Method for preparing and converting dactylis glomerata protoplast

Abstract

The invention relates to the field of protoplast preparation, in particular to a method for preparing and converting dactylis glomerata protoplast. The method comprises the following steps: S11, taking dactylis glomerata leaves, putting the dactylis glomerata leaves into a conical flask, and adding enzymatic hydrolysate for completely soaking; s12, the soaked dactylis glomerata is placed in a vacuum vessel to be vacuumized; s13, putting into a horizontal low-speed shaking table, and continuing enzymolysis in a dark place to obtain enzymatic hydrolysate containing protoplast; s14, flushing the tissue material fragments with a W5 solution for one to two times, reserving filtrate, and performing low-speed centrifugation to obtain turbid precipitates; s15, discarding the supernatant, washing with a W5 solution, and resuspending the protoplast; and S16, carrying out low-speed centrifugation, removing supernatant, and adding an MMG solution as required for resuspension. The enzymatic hydrolysate is adopted, the dactylis glomerata leaf is used as a material, the dactylis glomerata protoplast can be efficiently prepared, the yield, activity and transformation efficiency of the obtained protoplast are high, and the method has important basis and significance for subsequent dactylis glomerata gene function research and genetic character improvement research.

Description

Technical Field

[0001] This invention relates to the field of protoplast preparation technology, and in particular to a method for preparing and transforming orchard protoplasts. Background Technology

[0002] Orchardgrass, one of the world's four major economic grasses, originated in North Africa, Europe, and Central Asia. It boasts advantages such as rich nutrition, soft texture, good palatability, and stable biomass yield, making it widely used in forage cultivation and haymaking worldwide, and is considered an excellent forage crop. Orchardgrass also exhibits broad climate tolerance, primarily manifested in its good cold resistance, drought resistance, and adaptability, making it an important grass species for ecological engineering projects such as converting farmland back to grassland and establishing mixed vegetation, as well as for grassland construction. However, due to its poor heat tolerance and high summer mortality rate, its yield is severely affected. In recent years, with the development of orchardgrass functional genomics, molecular breeding has shown great potential for targeted improvement of specific traits. However, due to the complex genetic background of forage grasses and the difficulty in establishing tissue culture systems, there are currently no literature reports on the construction of orchardgrass genetic transformation systems. Therefore, developing an efficient protoplast isolation system for orchardgrass is of great significance for molecular biological research in orchardgrass.

[0003] Plant protoplasts are cells enclosed by a plasma membrane, obtained after the decomposition of plant cell walls. They readily take in exogenous genetic material such as DNA, chromosomes, organelles, and viral particles. Transient transformation of protoplasts can be used for subcellular localization, gene expression analysis, protein-protein interactions, and gene editing, making them ideal materials for developmental biology, cell biology, and cytogenetics research. Transient protoplast transformation technology does not require the integration of exogenous genes into the plant genome; it only requires introducing a recombinant vector containing the target gene into recipient cells, allowing for efficient gene expression or silencing within a short time. However, because protoplast preparation methods and conditions vary depending on the species and material type, optimization and development of isolation and transformation methods are necessary for specific species. Currently, however, efficient isolation and transformation methods for orchardgrass protoplasts are lacking. Summary of the Invention

[0004] The purpose of this invention is to address the problem of the lack of efficient separation and transformation methods for orchardgrass protoplasts in the prior art, and to propose a method for the preparation and transformation of orchardgrass protoplasts.

[0005] On the one hand, this invention proposes a method for preparing orchardgrass protoplasts, comprising the following steps:

[0006] S11. Take the leaves of the orchardgrass, put them into an Erlenmeyer flask and add the enzyme hydrolysate to soak them completely;

[0007] S12. Place the soaked duckgrass in a vacuum dish and vacuum it.

[0008] S13. Place the solution in a horizontal low-speed shaker in the dark for further enzymatic hydrolysis to obtain an enzymatic hydrolysate containing protoplasts.

[0009] S14. Rinse the tissue fragments with W5 solution once or twice, collect the filtrate, and centrifuge at low speed to see a turbid precipitate.

[0010] S15. Discard the supernatant, wash with W5 solution, and resuspend the protoplasts.

[0011] S16. Centrifuge at low speed, discard the supernatant, and resuspend in MMG solution as needed.

[0012] Preferably, in S11, the enzymatic hydrolysate is a mixture of cellulase, ionizing enzyme, mannitol, MES, KCl, CaCl2, BSA and β-mercaptoethanol.

[0013] Preferably, the specific composition of the enzymatic hydrolysate is: 1.5% cellulase, 0.8% ionase, 0.6M mannitol, 10mM MES, 10mM KCl, 10mM CaCl2, 0.11% BSA and 2mM β-mercaptoethanol, where M represents mol / L.

[0014] Preferably, in S11, the leaves of the orchardgrass are shredded and placed into an Erlenmeyer flask.

[0015] Preferably, in S14, the enzymatic hydrolysate obtained in S13 is filtered through a 70 μm filter membrane.

[0016] On the other hand, the present invention proposes a method for transforming orchard protoplasts, comprising the following steps:

[0017] S21. Mix the plasmid and protoplast cells, add PEG solution, and incubate at room temperature in the dark.

[0018] S22. After incubation, dilute the protoplasts with W5 solution, mix well to terminate the reaction, centrifuge, and discard the supernatant.

[0019] S23. After centrifugation, add W5 solution, mix slowly, and incubate in the dark for 15-20 hours. Centrifuge to collect protoplasts.

[0020] Preferably, the conversion efficiency of orchard protoplasts is greater than 75%, and the protoplast viability reaches more than 85%.

[0021] Compared with the prior art, the present invention has the following beneficial technical effects: using a specially prepared enzymatic hydrolysate with orchardgrass leaves as material, orchardgrass protoplasts can be prepared efficiently, and the yield, viability and transformation efficiency of the obtained protoplasts are high, which has important foundation and significance for subsequent research on the gene function and genetic trait improvement of orchardgrass. Attached Figure Description

[0022] Figure 1 This is a flowchart of the preparation method of oriental fescue protoplasts proposed in this invention;

[0023] Figure 2 A schematic diagram of the operation process for separating protoplasts of duckgrass;

[0024] Figure 3 This is a diagram showing the effect of enzymatic hydrolysate on the dissociation of orchardgrass leaves;

[0025] Figure 4 This is a flowchart of the transformation method for orchardgrass protoplasts proposed in this invention;

[0026] Figure 5 This is a schematic diagram showing the luminescence of transformed protoplasts in GFP, Bright Field, and Merged channels. Detailed Implementation

[0027] Example 1, as Figures 1-3 As shown, the present invention proposes a method for preparing orchardgrass protoplasts, comprising the following steps:

[0028] S11. Take fresh and tender orchard leaves, wash them three times with sterile water and dry them. Use a sterilized blade to cut the orchard leaves into uniform shreds of about 0.5-1mm and put them into a 50ml conical flask. Add the enzymatic hydrolysate and soak them completely. The weight / volume ratio of the shreds to the enzymatic hydrolysate is 1:10.

[0029] S12. Place the soaked filaments in a vacuum dish and evacuate them to prevent light exposure. The conical flask can be wrapped with tin foil to prevent light exposure. The pressure is -0.06 MPa and the time is 1 hour.

[0030] S13. Take the soaked filaments and place them in a low-speed shaker at 40 rpm in the dark for further enzymatic hydrolysis. The enzymatic hydrolysis temperature is 27-30℃. Stop shaking the shaker according to the enzymatic hydrolysis state to obtain an enzymatic hydrolysate containing duckweed protoplasts.

[0031] S14. Filter the enzymatic hydrolysate containing orchardgrass protoplasts through a 70μm filter membrane, then rinse the tissue fragments with 10ml of W5 solution, collect the filtrate, and centrifuge at 200×g for 5min to observe a turbid precipitate; W5 solution is a commonly used solution in biological experiments, especially in plant cell culture and tissue culture, and its main components include water, sucrose and phosphate buffer.

[0032] S15. Discard the supernatant of the centrifuged solution and resuspend the protoplasts in 10 ml of W5 solution.

[0033] Centrifuge at 200×g for 5 min, collect protoplasts, discard the supernatant, and suspend the protoplasts in 2-5 mL of MMG solution, depending on their condition.

[0034] Example 2, as Figure 4 and Figure 5 As shown, the present invention proposes a method for transforming orchard protoplasts, using protoplasts obtained in Example 1, specifically including the following steps:

[0035] S21. Adjust the plasmid concentration to be greater than 1 μg / μL. Add 5 μg of empty vector plasmid (pML-CAMBIA3300-GFP), 100 μL of protoplast, and an equal volume of PEG solution to a 2 mL round-bottom centrifuge tube. Mix gently and incubate at room temperature for 5 min in the dark.

[0036] S22. Add 0.5 mL of W5 solution to the incubated solution to dilute the protoplasts, mix well to terminate the reaction, centrifuge at 200×g for 2 min and then remove the supernatant.

[0037] S23. Add 0.5 mL of W5 solution to wash, mix slowly, and incubate in the dark for 15-20 h. Centrifuge to collect protoplasts, and observe the transformed protoplasts using a laser confocal microscope. Observe the luminescence of the protoplasts under GFP, Bright Field, and Merged channels to evaluate transformation efficiency and gene expression.

[0038] The conversion efficiency of orchard protoplasts is greater than 75%, and the protoplast viability reaches over 85%.

[0039] Example 3: A method for preparing and transforming orchard protoplasts proposed in this invention. Compared with Example 1 or Example 2, this example details W5 solution, MMG solution, enzymatic hydrolysate, and PEG solution.

[0040] In S11, the enzymatic hydrolysate is composed of cellulase, analyte, mannitol, MES, KCl, CaCl2, BSA, and β-mercaptoethanol. Specifically, the hydrolysate composition is: 1.5% cellulase, 0.8% analyte, 0.6M mannitol, 10mM MES, 10mM KCl, 10mM CaCl2, 0.11% BSA, and 2mM β-mercaptoethanol, where BSA is bovine serum albumin. The preparation method of the hydrolysate is as follows: 1.5% (w / v) cellulase, 0.8% (w / v) analyte, 0.6M mannitol, 10mM MES, 10mM KCl, 10mM CaCl2, 0.11% BSA, and 2mM β-mercaptoethanol. The enzyme hydrolysate was mixed with ES (pH=5.7) and 10 mM KCl, and pretreated in a 55℃ water bath for 10 min to inactivate non-specific enzymes. After the solution cooled to room temperature, 10 mM CaCl2, 0.11% (w / v) BSA, and 2 mM β-mercaptoethanol were added. In addition to cellulase and ionizing enzyme, the hydrolysate also contained mannitol, MES, KCl, CaCl2, BSA, and β-mercaptoethanol. These components stabilize osmotic pressure, maintain protoplast activity, and regulate pH, thus facilitating the preparation of orchardgrass protoplasts and improving their yield and activity. The results showed that using the described hydrolysate with orchardgrass leaves as material, orchardgrass protoplasts can be prepared efficiently, with high yield, activity, and transformation efficiency. This provides an important foundation for subsequent research on orchardgrass gene function and genetic trait improvement.

[0041] The W5 solution formulation is: 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 2 mM MES.

[0042] The MMG solution formula is: 0.4M mannitol, 15mM MgCl2, and 4mM MES. The pH is adjusted to 5.7, and the solution is sterilized at 110℃ before use.

[0043] The PEG solution was formulated as follows: 0.3M mannitol, 100mM CaCl2, and 20% (w / v) PEG4000.

[0044] In addition, W5 solution, MMG solution, enzymatic hydrolysate and PEG solution all need to be filtered through a 0.22μm microporous membrane for sterilization before use.

[0045] In summary, this invention achieves highly efficient and specific decomposition of the cell wall of orchardgrass leaves by combining the components and concentration ratios of the enzymatic hydrolysis solution. Cellulase is one of the main components in cell wall degradation, effectively promoting cell wall decomposition. The role of the dissociative enzyme is to degrade the middle lamella connecting adjacent cells, thus separating the cells. The addition of mannitol and MES can stabilize the external osmotic pressure, ensuring the stability and activity of protoplasts. KCl and CaCl2 provide an ionic environment, maintaining cell membrane potential and stability, and improving protoplast stability. The addition of BSA can reduce physical damage to the cell membrane and direct damage to the enzyme during enzymatic hydrolysis. Furthermore, mercaptoethanol can neutralize reactive oxygen species, maintaining the reduced thiol state of proteins, thereby achieving antioxidant effects and improving cell survival rate.

[0046] The core of protoplast transformation is to allow exogenous DNA to enter the protoplast and eventually the cell nucleus. PEG4000 is the key reagent, causing DNA to form microprecipitates on the cell membrane surface, increasing the local concentration. A 20% concentration of PEG4000 can promote the fusion of plasmid DNA with the protoplast membrane, helping to improve transformation efficiency. 0.3M mannitol maintains a hyperosmolar environment, preventing protoplast rupture. This PEG transformation solution, through a synergistic combination of physicochemical and biological methods, efficiently introduces exogenous DNA into plant protoplasts while relying on osmotic stabilizers to protect cell viability, thereby improving the success rate and stability of transformation and providing a more reliable foundation for subsequent gene expression analysis and functional studies.

[0047] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited thereto. Various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention.

Claims

1. A method for preparing orchardgrass protoplasts, characterized in that, Includes the following steps: S11. Take the leaves of the orchardgrass, put them into an Erlenmeyer flask and add the enzyme hydrolysate to soak them completely; S12. Place the soaked duckgrass in a vacuum dish and vacuum it. S13. Place the solution in a horizontal low-speed shaker in the dark for further enzymatic hydrolysis to obtain an enzymatic hydrolysate containing protoplasts. S14. Rinse the tissue fragments with W5 solution once or twice, collect the filtrate, and centrifuge at low speed to see a turbid precipitate. S15. Discard the supernatant, wash with W5 solution, and resuspend the protoplasts. S16. Centrifuge at low speed, discard the supernatant, and resuspend in MMG solution as needed.

2. The method for preparing orchardgrass protoplasts according to claim 1, characterized in that, In S11, the enzymatic hydrolysate is a mixture of cellulase, ionizing enzyme, mannitol, MES, KCl, CaCl2, BSA and β-mercaptoethanol.

3. The method for preparing and transforming orchardgrass protoplasts according to claim 2, characterized in that, The specific composition of the enzymatic hydrolysate is as follows: 1.5% cellulase, 0.8% ionase, 0.6M mannitol, 10mM MES, 10mM KCl, 10mM CaCl2, 0.11% BSA and 2mM β-mercaptoethanol, where M represents mol / L.

4. The method for preparing and transforming orchardgrass protoplasts according to claim 1, characterized in that, In S11, the leaves of the orchardgrass are shredded and placed into an Erlenmeyer flask.

5. The method for preparing and transforming orchardgrass protoplasts according to claim 1, characterized in that, In S14, the enzymatic hydrolysate obtained in S13 is filtered through a 70μm filter membrane.

6. A method for transforming orchardgrass protoplasts, using the protoplasts described in claim 1, characterized in that, Includes the following steps: S21. Mix the plasmid and protoplast cells thoroughly, add PEG solution, and incubate at room temperature in the dark. Education; S22. Dilute the protoplasts with W5 solution after incubation, mix well to terminate the reaction, and then... The heart goes to the highest purity; S23. After centrifugation, add W5 solution, mix slowly, and incubate in the dark for 15-20 h. Protoplasts were collected by centrifugation.

7. The method for transforming orchardgrass protoplasts according to claim 6, characterized in that, The conversion efficiency of orchard protoplasts is greater than 75%, and the protoplast viability reaches over 85%.

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