Trichoderma pseudokoningii DMPK1 and application thereof in preparation of conidium producing fungicide

Abstract

The invention belongs to the technical field of genetic engineering and microorganisms. The invention provides a technology for promoting spore production of trichoderma koningii in a liquid culture medium by editing a pk1 gene, and provides a strain of trichoderma koningii DMPK1 capable of rapidly producing conidia in the liquid culture medium, and the preservation number of the trichoderma koningii DMPK1 is CGMCC (China General Microbiological Culture Collection Center) NO.41236. The invention further provides a method for preparing the trichoderma koningii DMPK1. The strain DMPK1 has been preserved in China General Microbiological Culture Collection Center (CGMCC) at May 21, 2024, the center is called CGMCC for short, and the address of the center is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Under a liquid culture condition, the strain DMPK1 generates a large number of conidia within 2 days, and the sporulation quantity reaches 3.25 * 10 < 7 > cfu / mL.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and microbial biotechnology. Technical Background

[0002] Trichoderma is a filamentous fungus with good lignocellulose degradation ability and antagonism to various pathogenic microorganisms. In addition, some Trichoderma strains also have strong protein secretion ability, so they have important economic value. Trichoderma is widely used in biological control and straw degradation in agriculture, and is used to produce enzyme preparations such as cellulase, chitinase and protease in industry. In view of shelf life considerations, most Trichoderma agents used in agricultural production are Trichoderma conidia preparations. Compared with mycelium, conidia have strong stress resistance, stable biological properties, and are easy to store and transport. Obtaining a large number of conidia is a prerequisite for the factory production of Trichoderma agents. The preparation of Trichoderma conidia is mostly carried out by solid fermentation. However, solid fermentation has problems such as difficult control of nutrients, temperature, humidity, ventilation, pH value, etc. In addition, it is difficult to achieve consistency in terms of substrate water holding capacity, resulting in unstable Trichoderma spore production in solid fermentation, and even fermentation failure. In recent years, people have tried to achieve spore production in liquid fermentation of Trichoderma viride, Trichoderma harzianum, etc. by optimizing culture conditions (de Rezende LC et al., 2020). However, most naturally isolated Trichoderma strains have a weak ability to produce conidia under liquid fermentation conditions, or even do not produce conidia. Even if some Trichoderma strains can produce conidia through liquid fermentation, their fermentation cycle is often long and the yield of conidia is not high. Rapid and efficient preparation of Trichoderma spores through liquid fermentation is still one of the key factors restricting the industrialization of Trichoderma agents.

[0003] Like most filamentous fungi, the life cycle of Trichoderma strains includes the process of growth, development and morphological differentiation. The process of transition from hyphal growth to sporulation is not only affected by nutrients and environmental factors (such as temperature, humidity, osmotic pressure, ventilation, light, etc.), but also controlled by its own regulatory genes. Studies in the model fungus Aspergillus nidulans found that fluG, flbA, flbB flbC, flbD and flbE genes are involved in the formation of conidia (Adams et al., 1998). The expression of these genes is subject to complex and fine regulation, such as a cAMP-dependent protein kinase K inhibiting the production of conidia (Shimizu & Keller, 2001). External factors such as nutrients and environmental factors will play a role through this complex regulatory network. Although there is still a lack of research on the conidia formation system in Trichoderma, the development of genome sequencing and bioinformatics technology has provided us with the conditions for obtaining Trichoderma to produce a large number of sporulations under liquid fermentation conditions through directed genetic modification.

[0004] Trichoderma koningiopsis is a strain with the ability to degrade lignocellulose obtained by screening through Congo red staining and liquid shake flask fermentation. On a variety of substrates, the strain's ability to produce cellulase is better than that of Trichoderma reesei QM9414. After fermentation for 84 hours with microcrystalline cellulose as the only carbon source, the enzyme activities of filter paper cellulase, carboxymethyl cellulase, β-glucosidase and xylanase in the supernatant of Trichoderma koningiopsis were 1.72, 1.70, 6.35 and 1.12 times that of QM9414, respectively. However, the spore production performance of Trichoderma koningiopsis is not strong, and conidia can only be produced after long-term cultivation in liquid culture medium.

[0005] References:

[0006] de Rezende LC, de Andrade Carvalho AL, Costa LB, et al. Optimizing massproduction of Trichoderma asperelloides by submerged liquid fermentation and its antagonism against Sclerotinia sclerotiorum. World J MicrobiolBiotechnol. 2020, 36(8): 113.

[0007] Adams TH, Wieser JK, Yu JH. Asexual sporulation in Aspergillusnidulans. Microbiol Mol Biol Rev. 1998, 62(1): 35-54.

[0008] Shimizu K,Keller NP.Genetic involvement of a cAMP-dependent proteinkinase in a G protein signaling pathway regulating morphological and chemical transitions in Aspergillus nidulans.Genetics.2001,157(2):591-600. Summary of the invention

[0009] In view of the above reasons, the purpose of the present invention is to provide a strain of Trichoderma koningiopsis DMPK1, whose deposit number is CGMCC NO.41236. The strain DMPK1 has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee on May 21, 2024, which is referred to as CGMCC. The address of the center is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Compared with the wild strain of Trichoderma koningiopsis, the strain DMPK1 has the pk1 gene sequence knocked out; the pk1 gene sequence is shown in the sequence table SEQ ID NO.1.

[0010] The invention also provides the use of Trichoderma koningiopsis DMPK1 for rapidly producing conidia under liquid fermentation conditions.

[0011] The present invention also provides a method for constructing Trichoderma koningiopsis DMPK1, comprising the following steps:

[0012] Based on CRISPR / Cas9 technology, the plasmid pDpk1 for pk1 gene knockout was constructed.

[0013] The plasmid pDpk1 was introduced into Trichoderma pseudokoningii cells through PEG-mediated genetic transformation, and the strain DMPK1 with the pk1 gene knocked out was successfully obtained.

[0014] In a specific embodiment of the present invention, the construction method of the plasmid pDpk1 includes: using plasmid pUC19-AMA1-Hyg-Cas9 as the starting plasmid, adding a gRNA expression frame and homology arms; using the Trichoderma reesei 5S rRNA promoter and the Trichoderma pseudokoningii glycine tRNA promoter connected together as a dual promoter to drive gRNA expression, and using 6 Ts to terminate gRNA expression; using CRISPR Guide RNA Design Tools (www.benchling.com / crispr) to select a 20bp sequence for targeting the pk1 gene.

[0015] The pk1 gene knocked-out Trichoderma koningiopsis DMPK1 provided by the present invention can produce a large number of conidia under liquid culture conditions, and the conidia production amount reaches 3.25×10 7 cfu / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments or the description of the prior art will be described in detail below.

[0017] Figure 1 Schematic diagram of the construction strategy of the pDpk1 plasmid for pk1 gene knockout.

[0018] Figure 2 This is the pDpk1 plasmid map and restriction enzyme cutting verification diagram used for pk1 gene knockout.

[0019] Figure 3 This is a diagram of the construction and verification of the pk1 gene deletion mutant strain DMPK1.

[0020] Among them, (A) amplification primers are pk1 gene internal primers; (B) amplification primers are pk1 gene external primers. M: DNA marker; 1-3: pk1 gene deletion mutant DMPK1; P: plasmid pDpk1; WT: Trichoderma pseudokoningii wild type; H 2 O: negative control.

[0021] Figure 4 Growth of the wild-type strain (WT) and the pk1-deficient strain DMPK1.

[0022] Among them, (A) Growth on culture medium containing different carbon sources; (B) Comparison of colony diameters on culture medium containing different carbon sources. Data are expressed as Mean±SD (n=3), *p<0.05, **p<0.01, ***p<0.001.

[0023] Figure 5 Sporulation of the wild-type strain and the pk1 gene-deficient strain DMPK1 in solid culture medium.

[0024] Among them, (A) wild-type strain WT (B) DMPK1.

[0025] Figure 6 Sporulation of strain DMPK1 and the wild-type strain in liquid culture.

[0026] Among them, (A) the color of the culture after 48 hours. The left is the wild-type strain (WT), and the right is the pk1 deletion mutant DMPK1; (B) microscopic observation of the wild-type strain growing in liquid culture medium; (C) microscopic observation of the pk1 deletion strain DMPK1 growing in liquid culture medium.

[0027] Figure 7 Microscopic images of DMPK1 after culture in liquid culture medium for different time periods.

[0028] Among them, (A) the culture time is 10h; (B) the culture time is 28h; (C) the culture time is 30h. DETAILED DESCRIPTION

[0029] The invention provides a technical method for promoting spore production of Trichoderma pseudokoningii in a liquid culture medium through gene editing. The technical method comprises constructing an engineering strain DMPK1 with a pk1 gene deletion based on CRISPR / Cas9 technology.

[0030] The present invention also provides the use of the engineered strain DMPK1 in spore production in a liquid culture medium.

[0031] In the present invention, the strain used for pk1 gene knockout is Trichoderma koningiopsis 8985 (Li Peng et al., 2021).

[0032] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are based on conventional experimental conditions or the conditions recommended by the manufacturer's instructions.

[0033] The main experimental materials involved in the following examples are:

[0034] Table 1 Primer sequences used in the present invention

[0035]

[0036] Table 2 Primer sequences used in the present invention

[0037]

[0038] Table 3 Primer sequences used in the present invention

[0039]

[0040]

[0041] In the embodiments, the culture medium comprises the following components:

[0042] PDA medium (g / L): 200g potato, 20g glucose. Cut 200g peeled potato into small pieces, boil in boiling water for 30min, filter with eight layers of gauze, add glucose to the filtrate and make it up to 1L. PDA solid medium needs to add 1.5% agar powder. If agar powder is not added, it is PDB medium, and the pH is natural. High pressure steam sterilization at 115℃ for 30min.

[0043] LMM medium (g / L): 10g glucose, 1g yeast extract powder, 50mL 20×Nitrate salts, natural pH. Autoclave at 115℃ for 30min. Add 1mL 1000×Trace elements (filter sterilized) when using. 20×Nitrate Salts (g / L): NaNO 3 120 g, KCl 10.4 g, MgSO 4 7H 2 O10.4g, KH 2 PO 4 30.4g; 1000×Trace Elements (g / L): ZnSO 4 7H 2 O 22g, H 3 BO 3 11g, MnCl 2 ·4H 2 O 5g, FeSO 4 7H 2 O 1.6 g, CoCl 2 6H 2 O 1.6 g, CuSO 4 ·5H 2 O1.6g, (NH 4 ) 6 Mo 7 O 24 ·4H 2 O 1.1g, Na 4 EDTA 50g.

[0044] Protoplast transformation upper medium (g / L): 39 g potato broth, 218.6 g sorbitol, 0.5% agar powder, natural pH. High pressure steam sterilization at 115°C for 30 min.

[0045] Protoplast transformation substratum (g / L): 39 g potato broth, 218.6 g sorbitol, 1.5% agar powder, natural pH. High pressure steam sterilization at 115°C for 30 min.

[0046] Basic culture medium (g / L): KH 2 PO 4 15g, (NH 4 ) 2 SO 4 5g, MgSO 4 7H 2 O 0.6 g, CaCl 20.8g, urea 0.3g, pH 4.8. High pressure steam sterilization at 115℃, 30min. Add trace element solution (MnSO 4 ·H 2 O 0.0016g, FeSO 4 7H 2 O 0.0005g, ZnSO 4 7H 2 O 0.0014g, CoCl 2 0.0002 g filter sterilized).

[0047] Single carbon source medium (g / L): Add 20 g glucose / 20 g glycerol / 20 g lactose to the basal medium.

[0048] In the embodiments, the buffer comprises the following components:

[0049] Osmotic Medium:

[0050] MgSO 4 7H 2 O 1.2M

[0051] Phosphate buffer (pH 6.5) 0.01M

[0052] Use 1M Na 2 HPO 4 Adjust pH to 5.8.

[0053] Trapping Buffer:

[0054] Sorbitol 0.6M

[0055] Tris-HCl (pH 7.5) 0.1M

[0056] STC Buffer:

[0057] Sorbitol 1.2M

[0058] CaCl 2 0.01M

[0059] Tris-HCl (pH 7.5) 0.01M

[0060] PEG Buffer:

[0061] PEG4000 60%(w / v)

[0062] CaCl 2 0.05M

[0063] Tris-HCl (pH 7.5) 0.05M

[0064] The present invention will be further described below in conjunction with the embodiments.

[0065] Example 1

[0066] Construction of pk1 gene-deficient engineered strain DMPK1 based on CRISPR / Cas9 technology

[0067] (1) Construction of pDpk1 plasmid for pk1 gene knockout

[0068] The schematic diagram of plasmid construction strategy is shown in Figure 1 .

[0069] The pk1 gene (GenBank record PP505844) in Trichoderma koningiopsis was knocked out using CRISPR / Cas9-based gene editing technology. The amino acid sequence of the product encoded by the pk1 gene sequence is shown in SEQ ID NO.1 in the sequence table.

[0070] Plasmid pUC19-AMA1-Hyg-Cas9 contains the coding gene of Cas9 (the gene is 4,266 bp in length and contains sequences encoding the nuclear localization signal NLS and 3xFlag tag), and uses the promoter PgpdA from the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase gene to drive the expression of cas9 (GenBank record WYB68443) (Chen et al., 2020). The nucleotide sequence of the promoter PgpdA is shown in SEQ ID NO.2 in the sequence table.

[0071] In addition, the plasmid pUC19-AMA1-Hyg-Cas contains the autonomous replication sequence AMA1 (Aleksenko & Clutterbuck, 1997), so the plasmid can be introduced into Trichoderma koningiopsis in a non-integrated genome manner through PEG-mediated transformation.

[0072] Under resistance pressure, the plasmid can stably exist in the cell in a manner free from the genome; if the transformant is subcultured under non-resistance conditions, the autonomously replicating plasmid will be lost due to the lack of selection pressure, thus avoiding the integration of the plasmid into the genome and causing adverse effects on the strain.

[0073] Plasmid pUC19-AMA1-Hyg-Cas contains the hygromycin B phosphotransferase gene hyg. The antibiotic sensitivity test on Trichoderma koningiopsis showed that Trichoderma koningiopsis was sensitive to hygromycin B and could be used for the screening of transformants of Trichoderma koningiopsis.

[0074] Taking plasmid pUC19-AMA1-Hyg-Cas9 as the starting plasmid, after adding gRNA expression frame and homology arm, the plasmid pDpk1 for pk1 gene knockout was constructed. The gRNA expression frame sequence is shown in SEQ ID NO.3 in the sequence table. The homology arm includes pk1 upstream homology arm pk1-L and pk1 downstream homology arm pk1-R, the pk1 upstream homology arm pk1-L sequence is shown in SEQ ID NO.4 in the sequence table, and the pk1 downstream homology arm pk1-R sequence is shown in SEQ ID NO.5 in the sequence table.

[0075] A dual promoter was constructed by connecting the Trichoderma reesei 5S rRNA promoter and the Trichoderma pseudokoningiopsis glycine tRNA promoter in series to drive gRNA expression, and 6 Ts were used to terminate gRNA expression. A 20 bp sequence for targeting the pk1 gene was designed using CRISPR Guide Design Tools, 5'-CAAGTTAAAATAAGGCTAGT-3'. The Trichoderma reesei 5S rRNA promoter sequence is shown in SEQ ID NO.6 in the sequence list, and the Trichoderma pseudokoningiopsis glycine tRNA promoter sequence is shown in SEQ ID NO.7 in the sequence list.

[0076] Using Trichoderma reesei genomic DNA as a template, primers 5S rRNA-F / R were designed to amplify the 5S rRNA promoter sequence of Trichoderma reesei. Using Trichoderma koningiopsis genomic DNA as a template, primers tRNA-F / R were designed to amplify the glycine tRNA promoter sequence of Trichoderma koningiopsis. The two promoter sequences were amplified and connected into one fragment using primers gRNA-1-F / R to obtain the promoter sequence that drives gRNA expression.

[0077] Primers gRNA-2-F / R were designed to obtain the gRNA expression cassette by PCR amplification, which contained a 20 bp crRNA guide sequence targeting the knockout gene and a tracrRNA sequence for binding to the Cas9 protein. After recovering the fragment, primers gRNA-3-F / R were used to amplify the fragment by PCR and connect it to the promoter sequence driving gRNA expression to finally obtain a DNA fragment (total length 642 bp) containing the gRNA expression cassette coding sequence.

[0078] Using Trichoderma koningiopsis genomic DNA as a template, primers Pk1-1-F / R were designed, and a DNA fragment containing the upstream homologous arm of the pk1 gene (total length 1040 bp) was obtained by PCR amplification. Primers Pk1-2-F / R were designed, and a DNA fragment containing the downstream homologous arm of the pk1 gene (total length 1030 bp) was obtained by PCR amplification.

[0079] The plasmid pUC19-AMA1-Hyg-Cas9 was digested with SmaⅠ to obtain a linearized plasmid. The above fragment was connected to the linearized plasmid pUC19-AMA1-Hyg-Cas9 using the Gibson assembly method (Gibson et al., 2009) to obtain the plasmid pDpk1 ( Figure 1 ).

[0080] pDpk1 was digested with HindⅢ and three fragments were obtained, which were consistent with the theoretical size, proving that the plasmid was constructed correctly ( Figure 2 ).

[0081] (2) Construction of the pk1 gene-deficient strain DMPK1

[0082] Plasmid pDpk1 was introduced into Trichoderma koningiopsis cells by PEG-mediated protoplast transformation ( et al., 1987).

[0083] The invention comprises the preparation of protoplasts of Trichoderma pseudokoningii and the transformation of protoplasts of Trichoderma pseudokoningii mediated by PEG.

[0084] Preparation of Trichoderma pseudokoningii protoplasts:

[0085] ① Spread the glycerol stock solution of Trichoderma koningiopsis on PDA medium and place it in an incubator at 28°C and invert for 5 to 7 days to produce spores;

[0086] ② Use a sterile cotton swab to scrape the spores cultured on the plate and inoculate them into 50 mL of LMM mycelium germination medium. Cultivate at 28°C and 220 rpm for 10 to 12 hours. Observe under a microscope that the mycelium length is more than 5 times the spore diameter.

[0087] ③ Use a sterile 40μm filter to collect the mycelium and wash it with sterile water 2 to 3 times;

[0088] ④ Weigh 0.64g of Vinotaste, an enzyme for cell wall hydrolysis, into a 50ml centrifuge tube, add 10ml of Osmotic Medium, fully dissolve and filter to sterilize;

[0089] ⑤ Add 10 mL of filtered enzymatic solution to a sterilized 100 mL conical flask, resuspend the mycelium and add it to the enzymatic solution (mix thoroughly by blowing), and culture at 28°C and 100 rpm for 8 to 10 hours. When a large number of protoplasts are produced in the microscope field of view, stop the reaction and start collecting;

[0090] ⑥ After the protoplasts are formed, pour the enzymatic solution into a 50mL centrifuge tube and slowly add 10mL of Trapping buffer along the wall with a pipette to ensure upper and lower layers;

[0091] ⑦ Centrifuge at 4000 rpm for 18 min at 4°C, use a 1 mL pipette tip to draw the white protoplasts (middle layer) into a 50 mL centrifuge tube, add an equal volume of STC buffer, centrifuge at 4°C, 4000 rpm for 10 min, discard the supernatant, and obtain the protoplast precipitate;

[0092] ⑧The precipitate was resuspended in 5 mL STC buffer and centrifuged at 4000 rpm for 5 min;

[0093] ⑨Repeat step ⑧;

[0094] ⑩Discard the supernatant, resuspend the protoplasts in an appropriate amount of STC buffer and store at 4℃ for later use ( et al., 1987).

[0095] PEG-mediated transformation of Trichoderma pseudokoningii protoplasts:

[0096] ① Add about 5 μg of plasmid pDpk1 to 100 μL of Trichoderma pseudokoningii protoplast resuspension, mix gently, and place on ice for 50 min;

[0097] ② Slowly add 1.25 mL of PEG buffer, mix gently, and leave at room temperature for 20 minutes;

[0098] ③Add 5mL STC buffer and mix well;

[0099] ④ Each 1 mL was mixed with 5 mL of the upper culture medium for the transformation of Trichoderma pseudokoningii protoplasts (temperature below 50°C), and immediately spread on the lower culture medium for the protoplasts (the culture medium was supplemented with hygromycin B at a concentration of 200 μg / mL);

[0100] ⑤Cultivate at 28℃ for 4-7 days. When transformants emerge, transfer them to screening plates for cultivation.

[0101] Transformants were picked and their genomic DNA was extracted as templates for subsequent PCR verification. Internal primers (Pk1-iF / R, PCR verification with this primer should amplify a fragment of 992 bp for the wild-type strain, and no amplified band for the pk1 knockout strain) and external primers (Pk1-eF / R, PCR verification with this primer should amplify a fragment of 2677 bp for the wild-type strain, and a fragment of 1063 bp for the pk1 knockout strain) of the pk1 gene were designed.

[0102] The results showed that the band sizes of the three transformants were consistent with expectations, indicating that the knockout strain of the pk1 gene was successfully obtained ( Figure 3 ), and named the strain Trichoderma koningiopsis DMPK1. The strain DMPK1 was deposited in the General Microbiological Center of China Microbiological Culture Collection Administration on May 21, 2024, with the deposit number CGMCCNO.41236. The center is abbreviated as CGMCC, and the address of the center is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0103] Example 2

[0104] Sporulation of pk1 gene-deleted strain Trichoderma koningiopsis DMPK1 in liquid culture medium

[0105] (1) Growth and spore production of engineered strain Trichoderma koningiopsis DMPK1 in solid culture medium

[0106] The wild strain WT and the engineered strain Trichoderma koningiopsis DMPK1 were inoculated and cultured in solid culture media with different carbon sources, including composite culture medium PDA and basal culture medium with glucose / lactose / glycerol, placed at 28℃ and inverted to compare the mycelial growth ( Figure 4 ).

[0107] In addition, the wild strain WT and the engineered strain Trichoderma koningiopsis DMPK1 were inoculated onto PDA plates and cultured at 28°C for 7 days to observe the spore production of the strains.

[0108] It was found that compared with the wild-type strain WT, the engineered strain Trichoderma koningiopsis DMPK1 grew slowly and produced almost no spores when cultured on the composite medium PDA plate ( Figure 5 ).

[0109] (2) Sporulation of the engineered strain Trichoderma koningiopsis DMPK1 in liquid culture medium

[0110] The wild-type strain WT and the engineered strain Trichoderma koningiopsis DMPK1 with pk1 gene mutation were inoculated into PDB medium. In stark contrast to the phenomenon of no spore production on PDA solid plates, the engineered strain Trichoderma koningiopsis DMPK1 produced a large number of spores in the liquid environment after being cultured at 28°C and 220rpm for 48h, causing the culture medium to appear dark green. The bacterial liquids of the wild-type WT and the engineered strain Trichoderma koningiopsis DMPK1 were taken and observed under a microscope. A large number of oval spores appeared in the engineered strain Trichoderma koningiopsis DMPK1 under the microscope field of view ( Figure 6 ).

[0111] The experiment was designed to observe the spore production process of the engineered strain Trichoderma koningiopsis DMPK1 in a liquid environment. The spores (1.8×10 8 cfu / mL) was inoculated into 50 mL PDB medium, and cultured at 28°C and 220 rpm. Samples were taken every 2 hours from 10 hours for microscopic observation. Figure 7 As shown in the figure, most of the engineered strains of Trichoderma koningiopsis DMPK1 spores began to absorb water and swell at 10 hours, and germinated and grew germ tubes, which then spread into hyphae, and the hyphae stretched to form mycelium. At 28 hours, spores began to form at the top of the hyphae. At 30 hours, a large number of spores were produced. At this time, the spores were counted, about 3.25×10 7 cfu / mL.

[0112] In summary, the present invention obtains the engineered strain Trichoderma koningiopsis DMPK1 by knocking out the pk1 gene in Trichoderma koningiopsis. The engineered strain Trichoderma koningiopsis DMPK1 can produce spores under liquid culture conditions, and the number of spores reaches 3.25×10 7 cfu / mL.

[0113] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made on the basis of not departing from the main content of the present invention all belong to the scope of protection claimed by the present invention.

[0114] References

[0115] rook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual, Second Edition. Cold Spring Harbor Laboratory Press, New York, 1989.

[0116] Hao Z,Su X.Fast gene disruption in Trichoderma reesei using in vitroassembled Cas9 / gRNA complex.BMC Biotechnol,2019,19:2.

[0117] Aleksenko A, Clutterbuck AJ. Autonomous plasmid replication in Aspergillus nidulans: AMA1 and MATE elements. Fungal Genet Biol, 1997, 21: 373-387.

[0118] Chen C,Liu J,Duan C,Pan Y,Liu G.Improvement of the CRISPR-Cas9mediated gene disruption and large DNA fragment deletion based on a chimericpromoter in Acremonium chrysogenum.Fungal Genet Biol.2020,134:103279.

[0119] Gibson DG,Young L,Chuang R,et al.Enzymatic assembly of DNA moleculesup to several hundred kilobases.Nature Methods,2009,6:343-345.

[0120] M,Nevalainen HK, M,et al.A versatile transformation systemfor the cellulolytic filamentous fungus Trichoderma reesei.Gene,1987,61(2):155-164.

Claims

1. Trichoderma koningiopsis DMPK1, whose deposit number is CGMCC NO.41236.

2. Use of the Trichoderma koningiopsis DMPK1 described in claim 1 in the preparation of a conidia-producing bacterial agent.

3. The use of Trichoderma koningiopsis DMPK1 according to claim 2 in the preparation of a conidia-producing agent, characterized in that: The amount of conidia reached 3.25×10 7 cfu / mL.

4. The use of Trichoderma koningiopsis DMPK1 according to claim 2 in the preparation of a conidia-producing agent, characterized in that: The culture medium for producing conidia is PDB culture medium.

5. The method for constructing Trichoderma koningiopsis DMPK1 according to claim 1, characterized in that: The steps include: Based on CRISPR / Cas9 technology, plasmid pDpk1 was constructed; The plasmid pDpk1 was introduced into Trichoderma pseudokoningii cells through PEG-mediated protoplast transformation, and the engineered strain DMPK1 with the pk1 gene knocked out was successfully obtained.

6. The method for constructing Trichoderma koningiopsis DMPK1 according to claim 5, characterized in that: The construction method of the pDpk1 plasmid comprises: Using plasmid pUC19-AMA1-Hyg-Cas9 as the starting plasmid, add gRNA expression cassette and homology arms; The Trichoderma reesi 5S rRNA promoter and the Trichoderma pseudokoningii glycine tRNA promoter were connected together as a dual promoter to drive gRNA expression, and six Ts were used to terminate gRNA expression; A 20 bp sequence was selected for targeting the pk1 gene using CRISPR Guide RNA Design Tools.

7. The method for constructing Trichoderma koningiopsis DMPK1 according to claim 6, characterized in that: The pk1 gene sequence is shown in the sequence table SEQ ID NO.1.

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