Filamentous fungus genetic transformation system taking endogenous KatG1 gene as selection marker, method and application

Abstract

The invention discloses a filamentous fungus genetic transformation system taking an endogenous KatG1 gene as a selection marker, a method and application. The method comprises the following steps: carrying out linear DNA fragment construction on an endogenous Pgpd promoter, an endogenous KatG1 gene, a target promoter and a target gene of filamentous fungi, then transforming the constructed linear DNA fragment into filamentous fungi cells, and finally screening through a menadione-containing culture medium to obtain the directionally improved filamentous fungi strain containing the target gene. According to the invention, the endogenous KatG1 gene of the fungus is used as a selection marker gene and menadione is used as screening resistance to construct a novel selection marker based on an endogenous gene and a genetic transformation system without exogenous DNA (Deoxyribose Nucleic Acid) such as a carrier and the like, so that the method has the advantages of universality, high efficiency and safety; an environment-friendly solution is provided for directional genetic improvement of filamentous fungi, and the method has important commercial application potential in directional breeding of fungus strains in the fields of agriculture, industry and the like.

Description

Technical Field

[0001] This invention belongs to the field of fungal genetic engineering technology, and relates to a genetic transformation system, method and application of filamentous fungi using the endogenous KatG1 gene as a selection marker. Background Technology

[0002] Filamentous fungi, as an important microbial resource, play an indispensable role in many fields, including industry (such as enzyme preparations and organic acid fermentation), agriculture (such as biological control and plant growth promotion), and biological research (such as cell differentiation and secondary metabolic regulation). With the rapid development of genetic engineering technology, gene modification of filamentous fungi has become a core method for targeted improvement of their production performance, research on gene function, and development of novel applications. However, traditional fungal genetic transformation technology has a significant limitation and potential risks.

[0003] In conventional transformation procedures, to efficiently screen cells that have successfully integrated the exogenous target gene, the target gene is typically linked to a selectable marker gene and attached to a plasmid vector before co-transformation into the host. These selectable marker genes enable fungi to develop resistance to antibiotics (such as hygromycin, provided by the hygromycin phosphotransferase encoded by the hpt gene) or herbicides (such as phosphinic acid, provided by the bar gene). By adding the appropriate antibiotic or herbicide to the selective medium, untransformed wild-type cells are killed, while transformants carrying only the selectable marker gene survive and grow.

[0004] However, this genetic transformation strategy, which relies on antibiotic / herbicide resistance genes as selection markers combined with plasmid vectors, raises serious biosafety concerns in the application of genetically modified fungi, especially during environmental release or large-scale commercial applications. Once released into the environment, these exogenous resistance genes or plasmid DNAs may spread to other microorganisms (including potential pathogens or environmental microorganisms) through horizontal gene transfer. This not only exacerbates the spread of antibiotic resistance in the environment, threatening human and animal health, but may also cause unpredictable disruptions to ecological balance and biodiversity, thus significantly hindering the safety assessment and commercialization of related genetically modified fungal products. To overcome the biosafety risks posed by traditional selection markers, developing safe and efficient genetic transformation technologies without exogenous resistance selection markers and plasmid vectors is crucial, and breakthroughs are urgently needed in light of this critical need. Summary of the Invention

[0005] To overcome the biosafety risks associated with traditional selection markers and to develop safe and efficient exogenous resistance-free selection markers and plasmid vectors, this invention constructs the Pgpd-KatG1 selection marker system. The core principle of this system is the linear integration of the endogenous gene KatG1 (bifunctional catalase-peroxidase) with a highly efficient endogenous Pgpd promoter (derived from the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene) into fungal cells. On a selective medium containing an appropriate amount of menadione, successfully transformed fungal cells exhibit significant oxidative stress resistance due to high-level expression of the KatG1 enzyme, effectively decomposing reactive oxygen species generated by menadione, thus enabling normal growth and colony formation.

[0006] This system possesses versatility and application value, applicable to a variety of filamentous fungi of significant economic and ecological importance, including entomopathogenic fungi (such as *Beauveria bassiana* and *Metarhizium* spp.) and industrial microorganisms (such as *Trichoderma harzianum* and *Aspergillus niger*). In these diverse species, genetic transformation using the Pgpd-KatG1 system consistently yielded positive transformants exhibiting significantly enhanced tolerance on menadione-containing media. Therefore, the Pgpd-KatG1 system, as a safe, universally applicable, and exogenous DNA-free genetic transformation technology, holds broad application prospects in the field of filamentous fungal gene-directed modification, providing strong technical support for the development of environmentally friendly genetically modified strains.

[0007] Meanwhile, to improve transformation efficiency and expand the range of applicable strains, this invention employs electroporation. Compared to traditional methods relying on Agrobacterium-mediated transformation (ATMT), electroporation uses a physical high-voltage pulse to instantaneously create reversible pores in the cell membrane, allowing linear DNA to enter the cell more directly and efficiently. This method significantly shortens the time to obtain transformants and eliminates dependence on the complex and specific natural interaction mechanisms between Agrobacterium and fungi. This provides a more universal and efficient transformation pathway for strains that are insensitive to Agrobacterium (such as many yeasts) or that are difficult to co-culture with Agrobacterium (such as some specific filamentous fungi).

[0008] Therefore, one objective of this invention is to provide a linear DNA fragment comprising an endogenous Pgpd gene promoter from filamentous fungi and an endogenous KatG1 gene from the same fungi, wherein the endogenous Pgpd gene promoter drives the expression of the endogenous KatG1 gene in the host fungus. The KatG1 gene is a bifunctional catalase-peroxidase gene with oxidative stress resistance function, derived from endogenous genes in various species. In this invention, it serves as a selection marker gene, and the fungus overexpresses its own bifunctional enzyme gene KatG1 via its own Pgpd gene promoter, without containing any exogenous resistance marker genes (such as antibiotics or herbicides).

[0009] Furthermore, the aforementioned linear DNA fragment also includes a target promoter and a target gene, wherein the target promoter drives the expression of the target gene in the host fungus. This target gene can be a gene for targeted modification of filamentous fungi. Subsequent embodiments use the Trpc promoter and the EGFP reporter gene as the target promoter and target gene, respectively, to illustrate the screening method and its effects.

[0010] Preferably, the filamentous fungus is any one of Beauveria bassiana, Metarhizium anisopliae, Metarhizium acridum, Trichoderma harzianum, and Aspergillus niger.

[0011] Preferably, the sequence of the endogenous Pgpd gene promoter of *Beauveria bassiana* is shown in SEQ ID NO.1; the sequence of the endogenous Pgpd gene promoter of *Metarhizium anisopliae* is shown in SEQ ID NO.2; the sequence of the endogenous Pgpd gene promoter of *Metarhizium anisopliae* is shown in SEQ ID NO.2; the sequence of the endogenous Pgpd gene promoter of *Trichoderma harzianum* is shown in SEQ ID NO.3; and the sequence of the endogenous Pgpd gene promoter of *Aspergillus niger* is shown in SEQ ID NO.4.

[0012] Preferably, the nucleotide sequence of the endogenous KatG1 gene of *Beauveria bassiana* is shown in SEQ ID NO. 5, or the encoded amino acid sequence is shown in SEQ ID NO. 12; the nucleotide sequence of the endogenous KatG1 gene of *Metarhizium anisopliae* is shown in SEQ ID NO. 6, or the encoded amino acid sequence is shown in SEQ ID NO. 13; the nucleotide sequence of the endogenous KatG1 gene of *Metarhizium anisopliae* is shown in SEQ ID NO. 7, or the encoded amino acid sequence is shown in SEQ ID NO. 14; the nucleotide sequence of the endogenous KatG1 gene of *Trichoderma harzianum* is shown in SEQ ID NO. 8, or the encoded amino acid sequence is shown in SEQ ID NO. 15; and the nucleotide sequence of the endogenous KatG1 gene of *Aspergillus niger* is shown in SEQ ID NO. 9, or the encoded amino acid sequence is shown in SEQ ID NO. 16.

[0013] A second objective of this invention is to provide a Pgpd-KatG1 screening system comprising a linear DNA fragment as described in any one of claims 1-5, wherein the linear DNA fragment is transformed into a host fungus and then used as a selection transformant for resistance. This system uses the endogenous KatG1 gene as a selection marker gene and contains no exogenous resistance marker genes (such as antibiotics or herbicides) or plasmid vector DNA.

[0014] A third objective of this invention is to provide the application of the above-mentioned Pgpd-KatG1 screening system in the screening of fungal genetic transformation systems.

[0015] The fourth objective of this invention is to provide a gene-directed modified filamentous fungal strain without exogenous DNA, wherein the genome of the strain integrates the aforementioned linear DNA fragment and does not carry any exogenous resistance marker genes or plasmid vector DNA.

[0016] The fifth objective of this invention is to provide a method for genetic transformation of filamentous fungi using endogenous KatG1 as a selection gene, comprising the following steps:

[0017] 1) Construct the linear DNA fragment according to any one of claims 1-5;

[0018] 2) Preparation of competent cells of filamentous fungi;

[0019] 3) Transform the linear DNA fragment obtained in step 1) into the filamentous fungal competent cells prepared in step 2);

[0020] 4) The competent cells from step 3) were screened with menaquinone to obtain positive transformants expressing the endogenous KatG1 gene selection marker.

[0021] Preferably, the linear DNA fragment in step 1) is obtained by ligating the sequences through fusion PCR.

[0022] Preferably, the conversion in step 3) is an electrostatic conversion.

[0023] Preferably, the parameters of the electric shock conversion are: electric field strength 1.0-2.5kV / cm, capacitance 25-50μF, and resistance 200-600Ω.

[0024] Preferably, the screening with menadione in step 4) involves adding menadione to the culture medium, i.e., menadione is used as a screening resistant agent. The screening concentration of menadione can be the lowest inhibitory concentration against wild-type strains under the same culture conditions.

[0025] This invention provides a genetic transformation system, method, and application for filamentous fungi using the endogenous KatG1 gene as a selection marker. The system involves constructing a linear DNA fragment from the endogenous Pgpd promoter, the endogenous KatG1 gene, the target promoter, and the target gene of filamentous fungi. This fragment is then transformed into filamentous fungal cells, and finally, selected using a medium containing menadione, yields a targeted modified filamentous fungal strain containing the target gene. This invention utilizes the fungus's own endogenous KatG1 gene as a selection marker gene and menadione as a selection resistant molecule to construct a novel, safe selection marker-based genetic transformation system that does not contain exogenous DNA such as vectors. This system is universal, efficient, and safe, providing an environmentally friendly solution for the targeted genetic improvement of filamentous fungi and possessing significant commercial application potential in the targeted breeding of fungal strains in agriculture, industry, and other fields. Attached Figure Description

[0026] Figure 1 This invention relates to the determination of the antioxidant capacity of the Metarhizium anisopliae MaKatG1 overexpression strain;

[0027] Figure 2 The present invention screens the concentration of tolerance of Metarhizium anisopliae CQMa102 to menadione;

[0028] Figure 3 This is an agarose gel electrophoresis image of the PgpdM-MaKatG1-Trpc-EGFP DNA fragment of this invention;

[0029] Figure 4 PCR verification of the Metarhizium anisopliae CQMa102 transformant of this invention;

[0030] Figure 5 Fluorescence observation of the transformant CQMa102 of Metarhizium anisopliae in this invention;

[0031] Figure 6The growth of the wild-type strain and transformant of Metarhizium anisopliae CQMa102 in 1 / 4 SDAY medium with 40 μg / mL menopausal quinone was observed for the present invention.

[0032] Figure 7 The present invention screens the concentration of tolerance of Metarhizium anisopliae CQMa421 to menadione.

[0033] Figure 8 PCR verification of the Metarhizium anisopliae transformants of this invention;

[0034] Figure 9 For the observation of fluorescence of Metarhizium anisopliae transformants in this invention;

[0035] Figure 10 This invention is for screening the concentration of beauveria bassiana to tolerate menaquinone.

[0036] Figure 11 PCR verification of Beauveria bassiana transformants for this invention;

[0037] Figure 12 For the observation of Beauveria bassiana transformants in this invention;

[0038] Figure 13 This invention screens for the concentration tolerance of Trichoderma harzianum to menaquinone.

[0039] Figure 14 PCR verification of Trichoderma harzianum transformants in this invention;

[0040] Figure 15 For the observation of fluorescence of Trichoderma harzianum transformants in this invention;

[0041] Figure 16 This invention screens the concentration of Aspergillus niger to menaquinone for tolerance.

[0042] Figure 17 PCR verification of Aspergillus niger transformants for this invention;

[0043] Figure 18 Fluorescence observation of Aspergillus niger transformants for this invention. Detailed Implementation

[0044] The present invention will be described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples. Any modifications and variations made without departing from the spirit of the present invention should be included within the scope of the present invention. Unless otherwise specified, the experimental materials or reagents used in the following embodiments are commercially available.

[0045] The strains, culture media, and primers used in this embodiment are as follows:

[0046] Strains:

[0047] Beauveria bassiana ZJU435 (Chongqing Julixin Bioengineering Co., Ltd.);

[0048] Metarhizium anisopliae CQMa102 (Chongqing University Gene Engineering Research Center);

[0049] Metarhizium anisopliae CQMa421 (Chongqing Julixin Bioengineering Co., Ltd.);

[0050] Trichoderma harzianum XZN213-1 (Chongqing Julixin Bioengineering Co., Ltd.);

[0051] Aspergillus niger (isolated from the Biological Experiment Base of the Gene Engineering Research Center of Chongqing University, GenBank ID: PV946143).

[0052] Culture medium:

[0053] 1 / 4 SDAY medium (1g glucose, 0.5g yeast extract, 0.25g peptone, 1.8g agar powder, bring to 100mL, pH 6.4); Czapek's medium (0.3g sodium nitrate, 0.05g potassium chloride, 0.001g ferrous sulfate, 0.05g magnesium sulfate heptahydrate, 3g sucrose, 0.1g dipotassium hydrogen phosphate, bring to 100mL); PDA medium (20g potato, 2g glucose; 2g agar, bring to 100mL).

[0054] Primer information (Table 1, where lowercase letters represent homologous arm sequences):

[0055] Table 1 Primer sequence information

[0056]

[0057] Example 1: Genetic transformation of Metarhizium anisopliae using the Pgpd-KatG1 screening system

[0058] 1.1 Method

[0059] 1.1.1 Construction of Linear DNA Fragments

[0060] Using the genome and cDNA of *Metarhizium anisopliae* CQMa102 as templates, and primers PgpdM-F / PgpdM-R and MaKatG1-F / MaKatG1-R, the promoter fragment PgpdM of the *Metarhizium anisopliae* Pgpd gene and the sequence MaKatG1 of the *Metarhizium anisopliae* KatG1 gene were amplified, respectively. The Trpc and EGFP gene sequences, as shown in SEQ ID NO.10 and SEQ ID NO.11, were amplified from the PK2 plasmid (provided by the Gene Engineering Center of Chongqing University), using primers Trpc-F / Trpc-R and EGFP-F / EGFP-R, respectively. The KatG1, Trpc, and EGFP gene fragments containing homologous arms were amplified using primers P-MaKatG1 / MaKatG1-R, M-Trpc / Trpc-R, and T-EGFP / EGFP-R, respectively. Then, the PgpdM fragment and the three fragments containing homologous arms were fused by PCR. The fusion PCR reaction program is shown in Table 2. 2 μL of each fragment was added at a molar ratio of 1:1:1:1. 2 μL of primers PgpdM-F / EGFP-R were added, along with 25 μL of high-fidelity enzyme and ddH2O, to prepare a 50 μL total system for the fusion PCR reaction, yielding the PgpdM-MaKatG1-Trpc-EGFP linear DNA fragment. Using the obtained fusion PCR product as a template, PgpdM-F / EGFP-R was used as primers for further amplification to obtain a large number of linear DNA fragments. The reaction system and program are shown in Tables 3 and 4. PCR products were detected by 1% agarose gel electrophoresis, and observed and photographed using a gel imaging system.

[0061] Table 2 Fusion PCR Amplification Reaction Procedure

[0062]

[0063] Table 3 PCR amplification reaction system

[0064]

[0065] Table 4 PCR amplification reaction procedure

[0066]

[0067] 1.1.2 Preparation of competent cells of Metarhizium anisopliae

[0068] Mature spores of *Metarhizium anisopliae* CQMa102 cultured for 15 days were prepared with 0.05% Tween 80, imbibed overnight, and then inoculated into 1 / 4 SDAY liquid medium. The culture was incubated at 28°C with shaking at 200 rpm until germination rate >85%. The spores were centrifuged at 6500 rpm for 5 min at room temperature, and the supernatant was discarded. The spores were resuspended in PBS buffer and washed, then centrifuged at 6500 rpm for 5 min, repeated once. The spores were resuspended in 10 mL of enzyme digest (1 M sorbitol, 50 mM MMEs, 0.5% snailase, 0.5% lyase, 5 mM DTT, pH adjusted to 5.5), incubated at 28°C with shaking for 2.5 h, centrifuged at 200 rpm, 5000 rpm, 4°C for 5 min, and the supernatant was discarded. The spores were washed with pre-chilled 1 M sorbitol, centrifuged at 5000 rpm, 4°C for 5 min, and the supernatant was discarded, repeated once. The spores were further washed with pre-chilled sorbitol, centrifuged at 5000 rpm, 4°C for 5 min, and the supernatant was discarded. Pre-cooled sorbitol was used to resuspend competent spores to a final concentration of 1×10⁻⁶. 7 per mL.

[0069] 1.1.3 Electroconversion

[0070] Add 1 μg of linear plasmid to 100 μL of competent cells, incubate on ice for 5 min, then transfer to a 0.2 cm electroporation cuvette and perform electroporation transformation using the parameters (V = 2 kV, R = 600 Ω, C = 50 μF). Transfer the sample to a 1.5 mL centrifuge tube, add 800 μL of pre-chilled 1 / 4 SDAY liquid, and incubate at 28 °C, 200 rpm, shaking for 4 h. Centrifuge at 5000 rpm for 3 min and discard the supernatant. Resuspend in 1 mL ddH2O, and evenly spread 100 μL into 10 Czapek's plates containing menadione resistance. Incubate at 28 °C for 7–12 days.

[0071] 1.1.4 Transformer Screening

[0072] PCR Validation: Single colonies grown on menaquinone-resistant Czapek-Massachus medium from electroporation were picked and inoculated into new menaquinone-resistant Czapek-Massachus medium for secondary screening. After 3-5 days of growth, a small portion of each single colony was added to a centrifuge tube containing 400 μL of 1 / 4 SDAY liquid. The culture was incubated at 28°C for 2-3 days, followed by centrifugation at 12000 rpm for 10 min to collect fungal mycelia for micro-extraction validation. The steps were as follows: After freezing the fungal mycelial sample in liquid nitrogen, it was rapidly ground (60 Hz, 60 s, 3 times). Immediately, 400 μL of lysis buffer was added, and the mixture was thoroughly mixed with a pipette. Lysis was performed overnight at 37°C. 200 μL of potassium acetate was added to neutralize the lysis buffer. After incubation on ice for 10 min, the mixture was centrifuged at 12000 rpm for 10 min, and the supernatant was collected. An equal volume of pre-chilled isopropanol was added, and the mixture was incubated at 4°C for 30 min, centrifuged at 12000 rpm for 10 min, and the supernatant was discarded. Add 500 μL of 70% ethanol to wash the DNA, centrifuge at 12000 rpm for 2 min, discard the supernatant, and repeat once. Evaporate the ethanol, add 30 μL of ddH2O, centrifuge at 12000 rpm for 1 min to collect the DNA solution. Verify the transformants using primers EGFP-F / EGFP-R.

[0073] Fluorescence verification: Use an inoculation loop to take a small amount of colony and spread it on a glass slide with 10% glycerol, press it with a coverslip, and observe it under a fluorescence microscope.

[0074] 1.1.5 Validation of resistance in overexpression strains

[0075] To further verify the screening effect of menadione, spore suspensions of wild-type and transformants were prepared under aseptic conditions to a final concentration of 1×10⁻⁶. 6 The sample was taken at a concentration of 1 / 4 SDAY medium containing menaquinone and incubated at 28°C for 7 days. The growth of each strain was then observed.

[0076] 1.2 Results

[0077] 1.2.1 Specific sensitivity of Metarhizium anisopliae to menaquinone

[0078] Based on previous observations that overexpression of the *Metarhizium anisopliae* MaKatG1 gene exhibits specific sensitivity to menadione inhibition, the potential of menadione as a genetic transformation screening marker was further evaluated. Using the PK2 vector (available from the Chongqing University Gene Engineering Center) stored in the inventors' laboratory, an overexpressing strain of the MaKatG1 gene (OE strain) was constructed. Specifically, the *Metarhizium anisopliae* MaKatG1 gene fragment (as shown in SEQ ID NO. 6) was inserted into the PK2 vector, resulting in the plasmid PgpdM-KatG1-Bar. This plasmid functions as a PgpdM promoter (as shown in SEQ ID NO. 2) driving MaKatG1 gene expression, with the Bar gene as the resistance selection tag. Five overexpressing strains (OE-1, OE-11, OE-28, OE-33, OE-51) were obtained. The tolerance of the overexpressing strains to different oxidants was then tested, with a concentration of 1×10⁻⁶. 7 Wild-type (WT) strains, OE-1, OE-11, OE-28, OE-33, and OE-51 strains were prepared at a concentration of 1 / mL. 2 μL of each strain suspension was inoculated onto 1 / 4 SDAY plates supplemented with hydrogen peroxide (H₂O₂, final concentration 6 mmol / L) and menadione (MND, final concentration 0.076 mmol / L), respectively. After air-drying, the plates were incubated at 28°C in the dark. Data were observed and photographed at time intervals. Each treatment was repeated three times, and the experiment was repeated three times. Results are shown below. Figure 1 As shown, in the presence of hydrogen peroxide, the growth of both the MaKatG1 overexpressing strains (OE-1, OE-11, OE-28, OE-33, OE-51) and the wild-type strain (WT) was inhibited, but there was no significant difference in the inhibition rate between the two, indicating that increasing the expression level of MaKatG1 did not significantly change the sensitivity of Metarhizium anisopliae to hydrogen peroxide. In the presence of menadione, the growth of both MaKatG1 overexpressing strains and WT strains was inhibited. However, the growth inhibition rates of each OE strain (OE-1, OE-11, OE-28, OE-33, and OE-51 strains were 16.7±9.1, 21.6±3.3, 17.8±6.8, 29.6±9.1, and 30.3±7.3%, respectively) were significantly lower than those of WT (71.2±13.7%) (p<0.05). This indicates that increasing the expression level of MaKatG1 can reduce the sensitivity of Metarhizium anisopliae to menadione oxidants and shows specific sensitivity to menadione inhibition.

[0079] 1.2.2 Screening of Metarhizium anisopliae CQMa102 for tolerance concentration to menaquinone

[0080] To determine the appropriate concentration of menadione for use in the transformation process of *Metarhizium anisopliae*, the minimum inhibitory concentration (MIC) of menadione against wild-type *Metarhizium anisopliae* CQMa102 was determined. Prepare 1×10⁻⁶ [amount not specified]. 6 A spore suspension of 1 / mL was spread on 1 / 4 SDAY and Czapek-Dox Agar (CDA) media containing 0, 5, 10, 20, and 40 μg / mL menadione. Colony growth was observed after 7 days of incubation. The results showed that menadione inhibited the growth of *Metarhizium anisopliae* under both nutrient-rich and nutrient-poor conditions, with minimum inhibitory concentrations of 40 and 20 μg / mL, respectively. Figure 2 ) 1 / 4 SDAY medium and Czapek's medium containing 40 μg / mL and 20 μg / mL of menadione, respectively, were selected as subsequent screening media.

[0081] 1.2.3 Obtaining Linear DNA

[0082] Linear DNA fragments were constructed according to the method in 1.1.1, such as... Figure 3 As shown, the marker is 5000bp, and bands of approximately 5000bp were observed in lanes 1-3 of the samples. The actual size of the PgpdM-MaKatG1-Trpc-EGFP linear DNA fragment is 4752bp, which is consistent with the electrophoresis results, indicating that the linear DNA fragment was successfully constructed.

[0083] 1.2.4 Verification of positive transformants of Metarhizium anisopliae CQMa102

[0084] Electroporation was performed using Czapek-Doxorubicin resistant petri dishes containing 20 μg / mL menadione. Fourteen transformants were selected for secondary screening, genome extraction, and PCR verification. The results showed that six transformants exhibited a band around 800 bp. Figure 4 The actual length of EGFP was 726 bp, consistent with the electrophoresis results, indicating that the linear DNA fragment was transformed into *Metarhizium anisopliae*. Further fluorescence observation of the six transformants revealed green fluorescence in all six, while it was not observed in the wild-type strain (WT). Figure 5 It can be seen that PgpdM-MaKatG1-Trpc-EGFP was successfully transformed into Metarhizium anisopliae, with a positive rate of 42.85% and a transformation efficiency of 6 cells / μg DNA. 1.2.5 Validation of resistance in overexpression strains

[0085] To directly verify the screening of menaquinone, positive transformant #14 (MaKatG1) was randomly selected. OE Co-inoculated with WT on 1 / 4 SDAY plates containing 40 μg / mL menadione, the results showed that transformants grew normally while wild-type growth was significantly inhibited. Figure 6This further demonstrates that menaquinone can specifically screen for transformants overexpressing MaKatG1.

[0086] Example 2: Genetic transformation of Metarhizium anisopliae in scarab beetles using the Pgpd-KatG1 screening system

[0087] 2.1 Method

[0088] The experimental method was the same as in Example 1. The linear DNA fragment of Metarhizium anisopliae was PgpdM-421KatG1-Trpc-EGFP.

[0089] 2.2 Results

[0090] 2.2.1 Screening of Tolerance Concentrations of Metarhizium anisopliae CQMa421 to Menadione

[0091] To determine the appropriate concentration of menadione for use in the transformation process of *Metarhizium anisopliae*, the minimum inhibitory concentration (MIC) of menadione against wild-type *Metarhizium anisopliae* was determined. Prepare 1×10⁻⁶ [amount not specified]. 6 Spore suspensions of 1 / mL were spread on 1 / 4 SDAY medium containing 0, 100, 120, 140, 160, and 180 μg / mL menadione, and on Czapek's medium containing 0, 25, 50, 75, 100, and 125 μg / mL menadione. Colony growth was observed after 7 days of incubation. The results showed that menadione inhibited the growth of *Metarhizium anisopliae*, with minimum inhibitory concentrations of 140 and 100 μg / mL in 1 / 4 SDAY and Czapek's medium, respectively. Figure 7 ) 1 / 4 SDAY medium and Czapek's medium containing 140 μg / mL and 100 μg / mL menadione, respectively, were selected as subsequent screening media.

[0092] 2.2.2 Validation of Metarhizium anisopliae CQMa421 transformant

[0093] Electroporation was performed using Czapek-Doxorubicin resistant plates containing 100 μg / mL menadione. Ten transformants were selected for secondary screening, genome extraction, and PCR verification. The results showed that four transformants exhibited a band around 800 bp. Figure 8 The actual length of EGFP was 726 bp, consistent with the electrophoresis results, indicating that the linear DNA fragment was transformed into *Metarhizium anisopliae*. Further fluorescence observation of the four transformants revealed green fluorescence in all four, while it was not observed in the wild-type strain (WT). Figure 9 PgpdM-421KatG1-Trpc-EGFP was successfully transformed into Metarhizium anisopliae, with a positive transformation rate of 40% and a transformation efficiency of 4 cells / μg DNA.

[0094] Example 3: Genetic transformation in Beauveria bassiana using the Pgpd-KatG1 screening system

[0095] 3.1 Method

[0096] The experimental method was the same as in Example 1. The linear DNA fragment of Beauveria bassiana was PgpdB-BbKatG1-Trpc-EGFP.

[0097] 3.2 Results

[0098] 3.2.1 Screening of Beauveria bassiana concentrations for tolerance to menaquinone

[0099] To determine the appropriate concentration of menadione for use in the transformation process of Beauveria bassiana, the minimum inhibitory concentration (MIC) of menadione against wild-type Beauveria bassiana was determined. Prepare 1×10⁻⁶ [amount not specified]. 6 Spore suspensions of [number] cells / mL were spread on 1 / 4 SDAY medium containing 0, 20, 40, 60, and 80 μg / mL menadione and on Czapek's medium containing 0, 5, 10, 15, and 20 μg / mL. Colony growth was observed after 7 days of incubation. The results showed that menadione inhibited the growth of *Beauveria bassiana* under both nutrient-rich and nutrient-poor conditions, with minimum inhibitory concentrations of 80 and 15 μg / mL, respectively. Figure 10 ) 1 / 4 SDAY medium and Czapek's medium containing 80 μg / mL and 15 μg / mL menadione, respectively, were selected as subsequent screening media.

[0100] 3.2.2 Validation of Beauveria bassiana transformants

[0101] Electroporation was performed using Czapek-Doxorubicin resistant plates containing 15 μg / mL menaquinone. Four transformants were selected for secondary screening, genome extraction, and PCR verification. The results showed that only transformant #3 had a band around 800 bp. Figure 11 The actual length of EGFP was 726 bp, consistent with the electrophoresis results, indicating that the linear DNA fragment was transformed into Beauveria bassiana. Further observation of green fluorescence was observed in transformant #3, while it was not observed in the wild-type strain (WT). Figure 12 PgpdB-BbKatG1-Trpc-EGFP was successfully transformed into Beauveria bassiana, with a positive transformation rate of 25% and a transformation efficiency of 1 cell / μg DNA.

[0102] Example 4: Genetic transformation in Trichoderma harzianum using the Pgpd-KatG1 screening system

[0103] 4.1 Methods

[0104] The experimental method was the same as in Example 1. The linear DNA fragment of Trichoderma harzianum was PgpdT-ThKatG1-Trpc-EGFP.

[0105] 4.2 Results

[0106] 4.2.1 Screening of Trichoderma harzianum concentrations for tolerance to menaquinone

[0107] To determine the appropriate concentration of menadione for use in the transformation process of *Trichoderma harzianum*, the minimum inhibitory concentration (MIC) of menadione against wild-type *Trichoderma harzianum* was determined. Prepare 1×10⁻⁶ [amount not specified]. 6 Spore suspensions of 1 / mL were spread on 1 / 4 SDAY medium containing 0, 100, 200, 300, 400, and 500 μg / mL menadione and on Czapek's medium containing 0, 5, 10, 15, 20, and 25 μg / mL. Colony growth was observed after 7 days of incubation. The results showed that *Trichoderma harzianum* required high concentrations of menadione for complete inhibition on nutrient-rich 1 / 4 SDAY plates, with a minimum inhibitory concentration of 400 μg / mL. However, on nutrient-poor Czapek's plates, growth was only observed at 0 μg / mL. Figure 13 ) 1 / 4 SDAY medium and Czapek's medium containing 400 μg / mL and 5 μg / mL of menadione, respectively, were selected as subsequent screening media.

[0108] 4.2.2 Validation of Trichoderma harzianum transformants

[0109] Electroporation was performed using Czapek-Dox dishes resistant to menaquinone (5 μg / mL). One *Trichoderma harzianum* transformant was screened a second time, its genome extracted, and validated by PCR. The results showed a band around 800 bp. Figure 14 The actual length of EGFP was 726 bp, consistent with the electrophoresis results, indicating that the linear DNA fragment was transformed into *Trichoderma harzianum*. Further observation of green fluorescence was observed in transformant #1, while it was not observed in the wild-type strain (WT). Figure 15 PgpdT-ThKatG1-Trpc-EGFP was successfully transformed into Trichoderma harzianum with a positive transformation rate of 100% and a transformation efficiency of 1 cell / μg DNA.

[0110] Example 5: Genetic transformation of Aspergillus niger using the Pgpd-KatG1 screening system

[0111] 5.1 Method

[0112] The experimental method was the same as in Example 1. The linear DNA fragment of Aspergillus niger was PgpdA-AnKatG1-Trpc-EGFP.

[0113] 5.2 Results

[0114] 5.2.1 Screening of Aspergillus niger concentrations for tolerance to menaquinone

[0115] To determine the appropriate concentration of menadione for use during the transformation process of Aspergillus niger, the minimum inhibitory concentration (MIC) of menadione against wild-type Aspergillus niger was determined. Prepare 1×10⁻⁶ [amount not specified]. 6Spore suspensions of 1 / mL were spread on PDA medium containing 0, 25, 50, 75, 100, and 125 μg / mL menadione and on Czapek's medium containing 0, 10, 20, 30, 40, and 50 μg / mL. Colony growth was observed after 7 days of incubation. The results showed that the minimum inhibitory concentrations in PDA and Czapek's medium were 100 and 20 μg / mL, respectively. Figure 16 PDA medium and Czapek's medium containing 100 μg / mL and 20 μg / mL of menadione, respectively, were selected as subsequent screening media.

[0116] 5.2.2 Verification of Aspergillus niger transformants

[0117] Electroporation was performed using Czapek-Doxorubicin resistant plates containing 20 μg / mL menaquinone. One *Aspergillus niger* transformant was subjected to secondary screening, genome extraction, and PCR verification. The results showed a band around 800 bp. Figure 17 The actual length of EGFP was 726 bp, consistent with the electrophoresis results, indicating that the linear DNA fragment was transformed into Aspergillus niger. Further observation of green fluorescence was observed in transformant #1, while it was not observed in the wild-type strain (WT). Figure 18 PgpdA-AnKatG1-Trpc-EGFP was successfully transformed into Aspergillus niger with a positive transformation rate of 100% and a transformation efficiency of 1 cell / μg DNA.

[0118] In summary, constructing a linear DNA fragment from the endogenous KatG1 gene of filamentous fungi and overexpressing it in competent cells of filamentous fungi does not improve the resistance of filamentous fungi to hydrogen peroxide inhibition, but it can significantly improve the resistance of filamentous fungi under menadione inhibition conditions, enabling them to tolerate oxidative stress and grow. This method yields gene-directed modified fungal strains that are completely free of exogenous resistance markers and has strong universality, making them suitable as selection markers.

[0119] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art, and therefore will not be elaborated upon here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A linear DNA fragment, characterized in that, The device includes an endogenous Pgpd gene promoter from filamentous fungi and an endogenous KatG1 gene from the same filamentous fungus, wherein the endogenous Pgpd gene promoter drives the expression of the endogenous KatG1 gene in the host fungus.

2. The fragment as described in claim 1, characterized in that, It also includes a target promoter and a target gene, wherein the target promoter drives the expression of the target gene in a host fungus.

3. The fragment as described in claim 1, characterized in that, The filamentous fungi include any one of Beauveria bassiana, Metarhizium anisopliae, Metarhizium spp., Trichoderma harzianum, and Aspergillus niger.

4. The fragment as described in claim 3, characterized in that, The sequence of the endogenous Pgpd gene promoter of *Beauveria bassiana* is shown in SEQ ID NO.1; the sequence of the endogenous Pgpd gene promoter of *Metarhizium anisopliae* is shown in SEQ ID NO.2; the sequence of the endogenous Pgpd gene promoter of *Metarhizium anisopliae* is shown in SEQ ID NO.2; the sequence of the endogenous Pgpd gene promoter of *Trichoderma harzianum* is shown in SEQ ID NO.3; and the sequence of the endogenous Pgpd gene promoter of *Aspergillus niger* is shown in SEQ ID NO.

4.

5. The fragment as described in claim 3, characterized in that, The nucleotide sequence of the endogenous KatG1 gene of *Beauveria bassiana* is shown in SEQ ID NO. 5, or the encoded amino acid sequence is shown in SEQ ID NO. 12; the nucleotide sequence of the endogenous KatG1 gene of *Metarhizium anisopliae* is shown in SEQ ID NO. 6, or the encoded amino acid sequence is shown in SEQ ID NO. 13; the nucleotide sequence of the endogenous KatG1 gene of *Metarhizium anisopliae* is shown in SEQ ID NO. 7, or the encoded amino acid sequence is shown in SEQ ID NO. 14; the nucleotide sequence of the endogenous KatG1 gene of *Trichoderma harzianum* is shown in SEQ ID NO. 8, or the encoded amino acid sequence is shown in SEQ ID NO. 15; the nucleotide sequence of the endogenous KatG1 gene of *Aspergillus niger* is shown in SEQ ID NO. 9, or the encoded amino acid sequence is shown in SEQ ID NO.

16.

6. A Pgpd-KatG1 screening system, characterized in that, The product comprises a linear DNA fragment as described in any one of claims 1-5, wherein the linear DNA fragment is transformed into a host fungus and then selected for resistance selection using menadione.

7. The application of the screening system according to claim 6 in the screening of fungal genetic transformation systems.

8. A gene-directed modified filamentous fungal strain without exogenous DNA, characterized in that, The genome of the strain integrates the linear DNA fragment described in any one of claims 1-5.

9. A method for genetic transformation of filamentous fungi using endogenous KatG1 as a selection gene, characterized in that, Includes the following steps: 1) Construct the linear DNA fragment according to any one of claims 1-5; 2) Preparation of competent cells of filamentous fungi; 3) Transform the linear DNA fragment obtained in step 1) into the filamentous fungal competent cells prepared in step 2); 4) The competent cells from step 3) were screened with menaquinone to obtain positive transformants expressing the endogenous KatG1 gene selection marker.

10. The method as described in claim 9, characterized in that, Step 3) involves conversion to electroshock conversion.

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