Lithium acetate-polyethylene glycol mediated protoplast lipid nanoparticle delivery method

Abstract

The invention discloses a lithium acetate-polyethylene glycol mediated protoplast lipid nanoparticle delivery method, and belongs to the technical field of biology. LiAc is added into a protoplast conversion solution MMG for the first time, a PEG formula is improved by increasing the concentration of calcium ions (Ca < 2 + >), lipid nanoparticles (LNP) are successfully delivered into protoplasts of palm crops such as oil palm and coconut, mRNA of enhanced green fluorescent protein (eGFP) is wrapped in the nanoparticles, and the lipid nanoparticles can be used for preparing a biocarrier. The fluorescent protein can be directly expressed in the protoplast by taking mRNA as a template without a promoter. After the LNP is delivered to the palm crop protoplast by the method, a green fluorescence signal is observed and detected by a laser confocal microscope. The method can be used for directly delivering the target gene mRNA into the palm crop protoplast, and also can be used for delivering CRISPR / Cas system protein and the like for gene editing technology research.

Description

Technical Field

[0001] The 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 an application thereof. Background Art

[0002] Oil palm (Scientific name: Elaeis guineensis Jacq.), Coconut (scientific name: Cocos nucifera L.) and other palm crops are trees of the palm family, and their main production areas are Malaysia and Indonesia in Asia, western and central Africa, northern South America and Central America. At present, the selection and breeding of new varieties of palm crops such as coconut, oil palm, and betel nut is still mainly based on traditional hybrid breeding, which has problems of low efficiency and long cycle, and seriously hinders the breeding process of high-yield and high-quality breeding. With the development of biotechnology, the application of molecular technologies such as genetic transformation and gene editing based on protoplasts as the main platform in a variety of crops has promoted the development of new breeding technologies, but related research on palm crops such as coconut, oil palm, and betel nut has yet to be carried out.

[0003] Plant protoplasts are cell morphological structural units composed of cell membrane, cytoplasm, nucleus and organelles in addition to cell walls, and have the characteristics of rapid expression of exogenous genetic material. Therefore, transient expression based on protoplasts has been widely used in various fields of genetic research. In recent years, protoplast transformation technology has been greatly improved and has been widely used in the verification of gene editing systems and vector activity, which is of great significance for the directional transformation and precise editing of plant genomes.

[0004] Lipid nanoparticles (LNPs) are tiny particles composed of multiple lipid components, usually around 100 nm in diameter. The core function of these particles is to encapsulate and protect nucleic acid drugs, such as mRNA or siRNA, so that they can be effectively taken up and expressed by cells. The outer membrane of LNP is mainly composed of PEG lipids, which provide hydrophilicity. The inner membrane is mainly composed of neutral lipids, with cholesterol and neutral ionizable lipids in the middle. This structure enables LNP to be adsorbed to the negatively charged cell surface through electrostatic action, and then enter the cell through endocytosis. The cell membrane is usually negatively charged, and the nanoparticle lipids are positively charged. The electrostatic effect promotes the adsorption and fusion of LNP to the cell membrane. The anionic lipids of the cell membrane help release nucleic acids from LNP by neutralizing the charge of the cationic lipid carrier. LNP achieves effective delivery of nucleic acid drugs through its special structure and composition, as well as its interaction with the cell membrane. This delivery system not only protects nucleic acid drugs from degradation, but also improves their ability to be taken up and expressed by cells.

[0005] Although the technology of delivering exogenous nucleic acids to plant protoplasts through transient transformation technology 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. 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 to be carried out. Therefore, the establishment of an LNP delivery method suitable for protoplasts of tropical palm crops such as coconut, oil palm, and betel nut is a necessary supplement to plant protoplast transformation technology, which 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 above 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: Prepare 100 mL of MMG-LiAc solution by adding the ingredients in the table below directly, then dissolve them in a microwave oven or water bath, dilute to 100 mL with ultrapure water, and measure the pH value to 6.03 without adjustment. Then filter and sterilize with a 0.22 μm pore size filter membrane and store at 4°C.

[0014] Reagents Final concentration Mass / g MES 4mM 0.078096 D-Mannitol 0.4M 7.2868 <![CDATA[MgCl2·6H2O]]> 15mM 0.30495 <![CDATA[LiAc·2H2O]]> 0.1M 1.0202g The present invention also provides a method of preparing a calcium ion containing a high concentration of calcium ions (Ca 2+ ) polyethylene glycol (PEG-4000) solution formulation.

[0015] Specifically, the one containing high concentration of calcium ions (Ca 2+ ) polyethylene glycol (PEG-4000) solution was prepared according to the following table: ;

[0016] By using the above transformation solution, the present invention provides a lipid nanoparticle (LNP) for delivering enhanced green fluorescent protein (eGFP) mRNA to palm crop protoplasts.

[0017] The present invention adds LiAc to the plant protoplast transformation liquid MMG for the first time to improve the transformation efficiency.

[0018] Beneficial effects:

[0019] The present invention adds LiAc to the protoplast transformation solution MMG for the first time and increases the calcium ion (Ca 2+ ) concentration was improved to successfully deliver lipid nanoparticles (LNP) to the protoplasts of palm crops such as oil palm and coconut. The enhanced green fluorescent protein (eGFP) mRNA was encapsulated in the nanoparticles, and the fluorescent protein could be directly expressed in the protoplasts using mRNA as a template without a promoter. After LNP was delivered to the protoplasts of palm crops using the method of the present invention, a green fluorescent signal was detected by laser confocal microscopy. This method can be used to directly deliver target gene mRNA into the protoplasts of palm crops, and can also deliver CRISPR / Cas system proteins for gene editing technology research. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the lipid nanoparticle (LNP) structure.

[0021] Figure 2 Fluorescence detection of untransformed oil palm protoplasts (control).

[0022] Figure 3 Results of transient transformation of oil palm protoplasts (35s-GFP expression vector).

[0023] Figure 4 Delivery of lipid nanoparticles (LNPs) to oil palm protoplasts (eGFP-mRNA). DETAILED DESCRIPTION

[0024] In the following examples of the present invention, unless otherwise specified, conventional methods are used. The plant expression vector p35s-GFP in the following examples is preserved in this laboratory, and the lipid nanoparticles (LNPs) encapsulating eGFP-mRNA are purchased from GenScript Biotech Co., Ltd. The above biological materials are only used to repeat the relevant experiments of the present invention and cannot be used for other purposes.

[0025] Example 1

[0026] This embodiment provides a lipid nanoparticle (LNP) delivery method applicable to oil palm protoplasts, and the specific steps are as follows: Step 1: Prepare a MMG solution containing lithium acetate (LiAc) as follows: Prepare 100 mL of MMG-LiAc solution by adding the ingredients in the table below directly, then dissolve them in a microwave oven or water bath, dilute to 100 mL with ultrapure water, and measure the pH value to 6.03 without adjustment. Then filter and sterilize with a 0.22 μm pore size filter membrane and store at 4°C.

[0027]

[0028] Step 2: Prepare a high concentration of calcium ions (Ca 2+ ) polyethylene glycol (PEG-4000) solution as follows:

[0029] Prepare 10mL of working solution. Except for PEG (4000), the remaining stock solutions and ddH2O of the following system need to be added from the clean bench. Then place the solution in a 55℃ water bath for 10min, mixing it by inverting it several times. It can be stored at 4℃ for about a week. It is recommended to prepare it for immediate use.

[0030]

[0031] Step 3, preparing oil palm protoplasts. Take young leaves of sterile oil palm seedlings as materials, and separate and purify protoplasts according to a protoplast separation kit or other conventional methods.

[0032] Step 4: Transient transformation to deliver 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 protoplast solution and mix well; add 140 μL of the newly configured PEG (Ca 2+) solution, mix well and let stand at 28℃ for 30 min; add 1 mL of W5 culture medium to terminate the transformation reaction, centrifuge at 150 g for 3 min, and the centrifuge speed is 3; wash once with 1 mL of W5 culture medium, centrifuge to remove the supernatant, and finally add 1 mL of W5 culture medium and place the centrifuge tube flat at 28℃ for dark culture for 16 h.

[0033] Step 5: Fluorescence observation. Slowly lift the centrifuge tube and let it stand for 30 min, then centrifuge at 150 g, 3% acceleration, 3% deceleration for 3 min, remove the supernatant, retain about 100 μL of culture medium and mix with protoplasts, and observe with a confocal microscope or fluorescence microscope.

[0034] The schematic structure of lipid nanoparticles (LNPs) encapsulating enhanced green fluorescent protein (eGFP) mRNA is shown in Figure 1 .

[0035] The fluorescence detection results are shown in Figures 2 to 4 ,in Figure 2 represents untransformed protoplasts, serving as negative control; Figure 3 The protoplasts were transformed with the expression vector of GFP driven by 35s promoter as a positive control; Figure 4 Fluorescence was detected in the protoplasts of lipid nanoparticles (LNPs) encapsulating enhanced green fluorescent protein (eGFP) mRNA, indicating successful delivery and expression of GFP. Therefore, the method of the present invention can be used to deliver lipid nanoparticles (LNPs) to palm crop protoplasts, thereby providing a technical reference for the subsequent establishment of efficient delivery of lipid nanoparticles (LNPs) to palm crop protoplasts.

[0036] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

[0037] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by ordinary technicians in this technical field within the essential scope of the present invention should fall within the protection scope of the present invention.

Claims

1. Application of lithium acetate-polyethylene glycol mediated lipid nanoparticle delivery method in protoplast transformation, wherein the protoplasts are protoplasts of palm crops.

2. A lithium acetate-polyethylene glycol-mediated lipid nanoparticle delivery method for palm crop protoplasts, characterized in that: The following steps are involved: Protoplasts were isolated and purified from sterile young leaves of palm crops; the purified protoplasts were adjusted to a concentration of 2×10 5 Pieces / mL; First, take 40 μL of 0.15 mg / mL lipid nanoparticle solution and add it 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+ PEG-4000 solution was added, mixed and allowed to stand at 28°C for 30 min; 1 mL of W5 culture medium was added to terminate the transformation reaction, and the mixture was centrifuged at 150 g for 3 min, with the centrifuge speed increasing and decreasing at 3; 1 mL of W5 culture medium was used to wash the mixture once, centrifuged and the supernatant was removed, and finally 1 mL of W5 culture medium was added and the centrifuge tube was placed flat at 28°C for dark culture for 16 h; the centrifuge tube was slowly lifted and allowed to stand for 30 min, and then centrifuged at 150 g, speed increasing at 3, speed decreasing at 3 for 3 min, the supernatant was removed, and 100 μL of culture medium was retained and mixed with the protoplasts, and observed with a confocal microscope or a fluorescence microscope; The above-mentioned MMG solution containing lithium acetate is a solution containing 0.78~1 mg / mL of MES, 72.86~100 mg / mL of D-Mannitol, 3.04~10 mg / mL of MgCl2·6H2O, and 10.20~20 mg / mL of LiAc·2H2O, which is prepared by adjusting the pH value to 5.7~6.03 with KOH and filtering and sterilizing.

3. The delivery method according to claim 2, characterized in that Contains Ca 2+ The preparation method of PEG-4000 solution is: First, prepare 0.8M D-Mannitol and 2M CaCl2 stock solutions respectively: weigh 14.57 g of Mannitol and dissolve it in 100 ml of water, sterilize it by autoclave, and store it at room temperature; weigh 29.40 g of CaCl2.2H2O and dissolve it in 100 ml of water, sterilize it by autoclave, and store it at room temperature; Preparation method of 10 mL PEG working solution: weigh 4 g PEG-4000 and dissolve it in 5 mL ultrapure water, add 2.5 mL of 0.8 M D-Mannitol stock solution, add 2 mL of 2 M CaCl2 stock solution, then place in a 55°C water bath for 10 min to dissolve, invert several times during the process, and finally adjust the volume to 10 mL, which is a 400 mM CaCl2 solution. 2+ PEG-4000 conversion solution.

4. The delivery method according to claim 2, characterized in that The lipid nanoparticles are lipid nanoparticles encapsulating enhanced green fluorescent protein mRNA.

5. Use of the lipid nanoparticles according to claim 4 in protoplast transformation of palm crops.

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