A method for preparing protoplasts of serratia

Abstract

The present application relates to the technical field of protoplast preparation, and particularly relates to a preparation method of protoplast of selenomonas ruminantium; the method comprises the following steps: using a protoplast enzymolysis solution to enzymolyze selenomonas ruminantium; filtering by using a Miracloth filter membrane, placing a container for collecting filtrate on ice, and flushing by using a pre-cooled 0.6 M sorbitol solution; centrifuging the collected filtrate, collecting a precipitate, and cleaning the precipitate by using a pre-cooled 0.6 M sorbitol solution to remove the enzymolysis solution; and then resuspending the precipitate by using STC, centrifuging to collect the precipitate, and obtaining the protoplast; the present application establishes a corresponding enzymolysis system according to the characteristics of selenomonas ruminantium, the enzymolysis condition is mild and easy to control, the obtained protoplast is complete, stable, high in yield, and the quantity is as high as 1x10 8 / mL.

Description

Technical Field

[0001] This invention relates to the field of protoplast preparation technology, and specifically to a method for preparing protoplasts of uniform sclerotium. Background Technology

[0002] *Sclerotium sclerotiorum* is a fungus belonging to the class Agaricales, order Polyporaceae, and is a deuteromycete. It reproduces by producing sclerotia, typically consisting of hyphae and sclerotia (asexual state). In its sexual state, it produces sparse, white, smooth basidiosperms (fruiting bodies). Industrially, *Sclerotium sclerotiorum* is mainly used for the fermentation production of stearoglucan, which possesses various excellent physicochemical properties. *Sclerotium sclerotiorum* also produces lectins with anticancer activity, inhibiting the proliferation of human colon cancer, breast cancer, and ovarian cancer cells. Therefore, *Sclerotium sclerotiorum* is an industrial strain with significant development potential. Furthermore, its low cultivation cost, rapid growth, and ease of preservation and subculturing have enabled engineered fermentation, making it a promising candidate as a substrate cell for basidiomycete synthetic biology. Although research on stearoglucan has been conducted for over 40 years, effective transformation methods and genetic manipulation tools for *Sclerotium sclerotiorum* still require improvement due to its non-model filamentous nature. Choosing a suitable genetic transformation method for *Sclerotium sclerotiorum* remains a challenge in fungal genetic manipulation.

[0003] Currently, there is limited research in this field on the isolation and transformation of uniform sclerotium protoplasts, and there is no efficient method for preparing protoplasts. Methods for preparing uniform sclerotium protoplasts have drawbacks such as low isolation efficiency, difficulty in destroying and decomposing the cell wall, and low genetic transformation efficiency. Summary of the Invention

[0004] To address the shortcomings of the existing technology, this invention aims to provide a method for preparing protoplasts of *Sclerotium sclerotiorum*, thereby improving the extraction efficiency and quality of *Sclerotium sclerotiorum* protoplasts.

[0005] To solve the above problems, the present invention adopts the following technical solution: A method for preparing protoplasts of *Sclerotium uniflorum* involves enzymatically hydrolyzing *Sclerotium uniflorum* with a protoplast hydrolysate; filtering the solution using a Miracloth membrane; placing the container for collecting the filtrate on ice and rinsing it with a pre-cooled 0.6 M sorbitol solution; centrifuging the collected filtrate, collecting the precipitate, washing the precipitate with a pre-cooled 0.6 M sorbitol solution to remove the hydrolysate, and then resuspending the precipitate using STC centrifugation to collect the protoplasts.

[0006] Furthermore, the neatly arranged sclerotium was cultured using YMA medium.

[0007] Furthermore, the protoplast enzymatic hydrolysate includes 0.25 w / t% snail enzyme, cellulase, dissociation enzyme, and wall-lysing enzyme; the solution of the protoplast enzymatic hydrolysate is a 0.6 M sorbitol solution.

[0008] Furthermore, the ratio of the mass of the uniform sclerotium to the volume of the protoplast hydrolysate is 1:20.

[0009] Furthermore, the enzymatic hydrolysis conditions are 30°C and incubation in the dark with shaking for 3 hours.

[0010] Furthermore, the centrifugation conditions are 4℃, 3200 rpm / min, a rise / fall coefficient of 6, and centrifugation for 5 min.

[0011] Furthermore, the process includes adding 100 μL of protoplasts to a mixed solution of 5 μg pBARGPE1-mCherry plasmid, 50 μL PSTC, 5 μL 50 mM spermidine, and 5 μL 5 mg / mL heparin, and incubating on ice for 30 min; adding 1 mL SPTC buffer and incubating at room temperature for 20 min; transferring to a 50 mL centrifuge tube and adding 10-20 mL of TB3 medium, and incubating with shaking at 25 °C and 100 rpm for 12 h; concentrating the culture medium into a 1.5 mL EP tube, spreading it onto a YMA plate containing 0.15% basta, and incubating for 4-7 days; and transferring the transformants to YMA medium containing 0.15% basta for screening.

[0012] The beneficial effects of this invention are as follows: (1) The present invention uses mycelium on a plate as protoplast raw material, eliminating the need for further culture of mycelium in a liquid shake flask, which greatly shortens the protoplast preparation cycle.

[0013] (2) Considering that during the liquid culture of Sclerotium sclerotiorum, high-viscosity metabolites are produced, making it difficult to separate hyphae from viscous metabolites, this invention uses hyphae in solid plates as the material for protoplast preparation, which is convenient to obtain and greatly improves the operability of the protoplast preparation process.

[0014] (3) High yield: This invention establishes a corresponding enzymatic hydrolysis system tailored to the characteristics of uniform sclerotium. The enzymatic hydrolysis conditions are mild and easy to control, resulting in protoplasts with intact and stable morphology and high yield, reaching up to 1×10⁻⁶. 8 per mL. Attached Figure Description

[0015] Figure 1 This is a fluorescence image of the mycelium.

[0016] Figure 2The graph shows the effect of different enzyme types on protoplast release.

[0017] Figure 3 The graph shows the effect of different permeation stabilizers on protoplast release.

[0018] Figure 4 The graph shows the effect of different enzymatic hydrolysis times on the amount of protoplasts released. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to specific embodiments.

[0020] It should be noted that these embodiments are only used to illustrate the present invention and are not intended to limit the present invention. Simple improvements to the method under the premise of the present invention are all within the scope of protection claimed by the present invention.

[0021] Example 1 This invention provides a method for preparing protoplasts of *Sclerotium uniflorum*: *Sclerotium uniflorum* is enzymatically hydrolyzed using a protoplast hydrolysate; the solution is filtered using a Miracloth membrane, and the container for collecting the filtrate is placed on ice and rinsed with a 0.6 M sorbitol solution pre-cooled to 4 degrees Celsius; the collected filtrate is centrifuged, the precipitate is collected, and the precipitate is washed with a 0.6 M sorbitol solution pre-cooled to 4 degrees Celsius to remove the hydrolysate; the precipitate is then collected by STC resuspension centrifugation to obtain protoplasts.

[0022] Sclerotium moniliforme was cultured on YMA medium: holes were punched in the plates with well-grown Sclerotium moniliforme mycelia using the perforation method, and a 1 cm² piece of mycelia was inoculated into a new YMA solid culture dish and cultured for 3 days.

[0023] The protoplast hydrolysate includes 0.25 w / t% snail enzyme, cellulase, dissociation enzyme, and wall-lysing enzyme; the solution of the protoplast hydrolysate is a 0.6 M sorbitol solution.

[0024] Preparation of protoplast enzymatic hydrolysate: Weigh 50 mg of lysozyme, 50 mg of snailase, 50 mg of cellulase, and 50 mg of dissociative enzyme into a 50 mL centrifuge tube. Dissolve thoroughly in 20 mL of 0.6 M sorbitol solution. Filter through a 0.22 μm aqueous filter to obtain the protoplast enzymatic hydrolysate. Weigh 1 g of mycelia from the plate into the enzymatic hydrolysate.

[0025] Enzymatic hydrolysis: Incubate at 30℃ in the dark with shaking for 3 hours. The digestion effect can be observed under a microscope every hour.

[0026] Filtration was performed using a Miracloth membrane: The container for collecting the filtrate was placed on ice. Care should be taken in the following steps, as protoplasts are fragile and easily ruptured. After enzymatic digestion, the hydrolysate was filtered through a single layer of Miracloth membrane, and rinsed 2-3 times with a small amount of chilled 0.6 M sorbitol solution. All filtrate was then collected into 50 mL centrifuge tubes.

[0027] Washing: Centrifuge at 3200 rpm / min at 4℃, with a scaling factor of 6, for 5 min. Carefully discard the supernatant and collect the precipitate. Gently wash the protoplasts with 5 mL of pre-chilled 0.6 M sorbitol osmotic stabilizer (resuspend with a cut pipette tip), centrifuge at 3200 rpm at 4℃, with a scaling factor of 6, for 5 min. Carefully discard the supernatant and collect the precipitate. (6) Repeat step (5) again to remove residual enzymatic hydrolysate. (7) Place the protoplasts on ice and check their condition with a Zeiss Axio Observer. Calculate the protoplast concentration using a hemocytometer. (8) Subsequently, resuspend the protoplasts with pre-chilled STC, centrifuge at 3200 rpm / min at 4℃ for 5 min, and slowly pour the test tube, discarding the supernatant.

[0028] PEG-mediated protoplast transformation: (1) Take 100 μL of protoplasts, add 5 μg of plasmid (pBARGPE1-mCherry), 50 μL of pre-chilled PSTC, 5 μL of spermidine (50 mM), and 5 μL of heparin (5 mg / mL), then gently mix and incubate on ice for 30 min. (2) Gently add 1 mL of SPTC buffer and incubate at room temperature for 20 min. (3) Transfer the transformation mixture to a 50 mL centrifuge tube and add 10-20 mL of TB3 medium. (4) Incubate at 25 °C and 100 rpm for 12 h with shaking. (5) Concentrate the culture medium into 1.5 mL EP tubes, spread it onto YMA plates (containing 0.15% basta) containing the corresponding resistance, and incubate for 5-7 days to observe the growth. (6) Transfer the transformants to YMA medium (containing 0.15% basta) containing the corresponding resistance, and repeat the selection at least three times to screen for resistant transformants. (7) Extract mycelial DNA for PCR verification and observe the fluorescence expression of mycelia.

[0029] Example 1 Culture of neatly grown Sclerotium sclerotiorum: Puncture holes in the plate with neatly grown Sclerotium sclerotiorum mycelium using the punching method, take a 1 cm2 piece of mycelium and inoculate it into a new YMA solid culture dish, and culture for 3 days.

[0030] Protoplast preparation: (1) Preparation of enzymatic hydrolysate: Weigh 50 mg of lysozyme, 50 mg of snail enzyme, 50 mg of cellulase, and 50 mg of dissociative enzyme into a 50 mL centrifuge tube, add 20 mL of 0.6 M sorbitol solution, dissolve thoroughly, and filter through a 0.22 μm aqueous filter to obtain protoplast enzymatic hydrolysate. (2) Enzymatic hydrolysis: Weigh 1 g of mycelium from the plate into the enzymatic hydrolysate in (1), and culture at 30℃ in the dark with shaking for 3 h. The digestion effect can be checked under a microscope every 1 h. (3) Place the filtrate on ice. Be as careful as possible in the following steps, because protoplasts are relatively fragile and easily break. After digestion, pour the above enzymatic hydrolysate into a Miracloth filter membrane and filter, and add a small amount of cooled 0.6 M sorbitol solution to rinse 2-3 times. Collect all the filtrate into a 50 mL centrifuge tube. (4) Centrifuge at 3200 rpm / min at 4℃ for 5 min with a scaling factor of 6. Carefully discard the supernatant and collect the precipitate. (5) Gently wash the protoplasts with 5 mL of pre-cooled 0.6 M sorbitol osmotic stabilizer (resuspend with a cut pipette tip), centrifuge at 3200 rpm at 4℃ for 5 min with a scaling factor of 6. Carefully discard the supernatant and collect the precipitate. (6) Repeat step (5) again to remove residual enzymatic hydrolysate. (7) Place the protoplast solution on ice and check the state of the protoplasts with a Zeiss Axio Observer. Calculate the protoplast concentration using a hemocytometer. (8) Subsequently, resuspend the protoplasts with pre-cooled STC, centrifuge at 3200 rpm / min at 4℃ for 5 min, and slowly pour the test tube, discarding the supernatant.

[0031] PEG-mediated protoplast transformation: (1) Take 100 μL of protoplasts and add 5 μg of plasmid (pBARGPE1-mCherry), 50 μL of pre-chilled PSTC, 5 μL of spermidine (50 mM stock solution), and 5 μL of heparin (5 mg / mL). Mix gently and incubate on ice for 30 min. (2) Gently add 1 mL of SPTC buffer and incubate at room temperature for 20 min. (3) Transfer the transformation mixture to a 50 mL centrifuge tube and add 10-20 mL of TB3 medium. (4) Incubate at 25℃ and 100 rpm for 12 h with shaking. (5) Concentrate the culture medium into 1.5 mL EP tubes and spread it onto YMA plates (containing 0.15% basta) containing the corresponding resistance. Incubate for 5-7 days and observe the growth. (6) Transfer the transformants to YMA medium (containing 0.15% basta) containing the corresponding resistance. Repeat the selection at least three times. (7) Extract mycelial DNA for PCR verification, and observe the fluorescence expression of mycelia (see Figure 1 ).

[0032] Example 2 Modify the mycelial culture conditions of Example 1: Cut the YMA medium containing *Sclerotium sclerotiorum* that had grown for 3 days into mycelial blocks (approximately 1 mm × 1 mm). Collect the mycelial blocks into a 250 mL Erlenmeyer flask containing 100 mL of YMB medium and incubate at 30°C and 200 rpm for 48 hours. Use forceps to separate the fresh mycelia from the mycelial blocks and collect them into 50 mL centrifuge tubes. Resuspend the mycelia in 20 mL of 0.6 M sorbitol osmotic stabilizer, centrifuge for 5 minutes, and then discard the supernatant.

[0033] The mycelia obtained in this process contain a large amount of the sticky metabolite sclerotinia, which is difficult to remove and affects the enzymatic digestion efficiency of *Sclerotium tumefaciens*, reducing the amount of protoplasts released. Therefore, the preferred mycelial culture condition is cultivation in YMA culture dishes.

[0034] Example 3 Modify the formulation of the lyase in Example 1: Replace the lyase with 1% snail enzyme, 1% cellulase, 1% lyase, 1% lyase, 0.5% snail enzyme and 0.5% cellulase, 0.5% snail enzyme and 0.5% lyase, 0.5% cellulase and 0.5% lyase, or 0.33% snail enzyme + 0.33% cellulase + 0.33% lyase.

[0035] The remaining steps were performed using the same method as in Example 1.

[0036] See Figure 2 It can be seen that the optimal formulation is 0.25% snail enzyme + 0.25% cellulase + 0.25% dissociation enzyme + 0.25% wall-lysing enzyme. Under these conditions, the protoplasts are round, plump, regular, and uniform in shape, and have a high release rate.

[0037] Example 4 Modify the osmotic stabilizer culture in Example 1: Replace the osmotic stabilizer with 0.6 M NaCl, 0.6 M sucrose, 0.6 M KCl, 0.6 M MgSO4, and 0.6 M mannitol.

[0038] The remaining steps were performed using the same method as in Example 1.

[0039] See Figure 3 It can be seen that 0.6 M sorbitol is preferred as a penetration stabilizer.

[0040] Example 5 Change the enzymatic hydrolysis time in Example 1: Set the enzymatic hydrolysis time to 0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, 3.5 h and 4 h.

[0041] The remaining steps were performed using the same method as in Example 1.

[0042] See Figure 4 As can be seen, enzymatic hydrolysis for 3 hours is preferred.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described with reference to preferred embodiments, those skilled in the art should understand that various changes in form and detail can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A method for preparing protoplasts of uniform sclerotium, characterized in that, Protoplasts were enzymatically hydrolyzed using a protoplast hydrolysate. The solution was filtered through a Miracloth membrane, and the container collecting the filtrate was placed on ice and rinsed with pre-cooled 0.6 M sorbitol solution. The collected filtrate was centrifuged, the precipitate was collected, and the precipitate was washed with pre-cooled 0.6 M sorbitol solution to remove the hydrolysate. The precipitate was then resuspended using STC, centrifuged again, and the protoplasts were collected. The protoplast enzymatic hydrolysate comprises 0.25 w / t% snail enzyme, cellulase, dissociation enzyme, and wall-lysing enzyme; the solvent of the protoplast enzymatic hydrolysate is a 0.6 M sorbitol solution.

2. The preparation method according to claim 1, characterized in that, The uniform sclerotium was cultured using YMA medium.

3. The preparation method according to claim 1, characterized in that, The ratio of the mass of the uniform sclerotium to the volume of the protoplast hydrolysate is 1:

20.

4. The preparation method according to claim 1, characterized in that, The enzymatic hydrolysis conditions were 30°C and incubation in the dark with shaking for 3 hours.

5. The preparation method according to claim 1, characterized in that, The centrifugation conditions were 4℃, 3200 rpm / min, a rise / fall coefficient of 6, and centrifugation for 5 min.

6. The preparation method according to claim 1, characterized in that, The process also includes adding 100 μL of protoplasts to a mixed solution of 5 μg pBARGPE1-mCherry plasmid, 50 μL PSTC, 5 μL 50 mM spermidine, and 5 μL 5 mg / mL heparin, and incubating on ice for 30 min; adding 1 mL SPTC buffer and incubating at room temperature for 20 min; transferring to a 50 mL centrifuge tube and adding 10-20 mL TB3 medium, and incubating with shaking at 25 °C and 100 rpm for 12 h; concentrating the culture medium into a 1.5 mL EP tube, spreading it onto a YMA plate containing 0.15% basta, and incubating for 4-7 days; and transferring the transformants to YMA medium containing 0.15% basta for screening.

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