An embedding agent and a freeze-embedding method for plant spatial transcriptomics and spatial metabolomics analysis

Abstract

The application provides an embedding agent and a freezing embedding method for plant spatial transcriptomics and spatial metabolomics analysis, and belongs to the technical field of biotechnology. The embedding agent is composed of 2.5% hydroxypropyl methylcellulose and 7.5% polyvinylpyrrolidone in mass concentration, and the embedding agent is applied to plant spatial transcriptomics research for the first time, and fills the technical gap in the field. Meanwhile, the application realizes the combined application of spatial transcriptomics and spatial metabolomics in the same plant individual for the first time, and provides a systematic technical solution for plant spatial multi-omics research. Through the application, the spatiotemporal dynamic change of gene expression in the development process of plants can be accurately analyzed, and the metabolic pathway and key enzyme gene of natural products in medicinal plants are provided, which provides important technical support for the functional research and industrial development of medicinal plants.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an embedding agent and a freezing embedding method for plant spatial transcriptomics and spatial metabolomics analysis. BACKGROUND

[0002] Spatial multi-omics was one of the seven most anticipated technologies in 2022 by NATURE magazine. By combining different omics, a one-to-one correspondence between biomarkers and spatial location is achieved, obtaining a higher dimensional and higher resolution expression profile. Existing spatial multi-omics technologies mainly include spatial transcriptomics, spatial proteomics and spatial metabolomics. These technologies combine spatial information with molecular biology techniques, enabling the analysis of gene expression, protein distribution and metabolite changes while preserving the spatial location of cells or tissues, thereby revealing the complexity and dynamics of biological processes.

[0003] Spatial transcriptomics: a technology that captures the spatial information of RNA molecules, enabling the correlation of gene expression levels with spatial location. Spatial metabolomics: a technology that obtains spatial distribution information of metabolites through mass spectrometry imaging and other methods, enabling correlation analysis of metabolite content and spatial location. Existing spatial multi-omics technologies are mostly combined with pathological tissue sections, such as OCT-embedded pathological tissue sections, to perform related sequencing research on spatial proteomics and spatial transcriptomics on adjacent sections. By analyzing genes and metabolites in different regions of interest (ROI), researchers constructed a region-specific gene-metabolite network graph for gastric cancer.

[0004] Although spatial multi-omics technology has been widely used in histopathology, its application in plant research is still in its infancy. Currently, most plant spatial multi-omics research uses OCT embedding agent as the tissue fixation medium. Although OCT can effectively maintain tissue morphology, its potential interference in chemical composition becomes a major bottleneck in high-sensitivity analysis such as mass spectrometry imaging. Spatial metabolomics technology usually requires samples to have specific physical and chemical properties to obtain accurate data during mass spectrometry analysis. However, the polymer in the OCT-embedded sample may produce interference peaks during ionization, which will mask the true signal of metabolites, especially small molecule metabolites, affecting the detection accuracy and accuracy of metabolites, and increasing the difficulty of detection and data processing. The core limitation of existing technology is that the OCT embedding agent contains solvent residues, additives or unreacted monomers, which form additional ion signals during mass spectrometry ionization, i.e. background noise, severely hindering the accurate identification and quantification of small molecule metabolites, and affecting the accuracy and reliability of the entire spatial multi-omics analysis.

[0005] In view of the above, the present application is proposed. SUMMARY

[0006] One of the purposes of the present application is to provide an embedding agent for plant spatial transcriptomics and spatial metabolomics analysis to solve the technical problem that the plant embedding samples prepared by the OCT embedding agent contain solvent residues, additives or insufficiently reacted monomers, which can form additional ion signals, i.e. background noise, in the mass spectrometry ionization process, seriously hindering the accurate identification and quantification of small molecule metabolites, and further affecting the accuracy and reliability of spatial multi-omics analysis.

[0007] The second purpose of the present application is to provide a preparation method of the above-mentioned embedding agent.

[0008] The third purpose of the present application is the application of the above-mentioned embedding agent or the embedding agent prepared by the above-mentioned preparation method in plant spatial transcriptomics and spatial metabolomics analysis.

[0009] The fourth purpose of the present application is a plant freezing embedding method for spatial transcriptomics and spatial metabolomics analysis.

[0010] In order to achieve the above-mentioned purposes of the present application, the following technical solutions are adopted:

[0011] In the first aspect, the present application provides an embedding agent for plant spatial transcriptomics and spatial metabolomics analysis, which is composed of 2.5% hydroxypropyl methylcellulose and 7.5% polyvinylpyrrolidone in terms of mass concentration.

[0012] Further, the viscosity of the hydroxypropyl methylcellulose is 1000-6000 mPa.s.

[0013] Further, the molecular weight of the polyvinylpyrrolidone is 10000-100000.

[0014] In the second aspect, the present application provides a preparation method of the above-mentioned embedding agent, which comprises dissolving hydroxypropyl methylcellulose and polyvinylpyrrolidone in pure water according to the formula amount to obtain the embedding agent.

[0015] Further, after dissolving, it further comprises standing for at least 12 h.

[0016] Further, the dissolving conditions comprise stirring and dissolving at 60-70℃.

[0017] In the third aspect, the present application provides the application of the above-mentioned embedding agent or the embedding agent prepared by the above-mentioned preparation method in plant spatial transcriptomics and spatial metabolomics analysis.

[0018] Fourthly, the present invention provides a plant cryoembedding method for spatial transcriptomics and spatial metabolomics analysis, comprising immersing a plant sample in a pre-cooled embedding agent for cryoembedding to obtain a plant cryoembedding sample;

[0019] The embedding agent is the embedding agent described above or the embedding agent prepared by the above preparation method.

[0020] Furthermore, the step of immersing the plant sample in the pre-cooled embedding agent includes placing a portion of the plant sample in a portion of the pre-cooled embedding agent, and then covering another portion of the plant sample with another portion of the pre-cooled embedding agent, so that the plant sample is immersed in the pre-cooled embedding agent.

[0021] Furthermore, the freezing process also includes removing air bubbles from the embedding agent.

[0022] This invention provides an embedding medium for plant spatial transcriptomics and spatial metabolomics analysis. The medium is composed of a mixture of 2.5% hydroxypropyl methylcellulose and 7.5% polyvinylpyrrolidone (PVP), avoiding the influence of other adjuvants on embedding efficiency. This is the first time such an embedding medium has been applied to plant spatial transcriptomics research, filling a technological gap in this field. Furthermore, this invention is the first to achieve the combined application of spatial transcriptomics and spatial metabolomics in a single plant, providing a systematic technical solution for multi-omics spatial research in plants. Experiments demonstrate that, in terms of technical effectiveness, its application in plant embedding and subsequent spatial metabolomics and spatial transcriptomics research provides excellent section support while significantly reducing background signal interference during mass spectrometry ionization. In spatial transcriptomics research, it exhibits excellent gene capture capabilities and high-quality sequencing results. In spatial metabolomics research, it further optimizes the ionization efficiency of metabolite signals. Through this invention, the spatiotemporal dynamics of gene expression during plant development, as well as the metabolic pathways and key enzyme genes of natural products in medicinal plants, can be accurately analyzed, providing important technical support for the functional research and industrial development of medicinal plants. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This is a comparison diagram of the effects of frozen embedding of samples with different embedding agents in Example 1 of the present invention.

[0025] Figure 2A comparison of the permeation effects of frozen-embedded sample sections with different embedding agents provided in Application Example 2 of the present invention;

[0026] Figure 3 A comparison of DESI-MS detection results of frozen-embedded sample sections with different embedding agents provided in Application Example 2 of this invention;

[0027] Figure 4 Spatial metabolomics analysis diagram of paclitaxel in Taxus chinensis based on DESI-MS detection provided in Application Example 3 of this invention;

[0028] Figure 5 Spatial transcriptomics analysis diagram of Yunnan yew based on stereo-seq sequencing provided for application example 3 of the present invention. Detailed Implementation

[0029] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0030] Unless otherwise stated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification.

[0031] One aspect of this invention provides an embedding agent for plant spatial transcriptomics and spatial metabolomics analysis, which is composed of 2.5% hydroxypropyl methylcellulose and 7.5% polyvinylpyrrolidone by mass.

[0032] An embedding medium composed of 2.5% hydroxypropyl methylcellulose and 7.5% polyvinylpyrrolidone (PVP) avoids the impact of adding other adjuvants on embedding efficiency. This invention is the first to be applied to plant spatial transcriptomics research, filling a technological gap in this field. Simultaneously, this invention is the first to achieve the combined application of spatial transcriptomics and spatial metabolomics on the same plant, providing a systematic technical solution for multi-omics spatial research in plants. Experiments have demonstrated that, in terms of technical effectiveness, its application in plant embedding and subsequent spatial metabolomics and spatial transcriptomics research provides excellent section support while significantly reducing background signal interference during mass spectrometry ionization. In spatial transcriptomics research, it exhibits excellent gene capture capabilities and high-quality sequencing results; in spatial metabolomics research, it further optimizes the ionization efficiency of metabolite signals. Through this invention, the spatiotemporal dynamics of gene expression during plant development, as well as the metabolic pathways and key enzyme genes of natural products in medicinal plants, can be accurately analyzed, providing important technical support for the functional research and industrial development of medicinal plants.

[0033] In some specific embodiments, the viscosity of the hydroxypropyl methylcellulose is 1000-6000 mPa·s, preferably 4000 mPa·s.

[0034] In some specific embodiments, the molecular weight of the polyvinylpyrrolidone is 10,000 to 100,000, preferably 40,000.

[0035] According to another aspect of the present invention, a method for preparing the above-mentioned embedding agent is also provided, comprising dissolving hydroxypropyl methylcellulose and polyvinylpyrrolidone in pure water according to the formulation amount to obtain the embedding agent.

[0036] The embedding medium prepared by mixing hydroxypropyl methylcellulose (HPMC) and polyvinylpyrrolidone (PVP) in a specific formulation exhibits excellent embedding performance, providing good support for slide sections. Furthermore, the prepared embedded samples can be used simultaneously for spatial transcriptomics and spatial metabolomics analysis. This avoids the background signal generated during mass spectrometry ionization due to the presence of solvent residues, additives, or unreacted monomers in the embedded samples caused by multiple adjuvants in the embedding medium, which would affect the accuracy and reliability of spatial transcriptomics and spatial metabolomics analysis.

[0037] In some specific implementations, the solution is further allowed to stand for at least 12 hours after dissolution to remove bubbles.

[0038] In some specific embodiments, the dissolution conditions include stirring at 60–70°C.

[0039] According to another aspect of the present invention, the application of the above-described embedding agent or the embedding agent prepared by the above-described preparation method in plant spatial transcriptomics and spatial metabolomics analysis is also provided.

[0040] According to another aspect of the present invention, a plant cryoembedding method for spatial transcriptomics and spatial metabolomics analysis is also provided, comprising immersing a plant sample in a pre-cooled embedding agent for cryoembedding to obtain a plant cryoembedding sample; wherein the embedding agent is the embedding agent described above or the embedding agent prepared by the above preparation method.

[0041] Experiments have shown that plant frozen-embedded samples prepared with the embedding agent provided by this invention can be used for spatial transcriptomics analysis as well as spatial metabolomics analysis. Different spatial multi-omics analyses do not require the preparation of plant frozen-embedded samples obtained by embedding with different embedding agents.

[0042] Because the surface of plant samples is uneven, immersing the plant sample completely in the embedding medium at once can cause a large number of bubbles to accumulate around the plant sample, or the embedding medium may not make complete contact with the plant sample, thus affecting the embedding effect. In some specific embodiments, immersing the plant sample in pre-cooled embedding medium involves placing a portion of the plant sample in a portion of pre-cooled embedding medium, and then covering the other portion of the plant sample with another portion of pre-cooled embedding medium, thus immersing the plant sample in the pre-cooled embedding medium. Immersing in two parts can avoid the generation of a large number of bubbles.

[0043] In some specific embodiments, the freezing process also includes removing air bubbles from the embedding agent.

[0044] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] Reagents:

[0046] OCT embedding medium: SAKURA OCT frozen section embedding medium from the United States, catalog number 4583-118mL;

[0047] Carboxymethyl cellulose (CMC): Shanghai Maclean Biochemical Technology Co., Ltd., MW700000 (DS=0.9), 2500~4500mPa.s, C804618-25g;

[0048] Hydroxypropyl methylcellulose (HPMC): Shanghai Maclean Biochemical Technology Co., Ltd., 4000 mPa.s, H811102-100g;

[0049] Polyvinylpyrrolidone (PVP): Shanghai Yuanye Biotechnology Co., Ltd., K-30, molecular weight 40000, S30268-500g.

[0050] Example 1

[0051] An embedding agent for plant spatial transcriptomics and spatial metabolomics analysis comprises 2.5% hydroxypropyl methylcellulose (HPMC) and 7.5% polyvinylpyrrolidone (PVP). The HPMC has a viscosity of 4000 mPa·s, and the PVP has a molecular weight of 40000.

[0052] Comparative Example 1

[0053] Unlike Example 1, it includes 2.5% hydroxypropyl methylcellulose (HPMC) and 0.8% polyvinylpyrrolidone (PVP).

[0054] Comparative Example 2

[0055] Unlike Example 1, it includes 2.5% hydroxypropyl methylcellulose (HPMC) and 2.5% polyvinylpyrrolidone (PVP).

[0056] Example 2

[0057] A method for preparing an embedding agent for spatial transcriptomics and spatial metabolomics analysis of plants, wherein the formulation amount of Example 1 is used as an example, and the following method is followed: weigh the formulation amount of HPMC and PVP and place them in 100 mL of ultrapure water, heat and stir at 60-70°C until the powder is completely dissolved, mix evenly and let stand for 12 h to remove air bubbles.

[0058] Comparative Examples 3 and 4

[0059] Unlike Example 2, the preparation was carried out according to the formulation amounts of Comparative Examples 1 and 2, respectively.

[0060] The following application examples all use Yunnan yew stems as samples.

[0061] Application Example 1: Comparison of the effects of cryo-embedding with different embedding agents

[0062] The embedding agents prepared in Example 2 and Comparative Examples 3 and 4 were used. The following steps were performed for sample cryo-embedding: The samples were washed with sterile water, dried, and excess leaf tissue was removed. The stem segments were cut to appropriate sizes. The pre-cooled embedding agent was injected into the embedding cassette to 1 / 3 full. The sample was gently placed into the cassette containing the embedding agent, and the exposed tissue surface was covered with the embedding agent. It was confirmed that there were no air bubbles near the tissue; if air bubbles were present, they were removed with tweezers or a pipette tip. The sample placement and cut surfaces were marked. The embedding cassette was immediately placed on crushed dry ice until the embedding agent was completely frozen. The samples were then transferred to the freezer compartment of a cryostat for sectioning or temporarily stored in a -80°C freezer.

[0063] Cryosectioning: Set the cryostat's freezer temperature to -20℃ and the freeze head temperature to -15~-17℃. After equilibrating the embedded blocks obtained in the previous step in the freezer for 20 minutes, attach the embedded medicinal plant sample blocks to the sample holder and place them on the quick-freezing stage until the sample is completely fixed to the sample holder. Then transfer them to the sample holder of the microtome for fixation. Adjust the sectioning angle and appropriate thickness before sectioning. Collect the sections that meet the requirements and attach them to positive ion bonding slides.

[0064] The slicing effect of frozen embedded samples is as follows Figure 1 As shown, A and B represent embedded sample sections from Comparative Example 3, with a section thickness of 30 μm; C and D represent embedded sample sections from Comparative Example 4, with a section thickness of 20 μm; and E and F represent embedded sample sections from Example 2, with a section thickness of 20 μm. Both Comparative Example 4 and Example 2 maintain high tissue integrity at a section thickness of 20 μm. However, Comparative Example 4 produces more fragmented samples, while Example 2 exhibits better sample integrity. The thinnest embedded sample section in Comparative Example 3 is 30 μm; thinner sections lead to tissue fragmentation and difficulty in shaping. Therefore, the embedding agent in Example 2 provides better support for the plant tissue, resulting in less section warping during the slicing process, thus ensuring high fidelity and spatial resolution of the spatial omics sequencing results.

[0065] Application Example 2: Spatial transcriptomics and spatial metabolomics analysis of frozen-embedded sample sections with different embedding agents.

[0066] 1. Spatial transcriptomics analysis

[0067] Frozen-embedded sample sections prepared in Example 2 of Application Example 1 and frozen-embedded sample sections prepared with OCT embedding agent were used for spatial transcriptomics analysis, and the results are as follows. Figure 2As shown, A: Permeation results of OCT embedding material sections; B: Permeation results of embedding material sections from Example 2. It can be seen that the embedding material from Example 2 provides better support for plant tissues, resulting in better adhesion between the sections and the chip. This ensures that the tissues adhere firmly to the chip in subsequent experiments, thereby guaranteeing high fidelity and spatial resolution of the transcriptome sequencing results. Compared to traditional OCT embedding materials, OCT embedding materials are more prone to tissue separation from the embedding material, leading to missing data captured by the chip.

[0068] 2. Spatial metabolomics analysis

[0069] The frozen-embedded sample sections prepared in Example 2 of Application Example 1 and the frozen-embedded sample sections prepared with 2% CMC embedding agent were used for spatial metabolomics analysis, and the results are as follows. Figure 3 As shown, A and C represent the detection results of sections embedded with CMC, with mz values ​​of 104.1073 and 210.1127, respectively; B and D represent the detection results of sections embedded with the embedding agent from Example 2, with mz values ​​of 104.1073 and 210.1127, respectively. It can be seen that sections embedded with CMC are prone to detachment and warping of plant tissue. The embedding agent of Example 2 provides better support for plant tissue, resulting in better adhesion between the section and the slide, and relatively less background noise, thus ensuring the accuracy and spatial resolution of the DESI-MS detection results.

[0070] Application Example 3: Spatial Multi-omics Study of Yunnan Yew Stems

[0071] 1. The embedding agent prepared in Example 2 was selected.

[0072] 2. Embedding of medicinal plants: Take stem samples of Taxus yunnanensis, wash with sterile water, dry, remove excess leaf tissue, cut the stem segments to appropriate size, inject the pre-cooled embedding medium into the embedding cassette to 1 / 3 full, slowly place the sample into the embedding medium, and then cover the exposed tissue surface with the embedding medium. Confirm that there are no air bubbles near the tissue. If there are air bubbles, remove them with tweezers or pipette tip. Mark the sample placement position and cut surface, and immediately place the embedding cassette on crushed dry ice until the embedding medium is completely frozen. Transfer to the freezer compartment of a cryostat for sectioning or store temporarily in a -80℃ freezer.

[0073] 3. Cryosectioning: Set the cryostat's freezer temperature to -20℃ and the blast freezer temperature to -18~-20℃. After equilibrating the yew stem embedding blocks obtained in the previous step in the freezer for 20 minutes, use OCT embedding medium or other embedding medium to adhere the embedded yew stem embedding blocks to the sample holder. Place the sample holder on the blast freezer and allow it to stand until it is completely fixed to the sample holder. Then transfer it to the sample holder of the microtome for fixation. Adjust the sectioning angle and appropriate thickness before sectioning. Collect the sections that meet the requirements and attach them to positive ion-attached glass slides for subsequent spatial metabolomics analysis. The results are as follows: Figure 4 The image shows the spatial distribution of chemical components from the spatial metabolomics of *Taxus yunnanensis* analyzed by DESI-MS, with a spatial resolution of 20 μm × 20 μm. A represents 2-{4-[benzyl(ethyl)amino]benzylidene}malononitrile, and B represents 4-(3a,6a-dihydroxy-4-(4-hydroxy-3-methoxyphenyl)tetrahydro-1H,3H-furano[3,4-c]furan-1-yl)-2-methoxyphenyl beta-D-glucoside. tetrahydro-1H,3H-furo[3,4-c]furan-1-yl)-2-methoxyphenyl.beta.-D-glucopyranoside), C is 8-oxo-9-(3,4,5-trimethoxyphenyl)-5,5a,6,8,8a,9-hexahydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-5-yl hexopyranoside. Adjacent slices were agglutinated onto a Stereo-seq chip for spatial transcriptome analysis. The results are as follows: Figure 5As shown, this is the spatial transcriptome of *Taxus yunnanensis* based on stereo-seq sequencing. The left image is a spatial representation of cell clustering after normalization, and the right image is a spatial representation of cell clustering without normalization. Green represents medullary cells, xylem cells, and cambium cells; blue represents phloem cells; and other colors represent other undefined cells. It can be seen that the embedding agent in Example 2 provides excellent support for the *Taxus yunnanensis* stem segments, ensuring that the tissue adheres firmly to the chip in subsequent experiments, thereby ensuring high fidelity and spatial resolution of the transcriptome sequencing results.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A cryopreservation agent for plant spatial transcriptomics and spatial metabolomics analysis, characterized in that, It consists of 2.5% hydroxypropyl methylcellulose and 7.5% polyvinylpyrrolidone by mass. Hydroxypropyl methylcellulose and polyvinylpyrrolidone were dissolved in pure water to obtain an encapsulating agent; The viscosity of the hydroxypropyl methylcellulose is 1000~6000 mPa·s; The molecular weight of the polyvinylpyrrolidone is 10,000 to 100,000.

2. The method for preparing the cryo-embedding agent according to claim 1, characterized in that, This includes dissolving hydroxypropyl methylcellulose and polyvinylpyrrolidone in pure water according to the prescribed amounts to obtain an encapsulating agent.

3. The preparation method according to claim 2, characterized in that, After dissolving, it also includes standing for at least 12 hours.

4. The preparation method according to claim 2, characterized in that, The dissolution conditions include stirring at 60-70°C.

5. The application of the cryopreservation agent according to claim 1 or the cryopreservation agent prepared by the preparation method according to any one of claims 2 to 4 in plant spatial transcriptomics and spatial metabolomics analysis.

6. A method for cryopreservation of plants for spatial transcriptomics and spatial metabolomics analysis, characterized in that, This includes immersing plant samples in pre-cooled embedding medium and freezing them to obtain frozen-embedded plant samples; The embedding agent is the cryo-embedding agent according to claim 1 or the cryo-embedding agent prepared by the preparation method according to any one of claims 2 to 4.

7. The plant cryopreservation embedding method according to claim 6, characterized in that, The process of immersing the plant sample in the pre-cooled embedding medium includes placing a portion of the plant sample in a portion of the pre-cooled embedding medium, and then covering another portion of the plant sample with another portion of the pre-cooled embedding medium, so that the plant sample is immersed in the pre-cooled embedding medium.

8. The plant cryopreservation embedding method according to claim 7, characterized in that, The freezing process also includes removing air bubbles from the embedding agent.

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