Chitosan-iron nanoparticle leaf surface blocking and controlling agent as well as preparation method and application thereof

Abstract

The invention provides a chitosan-iron nanoparticle leaf surface resistance and control agent as well as a preparation method and application thereof, and belongs to the technical field of reduction of heavy metal pollution of crops. The preparation method comprises the following steps: dissolving chitosan in an organic solvent to obtain a chitosan solution, mixing the chitosan solution with ferrous sulfate, adding a sodium tripolyphosphate solution for crosslinking and stirring to obtain a solution containing precipitates, centrifuging, and collecting the precipitates to obtain the chitosan / ferrous sulfate composite material. The TPP and amino groups of the chitosan form ionic crosslinking through triphosphate groups, and Fe < 2 + > is successfully loaded on the surfaces of the chitosan nanoparticles. By complexing chitosan and Fe < 2 + >, the crystallinity of the material is reduced, and the adsorption activity of the amorphous structure is enhanced. Pot experiments show that by spraying the chitosan-iron nanoparticle leaf surface resistance and control agent on leaf surfaces, heavy metals of crops can be remarkably reduced, the antioxidant enzyme activity of plant tissues is improved, active oxygen is effectively removed, oxidative damage is relieved, and therefore plant growth is promoted, and the toxic effect of heavy metal stress on the crops is relieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of reducing heavy metal pollution of crops, and particularly relates to a chitosan-iron nanoparticle foliar control agent and a preparation method and application thereof. BACKGROUND

[0002] Cadmium (Cd) is one of the most harmful heavy metals to plants, animals and human bodies in nature, and is collectively referred to as a "five-toxic" element in soil together with heavy metals mercury (Hg), arsenic (As), chromium (Cr) and lead (Pb). Intake of food containing excessive cadmium, such as a large amount of cadmium-contaminated rice, wheat and other economic crops from soil, is the main way for humans to contact cadmium. Rice has the property of accumulating heavy metals, and when rice grows in soil with high heavy metal content, heavy metals are easily accumulated in roots, stems, leaves and seeds. Heavy metals are mainly accumulated in the surface layer of soil, especially the plough layer, so they are easily absorbed and transported by crops such as rice, and then transmitted through the food chain to cause harm to human health. The quality of crops is directly related to human health, and long-term contact of humans and animals with heavy metals will cause irreversible damage to their health.

[0003] In recent years, in the production of crops such as rice, foliar fertilization has been valued and widely used due to its high nutrient utilization rate and convenient application, which can regulate the absorption and tolerance of heavy metals by rice to a certain extent and promote the growth of vegetation. At present, foliar control agents mainly include organic foliar control agents, non-metal element foliar control agents and metal element foliar control agents. Therefore, it is of positive significance to develop foliar control agents suitable for crops.

[0004] However, chitosan-metal nanoparticle composites are currently mainly studied for crop growth and antibacterial applications, but few studies focus on their regulation of the absorption of heavy metals or metalloids by crops such as rice. SUMMARY

[0005] In view of this, the purpose of the present application is to provide a chitosan-iron nanoparticle foliar control agent and a preparation method and application thereof, which can effectively reduce the content of heavy metals in crops.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0007] The present application provides a preparation method of a chitosan-iron nanoparticle foliar control agent, comprising the following steps:

[0008] Dissolve chitosan in an organic solvent to obtain a chitosan solution, mix the chitosan solution with ferrous sulfate, add a sodium tripolyphosphate solution for cross-linking, stir to obtain a solution containing precipitates, centrifuge, collect the precipitates, and obtain the chitosan-iron nanoparticle foliar resistance control agent.

[0009] Preferably, the organic solvent is an acetic acid solution.

[0010] Preferably, the adding ratio of the chitosan, the organic solvent and the ferrous sulfate is (0.2-0.3) g:(20-30) mL:(0.02-0.03) g.

[0011] Preferably, the cross-linking temperature is 20-30 DEG C, the cross-linking time is 5-15 min, the stirring time is 15-25 min, the centrifugal speed is 13000-15000 rpm, and the centrifugal time is 15-25 min.

[0012] The application provides the chitosan-iron nanoparticle foliar resistance control agent prepared by the preparation method.

[0013] The application provides application of the chitosan-iron nanoparticle foliar resistance control agent in reducing heavy metals in crops.

[0014] The application provides application of the chitosan-iron nanoparticle foliar resistance control agent in preparing a foliar resistance control agent for reducing heavy metals in crops.

[0015] Preferably, the crops include rice, the heavy metals include cadmium, and the chitosan-iron nanoparticle foliar resistance control agent is used for reducing the cadmium content in roots, stems and grains of the rice.

[0016] The application provides a method for reducing heavy metals in crops, which comprises the following steps: mixing the chitosan-iron nanoparticle foliar resistance control agent with water to obtain a chitosan-iron nanoparticle foliar resistance control agent solution, and spraying the chitosan-iron nanoparticle foliar resistance control agent solution on leaves.

[0017] Preferably, the mass-volume ratio of the chitosan-iron nanoparticle foliar resistance control agent to water is 0.005-0.015 g:1000 mL, the spraying frequency is 2 times, the spraying dose of each time is 50 mL / 3 plants, and the spraying time is the heading stage and the filling stage.

[0018] Compared with the prior art, the application has the following beneficial effects:

[0019] The application provides a chitosan-iron nanoparticle foliar resistance control agent, a preparation method and application thereof. 2+Successfully loaded on the surface of chitosan nanoparticles. The complexation of chitosan and Fe 2+ The complexation of chitosan and Fe reduced the crystallinity of the material and enhanced the adsorption activity of the amorphous structure. Through pot experiment, it was shown that foliar spraying of chitosan-iron nanoparticle foliar resistance control agent could significantly reduce the heavy metals of crops, improve the antioxidant enzyme activity of plant tissues, effectively scavenge reactive oxygen species, reduce oxidative damage, thereby promoting the growth of plants and alleviating the toxic effects of heavy metal stress on crops. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The particle size distribution results of Ch-Fe, the left graph is the full size range distribution of Ch-Fe, and the right graph is the size distribution of Ch-Fe in the range of 100-250 nm;

[0021] Figure 2 The SEM images and EDS spectra of chitosan, ChNPs and Ch-Fe, wherein (a) is the SEM image of chitosan, (b) is the EDS spectrum of chitosan, (c) is the SEM image of ChNPs, (d) is the EDS spectrum of ChNPs, (e) is the SEM image of Ch-Fe, and (f) is the EDS spectrum of Ch-Fe;

[0022] Figure 3 (a) in the FTIR spectra of chitosan, ChNPs and Ch-Fe, (b) is the XRD spectrum of chitosan, ChNPs and Ch-Fe, (c) is the XPS wide scan spectrum of chitosan, ChNPs and Ch-Fe, and (d) is the Fe 2p spectrum of Ch-Fe;

[0023] Figure 4 The structural formula of Ch-Fe;

[0024] Figure 5 (a) in the influence of different treatment groups on the cadmium content of rice roots, (b) is the influence of different treatment groups on the cadmium content of rice stems, (c) is the influence of different treatment groups on the cadmium content of rice leaves, (d) is the influence of different treatment groups on the cadmium content of rice grains; wherein, compared with the CK group, different letters on different groups represent significant differences;

[0025] Figure 6 (a) in the influence of different treatment groups on the iron content of rice roots, (b) is the influence of different treatment groups on the iron content of rice stems, (c) is the influence of different treatment groups on the iron content of rice leaves, (d) is the influence of different treatment groups on the iron content of rice grains; wherein, compared with the CK group, different letters on different groups represent significant differences;

[0026] Figure 7(a) is the influence of different treatment groups on the POD activity of rice leaves, (b) is the influence of different treatment groups on the CAT activity of rice leaves; wherein different letters on different groups compared with the CK group represent significant differences;

[0027] Figure 8 (a) is the influence of different treatment groups on the expression level of cadmium transport related genes in rice roots, (b) is the influence of different treatment groups on the expression level of cadmium transport related genes in rice stems, (c) is the influence of different treatment groups on the expression level of cadmium transport related genes in rice leaves, wherein different letters on different groups compared with the CK group represent significant differences. DETAILED DESCRIPTION

[0028] The application provides a preparation method of a chitosan-iron nanoparticle foliar resistance control agent, comprising the following steps:

[0029] Dissolve chitosan in an organic solvent to obtain a chitosan solution, mix the chitosan solution with ferrous sulfate, add a sodium tripolyphosphate solution for crosslinking, stir to obtain a solution containing precipitates, centrifuge, collect the precipitates, and obtain the chitosan-iron nanoparticle foliar resistance control agent.

[0030] In the application, the chitosan-iron nanoparticle foliar resistance control agent (Ch-Fe) is sprayed on leaves by taking advantages of low cost, high activity, high solubility and plant growth promotion of chitosan nanoparticles, so as to promote plant growth, improve the stress resistance of rice, regulate the expression level of related genes, and reduce the accumulation of cadmium in rice grains, thereby ensuring food safety and human health.

[0031] In the application, chitosan is dissolved in an organic solvent to obtain a chitosan solution. The organic solvent is an acetic acid solution, and the concentration of the acetic acid solution is 0.5% to 1.5% by volume. The preparation method of the acetic acid solution with a concentration of 0.5% to 1.5% by volume is mixing 5mL of acetic acid with 333 to 1000mL. The dissolving is continuously stirred at 25±3 DEG C by using a magnetic stirrer until the solution is colorless and transparent.

[0032] In the present application, after obtaining the chitosan solution, the chitosan solution is mixed with ferrous sulfate, and a sodium tripolyphosphate solution is added for cross-linking, and then stirring is performed to obtain a solution containing precipitates. The addition ratio of the chitosan, the organic solvent and the ferrous sulfate is preferably (0.2-0.3) g : (20-30) mL : (0.02-0.03) g, further preferably (0.22-0.27) g : (23-28) mL : (0.022-0.028) g, and more preferably 0.25 g : 25 mL : 0.025 g. The temperature of the cross-linking is preferably 20-30℃, further preferably 22-28℃, and more preferably 24, 25, 26 or 27℃. The time of the cross-linking is preferably 5-15 min, further preferably 7-12 min, and more preferably 8, 9 or 10 min. The time of the stirring is preferably 15-25 min, further preferably 17-22 min, and more preferably 20 min.

[0033] In the present application, after obtaining the solution containing the precipitates, centrifugation is performed, and the precipitates are collected to obtain the chitosan-iron nanoparticle foliar resistance control agent. The rotation speed of the centrifugation is preferably 13000-15000 rpm, further preferably 13500-14500 rpm, and more preferably 14000 rpm. The time of the centrifugation is preferably 15-25 min, further preferably 17-23 min, and more preferably 20 min. After the precipitates are collected, drying the precipitates is further included before obtaining the chitosan-iron nanoparticle foliar resistance control agent, and the drying can be freeze-drying.

[0034] The chitosan-iron nanoparticle foliar resistance control agent of the present application is referred to as Ch-Fe, and has a particle size of 164 nm, an iron loading of 41.7 mg / g, and a complexing efficiency of 41.0%. The chitosan-iron nanoparticle foliar resistance control agent prepared in this way can be delivered to plants with high efficiency as an active ingredient. SEM-EDS and XPS analysis show that the TPP is ionically cross-linked with the amino groups of the chitosan through the triphosphate groups, and the Fe 2+ is successfully loaded on the surface of the nanoparticles; FTIR and XRD results show that the complexing of the chitosan and the Fe 2+ reduces the crystallinity of the material and enhances the adsorption activity of the amorphous structure.

[0035] In the present application, through the rice pot experiment, it is shown that the foliar spraying of Ch-Fe can respectively reduce the cadmium content in rice grains by 68.5%, which is superior to FeSO4 of the same concentration. Ch-Fe can provide essential nutrients such as Fe and enhance the bioavailability, improve the antioxidant enzyme activity of plant tissues, effectively remove active oxygen and reduce oxidative damage, thereby promoting the growth of plants and alleviating the toxic effects of cadmium stress on rice. In the chitosan-iron nanoparticle foliar control agent, iron and chitosan form a stable complex, which is more easily recognized and absorbed by the iron transport protein on the cell membrane of rice leaves. In addition, chitosan can protect iron from rapid oxidation, thereby maintaining the available form such as Fe(II), which is helpful for the absorption of iron by rice. Therefore, Ch-Fe can provide iron nutrition by forming a stable chitosan-iron complex, and improve the bioavailability of nutrients through the nano-size effect, which has a positive promoting effect on the growth and development of rice under cadmium stress. The increase of antioxidant enzyme activity helps to reduce the absorption and transport of cadmium, thereby alleviating the cadmium stress. Although FeSO4 can provide iron nutrition, it is not as effective as Ch-Fe in reducing the absorption of cadmium, because the chitosan-iron nanoparticle foliar control agent has higher biological activity and adsorption capacity.

[0036] The present application provides a chitosan-iron nanoparticle foliar control agent prepared by the above preparation method.

[0037] The present application provides the application of the above chitosan-iron nanoparticle foliar control agent in reducing heavy metals in crops.

[0038] The present application provides the application of the above chitosan-iron nanoparticle foliar control agent in preparing a foliar control agent for reducing heavy metals in crops.

[0039] In the present application, the crops include rice, such as "Jingliangyou 534". The heavy metals include cadmium; and the chitosan-iron nanoparticle foliar control agent is used to reduce the cadmium content in the roots, stems and grains of rice.

[0040] The present application provides a method for reducing heavy metals in crops, comprising the steps of: mixing the above chitosan-iron nanoparticle foliar control agent with water to obtain a chitosan-iron nanoparticle foliar control agent solution, and spraying the chitosan-iron nanoparticle foliar control agent solution on the leaves.

[0041] In the present application, the mass-volume ratio of chitosan-iron nanoparticle foliar control agent to water is 0.005-0.015 g:1000 mL; the spraying frequency is 2 times, and the dosage of each spraying is 50 mL / 3 plants; and the spraying timing is at the heading stage and the filling stage.

[0042] The application researches and finds that spraying Ch-Fe on the leaves can significantly reduce the cadmium content in rice grains, wherein the effect of spraying Ch-Fe on reducing the cadmium content in grains is better than that of spraying FeSO4 solution, and the higher the concentration is, the better the cadmium reduction effect is, in addition, spraying Ch-Fe on the leaves can also promote the accumulation of cadmium in rice leaves. Compared with spraying FeSO4 solution, spraying Ch-Fe can also significantly increase the iron content in grains and promote the accumulation of iron in grains. Spraying Ch-Fe on the leaves activates the antioxidant system of the roots, promotes the decomposition of hydrogen peroxide, and relieves the damage of active oxygen to plant tissues.

[0043] The technical solutions provided by the application will be described in detail below in combination with the embodiments, but they cannot be understood as limitations to the protection scope of the application.

[0044] In the following examples, the preparation method of 1% (v / v) acetic acid is as follows: 5mL of glacial acetic acid is measured with a graduated cylinder, and then it is placed in a beaker or a volumetric flask, and then distilled water is added to 500mL, and the solution is mixed uniformly by gently shaking or stirring.

[0045] 1% (w / v) sodium tripolyphosphate: 1.000g of sodium tripolyphosphate solid is weighed and placed in a beaker, 90mL of distilled water is added, and stirring is performed until complete dissolution, then the solution is transferred to a 100mL volumetric flask, water is added to the calibration line and shaken uniformly.

[0046] The application uses SPSS statistical software (version 22.0) for statistical analysis. First, the normality and homogeneity of variance of the data are tested, and then Duncan test is used to analyze the significant differences between different treatment groups (P<0.1).

[0047] Example 1

[0048] Preparation of chitosan nanoparticles (ChNPs): 0.25g of chitosan is dissolved in 25mL of 1% (v / v) acetic acid, and continuously stirred at room temperature (25±3℃) with a magnetic stirrer; after the solution becomes colorless and transparent, 1mL of 1% (w / v) sodium tripolyphosphate (TPP) solution is added dropwise, cross-linked at 25℃ for 10min, and then continuously stirred for 20min to stabilize it, to obtain a solution 1 containing precipitates; finally, the solution 1 containing precipitates is centrifuged at 14000rpm for 20min with an ultra-high speed centrifuge, the supernatant 1 is poured out, and the precipitates are freeze-dried with a freeze dryer to obtain ChNPs, which are stored at room temperature for further characterization and application.

[0049] Preparation of Chitosan-iron nanoparticles foliar control agent (Ch-Fe): 0.25 g of chitosan was dissolved in 25 mL of 1% (v / v) acetic acid, continuously stirred at room temperature (25±3℃) with a magnetic stirrer; after the solution became colorless and transparent, 0.025 g of ferrous sulfate (FeS04) powder was added to the obtained chitosan solution, and 1 mL of 1% (w / v) TPP solution was added dropwise, crosslinked at 25℃ for 10 min, and then continuously stirred for 20 min to stabilize it, to obtain a solution 2 containing precipitates; finally, the solution 2 containing precipitates was centrifuged at 14000 rpm for 20 min with an ultra-high-speed centrifuge, and the supernatant 2 and precipitates were collected, and the precipitates were freeze-dried with a freeze dryer to obtain Ch-Fe, which was stored at room temperature for further characterization and application. The supernatant 2 was used for the determination of complexing efficiency.

[0050] The prepared ChNPs and Ch-Fe were further characterized. At room temperature (25±3℃), the size of the samples was analyzed by nanoparticle analyzer with a scattering angle of 135°. For morphology analysis, the samples were smeared on conductive glue and gold sprayed, observed by scanning electron microscope (SEM) at 20 kV, and energy dispersive spectrometer (EDS) was used to observe the elemental composition and distribution of the samples. The surface functional groups present in the samples were identified by Fourier transform infrared spectroscopy (FTIR), 7.0-9.0 mg of dried sample was mixed with KBr, and the spectrum was scanned by FTIR spectrometer in the wave number range of 400-4000 cm -1 -1, with a resolution of 4 cm -1 -1. X-ray crystal diffractometer (XRD) was used to scan the samples from 5° to 90° at a scanning speed of 2° / min to analyze their structure and crystallinity. Finally, the surface elemental composition of the samples was determined by X-ray photoelectron spectrometer (XPS).

[0051] The particle size distribution of Ch-Fe is shown in Figure 1 , and the particle size of Ch-Fe prepared by the preparation method of the application is 164 nm (Dx50), at which time the absorption amount of plant leaf stomata to smaller nanoparticles is high, and the active ingredient can be transported to the plant with high efficiency.

[0052] The SEM images and EDS spectra of chitosan, ChNPs and Ch-Fe are shown in Figure 2 .

[0053] Chitosan presents an irregular flaky or block structure, the surface is rough, and the reticular structure is clearly visible, which may be due to the aggregation and crosslinking of chitosan molecular chains (see Figure 2(a)); the SEM images of ChNPs present a more compact spherical structure, which can be due to the electrostatic interaction or hydrogen bonding between particles (see Fig. 2 Figure 2 (c)); the SEM images of Ch-Fe can show more complex morphologies, such as spherical or fibrous structures, which can be due to the introduction of iron element that changes the crystallization behavior and aggregation state of the particles (see Fig. 2 Figure 2 (e)). The EDS spectra show that the chitosan contains carbon (C), oxygen (O) and nitrogen (N) elements, after cross-linking reaction, by adding sodium tripolyphosphate, the phosphorus (P) element is successfully loaded on the chitosan nanoparticles, by adding ferrous sulfate and sodium tripolyphosphate, the phosphorus (P) and iron (Fe) elements are successfully loaded on the chitosan (see Fig. 2 Figure 2 (b), Figure 2 (d), Figure 3 (f)) of the present application.

[0054] The FTIR spectra of chitosan, ChNPs and Ch-Fe are shown in Fig. 2 Figure 3 (a). The spectrum of chitosan shows a band at 3355 cm -1 and 2871 cm -1 , which can be attributed to the stretching vibration of typical —NH2 and —OH groups. The small peak at 1643 cm -1 can be assigned to the stretching vibration of —C=O group of acetyl amino in chitosan, while the small peak at 1023 cm -1 can be attributed to the stretching vibration of the second —OH group. Compared with chitosan, the FTIR spectra of ChNPs and Ch-Fe show new peaks, which can be the result of the interaction of chitosan with TPP and Fe 2+ . The FTIR spectra of ChNPs and Ch-Fe show that their peaks shift from 3355 cm -1 to 3204 cm -1 , corresponding to the combined peak of —NH2 and —OH groups stretching vibration in chitosan. The peak of bending vibration of chitosan —NH2 group at 1591 cm -1 shifts to 1535 cm -1 and 1530 cm -1 in ChNPs and Ch-Fe, respectively, indicating that the amino group of chitosan interacts with TPP. The peak at 1023 cm -1 shifts to 1065 cm -1 and 1064 cm -1 , respectively, indicating that the second —OH group is involved in the complexation. In addition, ChNPs and Ch-Fe both show the stretching vibration of C—O—C group at 892 cm -1 , which can be attributed to the glycosidic bond.

[0055] XRD spectra of chitosan, ChNPs and Ch-Fe ( Figure 3 Figure (b) shows that chitosan produces a primary characteristic diffraction peak at 2θ = 26.6°. The diffraction peaks for ChNPs and Ch-Fe are located at 2θ = 23.8° and 2θ = 24.0°, respectively. The broadening of the diffraction peaks indicates that the resulting nanoparticles are amorphous. During the interaction between chitosan and TPP, the interchain and intrachain bonds of the chitosan molecules disappear, resulting in a decrease in crystallinity. The decrease in crystallinity of Ch-Fe indicates that chitosan interacts with metal ions.

[0056] XPS determined the surface chemical composition of chitosan, ChNPs and Ch-Fe. Figure 3 As shown in (c), in the XPS broad spectrum, the P 2p peak appeared in the spectrum of ChNPs, indicating that TPP and chitosan macromolecules were successfully cross-linked. Based on the spectrum of ChNPs, the P 2p and Fe 2p peaks appeared in the spectrum of Ch-Fe, indicating that under the action of TPP, chitosan and Fe 2+ The complex formed Ch-Fe. The XPS fine spectrum of Ch-Fe ( Figure 4 As shown in (d)), spin-orbit splitting causes Fe(II) and Fe(III) to split into two peaks. The Fe 2p peaks at 710.24 eV and 724.08 eV belong to Fe(II), while those at 712.11 and 724.91 eV belong to Fe(II). The peak areas represent the relative abundances of Fe(II) and Fe(III), respectively. The results indicate that the Fe(II) and Fe(III) contents in Ch-Fe are 34% and 66%, respectively, with the elemental Fe accounting for 2.69% of the entire spectrum, which is consistent with the actual measured iron loading of 41.7 mg / g.

[0057] In summary, Ch-Fe is formed by the ionic interaction between the negatively charged triphosphate groups of TPP and the positively charged amine groups of chitosan (see Ingredients ).

[0058] Example 2

[0059] The Ch-Fe used in this example is the chitosan-iron nanoparticle foliar barrier agent prepared in Example 1.

[0060] The rice variety used in this example is "Jingliangyou 534". Uniformly sized rice seeds are selected, disinfected with 30% hydrogen peroxide for 30 minutes, washed three times with deionized water, then soaked in ultrapure water for 8 hours, placed on filter paper moistened with ultrapure water, and then sprayed with water at room temperature after the seeds turn white (about 48 hours). At the same time, the seeds are sown in a sand tray and placed in a light incubator for culture, with the culture conditions being: relative humidity 80%, light intensity 20000 lx, 28°C light treatment for 14 hours, then 23°C dark treatment for 10 hours, alternating light and dark treatment. During the culture process, the sand is irrigated with nutrient solution at regular intervals every day until it is fully saturated and moist. The rice nutrient solution formula is shown in Table 1. Before transplanting the rice seedlings, 4.5 kg of cadmium-contaminated soil sieved through a 10-mesh sieve is transferred to a PVC plastic bucket containing a rhizosphere bag, then 0.21 g / kg urea, 0.455 g / kg K2HPO4 and 0.036 g / kg KH2PO4 are added as compound fertilizer according to the weight of the cadmium-contaminated soil and immediately mixed evenly, and finally tap water is added to cover the soil surface by 2-3 cm, and the water level is balanced at room temperature for one week. When the seedlings in the sand tray grow to 3 leaves and 1 heart, they are transplanted into the PVC plastic buckets, with 3 seedlings per bucket.

[0061] The rice pot experiment is carried out in a temperature-controlled greenhouse, and the potting uses a PVC water bucket with a diameter of 29 cm and a height of 24 cm. The seedlings are divided into 5 treatment groups, with 3 replicates for each treatment. The treatment groups are set as follows: (1) CK: control group (no addition of any passivator), (2) Ch-Fe T1: spraying 5 mg / L Ch-Fe, (3) Ch-Fe T2: spraying 15 mg / L Ch-Fe, (4) FeSO4: spraying 5 mg / L FeSO4, (5) FeSO4: spraying 15 mg / L FeSO4. The rice is sprayed once at the heading stage and once at the filling stage, with 50 mL of solution sprayed per pot. The control group is sprayed with the same volume of water, and the spraying is done at 4 pm to achieve the best effect. Before spraying, the pH value of each solution is adjusted to 5.0-5.5 with 0.1 mol / L HCl or NaOH. When spraying, the solution is evenly sprayed on the leaves with a spray bottle, so that all the leaves are completely wet but not dripping, and a layer of tin paper is covered on the soil to prevent the solution from being sprayed into the soil.

[0062] Table 1 Rice nutrient solution formula

[0063] Concentration Volume (mL) 57.04 g / L KNO3 12.00 g / L 5.0 MgCl2.6H2O 18.00 g / L 1.0 <![CDATA[CaCl2·2H2O]]> 15.00 g / L 1.0 MgSO4.7H2O 33.46 g / L 1.0 KH2PO4 0.371 g / L 1.0 H3BO3 0.830 g / L 0.5 MnCl2.4H2O 14.95 mg / L 0.5 ZnSO4·7H2O 15.00 mg / L 0.2 CoCl2.6H2O 62.50 mg / L 0.1 [CuSO4·5H2O] 35.00 mg / L 0.1 NaMoO4·2H2O 4.38 g / L 0.2 EDTA-FeNa2 Sterile water 1.0 q.s. to 1 L / Figure 5

[0064] The whole plant of rice at the mature stage was collected and the husks were removed. The roots were soaked in 1% nitric acid solution for 15 min to remove the surface adsorbed metal ions, and the aboveground part was washed with deionized water for several times and then dried with absorbent paper. Then 0.5 g of rice roots were cut off for extracting root surface iron film, and the other parts were killed at 105°C for 30 min, and then dried at 60°C for 72 h to constant weight for subsequent analysis.

[0065] After the mature rice was washed, 0.5 g of fresh roots were cut off and placed in a 50 mL centrifuge tube, 40 mL of DCB extraction solution (DCB extraction solution was obtained by mixing 0.03 mol / L trisodium citrate and 0.125 mol / L sodium bicarbonate) was added and soaked for 10 min, then 0.5 g of sodium hydrosulfite was added, and oscillated at 220 r / min at room temperature for 50 min. After oscillation, the extracted white roots were washed with deionized water for 2-3 times, and dried at 60°C to constant weight. The dried rice plants were divided into four parts of white roots, stems, leaves and grains, and then cut into pieces with a ceramic scissors, 0.300 g of sample was taken into a polytetrafluoroethylene digestion tank, 8.7 mL of nitric acid and 1.3 mL of perchloric acid were added and placed overnight, and then digested with an electric heating plate until the solution volume was less than 2 mL and the color was light yellow or colorless. Finally, the solution was diluted to 25 mL with ultrapure water and mixed uniformly. The solution was filtered through a 0.45 μm filter membrane, and the cadmium content in the roots, stems, leaves and grains was determined by inductively coupled plasma mass spectrometry (ICP-MS).

[0066] As Figure 6As shown, cadmium in rice plants mainly accumulated in the roots, and the accumulation of cadmium in different rice tissues was in the order of root > stem > leaf > grain. Compared with the control group, the cadmium content of rice roots, stems and grains decreased after foliar spraying of Ch-Fe, while the cadmium content of leaves increased to varying degrees. At the mature stage, the cadmium content of the roots of Ch-Fe T1 and Ch-Fe T2 treatment groups decreased by 59.6% and 66.0% compared with the control group (CK), the cadmium content of the stems decreased by 50.0% and 46.6% compared with the control group, and the cadmium content of the leaves increased by 55.5% and 94.6% compared with the control group. The cadmium content of the grains of Ch-Fe T1, Ch-Fe T2, FeSO4 T1 and FeSO4 T2 treatment groups was 0.190 mg / kg, 0.176 mg / kg, 0.217 mg / kg and 0.195 mg / kg, respectively, which decreased by 65.8% and 68.4%, 61.0% and 64.9% compared with the control group, and the cadmium content of the grains of Ch-Fe T1, Ch-Fe T2 and FeSO4 T2 treatment groups was lower than the cadmium limit of rice (0.2 mg / kg) stipulated in the national food safety standard “Limits of Contaminants in Foods” (GB 2762-2017). The results showed that foliar spraying of Ch-Fe could significantly reduce the cadmium content in rice grains, and the effect of spraying Ch-Fe on reducing the cadmium content of grains was better than that of spraying FeSO4 solution, and the higher the concentration, the better the effect of reducing cadmium, in addition, foliar spraying of Ch-Fe could also promote the accumulation of cadmium in rice leaves.

[0067] The effects of different treatment groups on the iron content in each tissue of rice were as follows Figure 7Foliar spraying of Ch-Fe and FeSO4 decreased iron content in rice roots and increased it in stems and leaves. In rice grains, spraying Ch-Fe increased iron content, while spraying FeSO4 decreased it. Compared with the control, root iron content decreased by 6.51%, 37.5%, 42.9%, and 11.4% in the Ch-Fe T1, Ch-Fe T2, FeSO4 T1, and FeSO4 T2 treatments, while stem iron content increased by 61.8%, 76.9%, 53.5%, and 68.8%, and leaf iron content increased by 25.1%, 201%, 36.9%, and 29.6%. Grain iron content increased by 44.0% and 90.5% in the Ch-Fe T1 and Ch-Fe T2 treatments, while it decreased by 5.54% and 21.0% in the FeSO4 T1 and FeSO4 T2 treatments. The Ch-Fe T2 treatment group had the lowest iron content in the roots, while the stems, leaves, and grains had the highest iron content. At maturity, the distribution of iron accumulation in different rice tissues for the control, Ch-Fe T1, and FeSO4 T2 treatments was: roots > stems > leaves > grains; for the Ch-Fe T2 treatment group, the distribution was: leaves > stems > roots > grains; and for the FeSO4 T1 treatment group, the distribution was: stems > leaves > roots > grains. These results indicate that foliar application of Ch-Fe increased iron content in the stems, leaves, and grains of rice plants to varying degrees.

[0068] During the grain-filling stage, 0.5 g of fresh leaf tissue was collected from each treatment group and minced with ceramic scissors for measurement of peroxidase (POD) and catalase (CAT) activities. POD and CAT activities in rice roots and leaves were measured using assay kits developed by Isejiu Biotechnology Co., Ltd. (Lianyungang, Jiangsu Province), following the instructions provided by the respective kits.

[0069] Changes in POD activity in rice leaves Figure 7 As shown in (a), the changes in CAT activity are as follows ReactantsAs shown in (b), the POD activity of the Ch-Fe T1, Ch-Fe T2, FeSO4 T1 and FeSO4 T2 treatment groups increased by 83.7% and 98.2%, 46.6% and 58.7% respectively compared with the control group, and the CAT activity increased by 26.3% and 47.5%, 25.3% and 32.9% respectively. POD can promote the decomposition of hydrogen peroxide, thereby reducing the damage of a large amount of hydrogen peroxide accumulation in cells to cells. The main principle of CAT is to participate in the elimination of active oxygen in chloroplasts, mitochondria and peroxisomes, decompose hydrogen peroxide into water and O2, and reduce the peroxidation damage to cells. The results show that the POD and CAT activities of rice leaves sprayed with Ch-Fe and FeSO4 are significantly higher than those of the control group, among which the activity of the Ch-Fe T2 treatment group is higher than that of the Ch-Fe T1 treatment group, and the activity of the FeSO4 T2 treatment group is higher than that of the FeSO4 T1 treatment group. Moreover, under the same concentration, the effect of spraying Ch-Fe on promoting the activity of antioxidant enzymes is better than that of spraying FeSO4, which indicates that foliar spraying of Ch-Fe can effectively activate the antioxidant system of the roots, promote the decomposition of hydrogen peroxide, and alleviate the damage of active oxygen to plant tissues.

[0070] In the filling stage, 0.5 g of fresh root, stem and leaf tissues were cut from the control (CK), Ch-Fe T2 and FeSO4 T2 treatment groups after collection, and the above treatment groups were renamed as CK, Ch-Fe and FeSO4, and then immediately stored in a refrigerator at -80°C. The DNA was extracted by referring to the plant genomic DNA rapid extraction kit of Genview Biologic Engineering (Shanghai) Co., Ltd., and then the purity of the DNA was detected by an electrophoresis instrument (1.5% agarose, 1xTAE electrophoresis buffer) to ensure that the OD260 / OD280 value of each sample was within the normal range (1.9-2.1). According to the quantitative PCR reagent of Genview Biologic Engineering (Shanghai) Co., Ltd. and the fluorescence quantitative PCR instrument, the data was processed, the rice Actin1 gene was used as an internal reference gene, the melting curve analysis was performed at 65-95°C, and the relative expression amount of the target gene was calculated by 2-ΔΔCt value. The qPCR reaction system is shown in Table 1, and the primer sequence of the cadmium transport gene and the qPCR amplification conditions are shown in Tables 2-3.

[0071] Table 2 qPCR reaction system

[0072] Volume (μL) 2 x SG Excel Fast SYBR Mixture Forward primer (10 μmol / L) 5.0 Reverse primer (10 μmol / L) 0.2 DNA template 0.2 RNase-Free ddH2O 3.6 Total 1.0 Figure 8 10

[0073] Table 3 Primer sequence of cadmium transport gene

[0074]

[0075]

[0076] The qPCR amplification conditions were as follows: denaturation at 95°C for 3 min, followed by 45 annealing cycles at 95°C for 15 s, 60°C for 45 s, and finally extension at 60°C for 45 s.

[0077] Gene expression characteristics of rice roots such as Figure 8 As shown in (a), compared with the control group, the relative expression of the OsNramp1 gene decreased by 65.1% and 45.5% in the Chloride-Fe and FeSO₄ treatments, and the relative expression of the OsNramp5 gene decreased by 63.0% and 41.3%, respectively. This indicates that foliar spraying of metal-organic nanocomposite particles or inorganic ions significantly inhibited the expression of the OsNramp1 and OsNramp5 genes. Foliar spraying of chitosan-iron nanoparticles (Chloride-Fe treatment) was more effective than spraying of inorganic ions (FeSO₄ treatment), thereby inhibiting cadmium uptake from the soil by rice roots. The main function of the OsHMA3 gene, located on the tonoplast, is to transport cadmium from the cytoplasm into the vacuole, thereby inhibiting its transport to the aerial part. Studies have shown that increased OsHMA3 expression significantly reduces cadmium in rice roots. The relative expression levels of OsHMA3 genes in the Ch-Fe and FeSO4 treatment groups increased by 29.6% and 204% compared with the control group, indicating that foliar spraying of chitosan-iron nanoparticle foliar inhibitor or FeSO4 can significantly enhance the expression of OsHMA3 genes, thereby reducing the cadmium concentration in the roots.

[0078] The OsHMA2 gene mediates the transport of cadmium from rice roots to the aboveground parts and is an important transporter gene for loading cadmium into the xylem. The OsLCT1 gene is mainly expressed in the vascular bundle and can regulate the transport of cadmium into rice grains. Figure 8 As shown in (b), compared with the control group, the relative expression of OsHMA2 gene decreased by 66.3% and 59.7% in the Ch-Fe and FeSO4 treatment groups, and the relative expression of OsLCT1 gene decreased by 50.7% and 53.2% respectively. The results show that foliar spraying of Ch-Fe can significantly inhibit the relative expression of OsHMA2 and OsLCT1 genes in rice stems, thereby inhibiting the transport of cadmium from rice roots to the aboveground parts. ​ As shown in (c), compared with the control group, the relative expression of the OsHMA2 gene increased by 134% and 76.5% in the Ch-Fe and FeSO4 treatments, and the relative expression of the OsLCT1 gene increased by 36.8% and 12.9% in the Ch-Fe and FeSO4 treatments. In contrast to the stems, foliar application of Ch-Fe significantly increased the expression of the OsHMA2 and OsLCT1 genes in rice leaves. This suggests that more cadmium is being loaded into the xylem, promoting its accumulation in rice leaves, thereby inhibiting its transfer to the grain and reducing cadmium toxicity in the grain.

[0079] The above merely describes the preferred embodiments of the present application, and it should be pointed out that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as falling within the protection scope of the present application.

Claims

1. A method for preparing a chitosan-iron nanoparticle foliar barrier agent, characterized in that: The following steps are involved: Chitosan is dissolved in an organic solvent to obtain a chitosan solution, the chitosan solution is mixed with ferrous sulfate, a sodium tripolyphosphate solution is added for cross-linking, and the mixture is stirred to obtain a solution containing a precipitate. The mixture is centrifuged and the precipitate is collected to obtain a chitosan-iron nanoparticle foliar barrier agent.

2. The preparation method according to claim 1, characterized in that The organic solvent is acetic acid solution.

3. The preparation method according to claim 1, characterized in that The addition ratio of the chitosan, the organic solvent and the ferrous sulfate is (0.2-0.3) g: (20-30) mL: (0.02-0.03) g.

4. The preparation method according to claim 1, characterized in that The cross-linking temperature is 20-30° C., and the cross-linking time is 5-15 minutes; the stirring time is 15-25 minutes; the centrifugal speed is 13000-15000 rpm, and the centrifugal time is 15-25 minutes.

5. The chitosan-iron nanoparticle foliar barrier agent prepared according to the preparation method according to any one of claims 1 to 4.

6. Use of the chitosan-iron nanoparticle foliar barrier agent according to claim 5 in reducing heavy metals in crops.

7. Use of the chitosan-iron nanoparticle foliar barrier according to claim 5 in the preparation of a foliar barrier for reducing heavy metals in crops.

8. The use according to claim 6 or 7, characterized in that The crops include rice, the heavy metals include cadmium, and the chitosan-iron nanoparticle foliar barrier agent is used for reducing the cadmium content in roots, stems and grains of rice.

9. A method for reducing heavy metals in crops, characterized in that: The method comprises the following steps: mixing the chitosan-iron nanoparticle foliar barrier agent according to claim 5 with water to obtain a chitosan-iron nanoparticle foliar barrier agent solution, and spraying the chitosan-iron nanoparticle foliar barrier agent solution on leaves.

10. The method according to claim 9, characterized in that The mass volume ratio of the chitosan-iron nanoparticle foliar barrier agent to water is 0.005-0.015 g:1000 mL; the spraying frequency is 2 times, and the dosage of each spraying is 50 mL / 3 plants; the spraying time is the heading stage and the filling stage.

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