A recombinant expression vector and a method for improving heat resistance of lentinula edodes and resistance of trichoderma

Abstract

The present application relates to a kind of recombinant expression vectors and the method for improving lentinula edodes heat resistance and trichoderma resistance, the recombinant expression vector carries the gene of purpose as shown in SEQ ID NO.2, the method is into the lentinula edodes strain carrying LeV-HKB virus of the recombinant expression vector, the recombinant expression vector carrying the gene of purpose as shown in SEQ ID NO.2 can promote the growth of lentinula edodes mycelium and improve the heat resistance and trichoderma resistance of strain after thermal stress, using the method can simultaneously improve the heat resistance of lentinula edodes and the resistance to trichoderma, it is beneficial to reduce the economic loss caused by high temperature thermal stress or deep green trichoderma infection in lentinula edodes planting.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and more particularly to a recombinant expression vector and a method for improving the heat resistance of Lentinula edodes and the resistance to Trichoderma. BACKGROUND

[0002] Lentinula edodes is an edible fungus with important economic value, with umbrella-shaped caps and columnar stems. The edible part is the fruiting body, and it is the highest yield variety of edible fungi in the world (Document 1). However, Lentinula edodes strains generally carry fungal viruses (Document 2, Document 3). For example, in Chinese Lentinula edodes 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 the characteristics of latency, infected Lentinula edodes strains do not usually show symptoms under normal conditions, but when viral diseases occur, the wide and fast spread of viruses can cause huge yield losses. Existing Lentinula edodes detoxification methods mainly include chemical detoxification and protoplast regeneration detoxification, but chemical detoxification has a long cycle, limited virus removal, and affects the activity of Lentinula edodes strains. Protoplast regeneration detoxification has a lot of randomness and unstable detoxification effect.

[0003] The common disease in Lentinula edodes cultivation is green mold disease induced by Trichoderma pathogen. Studies have shown that high temperature can lead to a decrease in the resistance of Lentinula edodes mycelium to Trichoderma, and when a certain threshold is reached, it can cause the death of Lentinula edodes mycelium (Document 5), and the virus LeV-HKB further affects the resistance of Lentinula edodes mycelium to Trichoderma after high temperature stress. Currently and in the future, centralized stick production will be widely used for facility or factory production. In the centralized stick production of Lentinula edodes, the environmental conditions of high-density large-scale stick cultivation rooms are difficult to control, and a large amount of mycelium generates heat due to respiration, which can easily cause high-temperature burning of the mycelium and Trichoderma infection, resulting in huge economic losses. In facility or factory cultivation, when temperature control or insufficient ventilation and shading conditions occur, the local temperature in the cultivation shed can reach more than 37°C, and the temperature in the bag can reach 40°C. Under such high-temperature conditions, Lentinula edodes strains infected with LeV-HKB virus have poor heat resistance, and the growth rate of the mycelium decreases significantly after heat stress, and they are more susceptible to infection by the surrounding environment Trichoderma viride, which makes it difficult to form fruiting bodies, resulting in huge economic losses. Therefore, it is necessary to study a method that can promote the growth of Lentinula edodes strains after heat stress, especially the resistance to Trichoderma viride, to reduce the yield loss of Lentinula edodes strains after heat stress.

[0004] Document sources:

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

[0006] Document 2: Bian YP. Research Progress of Invasive and Competitive Diseases of Edible Fungus Mycelium. Journal of Edible Fungus, 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 meiotic stability of LeV-FMRI0339 among monokaryotic progeny. Plant Pathol J, 2013, 29(4): 460-464.

[0008] Document 4: Liu MJ, Shi J, Wang J, Guo MP, Shen GY, Bian YP, Xu ZY. Effect of Basidiospore-mediated Transmission on Population Formation of Lentinula edodes Double Segregant Virus LePV1. Microbiology Bulletin, 2020, 47(08): 2409-2416.

[0009] Document 5: Li FS, Ji BY, Han B, et al. Research Progress on Interaction Mode of Lentinula edodes and Trichoderma and Control of Green Mould Disease [J]. Journal of Edible Fungus, 2024, 31(03): 113-124. DOI: 10.16488 / j.cnki.1005-9873.2024.03.012. SUMMARY

[0010] In view of the above defects or improvement needs of the prior art, the present application provides a recombinant expression vector and a method for improving the heat resistance of Lentinula edodes and the resistance of Trichoderma, which aims to find that the recombinant plasmid carrying the target gene as shown in SEQ ID NO. 2 introduced into the Lentinula edodes strain carrying 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 Lentinula edodes strain carrying LeV-HKB virus has poor heat resistance, the mycelium grows slowly after heat stress, and its resistance to Trichoderma is reduced.

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

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

[0013] Preferably, the resistance gene of the recombinant expression vector comprises a 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 by the following method:

[0015] The target gene with a full length of 94 bp is synthesized according to the sequence shown in SEQ ID NO. 2, connected to a PUC57 plasmid, and the target gene fragment is amplified and recovered;

[0016] The plasmid with a Legpd promoter is used as the basic backbone of the target gene expression vector, and the recombinant expression vector is obtained by connecting the target gene fragment through homologous recombination after double enzyme digestion.

[0017] Preferably, the plasmid with a Legpd promoter of the recombinant expression vector is pCAMBIA1300, and the double enzyme digestion site is KpnI and EcoRI.

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

[0019] Preferably, the method comprises the step of transforming the recombinant expression vector with a resistance gene into a Lentinula edodes strain carrying LeV-HKB virus to prepare a transformant.

[0020] Preferably, the method comprises the step of transforming the recombinant expression vector into a Lentinula edodes strain by Agrobacterium-mediated transformation to prepare a transformant.

[0021] Preferably, the method comprises the step of adding a bacterial liquid of Agrobacterium containing the recombinant expression vector to a Lentinula edodes mycelium block for incubation.

[0022] Preferably, the method comprises the step of adding a bacterial liquid of Agrobacterium containing the recombinant expression vector to a Lentinula edodes mycelium block for incubation. 600 = 0.8-1.0, and the Lentinula edodes mycelium block is covered after adding the bacterial liquid, and incubated for 20-30 min.

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

[0024] The application provides a recombinant expression vector which carries a target gene shown as SEQ ID NO. 2, and the growth of Lentinula edodes mycelium and the heat resistance and Trichoderma resistance of the strain after heat stress can be promoted by transferring the recombinant expression vector into a Lentinula edodes strain carrying LeV-HKB virus.

[0025] In addition, the application provides a method for improving the heat resistance and Trichoderma resistance of Lentinula edodes, which comprises the step of transferring the recombinant expression vector into a Lentinula edodes strain carrying LeV-HKB virus to prepare a transformant, and the heat resistance and Trichoderma resistance of Lentinula edodes can be improved at the same time by using the method, which is beneficial to reducing economic losses caused by high-temperature heat stress or Trichoderma viride infection in a Lentinula edodes greenhouse. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is horizontal transmission of LeV-HKB between VI and VF-Hyg; Figure 1 A: phenotype comparison of VF and VF-Hyg; B: horizontal transmission of LeV-HKB; C: addition of Hyg to verify transmission;

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

[0028] Figure 3 is colony morphology and mycelium growth rate of VF-Hyg and VF-Hyg + virus under the conditions of 25 DEG C culture and 37 DEG C treatment for 48 h; Figure 3 A: colony morphology of VF-Hyg and VF-Hyg + virus under the conditions of 25 DEG C culture and 37 DEG C treatment for 48 h; B: mycelium growth rate;

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

[0030] Figure 5 is recombinant plasmid vector map and verification; Figure 5 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: E. coli liquid PCR identification bands; D: sequence similarity alignment result;

[0033] Figure 6 recombinant plasmid vector map; Figure 6M:2000bp Marker, 1, 2: 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 M:2000bp Marker, 2, 3: E. coli liquid PCR identification band; B: Sequence similarity alignment results; C: M:2000bp Marker, 1-10: Agrobacterium liquid PCR identification band;

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

[0037] Figure 9 RT-qPCR analysis of LeV-HKB ORFs in VI, VI-CK and transformants after normal temperature and heat treatment; Figure 9 A is RT-qPCR analysis of LeV-HKB ORFs in VI, VI-CK and OEPre transformants after normal temperature culture, B is RT-qPCR analysis of LeV-HKB ORFs in VI, VI-CK and OEPre transformants after heat treatment, C is RT-qPCR analysis of LeV-HKB ORFs in VI, VI-CK and STTM transformants after normal temperature culture, D is RT-qPCR analysis of LeV-HKB ORFs in VI, VI-CK and STTM transformants after heat treatment;

[0038] Figure 10 Colony morphology and mycelium growth rate of VI, VI-CK and OEPre transformants before and after heat stress; Figure 10 A: Colony morphology of VI, VI-CK and OEPre transformants; B: Mycelium growth rate of VI, VI-CK and OEPre transformants;

[0039] Figure 11 Colony morphology and mycelium growth rate of VI, VI-CK and STTM transformants before and after heat stress; Figure 11 A: Colony morphology of VI, VI-CK and STTM transformants; B: Mycelium growth rate of VI, VI-CK and STTM transformants;

[0040] Figure 12Fig. 2 is the colony morphology and infection ratio of VI, VI-CK and OEPre transformants against D. conicum before and after heat treatment; Figure 12 Fig. 3A is the colony morphology of VI, VI-CK and OEPre transformants against D. conicum before and after heat stress; Fig. 3B is the infection ratio of VI, VI-CK and OEPre transformants against D. conicum before and after heat stress;

[0041] Figure 13 Fig. 4 is the colony morphology and infection ratio of STTM transformants against D. conicum before and after heat treatment; Figure 13 Fig. 5A is the colony morphology of VI, VI-CK and STTM transformants against D. conicum before and after heat stress; Fig. 5B is the infection ratio of VI, VI-CK and STTM transformants against D. conicum before and after heat stress;

[0042] Figure 14 Fig. 6 is the colony morphology of STTM transformants against D. conicum before and after heat treatment; Figure 14 Fig. 7A is the colony morphology of VI, VI-CK and STTM transformants against D. conicum before and after heat stress; Fig. 7B is the infection ratio of VI, VI-CK and STTM transformants against D. conicum before and after heat stress; DETAILED DESCRIPTION

[0043] To further illustrate the technical means and effects adopted by the present application to achieve the predetermined object, the specific embodiments, structures, features and effects according to the present application are described in detail below in combination with the drawings and preferred embodiments.

[0044] The inventors found that, compared with normal temperature culture, the growth rate of the Lentinula edodes strain carrying the virus LeV-HKB decreased significantly after heat stress, and the resistance to D. conicum also decreased significantly.

[0045] Based on the construction of the recombinant expression vector, the inventors found that the recombinant plasmid carrying the target gene as shown in SEQ ID NO. 2 introduced into the Lentinula edodes strain carrying the virus LeV-HKB can promote the growth rate of the Lentinula edodes strain after heat stress, improve the heat resistance and resistance to D. conicum of the Lentinula edodes strain, and especially after the introduction of normal temperature culture after heat stress, the Lentinula edodes strain can still hinder the continuous infection of D. conicum, and compared with the Lentinula edodes strain carrying the virus LeV-HKB, the resistance of the Lentinula edodes strain to D. conicum can be significantly improved.

[0046] Based on this, the application provides a recombinant expression vector which carries a target gene as shown in SEQ ID NO. 2, the sequence of the SEQ ID NO. 2 is AATCGGCGCCctaTTTGCTATAAGTTGTTGTTGTTATGGTCTAATTTA AATATGGTCTAAAGAAGAAGAATAATCGGCGCCctaTTTGCTATAA, and is synthesized according to the following design:

[0047] In the two specific complementary sequences, 3 bases CTA are added respectively to form two incomplete complementary sequences, and the two incomplete complementary sequences are connected through a specific sequence of 48 nt to design the target sequence as shown in SEQ ID NO. 2 with a full length of 94 bp.

[0048] Preferably, the recombinant expression vector further contains a resistance gene, and 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 as shown in SEQ ID NO. 2, and is prepared according to the following method:

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

[0051] The plasmid with a Legpd promoter is used as a basic skeleton of the target gene expression vector, and after double enzyme digestion, the target gene fragment is connected through homologous recombination to obtain the recombinant expression vector.

[0052] For example, the plasmid pCAMBIA1300 with a Legpd promoter is used as a basic skeleton of the target gene expression vector, the double enzyme digestion site is KpnI and EcoRI, and the target gene fragment is connected through homologous recombination to obtain the recombinant expression vector.

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

[0054] In addition, the application also provides a method for simultaneously improving heat resistance of Lentinula edodes and Trichoderma resistance, which comprises the step of transferring the recombinant expression vector into a Lentinula edodes strain carrying LeV-HKB virus to prepare a transformant.

[0055] It is preferable to transform a recombinant expression vector carrying the resistance gene and the target gene as shown in SEQ ID NO.2 into a shiitake mushroom strain carrying the LeV-HKB virus to prepare transformants, which facilitates the screening of positive transformants through screening medium.

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

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

[0058] After the shiitake mushroom mycelium was activated in MYG medium, mycelial blocks of the same diameter were taken using a punch, rinsed with IM medium, and then placed in sterilized empty Petri dishes. The Agrobacterium tumefaciens culture solution (OD200) was then added after 4 hours of shaking. 600 =0.8) Pour into an empty Petri dish, cover the mycelial block and incubate for 20-30 min, shaking every 5 min; after incubation, slightly dry the mycelial block with filter paper, transfer it to 200 μmol / L AS Co-IM medium, and incubate at 25℃ for 3 days; after 3 days, wash the mycelial block several times with ddH2O, and soak the mycelial block in ddH2O containing 400 μg / mL cefotaximemycin for 20 min. After washing, transfer the mycelial block to MYG medium (containing 4 μg / mL hygromycin and 400 μg / mL cefotaximemycin) for direct screening to obtain positive transformants.

[0059] Experiments have confirmed that using this method to transform the recombinant expression vector described in this invention into a shiitake mushroom strain carrying the LeV-HKB virus can promote the growth of shiitake mushroom mycelium and enhance 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 Lentinula edodes strains SY1 and SY1-R8, and Trichoderma atroviride strain 92-1. SY1 Lentinula edodes strain is one of the main cultivated varieties in Hubei area, and dsRNA analysis shows that it carries LeV-HKB virus. In previous laboratory studies, the ribavirin chemical reagent was used to obtain the isogenic detoxification strain SY1-R8 (VF, Virus Free) of the SY1 strain (VI, Virus Infeted). According to the "NY / T1730-2009 Edible Fungus Strain Authenticity Identification ISSR Method", 10 ISSR primers were randomly selected to analyze the virus-carrying strain VI and the detoxified strain VF, and the results showed that the ISSR banding patterns before and after detoxification were completely consistent, indicating that ribavirin treatment did not affect the genetic information of the Lentinula edodes strain. Trichoderma atroviride strain 92-1 was isolated from a Lentinula edodes rotting stick by the laboratory, and the Trichoderma strain has been 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 preserved in the Institute of Applied Mycology of Huazhong Agricultural University.

[0062] Test medium: LB medium: Yeast extract 5g, Tryptone 10g, NaCl 10g, (solid medium plus Agar 20g), supplemented with deionized water (ddH2O) to 1000mL, and adjusted to pH 7.0 with NaOH.

[0063] Malt extract medium (MYG): Malt extract 20g, glucose 20g, Tryptone lg, yeast extract lg, (solid medium plus Agar 20g), supplemented with deionized water (ddH2O) to 1000mL.

[0064] Potato dextrose agar (PDA): Potato 250g, glucose 20g, Tryptone 5g, (solid medium plus Agar 20g), supplemented with deionized water (ddH2O) to 1000mL.

[0065] Minimal Media (MM): K-buffer 10 mL, M-N buffer 20 mL, 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, ddH2O to 1000 mL, adjust pH to 6.7-7.0 with HC1 or NaOH.

[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, ddH2O to 1000 mL, adjust pH to 6.7-7.0 with HC1 or NaOH.

[0067] Induction Media (IM): K-buffer 10 mL, M-N buffer 20 mL, 20% Glucose (w / v) 5 mL, 0.01% FeSO4(w / v) 10 mL, 20% (NH4)2SO4(w / v) 2.5 mL, 1% CaCl2·2H2O (w / v) 1 mL, 50% Glycerol (w / v) 10 mL, 7.808 g MES (40 mmol / L, MW 195.2), ddH2O to 1000 mL, adjust pH to 5.6 with HC1 or NaOH.

[0068] Co-IM: K-buffer 10 mL, M-N buffer 20 mL, 20% Glucose (w / v) 2.5 mL, 0.01% FeSO4(w / v) 10 mL, 20% (NH4)2SO4(w / v) 2.5 mL, 1% CaCl2·2H2O (w / v) 1 mL, 50% Glycerol (w / v) 10 mL, 7.808 g MES (40 mmol / L, MW 195.2), Agar 20 g, ddH2O to 1000 mL, adjust pH to 5.6 with HC1 or NaOH.

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

[0070] Table 1 Name and manufacturer of instruments used in the test

[0071]

[0072] Main reagents:

[0073] HLingene Agarose Gel Recovery PCR Purification Kit (Shanghai Huiling Biotechnology Co., Ltd.); 2x TaqPlus Master Mix II, IIQ RT SuperMix for qPCR and AceQTM Qpcr SYBR Green Master Mix (Invitrogen); restriction enzymes (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 purchased from Invitrogen (Nanjing) Co., Ltd.; miRNA Universal SYBR qPCR Master Mix purchased from Invitrogen (Nanjing) Co., Ltd.; primers purchased from Tianyi Huiyuan (Wuhan) Co., Ltd.

[0074] Real-time fluorescent quantitative PCR (RT-qPCR) analysis: Leactin was used as an internal reference gene, and RT-qPCR was used to detect gene expression. The total reaction system was 10 μL: cDNA 1 μL, AceQTM qPCR SYBR Green Master Mix 5 μL, primers (10 μM) 0.5 μL each, and ddH2O 3 μL. The amplification reaction used a two-step method: 95°C pre-denaturation for 5 min; 95°C for 10 s, 60°C for 30 s, 40 cycles; 95°C for 10 s; the final melting curve was set at 65°C-95°C, and the fluorescence signal was collected once every 0.5°C increase in temperature, with a collection time of 5 s. The relative expression of the gene was calculated by the 2-ΔΔCT method.

[0075]

[0076] All experiments were repeated 3-5 times, and the final data was shown as mean ± standard deviations (SD). The significance of the data was analyzed by SPSS software ("*" indicates p<0.05; "**" indicates p<0.01).

[0077] Example 1 Heat stress promotes the replication of viruses in virus-carrying Lentinula edodes strains

[0078] (1) LeV-HKB horizontal transmission

[0079] The empty plasmid pCAMBIA1300-g with Hyg resistance was introduced into E. coli competent cells Trans1-T1, and the plasmid was extracted and introduced into Agrobacterium, which was used to transform the virus-free Lentinula edodes strain VF. The strain was selected on MYG medium (4 μg / mL hygromycin was added) to obtain the virus-free transformant VF-Hyg strain with Hyg resistance. Agrobacterium-mediated transformation of Lentinula edodes mycelium was as follows:

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

[0081] After the shiitake mushroom mycelium was activated in MYG medium, mycelial blocks of the same diameter were taken using a punch, rinsed with IM medium, and then placed in sterilized empty Petri dishes. The Agrobacterium tumefaciens culture solution (OD200) was then added after 4 hours of shaking. 600 =0.8) Pour into an empty Petri dish, cover the mycelial block and incubate for 20 min, shaking every 5 min; after incubation, slightly dry the mycelial block with filter paper, transfer it to 200 μmol / L AS Co-IM medium, and incubate at 25℃ for 3 days; after 3 days, wash the mycelial block several times with ddH2O, and soak the mycelial block in ddH2O containing 400 μg / mL cefotaximemycin for 20 min. After washing, transfer the mycelial block to MYG medium (containing 4 μg / mL hygromycin and 400 μg / mL cefotaximemycin) for direct screening to obtain the non-toxic transformant VF-Hyg strain with Hyg resistance.

[0082] VF-Hyg was cultured at 25℃ and 37℃ for 48 h, with VF as the control. The phenotypic comparison results between VF and VF-Hyg are as follows: Figure 1 As shown in Figure A, there was no significant difference in mycelial growth rate, indicating that the empty vector pCAMBIA1300-g carrying Hyg resistance had no significant effect on the growth rate of the shiitake mushroom strain.

[0083] Horizontal transmission of the virus was achieved through three methods: simultaneous inoculation with VI and VF-Hyg on plates for 5 days; inoculation with VI for 3 days followed by inoculation with VF-Hyg for 2 days; and inoculation with VF-Hyg for 3 days followed by inoculation with VI for 2 days. Figure 1 (B) After the mycelia of VI and VF-Hyg have been in contact for 1 day, the mycelial blocks of VF-Hyg far from VI are picked and cultured on MYG medium supplemented with 4 μg / mL hygromycin. Figure 1As shown in Fig. 2C, the results showed that VI could not grow, while VF-Hyg+virus could grow normally, thereby verifying that the picked and re-introduced LeV-HKB block of VF-Hyg+virus was VF-Hyg+virus instead of 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, and the results are shown in Fig. 3A; and further verification was performed on the Hyg gene of strains VF-Hyg and VF-Hyg+virus and the ORFs of the virus by RT-qPCR, wherein the primer information used is shown in Table 2, and the results are shown in Fig. 3B. Figure 2 Figure 2 As shown in Fig. 3B, VF-Hyg+virus was used as a control, while the virus LeV-HKB was not contained in strain VF-Hyg, i.e., LeV-ORF1 and LeV-ORF2 were not detected in strain VF-Hyg, and it can be seen that among the three horizontal transmission of virus modes, only the mode of inoculating VI first and then inoculating VF-Hyg obtained the VF-Hyg+virus strain re-introduced with LeV-HKB.

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

[0086]

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

[0088] VF-Hyg was used as a control, and the test group was the virus-containing strain VF-Hyg+virus obtained by re-introducing LeV-HKB through mycelial horizontal transmission, and the effects of LeV-HKB on colony morphology and mycelial growth rate were verified, and the results of colony morphology are shown in Fig. 4A. Figure 3 As shown in Fig. 4A, under normal temperature culture conditions at 25°C, there was no significant difference in mycelial growth rate between VF-Hyg and VF-Hyg+virus; after heat stress treatment (37°C, 48h) and continued culture at 25°C, the growth rate of VF-Hyg and VF-Hyg+virus both decreased, but the mycelial growth rate of VF-Hyg was higher than that of VF-Hyg+virus; and the results of difference analysis of mycelial growth rate of each group are shown in Fig. 4B. Figure 3 As shown in Fig. 4B, after 48h of treatment at 37°C and continued culture 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 at 25°C.

[0089] Further analysis of the expression amount of LeV-HKB in VF-Hyg+virus before and after heat stress is shown in Fig. 5. Figure 4 ​As shown, after heat treatment, the ORFs expression of LeV-HKB of VF-Hyg+virus strain was up-regulated, and the expression of ORF1 was significantly increased, which indicated that heat stress could promote the replication of virus LeV-HKB in Lentinula edodes.

[0090] Example 2: Effect of different recombinant expression vectors on virus replication in Lentinula edodes strains

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

[0092] A sequence as shown in SEQ ID NO. 1 was synthesized, which was referred to as target gene 1, and was a non-coding sequence which was not matched to any sequence on the NCBI website. Primer 5 was used for primer design, and the homologous recombination primers used were designed by CE Design software, which had homologous arm sequences required for homologous recombination at both ends. The transformant verification primer was directly designed by SnapGene gene software, and the primers used were synthesized by Wuhan Tianyihuiyuan Biotechnology Co., Ltd. The specific primer sequence information is shown in Table 2. After the primer design was completed, the candidate gene was amplified with DNA as the template, and 1% agarose gel electrophoresis was used for detection. The San Prep column type DNA recovery kit was used to recover the candidate gene fragment, and the reaction product was transferred to the E. coli Trans1-T1 competent cells. A single colony was picked for verification, and was sent to the company for sequencing.

[0093] Construction of recombinant expression vector 1: the target gene DNA as shown in SEQ ID NO. 1 was first amplified by PCR, 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 Polymerase 1 (U / μL) 1 μL, template DNA 2 μL, and ddH2O was added to 50 μL. Reaction parameters: 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 61°C annealing for 40 s, 70°C extension for 1 min, 34 cycles, and 70°C extension for 10 min.

[0094] Restriction endonucleases EcoR1 and Kpn1 were used to completely digest the plasmid pCAMBIA1300-g at 37°C for 1 h, and the digestion system was as follows: pCAMBIA1300-g plasmid 30 μg, Kpn-Ⅰ and EcoR-Ⅰ each 2 μL, buffer 5 μL, and ddH2O was added to 50 μL. After the target gene and the plasmid were digested, agarose gel electrophoresis was used to detect the size of the target fragment, and the San Prep column type DNA recovery kit was used to recover the DNA fragment of the target gene and the linearized plasmid. The digested pCAMBIA1300-g and the target gene were connected by recombinant enzyme.

[0095] Reaction system: enzyme digestion linearized plasmid fragment pCAMBIA1300-g 4 μL, target gene 1 μL, 2x Basic Assembly Mix 5 μL, mix all solutions, place in PCR instrument, 50°C for 15 min. After the reaction, immediately place the reaction solution in ice for a few seconds, and then transfer the reaction solution to E. coli Trans1-T1 competent cells. Pick a single colony on LB medium (50 μg / mL Kan + ) and amplify the target gene fragment using the upstream and downstream primers of the target gene. Positive single colonies were cultured in liquid LB medium, and the bacterial solution was sent for sequencing to obtain a recombinant plasmid carrying the target gene shown in SEQ ID NO. 1, which is referred to as recombinant expression vector 1.

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

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

[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, and they were connected by a 48 nt specific sequence to design a target sequence STTM shown in SEQ ID NO. 2 with a full length of 94 bp: AATCGGCGCCctaTTTGCTATAAGTTGTTGTTGTTATGGTCTAATTTAATATGGTCTAAAGAAGAAGAATAATCGGCGCCctaTTTGCTATAA Figure 6 ​​​​Middle A), recorded as gene 2, was synthesized by Wuhan Tianyi Huiyuan Company and connected to PUC57 plasmid Figure 6 Middle B), the target sequence fragment was amplified from the recombinant plasmid PUC57-STTM and gel-recovered Figure 6 Middle C). The plasmid pCAMBIA1300 with Legpd promoter was used as the basic backbone of the target sequence expression vector as shown in SEQ ID NO. 2, and the double enzyme digestion sites were Kpnl and EcoRI. The gene fragment of the target sequence as shown in SEQ ID NO. 2 was connected by homologous recombination Figure 6 Middle D). After ligation, the E. coli Trans1-T1 competent cells were transformed, and the positive single colonies were detected by PCR. The positive single colonies were cultured in liquid LB medium, and the company was sequenced. The recombinant plasmid was named pCAMBIA1300-g-o-STTM, and was recorded as recombinant expression vector 2.

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

[0100] Table 3 primer information used for transformation

[0101]

[0102]

[0103] Note: For recombinant homologous primers, lowercase letters represent homologous arms, and uppercase letters are gene fragment primers.

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

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

[0106] After the Lentinula edodes mycelium was activated in the MYG medium, a puncher was used to take mycelial blocks with the same diameter, and the mycelial blocks were washed with the IM medium and placed in a sterile empty petri dish. The above-mentioned agrobacterium solution (OD = 0.8) after 4 h of shaking was poured into the empty petri dish, and the mycelial blocks were covered and incubated for 20 min, shaking every 5 min. The incubated mycelial blocks were slightly absorbed with filter paper and transferred to the Co-IM medium containing 200 μmol / L AS and cultured at 25°C for 3 d. After 3 d, the mycelial blocks were washed several times with ddH2O, and soaked in ddH2O containing 400 μg / mL cefotaxime for 20 min. The washed mycelial blocks were transferred to the MYG medium containing 4 μg / mL hygromycin and 400 μg / mL cefotaxime for direct screening.

[0107] Screening and identification of transformants: The mycelial growth was observed after culturing at 25°C for 5-10 d on the MYG medium containing 4 μg / mL hygromycin and 400 μg / mL cefotaxime. If the mycelium continued to grow, the mycelium was picked and cultured on the MYG medium containing hygromycin for further screening. After 3 rounds of screening, the mycelium that could still grow normally was regarded as a resistant transformant. The Lentinula edodes strain transformed with the recombinant expression vector 1 carrying the target gene shown in SEQ ID NO. 1 was an OEPre transformant, and the Lentinula edodes strain transformed with the recombinant expression vector 2 carrying the target sequence shown in SEQ ID NO. 2 was an STTM transformant. The mycelial DNA was extracted, and the target gene was verified.

[0108] The extraction method of Lentinula edodes mycelium DNA is as follows: a. 0.1 g of mycelium is ground with liquid nitrogen, transferred to a 1.5 mL centrifuge tube, 475 μL of 2% CTAB (preheated) and 25 μL of 1% SDS are added, and mixed well by inversion; b. 500 μL of PCI (phenol / chloroform / isoamyl alcohol = 25:24:1) is added, mixed well, and centrifuged at 12000 r / min for 10 min at 4°C, and the supernatant is transferred to a new 1.5 mL centrifuge tube; c. Repeat the above step until there is no obvious precipitate in the middle layer; d. Add 2 / 3 volume of isopropanol to the supernatant, mix well by inversion, precipitate at -20°C for 30 min, centrifuge at 12000 r / min for 10 min at 4°C, and discard the supernatant; e. Wash the precipitate with 1 mL of 70% ethanol, centrifuge at 12000 r / min for 5 min at 4°C, and discard the supernatant; f. Repeat the above 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 detection, use ultramicro spectrophotometer to detect the quality of extracted DNA, and store at -80°C for standby after measurement is completed.

[0109] The VI strain into which the empty vector pCAMBIA1300 is transferred is used as a control, named VI-CK, and the L. edodes Leactin gene is used as an internal reference to detect the expression amount of the target gene in the transformant OEPre and the expression amount of the target sequence in the transformant STTM by RT-qPCR technology. The quantitative results show that, compared with VI-CK, the expression amount of the target gene represented by SEQ ID NO. 1 in 10 OEPre transformants is significantly up-regulated (Fig. 1 Figure 8 C); for the artificially synthesized target sequence represented by SEQ ID NO. 2, it is almost undetectable in VI and VI-CK, but it is obviously expressed in 9 transformants STTM (Fig. 1 Figure 8 C).

[0110] Three transformants OEPre and transformants STTM are selected respectively to analyze the influence of the expression amount of two ORFs of the virus LeV-HKB in L. edodes strains after 25°C normal temperature culture and 37°C heat treatment for 48 h. The RT-qPCR results show that, under normal temperature culture conditions, there is no obvious difference in the expression amount of two ORFs of the virus in OEPre-7_25 and STTM-10_25 transformants compared with the control VI-CK_25 (Fig. 2 Figure 9 A, C). But after heat stress, the expression amount of two ORFs of the virus LeV-HKB in L. edodes strains into which recombinant expression vector 1 is transferred is significantly up-regulated, and the expression amount of two ORFs of the virus LeV-HKB in L. edodes strains into which recombinant expression vector 2 is transferred is significantly down-regulated (Fig. 2 Figure 9The results show that the recombinant expression vector 2 carrying the target sequence as shown in SEQ ID NO. 2 can inhibit the replication of virus LeV-HKB under heat stress conditions.

[0111] Example 3: Influence of different recombinant expression vectors on the growth of Lentinula edodes mycelium

[0112] Five OEPre transformants and five STTM transformants in Example 2 were selected respectively to analyze the influence of different recombinant expression vectors on the mycelium growth rate of Lentinula 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 respectively, and after 5 days of culture at 25°C, the colony phenotypes were observed and the mycelium growth rate was measured, as follows:

[0113] The colony phenotypes of the cultures on MYG solid medium under 25°C constant temperature and dark conditions for 5 days were observed and photographed. During the observation, the mycelium growth rate was detected by cross-line method, and the mycelium tip diameter r was measured with the colony center as the intersection. After the mycelium grew to an appropriate size, the colony diameter R was measured, the mycelium growth time n was recorded, and the growth rate was calculated. Five replicates were set for each strain. The average daily growth rate of the mycelium was calculated according to the formula v = (R-r) / n, and the results are shown in Figure 10 and Figure 11 .

[0114] With VI-CK as the control, the colony diameters of the five OEPre transformants were smaller, and the average daily growth rate of the mycelium was significantly lower than that of VI-CK. Among them, the colony diameters of OEPre-2 and OEPre-7 were the smallest, and the mycelium growth rate was the slowest. The colony diameters of the five STTM transformants were larger, and the average daily growth rate of the mycelium was significantly higher than that of VI-CK. After 5 days of culture at 25°C, 48 hours of treatment at 37°C, and 5 days of continuous culture at 25°C, it was found that the mycelium growth rate of the OEPre transformants decreased significantly, and the growth rates of OEPre-2 and OEPre-7 were the lowest Figure 10 . The mycelium growth rates of the five STTM transformants were higher than that of VI-CK Figure 11 , indicating that the recombinant expression vector 2 carrying the target sequence as shown in SEQ ID NO. 2 can promote the growth of Lentinula edodes mycelium, especially the growth of mycelium after heat stress, and that the recombinant expression vector 2 can improve the heat resistance of Lentinula edodes strain.

[0115] Example 4: Influence of different recombinant expression vectors on the resistance of Lentinula edodes

[0116] The transformant OEPre-7 in Example 2 was selected, and VI, VI-CK and the transformant OEPre-7 were cultured on MYG medium under 25℃ dark culture condition as a control group, and the shiitake fungus blocks were picked out after 10 days, and Trichoderma atroviride was inoculated at the center of the plate. The test group was treated at 37℃ for 48h after 8 days of dark culture at 25℃, and then Trichoderma atroviride was inoculated for infection, and the phenotype was observed after 5 days of Trichoderma atroviride infection.

[0117] The influence of different recombinant expression vectors on the ability of shiitake to resist Trichoderma was compared longitudinally, and it was found that the proportion of the area infected by Trichoderma to the colony area of the transformant OEPre-7 was larger than that of VI-CK under both normal temperature and heat treatment conditions, that is, the recombinant expression vector 1 reduced the resistance of shiitake strain to Trichoderma, and the resistance gap was more obvious after heat stress.

[0118] The influence of heat treatment on the ability of shiitake strain to resist Trichoderma was compared transversely, and it was found that heat treatment led to an increase in the proportion of the area infected by Trichoderma to the colony area of VI, VI-CK and OEPre-7, and the increase in the proportion of the area infected by Trichoderma to the colony area of OEPre-7 was greater. Figure 12 This result shows that the recombinant expression vector 1 has an inhibitory effect on the ability of shiitake strain to resist Trichoderma, and high temperature can significantly increase the expression amount of the target gene as shown in SEQ ID NO. 1 in shiitake strain, so that heat treatment further amplifies the resistance gap of the transformant OEPre-7 and VI-CK to Trichoderma atroviride.

[0119] The transformant STTM-10 in Example 2 was selected, and VI, VI-CK and the transformant STTM-10 were cultured on MYG medium under 25℃ dark culture condition as a control group, and the shiitake fungus blocks were picked out after 10 days, and Trichoderma atroviride was inoculated at the center of the plate, and the infection lasted for 5 days. The test group was treated at 37℃ for 48h after 8 days of dark culture at 25℃, and then Trichoderma atroviride was inoculated for infection for 5 days. The resistance of mycelium to Trichoderma atroviride was evaluated as follows:

[0120] Inoculate 1 piece of agaricus bisporus mycelium block with a diameter of about 9 mm in the center of MYG plate (diameter 90 mm), inoculate 10 plates for each strain, place in constant temperature at 25℃ in dark culture until the mycelium is full, then take out 5 plates and place in constant temperature at 37℃ in dark culture for 48h, the remaining 5 plates continue to be cultured at 25℃ in constant temperature in dark for 48h. After all the above treatments are completed, pick out the agaricus bisporus block and inoculate a piece of deep green trichoderma mycelium block with a diameter of about 9 mm in the center of the plate, with the mycelium facing up. After inoculation, all treatments are placed in constant temperature at 25℃ in dark culture, and the reaction after inoculating the deep green trichoderma mycelium block is observed. ImageJ 1.52a software is used to uniformly analyze and measure the area covered by the brown agaricus bisporus colony after inoculating the deep green trichoderma mycelium block. The ratio of deep green trichoderma mycelium to agaricus bisporus colony under different temperature treatments is calculated by the formula "brown agaricus bisporus colony ratio = brown agaricus bisporus colony covered area / plate area" to evaluate the resistance of agaricus bisporus strain to deep green trichoderma (the smaller the brown ratio, the stronger the resistance of agaricus bisporus to deep green trichoderma).

[0121] The longitudinal comparison of the effect of recombinant expression vector 2 on agaricus bisporus resistance to trichoderma found that under the two culture conditions of normal temperature and heat treatment, the proportion of trichoderma infection area to colony area of the transformant STTM-10 was lower than that of VI-CK, that is, recombinant expression vector 2 enhanced the resistance of agaricus bisporus strain to trichoderma, and the resistance gap was more obvious after heat treatment.

[0122] The horizontal comparison of the effect of heat treatment on agaricus bisporus strain resistance to trichoderma found that heat treatment led to an increase in the proportion of trichoderma infection of VI, VI-CK and STTM-10 colonies, and the increase in the proportion of trichoderma infection of STTM-10 colony was smaller Figure 13 ). These results show that the recombinant expression vector 2 carrying the sequence of interest as shown in SEQ ID NO. 2 can improve the resistance of agaricus bisporus strain to trichoderma after heat stress.

[0123] The agaricus bisporus mycelium cultured at normal temperature and after heat treatment was continued to be cultured at 25℃, and the observation was continued after trichoderma infection. It was found that after 20d of infection, the infected range of VI, VI-CK and transformant STTM-10 no longer expanded. Irregular green colonies of deep green trichoderma appeared in the center of VI and VI-CK colonies, and the mycelium not covered by deep green trichoderma at the periphery was darker. Compared with VI-CK, a clear stress ring was formed around the deep green trichoderma block in the center of STTM-10 colony, which prevented deep green trichoderma from continuing to infect the surrounding, and no green spores of deep green trichoderma were found in the stress ring. At the same time, the maximum proportion of agaricus bisporus strain infected by deep green trichoderma after 48h of 37℃ treatment was observed, and it was found that after 8d of infection, a large number of green spores of deep green trichoderma appeared on the colonies of VI and VI-CK, and the whole colony was almost completely infected. The range of STTM-10 infected by deep green trichoderma no longer expanded, and the spores of deep green trichoderma were yellowFigure 14 ). This result shows that heat treatment makes VI and VI-CK almost lose the resistance ability in the process of confrontation with T. atroviride eventually. STTM-10 can still form stress circle to hinder the continuous infection of T. atroviride, that is, it still maintains a certain resistance, thus further indicating that the recombinant expression vector 2 carrying the target sequence as shown in SEQ ID NO. 2 can increase the resistance of the Lentinula edodes strain to Trichoderma after heat stress.

[0124] The above merely describes preferred embodiments of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed as the above preferred embodiments, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed technical content to obtain equivalent embodiments with equivalent changes, as long as the changes or modifications do not deviate from the technical solution of the present application. Any modification, equivalent change and modification of the above embodiments made according to the technical essence of the present application still fall within the scope of the technical solution of the present application.

Claims

1. A recombinant expression vector, characterized in that, It carries the target gene as shown in SEQ ID NO.

2.

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

3. The recombinant expression vector as described in 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, ligated into the PUC57 plasmid, and the target gene fragment was amplified and recovered. Using a plasmid with the Legpd promoter as the basic backbone of the target gene expression vector, the target gene fragment was ligated through homologous recombination after double enzyme digestion to obtain a recombinant plasmid.

5. The recombinant expression vector as described in claim 4, characterized in that, The plasmid containing the Legpd promoter is pCAMBIA1300, and its double restriction sites are KpnI and EcoRI.

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

7. The method as described in claim 6, characterized in that, Transformants were prepared by transferring the recombinant expression vector as described in claim 2 into a shiitake mushroom strain carrying the LeV-HKB virus.

8. The method as described in claim 6 or 7, characterized in that, The recombinant expression vector was transformed into a shiitake mushroom strain via Agrobacterium-mediated transformation to prepare transformants.

9. The method as described in claim 8, characterized in that, The Agrobacterium-mediated transformation involves adding a bacterial solution of Agrobacterium containing the recombinant expression vector to a block of shiitake mushroom mycelium for incubation.

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

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