Preparation method of citric acid-coated copper-doped zinc oxide nano-enzyme and application of citric acid-coated copper-doped zinc oxide nano-enzyme in improvement of plant stress resistance

Abstract

The invention discloses a preparation method of citric acid-coated copper-doped zinc oxide nano-enzyme and application of the citric acid-coated copper-doped zinc oxide nano-enzyme in improvement of stress resistance of plants, and relates to a preparation method of citric acid-coated copper-doped zinc oxide nano-enzyme and application of the citric acid-coated copper-doped zinc oxide nano-enzyme in improvement of stress resistance of plants. The invention aims to solve the problem of low enzymatic activity of zinc oxide at present and relieve the growth pressure under plant stress by researching zinc oxide modification. The preparation method comprises the following steps: by taking zinc acetate and copper sulfate pentahydrate as raw materials, synthesizing the copper-doped zinc oxide nano-enzyme through high-temperature calcination, and then stirring to finish citric acid coating. The application method comprises the following steps: preparing the citric acid-coated copper-doped zinc oxide nano-enzyme into an aqueous solution, and applying the aqueous solution to wheat under saline-alkali stress. The prepared citric acid coated copper-doped zinc oxide nano-enzyme is good in superoxide dismutase activity and simple to prepare, superoxide anions can be removed, the growth vigor of plants under adversity stress is improved, and normal growth of the plants is promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of nanomaterials, and in particular to a preparation method of a citric acid-coated copper-doped zinc oxide nanozyme and application of the nanozyme in improving plant resistance to stress. Background Art

[0002] Food crops are the basic guarantee for human survival, providing the calories and nutrition required by most people in the world. Major food crops such as wheat, rice, corn, etc., ensure food security and promote agricultural development. Therefore, it is crucial to increase the yield of food crops. Among them, wheat is the world's largest staple food crop, providing rich carbohydrates and nutrients. It plays a key role in agricultural production, economic development and food security, and is an important food source that human life depends on.

[0003] In higher plants, saline-alkali stress is one of the most typical adverse environments, and about 1 billion hectares of land in the world are subject to saline-alkali stress. The salt content in saline-alkali soil is high, and many field crops are difficult to survive, which seriously affects food production and the ecological environment, and has become an urgent problem that hinders agricultural development. Therefore, it is particularly important to improve the utilization and management of saline-alkali land. Reactive oxygen, as a key signal molecule for regulating plant stress resistance, will be produced in large quantities in plants under saline-alkali stress environments to cope with environmental pressure. Usually, excessive reactive oxygen can cause damage to cell membranes and proteins, causing serious oxidative damage to plants. Therefore, the efficient removal of excess reactive oxygen is particularly important.

[0004] Nanozymes have great potential in plant stress and can effectively improve plant stress resistance by removing reactive oxygen. Compared with other types of nanozymes, metal oxide nanozymes are expected to solve the problem of limited plant growth and development in actual saline-alkali land and arid areas due to their simple synthesis process and easy large-scale production. In recent years, the invention patent CN118901729 A discloses the use of nano cerium oxide in improving the salt tolerance and yield of spinach, but has not conducted in-depth research on the enzyme activity (superoxide dismutase activity) that can remove reactive oxygen, nor has it explored the improvement of enzyme activity. The value of zinc oxide containing zinc elements in agricultural crop production is becoming increasingly prominent. Studies have shown that zinc oxide is not only an environmentally friendly and resistant material, but also can improve the overall development level of crops. The application of exogenous zinc oxide nanomaterials under salt stress has been shown to promote the growth of lupin plants. However, previous reports have shown that there are few studies on the design of high enzyme activity using metal oxide nanozymes in plants, so a synthesis method of high enzyme activity is needed to better cope with plant stress resistance. Summary of the invention

[0005] The purpose of the present invention is to solve the problem of low superoxide dismutase activity of zinc oxide nanozymes used for plant stress resistance. Accordingly, a method for preparing a citric acid-coated copper-doped zinc oxide nanozyme and its application in improving plant stress resistance are provided. This patent uses zinc acetate and copper sulfate pentahydrate as raw materials, prepares copper-doped zinc oxide nanozymes with high superoxide dismutase activity by high-temperature calcination, and coats them with citric acid to improve solubility and biocompatibility, thereby improving the growth of wheat under saline-alkali stress.

[0006] A method for preparing a citric acid-coated copper-doped zinc oxide nanozyme is specifically completed according to the following steps:

[0007] 1. Preparation process of copper-doped zinc oxide nanozyme: zinc acetate, copper sulfate pentahydrate and deionized water are mixed to obtain a mixture; magnetic stirring is performed at a speed of 300rpm / min to 500rpm / min for 6h to 10h and heated to 50℃ to 60℃ until completely dissolved to obtain a dissolved mixture solution; after the reaction is completed, the mixture solution is placed in a constant temperature drying oven to dry to obtain a white solid powder; the white solid powder is evenly ground and placed in a magnetic boat, and then placed in a tube furnace and calcined at 450℃ to 550℃ for 3h to 5h, and the calcination atmosphere is nitrogen to obtain a copper-doped metal oxidase solid after the reaction; the mass ratio of zinc acetate to copper sulfate pentahydrate in the step is 1mg: (4mg to 4.5mg), and the mass ratio of zinc acetate to deionized water in the step is 1mg: (20mL to 40mL);

[0008] 2. Preparation process of citric acid-coated copper-doped zinc oxide nanozyme: The copper-doped zinc oxide nanozyme solid after the reaction is dispersed in 15mL~25mL ethanol and aqueous solution (ethanol: water volume ratio = 3~5:1), and then mixed with 15mL~25mL ethanol and aqueous solution of 0.01mol / L~0.05mol / L citric acid (ethanol: water volume ratio = 3~5:1), and stirred at 20℃~30℃ for 2h~3h to obtain a mixture solution; the solution after the reaction is dried and ground to obtain citric acid-coated copper-doped zinc oxide nanozyme.

[0009] The application of a citric acid-coated copper-doped zinc oxide nanozyme in improving plant resistance to stress is specifically completed according to the following steps:

[0010] 1. Preparation of citric acid-coated copper-doped zinc oxide nanozyme dispersion: The citric acid-coated copper-doped zinc oxide nanozyme was dispersed in deionized water by ultrasonic vibration for 3 min to 10 min to prepare 30 mg / L to 480 mg / L citric acid-coated copper-doped zinc oxide nanozyme dispersion;

[0011] 2. Seed germination process: The wheat variety is conventional wheat sold in the local market, and seeds of the same size are selected for germination experiments; the wheat seeds are placed in an oven, dried at 25°C to 35°C for 12h to 24h, disinfected with 5% to 10% sodium hypochlorite for 10min to 15min and repeatedly rinsed with deionized water; the germination process of wheat is set in a light culture room, the room temperature is 20°C to 25°C, the relative humidity is 65% to 85%, and light is applied from 8:00 to 20:00 every day;

[0012] 3. Treatment process of wheat plant resistance to salt-alkali stress: The wheat plant growth process was set in a light culture room with a room temperature of 20℃~25℃, a relative humidity of 65%~85%, and light exposure from 8:00 to 20:00 every day; two neutral salts (sodium chloride and sodium sulfate) and two alkaline salts (sodium bicarbonate and sodium carbonate) were selected in the experiment, and mixed in proportion to simulate the salt-alkali stress environment. The molar ratio of each component was sodium chloride: sodium sulfate: sodium bicarbonate: sodium carbonate = 1:9:9:1; wheat seeds with consistent germination were selected, and the seeds were cultivated in a planting pot with 120g soil, with 5 to 10 germinated wheat seeds in each pot, and seven experimental groups were set up respectively: a blank control group, a salt-alkali stress group, and five material groups under salt-alkali stress environment (the material application concentration was 30mg / L~480m g / L), wherein the material is citric acid-coated copper-doped zinc oxide nanozyme, and the saline-alkali stress concentration is set to 120mmol / L~160mmol / L; in order to simulate the saline-alkali stress environment, three days before the start of the root experiment, the blank control group was applied with 10mL~15mL of water for three days, and the other groups were applied with 10mL~15mL of saline-alkali solution for three days, so that the soil salt content under saline-alkali stress reached 4g / kg~6g / kg; the root application experiment lasted for 5~7 days, the material group applied 10mL~15mL of material once a day, the blank control group and the saline-alkali stress group applied 10mL~15mL of water once a day, and the wheat growth was photographed and recorded, and the effect of citric acid-coated copper-doped zinc oxide nanozyme in improving the stress resistance of plants under saline-alkali stress was analyzed.

[0013] The advantages of the present invention are as follows: 1. The preparation method of the citric acid-coated copper-doped zinc oxide nanozyme of the present invention is simple to prepare, with only one step of reaction conditions being high-temperature calcination, and the reaction time being only 4 hours, and the remaining reactions are all completed under stirring conditions; 2. The superoxide dismutase activity of the prepared citric acid-coated copper-doped zinc oxide nanozyme is increased by 4 times compared with that when not doped, which indicates that the citric acid-coated copper-doped zinc oxide nanozyme can better remove superoxide anions; 3. The saline-alkali stressed land is alkaline, and citric acid, as an organic weak acid, can not only help regulate the pH of the soil after coating the copper-doped zinc oxide nanozyme, but also can be used as an organic acid to supply a carbon source for plants; 4. The citric acid coating can improve the biocompatibility of the material, making the citric acid-coated copper-doped zinc oxide nanozyme dispersion more stable, and it has been confirmed in experiments that it can alleviate the growth pressure of wheat under saline-alkali stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the X-ray photoelectron spectrum of the copper-doped zinc oxide nanozyme coated with citric acid in Example 1. Figure 1 (A) The overall spectrum shows that the elements contained in the material are oxygen (O), carbon (C), zinc (Zn), and copper (Cu). Figure 1 (B) is the C1s spectrum, the chemical bonds present are: carbon-oxygen double bond, carbon-oxygen single bond, carbon-carbon single bond, Figure 1 (C) is the O1s spectrum, the chemical bonds present are: carbon-oxygen single bond, carbon-oxygen double bond, zinc-oxygen single bond, Figure 1 (D) is the Zn 2p spectrum, the valence state of zinc is divalent, Figure 1 (E) is the Cu 2p spectrum, the valence state of copper element is mainly divalent;

[0015] Figure 2 The X-ray diffraction spectrum of the copper-doped zinc oxide nanozyme and its citric acid-coated embodiment 1 shows the successful coating of citric acid through the characteristic peaks of zinc oxide and citric acid. At the same time, no characteristic peaks of copper oxide and cuprous oxide were observed in the diffraction pattern of the copper-doped zinc oxide nanozyme. The presence of copper in the total spectrum of the X-ray photoelectron energy spectrum can confirm that the main body of zinc oxide is not destroyed after copper doping, which proves the successful doping of copper element.

[0016] Figure 3It is the UV-visible absorption spectrum of the copper-doped zinc oxide nanozyme coated with citric acid in Example 1. The reaction substrates used are riboflavin, disodium ethylenediaminetetraacetate, methionine and nitroblue tetrazolium. The designed comparative experimental conditions are: control group (light), control group (darkness), undoped zinc oxide nanozyme and citric acid-coated copper-doped zinc oxide nanozyme. It can be seen from the figure that compared with the curves under the light control and undoped conditions, the absorbance in the curve under the condition of citric acid-coated copper-doped zinc oxide nanozyme is lower. It is well known that the reaction In the system, the color developing reagent nitro blue tetrazolium undergoes a photoreduction reaction in the presence of riboflavin after illumination to produce a color developing substance with a maximum absorption peak at 560nm, and the nanomaterial with superoxide dismutase activity can inhibit the photoreduction reaction of nitro blue tetrazolium; and the intensity of the inhibition rate is proportional to the enzyme activity. From the degree of absorbance reduction, it can be concluded that the enzyme activity of the copper-doped zinc oxide nanozyme coated with citric acid is 4 times higher than that of the undoped zinc oxide; therefore, it is proved that the copper-doped zinc oxide nanozyme coated with citric acid has a higher superoxide dismutase activity;

[0017] Figure 4 This is a comparison diagram of wheat phenotypes under saline-alkali stress with citric acid-coated copper-doped zinc oxide nanozymes applied to the roots in Example 2, which is used to verify the stress resistance of citric acid-coated copper-doped zinc oxide nanozymes in improving plants under saline-alkali stress; the first group is a blank control group, the second group is a saline-alkali stress group, and the third to seventh material groups are 30 mg / L, 60 mg / L, 120 mg / L, 240 mg / L, and 480 mg / L citric acid-coated copper-doped zinc oxide nanozyme dispersions applied respectively under a saline-alkali stress environment. According to the growth conditions of wheat, it can be seen that on the fifth and seventh days, the third to seventh material groups can improve the growth of wheat under saline-alkali stress, among which the application concentrations of 120 mg / L and 240 mg / L are more significant in improving the growth of wheat under saline-alkali stress. DETAILED DESCRIPTION

[0018] Embodiment 1:

[0019] This embodiment is a method for preparing a citric acid-coated copper-doped zinc oxide nanozyme, which is specifically completed by the following steps:

[0020] 1. Preparation process of copper-doped zinc oxide nanozyme: 100 mg of zinc acetate, 415 mg of copper sulfate pentahydrate and 40 mL of deionized water are mixed to obtain a mixture; magnetic stirring is performed at a speed of 350 rpm / min for 8 hours until it is completely dissolved to obtain a dissolved mixture solution; after the reaction is completed, the mixture solution is placed in a constant temperature drying oven to dry to obtain a white solid powder; the white solid powder is evenly ground and placed in a magnetic boat, which is placed in a tubular furnace and calcined at 550° C. for 4 hours in a nitrogen atmosphere to obtain a copper-doped zinc oxide nanozyme solid after the reaction;

[0021] 2. Preparation process of citric acid-coated copper-doped zinc oxide nanozyme: The copper-doped zinc oxide nanozyme solid after the reaction is dispersed in 20 mL of ethanol and aqueous solution (ethanol: water volume ratio = 4:1), and then mixed with 20 mL of ethanol and aqueous solution of 0.05 mol / L citric acid (ethanol: water volume ratio = 4:1), and stirred at 30°C for 3 hours to obtain a mixture solution; the reaction solution is dried and ground to obtain citric acid-coated copper-doped zinc oxide nanozyme.

[0022] Embodiment 2:

[0023] This embodiment is an application process of a citric acid-coated copper-doped zinc oxide nanozyme in improving plant resistance to stress, which is specifically completed in the following steps:

[0024] 1. Preparation of citric acid-coated copper-doped zinc oxide nanozyme dispersion: The citric acid-coated copper-doped zinc oxide nanozyme was dispersed in deionized water by ultrasonic vibration for 10 min to prepare 30 mg / L, 60 mg / L, 120 mg / L, 240 mg / L, and 480 mg / L citric acid-coated copper-doped zinc oxide nanozyme dispersions;

[0025] 2. Seed germination process: The wheat variety is conventional wheat sold in the local market, and seeds of the same size are selected for germination experiments; the wheat seeds are placed in an oven, dried at 30°C for 24 hours, disinfected with 5% sodium hypochlorite for 15 minutes and repeatedly rinsed with deionized water; the germination process of wheat is set in a light culture room with a room temperature of 23°C and a relative humidity of 75%, and light is applied from 8:00 to 20:00 every day;

[0026] 3. Treatment process of wheat plant resistance to salt-alkali stress: The wheat plant growth process was set in a light culture room with a room temperature of 25°C and a relative humidity of 75%. Light was applied from 8:00 to 20:00 every day. Two neutral salts (sodium chloride and sodium sulfate) and two alkaline salts (sodium bicarbonate and sodium carbonate) were selected in the experiment and mixed in proportion to simulate the salt-alkali stress environment. The molar ratio of each component was sodium chloride: sodium sulfate: sodium bicarbonate: sodium carbonate = 1:9:9:1. Wheat seeds with consistent germination were selected and the seeds were cultivated in planting pots with 120g of soil. There were 10 germinated wheat seeds in each pot. Seven experimental groups were set up: a blank control group, a salt-alkali stress group, and five material groups under salt-alkali stress environment (the material application concentration was 30mg / L, 60mg / L, 120mg / L, 240mg / L, 480mg / L), wherein the material is citric acid-coated copper-doped zinc oxide nanozyme, and the saline-alkali stress concentration is set to 150mmol / L; in order to simulate the saline-alkali stress environment, the material was applied to the root experiment for three days before the start of the experiment. 15mL of water was applied to the blank control group for three days, and 15mL of saline-alkali solution was applied to the other groups for three days, so that the soil salt content under saline-alkali stress reached 6g / kg; the root application experiment lasted for 7 days, 15mL of material was applied once a day in the material group, and 15mL of water was applied once a day in the blank control group and the saline-alkali stress group, and the growth of wheat was photographed and recorded, and the effect of citric acid-coated copper-doped zinc oxide nanozyme in improving the resistance of plants under saline-alkali stress was analyzed.

Claims

1. A method for preparing citric acid-coated copper-doped zinc oxide nanozyme, characterized in that It is done in the following steps:

1. Preparation process of copper-doped zinc oxide nanozyme: zinc acetate, copper sulfate pentahydrate and deionized water are mixed to obtain a mixture; magnetic stirring is performed at a speed of 300rpm / min to 500rpm / min for 6h to 10h until it is completely dissolved to obtain a dissolved mixture solution; after the reaction is completed, the mixture solution is placed in a constant temperature drying oven to dry to obtain a white solid powder; the white solid powder is evenly ground and placed in a magnetic boat, which is placed in a tubular furnace and calcined at 450℃ to 550℃ for 3h to 5h to obtain a copper-doped zinc oxide nanozyme solid after the reaction; 2. Preparation process of citric acid-coated copper-doped zinc oxide nanozyme: The copper-doped zinc oxide nanozyme solid after the reaction is dispersed in 15mL~25mL ethanol and aqueous solution (ethanol: water volume ratio = 3~5:1), and then mixed with 15mL~25mL ethanol and aqueous solution of 0.01mol / L~0.05mol / L citric acid (ethanol: water volume ratio = 3~5:1), and stirred to obtain a mixture solution; the reaction solution is dried and ground to obtain citric acid-coated copper-doped zinc oxide nanozyme.

2. The method for preparing a citric acid-coated copper-doped zinc oxide nanozyme according to claim 1, characterized in that: The stirring temperature of the mixture in step 1 is 50°C to 60°C.

3. The method for preparing a citric acid-coated copper-doped zinc oxide nanozyme according to claim 1, characterized in that: The mass ratio of zinc acetate to copper sulfate pentahydrate in step 1 is 1 mg: (4 mg to 4.5 mg), and the mass ratio of zinc acetate to deionized water in the step is 1 mg: (20 mL to 40 mL).

4. The method for preparing a citric acid-coated copper-doped zinc oxide nanozyme according to claim 1, characterized in that: The muffle furnace calcination atmosphere in step 1 is nitrogen.

5. The method for preparing a citric acid-coated copper-doped zinc oxide nanozyme according to claim 1, characterized in that: The stirring time during the citric acid coating process in step 2 is 2 h to 3 h, and the stirring temperature is 20° C. to 30° C.

6. Use of a citric acid-coated copper-doped zinc oxide nanozyme prepared by the preparation method of claim 1 in improving plant resistance to stress, characterized in that It is done in the following steps:

1. Preparation of citric acid-coated copper-doped zinc oxide nanozyme dispersion: The citric acid-coated copper-doped zinc oxide nanozyme was dispersed in deionized water by ultrasonic vibration for 3 min to 10 min to prepare 30 mg / L to 480 mg / L citric acid-coated copper-doped zinc oxide nanozyme dispersion; 2. Seed germination process: The wheat variety is conventional wheat sold in the local market, and seeds of the same size are selected for germination experiments; the wheat seeds are placed in an oven, dried at 25°C to 35°C for 12h to 24h, disinfected with 5% to 10% sodium hypochlorite for 10min to 15min and repeatedly rinsed with deionized water; the germination process of wheat is set in a light culture room, the room temperature is 20°C to 25°C, the relative humidity is 65% to 85%, and light is applied from 8:00 to 20:00 every day; 3. Treatment process of wheat plant resistance to salt-alkali stress: The wheat plant growth process is set in a light culture room with a room temperature of 20℃~25℃, a relative humidity of 65%~85%, and light exposure from 8:00 to 20:00 every day; two neutral salts (sodium chloride and sodium sulfate) and two alkaline salts (sodium bicarbonate and sodium carbonate) are selected in the experiment, and mixed in proportion to simulate the salt-alkali stress environment. The molar ratio of each component is sodium chloride: sodium sulfate: sodium bicarbonate: sodium carbonate = 1:9:9:1; wheat seeds with consistent germination are selected, and the seeds are cultivated in a planting pot with 120g of soil, with 5 to 10 germinated wheat seeds in each pot, and seven experimental groups are set up respectively: one blank control group, one salt-alkali stress group, and five material groups under salt-alkali stress environment (the material application concentration is 30mg / L~480mg / L), and the other In the experiment, the material was citric acid-coated copper-doped zinc oxide nanozyme, and the saline-alkali stress concentration was set at 120mmol / L~160mmol / L; in order to simulate the saline-alkali stress environment, the material was applied with a solution for three days before the root experiment began, of which the blank control group was applied with 10mL~15mL of water once a day, and the other groups were applied with 10mL~15mL of saline-alkali solution once a day, so that the soil salt content under saline-alkali stress reached 4g / kg~6g / kg; the root application experiment lasted for 5~7 days, the material group was applied with 10mL~15mL of material once a day, and the blank control group and saline-alkali stress group were applied with 10mL~15mL of water once a day, and the wheat growth was photographed and recorded to analyze the effect of citric acid-coated copper-doped zinc oxide nanozyme in improving the stress resistance of plants under saline-alkali stress.

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