Beauvericin synthesis regulation related application of gene PP558224

Abstract

The invention provides a beauvericin synthesis regulation related application of a gene PP558224, and belongs to the technical field of biological control. The invention provides a basis for the synthesis principle of the beauvericin in the pathogenicity of the cordyceps sobolifera to the prodenia litura, and develops a new thought for the analysis of the pathogenic mechanism of the cordyceps sobolifera. The method can enrich gene function research contents of cordyceps sobolifera and entomogenous fungi, and assists in drawing a gene regulation network map; in view of various defects of chemical prevention and control of prodenia litura and biological prevention and control potential of cordyceps sobolifera, the synthetic amount of beauvericin of cordyceps sobolifera can be accurately regulated and controlled through a genetic engineering means, positive correlation of pathogenicity of cordyceps sobolifera to prodenia litura and expression quantity of the gene PP558224 is determined, and further accurate molecular breeding of the gene PP558224 is carried out, so that the prodenia litura can be effectively prevented and controlled. The development of efficient, environment-friendly and sustainable cordyceps sobolifera strains for biological prevention and control is facilitated, green prevention and control of agricultural pests are realized, and the yield and quality of crops are guaranteed.

Description

Technical Field

[0001] The present invention relates to the technical field of biological control, and particularly relates to the related applications of beauvericin synthesis regulation of gene PP558224. Background Art

[0002] As an entomogenous fungus with important medicinal value and ecological significance, Cordyceps cicadae has complex interaction relationships with various insects in nature. Among them, Spodoptera litura is one of many agricultural pests that cause serious damage to crops, and Cordyceps cicadae shows certain pathogenicity to Spodoptera litura, which makes it have potential application value in the field of biological control.

[0003] In the process of Cordyceps cicadae exerting its pathogenic effect on Spodoptera litura, numerous secondary metabolites play a key role. Beauvericin is one of the important toxins, which can interfere with the normal physiological functions of insect cells, such as affecting the permeability of cell membranes and disrupting the ion balance within cells, and thus has a significant impact on the growth, development, and survival of Spodoptera litura. It is one of the key material bases for Cordyceps cicadae to achieve pathogenicity to Spodoptera litura.

[0004] However, for a long time, there are still many ambiguities in the understanding of the synthesis principle of beauvericin produced by Cordyceps cicadae and its entire pathogenic mechanism. Although scientific researchers have carried out a large number of studies on the synthesis regulation of fungal secondary metabolites, the specific regulation mechanism of beauvericin synthesis in Cordyceps cicadae, especially the role of specific genes, remains to be further studied. Deeply analyzing the pathogenic mechanism of Cordyceps cicadae and clarifying the regulation principle of beauvericin synthesis can provide a solid theoretical support for the development of efficient biological control strategies based on Cordyceps cicadae and its secondary metabolites. Summary of the Invention

[0005] The purpose of the present invention is to provide the related applications of beauvericin synthesis regulation of gene PP558224, which provides an important basis for the beauvericin synthesis principle of the pathogenicity of Cordyceps cicadae to Spodoptera litura and a new idea for analyzing the pathogenic mechanism of Cordyceps cicadae.

[0006] In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides the application of Cordyceps cicadae histone deacetylase gene PP558224 in the regulation of beauvericin synthesis, and the nucleotide sequence of the Cordyceps cicadae histone deacetylase gene PP558224 is as shown in SEQ ID NO.1.

[0008] The present invention also provides the application of the cicada flower histone deacetylase gene PP558224 in regulating fungal metabolism, and the nucleotide sequence of the cicada flower histone deacetylase gene PP558224 is as shown in SEQ ID NO.1.

[0009] Preferably, genetic engineering means are used to knockout, weaken or overexpress the cicada flower histone deacetylase gene PP558224, thereby affecting the content of beauvericin in cicada flowers, and thus regulating fungal metabolism.

[0010] The present invention also provides the application of the cicada flower histone deacetylase gene PP558224 in the preparation of products for detecting the beauvericin synthesis ability of cicada flowers, and the nucleotide sequence of the cicada flower histone deacetylase gene PP558224 is as shown in SEQ ID NO.1.

[0011] The present invention also provides a primer set for detecting the pathogenicity of Spodoptera litura to cicada flowers, including an upstream primer with a nucleotide sequence as shown in SEQ ID NO.10 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.11.

[0012] The present invention also provides a method for detecting the pathogenicity of Spodoptera litura to cicada flowers, including the following steps:

[0013] Using the primer set to amplify the cicada flower gene. If a 1.8 kb band can be amplified, it indicates that the pathogenicity of Spodoptera litura is normal;

[0014] If a 1.8 kb band cannot be amplified, it indicates that the pathogenicity of Spodoptera litura is weak.

[0015] The present invention also provides a fungal gene knockout vector, which is a knockout vector targeting the cicada flower histone deacetylase gene PP558224, and the nucleotide sequence of the cicada flower histone deacetylase gene PP558224 is as shown in SEQ ID NO.1.

[0016] The present invention also provides a fungal gene overexpression vector, which is an overexpression vector targeting the cicada flower histone deacetylase gene PP558224, and the nucleotide sequence of the cicada flower histone deacetylase gene PP558224 is as shown in SEQ ID NO.1.

[0017] The present invention also provides the application of the above fungal gene knockout vector or fungal gene overexpression vector in the preparation of products for regulating the content of beauvericin in cicada flowers.

[0018] The present invention also provides a genetically engineered host cell, in which the cicada flower histone deacetylase gene PP558224 is overexpressed in the genome of the host cell, and the nucleotide sequence of the cicada flower histone deacetylase gene PP558224 is as shown in SEQ ID NO.1.

[0019] Advantages of the present invention:

[0020] The present invention focuses on the application of gene PP558224 in the regulation of beauvericin synthesis. By clarifying the role of this gene in this process, it provides a new idea for the analysis of the pathogenic mechanism of cicada flower, and the research on gene PP558224 enriches the research content on gene functions in cicada flower and even more extensive entomogenous fungi. By exploring its role in the regulation of beauvericin synthesis, the present invention can provide a reference basis for subsequent discovery of other genes that cooperate with it or have regulatory associations, contribute to drawing a larger and more detailed gene regulatory network map, and further explore the diverse functions of genes in the processes of entomogenous fungi adapting to the environment and interacting with hosts.

[0021] As a common and severely harmful agricultural pest, although current chemical control measures can control its population to a certain extent, they are often accompanied by many problems such as environmental pollution and enhanced pest drug resistance. Cicada flower has the pathogenicity to Spodoptera litura by producing secondary metabolites such as beauvericin, showing the potential of biological control. The present invention clarifies the application of gene PP558224 in the regulation of beauvericin synthesis. Based on this, in the future, it is expected to genetically engineer cicada flower to more precisely regulate the synthesis amount of beauvericin, enhance the pathogenic effect of cicada flower on Spodoptera litura, develop more efficient, environmentally friendly and sustainable biological control products, thereby effectively reducing the use of chemical pesticides, achieving green prevention and control of agricultural pests, and ensuring the yield and quality of crops. Description of the Drawings

[0022] Figure 1 For the phenotypes of Spodoptera litura larvae and the colony morphology of isolated strains, A: Spodoptera litura larvae inoculated with cicada flower for 10 days, with hyphae growing on the body surface; B: Colony morphology of isolated strains cultured on PDA medium for 14 days;

[0023] Figure 2 For the phenotypes of the PP558224 gene knockout mutant (A) and the wild type (B) cultured on antibiotic medium for 7 days;

[0024] Figure 3The lengths of the gene fragments of each sample amplified with the primer pair (Bbea-F1, Bbea-R1) are, from left to right: DL5000 DNA Marker (M), wild-type Cordyceps cicadae (1, 2), PP558224 gene knockout mutant (3, 4);

[0025] Figure 4 The lengths of the gene fragments of each sample amplified with the primer pair (Hyg-F, Hyg-R) are, from left to right: DL5000 DNA Marker (M), the third PCR product (1, 2), strain PP558224 gene knockout mutant (3, 4), wild-type Cordyceps sobolifera (5, 6);

[0026] Figure 5 The lengths of the gene fragments of each sample amplified with the primer pair (UP-F, Down-R) are, from left to right: DL5000 DNA Marker (M), the third PCR product (1), PP558224 gene knockout mutant (2), wild-type Cordyceps cicadae (3). Detailed implementation mode

[0027] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they cannot be construed as limiting the protection scope of the present invention.

[0028] The nucleotide sequence of the Cordyceps cicadae histone deacetylase gene PP558224 in the embodiment is:

[0029]

[0030] Example

[0031] 1 Materials and Methods

[0032] 1.1 Tested living materials

[0033] Tested Cordyceps cicadae strains: Cordyceps cicadae (strain number 022017-9) was provided by the Entomogenous Fungi Department of Zhejiang Institute of Subtropical Crops.

[0034] Tested Spodoptera litura larvae: The second-instar larvae of Spodoptera litura were provided by Jilin Haokang Biotechnology Co., Ltd. and were subcultured with artificial feed in an artificial climate chamber. The temperature of the insect rearing room was (24±1)°C, the relative humidity was 85%, and the light-dark ratio was 14h:10h.

[0035] 1.2 Culture media and reagents

[0036] Potato Dextrose Agar (PDA): 200 g peeled potatoes, 20 g glucose, 15 g agar, made up to 1 L with water, autoclaved at 121°C for 20 min.

[0037] Potato Dextrose Broth (PDB): 200 g peeled potatoes, 20 g glucose, made up to 1 L with water, autoclaved at 121°C for 20 min.

[0038] Bran culture-medium (BCM): 800 g oats, 200 g buckwheat, added with sterile water to submerge the wheat mixture, autoclaved at 121°C for 20 min.

[0039] Regenerated solid medium (RSM): 200 g peeled potatoes, 20 g glucose, 15 g agar, made up to 1 L with water, autoclaved at 121°C for 20 min, 127.5 g mannitol.

[0040] Regenerated liquid medium (RLM): 200 g peeled potatoes, 20 g glucose, made up to 1 L with water, autoclaved at 121°C for 20 min, 127.5 g mannitol.

[0041] Antibiotic medium: 200 g peeled potatoes, 20 g glucose, 15 g agar, made up to 1 L with water, autoclaved at 121°C for 20 min, and hygromycin was added to a concentration of 300 mg / mL when pouring the plates.

[0042] pKOV21 plasmid: used to obtain specific hyg fragment, with hygromycin resistance.

[0043] All reagents used in other experiments were of analytical grade.

[0044] 1.3 Experimental instruments

[0045] Artificial climate chamber (Zhejiang Top Yunnong Technology Co., Ltd.); clean workbench (VS-1300-L, Suzhou Antai Air Technology Co., Ltd.); constant temperature incubator (BMJ-400, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory); microwave oven (Galanz Microwave Oven Appliance Co., Ltd. P7021TP-6); PCR instrument (Thermo Fisher ABI SimpliAmp); high-speed refrigerated centrifuge (Shanghai Lu Xiangyi Centrifuge Instrument Co., Ltd.), etc.

[0046] Other experimental equipment: insect box, cotton balls, 200 mesh sieve.

[0047] 1.4 Determination of pathogenicity of Cordyceps sinensis strains

[0048] 1.4.1 Preparation of bacterial culture

[0049] The test strain was thawed at room temperature, inoculated on PDA medium, placed in a 24°C constant temperature incubator, and inverted for 3 days. Subsequently, colonies with good mycelium growth were selected, and some mycelium was streaked on a new PDA medium for subculture to separate single colonies. After 7-10 days of constant temperature culture at 24°C, spore powder was collected with a sieve in a clean bench, and the collected spore powder was dissolved with deionized water and 0.05% Tween-80 to prepare a concentration of 1×10 8 Spore powder suspension with a concentration of 100 / mL.

[0050] 1.4.2 Activation and isolation of cicada fungus strains

[0051] According to the inoculation and feeding of test insects in 1.4.3, the above bacterial solution was used to infect the larvae of Spodoptera litura, and the dead insects of Spodoptera litura were collected. The internal and surface tissues were extracted, and the cicada fungus strains were re-isolated and streaked on PDA medium for culture. After the uncontaminated single colonies grew, the hyphae of colonies from different sources were picked and repeatedly streaked on PDA plates to purify the strains until multiple single colonies were obtained. The strains with good growth status were selected as the purified strains, and the purified strains were re-identified by morphology and molecular identification.

[0052] The isolated cicada fungus strain was inoculated into PDB medium, mixed and transferred to a shaker at 25°C, 140rpm for culture for 3 days, then the bacterial solution was inoculated into BCM medium, mixed and cultured in a dark culture room at 25°C for 7 days, then transferred to a light culture room for light culture for 14 days, the fruiting bodies were harvested, and the spore powder of each strain was sieved.

[0053] 1.4.3 Inoculation and feeding of test insects

[0054] Referring to Zhang Shenglan's Spodoptera litura toxicity bioassay experiment (Zhang Shenglan 2022), the suspension stock solution was prepared with cicada fungus spore powder, deionized water, and Tween-80. After oscillation and mixing, the sample was dripped onto a blood cell counting plate to calculate the concentration of the stock solution. The spore suspension stock solution was diluted to 1×10 6 / mL, 1×10 7 / mL, 1×10 8 / mL, 1×10 9 The spore suspension with a concentration of 100 / mL and 0.05% Tween-80 were used as the control group.

[0055] Treat the test insects with the immersion method. Pick up the test insects with tweezers and dip them in the spore suspension for 2 seconds, then take them out and transfer them to the small grid of the insect box where feed and moistened cotton balls have been arranged in advance. Each treatment has 5 replicates, with 20 insects in each replicate. They are raised in an artificial climate box at 28°C, 80% humidity, and a light duration ratio (L:D) of 14h:10h. Change the feed every 24h and clean the feces and moistened cotton balls in the single grid that may be contaminated. Count the deaths of the test insects every day, poke the larvae lightly with a brush, and use no reaction and inability to crawl as the criterion for death. Stop recording 10 days after inoculation, and dispose of the test insects harmlessly.

[0056] 1.4.4 Determination of strain pathogenicity

[0057] After the larvae are inoculated, their behavior, symptoms of infection, and the number of diseased and dead larvae are observed and recorded daily, and the mortality rate is calculated. Excrement in the insect-raising container is promptly removed and the feed is replaced.

[0058] 1.4.5 Data processing

[0059] Mortality rate (%) = number of dead insects / total number of insects treated × 100%

[0060] Corrected mortality rate (%) = (treatment mortality rate - control mortality rate) / (1 - control mortality rate) × 100%

[0061] LC 50 , corrected mortality is used as an indicator for pest toxicity analysis, with the logarithm of concentration (c) as x and the probability value of mortality as y. The probability value analysis method is used to calculate the toxicity regression equation and LC 50 The values ​​were used to analyze the virulence of different cicada fungus strains.

[0062] 1.5 Construction of knockout mutant of histone deacetylase gene PP558224 in Cicadae

[0063] 1.5.1 Solution preparation

[0064] PTC buffer (100 mL): 60 g PEG, 0.735 g CaCl 2 .2H 2 O, 1 mL of 1 M Tris-HCl (pH = 7.5);

[0065] STC buffer (500 mL): 1093 g sorbitol, 5 mL of 1 M Tris-HCl (pH = 7.5), CaCl 2 ·2H 2 O 2.7745 g;

[0066] 0.5× TAE solution: 10 mL of 50× TAE solution, 990 mL of deionized water.

[0067] 1.5.2 Construction of the target vector

[0068] The primer sequences required for the construction of the target vector are shown in Table 1, and the primers were synthesized by Tsingke Biotechnology Co., Ltd.

[0069] Table 1 PCR amplification primer sequences used

[0070]

[0071]

[0072] Using Cordyceps cicadae cultured on PDA medium for 7 days as the material, the whole genome DNA of Cordyceps cicadae was extracted, and the method is described in the instruction manual. For the first PCR, the primer pairs (Up-F, Up-R) and (Down-F, Down-R) were used to amplify the genomic DNA of Cordyceps cicadae to obtain the upstream and downstream fragments of the target gene respectively; the primer pair (Hyg-F, Hyg-R) was used to amplify the pKOV21 plasmid to obtain the hygromycin resistance fragment.

[0073] The PCR amplification system is as follows: 5 μL of 10× Buffer; 4 μL of dNTP; 8 μL of template DNA; 1 μL of primer F; 1 μL of primer R; 0.25 μL of Ex Taq enzyme; finally, ddH2O was added to make up 50 μL.

[0074] PCR amplification conditions: pre-denaturation at 95°C for 3 min, then denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension time at 72°C was calculated according to the length of the amplification product (60 s / kb), 30 cycles were carried out, and finally thorough extension at 72°C for 5 min. The results were detected by 1% agarose gel electrophoresis.

[0075] For the second PCR, the upstream and downstream fragments of the target gene and the hygromycin resistance fragment were mixed in the same solution and added according to a mass ratio of 1:1:2, and the remaining components were added according to the conventional 50 μL PCR system.

[0076] The PCR amplification system is as follows: 5 μL of 10× Buffer; 4 μL of dNTP; 100 ng of upstream fragment; 100 ng of downstream fragment; 200 ng of hygromycin resistance fragment; 0.25 μL of Ex Taq enzyme; and finally make up to 50 μL with ddH2O.

[0077] PCR amplification conditions: Pre-denature at 94°C for 2 min, then denature at 94°C for 30 s, anneal at 55°C for 10 min, extend at 72°C for 5 min, perform 15 cycles, and finally extend thoroughly at 72°C for 10 min. Detect the results by 1% agarose gel electrophoresis.

[0078] For the third PCR, use the product of the second PCR as the template, and UP-F and Down-R as primers to amplify the "upstream - hygromycin - downstream" fragment. The PCR amplification system is as follows: 5 μL of 10× Buffer; 4 μL of dNTP; 8 μL of the second PCR product; 1 μL of primer F; 1 μL of primer R; 0.25 μL of Ex Taq enzyme; and finally make up to 50 μL with ddH 2 O.

[0079] PCR amplification conditions: Pre-denature at 95°C for 3 min, then denature at 95°C for 15 s, anneal at 55°C for 15 s, extend at 72°C for 4 min 30 s, perform 30 cycles, and finally extend thoroughly at 72°C for 5 min. Detect the results by 1% agarose gel electrophoresis.

[0080] Recover the product of the third PCR and store it at -20°C for later use.

[0081] 1.5.3 Protoplast preparation and transformation

[0082] Mycelium preparation: After activating the strain on the slant for 2 - 3 d, inoculate it into the PDB medium and culture it in a shaker for 3 - 4 d (25°C, 140 r / min). Centrifuge the bacterial liquid (5000 r / min, 10 min) and wash the mycelium precipitate three times with 0.7 mol / L NaCl solution. Finally, store the precipitate at 4°C to obtain the mycelium.

[0083] Lyse the mycelial cell wall: Prepare a 6 mg / mL snail enzyme digestion solution with 0.7 mol / L NaCl solution as the solvent. After filtering the mycelium to remove bacteria, add the prepared digestion solution and transfer it to a shaker for digestion (37°C, 150 r / min) for 4 - 4.5 h.

[0084] Protoplast filtration and washing: Sterile filter the enzymolysis product, discard the mycelium, centrifuge the filtrate at 4°C and 5000 r / min to retain the precipitate. Add pre-cooled STC buffer to make the volume up to 10 mL, centrifuge at 4°C and 5000 r / min for 10 min, then discard the supernatant. Repeat washing the precipitate twice with STC buffer. Finally, add 1 mL of STC buffer, gently pipette and mix the remaining liquid in the tube. Take a small amount of protoplast suspension on a hemocytometer and count under a microscope to determine the protoplast concentration of the mother solution. Then adjust the protoplast concentration to 10 8 cells / mL.

[0085] Protoplast transformation: Add 12 μg of the target vector to 850 μL of the adjusted protoplast solution and let it stand on ice for 25 min. After standing, dropwise add 1 mL of PTC buffer to stabilize the transformation environment and let it stand on ice for another 25 min. After standing, add it to 20 mL of RSM medium, transfer it to a shaker and culture overnight at 24°C and 100 r / min for 12 h to regenerate the cell wall.

[0086] 1.5.4 Screening and testing of transformants

[0087] Screening of transformants: Prepare RLM medium, pour the protoplasts after overnight culture into 150 mL of RLM medium cooled to semi-solid state, and evenly pour it onto a culture dish. Culture it in the dark at 24°C for 1 - 2 d until visible small colonies grow on the surface, cover it with an antibiotic medium for resistance screening. Culture it in the dark on the resistance medium for 7 d (room temperature 25°C), visible transformants grow, pick single colonies and transfer them to a new antibiotic medium for continued culture.

[0088] Testing of transformants: Re-inoculate the transformants on a new antibiotic medium, passage three times to stabilize their resistance. Extract DNA from the third-generation transformants and use primer pairs to test the knockout fragment to verify whether the genetic transformation system is established.

[0089] 1.5.5 Cultivation of fruiting bodies of strains

[0090] Inoculate the wild type and the screened mutant strains into PDB medium and culture them on a shaker (25°C, 140 r / min) for 3 d, then transfer them to BCM medium and culture them in the dark at 25°C for 7 d. After the mycelium covers the medium, transfer them to the fruiting body cultivation area and culture them under light at 25°C for 14 d. Harvest when the fruiting bodies grow to 4 - 7 cm, dry them in a drying oven at 60°C, crush them, and sieve them through a 200-mesh sieve until there are no large particles, then proceed with the subsequent determination of beauvericin content.

[0091] 1.5.6 Determination of beauvericin content

[0092] Determine the content of beauvericin according to the method of "Detection Method of Beauvericin in Cordyceps cicadae Fruit Bodies" (Chen Hui, 2018).

[0093] 2 Results and Analysis

[0094] 2.1 Activation and Isolation of Cordyceps cicadae Strains

[0095] For the prodenia litura cadavers after inoculating with Cordyceps cicadae spore suspension for 10 days, isolate and purify the mycelia on their body surfaces ( Figure 1 A). When the mycelia of the pathogenic fungi of the cadavers were cultured on a solid medium and cultured on PSD medium for 14 days, the colony diameter reached 60 - 72 mm, showing yellowish-white or light yellow, villous, with obvious concentric rings or radial lines, and slightly white on the back. The exudate was bead-shaped and colorless ( Figure 1 B). Through colony morphology analysis and molecular identification, it conformed to the colony morphology and sporulation structure characteristics of Cordyceps cicadae, and a total of four isolated and purified Cordyceps cicadae single-cell strains EX3.1, EX3.2, EX4.1, and EX4.2 were obtained. After separately culturing the isolated single-cell strains, take several of the outermost mycelia (the newly grown mycelia), store them in glycerol vials respectively, mark the storage time and strain number, and store them in a -80 °C refrigerator for later use.

[0096] 2.2 Pathogenicity of Cordyceps cicadae Strains to Prodenia litura

[0097] When infecting prodenia litura larvae with Cordyceps cicadae strains at different concentrations for 3 days, there was no significant difference in the corrected mortality of prodenia litura. The corrected mortality of prodenia litura increased significantly at 5 days and 10 days after inoculation with each strain. When 5 days after inoculating with Cordyceps cicadae, the mortality of larvae treated with a concentration of 1×10 9 per mL reached 0.36 - 0.47; the mortality of larvae treated with a concentration of 1×10 8 per mL was 0.28 - 0.39; the mortality of larvae treated with a concentration of 1×10 7 per mL was 0.25 - 0.33; the mortality of larvae treated with a concentration of 1×10 6 per mL was 0.15 - 0.3, indicating that with the increase in the concentration of the Cordyceps cicadae strain spore suspension, the mortality of inoculated prodenia litura increased significantly (Table 2). 4 - 6 days after inoculation was the peak period of the disease of prodenia litura larvae. The larvae gradually lost their motility, reduced food intake, and had slow body growth. After 8 - 10 days of inoculation, white mycelia began to appear on the body surfaces of some larvae.

[0098] The results of the median lethal concentration of each strain showed that the LC 50 of strain EX3.1 was 4.6×10 9 per mL, the LC 50 of strain EX3.2 was 2.9×10 8 per mL, and the LC of strain EX4.150 is 2.5×10 8 per mL, and the LC of strain EX4.2 50 is 5.4×10 9 per mL (Table 3). Among them, the lower the lethal concentration of strain EX4.1, the stronger its pathogenicity.

[0099] The above results indicate that Cordyceps cicadae has a certain pathogenicity to Spodoptera litura. Among them, strain EX4.2 has the weakest pathogenicity, and strain EX4.1 has the strongest pathogenicity. The strain is used for subsequent experiments to explore the pathogenic mechanism of Cordyceps cicadae against Spodoptera litura.

[0100] Table 2 Pathogenicity of different concentrations of Cordyceps cicadae strains to the 2nd instar larvae of Spodoptera litura

[0101]

[0102]

[0103] Note: The data in the table are mean ± standard deviation. After Tukey test, different lowercase letters indicate significant differences in the corrected mortality of Spodoptera litura among different spore suspension concentrations at the same time (P < 0.05)

[0104] Table 3 Regression equation of the pathogenicity of Cordyceps cicadae spore suspension to the 2nd instar larvae of Spodoptera litura

[0105] Strain Virulence regression equation <![CDATA[Correlation coefficient (R 2 )]]> <![CDATA[Lethal concentration LC 50 (cells / mL)]]> EX3.1 y = 0.0682x - 0.1591 0.9058 <![CDATA[4.6×10 9 > EX3.2 y = 0.0901x - 0.2637 0.9976 <![CDATA[2.9×10 8 > EX4.1 y = 0.0742x - 0.1235 0.9971 <![CDATA[2.5×10 8 <!-- 8 -->]]> EX4.2 y = 0.075x - 0.23 0.9947 <![CDATA[5.4×10 9 >

[0106] The 2.3PP558224 knockout mutant affects the pathogenicity of Cordyceps cicadae

[0107] 2.3.1 Resistance verification of the transformant and the wild-type strain

[0108] After culturing the PP558224 gene knockout mutant and the wild-type Cordyceps cicadae strain for 7 days, the wild-type could not grow on the medium containing hygromycin, while the mutant could grow normally ( Figure 2 ). After subculturing three times on the hygromycin medium, the PP558224 gene knockout mutant could grow normally, proving that it indeed has resistance to hygromycin B.

[0109] 2.3.2 Molecular verification of the knockout mutant and the wild-type strain

[0110] Take the PP558224 mutant cultured in PDA liquid medium for 3 days, filter the mycelium with a sterilized funnel and filter paper, quickly freeze it in liquid nitrogen, and obtain its genomic DNA with an Ezup column fungal genomic DNA extraction kit for PCR verification, using the plasmid as a positive control. After detection by 1% agarose gel electrophoresis, the band was single and clearly visible, indicating that the target gene was successfully knocked out in the PP558224 gene knockout mutant.

[0111] The wild-type Cordyceps cicadae and the mutant strain were amplified with the primer pair (Bbea-F1, Bbea-R1). The wild-type Cordyceps cicadae (1, 2) amplified bands of the correct length, while the PP558224 gene knockout mutant strain (3, 4) had no visible bands ( Figure 3 ), proving that the target gene PP558224 was absent in the mutant strain.

[0112] The wild-type Cordyceps cicadae and the mutant strain were amplified with the primer pair (Hyg-F, Hyg-R). The third PCR products (1, 2) amplified bands of the correct length, while the PP558224 gene knockout mutant strain (3, 4) amplified bands of the correct length, and the wild-type Cordyceps cicadae (5, 6) had no visible bands ( Figure 4 ), proving that the PP558224 gene knockout mutant strain had the HpH resistance fragment gene, and the wild-type Cordyceps cicadae had no HpH resistance gene.

[0113] The wild-type Cordyceps cicadae and the mutant strain were amplified with the primer pair (UP-F, Down-R). The bands amplified from the third PCR product (1) and the PP558224 gene knockout mutant strain (2) were shorter than the bands amplified from the wild-type Cordyceps cicadae (3), and the bands amplified from the PP558224 gene knockout mutant strain were the same length as the results of the third PCR ( Figure 5 ), proving that the target gene PP558224 fragment in the mutant strain had been replaced by the HpH resistance fragment.

[0114] In summary, the target gene PP558224 in the mutant strain has been successfully knocked out and replaced by the HpH resistance gene, which is used to study its effect on the pathogenicity of Cordyceps cicadae.

[0115] 2.3.3 The content of beauvericin in the PP558224 knockout mutant strain decreased

[0116] The wild-type Cordyceps cicadae and the PP558224 knockout mutant strain were cultured separately. The fruiting bodies were collected, dried and ground into powder, and the content of beauvericin was measured respectively. The experiment was repeated three times, and the measurement results are shown in Table 4.

[0117] The results of the significance test showed that the variance significance was 0.211, and there was no significant difference in variance. The mean significance < 0.01 was extremely significant. The average content of beauvericin in the wild-type Cordyceps cicadae was significantly higher than that in the mutant, indicating that the content of beauvericin in the PP558224 knockout mutant strain decreased significantly compared with the wild-type strain (Table 4). The Cordyceps cicadae histone deacetylase gene PP558224 may be involved in the synthesis of beauvericin in Cordyceps cicadae, and the content of beauvericin decreased significantly after knocking out this gene.

[0118] Table 4 The content of beauvericin obtained from the tested strains after high performance liquid chromatography-tandem mass spectrometry detection

[0119]

[0120] 2.3.4 PP558224 knockout mutant strain shows weakened pathogenicity

[0121] When inoculating Spodoptera litura larvae with different concentrations of strain EX4.1 and the mutant strain for 3 days, there was no significant difference in the corrected mortality rate of the larvae. When inoculating for 5 days and 10 days, the mortality rate of the larvae treated with strain EX4.1 was significantly higher than that of the mutant strain (Table 5), and the median lethal concentration of the mutant strain was higher. This indicates that compared with the wild-type strain, the Beauveria cicadae mutant strain with PP558224 knocked out shows weakened pathogenicity to Spodoptera litura (Table 6).

[0122] Table 5 Pathogenicity of Beauveria cicadae with different concentrations to the 2nd instar larvae of Spodoptera litura

[0123]

[0124] Table 6 Pathogenicity of Beauveria cicadae spore suspension to Spodoptera litura

[0125]

[0126] In summary, compared with the wild-type EX4.1, the knocked-out mutant strain shows weakened pathogenicity to Spodoptera litura (Tables 5 and 6), and the content of beauvericin in the mutant strain is significantly reduced (Table 4), indicating that the content of beauvericin in Beauveria cicadae is positively correlated with its pathogenicity to Spodoptera litura.

[0127] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Application of Cordyceps sinensis histone deacetylase gene PP558224 in regulating the synthesis of beauvericin, wherein the nucleotide sequence of Cordyceps sinensis histone deacetylase gene PP558224 is shown in SEQ ID NO.

1.

2. Application of the Cicadae Concolor histone deacetylase gene PP558224 in regulating fungal metabolism, wherein the nucleotide sequence of the Cicadae Concolor histone deacetylase gene PP558224 is shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that: Genetic engineering is used to knock out and weaken the cicada fungus histone deacetylase gene PP558224, thereby affecting the content of beauvericin in the cicada fungus, thereby regulating fungal metabolism.

4. Application of the cicada confusa histone deacetylase gene PP558224 in the preparation of a product for detecting the ability of cicada confusa to synthesize beauvericin, wherein the nucleotide sequence of the cicada confusa histone deacetylase gene PP558224 is shown in SEQ ID NO.

1.

5. A primer set for detecting pathogenicity of Spodoptera litura, characterized in that: It includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.10 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.

11.

6. A method for detecting the pathogenicity of cicada fungus to Spodoptera litura, characterized in that: The following steps are involved: The cicada fungus gene is amplified using the primer set of claim 5. If a 1.8 kb band can be amplified, it indicates that the pathogenicity to Spodoptera litura is normal; If the 1.8kb band cannot be amplified, it means that the pathogenicity to Spodoptera litura is weak.

7. A fungal gene knockout vector, characterized in that: The fungal gene knockout vector is a knockout vector targeting the histone deacetylase gene PP558224 of Cicadae Convolvulus, and the nucleotide sequence of the histone deacetylase gene PP558224 of Cicadae Convolvulus is shown in SEQ ID NO.

1.

8. A fungal gene overexpression vector, characterized in that: The fungal gene overexpression vector is an overexpression vector targeting the Cordyceps sinensis histone deacetylase gene PP558224, and the nucleotide sequence of the Cordyceps sinensis histone deacetylase gene PP558224 is shown in SEQ ID NO.

1.

9. Use of the fungal gene knockout vector according to claim 7 or the fungal gene overexpression vector according to claim 8 in preparing a product for regulating the content of Beauvericin in Cicadae.

10. A genetically engineered host cell, characterized in that The Cordyceps sinensis histone deacetylase gene PP558224 is overexpressed in the genome of the host cell, and the nucleotide sequence of the Cordyceps sinensis histone deacetylase gene PP558224 is shown in SEQ ID NO.1.

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