Genetically modified nano material without genotype dependence as well as preparation method and application of genetically modified nano material

Abstract

The invention discloses a genetype-dependence-free transgenic nano material as well as a preparation method and application thereof, and belongs to the technical field of novel nano materials. The preparation method of the transgenic nano material without genotype dependence comprises the following steps: dropwise adding an MES solution containing EDC and NHS into a suspension, then carrying out water bath ultrasonic treatment, then adjusting the pH value to 5-5.5, adding activated COOH-SWNTs into a chitosan acetic acid solution, and continuously carrying out shake culture at 30-35 DEG C to obtain the CS-fSWNTs nano material. In addition, the invention also provides an application of the pDNA-CS-fSWNTs nano material in expression of functional protein in plant chloroplast. According to the genetype-dependent-free transgenic nano material provided by the invention, target protein can be simply, conveniently and quickly expressed in chloroplast without depending on plant genotypes.

Description

Technical Field

[0001] The present invention relates to the technical field of novel nanomaterials, and in particular to a genotype-independent transgenic nanomaterial, a preparation method thereof, and an application thereof. Background Art

[0002] Plant chloroplasts offer the advantages of high protein expression levels, maternal inheritance, and polycistronic regulation. However, plant chloroplast transformation often relies on specialized equipment, such as gene guns, requires tissue culture, or is species-specific. New nanomaterials can overcome these three limitations. Among them, carbon nanotubes offer excellent penetrability, amenability to functional modification, and low cytotoxicity. Chitosan, a positively charged, biodegradable natural material, can adsorb plasmid DNA. Current applications of nanomaterial-mediated molecular delivery in plants are limited to delivering fluorescent proteins and silencing target genes. Research applications for expressing functional proteins in chloroplasts are lacking. Summary of the Invention

[0003] The purpose of the present invention is to overcome the above technical deficiencies, provide a genotype-independent transgenic nanomaterial and its preparation method and application, and solve the technical problem in the prior art of how to express the target protein in chloroplasts simply and quickly without relying on plant genotype.

[0004] To achieve the above technical objectives, the technical solution of the present invention provides a method for preparing a genetically modified nanomaterial, comprising dropwise adding an MES solution containing EDC and NHS to a COOH-SWNTs suspension, followed by water bath sonication, adjusting the pH to 5-5.5, and oscillating to obtain activated COOH-SWNTs, adding the activated COOH-SWNTs to a chitosan acetate solution, and continuing to oscillate and culture at 30-35°C to obtain the genetically modified nanomaterial.

[0005] In any embodiment, the COOH-SWNTs suspension is prepared by the following steps: mixing COOH-SWNTs with MES to obtain the COOH-SWNTs suspension.

[0006] In any embodiment, the water bath sonication time is 15-18 min.

[0007] In any embodiment, the activated COOH-SWNTs are obtained by shaking for 45-50 minutes; and / or, the transgenic nanomaterial is obtained by continuing shaking and culturing at 30-35° C. for 15-18 hours.

[0008] In addition, the present invention also provides a genetically modified nanomaterial prepared by the above preparation method.

[0009] The present invention also provides a method for preparing a pDNA-CS-fSWNTs nanomaterial, comprising the following steps: mixing the aforementioned transgenic nanomaterial with a pDNA solution to obtain the pDNA-CS-fSWNTs nanomaterial, wherein the pDNA comprises a plasmid containing sfGFP or a plasmid containing 6×his, respectively designated pCNT-sfGFP and pCNT-hIGF-1. The expression region sequence of pCNT-sfGFP is shown in SEQ ID NO. 1, and the expression region sequence of pCNT-hIGF-1 is shown in SEQ ID NO. 2.

[0010] In any embodiment, the mass ratio of the transgenic nanomaterial to the pDNA solution is (3-6):1.

[0011] In addition, the present invention also provides a pDNA-CS-fSWNTs nanomaterial, which is prepared by the above preparation method.

[0012] In addition, the present invention also proposes a pDNA-CS-fSWNTs nanomaterial prepared by the above preparation method or the use of the above pDNA-CS-fSWNTs nanomaterial in expressing functional proteins in plant chloroplasts.

[0013] In any embodiment, the pDNA-CS-fSWNTs nanomaterial is injected and infiltrated into the back of plant leaves, with 3-5 areas injected per leaf and 2-3 mL injected per leaf; and / or, the functional protein is a fluorescent protein and / or an insulin-like growth factor; and / or, the plant leaves are lettuce leaves or tobacco leaves.

[0014] Compared with the prior art, the beneficial effects of the present invention include: the preparation method of the transgenic nanomaterial proposed by the present invention, wherein an MES solution containing EDC and NHS is added dropwise to a COOH-SWNTs suspension, followed by water bath sonication, and then the pH is adjusted to 5-5.5, followed by oscillation culture to obtain activated COOH-SWNTs, the activated COOH-SWNTs are added to a chitosan acetic acid solution, and the oscillation culture is continued at 30-35°C to obtain the transgenic nanomaterial, the amino groups of chitosan and the carboxyl groups of the carboxylated carbon nanotubes undergo an amination reaction and cross-linking, and the chitosan can electrostatically adsorb plasmids, and the functionalized carbon nanotubes can load the plasmids, with each modification step, the material flow radius increases, the zeta potential changes, and the microscopic morphology changes, and the material is injected and penetrated into plant leaves in an MES buffer environment, and in the weakly alkaline environment of the chloroplast matrix, the charge adsorption of the plasmid chitosan decreases, resulting in the release of the plasmid, thereby expressing the target protein in the chloroplast, independently of the plant genotype, and expressing the target protein in the chloroplast simply and quickly. Increase transformation timeliness, reduce plant damage, and expand the application scope of nanomaterials in chloroplast transformation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a physical picture of carbon nanotubes before and after chitosan modification in Example 1 of the present invention.

[0016] Figure 2 This is the change in flow radius / zeta potential before and after the preparation of the pDNA-CS-fSWNTs nanomaterial in Example 1 of the present invention.

[0017] Figure 3 This is the microscopic morphology change of the pDNA-CS-fSWNTs nanomaterial before and after Example 1 of the present invention.

[0018] Figure 4 Schematic diagram of the GFP fusion vector and purification vector of Example 2 of the present invention.

[0019] Figure 5 This is a confocal microscopy image of Example 2 of the present invention after the pDNA-CS-fSWNTs nanomaterial delivered pCNT-sfGFP to lettuce leaves.

[0020] Figure 6 This is a protein analysis diagram of Example 2 of the present invention, in which pCNT-hIGF-1 is delivered by pDNA-CS-fSWNTs nanomaterials and injected into tobacco leaves and lettuce leaves. DETAILED DESCRIPTION

[0021] The "range" disclosed in this application is defined in the form of a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of a particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, i.e., any lower limit can be combined with any upper limit to form a range. For example, if a range of 60 to 120 and 80 to 110 is listed for a particular parameter, it is understood that a range of 60 to 110 and 80 to 120 is also expected. In addition, if the minimum range values ​​listed are 1 and 2, and if the maximum range values ​​listed are 3, 4, and 5, then the following ranges can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, and 2 to 5. In this application, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, wherein a and b are both real numbers. For example, a numerical range of "0-5" indicates that all real numbers between "0-5" are listed herein, and "0-5" is simply an abbreviation for these numerical combinations. Furthermore, stating that a parameter is an integer ≥ 2 is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0022] Unless otherwise specified, the terms "include" and "comprising" used in this application may be open-ended or closed-ended. For example, "include" and "comprising" may mean that other components not listed may also be included or that only the listed components are included.

[0023] Unless otherwise specified, the term "or" is used in this application to be inclusive. For example, the phrase "A or B" means "A, B, or both A and B." More specifically, the condition "A or B" is satisfied if any of the following conditions are met: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0024] This specific embodiment provides a method for preparing a genetically modified nanomaterial (i.e., CS-fSWNTs nanomaterial), comprising: adding an MES solution containing EDC and NHS dropwise to a COOH-SWNTs suspension, followed by water bath sonication for 15-18 minutes, adjusting the pH to 5-5.5, and oscillating for 45-50 minutes to obtain activated COOH-SWNTs; and adding the activated COOH-SWNTs to a chitosan acetic acid solution, followed by continued oscillation at 30-35°C for 15-18 hours to obtain the CS-fSWNTs nanomaterial.

[0025] In some embodiments, the COOH-SWNTs suspension is prepared by the following steps: mixing COOH-SWNTs with MES to obtain the COOH-SWNTs suspension.

[0026] This specific embodiment also provides a genetically modified nanomaterial, which is prepared by the above preparation method.

[0027] In addition, this specific embodiment also proposes a preparation method of a pDNA-CS-fSWNTs nanomaterial, comprising the following steps: mixing the above-mentioned CS-fSWNTs nanomaterial with a pDNA solution in a mass ratio of (3-6):1 to obtain the pDNA-CS-fSWNTs nanomaterial, wherein the pDNA includes a plasmid pCNT-sfGFP containing sfGFP or a plasmid pCNT-hIGF-1 containing 6×his, the pCNT-sfGFP expression region sequence is shown in SEQ ID NO.1, and the pCNT-hIGF-1 expression region sequence is shown in SEQ ID NO.2.

[0028] This specific embodiment also provides a pDNA-CS-fSWNTs nanomaterial, which is prepared by the above preparation method.

[0029] In addition, this specific embodiment also proposes an application of the pDNA-CS-fSWNTs nanomaterial prepared by the above preparation method or the above pDNA-CS-fSWNTs nanomaterial to express functional proteins in plant chloroplasts, comprising injecting the pDNA-CS-fSWNTs nanomaterial into the back of plant leaves, injecting 3-5 areas per leaf, and injecting 2-3 mL per leaf; the functional protein is a fluorescent protein and / or an insulin-like growth factor; the plant leaves are lettuce leaves or tobacco leaves.

[0030] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0031] In the present invention, references to “some embodiments”, “this embodiment”, examples, etc. describe a subset of all possible embodiments, but it can be understood that “some embodiments” may be the same subset or different subsets of all possible embodiments, and may be combined with each other without conflict.

[0032] If similar descriptions of "first / second" appear in the application documents, the following explanation is added. In the following description, the terms "first\second\third" are merely used to distinguish similar objects and do not represent a specific order of the objects. It can be understood that "first\second\third" can be interchanged with the specific order or sequence where permitted, so that the embodiments described herein can be implemented in an order other than that illustrated or described herein.

[0033] In this embodiment, the term "and / or" is merely a description of the association relationship between associated objects, indicating that three relationships may exist. For example, object A and / or object B may represent three situations: object A exists alone, object A and object B exist at the same time, and object B exists alone.

[0034] Below, the embodiment of the present application is described. The embodiment described below is exemplary and is only used to explain the present application, and is not to be construed as limiting the present application. Where specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. Reagents or instruments used that do not specify the manufacturer are conventional products that can be obtained commercially.

[0035] Example 1

[0036] Preparation of CS-fSWNTs nanomaterials:

[0037] (1) Add 25 mg of COOH-SWNTs (i.e., carboxylated single-walled carbon nanotubes) to 22.5 ml of 100 mM MES (i.e., 2-morpholinoethanesulfonic acid) to obtain a COOH-SWNTS suspension;

[0038] (2) 50 mg chitosan was dissolved in 25 ml 0.3% acetic acid;

[0039] (3) Add 125 mg of EDC (i.e., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 125 mg of NHS (i.e., N-hydroxysuccinimide) to 2.5 ml of 100 mM MES, dropwise add the mixture to the COOH-SWNTs suspension, sonicate in a water bath for 15 min, adjust the pH to 5, and activate the carboxyl groups by shaking at 180 rpm for 45 min. Ultrafiltration was performed at room temperature using a 100 kDa ultrafiltration tube at 4000 rpm. Resuspend the mixture in 0.3% acetic acid to a volume of 25 ml of activated COOH-SWNTs. Add the mixture to the chitosan acetic acid solution (25 ml) and shake at 180 rpm at 30°C for 16 h for amidation reaction. After returning to room temperature, sonicate with a 6 mm tip for 45 min (power 30%, 5 s on, 5 s off). Digest the mixture with a 100 kDa dialysis membrane for 3 days to remove free chitosan and store at 4°C.

[0040] Preparation of pDNA-CS-fSWNTs nanomaterials:

[0041] In MES buffer (20 mM MES, 10 mM MgCl2, pH 5.7), the CS-fSWNTs material (i.e., chitosan-functionalized single-walled carbon nanotubes) prepared in Example 1 was mixed with a pDNA solution at a mass ratio of 3:1 or 6:1 (w / w). When the CS-fSWNTs material (i.e., chitosan-functionalized single-walled carbon nanotubes) and the pDNA solution were mixed at a mass ratio of 6:1 (w / w), the CS-fSWNTs concentration was 3 mg / L. pDNA was added while stirring at a pDNA concentration of 0.5 mg / L. The stirring rate was 500 rpm and the mixture was stirred for 30 min to obtain a pDNA-CS-fSWNTs nanomaterial reaction system.

[0042] The solutions before and after modification of pDNA-CS-fSWNTs were analyzed using a Malvern ZSU3200 nanoparticle size analyzer to measure the flow radius and zeta potential of CS-fSWNTs, and the images of pDNA-CS-fSWNTs were observed using an atomic force microscope (AFM). The synthesized CS-fSWNTs can be stably stored at 4°C for more than 6 months.

[0043] like Figure 1As shown, the COOH-SWNTs suspension naturally aggregates and is unstable; after functional modification with chitosan, it has good dispersion and appears pure black; after the nanocomposite is loaded with plasmids, the working solution appears clear.

[0044] like Figure 2 As shown, the flow radius increases gradually from covalent cross-linking of SWNTs with chitosan to adsorption of DNA ( Figure 2 Left); Negatively charged carbon nanotubes become positively charged when cross-linked with chitosan, and the potential compromise change of the complex after negatively charged pDNA charge adsorption ( Figure 2 Right), C:P, i.e. CS-fSWNTs: pDNA.

[0045] like Figure 3 As shown, the initial carbon nanotube height is about 1.3 ± 0.1 nm (as shown in Figure 3 Left); CS-fSWNTs-pDNA clearly observed the aggregation of chitosan on the surface of carbon nanotubes under AFM. When the ratio of the two was 1:3 (such as Figure 3 The height is 72.4 ± 15.3 nm; when the ratio of pDNA to CS-fSWNT is 1:6 (e.g. Figure 3 Right), with a height distribution of 105.2±4.3 nm. Chitosan has obvious spherical and supercoiled structures belonging to plasmids. This functional loading is not uniform at the microscopic scale, including covalent cross-linking of chitosan, which makes the nanocomplex have an irregular and diverse structure.

[0046] Example 2

[0047] pDNA-CS-fSWNTs prepared from two plasmids pCNT-sfGFP and pCNT-hIGF-1 were delivered to plants.

[0048] Following the method of Example 1, pDNA-CS-fSWNTs were prepared from the pCNT-sfGFP and pCNT-hIGF-1 plasmids. A working solution of the pDNA-CS-fSWNTs nanomaterial was drawn up using a syringe at a ratio of 1:6. The needle was removed and the solution was slowly injected into the underside of tobacco or lettuce leaves. The pDNA-CS-fSWNTs nanomaterial working solution containing the pCNT-sfGFP plasmid was delivered to lettuce leaves, while the pDNA-CS-fSWNTs nanomaterial working solution containing the pCNT-hIGF-1 plasmid was delivered to lettuce and tobacco leaves. Three to five areas of each leaf were injected, with a total of 2-3 ml injected per leaf. The plant leaves can be punctured to enhance the penetration of the nanocomplex.

[0049]

[0050]

[0051] Figure 4 The gene element composition of the expression region of two plasmids, pCNT-sfGFP and pCNT-hIGF-1, was described.

[0052] Fluorescence expression analysis or protein analysis after injection of pDNA-CS-fSWNTs nanoparticle solution into plants

[0053] (1) If the transformed plasmid is pCNT-sfGFP, use a drill to sample the marked area of ​​the leaf 48 hours after injection, quickly separate the leaf with tweezers and place it on a glass slide, add two drops of deionized water, press the cover glass, and place it on the stage of a fluorescence confocal microscope. A relatively uniform GFP signal is observed under a laser confocal microscope (see Figure 5 ), Confocal image of the GFP fusion vector delivered by pDNA-CS-fSWNTs nanomaterials. The GFP signal is evenly distributed in the field of view, and statistics show that 76.7±8.3% of the GFP signal is co-localized with the chloroplast.

[0054] (2) If the transformed plasmid is pCNT-hIGF-1, harvest the leaves of the plant 48 hours after injection. Quickly freeze the leaves in liquid nitrogen and grind them into powder. Add protein extract to the powder and mix well (1 ml of protein extract for every 240 mg of fresh weight leaf sample). Transfer the homogenate to a centrifuge tube and incubate at 4°C for 1 hour. Ultrasonicate for 5 minutes using a Mio UCS-650 ultrasonic disruptor (2 mm tip) at a cycle of 5 seconds on and 10 seconds off. Centrifuge at 15,000 g for 10 minutes at 4°C. Aspirate the supernatant and store at -80°C for quantification and protein purification.

[0055] Adjust the protein concentration of all samples to 2 μg / ul with PBS. Take 30 μg of protein from each sample, add 1 / 4 volume of 5X protein loading buffer to mix, and heat at 100°C for denaturation for 5 min. Load the sample into the wells of a 12% (wt / v) SDS-PAGE precast gel. Set the initial current to 90 V until bromophenol blue runs in a horizontal straight line, then increase the current to 150 V until the dye reaches the bottom of the gel. Take out the gel sample and place it in a box of appropriate size, add Coomassie brilliant blue dye and place it on a shaker to slowly shake for 2 hours, then pour out the dye solution, add ddH2O and shake for 20 minutes, wash three times, or place it in a 20% (v / v) ethanol solution and shake overnight for destaining. After the gel surface is destained, observe the protein band results using a protein gel imager ( Figure 6Immunoblotting was performed using rabbit anti-IGF-1 (1:4000) (Abcam, Cambridge, UK) and goat anti-rabbit IgG HRP (1:4000) (Southern Biotechnology, Birmingham, AL). Protein markers were pre-stained with YAMA-2 dual-color markers, and the chemiluminescent signal emitted by HRP was developed on X-ray film. Figure 6 As shown, after pDNA-CS-fSWNTs delivery, 20 mg of freeze-dried tobacco leaves (left) and lettuce (right) were extracted and separated by SDS-PAGE. The 14.3 kDa band is the target protein hIGF-1 of the predicted size, and the corresponding anti-hIGF-1 Western blot bands are shown below. Figure 6 Middle, M: Marker, CP: CS-fSWNTs-pDNA injection, pDNA: pDNA injection only.

[0056] The formula of the protein extract is shown in Table 1.

[0057] Table 1

[0058]

[0059] The pDNA-CS-fSWNTs nanomaterial proposed by the present invention expresses fluorescent protein in plant leaves within 2-4 days, and expresses insulin-like growth factor (hIGF-1) in plant leaves within 2-4 days.

[0060] The specific embodiments of the present invention described above do not limit the scope of protection of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a genotype-independent transgenic nanomaterial, characterized in that: The MES solution containing EDC and NHS was added dropwise to the COOH-SWNTs suspension, followed by water bath sonication, and then the pH was adjusted to 5-5.5, and then oscillated to obtain activated COOH-SWNTs. The activated COOH-SWNTs were added to the chitosan acetate solution, and the shaking culture was continued at 30-35°C to obtain the genetically modified nanomaterials.

2. The method for preparing a genetically modified nanomaterial according to claim 1, characterized in that: The COOH-SWNTs suspension is prepared by the following steps: mixing COOH-SWNTs with MES to obtain the COOH-SWNTs suspension.

3. The method for preparing a genetically modified nanomaterial according to claim 1, wherein: The water bath ultrasound time is 15-18 min.

4. The method for preparing a genetically modified nanomaterial according to claim 1, wherein: The activated COOH-SWNTs are obtained by shaking for 45-50 minutes; and / or, the transgenic nanomaterial is obtained by continuing shaking culture at 30-35° C. for 15-18 hours.

5. A genetically modified nanomaterial, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 4.

6. A method for preparing pDNA-CS-fSWNTs nanomaterials, characterized in that: The method comprises the following steps: mixing the transgenic nanomaterial according to claim 5 with a pDNA solution to obtain the pDNA-CS-fSWNTs nanomaterial, wherein the pDNA comprises a plasmid containing sfGFP or a plasmid containing 6×his, the expression region sequence of the plasmid containing sfGFP is shown as SEQ ID NO.1, and the expression region sequence of the plasmid containing 6×his is shown as SEQ ID NO.

2.

7. The method for preparing the pDNA-CS-fSWNTs nanomaterial according to claim 6, characterized in that: The mass ratio of the transgenic nanomaterial to the pDNA solution is (3-6):

1.

8. A pDNA-CS-fSWNTs nanomaterial, characterized in that: Prepared by the preparation method according to any one of claims 6 to 7.

9. Use of the pDNA-CS-fSWNTs nanomaterial prepared by the preparation method according to any one of claims 6 to 7 or the pDNA-CS-fSWNTs nanomaterial according to claim 8 in expressing functional proteins in plant chloroplasts.

10. The use according to claim 9, characterized in that The pDNA-CS-fSWNTs nanomaterial is injected into the back of plant leaves, with 3-5 areas injected on each leaf and 2-3 mL injected on each leaf; and / or the functional protein is a fluorescent protein and / or an insulin-like growth factor; and / or the plant leaves are lettuce leaves or tobacco leaves.

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