Efficient preparation and transformation method of immature xylem protoplast of catalpa bungei

Abstract

The invention relates to a method for efficiently preparing and converting immature xylem protoplast of catalpa bungei. The invention particularly relates to an enzyme solution for preparing immature xylem protoplast of catalpa bungei as well as a preparation method and a conversion method of the enzyme solution. The preparation method provided by the invention can realize efficient preparation and conversion of the protoplast.

Description

Technical Field

[0001] This invention belongs to the field of plant protoplast preparation technology, specifically, it relates to a method for preparing and efficiently converting immature xylem protoplasts from Catalpa trees. Background Technology

[0002] The Chinese catalpa (scientific name: *Catalpa bungei* CAMey), a perennial tree widely cultivated in China, is highly valued for its straight trunk, adaptability, and hard wood, making it an excellent timber for construction and furniture. Its beautiful shape and abundant foliage contribute to sound insulation, noise reduction, and dust trapping. The catalpa's vibrant flowers and short flowering period, along with its non-wind-borne pollen, have no negative impact on air quality, making it increasingly popular in landscaping projects in recent years. Furthermore, the catalpa possesses rich medicinal and edible value. Its leaves and bark can be used medicinally to treat various ailments. The seeds are rich in nutrients and can be processed into food.

[0003] The state attaches great importance to the development of the catalpa industry, and the "National Reserve Forest Construction Plan" designates catalpa as an important timber species with broad market prospects. However, the long growth cycle and slow maturity of catalpa trees lead to relative scarcity of resources, making the breeding of superior varieties an urgent task. Traditional catalpa breeding methods face challenges such as long cycles and difficulty in effectively integrating superior traits.

[0004] Against this backdrop, plant protoplast isolation and transformation technology, as an advanced biotechnology, has been successfully applied in numerous plants, playing a crucial role in advancing plant science research and breeding. Currently, most protoplast preparations focus on young herbaceous plants, while reports on protoplast preparation from woody plants are scarce. The main challenge lies in maintaining the osmotic pressure of the protoplasts, which can lead to cell rupture. Furthermore, the high degree of cellulose and lignification in the immature xylem of woody plants further complicates protoplast preparation.

[0005] Therefore, developing an efficient and feasible method for the preparation and transformation of immature xylem protoplasts from Catalpa trees is of great significance for accelerating the breeding process of Catalpa trees and deepening scientific research. Summary of the Invention

[0006] This invention provides an efficient method for the preparation and transformation of immature xylem protoplasts from Catalpa trees.

[0007] In a first aspect of the invention, an enzyme solution for preparing immature xylem protoplasts of Catalpa tree is provided, the enzyme solution comprising the following components:

[0008] Cellulase R-10 2 w / v%, macerozyme R-10 2 w / v%, pectolase Y-23 3 w / v%, 0.5 M mannitol, 10 mM morpholine ethanesulfonic acid, 5 mM glucose, 5 mM MgSO4, 3 mM sodium succinate, polyvinylpyrrolidone 1 w / v%, 10 mM CaCl2, BSA 0.1 w / v%, and β-mercaptoethanol 0.3 v / v.

[0009] In another preferred embodiment, the mass ratio of cellulase R-10, dissociative enzyme R-10, and pectinase Y-23 is 1:1:1.5.

[0010] In another preferred embodiment, the pH value of the enzyme solution is 5.6 to 5.8, more preferably 5.7.

[0011] In another preferred embodiment, the cellulase is cellulase R-10 with an enzyme activity of 500 U / g.

[0012] In another preferred embodiment, the analyte is analyte R-10 with an enzyme activity of 800 U / g.

[0013] In another preferred embodiment, the pectinase is pectinase Y-23 with an enzyme activity of 1000 U / g.

[0014] In another preferred embodiment, the cellulase, dissociative enzyme, and pectinase are branded as Yakult.

[0015] In a second aspect of the present invention, a method for preparing an enzyme solution as described in the first aspect of the present invention is provided, comprising the following steps:

[0016] (s1) Prepare 8 ml of stock solution containing 0.5 M mannitol, and add 10 mM morpholine ethanesulfonic acid and 5 mM glucose in sequence. Shake at room temperature to dissolve and adjust the pH to 5.6-5.8.

[0017] (s2) Add 2 w / v% cellulase R-10, 2 w / v% macerozyme R-10 and 3 w / v% pectolase Y-23;

[0018] (s3) After stirring and dissolving, heat in a water bath at 55°C for 10 minutes, cool naturally to room temperature, and then add 5mM MgSO4, 3mM sodium succinate, 1w / v% polyvinylpyrrolidone, 10mM CaCl2, 0.1w / v% BSA and 0.3v / v% β-mercaptoethanol.

[0019] (s4) Make up to 10ml, then store the enzyme digest in the dark for later use.

[0020] In a third aspect of the invention, the use of the enzyme solution as described in the first aspect of the invention or the enzyme solution prepared by the preparation method described in the second aspect of the invention in the preparation of immature xylem protoplasts of Catalpa tree is provided.

[0021] In a fourth aspect of the invention, a method for preparing immature xylem protoplasts of Catalpa tree is provided, comprising the steps of:

[0022] (s1) Provide immature xylem tissue of Catalpa tree, wherein the xylem tissue is the surface tissue of a stripped Catalpa seedling stem segment;

[0023] (s2) Equilibration: The xylem tissue was placed in a 0.4-0.6M mannitol solution for equilibration for 10-30 min, and then centrifuged at 100g for 5 min.

[0024] (s3) Enzymatic hydrolysis: The mannitol solution in the centrifuged solution is removed, and the enzyme solution described in the first aspect of this invention is added to the treated tissue. Enzymatic hydrolysis is performed under dark conditions (dark conditions mean protection from light, using a brown digestion dish wrapped in aluminum foil) to obtain enzymatically hydrolyzed cell solution; and

[0025] (s4) Release: Add W5 solution with 1 volume of the enzymatic hydrolysate to the enzymatic hydrolysate and release for 8-10 min;

[0026] (s5) After filtering the released cell fluid using a cell sieve, repeat step (s4) to obtain a second released cell fluid.

[0027] (s6) The cell sap after the secondary release is filtered using a cell sieve, and the filtrate is centrifuged to remove the supernatant, thereby obtaining the immature xylem protoplasts of the catalpa tree.

[0028] In another preferred embodiment, in step (s1), the ratio of the enzyme solution to the xylem tissue is 5-10 ml: 2-3 g.

[0029] In another preferred embodiment, the stem segment of the catalpa seedling is taken from a catalpa seedling cultivated to a height of 80cm. The 40cm section above the root is selected, and the segment is cut to a length and width of 1cm. Specifically, this involves scraping the surface tissue of the main stem of the stripped segment using a sterilized scalpel. Preferably, a new, sharp scalpel is used, and the force applied is gentle to prevent the generation of excessive broken cells and cell sap. Excessive cell sap can negatively impact the subsequent survival and transformation of protoplasts.

[0030] In another preferred embodiment, in step (s3), the enzymatic hydrolysis temperature is 24°C to 28°C, preferably 26°C.

[0031] In another preferred embodiment, in step (s3), the enzymatic hydrolysis time is 12 to 16 hours.

[0032] In another preferred embodiment, during step (s3), the rotation speed of the enzymatic hydrolysis shaker is 30-50 r / min, preferably 40 r / min.

[0033] In another preferred embodiment, during enzymatic hydrolysis, the shaking speed is 30–50 r / min, the hydrolysis time is 12–16 h, and the hydrolysis temperature is 24 °C–28 °C.

[0034] In another preferred embodiment, after adding the enzyme solution described in the first aspect of the present invention to the treated tissue in step (s3), the mixture of enzyme solution and tissue is further evacuated for 30 minutes.

[0035] In another preferred embodiment, in step (s3), after obtaining the enzymatically hydrolyzed cell solution, a drop of the enzymatically hydrolyzed cell solution is examined under a microscope. If the cells are round and shiny, it indicates that the cells are in a healthy state, and step (s4) is continued; if the cells are flat and black, they are discarded, and step (s1) is restarted.

[0036] In another preferred embodiment, in step (s4), the release is performed on a horizontal rocker at a speed of 80 r / min.

[0037] In another preferred embodiment, in step (s4), the release time is 9 to 11 minutes, preferably 10 minutes.

[0038] In another preferred embodiment, the W5 solution comprises the following components: 2 mM morpholine ethanesulfonic acid, 5 mM glucose, 154 mM NaCl, 140 mM CaCl2, 5 mM KCl, 15 mM MgSO4, 3 mM sodium succinate, 5 mM sorbitol and 5 mM CuSO4.

[0039] In another preferred embodiment, the pH value of the W5 solution is 5.7.

[0040] In another preferred embodiment, the cell sieve is a cell sieve that has been rinsed with 1-2 ml of W5 solution, and the pore size of the cell sieve is 35-45 μm.

[0041] In another preferred embodiment, step (s4) includes the following steps: washing the enzymatic hydrolysate with one volume of W5 solution into an Erlenmeyer flask wrapped with tin foil, releasing the protoplasts on a horizontal shaker at 80 r / min for 10 min, filtering into another new 50 ml round-bottom centrifuge tube, repeating once, and collecting a total of two volumes of the W5 filtrate of the washed enzymatic hydrolysate.

[0042] Preferably, two tubes of protoplast filtrate are collected, and the protoplasts from the second tube are used for subsequent transformation. The enzymes in the first tube are released earlier than those in the second tube, and the enzyme solution contains a large number of cell fragments, which has a significant impact on the survival of protoplast cells and transformation efficiency.

[0043] In another preferred embodiment, the centrifugation speed is 100 g / min, the acceleration / deceleration rate is 1, the centrifugation time is 5 min, and the temperature is room temperature; after centrifugation, the supernatant is slowly aspirated with a 1 ml pipette, leaving 1.5 to 2.5 ml of supernatant; 1 ml of W5 solution is slowly added, and the mixture is gently shaken to disperse the protoplasts.

[0044] In another preferred embodiment, the tips of the pipettes are trimmed with scissors, and the burrs are removed by burning off the tips with an alcohol lamp to prevent the pipette from drawing liquid too quickly and damaging the protoplasts.

[0045] In a fifth aspect of the invention, a method for efficient conversion of immature xylem protoplasts of Catalpa tree is provided, comprising the steps of:

[0046] (i) Obtain protoplasts prepared by the method described in the second aspect of the present invention, and adjust the protoplasts with MMG solution to obtain a concentration of (2-4)×10⁻⁶. 6 A protoplast suspension of 1 protoplast per ml was prepared; and the protoplast suspension was placed in the dark and kept at room temperature for 60–90 min.

[0047] (ii) Centrifuge at room temperature, remove the supernatant with a pipette, resuspend in MMG solution to obtain a concentration of (2-4)×10⁻⁶. 6 Protoplast suspension per ml;

[0048] (iii) Mix the protoplast suspension obtained in step (ii) with the plasmid containing the target gene, add PEG-CaCl2 solution, mix gently, and incubate at room temperature in the dark for 10-20 min.

[0049] (iv) Add W5 solution, gently invert the container to stop the conversion, then centrifuge and remove the supernatant;

[0050] (v) Resuspend the transformed protoplasts in WI solution and incubate in the dark at 28°C for 12-16 h.

[0051] In another preferred embodiment, the PEG-CaCl2 solution is prepared by mixing 40% PEG4000, 0.5M mannitol and 100mM CaCl2, and placing the mixture in a water bath at 55°C for more than 1 hour to obtain the PEG-CaCl2 solution.

[0052] In another preferred embodiment, in step (iii), the volume of the protoplast suspension obtained in step (ii) is 100 μl. This allows for the performance of most experiments, including protein subcellular localization and bimolecular fluorescence complementation assays. The specific dosage can be adjusted in real-time according to the experiment.

[0053] In another preferred embodiment, the total amount of the plasmid containing the target gene is 1 to 5 μg, and the volume ratio of the plasmid to the protoplast suspension is (1 to 2): 10.

[0054] In another preferred embodiment, the plasmid is extracted using a large-scale extraction kit from QIAGEN. This kit effectively removes bacterial endotoxins without damaging the protoplast.

[0055] In another preferred embodiment, the plasmid is free of endotoxin.

[0056] In another preferred embodiment, the MMG solution comprises the following components: 0.5 M Mannitol, 5 m M glucose, 15 m M magnesium chloride, and 4 m M morpholine ethanesulfonic acid.

[0057] In another preferred embodiment, the pH value of the MMG solution is 5.7.

[0058] In another preferred embodiment, the W5 solution comprises the following components: 2 mM morpholine ethanesulfonic acid, 5 mM glucose, 154 mM NaCl, 140 mM CaCl2, 5 mM KCl, 15 mM MgSO4, 3 mM sodium succinate, 5 mM sorbitol and 5 mM CuSO4.

[0059] In another preferred embodiment, the pH value of the W5 solution is 5.7.

[0060] In another preferred embodiment, the WI solution comprises the following components: 0.5 M mannitol, 5 m M glucose, 4 m M KCl, and 4 m M morpholine ethanesulfonic acid.

[0061] In another preferred embodiment, the pH value of the WI solution is 5.7.

[0062] In another preferred embodiment, in steps (iii) and (v), the culture vessel is a 24-well cell culture plate rinsed with 5% fetal bovine serum solution.

[0063] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0064] Figure 1 The images show (A) immature xylem protoplasts of Catalpa tree separated using unoptimized enzymatic hydrolysate in Example 1, and (B) the separation effect of optimized immature xylem protoplasts of Catalpa tree; images under 40x magnification, scale bar at 50 μm; red markers indicate intact protoplasts in the same field of view.

[0065] Figure 2 The image shows the laser confocal microscopy detection of the exogenous gene GFP in protoplasts of immature xylem tissue extracted from Catalpa elata in Example 2. Bright field represents the observation results of transformed Catalpa elata protoplasts under bright field conditions, with a scale bar of 100 μm. (A) shows the laser confocal microscopy detection of the exogenous gene GHD7-GFP in Catalpa elata protoplasts treated with the optimized enzyme solution (enzyme solution B), and (B) shows the laser confocal microscopy detection of the exogenous gene GHD7-GFP in Catalpa elata protoplasts treated with the unoptimized enzyme solution (enzyme solution A).

[0066] Figure 3 The results of Western blot analysis of the exogenous protein gene (GHD7) extracted from immature xylem tissue of Catalpa tree in Example 3 are shown.

[0067] Figure 4 The results of an immunoprecipitation assay show that immature xylem tissue extracted from Catalpa bungei in Example 4 expressed two tag proteins (BZIP63-GFP and BZIP63-FLAG) and their interaction was subsequently detected.

[0068] Figure 5 and Figure 6 Micrographs of protoplasts prepared using enzyme hydrolysate B and enzyme hydrolysate A are shown (randomly selected regions).

[0069] Figures 7-14 Micrographs of protoplasts prepared using the enzymatic hydrolysates of Comparative Examples 1-8 are shown (randomly selected regions).

[0070] Note: Figures 5-14 The microscope images were taken at the same magnification, and the randomly selected areas were all the same size. The areas marked by the red arrows in the images are the prepared protoplasts. Detailed Implementation

[0071] Through extensive and in-depth research and screening, the inventors discovered an enzyme solution and a method for the efficient preparation of immature xylem protoplasts from Catalpa bungei. Using this enzyme solution or method, immature xylem protoplasts from Catalpa bungei were efficiently prepared, exhibiting good cell viability. Based on these protoplasts, efficient transformation of exogenous target genes was achieved, with a transformation efficiency of approximately 50%. This invention is based on these findings.

[0072] the term

[0073] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0074] As used in this article, "protoplast" refers to the part of a plant cell remaining after the cell wall has been removed experimentally. It includes the cell membrane, cytoplasm, and nucleus. If the cell wall is not completely removed, the resulting structure is called a protoplast sphere. Furthermore, animal cells, lacking a cell wall, can also be considered protoplasts. The term protoplast originates from protoplasm, which refers to the collective living matter that makes up a cell.

[0075] As used in this article, "enzyme solution" and "enzyme hydrolysate" have the same meaning and can be used interchangeably. They both refer to enzyme solutions that can be used to prepare immature xylem protoplasts of Catalpa trees.

[0076] Preparation of immature xylem protoplasts

[0077] The xylem is one of the vascular tissues of a plant, and its main functions include: transporting water and inorganic salts (from the roots upwards to the stems, leaves, etc.), providing mechanical support (keeping the plant upright), and storing nutrients (some xylem cells can store substances such as starch).

[0078] The cell walls of xylem tissue cells are highly lignified, and xylem tissue contains few living cells while containing a large number of dead cells and their metabolites. Therefore, protoplasts prepared from xylem tissue are more prone to rupture. Furthermore, lignified cells become dead cells after maturation (such as vessels and fibers), with only a few xylem parenchyma cells surviving, resulting in a scarce number of usable protoplasts. Although immature xylem has a lower degree of lignification and thinner cell walls than mature xylem, the preparation of protoplasts from immature xylem of catalpa trees, a woody plant with secondary growth, is more difficult than that from young herbaceous plants such as Arabidopsis thaliana and grasses with only primary growth such as rice.

[0079] Therefore, the preparation of immature xylem protoplasts requires a comprehensive balance between enzymatic hydrolysis efficiency and cell activity, which is significantly more difficult than that of non-lignified leaves and low-lignified root tips.

[0080] Generally, prepared protoplasts are used for subsequent transformations. Furthermore, transfection efficiency is typically observed using fluorescence to determine cell viability. However, the method of this invention uses two plasmids for transformation and verifies protein-protein interactions. The results demonstrate that the protoplasts of this invention not only exhibit good viability but also demonstrate high expression efficiency of the target plasmid.

[0081] The main advantages of this invention include:

[0082] (a) Using the enzyme solution for protoplast preparation of the present invention, the resulting immature xylem tissue protoplasts from *Catalpa bungei* exhibit good integrity, high yield, and strong activity. The preparation efficiency reaches 3.2 × 10⁻⁶. 6 Protoplasts per ml. See attached micrographs for details of the prepared protoplasts. Figure 5 .

[0083] (b) The protoplasts prepared by the method of this invention can be transformed using the efficient transformation method of this invention, achieving a high transformation efficiency of 40% to 50%. This is sufficient to support subsequent biochemical and molecular biological research and has excellent application prospects.

[0084] (c) The enzyme hydrolysate, MMG, W5 and WI solutions used in this invention all contain a certain amount of glucose, which can provide energy for protoplasts and improve the survival rate of protoplasts during extraction and incubation.

[0085] (d) The protoplast preparation method and transformation method of the present invention have potential application value in exogenous gene expression, subcellular localization of functional genes, immunoprecipitation screening and verification of interacting proteins, and breeding of new varieties of Catalpa.

[0086] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0087] Experimental materials

[0088] The pUC-35S-GFP and pUC-35S-FLAG vectors used in the embodiments of this application are conventional plant expression vectors;

[0089] Cellulase R-10, dissociation enzyme R-10 and pectinase Y-23 were purchased from Yakult.

[0090] Among them, the cellulase is cellulase R-10 with an enzyme activity of 500 U / g, the cleavage enzyme is cleavage enzyme R-10 with an enzyme activity of 800 U / g, and the pectinase is pectinase Y-23 with an enzyme activity of 1000 U / g.

[0091] PEG4000 was purchased from Sigma.

[0092] FLAG antibody and GFP antibody were purchased from ABclonal Biotechnology Co., Ltd.

[0093] WB / IP lysis buffer, Protein A / G agarose packing material, and ECL chemiluminescence detection reagent were purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.

[0094] Example 1: Isolation and transformation of protoplasts from immature xylem tissue of Catalpa tree

[0095] 1.1 Isolation of protoplasts

[0096] (1) Take the tissue containing immature xylem from the main stem of the catalpa tree after peeling the bark, and immediately put it into 0.5M mannitol for 10 minutes to prepare the enzymatic hydrolysate; mannitol is a necessary condition for maintaining cell osmotic pressure. The protoplast cells of different species and tissues are different in size and have different osmotic pressure. After screening, the optimal concentration of mannitol used for the catalpa tree in this invention is 0.5M.

[0097] The unoptimized enzyme solution (enzyme solution A) included 1.0 w / v of cellulase R-10, 1.0 w / v of macerozyme R-10, 0.6 M mannitol, 10 m M morpholinoethanesulfonic acid, 10 m M CaCl2, and 0.1% w / v% BSA; the pH of the enzyme solution was 5.7.

[0098] The optimized enzyme solution (enzyme solution B) comprises 2 w / v of cellulase R-10, 2 w / v of macerozyme R-10, 3 w / v of pectinase Y-23, 0.5 M mannitol, 5 m M glucose, 10 m M morpholine ethanesulfonic acid, 5 m M MgSO4, 3 m M sodium succinate, 1 w / v% polyvinylpyrrolidone, 10 m M CaCl2, 0.1 w / v% BSA, and 0.3 v / v% β-mercaptoethanol; the pH of the enzyme solution is 5.7.

[0099] (2) After removing the mannitol solution, the treated tissue fragments were added to an enzyme solution placed at 26°C for light-protected enzymatic hydrolysis; the tissue was then slowly shaken on a shaker for 15 hours at a speed of 50 r / min.

[0100] (3) Release protoplasts on a horizontal shaker at 80 r / min, add 1 volume of W5 of enzyme hydrolysate and release for 8-10 min;

[0101] The above W5 solution contains 2 mM morpholine ethanesulfonic acid, 5 mM glucose, 154 mM NaCl, 140 mM CaCl2, 5 mM KCl, 15 mM MgSO4, 3 mM sodium succinate, 5 mM sorbitol, and 5 mM CuSO4; the pH value is 5.7.

[0102] (4) Filter the enzymatically digested cell solution using a cell sieve and filter it into a 50ml round-bottom centrifuge tube.

[0103] (5) Wash the remaining enzyme hydrolysate after filtering the enzyme hydrolysate in step (3) with W5 solution of 1 volume of enzyme hydrolysate into the above-mentioned triangular flask wrapped with tin foil. Release the protoplasts for 10 min at 80 r / min on a horizontal shaker. Filter into another new 50 ml round bottom centrifuge tube. Repeat the release once and collect it into another centrifuge tube. Collect a total of 2 volumes of W5 filtrate for washing the enzyme hydrolysate.

[0104] (6) Centrifuge the protoplast suspensions obtained from the two releases to obtain two tubes of protoplasts from immature xylem tissue of Catalpa tree;

[0105] The centrifugation speed was 100 g / min, the acceleration / deceleration rate was 1, and the centrifugation time was 5 min at room temperature.

[0106] (7) After centrifugation, use a 1ml pipette to slowly remove the supernatant, leaving 1.5 to 2.5ml of supernatant; slowly add 1ml of W5 solution and gently shake to disperse the protoplasts.

[0107] Among them, the immature xylem protoplasts (second release) of Catalpa tree separated using unoptimized and optimized enzymatic hydrolysates were respectively as follows: Figure 1 As shown. Among them, Figure 1 A represents protoplasts obtained from unoptimized enzymatic digestion, indicating a low number of protoplasts; while Figure 1 B represents the protoplasts obtained from digestion with the optimized enzymatic hydrolysate, showing a significant increase in protoplast number. Cell counts were 35 (unoptimized) and 94 (optimized). This result indicates that the optimized enzymatic hydrolysate can significantly improve cell separation from the immature xylem of *Catella macrantha*. Figure 1 A and Figure 1 B represents observations from a single field of view; the other field-of-view observations are similar. Figure 1 A and Figure 1 The case of B is similar. Therefore, Figure 1 A and Figure 1 The results observed by B can be considered representative.

[0108] 1.2 Transformation

[0109] (8) Take 10 μl of protoplast suspension, count the cells using a hemocytometer, and adjust the protoplast cell concentration to (2-4) × 10⁻⁶ using MMG solution. 6 The protoplast suspension was prepared at 1 / ml and stored in the dark for 60–90 minutes at room temperature.

[0110] The MMG solution comprises 0.5M Mannitol, 5mM glucose, 15mM magnesium chloride, 4mM morpholine ethanesulfonic acid, and has a pH of 5.7.

[0111] (9) Centrifuge at 100 g / min with a speed of 1 for 5 min at room temperature. Aspirate the supernatant with a 1 ml pipette, resuspend the cells in MMG, and adjust the protoplast concentration to (2-4) × 10⁻⁴. 6 per ml.

[0112] (10) Mix with a plasmid containing the target gene (expressing GFP), add PEG-CaCl2 solution, mix gently, and incubate in the dark at room temperature for 10-20 min.

[0113] The preparation method of the PEG-CaCl2 is as follows: preferably PEG4000 (Sigma), the concentration of PEG4000 is 40%, 0.5M Mannitol, 100mM CaCl2, and placed in a water bath at 55℃ for more than 1 hour.

[0114] The total amount of the exogenous DNA plasmid target plasmid is 1-5 μg and the volume ratio of it to the protoplast is (1-2):10; the same volume of the PEG-CaCl2 solution is added to the suspension.

[0115] (11) Add W5 solution, gently invert the container to stop the transformation, then centrifuge at 100g / min and remove the supernatant (PEG-CaCl2 plays a role in mediating the entry of plasmids into cells during protoplast transformation. After the transformation is completed, PEG-CaCl2 in the cell culture medium needs to be removed, so the reaction solution is diluted with W5 and centrifuged).

[0116] (12) Resuspend the protoplasts in WI solution and incubate them in the dark at 28°C for 12-16 h (the transformed protoplasts need to be further cultured to express the transformed plasmid, so WI solution is required for culture); then centrifuge and remove the supernatant;

[0117] The WI solution contains 0.5M Mannitol, 5mM glucose, 4mM KCl, 4mM morpholine ethanesulfonic acid, and has a pH of 5.7.

[0118] The culture containers were selected as 24-well cell culture plates rinsed with 5% fetal bovine serum solution.

[0119] The preparation steps for exogenous DNA plasmids are as follows:

[0120] (1) pUC-35S-FLAG and pUC-35S-GFP vector plasmids were obtained by double digestion with SalI and BamHI.

[0121] (2) The sequence of the nuclear marker gene GHD7 was obtained by high-fidelity enzyme amplification;

[0122] (3) The target gene was constructed into the vectors pUC-35S-FLAG and pUC-35S-GFP by homologous recombination.

[0123] (4) The vector was transformed into Escherichia coli Top10, and then plasmid extraction was performed to obtain plasmids with no endotoxin, high purity and high concentration.

[0124] Example 2: Microscopic observation and analysis

[0125] The culture medium of protoplasts transformed with exogenous DNA plasmid obtained in Example 1 was taken, centrifuged at 100g / min for 2min with a speed of 1, and the remaining 100μl of supernatant was taken directly into a concave glass slide for observation using a laser confocal microscope. The transformation efficiency was statistically analyzed.

[0126] The results are as follows Figure 2 As shown, under a fluorescence microscope, cells expressing GFP protein appear green in the fluorescence channel, while untransformed cells do not fluoresce. The transformation efficiency was obtained by combining the fluorescence field and the bright field field.

[0127] in, Figure 2 A shows that the transformation efficiency (number of luminescent cells / total number of cells) of *Catella macrantha* protoplasts treated with the optimized enzyme solution (enzyme solution B) was 40%–50% (the cell counter showed approximately 90 luminescent cells and approximately 190 total cells). Figure 2 B shows that the transformation efficiency (number of luminescent cells / total number of cells) of the catalpa protoplasts treated with the unoptimized enzyme solution (enzyme solution A) is about 10% (the cell counter shows about 15 luminescent cells and about 140 total cells).

[0128] The results indicate that the protoplasts prepared from the optimized enzymatic hydrolysate have good activity and high conversion efficiency.

[0129] Example 3: Western blot detection of protein expression

[0130] The protein in the protoplasts transformed with exogenous DNA plasmid (GHD7-GFP) obtained in Example 1 was extracted using WB / IP lysis buffer, and the expression level of GFP fusion protein was detected by WB.

[0131] The results are as follows Figure 3 As shown, the target protein was detected under different loading amounts of protoplast protein extract. The single and darker bands indicated high GFP expression abundance and specificity of the target protein expression.

[0132] Example 4: Co-IP detection of protein interactions

[0133] (1) Incubate 500-1000 μg of the protein extracted in Example 3 with 3-5 μl of antibody (FLAG) and rotate at 4°C for 1 h;

[0134] (2) Add 50 μl of Protein A / G agarose packing material washed with lysis buffer, and mix at 4°C for 1-3 h.

[0135] (3) Wash the Protein A / G agarose packing material three times with lysis buffer;

[0136] (4) Resuspend the precipitate in 20–40 μl of 5*SDS loading buffer. Vortex and then microcentrifuge for 30 s;

[0137] (5) Heat the sample to 95-100℃ for 5 min, and centrifuge at 14000g / min for 1 min;

[0138] (6) WB detection of Co-IP results.

[0139] The results are as follows Figure 4 As shown, FLAG antibody detection revealed clear bands in the total protein extract (input) samples, indicating successful co-transformation. Western blot analysis of the IP-derived products using FLAG antibody also showed clear bands, confirming successful IP.

[0140] GFP antibody detection showed that all total protein extract (input) samples had clear bands, indicating successful co-transformation. Western blot analysis of the IP-derived products using the GFP antibody revealed protein-protein interaction between the GFP-tagged BZIP63 protein (BZIP63-GFP) and the Flag-tagged BZIP63 protein (BZIP63-FLAG). BZIP63 is a transcription factor that can form protein dimers. The construction of BZIP63-GFP and BZIP63-FLAG was the same as the construction process of the exogenous gene GHD7 fusion plasmid with GFP in Example 1.

[0141] Therefore, the protoplasts prepared by the method of the present invention have high transformation efficiency and can achieve protein expression.

[0142] Example 5: Comparison of protoplasts prepared with different enzyme solutions

[0143] Different tissue types were treated with enzyme solutions of varying compositions, release solution W5, and suspension MMG to prepare and transform protoplasts. See Example 1 for the specific preparation and transformation process.

[0144] Among them, the immature xylem tissue of Catalpa trees is selected from the stem segments of Catalpa seedlings, specifically from Catalpa seedlings that are 80cm tall, with 40cm above the root, and the cut size is 1cm in length and width.

[0145] Rice stem segment selection: Stem segments with a growth period of about 15 days, a plant height of 15 cm, and a root portion of about 5 cm were selected as samples for preparation.

[0146] Specific preparation examples, the final number of protoplast cells obtained, and the protoplast transformation efficiency data are shown in Table 1 below. A microscopic field-of-view image of protoplasts prepared using enzyme hydrolysate B and enzyme hydrolysate A is detailed below. Figure 5 and Figure 6 For details of a field of view of protoplasts prepared using the enzymatic digests of Comparative Examples 1-8 at the same microscope magnification, please refer to [link to relevant documentation]. Figures 7-14 . Figures 5-14 Their field of vision is the same.

[0147] Table 1

[0148]

[0149]

[0150]

[0151]

[0152] As shown in Table 1 above, the optimized enzymatic hydrolysate of the present invention, namely enzymatic hydrolysate B, has the best protoplast preparation effect and the best conversion efficiency.

[0153] Furthermore, the results from enzymatic hydrolysate A and Comparative Example 1 show that the preparation of protoplasts from immature xylem tissue of the woody plant *Cathaya argyrophylla* is more difficult than that from stem segments of the grass plant *Rhizoma Citrus reticulata*. Under the same conditions of enzymatic hydrolysate, release solution, and suspension, fewer protoplasts were obtained from immature xylem tissue, and the conversion efficiency was also lower. Therefore, the enzyme solution and preparation method of the present invention have extremely significant protoplast preparation efficiency, and the prepared protoplasts also have extremely excellent conversion efficiency.

[0154] Specifically, as shown in Table 1 above, the enzyme solutions used in Comparative Examples 7 and 8 were the same as those in enzyme hydrolysate B. The only difference between the enzyme hydrolysates in Comparative Examples 7 and 6 was that Comparative Example 7 contained 3 mM sodium succinate and 1 w / v% polyvinylpyrrolidone, while the protoplast preparation rate in Comparative Example 7 was 1.9 × 10⁻⁶. 6 However, this is in contrast to proportion 6 (1.3 × 10⁻⁶). 6 The yield is approximately 1.46 times that of the original solution. Therefore, the enzymatic hydrolysate B of this invention has an unexpectedly high protoplast preparation rate.

[0155] Furthermore, the protoplast preparation rate corresponding to the enzymatic hydrolysate B of this invention is 3.2 × 10⁻⁶. 6 The protoplast preparation rate corresponding to Comparative Example 8 was 2.6 × 10⁻⁶. 6 The only difference between the two is that the preparation process release solution W5 corresponding to enzymatic hydrolysate B contains 15 mM MgSO4 and 5 mM CuSO4. Surprisingly, the protoplast preparation efficiency corresponding to enzymatic hydrolysate B is 1.23 times that of Comparative Example 8. Therefore, the combination of enzymatic hydrolysate B and the specific release solution W5 in this invention can unexpectedly improve the protoplast preparation efficiency.

[0156] discuss

[0157] This invention optimizes the preparation method of protoplasts from woody plant tissues, using a specific ratio of PEG / CaCl2 to mediate the entry of exogenous DNA plasmids into protoplasts and express exogenous genes. It is applied to immature xylem tissues of the woody plant *Cathaya argyrophylla*, solving the problems of excessively long preparation processes and low efficiency in protoplast preparation in woody plants, and improving protoplast transformation efficiency.

[0158] The present invention provides a composition for preparing immature xylem protoplasts of Catalpa tree, comprising pectinase and mercaptoethanol, wherein the pectinase content is 3% by mass / volume and the mercaptoethanol content is 0.3% by volume. This method can efficiently extract up to 10 immature xylem protoplasts from Catalpa tree. 6The method can improve the efficiency of introducing exogenous genes into catalpa protoplasts by 50%, which has great application value for cultivating new catalpa germplasm through genetic engineering.

[0159] The method used in this invention to process immature xylem tissue of catalpa trees, combined with a sharp new surgical blade and 0.5M mannitol to balance the scraped tissue, allows the catalpa tissue to gradually adapt to the subsequent osmotic environment and effectively reduces cell debris and cytosap generated during the processing. Cytosap contains various hydrolytic enzymes that negatively impact protoplast survival, which is crucial for determining protoplast activity and transformation efficiency.

[0160] In the process of preparing immature xylem protoplasts of the catalpa tree, this invention uses an optimized enzyme solution and adds additional pectinase Y-23, making it more suitable for the enzymatic hydrolysis of the surface tissue of the main trunk after the bark of the catalpa stem segment is peeled. With specific enzymatic hydrolysis conditions and methods for releasing protoplasts, a large number of highly active and transparent protoplasts can be obtained.

[0161] The protoplasts prepared using this invention, under the preferred PEG4000-mediated transformation, can efficiently transform endotoxin-free exogenous DNA plasmids into protoplasts, with a transformation efficiency of up to 50%. Figure 2 The cell counter showed approximately 90 luminescent cells (out of a total of approximately 190 cells), demonstrating the widespread expression of exogenous genes by protoplasts.

[0162] The enzyme hydrolysate, MMG, W5 and WI solutions used in this invention all contain a certain amount of glucose, which can provide energy for protoplasts and improve the survival rate of protoplasts during extraction and incubation.

[0163] The protoplast preparation and transformation methods of the present invention have potential application value in exogenous gene expression, subcellular localization of functional genes, immunoprecipitation screening and verification of interacting proteins, and breeding of new varieties of Catalpa trees.

[0164] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for preparing immature xylem protoplasts from Catalpa trees, characterized in that, Including the following steps: (s1) Provide immature xylem tissue of Catalpa tree, wherein the xylem tissue is the surface tissue of a stripped Catalpa seedling stem segment; (s2) Equilibration: The xylem tissue was placed in a 0.4-0.6M mannitol solution for equilibration for 10-30 min, and then centrifuged at 100g for 5 min. (s3) Enzymatic hydrolysis: Remove the mannitol solution from the centrifuged solution and add the enzyme solution to the treated tissue. Perform enzymatic hydrolysis in the dark to obtain the enzymatic cell slurry. The enzyme solution comprises the following components: Cellulase R-10 2 w / v%, macerozyme R-10 2 w / v%, pectolase Y-23 3 w / v%, 0.5 M mannitol, 10 m M morpholine ethanesulfonic acid, 5 m M glucose, 5 m M MgSO4, 3 m M sodium succinate, polyvinylpyrrolidone 1 w / v%, 10 m M CaCl2, BSA 0.1 w / v%, and β-mercaptoethanol 0.3 v / v%. (s4) Release: Add W5 solution with 1 volume of the enzymatic hydrolysate to the enzymatic hydrolysate and release for 8-10 min; The W5 solution comprises the following components: 2 mM morpholine ethanesulfonic acid, 5 mM glucose, 154 mM NaCl, 140 mM CaCl2, 5 mM KCl, 15 mM MgSO4, 3 mM sodium succinate, 5 mM sorbitol and 5 mM CuSO4. (s5) After filtering the released cell slurry using a cell sieve, repeat step (s4) to obtain a second-released cell slurry; and (s6) The cell sap after the secondary release is filtered using a cell sieve, and the filtrate is centrifuged to remove the supernatant, thereby obtaining the immature xylem protoplasts of the catalpa tree.

2. The method as described in claim 1, characterized in that, The catalpa seedling stem segments were taken from catalpa seedlings that were cultivated to a height of 80cm, with 40cm above the root selected and cut to a length and width of 1cm.

3. The method as described in claim 1, characterized in that, In step (s3), the enzymatic hydrolysis temperature is 24℃~28℃, and the enzymatic hydrolysis time is 12~16h.

4. The method as described in claim 1, characterized in that, The pH value of the W5 solution is 5.

7.

5. An enzyme solution for preparing immature xylem protoplasts of Catalpa trees, characterized in that, The enzyme solution comprises the following components: Cellulase R-10 2 w / v%, macerozyme R-10 2 w / v%, pectolase Y-23 3 w / v%, 0.5 M mannitol, 10 mM morpholine ethanesulfonic acid, 5 mM glucose, 5 mM MgSO4, 3 mM sodium succinate, polyvinylpyrrolidone 1 w / v%, 10 mM CaCl2, BSA 0.1 w / v%, and β-mercaptoethanol 0.3 v / v.

6. The enzyme solution according to claim 1, characterized in that, The pH value of the enzyme solution is 5.6 to 5.

8.

7. The enzyme solution according to claim 1, characterized in that, The cellulase is cellulase R-10 with an activity of 500 U / g, the cleavage enzyme is cleavage enzyme R-10 with an activity of 800 U / g, and the pectinase is pectinase Y-23 with an activity of 1000 U / g.

8. The method for preparing the enzyme solution as described in claim 5, characterized in that, Includes the following steps: (s1) Prepare 8 ml of stock solution containing 0.5 M mannitol, and add 10 mM morpholine ethanesulfonic acid and 5 mM glucose in sequence. Shake at room temperature to dissolve and adjust the pH to 5.6-5.

8. (s2) Add 2 w / v% cellulase R-10, 2 w / v% macerozyme R-10 and 3 w / v% pectolase Y-23; (s3) After stirring and dissolving, heat in a 55°C water bath for 10 minutes, then cool naturally to room temperature. Add 5 mM MgSO4, 3 mM sodium succinate, 1 w / v% polyvinylpyrrolidone, 10 mM CaCl2, 0.1 w / v% BSA, and 0.3 v / v% β-mercaptoethanol; and (s4) Make up to 10ml, then store the enzyme digest in the dark for later use.

9. The use of the enzyme solution as described in claim 5 or the enzyme solution prepared by the preparation method as described in claim 8 in the preparation of immature xylem protoplasts of Catalpa tree.

10. A method for efficient transformation of immature xylem protoplasts from Catalpa trees, characterized in that, Including the following steps: (i) Obtain protoplasts prepared by the method of claim 1, and adjust the protoplasts with MMG solution to obtain a concentration of (2-4)×10⁻⁶. 6 A protoplast suspension of 1 protoplast per ml was prepared; and the protoplast suspension was placed in the dark and kept at room temperature for 60–90 min. The MMG solution comprises the following components: 0.5M Mannitol, 5mM glucose, 15mM magnesium chloride, and 4mM morpholine ethanesulfonic acid. (ii) Centrifuge at room temperature, remove the supernatant with a pipette, resuspend in MMG solution to obtain a concentration of (2-4)×10⁻⁶. 6 Protoplast suspension per ml; (iii) Mix the protoplast suspension obtained in step (ii) with the plasmid containing the target gene, add PEG-CaCl2 solution, mix gently, and incubate at room temperature in the dark for 10-20 min. (iv) Add W5 solution, gently invert the container to stop the conversion, then centrifuge and remove the supernatant; The W5 solution comprises the following components: 2 mM morpholine ethanesulfonic acid, 5 mM glucose, 154 mM NaCl, 140 mM CaCl2, 5 mM KCl, 15 mM MgSO4, 3 mM sodium succinate, 5 mM sorbitol, and 5 mM CuSO4; and (v) Resuspend the transformed protoplasts in WI solution and incubate in the dark at 28°C for 12-16 h.

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