A method for high-throughput and rapid detection of the metabolic activity of glutathione S-transferase on herbicides based on GSH-DTNB color development

Abstract

The present invention discloses a method for high-throughput and rapid detection of the metabolic activity of glutathione S-transferase on herbicides based on GSH-DTNB color development. The method is as follows: Take purified GST protein and herbicide solution, use GSH solution as the reaction initiator to start the reaction. After incubation, add DTNB solution as the reaction terminator to terminate the reaction to obtain a reaction system; Use an equal volume of purified eluent to replace the purified GST protein in the reaction system as a blank control system; Detect the A 412 absorbance value of the reaction system and the blank control system, quantify the GSH concentration in the reaction system and the blank control system, and calculate the metabolic rate of glutathione S-transferase on the herbicide. This method not only improves the detection efficiency and accuracy, but also reduces the detection cost, providing an important reference for the development of more subsequent rapid detection methods based on GSH-DTNB.

Description

Technical Field

[0001] The present invention is applicable to the technical field of plant protection, and relates to a method for high-throughput rapid detection of the metabolic activity of glutathione S-transferase against herbicides based on GSH-DTNB color development, which is used for rapidly detecting the metabolic efficiency of plant glutathione S-transferase against different herbicides. Background Art

[0002] Glutathione S-transferase (GST) catalyzes the glutathione conjugation reaction and participates in physiological activities such as the regulation of intracellular redox balance, cell signal transduction, and the detoxification metabolism of exogenous harmful compounds. With the rapid development of genomics and bioinformatics, genome-wide identification and annotation analysis of the GST gene family in various plants have been carried out, accelerating the research on the localization and member classification of GST genes. For example, Jain and Sappl et al. systematically identified and annotated the GST genes in three plants, Arabidopsis thaliana, Oryza sativa, and Populus tomentosa, revealing the diversity of GST members in different plants. There are 53 GST members in Arabidopsis thaliana, 79 in Oryza sativa, and as many as 81 in Populus tomentosa. This diversity reflects the expansion of the GST gene family during plant evolution and its ability to adapt to different environments. Further findings show that in terms of subfamily classification, plants have four special GST categories: phi (F), tau (U), dehydroascorbate reductase (DHAR), and lambda (L). GST members in the same subfamily usually have high conservation in gene sequence and protein structure, which is beneficial for the identification, classification, model construction, and activity prediction of family members. So far, although a large number of GST members in different plants have been identified and annotated, only the functions of a few members have been analyzed in detail.

[0003] Pelon et al. found that phi (F) and tau (U) in the GST gene family are involved in the detoxification metabolism of herbicides. Their large number of members is of great significance in the fields of weed resistance and crop breeding research. They directly and effectively detoxify the active molecules of herbicides by catalyzing the glutathione (GSH) conjugation reaction. In this process, GST can catalyze the substitution of halogen elements such as chlorine (-Cl) by GSH, or catalyze the nucleophilic attack of the thiol group of GSH on the ether bond (-O-) in the herbicide molecule, causing the molecular structure of the herbicide compound to break, generating a metabolite combined with a hydroxyl group-containing product and GSH, and destroying the entire molecular structure of the herbicide. At the same time, these reactions also increase the hydrophilicity of the herbicide metabolite, making it easier to be transported to the vacuole for isolation or excreted through the transporter in the plant, thus effectively reducing the toxic effect of the herbicide on the plant.

[0004] The metabolic functions and mechanisms of action of some GST members in plants have been well elucidated. Prade et al. demonstrated the protein crystal structures of the herbicide molecules (FE-4053, atrazine) bound to the detoxifying enzymes AraGST (1bx9) or ZmGSTI (1bye), providing ideal crystal conformations for in-depth analysis of the catalytic sites. Dücker et al. used isotope labeling and liquid chromatography tandem radioactive detectors to deeply analyze the degradation of flufenacet in phase III metabolism and found that GST-catalyzed flufenacet metabolism could significantly reduce the sensitivity of Alopecurus myosuroides Huds. and Lolium spp., further confirming the important role of GST in mediating weed herbicide resistance. With the development of transcriptome sequencing technology, it is possible to accurately analyze the expression levels of GST gene family members at various growth and development stages of plants, enabling the selection of differentially expressed GST members for in vitro enzyme activity reaction experiments and deeper understanding of the GST-catalyzed metabolism and corresponding product structures through HPLC-DAD or liquid chromatography tandem mass spectrometry (LC-MS / MS) detection. However, although this research process can accurately establish the metabolic relationship between metabolic enzymes and herbicide active molecules, the equipment and consumables required for the entire detection system are costly, and the detection process is time-consuming and relatively inefficient. Therefore, there is an urgent need to develop a high-throughput analytical method for detecting GST enzyme-catalyzed herbicide metabolism to accelerate the study of plant GST gene functions.

[0005] DTNB, whose full name is 5,5'-dithiobis-(2-nitrobenzoic acid), is a colorless or light yellow powder and is widely used in the fields of medical and plant pathology detection as an important reagent for evaluating the content of free glutathione (GSH) in biological samples. The color development principle of DTNB is based on the substitution reaction between disulfide bonds (-S-S-) and sulfhydryl groups (-SH), and it can react rapidly with GSH at room temperature, releasing one molecule of GS-TNB and one molecule of TNB. 2- . TNB 2- is relatively stable in neutral and weakly alkaline environments (pH = 7.2 - 8.0) and has a maximum absorption peak at a light wavelength of 412 nm (14,150 M -1 cm -1), which can be detected by a microplate reader at the corresponding optical wavelength. Utilizing this property, in the GST-catalyzed herbicide metabolism reaction system, DTNB is introduced to bind the remaining free GSH in the reaction, prompting the reaction to terminate rapidly; the microplate reader is used to detect the color development of the reaction system, and the metabolism amount of herbicide molecules is calculated, and then under certain conditions, accurate and efficient qualitative and quantitative functional analysis of plant GST members is carried out. The entire process from initial sample loading to final detection of the reaction can be completed within two hours, and the efficiency and cost are significantly lower than the analysis method of the liquid chromatography-mass spectrometry system. Summary of the Invention

[0006] The object of the present invention is to provide a high-throughput enzyme activity assay method for detecting the herbicide metabolism ability of glutathione S-transferase GST based on the GSH-DTNB color reaction.

[0007] The object of the present invention can be achieved by the following technical solutions:

[0008] In the first aspect, the present invention claims a detection system for high-throughput and rapid detection of the herbicide metabolism activity of glutathione S-transferase GST based on GSH-DTNB color development. The detection system includes a reaction system and a blank control system. The reaction system contains: purified glutathione S-transferase GST protein, herbicide solution, reaction initiator GSH solution, and reaction terminator DTNB solution; an equal volume of purified eluent is used to replace the purified GST protein in the reaction system as the blank control system.

[0009] Furthermore, in the above detection system, the reaction system contains: 189 μL of 0.25 mg / mL purified GST protein, 1 μL of 50 mM herbicide solution, the reaction is initiated with 10 μL of 5 mM GSH solution, after incubation at 29 - 31 °C in the dark, 10 μL of 20 mM DTNB solution is added as the reaction terminator; 189 μL of purified eluent is used to replace the purified GST protein in the reaction system as the blank control.

[0010] Even further, the aforementioned herbicide solution uses DMSO as the solvent, the aforementioned GSH solution uses ddH2O as the solvent, and the aforementioned DTNB solution uses DMSO as the solvent. The aforementioned purified eluent is a salt solution containing 50 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, and pH = 7.5.

[0011] In the second aspect, the present invention claims the application of the reagents in the above detection system in the preparation of a kit for high-throughput and rapid detection of the herbicide metabolism activity of glutathione S-transferase.

[0012] In a third aspect, the present invention claims protection for a kit for high-throughput rapid detection of the metabolic activity of glutathione S-transferase against herbicides based on GSH-DTNB color development, and the kit contains the reagents in the aforementioned detection system.

[0013] In a fourth aspect, the present invention claims protection for a method for high-throughput rapid detection of the metabolic activity of glutathione S-transferase against herbicides based on GSH-DTNB color development. The method is as follows: Take purified GST protein and herbicide solution, use GSH solution as the reaction initiator to start the reaction. After dark incubation, add DTNB solution as the reaction terminator to terminate the reaction to obtain a reaction system; Use an equal volume of purified eluent to replace the purified GST protein in the reaction system as a blank control system; Detect the A 412 absorbance value, quantify the GSH concentration in the reaction system and the blank control system, and calculate the metabolic rate of glutathione S-transferase against the herbicide.

[0014] The formula for calculating the metabolic rate is as follows:

[0015] Herbicide metabolic rate (%) = (1 - (GSH residue in the reaction system) / (GSH residue in the blank control system)) × 100%

[0016] Furthermore, in the above method, take 189 μL of 0.25 mg / mL purified GST protein and 1 μL of 50 mM herbicide solution, use 10 μL of 5 mM GSH solution as the reaction initiator to start the reaction. After dark incubation at 29 - 31 °C, add 10 μL of 20 mM DTNB solution as the reaction terminator to terminate the reaction to obtain a reaction system; Use 189 μL of purified eluent to replace the purified GST protein in the reaction system as a blank control system.

[0017] Even further, in the above method, the herbicide solution uses DMSO as the solvent, the GSH solution uses ddH2O as the solvent, and the DTNB solution uses DMSO as the solvent. The purified eluent is a salt solution containing 50 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, and pH = 7.5.

[0018] In the specific implementation manner of the present invention, oscillating and mixing the reaction system can accelerate the color development reaction. After oscillating and mixing for 5 min, detect the absorbance (A 412 ) at 25 °C using an enzyme-linked immunosorbent assay reader.

[0019] The technical principle of the method of the present invention is as follows: during the reaction process of the reaction system, glutathione S-transferase (GST) catalyzes the binding of the herbicide to glutathione (GSH) to generate a conjugate of the herbicide molecule and GSH (GS-Herb). After the reaction of the herbicide, GST, and GSH, DTNB is added to bind to the remaining GSH in the reaction system to form a GS-TNB conjugate, and at the same time, detectable yellow TNB is generated. 2+ This product has a maximum absorption peak at a wavelength of 412 nm. By measuring the absorbance of the reaction product and referring to the standard curve, the content of the remaining GSH in the reaction system can be obtained. By calculating the change in GSH before and after the reaction, the ability of GST to metabolize the herbicide can be determined.

[0020] Advantages of the present invention:

[0021] I. High detection efficiency

[0022] 1. High-throughput processing: This method can simultaneously process multiple samples through automated instruments and microplate technology, significantly improving the detection efficiency. This is of great significance for large-scale sample screening and real-time monitoring.

[0023] 2. Simple experimental operation: The basic reaction conditions and the operation process based on 96-well plates have been established for this method, the reaction system is simplified, and no complex sample pretreatment operations are required during detection.

[0024] 3. Shortened detection time: Compared with the traditional high-performance liquid chromatography-mass spectrometry (HPLC) method, this method simplifies the operation steps and shortens the detection time. For the traditional HPLC method, as the number of samples increases, the detection time increases exponentially. However, for this method in a 96-well plate system, as the number of samples increases, the required time is basically the same, that is, from reaction to detection, it can be completed within two hours.

[0025] II. High detection accuracy

[0026] 1. Taking some samples and comparing them with the traditional HPLC method, there is no significant difference in the detected metabolic rate.

[0027] 2. Standardized operation: This method adopts standardized operation steps and conditions to ensure the accuracy and comparability of the detection results.

[0028] 3. Reduced human error: The one-step reaction system reduces the error caused by human operation and improves the reliability of the detection results.

[0029] III. Low detection cost

[0030] 1. Reduced reagent consumption: Since the reaction volume is in the microliter range, the required amounts of protein samples and reagents are relatively small, thus reducing the reagent consumption and detection cost.

[0031] 2. Reduce instrument wear: The present invention can detect multiple samples at the same time, and a large amount of sample data can be obtained through a single instrument detection, thereby reducing the wear of the instrument and further reducing the detection cost.

[0032] IV. Wide application scenarios

[0033] 1. Basic research: This method provides a powerful tool for in-depth study of the mechanism of GST-metabolized herbicides, helps to reveal the impact of herbicides on organisms and clarify its catalytic mechanism.

[0034] 2. Applied research: This method has broad application prospects in the fields of pesticide residue detection, environmental monitoring, food safety, etc., and can provide technical support for scientific research, risk assessment and supervision in related fields.

[0035] 3. Promote technological innovation: The establishment of this method promotes technological innovation and development in related fields, and promotes the emergence and application of new technologies and new methods.

[0036] In summary, the establishment of a high-throughput rapid detection method for glutathione S-transferase-metabolized herbicides based on GSH-DTNB colorimetry is of great significance. It not only improves the detection efficiency and accuracy, reduces the detection cost, but also provides an important reference for the development of more subsequent rapid detection methods based on GSH-DTNB. Brief description of the drawings

[0037] Figure 1 : Schematic diagram of the GSH-DTNB colorimetric reaction for analyzing the activity of herbicide-metabolizing enzymes. During the reaction process, glutathione S-transferase (GST) catalyzes the binding of herbicides to glutathione (GSH) to form a conjugate of herbicide molecule and GSH (GS-Herb). After the reaction of herbicide, GST and GSH, DTNB is added to bind to the remaining GSH in the reaction system to form a GS-TNB conjugate, and at the same time, detectable yellow TNB is generated 2+ , and this product has a maximum absorption peak at a wavelength of 412 nm. By measuring the absorbance of the reaction product and referring to the standard curve, the content of the remaining GSH in the system can be obtained. By calculating the change in GSH before and after the reaction, the ability of GST to metabolize herbicides can be clarified.

[0038] Figure 2 : GSH-DTNB colorimetric standard curve. By constructing the GSH-DTNB standard curve, the upper and lower limits of the GSH concentration in the reaction system are determined.

[0039] Figure 3: Optimization effect diagram of three substrate reaction combinations. Considering the stability of the herbicide stock solution and the negative impact of its turbidity on detection, DMSO was selected as the solvent in this method. When the stock solution concentration increases with the actual concentration of each group of agents being the same, the turbidity state tends to become clear ( Figure 3 A in). In group A, there were no significant visual differences between the treatment group of the chloroacetamide herbicide stock solution and the control group (added DMSO) before and after adding DTNB for color development. Therefore, group A was selected as the candidate system ( Figure 3 A, B in).

[0040] Figure 4 : Absorbance detection results of three substrate reaction combinations. When the actual concentration of each group of agents is the same, before adding the DTNB color-developing solution, the absorbance (A 412 ) of the system of the chloroacetamide herbicide stock solution decreased, and there were low-level fluctuations in the absorbance (A 412 ) among different types of herbicides. However, after adding DTNB for color development, there were no significant differences between the treatment group containing the agent and the control group, indicating that TNB 2- provided excellent color development and masking effects.

[0041] Figure 5 : Experimental results of exploring the optimal reaction temperature of OsGSTs. By incubating the purified OsGSTs at different temperatures and detecting the change of absorbance A 412 in each group, it was concluded that the optimal reaction temperature of this group was 29 - 31 °C.

[0042] Figure 6 : Effect diagram of the influence of DMSO solvent on the color development of OsGSTs. Except for the purified empty control EV, no significant influence was detected after adding DMSO among various samples, excluding the potential denaturation or turbidity influence of the solvent on the purified GST.

[0043] Figure 7 : High-throughput analysis results of the metabolism of herbicide-related compounds by OsGSTUs.

[0044] Figure 8 : Comparison chart of the metabolism rate and accuracy of three chloroacetamide herbicides analyzed by HPLC method and DNTB method. By comparing the metabolism rates of alachlor, acetochlor, and S-metolachlor detected by the DTNB method and the HPLC method under the same system, it was found that there were no significant differences in the metabolism rate results obtained by the two methods. Specific implementation method

[0045] The method of the present invention will be described below through specific embodiments. However, the present invention is not limited thereto, and any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, can be obtained from commercial channels.

[0046] Example 1

[0047] I. Experimental materials

[0048] 1. Chemical reagents

[0049] Analytical grade sodium dihydrogen phosphate, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), HPLC grade dimethyl sulfoxide (DMSO), and glacial acetic acid were purchased from Macklin Biochemical Co., Ltd. in Shanghai, China. HPLC grade acetonitrile was purchased from Guangzhou Guanghua Technology Co., Ltd. Reduced glutathione (GSH) was purchased from Shanghai BBI Life Sciences Co., Ltd.

[0050] 2. In vitro expression and purification of candidate OsGSTs

[0051] Four OsGSTs (GSTU1: Os03g0785900, GSTU4: Os10g0528300, GSTU33: Os10g0365200, and GSTU50: Os10g0530900) were selected for metabolic profile analysis. RNA was extracted from rice plants and reverse transcribed into cDNA as a template for amplification. The primers and restriction enzyme sites used are shown in Table 1:

[0052] Table 1 Primers and restriction enzyme sites

[0053]

[0054] F represents the forward primer, and R represents the reverse primer. The underlined part marks the restriction enzyme sites.

[0055] OsGSTs vector construction scheme: Recombinant expression was carried out in Escherichia coli BL21 strain (purchased from Nanjing Novozymes Biotech Co., Ltd.) using the pET-28a(+) system. After induced expression, the cells were collected by centrifugation, incubated with Hammer Super Lysis Buffer (purchased from ACE Bio) at 4°C for 20 min, and then centrifuged at 12,000 ×g for 20 min at 4°C to collect the crude protein extract. The purification of OsGSTs crude enzyme adopted the purification scheme of a His-tagged protein purification kit resistant to reducing chelating agents (purchased from Beyotime Biotechnology Co., Ltd.).

[0056] II. Establishment and optimization of the reaction system

[0057] To simplify the preparation process, the components in the reaction system mainly include purified OsGSTs protein, herbicide, and GSH. After the reaction reaches the set time, DTNB is added to terminate the reaction, and the reaction system is detected using a microplate reader. To confirm the stability of the entire system, the following parameters need to be considered: the detection concentration range of glutathione, the interference of the low solubility of the herbicide, the interference of the solvent, the denaturation of the protein, etc.

[0058] 1. Determine the appropriate working concentration range of glutathione

[0059] By establishing a glutathione standard curve, the appropriate working and detection concentrations of glutathione are obtained. Prepare a glutathione stock solution of 50 mM and dilute it to 5 mM with ddH2O. Add 190 μL of purified elution buffer (containing 50 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH = 7.5) to a 96-well plate. After gradient dilution, add 10 μL of GSH to the template buffer, and the system concentrations are 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, and 0 μM, respectively. Add 10 μL of DTNB (20 mM, with DMSO as the solvent) to react completely with glutathione. Since the total volume changes slightly after adding DTNB, the actual final concentrations are adjusted to 476.19, 238.09, 119.05, 59.52, 29.76, 14.88, 7.44, and 0 μM. The absorbance (A 412 ) is detected at 25 °C using an Infinite ® 200 PRO microplate reader (Tecan, Switzerland). Three replicated standard curves are established. The absorbance values of the standard curves are analyzed, fitted, and plotted for a linear relationship in Excel 2019 (Microsoft, USA) software.

[0060] 2. Analysis of the influence of the turbidity degree of the herbicide on the reaction system and optimization scheme

[0061] To investigate the turbidity effect after adding the herbicide stock solution into the system, using DMSO as the solvent, five commonly used chloroacetamide herbicides with low water solubility were selected: alachlor, acetochlor, pretilachlor, butachlor, and S-metolachlor for the turbidity degree test. An initial reaction system was established: with different volumes of purified eluent (180, 185, 189 μL) (the purified eluent contains 50 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH = 7.5) as the background, the corresponding concentration of chloroacetamide herbicide stock solution (10 μL, 5 mM; 5 μL, 10 mM; 1 μL, 50 mM) was added to generate a colorimetric system with the same final concentration and total volume of the agent. Finally, 10 μL of GSH solution (5 mM, with ddH2O as the solvent) and 10 μL of DTNB solution were added for color development (20 mM, with DMSO as the solvent). Three replicates were set, and at 25 °C, the absorbance (A 412 ) was detected with a microplate reader, and at the same time, the turbidity effect of the herbicide and the anti-interference of DTNB on color development were evaluated.

[0062] 3. Analysis of the Optimal Reaction Conditions for OsGSTs

[0063] In vitro enzymatic reactions are usually carried out continuously in batches. Without an appropriate reaction temperature, proteins may precipitate and denature, affecting the final detection results. 100 μL of purified OsGST1, 4, 33, and 50 were incubated at gradient temperatures (27, 29, 31, 33, 35, and 37 °C) of a VeritiPro PCR instrument (Applied Biosystems, USA) for 1.5 h. The experiment was repeated 3 times, and the absorbance (A 412 ) was detected with a microplate reader at 25 °C to observe the denaturation of different OsGSTs.

[0064] To exclude the influence of DMSO and the possibility of protein denaturation by organic solvents, 189 μL of purified OsGST1, 4, 33, and 50 were used to replace the above-mentioned purified eluent blank control to detect the system (A 412 ). The test was repeated 3 times in total.

[0065] 4. Metabolism Analysis of Candidate OsGSTs on Herbicides

[0066] 4.1 High-Throughput Enzyme Activity Assay of the OsGSTs Metabolism Profile

[0067] Based on the above reaction system, the metabolic effects of 4 OsGSTs on 29 herbicide-related compounds were studied and analyzed. First, 189 μL of purified OsGSTU1, 4, 33, and 50 (0.25 mg / mL) were added to a 96-well plate, and then 1 μL of the herbicide-related compound stock solution (with DMSO as the solvent, 50 mM) was added. At the same time, 10 μL of GSH (5 mM, ddH2O) was added to initiate the reaction. A purified elution solution with the same volume as the purified OsGSTs (containing 50 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH = 7.5) was set as a control. After culturing at 29 °C for 1 h, 10 μL (20 mM, with DMSO as the solvent) of DTNB was added to terminate the color development. Three replicates were performed. The absorbance (A 412 ) was measured using a microplate reader at 25 °C. The concentration of GSH after the reaction was quantified using the above standard curve.

[0068] The calculation formula for the metabolic rate is as follows:

[0069] Herbicide metabolic rate (%) = (1 - (residual amount of GSH in the treatment group) / (residual amount of GSH in the control group)) × 100%

[0070] 4.2 Comparison of the detection effects of DTNB and HPLC on chloroacetamide herbicides

[0071] Alachlor, acetochlor, and S-metolachlor were selected as the detection objects, and the results obtained by the GSH-DTNB method and the traditional HPLC detection method were compared. The reaction system after DTNB method detection was subjected to sample pretreatment. The high-performance liquid chromatography instrument was Agilent HPLC 1260 infinity, equipped with a ZORBAX Eclipse XDB-C18 chromatographic column (5 μm, 4.6 × 250 mm, Agilent, USA). The mobile phase was acetonitrile / 0.5% acetic acid aqueous solution (v / v) (80:20), and the flow rate was 1.0 mL / min. A diode array detector (DAD) was used to analyze the unreacted herbicide at a wavelength of 230 nm, and a standard curve was established for quantitative analysis. By analyzing and comparing the metabolic rates measured by the two methods, the accuracy of the GSH-DTNB method and its consistency with the traditional method were judged.

[0072] 4.3 Data statistical analysis

[0073] Statistics and plotting were performed on GraphPad Prism 10.0. One-way analysis of variance (Tukey test) was used to analyze the effect of the insolubility of herbicides in the reaction system before and after the addition of DTNB.

[0074] The effects of OsGSTs protein denaturation on the reaction system under different conditions were analyzed by two-way ANOVA (Turkey test). One-way ANOVA (Turkey test) was used to analyze the high-throughput enzyme activity data. Two-way ANOVA (Sidak test) was used to analyze the comparison results of the HPLC-DTNB method (alachlor, acetochlor, and S-metolachlor).

[0075] III. Experimental Conclusions

[0076] 1. A sensitive GSH-DTNB colorimetric reaction system was constructed and optimized

[0077] The principle of detecting the metabolism amount of herbicides based on the GSH-DTNB colorimetric reaction is as Figure 1 shown. By constructing a GSH-DTNB standard curve, the upper and lower limits of the GSH working concentration were determined. When the GSH reaction concentration was 500 μM (detection concentration = 476.19 μM, 210 μL), the linear relationship with the absorbance (A 412 ) tended to be a quadratic equation (y = 36.333x 2 + 36.104x - 16.034, R 2 = 0.9951), and the highest concentration point deviated from the linear relationship, as Figure 2 shown. Therefore, the upper limit of the GSH concentration was adjusted to 250 μM ( Figure 2 ). In the prepared GSH standard curve, the color development effect was obvious, and the gradient change of the color was clearly visible ( Figure 2 ).

[0078] Similarly, in order to fully react with the glutathione substrate, the final concentration of the test-added herbicide-related compound could be set at 250 μM. Considering the stability of the herbicide stock solution and the negative impact of the solvent on the enzyme, DMSO was selected as the solvent in this study. When the final concentrations were the same, as the concentration of the stock solution increased, the turbidity state tended to be clear (A in Figure 3 ). In group A, when controlling the final concentration of DMSO to be less than 0.5% (v / v), whether DTNB was added for coloring or not, there was no obvious difference between the system with the chloroacetamide herbicide added and the DMSO solvent control. Therefore, the solvent parameters of group A were preliminarily determined (A, B in Figure 3 ).

[0079] Compared with the control DMSO, the absorbance (A 412 ) of the system decreased after adding the herbicide stock solution, and the absorbance (A 412 ) fluctuated among different herbicides (especially in the alachlor and butachlor systems) (A in Figure 4 ). However, after DTNB color development, the absorbance values (A 412) There was no significant difference in the numerical values, indicating that TNB 2- provided a good masking effect for the reaction system, and this method had a certain ability to resist turbidity interference ( Figure 4 B in

[0080] In terms of protein denaturation, when the reaction temperature was 27 °C or incubated for 1.5 h in the range of 33 - 37 °C, precipitation or turbidity occurred in the OsGSTU33 protein, resulting in a significant difference in the measured A 412 value ( Figure 5 ). Therefore, 29 °C was selected as the appropriate reaction temperature in this study. Except for the purified EV, no significant difference was detected after adding DMSO to each purified OsGST protein sample, so the potential denaturation or turbidity effect of the purified OsGST protein caused by the solvent was excluded ( Figure 6 ).

[0081] According to the above experimental results, the whole protocol was set as follows: 189 μL of purified GST (using the aforementioned purified eluent as a control), 1 μL of 50 mM herbicide stock solution, and the reaction was initiated with 10 μL of 5 mM GSH. After incubating in the dark at 29 °C for 1 h, 10 μL of 20 mM DTNB (diluted with DMSO) was added as a reaction terminator. Oscillating and mixing could accelerate the color reaction, and the absorbance value (A 412 ) was measured after mixing for 5 min.

[0082] 2. Comparison of the accuracy and efficiency between the high-throughput GSH-DTNB colorimetric assay method and the HPLC method

[0083] To detect the metabolic profiles of selective OsGSTs, 29 widely used herbicide-related compounds were selected in this study for high-throughput enzyme activity assays ( Figure 7 ).

[0084] Compared with CK, both OsGSTU1 and OsGSTU4 can metabolize five chloroacetamide herbicides. OsGSTU4 has the highest metabolism rates for alachlor (52.37%) and acetochlor (50.86%), followed by S-metolachlor (30.46%), butachlor (30.00%) and pretilachlor (28.56%). Although there are significant differences in the metabolism of the five chloroacetamide herbicides by OsGSTU1, the overall corresponding metabolism rates are all less than 10%, indicating a relatively low metabolism level. Meanwhile, OsGSTU50 has a weak metabolism ability for chloroacetamide herbicides. In addition, OsGSTU4 can significantly metabolize two acetyl-CoA carboxylase (ACCase) inhibitor herbicides with an aryloxyphenoxypropionate (AOPP) structure (fenoxaprop-P-ethyl: 11.95%; pinoxaden: 11.12%). OsGSTU1, 4 and 50 can all effectively metabolize quinclorac, with metabolism rates of 6.8%, 15.1% and 20.3% respectively. The safener fenclorim can also be metabolized by OsGSTU1 and OsGSTU4, with a metabolism rate of approximately 10% ( Figure 7 ).

[0085] To compare the results of the metabolism rate determined by the HPLC method and the DTNB method, the enzyme activity samples of alachlor, acetochlor and S-metolachlor after DTNB detection were subjected to chromatographic sample pretreatment operations, filtered with a 0.22 μm organic phase filter membrane, and the samples were analyzed by HPLC. The comparative analysis results found that there were no significant differences in the metabolism rate values obtained by the two methods, indicating that the two methods had consistency in the detection of this enzyme activity system. ( Figure 8 ).

[0086] Thus, this innovative high-throughput detection technology based on the GSH-DTNB color reaction has demonstrated many outstanding advantages in evaluating the metabolism of herbicides catalyzed by glutathione S-transferase (GST). Specifically: This method relies on the highly specific and sensitive reaction between DTNB and GSH, and TNB 2-Excellent stability at the appropriate pH value ensures the reliability of the detection results. More prominently, by constructing a reaction system in a 96-well plate, this technology achieves efficient batch detection, enabling easy processing of multiple samples in a single experiment and significantly improving the detection throughput. Compared with the traditional HPLC method, this innovation not only eliminates the complex sample pretreatment and chromatographic separation steps but also shortens the entire detection process to within two hours, greatly shortening the detection cycle. In addition, the requirements for experimental instruments are more lenient for this technology, without the need for expensive equipment such as high-precision liquid chromatographs and mass spectrometers, thus reducing the experimental cost and improving the popularity and application convenience of the technology. In summary, this high-throughput technology for detecting glutathione S-transferase metabolism of herbicides based on the GSH-DTNB color reaction demonstrates significant advantages in terms of simplicity of operation, detection efficiency, cost-effectiveness, sensitivity, specificity, and wide applicability, providing strong technical support for the study of plant glutathione S-transferase GST-catalyzed herbicide metabolism.

Claims

1. A detection system for high-throughput and rapid detection of the metabolic activity of glutathione S-transferase GST on herbicides based on GSH-DTNB color development, characterized in that, The detection system includes a reaction system and a blank control system. The reaction system contains: 189 μL of 0.25 mg / mL purified GST protein, 1 μL of 50 mM herbicide solution, and the reaction is initiated with 10 μL of 5 mM GSH solution. After incubation at 29 - 31°C in the dark, 10 μL of 20 mM DTNB solution is added as a reaction terminator; 189 μL of purified eluent is used to replace the purified GST protein in the reaction system as a blank control; The herbicide solution uses DMSO as a solvent, the GSH solution uses ddH2O as a solvent, and the DTNB solution uses DMSO as a solvent; The purified eluent is a salt solution containing 50 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, and pH = 7.5; The herbicide is at least one of chloroacetamide herbicides, aryloxyphenoxypropionate herbicides, quinclorac, fenclorim, tribenuron-methyl, 2,4-D, pyraflufen-ethyl, sulfosulfuron, mesosulfuron-methyl, quinclorac acid, propanil, imazamox, pyrazosulfuron-ethyl, mesotrione, penoxsulam, isoproturon, halosulfuron-methyl, mesotrione, bensulfuron-methyl, nicosulfuron, imazethapyr, sulfentrazone, quizalofop-p-ethyl, and flazasulfuron.

2. Use of the reagents in the detection system according to claim 1 in the preparation of a kit for high-throughput rapid detection of the metabolic activity of glutathione S-transferase against herbicides; The herbicide is at least one of chloroacetamide herbicides, aryloxyphenoxypropionate herbicides, quinclorac, fenclorim, tribenuron-methyl, 2,4-D, pyraflufen-ethyl, sulfosulfuron, mesosulfuron-methyl, quinclorac acid, propanil, imazamox, pyrazosulfuron-ethyl, mesotrione, penoxsulam, isoproturon, halosulfuron-methyl, mesotrione, bensulfuron-methyl, nicosulfuron, imazethapyr, sulfentrazone, quizalofop-p-ethyl, and flazasulfuron.

3. A kit for high-throughput and rapid detection of the metabolic activity of glutathione S-transferase (GST) against herbicides based on GSH-DTNB color development, characterized in that, The kit contains the reagents in the detection system according to claim 1.

4. A method for high-throughput and rapid detection of the metabolic activity of glutathione S-transferase (GST) on herbicides based on GSH-DTNB color development, characterized in that, The method is as follows: Take 189 μL of 0.25 mg / mL purified GST protein and 1 μL of 50 mM herbicide solution, use 10 μL of 5 mM GSH solution as the reaction initiator to start the reaction. After incubation at 29 - 31 °C in the dark, add 10 μL of 20 mM DTNB solution as the reaction terminator to terminate the reaction to obtain a reaction system; use 189 μL of purified eluent to replace the purified GST protein in the reaction system as a blank control system; detect the A 412 absorbance value, quantify the GSH concentration in the reaction system and the blank control system, and calculate the metabolic rate of glutathione S-transferase to the herbicide; The herbicide solution uses DMSO as a solvent, the GSH solution uses ddH2O as a solvent, and the DTNB solution uses DMSO as a solvent; The purified eluent is a salt solution containing 50 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, and pH = 7.5; The herbicide is at least one of chloroacetamide herbicides, aryloxyphenoxypropionate herbicides, quinclorac, fenclorim, tribenuron-methyl, 2,4-D, pyraflufen-ethyl, sulfosulfuron, mesosulfuron-methyl, quinclorac acid, propanil, imazamox, pyrazosulfuron-ethyl, mesotrione, penoxsulam, isoproturon, halosulfuron-methyl, mesotrione, bensulfuron-methyl, nicosulfuron, imazethapyr, sulfentrazone, quizalofop-p-ethyl, and flazasulfuron.

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