Polynucleotide molecule for regulating resistance of sphaeropsis fuliginea to hymexazol and application thereof
By identifying key transcription factors SsAOD2 and SsAOD5 in Sclerotinia sclerotiorum, overexpression transformants were constructed to regulate Ssaox expression, thus solving the problem of Sclerotinia sclerotiorum resistance to pyraclostrobin and significantly enhancing the strain's sensitivity to the agent.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- TIANJIN AGRICULTURE COLLEGE
- Filing Date
- 2026-03-11
- Publication Date
- 2026-06-12
Smart Images
Figure CN122189031A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of genetic engineering technology, and more specifically to a polynucleotide molecule that regulates the resistance of Sclerotinia sclerotiorum to pyraclostrobin and its application. Background Technology
[0002] Sclerotinia sclerotiorum is an important plant pathogenic fungus that can cause sclerotinia rot in various crops. Pyraclostrobin is a novel fungicide that shows promising application prospects in controlling Sclerotinia sclerotiorum. However, with continued use of the fungicide, the pathogen may develop resistance.
[0003] Studies have shown that the resistance of Sclerotinia sclerotiorum to pyraclostrobin is related to the activation of its alternative oxidase (AOX) pathway, thereby reducing the toxicity of the drug.
[0004] Previous studies have cloned the alternative oxidase gene Ssaox from Sclerotinia sclerotiorum and confirmed that it plays a key role in drug resistance.
[0005] Ssaox gene expression may be influenced by upstream regulatory factors. Therefore, providing a polynucleotide molecule that regulates Sclerotinia sclerotiorum resistance to pyraclostrobin and its applications is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0006] In view of this, the present invention provides a polynucleotide molecule that regulates the resistance of *Sclerotinia sclerotiorum* to pyraclostrobin and its application. The present invention identifies and verifies key transcription factors mediating pyraclostrobin resistance in *Sclerotinia sclerotiorum*, elucidates the molecular mechanism by which these factors regulate Ssaox expression, thereby revealing a resistance signaling pathway and providing a theoretical basis and core molecular target for developing new resistance diagnosis and control technologies targeting this pathway. Simultaneously, by utilizing the polynucleotide molecules SsAOD2 and SsAOD5 that regulate *Sclerotinia sclerotiorum* resistance to pyraclostrobin, the present invention aims to develop a new targeted disease control method that can effectively reduce existing resistance in *Sclerotinia sclerotiorum*, overcoming the limitations of existing resistance control methods that only address the symptoms (relying solely on rotating medications). By directly intervening in the upstream regulatory hub of resistance development, the present invention provides a novel and clearly targeted technical solution for controlling non-targeted resistance mediated by AOX.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] A polynucleotide molecule that regulates the resistance of Sclerotinia sclerotiorum to pyraclostrobin, wherein the polynucleotide molecule is selected from any of the following: (a) A nucleotide encoding the amino acid sequence shown in SEQ ID NO: 2; (b) Nucleotides encoding the amino acid sequence shown in SEQ ID NO: 4; (c) The nucleotide sequence shown in SEQ ID NO: 1; (d) The nucleotide sequence shown in SEQ ID NO: 3.
[0009] The present invention also provides a Sclerotinia sclerotiorum transformant, wherein the transformant integrates the above-mentioned polynucleotide molecules into its genome, and the above-mentioned polynucleotide molecules are overexpressed in the transformant, thereby significantly enhancing the resistance level of pyraclostrobin.
[0010] The present invention also provides a method for reducing or inhibiting the resistance of Sclerotinia sclerotiorum to pyraclostrobin, comprising downregulating or inhibiting the expression of the above-mentioned polynucleotide molecules in Sclerotinia sclerotiorum or the activity of the proteins they encode.
[0011] Preferred method: Downregulation or suppression is achieved through any of the following means: (a) Applying RNA interference molecules to Sclerotinia sclerotiorum to specifically silence the above-mentioned polynucleotide molecules; (b) Applying a small molecule inhibitor to Sclerotinia sclerotiorum that specifically binds to and inhibits the activity of the protein shown in SEQ ID NO: 2 or SEQ ID NO: 4.
[0012] Preferred: Used to enhance the control effect of pyraclostrobin.
[0013] The present invention also provides the application of the above-mentioned polynucleotide molecule or the *Sclerotinia sclerotiorum* obtained by the above method in the study of agents that inhibit the resistance of *Sclerotinia sclerotiorum* to pyraclostrobin.
[0014] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a polynucleotide molecule for regulating the resistance of Sclerotinia sclerotiorum to pyraclostrobin and its application, and the technical effects achieved are as follows: This invention clarifies the positive regulatory role of transcription factors SsAOD2 and SsAOD5 in Ssaoxeroides resistance to pyraclostrobin. By constructing overexpression transformants and combining them with a dual-luciferase reporter system, it was confirmed that the transcriptional levels of SsAOD2 and SsAOD5 were significantly positively correlated with Ssaox expression and strain resistance levels. Knockout of SsAOD2 and SsAOD5 downregulated Ssaox expression and increased sensitivity to pyraclostrobin; overexpression of SsAOD2 and SsAOD5 upregulated Ssaox expression and decreased sensitivity to pyraclostrobin. This provides a theoretical basis for developing novel control strategies. Attached Figure Description
[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0016] Appendix Figure 1 This is a schematic diagram illustrating the cis-acting element of the Ssaox promoter provided by the present invention.
[0017] Appendix Figure 2 This is a phylogenetic tree construction diagram of SsAOD2 and SsAOD5 provided by the present invention.
[0018] Appendix Figure 3 The predicted structural domain diagrams of the SsAOD2 and SsAOD5 proteins of Sclerotium sclerotiorum provided by this invention.
[0019] Appendix Figure 4 Subcellular localization maps of SsAOD2 and SsAOD5 provided by this invention.
[0020] Appendix Figure 5 The relative expression levels of Ssaox, SsAOD2, and SsAOD5 in the pyraclostrobin-sensitive parent strain and resistant mutant provided by this invention are shown in the figure.
[0021] Appendix Figure 6 The relative expression levels of Ssaox, SsAOD2, and SsAOD5 in the pyraclostrobin-sensitive parent strain and the Ssaox knockout mutant provided by this invention are shown in the figure.
[0022] Appendix Figure 7 The diagram shows the relative expression levels of Ssaox, SsAOD2, and SsAOD5 in the sensitive parental strain and the Ssaox overexpression mutant provided by this invention.
[0023] Appendix Figure 8 The Ssaox knockout transformants and overexpression transformants provided by this invention are compared with the original strain in EC50. 50 Value comparison chart.
[0024] Appendix Figure 9The diagrams provided by this invention illustrate dual-luciferase activity analysis, where: Left: Dual-luciferase activity of the susceptible parent strain SBH1 of *Sclerotinia sclerotiorum*; Right: Dual-luciferase activity of the highly resistant mutant RH1-3 of *Sclerotinia sclerotiorum*; SK is an empty vector, a plasmid without the target transcription factor gene, serving as a negative control; SsAOD2+promoter+luc: Experimental plasmid containing the SsAOD2 transcription factor gene and the target promoter Ssaox linked to the firefly luciferase; SsAOD5+promoter+luc: Experimental plasmid containing the SsAOD5 transcription factor gene and the target promoter Ssaox linked to the firefly luciferase; SsAOD2+SsAOD5+promoter+luc: Experimental plasmid containing both the SsAOD2 and SsAOD5 transcription factor genes and the target promoter Ssaox linked to the firefly luciferase.
[0025] Appendix Figure 10 The image shows the identification of the recombinant plasmid pAOXpro-AbAi provided by this invention.
[0026] Appendix Figure 11 This is a screening diagram of the self-activation concentration of the pAOXpro-AbAi yeast bait carrier provided by the present invention.
[0027] Appendix Figure 12 This is a point-to-point verification diagram of Ssaox yeast interaction provided by the present invention.
[0028] Appendix Figure 13 The relative expression levels of Ssaox, SsAOD2, and SsAOD5 in the sensitive parent strain of Sclerotium sclerotiorum and the SsAOD2 knockout mutant provided by this invention are shown in the figure.
[0029] Appendix Figure 14 The relative expression levels of Ssaox, SsAOD2, and SsAOD5 in the sensitive parent strain of Sclerotium sclerotiorum and the SsAOD5 knockout mutant provided by this invention are shown in the figure.
[0030] Appendix Figure 15 The diagram shows the relative expression levels of Ssaox, SsAOD2, and SsAOD5 in the sensitive parent strain of Sclerotium sclerotiorum and the SsAOD2 overexpression mutant provided by this invention.
[0031] Appendix Figure 16 The diagram shows the relative expression levels of Ssaox, SsAOD2, and SsAOD5 in the sensitive parent strain of Sclerotium sclerotiorum and the SsAOD5 overexpression mutant provided by this invention.
[0032] Appendix Figure 17 EC2 of the SsAOD2 and SsAOD5 knockout and overexpression transformants provided by this invention with the original strain 50value. Detailed Implementation
[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0034] This invention discloses a polynucleotide molecule that regulates the resistance of Sclerotinia sclerotiorum to pyraclostrobin and its application.
[0035] Unless otherwise specified, the experimental methods used in these examples are conventional methods, such as double enzyme digestion. When the plasmid concentration is 100 ng / μL, the double enzyme digestion reaction system (Takara) is prepared as shown in Table 1 below:
[0036] The prepared reaction solution was placed at 37 °C and reacted for 2 h, then placed on ice at 4 °C.
[0037] Ligation and recombination were performed according to the instructions of the ClonExpress® II One Step Cloning Kit (Vazyme). When the plasmid vector concentration was 100 ng / μL and the target gene concentration was 40 ng / μL, the reaction system was as shown in Table 2 below:
[0038] The prepared reaction solution was placed at 37 °C and allowed to react for 30 min. Then it was immediately placed on ice at 4 °C.
[0039] Real-time quantitative PCR Dilute the cDNA 10-fold and prepare a 20 μL reaction mixture (Takara) in an 8-tube RNase-Free Microtube (see Table 3).
[0040] The prepared reaction solution was placed in a Thermo Fisher Scientific QuantStudio™ 1 real-time PCR detection system under the following reaction conditions: 95℃ pre-denaturation for 30 sec, 40 cycles: 95℃ denaturation for 5 sec, 57℃ annealing for 30 sec and 72℃ extension for 15 sec; a melting curve was plotted: for every 0.5℃ increase from 55℃ to 95℃, the reaction time was 10 sec.
[0041] Amplification procedures and systems: The reaction solution (Vazyme) was prepared on ice, and the system is shown in Table 4 below:
[0042] Place the prepared reaction solution on a Bio-rad C1000 thermal cycle PCR instrument, and amplification conditions are shown in Table 5 below:
[0043] Unless otherwise specified, all experimental materials used in the examples were purchased from commercial sources. Primers used in the examples are listed in Tables 6 and 7.
[0044]
[0045] Example 1 Promoter cloning and cis-acting element analysis of the bypass oxidase gene Ssaox Using specific primers AOX-F / AOX-R, the genomic DNA of the susceptible parent strain SBH1 of Sclerotinia sclerotiorum (strain nomenclature refers to Mitochondrial AOX Activation Drives Pyribencarb Resistance in Sclerotiniasclerotiorum: Cellular Trade-Offs and Nontarget Site Mechanisms, Journal of Agricultural and Food Chemistry, 2025) was used as a template to clone the promoter sequence 2.0 kb upstream of the Ssaox gene (GenBank: CP017817.1).
[0046] Predictive analysis of the cis-acting elements of promoters (PlantCARE website (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ).
[0047] Results: Cis-acting element prediction revealed that the Ssaox promoter contains 12 core cis-acting elements (TATA-box), 43 enhancing elements (CAAT-box), multiple hormone regulatory elements (two abscisic acid response elements (ABRE, ACGTG) and five methyl jasmonate response elements (CGTCA)), and multiple protein-binding elements. It also contained a typical bypass oxidase inducible motif (AIM: cgggccacacgtcccgg), providing a structural basis for transcription factor recognition. Figure 1 ).
[0048] Example 2 Cloning, subcellular localization and gene expression analysis of transcription factors AOD2 / AOD5 in Sclerotium sclerotiorum
[0049] Results: Phylogenetic analysis showed that SsAOD2 / SsAOD5 is most closely related to homologous proteins in Sclerotinia trifoliorum. Figure 2 ) Protein structure analysis: Conserved domain analysis (using online SMART software) confirmed that both SsAOD2 and SsAOD5 proteins contain typical zinc cluster transcription factor DNA-binding domains. Figure 3 The GLA4 domain and the signal-sensing PAS domain are structurally highly similar to homologous proteins in model fungi, revealing that their functions are highly conserved. Figure 3 ).
[0050] Subcellular localization analysis and expression regulation association of transcription factors SsAOD2 / SsAOD5: Specific primers were designed to amplify SsAOD2 and SsAOD5 (Y-SsAOD2F / R; Y-SsAOD5F / R). The purified and recovered full-length coding sequences (CDS) of the SsAOD2 and SsAOD5 gene regions were then ligated to the pCAMBIA1302-GFP (Qingke Biotechnology) vector backbone, which had been digested with SacI and KpnI and purified.
[0051] SsAOD2 and SsAOD5 were constructed into the pCAMBIA1302-GFP vector, respectively. The subcellular localization vectors pCAMBIA1302-SsAOD2-GFP and pCAMBIA1302-SsAOD5-GFP were transiently transformed into *Nicotiana benthamiana* leaves using a transient transformation method. The distribution of the SsAOD2 and SsAOD5 fusion GFP expressed proteins was detected using fluorescence microscopy and photographed.
[0052] Results: SsAOD2 and SsAOD5 are located in the nucleus and cytoplasm, providing the cellular basis for their transcriptional regulatory functions. Figure 4 ).
[0053] Expression association analysis: Using the susceptible parent strain SBH1 of *Sclerotinia sclerotiorum* as the control strain and the β-tubulin gene as the internal reference gene, the expression association of the highly resistant mutant RH1-3 of pyraclostrobin was determined (strain nomenclature follows *Mitochondrial AOX ActivationDrives Pyribencarb Resistance in *Sclerotinia sclerotiorum*: Cellular Trade-Offs and Nontarget Sites). The transcriptional levels of Ssaox, SsAOD2, and SsAOD5 in Ssaox gene knockout transformants (ΔSsAox-R3-7 and ΔSsAox-R3-12, obtained from RH1-3 using conventional PEG-mediated protoplast transformation) and two overexpression transformants (OESsaox-S1-1 and OESsaox-S1-2, obtained from SBH1 using conventional Agrobacterium-mediated transformation) were analyzed to verify the role of transcription factors SsAOD2 and SsAOD5 in regulating Ssaox expression.
[0054] Results: The transcriptional levels of the three genes Ssaox, SsAOD2, and SsAOD5 under different genetic backgrounds were compared using a qPCR system (primers: AoxRTF / AoxRTR; SsAOD2RTF / SsAOD2RTR; SsAOD5RTF / SsAOD5RTR). The results showed that ( Figure 5 , 6 7. Note: Different lowercase letters indicate significant differences between treatments (P<0.05); the error bar is the standard error (±SD) of three biological replicates. The expression of the three genes showed a highly synergistic trend and was closely related to the strain resistance level: ① In the pyraclostrobin highly resistant mutant (RH1-3), the transcriptional levels of all three genes were significantly upregulated compared to SBH1 (P<0.05); ② In the Ssaox gene knockout mutants (ΔSsAox-R3-7 and ΔSsAox-R3-12), the transcriptional levels of all three genes were significantly downregulated compared to RH1-3 (P<0.05); ③ In the Ssaox overexpression mutants (OESsaox-S1-1 and OESsaox-S1-2), the transcriptional levels of all three genes were significantly upregulated compared to SBH1 (P<0.05). The sensitivity results of Ssaox knockout and overexpression mutants to pyraclostrobin are as follows: Figure 8 As shown: Compared with the original strain RH1-3, the two knockout transformants ΔSsaox-R3-7 and ΔSsaox-R3-12 exhibited significantly reduced resistance to pyraclostrobin, decreasing by 99.87% and 99.85%, respectively. EC50 50 The values were 0.0067 μg / mL and 0.0077 μg / mL, respectively; compared with the original strain SBH1, the two overexpression transformants OESsaox-S1-1 and OESsaox-S1-2 showed significantly enhanced resistance to pyraclostrobin, with resistance increasing by 194.04 and 77.87 times, respectively, EC50. 50 The values were 4.6182 μg / mL and 1.8532 μg / mL, respectively.
[0055] In summary, the transcriptional levels of SsAOD2 and SsAOD5 were significantly positively correlated with the expression of Ssaox and the strain's resistance level, providing important expression profile evidence that the three are in the same regulatory pathway and may have a synergistic relationship.
[0056] Example 3 Verification of the binding of transcription factors SsAOD2 / SsAOD5 to the Ssaox promoter Dual luciferase reporter (DLR) assay: To determine binding activity, specific primers (AOXpro-F / AOXpro-R) with homologous arms were designed based on the known sequence of the 2000 bp region upstream of the start codon of the Ssaox gene. The primers contained recognition sites for the restriction endonucleases EcoRV (recognition sequence GATATC) and SpeI (recognition sequence ACTAGT). Using genomic DNA from the susceptible parent strain SBH1 of *Sclerotinia sclerotiorum* as a template, PCR amplification was performed using high-fidelity DNA polymerase (2×Phanta Flash Master Mix (Dye Plus), Novizan). The PCR products were separated and verified by 1% agarose gel electrophoresis, confirming the amplification of the expected 2000 bp fragment. Subsequently, the target band was excised and the PCR product was purified using a gel extraction kit. The pGreen II 0800-LUC vector (Beijing Cooler Master Technology Co., Ltd.) was double-digested with EcoRV and SpeI, respectively. The purified PCR product fragment was ligated into the pGreen II 0800-LUC vector as a reporter; the 35S promoter drove the Renilla reniformis (REN) reporter gene in the same vector as an internal control. The full-length coding sequences of the SsAOD2 and SsAOD5 genes were obtained from the NCBI database, with sequences identical to those in Example 2. Specific primers (S-SsAOD2-F, S-SsAOD2-R; S-SsAOD5-F, S-SsAOD5-R) were designed for the two genes, containing the restriction endonucleases SacI (recognition sequence GAGCTC) and HindIII (recognition sequence AAGCTT) at the recognition sites.
[0057] Total RNA (Takara) was extracted from the susceptible parental strain SBH1 of *Sclerotinia sclerotiorum*. cDNA was synthesized using a two-step method following the instructions of the Takara Bio PrimeScript™ FAST RT reagent Kit with gDNA Eraser. RT-PCR amplification was performed using the cDNA as a template to obtain CDS fragments of SsAOD2 and SsAOD5. The PCR products were purified after verification by agarose gel electrophoresis. The pGreen II 62-SK vector (Beijing Coollab Technology Co., Ltd.) was double-digested with SacI and HindIII. The purified CDS fragments were ligated into the digested pGreen II 62-SK vector and transformed into *E. coli* DH5α competent cells for screening. Positive clones were selected for initial colony PCR screening, and plasmid DNA was extracted. This plasmid served as the effector plasmid. The pGreen II 62-SK empty vector served as a negative control. All sequence-verified reporter gene plasmids and effector gene plasmids were used for subsequent transient expression experiments. The recombinant plasmid was transformed into Agrobacterium strain GV3101 and used for tobacco leaf transformation. Two days later, the values of firefly luciferase LUC and REN were detected using a dual-luciferase reporter gene assay kit (Yisheng Biotechnology). The ratio of LUC to REN was calculated to reflect the transcriptional regulatory activity of SsAOD2 / SsAOD5 on Ssaox.
[0058] Results: When the effector carried SsAOD2 or SsAOD5 from SBH1, the Ssaox promoter activity was increased to 2-fold and 5-fold compared to the control, respectively; when both were co-expressed, the activity was further increased to approximately 5.5-fold (P<0.01), showing a synergistic enhancement effect. When the effector carried SsAOD2 or SsAOD5 from RH1-3, both single and co-expression significantly activated the reporter gene (1.5-fold, 2-fold, and 3-fold compared to the control, respectively). Figure 9 Note: Different lowercase letters indicate significant differences between treatments (P<0.05); the error bar is the standard error (±SD) of three biological replicates. This result confirms that SsAOD2 / SsAOD5 has the ability to co-activate transcription of the Ssaox promoter.
[0059] Yeast one-hybrid and transcriptional activation analysis: a. Construction and identification of the bait carrier pAOXpro-AbAi Based on previous promoter and transcription factor analyses, the -2000bp-0bp region of the Ssaox promoter was selected for yeast one-hybrid experiments. Using the Ssaox promoter sequence and the genomic DNA of the susceptible parent strain SBH1 (a *Sclerotinia sclerotiorum*) as a template, sequence-specific primers (J-AOXpro-F, J-AOXpro-R) with the restriction endonucleases KpnI and XhoI were designed. The Ssaox promoter fragment was amplified by high-fidelity polymerase chain reaction (PCR). The vector pAbAi was double-digested with KpnI and XhoI. The purified Ssaox promoter fragment was ligated into the digested pAbAi vector. The ligation product was transformed into competent *E. coli* DH5α cells to obtain the recombinant plasmid pAOXpro-AbAi. Linearization of the yeast one-hybrid pAbAi vector often uses the BtsbI restriction enzyme because its recognition site is downstream of the AbA resistance gene, enabling efficient genome integration. The recombinant plasmid was linearized using BtsbI restriction enzyme, and the linearized plasmid was transformed into Y1HGold yeast competent cells via LiAc / PEG method. The integration site was amplified using Matchmaker Insert Check PCR Mix 1, which contains the universal pAbAi primers AbAi-F (5′-TACGATTAGTATCCGGTAAG-3′) and AbAi-R (5′-TTCTTCCCTCGTCGTTCAGC-3′). The expected positive band size should be the sum of the inserted Ssaox promoter fragment (2000 bp) and the 466 bp flanking sequence upstream of the AbA resistance gene on the pAbAi vector, i.e., 2466 bp. Figure 10 By verifying the band size of the PCR products using agarose gel electrophoresis, Y1HGold-pAOXpro-AbAi reporter strains that successfully integrate the Ssaox promoter can be screened. These reporter strains can then be used in subsequent yeast one-hybrid experiments to screen transcription factors that interact with the Ssaox promoter region or to verify specific DNA-protein interactions using candidate transcription factor plasmids.
[0060] b. AbA background concentration screening The obtained recombinant plasmid pAOXpro-AbAi was linearized by enzyme digestion and then transformed into Y1HGold yeast competent cells (Beijing Cooler Master Technology Co., Ltd.) to obtain a positive bait strain. The resuspended bacterial solution was spread on solid medium containing different concentrations of SD / -Ura, and the AbA background concentration was screened by observing the yeast growth. The AbA concentrations were set to 0 ng / mL, 25 ng / mL, 50 ng / mL, and 75 ng / mL, respectively.
[0061] The results showed that in OD 600=0.002, which is just enough to prevent growth on SD / -Ura with AbA (25 ng / mL) plates. Therefore, the minimum concentration of AbA to inhibit yeast growth is 25 ng / mL. Figure 11 P53-AbAi was the negative control, and P53-AbAi+pGADT7-p53 was the positive control (both provided by Beijing Coollab Technology Co., Ltd.). Subsequent experiments used an AbA concentration of 25 ng / mL to fully inhibit yeast growth.
[0062] c. Yeast mono-hetero-interaction screening The cDNA of the susceptible parent strain SBH1 of *Sclerotinia sclerotiorum* was used as a template. EcoRI and XhoI restriction endonuclease recognition sites were introduced into the amplification primers (J-SsAOD2-F / J-SsAOD2-R; J-SsAOD5-F / J-SsAOD5-R). The amplified CDS fragments of SsAOD2 and SsAOD5 were ligated into the yeast one-hybrid prey vector pGADT7 (Beijing Coollab Technology Co., Ltd.) after double digestion with the same enzymes (EcoRI and XhoI). The pGADT7 vector contains the GAL4 activation domain (AD), therefore, the successful ligation product will produce a transcription factor protein fused to express AD. The ligation product was transformed into *E. coli* DH5α competent cells for amplification and screening, ultimately obtaining positive recombinant plasmids SsAOD2-AD and SsAOD5-AD. After extensive replication in *E. coli*, these plasmids were extracted and purified for subsequent yeast transformation experiments. Recombinant prey vector plasmids SsAOD2-AD, SsAOD5-AD, and empty vector pGADT7-AD (Beijing Coollab Technology Co., Ltd.) (negative control) were transformed into bait vector pAOXpro-AbAi yeast competent cells and plated on SD / -Leu / AbA 25 ng / mL solid medium. The results showed that the empty vector pGADT7-AD could not grow normally in SD / -Leu / AbA 25 medium, while transcription factors SsAOD2-AD and SsAOD5-AD grew normally in SD / -Leu / AbA 25 medium. To further verify the interaction, positive yeast competent cells were serially diluted 10-fold. After 10-fold, 100-fold, 1000-fold, and 10000-fold dilutions, the OD values were measured. 600 The values were 0.2, 0.02, 0.002, and 0.0002, respectively. These dilutions correspond to 10 times the original sample was diluted. - ¹、10 - ²、10 - ³ and 10 -4 gradient.
[0063] See, Figure 12The final results showed that the SsAOD2 and SsAOD5 transcription factors interact with the Ssaox gene promoter, and this interaction can activate the expression of the reporter gene Ssaox.
[0064] Example 4 Construction of SsAOD2 / SsAOD5 single gene knockout and overexpression mutant vector Construction of SsAOD2 / SsAOD5 single-gene knockout vectors: Using DNA from the susceptible parent strain SBH1 of *Sclerotinia sclerotiorum* as a template, the upstream and downstream fragments of the SsAOD2 gene were amplified using specific primers (SsAOD2up-F / SsAOD2up-R and SsAOD2 down-F / SsAOD2 down-R), each 1000 bp in length. The upstream and downstream SsAOD2 vectors pUHPE1-4-SsAOD2 and pUHTE1-3-SAOD2 were constructed, replacing the SsAOD2 gene with the hygromycin resistance gene HPTII. Finally, using these two plasmids as templates, linear DNA fragments SsAOD2-5'-HY and YG-SsAOD2-3' for transformation were amplified using primer pairs M13YF / HYR and YGF / M13YR. These fragments were purified by gel electrophoresis and used for protoplast transformation. The upstream and downstream fragments of the SsAOD5 gene were amplified using specific primers (SsAOD5 up-F / SsAOD5up-R and SsAOD5 DF / (SsAOD5 DR:)). The remaining methods were the same as above. Linear DNA fragments SsAOD5-5'-HY and YG-SsAOD5-3' were amplified for transformation. These fragments were purified by gel electrophoresis and used for protoplast transformation. Protoplast preparation: *Sclerotinia sclerotiorum* was inoculated onto PDA medium lined with cellophane and cultured at 23 °C for 36 h. The hyphae were finely cut with a sterile blade and scraped into 100 mL of PDB. The culture was carried out at 28 °C and 180 rpm for 24 h until spherical hyphae appeared. The hyphae were collected by filtering through a sterile funnel lined with three layers of lens paper, washed twice with sterile water, and twice with 0.7 M NaCl solution. The hyphae were slightly air-dried, transferred to 10 mL centrifuge tubes, weighed, and 2 mL of protoplast preparation solution was added. The culture was carried out at 28 °C for 24 h. Incubate at 120 rpm for 1-2 h at ℃, stopping when sufficient protoplasts have formed. Collect protoplasts by filtration through six layers of lens paper and wash with an appropriate amount of 0.7 M NaCl solution. Collect the filtrate and centrifuge at 6000 rpm for 10 min at 4℃, carefully discarding the supernatant. Resuspend the precipitate in an appropriate amount of STC, centrifuge again at 6000 rpm for 10 min at 4℃, carefully discarding the supernatant and retaining 1 mL of STC suspension to control the protoplast concentration at 1×10⁻⁶. 7Approximately [number] samples are prepared for subsequent experiments. Protoplast transformation process: Take 500 μL of protoplasts, add 5 μg each of upstream and downstream fragments, mix well, and incubate on ice for 40 min. Add protoplast transformation buffer solution TB, mix gently, and incubate at room temperature in the dark for 30 min. Take 500 μL of the protoplast mixture and add it to 50 mL of RM medium (melted and cooled to 60 ℃). Shake to mix, pour into 9 cm diameter culture dishes and air dry (repeat three times). Incubate at 23 ℃ upright in the dark for 1–2 days. When a large number of protoplasts have germinated, cover each dish with RM medium containing 50 μg / mL hygromycin, and continue incubating at 23 ℃ upright in the dark. Approximately 7 days later, hyphae emerged from the double-layer RM medium. Single hyphae were picked and transferred to PDA containing 25 μg / mL. After hyphae growth, single hyphae were continued to be picked and transferred to hygromycin PDA plates. After 8 generations of hygromycin selection, the SsAOD2 and SsAOD5 genes were replaced by the hygromycin resistance gene HPTII after homologous recombination. DNA was extracted from the transformants for PCR verification. The corresponding primers UF-2 / PR and UF-5 / PR amplified the upstream fragments of the SsAOD2 and SsAOD5 genes (L+PtrpC), TF / DR-2 and TF / DR-5 amplified the downstream fragments of the SsAOD2 and SsAOD5 genes (R+TtrpC), YGF / HYR amplified the hygromycin resistance gene fragment HPTII, OESsAOD2F / OESsAOD2R amplified the target gene fragment (SsAOD2), and OESsAOD5F / OESsAOD5R amplified the target gene fragment (SsAOD2). The target gene fragment (SsAOD5) was amplified. RNA was then extracted for reverse transcription and qRT-PCR to detect the expression level of the target gene. Two knockout transformants, ΔSsAOD2 and ΔSsAOD5, were ultimately obtained.
[0065] Overexpression vector construction: Using the cNDA of the susceptible parent strain SBH1 of *Sclerotinia sclerotiorum* as a template, the target fragment OESsAOD2 was obtained by gene amplification using specific primers (OE-SsAOD2-F / OE-SsAOD2-R). The pCETNS-4 plasmid vector was digested with restriction endonucleases SmaI and SacI, and then the target gene fragment was ligated to the double-digested vector plasmid using a seamless cloning kit (Novizan) to construct the overexpression vector pCETNS-4-OESsAOD2. The target fragment OESsAOD5 was obtained by amplification using specific primers (OE-SsAOD5-F / OE-SsAOD5-R), and the overexpression vector pCETNS-4-OESsAOD5 was constructed using the same method. The pCETNS-4-OESsAOD2 and pCETNS-4-OESsAOD5 vector plasmids were ligated and transformed into competent E. coli cells. Single colonies were picked, and bacterial culture PCR was performed using primers OESsAOD2F / OESsAOD2R and OESsAOD5F / OESsAOD5R. Fragments with the correct band size were selected and sent to Qingke Biotechnology for sequencing. Plasmids were extracted from the bacterial cultures with correct sequencing results and transformed into Agrobacterium EHA105 competent cells. The cells were incubated upside down at 28 ℃ for 2 days. Single colonies were picked, and bacterial culture was performed using primers OESsAOD2F / OESsAOD2R and OESsAOD5F / OESsAOD5R to obtain overexpression vectors for transforming Sclerotinia sclerotiorum. After transformation into SBH1, two stable overexpression transformants, OESsAOD2 and OESsAOD5, were obtained after 10 generations of continuous screening on PDAs containing G418 antibiotic.
[0066] Results: Using the susceptible parental strain SBH1 of *Sclerotinia sclerotiorum* as a control and the β-tubulin gene as an internal reference, the expression levels of Ssaox, SsAOD2, and SsAOD5 in the SsAOD2 / SsAOD5 gene knockout transformants ΔSsAOD2 and ΔSsAOD5 and the two overexpression transformants OESsAOD2 and OESsAOD5, as well as their sensitivity to pyraclostrobin, were determined. The results showed ( Figure 13 , 14 15, 16, Note: Different letters indicate significant differences between treatments (P<0.05); the error bar is the standard error (±SD) of 3 biological replicates: ① In ΔSsAOD2 and ΔSsAOD5, Ssaox expression was significantly downregulated compared to SBH1 (P<0.05, Figure 13 , 14 EC of ΔSsAOD2 and ΔSsAOD5 50 The values were 0.0109 μg / mL and 0.0083 μg / mL, respectively, which were significantly lower than those of SBH1 (0.0375 μg / mL). Figure 17① The knockout mutant showed increased sensitivity to the drug; ② In OESsAOD2 and OESsAOD5, Ssaox expression was significantly upregulated compared to SBH1 (P<0.05). Figure 14 , 15 Resistance increased by 10.18 and 9.61 times respectively, EC 50 The values were 0.3817 μg / mL and 0.3230 μg / mL, respectively. Figure 17 This indicates that SsAOD2 / SsAOD5 positively regulates Ssaox expression and participates in the formation of resistance in Sclerotinia sclerotiorum to pyraclostrobin.
[0067] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.
[0068] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A polynucleotide molecule that regulates the resistance of Sclerotinia sclerotiorum to pyraclostrobin, characterized in that, The polynucleotide molecule is selected from any of the following: (a) A nucleotide encoding the amino acid sequence shown in SEQ ID NO: 2; (b) Nucleotides encoding the amino acid sequence shown in SEQ ID NO: 4; (c) The nucleotide sequence shown in SEQ ID NO: 1; (d) The nucleotide sequence shown in SEQ ID NO:
3.
2. A Sclerotinia sclerotiorum transformant, characterized in that, The transformant integrates the polynucleotide molecule of claim 1 into its genome, and the polynucleotide molecule is overexpressed in the transformant to significantly enhance the resistance level of pyraclostrobin.
3. A method for reducing or inhibiting the resistance of Sclerotinia sclerotiorum to pyraclostrobin, characterized in that, This includes downregulating or inhibiting the expression of the polynucleotide molecule of claim 1 in Sclerotium sclerotiorum or the activity of its encoded protein.
4. The method as described in claim 3, characterized in that, The downregulation or suppression is achieved in any of the following ways: (a) Applying an RNA interference molecule capable of specifically silencing the polynucleotide molecule of claim 1 to *Sclerotinia sclerotiorum*; (b) Applying a small molecule inhibitor to Sclerotinia sclerotiorum that specifically binds to and inhibits the activity of the protein shown in SEQ ID NO: 2 or SEQ ID NO:
4.
5. The method as described in claim 4, characterized in that, Used to enhance the control effect of pyraclostrobin.
6. The use of the polynucleotide molecule of claim 1 or the *Sclerotinia sclerotiorum* obtained by the method of claims 3-5 in the study of agents that inhibit *Sclerotinia sclerotiorum* resistance to pyraclostrobin.