Plant alpha-ketoglutaric acid / Fe (II) dependent dioxygenase enzyme activity detection reagent, kit and application

Abstract

The invention discloses an enzyme activity detection reagent for plant alpha-ketoglutaric acid / Fe (II) dependent dioxygenase, a kit and application, and belongs to the technical field of biology. The enzyme activity detection reagent comprises a reagent I and a reagent II, wherein the reagent I comprises the following components: 1-5 mM of dithiothreitol, 0.1-1 mM of oxoglutaric acid, 0.1-1 mM of sodium ascorbate and 0.01-0.1 mg / mL of catalase, and sterilized distilled water is used as a solvent; the reagent II is prepared from 2 to 3 mg of FeSO4. 7H2O solid. Reagents for enzyme catalytic reaction are mixed according to a specific proportion to prepare the reagent I and the reagent II, the reagent I and the reagent II are directly subjected to one-time mixed reaction with a sample to be detected and a substrate during use, the operation is simple, convenient and rapid, the problems of errors, omission and the like caused by mixing reaction components for multiple times are avoided, and the detection accuracy is improved. And the method can also be used for detecting the enzyme activity of alpha-ketoglutaric acid / Fe (II) dependent dioxygenase of various plants, and has universality.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an enzyme activity detection reagent, a kit and applications of plant alpha-ketoglutarate / Fe(II)-dependent dioxygenase. Background Art

[0002] There are a large number of small molecule compounds in plants that are synthesized and metabolized in complex and varied ways. Among them, oxygenation (hydroxylation) is an important synthesis and metabolism method. Hydroxylation in plants is mainly catalyzed by two enzymes, namely cytochrome P450 oxygenases (CYPs) and α-ketoglutarate / Fe(Ⅱ)-dependent dioxygenases (2–oxoglutarate-dependent dioxygenases, 2OGDs).

[0003] The number of 2OGD proteins in different plants varies greatly, with 130 in Arabidopsis, 41 in Chlamydomonas, 142 in spruce, and 114 in rice. Kai et al. identified genes related to scopoletin synthesis in Arabidopsis and detected the accumulation of scopoletin in several T-DNA insertion mutants. Studies have shown that before the formation of the scopoletin lactone ring, the 2OGD protein F6'H ortho-hydroxylates feruloyl-CoA instead of catalyzing the production of ferulic acid products. In 2018, Siwinska identified a 2OGD protein with a unique function in Arabidopsis, namely scopoletin 8-hydroxylase (S8H), which can catalyze scopoletin to produce fraxetin (Siwinska et al., 2018). In cotton, the biosynthesis of the sesquiterpenoid secondary metabolite gossypol requires 2ODD-1 to catalyze the production of furanyl carrageenanedione. Salicylic acid-3-hydroxylase and salicylic acid-5-hydroxylase in Arabidopsis were discovered in 2013 and 2017, respectively, and jasmonic acid hydroxylase was discovered in 2017. 2OGD proteins in plants metabolize plant hormones and regulate plant disease resistance and various physiological activities. In recent years, it has been a hot topic in biological research.

[0004] 2OGD is a type of iron-containing protein that does not contain heme and is often located in the cytoplasm. It requires α-ketoglutarate and molecular oxygen as co-substrates, and uses ferrous ions as cofactors to convert α-ketoglutarate into succinate and carbon dioxide, catalyzing the hydroxylation of the substrate. At present, when detecting the activity of 2OGD-type proteases, it is necessary to add each substrate of the reaction, such as α-ketoglutarate, ferrous ions, etc., one by one. The reaction is cumbersome, the operation is complicated, and it is time-consuming. It is also very easy to make mistakes and omissions in the operation; some reactions require the addition of strong acid to terminate the reaction. In addition, the current detection method can only detect specific 2OGD, which is not universal and is not conducive to high-throughput drug screening. Therefore, it is very necessary to provide a simple and efficient general method for 2OGD-type proteases. Summary of the invention

[0005] The purpose of the present invention is to provide a plant α-ketoglutarate / Fe (II) dependent dioxygenase enzyme activity detection reagent, kit and application, so as to solve the problems existing in the above-mentioned prior art. The present invention mixes the reagents of the enzyme catalytic reaction according to a specific ratio to prepare reagent I and reagent II, which can be directly mixed with the sample to be tested and the substrate for a one-time reaction when used. The operation is simple and quick, and the problems of errors and omissions caused by multiple mixing of reaction components are avoided.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a plant α-ketoglutarate / Fe (II) dependent dioxygenase enzyme activity detection reagent, the enzyme activity detection reagent comprises a reagent I and a reagent II, the components of the reagent I are: 1-5mM dithiothreitol, 0.1-1mM oxoglutaric acid, 0.1-1mM sodium ascorbate, 0.01-0.1mg / mL catalase, and sterile distilled water as a solvent;

[0008] Reagent II includes FeSO4·7H2O solid, the content of which is 2-3 mg.

[0009] Optionally, the reagent II can be dissolved in distilled water and used immediately upon preparation.

[0010] Optionally, the concentration of reagent II is 2-3 mg / mL.

[0011] The present invention also provides a kit for detecting the enzyme activity of plant α-ketoglutarate / Fe(II)-dependent dioxygenase, comprising the enzyme activity detection reagent.

[0012] More preferably, the enzyme activity detection kit of the present invention comprises reagent I and reagent II, wherein reagent I comprises: 5 mM dithiothreitol, 1 mM oxoglutaric acid, 1 mM sodium ascorbate, 0.1 mg / mL catalase and sterile distilled water, and the total volume after mixing the above components is 50 μL, and placed in an enzyme-free 1.5 mL centrifuge tube for standby use.

[0013] Reagent II is: FeSO4·7H2O solid, content is 2.78mg, placed in an enzyme-free 1.5mL centrifuge tube, ready for use. Add 1mL of distilled water before use, ready for use.

[0014] The present invention also provides a method for detecting the activity of plant α-ketoglutarate / Fe(II)-dependent dioxygenase based on the enzyme activity detection reagent, comprising the following steps:

[0015] The sample to be tested, reagent II, substrate and reaction buffer are added to the enzyme-free centrifuge tube containing the reagent I, and an enzyme-catalyzed reaction is performed to produce a product. The enzyme activity of the sample to be tested is determined based on the amount of the product.

[0016] Optionally, the substrate includes jasmonic acid and salicylic acid. The amount of the substrate and the enzyme catalytic reaction conditions are determined according to the properties of the sample to be tested. The reagents I and II prepared by the present invention can accurately and efficiently detect different enzymes.

[0017] Optionally, the ratio of the reagent I, the sample to be tested, and the reagent II is 50 μL: (1-5) μg: (1-4) μL.

[0018] The present invention also provides application of the enzyme activity detection reagent or the enzyme activity detection kit or the method in detecting the enzyme activity of plant α-ketoglutarate / Fe(II)-dependent dioxygenase.

[0019] The present invention discloses the following technical effects:

[0020] The kit of the present invention is composed of reagent I and reagent II, wherein reagent I is prepared by premixing dithiothreitol, oxoglutaric acid, sodium ascorbate, catalase and sterilized distilled water in a specific ratio, and reagent II is prepared by mixing FeSO4·7H2O solid and distilled water in a specific ratio. Utilizing the kit of the present invention, reagent I and reagent II are directly mixed with the 2OGD protein to be tested and the substrate for enzyme catalysis reaction, and the enzyme activity of the 2OGD protein to be tested is determined by detecting the amount of the enzyme reaction product. This method avoids the defect of complicated operation of adding different reagents one by one in the prior art. The detection method of the present invention is simple and quick to operate, and also avoids the problems of errors and omissions caused by mixing into each component of the reaction for many times. The kit of the present invention can detect a variety of 2OGD proteins and has universality. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The high performance liquid chromatogram of the reaction product of AhS5H2 and salicylic acid is 2,5-dihydroxybenzoic acid (2,5-DHBA);

[0023] Figure 2 The extracted ion chromatogram of the reaction product of AhJAO1 and jasmonic acid is 12-hydroxyjasmonic acid (12-OH-JA);

[0024] Figure 3 The high performance liquid chromatogram of the reaction product of AhS5H1 and salicylic acid is 2,5-dihydroxybenzoic acid (2,5-DHBA);

[0025] Figure 4 is the rate of product formation of the reaction between AhS5H1 and salicylic acid at different temperatures. DETAILED DESCRIPTION

[0026] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0027] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0029] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0030] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0031] Example 1 Reaction kit for detecting plant α-ketoglutarate / Fe(Ⅱ) dependent dioxygenase activity

[0032] The kit includes reagent I and reagent II, wherein reagent I contains 5 mM dithiothreitol, 1 mM oxoglutaric acid, 1 mM sodium ascorbate, 0.1 mg / mL catalase, and the solvent is sterile distilled water, and the total volume after the above components are mixed is 50 μL. The reagents are placed in a 1.5 mL centrifuge tube without enzyme for later use.

[0033] Reagent II is: FeSO4·7H2O solid, content is 2.78mg, placed in an enzyme-free 1.5mL centrifuge tube, ready for use. Add 1mL of distilled water before use, ready for use.

[0034] Example 2 Reaction kit for detecting plant α-ketoglutarate / Fe(Ⅱ) dependent dioxygenase activity

[0035] The kit includes reagent I and reagent II, wherein reagent I contains 1 mM dithiothreitol, 0.1 mM oxoglutaric acid, 0.1 mM sodium ascorbate, 0.01 mg / mL catalase, and the solvent is sterile distilled water. The total volume of the above components after mixing is 50 μL. The reagents are placed in a 1.5 mL centrifuge tube without enzyme for later use.

[0036] Reagent II is: FeSO4·7H2O solid, content is 2mg, placed in an enzyme-free 1.5mL centrifuge tube, ready for use. Add 1mL of distilled water before use, ready for use.

[0037] Example 3 Reaction kit for detecting plant α-ketoglutarate / Fe(Ⅱ) dependent dioxygenase activity

[0038] The kit includes reagent I and reagent II, wherein reagent I contains 2.5 mM dithiothreitol, 0.3 mM oxoglutaric acid, 0.5 mM sodium ascorbate, and 0.03 mg / mL catalase, which are dissolved in sterile distilled water, and the total volume after mixing the above components is 50 μL. The reagents are placed in a 1.5 mL centrifuge tube without enzyme for later use.

[0039] Reagent II is: FeSO4·7H2O solid, content is 3mg, placed in an enzyme-free 1.5mL centrifuge tube for use. Add 1mL of distilled water before use.

[0040] Application Examples

[0041] The kit prepared in Example 1 of the present invention was used to detect 2OGD proteases. Prepare the protein expressed and purified by Escherichia coli in prokaryotic cells in advance. The optimal substrate content, pH, reaction temperature, reaction time, protein, and buffer solution required for each protein are different. The "single factor method" was used to screen and optimize the reaction conditions before the formal reaction. See the literature "Liang, B.; Wang, H.; Yang, C.; Wang, L.; Qi, L.; Guo, Z.; Chen, X. Salicylic Acid Is Required for Broad-Spectrum Disease Resistance in Rice. Int. J. Mol. Sci. 2022, 23, 1354.".

[0042] ① Taking the detection of salicylic acid hydroxylase AhS5H2 in peanut as an example, the kit prepared in Example 1 was used to carry out the reaction.

[0043] 10 mM salicylic acid was used as the substrate for enzyme activity detection. 5 μg of protein expressed and purified by E. coli was prepared before the reaction. 1 mL of sterile water was added to the centrifuge tube containing reagent II, mixed thoroughly and placed on ice.

[0044] Enzyme activity reaction process: Add 5 μg AhS5H2 protein to the centrifuge tube containing reagent I, add 4 μL reagent II, add substrate to make the final concentration 10 mM, add phosphate buffer solution with a pH of 6.8 to make the concentration 50 mM. Use distilled water to make up the volume to 100 μL. React at 40°C for 30 minutes.

[0045] After the reaction was completed, the mixture was placed in a boiling water bath for 1 min, and the amount of the product was directly detected by high performance liquid chromatography after cooling. The product was analyzed by a Waters 2695 liquid chromatography system (Waters Technology (Shanghai) Co., Ltd.) equipped with a Dima C18 column (250×4.6 mm, Beijing Dima Technology Co., Ltd.). The mobile phase for detection was acetonitrile and water. The chromatographic conditions were: a flow rate of 1 mL / min, an acetonitrile ratio of 12%, maintained for 15 min, and then the acetonitrile ratio was increased to 75% within 8 min; then the acetonitrile ratio was increased to 95% within 1 min; then restored to 12%. The detection wavelength of salicylic acid was set to 230 nm.

[0046] The product generated by AhS5H2 catalyzing salicylic acid is 2,5-dihydroxybenzoic acid. Figure 1. Using 10mM salicylic acid and 5μg protein, the concentration of the product after 30min reaction was 958.08±118.83ng / mL, and the enzyme activity was evaluated by the product generation rate. The enzyme activity of this reaction was 638.72±52.81ng product / mg protein / min. Five technical replicates were performed at the same time, and the relative average deviation RSD (%) of the enzyme activity was determined to be 8.27%.

[0047] ② Taking the detection of jasmonic acid hydroxylase AhJAO1 in peanut as an example, the kit prepared in Example 3 was used for the reaction.

[0048] 20 mM jasmonic acid was used as the substrate for enzyme activity detection. 5 μg of rice jasmonic acid hydroxylase protein expressed and purified by E. coli was prepared before the reaction. 1 mL of sterile water was added to the centrifuge tube containing reagent II, mixed thoroughly and placed on ice.

[0049] Enzyme activity reaction process: Add 5 μg AhJAO1 protein to a centrifuge tube containing reagent I, add 4 μL reagent II solution, add substrate to a final concentration of 20 mM, and add Tris-HCl buffer solution with a pH of 7.5 to a concentration of 50 mM. Use distilled water to make up the volume to 100 μL. React at 30°C for 2 hours. After the reaction, extract twice with an equal volume of ethyl acetate, blow dry with nitrogen, dissolve with 50 μL 90% methanol, and detect with Agilent liquid chromatography-mass spectrometry (LC-MS / MS, 1260-Qtof-6520B, Agilent, USA), inject 5 μL, and separate the compounds via a C18 column (Phenomenex, Luna 3u C18100A, 2.0×150mm, 3.0 μm) under the following conditions:

[0050] The flow rate was 0.25 mL / min, the mobile phases were water and acetonitrile, both containing 0.05% acetic acid, the initial mobile phase acetonitrile ratio was 5%, 5% acetonitrile from 0 to 2 min, 25% at 10 min, 70% at 40 min, 95% at 43 min, 95% at 50 min, and 53-60 min, the acetonitrile ratio was restored to 5%.

[0051] The mass spectrometry detection conditions were as follows: drying gas was N2, flow rate was 11 L / min, temperature was 340°C, Nebulizer was 40 psi, capillary inlet and outlet voltages were 3200 V and 140 V, respectively, and secondary mass spectrometry collision voltages were 10 V and 20 V.

[0052] The product of jasmonic acid catalyzed by AhJAO1 is 12-hydroxyjasmonic acid, as shown in the chromatogram. Figure 2After 2 hours, 20mM jasmonic acid was catalyzed by 5μg protein, and the concentration of the product was 65.03±11.55ng / mL. The enzyme activity was evaluated by the product generation rate. The enzyme activity of this reaction was 10.84±0.32ng product / mg protein / min. Five technical replicates were performed at the same time, and the RSD (%) of the enzyme activity was determined to be 2.96%.

[0053] ③ Taking the detection of the reaction activity of peanut salicylic acid hydroxylase AhS5H1 at different temperatures as an example, the kit prepared in Example 1 was used for the reaction.

[0054] 10 mM salicylic acid was used as the substrate for enzyme activity detection. 5 μg of protein expressed and purified by E. coli was prepared before the reaction. 1 mL of sterile water was added to the centrifuge tube containing reagent II, mixed thoroughly and placed on ice.

[0055] Enzyme activity reaction process: Add 5μg AhS5H1 protein to the centrifuge tube containing reagent I, add 4μL reagent II, add substrate to make the final concentration of 10mM, and add phosphate buffer solution with a pH of 6.8 to make the concentration of 50mM. Use distilled water to make the volume up to 100μL. React at 25℃, 30℃, 35℃, 40℃, and 45℃ respectively, and the reaction time is 30min. After the reaction is completed, boil in a boiling water bath for 1min, and after cooling, directly detect the amount of the product by high performance liquid chromatography. The detection method and enzyme activity algorithm are the same as those in Application Example ①. The product generated by AhS5H1-catalyzed salicylic acid is 2,5-dihydroxybenzoic acid. See the chromatogram Figure 3 At different temperatures, the content of reaction products is different, and the amount of reaction products at 40℃ is the highest (see Figure 4 ), the enzyme activity was 282.36±14.63 ng product / mg protein / min. Five technical replicates were performed at the same time, and the RSD (%) of the enzyme activity was 5.18%.

[0056] Comparative Example 1

[0057] The difference from application example ① is that the substrate, oxoglutaric acid and FeSO4·7H2O solution are added to the sample to be tested in sequence, and then dithiothreitol, sodium ascorbate and catalase are added, and then the reaction is carried out. The other steps of the method are the same.

[0058] Using 10mM salicylic acid and 5μg protein, the concentration of the product after 30min reaction was 793.58±143.18ng / mL, and the enzyme activity was evaluated by the product generation rate. The enzyme activity of this reaction was 529.05±95.45ng product / mg protein / min. Five technical replicates were performed at the same time, and the RSD (%) of the enzyme activity was determined to be 18.04%.

[0059] Comparative Example 2

[0060] The difference from Application Example ① is that dithiothreitol is not included in reagent I. The other steps of the method are the same.

[0061] Using 10mM salicylic acid and 5μg protein, the concentration of the product after 30min reaction was 527.20±54.33ng / mL, and the enzyme activity was evaluated by the product generation rate. The enzyme activity of this reaction was 351.47±36.22ng product / mg protein / min. Five technical replicates were performed at the same time, and the RSD (%) of the enzyme activity was determined to be 10.31%.

[0062] Comparative Example 3

[0063] The difference from Application Example ① is that sodium ascorbate is not included in Reagent I. The other steps of the method are the same.

[0064] Using 10mM salicylic acid and 5μg protein, the concentration of the product after 30min reaction was 468.95±49.25ng / mL, and the enzyme activity was evaluated by the product generation rate. The enzyme activity of this reaction was 312.63±32.83ng product / mg protein / min. Five technical replicates were performed at the same time, and the RSD (%) of the enzyme activity was determined to be 10.50%.

[0065] Comparative Example 4

[0066] The difference from Application Example ① is that catalase is not included in reagent I. The other steps of the method are the same.

[0067] Using 10mM salicylic acid and 5μg protein, the concentration of the product after 30min reaction was 601.34±67.57ng / mL, and the enzyme activity was evaluated by the product generation rate. The enzyme activity of this reaction was 400.89±45.05ng product / mg protein / min. Five technical replicates were performed at the same time, and the RSD (%) of the enzyme activity was determined to be 11.24%.

[0068] Comparative Example 5

[0069] The difference from Application Example ① is that catalase and sodium ascorbate are not included in reagent I. The other steps of the method are the same.

[0070] Using 10mM salicylic acid and 5μg protein, the concentration of the product after 30min reaction was 347.62±45.47ng / mL, and the enzyme activity was evaluated by the product generation rate. The enzyme activity of this reaction was 231.75±30.32ng product / mg protein / min. Five technical replicates were performed at the same time, and the RSD (%) of the enzyme activity was determined to be 13.08%.

[0071] It can be seen from the above examples and comparative examples that the detection of enzyme activity using the kit of the present invention has higher stability and accuracy, and good repeatability compared to the conventional method of adding reagents one by one. In addition, the kit of the present invention obtained by screening and optimizing specific reagents is more conducive to the hydroxylation reaction of the reaction, which is conducive to product formation, thereby improving the efficiency of enzyme activity detection.

[0072] It can be seen from the above-described embodiments and application examples that in order to judge whether a certain enzyme in a plant has a certain endogenous compound that catalyzes metabolism by hydroxylation, this kit can be used to react. A small amount of dissolved oxygen and α-ketoglutaric acid contained in the premix solution constructed by reagent I and reagent II of the present invention react with substrate to be measured under the assistance of ferrous ions, α-ketoglutaric acid undergoes decarboxylation reaction, generates succinate and carbon dioxide, and a certain position of the substrate is then added with hydroxyl, and catalase and sodium ascorbate are also included in the reaction system, enzyme oxidation can be suppressed, and the presence of dithiothreitol can also avoid the intramolecular or intermolecular disulfide bond formed between the cysteine ​​in the protein during the reaction, and the reaction system that buffer can provide stable pH is conducive to the smooth reaction. After reacting through the kit provided by the present invention, select suitable detection mode for the characteristics of 2OGD compounds to be measured to detect the product, determine enzyme activity. Solve that traditional reactions need to prepare many reagents, add the cumbersome and error problems brought one by one.

[0073] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A plant α-ketoglutarate / Fe(Ⅱ)-dependent dioxygenase enzyme activity detection reagent, characterized in that: The enzyme activity detection reagent includes reagent I and reagent II, wherein the components of reagent I are: 1-5 mM dithiothreitol, 0.1-1 mM oxoglutaric acid, 0.1-1 mM sodium ascorbate, 0.01-0.1 mg / mL catalase, and sterile distilled water as a solvent; Reagent II includes FeSO4·7H2O solid, the content of which is 2-3 mg.

2. The enzyme activity detection reagent according to claim 1, characterized in that When the reagent II is used, it is dissolved in distilled water and used immediately after preparation.

3. The enzyme activity detection reagent according to claim 1, characterized in that The concentration of the reagent II is 2-3 mg / mL.

4. A kit for detecting the activity of plant α-ketoglutarate / Fe(II)-dependent dioxygenase, characterized in that: The invention comprises the enzyme activity detection reagent according to any one of claims 1 to 3.

5. A method for detecting the activity of plant α-ketoglutarate / Fe(II)-dependent dioxygenase based on the enzyme activity detection reagent according to any one of claims 1 to 3, characterized in that: The following steps are involved: The sample to be tested, reagent II, substrate and reaction buffer are added to the enzyme-free centrifuge tube containing the reagent I, and an enzyme-catalyzed reaction is performed to produce a product. The enzyme activity of the sample to be tested is determined based on the amount of the product.

6. The method according to claim 5, characterized in that The substrates include jasmonic acid and salicylic acid.

7. The method according to claim 5, characterized in that The ratio of the reagent I, the sample to be tested, and the reagent II is 50 μL: (1-5) μg: (1-4) μL.

8. Use of the enzyme activity detection reagent according to any one of claims 1 to 3, the enzyme activity detection kit according to claim 4, or the method according to any one of claims 5 to 7 in detecting the enzyme activity of plant α-ketoglutarate / Fe(II)-dependent dioxygenase.

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