Lithium acetate-polyethylene glycol mediated delivery of plasmid dna into yeast spheroplasts

Abstract

The application discloses a lithium acetate-polyethylene glycol mediated protoplast lipid nanoparticle delivery method and belongs to the technical field of biology. 2+ )concentration to improve the PEG formula, successfully delivers the lipid nanoparticle (LNP) to the protoplast of oil palm, coconut and other palm crops, the mRNA of the nanoparticle is wrapped with enhanced green fluorescent protein (eGFP), and the fluorescent protein can be directly expressed in the protoplast with the mRNA as a template without a promoter. After the LNP is delivered to the protoplast of the palm crops by the method, green fluorescent signals are detected by laser confocal microscopy. The method can be used for directly delivering target gene mRNA to the protoplast of the palm crops, and can also deliver CRISPR / Cas system protein and the like for gene editing technology research.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to a lithium acetate (LiAc)-polyethylene glycol (PEG-4000)-mediated lipid nanoparticle (LNP) delivery method suitable for protoplasts of palm crops such as oil palm, coconut, and betel nut, and its application. Background Art

[0002] Oil palm (scientific name: Elaeis guineensis Jacq.), Cocos nucifera (scientific name: Cocos nucifera Palm crops such as (L.) are trees in the Arecaceae family, primarily grown in Malaysia and Indonesia in Asia, western and central Africa, and northern and Central South America. Currently, the selection and breeding of new varieties of palm crops like coconut, oil palm, and betel nut still relies primarily on traditional hybridization, which suffers from low efficiency and long production cycles, severely hindering the development of high-yield, high-quality varieties. Advances in biotechnology have led to the application of molecular techniques such as protoplast-based genetic transformation and gene editing to a variety of crops, promoting the development of new breeding technologies. However, relevant research on palm crops like coconut, oil palm, and betel nut remains underway.

[0003] Plant protoplasts are cellular structural units composed of a cell membrane, cytoplasm, nucleus, and organelles, in addition to the cell wall. They are characterized by their rapid expression of exogenous genetic material. Therefore, protoplast-based transient expression has been widely applied in various fields of genetic research. In recent years, protoplast transformation technology has significantly advanced and is widely used to validate gene editing systems and vector activity, with significant implications for the targeted modification and precise editing of plant genomes.

[0004] Lipid nanoparticles (LNPs) are tiny particles composed of multiple lipid components, typically around 100 nm in diameter. Their core function is to encapsulate and protect nucleic acid therapeutics, such as mRNA or siRNA, enabling efficient cellular uptake and expression. The outer membrane of LNPs is primarily composed of PEG lipids, providing hydrophilicity. The inner membrane is primarily composed of neutral lipids, with cholesterol and neutral ionizable lipids in the center. This structure enables LNPs to electrostatically adhere to negatively charged cell surfaces and subsequently enter cells via endocytosis. Cell membranes are typically negatively charged, while the nanoparticle lipids are positively charged. This electrostatic interaction facilitates LNP adsorption and fusion with cell membranes. Anionic lipids in the cell membrane neutralize the charge of the cationic lipid carrier, facilitating nucleic acid release from the LNP. LNPs achieve efficient delivery of nucleic acid therapeutics through their unique structure and composition, as well as their interaction with cell membranes. This delivery system not only protects nucleic acid therapeutics from degradation but also enhances their cellular uptake and expression.

[0005] Although the technology of delivering exogenous nucleic acids to plant protoplasts through transient transformation has been widely used in many species, the protoplast transformation technology for tropical palm crops such as coconut, oil palm, and betel nut is still in its infancy and the transformation efficiency is low. Moreover, the research on the delivery technology of LNP in the protoplasts of tropical palm crops such as coconut, oil palm, and betel nut is still under development. Therefore, the establishment of LNP delivery methods suitable for the protoplasts of tropical palm crops such as coconut, oil palm, and betel nut is a necessary supplement to plant protoplast transformation technology and has positive significance for the development of tropical palm crop breeding technology. Summary of the Invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide a lipid nanoparticle (LNP) delivery method and application suitable for palm crop protoplasts.

[0007] In order to solve the above technical problems, the present invention provides an application of a multi-substance solution in palm crop protoplast transformation.

[0008] Specifically, the present invention provides an application of an MMG solution containing lithium acetate (LiAc) in the transformation of palm crop protoplasts.

[0009] Provides a high concentration of calcium ions (Ca 2+ Application of polyethylene glycol (PEG-4000) solution in protoplast transformation of palm crops.

[0010] More preferably, the two solutions are used together in the transformation of palm crop protoplasts.

[0011] Furthermore, a lithium acetate (LiAc)-polyethylene glycol (PEG-4000)-mediated lipid nanoparticle (LNP) delivery system suitable for palm crop protoplasts is provided.

[0012] The present invention also provides a formula of an MMG solution containing lithium acetate (LiAc).

[0013] Specifically, the MMG solution containing lithium acetate (LiAc) is prepared according to the following table:

[0014] Prepare 100 mL of MMG-LiAc solution by adding the ingredients listed in the table below. Dissolve in a microwave or water bath. Add ultrapure water to 100 mL. pH 6.03 (no adjustment required). Sterilize by filtration through a 0.22 μm filter and store at 4°C.

[0015] Reagents Final concentration Mass / g MES 4mM 0.078096 D-Mannitol 0.4M 7.2868 MgCl2.6H2O 15mM 0.30495 LiAc 2H2O 0.1M 1.0202g

[0016] The present invention also provides a method containing high concentration of calcium ions (Ca 2+) polyethylene glycol (PEG-4000) solution formulation.

[0017] Specifically, the one containing high concentration of calcium ions (Ca 2+ The polyethylene glycol (PEG-4000) solution was prepared according to the following table:

[0018] ;

[0019]

[0020] By utilizing the above transformation solution, the present invention provides a lipid nanoparticle (LNP) for delivering encapsulated enhanced green fluorescent protein (eGFP) mRNA to palm crop protoplasts.

[0021] The present invention is the first to add LiAc into the plant protoplast transformation liquid MMG to improve the transformation efficiency.

[0022] Beneficial effects:

[0023] The present invention adds LiAc to the protoplast transformation solution MMG for the first time and increases the calcium ion (Ca 2+ ) concentration, successfully delivered lipid nanoparticles (LNPs) into the protoplasts of palm crops such as oil palm and coconut. These nanoparticles encapsulated mRNA for enhanced green fluorescent protein (eGFP), enabling direct expression of the fluorescent protein within the protoplasts using the mRNA as a template without the need for a promoter. Using the method of this invention, green fluorescent signals were detected using laser confocal microscopy after LNP delivery into palm crop protoplasts. This method can be used to directly deliver target gene mRNA into palm crop protoplasts, as well as to deliver CRISPR / Cas system proteins for gene editing research. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the lipid nanoparticle (LNP) structure.

[0025] Figure 2 Fluorescence detection of untransformed oil palm protoplasts (control).

[0026] Figure 3 Transient transformation results of oil palm protoplasts (35s-GFP expression vector).

[0027] Figure 4 Delivery of lipid nanoparticles (LNPs) to oil palm protoplasts (eGFP-mRNA). DETAILED DESCRIPTION

[0028] Unless otherwise noted, the following examples of the present invention follow conventional methods. The plant expression vector p35s-GFP used in these examples was maintained in our laboratory, and lipid nanoparticles (LNPs) encapsulating eGFP-mRNA were purchased from GenScript Biotech Co., Ltd. These biological materials were used solely for reproducing the experiments described herein and should not be used for other purposes.

[0029] Example 1

[0030] This example provides a lipid nanoparticle (LNP) delivery method applicable to oil palm protoplasts, and the specific steps are as follows:

[0031] Step 1: Prepare a MMG solution containing lithium acetate (LiAc) as follows:

[0032] Prepare 100 mL of MMG-LiAc solution by adding the ingredients listed in the table below. Dissolve in a microwave or water bath. Add ultrapure water to 100 mL. pH 6.03 (no adjustment required). Sterilize by filtration through a 0.22 μm filter and store at 4°C.

[0033]

[0034] Step 2: Prepare a solution containing high concentration of calcium ions (Ca 2+ ) polyethylene glycol (PEG-4000) solution as follows:

[0035]

[0036] Prepare 10 mL of working solution. Add all stock solutions (except PEG (4000)) and ddH2O from a clean hood. Incubate the solution in a 55°C water bath for 10 minutes, mixing by inverting several times. Store at 4°C for approximately one week. Prepare the solution immediately before use.

[0037]

[0038] Step 3: Prepare oil palm protoplasts. Take young leaves of sterile oil palm seedlings as materials, and isolate and purify protoplasts using a protoplast isolation kit or other conventional methods.

[0039] Step 4: Transient transformation and delivery of lipid nanoparticles to oil palm protoplasts. The purified protoplasts were adjusted to a concentration of 2×10 5 / mL; first take 40 μL of 0.15 mg / mL LNP solution and add it to a new 2mL round-bottom centrifuge tube, then add 100 μL of the adjusted concentration of protoplast solution and mix well; add 140 μL of the newly prepared PEG (Ca 2+) solution, after mixing, standing at 28°C for 30 min; adding 1 mL of W5 culture solution to terminate the conversion reaction, 150 g centrifugation for 3 min, the centrifuge speed up, the speed down is 3; again with 1 mL of W5 culture solution washing once, centrifugation to remove supernatant, finally adding 1 mL of W5 culture solution to the centrifuge tube flat to 28°C under dark culture 16 h.

[0040] Step five, fluorescence observation. Slowly lift the centrifuge tube first stationary 30 min, then centrifugation at 150 g, speed up 3, speed down 3 for 3 min, remove supernatant, reserve about 100 μL culture solution mixed with protoplast, observed with confocal microscope or fluorescence microscope.

[0041] The schematic structure of the lipid nanoparticles (LNP) wrapped enhanced green fluorescent protein (eGFP) mRNA is shown in Figure 1 .

[0042] The fluorescence detection results are shown in Figures 2 to 4 , wherein Figure 2 represents unconverted protoplast, as a negative control; Figure 3 is the 35s promoter driven GFP expression vector transformed protoplast, as a positive control; Figure 4 represents the protoplast delivered by the lipid nanoparticles (LNP) wrapped enhanced green fluorescent protein (eGFP) mRNA detects fluorescence, indicating successful delivery and expression of GFP. Therefore, the method described in the present application can be used to deliver lipid nanoparticles (LNP) to palm crop protoplasts, thereby providing a technical reference for subsequent establishment of efficient delivery of lipid nanoparticles (LNP) to palm crop protoplasts.

[0043] Obviously, the above examples are only examples for clarity, and not limited to the embodiments. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to exhaust all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

[0044] Of course, the above description is not a limitation of the present application, and the present application is not limited to the above examples. Those skilled in the art, within the essential scope of the present application, make changes, modifications, additions or substitutions, should belong to the protection scope of the present application.

Claims

1. Application of a lithium acetate-polyethylene glycol 4000-mediated lipid nanoparticle delivery method in protoplast transformation, wherein the protoplasts are protoplasts of palm crops; the delivery method comprises the following steps: Protoplasts were isolated from sterile young leaves of palm crops and purified. The purified protoplasts were adjusted to a concentration of 2×10 5 / mL; First, add 40 μL of 0.15 mg / mL lipid nanoparticle solution to a new 2 mL round-bottom centrifuge tube, then add 100 μL of the adjusted protoplast solution and mix well; Add 140 μL of freshly prepared Ca-containing 2+ 4000 solution, mix well, and let it stand at 28°C for 30 minutes; add 1 mL of W5 culture medium to terminate the transformation reaction, centrifuge at 150g for 3 minutes, and centrifuge at both speeds of 3; wash once with 1 mL of W5 culture medium, centrifuge and remove the supernatant, and finally add 1 mL of W5 culture medium. Place the centrifuge tube flat at 28°C and incubate in the dark for 16 hours; slowly lift the centrifuge tube and let it stand for 30 minutes, then centrifuge at 150g, speed of 3, speed of 3 for 3 minutes, remove the supernatant, retain 100 μL of culture medium and mix with the protoplasts, and observe with a confocal microscope or fluorescence microscope; The lithium acetate-containing MMG solution is prepared by adjusting the pH value to 5.7-6.03 with KOH and then sterilizing by filtration. Contains Ca 2+ The preparation method of polyethylene glycol 4000 solution is as follows: First, prepare 0.8M D-Mannitol and 2M CaCl2 stock solutions respectively; weigh 4 g of polyethylene glycol 4000 and dissolve it in 5 mL of ultrapure water, add 2.5 mL of 0.8M D-Mannitol stock solution and 2 mL of 2M CaCl2 stock solution, then dissolve in a 55°C water bath for 10 minutes, inverting several times during the dissolution, and finally adjust the volume to 10 mL, which is a solution containing 400mM CaCl2. 2+ polyethylene glycol 4000 conversion solution; The lipid nanoparticles are lipid nanoparticles that encapsulate enhanced green fluorescent protein (eGFP) mRNA.

Images