A Lectin gene that enhances the resistance of Gummy Lecithia hyphae to Trichoderma viride and its application in cultivation.

Abstract

This invention belongs to the field of agricultural genetic engineering technology, specifically relating to a lectin gene that enhances the resistance of *Trichoderma viride* hyphae to *Trichoderma viride* and its application in cultivation. This invention obtains the mutant Mu-Lectin gene by site-directed mutagenesis of the lectin gene shown in SEQ ID NO:1 (T34W and N40Q), with its nucleotide sequence shown in SEQ ID NO:2, encoding the mutant Mu-Lectin protein shown in SEQ ID NO:3. The recombinant expression vector Mu-Lectin-pCAMBIA1300 was constructed and transformed into *Lectinus colliformis* via Agrobacterium-mediated transformation to obtain a genetically engineered strain overexpressing Mu-Lectin. Experimental results show that the infection rate of *Trichoderma viride* by *Lectinus colliformis* overexpressing Mu-Lectin is significantly lower than that of the wild-type strain and the control strain overexpressing the unmutated lectin gene. This invention optimizes the binding ability of the lectin protein to *Trichoderma viride* N-acetylglucosamine through site-directed mutagenesis, significantly improving the resistance of *Lectinus colliformis* to *Trichoderma viride*, and can be applied to disease-resistant breeding and green cultivation of *Lectinus colliformis*.

Description

Technical Field

[0001] This invention belongs to the field of agricultural genetic engineering technology, specifically relating to a Lectin gene that enhances the resistance of Gastrodia elata hyphae to Trichoderma viride and its application in planting. Background Technology

[0002] Trichoderma viride is a common pathogen in the cultivation of Gynostemma pentaphyllum, widely distributed in soil, air, and plant debris. It can spread through air, water, and tools, and germinates rapidly under high temperature and humidity conditions. Its mycelial growth rate is significantly faster than that of Gynostemma pentaphyllum, leading to rapid invasion of the culture medium. Furthermore, during its growth, Trichoderma viride secretes secondary metabolites such as mycotoxins and viridin, which damage the cell membrane structure of Gynostemma pentaphyllum hyphae, inhibiting its growth and severely impacting the yield and quality of Gynostemma pentaphyllum.

[0003] Currently, the control of *Trichoderma viride* infection during the cultivation of *Cladosporium* var. *gelatinosa* still relies primarily on chemical control. However, long-term use of chemical agents alone can easily lead to drug resistance in *Trichoderma*, and chemical residues pose safety risks. Breeding disease-resistant varieties has become an effective solution; however, traditional breeding methods are time-consuming, inefficient, and difficult to directly introduce disease-resistant genes. With the development of molecular biology techniques, breeding disease-resistant edible fungi varieties through genetic engineering has become a research hotspot.

[0004] Lectin is a lectin found in edible fungi, and members of this family play crucial roles in plant disease resistance, stress tolerance, and growth regulation. However, there are no reports on its application in the cultivation of *Trichoderma viride*. Lectin specifically recognizes and binds to N-acetylglucosamine and β-glucan in *Trichoderma viride*, reducing its infectivity to the host. Improving lectin's binding ability to *Trichoderma viride* through site-directed mutagenesis is an effective way to enhance host resistance. Therefore, researching how to improve lectin's resistance to *Trichoderma viride* through site-directed mutagenesis of the lectin gene has significant theoretical and practical value. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention uses a vector carrying the Lectin gene shown in SEQ ID NO:1 as a template. Specific primers were designed to perform T34W at the N-acetylglucosamine binding region of the Lectin gene and the pathogen *Trichoderma viride*. A site-directed mutation at N40Q was performed to eliminate glycosylation interference, and the binding pocket was optimized to obtain the Mu-Lectin gene as shown in SEQ ID NO:2 and the Mu-Lectin protein as shown in SEQ ID NO:3. Experiments show that overexpression of the Mu-Lectin gene significantly reduces the *Trichoderma viride* infection rate compared to overexpression of wild-type Lectin and wild *Trichoderma colloidalis*. This invention provides a new gene resource and technical solution for the control of *Trichoderma colloidalis*.

[0006] On one hand, the present invention provides a gene that enhances the resistance of Gynostemma pentaphyllum hyphae to Trichoderma viride. The gene is a mutant Mu-Lectin gene, the nucleotide sequence of which is shown in SEQ ID NO:2. The mutant Mu-Lectin gene is obtained by site-directed mutation based on the wild-type Lectin gene.

[0007] Specifically, the mutant Mu-Lectin gene, compared to the wild-type Lectin gene shown in SEQ ID NO:1, has its 34th codon mutated from ACC to TGG, and its 40th codon mutated from AAC to CAA.

[0008] A mutant Mu-Lectin protein that enhances the resistance of Gynostemma pentaphyllum hyphae to Trichoderma viride is also provided. The amino acid sequence of the mutant Mu-Lectin protein is shown in SEQ ID NO:3. The mutant Mu-Lectin protein is encoded by the mutant Mu-Lectin gene described in this invention.

[0009] A recombinant expression vector is also provided, wherein the recombinant expression vector contains the mutant Mu-Lectin gene described in this invention.

[0010] As a preferred technical solution, the recombinant expression vector is Mu-Lectin-pCAMBIA1300, which contains the mutant Mu-Lectin gene of the present invention cloned into the empty pCAMBIA1300 vector.

[0011] A genetically engineered strain is also provided to enhance the resistance of Gastrodia elata hyphae to Trichoderma viride. The genome of the genetically engineered strain integrates the recombinant expression vector described in this invention and overexpresses the mutant Mu-Lectin protein.

[0012] As a preferred technical solution, the genetically engineered strain is *Amanita muscaria* overexpressing the mutant Mu-Lectin protein, which is obtained by introducing the recombinant expression vector into wild-type *Amanita muscaria* through Agrobacterium-mediated genetic transformation.

[0013] It also provides the application of the mutant Mu-Lectin gene described in this invention in improving the resistance of Gynostemma pentaphyllum to Trichoderma viride.

[0014] Also provided is a breeding method for improving the resistance of *Trichoderma viride* to *Trichoderma harzianum*, the method comprising the following steps: introducing the mutant Mu-Lectin gene of the present invention into *Trichoderma harzianum* to overexpress it, wherein the nucleotide sequence of the mutant Mu-Lectin gene is shown in SEQ ID NO:2.

[0015] Furthermore, the present invention also provides the application of the genetically engineered strain described herein in the cultivation of Trichoderma viride, the application comprising the following steps: When the genetically engineered strain described in this invention is inoculated into a cultivation medium and fruiting management is carried out under conventional gelatinous lepidopteran cultivation conditions, the resulting gelatinous lepidopteran fruiting bodies show a significantly lower infection rate of Trichoderma viride than wild-type gelatinous lepidopteran.

[0016] While antifungal effects of lectin have been reported in plants, the strength of these resistance effects is generally moderate and involves complex host-pathogen interactions. Given the significant differences in immune mechanisms between edible fungi and plants (such as the unique cellular structure and growth patterns of mycelium) and the lack of systemic acquired resistance (SAR) pathways in edible fungi, those skilled in the art cannot simply extrapolate knowledge of plant resistance to the overexpression of lectin in edible fungi. In particular, the T34W and N40Q double mutations, with the 34th amino acid changed from threonine (Thr) to tryptophan (Trp), introduce a larger aromatic side chain, enhancing the hydrophobic interaction and π-stacking ability with N-acetylglucosamine in the *Trichoderma viride* cell wall, thus improving the specific recognition and binding affinity of lectin to the pathogen. The 40th amino acid, changed from asparagine (Asn) to glutamine (Gln), removes the potential N-glycosylation site (Asn-X-Ser / Thr motif), avoiding the steric hindrance effect of glycosylation modification on the binding pocket, making the protein structure more stable and improving binding efficiency. T34W enhances binding strength, and N40Q eliminates steric interference. Together, they optimize the conformation of the binding pocket between the lectin protein and the *Trichoderma viride* surface receptor, thereby more effectively blocking the pathogen's infection process and synergistically improving the host's resistance to *Trichoderma viride*. This has a specific effect independent of known conventional experimental rules, an effect that is completely unpredictable in existing technologies. (1) In this invention, the mutant Mu-Lectin gene was obtained by site-directed mutagenesis of the Lectin gene at T34W and N40Q. The results of the plate confrontation test showed that the infection rates of Mu-Lectin-overexpressing Lectin strains (OE1, OE5, OE7) against Trichoderma viride were 0.35, 0.41 and 0.38 for the wild type, respectively, which were significantly lower than those of the control strains overexpressing wild-type Lectin (infection rates of 0.71 and 0.80) and the empty vector control (1.05), indicating that site-directed mutagenesis greatly enhances the resistance of Lectin strains to Trichoderma viride.

[0017] (2) This invention overcomes the technical bottleneck of conventional overexpression. Simply overexpressing the wild-type Lectin gene can only reduce the infection rate to a moderate degree (about 29%). However, this invention introduces a two-site mutation through molecular design, which increases the disease resistance effect to more than 65%, achieving unexpected technical results.

[0018] (3) This invention is the first to apply the rationally designed mutant Mu-Lectin gene to the breeding of disease-resistant edible fungi, providing a new genetic engineering strategy for the biological control of Trichoderma viride in Gynostemma pentaphyllum and similar edible fungi.

[0019] In summary, this invention significantly improves the resistance of *Trichoderma viride* to *Trichoderma* by optimizing the functional domain of the lectomyces through site-directed mutagenesis, filling the gap in the current lack of disease resistance genes in *Trichoderma viride* and providing a new technical means for *Trichoderma viride* to resist *Trichoderma*. Attached Figure Description

[0020] Figure 1 This is a graph showing the statistical results of the infection rate of Trichoderma viride, a type of agaric fungus. Detailed Implementation

[0021] To enable those skilled in the art to better understand the technical solution, the present invention will be described in detail below with reference to embodiments. The description in this part is only exemplary and explanatory, and should not be used to limit the scope of protection of the present invention in any way.

[0022] Example 1

[0023] This embodiment uses the Lectin gene sequence shown in SEQ ID NO:1 as a template. Site-directed mutagenesis is performed on the AP2 domain, the core functional region of the Lectin gene. After T34W and N40Q mutations in the Lectin gene shown in SEQ ID NO:1, the nucleotide sequence of the target gene Mu-Lectin after site-directed mutagenesis is shown in SEQ ID NO:2, and the corresponding amino acid sequence of the protein is shown in SEQ ID NO:3. The specific amino acid sequence is as follows: MFLPKPGVYRLVNVQSNTALDLSEGDGKSFIGWWKHDNPQQQFVFTPLGHGGGYLIQSAWNGNYATIEDGICTGTAVVGSGFPVTWALEDVTPEMHETHAPRSNGSCFRIRWPNSRYVFDLEGYGCDKDGTRIQLAYEQNPV HPCQVWRFEEISYTVPLTCGVNVDAATPLAKFDPGLEVPSGWLVDDLEDESDEDSDYGEFQDASEDGLELELISAVTPINSTPTNKEDKEVEKYKIGRFFDYSDEEEKGRLRQSQMTGIMTTSSTGMTTMVYKDMYCTKQVH.

[0024] Using the previously obtained Lectin gene sequence as shown in SEQ ID NO:1 as a template, point mutations were introduced by overlap extension PCR to obtain Mu-Lectin, and then the overexpression vector Mu-Lectin-pCAMBIA1300 was constructed.

[0025] 1.1 First round of PCR Using the Lectin gene shown in SEQ ID NO:1 as a template, fragment A was amplified by PCR with Lectin-F-BamHI (upstream primer) and Lectin-Mut-R (downstream primer); fragment B was amplified by PCR with Lectin-Mut-F (upstream primer) and Lectin-R-HindIII (downstream primer). The sequences of Lectin-F-BamHI, Lectin-Mut-R, Lectin-Mut-F, and Lectin-R-HindIII are shown below: Lectin-F-BamHI (SEQ ID NO:4): CGCGGATCCATGTTTTTACCCAAACCGGG; Lectin-Mut-R (SEQ ID NO:5): GGTTGTCGTGCTTCCAC.

[0026] Lectin-Mut-F (SEQ ID NO: 6): TGGAAGCACGACAACCCACAA; Lectin-R-HindIII (SEQ ID NO:7): CCCAAGCTTTCAGTGCACTTGCTTCGTG.

[0027] The reaction system is shown in Table 1: Table 1. First-round PCR reaction system

[0028] Perform PCR according to the following procedure: Initial denaturation stage: 98℃ pre-denaturation for 2-5 min, 1 cycle.

[0029] Cyclic amplification phase (20 cycles in total): Denaturation: 98℃, 15s Annealing: 55-59℃, 30s Extension: 72℃, 15s Final extension stage: 72℃, incubate for 5 min, then store the amplification product at 4℃ for later use.

[0030] PCR amplification products were detected by agarose gel electrophoresis to confirm the amplification of single bands of the correct size. Fragment A and fragment B of the PCR amplification products were purified and recovered separately using a gel extraction kit (such as the Omega Gel Extraction Kit), and the concentrations of the purified fragments A and B were determined.

[0031] 1.2 Overlap Extension PCR Fusion The purified fragments A and B were mixed and subjected to 16 cycles of PCR without primers. Annealing and extension of the overlapping regions resulted in the full-length mutant gene. Then, external primers Lectin-F-BamHI and Lectin-R-HindIII were added for a second round of PCR amplification of the full-length product, yielding the full-length PCR product carrying the mutation site (Mu-Lectin).

[0032] 1.3 Construction of Mu-Lectin-pCAMBIA1300 overexpression vector The empty vector pCAMBIA1300 and the Mu-Lectin obtained from step 1.2 were double-digested with XbaI and BamHI. The double-digested Mu-Lectin was ligated into the double-digested pCAMBIA1300 empty vector and transformed into DH5α E. coli competent cells. The cells were plated on LB agar plates containing kanamycin antibiotic, and positive clones were screened. Positive clones were verified by colony PCR. Colonies identified as positive by PCR were sent to a biotechnology company for sequencing to ensure the complete correctness of the Mu-Lectin gene coding region, especially the successful mutation of the T34W and N40Q sites into the target amino acids, without the introduction of any other unexpected mutations. The sequencing results are shown in SEQ ID NO:2, and the specific sequence is as follows: ATGTTTTTACCCAAACCGGGAGTCTATCGGCTCGTCAATGTCCAAAGCAACACAGCTCTTGACCTTTCGGAGGGAGACGGAAAGAGCTTCATAGGCTGGTGGAAGCACGACAACCCACAACAACAATTTGTCTTCACCCCTCTCGGTCACGGTGGAGGATACCTCATCCAAAGCGCATGGAACGGTAACTATGCCACAATCGAAGATGGAATCTGTACGGGAACCGCAGTGGTGGGGAGTGGGTTTCCTGTTACATGGGCTCTGGAAGACGTTACACCTGAAATGCATGAAACGCACGCTCCTCGAAGTAACGGATCCTGCTTCCGAATTCGCTGGCCGAATAGTCGGTATGTGTTTGATTTGGAAGGATATGGATGCGATAAGGATGGAACGAGGATCCAACTCGCTTACGAGCAAAATCCAGTCCATCCATGTCAAGTTTGGCGATTCGAGGAGATATCATATACTGTTCCTCTGACTTGCGGCGTGAACGTGGACGCCGCTACTCCTCTCGCTAAATTTGATCCTGGATTAGAGGTGCCCTCAGGTTGGTTAGTTGATGATTTAGAGGACGAATCGGATGAGGATTCGGACTACGGTGAATTCCAAGACGCATCGGAAGACGGGCTCGAGCTCGAGCTCATCTCGGCTGTAACGCCAATCAATTCTACTCCAACCAATAAAGAAGACAAGGAGGTCGAGAAATATAAGATTGGGCGATTCTTTGACTATTCAGACGAAGAAGAGAAAGGGAGATTGAGGCAGTCTCAGATGACTGGTATCATGACTACTAGCTCAACGGGCATGACTACAATGGTGTACAAGGACATGTATTGCACGAAGCAAGTGCACTGA。

[0033] Sequencing results showed that the expected T34W (ACC->TGG) and N40Q (AAC->CAA) mutations were successfully introduced. The rest of the inserted gene was completely identical to the wild-type sequence (SEQ ID NO:1), with no other nucleotide variations, indicating that the Mu-Lectin-pCAMBIA1300 overexpression vector was successfully obtained. This Mu-Lectin-pCAMBIA1300 expression vector carries the Mu-Lectin gene, and its corresponding amino acid sequence is shown in SEQ ID NO:3.

[0034] Using primers Lectin-F-BamHI (SEQ ID NO:4) and Lectin-R-HindIII (SEQ ID NO:7), the Lectin gene shown in SEQ ID NO:1 was amplified and then double-digested with the pCAMBIA1300 vector. The resulting vector was then ligated to construct the Lectin-pCAMBIA1300 vector carrying the wild-type gene for later use.

[0035] Example 2

[0036] In this embodiment, Agrobacterium-mediated genetic transformation was used to cultivate Mu-Lectin-pCAMBIA1300 overexpressing Agaricus colloidus.

[0037] Using the freeze-thaw method, the Mu-Lectin-pCAMBIA1300 overexpression vector and the empty vector pCAMBIA1300 (CK1) plasmid were transformed into Agrobacterium competent cells EHA105. At the same time, the Lectin-pCAMBIA1300 plasmid carrying the wild-type gene was set as CK2. The steps are as follows: 1) Remove the preserved EHA105 competent cells from the -80℃ freezer and place them on ice while they are in an ice-water mixture; 2) Add approximately 1 μg of the constructed vector plasmid to 100 μL of competent cells and mix gently; 3) Incubate on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and in an ice bath for 5 min; 4) Add 700 μL of antibiotic-free LB liquid medium and incubate at 28℃ and 200 rpm for 2-3 h; 5) Centrifuge at 6000 rpm for 1 min, collect approximately 200 μL of supernatant, gently resuspend the bacterial blocks, and spread them onto LB agar plates containing 50 μg / mL Kan+ and 20 μg / mL Rif; 6) Incubate at 28℃ for 60 h; PCR-positive single clones are shaken to OD. 600 The value was approximately 2.0. The bacteria were preserved in an 80°C ultra-low temperature freezer with an equal volume of 50% glycerol for later use. The Lectin-pCAMBIA1300 was constructed using conventional methods.

[0038] Using wild-type Gynostemma pentaphyllum as the recipient, Agrobacterium-mediated genetic transformation was performed as follows: [The following text appears to be a separate, unrelated section:] ... Agrobacterium EHA105 containing the linear vector plasmid empty vector pCAMBIA1300, recombinant Mu-Lectin-pCAMBIA1300, and recombinant plasmid Mu-Lectin-pCAMBIA1300 was streaked onto LB acclimatization plates (50 μg / mL Kan+ and 20 μg / mL Rif) for 2 days. Single colonies were picked and cultured in 1 mL of liquid LB (50 μg / mL Kan+ + 20 μg / mL Rif) at 28 °C and 200 rpm for 48 h. The culture was then transferred to 100 mL of basal medium MM (50 μg / mL Kan+) and cultured at 28 °C and 200 rpm for 24 h. After centrifugation at 4 °C and 5000 rpm for 10 min, the supernatant was discarded, and the bacterial culture was collected. The bacterial cells were resuspended in an appropriate amount of induction medium IM to achieve the initial OD. 600 Approximately 0.4, add acetylsuccinone (AS) to a final concentration of 200 μmol / L, and incubate at 28℃ and 220 r / min for 4-6 h to allow OD to develop. 600 The value was approximately 0.8. After the mycelia of *Gnaphalium affine* were activated on MYG culture dishes for 7 days, mycelial blocks with a diameter of 5 mm were collected using a sterilizing pipette tip and then analyzed using the aforementioned OD... 600 Immerse the Agrobacterium tumefaciens in a solution of approximately 0.8 for 25 minutes, shaking well every 5 minutes. Then, blot dry the solution with sterile filter paper and inoculate the mycelial blocks into co-culture medium (200 μmol / L AS), incubating at 25°C for 2 days. After 2 days of co-culture, remove the mycelial blocks from the medium, wash 2-3 times with sterile ddH2O, and soak in sterile water containing 400 μg / mL cefotaxime (Cef) for 15 minutes, shaking well every 5 minutes to inhibit Agrobacterium growth. Blot dry the surface moisture of the mycelial blocks with sterile filter paper and transfer them to MYG culture dishes containing 70 μg / mL hygromycin and 400 μg / mL Cef, incubating at 25°C for 15 days for the first selection. After the first selection, punch holes in the germinated mycelia using a sterile pipette tip and transfer them to MYG medium containing 4 μg / mL hygromycin, incubating at 25°C for 7-10 days for the second selection. The strains that could still grow after five identical screenings were identified as Mu-Lectin-pCAMBIA1300 overexpressing Agaricus mucilaginus.

[0039] The basal culture medium (Minimal Media, MM) consisted of 10 mL K-buffer, 20 mL MN buffer, and 20% glucose. (w / v) 10mL, 0.01% FeSO4 (w / v) 10mL, 20% (NH4)2SO4 (w / v) 2.5mL, 1% CaCl2·2H2O (w / v) 1mL, Add 1 L of ddH2O and adjust the pH to 6.7-7.0 using H3PO4 or NaOH.

[0040] The induction medium (IM) consisted of 10 mL K-buffer, 20 mL MN buffer, and 20% glucose. Sugar (w / v) 5mL, 0.01% FeSO4 (w / v) 10mL, 20% (NH4)2SO4 (w / v) 2.5mL, 1% CaCl2·2H2O (w / v) 1mL, 50% glycerol (w / v) 10mL, 7.808g MES (40mmol / L, MW 195.2), ddH2O 1L, adjusted with H3PO4 or NaOH. The pH is 5.5.

[0041] The co-induction media (Co-IM) consisted of 10 mL K-buffer, 20 mL MN buffer, 2.5 mL 20% glucose (w / v), 10 mL 0.01% FeSO4 (w / v), 2.5 mL 20% (NH4)2SO4 (w / v), 1 mL 1% CaCl2·2H2O (w / v), 10 mL 50% glycerol (w / v), 7.808 g MES (40 mmol / L, MW 195.2), 20 g agar, and 1 L ddH2O, with the pH adjusted to 5.5 using H3PO4 or NaOH.

[0042] Example 3

[0043] This example demonstrates the identification of Mu-Lectin-pCAMBIA1300 overexpression of Agaricus colloidus as a positive bacterium.

[0044] cDNA was extracted from *Agaricus colloides* overexpressing Mu-Lectin-pCAMBIA1300. Positive transformants were identified by quantitative real-time PCR (qRT-PCR) using Mu-Lectin-pCAMBIA1300 overexpression. The primers for qRT-PCR were Mu-Lectin-F and Mu-Lectin-R. The sequence information of Mu-Lectin-F and Mu-Lectin-R is as follows: qRT-PCR-Mu-Lectin-F (SEQ ID NO:8): TGGTGGAAGCACGACAACC; qRT-PCR-Mu-Lectin-R (SEQ ID NO:9): TGGCATAGTTACCGTTCCATGCG.

[0045] The qRT-PCR reaction system consisted of: 5 μL of AceQ™ qPCR SYBR Green Master Mix, and primers (10 μM each). 0.5 μL, cDNA 3 μL, ddH2O 1 μL. Reaction parameters: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 10 s, 62℃ annealing for 30 s, 72℃ extension for 30 s, cycle number 40; melting curve temperature set from 65℃ to 95℃, data read every 0.5℃. Positive Mu-Lectin-pCAMBIA1300 overexpressing *Amanita muscaria* strains were identified by qRT-PCR amplification of the correct fragment (100 bp) and sequencing results showing correct sequence (containing mutant bases). Three positive Mu-Lectin-pCAMBIA1300 overexpressing *Amanita muscaria* strains (Mu-Lectin-pCAMBIA1300-OE1, Mu-Lectin-pCAMBIA1300-OE5, and Mu-Lectin-pCAMBIA1300-OE7) were obtained.

[0046] The same method was used to identify positive *Agaricus colloidis* bacteria overexpressing Lectin-pCAMBIA1300, using primers qRT-PCR-Lectin-F and qRT-PCR-Lectin-R. The primer information is as follows: qRT-PCR-Lectin-F (SEQ ID NO:10):TCTCGGTCACGGTGGAGGATAC; qRT-PCR-Lectin-R (SEQ ID NO: 11): TCGGCCAGCGAATTCGG.

[0047] The qRT-PCR amplified fragment (200bp) was correctly sequenced. After sequencing, the sequencing results (without mutant bases) were correct and identified as positive. A total of 2 strains of Lectin-pCAMBIA1300 overexpressing Agaricus colloidus were obtained and labeled as (OE'-1 and OE'-2). Example 4

[0048] This example is to determine the resistance levels of three strains of *Trichoderma viride* overexpressing Mu-Lectin-pCAMBIA1300-OE1 (OE1), Mu-Lectin-pCAMBIA1300-OE5 (OE5), and Mu-Lectin-pCAMBIA1300-OE7 (OE7) obtained from Example 2, as well as wild-type *Trichoderma viride* (WT), Lectin-pCAMBIA1300-OE'-1 (OE'-1), Lectin-pCAMBIA1300-OE'-2 (OE'-2), and *Trichoderma viride* carrying pCAMBIA1300 (empty vector) to *Trichoderma viride* on plates. 90 mm diameter petri dishes were used. Approximately 20 mL of PDA medium was poured into each dish. An 8 mm diameter block of *Trichoderma viride* mycelium was inoculated at the center of each dish. After culturing at 25°C for 10 days, the central *Trichoderma viride* mycelium block was removed, and a 5-day-old *Trichoderma viride* spore of the same size was inoculated at the same location. The dishes were then returned to 25°C for further incubation. Five days after inoculation with *Trichoderma viride*, the interaction between *Trichoderma viride* and *Trichoderma viride* mycelium before and after the confrontation was observed and statistically analyzed. The area of ​​the central lesion was measured using ImageJ software. The *Trichoderma viride* infection rate was calculated using the following formula to quantify the resistance of the *Trichoderma viride* strain to *Trichoderma viride*. A higher *Trichoderma viride* infection rate indicates a lower resistance value. Each strain was tested in triplicate. The test results are as follows: Figure 1 As shown, the formula for calculating the infection rate of *Trichoderma viride* is as follows:

[0049] Depend on Figure 1 It was found that the infection rates of *Trichoderma viride* in the three lines OE1, OE5, and OE7 overexpressing the Mu-Lectin gene were significantly lower than those in the wild type, and lower than those in OE'-1 and OE'-2. This indicates that overexpression of *Trichoderma viride* by Mu-Lectin-pCAMBIA1300-OE1 (OE1), Mu-Lectin-pCAMBIA1300-OE5, and Mu-Lectin-pCAMBIA1300-OE7 significantly enhanced resistance to *Trichoderma viride*. This method can be applied to improve resistance to *Trichoderma viride* in *Trichoderma viride* cultivation.

[0050] It should be noted that, in this document, the terms "comprising," "including," and any other variations are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Specific examples have been used in this document to illustrate the principles and implementation methods of the present invention. These examples are merely for the purpose of helping to understand the method and core ideas of the present invention. The above descriptions are only preferred embodiments of the present invention. It should be pointed out that, due to the limitations of written expression and the objective existence of infinite specific structures, those skilled in the art can make several improvements, modifications, or variations without departing from the principles of the present invention, and can also combine the above technical features in an appropriate manner. These improvements, modifications, variations, or combinations, or the direct application of the concept and technical solution of the present invention to other situations without modification, should all be considered within the scope of protection of the present invention.

Claims

1. A gene that enhances the resistance of *Trichoderma viride* hyphae to *Trichoderma viride*, characterized in that, The gene is a mutant Mu-Lectin gene, and the nucleotide sequence of the mutant Mu-Lectin gene is shown in SEQ ID NO:

2.

2. The gene for enhancing resistance of *Trichoderma viride* hyphae to *Trichoderma viride* according to claim 1, characterized in that: The mutant Mu-Lectin gene, compared to the wild-type Lectin gene shown in SEQ ID NO:1, has its 34th codon changed from ACC to TGG, and its 40th codon changed from AAC to CAA.

3. A mutant Mu-Lectin protein that enhances the resistance of *Trichoderma viride* hyphae to *Gnaphalium affine*, characterized in that... The amino acid sequence of the mutant Mu-Lectin protein is shown in SEQ ID NO:3, and the mutant Mu-Lectin protein is encoded by the mutant Mu-Lectin gene as described in claim 1.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the mutant Mu-Lectin gene as described in claim 1.

5. The recombinant expression vector according to claim 4, characterized in that, The recombinant expression vector is Mu-Lectin-pCAMBIA1300, which contains the mutant Mu-Lectin gene of claim 1 cloned into the empty pCAMBIA1300 vector.

6. A genetically engineered strain that enhances the resistance of *Trichoderma viride* hyphae to *Gnaphalium affine*, characterized in that, The genetically engineered strain has the recombinant expression vector of claim 4 or 5 integrated into its genome, and overexpresses the mutant Mu-Lectin protein.

7. The genetically engineered strain according to claim 6, characterized in that, The genetically engineered strain is a type of Agaricus collodion that overexpresses the mutant Mu-Lectin protein, obtained by introducing the recombinant expression vector into wild-type Agaricus collodion through Agrobacterium-mediated genetic transformation.

8. The application of the mutant Mu-Lectin gene as described in claim 1 in improving the resistance of Gynostemma pentaphyllum to Trichoderma viride.

9. A breeding method for improving the resistance of *Trichoderma viride* to *Trichoderma viride*, characterized in that, The method includes the following steps: introducing the mutant Mu-Lectin gene of claim 1 into *Amanita muscaria* to overexpress it, wherein the nucleotide sequence of the mutant Mu-Lectin gene is shown in SEQ ID NO:

2.

10. The application of the genetically engineered strain according to claim 6 in the cultivation of *Trichoderma viride*, characterized in that, The application includes the following steps: The genetically engineered strain described in claim 6 was inoculated into a cultivation medium and fruiting management was carried out under conventional gelatinous scaly mushroom cultivation conditions. The resulting gelatinous scaly mushroom fruiting bodies showed a significantly lower infection rate of Trichoderma viride than the wild-type gelatinous scaly mushroom.

Images