Human ginseng permeabilization liquid as well as preparation method and application thereof

Abstract

The invention provides a human penetration solution and a preparation method and application thereof, and relates to the technical field of space transcriptome sequencing, the human penetration solution comprises cellulase with the concentration of 0.01-0.03 g / ml, pectinase with the concentration of 0.005-0.02 g / ml, macerozyme with the concentration of 0.002-0.006 g / ml, Ca < 2 + > with the concentration of 8-12 mM and pepsase with the concentration of 0.1-0.3 g / ml, and the cellulase, the pectinase and the macerozyme mainly aim at various main components of plant cell walls; the pepsase can weaken the influence of protein in cells on RNA binding and cell adhesion and promote RNA release; meanwhile, Ca < 2 + > contributing to activating the enzyme is added. Through mutual cooperation of cellulase, pectinase, macerozyme, Ca < 2 + > and pepsase, the permeabilization effect of the human permeabilization liquid on ginseng tissues is improved, and the requirement of space transcriptome sequencing is met.

Description

Technical Field

[0001] The present invention relates to the technical field of spatial transcriptome sequencing, and in particular to a ginseng permeabilization solution, a preparation method thereof and an application thereof. Background Art

[0002] Ginseng is a perennial herb belonging to the genus Panax in the Araliaceae family. As a precious Chinese herbal medicine, it has been used in China for thousands of years and is known as the "king of herbs". The fleshy root of ginseng is a tonic medicine that is suitable for adjusting blood pressure, restoring heart function, neurasthenia and physical weakness. It also has the effects of expectoration, stomachic, diuresis and excitement. The stems, leaves, flowers, fruits and processing by-products of ginseng are all raw materials for light industry, and can be processed into products such as tobacco, wine, tea, crystals, and pastes containing ginseng ingredients. Ginseng cultivation is an important project for farmers' diversified operations. The taproot of ginseng is the main medicinal part of ginseng and contains a variety of ingredients that are beneficial to the human body, such as ginsenosides, polysaccharides, lipids, etc. Scientific research on the growth and development of the taproot of ginseng and the distribution and production rules of its secondary metabolites will help improve ginseng breeding capabilities, establish ginseng quality standards, and ensure the stability and effectiveness of medicinal materials. In addition, when ginseng is planted continuously on the same piece of land, even under normal management conditions, the yield will decrease, the quality will deteriorate, and the growth condition will deteriorate, which is called continuous cropping disorder. Research on the production of ginseng root growth and development secretions and their autotoxic activity, as well as the changes in the rhizosphere microecology, is also of great significance for understanding the mechanism of continuous cropping disorder and finding solutions.

[0003] Transcriptomics is an important method that uses all RNA molecules in cells as research objects, and uses RNA capture sequencing and bioinformatics analysis to study the regulation of genes under different biological conditions and how gene regulation affects cell function and disease development. Spatial transcriptomics further combines transcriptomics and spatial information to obtain the spatial distribution of gene expression, further reveal the spatial distribution and interaction relationship of cells at the tissue level, and understand the functional response of cells in their microenvironment. It has been widely used in the field of animal research. However, the application of spatial transcriptomics methods in the field of plant research is still relatively limited. The main reasons are as follows: from the perspective of bioinformatics analysis, the genome and transcriptome of plants are more complex and diverse than those of animals, and there is a lack of a large enough database to meet the requirements of data mining; from the perspective of experimental operation, the cell wall of plant tissues will hinder the release of RNA in cells and reduce the capture efficiency of spatial transcriptomes. In addition, the differences in the composition of cell walls of different types of plants and the heterogeneity of different organ parts of the same plant make it difficult to form a unified processing method. Therefore, different types of permeabilization strategies need to be developed for different types of plant tissues to improve the capture strategy of plant tissue RNA.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide a ginseng permeabilization solution to solve the technical problem in the prior art of lacking a permeabilization solution for ginseng tissue permeabilization in spatial transcriptome analysis.

[0006] A second object of the present invention is to provide a method for preparing the above-mentioned ginseng permeabilization solution.

[0007] The third object of the present invention is to provide the use of the above-mentioned ginseng permeabilization solution or the ginseng permeabilization solution prepared by the above-mentioned preparation method in the permeabilization of ginseng tissue.

[0008] A fourth object of the present invention is to provide a method for permeabilizing ginseng tissue.

[0009] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0010] In a first aspect, the present invention provides a ginseng permeabilization solution, comprising 0.01-0.03 g / ml of cellulase, 0.005-0.02 g / ml of pectinase, 0.002-0.006 g / ml of analyte, 8-12 mM of Ca 2+ and 0.1-0.3 g / ml of pepsin.

[0011] Furthermore, the pH of the ginseng permeabilization solution is 2.5-3.5, preferably 3.0.

[0012] Furthermore, the solvent of the ginseng permeabilization solution is an enzyme dissolving solution;

[0013] Preferably, the enzyme dissolution solution comprises 0.8 M mannitol, 20 mM 2-(N-morpholino)ethanesulfonic acid and 10 mM potassium chloride;

[0014] Preferably, the pH of the enzyme dissolving solution is 5 to 5.5.

[0015] In a second aspect, the present invention provides a method for preparing the above-mentioned ginseng permeabilization solution, comprising adding pepsin according to the formula amount to a liquid containing cellulase, pectinase, macerate and Ca 2+ in solution.

[0016] Further, the pepsin is added in the form of a pepsin stock solution;

[0017] Preferably, the pH of the pepsin stock solution is 1.5-2.5.

[0018] Furthermore, the composition contains cellulase, pectinase, macerate and Ca 2+The method for preparing the solution comprises adding cellulase, pectinase, macerate and Ca according to the formula amount. 2+ Prepared by dissolving in enzyme dissolving solution;

[0019] Preferably, the method comprises dissolving cellulase, pectinase and macerate in enzyme dissolving solution according to the formula amount and then adding Ca 2+ Prepared.

[0020] Furthermore, the preparation method of the enzyme dissolving solution comprises dissolving mannitol, 2-(N-morpholino)ethanesulfonic acid and potassium chloride in water according to the formula amount;

[0021] Preferably, the method for preparing the enzyme dissolution solution further comprises adjusting the pH to 5 to 5.5.

[0022] In a third aspect, the present invention provides use of the above-mentioned ginseng permeabilization solution or the ginseng permeabilization solution prepared by the above-mentioned preparation method in permeabilization of ginseng tissue.

[0023] In a fourth aspect, the present invention provides a method for permeabilizing ginseng tissue, comprising incubating permeabilized ginseng tissue with the ginseng permeabilization solution or the ginseng permeabilization solution prepared by the preparation method described above;

[0024] Preferably, the incubation method comprises adding ginseng permeabilization solution to the ginseng tissue.

[0025] Furthermore, the incubation time is 6 to 30 minutes, preferably 24 minutes;

[0026] Preferably, the incubation temperature is 35-40°C, preferably 37°C.

[0027] Preferably, the method further comprises removing the ginseng permeabilization solution after incubation.

[0028] The present invention provides a ginseng permeabilization solution, wherein cellulase, pectinase and maceration enzyme are mainly targeted at various main components of plant cell walls such as cellulose, pectin and hemicellulose; pepsin helps the diffusion of target molecules inside the tissue and helps capture RNA in cells far away from the capture point. Pepsin can weaken the effect of protein binding to RNA and cell adhesion in cells, and promote RNA release; at the same time, Ca2+, which helps to activate the enzyme, is added. 2+ . Through cellulase, pectinase, maceration enzyme, Ca 2+ It cooperates with pepsin to improve the permeabilization effect of ginseng permeabilization solution on ginseng tissue, meeting the needs of spatial transcriptome sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] 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.

[0030] Figure 1 A comparison chart of the permeabilization effects of different ginseng permeabilization solutions provided in Application Example 1 of the present invention;

[0031] Figure 2 This is a comparison chart of the permeabilization effects of ginseng tissue after being treated with the ginseng permeabilization solution provided in Application Example 2 of the present invention for different time periods. DETAILED DESCRIPTION

[0032] 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.

[0033] 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.

[0034] The present invention provides a ginseng permeabilization solution, comprising 0.01-0.03 g / ml of cellulase, 0.005-0.02 g / ml of pectinase, 0.002-0.006 g / ml of analyte, 8-12 mM of Ca 2+ and 0.1-0.3 g / ml of pepsin.

[0035] The cellulase, pectinase and maceration enzyme in the ginseng permeabilization solution provided by the present invention mainly target various main components of plant cell walls such as cellulose, pectin and hemicellulose; pepsin can weaken the effect of protein binding to RNA and cell adhesion in cells, and promote RNA release; at the same time, Ca2+, which helps to activate the enzyme, is added. 2+ . Through cellulase, pectinase, maceration enzyme, Ca 2+ It cooperates with pepsin to improve the permeabilization effect of ginseng permeabilization solution on ginseng tissue, meeting the needs of spatial transcriptome sequencing.

[0036] Among them, Ca 2+ It may be selected from calcium chloride, calcium hydrogen phosphate or calcium nitrate.

[0037] In some specific embodiments, the pH of the ginseng permeabilization solution is 2.5 to 3.5. Specifically, the pH of the ginseng permeabilization solution can be, but is not limited to, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 or 3.5, or any value between 2.5 and 3.5. Experiments have shown that the pH is best at 3.0. In some specific embodiments, the pH of the ginseng permeabilization solution is preferably 3.0.

[0038] In some specific embodiments, the solvent of the ginseng permeabilization solution is an enzyme dissolving solution, which is used to maintain the activity of the enzyme and reduce the stimulation of the permeabilization solution on the plant tissue, so as to further improve the permeabilization effect on the plant tissue.

[0039] In some specific embodiments, the enzyme dissolution solution comprises 0.8 M mannitol, 20 mM 2-(N-morpholino)ethanesulfonic acid and 10 mM potassium chloride.

[0040] Mannitol is used to adjust the osmotic pressure and stabilize the enzyme activity to prevent the enzyme from being inactivated in a high or low pH environment; 2-(N-morpholino)ethanesulfonic acid (MES) is added to better control the pH value of the enzyme solution so that it is in a range that is most favorable for enzyme activity; potassium chloride assists mannitol in stabilizing enzyme activity. The various components work together to maintain the osmotic pressure balance inside and outside the cell and stabilize the enzyme activity, ensuring that the enzyme maintains high activity during the permeabilization process and improving the permeabilization effect.

[0041] In some specific embodiments, the pH of the enzyme dissolution solution is 5 to 5.5.

[0042] Specifically, the pH of the enzyme dissolving solution may be, but is not limited to, 5, 5.1, 5.2, 5.3, 5.4 or 5.5, and may be any value between 5 and 5.5.

[0043] According to another aspect of the present invention, a method for preparing the above-mentioned ginseng permeabilization solution is also provided, comprising adding pepsin according to the formula amount to a mixture containing cellulase, pectinase, macerate and Ca 2+ in solution.

[0044] Contains cellulase, pectinase, macerate and Ca 2+ The solution has formed a stable solution environment, and the preparation method of adding pepsin can maintain the stability of the pH and temperature conditions in the solution where pepsin is located, avoiding the decrease in enzyme activity due to changes in pH and temperature when pepsin is dissolved with other enzymes. This preparation method can ensure that each enzyme works under optimal conditions, thereby improving the overall permeabilization effect.

[0045] In some specific embodiments, the pepsin is added in the form of a pepsin stock solution.

[0046] In some specific embodiments, the pH of the pepsin stock solution is 1.5-2.5.

[0047] Specifically, the pH of the pepsin stock solution can be, but is not limited to, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4 or 2.5, or any value between 1.5 and 2.5. In some specific embodiments, the pH of the pepsin stock solution is preferably 2.0.

[0048] In some specific embodiments, the composition contains cellulase, pectinase, macerator and Ca 2+ The preparation method of the solution comprises adding cellulase, pectinase, macerate and Ca according to the formula amount. 2+ The enzyme dissolving solution is used as a solvent to provide an environment for various enzymes to stabilize the enzyme activity, and can maintain the osmotic pressure balance during permeabilization, thereby improving the permeabilization effect. In order to further exert the activation effect of metal ions on enzymes, in some specific embodiments, the cellulase, pectinase and macerator are dissolved in the enzyme dissolving solution according to the formula amount, and then Ca is added. 2+ Prepared.

[0049] In some specific embodiments, the preparation method of the enzyme dissolution solution comprises dissolving mannitol, 2-(N-morpholino)ethanesulfonic acid and potassium chloride in water according to the formula amount. In some specific embodiments, the preparation method of the enzyme dissolution solution further comprises adjusting the pH to 5-5.5.

[0050] According to another aspect of the present invention, there is also provided use of the above-mentioned ginseng permeabilization solution or the ginseng permeabilization solution prepared by the above-mentioned preparation method in permeabilization of ginseng tissue.

[0051] According to another aspect of the present invention, a method for permeabilizing ginseng tissue is provided, comprising incubating permeabilized ginseng tissue with the above-mentioned ginseng permeabilization solution or the ginseng permeabilization solution prepared by the above-mentioned preparation method.

[0052] In some specific embodiments, the incubation method includes adding ginseng permeabilization solution to the ginseng tissue so that the ginseng tissue is fully in contact with the ginseng permeabilization solution and the incubation is complete. In actual operation, the ginseng permeabilization solution is added to the transcription chip with the ginseng tissue attached so that the ginseng permeabilization solution is added to the ginseng tissue.

[0053] In some specific embodiments, the incubation time is 6 to 30 minutes.

[0054] Specifically, the incubation time may be, but is not limited to, 6 min, 8 min, 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min or 30 min, or any value between 6 and 30 min. In some specific embodiments, the incubation time is preferably 24 min.

[0055] In some specific embodiments, the incubation temperature is 35-40°C.

[0056] Specifically, the incubation temperature may be, but is not limited to, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, or any temperature between 35 and 40°C. In some specific embodiments, the incubation temperature is preferably 37°C.

[0057] In some specific embodiments, the incubation further comprises removing the ginseng permeabilization solution.

[0058] 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.

[0059] Example 1

[0060] A ginseng permeabilization solution, comprising 0.02 g / ml cellulase, 0.01 g / ml pectinase, 0.04 g / ml macerate, 10 mM Ca 2+ and 0.2 g / ml pepsin, the solvent is enzyme dissolving solution, and the pH is 3.0.

[0061] The enzyme dissolution solution included 0.8 M mannitol, 20 mM 2-(N-morpholino)ethanesulfonic acid and 10 mM potassium chloride, and the pH was 5.3.

[0062] Comparative Example 1

[0063] Different from Example 1, no pepsin was added.

[0064] Comparative Example 2

[0065] Different from Example 1, only pepsin was included at a concentration of 0.02 g / ml and the pH was 3.0.

[0066] Example 2

[0067] A method for preparing a ginseng permeabilization solution, comprising the steps of:

[0068] 1. Prepare enzyme solution: dissolve mannitol, MES and potassium chloride in NF water according to the formula, adjust the pH and set aside.

[0069] 2. Solution A: Add cellulase, pectinase and macerate into the enzyme solution according to the formula, heat in a 55°C water bath for 10 min, cool to room temperature after dissolution, and then add calcium chloride to obtain solution A.

[0070] 3. Solution B: 2% pepsin stock solution at pH = 2.

[0071] 4. Add solution B into solution A according to the formula amount to obtain ginseng permeabilization solution.

[0072] Comparative Example 3

[0073] Different from Example 2, the following steps are followed according to the formula amount of Comparative Example 1: cellulase, pectinase and maceration enzyme are added to the enzyme dissolution solution according to the formula amount, heated in a 55°C water bath for 10 minutes, cooled to room temperature after dissolution, and then calcium chloride is added to obtain a permeabilization solution.

[0074] Comparative Example 4

[0075] Different from Example 2, the following steps were followed according to the formulation of Comparative Example 2: Solution B was added to a 0.01 M hydrochloric acid solution to obtain a permeabilization solution with a pH of 3.0.

[0076] The following plant samples are all three-year-old garden ginseng.

[0077] Application Example 1

[0078] The permeabilization of ginseng tissue was performed using the permeabilization solutions prepared in Example 2, Comparative Example 3 and Comparative Example 4 according to the following procedures:

[0079] 1. Slice preparation: Fix the tissues of various parts of ginseng and embed them with OCT embedding medium, and perform frozen sections in a -20 slicer.

[0080] 2. Tissue slice and chip processing: 10-20μm tissue slices were attached to the BGI spatiotemporal permeabilization chip. The chip was placed in a 37°C oven for 5 minutes, and then transferred to -20°C pre-cooled methanol for fixation for 30 minutes. The chip was removed and placed in a fume hood to wait for the methanol on the chip and tissue surface to dry completely.

[0081] 3. Permeabilization: Add 150 μL of permeabilization solution to the chip surface and incubate at 37°C for 24 min. After incubation, use a pipette to remove the permeabilization solution, add 200 μL of 0.1×SSC (containing 5% RI) to wash the chip surface, and remove the solution on the chip surface.

[0082] 4. Reverse transcription reaction. Add 100 μL of fluorescent RT QC mix solution and incubate at 42 °C for 16 h. Use a pipette to remove the RT QC mix solution, add 400 μL of 0.1×SSC to wash the chip surface twice, and remove the solution on the chip surface.

[0083] 5. Tissue removal. Add 400μL TR mix solution and incubate at 55 for 1h. Use a pipette to remove the TR mix solution, add 400μL 0.1×SSC to wash the chip surface twice, and remove the solution on the chip surface. Add 400μL Nuclease-FreeWater to clean the salt on the chip surface, remove the solution on the chip surface, and use an air can to completely dry the chip surface.

[0084] 6. Fluorescence photography. Use a fluorescence microscope (with stitching function), select the epifluorescence scanning mode, manually select the TRITC channel, and select a 10x lens to scan the entire chip. When the tissue is removed cleanly and the same imaging conditions (including brightness and exposure conditions) are maintained, the optimal permeabilization time is determined by the complete tissue morphology, the strongest fluorescence value, and no diffusion. The specific results are as follows: Figure 1 As shown, from left to right are Comparative Example 4, Comparative Example 3 and Example 2. It can be seen that Example 2 has better permeability to ginseng tissue than Comparative Examples 3 and 4, indicating that the RNA release effect is better.

[0085] Application Example 2

[0086] Different from Application Example 1, the ginseng permeabilization solution prepared in Example 2 was used, and the permeabilization and incubation treatments were performed for 6 minutes, 12 minutes, 18 minutes, and 24 minutes, respectively. Figure 2 As shown, the incubation time from left to right is 6min, 12min, 18min and 24min. It can be seen that clear tissue fluorescence signals can be obtained when the treatment time is 12-24min, indicating that the patented permeabilization solution can permeabilize the tissue cell membrane and cell wall to release RNA completely. Among them, the tissue fluorescence signal and tissue veins are the clearest at 24min.

[0087] 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. A ginseng permeabilization solution, characterized in that: It includes 0.01-0.03 g / ml cellulase, 0.005-0.02 g / ml pectinase, 0.002-0.006 g / ml macerate, 8-12 mM Ca 2+ and 0.1-0.3 g / ml of pepsin.

2. The ginseng permeabilization solution according to claim 1, characterized in that The pH of the ginseng permeabilization solution is 2.5-3.5, preferably 3.

0.

3. The ginseng permeabilization solution according to claim 1, characterized in that The solvent of the ginseng permeabilization solution is an enzyme dissolving solution; Preferably, the enzyme dissolution solution comprises 0.5-1.0 M mannitol, 18-23 mM 2-(N-morpholino)ethanesulfonic acid and 8-12 mM potassium chloride; Preferably, the pH of the enzyme dissolving solution is 5 to 5.

5.

4. The method for preparing the ginseng permeabilization solution according to any one of claims 1 to 3, characterized in that: The method comprises adding pepsin according to the formula amount to a mixture containing cellulase, pectinase, macerate and Ca 2+ in solution.

5. The preparation method according to claim 4, characterized in that: The pepsin is added in the form of a pepsin stock solution; Preferably, the pH of the pepsin stock solution is 1.5-2.5, preferably 2.

6. The preparation method according to claim 4, characterized in that: The invention contains cellulase, pectinase, macerate and Ca 2+ The preparation method of the solution comprises adding cellulase, pectinase, macerate and Ca according to the formula amount. 2+ Prepared by dissolving in enzyme dissolving solution; Preferably, the method comprises dissolving cellulase, pectinase and macerate in enzyme dissolving solution according to the formula amount and then adding Ca 2+ Prepared.

7. The preparation method according to claim 6, characterized in that: The preparation method of the enzyme dissolving solution comprises dissolving mannitol, 2-(N-morpholino)ethanesulfonic acid and potassium chloride in water according to the formula amount; Preferably, the method for preparing the enzyme dissolution solution further comprises adjusting the pH to 5 to 5.

5.

8. Use of the ginseng permeabilization solution according to any one of claims 1 to 3 or the ginseng permeabilization solution prepared by the preparation method according to any one of claims 4 to 7 in permeabilization of ginseng tissue.

9. A method for permeabilizing ginseng tissue, characterized in that: The method comprises incubating permeabilized ginseng tissue with the ginseng permeabilization solution according to any one of claims 1 to 3 or the ginseng permeabilization solution prepared by the preparation method according to any one of claims 4 to 7; Preferably, the incubation method comprises adding ginseng permeabilization solution to the ginseng tissue.

10. The permeabilization method according to claim 9, characterized in that: The incubation time is 6 to 30 minutes, preferably 24 minutes; Preferably, the incubation temperature is 35-40°C, preferably 37°C; Preferably, the incubation further comprises removing the ginseng permeabilization solution.

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