MNP-mediated DNA plasmid transient plant transformation system and application thereof
By using PEI-Fe3O4 nanoparticle carriers to bind DNA plasmids, an MNP-mediated transient plant transformation system mediated by DNA plasmids was constructed, solving the problem of efficient gene transformation in plants such as scallions and achieving a simple transient transformation effect, which is suitable for biological breeding and scientific research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-03
- Publication Date
- 2026-04-21
AI Technical Summary
Existing gene transformation methods for monocotyledonous plants such as scallions are inefficient, and traditional methods are difficult to achieve efficient and safe gene transformation. Nanoparticle-mediated transformation systems need further improvement.
Using polyethyleneimine-coated Fe3O4 nanoparticles (PEI-Fe3O4) as a carrier, they were combined with DNA plasmids to achieve transient transformation of plant cells such as scallions and tobacco through simple methods such as injection and infiltration. MNP-mediated transient plant transformation system of DNA plasmids was formed using MES buffer.
It enables efficient and convenient instantaneous transformation of different plant cells, simplifies the operation process, improves transformation efficiency, and is suitable for biological breeding and scientific research.
Smart Images
Figure CN121896271A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to an MNP-mediated transient plant transformation system for DNA plasmids and its applications. Background Technology
[0002] Currently, the main breeding methods for scallions are artificial selection and hybridization breeding. These traditional breeding methods are inefficient and time-consuming. Therefore, constructing a scallion transformation system and obtaining higher-quality germplasm through molecular breeding is crucial for scallion production.
[0003] Gene transformation is a crucial step in plant functional genomics research and molecular breeding. Traditional plant genetic transformation methods include Agrobacterium tumefaciens infection, particle bombardment, and viral infection. These methods have been widely used in model plants and annual or biennial herbaceous plants. However, transformation of some monocotyledonous plants using Agrobacterium-mediated transformation is extremely difficult. Therefore, developing safe, efficient, and versatile gene transformation methods has been a hot topic in genetic engineering, molecular biology, and genetic breeding. In recent years, breakthroughs in nanotechnology in genetic engineering have provided new and more advantageous tools for plant genetic transformation. Nanoparticles (NPs) have been developed as gene delivery vectors and have been applied to species such as tobacco, maize, Arabidopsis thaliana, and onion. Previous studies have reported that carbon dots (CDs) can carry plasmids for transient transfection of wheat via seed soaking, constructing an efficient and convenient transient wheat transformation system. However, current nanoparticle-mediated transformation systems still need further development and improvement, and other different nanoparticle transformation vectors require further exploration.
[0004] Fe3O4 nanoparticles (MNPs) are a commonly used type of magnetic nanoparticle, possessing stable chemical properties, high catalytic activity, good magnetic responsiveness, and biocompatibility. In plant research, positively modified magnetic nanoparticles have been shown to deliver plasmids into pollen cells.
[0005] This invention aims to develop a transient transformation system mediated by PEI-coated Fe3O4 nanoparticles (MNPs) for scallion protoplasts, scallion seeds, tobacco leaves, and scallion stem discs. This system utilizes commercially available nanomaterials that can directly bind to DNA plasmids and achieve transient transformation of different plant cells through simple methods such as injection and infiltration, offering high efficiency and convenience. Summary of the Invention
[0006] The purpose of this invention is to provide an MNP-mediated transient plant transformation system for DNA plasmids and its application, in order to solve the problems existing in the prior art.
[0007] To achieve the above objectives, the present invention provides the following solution: This invention provides an MNP-mediated transient plant transformation system using DNA plasmids, the system comprising the following components at the following concentrations: MNP 0.03 mg / mL-0.07 mg / mL, DNA (plasmid DNA) 100 ng / μL-0.2 mg / mL and MESBuffer 10 mM.
[0008] Optionally, the transient plant transformation system for DNA plasmids includes components at the following concentrations: MNP 0.05mg / mL, DNA 190 ng / μL-0.2 mg / mL and MES Buffer 10 mM; Alternatively, MNP 0.04 mg / mL, DNA 0.2 mg / mL, and MES Buffer 10 mM; Alternatively, MNP 0.06 mg / mL, DNA 120 ng / μL, and MES Buffer 10 mM; Alternatively, MNP 0.06 mg / mL, DNA 150 ng / μL, and MES Buffer 10 mM.
[0009] Optionally, the plants include scallions and tobacco.
[0010] This invention provides the application of the above-described transient plant transformation system for DNA plasmids in the preparation of products for transient plant transformation of DNA plasmids.
[0011] This invention provides a product for transient plant transformation of DNA plasmids, the product comprising the aforementioned transient plant transformation system of DNA plasmids.
[0012] This invention provides the application of the above-described DNA plasmid transient plant transformation system or the above-described product in the transient transformation of plants using DNA plasmids.
[0013] The present invention provides a method for transient transformation of plants with DNA plasmids, the method comprising the steps of infecting explants with the above-described DNA plasmid transient plant transformation system or the above-described product to obtain DNA plasmid transiently transformed plants.
[0014] Optionally, the plants include scallions and tobacco.
[0015] Optionally, the explant may include one or more of the following: protoplast, leaf, seed, and stem disc.
[0016] Optionally, when the explant is a protoplast, the infection includes the steps of mixing the protoplast with the above-mentioned DNA plasmid transient plant transformation system or the above-mentioned product, and then performing magnetic treatment and incubation treatment in sequence. When the explant is a leaf, the infection includes the step of injecting the leaf with the above-mentioned transient plant transformation system with DNA plasmid or the above-mentioned product and culturing it. When the explant is a seed, the infection includes mixing the seed with the above-mentioned DNA plasmid transient plant transformation system or the above-mentioned product, and sequentially performing static treatment, incubation treatment, vacuum treatment and culture. When the explant is a stem disc, the infection includes the step of immersing the stem disc in the aforementioned DNA plasmid transient plant transformation system or the aforementioned product for cultivation. The present invention discloses the following technical effects: This invention marks the first discovery that polyethyleneimine (PEI)-coated Fe3O4 nanoparticles (PEI-Fe3O4) can serve as an infection component. PEI-Fe3O4 is a commercially available product with a stable source. Furthermore, PEI-Fe3O4 can be used to infect various explants. In summary, this system utilizes commercially available nanomaterials to directly bind to DNA plasmids, achieving instantaneous transformation of different plant cells through simple methods such as injection and infiltration, which is highly efficient and convenient. Therefore, the DNA plasmid instantaneous plant transformation system provided by this invention can be applied to biological breeding and scientific research, effectively solving the problem of lacking transgenic systems for functional gene research in onion molecular breeding. Attached Figure Description
[0017] 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.
[0018] Figure 1 TEM image of MNPs (left) and MNPs-DNA complex (right); Figure 2 Size distribution diagram of MNPs and MNPs-DNA complex; Figure 3 The zeta potentials of MNPs and the MNPs-DNA complex are shown; where the error bars represent the standard deviations of three replicates. Figure 4 Electrophoresis diagram of MNPs binding to plasmid DNA; Figure 5For MNPs-mediated transient transformation of tobacco leaves; Figure 6 Iron oxide nanoparticles mediated the transformation of leek protoplasts; Figure 7 Transient conversion of scallion seeds mediated by iron oxide nanoparticles; Figure 8 Transient transformation of scallion roots mediated by iron oxide nanoparticles; Figure 9 The instantaneous conversion efficiency of scallion roots mediated by iron oxide nanoparticles. Detailed Implementation
[0019] 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.
[0020] 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.
[0021] 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.
[0022] 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 obvious to those skilled in the art. This specification and embodiments are merely exemplary.
[0023] 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.
[0024] Experimental materials: Magnetic nanoparticles: The core conversion reagent used in this invention is Fe3O4 nanoparticles coated with polyethyleneimine (PEI) (PEI-Fe3O4), purchased from Ruixi Biotechnology Co., Ltd., catalog number R-CEI10010.
[0025] DNA plasmid: The plasmid used in this invention is GFP-1305.4 (CmBr confers fruitbitterness under CPPU treatment in melon) DOI 10.1111 / pbi.14399 This plasmid carries a green fluorescent protein (GFP) reporter gene. Those skilled in the art will understand that any other target DNA plasmid (whether a recombinant plasmid or a tagged empty vector plasmid) can be used in this transformation system.
[0026] Buffer system: Use 10 mM MES buffer as the base solution for the entire transformation system.
[0027] Plant material: Tobacco: Nicotiana benthamiana ( Nicotiana benthamiana The healthy, disease-free young leaves; Scallions: The variety is 'Zhangqiu Dawutong', using fresh tissue for protoplast transformation and healthy, plump seeds for seed transformation.
[0028] Magnetic field device: The magnetic field used for the transformation of scallion protoplasts is provided by a commercially available circular magnetic plate.
[0029] Example 1: Preparation and characterization of the conversion complex The transformation complex is a mixture of PEI-Fe3O4 nanoparticles, DNA plasmid, and MES buffer. The general preparation method is as follows: 1 μl of a 1.2 mg / ml PEI-Fe3O4 nanoparticle solution is added to 7.9 μl of ddH2O and vortexed to ensure thorough dispersion. Then, 1 μL of a 1000 ng / μl DNA plasmid solution (e.g., GFP-1305.4 plasmid) is added. Finally, 0.1 μl of 1 mM MES buffer is added, and the nanoparticles are gently pipetted or briefly vortexed (e.g., 1000 rpm for 10-15 seconds) to allow for electrostatic adsorption binding between the nanoparticles and DNA. The mixture is incubated at room temperature for 10-15 minutes to complete the assembly of the nanoparticle-DNA complex (MNPs-DNA or pDNA-MNPs). A control group without added DNA plasmid is used. The size and potential changes of the complex formed by the binding of MNPs and pDNA were investigated, as well as by agarose gel electrophoresis experiments (the steps for preparing MNPs-DNA in the agarose gel electrophoresis experiment are the same as before, the only difference being that the mass ratio of DNA plasmid to PEI-Fe3O4 nanoparticles is 1:0, 1:0.5, 1:0.8, 1:1 or 1:1.2). Figures 1-4 This indicates that the two do indeed combine.
[0030] Example 2 Instantaneous transformation of Tobacco Benedict leaves Preparation of infection solution: Prepare the infection solution in sterile centrifuge tubes to the following final concentrations: PEI-Fe3O4 nanoparticles: 0.05 mg / mL; GFP-1305.4 DNA plasmid: 190 ng / μL; buffer: 10 mM MME Buffer (pH 5.7).
[0031] Mix thoroughly according to the above-described "Preparation of Conversion Complex" method.
[0032] Transformation procedure: Draw up the infection solution using a 1 mL sterile syringe (no needle required). Gently hold the underside of a Nicotiana benthamiana leaf with your fingers, press the syringe tip against the lower epidermis of the leaf, and slowly apply pressure to inject the infection solution into the air cells (i.e., interstitial spaces of the mesophyll tissue) on the underside of the leaf, denoted as GFP-pDNA+MNPs. Simultaneously, use an infection solution containing only 190 ng / μL of GFP-1305.4 DNA plasmid (pDNA) and 10 mM MES as a control, denoted as GFP-pDNA.
[0033] Cultivation and Detection: The treated tobacco plants were placed in a 22℃ light incubator and cultured for 36 hours. Observations were then performed, and the results are as follows: Figure 5As shown in the figure. The results showed that the green fluorescent protein (GFP) signal could be observed under a confocal microscope after 36 h.
[0034] Example 3: Transformation of protoplasts from 'Zhangqiu Sycamore' Protoplast preparation: Protoplasts were isolated from fresh tissue of 'Zhangqiu Dawutong' using a conventional enzymatic hydrolysis method and resuspended in a suitable osmotic stabilizer (a solution containing 0.6 M mannitol). Specifically, a combination of cellulase and dissociative enzyme was used to isolate the protoplasts. The enzymatic hydrolysate consisted of the following components: 20 mmol / L KCl, 20 mmol / L MES (pH 5.8), 10 mmol / L CaCl₂, 1.0 g / L BSA, 10 g / L Cellulase R-10, and 7 g / L Macerozyme R-10. These components were mixed to prepare 10 mL of hydrolysate. Tender leaves were selected from healthy scallion plants, rinsed with ddH₂O, wiped dry with absorbent paper, and placed in petri dishes to air dry. They were then quickly cut into small fragments 0.5–1.0 mm wide using a sharp double-edged blade. 0.5 g of the fragments was weighed out. The leaf fragments were thoroughly mixed with the freshly prepared enzyme solution and placed in a dark environment at 26°C. After vacuum treatment for 0.5 h, the mixture was gently shaken at 50 r / min for 4-10 h to carry out enzymatic hydrolysis until the protoplasts were fully released.
[0035] After enzymatic hydrolysis, the mixture was filtered through a 70 pm cell filter to remove undigested tissue fragments and other impurities. The filtered liquid was collected in 50 mL round-bottom centrifuge tubes. An equal volume of pre-chilled W5 solution (containing 4 mmol / L MES, 154 mmol / L NaCl, 125 mmol / L CaCl, 5 mmol / L KCl, pH 5.7) was added to the filtered protoplast suspension, and the mixture was centrifuged at 500 r / min for 4 min, gently discarding the supernatant. The protoplasts were resuspended in 5 mL of W5 solution to the resulting precipitate, incubated on ice in the dark for 30 min, and the supernatant was discarded. The precipitate was then resuspended in 1 mL of MMG solution (containing 0.4 mol / L mannitol, 15 mmol / L MgCl, and 4 mmol / L MES). The solution was adjusted to a density of 1 × 10⁻⁶. 6 pcs·mL −1 Protoplast suspension.
[0036] Preparation of infection solution: Take 200 μL of the prepared protoplast suspension and add each component directly to the following final concentrations: PEI-Fe3O4 nanoparticles: 0.04 mg / mL; GFP-1305.4 DNA plasmid: 0.2 mg / mL; buffer: 10 mM MME Buffer (pH 5.7).
[0037] Gently invert the centrifuge tube 8-10 times to thoroughly mix the infection solution with the protoplasts.
[0038] Conversion procedure: Place the mixed centrifuge tubes on a pre-cooled magnetic plate, gently invert and shake the magnetic plate and centrifuge tubes together 8-10 times, and then let stand for 3 minutes.
[0039] Culture and Detection: Centrifuge tubes were removed from the magnetic plate, wrapped in aluminum foil to protect from light, and incubated in the dark at 25°C for 16 hours, denoted as GFP-pDNA+MNPs. A control containing only 0.2 mg / mL GFP-1305.4 DNA plasmid (pDNA) and 10 mM MES was used, denoted as GFP-pDNA. After incubation, observations were performed, and the results are as follows: Figure 6 As shown in the figure. The results show that the expression of green fluorescent protein (GFP) in protoplasts can be directly observed under a fluorescence microscope.
[0040] Example 4: Transformation of Zhangqiu Sycamore Seeds Seed pretreatment: Select plump 'Zhangqiu Dawutong' seeds and lightly abrade the seed coat with sandpaper or emery to facilitate the penetration of the conversion solution.
[0041] Preparation of inoculum: Prepare the inoculum to the following final concentration: PEI-Fe3O4 nanoparticles: 0.06 mg / mL; GFP-1305.4 DNA plasmid: 120 ng / μL; buffer: 10 mM MME Buffer (pH 5.7).
[0042] Conversion steps: 1. Soak the abraded seeds completely in the inoculum.
[0043] 2. Let stand at 4℃ for 12 hours.
[0044] 3. Transfer the system to a 37°C water bath and incubate for another 30 minutes.
[0045] 4. Remove the seeds and some of the inoculum, place them in a vacuum desiccator, and evacuate them at 600 MPa for 20 minutes. Then slowly release the vacuum to allow the pressure to return to normal.
[0046] 5. Repeat the above vacuuming steps once.
[0047] 6. Place the system in a 4°C environment overnight (approximately 12-16 hours), and label it as GFP-pDNA+MNPs. Simultaneously, use an infection solution containing only 120 ng / μL of GFP-1305.4 DNA plasmid (pDNA) and 10 mM MES as a control, labeled as GFP-pDNA.
[0048] Cultivation and Detection: After transformation treatment, seeds were removed, lightly rinsed with sterile water, and sown in moist nutrient soil. Four days after seedling emergence, samples were taken to detect the integration and expression of the exogenous gene. The results are as follows: Figure 7 As shown in the figure. The results showed that green fluorescent protein (GFP) signals could be observed in seedling leaves under a confocal microscope.
[0049] Example 5: Instantaneous transformation of the root system of a living *Firmiana simplex* var. *zhangqiuensis*. Pretreatment of Zhangqiu Sycamore: Healthy seedlings grown in hydroponics or substrate under standard conditions until one month of age.
[0050] Preparation of inoculum: Prepare the inoculum to the following final concentration: PEI-Fe3O4 nanoparticles: 0.06 mg / mL; GFP-1305.4 DNA plasmid: 150 ng / μL; buffer: 10 mM MES Buffer (pH 5.7).
[0051] Conversion steps: 1. Material pretreatment: Select uniformly growing scallion seedlings, and use a sterile blade to cut off the root system at the base of the stem plate, leaving the intact stem plate and above-ground plant.
[0052] 2. Immersion treatment: Immerse the stem plate of the scallion treated above directly in the prepared immersion solution, ensuring that the stem plate is completely submerged.
[0053] 3. Co-culture: Containers containing the material were placed in an incubator at 25°C under normal light conditions and incubated statically for 48 hours, denoted as GFP-pDNA+MNPs. Simultaneously, an infection solution containing only 150 ng / μL of GFP-1305.4 DNA plasmid (pDNA) and 10 mMMES was used as a control, denoted as GFP-pDNA.
[0054] 4. Detection and Analysis: After cultivation, remove the newly generated root system and gently rinse it with buffer solution. It can then be observed directly, and the results are as follows: Figure 8 As shown in the figure. The results show that GFP expression can be observed using confocal microscopy.
[0055] 5. Results: Using the above method, reporter gene (e.g., GFP) expression signals can be observed in the root tips and elongation zones of scallion roots after 48 hours of co-culture, proving that the exogenous gene has been successfully introduced and expressed. The transformation rate is as follows: Figure 9 As shown (conversion rate = number of positive roots / total number of roots × 100%), the conversion rate of GFP-pDNA+MNPs is significantly higher than that of GFP-pDNA, approximately 68%.
[0056] In summary, this method eliminates the need for protoplast preparation and avoids the use of Agrobacterium, allowing direct manipulation of living root systems. The process is simple, fast, and highly reproducible, providing a powerful tool for biological research on scallion roots.
[0057] 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 transient plant transformation system mediated by MNP-mediated DNA plasmids, characterized in that, The transient plant transformation system for DNA plasmids comprises the following components at the following concentrations: MNP 0.03 mg / mL-0.07 mg / mL, DNA 100 ng / μL-0.2 mg / mL and MES Buffer10 mM.
2. The transient plant transformation system using DNA plasmids according to claim 1, characterized in that, The transient plant transformation system for DNA plasmids comprises the following components at the following concentrations: MNP 0.05mg / mL, DNA 190 ng / μL-0.2 mg / mL and MES Buffer 10 mM; Alternatively, MNP 0.04 mg / mL, DNA 0.2 mg / mL, and MES Buffer 10 mM; Alternatively, MNP 0.06 mg / mL, DNA 120 ng / μL, and MES Buffer 10 mM; Alternatively, MNP 0.06 mg / mL, DNA 150 ng / μL, and MES Buffer 10 mM.
3. The transient plant transformation system using DNA plasmids according to claim 1, characterized in that, The plants mentioned include scallions and tobacco.
4. The use of the DNA plasmid transient plant transformation system according to any one of claims 1-3 in the preparation of products for DNA plasmid transient plant transformation.
5. A product for transient plant transformation of DNA plasmids, characterized in that, The product includes the DNA plasmid transient plant transformation system as described in claim 1 or 2.
6. The use of the DNA plasmid transient plant transformation system according to any one of claims 1-3 or the product according to claim 5 in the transient transformation of plants with DNA plasmids.
7. A method for transient transformation of plants using DNA plasmids, characterized in that, The method includes the step of infecting explants with the DNA plasmid transient plant transformation system according to any one of claims 1-3 or the product according to claim 5 to obtain DNA plasmid transiently transformed plants.
8. The method according to claim 7, characterized in that, The plants mentioned include scallions and tobacco.
9. The method according to claim 7, characterized in that, The explants include one or more of the following: protoplasts, leaves, seeds, and stem discs.
10. The method according to claim 9, characterized in that, When the explant is a protoplast, the infection includes the steps of mixing the protoplast with the DNA plasmid transient plant transformation system according to any one of claims 1-3 or the product according to claim 5, and then performing magnetic treatment and incubation treatment in sequence. When the explant is a leaf, the infection includes the step of injecting the leaf with the DNA plasmid transient plant transformation system according to any one of claims 1-3 or the product according to claim 5, and then culturing it. When the explant is a seed, the infection includes mixing the seed with the DNA plasmid transient plant transformation system according to any one of claims 1-3 or the product according to claim 5, and sequentially performing static treatment, incubation treatment, vacuum treatment and culture. When the explant is a stem disc, the infection includes the step of immersing the stem disc in the DNA plasmid transient plant transformation system of any one of claims 1-3 or the product of claim 5 for cultivation.