Plant single cell suspension, preparation method and application thereof

Abstract

The present invention relates to the preparation of plant single cells, and specifically to a plant single cell suspension, a preparation method and an application thereof. The preparation method comprises the following steps: (1) selecting plant seedlings with good growth conditions, placing the selected seedlings in a pre-plasmolysis solution, and taking the middle leaf sheath part after the treatment; (2) performing enzymatic hydrolysis on the treated leaf sheath part protoplast hydrolysate; (3) purifying the protoplasts after the enzymatic hydrolysis to obtain a plant single cell suspension. The present invention can efficiently and massively prepare plant protoplasts, and the prepared protoplasts have a high survival rate and can be used for subsequent research. For example, plant biological breeding, protein subcellular localization, protein purification and other aspects.

Description

Technical Field

[0001] The present invention relates to the field of plant single cells, and mainly to a plant single cell suspension, a preparation method and an application thereof. Background Art

[0002] Plant protoplast refers to the special protoplasm part within the plant cell wall, that is, the part of the cell material that can be separated from the cell wall through plasmolysis. In other words, it is the "naked cell" wrapped by the plasma membrane obtained after decomposing the plant cell wall by a certain method.

[0003] Plant protoplasts are generally complicated to operate due to the lack of cell wall protection. However, it is precisely because of the special state of protoplasts without cell walls that they are easier to absorb external genetic material. This characteristic makes protoplasts have a wide range of application value. At present, the protoplast system has been widely used in the research of physiology, biochemistry, genetics, molecular biology, genomics, proteomics, and metabolomics. Therefore, it is very important to obtain protoplasts with high survival rate and high activity. Summary of the invention

[0004] In view of the deficiencies of the prior art, the present invention aims to provide a plant single cell suspension, a preparation method and applications thereof.

[0005] In a first aspect, the present invention provides a method for preparing a plant single cell suspension, and the specific preparation steps are as follows:

[0006] S1. Cut plant seedlings with good growth and a length of 1-6 cm, and place the plant seedlings in the pre-plasmolysis treatment solution (the pre-plasmolysis treatment solution should be sufficient to immerse the plant seedlings) for 5-20 minutes under dark conditions, wherein the plant seedlings can be wheat seedlings, corn seedlings, etc.;

[0007] S2. After the pre-plasmolysis is completed, the precipitate is filtered and collected to obtain the pre-enzyme material, and the pre-enzyme material is added to the enzymolysis solution for enzymolysis in a dark environment for 2 to 5 hours;

[0008] S3. Mix the enzymatic solution with W5 washing solution, centrifuge for 1-2 times, and collect the precipitate. Mix the precipitate with W5 washing solution to obtain a purified single cell suspension. The formula of W5 washing solution is: CaCl 2 ·2H 2 O 125.0mmol / L, NaCl 154.0mmol / L, KCl 5.0 mmol / L, glucose 5.0 mmol / L, MES 5.0 mmol / L, pH 5.8.

[0009] The pre-plasmolysis solution in step S1 is a dispersion of 0.25-0.5 M mannitol and 0.4-1.2% proline-sodium alginate-zein nanoparticles, wherein the volume ratio of the two is 2.5:1.

[0010] The preparation method of proline-sodium alginate-zein nanoparticles is as follows: 50 mg of proline and 1.0 g of zein are dissolved in 100 ml of 70% ethanol solution by mass, 100 mg of sodium alginate is weighed and dissolved in 300 ml of distilled water, the prepared proline ethanol solution is dripped into the sodium alginate solution at a rate of 0.1 ml / min, and stirring is performed while dripping. After the dripping is completed, the ethanol is evaporated by a rotary evaporator, and the nanopowder is obtained by freeze-drying.

[0011] The solute and concentration of the enzymolysis solution in step S2 are 0.25-0.7M mannitol, 0.8%-2.1% cellulase, 0.8%-1.5% pectinase, 15-25mM MES, 5-15mM calcium chloride, 8-25mM potassium chloride, 0.6%-2% ascorbic acid, 0.4%-1.2% proline-sodium alginate-zein nanoparticles, the solvent is water, and the pH is adjusted to 5.6-5.8. Cellulase and pectinase are purchased from Beijing Solebow Technology Co., Ltd., and ascorbic acid is purchased from Shanghai McLean Biochemical Technology Co., Ltd.

[0012] The weight-to-volume ratio of the pre-enzymatic hydrolysis material to the enzymatic hydrolysis solution in step S2 is 1 g:4 ml.

[0013] In step S3, the volume ratio of the enzymatic hydrolysis solution to the W5 washing solution is 1: (1-3), and the volume ratio of the obtained precipitate mass to the W5 washing solution is 1g: (3-4)ml. The centrifugation temperature is 3-5°C, the rotation speed is 600-800rpm, and the centrifugation time is 1-2min.

[0014] In the second aspect, the present invention can efficiently and massively prepare plant protoplasts, and the prepared protoplasts have a high survival rate and can be used for subsequent research, such as plant biological breeding, protein subcellular localization, protein purification, etc.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] 1. Pre-plasmolysis treatment can make the inner and outer layers of the cell wall fully contact with the enzymatic hydrolysis solution, thereby increasing the enzymatic hydrolysis rate. However, pre-plasmolysis treatment will inevitably cause the death of plant cells. Therefore, adding nanomaterials to the pretreatment solution can effectively reduce cell death and improve the survival rate of plant cells.

[0017] 2. Add ascorbic acid to the enzymatic hydrolysis solution. Adding an appropriate concentration of ascorbic acid can remove ROS generated during the enzymatic hydrolysis process, which can cause protoplast exhaustion, decreased vitality, and death. At the same time, the added nanomaterials can regulate the expression of related genes to achieve an antioxidant effect, while also improving the integrity of the cell membrane and protecting the cell membrane structure during the enzymatic hydrolysis process. The two can act at the same time to enhance the antioxidant capacity of cells and reduce the loss of electrolytes in the cell membrane caused by the enzymatic hydrolysis process, thereby improving the survival rate of plant cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The figure is a bar graph showing the number of protoplasts in each sample.

[0019] Figure 2 The graph shows the protoplast activity results in each sample.

[0020] Figure 3 This is the result graph of malondialdehyde content in protoplasts.

[0021] Figure 4 This is a characterization diagram of the proline-sodium alginate-zein nanoparticles prepared in Example 1. DETAILED DESCRIPTION

[0022] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the following embodiments and features in the embodiments can be combined with each other without conflict.

[0023] Example 1

[0024] A plant single cell suspension, the specific preparation steps of which are as follows:

[0025] S1. Cut off the leaves of wheat seedlings with good growth and length of 5 cm, remove the roots and leaf tips, and place the remaining parts in the pre-plasmolysis solution in the dark for 15 minutes;

[0026] S2. After the pre-plasmolysis is completed, the precipitate is filtered and collected to obtain the pre-enzyme material, and the pre-enzyme material is added to the enzymolysis solution for enzymolysis in a dark place for 3 hours;

[0027] S3. The enzymatic solution was mixed with W5 washing solution, centrifuged twice, and the precipitate was collected. The precipitate was mixed with W5 washing solution to obtain a purified single cell suspension. The formula of W5 washing solution is: CaCl2 ·2H 2 O 125.0mmol / L, NaCl 154.0mmol / L, KCl 5.0 mmol / L, glucose 5.0 mmol / L, MES 5.0 mmol / L, pH 5.8.

[0028] The pre-plasmolysis solution in step S1 is a dispersion of 0.4M mannitol and 0.8% proline-sodium alginate-zein nanoparticles, wherein the volume ratio of the two is 2.5:1.

[0029] The preparation method of proline-sodium alginate-zein nanoparticles is as follows: 50 mg of proline and 1.0 g of zein are dissolved in 100 ml of 70% ethanol solution by mass to obtain a proline-zein ethanol solution, 100 mg of sodium alginate is weighed and dissolved in 300 ml of distilled water, and the prepared proline-zein ethanol solution is dripped into the sodium alginate solution at a rate of 0.1 ml / min, while stirring while dripping, and after the dripping is completed, the ethanol is evaporated by a rotary evaporator, and then freeze-dried to obtain a nano powder; the obtained proline-sodium alginate-zein powder is ultrasonically dispersed in water to prepare a 0.8% proline-sodium alginate-zein dispersion. The SEM of the proline-sodium alginate-zein nanoparticles obtained in this example is as follows Figure 4 As shown. Figure 4 It can be seen from the SEM image that the particle size of the proline-sodium alginate-zein nanoparticles is 200-300nm, the nanoparticles are spherical, and the size distribution is uniform, which shows that the obtained nanoparticles have strong stability and can be better used in the preparation of single cell suspensions.

[0030] The solute and concentration of the enzymatic hydrolyzate in step S2 are 0.5M mannitol, 1.3% cellulase, 1.2% pectinase, 18mM MES, 11mM calcium chloride, 20mM potassium chloride, 1.3% ascorbic acid, 0.8% proline-sodium alginate-zein nanoparticles, the solvent is water, and the pH is adjusted to 5.6.

[0031] The weight-to-volume ratio of the pre-enzymatic material to the enzymatic solution described in step S2 is 1 g:4 ml.

[0032] In step S3, the volume ratio of the enzymatic hydrolysis solution to the W5 washing solution is 1:2, and the volume ratio of the obtained precipitate mass to the W5 washing solution is 1g:3ml. The centrifugation temperature is 3°C, the speed is 800rpm, and the centrifugation time is 2min. The zein in this experiment was purchased from Tianjin Solomon Biotechnology Co., Ltd.; L-proline, sodium alginate, etc. were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0033] Example 2

[0034] The preparation method of Example 2 is substantially the same as that of Example 1, except that the mass fraction of the mixed solution loaded with proline-sodium alginate-zein nanoparticles added to the pre-plasmolysis solution and the enzymatic hydrolysis solution is 1.0%.

[0035] Comparative Example 1

[0036] The preparation method of Comparative Example 1 is basically the same as that of Example 1, except that 0.4 M mannitol and 0.8% dispersion of sodium alginate-zein nanoparticles are added to the pre-plasmolysis solution in step S1 of Comparative Example 1.

[0037] The preparation method of the dispersion is as follows: 1.0g of zein is dissolved in 100ml of 70% ethanol solution, 100mg of sodium alginate is weighed and dissolved in 300ml of distilled water, the prepared zein ethanol solution is dripped into the sodium alginate solution at a rate of 0.1ml / min, and stirring is performed while dripping. After the dripping is completed, the ethanol is evaporated by a rotary evaporator, and the nano powder is obtained by freeze-drying; the obtained sodium alginate-zein powder is ultrasonically dispersed in water to prepare a 0.8% sodium alginate-zein dispersion.

[0038] Comparative Example 2

[0039] The preparation method of Comparative Example 2 is basically the same as that of Example 1, except that the pre-plasmolysis solution in step S1 of Comparative Example 2 consists of 0.4 M mannitol and 0.8% proline aqueous solution, wherein the volume ratio of the two is 2.5:1.

[0040] Comparative Example 3

[0041] The preparation method of Comparative Example 3 is substantially the same as that of Example 1, except that ascorbic acid is not added to the components of the enzymatic hydrolysate in step S2 of the comparative example.

[0042] Comparative Example 4

[0043] The preparation method of Comparative Example 4 is substantially the same as that of Example 1, except that no proline-sodium alginate-zein nanoparticles are added to the components of the enzymatic hydrolysate in step S2 of the comparative example.

[0044] Comparative Example 5

[0045] The preparation method of Comparative Example 5 is basically the same as that of Example 1, except that the proline-sodium alginate-zein nanoparticles are replaced with sodium alginate-zein powder of the same mass in the composition of the enzymatic hydrolyzate in step S2 of the comparative example. The preparation method is the same as that in Comparative Example 1.

[0046] Comparative Example 6

[0047] The preparation method of Comparative Example 6 is basically the same as that of Example 1, except that only 0.4 M mannitol is added to the composition of the pre-plasmolysis solution in step S1 of Comparative Example 6.

[0048] In step S2, no ascorbic acid and no proline-sodium alginate-zein nanoparticles are added to the enzymatic hydrolysate.

[0049] Wheat protoplast number detection

[0050] (1) Sample selection

[0051] The wheat single cell suspension prepared in Examples 1-2 and Comparative Examples 1-6 of the present invention.

[0052] (2) Experimental methods

[0053] Take 1 ml of wheat single cell suspension and add 10 times the volume of W5 solution to fully suspend it. Take a small amount of suspension and drop it into the counting slot of 0.1 mm, 25×16 type blood cell counting plate. Observe under an ordinary optical microscope and count the number of protoplasts in the upper left, lower left, upper right, lower right and middle 5 squares (a total of 80 small squares). According to the formula: protoplast number / mL = protoplast number in 80 small squares / 80×400×10 4 × dilution factor to calculate the total amount of protoplasts. Each sample was counted 3 times and 3 samples were taken for each experiment.

[0054] (3) Experimental results

[0055] Table 1 Number of wheat protoplasts in each sample

[0056]

[0057] Table 1 shows the number of wheat protoplasts in each sample. It can be clearly seen from the data in the table that the number of protoplasts in Example 1-2 is greater than the number of protoplasts in other samples. Among them, the number of protoplasts in the comparative example 1 in which the pre-plasmolysis solution was replaced with alginate-zein nanoparticle dispersion was (1.6±0.2)×10 7 , and the number of protoplasts in Comparative Example 2 was (1.7±0.2)×10 7 , the number of protoplasts in Comparative Examples 1 and 2 is much smaller than that in Example 1. As can be seen from the data, the proline-sodium alginate-zein nanoparticle dispersion is helpful for the pre-plasmolysis process. The reason may be that the nanoparticles can enter the interior of the plant cell through the cell membrane, protect the cell membrane, and prevent the imbalance of osmotic pressure inside and outside the plant cell during the pre-plasmolysis of the cell, causing cell death. Secondly, from the data comparison of Comparative Examples 3 to 6 and Example 1, it can be seen that adding ascorbic acid and proline-sodium alginate-zein nanoparticle dispersion in the enzymolysis solution is helpful for the preparation of protoplasts. This and ascorbic acid can regulate the expression of antioxidant genes and regeneration genes, can better ensure the preparation of protoplasts, and ascorbic acid and proline-sodium alginate-zein nanoparticle dispersion have a synergistic effect, and both protect the cell membrane from being destroyed at the same time.

[0058] Protoplast activity assay

[0059] (1) Sample selection

[0060] The wheat single cell suspension prepared in Examples 1-2 and Comparative Examples 1-6 of the present invention.

[0061] (2) Experimental methods

[0062] Protoplast viability was determined by staining with 0.01% fluorescein diacetate (FDA), and the number of protoplasts emitting green fluorescence and the total number of protoplasts were counted using a fluorescence phase contrast microscope (Zeiss, Axio Imager A1). Protoplast viability was expressed as the percentage of viable protoplasts in a field of view to the total number of protoplasts in the field of view. Three representative fields of view were selected for statistics, and the average value was taken.

[0063] Protoplast viability = (number of protoplasts emitting green fluorescence / total number of protoplasts) × 100%.

[0064] (3) Experimental results

[0065] Table 2 shows the wheat protoplast activity in each sample

[0066]

[0067] Table 2 shows the activity of wheat protoplasts in each sample. Protoplasts have consistency and are relatively uniform single-cell populations with cell walls removed. Protoplasts are in the same separation cycle, and the expression of the introduced exogenous genes has good synchronization; at the same time, long-term tissue culture is not required, and the preparation and detection process only takes 2 days. The protoplast transient expression system provides a convenient and effective experimental system for studying the regulation of gene subcellular localization and gene expression, so the activity of protoplasts is crucial. From the data in Table 2, it can be seen that the protoplast activity of Example 1-2 is higher than that in the comparative example, which is related to the properties of ascorbic acid. Ascorbic acid can remove the active oxygen and polyunsaturated fatty acid free radicals produced in plants, and can also maintain redox balance, which helps to enhance cell activity. Secondly, proline-sodium alginate-zein nanoparticles enter between the cell wall and the cell membrane, and the proline released by them synergizes with ascorbic acid to ensure the internal balance of plant cells, so that wheat protoplasts maintain a high cell activity.

[0068] Malondialdehyde content determination

[0069] (1) Sample selection

[0070] The wheat single cell suspension prepared in Examples 1-2 and Comparative Examples 1-6 of the present invention.

[0071] (2) Experimental methods

[0072] Weigh the same mass of wheat single-cell suspension, extract it with 5% trichloroacetic acid, boil it in a water bath (add 0.67% TBA), take the supernatant and measure the absorbance at 450nm, 532nm, and 600nm using a spectrophotometer to calculate the MDA content.

[0073] Experimental Results

[0074] Table 3 shows the malondialdehyde content of wheat protoplasts in each sample

[0075]

[0076] Table 3 shows the malondialdehyde content of wheat protoplasts in each sample. MDA is one of the most important products of plant cell membrane lipid peroxidation. Increased MDA content can aggravate the damage of cell membrane. Therefore, MDA content is often used as an important indicator in plant aging physiology and resistance physiology research. From the data of comparative examples 1-6, it can be seen that ascorbic acid and proline-sodium alginate-zein nanoparticle dispersion can remove the biological toxicity of some chemical agents. This is because biological toxicity is caused by the destruction of cell membrane integrity, damage to organelles such as chloroplasts and mitochondria due to membrane lipid peroxidation, and oxidative stress can also cause factors such as protein and nucleic acid denaturation gene toxicity. Ascorbic acid and proline-sodium alginate-zein nanoparticle dispersion work together to protect the integrity of cell membranes and the normal operation of organelles such as chloroplasts and mitochondria.

Claims

1. A method for preparing a plant single cell suspension, characterized in that: The specific preparation steps are as follows: S1. Cut the plant seedlings with good growth and length of 1-6 cm, and place the plant seedlings in the pre-plasmolysis solution in the dark for 5-20 minutes; S2. After the pre-plasmolysis is completed, the precipitate is filtered and collected to obtain the pre-enzyme material, and the pre-enzyme material is added to the enzymolysis solution for enzymolysis in a dark environment for 2 to 5 hours; S3. The enzymatically hydrolyzed solution was mixed with W5 washing solution, and then centrifuged 1-2 times to obtain a precipitate, and the precipitate was mixed with W5 washing solution to obtain a purified single cell suspension; The pre-plasmolysis solution in step S1 is a dispersion of 0.25-0.5M mannitol and 0.4%-1.2% proline-sodium alginate-zein nanoparticles, wherein the volume ratio of the two is 2.5:1; The preparation method of the proline-sodium alginate-zein nanoparticles is as follows: 50 mg of proline and 1.0 g of zein are dissolved in 100 ml of 70% ethanol solution by mass to obtain a proline-zein ethanol solution; 100 mg of sodium alginate is weighed and dissolved in 300 ml of distilled water; the prepared proline-zein ethanol solution is dripped into the sodium alginate solution at a rate of 0.1 ml / min, and the mixture is stirred while dripping; after the dripping is completed, the ethanol is evaporated by a rotary evaporator, and the proline-sodium alginate-zein nanoparticles are obtained by freeze drying; the obtained proline-sodium alginate-zein nanoparticles are ultrasonically dispersed in water to prepare a 0.4%-1.2% proline-sodium alginate-zein dispersion; The formula of the W5 washing solution is: CaCl2·2H2O 125.0 mmol / L, NaCl 154.0 mmol / L, KCl 5.0 mmol / L, glucose 5.0 mmol / L, MES 5.0 mmol / L, pH 5.

8.

2. The method for preparing a plant single cell suspension according to claim 1, characterized in that: The solute of the enzymatic hydrolyzate described in step S2 is 0.25-0.7M mannitol, 0.8%-2.1% cellulase, 0.8%-1.5% pectinase, 15-25mM MES, 5-15mM calcium chloride, 8-25mM potassium chloride, 0.6%-2% ascorbic acid, 0.4%-1.2% proline-sodium alginate-zein nanoparticles, the solvent is water, and the pH is adjusted to 5.6-5.

8.

3. The method for preparing a plant single cell suspension according to claim 1, characterized in that: The weight-to-volume ratio of the pre-enzymatic hydrolysis material to the enzymatic hydrolysis solution in step S2 is 1 g:4 ml.

4. The method for preparing a plant single cell suspension according to claim 1, characterized in that: The volume ratio of the solution after enzymatic hydrolysis in step S3 to the W5 washing solution is 1:(1-3), and the volume ratio of the obtained precipitate mass to the W5 washing solution is 1g:(3-4)ml.

5. The method for preparing a plant single cell suspension according to claim 1, characterized in that: The centrifugation temperature in step S3 is 3-5°C, the rotation speed is 600-800 rpm, and the centrifugation time is 1-2 min.

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