CN115386530A - Rapid preparation and transformation method of plant leaf protoplast, kit and application - Google Patents
Rapid preparation and transformation method of plant leaf protoplast, kit and application Download PDFInfo
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Abstract
The invention provides a method for rapidly preparing and transforming plant leaf protoplasts, which adopts a reagent for preparing and transforming the protoplasts, wherein the reagent comprises WQ1 enzymolysis liquid, WQ2 preservation liquid, WQ3 resuspension liquid and WQ4 transformation liquid, and the method comprises the steps of enzymolysis, purification, resuspension, transformation, rinsing and culture. The invention also provides a kit and a transformation kit for rapidly preparing the plant leaf protoplast, a method for rapidly preparing the plant leaf protoplast and application of the kit in the preparation of the plant leaf protoplast, and a method and a kit for rapidly transforming the plant leaf protoplast and application in the transformation of the plant leaf protoplast. The protoplast obtained by the method has good integrity, high transformation efficiency, strong experimental repeatability and difficult damage after long-term storage, can complete the preparation and transformation processes of the high-quality protoplast within 1.5 hours, breaks through the time limit of the preparation and transformation of the existing plant protoplast at home and abroad, and has wide application range and high application value.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a method for quickly preparing and transforming plant leaf protoplasts, a kit and application thereof.
Background
Protoplasts are viable plant cells that are coated with a cytoplasmic membrane after removal of the cell wall. The protoplast has the characteristics of simple structure, good development synchronism and easy introduction of exogenous genetic materials, and is a good material for plant gene transient transformation. Transient transformation of protoplasts is an important method for the study of transient expression of plant genes, promoter activity analysis, interaction between transcription factors and cis-acting elements, interaction between proteins, and subcellular localization of proteins. However, the inventor of the present application finds, through research, that the existing plant leaf protoplast preparation and transformation methods at home and abroad have at least a plurality of the following disadvantages:
a large amount of plant material is required: for example, 20 leaves are required to be taken for experiments in the preparation method of rape mesophyll protoplast disclosed in CN111676183A, which is not suitable for the preparation and transformation of protoplast of rare plant materials.
Large amounts of reagents need to be used: for example, a large amount of reagents such as 20mL of enzymolysis solution and the like are needed for an Arabidopsis thaliana mesophyll cell protoplast disclosed in CN109355246B, and the preparation method and the application thereof are high in cost.
The universality is poor: for example, the arabidopsis thaliana mesophyll cell protoplast disclosed in CN109355246B, the preparation method and the application thereof, the method for rapidly obtaining halophyte leaf protoplast disclosed in CN105316276B, and the preparation method of the rape mesophyll protoplast disclosed in CN111676183A can be only applied to one plant leaf.
Poor stability: when experiments of different batches are carried out by the same method, the instantaneous conversion efficiency difference of protoplasts of different batches is large, and the stability of experimental results is poor.
The protoplast preparation speed is slow: in the existing method, the preparation speed of the protoplast can be reduced by various factors such as complicated material taking method, long cell wall enzymolysis time, complex protoplast purification steps and the like.
The protoplast has short preservation time: the protoplast cannot be completely preserved for a long time due to the poor quality of the protoplast or the unstable property of the protoplast preservation solution.
The protoplast yield is low: plant material selection, plant material processing, cell wall digestion, etc., can reduce protoplast yield.
Protoplasts are subject to rupture: the protoplast is easily broken during the preparation process, or is broken in a large amount after transient transformation, or is easily broken during the continuous culture process after transformation.
The operation process is complex: in the existing method, the sample needs to be subjected to vacuum infiltration, a centrifuge tube and a culture vessel need to be replaced for multiple times, and various protoplast containers need to be pretreated by BSA solution, so that the method has the advantages of multiple operation steps, complex transformation process and high cost.
Low conversion efficiency: the conversion efficiency of the protoplast can be reduced due to the defects of extraction and preservation of high-quality plasmids, preparation of the protoplast, control of conversion environmental conditions, stability of reagents, activity of the protoplast and the like.
Dependence on the inlet reagent: the existing methods for preparing and transforming Protoplast, such as Wang et al (2022) [ An Efficient and Universal Protocol Isolation Protocol Suitable for vector Gene Expression Analysis and Single-Cell RNA sequencing. Int.J.mol.Sci.2022;23 3419 ] the method reported depends on imported reagents, but the imported reagents are difficult to purchase, high in cost and long in shelf life, and have a lot of inconvenience in the actual popularization and application process.
The types of reagents are more: in the preparation and transformation processes of the protoplast, a plurality of reagents are used, which not only increases the difficulty of reagent preparation, but also influences the activity of the protoplast.
Reagents cannot be stored for a long time: in the existing method, all prepared reagents cannot be effectively stored for a long time, and a plurality of reagents must be prepared as they are, so that the method is not suitable for repeated experiments of multiple batches.
Therefore, how to innovatively develop a method for quickly preparing and transforming plant leaf protoplasts, a kit and application which can overcome the defects of the prior art is of great significance.
Disclosure of Invention
Aiming at various technical problems in the prior art, the invention provides a rapid preparation and transformation method of plant leaf protoplast, and the protoplast obtained by the method has good integrity, high transformation efficiency, strong experimental repeatability and difficult damage after long-term storage.
In order to solve the technical problems, the invention adopts the following technical scheme:
the method adopts a protoplast preparation and transformation reagent, the reagent comprises WQ1 enzymolysis solution, WQ2 preservation solution, WQ3 resuspension and WQ4 transformation solution, the WQ1 enzymolysis solution consists of 1.5 percent (w/v) of cellulase R-10, 0.5 percent (w/v) of eductase R-10, 500mM of D-Mannitol, 20mM of MES, 10mM of KCl and 5mM of CaCl 2 ·2H 2 O, 0.2% (w/v) BSA and sterile ultrapure water, wherein the pH value of the WQ1 enzymatic hydrolysate is 5.6-5.8, and the solution is stored for a long time at the temperature of-80 to-20 ℃; the WQ2 preservation solution is prepared from 154mM NaCl and 125mM CaCl 2 ·2H 2 O, 0.2mM MES, 5mM KCl, 2mM sucrose and sterile ultrapure water, wherein the pH value of the WQ2 preservation solution is 5.6-5.8, and the solution is preserved for a long time at the temperature of-80 to-20 ℃ after filtration sterilization; the WQ3 heavy suspension is prepared from 20mM MgCl 2 ·6H 2 O, 400mM D-Mannitol, 1mM MES and sterile ultrapure water, wherein the pH value of the WQ3 resuspension is 5.6-5.8, and the suspension is stored for a long time at the temperature of-80 to-20 ℃ after being filtered and sterilized; the WQ4 transformation solution is prepared from 100mM CaCl 2 ·2H 2 O, 200mM D-Mannitol, 2mMMES, 40% (w/v) PEG4000 and sterile ultrapure water, wherein the pH value of the WQ4 conversion solution is 5.6-10.0, and the WQ4 conversion solution can be prepared as it is or can be effectively stored for two days at 4 ℃; the method comprises the following steps:
(1) Enzymolysis: 1 leaf with 1cm area is cut from the plant with normal growth 2 The flat blade of (1), fix the one side of upper epidermis of the blade in the hard adhesive tape of the inelasticity, tear off the epidermis with the sticky tape or meticulous tweezers, remove the surplus sticky tape around the blade, submerge the sticky tape of adhering the blade in 2mL aseptic round bottom centrifuge tube containing 600 uL WQ1 enzymolysis solution; fixing the centrifuge tube to a shaking table with the rotating speed of 45rpm and the temperature of 24-28 ℃, shaking for enzymolysis for 20-30 min in a dark place, taking out the centrifuge tube every 5-10 min during the enzymolysis, and manually shaking for 5-10 s; taking out the adhesive tape by using tweezers, placing the centrifugal tube in a centrifugal machine, and centrifuging for 1-2 min under the conditions that the temperature is 4 ℃, the centrifugal force is 100g, the rising speed is 3 and the falling speed is 3; gently sucking off the supernatant, and precipitating to obtain an unpurified protoplast;
(2) And (3) purification: slowly adding 2mL of WQ2 preservation solution into the centrifuge tube for collecting the protoplast in the step (1) along the tube wall, and slowly reversing the centrifuge tube to resuspend the protoplast; centrifuging for 1-2 min at 4 deg.C, centrifugal force of 100g, speed of 3 g, and speed of 3 d; slowly sucking the supernatant, slowly adding 2mL WQ2 preservative solution along the tube wall, and slowly reversing the centrifuge tube to resuspend the protoplast; centrifuging for 1-2 min at 4 deg.C, centrifugal force of 100g, speed of 3 g, and speed of 3 d; gently sucking and removing the supernatant, slowly adding 1mL WQ2 preservative solution along the tube wall, gently shaking and resuspending the protoplast, standing on ice for 30min, gently sucking and removing the supernatant, precipitating to obtain purified leaf protoplast, and checking the state of the protoplast under a microscope;
(3) Resuspending: centrifuging the purified protoplast for 1-2 min at the temperature of 4 ℃, the centrifugal force of 100g, the speed of rise of 3 and the speed of fall of 3, and precipitating the protoplast; slightly sucking the supernatant, adding 100-200 mu L of WQ3 heavy suspension to heavily suspend the protoplast, adjusting the density of the protoplast to be optimal, and standing on ice for 5-10 min;
(4) And (3) transformation: adding 10-20 mu L of purified plasmid with the concentration of 1-3 mu g/mu L into the suspended protoplast in the step (3) along the tube wall of a centrifuge tube, and shaking gently and mixing uniformly; slowly adding 110-220 mu L of WQ4 conversion solution along the tube wall at a constant speed, and quickly and uniformly mixing by shaking; incubating for 3-5 min in a dark environment at 24-28 ℃; slowly adding 1mL of WQ2 preservative solution along the tube wall, and slowly inverting the centrifuge tube to mix the sample to terminate the conversion;
(5) Rinsing: centrifuging the protoplast mixed solution after the conversion is ended for 1-2 min at the temperature of 24-28 ℃, the centrifugal force of 100g, the acceleration of 3 and the deceleration of 3, and precipitating the protoplast; slowly sucking off the supernatant, slowly adding 2mL WQ2 preservation solution along the tube wall at a constant speed, and slowly reversing the centrifuge tube to resuspend the protoplast; centrifuging for 1-2 min at 24-28 deg.c, centrifugal force of 100g, raising speed of 3 and lowering speed of 3 to precipitate protoplast; slowly sucking the supernatant, slowly adding 2mL WQ2 preservation solution along the tube wall at a constant speed, and slowly reversing the centrifuge tube to resuspend the protoplast; removing supernatant, keeping precipitate, slowly adding 500 μ L WQ2 preservation solution along the tube wall at uniform speed, shaking gently, and mixing;
(6) Culturing: and (4) performing static culture on the protoplast rinsed in the step (5) at the temperature of 24-28 ℃ for 12-48 h in a dark place, and observing and analyzing under a fluorescence microscope.
Further, in order to obtain more protoplasts in the step (1), 600. Mu.L of WQ1 enzymolysis solution is added for each 1 leaf.
Further, 100-200 μ L of WQ3 heavy suspension is added in the step (3) to re-suspend the protoplast, so that the density of the protoplast is optimally adjusted to 1 × 10 3 ～1×10 7 Individual cells/mL.
Further, the plasmids in the step (4) are converted into a single plasmid, two or three plasmids, if only one plasmid is converted, 10 to 30 mu g of plasmids are added; if the two plasmids are transformed together, 10-15 mu g of the purified plasmids are mixed and added; if the three plasmids are transformed together, 10 mu g of the purified plasmids are mixed and added; and, the volume of the WQ4 transformation solution added in step (4) should be the sum of the volume of the WQ3 resuspension added in step (3) and the volume of the plasmid added in step (4).
Further, when the WQ2 preservation solution is added for the last time in the step (5), the ratio of ampicillin: WQ2 preservation liquid =1:1000 (v/v) ampicillin was added to the WQ2 stock solution at a concentration of 100 mg/mL.
Further, the plant is a monocotyledon or a dicotyledon.
Further, the plant is a dicotyledonous plant including but not limited to arabidopsis, soybean, chinese cabbage, rape, sweet wormwood, potato, sweet potato, tomato, tobacco, spinach, celery, peanut, mint.
The invention also provides a kit for rapidly preparing the plant leaf protoplast and a transformation kit, wherein the kit contains at least one component of the WQ1 enzymolysis liquid, the WQ2 preservation liquid, the WQ3 resuspension liquid and the WQ4 transformation liquid.
The invention also provides a method for rapidly preparing the plant leaf protoplast and application of the kit for rapidly preparing the plant leaf protoplast in preparation of the plant leaf protoplast.
The invention further provides a plant leaf protoplast rapid transformation method and application of the plant leaf protoplast rapid transformation kit in plant leaf protoplast transformation.
Compared with the prior art, the rapid preparation and transformation method of the plant leaf protoplast, the kit and the application have the following beneficial effects:
1. the invention discloses a rapid preparation and transformation method of plant leaf protoplast, a kit and application thereof for the first time, has the advantages of less samples, high yield, simple operation, high speed, low cost, long reagent storage time, good protoplast integrity, high transformation efficiency, strong experimental repeatability and difficult damage after long-term storage, is universal in various plants, and overcomes various defects in the prior art.
2. The invention discloses a method for completing the preparation and transformation process of high-quality protoplast within 1.5 hours for the first time, breaks through the time limit of the preparation and transformation of the existing plant protoplast at home and abroad, has wide application range and high application value, and is suitable for popularization. The key technical features of the method are the following seven aspects, firstly, the method of the invention is usedThe reagents do not need to be prepared as before, so that the reagent preparation process before the experiment is omitted; secondly, the centrifuge tube does not need to be replaced in the whole process of the method, and the operation process is simple; third, the process of the present invention requires only about 1cm 2 The plant leaves do not need to draw a large amount of materials; fourthly, in the enzymolysis step, the lower epidermis of the flat leaf blade is torn off, so that mesophyll cells can fully contact WQ1 enzymolysis liquid, and the leaf blade is manually and lightly shaken once every certain time during the enzymolysis period, so that the cells which are subjected to enzymolysis on the surface of the leaf blade are quickly released into the enzymolysis liquid, the contact area between the cells which are not subjected to full enzymolysis and the enzymolysis liquid is increased, and the enzymolysis time is shortened to be within 30 minutes from 4-6 hours of the traditional method; fifthly, in the enzymolysis step, one side of the upper surface skin of the leaf is fixed on an inelastic hard adhesive tape, the protoplast is released into the enzymolysis liquid, the plant tissue which is not subjected to enzymolysis is still adhered to the adhesive tape, and a large amount of impurities can be removed by taking out the adhesive tape, so that the filtering and impurity removing process in the traditional method is omitted; sixthly, in the transformation step, under the condition of not influencing the transformation efficiency, the light-shielding incubation time is successfully shortened from 30 minutes of the traditional method to 3-5 minutes; seventh, in the method of the present invention, the amount of the reagent used is greatly reduced, thereby reducing the number of pipetting times, and furthermore, the method of the present invention shortens the time per centrifugation. Therefore, the preparation and transformation process of the high-quality protoplast can be completed within 1.5 hours.
3. The invention discloses a method for completing 1-time protoplast preparation and transformation only by using 1 centrifugal tube (namely, the centrifugal tube does not need to be replaced) and 1 leaf in the whole process for the first time, and the dosage of an experimental reagent is less than 15mL each time, so that the cost is saved, and an important basis is provided for developing high-throughput protoplast preparation and transformation in the future.
4. The reagent is a domestic reagent or an imported reagent, and the domestic reagent comprises D-Mannitol, MES, BSA, naCl, KCl and MgCl 2 ·6H 2 O、CaCl 2 .2H 2 O, PEG4000, sucrose, imported reagents including cellulase R-10 and isolation enzyme R-10, so more than 80% of the used reagents are domestic reagents, the technical limitation that the traditional method depends on imported reagents is overcome, the reagents are convenient to purchase, and the consumption is reducedCost; moreover, the domestic reagent in the method is replaced by the imported reagent with the same or better quality, the same or similar effect can be achieved, the universality is strong, and the applicability is wide; the invention also provides a method for effectively preserving the reagent for a long time, which saves the reagent preparation step before each experiment, saves time, and improves the experiment repeatability by using the same batch of prepared reagent.
5. The method does not need to use BSA solution to pretreat various containers, and has ideal effect, high repeatability and simple operation.
6. The method provides a powerful tool for carrying out large-scale cell culture of plants, introducing exogenous genes, sequencing single cells and genetic improvement of plant germplasm resources.
7. The method provides a new technology for the researches of plant gene transient expression, promoter activity analysis, interaction of transcription factors and cis-acting elements, interaction between proteins, protein subcellular localization and the like.
Drawings
FIG. 1 is a diagram showing the effect of the method of the present invention in the preparation and transformation of Arabidopsis thaliana leaf protoplasts, wherein A: purified protoplasts; b: and (5) detecting the protoplast transformation effect.
FIG. 2 is a diagram showing the effect of the method of the present invention in the cultivation of Arabidopsis thaliana leaf protoplasts, wherein A: culturing for 4 days after transformation; b: the cells were cultured for 8 days after transformation.
Detailed Description
In order to make the technical means, the creation features, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the accompanying drawings and the embodiments. The following examples are intended only to illustrate the invention and should not be construed as limiting it; the experimental methods in the following examples are all conventional methods; the plant material and the pAN580 plasmid used in the examples described below were provided by the Rice research institute, university of southwestern; reagents D-Mannitol, MES, BSA, naCl, KCl, mgCl made in the examples below 2 ·6H 2 O、CaCl 2 .2H 2 O, PEG4000 and sucrose were purchased from Biotechnology engineering (Shanghai) GmbH; the following embodimentsThe imported reagents cellulase R-10 and macerozyme R-10 in the examples were purchased from Yakult; the instrumentation used in the examples described below is conventional in molecular biology laboratories. Modifications or substitutions to methods, steps or conditions of the invention by those skilled in the art without departing from the spirit and substance of the invention are within the scope of the invention.
The invention provides a method for rapidly preparing and transforming plant leaf protoplasts, which adopts a reagent for preparing and transforming the protoplasts, wherein the reagent comprises WQ1 enzymolysis liquid, WQ2 preservation liquid, WQ3 resuspension and WQ4 transformation liquid, and the WQ1 enzymolysis liquid comprises 1.5% (w/v) of cellulase R-10, 0.5% (w/v) of eductase R-10, 500mM of D-Mannitol, 20mM of MES, 10mM of KCl and 5mM of CaCl 2 ·2H 2 O, 0.2% (w/v) BSA and sterile ultrapure water, wherein the pH value of the WQ1 enzymolysis solution is 5.6-5.8, and the solution is stored for a long time at the temperature of-80 to-20 ℃; the WQ2 preservation solution is prepared from 154mM NaCl and 125mM CaCl 2 ·2H 2 O, 0.2mM MES, 5mM KCl, 2mM sucrose and sterile ultrapure water, wherein the pH value of the WQ2 preservation solution is 5.6-5.8, and the solution is preserved for a long time at the temperature of-80 to-20 ℃ after filtration sterilization; the WQ3 heavy suspension is prepared from 20mM MgCl 2 ·6H 2 O, 400mM D-Mannitol, 1mM MES and sterile ultrapure water, wherein the pH value of the WQ3 resuspension is 5.6-5.8, and the suspension is stored for a long time at the temperature of-80 to-20 ℃ after being filtered and sterilized; the WQ4 transformation solution is prepared from 100mM CaCl 2 ·2H 2 O, 200mM D-Mannitol, 2mM MES, 40% (w/v) PEG4000 and sterile ultrapure water, wherein the pH value of the WQ4 conversion solution is 5.6-10.0, and the WQ4 conversion solution can be prepared for use or can be effectively stored for two days at 4 ℃; the method comprises the following steps:
(1) Enzymolysis: cutting 1 leaf from normal growth plant with leaf area of about 1cm 2 The method comprises the steps of (1) flattening the leaves, fixing one side of the upper surface skin of each leaf on an inelastic hard adhesive tape, tearing off the upper surface skin by using an adhesive tape or fine tweezers, removing redundant adhesive tapes around the leaves, and immersing the adhesive tapes adhered to the leaves in a 2mL sterile round-bottom centrifuge tube containing 600 mu L WQ1 enzymatic hydrolysate; fixing the centrifuge tube to a shaker at a rotation speed of 45rpm and a temperature of 24-28 DEG CShaking and performing enzymolysis for 20-30 min in the dark, taking out the centrifuge tube every 5-10 min, and manually shaking for 5-10 s; taking out the adhesive tape by using tweezers, placing the centrifugal tube in a centrifugal machine, and centrifuging for 1-2 min under the conditions that the temperature is 4 ℃, the centrifugal force is 100g, the rising speed is 3 and the falling speed is 3; gently sucking off the supernatant, and precipitating to obtain an unpurified protoplast;
(2) And (3) purification: slowly adding 2mL WQ2 preservative solution into the centrifuge tube for collecting the protoplast in the step (1) along the tube wall, and slowly reversing the centrifuge tube to resuspend the protoplast; centrifuging for 1-2 min under the conditions of the temperature of 4 ℃, the centrifugal force of 100g, the speed of rise of 3 and the speed of fall of 3; slowly sucking the supernatant, slowly adding 2mL WQ2 preservative solution along the tube wall, and slowly reversing the centrifuge tube to resuspend the protoplast; centrifuging for 1-2 min under the conditions of the temperature of 4 ℃, the centrifugal force of 100g, the speed of rise of 3 and the speed of fall of 3; slowly sucking to remove supernatant, slowly adding 1mL WQ2 preservative solution along the tube wall, gently shaking to resuspend the protoplast, standing on ice for 30min, slightly sucking to remove supernatant, precipitating to obtain purified leaf protoplast, and examining the state of the protoplast under a microscope;
(3) Resuspending: centrifuging the purified protoplast for 1-2 min at the temperature of 4 ℃, the centrifugal force of 100g, the speed of rise of 3 and the speed of fall of 3, and precipitating the protoplast; gently sucking the supernatant, adding 100-200 mu L of WQ3 heavy suspension to resuspend the protoplast according to the requirement of the next research, adjusting the density of the protoplast to be optimal, and standing on ice for 5-10 min;
(4) And (3) transformation: adding 10-20 mu L of purified plasmid with the concentration of 1-3 mu g/mu L into the resuspended protoplast in the step (3) along the tube wall of the centrifuge tube, and shaking gently and mixing uniformly; slowly adding 110-220 mu L of WQ4 conversion solution along the tube wall at a constant speed, and quickly and uniformly shaking; incubating for 3-5 min in a dark environment at 24-28 ℃; slowly adding 1mL of WQ2 preservative solution along the tube wall, and slowly inverting the centrifuge tube to mix the sample to terminate the conversion;
(5) Rinsing: centrifuging the protoplast mixed solution after the conversion is ended for 1-2 min under the conditions that the temperature is 24-28 ℃, the centrifugal force is 100g, the speed is increased to 3 and the speed is decreased to 3, and precipitating the protoplast; slowly sucking the supernatant, slowly adding 2mL WQ2 preservation solution along the tube wall at a constant speed, and slowly reversing the centrifuge tube to resuspend the protoplast; centrifuging for 1-2 min at 24-28 deg.c, centrifugal force of 100g, raising speed of 3 and lowering speed of 3 to precipitate protoplast; slowly sucking the supernatant, slowly adding 2mL WQ2 preservation solution along the tube wall at a constant speed, and slowly reversing the centrifuge tube to resuspend the protoplast; removing supernatant, keeping precipitate, slowly adding 500 μ L WQ2 preservation solution along the tube wall at uniform speed, shaking gently, and mixing;
(6) Culturing: and (4) performing static culture on the protoplast rinsed in the step (5) at the temperature of 24-28 ℃ for 12-48 h in a dark place, and observing and analyzing under a fluorescence microscope.
As a specific embodiment, the WQ1 enzymolysis solution, the WQ2 preservation solution and the WQ3 resuspension can be effectively preserved for a long time at the temperature of between 80 ℃ below zero and 20 ℃ below zero, and the WQ4 conversion solution does not need to be prepared as it is, so that the reagent preparation step before each experiment is omitted, the time is saved, and the reagent preparation in the same batch is used, so that the experiment repeatability can be improved, and the comparative analysis of different experimental groups is facilitated; before the WQ1 enzymolysis liquid and the WQ4 conversion liquid are used, the temperature should be balanced to 24-28 ℃.
As a specific embodiment, in the step (1), in order to obtain more protoplasts, the number of leaves can be increased, and the amount of WQ1 enzymolysis solution needs to be increased proportionally, wherein the optimal proportion is that 600 μ L of WQ1 enzymolysis solution is added for each 1 leaf, and a larger-capacity sterile centrifuge tube needs to be replaced when the total volume exceeds 2 mL; further, if multiple transformation experiments are needed, the unpurified protoplasts are dispensed into different 2mL sterile round-bottom centrifuge tubes according to the amount of 600. Mu.L protoplast transformed for 1 time, and then the next step is performed.
As a specific embodiment, in the step (1), the leaves are fixed by using an inelastic hard adhesive tape, and after enzymolysis of mesophyll cells is completed, plant materials which are difficult to perform enzymolysis still adhere to the adhesive tape, so that the adhesive tape is taken out by using tweezers after enzymolysis, that is, most impurities are removed, and a filtering step is not required; in the enzymolysis process, the centrifugal tube is taken out every 5-10 min and manually shaken for 5-10 s, so that mesophyll cells which do not finish cell wall enzymolysis fully contact with the enzymolysis liquid, and the enzymolysis process is accelerated.
As a specific example, compared with a 10-50 mL centrifuge tube used in the traditional method, in the step (2), the method of the invention completes the purification step in the 2mL centrifuge tube, so that the damage to the protoplast can be effectively reduced, and the container volume is small, and the reagent dosage is reduced.
As a specific example, in the step (3), depending on the purpose of the study and the density of the desired protoplast, the density of the protoplast can be optimally adjusted to 1X 10 by resuspending the protoplast with 100. Mu.L to 200. Mu.L of the WQ3 resuspension solution 3 ～1×10 7 Individual cells/mL.
As a specific example, the high-purity plasmid in step (4) helps to improve transformation efficiency, and the plasmid transformation may be single plasmid transformation, or two or three plasmid transformations, with different plasmid dosages for different transformation modes; if only one plasmid is transformed, 10-30 mug of plasmid is added; if the two plasmids are transformed together, respectively taking 10-15 mu g of the purified plasmids to mix and add; if the three plasmids are transformed together, 10 mu g of the purified plasmids are mixed and added; under the same other conditions, increasing the plasmid concentration within a certain range can improve the protoplast transformation efficiency.
As specific examples, in the steps (3) to (4), the volume of the WQ4 transformation solution added in step (4) should be the sum of the volume of the WQ3 resuspension added in step (3) and the volume of the plasmid added in step (4), for example, 100 μ L of the WQ3 resuspension is added in step (3), and 10 μ L of the plasmid is added in step (4), and then 110 μ L of the WQ4 transformation solution is added in step (4); if 200 mu L of WQ3 resuspension is added in the step (3) and 10 mu L of plasmid is added in the step (4), 210 mu L of LWQ4 transformation liquid is added in the step (4); if 200. Mu.L of the WQ3 resuspension is added in the step (3) and 20. Mu.L of the plasmid is added in the step (4), 220. Mu.L of the transformation solution of the WQ4 is added in the step (4). By adopting the technical scheme in the embodiment, the osmotic pressure of the conversion system can be balanced, and the conversion efficiency is improved.
As a specific example, when the WQ2 preservative solution is added for the last time in the step (5), the ratio of ampicillin: WQ2 preservation liquid =1:1000 (v/v) ampicillin was added to the WQ2 stock solution at a concentration of 100mg/mL, thereby effectively preventing bacterial growth during the culture.
As a specific embodiment, the whole process of the method is carried out in the same 2mL centrifuge tube, so that the step (6) can be directly carried out in the original centrifuge tube during the culture of the protoplast without replacing the original centrifuge tube with a cell culture plate; of course, it is within the scope of the present invention to transfer protoplasts to cell culture plates or other cell culture vessels to achieve the same or similar results.
As a specific example, the plant is a monocot or a dicot. As a preferred example, the plant is a dicotyledonous plant including, but not limited to, arabidopsis, soybean, bok choy, canola, sweet wormwood, potato, sweet potato, tomato, tobacco, spinach, celery, peanut, mint. In one embodiment of the invention, the plant is arabidopsis thaliana.
The invention also provides a kit and a transformation kit for rapidly preparing the plant leaf protoplast, wherein the kit contains at least one component of the WQ1 enzymolysis liquid, the WQ2 preservation liquid, the WQ3 resuspension and the WQ4 transformation liquid, namely the kit contains any one component, any two components, any three components or four components of the WQ1 enzymolysis liquid, the WQ2 preservation liquid, the WQ3 resuspension or the WQ4 transformation liquid.
The invention also provides a method for rapidly preparing the plant leaf protoplast and application of the kit for rapidly preparing the plant leaf protoplast in preparation of the plant leaf protoplast.
The invention also provides a method for quickly transforming the plant leaf protoplast and application of the kit for quickly transforming the plant leaf protoplast in the transformation of the plant leaf protoplast.
Example 1: preparation of plant leaf protoplast and preparation of transformation reagent
(1) Preparing WQ1 enzymolysis liquid: the WQ1 enzymolysis solution comprises 1.5% (w/v) of cellulase R-10, 0.5% (w/v) of macerozyme R-10, 500mM of D-Mannitol, 20mM of MES, 10mM of KCl and 5mM of CaCl 2 ·2H 2 O, 0.2% (w/v) BSA and sterile ultrapure water, the pH value is 5.6-5.8, the filtration sterilization is not needed, and the prepared product is stored for a long time at the temperature of-80 to-20 ℃.
(2) Preparing a WQ2 preservation solution: the WQ2 preservation solution is prepared from 154mM NaCl and 125mM CaCl 2 ·2H 2 O, 0.2mM MES, 5mM KCl, 2mM sucrose and sterile ultrapure water, the pH value is 5.6-5.8, and the mixture is stored for a long time at the temperature of minus 80-minus 20 ℃ after being filtered and sterilized.
(3) Preparing a WQ3 heavy suspension: the WQ3 heavy suspension is prepared from 20mM MgCl 2 ·6H 2 O, 400mM D-Mannitol, 1mM MES and sterile ultrapure water, the pH value is 5.6 to 5.8, and the mixture is stored for a long time at the temperature of between 80 ℃ below zero and 20 ℃ below zero after being filtered and sterilized.
(4) Preparing WQ4 conversion solution: the WQ4 transformation solution is prepared from 100mM CaCl 2 ·2H 2 O, 200mM D-Mannitol, 2mM MES, 40% (w/v) PEG4000 and sterile ultrapure water, the pH value is 5.6-10.0, the WQ4 conversion solution can be prepared for use, and can be effectively stored for two days at the temperature of 4 ℃.
Example 2: preparation and transformation of arabidopsis thaliana leaf protoplast
Preparing and transforming arabidopsis thaliana leaf protoplast:
(1) The reagents were prepared as described in example 1 of the present invention.
(2) Enzymolysis: 1 leaf with the area of about 1cm is cut from normal 3-4 weeks of cultivated Arabidopsis thaliana rosette leaves 2 The flat blade of (1), fix the one side of upper epidermis of the blade in the hard adhesive tape of the inelasticity, tear off the epidermis with the sticky tape or meticulous tweezers, remove the surplus sticky tape around the blade, submerge the sticky tape of adhering the blade in 2mL aseptic round bottom centrifuge tube containing 600 uL WQ1 enzymolysis solution; fixing the centrifuge tube to a shaker with the rotation speed of 45rpm and the temperature of 24 ℃, shaking and performing enzymolysis for 25min in the dark, taking out the centrifuge tube every 10min, and manually shaking for 5s; taking out the adhesive tape with tweezers, placing the centrifuge tube in a centrifuge, and centrifuging for 2min at 4 deg.C, 100g centrifugal force, 3 rising speed and 3 falling speed; gently sucking off the supernatant, and precipitating to obtain an unpurified protoplast;
(3) And (3) purification: slowly adding 2mL of WQ2 preservation solution into the centrifuge tube for collecting the protoplast in the step (2) along the tube wall, and slowly reversing the centrifuge tube to resuspend the protoplast; centrifuging at 4 deg.C, centrifugal force of 100g, acceleration of 3, and deceleration of 3 for 2min; slowly sucking the supernatant, slowly adding 2mL WQ2 preservative solution along the tube wall, and slowly reversing the centrifuge tube to resuspend the protoplast; centrifuging at 4 deg.C, centrifugal force of 100g, acceleration of 3, and deceleration of 3 for 2min; slowly sucking to remove supernatant, slowly adding 1mL WQ2 preservative solution along the tube wall, gently shaking to resuspend the protoplast, standing on ice for 30min, slightly sucking to remove supernatant, precipitating to obtain purified leaf protoplast, and examining the state of the protoplast under a microscope;
(4) Resuspending: centrifuging the purified protoplast for 2min at 4 deg.C under the conditions of centrifugal force of 100g, rising speed of 3 and falling speed of 3, and precipitating the protoplast; gently sucking off the supernatant, adding 200 μ L WQ3 heavy suspension to resuspend the protoplast, adjusting the density of the protoplast to the optimum, and standing on ice for 10min;
(5) And (3) transformation: adding 20 mu L of pAN580 plasmid (containing a complete 35S promoter-GFP coding frame-terminator gene expression cassette) with the concentration of 1 mu g/mu L after purification into the protoplast after the resuspension in the step (4) along the tube wall of a centrifuge tube, and shaking gently and mixing uniformly; slowly adding 220 μ L WQ4 conversion solution along the tube wall at uniform speed, and quickly shaking and mixing; incubating for 5min at 24 deg.C in dark; slowly adding 1mL of WQ2 preservative solution along the tube wall, and slowly inverting the centrifuge tube to mix the sample to terminate the conversion;
(6) Rinsing: centrifuging the protoplast mixture solution at 24 deg.C under centrifugal force of 100g, 3% of rising speed and 3% of falling speed for 2min to precipitate protoplast; slowly sucking the supernatant, slowly adding 2mL WQ2 preservation solution along the tube wall at a constant speed, and slowly reversing the centrifuge tube to resuspend the protoplast; centrifuging at 24 deg.C under centrifugal force of 100g, 3 g and 3 g for 2min to precipitate protoplast; slowly sucking the supernatant, slowly adding 2mL WQ2 preservation solution along the tube wall at a constant speed, and slowly reversing the centrifuge tube to resuspend the protoplast; removing supernatant, keeping precipitate, slowly adding 500 μ L WQ2 preservation solution along the tube wall at constant speed, and shaking gently to mix well;
(7) Culturing: and (5) performing static culture on the protoplast rinsed in the step (6) at 24 ℃ in the dark for 16h, and observing and analyzing under a fluorescence microscope.
(II) analyzing results:
(1) The preparation effect of the arabidopsis thaliana leaf protoplast is shown in figure 1A, the yield of the protoplast is high, and the density of the purified protoplast is more than 110 8 The protoplast integrity rate is higher than 90% per mL, and the result is stable after repeated times.
(2) The transformation effect of the arabidopsis leaf protoplast is shown in fig. 1B, the integrity of the protoplast is good, the transformation efficiency is higher than 95%, strong GFP fluorescent signals exist in different organelles, the result is stable after repeated times, and obvious GFP signals can still be observed after the arabidopsis leaf protoplast is continuously cultured for 48 hours.
(3) As shown in figure 2, the arabidopsis thaliana leaf protoplast is transformed by the plasmid and is continuously cultured for 8 days, the integrity rate of the protoplast is still more than 90%, and the culture effect is stable.
(4) The WQ1 enzymolysis liquid, the WQ2 preservation liquid and the WQ3 resuspension liquid are preserved for 1 year at the temperature of minus 80 ℃ and then are used in the experiment, and the obvious difference between the preparation effect of the protoplast and the instantaneous transformation effect is found, which indicates that the method has good stability.
Finally, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. The method for rapidly preparing and transforming plant leaf protoplasts is characterized in that a protoplast preparation and transformation reagent is adopted in the method, the reagent comprises WQ1 enzymolysis liquid, WQ2 preservation liquid, WQ3 resuspension and WQ4 transformation liquid, and the WQ1 enzymolysis liquid comprises 1.5% (w/v) of cellulase R-10, 0.5% (w/v) of maceration enzyme R-10, 500mM of D-Mannitol, 20mM of MES, 10mM of KCl and 5mM of CaCl 2 ·2H 2 O, 0.2% (w/v) BSA and sterile ultrapure water, wherein the pH value of the WQ1 enzymatic hydrolysate is 5.6-5.8, and the solution is stored for a long time at the temperature of-80 to-20 ℃; the WQ2 preservation solution is prepared from 154mM NaCl and 125mM CaCl 2 ·2H 2 O, 0.2mM MES, 5mM KCl, 2mM sucrose and sterile ultrapure water, wherein the pH value of the WQ2 preservation solution is 5.6-5.8, and the preservation solution is preserved for a long time at the temperature of-80 to-20 ℃ after filtration sterilizationStoring; the WQ3 heavy suspension is prepared from 20mM MgCl 2 ·6H 2 O, 400mM D-Mannitol, 1mM MES and sterile ultrapure water, wherein the pH value of the WQ3 resuspension is 5.6-5.8, and the suspension is stored for a long time at the temperature of-80 to-20 ℃ after being filtered and sterilized; the WQ4 transformation solution is prepared from 100mM CaCl 2 ·2H 2 O, 200mM D-Mannitol, 2mM MES, 40% (w/v) PEG4000 and sterile ultrapure water, wherein the pH value of the WQ4 conversion solution is 5.6-10.0, and the WQ4 conversion solution can be prepared for use or can be effectively stored for two days at 4 ℃; the method comprises the following steps:
(1) Enzymolysis: 1 leaf with 1cm area is cut from the plant with normal growth 2 The method comprises the steps of (1) flattening the leaves, fixing one side of the upper surface skin of each leaf on an inelastic hard adhesive tape, tearing off the upper surface skin by using an adhesive tape or fine tweezers, removing redundant adhesive tapes around the leaves, and immersing the adhesive tapes adhered to the leaves in a 2mL sterile round-bottom centrifuge tube containing 600 mu L WQ1 enzymatic hydrolysate; fixing the centrifuge tube to a shaking table with the rotating speed of 45rpm and the temperature of 24-28 ℃, shaking for enzymolysis for 20-30 min in a dark place, taking out the centrifuge tube every 5-10 min during the enzymolysis, and manually shaking for 5-10 s; taking out the adhesive tape by using a pair of tweezers, placing the centrifugal tube in a centrifugal machine, and centrifuging for 1-2 min under the conditions that the temperature is 4 ℃, the centrifugal force is 100g, the rising speed is 3 and the falling speed is 3; gently sucking off the supernatant, and precipitating to obtain an unpurified protoplast;
(2) And (3) purification: slowly adding 2mL of WQ2 preservation solution into the centrifuge tube for collecting the protoplast in the step (1) along the tube wall, and slowly reversing the centrifuge tube to resuspend the protoplast; centrifuging for 1-2 min under the conditions of the temperature of 4 ℃, the centrifugal force of 100g, the speed of rise of 3 and the speed of fall of 3; gently sucking off the supernatant, slowly adding 2mL WQ2 preservative solution along the tube wall, and slowly reversing the centrifuge tube to resuspend the protoplast; centrifuging for 1-2 min under the conditions of the temperature of 4 ℃, the centrifugal force of 100g, the speed of rise of 3 and the speed of fall of 3; gently sucking and removing the supernatant, slowly adding 1mL WQ2 preservative solution along the tube wall, gently shaking and resuspending the protoplast, standing on ice for 30min, gently sucking and removing the supernatant, precipitating to obtain purified leaf protoplast, and checking the state of the protoplast under a microscope;
(3) Resuspending: centrifuging the purified protoplast for 1-2 min at the temperature of 4 ℃, the centrifugal force of 100g, the speed of rise of 3 and the speed of fall of 3, and precipitating the protoplast; slightly sucking the supernatant, adding 100-200 mu L of WQ3 heavy suspension to heavily suspend the protoplast, adjusting the density of the protoplast to be optimal, and standing on ice for 5-10 min;
(4) And (3) transformation: adding 10-20 mu L of purified plasmid with the concentration of 1-3 mu g/mu L into the resuspended protoplast in the step (3) along the tube wall of the centrifuge tube, and shaking gently and mixing uniformly; slowly adding 110-220 mu L of WQ4 conversion solution along the tube wall at a constant speed, and quickly and uniformly shaking; incubating for 3-5 min in a dark environment at 24-28 ℃; slowly adding 1mL of WQ2 preservative solution along the tube wall, and slowly inverting the centrifuge tube to mix the sample to terminate the conversion;
(5) Rinsing: centrifuging the protoplast mixed solution after the conversion is ended for 1-2 min at the temperature of 24-28 ℃, the centrifugal force of 100g, the acceleration of 3 and the deceleration of 3, and precipitating the protoplast; slowly sucking the supernatant, slowly adding 2mL WQ2 preservation solution along the tube wall at a constant speed, and slowly reversing the centrifuge tube to resuspend the protoplast; centrifuging for 1-2 min at 24-28 deg.c, centrifugal force of 100g, raising speed of 3 and lowering speed of 3 to precipitate protoplast; slowly sucking off the supernatant, slowly adding 2mL WQ2 preservation solution along the tube wall at a constant speed, and slowly reversing the centrifuge tube to resuspend the protoplast; removing supernatant, keeping precipitate, slowly adding 500 μ L WQ2 preservation solution along the tube wall at constant speed, and shaking gently to mix well;
(6) Culturing: and (4) performing static culture on the protoplast rinsed in the step (5) at the temperature of 24-28 ℃ for 12-48 h in a dark place, and observing and analyzing under a fluorescence microscope.
2. The method for rapid preparation and transformation of plant leaf protoplasts as claimed in claim 1, wherein 600. Mu.L of WQ1 hydrolysate is added for each 1 leaf to obtain more protoplasts in step (1).
3. The method for rapid preparation and transformation of plant leaf protoplast according to claim 1, wherein 100-200 μ L of WQ3 resuspension is added in step (3) to resuspend the protoplast such that the density of the protoplast is optimally adjusted to 1 x 10 3 ～1×10 7 Individual cells/mL.
4. The method for rapid preparation and transformation of plant leaf protoplasts as claimed in claim 1, wherein the plasmids in step (4) are transformed into a single plasmid, two or three plasmids, and if only one plasmid is transformed, 10-30 μ g of plasmid is added; if the two plasmids are transformed together, 10-15 mu g of the purified plasmids are mixed and added; if the three plasmids are transformed together, 10 mu g of the purified plasmids are mixed and added; and, the volume of the WQ4 transformation solution added in step (4) should be the sum of the volume of the WQ3 resuspension added in step (3) and the volume of the plasmid added in step (4).
5. The method for rapid preparation and transformation of plant leaf protoplasts according to claim 1, wherein the final addition of the WQ2 preservation solution in step (5) is performed according to the following formula: WQ2 preservation liquid =1:1000 (v/v) ampicillin was added to the WQ2 stock solution at a concentration of 100 mg/mL.
6. The method for rapid preparation and transformation of plant leaf protoplasts as claimed in claim 1, wherein the plant is a monocot or a dicot.
7. The method for rapid preparation and transformation of plant leaf protoplasts according to claim 6, wherein the plant is a dicotyledonous plant including but not limited to Arabidopsis, soybean, cabbage, canola, sweet wormwood, potato, sweet potato, tomato, tobacco, spinach, celery, peanut, mint.
8. The kit for rapidly preparing plant leaf protoplasts and the kit for transforming the same, wherein the kit comprises at least one component selected from the group consisting of WQ1 enzymolysis liquid, WQ2 preservation liquid, WQ3 resuspension liquid and WQ4 transformation liquid as defined in claim 1.
9. The method for rapidly preparing plant leaf protoplasts according to claim 1 and the use of the kit for rapidly preparing plant leaf protoplasts according to claim 8 in the preparation of plant leaf protoplasts.
10. The method for rapid transformation of plant leaf protoplasts according to claim 1 and the use of the kit for rapid transformation of plant leaf protoplasts according to claim 8 for transformation of plant leaf protoplasts.
Priority Applications (1)
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| CN116042503A (en) * | 2022-12-28 | 2023-05-02 | 河北农业大学 | Method for rapidly extracting high-purity celery cabbage protoplast |
| CN116676250A (en) * | 2023-03-20 | 2023-09-01 | 海南大学 | Separation and transformation method of passion fruit mesophyll protoplast |
| CN116987657A (en) * | 2023-07-20 | 2023-11-03 | 四川农业大学 | Celery protoplast and preparation method and application thereof |
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| CN114480248A (en) * | 2022-01-18 | 2022-05-13 | 南通大学 | Preparation method and application of sugarcane stem tissue protoplast |
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| CN116676250A (en) * | 2023-03-20 | 2023-09-01 | 海南大学 | Separation and transformation method of passion fruit mesophyll protoplast |
| CN116987657A (en) * | 2023-07-20 | 2023-11-03 | 四川农业大学 | Celery protoplast and preparation method and application thereof |
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