A vomitoxin detoxification enzyme, its encoding gene, and its applications

Abstract

This invention discloses a vomitoxin detoxification enzyme, its encoding gene, and its applications, belonging to the field of molecular biology. This invention discovered a wheat SOT gene, TaSOT2b. The TaSOT2b protein was obtained through in vitro induced expression, and its amino acid sequence is shown in SEQ ID NO.1. This invention found that this protein possesses in vitro enzymatic activity, capable of modifying DON into SON-3-sulfate, and can act as a vomitoxin detoxification enzyme. It has potential application value and reference significance for the scientific prevention and control of wheat scab and DON toxin contamination, and has broad application prospects in agriculture, feed, and industry.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a vomitoxin detoxification enzyme, its encoding gene, and its applications. Background Technology

[0002] Vomitoxin, also known as deoxynivalenol (DON), chemically named 12,13-epoxy-3α,7α,15-trihydroxytrichosporon-9-en-8-one, is named for the vomiting it causes. It is primarily produced by Fusarium fungi such as *Fusarium graminearum* and *Fusarium culmorum* when they infect grains like wheat, barley, oats, and corn. It is one of the major mycotoxins contaminating grains, feed, and food, seriously affecting the health of humans and livestock. Therefore, reducing or eliminating its toxicity is crucial. Thus, isolating genes or enzymes that can efficiently remove vomitoxin and using in vitro enzymatic treatment of toxin-contaminated grain products will meet the detoxification needs of the feed, food, and pharmaceutical industries.

[0003] DON-sulfate is a low-toxicity DON toxin-modified product produced in wheat under natural disease conditions and simulated inoculation (DON treatment). Current toxicological studies of naturally occurring DON-sulfate indicate that it possesses low toxicity and is a non-masked toxin, and can be considered a DON detoxification product. Sulfation metabolism in organisms is generally catalyzed by sulfotransferases (SOTs) catalyzing the binding of small molecule compounds to sulfonic acid groups provided by 3'-phosphoadenosine 5'-phosphosulfate (PAPS). SOT is a key enzyme involved in sulfation reactions and plays an important role in plant growth, development, and adaptation to stress. However, in studies related to wheat detoxification modification, there are no reports on SOT-catalyzed DON toxin sulfation. Therefore, in-depth research into wheat genes encoding SOT related to DON modification and developing their application value is of significant research importance. Summary of the Invention

[0004] To address the aforementioned limitations of existing technologies, the present invention aims to provide a vomitoxin detoxification enzyme, its encoding gene, and its applications. This invention is the first to discover a wheat SOT protein capable of modifying DON into SON-3-sulfate. The gene encoding this protein is located on chromosome 2B and named TaSOT2b. The TaSOT2b protein was obtained through in vitro induced expression, and its in vitro enzyme activity was measured using high-performance liquid chromatography-mass spectrometry (HPLC-MS). The results showed that it possesses the ability to modify DON into SON-3-sulfate, making it a potential vomitoxin detoxification enzyme with potential application value and reference significance for the scientific prevention and control of wheat scab and DON toxin contamination.

[0005] This invention is achieved through the following technical solution:

[0006] In a first aspect, the present invention provides a vomitoxin detoxification enzyme, said vomitoxin detoxification enzyme being any of the proteins shown in (A1)-(A3) below:

[0007] (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1 of the sequence listing;

[0008] (A2) A protein derived from SEQ ID NO.1 with one or more amino acid residues substituted and / or deleted and / or added, and which is associated with the detoxification performance of vomitoxin;

[0009] (A3) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein defined in (A1) or (A2).

[0010] To facilitate the purification of proteins in (A1) or (A2), a tag can be attached to the amino or carboxyl terminus of the protein in (A1) or (A2). The tag can be Poly-Arg (typically 6 RRRRR), Poly-His (typically 6 HHHHHH), FLAG (DYKDDDDK), Strep-tag II (WSHPQFEK), or c-myc (EQKLISEEDL).

[0011] In a second aspect, the present invention provides a gene encoding the above-mentioned vomitoxin detoxification enzyme.

[0012] Preferably, the encoding gene is a nucleic acid molecule as shown in i), ii), or iii):

[0013] i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2;

[0014] ii) Nucleic acid molecules that have 75% or more nucleotide sequence identity with i) and express the same functional protein;

[0015] iii) Nucleic acid molecules other than i) encoding the amino acid sequence shown in SEQ ID NO.1.

[0016] The term "identity" used here refers to sequence similarity to natural nucleic acid sequences. Identity can be evaluated using computer software, such as the BLAST algorithm (Altschul et al. 1990. Journal of Molecular Biology 215:403-410; Karlin and Altschul. 1993. Proceedings of the National Academy of Sciences 90:5873-5877).

[0017] In a third aspect, the present invention provides a recombinant expression vector, a transgenic cell line, or a genetically engineered bacterium containing the above-described encoding gene.

[0018] In a fourth aspect, the present invention provides the use of the above-mentioned vomitoxin detoxifying enzyme in either (1) or (2) below:

[0019] (1) Degrades vomitoxin;

[0020] (2) Preparation of detoxification products for vomitoxin.

[0021] In the above applications, the vomitoxin detoxification enzyme catalyzes the reaction of the C3 hydroxyl group of vomitoxin with the sulfonic acid group to generate a sulfate derivative.

[0022] In a fifth aspect, the present invention provides the use of the encoding gene of the above-mentioned vomitoxin detoxification enzyme, a recombinant expression vector containing the encoding gene, a transgenic cell line or genetically engineered bacteria in at least one of the following (1)-(3):

[0023] (1) Production of vomitoxin detoxification enzymes;

[0024] (2) Improve plant resistance to DON;

[0025] (3) Cultivate plant varieties resistant to Fusarium head blight.

[0026] In the above applications, the plant is preferably wheat.

[0027] A sixth aspect of the present invention provides a method for reducing the toxicity of vomitoxin, comprising the step of contacting the above-mentioned vomitoxin detoxifying enzyme with a sample to be treated under enzymatic reaction conditions.

[0028] Preferably, the sample to be treated contains a sulfonic acid group donor; or a sulfonic acid group is added to the sample to be treated.

[0029] More preferably, the donor of the sulfonic acid group is 3'-phosphoadenosine-5'-phosphorylsulfate (PAPS).

[0030] The beneficial effects of this invention are:

[0031] This invention reveals for the first time the sulfation reaction occurring in wheat organisms. A wheat sulfotransferase gene capable of degrading vomitoxin was discovered, and its activity was demonstrated in vitro. Experimental results show that DON can undergo a sulfation binding reaction catalyzed by the TaSOT2b protein. This experiment provides important data on the sulfation metabolism of DON in wheat both in vivo and in vitro, and has potential application value and reference significance for the scientific prevention and control of wheat scab and DON toxin contamination. Attached Figure Description

[0032] Figure 1 Differences in the expression levels of sulfotransferase genes in wheat after inoculation with Fusarium graminearum.

[0033] Figure 2 Colony PCR (A) and double enzyme digestion (B) detection of prokaryotic expression vector pET28a-TaSOT2b; M.DL2000 label (bp); 1-2: TaSOT2b colony PCR; 3. Enzyme digestion products; 4. Undigested empty vector.

[0034] Figure 3 Expression of pET28a-TaSOT2b fusion protein at different induction temperatures and times; where M is protein label; 0 is pre-induction sample; pET28a-TaSOT2b was induced at 1–4.25℃ for 4, 8, 10, and 12 h; pET28a-TaSOT2b was induced at 5–8.20℃ for 4, 8, 10, and 12 h.

[0035] Figure 4 Solubility analysis and purification of recombinant protein pET28a-TaSOT2b; M. Protein labeling; 1. Pre-induction sample; 2. Post-induction sample; 3. Ultrasonic disruption of supernatant; 4. Ultrasonic disruption of precipitate; 5. Protein purification; 6-7. Protein concentration.

[0036] Figure 5 Western blot analysis.

[0037] Figure 6 : Sample material from wheat ears; Note: From left to right: water-collected *Phyllostachys edulis*, bacteria-inoculated *Phyllostachys edulis*, water-collected Fielder, bacteria-inoculated Fielder.

[0038] Figure 7 TaSOT2b three-level structure prediction.

[0039] Figure 8 : Molecular docking simulation conformation of TaSOT2b protein with DON.

[0040] Figure 9 Peak diagram of wheat sample detection; Note: A: Detection of DON and DON-3-S in wheat ears after water inoculation and bacterial inoculation; B: Detection of DON and DON-3-S in wheat leaves after water inoculation and bacterial inoculation.

[0041] Figure 10 Mass spectrum of DON characteristic ions detected in wheat samples.

[0042] Figure 11 Mass spectrum of characteristic ions of DON-3-S in wheat sample.

[0043] Figure 12 Analysis of TaSOT2b enzyme activity during DON sulfation. Detailed Implementation

[0044] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0045] As mentioned earlier, sulfotransferases (SOTs) in plants and animals are key genes involved in sulfation reactions. However, the number of plant SOT genes with functional characterization is currently limited, and no related genes have been reported in wheat studies. SOT is a superfamily of genes whose members participate in phase II metabolism, catalyzing the sulfation of various endogenous and exogenous complexes and playing a detoxification role. Their substrates are very broad, but the specific substrates catalyzed by each plant SOT are usually not accurately predicted based solely on amino acid sequence similarity.

[0046] Based on this, this invention utilizes the Ensembl Plants plant genome database, combined with the NCBI wheat transcriptome database, to screen all wheat genes currently annotated as sulfotransferases based on their expression levels after inoculation with *Fusarium graminearum*. The gene most significantly induced by *Fusarium graminearum* was identified, and because it is located on chromosome 2B, it was named TaSOT2b. The CDS sequence of this gene was amplified from the cDNA of leaves of the wheat scab-resistant variety *Wangshuibai*, and sequencing revealed the CDS sequence of TaSOT2b as shown in SEQ ID NO.2; the amino acid sequence of the encoded TaSOT2b protein is shown in SEQ ID NO.1.

[0047] This invention utilizes multiple online tools to analyze the amino acid sequence of the TaSOT2b protein. The results show that the TaSOT2b protein has a molecular weight of 41.14 kDa, classifying it as a hydrophilic protein. Furthermore, the TaSOT2b protein lacks a signal peptide and transmembrane domain, indicating that it is not a membrane protein or transport protein, but rather a cytoplasmic sulfonyltransferase modified with a small molecule compound. Three-dimensional homology modeling of the protein was performed using the SWISS-MODEL website, and semi-flexible docking simulation of TaSOT2b and DON was conducted using AutoDock 4.2.6 software. The good binding affinity between the substrate molecule DON and the protein molecule TaSOT2b during the docking process indicates that they have an interaction capability.

[0048] To obtain a large quantity of sulfonyltransferase in a short time for subsequent modification reactions and to study the sulfonation mechanism of DON toxin, this invention uses an E. coli prokaryotic expression system for in vitro recombinant protein production. After experimenting with induction temperature and time, the optimal induction conditions for TaSOT2b protein were established as: 20℃ for 12 h. SDS-PAGE electrophoresis and Western blot confirmed that the protein induced in this system was indeed TaSOT2b.

[0049] To investigate the ability of TaSOT2b protein to modify DON and to facilitate its large-scale application in the detoxification of DON-contaminated wheat, a stable and reliable in vitro enzymatic reaction is needed to simulate the sulfation reaction of DON in vitro. The characteristic ion fragment m / z 345 of DON-3-sulfate is currently the only method for distinguishing the DON sulfate products at positions 3 and 15 by mass spectrometry. This invention, through an in vitro enzymatic reaction system and a high-resolution mass spectrometry platform, demonstrates that TaSOT2b possesses enzymatic activity and can modify the toxic site 3 of DON into DON-3-sulfate, thus exhibiting the ability to detoxify DON.

[0050] This invention establishes an in vitro enzymatic reaction system for sulfonyltransferases, which provides a theoretical basis for improving the activity of TaSOT2b detoxification enzyme and developing detoxification enzyme preparations.

[0051] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0052] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein:

[0053] The wheat varieties Wangshuibai (highly resistant to Fusarium head blight) and Fielder (susceptible to Fusarium head blight), and the wild-type Fusarium graminearum strain PH-1, are available to the public from the applicant for use in replicating this invention.

[0054] The primer sequences used in this embodiment of the invention are as follows:

[0055]

[0056]

[0057] Example 1: Screening of candidate sulfonyltransferase genes

[0058] To obtain endogenous wheat genes encoding sulfotransferases that may be involved in DON toxin sulfate modification, all wheat genes annotated as sulfotransferases were downloaded from the Ensembl Plants genome database (http: / / plants.ensembl.org / ). Transcriptome data on wheat expression levels after Fusarium graminearum inoculation (accession number CM-82036) from NCBI (https: / / www.ncbi.nlm.nih.gov / ) were analyzed to screen for genes induced by Fusarium graminearum and sorted according to expression levels. TraesCS2B02G200100 showed the most significant difference in expression level after Fusarium graminearum inoculation, with a significant increase in expression level 50 h after inoculation. Figure 1 Therefore, this gene was selected as a candidate gene for further research and, according to convention, was named TaSOT2b based on its location on chromosome 2B.

[0059] Example 2: Cloning of the full-length CDS of TaSOT2b from the water lily

[0060] 1. Extraction and reverse transcription of RNA from leaves of *Bletilla striata*

[0061] a. Extract total RNA from *Pleurotus ostreatus* using the Biospin kit (Tiangen Biotech Co., Ltd.)

[0062] To minimize the degradation of plant sample RNA due to RNase contamination, RNase-free equipment was used. Total RNA was extracted from wheat cultivar *Wangshui Bai* leaves following the instructions of the plant total RNA extraction kit. The samples were pre-cooled at 4°C before operation and maintained at low temperature throughout the experiment. After RNA extraction, fresh electrophoresis buffer was used for agarose gel electrophoresis. The electrophoresis voltage was set to 180V for 15 minutes. If 2-3 clear bands were observed in the electrophoresis result, the extracted RNA was of good quality. The operating steps are as follows:

[0063] (1) Wheat leaf samples were thoroughly pulverized in liquid nitrogen using a vibratory grinder and temporarily stored in liquid nitrogen. 2% β-mercaptoethanol was added to Lysis AG.

[0064] (2) Add an appropriate amount of plant sample to small steel balls and freeze it in liquid nitrogen. Grind it into powder using a ball mill, add Lysis AG solution, and immediately shake vigorously until there are no obvious particles.

[0065] (3) Centrifuge at 12000rpm for 5 minutes at 4℃, and transfer the supernatant to an RNase-Free centrifuge tube.

[0066] (4) Add 1 / 2 volume of anhydrous ethanol to the supernatant and mix immediately. Transfer the mixture to a Spin Column and centrifuge at 12,000 rpm for 1 min at 4 °C. Pipette 500 μL of Wash Buffer containing anhydrous ethanol into the Spin Column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid from the outer tube again.

[0067] (5) Add 50 μL of DNase I solution to the center of the Spin Column membrane and let it stand for 15 min.

[0068] (6) Take 600 μL of PG Buffer and centrifuge at 12000 rpm for 0.5 min. Then add 500 μL of Wash Buffer and centrifuge at 12000 rpm for 30 s. Add 250 μL of Wash Buffer and repeat the centrifugation. After discarding the waste liquid in the outer tube, centrifuge at 4℃ and 12000 rpm for 1 min to completely dry the liquid.

[0069] (7) Transfer the Spin Column to a new tube, add 50 μL of Relution Buffer, let stand for 2 min, and then centrifuge at 12000 rpm for 1 min to obtain total RNA.

[0070] b. Use RT-PCR SuperMix to reverse transcribe RNA to synthesize cDNA.

[0071] Add the following to the PCR tube in sequence: 1 μL Oligo(dT) Primer, 10 μL 2×TS Reaction Mix, 1 μg RNA, 1 μL TransScript RT / RI Enzyme Mix, 1 μL gDNA Remover, and RNase-free Water to a total volume of 20 μL.

[0072] 2. TaSOT2b gene CDS cloning and electrophoresis detection

[0073] Using cDNA from *Pleioblastus sylvestris* leaves as a template, the CDS sequence of the TaSOT2b gene was amplified using TaSOT1-F / R primers.

[0074] PCR reaction system: 2×PhantaMax Buffer 25μL, 10mM dNTP Mix 1μL, TaSOT1-F (10μM) 2μL, TaSOT1-R (10μM) 2μL, template cDNA 2μL, PhantaMax Super-Fidelity DNA Polymerase 2μL, ddH2O 18μL.

[0075] PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 64℃ annealing for 30 s, 72℃ extension for 1 min 20 s, 35 cycles; 72℃ final extension for 5 min.

[0076] Agarose gel electrophoresis detection:

[0077] Gel preparation: Pour 25 mL of 1×TAE electrophoresis buffer and 0.25 g of agar powder into an Erlenmeyer flask and shake well. Heat until the agar is completely dissolved. After cooling slightly, immediately add 2.5 μL of nucleic acid dye and shake quickly. Pour into a gel casting plate and let stand and cool for about 20 minutes to solidify the gel. The 1% gel is now ready.

[0078] Sample loading: Carefully transfer the gel to the electrophoresis tank. Add an appropriate amount of loading buffer to the PCR product, mix well by pipetting, and then load the sample.

[0079] Gel running: Turn on the power, adjust the voltage to 120V, and electrophoresis for about 25 minutes before turning off the power. Place the gel under a gel imaging system for detection, take pictures, and save the images.

[0080] 3. PCR product purification and sequencing vector ligation

[0081] (1) Agarose gel DNA recovery: The gel band containing the target gene was rapidly excised under UV light, and the product was purified and recovered using a kit. The gel was placed in a 1.5 mL centrifuge tube, PG Buffer was added, and the mixture was heated at 50 °C until the gel was completely melted. The gel was then transferred to a spin column, centrifuged at 10000 × g for 1 min, 450 μL of PG Buffer was added, and the mixture was centrifuged at 10000 × g for 1 min. The washing was repeated once. The centrifuge speed was increased to 13000 × g, and the empty column was centrifuged for 2 min to remove any remaining waste liquid from the spin column. The spin column was transferred to a new tube, and 20 μL of Elution Buffer was carefully aspirated and dropped into the center of the membrane. The tube was incubated at 37 °C for 2 min. The DNA was collected by centrifugation at 13000 × g for 1 min and stored at -20 °C.

[0082] (2) Ligation of the recovered product with the pEASY-Blunt sequencing vector:

[0083] Table 1: Sequencing vector ligation system

[0084]

[0085] 4. Preparation of competent Escherichia coli cells

[0086] (1) Streak DH5α / BL21 competent cells on LA plates, pick newly activated single colonies and activate them in LB for 12 h; take 5 mL and inoculate into 250 mL LB (1:50), incubate at 37℃ and 200 rpm until the OD value reaches 0.50. 600 The concentration is approximately 0.5. Heat the centrifuge tubes in an ice bath for 15 minutes, while simultaneously pre-cooling the centrifuge tubes, 0.1M MgCl2, and 0.1M CaCl2.

[0087] (2) Centrifuge the bacterial solution at 4℃ and 6000rpm for 10min, collect the precipitate, and take 50mL of pre-cooled 0.1MMgCl2 to fully suspend the bacterial precipitate.

[0088] (3) Centrifuge the bacterial culture at 6000 rpm for 10 min, collect the precipitate, add 125 mL of pre-cooled 0.1 M CaCl2 to fully suspend the bacterial precipitate, and immediately place it on an ice bath for 20 min.

[0089] (4) Centrifuge at 6000rpm for 10min at 4℃ to collect the precipitate, suspend the bacterial cells in 0.1M CaCl2, add 5mL of 50% glycerol, dispense into aliquots and freeze in liquid nitrogen at -80℃.

[0090] 5. Conversion of the linker products

[0091] (1) Thaw competent cells on ice for a few minutes, add ligation product and mix well, then place on ice again and wait for 30 minutes.

[0092] (2) Place the centrifuge tubes on a floating plate and incubate at a constant temperature of 42°C for 1 min 30 s, then immediately cool on ice for 5 min.

[0093] (3) Add 900 μL of LB solution, place it in a constant temperature shaker, set the parameters to 37℃ and 220 rpm, and incubate for about 60 min before taking it out.

[0094] (4) Spread 100 μL of bacterial solution evenly on Kan-resistant LB plates, seal with sealing film and incubate overnight at 37°C.

[0095] (5) Select a single colony for PCR verification. Use M13-F / R primers and preliminarily detect whether the target DNA fragment has been transferred into E. coli by electrophoresis bands.

[0096] 6. Screening and sequencing of positive clones

[0097] Positive clones were screened by colony PCR using 2×Taq Mix DNA polymerase. The reaction mixture (excluding the template) was added sequentially to 200 μL PCR tubes (operated on ice). Colonies were picked and streaked onto antibiotic plates for preservation. Then, a centrifuge tube was inserted, and the remaining colonies were mixed with the mixture using a toothpick. The centrifuge tube was tightly capped, centrifuged, and then placed in a PCR instrument. The PCR program was set for the PCR reaction. The PCR reaction mixture (11 μL) is as follows:

[0098] Table 2: Colony PCR Reaction System

[0099]

[0100] PCR reaction program: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 3 min, 30 cycles; 72℃ final extension for 10 min.

[0101] Based on the sequence information of Chinese spring wheat varieties published by Ensembl Plants, the mRNA reference sequence of TaSOT2b was obtained, and specific primer pairs were designed accordingly. RNA was extracted from the leaves of the highly resistant wheat scab variety Wangshuibai, and cDNA was obtained through reverse transcription. Finally, the CDS sequence of TaSOT2b was amplified using specific primers. Sequencing revealed that the full-length CDS sequence of TaSOT2b is 1110 bp (including the start codon and stop codon, as shown in SEQ ID NO.2), encoding a total of 369 amino acids.

[0102] Example 3: Expression and purification of TaSOT2b protein

[0103] 1. Test method:

[0104] 1.1 Construction of the pET28a-TaSOT2b expression vector

[0105] 1.1.1 Construction of the pET28a-TaSOT2b expression vector

[0106] The wheat sulfotransferase SOT gene sequence was obtained by referring to wheat transcriptome data and NCBI (http: / / www.ncbi.nlm.nih.gov / ) BLAST, and CDS cloning primers were designed (Table 1). PCR system: ddH2O 18 μL, 10×PhantaMax Buffer 25 μL, 10 mmol·L⁻¹ dNTPs 1 μL, TaSOT1-F / R 2 μL, cDNA 2 μL, and PhantaMax Super-Fidelity DNA Polymerase 1 μL. Reaction program: 94℃ 5 min; 94℃ 30 s, 64℃ 30 s, 72℃ 1 min 20 s, 35 cycles; 72℃ 5 min.

[0107] The target gene fragment was ligated to the pEASY sequencing vector, transformed into DH5α plates, and cultured overnight at 37°C. The correct target gene fragment was confirmed by colony PCR and sequencing. The correctly sequenced plasmid DNA was extracted and amplified by PCR using pEASY-TaSOT2b as a template and TaSOT2-F / R as primers. After electrophoresis, the target fragment was recovered from the gel and double-digested with NotI-HF and EcoRI-HF restriction endonucleases, respectively. The reaction mixture consisted of 37 μL ddH2O, 5 μL CutSmart Buffer, 5 μL pET28a PCR product, and 1 μL restriction endonuclease. After vortexing and centrifugation, the mixture was incubated at 37°C for 2 h and then inactivated at 65°C for 20 min.

[0108] The gene fragment and vector were ligated overnight at 16°C using T4 DNA ligase. The ligation product was transformed into DH5α and cultured overnight. Positive plasmids were identified by colony PCR, and further verification was performed by sequencing of positive transformants. Sequence alignment confirmed the sequencing results. The plasmid pET28a-TaSOT2b was transformed into BL21(DE3), plated on Kansei LB plates, and incubated overnight at 37°C. Positive plasmids were identified again by colony PCR, and the plasmids were ready for prokaryotic expression.

[0109] 1.2 Plasmid Extraction

[0110] (1) Centrifuge the overnight cultured bacterial solution at 10,000 rpm for 30 seconds and collect the precipitate.

[0111] (2) Take 250 μL of Resuspension Buffer to fully suspend the bacterial precipitate.

[0112] (3) Add 250 μL of Lysis Buffer and shake up and down to fully lyse. Then add 350 μL of Neutralization Buffer and mix thoroughly until white flocculent material appears in the tube.

[0113] (4) Centrifuge at 13,000 rpm for several minutes until a white flocculent precipitate forms. Transfer the supernatant to a spin column, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid.

[0114] (5) Pipette 650 μL of Wash Buffer into a Spin column, centrifuge at 12000 g for 1 min, and discard the waste liquid in the tube. Repeat this step once.

[0115] (6) Centrifuge again at 12000g for 1min, remove the residual liquid, and transfer the spin column to a clean 1.5mL centrifuge tube.

[0116] (7) Add 50 μL of solution buffer to elute the plasmid DNA and let stand for one minute. Centrifuge at 12000g for 1 minute, collect the plasmid DNA, and store at -20℃.

[0117] 1.3 Enzyme digestion and product recovery

[0118] (1) Double digestion: The pEASY vector containing the TaSOT2b gene and the prokaryotic expression vector pET-28a were digested with NotI and EcoRI, respectively, and incubated overnight at 37°C. The digestion products were then recovered by gel extraction. The digestion system is as follows (50 μL):

[0119] Table 3: Enzyme digestion system

[0120]

[0121] (2) Ligate the target fragment to the prokaryotic expression vector using T4 ligase. The ligation procedure is: 16℃, 3h. The ligation system is as follows (10μL):

[0122] Table 4: T4 ligase ligation system

[0123]

[0124] (3) Transformation and verification of E. coli and sequencing. PCR verification and sequencing were performed using universal primers T7-F / R.

[0125] 1.4 qRT-PCR analysis

[0126] The reaction mixture was added to a qPCR tube and placed in a real-time PCR instrument for amplification, with three replicates per sample. Reaction volume (20 μL)

[0127] Table 5: qRT-PCR reaction system

[0128]

[0129] Reaction program: 95℃ for 3 min; 95℃ for 5 s, 60℃ for 30 s, 40 cycles.

[0130] 1.5 Induced protein expression

[0131] Positive clonal colonies were placed in 2 mL of Kan-resistant LB medium and incubated overnight at 37°C and 220 rpm. The culture was then expanded at a 1:100 ratio and incubated at 37°C and 220 rpm until the OD reached the target value. 600 At approximately 0.6, 100 μL of culture was collected and temporarily stored at 4°C as a negative control. 1 mL of IPTG was added to the remaining culture medium to induce protein expression. After induction at 20°C and 25°C for 4 h, 8 h, 10 h, and 12 h respectively, 100 μL of the bacterial culture was collected each time and temporarily stored at 4°C for subsequent SDS-PAGE electrophoresis. After complete induction, 100 μL of 2×SDS PAGE loading buffer was added to suspend the previously stored precipitate. The mixture was centrifuged multiple times and boiled at 100°C for 8 min to denature the protein. 30 μL of the sample was taken for electrophoresis. After electrophoresis, the gel was submerged in Coomassie Brilliant Blue and placed on a low-speed shaker for 2 h to stain the protein. After destaining, the gel was photographed using a gel imager to analyze the optimal induction conditions for the protein. The SDS-PAGE gel preparation is shown in the table below:

[0132] Table 6: 12% Separating Gel Solution for SDS-PAGE Gel

[0133]

[0134] After adding all components, mix them well. Use a 1mL pipette tip to add the gel solution to the gel casting plate in several batches. Then add 2mL of water to seal the gel. After the gel solidifies, pour out the water and blot dry with filter paper.

[0135] Table 7: Preparation of 5% Stacking Solution for SDS-PAGE Gel

[0136]

[0137] After mixing all components, add them to the separating gel, insert a 1mm electrophoresis comb vertically, and let it stand for 20 minutes to solidify.

[0138] The recombinant protein was induced using the optimal induction conditions for the SOT protein. After induction, the sample was centrifuged at 5000 rpm for 10 min, the precipitate was collected, 5 mL of PBS was added to resuspend the precipitate, and the sample was centrifuged at 5000 rpm for 5 min to collect the precipitate. The precipitate was then frozen at -20℃ for 30 min. 15 mL of PBS was used to resuspend the precipitate, and 30 μL of protease inhibitor (PMSF) was added to the solution. To ensure low-temperature operation, the centrifuge tube containing the bacterial culture was placed on ice to prevent protein denaturation due to temperature rise during disruption. The ice was kept above the liquid surface. The bacterial culture was disrupted using an ultrasonic cell disruptor (programmed with 3-second intervals and 5-second intervals, for a total of 5 min per cycle). Sonication was stopped when the bacterial culture changed from turbid to clear. The sample was centrifuged at 5000 rpm for 10 min, the crude enzyme solution was collected, and temporarily stored at 4℃.

[0139] 1.6 Protein Purification

[0140] a. Cobalt resin affinity chromatography

[0141] (1) Place the crude protein enzyme solution obtained in the previous step on ice.

[0142] (2) Shake thoroughly to mix the TALON His-Tag purification resin. Pipette 1 mL into an affinity chromatography tube, then add 1 mL of PBS equilibration buffer to the resin gel for rinsing. The buffer will flow out naturally from the bottom of the chromatography column.

[0143] (3) Rinse once with PBS equilibration buffer.

[0144] (4) Add an appropriate amount of crude enzyme solution to the affinity chromatography column at a ratio of 1:8 between gel and crude enzyme solution, cover the upper and lower caps, and place the mixed solution at 4°C and invert it for 4 hours to allow the protein and gel to fully combine.

[0145] (5) Open the bottom cap of the column tube to allow the liquid to drain naturally into the waste liquid container. Close the bottom cap tightly, add 1 mL of non-denaturing washing buffer, and invert the column at 4°C for 10 min.

[0146] (6) Open the bottom cap of the chromatography column to allow the liquid to drain naturally. After tightening the bottom cap, add 1 mL of non-denaturing elution buffer to elute the target protein. Invert the column at 4°C for 15 min.

[0147] (7) Open the bottom cap, collect the liquid into a clean centrifuge tube, obtain the purified protein, and detect and record the protein concentration.

[0148] b. Ultrafiltration Concentration

[0149] Insert the Amicon-0.5 ultrafiltration tube into the centrifuge tube. First, soak the ultrafiltration tube in 70% ethanol solution for 5 min, rinse with distilled water, add 500 μL of 0.1N NaOH solution, centrifuge at 14000g for 5 min, then wash with non-denaturing elution buffer and centrifuge to dry. Add 500 μL of protease solution to the inner tube of the ultrafiltration tube, centrifuge at 14000g for 20 min at 10℃, discard the liquid in the centrifuge tube, add 450 μL of non-denaturing elution buffer to the inner tube of the ultrafiltration tube, centrifuge at 14000g for 20 min to concentrate, repeat the washing twice to achieve desalting and fully reduce the concentration of impurities in the protein. To recover the concentrated protein, invert the ultrafiltration tube into the centrifuge tube. Place it in the centrifuge, with the open cap facing the center of the rotor, rotate to close the centrifuge cap, centrifuge at 1000g for 2 minutes to transfer the concentrated protein retained in the inner tube of the ultrafiltration tube to the filtrate collection tube. Transfer all protein solution from the collection tube into a clean PCR tube, detect the concentrated protein concentration, and load the sample for detection by SDS electrophoresis.

[0150] 1.7 Western blot detection of TaSOT2b protein

[0151] (1) Clean the glue-making glass clamp, dry it with filter paper, align the glass plates and assemble them on the glue-making frame. Add an appropriate amount of water to the assembled glue-making plate and let it stand for 1 minute. Check the sealing of the glue-making plate. If no water droplets leak out, it proves that the sealing is good. Pour out the water.

[0152] (2) Prepare a 12% separating gel solution, adding the same reagents as in Table 6.

[0153] (3) Prepare a 5% stacking gel solution, adding reagents as shown in Table 7.

[0154] (4) After adding TEMED, quickly mix the solution and transfer the entire mixture to the glue-making plate. Install the glue-making comb vertically and wait for about 25 minutes for the glue to solidify completely.

[0155] (5) While waiting for the gel to solidify, boil the protein sample. Add 24 μL of sample and 6 μL of loading buffer to the PCR tube and mix thoroughly. Heat at 100°C for about 8 minutes to denature the protein. After boiling, immediately place the protein on ice.

[0156] (6) Vertically pull out the gel casting comb, fix the gel casting plate into the electrophoresis tank, and add an appropriate amount of 1×Running Buffer electrophoresis buffer.

[0157] (7) Add 5 μL of protein marker and 20 μL of protein sample to the dispensing well in sequence.

[0158] (8) Turn on the power and set the electrophoresis apparatus voltage to 60V. After about 30 minutes, you can see the protein dye reaching the bottom of the stacking gel. At this time, increase the voltage to 120V. After about 60 minutes, the dye will reach the bottom of the separating gel. Turn off the power and stop electrophoresis.

[0159] (9) Take out the gel-making plate, gently pry the two glass plates apart, and use the gel-cutting plate to neatly cut off the separating gel.

[0160] (10) Cut a PVDF membrane to the same size as the gel, immerse it in methanol solution for 30 seconds, then immediately transfer it to the transfer buffer for 30 seconds, simultaneously processing 6 sheets of filter paper and a buffer sponge. Stack the buffer sponge, 3 sheets of filter paper, separating gel, 3 sheets of filter paper, and buffer sponge onto the transfer plate in sequence, pressing each layer firmly to prevent air bubbles from forming, and seal the clamps. Set the electrophoresis apparatus current to a constant 400 mA, and complete the transfer in approximately 45 minutes.

[0161] (11) Immunohistometry. After transfer, the PVDF membrane was washed three times with PBST. The PVDF membrane was placed in blocking buffer containing skim milk powder and gently shaken on a shaker for about 1 hour. Primary antibody was added at a ratio of 1:5000, and the membrane was sealed for about one hour. Then, the PVDF membrane was treated three more times with PBST. Secondary antibody was added at a ratio of 1:10000 to PBST containing skim milk powder for blocking. After 1 hour, the PVDF membrane was treated three times with PBST solution.

[0162] (12) Use a gel imaging system to observe the transfer results and take pictures to record them.

[0163] 2. Test Results:

[0164] 2.1 Construction of the prokaryotic expression vector pET28a-TaSOT2b

[0165] The target fragment was recovered and purified. A prokaryotic expression vector was constructed to induce large-scale expression of TaSOT2b. The obtained protein was purified using cobalt resin affinity chromatography, utilizing the characteristics of the His tag. An in vitro enzymatic reaction system was established to catalyze the sulfation of DON by TaSOT2b protein. The target fragment TaSOT2b and the prokaryotic expression vector pET-28a were double-digested with NotI-HF and EcoRI-HF restriction endonucleases, respectively. The digested gene fragment and vector were then directly ligated using T4 ligase, and transformed into DH5α to obtain the pET28a-TaSOT2b recombinant expression vector. Positive clones were selected using kanamycin resistance screening, and colony PCR was performed for identification. Figure 2 A) and double enzyme digestion detection ( Figure 2 B) The activated positive clone bacterial solution was sent for sequencing (Sangon Biotech Co., Ltd.). The sequence alignment results were consistent with the previous results, indicating that the pET28a-TaSOT2b prokaryotic expression vector has been successfully constructed.

[0166] 2.2 Expression and purification of TaSOT2b protein

[0167] To explore the optimal expression environment for the recombinant protein, pET28a-TaSOT2b was transformed into BL21 cells, screened on Kan-resistant medium, and incubated overnight at 37°C. Colony PCR verification showed that the amplified electrophoretic bands were single and specific, and the size of the amplified bands was consistent with the size of the target gene, indicating successful transformation into BL21 competent cells. Single colonies with the correct band were picked and cultured in LB (Kan-resistant) medium at 37°C with shaking at 200 rpm for 8 hours. IPTG was then added, and induction was performed at 20°C and 25°C for 4h, 8h, 10h, and 12h, respectively. SDS-PAGE electrophoresis analysis was then used to determine the optimal induction conditions for this recombinant protein. Figure 3 ).

[0168] To further understand the solubility and purification of TaSOT2b protein, the optimal induction conditions for this SOT protein, determined by the above experiments, were used. IPTG was added, and the mixture was incubated overnight at 20℃ and 150 rpm with shaking. After ultrasonic disruption and centrifugation, the supernatant and precipitate were analyzed by SDS-PAGE electrophoresis. The results showed that the target protein was present in both the supernatant and the precipitate. Since high-purity soluble protein is required for protein activity studies, further purification was necessary. High-purity TaSOT2b protein was obtained using cobalt resin affinity chromatography and ultrafiltration concentration. The purified protein was then analyzed by SDS-PAGE gel electrophoresis. The results showed that the pET28a-TaSOT2b recombinant protein had a distinct band at approximately 45 kDa, consistent with the expected size. Figure 4 It can be observed that the amount of protein increased significantly after concentration, indicating that the purification was relatively successful.

[0169] 2.3 Western blot detection of TaSOT2b protein

[0170] To further verify whether the protein obtained after expression was the target protein, Western blotting was necessary to confirm the protein purification status. Mouse anti-His6 was selected as the primary antibody, and goat anti-mouse was used as the secondary antibody. A series of steps were performed, including PVDF membrane transfer, blocking with skim milk powder, adding primary antibody, and adding secondary antibody. The results showed a clear specific band at 45 kDa in the purified eluent, consistent with the theoretical molecular weight, indicating that the TaSOT2b protein had been successfully expressed and purified in *E. coli*. Figure 5 ).

[0171] Example 4: TaSOT2b protein activity assay

[0172] 1. Test Methods

[0173] 1.1 Preparation of wheat inoculated samples

[0174] 1.1.1 Inoculation of wheat ears

[0175] (1) Wheat inoculated samples were used as positive controls. The wheat samples were inoculated using the single-floret drip method. Fielder and Wangshuibai wheat materials at the flowering stage were used. The lower and middle spikelets were marked with a marker pen, and two florets were inoculated per spike, with 20 μL inoculated per floret. Three replicates were performed for each wheat material. The samples were sprayed with water and covered with resealable bags to maintain moisture. Obvious disease symptoms were observed in the wheat ears on day 12 after inoculation with Fusarium graminearum. Figure 6 The samples were quick-frozen in liquid nitrogen and stored at -80°C for subsequent detection of DON and DON-sulfate in wheat.

[0176] (2) Extraction method of DON and DON-Sulfate from wheat samples. After grinding the spikelet samples, add 1 mL of 75% methanol-water mixture, sonicate at low temperature for 30 min, centrifuge at 13000 rpm for 5 min, collect the supernatant into a new tube, and concentrate under vacuum to a gel state. Before injection, resuspend the sample in 100 μL of 20% acetonitrile-water mixture, filter it through a 0.22 μm filter membrane into a vial, and prepare for LC-MS / MS determination of DON and its derivatives.

[0177] 1.1.2 Inoculation of wheat detached leaves

[0178] (1) Select an appropriate amount of wheat seeds, disinfect and clean them with 0.1% sodium hypochlorite solution, and place them evenly on a petri dish containing moistened filter paper with the ventral groove facing down. Incubate at 4℃ for 24 hours and then incubate in the dark at room temperature for 3 days.

[0179] (2) Transplant the sprouted seedlings into flower pots, cover them with soil and cultivate them at 20℃~24℃, control the light for 16 hours, and harvest the material when the seedlings reach the three-leaf stage.

[0180] (3) Cut wheat leaves of uniform size and length with sterilized scissors. Immediately transfer the cut leaves to a 96-well cell culture plate that has been filled with 1 / 2 MS liquid culture medium, so that both ends of the wound are fully immersed in the liquid culture medium and the leaf face up in an arc shape.

[0181] (4) Use a small pipette tip to create a slight wound in the middle of the leaf, and draw 3 μL of Fusarium graminearum solution to drip onto the wound, ensuring that the wound size is consistent on each leaf.

[0182] (5) Place the cell culture plate horizontally in a plastic container containing an appropriate amount of water to maintain humidity. Seal the container opening with a thin film, control the temperature at 25℃, and incubate under light for 16 hours. Take samples at certain time intervals for later use.

[0183] 1.2 LC-MS / MS determination of enzyme activity

[0184] Thermo TSQ Vantage LC-MS / MS triple quadrupole system and Accela 1250LC system were used for the tests. Xcalibur software (version 3.0) was used for data acquisition, and LCquan was used for data evaluation. The mass spectrometer was equipped with a heated electrospray ionization (hESI) interface, which operated in negative ionization mode. Nitrogen was used as the drying gas, and argon as the collision gas. The purge valve was opened, and flow paths A and B were adjusted to 50% each to purge air bubbles. After this, flow paths C and D were each adjusted to 50% to purge air bubbles. The purge valve was then closed. The column pressure was equilibrated according to the required mobile phase ratio according to the detection method. After approximately 20 minutes of column pressure stabilization, the needle was washed, and the sample was injected. After the sample was run, the flow paths were flushed, and the instrument was placed in standby mode.

[0185] Instrumentation: Triple quadrupole tandem mass spectrometer; Mobile phase A: 20 mM ammonium acetate solution; Mobile phase B: acetonitrile (containing 20 mM ammonium acetate), flow rate: 600 μL / min. Elution temperature: 30℃, elution time: 14 min.

[0186] Elution gradient: 0–0.5 min, A = 95%, B = 5%; 0.5–6 min, B increases to 15%; 6–9 min, B increases to 100%, held for 2.0 min, then reequilibrate at A = 95% for 3.0 min. Ion spray voltage was set to -4000V, and collision energy was -50V.

[0187] Based on the DON and DON derivative detection platform established in our laboratory, and referring to the DON-sulfate detection method established by Warth et al., the mass-to-charge ratio parameters of DON and DON-sulfate ion fragments were input for detection. The platform exhibits higher responsivity in negative ion mode. DON-3-sulfate and DON-15-sulfate are two isomers. The difference between them is that an ion fragment at m / z 345 can be observed in the mass spectrum of DON-3-sulfate, while this fragment is not present in DON-15-sulfate (Warth et al., 2015). Based on this, the enzyme-catalyzed reactants and inoculated wheat samples were detected and identified.

[0188] Table 8: Mass Spectrometry Parameters of DON and DON-Sulfate

[0189]

[0190] 2. Test Results:

[0191] 2.1 Physicochemical Properties Analysis of TaSOT2b Protein

[0192] The basic biochemical properties of the protein encoded by TaSOT2b were predicted using the ProtParam tool. The results showed that the TaSOT2b protein has a molecular weight of 41140.49 kDa and a theoretical isoelectric point (PI) of 8.43, indicating that it is a basic protein. The instability index was 45.72, indicating that the protein possesses instability. The protein's lipophilicity index was 81.95, and its hydrophilicity value was -0.159, indicating that the protein is hydrophilic. The functional domains of the protein sequence were estimated using SMART, revealing the presence of a sulfotransfer-3 domain. The phosphorylation sites of the protein were predicted using NetPhos. The results showed that the TaSOT2b protein has 28 phosphorylation sites, including 15 Ser, 10 Thr, and 3 Tyr, indicating that phosphorylation modification is crucial to the structure and function of the TaSOT2b protein. By using the SignaIP model to analyze the signal peptide and transmembrane structure of the target protein, it can be found that the TaSOT2b protein does not contain a signal peptide or transmembrane structure, does not produce membrane proteins or transport proteins, but has the characteristics of cytoplasmic sulfotransferase.

[0193] 2.2 TaSOT2b protein structure analysis

[0194] Three-dimensional structural homology modeling of the TaSOT2b protein sequence was performed using the SWISS-MODEL online platform. Figure 7 The TaSOT2b protein and DON molecule were simulated docking using AutoDock 4.2.6 and MGL Tools-1.5.6 software. Figure 8 The docking was performed 50 times using a semi-flexible docking method. The docking results of the optimal conformation showed that the docking binding energy between the two was -5.52 kcal / mol, which is less than -1.2, indicating that the docking results were relatively good. This demonstrates that there is a good binding ability between the substrate small molecule DON and the protein macromolecule TaSOT2b.

[0195] 2.3 Establishment of LC-MS / MS detection method

[0196] To rapidly and accurately detect the activity of TaSOT2b protein, we utilized the DON and DON derivative detection platform previously established in our laboratory and selected LC-MS / MS for detection. This platform can accurately detect DON and DON derivatives, including DON-sulfate, in wheat samples inoculated with Fusarium graminearum. Due to limited research on DON-sulfate and the lack of standard samples, to ensure accurate detection of the enzymatic reaction products, we first used the LC-MS / MS detection platform to detect the inoculated wheat samples. The results showed that DON and DON-3-S (DON-3-sulfate) were detected in both the ears and leaves of wheat treated with Fusarium graminearum. Figure 9 The peak times for DON were 2.79 and 2.80, and for DON-3-S, they were 2.24 and 2.28. The characteristic ion fragments of DON-3-S were detected at m / z 345, 163, and 97. Figure 10 , Figure 11 Neither DON nor DON-3-S was detected in the water-treated wheat material, verifying that the detection method can be used to detect the enzyme activity of TaSOT2b protein.

[0197] 2.4 LC-MS / MS detection of protein activity

[0198] TaSOT2b encodes a putative TaSOT protein, the activity of which has not yet been confirmed. Based on the sulfation reaction principle, PAPS was used as the sulfonic acid donor, DON as the substrate, and purified TaSOT2b protein was added. The final volume was adjusted to 50 μL with Tris-HCl buffer. The enzymatic reaction products were qualitatively analyzed using an ultra-high performance TSQ-Quantiva liquid chromatography-mass spectrometry (LC-MS / MS) system to detect DON and its derivatives. Reaction substrate: DON toxin standard samples were dissolved in acetonitrile to a concentration of 5000 ppm and stored at -20°C for later use. SO3 -Donor: Lithium 3′-phosphoadenosine-5′-phosphoryl sulfate hydrate (PAPS, Sigma-Aldrich, https: / / www.sigmaaldrich.cn / ) was used as the sulfate donor. The PAPS stock solution was diluted with purified water to a concentration of 250 μM. In a mixture containing 1 μL PAPS, purified TaSOT2b protein, 1 μL substrate DON, and 50 mM Tris-HCl buffer (final volume 100 mL), the reaction was allowed to proceed at 20 °C with shaking at 500 rpm for 12 h. After concentration using a vacuum concentrator, the solution was immediately stored at -80 °C. 100 μL of a 20% acetonitrile solution was added to the concentrated sample and mixed thoroughly. The solution was filtered through a 0.22 μm filter into a vial for LC-MS / MS analysis. Mass spectrometry analysis revealed the characteristic ion of DON-3-sulfate at m / z 345, with DON and DON-3-S characteristic ion peaks detected at 2.79 min and 2.23 min, respectively. DON-3-S was not detected in the reaction mixture without the addition of TaSOT2b. Figure 12 A), but DON-3-S (a) can be observed in the reaction mixture containing TaSOT2b after 12 hours of reaction. Figure 12 (B) This indicates that the TaSOT2b protein can, to some extent, convert DON into the less toxic DON-3-S form, which also suggests the potential role of the TaSOT2b protein in FHB resistance. Preliminary confirmation shows that the TaSOT2b protein possesses sulfotransferase activity. Analysis reveals that the TaSOT2b protein has enzymatic activity, and its mediated DON sulfation occurs at the 3-position of DON, producing DON-3-sulfate.

[0199] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of vomitoxin detoxifying enzyme in the preparation of vomitoxin detoxified products, characterized in that, The vomitoxin detoxification enzyme is a protein as shown in (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1 of the sequence listing; (A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein defined in (A1).

2. The application according to claim 1, characterized in that, The vomitoxin detoxification enzyme catalyzes the reaction of the C3 hydroxyl group of vomitoxin with a sulfonic acid group to generate a sulfate derivative.

3. Application of recombinant expression vectors, transgenic cell lines or genetically engineered bacteria containing genes encoding vomitoxin detoxification enzymes in the production of vomitoxin detoxification enzymes; The gene encoding the vomitoxin detoxification enzyme is a nucleic acid molecule as shown in i) or ii) below: i) The nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.2; ii) Nucleic acid molecules other than those in i) that encode the amino acid sequence shown in SEQ ID NO.

1.

4. A method for reducing the toxicity of vomitoxin, characterized in that, The method is not used for the diagnosis or treatment of disease, and the method includes the step of contacting a vomitoxin detoxification enzyme with a sample to be treated under enzymatic reaction conditions; The vomitoxin detoxification enzyme is a protein as shown in (A1) or (A2) below: (A1) A protein consisting of the amino acid sequence shown in SEQ ID NO.1 of the sequence listing; (A2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein defined in (A1).

5. The method according to claim 4, characterized in that, The sample to be treated contains a sulfonic acid group donor; or a sulfonic acid group is added to the sample to be treated.

6. The method according to claim 5, characterized in that, The donor for the sulfonic acid group is 3'-adenosine-5'-phosphosulfate.

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