A prothioconazole methyltransferase gene proS38 and its application

Abstract

This invention belongs to the field of environmental biotechnology, specifically, it relates to a prothioconazole methyltransferase gene. proS38 And its applications. This invention, through analysis, screening, identification, and verification, discovered... Sphingomonas Prothiozoxystrobin methyltransferase in sp. AJ-1 strain proS38 The gene and its protein expression product, prothioconazole methyltransferase, have been shown to have a highly efficient and specific degradation catalytic ability for the widely used pesticide prothioconazole, laying the foundation for the biodegradation of prothioconazole.

Description

Technical Field

[0001] This invention belongs to the field of environmental biotechnology, specifically, it relates to a prothioconazole methyltransferase gene. proS38 And its applications. Background Technology

[0002] Prothioconazole is a novel triazole fungicide developed in 2004. It has one chiral carbon atom and consists of a pair of enantiomers. Prothioconazole is mainly used to control numerous diseases in cereal crops such as wheat, barley, rapeseed, peanuts, rice, and legumes. It has good control effects on almost all cereal diseases, such as powdery mildew, sheath blight, wilt, leaf spot, rust, sclerotinia rot, net blight, and clouding. It can also control soil-borne diseases of rapeseed and peanuts, such as sclerotinia rot, as well as major foliar diseases, such as gray mold, black spot, brown spot, black shank, sclerotinia rot, and rust. However, the widespread use of prothioconazole poses a potential threat to the ecological environment and human health. The European Food Safety Authority (EFSA) has confirmed the potential acute risks of prothioconazole and established maximum residue limits (MRLs) of 0.02-0.1 mg / kg for crops such as corn, sugar beets, potatoes, and peanuts. Canada has set the MRL for prothioconazole in sunflowers at 0.2 mg / kg. Studies by Zhai et al. found that the EC50s of prothioconazole against Daphnia magna, Chlorella vulgaris, and lemurs were 2.68, 9.33, and 1.85 mg / L, respectively. The 96-h LD50 of prothioconazole in zebrafish embryos was 1.70 mg / L; concentrations exceeding 0.43 mg / L led to developmental toxicity and cardiovascular dysplasia in zebrafish. Junwei Huang et al. published (Science of the Total Environment 851(2022) 158220, DOI: 10.1016 / j.scitotenv.2022.158220) a microorganism, Sphingomonas AJ-1, which degrades prothioconazole. Sphingomonas sp. This bacterium (AJ-1) reduces the toxicity of prothioconazole by methylating the C=S bond of the prothioconazole to generate methylated prothioconazole. However, to further improve enzymatic methods and obtain strains with stronger degradation capabilities, it is necessary to conduct further research and exploration on the methylation-related genes and enzymes involved in the degradation of prothioconazole by these strains. Summary of the Invention

[0003] To address the related problems in the prior art, this invention, through further in-depth research, discovered [the virus] from Sphingomonas AJ-1 (… Sphingomonas sp. The prothioconazole methyltransferase gene in AJ-1 proS38The ability of it to degrade prothioconazole was verified, laying the foundation for obtaining stronger prothioconazole through enzymatic degradation or mutation screening, thus completing this invention.

[0004] The technical solution of the present invention is as follows:

[0005] In one aspect, this invention discloses a prothioconazole methyltransferase gene. proS38 The gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0006] In one aspect, the present invention discloses a prothioconazole methyltransferase ProS38. ， Its amino acid sequence is shown in SEQ ID NO.2.

[0007] In one aspect, the present invention discloses an expression vector containing the prothioconazole methyltransferase gene. proS38 Gene. The expression vector can be a prokaryotic expression vector or a eukaryotic expression vector; preferably, the expression vector is a prokaryotic expression vector.

[0008] In one aspect, the present invention discloses a bacterial strain containing an expression vector containing a prothioconazole methyltransferase gene. proS38 Gene.

[0009] In one embodiment, the strain is Escherichia coli.

[0010] In one aspect, the present invention discloses a solution containing prothioconazole methyltransferase. proS38 Application of strains with gene expression vectors in the degradation of prothioconazole.

[0011] In one aspect, the present invention discloses the application of prothioconazole methyltransferase ProS38 in the degradation of prothioconazole.

[0012] In one aspect, the present invention discloses an improvement Sphingomonas A method for enhancing the ability of sp. AJ-1 to degrade prothioconazole, the method comprising: Sphingomonas sp. AJ-1 proS38 Gene expression levels.

[0013] In one aspect, the present invention also discloses a formulation for degrading prothioconazole, said formulation containing a prothioconazole methyltransferase gene. proS38 The strain containing the gene expression vector; or the preparation containing prothioconazole methyltransferase ProS38.

[0014] In one aspect, the present invention also discloses an immobilized enzyme preparation, wherein the immobilized enzyme is prothioconazole methyltransferase ProS38. Beneficial effects

[0015] This invention, through bioinformatics analysis and in vitro screening and identification, has discovered prothioconazole methyltransferase. proS38 The gene and its protein expression product, prothioconazole methyltransferase, were investigated, demonstrating its highly efficient and specific degradation catalytic ability for prothioconazole, laying the foundation for the development of corresponding biodegradation reagents or highly efficient degrading bacteria. Attached Figure Description

[0016] Figure 1 The interaction site and reaction equation for the degradation of prothioconazole (PTC) by strain AJ-1 are shown in the figure. As can be seen from the figure, strain AJ-1 breaks the double bond between the sulfur atom and the triazole ring in prothioconazole, and adds a methyl group to the sulfur atom. Corresponding to the structural formula of prothioconazole, it can be seen that the degradation process of prothioconazole in strain AJ-1 is a methylation process, and the product is methylated prothioconazole (Prothioconazole-S-methyl, PTM).

[0017] Figure 2 Methyltransferase gene proS38 The expression and functional verification strategy diagram in BL21(pET-32a(+)).

[0018] Figure 3 Methyltransferase gene proS38 PCR detection, where A represents PCR amplification. proS38 As a result, B is used to construct pET- proS38 Expression plasmid, transformed into E. coli After being placed in BL21 (DE3) competent cells, the positive clones with correct sequencing were amplified by PCR for detection.

[0019] Figure 4 : E. coil BL21 (DE3) (pET- proS38 Liquid phase diagram of PTC degradation.

[0020] Figure 5 SDS-PAGE gel image of the fusion protein His-ProS38.

[0021] Figure 6 ProS38 pairs ( Rac )- / ( S )- / ( R Degradation of )-PTC.

[0022] Figure 7 ProS38 degradation ( S )- / ( R Enzyme kinetics of )-PTC.

[0023] Figure 8 Liquid phase diagram of PTC degradation by ProS38.

[0024] Figure 9 Mass spectrum of ProS38 degradation products of PTC, where A is the product of ProS38 degradation ( Rac A is the HPLC chromatogram of PTC; B is the ion chromatogram; C is the primary mass spectrum; D is the secondary mass spectrum. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments and experimental examples are commercially available. Unless otherwise stated, all reagents used in this invention are analytical grade reagents. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention.

[0026] Example 1: Prothioconazole methyltransferase proS38 Gene cloning and expression vector construction 1.1 Bioinformatics analysis to determine the methyltransferase gene sequence

[0027] extract Sphingomonas DNA of sp. AJ-1 was extracted and sequenced to obtain its genomic data, which was then analyzed using Uedit32 software. Sphingomonas Using sp. AJ-1 genome sequencing data, gene sequences annotated as methyltransferases were identified. Then, the ORF Finder tool (https: / / www.ncbi.nlm.nih.gov / orffinder / ) was used to perform open reading frame (ORF) analysis on the upstream and downstream sequences to identify potential protein-coding regions. Finally, these sequences were submitted to NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) for alignment analysis to further verify their function and homology, and to identify the corresponding methyltransferase genes.

[0028] 1.2 Prothioconazole methyltransferase gene proS38 Cloning

[0029] With strain AJ-1 ( SphingomonasUsing sp. AJ-1 genomic DNA as a template, the methyltransferase gene (prothioconazole methyltransferase gene) was amplified by PCR using a 2 × phanta®Max Master Mix kit. proS38 The nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. A 50 μL system was used according to the kit instructions. The components are as follows (Table 1):

[0030] Table 1 PCR amplification system

[0031]

[0032] The primers are as follows:

[0033] proS38 -32a-F: gccatggctgatatcggatccGACGATGCTGGCGAGAATCT (SEQ ID NO.3)

[0034] proS38 -32a-R: gtggtggtggtggtgctcgagAACGCTGGGCTGACCAG (SEQ ID NO.4)

[0035] The amplification procedure is as follows (Table 2):

[0036] Table 2 PCR amplification program

[0037]

[0038] After amplification, the amplification products were detected by gel electrophoresis to confirm whether the target band was a single target band. Subsequently, the agarose gel containing the target band was cut and purified by gel recovery using the FastPure® Gel DNAExtraction Mini Kit (Nanjing Novizan Biotechnology Co., Ltd.) according to the instructions.

[0039] (1) Add Buffer GDP, which is approximately the same volume as the cut gel, and heat in a 55°C water bath for about 10 minutes. To ensure complete gel dissolution, invert and mix 2-3 times during the water bath to accelerate the sol.

[0040] (2) Briefly separate the centrifuge tube to collect the solution on the tube wall, place the FastPure DNA Mini Columns-G adsorption column in a Collection Tube 2 mL collection tube, transfer all the sol solution to the adsorption column, and centrifuge at 12000 rpm for 1 min;

[0041] (3) Discard the filtrate and place the adsorption column in the collection tube. Add 300 μL of Buffer GDP to the adsorption column, let stand for 1 min, and then centrifuge at 12000 rpm for 1 min;

[0042] (4) Discard the filtrate and place the adsorption column in the collection tube. Add 700 μL of Buffer GW mixed with anhydrous ethanol to the adsorption column, let stand for 1 min, and then centrifuge at 12000 rpm for 1 min. To ensure that the salt can be completely removed and to eliminate its influence on subsequent experiments, repeat this operation once.

[0043] (5) Discard the filtrate, place the adsorption column back into the collection tube, and centrifuge at 12,000 rpm for 2 min. Open the lid and place it in a fume hood to allow the residual ethanol to evaporate completely;

[0044] (6) Elution of DNA: Transfer the adsorption column to a new 1.5 mL centrifuge tube, add 30 μL of sterile ddH2O to the center of the adsorption column membrane, let stand at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, and discard the adsorption column.

[0045] (7) The recovered PCR product fragments were detected by agarose gel electrophoresis, and the concentration and purity were detected by NanoDrop One. The product was stored at -20 °C.

[0046] 1.3 Methyltransferase proS38 Construction of exogenous expression vectors

[0047] Methyltransferase gene constructed using homologous recombination. proS38 The exogenous recombinant expression vector, the specific steps are as follows:

[0048] (1) Vector linearization: The pET32a(+) expression vector (preserved in our laboratory) was double-digested with QuickCut™ BamHI and QuickCut™ Xho I (Takara). The digestion was carried out in a water bath at 37 ℃ for 1 h. The digestion system is shown in Table 3.

[0049] Table 3. Double enzyme digestion system of pET-32a (+) expression vector

[0050]

[0051] (2) Enzyme digestion gel recovery and purification: After enzyme digestion, the digestion products were detected using an electrophoresis system and a gel imaging system. The target plasmid fragment was excised and purified using FastPure gel electrophoresis. ®The DNA was recovered and purified using the Gel DNA Extraction Mini Kit. Subsequently, the recovered pET32a (+) expression vector digestion fragments were analyzed by agarose gel electrophoresis, and the concentration and purity were determined using NanoDrop One.

[0052] (3) Homologous recombination: ClonExpress was used. ® The II One Step Cloning Kit is used to process the recovered pET32a(+) expression vector fragments and... proS38 Homologous recombination was performed on the gene PCR amplification fragments, and the reaction system is shown in Table 4. After the system was prepared, it was reacted in a water bath at 37 ℃ for 30 min.

[0053] Table 4 proS38 Homologous recombination reaction system of exogenous expression vector

[0054]

[0055] (4) Transformation:

[0056] 1) The recombinant product was taken from -80 °C E. coli BL21 (DE3) competent cells were placed on ice;

[0057] 2) After the competent cells have thawed (about 6 min), add 10 μL of the recombinant product to the competent cells and gently tap the bottom of the tube to mix. Place the tube on ice and let it stand for 30 min.

[0058] 3) Heat stimulate in a 42 ℃ water bath for 45 s, then quickly return to ice and place in ice for 2 min;

[0059] 4) Add 700 μL of antibiotic-free LB medium and mix well;

[0060] 5) After shaking culture in a constant temperature shaker at 37 ℃ for 1 h (220 rpm), centrifuge at 5000 rpm for 1 min, discard 500 μL of supernatant, resuspend the bacterial cells, and take 100 μL and spread evenly on LB agar plates containing Amp.

[0061] 6) Invert the container and incubate overnight at 37 ℃ for 12 h.

[0062] (5) Validation of positive clones

[0063] 1) After the colonies have grown on the transformation plate, pick 3 single clones and place them in 4 mL of AmpLB medium containing 100 mg / L. Place the test tubes in a constant temperature shaker at 37 ℃ and shake for 5 h (220 rpm).

[0064] 2) Take 200 μL of bacterial culture for sequencing verification, and temporarily store the remaining bacterial culture at 4 ℃.

[0065] 3) After the sequencing results are correctly matched, resuspend the preserved bacterial solution, take 700 μL of the bacterial solution and add an equal volume of 35% sterile glycerol, and store it in a -80 ℃ freezer.

[0066] Example 2: Containing prothioconazole methyltransferase gene proS38 Functional verification of recombinant strains

[0067] The correctly sequenced recombinant expression strain was inoculated into 20 mL of LB liquid medium, and Amp was added to a final concentration of 50 mg / L, along with 40 μM ( ) Rac PTC and 0.5 mM IPTG were incubated at 37 ℃ and 220 rpm for 12 h in a shaker. 600 μL of the culture medium was taken and an equal volume of methanol was added. The mixture was thoroughly mixed by inversion and centrifuged at 12000 rpm for 2 min. The supernatant was filtered through a 0.22 μm nylon filter membrane, and the concentration of PTC in the culture medium was detected by high performance liquid chromatography (HPLC).

[0068] The high-performance liquid chromatography (HPLC) method for detecting PTC was as follows: the chromatographic column was an Agilent Eclipse XDB C18 (4.6 mm × 250 mm; 5 μm); the mobile phase was acetonitrile: 0.1% formic acid aqueous solution = 73:27 (v / v); the detection wavelength was 193 nm; the injection volume was 20 μL; the flow rate was 1 mL / min; and the column temperature was 30℃.

[0069] ( Rac )- / ( S )- / ( R HPLC detection conditions for chiral separation of PTC and its metabolites: chromatographic column: CHIRALCEL ® OD-RH (4.6 mm × 150 mm; 5 μm); mobile phase: acetonitrile: 0.1% formic acid aqueous solution = 60:40 (v / v); detection wavelength: 200 nm; injection volume: 20 μL; flow rate: 0.6 mL / min; column temperature: 25 ℃.

[0070] HPLC detection results are as follows Figure 4 As shown, E. coli BL21 (pET- proS38 The PTC levels in the culture medium decreased, accompanied by the formation of new substance peaks. E. coli BL21 (pET- proS38 The peak height of the characteristic PTC peak (RT=5.1 min) in the culture medium decreased by 69%, and a new product peak appeared at 9.3 min, indicating that... E. coli BL21 (pET- proS38 It can degrade PTC and generate products. Therefore, it has been preliminarily determined that... proS38 This is the functional gene in strain AJ-1 that catalyzes PTC methylation.

[0071] Example 3: Inducible expression, purification, and degradation ability test of prothioconazole methyltransferase Pro38

[0072] 3.1 Obtaining the methyltransferase ProS38 protein

[0073] Five mL of the activated recombinant bacterial culture was inoculated into 500 mL of LB medium containing Amp at a ratio of 1:100, and cultured at 37 °C and 220 rpm until the bacterial culture reached OD. 600 When the value reaches 0.5-0.8, [the following is a direct translation of the original text:] to E. coli BL21 (pET- proS38 IPTG was added to the bacterial culture to a final concentration of 0.5 mM and incubated in a shaker at 16 ℃ for 12 h. The bacterial pellet was then collected by centrifugation at 12000 rpm for 15 min at 4 ℃. The cells were washed twice with 50 mM Tris-HCl (pH 7.4) buffer, and finally resuspended in 35 mL of the same buffer. After cell disruption by sonication, the supernatant was collected by centrifugation at 8000 rpm for 30 min, which was the crude ProS38 enzyme solution. The crude enzyme solution was purified using a Ni-TED 6FF pre-packed gravity column in a chromatography cabinet at 4 ℃ to obtain the methyltransferase ProS38 protein.

[0074] See results Figure 5 SDS-PAGE results showed that the purified ProS38 enzyme was eluted in 500 mM imidazole buffer with a single band, and the ProS38 value was approximately 50 kDa, consistent with the theoretical value (49.59 kDa).

[0075] 3.2 Degradation by methyltransferase proS38 ( Rac )- / ( S )- / ( R Comparison of PTC capabilities

[0076] Add 20 μM (5 ng / μL ProS38) to 500 μL of enzyme reaction solution. Rac )-PTC、( S )-PTC、( RThe reaction was carried out in a 30 °C water bath with 1.5 mM SAM and 1-PTC. Samples were taken every 5 min until 25 min, and an equal volume of acetonitrile was added to terminate the enzyme reaction.

[0077] The enzyme reaction solution used to terminate the reaction was vortexed, centrifuged at 12,000 rpm for 2 min, and filtered through a 0.22 μm organic phase filter. The concentrations of substrate and product were determined by HPLC. All treatments were performed in triplicate.

[0078] The results showed that 5 μg / mL of ProS38 purified enzyme had an effect on ( ) within 22 min. S The degradation rate of )-PTC was 96.99%, while that of ( Rac The degradation rate of )-PTC was 71.78%, which is beneficial to ( S The degradation rate of PTC was 73.81%. Figure 6 In the middle A), the EF value increased from 0.49s to 0.97 within 160 min. Figure 6 (B), indicating that ProS38 selectively and preferentially degrades ( S )-PTC, which is consistent with the selectivity of strain AJ-1 in degrading PTC enantiomers.

[0079] 3.3 Determination of Enzymatic Parameters of ProS38

[0080] Will E. coli BL21 (pET- proS38 The recombinant strains were inoculated into 500 mL LB liquid medium containing 100 mg / L Amp, and 0.5 mM IPTG was added. After induction culture at 16 °C and 180 rpm for 12 h, the protein was purified.

[0081] Add 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, and 280 μM of ( ) to a 500 μL Tris-HCl enzymatic reaction system containing 5 μg / mL ProS38. S )-PTC was reacted in a 30 ℃ water bath for 10 min, and at concentrations of 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360 and 380 μM. R The PTC-PTC mixture was reacted in a 30 °C water bath for 30 min. An equal volume of acetonitrile was added to the reaction mixture, and the mixture was thoroughly mixed by inverting to terminate the enzymatic reaction. The mixture was centrifuged at 12000 rpm for 2 min, and the supernatant was filtered through a 0.22 μm oil-based filter membrane. The concentrations of PTC and the product PTM were determined by HPLC.

[0082] The substrate concentration and reaction rate in the reaction were nonlinearly fitted using GraphPad Prism software (GraphPad Software Inc., San Diego, CA) to obtain... K m , V max and k cat Values. All processes are set up with three parallel groups.

[0083] Enzyme kinetic analysis showed that ProS38 has the effect of ( S )-PTC catalytic efficiency ( k cat / K m ) is 0.05 min -1 μM -1 ( Figure 7 (A), for () R -PTC k cat / K m 0.01 min -1 μM -1 ( Figure 7 (B), namely ProS38 pair ( S The catalytic efficiency of )-PTC is ( R )-PTC is 5 times that of PTC.

[0084] 3.4 Substrate profile of prothioconazole methyltransferase ProS38

[0085] In a 500 μL Tris-HCl enzymatic reaction system (containing 50 μg / mL ProS38 pure enzyme and 1 mM SAM), 10 mg / L of triazole fungicides such as propiconazole, difenoconazole, tebuconazole, tricyclazole, triadimefon, and triadimefon were added respectively, and the reaction was carried out at 30 °C for 48 h. The degradation of each substrate was detected by HPLC.

[0086] HPLC detection method for propiconazole: mobile phase: methanol:water = 65:35 (v / v); detection wavelength: 230 nm; injection volume: 20 μL; flow rate: 1 mL / min; column temperature: 40 ℃.

[0087] HPLC detection method for difenoconazole: mobile phase: acetonitrile:methanol:0.1% phosphoric acid solution = 50:20:30 (v / v); detection wavelength: 236 nm; injection volume: 10 μL; flow rate: 1 mL / min; column temperature: 35 ℃.

[0088] HPLC detection method for tebuconazole: mobile phase: methanol:water = 80:20 (v / v); detection wavelength: 223 nm; injection volume: 10 μL; flow rate: 0.8 mL / min; column temperature: 30 ℃.

[0089] HPLC detection method for tricyclazole: mobile phase: methanol:water = 45:55 (v / v); detection wavelength: 226 nm; injection volume: 20 μL; flow rate: 1.0 mL / min; column temperature: 30 ℃.

[0090] HPLC detection method for triazolol and triazolone: ​​mobile phase: acetonitrile: 0.1% formic acid aqueous solution = 85:15 (v / v); detection wavelength: 220 nm; injection volume: 5 μL; flow rate: 0.8 mL / min; column temperature: 25 ℃.

[0091] As shown in Table 5, among the substrates degraded by ProS38, except for PTC, ProS38 could not degrade triazole fungicides such as propiconazole, difenoconazole, tebuconazole, tricyclazole, triadimefon, and triadimefon. Based on the structural formulas of each substance, this indicates that ProS38 is a methyltransferase with selective action site on the substrate, and its action atom is a sulfur atom.

[0092] Table 5. Substrate spectrum of ProS38

[0093]

[0094] Note: "+" indicates that the material has degradation ability; "-" indicates that it does not have degradation ability.

[0095] 3.5 Study on the metabolic pathway of PTC degradation by ProS38

[0096] After terminating the reaction with acetonitrile, the ProS38 enzyme reaction solution was vortexed, centrifuged at 12,000 rpm for 2 min, filtered through a 0.22 μm organic phase filter, and the product was detected by HPLC and UPLC-Q-TOF-MS.

[0097] Results: The HPLC chromatogram shows that ProS38 preferentially degrades ( S )-PTC ( Figure 8 ). For ProS38 catalysis ( Rac The products generated by )-PTC were identified by mass spectrometry, and the results are as follows: Figure 9 As shown, an ion current peak appears at 23.46 min ( Figure 9 (A), corresponding to the first-order mass spectrum ( Figure 9 The molecular ion peak was detected in (B) m / z =358.0541 (M+CH2+H) +This corresponds to the relative molecular weight of PTM. Then, further secondary mass spectrometry analysis is performed. Figure 9 (C), and a fragment peak was found. m / z =(M+H) + =125.0154, which is consistent with the molecular weight of methylated prothioconazole after the loss of one o-chlorobenzyl group. This is consistent with the degradation of strain AJ-1 ( Rac The results are consistent with the identification of the metabolites of )-PTC. These results indicate that ProS38 catalyzes ( Rac PTM is generated by PTC.

[0098] The above description, in conjunction with specific embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention, and all such deductions or substitutions should be considered to fall within the scope of protection defined by the claims submitted herein.

Claims

1. A prothioconazole methyltransferase gene proS38 Genes, characterized by, The proS38 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A prothioconazole methyltransferase ProS38 ， It is characterized by having an amino acid sequence as shown in SEQ ID NO.

2.

3. An expression carrier, characterized in that, The expression vector contains the prothioconazole methyltransferase according to claim 1. proS38 Gene.

4. The expression vector according to claim 3, characterized in that, The expression vector is a prokaryotic expression vector.

5. A strain, characterized in that, The strain contains an expression vector containing the prothioconazole methyltransferase as described in claim 1. proS38 Gene.

6. Contains prothioconazole methyltransferase proS38 The application of strains with gene expression vectors in the degradation of prothioconazole is characterized by, The proS38 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

7. The application according to claim 6, characterized in that, The strain was Escherichia coli. E. coli BL21.

8. The application of prothioconazole methyltransferase ProS38 in the degradation of prothioconazole, characterized in that, The amino acid sequence of prothioconazole methyltransferase ProS38 is shown in SEQ ID NO.

2.

9. A formulation for degrading prothioconazole, characterized in that, The formulation contains prothioconazole methyltransferase. proS38 The strain containing the gene expression vector; or the preparation containing prothioconazole methyltransferase PoS38; wherein, the proS38 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the amino acid sequence of the prothioconazole methyltransferase PoS38 is shown in SEQ ID NO.

2.

10. An immobilized enzyme preparation, characterized in that, The immobilized enzyme in the immobilized enzyme preparation is prothioconazole methyltransferase ProS38, whose amino acid sequence is shown in SEQ ID NO.2.

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