Brassica plant leaf protoplast preparation and transformation method

Abstract

The invention relates to the field of plant genetic engineering, in particular to a method suitable for preparation and transformation of leaf protoplasts of all plants under brassica. By changing the selection and dosage of the enzyme and the formula of the enzymolysis buffer solution, the protoplast preparation requirements of different plant leaves under brassica are met. By changing the formula of the MMG solution and the conversion solution and the conversion method, the protoplast conversion requirements of leaves of different plants belonging to brassica are met. The method can be suitable for leaves of different degrees, special treatment is not needed, and the protoplast conversion efficiency of leaves of different plants under brassica is improved through a simple and convenient centrifugal mode.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to a method for preparing and transforming protoplasts of Brassica plant leaves. Background Art

[0002] Protoplast transient gene expression system has been widely used in plant gene function research and genetic improvement due to its advantages of simple operation, short transformation cycle and high transformation efficiency. However, in Brassica vegetable crops, the efficiency of protoplast preparation and transformation is relatively low.

[0003] With reference to the Chinese invention patent with application number CN201710916783.1, a method for separating and transforming rape protoplasts is disclosed, which specifically includes using rape cotyledons or true leaves as raw materials, analyzing the main factors affecting the separation of rape leaf flesh protoplasts and the transformation of target genes, determining the system suitable for enzymatic hydrolysis, separation, purification and transformation of rape, and finally using rape protoplasts as receptors, using PEG mediation at a suitable concentration, transferring the expression vector integrated with the exogenous gene into the rape protoplasts, and after optimized cultivation, the exogenous gene is successfully expressed in the rape protoplasts. With reference to the Chinese invention patent with application number CN201910840640.6, a method for preparing protoplasts and genetic transformation of Chinese cabbage is disclosed, including the step of obtaining Chinese cabbage protoplasts from yellow inner leaves of Chinese cabbage.

[0004] The following disadvantages exist in the prior art: 1. There are few optimization methods for the preparation and transformation of protoplasts of Brassica plants, and only the protoplasts of a single plant under the genus Brassica can be prepared and transformed, which is not applicable to all plants under the genus Brassica. 2. The selected materials must be seedlings, and the materials need to be pretreated with tape. 3. After enzymatic hydrolysis, it needs to be purified with sucrose solution, and the experimental conditions are demanding and the operation is complicated. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for preparing and transforming leaf protoplasts applicable to all Brassica plants, which ensures that experimental materials can be quickly obtained, the experimental operation process is simple, and the transformation efficiency is improved.

[0006] The present application provides a method for preparing and transforming leaf protoplasts of Brassica plants, comprising the following steps:

[0007] Step S1: 1 g of fresh and healthy leaves of plants of the genus Brassica (cabbage, rapeseed, Chinese cabbage, mustard greens, green vegetables) are selected, rinsed with running water, wiped dry with absorbent paper, and cut into 0.5-1 mm strips with a sharp blade parallel to the main veins;

[0008] Step S2: placing the above materials into 20 mL of enzymatic hydrolysis solution and performing enzymatic hydrolysis at 25-28° C. in the dark, wherein the enzymatic hydrolysis solution comprises: 0.75 wt % cellulase, 0.2 wt % pectinase, 0.6 M mannitol, 20 mM KCl, 20 mM MES with a pH of 5.7, 10 mM CaCl2, and 0.1 wt % BSA;

[0009] Step S3: After 5 h of enzymolysis, an equal volume of W5 solution (4 mM MES, 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES (pH 5.7)) was added to the enzymolysis mixture, and the protoplasts were washed by centrifugation. After the supernatant was removed by centrifugation, the protoplasts were resuspended with 10 mL of W5 solution. The resulting green solution was the purified protoplasts.

[0010] Step S4: Place the green solution on ice and keep it away from light for 30 minutes;

[0011] Step S5: Remove the W5 solution by centrifugation, and resuspend the protoplast pellet with MMG solution (0.4 M Mannitol, 15 mM MgCl2 and 4 mM MES (pH 5.7)). The cell density after resuspension is 1.5-2×10 5 cells / mL, for future use;

[0012] Step S6: Protoplast transformation: The transformation system is 10 μg plasmid, 100 μL protoplast-MMG suspension, 110 μL 40% PEG-4000. After gentle mixing, incubate in a 37°C water bath for 6 min, then add 500 μL W5 solution to resuspend and terminate the transformation; centrifuge and discard the supernatant, add 500 μL W5 solution to resuspend, and culture at room temperature for 12-16 h.

[0013] Technical effects and advantages of the present invention:

[0014] 1. The method of the present invention is applicable to leaves of different degrees and does not require special treatment. Through a simple centrifugation method, the protoplast transformation efficiency of leaves of different plants under the genus Brassica is increased.

[0015] 2. The present invention meets the protoplast preparation requirements of different plant leaves under the genus Brassica by changing the selection and dosage of enzymes and the formula of enzymolysis buffer. The protoplast transformation requirements of different plant leaves under the genus Brassica are met by changing the formula of MMG solution and transformation liquid and the transformation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is an operational flow chart of the preparation and transformation of Brassica protoplasts using Brassica as an example provided in the examples of the present application;

[0017] Figure 2Schematic diagram of bright field and fluorescence of plants under the genus Brassica provided in the examples of the present application;

[0018] Figure 3 This is the result of protoplast microscopy of Brassica rapa (taking Brassica napus as an example) provided in the examples of the present application. DETAILED DESCRIPTION

[0019] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The embodiments of the present invention are provided for the purpose of illustration and description, and are not intended to be exhaustive or to limit the present invention to the disclosed forms. Many modifications and variations will be apparent to those of ordinary skill in the art. The embodiments are selected and described in order to better illustrate the principles and practical applications of the present invention, and to enable those of ordinary skill in the art to understand the present invention and thereby design various embodiments with various modifications suitable for specific uses.

[0020] Example 1

[0021] In this embodiment, a method for preparing protoplasts from Brassica leaves is provided. Figure 1 , taking Brassica as an example:

[0022] The following steps are involved:

[0023] S1: Select 1 g of fresh and healthy leaves of plants under the genus Brassica, rinse them with running water, wipe them dry with absorbent paper, and cut them into 0.5-1 mm strips with a sharp blade parallel to the main veins; plants under the genus Brassica include but are not limited to cabbage, rapeseed, cabbage, mustard, and greens.

[0024] S2: Place the above-mentioned strip leaves into 20 mL of enzymatic hydrolysis solution and perform enzymatic hydrolysis at 25-28°C in the dark; the formula of the enzymatic hydrolysis solution is: 0.75wt% cellulase, 0.2wt% pectinase, 0.6M mannitol, 20mM KCl, 20mM MES with a pH of 5.7, 10mM CaCl2, and 0.1wt% BSA.

[0025] S3: After 5 hours of enzymatic hydrolysis, an enzymatic hydrolysis mixture is formed. An equal volume of W5 solution is added to the enzymatic hydrolysis mixture and the protoplasts are washed by centrifugation. After centrifugation to remove the supernatant, the protoplasts are resuspended with 10 mL of W5 solution. The resulting green solution is the purified protoplasts. The formula of the W5 solution is: 4 mM MES, 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 2 mM MES with a pH of 5.7.

[0026] S4: separating the purified protoplasts from the W5 solution.

[0027] S4.1: Place the green solution obtained in step S3 on ice and keep it away from light for 30 minutes;

[0028] S4.2: Remove W5 solution by centrifugation and resuspend the protoplast pellet with MMG solution. The cell density after resuspension is 1.5-2×10 5 The formula of the MMG solution is: 0.4M mannitol, 15mM MgCl2 and 4mM MES with a pH of 5.7.

[0029] Example 2

[0030] According to the purified protoplasts obtained in Example 1, a method for transforming protoplasts of Brassica leaves is provided, comprising the following steps:

[0031] 1) Take the protoplasts prepared in S4.2;

[0032] 2) The transformation system is 10 μg plasmid, 100 μL protoplast-MMG suspension, 110 μL 40% PEG-4000. After gentle mixing, incubate in a 37°C water bath for 6 min, and then add 500 μL W5 solution to resuspend and terminate the transformation;

[0033] 3) Centrifuge and discard the supernatant, add 500 μL W5 solution for re-suspending, and incubate at room temperature for 12-16 hours.

[0034] This application reduces the amount of cellulase and cleavage enzyme, increases the mannitol content of the enzymatic buffer, and thus optimizes the osmotic pressure, so that this method can successfully prepare all plants under the Brassica genus, which is a significant improvement over the prior art that can only enzymatically hydrolyze one plant alone. The Brassica genus includes a variety of important vegetables and oil crops, such as Chinese cabbage, Brassica napus, purple cabbage, green cabbage, lettuce, mustard, Chinese cabbage, and Shanghai green. Establishing a universal protoplast preparation and transformation method will make the study of gene function and genetic improvement of these crops more convenient and efficient.

[0035] refer to Figure 2 As shown, a bright field image and a fluorescence image of plants under the genus Brassica are provided, including a bright field image of Brassica rape, a fluorescence image of Brassica rape, a bright field image of Brassica juncea, a fluorescence image of Brassica juncea, a bright field image of purple cabbage, a fluorescence image of purple cabbage, a bright field image of green cabbage, a fluorescence image of green cabbage, a bright field image of Chinese cabbage, and a fluorescence image of Chinese cabbage. The method can successfully prepare protoplasts under the genus and successfully transform them with an efficiency of 30%.

[0036] refer to Figure 3 As shown, a 400× Brassica (Brassica napus) protoplast microscopy result is provided. Using 10 μg of endotoxin-free pBI221-EGFP to transform into 100 uL Brassica (Brassica napus) protoplasts, the transformation efficiency can reach 37.00±6.26%.

[0037] In the present application, cellulase and cleavage enzymes are used to enzymatically hydrolyze the cell wall and prepare protoplasts. Through the mediation of Polyethyleneglycol (PEG), the exogenous gene is transferred into the protoplasts of the Brassica plant and transiently expressed. In this embodiment, because the enzymatic hydrolysis solution and enzymatic hydrolysis method are used, a large amount of plant fragments will not be generated. At the same time, the present application reduces the amount of enzymes used, adjusts the osmotic pressure of the enzymatic hydrolysis buffer, and combines slow centrifugation conditions to avoid the generation of protoplast fragments or plant tissue residues, so there is no need for the cumbersome purification steps involved in the prior art, which improves simplicity.

[0038] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field and related fields without creative work should fall within the scope of protection of the present invention. The structures, devices and operating methods not specifically described and explained in the present invention are implemented according to the conventional means in the field unless otherwise specified and limited.

Claims

1. A method for preparing protoplasts from Brassica leaves, characterized in that: The following steps are involved: S1: Select 1g of fresh and healthy leaves of Brassica plants, rinse them with running water, wipe them dry with absorbent paper, and cut them into 0.5-1mm strips with a sharp blade parallel to the main veins; S2: Place the above-mentioned strip leaves into 20 mL of enzymatic hydrolysis solution and perform enzymatic hydrolysis at 25-28°C in the dark; S3: After 5 hours of enzymatic hydrolysis, an enzymatic hydrolysis mixture is formed. An equal volume of W5 solution is added to the enzymatic hydrolysis mixture and the protoplasts are washed by centrifugation. After the supernatant is removed by centrifugation, the protoplasts are resuspended with 10 mL of W5 solution. The resulting green solution is the purified protoplasts. S4: separating the purified protoplasts from the W5 solution.

2. The method for preparing protoplasts from Brassica leaves according to claim 1, characterized in that: The step S4 specifically includes: S4.1: Place the green solution obtained in step S3 on ice and keep it away from light for 30 minutes; S4.2: Remove W5 solution by centrifugation and resuspend the protoplast pellet with MMG solution. The cell density after resuspension is 1.5-2×10 5 cells / mL, for future use.

3. The method for preparing protoplasts from Brassica leaves according to claim 1, characterized in that: The formula of the enzymatic hydrolysis solution is: 0.75wt% cellulase, 0.2wt% pectinase, 0.6M mannitol, 20mM KCl, 20mM MES with a pH of 5.7, 10mM CaCl2, and 0.1wt% BSA.

4. The method for preparing protoplasts from Brassica leaves according to claim 1, characterized in that: The formula of the W5 solution is: 4 mM MES, 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES with a pH of 5.

7.

5. The method for preparing protoplasts from Brassica leaves according to claim 2, characterized in that: The formula of the MMG solution is: 0.4M mannitol, 15mM MgCl2 and 4mM MES with a pH of 5.

7.

6. The method for preparing protoplasts from Brassica leaves according to claim 1, characterized in that: The plants under the genus Brassica include but are not limited to cabbage, rapeseed, Chinese cabbage, mustard, and green vegetables.

7. A method for transforming protoplasts from Brassica leaves, characterized in that: The method comprises the purified protoplasts according to any one of claims 1 to 6.

8. The method for transforming protoplasts from Brassica leaves according to claim 7, characterized in that: The following steps are involved: 1) Take the protoplasts prepared in S4.2; 2) The transformation system is 10 μg plasmid, 100 μL protoplast-MMG suspension, 110 μL 40% PEG-4000. After gentle mixing, incubate in a 37°C water bath for 6 min, and then add 500 μL W5 solution to resuspend and terminate the transformation; 3) Centrifuge and discard the supernatant, add 500 μL W5 solution for re-suspending, and incubate at room temperature for 12-16 hours.

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