Application of afdapa gene in screening drugs for preventing and treating aspergillus flavus

Abstract

The application discloses afdapa The application of a gene in screening and preventing and treating aspergillus flavus belongs to the field of agricultural biotechnology. In view of the technical difficulties of the existing prevention and treatment lacking early anti-adversity targets for the niche expansion (such as the infection of emerging host kiwifruit to cause fruit rot disease) of aspergillus flavus, the application first confirms that a DapA synthase gene afdapa and its coded protein are core mechanisms for regulating the survival and pathogenic colonization of aspergillus flavus under extreme environments (high temperature, strong alkali and high osmosis). Based on the core target, the application establishes a standardized in-vitro enzymatic reaction drug screening model, and provides afdapa a gene deletion mutant ( delta afdapa ) for constructing a drug efficacy evaluation model. The application fundamentally weakens the environmental adaptability of the pathogenic bacteria, and provides a brand-new underlying target and industrialization screening platform for developing a safe and highly specific new type of anti-aspergillus flavus green drug.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to the L-2,3-dihydropyridinedicarboxylic acid (DHD) synthase gene. afdapa As a target for the prevention and control of aflatoxin and its novel applications in screening drugs for the prevention and control of aflatoxin. Background Technology

[0002] Aspergillus flavus ( Aspergillus flavus Aspergillus flavus is a widespread and important global pathogenic fungus that traditionally infects major crops such as corn and peanuts. However, in recent years, with changes in climate and ecological environment, the host niche of Aspergillus flavus has been continuously expanding. The inventors' team's previous research first discovered that Aspergillus flavus can cross its original host boundaries, infecting kiwifruit and causing severe fruit rot (Plant Disease 2022, 106(7): 1990). Diseases caused by Aspergillus flavus not only lead to serious economic losses, but some strains also produce highly carcinogenic aflatoxins. Aspergillus flavus has extremely strong environmental adaptability, and its spores exhibit amazing tolerance to extreme temperatures, acidity, alkalinity, or hypertonic environments, making traditional control methods ineffective in dealing with this ever-expanding threat of new diseases.

[0003] Currently, the control of Aspergillus flavus and the agricultural or postharvest diseases it causes mainly relies on chemical fungicides. Long-term use of chemical pesticides not only leads to increased drug resistance in pathogens, but also results in pesticide residues that fail to meet consumers' demands for green and safe food. Therefore, developing safe, efficient, and highly specific novel biological control targets and drug screening targets by addressing the key survival genes of the pathogens themselves has become an urgent need for the sustainable development of the industry.

[0004] Dihydropyridine dicarboxylic acid synthase (DapA) is a key enzyme in bacteria, catalyzing the production of 2,6-pyridine dicarboxylic acid (DPA) and lysine. DPA is known to be a core component in maintaining the stress resistance of bacterial spores. However, fungi primarily rely on the 2-aminoadipic acid (AAA) pathway to synthesize lysine, and the specific function of DapA homologs in fungi has long been controversial. Although the sequence information of this enzyme has been publicly disclosed, there are currently no research reports on whether DapA synthase in plant pathogenic fungi (especially Aspergillus flavus) participates in the integrated process regulating their resistance to environmental stress and pathogenicity to specific hosts (such as typical kiwifruit). Furthermore, no technology has been publicly disclosed for using it as a target for screening drugs to control Aspergillus flavus and related diseases.

[0005] Current research on molecular targets of Aspergillus flavus mainly focuses on genes related to aflatoxin synthesis (such as...). aflR , aflSThere is insufficient attention paid to the molecular mechanisms by which fungi adapt to the host surface and complex external environment (such as temperature and osmotic pressure fluctuations) in the early stages of infection, particularly regarding late-stage secreted proteins (such as Aspergillus flavus DapA synthase gene). Currently, no existing technology has been developed to synthesize this gene. afdapa This gene serves as a core target for screening drugs to control aflatoxin. However, the industry lacks standardized, high-throughput drug evaluation models based on this early stress-resistance gene or its mutant strains. There is an urgent need to fill this technological gap in order to fundamentally weaken the environmental adaptability of aflatoxin and develop efficient biocontrol strategies. Summary of the Invention

[0006] To address the threat of novel diseases arising from the expansion of Aspergillus flavus's host niche (such as postharvest rot in kiwifruit), and the lack of existing control techniques targeting early environmental adaptations of the pathogen, this invention aims to provide the Aspergillus flavus DapA synthase gene (…). afdapa This invention aims to reveal novel applications of [a substance] in screening drugs for the prevention and treatment of aflatoxin. afdapa To investigate the core functions of genes in the stress resistance and pathogenicity of Aspergillus flavus, a novel method for screening anti-Aspergillus flavus drugs and an evaluation model for mutant strains will be established, providing a foundation for the development of highly specific and environmentally friendly antifungal agents. Technical solution

[0007] The primary technical solution of this invention is to provide Aspergillus flavus ( Aspergillus flavus DapA synthase gene afdapa Or the application of its encoded proteins as targets in screening drugs for the prevention and treatment of aflatoxin.

[0008] The afdapa The nucleotide sequence of the gene is shown in SEQ ID NO: 1; the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2.

[0009] ATGGGCTCCCACGTCACCCCGCAACCCCACGTCCCCAAAGCCGGGGTATGGTGTCCCGCCATTACATTCTTCGACCACAGCACCGACACAATCGACTTCGACGCACAAAAGAAATACTACTCCTACCTCTCCAAGACCGGACTAGCGGGCCTAGTCATCCTCGGCACAAACTCCGAGGCCTTCCTCCTCACCCGCGAAGAGCGTGCCCAATGTATCGCTGCCGCTCGCGAAGCCGTCGGTCCCGATTTCCCGCTCATGGCCGGTGTGGGTGCCCACTCCACAAAGCAGGTGCTTGAGTTAGCGCATGATGCTGCGGGTGCGGGTGCGAATTACTTGCTTGTTCTCCCACCGGCGTATTTCGGAAAGGCGACGACGCCGGCCGTTGTGAAGAAATTCTTTGCGGATGTGGCGCGTCAGTCGCCGTTGCCGGTTGTTGTGTACAACTTCCCTGGGGTGTGTAATGGGGTTGATCTTGATTCGGAGACTATTACGGCTATTGTGCGCGAGTCTGCTGCCAGCCGGGGAGATGGAAAGAGTAATGTGGTGGGTGTGAAGTTGACTTGTGCTTCGGTGGGTAAGATTACGAGGTTGGCGGCCACACTTAAGCCGGAGGAGTTTGCTGTTTATGGCGGGCAGTGTGATTTCTTGATTGGAGGGCTTAGTGTTGGATCTGCTGGGTGCATTGGGGCGTTTGCCAATGTGTTCCCCAAAACTTCGGCCAAGATTTATGAGTTGTATAAGGCCGGGAAGGTGGCGGAGGCCTTGGACTTGCAGCAGAAGGCGGCGCTAGCGGAGAGTCCCTGCAAGAGTGGCATTGCGTCTACTAAATATGCTGCCGCAATTTACTCGGCGCCCTTGGCCGGAATTGAGGGGGCCGAGGAGAAGGCGAAGCCGAGGACGCCGTACGAGGAACCCGGAGAGGGTGCTAAGAAGACGGTGAGGGAGTTGATGGACTCGGTAGCCAAGCTTGAAGTTAGCATCTAG(SEQ ID NO.1) MGSHVTPQPHVPKAGVWCPAITFFDHSTDTIDFDAQKKYYSYLSKTGLAGLVILGTNSEAFLLTREERAQCIAAAREAVGPDFPLMAGVGAHSTKQVLELAHDAAGAGANYLLVLPPAYFGKATTPAVVKKFFADVARQSPLPVVVYNFPGVCNGVDLDSETITAI VRESAASRGDGKSNVVGVKLTCASVGKITRLAATLKPEEFAVYGGQCDFLIGGLSVGSAGCIGAFANVFPKTSAKIYELYKAGKVAEALDLQQKAALAESPCKSGIASTKYAAAIYSAPLAGIEGAEEKAKPRTPYEEPGEGAKKTVRELMDSVAKLEVSI* (SEQ ID NO.2).

[0010] Furthermore, the present invention provides a method for screening potential drugs for the prevention and treatment of aflatoxin based on the target, comprising the following steps: (1) establishing a system containing aflatoxin afdapa (1) An in vitro enzymatic reaction system for gene-encoded protein; (2) Contacting the candidate compound with the enzymatic reaction system; (3) Detecting the catalytic activity of DapA synthase in the system; (4) If the candidate compound can significantly reduce or inhibit the catalytic activity of the protein compared with the control group without the addition of the candidate compound, it is selected as a potential candidate drug for the prevention and treatment of aflatoxin.

[0011] The inventors' team has for the first time isolated pathogenic Aspergillus flavus strain NJC03 from Chinese kiwifruit. (First Report of) Aspergillus flavus (Causing Fruit Rot on Kiwifruit in China). Based on the analysis of the spore components of this strain, it is speculated that it carries a DapA synthase expression gene and may maintain its extremely high stress resistance by regulating potential DPA synthesis or related metabolic pathways. Therefore, this invention constructs... afdapa Gene knockout mutants ( Δ afdapa ) and replenishment strains ( Δafdapa::afdapa Extensive environmental stress testing has shown that the absence of... afdapa The gene not only causes morphological abnormalities in spores, but also devastates the survival ability of Aspergillus flavus in high-temperature (40°C-60°C), strongly alkaline (pH 10-13), and hypertonic (0.5-1.5 M NaCl) environments. In vitro inoculation experiments further confirmed that the deletion of this gene significantly deprives the pathogen of its ability to colonize and cause disease on kiwifruit.

[0012] The present invention afdapa The nucleotide sequence of the gene can be obtained by searching the NCBI (National Center for Biotechnology Information) database for accession number XM_041286310.1. The amino acid sequence obtained by transcription and translation of this nucleic acid molecule is also the sequence disclosed in the annotation information corresponding to this accession number. Those skilled in the art can directly obtain the complete nucleotide sequence and corresponding amino acid sequence of the gene by entering this accession number on the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). The above sequence is used as part of this invention for illustration. afdapa Molecular characteristics of genes.

[0013] Compared with the prior art, the present invention has the following significant advancements and technical effects: This has opened up entirely new underlying targets for pharmaceutical research: the first confirmation of aflatoxin's role in drug development. afdapa Genes are key to regulating the environmental adaptability and early pathogenicity of pathogens. Compared to targets that only inhibit toxin synthesis, drugs screened targeting this gene can fundamentally block the survival and colonization of pathogens on the fruit surface.

[0014] This invention provides a screening system with significant industrial value: It constructs a screening system based on... afdapa Protein activity screening methods and deletion mutants ( Δafdapa Phenotypic evaluation model. This provides a complete patented technology platform for domestic and foreign pharmaceutical / pesticide companies to conduct high-throughput drug screening for kiwifruit fruit rot disease.

[0015] Potentially high biosafety: Due to significant evolutionary differences between the metabolic pathways involved in DapA synthase in fungi and those in mammals, targeting... afdapa Inhibitors developed through gene or protein targeting are expected to exhibit extremely high safety for humans and the ecological environment, which is highly compatible with the development needs of green agriculture. Attached Figure Description

[0016] Figure 1 Wild-type Aspergillus flavus NJC03, afdapa Gene deletion mutant strains ( Δafdapa ) and replenishment strains ( Δ afdapa::afdapa Survival assessment of the colony under different temperature stresses (28°C, 40°C, 50°C, 60°C); where A is the colony growth phenotype and B is the colony count statistical analysis graph.

[0017] Figure 2 Wild-type NJC03, deletion mutant strain Δafdapa and replenishment strains Δafdapa::afdapaSurvival assessment under different alkaline environments (pH=8, 9, 10, 11, 12, 13); where A is the colony growth phenotype and B is the colony count statistical analysis graph.

[0018] Figure 3 Wild-type NJC03, deletion mutant strain Δafdapa and replenishment strains Δafdapa::afdapa Survival assessment under different salt concentrations (0 M, 0.5 M, 1 M, 1.5 M NaCl); where A is the colony growth phenotype and B is the colony count statistical analysis graph.

[0019] Figure 4 Wild-type NJC03, deletion mutant strain Δafdapa and replenishment strains Δafdapa::afdapa Scanning electron microscope (SEM) images of spore morphology and statistical analysis of spore length and width dimensions.

[0020] Figure 5 After temperature stress, wild-type NJC03 and deletion mutant strains Δafdapa Phenotypic characteristics of colonization on kiwifruit (days 3, 4, and 5 post-inoculation).

[0021] Figure 6 After alkali stress, wild-type NJC03 and deletion mutant strains Δafdapa Phenotypic characteristics of colonization on kiwifruit (days 3, 4, and 5 post-inoculation).

[0022] Figure 7 After salt stress, wild-type NJC03 and deletion mutant strains Δafdapa Phenotypic characteristics of colonization on kiwifruit (days 3, 4, and 5 post-inoculation).

[0023] Figure 8 Wild-type NJC03 and deletion mutant strains Δafdapa Statistical analysis of lesion diameters on the surface of kiwifruit after different temperature, pH, and salt concentration stresses; where AC represents the temperature stress group, DF represents the alkali stress group, and HJ represents the salt stress group. Detailed Implementation

[0024] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0027] The following embodiments 1-5 of the present invention aim to construct afdapa Gene deletion mutant strains ( Δafdapa ), confirming at the phenotypic level (resistance and pathogenicity) afdapa The effectiveness of this core target in controlling kiwifruit rot; Example 6 aims to disclose how to use this target for high-throughput screening of potential antifungal drugs. Example 1

[0028] Aspergillus flavus afdapa Construction of gene deletion mutants and assessment of their survival ability under temperature stress Constructed using Split-marker homologous recombination technology afdapa Gene knockout mutants ( Δafdapa Using the genomic DNA of wild-type Aspergillus flavus strain NJC03 as a template, the following were amplified separately. afdapa Homologous arm sequences of the upstream 1072 bp (fragment A) and downstream 888 bp (fragment B) of the gene were extracted. Using plasmid PDL2 as a template, the hygromycin resistance gene hph (fragment H1 764 bp, fragment H2 930 bp) was amplified segmentally. Fragment A was fused with fragment H1, and fragment B with fragment H2 using overlap extension PCR to obtain a complete homologous recombination fragment containing the hph selection marker. The fusion fragment was transformed into Aspergillus flavus protoplasts via PEG-mediated transformation. Hygromycin B resistance selection, PCR, and sequencing verification confirmed the presence of the fusion fragment. afdapa Gene knockout strains Δafdapa .

[0029] Replenishment plant ( Δafdapa::afdapa Construction of ) : Using wild-type Aspergillus flavus genomic DNA as a template, amplification of ) containing afdapa The promoter region and the complete open reading frame (984 bp) DNA fragment were cloned into the pKNT vector and transformed into... Δafdapa Protoplasts were selected for G418 resistance, and the results were verified by PCR and sequencing to confirm the availability of replacement strains. Δafdapa::afdapa .

[0030] PDA medium (formulation: 200 g potato, 20 g glucose, 15 g agar powder, water to 1 L; sterilized at 121°C for 20 min) was used to obtain wild-type NJC03 and deletion mutant strains. Δafdapa and replant Δafdapa::afdapa The spore suspension was adjusted to a spore concentration of 1×10⁻⁶. 51 spore / mL. Spread 100 μL of bacterial culture onto a PDA plate and pretreat at 28°C, 40°C, 50°C and 60°C for 24 h. Then, incubate at 28°C for 2-3 days. After single cells grow, count and record their morphology. Set up 3 replicates for each group.

[0031] like Figure 1 As shown, under a suitable growth temperature of 28°C, wild-type NJC03 (isolated from kiwifruit in the inventor's laboratory and reported in Plant Disease 2022, 106(7): 1990, and available to the public from the Agricultural Microbial Application Technology Team of Nantong University) and mutant strains... Δafdapa and replant Δafdapa::afdapa There was no significant difference in colony count; however, when the temperature rose to 40°C, 50°C, and 60°C, the mutant strains showed... Δafdapa The colony count of the strain was precipitously lower than that of the wild type, while the survival rate of the replacement strains recovered to the level of the wild type. The results indicate that... afdapa Gene expression has an absolute regulatory effect on the survival ability of Aspergillus flavus under medium and high temperature stress. Example 2

[0032] Aspergillus flavus afdapa Survival assessment of gene deletion mutants under alkaline stress Get 1×10 5 spores / mL wild-type NJC03, deletion mutant Δafdapa and replant Δafdapa:: afdapa For the spore suspension, take 100 μL of bacterial suspension into a centrifuge tube, and add 900 μL of buffer solution prepared with sterile water and adjusted to pH 8, 9, 10, 11, 12, and 13 respectively. After stress treatment in a 28°C incubator for 24 h, take 100 μL and spread it on a PDA plate. Incubate at 28°C for 2-3 days, count, and repeat 3 times for each group.

[0033] like Figure 2 As shown, in a weakly alkaline environment with pH 8-9, there was no significant difference in the number of viable colonies among the three strains; when pH ≥10, the mutant strain... Δafdapa The colony count showed a significant decreasing trend with increasing pH, and the survival rate was significantly lower than that of the wild type in the pH range of 10-13. The recombinant strains recovered to a similar survival level. These results confirmed that... afdapa Gene deletions significantly impair the survival tolerance of pathogens in highly alkaline environments. Example 3

[0034] Aspergillus flavus afdapa Survival assessment of gene deletion mutants under hyperosmolar (salt) stress Get 1×10 5 spores / mL wild-type NJC03, deletion mutant Δafdapa and replant Δafdapa:: afdapa Spore suspensions were prepared by adding 100 μL of bacterial suspension to 900 μL of sterile NaCl solutions with concentrations of 0 M, 0.5 M, 1 M, and 1.5 M, respectively. After stress treatment at 28°C for 24 h, 100 μL of each solution was spread onto PDA plates and incubated at 28°C for 2-3 days. The spores were counted, with 3 replicates per group.

[0035] like Figure 3 As shown, in a 0 M NaCl (salt-free) environment, there was no significant difference in colony growth among the three strains; however, when the NaCl concentration increased to 0.5 M, 1 M, and 1.5 M, the mutant strain... Δafdapa The colony count decreased significantly with increasing salt concentration, showing a highly significant disadvantage compared to the wild type. The results confirmed that... afdapa The gene is the core resistance gene of Aspergillus flavus in response to high osmotic pressure (high salt) stress. Example 4

[0036] Wild type and afdapa Comparative evaluation of spore morphology characteristics of gene deletion mutants Wild-type NJC03 and deletion mutant strains cultured on PDA medium for 5 days were picked using a sterile inoculation loop. Δ afdapa and replant Δafdapa::afdapa The bacterial cells were fixed in 4% paraformaldehyde solution, air-dried, and then dehydrated using a gradient of alcohols. After critical point drying and gold sputtering, the spore size was observed and measured using a scanning electron microscope (SEM).

[0037] like Figure 4 As shown, the wild-type NJC03 spores are regular in morphology, uniform in size, and have an intact surface structure; while the mutant strain... Δ afdapa The spores showed significant aberrations, exhibiting uneven size and severely compromised surface structure integrity; the spore characteristics of the recombinant strain were completely restored. This result objectively validates the targeted inhibition... afdapa Genes can directly disrupt the physical structural integrity of the reproductive organs of pathogens. Example 5

[0038] afdapa The impact of gene deletion on the pathogenicity and colonization ability of Aspergillus flavus in an emerging host (kiwifruit). Spore suspensions of wild-type and deletion mutant strains were prepared and subjected to the three stress treatments (temperature, alkali, and salt) described in Examples 1-3 for 24 h. Healthy kiwifruit fruits with sterilized surfaces were punctured to cause injury, and 20 μL of the treated bacterial suspension was dripped into the wounds. The fruits were sealed and incubated at 28°C and 70% relative humidity. On days 3, 4, and 5, the epidermis was peeled off and the diameter of the lesions was measured.

[0039] like Figures 5 - 8 As shown, (1) Temperature stress group: after pretreatment at 40°C-60°C, inoculation Δafdapa The diameter of lesions on kiwifruit fruits in the mutant strain was significantly smaller than that in the wild type, and the rate of expansion was significantly delayed. (2) Alkali stress group: After pretreatment with pH ≥ 10, Δafdapa The colonization ability of the mutant strain on kiwifruit was significantly reduced, and its pathogenicity was greatly weakened. (3) Salt stress group: After pretreatment with 0.5 M-1.5 M NaCl, the mutant strain was inoculated. Δafdapa The lesions on the mutant kiwifruit hardly expand.

[0040] Overall conclusion: Targeted afdapa The gene can fundamentally undermine the environmental adaptability of pathogens, causing them to lose their ability to colonize and infect on the surface of kiwifruit, thus confirming that the gene is an excellent target for biological control. Example 6

[0041] based on afdapa In vitro screening model and operating procedures for drugs targeting aflatoxin This embodiment provides a method using Aspergillus flavus. afdapa Specific operational procedures for constructing a standardized in vitro screening model for the gene-encoded protein (AfDapA) and conducting high-throughput screening of potential antifungal drugs: (1) High-efficiency expression and purification of target proteins Contains Aspergillus afdapa The recombinant expression plasmid containing the complete coding region of the gene (NCBI accession number: XM_041286310.1) was transformed into an E. coli expression host. After IPTG (isopropyl-β-D-thiogalactoside) induction, bacterial cells were collected and lysed. The expression product was purified using affinity chromatography (such as a Ni-NTA column). After dialysis to remove salts, a high-purity recombinant AfDapA synthase protein with catalytic activity was obtained as a reserve of target molecules for drug screening.

[0042] (2) Standardized construction of in vitro enzyme-catalyzed reaction system A microplate-level reaction system for DapA enzyme activity assay was constructed in vitro to meet the needs of high-throughput screening. The standard reaction system included: pH 7.0 and PBS buffer, L-aspartic semialdehyde (ASA) at a final substrate concentration of 5 mM and pyruvate, and an appropriate amount of recombinant AfDapA protein purified in step (1) was added. The reaction was carried out at 28°C for 2 h, and the pyruvate content was detected.

[0043] (3) Candidate drug screening and real-time activity monitoring Different types of candidate compound libraries (dissolved in trace amounts of DMSO, with the final DMSO concentration not exceeding the system's tolerance threshold) were added to the above enzymatic reaction system. The microplate was placed in a microplate reader, and the pyruvate content in the reaction system was detected at 520 nm using a pyruvate assay kit (Solarbio, BC220). A blank control group (containing only an equal amount of solvent) without candidate compounds was set up. An inhibition concentration curve was plotted with the pyruvate consumption as the ordinate and the candidate compound concentration as the abscissa, and the IC50 of each candidate compound was calculated. 50 .

Claims

1. Aspergillus flavus ( Aspergillus flavus DapA synthase gene afdapa Or the use of its encoded proteins as targets in screening or evaluating drugs for the prevention and treatment of aflatoxin.

2. The application according to claim 1, characterized in that, The afdapa The nucleotide sequence of the gene is shown in SEQ ID NO: 1; the amino acid sequence of the encoded protein is shown in SEQ ID NO:

2.

3. The application according to claim 1 or 2, wherein the aflatoxin control agent is used to control agricultural diseases or postharvest diseases of fruit caused by aflatoxin infection, wherein the postharvest diseases of fruit include fruit rot caused by aflatoxin infection of the emerging host kiwifruit.

4. A method based on Aspergillus flavus afdapa A method for screening candidate drugs for the prevention and treatment of aflatoxin by gene target selection, characterized in that, Includes the following steps: (1) Provides products containing Aspergillus flavus afdapa Determination system for gene-encoded proteins; (2) Contact the candidate compound with the assay system; (3) Detecting Aspergillus flavus in the assay system afdapa Catalytic activity of gene-encoded proteins; (4) Compared with the control group without the addition of the candidate compound, if the candidate compound can reduce or inhibit the aflatoxin afdapa The catalytic activity of the gene-encoded protein led to its selection as a potential candidate drug for the prevention and treatment of aflatoxin.

5. The method according to claim 4, characterized in that, The assay system described in step (1) is an in vitro enzymatic reaction system containing recombinant purified Aspergillus flavus. afdapa The gene encodes a protein, a buffer solution, the substrate L-aspartic semialdehyde, and pyruvate.

6. The method according to claim 4 or 5, characterized in that, In step (3), the rate of increase of the characteristic absorption peak when dihydropyridine dicarboxylic acid is generated in the assay system is monitored, or an enzyme-coupled colorimetric assay is used to quantitatively assess the aspergillosis in real time. afdapa The catalytic reaction rate of gene-encoded proteins.

7. The method according to claim 4, characterized in that, In step (4), the reduction or inhibition refers to the effect of the candidate compound on Aspergillus flavus. afdapa The inhibition rate of the catalytic activity of the gene-encoded protein is greater than 50%.

8. The method according to any one of claims 3-7, characterized in that, The aflatoxin control agent is used to prevent and control agricultural diseases or postharvest diseases of fruit caused by aflatoxin infection, including fruit rot caused by aflatoxin infection of the emerging host kiwifruit.

9. Aspergillus flavus afdapa Application of gene deletion mutants in constructing a drug efficacy model for evaluating the prevention and treatment of aflatoxin.

10. The application according to claim 9, or the method according to claim 3, characterized in that, The aflatoxin control agent is used to prevent and control agricultural diseases or postharvest diseases of fruit caused by aflatoxin infection, including fruit rot caused by aflatoxin infection of the emerging host kiwifruit.

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