Separation and transformation method of grape protoplast

Abstract

The invention discloses a grape protoplast separation and transformation method, and belongs to the technical field of protoplast separation and transformation. According to the method, grape calluses are used as materials, and a protoplast preparation technology which is feasible all year round, high in efficiency and large in yield is developed. Compared with the prior art, the method has the advantages that a large amount of high-activity protoplasts can be continuously and stably obtained by utilizing the grape calluses, the operation is simple and convenient, the instantaneous transformation of genes is easier to realize, and the transformation efficiency is obviously improved. The method disclosed by the invention can be effectively used for subcellular localization and bimolecular fluorescence interaction (BiFC) verification of grape body genes, and the accuracy and reliability of grape protein function localization and interaction analysis thereof are greatly improved. The invention provides powerful technical support for grape gene function analysis and precise gene editing research, and has wide application prospect and important scientific research value.

Description

Technical Field

[0001] This invention relates to the field of protoplast separation and transformation technology, and in particular to a method for separating and transforming grape protoplasts. Background Technology

[0002] Grapes (Vitis L.) are a globally important economic fruit tree, widely used in various industries such as winemaking, fresh consumption, drying, and juice production. Since the 1970s, modern biotechnology, such as cell engineering and genetic engineering, has been continuously developing in crop breeding, propelling crop improvement from traditional phenotypic selection to the molecular level. However, due to the long life cycle, immature genetic transformation system, and complex genome structure of grapes, functional gene research has lagged behind, making it difficult to achieve precise variety improvement through genetic engineering. Therefore, there is an urgent need to construct a rapid and efficient transient gene expression system to support the development needs of grape molecular breeding and functional gene research.

[0003] Plant protoplasts are naked cellular structures after the removal of their cell walls. Lacking a cell wall barrier, they can efficiently take up exogenous DNA, making them ideal recipient materials for genetic engineering manipulation (especially transient expression). The isolation and purification of protoplasts are not only crucial steps in constructing transient expression systems but also fundamental to protoplast fusion and plant regeneration research. This system offers advantages such as ease of operation, rapid expression, and high efficiency, and is widely used in transient gene expression, protein subcellular localization, protein-protein interaction analysis, and enzyme activity detection. It is also a commonly used platform for validating the feasibility of functional vectors.

[0004] Current grape protoplast preparation systems primarily rely on leaves as explants, and these systems have high requirements for the physiological state of the donor leaves. Generally, the second to fourth unopened young leaves from the top of the plant are collected to ensure a relatively loose cell wall structure, thereby increasing protoplast yield. However, this material selection limits the timeliness and reproducibility of experiments, hindering their widespread adoption in routine experiments. Furthermore, the application of existing systems is mostly limited to morphological observation at the cellular level, such as fluorescent staining to trace organelle structures or detect membrane integrity, and their potential in gene function analysis has not been fully realized. Particularly in the field of protein-protein interaction research, the grape protoplast system lacks systematic technical exploration and optimization.

[0005] Therefore, how to construct a more efficient, stable, and widely applicable method for protoplast isolation and transformation is a technical bottleneck that needs to be overcome in current grape functional gene research and molecular breeding. Summary of the Invention

[0006] The purpose of this invention is to provide a method for the isolation and transformation of grape protoplasts, thereby solving the problems existing in the prior art. This is achieved by establishing an optimized PEG-Ca...2+ This invention significantly improves the efficiency of exogenous plasmid delivery through a highly efficient process mediated by the present invention. It can widely support molecular biological analyses such as protein-protein interaction (BiFC) and subcellular localization, and successfully avoids the high dependence of traditional leaf protoplast systems on developmental stages. It provides a stable, efficient and reproducible in vitro experimental platform for grape functional gene research.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] In a first aspect, the present invention provides a method for isolating grape protoplasts, comprising the following steps:

[0009] Take callus tissue and place it in protoplast enzymatic hydrolysis solution, and incubate at 25-35℃ for 5-17 hours for static enzymatic hydrolysis.

[0010] Remove the enzymatically hydrolyzed tissue, add W5 solution and mix, filter, collect the filtrate, centrifuge, and discard the supernatant;

[0011] Resuspend in W5 solution, centrifuge, discard the supernatant, and repeat the operation.

[0012] Resuspend in W5 solution, let stand, centrifuge, and discard the supernatant; resuspend in MMG solution to obtain protoplast suspension.

[0013] Preferably, the static enzymatic hydrolysis is performed at a temperature of 30°C for 10-12 hours.

[0014] Preferably, the mass-to-volume ratio of the callus tissue to the protoplast enzymatic hydrolysate is 8-10 g: 10 mL.

[0015] Preferably, the protoplast enzymatic hydrolysate comprises the following components: 1-3% cellulase, 1-3% dissociation enzyme, 5-15mM CaCl2, 1-9mM 2-morpholinoethanesulfonic acid, and 0.1-0.9M mannitol.

[0016] Preferably, the enzymatically digested tissue is mixed with 1-5 mL of W5 solution, filtered, and centrifuged at 100-200 g for 2-6 min at room temperature, and the supernatant is discarded.

[0017] Preferably, the protoplast suspension is resuspended in 1-3 times the volume of the W5 solution, centrifuged at 100-200g for 2-6 minutes at room temperature, the supernatant is discarded, and the operation is repeated; then, the protoplast suspension is resuspended in 1-3 times the volume of the W5 solution, allowed to stand on ice for 30 minutes, centrifuged at 100-200g for 2-6 minutes at 4°C, the supernatant is discarded; and then resuspended in 50-200μL of MMg solution to obtain the protoplast suspension.

[0018] Secondly, the present invention also provides a method for transforming grape protoplasts, comprising the following steps:

[0019] The plasmid and the protoplast suspension were mixed at a mass-to-volume ratio of 10-12 μg:110 μL to obtain suspension A. Suspension A was mixed with PEG solution at a volume ratio of 1-2:1-2 and allowed to stand for 10-50 min. W5 solution was added at a volume ratio of 4-6 times that of PEG solution to stop the reaction. The mixture was centrifuged at 25°C and 100-200g for 1-3 min, and the supernatant was discarded. The mixture was resuspended in 0.5-1.5 mL of WI solution and transferred to a cell culture plate. The cells were cultured in the dark at 20-24°C for 18-24 h to obtain protoplast cells that successfully expressed the target gene.

[0020] Preferably, the PEG solution comprises the following components: 40% PEG4000, 0.2M Mannitol, and 0.1M CaCl2 by mass concentration.

[0021] Preferably, the centrifugation conditions are 25°C, 150g, centrifugation for 2 minutes.

[0022] Preferably, the WI solution comprises the following components: 4 mM MES, 0.5 M Mannitol, and 20 mM KCl.

[0023] The present invention discloses the following technical effects:

[0024] (1) Efficiency and visibility: A high-efficiency, fast, convenient and fluorescence visualization technology platform has been established to support intuitive detection of gene transformation effects.

[0025] (2) Strong adaptability to functional studies: This system is particularly suitable for a variety of molecular biology experiments such as preliminary screening of gene function, subcellular localization, and protein-protein interactions (such as BiFC), and has good scalability and applicability.

[0026] (3) High repeatability and material universality: The material source is stable and easy to obtain, which avoids the strict requirements on the development state of the donor leaf during the preparation of leaf protoplasts, and improves the operational stability and data consistency of the experiment.

[0027] This invention uses grape callus as the starting material, efficiently separates and obtains protoplasts through enzymatic hydrolysis, and combines it with PEG-Ca 2+ A mediated transformation method was used to introduce the target exogenous plasmid into protoplasts. Fluorescence signals were detected using laser confocal microscopy, successfully validating the transient expression of the exogenous gene in grape cells. This system overcomes the problems of strong dependence on leaf development stages and poor reproducibility in traditional grape protoplast preparation processes, providing a reliable platform for rapid validation of grape functional genes.

[0028] In summary, this invention constructs a grape protoplast isolation and transformation technology system with universality and practical value, which can significantly improve the efficiency of grape functional gene research and promote the in-depth development of grape molecular breeding and gene editing. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 A process flow diagram for the isolation and transformation of grape protoplasts;

[0031] Figure 2 The effects of grape leaves and callus on protoplast state are shown; where A represents protoplasts isolated from grape leaves, B represents protoplasts isolated from grape callus, and C represents the number of 10 protoplasts in the microscope field of view.

[0032] Figure 3 The effect of different enzymatic hydrolysis methods on protoplast state; where A represents the hydrolysis process performed at 30℃ on a shaker for 30-40 rpm. -1 B is the low-speed shaking treatment, C is the treatment of enzymatic digestion in a 30℃ incubator, and D is the number of 10 protoplasts in the microscope field of view.

[0033] Figure 4 To verify the interaction between VaTIP1-1 and VaATG8f in grapes; where A is the fluorescence detection map of the interaction between VaTIP1-1 and VaATG8f in the endoplasmic reticulum (ER), and B is the quantitative analysis map of the fluorescence colocalization intensity of the VaTIP1-1 and VaATG8f interaction region and the ER marker gene. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] This invention discloses a method for the isolation and transformation of grape protoplasts, the process flow diagram of which is shown below. Figure 1 The method is detailed below.

[0040] Grape source: Young shoots and leaves and callus tissue of the European grape variety 'Cabernet Sauvignon' were used as experimental materials.

[0041] Source of induced callus: The callus was induced from the leaves of the European grape variety 'Cabernet Sauvignon'.

[0042] The proliferation medium consists of the following components: 4.43 g·L⁻¹ -1 MS, 30 g·L -1 Sucrose, 2 mg·L -1 6-BA, 0.5 mg·L - 1 NAA, 3g·L -1 Plant-based gel, 0.5 g·L -1 Activated carbon, pH=5.8.

[0043] Each 10 mL of enzymatic hydrolysate contains the following components: 2% (w / v) cellulase, 1% (w / v) macerozyme, 10 mM CaCl2, 5 mM 2-morpholinoethanesulfonic acid (MES), and 0.5 M mannitol, pH = 5.7; it is used after being sterilized by filtration through a 0.45 μm filter membrane.

[0044] Each 10 mL W5 solution contains the following components: 2 mM MES, 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, pH = 5.7; it is used after being sterilized by filtration through a 0.45 μm filter membrane.

[0045] Each 10 mL WI solution contains the following components: 4 mM MES, 0.5 M Mannitol, 20 mM KCl, pH = 5.7; it is used after being sterilized by filtration through a 0.45 μm filter membrane.

[0046] Each 10 mL PEG solution contains the following components: 40% (w / v) PEG4000, 0.2 M Mannitol, 0.1 M CaCl2, pH = 5.7; used after sterilization by filtration through a 0.45 μm filter membrane.

[0047] Each 10 mL MMg solution contains the following components: 0.4 M Mannitol, 4 mM MES, 15 mM MgCl2, pH = 5.7; it is used after being sterilized by filtration through a 0.45 μm filter membrane.

[0048] Plasmid preparation and concentration: 200 μL of *E. coli* bacterial culture containing the fluorescently labeled vector was transferred to 50 mL of LB liquid medium containing Kan resistance and cultured for 12-16 h. Following the instructions of the Tiangen plasmid mini-prep kit, the 50 mL culture was divided into 10 portions for extraction. The extracted plasmids were eluted with ddH2O and collected in 1.5 mL centrifuge tubes. 1 / 10 volume of 3M NaAc (pH = 5.2) and 2 volumes of pre-cooled anhydrous ethanol were added and mixed well. The mixture was incubated at -20℃ for 2 h. The tubes were then centrifuged at 12000 rpm for 10 min at 4℃, and the supernatant was discarded. 1 mL of 70% anhydrous ethanol was added, and the tubes were gently washed. The tubes were centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. This washing step was repeated once. After discarding the supernatant, the tubes were allowed to stand at room temperature for 10-15 min to evaporate any residual alcohol. Add 50 μL of ddH2O, determine the plasmid concentration using a Nanodrop 2000 / 2000c spectrophotometer, adjust the plasmid concentration to 2000-2500 ng / μL, and store at -20℃.

[0049] Example 1: Isolation and Transformation of Grape Protoplasts

[0050] A method for isolating and transforming grape protoplasts includes the following steps:

[0051] (1) Preparation of grape callus: The pre-induced callus was placed in a proliferation medium for subculture and cultured in the dark at 25°C for 2-3 weeks to obtain high-quality material for protoplast isolation.

[0052] (2) Protoplast isolation: Add 8-10g of callus tissue to 10mL of protoplast enzymatic hydrolysis solution, gently shake to disperse the callus tissue, and incubate at 30℃ for 10-12h. Remove the hydrolyzed tissue, add 5mL of W5 solution, gently shake, and filter through 100-mesh gauze. Transfer the obtained protoplasts to a 50mL centrifuge tube, centrifuge at 200g for 2min at room temperature, and discard the supernatant. Add 2 times the volume of pre-cooled W5 solution to resuspend the protoplasts, centrifuge at 200g for 2min at room temperature, and discard the supernatant; repeat this step; add 2 times the volume of W5 solution again to resuspend the protoplasts, and incubate on ice for 30min. Centrifuge at 200g for 2min at 4℃, discard the supernatant, add MMG solution (100-150μL) to resuspend the protoplasts to obtain a protoplast suspension. Take 20μL for microscopic examination to observe the mass and concentration of protoplasts.

[0053] (3) Protoplast transformation: Take plasmid (10-12 μg) into a 2.0 mL centrifuge tube, and take 110 μL of the protoplast suspension prepared above. Gently mix to obtain suspension A. Add an equal volume (based on the volume of suspension A) of PEG solution and gently mix. Incubate for 30 min. Add 4 times the volume (based on the volume of PEG solution) of W5 solution and mix. Invert the tube to terminate the reaction. Centrifuge at 25℃ and 150g for 2 min, discard the supernatant, and resuspend the reaction system in 1 mL of WI solution. Place the above reaction system in a cell culture plate rinsed with 0.005 g / mL BSA (pre-added with 1 mL of W5 solution) and culture at 22℃ in the dark for 18-24 h to obtain protoplast cells that successfully express the target gene. Observe using a Leica laser confocal microscope.

[0054] Example 2: Optimization of Experimental Conditions

[0055] 1. The Influence of Different Tissue Materials on Protoplast State

[0056] The experiment used young shoots and leaves of the European grape variety 'Cabernet Sauvignon' and callus tissue as materials. For grape leaves, the leaf surface was washed with clean water and rinsed for 30 minutes, then the leaves were cut into regular small strips. For grape callus tissue, highly soft and hydrated callus tissue was selected. Following experimental step (2) in Example 1, the materials were placed in an enzymatic hydrolysate to separate protoplasts. After separation, 20 μL was taken for microscopic examination to observe the mass and concentration of protoplasts.

[0057] Results analysis: See results below. Figure 2 .Depend on Figure 2 The results from the AB analysis show that using grape callus tissue as the material for protoplast isolation yields a significantly higher number of intact protoplasts than using grape leaves. Therefore, grape callus tissue was ultimately chosen as the material for protoplast isolation.

[0058] 2. Effects of different enzymatic hydrolysis methods on protoplast state

[0059] The experiment used callus tissue from the European grape variety 'Cabernet Sauvignon' as material. The enzymatic hydrolysis method was set as follows: the callus tissue was added to the protoplast enzymatic hydrolysis solution and placed on a shaker at 30℃ for 30-40 rpm. -1 Enzymatic hydrolysis was performed by low-speed shaking for 10-12 hours, or by incubation at 30°C for 10-12 hours. Subsequent experimental procedures were the same as step (2) in Example 1. After separation, 20 μL was taken for microscopic examination to observe the mass and concentration of protoplasts.

[0060] Results analysis: See results below. Figure 3 .Depend on Figure 3 The results from the AB analysis showed that the number of intact protoplasts obtained by enzymatic hydrolysis in a 30℃ incubator for 10-12 hours was significantly higher than that obtained by low-speed shaking at 30℃ for 10-12 hours. Therefore, the method of enzymatic hydrolysis in a 30℃ incubator for 10-12 hours was ultimately chosen for the material.

[0061] Example 3: Verification of bimolecular fluorescence interaction using grape callus protoplasts

[0062] Plasmids pSPYNE-VaTIP1-1 and pSPYCE-VaATG8f were extracted and purified to a concentration of 2 μg / μL. 110 μL of protoplasts and an equal volume of PEG solution (protoplast volume + plasmid volume) were added sequentially, gently mixed, and incubated at room temperature for 30 min. The reaction was then terminated by adding 4 times the volume of PEG solution in W5 solution, and gently mixed again. After centrifugation at 200g for 2 min, the supernatant was discarded, and the protoplasts were resuspended in 1 mL of W5 solution. The mixture was incubated at room temperature in the dark for 12–16 h. After incubation, 20 μL of protoplasts was placed on a glass slide, covered with a coverslip, and fluorescence was observed using a laser confocal microscope. The excitation and emission wavelengths of green fluorescent protein (GFP) and red fluorescent protein (mCherry) were observed to be 470 / 510 nm and 538 / 584 nm, respectively.

[0063] Results analysis: See results below. Figure 4The results showed that pSPYNE-VaTIP1-1 and pSPYCE-VaATG8f interacted and produced yellow fluorescence in callus protoplasts, indicating that VaTIP1-1 and VaATG8f interact in cell protoplasts. To verify whether their interaction fluorescence was localized to the endoplasmic reticulum (ER), pSPYNE-VaTIP1-1, pSPYCE-VaATG8f, and the ER marker were co-expressed in callus protoplasts. The results showed that their interaction yellow fluorescence and the ER marker red fluorescence were highly co-localized. Figure 4 In the middle A), quantitative fluorescence data also confirmed the high colocalization of yellow fluorescence (YFP) and red fluorescence (mCherry), indicating that VaTIP1-1 and VaATG8f interact in the endoplasmic reticulum. Figure 4 (B)

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for isolating grape protoplasts, characterized in that, Includes the following steps: Take callus tissue and place it in protoplast enzymatic hydrolysis solution, and incubate at 25-35℃ for 5-17 hours for static enzymatic hydrolysis. Remove the enzymatically hydrolyzed tissue, add W5 solution and mix, filter, collect the filtrate, centrifuge, and discard the supernatant; Resuspend in W5 solution, centrifuge, discard the supernatant, and repeat the operation. Resuspend in W5 solution, let stand, centrifuge, and discard the supernatant; resuspend in MMG solution to obtain protoplast suspension.

2. The separation method according to claim 1, characterized in that, The static enzymatic hydrolysis was carried out at a temperature of 30°C for 10-12 hours.

3. The separation method according to claim 1, characterized in that, The mass-to-volume ratio of the callus tissue to the protoplast enzymatic hydrolysate is 8-10 g: 10 mL.

4. The separation method according to claim 1, characterized in that, The protoplast enzymatic hydrolysate comprises the following components: 1-3% cellulase, 1-3% dissociation enzyme, 5-15mM CaCl2, 1-9mM 2-morpholinoethanesulfonic acid, and 0.1-0.9M mannitol.

5. The separation method according to claim 1, characterized in that, After enzymatic hydrolysis, the tissue was mixed with 1-5 mL of W5 solution, filtered, and centrifuged at 100-200 g for 2-6 min at room temperature. The supernatant was then discarded.

6. The separation method according to claim 1, characterized in that, Resuspend the protoplast in 1-3 times its volume of the W5 solution, centrifuge at 100-200g for 2-6 minutes at room temperature, discard the supernatant, and repeat the operation; then resuspend in 1-3 times its volume of the W5 solution, incubate on ice for 30 minutes, centrifuge at 100-200g for 2-6 minutes at 4℃, discard the supernatant; resuspend in 50-200μL of MMg solution to obtain a protoplast suspension.

7. A method for transforming grape protoplasts, characterized in that, Includes the following steps: The plasmid and the protoplast suspension described in claim 1 are mixed at a mass-to-volume ratio of 10-12 μg:110 μL to obtain suspension A. Suspension A is mixed with PEG solution at a volume ratio of 1-2:1-2 and allowed to stand for 10-50 min. 4-6 times the volume of W5 solution relative to the PEG solution is added and mixed to terminate the reaction. The mixture is centrifuged at 25°C and 100-200g for 1-3 min, and the supernatant is discarded. 0.5-1.5 mL of WI solution is added to resuspend the protoplast, which is then transferred to a cell culture plate and cultured in the dark at 20-24°C for 18-24 h to obtain protoplast cells that successfully express the target gene.

8. The conversion method according to claim 7, characterized in that, The PEG solution comprises the following components: 40% PEG4000, 0.2M Mannitol, and 0.1M CaCl2.

9. The conversion method according to claim 7, characterized in that, The centrifugation conditions were: 150g at 25°C for 2 minutes.

10. The conversion method according to claim 7, characterized in that, The WI solution comprises the following components: 4 mM MES, 0.5 M Mannitol, and 20 mM KCl.

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