CN106554935A - A kind of detached method of tomato leaf Guard Cell Protoplasts - Google Patents

Abstract

The invention discloses a method for separating guard cell protoplasts from tomato leaves. The method is to enzymolyze the epidermis tissue of tomato leaves containing guard cells to extract the guard cell protoplasts. The invention successfully separates the protoplasts of the guard cells of tomato leaves by improving the types and concentrations of relevant enzymes in the enzyme liquid and the temperature and rotating speed conditions required for enzymolysis. The method of the invention is especially suitable for tomato, and the obtained protoplast has better activity, which lays a foundation for further research on cell induction and signal transduction of guard cells.

Description

A method for isolating protoplasts of tomato leaf guard cells

technical field

The invention relates to a method for isolating protoplasts of tomato leaf guard cells and belongs to the field of biotechnology.

Background technique

Plant stomata are surrounded by a pair of guard cells, which control the exchange of gas between plants and the external environment. In gas metabolism such as carbon assimilation, respiration, and transpiration, stomata become passages for air and water vapor, and their throughput is determined by guard cells. It is very important in physiology to regulate the opening and closing of cells.

Stomatal closure is mainly affected by the turgor of guard cells. With the in-depth research on guard cells, extracting guard cell protoplasts has almost become a necessary means. Related studies have used patch clamp technology to study the electrophysiology of plant leaf guard cells under ABA stimulation, which will help to further improve the signal transduction network in plant cells under drought conditions. Therefore, guard cells have become a good model system in the study of cell induction and signal transduction, and a very effective experimental system for the study of stomatal physiology. But at present, there are not many plants that have successfully isolated guard cell protoplasts, and there is no method system established in tomato, and some current isolation methods are used to separate guard cell protoplasts from tomato leaf epidermis, and protoplasts in good condition cannot be obtained, so they cannot be used questions about other experiments.

Contents of the invention

Based on the existing problems in the prior art, the purpose of this invention is to provide a method for the isolation of tomato leaf guard cell protoplasts, which is suitable for isolating guard cell protoplasts from the tomato leaf epidermis and obtaining protoplasts in good condition. A good foundation has been laid.

The purpose of the present invention is achieved through the following technical solutions:

The embodiment of the present invention provides a method for isolating protoplasts of tomato leaf guard cells, comprising:

Step 1, preparing a solution for separation, the solution comprising: basic medium 1, basic medium 2, enzyme liquid 1 and enzyme liquid 2;

Step 2, obtaining vigorously growing tomato seedling leaves as raw materials, putting them into a homogenizer containing cold distilled water for homogenization treatment, and obtaining tomato leaf homogenate;

Step 3, filter the homogenate with a 100 μm nylon mesh, rinse with water and then rinse with the basic medium 1, transfer the obtained epidermis into the enzyme solution 1, and shake at 27°C in a water bath at 100 rpm The first step of enzymatic hydrolysis reaction is carried out on the bed, and the reaction time of the first step is 30 minutes to 35 minutes;

Step 4, in the first enzymolysis reaction of step 3, when more than 80% of the guard cells slightly swell and the stomata are full ovals, the first enzymolysis reaction is terminated, and the first enzymolysis reaction is filtered through a 65 μm nylon mesh before using the basic Rinse the medium two, transfer the obtained epidermis into the enzyme solution medium two, and carry out the second enzymolysis reaction on a water bath shaker at 25°C and 70 rpm. The second enzymatic hydrolysis reaction time is 2.5h. During the two-step enzymatic hydrolysis reaction, use a microscope to observe the dissociated protoplasts. If the dissociated protoplasts are spherical and full in shape, stop the second enzymatic hydrolysis reaction;

Step 5, the reaction solution after the second step of enzymolysis in step 4 is filtered with a 30 μm nylon mesh, washed with the basic medium two, the filtrate is collected, centrifuged at 450g for 7min, the supernatant is removed, and the The above-mentioned basic medium 2 was blown and mixed, centrifuged with 450g for 7min, and then repeated centrifuged washing once. Finally, the precipitated guard cell protoplasts were suspended with 2mL of the basic medium 2, and stored at 4°C to obtain the isolated tomato leaf guard. Cell protoplast suspension.

It can be seen from the above-mentioned technical scheme provided by the present invention that the separation method provided by the embodiment of the present invention has the beneficial effects of: the method improves the type and concentration of the relevant enzymes in the enzyme solution and the temperature and rotational speed conditions required for enzymatic hydrolysis, The protoplasts of the guard cells of tomato leaves were successfully isolated, and the activity of the isolated protoplasts was relatively good, which laid the foundation for further research on the cell induction and signal transduction of guard cells.

Description of drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the accompanying drawings that need to be used in the description of the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For Those of ordinary skill in the art can also obtain other drawings based on these drawings on the premise of not paying creative efforts.

Fig. 1 is the image of the enzymolysis process of tomato leaf epidermal cells provided by the embodiment of the present invention, wherein, a picture is the image of enzymatic liquid first enzymolysis for 30 minutes; b picture is the image of enzymatic liquid two enzymolysis 1.5h; c picture is the image of enzyme liquid Two-hour enzymatic hydrolysis image; d picture is the enzymatic solution two-enzymatic hydrolysis 2.5h image, total enzymatic hydrolysis 3h;

Among Fig. 2, a picture is the guard cell protoplast image before the purification that the present invention method separates, and b picture is the protoplast image after the purification that the present invention method separates;

Fig. 3 is a detection image of protoplast activity of guard cells separated by the method of the present invention.

detailed description

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the specific content of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

An embodiment of the present invention provides a method for isolating guard cell protoplasts from tomato leaves, which is a method of enzymatically hydrolyzing tomato leaves containing guard cells to obtain guard cell protoplasts therein, including:

Step 1, preparing a solution for separation, the solution comprising: basic medium 1, basic medium 2, enzyme liquid 1 and enzyme liquid 2;

Step 2, obtaining 8-10 parts by weight of vigorously growing tomato seedling leaves as raw materials, putting them into a homogenizer containing 150 parts by weight of cold distilled water for homogenization treatment, and obtaining homogenate of tomato leaves; such as about 10 grams of tomato Seedling leaves, homogenized with 150ml cold distilled water;

Step 3, filter the homogenate with a 100 μm nylon mesh, rinse with water and then rinse with the basic medium 1, transfer the obtained epidermis into the enzyme solution 1, and shake at 27°C in a water bath at 100 rpm The first step of enzymatic hydrolysis reaction is carried out on the bed, and the reaction time of the first step is 30 minutes to 35 minutes;

Step 4, in the first enzymolysis reaction of step 3, when more than 80% of the guard cells slightly swell and the stomata are full ovals, the first enzymolysis reaction is terminated, and the first enzymolysis reaction is filtered through a 65 μm nylon mesh before using the basic Rinse the medium two, transfer the obtained epidermis into the enzyme solution medium two, and carry out the second enzymolysis reaction on a water bath shaker at 25°C and 70 rpm. The second enzymatic hydrolysis reaction time is 2.5h. During the two-step enzymatic hydrolysis reaction, use a microscope to observe the dissociated protoplasts. If the dissociated protoplasts are spherical and full in shape, stop the second enzymatic hydrolysis reaction;

Step 5, the reaction solution after the second step of enzymolysis in step 4 is filtered with a 30 μm nylon mesh, washed with the basic medium two, the filtrate is collected, centrifuged at 450g for 7min, the supernatant is removed, and the The above-mentioned basic medium 2 was blown and mixed, centrifuged with 450g for 7min, and then repeated centrifuged washing once. Finally, the precipitated guard cell protoplasts were suspended with 2mL of the basic medium 2, and stored at 4°C to obtain the isolated tomato leaf guard. Cell protoplast suspension.

In step 1 of the above method,

The basic medium one is: 0.3mol·L ^-1 mannitol, 5mmol·L ^-1 Mes, 0.5mmol·L ^-1 , CaCl ₂ , 0.5mmol·L ^-1 MgCl ₂ , 10μmol·L ^-1 KH ₂ PO _4. pH5.5 (KOH);

The basic medium two is: 0.4mol·L ^-1 mannitol, 5mmol·L ^-1 Mes, 0.5mmol·L ^-1 , CaCl ₂ , 0.5mmol·L ^-1 MgCl ₂ , 10μmol·L ^-1 KH ₂ PO _4. pH5.5 (KOH);

The enzyme liquid one is: cellulase RS with a mass concentration of 1% (m/v), polyvinylpyrrolidone-40 with a mass concentration of 0.1% (m/v), pectin with a mass concentration of 0.01% (m/v) Enzyme Y-23, bovine serum albumin with a mass concentration of 0.25% (m/v), 0.5mmol·L ^-1 ascorbic acid, and the solvent is the basic medium one;

The enzyme liquid two is: cellulase RS with a mass concentration of 1.5% (m/v), pectinase Y-23 with a mass concentration of 0.02% (m/v), and bovine enzyme with a mass concentration of 0.25% (m/v). Serum albumin, 0.5mmol·L ^-1 ascorbic acid, and the solvent is the basic medium two.

In step 2 of the above method, put it into a homogenizer containing cold distilled water for homogenization treatment: put the selected tomato seedling leaves (about 10 grams) into a homogenizer containing 150 mL of cold distilled water for homogenization for 1 minute , the rotating speed is 8000～10000rpm.

The above method also includes: step 5, purifying the obtained isolated tomato leaf guard cell protoplast suspension, including:

Using Histopaque-1077 as the purified solution, absorb the purified solution and inject it into the protoplast suspension, spread the protoplast suspension on the purified solution, and centrifuge for 15 minutes under the condition of 100g density gradient;

Use a pipette to suck out the protoplasts in the upper layer, put them into another clean centrifuge tube, add an appropriate amount of the basic medium two washing solution, centrifuge at 450 g for 7 minutes, discard the supernatant, and obtain the tomato leaf guard cell protoplasts.

In the above method, during the second step of the enzymatic hydrolysis reaction in step 4, use a microscope to observe the dissociated protoplasts, that is, observe the actual effect of the guard cell enzymatic hydrolysis, and take pictures according to the instructions of the Zeiss fluorescence microscope Axio Observer D1;

The method of the invention is suitable for tomato leaves, and the obtained guard cell protoplasts have regular shape and good activity, which lays a foundation for further research on the physiological, biochemical and signal transduction aspects of the guard cells in tomato leaves.

Embodiments of the present invention will be further described in detail below in conjunction with the accompanying drawings.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Embodiment 1, the separation of tomato leaf guard cell protoplast:

(1) Prepare the solution for separation

11. Basic medium 1: 0.3mol·L ^-1 mannitol, 5mmol·L ^-1 Mes, 0.5mmol·L ^-1 , CaCl ₂ , 0.5mmol·L ^-1 MgCl ₂ , 10μmol·L ^-1 KH ₂ PO ₄ , pH5.5 (KOH).

12. Basic medium two: 0.4mol·L ^-1 mannitol, 5mmol·L ^-1 Mes, 0.5mmol·L ^-1 , CaCl ₂ , 0.5mmol·L ^-1 MgCl ₂ , 10μmol·L ^-1 KH ₂ PO ₄ , pH5.5 (KOH).

13. Enzyme solution one: 1% (m/v) cellulase RS, 0.1% (m/v) polyvinylpyrrolidone-40, 0.01% (m/v) pectinase Y-23, 0.25% (m/v) v) Bovine serum albumin, 0.5mmol·L ^-1 ascorbic acid, the solvent is basic medium one.

14. Enzyme solution two: 1.5% (m/v) cellulase RS, 0.02% (m/v) pectinase Y-23, 0.25% (m/v) bovine serum albumin, 0.5mmol·L ^-1 Ascorbic acid, solvent as basic medium II.

(2) Explanation of the role of the relevant components in the solution

Adding a certain concentration of MES, mannitol, Ca ²⁺ and K ⁺ to the enzyme solution can stabilize the plasma membrane and stabilize the osmotic pressure.

(3) Concrete steps of protoplast isolation:

21. Material selection: Take vigorously growing tomato seedling leaves. The seedling age and leaf age have a great relationship with the yield and vitality of protoplasts. The leaves should not be too old or too tender.

22. Put the selected tomato leaves (about 10 grams) into a homogenizer containing 150 mL of cold distilled water and homogenize for 1 minute at a speed of 8000-10000 rpm.

23. Then filter the homogenate with a 100 μm nylon mesh, rinse with water and then rinse with the basic medium 1, transfer the obtained epidermis into the enzyme solution 1, and enzymolyze it on a water bath shaker at 100 rpm at 27°C for about 30 minutes. The effect of enzymatic hydrolysis is shown in Figure 1a.

24. In the above enzymatic hydrolysis, when more than 80% of the guard cells swell slightly and the stomata are full ovals, stop the first step of enzymatic hydrolysis, filter with 65 μm nylon mesh, and then rinse with basic medium two. The obtained epidermis was transferred into the enzyme solution II, and enzymatically hydrolyzed on a water-bath shaker at 70 rpm at 25°C for about 2.5 hours. During the enzymatic hydrolysis process, observe the dissociated status of the protoplasts under a microscope in time. If the dissociated protoplasts are spherical and full in shape, the second enzymatic hydrolysis reaction is terminated. See b-d in Figure 1 for the effect of enzymatic hydrolysis.

25. The reaction solution after the above enzymatic hydrolysis was filtered with a 30 μm nylon mesh, rinsed with the basic medium 2, collected the filtrate, centrifuged at 450g for 7 minutes, carefully discarded the supernatant, added the basic medium 2 and carefully blown and mixed, centrifuged with 450g for 7 minutes, and then repeated the centrifugation Wash once. Finally, suspend the precipitated guard cell protoplasts with about 2 mL of basic medium 2, and store them at 4°C until the next step. The effect of protoplast rough fractionation is shown in Figure 2a.

Purification of tomato leaf guard cell protoplasts:

31. Purification is based on the density gradient centrifugation method, and the purified solution is Histopaque-1077.

32. The purification steps are as follows: use a straw to absorb the purified solution and carefully inject it into the protoplast suspension, spread the suspension on the purified solution, centrifuge at 100 g for 15 minutes, and the protoplasts in good state will float due to the effect of density gradient centrifugation Between the purified solution and the basic medium, broken cell debris sinks to the bottom of the tube. Carefully aspirate the protoplasts in the upper layer with a pipette and put them into another clean centrifuge tube. Add an appropriate amount of basic medium two washing solution, centrifuge at 450g for 7min, and discard the supernatant. The precipitate was suspended with Basic Medium Solution II. The purification effect is shown in Figure 2b.

Detection of protoplast activity in guard cells of tomato leaves:

41. Fluorescein diacetate (FDA) was used as the activity detection indicator, and the FDA storage solution was 10% (m/v) acetone solution, stored at 4°C in the dark, and the concentration of the working solution was 0.01%.

42. Detection steps: draw the purified protoplast suspension on the glass slide, add the dye and mix well, keep it at room temperature for 5 minutes in the dark, and observe directly with the blue excitation block in the fluorescence microscope. Protoplasts that glow green after FDA staining are viable. Activity detection is shown in Figure 3.

The above is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person familiar with the technical field can easily conceive of changes or changes within the technical scope disclosed in the present invention. Replacement should be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the protection scope of the claims.

Claims (4)

1. a method for tomato leaf guard cell protoplast separation, is characterized in that, comprises: Step 1, preparing a solution for separation, the solution comprising: basic medium 1, basic medium 2, enzyme liquid 1 and enzyme liquid 2; Step 2, obtaining vigorously growing tomato seedling leaves as raw materials, putting them into a homogenizer containing cold distilled water for homogenization treatment, and obtaining tomato leaf homogenate; Step 3, filter the homogenate with a 100 μm nylon mesh, rinse with water and then rinse with the basic medium 1, transfer the obtained epidermis into the enzyme solution 1, and shake at 27°C in a water bath at 100 rpm The first step of enzymatic hydrolysis reaction is carried out on the bed, and the reaction time of the first step is 30 minutes to 35 minutes; Step 4, in the first enzymolysis reaction of step 3, when more than 80% of the guard cells slightly swell and the stomata are full ovals, the first enzymolysis reaction is terminated, and the first enzymolysis reaction is filtered through a 65 μm nylon mesh before using the basic Rinse the medium two, transfer the obtained epidermis into the enzyme solution medium two, and carry out the second enzymolysis reaction on a water bath shaker at 25°C and 70 rpm. The second enzymatic hydrolysis reaction time is 2.5h. During the two-step enzymatic hydrolysis reaction, use a microscope to observe the dissociated protoplasts. If the dissociated protoplasts are spherical and full in shape, stop the second enzymatic hydrolysis reaction; Step 5, the reaction solution after the second step of enzymolysis in step 4 is filtered with a 30 μm nylon mesh, washed with the basic medium two, the filtrate is collected, centrifuged at 450g for 7min, the supernatant is removed, and the The above-mentioned basic medium 2 was blown and mixed, centrifuged with 450g for 7min, and then repeated centrifuged washing once. Finally, the precipitated guard cell protoplasts were suspended with 2mL of the basic medium 2, and stored at 4°C to obtain the isolated tomato leaf guard. Cell protoplast suspension. 2. the method for a kind of tomato leaf guard cell protoplast separation according to claim 1, is characterized in that, in described step 1, The basic medium one is: 0.3mol·L ^-1 mannitol, 5mmol·L ^-1 Mes, 0.5mmol·L ^-1 , CaCl ₂ , 0.5mmol·L ^-1 MgCl ₂ , 10μmol·L ^-1 KH ₂ PO _4. pH5.5 (KOH); The basic medium two is: 0.4mol·L ^-1 mannitol, 5mmol·L ^-1 Mes, 0.5mmol·L ^-1 , CaCl ₂ , 0.5mmol·L ^-1 MgCl ₂ , 10μmol·L ^-1 KH ₂ PO _4. pH5.5 (KOH); The enzyme liquid one is: cellulase RS with a mass concentration of 1% (m/v), polyvinylpyrrolidone-40 with a mass concentration of 0.1% (m/v), pectin with a mass concentration of 0.01% (m/v) Enzyme Y-23, bovine serum albumin with a mass concentration of 0.25% (m/v), 0.5mmol·L ^-1 ascorbic acid, and the solvent is the basic medium one; The enzyme liquid two is: cellulase RS with a mass concentration of 1.5% (m/v), pectinase Y-23 with a mass concentration of 0.02% (m/v), and bovine enzyme with a mass concentration of 0.25% (m/v). Serum albumin, 0.5mmol·L ^-1 ascorbic acid, and the solvent is the basic medium two. 3. the method for a kind of tomato leaf guard cell protoplast separation according to claim 1 and 2 is characterized in that, in described step 2, put into the homogenizer that contains cold distilled water and carry out homogenization process as: 8-10 parts by weight of the selected tomato seedling leaves are put into a homogenizer containing 150 parts by weight of cold distilled water and homogenized for 1 minute at a speed of 8000-10000 rpm. 4. the method for a kind of tomato leaf guard cell protoplast separation according to claim 1 or 2, is characterized in that, also comprises: Step 6, the tomato leaf guard cell protoplast suspension that obtains separation is purified ,include: Using Histopaque-1077 as the purified solution, absorb the purified solution and inject it into the protoplast suspension, spread the protoplast suspension on the purified solution, and centrifuge for 15 minutes under the condition of 100g density gradient; Use a pipette to suck out the protoplasts in the upper layer, put them into another clean centrifuge tube, add an appropriate amount of the basic medium two washing solution, centrifuge at 450 g for 7 minutes, discard the supernatant, and obtain the tomato leaf guard cell protoplasts.