A method for genetic transformation of Efibula sp. ET39 fungal protoplasts

Abstract

The present invention discloses a Fibula sp.ET39 fungal protoplast genetic transformation method. The present invention first established Fibula The genetic transformation method of fungal protoplasts of sp.ET39 was carried out by using a mixed enzyme solution prepared by Driselase, Lysing Enzymes and Lywallzyme in a mass ratio of 1:1:1. Under the conditions of 0.6~0.7M MgSO4 buffer, 30℃ and enzymatic hydrolysis for 3 h, a high yield of protoplasts of up to 2.28×10 7 This method has the characteristics of high transformation efficiency (6-8 transformants / μg DNA) and short transformation cycle (1-2 weeks), which can be used for future Fibula It provides strong support for the research on the gene function and active metabolites of sp.ET39 fungus.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for genetic transformation of Efibula sp. ET39 fungal protoplasts. Background Art

[0002] Microbial metabolites, characterized by diverse chemical structures and significant biological activity, are an important source for the discovery of pesticide lead compounds. Chinese patent application CN118440825A discloses that the Basidiomycetes fungus Efibula sp. ET39 produces secondary metabolites Efi-A, Efi-B, and Efi-C, which exhibit immune-inducing activity. These compounds have novel structures and can significantly enhance plant disease resistance while having no inhibitory effects on plant growth, demonstrating promising application prospects. However, their development faces numerous challenges, including low yields, complex separation systems, and difficulty in scalable production.

[0003] In recent years, addressing the problem of low fermentation yields of natural products through biosynthesis research has become a hot topic. Elucidating the biosynthetic pathways within microbial metabolites at the molecular level is crucial for achieving efficient and targeted synthesis of active natural products. Therefore, establishing a genetic transformation system for Efibula sp. ET39 is a crucial technical foundation for studying the biosynthesis of active metabolites.

[0004] Despite recent reports establishing genetic transformation methods for fungi such as Ustilago officinalis from the Basidiomycetes, these methods have faced challenges, including the discontinuation of the lytic enzyme (Sigma Aldrich, L1412), a long transformation cycle (2-3 weeks), and low transformation efficiency. Therefore, establishing a protoplast preparation and genetic transformation method for Efibula sp. ET39 is of great significance for future biosynthesis research and the efficient and targeted synthesis of active products. This will help overcome bottlenecks in the application of natural products and provide a solid theoretical foundation for the development and application of novel plant immune elicitors. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention aims to provide a method for genetic transformation of Efibula sp. ET39 fungal protoplasts.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a method for genetic transformation of Efibula sp. ET39 (also known as E. sp. ET39) fungal protoplasts, comprising the following steps:

[0008] (1) Mycelial culture and pretreatment: Efibula sp. ET39 was inoculated into the culture medium and the supernatant was removed by centrifugation, while the mycelial culture was retained.

[0009] (2) Preparation of enzyme solution: dissolve the lysing enzyme in 0.6-0.7 M MgSO4 solution, filter and sterilize, and collect the filtrate; the lysing enzyme is one or a mixed combination of Driselase, Lysing Enzymes and Lywallzyme;

[0010] (3) Preparation and transformation of protoplasts: add the enzyme solution of step (2) to the mycelium pretreated in step (1), place it in a shaker at 25℃~30℃, 70-100 rpm, and perform enzymolysis for 2~4 hours; after the enzymolysis is completed, remove the supernatant, resuspend the precipitate with STC aqueous solution, wash, and resuspend the precipitate with an appropriate amount of STC solution to obtain a purified protoplast suspension; take the plasmid containing the target gene and mix it with the protoplast suspension to obtain a first protoplast suspension, and let it stand at room temperature for 25-35 minutes; then add PTC solution to it to obtain a second protoplast suspension, and let it stand at room temperature for 25-35 minutes;

[0011] (4) Protoplast regeneration: The second protoplast suspension in step (3) is added to the regeneration medium and cultured until regenerated transformants grow.

[0012] Furthermore, step (1) Efibula sp. ET39 has been disclosed in the prior art (CN118440825A).

[0013] Furthermore, the mass dosage of the mycelium after the pretreatment in step (1) is 100 to 200 mg / mL based on the volume of the enzyme solution.

[0014] Furthermore, the Efibula sp. ET39 in step (1) was inoculated into PDB medium and cultured under the following conditions: 25°C to 30°C, 70-100 rpm for 2-3 days.

[0015] Furthermore, the components of the PDB culture medium in step (1) include: 200 g / L potato and 20 g / L glucose, and the solvent is water.

[0016] Furthermore, in step (2), the lysing enzymes are Driselase, Lysing Enzymes and Lywallzyme in a mass ratio of 0.5~3:0.5~3:0.5~3.

[0017] Furthermore, in step (2), the lysing enzymes are Driselase, Lysing Enzymes and Lywallzyme in a mass ratio of 1:1:1.

[0018] Furthermore, in step (2), filtration sterilization is performed using a 0.22 μm microporous filter membrane.

[0019] Furthermore, in step (2), the MgSO4 buffer solution is prepared by dissolving a formulated amount of MgSO4 in water and then adjusting the volume.

[0020] According to the technical solution of the present application, the lysing enzyme combination, enzymatic hydrolysis time, and enzymatic hydrolysis temperature significantly affect the preparation quality of Efibula sp. ET39 fungal protoplasts. When using a combination of Driselase, Lysing Enzymes, and Lywallzyme in a mass ratio of 1:1:1, a high yield of protoplasts can be obtained under the conditions of 0.6 M MgSO4 buffer, 30°C, and enzymatic hydrolysis for 3 h, with a protoplast yield of 2.28×10 7 / mL; using a single enzyme or a dual enzyme combination, the amount of protoplasts is small.

[0021] Furthermore, the STC solution in step (3) comprises 200 g / L sucrose, 5.54 g / L CaCl2 and 50 mL Tris-HCl (pH = 8.0), and the solvent is ultrapure water; the PTC solution comprises 60 g PEG4000, which is diluted to 100 mL with STC solution.

[0022] Furthermore, the plasmid in step (3) also contains an antibiotic marker gene, wherein the marker gene is one or a combination of hygromycin, bleomycin and G418 resistance expression genes.

[0023] Furthermore, the specific steps of protoplast regeneration in step (4) are as follows: the second protoplast suspension in step (3) is added to a liquid regeneration medium for overnight recovery, and then added to a solid regeneration medium, mixed and evenly coated on a plate, and after the plate is dried, a layer of liquid regeneration medium containing antibiotics is coated, and the plate is cultured upright until regenerated transformants grow.

[0024] Furthermore, in step (4), the liquid regeneration medium comprises the following components: 3 g / L yeast extract, 3 g / L tyrosine hydrolysate, 200 g / L sucrose, and 15 g / L agar (agar needs to be added to the solid regeneration medium), and the solvent is ultrapure water.

[0025] Furthermore, in step (4), the overnight recovery conditions are: 25-30°C, 70-100 rpm, and dark culture for 12-18 h.

[0026] Furthermore, in step (4), the protoplast regeneration conditions are: upright culture in an incubator at 25°C to 30°C for 5-7 days.

[0027] The applicant used the exogenous plasmid pYF11-eGFP carrying the green fluorescent protein gene to conduct verification referring to the aforementioned protoplast transformation and regeneration method. The results showed that the genetic transformation method provided by the present invention can obtain E. sp. ET39 fungal recombinant strains, thereby verifying the feasibility of the method of the present invention.

[0028] Beneficial effects

[0029] The present invention provides, for the first time, a method for genetic transformation of Efibula sp. ET39 fungal protoplasts, which has the following beneficial effects: the present invention establishes, for the first time, a method for genetic transformation of Efibula sp. ET39 fungal protoplasts, using a mixed lysing enzyme solution prepared with Driselase, Lysing Enzymes, and Lywallzyme, under the conditions of a mass ratio of 1:1:1, 0.6 MMgSO4 buffer, 30°C, and enzymatic hydrolysis for 3 h, to obtain a high yield of protoplasts, which can reach 2.28×10 7 This method has the characteristics of high transformation efficiency (6-8 transformants / μg DNA) and short transformation cycle (1-2 weeks), which can provide strong support for future research on gene function and active metabolites of Efibula sp. ET39 fungus. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Efibula sp. ET39 fungal protoplasts and DAPI staining microscopy, Figure 1 Panel a shows the release of protoplasts from strain ET39; Figure 1 Figure b shows the protoplast cells under bright field fluorescence microscope; Figure 1 Figure c shows protoplast cells stained with DAPI under fluorescence microscopy.

[0031] Figure 2 Results of the sensitivity test of Efibula sp. ET39 fungus to different concentrations of hygromycin B, bleomycin and G418.

[0032] Figure 3 This is a gel image of PCR verification of the recombinant strain Efibula sp. ET39 in Example 7.

[0033] Figure 4 This is a fluorescence microscopy image of the recombinant strain Efibula sp. ET39 in Example 7. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below with reference to the examples. Reagents or instruments used without manufacturer's indication are considered to be conventional products that can be purchased on the market.

[0035] The supplier of Driselase is Sigma Aldrich, the supplier of Lywallzyme is Guangdong Bide Biotechnology Co., Ltd., and the supplier of Lysing Enzymes is Nanjing Luqianyou Biotechnology Co., Ltd.

[0036] The components of PDB culture medium are: 200 g / L potato and 20 g / L glucose, and the solvent is water.

[0037] The STC solution consists of 200 g / L sucrose, 5.54 g / L CaCl2, and 50 mL Tris-HCl (pH = 8.0), and the solvent is ultrapure water. The PTC solution consists of 60 g PEG4000, which is diluted to 100 mL with STC solution.

[0038] The liquid regeneration medium consists of 3 g / L yeast extract, 3 g / L tyrosine hydrolysate, 200 g / L sucrose, and 15 g / L agar. The solvent is ultrapure water (agar needs to be added to the solid regeneration medium).

[0039] Example 1 Preparation of Efibula sp. ET39 fungal protoplasts

[0040] (1) Mycelial culture and purification: Efibula sp. ET39 was cultured in a 30°C incubator for 5 days. Twenty pieces of E. sp. ET39 mycelial fragments were taken with a cork punch and inoculated into a flask containing PDB medium at a volume of 150 mL / 250 mL. The mixture was cultured at 30°C and 70 rpm for 2-3 days. After the culture was completed, the mycelia were collected by centrifugation at 8000 rpm for 5 minutes, washed 2-3 times with 0.6 MMgSO4 solution, and the supernatant was removed by centrifugation to obtain pretreated mycelia. The resulting mycelia were used to prepare protoplasts.

[0041] (2) Preparation of enzyme solution: Weigh 5 mg of Driselase, Lysing Enzymes, and Lywallzyme enzymes in a mass ratio of 1:1:1, dissolve each in 1 mL of 0.6 M MgSO4, filter through a 0.22 μm microporous filter membrane for sterilization, and store for later use.

[0042] (3) Preparation and transformation of protoplasts: 1 mL of the enzyme solution prepared in step (2) was added to 100 mg of E. sp. ET39 mycelium prepared in step (1), and the mixture was shaken at 30°C and 70 rpm for 3 h. After the enzymatic hydrolysis, the majority of the mycelium was removed by filtration using a filter cloth, and the filtrate was collected and centrifuged at 4000 rpm for 10 min. The precipitate was resuspended in 0.6 M MgSO4 and washed 2–3 times to obtain purified protoplasts.

[0043] The hyphae of Efibula sp. ET39 fungus were lysed using lytic enzymes. During the enzymatic hydrolysis, protoplasts of various sizes were released from the hyphae of E. sp. ET39 fungus ( Figure 1 E. sp ET39 fungal protoplasts are mononuclear cells with a diameter of less than 20 μm ( Figure 1 Figure b in the figure, Figure 1 (Figure c in the figure) This characteristic helps improve the efficiency of genetic transformation of mycelium. Based on this, 8 μL of protoplast solution was aspirated and dropped onto a glass slide. 2 μL of DAPI solution was added and stained for 5-10 minutes. The protoplasts were then observed under a fluorescence microscope (ZEISS HAL-100). As shown, upon entering the protoplasts, DAPI specifically binds to the DNA within them. Under fluorescence excitation, the DAPI-bound DNA emits blue fluorescence.

[0044] Example 2 Effect of Lytic Enzyme Combination on the Number of Protoplasts Produced During Protoplast Preparation

[0045] (1) Mycelial culture and purification: Efibula sp. ET39 was cultured in a 30°C incubator for 5 days. Twenty pieces of E. sp. ET39 mycelial fragments were taken with a cork punch and inoculated into a flask containing PDB medium at a volume of 150 mL / 250 mL. The mixture was cultured at 30°C and 70 rpm for 2-3 days. After the culture was completed, the mycelia were collected by centrifugation at 8000 rpm for 5 minutes, washed 2-3 times with 0.6 MMgSO4 solution, and the supernatant was removed by centrifugation to obtain pretreated mycelia. The resulting mycelia were used to prepare protoplasts.

[0046] (2) Preparation of enzyme solution: Weigh Driselase, Lysing Enzymes and Lywallzyme according to Table 1, dissolve each in 1 mL of 0.6 M MgSO4, filter through a 0.22 μm microporous filter membrane for sterilization, and store for later use.

[0047] (3) Preparation and purification of protoplasts: 1 mL of the enzyme solution prepared in step (2) was added to 100 mg of E. sp. ET39 fungal mycelium prepared in step (1), and the mixture was placed in a shaker at 30°C and 70 rpm for 3 h. After the enzymatic hydrolysis, most of the mycelium was removed by filtration with a filter cloth, and the filtrate was collected and centrifuged at 4000 rpm for 10 min. The precipitate was resuspended in 0.6 M MgSO4 and washed 2 to 3 times to obtain purified protoplasts. The yield was calculated using a hemocytometer.

[0048] The experimental results showed (Table 1) that when the mass ratio of Driselase, Lysing Enzymes and Lywallzyme was 1:1:1, the yield of Efibula sp. ET39 fungal protoplasts was the highest, which was significantly higher than the lysis effect of a single enzyme or two enzymes.

[0049]

[0050] Example 3 Effect of enzymatic hydrolysis time on the number of protoplasts produced during protoplast preparation

[0051] The enzymolysis time affects the number of protoplasts generated. Based on the enzyme combination screening in Example 2, the enzymolysis time was optimized to investigate the effect of the enzymolysis time on the number of protoplasts generated.

[0052]

[0053] The results are shown in Table 2. When using Driselase, Lysing Enzymes, and Lywallzyme at a mass ratio of 1:1:1, the amount of protoplasts released increased with increasing enzymatic hydrolysis time. The number of protoplasts reached its peak after 3 h of hydrolysis. After 3.0 h, the amount of protoplasts digested exceeded the amount released, and the number began to decline. Because prolonged enzymatic hydrolysis can damage protoplasts, approximately 3.0 h of hydrolysis is the optimal time for protoplast genetic transformation.

[0054] Example 4 Effect of enzymatic hydrolysis temperature on the number of protoplasts produced during protoplast preparation

[0055] Temperature has a great influence on enzyme activity, and a suitable temperature can enable it to exert normal activity. Based on Example 3, the effect of enzymolysis temperature on the number of protoplasts generated was investigated.

[0056]

[0057] The results are shown in Table 3. When the enzymatic hydrolysis temperature was 30°C, the activity of the three enzyme combination of Driselase, Lysing Enzymes and Lywallzyme was the highest, the protoplast yield was the highest, and was significantly higher than that of other groups.

[0058] Example 5 Effect of buffer solution on the number of protoplasts produced during protoplast preparation

[0059] Different protoplasts have different osmotic stability and ion balances, resulting in significant variability in yields in different buffers. Buffers containing three different ionic components—NaCl, CaCl₂, and MgSO₄—were selected and adjusted to 0.6 M and 0.7 M, respectively, to dissolve the aforementioned lyase.

[0060]

[0061] The experimental results are shown in Table 4. Compared with other buffers, 0.6 M MgSO4 buffer produced the best protoplast effect.

[0062] Example 6 Evaluation of the Susceptibility of Efibula sp. ET39 to Antibiotics

[0063] To determine the tolerance of Efibula sp. ET39 (also known as E.sp. ET39) to hygromycin B, bleomycin, and G418 and the tolerance concentrations, E.sp. ET39 fungal blocks were sequentially inoculated onto PDA plates containing 0, 25, 50, 75, 100, 150, and 200 μg / mL antibiotics and placed in a constant temperature incubator at 30°C. After 5 days of incubation, the growth of E.sp. ET39 fungi on the culture media with different concentrations of hygromycin B, bleomycin, and G418 were observed to determine the optimal antibiotic concentration for inhibiting E.sp. ET39.

[0064] like Figure 2 As shown, E. sp. ET39 is highly sensitive to hygromycin B, with 50 μg / mL of hygromycin B inhibiting its growth. E. sp. ET39 is less sensitive to bleomycin and G418 than to hygromycin B. 200 μg / mL of G418 completely inhibited its growth, and this concentration can be used for subsequent resistance screening.

[0065] Example 7 Genetic Transformation of Efibula sp. ET39 Fungal Protoplasts

[0066] (1) Protoplast transformation: The purified Efibula sp. ET39 protoplasts from Example 1 were resuspended in STC solution; 2 μg of pYF11-eGFP plasmid was mixed with the protoplast suspension to obtain a first protoplast suspension, which was allowed to stand at room temperature for 30 min; 1 mL of PTC solution was then added to the suspension to obtain a second protoplast suspension, which was allowed to stand at room temperature for 30 min;

[0067] (2) Protoplast regeneration: The second protoplast suspension in step (1) was added to the liquid regeneration medium for overnight recovery, and then added to the solid regeneration medium. Mix well and gently spread evenly on the plate. After the plate is dried, a layer of regeneration medium containing G418 was applied. The plate was cultured in the dark at 30°C for 5-7 days until regenerated transformants grew.

[0068] The protoplast-regenerated single colonies grew on the culture medium, yielding 12-16 clones, or 6-8 transformants per μg of DNA. Subsequently, the genomes of the single colonies were extracted and verified by PCR (Table 5). PCR results showed that a fragment of the reporter gene eGFP could be amplified from these protoplast-regenerated single colonies ( Figure 3 ), indicating that the pYF11-eGFP plasmid was successfully introduced into E. sp. ET39 fungi. Based on this, hyphae from a single colony of the regenerated strain were picked and observed under a fluorescence microscope. The hyphae of the transgenic strain in the control group did not detect green fluorescence, while the hyphae of the protoplast-regenerated strain showed green fluorescence ( Figure 4 ). The experimental results show that the genetic transformation method provided by the present invention can obtain E. sp. ET39 fungal recombinant strains, thereby verifying the feasibility of the method of the present invention.

[0069]

[0070] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A genus of leather without locks ( Efibula sp.) ET39 fungal protoplast genetic transformation method, characterized in that, The method comprises the following steps: (1) Mycelium cultivation and pretreatment: Efibula sp. ET39 was inoculated into the culture medium, the supernatant was removed by centrifugation, and the hyphae were retained; (2) Preparation of enzyme solution: Dissolve 5 mg each of Driselase, Lysing Enzymes, and Lywallzyme in 1 mL of 0.6-0.7 M MgSO4 buffer, filter and sterilize for later use; (3) Preparation and transformation of protoplasts: Add the enzyme solution of step (2) to 100 mg of mycelium pretreated in step (1), and perform enzymolysis at 25°C to 30°C and 70-100 rpm for 2-4 hours. After the enzymolysis is completed, filter, resuspend the precipitate with STC aqueous solution, and wash to obtain a protoplast suspension; mix the protoplast suspension with a plasmid containing the target gene to obtain a first protoplast suspension; then add PTC solution to the first protoplast solution to obtain a second protoplast suspension; (4) Protoplast regeneration: the second protoplast suspension in step (3) is added to the regeneration medium and cultured until regenerated transformants grow; In step (1) Efibula sp. ET39 was inoculated into PDB medium and cultured at 25°C–30°C and 70–100 rpm for 2–3 days.

2. according to claim 1 Efibula sp. ET39 fungal protoplast genetic transformation method, characterized in that, The components of PDB culture medium are: 200 g / L potato and 20 g / L glucose, and the solvent is water.

3. according to claim 1 Efibula sp. ET39 fungal protoplast genetic transformation method, characterized in that, In step (3), the STC solution consists of 200 g / L sucrose, 5.54 g / L CaCl2 and 50 mL Tris-HCl, pH=8.0, and the solvent is ultrapure water; the PTC solution consists of 60 g PEG4000, which is diluted to 100 mL with STC solution.

4. according to claim 1 Efibula sp. ET39 fungal protoplast genetic transformation method, characterized in that, In step (3), the plasmid further contains an antibiotic marker gene, which is one or a combination of hygromycin, bleomycin and G418 resistance expression genes.

5. according to claim 4 Efibula sp. ET39 fungal protoplast genetic transformation method, characterized in that, The specific steps of protoplast regeneration in step (4) are as follows: the second protoplast suspension in step (3) is added to the liquid regeneration medium for overnight recovery, and then added to the solid regeneration medium, mixed and evenly coated on the plate, and after the plate is dried, a layer of liquid regeneration medium containing antibiotics is coated on it, and the plate is cultured upright until regenerated transformants grow.

6. according to claim 5 Efibula sp. ET39 fungal protoplast genetic transformation method, characterized in that, In step (4), the liquid regeneration medium comprises the following components: 3 g / L yeast extract, 3 g / L tyrosine hydrolysate, 200 g / L sucrose and 15 g / L agar, and the solvent is ultrapure water.

7. according to claim 5 Efibula sp. ET39 fungal protoplast genetic transformation method, characterized in that, In step (4), the conditions for overnight recovery are: 25-30°C, 70-100 rpm, and dark culture for 12-18 h.

8. according to claim 5 Efibula sp. ET39 fungal protoplast genetic transformation method, characterized in that, In step (4), the regeneration conditions are: upright culture in an incubator at 25°C to 30°C for 5-7 days.

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