Rapid extraction method of cherry leaf protoplast

Abstract

The invention discloses a rapid extraction method of cherry seedling leaf protoplast and application of the cherry seedling leaf protoplast in protein subcellular localization analysis, and belongs to the technical field of fruit tree molecular breeding. According to the method, tender leaves of cherry seedlings are adopted as materials, and the enzymolysis condition is optimized, so that the protoplast is prevented from being damaged by polysaccharides and polyphenols. Compared with an existing cherry protoplast preparation technology, the method has the advantages that the material culture period is effectively shortened, the operation difficulty is simplified, the preparation means are enriched, a large amount of protoplast can be efficiently prepared and used for subcellular localization analysis, and the homologous transformation efficiency of cherries and the accuracy of protein localization are improved.

Description

Technical Field

[0001] The invention belongs to the technical fields of molecular biotechnology and genetic engineering, and particularly relates to a method for efficiently preparing protoplasts of a Chinese cherry 'Agate Red'. Background Art

[0002] Chinese cherry (Cerasus pseudocerasus (Lindl.) G.Don), commonly known as cherry or small cherry, belongs to the genus Cerasus in the subfamily Prunoideae of the Rosaceae family. It is a unique stone fruit crop native to my country with a cultivation history of over 3,000 years. Known as the "first fruit of spring" for its early flowering and ripening, Chinese cherry has a high economic value. The Agate Red Cherry (Cerasus pseudocerasus Lindl.) is a high-quality local specialty of Guizhou Province and has high economic value.

[0003] In current basic research and breeding efforts, the inability to establish a stable genetic regeneration system for cherry species has hampered research at the cellular level and related to genetic engineering. Therefore, establishing an efficient cherry protoplast transformation system, using cherry protoplasts to regenerate complete plants, will provide technical support for subsequent cherry research.

[0004] Protoplast technology first emerged in model plants such as tobacco and Arabidopsis. Protoplast culture has become a key tool for analyzing target gene function, protein and organelle localization, protein-protein interactions, and cellular metabolic processes. However, due to the limitations of traditional fruit tree breeding, such as long production cycles, large land requirements, and low hybridization efficiency, it is difficult to breed high-quality new varieties that meet human needs in the short term. Therefore, fruit trees are also an important tool in the field of fruit tree molecular breeding technology. Efficient and rapid preparation of highly viable protoplasts is crucial for establishing a stable protoplast transient expression system.

[0005] In stone fruit trees, selecting the right protoplast isolation material is a key factor in determining the success of protoplast culture. In theory, living cells from any plant can serve as a source of protoplasts. Therefore, this study aimed to optimize the enzymatic hydrolysis process for efficient cherry protoplast preparation. Summary of the Invention

[0006] In view of the above problems, the object of the present invention is to provide a method for extracting protoplasts from cherry leaves and to improve the efficiency of protoplast extraction.

[0007] The technical solution of the present invention is: a method for rapidly extracting protoplasts from cherry leaves, comprising the following steps:

[0008] (1) Cultivation of agate red cherry seedlings: After agate red cherry seeds germinate, they are potted in a greenhouse and grown to a seedling age of 20 days; (2) Young leaves of the cherry seedlings without damage or disease are selected from step (1), cut into thin strips, and added to an enzymatic hydrolysis solution; (3) The young leaf strips added with the enzymatic hydrolysis solution in step (2) are enzymatically hydrolyzed for 10-18 hours; the state of the protoplasts is observed every 2 hours; W5 solution is added to terminate the enzymatic hydrolysis, and the enzymatic hydrolysis solution is filtered through a sterile cell sieve to remove unhydrolyzed leaves and broken cell impurities, and the plant tissue is gently crushed to release more protoplasts, and the filtrate is collected in a sterile centrifuge tube; (4) The sterile centrifuge tube in step (3) is centrifuged at room temperature, the supernatant is removed, the W5 solution is added, the mixture is slowly pipetted and mixed, and an ice bath is performed; the mixture is centrifuged again, the supernatant is removed, and the mixture is resuspended with MMG solution to 1-3×105 cells / ml to obtain purified protoplasts.

[0009] The size of the young leaves used in step (2) is 20 mm×5 mm, and the size of the shredded leaves is 0.5-1 mm.

[0010] The enzymatic hydrolysis conditions in step (3) are 24° C., 60 rpm in the dark; the amount of W5 solution used is 5 ml, and the specification of the sterile cell sieve is 200 mesh; wherein the W5 aqueous solution is a sterile solution composed of a solute and a solvent.

[0011] In step (4), the first centrifugation time is 5 minutes, and the second centrifugation time is 3 minutes; the amount of W5 added is 20 ml; the ice bath time is 30 minutes; the pipette tip used for pipetting and mixing is a sheared pipette tip; the MMG aqueous solution is a sterile pre-cooled solution composed of solute and solvent.

[0012] The method further comprises: staining the purified protoplasts obtained in step (4), gently mixing 1 ml of protoplasts with 5 μl of DAPI, and culturing the cells at room temperature for 10 minutes; and observing under a microscope after the culturing is completed.

[0013] Beneficial effects of the present invention:

[0014] (1) The present invention utilizes young leaves of cherry seedlings to prepare protoplasts. The materials are not restricted by seasons and do not require sterile tissue culture, so the materials are relatively easy to obtain.

[0015] (2) The present invention establishes a corresponding enzymatic hydrolysis system based on the characteristics of cherry leaves, and the protoplasts obtained are complete and stable in morphology, with high yield, and the number can reach 1.6×10 7 / g·FW, and the protoplast activity was as high as over 81.2%.

[0016] (3) The protoplast isolation technology proposed in the present invention is characterized by its ease of operation and strong applicability. The purification process is simple and easy to implement, effectively reducing the content of cell debris and other impurities, thereby significantly improving the purity of the protoplasts.

[0017] (4) The present invention has certain reference significance for the isolation of protoplasts from woody plants. It provides a solid technical foundation for the subsequent in-depth research on protein subcellular localization and transient gene expression using protoplasts. Through this method, researchers can more quickly and accurately explore the molecular mechanisms within plant cells and promote scientific progress in related fields.

[0018] (5) Compared with the protoplast preparation method described in "Method for Transforming Sweet Cherry with Target Gene and Its Application in Transient Transformation of Sweet Cherry Protoplasts", the superiority of the protoplast preparation method for Agate Red Cherry is reflected in the materials, operation, and yield and vitality. This method uses young leaves of Agate Red Cherry, which are easy to obtain and have a short cycle. The operation steps are simpler and the enzymatic hydrolysis conditions are mild and easy to control.

[0019] Convenience of material acquisition: Agate Red Cherry uses young leaves from 20-day-old seedlings. Compared with sweet cherries, which use the pulp of fruits at a specific developmental stage (10-40DAFB), leaf materials are easier to obtain. There is no need to wait for fruit development and is not restricted by the seasonality of fruits. Materials can be obtained at any time for experiments.

[0020] Material processing complexity: Sweet cherry pulp requires multiple steps, including fruit disinfection, seed coat removal, callus induction, and multiple subcultures. This is a tedious and time-consuming process. Agate red cherry leaves only require cleaning, disinfection, deveining, and shredding. This reduces the number of processing steps and is simple, allowing for rapid production of material for enzymatic hydrolysis.

[0021] The enzymatic hydrolysis conditions are mild and easy to control: the enzymatic hydrolysis of Agate Red Cherry leaves is carried out in the dark at 24℃ and 60rpm for 14 hours. The enzymatic hydrolysis of sweet cherry suspended cells must first be treated with CPW13M and then incubated with a specific enzyme solution at weak light, 22-24℃, and 50r / min for 16-20 hours. The enzymatic hydrolysis conditions of Agate Red Cherry are milder, have low equipment requirements, and are easy to control.

[0022] Simplified process: Purification of Agate Red Cherry protoplasts requires only two centrifugation washes, whereas sweet cherry protoplasts require multiple centrifugation and washes, and involve multiple solutions (CPW13M, CPW25S, etc.). The Agate Red Cherry process is simplified, reducing steps and time, and minimizing contamination risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the morphology of the purified cherry leaf protoplasts under a 40× optical microscope;

[0024] Figure 2This is a morphological image of the cherry leaf protoplasts after staining under a 40× optical microscope. DETAILED DESCRIPTION

[0025] The present invention is described in detail below with reference to the embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several adjustments and improvements can be made without departing from the present invention.

[0026] Before the following experiments, the following preliminary preparations should be performed:

[0027] Experimental tools such as blades, cell sieves, culture dishes, filter membranes, centrifuge tubes, and pipette tips must be sterilized at 121°C for 40 minutes; the clean bench must be wiped with 75% alcohol, irradiated with ultraviolet light for 20 minutes, and ventilated for 10 minutes.

[0028] The enzymatic hydrolysate with the corresponding formula is shown in the attached table. The pH is adjusted to 5.7 and sterilized by filtration through a 0.45 μm filter membrane.

[0029] Appendix 1 Enzyme hydrolysate formula

[0030]

[0031] Example 1:

[0032] 1. Material handling

[0033] Cultivation of Agate Red Cherry Seedlings: After the Agate Red Cherry seeds germinate, they are planted in pots in the greenhouse and grown to 20 days old.

[0034] 2. Enzymatic hydrolysis material selection

[0035] Select young leaves that are undamaged and disease-free and are about 20mm×5mm in size. Wash them twice with sterile water in a clean bench, then disinfect them with 75% alcohol for 30s, wash them with sterile water 4-5 times, remove the main veins of the leaves, cut them into 0.5-1mm thin strips, and completely immerse them in enzymatic hydrolysate. The composition of the enzymatic hydrolysate is shown in Table 1. Prepare and use it immediately.

[0036] 3. Leaf enzymatic hydrolysis

[0037] The enzymatic hydrolysis was carried out at 24°C and 60 rpm in the dark for 14 h; the protoplast status was observed at 2-h intervals; 5 ml of W5 solution was added to terminate the enzymatic hydrolysis. The composition of W5 solution is shown in Appendix 2. The enzymatic hydrolysis solution was filtered through a 200-mesh sterile cell sieve to remove impurities such as unenzymatically hydrolyzed leaves and broken cells, and the plant tissue was gently crushed to release more protoplasts. The filtrate was collected in a sterile centrifuge tube.

[0038] Appendix 2: Composition of W5 solution

[0039]

[0040] 4. Protoplast Purification

[0041] The obtained protoplasts were placed in a sterile centrifuge tube, centrifuged at 100 × g, speed up and down 2, centrifuged at room temperature for 5 min, removed the supernatant, added 20 ml of W5 solution, and ice bathed for 30 min; centrifuged at 100 × g, speed up and down 2, centrifuged at room temperature for 3 min, removed the supernatant, and resuspended in MMG solution to 1-3 × 10 5 The composition of the MMG solution is shown in Table 3. Gently pipette the purified protoplasts evenly, then pipette 10 μl onto a hemocytometer. Cover with a coverslip and count under an inverted microscope. Count three times and take the average. Calculate the protoplast yield per gram of leaves based on the amount of leaves added to the enzyme solution. The protoplasts obtained are intact, stable, and have a high yield, reaching 1.6×10 7 / g·FW, and the protoplast activity was as high as over 81.2%.

[0042] Table 3: Composition of MMG solution

[0043]

[0044] 5. Protoplast Staining

[0045] The purified protoplasts were stained by gently flicking 1 ml of protoplasts with 5 μl of DAPI. The cells were then incubated at room temperature for 10 minutes. Microscopic examination of the DAPI-stained protoplasts revealed multiple blue fluorescent protoplast structures. The protoplasts were mostly round or nearly round in shape, with some individual protoplasts exhibiting varying fluorescence intensities. No obvious abnormal morphology or unusual fluorescence aggregation was observed, indicating that the protoplasts were structurally intact under the staining conditions, and the cell nuclei were effectively stained and visualized.

Claims

1. A method for rapid extraction of cherry leaf protoplasts, characterized in that: The method comprises the following steps: (1) cultivating agate red cherry seedlings: after the agate red cherry seeds germinate, they are potted in a greenhouse and grown to a 20-day-old seedling age; (2) Select undamaged and disease-free young leaves of the cherry seedlings in step (1), cut them into thin strips, and add them to the enzymatic hydrolyzate; (3) The young leaf filaments to which the enzymatic hydrolysis solution was added in step (2) were enzymatically hydrolyzed for 10-18 hours; the protoplast status was observed at intervals of 2 hours; W5 solution was added to terminate the enzymatic hydrolysis, and the enzymatic hydrolysis solution was filtered through a sterile cell sieve to remove unhydrolyzed leaves and ruptured cell impurities, and the plant tissue was gently crushed to release more protoplasts, and the filtrate was collected in a sterile centrifuge tube; (4) The sterile centrifuge tube in step (3) was centrifuged at room temperature, the supernatant was removed, W5 solution was added, and the mixture was mixed by slowly pipetting. The mixture was placed in an ice bath. Centrifuge again, the supernatant was removed, and the mixture was resuspended in MMG solution to a concentration of 1-3 × 10 5 / ml to obtain purified protoplasts.

2. A method for rapid extraction of cherry leaf protoplasts according to claim 1, characterized in that: The size of the young leaves used in step (2) is 20 mm × 5 mm, and the size of the shredded leaves is 0.5-1 mm.

3. A method for rapid extraction of cherry leaf protoplasts according to claim 1, characterized in that: The enzymatic hydrolysis conditions in step (3) are 24° C., 60 rpm in the dark; the amount of W5 solution used is 5 ml, and the specification of the sterile cell sieve is 200 mesh; wherein the W5 aqueous solution is a sterile solution composed of solute and solvent.

4. A method for rapid extraction of cherry leaf protoplasts according to claim 1, characterized in that: In step (4), the first centrifugation time was 5 min, and the second centrifugation time was 3 min; the amount of W5 added was 20 ml; the ice bath time was 30 min; the pipette tip used for pipetting and mixing was a sheared pipette tip; The MMG aqueous solution is a sterile pre-cooled solution consisting of a solute and a solvent.

5. A method for rapid extraction of cherry leaf protoplasts according to claim 1, characterized in that: Also includes: The purified protoplasts obtained in step (4) were stained by gently flicking 1 ml of protoplasts and 5 μl of DAPI to mix well. The cells were cultured at room temperature for 10 min. After the culture was completed, microscopic observation was performed.

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