Method for rapidly detecting metabolic activity of glutathione S-transferase to herbicide based on GSH-DTNB color development and high throughput

Abstract

The invention discloses a method for rapidly detecting the metabolic activity of glutathione S-transferase to herbicides on the basis of GSH-DTNB (glutathione S-transferase) color development and high throughput, which comprises the following steps: taking purified GST protein and herbicide solution, starting reaction by taking GSH solution as reaction initiator, incubating, and adding DTNB solution as reaction terminator to terminate reaction to obtain a reaction system; replacing the purified GST protein in the reaction system with an isovolumetric purified eluent to serve as a blank control system; detecting the A412 light absorption values of the reaction system and the blank control system, quantifying the GSH concentration in the reaction system and the blank control system, and calculating the metabolic rate of the glutathione S-transferase to the herbicide. The method not only improves the detection efficiency and accuracy, but also reduces the detection cost, and provides important reference for development of more subsequent GSH-DTNB-based rapid detection methods.

Description

Technical Field

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

[0002] Glutathione S-transferase (GST) catalyzes glutathione conjugation reactions and participates in physiological activities such as intracellular redox balance regulation, cell signal transduction, and detoxification metabolism of exogenous harmful compounds. With the rapid development of genomics and bioinformatics, the GST gene family of various plants has been identified and annotated at the whole genome level, which has accelerated the localization of GST genes and member classification. For example, Jain and Sappl et al. systematically identified and annotated GST genes in three plants, Arabidopsis, rice, and Populus tomentosa, revealing the diversity of GST members in different plants. There are 53 GST members in Arabidopsis, 79 in rice, 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. It was further found that in terms of subfamily classification, plants have four special GST categories: phi (F), tau (U), dehydroascorbate reductase (DHAR), and lambda (L). GST members of the same subfamily are usually highly conserved in gene sequence and protein structure, which is conducive to the identification, classification, model construction and activity prediction of family members. So far, although a large number of GST members from different plants have been identified and annotated, the functions of only a few of them 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. The number of its members is large, and it is of great significance in the field of weed resistance and crop breeding research; they directly and effectively detoxify the active molecules of herbicides by catalyzing the glutathione (GSH) binding reaction. In this process, GST can catalyze the replacement 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, so that the molecular structure of the herbicide compound is broken, and a metabolite containing a hydroxyl product and GSH is generated, which destroys the molecular structure of the entire herbicide. At the same time, these reactions will also increase the hydrophilicity of the herbicide metabolites, making it easier to be transported to the vacuole for isolation or excretion through the transporter protein in the plant body, thereby effectively reducing the toxic effects of herbicides on plants.

[0004] The metabolic functions and mechanisms of some GST members in plants have been well understood. Prade et al. demonstrated the protein crystal structure of the herbicide molecule (FE-4053, atrazine) bound to the detoxification enzyme AraGST (1bx9) or ZmGSTI (1bye), providing an ideal crystal conformation for in-depth analysis of the catalytic site. Dücker et al. used isotope labeling and liquid phase tandem radioactive detector to deeply analyze the decomposition of flufenacet in phase III metabolism and found that GST-catalyzed flufenacet metabolism can significantly reduce the sensitivity of Alopecurus myosuroides Huds. and ryegrass (Lolium spp.), further confirming the important role of GST in mediating weed resistance. With the development of transcriptome sequencing technology, the expression levels of GST gene family members can be accurately analyzed at various growth and development stages of plants. This makes it possible to select GST members with differential expression for in vitro enzyme activity reaction experiments, and to have a deeper understanding of GST catalytic metabolism and the corresponding product structure through HPLC-DAD or liquid chromatography mass spectrometry tandem analysis (LC-MS / MS) detection. However, although this research process can accurately establish the metabolic association between metabolic enzymes and herbicide active molecules, the equipment and consumables required for the entire detection system are expensive, and the detection process is time-consuming and relatively inefficient. Therefore, it is urgent to develop an analytical method for high-throughput detection of GST enzyme-catalyzed herbicide metabolism to accelerate the study of plant GST gene function.

[0005] DTNB stands for 5,5'-dithiobis-(2-nitrobenzoic acid), a colorless or light yellow powder. It is an important reagent for evaluating the content of free glutathione (GSH) in biological samples and is widely used in the fields of medical and plant pathology detection. The color development principle of DTNB is based on the substitution reaction between disulfide bonds (-SS-) and sulfhydryl groups (-SH). It can react quickly with GSH at room temperature to release one molecule of GS-TNB and one molecule of TNB. 2- . TNB 2- It is relatively stable in neutral and weakly alkaline environments (pH = 7.2 ~ 8.0), and has a maximum absorption peak at a wavelength of 412 nm (14,150 M -1 cm -1), which can be detected by an enzyme marker at the corresponding light wavelength. Taking advantage of this feature, in the GST-catalyzed herbicide metabolism reaction system, DTNB is introduced to bind to the remaining free GSH in the reaction, prompting the reaction to terminate quickly; the color development of the reaction system is detected by an enzyme marker, and the metabolic amount of the herbicide molecule is calculated, and then the plant GST members are accurately and efficiently qualitatively and quantitatively analyzed under certain conditions. The entire process from initial sample loading to final detection 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 determination method for detecting the metabolic ability of glutathione S-transferase GST on herbicides based on GSH-DTNB colorimetric reaction.

[0007] The purpose of the present invention can be achieved through the following technical solutions:

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

[0009] Further, the reaction system described in the above detection system comprises: 189 μL 0.25 mg / mL purified GST protein, 1 μL 50 mM herbicide solution, 10 μL 5 mM GSH solution to start the reaction, incubate in the dark at 29-31°C, and then add 10 μL 20 mM DTNB solution as a reaction terminator; 189 μL purified eluate is used to replace the purified GST protein in the reaction system as a blank control.

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

[0011] In a second aspect, the present invention seeks to protect the use of the reagents in the above detection system in the preparation of a kit for high-throughput rapid detection of the metabolic activity of glutathione S-transferase on herbicides.

[0012] In a third aspect, the present invention claims a kit for rapid detection of the metabolic activity of glutathione S-transferase on herbicides based on GSH-DTNB colorimetric high-throughput, wherein the kit comprises the reagents in the aforementioned detection system.

[0013] In a fourth aspect, the present invention claims a method for rapid detection of glutathione S-transferase metabolic activity of herbicides based on GSH-DTNB color development. The method comprises: taking purified GST protein and herbicide solution, starting the reaction with GSH solution as a reaction initiator, incubating in the dark, adding DTNB solution as a reaction terminator to terminate the reaction to obtain a reaction system; replacing the purified GST protein in the reaction system with an equal volume of purified eluate as a blank control system; detecting the A of the reaction system and the blank control system. 412 The absorbance value was used to quantify the GSH concentration in the reaction system and the blank control system and to calculate the metabolic rate of the herbicide by glutathione S-transferase.

[0014] The metabolic rate is calculated as follows:

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

[0016] Furthermore, in the above method, 189 μL of 0.25 mg / mL purified GST protein and 1 μL of 50 mM herbicide solution were taken, and the reaction was initiated with 10 μL of 5 mM GSH solution as a reaction initiator. After incubation in the dark at 29-31°C, 10 μL of 20 mM DTNB solution was added as a reaction terminator to terminate the reaction to obtain a reaction system; 189 μL of purified eluate was used to replace the purified GST protein in the reaction system as a blank control system.

[0017] Furthermore, in the above method, 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 purification eluent contains 50 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, and a salt solution with a pH of 7.5.

[0018] In a specific embodiment of the present invention, the reaction system is shaken and mixed to accelerate the color reaction. After shaking and mixing for 5 minutes, the absorbance (A) is detected by an enzyme marker at 25°C. 412 ).

[0019] The technical principle of the method of the present invention is: during the reaction process of the reaction system, glutathione S-transferase (GST) catalyzes the combination of herbicide and glutathione (GSH) to generate a herbicide molecule and GSH conjugate (GS-Herb). After the herbicide, GST and GSH react, DTNB is added to combine with the remaining GSH in the reaction system to form a GS-TNB conjugate, and at the same time, a detectable yellow TNB is generated. 2+ The product has a maximum absorption peak at 412 nm. By measuring the absorbance of the reaction product and referring to the standard curve, the content of residual 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 herbicides can be clarified.

[0020] Beneficial effects of the present invention:

[0021] 1. High detection efficiency

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

[0023] 2. Simple experimental operation: This method has established basic reaction conditions and 96-well plate-based operation procedures, the reaction system has been simplified, and no complicated sample pretreatment operations are required during detection.

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

[0025] 2. High detection accuracy

[0026] 1. The metabolic rates detected by this method were compared with those by the traditional liquid chromatography-mass spectrometry HPLC method on some samples, and there was no significant difference.

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

[0028] 3. Reduce human errors: The one-step reaction system reduces the errors caused by human operation and improves the reliability of the test results.

[0029] 3. Low testing cost

[0030] 1. Reduce reagent consumption: Since the reaction volume is at the microliter level, the amount of protein sample and reagent required is relatively small, thereby reducing reagent consumption and detection costs.

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

[0032] 4. Wide application scenarios

[0033] 1. Basic research: This method provides a powerful tool for in-depth research on the mechanism of GST metabolism of herbicides, which helps to reveal the effects of herbicides on organisms and clarify their 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 has promoted technological innovation and development in related fields, and promoted the continuous emergence and application of new technologies and methods.

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

[0037] Figure 1 :Schematic diagram of the GSH-DTNB colorimetric reaction for the analysis of herbicide metabolic enzyme activity. During the reaction, glutathione S-transferase (GST) catalyzes the binding of herbicides to glutathione (GSH) to generate a herbicide molecule and GSH conjugate (GS-Herb). After the herbicide, GST and GSH react, DTNB is added to bind to the remaining GSH in the reaction system to form a GS-TNB conjugate, and at the same time, a detectable yellow TNB is generated. 2+ The product has a maximum absorption peak at 412 nm. By measuring the absorbance of the reaction product and referring to the standard curve, the content of GSH remaining 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 color development standard curve. By constructing the GSH-DTNB standard curve, the upper and lower limits of GSH concentration in the reaction system were determined.

[0039] Figure 3:The optimization effect diagram of three substrate reaction combinations. Considering the stability of the herbicide mother solution and the negative impact of its turbidity on the detection, this method uses DMSO as the solvent. When the actual concentration of each group of drugs is consistent, when the mother solution concentration increases, the turbidity state tends to be clear ( Figure 3 In Group A, there was no significant visual difference between the treatment group containing chloroacetamide herbicide stock solution and the control group (adding DMSO) before and after adding DTNB for color development. Therefore, Group A was selected as the candidate system ( Figure 3 A, B in it).

[0040] Figure 4 : Absorbance test results of three substrate reaction combinations. When the actual concentrations of each group of agents are consistent, the absorbance of the system containing chloroacetamide herbicide mother solution (A 412 ) decreased, and the absorbance (A 412 ) had low-level fluctuations, but after adding DTNB for color development, there was no significant difference between the treatment group containing the drug and the control group, indicating that TNB 2- Provides excellent color rendering and masking effects.

[0041] Figure 5 :Explore the results of the experiment on the optimal reaction temperature of OsGSTs. Purified OsGSTs were incubated at different temperatures and the absorbance values ​​A of each group were detected. 412 The optimum reaction temperature of this group was found to be 29-31℃.

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

[0043] Figure 7 : Results of high-throughput analysis of herbicide-related compounds metabolized by OsGSTUs.

[0044] Figure 8 :Comparison of the metabolic rate and accuracy of three chloroacetamide herbicides analyzed by HPLC and DNTB. By comparing the metabolic rate of alachlor, acetochlor and S-isopropylacetamide by DTNB and HPLC under the same system, it was found that there was no significant difference in the metabolic rate results obtained by the two methods. DETAILED DESCRIPTION

[0045] The method of the present invention is described below by means of specific examples, but the present invention is not limited thereto, and any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0046] Example 1

[0047] 1. 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 Shanghai MacLean Company, 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 profiling analysis, and RNA was extracted from rice plants and reverse transcribed into cDNA as a template for amplification. The primers and restriction sites used are shown in Table 1:

[0052] Table 1 Primers and restriction sites

[0053]

[0054] F represents the forward primer, and R represents the reverse primer. The underline marks the restriction site.

[0055] OsGSTs vector construction scheme: The pET-28a (+) system was used for recombinant expression in Escherichia coli BL21 strain (purchased from Nanjing Novozyme Biotechnology Co., Ltd.). After induction of 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 the crude OsGSTs enzyme was carried out using the purification scheme of the reduction-resistant chelating agent His tag protein purification kit (purchased from Beyotime Biotechnology Co., Ltd.).

[0056] 2. Establishment and Optimization of Reaction System

[0057] In order 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 an ELISA instrument. To confirm the stability of the entire system, the following parameters need to be considered: the detection concentration range of glutathione, the low solubility interference 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 were obtained. Prepare a 50 mM glutathione stock solution and dilute it to 5 mM with ddH2O. Add 190 μL of purified eluate (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. 10 μL DTNB (20 mM, DMSO as solvent) was added to react completely with glutathione. Since the total volume changed slightly after adding DTNB, the actual final concentrations were adjusted to 476.19, 238.09, 119.05, 59.52, 29.76, 14.88, 7.44 and 0 μM. 412 ) Use Infinite ® The detection was performed using a 200 PRO microplate reader (Tecan, Switzerland) at 25°C. Three replicate standard curves were established. The absorbance values ​​of the standard curves were analyzed in Excel 2019 (Microsoft, USA), and the linear relationship was fitted and plotted.

[0060] 2. Analysis and optimization of the effect of herbicide turbidity on the reaction system

[0061] In order to explore the turbidity effect of herbicide mother solution after adding to the system, five commonly used low water-soluble chloroacetamide herbicides: alachlor, acetochlor, pretilachlor, butachlor and S-isopropyl chloramine were selected for turbidity test with DMSO as solvent. The initial reaction system was established: with different volumes of purified eluent (180, 185, 189 μL) (purified eluent containing 50mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH=7.5) as the background, the corresponding concentration of chloroacetamide herbicide mother solution (10 μL, 5 mM; 5 μL, 10 mM; 1 μL, 50 mM) was added to generate a color system with the same final concentration and total volume of the agent. Finally, 10 μL of GSH solution (5 mM, ddH2O as solvent) and 10 μL of DTNB solution (20 mM, DMSO as solvent) were added for color development. Set up 3 replicates, and use a microplate reader to detect the absorbance before and after the addition of GSH and DTNB at 25°C (A 412 ), and simultaneously evaluate the turbidity effect of the herbicide and the anti-interference of DTNB on color development.

[0062] 3. Analysis of suitable reaction conditions for OsGSTs

[0063] In vitro enzymatic reactions are usually carried out in batches. If the reaction temperature is not appropriate, the protein may precipitate and denature, affecting the final detection effect. 100 μL of purified OsGST1, 4, 33 and 50 were incubated at a gradient temperature (27, 29, 31, 33, 35 and 37°C) in a VeritiPro PCR instrument (Applied Biosystems, USA) for 1.5 h. The experiment was repeated 3 times and the absorbance (A) was measured with an ELISA reader at 25°C. 412 ), observe the denaturation of different OsGSTs.

[0064] In order 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 instead of the blank control of the above purified eluent to detect the system (A 412 The experiment was repeated 3 times.

[0065] 4. Metabolic analysis of candidate OsGSTs towards herbicides

[0066] 4.1 High-throughput enzyme activity assay for OsGSTs metabolic profile

[0067] According to the above reaction system, the metabolic effects of four 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 herbicide-related compound stock solution (DMSO as solvent, 50 mM) was added. At the same time, 10 μL of GSH (5 mM, ddH2O) was added to start the reaction. The same volume of purified eluate (containing 50 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH=7.5) as the purified OsGSTs was set as a control. After incubation at 29°C for 1 hour, 10 μL (20 mM, with DMSO as solvent) DTNB was added to terminate the color development. This was repeated three times. The absorbance (A 412 ). The concentration of GSH after the reaction was quantified using the standard curve mentioned above.

[0068] The metabolic rate is calculated as follows:

[0069] Herbicide metabolism rate (%) = (1-(GSH residue in treatment group) / (GSH residue in 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 GSH-DTNB method and traditional HPLC detection method were compared. The reaction system after DTNB detection was subjected to sample pretreatment. The HPLC instrument was Agilent HPLC 1260 infinity, equipped with a ZORBAX Eclipse XDB-C18 column (5 μm, 4.6 × 250mm, 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. The unreacted herbicides were analyzed by diode array detector (DAD) at a wavelength of 230 nm, and a standard curve was established for quantitative analysis. The accuracy of the GSH-DTNB method and its consistency with the traditional method were determined by analyzing and comparing the metabolic rates measured by the two methods.

[0072] 4.3 Data Statistical Analysis

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

[0074] Two-way ANOVA (Turkey test) was used to analyze the effect of OsGSTs protein denaturation on the reaction system under different conditions. 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 comparative results of HPLC-DTNB method (alachlor, acetochlor and S-metolachlor).

[0075] 3. Experimental Conclusion

[0076] 1. Constructed and optimized a sensitive GSH-DTNB colorimetric reaction system

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

[0078] Similarly, in order to fully react with glutathione substrate, the final concentration of herbicide-related compounds added to the experiment can be set to 250 μM. Considering the stability of the herbicide mother solution and the negative effect of the solvent on the enzyme, DMSO was selected as the solvent in this study. When the final concentration is consistent, as the concentration of the mother solution increases, the turbidity state tends to be clear ( Figure 3 In group A, when the final concentration of DMSO was controlled to be less than 0.5% (v / v), there was no significant difference between the system with chloroacetamide herbicide added and the DMSO solvent control, regardless of whether DTNB was added for coloring. Therefore, the solvent parameters of group A were preliminarily determined ( Figure 3 A, B in it).

[0079] Compared with the control DMSO, the absorbance of the system after adding the herbicide mother solution (A 412 ) decreased, and the absorbance (A 412 ) fluctuates ( Figure 4 However, after DTNB color development, the absorbance values ​​of each group (A 412) values ​​were not significantly different. This indicates that TNB 2- It provides a good masking effect for the reaction system, and the method has a certain ability to resist turbidity interference ( Figure 4 B in the figure).

[0080] In terms of protein denaturation, when the reaction temperature was 27°C or incubated at 33-37°C for 1.5 h, OsGSTU33 protein precipitated or became turbid, which made the measured A 412 There are significant differences in the values ​​( 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, thus excluding the potential denaturation or turbidity of the purified OsGST protein caused by the solvent ( Figure 6 ).

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

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

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

[0084] Compared with CK, both OsGSTU1 and OsGSTU4 could metabolize five chloroacetamide herbicides. OsGSTU4 had the highest metabolic rates for alachlor (52.37%) and acetochlor (50.86%), followed by S-isopropylamine (30.46%), butachlor (30.00%) and pretilachlor (28.56%). Although OsGSTU1 had significant differences in the metabolism of the five chloroacetamide herbicides, the overall corresponding metabolic rates were less than 10%, indicating a low metabolic level. Meanwhile, OsGSTU50 had a weak metabolic capacity for chloroacetamide herbicides. In addition, OsGSTU4 could significantly metabolize two acetyl-CoA carboxylase (ACCase) inhibitor herbicides with aryloxyphenoxypropionate (AOPP) structures (fenoxadone: 11.95%; oxadiazine: 11.12%). OsGSTU1, 4 and 50 can effectively metabolize dichloroquinoline, with metabolic rates of 6.8%, 15.1% and 20.3% respectively. The safener fenthiocarb can also be metabolized by OsGSTU1 and OsGSTU4, with a metabolic rate of about 10% ( Figure 7 ).

[0085] In order to compare the results of metabolic rate determination by HPLC and DTNB, the enzyme activity samples of acetochlor, acetochlor and S-isopropylacetochlor after DTNB detection were subjected to chromatographic sample pretreatment, filtered with a 0.22 μm organic phase filter membrane, and the samples were analyzed by HPLC. The results of the comparative analysis showed that there was no significant difference in the metabolic rate values ​​obtained by the two methods, indicating that the two methods were consistent in detecting the enzyme activity system. ( Figure 8 ).

[0086] It can be seen that this innovative high-throughput detection technology based on the GSH-DTNB colorimetric reaction has shown 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-The excellent stability at the appropriate pH value ensures the reliability of the test results. More importantly, by constructing a reaction system in a 96-well plate, the technology achieves efficient batch detection, making it easy to process multiple samples in a single experiment, 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 technology has more relaxed requirements for experimental instruments, and does not require expensive equipment such as high-precision liquid chromatographs and mass spectrometers, thereby reducing experimental costs and improving the popularity and ease of application of the technology. In summary, this technology for high-throughput detection of glutathione S-transferase-metabolized herbicides based on the GSH-DTNB colorimetric reaction has shown significant advantages in terms of ease 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 based on GSH-DTNB colorimetric high-throughput rapid detection of glutathione S-transferase GST's metabolic activity on herbicides, characterized in that: The detection system includes a reaction system and a blank control system. The reaction system includes: 189 μL of 0.25 mg / mL purified GST protein, 1 μL of 50 mM herbicide solution, 10 μL of 5 mM GSH solution to start the reaction, incubate in the dark at 29-31°C, and then add 10 μL of 20 mM DTNB solution as a reaction terminator; 189 μL of purified eluate is used to replace the purified GST protein in the reaction system as a blank control.

2. The detection system according to claim 1, characterized in that 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.

3. The detection system according to claim 1, characterized in that The purification eluent is a salt solution containing 50 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, and pH=7.

5.

4. Use of the reagents in the detection system of claim 1, 2 or 3 in the preparation of a kit for high-throughput rapid detection of glutathione S-transferase metabolic activity on herbicides.

5. A kit for rapid detection of glutathione S-transferase GST metabolic activity of herbicides based on GSH-DTNB colorimetric high-throughput detection, characterized in that: The kit comprises the reagents in the detection system according to claim 1, 2 or 3.

6. A method for rapid detection of glutathione S-transferase GST metabolic activity on herbicides based on GSH-DTNB colorimetric high-throughput, characterized in that: The method comprises the following steps: taking purified GST protein and herbicide solution, starting the reaction with GSH solution as a reaction initiator, incubating in the dark, and then adding DTNB solution as a reaction terminator to terminate the reaction to obtain a reaction system; replacing the purified GST protein in the reaction system with an equal volume of purified eluate as a blank control system; and detecting the A of the reaction system and the blank control system. 412 The absorbance value was used to quantify the GSH concentration in the reaction system and the blank control system and to calculate the metabolic rate of the herbicide by glutathione S-transferase.

7. The method according to claim 6, characterized in that Take 189 μL of 0.25 mg / mL purified GST protein and 1 μL of 50 mM herbicide solution, start the reaction with 10 μL of 5 mM GSH solution as a reaction initiator, incubate in the dark at 29-31°C, and then add 10 μL of 20 mM DTNB solution as a reaction terminator to terminate the reaction to obtain a reaction system; 189 μL of purified eluate was used to replace the purified GST protein in the reaction system as a blank control system.

8. The method according to claim 6 or 7, characterized in that: 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.

9. The method according to claim 6 or 7, characterized in that The purification eluent is a salt solution containing 50 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, and pH=7.5.

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