CN109825529A - A kind of preparation method of LDH nano particle and nucleic acid molecules conjugate - Google Patents
A kind of preparation method of LDH nano particle and nucleic acid molecules conjugate Download PDFInfo
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Abstract
The invention belongs to nano material and field of biotechnology, the preparation method and its application in Genetic Transformation in Higher Plants of specially a kind of LDH nano particle and nucleic acid molecules conjugate.Pass through the magnalium solution and sodium hydroxide solution of mixed preparing, and water-bath 8h and ultrasonic wave combined treatment, LDH nano particle obtained has good dispersion, the strong feature of stability, Tween-20 can effectively prevent nano particle that aggregation occurs in LDH nano particle and nucleic acid molecules coupling process and be formed and be precipitated, and improve LDH nano particle and nucleic acid molecules conjugate imports plant cell ability；Genetic Transformation in Higher Plants is carried out using LDH nano particle and nucleic acid molecules conjugate, it is easy to operate, at low cost, do not need special instrument；It is not limited by exogenous nucleic acid molecule type, size and acceptor material genotype；The importing of foreign gene can be realized in Seed Germination, infect the experimental implementation process with complexity such as tissue cultures so as to avoid Agrobacterium, shorten the time for obtaining genetic transformation material.
Description
Technical field
The invention belongs to nano material and field of biotechnology, specially a kind of LDH nano particle and nucleic acid molecules are coupled
The preparation method of object and its application in Genetic Transformation in Higher Plants.
Background technique
Plant cell is characterized in that cell wall is mainly made of cellulose and pectin polysaccharide, and effective protection plant cell is not
By the infiltration of the substances such as external inorganic particle or the detergent for being attached to cell surface.Therefore, the genetic transformation in plant is very
Dependent on agrobcterium-mediated transformation is based in big degree, this method is seriously limited by genotype, and mesh
Before be only applicable to most of dicotyledon and few part monocotyledon.In recent years, inorganic nanoparticles are as nanometer
The conveyer of grade causes the extensive concern of people.However, to hinder nano particle in many cases thin in plant for cell wall
Application in born of the same parents.For example, multi-walled carbon nanotube (MWCNTs), mesoporous silica nano-particle (MSN), titanium dioxide and oxidation
Cerium nano particle all fails to penetrate plant cell.Some nano particles can enter plant cell, but show higher thin
Cellular toxicity damages plasma membrane and cytoskeleton.
Layered double hydroxide, abbreviation LDH (Layered double hydroxide), generally comprises two kinds and two
Kind or more metal ion, be a kind of new function material with layer structure and property.LDH not only have high yin from
The features such as sub- exchange capacity and active either high redox, and low manufacture cost, pollution less, therefore have extensive purposes.By
In unique structure and property, the nuclear-shell structured nano-composite material based on LDH has many outstanding physical and chemical performances, such as
High surface area, magnetism and porous structure etc..These performances make they catalysis, drug conveying, purification of waste water, energy conversion and
Storage and supercapacitor etc. suffer from potential application value.
Therefore in field of genetic transformation, using LDH nano particle as conveyer, its conveying activity is played, by nucleic acid molecules
It is importing directly into plant cell body, reduces the injury to plant cell itself, and do not limited by cell wall and genotype,
It will have broad application prospects.However, the existing generally existing bio-compatibility of LDH nano particle is bad, bearer capabilities are low,
The problems such as performance is unstable, thus cause transformation efficiency low.Therefore how to prepare high performance LDH nano particle and be assembled into
Effective LDH nano particle and nucleic acid molecules conjugate become the key problem for realizing its application.
In addition, at present for the nucleic acid molecules of small fragment, such as dsRNA, it is already possible to be realized and be planted using LDH nano particle
The importing of object cell, and certain effect (Mitter N, Worrall E is above produced in homologous plant virus RNA interference application
A, Robinson K E, et al., Clay nanosheets for topical delivery of RNAi for
Sustained protection against plant viruses [J] .Nat Plants, 2017.3:16207.；WO
2015/089543 A1).But in the successful importing of the large fragments such as plasmid vector, there are no researchs to report.Therefore, to substitution
Or the deficiency of traditional Agrobacterium-mediated genetic transformation mode is made up, the win or lose that large fragment imports is similarly important.
Summary of the invention
To solve the above problems, the present invention provides the preparation method of a kind of LDH nano particle and nucleic acid molecules conjugate,
The following steps are included:
1) magnalium solution is prepared；
2) sodium hydroxide solution is prepared；
3) according to 1: 4 volume ratio mix above-mentioned steps 1) and step 2) in acquisition magnalium solution and sodium hydroxide it is molten
Liquid is collected, washs and is resuspended precipitating, obtains nano particle emulsion；
4) nano particle emulsion obtained in the step 3) is subjected to water bath processing；
5) by above-mentioned steps 4) in nano particle emulsion after water bath processing carry out ultrasonication, be made LDH nanometers
Particle；
6) by above-mentioned steps 5) made from LDH nano particle coupling is mixed with nucleic acid molecules；
7) Tween-20 is added, and stands.
Specifically, magnalium solution is by MgCl in the step 1)2And AlCl3It is dissolved in deionized water acquisition, the magnalium is molten
MgCl in liquid2And AlCl3Concentration be respectively 0.029g/mL and 0.024g/mL；
Specifically, sodium hydroxide solution is dissolved in deionized water by NaOH and obtains, and the sodium hydroxide is molten in the step 2)
The concentration of liquid is 0.006g/mL；
Specifically, magnalium solution is added in 3~10sec in the NaOH solution of stirring in the step 3), then after
Continuous stirring 30min, the method for collecting precipitating are that 8000rpm is centrifuged 10min.
Specifically, the method for water bath processing is 100 DEG C of 4~15h of water-bath in the step 4)；It is wherein preferred, the water
The method of bath processing is 100 DEG C of water-bath 8h.
Specifically, ultrasonication mode is ultrasonication 3 times, each 5min in the step 4).
Specifically, the LDH nano particle and nucleic acid molecules are carried out according to mass ratio 1: 1~1: 6 in the step 6)
Mixing coupling.
Specifically, the nucleic acid molecules are the threadiness or circular nucleic acid molecules of 1.832-13.559kb in the step 6)；
Specifically, the nucleic acid molecules include eGFP gene (SEQ ID NO.1).
Specifically, the final concentration of the Tween-20 reaches 0.1% in the step 7).
The present invention also provides a kind of LDH nano particles being prepared according to above-mentioned any one the method and nucleic acid point
Application of the sub- conjugate in Genetic Transformation in Higher Plants, it is characterised in that: it is described by LDH nano particle and nucleic acid molecules conjugate with
Plant cell co-incubation makes nucleic acid molecules import plant cell.
Specifically, the cell is epidermal cell or root-tip cells.
Specifically, the plant is dicotyledon, including mung bean or monocotyledon, including onion, rice, cucumber.
Compared with prior art, the beneficial effects of the present invention are:
1) through the invention in provide preparation method be made LDH nano particle good dispersion, stability is strong, water-bath 8h and
The combination of ultrasonication can enhance the dispersion degree of LDH nano particle；
2) Tween-20 can effectively prevent nano particle to assemble in LDH nano particle and nucleic acid molecules coupling process
And precipitating is formed, it improves LDH nano particle and nucleic acid molecules conjugate imports plant cell ability；
3) it is provided in the present invention using LDH nano particle compared with the method for the couplet mediated genetic transformation of nucleic acid molecules
The genetic transforming methods such as traditional particle gun, mediated by agriculture bacillus, it is easy to operate, it is at low cost, do not need special instrument；
4) it in the Transformation Application, is not limited by exogenous nucleic acid molecule type, size and acceptor material genotype；
5) importing that foreign gene can be thus achieved in Seed Germination, infects and is organized so as to avoid Agrobacterium
The experimental implementation of the processes complexity such as culture shortens the time for obtaining genetic transformation material.
Detailed description of the invention
The Tydall phenomenon of LDH nano granule suspension in Fig. 1 embodiment of the present invention 1, wherein Figure 1A~Fig. 1 H be respectively
The nano granule suspension of distinct methods preparation, number is NP-1~NP-8.
LDH nano particle carries out agar to the struck capacity of 13.559kb cyclic plasmid molecule in Fig. 2 embodiment of the present invention 2
Sugared detected through gel electrophoresis, wherein Fig. 2A~Fig. 2 H is respectively NP-1~NP-8 nano granule suspension that prepared by totally 8 kinds of methods；Often
The swimming lane of a method is from left to right followed successively by Marker, plasmid and nanoparticles solution according to 1: 0,1: 1,1: 2,1: 3,1: 4,1:
5,1: 6 ratio mixes.
The cyclic plasmid map that transient expression imports in Fig. 3 embodiment of the present invention 3, it includes green fluorescent protein (GFP)
Expression cassette.
Fig. 3 A 10.550kb cyclic plasmid map, each element English and each abbreviation meaning are listed below:
The area RB T-DNA repeat T-DNA right border sequence
Nos terminator nopaline syntase terminator
Mgfp5 GFP gene
CaMV 35S promoter cauliflower mosaic virus 35 S promoter
LacZ beta-galactosidase gene
MCS multiple cloning sites
LacZ promoter beta-galactosidase gene promoter
CaMV 35S promoter cauliflower mosaic virus 35 S promoter
HygR hygromycin resistance sequence
CaMV poly (A) signal cauliflower mosaic virus terminator
The area LB T-DNA repeat T-DNA left margin sequence
KanR kalamycin resistance sequence
The sintering ori pBR322
Bom pBR322 skeleton area
The sintering pVS1 oriV pVS1
PVS1 RepA pVS1 replicon
The transcription initiation region pVS1 StaA pVS1
Fig. 3 B 13.559kb cyclic plasmid map, each element English and each abbreviation meaning are listed below:
The area RB T-DNA right border sequence
3 ' UTR nopaline syntase terminator of Nos
Second gusA exon gusA second exon of gene
First gus exon gus first exon of gene
CaMV35S cauliflower mosaic virus 35 S promoter
D35S 35S promoter
EGFP eGFP gene
3 ' UTR cauliflower mosaic virus terminator of CaMV
D35S 35S promoter
Hygromycin R hygromycin resistance sequence
3 ' UTR cauliflower mosaic virus terminator of CaMV
The area LB T-DNA left margin sequence
Kanamycin R kalamycin resistance sequence
The sintering pBR322-ori pBR322
PBR322-bom pBR322 skeleton area
PVS1-REP pVS1 replicon
The transcription initiation region pVS1-STA pVS1
Expression and localization result of 10 times of microscopic observation GFP albumen in onion epidermis cell in Fig. 4 embodiment of the present invention 3.
Fig. 4 A, Fig. 4 C and Fig. 4 E: dark field；Fig. 4 B, Fig. 4 D and Fig. 4 F: light field；Fig. 4 A, the conversion of Fig. 4 B:1.832kb threadiness nucleic acid molecules；
Fig. 4 C, the conversion of Fig. 4 D:10.550kb circular nucleic acid molecules；Fig. 4 E, the conversion of Fig. 4 F:13.559kb circular nucleic acid molecules.
Nano particle and nucleic acid molecules carry out the effect that Tween-20 is added in coupling process in Fig. 5 embodiment of the present invention 3.
For Tween-20 is not added in coupling process in the test tube of the left side, nano particle deposits quickly；For in coupling process in the test tube of the right
Tween-20 is added, the nanoparticles stable after coupling is preferable, does not deposit.
Expression of results of the eGFP gene of 4 times of microscopic observations in cucumber organization of root tips in Fig. 6 embodiment of the present invention 4.Fig. 6 A:
Dark field；Fig. 6 B: light field.Organization of root tips is handled by 13.559kb circular nucleic acid molecules and nano particle conjugate infected liquid.
Specific embodiment
Defined below and method is provided preferably to define the application and instruct this field general in the application practice
Logical technical staff.Unless otherwise mentioned, term understands according to the common usage of person of ordinary skill in the relevant.It is cited herein
All patent documents, academic paper, professional standard and other public publications etc., full content therein is integrally incorporated herein
As reference.
Unless otherwise stated, nucleic acid is write from left to right with 5 ' to 3 ' directions；Amino acid sequence is with amino to carboxyl direction
It writes from left to right.Amino acid can use its generally known three letter symbols or IUPAC-IUB biological chemical name in this paper
The one-letter symbol that the committee is recommended indicates.Likewise it is possible to indicate nucleotide with the single-letter code usually received.Digital model
Enclose the number including limiting the range.As used herein, " nucleic acid " includes being related to the dezyribonucleoside of single-stranded or double-stranded form
Acid or ribonucleotide polymer, and unless otherwise limitation, including the known analog with natural nucleotide fundamental property
(for example, peptide nucleic acid), the analog in a manner of as naturally occurring ucleotides with single-chain nucleic acid to hybridize.
" transgenosis " refer to its genome because heterologous nucleic acids (such as recombinant dna construct) there are due to change appoint
What cell, cell line, callus, tissue, plant part or plant.The term as used herein " transgenosis " includes that those are initial
Transgenic event and those of generated from initial transgenic event by sexual hybridization or vegetative propagation, and do not contain
Lid is by conventional plant breeding method or passes through abiogenous event (such as random allogamy, non-recombinant virus infection, non-
Recombinant bacteria conversion, non-recombinant swivel base or spontaneous mutation) genome (outside chromosome or chromosome) that carries out changes.
In some embodiments, further include nucleotide sequence and its coding amino acid sequence segment.Such as this paper institute
With term " segment " refers to the one of a part of the nucleotide sequence of the polynucleotides of embodiment or the amino acid sequence of polypeptide
Part.The segment of nucleotide sequence can encode protein fragments, and the protein fragments retain the life of natural or corresponding full-length proteins
Object activity, and thus have protein active.Mutant protein includes the bioactive fragment of native protein, and it includes retain day
The continuous amino acid residue of right biological activity of albumen.Some embodiments further include that the plant cell of conversion or transgenosis are planted
Object, it includes the nucleotide sequences of at least one embodiment.In some embodiments, plant is converted using expression vector,
The expression vector include at least one embodiment nucleotide sequence and be operably connected with it in plant cell
The promoter of middle driving expression.The plant cell of conversion and genetically modified plants indicate the plant in genome comprising heterologous polynucleotide
Object cell or plant.In general, the heterologous polynucleotide is in the plant cell of conversion or the genome of genetically modified plants
It steadily integrates, so that the polynucleotides are passed to offspring.It can be by the heterologous polynucleotide either individually or as table
A part up to carrier is integrated into genome.In some embodiments, this application involves plant include plant cell, plant
Protoplast, the plant cell tissue cultures that plant can be regenerated, plant callus, agglomerate and plant cell,
Its part for complete plant or plant, such as embryo, pollen, ovule, seed, leaf, flower, branch, fruit, kernel, fringe, fringe
Axis, shell, stalk, root, the tip of a root, anther etc..The application further include derived from the application genetically modified plants or its filial generation and thus
At least partly comprising the application nucleotide sequence plant cell, protoplast, tissue, callus, embryo and flower,
Stem, fruit, leaf and root.
" plant " includes to whole plant, plant organ, plant tissue, seed and plant cell and their filial generation
Index.Plant cell include but is not limited to come from seed, suspension culture, plumule, meristematic region, callus, leaf, root, seedling,
Gametophyte, sporinite, pollen and the cell of microspore.
In this application, word "include", "comprise" or its variant are interpreted as except described element, number or step
It outside, also include other elements, number or step.
Technical solution of the present invention is described further below, however, it is not limited to this, all using to the technology of the present invention
Scheme is replaced or is modified on an equal basis, without departing from the technology of the present invention principle and purport, should all cover the present invention claims protection model
It encloses.
The preparation of embodiment 1:LDH nano particle
The preparation process of LDH nano particle are as follows:
1) MgCl is weighed20.29g, AlCl30.24g is dissolved in 10ml deionized water.
2) NaOH 0.24g is weighed again, is dissolved in 40ml deionized water, places the beaker on magnetic stirring apparatus, and adjustment turns
Speed avoids bubble from generating as far as possible.
3) 10ml magnalium solution is added in 3sec, 5sec, 10sec to the NaOH solution in stirring respectively, continues to stir
30min.Solution after stirring pours into 2 50ml centrifuge tubes respectively, and 8000rpm is centrifuged 10min and collects precipitating, with 40ml deionization
Precipitating is resuspended in water, and repeated washing is primary after centrifugation.
4) emulsion being resuspended is transferred to vial with cover, in 100 DEG C of difference water-bath 4h, 8h, 12h or 16h,
It takes later or does not take ultrasonic wave to be handled, processing mode is 5min/ times, coprocessing 3 times.
5) according to water bath time with whether 8 kinds of different preparation methods such as the following table 1 are arranged in ultrasonication altogether.Finally, will
The obtained nano particle of different disposal (nano particle, NP) suspension is using Tydall method detection particles in solution
Size and degree of scatter form a straight optical path, illustrate that nano particle is smaller if red laser linearly passes through solution,
And be uniformly dispersed, see Fig. 1.
The different preparation methods of 1 nano particle of table
As shown in Figure 1, the obtained nano granule suspension of different preparation methods, is observed using infrared radiation and is suspended
The Tydall phenomenon of liquid can tentatively judge the granular size and dispersed homogeneous degree of nano particle.NP-1 to NP-8 is not Tongfang
The nano granule suspension of method preparation.
The result shows that magnalium solution, which is poured into the speed of NaOH solution, to be influenced the size of final nano particle and little,
Therefore it chooses in 3~5sec and magnalium solution is added preferably.The time of water-bath is affected to NP size and the uniformity,
The water bath time of 8h is better than 4h, 12h or 15h.Ultrasonication can be further improved the quality of NP, the obtained suspension of NP-6
Liquid Tydall phenomenon is the most obvious.
The preparation of embodiment 2:LDH nano particle and nucleic acid molecules conjugate and LDH nano particle are to nucleic acid molecules
Struck capacity test
The DNA molecular for preparing different size and type respectively mixes with NP solution according to different volumes ratio, passes through agarose
The method of gel judges whether DNA molecular is all loaded by NP.
In the present embodiment, be prepared for respectively 1.832kb linear nucleic acid molecule, 10.550kb linear plasmid segment,
The cyclic plasmid molecule of 10.550kb cyclic plasmid molecule, the linear plasmid segment of 13.559kb size and 13.559kb size,
The linear plasmid segment or cyclic plasmid molecular sequences are identical, contain green fluorescent protein (GFP) expression cassette, see Fig. 3.Its
Middle cyclic plasmid is taken alkaline lysis to extract from Escherichia coli and is obtained, which contains pD1301s or pCAMBIA1302
Plasmid.Wherein pD1301s plasmid joined eGFP gene between the PstI and SalI of multiple cloning sites, which is opened by 35S
Mover regulating and expressing.The cyclic plasmid of extraction obtains linear plasmid after BamHI digestions, and digestion illustrates according to producer
Method described in book is operated.1.832kb linear nucleic acid molecule is manually to synthesize cyclic plasmid pD1301s-eGFP warp
It crosses HindIII and EcoRI digestion and recycles gained DNA molecular later.
DNA solution and NP suspension are mixed according to 1: 0,1: 1,1: 2,1: 3,1: 4,1: 5,1: 6 ratio respectively
It closes, uses 1% agarose gel electrophoresis, delivered payload capability of the detection nano particle to DNA molecular later.Nano particle and DNA divide
After son combines, charge will be neutralized, and will be assembled to form bigger particle between single crystal particles, be relatively large in diameter, no
The hole of gel can be passed through as DNA molecular, will be left in loading wells during electrophoresis.If only part DNA points
Son is combined by nano particle, and the DNA molecular not being loaded can migrate under the action of voltage to anode, forms electrophoretic band,
See Fig. 2.Nano particle can be calculated by this method to the struck capacity of specific DNA molecular.
The result shows that the nano particle being ultrasonically treated significantly improves the struck capacity of 13.559kb plasmid.Its
Middle NP-6 reaches 326ng/ μ l to the struck capacity highest of cyclic plasmid molecule, and NP-2 sample holds the loading of identical plasmid
Amount is only 125ng/ μ l.For linear plasmid molecule, the useful load of NP-6 sample is also significantly greater than NP-2 sample, specific to join
It is shown in Table 2.
Struck capacity (ng/ μ L) of the different nano particles of table 2 to different DNA samples
To 1.832kb linear molecule, 10.550kb is linear and the delivered payload capability test data of ring molecule also it can be concluded that
Same conclusion.
The above result shows that prepared by the method LDH nano particle to the delivered payload capability of long-fragment nucleic acid molecule very
It is prominent.It is well known that plant conversion carrier molecular weight is generally above 10kb, a large amount of loadings of this kind of plasmid molecule are made
The Genetic Transformation in Higher Plants of LDH nanoparticle mediated is possibly realized.
Transient expression of the embodiment 3:LDH nanoparticle mediated GFP gene in onion epidermis cell
Studies have shown that the metallic particles that diameter is 20~50nm can be entered plant cell, therefore nano particle by endocytosis
Enter the good carrier of plant cell as foreign protein or nucleic acid molecules.Therefore, the present embodiment attempt using nano particle as
1.832kb linear molecule, 10.550kb cyclic plasmid and 13.559kb cyclic plasmid are imported onion epidermis cell by carrier, are passed through
The transient expression of fluorescence signal detection GFP gene.Wherein the cyclic plasmid map of 10.550 kb and 13.559kb is shown in Fig. 3,
The linear fragment of 1.832kb is cyclic plasmid pD1301-eGFP by recycling gained DNA after HindIII and EcoRI digestion
Molecule, coded sequence are shown in SEQ ID NO.2.
Specific experimental method is as follows:
1) endepidermis is torn on superclean bench with tweezers after surface sterilization, is cut into 1cm by fresh onion2's
Fritter is laid on MS minimal medium, preculture 6h；
2) each 200 μ l of NP-2 and NP-6 sample is taken, is diluted to 1ml with aseptic deionized water, the matter that total amount is 60 μ g is added
Grain DNA, it is another that 1.0 μ L of Tween-20, i.e. final concentration of the 0.1% of Tween-20 is added to prevent nano particle from assembling, it mixes
15 minutes are stood after even；
3) onion epidermis Jing Guo preculture is put among the eppendorf pipe containing NP solution, is placed 30 minutes,
Period overturns centrifuge tube several times；
4) it takes out onion epidermis and is placed in suck dry moisture on aseptic filter paper with sterile water wash, be laid on MS culture medium,
Dark culture uses fluorescence microscope afterwards for 24 hours.
As seen from Figure 4, fluorescence signal accumulates in nucleus, shows that LDH nano particle can be carried containing GFP base
The exogenous plasmid molecule of cause enters cell.
In infected liquid preparation process, be added Tween-20 (final concentration 0.1%) can effectively prevent nano particle occur aggregation and
Precipitating is formed, while not influencing nano particle again to the delivered payload capability (Fig. 5) of plasmid molecule.
Compared with NP-6, other NP processing or the processing of Tween-20 is added without not it is observed that onion epidermis cell
Fluorescence signal.
Expression of the embodiment 4:LDH nanoparticle mediated GFP gene in plant root tip
The present embodiment is attempted foreign gene being transferred to the seed in sprouting by the mediation of nano particle, turns base to obtain
Because of plant.The infected liquid containing NP-6 nano particle and cyclic plasmid is prepared according to the method in a upper embodiment, is added simultaneously
Tween-20 makes its final concentration reach 0.1%.Rice, mung bean, cucumber seeds each 40 are selected, every 10 dresses after sterile-processed
The vial for entering a 20ml capacity, being separately added into 1ml sterile water, 1ml, only the solution containing nano particle, 1ml only contain matter
The solution and 1ml infected liquid of grain, cover being placed in 28 DEG C of incubators for ventilative lid and are cultivated.According to moisture absorption feelings
Sterile water is replenished in time in condition, and the tip of a root of germination seed is cut after 7 days in fluorescence microscopy microscopic observation.
The result shows that the rice impregnated by infected liquid, mung bean, the cucumber tip of a root have apparent fluorescence signal, and it is other
Processing does not have then.Wherein the tip of a root fluorescence signal of cucumber seeds is most strong, sees Fig. 6, may compared with its root tissue tender spy
Property is related.Tween-20 processing as plasmid vector or is added without using the nano particle of the other methods preparation except NP-6,
Expression signal of the GFP in organization of root tips is not observed under identical experiment condition, this is also demonstrated at NP-6 and Tween-20
Reason is to implement the best preparation method of the LDH nano particle and nucleic acid molecules conjugate of genetic transformation.
Claims (13)
1. a kind of preparation method of LDH nano particle and nucleic acid molecules conjugate, which comprises the following steps:
1) magnalium solution is prepared；
2) sodium hydroxide solution is prepared；
3) above-mentioned steps 1 are mixed) and the middle magnalium solution and sodium hydroxide solution obtained of step 2), it collects, wash and is resuspended and sink
It forms sediment, obtains nano particle emulsion；
4) by above-mentioned steps 3) obtained in nano particle emulsion carry out water bath processing；
5) by above-mentioned steps 4) in nano particle emulsion after water bath processing carry out ultrasonication, LDH nano particle is made；
6) by above-mentioned steps 5) made from LDH nano particle coupling is mixed with nucleic acid molecules；
7) Tween-20 is added, and stands.
2. the preparation method of LDH nano particle and nucleic acid molecules conjugate according to claim 1, it is characterised in that: institute
It states in step 1), the magnalium solution is by MgCl2And AlCl3It is dissolved in deionized water acquisition, MgCl in the magnalium solution2With
AlCl3Concentration be respectively 0.029g/mL and 0.024g/mL.
3. the preparation method of LDH nano particle and nucleic acid molecules conjugate according to claim 1, it is characterised in that: institute
It states in step 2), the sodium hydroxide solution is dissolved in deionized water by NaOH and obtains, and the concentration of the sodium hydroxide solution is
0.006g/mL。
4. the preparation method of LDH nano particle and nucleic acid molecules conjugate according to claim 1, it is characterised in that: institute
It states in step 3), magnalium solution is added in the NaOH solution of stirring in 3~10sec according to 1: 4 volume ratio, is then proceeded to
30min is stirred, the method for collecting precipitating is that 8000rpm is centrifuged 10min.
5. the preparation method of LDH nano particle and nucleic acid molecules conjugate according to claim 1, it is characterised in that: institute
It states in step 4), the method for water bath processing is 100 DEG C of 4~15h of water-bath.
6. water-bath processing method according to claim 5, it is characterised in that: the method for the water bath processing is 100 DEG C of water
Bathe 8h.
7. the preparation method of LDH nano particle and nucleic acid molecules conjugate according to claim 1, it is characterised in that: institute
It states in step 5), the ultrasonication mode is ultrasonication 3 times, each 5min.
8. the preparation method of LDH nano particle and nucleic acid molecules conjugate according to claim 1, it is characterised in that: institute
It states in step 6), the LDH nano particle carries out mixing coupling according to mass ratio 1: 1~1: 6 with nucleic acid molecules.
9. the preparation method of LDH nano particle and nucleic acid molecules conjugate according to claim 1, it is characterised in that: institute
It states in step 6), the nucleic acid molecules are the threadiness or circular nucleic acid molecules of 1.832-13.559kb.
10. nucleic acid molecules according to claim 9, it is characterised in that: the nucleic acid molecules include eGFP gene.
11. the preparation method of LDH nano particle and nucleic acid molecules conjugate according to claim 1, it is characterised in that: institute
It states in step 7), final concentration of the 0.1% of the Tween-20.
12. a kind of LDH nano particle and nucleic acid molecules being prepared according to any one of claim 1~11 the method
Application of the conjugate in Genetic Transformation in Higher Plants, it is characterised in that: by the LDH nano particle and nucleic acid molecules conjugate and plant
Object cell co-culture makes nucleic acid molecules import plant cell.
13. application according to claim 12, it is characterised in that: the cell is epidermal cell or root-tip cells；It is described
Plant is dicotyledon, including mung bean；The plant is monocotyledon, including onion, rice and cucumber.
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