CN119162078A - Plant single cell suspension, preparation method and application thereof - Google Patents
Plant single cell suspension, preparation method and application thereof Download PDFInfo
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- 239000006285 cell suspension Substances 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 24
- 241000196324 Embryophyta Species 0.000 claims abstract description 46
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 9
- 230000007071 enzymatic hydrolysis Effects 0.000 claims abstract description 6
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims abstract description 6
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- 229910052708 sodium Inorganic materials 0.000 claims description 33
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- 229930195725 Mannitol Natural products 0.000 claims description 10
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- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 7
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- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 5
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- NNMHDOLBLHOTNT-WCCKRBBISA-N ethanol;(2s)-pyrrolidine-2-carboxylic acid Chemical compound CCO.OC(=O)[C@@H]1CCCN1 NNMHDOLBLHOTNT-WCCKRBBISA-N 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
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Abstract
本发明涉及到植物单细胞的制备，具体涉及植物单细胞悬液、制备方法及其应用，制备方法包括以下步骤：（1）选取生长状况良好的植物幼苗，将选取的幼苗放入预质壁处理液中，处理后，取中间叶鞘部分；（2）对处理好的叶鞘部分原生质体酶解液进行酶解处理；（3）对酶解后的原生质体进行纯化处理，得到植物单细胞悬液。本发明可高效、大量的制备植物原生质体，制备得到的原生质体存活率高，可供进行后续研究。例如植物生物育种、蛋白亚细胞定位、蛋白纯化等方面。
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 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
The invention relates to the field of single cells of plants, and mainly relates to a single cell suspension of the plants, a preparation method and application thereof.
Background
Plant protoplasts (Protoplast) refer to a specific cytoplasmic fraction within the plant cell wall, i.e., the fraction of cellular material that can be separated from the cell wall by plasma wall separation, i.e., the "naked cells" that are surrounded by plasma membranes after breaking down the plant cell wall by a defined method.
Plant protoplasts generally have complex related operations due to lack of protection of cell walls, but the protoplasts are easier to absorb outside genetic materials due to the fact that the protoplasts do not have the special state of cell walls, and the characteristic makes the protoplasts have wide application values. At present, the protoplast system has been widely applied to the research of physiology, biochemistry, genetics, molecular biology, genomics, proteomics and metabonomics, so that the acquisition of protoplasts with high survival rate and high activity is important.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a plant single-cell suspension, a preparation method and application thereof.
In a first aspect, the invention provides a preparation method of a single-cell suspension of a plant, which comprises the following specific preparation steps:
s1, cutting plant seedlings with good growth conditions and the length of 1-6cm, standing the plant seedlings in a pre-wall treatment liquid (the pre-wall treatment liquid is used for immersing young plants of the plant) for 5-20 min in dark conditions, wherein the plant seedlings can be wheat seedlings, corn seedlings and the like;
s2, filtering after the pretreatment of the pre-material wall is finished, collecting sediment to obtain a pre-enzymolysis material, adding the obtained pre-enzymolysis material into an enzymolysis liquid, and carrying out enzymolysis in the dark for 2-5 hours;
S3, mixing the solution subjected to enzymolysis with the W5 washing liquid, centrifuging for 1-2 times, taking a precipitate, and mixing the precipitate with the W5 washing liquid to obtain the purified single-cell suspension. Wherein. The formula of the W5 lotion comprises CaCl 2·2H2 O125.0 mmol/L, naCl 154.0.0 mmol/L, KCl 5.0.0 mmol/L, glucose 5.0mmol/L, MES 5.0mmol/L and pH 5.8.
The pretreatment liquid in the step S1 is a dispersion liquid of 0.25-0.5M mannitol and 0.4-1.2% proline-sodium alginate-zein nano particles, wherein the volume ratio of the pretreatment liquid to the dispersion liquid is 2.5:1.
The preparation method of the proline-sodium alginate-zein nano-particles comprises the steps of dissolving 50mg of proline and 1.0g of zein in 100ml of ethanol solution with the mass fraction of 70%, weighing 100mg of sodium alginate, dissolving in 300ml of distilled water, dripping the prepared proline ethanol solution into the sodium alginate solution at the rate of 0.1ml/min, stirring while dripping, evaporating ethanol by a rotary evaporator after dripping is completed, and freeze-drying to obtain nano-powder.
The solute of the enzymolysis liquid in the step S2 has the concentration of 0.25-0.7M mannitol, 0.8-2.1% of cellulase, 0.8-1.5% of pectase, 15-25 mM MES, 5-15 mM calcium chloride, 8-25 mM potassium chloride, 0.6-2% of ascorbic acid, 0.4-1.2% of proline-sodium alginate-zein nano particles, and the solvent is water, and the pH is adjusted to 5.6-5.8. Wherein the cellulase and the pectase are all purchased from Beijing Soy Bao technology Co., ltd, and the ascorbic acid is purchased from Shanghai Michelin Biochemical Co., ltd.
The weight-volume ratio of the pre-enzymolysis material to the enzymolysis liquid in the step S2 is 1g:4ml.
In the step S3, the volume ratio of the enzymolysis completion liquid to the W5 washing liquid is 1 (1-3), and the volume ratio of the obtained precipitation mass to the W5 washing liquid is 1g (3-4) ml. The centrifugal temperature is 3-5 ℃, the rotating speed is 600-800 rpm, and the centrifugal time is 1-2 min.
In the second aspect, the invention can prepare plant protoplasts in high efficiency and large quantity, and the prepared protoplasts have high survival rate and can be used for subsequent research. Such as plant biological breeding, protein subcellular localization, protein purification, and the like.
Compared with the prior art, the invention has the following beneficial effects:
1. The pretreatment can make the inner and outer layers of the cell wall fully contact with the enzymolysis liquid, so that the enzymolysis speed is improved, but the pretreatment inevitably causes death of plant cells, so that the addition of the nano material in the pretreatment liquid can effectively reduce the death of the cells and improve the survival rate of the plant cells.
2. Adding ascorbic acid into the enzymolysis liquid. The addition of ascorbic acid at a suitable concentration can scavenge ROS produced during the enzymatic hydrolysis process, which can cause the exhaustion, decrease in viability and death of protoplasts. The added nano material can regulate and control the expression quantity of related genes to achieve the antioxidation effect, and can also improve the integrity of cell membranes and protect the cell membrane structure in the enzymolysis process. The two can act simultaneously, so that the oxidation resistance of the cells can be enhanced, and the loss of electrolyte in cell membranes caused in the enzymolysis process can be reduced, thereby improving the survival rate of the plant cells.
Drawings
FIG. 1 is a bar graph of the number of protoplasts in each sample.
FIG. 2 is a graph showing the results of protoplast activity in each sample.
FIG. 3 is a graph showing the results of malondialdehyde content in protoplasts.
Fig. 4 is a graph showing characterization of proline-sodium alginate-zein nanoparticles prepared in example 1.
Detailed Description
Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention. It should be noted that the following embodiments and features in the embodiments may be combined with each other without conflict.
Example 1
A plant single cell suspension is prepared by the following steps:
s1, cutting wheat seedling leaves with good growth condition and 5cm length, removing roots and leaf tips, standing the rest part in a pretreatment liquid for 15min in dark condition;
S2, filtering after the pretreatment of the pre-plasma wall is finished, collecting sediment to obtain a pre-enzymolysis material, adding the obtained pre-enzymolysis material into an enzymolysis liquid, and carrying out enzymolysis in a dark place for 3 hours;
s3, mixing the solution subjected to enzymolysis with the W5 washing liquid, centrifuging for 2 times, taking a precipitate, and mixing the precipitate with the W5 washing liquid to obtain the purified single-cell suspension. Wherein. The formula of the W5 lotion comprises CaCl 2·2H2 O125.0 mmol/L, naCl 154.0.0 mmol/L, KCl 5.0.0 mmol/L, glucose 5.0mmol/L, MES 5.0mmol/L and pH 5.8.
Wherein the pre-wall treatment liquid in the step S1 is a dispersion liquid of mannitol with the concentration of 0.4M and proline-sodium alginate-zein nano particles with the concentration of 0.8%, and the volume ratio of the mannitol to the proline-sodium alginate-zein nano particles is 2.5:1.
The preparation method of the proline-sodium alginate-zein nano-particles comprises the steps of dissolving 50mg of proline and 1.0g of zein in 100ml of ethanol solution with the mass fraction of 70% to obtain proline-zein ethanol solution, weighing 100mg of sodium alginate, dissolving the prepared proline-zein ethanol solution in 300ml of distilled water, dropwise adding the prepared proline-zein ethanol solution into the sodium alginate solution at the rate of 0.1ml/min, stirring while dropwise adding, evaporating ethanol by using a rotary evaporator after dropwise adding is completed, freeze-drying to obtain nano-powder, and ultrasonically dispersing the obtained proline-sodium alginate-zein powder in water to obtain the 0.8% sodium alginate proline-zein dispersion liquid. SEM of the proline-sodium alginate-zein nanoparticles obtained in this example is shown in FIG. 4. As can be seen from the SEM image of FIG. 4, the particle size of the proline-sodium alginate-zein nanoparticle is 200-300nm, the nanoparticle is spherical and the size distribution is uniform, which indicates that the obtained nanoparticle has stronger stability and can be better applied to the preparation of single cell suspension.
The solute and concentration of the enzymolysis liquid in the step S2 are 0.5M mannitol, 1.3% of cellulase, 1.2% of pectase, 18mM MES, 11mM calcium chloride, 20mM potassium chloride, 1.3% of ascorbic acid and 0.8% of proline-sodium alginate-zein nano particles, the solvent is water, and the pH is adjusted to 5.6.
The weight-volume ratio of the pre-enzymolysis material to the enzymolysis liquid in the step S2 is 1g:4ml.
In the step S3, the volume ratio of the enzymolysis completion liquid to the W5 washing liquid is 1:2, and the volume ratio of the obtained precipitation mass to the W5 washing liquid is 1g:3ml. The centrifugal temperature is 3 ℃, the rotating speed is 800rpm, and the centrifugal time is 2min. The zein in the experiment is purchased from Tianjin solomo biotechnology Co., ltd, and L-proline, sodium alginate and the like are purchased from Shanghai Ala Biochemical technology Co., ltd.
Example 2
The preparation method of example 2 is basically the same as that of example 1, except that the mass fraction of the mixture of the pre-plasmolysis solution and the proline-sodium alginate-zein nanoparticle-loaded mixed solution added to the enzymolysis solution is 1.0%.
Comparative example 1
The preparation method of comparative example 1 was substantially the same as in example 1, except that 0.4M mannitol, 0.8% sodium alginate-zein nanoparticle dispersion was added to the pre-wall separation solution in step S1 of comparative example 1.
The preparation method of the dispersion liquid comprises the steps of dissolving 1.0g zein in 100ml of ethanol solution with the mass fraction of 70%, weighing 100mg sodium alginate, dissolving in 300ml of distilled water, dropwise adding the prepared zein ethanol solution into the sodium alginate solution at the rate of 0.1ml/min, stirring while dropwise adding, evaporating ethanol by a rotary evaporator after dropwise adding is completed, and freeze-drying to obtain nano powder, and ultrasonically dispersing the obtained sodium alginate-zein powder in water to obtain the 0.8% sodium alginate-zein dispersion liquid.
Comparative example 2
The preparation method of comparative example 2 is basically the same as that of example 1, except that the composition of the pre-wall separation liquid in step S1 of comparative example 2 is 0.4M mannitol and 0.8% proline aqueous solution, wherein the volume ratio of the two is 2.5:1.
Comparative example 3
The preparation method of comparative example 3 was substantially the same as in example 1, except that no ascorbic acid was added to the constituent components of the enzymatic hydrolysate in step S2 of comparative example.
Comparative example 4
The preparation method of comparative example 4 is basically the same as that of example 1, except that proline-sodium alginate-zein nanoparticles are not added to the constituent components of the enzymatic hydrolysate in step S2 of comparative example.
Comparative example 5
The preparation method of comparative example 5 is basically the same as that of example 1, except that proline-sodium alginate-zein nanoparticles are replaced with sodium alginate-zein powder of the same mass in the composition of the enzymatic hydrolysate in step S2 of comparative example. The preparation method is the same as that in comparative example 1.
Comparative example 6
Comparative example 6 was prepared in substantially the same manner as in example 1 except that only 0.4M mannitol was added to the composition of the pre-wall separation liquid in step S1 of comparative example 6.
In the step S2, ascorbic acid and proline-sodium alginate-zein nano particles are not added into the enzymolysis liquid.
Wheat protoplast number detection
(1) Sample selection
The wheat single cell suspensions prepared in examples 1-2 and comparative examples 1-6 of the present invention.
(2) Experimental method
1Ml of wheat single-cell suspension is taken, and 10 times of W5 solution is added for full suspension. A small amount of suspension was dropped into a 0.1 mm,25×16 type hemocytometer counting chamber, and observed under a common optical microscope, the number of protoplasts was counted in 5 middle squares (total 80 small squares) of the upper left, lower left, upper right, lower right and middle. The total amount of protoplasts was calculated according to the formula number of protoplasts/mL=number of protoplasts in 80 cells/80X 400X 10 4 X dilution. Each sample was counted in 3 replicates and 3 samples were taken per experiment.
(3) Experimental results
Table 1 number of wheat protoplasts in each sample
Table 1 shows the number of wheat protoplasts in each sample. As is evident from the data in the table, the number of protoplasts in examples 1-2 is greater than the number of protoplasts in the other samples. Among them, the number of protoplasts of comparative example 1, in which the dispersion of sodium alginate-zein nanoparticles was replaced with the dispersion of pre-wall separation liquid, was (1.6.+ -. 0.2). Times.10 7, and the number of protoplasts of comparative example 2, in which the number of protoplasts was (1.7.+ -. 0.2). Times.10 7, were much smaller than those of example 1. From the data, the proline-sodium alginate-zein nanoparticle dispersion liquid was helpful for the pre-wall separation process. The reason for this may be that the nanoparticles can enter the inside of plant cells through the cell membrane, protect the cell membrane, and prevent the cell death caused by the imbalance of the osmotic pressure inside and outside the plant cells during the separation of the cell pre-plasma wall. Secondly, as can be seen from the comparison of the data of comparative examples 3-6 and example 1, the addition of ascorbic acid and proline-sodium alginate-zein nanoparticle dispersion in the enzymatic hydrolysate is helpful for the preparation of protoplasts. The ascorbic acid and the ascorbic acid can regulate and control the expression of antioxidant genes and regeneration genes, can better ensure the preparation of protoplasts, and simultaneously have synergistic effect with proline-sodium alginate-zein nanoparticle dispersion liquid, and simultaneously protect cell membranes from being damaged.
Protoplast activity assay
(1) Sample selection
The wheat single cell suspensions prepared in examples 1-2 and comparative examples 1-6 of the present invention.
(2) Experimental method
Protoplast viability assay the number of green fluorescing protoplasts and total number of protoplasts were counted using a fluorescence phase contrast microscope (Zeiss, axio image A1) stained with 0.01% diacetic Fluorescein (FDA), and protoplast viability was expressed as a percentage of viable protoplasts in one field of view relative to the total number of protoplasts in that field of view, and 3 representative fields were counted and averaged.
Protoplast viability= (number of green fluorescing protoplasts/total number of protoplasts) ×100%.
(3) Experimental results
Table 2 shows wheat protoplast activity in each sample
Table 2 shows the wheat protoplast activity in each sample. Protoplasts are consistent and a relatively uniform single cell population with cell walls removed. The protoplast is in the same separation period, the introduced exogenous gene expression has better synchronism, and meanwhile, long-time tissue culture is not needed, and the preparation and detection process only needs 2d time. The protoplast transient expression system provides a convenient and effective experimental system for researching the subcellular localization of genes and the regulation and control of gene expression, so that the activity of the protoplast is important. As can be seen from the data in Table 2, the protoplast activity of examples 1-2 was higher than that of the comparative example, which is related to the nature of ascorbic acid. The ascorbic acid can remove active oxygen and polyunsaturated fatty acid free radicals generated in plants, maintain redox balance, and promote cell activity. Secondly, proline-sodium alginate-zein nano particles enter between cell walls and cell membranes, and proline released by the proline-sodium alginate-zein nano particles and ascorbic acid have a synergistic effect, so that the internal balance of plant cells is ensured, and the wheat protoplast maintains higher cell activity.
Malondialdehyde content determination
(1) Sample selection
The wheat single cell suspensions prepared in examples 1-2 and comparative examples 1-6 of the present invention.
(2) Experimental method
The wheat single cell suspension with the same mass is weighed, extracted by 5% trichloroacetic acid, boiled in water bath (0.67% TBA is added), and the supernatant is measured for absorbance at 450nm, 532nm and 600nm by a spectrophotometry method, so as to calculate the MDA content.
Experimental results
Table 3 shows the malondialdehyde content of wheat protoplasts in each sample
Table 3 shows the malondialdehyde content of wheat protoplasts in each sample. Malondialdehyde is one of the most important products of lipid peroxidation of plant cell membranes, and increased levels of malondialdehyde exacerbate cell membrane damage, so malondialdehyde levels are often used as an important indicator in physiological studies of plant senescence and resistance. From the data of comparative examples 1-6, it can be seen that ascorbic acid as well as proline-sodium alginate-zein nanoparticle dispersions are able to detoxify some chemicals. This is because biotoxicity is caused by factors such as membrane integrity damage caused by peroxidation of membrane lipid, damage to organelles such as chloroplasts and mitochondria, oxidative stress, and protein and nucleic acid denaturation genotoxicity. And the ascorbic acid and the proline-sodium alginate-zein nanoparticle dispersion liquid cooperate to protect the integrity of cell membranes and the normal operation of organelles such as chloroplasts, mitochondria and the like.
Claims (9)
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| US20110247100A1 (en) * | 2010-03-31 | 2011-10-06 | Dow Agrosciences Llc | Plant peptide gamma-zein for delivery of biomolecules into plant cells |
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| CN117247906A (en) * | 2023-09-20 | 2023-12-19 | 西安交通大学 | A kind of receptor-high-expressing cell membrane drug screening material based on magnetic nanoparticles |
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| US20110247100A1 (en) * | 2010-03-31 | 2011-10-06 | Dow Agrosciences Llc | Plant peptide gamma-zein for delivery of biomolecules into plant cells |
| US20150004102A1 (en) * | 2012-02-13 | 2015-01-01 | Bionanoplus, S.L. | Nanoparticles comprising a vegetable hydrophobic protein and a water miscible non-volatile organic solvent and uses thereof |
| CN115927157A (en) * | 2023-01-13 | 2023-04-07 | 广西壮族自治区药用植物园 | Preparation method of corydalis saxicola bunting leaf protoplast |
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