Preparation method of Zr metal organic framework nano-enzyme with phosphatase activity and application of Zr metal organic framework nano-enzyme in relieving plant low-phosphorus stress

Abstract

The invention discloses a preparation method of a Zr metal organic framework nano-enzyme with phosphatase activity and application of the Zr metal organic framework nano-enzyme in relieving low-phosphorus stress of plants, and relates to a method for preparing the Zr metal organic framework nano-enzyme and application of the Zr metal organic framework nano-enzyme in relieving low-phosphorus stress of plants by utilizing the phosphatase activity of the Zr metal organic framework nano-enzyme. The invention aims to solve the problems that precise fertilization is difficult and the environment is easy to pollute in the process of relieving low phosphorus stress of plants by applying a phosphate fertilizer at present. The preparation method comprises the following step: synthesizing the Zr metal organic framework nano-enzyme with phosphatase activity and ultra-small size from Zr6 clusters and 2-fluoroterephthalic acid by adopting a solvent induction method. The application method comprises the following steps: pouring the aqueous dispersion of the Zr metal organic framework nano-enzyme into phosphorus-deficient soil, and mineralizing organophosphorus release plants in the soil by virtue of the phosphatase activity of the Zr metal organic framework nano-enzyme, so that low-phosphorus stress can be relieved by utilizing the organophosphorus release plants. The prepared Zr metal organic framework nano-enzyme is good in phosphatase activity and high in stability, and the established method for relieving plant low-phosphorus stress is convenient and efficient.

Description

Technical Field

[0001] This invention relates to the preparation of a metal-organic framework nanozyme with phosphatase activity and its application in alleviating low phosphorus stress in plants. Background Technology

[0002] Phosphorus is one of the three essential nutrients for plant growth, playing a crucial role in regulating metabolism and the synthesis of genetic material. Plants can utilize phosphorus in the form of soluble phosphates (such as PO42-). 3- and HPO4 2- Phosphorus exists primarily in the form of organic phosphorus, derived from soil humus and phosphorus-containing minerals. However, most phosphorus in the soil exists in the form of organic phosphorus, which cannot be directly utilized by plants. Low phosphorus stress typically occurs in soil environments affected by floods and fires, inhibiting plant growth, reducing yield and quality, and has become a focus of national economic and rare plant protection efforts. Plants usually alleviate mild low phosphorus stress by releasing organic acids and phosphatases to decompose organic phosphorus in the soil. When facing severe phosphorus deficiency, relying solely on plant self-regulation is insufficient. Currently, phosphate fertilizers are commonly applied, but this often leads to over-fertilization due to overestimating plant uptake, resulting in eutrophication of surface water sources. Invention patent CN201010289297.X discloses a growth regulator containing 24-epibrassinolide, ethanol, and water for alleviating low phosphorus stress in cucumbers. However, this method alleviates low phosphorus stress indirectly by regulating the crop rather than directly supplying usable phosphate. Therefore, the agricultural sector increasingly needs more ecological and convenient methods to continuously protect plants from low phosphorus stress.

[0003] With the development of nanotechnology, nanozymes that mimic the function of phosphatases can not only catalyze the decomposition of phosphoryl-containing organic phosphorus into PO4 that can be utilized by plants. 3- Moreover, its stability and low cost make it a potential nano-fertilizer to help plants resist low phosphorus stress. Metal-organic frameworks (MOFs) are versatile porous crystalline materials, and their tunable and ordered structure makes them ideal for use as nanozymes. Metal nodes in MOFs act as active centers, and ligands act as molecular bridges, thus jointly mimicking a pattern similar to the catalytic center and cofactors in natural enzymes. Metal-ligand interactions provide chemoregioselectivity for the enzyme-like catalytic operation of MOF nanozymes. The ligand's intervention on the charge density near the central metal ion controls the Lewis acidity, which is directly related to the expression of phosphatase activity, significantly affecting the affinity and cleavage of the nanozyme for phosphorus-containing substrates. Therefore, obtaining satisfactory phosphatase activity by modifying ligands for MOF nanozymes is fundamental to enabling them to serve as effective nano-fertilizers to alleviate low phosphorus stress in plants. Summary of the Invention

[0004] The purpose of this invention is to develop a novel method to alleviate low phosphorus stress in plants, meeting the needs of precision agriculture. Based on this, a method for preparing a metal-organic framework nanozyme with phosphatase activity and its application in alleviating low phosphorus stress in plants is proposed. This patent focuses on plant physiological environment and efficient phosphate substrate mass transfer, using a solvent-induced method combined with ligand engineering to prepare an ultra-small Zr metal-organic framework nanozyme with good phosphatase activity. Using Arabidopsis thaliana as a model plant, studies have shown that this nanozyme can mineralize fixed organic phosphorus in low-phosphorus soils, releasing plant-available PO4. 3- It effectively alleviated low phosphorus stress.

[0005] A method for preparing a Zr metal-organic framework nanozyme with phosphatase activity is specifically carried out according to the following steps:

[0006] I. Synthesis of Zr6 cluster: Zirconium chloride was dissolved in a mixed solution of acetic acid and isopropanol; the mixture was reacted at 110℃ ~ 130℃ and a stirring rate of 600 rpm for 50 min ~ 70 min; after the reaction, it was cooled to room temperature; the reaction product was centrifuged at 10000 rpm for 10 min, and the solid product was collected; the product was washed three times with acetone and then vacuum dried at 60℃ for 8 h. The white powder obtained after drying was Zr6 cluster; the mass of zirconium chloride in the steps was 0.9 g ~ 1.2 g; the volume ratio of acetic acid to isopropanol in the steps was 1 mL : (1.5 mL ~ 1.8 mL);

[0007] II. Synthesis process of Zr metal-organic framework nanozyme: Zr6 clusters were dispersed in a mixed solution of acetic acid and deionized water and stirred at 600 rpm at 25°C until clear; this mixed solution was then added simultaneously with 50 mg to 60 mg of 2-fluoroterephthalic acid to 75 mL to 90 mL of ethanol; the mixture was stirred at 600 rpm at 25°C for 100 to 140 min; the product was centrifuged at 14500 rpm for 60 min; the product was washed three times with a mixed solution of ethanol and acetone and then vacuum dried at 60°C for 8 h. The white powder obtained after drying was the Zr metal-organic framework nanozyme; the mass of the Zr6 clusters in the steps was 65 mg to 80 mg; the volume ratio of acetic acid to deionized water in the steps was 1 mL:(1 mL to 2 mL); the volume ratio of ethanol to acetone in the steps was 1 mL:(1 mL to 1.5 mL);

[0008] III. Identification of Phosphatase Activity of Zr Metal-Organic Framework Nanozymes: 50 μL ~ 100 μL of 1.5 mg / mL ~ 2 mg / mL disodium p-nitrophenyl phosphate aqueous solution was mixed with 25 μL ~ 40 μL of 1.5 mg / mL ~ 2 mg / mL Zr metal-organic framework nanozyme aqueous dispersion; the mixture was diluted to 1.4 mL ~ 1.6 mL with 10 mM Tris-HCl solution at pH=9; after incubation at 40℃ for 15 min ~ 25 min, the absorption spectrum of the system was measured by UV-Vis spectrophotometer; the phosphatase performance of Zr metal-organic framework nanozymes was evaluated by the absorption peak near 405 nm.

[0009] The application process of a Zr metal-organic framework nanozyme with phosphatase activity to alleviate low phosphorus stress in plants is carried out in the following steps:

[0010] I. Cultivation of Arabidopsis thaliana: The culture soil is repeatedly soaked with deionized water until the PO4 content in the soil is reduced. 3- The concentration of phosphorus was below 3 mg / kg to create a low-phosphorus environment; Arabidopsis seeds were disinfected with 5% sodium hypochlorite solution and then rinsed three times with deionized water; Arabidopsis seeds were vernalized at 4℃ for three days; Arabidopsis seeds were planted in normal culture soil and cultured for one week before being transplanted into separate pots; Arabidopsis seedlings were transplanted into normal culture soil, low-phosphorus culture soil, and low-phosphorus culture soil with 1 mL ~ 2 mL of Zr metal-organic framework nanozyme at a concentration of 60 μg / mL, and cultured for another two weeks; Arabidopsis were grown in an incubator at 22℃ with a light / dark cycle of 16 h / 8 h.

[0011] II. Analysis of the effect of Zr metal-organic framework nanozymes on alleviating low phosphorus stress in Arabidopsis thaliana: Roots and leaves of Arabidopsis thaliana collected in step one were analyzed; phenotypic information of taproots, lateral roots, and adventitious roots of Arabidopsis thaliana under different culture conditions was collected; phenotypic information of leaf color, shape, and size of Arabidopsis thaliana under different culture conditions was collected; Arabidopsis thaliana leaves were washed several times with deionized water and then frozen with liquid nitrogen; the leaves were ground in a pre-frozen mortar in the dark; 50 mg ~ 150 mg of leaf powder was mixed with 8 mL ~ 12 mL of acetone; the mixture was shaken in an ice bath for 1.5 h ~ 2 h until the powder faded; the mixture was centrifuged at 5000 rpm for 10 min at 4°C; the absorption spectrum of the supernatant was measured using a UV-Vis spectrophotometer, and the total chlorophyll content was calculated based on the absorbance values ​​at 645 nm and 663 nm.

[0012] Advantages of this invention: 1. The developed Zr metal-organic framework nanozyme is small in size and possesses strong phosphatase activity, showing good potential in biocompatibility and catalytic substrate mass transfer; 2. The Zr metal-organic framework nanozyme exhibits strong stability, maintaining 90% of its catalytic performance within 30 days; 3. The Zr metal-organic framework nanozyme releases plant-available PO4 from organic phosphorus fixed in mineralized soil. 3- 4. The proposed method for alleviating low phosphorus stress is simple to operate and easy to use on a large scale. Attached Figure Description

[0013] Figure 1 This is a transmission electron microscope image of the Zr metal-organic framework nanozyme from Example 1; Figure 1 It can be seen that the Zr metal-organic framework nanozymes are smaller than 10 nm in size and well dispersed;

[0014] Figure 2 This is the X-ray photoelectron spectrum of the Zr metal-organic framework nanozyme in Example 1; Figure 2 The medium-carbon quantum dot nanozyme exhibited five strong peaks at 184.3 eV, 284.6 eV, 333.2 eV, 530.8 eV, and 686.8 eV, which were attributed to Zr 3d, C 1s, Zr 3p, O 1s, and F 1s, respectively, indicating that the ligand and Zr6 cluster were successfully assembled into a Zr metal-organic framework nanozyme.

[0015] Figure 3 This is a graph showing the catalytic properties of the Zr metal-organic framework nanozyme in Example 1 using disodium p-nitrophenyl phosphate as a substrate; in the graph, a represents the Zr metal-organic framework nanozyme and the disodium p-nitrophenyl phosphate system, and b represents the disodium p-nitrophenyl phosphate system; from Figure 3 It is known that Zr metal-organic framework nanozymes can hydrolyze disodium p-nitrophenyl phosphate and release a chromogenic substrate with a characteristic peak near 405 nm.

[0016] Figure 4 The left figure shows the phenotypic analysis of roots during the process of Zr metal-organic framework nanozymes alleviating low phosphorus stress in Arabidopsis thaliana in Example 2; Figure 4 The right figure corresponds to Figure 4 The left figure shows the differences in root length under different cultivation conditions. Figure a represents the root length of *Arabidopsis thaliana* in normal soil; figure b represents the root length of *Arabidopsis thaliana* under low phosphorus stress; figure c represents the root length of *Arabidopsis thaliana* cultivated in low phosphorus soil after 14 days with Zr metal-organic framework nanozymes added; figure d represents the root length of *Arabidopsis thaliana* cultivated in low phosphorus soil after 18 days with Zr metal-organic framework nanozymes added; and figure e represents the root length of *Arabidopsis thaliana* cultivated in low phosphorus soil after 21 days with Zr metal-organic framework nanozymes added. Figure 4It can be seen that Zr metal-organic framework nanozymes effectively restore the taproot length of Arabidopsis thaliana in low phosphorus environments and reduce the density of lateral roots and adventitious roots.

[0017] Figure 5 The left figure shows the phenotypic analysis of leaves during the process of Zr metal-organic framework nanozymes alleviating low phosphorus stress in Arabidopsis thaliana in Example 2; Figure 5 The right figure corresponds to Figure 5 The left figure shows the difference in total chlorophyll content in leaves under different cultivation conditions. Figure a represents the total chlorophyll content in Arabidopsis leaves cultivated in normal soil, figure b represents the total chlorophyll content in Arabidopsis leaves cultivated under low phosphorus stress, and figure c represents the total chlorophyll content in Arabidopsis leaves after 21 days of cultivation with Zr metal-organic framework nanozymes added to low phosphorus soil. Figure 5 It can be seen that Zr metal-organic framework nanozymes effectively alleviated the slowed development, purple discoloration of leaves, and decrease in total chlorophyll content caused by low phosphorus environment in Arabidopsis leaves. Detailed Implementation

[0018] Example 1:

[0019] This embodiment describes a method for preparing a Zr metal-organic framework nanozyme with phosphatase activity, specifically carried out according to the following steps:

[0020] I. Synthesis of Zr6 cluster: 1 g of zirconium chloride was dissolved in a mixed solution of 1.5 mL acetic acid and 2.5 mL isopropanol; the mixture was reacted at 120 °C and 600 rpm for 60 min; after the reaction was completed, it was cooled to room temperature; the reaction product was centrifuged at 10000 rpm for 10 min, and the solid product was collected; the product was washed three times with acetone and then vacuum dried at 60 °C for 8 h. The powder obtained after drying was Zr6 cluster.

[0021] II. Synthesis process of Zr metal-organic framework nanozyme: 75 mg of Zr6 cluster was dispersed in a mixed solution of 0.5 mL acetic acid and 1.25 mL deionized water, and stirred at 600 rpm at 25 °C until clear; this mixed solution and 55 mg of 2-fluoroterephthalic acid were added to 80 mL of ethanol; the mixture was stirred at 600 rpm at 25 °C for 120 min; the product was centrifuged at 14500 rpm for 60 min; the product was washed three times with a mixed solution of 5 mL ethanol and 5 mL acetone, and then vacuum dried at 60 °C for 8 h. The powder obtained after drying was Zr metal-organic framework nanozyme.

[0022] III. Identification of Phosphatase Activity of Zr Metal-Organic Framework Nanozymes: 70 μL of 2 mg / mL disodium p-nitrophenyl phosphate aqueous solution was mixed with 30 μL of 2 mg / mL Zr metal-organic framework nanozyme aqueous dispersion; the mixture was diluted to 1.5 mL with 10 mM Tris-HCl solution at pH 9; after incubation at 40 °C for 20 min, the absorption spectrum of the system was measured by UV-Vis spectrophotometer; the phosphatase performance of Zr metal-organic framework nanozymes was evaluated by the absorption peak near 405 nm.

[0023] Example 2:

[0024] This embodiment describes the application process of a Zr metal-organic framework nanozyme with phosphatase activity to alleviate low phosphorus stress in plants, specifically completed according to the following steps:

[0025] I. Cultivation of Arabidopsis thaliana: The culture soil is repeatedly soaked with deionized water until the PO4 content in the soil is reduced. 3- The concentration of phosphorus was below 3 mg / kg to create a low-phosphorus environment; Arabidopsis seeds were disinfected with 5% sodium hypochlorite solution and then rinsed three times with deionized water; Arabidopsis seeds were vernalized at 4℃ for three days; Arabidopsis seeds were planted in normal culture soil and cultured for one week before being transplanted into separate pots; Arabidopsis seedlings were transplanted into normal culture soil, low-phosphorus culture soil, and low-phosphorus culture soil with 1.5 mL of 60 μg / mL Zr metal-organic framework nanozyme added, and cultured for another two weeks; Arabidopsis were grown in an incubator at 22℃ with a light / dark cycle of 16 h / 8 h.

[0026] II. Analysis of the effect of Zr metal-organic framework nanozymes on alleviating low phosphorus stress in Arabidopsis thaliana: Roots and leaves of Arabidopsis thaliana collected in step one were analyzed; phenotypic information of taproots, lateral roots, and adventitious roots of Arabidopsis thaliana under different culture conditions was collected; phenotypic information of leaf color, shape, and size of Arabidopsis thaliana under different culture conditions was collected; Arabidopsis thaliana leaves were washed several times with deionized water and then frozen with liquid nitrogen; the leaves were ground in a pre-frozen mortar in the dark; 100 mg of leaf powder was mixed with 10 mL of acetone; the mixture was shaken in an ice bath for 2 h until the powder faded; the mixture was centrifuged at 5000 rpm for 10 min at 4°C; the absorption spectrum of the supernatant was measured using a UV-Vis spectrophotometer, and the total chlorophyll content was calculated based on the absorbance values ​​at 645 nm and 663 nm.

Claims

1. A method for preparing a Zr metal-organic framework nanozyme with phosphatase activity, characterized in that... It was accomplished in the following steps: I. Synthesis of Zr6 cluster: Zirconium chloride was dissolved in a mixed solution of acetic acid and isopropanol; the mixture was reacted at 110℃ ~ 130℃ and a stirring rate of 600 rpm for 50 min ~ 70 min; after the reaction, it was cooled to room temperature; the reaction product was centrifuged at 10000 rpm for 10 min, and the solid product was collected; the product was washed three times with acetone and then vacuum dried at 60℃ for 8 h. The white powder obtained after drying was Zr6 cluster; the mass of zirconium chloride in the steps was 0.9 g ~ 1.2 g; the volume ratio of acetic acid to isopropanol in the steps was 1 mL : (1.5 mL ~ 1.8 mL); II. Synthesis process of Zr metal-organic framework nanozyme: Zr6 clusters were dispersed in a mixed solution of acetic acid and deionized water and stirred at 600 rpm at 25°C until clear; this mixed solution was then added simultaneously with 50 mg to 60 mg of 2-fluoroterephthalic acid to 75 mL to 90 mL of ethanol; the mixture was stirred at 600 rpm at 25°C for 100 to 140 min; the product was centrifuged at 14500 rpm for 60 min; the product was washed three times with a mixed solution of ethanol and acetone and then vacuum dried at 60°C for 8 h. The white powder obtained after drying was the Zr metal-organic framework nanozyme; the mass of the Zr6 clusters in the steps was 65 mg to 80 mg; the volume ratio of acetic acid to deionized water in the steps was 1 mL:(1 mL to 2 mL); the volume ratio of ethanol to acetone in the steps was 1 mL:(1 mL to 1.5 mL); III. Identification of Phosphatase Activity of Zr Metal-Organic Framework Nanozymes: 50 μL ~ 100 μL of 1.5 mg / mL ~ 2 mg / mL disodium p-nitrophenyl phosphate aqueous solution was mixed with 25 μL ~ 40 μL of 1.5 mg / mL ~ 2 mg / mL Zr metal-organic framework nanozyme aqueous dispersion; the mixture was diluted to 1.4 mL ~ 1.6 mL with 10 mM Tris-HCl solution at pH=9; after incubation at 40℃ for 15 min ~ 25 min, the absorption spectrum of the system was measured by UV-Vis spectrophotometer; the phosphatase performance of Zr metal-organic framework nanozymes was evaluated by the absorption peak near 405 nm.

2. The application process of using the Zr metal-organic framework nanozyme with phosphatase activity as described in claim 1 to alleviate low phosphorus stress in plants, characterized in that... It was accomplished in the following steps: I. Cultivation of Arabidopsis thaliana: The culture soil is repeatedly soaked with deionized water until the PO4 content in the soil is reduced. 3- The concentration of phosphorus was below 3 mg / kg to create a low-phosphorus environment; Arabidopsis seeds were disinfected with 5% sodium hypochlorite solution and then rinsed three times with deionized water. Arabidopsis seeds were vernalized at 4℃ for three days; Arabidopsis seeds were planted in normal potting soil and cultured for one week before being transplanted into individual pots; Arabidopsis seedlings were transplanted into normal potting soil, low-phosphorus potting soil, and low-phosphorus potting soil supplemented with 1-2 mL of Zr metal-organic framework nanozyme at a concentration of 60 μg / mL, and cultured for another two weeks; Arabidopsis were grown in an incubator at 22℃ with a light / dark cycle of 16h / 8h. II. Analysis of the effect of Zr metal-organic framework nanozymes on alleviating low phosphorus stress in Arabidopsis thaliana: Roots and leaves of Arabidopsis thaliana collected in step one were analyzed; phenotypic information of taproots, lateral roots, and adventitious roots of Arabidopsis thaliana under different culture conditions was collected; phenotypic information of leaf color, shape, and size of Arabidopsis thaliana under different culture conditions was collected; Arabidopsis thaliana leaves were washed several times with deionized water and then frozen with liquid nitrogen; the leaves were ground in a pre-frozen mortar in the dark; 50 mg ~ 150 mg of leaf powder was mixed with 8 mL ~ 12 mL of acetone; the mixture was shaken in an ice bath for 1.5 h ~ 2 h until the powder faded; the mixture was centrifuged at 5000 rpm for 10 min at 4°C; the absorption spectrum of the supernatant was measured using a UV-Vis spectrophotometer, and the total chlorophyll content was calculated based on the absorbance values ​​at 645 nm and 663 nm.

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