Method for relieving toxic effect of tetrabromobisphenol A on rice by utilizing phosphorus source

Abstract

According to the method for relieving the toxic effect of tetrabromobisphenol A on rice by utilizing the phosphorus source, 22.4 mg / L monopotassium phosphate (KH2PO4) is taken as a core concentration parameter, and the phosphorus absorption efficiency and stress resistance of seedlings are remarkably improved through rhizosphere liquid application supplement. The method is strictly based on laboratory verification data, is suitable for planting the rice in the farmland polluted by the tetrabromobisphenol A, and is simple and convenient to operate and low in cost.

Description

Technical Field

[0001] The present invention belongs to the technical field of farmland organic pollution remediation, and specifically relates to a method for alleviating the toxic effect of tetrabromobisphenol A on rice by using a phosphorus source. Background Art

[0002] Tetrabromobisphenol A (TBBPA), as a highly efficient brominated flame retardant, is widely used in fields such as electronic devices, plastic products, and building materials. However, TBBPA has environmental persistence, bioaccumulation, and potential ecological toxicity, and can enter farmland soil through industrial wastewater discharge, e-waste dismantling, and plastic degradation, and remain in the environment for a long time. Research shows that the detected concentration of TBBPA in farmland soil can reach the level of μg / kg to mg / kg, posing a serious threat to rice growth.

[0003] In the rice planting system, TBBPA can be absorbed by plants through the roots and migrate in the plant body, interfering with its normal physiological metabolism. Specifically, it inhibits the growth of seedling plant height, root length, and biomass; destroys the root cell structure, leading to increased membrane lipid peroxidation and oxidative stress damage; interferes with photosynthesis efficiency, mineral nutrient absorption, and endogenous hormone balance, and ultimately reduces the yield and quality of rice.

[0004] Currently, the remediation technologies for TBBPA-contaminated soil mainly include microbial degradation, chemical oxidation, adsorption and fixation, and phytoremediation. For example, certain specific strains can degrade TBBPA, but due to environmental conditions, the remediation efficiency is low; while applying biochar or clay minerals can adsorb TBBPA, but it may affect soil fertility. In addition, although transgenic technology can enhance the TBBPA tolerance of plants, there are controversies over gene drift and ecological safety, making it difficult to be applied on a large scale.

[0005] Phosphorus (P), as a core element for plant growth and development, participates in energy metabolism, nucleic acid synthesis, and the regulation of cell membrane stability. Existing research has confirmed that exogenous phosphorus supplementation can alleviate the toxicity of heavy metals (such as cadmium and lead) to plants, and can also enhance the fixation of organic pollutants by regulating cell wall components (such as pectin and lignin). However, there is no report on the study of exogenous phosphorus regulating rice's response to TBBPA stress, and its potential mechanisms (such as antioxidant defense, pollutant binding, and transport inhibition) are not clear. Therefore, developing an efficient, economical, and environmentally friendly TBBPA toxicity alleviation technology based on exogenous phosphorus supplementation is of great significance for the safe utilization of polluted farmland and the sustainable production of rice. Summary of the Invention

[0006] In view of the problems that the pollution of TBBPA severely inhibits the growth of rice, the existing remediation methods (such as microbial degradation, adsorption and fixation, etc.) are inefficient, or there are ecological risks, etc., the present invention provides an innovative method for alleviating the toxicity of TBBPA through exogenous phosphorus supplementation to solve the following technical problems: 1) TBBPA stress leads to obstacles in rice nutrient absorption, especially the lack of key elements such as phosphorus (P), calcium (Ca), zinc (Zn), etc.; 2) TBBPA-induced oxidative stress and root damage inhibit the growth of rice; 3) It is difficult for existing remediation technologies to balance high efficiency, economy and environmental friendliness.

[0007] To solve the above technical problems, the technical solution proposed in this application is:

[0008] The present invention provides a method for using a phosphorus source to alleviate the toxic effect of tetrabromobisphenol A on rice. During the seedling stage of rice, potassium dihydrogen phosphate (KH2PO4) is used as the phosphorus source and supplemented to the rice growth environment at a concentration of 22.4 mg / L.

[0009] Furthermore: The supplementation method of potassium dihydrogen phosphate is rhizosphere liquid application, which specifically includes the following steps:

[0010] Calculate the irrigation amount according to the soil water content to ensure that the concentration of potassium dihydrogen phosphate in the rhizosphere solution is 22.4 mg / L.

[0011] Furthermore: The pollution concentration of tetrabromobisphenol A in the rice growth environment is 4000 - 6000 μg / L.

[0012] Furthermore: Potassium dihydrogen phosphate is dissolved and applied synchronously with the conventional nutrient solution.

[0013] Furthermore: The method increases the expression level of phosphorus transporter proteins (PTPs) in the roots of rice seedlings by 35% - 50%.

[0014] Furthermore: The method restores the biomass of rice seedlings under TBBPA stress to 85% - 95% of the non-polluted state, and reduces the leaf withering rate by 60% - 70%.

[0015] Furthermore: The irrigation amount for rhizosphere liquid application is 20 - 30 m 3 / mu.

[0016] On the other hand, this application also claims the protection of an application according to the method described in any one of the foregoing, which is used for paddy rice planting in farmland with a tetrabromobisphenol A pollution concentration of 4000 - 6000 μg / L to reduce the toxic effect of tetrabromobisphenol A on rice.

[0017] Compared with the prior art, the method for alleviating the toxicity of tetrabromobisphenol A through exogenous phosphorus supplementation during the seedling stage of rice in the present invention has achieved the following beneficial technical effects:

[0018] 1) High efficiency: The supplementation of exogenous phosphorus restores the biomass and plant height of rice under TBBPA stress;

[0019] 2) Economy: Only a low concentration of phosphorus source (22.4 mg / L) needs to be added, with low cost and simple operation;

[0020] 3) Environmentally friendly: Avoid introducing exogenous microorganisms or chemical reagents, without ecological risks. Brief Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 It shows the effects of exogenous phosphorus on the overall growth and plant height of rice;

[0023] Figure 2 It shows the recovery of rice seedlings after the supplementation of phosphorus source;

[0024] Figure 3 It shows the change multiples of the contents of 12 nutrient elements in rice seedlings after the supplementation of phosphorus source, compared with the BK group;

[0025] Figure 4 It shows the changes in the concentration of phosphorus element (P) and phosphorus transporter concentration (PTPs) in rice seedlings after the supplementation of phosphorus source, where A is the content of phosphorus element; B is the phosphorus transporter. Detailed Embodiments

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

[0027] The core of the present invention is to regulate the physiological metabolism of rice through the supplementation of exogenous phosphorus, specifically including: Selection and addition of phosphorus source: Add 22.4 mg / L potassium dihydrogen phosphate to the conventional nutrient solution of rice. This concentration has been experimentally verified to significantly relieve the toxicity of TBBPA and does not affect the normal growth of rice (see Figure 1 ).

[0028] The mechanism of action of this application is as follows:

[0029] 1) Nutrient absorption regulation: Promote the expression of PTPs and restore the absorption of phosphorus and other nutrient elements (such as Ca, Zn, Fe, Cu) (see Figure 3 , Figure 4 );

[0030] 2) Physiological repair: Alleviate the oxidative damage caused by TBBPA, repair the root cell structure, and increase the biomass and plant height (see Figure 2 );

[0031] The innovation of this application lies in: for the first time, it is proposed to directly regulate rice nutrient metabolism by exogenous phosphorus supplementation to alleviate the toxicity of TBBPA; clarify the molecular mechanism of phosphorus-mediated up-regulation of PTPs expression and restoration of multi-element absorption; provide a farmland restoration plan that can be applied on a large scale, filling the research gap in the interaction between organic pollutants and plant phosphorus metabolism.

[0032] Example 1: Laboratory hydroponic culture

[0033] I. Materials and methods

[0034] 1. Rice planting and cultivation

[0035] The rice variety used was Nipponbare (Oryza sativa Japonica.cv.Nipponbare), and the seeds were purchased from Nanjing Agricultural University. The seeds were disinfected to remove surface impurities and pathogens, and then soaked in a beaker filled with sterile water at 37 °C for 24 h to promote seed germination. Then, the soaked seeds were placed in the dark at 30 °C for 3 d of germination. During germination, the sterile water was changed daily to keep it clean. The germinated seeds were transplanted into a 50% concentration of Hoagland nutrient solution and further cultured in an intelligent light incubator. The light cycle was 16 h (28 °C), and the dark cycle was 8 h (25 °C). After 2 weeks of culture, seedlings with uniform growth were selected and transferred to brown glass bottles for continued culture until they were 4 weeks old.

[0036] 2. Addition of phosphorus source

[0037] According to the growth requirements of rice and the phosphorus content in the hydroponic nutrient solution, an appropriate amount of potassium dihydrogen phosphate (KH2PO4) was added to the Hoagland nutrient solution exposed to 5000 μg / L TBBPA to make the concentration of the phosphorus source in the Hoagland nutrient solution reach the set optimized range. In the hydroponic system, the phosphorus source concentration can be set to 22.4 μg / L, and this phosphorus source concentration will not affect the normal growth of rice, as Figure 1 shown.

[0038] 3. Determination of the expression level of phosphorus transporter proteins (PTPs)

[0039] Take the roots and shoots of 2 fresh rice seedlings, cut them into small pieces, put them into a 2 mL centrifuge tube, add 1 mL of PBS buffer and 2 mm zircon beads. Homogenize for 3 min using TissueLyser II (QIAGEN, Hilden, China), and then centrifuge at 8000×g (relative centrifugal force) for 15 min at 4 °C using a high-speed centrifuge (Eppendorf 5810R, Germany) at 4 °C. Take the supernatant and dilute it to an appropriate concentration. Use a commercial plant phosphorus transporter (PTPs) ELISA kit provided by Jingmei Biotechnology Company in Jiangsu Province to quantitatively analyze the content of PTPs in rice seedlings.

[0040] 4. Determination of nutrient element content

[0041] Determine the contents of 12 trace elements (including phosphorus P, potassium K, calcium Ca, magnesium Mg, iron Fe, manganese Mn, zinc Zn, copper Cu, boron B, cobalt Co, chromium Cr, and nickel Ni) in the roots and shoots of rice. The specific steps are as follows:

[0042] 1) Sample pretreatment: Collect an appropriate amount of rice root and shoot samples, thoroughly rinse them multiple times with ultrapure water, and then use a freeze dryer to perform vacuum freeze-drying treatment on the samples.

[0043] 2) Sample digestion: Adopt the strong acid method. The specific operation is as follows: Add 1 mL of concentrated nitric acid (69% w / w) and 250 μL of hydrogen peroxide (30% w / w) to the sample, mix well, and then place it in a water bath at 90 °C for heating and reaction for 6 h to ensure that the organic matter in the sample is completely digested and converted into a dissolved state suitable for ICP-MS determination. 3) ICP-MS analysis: The digested sample solution is analyzed by ICP-MS (Agilent 8800, USA) to determine the content of trace elements in the roots and shoots. The operating parameters of ICP-MS are shown in Table 1.

[0044] Table 1 Instrument parameters of ICP-MS

[0045]

[0046] 5. Data processing

[0047] All experimental data were statistically analyzed using IBM SPSS Statistics 22 and Origin 2024b software. The significance test of the data was performed using one-way analysis of variance (ANOVA), and Tukey's post hoc test was combined to further evaluate the differences between groups. When the p-value was less than 0.05, it indicated that there were significant differences between the experimental groups.

[0048] 6. Quality assurance and control

[0049] To ensure the accuracy and reliability of the experimental results, strict quality assurance and control (QA / QC) procedures were implemented throughout this study. All glassware used in the experiments was rigorously cleaned and processed before use. The specific process was as follows: First, the glassware was soaked in a 10% nitric acid solution by mass to remove possible contaminants; then it was thoroughly rinsed with deionized water to ensure no residues; finally, the cleaned glassware was dried in a muffle furnace (Neytech, 3-1750, USA) at a high temperature of 400 °C to ensure it was completely clean and contamination-free. To avoid potential interference of iron scissors on the determination results of plant elements, all tools used for cutting plant samples in this study were made of Teflon material.

[0050] II. Experimental Results

[0051] 1. Supplementary exogenous phosphorus effectively alleviated the growth inhibition of rice

[0052] Supplementary exogenous phosphorus significantly alleviated the rust spots and withering of rice leaves (see Figure 2 ), and the seedling biomass and plant height were also restored.

[0053] 2. Supplementary exogenous phosphorus effectively promoted the absorption of nutrient elements by rice

[0054] Element content analysis (see Figure 3 ) showed that compared with the 5000 μg / L TBBPA exposure group, the phosphorus source supplementation promoted the recovery of the absorption of 12 essential nutrient elements, especially P, Ca, Zn, Fe, and Cu, and their concentrations increased significantly, with the increase multiples being 1, 0.8, 0.6, 0.6, and 2 respectively, and the concentrations of the other seven elements also increased slightly. It is worth noting that the elements whose absorption was inhibited in the 5000 μg / L TBBPA exposure group, such as P, K, Ca, Mg, B, Mn, and Co, had enhanced absorption after the supplementary exogenous phosphorus. The absorption of key elements such as Ca, Zn, Fe, and Cu was significantly improved. Ca plays an important role in plants, involving the stability of cell walls and signal transduction; while Zn, Fe, and Cu are key trace elements for plants to cope with environmental stresses, participating in antioxidant defense, stress response, and metabolic regulation; in addition, elements such as P, K, and Ca play important roles in restoring plant biomass because they are involved in biomolecule synthesis and energy metabolism. It can be seen that plants can respond to the stress of environmental pollutants by flexibly regulating their nutrient absorption mechanisms to promote the absorption of essential nutrient elements by rice.

[0055] 3. Effects of added phosphorus source on phosphorus transporters and phosphorus content in rice seedlings

[0056] P plays a key role in ATP synthesis, phospholipid metabolism, and nucleic acid synthesis. In addition, as a key component of energy transfer and signal transduction pathways, the homeostasis of P is strictly regulated by PTPs. In this study, as Figure 4 shown, after exposure to 5000 μg / L TBBPA, the phosphorus content of rice decreased, while the level of PTPs increased significantly. After exogenous phosphorus supplementation (5000 + P), the concentration of PTPs was not significantly different from that of the 5000 μg / L TBBPA group. However, the P content increased significantly (p < 0.05) and exceeded the level of the control group. This indicates that exogenous phosphorus supplementation can enhance P absorption and alleviate phosphorus deficiency symptoms caused by TBBPA.

[0057] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for alleviating the toxic effect of tetrabromobisphenol A on rice by using a phosphorus source, characterized in that: During the rice seedling stage, potassium dihydrogen phosphate (KH2PO4) was used as the phosphorus source and supplemented into the rice growth environment at a concentration of 22.4 mg / L.

2. The method according to claim 1, wherein: The method of supplementing potassium dihydrogen phosphate was rhizosphere liquid application, which specifically included the following steps: Calculate the irrigation amount according to the soil water content to ensure that the concentration of potassium dihydrogen phosphate in the rhizosphere solution was 22.4 mg / L.

3. The method according to claim 1 or 2, characterized in that: The pollution concentration of tetrabromobisphenol A in the rice growth environment was 4000 - 6000 μg / L.

4. The method according to any one of claims 1 to 3, characterized in that: Potassium dihydrogen phosphate was dissolved and applied synchronously with the conventional nutrient solution.

5. The method according to any one of claims 1 to 3, characterized in that: The method increased the expression level of phosphorus transporter proteins (PTPs) in the roots of rice seedlings by 35% - 50%.

6. The method according to any one of claims 1-3, characterized in that: The method restored the biomass of rice seedlings under tetrabromobisphenol A stress to 85% - 95% of the non-polluted state and reduced the yellowing rate of leaves by 60% - 70%.

7. The method according to claim 2, wherein: The irrigation amount of the rhizosphere liquid application is 20 - 30 m 3 / mu.

8. An application of the method according to any one of claims 1 to 3, characterized in that: It was used for paddy rice planting in farmland with a tetrabromobisphenol A pollution concentration of 4000 - 6000 μg / L to reduce the toxic effect of tetrabromobisphenol A on rice.

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