Embedding medium for plant space transcriptomics and space metabonomics analysis and freezing embedding method

Abstract

The invention provides an embedding medium for plant space transcriptomics and space metabonomics analysis and a freezing embedding method, and belongs to the technical field of biology. The embedding medium is composed of hydroxypropyl methyl cellulose with the mass concentration of 2.5% and polyvinylpyrrolidone with the mass concentration of 7.5%, is applied to plant space transcriptomics research for the first time, and fills the technical blank in the field. Meanwhile, combined application of space transcriptomics and space metabonomics in the same plant individual is achieved for the first time, and a systematic technical solution is provided for plant space multi-omics research. By means of the method, spatial-temporal dynamic changes of gene expression in the plant development process and metabolic pathways and key enzyme genes of natural products in the medicinal plants can be accurately analyzed, and important technical support is provided for functional research and industrial development of the medicinal plants.

Description

Technical Field

[0001] The invention 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 Art

[0002] "Spatial multi-omics" was named one of the seven most anticipated technologies by NATURE magazine in 2022. Combining different omics, one-to-one correspondence between biomarkers and spatial locations is achieved to obtain expression maps with higher dimensions and higher resolution. Existing spatial multi-omics technologies mainly include three categories: spatial transcriptomics, spatial proteomics, and spatial metabolomics. These technologies combine spatial information with molecular biology techniques, and can analyze gene expression, protein distribution, and metabolite changes in cells or tissues while preserving their spatial locations, thereby revealing the complexity and dynamics of biological processes.

[0003] Spatial transcriptomics: a technology that captures the spatial information of RNA molecules to associate gene expression levels with spatial locations. Spatial metabolomics: a technology that obtains metabolite spatial distribution information through mass spectrometry imaging and other methods, which can realize the correlation analysis between metabolite content and spatial location. Existing spatial multi-omics technologies are mostly combined with pathological tissue sections, such as using OCT to embed pathological tissue sections, and using adjacent sections to conduct spatial proteomics and spatial transcriptomics related sequencing studies. By analyzing genes and metabolites in different regions of interest (ROI), researchers have constructed a gene-metabolite network map specific to gastric cancer regions.

[0004] Although spatial multi-omics technology has been widely used in the field of tissue pathology, its application in the field of plant research is still in its infancy. At present, most plant spatial multi-omics studies use OCT embedding agents as tissue fixation media. Although OCT can effectively maintain tissue morphology, the potential interference of its chemical composition has become 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 in order to obtain accurate data during mass spectrometry analysis. However, in the mass spectrometry analysis process, the polymers in the OCT-embedded samples may produce interference peaks during the ion ionization process. These interference peaks will mask the true signals of metabolites, especially small molecule metabolites, thereby affecting the detection precision and accuracy of metabolites, thereby increasing the difficulty of detection and data processing. The core limitation of the existing technology is that the solvent residues, additives or inadequately reacted monomers contained in the OCT embedding agent form additional ion signals during the mass spectrometry ionization process, the so-called background noise, which seriously hinders the accurate identification and quantification of small molecule metabolites, and thus affects the accuracy and reliability of the entire spatial multi-omics analysis.

[0005] In view of this, the present invention is proposed. Summary of the invention

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

[0007] A second object of the present invention is to provide a method for preparing the above-mentioned embedding agent.

[0008] The third object of the present invention is to use the embedding agent or the embedding agent prepared by the above preparation method in plant spatial transcriptomics and spatial metabolomics analysis.

[0009] A fourth object of the present invention is to provide a plant freezing embedding method for spatial transcriptomics and spatial metabolomics analysis.

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

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

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

[0013] Furthermore, the molecular weight of the polyvinyl pyrrolidone is 10,000 to 100,000.

[0014] In a second aspect, the present invention provides a method for preparing the embedding agent, comprising dissolving hydroxypropyl methylcellulose and polyvinyl pyrrolidone in pure water according to the formula amount to obtain the embedding agent.

[0015] Furthermore, after dissolving, the method further comprises standing for at least 12 hours.

[0016] Furthermore, the dissolving conditions include stirring and dissolving at 60-70°C.

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

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

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

[0020] Furthermore, immersing the plant sample in the precooled embedding medium includes placing a portion of the plant sample in a portion of the precooled embedding medium, and then covering another portion of the precooled embedding medium on another portion of the plant sample, so that the plant sample is immersed in the precooled embedding medium.

[0021] Furthermore, the method further includes removing bubbles in the embedding medium before freezing.

[0022] The present invention provides an embedding agent for plant spatial transcriptomics and spatial metabolomics analysis, which is composed of a mixture of hydroxypropyl methylcellulose with a mass concentration of 2.5% and polyvinyl pyrrolidone with a mass concentration of 7.5%. It avoids adding other auxiliary components in the embedding agent to affect the embedding effect, and is applied to plant spatial transcriptomics research for the first time, filling the technical gap in this field. At the same time, the present invention realizes the combined application of spatial transcriptomics and spatial metabolomics in the same plant individual for the first time, providing a systematic technical solution for plant spatial multi-omics research. Experiments have shown that in terms of technical effects, it is applied to plant embedding and subsequent spatial metabolomics and spatial transcriptomics research, which can provide good slice support while significantly reducing the background signal interference in the mass spectrometry ionization process; in spatial transcriptomics research, it shows excellent gene capture ability and high-quality sequencing effect; in spatial metabolomics research, the ionization efficiency of metabolite signals is further optimized. Through the present invention, the spatiotemporal dynamic changes 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 A comparison chart of the effects of frozen embedded sample sections in different embedding agents provided in Application Example 1 of the present invention;

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

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

[0027] Figure 4 The spatial metabolomics analysis diagram of paclitaxel in Taxus yunnanensis based on DESI-MS detection provided in Application Example 3 of the present invention;

[0028] Figure 5 This is a spatial transcriptomic analysis diagram of Taxus yunnanensis based on stereo-seq sequencing provided in Application Example 3 of the present invention. DETAILED DESCRIPTION

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

[0030] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0031] On one hand, the present invention provides an embedding agent for plant spatial transcriptomics and spatial metabolomics analysis, which is composed of 2.5% hydroxypropyl methylcellulose and 7.5% polyvinyl pyrrolidone in a mass concentration.

[0032] The embedding agent composed of a mixture of 2.5% hydroxypropyl methylcellulose and 7.5% polyvinyl pyrrolidone in a mass concentration avoids the addition of other auxiliary components in the embedding agent to affect the embedding effect. It is used for the first time in plant spatial transcriptomics research, filling the technical gap in this field. At the same time, the present invention realizes the combined application of spatial transcriptomics and spatial metabolomics in the same plant individual for the first time, providing a systematic technical solution for plant spatial multi-omics research. Experiments have shown that in terms of technical effects, its application in plant embedding and subsequent spatial metabolomics and spatial transcriptomics research can provide good slice support while significantly reducing background signal interference in the mass spectrometry ionization process; in spatial transcriptomics research, it shows excellent gene capture ability and high-quality sequencing effect; in spatial metabolomics research, the ionization efficiency of metabolite signals is further optimized. Through the present invention, the spatiotemporal dynamic changes 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 polyvinyl pyrrolidone is 10,000 to 100,000, preferably 40,000.

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

[0036] The embedding agent prepared by mixing hydroxypropyl methylcellulose (HPMC) and polyvinyl pyrrolidone (PVP) in a certain formula has a good embedding effect and can provide good slice support. The prepared embedded samples can be simultaneously applied to spatial transcriptomics and spatial metabolomics analysis, avoiding the presence of solvent residues, additives or inadequately reacted monomers in the embedded samples due to the presence of multiple additives in the embedding agent, which will cause the background signal generated during the mass spectrometry ionization process, thereby affecting the accuracy and reliability of spatial transcriptomics and spatial metabolomics analysis.

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

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

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

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

[0041] Experiments have shown that the frozen plant embedded samples prepared with the embedding agent provided by the present invention can be applied to spatial transcriptomics analysis and spatial metabolomics analysis, and there is no need to prepare frozen plant embedded samples obtained by embedding with different embedding agents in different spatial multi-omics analyses.

[0042] Due to the unevenness of the plant sample surface, when the plant sample is completely immersed in the embedding agent at one time, a large number of bubbles will be generated and gathered around the plant sample, or the embedding agent and the plant sample cannot be completely in contact, thereby affecting the embedding effect. In some specific embodiments, immersing the plant sample in the precooled embedding agent includes placing a portion of the plant sample in a portion of the precooled embedding agent, and then covering another portion of the precooled embedding agent on another portion of the plant sample, so that the plant sample is immersed in the precooled embedding agent. Completing the immersion in two parts can avoid the generation of a large number of bubbles.

[0043] In some specific embodiments, the step of removing bubbles in the embedding medium before freezing is also included.

[0044] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] Reagents:

[0046] OCT embedding medium: American Sakura OCT frozen section embedding medium, SAKURA, product number is 4583-118mL;

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

[0048] Hydroxypropyl methylcellulose (HPMC): Shanghai McLean Biochemical Technology Co., Ltd., 4000mPa.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% polyvinyl pyrrolidone (PVP), wherein the hydroxypropyl methylcellulose (HPMC) has a viscosity of 4000 mPa.s and the polyvinyl pyrrolidone (PVP) has a molecular weight of 40000.

[0052] Comparative Example 1

[0053] The difference from Example 1 is that 2.5% of hydroxypropyl methylcellulose (HPMC) and 0.8% of polyvinyl pyrrolidone (PVP) are included.

[0054] Comparative Example 2

[0055] The difference from Example 1 is that 2.5% of hydroxypropyl methylcellulose (HPMC) and 2.5% of polyvinyl pyrrolidone (PVP) are included.

[0056] Example 2

[0057] A method for preparing an embedding agent for plant spatial transcriptomics and spatial metabolomics analysis is prepared according to the formula amounts of Example 1, taking 100 ml as an example, according to the following method: weighing the formula amounts of HPMC and PVP and placing them in 100 mL of ultrapure water, heating and stirring at 60-70° C. until the powder is completely dissolved, mixing evenly and letting it stand for 12 hours to remove bubbles.

[0058] Comparative Examples 3 and 4

[0059] The difference from Example 2 is that the preparation is carried out according to the formula amounts of Comparative Examples 1 and 2 respectively.

[0060] The following application examples all use the stems of Taxus yunnanensis as samples.

[0061] Application Example 1 Comparison of freezing embedding effects of different embedding agents

[0062] The embedding agents prepared in Example 2 and Comparative Examples 3 and 4 were selected. The samples were frozen and embedded in the following steps: the samples were washed with sterile water, the water was absorbed, the excess leaf tissue was removed, the stem segments were cut to a suitable size, the embedding agent prepared after precooling on ice was injected into the embedding box to 1 / 3, the samples were slowly placed in the embedding box containing the embedding agent, and the exposed tissue surface was covered with the embedding agent, and it was confirmed that there were no bubbles near the tissue. If there were bubbles, they were picked out with tweezers or a gun tip, the sample placement position and the cut surface were marked, and the embedding box was immediately placed on the crushed dry ice until the embedding agent was completely frozen, and the samples were transferred to the freezer of the cryostat for slicing or placed in a -80°C refrigerator for temporary storage.

[0063] Frozen section: The freezing chamber temperature of the cryostat is set at -20°C, and the freezing head temperature is set at -15 to -17°C. After the embedded blocks obtained in the previous step are balanced in the freezing chamber for 20 minutes, the embedded blocks of medicinal plant samples are bonded to the sample holder, placed on the quick freezing table and left to stand until the samples are completely fixed to the sample holder, and then transferred to the sample holder of the slicer for fixation. Adjust the angle and appropriate thickness of the slices, and then slice them. Collect the slices that meet the requirements and adhere them to the positive ion adhesion slide.

[0064] The sectioning effect of frozen embedded samples is as follows Figure 1 As shown, A and B: embedded sample slices of comparative example 3, with a slice thickness of 30 μm; C and D: embedded sample slices of comparative example 4, with a slice thickness of 20 μm; E and F: embedded sample slices of example 2, with a slice thickness of 20 μm. Comparative example 4 and example 2 can ensure a high degree of tissue integrity at a slice thickness of 20 μm, but the sample of comparative example 4 has more fragments, and the sample of example 2 has better integrity, while the embedded sample slices of comparative example 3 are as thin as 30 μm, and thinner slices will cause tissue fragmentation and difficulty in forming. Therefore, the embedding agent of example 2 can play a better supporting role for plant tissues, and there are fewer cases of slice warping during the slicing process, which can ensure the high fidelity and spatial resolution of spatial omics sequencing results.

[0065] Application Example 2 Spatial transcriptomic analysis and spatial metabolomic analysis of frozen embedded sample sections with different embedding agents

[0066] 1. Spatial transcriptomic analysis

[0067] The frozen embedded sample slices prepared in Example 2 of Application Example 1 and the frozen embedded sample slices prepared with OCT embedding agent were respectively used for spatial transcriptomics analysis. The results are as follows: Figure 2As shown, A: OCT embedding agent slice permeabilization result; B: Example 2 embedding agent slice permeabilization result. It can be seen that the embedding agent of Example 2 can provide better support for plant tissues, and the slices and chips have better adhesion, which can ensure that the tissues are firmly attached to the chip in subsequent experiments, thereby ensuring the high fidelity and spatial resolution of the transcriptome sequencing results. Compared with traditional OCT embedding agents, OCT is more likely to cause separation of tissues and embedding agents, resulting in missing chip capture data.

[0068] 2. Spatial metabolomics analysis

[0069] The frozen embedded sample slices prepared in Example 2 of Application Example 1 and the frozen embedded sample slices prepared with 2% CMC embedding agent were used for spatial metabolomics analysis. The results are as follows: Figure 3 As shown, A and C: CMC embedding agent slice test results, mz are 104.1073 and 210.1127 respectively; B and D: Example 2 embedding agent slice test results, mz are 104.1073 and 210.1127 respectively. It can be seen that CMC embedded slices are prone to cause plant tissue to fall off and warp, and the embedding agent of Example 2 can play a better supporting role for plant tissue, the adhesion between the slice and the slide is better, and the background noise generated is relatively small, thereby ensuring the accuracy and spatial resolution of the DESI-MS test results.

[0070] Application Example 3 Spatial Multi-omics Study on the Stems of Taxus yunnanensis

[0071] 1. Use the embedding agent prepared in Example 2.

[0072] 2. Embedding of medicinal plants: Take stem samples of Taxus yunnanensis, wash them with sterile water, dry them, remove excess leaf tissue, cut the stem segments to a suitable size, inject the prepared embedding agent that has been pre-cooled on ice into the embedding box to 1 / 3, slowly put the sample into the embedding box containing the embedding agent, and then cover the exposed tissue surface with the embedding agent. Make sure there are no bubbles near the tissue. If there are bubbles, pick them out with tweezers or a gun tip, mark the sample placement and section, and immediately place the embedding box on crushed dry ice until the embedding agent is completely frozen, transfer it to the freezer of a cryostat for slicing or store it in a -80℃ refrigerator.

[0073] 3. Frozen sections: Set the freezing chamber temperature of the cryostat to -20°C, and the quick-freezing table temperature to -18 to -20°C. After balancing the yew stem segment embedding block obtained in the previous step in the freezer for 20 minutes, use OCT embedding agent or other embedding agent to bond the embedded yew stem segment embedding block to the sample holder, place it on the quick-freezing table to let the sample completely fixed to the sample holder, and then transfer it to the sample holder of the slicer to fix it. Adjust the angle and appropriate thickness of the slices, then slice, collect the slices that meet the requirements and adhere them to the positive ion adhesion slide for subsequent spatial metabolomics analysis. The results are as follows: Figure 4 The figure shows the spatial distribution of chemical components of Taxus yunnanensis spatial metabolomics based on DESI-MS detection, with a spatial resolution of 20 μm × 20 μm, where A is 2-{4-[Benzyl(ethyl)amino]benzylidene}malononitrile, B is 4-(3a,6a-dihydroxy-4-(4-hydroxy-3-methoxyphenyl)tetrahydro-1H,3H-furano[3,4-c]furan-1-yl)-2-methoxyphenyl.beta.-D-glucoside The adjacent sections were attached to the Stereo-seq chip for spatial transcriptome analysis. The results are shown in Figure 2. Figure 5As shown, it is the spatial transcriptome of Taxus yunnanensis based on stereo-seq sequencing. The left picture is the spatial display of the cell clustering diagram after Normalization, and the right picture is the spatial display of the cell clustering diagram without Normalization, where 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 of Example 2 can play a good supporting role for the stem segment of Taxus yunnanensis, ensuring that the tissue is firmly attached to the chip in subsequent experiments, thereby ensuring the 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, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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. An embedding agent for plant spatial transcriptomics and spatial metabolomics analysis, characterized in that: It is composed of 2.5% hydroxypropyl methylcellulose and 7.5% polyvinyl pyrrolidone in a mass concentration.

2. The embedding agent according to claim 1, characterized in that The viscosity of the hydroxypropyl methylcellulose is 1000-6000 mPa.s.

3. The embedding agent according to claim 2, characterized in that The molecular weight of the polyvinyl pyrrolidone is 10,000 to 100,000.

4. The method for preparing the embedding agent according to any one of claims 1 to 3, characterized in that: The method comprises dissolving hydroxypropyl methylcellulose and polyvinyl pyrrolidone in pure water according to the formula amount to obtain an embedding agent.

5. The preparation method according to claim 4, characterized in that: After dissolution, the mixture is allowed to stand for at least 12 hours.

6. The preparation method according to claim 4, characterized in that: The dissolving conditions include stirring and dissolving at 60-70°C.

7. Use of the embedding agent according to any one of claims 1 to 3 or the embedding agent prepared by the preparation method according to any one of claims 4 to 6 in plant spatial transcriptomics and spatial metabolomics analysis.

8. A plant freezing embedding method for spatial transcriptomics and spatial metabolomics analysis, characterized in that: The method comprises immersing the plant sample in a pre-cooled embedding medium and freezing it to obtain a plant frozen embedding sample; The embedding agent is the embedding agent described in any one of claims 1 to 3 or the embedding agent prepared by the preparation method described in any one of claims 4 to 6.

9. The plant freezing embedding method according to claim 8, characterized in that: The step of immersing the plant sample in the precooled embedding medium includes placing a portion of the plant sample in a portion of the precooled embedding medium, and then covering another portion of the precooled embedding medium on another portion of the plant sample, so that the plant sample is immersed in the precooled embedding medium.

10. The plant freezing embedding method according to claim 8, characterized in that: The step of removing bubbles from the embedding medium before freezing is also included.

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