Polyamine carbon dots, preparation method thereof and application of polyamine carbon dots in improvement of corn resistance

Abstract

The invention discloses a polyamine carbon dot as well as a preparation method and application thereof in improvement of corn resistance, and belongs to the technical field of nano agricultural regulation and control. The polyamine carbon dots are subjected to hydrothermal synthesis, the size of the PEICDs is 2.3 nm, the quantum yield of the carbon dots can be increased, carbon dioxide in the atmosphere can be captured and converted into bicarbonate radicals, and it is verified through in-vitro experiments that the PEICDs can improve the carbon dioxide conversion product 3-phosphoglycerate in vitro; under the stress of drought and salt, the polyethyleneimine functionalized carbon dot solution is applied to the leaf surfaces of the crops, so that the growth and development of the crops can be improved by remarkably improving the net photosynthetic rate and the intercellular carbon dioxide concentration, and the adaptability of the crops to the environment is further improved.

Description

Polyamine carbon dots, their preparation methods, and their application in enhancing maize resistance Technical Field

[0001] This invention relates to polyamine carbon dots, their preparation methods, and their application in enhancing maize resistance, belonging to the field of nano-agricultural regulation technology. Background Technology

[0002] The prevalence of environmental stresses (drought and salinization) is intensifying due to frequent high temperatures, soil degradation, and climate change. In arid and semi-arid regions, evaporation and water consumption from plant roots lead to increased solute concentrations in the soil, resulting in rising soil salinity levels. Globally, approximately 12.6% (5.43 million square kilometers) of arid areas suffer from land degradation due to drought, and soil salinity in arable and irrigated agricultural land already accounts for 20% of the total area, with the proportion reaching as high as 33% in irrigated agricultural land. Therefore, drought and salinization constitute serious abiotic stress factors, hindering crop yields and threatening global food security.

[0003] Drought and salinization are among the most common problems globally, negatively impacting crop yields by reducing net photosynthetic efficiency through altered carbon allocation and metabolic processes in plants. On one hand, they severely affect the light-reaction phase, reducing photosynthetic pigment production and disrupting the photosynthetic electron chain, leading to the effects of drought and salinization stress. On the other hand, they reduce the activity of carbon-fixing enzymes in the Calvin cycle, inhibiting crop growth.

[0004] In recent years, many studies have reported on improving crop resistance through nanomaterials, among which carbon dots have been widely studied due to their high biocompatibility and environmental friendliness. However, most studies on the effects of carbon dots on photosynthesis focus on the light reaction stage, aiming to enhance photosynthesis by increasing light utilization and electron transport rates, thereby improving crop resistance, but the improvement effect remains limited. Summary of the Invention

[0005] [Technical Issue] Currently reported carbon dots in the field of nano-agriculture are usually based on improving photosynthesis by increasing light utilization and electron transport rates, thereby enhancing crop resistance. However, the effect of these carbon dots on improving resistance is still limited, restricting their widespread application. Based on this, the purpose of this invention is to provide a polyamine carbon dot (polyethyleneimine functionalized carbon dot), its preparation method, and its application in improving maize resistance. This polyethyleneimine functionalized carbon dot can effectively improve the photoelectric properties and carbon dioxide capture capacity of the carbon dot, enhance the crop's photosynthetic capacity and carbon fixation capacity, thereby improving crop photosynthesis, increasing crop yield under drought and salt stress, and enhancing the crop's environmental resistance.

[0006] [Technical Solution] To achieve the above objectives, the following technical solution is provided: The first objective of this invention is to provide a method for preparing polyethyleneimine functionalized carbon dots for enhancing crop resistance. The method includes the following steps: mixing polyethyleneimine, citric acid, and water evenly, then transferring the solution to a stainless steel autoclave for hydrothermal reaction, followed by dialysis and drying to obtain the final product.

[0007] In one embodiment, the homogenization is achieved by ultrasonic mixing in an ultrasonic bath.

[0008] In one embodiment, the ultrasound duration is 5-10 minutes.

[0009] In one embodiment, the mass ratio of polyethyleneimine to citric acid is 8 to 10:1.

[0010] In one embodiment, the mass ratio of polyethyleneimine, citric acid, and water is 8~10:1:2~5.

[0011] In one embodiment, the water is deionized water.

[0012] In one embodiment, the stainless steel autoclave is a stainless steel autoclave lined with Teflon 126.

[0013] In one embodiment, the hydrothermal reaction conditions are: temperature of 180~250℃ and time of 8~15h.

[0014] In one embodiment, the hydrothermal reaction is carried out under the following conditions: temperature 200°C and time 10 h.

[0015] In one embodiment, the dialysis bag used for the dialysis is a MW2000 dialysis bag.

[0016] In one embodiment, the dialysis time is 36-72 hours.

[0017] In one implementation, the dialysis time is 48 hours.

[0018] A second objective of this invention is to provide a polyethyleneimine functionalized carbon dot prepared by the method described above.

[0019] In one embodiment, the polyethyleneimine functionalized carbon dots (PEICDs) are spherical.

[0020] In one embodiment, the size of the polyethyleneimine functionalized carbon dots (PEICDs) is 2-3 nm.

[0021] In one embodiment, the polyethyleneimine functionalized carbon dots (PEICDs) have a size of 2.3 nm.

[0022] A third objective of this invention is to provide an application of the aforementioned polyethyleneimine functionalized carbon dots in the agricultural field.

[0023] A fourth objective of this invention is to provide an application of the above-described polyethyleneimine functionalized carbon dots in enhancing crop resistance.

[0024] In one embodiment, the resistance includes any one of drought stress, salt stress, freezing stress, and heat wave stress.

[0025] In one embodiment, the crops include wheat, soybeans, corn, rice, tomatoes, leafy greens, etc.

[0026] The fifth objective of this invention is to provide a method for improving photosynthesis in maize under stress, wherein the method involves applying a PEICDs solution to the leaves during the maize seedling stage.

[0027] In one implementation, the corn seedling stage is the stage when the corn reaches the "three leaves and one heart" stage.

[0028] In one embodiment, the foliar application refers to foliar spraying.

[0029] In one embodiment, the PEICDs solution is a suspension formed by dissolving PEICDs in water.

[0030] In one embodiment, the concentration of the PEICDs solution is 10~30 mg / L.

[0031] In one embodiment, the concentration of the PEICDs solution is 10 mg / L.

[0032] In one embodiment, the amount of PEICDs solution applied is 2-3 mL / plant. In another embodiment, the PEICDs solution is applied in 1-5 applications; preferably in 1-3 applications.

[0033] In one embodiment, the PEICDs solution is applied for 7 consecutive days, starting from the "three leaves and one heart" stage of the corn.

[0034] In one implementation, the stress refers to drought stress and / or salt stress.

[0035] The sixth objective of this invention is to provide a method for promoting maize growth under stress, wherein the method involves applying a PEICDs solution to the leaves during the maize seedling stage.

[0036] In one implementation, the corn seedling stage is the stage when the corn reaches the "three leaves and one heart" stage.

[0037] In one embodiment, the foliar application refers to foliar spraying.

[0038] In one embodiment, the PEICDs solution is a suspension formed by dissolving PEICDs in water.

[0039] In one embodiment, the concentration of the PEICDs solution is 10~30 mg / L.

[0040] In one embodiment, the concentration of the PEICDs solution is 10 mg / L.

[0041] In one embodiment, the amount of PEICDs solution applied is 2-3 mL / plant. In another embodiment, the PEICDs solution is applied in 1-5 applications; preferably in 1-3 applications.

[0042] In one embodiment, the PEICDs solution is applied for 7 consecutive days, starting from the "three leaves and one heart" stage of the corn.

[0043] In one implementation, the stress refers to drought stress and / or salt stress.

[0044] Beneficial effects: The polyethyleneimine functionalized carbon dots (PEICDs) of the present invention, wherein the functional polymer polyethyleneimine (PEI) can not only act as a passivator to enhance the photoelectron conversion of CDs, but also capture carbon dioxide through its rich polyamine branches, thereby improving crop photosynthesis, so as to increase crop yield under drought and salt stress and enhance the environmental resistance of crops; (1) The primary and secondary amines on the surface of PEICDs can adsorb carbon dioxide, form ammonium carbamate and ammonium carbamate, and finally hydrolyze into bicarbonate. In C4 crops, phosphoenolpyruvate carboxylase (PEPCase) initiates the carboxylation reaction in mesophyll cells, and phosphoenolpyruvate (PEP) is converted into HCO3- The substrate is carboxylated to generate tetracarbonate. The compound then enters the vascular bundle region, where CO2 is released for Rubisco, ultimately producing the metabolite 3-PGA; the accessibility of PEICDs is enhanced to obtain higher CO2 adsorption efficiency; (2) Polyethyleneimine functionalized carbon dots of the present invention: compared with undoped carbon dots, polyethyleneimine still maintains the nanoscale size and increases the quantum yield by 8.9 times and the fluorescence intensity by nearly 10 times; (3) Polyethyleneimine functionalized carbon dots of the present invention can absorb carbon dioxide and convert it into bicarbonate, and in vitro, it is catalyzed to the dark reaction stage product 3-phosphoglyceric acid (3-PGA), which is increased by 3.2 times, thereby increasing the biomass of crops; (4) Compared with ordinary carbon dots, the polyethyleneimine functionalized carbon dots used in the present invention enable plants to utilize light and fix carbon more effectively, thereby increasing the photosynthetic rate and intercellular carbon dioxide concentration, ultimately increasing the dry and fresh weight under stress and enhancing crop resistance. Attached Figure Description

[0045] Figure 1 shows transmission electron microscopy images of carbon dots prepared in Example 1 and Comparative Example 1; (a) Comparative Example 1; (b) Example 1; Figure 2 shows particle size distribution of carbon dots prepared in Example 1 and Comparative Example 1; (a) Comparative Example 1; (b) Example 1; Figure 3 shows fluorescence spectra and quantum yield of carbon dots prepared in Example 1 and Comparative Example 1; Figure 4 shows the carbon-13 diagram of polyethyleneimine functionalized carbon dots prepared in Example 1; Figure 5 shows the in vitro carbon dioxide fixation data of polyethyleneimine functionalized carbon dots prepared in Example 1; Figure 6 shows the photosynthetic rate data of maize seedlings under different stress conditions; (a) drought; (b) salt stress; Figure 7 shows the intercellular carbon dioxide concentration data of maize seedlings under different stress conditions; (a) drought; (b) salt stress; Figure 8 shows the dry and fresh weight data of maize seedlings under different stress conditions; (ab) drought; (cd) salt stress. Detailed Implementation

[0046] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The specific embodiments described below further illustrate the present invention.

[0047] The material source involved in this invention is: polyethyleneimine (PEI), branched type, purity 99%, molecular weight 1800.

[0048] Citric acid with a purity of 99.5% was purchased from Yamada Pharmaceutical Co., Ltd., China. Example 1: Preparation of Polyethyleneimine Functionalized Carbon Dots (PEICDs) was carried out via a hydrothermal method using polyethyleneimine and citric acid as precursors. The specific synthesis process was as follows: Polyethyleneimine (900 mg), citric acid (96 mg), and Milli-Q water (30 mL) were mixed in an ultrasonic bath for 10 minutes; the solution was then transferred to a Teflon-lined stainless steel autoclave (50 mL), heated at 200°C for 10 hours, impurities were removed using a dialysis tube (2000 D), and the mixture was freeze-dried to obtain PEICDs.

[0049] The preparation of carbon dots in Comparative Example 1 was as follows: Citric acid (96 mg) and deionized water (30 mL) were mixed in an ultrasonic bath for 10 minutes; then the solution was transferred to a Teflon-lined stainless steel autoclave (50 mL), heated at 200 °C for 10 hours, impurities were removed by dialysis tube (2000 D), and freeze-dried to obtain CDs.

[0050] Comparative Example 2: Citric acid (96 mg), ethylenediamine (0.5 mL), and deionized water (30 mL) were mixed in an ultrasonic bath for 10 minutes; the solution was then transferred to a Teflon-lined stainless steel autoclave (50 mL), heated at 200 °C for 10 hours, impurities were removed by dialysis tube (2000 D), and the solution was freeze-dried to obtain NH2-CDs.

[0051] Comparative Example 3: Citric acid (96 mg), polyacrylic acid (0.2 g), and deionized water (30 mL) were mixed in an ultrasonic bath for 10 minutes; the solution was then transferred to a Teflon-lined stainless steel autoclave (50 mL), heated at 200 °C for 10 hours, impurities were removed by dialysis tube (2000 D), and the mixture was freeze-dried to obtain PNDs.

[0052] Material Characterization 1. The carbon dots prepared in Example 1 and Comparative Example 1 were observed by transmission electron microscopy; the results are shown in Figure 1. Both the carbon dots and the polyethyleneimine functionalized carbon dots have a uniform spherical distribution.

[0053] 2. Particle size analysis was performed on the carbon dots prepared in Example 1 and Comparative Example 1. The results are shown in Figure 2. As can be seen from the results in Figure 2, the average particle size of the carbon dots is 2.7 nm and the average particle size of the PEICDs is 2.3 nm.

[0054] 3. The fluorescence intensity test and quantum yield calculation results of the carbon dots prepared in Example 1 and Comparative Example 1 are shown in Figure 3. The quantum yield of CDs is 4.4%, and the quantum yield of PEICDs is 39.3%, with the fluorescence intensity being nearly ten times stronger.

[0055] 4. The carbon content of carbon dioxide introduced into PEICDs was obtained by nuclear magnetic resonance spectroscopy. 13 C) Spectroscopic analysis of the nuclear magnetic resonance spectrum, the results of which are shown in Figure 4, show a bicarbonate peak at 160 ppm.

[0056] 5. Organic matter content was determined using liquid chromatography-triple quadrupole mass spectrometry (LC-MS / MS). The test subject was 3-phosphoglycerate (3-PGA), a carbon dioxide fixation product simulated by the in vitro Rubisco enzyme. 99.9% carbon dioxide gas was passed through either 5 mL of 5 mg / mL PEICDs solution or 187 mL of pure water (both pretreated with nitrogen to remove carbon dioxide) at room temperature for 5 minutes. These carbon dioxide-rich solutions were used as substrates for the carboxylation reaction of partially purified Rubisco. In a 1.5 mL test tube, 905 μL of a carbon dioxide-free reaction mixture (0.1 M N-diaglycine, pH 8.2, containing 5 mM MgCl2), 25 μL of Rubisco enzyme solution, and 20 μL of 0.5 M NaHCO3, 5 mg / mL PEICDs solution containing carbon dioxide, or pure water were added. These mixtures were incubated at 25°C for 10 minutes to activate Rubisco. Then, 50 μL of 6 mol RuBP was added to start the reaction, and the reaction was terminated after 6 minutes. 200 μL of formic acid was then added, and the mixture was tested. The results, as shown in Figure 5, indicate that the content of 3-PGA increased by 2.77 times in the presence of PEICDs.

[0057] Example 2: The effects of CDs and PEICDs on the growth-promoting and photosynthetic effects of maize seedlings under stress were investigated. The specific process is as follows: Soil was taken from Jiangsu Province, China. The soil was filtered through a 5.0 mm sieve to remove large pieces of plant debris and impurities. Each treatment had 5 replicates, and each pot contained 500 grams of soil. The normal soil moisture content was 70%, the dry soil moisture content was 40%, and the soil salinity was 0.25%. Maize seeds (Maize, Zhengdan 958 variety) were cultured in conventional soil for 3 days before being transplanted to treatment and control groups. Seedlings continued to grow in a greenhouse for 18 days under conditions of 20 / 15°C diurnal temperature variation and 18 / 6-hour light / dark cycles. The Non-CK (no stress) group was in conventional soil with 70% soil moisture; the control group was under drought stress with 40% soil moisture; and the control group was under salt stress with 70% soil moisture and 0.25% soil salinity (2.5g sodium chloride per kg of soil). Treatment group 1 was under drought stress with 40% soil moisture + CDs; treatment group 2 was under drought stress with 40% soil moisture + PEICDs; treatment group 1 was under salt stress with 0.25% soil salinity + CDs; treatment group 2 was under salt stress with 0.25% soil salinity + PEICDs. CDs and PEICDs were prepared at 10 mg / L. For the nano-aqueous solution, when the corn seedlings are in the three-leaf and one-core stage, spray 5 mL of nano-aqueous solution on the leaves of each pot (2 plants) for seven consecutive days, once a day.

[0058] The results are shown in Figures 6-8. Compared with the control group (CK), foliar application of PEICDs increased the photosynthetic rate of maize seedlings by 67.2% and by 85.2% under salt stress. Under drought stress, foliar application of PEICDs increased the intercellular carbon dioxide concentration of maize seedlings by 101.0% and by 109.8% under salt stress. Under drought stress, foliar application of PEICDs increased the aboveground and root fresh weight of maize seedlings by 35.5% and 41.9%, respectively, and by 106.3% and 67.3% under salt stress. Under drought stress, foliar application of PEICDs increased the aboveground and root dry weight of maize seedlings by 46.2% and 50.0%, respectively, and by 96.9% and 72.7% under salt stress.

[0059] Example 3: Referring to Example 2, the number of times the PEICDs nano-water solution was sprayed per day in the drought stress treatment group was adjusted. Other parameters and conditions were the same as in Example 2. The photosynthetic parameters of maize seedlings were measured, and the results are shown in Table 1. Table 1. Results of photosynthetic parameters of maize seedlings

[0060] Example 4: Referring to Example 2, the application method of PEICDs nano-aqueous solution in the drought stress treatment group was adjusted, while other parameters and conditions remained the same as in Example 2. The photosynthetic parameters of maize seedlings were measured, and the results are shown in Table 2. Table 2. Results of photosynthetic parameters of maize seedlings.

[0061] Comparative Example 4 replaced PEICDs in Example 2 with NH2-CDs 10 mg / L in Comparative Example 2, with other parameters and conditions being the same as in Example 1; taking drought stress as an example; the results are shown in Table 3.

[0062] Table 3. Results of photosynthetic parameters of maize seedlings

[0063] Comparative Example 5 replaced the PEICDs in Example 3 with 10 mg / L of polyacrylic acid carbon dots from Comparative Example 3, while other parameters and conditions were the same as in Example 1; drought stress was used as an example; the results are shown in Table 4.

[0064] Table 4. Results of photosynthetic parameters of maize seedlings

[0065] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A method for preparing polyethyleneimine functionalized carbon dots for enhancing crop resistance, characterized in that, The method includes the following steps: mixing polyethyleneimine, citric acid and water evenly, then transferring the solution to a stainless steel autoclave for hydrothermal reaction, followed by dialysis and drying to obtain the final product.

2. The method according to claim 1, characterized in that, The mass ratio of polyethyleneimine to citric acid is 8~10:

1.

3. The method according to claim 1, characterized in that, The mass ratio of polyethyleneimine, citric acid and water is 8~10:1:2~5.

4. The method according to claim 1, characterized in that, The conditions for the hydrothermal reaction are: temperature 180~250℃, time 8~15h.

5. The method according to claim 1, characterized in that, The conditions for the hydrothermal reaction are: temperature 200℃ and time 10 h.

6. The method according to claim 1, characterized in that, The dialysis time is 36-72 hours.

7. Polyethyleneimine functionalized carbon dots prepared by the method according to any one of claims 1 to 6.

8. The polyethyleneimine functionalized carbon dots according to claim 7, characterized in that, The polyethyleneimine functionalized carbon dots (PEICDs) are spherical.

9. The polyethyleneimine functionalized carbon dots according to claim 7, characterized in that, The size of the polyethyleneimine functionalized carbon dots (PEICDs) is 2-3 nm.

10. The polyethyleneimine functionalized carbon dots according to claim 7, characterized in that, The size of the polyethyleneimine functionalized carbon dots (PEICDs) is 2.3 nm.

11. The application of the polyethyleneimine functionalized carbon dots according to any one of claims 7 to 10 in the agricultural field.

12. The application of the polyethyleneimine functionalized carbon dots according to any one of claims 7 to 10 in enhancing crop resistance.

13. A method for improving photosynthesis in maize under stress, characterized in that, The method involves applying a PEICDs solution to the leaves during the corn seedling stage.

14. The method according to claim 13, characterized in that, The corn seedling stage refers to the stage when the corn reaches the "three leaves and one heart" stage.

15. The method according to claim 13, characterized in that, Foliar application refers to foliar spraying.

16. The method according to claim 13, characterized in that, The concentration of the PEICDs solution is 10~30 mg / L.

17. The method according to claim 13, characterized in that, The concentration of the PEICDs solution is 10 mg / L.

18. The method according to claim 13, characterized in that, The amount of PEICDs solution applied is 2-3 mL per plant.

19. The method according to claim 13, characterized in that, The PEICDs solution is applied in 1 to 5 applications; preferably in 1 to 3 applications.

20. The method according to claim 13, characterized in that, The stresses referred to are drought stress and / or salt stress.

21. A method for promoting maize growth under stress, characterized in that, The method involves applying a PEICDs solution to the leaves during the corn seedling stage.

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