Use of biological material related to SDR32 gene in regulating yield and / or high temperature tolerance of Auricularia genus edible fungi

Abstract

The application relates to application of a biological material related to an SDR32 gene in regulating yield and / or high-temperature tolerance of Pleurotus edulis. The biological material plays a role in improving the yield and / or high-temperature tolerance of the fungus by overexpressing SDR32 in the edible fungus of the genus Pleurotus. Experimental results show that the expression amount of the SDR32 gene in the overexpression strain is significantly higher than that in the wild type strain, the yield of the strain overexpressing the SDR32 gene during fruiting is significantly improved compared with the wild type strain, and the high-temperature tolerance of the strain overexpressing the SDR32 gene is significantly enhanced compared with the wild type strain.

Description

Technical Field

[0001] This invention belongs to the field of edible fungi cultivation technology, specifically relating to the application of a biomaterial related to the SDR32 gene in regulating the yield and / or heat resistance of Pleurotus eryngii edible fungi. Background Technology

[0002] my country is a major producer, consumer, and exporter of edible fungi. In recent years, the overall output value of the edible fungi industry has maintained a continuous growth trend, making edible fungi the "fifth largest crop" in my country after grain, oilseeds, vegetables, and fruits. However, problems such as climate change and insufficient technological innovation leading to a scarcity of superior germplasm resources have constrained the improvement of the quality and yield of edible fungi. Therefore, cultivating high-quality and high-yield superior germplasm for edible fungi is an important goal for the high-quality development of my country's edible fungi industry. (Oyster mushroom...) Pleurotusostreatus Oyster mushrooms are an important edible fungus. Their fruiting bodies are typically fan-shaped or shell-shaped, with a smooth surface and colors ranging from grayish-white to dark gray. They are known for their soft and delicious taste, making them popular with consumers. Oyster mushrooms are low-temperature, temperature-dependent fruiting fungi. The optimal growth temperature for mycelium is 25-27℃. Under high-temperature stress above 30℃, the mycelium produces a large amount of reactive oxygen species, further causing oxidative damage to cellular components such as DNA and proteins, and reducing the mycelium's resistance to contaminating microorganisms, thus affecting the yield and quality of oyster mushrooms. To address the problems of oyster mushrooms' intolerance to high temperatures and low biological efficiency, creating new high-temperature resistant, high-quality, and high-yield oyster mushroom materials is a key means to solve the problems restricting the development of its industry.

[0003] The short-chain alcohol dehydrogenase / reductase (SDR) superfamily is a core class of oxidoreductases. Through NAD(P)H-dependent catalysis, they regulate a series of key biochemical reactions, thus participating extensively and profoundly in core life processes such as growth, development, physiological metabolism, and morphogenesis. However, research on the role of SDRs in regulating yield in edible fungi remains lacking.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] In view of the problems existing in the prior art, one of the objectives of the present invention is to provide the application of biomaterials related to the SDR32 gene in regulating the yield and / or heat resistance of Pleurotus eryngii edible fungi.

[0006] The second objective of this invention is to provide a method for increasing the yield and / or heat resistance of edible fungi of the genus Pleurotus.

[0007] The third objective of this invention is to provide a genetically engineered edible fungus strain of the genus Pleurotus.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The first aspect of this invention provides the application of SDR32 gene-related biomaterials in regulating the yield and / or heat resistance of Pleurotus eryngii edible fungi. The regulation of Pleurotus eryngii yield and / or heat resistance refers to increasing the yield and / or heat resistance of Pleurotus eryngii edible fungi by overexpressing the SDR32 gene through the biomaterials. The biomaterials are any one of (A) to (D) below:

[0010] (A) The nucleotide sequence encoding the SDR32 amino acid sequence shown in Sequence 2 of the Sequence Listing;

[0011] (B) An expression cassette containing the nucleotide sequence described in (A);

[0012] (C) A recombinant vector containing the nucleotide sequence described in (A) or a recombinant vector containing the expression cassette described in (B);

[0013] (D) A recombinant microorganism containing the nucleotide sequence described in (A), or the expression cassette described in (B), or the recombinant vector described in (C).

[0014] In the application of the first aspect described above, as an optional implementation, the nucleotide sequence of the SDR32 amino acid sequence shown in Sequence 2 of the encoding sequence listing is as shown in Sequence 1 of the sequence listing.

[0015] In the application of the first aspect above, as an optional implementation, the promoter controlling the transcription of the nucleotide sequence (A) in the expression cassette or the recombinant vector is a GPD promoter from basidiomycetes; preferably, the GPD promoter from basidiomycetes is a GPD promoter from oyster mushrooms.

[0016] In the application of the first aspect described above, as an optional implementation, the host bacterium of the recombinant microorganism is Agrobacterium.

[0017] In the application of the first aspect above, as an optional implementation, the recombinant vector is obtained by inserting the expression cassette into the multiple cloning site of the base vector, wherein the base vector is obtained by replacing the promoter in the plant binary expression vector with the GPD promoter in the basidiomycetes.

[0018] In the application of the first aspect above, as an optional implementation, the edible fungus of the genus Pleurotus is Pleurotus ostreatus.

[0019] A second aspect of the present invention provides a method for increasing the yield and / or heat resistance of edible fungi of the genus Pleurotus, comprising the following steps:

[0020] S1: Introduce the biological material described in the first aspect above into the Pleurotus genus edible fungus strain;

[0021] S2: Screen out successfully transformed transgenic Pleurotus ostreatus edible fungi strains from the strains obtained in step S1;

[0022] S3: Obtain fruiting bodies of edible fungi of the genus Pleurotus using transgenic strains of Pleurotus.

[0023] In the second aspect of the method described above, as an optional implementation, the edible fungus of the genus Pleurotus is Pleurotus ostreatus.

[0024] In the second aspect of the method described above, as an optional implementation, the introduction of the biomaterial described in the first aspect into the Pleurotus ostreatus edible fungus strain is accomplished using Agrobacterium transformation.

[0025] A third aspect of this invention provides a genetically engineered edible fungus strain of the genus Pleurotus, which is obtained by the following method:

[0026] Sa: Introduce the biological material described in the first aspect above into the edible fungal strain of Pleurotus ostreatus to be modified;

[0027] Sb: Select successfully transformed transgenic Pleurotus ostreatus edible fungi strains from the strains obtained in step Sa as genetically engineered Pleurotus ostreatus edible fungi strains.

[0028] In the genetically engineered edible fungi strains of the genus Pleurotus described in the third aspect above, as a preferred embodiment, the edible fungi strain of the genus Pleurotus is a oyster mushroom strain.

[0029] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows:

[0030] This invention provides the application of SDR32 gene-related biomaterials in regulating the yield and / or heat resistance of Pleurotus ostreatus edible fungi. By introducing or importing the SDR32 gene into Pleurotus ostreatus edible fungi, the SDR32 gene is overexpressed in Pleurotus ostreatus edible fungi, thereby increasing the yield of the fungi. Moreover, the overexpression of the gene also improves the heat resistance of the Pleurotus ostreatus edible fungi.

[0031] This invention utilizes molecular biology and genetic engineering techniques to successfully clone the SDR32 encoding gene from *Pleurotus ostreatus* using molecular cloning. An overexpression vector for SDR32 was constructed, and this vector was then transformed into *Pleurotus ostreatus* strains using Agrobacterium-mediated transformation. Experimental results showed that the expression level of the SDR32 gene in the overexpression strain was significantly higher than that in the wild-type strain. The recovery growth rate of the overexpression strain after heat stress at 35℃ and 40℃ was significantly higher than that of the wild-type strain, and the area of ​​mycelial damage was smaller. Furthermore, the fruiting yield of the strain overexpressing the SDR32 gene was significantly higher than that of the wild-type strain, confirming that SDR32 has a positive regulatory effect on the yield of *Pleurotus ostreatus*. This invention not only provides a new strategy for creating high-yielding *Pleurotus ostreatus* strains but also lays a theoretical foundation for the molecular genetic improvement of high-quality *Pleurotus ostreatus* germplasm, demonstrating significant application potential and market prospects. Attached Figure Description

[0032] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0033] Figure 1 for Po Agarose gel electrophoresis image of SDR32 cDNA PCR amplification products, where lane M is the DNA marker with a molecular weight of 2000; lanes 2 and 3 are respectively... Po SDR32 cDNA positive amplification product.

[0034] Figure 2 To build successfully Po -OESDR32 vector spectrum, where the red fragment represents the target... Po SDR32 gene.

[0035] Figure 3 The image shows an agarose gel electrophoresis image of the PCR amplification products of the *Pleurotus ostreatus* strain ACCC 50596 obtained by transforming it with the overexpression vector. In the image, M: DL2000 marker; 1: OE-SDR32 plasmid amplification product as a positive control; 2: wild-type *Pleurotus ostreatus* strain amplification product as a negative control; 3: ddH2O amplification product as a blank control; and 4-16: amplification products of the transformants.

[0036] Figure 4For Pleurotus ostreatus ACCC 50596 strain (WT) and overexpression Po In transgenic strains of the SDR32 gene (OESDR-18, OESDR-22, OESDR-26) Po The relative expression level of the SDR32 gene.

[0037] Figure 5 For Pleurotus ostreatus ACCC 50596 strain (WT) and overexpression Po The recovery and statistical analysis of transgenic strains of the SDR32 gene (OE-SDR-18, OE-SDR-22, OE-SDR-26) after heat stress at 35℃ and 40℃ are presented. In the figures, A shows the images of the recovery after heat stress at 35℃ and 40℃, B shows the statistical analysis results of the colony diameter after heat stress at 35℃, and C shows the statistical analysis results of the colony diameter after heat stress at 40℃.

[0038] Figure 6 For Pleurotus ostreatus ACCC 50596 strain (WT) and overexpression Po The phenotype of transgenic strains of the SDR32 gene (OE-SDR32-18, OE-SDR32-22, OE-SDR32-26) at the same time of fruiting.

[0039] Figure 7 For the Oyster mushroom ACCC 50596 strain and overexpression Po Fruiting yield and statistical analysis of transgenic strains of the SDR32 gene (OE-SDR-18, OE-SDR-22, OE-SDR-26).

[0040] Figure 8 For the Oyster mushroom ACCC 50596 strain and overexpression Po Biological efficiency and statistical analysis of transgenic strains of the SDR32 gene (OE-SDR-18, OE-SDR-22, OE-SDR-26).

[0041] Figure 9 For the Oyster mushroom ACCC 50596 strain and overexpression Po Cap diameter and statistical analysis of transgenic strains of the SDR32 gene (OE-SDR-18, OE-SDR-22, OE-SDR-26).

[0042] Figure 10 For the Oyster mushroom ACCC 50596 strain and overexpression Po Stipe length and statistical analysis of transgenic strains of the SDR32 gene (OE-SDR-18, OE-SDR-22, OE-SDR-26).

[0043] In the statistical analysis graph, * indicates that the transgenic strain has a significant difference compared with WT. The more * there are, the more significant the difference. Among them, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. Detailed Implementation

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Those skilled in the art should understand that the embodiments described are merely illustrative of the invention and should not be considered as specific limitations thereof. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0045] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0046] This invention provides a biomaterial related to the SDR32 gene, wherein the biomaterial is any one of (A) to (D) below:

[0047] (A) The nucleotide sequence encoding the amino acid sequence of the SDR32 transcription factor shown in Sequence 2 of the sequence listing;

[0048] (B) An expression cassette containing the nucleotide sequence described in (A);

[0049] (C) A recombinant vector containing the nucleotide sequence described in (A) or a recombinant vector containing the expression cassette described in (B);

[0050] (D) A recombinant microorganism containing the nucleotide sequence described in (A), or the expression cassette described in (B), or the recombinant vector described in (C).

[0051] SDR32 shown in sequence 2 of the sequence list ( Po The SDR32 amino acid sequence consists of 256 amino acids.

[0052] Preferably, the nucleotide sequence of the SDR32 amino acid sequence shown in Sequence 2 of the coding sequence listing is as shown in Sequence 1 of the sequence listing, totaling 771 nucleotide sequences, which are derived from oyster mushrooms (Pleurotus ostreatus). Pleurotusostreatus It is obtained from the mycelium of ACCC 50596, and can also be obtained through artificial synthesis.

[0053] The expression cassette includes a promoter, a target gene, and a terminator. The target gene is the oyster mushroom gene SDR32 (PoSDR32), which is the nucleotide sequence encoding the SDR32 amino acid sequence shown in Sequence 2 of the sequence listing. The promoter can be the GPD promoter from Basidiomycetes. This promoter is beneficial for the expression of the target gene or other selection genes in Basidiomycetes strains and has good compatibility with Pleurotaceae, especially Pleurotus genus edible fungi, which is conducive to the expression of the target gene therein.

[0054] Preferably, the GPD promoter in the basidiomycete is the GPD promoter of Pleurotus ostreatus, specifically, the nucleotide sequence of the GPD promoter has the NCBI accession number KY924471.2. This promoter nucleotide sequence can be synthesized artificially or cloned directly from Pleurotus ostreatus strains using conventional methods.

[0055] The recombinant vector is obtained by inserting the expression cassette into the multiple cloning site of a base vector. The base vector can be any commercially available vector suitable for transforming Basidiomycetes strains and conducive to the expression of the target gene therein. The base vector can also be a commercially available vector used in plants modified to be suitable for transforming Basidiomycetes strains; for example, the base vector is obtained by replacing the 35S promoter or other promoters in a plant binary expression vector with the GPD promoter from Basidiomycetes. Typically, but not limited to, the plant binary expression vector is a pCAMBIA series vector, such as the commercially available pCAMBIA1300.

[0056] The host bacteria of the recombinant microorganism can be a conventional strain used for transgenic research, such as Agrobacterium, more specifically Agrobacterium GV3101.

[0057] The biomaterial of the present invention, when transferred into the edible fungus Basidiomycota, leads to overexpression of the SDR32 gene in the host, thereby increasing the host's yield and / or heat resistance.

[0058] The specific embodiments of the present invention also provide the above-mentioned biological materials for regulating edible fungi of the genus *Pleurotus*, particularly those of the family *Pleurotus*, and more particularly those of the genus *Pleurotus*. Pleurotus Edible fungi, most notably oyster mushrooms ( Pleurotusostreatus Applications related to yield and / or heat resistance: After the biomaterial is successfully transferred into edible fungi, the SDR32 gene will be overexpressed, thereby increasing the yield and / or heat resistance of edible fungi.

[0059] The edible fungi of the genus Pleurotus can include oyster mushrooms, king oyster mushrooms, and white lingzhi mushrooms.

[0060] The present invention also provides a method for regulating the yield and / or heat resistance of edible fungi of the genus *Pleurotus*, particularly those of the family Pleurotaceae, and even more particularly those of the genus *Pleurotus*, comprising the following steps:

[0061] S1: Introduce the biological material into the edible fungal strain;

[0062] S2: Screen out successfully transformed transgenic edible fungi strains from the strains obtained in step S1;

[0063] S3: Obtain fruiting bodies of Pleurotus eryngii edible fungi using transgenic edible fungal strains.

[0064] This method can significantly improve the yield and / or heat resistance of edible fungi of the Basidiomycota family, especially those of the Pleurotaceae family, and even more so those of the Pleurotaceae genus.

[0065] The edible fungi of the genus Pleurotus include oyster mushrooms, king oyster mushrooms, and white lingzhi mushrooms;

[0066] The introduction of biological material into the edible fungal strain was accomplished using the Agrobacterium-mediated transformation method.

[0067] The screening described in step S2 refers to using the genome of the strain obtained in step S1 as a template to identify successfully transformed transgenic edible fungal strains through PCR. The nucleotide sequences of the primer pairs used in PCR can be designed according to conventional methods. Typically, but not limited to, the nucleotide sequences of the primer pairs used in PCR are shown in sequences 5 and 6 in the sequence listing.

[0068] The present invention also provides a genetically engineered basidiomycete edible fungus strain, particularly a strain of Pleurotaceae edible fungus, and more particularly a strain of Pleurotus genus edible fungus, which is obtained by the above method, such as steps S1 and S2 of the above method.

[0069] The edible fungal strains of the genus Pleurotus include oyster mushroom strains, king oyster mushroom strains, and white lingzhi mushroom strains.

[0070] In this invention, the edible fungi overexpressing the SDR32 gene have a significantly higher yield than the wild type when fruiting, which can be specifically reflected in a significant increase in cap diameter, stipe length, and fruiting body weight.

[0071] After high-temperature stress treatment, the edible fungal strains overexpressing the SDR32 gene in this invention exhibited better recovery and growth ability compared to wild-type strains, with significantly longer mycelial lengths. The high-temperature stress treatment can be carried out at temperatures higher than the conventional culture temperature for edible fungal strains; for oyster mushrooms, high-temperature stress treatment can be performed at temperatures above 35°C.

[0072] The genetically engineered basidiomycete edible fungus strains provided by this invention are characterized by high yield and high temperature resistance. The culture methods and conditions for these genetically engineered basidiomycete edible fungus strains can be the same as those for the corresponding wild-type edible fungus strains.

[0073] The embodiments of the present invention described Po Both SDR32 gene and SDR32 gene refer to the nucleotides shown in sequence 1 of the sequence listing.

[0074] The technical solution and effects of the present invention will be described in detail below using the edible fungus Pleurotus ostreatus as an example.

[0075] Example 1: Po Cloning of the SDR32 gene

[0076] The genes used in this invention are Po SDR32, whose nucleotide sequence is shown in Sequence 1 of the sequence listing.

[0077] Obtaining genes Po Specific steps for SDR32:

[0078] Step 1: Collect oyster mushroom ACCC50596 (deposited at the China Agricultural Microbial Culture Collection Center, website: http: / / www.accc.org.cn / , this strain was purchased from the center) mycelium grown on PDA medium for 7 days, and extract total RNA from oyster mushroom ACCC 50596 according to the EZNA Fungal RNA Kit (Omega, USA) instructions. Reverse transcription was performed to obtain cDNA according to the HiScript® II 1st Strand cDNA Synthesis Kit (Vezyme, China) instructions.

[0079] Step 2: According to Po Primers were designed using the CDS sequence of SDR32. The upstream and downstream primer sequences are as follows:

[0080] Po SDR32-F: 5'-ATTTCACTGACCTGGGGATCCATGGCCGCTCTTCTCTCTACTTC-3' (as shown in sequence 3 in the sequence listing);

[0081] Po SDR32-R: 5'-AGCTTGCATGCCAATTCTAGAAACGACGAAGCCACCGTTG-3' (as shown in sequence 4 in the sequence listing);

[0082] Using cDNA from Pleurotus ostreatus ACCC 50596 as a template, PCR amplification was performed according to the following reaction system and conditions.

[0083] Reaction system: 25 μL, including 1 μL of cDNA, Po SDR32-F 1 μL Po SDR32-R 1 μL, 2 × TaqPlus Master Mix 12.5 μL, deionized water 9.5 μL.

[0084] Reaction conditions: pre-denaturation at 95℃ for 3 min; 95℃ for 30 sec, 62℃ for 30 sec, 72℃ for 1 min, for a total of 30 cycles; extension at 72℃ for 10 min.

[0085] Finally obtained Po The full-length CDS PCR product of SDR32 was subjected to 1% agarose gel electrophoresis, and the target gene was... Po The cDNA agarose gel electrophoresis results of SDR32 are as follows: Figure 1 As shown.

[0086] Step 3: The PCR products were recovered using the FastPure Gel DNA Extraction Mini Kit (Vezyme, China).

[0087] in addition, Po The SDR32 gene can also be obtained through artificial synthesis.

[0088] Example 2 Construction of overexpression vector

[0089] Step 1: The gene obtained from gel extraction and recovery in Example 1 Po The SDR32 target fragment was ligated into the pEASY®-Blunt ZeroCloning Vector (TransGen, China), and the ligation product was then transformed into Escherichia coli (DH5α) competent cells. The cells were screened on LB solid medium containing kanamycin sulfate, and single clones were picked and detected by bacterial PCR.

[0090] Step 2: Sequencing the correct sequence PoThe CDS sequence of SDR32 was recombined with the basic vector OE-GFP. Specifically, the OE-GFP vector was double-digested and purified using BamHI and XbaI (Takara, Japan) restriction endonucleases. Following the manufacturer's instructions, the CDS sequence of PoSDR32 was ligated into the double-digested linearized plasmid using the ClonExpress II One Step Cloning Kit (Vezyme, China). The ligation product was transformed into competent E. coli cells. Positive clones were then screened and identified. Finally, the plasmid with the completely correct sequence was obtained by DNA sequencing, which is the overexpression vector. Po -OESDR32, its spectrum is as follows Figure 2 As shown.

[0091] The basic vector OE-GFP was obtained as follows: the GPD promoter-encoding gene of *Pleurotus ostreatus* was cloned, then the hygromycin resistance gene (HygR) promoter in the commercially available original pCAMBIA1300 vector was replaced with the GPD promoter, and the GPD promoter-encoding gene and the GFP-encoding gene were referenced at the multiple cloning site to generate an expression module driven by the GPD promoter. The nucleotide sequence of the OE-GFP vector is shown in Sequence 9 of the sequence listing.

[0092] Example 3: The target oyster mushroom fungus strain was transformed using Agrobacterium-mediated transformation and the successfully transgenic strain was identified.

[0093] Step 1: Refer to the Efficom GV3101 Chemically Competent Cell (Genesand, China) instruction manual to overexpress the vector. Po -OESDR32 was introduced into competent Agrobacterium tumefaciens GV3101 cells. Single clones were selected and subjected to culture PCR on LB agar containing kanamycin sulfate and rifampin.

[0094] Step 2: [The text appears to be incomplete and contains several grammatical errors. A more accurate translation would require the full context.] Po Agrobacterium tumefaciens with the OESDR32 vector was inoculated into 100 mL of LB medium and cultured at 28°C and 180 rpm for 14 h. The cells were then collected by centrifugation at 4500 rpm for 10-15 min at 4°C. The cells were washed once with 50 mL of IM medium and then resuspended in 24 mL of IM medium. The cells were then induced to grow at 28°C and 90 rpm in the dark for 5 h.

[0095] Table 1 Composition of IM culture medium

[0096]

[0097]

[0098] Step 3: Inoculate the oyster mushroom mycelium (ACCC 50596, purchased from the China Agricultural Microbial Culture Collection Center, website: http: / / www.accc.org.cn / ) into PDA medium, incubate at 25℃ for 10 days, then punch holes with a 5 mm punch and inoculate into CYM medium for static incubation for 2 days.

[0099] Step 4: After rinsing the mycelial blocks from Step 3 with deionized water, co-culture them with the Agrobacterium tumefaciens cultured in Step 2 (induction culture in the dark for 5 h) at 28℃ for 5 h. After the culture is complete, rinse the Agrobacterium tumefaciens with deionized water, inoculate the oyster mushroom mycelial blocks onto IM solid plates, seal them, and incubate at 28℃ for 5 days.

[0100] Step 5: After 5 days of cultivation, the oyster mushroom blocks obtained in Step 4 were inoculated onto CYM solid medium. Corresponding screening agents were added to the medium (100 μL of 50 mg / mL hygromycin and 600 μL of 200 mg / mL cefotaxime sodium per 400 mL CYM). After 20 days of cultivation, the desired oyster mushrooms were obtained. Po Oyster mushroom strain transformed with the SDR32 gene.

[0101] Step 6: Use amplification primers Po SDR32-tF and Po SDR32-tR was used to identify the transformed oyster mushroom strain using PCR to determine whether the transformation was successful. Among other things:

[0102] Po The nucleotide sequence of SDR32-tF is: 5'-TCGGTTTCCACTATCGGCGAGTACTTCTACACA-3' (as shown in sequence 5 of the sequence listing);

[0103] Po The nucleotide sequence of SDR32-tR is: 5'-TCTCGTGCTTTCAGCTTCGATGTAGGAGGG-3' (as shown in sequence 6 of the sequence listing).

[0104] Reaction system: 25 μL, including: 1 μL template, Po SDR32-tF 1 μL Po SDR32-tR 1 μL, 2 ×Taq Plus Master Mix 12.5 μL, Deionized Water 9.5 μL.

[0105] Reaction conditions: pre-denaturation at 95℃ for 3 min; 95℃ for 30 sec, 62℃ for 30 sec, 72℃ for 1 min, for a total of 30 cycles; extension at 72℃ for 10 min.

[0106] The agarose gel electrophoresis results of PCR amplification of the positive strains are as follows: Figure 3 As shown, the product size is approximately 700 bp.

[0107] After identification, three transgenic oyster mushroom strains were obtained after transforming strain ACCC 50596, and were named: OE-SDR-18 (also represented as OESDR-18), OE-SDR-22 (also represented as OESDR-22), and OE-SDR-26 (also represented as OESDR-26).

[0108] Step 7: Use q to test for the above-mentioned positive strains. Po SDR32-F 5'-TCATACGTTGCTTCCAAGGG-3' (as shown in sequence 7 of the sequence listing) and q Po RT-qPCR identification was performed using primers SDR32-R 5'-CTTGATAATGTGTTCGGGCTTTC-3' (as shown in sequence 8 in the sequence listing) to identify *Pleurotus ostreatus* strain ACCC 50596 (WT) and transgenic strains obtained from it. Po The relative expression level of the SDR32 gene is as follows: Figure 4 As shown (the relative expression levels of each strain in the figure are based on statistical results from three replicates), among the three transgenic strains... Po The relative expression levels of the SDR32 gene were significantly higher than those of the wild-type strain.

[0109] Example 4: Determination of the heat sensitivity of the mycelium of the successfully transformed oyster mushroom strain

[0110] The heat sensitivity of the transgenic *Pleurotus ostreatus* strains identified by PCR and qPCR was determined at 35°C and 40°C. Po SDR32 gene overexpressing strain and oyster mushroom strain ACCC 50596 were subjected to recovery culture after being subjected to stress at different temperatures. Fresh mycelia of oyster mushroom strain ACCC 50596 and the corresponding transgenic strain were inoculated into 90 mm PDA plates using a 5 mm punch. After culturing at 28℃ for 4 days, the growth diameter was recorded by streaking. The plates were then subjected to high-temperature stress at 35℃ and 40℃ for 48 h, respectively, and then transferred to a 28℃ incubator for 2-4 days, during which the growth diameter was recorded and photographed. After 2 days of high-temperature stress at 35℃, a 2-day recovery treatment was performed, and the colony growth diameter was recorded. Three replicates were set up, such as... Figure 5 A and Figure 5 As shown in B, PoThe transgenic strain overexpressing the SDR32 gene showed a significantly higher growth rate than the oyster mushroom strain ACCC50596; after 2 days of high-temperature stress at 40℃, followed by a 4-day recovery treatment, the colony diameter was recorded in triplicate. Figure 5 A and Figure 5 As shown in C, compared to the wild-type strain... Po SDR32 gene overexpression strains exhibit more complete colony edges and larger colony diameters, i.e. Po Transgenic strains overexpressing the SDR32 gene exhibited a higher recovery growth rate. In summary, Po The SDR32 gene plays a positive regulatory role in the recovery growth of oyster mushrooms under heat stress. Po Strains overexpressing the SDR32 gene exhibit stronger resistance to heat stress than the oyster mushroom strain ACCC 50596.

[0111] Example 5: Determination of agronomic traits of successfully transformed oyster mushroom strains

[0112] The transgenic *Pleurotus ostreatus* strains identified by PCR and qPCR were subjected to fruiting and agronomic trait testing. Fresh mycelia of *Pleurotus ostreatus* strain ACCC 50596 (activated for 5 days) and the corresponding transgenic strain were inoculated into 180 g culture bottles using a 5 mm punch. The bottles were incubated in the dark at 25°C for approximately 20 days. After the mycelia had fully colonized the bottles, incubation continued for another 2 days. The bottles were then transferred to an intelligent mushroom cultivation box for fruiting under the following conditions: temperature 15-18°C, humidity 85-90%, CO2 concentration 500 mg / kg, and light intensity greater than 200 lux, with 12 hours of light followed by 12 hours of darkness daily to ensure normal fruiting body development. Harvesting was conducted after a total of 28 days of cultivation. Yield was measured using a balance after harvesting. Nine of the best-growing samples were then selected, and the cap diameter and stipe length were measured using calipers and statistically analyzed.

[0113] like Figure 6 As shown, from the perspective of the growth status of mushrooms after different cultivation days, when the cultivation period is the same, Po The transgenic strain overexpressing the SDR32 gene grew significantly faster than the wild-type oyster mushroom ACCC 50596. Po The yield of transgenic strains overexpressing the SDR32 gene is as follows: Figure 7 As shown, biological efficiency is as follows Figure 8 As shown, the diameter of the cap is as follows Figure 9 As shown, the length of the stipe is as follows Figure 10 As shown.

[0114] In this embodiment, biological efficiency is defined as (fresh mushroom yield / dry weight of substrate) × 100%.

[0115] The cultivation medium formula in the cultivation bottle by weight percentage is: 94% cottonseed hulls, 5% wheat bran, 1% lime, with a moisture content of 65%. Each cultivation bottle contains 180 g of cultivation medium and is sterilized at 126℃ for 2 hours.

[0116] As can be seen from the above embodiments, the present invention has identified and cloned *Pleurotus ostreatus* strain ACCC 50596. Po The SDR32 gene was overexpressed using a vector constructed through genetic engineering and molecular biology techniques. Overexpression was achieved via Agrobacterium-mediated transformation. Po Transgenic oyster mushrooms with the SDR32 gene. Overexpression was achieved. Po Transgenic oyster mushrooms with the SDR32 gene showed significantly improved agronomic traits compared to wild-type oyster mushroom strains, including:

[0117] The transgenic strain obtained based on Pleurotus ostreatus ACCC 50596 showed a yield increase of 21.33%-26.30% compared to the wild-type strain. Overexpression Po The SDR32 gene can significantly improve the original biological efficiency of the oyster mushroom ACCC 50596 strain from 62.26%, and can increase it to a maximum of 78.63%.

[0118] from Figure 9 and Figure 10 It is evident that the cap diameter and stipe length of the transgenic strain after fruiting are significantly higher than those of the wild-type oyster mushroom strain ACCC 50596.

[0119] The above indicates that the present invention identifies and clones the oyster mushroom from ACCC 50596. Po The SDR32 gene can be used to improve the yield and biological efficiency of Pleurotus ostreatus edible fungi, especially oyster mushrooms, providing a high-quality pathway for the genetic breeding and selection of superior germplasm of Pleurotus ostreatus edible fungi, especially oyster mushrooms. It has good application prospects in the cultivation and production of Pleurotus ostreatus edible fungi, especially oyster mushrooms.

[0120] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of SDR32 gene-related biomaterials in regulating the yield and / or heat resistance of edible fungi of the genus Pleurotus, characterized in that, The regulation of Pleurotus eryngii yield and / or heat resistance refers to increasing the yield and / or heat resistance of Pleurotus eryngii edible fungi by overexpressing the SDR32 gene in the Pleurotus eryngii edible fungi through the biological material described herein, wherein the biological material is any one of the following (A) to (D): (A) The nucleotide sequence encoding the SDR32 amino acid sequence shown in Sequence 2 of the Sequence Listing; (B) An expression cassette containing the nucleotide sequence described in (A); (C) A recombinant vector containing the nucleotide sequence described in (A) or a recombinant vector containing the expression cassette described in (B); (D) A recombinant microorganism containing the nucleotide sequence described in (A), or the expression cassette described in (B), or the recombinant vector described in (C); The edible fungus mentioned is Pleurotus ostreatus.

2. The application according to claim 1, characterized in that, The nucleotide sequence of the SDR32 amino acid sequence shown in Sequence 2 of the coding sequence listing is as shown in Sequence 1 of the sequence listing.

3. The application according to claim 1, characterized in that, The promoter controlling the transcription of the nucleotide sequence (A) in the expression cassette or the recombinant vector is the GPD promoter from Basidiomycetes; And / or, the host bacterium of the recombinant microorganism is Agrobacterium.

4. The application according to claim 3, characterized in that, The GPD promoter in the basidiomycete is the GPD promoter of Pleurotus ostreatus.

5. The application according to any one of claims 1-4, characterized in that, The recombinant vector is obtained by inserting the expression cassette into the multiple cloning site of the base vector, which is obtained by replacing the promoter in the plant binary expression vector with the GPD promoter in basidiomycetes.

6. A method for increasing the yield and / or heat resistance of edible fungi of the genus Pleurotus, characterized in that, Includes the following steps: S1: Introduce the biological material as described in any one of claims 1 to 5 into the Pleurotus genus edible fungus strain; S2: Screen out successfully transformed transgenic Pleurotus ostreatus edible fungi strains from the strains obtained in step S1; S3: Obtain fruiting bodies of edible fungi of the genus Pleurotus using transgenic strains of Pleurotus spp.; The edible fungus mentioned is Pleurotus ostreatus.

7. The method as described in claim 6, characterized in that, The introduction of the biomaterial as described in any one of claims 1 to 5 into the Pleurotus erythrorhizon edible fungal strain is accomplished using the Agrobacterium tumefaciens transformation method.

8. A genetically engineered edible fungus strain of the genus Pleurotus, characterized in that, Obtained through the following method: Sa: Introduce the biomaterial as described in any one of claims 1 to 5 into the edible fungal strain of Pleurotus ostreatus to be modified; Sb: Select the successfully transformed transgenic Pleurotus ostreatus edible fungi strains from the strains obtained in step Sa as genetically engineered Pleurotus ostreatus edible fungi strains. The edible fungal strain of the genus Pleurotus to be modified is a Pleurotus ostreatus strain.

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