Preparation method and application of pepper placenta protoplast single-cell suspension

Abstract

The invention discloses a preparation method and application of a pepper placenta protoplast single-cell suspension. The preparation method disclosed by the invention comprises the following steps: acquiring a pepper fetal base tissue, transferring the pepper fetal base tissue into enzymatic hydrolysate for digestion, adding a DB buffer solution to terminate digestion, and collecting digestive juice; enabling the digestive juice to pass through a cell sieve, collecting filtrate to obtain a crude extracting solution of the pepper placenta protoplast single cell, then performing centrifugal treatment on the crude extracting solution of the pepper placenta protoplast single cell, collecting precipitates, resuspending the precipitates by using the DB buffer solution, and repeating the steps of centrifuging and resuspending to obtain the pepper placenta protoplast single cell suspension. The method disclosed by the invention is suitable for preparing the placenta protoplast single-cell suspensions of a plurality of pepper varieties, and the prepared placenta protoplast single-cell suspensions are complete in cell morphology and high in density, can meet the on-machine requirements of the quality of the cell suspensions of various high-throughput single-cell sequencing platforms and subsequent related experiments of the protoplast, and can be used for preparing the placenta protoplast single-cell suspensions. And the method has extremely high application value.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a preparation method and application of a pepper placenta protoplast single cell suspension. Background Art

[0002] As an important vegetable crop, pepper is rich in vitamins, carotenoids, capsaicin, organic acids and other effective ingredients. Capsaicin has many uses in the fields of pharmaceuticals, cosmetics and agricultural industries. Capsaicinoids are formed by the condensation of vanillin (derived from phenylalanine) and branched fatty acids (valine or leucine precursors). They usually accumulate in the placenta tissue of pepper fruit. In recent years, the development of molecular biology and biotechnology has promoted the research on the biosynthesis and regulation of capsaicinoids, and provided opportunities for the cultivation of new pepper varieties with higher capsaicinoid content. Single cell RNA sequencing (scRNA-Seq), as a new technology for sequencing transcriptomes at the single cell level, is the main technical means to identify heterogeneity between different cells. scRNA-Seq provides genetic difference information at the cellular level. By comparing the differences between the transcriptomes of various types of cells, pepper varieties with higher capsaicinoid content can be screened. However, in practical applications, the presence of cell walls in plants makes protoplast preparation difficult, which limits its application.

[0003] In related technologies, most of the species studied in plant single-cell transcriptome sequencing are model plants such as Arabidopsis, tobacco, and rice. However, there are currently no reports on the preparation of pepper placenta protoplast single-cell suspensions for single-cell transcriptome sequencing applications. The reason is that plants live in different environments and usually exhibit completely different growth and development characteristics, resulting in very large differences in physiology, biochemistry, structure, and function between different species. Related studies have found that conventional plant dissociation protocols are not suitable for the preparation of pepper placenta protoplast single-cell suspensions. It is speculated that this is because the cell wall density is too high, which can easily damage the cells during the removal process. Therefore, further research on pepper placenta at the cellular level is limited. At the same time, there are also differences in cells between different pepper varieties.

[0004] Based on this, in order to better study the placenta of pepper, it is necessary to establish a method for preparing single-cell suspension of pepper placenta protoplasts with high separation efficiency, good cell concentration and cell activity, and applicable to multiple pepper varieties, so as to promote the application of single-cell sequencing technology in pepper. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes a method for preparing a single cell suspension of pepper placenta protoplasts and its application. The method of the present invention is applicable to the preparation of single cell suspensions of placenta protoplasts of multiple pepper varieties, and the prepared single cell suspension of placenta protoplasts has complete cell morphology and high density, which can meet the cell suspension quality requirements of various high-throughput single-cell sequencing platforms and subsequent related experiments of protoplasts, and has extremely high application value.

[0006] The first aspect of the present invention provides a method for preparing a single cell suspension of pepper placenta protoplasts, comprising the following steps:

[0007] S1. Obtain pepper placenta tissue and transfer it to enzymatic solution for digestion, then add DB buffer to terminate digestion and collect digestion solution;

[0008] S2, passing the digestion solution through a cell sieve, collecting the filtrate, and obtaining a crude single-cell extract of pepper placenta protoplasts;

[0009] S3, centrifuging the pepper placenta protoplast single cell crude extract, collecting the precipitate and resuspending it in the DB buffer, repeating the centrifugation and resuspending steps to obtain a pepper placenta protoplast single cell suspension;

[0010] The enzymatic hydrolysate comprises 15-25 mg / mL cellulase, 8-12 mg / mL macerate, 2-8 mg / mL snail enzyme, 1.5-5 mg / mL pectinase, 80-120 mg / mL mannitol, 2-8 mg / mL bovine serum albumin, 15-25 mM MES, 8-12 mM KCl and 0.2-0.8 mM dithiothreitol;

[0011] The DB buffer contains 80-120 mg / mL mannitol, 2-8 mg / mL bovine serum albumin and 8-12 mM KCl.

[0012] The preparation method according to the embodiment of the present invention has at least the following beneficial effects:

[0013] (1) The present invention greatly improves the integrity and cell activity of pepper placenta protoplasts by synergistically combining cellulase, macerate, snail enzyme and pectinase, wherein cellulase is mainly used to decompose cellulose in the cell wall. Pepper placenta protoplasts contain a large amount of cellulose components. Cellulase can hydrolyze the β-1,4-glycosidic bonds in cellulose, promote cellulose degradation, and reduce the hardness and structural compactness of pepper placenta; while macerate can hydrolyze the macromolecular pectin in pepper placenta tissue into smaller fragments, which not only helps to promote the degradation of pectin, but also can make the pepper placenta soft by destroying the pectin structure in the cell wall. On this basis, further adding pectinase can completely decompose the pectin in the pepper placenta tissue into smaller molecules, such as galacturonic acid, etc., while snail enzyme can synergistically act on the degradation of pectin and cellulose through multiple pathways, further promoting the digestion of pepper placenta. Therefore, under layer-by-layer decomposition, the cell walls of pepper placenta cells are "gently" removed. The use of the enzymatic hydrolyzate of the present invention can improve the removal rate of pepper placenta cell walls, while also ensuring that its protoplasts are not excessively decomposed and damaged by a single component to maintain cell activity.

[0014] (2) In the preparation process of the pepper placenta protoplast single cell suspension of the present invention, mannitol, dithiothreitol, bovine serum albumin, MES and KCl and other components are also coordinated in the enzymatic hydrolyzate, wherein mannitol and KCl help to regulate the osmotic pressure of the system, maintain the stability of the cell membrane, and prevent the protoplasts from losing water and shrinking during the wall removal process; while an appropriate amount of bovine serum albumin helps to reduce the mechanical damage and oxidative damage to the protoplasts during the separation process, and can reduce the aggregation between protoplasts by preventing the interaction of cell surface charges, and maintain the dispersibility of the protoplasts; dithiothreitol mainly reduces the disulfide bonds in the cell wall decomposing enzymes, enhances its hydrolysis ability, helps to accelerate the degradation process of the cell wall, makes the cell wall in the pepper placenta easier to be enzymatically hydrolyzed, thereby improving the separation efficiency of the protoplasts. In addition, dithiothreitol can also protect the proteins and enzymes in the protoplasts, prevent them from being oxidized and degraded, and prolong the survival time of the protoplasts.

[0015] (3) In the preparation of pepper placenta protoplast single cell suspension, DB buffer is also combined, which contains mannitol, bovine serum albumin and KCl. Mannitol as its main component can help maintain cell hydration by providing external osmotic pressure, prevent protoplast dehydration and shrinkage, and help provide protoplast stability.

[0016] (4) The method for preparing a single cell suspension of pepper placenta protoplasts of the present invention is applicable to the preparation of single cell suspensions of placenta protoplasts of multiple pepper varieties, and the prepared single cell suspension of placenta protoplasts has complete cell morphology and high density, and can meet the requirements of cell suspension quality on various high-throughput single-cell sequencing platforms and subsequent related experiments on protoplasts, and has extremely high application value. In addition, the solutions used in all steps of the present invention do not contain reagents containing Ca ions and Mg ions, which helps to reduce cell agglomeration and improve the efficiency of subsequent protoplast capture, and the solutions used in all steps of the present invention have few added components, which helps to reduce the preparation cost.

[0017] In some embodiments of the present invention, in step S1, obtaining pepper placenta tissue includes: selecting healthy pepper fruits, taking out pepper placenta tissue, washing it in enzyme-free water, and then cutting the pepper placenta tissue for later use.

[0018] In some embodiments of the present invention, the varieties of peppers include any one of Zhangshugang, red pepper, sweet pepper, Erjingtiao, screw pepper, colored pepper, pointed pepper, wrinkled pepper, millet pepper, Chaotian pepper, and lantern pepper.

[0019] In some embodiments of the present invention, the cutting includes cutting into strips with a width of 1 to 2 mm.

[0020] In some embodiments of the present invention, the cutting should be immediately transferred to a culture dish containing enzyme-free water to prevent drying; after all pepper placenta tissues are cut, they are rinsed with enzyme-free water for 3 to 5 times, and then the enzymatic solution is added.

[0021] In some embodiments of the present invention, the cellulase is cellulase R-10; the macerate is macerate R-10.

[0022] In some embodiments of the present invention, the enzymatic hydrolysis solution comprises 15-20 mg / mL cellulase R-10, 8-10 mg / mL macerate R-10, 2-5 mg / mL snail enzyme, 1.5-3 mg / mL pectinase, 80-100 mg / mL mannitol, 2-5 mg / mL bovine serum albumin, 15-20 mM MES, 10-12 mM KCl and 0.2-0.5 mM dithiothreitol.

[0023] In some embodiments of the present invention, the DB buffer comprises 80-100 mg / mL mannitol, 2-5 mg / mL bovine serum albumin and 8-10 mM KCl.

[0024] In some embodiments of the present invention, the enzymatic hydrolysate and the DB buffer are prepared and sterilized by filtration with a 0.22 μm sterile syringe filter, and are shaken and mixed before use. They are prepared and used immediately.

[0025] In some embodiments of the present invention, in step S1, the digestion temperature is 28±3°C.

[0026] In some embodiments of the present invention, the digestion time is 1 to 3 hours.

[0027] In some embodiments of the present invention, the rotation speed of the shaker during the digestion process is 30 to 80 rpm.

[0028] In some embodiments of the present invention, in step S1, during the termination of digestion, the volume of the DB buffer is 25% to 50% of the volume of the enzymatic hydrolysis solution.

[0029] In some embodiments of the present invention, in step S2, the pore diameter of the cell sieve is 20-120 μm.

[0030] In some embodiments of the present invention, the mesh diameter of the cell sieve is 40-100 μm.

[0031] In some embodiments of the present invention, the cell sieve comprises sequentially passing through a cell sieve having a sieve hole diameter of 90 to 120 μm and a sieve hole diameter of 20 to 60 μm.

[0032] In some embodiments of the present invention, in step S2, the centrifugation temperature is 2±6°C.

[0033] In some embodiments of the present invention, the centrifugal rotation speed is 600-800 rpm.

[0034] In some embodiments of the present invention, the centrifugation time is 3 to 8 minutes.

[0035] In some embodiments of the present invention, in step S2, the resuspending comprises: slowly adding the DB buffer to the precipitate, and then gently pipetting to mix.

[0036] In some embodiments of the present invention, in step S2, the number of repetitions is 1 to 3 times.

[0037] The second aspect of the present invention provides a pepper placenta protoplast single cell suspension, which is prepared by the method for preparing the pepper placenta protoplast single cell suspension according to any one of the first aspects.

[0038] The third aspect of the present invention provides a method for preparing a single-cell suspension of pepper placenta protoplasts as described in any one of the first aspect or a use of the single-cell suspension of pepper placenta protoplasts as described in the second aspect in single-cell sequencing or flow cytometry analysis.

[0039] The placenta protoplast single cell suspension prepared by the method of the present invention has complete cell morphology and high density, can meet the cell suspension quality requirements of various high-throughput single-cell sequencing platforms and subsequent protoplast related experiments, and has extremely high application value.

[0040] Other features and advantages of the present invention will be set forth in the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0042] Figure 1 This is a flow chart of the method for preparing a single cell suspension of pepper placenta protoplasts of the present invention;

[0043] Figure 2 The diagram is a diagram of different varieties of peppers of the present invention, wherein A is Zhangshugang, B is red pepper, C is sweet pepper, D is Erjingtiao, and E is screw pepper;

[0044] Figure 3 The placenta and tissue sampling sites of different varieties of peppers of the present invention, wherein A is Zhangshugang, B is red pepper, C is sweet pepper, D is Erjingtiao, and E is screw pepper;

[0045] Figure 4 This is a microscopic observation of a protoplast single cell suspension after washing and resuspension in Example 1 of the present invention, with a scale of 100 μm;

[0046] Figure 5 This is a microscopic observation of a protoplast single cell suspension after washing and resuspension in Example 2 of the present invention, with a scale of 100 μm;

[0047] Figure 6 This is a microscopic observation of a protoplast single cell suspension after washing and resuspension in Example 3 of the present invention, with a scale of 100 μm;

[0048] Figure 7 This is a microscopic observation picture of a protoplast single cell suspension after washing and resuspension in Example 4 of the present invention, with a scale of 50 μm;

[0049] Figure 8 This is a microscopic observation of a protoplast single cell suspension after washing and resuspension in Example 5 of the present invention, with a scale of 100 μm;

[0050] Fig. 9 This is a microscopic observation of a protoplast single cell suspension after washing and resuspension in Example 6 of the present invention, with a scale of 100 μm;

[0051] Fig.10 This is a microscopic observation of a protoplast single cell suspension after washing and resuspension in Comparative Example 1 of the present invention, with a scale of 100 μm;

[0052] Fig.11 This is a microscopic observation of a protoplast single cell suspension after washing and resuspension in Comparative Example 2 of the present invention, with a scale of 100 μm;

[0053] Fig.12 This is a microscopic observation of a protoplast single cell suspension after washing and resuspension in Comparative Example 3 of the present invention, with a scale of 100 μm;

[0054] Fig.13 This is a microscopic observation of a protoplast single cell suspension after washing and resuspension in Comparative Example 4 of the present invention, with a scale of 100 μm;

[0055] Fig.14 This is a microscopic observation picture of the protoplast single cell suspension after washing and resuspending in Comparative Example 5 of the present invention, and the scale is 100 μm. DETAILED DESCRIPTION

[0056] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0057] The words "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0058] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0059] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the associated listed items.

[0060] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0061] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0062] Example 1: Zhangshu Port

[0063] This embodiment provides a method for preparing a single cell suspension of placental protoplasts of pepper of the Zhangshugang variety. The specific flow chart is as follows: Figure 1 As shown, including the following:

[0064] (1) Preparation of enzymatic solution and DB buffer:

[0065] The raw materials for preparing the enzymatic hydrolysate used in this example are shown in Table 1:

[0066] Table 1: Enzyme digestion solution

[0067]

[0068]

[0069] The pH of the enzymatic hydrolyzate prepared above is 5.6-5.8.

[0070] The buffer used in this example is DB buffer, which contains 90 mg / mL mannitol (Shanghai), 5 mg / mL bovine serum albumin (Miltenyi Biotec) and 10 mM KCl (Shanghai). Enzyme-free water is used to make up to 50 mL during the preparation process, and the pH is 5.6-5.8.

[0071] The prepared solution was filtered through a 0.22 μm sterile syringe filter for sterilization, shaken and mixed before use, and pre-cooled on ice for use immediately after preparation.

[0072] (2) Obtaining pepper placental tissue:

[0073] Select healthy pepper fruits with no skin damage. The pepper variety is Zhangshugang (such as Figure 2), remove the pepper pulp and pepper seeds with a blade and tweezers, take out the pepper placenta and place it in enzyme-free water, then use a sharp double-sided blade to evenly cut the pepper placenta into 1-2 mm strips, and immediately transfer about 0.1-0.2 g of the cut pepper placenta tissue to a culture dish containing enzyme-free water (as shown in Figure 1). Figure 3 Prevent it from drying out and keep it aside.

[0074] (3) Digestion and treatment of pepper placenta tissue:

[0075] After all pepper placenta tissues are cut, wash them with enzyme-free water for 3 to 5 times and then transfer them to a sterile culture dish containing 5 mL of the above enzymatic hydrolyzate. Incubate them in a constant temperature water bath shaker set at 28°C and 50 rpm in the dark for 2.5 h to obtain the digestion solution and digested pepper placenta tissue.

[0076] (4) Obtaining a crude single-cell extract of pepper placenta protoplasts:

[0077] Take out the culture dish containing the digestion solution and digested pepper placenta tissue from the constant temperature water bath shaker, add 2 mL of the prepared DB buffer to terminate the digestion reaction, and use a 1 mL wide-mouthed pipette tip to gently suck the digestion solution to blow away the cell clumps and release more cells. Use a wide-mouthed pipette tip to transfer the digestion solution to a cell sieve, and slowly filter through 100 μm and 40 μm cell sieves in turn, and add 2 mL of the above DB buffer to the cell sieve to rinse the tissue residue, discard the remaining pepper placenta tissue residue after digestion, collect the filtered pepper placenta protoplast single cell crude extract, and set aside.

[0078] (5) Obtaining pepper placenta protoplast single cell suspension:

[0079] The crude single-cell fluid of pepper placenta protoplasts obtained above was transferred to a 15-mL centrifuge tube, and centrifuged in a low-temperature horizontal centrifuge at 4°C, 700 rpm, 5 min, and a lifting rate of 4 rpm / s using the sedimentation centrifugation method. The supernatant was removed and the precipitate was retained. An appropriate amount of the above-mentioned DB buffer was added and centrifuged in the same manner. The mixture was washed twice, the supernatant was removed, and 200 μL of the above-mentioned DB buffer was added. The mixture was gently blown to mix well to obtain a single-cell suspension of pepper placenta protoplasts.

[0080] The single cell suspension of pepper placenta protoplasts obtained above was observed under a microscope. Figure 4 As shown, it is shown that the method of the present invention can obtain pepper placenta protoplast single cells with complete morphology, and the protoplasts have high density and few impurities, which can meet the cell suspension quality requirements of various high-throughput single-cell sequencing platforms.

[0081] Example 2: Red pepper

[0082] This embodiment provides a method for preparing a single cell suspension of placental protoplasts of red pepper, which specifically includes the following contents:

[0083] (1) Preparation of enzymatic solution and DB buffer:

[0084] The preparation method of the enzymatic hydrolysate and DB buffer in this example is prepared by referring to Example 1.

[0085] (2) Obtaining the placental tissue of red pepper:

[0086] Choose healthy red peppers with intact skin (such as Figure 2 Use a blade and tweezers to remove the red pepper pulp and red pepper seeds, take out the red pepper placenta and put it in enzyme-free water, then use a sharp double-sided blade to evenly cut the red pepper placenta into 1-2 mm strips. The cut red pepper placenta tissue will be immediately transferred to a culture dish containing enzyme-free water (as shown in Figure 1). Figure 3 Prevent it from drying out and keep it aside.

[0087] (3) Digestion of red pepper placenta tissue:

[0088] After all the placenta tissues of the red pepper are cut, they are washed with enzyme-free water for 3 to 5 times and then transferred to a sterile culture dish containing 5 mL of the above enzymatic hydrolyzate. They are incubated in a constant temperature water bath shaker set at 28°C and 50 rpm in the dark for 2.5 h to obtain the digestion solution and the digested placenta tissues of the red pepper.

[0089] (4) Obtaining a crude single-cell extract of red pepper placenta protoplasts:

[0090] The culture dish containing the digestion solution and the digested red pepper placenta tissue was taken out from the constant temperature water bath shaker, 2 mL of the prepared DB buffer was added to terminate the digestion reaction, and the digestion solution was gently sucked and beaten with a 1 mL wide-mouthed pipette tip to disperse the cell clumps and release more cells. The digestion solution was transferred to a cell sieve with a wide-mouthed pipette tip, and slowly filtered through 100 μm and 40 μm cell sieves in turn, and 2 mL of the above DB buffer was added to the cell sieve to rinse the tissue residue, the remaining red pepper placenta tissue residue after digestion was discarded, and the filtered red pepper placenta protoplast single cell crude extract was collected for later use.

[0091] (5) Obtaining a single cell suspension of red pepper placenta protoplasts:

[0092] The crude single-cell liquid of the placenta protoplasts of red pepper obtained above was transferred to a 15 mL centrifuge tube, and centrifuged in a low-temperature horizontal centrifuge at 4 ° C, 700 rpm, 5 min and a lifting rate of 4 rpm / s using the sedimentation centrifugation method. Then the supernatant was removed and the precipitate was retained, and an appropriate amount of the above-mentioned DB buffer was added and centrifuged in the same way. The mixture was washed twice, the supernatant was removed, and 200 μL of the above-mentioned DB buffer was added. The mixture was gently blown and mixed to obtain a single-cell suspension of the placenta protoplasts of red pepper.

[0093] The single cell suspension of the protoplasts of the placenta of red pepper obtained above was observed under a microscope. Figure 5 As shown, it is shown that the single cell morphology of the red pepper placenta protoplasts obtained by the method of the present invention is complete, high in density, and less in impurities, and can meet the cell suspension quality requirements of various high-throughput single-cell sequencing platforms.

[0094] Example 3: Bell Pepper

[0095] This embodiment provides a method for preparing a single cell suspension of placental protoplasts of bell pepper, which specifically includes the following contents:

[0096] (1) Preparation of enzymatic solution and DB buffer:

[0097] The preparation method of the enzymatic hydrolysate and DB buffer in this example is prepared by referring to Example 1.

[0098] (2) Obtaining sweet pepper placenta tissue:

[0099] Choose healthy bell peppers with undamaged skin (such as Figure 2 Use a blade and tweezers to remove the pepper pulp and seeds, take out the pepper placenta and place it in enzyme-free water. Then use a sharp double-sided blade to evenly cut the pepper placenta into 1-2 mm strips. The cut pepper placenta tissue will be immediately transferred to a culture dish containing enzyme-free water (as shown in C). Figure 3 Prevent it from drying out and keep it aside.

[0100] (3) Digestion and processing of sweet pepper placenta tissue:

[0101] After all the bell pepper placenta tissues are cut, wash them with enzyme-free water for 3 to 5 times, then transfer them to a sterile culture dish containing 5 mL of the above enzymatic hydrolyzate, and incubate them in a constant temperature water bath shaker set at 28°C and 50 rpm in the dark for 2.5 h to obtain the digestion solution and the digested bell pepper placenta tissue.

[0102] (4) Obtaining a crude single-cell extract of bell pepper placenta protoplasts:

[0103] Take out the culture dish containing the digestion solution and the digested sweet pepper placenta tissue from the constant temperature water bath shaker, add 2 mL of the prepared DB buffer to terminate the digestion reaction, and use a 1 mL wide-mouthed pipette tip to gently suck the digestion solution to disperse the cell clumps and release more cells. Use a wide-mouthed pipette tip to transfer the digestion solution to a cell sieve, and slowly filter through 100 μm and 40 μm cell sieves in turn, and add 2 mL of the above DB buffer to the cell sieve to rinse the tissue residue, discard the remaining sweet pepper placenta tissue residue after digestion, collect the filtered sweet pepper placenta protoplast single cell crude extract, and set aside.

[0104] (5) Obtaining a single cell suspension of bell pepper placenta protoplasts:

[0105] The single-cell crude liquid of the bell pepper placenta protoplasts obtained above was transferred to a 15 mL centrifuge tube, and centrifuged in a low-temperature horizontal centrifuge at 4°C, 700 rpm, 5 min, and a lifting rate of 4 rpm / s using the sedimentation centrifugation method. The supernatant was removed and the precipitate was retained. An appropriate amount of the above-mentioned DB buffer was added and centrifuged in the same way. The mixture was washed twice, the supernatant was removed, and 200 μL of the above-mentioned DB buffer was added. The mixture was gently blown and mixed to obtain a single-cell suspension of the bell pepper placenta protoplasts.

[0106] The single cell suspension of bell pepper placenta protoplasts obtained above was observed under a microscope. Figure 6 As shown, it is shown that the single-cell morphology of the sweet pepper placenta protoplasts obtained by the method of the present invention is complete, and the density is high and the impurities are few, which can meet the cell suspension quality requirements of various high-throughput single-cell sequencing platforms.

[0107] Embodiment 4: Vitex truncatula

[0108] This embodiment provides a method for preparing a single cell suspension of placental protoplasts of Capsicum annuum (Ciprinus erinaceus), which specifically includes the following contents:

[0109] (1) Preparation of enzymatic solution and DB buffer:

[0110] The preparation method of the enzymatic hydrolysate and DB buffer in this example is prepared by referring to Example 1.

[0111] (2) Obtaining pepper placental tissue:

[0112] Choose healthy peppers with undamaged skin (such as Figure 2 The pepper variety is Erjingtiao. The pepper pulp and pepper seeds are removed with a blade and tweezers, and the pepper placenta is taken out and placed in enzyme-free water. Then, a sharp double-sided blade is used to evenly cut the pepper placenta into strips of 1 to 2 mm. The cut pepper placenta tissue is immediately transferred to a culture dish containing enzyme-free water (such as Figure 3 Prevent it from drying out and keep it aside.

[0113] (3) Digestion and treatment of pepper placenta tissue:

[0114] After all pepper placenta tissues are cut, wash them with enzyme-free water for 3 to 5 times and then transfer them to a sterile culture dish containing 5 mL of the above enzymatic hydrolyzate. Incubate them in a constant temperature water bath shaker set at 28°C and 50 rpm in the dark for 2.5 h to obtain the digestion solution and digested pepper placenta tissue.

[0115] (4) Obtaining a crude single-cell extract of pepper placenta protoplasts:

[0116] Take out the culture dish containing the digestion solution and digested pepper placenta tissue from the constant temperature water bath shaker, add 2 mL of the prepared DB buffer to terminate the digestion reaction, and use a 1 mL wide-mouthed pipette tip to gently suck the digestion solution to blow away the cell clumps and release more cells. Use a wide-mouthed pipette tip to transfer the digestion solution to a cell sieve, and slowly filter through 100 μm and 40 μm cell sieves in turn, and add 2 mL of the above DB buffer to the cell sieve to rinse the tissue residue, discard the remaining pepper placenta tissue residue after digestion, collect the filtered pepper placenta protoplast single cell crude extract, and set aside.

[0117] (5) Obtaining pepper placenta protoplast single cell suspension:

[0118] The crude single-cell fluid of pepper placenta protoplasts obtained above was transferred to a 15-mL centrifuge tube, and centrifuged in a low-temperature horizontal centrifuge at 4°C, 700 rpm, 5 min, and a lifting rate of 4 rpm / s using the sedimentation centrifugation method. The supernatant was removed and the precipitate was retained. An appropriate amount of the above-mentioned DB buffer was added and centrifuged in the same manner. The mixture was washed twice, the supernatant was removed, and 200 μL of the above-mentioned DB buffer was added. The mixture was gently blown to mix well to obtain a single-cell suspension of pepper placenta protoplasts.

[0119] The single cell suspension of pepper placenta protoplasts obtained above was observed under a microscope. Figure 7 As shown, it is shown that the pepper placenta protoplast single cell morphology obtained by the method of the present invention is complete, high in density, and less in impurities, and can meet the cell suspension quality requirements of various high-throughput single-cell sequencing platforms.

[0120] Example 5: Screw pepper

[0121] This embodiment provides a method for preparing a single cell suspension of placental protoplasts of Capsicum annuum, which specifically includes the following contents:

[0122] (1) Preparation of enzymatic solution and DB buffer:

[0123] The preparation method of the enzymatic hydrolysate and DB buffer in this example is prepared by referring to Example 1.

[0124] (2) Obtaining the placental tissue of screw pepper:

[0125] Choose healthy growing screw peppers with undamaged skin (such as Figure 2 Use a blade and tweezers to remove the pepper pulp and seeds, take out the pepper placenta and place it in enzyme-free water. Then use a sharp double-sided blade to evenly cut the pepper placenta into 1-2 mm strips. The cut pepper placenta tissue will be immediately transferred to a culture dish containing enzyme-free water (as shown in Figure E). Figure 3 Prevent it from drying out and keep it aside.

[0126] (3) Digestion and treatment of the placenta tissue of screw pepper:

[0127] After all the placenta tissues of the screw pepper are cut, wash them with enzyme-free water for 3 to 5 times, then transfer them to a sterile culture dish containing 5 mL of the above-mentioned enzymatic hydrolyzate, and incubate them in a constant temperature water bath shaker set at 28°C and 50 rpm in the dark for 2.5 hours to obtain the digestion solution and the digested placenta tissue of the screw pepper.

[0128] (4) Obtaining a crude single-cell extract of the placenta protoplasts of pepper:

[0129] Take out the culture dish containing the digestion solution and the digested screw pepper placenta tissue from the constant temperature water bath shaker, add 2 mL of the prepared DB buffer solution to terminate the digestion reaction, and use a 1 mL wide-mouthed pipette tip to gently suck the digestion solution to disperse the cell clumps and release more cells. Use a wide-mouthed pipette tip to transfer the digestion solution to a cell sieve, and slowly filter through 100 μm and 40 μm cell sieves in turn, and add 2 mL of the above DB buffer solution to the cell sieve to rinse the tissue residue, discard the remaining screw pepper placenta tissue residue after digestion, collect the filtered screw pepper placenta protoplast single cell crude extract, and set aside.

[0130] (5) Obtaining a single cell suspension of the placenta protoplasts of pepper:

[0131] The crude single-cell liquid of the placenta protoplasts of the screw pepper obtained above was transferred to a 15 mL centrifuge tube, and centrifuged in a low-temperature horizontal centrifuge at 4°C, 700 rpm, 5 min and a lifting rate of 4 rpm / s using the sedimentation centrifugation method. The supernatant was then removed and the precipitate was retained. An appropriate amount of the above-mentioned DB buffer was added and centrifuged in the same manner. The mixture was washed twice, the supernatant was removed, and 200 μL of the above-mentioned DB buffer was added. The mixture was gently blown and mixed to obtain a single-cell suspension of the placenta protoplasts of the screw pepper.

[0132] The single cell suspension of the protoplasts of the screw pepper obtained above was observed under a microscope. Figure 8 As shown, it is shown that the single-cell morphology of the placenta protoplasts of screw pepper obtained by the method of the present invention is complete, and the density is high and the impurities are few, which can meet the cell suspension quality requirements of various high-throughput single-cell sequencing platforms.

[0133] Example 6: Zhangshu Harbor

[0134] This embodiment provides a method for preparing a single cell suspension of placental protoplasts of a pepper of the Zhangshugang variety, comprising the following contents:

[0135] (1) Preparation of enzymatic solution and DB buffer:

[0136] The raw materials for preparing the enzymatic hydrolysate used in this example are shown in Table 2:

[0137] Table 2: Enzymatic solution

[0138] Where to buy Content (final concentration) Cellulase R-10 Yakult(MX7351) 24 mg / mL Mesozyme R-10 Yakult(MX7352) 12mg / mL Snail enzyme Source Leaf Biotechnology (S10083) 2mg / mL Pectinase Y-23 Yakult(MX7354) 6mg / mL Mannitol Hushi(63008816) 100mg / mL BSA Miltenyi Biotec (130-091-376) 5mg / mL MES Hushi(30123760) 20mM KCl Hushi(10016318) 10mM Dithiothreitol (DTT) Solarbio(D8220) 0.5mM Enzyme-free water - Make up to 5mL

[0139] The pH of the enzymatic hydrolyzate prepared above is 5.6-5.8.

[0140] The buffer used in this example is DB buffer, which contains 90 mg / mL mannitol (Shanghai), 5 mg / mL bovine serum albumin (Miltenyi Biotec) and 10 mM KCl (Shanghai). Enzyme-free water is used to make up to 50 mL during the preparation process, and the pH is 5.6-5.8.

[0141] The prepared solution was filtered through a 0.22 μm sterile syringe filter for sterilization, shaken and mixed before use, and pre-cooled on ice for use immediately after preparation.

[0142] (2) Obtaining pepper placental tissue:

[0143] Select healthy pepper fruits with intact skin. The pepper variety is Zhangshugang. Use a blade and tweezers to remove the pepper pulp and seeds, then take out the pepper placenta and place it in enzyme-free water. Then use a sharp double-sided blade to evenly cut the pepper placenta into 1-2 mm strips. Immediately transfer about 0.1-0.2 g of the cut pepper placenta tissue to a culture dish containing enzyme-free water to prevent drying and set aside.

[0144] (3) Digestion and treatment of pepper placenta tissue:

[0145] After all pepper placenta tissues are cut, wash them with enzyme-free water for 3 to 5 times and then transfer them to a sterile culture dish containing 5 mL of the above enzymatic hydrolyzate. Incubate them in a constant temperature water bath shaker set at 28°C and 50 rpm in the dark for 2.5 h to obtain the digestion solution and digested pepper placenta tissue.

[0146] (4) Obtaining a crude single-cell extract of pepper placenta protoplasts:

[0147] Take out the culture dish containing the digestion solution and digested pepper placenta tissue from the constant temperature water bath shaker, add 2 mL of the prepared DB buffer to terminate the digestion reaction, and use a 1 mL wide-mouthed pipette tip to gently suck the digestion solution to blow away the cell clumps and release more cells. Use a wide-mouthed pipette tip to transfer the digestion solution to a cell sieve, and slowly filter through 100 μm and 40 μm cell sieves in turn, and add 2 mL of the above DB buffer to the cell sieve to rinse the tissue residue, discard the remaining pepper placenta tissue residue after digestion, collect the filtered pepper placenta protoplast single cell crude extract, and set aside.

[0148] (5) Obtaining pepper placenta protoplast single cell suspension:

[0149] The crude single-cell fluid of pepper placenta protoplasts obtained above was transferred to a 15-mL centrifuge tube, and centrifuged in a low-temperature horizontal centrifuge at 4°C, 700 rpm, 5 min, and a lifting rate of 4 rpm / s using the sedimentation centrifugation method. The supernatant was removed and the precipitate was retained. An appropriate amount of the above-mentioned DB buffer was added and centrifuged in the same manner. The mixture was washed twice, the supernatant was removed, and 200 μL of the above-mentioned DB buffer was added. The mixture was gently blown to mix well to obtain a single-cell suspension of pepper placenta protoplasts.

[0150] The single cell suspension of pepper placenta protoplasts obtained above was observed under a microscope. Fig. 9 As shown, it is shown that the method of the present invention can obtain pepper placenta protoplast single cells with complete morphology and high density.

[0151] Comparative Example 1:

[0152] This comparative example provides a method for preparing a single cell suspension of placental protoplasts of a pepper of the Zhangshugang variety, which specifically includes the following contents:

[0153] (1) Preparation of enzymatic solution and DB buffer:

[0154] The raw materials for preparing the enzymatic hydrolysate used in this comparative example are shown in Table 3, specifically as follows:

[0155] Table 3: Enzymatic solution

[0156] Where to buy Content (final concentration) Cellulase R-10 Yakult(MX7351) 20mg / mL (originally recorded as 2%) Mesozyme R-10 Yakult(MX7352) 3mg / mL (originally recorded as 0.3%) Mannitol Hushi(63008816) 72mg / mL (originally recorded as 0.4M) BSA Miltenyi Biotec 3mg / mL (originally recorded as 0.3%) MES Hushi(30123760) 0.3mg / mL (originally recorded as 0.03%) CaCl2 Hushi 8mM Enzyme-free water - Make up to 5mL

[0157] The pH of the enzymatic hydrolyzate prepared above is 5.6-5.8.

[0158] The buffer used in this comparative example is DB buffer, and its preparation method refers to Example 1.

[0159] The prepared solution was filtered through a 0.22 μm sterile syringe filter for sterilization, shaken and mixed before use, and pre-cooled on ice for use immediately after preparation.

[0160] (2) Obtaining pepper placental tissue:

[0161] Select healthy pepper fruits with undamaged skin. The pepper variety is Zhangshugang. Use a blade and tweezers to remove the pepper pulp and seeds, then take out the pepper placenta and place it in enzyme-free water. Then use a sharp double-sided blade to evenly cut the pepper placenta into 1-2 mm strips. Immediately transfer the cut pepper placenta tissue to a culture dish containing enzyme-free water to prevent drying and set aside.

[0162] (3) Digestion and treatment of pepper placenta tissue:

[0163] After all pepper placenta tissues are cut, wash them with enzyme-free water for 3 to 5 times and then transfer them to a sterile culture dish containing 5 mL of the above enzymatic hydrolyzate. Incubate them in a constant temperature water bath shaker set at 28°C and 50 rpm in the dark for 2.5 h to obtain the digestion solution and digested pepper placenta tissue.

[0164] (4) Obtaining a crude single-cell extract of pepper placenta protoplasts:

[0165] Take out the culture dish containing the digestion solution and digested pepper placenta tissue from the constant temperature water bath shaker, add 2 mL of the prepared DB buffer to terminate the digestion reaction, and use a 1 mL wide-mouthed pipette tip to gently suck the digestion solution to blow away the cell clumps and release more cells. Use a wide-mouthed pipette tip to transfer the digestion solution to a cell sieve, and slowly filter through 100 μm and 40 μm cell sieves in turn, and add 2 mL of the above DB buffer to the cell sieve to rinse the tissue residue, discard the remaining pepper placenta tissue residue after digestion, collect the filtered pepper placenta protoplast single cell crude extract, and set aside.

[0166] (5) Obtaining pepper placenta protoplast single cell suspension:

[0167] The crude single-cell fluid of pepper placenta protoplasts obtained above was transferred to a 15-mL centrifuge tube, and centrifuged in a low-temperature horizontal centrifuge at 4°C, 700 rpm, 5 min, and a lifting rate of 4 rpm / s using the sedimentation centrifugation method. The supernatant was removed and the precipitate was retained. An appropriate amount of the above-mentioned DB buffer was added and centrifuged in the same manner. The mixture was washed twice, the supernatant was removed, and 200 μL of the above-mentioned DB buffer was added. The mixture was gently blown to mix well to obtain a single-cell suspension of pepper placenta protoplasts.

[0168] The single cell suspension of pepper placenta protoplasts obtained above was observed under a microscope. Fig.10 shown.

[0169] Comparative Example 2:

[0170] This comparative example provides a method for preparing a single cell suspension of placental protoplasts of a pepper of the Zhangshugang variety, which specifically includes the following contents:

[0171] (1) Preparation of enzymatic solution and DB buffer:

[0172] The raw materials for preparing the enzymatic hydrolysate used in this comparative example are shown in Table 4. The difference between the enzymatic hydrolysate and the enzymatic hydrolysate of Example 1 is that the snail enzyme is replaced by cellulase R-10, and the other contents are the same:

[0173] Table 4: Enzymatic solution

[0174]

[0175]

[0176] The pH of the enzymatic hydrolyzate prepared above is 5.6-5.8.

[0177] The buffer used in this comparative example is DB buffer, and its preparation method refers to Example 1.

[0178] The prepared solution was filtered through a 0.22 μm sterile syringe filter for sterilization, shaken and mixed before use, and pre-cooled on ice for use immediately after preparation.

[0179] (2) Obtaining pepper placental tissue:

[0180] Select healthy pepper fruits with undamaged skin. The pepper variety is Zhangshugang. Use a blade and tweezers to remove the pepper pulp and seeds, then take out the pepper placenta and place it in enzyme-free water. Then use a sharp double-sided blade to evenly cut the pepper placenta into 1-2 mm strips. Immediately transfer the cut pepper placenta tissue to a culture dish containing enzyme-free water to prevent drying and set aside.

[0181] (3) Digestion and treatment of pepper placenta tissue:

[0182] After all pepper placenta tissues are cut, wash them with enzyme-free water for 3 to 5 times and then transfer them to a sterile culture dish containing 5 mL of the above enzymatic hydrolyzate. Incubate them in a constant temperature water bath shaker set at 28°C and 50 rpm in the dark for 2.5 h to obtain the digestion solution and digested pepper placenta tissue.

[0183] (4) Obtaining a crude single-cell extract of pepper placenta protoplasts:

[0184] Take out the culture dish containing the digestion solution and digested pepper placenta tissue from the constant temperature water bath shaker, add 2 mL of the prepared DB buffer to terminate the digestion reaction, and use a 1 mL wide-mouthed pipette tip to gently suck the digestion solution to blow away the cell clumps and release more cells. Use a wide-mouthed pipette tip to transfer the digestion solution to a cell sieve, and slowly filter through 100 μm and 40 μm cell sieves in turn, and add 2 mL of the above DB buffer to the cell sieve to rinse the tissue residue, discard the remaining pepper placenta tissue residue after digestion, collect the filtered pepper placenta protoplast single cell crude extract, and set aside.

[0185] (5) Obtaining pepper placenta protoplast single cell suspension:

[0186] The crude single-cell fluid of pepper placenta protoplasts obtained above was transferred to a 15-mL centrifuge tube, and centrifuged in a low-temperature horizontal centrifuge at 4°C, 700 rpm, 5 min, and a lifting rate of 4 rpm / s using the sedimentation centrifugation method. The supernatant was removed and the precipitate was retained. An appropriate amount of the above-mentioned DB buffer was added and centrifuged in the same manner. The mixture was washed twice, the supernatant was removed, and 200 μL of the above-mentioned DB buffer was added. The mixture was gently blown to mix well to obtain a single-cell suspension of pepper placenta protoplasts.

[0187] The single cell suspension of pepper placenta protoplasts obtained above was observed under a microscope. Fig.11 shown.

[0188] Comparative Example 3:

[0189] This comparative example provides a method for preparing a single cell suspension of placental protoplasts of a pepper of the Zhangshugang variety, which specifically includes the following contents:

[0190] (1) Preparation of enzymatic solution and DB buffer:

[0191] The raw materials for preparing the enzymatic hydrolysate used in this comparative example are shown in Table 5. The difference between the enzymatic hydrolysate and the enzymatic hydrolysate of Example 1 is that pectinase Y-23 is replaced by macerate R-10, and the other contents are the same:

[0192] Table 5: Enzymatic solution

[0193] Where to buy Content (final concentration) Cellulase R-10 Yakult(MX7351) 20mg / mL Mesozyme R-10 Yakult(MX7352) 12.5 mg / mL Snail enzyme Source Leaf Biotechnology (S10083) 5mg / mL Mannitol Hushi(63008816) 90mg / mL BSA Miltenyi Biotec 5mg / mL MES Hushi(30123760) 20mM KCl Hushi(10016318) 10mM DTT Solarbio(D8220) 0.5mM Enzyme-free water - Make up to 5mL

[0194] The pH of the enzymatic hydrolyzate prepared above is 5.6-5.8.

[0195] The buffer used in this comparative example is DB buffer, and its preparation method refers to Example 1.

[0196] The prepared solution was filtered through a 0.22 μm sterile syringe filter for sterilization, shaken and mixed before use, and pre-cooled on ice for use immediately after preparation.

[0197] (2) Obtaining pepper placental tissue:

[0198] Select healthy pepper fruits with undamaged skin. The pepper variety is Zhangshugang. Use a blade and tweezers to remove the pepper pulp and seeds, then take out the pepper placenta and place it in enzyme-free water. Then use a sharp double-sided blade to evenly cut the pepper placenta into 1-2 mm strips. Immediately transfer the cut pepper placenta tissue to a culture dish containing enzyme-free water to prevent drying and set aside.

[0199] (3) Digestion and treatment of pepper placenta tissue:

[0200] After all pepper placenta tissues are cut, wash them with enzyme-free water for 3 to 5 times and then transfer them to a sterile culture dish containing 5 mL of the above enzymatic hydrolyzate. Incubate them in a constant temperature water bath shaker set at 28°C and 50 rpm in the dark for 2.5 h to obtain the digestion solution and digested pepper placenta tissue.

[0201] (4) Obtaining a crude single-cell extract of pepper placenta protoplasts:

[0202] Take out the culture dish containing the digestion solution and digested pepper placenta tissue from the constant temperature water bath shaker, add 2 mL of the prepared DB buffer to terminate the digestion reaction, and use a 1 mL wide-mouthed pipette tip to gently suck the digestion solution to blow away the cell clumps and release more cells. Use a wide-mouthed pipette tip to transfer the digestion solution to a cell sieve, and slowly filter through 100 μm and 40 μm cell sieves in turn, and add 2 mL of the above DB buffer to the cell sieve to rinse the tissue residue, discard the remaining pepper placenta tissue residue after digestion, collect the filtered pepper placenta protoplast single cell crude extract, and set aside.

[0203] (5) Obtaining pepper placenta protoplast single cell suspension:

[0204] The crude single-cell fluid of pepper placenta protoplasts obtained above was transferred to a 15-mL centrifuge tube, and centrifuged in a low-temperature horizontal centrifuge at 4°C, 700 rpm, 5 min, and a lifting rate of 4 rpm / s using the sedimentation centrifugation method. The supernatant was removed and the precipitate was retained. An appropriate amount of the above-mentioned DB buffer was added and centrifuged in the same manner. The mixture was washed twice, the supernatant was removed, and 200 μL of the above-mentioned DB buffer was added. The mixture was gently blown to mix well to obtain a single-cell suspension of pepper placenta protoplasts.

[0205] The single cell suspension of pepper placenta protoplasts obtained above was observed under a microscope. Fig.12 shown.

[0206] Comparative Example 4:

[0207] This comparative example provides a method for preparing a single cell suspension of placental protoplasts of a pepper of the Zhangshugang variety, which specifically includes the following contents:

[0208] (1) Preparation of enzymatic solution and DB buffer:

[0209] The raw materials for preparing the enzymatic hydrolysate used in this comparative example are shown in Table 6. The difference between the enzymatic hydrolysate and the enzymatic hydrolysate in Example 1 is that DTT is not contained, and the other contents are the same:

[0210] Table 6: Enzyme digestion solution

[0211]

[0212]

[0213] The pH of the enzymatic hydrolyzate prepared above is 5.6-5.8.

[0214] The buffer used in this comparative example is DB buffer, and its preparation method refers to Example 1.

[0215] The prepared solution was filtered through a 0.22 μm sterile syringe filter for sterilization, shaken and mixed before use, and pre-cooled on ice for use immediately after preparation.

[0216] (2) Obtaining pepper placental tissue:

[0217] Select healthy pepper fruits with undamaged skin. The pepper variety is Zhangshugang. Use a blade and tweezers to remove the pepper pulp and seeds, then take out the pepper placenta and place it in enzyme-free water. Then use a sharp double-sided blade to evenly cut the pepper placenta into 1-2 mm strips. Immediately transfer the cut pepper placenta tissue to a culture dish containing enzyme-free water to prevent drying and set aside.

[0218] (3) Digestion and treatment of pepper placenta tissue:

[0219] After all pepper placenta tissues are cut, wash them with enzyme-free water for 3 to 5 times and then transfer them to a sterile culture dish containing 5 mL of the above enzymatic hydrolyzate. Incubate them in a constant temperature water bath shaker set at 28°C and 50 rpm in the dark for 2.5 h to obtain the digestion solution and digested pepper placenta tissue.

[0220] (4) Obtaining a crude single-cell extract of pepper placenta protoplasts:

[0221] Take out the culture dish containing the digestion solution and digested pepper placenta tissue from the constant temperature water bath shaker, add 2 mL of the prepared DB buffer to terminate the digestion reaction, and use a 1 mL wide-mouthed pipette tip to gently suck the digestion solution to blow away the cell clumps and release more cells. Use a wide-mouthed pipette tip to transfer the digestion solution to a cell sieve, and slowly filter through 100 μm and 40 μm cell sieves in turn, and add 2 mL of the above DB buffer to the cell sieve to rinse the tissue residue, discard the remaining pepper placenta tissue residue after digestion, collect the filtered pepper placenta protoplast single cell crude extract, and set aside.

[0222] (5) Obtaining pepper placenta protoplast single cell suspension:

[0223] The crude single-cell fluid of pepper placenta protoplasts obtained above was transferred to a 15-mL centrifuge tube, and centrifuged in a low-temperature horizontal centrifuge at 4°C, 700 rpm, 5 min, and a lifting rate of 4 rpm / s using the sedimentation centrifugation method. The supernatant was removed and the precipitate was retained. An appropriate amount of the above-mentioned DB buffer was added and centrifuged in the same manner. The mixture was washed twice, the supernatant was removed, and 200 μL of the above-mentioned DB buffer was added. The mixture was gently blown to mix well to obtain a single-cell suspension of pepper placenta protoplasts.

[0224] The single cell suspension of pepper placenta protoplasts obtained above was observed under a microscope. Fig.13 shown.

[0225] Comparative Example 5:

[0226] This comparative example provides a method for preparing a single cell suspension of placental protoplasts of a pepper of the Zhangshugang variety, which specifically includes the following contents:

[0227] (1) Preparation of enzymatic solution and DB buffer:

[0228] The enzymatic hydrolyzate used in this comparative example is the same as that in Example 1, and the buffer used is DB buffer. The difference between it and the buffer in Example 1 is that mannose is replaced by glucose, specifically comprising 90 mg / mL glucose (Shanghai), 5 mg / mL bovine serum albumin (Miltenyi Biotec) and 10 mM KCl (Shanghai), and enzyme-free water is used to make up to 50 mL during the preparation process, and the pH is 5.6-5.8.

[0229] The prepared solution was filtered through a 0.22 μm sterile syringe filter for sterilization, shaken and mixed before use, and pre-cooled on ice for use immediately after preparation.

[0230] (2) Obtaining pepper placental tissue:

[0231] Select healthy pepper fruits with undamaged skin. The pepper variety is Zhangshugang. Use a blade and tweezers to remove the pepper pulp and seeds, then take out the pepper placenta and place it in enzyme-free water. Then use a sharp double-sided blade to evenly cut the pepper placenta into 1-2 mm strips. Immediately transfer the cut pepper placenta tissue to a culture dish containing enzyme-free water to prevent drying and set aside.

[0232] (3) Digestion and treatment of pepper placenta tissue:

[0233] After all pepper placenta tissues are cut, wash them with enzyme-free water for 3 to 5 times and then transfer them to a sterile culture dish containing 5 mL of the above enzymatic hydrolyzate. Incubate them in a constant temperature water bath shaker set at 28°C and 50 rpm in the dark for 2.5 h to obtain the digestion solution and digested pepper placenta tissue.

[0234] (4) Obtaining a crude single-cell extract of pepper placenta protoplasts:

[0235] Take out the culture dish containing the digestion solution and digested pepper placenta tissue from the constant temperature water bath shaker, add 2 mL of the prepared DB buffer to terminate the digestion reaction, and use a 1 mL wide-mouthed pipette tip to gently suck the digestion solution to blow away the cell clumps and release more cells. Use a wide-mouthed pipette tip to transfer the digestion solution to a cell sieve, and slowly filter through 100 μm and 40 μm cell sieves in turn, and add 2 mL of the above DB buffer to the cell sieve to rinse the tissue residue, discard the remaining pepper placenta tissue residue after digestion, collect the filtered pepper placenta protoplast single cell crude extract, and set aside.

[0236] (5) Obtaining pepper placenta protoplast single cell suspension:

[0237] The crude single-cell fluid of pepper placenta protoplasts obtained above was transferred to a 15-mL centrifuge tube, and centrifuged in a low-temperature horizontal centrifuge at 4°C, 700 rpm, 5 min, and a lifting rate of 4 rpm / s using the sedimentation centrifugation method. The supernatant was removed and the precipitate was retained. An appropriate amount of the above-mentioned DB buffer was added and centrifuged in the same manner. The mixture was washed twice, the supernatant was removed, and 200 μL of the above-mentioned DB buffer was added. The mixture was gently blown to mix well to obtain a single-cell suspension of pepper placenta protoplasts.

[0238] The single cell suspension of pepper placenta protoplasts obtained above was observed under a microscope. Fig.14 shown.

[0239] Assay Example: Cell Yield and Viability Assay

[0240] In this test example, the protoplast yield and cell activity of the pepper placenta protoplast single cell suspension obtained by the above method are tested. The specific method is as follows:

[0241] 9 μL of the above protoplast single cell suspension and 3 μL of 0.4% trypan blue staining solution were respectively taken in a centrifuge tube and mixed evenly, and stained for 1 minute; then 10 μL of trypan blue single cell mixture was taken on a hemocytometer plate, and the number of pepper placenta protoplasts was counted under an optical microscope, and the cell activity was calculated. The cell activity calculation formula is:

[0242] Cell activity (%) = number of living cells / total number of cells × 100%.

[0243] The test results are shown in Table 7 below.

[0244] Table 7: Protoplast yield and activity test results

[0245]

[0246] The test results show that the pepper placenta protoplast single cell suspension prepared by the preparation method of the present invention has a high protoplast yield and good cell activity, and can meet the cell suspension quality requirements of various high-throughput single-cell sequencing platforms and subsequent protoplast-related experiments.

[0247] Compared with Example 1 of the present invention, Comparative Example 1 uses conventional enzymolysis solution for enzymolysis, and the results show that its protoplast yield and activity are significantly reduced, which is speculated to be related to incomplete cell wall decomposition. In Comparative Example 1, only cellulase R-10 and macerate R-10 are added, and it is difficult to completely decompose the cell wall. Further, snail enzyme and pectinase are not added in Comparative Example 2 and Comparative Example 3, and DTT is not added in Comparative Example 4, and the results show that the yield and activity of its protoplasts are reduced, which is speculated to be caused by excessive local reduction of the cell wall or oxidation of the protoplasts. And in Comparative Example 5, the mannitol in the buffer is replaced with conventional glucose, and the results show that it is not conducive to the improvement of protoplast yield and cell activity.

[0248] In summary, the present invention provides a method for preparing a single-cell suspension of pepper placenta protoplasts. By adopting the method for preparing a single-cell suspension of pepper placenta protoplasts provided by the present invention, a large number of protoplasts can be obtained quickly and efficiently, which is beneficial to reduce the external damage of the pepper placenta protoplasts during the digestion process as much as possible, thereby ensuring the integrity of the cells; at the same time, the digestion time of the present invention is relatively short, which greatly reduces the probability of RNA degradation and digestion fluid contamination, and ensures the stability and reliability of subsequent data analysis.

[0249] In addition, the solutions used in all steps of the present invention do not contain reagents containing Ca ions and Mg ions, which helps to reduce cell agglomeration and improves the efficiency of subsequent protoplast capture. In addition, the solutions used in all steps of the present invention have few added components, which helps to reduce the preparation cost.

[0250] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for preparing a pepper placenta protoplast single cell suspension, characterized in that: The following steps are involved: S1. Obtain pepper placenta tissue and transfer it to enzymatic solution for digestion, then add DB buffer to terminate digestion and collect digestion solution; S2, passing the digestion solution through a cell sieve, collecting the filtrate, and obtaining a crude single-cell extract of pepper placenta protoplasts; S3, centrifuging the pepper placenta protoplast single cell crude extract, collecting the precipitate and resuspending it in the DB buffer, repeating the centrifugation and resuspending steps to obtain a pepper placenta protoplast single cell suspension; The enzymatic hydrolysate comprises 15-25 mg / mL cellulase, 8-12 mg / mL macerate, 2-8 mg / mL snail enzyme, 1.5-5 mg / mL pectinase, 80-120 mg / mL mannitol, 2-8 mg / mL bovine serum albumin, 15-25 mM MES, 8-12 mM KCl and 0.2-0.8 mM dithiothreitol; The DB buffer contains 80-120 mg / mL mannitol, 2-8 mg / mL bovine serum albumin and 8-12 mM KCl.

2. The preparation method according to claim 1, characterized in that: In step S1, the method of obtaining pepper placenta tissue includes: selecting healthy pepper fruits, taking out pepper placenta tissue, washing it in enzyme-free water, and then cutting the pepper placenta tissue for later use.

3. The preparation method according to claim 2, characterized in that: In step S1, the variety of peppers includes any one of zhangshugang, red pepper, sweet pepper, erjingtiao, screw pepper, colored pepper, pointed pepper, wrinkled pepper, millet pepper, chili pepper, and lantern pepper.

4. The preparation method according to claim 1, characterized in that: In step S1, the digestion temperature is 28±3°C; And / or, the digestion time is 1 to 3 hours; And / or, the rotation speed of the shaker during the digestion process is 30 to 80 rpm.

5. The preparation method according to claim 1, characterized in that: In step S1, during the digestion termination process, the volume of the DB buffer is 25% to 50% of the volume of the enzymatic hydrolyzate.

6. The preparation method according to claim 1, characterized in that: In step S2, the mesh diameter of the cell sieve is 20 to 120 μm; Preferably, the mesh diameter of the cell sieve is 40 to 100 μm; Preferably, the cell sieve comprises a cell sieve having a sieve hole diameter of 90 to 120 μm and a cell sieve having a sieve hole diameter of 20 to 60 μm.

7. The preparation method according to claim 1, characterized in that: In step S2, the centrifugation temperature is 2±6°C; And / or, the centrifugal speed is 600-800 rpm; And / or, the centrifugation time is 3 to 8 minutes.

8. The preparation method according to claim 1, characterized in that: In step S2, the number of repetitions is 1 to 3 times.

9. A pepper placenta protoplast single cell suspension, characterized in that: The method for preparing the pepper placenta protoplast single cell suspension according to any one of claims 1 to 8 is adopted.

10. The method for preparing the pepper placenta protoplast single cell suspension according to any one of claims 1 to 8 or the use of the pepper placenta protoplast single cell suspension according to claim 9 in single cell sequencing or flow cytometry analysis.

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