Application of transcription factor myb1 in improving hyperparasitism ability of trichoderma harzianum

Abstract

The invention relates to the technical field of microbial genetic engineering, in particular to application of a transcription factor myb1 in improving the hyperparasitism ability of trichoderma harzianum. The nucleotide sequence of the transcription factor myb1 is as shown in SEQ ID NO. 1, and the amino acid sequence of the transcription factor myb1 is as shown in SEQ ID NO. 2. The transcription factor myb1 is overexpressed in the trichoderma harzianum, so that the response of the trichoderma harzianum to a staphylococcus cinereus signal is remarkably improved, and the hyperparasitism capability of the trichoderma harzianum is improved. According to the invention, the regulation function of MYB1 homologous protein in hyperparasitic fungi is focused for the first time, and a research basis is provided for deep understanding of biological functions of MYB family transcription factors.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial genetic engineering, and more particularly to the application of transcription factor myb1 in improving the parasitic ability of Trichoderma harzianum. Background Art

[0002] MYB family transcription factors are a highly conserved class of regulatory proteins in eukaryotes. Their core characteristic is a highly conserved DNA-binding domain composed of approximately 50 amino acids folded into three α-helices. Since their initial identification in avian myeloblastosis virus, the functional diversity of MYB family members in eukaryotes has been gradually revealed, particularly in filamentous fungi, where they have been shown to play a central role in regulating key life processes such as growth and development, environmental responses, and secondary metabolism.

[0003] Recent studies have shown that the MYB family transcription factors of filamentous fungi exert multidimensional functions by targeting different biological processes: at the morphological development level, Aspergillus FlbD regulates asexual reproduction by activating genes related to conidia formation, while Fusarium graminearum GzMYT1 / GzMYT2 are specifically involved in the developmental regulation of sexual fruiting bodies; in pathogenicity regulation, the rice blast fungus MoMYB1 significantly enhances the host infection ability by mediating the differentiation of infected hyphae and the secretion of effector proteins; in addition, its core position in secondary metabolism has also been confirmed. For example, overexpression of the GzMyt2 gene in Fusarium graminearum will affect the production of fungal toxins.

[0004] While the roles of MYB family transcription factors in fungal pathogenicity and metabolic regulation have been partially elucidated, their functional mechanisms in the hyperparasitic behavior of biocontrol fungi remain largely unknown. Hyperparasitic fungi achieve biocontrol by recognizing, infecting, and degrading pathogenic fungi, making them a valuable resource for sustainable agriculture. However, the key molecular components that regulate these interactions are less well understood.

[0005] Therefore, it is of great significance to develop relevant key molecular elements and apply them to enhance the re-hosting ability of biocontrol fungi. Summary of the Invention

[0006] In response to the above problems, the present invention provides a method for improving the heavy parasitic ability of Trichoderma harzianum and its application.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The application of the transcription factor myb1 in improving the parasitic ability of Trichoderma harzianum. The nucleotide sequence of the transcription factor myb1 is shown in SEQ ID NO.1.

[0009] Preferably, the amino acid sequence of the transcription factor myb1 is shown as SEQ ID NO.2.

[0010] Preferably, the response of Trichoderma harzianum to the Botrytis cinerea signal is enhanced by overexpression of the transcription factor myb1.

[0011] Another object of the present invention is to provide a method for improving the parasitic ability of Trichoderma harzianum, comprising: overexpressing the transcription factor myb1 in Trichoderma harzianum, wherein the nucleotide sequence of the transcription factor myb1 is shown in SEQ ID NO.1.

[0012] Preferably, the nucleotide sequence of the transcription factor myb1 is shown as SEQ ID NO.1.

[0013] Preferably, the amino acid sequence of the transcription factor myb1 is shown as SEQ ID NO.2.

[0014] Compared with the prior art, the present invention can achieve the following beneficial effects:

[0015] The present invention successfully identified a new transcription factor MYB1 that directly regulates the biocontrol mechanism of Trichoderma harzianum. Through plate antagonism experiments, it was found that overexpression of myb1 can greatly enhance the antibacterial ability, and the overexpression strain can completely cover the surface of the Botrytis cinerea colony, while the uracil-deficient ΔpyrG strain of Trichoderma harzianum can only cover a small part of the colony surface. Under liquid shake flask and confrontation growth conditions, the whole genome expression differences caused by myb1 overexpression were analyzed, and it was found that overexpression of myb1 upregulated the expression levels of multiple biocontrol-related or potentially related genes. Overexpression of myb1 in Trichoderma harzianum can enhance the strain's ability to parasitize plant pathogenic fungi and activate the expression of biocontrol-related or potentially related genes. The present invention focuses on the regulatory function of MYB1 homologous proteins in parasitic fungi for the first time, providing a research basis for in-depth understanding of the biological functions of MYB family transcription factors. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Electrophoretogram of PCR verification of the uracil-deficient Δpyr4 strain of Trichoderma harzianum, wherein, M: Marker, T22: Trichoderma harzianum T22 wild-type strain, Δpyr4-1: pyrG gene knockout transformant 1, Δpyr4-2: pyrG gene knockout transformant 2, Δpyr4-3: pyrG gene knockout transformant 3; U: upstream fragment verification of the pyr4 knockout cassette of the transformant, D: downstream fragment verification of the pyr4 knockout cassette of the transformant, CDS: coding region fragment verification of the pyr4 transformant;

[0017] Figure 2: Electrophoretogram of PCR verification of the myb1 overexpression mutant strain of Trichoderma harzianum, where M: Marker, +: amplification using the expression cassette as a template, -: amplification using T22 as a template, 1: amplification using transformant 1 as a template, 2: amplification using transformant 2 as a template, 3: amplification using transformant 3 as a template;

[0018] Figure 3 : The results of the plate confrontation between the myb1 overexpression mutant strain and Botrytis cinerea, NC: control group (inoculated with Botrytis cinerea only), 0: the plate confrontation phenotype of the wild-type Trichoderma harzianum pyr4 gene knockout mutant strain ΔpyrG, 1: the plate confrontation phenotype of the Trichoderma harzianum myb1 overexpression mutant strain OEmyb1-1, 2: the plate confrontation phenotype of the Trichoderma harzianum myb1 overexpression mutant strain OEmyb1-2, 3: the Trichoderma harzianum myb1 overexpression mutant strain OEmyb1-3. DETAILED DESCRIPTION

[0019] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0020] The reagents involved in the embodiments of the present invention were all purchased from commercial channels, and the methods not mentioned are conventional experimental methods and will not be described in detail here.

[0021] The following reagents are illustrative:

[0022] Trichoderma harzianum T22 (deposit number ATCC 20847) and Trichoderma reesei QM9414 (deposit number ATCC 26921) are deposited in the American Type Culture Collection (ATCC) and are available for purchase from the corresponding depository institutions.

[0023] The composition of conversion solution S1: 200 g / L sorbitol, 13.6 g / L KH2PO4, pH adjusted to 8.0.

[0024] The composition of conversion solution S2: 182 g / L sorbitol, 5.55 g / L CaCl2, 1.21 g / L Tris, pH adjusted to 7.5.

[0025] The composition of transformation solution S3: 25% (w / v) PEG 6000, 5.55 g / L CaCl2, 1.21 g / L Tris, pH adjusted to 7.5.

[0026] The composition of the transformation medium: 182 g / L sorbitol, 15 g / L glucose, 3 g / L Na3C6H5O7·2H2O, 6 g / L (NH4)2SO4, 10 g / L KH2PO4, 1 g / L MgSO4·7H2O, 100 μL / L trace element stock solution (50 g / L FeSO4·7H2O, 17 g / L MnSO4·H2O, 14 g / L ZnSO4·2H2O, 20 g / L CoCl2), 0.6% agarose.

[0027] The Botrytis cinerea strain is preserved by the Center for Plant Functional Components Research, Tobacco Research Institute, Chinese Academy of Agricultural Sciences. The strain collection number is BC-14. The public can obtain it from this collection institution for scientific research.

[0028] Example 1 Acquisition of myb1 gene

[0029] The MYB-type transcription factor protein sequence of the whole genome of Trichoderma harzianum was downloaded from the JGI (Joint Genome Institute, U.S. Department of Energy Joint Genome Institute) database, and by comparing it with the homologous protein sequence of similar species, it was found that the protein numbered TriharT22_1|467969 (named MYB1) was incorrectly annotated, and the inventors corrected the myb1 gene sequence encoded by it. The sequence of the corrected myb1 gene is shown in SEQ ID NO.1, and its DNA is 1069bp in length and has no introns. The gene encodes a protein comprising 363 amino acids (specific sequence is shown in SEQ ID NO.2), which respectively comprises two MYB-type DNA binding domains and two low complexity domains.

[0030] Example 2 Construction of myb1 overexpression cassette

[0031] 1. Cloning of genetic elements

[0032] (1) Cloning of the coding region and terminator sequence of the transcription factor myb1

[0033] The genome of Trichoderma harzianum T22 was used as a template, and Ptef1-OEmyb1-F (5'-TACATCACACAAACCGTCAAAATGACCGCCGTCTCTCCTC-3', SEQ ID NO. 3) and Ptef1-OEmyb1-R (5'-CGCGTTCTCGAGGAAGTTGATTTCCATCCAAGCCAAGC-3', SEQ ID NO. 4) were used as primers for PCR amplification. The PCR amplification system was as follows: 25 μL buffer, 10 mM dNTP 1μL, 2μL of upstream / downstream primers with a concentration of 10ng / μL, 1μL of high-fidelity DNA polymerase, 1μL of DNA template with a concentration of 100ng / μL, and ddH2O to 50μL; the PCR amplification program is as follows: pre-denaturation at 95℃ for 3min; denaturation at 95℃ for 15s, annealing at 58℃ for 15s, extension at 72℃ for 3min, 30 cycles; complete extension at 72℃ for 5min; cooling at 4℃, and detection of PCR product by agarose gel electrophoresis. The size is 1644bp, which is the correct target fragment amplified.

[0034] (2) Cloning of the tef1 promoter

[0035] The genome of Trichoderma reesei QM9414 was used as a template, and Ptef1-F (5'-CCGAGTTTGCTGGCTACTTAC-3', SEQ ID NO. 5) and Ptef1-R (5'-TTTGACGGTTTGTGTGATGTAGC-3', SEQ ID NO. 6) were used as primers for PCR amplification. The PCR amplification system was as follows: 25 μL buffer, 10 mM dNTP 1μL, 2μL of upstream / downstream primers at a concentration of 10ng / μL, 1μL of high-fidelity DNA polymerase, 1μL of DNA template at a concentration of 100ng / μL, and ddH2O to 50μL; the PCR amplification program is as follows: pre-denaturation at 95℃ for 3min; denaturation at 95℃ for 15s, annealing at 58℃ for 15s, extension at 72℃ for 3min, 30 cycles; complete extension at 72℃ for 5min; cooling at 4℃, and detection of PCR product by agarose gel electrophoresis. The size is 1206bp, which is the correct target fragment amplified.

[0036] (3) Cloning with pyrG screening marker

[0037] The Aspergillus nidulans pyrG plasmid (the 4722 to 6119 fragment of the gene with the NCBI GenBank database accession number FJ868796.1, which can be obtained by gene synthesis) was used as a template, and pyrG-F (5'-CAACTTCCTCGAGAACGCG-3', SEQ ID NO. 7) and pyrG-R (5'-CCCTTTTAGTCAATACCGTTAC-3', SEQ ID NO. 8) were used for PCR amplification. The PCR amplification system was as follows: 25 μL buffer, 10 mM dNTP 1μL, 2μL of upstream / downstream primers at a concentration of 10ng / μL, 1μL of high-fidelity DNA polymerase, 1μL of DNA template at a concentration of 100ng / μL, and ddH2O to 50μL; the PCR amplification program is as follows: pre-denaturation at 95℃ for 3min; denaturation at 95℃ for 15s, annealing at 58℃ for 15s, extension at 72℃ for 3min, 30 cycles; complete extension at 72℃ for 5min; cooling at 4℃, and detection of PCR product by agarose gel electrophoresis. The size is 1397bp, which is the correct target fragment amplified.

[0038] 2. Construction of Overexpression Cassette

[0039] After the three fragments obtained by (1), (2) and (3) above were purified and recovered by PCR products, fusion PCR was performed according to the molar ratio of 1:2:1. The fusion PCR amplification system was as follows: 12.5 μL buffer, 0.5 μL 10 mM dNTP, 0.5 μL tef1 promoter, 0.5 μL pyrG screening marker, 1 μL myb1 coding region and terminator fragment, 0.5 μL high-fidelity DNA polymerase, and ddH2O to 25 μL. The fusion PCR program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 50℃ annealing for 4 min, 72℃ extension for 4 min, 15 cycles; 72℃ complete extension for 5 min; 4℃ cooling for 5 min. After the fusion PCR was completed, Ptef1-NF (5'-ACACATCTGAGCAGTACTATCAAAG-3', SEQ ID The myb1 overexpression cassette was amplified by PCR using pyrG-R (5'-CCCTTTTAGTCAATACCGTTAC-3', SEQ ID NO. 8). The PCR amplification system consisted of 25 μL of buffer, 1 μL of 10 mM dNTPs, 2 μL of upstream and downstream primers at 10 ng / μL, 1 μL of high-fidelity DNA polymerase, 1 μL of DNA template at 100 ng / μL, and ddH2O to 50 μL. The PCR amplification procedure was as follows: pre-denaturation at 95°C for 3 min; 30 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 3 min; complete extension at 72°C for 5 min; and cooling at 4°C. The PCR product was detected by agarose gel electrophoresis, and the size was 1397 bp, indicating the correct target fragment.

[0040] Example 3 Construction of ΔpyrG strain

[0041] To obtain the pyrG gene selection marker that can be used when overexpressing myb1, it is necessary to first use the wild-type T. harzianum T22 strain as the starting strain and knock out the pyrG gene to obtain a uracil-deficient ΔpyrG strain. The specific process is as follows:

[0042] 1. Construction of the ΔpyrG knockout cassette

[0043] PCR amplification was performed using the genome of Trichoderma harzianum T22 as a template and primers pyr4-UF (5'-GCGTTGTGGCGTTAGAAGTT-3', SEQ ID NO. 10) and pyr4-UR (5'-GCTTTTAGTTTTCTCCTTCCTGGTCTTTGCGTGCTAGACT-3', SEQ ID NO. 11). The PCR amplification system and procedure were the same as those in Example 2(1). The PCR product was detected by agarose gel electrophoresis and was 1988 bp in size, indicating that the target fragment 1 was amplified correctly.

[0044] PCR amplification was performed using the genome of Trichoderma harzianum T22 as a template and primers pyr4-DF (5'-AGTCTAGCACGCAAAGACCAGGAAGGAGAAAACTAAAAGC-3', SEQ ID NO. 12) and pyr4-DR (5'-GGTCAATGTACATCCTGGCG-3', SEQ ID NO. 13). The PCR amplification system and procedure were the same as those in Example 2(1). The PCR product was detected by agarose gel electrophoresis and was 2015 bp in size, indicating that the target fragment 2 was amplified correctly.

[0045] After fragment 1 and fragment 2 were purified and recovered through PCR product purification, fusion PCR was performed at a molar ratio of 1:1. The fusion PCR amplification system was as follows: 12.5 μL of buffer, 0.5 μL of 10 mM dNTP, 10.5 μL of fragment, 20.5 μL of fragment, 0.5 μL of high-fidelity DNA polymerase, and ddH2O added to 25 μL. The fusion PCR program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 50°C for 4 min, and extension at 72°C for 4 min, for 15 cycles; complete extension at 72°C for 5 min; cooling at 4°C for 5 min. After the fusion PCR was completed, a fusion PCR product was obtained.

[0046] PCR amplification was performed using the fusion PCR product as a template and pyr4-NF (5'-AAAAGGACCAAGTTTCGGGC-3', SEQ ID NO. 14) and pyr4-NR (5'-CAGAAAGCAGAGCGTGGAC-3', SEQ ID NO. 15) as primers. The PCR amplification system and procedure were the same as those in Example 2(1). The PCR products were detected by agarose gel electrophoresis, and the sizes were 3691 bp, indicating that the target fragment 3 was amplified correctly.

[0047] 2. Construction of the ΔpyrG strain

[0048] (1) Preparation of competent cells

[0049] The spore suspension of Trichoderma harzianum T22 was inoculated onto PDA medium and cultured at a constant temperature of 28°C for 24h. The mycelium was scraped and transferred to 50mL of PDB medium. After mixing by pipetting, the culture was placed on a shaker at 28°C and 200rpm for 12h. The culture was then filtered through a Buchner funnel, rinsed twice with distilled water, rinsed once with transformation liquid S1, and then transferred to S1 transformation liquid containing 0.5% of Trichoderma harzianum lytic enzyme (Sigma). The culture was shaken at 30°C and 100rpm for 4h. After the enzymatic hydrolysis, an equal volume of transformation liquid S1 was added, mixed by pipetting, filtered, the filter residue was taken, and centrifuged at 4°C and 3500rpm for 10min. The supernatant was taken and 10mL of pre-cooled transformation liquid S2 was added, mixed by pipetting, and centrifuged at 4°C and 3500rpm for 10min. The supernatant was poured out, 200μL of transformation liquid S2 was added, and the protoplasts obtained after mixing by pipetting were the Trichoderma harzianum T22 competent cells.

[0050] (2) Chemical transformation

[0051] Prepare a mixture of 100 μL of the Trichoderma harzianum T22 competent cells, 5 μL of the ΔpyrG knockout cassette, and 25 μL of transformation solution S3 on ice. After incubation on ice for 20 minutes, add 1 mL of transformation solution S3. After standing at room temperature for 5 minutes, add 2 mL of transformation solution S2 to terminate the reaction. Pour the entire transformation system into 50 mL of transformation medium containing 5-fluoroorotic acid (2 mg / ml) and 0.5% uracil and allow colonies to grow.

[0052] (3) Verification of ΔpyrG transformants

[0053] The grown colonies were picked to extract genomes and used as samples for PCR detection. Three sequences were amplified respectively. Specifically, pyr4-UF (5'-GCGTTGTGGCGTTAGAAGTT-3', SEQ ID NO.10) and pyr4-YZR (5'-GGGCAGCCTTATTACCAGAC-3', SEQ ID NO.16) were used as primers for PCR amplification. The PCR amplification system and amplification procedure were the same as those in Example 2(1), and PCR product 1 was obtained. pyr4-YZF (5'-GGGCAGCCTTATTACCAGAC-3', SEQ ID NO.17) and pyr4-DR (5'-GGTCAATGTACATCCTGGCG-3', SEQ ID NO.13) were used as primers for PCR amplification. The PCR amplification system and amplification procedure were the same as those in Example 2(1), and PCR product 2 was obtained. pyr4-CDS-YZF (5'-CTCCCCGTTATCTCTCGTCC-3', SEQ ID PCR amplification was performed using primers 5′-GCCTGGGAATAGTGACTCGTC-3′, SEQ ID NO. 18) and pyr4-CDS-YZR (5′-GCCTGGGAATAGTGACTCGTC-3′, SEQ ID NO. 19). The PCR amplification system and procedure were the same as those in Example 2 (1), and PCR product 3 was obtained.

[0054] The above PCR products were detected by agarose gel electrophoresis (see the electrophoresis results in Figure 1 ), the size of PCR product 1 was 2577 bp, the size of PCR product 2 was 2448 bp, and the electrophoresis diagram of PCR product 3 showed no bands, indicating that the pyr4 gene was successfully knocked out. The corresponding strain was recorded as the uracil-deficient ΔpyrG strain.

[0055] Example 4 Construction of myb1 overexpression mutant strain

[0056] Based on the uracil-deficient ΔpyrG strain obtained in Example 3, the myb1 overexpressing strain OEmyb1 was constructed using the pyr4 gene in Aspergillus nidulans, which is homologous to the pyrG gene of Trichoderma harzianum, as a selection marker. The construction process is as follows:

[0057] 1. Preparation of Competent Cells

[0058] The spore suspension of uracil-deficient ΔpyrG was inoculated onto PDA medium and cultured at 28°C for 24 h. The mycelia were scraped and transferred into 50 mL of PDB medium. After aspiration and mixing, the culture was placed in a shaking incubator at 28°C and 200 rpm for 12 h. The culture was then filtered through a Buchner funnel, rinsed twice with distilled water, rinsed once with transformation solution S1, and transferred into S1 transformation solution containing 0.5% lytic enzyme from Trichoderma harzianum (Sigma). The culture was incubated at 30°C and 100 rpm. After enzymatic hydrolysis, shake on a shaker at rpm for 4 h; after the enzymatic hydrolysis, add an equal volume of transformation solution S1, pipet and mix, filter, take the filter residue, and centrifuge at 4°C and 3500 rpm for 10 min; take the supernatant, add 10 mL of pre-cooled transformation solution S2, pipet and mix, and centrifuge at 4°C and 3500 rpm for 10 min; take the supernatant and pour it out, add 200 μL of transformation solution S2, pipet and mix to obtain the protoplasts, which are the uracil-deficient ΔpyrG competent cells.

[0059] 2. Chemical transformation

[0060] Prepare a mixture on ice by combining 100 μL of competent cells of the uracil-deficient ΔpyrG strain obtained above, 5 μL of the myb1 overexpression cassette DNA fragment, and 25 μL of Transformation Solution S3. Incubate the mixture on ice for 20 minutes, then add 1 mL of Transformation Solution S3. After incubating at room temperature for 5 minutes, add 2 mL of Transformation Solution S2 to terminate the reaction. Pour the entire transformation mixture into 50 mL of transformation medium and incubate until colonies develop.

[0061] 3. Monoclonal Screening

[0062] Grown colonies were picked and the genome was extracted as samples for PCR detection. PCR detection was performed on the above samples using Ptef1-YZF (5'-CATACCCGGTTCAAGCATCC-3', SEQ ID NO. 20) and OE-myb1-YZR (5'-AATTCCCAAACAAAACGCTGC-3', SEQ ID NO. 21) as primers. The PCR amplification system was as follows: 25 μL buffer, 1 μL of 10 mM dNTPs, 2 μL of upstream / downstream primers each at a concentration of 10 ng / μL, 1 μL of high-fidelity DNA polymerase, 1 μL of DNA template at a concentration of 100 ng / μL, and ddH2O to 50 μL. The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; 30 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 15 s, and extension at 72°C for 2 min; complete extension at 72°C for 5 min; and cooling at 4°C for 5 min. The amplified fragment was 1560 bp in length, which was consistent with the theoretical size. This indicated that the myb1 overexpression mutant strain was successfully constructed.

[0063] Example 5 Detection of the re-transmission ability of myb1 overexpression mutant strains

[0064] Inoculate the Botrytis cinerea on one side of the PDA plate medium and culture at 28℃ for 3-4 days. Then, use an agar gel puncher with a diameter of 0.5 cm to randomly punch bacterial blocks at the edge of the colony. 7 A spore suspension of the myb1 overexpressing mutant strain OEmyb1 of Trichoderma harzianum and a 0.5 mm diameter piece of Botrytis cinerea were inoculated on both sides of a PDA plate, 5.5 cm apart. Three biological parallels (respectively designated as OEmyb1-1, OEmyb1-2, and OEmyb1-3) were used. An equal amount of the uracil-deficient ΔpyrG strain of Trichoderma harzianum was used as a negative control, and only the piece of Botrytis cinerea was inoculated on the plate as a blank control. After culturing at 28°C in the dark for 7 days, the phenotype of the opposing plates was observed. The results are as follows: Figure 3 shown.

[0065] Depend on Figure 3 It can be seen that the three mutants OEmyb1-1, OEmyb1-2 and OEmyb1-3 obtained after overexpression have significantly improved parasitic abilities and can achieve full coverage of the gray mold colony.

[0066] Example 6 Verification of Overexpression of Myb1 to Enhance the Response of Trichoderma harzianum to Botrytis cinerea Signals

[0067] Transcriptomic analysis was performed on wild-type T. harzianum T22 strain and T. harzianum myb1 overexpression mutant strain OEmyb1 under independent and opposing growth conditions on PDA plates. It was found that among the differentially expressed genes in the uracil-deficient ΔpyrG strain and T. harzianum myb1 overexpression mutant strain under opposing growth conditions, several biocontrol-related or potentially related genes were upregulated (see Table 1 for details), including: (1) fungal cell wall degrading enzymes (chitinase and glucanase); (2) proteins containing the Ecp2 effector protein domain. In Cladosporium fulvum, ECP1 and ECP2 act as plant pathogen effector proteins that are secreted extracellularly and targeted to the plant cell apoplast to function; and (3) nitrogen metabolism repressor-like NmrA protein. In the plant pathogen Verticillium dahliae, knockout of nmrA not only resulted in slower mycelial growth and reduced melanin production in the mutant strain, but also weakened its ability to infect cotton and Arabidopsis thaliana.

[0068]

[0069]

[0070] In summary, overexpression of myb1 in Trichoderma harzianum can enhance the strain's ability to parasitize plant pathogenic fungi and activate the expression of biocontrol-related or potentially related genes.

[0071] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0072] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of the transcription factor myb1 in enhancing the parasitic ability of Trichoderma harzianum is characterized in that: The nucleotide sequence of the transcription factor myb1 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The amino acid sequence of the transcription factor myb1 is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that Overexpression of the transcription factor myb1 enhances the response of Trichoderma harzianum to Botrytis cinerea signals.

4. A method for improving the parasitic ability of Trichoderma harzianum, characterized in that: include: The transcription factor myb1 was overexpressed in Trichoderma harzianum. The nucleotide sequence of the transcription factor myb1 is shown in SEQ ID NO.

1.

5. The method according to claim 4, characterized in that The amino acid sequence of the transcription factor myb1 is shown in SEQ ID NO.2.

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