Genetic transformation method of PEG-mediated penicillium sclerotiorum strain protoplast and application of genetic transformation method

Abstract

The invention discloses a genetic transformation method of PEG (Polyethylene Glycol) mediated penicillium sclerotiorum strain protoplast and application of the genetic transformation method. The method comprises the following steps: (1) culturing penicillium sclerotiorum to produce spores, germinating hyphae, and collecting and cleaning fresh penicillium sclerotiorum hyphae; (2) compounding Lysing Enzymes, Yatase, cellulase and pectinase to prepare a protoplast enzymatic hydrolysate, and then adding the protoplast enzymatic hydrolysate into the penicillium sclerotiorum hypha for enzymolysis to obtain a penicillium sclerotiorum protoplast; and (3) adding plasmids to be transformed into the penicillium sclerotiorum protoplast, and carrying out PEG (Polyethylene Glycol) mediated transformation and resistance screening to obtain a penicillium sclerotiorum protoplast transformant. According to the method, the number and regeneration rate of the protoplast can be remarkably increased, and the enzymolysis time can be effectively shortened.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a PEG-mediated genetic transformation method for protoplasts of Penicillium sclerotiorum strains and its application. Background Art

[0002] Plant endophytic fungi are widely distributed and diverse in species, with significant biodiversity. They are rich resources of bioactive natural products, and also a new source of plant chemical components and important microbial resources. A large number of studies have shown that medicinal plant endophytes have strong biological activities and can produce a large number of substances with potential uses in agriculture, industry and modern medicine, such as new antibiotics, antifungal drugs, immunosuppressants and anticancer compounds, showing great development potential in the fields of medicine and agriculture.

[0003] Penicillium sclerotiorum is a plant endophytic fungus isolated from the rhizome of the Chinese medicinal plant Portulaca oleracea. It shows extremely strong biological activities in the field of agricultural pest control, such as being able to effectively control bacterial wilt, rice blast and parasitic nematodes of solanaceous crops, and has the potential to become a new biocontrol agent. Due to the relatively weak research foundation on the action mechanism of Penicillium sclerotiorum, the lack of an effective genetic transformation system is an important reason restricting its in-depth research. Therefore, establishing an efficient, simple and stable genetic transformation system and method is of great significance for the visual dynamic monitoring and mechanism exploration of Penicillium sclerotiorum strains. Summary of the Invention

[0004] The purpose of the present invention is to overcome the disadvantages and deficiencies of the prior art and provide a PEG-mediated genetic transformation method for protoplasts of Penicillium sclerotiorum strains.

[0005] Another purpose of the present invention is to provide the application of the PEG-mediated genetic transformation method for protoplasts of Penicillium sclerotiorum strains.

[0006] The purpose of the present invention is achieved by the following technical solutions:

[0007] A PEG-mediated genetic transformation method for protoplasts of Penicillium sclerotiorum strains includes the following steps:

[0008] (1) Collect Penicillium sclerotiorum hyphae

[0009] Inoculate Penicillium sclerotiorum on a plate for spore production, then pour the spore-producing Penicillium sclerotiorum culture into a medium for hyphal germination, and then collect and wash the fresh Penicillium sclerotiorum hyphae;

[0010] (2) Prepare Penicillium sclerotiorum protoplasts

[0011] ① Prepare the protoplast enzymatic hydrolysate:

[0012] The formula of the protoplast enzymatic hydrolysis solution is as follows: Lysing Enzymes 0 - 30 mg, Yatalase 5 - 20 mg, cellulase 0 - 30 mg, pectinase 5 - 20 mg, mycelium washing solution (Osmotic medium) 20 mL; among them, the total amount of enzyme added is controlled to be 60 - 70 mg;

[0013] ② Enzymatic hydrolysis: Add the prepared protoplast enzymatic hydrolysis solution to the sclerotium Penicillium mycelium collected in step (1) for enzymatic hydrolysis until the cell wall ruptures and protoplasts are released, then filter with a filter cloth, centrifuge and wash with STC solution (STC buffer), and resuspend with STC solution to obtain Penicillium sclerotiorum protoplasts;

[0014] (3) Transformation of Penicillium sclerotiorum protoplasts

[0015] Add the plasmid to be transformed (transfection plasmid) to the Penicillium sclerotiorum protoplasts obtained in step (2), then add PEG (polyethylene glycol) for mediated transformation, and screen with antibiotics to obtain Penicillium sclerotiorum protoplast transformants.

[0016] The Penicillium sclerotiorum described in step (1) is preferably Penicillium sclerotiorum jyscaumcx01, and its preservation number is: GDMCC No.60249.

[0017] The sporulation of the Penicillium sclerotiorum described in step (1) can be carried out according to the conventional methods in the art, or refer to the method described in the Chinese patent (application number: 202210053325.0, title: "A method for solid fermentation sporulation of Penicillium sclerotiorum and its application") for sporulation; preferably, it is achieved through the following steps: inoculate the Penicillium sclerotiorum strain on a culture medium plate, culture in the dark at 25 - 28 °C for about 5 - 12 days (preferably 5 - 7 days) for sporulation, and then collect the Penicillium sclerotiorum spores.

[0018] The plate described in step (1) is preferably a PDA plate.

[0019] The culture medium described in step (1) is preferably LB medium.

[0020] In step (1), the conditions for mycelial germination are: culture in a shaker at 28 °C and 180 rpm for 14 - 15 h.

[0021] In step (1), the collection and cleaning of fresh Penicillium sclerotiorum mycelium are achieved through the following steps: collect the fresh mycelium formed after culture by centrifugation, then mix and shake with 0.2% (w / v) Tween - 80 solution and let it stand for 20 - 30 min, then discard the supernatant, add an equal volume of STC solution for washing, discard the supernatant after washing, and add an equal volume of STC solution again for washing to obtain Penicillium sclerotiorum mycelium.

[0022] The preferred centrifugation conditions are: centrifugation at 4°C and 4000 rpm for 8 - 10 min.

[0023] The formulation of the STC solution is as follows: 1.2M sorbitol, 50mM Tris - HCl pH 8.0, 50mM CaCl2.

[0024] The preferred formulation of the protoplast enzymatic hydrolysis solution in step (2) is: 10 - 30 mg of Lysing Enzymes, 5 - 20 mg of Yatalase, 10 - 30 mg of cellulase, 5 - 20 mg of pectinase, 20 mL of mycelium washing solution (Osmotic medium); among them, the total amount of enzyme added is controlled to be 60 - 70 mg.

[0025] A further preferred formulation of the protoplast enzymatic hydrolysis solution in step (2) is: 15 - 30 mg of Lysing Enzymes, 5 - 10 mg of Yatalase, 10 - 20 mg of cellulase, 10 - 20 mg of pectinase, 20 mL of mycelium washing solution (Osmotic medium); among them, the total amount of enzyme added is controlled to be 60 mg.

[0026] An even further preferred formulation of the protoplast enzymatic hydrolysis solution in step (2) is: 30 mg of Lysing Enzymes, 10 mg of Yatalase, 10 mg of cellulase, 10 mg of pectinase.

[0027] The protoplast enzymatic hydrolysis solution in step (2) is prepared by the following method: Add Lysing Enzymes, Yatalase, cellulase and pectinase to the mycelium washing solution (Osmotic medium), mix well and place on a shaker for cultivation, and then filter and sterilize with a filter membrane to obtain it.

[0028] The conditions for the shaker cultivation are: cultivation on a shaker at 28 - 30°C and 100 - 150 rpm for 0.5 - 1.5 h; preferably: cultivation on a shaker at 30°C and 120 rpm for 1 h.

[0029] The preferred filter membrane is a 0.22μm filter membrane.

[0030] The formulation of the mycelium washing solution (Osmotic medium) in step (2) is as follows: 295.8 g of MgSO4·7H2O, 0.46 g of Na2HPO4, 1.06 g of NaH2PO4·2H2O, made up to 1000 mL with distilled water, pH 5.8.

[0031] The preparation method of the mycelium washing solution (Osmotic medium) described in step (2) is as follows: First, dissolve 295.8 g of MgSO4·7H2O in 600 mL of distilled water to obtain solution I; then dissolve 0.46 g of Na2HPO4 and 1.06 g of NaH2PO4·2H2O in no more than 50 mL of distilled water respectively to obtain solution II and solution III; then add solution II and solution III to solution I respectively, stir continuously during the addition process, and add distilled water to make up to 1000 mL. Finally, adjust the pH value to 5.8 with Na2HPO4 solution to obtain it.

[0032] The volume ratio of the sclerotial Penicillium mycelium to the protoplast enzymolysis solution described in step (2) is 1:10 - 15; preferably 1:10.

[0033] The enzymolysis conditions described in step (2) are: enzymolysis at 28 ± 1 °C, 100 - 150 rpm for 4 - 7 h; preferably: enzymolysis at 28 ± 1 °C, 100 - 150 rpm for 4 - 6 h; more preferably: enzymolysis at 28 °C, 100 rpm for 6 h.

[0034] The filter cloth described in step (2) is a magic filter cloth; preferably Miracloth magic filter cloth (Solarbio, product number: 475855-1R), with a pore size of 40 μm.

[0035] The formula of the STC solution described in step (2) is as follows: 1.2 M sorbitol, 50 mM Tris-HCl pH 8.0, 50 mM CaCl2.

[0036] The centrifugal washing described in step (2) is to wash with STC solution more than once, and the STC solution needs to be ice-bathed in advance.

[0037] The centrifugation conditions described in step (2) are: centrifuge at 4 °C, 4000 - 5000 rpm for 10 min; preferably: centrifuge at 4 °C, 5000 rpm for 10 min.

[0038] The concentration of the sclerotial Penicillium protoplasts described in step (2) is (6 - 25)×10 6 protoplasts / mL; preferably (15 - 25)×10 6 protoplasts / mL; more preferably (18 - 24)×10 6 protoplasts / mL.

[0039] In the described PEG-mediated genetic transformation method of Penicillium sclerotiorum protoplasts, after step (2) and before step (3), there is also a step of regenerating Penicillium sclerotiorum protoplasts; specifically: Dilute the Penicillium sclerotiorum protoplasts obtained in step (2) with STC solution, coat them on the Bottom Agar medium, and place them in an incubator at a constant temperature of 28 ± 1 °C for 2 - 3 days.

[0040] The dilution multiple described can be adjusted according to the actual situation. For example, for the Penicillium sclerotiorum protoplasts obtained in step (2), they can be diluted 400 - 1500 times with STC solution (control the concentration after dilution to be about 1.5×10 6 ~1.6×10 6 protoplasts / mL), and then coat them on the Bottom Agar medium.

[0041] The formula of the described Bottom Agar medium is: 3 g of yeast extract, 3 g of acid-hydrolyzed casein, 200 g of sucrose, 10 g of agar, and add distilled water to 1000 mL.

[0042] The PEG solution in step (3) is a PEG 8000 solution; preferably, the PEG 8000 solution is prepared with STC solution.

[0043] The addition amount of the PEG solution described in step (3) can be added according to actual needs, and preferably, it is calculated according to its final concentration in the system being 20% by mass.

[0044] The transformation of the Penicillium sclerotiorum protoplasts in step (3) is preferably achieved through the following steps:

[0045] Add the plasmid to be transformed to the Penicillium sclerotiorum protoplasts obtained in step (2), mix gently and place on ice for 20 ± 1 minutes; then add the PEG (polyethylene glycol) solution, let it stand on ice for 20 ± 1 minutes, after standing, transfer the mixed system to the TB3 medium containing ampicillin and culture for 12 - 14 h, then pour the cultured system onto the PDA plate containing ampicillin (Amp) and hygromycin (Hyg) and incubate it upside down until fresh mycelia are formed, and finally pick the mycelia to the PDA medium containing ampicillin and hygromycin and screen more than twice to obtain protoplast transformants.

[0046] The transformation plasmid described is preferably the expression vector pCT-74 (containing Amp and Hyg resistance genes).

[0047] The dosage (access amount) of the transformation plasmid described can be adjusted according to the actual situation. For example, about 20 μg of the transformation plasmid can be added to 4×10 6 Penicillium sclerotiorum protoplasts.

[0048] The formula of the TB3 medium is as follows: 3 g of yeast extract, 3 g of acid-hydrolyzed casein, 200 g of sucrose, and the volume is made up to 1000 mL with distilled water.

[0049] The concentration of ampicillin in the TB3 medium containing ampicillin is 50 mg / L.

[0050] The concentration of ampicillin in the PDA medium (PDA plate) containing ampicillin and hygromycin is 50 mg / L, and the concentration of hygromycin is 70 mg / L.

[0051] Furthermore, the hygromycin described in the present invention is all hygromycin B.

[0052] The conditions for inverted culture are: inverted culture at 28 °C for 5 - 7 days until a large number of spores are visible.

[0053] Application of the PEG-mediated genetic transformation method of Penicillium sclerotiorum protoplasts in the preparation of Penicillium sclerotiorum protoplasts.

[0054] The present invention has the following advantages and effects compared with the prior art:

[0055] 1. The present invention provides a PEG-mediated genetic transformation method for the preparation and regeneration of Penicillium sclerotiorum protoplasts, aiming to solve the problems of incomplete lysis of the strain cell wall (incomplete removal of the cell wall), low protoplast transformation efficiency, and visual monitoring. The present invention realizes the PEG-mediated transformation of Penicillium sclerotiorum protoplasts, establishes a complete set of genetic transformation and screening methods, obtains Penicillium sclerotiorum protoplasts, which have a very high regeneration rate after culture, and at the same time obtains a large number of transformed strains with high-intensity expression of hygromycin resistance protein and green fluorescent protein, and all can be stably inherited.

[0056] 2. The present invention controls conditions such as the spore germination time, hyphal growth time, protoplast lysis solution, lysis time, and the addition amount of vector plasmid of Penicillium sclerotiorum, and transforms the exogenous DNA fragment containing resistance and screening tags into the Penicillium sclerotiorum genome, so as to obtain better transformation effects and transformation rates, providing a certain experimental basis for the transformation of Penicillium sclerotiorum strains and the study of plant interactions.

[0057] 3. The present invention selects a combination of multiple enzymes (Lysing Enzymes, Yatalase, cellulase, and pectinase) as the protoplast lysis solution for enzymatic hydrolysis, which can significantly increase the protoplast quantity and regeneration rate, and can effectively shorten the enzymatic hydrolysis time. Description of the Drawings

[0058] Figure 1 It is a germination process diagram of Penicillium sclerotiorum spores; among them, a, b, and c are the 14th h, 15th h, and 16th h of germination respectively.

[0059] Figure 2 Photographs of protoplasts under a microscope after enzymatic hydrolysis, showing three different fields of view.

[0060] Figure 3 Diagram of the regeneration of protoplasts on Bottom Agar medium; where a is the STC dilution group; b is the sterile water dilution group; c is the sterile water control group.

[0061] Figure 4 Diagram of the growth of Penicillium sclerotiorum on hygromycin resistance plates with different concentrations.

[0062] Figure 5 Diagram of the growth of Penicillium sclerotiorum transformants on PDA plates; where a is the growth of Penicillium sclerotiorum transformants on a 70 mg / mL hygromycin-resistant PDA plate; b is the growth of the wild-type Penicillium sclerotiorum strain on a PDA plate without hygromycin addition (positive control); c is the growth of wild-type Penicillium sclerotiorum on a 70 mg / mL hygromycin-resistant PDA plate (negative control).

[0063] Figure 6 Agarose gel electrophoresis diagram of some Penicillium sclerotiorum transformants (in the figure, M: DL5000, lane 1: negative control of the fluorescent protein GFP gene; lane 2: amplified fragment (720 bp) of some Penicillium sclerotiorum transformants labeled with fluorescent protein; lane 3: amplified fragment (146 bp) of some Penicillium sclerotiorum transformants amplified with Penicillium sclerotiorum-specific primers; lane 4: amplified fragment (1026 bp) of some Penicillium sclerotiorum transformants labeled with the hygromycin resistance gene).

[0064] Figure 7 Diagram of the morphology of wild-type and transformants of Penicillium sclerotiorum under blue light excitation in a fluorescence microscope; where a is the observation result of the dark-field transformant under blue excitation light; b is the bright-field observation result of the transformant under the same field of view and blue excitation light; c is the bright-field observation result of the transformant without blue excitation light in the same field of view; d is the dark-field observation result of the transformant without blue excitation light in the same field of view; e is the bright-field observation result of the wild-type under blue excitation light; f is the bright-field observation result of the wild-type without blue excitation light in the same field of view.

[0065] Figure 8 Statistical diagram of the number of protoplasts per milliliter of solution.

[0066] Figure 9 Diagram of the most representative protoplast morphology under different enzymatic hydrolysis solution treatment conditions.

[0067] Figure 10Diagram of hyphae at different enzymatic hydrolysis times; among them, a is the hypha situation at the start of enzymatic hydrolysis; b is the hypha situation after 2 hours of enzymatic hydrolysis; c is the hypha situation after 4 hours of enzymatic hydrolysis; d is the hypha situation after 5 hours of enzymatic hydrolysis.

[0068] Figure 11 Diagram of protoplast release at different enzymatic hydrolysis times; among them, a is the protoplast release situation at 2 hours when enzymatic hydrolysis starts; b is the protoplast release situation after 3 hours of enzymatic hydrolysis; c is the protoplast release situation after 4 hours of enzymatic hydrolysis; d is the protoplast release situation after 6 hours of enzymatic hydrolysis. Specific implementation mode

[0069] The present invention will be further described in detail below in conjunction with embodiments, but the implementation modes of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. The test methods without specific experimental conditions in the following embodiments are usually carried out according to conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained through commercial channels.

[0070] The strain in the present invention is Penicillium sclerotiorum jyscaumcx01, and its preservation number is: GDMCC No. 60249, which has been disclosed in a Chinese patent (application number: 201711279355.9, title: Penicillium sclerotiorum of Portulaca oleracea and its application in preparing drugs against Ralstonia solanacearum), and is a strain isolated, screened, identified and preserved from the rhizome of Portulaca oleracea.

[0071] The preparation methods of the main reagents and culture media involved in the embodiments of the present invention are as follows:

[0072] 1. Hypha washing solution (Osmotic medium): Weigh 295.8 g of MgSO4·7H2O, 0.46 g of Na2HPO4, and 1.06 g of NaH2PO4·2H2O respectively. First, dissolve MgSO4·7H2O with 600 mL of distilled water, then dissolve Na2HPO4 and NaH2PO4·2H2O with no more than 50 mL of distilled water respectively, and then add them to the dissolved MgSO4·7H2O solution respectively. Stir continuously during the addition process, make up the volume to 1000 mL with distilled water, and finally adjust the pH value to 5.8 with 1 M Na2HPO4 solution, and place it at room temperature for later use;

[0073] 2. STC solution (STC buffer): 1.2 M sorbitol, 50 mM Tris-HCl pH 8.0, 50 mM CaCl2;

[0074] 3. PDA medium: Purchased from Guangdong Huankai Microbial Sci-Tech Co., Ltd., containing 300 g of potato, 20 g of glucose, and 15 g of agar per liter;

[0075] 4. LB medium: Purchased from Guangdong Huankai Microbial Sci-Tech Co., Ltd., containing 10 g of tryptone, 5 g of yeast extract, and 5 g of sodium chloride per liter;

[0076] 5. Bottom Agar medium: 3 g of yeast extract, 3 g of acid-hydrolyzed casein, 200 g of sucrose, 10 g of agar, made up to 1000 ml with distilled water;

[0077] 6. TB3 medium: 3 g of yeast extract, 3 g of acid-hydrolyzed casein, 200 g of sucrose, made up to 1000 mL with distilled water.

[0078] The information of the enzymes involved in the embodiments of the present invention is as follows:

[0079] (1) Lysing Enzymes was purchased from Nanjing Dulai Biotech Co., Ltd. (lysozyme, activity: ≥200 u / mg);

[0080] (2) The enzyme Yatalase for preparing protoplasts of filamentous fungi was purchased from takara company (product number: T017; having Chitinase and Chitobiase enzyme activities, among which Chitinase activity: above 50 U / g powder; Chitobiase activity: above 500 U / g powder; cell wall lysis activity: about 10000 U / g powder);

[0081] (3) Cellulase was purchased from Feijing Biotech Co., Ltd. (PH9018 cellulase, CAS number: 9012-54-8; activity: ≥400 u / mg for protein);

[0082] (4) Pectinase was purchased from Feijing Biotech Co., Ltd. (PH1561 pectinase, CAS number: 9032-75-1; activity: ≥500 u / mg).

[0083] Example 1

[0084] A genetic transformation method for preparing and regenerating protoplasts of Penicillium sclerotiorum mediated by PEG, specifically comprising the following steps:

[0085] (1) Accumulation of fresh spores of Penicillium sclerotiorum

[0086] Inoculate the Penicillium sclerotiorum jyscaumcx01 strain preserved in the -80°C refrigerator on a PDA plate (90*15 mm), and incubate it in the dark at 28°C for about 5 - 7 days for spore production (the states at the 14th, 15th, and 16th hours of spore germination of Penicillium sclerotiorum are shown inFigure 1 )

[0087] (2) Collection of Penicillium sclerotiorum hyphae

[0088] Chop the culture medium and culture in the whole plate and pour all of them into LB medium. Collect the fresh hyphae formed after culturing on a shaker at 28 °C and 180 rpm for 14 - 15 h into a 50 mL sterile centrifuge tube. Centrifuge at 4 °C and 4000 rpm for 10 min in a refrigerated centrifuge. After collecting the hyphae, mix and shake with 3 - 5 mL of 0.2% (w / v) Tween - 80 solution (sterilized by filtration through a 0.22 μm filter membrane), let it stand for 20 - 30 min, then discard the supernatant, add an equal volume of STC solution (the STC solution needs to be pre - ice - bathed, the same below) for washing. After the washing is completed, discard the supernatant and add an equal volume of STC solution for washing once again; After collecting the hyphae and allowing them to settle naturally in the centrifuge tube, the stacked volume corresponds to the 25 mL mark on the centrifuge tube, with a total hyphal amount of 25 mL;

[0089] (3) Preparation of Penicillium sclerotiorum protoplasts

[0090] ① The preparation method of the protoplast enzyme solution is as follows: Weigh 30 mg of Lysing Enzymes, 10 mg of Yatalase, 10 mg of cellulase, and 10 mg of pectinase, and dissolve them successively in 20 mL of hyphal washing solution (Osmotic medium). After vortex - mixing, transfer it to a centrifuge tube, culture it on a shaker at 30 °C and 120 rpm for 1 h, then take it out and filter it through a 0.22 μm filter membrane for sterilization, and store it in a 4 °C refrigerator for later use.

[0091] ② Pour the fresh hyphae treated in the above step (2) (pipette 1 mL of hyphae for each group) into 10 mL of the protoplast enzyme solution, fully resuspend it, and then transfer it to a sterilized 50 mL conical flask. Then place it in a constant - temperature shaking incubator at 28 °C and 100 rpm for enzymatic hydrolysis for 6 h; Set up three replicates for the experiment;

[0092] ③ Filter the protoplasts in the conical flask through two layers of Miracloth (475855 - 1R Miracloth produced by Solarbio) into a sterilized 50 mL centrifuge tube, and centrifuge at 4 °C and 4000 - 5000 rpm in a refrigerated centrifuge for 10 min;

[0093] ④ Gently pipette the fresh protoplasts into a 50 mL sterilized centrifuge tube, and at the same time add 3 times the volume of ice - bathed STC solution to resuspend them. Place it in a refrigerated centrifuge at 4 °C and 5000 rpm for centrifugation for 10 min. After centrifugation, remove most of the supernatant, and then add an appropriate amount of ice - bathed STC solution to resuspend the protoplasts to a concentration of 2×10 7cells / mL; The protoplasts observed under the microscope after enzymolysis are as Figure 2 shown;

[0094] ⑤ Aliquot the protoplasts into 2 mL centrifuge tubes and store them at -20 °C, 200 μL in each centrifuge tube.

[0095] (4) Regeneration of Penicillium sclerotiorum protoplasts

[0096] Take 20 μL of the aliquoted protoplasts, dilute them 1500-fold with sterile water and STC solution respectively, pipette 20 μL of the diluted solution and spread it evenly on the Bottom Agar medium, and incubate it in a constant temperature incubator at 28 °C for 2 days, using sterile water treatment as a control. The experiment is set up with three replicates. Observe the growth of protoplasts on the plate ( Figure 3 ). Calculate the protoplast regeneration rate and the amount of effective protoplasts according to the formula. Among them, the calculation formula of the regeneration rate (%) is as follows:

[0097] Regeneration rate = (Number of colonies grown on the Bottom Agar medium - Number of colonies grown after sterile water treatment) / (Volume of protoplast spread × Protoplast yield / 1500) × 100%.

[0098] (5) Screening of hygromycin resistance concentration of Penicillium sclerotiorum

[0099] Inoculate the Penicillium sclerotiorum jyscaumcx01 strain on PDA plates containing hygromycin (all hygromycin used is hygromycin B, the same below) at concentrations of 0, 10, 30, 50, 70, 90, and 100 mg / L respectively, and incubate them in a constant temperature incubator at 28 °C for 7 days, and observe the growth of the strains. The experiment is set up with three replicates. The results are as Figure 4 shown. It is observed that the growth of the Penicillium sclerotiorum strain can be inhibited when the hygromycin concentration is 70 mg / mL, so it is used as the transformant screening concentration.

[0100] (6) Transformation of Penicillium sclerotiorum protoplasts

[0101] 1) Pipette 20 μg of the expression vector pCT-74 (purchased from Fenghui Biotech Co., Ltd., containing ampicillin and hygromycin resistance genes) into 200 μL of the Penicillium sclerotiorum protoplasts prepared in step (3), mix gently and place on ice for 20 minutes;

[0102] 2) Add 1.25 mL of 20% (w / v) PEG 8000 solution prepared with STC buffer as a solvent, let it stand on ice for 20 minutes, then transfer the liquid to 20 mL of sterile TB3 medium containing 50 mg / L Amp (ampicillin) antibiotic, and shake it overnight on a constant temperature shaker at 28 °C and 120 rpm;

[0103] 3) Pour the cultured protoplasts into PDA medium containing 50 mg / L ampicillin and 70 mg / L hygromycin, and culture them in a constant temperature incubator at 28°C for 7 days (invert the culture dish on the second day);

[0104] 4) After culturing for 7 days, pick the hyphae into a new PDA medium containing 50 mg / L ampicillin and 70 mg / L hygromycin and screen twice. Those that can regenerate are transformants.

[0105] Example 2

[0106] (1) DNA extraction and gel electrophoresis verification:

[0107] 1) Collect the untransformed wild-type Sclerotinia sclerotiorum jyscaumcx01 from PDA and the transformed fungal hyphae (transformants with GFP label) finally obtained in step (6) of Example 1 above for DNA extraction, and then perform PCR amplification. Among them, the amplification primers for the GFP gene are: forward 5′-gcgacgtaaacggccacaag-3′, reverse 5′-ccagcaggaccatgtgtgatcg-3′ primers; the amplification primers for the Hyg gene (the amplified fragment is 1000 bp): forward 5′-atgaaaaagcctgaactcaccgcgacgtctgtcgagaagt-3′; reverse 5′-ctattcctttgccctcggacgagtgctggggcgtcggtttc-3′. The amplification program is: 94°C for 1 min, 53°C for 2 min, 72°C for 1 - 5 min, 35 cycles, 72°C for 5 min.

[0108] 2) Perform gel electrophoresis in 1% agarose TBE, stain with ethidium bromide, and observe the bands under ultraviolet light. Among them, there are no hygromycin and GFP protein fragments in the wild-type strain, and bands are generated after amplification with the specific primers of Sclerotinia sclerotiorum, hygromycin gene primers, and GFP primers for the Sclerotinia sclerotiorum transformants, indicating successful transformation ( Figure 6 )

[0109] 3) Inoculate the hyphae of the Sclerotinia sclerotiorum transformants onto a PDA plate containing 70 mg / L hygromycin, and it is found that they can grow normally. Among them, inoculating the wild-type Sclerotinia sclerotiorum strain onto a PDA plate without hygromycin is used as a positive control, and inoculating the wild-type strain onto a PDA plate containing 70 mg / L hygromycin is used as a negative control ( Figure 5 )

[0110] (2) Microscopic examination

[0111] Hyphal filaments of the Penicillium sclerotiorum transformants and the wild-type Penicillium sclerotiorum strain were picked. Using blue light as the excitation light source under a fluorescence microscope, it was observed that the hyphal filaments of the transformants emitted green fluorescence, while the wild-type did not fluoresce, indicating successful transformation. Figure 7 )

[0112] Example 3

[0113] In this example, the genetic transformation of Penicillium sclerotiorum protoplasts was carried out by adjusting the composition and dosage of the protoplast enzymolysis solution. The specific steps are as follows:

[0114] (1) Accumulation of fresh spores of Penicillium sclerotiorum

[0115] The Penicillium sclerotiorum jyscaumcx01 strain stored in a -80°C refrigerator was inoculated on a PDA plate (90*15 mm) and cultured in the dark at 28°C for about 5 - 7 days for sporulation.

[0116] (2) Collection of Penicillium sclerotiorum hyphal filaments

[0117] The medium and culture in the whole plate were chopped and all poured into LB medium. The fresh hyphal filaments formed after culturing on a shaker at 28°C and 180 rpm for 14 - 15 h were collected in a 50 mL sterile centrifuge tube. Centrifuged at 4°C and 4000 rpm for 10 min in a refrigerated centrifuge. After collecting the hyphal filaments, they were mixed and shaken with 3 - 5 mL of 0.2% (w / v) Tween-80 solution (filtered and sterilized through a 0.22 μm filter membrane), left standing for 20 - 30 min, then the supernatant was discarded, and an equal volume of STC solution (the STC solution needs to be ice-bathed in advance, the same below) was added for washing. After the washing was completed, the supernatant was discarded, and an equal volume of STC solution was added again for washing once. After the hyphal filaments were allowed to settle naturally in the centrifuge tube, the accumulated volume corresponded to the 25 mL mark on the centrifuge tube, with a total hyphal filament amount of 25 mL.

[0118] (3) Preparation of Penicillium sclerotiorum protoplasts

[0119] ① Preparation of the protoplast enzymolysis solution: The experiment was divided into 25 groups, that is, 25 kinds of protoplast enzymolysis solutions were prepared (the total enzyme addition amount in treatment 1 was 70 mg, and the total enzyme amount in the remaining treatment groups was 60 mg). The specific grouping is shown in Table 1, and the specific preparation method is the same as step (3)① of Example 1.

[0120] Table 1

[0121]

[0122]

[0123] ② Transfer the collected sclerotial Penicillium mycelium above (pipette 1 mL of mycelium for each group) to 10 mL of protoplast enzymolysis solution for enzymolysis until the cell wall ruptures. Then transfer the mycelium and enzymolysis solution to a sterile 50 mL conical flask and enzymolyze at 28 °C and 100 rpm in a constant temperature shaking incubator for 6 h. The experiment is set up with three replicates.

[0124] ③ Filter the enzymolysis solution containing Penicillium sclerotiorum protoplasts through two layers of Magic Filter Cloth (475855-1R Miracloth produced by Solarbio). After adding three volumes of STC solution and resuspending and centrifuging once, carefully remove the supernatant with a pipette, leaving about 7 mL. Resuspend gently by pipetting up and down and take samples for storage in a -20 °C refrigerator.

[0125] (4) Protoplast counting

[0126] Count the protoplasts in the enzymolysis treatment groups under 25 different conditions. Take 50 μL of samples from each group and measure the number 3 times using the 4-point method (25*25 grids). Record the data (Table 2, Figure 8 ) and the protoplast morphology ( Figure 9 ).

[0127] Table 2

[0128]

[0129]

[0130]

[0131] (5) Protoplast regeneration rate

[0132] Select the treatment groups with the highest number of protoplasts for regeneration. Take 20 μL of samples from each group and dilute them 1500 times with STC solution and sterile water respectively. Then pipette 20 μL of the diluted solution and spread it evenly on the Bottom Agar medium three times. Incubate in a 28 °C constant temperature incubator for 2 days, with sterile water treatment as the control. Calculate the protoplast regeneration rate using the same method as in Example 1. Record the data (Table 3) and observe the growth of protoplasts on the plate.

[0133] Table 3

[0134] Processing First coating regeneration rate Second coating regeneration rate Third coating regeneration rate Average regeneration rate Treatment 1 10.53％ 10.53％ 44.22％ 21.76％ Treatment 2 16.38％ 85.99％ 58.70％ 53.69％ Treatment 3 30.91％ 65.47％ 63.65％ 53.43％ Treatment 5 13.48％ 8.98％ 0 4.49％ Treatment 15 40.74％ 92.59％ 62.96％ 65.43％ Treatment 20 46.23％ 57.79％ 0 34.67％

[0135] (6) Effects of different enzymolysis times on Penicillium sclerotiorum mycelium

[0136] In the above treatment 2, at the start of enzymolysis and at the enzymolysis time points of 2 h, 4 h, and 5 h, use a 20-fold microscope to check the mycelium condition; and observe the protoplast release at the enzymolysis time points of 2 h, 3 h, 4 h, and 6 h.

[0137] The hypha condition is as follows Figure 10 shown. After 2 hours of enzymatic hydrolysis, the hyphae began to collapse and expose the interior. After 4 hours of enzymatic hydrolysis, the outer wall of the hyphae became transparent and protoplasts could be seen. After 5 hours of enzymatic hydrolysis, the obvious overlap of hyphae decreased and became smaller fragments. The release of protoplasts is as follows Figure 11 shown. After 2 hours of enzymatic hydrolysis, protoplasts could be observed. After 3 hours of enzymatic hydrolysis, a small amount of protoplasts could be seen. After 4 hours of enzymatic hydrolysis, more protoplasts could be seen. After 6 hours of enzymatic hydrolysis, a large number of protoplasts could be seen, and at the same time, a small number of protoplasts were broken.

[0138] In summary, according to the above results, it can be seen that the types and dosages of enzymes used in the protoplast enzymatic hydrolysis solution directly affect the number of protoplasts and the regeneration rate of protoplasts. Among them, Treatment 2 (containing 4 enzymes: Lysing Enzymes 30 mg, Yatalase 10 mg, cellulase 10 mg, pectinase 10 mg) has the best effect (more protoplasts, higher regeneration rate, and shorter enzymatic hydrolysis time). However, the number of protoplasts and the regeneration rate of only using two enzymes (Treatments 16, 18, 21) or single enzymatic hydrolysis (Treatments 24, 25) are significantly lower than those of the present invention. In addition, Yatalase is the main contributor to enzymatic hydrolysis, but the proportion should not be too much; Lysing Enzymes is the second contributor to enzymatic hydrolysis, which can make up for the enzymatic hydrolysis effect of less Yatalase; cellulase mainly plays a role in destroying the hypha structure, enabling Yatalase and Lysing Enzymes to hydrolyze better and improving the enzymatic hydrolysis efficiency; pectinase itself has no effect on hydrolyzing the hypha cell wall and mainly plays a role in removing the peripheral obstacles and improving the accessibility of other enzyme substrates.

[0139] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A PEG-mediated genetic transformation method for protoplasts of Penicillium sclerotiorum strains, characterized in that, It includes the following steps: (1) Collect Penicillium sclerotiorum hyphae Inoculate Penicillium sclerotiorum on a plate for spore production, then pour the spore-producing Penicillium sclerotiorum culture into a medium for hyphal germination, and then collect and wash the fresh Penicillium sclerotiorum hyphae; (2) Prepare Penicillium sclerotiorum protoplasts ① Prepare the protoplast enzymatic hydrolysis solution: The formula of the protoplast enzymatic hydrolysis solution is as follows: Lysing Enzymes 0 - 30 mg, Yatalase 5 - 20 mg, cellulase 0 - 30 mg, pectinase 5 - 20 mg, hyphal washing solution 20 mL; among them, the total amount of enzyme added is controlled to be 60 - 70 mg; ② Enzymatic hydrolysis: Add the prepared protoplast enzymatic hydrolysis solution to the Penicillium sclerotiorum hyphae collected in step (1) for enzymatic hydrolysis until the cell wall ruptures and protoplasts are released, then filter with a filter cloth, centrifuge and wash with STC solution, and then resuspend with STC solution to obtain Penicillium sclerotiorum protoplasts; (3) Transformation of Penicillium sclerotiorum protoplasts Add the plasmid to be transformed to the Penicillium sclerotiorum protoplasts obtained in step (2), then add PEG-mediated transformation, and after antibiotic screening, obtain Penicillium sclerotiorum protoplast transformants.

2. The method according to claim 1, wherein: The Penicillium sclerotiorum described in step (1) is Penicillium sclerotiorum jyscaumcx01, and its preservation number is: GDMCC No. 60249.

3. The method according to claim 1, wherein: The formula of the protoplast enzymatic hydrolysis solution described in step (2) is: Lysing Enzymes 10 - 30 mg, Yatalase 5 - 20 mg, cellulase 10 - 30 mg, pectinase 5 - 20 mg, hyphal washing solution 20 mL; among them, the total amount of enzyme added is controlled to be 60 - 70 mg; The formula of the hyphal washing solution described in step (2) is as follows: MgSO4·7H2O 295.8 g, Na2HPO4 0.46 g, NaH2PO4·2H2O 1.06 g, make up the volume to 1000 mL with distilled water, pH 5.8; The conditions for enzymatic hydrolysis described in step (2) are: 28 ± 1 °C, 100 - 150 rpm for enzymatic hydrolysis for 4 - 7 h; The volume ratio of the Penicillium sclerotiorum hyphae to the protoplast enzymatic hydrolysis solution described in step (2) is 1:10 - 15.

4. The method according to claim 3, wherein: The formula of the protoplast enzymatic hydrolysis solution described in step (2) is: Lysing Enzymes 15 - 30 mg, Yatalase 5 - 10 mg, cellulase 10 - 20 mg, pectinase 10 - 20 mg, hyphal washing solution 20 mL; among them, the total amount of enzyme added is 60 mg; The conditions for enzymatic hydrolysis described in step (2) are: 28 °C, 100 rpm for enzymatic hydrolysis for 6 h; The volume ratio of the Penicillium sclerotiorum hyphae to the protoplast enzymatic hydrolysis solution described in step (2) is 1:

10.

5. The method according to claim 1, wherein: The filter cloth described in step (2) is a magic filter cloth; The formulation of the STC solution described in step (2) is as follows: 1.2 M sorbitol, 50 mM Tris-HCl pH 8.0, 50 mM CaCl2.

6. The method according to claim 1, wherein: After step (2) and before step (3), there is also a step of regenerating Penicillium sclerotiorum protoplasts; specifically: the Penicillium sclerotiorum protoplasts obtained in step (2) are diluted with the STC solution, spread on the Bottom Agar medium, and cultured in an incubator at a constant temperature of 28 ± 1 °C for 2 - 3 days.

7. The method according to claim 1, wherein: The transformation of the Penicillium sclerotiorum protoplasts described in step (3) is achieved through the following steps: Add the plasmid to be transformed into the Penicillium sclerotiorum protoplasts obtained in step (2), gently mix and place on ice for 20 ± 1 minute; then add the PEG solution, let it stand on ice for 20 ± 1 minute, after standing, transfer the mixed system to the TB3 medium containing ampicillin and culture for 12 - 14 h, then pour the cultured system into the PDA plate containing ampicillin and hygromycin and incubate it upside down until fresh mycelia are formed, and finally pick the mycelia into the PDA medium containing ampicillin and hygromycin and screen more than twice to obtain protoplast transformants; The concentration of ampicillin in the TB3 medium containing ampicillin is 50 mg / L; The concentration of ampicillin in the PDA medium containing ampicillin and hygromycin is 50 mg / L, and the concentration of hygromycin is 70 mg / L; The PEG solution described in step (3) is a PEG 8000 solution; The addition amount of the PEG solution described in step (3) is calculated according to its final concentration in the system being 20% by mass percentage.

8. The method according to claim 1, wherein: In step (1), collecting and washing fresh Penicillium sclerotiorum mycelia is achieved through the following steps: the fresh mycelia formed after culture are collected by centrifugation, then mixed and shaken with a 0.2% (mass fraction) Tween-80 solution and left standing for 20 - 30 min, then discard the supernatant, add an equal volume of STC solution for washing, after washing, discard the supernatant, and add an equal volume of STC solution for washing again to obtain Penicillium sclerotiorum mycelia; The protoplast enzymolysis solution described in step (2) is prepared by the following method: add Lysing Enzymes, Yatalase, cellulase and pectinase to the mycelia washing solution, mix well and place it in a shaker for culture, and then filter and sterilize it with a filter membrane to obtain it; The conditions for the shaker culture are: culture in a shaker at 28 - 30 °C and 100 - 150 rpm for 0.5 - 1.5 h; The filter membrane is a 0.22 μm filter membrane; The preparation method of the mycelium washing solution described in step (2) is as follows: First, dissolve 295.8 g of MgSO4·7H2O in 600 mL of distilled water to obtain solution I; then dissolve 0.46 g of Na2HPO4 and 1.06 g of NaH2PO4·2H2O in no more than 50 mL of distilled water respectively to obtain solution II and solution III; then add solution II and solution III to solution I respectively, stir continuously during the addition process, and add distilled water to make up the volume to 1000 mL. Finally, adjust the pH value to 5.8 with Na2HPO4 solution to obtain the solution.

9. The method according to claim 1, wherein: The plate described in step (1) is a PDA plate; The culture medium described in step (1) is an LB culture medium; In step (1), the conditions for mycelium germination are: culturing in a shaker at 28 °C and 180 rpm for 14 - 15 h; The conditions for centrifugation described in step (2) are: centrifuging at 4 °C and 4000 - 5000 rpm for 10 min.

10. Use of the PEG-mediated genetic transformation method of Penicillium sclerotiorum protoplasts according to any one of claims 1 - 9 in the preparation of Penicillium sclerotiorum protoplasts.

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