Application of nitrogen-doped carbon quantum dots in improvement of cold resistance of crops

Abstract

The invention belongs to the technical field of agriculture, and particularly relates to application of nitrogen-doped carbon quantum dots to improvement of cold resistance of crops. The preparation method of the nitrogen-doped carbon quantum dots comprises the following steps: uniformly mixing citric acid, urea and N, N-dimethylformamide, and carrying out solvothermal reaction and post-treatment to obtain the nitrogen-doped carbon quantum dots. Research finds that after crop seeds are treated by using the nitrogen-doped carbon quantum dots prepared by a specific method, the germination rate and growth vigor of the seeds under cold stress can be obviously improved, the oxidative stress of the crop seeds under the cold stress can be relieved, the oxidation resistance of crops can be improved, and meanwhile, the nitrogen-doped carbon quantum dots have good application prospects. The nitrogen-doped carbon quantum dots can also up-regulate expression of cold-resistant genes such as ICE1 genes, OST1 genes or DREB1 genes in crop seeds, and can relieve harm caused by cold damage in the crop growth process.

Description

Technical Field

[0001] The present invention belongs to the technical field of agriculture. More specifically, it relates to the application of nitrogen-doped carbon quantum dots in improving the cold tolerance of crops. Background Art

[0002] Maize is a temperature-sensitive crop with extremely high temperature requirements during growth and development. Low-temperature stress is an important environmental factor limiting maize seed germination and seedling growth. In actual production, when maize seeds encounter low temperatures of 0-12°C during the imbibition period, imbibition chilling injury occurs, resulting in seed death or delayed emergence, ultimately leading to a decrease in field emergence rate. In recent years, with the emergence of extreme weather, cold weather has become more uncertain, and low temperatures occur frequently. Especially during the seedling germination and early development stages, low-temperature stress seriously affects the emergence rate and seedling vigor of maize seeds, and increases the chance of soil-borne diseases, severely reducing the yield of maize. Therefore, improving the cold tolerance of crops during the seed stage and maintaining their healthy vegetative growth is crucial.

[0003] In traditional agricultural production, in order to increase the cold tolerance and yield of crops, large amounts of chemical fertilizers are often applied; however, in actual production, the utilization efficiency of chemical fertilizers by crops is often low; at the same time, the large-scale application of chemical fertilizers not only increases agricultural costs but also causes great pollution to the environment. In addition, using plant growth regulators to improve plant cold tolerance is also a feasible strategy. However, the high cost and low absorption efficiency of exogenous plant growth regulators still make it difficult to be widely applied in field actual production. Using gene editing and breeding techniques to select cold-tolerant plant varieties provides certain potential for improving plant cold tolerance. However, the long breeding cycle and public concerns about gene safety also limit the development and application of such technologies. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art and provide the application of nitrogen-doped carbon quantum dots in improving the cold tolerance of crop seeds.

[0005] The object of the present invention is to provide a method for improving the cold tolerance of crop seeds.

[0006] The above object of the present invention is achieved by the following technical solutions:

[0007] The present invention provides the application of nitrogen-doped carbon quantum dots in improving the cold tolerance of crop seeds. The preparation method of the nitrogen-doped carbon quantum dots is as follows: Mix citric acid, urea, and N,N-dimethylformamide, perform a solvothermal reaction, and perform post-treatment to obtain nitrogen-doped carbon quantum dots.

[0008] Preferably, the crop seeds are maize seeds.

[0009] Preferably, improving the cold tolerance of crop seeds means improving the germination rate of crop seeds under cold stress.

[0010] Preferably, improving the cold tolerance of crop seeds means up-regulating the expression of cold tolerance genes.

[0011] Preferably, the cold tolerance gene is any one or more of ICE1 gene, OST1 gene or DREB1 gene

[0012] Preferably, improving the cold tolerance of crop seeds means improving the antioxidant property of crop seeds under cold stress.

[0013] Preferably, the temperature of the solvothermal reaction is 160 °C to 180 °C.

[0014] Preferably, the time of the solvothermal reaction is 4 to 6 h.

[0015] Preferably, the mixing ratio of citric acid, urea and N,N-dimethylformamide is 1 - 2 g : 2 - 4 g : 10 mL.

[0016] Preferably, the post-treatment is as follows: the solution after the solvothermal reaction is mixed with ethanol, and the precipitate is taken and dried.

[0017] Preferably, the volume ratio of the solution after the solvothermal reaction to ethanol is 1 : 1 - 3.

[0018] Furthermore, the present invention also provides a method for improving the cold tolerance of crop seeds, which is to soak crop seeds in a nitrogen-doped carbon quantum dot solution;

[0019] The preparation method of the nitrogen-doped carbon quantum dots is as follows: citric acid, urea and N,N-dimethylformamide are mixed evenly, followed by a solvothermal reaction and post-treatment to obtain the product;

[0020] The concentration of the nitrogen-doped carbon quantum dot solution is 600 mg / L to 800 mg / L.

[0021] Preferably, the mass-volume ratio of the crop seeds to the nitrogen-doped carbon quantum dot solution is 2 g : 2 - 4 mL.

[0022] The present invention has the following beneficial effects:

[0023] The present invention provides the application of nitrogen-doped carbon quantum dots in improving the cold tolerance of crop seeds. The research of the present invention finds that after crop seeds are treated with nitrogen-doped carbon quantum dots prepared by a specific method, the germination rate and growth of the seeds under cold stress can be significantly improved, the oxidative stress of crop seeds under cold stress can be alleviated, the antioxidant property of crops can be improved. At the same time, the nitrogen-doped carbon quantum dots of the present invention can also up-regulate the expression of cold tolerance genes such as ICE1 gene, OST1 gene or DREB1 gene in crop seeds, and can alleviate the harm caused by cold damage during the growth process of crops. Description of the Drawings

[0024] Figure 1 It is the preparation flow chart of nitrogen-doped carbon quantum dots and related structure characterization diagrams.

[0025] Figure 2 It is the growth phenotype diagram of corn seeds after being treated with carbon quantum dots at different concentrations under cold stress. Among them, Figure 2 Figure A in it is the control group treated with clear water, Figure 2 Figure B in it is the treatment with nitrogen-doped carbon quantum dots at a concentration of 200 mg / L, Figure 2 Figure C in it is the treatment with nitrogen-doped carbon quantum dots at a concentration of 400 mg / L, Figure 2 Figure D in it is the treatment with nitrogen-doped carbon quantum dots at a concentration of 600 mg / L, Figure 2 Figure E in it is the treatment with nitrogen-doped carbon quantum dots at a concentration of 800 mg / L.

[0026] Figure 3 It is the diagram of relevant data on the growth trend of corn seeds after being treated with carbon quantum dots at different concentrations under cold stress.

[0027] Figure 4 It is the phenotype diagram of corn seeds induced by nitrogen-doped carbon quantum dots under cold stress.

[0028] Figure 5 It is the germination rate diagram of corn seeds induced by nitrogen-doped carbon quantum dots under cold stress.

[0029] Figure 6 It is the diagram of the contents of proline and malondialdehyde in corn seeds induced by nitrogen-doped carbon quantum dots under cold stress.

[0030] Figure 7 It is the diagram of the determination of the reactive oxygen species content in corn seeds induced by nitrogen-doped carbon quantum dots under cold stress.

[0031] Figure 8 It is the diagram of the determination of the antioxidant enzyme activity in corn seeds induced by nitrogen-doped carbon quantum dots under cold stress.

[0032] Figure 9 It is the diagram of the expression levels of relevant cold stress genes in corn seeds induced by nitrogen-doped carbon quantum dots under cold stress. Detailed Embodiments

[0033] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0035] Example 1

[0036] The nitrogen-doped carbon quantum dots were prepared by the following method:

[0037] Dissolve 1 g of citric acid and 2 g of urea in 10 mL of N,N-dimethylformamide, and carry out hydrothermal reaction at 160 °C for 6 h. Cool to room temperature to obtain a dark brown solution; then mix the solution with 20 mL of ethanol, dispense the mixed solution into 2 mL Eppendorf tubes, place them in a centrifuge, centrifuge at 16,000 rpm for 10 min, centrifuge twice, retain the precipitate, and dry it.

[0038] Material characterization:

[0039] The prepared N-CDs were subjected to transmission microscopy imaging (TEM), potential measurement, X-ray diffraction analysis (XRD), and X-ray photoelectron (XPS) experiments.

[0040] The experimental results are as Figure 1 shown. It can be seen from Figure 1 that the N-CDs prepared in this example appeared spherical in appearance, with an average particle size of 1-7 nm and a potential of -38.5 ± 0.67 Mv.

[0041] Example 2

[0042] The nitrogen-doped carbon quantum dots were prepared by the following method:

[0043] Dissolve 2 g of citric acid and 4 g of urea in 10 mL of N,N-dimethylformamide, and carry out hydrothermal reaction at 160 °C for 6 h. Cool to room temperature to obtain a dark brown solution; then mix the solution with 20 mL of ethanol, dispense the mixed solution into 2 mL Eppendorf tubes, place them in a centrifuge, centrifuge at 16,000 rpm for 10 min, centrifuge twice, retain the precipitate, and dry it.

[0044] Comparative Example 1

[0045] The nitrogen-doped carbon quantum dots were prepared by the following method:

[0046] Dissolve 3.1 g of citric acid (from Sigma-Aldrich) and 1.0 mL of ethylenediamine (from Sigma-Aldrich) in 30 mL of deionized water, and then react at 200 °C for 8 hours. The solid CDs were obtained by rotary evaporation and freeze-drying, denoted as H2O-CDs.

[0047] Effect Example 1

[0048] Experimental material: N-CDs prepared in Example 1.

[0049] Identification of the ability of nitrogen-doped carbon quantum dots to scavenge reactive oxygen species in vitro. The specific detection methods are as follows (the concentration of the used N-CDs is 800 mg / L, prepared with water):

[0050] For the determination of hydrogen peroxide (H2O2) content, the "Hydrogen Peroxide Content Assay Kit" (A064-1-1, Nanjing Jiancheng Bioengineering Institute) is used. According to the principle that hydrogen peroxide can react with molybdic acid to form a complex with a characteristic absorption peak at 405 nm, the efficiency of the material to scavenge hydrogen peroxide can be calculated. Samples are added according to the sample addition procedure in the instruction manual. After mixing, the absorbance value is measured at a wavelength of 405 nm and a light path of 1 cm.

[0051] For the determination of superoxide anion (·O2 - ) content, the "Superoxide Anion Content Assay Kit" (Solarbio Science & Technology Co., Ltd., Beijing) is used and carried out according to the instruction manual.

[0052] Based on the principle of the Fenton reaction, Fe 2+ and H2O2 reagents are mixed to generate hydroxyl radicals, and the soaking agent is added to this system. The change in the absorbance value per unit time is measured at 240 nm, and the hydroxyl radical scavenging efficiency of the sulfur element-containing soaking agent is calculated.

[0053] Experimental results: The scavenging rates of N-CDs to scavenge reactive oxygen species in vitro, the scavenging rates of hydroxyl radicals, superoxide anions and hydrogen peroxide are 71.24 ± 1.17%, 29.65 ± 2.10% and 27.79 ± 5.32% respectively. This indicates that N-CDs can act as a nanozyme.

[0054] Effect Example 2

[0055] Experimental materials: N-CDs prepared in Example 1

[0056] Experimental method: Soaking treatment of corn seeds. The corn seeds of Zhengdan 958 variety are selected. The N-CDs prepared in Example 1 are diluted with water to soaking agents with mass concentrations of 200, 400, 600, and 800 mg / L. The mass-volume ratio of corn seeds to the soaking agent is 2 g: 3 mL. Soaking with water is used as a control (HP, Hydropriming). The soaked corn seeds are evenly placed in a germination box, with 40 seeds in each germination box (5×8), and 3 groups of replicates are set. Subsequently, the germination box is placed in a low-temperature incubator with 16 h of light, 8 h of darkness, and a temperature of 12 °C. After five days of low-temperature treatment, it is restored to normal temperature for 5 days, and the seed phenotypes are observed.

[0057] Experimental results: Figure 2Phenotype diagrams of corn seeds after soaking with N-CDs at different concentrations, followed by 5 days of low-temperature treatment and 5 days of treatment at natural normal temperature. It can be seen from the figure that after soaking with the N-CDs prepared in Example 1, the growth of corn seeds is better. Figure 3 For the stem height, fresh weight, main root length, and overground part of corn seeds. It can be seen from the figure that when the concentration of N-CDs is 600-800 mg / L, it can significantly promote the growth of corn after low-temperature stress.

[0058] Effect Example 3

[0059] Nitrogen-doped carbon quantum dots improve the growth and germination rate of corn under cold stress.

[0060] Experimental materials: N-CDs prepared in Example 1 and H2O-CDs prepared in Comparative Example 1.

[0061] Experimental method: Soaking treatment of corn seeds. The variety of corn seeds selected is Zhengdan 958. The carbon quantum dot nanomaterials prepared in Example 1 and Comparative Example 1 are diluted with water to an immersion agent with a mass concentration of 800 mg / L. The mass-volume ratio of corn seeds to the immersion agent is 2 g: 3 mL. Soaking with water is used as a control (HP, Hydropriming). The soaked corn seeds are evenly placed in a germination box, with 40 seeds (5×8) in each germination box, and 3 replicates are set. Subsequently, the germination box is placed in a low-temperature incubator with 16 h of light, 8 h of darkness, and a temperature of 12 °C. After 5 days of low-temperature treatment, the number of germinated seeds is counted, and the germination rate (Exposure rate) is calculated.

[0062] Experimental results: Figure 4 Phenotype diagrams of corn seeds after different soaking treatments, followed by 5 days of low-temperature treatment. It can be seen from the figure that the yellow corn seeds grow better after soaking with the nitrogen-doped carbon quantum dots prepared in Example 1 and then low-temperature cultivation for five days. After soaking with the nitrogen-doped carbon quantum dots prepared in Comparative Example 1 and then low-temperature cultivation for five days, the growth of yellow corn seeds is significantly worse than that of the corn seeds treated with the materials in Example 1.

[0063] Figure 5 Statistics of the germination rate of corn seeds after different soaking treatments, followed by 5 days of low-temperature treatment. It can be seen from the figure that after soaking with Example 1 (N-CDs), corn still has a good germination rate under cold stress, while after soaking with Comparative Example 1 (H2O-CDs), the germination rate of corn under cold stress is significantly lower than that of Example 1 (p<0.05).

[0064] The above results indicate that only the nitrogen-doped carbon quantum dots prepared by the preparation method described in Embodiment 1 of the present invention have a better effect of improving the growth and germination rate of corn seeds under cold stress.

[0065] Effect Example 4

[0066] The quasi-enzyme of nitrogen-doped carbon quantum dots reduces proline and malondialdehyde in corn seeds.

[0067] Sample material: Each group of corn seeds after five days of low-temperature treatment in Effect Example 3.

[0068] Determination of proline content:

[0069] Prepare a proline standard curve: First, prepare proline standard solutions with concentrations of 3, 4, 5, 6, 7, and 8 μg / mL respectively. Take 6 stoppered test tubes, add 2 mL of the standard solution with different concentrations to each test tube, then add 2 mL of 2.5% acidic ninhydrin solution and 2 mL of glacial acetic acid, and heat in a boiling water bath for 30 min. After heating, take out the test tubes and cool them, then add 4 mL of toluene, shake and extract for 30 s, let it stand, absorb the upper red proline toluene solution, use toluene as a blank control, and colorimetric at a wavelength of 520 nm to prepare a proline standard curve.

[0070] Take the corn seeds after five days of low-temperature treatment in Effect Example 3, wipe off the residual moisture on the seeds, and grind them into powder at low temperature with a grinding machine (2 large steel balls, frequency 25 s -1 , time 35 s, 2 times). Weigh 0.05 g of the sample and set 6 replicates for each treatment in a 2 mL EP tube, and set a blank. Add 1 mL of 3% sulfosalicylic acid solution to the EP tube, extract by shaking in a 90 °C water bath for 10 min, centrifuge at 25 °C × 12000 rpm for 10 min. In a new 2 mL EP tube, add 150 μL of the supernatant, 150 μL of distilled water, 150 μL of glacial acetic acid, 300 μL of acidic ninhydrin, and 400 μL of toluene in sequence, shake for 1 min, wait for the solution to layer, read the absorbance value A at 520 nm, and calculate the proline content according to the following formula:

[0071] Proline (Pro) content = 18.11×(ΔA - 0.0024) / W×D, where ΔA = A determination - A blank, D is the dilution factor, and W is the sample mass (g).

[0072] Determination of malondialdehyde (MDA) content: Take the corn seeds after 5 days of low-temperature treatment in Effect Example 3, wipe off the residual moisture on the seeds, and grind them into powder at low temperature with a sample mill (2 large steel balls, frequency 25 s⁻¹, time 35 s, 2 times). Weigh 0.05 g of the sample into a 2 mL EP tube, quickly add 1.5 mL of 5% trichloroacetic acid (TCA) to make a homogenate, and then centrifuge at 3000 r / min at 4 °C for 10 min. Take 0.7 mL of the supernatant into a centrifuge tube, add 0.7 mL of 0.67% thiobarbituric acid (TBA), mix well, boil in a water bath at 100 °C for 30 min, and centrifuge again after cooling. Measure the absorbance values of the supernatant at 532 nm and 600 nm respectively. The calculation formula is: ΔA = A532 - A600, MDA content (nmol / g) = 32.3 × ΔA ÷ W; W is the sample mass, g.

[0073] The test results are as Figure 6 shown. Compared with the control group, after 5 days of low-temperature stress, the MDA content of the corn seeds treated with the nitrogen-doped carbon quantum dots prepared in Example 1 decreased significantly. At the same time, among the physiological indexes for evaluating plant cold resistance, proline is also one of the evaluation indexes. It generally exists in plants in a free state and can maintain cell turgor pressure and reduce the harm of low temperature to plants. After 5 days of low temperature, the proline content of the corn seeds treated with N-CDs prepared in Example 1 decreased significantly. The above results show that the nitrogen-doped carbon quantum dots described in the present invention can better relieve the low-temperature stress of corn seeds and reduce the harm of low temperature to plants.

[0074] Effect Example 5

[0075] Sample material: The corn seeds in each group after 5 days of low-temperature treatment in Effect Example 3 were used as the samples to be tested.

[0076] The determination of hydrogen peroxide (H₂O₂) content used a "hydrogen peroxide content determination kit" (A064-1-1, Nanjing Jiancheng Bioengineering Institute Co., Ltd.). According to the fact that hydrogen peroxide can react with molybdic acid to form a complex with a characteristic absorption peak at 405 nm, the efficiency of the material to scavenge hydrogen peroxide can be calculated. Add samples according to the sample addition process in the instruction manual, mix well, and measure the absorbance value at a wavelength of 405 nm and an optical path of 1 cm.

[0077] The determination of superoxide anion (·O₂ - ) content used a "superoxide anion content determination kit" (Solarbio Science & Technology Co., Ltd., Beijing) and was carried out according to the instruction manual.

[0078] The soluble protein content of corn seeds was determined using the Nanjing Jiancheng kit (A045-2-2). The specific operation is as follows: Accurately weigh 0.1 g of fresh sample in a 2 mL grinding tube, and add PBS buffer (pH 7.0 - 7.4) according to the ratio of weight (g) to volume (mL) = 1:9. At low temperature, use a grinder (50 Hz, 180 sec) to grind the leaves into a homogenate. Centrifuge the homogenate to obtain the supernatant for use (2500 r / min, 4 °C, 10 min). Add 3 mL of Coomassie Brilliant Blue solution and 50 μL of sample supernatant into a new centrifuge tube. Then, mix the solution in the centrifuge tube well, let it stand for 10 min, and then measure the absorbance value at 595 nm using a UV spectrophotometer to calculate the protein content.

[0079] Figure 7 The results showed that after 5 days of low-temperature treatment, N-CDs significantly reduced the contents of hydrogen peroxide (H2O2) and superoxide anion (·O2 - ) in corn seeds, indicating that N-CDs can maintain the reactive oxygen species homeostasis in corn seeds and alleviate the toxic effect of excessive ROS concentration on cells under low-temperature stress. In addition, we also measured the soluble protein content in corn seeds, and the results showed that after 5 days of low-temperature stress, N-CDs significantly increased the soluble protein content in corn seeds.

[0080] Effect Example 6

[0081] Experimental materials: The corn seeds of each group after 5 days of low-temperature treatment in Effect Example 3 were used as test samples.

[0082] Preparation of enzyme extraction solution: The samples were extracted with 0.1 M PBS (containing 1 mM EDTA and 1% PVPP) at pH = 7.5, ground into a homogenate according to the ratio of sample: extraction solution = 1:10 (for example, 0.2 g of fresh sample was extracted with 2 mL of extraction solution), centrifuged the homogenate (15000 g, 20 min, 4 °C), and the supernatant was aliquoted (100 μL per tube, a total of 6 - 8 tubes), and stored at -80 °C for later use. This enzyme extraction solution can be used for the determination experiments of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD).

[0083] The determination of peroxidase (POD) was carried out according to the method described by Li Hesheng (Li Hesheng. Experimental Plant Physiology [M]. Beijing: Higher Education Press, 2003. 56 - 58.) with slight modifications:

[0084] Prepare the reaction mixture: Take 200 mL of sodium phosphate buffer (PBS, 0.2 M, pH = 6), add 0.076 mL of the original guaiacol (2-methoxyphenol) solution, heat and stir to dissolve, and then add 0.112 mL of 30% H2O2 after cooling. Enzyme activity assay: Take 3 mL of the above reaction solution, add 50 μL of the enzyme extract, mix well, and zero the absorbance at 470 nm of the mixture with PBS as the control. Measure for a total of 180 s, reading once every 30 s. An increase in absorbance of 0.01 per minute is defined as 1 enzyme activity unit (U). Calculate the peroxidase activity according to the following formula:

[0085] POD activity (U / g·min) = (ΔA470 * V) / (W * VT * T * 0.01)

[0086] Where: ΔA470 is the change in absorbance during the reaction time; W is the fresh sample mass of the sample (g); T is the reaction time; V is the volume of the enzyme solution used in the measurement (mL); VT is the total volume of the extracted enzyme solution (mL).

[0087] The catalase (CAT) assay was carried out according to Li Hesheng (Li Hesheng. Plant Physiology Experiments [M]. Beijing: Higher Education Press, 2003. 56 - 58.) with slight modifications:

[0088] Prepare the reaction mixture: Take 200 mL of sodium phosphate buffer (0.15 M, pH = 7), add 0.3092 mL of 30% H2O2 stock solution, and shake well. Enzyme activity assay: Take 3 mL of the above reaction solution, add 20 μL of the enzyme extract, zero the absorbance with the buffer as the control, and measure the absorbance of the mixture at 240 nm. Measure for a total of 180 s, reading once every 30 s. A decrease in absorbance of 0.01 per minute is defined as 1 enzyme activity unit (U). Calculate the CAT activity according to the following formula:

[0089] CAT activity (U / g·min) = (ΔA240 * V) / (W * VT * T * 0.01)

[0090] Where: ΔA240 is the average absorbance during the reaction time; W is the fresh sample mass of the sample (g); T is the reaction time; V is the volume of the enzyme solution used in the measurement (mL); VT is the total volume of the extracted enzyme solution (mL).

[0091] Superoxide dismutase (SOD) assay: The superoxide dismutase (SOD) assay was carried out according to Wang Xuekui (Wang Xuekui. Principles and Techniques of Plant Physiological and Biochemical Experiments. 2nd Edition [M]. Higher Education Press, 2006):

[0092] Add 1.5 mL of 0.05 mol / L PBS, 0.3 mL of 0.13 mol / L methionine solution, 0.3 mL of 0.75 mmol / L NBT solution, 0.3 mL of EDTA-Na2 solution, 0.3 mL of 0.02 mmol / L riboflavin solution into a 5 mL test tube. Add 0.05 mL of enzyme extract (for the light control tube and the dark control tube, replace the enzyme solution with sodium phosphate buffer), and finally add 0.25 mL of distilled water to make the total volume 3 mL. After mixing, place the dark control tube in the dark, and react the other tubes under 4000 lx sunlight for 20 min. Measure the absorbance of each tube at 560 nm. Calculate the SOD activity according to the following formula:

[0093] Total SOD activity (U / g) = (ALC - AE) * V / (0.5 * (ALC - ADC * W * VT))

[0094] Where: ALC is the absorbance of the light control tube; ADC is the absorbance of the dark control tube; AE is the absorbance of the sample tube; V is the total volume of the sample solution (mL); VT is the sample volume used during measurement (mL); W is the fresh weight of the sample (g).

[0095] Figure 8 The results show that after 5 days of low temperature treatment of maize seeds, the SOD enzyme activity of maize seeds treated with N-CDs was significantly increased compared with that of the Control treated with water. Among them, the average SOD enzyme activity of the Control treatment group was 292.5 U / g FW, and the average SOD enzyme activity of the N-CDs treatment group was 656.89 U / g FW (FW represents the fresh weight of the sample), which was increased by 124.58% compared with the Control. After 5 days of low temperature stress, the POD enzyme activity in maize seeds treated with N-CDs was significantly increased. The average enzyme activity of the Control treatment group was 337.83 U / g / min FW, and the average enzyme activity of the N-CDs treatment group was 437.45 U / g FW, which was increased by 29.49% compared with the Control. At the same time, after 5 days of low temperature stress, the CAT enzyme activity of maize seeds in the N-CDs treatment group was significantly increased. The average enzyme activity of the Control treatment group was 254.96 U / g / min FW, and the average enzyme activity of the N-CDs treatment group was 317.74 U / g FW, which was increased by 24.62% compared with the Control. Therefore, N-CDs can increase the antioxidant enzyme activity in maize seeds.

[0096] Effect Example 7

[0097] Experimental materials: Maize seeds of each group after 5 days of low temperature treatment in Effect Example 3 were used as the test samples.

[0098] Experimental method: Maize seeds after 5 days of cold stress were selected as samples, quickly frozen in liquid nitrogen, and stored at -80 °C. The target gene sequence was queried using NCBI (https: / / www.ncbi.nlm.nih.gov / ) and primers were designed. The specific primer sequences are shown in Table 1. In this invention, all total RNA extractions were performed using a plant total RNA extraction kit (RN38, Aidlab, Beijing). cDNA synthesis was carried out using TRUEscript First Strand cDNA Synthesis (PC5402, Aidlab, Beijing). Finally, qRT-PCR was performed using the SYBRGreen qPCR Mix kit (PC3302, Aidlab, Beijing) on a Bio-Rad 298CFX Connect Real-Time PCR System (Bio-Rad, California, USA). There were four replicates for each treatment. According to the 2 -ΔΔCt -ΔΔCt method to analyze the relative gene expression levels.

[0099] Table 1 Primer sequences of ICE1, OST1, and DREB1

[0100]

[0101] ICE1 can activate the expression of the CBF gene. ICE1 encodes a bHLH transcription factor similar to MYC, which can specifically bind to the MYC response element of the CBF3 promoter and induce the transcriptional expression of the downstream cold-resistant gene COR regulated by CBF / DREB1, helping plants synthesize antifreeze proteins, osmotic adjustment substances (such as proline), etc., thereby improving the cold resistance of plants. Both OST1 and DREB1 can regulate the cold stress response and thus enhance the cold resistance of plants. Figure 9 The data show that N-CDs treatment can promote the expression of these three genes to a certain extent. Compared with the Control, the expression level of ICE1 increased by 16 times, the expression level of OST1 increased by 2.3 times, and the expression level of DREB1 increased by 4.5 times.

[0102] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of nitrogen-doped carbon quantum dots in improving the cold tolerance of crop seeds, characterized in that: The preparation method of the nitrogen-doped carbon quantum dots is as follows: citric acid, urea and N,N-dimethylformamide are mixed evenly, solvent thermal reaction is performed, and post-treatment is performed to obtain the nitrogen-doped carbon quantum dots.

2. The application according to claim 1, characterized in that: The crop seeds are corn seeds.

3. The application according to claim 1, characterized in that: The method of improving the cold tolerance of crop seeds is to improve the germination rate of crop seeds under cold stress.

4. The use according to claim 1, characterized in that: The method of improving the cold tolerance of crop seeds is to up-regulate the expression of cold tolerance genes.

5. The use according to claim 1, characterized in that: The method of improving the cold tolerance of crop seeds is to improve the antioxidant capacity of crop seeds under cold stress.

6. The use according to claim 1, characterized in that: The temperature of the solvent thermal reaction is 160°C to 180°C.

7. The use according to claim 1, characterized in that: The mixing ratio of the citric acid, urea and N,N-dimethylformamide is 1-2 g: 2-4 g: 10 mL.

8. The use according to claim 1, characterized in that: The post-treatment is as follows: the solution after the solvent thermal reaction is mixed with ethanol, and the precipitate is taken and dried.

9. A method for improving the cold tolerance of crop seeds, characterized in that: soaking crop seeds in a nitrogen-doped carbon quantum dot solution; The preparation method of the nitrogen-doped carbon quantum dots is as follows: citric acid, urea and N,N-dimethylformamide are mixed, subjected to solvent thermal reaction, and post-treated to obtain; The concentration of the nitrogen-doped carbon quantum dot solution is 600 mg / L to 800 mg / L.

10. The method according to claim 9, characterized in that: The mass volume ratio of the crop seeds and the nitrogen-doped carbon quantum dot solution is 2 g: 2-4 mL.

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