Recombinant expression vector and method for improving heat resistance and trichoderma resistance of lentinus edodes

Abstract

The invention relates to a recombinant expression vector and a method for improving heat resistance and trichoderma resistance of lentinus edodes, the recombinant expression vector carries a target gene as shown in SEQ ID NO.2, and the method comprises the following steps: transferring the recombinant expression vector into a lentinus edodes strain carrying LeV-HKB virus, the recombinant expression vector carrying the target gene as shown in SEQ ID NO.2 can promote the growth of mushroom hyphae and improve the heat resistance and trichoderma resistance of strains subjected to heat stress, the heat resistance and trichoderma resistance of mushrooms can be improved at the same time by adopting the method, and economic losses caused by high-temperature heat stress or trichoderma atroviride infection in mushroom planting can be reduced.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a recombinant expression vector and a method for improving the heat resistance of shiitake mushrooms and the resistance of trichoderma. Background Art

[0002] Lentinula edodes is an edible mushroom with important economic value, with an umbrella-shaped cap and a columnar stipe, etc., and the edible part is its fruiting body, which is the variety with the highest annual output of edible mushrooms in the world (document 1). However, Lentinula edodes strains generally carry fungal viruses (document 2, document 3), such as in Chinese Lentinula germplasm resources, the most common virus carried by Asian cultivated Lentinula edodes strains is LeV-HKB (document 3, document 4). Although most Lentinula edodes viruses have latent characteristics, the Lentinula edodes strains infected with viruses will not show symptoms under normal circumstances, but when the virus disease occurs, due to the wide range of virus transmission, the transmission speed is fast, thereby causing huge yield losses. Existing Lentinula edodes detoxification methods mainly include chemical detoxification and protoplast regeneration detoxification, but the chemical detoxification cycle is long, the virus species are limited and the activity of the Lentinula edodes strain itself is affected, and there is a large randomness in the protoplast regeneration detoxification, and the detoxification effect is unstable.

[0003] A common disease in shiitake cultivation is green mold disease caused by pathogenic fungi of the genus Trichoderma. Studies have shown that high temperature can cause the resistance of shiitake mycelium to Trichoderma to decrease, and reaching a certain threshold can cause the death of shiitake mycelium (Reference 5), and the virus LeV-HKB will further affect the resistance of shiitake mycelium to Trichoderma after being subjected to high temperature stress. At present and in the future, shiitake cultivation will generally adopt centralized stick making for facility-based or factory-based production. In the centralized stick making of shiitake, the environmental conditions of the high-density and large-scale mushroom stick culture room are difficult to control. In addition, a large amount of mycelium releases heat due to respiration, which can easily burn the mycelium at high temperatures and be infected by Trichoderma, causing huge economic losses. In facility or factory cultivation, when temperature control or ventilation and shading conditions are insufficient, the local temperature in the cultivation shed can reach above 37°C, and the temperature in the fungus bag can reach 40°C. Under such high temperature conditions, the heat resistance of the shiitake mushroom strain infected with the LeV-HKB virus is poor, and the mycelial growth rate decreases significantly after heat stress, and it is more susceptible to infection by dark green Trichoderma in the surrounding environment, making it difficult to form fruiting bodies, which will cause huge economic losses. Therefore, a method for promoting the mycelial growth of shiitake mushroom strains after heat stress, especially enhancing resistance to dark green Trichoderma, is studied to reduce the yield loss of shiitake mushroom cultivation after heat stress.

[0004] Source:

[0005] Document 1: Royse DJ, Baars J, Tan Q. Current Overview of Mushroom Production in the World. John Wiley & Sons, Ltd, 2017.

[0006] Reference 2: Bian Yinbing. Research progress on infectious and competitive diseases of edible fungi mycelium. Journal of Edible Fungi, 2013, 20(2): 1-7.

[0007] Document 3: Kim J, Yun S, Park S, Ko H, Kim D. Occurrence of dsRNA mycovirus (LeV-FMRI0339) in the edible mushroom Lentinula edodes and mei-otic stability of LeV-FMRI0339 among monokaryotic progeny. Plant Pathol J, 2013, 29(4): 460-464.

[0008] Reference 4: Liu Mingjie, Shi Jing, Wang Jinjie, Guo Mengpei, Shen Guiyu, Bian Yinbing, Xu Zhangyi. Effects of basidiospore-mediated transmission on the population formation of Lentinula edodes bipartite virus LePV1. Bulletin of Microbiology, 2020, 47(08): 2409-2416.

[0009] Reference 5: Li Fengshuo, Ji Baoying, Han Bing, et al. Research progress on the interaction between Lentinula edodes and Trichoderma and the prevention and control of green mold disease [J]. Journal of Edible Fungi, 2024, 31(03): 113-124. DOI: 10.16488 / j.cnki.1005-9873.2024.03.012. Summary of the invention

[0010] In view of the above defects or improvement needs of the prior art, the present invention provides a recombinant expression vector and a method for improving the heat resistance and Trichoderma resistance of Lentinus edodes. The purpose is to find that transferring a recombinant plasmid carrying the target gene as shown in SEQ ID NO.2 into a Lentinus edodes strain carrying the LeV-HKB virus can promote the growth of mycelium after heat stress and enhance its resistance to Trichoderma, thereby solving the technical problems that the existing Lentinus edodes strain carrying the LeV-HKB virus has poor heat resistance, slow mycelium growth after heat stress, and reduced resistance to Trichoderma.

[0011] To achieve the above object, according to one aspect of the present invention, a recombinant expression vector is provided, which carries the target gene as shown in SEQ ID NO.2.

[0012] Preferably, the recombinant expression vector further contains a resistance gene.

[0013] Preferably, the resistance gene in the recombinant expression vector includes the Hyg gene.

[0014] Preferably, the recombinant expression vector is a recombinant plasmid carrying the target gene as shown in SEQ ID NO.2, which is prepared according to the following method:

[0015] According to the sequence shown in SEQ ID NO.2, a 94 bp target gene was synthesized, connected into the PUC57 plasmid, and the target gene fragment was amplified and recovered;

[0016] The plasmid with Legpd promoter was used as the basic skeleton of the target gene expression vector. After double restriction digestion, the target gene fragments were connected through homologous recombination to obtain the recombinant expression vector.

[0017] Preferably, in the recombinant expression vector, the plasmid carrying the Legpd promoter is pCAMBIA1300, and the double restriction enzyme cutting sites are KpnI and EcoRI.

[0018] According to another aspect of the present invention, a method for simultaneously promoting the growth of Lentinus edodes mycelium and enhancing the resistance of Trichoderma after heat stress is provided, which comprises the step of transferring the recombinant expression vector of the present invention into a Lentinus edodes strain carrying the LeV-HKB virus to prepare transformants.

[0019] Preferably, the method comprises transferring the recombinant expression vector carrying the resistance gene into a Lentinus edodes strain carrying the LeV-HKB virus to prepare transformants.

[0020] Preferably, in the method, the recombinant expression vector is introduced into a Lentinus edodes strain through Agrobacterium-mediated transformation to prepare transformants.

[0021] Preferably, in the method, the Agrobacterium-mediated transformation is to add the bacterial solution of Agrobacterium containing the recombinant expression vector into the shiitake mycelium block for incubation.

[0022] Preferably, in the method, the OD of the Agrobacterium bacterial solution is 600 =0.8~1.0, add the bacterial solution to cover the mushroom mycelium blocks and incubate for 20~30min.

[0023] In general, the above technical solutions conceived by the present invention can achieve the following beneficial effects compared with the prior art:

[0024] The present invention provides a recombinant expression vector, which carries the target gene shown in SEQ ID NO.2. Transferring the recombinant expression vector into a Lentinus edodes strain carrying the LeV-HKB virus can promote the growth of Lentinus edodes mycelium and improve the heat resistance and Trichoderma resistance of the strain after heat stress.

[0025] In addition, the method for improving the heat resistance and Trichoderma resistance of shiitake mushrooms provided by the present invention comprises the step of transferring the recombinant expression vector into a shiitake mushroom strain carrying the LeV-HKB virus to prepare a transformant. The method can simultaneously improve the heat resistance and Trichoderma resistance of shiitake mushrooms, which is beneficial to reducing the economic losses caused by high temperature heat stress or dark green Trichoderma infection in greenhouse cultivation of shiitake mushrooms. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the horizontal transmission of LeV-HKB between VI and VF-Hyg; Figure 1 A: Phenotypic comparison of VF and VF-Hyg; B: Horizontal infection of LeV-HKB; C: Addition of Hyg to verify infection;

[0027] Figure 2 It is the gel electrophoresis and RT-qPCR verification of VF-Hyg+virus; Figure 2 A is gel electrophoresis, and B is RT-qPCR verification;

[0028] Figure 3 The colony morphology and hyphal growth rate of VF-Hyg and VF-Hyg+virus under the conditions of 25℃ culture and 37℃ treatment for 48h; Figure 3 A: Colony morphology of VF-Hyg and VF-Hyg+virus under 25℃ and 37℃ treatment for 48h; B: Mycelial growth rate;

[0029] Figure 4 is the expression level of LeV-HKB ORFs;

[0030] Figure 5 It is the map and verification of recombinant plasmid vector; Figure 5 Middle A: recombinant plasmid vector map;

[0031] B: M: 1kb Marker, 1: original plasmid without enzyme digestion, 2-3: linear plasmid after double enzyme digestion;

[0032] C: M: 2000bp Marker, 3-10 and 12-20: PCR identification bands of E. coli culture; D: Sequence similarity comparison results;

[0033] Figure 6 Recombinant plasmid vector map; Figure 6A: STTM target sequence; B: PUC57-STTM recombinant plasmid; C: M: 2000bp Marker, 1, 2: PUC57-STTM recombinant plasmid;

[0034] D: Recombinant plasmid vector map;

[0035] Figure 7 Recombinant plasmid verification; Figure 7 A: M: 2000bp Marker, 2, 3: Escherichia coli PCR identification bands; B: sequence similarity comparison results; C: M: 2000bp Marker, 1-10: Agrobacterium tumefaciens PCR identification bands;

[0036] Figure 8 is RT-qPCR analysis of OEPre and STTM transformants; Figure 8 A: the expression level of the target gene shown in SEQ ID NO.1 in the OEPre transformant; C: the expression level of the target sequence shown in SEQ ID NO.2 in the STTM transformant;

[0037] Fig. 9 is RT-qPCR analysis of LeV-HKB ORFs in VI, VI-CK, and transformants after room temperature and heat treatment; Fig. 9 A is the RT-qPCR analysis of LeV-HKB ORFs in VI, VI-CK and OEPre transformants cultured at room temperature, B is the RT-qPCR analysis of LeV-HKB ORFs in VI, VI-CK and OEPre transformants treated with heat, C is the RT-qPCR analysis of LeV-HKB ORFs in VI, VI-CK and STTM transformants cultured at room temperature, and D is the RT-qPCR analysis of LeV-HKB ORFs in VI, VI-CK and STTM transformants treated with heat;

[0038] Fig.10 is the colony morphology and hyphal growth rate of VI, VI-CK and OEPre transformants before and after heat stress; Fig.10 A: Colony morphology of VI, VI-CK and OEPre transformants; B: Mycelial growth rate of VI, VI-CK and OEPre transformants;

[0039] Fig.11 The colony morphology and hyphal growth rate of VI, VI-CK and STTM transformants before and after heat stress; Fig.11 A: Colony morphology of VI, VI-CK and STTM transformants; B: Mycelial growth rate of VI, VI-CK and STTM transformants;

[0040] Fig.12is the colony morphology and infection ratio of OEPre transformants against Trichoderma aureum before and after heat treatment; Fig.12 A: Colony morphology of VI, VI-CK and OEPre transformants resistant to Trichoderma viride before and after heat stress; B: Infection ratio of VI, VI-CK and OEPre transformants to Trichoderma viride before and after heat stress;

[0041] Fig.13 The colony morphology and infection ratio of STTM transformants against Trichoderma aureum before and after heat treatment; Fig.13 A: Colony morphology of VI, VI-CK and STTM transformants resistant to dark green Trichoderma before and after heat stress; B: Infection ratio of dark green Trichoderma in VI, VI-CK and STTM transformants before and after heat stress;

[0042] Fig.14 It is the colony morphology of STTM transformants resistant to Trichoderma aureum before and after heat treatment; Fig.14 A: Mycelium was cultured at 25℃ for 10 days and inoculated with dark green Trichoderma for 20 days; B: Mycelium was cultured at 25℃ for 8 days, treated at 37℃ for 48 hours, and inoculated with dark green Trichoderma for 8 days; the part in the red circle is the area of ​​​​Shiitake mushrooms infected with dark green Trichoderma. DETAILED DESCRIPTION

[0043] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0044] The inventors simulated heat stress to study the effects of heat stress on the mycelial growth and Trichoderma resistance of Lentinus edodes strains carrying the virus LeV-HKB before and after heat stress, and found that compared with normal temperature culture, the mycelial growth rate of Lentinus edodes decreased significantly after heat stress, and the resistance to dark green Trichoderma was significantly reduced.

[0045] The present invention is based on the construction of a recombinant expression vector, and it is found that the recombinant plasmid carrying the target gene shown in SEQ ID NO.2 is transferred into a shiitake mushroom strain carrying the virus LeV-HKB, which can promote the growth rate of the shiitake mushroom strain after heat stress, improve the heat resistance of the shiitake mushroom strain and the resistance to dark green Trichoderma, especially after the heat stress is transferred to normal temperature culture, the shiitake mushroom strain can still hinder the continuous infection of dark green Trichoderma, and compared with the shiitake mushroom strain carrying the virus LeV-HKB in the control group, the resistance of the shiitake mushroom strain to dark green Trichoderma can be significantly improved.

[0046] Based on this, the present invention provides a recombinant expression vector carrying a target gene as shown in SEQ ID NO.2, wherein the sequence of SEQ ID NO.2 is AATCGGCGCCctaTTTGCTATAAGTTGTTGTTGTTATGGTCTAATTTA AATATGGTCTAAAGAAGAAGAATAATCGGCGCCctaTTTGCTATAA, and is designed and synthesized as follows:

[0047] Three bases of CTA were added to each of the two specific complementary sequences to form two incomplete complementary sequences, which were connected through a 48 nt specific sequence to design a target sequence with a total length of 94 bp as shown in SEQ ID NO.2.

[0048] Preferably, the recombinant expression vector further contains a resistance gene. In some embodiments, the resistance gene is a Hyg gene.

[0049] In some embodiments, the recombinant expression vector is a recombinant plasmid carrying the target gene shown in SEQ ID NO.2, which is prepared as follows:

[0050] According to SEQ ID NO.2, a target gene with a full length of 94 bp was synthesized and connected into the PUC57 plasmid, and the target gene fragment was amplified from the recombinant plasmid and recovered;

[0051] The plasmid with Legpd promoter was used as the basic skeleton of the target gene expression vector. After double restriction digestion, the target gene fragments were connected through homologous recombination to obtain the recombinant expression vector.

[0052] For example, the plasmid pCAMBIA1300 with Legpd promoter is used as the basic skeleton of the target gene expression vector, the double restriction sites are KpnI and EcoRI, and the target gene fragments are connected by homologous recombination to obtain a recombinant expression vector.

[0053] Verification of the recombinant expression vector: The recombinant expression vector was transferred into the competent Escherichia coli cells Trans1-T1, and a single colony was picked and cultured in LB (50 μg / mL Kan+) liquid medium for shaking. The target gene fragment (a target band of about 100 bp) was verified by bacterial liquid PCR, and part of the bacterial liquid was sequenced and compared with the target gene sequence shown in SEQ ID NO.2.

[0054] In addition, the present invention also provides a method for simultaneously improving the heat resistance and Trichoderma resistance of shiitake mushrooms, which comprises the step of transferring the recombinant expression vector of the present invention into a shiitake mushroom strain carrying the LeV-HKB virus to prepare transformants.

[0055] Preferably, the recombinant expression vector carrying the resistance gene and the target gene as shown in SEQ ID NO.2 is transferred into the shiitake mushroom strain carrying the LeV-HKB virus to prepare transformants, which is conducive to screening out positive transformants through screening culture medium.

[0056] In some embodiments, the recombinant expression vector is transformed by Agrobacterium-mediated transformation, specifically:

[0057] The Agrobacterium containing the recombinant plasmid carrying the resistance gene and the target gene shown in SEQ ID NO.2 was streaked for 1 day, and a single colony was picked and placed in 1mL LB (50μg / mL Kan+) liquid medium, and cultured overnight at 28℃, 200rpm. Then the cultured Agrobacterium culture was added to 100mL MM (50μg / mL Kan+) liquid medium, and cultured at 28℃, 200rpm until the OD value was about 0.8. The culture was collected, centrifuged at 4℃, 5000r / min for 10min, the supernatant was removed, and the precipitate was resuspended with an equal volume of IM culture medium, and the OD of the culture was measured. 600 and diluted to OD 600 =0.4. Add AS to a final concentration of 200 μmol / L and culture at 200 r / min and 28°C for 4 h until OD 600 =0.8~1.0.

[0058] After the mycelium of Lentinus edodes was activated in MYG medium, a mycelium block of the same diameter was taken out with a cork puncher, and washed with IM medium. The washed mycelium block was placed in a sterilized empty plate. The Agrobacterium liquid (OD 600 =0.8) was poured into an empty plate, the mycelial block was covered and incubated for 20-30 min, and shaken every 5 min; the incubated mycelial block was slightly dried with filter paper, transferred to 200 μmol / L AS Co-IM medium, and cultured at 25°C for 3 d; after 3 d, the mycelial block was rinsed with ddH2O several times, and the mycelial block was immersed in ddH2O containing 400 μg / mL cefotaxime for 20 min, and the washed mycelial block was transferred to MYG (containing 4 μg / mL hygromycin and 400 μg / mL cefotaxime) medium for direct screening to obtain positive transformants.

[0059] Experiments have confirmed that using this method to transfer the recombinant expression vector of the present invention into a Lentinus edodes strain carrying the LeV-HKB virus can promote the growth of Lentinus edodes mycelium and improve the heat resistance and Trichoderma resistance of the strain after heat stress.

[0060] The following are examples

[0061] Test strains: The strains used in the following examples are Lentinus edodes strains SY1 and SY1-R8, and Trichoderma atroviride strain 92-1. SY1 Lentinus edodes strain is one of the main Lentinus edodes varieties in Hubei Province, and dsRNA analysis shows that it carries the LeV-HKB virus. In the laboratory's preliminary research, the chemical reagent ribavirin detoxification method was used to obtain the isogenic virus-free strain SY1-R8 strain (VF, Virus Free) of the SY1 strain (VI, Virus Infeted). According to the "NY / T1730-2009 ISSR Method for Authenticity Identification of Edible Fungi", 10 ISSR primers were randomly selected to analyze the virus-infected strain VI and the virus-free strain VF. The results showed that the ISSR banding patterns before and after detoxification were completely consistent, indicating that the ribavirin treatment did not affect the genetic information of the Lentinus edodes strain. The dark green Trichoderma (Trichoderma atroviride) strain 92-1 was isolated from a rotten mushroom stick by our laboratory. The Trichoderma strain was previously identified by morphology and ITS sequence (reference: Liu S, Xie J, Cheng J, Li B, Chen T, Fu Y, Li G, Wang M, Jin, Wan H, Jiang D. Fungal DNA virus infects a mycophagous insect and utilizes it as a transmission vector. Proc Natl Acad Sci USA, 2016, 113: 12803-12808). All the above strains are deposited in the Institute of Applied Mycology, Huazhong Agricultural University.

[0062] Test medium: LB medium: 5 g yeast extract, 10 g peptone, 10 g NaCl, (solid medium plus Agar 20 g), add deionized water (ddH2O) to 1000 mL, and adjust the pH to 7.0 with NaOH.

[0063] Malt extract medium (MYG): 20 g malt extract, 20 g glucose, 1 g peptone, 1 g yeast extract (solid medium plus 20 g Agar), add deionized water (ddH2O) to 1000 mL.

[0064] Potato Dextrose Agar PDA: 250 g potatoes, 20 g glucose, 5 g tryptone (solid medium plus 20 g agar), add deionized water (ddH2O) to 1000 mL.

[0065] Minimal Media MM: K-buffer 10mL, MN buffer 20mL,

[0066] 20% Glucose (w / v) 10 mL, 0.01% FeSO4 (w / v) 10 mL, 20% (NH4)2SO4 (w / v) 2.5 mL, 1% CaCl2·2H2O (w / v) 1 mL, add deionized water (ddH2O) to 1000 mL, adjust the pH to 6.7-7.0 with HCl or NaOH.

[0067] Induction Media IM: K-buffer 10mL, MN buffer 20mL, 20% Glucose (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), add deionized water (ddH2O) to 1000mL, adjust the pH to 5.6 with HCl or NaOH.

[0068] Co-culture medium (Co-IM): K-buffer 10mL, MN buffer 20mL, 20% Glucose (w / v) 2.5mL, 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), Agar 20g, add deionized water (ddH2O) to 1000mL, adjust the pH to 5.6 with HCl or NaOH.

[0069] The instruments and equipment used are shown in Table 1.

[0070] Table 1 Names and manufacturers of instruments used in the test

[0071]

[0072] Main reagents:

[0073] HLingene agarose gel recovery PCR purification kit (Shanghai Huiling Biotechnology Co., Ltd.); 2×TaqPlus Master MixⅡ, IIQ RT SuperMix for qPCR and AceQTM QPCR SYBR Green Master Mix (Venozyme Biotechnology Co., Ltd.); restriction endonucleases (Thermo Scientific, USA); hygromycin B (Hyg B) (Roche, Switzerland). RNAiso Plus total RNA extraction kit (Takara); miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) kit was purchased from Novazon Biotechnology (Nanjing); miRNA Universal SYBR qPCR Master Mix was purchased from Novazon Biotechnology (Nanjing); primers were purchased from Tianyi Huiyuan (Wuhan).

[0074] Real-time fluorescence quantitative PCR (RT-qPCR) analysis: Leactin was used as the internal reference gene, and the gene expression was detected by RT-qPCR technology. The total quantitative reaction system was 10 μL: cDNA 1 μL, AceQTM qPCR SYBR Green Master Mix 5 μL, primers (10 μM) 0.5 μL each, ddH2O 3 μL. The amplification reaction used a two-step method: 95°C pre-denaturation for 5 min; 95°C

[0075] 10s, 60℃30s, 40 cycles; 95℃10s; the final melting curve was set at 65℃-95℃, and the fluorescence signal was collected every 0.5℃ increase in temperature for 5s. The relative expression of genes was calculated using the 2-ΔΔCT method.

[0076] All experiments were repeated 3-5 times, and the final data were presented as mean±standard deviation (SD). SPSS software was used to analyze the significance of the data ("*" indicates p<0.05; "**" indicates p<0.01).

[0077] Example 1 Heat stress promotes virus replication in poisonous shiitake mushroom strains

[0078] (1) LeV-HKB horizontal infection

[0079] The empty plasmid pCAMBIA1300-g with Hyg resistance was transferred into the competent cell Trans1-T1 of Escherichia coli, and the plasmid was extracted and transferred into Agrobacterium, and the detoxified edodes strain VF was transformed, and the non-toxic transformant VF-Hyg strain with Hyg resistance was obtained by screening on MYG medium (added with 4 μg / mL hygromycin), and the transformation of edodes mycelium mediated by Agrobacterium was as follows:

[0080] Agrobacterium containing empty plasmid was streaked and cultured for 1 day, and single colonies were picked and placed in 1 mL LB (50 μg / mL Kan + ) liquid medium, cultured overnight at 28°C, 200 rpm, and then the cultured Agrobacterium was added to 100 mL MM (50 μg / mL Kan + ) liquid medium, 28℃, 200rpm culture until OD value is about 0.8. Collect the bacterial solution, centrifuge at 4℃, 5000r / min for 10min, remove the supernatant, resuspend the precipitate with an equal volume of IM culture medium, and measure the OD value of the bacterial solution at 600nm 600 and diluted to OD 600 =0.4. Add AS to a final concentration of 200 μmol / L and culture at 200 r / min and 28°C for 4 h until OD 600 =0.8.

[0081] After the mycelium of Lentinus edodes was activated in MYG medium, a mycelium block of the same diameter was taken out with a cork puncher, and washed with IM medium. The washed mycelium block was placed in a sterilized empty plate. The Agrobacterium liquid (OD 600 =0.8) was poured into an empty plate, the mycelial block was covered and incubated for 20 min, and shaken every 5 min; the incubated mycelial block was slightly dried with filter paper, transferred to 200 μmol / L AS Co-IM medium, and cultured at 25°C for 3 d; after 3 d, the mycelial block was rinsed with ddH2O several times, and the mycelial block was immersed in ddH2O containing 400 μg / mL cefotaxime for 20 min, and the washed mycelial block was transferred to MYG (containing 4 μg / mL hygromycin and 400 μg / mL cefotaxime) medium for direct screening to obtain the non-toxic transformant VF-Hyg strain with Hyg resistance.

[0082] VF-Hyg was cultured at 25°C and 37°C for 48 h, with VF as the control. The phenotypes of VF and VF-Hyg were compared as shown in Figure 1 As shown in A, there was no significant difference in mycelial growth rate, indicating that the empty plasmid pCAMBIA1300-g carrying Hyg resistance had no significant effect on the growth rate of Lentinus edodes strains.

[0083] The virus was horizontally transmitted by inoculating VI and VF-Hyg on the plate at the same time and culturing for 5 days; inoculating VI for 3 days and then inoculating VF-Hyg for 2 days; inoculating VF-Hyg for 3 days and then inoculating VI for 2 days. Figure 1 (B) After the mycelia of VI and VF-Hyg were in contact for 1 day, the mycelia of VF-Hyg away from VI were selected and cultured on MYG medium supplemented with 4 μg / mL hygromycin. Figure 1As shown in C, the results showed that VI could not grow, while VF-Hyg+virus could grow normally, thus verifying that the bacterial block picked and re-transferred into LeV-HKB was VF-Hyg+virus rather than VI.

[0084] RT-PCR analysis was performed on the Hyg gene of strains VF-Hyg and VF-Hyg+virus and the RdRp gene of LeV-HKB in strain VF-Hyg+virus. The results are as follows Figure 2 As shown in A; and the Hyg gene and virus ORFs of strains VF-Hyg and VF-Hyg+virus were further verified by RT-qPCR, where the primer information used is shown in Table 2, and the results are shown in Figure 2 As shown in B, VF-Hyg+virus was used as the control, and the strain VF-Hyg did not contain the virus LeV-HKB, that is, LeV-ORF1 and LeV-ORF2 were not detected in the VF-Hyg strain. It can be seen that among the three methods of horizontal virus transmission, only the method of inoculating VI first and then VF-Hyg obtained the VF-Hyg+virus strain that was re-transferred into LeV-HKB.

[0085] Table 2 Primer information used for VI-Hyg+virus verification

[0086]

[0087] Note: For recombination homology primers, lowercase letters represent homology arms, and uppercase letters represent gene fragment primers.

[0088] Using VF-Hyg as the control, the experimental group was the virus-carrying strain VF-Hyg+virus obtained by horizontal transmission of hyphae and re-transfer into LeV-HKB to verify the effect of LeV-HKB on colony morphology and hyphae growth rate. The results of colony morphology are shown in Figure 3 As shown in A, under the condition of 25℃ room temperature culture, there was no significant difference in the mycelial growth rate between VF-Hyg and VF-Hyg+virus; after heat stress treatment (37℃, 48h), the mycelial growth rate of VF-Hyg and VF-Hyg+virus decreased when cultured at 25℃, but the mycelial growth rate of VF-Hyg was higher than that of VF-Hyg+virus; the results of the difference analysis of mycelial growth rate of each group are shown in Figure 3 As shown in B, after being treated at 37°C for 48 h and then cultured at 25°C, the mycelial growth rate of VF-Hyg was significantly higher than that of VF-Hyg+virus, while there was no significant difference between the two under normal temperature culture conditions of 25°C.

[0089] The expression level of LeV-HKB of VF-Hyg+virus before and after heat stress was further analyzed, and the quantitative analysis results were as follows Figure 4As shown, after heat treatment, the ORFs expression level of LeV-HKB in the VF-Hyg+virus strain was upregulated, among which the expression level of ORF1 increased significantly. It can be seen that heat stress can promote the replication of the virus LeV-HKB in shiitake mushrooms.

[0090] Example 2 Effects of different recombinant expression vectors on viral replication in Lentinus edodes strains

[0091] (1) Construction and verification of recombinant expression vector 1

[0092] Synthesize a sequence as shown in SEQ ID NO.1, record it as target gene 1, which is a non-coding sequence, and no sequence is aligned to it on the NCBI website. Primer5 was used to design primers, and the homologous recombination primers used were designed using CE Design software, with homologous arm sequences required for homologous recombination at both ends. Transformant verification primers were directly designed using SnapGene gene software, and all primers used were synthesized by Wuhan Tianyi Huiyuan Biotechnology Co., Ltd., and specific primer sequence information is shown in Table 2. After the primer design is completed, DNA is used as a template to amplify the candidate gene, and 1% agarose gel electrophoresis is used for detection. The candidate gene fragment is recovered using the San Prep column DNA recovery kit, and the reaction product is transferred to Escherichia coli Trans1-T1 competent cells, a single colony is picked for verification, and sent to the company for sequencing.

[0093] Construction of recombinant expression vector 1: First, PCR amplification of the target gene DNA shown in SEQ ID NO.1 was performed, and the amplification reaction system (50 μL) was as follows: 2×Phanta Max Buffer 25 μL, primers (10 μM) 2 μL each, dNTP Mix 1 μL, PhantaMax Super-Fidelity DNA Polymerase1 (U / μL) 1 μL, template DNA 2 μL, and ddH2O was added to 50 μL. Reaction parameters: 95°C pre-deformation for 5 min, 95°C denaturation for 30 s, 61°C annealing for 40 s, 70°C extension for 1 min, cycle number 34, 70°C extension for 10 min.

[0094] The plasmid pCAMBIA1300-g was completely digested with restriction endonucleases EcoR1 and Kpn1 at 37°C for 1 h. The digestion system was as follows: 30 μg of pCAMBIA1300-g plasmid, 2 μL each of Kpn-Ⅰ and EcoR-Ⅰ, 5 μL of buffer, supplemented with ddH2O to 50 μL. After the digestion of the target gene and plasmid, the size of the target fragment was checked by agarose gel electrophoresis, and the DNA fragment of the target gene and the linearized plasmid were recovered using the San Prep column DNA recovery kit. The digested pCAMBIA1300-g and the target gene were connected by recombinase.

[0095] Reaction system: 4 μL of linearized plasmid fragment pCAMBIA1300-g, 1 μL of target gene, 5 μL of 2×BasicAssembly Mix, mix all the solutions, place in a PCR instrument, and react at 50°C for 15 minutes. After the reaction is completed, immediately place the reaction solution in ice to cool for a few seconds, and then transform the reaction solution into E. coli Trans1-T1 competent cells. Pick up LB medium (50 μg / mL Kan + ) and amplified the target gene fragment using the upstream and downstream primers of the target gene. The positive single colony was expanded in liquid LB medium, and the bacterial solution was sent to the company for sequencing. The recombinant plasmid with the target gene shown in SEQ ID NO.1 was obtained and recorded as recombinant expression vector 1.

[0096] The plasmid pCAMBIA1300-g with Legpd promoter was used as the basic backbone of the recombinant expression vector in VI. The original backbone was double-digested with KpnI and EcoRI to obtain a linearized vector ( Figure 5 B), the linear vector and the target gene fragment shown in SEQ ID NO.1 are connected by homologous recombination ( Figure 5 After the constructed recombinant plasmid was transferred into E. coli competent cells Trans1-T1, a single colony on the plate was picked up and cultured in LB liquid (50 μg / mL Kan+) for 1 day. The target gene fragment was verified by bacterial liquid PCR. The PCR results showed that 17 bacterial liquids had a bright target band of about 600 bp ( Figure 5 C). A portion of the bacterial solution was sent to the company for sequencing, and the sequencing results were compared with the target gene sequence shown in SEQ ID NO.1 of the genome sequenced monokaryotic strain W1-26, and the similarity was 100% ( Figure 5 D), indicating that the recombinant expression vector 1 carrying the target gene shown in SEQ ID NO.1 has been successfully constructed.

[0097] (2) Construction and verification of recombinant expression vector 2

[0098] Construction of recombinant expression vector 2: Three bases CTA were added to each of the two specific complementary sequences to form two incomplete complementary sequences, which were connected through a 48 nt specific sequence to design a target sequence STTM with a total length of 94 bp as shown in SEQ ID NO.2: AATCGGCGCCctaTTTGCTATAAGTTGTTGTTGTTATGGTCTAATTTA AATATGGTCTAAAGAAGAAGAATAATCGGCGCCctaTTTGCTATAA ( Figure 6A), denoted as target gene 2, was synthesized by Wuhan Tianyi Huiyuan Company and linked into the PUC57 plasmid ( Figure 6 B), the target sequence fragment was amplified from the recombinant plasmid PUC57-STTM and recovered by gel ( Figure 6 C). The plasmid pCAMBIA1300 with Legpd promoter was used as the basic skeleton of the expression vector of the target sequence shown in SEQ ID NO.2, and the double restriction sites were KpnI and EcoRI. The gene fragments of the target sequence shown in SEQ ID NO.2 were connected by homologous recombination ( Figure 6 After ligation, the cells were transformed into competent E. coli Trans1-T1 cells. The positive single colonies were detected by PCR. The positive single colonies were expanded and cultured in liquid LB medium and sent to the company for sequencing. The recombinant plasmid was named pCAMBIA1300-go-STTM and recorded as recombinant expression vector 2.

[0099] The recombinant plasmid pCAMBIA1300-go-STTM was transferred into the competent E. coli cell Trans1-T1, and a single colony was picked and cultured in LB (50 μg / mL Kan+) liquid medium. The gene fragment of the target sequence shown in Legpd+SEQ ID NO.2 was verified by bacterial liquid PCR. The PCR results showed that the two bacterial liquids had a bright target band of about 100 bp ( Figure 7 A). A portion of the bacterial solution was sent to the company for sequencing, and the sequencing results were compared with the sequence shown in the designed SEQ ID NO.2, and the similarity was 100% ( Figure 7 B), indicating that the recombinant expression vector 2 carrying the target sequence shown in SEQ ID NO.2 has been successfully constructed. The recombinant plasmid was extracted and transferred into Agrobacterium EHA105, and 10 single colonies were picked and shaken (50 μg / mL Kan+, Rif), and the bacterial solution was verified by PCR ( Figure 7 Middle C).

[0100] Table 3 Primer information used for transformation

[0101]

[0102]

[0103] Note: For recombination homology primers, lowercase letters represent homology arms, and uppercase letters represent gene fragment primers.

[0104] (3) Agrobacterium-mediated transformation of Lentinus edodes mycelium to prepare transformants

[0105] Agrobacterium containing recombinant plasmids (recombinant expression vector 1 or recombinant expression vector 2) was streaked and cultured for 1 day, and a single colony was picked and placed in 1 mL LB (50 μg / mL Kan+) liquid medium, and cultured overnight at 28°C and 200 rpm. Then, the cultured Agrobacterium culture was added to 100 mL MM (50 μg / mL Kan+) liquid medium, and cultured at 28°C and 200 rpm until the OD value was about 0.8. The culture was collected, centrifuged at 4°C and 5000 r / min for 10 min, the supernatant was removed, and the precipitate was resuspended with an equal volume of IM culture medium, and the OD of the culture was measured. 600 and diluted to OD 600 =0.4. Add AS to a final concentration of 200 μmol / L and culture at 200 r / min and 28°C for 4 h until OD 600 =0.8.

[0106] After the activation of Lentinus edodes hyphae in MYG medium, use a cork puncher to take out the same diameter of the mycelium block, rinse it with IM medium, and put the washed mycelium block into a sterilized empty plate; pour the Agrobacterium culture solution (OD=0.8) after shaking for 4 hours into the empty plate, cover the mycelium block and incubate it for 20 minutes, shaking it every 5 minutes; the incubated mycelium block is slightly dried with filter paper, transferred to 200μmol / L AS Co-IM medium, and cultured at 25℃ for 3 days; after 3 days, rinse the mycelium block with ddH2O several times, and soak the mycelium block in ddH2O containing 400μg / mL cefotaxime for 20 minutes, and the washed mycelium block is transferred to MYG (containing 4μg / mL hygromycin and 400μg / mL cefotaxime) medium for direct screening.

[0107] Transformant screening and identification: Culture on MYG medium (containing 4 μg / mL hygromycin and 400 μg / mL cefotaxime) at 25°C for 5-10 days, and observe the growth of mycelium. If the mycelium continues to grow, pick the mycelium to MYG medium containing hygromycin for further screening. Those that can still grow normally after 3 rounds of screening are considered resistant transformants. Among them, the shiitake mushroom strains transferred with the recombinant expression vector 1 carrying the target gene shown in SEQ ID NO.1 are OEPre transformants, and the shiitake mushroom strains transferred with the recombinant expression vector 2 carrying the target sequence shown in SEQ ID NO.2 are STTM transformants. Extract mycelium DNA and verify the target gene.

[0108] The method for extracting DNA from Lentinus edodes mycelium is as follows: a. Grind 0.1 g of mycelium with liquid nitrogen, transfer to a 1.5 mL centrifuge tube, add 475 μL 2% CTAB (preheated) and 25 μL 1% SDS, and mix by inversion; b. Add 500 μL PCI (phenol / chloroform / isoamyl alcohol = 25:24:1), mix well, centrifuge at 12000 r / min and 4°C for 10 min, and take the supernatant to a new 1.5 mL centrifuge tube; c. Repeat the previous step until there is no obvious precipitation in the middle layer; d. Add 2 / 3 volume of isopropanol to the supernatant, mix by inversion, precipitate at -20°C for 30 min, centrifuge at 12000 r / min and 4°C for 10 min, and discard the supernatant; e. Add 1 mL 70% ethanol to wash the precipitate, 12000 r / min Centrifuge at 4°C for 5 min and discard the supernatant; f. Repeat the previous step, dry the precipitate on a clean bench at room temperature, add appropriate amount of ddH2O and 1% RNase (Vazyme), and dissolve the precipitate at 37°C for 1 h; g. DNA quality test: use an ultra-micro spectrophotometer to test the quality of the extracted DNA, and after the measurement is completed, store it at -80°C for future use.

[0109] The VI strain transformed with the empty vector pCAMBIA1300 was used as a control, named VI-CK, and the Lentinus edodes Leactin gene was used as an internal reference. The expression of the target gene in the transformant OEPre and the expression of the target sequence in the transformant STTM were detected by RT-qPCR technology. The quantitative results showed that compared with VI-CK, the expression of the target gene shown in SEQ ID NO.1 in 10 OEPre transformants was significantly upregulated ( Figure 8 A); the synthetic target sequence as shown in SEQ ID NO.2 was almost undetectable in VI and VI-CK, but was clearly expressed in 9 transformants STTM ( Figure 8 Middle C).

[0110] Three transformants OEPre and STTM were selected to analyze the effects of 25℃ room temperature culture and 37℃ heat treatment for 48h on the expression of two ORFs of the virus LeV-HKB in the shiitake mushroom strain. RT-qPCR results showed that under room temperature culture conditions, compared with the control VI-CK_25, there was no significant difference in the expression of the two ORFs of the virus in OEPre-7_25 and STTM-10_25 transformants ( Fig. 9 However, after heat stress, the expression levels of the two ORFs of the virus LeV-HKB in the edodes strains transferred with recombinant expression vector 1 were significantly upregulated, while the expression levels of the two ORFs of the virus LeV-HKB in the edodes strains transferred with recombinant expression vector 2 were significantly downregulated ( Fig. 9B and D), indicating that the recombinant expression vector 2 carrying the target sequence shown in SEQ ID NO.2 can inhibit the replication of the virus LeV-HKB under heat stress conditions.

[0111] Example 3 Effects of different recombinant expression vectors on the growth of Lentinus edodes mycelium

[0112] The five OEPre transformants and five STTM transformants in Example 2 were selected to analyze the effects of different recombinant expression vectors on the mycelial growth rate of Lentinus edodes strain VI. VI, VI-CK, OEPre transformants OEPre-2, OEPre-4, OEPre-7, OEPre-9, OEPre-11, and STTM transformants STTM-1, STTM-3, STTM-5, STTM-6, STTM-10 were cultured on MYG medium, and the colony phenotypes were observed and the mycelial growth rate was measured after culturing at 25° C. for 5 days, as follows:

[0113] The phenotype of the colonies cultured for 5 days at 25°C in the dark was observed on MYG solid medium, and photographed and recorded. During the observation process, the cross-line method was used to detect the growth rate of the hyphae. The center of the bacterial block was used as the intersection to draw a cross and measure the diameter r of the hyphae tip. After the hyphae grew to an appropriate size, the colony diameter R was measured, and the number of days of hyphae growth was recorded as n. The growth rate was calculated, and 5 replicates were set for each strain. The average daily growth rate of the hyphae was calculated according to the formula ν = (R-r) / n. The results are as follows Fig.10 and Fig.11 shown.

[0114] Taking VI-CK as the control, the colony diameters of the five OEPre transformants were smaller, and the average daily growth rate of hyphae was significantly lower than that of VI-CK. Among them, OEPre-2 and OEPre-7 had the smallest colony diameters and the slowest hyphae growth rates. The colony diameters of the five STTM transformants were larger, and the average daily growth rate of hyphae was significantly higher than that of VI-CK. After culturing at 25℃ for 5 days, 37℃ for 48 hours, and then culturing at 25℃ for 5 days, it was found that the hyphae recovery growth rate of the OEPre transformants decreased significantly, and the growth rates of OEPre-2 and OEPre-7 were the lowest ( Fig.10 ); the mycelial growth rate of the five STTM transformants was higher than that of VI-CK ( Fig.11 ), indicating that the recombinant expression vector 2 carrying the target sequence shown in SEQID NO.2 can promote the growth of Lentinus edodes mycelium, especially the growth of mycelium after heat stress, indicating that the recombinant expression vector 2 can improve the heat resistance of Lentinus edodes strains.

[0115] Example 4 Effects of different recombinant expression vectors on resistance of Lentinus edodes

[0116] Transformant OEPre-7 in Example 2 was selected, and the control group was cultured in the dark at 25°C. VI, VI-CK and transformant OEPre-7 were cultured on MYG medium, and the mushroom blocks were removed after 10 days, and dark green Trichoderma was inoculated in the center of the plate. The experimental group was cultured in the dark at 25°C for 8 days, treated at 37°C for 48 hours, and then inoculated with dark green Trichoderma, and the phenotype was observed after 5 days of Trichoderma infection.

[0117] A longitudinal comparison of the effects of different recombinant expression vectors on the ability of Lentinus edodes to resist Trichoderma revealed that under both normal temperature and heat treatment culture conditions, the proportion of the area infected by Trichoderma to the colony area of ​​the transformant OEPre-7 was greater than that of VI-CK, that is, the introduction of recombinant expression vector 1 reduced the resistance of Lentinus edodes strains to Trichoderma, and the resistance gap was more obvious after heat stress.

[0118] A horizontal comparison of the effects of heat treatment on the resistance of Lentinus edodes strains to Trichoderma revealed that heat treatment increased the proportion of Trichoderma infection in colonies VI, VI-CK, and OEPre-7, with the increase in OEPre-7 being greater ( Fig.12 ). This result indicates that the recombinant expression vector 1 has an inhibitory effect on the resistance of Lentinus edodes strains to Trichoderma, and high temperature can significantly increase the expression level of the target gene shown in SEQ ID NO.1 in Lentinus edodes strains. Therefore, heat treatment further amplifies the resistance gap between transformants OEPre-7 and VI-CK to dark green Trichoderma.

[0119] Transformant STTM-10 in Example 2 was selected, and the control group was cultured in the dark at 25°C. VI, VI-CK and transformant STTM-10 were cultured on MYG medium respectively. After 10 days, the mushroom blocks were picked out, and dark green Trichoderma was inoculated in the center of the plate and infected for 5 days. The test group was cultured in the dark at 25°C for 8 days, treated at 37°C for 48 hours, and then inoculated with dark green Trichoderma for 5 days. The dark green Trichoderma resistance of the mycelium was evaluated as follows:

[0120] Inoculate a 9mm diameter shiitake mycelium block in the center of the MYG plate (90mm in diameter), inoculate 10 plates for each strain, and place them at a constant temperature and dark place at 25℃ for incubation until the mycelium is fully grown. Take out 5 of the plates and place them at a constant temperature and dark place at 37℃ for incubation for 48h, and continue to culture the remaining 5 plates at a constant temperature and dark place at 25℃ for 48h. After all the above treatments are completed, pick out the shiitake mycelium blocks, and inoculate a dark green Trichoderma mycelium block with a diameter of about 9mm in the center of the plate, with the mycelium facing up. After inoculation, all treatments are placed at a constant temperature and dark place at 25℃ for incubation, and the reaction after inoculation of dark green Trichoderma blocks is observed. ImageJ 1.52a software was used to uniformly analyze and measure the area covered by the browning of the shiitake colony after inoculation of dark green Trichoderma blocks. The formula "browning ratio of shiitake colonies = area covered by browning of shiitake colonies / plate area" was used to calculate the ratio of dark green Trichoderma infecting shiitake colonies under different temperature treatments, which was used to evaluate the resistance of shiitake strains to dark green Trichoderma (the smaller the browning ratio, the stronger the resistance of shiitake to dark green Trichoderma).

[0121] A longitudinal comparison of the effects of recombinant expression vector 2 on the resistance of shiitake mushrooms to Trichoderma revealed that under both normal temperature and heat treatment culture conditions, the proportion of the area infected by Trichoderma to the colony area of ​​the transformant STTM-10 was lower than that of VI-CK, indicating that the recombinant expression vector 2 enhanced the resistance of shiitake mushrooms to Trichoderma, and the resistance gap was also more obvious after heat treatment.

[0122] A horizontal comparison of the effects of heat treatment on the resistance of Lentinus edodes strains to Trichoderma revealed that heat treatment increased the proportion of Trichoderma infection in colonies VI, VI-CK, and STTM-10, while the increase in the proportion of infection in the transformant STTM-10 colony was smaller ( Fig.13 ). These results indicate that the recombinant expression vector 2 carrying the target sequence shown in SEQ ID NO.2 can enhance the resistance of Lentinus edodes strains to Trichoderma after heat stress.

[0123] The mycelia of Lentinus edodes cultured at room temperature and heat-treated were continued to be cultured at 25℃, and the infection of Trichoderma was continuously observed. It was found that after 20 days of infection, the infected range of VI, VI-CK and transformant STTM-10 no longer expanded. Irregular green colonies of dark green Trichoderma appeared in the center of the colonies of VI and VI-CK, and the mycelia that were not completely covered by dark green Trichoderma were darker in color. Compared with VI-CK, a clear stress circle was formed around the dark green Trichoderma block in the center of the STTM-10 colony, preventing dark green Trichoderma from continuing to infect the surrounding area, and no green spores of dark green Trichoderma were found in the stress circle. At the same time, the maximum proportion of Lentinus edodes strains infected by dark green Trichoderma after being treated at 37℃ for 48 hours was observed. It was found that after 8 days of infection, a large number of green spores of dark green Trichoderma appeared on the colonies of VI and VI-CK, and the entire colony was almost completely infected. However, STTM-10 was no longer infected by dark green Trichoderma, and the spores of dark green Trichoderma were yellow ( Fig.14 ). This result shows that heat treatment makes VI and VI-CK almost lose their resistance in the process of confrontation with dark green Trichoderma. However, STTM-10 can still form a stress circle that hinders the continuous infection of dark green Trichoderma, that is, it can still maintain a certain resistance, thus further indicating that the recombinant expression vector 2 carrying the target sequence shown in SEQ ID NO.2 can increase the resistance of Lentinula edodes strains to Trichoderma after heat stress.

[0124] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A recombinant expression vector, characterized in that: Carrying the target gene shown in SEQ ID NO.

2.

2. The recombinant expression vector according to claim 1, characterized in that It also contains resistance genes.

3. The recombinant expression vector according to claim 2, characterized in that The resistance gene includes the Hyg gene.

4. The recombinant expression vector according to any one of claims 1 to 3, characterized in that: The recombinant plasmid carrying the target gene shown in SEQ ID NO.2 was prepared according to the following method: The target gene was synthesized according to the sequence shown in SEQ ID NO.2, connected into the PUC57 plasmid, and the target gene fragment was amplified and recovered; The plasmid with Legpd promoter was used as the basic skeleton of the target gene expression vector. After double restriction digestion, the target gene fragments were connected through homologous recombination to obtain the recombinant plasmid.

5. The recombinant expression vector according to claim 4, characterized in that The plasmid carrying the Legpd promoter is pCAMBIA1300, and its double restriction enzyme cutting sites are KpnI and EcoRI.

6. A method for improving the heat resistance of shiitake mushrooms and the resistance of Trichoderma, characterized in that: The method comprises the step of transferring the recombinant expression vector as claimed in any one of claims 1 to 5 into a shiitake mushroom strain carrying the LeV-HKB virus to prepare transformants.

7. The method according to claim 6, characterized in that The recombinant expression vector as claimed in claim 2 is transferred into a Lentinus edodes strain carrying the LeV-HKB virus to prepare transformants.

8. The method according to claim 6 or 7, characterized in that The recombinant expression vector is transferred into the shiitake mushroom strain through Agrobacterium-mediated transformation to prepare transformants.

9. The method according to claim 8, characterized in that The Agrobacterium-mediated transformation is to add the bacterial solution of Agrobacterium containing the recombinant expression vector into the shiitake mycelium block for incubation.

10. The method according to claim 9, characterized in that The OD of the Agrobacterium bacterial solution 600 =0.8~1.0, add the bacterial solution to cover the mushroom mycelium blocks and incubate for 20~30min.

Images