared to the non-desiccated cotyledons (NC) (table 2), and was all up-regulated. As shown in table 2, quinic acid, which is the most fold different, was up-regulated to 46.71 fold; there were also 3 carbohydrates that were also significantly elevated, with the glucose upregulated the most, 16.09 times the original; arginine, one of the highest N/C amino acids in plants, was also significantly up-regulated, up-regulated to 6.95-fold of the original.
TABLE 2 significant differential metabolites between Desiccated Cotyledons (DC) and undried cotyledons (NC)
② differential metabolite (DR vs NR) of anhydrated and undried radicles
The content of 5 metabolites in the Desiccated Radicles (DR) was significantly changed compared to the undried radicles (NR) (table 3). Arginine, fructose, glucose and quinic acid are obviously up-regulated to 6.64, 4.02, 5.78 and 3.10 times of the original levels respectively; sucrose was significantly down-regulated to 0.25 of the original.
TABLE 3 significant differential metabolites between Desiccated (DR) and undried (NR) radicles
③ differential metabolite of dried cotyledon and undried radicle (DC vs NR)
There were 7 significant increases and 1 significant decrease in Desiccated Cotyledons (DCs) compared to the undried radicles (Table 4). As shown in table 4, quinic acid, glucose, arginine, sucrose, inositol, glutamate, and 4-aminobutyric acid were upregulated to 3.21, 2.05, 3.81, 2.38, 1.97, 1.58, and 4.80 fold, respectively; while glutamine was down-regulated to 0.40.
TABLE 4 significant differential metabolites between Desiccated Cotyledons (DC) and undried radicles (NR)
Fourthly differential metabolite of anhydrated radicle and undried cotyledon (DR vs NC)
The content of 7 substances in the anhydrated radicles (DR) changed significantly compared to the anhydrated cotyledons (NC) (table 5). Wherein, the content of 6 substances is up-regulated, and arginine, choline, fructose, glucose, quinic acid and inositol are up-regulated to 12.12, 1.83, 19.84, 45.37, 45.09 and 2.00 respectively; while the sucrose content was reduced to 0.29.
TABLE 5 significant differential metabolites between anhydrated radicles (DR) and undried cotyledons (NC)
(2) Screening of labeled metabolites with strong and weak somatic embryo germination capacity
By comparative analysis of the dried groups (DC and DR) and the undried groups (NC and NR) metabolome, the arginine, glucose and quinic acid content was found to be higher in the dried groups than in the undried groups (fig. 5). Therefore, the three differential metabolites of arginine, glucose and quinic acid can be used as labeled metabolites for judging the germination capacity of the spruce embryo with rough branches, and when the content is increased after drying treatment, the germination capacity of the spruce embryo with rough branches is enhanced.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (4)
1. A method for screening spruce embryo germination marker metabolites from spruce branches based on an NMR technology is characterized by comprising the following steps: drying the spruce embryo with a filter paper method, separating cotyledons and radicles of the non-dried embryo and cotyledons and radicles of the dried embryo, extracting, freeze-drying and redissolving metabolites of different embryo tissues, carrying out NMR detection, processing metabolic group data and screening labeled metabolites;
the drying treatment method comprises the following steps: placing somatic embryo of spruce with thick branch on filter paper, and performing 16h photoperiod at 24 + -1 deg.C and 15-18 μmol/m2S, culturing for 7-14 days under the condition of illumination intensity to obtain a dried somatic embryo;
the separation step comprises the steps of cutting cotyledons and radicles of the dried embryos under a stereomicroscope, and simultaneously cutting cotyledons and radicles of the embryos which are not dried; placing the cut target tissue in a freezing tube, and freezing and storing;
the extraction method of the somatic embryo tissue metabolite comprises the following steps: adding 50% (v/v) methanol solution as extractant into the sample after low-temperature grinding, performing ultrasonic extraction under the condition of amplitude of 93 μm, performing ultrasonic extraction for 4 seconds, intermittently for 3 seconds, and circulating for 16 times; centrifuging, taking supernatant fluid in a centrifuge tube, and drying by using nitrogen;
the freeze-drying and re-dissolving method comprises the following steps: adding pure water into the freeze-dried sample, adding the ACDSS reagent after vortex, uniformly mixing by vortex again, centrifuging and taking supernatant as an extracting solution for NMR detection;
the treatment method of the metabolome data is as follows:
(1) data preprocessing: will be provided with1Introducing the H NMR free induction decay signal into Chenomx NMR suit software, automatically performing Fourier transform, adjusting the phase, correcting the base line by DSS-d6Peaks as overall spectral chemical positionsShifting standard, performing inverse convolution operation, adjusting spectrum peak shape, and adjusting spectrum peak shape according to the standard1Correlation of signals in H NMR spectra, in DSS-d6The peak concentration and the spectrum peak area are taken as standards, signals of 24 sample spectrograms are compared and analyzed one by combining a Chenomx self-contained database, and then metabolites and corresponding absolute value concentrations are led out to an EXCEL table to obtain a metabolite variable matrix;
(2) chemometric analysis: performing subsequent principal component analysis and partial least square method-discriminant analysis by using the variable matrix as source data, and discriminating differentially accumulated metabolites from complex data through comparative analysis of the inter-group metabolites;
cotyledon and radicle of the somatic embryo before and after desiccation of the spruce are separately detected and analyzed, and arginine, glucose and/or quinic acid are screened out through the metabolite difference analysis of the cotyledon and radicle before and after desiccation and can be used as a labeled metabolite for judging the germination capacity of the spruce somatic embryo.
2. The method according to claim 1, wherein the specific parameter conditions for NMR detection are:
the application of the NMR technology in screening the spruce embryo germination marker metabolites is characterized in that the marker metabolites are arginine, glucose and/or quinic acid.
4. Application of arginine, glucose and/or quinic acid as marker metabolite for somatic embryo germination of picea crassifolia.
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