Efibula sp. ET39 fungus protoplast genetic transformation method

Abstract

The invention discloses a genetic transformation method for a protoplast of an Efibula sp.ET39 fungus. The genetic transformation method of the Efibulla sp.ET39 fungus protoplast is established for the first time, a mixed enzyme solution prepared from Drisease, Lysing Enzymes and Lywallzyme is adopted, the mass ratio of the mixed enzyme solution to the Lysing Enzymes to the Lywallzyme is 1: 1: 1, the protoplast with the high yield can be obtained under the conditions that an MgSO4 buffer solution with the concentration being 0.6-0.7 M is adopted, the temperature is 30 DEG C, and enzymolysis is conducted for 3 h, and the protoplast can reach 2.28 * 10 < 7 > / mL; the method has the characteristics of high transformation efficiency (6-8 transformants / mu g DNA), short transformation time period (1-2 weeks) and the like, and can provide powerful support for research work of gene functions and active metabolites of Efibula sp.ET39 fungi in the future.

Description

Technical Field

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

[0002] Microbial metabolites have the characteristics of diverse chemical structures and significant biological activities, and are an important source for the discovery of pesticide lead compounds. Chinese Patent Application CN118440825A discloses that the basidiomycete fungus Efibula sp. ET39 can produce a class of secondary metabolites Efi-A, Efi-B, and Efi-C with immune-inducing and resistance activities. These compounds have novel structures, can significantly enhance the disease resistance activity of plants, and have no inhibitory effect on plant growth, showing good application prospects. However, their development faces many problems such as low yield, complex separation system, and difficulty in large-scale preparation.

[0003] In recent years, using biosynthetic research to solve problems such as low fermentation yield of natural products has become a research hotspot. Taking microbial metabolites as the research object and clarifying their biosynthetic pathways in strains at the molecular level helps to achieve the efficient and directional synthesis of bioactive natural products. Thus, the establishment of a genetic transformation system for the Efibula sp. ET39 strain is an important technical basis for studying the biosynthesis of bioactive metabolites.

[0004] In recent years, although relevant reports have established genetic transformation methods in fungi such as basidiomycete smut fungi, there are problems such as the discontinuation of the lyase (product number Sigma Aldrich, L1412), long time period (2 - 3 weeks), and low transformation efficiency. Therefore, constructing a method for the preparation and genetic transformation of Efibula sp. ET39 protoplasts is of great significance for future biosynthetic research and the efficient and directional synthesis of bioactive products, helps to break through the bottleneck of the application of natural products, and provides a solid theoretical basis for the development and application of new plant immune-inducing agents. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide a method for genetic transformation of protoplasts of the fungus Efibula sp. ET39.

[0006] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

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

[0008] (1)Cultivation and pretreatment of hyphae: Efibula sp. ET39 was inoculated into the medium for cultivation, and the supernatant was removed by centrifugation, and the hyphae were retained;

[0009] (2)Preparation of enzyme solution: Take lysing enzyme and dissolve it 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 in step (2) to the pretreated hyphae in step (1), place it in a shaker at 25℃ - 30℃, 70 - 100 rpm, and enzymolyze for 2 - 4 h; 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 the first protoplast suspension, and let it stand at room temperature for 25 - 35 min; then add PTC solution to it to obtain the second protoplast suspension, and let it stand at room temperature for 25 - 35 min;

[0011] (4)Regeneration of protoplasts: Add the second protoplast suspension in step (3) to the regeneration medium and culture until regenerated transformants grow out.

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

[0013] Further, the mass dosage of the pretreated hyphae in step (1) is 100 - 200 mg / mL based on the volume of the enzyme solution.

[0014] Further, Efibula sp. ET39 in step (1) was inoculated in PDB medium for cultivation, and the cultivation conditions were: 25℃ - 30℃, 70 - 100 rpm for 2 - 3 d.

[0015] Even further, the composition of the PDB medium in step (1) includes: 200 g / L of potato and 20 g / L of glucose, and the solvent is water.

[0016] Further, the lysing enzyme in step (2) is Driselase, Lysing Enzymes, and Lywallzyme, and the mass ratio is 0.5 - 3:0.5 - 3:0.5 - 3.

[0017] Even further, the lysing enzyme in step (2) is Driselase, Lysing Enzymes, and Lywallzyme, and the mass ratio is 1:1:1.

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

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

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

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

[0022] Furthermore, the plasmid in step (3) also contains an antibiotic marker gene, and 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: Add the second protoplast suspension in step (3) to the liquid regeneration medium for overnight resuscitation, then add it to the solid regeneration medium, mix well, spread evenly, pour the plate, and after the plate dries, spread a layer of liquid regeneration medium containing antibiotics, and incubate upright until regenerated transformants grow out.

[0024] Even further, in step (4), the composition of the liquid regeneration medium includes: 3 g / L of yeast extract, 3 g / L of tyrosine hydrolyzate, 200 g / L of sucrose, and 15 g / L of agar (agar needs to be added to the solid regeneration medium), and the solvent is ultrapure water.

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

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

[0027] The applicant used the exogenous plasmid pYF11-eGFP carrying the green fluorescent protein gene to verify according to the aforementioned protoplast transformation and regeneration method. The results showed that the genetic transformation method provided by the present invention could obtain the recombinant strain of E. sp. ET39 fungus, thus verifying the feasibility of the method of the present invention.

[0028] Beneficial effects

[0029] The present invention first provides a genetic transformation method for protoplasts of Efibula sp. ET39 fungus, which has the following beneficial effects: The present invention first established a genetic transformation method for protoplasts of Efibula sp. ET39 fungus. 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 M MgSO4 buffer, 30 °C, and enzymatic hydrolysis for 3 h, protoplasts with a relatively high yield can be obtained, up to 2.28×10 7 cells / mL; this method has the characteristics of high transformation efficiency (6-8 transformants / μg DNA), short transformation time period (1-2 weeks), etc., and can provide strong support for future research on the gene function and active metabolites of Efibula sp. ET39 fungus. Description of the drawings

[0030] Figure 1 Microscopic examination diagrams of protoplasts of Efibula sp. ET39 fungus and their DAPI staining, where Figure 1 Figure a in it shows the release of protoplasts from strain ET39; Figure 1 Figure b in it shows the protoplast cells under the bright field of a fluorescence microscope; Figure 1 Figure c in it shows the protoplast cells of a DAPI-stained fluorescence microscope.

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

[0032] Figure 3 Gel diagram of PCR verification of the recombinant strain of Efibula sp. ET39 in Example 7.

[0033] Figure 4 Microscopic examination diagram of the fluorescence microscope of the recombinant strain of Efibula sp. ET39 in Example 7. Detailed implementation manners

[0034] The present invention will be further described in detail below with reference to the embodiments. Reagents or equipment not indicating the manufacturer are regarded as conventional products that can be purchased on the market.

[0035] Driselase is supplied by Sigma Aldrich, Lywallzyme is supplied by Guangdong Bide Biotechnology Co., Ltd., and Lysing Enzymes is supplied by Nanjing Luqianyou Biotechnology Co., Ltd.

[0036] The composition of PDB medium: 200 g / L of potato and 20 g / L of glucose, with water as the solvent.

[0037] The composition of STC solution: 200 g / L of sucrose, 5.54 g / L of CaCl2 and 50 mL of Tris-HCl (pH = 8.0), with ultrapure water as the solvent; the composition of PTC solution: 60 g of PEG4000, made up to 100 mL with STC solution.

[0038] The composition of liquid regeneration medium: 3 g / L of yeast extract, 3 g / L of tyrosine hydrolyzate, 200 g / L of sucrose and 15 g / L of agar, with ultrapure water as the solvent (agar needs to be added for solid regeneration medium).

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

[0040] (1) Cultivation and purification of hyphae: The Efibula sp. ET39 fungus was statically cultured in an incubator at 30 °C for 5 d. Twenty hyphal blocks of E. sp. ET39 were taken with a punch and inoculated into a 250 mL Erlenmeyer flask containing PDB medium with a liquid loading of 150 mL / 250 mL, and cultured at 30 °C and 70 rpm for 2 - 3 d. After the cultivation, it was centrifuged at 8000 rpm for 5 min to collect the hyphae, and the hyphae were washed 2 - 3 times with 0.6 M MgSO4 solution, and the supernatant was removed by centrifugation to obtain the pretreated hyphae; the obtained hyphae were used for the preparation of protoplasts.

[0041] (2) Preparation of enzyme solution: Weigh 5 mg of Driselase, Lysing Enzymes and Lywallzyme enzymes respectively according to the mass ratio of 1:1:1, and then dissolve them separately with 1 mL of 0.6 M MgSO4, filter and sterilize them with a 0.22 μm microporous membrane and store for later use.

[0042] (3)Protoplast preparation and transformation: Add 1 mL of the enzyme solution prepared in step (2) to 100 mg of the E. sp. ET39 fungal mycelia prepared in step (1), place it in a shaker at 30 °C, and enzymatically digest for 3 h at 70 rpm. After the enzymatic digestion, filter through a filter cloth to remove most of the mycelia, collect the filtrate, centrifuge the filtrate at 4000 rpm for 10 min, resuspend the precipitate in 0.6 M MgSO4, and wash 2 - 3 times to obtain purified protoplasts.

[0043] Use lysing enzymes to lyse the mycelia of Efibula sp. ET39 fungi. During enzymatic digestion, protoplast cells of different sizes are released from the mycelia of E. sp. ET39 fungi ( Figure 1 Figure a in Figure 1 . The protoplasts of E. sp ET39 fungi are mononuclear cells with a diameter of less than 20 μm ( Figure 1 Figure b in,

[0044] Example 2 Effect of the combination of lysing enzymes on the number of protoplasts generated during protoplast preparation

[0045] (1)Culture and purification of mycelia: Efibula sp. ET39 fungi are statically cultured in an incubator at 30 °C for 5 d. Use a punch to take 20 pieces of E. sp. ET39 fungal mycelia blocks and inoculate them into a triangular flask containing PDB medium. The liquid loading volume is 150 mL / 250 mL, and culture at 30 °C and 70 rpm for 2 - 3 d. After the culture, centrifuge at 8000 rpm for 5 min, collect the mycelia, wash the mycelia 2 - 3 times with 0.6 M MgSO4 solution, centrifuge to remove the supernatant, and obtain pretreated mycelia; the obtained mycelia are used for protoplast preparation.

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

[0047] (3)Preparation and purification of protoplasts: Add 1 mL of the enzyme solution prepared in step (2) to 100 mg of the E. sp. ET39 fungal mycelia prepared in step (1), place it in a shaker at 30 °C, and enzymatically digest for 3 h at 70 rpm. After the enzymatic digestion is completed, filter through a filter cloth to remove most of the mycelia, collect the filtrate, centrifuge the filtrate at 4000 rpm for 10 min, resuspend the precipitate in 0.6 M MgSO4, wash 2 - 3 times to obtain purified protoplasts, and calculate the yield using a hemocytometer.

[0048] The experimental results show (Table 1) that under the condition of a mass ratio of 1:1:1 of the three enzymes Driselase, Lysing Enzymes, and Lywallzyme, the yield of protoplasts of the Efibula sp. ET39 fungus is the highest, significantly higher than the lysis effect of single enzyme or two enzymes.

[0049]

[0050] Example 3 Effect of enzymatic digestion time on the number of protoplasts generated during the protoplast preparation process

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

[0052]

[0053] The results are shown in Table 2. When using Driselase, Lysing Enzymes, and Lywallzyme with a mass ratio of 1:1:1, as the enzymatic digestion time extends, the release amount of protoplasts continuously increases. When the enzymatic digestion time is 3 h, the number of protoplasts reaches the maximum. After 3.0 h, the digestion amount of protoplasts is greater than the release amount, so its number begins to decline. Since too long enzymatic digestion time will damage the protoplasts, the best time for protoplast genetic transformation is about 3.0 h of enzymatic digestion.

[0054] Example 4 Effect of enzymatic digestion temperature on the number of protoplasts generated during the protoplast preparation process

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

[0056]

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

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

[0059] The osmotic pressure stabilities of different protoplasts are different, and the ionic balances contained are also different. Therefore, the yield differences in different buffer solutions are relatively large. Three buffer solutions with different ionic components of NaCl, CaCl2, and MgSO4 were selected, and their concentrations were respectively adjusted to 0.6 M and 0.7 M for dissolving the above-mentioned lytic enzymes.

[0060]

[0061] The experimental results are shown in Table 4. The 0.6 M MgSO4 buffer solution has the best effect on generating protoplasts compared with other buffer solutions.

[0062] Example 6 Sensitivity assessment of Efibula sp. ET39 fungus to antibiotics

[0063] To determine whether the Efibula sp. ET39 (also known as E. sp. ET39) fungus is tolerant to hygromycin B, bleomycin, and G418 and the tolerance concentrations, the E. sp. ET39 fungal blocks were successively inoculated on 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 culturing for 5 days, observe the growth of E. sp. ET39 fungus on the culture media with different concentrations of hygromycin B, bleomycin, and G418 to determine the optimal antibiotic concentration for inhibiting E. sp. ET39 fungus.

[0064] As Figure 2 shown, the E. sp. ET39 fungus is highly sensitive to hygromycin B, and 50 μg / mL of hygromycin B can inhibit the growth of E. sp. ET39 fungus. The sensitivity of E. sp. ET39 fungus to bleomycin and G418 is lower than that to hygromycin B, and 200 μg / mL of G418 can completely inhibit the growth of E. sp. ET39 fungus, and this concentration can be used for subsequent resistance screening.

[0065] Example 7 Genetic transformation of protoplasts of Efibula sp. ET39 fungus

[0066] (1)Transformation of protoplasts: Resuspend the purified Efibula sp. ET39 protoplasts in Example 1 with STC solution; Take 2 μg of pYF11-eGFP plasmid and mix it with the protoplast suspension to obtain the first protoplast suspension, and let it stand at room temperature for 30 min; Then add 1 mL of PTC solution to it to obtain the second protoplast suspension, and let it stand at room temperature for 30 min;

[0067] (2)Regeneration of protoplasts: Add the second protoplast suspension in step (1) to the liquid regeneration medium for overnight resuscitation, and then add it to the solid regeneration medium, mix well, gently spread it evenly, pour the plate, and after the plate is dried, spread another layer of regeneration medium containing G418, and culture it in the dark at 30 °C for 5 - 7 d until the regenerated transformants grow out.

[0068] Single colony strains of protoplast regeneration grow on the medium, and a total of 12 - 16 clones grow out, that is, 6 - 8 transformants / μg DNA. Subsequently, the genomic DNA was extracted from the single colonies and verified by PCR (Table 5). The results of the PCR experiment showed that in these single colony strains obtained by protoplast regeneration, the fragment of the reporter gene eGFP could be amplified ( Figure 3 ), indicating that the pYF11-eGFP plasmid was successfully introduced into the E. sp. ET39 fungus. On this basis, the hyphae of the single colonies of the regenerated strains were picked and observed under a fluorescence microscope. The hyphae of the transgenic strains in the control group did not detect green fluorescence, while the hyphae in the protoplast regenerated strains showed green fluorescence ( Figure 4 ). The experimental results showed that the recombinant strains of E. sp. ET39 fungus could be obtained by using the genetic transformation method provided by the present invention, thus verifying the feasibility of the method of the present invention.

[0069]

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

Claims

1. A Efibula genetic transformation method for protoplasts of sp. ET39 fungi, characterized in that The method includes the following steps: (1)Cultivation and pretreatment of hyphae: Efibula Inoculate ET39 sp. into the medium for cultivation, centrifuge to remove the supernatant, and retain the hyphae; (2) Preparation of enzyme solution: Dissolve lysing enzymes in a buffer solution of 0.6 - 0.7 M MgSO4, filter and sterilize for later use. The lysing enzymes are composed of Driselase, Lysing Enzymes, and Lywallzyme prepared according to a mass ratio of 0.5 - 3:0.5 - 3:0.5 - 3; (3) Preparation and transformation of protoplasts: Add the enzyme solution from step (2) to the mycelia pretreated in step (1), and carry out enzymatic hydrolysis at 25°C - 30°C and 70 - 100 rpm for 2 - 4 h. After enzymatic hydrolysis, 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) Regeneration of protoplasts: Add the second protoplast suspension in step (3) to a regeneration medium and culture until regenerated transformants grow out.

2. According to claim 1 Efibula method for genetic transformation of protoplasts of sp. ET39 fungi, characterized in that In step (1), Efibula sp. ET39 was inoculated into PDB medium for cultivation under the following conditions: 25°C to 30°C, 70 - 100 rpm for 2 - 3 days.

3. According to claim 2 Efibula The genetic transformation method of sp. ET39 fungal protoplasts, characterized in that Composition of PDB medium: 200 g / L of potato and 20 g / L of glucose, with water as the solvent.

4. According to claim 1 Efibula A genetic transformation method for protoplasts of sp. ET39 fungi, characterized in that In step (2), the lysing enzymes are composed of Driselase, Lysing Enzymes, and Lywallzyme mixed according to a mass ratio of 1:1:

1.

5. According to claim 1 Efibula a genetic transformation method for protoplasts of sp. ET39 fungi, characterized in that In step (3), the composition of the STC solution: 200 g / L of sucrose, 5.54 g / L of CaCl2, and 50 mL of Tris - HCl, pH = 8.0, with ultrapure water as the solvent; The composition of the PTC solution: 60 g of PEG4000, made up to 100 mL with STC solution.

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

7. According to claim 6, Efibula the genetic transformation method of sp. ET39 fungal protoplasts, characterized in that, The specific steps for protoplast regeneration in step (4) are as follows: Add the second protoplast suspension in step (3) to a liquid regeneration medium for overnight resuscitation, then add it to a solid regeneration medium, mix well, spread evenly, pour the plate, and after the plate dries, spread a layer of liquid regeneration medium containing antibiotics, and incubate upright until regenerated transformants grow out.

8. According to claim 7, Efibula a genetic transformation method of sp. ET39 fungal protoplasts, characterized in that, In step (4), the composition of the liquid regeneration medium: 3 g / L of yeast extract, 3 g / L of tyrosine hydrolysate, 200 g / L of sucrose, and 15 g / L of agar, with ultrapure water as the solvent.

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

10. According to claim 7 Efibula A genetic transformation method for protoplasts of sp. ET39 fungi, characterized in that In step (4), the conditions for regeneration are: Incubate upright in an incubator at 25°C - 30°C for 5 - 7 d.

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