An exogenous gene delivery system based on carbon dot nanoparticles

Abstract

The present invention relates to an exogenous gene delivery system based on carbon dot nanoparticles. The preparation method of the carbon dot nanoparticles of the present invention is to electrolyze a graphite rod with a voltage of 15V - 20V to obtain an electrolyte solution, filter and dialyze the electrolyte solution to obtain a carbon dot solution; add an activator to the carbon dot solution for the first ice-water bath reaction, then add polyethyleneimine and carry out the second ice-water bath reaction, and obtain carbon dot nanoparticles after dialysis. Mixing the carbon dot nanoparticles of the present invention with a vector carrying an exogenous gene can prepare the exogenous gene delivery system of the present invention, and transgenic plants can be achieved by coating or soaking; moreover, the carbon dot nanoparticles of the present invention are tubular, so the exogenous gene delivery system can enter the seed embryo cells and carry the exogenous gene when the plant grows, and transgenic plants can be achieved from the root source.

Description

Technical Field

[0001] The present invention relates to the field of transgenic technology, and in particular to an exogenous gene delivery system based on carbon dot nanoparticles. Background Art

[0002] Genetic transformation technology can quickly endow crops with ideal genetic traits, improve crop productivity while greatly shortening the breeding period, and is an effective way to achieve precise creation of excellent germplasms. Since the Americans first successfully obtained the first transgenic tobacco plant using the Agrobacterium tumefaciens T-DNA mediated method 40 years ago, most of the known transgenic plants have been obtained by this method, and the related core technologies and patents are in the hands of a few American companies. For example, in the transgenic seed market of important crops such as corn, soybean, and cotton, Monsanto Company of the United States accounts for 70%-100% of the share, and more than 90% of the transgenic seeds globally use the patents of this company. If China conducts precise creation of germplasm resources through traditional genetic transformation methods, it cannot bypass the patent barriers set by the United States for us.

[0003] Agrobacterium tumefaciens is a Gram-negative bacterium commonly present in soil, and it can chemotactically infect the wounded parts of plants under natural conditions. The Agrobacterium tumefaciens T-DNA mediated method is to insert the target gene into the modified T-DNA region, and achieve the transfer and integration of exogenous genes into plant cells by means of the infection of Agrobacterium tumefaciens, and then regenerate transgenic plants through cell and tissue culture techniques. The whole process requires in vitro culture of cells or tissues, so it is impossible to achieve transgenic of living plants, and the Agrobacterium tumefaciens T-DNA mediated method only achieves transgenic of plant callus, with limited receptors, and it is also easy to induce gene mutations during the culture process of callus. Therefore, there is an urgent need to seek a genetic transformation technology that can perform transgenic on living plants and the transgenic receptors are not limited. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problems in the prior art that it is impossible to perform transgenic on living plants and the transgenic receptors are limited.

[0005] To solve the above technical problems, the present invention provides a gene delivery system based on carbon dot nanoparticles. An electrolyte solution is obtained by electrolyzing a graphite rod under a voltage of 15V - 20V, and the electrolyte solution is filtered and dialyzed to obtain a carbon dot solution. An activator is added to the carbon dot solution for an ice - water bath reaction, and then PEI is added for an ice - water bath reaction. After dialysis, carbon dot nanoparticles are obtained. Mixing the carbon dot nanoparticles with a vector carrying an exogenous gene can obtain the gene delivery system of the present invention. The gene delivery system of the present invention can achieve transgenic modification of living plants by coating or soaking methods, and the receptors are not restricted. Moreover, the carbon dot nanoparticles of the present invention are tubular and have a diameter of about 50nm. Therefore, the gene delivery system of the present invention can not only enter roots, stems, leaves, flowers and fruits, but also enter seed embryo cells, carrying exogenous genes when the plants grow, and can achieve transgenic modification of plants from the root source.

[0006] The first object of the present invention is to provide a method for preparing carbon dot nanoparticles, comprising the following steps:

[0007] S1. Electrolyze a graphite rod under a voltage of 15V - 20V to obtain an electrolyte solution, and dialyze the electrolyte solution to obtain a carbon dot solution;

[0008] S_{2}. Add an activator to the carbon dot solution for a first ice - water bath reaction, then add PEI for a second ice - water bath reaction, and obtain the carbon dot nanoparticles after dialysis.

[0009] Further, the molecular weight of the PEI is 600D - 1000D, preferably 600D.

[0010] PEI is a common transfection reagent. However, when using PEI to transfect plants, very few plant cell lines can be utilized, and it is also very difficult to grow into plants. The most transfected by PEI is protoplasts. Isolating protoplasts from plant tissues and using PEI as a transfection reagent can achieve transient exogenous gene expression in protoplasts. However, protoplasts cannot self - replicate and cannot achieve transgenic modification of the whole plant. In addition, the toxicity of PEI is related to its molecular weight. The PEI selected in the present invention is 600D - 1000D, which can reduce the toxicity of PEI to cells.

[0011] Further, the activator includes N - ethyl - N - (3 - dimethylaminopropyl) carbodiimide hydrochloride. The activator is used to activate the carboxyl groups on the surface of carbon dots to facilitate the coupling of carbon dots with PEI.

[0012] Further, the time of the first ice - water bath is 1.5 - 3 hours, and the time of the second ice - water bath is 3 - 5 hours.

[0013] Furthermore, the mass ratio of the carbon dot solution to the PEI is (1 - 3):(3 - 7). Excessive PEI will cause precipitation of carbon dots, failing to exert the effect of carbon dot nanoparticles and even being toxic to plants.

[0014] The second object of the present invention is to provide a carbon dot nanoparticle prepared by the above preparation method.

[0015] Due to the rich carboxyl groups on its surface, carbon dots are electronegative. After binding with PEI through an amide reaction, the surface charge can be reduced, thereby reducing the toxicity of PEI. Therefore, the carbon dot nanoparticles not only have the ability of PEI to bind and protect DNA but also have excellent biocompatibility of carbon dots.

[0016] The third object of the present invention is to provide an exogenous gene delivery system based on the above carbon dot nanoparticles. The exogenous gene delivery system includes the carbon dot nanoparticles and a plasmid carrying an exogenous gene.

[0017] Furthermore, the vector includes a plasmid.

[0018] Furthermore, the concentration of the plasmid is 5 - 10 ng / μL.

[0019] The fourth object of the present invention is to provide an application of the above exogenous gene delivery system in the preparation of transgenic plants.

[0020] Furthermore, the application is to soak or coat the plant organs with the exogenous gene delivery system.

[0021] Furthermore, the plant organs include roots, stems, leaves, flowers, fruits, and seeds.

[0022] Advantages of the present invention:

[0023] In the present invention, an electrolyte solution is obtained by electrolyzing a graphite rod with a voltage of 15V - 20V, and the electrolyte solution is filtered and dialyzed to obtain a carbon dot solution; an activator is added to the carbon dot solution and reacted in an ice - water bath, then PEI is added and reacted in an ice - water bath, and carbon dot nanoparticles are obtained after dialysis. Mixing the carbon dot nanoparticles with a vector carrying an exogenous gene can obtain the exogenous gene delivery system of the present invention. The carbon dot nanoparticles of the present invention can achieve transgenic modification of living plants by coating or soaking methods, and the recipients are not restricted; moreover, the carbon dot nanoparticles of the present invention are tubular and have a diameter of about 50 nm, so they can enter the seed embryo cells and carry the exogenous gene when the plant grows, enabling transgenic modification of plants from the root source. Description of the Drawings

[0024] To make the content of the present invention easier to be clearly understood, the following further describes the present invention in detail according to specific embodiments of the present invention in combination with the accompanying drawings, wherein

[0025] Figure 1 is the scanning electron microscope image of carbon dots;

[0026] Figure 2 is the XPS result image of carbon dots;

[0027] Figure 3 is the scanning electron microscope image of Smart-CD carbon dot nanoparticles;

[0028] Figure 4 is the XPS result image of Smart-CD carbon dot nanoparticles;

[0029] Figure 5 is the experimental result image of Examples 2-4;

[0030] Figure 6 is the experimental result image of Example 5;

[0031] Figure 7 is the experimental result image of Example 6;

[0032] Figure 8 is the electron microscope scanning image of CDP in Comparative Example 1;

[0033] Figure 9 is the experimental result image of Comparative Example 1. Specific Embodiments

[0034] The following further describes the present invention in combination with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the examples given are not intended to limit the present invention.

[0035] Example 1: Preparation of Carbon Dot Nanoparticles (Smart-CD)

[0036] (1) Place two graphite rods parallel in ultrapure water, apply a DC voltage of 15v - 20v on both sides. After electrolyzing for several weeks, when one of the graphite rods is completely etched, centrifuge the solvent to obtain the supernatant, and then dialyze the supernatant with a 2500Da cellulose filter membrane against deionized water for 1 - 3 days to obtain a carbon dot solution.

[0037] (2) Add 30 - 50 mg of N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride to 5 - 15 mL of a carbon dot solution with a concentration of 1 mg / mL, react in an ice-water bath for 1.5 - 3 hours, then add 5 mL of a PEI aqueous solution with a concentration of 3 - 7 mg / mL, react in an ice-water bath for 3 - 5 hours, and then dialyze in ultrapure deionized water using a 500 - 1000 KD cellulose semipermeable membrane for 72 hours to finally obtain a Smart-CD solution.

[0038] Example 2: Delivery of foreign genes into corn seeds by Smart-CD

[0039] (1) Prepare a Smart-CD transgenic mixture: Thoroughly mix the solutions shown in Table 1 and incubate at 37 °C for 30 minutes (morpholineethanesulfonic acid is used as a buffer and glycerol is used to maintain the osmotic pressure of plant cells).

[0040] Table 1 Composition ratio of the Smart-CD transgenic mixture in Example 2

[0041] Smart-CD solution 80 μL Plasmid containing green fluorescent protein (GFP) gene (5 mg / mL) 60 μL 10% glycerol 200 μL 0.02 M morpholineethanesulfonic acid (MES), pH 5.8 200 μL <![CDATA[ddH2O]]> 3460 μL Total 4000 μL

[0042] (2) Soak the newly germinated corn seeds in the above mixture. After soaking for 48 h, take out the seeds and let them develop normally. After they grow roots, transplant them into the soil. Select some root tips and leaves and observe them with the aid of a fluorescence microscope. As Figure 5 shown in a, obvious green fluorescence can be observed in both the root tips and leaves developed from the seeds soaked in the mixture. Select the leaves at the 6th day and the 8th day of development. After freezing in liquid nitrogen and then breaking the cells, extract proteins with RIPA and perform western blotting detection. As Figure 5 shown in b, specific GFP protein bands can be detected in the leaves developed from the seeds soaked in the mixture. All these indicate that Smart-CD has successfully delivered plasmid DNA into cells and successfully expressed it.

[0043] Example 3: Delivery of foreign genes into tobacco leaves by Smart-CD

[0044] (1) Prepare a Smart-CD transgenic mixture: Thoroughly mix the solutions shown in Table 2 and incubate at 37 °C for 30 minutes.

[0045] Table 2 Composition ratio of the Smart-CD transgenic mixture in Example 3

[0046]

[0047]

[0048] (2) Immerse tobacco seeds in the above solution for 48 hours until the seeds germinate. Sow the seeds on 1 / 2 MS medium. After the cotyledons are fully extended, transplant them into the soil and continue to grow in a light incubator for 30 days. Select some leaves and use a fluorescence microscope to detect the GFP expression. As Figure 5 shown in c, obvious green fluorescence can be seen in the leaves under the microscope, indicating that Smart-CD successfully delivered plasmid DNA into the cells and was successfully expressed.

[0049] Example 4: Exogenous gene delivery to rice seeds by Smart-CD

[0050] (1) Prepare the Smart-CD transgenic mixture: Thoroughly mix the solution shown in Table 3 and incubate at 37 °C for 30 minutes.

[0051] Table 3 Composition ratio of the Smart-CD transgenic mixture in Example 4

[0052] Smart-CD solution 20 μL Plasmid containing hygromycin (HYG) gene (5 mg / mL) 15 μL 10% glycerol 50 μL 0.02 M morpholineethanesulfonic acid (MES), pH 5.8 50 μL <![CDATA[ddH2O]]> 865 μL Total 1000 μL

[0053] (2) After shelling the rice seeds, soak them until the seeds show white tips. Then soak the seeds with white tips in the above mixture for 48 h. After taking them out, cover them with gauze for germination until they develop normally. Cut the leaves, grind them in liquid nitrogen, and use the SDS method to extract genomic DNA for PCR detection of the integration of the HYG gene. The results are as Figure 5 shown in d (PCA is the positive control, CD-plasmid 1 and CD-plasmid 2 represent two individual plants). A single specific band of HYG can be detected in the leaves developed after soaking with the mixture. It indicates that Smart-CD successfully delivered the plasmid into the plant genome.

[0054] Example 5: Exogenous gene delivery to soybean and sesbania seeds by Smart-CD

[0055] (1) Prepare the Smart-CD transgenic mixture: Thoroughly mix the solution shown in Table 4 and incubate at 37 °C for 30 minutes.

[0056] Table 4 Composition ratio of the Smart-CD transgenic mixture in Example 5

[0057]

[0058]

[0059] (2) Soybean or Sesbania seeds were first soaked in sterile water for 24 hours. After sucking dry the surface moisture, the seeds were soaked in the above-mentioned mixture for 48 - 72 hours, and then placed in a moist petri dish for germination. When the length of the germ reached 1 cm, they were transferred to pots for cultivation. After the true leaves grew out, the leaves were cut to extract DNA, and the GFP gene was detected by PCR as Figure 6 shown in a. Positive bands were amplified in both soybeans and Sesbania in the transgenic mixture treatment group, indicating that the foreign gene had been delivered into soybean and Sesbania plants. Further, western blotting was performed using a GFP-specific antibody, and the results were as Figure 6 shown in b (soybean), 6C (Sesbania). The treatment group highly expressed the GFP protein effectively and highly expressed it at all growth stages, suggesting that the foreign DNA had been stably integrated into plant cells and highly expressed.

[0060] Example 6: Delivery of foreign genes into soybean, Agropyron cristatum, Medicago sativa, cucumber, rapeseed, radish, eggplant, and potato seeds using Smart-CD

[0061] (1) Preparation of Smart-CD transgenic mixture: The solutions shown in Table 5 were fully mixed and incubated at 37 °C for 30 minutes.

[0062] Table 5 Composition ratio of Smart-CD transgenic mixture in Example 6

[0063] Smart-CD solution 20 μL Plasmid containing green fluorescent protein (GFP) gene (5 mg / mL) 15 μL 10% glycerol 50 μL 0.02 M morpholineethanesulfonic acid (MES), pH 5.8 50 μL <![CDATA[ddH2O]]> 865 μL Total 1000 μL

[0064] (2) Seeds of soybean, Agropyron cristatum, Medicago sativa, cucumber, rapeseed, radish, eggplant, potato, etc. were first soaked in sterile water for 24 hours. After sucking dry the surface moisture, the seeds were soaked in the above-mentioned mixture for 48 - 72 hours, and then placed in a moist petri dish for germination. When the length of the germ reached 1 cm, they were transferred to a hydroponic box for cultivation, and the culture solution component was 1 / 2MS. After 7 - 20 days, when the cotyledons were fully unfolded, the luciferase substrate (purchased from Jiangsu Beyotime Biotechnology Co., Ltd.) was applied to the surface of the seedlings, and photos were taken one hour later. As Figure 7 shown, obvious fluorescence signals were detected on the whole plant of each species, indicating that the foreign gene had been stably transferred into the plant and highly expressed.

[0065] Comparative Example 1

[0066] A foreign gene delivery system based on CDP was prepared and used for delivering foreign genes into seeds.

[0067] (1) Two graphite rods were placed parallel in ultrapure water, and a DC voltage of 25v - 30v was applied on both sides. After electrolysis for several weeks, when one of the graphite rods was completely corroded, the solvent was centrifuged to obtain the supernatant, and then the supernatant was dialyzed against deionized water using a 2500Da cellulose filter membrane for 1 - 3 days to obtain a carbon dot solution;

[0068] (2) Add 30 - 50 mg of N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride to 5 - 15 mL of a carbon dot solution with a concentration of 1 mg / mL, react for 1.5 - 3 hours, then add 5 mL of a PEI aqueous solution with a concentration of 5 mg / mL. After reacting for 3 - 5 hours, use a cellulose semi-permeable membrane to dialyze in ultrapure deionized water to remove the residual PEI, and finally obtain a CDP solution; among them, the molecular weight of PEI in CDP is 10 KD, and the TEM scan image of CDP is as Figure 8 shown.

[0069] Soak rice seeds with the CDP transgenic mixed reagent and the Smart-CD transgenic mixed reagent shown in Table 6 respectively. After seven days of germination, collect the leaves. After extracting the leaf DNA, perform PCR detection on the hygromycin gene fragment on the plasmid. The results show that the transformation efficiency of Smart-CD is 100%, while CDP cannot transform the exogenous plasmid through seeds. In addition, soak rice seeds with the CDP transgenic mixed reagent with a GFP plasmid. The western blotting detection results of the proteins extracted from the germinated seedlings also show that the seed transformation of CDP is not successful (as Figure 9 shown).

[0070] Table 6 The ratio of the Smart-CD / CDP transgenic mixture in Comparative Example 1

[0071] Smart-CD solution / CDP solution 20 μL Plasmid containing hygromycin (HYG) gene (5 mg / mL) 15 μL 10% glycerol 50 μL 0.02 M morpholineethanesulfonic acid (MES), pH 5.8 50 μL <![CDATA[ddH2O]]> 865 μL Total 1000 μL

[0072] Obviously, the above embodiments are merely examples given for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or alterations can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or alterations derived therefrom are still within the protection scope of the present invention.

Claims

1. A preparation method of carbon dot nanoparticles, characterized in that, It includes the following steps: S1. Apply a voltage of 15V - 20V to a graphite rod for electrolysis to obtain an electrolyte solution, and dialyze the electrolyte solution to obtain a carbon dot solution; S2. Add an activator to the carbon dot solution and conduct a first ice-water bath reaction, add polyethyleneimine and conduct a second ice-water bath reaction, and obtain the carbon dot nanoparticles after dialysis, wherein the molecular weight of the polyethyleneimine is 600D - 1000D.

2. The preparation method according to claim 1, characterized in that, The activator includes N-ethyl-N-(3-dimethylaminopropyl) carbodiimide hydrochloride.

3. The preparation method according to claim 1, characterized in that, The mass ratio of the carbon dot solution to the polyethyleneimine is (1 - 3):(3 - 7).

4. A carbon dot nanoparticle prepared by the preparation method according to any one of claims 1 - 3.

5. An exogenous gene delivery system based on the carbon dot nanoparticles described in claim 4, characterized in that, The exogenous gene delivery system is prepared by mixing the carbon dot nanoparticles and a vector carrying an exogenous gene.

6. The exogenous gene delivery system according to claim 5, wherein The vector includes a plasmid.

7. Use of the exogenous gene delivery system according to any one of claims 5 - 6 in the preparation of transgenic plants.

8. The application according to claim 7, characterized in that, The use is to soak or coat the plant organs with the exogenous gene delivery system.

9. The application according to claim 8, wherein The plant organs include roots, stems, leaves, flowers, fruits and seeds.

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