Preparation and instantaneous conversion method of taraxacum mongolicum protoplast

Abstract

The invention relates to a protoplast, in particular to a preparation and instantaneous conversion method of a taraxacum mongolicum protoplast. According to the method, separation parameters, including a leaf pretreatment mode, an enzymolysis system, an osmotic pressure regulator and the like, of the leaf protoplast of the tarragon are systematically optimized, and a preparation scheme of the first protoplast of the tarragon is successfully constructed; by optimizing the plasmid transfection concentration, a PEG-mediated transient transformation system of the taraxacum heteropterum protoplast is established; the system is used for verifying subcellular localization of a photosynthetic key enzyme SaPEPC1 and a transcription factor SabHLH169 in a single-cell C4 pathway of the suaeda variegata, a key technical support is provided for subsequently revealing a complex regulation mechanism of development of a single-cell C4 system, and meanwhile, a novel technical platform is also established for research on stress-resistant gene functions of halophyte.

Description

Technical Field

[0001] The present invention relates to protoplasts, and particularly to a method for preparing and transiently transforming protoplasts of Suaeda heteroptera Background Art

[0002] As a functional unit that still maintains physiological activity after removing the cell wall, plant protoplasts have become an important experimental system for plant molecular biology research due to their unique biological characteristics. Its technical advantages are mainly reflected in two aspects: the absence of the cell wall makes protoplasts prone to cell fusion, providing convenience for somatic hybridization and the cultivation of hybrid plants; they can directly absorb macromolecules such as exogenous DNA, microorganisms, and organelles, making them an ideal receptor system for plant genetic transformation, somatic clone variation, and mutant screening. The increasingly mature protoplast transient transformation system has supported multi-dimensional plant molecular biology research. The application scope of this technical platform has expanded from early subcellular localization to research fields such as molecular interaction analysis (such as BiFC, ChIP, dual-luciferase reporter gene detection), gene editing, epigenetic regulation, and single-cell transcriptome sequencing.

[0003] Although transient transformation systems for protoplasts of many plants have been successively established, many challenges still exist in the construction process. Due to significant biological differences between different species, establishing a transient transformation system for protoplasts of a new species often requires systematic optimization of various experimental parameters, and preparing sufficient amounts of highly active protoplasts is a prerequisite for successfully establishing a transformation system. During the isolation stage, the absence of the cell wall makes protoplasts extremely fragile, and any minor change in conditions can significantly affect the yield and activity of protoplasts, including but not limited to: internal factors such as the tissue part of plant materials, pretreatment methods, growth stage, and physiological state; and external conditions such as the enzymatic hydrolysis system (types and concentration ratios of cellulase and macerozyme), osmotic pressure regulators (types and concentrations), buffer systems (pH value and ionic strength), separation time, and temperature. During the transformation stage, the transient transformation efficiency is also restricted by both species specificity and experimental parameters, and systematic optimization mainly focuses on the delivery conditions of genetic materials, such as the total amount of plasmid, PEG concentration, and incubation time.

[0004] Suaeda aralocaspica belongs to the genus Suaeda of the family Amaranthaceae. It is an annual herbaceous halophyte widely distributed in saline and arid environments and has extremely strong stress resistance. This species has evolved a unique single-cell C4 photosynthetic pathway, which can complete the C4 and C3 cycles in the two polar regions within a single cell respectively, showing significant high photosynthetic efficiency characteristics. This unique photosynthetic mechanism and outstanding environmental adaptability make Suaeda aralocaspica an ideal model plant for studying plant photosynthesis and stress adaptation. However, there are significant technical bottlenecks in the current research on this species. Due to the lack of an efficient genetic transformation system and molecular manipulation techniques at the cellular level, existing research has mostly focused on the physiological and ecological aspects, greatly limiting the analysis of the molecular regulatory network for the formation of its single-cell C4 photosynthetic structure. Summary of the Invention

[0005] Object of the Invention: The present invention provides a method for preparing protoplasts of Suaeda aralocaspica and transient transformation. By optimizing the composition of the lysis solution, the material pretreatment steps and the transformation conditions, it provides protoplasts with high activity and high integrity for subsequent gene function research.

[0006] Technical Solution

[0007] A method for preparing protoplasts of Suaeda aralocaspica, characterized in that it is implemented according to the following steps

[0008] Take 1 g of the club-shaped leaves of Suaeda aralocaspica. After pretreatment, place them in the lysis solution and incubate in the dark for 4 h. After the incubation ends, filter and centrifuge for 4 min to obtain protoplasts.

[0009] As a preferred mode:

[0010] The lysis solution is prepared by the following steps: Dissolve 0.75% w / v cellulase R-10 and 0.25% w / v macerozyme R-10 in CS buffer, heat at 55 °C for 10 min. After the enzyme solution is cooled to room temperature, add 0.1% w / v bovine serum albumin to obtain the lysis solution.

[0011] The CS buffer consists of 1.3 M glucose, 25 mM HEPES-KOH with pH 6.5, 5 mM MgCl2, and 125 mM CaCl2.

[0012] The method is characterized in that the incubation temperature is 4 °C.

[0013] The pretreatment is to pre-cut and peel the club-shaped leaves of Suaeda aralocaspica horizontally and longitudinally.

[0014] A transient transformation method for Suaeda aralocaspica protoplasts, characterized in that the Suaeda aralocaspica protoplasts are resuspended with W5 buffer and then incubated on ice for 30 min; after incubation, the supernatant is removed, and the protoplasts are resuspended with MMS buffer, and 5 μg of pCAMBIA2300-eGFP plasmid with a concentration of 2400 ng / μL and 40% PEG4000 solution are added for transformation.

[0015] Beneficial effects

[0016] Suaeda aralocaspica is a typical halophyte, mainly distributed in the saline deserts of Central Asia and the arid and semi-arid regions of northwestern China. To adapt to the extreme saline and arid environment, its leaves have evolved unique morphological and anatomical structures. The leaf surface is covered with a dense waxy layer and salt crystals, the leaf has a high water content and contains a large amount of palisade tissue, and the cell wall is thickened and rich in polysaccharides and other substances. However, conventional methods cannot provide high-quality protoplasts. For example: in the actual operation process, there is a significant adhesion phenomenon in the enzymatic hydrolysis products, making it difficult to effectively separate the protoplasts; the leaves are completely digested but the protoplast yield is low, and a large number of protoplasts are still squeezed in the leaf palisade tissue and accompanied by chloroplast leakage, mostly vacuoles; the protoplast fragments become more; during the separation process, the palisade tissue cells show browning, and the above problems are closely related to the enzyme type and concentration, osmotic pressure regulator type and concentration, temperature, leaf cutting method, etc. (see Figure 7 A-C).

[0017] The transformation of plant cells into protoplasts requires two key steps: enzymatic hydrolysis of the cell wall and disruption of cell adhesion. As a non-specific catalyst, biological enzymes may undergo non-specific reactions with cell membrane and other components while degrading the cell wall, resulting in protoplast damage and affecting its activity and subsequent experimental effects. This limitation has prompted researchers to continuously optimize the enzymatic hydrolysis conditions to achieve efficient separation of protoplasts while maximizing their integrity. In the present invention, cellulase R-10 and macerozyme R-10 are preferably selected as the enzymes for separating protoplasts, which maximally protects the integrity of the protoplasts without damage. The concentration of the separating enzyme has a significant impact on the separation efficiency of protoplasts. In the present invention, when the concentration of the separating enzyme is too high (3%), the separating enzyme will precipitate and inactivate in the CS buffer, and at the same time cause the enzymatic hydrolysis material of Suaeda aralocaspica leaves to turn yellow, seriously affecting the separation and counting of protoplasts ( Figure 8 ); while too low enzyme concentration will result in incomplete degradation of the cell wall and insufficient release of protoplasts ( Figure 9 ). Thus, optimizing the concentration combination of the separating enzyme is crucial for establishing an efficient protoplast separation system. In the present invention, the optimal protoplast separation enzyme concentration and combination for Suaeda aralocaspica are 0.75% cellulase R-10 and 0.25% macerozyme R-10, which may be related to the speciality of its cell wall components or the closely arranged characteristics of photosynthetic cells.

[0018] After enzymatic hydrolysis and separation, the maintenance of the integrity and physiological activity of the protoplast cell membrane depends on a suitable osmotic pressure buffer environment. The selection of osmotic pressure regulators and the optimization of their concentrations are one of the key parameters in the protoplast separation process. Common osmotic pressure regulators include sucrose, glucose, sorbitol, and mannitol, etc., among which mannitol is widely used in the separation of most plant protoplasts. Research shows that there are significant differences in the requirements of different plant species for osmotic pressure regulators, which may be related to factors such as cell wall structure and intracellular osmotic pressure characteristics. For example, 0.4M mannitol is usually used for the separation of Arabidopsis thaliana protoplasts, while 0.7M mannitol is more suitable for the separation of Areca catechu leaf protoplasts, which further emphasizes the necessity of optimizing osmotic pressure conditions for different plant materials. In order to better simulate the osmotic pressure of the intracellular environment of Suaeda aralocaspica, it was found through a dew point osmometer in this study that the osmotic pressure of the homogenate of the fleshy leaves of Suaeda aralocaspica was stable at about 1.3M. 1.3M and 0.7M were selected as the reference concentrations in this study, and the effects of mannitol, sucrose, glucose, and sorbitol on the separation efficiency of Suaeda aralocaspica protoplasts were systematically compared. The results showed that when glucose was used as the osmotic pressure regulator, the protoplast yield was the highest and the stability was the best. On this basis, we further optimized the glucose concentration gradient and found that 1.3M was the optimal concentration for the separation of Suaeda aralocaspica protoplasts. This result was highly consistent with the measurement data of the osmotic pressure detector, confirming that this concentration could effectively maintain the physiological activity and structural integrity of the protoplasts.

[0019] Effect of temperature: Under room temperature conditions, serious browning occurred in the protoplasts, so it was changed to 4°C.

[0020] The diversity of the leaf structure and the specificity of cell arrangement in higher plants significantly affect the protoplast separation efficiency. Optimizing the leaf pretreatment method is crucial for improving the enzymatic hydrolysis effect and protecting the integrity of the leaf tissue. In this study, the leaf pretreatment method was specifically optimized. In the preliminary experiment, the leaf pretreatment was carried out by the cutting method. Although protoplasts were successfully separated, the yield was low. To improve the enzymatic hydrolysis efficiency, we added the treatment step of removing the epidermis on the basis of the cutting method. The results showed that removing the epidermis significantly improved the penetration efficiency of the enzyme solution and greatly increased the protoplast yield. Based on the above results, we finally determined the optimal pretreatment plan for the separation of Suaeda aralocaspica leaf protoplasts as follows: first remove the leaf epidermis, and then carry out transverse and longitudinal cutting. This optimized plan fully considers the structural characteristics of Suaeda aralocaspica leaves and significantly improves the protoplast yield while ensuring cell viability.

[0021] The PEG-mediated transformation method is a technique widely used in the genetic transformation of plants and microorganisms and is also the main method for transfecting protoplasts. It should be noted that there are huge differences in the transfection efficiency and required conditions of protoplasts from different species. During the transfection of Bienertia sinuspersici protoplasts, the total amount of exogenous DNA added is positively correlated with the transfection efficiency, and when the total plasmid amount reaches 5 μg, the highest transfection efficiency can reach 80%. Optimization experiments on the high-throughput transient transformation system of rice protoplasts showed that an optimal transfection efficiency can be achieved with a 20% PEG concentration in combination with a total plasmid amount of 10 μg. The experimental results of this study showed that when the total plasmid amount remains the same, the plasmid concentration has a significant impact on the transfection efficiency of Suaeda aralocaspica protoplasts. Furthermore, the transfection efficiencies at three concentration gradients of 600, 1000, and 2400 ng / μL were systematically compared, and it was found that the transfection efficiency was optimal when the plasmid concentration reached 2400 ng / μL, which was 3 times higher than that at 300 ng / μL. This plasmid concentration dependence may be related to the PEG-mediated cell membrane permeability threshold, and high-concentration plasmids are more likely to form DNA aggregates.

[0022] In summary, through systematic optimization of the parameters for isolating Suaeda aralocaspica leaf protoplasts, including the pretreatment method, enzymatic hydrolysis system, and osmotic pressure regulator, etc., the first protoplast preparation protocol for Suaeda aralocaspica was successfully constructed; by optimizing the plasmid transfection concentration, a PEG-mediated transient transformation system for Suaeda aralocaspica protoplasts was established; and the subcellular localization of the photosynthetic key enzyme SaPEPC1 and the transcription factor SabHLH169 in the single-cell C4 pathway of Suaeda aralocaspica was verified using this system, providing key technical support for revealing the complex regulatory mechanism of single-cell C4 system development, and also establishing a new technical platform for the study of the functions of stress-resistant genes in halophytes. Description of the Drawings

[0023] Figure 1 Optimization of the isolation conditions of Suaeda aralocaspica protoplasts; among them, A, the protoplast extraction efficiency at different concentrations of cellulase R-10; B, the protoplast extraction efficiency at different concentrations of macerozyme R-10; C, the determination of the osmotic pressure of each concentration of osmotic pressure regulator and Suaeda aralocaspica leaves; D, the protoplast isolation efficiency of different concentrations and different types of osmotic pressure regulators; E, comparison of the protoplast isolation efficiency under different concentrations of glucose osmotic pressure; CR: cellulase R-10; MR: macerozyme R-10; OS: osmotic pressure regulator; different lowercase letters represent significant differences in the protoplast yield between different treatments (P<0.05). Each value represents the mean ± standard deviation of three replicates;

[0024] Figure 2, Schematic diagram of the extraction of Suaeda heteroptera protoplasts; A. Sampling plants of Suaeda heteroptera; B. Transverse and longitudinal cutting of leaves; C. Transverse and longitudinal cutting combined with peeling treatment; D. Schematic diagram of leaf digestion; E. Photosynthetic cells in the palisade tissue of leaves; F. Observation of isolated Suaeda heteroptera protoplasts under a 10× microscope; G. Observation of isolated protoplasts under a 40× microscope; H. Yield of Suaeda heteroptera protoplasts under different leaf pretreatment conditions; I. FDA staining of Suaeda heteroptera protoplasts; ** indicates that there are significant differences in protoplast yields between different treatments (P<0.001); ER: Treatment of removing leaf epidermis; WER: Treatment of not removing leaves; Bar = 50 μm; Each value represents the mean ± standard deviation of three replicates.

[0025] Figure 3 , Fluorescent staining of subcellular organelles in protoplasts; A. FDA staining; B. DAPI staining; C. Rhodamine 123 staining; D. Autofluorescence of chloroplasts; Merge: Overlap of each channel.

[0026] Figure 4 , Transformation of Suaeda heteroptera protoplasts with pCAMBIA2300-eGFP plasmid; A: Observation of transfected protoplasts under a 10× microscope; B: Observation of protoplasts after transfection under a 20× microscope; C: Observation of protoplasts after transfection from another perspective under a 20× microscope; GFP: Green fluorescent protein; Autofluorescence: Chloroplast autofluorescence channel; Bright field: Bright field; Merge: Overlap of each channel.

[0027] Figure 5 , Effect of plasmid concentration on transfection efficiency; A-D. GFP imaging of Suaeda heteroptera protoplasts after transformation at different plasmid concentrations; E. Transformation efficiency of Suaeda heteroptera protoplasts at different plasmid concentrations. Different lowercase letters represent significant differences in protoplast yields between different treatments (P<0.05); Bar = 10 μm; Each value represents the mean ± standard deviation of three replicates.

[0028] Figure 6 , Subcellular localization of SaPEPC1 and SabHLH169 in Suaeda heteroptera protoplasts; A. Subcellular localization of SabHLH169; B. Subcellular localization of SaPEPC1; GFP: Green fluorescent protein; Autofluorescence: Chloroplast autofluorescence channel; DAPI: Nuclear localization marker; Bright field: Bright field; Merge: Overlap of each channel. Bar = 20 μm.

[0029] Figure 7, problems occurred in the actual operation of isolating Suaeda heteroptera protoplasts using conventional methods; A shows the adhesion phenomenon of protoplasts under the action of various enzymes, B shows the browning phenomenon of photosynthetic cells in the palisade tissue, C shows that although the leaf digestion is complete, the protoplast yield is low, a large number of protoplasts are still squeezed in the leaf tissue, and most of the vacuoles with chloroplast leakage.

[0030] Figure 8 , 3 Microscopic examination results of Suaeda heteroptera protoplasts under the enzymatic hydrolysis combination of 6% cellulase R-10 and 2% macerozyme R-10.

[0031] Figure 9 Microscopic examination results of Suaeda heteroptera protoplasts under the enzymatic hydrolysis combination of 0.25% cellulase R-10 and 0.2% macerozyme R-10.

[0032] Figure 10 Flow chart of the isolation and transformation of Suaeda heteroptera protoplasts. Detailed implementation method

[0033] Plant materials:

[0034] The naturally mature seeds of Suaeda heteroptera were collected from near the Wujiaqu 103 Regiment North Sand Dune Desert Control Station on the edge of the Gurbantunggut Desert in Xinjiang in 2017 (44°19'N, 86°57'E; 429 m altitude). In this experiment, the mature and plump brown seeds among the heteromorphic seeds of Suaeda heteroptera were selected and sown in a mixed substrate composed of nutrient soil, vermiculite, and perlite (3:1:1). The 1 / 2 Hoagland nutrient solution was watered once a week, and the plants were cultured in a plant cultivation room at a temperature of 24 - 30 °C, with a 16 h light / 8 h dark cycle, a relative humidity of 20 - 30%, and a light intensity of 120 - 150 μmol·m-2·s-1.

[0035] Cellulase R-10

[0036] Main components: mainly composed of cellulase complex, including endo-β-1,4-glucanase (EGase), exo-β-1,4-glucanase (CBH), and β-glucosidase (BG).

[0037] Action target: specifically hydrolyze the β-1,4-glycosidic bond of the cellulose skeleton and destroy the fiber network structure of the plant cell wall.

[0038] Application scenario: when used alone, it mainly degrades cellulose components and is suitable for cellulose hydrolysis experiments or plant cell wall treatment.

[0039] Macerozyme R-10

[0040] Core components: mainly dominated by pectinase, and also contains xylanase and a small amount of cellulase.

[0041] Key function: preferentially cleave the α-1,4-glycosidic bond in the middle lamella of plants and decompose polygalacturonic acid in pectin.

[0042] Application scenario: It is a key enzyme for protoplast preparation by disrupting the intercellular cementing substance. Electron microscopy studies show that its dissolution efficiency for the middle lamella can reach over 85%.

[0043] Example 1 Establishment of an efficient isolation system for Suaeda heteroptera protoplasts

[0044] In view of the leaf structure characteristics of Suaeda heteroptera, the present invention significantly improves the yield and activity of protoplasts by synergistically optimizing the enzymatic hydrolysis system and osmotic pressure regulation parameters, specifically including optimizing the concentrations of cellulase R-10 and macerozyme R-10, the types and concentrations of osmotic pressure regulators, and the leaf pretreatment methods, etc.

[0045] 1. Preparation of Suaeda heteroptera protoplasts

[0046] The isolation and purification of Suaeda heteroptera protoplasts refer to the method for extracting protoplasts from the green tissue cells of Bienertia sinuspersici, a plant of the genus Bienertia with a single-cell C4 photosynthetic pathway, and modifications are made (see reference: LUNG S C, YOGADASAN N, YANAGISAWA M, et al. Protoplast Isolation and Transfection in the Single-Cell C(4) Species Bienertia sinuspersici [J]. Methods Mol Biol, 2022, 2464(21-8.).

[0047] Take 1 g of the rod-shaped leaves of Suaeda heteroptera, remove the lower epidermis, and after pretreatment, place them in the prepared lysis solution, where the lysis solution is composed of 1.5% cellulase R-10 and 0.5% macerozyme dissolved in CS buffer (the components are shown in Table 1). Use a vacuum pump to evacuate for 20 min, and then incubate in the dark for 4 h. After the incubation, filter the liquid through a 100-mesh gauze, and rinse the gauze with CS buffer to collect the protoplasts remaining in the undigested leaf tissue. Use a horizontal rotor to centrifuge the collected liquid at 800 rpm for 4 min, discard the supernatant, and resuspend the precipitate with CS buffer to obtain purified protoplasts for subsequent experiments. Take 10 μL of the purified protoplasts and add them to a hemocytometer to measure the protoplast density.

[0048] Table 1 Components of CS buffer

[0049]

[0050] 2. Optimization of Cellulase R-10 and Macerozyme R-10

[0051] To meet the requirements of subsequent transformation experiments of Suaeda aralocaspica protoplasts and improve the protoplast isolation efficiency, optimizing the protoplast isolation conditions of Suaeda aralocaspica is the key. Therefore, the present invention systematically optimized the protoplast isolation conditions of Suaeda aralocaspica, and focused on investigating the effect of enzyme concentration combinations on the protoplast yield.

[0052] Dissolve a certain concentration of Cellulase R-10 (Yakult, Japan) and a certain concentration of Macerozyme R-10 (Yakult, Japan) in CS cell stabilization buffer to prepare an enzyme solution, and heat it in a water bath at 55 °C for 10 min. After incubation, the enzyme solution should be clear and brown. After the enzyme solution is cooled to room temperature, add 0.1% (w / v) bovine serum albumin (Coolaber, China). Thus, the lysis solution is prepared.

[0053] Results:

[0054] First, under the condition of fixing the concentration of Macerozyme R-10 at 0.5% w / v, the enzymatic hydrolysis efficiency of different concentrations (0.5%, 0.75%, 1%, 1.5% w / v) of Cellulase R-10 was detected. The results showed that when the concentration of Cellulase R-10 was 0.75%, the protoplast yield was the highest, reaching 6.8×10 4 cells / g·FW ( Figure 1 A), so this concentration was determined as the optimal concentration. Subsequently, under the condition of fixing the concentration of Cellulase R-10 at 0.75%, the concentration gradient of Macerozyme R-10 (0.125%, 0.25%, 0.5% w / v) was further optimized, and it was found that when the concentration of Macerozyme R-10 was 0.25%, the protoplast yield was significantly increased to 8×10 4 cells / g·FW ( Figure 1 B).

[0055] 3. Optimization of Osmotic Pressure Regulators in CS Buffer

[0056] Set different types of osmotic pressure regulators (mannitol, sorbitol, sucrose, and glucose) and different concentration gradients of glucose (0.4 M, 0.7 M, 1.0 M, 1.3 M, 1.6 M, and 1.9 M) to conduct protoplast isolation experiments.

[0057] Specifically as follows: First, select healthy Suaeda aralocaspica plants at 3-4 months old, collect about 0.1 g of mature leaves with plump morphology, homogenize them using a tissue homogenizer, centrifuge the homogenate instantaneously using a tabletop centrifuge, take 100 μL of the supernatant, and measure the osmotic pressure reference value of Suaeda aralocaspica leaves using an osmometer.

[0058] Based on the reference osmotic pressure value of Suaeda aralocaspica leaves, the types and concentrations of osmotic regulators in the CS buffer solution of Suaeda aralocaspica were adjusted, and the protoplast isolation experiment was carried out with reference to Step 1. After separation and purification of each group, 10 μL of protoplasts were taken, and the density was measured using a hemocytometer combined with an automatic cell counter to explore the effects of different conditions on the protoplast isolation effect of Suaeda aralocaspica, providing appropriate separation condition parameters for subsequent protoplast-related experiments.

[0059] Results:

[0060] To explore the optimal osmotic pressure environment for protoplast isolation of Suaeda aralocaspica, the specific osmotic pressure values corresponding to different concentration gradients (0.4 M, 0.7 M, 1.3 M) of osmotic regulators were detected ( Figure 1 C), and the results showed that 1.3 M was closer to the true osmotic pressure (about 1.2 M) of the Suaeda aralocaspica leaf homogenate. Based on this reference value, combined with the optimal osmotic pressure (0.7 M) for protoplast isolation of Bienertia sinuspersici in the genus Bienertia with a single-cell C4 photosynthetic pathway, the effects of different types of osmotic regulators on the protoplast isolation effect of Suaeda aralocaspica were further compared. The results showed that when the concentrations of glucose and sorbitol were both 1.3 M, the protoplast yield reached the peak. However, the data dispersion degree (CV = 29%) of the sorbitol treatment group was higher than that of the glucose group (CV = 1.5%), indicating that glucose has more stable osmotic regulation characteristics ( Figure 1 D). On this basis, a glucose concentration gradient experiment of 0.4 - 1.9 M was set up, and it was found that when the glucose concentration was 1.3 M, the highest protoplast yield reached 9×10 4 cells / g·FW, which was significantly higher than that of other concentration treatment groups ( Figure 1 E).

[0061] 4. Optimization of leaf pretreatment methods

[0062] Suaeda aralocaspica has typical morphological characteristics of halophyte leaves, which are fleshy rod-shaped leaves with relatively thick leaves and cutin membranes ( Figure 2 A), and the photosynthetic cells arranged in a palisade shape inside the leaves are highly dense ( Figure 2 E). This special anatomical structure puts forward higher requirements for the enzymolysis efficiency. In order to make the photosynthetic cell structure inside the leaves become loose and better contact with the enzyme solution, the present invention explored the effects of different leaf pretreatment methods on the protoplast yield.

[0063] Results:

[0064] Only transverse and longitudinal cutting treatment was carried out on the leaves of Suaeda aralocaspica ( Figure 2 B). Although protoplasts were successfully isolated, the yield was not ideal. By optimizing the leaf pretreatment method, the leaf epidermis peeling step was added on the basis of transverse and longitudinal cutting ( Figure 2C), due to the increased enzymatic contact area, the yield of treated protoplasts was significantly increased to 1.1×10 5 cells / g·FW( Figure 2 H). Under a 20-fold microscopic field of view, more Suaeda heteroptera protoplasts could be observed, and most of the protoplasts showed no significant shrinkage or rupture, indicating that the leaf pretreatment method combining mechanical cutting and epidermal peeling could effectively improve the enzymatic hydrolysis efficiency of Suaeda heteroptera protoplasts while maintaining cell integrity( Figure 2 F, 2G). FDA staining showed that the Suaeda heteroptera protoplasts were successfully stained and showed bright green fluorescence, indicating that the isolated protoplasts had good activity. Counting the stained and unstained cells in the field of view showed that the viability of the isolated protoplasts reached 64%( Figure 2 I).

[0065] Based on the above experimental results, the present invention determined the optimal conditions for isolating Suaeda heteroptera protoplasts: the rod-shaped leaves of Suaeda heteroptera were pre-cut transversely and longitudinally and peeled, 1.3M glucose was used as an osmotic pressure regulator, the enzymatic hydrolysis system contained 0.75% cellulase R-10 and 0.25% macerozyme R-10, and the incubation temperature was 4°C. Under these conditions, protoplasts with high yield and good integrity can be isolated, providing stable experimental materials for subsequent functional genomics research.

[0066] Example 2 Detection of the viability of Suaeda heteroptera protoplasts

[0067] To deeply explore the biological characteristics of Suaeda heteroptera leaf protoplasts and evaluate their feasibility for subsequent experiments, the viability of Suaeda heteroptera protoplasts was detected.

[0068] Fluorescein diacetate (FDA) was used to detect the viability of Suaeda heteroptera protoplasts. 1 mL of resuspended protoplasts was aspirated and placed in a 2 mL centrifuge tube pre-wrapped with tin foil. 2 μL of 1% DAPI staining solution, 2 μL of 0.2% FDA staining solution, and 2 μL of 1% rhodamine 123 staining solution were added respectively, and stained in the dark for 15 min. After staining, 50 μL was aspirated and placed on a glass slide, and observed under a Nikon A1R HD25 high-resolution laser confocal microscope (Nikon Ti-2, Japan) (A1 mode: excitation light wavelength 488 nm, emission light wavelength 510 - 550 nm). Viable protoplasts would emit green fluorescence, and the number of protoplasts emitting green fluorescence was counted. The ratio of the number of viable protoplasts to the total number of protoplasts in the same field of view was the protoplast viability.

[0069] Results:

[0070] To evaluate the subcellular structural integrity and cell viability of Suaeda heteroptera Koidz. leaf protoplasts, this study used a variety of fluorescent dyes to stain and analyze the purified protoplasts. The results showed that the autofluorescence of chloroplasts could be clearly observed at an excitation wavelength of 640 nm, and it coincided with the chloroplasts in the bright field, proving that the chloroplast structure of the isolated Suaeda heteroptera Koidz. protoplasts was intact( Figure 3 D). Observing individual protoplasts stained with FDA, the morphological distribution of protoplast cell channels could be preliminarily observed( Figure 3 A). The DAPI staining results showed that the nucleus presented typical blue fluorescence( Figure 3 B), spatially separated from the autofluorescence signal of chloroplasts. The rhodamine 123 staining results showed that mitochondria were successfully stained, presenting a punctate green fluorescence distribution, and this fluorescence signal did not coincide with the autofluorescence of chloroplasts( Figure 3 C). The above results together indicated that the subcellular organelle structure of Suaeda heteroptera Koidz. leaf protoplasts isolated by enzymatic digestion was intact, with good cell viability, and could meet the strict requirements of subsequent experiments.

[0071] Example 3 Establishment and application of the transfection system for Suaeda heteroptera Koidz. protoplasts

[0072] 1. PEG-mediated transient transformation of Suaeda heteroptera Koidz. protoplasts

[0073] Protoplast resuspension and preparation: Resuspend the purified protoplasts with W5 buffer, and then place them on ice for static incubation for 30 min. After the incubation ends, remove the supernatant, and then resuspend the protoplasts with MMS buffer.

[0074] Transformation operation: Take 500 μL of the resuspended protoplasts and add them to a new 2 mL centrifuge tube. Add 5 μg of the empty vector control (pCAMBIA2300-eGFP) plasmid to the tube, and gently tap the centrifuge tube to mix the contents. Then, add 110 μL of 40% PEG4000 solution, invert the centrifuge tube 6 - 8 times up and down, and then let it stand at room temperature for 15 - 30 min. This step aims to initiate the transformation process.

[0075] Termination of transformation and subsequent culture: After the transformation is initiated, add 800 μL of W5 buffer and elute twice, gently inverting the centrifuge tube 6 - 8 times each time to stop the transformation. Then incubate at room temperature for 20 min. After the incubation ends, remove the supernatant, and resuspend the protoplasts with WI buffer. Finally, transfer the resuspended protoplasts to a 35 mm cell culture dish and culture them for 16 h under the conditions of a temperature of 24 °C and a light intensity of 22 μmol·m-2·s-1.

[0076] Evaluation of transfection efficiency: Use a 200 μL pipette tip with the tip cut off to aspirate 30 μL of the transformed Suaeda aralocaspica protoplasts, drop them onto a glass slide, cover with a coverslip, and observe the transfection of protoplasts under a Nikon A1R HD25 high-resolution laser confocal microscope (Nikon Ti-2, Japan) (A1 mode: excitation light wavelength 488 nm, emission light wavelength 510 - 550 nm). The successfully transformed protoplasts in every 30 μL.

[0077] Table 2 Components of each transformation reagent

[0078]

[0079] Results:

[0080] GFP was introduced into Suaeda aralocaspica protoplasts, emitting green fluorescence, evenly distributed in the cytoplasm and nucleus, without overlap with the autofluorescence of Suaeda aralocaspica chloroplasts, indicating that the pCAMBIA2300-eGFP plasmid has been successfully transformed into Suaeda aralocaspica protoplasts, and the gene expression program has been initiated inside the protoplasts, successfully expressing the GFP protein ( Figure 4 A - C).

[0081] 2. Optimization of the transient transformation system of Suaeda aralocaspica protoplasts

[0082] To optimize the transformation system of Suaeda aralocaspica protoplasts, under the condition of fixing the total amount of pCAMBIA2300-eGFP plasmid (5 μg), the effects of different plasmid concentration gradients (600, 1000, 1900, 2400 ng / μL) on the transfection efficiency were systematically investigated. The same batch of extracted Suaeda aralocaspica protoplasts was used for parallel transformation in the experiment. The results showed that when the endotoxin-free plasmid concentration reached 2400 ng / μL, the highest transfection efficiency of protoplasts could reach 75%, significantly better than other concentration treatment groups ( Figure 5 ). This result determined the optimal plasmid usage concentration for subsequent research on the transformation of Suaeda aralocaspica protoplasts.

[0083] Example 4 Subcellular localization of Suaeda aralocaspica protoplasts

[0084] To test the reliability of the transient transformation system of Suaeda aralocaspica protoplasts established in this invention, the key photosynthetic enzyme SaPEPC1 and the transcription factor SabHLH169 of Suaeda aralocaspica were selected for subcellular localization analysis. The endotoxin-free plasmids pSuper1300-SaPEPC1-eGFP and pSuper1300-SabHLH169-eGFP (attached Figure 1 ) that were correctly identified by enzyme digestion were transferred into Suaeda aralocaspica protoplasts, and their localization was observed.

[0085] Results:

[0086] The green fluorescence of the SabHLH169-eGFP fusion protein overlaps with the nuclear signal labeled by DAPI and does not coincide with the autofluorescence of chloroplasts ( Figure 6 A), and the SaPEPC1-eGFP fusion protein has strong fluorescence signals in the cell membrane, cytoplasm, and chloroplasts ( Figure 6 B).

Claims

1. A method for preparing protoplasts of Phragmites australis, characterized in that: Follow the steps below to implement Take 1 g of leaves of Pleurotus eryngii, put them in lysis solution after pretreatment, incubate them in dark for 4 hours, filter them after incubation, centrifuge them for 4 minutes, and obtain protoplasts.

2. The method according to claim 1, characterized in that The lysate is prepared by the following steps: 0.75% w / v cellulase R-10 and 0.25% w / v cleavage enzyme R-10 are dissolved in CS buffer, heated at 55°C for 10 minutes, and after the enzyme solution is cooled to room temperature, 0.1% w / v bovine serum albumin is added to obtain the lysate.

3. The method according to claim 1, characterized in that The CS buffer consists of 1.3 M glucose, 25 mM HEPES-KOH (pH 6.5), 5 mM MgCl2, and 125 mM CaCl2.

4. The method according to claim 1, characterized in that: The incubation temperature was 4°C.

5. The method according to claim 1, characterized in that The pretreatment is to perform cross-cutting and longitudinal cutting on the rod-shaped leaves of the Pleurotus eryngii and peel off the epidermis in advance.

6. A method for transient transformation of protoplasts of Phragmites australis, characterized in that: The protoplasts of the P. sphaerocephala described in claim 1 are resuspended with W5 buffer, and then placed on ice for incubation for 30 minutes; after the incubation is completed, the supernatant is removed, and the protoplasts are resuspended with MMS buffer, and 5 μg of pCAMBIA2300-eGFP plasmid with a concentration of 2400 ng / μL and 40% PEG4000 solution are added for transformation; Among them: W5 buffer is 125mM CaCl2, 2mM MES-KOH, 5mM KCl, 154mM NaCl, and the balance is water; MMS buffer is 15mM MgCl2, 4mM MES-KOH, 1.0M glucose, and the balance is water.

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