Carbon quantum dot and small nucleic acid nanocomposite and application thereof

Abstract

The invention provides a carbon quantum dot and small nucleic acid nanocomposite, which is formed by wrapping the outer layer of a positively charged carbon quantum dot with negatively charged siRNA through electrostatic interaction, the carbon quantum dot is a histidine-doped chitosan quantum dot or arginine-doped chitosan quantum dot, chitosan is dissolved in an acetic acid solution, and the small nucleic acid and the chitosan are mixed to form the nanocomposite. The preparation method comprises the following steps: continuously adding amino acid, stirring until the amino acid is dissolved, treating for 6-8 hours at the temperature of 100-180 DEG C to obtain a mixture, cooling the mixture to room temperature, centrifuging, passing through a filter membrane with the pore diameter of 0.22-0.45 mu m, dialyzing the obtained filtrate in ultrapure water for 8-12 hours by using a dialysis bag with the MWCO value of 800-1000Da, freeze-drying the residual dialysate in the dialysis bag to obtain carbon quantum dots, mixing the carbon quantum dots with free siRNA, and performing freeze-drying on the carbon quantum dots to obtain the siRNA. Incubating at room temperature for 30 minutes to 1 hour to obtain a nano compound of the carbon quantum dots and the small nucleic acid; according to the invention, the drug stability and targeting effect can be improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical materials, and particularly relates to a small nucleic acid delivery system of carbon quantum dots and application thereof in anti-plant virus. BACKGROUND

[0002] Plant viruses can infect a variety of crops, resulting in reduced crop yield and economic losses, and pose a persistent and serious threat to global agricultural production. According to statistics, the annual crop loss caused by plant viruses is as high as tens of billions of dollars. At present, the prevention and control of plant virus diseases mainly relies on agricultural management measures, such as strengthening fertilizer and water management, increasing the application of organic and biological bacterial fertilizers to improve the soil environment, and improving the disease resistance of crops. In addition, chemical control of the transmission medium is also a common means, such as using pesticides to control the population of aphids, whiteflies and other pests to block the transmission route of the virus. However, the overuse of chemical pesticides not only causes environmental pollution and ecological imbalance, but also may lead to the development of pesticide resistance in pests, further exacerbating the difficulty of preventing and controlling the virus disease.

[0003] Traditional plant transformation methods mainly include Agrobacterium-mediated gene transformation, particle bombardment, electroporation, PEG-mediated delivery and viral vector delivery, etc. Agrobacterium-mediated transformation is one of the most widely used methods for plant nucleic acid delivery. Agrobacterium mainly enters plant cells through infiltration or co-culture with plant bodies. The advantages of Agrobacterium-mediated gene transformation are simple operation, low cost and high reproducibility; however, the application efficiency of Agrobacterium-mediated gene transformation in monocotyledonous plants (such as wheat and rice) is low, and the random integration of exogenous DNA into the plant genome by Agrobacterium leads to uncontrollability. Particle bombardment, also known as gene gun method, particle bombardment as a direct delivery method uses high-speed microprojectiles coated with exogenous DNA to accelerate gold or tungsten through plant cell walls to introduce into plant tissues or cells. The main advantages of particle bombardment method include wide application range, ability to transform various tissue or cell types, and ability to deliver RNA or protein, etc. However, particle bombardment method also has some limitations, including the need for expensive equipment and consumables, complex integration mode of exogenous genes, etc. Electroporation is a transformation method based on the action of electric field, which induces the formation of transient pores on the plasma membrane of prokaryotic and eukaryotic cells, thereby realizing the transmembrane transport of micromolecules or macromolecules in and out of cells. Electroporation has shown significant advantages in plant cell gene delivery: fast operation, low cost, high efficiency and suitable for single cell or cell cluster transformation. However, electroporation technology also has some limitations: the range of recipient species is narrow, especially for plant cells with thick cell walls, the transformation efficiency is significantly reduced, and strong electric field pulses may cause degradation of naked DNA. These limiting factors to some extent restrict the application of electroporation technology in plant genetic engineering.

[0004] With the development of nanomaterial-mediated exogenous gene delivery technology in recent years, it is expected to change the difficulties faced by traditional plant nucleic acid delivery. Nanomaterials have been widely used as gene delivery carriers in animal research, but their application in plants is still in the initial exploration stage. Studies have shown that the pore size of plant cell wall has a certain size limit, usually less than 20 nm in at least one dimension. Carbon quantum dots (CDs) are a kind of zero-dimensional carbon material with a diameter of less than 10 nm, which are widely used in the fields of biomedicine, photocatalysis and other fields due to their excellent photoluminescence properties. The stability and fluorescence properties of CDs significantly enhance their uptake, transfer and accumulation ability in plant cells. The development of CDs-based small nucleic acid plant delivery system not only can broaden the application direction of CDs, but also bring new strategies for new plant nucleic acid delivery, optimize plant genetic engineering technology means, promote the application of plant siRNA biological pesticides, and have important significance for the efficient and sustainable development of agriculture. SUMMARY

[0005] Carbon quantum dots have wide application prospects in many fields, including biomedical, chemical analysis, etc. Currently, existing scientific research mainly focuses on the interaction between carbon nanomaterials and mammalian cells, and the research on using them as carriers to deliver exogenous genes to plant cells is still in the exploratory stage. If carbon dot materials can be effectively used as carriers to successfully deliver small nucleic acid drugs to plant cells, it will open up a new application approach for plant gene editing and disease treatment.

[0006] The present application aims to provide a carbon quantum dot small nucleic acid delivery system and its preparation method and application in resisting plant viruses. The negatively charged siRNA is adsorbed on the surface of the carbon quantum dot to form a nanocomposite through electrostatic adsorption, which not only improves the stability of siRNA in the plant body, but also realizes the precise delivery of siRNA, providing strong support for plant gene therapy research.

[0007] The technical solutions of the present application are as follows: The present application provides a carbon quantum dot and small nucleic acid nanocomposite, which is formed by wrapping negatively charged siRNA to the outer layer of positively charged carbon quantum dots through electrostatic interaction. The carbon quantum dots are histidine-doped chitosan quantum dots or arginine-doped chitosan quantum dots, and the preparation method is as follows: (1) chitosan (preferably chitosan with a degree of deacetylation greater than 95%) is dissolved in acetic acid solution, amino acid is continuously added and stirred until dissolved, and the mixture is treated at 100-180°C for 6-8h, the mixture is cooled to room temperature, centrifuged, and then filtered through a filter membrane with a pore size of 0.22-0.45 μm, the obtained filtrate is dialyzed in ultra-pure water for 8-12h using a dialysis bag with a MWCO value of 800-1000Da, the remaining dialysis solution in the dialysis bag is freeze-dried, and carbon quantum dots are obtained, the amino acid is histidine or arginine, and the mass ratio of chitosan, acetic acid in acetic acid solution, and amino acid is 1-10:5-10:1 (4°C storage); (2) the carbon quantum dots of step (1) are mixed with free siRNA at a mass ratio of 50-300:1 (preferably 198:1), incubated at room temperature for 30 min-1 h, (siRNA is wrapped outside the carbon quantum dots by electrostatic interaction), and the nanocomposite of carbon quantum dots and small nucleic acids is obtained.

[0008] Further, in step (1), chitosan is dissolved in acetic acid solution, amino acid is continuously added and stirred until dissolved, and the mixture is treated at 180°C for 8h to obtain the mixture.

[0009] Further, in step (1), the MWCO value of the dialysis bag is 1000Da, and the dialysis time is 8h.

[0010] Further, the amino acid of step (1) is histidine.

[0011] Further, the mass ratio of chitosan, acetic acid in acetic acid solution, and amino acid of step (1) is 10:20:3.

[0012] Further, in step (2), the carbon quantum dots are mixed with free siRNA at a mass ratio of 198:1.

[0013] Further, the siRNA of step (2) is selected from the group consisting of the following double-stranded RNAs: (a) a double-stranded RNA with a sense strand of SEQ ID NO. 7 and an antisense strand of SEQ ID NO. 8; (b) a double-stranded RNA with a sense strand of SEQ ID NO. 9 and an antisense strand of SEQ ID NO. 10; (c) a double-stranded RNA with a sense strand of SEQ ID NO. 11 and an antisense strand of SEQ ID NO. 12; (d) a double-stranded RNA with a sense strand of SEQ ID NO. 21 and an antisense strand of SEQ ID NO. 22.

[0014] Further, the preparation method is as follows: (1) Dissolve chitosan in 1% acetic acid solution, continue to add histidine and stir until dissolved, treat at 180℃ for 8h to obtain a mixture, cool the mixture to room temperature, centrifuge and pass through a filter membrane with a pore size of 0.22μm, dialyze the obtained filtrate in an ultra-pure water dialysis bag with a MWCO value of 1 kDa for 8h, freeze-dry the remaining dialysis solution in the dialysis bag to obtain carbon quantum dots, and the mass ratio of the chitosan, acetic acid in the acetic acid solution and histidine is 10:20:3; (2) Mix the carbon quantum dots obtained in step (1) with free siRNA at a mass ratio of 198:1, incubate at room temperature for 30min, and the siRNA is wrapped outside the carbon quantum dots by electrostatic interaction to obtain the nanocomposite of carbon quantum dots and small nucleic acids.

[0015] The application also provides a nanocomposite of carbon quantum dots and small nucleic acids for use in plant gene silencing.

[0016] Further, the application method is as follows: Inject the nanocomposite of carbon quantum dots and small nucleic acids into the cytoplasm of plant cells for target gene silencing.

[0017] The small nucleic acid plant delivery system based on CDs can be applied to plant gene delivery, and is specifically used for plant gene transformation, gene editing, etc.

[0018] In addition, the small nucleic acid plant delivery system based on CDs can also be applied to plant gene silencing, and is specifically used for exogenous GFP gene silencing and TRV virus RdRP gene silencing, or for endogenous PDS gene and CHLH gene silencing.

[0019] The technical principle of the application includes: Carbon quantum dots (CDs) are considered as a kind of quantum dot "green alternative" with non-toxic, low cost and good biocompatibility. Through hydrothermal reaction, CDs are synthesized by taking chitosan as a carbon source, and amino acids (histidine, arginine and aspartic acid) are introduced on the surface of the CDs, so that the CDs have pH response characteristics. By electrostatic adsorption, the negatively charged small interfering RNA (siRNA) is combined to achieve the effect of extracellular binding and intracellular release. In addition, the diameter of CDs is less than 10 nanometers, and this size helps to efficiently internalize into intact plant cells, realizing precise delivery of nucleic acids.

[0020] Small interfering RNA (siRNA) controls plant diseases and insect pests by specifically inhibiting the expression of genes related to pathogens and insect pests. This technology uses siRNA molecules to mediate gene silencing, providing an efficient, sustainable and environmentally friendly solution. Compared with traditional pesticides, siRNA interference technology significantly reduces the dependence on chemical pesticides, providing a more sustainable solution for agricultural production. By designing and synthesizing siRNA molecules matching the gene sequences related to pathogens and insect pests, they can be introduced into plant cells to specifically inhibit the expression of target genes. This inhibition can block the key steps of pathogen infection and insect pest development, effectively controlling plant diseases and insect pests.

[0021] Although siRNA interference technology has broad application prospects in plant disease and insect pest control, due to the degradability of naked siRNA, low transfection efficiency and non-specific biological distribution, its therapeutic effect is not ideal. Therefore, it is urgent to develop new carriers to improve the delivery efficiency of siRNA in plant cells. CDs with positive charges on the surface of PEI as a passivation agent can adsorb negatively charged siRNA through electrostatic interaction and effectively protect it from enzymatic degradation in vivo, thereby achieving efficient loading and precise release of siRNA into intact plant cells.

[0022] The beneficial effects of the present application are: 1. CDs are formed by carbonization of chitosan and amino acids by hydrothermal method, which exist stably in aqueous solution and remain stable after long-term storage.

[0023] 2. The nucleic acid functionalized carbon quantum dot nucleic acid carrier system of the application can load DNA, RNA and other nucleic acid substances. It can prevent nucleic acid substances from being degraded by nucleases, thereby maintaining their biological activity. This nucleic acid carrier is relatively stable and has good biological safety.

[0024] 3. The size of carbon quantum dot material is 1-10 nm nanoparticles, which is smaller than the exclusion range of plant cell wall. As a "green" substitute for quantum dots, carbon quantum dots have small particle size, non-toxicity, low cost and good biocompatibility, and play a role in plant gene delivery.

[0025] 4. Gene therapy drugs have advantages such as high specificity, rich target points and long-acting, but the instability and negative charge of nucleic acids make it difficult to enter cells. To address the above, loading gene therapy drugs with CDs can improve drug stability and targeting effect. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The synthesis route map of CDs@siRNA nanogene delivery system in the present application.

[0027] Figure 2 Particle size, potential map of the carbon dots related in embodiment 1 of the present application; (A) potential distribution map of the carbon dots (carbon quantum dots); (B) particle size distribution map of the carbon dots.

[0028] Figure 3 UV-visible absorption spectrum map of the carbon dots related in embodiment 1 of the present application.

[0029] Figure 4 Fluorescence, infrared spectrum map of the carbon dots related in embodiment 1 of the present application; (A) fluorescence spectrum map of the carbon dots; (B) infrared spectrum map of the carbon dots and chitosan solution.

[0030] Figure 5 Fluorescence spectrum map of the carbon dots related in embodiment 2 of the present application before and after loading siRNA.

[0031] Figure 6 siRNA loading condition and pH response condition of the carbon dots related in embodiment 2 of the present application; (A) siRNA release efficiency map of the carbon dots and siRNA under different pH; (B) loading efficiency map of the carbon dots and siRNA under pH 5.5.

[0032] Figure 7 Agarose gel electrophoresis map of siRNA protection condition of the carbon dots related in embodiment 2 of the present application.

[0033] Figure 8 Uptake condition of Cy3-siRNA in trans-GFP type tobacco leaves mediated by the carbon dots related in embodiment 3 of the present application.

[0034] Figure 9 First day leaf treated by the carbon dots related in embodiment 4 of the present application (SDS solution group as positive control); Figure 10 qPCR analysis result map of plant stress gene NbrbohB in embodiment 4 of the present application.

[0035] Figure 11 qPCR analysis result map of plant exogenous gene GFP mRNA level in embodiment 5 of the present application.

[0036] Figure 12 Representative CLSM image of GFP transgenic tobacco leaves treated by the carbon dots related in embodiment 5 of the present application after 3 days.

[0037] Figure 13 GFP protein expression condition of the GFP transgenic tobacco leaves treated by the carbon dots related in embodiment 5 of the present application after 3 days.

[0038] Figure 14 qPCR analysis result map of plant endogenous gene PDS gene mRNA level in embodiment 5 of the present application.

[0039] Figure 15 Figure of qPCR analysis results of CHLH gene mRNA level in plants of embodiment 5 of the present application.

[0040] Figure 16 Figure of leaf phenotype after 10 days of treatment with the relevant carbon dots in embodiment 5 of the present application.

[0041] Figure 17 Figure of qPCR analysis results of TRV virus RdRp gene mRNA level in embodiment 6 of the present application

[0042] Figure 18 Representative CLSM image of wild type tobacco leaves infected with TRV virus after 3 days of treatment with the relevant carbon dots in embodiment 6 of the present application.

[0043] Figure 19 TRV-GFP protein expression after 3 days of treatment with the relevant carbon dots in embodiment 6 of the present application. DETAILED DESCRIPTION

[0044] The above content of the present application is further described in detail through the specific embodiments of the examples. However, it should not be understood that the scope of the above subject matter of the present application is limited to the following examples. Any modifications made without departing from the spirit and principles of the present application, and equivalent replacements or improvements made according to the ordinary technical knowledge and common means in the art, should be included in the protection scope of the present application.

[0045] Example 1: Preparation and characterization of CDs@siRNA nanogene delivery system

[0046] p)}and stirred until dissolved with a magnetic stirrer; 1) Four kinds of carbon dots (carbon quantum dots) are designed in the present application: CD, His-CD, Arg-CD, and Asp-CD, all of which are prepared by hydrothermal method. The specific steps are as follows: 0.5 g of chitosan powder is weighed using an electronic balance and dissolved in 50 mL of 2% acetic acid solution, and stirred for 30 min using a magnetic stirrer (the stirring temperature is set to 50 ℃) to obtain a chitosan solution. Then, 0.15 g of histidine, 0.15 g of arginine, and 0.15 g of aspartic acid are weighed using an electronic balance and added to three portions of the chitosan solution, respectively, and continue to stir for 30 min to obtain three mixed solutions.

[0047] 2) Take 20 ml of each of the three mixed solutions obtained after stirring in step (1) and one untreated chitosan solution and place them in a polytetrafluoroethylene liner, and heat in an oven at 180 ℃ for 8 h; 3) After the mixture is cooled to room temperature, centrifugation (5000g for 15 min) is performed and then the filtrate is filtered through a 0.22 μm filter membrane; 4) The filtrate is dialyzed in an ultra-pure water dialysis bag with a MWCO value of 1 kDa for 8 h; 5) The remaining dialysate is subjected to lyophilization, and then the lyophilized sample is collected for weighing and quantification. Subsequently, ultra-pure water is added to resuspend the sample into a solution state of 2.2 mg / ml, to obtain Arg-CD (arginine-doped chitosan quantum dots), His-CD (histidine-doped chitosan quantum dots), Asp-CD (aspartic acid-doped chitosan carbon quantum dots) and CD (chitosan carbon quantum dots) solutions (2.2 mg / ml), respectively. The above four CDs (carbon quantum dots) solutions (2.2 mg / ml) can also be resuspended to prepare other desired concentrations, and are stored at 4°C for subsequent experimental operations.

[0048] 6) 90 μL (i.e. 198 μg CDs) of the CDs solution obtained in step 5) is mixed with a free siRNA solution (10 μL of 8 μM, i.e. 1 μg siRNA, which is an NCRNA without silencing function, commercially available from Shanghai Jimabio Pharmaceutical Technology Co., Ltd., 1 OD 260 / tube, and the product is dried by vacuum centrifugation to obtain a dry powder. To prepare a 20 μM solution, 125 μl of DEPC water needs to be added to each tube, and the DEPC water is an attached product) at a mass ratio of 198:1, and incubated at room temperature for 30 min. The siRNA is wrapped outside the CDs by electrostatic interaction to obtain a carbon quantum dot and small nucleic acid nanocomposite, which is denoted as CDs@siRNA. A histidine-doped chitosan quantum dot and small nucleic acid nanocomposite is denoted as His-CD@siRNA, an arginine-doped chitosan quantum dot and small nucleic acid nanocomposite is denoted as Arg-CD@siRNA, a chitosan carbon quantum dot and small nucleic acid nanocomposite is denoted as CD@siRNA, and an aspartic acid-doped chitosan carbon quantum dot and small nucleic acid nanocomposite is denoted as Asp-CD@siRNA.

[0049] Particle size and potential detection

[0050] The potential and size of the carbon quantum dots and the nanocomposites obtained in the present embodiment are detected by Zeta and DLS, as shown in Table 1. Figure 2As shown in (A), the potentials of His-CD (histidine-doped chitosan quantum dots) before and after loading siRNA are 30 mv and 27 mv respectively; the potentials of Arg-CD (arginine-doped chitosan quantum dots) before and after loading siRNA are 42 mv and 37 mv respectively; the potentials of CD (chitosan carbon quantum dots) before and after loading siRNA are 19 mv and 14 mv respectively; the potentials of Asp-CD (aspartic acid-doped chitosan carbon quantum dots) before and after loading siRNA are 5 mv and 1.2 mv respectively; as shown in (B), the particle sizes of His-CD before and after loading siRNA are 7.5 nm and 11.7 nm respectively; the particle sizes of Arg-CD before and after loading siRNA are 11.7 nm and 15.6 nm respectively; the particle sizes of CD before and after loading siRNA are 18.1 nm and 24 nm respectively; the particle sizes of Asp-CD before and after loading siRNA are 105 nm and 122 nm respectively. Figure 2 As shown in (A), the potentials of His-CD (histidine-doped chitosan quantum dots) before and after loading siRNA are 30 mv and 27 mv respectively; the potentials of Arg-CD (arginine-doped chitosan quantum dots) before and after loading siRNA are 42 mv and 37 mv respectively; the potentials of CD (chitosan carbon quantum dots) before and after loading siRNA are 19 mv and 14 mv respectively; the potentials of Asp-CD (aspartic acid-doped chitosan carbon quantum dots) before and after loading siRNA are 5 mv and 1.2 mv respectively; as shown in (B), the particle sizes of His-CD before and after loading siRNA are 7.5 nm and 11.7 nm respectively; the particle sizes of Arg-CD before and after loading siRNA are 11.7 nm and 15.6 nm respectively; the particle sizes of CD before and after loading siRNA are 18.1 nm and 24 nm respectively; the particle sizes of Asp-CD before and after loading siRNA are 105 nm and 122 nm respectively.

[0051] UV-visible absorption spectrum analysis

[0052] As shown in (A), the potentials of His-CD (histidine-doped chitosan quantum dots) before and after loading siRNA are 30 mv and 27 mv respectively; the potentials of Arg-CD (arginine-doped chitosan quantum dots) before and after loading siRNA are 42 mv and 37 mv respectively; the potentials of CD (chitosan carbon quantum dots) before and after loading siRNA are 19 mv and 14 mv respectively; the potentials of Asp-CD (aspartic acid-doped chitosan carbon quantum dots) before and after loading siRNA are 5 mv and 1.2 mv respectively; as shown in (B), the particle sizes of His-CD before and after loading siRNA are 7.5 nm and 11.7 nm respectively; the particle sizes of Arg-CD before and after loading siRNA are 11.7 nm and 15.6 nm respectively; the particle sizes of CD before and after loading siRNA are 18.1 nm and 24 nm respectively; the particle sizes of Asp-CD before and after loading siRNA are 105 nm and 122 nm respectively. Figure 3

[0053] Fluorescence spectrum analysis

[0054] As shown in (A), the potentials of His-CD (histidine-doped chitosan quantum dots) before and after loading siRNA are 30 mv and 27 mv respectively; the potentials of Arg-CD (arginine-doped chitosan quantum dots) before and after loading siRNA are 42 mv and 37 mv respectively; the potentials of CD (chitosan carbon quantum dots) before and after loading siRNA are 19 mv and 14 mv respectively; the potentials of Asp-CD (aspartic acid-doped chitosan carbon quantum dots) before and after loading siRNA are 5 mv and 1.2 mv respectively; as shown in (B), the particle sizes of His-CD before and after loading siRNA are 7.5 nm and 11.7 nm respectively; the particle sizes of Arg-CD before and after loading siRNA are 11.7 nm and 15.6 nm respectively; the particle sizes of CD before and after loading siRNA are 18.1 nm and 24 nm respectively; the particle sizes of Asp-CD before and after loading siRNA are 105 nm and 122 nm respectively. Figure 4

[0055] Infrared spectrum analysis

[0056] As shown in (A), the potentials of His-CD (histidine-doped chitosan quantum dots) before and after loading siRNA are 30 mv and 27 mv respectively; the potentials of Arg-CD (arginine-doped chitosan quantum dots) before and after loading siRNA are 42 mv and 37 mv respectively; the potentials of CD (chitosan carbon quantum dots) before and after loading siRNA are 19 mv and 14 mv respectively; the potentials of Asp-CD (aspartic acid-doped chitosan carbon quantum dots) before and after loading siRNA are 5 mv and 1.2 mv respectively; as shown in (B), the particle sizes of His-CD before and after loading siRNA are 7.5 nm and 11.7 nm respectively; the particle sizes of Arg-CD before and after loading siRNA are 11.7 nm and 15.6 nm respectively; the particle sizes of CD before and after loading siRNA are 18.1 nm and 24 nm respectively; the particle sizes of Asp-CD before and after loading siRNA are 105 nm and 122 nm respectively. Figure 4

[0057] Table 1 Negative control NC RNA sequence

[0058] Example 2: Test of the loading capacity and protection capacity of CDs on siRNA ​​​​​

[0059] CDs siRNA loading capacity test

[0060] The fluorescence quenching of CDs before and after loading siRNA was determined by fluorescence spectrophotometer. The CDs prepared in Example 1 (90 μL of each, i.e. 198 μg of CDs) and 10 μL of 8 μM Cy3-siRNA (i.e. Cy3-labeled siRNA, 1 μg mg of Cy3-siRNA) were incubated at a mass ratio of 198:1 for 30 min. The fluorescence change before and after loading was determined under excitation at the optimal wavelength of Cy3-siRNA, 520 nm. As shown in Figure 5 , the fluorescence of His-CD after binding with siRNA was consistent with that of His-CD, and the signal peak of Cy3-siRNA could still be observed after Asp-CD combined with siRNA, indicating that fluorescence shielding phenomenon would occur after Cy3-siRNA combined with CDs. Therefore, the specific loading efficiency could be determined by observing the quenching of Cy3-siRNA. By changing the pH value of the CDs solution, five pH buffers of pH=4, pH=5.5, pH=6.5, pH=7.4 and pH=8 were selected, and the CDs solution and Cy3-labeled siRNA solution were prepared into nanoparticles according to the above method. The fluorescence intensity at 610 nm emission under excitation at 520 nm was determined and recorded on the microplate reader, and the release amount of siRNA was calculated according to the standard curve. As shown in Figure 6 , (A) The binding efficiency of His-CD, Arg-CD and CD with nucleic acid under acidic conditions (pH 4-6) was greater than 90%, and the binding efficiency of Asp-CD was about 80%. His-CD, Asp-CD and CD started to release under neutral / alkaline addition, while Arg-CD still had strong binding ability at pH 7. (B) is the loading effect of CDs with siRNA at different ratios at pH 5.5. The results showed that the synthesized CDs had pH response characteristics and good siRNA loading capacity.

[0061] CDs siRNA protection capacity test

[0062] The RNase A stability of CDs nanocomposites loaded with siRNA was evaluated. Naked siRNA and CDs@siRNA were incubated with RNase A solution at 37 ℃ for 30 min. As shown in Figure 7As shown, the rapid degradation of Naked siRNA was verified by 3% agarose gel electrophoresis at 150 V for 30 min. However, the RNase A stability of CDs@siRNA was significantly improved compared with Naked siRNA. The results showed that the synthesized CDs could effectively protect siRNA from RNase A degradation.

[0063] Table 2 Cy3-NCRNA sequence

[0064] Example 3: Internalization of CDs@siRNA nanocomplexes in mature plant leaves

[0065] To better locate the intracellular location of the nanocomplex, we used a 16C Nicotiana benthamiana plant transfected with GFP green fluorescence in the cytoplasm (a gift from Fujian Agricultural University). Cy3-labeled siRNA was used as a tracer molecule. A total volume of 100 μΐ, of nanocomplex solution (His-CD@Cy3-siRNA, Arg-CD@Cy3-siRNA, CD@Cy3-siRNA, and Asp-CD@Cy3-siRN) was prepared by incubating 90 μΐ, of 2.2 mg / mL carbon dot solution (CD, His-CD, Arg-CD, and Asp-CD) with 10 μΐ, of 2 μΜ Cy3-siRNA (the same sequence as in Example 2) solution at room temperature for 30 min. A total volume of 100 μΐ, of carbon dot solution alone was prepared by mixing 90 μΐ, of 2.2 mg / mL carbon dot solution with 10 μΐ, of water at room temperature for 30 min. A total volume of 100 μΐ, of free nucleic acid solution (Cy3-siRNA) was prepared by mixing 10 μΐ, of 2 μΜ Cy3-siRNA solution with 90 μΐ, of water at room temperature for 30 min. Each 100 μΐ, of Cy3-siRNA solution, His-CD@Cy3-siRNA, Arg-CD@Cy3-siRNA, CD@Cy3-siRNA, and Asp-CD@Cy3-siRNA was injected into the back of the intact plant leaves. After 24 hours of incubation, the infiltrated tobacco plant leaves were observed by Leica DMI-8 confocal microscopy to determine the intracellular location of the CDs@siRNA nanocomplex. As shown in Figure 6, the CDs@siRNA nanocomplexes were mainly located in the cytoplasm of the plant cells, and the CDs@siRNA nanocomplexes were not found in the nucleus of the plant cells. The results showed that the CDs@siRNA nanocomplexes were successfully internalized into the plant cells. Figure 8As shown, there is a high degree of colocalization between intracellular (cytoplasmic) GFP and Cy3 fluorescence in His-CD@Cy3-RNA infiltrated leaves, which confirms that CDs-CD@siRNA is effectively internalized into the cytoplasm of intact plant cells. In contrast, there is little colocalization between GFP and Cy3 fluorescence in leaves infiltrated with Arg-CD@Cy3-RNA, CD@Cy3-RNA, and Cy3-RNA and Asp-CD@Cy3-RNA, and Cy3 fluorescence in leaves infiltrated with Cy3-RNA and Asp-CD@Cy3-RNA mainly appears around stomata, indicating that free siRNA cannot be effectively internalized into intact plant cells. In plant cells, the vacuole is a typical organelle that plays multiple functions in plants. The vacuole is filled with water and occupies 80% of the cell volume, so the shape of the fluorescence localization follows the contour morphology of the vacuole.

[0066] The carbon dot nanomaterial-based plant nucleic acid delivery system shows higher delivery efficiency and stability. Carbon dot nanomaterials can effectively protect nucleic acids from the external environment and more accurately deliver nucleic acids to the interior of plant cells. Therefore, this system has potential advantages in plant gene transformation and gene editing applications.

[0067] Example 4: Safety assay of CDs in N. benthamiana

[0068] Observation of leaf phenotype 1 day after injection

[0069] 100ul of water, free siRNA solution (the same as in Example 1), His-CD solution (2.2 mg / ml), NC (Arg) solution (2 mg / ml), NC (CD) solution (2 mg / ml), His-CD@siRNA (the same as in Example 1), Arg-CD@siRNA (the same as in Example 1), CD@iRNA (the same as in Example 1) were injected into different areas of WT wild-type N. benthamiana leaves, respectively. The above nanocomposite solutions were prepared by mixing 90 μl of 2.2 mg / ml CDs solution with 10 μl of 8 μM siRNA solution, at this time the mass ratio of CDs to siRNA was 198:1. Since SDS can cause damage to the leaves, 1% SDS solution was injected into the leaves as a positive control. 100 μL of the above nanocomposite solutions and 1% SDS were injected into wild-type N. benthamiana leaves, respectively, and the phenotype 1 day after injection of different nanoparticles (carbon quantum dots) and the phenotype 3, 6, 24 h after treatment with 1% SDS solution were observed. As shown in Figure 9 As shown in FIG. 6, compared with the SDS solution positive control group, the CDs@siRNA complex solution group leaves had no obvious damage and were in good condition, while the leaves in the SDS solution group had obvious damage and were shriveled.

[0070] qPCR analysis of plant stress gene NbrbohB

[0071] The above water, free siRNA solution, His-CD, Arg-CD, CD, His-CD@siNC, Arg-CD@siNC, CD@siNC infiltrated leaf tissues for 3h and 1% SDS infiltrated leaf tissues for 3h, 6h, 24h were taken respectively, and total RNA was extracted by Trizol method, and cDNA was obtained by reverse transcription using Hiscript IV All-in-On Ultra RT SuperMix reagent, The obtained cDNA was quantitatively detected for stress gene NbrbohB expression of carbon dot samples by SYBR Premix Ex Taq II (Yeasen Biotech) kit, and data analysis was performed by real-time PCR system. The gene is commonly used in plant research to reflect biological or abiotic stress. The gene expression level was normalized to the value of the internal reference EFl, and the relative expression amount was determined by the relative CT method. As shown in Figure 10 compared with the water solution negative control group, the gene expression levels of CDs group and CDs@siRNA group had no obvious up-regulation change, while the gene expression level of 1% SDS-3h solution positive control group had obvious up-regulation change, and 1% SDS solution 6h appeared a downward trend until 24h consistent with the Control group (water solution), indicating that NbrbohB gene can be used as an indicator for early damage of plant leaves to judge the damage of leaves.

[0072] Table 3 NbrbohB gene primer sequence

[0073] Example 5: CDs@siRNA mediated gene silencing in intact plants

[0074] On the basis of verifying that siRNA can be successfully delivered to intact plant cells, the present study used this method to carry out gene silencing experiments. Based on the wide application of the GFP gene in transient expression analysis, the present study selected GFP as a reporter gene. The experiment designed siGFP specifically targeting GFP mRNA and non-specific siNC (the sequence is the same as the NCRNA without silencing function in Example 1) as a negative control. 90 μL of 2.2 mg / mL carbon dot solution (CD, His-CD, Arg-CD) was incubated with 10 μL of 8 μM siGFP solution at room temperature for 30 min to prepare a total volume of 100 μL of nanocomposite solution. 90 μL of 2.2 mg / mL carbon dot solution (CD, His-CD, Arg-CD) was incubated with 10 μL of 8 μM siNC solution at room temperature for 30 min to prepare a total volume of 100 μL of negative control nanocomposite solution. 90 μL of water was incubated with 10 μL of 8 μM siRNA at room temperature for 30 min to prepare a free siRNA solution. The experimental plants were 16C strain of Nicotiana benthamiana, and 100 μL of water, free siRNA, CDs@siNC (CD@siNC, His-CD@siNC, Arg-CD@siNC) and CDs@siGFP (CD@siGFP, His-CD@siGFP, Arg-CD@siGFP) (the siRNA used targets the GFP gene) were injected, respectively. Leaf samples were collected at 1, 3, 7 and 14 days after injection, total RNA was extracted and reverse transcription-quantitative PCR (RT-qPCR) analysis was performed. The SYBR®PremixEx TaqTM II (Yeasen Biotech) kit was used to quantitatively detect the silencing efficiency of the exogenous GFP gene, and the data was analyzed by a real-time fluorescence quantitative PCR system. The experimental results showed that compared with the free siRNA group and the CDs@siNC group, the CDs@siGFP treatment group significantly inhibited the expression level of GFP mRNA Figure 11 ), while the first two groups of treatments had no significant effect on the expression of GFP mRNA.

[0075] The treated GFP tobacco leaves were imaged by Leica DMI-8 confocal microscope, and the GFP green fluorescence intensity was observed, as shown in Figure 12 . All NC and free siRNA treated leaves had no obvious effect on GFP expression, while His-CD@siGFP, Arg-CD@siGFP treated leaves had significantly reduced GFP expression after 3 days of infiltration, and CD@siGFP treated leaves had weakly reduced GFP expression after 3 days of infiltration.

[0076] The expression of GFP protein in the whole leaf mediated by CDs@siRNA was evaluated by Western blot. On the 3rd day after injection, the leaves were collected, cut, ground, and total proteins of the plant leaves were extracted with plant RIPA lysis buffer. The protein concentration was calculated by BCA kit, the protein loading amount was determined, and then 8% SDS-PAGE electrophoresis was performed on the protein sample using running buffer. The separated proteins were transferred to PVDF membrane in turn, blocked with skimmed milk powder for 120 min, and then incubated with the primary antibody at 4°C overnight. After washing with TBST for 3 times, the secondary antibody labeled with HRP was incubated at room temperature for 2 h, and finally the luminescence solution was prepared by using the ECL luminescence detection kit, and the imaging was performed in the dark for 1 min. As shown in Figure 13 As shown in Fig. 9, only His-CD@siGFP and Arg-CD@siGFP caused a decrease in the level of GFP protein, which was consistent with the result of CLSM.

[0077] The above experimental results show that the CDs pathway for delivering siGFP can cause silencing of the exogenous gene GFP in the whole plant cell.

[0078] In order to study the application of gene silencing in plant endogenous genes, two siRNA sequences targeting the PDS gene and the CHLH gene of N. benthamiana were designed to verify the endogenous gene silencing. siPDS and siCHLH were combined with CD, His-CD, Arg-CD and - to form CDs@siRNA nanocomplexes, and siNC was combined with CD, His-CD, Arg-CD to form CDs@siNC nanocomplexes as negative control. 100 μL of water, free siRNA, CDs@siNC (CD@siNC, His-CD@siNC, Arg-CD@siNC) and CDs@siRNA were injected into wild-type plants of N. benthamiana, respectively. For PDS gene silencing detection, total RNA was extracted from the treated leaf tissues on the 1st, 3rd, 7th and 14th day after injection, and then RT-qPCR analysis was performed to detect PDS mRNA transcripts. The endogenous PDS gene silencing was quantitatively detected by SYBR®Premix Ex TaqTM II (Yeasen Biotech) kit, and the data analysis was performed by real-time PCR system. For CHLH gene silencing detection, total RNA was extracted from the treated leaf tissues on the 1st, 3rd, 7th and 14th day after injection, and then RT-qPCR analysis was performed to detect CHLH mRNA transcripts. The results are shown in Figure 14-15As shown in the table, the free siPDS group, the free siCHLH group and the CDs@siNC group had no significant effect on the expression levels of the endogenous PDS mRNA and the endogenous CHLH mRNA after treatment, however, the His-CD@siPDS group significantly reduced the expression level of the PDS mRNA 1 day and 3 days after treatment; the CDs@siPDS reduced the expression of the PDS mRNA on the third day after treatment; the Arg-CD@siRNA (PDS) group did not significantly reduce the expression level of the PDS mRNA 1-14 days after treatment. The CHLH mRNA expression reduced consistently on the third day after treatment with the His-CD@siCHLH and lasted for 7 days after treatment, while the Arg-CD@siCHLH and the CDs@siCHLH did not reduce the expression level of the CHLH mRNA 1-14 days after treatment.

[0079] It was found through research that the expression of the PDS gene and the CHLH gene is related to the synthesis of chlorophyll in the leaves of the plant, and if the expression of the PDS gene and the CHLH gene is down-regulated, the leaves will have yellow and white spots or patches, which indicates that the accumulation of chlorophyll is reduced. Therefore, the expression of the endogenous PDS gene and the CHLH gene can be determined by observing the phenotype of the leaves after injection of the nanocomposite. Figure 16 As shown in the table, the leaves near the injection site turned yellow 10 days after injection of the His-CD material group, and the expression of the PDS gene and the CHLH gene was down-regulated.

[0080] The above experimental results show that the free siPDS, the free siCHLH and the CDs@siNC (non-coding RNA sequence) are ineffective for silencing the endogenous PDS gene and the endogenous CHLH gene, while the CDs@siPDS nanocomposite described in the present application has a silencing effect on the endogenous PDS gene, resulting in a significant down-regulation of the expression level, and the CDs@siCHLH nanocomposite has a silencing effect on the endogenous CHLH gene, resulting in a significant down-regulation of the expression level. In addition, a significant yellowing and whitening phenomenon was observed on the leaves, which indicates that the CDs@siRNA (sequence specifically targeting a specific gene) nanocomposite can effectively deliver siRNA into the plant cells of N. benthamiana and exert an RNAi effect.

[0081] Table 4: Sequences of the GFP, PDS, CHLH, NC gene interference RNA and primers

[0082] Example 6: CDs@siRNA-mediated gene silencing in intact plants

[0083] The siRNA was confirmed to be delivered into plant cells and successfully achieved gene silencing. Similarly, we designed a siRNA sequence targeting the RNA-dependent RNA polymerase (RdRp) of TRV virus (siRdRp) and a non-targeting siNC (the sequence is the same as the NCRNA without silencing function in Example 1) as a negative control. 90 μL of 2.2 mg / mL His-CD solution was incubated with 10 μL of 7.5 μM (1 ug) siRdRp solution at room temperature for 30 min to prepare a total volume of 100 μL of nanocomposite solution. 90 μL of 2.2 mg / mL His-CD solution was incubated with 10 μL of 7.5 μM siNC solution at room temperature for 30 min to prepare a total volume of 100 μL of negative control nanocomposite solution. 90 μL of water was incubated with 10 μL of 7.5 μM siRdRp at room temperature for 30 min to prepare a free siRNA solution. In the experiment, 100 μL of water, free siRNA, His-CD@siNC and His-CD@siRdRp were injected into wild-type N. benthamiana plants. One day after nanoparticle injection, Agrobacterium carrying the TRV virus plasmid was injected into the leaf tissue that had been injected with nanoparticles for infection. After 1, 3, and 7 days of TRV virus injection, total RNA was extracted from the treated leaf tissue, and then reverse transcription-quantitative PCR (RT-qPCR) analysis was performed to detect RdRp mRNA transcripts. RdRp mRNA gene silencing was determined by SYBR®Premix Ex TaqTM II (Yeasen Biotech) kit, and data analysis was performed by real-time PCR system. The results are shown in Figure 17 As shown, there was no effect on the expression level of RdRp mRNA in the leaves treated with free siRNA and His-CD@siNC groups at 1-7 days, while the expression level of RdRp mRNA was significantly reduced in the leaves treated with His-CD@siRdRp group at 4 days.

[0084] Because of the GFP protein contained in the TRV virus, the treated tobacco leaves could be imaged by Leica DMI-8 confocal microscope to observe the GFP green fluorescence intensity, and the results are shown in Figure 18 As shown, all NC and free siRNA treated leaves had no obvious effect on GFP expression, while the GFP expression in the leaves treated with His-CD@siRdRp was significantly reduced after 3 days of TRV virus infiltration.

[0085] The TRV virus prevention mediated by His-CD@siRdRp in the whole leaf was evaluated by Western blot. The TRV virus was infected one day after the injection of His-CD@siRdRp, and the leaves were collected 3 days after the virus infection, cut, ground, and extracted with a plant RIPA lysis buffer to obtain total proteins of the plant leaves. The protein concentration was calculated by using a BCA kit to determine the protein loading amount, and then the protein samples were separated and detected by 8 % SDS-PAGE electrophoresis using a protein running buffer. The separated proteins were transferred to a PVDF membrane in turn, blocked with skimmed milk powder for 120 min, and then incubated with a primary antibody at 4 ℃ overnight. After washing with TBST for 3 times, a HRP-labeled secondary antibody was incubated at room temperature for 2 h, and finally a luminescence solution was prepared by using an ECL luminescence detection kit, and the imaging was performed by avoiding light for 1 min. As shown in Figure 19 As shown in FIG. 9, only His-CD@siRdRp can reduce the TRV-GFP protein level, which is consistent with the CLSM display result, indicating that the TRV virus is inhibited.

[0086] The above experimental results show that the CDs pathway delivery of siRdRp described in the present application can play an antiviral role by prevention.

[0087] Table 5: RdRp gene interference RNA and primer sequences

[0088] In summary, this new type of carbon dot nanomaterial has obvious advantages in the delivery of nucleic acids in the field of plants. Compared with the traditional Agrobacterium-mediated delivery method, the carbon dot nanomaterial can achieve precise delivery of nucleic acids, thereby reducing the side effects caused by non-specific delivery of exogenous DNA randomly integrated into the plant genome and reducing the occurrence of undesirable agronomic traits. In addition, this new type of carbon nanomaterial improves the transformation efficiency and silencing ability, further highlighting its superior applicability in the field of plant science.

Claims

1. A nanocomplex of carbon quantum dots and a small nucleic acid, characterized in that, The siRNA is wrapped to the outer layer of the positively charged carbon quantum dots by electrostatic interaction to form a negatively charged siRNA, and the carbon quantum dots are histidine-doped chitosan quantum dots or arginine-doped chitosan quantum dots, and the preparation method is as follows: (1) chitosan is dissolved in acetic acid solution, and amino acid is continuously added and stirred until dissolved, and the mixture is treated at 100-180℃ for 6-8h to obtain a mixture, the mixture is cooled to room temperature, centrifuged, and then filtered through a filter membrane with a pore size of 0.22-0.45 μm, the obtained filtrate is dialyzed in ultrapure water for 8-12h using a dialysis bag with a MWCO value of 800-1000Da, the remaining dialysate in the dialysis bag is freeze-dried to obtain carbon quantum dots, and the mass ratio of chitosan, acetic acid in acetic acid solution and amino acid is 1-10:5-10:1; (2) the carbon quantum dots obtained in step (1) are mixed with free siRNA at a mass ratio of 50-300:1, and incubated at room temperature for 30min-1h to obtain the nanocomposite of carbon quantum dots and small nucleic acids.

2. The carbon quantum dots and small nucleic acid nanocomplex of claim 1, wherein, In step (1), chitosan is dissolved in acetic acid solution, and amino acid is continuously added and stirred until dissolved, and the mixture is treated at 180℃ for 8h to obtain a mixture.

3. The carbon quantum dots and small nucleic acid nanocomplex of claim 1, wherein, In step (1), the MWCO value of the dialysis bag is 1000Da, and the dialysis time is 8h.

4. The carbon quantum dots and small nucleic acid nanocomplex of claim 1, wherein, In step (1), the amino acid is histidine.

5. The carbon quantum dots and small nucleic acid nanocomplex of claim 1, wherein, In step (1), the mass ratio of chitosan, acetic acid in acetic acid solution and amino acid is 10:20:

3.

6. The carbon quantum dots and small nucleic acid nanocomplex of claim 1, wherein, In step (2), the carbon quantum dots are mixed with free siRNA at a mass ratio of 198:

1.

7. The carbon quantum dots and small nucleic acid nanocomplex of claim 1, wherein, The siRNA in step (2) is selected from the group consisting of the following double-stranded RNAs: (a) double-stranded RNA with a sense strand of SEQ ID NO. 7 and an antisense strand of SEQ ID NO. 8; (b) double-stranded RNA with a sense strand of SEQ ID NO. 9 and an antisense strand of SEQ ID NO. 10; (c) double-stranded RNA with a sense strand of SEQ ID NO. 11 and an antisense strand of SEQ ID NO. 12; (d) double-stranded RNA with a sense strand of SEQ ID NO. 21 and an antisense strand of SEQ ID NO.

22.

8. The carbon quantum dots and small nucleic acid nanocomplex of claim 1, wherein, The preparation method is as follows: (1) chitosan is dissolved in 1% acetic acid solution, and histidine is continuously added and stirred until dissolved, and the mixture is treated at 180℃ for 8h to obtain a mixture, the mixture is cooled to room temperature, centrifuged, and then filtered through a filter membrane with a pore size of 0.22 μm, the obtained filtrate is dialyzed in ultrapure water for 8h using a dialysis bag with a MWCO value of 1 kDa, and the remaining dialysate in the dialysis bag is freeze-dried to obtain carbon quantum dots, and the mass ratio of chitosan, acetic acid in acetic acid solution and histidine is 10:20:3; (2) the carbon quantum dots obtained in step (1) are mixed with free siRNA at a mass ratio of 198:1, and incubated at room temperature for 30min, and siRNA is wrapped to the outer layer of the carbon quantum dots by electrostatic interaction to obtain the nanocomposite of carbon quantum dots and small nucleic acids.

9. The use of the nanocomplex of carbon quantum dots and small nucleic acid in claim 1 in plant gene silencing.

10. Use according to claim 9, wherein the compound is ###0002### The application method is as follows: The nanocomplex of carbon quantum dots and small nucleic acid is injected into the cytoplasm of plant cells for target gene silencing.

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