CN109825529B - Preparation method of LDH (layered double hydroxide) nanoparticle and nucleic acid molecule conjugate - Google Patents

Abstract

The invention belongs to the technical field of nanometer materials and biology, and particularly relates to a preparation method of a conjugate of LDH (layered double hydroxide) nanometer particles and nucleic acid molecules and application of the conjugate in plant genetic transformation. Through mixing the prepared magnesium-aluminum solution and the sodium hydroxide solution, and performing water bath for 8 hours and ultrasonic wave combined treatment, the prepared LDH nano-particles have the characteristics of good dispersibility and strong stability, and the Tween-20 can effectively prevent the aggregation of the nano-particles to form precipitates in the process of coupling the LDH nano-particles with nucleic acid molecules, so that the capability of introducing the LDH nano-particles and the nucleic acid molecule conjugate into plant cells is improved; the LDH nano-particles and the nucleic acid molecule conjugate are used for carrying out plant genetic transformation, the operation is simple, the cost is low, and no special instrument is needed; the method is not limited by the type and the size of exogenous nucleic acid molecules and the genotype of receptor materials; the introduction of exogenous genes can be realized in the seed germination process, thereby avoiding complicated experimental operation processes such as agrobacterium infection, tissue culture and the like and shortening the time for obtaining genetic transformation materials.

Description

Preparation method of LDH (layered double hydroxide) nanoparticle and nucleic acid molecule conjugate

Technical Field

The invention belongs to the technical field of nanometer materials and biology, and particularly relates to a preparation method of a conjugate of LDH (layered double hydroxide) nanometer particles and nucleic acid molecules and application of the conjugate in plant genetic transformation.

Background

Plant cells are characterized in that the cell wall is mainly composed of cellulose and pectic polysaccharides, and the plant cells are effectively protected from penetration by foreign inorganic particles or detergents attached to the cell surface. Thus, gene transformation in plants relies heavily on methods based on Agrobacterium-mediated genetic transformation, which is severely genotypically restricted and is currently only applicable to most dicotyledonous and very few monocotyledonous plants. In recent years, inorganic nanoparticles have attracted much attention as a nano-sized conveyor. However, the cell wall in many cases prevents the use of nanoparticles in plant cells. For example, multi-walled carbon nanotubes (MWCNTs), mesoporous Silica Nanoparticles (MSNs), titanium dioxide, and cerium oxide nanoparticles have failed to penetrate plant cells. Some nanoparticles can enter plant cells, but show high cytotoxicity, causing damage to plasma membranes and cytoskeletons.

Layered Double Hydroxides (LDH) generally comprise two or more metal ions, and are novel functional materials with Layered structures and special properties. LDH not only has the characteristics of high anion exchange capacity, high redox activity and the like, but also has low manufacturing cost and less pollution, thereby having wide application. Due to unique structure and properties, the core-shell structure nanocomposite material based on LDH has many excellent physicochemical properties, such as high surface area, magnetism, porous structure and the like. These properties make them potentially useful in catalysis, drug delivery, wastewater purification, energy conversion and storage, and supercapacitors.

Therefore, in the field of genetic transformation, the LDH nano-particles are used as a conveyor to exert the conveying activity, and the nucleic acid molecules are directly introduced into plant cells, so that the damage to the plant cells is reduced, the LDH nano-particles are not limited by cell walls and genotypes, and the LDH nano-particles have wide application prospects. However, the existing LDH nanoparticles generally have the problems of poor biocompatibility, low carrier capacity, unstable performance, and the like, thereby resulting in low transformation efficiency. Therefore, how to prepare high-performance LDH nanoparticles and load the LDH nanoparticles into effective conjugates with nucleic acid molecules become core problems for realizing the application of the LDH nanoparticles.

In addition, LDH nanoparticles are currently available for the introduction of small fragments of nucleic acid molecules, such as dsRNA, into plant cells and have been shown to have some effect in the application of homologous plant viral RNA interference (Mitter N, worrall E A, robinson K E, et al, clay nanosheets for topical delivery of RNAi for substained protection against plant viruses [ J ]. Nat Plants,2017.3 16207.; WO 2015/089543 A1). However, there has been no report on successful introduction of large fragments such as plasmid vectors. Therefore, to replace or compensate for the deficiencies of the traditional Agrobacterium-mediated genetic transformation approach, the success or failure of large fragment introduction is also important.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for preparing a conjugate of LDH nanoparticles and a nucleic acid molecule, comprising the steps of:

1) Preparing a magnesium-aluminum solution;

2) Preparing a sodium hydroxide solution;

3) According to the following steps: 4) mixing the magnesium-aluminum solution and the sodium hydroxide solution obtained in the step 1) and the step 2) in a volume ratio, collecting, washing and resuspending precipitate to obtain a nanoparticle emulsion;

4) Carrying out water bath treatment on the nanoparticle emulsion obtained in the step 3);

5) Carrying out ultrasonic treatment on the nano-particle emulsion treated by the water bath in the step 4) to prepare LDH nano-particles;

6) Mixing and coupling the LDH nano-particles prepared in the step 5) with nucleic acid molecules;

7) Adding Tween-20, and standing.

Specifically, in the step 1), the magnesium-aluminum solution is prepared from MgCl ₂ And AlCl ₃ Dissolving in deionized water to obtain MgCl in the magnesium-aluminum solution ₂ And AlCl ₃ The concentrations of (a) are 0.029g/mL and 0.024g/mL respectively;

specifically, in the step 2), a sodium hydroxide solution is obtained by dissolving NaOH in deionized water, and the concentration of the sodium hydroxide solution is 0.006g/mL;

specifically, in the step 3), the magnesium-aluminum solution is added into the stirred NaOH solution within 3-10 sec, then stirring is continued for 30min, and the method for collecting the precipitate is centrifugation at 8000rpm for 10min.

Specifically, in the step 4), the water bath treatment method is water bath at 100 ℃ for 4-15 h; preferably, the water bath treatment method is a water bath at 100 ℃ for 8 hours.

Specifically, in the step 4), the ultrasonic treatment mode is ultrasonic treatment for 3 times, and each time is 5min.

Specifically, in the step 6), the LDH nanoparticles are mixed and coupled with the nucleic acid molecules in a mass ratio of 1.

Specifically, in the step 6), the nucleic acid molecule is a linear or circular nucleic acid molecule with the length of 1.832-13.559 kb; specifically, the nucleic acid molecule comprises an eGFP gene (SEQ ID NO. 1).

Specifically, in the step 7), the final concentration of the tween-20 reaches 0.1%.

The invention also provides an application of the LDH nano-particles prepared by the method in any one of the above aspects and the nucleic acid molecule conjugate in plant genetic transformation, which is characterized in that: the LDH nanoparticles and the nucleic acid molecule conjugates are co-cultured with plant cells to introduce the nucleic acid molecules into the plant cells.

Specifically, the cell is an epidermal cell or a root tip cell.

Specifically, the plant is dicotyledonous plant, including mung bean and cucumber; or monocotyledons including onion and rice.

Compared with the prior art, the invention has the beneficial effects that:

1) The LDH nano-particles prepared by the preparation method provided by the invention have good dispersibility and strong stability, and the dispersion degree of the LDH nano-particles can be enhanced by the combination of water bath for 8h and ultrasonic treatment;

2) The Tween-20 can effectively prevent the nanoparticles from aggregating to form precipitates in the process of coupling the LDH nanoparticles with the nucleic acid molecules, and the capability of introducing the LDH nanoparticles and the nucleic acid molecule conjugates into plant cells is improved;

3) Compared with the traditional genetic transformation methods such as gene gun and agrobacterium mediation, the method for mediating genetic transformation by using the LDH nano-particles and the nucleic acid molecule conjugates provided by the invention has the advantages of simple operation, low cost and no need of special instruments;

4) The transformation application is not limited by the types, sizes and receptor material genotypes of exogenous nucleic acid molecules;

5) The introduction of exogenous genes can be realized in the seed germination process, thereby avoiding complicated experimental operations in the processes of agrobacterium infection, tissue culture and the like and shortening the time for obtaining genetic transformation materials.

Drawings

FIG. 1 Tydall phenomenon of LDH nanoparticle suspension in example 1 of the present invention, wherein FIGS. 1A to 1H are nanoparticle suspensions prepared by different methods, numbered NP-1 to NP-8, respectively.

FIG. 2 the loading capacity of LDH nanoparticles for 13.559kb circular plasmid molecules in example 2 of the present invention was checked by agarose gel electrophoresis, in which FIGS. 2A-2H are nanoparticle suspensions prepared by 8 methods, NP-1 to NP-8, respectively; lanes for each method are Marker, plasmid and nanoparticle solutions in order from left to right according to 1: 0. 1: 1. 1: 2. 1: 3. 1: 4. 1: 5. 1: and 6, mixing in proportion.

FIG. 3 is a map of a circular plasmid introduced for transient expression in example 3 of the present invention, which contains a Green Fluorescent Protein (GFP) expression cassette.

FIG. 3A 10.550kb circular plasmid map, elements in English and abbreviations are as follows:

RB T-DNA repeat T-DNA region right border sequence

Nos terminator of betaine synthetase

mgfp5 GFP gene

CaMV35S promoter cauliflower mosaic virus 35S promoter

LacZ beta-galactosidase gene

MCS multiple cloning site

LacZ promoter beta-galactosidase gene promoter

CaMV35S promoter cauliflower mosaic virus 35S promoter

HygR hygromycin resistance sequence

CaMV poly (A) signal cauliflower mosaic virus terminator

Left border sequence of LB T-DNA repeat T-DNA region

kanR kanamycin resistance sequence

ori pBR322 initiation region

bom pBR322 framework regions

pVS1 oriV pVS1 initiation region

pVS1 RepA pVS1 replicon

pVS1 StaA pVS1 transcriptional initiation region

FIG. 3B 13.559kb circular plasmid map, elements English and abbreviations are listed below:

RB T-DNA region right border sequence

Nos 3' UTR nopaline synthase terminator

second exon of second gusA exon

first exon of first gus exon gus gene

CaMV35S cauliflower mosaic virus 35S promoter

D35S 35S promoter

eGFP eGFP gene

CaMV 3' UTR cauliflower mosaic virus terminator

D35S 35S promoter

hygromycin R hygromycin resistance sequence

CaMV 3' UTR cauliflower mosaic virus terminator

Left border sequence of LB T-DNA region

kanamycin R kanamycin resistance sequence

pBR322-ori pBR322 initiation region

pBR322-bom pBR322 framework regions

pVS1-REP pVS1 replicon

pVS1-STA pVS1 transcriptional initiation region

FIG. 4 shows the results of localization of expression of GFP in epidermal cells of onion under a 10-fold microscope in example 3 of the present invention. Fig. 4A, 4C, and 4E: a dark field; fig. 4B, 4D, and 4F: bright field; fig. 4A and 4B:1.832kb linear nucleic acid molecule; fig. 4C and 4D:10.550kb circular nucleic acid molecule; fig. 4E, 4F:13.559kb circular nucleic acid molecule.

FIG. 5 shows the effect of adding Tween-20 during the coupling of the nanoparticles with nucleic acid molecules in example 3 of the present invention. Tween-20 is not added in the left test tube in the coupling process, and the nanoparticles are quickly deposited; tween-20 is added in the right test tube in the coupling process, and the coupled nanoparticles have good stability and are not deposited.

FIG. 6 shows the expression results of eGFP gene in cucumber root tip tissue under 4-fold observation in example 4 of the present invention. FIG. 6A: a dark field; FIG. 6B: bright field. The root tip tissue is treated by an infection solution of 13.559kb circular nucleic acid molecules and nanoparticle conjugates.

Detailed Description

The following definitions and methods are provided to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. All patent documents, academic papers, industry standards and other publications, etc., cited herein are incorporated by reference in their entirety.

Unless otherwise indicated, nucleic acids are written from left to right in the 5 'to 3' direction; amino acid sequences are written from left to right in the amino to carboxyl direction. Amino acids may be referred to herein by their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Similarly, nucleotides may be represented by commonly accepted single-letter codes. Numerical ranges include the numbers defining the range. As used herein, "nucleic acid" includes reference to deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form, and unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) having the basic properties of natural nucleotides that hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides.

By "transgenic" is meant any cell, cell line, callus, tissue, plant part or plant whose genome has been altered by the presence of a heterologous nucleic acid (such as a recombinant DNA construct). The term "transgene" as used herein includes those initial transgenic events as well as those generated by sexual crosses or asexual propagation from the initial transgenic events and does not encompass genomic (chromosomal or extra-chromosomal) alteration by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.

In some embodiments, fragments of the nucleotide sequences and the amino acid sequences encoded thereby are also included. As used herein, the term "fragment" refers to a portion of the nucleotide sequence of a polynucleotide or a portion of the amino acid sequence of a polypeptide of an embodiment. Fragments of the nucleotide sequences may encode protein fragments that retain the biological activity of the native or corresponding full-length protein, and thus have protein activity. Mutant proteins include biologically active fragments of the native protein that comprise contiguous amino acid residues that retain the biological activity of the native protein. Some embodiments also include a transformed plant cell or transgenic plant comprising the nucleotide sequence of at least one embodiment. In some embodiments, plants are transformed with an expression vector comprising at least one embodiment of the nucleotide sequence and operably linked thereto a promoter that drives expression in plant cells. Transformed plant cells and transgenic plants refer to plant cells or plants that comprise a heterologous polynucleotide within their genome. Generally, the heterologous polynucleotide is stably integrated within the genome of the transformed plant cell or transgenic plant such that the polynucleotide is transmitted to progeny. The heterologous polynucleotide may be integrated into the genome alone or as part of an expression vector. In some embodiments, the plants to which the present application relates include plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells, which are whole plants or parts of plants, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, nuts, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. The present application also includes plant cells, protoplasts, tissues, calli, embryos, and flowers, stems, fruits, leaves, and roots derived from the transgenic plants of the present application or progeny thereof, and thus comprising at least in part the nucleotide sequences of the present application.

"plant" includes reference to whole plants, plant organs, plant tissues, seeds, and plant cells, and progeny of same. Plant cells include, but are not limited to, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores.

In this application, the words "comprise", "comprising" or variations thereof are to be understood as embracing elements, numbers or steps in addition to those described.

The technical solutions of the present invention are further described below, but not limited thereto, and all the technical solutions of the present invention should be equally replaced or modified without departing from the technical principles and the spirit of the present invention, and the protection scope of the present invention is covered.

Example 1: preparation of LDH nanoparticles

The preparation process of the LDH nano-particles comprises the following steps:

1) Weighing MgCl ₂ 0.29g，AlCl ₃ 0.24g, dissolved in 10ml of deionized water.

2) Then 0.24g of NaOH is weighed and dissolved in 40ml of deionized water, the beaker is placed on a magnetic stirrer, and the rotating speed is adjusted to avoid bubbles as much as possible.

3) 10ml of magnesium aluminum solution was added to the NaOH solution while stirring for 3sec, 5sec, and 10sec, respectively, and stirring was continued for 30min. The stirred solution was poured into 2 50ml centrifuge tubes, centrifuged at 8000rpm for 10min to collect the precipitate, resuspended in 40ml deionized water, and washed once more after centrifugation.

4) Transferring the resuspended emulsion to a glass bottle with a cover, performing water bath at 100 deg.C for 4h, 8h, 12h or 16h, and optionally treating with ultrasonic wave for 3 times (5 min/time).

5) A total of 8 different preparation methods were set up depending on the bath time and whether sonication was carried out or not, as shown in Table 1 below. Finally, the Nanoparticle (NP) suspensions obtained from different treatments were examined for particle size and degree of dispersion in the solution by Tydall method, and if the red laser light passed through the solution in a straight line, a straight light path was formed, which indicates that the nanoparticles were small and uniformly dispersed, as shown in fig. 1.

Table 1 different preparation methods of nanoparticles

As shown in fig. 1, the Tydall phenomenon of the suspension obtained by different preparation methods is observed by infrared irradiation, so that the particle size and dispersion uniformity of the nanoparticles can be preliminarily determined. NP-1 to NP-8 were nanoparticle suspensions prepared by different methods.

The result shows that the speed of pouring the magnesium-aluminum solution into the NaOH solution has little influence on the size of the final nano-particles, so the magnesium-aluminum solution is preferably added within 3-5 sec. The time of the water bath has a great influence on the size and uniformity of the NP, and the water bath time of 8h is better than 4h, 12h or 15h. Sonication can further improve the quality of NP, with Tydall being the most evident for the suspension obtained with NP-6.

Example 2: preparation of LDH nanoparticles and nucleic acid molecule conjugates and LDH nanoparticles loading capacity test for nucleic acid molecules

Preparing DNA molecules with different sizes and types, mixing the DNA molecules with NP solution according to different volume ratios, and judging whether all the DNA molecules are loaded by NP by an agarose gel method.

In this example, a 1.832kb linear nucleic acid molecule, a 10.550kb linear plasmid fragment, a 10.550kb circular plasmid molecule, a 13.559kb linear plasmid fragment and a 13.559kb circular plasmid molecule, respectively, were prepared, which have the same sequence and all contain a Green Fluorescent Protein (GFP) expression cassette, as shown in FIG. 3. Wherein the circular plasmid is extracted from Escherichia coli by alkaline lysis method, and the Escherichia coli contains pD1301s or pCAMBIA1302 plasmid. Wherein the pD1301S plasmid adds an eGFP gene between PstI and SalI of a multiple cloning site, and the gene is regulated and expressed by a 35S promoter. The extracted circular plasmid was digested with BamHI to obtain a linear plasmid, which was then digested as described in the manufacturer's instructions. The 1.832kb linear nucleic acid molecule is a DNA molecule artificially synthesized into a circular plasmid pD1301s-eGFP and recovered after HindIII and EcoRI digestion.

The DNA solution and NP suspension were mixed according to the ratio of 1: 0. 1: 1. 1: 2. 1: 3. 1: 4. 1: 5. 1:6, and then detecting the loading capacity of the nanoparticles on the DNA molecules by using 1% agarose gel electrophoresis. After the nano particles are combined with the DNA molecules, the charges of the nano particles are neutralized, single crystal particles are gathered to form larger particles, the diameter of the particles is larger, the particles cannot penetrate through the pores of the gel like the DNA molecules, and the particles are left in sample application holes in the electrophoresis process. If only a part of the DNA molecules are bound by the nanoparticles, the DNA molecules that are not loaded will migrate to the positive electrode under the action of the voltage, forming an electrophoretic band, see FIG. 2. The loading capacity of the nanoparticles for a particular DNA molecule can be calculated by this method.

The result shows that the loading capacity of the nanoparticles treated by ultrasonic waves to the 13.559kb plasmid is obviously improved. The loading capacity of NP-6 on the circular plasmid molecule was the highest, reaching 326 ng/. Mu.l, while the loading capacity of NP-2 sample on the same plasmid was only 125 ng/. Mu.l. For the linear plasmid molecules, the loading of the NP-6 samples was also significantly higher than the NP-2 samples, see Table 2 for details.

TABLE 2 Loading capacity (ng/. Mu.L) of different nanoparticles for different DNA samples

The same conclusions can be drawn from the data for the load capacity test for linear molecules of 1.832kb, linear and circular molecules of 10.550 kb.

The results show that the loading capacity of the LDH nano-particles prepared by the method for the long-fragment nucleic acid molecules is very outstanding. It is well known that plant transformation vectors typically have molecular weights in excess of 10kb, and that large loadings of such plasmid molecules make possible LDH nanoparticle-mediated genetic transformation of plants.

Example 3: LDH nano-particle mediated transient expression of GFP gene in onion epidermal cells

Research shows that the metal particles with the diameter of 20-50 nm can be endocytosed into plant cells, so that the nanoparticles become good carriers for foreign proteins or nucleic acid molecules to enter the plant cells. Therefore, this example attempted to introduce a 1.832kb linear molecule, a 10.550kb circular plasmid and a 13.559kb circular plasmid into onion epidermal cells using nanoparticles as vectors, and detect transient expression of the GFP gene by fluorescent signals. Wherein 10.550kb and 13.559kb circular plasmid map is shown in FIG. 3,1.832kb linear fragment is circular plasmid pD1301-eGFP which is digested by HindIII and EcoRI, and the obtained DNA molecule is recovered, and the coding sequence is shown in SEQ ID NO.2.

The specific experimental method is as follows:

1) After surface sterilization of fresh onion, the inner skin was torn off on a superclean bench with tweezers and cut into 1cm ² The small blocks are laid on an MS basic culture medium and are pre-cultured for 6 hours;

2) Taking 200 mu L of each NP-2 sample and NP-6 sample, diluting the samples to 1ml by using sterile deionized water, adding plasmid DNA with the total amount of 60 mu g, adding 1.0 mu L of Tween-20 to prevent the nanoparticles from aggregating, namely the final concentration of the Tween-20 is 0.1%, uniformly mixing, and standing for 15 minutes;

3) Placing the precultured onion epidermis into an eppendorf tube containing NP solution, and placing for 30 minutes, wherein the centrifuge tube is inverted for several times;

4) Taking out onion epidermis, cleaning with sterile water, placing on sterile filter paper, sucking water, spreading on MS culture medium, dark culturing for 24 hr, and observing with fluorescence microscope.

As can be seen in fig. 4, the fluorescent signal accumulated in the nucleus, indicating that the LDH nanoparticles can carry the exogenous plasmid molecule containing the GFP gene into the cell.

In the preparation process of the infection liquid, tween-20 (with the final concentration of 0.1%) is added, so that the nanoparticles can be effectively prevented from aggregating to form precipitates, and the loading capacity of the nanoparticles on plasmid molecules is not influenced (figure 5).

Compared with NP-6, no fluorescence signal of onion epidermal cells was observed in the other NP treatments or the treatment without Tween-20.

Example 4: LDH nanoparticles mediate expression of GFP gene in plant root tips

This example attempts to transfer exogenous genes into germinating seeds via nanoparticle mediation to obtain transgenic plants. An infection solution containing NP-6 nanoparticles and circular plasmid was prepared according to the method of the previous example, and Tween-20 was added thereto to give a final concentration of 0.1%. Selecting 40 seeds of rice, mung bean and cucumber, sterilizing, filling 10 seeds into a glass bottle with the capacity of 20ml, adding 1ml of sterile water, 1ml of solution only containing nano particles, 1ml of solution only containing plasmids and 1ml of infection solution, covering with a ventilated cover, and culturing in an incubator at 28 ℃. And supplementing sterile water in time according to the water absorption condition, cutting the root tips of the germinated seeds after 7 days, and observing under a fluorescent microscope.

The results show that the root tips of the rice, the mung bean and the cucumber soaked in the staining solution have obvious fluorescent signals, while other treatments do not. Among them, the cucumber seed has the strongest root tip fluorescence signal, as shown in fig. 6, and may be related to the tender characteristics of its root tissue. Using nanoparticles prepared by methods other than NP-6 as plasmid vectors or without Tween-20 treatment, no expression signal of GFP in root tip tissue was observed under the same experimental conditions, which also demonstrates that NP-6 and Tween-20 treatment is the best preparation protocol for performing genetically transformed LDH nanoparticles and nucleic acid molecule conjugates.

Claims (12)

1. A method for preparing a LDH nanoparticle-nucleic acid molecule conjugate, comprising the steps of:

1) Preparing a magnesium-aluminum solution;

2) Preparing a sodium hydroxide solution;

3) Mixing the magnesium-aluminum solution obtained in the step 1) and the step 2) with a sodium hydroxide solution, collecting, washing and resuspending the precipitate to obtain a nanoparticle emulsion;

4) Carrying out water bath treatment on the nanoparticle emulsion obtained in the step 3), wherein the water bath treatment method is water bath at 100 ℃ for 4-15 h;

5) Carrying out ultrasonic treatment on the nano-particle emulsion subjected to water bath treatment in the step 4) to prepare LDH nano-particles; the ultrasonic treatment mode is ultrasonic treatment for 3 times, and each time is 5min;

6) Mixing and coupling the LDH nano-particles prepared in the step 5) with nucleic acid molecules;

7) Tween-20 was added to a final concentration of 0.1% and left to stand.

2. A method for preparing LDH nanoparticles and nucleic acid molecule conjugates as claimed in claim 1, characterized in that: in the step 1), the magnesium-aluminum solution is prepared from MgCl ₂ And AlCl ₃ Dissolving in deionized water to obtain MgCl in the magnesium-aluminum solution ₂ And AlCl ₃ The concentrations of (a) are 0.029g/mL and 0.024g/mL, respectively.

3. A method for preparing LDH nanoparticles with nucleic acid molecule conjugates as claimed in claim 1, characterized in that: in the step 2), the sodium hydroxide solution is obtained by dissolving NaOH in deionized water, and the concentration of the sodium hydroxide solution is 0.006g/mL.

4. A method for preparing LDH nanoparticles with nucleic acid molecule conjugates as claimed in claim 1, characterized in that: in the step 3), the steps are as follows: 4, adding the magnesium-aluminum solution into the stirred NaOH solution within 3-10 sec, then continuously stirring for 30min, and collecting the precipitate by centrifuging at 8000rpm for 10min.

5. The water bath treatment method according to claim 1, characterized in that: the water bath treatment method is a water bath at 100 ℃ for 8h.

6. A method for preparing LDH nanoparticles with nucleic acid molecule conjugates as claimed in claim 1, characterized in that: in the step 6), the LDH nanoparticles and the nucleic acid molecules are subjected to mixed coupling according to the mass ratio of 1.

7. A method for preparing LDH nanoparticles and nucleic acid molecule conjugates as claimed in claim 1, characterized in that: in the step 6), the nucleic acid molecule is a linear or circular nucleic acid molecule with 1.832-13.559 kb.

8. The nucleic acid molecule of claim 7, wherein: the nucleic acid molecule comprises an eGFP gene.

9. Use of LDH nanoparticles prepared according to the method of any one of claims 1 to 8 in the genetic transformation of plant cells in conjunction with a nucleic acid molecule conjugate.

10. Use according to claim 9, characterized in that: the plant is a dicotyledonous plant or a monocotyledonous plant.

11. Use according to claim 10, characterized in that: the dicotyledonous plant is any one of mung bean or cucumber; the monocotyledon is any one of onion and rice.

12. Use according to claim 11, characterized in that: the cells are epidermal cells or root tip cells.

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