Application of CYP51 protein of botryosphaeria dothidea in resistance to tebuconazole

Abstract

The application provides application of CYP51 protein of Botryosphaeria dothidea in anti-azoles, and belongs to the field of agricultural biotechnology. The application provides application of a reagent for detecting mutation of CYP51 gene sequence or CYP51 protein in Botryosphaeria dothidea in evaluation of azole resistance of Botryosphaeria dothidea. Whether mutation occurs to the 406~408bp position of the CYP51 gene or the 136th tyrosine of the CYP51 protein can effectively evaluate the azole resistance of Botryosphaeria dothidea. The application provides a model of Botryosphaeria dothidea with high azole resistance and a construction method thereof. The model can be used for research and preparation of drugs for prevention and treatment of Botryosphaeria dothidea. The application provides an identification method for azole-resistant Botryosphaeria dothidea and application of the identification method in development monitoring of azole resistance of Botryosphaeria dothidea. The application has important significance for agricultural disease prevention and control and rational use of pesticides, and helps to improve the sustainability and economic benefits of agricultural production.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to the application of Staphylococcus aureus CYP51 protein in anti-tebuconazole. Background Technology

[0002] Staphylococcus aureus ( Botryosphaeria dothidea *Botrytis cinerea* is a widespread plant pathogen with an extremely broad host range, capable of infecting various fruit trees and often causing a variety of serious diseases, such as soft rot, branch blight, canker, leaf spot, black rot, ring rot, and gummosis. For example, in kiwifruit cultivation, soft rot caused by *Botrytis cinerea* is one of the main diseases affecting fruit quality and taste, and in severe cases, it can cause huge economic losses to the kiwifruit industry. In grape cultivation, *Botrytis cinerea* can cause problems such as canker on grape branches and rotting of fruit, reducing grape yield and quality, and affecting its commercial value. In pomegranate cultivation, *Botrytis cinerea* is also one of the pathogens causing pomegranate dry rot, mainly affecting the thorns and forks of branches older than two years. In the early stage of the disease, the lesions are light brown, round or oval, and saddle-shaped lesions easily appear on the bark of the branches. In the later stage, the diseased parts lose water, become sunken, dry and hardened, and turn grayish-brown or dark brown, seriously affecting the vigor of the pomegranate tree and fruit yield.

[0003] Tebuconazole, a systemic triazole fungicide, is widely used in agricultural production to control various fungal diseases caused by pathogens such as *Botrytis cinerea*, due to its protective, curative, and eradicative effects. Its mechanism of action primarily involves inhibiting the demethylation of sterols within the pathogenic fungus, thereby hindering biofilm formation and exerting its fungicidal activity. In practical applications, tebuconazole has shown good control effects against various diseases such as apple leaf spot, wheat powdery mildew, Fusarium head blight, sheath blight, and rust. However, with the long-term and large-scale use of tebuconazole in agricultural production, the problem of *Botrytis cinerea* resistance to tebuconazole has become increasingly prominent, posing a significant challenge to agricultural production. The development of resistance not only leads to a substantial decrease in the control efficacy of tebuconazole against *Botrytis cinerea*, making the disease difficult to control effectively and increasing the risk of outbreaks, but also prompts farmers to continuously increase the dosage and frequency of application to achieve control effects. This not only increases agricultural production costs but also leads to increased pesticide residues, affecting the quality and safety of agricultural products. Summary of the Invention

[0004] To address the problems existing in the prior art, the first objective of this invention is to provide a reagent for detecting CYP51 gene sequence mutations or CYP51 protein mutations in Staphylococcus aureus and its application in assessing tebuconazole resistance in Staphylococcus aureus.

[0005] A second objective of this invention is to provide a tebuconazole-resistant Staphylococcus aureus model, its construction method, and its application in the development of drugs for the prevention and treatment of Staphylococcus aureus.

[0006] A third objective of this invention is to provide a method for identifying drug-resistant Staphylococcus aureus and its application in monitoring the development of tebuconazole resistance in Staphylococcus aureus.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of a reagent for detecting CYP51 gene sequence mutations in Staphylococcus aureus in assessing tebuconazole resistance in Staphylococcus aureus, wherein the mutation location is 406-408 bp, and the sequence before mutation is TAC or its degenerate sequence.

[0008] This invention provides a reagent for detecting mutations in the CYP51 protein in Staphylococcus aureus and its application in assessing tebuconazole resistance in Staphylococcus aureus, wherein the mutation occurs at position 136, and the original protein is tyrosine.

[0009] Preferably, the protein is mutated from tyrosine to phenylalanine or histidine.

[0010] This invention provides a method for constructing a tebuconazole-resistant Staphylococcus aureus model, the method comprising: performing a point mutation on the 136th tyrosine residue of the CYP51 protein in Staphylococcus aureus.

[0011] Preferably, the CYP51 protein in the *Staphylococcus aureus* is mutated at position 136 to phenylalanine or histidine.

[0012] Preferably, the point mutation method includes drug induction, ultraviolet induction, or PCR-mediated induction.

[0013] This invention provides a tebuconazole-resistant Staphylococcus aureus model constructed using the aforementioned method.

[0014] This invention provides the application of the tebuconazole-resistant Staphylococcus aureus model in the development of drugs for the prevention and treatment of Staphylococcus aureus.

[0015] This invention provides a method for identifying drug-resistant Staphylococcus aureus, comprising the following steps: detecting the 136th amino acid of the Staphylococcus aureus CYP51 protein; when the 136th amino acid is tyrosine, Staphylococcus aureus is sensitive to tebuconazole; when the 136th amino acid is phenylalanine or histidine, Staphylococcus aureus is resistant to tebuconazole.

[0016] This invention provides the application of the identification method in monitoring the development of tebuconazole resistance in Staphylococcus aureus.

[0017] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: This invention is the first to discover that the key to Staphylococcus aureus' resistance to tebuconazole lies in a specific change in the tyrosine residue 136 of its CYP51 protein. Mutations at tyrosine residue 136 of the CYP51 protein in resistant strains alter the structure of the active site of the CYP51 protein, reducing the binding affinity of tebuconazole to the CYP51 protein. This prevents tebuconazole from effectively inhibiting the demethylation process of sterols in Staphylococcus aureus, thus leading to resistance. Therefore, the tyrosine residue 136 of the CYP51 protein can serve as a key indicator for determining whether Staphylococcus aureus possesses resistance to tebuconazole, providing an important molecular target for the identification of resistant strains and the monitoring of resistance development. Attached Figure Description

[0018] Figure 1 PCR amplification results of CYP51 gene fragment.

[0019] Figure 2 : Blast alignment results of CYP51 fragment.

[0020] Figure 3 Results of the second round of Tail-PCR for the 5' and 3' ends of the CYP51 gene.

[0021] Figure 4 Results of the third round of Tail-PCR at the 5' and 3' ends of the CYP51 gene.

[0022] Figure 5 Results of the fourth round of Tail-PCR at the 5' and 3' ends of the CYP51 gene.

[0023] Figure 6 Blast comparison results of the full-length amino acid sequence of Bdcyp51.

[0024] Figure 7 Differences in Bdcyp51 gene expression levels between susceptible strain TS1 and resistant strain TS1-F30.

[0025] Figure 8 PCR gel electrophoresis of Bdcyp51 gene with different point mutations.

[0026] Figure 9 PCR gel electrophoresis of Bdcyp51 mature peptide recombinant plasmid colonies.

[0027] Figure 10 Plate screening of yeast transformants using methanol phenotype. Detailed Implementation

[0028] This invention provides a reagent for detecting CYP51 gene sequence mutations or CYP51 protein mutations in *Staphylococcus aureus* in the assessment of tebuconazole resistance in *Staphylococcus aureus*. By detecting whether a mutation occurs at position 406-408 bp of the CYP51 gene or at tyrosine position 136 of the CYP51 protein, tebuconazole resistance in *Staphylococcus aureus* can be effectively assessed. The sequence before the mutation at position 406-408 bp of the CYP51 gene described in this invention is a TAC or its degenerate sequence, corresponding to tyrosine position 136 of the CYP51 protein. As an optional embodiment, the protein mutation described in this invention involves a mutation at tyrosine position 136 to phenylalanine or histidine.

[0029] As an optional implementation, the reagents of the present invention include allele-specific PCR reagents, real-time PCR probe reagents, high-throughput sequencing reagents, and protein chip reagents.

[0030] This invention provides a method for constructing a tebuconazole-resistant Staphylococcus aureus model, the method comprising: point mutation of tyrosine residue 136 of the CYP51 protein in Staphylococcus aureus. Preferably, the tyrosine residue 136 of the CYP51 protein in Staphylococcus aureus is mutated to phenylalanine or histidine. As an optional embodiment, the point mutation method of this invention is an artificially induced mutation method, including drug induction, ultraviolet induction, or PCR-mediated mutation.

[0031] This invention provides a tebuconazole-resistant Staphylococcus aureus model constructed using the aforementioned method. When the tyrosine residue at position 136 of the CYP51 protein in Staphylococcus aureus is mutated to phenylalanine, its tebuconazole resistance can be increased by 5.69 times, EC50. 50 (95% CL) can reach 0.0614 μg·mL -1 When the tyrosine residue at position 136 of the CYP51 protein in Staphylococcus aureus is mutated to histidine, its tebuconazole resistance can be increased by 8.26 times, EC50. 50 (95% CL) can reach 0.0892 μg·mL -1 .

[0032] This invention provides the application of the tebuconazole-resistant Staphylococcus aureus model in the development of drugs to control Staphylococcus aureus, using the tebuconazole-resistant Staphylococcus aureus model strain to develop or prepare novel pesticides.

[0033] This invention provides a method for identifying resistant *Staphylococcus aureus*, comprising the following steps: detecting the 136th amino acid of the *Staphylococcus aureus* CYP51 protein; when the 136th amino acid is tyrosine, *Staphylococcus aureus* is sensitive to tebuconazole; when the 136th amino acid is phenylalanine or histidine, *Staphylococcus aureus* is resistant to tebuconazole. This invention uses the 136th tyrosine of the CYP51 protein as a key indicator for determining whether *Staphylococcus aureus* is resistant to tebuconazole, providing an important molecular target for the identification of resistant strains and the monitoring of resistance development. It can be used for monitoring the development of tebuconazole resistance in *Staphylococcus aureus*, which is of great significance for agricultural disease control and the rational use of pesticides.

[0034] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0035] Unless otherwise specified, the following embodiments are all conventional methods.

[0036] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0037] Example 1 1. Experimental materials 1.1. Grape spore-forming strains: Grape spore-forming strain TS1 (disclosed in "Fu Li, Qu Jianlu, Wu Haibin, et al. Physiological and biochemical characteristics of UV-TS1-10, a tebuconazole-resistant mutant of apple ring rot fungus [J]. Plant Protection, 2016, 42(06): 51-57.") was selected. The EC50 of tebuconazole was tested. 50 The concentration of tebuconazole was 0.0111 μg / mL (as a sensitive strain). TS1-F30 was a sensitive strain. TS1 was continuously cultured for 30 generations on medium containing 10 μg / mL tebuconazole (with added tebuconazole technical). The EC50 of tebuconazole was measured to be 0.0111 μg / mL. 50 The high-level resistant strain with a concentration of 0.6529 μg / mL and a resistance multiple of 58.82 was selected as the resistant strain.

[0038] 1.2. Strains and plasmids: Competent Escherichia coli ( Escherichia coli Trans1-T1, pEASY-T1, and the carrier were all purchased from TransGen; DEPC, Tris, IPTG, X-gal, Kana, and Amp were purchased from Sigma.

[0039] 1.3 Enzymes and biochemical reagents: Takara Taq and DNA Marker DL2000 were purchased from Takara; Easy Pure Plasmid Miniprep Kit and EasyPure Quick Gel Extraction Kit were purchased from TransGen; Fungal DNA Kit and Fungal RNA Kit were purchased from OMEGA; CTAB, Tris, EDTA, β-mercaptoethanol, PVP-360, NaCl, chloroform, isoamyl alcohol, anhydrous ethanol, isopropanol, NaAC, and HCl were all domestically produced analytical grade reagents.

[0040] 1.4 Instruments: Biometra PCR instrument, BIO-RAD CFX Connect™ real-time PCR instrument, gel imaging system (Bio-Rad), refrigerated centrifuge (Eppendorf centrifuge 5810R), constant voltage and constant current electrophoresis apparatus (Beijing Liuyi Instrument Factory), electric thermostatic water bath (Shanghai Boxun Industrial Co., Ltd.), vertical pressure steam sterilizer (Shanghai Boxun Industrial Co., Ltd.), vortex mixer, full-temperature shaking incubator, UV-Vis spectrophotometer.

[0041] 1.5. Culture medium: LB medium: 10g tryptone, 5g yeast extract, 5g sodium chloride, adjust pH to 7.0-7.7, and bring volume to 1000ml.

[0042] PDA medium: 200g potato, 20g glucose, 15g agar, bring to a final volume of 1000ml.

[0043] PD medium: 200g potato, 20g glucose, bring to a final volume of 1000ml.

[0044] 1.6 Test buffer solution: (1) 0.5M EDTA: 18.61g of EDTA·H2O was added to 80mL of water, stirred vigorously with a magnetic stirrer, and the pH value was adjusted to 8.0 with NaOH (about 2g). The volume was brought to 100mL, dispensed, and autoclaved.

[0045] (2) 5×TBE buffer: Tris 54g, boric acid 27.5g, and add 0.5mol / L EDTA (pH 8.0) 20mL, and make up to 1000mL.

[0046] (3) 1mol / L Tris-HCl: Dissolve 12.1g of Tris base in 80mL of water, adjust the pH to 8.0 with concentrated hydrochloric acid, mix well, and bring the volume to 100mL.

[0047] (4) X-gal stock solution: Dissolve X-gal in dimethylformamide to prepare a solution with a concentration of 20 mg / mL, put it into a glass bottle or polypropylene tube, wrap it with tin foil, and store it at -20℃.

[0048] (5) IPTG (20%, w / v) solution: Dissolve 2g IPTG in 8mL of water and bring the volume to 10mL. Sterilize using a 0.22µm filter. Aliquot into 1mL portions and store at -20℃.

[0049] (6) Amp (50mg / mL) solution: Weigh 1g of Amp and dissolve it in 20mL of deionized water. Filter the solution through a 0.22µm filter to remove bacteria and dispense into smaller containers. Store at -20℃ before use.

[0050] (7) Kana (50 mg / mL) solution: Weigh 1 g of Kana and dissolve it in 20 mL of deionized water. Filter the solution through a 0.22 µm filter to remove bacteria and dispense into smaller containers. Store at -20 °C for use.

[0051] 2. Experimental Methods 2.1 Extraction of Staphylococcus aureus genomic DNA After culturing *S. truncatula* strain TS1 on a PDA at 25°C for 4 days, 5 mm diameter mycelial discs were prepared and inoculated into PD culture medium. After shaking (120 r / min) for 7 days, the culture was filtered, washed twice with distilled water, and then freeze-dried for later use. DNA was extracted from *S. truncatula* hyphae using the EZNA Fungal DNA Mini Kit.

[0052] 2.2 Primer Design and Synthesis Degenerate primers were designed and synthesized based on conserved regions of the CYP51 gene sequences of other published plant pathogenic fungi. DNA fragments of the CYP51 gene from *Staphylococcus aureus* TS1 were amplified by PCR. All primers were synthesized by Shanghai Yingweijieji Trading Co., Ltd., and the primer sequences are as follows: Table 1 Primers for amplifying the CYP51 gene fragment of Staphylococcus aureus

[0053] Note: In the table, Y=C / T, N=A / C / G / T, V=A / C / G, D=A / G / T, H=A / C / T, S=C / G. Y, N, V, D, H, and S are degenerate sites used in primer design. Degenerate sequences designed in this way refer to sequences at these specific positions that are not a single base (A / T / G / C), but a mixture of multiple bases. This is mainly because the amino acid sequence of the protein at this site is conserved while the base sequence is unknown, and it can be used to amplify unknown sequences.

[0054] 2.3 PCR amplification of the CYP51 gene fragment PCR reaction system: A 50 μL reaction mixture consisted of: 32.5 μL ddH2O, 5.0 μL 10× buffer, 4.0 μL MgCl2 (25 mM), 4.0 μL dNTP Mixture (each 2.5 mM), 1.0 μL F Primer (10 µM), 1.0 μL R Primer (10 µM), 2.0 μL DNA, and 0.5 μL TaqE. Amplification was performed on a PCR instrument using the following program: Table 2 PCR reaction procedure

[0055] 2.4 Retrieval and Linking of Target Fragments 50 µL of the entire reaction product was subjected to electrophoresis, and the results are as follows: Figure 1 As shown in the figure, the lanes from left to right are: lane 1 represents the marker, and lanes 2 to 7 are all CYP51 gene fragment amplification products.

[0056] Two retrievable target bands were obtained using JB3 F / R, with fragment sizes of approximately 500 bp and 750 bp, respectively. After fragment recovery, ligation, transformation, and sequencing, a 589 bp fragment was obtained, as shown in SEQ ID No. 7. The fragment blast results are consistent with... Neofusicoccum parvum UCRNP2 has a 71% possible P450 gene homology, and the CYP51 fragment Blast alignment results are as follows. Figure 2 As shown.

[0057] The steps for recovering the target fragment are as follows: (1) Cut off the product from the agarose gel electrophoresis, put it into a clean centrifuge tube, and weigh it. For example, if the gel is 100 mg, it can be regarded as 100 µL, and so on.

[0058] (2) Add 3 times the volume of GSB solution and sol-gel in a 55°C water bath (ensure the gel block is completely melted). After the gel is completely melted, observe the color of the solution (to increase the amount of DNA recovered, 1 volume of isopropanol can be added to the melted gel).

[0059] (3) Add the melted gel to the adsorption column, centrifuge at 1000×g for 1 min after 1 min, and discard the effluent.

[0060] (4) Add 650 µL of solution WB, centrifuge at 1000×g for 1 min after 1 min, and discard the effluent.

[0061] (5) Centrifuge at 1000×g for 1 min and discard the remaining WB.

[0062] (6) Place the adsorption column in a clean centrifuge tube, add 30 µL of deionized water to the center of the column, and let it stand at room temperature for 1 min.

[0063] (7) Centrifuge at 1000×g for 1 min to elute DNA. Use the eluted DNA for ligation transformation.

[0064] The ligation and transformation steps of the target product are as follows: (1) Add the following to a microcentrifuge tube in sequence: 4 µL of the recovered product and 1 µL of pEASY-T3 Cloning Vector. Mix gently and react at room temperature for 5 min. After the reaction is complete, place the centrifuge tube on ice.

[0065] (2) Add 50 µL of TransT1 competent cells to the ligation product, mix gently, and incubate on ice for 30 min.

[0066] (3) Heat shock in a 42 ℃ water bath for 90 s, then immediately place on ice for 2 min.

[0067] (4) Add 600 µL of LB equilibrated to room temperature, incubate at 200 rpm and 37 °C for 1 h.

[0068] (5) Add 4 µL of 500 mM IPTG and 40 µL of 40 mg / mL X-gal to the melted LB and pour the LB plate.

[0069] (6) After incubating the bacterial culture for 1 h, centrifuge and discard the supernatant. Take 200 µL, plate it, and incubate it overnight. After taking it out, observe the blue and white spots and mark them.

[0070] (7) Pick white colonies into 5 mL of LB liquid medium containing Amp antibiotic, shake at 37 ℃ and 250 rpm for 8-10 h, and remove them when the OD value is 0.6-0.8 for PCR colony verification.

[0071] The strains that were verified as positive clones were sent to Shanghai Boshan Biotechnology Co., Ltd. for sequencing.

[0072] 2.5 Full-length cloning of the Staphylococcus aureus CYP51 gene Based on the known sequence of the target gene fragment, specific primers were designed and randomly combined with seven short, random primers with low Tm values ​​for TAIL-PCR to amplify the 5' or 3' flanking sequences. The primers are as follows: Table 3 Primers for TAIL-PCR amplification of the full-length gene of Staphylococcus aureus

[0073] Note: In the table, W = A / T, S = C / G, N = A / C / G / T, and M represents hypoxanthine, which pairs with A / T / C / G.

[0074] Using Staphylococcus aureus TS1 genomic DNA as a template, PCR amplification was performed according to the following system: 50 μL reaction volume consisted of: 28.5 μL ddH2O, 5.0 μL 10× buffer, 4.0 μL MgCl2 (25 mM), 8.0 μL dNTP™ extract (each 2.5 mM), 1.0 μL AD Primer (100 µM), 1.0 μL Specific Primer (10 µM), 2.0 μL DNA, and 0.5 μL TaqE.

[0075] The reaction procedure is carried out in three steps: Step 1: Using genomic DNA as a template, react the target gene-specific primers SEQ ID No. 11 and SEQ ID No. 12 with seven different AD primers, respectively, under the following conditions: Table 4. First-step reaction procedure

[0076] Step 2: Using the PCR product from Step 1 diluted 100 times as a template, react SEQ ID No. 10 and SEQ ID No. 13 with seven different AD primers, respectively, under the following conditions: Table 5. Second step reaction procedure

[0077] The results of the second round of Tail-PCR at the 5' and 3' ends of the CYP51 gene are as follows: Figure 3 As shown in the figure, the lanes from left to right represent the second-round products of SEQ ID No. 10 and AD1-AD7 respectively, the Marker, and the second-round products of SEQ ID No. 13 and AD1-AD7 respectively.

[0078] The third step involves using the PCR product from the second step diluted 100 times as a template, and reacting SEQ ID No. 9 and SEQ ID No. 14 with seven different AD primers under the same conditions as the second step, until a single band of the predicted fragment size is amplified.

[0079] The results of the third round of Tail-PCR at the 5' and 3' ends of the CYP51 gene are as follows: Figure 4As shown in the figure, the lanes from left to right represent the third-round products of SEQ ID No. 9 and AD1-AD7 respectively, the marker, and SEQ ID No. 14 and AD1-AD7 respectively. The product with a single, clear, and bright band is recovered for the next reaction.

[0080] Finally, the target gene fragment obtained by the above PCR was enhanced. Using the PCR product diluted 100-fold in step 3 as a template, primers SEQ ID No. 15 and AD7 were used for reaction, and primers SEQ ID No. 8 and AD2 were used for reaction. Each reaction was repeated 6 times.

[0081] The PCR reaction program was as follows: pre-denaturation at 94℃ for 4 min, followed by the following cycles: denaturation at 94℃ for 1 min, annealing at 55℃ for 45 s, extension at 72℃ for 45 s, for a total of 35 cycles, and a final extension at 72℃ for 10 min. The reaction mixture consisted of: 32.5 μL ddH2O, 5.0 μL 10× buffer, 4.0 μL MgCl2 (25 mM), 4.0 μL dNTPs (each 2.5 mM), 1.0 μL Primer1 (10 µM), 1.0 μL Primer2 (10 µM), 2.0 μL DNA, and 0.5 μL TaqE.

[0082] 50 µL of the entire reaction product was subjected to electrophoresis to confirm the presence of the target band. The results of the fourth round of Tail-PCR for the 5' and 3' ends of the CYP51 gene are as follows. Figure 5 As shown in the figure, the lanes from left to right represent the 5' end of the Bdcyp51 gene with six repeats of primer AD7, the marker, and the 3' end of the Bdcyp51 gene with six repeats of primer AD2. The fragments amplified from the 5' end to AD7 and the 3' end to AD2 were excised from the gel, recovered, ligated, transformed, and sequenced (same as step 2.4), yielding the full-length CYP51 gene of 1607 bp (as shown in SEQ ID No. 23), of which 443 bp-504 bp are intron sequences.

[0083] 2.6 Extraction of total RNA and synthesis of cDNA from grape variegated strains TS1 and TS1-F30 After culturing grape ablation strains (TS1, TS1-F30) on a PDA at 25°C for 4 days, they were made into 5 mm diameter fungal discs (hereinafter referred to as fungal discs), which were then inoculated into PD culture medium and cultured with shaking (120 r / min) for 7 days. After filtration, the discs were washed twice with distilled water and then freeze-dried for later use.

[0084] Total RNA was extracted from grape spore strains TS1 and TS1-F30 using the OMEGA Fungal RNA Kit. 2.5 μL of RNA sample was taken, and the relative absorbance values ​​of A260, A280, and A230 were measured using a UV spectrophotometer. High-purity RNA with an A260 / A280 ratio between 1.7 and 2.0 and an A260 / A230 ratio between 1.8 and 2.2 was selected.

[0085] Reverse transcription kit: High speed-strand cDNA synthesis Plus Kit (KOFITE (Hong Kong) International Trading Co., Ltd. LBQ7513) Prepare the following mixture in an RNase-free centrifuge tube: Table 6 Reverse Transcription Procedure

[0086] The reverse transcription reaction conditions are set as follows: Table 7 Reverse Transcription PCR Reaction Procedure

[0087] 2.7 Amplification of the full-length cDNA gene sequence of Staphylococcus aureus CYP51 Full-length amplification primers were synthesized based on the 5' and 3' end sequences of the obtained Bdcyp51 gene sequence. All primers were synthesized by Shanghai Invitrogen Trading Co., Ltd., and the primer sequences are as follows: Table 8 Primers for full-length amplification of the Staphylococcus aureus CYP51 cDNA gene

[0088] The PCR reaction system was as follows: a 50 μL reaction volume consisted of: 32.5 μL ddH2O, 5.0 μL 10× buffer, 4.0 μL MgCl2 (25 mM), 4.0 μL dNTP Mixture (each 2.5 mM), 1.0 μL SEQ ID No.24 (10 µM), 1.0 μL SEQ ID No.25 (10 µM), 2.0 μL DNA, and 0.5 μL TaqE. Amplification was performed on a PCR instrument using the following program: Table 9. PCR reaction procedure for the full-length cDNA gene of Staphylococcus aureus CYP51

[0089] 50 µL of the entire reaction product was subjected to electrophoresis, and the target band was excised and recovered via gel extraction (same as step 2.4), and then sent for sequencing. The full-length ORF of the gene is 1578 bp (as shown in SEQ ID No. 26), encoding 525 amino acids (as shown in SEQ ID No. 27), and the predicted size of the encoded product is approximately 59 kDa. It was named Bdcyp51.

[0090] The full-length amino acid sequence of Bdcyp51 was compared with BLAST, and the results are as follows: Figure 6 As shown, the results indicate that the CYP51 sequence with high amino acid sequence homology to the Bdcyp51 gene is: Macrophomina phaseolina MS6 (GeneBank No: EKG14983), Neofusicoccum parvum UCRNP2 (GeneBank No:XP_007581984), Paraphaeosphaeria sporulosa (GeneBank No: XP_018030695), sequence alignment was performed using DNAMAN software, and the sequence homology reached 87.81%. The Cypx functional domain, the six conserved domains SRS1~6 specific to the P450 family, and the heme cofactor binding region (-FGxGxHxcCxGxxFA-), the -YxxF / L(i)xxPxFGxxVxF / YD / a- of SRS1 and the -GQ / hHT / sS- of SRS4 are highly conserved and are identification markers for members of the CYP51 family.

[0091] 2.7 qRT-PCR detection of CYP51 gene in TS1 and TS1-F30 RNA was extracted from the sensitive strain TS1 and the resistant strain TS1-F30 and cDNA was reverse transcribed. qRT-PCR specific primers were designed using the known CYP51 gene sequence, and qRT-PCR primers were designed using the known housekeeping genes β-tubulin, ITS, and EF-1 sequences for real-time quantitative PCR amplification.

[0092] Table 10 Primer Design for CYP51 Gene qRT-PCR

[0093] The reaction system is as follows: 50 μL system, 25 μL Power SYBR Green PCR Master Mix, 0.5 μL forward primer, 0.5 μL reverse primer, 5.0 μL cDNA template, and 19.0 μL RNase-free ddH2O.

[0094] The PCR program was as follows: 95℃ for 3 min; 95℃ for 10 s, 55℃ for 20 s, 72℃ for 20 s, 75℃ for 5 s, for a total of 40 cycles; finally, melting curves were prepared at 65–95℃. The results were analyzed using the system software configured for the instrument to obtain the cycle threshold (Ct) for each internal reference gene. 2 –△△CT The method was used to perform relative quantitative analysis of gene expression differences in the samples.

[0095] The first step is to use a reference gene to calibrate the calibration sample and the sample to be tested: △CT (calibration sample) = Genel (Mean CT)1 - Reference gene CT (Mean CT)1 △CT (sample to be tested) = Genel (Mean CT)² - Reference gene CT (Mean CT)² The second step is to normalize the ΔCT of the calibration sample and the sample to be tested: △△CT = △CT(sample to be tested) – △CT(calibration sample) Step 3, expression difference calculation: 2 –△△CT = Normalized expression ratio (relative expression level).

[0096] The results of the differential expression analysis of the Bdcyp51 gene in the susceptible strain TS1 and the resistant strain TS1-F30 are as follows: Figure 7 As shown in the qRT-PCR results, the relative expression level of the Bdcyp51 gene in the resistant strain TS1-F30 did not increase, but instead decreased, with a relative expression level of 0.8568.

[0097] Example 2 To investigate the effect of point mutations on the Bdcyp51 gene (SEQ ID No. 26), artificial point mutations were used to study the anti-tebuconazole molecule in the Bdcyp51 gene.

[0098] The amino acid sequence of BdCYP51 (SEQ ID No. 27) was modified by point mutagenesis, with Y (TAC) at position 136 mutated to F (TTC), Y (TAC) at position 136 mutated to H (CAC), F (TTC) at position 149 mutated to L (CTC), I (ATC) at position 278 mutated to S (AGC), and L (CTC) at position 334 mutated to H (CAC). Primers were designed based on the restriction site of the eukaryotic expression vector pPIC9K, and the primers are as follows: Table 11 Primers for Bdcyp51 gene point mutation and eukaryotic vector construction

[0099] Note: 5'AOX and 3'AOX are the validation primer pairs in the Pichia pastoris expression system.

[0100] Using the full-length cDNA of the Bdcyp51 gene and the PCR transformation products of each mutant gene sequence as templates, PCR amplification was performed using primers CYP51-F / CYP51-R, 136-1f / 136-1r (corresponding to 136Y / F), 136-2f / 136-2r (corresponding to 136Y / H), 149f / 149r (corresponding to 149F / L), 278f / 278r (corresponding to 278I / S), and 334f / 334r (corresponding to 334L / H) (using a point mutation kit, Mut Express II Fast Mutagenesis Kit V2, Nanjing Novozymes). The Bdcyp51 gene expression sequence was obtained, with a product size of approximately 1578 bp. Gel electrophoresis of PCR amplification of different point mutation genes of Bdcyp51 is shown below. Figure 8 As shown in the figure, M: DL-2000; 1: Bdcyp51; 2: 136Y / F; 3: 136Y / H; 4: 149F / L; 5: 278I / S; 6: 334L / H.

[0101] After recovering the target gene fragment (same as step 2.4 in Example 1), it was ligated to the pPIC9K vector via the AOX restriction site (the full-length cDNA fragment of the Bdcyp51 gene was ligated between the AOX sites of the known pPIC9K vector), and transformed into E. coli DH5α. Positive clones were screened by blue-white screening and recombinant colony PCR identification, and after sequencing identification, they were applied to eukaryotic expression.

[0102] The selected positive clones were expanded and cultured, and then plasmids were extracted using a rapid plasmid extraction kit. PCR amplification was performed using the 5'AOX1 primer and the 3'AOX1 primer on the pPIC9K vector.

[0103] PCR reaction system: The 50 μL reaction system consisted of: 32.5 μL ddH2O, 5.0 μL 10× buffer, 4.0 μL MgCl2 (25 mM), 4.0 μL dNTP Mixture (each 2.5 mM), 1.0 μL SEQ ID No.47 (10 µM), 1.0 μL SEQ ID No.48 (10 µM), 2 μL DNA, and 0.5 μL TaqE. Amplification was performed on a PCR instrument using the following program: Table 12 PCR reaction procedure

[0104] Gel electrophoresis shows bands as follows Figure 9 As shown, the amplification products of the characteristic primers 5'AOX1 and 3'AOX1 contain a portion of the pPIC9K vector fragment, which is relatively large (approximately 2.0 kb) and appears later in the sequence; when the empty vector is used as a template, the PCR product size is only 492 bp. These results indicate that the Bdcyp51 gene fragment has been inserted into the pPIC9K vector.

[0105] Screening and identification of BdCYP51 yeast transformants: The recombinant expression plasmid pPIC9K / cyp51 and the empty pPIC9K plasmid were screened and identified using restriction endonucleases. Sac After linearization, GS115 yeast competent cells were transformed using electroporation. The transformed cells were first grown on histidine-deficient basal medium, and colonies appeared after 3-4 days, yielding more than 50 clones. Forty-six single colonies were selected and screened for positive transformants on MD / MM plates. Because the yeast recipient strain GS115 is a histidine auxotroph (His+Mut+), and the pPIC9K vector is a yeast integrative vector containing the histidine dehydrogenase gene (HIS4) but not the yeast replication factor, only recombinants with the pPIC9K vector integrated into the yeast chromosome could grow on histidine-deficient MD basal medium. Yeast transformants were screened using methanol phenotypic plate assays as follows: Figure 10 As shown.

[0106] Interaction assay of Bdcyp51 point mutant expressed protein with tebuconazole: GS-pPIC9K / Bdcyp51 transformants (5 mutants and 1 non-mutant) from validated resistant and susceptible strains were added to 20 ml of YPD medium and incubated at 28°C until OD500. 600 Value 1.1. 10 μl of the solution was added to YPD liquid medium containing different concentrations of tebuconazole (0, 0.125, 0.25, 0.5, 1, 2 μg / ml) and cultured further. With no treatment as a control, the growth of transformants under different concentrations was measured, and the sensitivity of different transformants to tebuconazole was calculated. The experimental results are shown in the table below. Table 13 Results of tebuconazole sensitivity assay for different BdCYP51 point mutant proteins

[0107] The results showed that the GS-pPIC9K / Bdcyp51 unmutated gene expressed the EC protein. 50 The value is: 0.0108 μg·mL -1 The protein EC expressed by GS-pPIC9K / Bdcyp51-136Y / H 50 It is 0.0892 μg·mL -1The highest value was found in the GS-pPIC9K / Bdcyp51-136Y / F protein EC expression. 50 It is 0.0614 μg·mL -1 EC at the remaining four mutation sites 50 The values ​​also changed, but not significantly compared to the original gene. This indicates that the Y mutation at position 136 has the greatest impact on the tebuconazole resistance of the Bdcyp51 gene.

[0108] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a reagent for detecting CYP51 gene sequence mutations in Staphylococcus aureus in assessing tebuconazole resistance in Staphylococcus aureus, characterized in that, The mutation location of the gene sequence is 406~408bp, and the sequence before the mutation is TAC or its degenerate sequence.

2. The application of a reagent for detecting CYP51 protein mutations in Staphylococcus aureus in assessing tebuconazole resistance in Staphylococcus aureus, characterized in that, The protein mutation occurred at position 136, where the original residue was tyrosine.

3. The application according to claim 2, characterized in that, The protein was mutated from tyrosine to phenylalanine or histidine.

4. A method for constructing a tebuconazole-resistant Staphylococcus aureus model, characterized in that, The method includes: performing a point mutation on the 136th tyrosine residue of the CYP51 protein in Staphylococcus aureus.

5. The construction method according to claim 4, characterized in that, The CYP51 protein in the *Staphylococcus aureus* was mutated at position 136 to either phenylalanine or histidine.

6. The construction method according to claim 4, characterized in that, The point mutation methods include drug induction, ultraviolet induction, or PCR-mediated induction.

7. The tebuconazole-resistant Staphylococcus aureus model constructed by the construction method described in claims 4 to 6.

8. The application of the tebuconazole-resistant Staphylococcus aureus model described in claim 7 in the development of drugs for the prevention and treatment of Staphylococcus aureus.

9. A method for identifying drug-resistant Staphylococcus aureus, characterized in that, The procedure includes the following steps: detecting the 136th amino acid of the Staphylococcus aureus CYP51 protein; when the 136th amino acid is tyrosine, Staphylococcus aureus is sensitive to tebuconazole; when the 136th amino acid is phenylalanine or histidine, Staphylococcus aureus is resistant to tebuconazole.

10. The application of the identification method according to claim 9 in monitoring the development of tebuconazole resistance in Staphylococcus aureus.

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