CN116064448A - Vomitoxin detoxification enzyme and encoding gene and application thereof - Google Patents

Abstract

The invention discloses vomitoxin detoxification enzyme, and a coding gene and application thereof, and belongs to the technical field of molecular biology. The research of the invention discovers a wheat SOT gene TaSOT2b. The TaSOT2b protein is obtained through in vitro induction expression, and the amino acid sequence of the TaSOT2b protein is shown as SEQ ID NO. 1. The invention discovers that the protein has in-vitro enzyme activity, can modify DON into SON-3-sulfate, and can be used as vomitoxin detoxification enzyme. Has potential application value and reference significance for scientifically preventing and treating wheat scab and DON toxin pollution, and has wide application prospect in the fields of agriculture, feed, industry and the like.

Description

Vomitoxin detoxification enzyme and encoding gene and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to vomitoxin detoxification enzyme, and a coding gene and application thereof.

Background

Vomitoxin (DON), also known as deoxynivalenol (12, 13-epoxy-3 alpha, 7 alpha, 15-trihydroxy trichothecene-9-8 ketone), is named as vomitoxin because of vomitoxin which can cause vomit when ingested, is mainly produced when wheat, barley, oat, corn and other grains are infected by fusarium graminearum (Fusarium graminearum), fusarium flavum (Fusarium culmorum) and the like, is one of main mycotoxins of grains, feeds and foods, seriously affects the health of people and livestock, and is particularly important to reduce or remove the toxicity. Therefore, the genes or enzymes capable of efficiently removing the vomitoxin are separated, and the cereal products polluted by the toxin are treated by in vitro enzyme catalysis, so that the detoxication requirement of the feed industry, the food industry and the medicine industry on the vomitoxin can be met.

DON-sulfate conjugates (DON-sulfate) are a low-toxic DON toxin modification product produced in wheat under natural pathogen conditions and simulated inoculation (DON treatment). At present, the results of the toxicology studies on naturally occurring DON-sulfate show that DON-sulfate has the characteristics of low toxicity and non-masking type toxins and can be regarded as the detoxification product of DON. Sulfation metabolism in organisms is typically accomplished by a Sulfotransferase (SOT) that catalyzes the binding of small molecule compounds to sulfonic acid groups provided by 3'-phosphoadenosine 5' -phosphosulfate (PAPS). SOT is a key enzyme involved in the sulfation reaction and plays an important role in plant growth, development and stress adaptation. However, in the related studies of wheat detoxification modification, there is no report about SOT-catalyzed DON toxin sulfation. Therefore, the research significance is important in deep mining of SOT-encoding genes related to DON modification in wheat and development of application value of the SOT-encoding genes.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide vomitoxin detoxification enzyme and encoding genes and application thereof. The research of the invention discovers a wheat SOT protein with the capability of modifying DON into SON-3-sulfate for the first time, a gene encoding the protein is located on a 2B chromosome, and the gene is named as TaSOT2B. The TaSOT2b protein is obtained through in vitro induced expression, and the in vitro enzyme activity is measured by utilizing an ultra-high performance liquid chromatography-high resolution mass spectrum (High performance liquid chromatography-mass spectrometer, HPLC-MS), so that the DON modified SON-3-sulfate can be used as vomitoxin detoxification enzyme, and has potential application value and reference significance for scientifically preventing and treating wheat scab and DON toxin pollution.

The invention is realized by the following technical scheme:

in a first aspect of the present invention, there is provided a vomitoxin detoxification enzyme which is a protein as shown in any one of (A1) to (A3) below:

(A1) A protein consisting of an amino acid sequence shown as SEQ ID NO.1 in a sequence table;

(A2) A protein which is derived from the SEQ ID NO.1 and is related to the detoxication performance of vomit toxin through the substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence shown as the SEQ ID NO.1 in the sequence table;

(A3) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in (A1) or (A2) with a tag.

Wherein, in order to facilitate purification of the protein in (A1) or (A2), a tag may be attached to the amino-terminus or the carboxyl-terminus of the protein of (A1) or (A2). The tag may be Poly-Arg (typically 6 RRRRRs), poly-His (typically 6 HHHHHH), FLAG (DYKDDDDK), strep-tag II (WSHPQFEK) or c-myc (EQKLISEEDL).

In a second aspect of the present invention, there is provided a gene encoding the vomitoxin detoxification enzyme described above.

Preferably, the coding gene is a nucleic acid molecule as set forth in i) or ii) or iii) below:

i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 2;

ii) a nucleic acid molecule which has 75% or more identity to the nucleotide sequence of i) and which expresses the same functional protein;

iii) A nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 1.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. Identity can be assessed using computer software, for example, using the BLAST algorithm (Altschul et al 1990.Journal of Molecular Biology 215:215-403-410;Karlin and Altschul.1993.Proceedings of the National Academy of Sciences 90:5873-5877).

In a third aspect of the present invention, there is provided a recombinant expression vector, transgenic cell line or genetically engineered bacterium comprising the above-described coding gene.

In a fourth aspect of the invention, there is provided the use of a vomitoxin detoxification enzyme as defined in (1) or (2) below:

(1) Degrading vomitoxin;

(2) Preparing vomitoxin detoxified products.

In the application, the vomitoxin detoxification enzyme catalyzes the reaction of hydroxyl at the C3 position of vomitoxin with a sulfonic acid group to produce a sulfate derivative.

In a fifth aspect, the present invention provides an application of the coding gene of the vomitoxin detoxification enzyme, a recombinant expression vector containing the coding gene, a transgenic cell line or a genetically engineered bacterium in at least one of the following (1) - (3):

(1) Producing vomitoxin detoxification enzyme;

(2) Improving the resistance of plants to DON;

(3) Cultivating scab-resistant plant variety.

In the above application, the plant is preferably wheat.

In a sixth aspect of the invention, there is provided a method of reducing vomitoxin toxicity comprising: and contacting the vomitoxin detoxification enzyme with the sample to be treated under the condition of enzymatic reaction.

Preferably, the sample to be treated contains a donor of sulfonic acid groups; or adding a sulfonic acid group to the sample to be treated.

More preferably, the sulfonic acid group donor is 3'-phosphoadenosine 5' -phosphosulfate (PAPS).

The invention has the beneficial effects that:

the present invention discloses for the first time the sulfation reactions that occur in wheat organisms. A wheat sulfotransferase gene capable of degrading vomitoxin is excavated, the activity of the wheat sulfotransferase gene is proved in vitro, and test results prove that DON can generate sulfation binding reaction under the catalysis of TaSOT2b protein. The test provides important data for the sulfation metabolism of DON in the wheat body and in vitro to a certain extent, and has potential application value and reference significance for scientifically preventing and treating wheat scab and DON toxin pollution.

Drawings

Fig. 1: sulfotransferase gene expression level difference after wheat inoculation with fusarium graminearum.

Fig. 2: colony PCR (A) and double digestion (B) detection of prokaryotic expression vector pET28a-TaSOT 2B; wherein M.DL2000 tag (bp); 1-2 TaSOT2b colony PCR;3. enzyme cutting of the product; 4. the carrier was not cut empty.

Fig. 3: expressing pET28a-TaSOT2b fusion proteins at different induction temperatures and times; wherein M.protein is labeled; 0. inducing a pre-sample; inducing pET28a- TaSOT2b 4,8,10 and 12 hours at the temperature of 1-4.25 ℃; and (3) inducing pET28a- TaSOT2b 4,8,10 and 12 hours at the temperature of 5-8.20 ℃.

Fig. 4: solubility analysis and purification of recombinant protein pET28a-TaSOT2 b; wherein, protein label; 1. inducing a pre-sample; 2. performing post-induction sampling; 3. crushing the supernatant by ultrasonic waves; 4. crushing and precipitating by ultrasonic waves; 5. purifying the protein; 6-7, concentrating the protein.

Fig. 5: western blot analysis.

Fig. 6: wheat ear sampling material; and (3) injection: the steps are as follows from left to right: the water receiving color is white, the bacteria receiving color is white, the water receiving field is an bacteria receiving field.

Fig. 7: taSOT2b tertiary structure prediction.

Fig. 8: the molecular docking of the TaSOT2b protein with DON mimics the conformation.

Fig. 9: detecting a peak graph of the wheat sample; and (3) injection: a, detecting DON and DON-3-S under the conditions of water receiving and bacteria receiving of wheat ears; and B, detecting DON and DON-3-S under the condition that the wheat leaves are water-receiving and bacteria-receiving.

Fig. 10: and detecting DON characteristic ion mass spectrogram of the wheat sample.

Fig. 11: and detecting DON-3-S characteristic ion mass spectrogram of the wheat sample.

Fig. 12: analysis of TaSOT2b enzyme Activity during DON sulfation.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the present application. Unless defined otherwise, 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 belongs.

As described above, the in vivo sulfotransferase SOT of animals and plants is a key gene involved in the sulfation reaction, but the number of plant SOT genes functionally characterized so far is small, and no related genes are reported in the research of wheat. SOT is a gene superfamily, family members participate in phase II metabolism in a organism, catalyze a plurality of endogenous and exogenous complexes to carry out sulfation metabolism, and have detoxification function, and the substrates are quite wide, but specific substrates catalyzed by each SOT of plants can not be accurately predicted only according to the similarity of amino acid sequences.

Based on this, the present invention utilizes plant genome database Ensembl Plants in combination with NCBI wheat transcriptome database to select the most significant gene induced by Fusarium graminearum from all genes annotated as sulfotransferase in wheat at present, according to the expression level of Fusarium graminearum inoculated, and the gene is named as TaSOT2B because it is located on 2B chromosome. Amplifying the CDS sequence of the gene from the wheat scab resistant variety Wang shui leaf cDNA, and sequencing to obtain the CDS sequence of TaSOT2b as shown in SEQ ID NO. 2; the amino acid sequence of the encoded TaSOT2b protein is shown as SEQ ID NO. 1.

The invention utilizes a plurality of online tools to analyze the amino acid sequence of the TaSOT2b protein, and the result shows that the molecular weight of the TaSOT2b protein is 41.14kDa, belongs to hydrophilic protein, and the TaSOT2b protein does not have signal peptide and transmembrane structural domain, which indicates that the protein does not belong to membrane protein or transport protein, and belongs to cytoplasmic sulfotransferase of modified small molecular compound. The three-dimensional structure homology modeling is carried out on the protein through the SWISS-MODEL website, and the TaSOT2b and DON are subjected to semi-flexible simulation butt joint by utilizing Autodock 4.2.6 software, so that the binding capacity between the substrate micromolecule DON and the protein molecule TaSOT2b in the butt joint process is good, and the interaction capacity of the substrate micromolecule DON and the protein molecule TaSOT2b is demonstrated.

In order to obtain a large amount of sulfotransferase for the establishment of subsequent modification reaction and the research of the sulfonation reaction mechanism of DON toxin in a short time, the invention adopts an escherichia coli prokaryotic expression system to carry out the production of recombinant proteins in vitro. Through fumbling of the induction temperature and induction time conditions, the optimal TaSOT2b protein induction conditions are established as follows: induction was carried out at 20℃for 12h. The protein induced by the phylogenetic system is verified to be TaSOT2b by SDS-PAGE electrophoresis detection and western-blot.

In order to study the ability of TaSOT2b protein to modify DON and for subsequent large-scale application of the enzyme in DON-contaminated wheat detoxification, it is necessary to establish a stable and reliable in vitro enzymatic reaction that mimics the sulfation reaction of DON in vitro. The characteristic ion fragment m/z 345 of DON-3-sulfate is the only mode for distinguishing the sulfate products at the 3 rd and 15 th positions of DON through mass spectrometry detection, and is obtained through an in-vitro enzymatic reaction system and a high-resolution mass spectrometry detection platform, and TaSOT2b has the enzymatic activity, can modify the 3 rd toxic site of DON into DON-3-sulfate and has the capability of detoxification of DON.

The invention establishes a sulfotransferase in-vitro enzymatic reaction system, and the reaction system provides theoretical basis for activity improvement of TaSOT2b detoxification enzyme and research and development of a detoxification enzyme preparation.

In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention are all conventional in the art and are commercially available. The experimental procedure, without specifying the detailed conditions, was carried out according to the conventional experimental procedure or according to the operating instructions recommended by the suppliers. Wherein:

wheat species are Walker (highly resistant to scab), fielder (gibberellic disease), F.graminearum) wild Fusarium graminearum strain PH-1, publicly available from applicant, for use in the repetition of the present invention.

The primer sequences used in the examples of the present invention are as follows:

example 1: screening of candidate sulfotransferase genes

To obtain the endogenous gene encoding the sulfotransferase of wheat that may be involved in sulfate modification of DON toxin, all genes annotated as the sulfotransferase in wheat were downloaded locally from the genome database Ensembl Plants (http:// Plants. Ensembl. Org /). Transcriptome data (accession number is CM-82036) of the expression level of wheat inoculated with Fusarium graminearum in NCBI (https:// www.ncbi.nlm.nih.gov /) are combined for analysis, and genes which can be induced to express by Fusarium graminearum are screened and sequenced according to the expression level. The difference in expression levels of TraesCS2B02G200100 was found to be most pronounced after inoculation with Fusarium graminearum, and the expression level was significantly elevated after 50h inoculation (FIG. 1). The gene was then used as a candidate gene for subsequent studies and was conventionally designated as TaSOT2B, as well as its characteristic location on the 2B chromosome.

Example 2: clone of Wangshuibai TaSOT2b full-length CDS

1. Extraction and reverse transcription of Wangshui white leaf RNA

a. Extraction of Hospital RNA with Biospin kit (Tiangen Biotechnology Co., ltd.)

In order to avoid degradation of RNA of the plant sample after being polluted by RNase as much as possible, RNaseF equipment is used; and extracting total RNA of the wheat variety Wangshuibai leaves by referring to the operation guideline of the plant total RNA extraction kit. Pre-cooling by a centrifuge at the temperature of 4 ℃ before operation, and performing low-temperature operation in the whole test process; when agarose gel electrophoresis detection is carried out after RNA extraction is finished, fresh electrophoresis liquid is replaced, 180V voltage is set for 15min, and if electrophoresis results have 2-3 clear bands, the quality of the extracted RNA is better. The operation steps are as follows:

(1) Thoroughly crushing the wheat leaf samples by using a vibration mill under the condition of liquid nitrogen in advance, and temporarily preserving the wheat leaf samples by using liquid nitrogen. To Lysis AG 2% β -mercaptoethanol was added.

(2) And adding a proper amount of plant samples into small steel balls, quick-freezing in liquid nitrogen, grinding into powder through a ball mill, adding a Lysis AG solution, and immediately and severely oscillating until no obvious particle shape exists.

(3) Centrifuge at 12000rpm for 5min at 4℃and aspirate the supernatant into an RNase-Free centrifuge tube.

(4) Adding 1/2 of the supernatant volume of absolute ethanol, and immediately mixing. The mixture was transferred to Spin Column and centrifuged at 12000rpm at 4℃for 1min. mu.L of Wash Buffer containing absolute ethanol was pipetted into Spin Column, centrifuged at 12000rpm, and the outer tube waste was decanted again.

(5) 50. Mu.L DNase I solution was added to the center of Spin Column membrane and allowed to stand for 15min.

(6) mu.L PG Buffer was pipetted and centrifuged at 12000rpm for 0.5min. Then, 500. Mu.L of Wash Buffer was added thereto, and the mixture was centrifuged at 12000rpm for 30s. Centrifugation was repeated by adding 250. Mu.L Wash Buffer. Pouring out the waste liquid of the outer tube, centrifuging at 12000rpm for 1min at 4 ℃ in a centrifuge, and thoroughly spin-drying.

(7) Spin Column was changed to a new tube, 50. Mu. L RElution Buffer was added, and after 2min of standing, total RNA was obtained by centrifugation at 12000rpm for 1min.

b. The RNA was reverse transcribed using RT-PCR Supermix to synthesize cDNA.

Sequentially adding the following components into a PCR tube: mu.L Oligo (dT) Primer, 10. Mu.L 2 XTS Reaction Mix, 1. Mu.g RNA, 1. Mu.L Transscript RT/RI Enzyme Mix, 1. Mu.L gDNA reverse, RNase-free Water was supplemented to 20. Mu.L of the total system.

2. CDS cloning and electrophoresis detection of TaSOT2b gene

The CDS sequence of the TaSOT2b gene is amplified by using the cDNA of the water white leaf as a template and a TaSOT1-F/R primer.

PCR reaction system: 2X Phanta Max Buffer. Mu.L, 10mM dNTP Mix 1. Mu.L,TaSOT1-F (10. Mu.M) 2. Mu. L, taSOT1-R (10. Mu.M) 2. Mu.L, template cDNA 2. Mu.L, phanta Max Super-Fidelity DNA Polymerase 2. Mu. L, ddH ₂ O 18μL。

PCR reaction procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 64℃for 30s, elongation at 72℃for 1min for 20s,35 cycles; finally, the extension is carried out for 5min at 72 ℃.

Agarose gel electrophoresis detection:

and (3) glue preparation: 25mL of 1 xTAE electrophoresis buffer and 0.25g of agar powder are respectively poured into a conical flask to shake evenly, the agar is heated until the agar is completely dissolved, 2.5 mu L of nucleic acid dye is immediately added after the cooling, the mixture is quickly shaken evenly, poured into a gel plate, and left stand for cooling for 20min to solidify, thus the gel of 1% is prepared.

Loading: the gel was carefully transferred to the electrophoresis tank. And adding a proper amount of Loading Buffer into the PCR product, sucking and beating, mixing uniformly and Loading.

Running glue: the power supply is turned on, the voltage is regulated to 120V, and the power supply is turned off after electrophoresis for 25 min. And (5) placing the gel under a gel imager for detection and shooting and storing pictures.

3. PCR product purification and sequencing vector ligation

(1) Agarose gel DNA recovery: the gel tape of the target gene is cut off rapidly under ultraviolet light, and the product is purified and recovered by the kit. Putting the gel into a 1.5mL centrifuge tube, adding a PGBuffer, heating at a constant temperature of 50 ℃ until the gel is completely melted, transferring into a Spin Column, centrifuging for 1min at 10000 Xg, adding a 450 MuLSPWBuffer, centrifuging for 1min at 10000 Xg, and washing repeatedly; and (3) increasing the rotating speed of the centrifugal machine to 13000 Xg for 2min by empty Column centrifugation, and Spin-drying Spin Column residual waste liquid. Spin Column was transferred to a fresh tube, carefully blotted with 20. Mu.L of ElutronBuffer in the center of the membrane and allowed to stand at 37℃for 2min. The DNA was collected by centrifugation at 13000 Xg for 1min and stored at-20 ℃.

(2) The recovered product was ligated with pEASY-Blunt sequencing vector:

table 1: sequencing vector ligation system

4. Preparation of competent cells of E.coli

(1) Streaking DH5 alpha/BL 21 competence on a LA plate, and picking a newly activated single colony to activate for 12 hours in LB; 5mL was inoculated into 250mL LB (1:50), and the OD was cultured at 37℃at 200rpm to OD ₆₀₀ About 0.5, ice bath for 15min, precooling centrifuge tube, 0.1M MgCl ₂ 、0.1M CaCl ₂ 。

(2) Centrifuging the bacterial liquid at 6000rpm of 4 ℃ for 10min, collecting precipitate, and sucking 50mL of precooled 0.1MMgCl ₂ Sufficiently suspending the bacterial precipitate.

(3) Centrifuging the bacterial liquid at 6000rpm for 10min, collecting precipitate, adding 125mL of pre-cooled 0.1M CaCl ₂ The bacterial cells were fully suspended for precipitation, and immediately ice-bath for 20min.

(4) Centrifuging at 6000rpm at 4deg.C for 10min, collecting precipitate, and collecting 0.1M CaCl ₂ Suspending thallus, adding 5mL of 50% glycerol, subpackaging, and quick freezing with liquid nitrogen at-80deg.C.

5. Conversion of ligation products

(1) Thawing competent cells on ice for several minutes, adding the ligation product, mixing well, and placing on ice again for 30min.

(2) The centrifuge tube was placed on a floating plate and incubated at 42℃for 1min 30s, immediately cooled on ice for 5min.

(3) Adding 900 μl of LB solution, placing into a constant temperature shaking table, setting parameters of 37deg.C, 220rpm, culturing for about 60min, and taking out.

(4) 100. Mu.L of the bacterial liquid was uniformly spread on a Kan-resistant LB plate, and after sealing with a sealing film, the plate was cultured overnight in an incubator at 37 ℃.

(5) And (3) selecting a single colony for PCR verification, and using M13-F/R as a primer to preliminarily detect whether the target DNA fragment is transferred into the escherichia coli through an electrophoresis band.

6. Positive clone screening and sequencing

Positive clones were screened by colony PCR using 2 XTaq MixDNA polymerase. Sequentially adding the reaction system (except for a colony template) into a 200 mu L PCR tube (on-ice operation), picking the bacterial colony, streaking and storing bacteria on a resistance flat plate, then stirring the toothpick inserted into a centrifuge tube to uniformly mix the rest bacterial colony and the system in the tube, tightly covering a cover of the centrifuge tube, vibrating and centrifuging, then placing the mixture into a PCR instrument, and setting a PCR program to perform PCR reaction. The PCR reaction was as follows (11. Mu.L):

table 2: colony PCR reaction system

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

And (3) obtaining an mRNA reference sequence of TaSOT2b according to the sequence information of the Chinese spring variety wheat disclosed by Ensembl Plants, and designing a specific primer pair according to the mRNA reference sequence. Extracting RNA of a wheat scab variety Wangshui leaf with high resistance, obtaining cDNA after reverse transcription, and finally amplifying by using a specific primer to obtain a CDS sequence of TaSOT2b. The CDS sequence of TaSOT2b is obtained through sequencing to have the total length of 1110bp (comprising an initiation codon and a termination codon as shown in SEQ ID NO. 2), and the total code is 369 amino acids.

Example 3: expression and purification of TaSOT2b protein

1. The test method comprises the following steps:

1.1 Construction of pET28a-TaSOT2b expression vector

1.1.1 Construction of pET28a-TaSOT2b expression vector

CDS cloning primers were designed (Table 1) with reference to wheat transcriptome data in combination with NCBI (http:// www.ncbi.nlm.nih.gov /) BLAST to obtain the wheat sulfotransferase SOT gene sequence. PCR system: ddH ₂ O18. Mu.L, 10X Phanta Max Buffer. Mu.L, 10 mmol.L-1 dNTPs 1. Mu. L, taSOT 1-F/R2. Mu. L, cDNA 2. Mu.L and Phanta Max Super-Fidelity DNA Polymerase. Mu.L. The reaction procedure: 94 ℃ for 5min; cycling for 35 times at 94 ℃ for 30s,64 ℃ for 30s and 72 ℃ for 1min for 20 s; and at 72℃for 5min.

Connecting the target gene fragment with a pEASY sequencing vector, transforming DH5 alpha coating, culturing at 37 ℃ overnight, and obtaining the correct target gene fragment through colony PCR verification and sequencing verification. Extracting plasmid DNA with correct sequencing, and carrying out PCR amplification by taking plasmid pEASY-TaSOT2b as a template and taking TaSOT2-F/R as a primer. After electrophoresis detection, the gel is cut backThe target fragment was obtained, and the target fragment and the vector pET28a were digested with NotI-HF and EcoRI-HF restriction enzymes, respectively, in the following reaction system: ddH ₂ O37 mu L, cutSmart Buffer 5 mu L, PCR product pET28a carrier 5 mu L and restriction enzyme 1 mu L each, and the components in the system are mixed uniformly by vibration and centrifuged, then reacted for 2h at 37 ℃ and inactivated for 20min at 65 ℃.

The gene fragment was ligated to the vector overnight at 16℃under the action of T4 DNA ligase. The ligation products were transformed into DH 5. Alpha. Overnight and positive plasmids were identified by colony PCR screening and further verified by sequencing positive transformants. Sequence alignment confirmed sequencing results, plasmid pET28a-TaSOT2b was transformed to BL21 (DE 3), plated on Kan-resistant LB plates overnight at 37℃and positive plasmids were again identified by colony PCR in preparation for prokaryotic expression.

1.2 plasmid extraction

(1) The overnight cultured bacterial liquid was centrifuged at 10000rpm for 30s, and the precipitate was collected.

(2) The bacterial pellet was well suspended by pipetting 250 mu L Resuspension Buffer.

(3) 250. Mu.L of Lysis Buffer was added and the mixture was well lysed by shaking up and down. Then 350 mu L Neutralization Buffer was added and thoroughly mixed until white floc was present in the tube.

(4) Centrifuge in a centrifuge at 13000rpm for several minutes until a white floc forms a tight precipitate. The supernatant was transferred to Spin column and centrifuged at 12000rpm for 1min, and the waste liquid was decanted.

(5) 650 μL Wash Buffer was pipetted into Spin column and centrifuged at 12000g for 1min to pour out the waste solution. This step was repeated once.

(6) The residue was spun off by centrifugation at 12000g for a further 1min and Spin column transferred to a clean 1.5mL centrifuge tube.

(7) The plasmid DNA was eluted by adding 50. Mu. LElute Buffer and allowed to stand for one minute. The plasmid DNA was collected by centrifugation at 12000g for 1min and stored at-20 ℃.

1.3 cleavage and recovery of products

(1) Double enzyme cutting: the pEASY vector and the prokaryotic expression vector pET-28a connected with the TaSOT2b gene are respectively digested with Not I and EcoR I, digested in water bath at 37 ℃ overnight, and the digested product glue is recovered. The cleavage system was as follows (50 μl):

table 3: enzyme cutting system

(2) Ligation of the fragment of interest with the prokaryotic expression vector using T4 ligase, reaction ligation procedure: 16 ℃ for 3h. The ligation system was as follows (10 μl):

table 4: t4 ligase ligation system

(3) And (5) verifying and sequencing the transformed escherichia coli. PCR verification and sequencing were performed using the universal primer T7-F/R.

1.4 qRT-PCR analysis

The reaction system was added to a qPCR tube and placed on a fluorescent quantitative PCR instrument for amplification, 3 replicates per sample. Reaction system (20. Mu.L)

Table 5: qRT-PCR reaction system

The reaction procedure: 3min at 95 ℃;95℃for 5s, 60℃for 30s,40 cycles.

1.5 Induction of protein expression

The positive clone colony was placed in 2mL Kan-resistant LB medium and cultured overnight at 37℃at 220 rpm; according to 1:100 proportion expansion culture bacterial liquid, culturing at 37 ℃ and 220rpm until OD ₆₀₀ About 0.6. Mu.L of the culture was aspirated and temporarily stored at 4℃for use as a negative control. Adding 1mL of IPTG to the residual culture solution to induce protein expression, respectively inducing at 20 ℃ and 25 ℃ for 4h, 8h, 10h and 12h, taking 100 mu L of each of the collected bacterial solutions, and temporarily storing at 4 ℃ for later use for detection by SDS-PAGE. After the induction is completed, 100 mu L of sediment which is preserved before 2X SDS PAGE loading Buffer suspension is added, and the mixture is centrifugally mixed for many times and boiled at 100 ℃ for 8 timesThe protein was denatured by min. Taking 30 mu L of sample for electrophoresis detection, placing coomassie brilliant blue on a low-speed shaking table after the coomassie brilliant blue is finished to dye the protein for 2 hours, and photographing on a gel viewing instrument after decolorization to analyze the optimal induction condition of the protein. SDS-PAGE gels were prepared as follows:

table 6: SDS-PAGE gel 12% separation gel solution

After the components are added, the components are mixed uniformly, the gel solution is added into a gel plate by a 1mL suction head for several times, then 2mL water seal is added, after the gel is solidified, water is poured out and the gel solution is sucked by filter paper.

Table 7: SDS-PAGE gel 5% gel concentrate solution configuration

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Mixing the above components, adding into separating gel, vertically inserting into 1mm electrophoresis comb, standing for 20min, and solidifying.

Inducing recombinant protein under optimal induction conditions by using the SOT protein, centrifuging the induced sample at 5000rpm for 10min, collecting precipitate, adding 5mL PBS, suspending the precipitate, centrifuging at 5000rpm for 5min, collecting precipitate, and freezing at-20deg.C for 30min. Sucking 15mL of PBS, suspending and precipitating, adding 30 mu L of protease inhibitor (PMSF) into the solution, placing a centrifuge tube filled with bacterial liquid on ice to prevent protein from heating and denaturing in the breaking process, covering ice blocks above the liquid level, breaking the bacterial liquid by an ultrasonic cytobreaker, setting the procedure to work for 3s, and performing ultrasonic treatment for 5min at intervals of 5s, and stopping ultrasonic treatment after the bacterial liquid becomes clear from turbidity. Centrifuging at 5000rpm for 10min, collecting crude enzyme solution, and temporarily storing at 4deg.C.

1.6 protein purification

a. Cobalt resin affinity chromatography

(1) And (3) placing the crude protein enzyme solution obtained in the previous step on ice.

(2) The TALON His-Tag purified resin was thoroughly mixed by shaking. 1mL of the buffer solution is sucked into the affinity chromatography tube, 1mL of PBS balance buffer solution is added into the resin gel for washing, and the buffer solution naturally flows out from the lower opening of the chromatography column tube.

(3) The PBS equilibration buffer was repeated one time.

(4) According to gel and crude enzyme solution 1:8, adding a proper amount of crude enzyme solution into an affinity chromatography column, covering an upper cover and a lower cover, and putting the mixed solution into a condition of 4 ℃ to turn over for 4 hours up and down so that the protein and the gel are fully combined.

(5) The lower cover of the column tube is opened to naturally discharge the liquid into the waste liquid cylinder, the lower cover is closed, 1mL of non-denatured washing buffer solution is added, and the column tube is turned up and down at 4 ℃ for 10min.

(6) Opening the lower cover of the chromatographic column tube to naturally discharge the liquid, closing the lower cover, adding 1mL of non-denaturing eluting buffer solution to elute the target protein, and turning over at 4 ℃ for 15min.

(7) And opening the lower cover, collecting the liquid into a clean centrifuge tube, obtaining purified protein, and detecting and recording the protein concentration.

b. Ultrafiltration concentration

Amicon-0.5 ultrafiltration tubes were inserted into the centrifuge tube. The ultrafiltration tube is soaked in 70% ethanol solution for 5min, washed with distilled water, added with 500 mu L of 0.1N NaOH solution, centrifuged for 5min at 14000g, washed with non-denaturing elution buffer and spin-dried. Adding 500 mu L protease solution into the ultrafiltration inner tube, centrifuging for 20min at 14000g in a centrifuge at 10deg.C, discarding the liquid in the liquid receiving centrifuge tube, adding 450 mu L non-denaturing elution buffer into the ultrafiltration inner tube, centrifuging for concentrating for 20min at 14000g, and repeatedly washing for 2 times to achieve desalting effect and fully reduce the concentration of impurities in protein. To recover the concentrated protein, the ultrafiltration tube was placed in a centrifuge tube. Put in a centrifuge, the direction of opening the lid was concentrated in the center of the rotor, the centrifuge lid was spin-covered, and 1000g was centrifuged for 2 minutes, so that the protein concentrate trapped in the ultrafiltration inner tube was transferred to the filtrate collection tube. And (3) sucking all protein liquid in the collecting pipe into a clean PCR tube, detecting the concentration of the concentrated protein, and loading the sample and detecting the sample through SDS electrophoresis.

Western blot detection of 1.7TaSOT2b protein

(1) And cleaning the glue-making glass clamping plate, sucking water by using filter paper, assembling the aligned glass plates on a glue-making frame, adding a proper amount of water into the assembled glue-making plate, standing for 1min, detecting the tightness of the glue-making plate, ensuring that the tightness is good without water dripping, and pouring the water.

(2) A12% solution of the separation gel was prepared and the reagents were added as in Table 6.

(3) A5% concentrated gum solution was prepared and the reagents were added as in Table 7.

(4) And finally, after TEMED is added, the solution is quickly mixed, the mixed solution is completely transferred into a glue making plate, the glue making comb is vertically installed, and the glue is completely solidified after about 25 minutes.

(5) When the gel is fixed, the protein sample is boiled. 24. Mu.L of sample and 6. Mu.L of loading buffer were added to the PCR tube and mixed well, and the protein was denatured by heating at 100℃for about 8 minutes, and immediately after boiling, the protein was placed on ice.

(6) The glue making comb is vertically pulled out, the glue making plate is fixed in an electrophoresis tank, and a proper amount of 1 Xrunning Buffer electrophoresis Buffer solution is added

(7) To the dispensing wells, 5. Mu.L of protein Marker and 20. Mu.L of protein sample were added sequentially.

(8) The power supply is switched on, the voltage of the electrophoresis apparatus is set to be 60V, the protein dye can be seen to reach the bottom of the concentrated gel after about 30min, the voltage is increased to 120V at the moment, and the electrophoresis is stopped after the dye reaches the bottom of the separation gel for about 60 min.

(9) And taking out the glue making plate, slightly prying the two glass plates apart, and neatly cutting the separating glue by using the glue cutting plate.

(10) PVDF film with the same size as the glue is cut, immersed in methanol solution for 30s, immediately transferred to transfer solution for 30s, and 6 pieces of filter paper and buffer sponge are simultaneously processed. The buffer sponge, 3 pieces of filter paper, separating glue, 3 pieces of filter paper and the buffer sponge are sequentially overlapped on the film transfer plate, and the film transfer plate is compacted layer by layer to prevent bubbles from appearing, and the clamp is sealed. Setting the current of the electrophoresis apparatus to be 400mA, and finishing film transfer about 45 min.

(11) And (5) immune hybridization. After completion of the transfer, the PVDF membrane was washed three times with PBST. The PVDF film is placed in a sealing liquid added with skim milk powder, slowly shaken on a shaking table for about 1h, primary antibody is added in a ratio of 1:5000, sealing is carried out for about one hour, and PBST is used for treating the PVDF film for three times. Adding secondary antibody into PBST containing skimmed milk powder at a ratio of 1:10000, and sealing. After 1h, PVDF membranes were treated three times with PBST solution.

(12) The film transfer result was observed with a gel imager, and recorded by photographing.

2. Test results:

2.1 construction of prokaryotic expression vector pET28a-TaSOT2b

The target fragment was recovered and purified. By constructing a prokaryotic expression vector, inducing mass expression of TaSOT2b, purifying the obtained protein by using a cobalt resin affinity chromatography by utilizing the characteristics of His labels, and establishing an in vitro enzymatic reaction system of the TaSOT2b protein for catalyzing DON to be sulfated. The target fragment TaSOT2B and the prokaryotic expression vector pET-28a are subjected to double digestion by using NotI-HF and EcoRI-HF restriction enzymes successively, then the gene fragment and the vector which are subjected to the same digestion treatment are directly connected by using T4 ligase, DH5 alpha is transformed to obtain the recombinant expression vector pET28a-TaSOT2B, positive clones are screened by kanamycin resistance to carry out colony PCR identification (figure 2A) and double digestion detection (figure 2B), the activated positive clone bacterial liquid is sent to sequencing (biological company, the sequence comparison result is consistent with the previous result, and the fact that the prokaryotic expression vector pET28a-TaSOT2B is successfully constructed is shown.

2.2 Expression and purification of TaSOT2b protein

To explore the optimal expression environment of the recombinant protein, pET28a-TaSOT2b was transferred to BL21, plated on Kan-resistant medium, and cultured upside down at 37℃overnight. The colony PCR verification shows that the amplified electrophoresis band is single and specific, and the size of the amplified electrophoresis band is consistent with the size of the target gene, which indicates that BL21 competent cells have been successfully transferred. The single colony of correct band was picked up and placed in LB (Kan resistant) medium, after shaking culture for 8 hours at 37℃and 200rpm, IPTG was added, and after induction for 4h, 8h, 10h and 12h at 20℃and 25℃respectively, samples were taken and SDS-PAGE electrophoresis detection analysis determined the optimal induction conditions for the recombinant protein (FIG. 3).

In order to further understand the solubility and purification condition of the TaSOT2b protein, the optimal induction condition of the SOT protein obtained by the test is adopted, IPTG is added, shaking culture is carried out at 20 ℃ and 150rpm overnight, after ultrasonic crushing and centrifugation, the supernatant and the precipitate are respectively detected by SDS-PAGE electrophoresis, and the result shows that the target protein exists in the supernatant and the precipitate. Since the research of protein activity requires high-purity soluble protein, the protein needs to be continuously purified, and the TaSOT2b protein with higher purity is obtained by utilizing a cobalt resin affinity chromatography method and an ultrafiltration concentration method, and SDS-PAGE protein gel electrophoresis detection is carried out after the purified protein is obtained. The results showed that the pET28a-TaSOT2b recombinant protein had a distinct band at a molecular weight of about 45kDa, consistent with the expected size (FIG. 4), and a significant increase in the amount of protein after concentration was found, indicating that purification was more successful.

2.3 Western blot detection of TaSOT2b protein

To further examine whether the protein obtained after expression is the target protein, protein purification must be identified by Western Blot. The murine anti-His 6 was selected as the primary antibody, the goat anti-mouse was selected as the secondary antibody, and the results showed that a distinct specific band at 45kDa was seen in the purified eluate by a series of steps of transferring PVDF membrane, blocking with skimmed milk powder, adding primary antibody, adding secondary antibody, etc., consistent with the theoretical molecular weight, indicating that the TaSOT2b protein had been successfully expressed and purified in E.coli (FIG. 5).

Example 4: taSOT2b protein Activity assay

1. Test method

1.1 preparation of wheat germ sample

1.1.1 wheat ear inoculation

(1) The wheat inoculation sample is measured as a positive control, and the wheat sample inoculation method is wheat Shan Hua drip inoculation. Wheat materials in the flowering stage, namely field, are expected to be white, the middle and lower spikes are taken and marked by marker points, 2 florets are inoculated per spike, 20 mu L of each floret is inoculated, and 3 times of each wheat material are repeated. Spraying water from the watering can, and sleeving a self-sealing bag to preserve moisture. Obvious onset symptoms of wheat ears can be observed on day 12 after fusarium graminearum inoculation (figure 6), and the wheat ears are stored at-80 ℃ after liquid nitrogen quick freezing and used for detecting DON and DON-sulfate in subsequent wheat.

(2) DON and DON-Sulfate extraction method in wheat samples. After grinding the spikelet sample, 1mL of 75% methanol water was added, sonicated at low temperature for 30min, centrifuged at 13000rpm for 5min, the supernatant was taken into a fresh tube and concentrated in vacuo to gel. Before sample injection, 100. Mu.L of 20% acetonitrile water was taken up to resuspend the sample, filtered through a 0.22 μm filter into sample injection vials, and prepared for LC-MS/MS measurement of DON and its derivatives.

1.1.2 wheat in vitro leaf blade inoculation

(1) Selecting a proper amount of wheat seeds, sterilizing and cleaning the wheat seeds by using a 0.1% sodium hypochlorite solution, uniformly placing the wheat seeds on a culture dish with wetted filter paper, culturing the wheat seeds with the abdominal ditch facing downwards at the temperature of 4 ℃ for 24 hours, and shading and culturing the wheat seeds for 3 days at room temperature;

(2) Transplanting the germinated seedlings to a flowerpot for soil covering culture, controlling illumination for 16 hours at 20-24 ℃, and obtaining materials when the seedlings are cultured to a trefoil stage;

(3) Cutting wheat leaves with uniform size and equal length by using sterilized scissors, immediately transferring the cut leaves into a 96-hole cell culture plate in which a 1/2MS liquid culture medium is placed, fully immersing two ends of a wound into the liquid culture medium, and enabling the right side of the leaves to face upwards to form an arc shape;

(4) Manufacturing a slight wound in the middle of each blade by using a small-size pipetting gun head, sucking 3 mu L of fusarium graminearum liquid drops to the wound, and controlling the wound size of each blade to be consistent;

(5) The cell culture plate is horizontally placed in a plastic container containing proper amount of water to ensure humidity, a layer of film is sealed at the mouth of the container, the temperature is controlled at 25 ℃, and the culture is carried out under illumination for 16 hours. Sampling at certain time intervals for standby.

1.2LC-MS/MS determination of enzyme Activity

Thermo TSQ Vantage LC-MS/MS triple quadrupole system and Accela 1250LC system. Data acquisition was performed using Xcalibur software (version 3.0), while data evaluation was performed using LCquan. Mass spectrometers are equipped with a heated electrospray (hESI) interface that operates in negative ionization mode. Nitrogen as the dry gas and argon as the collision gas. And opening the purge valve, regulating the flow paths A and B to 50% respectively to discharge bubbles, discharging 50% of bubbles from the flow paths C and D after the completion, and closing the purge valve after the completion. And (3) regulating the required flow phase ratio according to a detection method, balancing the column pressure of the chromatographic column, washing the needle after the column pressure is stabilized for about 20min, and carrying out sample injection after the completion. And after the sample is run, the flow path is flushed, so that the instrument is in a standby state.

Instrument: triple quadrupole tandem mass spectrometer; mobile phase a:20mM ammonium acetate solution; b: acetonitrile (containing 20mM ammonium acetate) at a flow rate of 600. Mu.L/min. The elution temperature was 30℃and the elution time was 14min.

Elution gradient: 0 to 0.5min, a=95%, b=5%; 0.5-6 min, B rises to 15%; 6-9 min, b was raised to 100%, held for 2.0min, and then rebalanced at a=95% for 3.0min. The ion spray voltage was set at-4000V and the collision energy at-50V.

According to DON and DON derivative detection platform established in the laboratory, DON and DON-sulfate ion fragment mass-charge ratio parameters are input for detection according to a DON-sulfate detection method established by Warth et al, and the DON and DON-sulfate ion fragment mass-charge ratio parameters have higher responsivity in a negative ion mode, wherein DON-3-sulfate and DON-15-sulfate are two isomers, and the difference between the DON-3-sulfate and DON-15-sulfate is that ion fragments at m/z 345 can be observed in a mass spectrogram of DON-3-sulfate, and DON-15-sulfate does not exist in the fragments (Warth et al, 2015), so that detection and identification of enzymatic reactants and inoculated wheat samples are carried out according to the detection and identification.

Table 8: DON and DON-sulfate mass spectrometry parameters

2. Test results:

2.1 Analysis of TaSOT2b protein physicochemical Properties

The ProtParam tool was used to predict the basic biochemical properties of the protein encoded by TaSOT2b. The results showed that the TaSOT2b protein had a molecular weight of 41140.49kDa and a theoretical isoelectric Point (PI) of 8.43, representing that the protein was an alkaline protein. The instability index was 45.72, indicating that the protein had a factor of instability. The protein has a lipid solubility index of 81.95 and a hydrophilicity value of-0.159, indicating that the protein has hydrophilicity. The functional domain of the protein sequence was estimated by using SMART and the result showed that the protein had a sulfotransferase functional domain (Sulfotransfer-3 domain). The protein phosphorylation site was predicted using NetPhos. The results show that there are 28 phosphorylation sites of the TaSOT2b protein, wherein Ser15, thr 10 and Tyr 3, which indicate that the modification of the phosphorylation sites is very important for the structure or function of the TaSOT2b protein. By analyzing the signal peptide and the transmembrane structure in the target protein using the SignalP model, it was found that the TaSOT2b protein did not include the signal peptide and the transmembrane structure, and did not produce the membrane protein and the transport protein, but had the characteristics of cytoplasmic sulfotransferase.

2.2 TaSOT2b protein structural analysis

Three-dimensional structural homology modeling was performed on the TaSOT2b protein sequence by SWISS-MODEL on-line platform (FIG. 7), and simulated docking was performed on the TaSOT2b protein and DON molecule using Autodock 4.2.6 and MGL Tools-1.5.6 software (FIG. 8). 50 times of butt joint are performed in a semi-flexible butt joint mode, and the butt joint result of the optimal conformation shows that the butt joint binding energy of the two is-5.52 kcal/mol which is smaller than-1.2, and the butt joint result is good, so that the good binding capacity between the substrate micromolecule DON and the protein macromolecule TaSOT2b is proved.

2.3 LC-MS/MS detection method establishment

In order to rapidly and accurately detect the activity of the TaSOT2b protein, an LC-MS/MS method is selected to detect the activity of the TaSOT2b protein by using a DON and DON derivative detection platform established before the laboratory. The platform can accurately detect DON and derivatives of DON in wheat samples after being inoculated with fusarium graminearum, wherein DON-sulfate is also included. Because of the lack of standard samples in the prior art, in order to ensure accurate detection of enzymatic reaction products, the detection of the bacterial wheat sample is firstly carried out by using an LC-MS/MS detection platform, and the result shows that DON and DON-3-S (DON-3-sulfate) can be detected by both wheat ears and leaves treated by fusarium graminearum (FIG. 9), the peak time of DON is 2.79 and 2.80 respectively, the peak time of DON-3-S is 2.24 and 2.28 respectively, and the characteristic ion fragments of DON-3-S are detected to be m/z 345,163,97 (FIG. 10 and FIG. 11); neither DON nor DON-3-S was detected in the water-receiving wheat material, verifying that this detection method could be used to detect the enzymatic activity of the TaSOT2b protein.

2.4 LC-MS/MS detection of protein Activity

TaSOT2b encodes a putative TaSOT protein, the activity of which has not been demonstrated. According to the principle of sulfation reaction, PAPS is taken as a sulfonic donor, DON is taken as a substrate, purified TaSOT2b protein is added, finally, a Tris-HCl buffer solution is used for complementing the system to 50 mu L, and a DON and DON derivative detection platform is used for carrying out qualitative analysis on enzymatic reaction products in an ultra-efficient TSQ-Quantiva liquid chromatography-mass spectrometer (Ultimate TSQ Quantiva). The reaction substrate: the DON toxin standard sample is dissolved with acetonitrile to a concentration of 5000ppm and stored at-20 ℃ for later use. SO (SO) ₃ ^- Donor: the PAPS stock solution was diluted with purified water to a concentration of 250. Mu.M using lithium 3 '-phosphoadenosine-5' -phosphosulfate salt hydrate (PAPS, sigma-Aldrich, https:// www.sigmaaldrich.cn /) as the sulfate donor. In a mixture (final volume 100 mL) containing 1. Mu.L of PAPS, taSOT2b purified protein, 1. Mu.L of substrate DON and 50mM Tris-HCl buffer, the reaction was allowed to shake at 20℃for 12h at 500rpm, and immediately frozen at-80℃after spin-concentration in a vacuum concentrator. Prepared 20% acetonitrile water, and 100 μl of the mixture was pipetted into the concentrated sample and mixed well by shaking. Filtered through a 0.22 μm filter into a sample vial in preparation for LC-MS/MS measurement. In the mass spectrum detection, characteristic ions m/z 345 of DON-3-sulfate are found, and DON-3-S characteristic ion peaks are detected at 2.79min and 2.23min respectively. DON-3-S was not detected in the reaction mixture without TaSOT2B added (FIG. 12A), but DON-3-S was observed in the reaction mixture containing TaSOT2B after 12 hours of reaction (FIG. 12B), which illustrates that the TaSOT2B protein could convert DON to the less toxic form of DON-3-S to some extent, which also illustrates the potential significance of the TaSOT2B protein in FHB resistance. Preliminary confirmation of the activity of TaSOT2b protein as sulfotransferase. It can be analyzed that the TaSOT2b protein has enzymatic activity, the mediated sulfation of DON occurs at position 3 of DON, and the product is DON-3-sulfate.

The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims (10)

1. A vomitoxin detoxification enzyme, wherein the vomitoxin detoxification enzyme is a protein as set forth in any one of (A1) - (A3) below:

(A1) A protein consisting of an amino acid sequence shown as SEQ ID NO.1 in a sequence table;

(A2) A protein which is derived from the SEQ ID NO.1 and is related to the detoxication performance of vomit toxin through the substitution and/or deletion and/or addition of one or more amino acid residues of the amino acid sequence shown as the SEQ ID NO.1 in the sequence table;

(A3) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in (A1) or (A2) with a tag.

2. A gene encoding a vomitoxin detoxification enzyme according to claim 1.

3. The coding gene according to claim 2, wherein the coding gene is a nucleic acid molecule as set forth in i) or ii) or iii) below:

i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 2;

ii) a nucleic acid molecule which has 75% or more identity to the nucleotide sequence of i) and which expresses the same functional protein;

iii) A nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 1.

4. A recombinant expression vector, transgenic cell line or genetically engineered bacterium comprising the coding gene of claim 2.

5. Use of a vomitoxin detoxification enzyme according to claim 1 in (1) or (2) as follows:

(1) Degrading vomitoxin;

(2) Preparing vomitoxin detoxified products.

6. The use according to claim 5, wherein the vomitoxin detoxification enzyme catalyzes the reaction of the hydroxyl group at the C3 position of the vomitoxin with a sulfonic acid group to produce a sulfate derivative.

7. Use of the coding gene of claim 2 or 3 or the recombinant expression vector containing the coding gene of claim 4, a transgenic cell line or a genetically engineered bacterium in at least one of the following (1) - (3):

(1) Producing vomitoxin detoxification enzyme;

(2) Improving the resistance of plants to DON;

(3) Cultivating a scab-resistant plant variety;

preferably, the plant is preferably wheat.

8. A method of reducing vomitoxin toxicity comprising: a step of contacting the vomitoxin detoxification enzyme of claim 1 with a sample to be treated under enzymatic reaction conditions.

9. The method according to claim 8, wherein the sample to be treated comprises a donor of sulfonic acid groups; or adding a sulfonic acid group to the sample to be treated.

10. The method of claim 9, wherein the sulfonic acid group donor is adenosine 3 '-phosphate-5' -phosphosulfate.

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