Method for enhancing plant salt stress resistance based on polyoxometallate-rhizosphere bacterium composite system

Abstract

The invention discloses a method for enhancing plant salt stress resistance based on a polyoxometallate-rhizosphere bacterium composite system. The method comprises the following steps: step 1, preparing a polyoxometallate nanocluster with enzyme-like activity; step 2, carrying out surface modification on the polyoxometallate by adopting polyethyleneimine; step 3, loading the modified polyoxometallate on the surfaces of rhizosphere microorganisms to construct a polyoxometallate-rhizosphere bacterium composite system; 4, applying the composite system to plants under the salt stress condition so as to relieve salt stress and promote plant growth. The method has the advantages that the method for regulating and controlling the salt stress resistance of the plants is simple and convenient to operate and high in practicability; by synergistically exerting the effects of removing active oxygen and promoting growth of rhizosphere microorganisms of the polyoxometallate, oxidative damage induced by salt stress is effectively reduced; the method is good in biocompatibility and suitable for plant growth regulation and control under the salt stress condition.

Description

Technical Field

[0001] This invention belongs to the field of bio-nanomaterials and agricultural environmental remediation technology, specifically relating to a composite material (PEI-Mo-POM@Bs) constructed based on polyethyleneimine-modified molybdenum-based polyacid nanoclusters and Bacillus subtilis, and its method for promoting plant growth and alleviating salt stress. Background Technology

[0002] Soil salinization is a significant environmental factor restricting plant growth and ecosystem stability. Salt stress can induce excessive accumulation of reactive oxygen species (ROS) in plants through osmotic stress and ion toxicity, leading to oxidative damage to cells and inhibiting normal plant growth and development. Dandelion (Taraxacum officinale) is a perennial herb with a certain degree of environmental adaptability and has potential applications in ecological restoration and saline-alkali land utilization. However, under salt stress, dandelion seedlings generally exhibit problems such as inhibited growth, poor root development, and aggravated oxidative damage, indicating room for improvement in their salt tolerance. Existing methods for alleviating plant salt stress mainly include exogenous regulation and the use of rhizosphere growth-promoting microorganisms, but single microbial systems are prone to insufficient stability and functional decline in high-salt environments. Meanwhile, although polyoxometalates have good ROS scavenging capabilities, their stability and applicability in biological environments remain limited. Therefore, there is an urgent need to develop a technical method that can synergistically leverage the antioxidant properties of polyoxometalates and the growth-promoting effects of rhizosphere microorganisms to effectively enhance the growth performance of dandelion under salt stress conditions. This invention is proposed against this backdrop. Summary of the Invention

[0003] The purpose of this invention is to construct a PEI-Mo-POM@Bs composite system by loading positively charged polyethyleneimine (PEI) modified Mo-POM onto the surface of negatively charged Bacillus subtilis (Bs) using electrostatic self-assembly technology. This system is used to scavenge reactive oxygen species generated by plant stress and promote the growth of plants in saline-alkali land.

[0004] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0005] A method for enhancing plant salt stress resistance based on a polyoxometalate-rhizosphere bacteria complex system, the method comprising the following steps:

[0006] Step 1: Synthesis of Mo-POM Nanoclusters

[0007] Under stirring conditions, the phosphomolybdic acid solution was mixed with the reducing agent and reacted for 2-5 h until the solution turned dark blue or dark green. Unreacted substances were removed by dialysis to obtain a Mo-POM nanocluster solution with enzyme-like activity.

[0008] Step 2: Preparation of PEI-modified Mo-POM

[0009] Polyethyleneimine (PEI, molecular weight 25000 Da) was dissolved in deionized water to prepare a PEI solution of 0.5~5 mg / mL; Mo-POM solution was added to the PEI solution under stirring and reacted for 30~60 min. Free PEI was removed by dialysis to obtain a PEI-Mo-POM modified solution with a positively charged surface.

[0010] Step 3: Construction of the PEI-Mo-POM@Bs composite system

[0011] After Bacillus subtilis was cultured to the logarithmic growth phase, the bacterial cells were collected and washed with buffer. The bacterial cells were mixed with PEI-Mo-POM solution and shaken at room temperature for 30-60 min to allow nanoclusters to be loaded onto the bacterial cell surface through electrostatic adsorption. The PEI-Mo-POM@Bs composite system was obtained by centrifugation.

[0012] Step 4: Relief of Salt and Alkali Stress in Plants

[0013] Mo-POM, PEI-Mo-POM, Bacillus subtilis (Bs), and PEI-Mo-POM@Bs were dispersed in deionized water or buffer solution to prepare treatment solutions with a concentration of 200 μg / mL. These solutions were then applied to dandelion seedlings under NaCl salt stress conditions via root application or root soaking. After culturing for 7–14 days, plant growth indicators and oxidative stress-related physiological indicators under different treatments were measured to evaluate their salt stress resistance.

[0014] The advantages of this invention over the prior art are as follows:

[0015] (1) It combines the enzyme-like activity of Mo-POM with the bio-promoting ability of Bacillus subtilis to synergistically alleviate salt stress.

[0016] (2) Rapid electrostatic self-assembly is achieved by utilizing the positive charge of PEI and the negative charge on the surface of bacteria, without the need for complex chemical cross-linking agents, and with good biocompatibility.

[0017] (3) Experiments show that within a certain concentration range, the composite system can significantly reduce the ROS intensity in stressed plants and restore the plant growth phenotype. Attached Figure Description

[0018] Figure 1 The image shows a scanning electron microscope (SEM) image of the Mo-POM nanoclusters obtained in the example.

[0019] Figure 2 The diagram shows the change in Zeta potential of the Mo-POM modified materials before and after the example.

[0020] Figure 3 Scanning electron microscope (SEM) images of the PEI-Mo-POM@Bs composite material obtained in the example;

[0021] Figure 4 The examples show a comparison of the Zeta potentials of different materials.

[0022] Figure 5 The UV-Vis spectra of free radicals in PEI-Mo-POM@Bs composite materials of different concentrations are shown in the examples.

[0023] Figure 6 This study evaluates the biocompatibility of PEI-Mo-POM@Bs with plants in the example.

[0024] Figure 7 The following is a comparison of the phenotypes of dandelion seedlings under different treatment conditions in the examples.

[0025] Figure 8 The following are statistical results of the root length of dandelion seedlings in different treatment groups in the example.

[0026] Figure 9 The following are the results of the detection of reactive oxygen species levels in dandelion leaves in the example. Detailed Implementation

[0027] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments. However, the experimental conditions and setting parameters therein should not be regarded as limitations on the basic technical solution of the present invention. Any modifications or equivalent substitutions to the technical solution of the present invention that do not depart from the spirit and content of the technical solution of the present invention should be covered within the protection scope of the present invention.

[0028] Step 1: Synthesis of Mo-POM Nanoclusters

[0029] Under stirring, phosphomolybdic acid solution was mixed with gallic acid and stirred vigorously. Then, 7.5 wt% sodium carbonate solution was added and the reaction was continued at room temperature for 5 h until the solution color turned dark blue or dark green. Unreacted substances were removed by dialyzing for 3 days and then freeze-dried to obtain Mo-POM nanoclusters.

[0030] Step 2: Preparation of PEI-modified Mo-POM

[0031] (1) Dissolve polyethyleneimine (PEI, molecular weight 25000 Da) in deionized water to prepare an aqueous solution of PEI with a mass concentration of 0.5~5 mg / mL.

[0032] (2) Under vigorous stirring, the Mo-POM solution obtained in step one is added dropwise to the PEI solution to utilize the amino group of PEI to combine with Mo-POM.

[0033] (3) After reacting for 30 minutes, the free PEI was removed by dialysis through a dialysis bag (molecular weight cutoff 3500 Da) to obtain a PEI-Mo-POM modified solution with a positive charge on the surface.

[0034] Step 3: Construction of the PEI-Mo-POM@Bs composite system

[0035] (1) Bacillus subtilis was cultured in LB liquid medium with shaking until the logarithmic growth phase (OD). 600 (≈0.6~0.8), centrifuge to collect bacterial cells, and wash three times with PBS buffer to remove culture medium residue.

[0036] (2) The washed Bs cells were suspended in PBS, and the PEI-Mo-POM solution prepared in step 2 was added. The mixture was shaken on a shaker (100~150 rpm) for 30~60 minutes in the dark at room temperature to load the nanoclusters onto the surface of the cells using electrostatic adsorption.

[0037] (3) Centrifuge (5000~8000 rpm), discard the supernatant, and collect the precipitate to obtain the PEI-Mo-POM@Bs composite material.

[0038] Step 4: Application of plant salt-alkali stress relief

[0039] Mo-POM, PEI-Mo-POM, Bacillus subtilis (Bs), and PEI-Mo-POM@Bs were dispersed in deionized water or buffer solution to prepare treatment solutions with a concentration of 200 μg / mL. These solutions were applied to dandelion seedlings under NaCl salt stress via root irrigation or root soaking. After a certain period of cultivation, plant growth and physiological indicators related to oxidative stress were assessed.

[0040] Example:

[0041] A method for enhancing plant salt stress resistance based on a polyoxometalate-rhizosphere bacteria complex system is specifically carried out according to the following steps:

[0042] I. Synthesis of Mo-POM Nanoclusters

[0043] Dissolve 100 mg of gallic acid in 6 mL of deionized water. After complete dissolution, add 1 mL of H₃O₂. 40 PMo 12Add .xH2O (70 mg / mL) and stir vigorously for 5 min. Then add 3.0 mL of Na2CO3 solution (7.5 wt%), and let the entire system stand for 5 h with continuous stirring. Then, place the dark green solution in a dialysis bag (M... W The purified aqueous solution was freeze-dried after being immersed in water containing 3500 Da for 3 days to obtain the desired Mo-POM nanoclusters. Figure 1 The scanning electron microscope (SEM) image of the Mo-POM nanoclusters shows that the prepared Mo-POM exhibits a uniformly dispersed nanocluster structure with a particle size in the nanoscale range, indicating that a structurally stable polyoxometalate nanomaterial has been successfully obtained.

[0044] II. Preparation of PEI-modified Mo-POM

[0045] To prepare the PEI solution, accurately weigh polyethyleneimine (PEI, molecular weight 25000 Da), dissolve it in deionized water, and mix thoroughly to prepare a PEI aqueous solution with a mass concentration of 0.5–5 mg / mL. Then, adjust the pH of the PEI aqueous solution to 7.0–7.4 using a 0.1 mol / L hydrochloric acid solution.

[0046] Under ambient temperature and in the dark, the PEI solution was vigorously stirred (800-1000 rpm). The Mo-POM solution prepared in step one was added dropwise to the PEI solution at a rate of 0.5-1.0 mL / min. After the addition was complete, the reaction was continued with stirring for 30-60 minutes.

[0047] The above reaction mixture was transferred to a dialysis bag (molecular weight cutoff 3500 Da) and dialyzed for 12-24 hours to remove unbound free PEI. After that, it was lyophilized to obtain a PEI-Mo-POM modified solution with a positively charged surface and good dispersibility. Figure 2 The diagram shows the change in Zeta potential of the material before and after Mo-POM modification. It can be seen that after PEI modification, the surface potential of the material changes from negative to positive, indicating that PEI was successfully modified on the Mo-POM surface.

[0048] III. Construction of PEI-Mo-POM@Bs Composite System

[0049] Cell culture and pretreatment: Activated Bacillus subtilis was inoculated into fresh LB liquid medium and cultured with shaking at 37°C and 180 rpm until the logarithmic growth phase (OD600≈0.6~0.8). The bacterial suspension was centrifuged at 4°C and 5000-6000 rpm for 5~8 minutes to collect the bacterial pellet, which was then washed three times with sterile PBS buffer (pH 7.4). Finally, the pellet was resuspended in PBS and the bacterial concentration was adjusted to OD600=1.0.

[0050] Electrostatic self-assembly: A certain volume (e.g., 5-10 mL) of Bs cell suspension with adjusted concentration is placed in a reaction vessel, and the PEI-Mo-POM modified solution prepared in step two is added at a volume ratio of 1:1 to 1:3. Under room temperature and in the dark, the mixture is incubated on a shaker at 100-150 rpm for 30-60 minutes.

[0051] Purification and Collection: After incubation, the reaction mixture was centrifuged at 5000-6000 rpm for 5-8 minutes, and the supernatant was discarded to remove unbound free nanomaterials. After washing with sterile PBS, the purified PEI-Mo-POM@Bs composite material was obtained. Figure 3 The image shows a scanning electron microscope (SEM) image of the PEI-Mo-POM@Bs composite material. It can be seen that the PEI-Mo-POM nanomaterials are uniformly loaded on the surface of the bacteria, and the bacterial structure remains intact. Figure 4 The figure shows a comparison of the Zeta potentials of Mo-POM, PEI-Mo-POM, Bacillus subtilis (Bs), and PEI-Mo-POM@Bs. The figure shows that the surface of Mo-POM is negatively charged, but its surface potential becomes positively charged after modification with PEI. The surface of Bacillus subtilis carries a negative charge, and after being combined with PEI-Mo-POM, the surface potential of the composite system changes significantly, indicating that PEI-Mo-POM is successfully loaded onto the bacterial surface through electrostatic interaction, thus confirming the formation of the composite system from an electrical perspective.

[0052] IV. Evaluation of the oxidation resistance of PEI-Mo-POM@Bs composites with different concentrations

[0053] The PEI-Mo-POM@Bs composite material synthesized in step three was serially diluted with deionized water to obtain composite material suspensions of different concentrations (10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, and 50 μg / mL). 0.6 mL of each composite material suspension was taken, and 1.6 mL of freshly prepared ABTS free radical working solution was added to each. The mixture was vortexed and then reacted at room temperature in the dark for 10 min. Absorbance was immediately measured at a wavelength of 734 nm. The instrument used was a UV-Vis spectrophotometer (UV-2600) purchased from Shimadzu Enterprise Management (China) Co., Ltd. Figure 5 The UV-Vis absorption spectra of PEI-Mo-POM@Bs composites at different concentrations for ABTS radicals show that the scavenging ability of the composite material significantly increases with increasing concentration, exhibiting a clear dose-dependent effect. The scavenging rate reaches its maximum at a concentration of 50 μg / mL, indicating that the PEI-Mo-POM@Bs composite material possesses excellent free radical scavenging activity.

[0054] V. Biocompatibility Testing of Composite Materials

[0055] Different concentrations (50–400 μg / mL) of the composite material were added dropwise to the surface of fresh dandelion leaves, with deionized water as a control. After 3–5 days of incubation, [the following was observed]: Figure 6 It can be seen that within the concentration range of 50–200 μg / mL, the leaf surface remained bright green, without necrotic spots or yellowing, consistent with the control group, demonstrating good biocompatibility. When the concentration increased to 300–400 μg / mL, slight yellowing and tissue dehydration and wilting appeared in the leaf contact area, indicating that high concentrations of the material have certain contact toxicity. In conclusion, this composite material exhibits excellent biocompatibility at concentrations ≤200 μg / mL.

[0056] VI. Relief of Salt and Alkali Stress in Plants

[0057] Mo-POM, PEI-Mo-POM, Bacillus subtilis (Bs), and PEI-Mo-POM@Bs were dispersed in deionized water or buffer solution to prepare treatment solutions with a concentration of 200 μg / mL. For dandelion seedlings under NaCl salt stress, the treatment solutions were applied by root irrigation or root soaking. After a certain period of cultivation, plant growth and physiological indicators related to oxidative stress were measured. Figure 7 The phenotypic comparison of dandelion seedlings under different treatment conditions shows that the growth status of dandelion seedlings treated with PEI-Mo-POM@Bs was significantly improved. Figure 8 Statistical results of root length of dandelion seedlings in different treatment groups. Figure 9 The results show the levels of reactive oxygen species in dandelion leaves.

Claims

1. A method for enhancing plant salt stress resistance based on a polyoxometalate-rhizosphere bacteria complex system, characterized in that: The method steps are as follows: Step 1: Synthesis of Mo-POM Nanoclusters Under stirring, phosphomolybdic acid solution was mixed with gallic acid and stirred vigorously. Then, 7.5 wt% sodium carbonate solution was added and the reaction was continued at room temperature for 5 h until the solution color turned dark blue or dark green. Unreacted substances were removed by dialysis for 3 days and then freeze-dried to obtain Mo-POM nanoclusters. Step 2: Preparation of PEI-modified Mo-POM (1) Polyethyleneimine (PEI, molecular weight 600-25000 Da) was dissolved in deionized water to prepare an aqueous solution with a mass concentration of 0.5-5 mg / mL. Subsequently, the pH of the PEI aqueous solution was adjusted to 7.0-7.4 using 0.1 mol / L hydrochloric acid solution; (2) Under vigorous stirring, the Mo-POM solution obtained in step one is added dropwise to the PEI solution to utilize the amino groups of PEI to combine with Mo-POM; (3) After reacting for 30 minutes, the free PEI was removed by dialysis through a dialysis bag (molecular weight cutoff 3500 Da) to obtain a PEI-Mo-POM modified solution with a positive charge on the surface; Step 3: Construction of the PEI-Mo-POM@Bs composite system (1) Bacillus subtilis was cultured in LB liquid medium with shaking until the logarithmic growth phase (OD). 600 (≈0.6~0.8), collect the bacterial cells by centrifugation, and wash three times with PBS buffer to remove culture medium residue; (2) The washed Bs cells were suspended in PBS, and the PEI-Mo-POM solution prepared in step 2 was added. The cells were shaken on a shaker (100-150 rpm) at room temperature in the dark for 30-60 minutes to load the nanoclusters onto the surface of the cells by electrostatic adsorption. (3) Centrifuge (5000-8000 rpm), discard the supernatant, and collect the precipitate to obtain the PEI-Mo-POM@Bs composite material; Step 4: Relief of Salt and Alkali Stress in Plants Mo-POM, PEI-Mo-POM, Bacillus subtilis (Bs), and PEI-Mo-POM@Bs were dispersed in deionized water or buffer solution to prepare treatment solutions with a concentration of 200 μg / mL. For dandelion seedlings under NaCl salt stress, the treatment solutions were applied by root irrigation or root soaking. After a certain period of cultivation, plant growth and physiological indicators related to oxidative stress were measured.

2. The method for enhancing plant salt stress resistance based on a polyoxometalate-rhizosphere bacteria complex system according to claim 1, characterized in that, In step one, the phosphomolybdic acid and gallic acid are mixed and stirred vigorously. Then, a sodium carbonate solution with a mass fraction of 7.5 wt% is added, and the mixture is reacted at room temperature for 5 hours until the solution turns dark blue or dark green.

3. The method for enhancing plant salt stress resistance based on a polyoxometalate-rhizosphere bacteria complex system according to claim 1, characterized in that, The product obtained from the reaction in step one needs to be dialyzed for 3 days to remove unreacted substances, and then freeze-dried to obtain Mo-POM nanoclusters.

4. The method for enhancing plant salt stress resistance based on a polyoxometalate-rhizosphere bacteria complex system according to claim 1, characterized in that, In step two, the molecular weight of the polyethyleneimine is 600~25000 Da, the mass concentration is 0.5~5 mg / mL, and the pH value of the PEI aqueous solution is adjusted to 7.0~7.4 using 0.1 mol / L hydrochloric acid solution.

5. The method for enhancing plant salt stress resistance based on a polyoxometalate-rhizosphere bacteria complex system according to claim 1, characterized in that, In step two, the modification reaction time is 30-60 minutes, and after the reaction, the solid precipitate is obtained by freeze-drying.

6. The method for enhancing plant salt stress resistance based on a polyoxometalate-rhizosphere bacteria complex system according to claim 1, characterized in that, The rhizosphere bacteria mentioned in step three are Bacillus subtilis, and their OD values ​​are [missing information] when cultured to the logarithmic growth phase. 600 It ranges from 0.6 to 0.

8.

7. The method for enhancing plant salt stress resistance based on a polyoxometalate-rhizosphere bacteria complex system according to claim 1, characterized in that, In step three, Bacillus subtilis and PEI-Mo-POM solution are shaken at 100-150 rpm for 30-60 minutes at room temperature in the dark, so that the polyoxometalate is loaded onto the surface of the bacteria through electrostatic adsorption.

8. The method for enhancing plant salt stress resistance based on a polyoxometalate-rhizosphere bacteria complex system according to claim 1, characterized in that, The compound system described in step four is applied at a concentration of 200 μg / mL and is applied to plants under salt stress conditions by root irrigation or root soaking.

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