Preparation method of fresh taxus chinensis stem and leaf protoplast and special reagent thereof

Abstract

The application discloses a preparation method of fresh taxus chinensis stem and leaf protoplasts, which comprises a preparation process and a purification process. The preparation method of the taxus chinensis stem and leaf protoplasts is simple and easy to operate, raw materials are easy to obtain, the price is low, reagent components have good biocompatibility, have no harmful components, and are safe and environmentally friendly. The application provides a simple and rapid enzyme hydrolysis preparation method of the taxus chinensis stem and leaf protoplasts, vacuum filtration is adopted to accelerate the permeation of the enzyme hydrolysate and improve the enzyme hydrolysis efficiency, so that the protoplasts can be quickly obtained. Meanwhile, the high-purity separation of the protoplasts is carried out by combining with the density gradient sedimentation of iodixanol solution, and the uniform size and complete morphology of the protoplasts are obtained, which provides convenience for subsequent scientific researches such as transformation and single-cell transcriptome sequencing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant cells, and particularly relates to a preparation method of fresh yew stem and leaf protoplast and special reagents thereof. BACKGROUND

[0002] Yew is a world-recognized natural rare anticancer plant on the brink of extinction. Due to the slow growth of yew under natural conditions and poor regeneration, a large-scale yew raw material forest base has not been formed worldwide.

[0003] At present, for the problems of slow growth and low seed germination rate of yew, protoplast preparation, protoplast culture and callus induction are a good solution. Protoplast has uniformity, is a relatively uniform single cell population without cell walls. And the protoplast has the same separation cycle, and the expression of the introduced exogenous gene has good synchronicity; at the same time, long-term tissue culture is not required, and the preparation and detection process is short in time. In addition, through the protoplast transient expression system and transcriptome sequencing, researches on rapid plant propagation, metabolic products, generation of polyploidy and plant signal transduction mechanism can be carried out. SUMMARY

[0004] In view of the deficiencies in the prior art, the purpose of the present application is to provide a preparation method of fresh yew stem and leaf protoplast. The protoplast preparation and purification method provided by the present application has the advantages of short operation time, simple process, high yield, clean background and maximum reduction of protoplast rupture and RNA degradation.

[0005] In order to achieve the above purpose, the present application adopts the following technical means:

[0006] The first aspect of the present application is to provide a preparation method of fresh yew stem and leaf protoplast, which comprises a preparation process and a purification process, and the specific steps are as follows:

[0007] I. Preparation process:

[0008] S1, 0.5-1g of fresh yew tissue is taken, washed and then absorbed dry;

[0009] S2, the fresh yew tissue is then placed in a culture dish, and an enzyme solution is added, the volume-to-mass ratio of the enzyme solution to the fresh yew tissue being 10mL:1g, the fresh yew tissue is cut into about 0.1-0.5mm filaments on ice with a blade;

[0010] S3, vacuum filtration for 30min, then shaking enzyme hydrolysis at 28℃ for 4 hours in the dark, the shaking frequency being 40rpm, to obtain the enzyme-hydrolyzed tissue;

[0011] II. Purification process:

[0012] S4, filtering the enzymolyzed tissue using a 200-mesh filter screen, centrifuging at 4°C and discarding the supernatant to obtain a precipitate;

[0013] S5, adding a washing solution to the precipitate obtained in step S4 to resuspend the precipitate to obtain a protoplast resuspension; wherein the volume ratio of the washing solution to the precipitate is 5-15 mL:1 g;

[0014] S6, taking another centrifuge tube, adding a same volume of a purification reagent to the resuspension, and pouring the protoplast resuspension into the purification reagent to obtain a mixture;

[0015] S7, centrifuging the mixture at 4°C, taking the middle protoplast layer, and adding a washing solution; wherein the volume ratio of the washing solution to the protoplast layer is about 10:1;

[0016] S8, centrifuging at 4°C, discarding the supernatant, and resuspending the precipitate with a mannitol solution to obtain the protoplast of the fresh Taxus tissue; wherein the volume ratio of the resuspension to the protoplast is 10 mL:1 g.

[0017] The second aspect of the present application provides a special reagent for the preparation method of the fresh Taxus stem and leaf protoplast according to the first aspect of the present application, which comprises an enzymolysis solution, a washing solution, and a purification reagent, wherein the purification reagent comprises an iodixanol solution, which is prepared by mixing iodixanol and nuclease-free water at a volume ratio of 1:1.

[0018] Preferably, the enzymolysis solution comprises cellulase, hemicellulase, lyase, mannitol, an anion, a metal cation, an ionic buffer, BSA, and beta-mercaptoethanol; and the washing solution comprises a metal halide and an ionic buffer, wherein the metal halide is composed of sodium chloride, calcium chloride, and potassium chloride, and the ionic buffer is MES.

[0019] Preferably, the anion is selected from one of chloride, nitrate, and sulfate, and is preferably chloride.

[0020] Preferably, the cation is composed of sodium ion, potassium ion, and magnesium ion.

[0021] Preferably, the cellulase is cellulase R-10, which is used to catalyze the hydrolysis of cellulose molecules to generate cellooligosaccharides, cellobiose, and glucose. In addition, the cellulase can also cleave the endo-1,4-beta-D-glycosidic bonds in cellulose, lichenin, barley glucan, and cellotriose to cellotetrose.

[0022] The hemicellulase is used to hydrolyze polysaccharides constituting the plant cell membrane, except for cellulose and pectin substances;

[0023] The dissociative enzyme is dissociative enzyme P, which is used to crack the cell wall of plant protoplast before the separation of cell organs.

[0024] The mannitol is a high-osmotic tissue dehydrating agent, which can increase the osmotic pressure of the enzymatic solution and avoid the rupture of the protoplast due to water absorption;

[0025] The ionic buffer is a zwitterionic buffer; further, the ionic buffer is MES, which has a pKa value close to the physiological pH value, good water solubility, and is not easy to dissolve in other solvents and not easy to chelate salt ions;

[0026] The sodium ions, chloride ions and potassium ions have an influence on the osmotic pressure on both sides of the membrane;

[0027] The β-mercaptoethanol is used as an antioxidant to prevent the oxidation of disulfide bonds;

[0028] The BSA is used to maintain the osmotic pressure, play a pH buffering and carrier role, and protect the cell nucleus;

[0029] The iodixanol is a contrast agent, which has the advantages of non-ionic, non-toxic to cells and metabolic inertness, and under the action of a certain centrifugal force, the particles will respectively settle at a certain speed, and different bands will be formed on the density gradient area, thereby playing a purifying purpose.

[0030] Further, the enzymatic solution comprises 0.40-0.50 parts by weight of cellulase, 0.20-0.30 parts by weight of hemicellulase, 0.10-0.15 parts by weight of dissociative enzyme, 2.5-3.0 parts by weight of mannitol, 0.65-0.70 parts by weight of potassium chloride, 1.90-1.95 parts by weight of MES, 0.4-0.6 parts by weight of calcium chloride, 0.5-3.5 parts by weight of BSA and 0.014-0.020 parts by weight of β-mercaptoethanol.

[0031] Further, the cleaning solution comprises 0.3-0.5M of sodium chloride, 5-15mM of calcium chloride, 15-25mM of potassium chloride and 5-15mM of MES.

[0032] Further, the preparation method of the enzymatic solution is as follows: the cellulase, hemicellulase, dissociative enzyme, mannitol, potassium chloride and MES are added to nuclease-free water, and heated to dissolve at 50-60°C, and then cooled to room temperature with ice blocks, and then the calcium chloride, BSA and β-mercaptoethanol are added, and the nuclease-free water is used to make up the volume, so that the enzymatic solution is obtained;

[0033] Further, the cleaning solution is prepared by adding sodium chloride, calcium chloride, potassium chloride and MES into nuclease-free water, dissolving, adjusting pH to 5.7 by potassium hydroxide, and constant volume with nuclease-free water, to obtain the cleaning solution, and storing at 4 DEG C for standby.

[0034] Advantages of the present application

[0035] Compared with the prior art, the present application has the following advantages:

[0036] (1) The present application provides a preparation method of yew stem and leaf protoplast, which is simple and easy to operate, and the raw materials are easy to obtain, low in price, and the reagent components have good biocompatibility, no harmful ingredients, and are safe and environmentally friendly.

[0037] (2) The present application provides a simple and rapid preparation method of yew stem and leaf protoplast enzymolysis, which uses vacuum filtration to accelerate the penetration of the enzyme solution and improve the enzymolysis efficiency, so that the protoplast can be quickly obtained. At the same time, the high-purity separation of the protoplast is carried out by combining the density gradient sedimentation of iodixanol solution, and the uniform size and complete morphology of the protoplast are obtained, which provides convenience for subsequent transformation, single cell transcriptome sequencing and other scientific research.

[0038] (3) The present application also provides a special reagent for the preparation method of fresh yew stem and leaf protoplast, which is used for the enzymolysis, extraction and purification of yew stem and leaf protoplast, and the obtained protoplast has complete structure and stable gene expression, and can be further used for gene detection and research. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The present application shows the protoplast microscope result graph of yew stem enzymolysis digestion for 2 hours in embodiment 1;

[0040] Figure 2 The present application shows the protoplast microscope result graph of yew stem enzymolysis digestion for 3 hours in embodiment 1;

[0041] Figure 3 The present application shows the protoplast microscope result graph of yew stem enzymolysis digestion for 4 hours in embodiment 1;

[0042] Figure 4 The present application shows the protoplast microscope result graph of yew stem enzymolysis for 3 hours in enzymolysis solution A in embodiment 2;

[0043] Figure 5 The present application shows the protoplast microscope result graph of yew stem enzymolysis for 3 hours in enzymolysis solution B in embodiment 2;

[0044] Figure 6 The present application shows the protoplast microscope result graph of yew stem enzymolysis for 4 hours in enzymolysis solution A in embodiment 2;

[0045] Figure 7 The protoplast microscopy results of yew stems enzymatically hydrolyzed in enzymatic hydrolyzate B for 4 hours in Example 2 of the present invention are shown;

[0046] Figure 8 The protoplast microscopy results of yew stems enzymatically hydrolyzed in enzymatic hydrolyzate A-1 for 3 hours in Example 3 of the present invention are shown;

[0047] Figure 9 The protoplast microscopy results of yew stems enzymatically hydrolyzed in enzymatic hydrolyzate A-2 for 3 hours in Example 3 of the present invention are shown;

[0048] Figure 10 The protoplast microscopy results of yew stems enzymatically hydrolyzed in enzymatic hydrolyzate A-1 for 4 hours in Example 3 of the present invention are shown;

[0049] Figure 11 The protoplast microscopy results of yew stems enzymatically hydrolyzed in enzymatic hydrolyzate A-2 for 4 hours in Example 3 of the present invention are shown;

[0050] Figure 12 The protoplasm microscopy results of yew stems obtained by vacuum filtration for 10 minutes and enzymatic hydrolysis for 1 hour in Example 4 of the present invention are shown;

[0051] Figure 13 The protoplasm microscopy results of yew stems obtained by vacuum filtration for 30 minutes and enzymatic hydrolysis for 1 hour in Example 4 of the present invention are shown;

[0052] Figure 14 The protoplasm microscopy results of yew stems obtained by vacuum filtration for 60 minutes and enzymatic hydrolysis for 1 hour in Example 4 of the present invention are shown;

[0053] Figure 15 The protoplasm microscopy results of yew stems subjected to vacuum filtration for 30 minutes and then enzymatic hydrolysis for 4 hours in Example 4 of the present invention are shown;

[0054] Figure 16 The protoplasm microscopy results of yew stems subjected to vacuum filtration for 60 minutes and then enzymatic hydrolysis for 4 hours in Example 4 of the present invention are shown;

[0055] Figure 17 The microscopic results of the protoplasm after the yew stems were purified with iodixanol solution in Example 5 of the present invention are shown;

[0056] Figure 18 The microscopic results of protoplasm from yew stems not purified with iodixanol solution in Example 5 of the present invention are shown;

[0057] Figure 19 The microscopic results of the protoplasts of the yew stems in Example 6 of the present invention are shown;

[0058] Figure 20 A prokaryotic microscopic counting result chart of Taxus yunnanensis stem of the present application embodiment 6 is shown;

[0059] Figure 21 A prokaryotic single cell transcriptome sequencing result of Taxus yunnanensis stem of the present application embodiment 6 is shown;

[0060] Figure 22 A prokaryotic microscopic counting result chart of Taxus yunnanensis leaf of the present application embodiment 7 is shown;

[0061] Figure 23 A prokaryotic microscopic counting result chart of Taxus yunnanensis leaf of the present application embodiment 7 is shown;

[0062] Figure 24 A prokaryotic single cell transcriptome sequencing result of Taxus yunnanensis leaf of the present application embodiment 7 is shown. DETAILED DESCRIPTION

[0063] Unless otherwise indicated, all parts and percentages expressed herein are based upon weight and all tests and measurements are conducted synchronously with the filing date of the present application. To the extent that any patent, patent application, or publication is cited in this application, the same is hereby incorporated by reference in its entirety into the present application, and the equivalent same family of patents is hereby incorporated by reference, particularly with respect to the definitions of synthetic techniques, products and processing designs, polymers, comonomers, initiators or catalysts, etc. in the art disclosed in these documents. If the definition of a specific term in the prior art is inconsistent with any definition provided in the present application, the term is defined as provided in the present application.

[0064] Numerical ranges expressed in the present application as comprising one or more endpoints are approximate, and thus can include numbers that are not exact endpoints. A range includes all values from the lower to the upper value, incremented by one unit, unless otherwise stated. For example, if a component, physical or other property (such as a molecular weight, melt index, etc.) is stated to be from 100 to 1000, it is intended that all individual values, such as 100, 101, 102, etc., and sub-ranges, such as 100 to 166, 155 to 170, 198 to 200, etc., are expressly enumerated. For ranges containing values less than one or containing fractional numbers (such as 1.1, 1.5, etc.), one unit is considered to be 0.0001, 0.001, 0.01 or 0.1 as appropriate. For ranges containing fewer than ten (e.g., 1 to 5) decimal digits, one unit is typically considered to be 0.1. These are only examples of what is meant by a range, and all possible combinations of numerical values between the lowest and highest value enumerated, are to be considered to be expressly stated in the present application.

[0065] When used with respect to chemical compounds, unless expressly stated otherwise, the singular includes all isomeric forms and vice versa (e.g., "hexane" includes all isomers of hexane, individually or collectively). In addition, nouns using "a," "an," or "the" also include their plural forms unless expressly stated otherwise.

[0066] The terms "comprising", "including", "having" and their derivatives do not exclude the presence of any other components, steps or processes, and are irrelevant to whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, all compositions using the terms "comprising", "including", or "having" in this application may include any additional additives, excipients or compounds unless expressly stated otherwise. In contrast, the term "essentially consisting of" excludes any other components, steps or processes from the scope of any subsequent description of the term, except those necessary for operational performance. The term "consisting of" does not include any components, steps or processes that are not specifically described or listed. Unless expressly stated otherwise, the term "or" refers to the listed members alone or in any combination.

[0067] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments.

[0068] Example

[0069] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.

[0070] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials they cite are hereby incorporated by reference.

[0071] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.

[0072] Example 1

[0073] (1) Preparation of enzymatic hydrolysate: The enzymatic hydrolysate was prepared as shown in Table 1 below.

[0074] Table 1. Preparation of enzymatic hydrolysate

[0075]

[0076]

[0077] (2) The preparation process of the Taxus chinensis stem protoplast is as follows:

[0078] S1, 0.5 g of fresh Taxus chinensis stem is taken, washed and then dried;

[0079] S2, the stem is placed in a culture dish, and an enzyme solution is added, the volume-to-mass ratio of the enzyme solution to the tissue is 10 mL:1 g, and the tissue is cut into about 0.1-0.5 mm filaments on ice with a blade;

[0080] S3, vacuum filtration is performed for 30 min, and then the enzyme hydrolysis is performed for 4 hours at 28°C in the dark, the shaking frequency is 40 rpm, 10 μL of the enzyme solution is taken every hour to observe the protoplast release result under a microscope, and the result is shown in Figures 1-3 .

[0081] The experimental result is as follows: no protoplast is released under a microscope after 1 and 2 hours of enzyme hydrolysis, a small amount of protoplast is released after 3 hours of enzyme hydrolysis, but the protoplast morphology is broken, and part of the protoplast is released after 4 and 5 hours of enzyme hydrolysis, but the morphology is not complete and part of the protoplast is broken, and the enzyme amount in the enzyme solution needs to be increased to accelerate the enzyme hydrolysis and digestion to ensure the release of the protoplast.

[0082] Example 2

[0083] (1) Preparation of the enzyme solution: the enzyme solution A and the enzyme solution B are prepared according to Table 2 and Table 3 shown below

[0084] Table 2, preparation of the enzyme solution A

[0085]

[0086]

[0087] Table 3, preparation of the enzyme solution B

[0088]

[0089] (2) The preparation process of the Taxus chinensis stem protoplast using the enzyme solution A and the enzyme solution B is as follows:

[0090] The enzyme solution A and the enzyme solution B are used respectively to perform the experiment according to the preparation process of the Taxus chinensis stem protoplast in Example 1, 10 μL of the enzyme solution is taken every hour to observe the protoplast release result under a microscope, and the result is shown in Figures 4-7 .

[0091] The experimental results are as follows: after enzymolysis for 1, 2 and 3 hours, some protoplasts are released in the enzyme solution A and B, and the protoplasts are in a complete state; after enzymolysis for 4 and 5 hours, the protoplasts are obviously released in the enzyme solution A and B, and there is no significant difference in the number of the protoplasts between the two enzyme solutions, but the protoplasts are broken. It is indicated that the enzyme components in the enzyme solution are sufficient for the enzymolysis and digestion of the cell wall and the cell, but the osmotic pressure is too low, which leads to the easy breaking of the protoplasts.

[0092] Example 3

[0093] (1) Preparation of the enzyme solution: the enzyme solution A-1 and the enzyme solution A-2 are prepared according to Table 3' and Table 4 shown below

[0094] Table 3', Preparation of the enzyme solution A-1

[0095]

[0096] Table 4, Preparation of the enzyme solution A-2

[0097]

[0098]

[0099] (2) Preparation of the Taxus stem protoplasts by using the enzyme solution A-1 and the enzyme solution A-2

[0100] The enzyme solution A-1 and the enzyme solution A-2 are used respectively to carry out the preparation process of the Taxus stem protoplasts according to the preparation process of the Taxus stem protoplasts in Example 1, 10 μL of the enzyme solution is taken every hour to observe the release of the protoplasts under a microscope, and the results are shown in Table 6. Figures 8-11 .

[0101] The experimental results are as follows: after enzymolysis for 1, 2 and 3 hours, the amount of the released protoplasts in the enzyme solution A-1 and the enzyme solution A-2 is not different and is a small amount, and the amount is less than that in Example 2; after enzymolysis for 4 hours, the amount of the protoplasts in the enzyme solution A-2 is obviously more than that in the enzyme solution A-1, and the protoplasts are in a complete state. It is indicated that the osmotic pressure in the enzyme solution A-1 is slightly high, which is not conducive to the penetration of the enzyme solution into the intercellular space and the intercellular enzymolysis and digestion; the enzyme solution A-2 can ensure the protoplast form and does not obviously hinder the penetration of the enzyme solution, and the penetration of the enzyme solution can be improved by vacuum filtration.

[0102] Example 4

[0103] (1) Preparation of the enzyme solution: the enzyme solution is prepared according to Table 5 shown below

[0104] Table 5, Preparation of the enzyme solution

[0105]

[0106]

[0107] (2) The preparation process of yew stem protoplasts using enzyme solution:

[0108] S1, 0.5 g of fresh yew stem was taken respectively, washed and then absorbed water;

[0109] S2, the stems were placed in a culture dish respectively, and an appropriate amount of enzyme solution was added, the volume-to-mass ratio of enzyme solution to tissue was 10:1 (ml / g), and the tissue was cut into about 0.1-0.5 mm filaments on ice with a blade,

[0110] S3, vacuum filtration for 60 min, 30 min and 10 min respectively, and then enzymolysis for 4 hours at 28°C in the dark, the shaking frequency was 40 rpm, and the enzyme solution was taken every hour to observe the release of protoplasts under a microscope, as shown in Figures 12-16 .

[0111] Experimental results: after 3 and 4 hours of enzymolysis, the number of protoplasts after vacuum filtration for 30 min and 60 min was more than that after vacuum filtration for 10 min; but after 4 hours of enzymolysis, there was no obvious difference in the morphology and number of protoplasts after vacuum filtration for 60 min and 30 min, and considering the preparation time, protoplast release speed and protoplast morphology, vacuum filtration for 30 min was selected.

[0112] Example 5

[0113] (1) Preparation of enzyme solution and washing solution: the enzyme solution and washing solution were prepared according to Table 6 and Table 7 as shown below;

[0114] Table 6, preparation of enzyme solution

[0115]

[0116]

[0117] Table 7, preparation of washing solution

[0118]

[0119] Preparation of purified reagent: iodixanol and nuclease were mixed according to the volume ratio of iodixanol to nuclease 1:1 to obtain an iodixanol solution.

[0120] (2) The preparation method of fresh yew stem protoplasts includes a preparation process and a purification process, and the specific steps are as follows:

[0121] I. Preparation process:

[0122] S1, 1.0 g of fresh yew stem or leaf was taken, washed and then absorbed water;

[0123] S2, place the stems in a culture dish, add enzyme solution, the volume to mass ratio of enzyme solution to tissue is 10 mL: 1 g, cut the fresh Taxus stems, leaves and other tissues into about 0.1-0.5 mm filaments on ice with a blade;

[0124] S3, vacuum filtration for 30 min, then shake and enzymatically digest for 4 hours at 28°C in the dark, with a shaking frequency of 40 rpm, to obtain the enzymatically digested tissue;

[0125] II. Purification process:

[0126] S4, filter the enzymatically digested tissue using a 200-mesh filter screen, then centrifuge at 4°C at 300 rpm for 5 min and discard the supernatant, to obtain the precipitate;

[0127] S5, add a washing solution to the precipitate obtained in step S4 to resuspend it, to obtain a protoplast resuspension; wherein the volume to mass ratio of the washing solution to the precipitate is 10 mL: 1 g;

[0128] S6, take another centrifuge tube, add an iodixanol solution of the same volume as the resuspension, and gently pour the protoplast resuspension into the iodixanol solution to obtain a mixture;

[0129] S7, centrifuge the mixture at 4°C at 400 rpm for 5 min, take 10 μL from each layer, and observe them under a microscope, respectively, to see Figures 17-18 .

[0130] Experimental results: the protoplasts purified using a 50% iodixanol solution are concentrated in the middle layer and have fewer impurities; the unpurified protoplasts and impurities cannot be separated.

[0131] Example 6 Preparation, purification and single-cell transcriptome sequencing of fresh Taxus stem protoplasts

[0132] (1) Preparation of enzyme solution and washing solution: prepare the enzyme solution and washing solution according to Tables 8 and 9, respectively;

[0133] Table 8, preparation of enzyme solution

[0134]

[0135] Table 9, preparation of washing solution

[0136]

[0137] (2) Preparation method of fresh Taxus stem protoplasts, the specific steps are as follows:

[0138] I. Preparation process:

[0139] S1, take 0.5 g of fresh Taxus stems, wash and then absorb the water;

[0140] S2, the stem or leaf tissue and the like are placed in a culture dish, and an enzymatic solution is added, with a volume-mass ratio of the enzymatic solution to the tissue being 10 mL:1 g. The fresh Taxus stem, leaf and the like are cut into filaments of about 0.1-0.5 mm on ice using a blade;

[0141] S3, vacuum filtration is performed for 30 min, and then the enzymatic hydrolysis is performed for 4 hours at 28°C in the dark with shaking at a frequency of 40 rpm, to obtain the enzymatically hydrolyzed tissue;

[0142] II. Purification process:

[0143] S4, the enzymatically hydrolyzed tissue is filtered using a 200-mesh filter screen, and then centrifuged at 300 rpm for 5 min at 4°C, and the supernatant is discarded, to obtain a precipitate;

[0144] S5, the precipitate obtained in step S4 is resuspended by adding a washing solution, to obtain a protoplast resuspension; wherein the volume-mass ratio of the washing solution to the precipitate is 10 mL:1 g;

[0145] S6, another centrifuge tube is taken, and an equal volume of iodixanol solution is added to the protoplast resuspension, and the protoplast resuspension is gently added to the iodixanol solution, to obtain a mixture;

[0146] S7, the mixture is centrifuged at 400 rpm for 5 min at 4°C, and the middle protoplast layer is taken and added to a washing solution; wherein the volume ratio of the washing solution to the protoplast layer is about 10:1;

[0147] S8, centrifugation is performed at 300 rpm for 5 min at 4°C, and then the supernatant is discarded, and the precipitate is resuspended by adding a 8% mannitol solution, to obtain the protoplasts of the Taxus stem; wherein the volume-mass ratio of the resuspension to the protoplasts is 10 mL:1 g;

[0148] The protoplasts are observed under a microscope to observe the protoplast morphology, and are counted using a trypan blue microscope.

[0149] (3) 20,000 of the protoplasts prepared above are subjected to single-cell transcriptome sequencing, and the results are shown in Table 3. Figures 19-21 .

[0150] Experimental results: The enzymatically released and purified protoplasts are morphologically complete, and the number and integrity of the protoplasts meet the requirements of single-cell transcriptome sequencing; the sequencing results show that 8,469 cells are actually captured, with an average number of Reads being 11,661, and the proportion of high-quality reads being 94.5%.

[0151] Example 7: Preparation and purification of fresh Taxus leaf protoplasts and single-cell transcriptome sequencing

[0152] (2) Preparation of enzymatic hydrolysis solution and cleaning solution: The enzymatic hydrolysis solution and cleaning solution were prepared as shown in Table 10 and Table 11 below, respectively;

[0153] Table 10, Preparation of Enzyme Hydrolysate

[0154]

[0155] Table 11. Preparation of cleaning solution

[0156]

[0157] (2) Preparation method of fresh yew leaf protoplasts, the specific steps are as follows:

[0158] 1. Preparation process:

[0159] S1, take 0.5g fresh yew leaves, wash and dry them;

[0160] S2, placing leaves and other tissues in a culture dish, adding enzymatic hydrolysis solution, with the volume-to-mass ratio of enzymatic hydrolysis solution to tissue being 10 mL:1 g, and cutting fresh yew stems, leaves and other tissues into approximately 0.1-0.5 mm thin strips with a blade on ice;

[0161] S3, vacuum filtration for 30 min, followed by enzymatic hydrolysis at 28°C in the dark for 4 h at a shaking frequency of 40 rpm to obtain the enzymatically hydrolyzed tissue;

[0162] 2. Purification process:

[0163] S4, filter the enzymatically hydrolyzed tissue using a 200-mesh filter, then centrifuge at 300 rpm for 5 min at 4°C and discard the supernatant to obtain the precipitate;

[0164] S5, adding a cleaning solution to the precipitate obtained in step S4 to resuspend the precipitate to obtain a native body resuspension; wherein the volume mass ratio of the cleaning solution to the precipitate is cleaning solution:precipitate = 10 mL:1 g;

[0165] S6, take another centrifuge tube, add the same volume of iodixanol solution as the resuspension, and gently add the native body resuspension to the iodixanol solution at an angle to obtain a mixture;

[0166] S7, centrifuging the mixture at 400 rpm for 5 min at 4°C, taking the middle protoplast layer, and adding a washing solution; wherein the volume ratio of the washing solution to the protoplast layer is about 10:1;

[0167] S8, centrifuging at 300 rpm for 5 min at 4°C, discarding the supernatant, and resuspending the precipitate in 8% mannitol solution to obtain the yew stem protoplasts; wherein the volume-to-mass ratio of the resuspension solution to the protoplasts is 10 mL:1 g;

[0168] The protoplasts were observed under a microscope, and the protoplast morphology was observed and counted using trypan blue.

[0169] (3) 20,000 protoplasts prepared above were subjected to single-cell transcription sequencing, and the results are shown in Table 3. Figures 22-24 .

[0170] Experimental results: The enzyme-released and purified protoplasts were morphologically intact, and the number and integrity of the protoplasts met the requirements of single-cell transcription sequencing; the sequencing results showed that 7,339 cells were actually captured, the average number of cell reads was 35,251, and the proportion of high-quality reads was 93.2%.

[0171] All the documents mentioned in the present application are incorporated herein by reference as if each document were individually incorporated by reference. In addition, it should be understood that various changes and modifications can be made to the present application by those skilled in the art upon reading the above description of the present application, and such equivalent forms are also within the scope of the appended claims.

Claims

1. An application of fresh yew stem and leaf protoplasts in single-cell transcriptome sequencing, characterized in that: The method for obtaining fresh yew stem and leaf protoplasts includes a preparation process and a purification process, and the specific steps are as follows:

1. Preparation process: S1, take 0.5-1g of fresh yew tissue, wash it and dry it; S2, fresh yew tissue was placed in a culture dish, enzyme solution was added, and fresh yew tissue was cut into thin strips of about 0.1-0.5 mm using a blade on ice; S3, vacuum filtration for 30 min, followed by enzymatic hydrolysis at 28°C in the dark for 4 h at a shaking frequency of 40 rpm to obtain the enzymatically hydrolyzed tissue; The enzymatic hydrolysis solution includes 0.40-0.50 parts by weight of cellulase, 0.20-0.30 parts by weight of hemicellulase, 0.10-0.15 parts by weight of macerate, 2.5-3.0 parts by weight of mannitol, 0.65-0.70 parts by weight of potassium chloride, 1.90-1.95 parts by weight of MES, 0.4-0.6 parts by weight of calcium chloride, 0.5-3.5 parts by weight of BSA and 0.014-0.020 parts by weight of β-mercaptoethanol; 2. Purification process: S4, filter the enzymatically digested tissue using a 200-mesh filter, then centrifuge at 4°C and discard the supernatant to obtain a precipitate; S5, adding a cleaning solution to the precipitate obtained in step S4 to resuspend it, thereby obtaining a native body resuspension; S6, take another centrifuge tube, add the same volume of purification reagent as the resuspension, and add the native body resuspension to the purification reagent at an angle to obtain a mixture; S7, the mixture was centrifuged at 4°C, the middle protoplast layer was taken, and washing solution was added; S8, centrifuge at 4°C, discard the supernatant, and add mannitol solution to resuspend the precipitate to obtain the protoplasts of fresh yew tissue; The cleaning solution includes 0.3-0.5M sodium chloride, 5-15mM calcium chloride, 15-25mM potassium chloride and 5-15mM MES; The purification reagent includes iodixanol solution.

2. The use according to claim 1, characterized in that In step S2, the volume mass ratio of the enzymatic hydrolyzate to fresh yew tissue is 10 mL:1 g; in step S5, the volume mass ratio of the cleaning solution to the precipitate is cleaning solution:precipitate = (5-15) mL:1 g; in step S7, the volume mass ratio of the cleaning solution to the protoplast layer is 10:1; in step S8, the volume mass ratio of the resuspension solution to the protoplast is 10 mL:1 g.

3. The use according to claim 1, characterized in that The iodixanol solution is prepared by mixing iodixanol and nuclease-free water in a volume ratio of 1:1.

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