CN105441484B - The method that the houghite ultrathin nanometer piece load bioactive molecule of lactate intercalation imports plant cell - Google Patents
The method that the houghite ultrathin nanometer piece load bioactive molecule of lactate intercalation imports plant cell Download PDFInfo
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Abstract
The invention discloses the methods that a kind of houghite ultrathin nanometer piece of lactate intercalation load bioactive molecule imports plant cell, and the acrylic/hydrotalcite-like nano material of synthesizing lactic acid root cutting layer, removing obtains two-dimensional nano piece in water；The identification of the phenetic analysis such as XRD is carried out to LDH-lactate-NS；With medium treatment model plant material BY-2 cell and arabidopsis containing various concentration LDH-lactate-NS, the safe concentration that LDH-lactate-NS load bioactive molecule imports plant cell is obtained；Bioactive molecule and LDH-lactate-NS are coupled with different mass ratioes, obtain nanometer sheet-bioactive molecule compound of optimal absorption ratio；Nanometer sheet-bioactive molecule compound of optimal absorption ratio is incubated for altogether with plant cell, is imported bioactive molecule in complete live plant cell with optimal incubation time.
Description
Technical field
The invention belongs to bioactive molecule technical fields, are related to a kind of houghite ultrathin nanometer piece of lactate intercalation
Load the method that bioactive molecule imports plant cell.
Background technique
Acidic bio bioactive molecule is being imported plant cell this aspect by bioactive molecule leading-in technique, still by height
The limitation of effect property, economy and ease of handling etc. is still to carry out active somatic cell to wider plant species to examine in real time
One of bottleneck of survey technology.Correlative study once reported that researcher was imported DNA molecular using materials such as porous silicon nanoparticles
In plant protoplast.Not only cumbersome, yield is lower however, when preparing protoplast, obtained protoplast is highly brittle
It is weak, easily rupture.Therefore, researchers, which are exploring always one kind, can be introduced directly into bioactive molecule intact live plant
The ideal carrier of cell.
As a kind of novel bioactive molecule carrier, inorganic nano material is just easy to operate with its, widely applicable etc.
Attention of the advantage by researchers.Wherein, acrylic/hydrotalcite-like nano piece (layered double hydroxide, LDH) is opposite
For other nano materials, have synthetic method simple, the advantage that raw material are easy to get.It especially can also be by its piece
Layer body structure surface carries out the special sex modification of functional group, further develops its application potential.
Therefore, this research is developed by the houghite of inorganic nano material lactate intercalation a kind of that biology is living for the first time
Property molecule import the technology of intact live plant cell, this can not only provide strong tool for basic biological study, also general
It is of great significance in terms of the genetic modification of various crop and economy of forestry tree species and genetic breeding.
Summary of the invention
The object of the present invention is to provide a kind of houghite ultrathin nanometer pieces of lactate intercalation to load bioactive molecule
The method for importing plant cell solves the prior art and is difficult to bioactive molecule importing asking in intact live plant cell
Topic.
The technical scheme adopted by the invention is that a kind of houghite ultrathin nanometer piece load biology of lactate intercalation is living
Property molecule import plant cell method, follow the steps below:
Step 1, the acrylic/hydrotalcite-like nano material MgAl-lactate LDH of synthesizing lactic acid root cutting layer, removing obtains in water
Two-dimensional nano piece LDH-lactate-NS；
Step 2, the phenetic analysis such as XRD are carried out to LDH-lactate-NS to identify；
Step 3, with buffer processing BY-2 cell and arabidopsis containing various concentration LDH-lactate-NS, it is obtained
Load the safe concentration that bioactive molecule imports plant cell；
Step 4, bioactive molecule and LDH-lactate-NS are coupled with different mass ratioes；
Step 5, two-dimensional nano piece-bioactive molecule compound is incubated for altogether with plant cell, bioactive molecule
It imports in plant cell.
Further, the step 1 specifically follows the steps below: the magnalium for being 0.5:1-6:1 by magnesium/al mole ratio
Lactate dissolution under the conditions of 30-100 DEG C in 125mL distilled water is configured to solution A and is placed in separatory funnel；Separately it will be greater than
It is dissolved in 25mL distilled water in the Pfansteihl of the mole of aluminium, is configured to solution B and is placed in three neck round bottom；It is fast at room temperature
Speed stirring instills solution A in solution B dropwise, controls its pH system with sodium hydroxide solution in titration process, and maintenance pH value is
8-12；After to be titrated, continue to be vigorously stirred and be aged 5-48h, obtain opalescent mixture, passes through centrifugation side with distilled water
Method is washed repeatedly until supernatant becomes cloudy；Sample after centrifugation is divided into two parts, it is dry that portion is placed in room temperature in vacuum oven
It is dry, it is ground after dry, gained powder is MgAl-lactate LDH；Another is placed in a certain amount of distilled water, is vigorously stirred
Until becoming transparence without precipitating obtains suspension C, the concentration of suspension C by MgAl-lactate LDH mass after drying and
The content of distilled water is calculated, and is denoted as LDH-lactate-NS, overall process is in N2Protection is lower to be carried out.
Further, the step 2 specifically follows the steps below: by the LDH-lactate-NS drop of removing in copper mesh
The imaging of upper progress TEM (transmission electron microscope), and dry LDH-lactate-NS carry out SEM (scanning electron microscope) at
LDH-lactate-NS is placed in pallet using gel method and obtains XRD characteristic spectrum by picture, and with laser irradiation LDH-lactate-NS
Colloid detects Tyndall phenomenon.
Further, in the step 4, LDH-lactate-NS: negatively charged bioactive molecule DNA (deoxidation core
Ribosomal ribonucleic acid) mass ratio be 3:1 when, LDH-lactate-NS: negatively charged bioactive molecule FITC (isosulfocyanic acid fluorescence
Element) mass ratio be 5:1 when, can adsorb completely.
Further, the step 5 specifically follows the steps below: the concentration control of LDH-lactate-NS is existed
100ug/ml or less is used as working concentration, with DNA:LDH-lactate-NS mass ratio 1:3 or more, with FITC:LDH-
Lactate-NS mass ratio 1:5 or more, incubation time 1h import bioactive molecule in plant cell.
The beneficial effects of the present invention are: passing through idol using the two-dimensional nano piece (LDH-lactate-NS) removed in water for the first time
Join bioactive molecule and obtain nanometer sheet-bioactive molecule compound, with optimal absorption ratio and incubation time and plant cell
It is incubated for altogether and imports bioactive molecule in complete live plant cell.(1) removing obtains LDH-lactate- in water
NS two-dimensional nano piece ensure that solvent to cytotoxic, and DMSO/ first can only be dissolved in by overcoming existing bioactive molecule carrier
In the toxic solvents such as alcohol；(2) by experimental exploration, LDH-lactate-NS two-dimensional nano piece adsorbed bioactive molecule is utilized
Characteristic compensates for longer disadvantage of existing bioactive molecule carrier adsorption bioactive molecule time, substantially reduces examination
The time is tested, provides more convenience and efficiently method for living body real-time detection；(3) bioactive molecule is born by optimization
It carries and importing bioactive molecule is to the condition of active somatic cell, it can be by LDH-lactate-NS two-dimensional nano piece efficient absorption
Bioactive molecule simultaneously effectively imports in intact live plant cell, has surmounted the function pole of existing bioactive molecule carrier
Limit.
Detailed description of the invention
Fig. 1 is LDH-lactate-NS characterization: where A is the XRD characterization of LDH-lactate-NS；B is LDH-
The FE-SEM of lactate-NS is characterized；C is the Tyndall phenomenon of LDH-lactate-NS；D is the HR- of LDH-lactate-NS
TEM characterization.
Fig. 2 is the BY-2 cell of PI dyeing various concentration LDH-lactate-NS processing: where A-C is that PI dyes 25 μ g/
The BY-2 cell of ml LDH-lactate-NS processing；D-F is that the BY-2 of PI dyeing 50 μ g/ml LDH-lactate-NS processing is thin
Born of the same parents；G-I is the BY-2 cell that PI dyes 100 μ g/ml LDH-lactate-NS processing；J-L is that PI dyes 300 μ g/ml LDH-
The BY-2 cell of lactate-NS processing.Length of the scale is 100 μm.
Fig. 3 is seedling root long, plumular axis length on the culture medium of LDH-lactate-NS containing various concentration: where A is quasi- south
The root long of mustard seedling；B is the plumular axis length of Arabidopsis thaliana Seedlings.* indicates that P≤0.01, * * * indicate P≤0.001.
Fig. 4 is adsorption capacity of the LDH-lactate-NS to DNA molecular.
Fig. 5 is Zeta-potential data.
Fig. 6 is that LDH-lactate-NS load FITC imports BY-2 cell: A is light field；B is fluorogram；C is under high power lens
DIC figure；D is the fluorogram under high power lens；E is the stacking chart of C and D.Length of the scale is respectively 100 μm (A and B) and 20 μm
(C, D and E).
Fig. 7 is that LDH-lactate-NS load ssDNA-FITC imports BY-2 cell: A is background fluorescence figure；B is LDH-
Lactate-NS loads ssDNA-FITC and imports the fluorogram after BY-2 cell；C is the three-dimensional imaging to A figure fluorescence field；D is pair
The three-dimensional imaging of B figure fluorescence field；E is the gray value of A and B figure fluorescence field；F is the light field of corresponding B figure cell.Length of the scale is 30
μm。
Fig. 8 is that LDH-lactate-NS load FITC imports arabidopsis cell, and length of the scale is 30 μm.
Fig. 9 is that LDH-lactate-NS load ssDNA-FITC imports arabidopsis cell, and length of the scale is 30 μm.
Specific embodiment
The following describes the present invention in detail with reference to the accompanying drawings and specific embodiments.
A kind of houghite ultrathin nanometer piece load bioactive molecule of lactate intercalation of the present invention imports plant cell
Method, follow the steps below:
Step 1, houghite (MgAl-lactate LDH) nano material of synthesizing lactic acid root cutting layer, removing obtains in water
It obtains two-dimensional nano piece (LDH-lactate-NS):
By magnesium/al mole ratio be 0.5:1-6:1 magnalium lactate in 125mL distilled water under the conditions of 30-100 DEG C it is molten
Solution is configured to solution A and is placed in separatory funnel；The Pfansteihl of the mole of the another aluminium that will be greater than or equal to is dissolved in 25mL distilled water, is matched
Solution B is made to be placed in three neck round bottom flask；Quickly stirring instills solution A in solution B dropwise at room temperature, in titration process
Its pH system is controlled with sodium hydroxide solution, maintenance pH value is 8-12；After to be titrated, continue to be vigorously stirred and be aged 5-
48h obtains opalescent mixture, is washed repeatedly by centrifugal method until supernatant becomes cloudy with distilled water；After centrifugation
Sample is divided into two parts, and portion is placed in drying at room temperature in vacuum oven, grinds after dry, and gained powder is MgAl-lactate
LDH；Another is placed in a certain amount of distilled water, is vigorously stirred and obtains suspension C up to becoming transparence without precipitating, suspended
The concentration of liquid C is calculated by MgAl-lactate LDH mass after drying and the content of distilled water, is denoted as LDH-lactate-
NS, overall process is in N2Protection is lower to be carried out.
Step 2: the phenetic analysis such as XRD being carried out to LDH-lactate-NS and are identified: by the LDH-lactate-NS drop of removing
TEM imaging is carried out on copper mesh, and is dried LDH-lactate-NS and carried out SEM imaging.Using gel method by LDH-lactate-
NS is placed in pallet and obtains XRD characteristic spectrum, and detects Tyndall phenomenon with laser irradiation LDH-lactate-NS colloid.
As shown in Figure 1, XRD is the result shows that obtain the material (Figure 1A) of proper characteristics.The material that synthesis is obtained carries out
FE-SEM imaging, the results showed that, it is multilayer pile stack structure (Figure 1B) before removing.By removing in water, it is existing to produce dindar
As illustrating lamella size (Fig. 1 C) between 1nm~100nm.HR-TEM imaging obtains two dimension after showing removing in water
The LDH-lactate-NS (Fig. 1 D) of structure.Using the two-dimensional nano piece LDH-lactate-NS removed in water as carrier in this research
Load bioactive molecule.
Step 3, it with medium treatment BY-2 cell and arabidopsis containing various concentration LDH-lactate-NS, explores
Toxicological effect of the LDH in plant cell establishes LDH-lactate-NS to the toxicological mechanism model of plant cell, is subsequent
Load test provides reference:
As shown in Figure 2.Propidium iodide (PI) dyestuff is a kind of dyestuff that red fluorescence is issued in conjunction with DNA.
However, PI dyestuff can not penetrate the complete cell membrane of living cells.Using this characteristic, with the LDH-lactate-NS of various concentration
(concentration is respectively 25/50/100/300ug/ml) processing BY-2 cell simultaneously carries out PI dyeing.The results show that working as LDH-
When the concentration of lactate-NS is 100ug/ml, faint fluorescence intracellular is detected.However, the concentration of LDH-lactate-NS is high
When up to 300ug/ml, extremely strong fluorescence intracellular is detected.Illustrating LDH-lactate-NS concentration, the following are loads in 100ug/ml
The safe concentration of bioactive molecule.
As shown in Figure 3.Preparing the culture medium containing various concentration LDH-lactate-NS, (concentration is respectively 25/50/100/
300ug/ml), by the sowing of arabidopsis seed on the culture medium.After seed is sprouted, the root long and plumular axis of seedling (6 days) are counted
Length.The result shows that the concentration of LDH-lactate-NS is in 100ug/ml or less, without apparent inhibiting effect.And
When 300ug/ml, inhibitory effect is extremely significant.Illustrating LDH-lactate-NS concentration, the following are load biologies in 100ug/ml
The safe concentration of bioactive molecule.
Step 4, bioactive molecule and LDH-lactate-NS are coupled with different mass ratioes, are obtained optimal
Absorption ratio:
DNA and LDH-lactate-NS is incubated for altogether by different quality ratio (1:1,1:2,1:3,1:4 and 1:5) and obtains LDH-
DNA conjugate.As shown in figure 4, using independent DNA as positive control, using the electrophoresis hole of not DNA as negative control, to its into
Row gray analysis can adsorb completely when result proves that LDH-DNA mass ratio is 3:1.
Such as Fig. 5 Zeta-potential, statistics indicate that, FITC is negatively charged bioactive molecule, LDH-Lactate
It is positively charged bioactive molecule.By FITC and LDH-lactate-NS by different quality ratio (1:1,1:2,1:3,1:4 and
It 1:5) is incubated for 5min altogether and obtains LDH-FITC conjugate.When LDH-lactate-NS:FITC mass ratio is 5:1 as the result is shown, electricity
Lotus is 0, shows to adsorb completely when its mass ratio is 5:1.
Step 5, the method being incubated for altogether by nanometer sheet-bioactive molecule compound and plant cell, utilizes two wieners
Rice piece carrier imports bioactive molecule in plant cell:
It is according to toxicity test as a result, the concentration control of LDH-lactate-NS is dense as work in 100ug/ml or less
Degree.Meanwhile according to adsorption test as a result, with DNA:LDH-lactate-NS mass ratio 1:3 or more, with FITC:LDH-
Lactate-NS mass ratio 1:5 or more is as work absorption than carrying out load test.
Optimal situation is: the FITC:LDH-lactate-NS mass ratio used is 1:6, DNA:LDH-lactate-NS matter
Amount is than being 1:5, final concentration of 10ug/ml, incubation time 1h.By confocal fluorescent microscopic, detected from cytoplasm
Apparent fluorescence.
As shown in Figure 6 and Figure 7, under above method and experimental condition, by LDH-Lactate-NS-FITC compound with
After BY-2 suspension cell and arabidopsis are incubated for altogether, strong fluorescence intracellular is detected using confocal, illustrates the nanometer sheet
High-efficient carrier FITC dyestuff has imported in BY-2 cell and arabidopsis cell.Moreover, LDH-Lactate-NS can also be loaded
SsDNA-FITC is imported in BY-2 cell (such as Fig. 8) and arabidopsis cell (such as Fig. 9), has further been convincingly demonstrated us and has been explored
Biologically functional molecule efficiently effectively can be imported complete live plant cell using LDH-Lactate-NS by the method for acquisition
In.
Claims (2)
1. the method that a kind of houghite ultrathin nanometer piece load bioactive molecule of lactate intercalation imports plant cell,
It is characterized in that, follows the steps below:
Step 1, the acrylic/hydrotalcite-like nano material MgAl-lactate LDH of synthesizing lactic acid root cutting layer, removing obtains two dimension in water
Nanometer sheet LDH-lactate-NS；
Step 2, the LDH-lactate-NS drop of removing is subjected on copper mesh transmission electron microscope imaging, and dries LDH-
Lactate-NS is scanned Electron Microscope images, and LDH-lactate-NS is placed in pallet using gel method and obtains XRD spy
Sign spectrum, and Tyndall phenomenon is detected with laser irradiation LDH-lactate-NS colloid；
Step 3, with buffer processing BY-2 cell and arabidopsis containing various concentration LDH-lactate-NS, its load is obtained
The safe concentration of bioactive molecule importing plant cell；
Step 4, bioactive molecule and LDH-lactate-NS are coupled with different mass ratioes；
Step 5, two-dimensional nano piece-bioactive molecule compound is incubated for altogether with plant cell, bioactive molecule is imported
In plant cell；
In the step 4, LDH-lactate-NS: when negatively charged bioactive molecule DNA mass ratio is 3:1, LDH-
Lactate-NS: it when negatively charged bioactive molecule FITC mass ratio is 5:1, can adsorb completely；
The step 5 specifically follows the steps below:
The concentration control of LDH-lactate-NS is regard as working concentration in 100ug/ml or less, with DNA:LDH-lactate-NS
Mass ratio 1:3 or more, with FITC:LDH-lactate-NS mass ratio 1:5 or more, incubation time 1h, i.e., by bioactivity point
Son imports in plant cell.
2. a kind of houghite ultrathin nanometer piece load bioactive molecule of lactate intercalation according to claim 1 is led
Enter the method for plant cell, which is characterized in that the step 1 specifically follows the steps below:
The magnalium lactate that magnesium/al mole ratio is 0.5:1-6:1 is dissolved under the conditions of 30-100 DEG C in 125mL distilled water and is matched
Solution A is made to be placed in separatory funnel；The Pfansteihl of the mole of the another aluminium that will be greater than or equal to is dissolved in 25mL distilled water, is configured to
Solution B is placed in three neck round bottom；Quickly stirring instills solution A in solution B dropwise at room temperature, and hydrogen is used in titration process
Sodium hydroxide solution controls its pH system, and maintenance pH value is 8-12；After to be titrated, continue to be vigorously stirred and be aged 5-48h, obtain
To opalescent mixture, washed repeatedly by centrifugal method until supernatant becomes cloudy with distilled water；By the sample after centrifugation point
It is two parts, portion is placed in drying at room temperature in vacuum oven, grinds after dry, and gained powder is MgAl-lactate LDH；Separately
Portion is placed in a certain amount of distilled water, is vigorously stirred and is obtained suspension C up to becoming transparence without precipitating, suspension C's
Concentration is calculated by MgAl-lactate LDH mass after drying and the content of distilled water, is denoted as LDH-lactate-NS, entirely
Process is in N2Protection is lower to be carried out.
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| US11591234B2 (en) | 2017-03-17 | 2023-02-28 | Setolas Holdings, Inc. | Microparticulate hydrotalcite, method for producing same, resin composition of same, and suspension of same |
| CN109060664B (en) * | 2018-06-29 | 2021-03-26 | 苏州同阳科技发展有限公司 | Plant growth process monitoring method based on laser speckle technology |
| CN108906057B (en) * | 2018-07-04 | 2021-10-29 | 北京石油化工学院 | Nano-scale intercalated hydrotalcite catalyst and preparation method and application thereof |
| CN109825529B (en) * | 2018-08-31 | 2023-01-10 | 武汉隽秀园艺科技有限公司 | Preparation method of LDH (layered double hydroxide) nanoparticle and nucleic acid molecule conjugate |
| CN113717988A (en) * | 2021-08-17 | 2021-11-30 | 广东省林业科学研究院 | Method for directionally creating plant mutant |
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