A method for preparing and regenerating protoplasts of Tremella fuciformis spores

Abstract

This invention discloses an efficient method for preparing and regenerating *Tremella fuciformis* spore protoplasts. The method includes steps such as *Tremella fuciformis* spore activation culture, seed culture, protoplast preparation, and protoplast regeneration. This invention utilizes a 30 mg / mL composite enzymatic hydrolysate (a 4:1 mass ratio of lysozyme to lysozyme, 0.6 mol / L potassium chloride as the osmotic stabilizer, and pH 5) to prepare a bacterial cell concentration of 3.6 × 10⁻⁶ cells / mL. 8 The cell walls of *Tremella fuciformis* spores at a density of 1 spore / mL were enzymatically hydrolyzed at 26°C and then regenerated on a regeneration medium containing 0.8 mol / L sucrose and a growth promoter (20 mg / L VVB1 + 500 mg / L bovine serum albumin). The number of protoplasts prepared using this method can reach 1.14 × 10⁻⁶. 8 The regeneration rate reached 43.52%, with a yield of [number] spores / mL. This invention provides a highly efficient and stable method for the preparation and regeneration of *Tremella fuciformis* spore protoplasts. The method is simple to operate, highly reproducible, and can provide high-quality protoplasts for subsequent genetic transformation of *Tremella fuciformis* spores.

Description

Technical Field

[0001] This invention belongs to the field of fungal protoplast preparation and regeneration technology, specifically relating to a method for preparing and regenerating Tremella fuciformis spore protoplasts. Background Technology

[0002] Tremella fuciformis, a fungus belonging to the phylum Basidiomycota, order Tremellales, and family Tremellaceae, is a traditional Chinese medicinal and edible fungus. It is rich in polysaccharides, proteins, amino acids, and other active ingredients, possessing high nutritional and medicinal value. Tremella polysaccharides, the main active ingredient in Tremella fuciformis, have been shown to possess antioxidant, immunomodulatory, hypoglycemic, and antitumor activities. Deep fermentation of Tremella fuciformis spores and protoplast-based genetic breeding are currently popular research areas; however, research using molecular biology techniques is still in its early stages.

[0003] Efficient protoplast preparation technology is a crucial foundation for establishing the molecular genetic system of edible fungi. The preparation and regeneration of protoplasts play a vital role in genetic transformation, cell fusion, trait improvement, and gene recombination. However, due to the extremely complex composition and considerable thickness of the cell wall of *Tremella fuciformis* spores, using a single type of enzyme often results in poor enzymatic hydrolysis, long hydrolysis times, low protoplast viability, and low protoplast formation and regeneration rates. Combining multiple functional degrading enzymes can improve the enzymatic hydrolysis efficiency of *Tremella fuciformis* spore cell walls, shorten protoplast preparation time, and increase the regeneration rate of *Tremella fuciformis* spore protoplasts. Therefore, this invention aims to develop an easy-to-operate, time-efficient, and highly effective method for preparing *Tremella fuciformis* spore protoplasts, providing technical support for future theoretical research at the molecular level. Summary of the Invention

[0004] The technical problem to be solved by the present invention is that the existing methods for preparing Tremella fuciformis protoplasts are inefficient and have a low regeneration rate due to the thick cell walls of the spores themselves. The present invention provides a method for preparing and regenerating Tremella fuciformis protoplasts.

[0005] To address the aforementioned technical problems, this invention targets the glycosidic bond composition of the polysaccharide in the cell wall of *Tremella fuciformis* spores. It employs a complex enzyme system to treat *Tremella fuciformis* spores, then separates protoplasts from the enzymatic hydrolysate. Finally, by optimizing regeneration culture conditions, the preparation rate and regeneration rate are significantly improved. The specific technical solution adopted is as follows:

[0006] A method for preparing and regenerating spore protoplasts of Tremella fuciformis includes the following steps:

[0007] (1) Activation culture of Tremella fuciformis spores: Tremella fuciformis spore colonies were picked and activated on PDA solid medium to obtain activated Tremella fuciformis spores;

[0008] (2) Tremella fuciformis spore seed culture: The activated Tremella fuciformis spores in step (1) are inoculated into the seed culture medium for fermentation culture to obtain Tremella fuciformis spore suspension. The Tremella fuciformis spore suspension is centrifuged to obtain the precipitate, which is the Tremella fuciformis spore mycelium.

[0009] (3) Preparation of enzymatic hydrolysate of tremella spore protoplasts: The tremella spores obtained in step (2) were washed with a osmotic stabilizer, centrifuged to remove the supernatant, and then resuspended in a compound enzymatic hydrolysate for enzymatic hydrolysis to prepare tremella spore protoplast enzymatic hydrolysate.

[0010] (4) Regeneration of tremella spore protoplasts: Centrifuge the enzymatic hydrolysate of tremella spore protoplasts obtained in step (3) to remove the supernatant and separate the protoplasts. Wash and dilute with a osmotic stabilizer to prepare a protoplast suspension. Spread the protoplast suspension on a regeneration medium and culture to form regenerated colonies.

[0011] The spores mentioned include, but are not limited to, spores scattered from the fruiting bodies of Tremella fuciformis, spores held by fungal culture preservation institutions and profit-making institutions of companies, spores used in laboratories, and spore transformants obtained by molecular biology methods. All fungal cells that fall within the category of spores in the prior art are applicable to this invention.

[0012] In some embodiments of the present invention, the tremella spores are yeast-like tremella spores. This strain was isolated from the fruiting body of tremella in Gutian, Fujian on March 21, 2021, and was deposited at the China General Microbiological Culture Collection Center on January 14, 2022. The strain number is S-41, the classification name is Tremella fuciformis, the accession number is CGMCC No.40060, and the deposit address is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0013] In step (1), the activation culture is carried out under the following conditions: 24-30℃ for 5-8 days.

[0014] In step (2), the fermentation culture is carried out under the following conditions: 20-32℃, 150-200rpm, for 3-5 days.

[0015] In step (2), the seed culture medium has the following formula: glucose 5-30 g / L, yeast powder 1-5 g / L, peptone 1-5 g / L, magnesium sulfate 0.1-2 g / L, potassium dihydrogen phosphate 1-10 g / L, dipotassium hydrogen phosphate 0.1-2 g / L, and water as the solvent.

[0016] In step (3) or step (4), the osmotic stabilizer is a 0.4-1.2 mol / L potassium chloride solution prepared with 0.5-1.0 mol / L sodium phosphate buffer as solvent, with pH = 5-8.

[0017] Preferably, the osmotic stabilizer is a 0.4–1.2 mol / L potassium chloride solution prepared with 1 mol / L sodium phosphate buffer as solvent, with a pH of 5–8.

[0018] Specifically, the 1 mol / L sodium phosphate buffer solution is formulated as follows: Weigh 136.1 g of sodium dihydrogen phosphate and 176.2 g of disodium hydrogen phosphate, dissolve them separately in ultrapure water, and bring the volume to 1 L. Mix 1 mol / L sodium dihydrogen phosphate and 1 mol / L disodium hydrogen phosphate in the following proportions to prepare 1 mol / L sodium phosphate buffer solutions with pH values ​​of 5 (1.0:99.0), 6 (12.0:88.0), 7 (57.7:42.3), and 8 (93.2:6.8) for later use.

[0019] In step (3), the resuspension involves a resuspension of 10-1 spores. 8 ~10 10 spores / mL.

[0020] In step (3), the composite enzymatic hydrolysate includes a wall-lysing enzyme, a collapse enzyme, and an osmotic stabilizer, and is prepared as follows: the wall-lysing enzyme and the collapse enzyme are weighed into the osmotic stabilizer at a mass ratio of 1:1 to 9:1, dissolved, and then sterilized by passing through a 0.22 μm aqueous membrane for later use. Preferably, the mass ratio of the wall-lysing enzyme to the collapse enzyme is 4:1.

[0021] Specifically, the total enzyme concentration of the compound enzymatic hydrolysate is 10-30 mg / mL, and the pH is 5-8; preferably, the total enzyme concentration is 30 mg / mL, and the pH is 5.

[0022] In step (3), the enzymatic hydrolysis is carried out under the following conditions: 26-35℃, 200rpm for 2-8h, with the preferred conditions being 26℃, 200rpm for 4h.

[0023] In step (4), the regeneration culture medium has the following formula: maltose 5-20 g / L, glucose 1-10 g / L, yeast powder 1-5 g / L, peptone 1-5 g / L, KH2PO4 1-10 g / L, magnesium sulfate 0.1-2 g / L, agar 15-20 g / L, regeneration stabilizing agent 0.4-1.2 mol / L, growth promoter 10-1000 mg / L, and water as the solvent.

[0024] Specifically, the regeneration stabilizer is any one of potassium chloride, sucrose, mannitol, and sorbitol, with sucrose being preferred.

[0025] Specifically, the growth promoter is any one or a combination of two of 10-50 mg / L VB1 and 100-1000 mg / L bovine serum albumin, preferably 10-50 mg / L VB1 + 100-1000 mg / L bovine serum albumin.

[0026] In step (4), the culture conditions are: inverted culture at 20-32℃ for 10-15 days. The preferred culture conditions are: inverted culture at 26℃ for 10-15 days.

[0027] In some embodiments of the present invention, the number of protoplasts prepared using the above method can reach 1.14 × 10⁻⁶. 8 The regeneration rate reached 43.52% with a density of cells / mL.

[0028] Beneficial effects: This invention targets the glycosidic bond composition of polysaccharides in the cell wall of Tremella fuciformis spores, uses a complex enzyme system to treat Tremella fuciformis spores, and optimizes the preparation and regeneration reaction conditions, thereby obtaining Tremella fuciformis spore protoplasts with high preparation and regeneration rates. This method can be widely applied to the mononuclear preparation, genetic breeding, and variety improvement of Tremella fuciformis, promoting the development of Tremella fuciformis spores in molecular biology. Attached Figure Description

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0030] Figure 1 Morphology of untreated Tremella fuciformis spores.

[0031] Figure 2 Morphology of protoplasts of Tremella fuciformis spores after enzymatic hydrolysis.

[0032] Figure 3 Effects of different factors on the preparation of spore protoplasts from Tremella fuciformis: (a) cell concentration; (b) pH; (c) concentration of the compound enzyme hydrolysate; (d) enzymatic hydrolysis temperature.

[0033] Figure 4 The effect of different types of regeneration stabilizers on protoplast regeneration rate in regeneration culture medium.

[0034] Figure 5 The effect of different concentrations of regeneration stabilizer (sucrose) on protoplast regeneration rate.

[0035] Figure 6Colonies formed by protoplasts on regeneration media containing different concentrations of regeneration stabilizer (sucrose): (a) no regeneration stabilizer added; (b) 0.4 mol / L regeneration stabilizer (sucrose) added; (c) 0.6 mol / L regeneration stabilizer (sucrose) added; (d) 0.8 mol / L regeneration stabilizer (sucrose) added; (e) 1 mol / L regeneration stabilizer (sucrose) added; (f) 1.2 mol / L regeneration stabilizer (sucrose) added.

[0036] Figure 7 The effect of different types of growth promoters on protoplast regeneration rate. Detailed Implementation

[0037] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0038] In the following examples, the 1 mol / L sodium phosphate buffer solution is formulated as follows: 136.1 g of sodium dihydrogen phosphate and 176.2 g of disodium hydrogen phosphate are weighed and dissolved separately in ultrapure water, and the volume is adjusted to 1 L. 1 mol / L sodium dihydrogen phosphate and 1 mol / L disodium hydrogen phosphate are mixed in the following proportions to prepare 1 mol / L sodium phosphate buffer solutions with pH values ​​of 5 (1.0:99.0), 6 (12.0:88.0), 7 (57.7:42.3), and 8 (93.2:6.8), for later use.

[0039] In the following examples, the 0.6 mol / L potassium chloride solution is formulated as follows: 44.7 g of potassium chloride is weighed and dissolved in 1 mol / L sodium phosphate buffer (pH 5, 6, 7, 8).

[0040] In the following examples, the composite enzymatic hydrolysate is prepared as follows: the wall-dissolving enzyme and the wall-breaking enzyme are weighed at a mass ratio of 4:1 in 0.4-1.2 mol / L osmotic stabilizer to prepare a 10-30 mg / mL composite enzymatic hydrolysate. After dissolution, the solution is sterilized by passing it through a 0.22 μm aqueous membrane for later use.

[0041] In the following examples, the lysozyme was purchased from the Guangdong Institute of Microbiology, with a specification of ≥200 u / mg; the catalytic enzyme was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a specification of ≥100 u / mg. Unit of enzyme activity is defined as the amount of enzyme required to increase ΔA800 by 0.001 per minute in 3 mL of a 3 mL reaction mixture using suspended yeast as a substrate at 25°C and pH 7.5.

[0042] In the following examples, the tremella mentioned is a yeast-like tremella spore. This strain was isolated from the fruiting body of tremella in Gutian, Fujian on March 21, 2021, and was deposited at the China General Microbiological Culture Collection Center on January 14, 2022. The strain number is S-41, the classification name is Tremella fuciformis, the accession number is CGMCCNo.40060, and the deposit address is No.3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0043] Example 1: Activation and culture of Tremella fuciformis spores

[0044] (1) Activation culture of Tremella fuciformis spores by streak plating: Tremella fuciformis spores were stored at -80℃ in 40% glycerol (glycerol:water = 40:60, v / v). A small amount of Tremella fuciformis spore suspension was taken from the glycerol tube with an inoculation loop and streaked on PDA solid medium. The medium was then incubated upside down in an incubator at 24-30℃ for 5-8 days to obtain activated Tremella fuciformis spores.

[0045] The PDA solid culture medium has the following formula: 200 g / L potato extract, 20 g / L glucose, 20 g / L agar, and the remainder is water.

[0046] (2) Tremella fuciformis spore seed culture: Select a single colony of activated Tremella fuciformis spores from step (1) and inoculate it into a seed culture medium. Ferment in a shake flask for 3-5 days at a temperature of 20-32℃ and a rotation speed of 150-200 rpm to obtain a Tremella fuciformis spore suspension. Figure 1 ).

[0047] The seed culture medium has the following formula: glucose 5-30 g / L, yeast powder 1-5 g / L, peptone 1-5 g / L, magnesium sulfate 0.1-2 g / L, potassium dihydrogen phosphate 1-10 g / L, dipotassium hydrogen phosphate 0.1-2 g / L, and the remainder is water.

[0048] Example 2: Effect of initial concentrations of different Tremella fuciformis spores on enzymatic hydrolysis efficiency

[0049] A 0.6 mol / L potassium chloride solution with a pH of 5.0 was prepared using a 1 mol / L sodium phosphate buffer (a mixture of 1 mol / L sodium dihydrogen phosphate and 1 mol / L disodium hydrogen phosphate, pH 5.0) as an osmotic stabilizer. A 20 mg / mL composite enzymatic hydrolysate was then prepared using the osmotic stabilizer and sterilized by passing the hydrolysate through a 0.22 μm aqueous membrane for later use.

[0050] The *Tremella fuciformis* spore suspension obtained from shake-flask fermentation in Example 1 was collected and centrifuged at 3,000 rpm for 10 min to obtain *Tremella fuciformis* spore cells. The *Tremella fuciformis* spore cells were washed three times with an osmotic stabilizer, centrifuged at 3,000 rpm for 10 min to remove the supernatant, and resuspended in a prepared 20 mg / mL composite enzyme digest to achieve an initial concentration of 3.6 × 10⁻⁶. 8 3.6×10 9 2.2×10 10 The spores were collected at a concentration of 1 spore / mL and placed in a shaker at 26℃ for 2-8 hours to prepare the enzymatic hydrolysate of Tremella fuciformis spore protoplasts. Figure 2 ).

[0051] 10 μL of the prepared *Tremella fuciformis* spore protoplast hydrolysate was dropped onto a hemocytometer for counting to determine the number of protoplasts at different initial concentrations of *Tremella fuciformis* spores. The formula for calculating the number of protoplasts was: A (cells / mL) = (number of cells in 80 small squares / 80) × 400 × 10000 × dilution factor. Where A represents the number of protoplasts.

[0052] The result is Figure 3 (a) It can be seen that when the initial concentration of Tremella fuciformis spores is 3.6 × 10⁻⁶, 9 cells / mL or 2.2 × 10⁻⁶ 10 At a concentration of 3.6 × 10⁶ cells / mL, the enzymatic hydrolysis efficiency of protoplasts is low. 8 The highest enzymatic hydrolysis efficiency was achieved when the concentration of enzymes was 7.95 × 10⁻⁶ / mL, yielding 7.95 × 10⁻⁶ cells / mL after 4 hours of enzymatic hydrolysis. 7 One protoplast per mL.

[0053] Example 3: Effect of different pH stabilizing agents (i.e., enzymatic hydrolysis systems with different pH values) on enzymatic hydrolysis

[0054] Based on Example 2, the effect of different pH stabilizing agents (i.e., enzymatic hydrolysis systems with different pH values) on enzymatic hydrolysis was studied. The specific experimental steps are as follows:

[0055] Potassium chloride solutions with a concentration of 0.6 mol / L and pH values ​​of 5, 6, 7, and 8 were prepared using 1 mol / L sodium phosphate buffer (a mixture of 1 mol / L sodium dihydrogen phosphate and 1 mol / L disodium hydrogen phosphate, with pH values ​​of 5, 6, 7, and 8) as osmotic stabilizers. 20 mg / mL composite enzymatic hydrolysates were prepared using osmotic stabilizers with different pH values. The composite enzymatic hydrolysates were sterilized by passing through a 0.22 μm aqueous membrane for later use.

[0056] The *Tremella fuciformis* spore suspension obtained from shake-flask fermentation in Example 1 was collected and centrifuged at 3,000 rpm for 10 min to obtain *Tremella fuciformis* spore cells. The *Tremella fuciformis* spore cells were washed five times with osmotic stabilizers of different pH values, centrifuged at 2,000 rpm for 20 min to remove the supernatant, and resuspended in a prepared 20 mg / mL composite enzyme digest to an initial concentration of 3.6 × 10⁻⁶. 8 1 spore / mL. Place it in a shaker at 26℃ and enzymatically hydrolyze at 200rpm for 4-16h to prepare the tremella spore protoplast hydrolysate.

[0057] 10 μL of the prepared *Tremella fuciformis* spore protoplast enzymatic hydrolysate was dropped onto a hemocytometer for counting, and the number of protoplasts was determined under different pH stabilizing agents (i.e., enzymatic hydrolysis systems with different pH values). The formula for calculating the number of protoplasts is: A (cells / mL) = (number of cells in 80 small squares / 80) × 400 × 10000 × dilution factor. Where A represents the number of protoplasts.

[0058] The result is Figure 3 (b) It can be seen that when the pH value of the enzymatic hydrolysis system is 5, 6, 7, and 8, 7.95 × 10⁻⁶ g / L can be obtained after 4 hours of enzymatic hydrolysis. 7 5.75×10 7 1.25×10 7 0.2×10 7 One protoplast per mL. Therefore, the optimal pH for enzymatic hydrolysis of the cell wall of *Tremella fuciformis* spores using the complex enzyme system is 5.

[0059] Example 4: Effect of different concentrations of compound enzyme hydrolysate on enzymatic hydrolysis

[0060] Based on Example 3, the effect of different concentrations of the composite enzyme hydrolysate on enzymatic hydrolysis was studied. The specific experimental steps are as follows:

[0061] A 0.6 mol / L potassium chloride solution with a pH of 5.0 was prepared using a 1 mol / L sodium phosphate buffer (a mixture of 1 mol / L sodium dihydrogen phosphate and 1 mol / L disodium hydrogen phosphate, pH 5.0) as an osmotic stabilizer. Composite enzyme hydrolysates of 10, 20, and 30 mg / mL were prepared using the osmotic stabilizer. The composite enzyme hydrolysates were then sterilized by passing them through a 0.22 μm aqueous membrane for later use.

[0062] The *Tremella fuciformis* spore suspension obtained from shake-flask fermentation in Example 1 was collected and centrifuged at 3,000 rpm for 10 min to obtain *Tremella fuciformis* spore cells. The *Tremella fuciformis* spore cells were washed twice with an osmotic stabilizer, centrifuged at 4,000 rpm for 10 min to remove the supernatant, and resuspended in different concentrations of prepared compound enzyme hydrolysate to achieve an initial concentration of 3.6 × 10⁻⁶. 81 spore / mL. Place it in a shaker at 26℃ and enzymatically hydrolyze at 200rpm for 2-6 hours to prepare the Tremella fuciformis protoplast hydrolysate.

[0063] 10 μL of the prepared *Tremella fuciformis* spore protoplast enzymatic hydrolysate was dropped onto a hemocytometer for counting, and the number of protoplasts was determined at different concentrations of the combined enzymatic hydrolysate. The formula for calculating the number of protoplasts was: A (cells / mL) = (number of cells in 80 small squares / 80) × 400 × 10000 × dilution factor. Where A represents the number of protoplasts.

[0064] The result is Figure 3 (c) It can be seen that when the concentration of the compound enzyme hydrolysate is 10 mg / mL, 20 mg / mL, and 30 mg / mL, 6.50 × 10⁶ mg / mL can be obtained after 4 hours of enzymatic hydrolysis. 7 7.95×10 7 9.90×10 7 One protoplast per mL. Therefore, the optimal concentration of the compound enzymatic hydrolysate is 30 mg / mL.

[0065] Example 5: Effect of different enzymatic hydrolysis temperatures on enzymatic hydrolysis

[0066] Based on Example 4, the effect of different enzymatic hydrolysis temperatures on enzymatic hydrolysis was studied. The specific experimental steps are as follows:

[0067] A 0.6 mol / L potassium chloride solution with a pH of 5 was prepared using a 1 mol / L sodium phosphate buffer (a mixture of 1 mol / L sodium dihydrogen phosphate and 1 mol / L disodium hydrogen phosphate, pH 5.0) as an osmotic stabilizer. A 30 mg / mL composite enzyme hydrolysate was prepared using the osmotic stabilizer. The composite enzyme hydrolysate was sterilized by passing it through a 0.22 μm aqueous membrane for later use.

[0068] The *Tremella fuciformis* spore suspension obtained from shake-flask fermentation in Example 1 was collected and centrifuged at 3,000 rpm for 10 min to obtain *Tremella fuciformis* spore cells. The *Tremella fuciformis* spore cells were washed three times with an osmotic stabilizer, centrifuged at 3,000 rpm for 10 min to remove the supernatant, and resuspended in a prepared 30 mg / mL composite enzyme digest to achieve an initial concentration of 3.6 × 10⁻⁶. 8 1 spore / mL. The spores were placed in shakers at 26, 30, and 35°C and enzymatically hydrolyzed at 200 rpm for 2-6 hours to prepare the tremella spore protoplast hydrolysate.

[0069] 10 μL of the prepared *Tremella fuciformis* spore protoplast enzymatic hydrolysate was dropped onto a hemocytometer for counting, and the number of protoplasts was determined at different enzymatic hydrolysis temperatures. The formula for calculating the number of protoplasts is: A (cells / mL) = (number of cells in 80 small squares / 80) × 400 × 10000 × dilution factor. Where A represents the number of protoplasts.

[0070] The result is Figure 3 (d) It can be seen that when the enzymatic hydrolysis temperatures are 26, 30, and 35℃, 1.14 × 10⁻⁶ ppm can be obtained after 4 hours of enzymatic hydrolysis. 8 1.18×10 8 9.7×10 7 One protoplast per mL. There was almost no difference in protoplast preparation efficiency between enzymatic hydrolysis at 26°C and 30°C; therefore, the preferred hydrolysis temperature was 26°C.

[0071] Example 6: Effect of regeneration culture media containing different regeneration stabilizers on the regeneration rate of Tremella fuciformis spore protoplasts

[0072] The enzymatic hydrolysate of Tremella fuciformis spore protoplasts prepared at 26°C in Example 5 was centrifuged at 3,000 rpm for 5 min, then washed 3–5 times with a 0.6 mol / L potassium chloride solution at pH 5.0, and diluted to obtain a solution containing approximately 10 3 The protoplast suspension of *Tremella fuciformis* spores was counted using a hemocytometer. 100 μL of the diluted protoplast suspension was spread onto regeneration medium containing different regeneration stabilizers and incubated upside down at 26°C for 10–15 days. The number of regenerated colonies on the regeneration medium was counted, and the protoplast regeneration rate was calculated.

[0073] The regeneration culture medium has the following formula: maltose 10g / L, glucose 10g / L, yeast powder 2g / L, peptone 2g / L, KH2PO4 4.6g / L, magnesium sulfate 0.5g / L, agar 20g / L, and regeneration stabilizer (potassium chloride, sucrose, mannitol, sorbitol) 0.6mol / L.

[0074] The formula for calculating the protoplast regeneration rate is: C(%) = B*10 / A×100%. Where: A represents the number of protoplasts; B represents the number of colonies regenerated on the regeneration medium; and C represents the protoplast regeneration rate.

[0075] The result is Figure 4 It can be seen that when the regeneration stabilizer in the regeneration medium is selected as 0.6 mol / L potassium chloride, sucrose, mannitol or sorbitol, the protoplast regeneration rate of Tremella fuciformis spores is 7.77%, 15.93%, 14.07% and 13.05%, respectively. Therefore, sucrose is the most effective osmotic stabilizer when added to the regeneration medium.

[0076] Example 7: Effect of adding different concentrations of sucrose stabilizing agent to the regeneration medium on the regeneration rate of Tremella fuciformis spore protoplasts

[0077] The enzymatic hydrolysate of Tremella fuciformis spore protoplasts prepared at 26°C in Example 5 was centrifuged at 3,000 rpm for 5 min, then washed 3–5 times with a 0.6 mol / L potassium chloride solution at pH 5.0, and diluted to obtain a solution containing approximately 10 3 The number of spore protoplasts per ml of *Tremella fuciformis* was counted using a hemocytometer. 100 μL of the diluted *Tremella fuciformis* spore protoplast suspension was spread onto regeneration medium containing different concentrations of regeneration stabilizer and incubated upside down at 26°C for 10–15 days. The number of regenerated colonies on the regeneration medium was counted, and the protoplast regeneration rate was calculated.

[0078] The regeneration culture medium has the following formula: maltose 10g / L, glucose 10g / L, yeast powder 2g / L, peptone 2g / L, KH2PO4 4.6g / L, magnesium sulfate 0.5g / L, agar 20g / L, and regeneration stabilizer (sucrose) 0.4~1.2mol / L.

[0079] The formula for calculating the protoplast regeneration rate is: C(%) = B*10 / A×100%. Where: A represents the number of protoplasts; B represents the number of colonies regenerated on the regeneration medium; and C represents the protoplast regeneration rate.

[0080] The result is Figure 5 It can be seen that when the concentration of the osmotic stabilizer sucrose in the regeneration medium was 0.4, 0.6, 0.8, 1, and 1.2 mol / L, the regeneration rates of protoplasts were 12.24%, 15.93%, 32.64%, 15.93%, and 11.01%, respectively. Figure 6 It can also be visually observed that the highest number of colonies were formed when 0.8 mol / L of sucrose, a regeneration and permeation stabilizer, was added to the regeneration medium. Therefore, the regeneration rate of Tremella fuciformis protoplasts was highest when the concentration of sucrose, a regeneration and permeation stabilizer, in the regeneration medium was 0.8 mol / L.

[0081] Example 8: Effect of adding a promoter to the regeneration medium on the regeneration rate of Tremella fuciformis spore protoplasts

[0082] The enzymatic hydrolysate of Tremella fuciformis spore protoplasts prepared at 26°C in Example 5 was centrifuged at 3,000 rpm for 5 min, then washed 3–5 times with a 0.6 mol / L potassium chloride solution at pH 5.0, and diluted to obtain a solution containing approximately 10 3 The protoplast suspension of *Tremella fuciformis* spores was counted using a hemocytometer. 100 μL of the diluted protoplast suspension was spread onto regeneration medium supplemented with different promoters (VB1 and bovine serum albumin), and incubated upside down at 26°C for 10–15 days. The number of regenerated colonies on the regeneration medium was counted, and the protoplast regeneration rate was calculated.

[0083] The regeneration culture medium has the following formula: maltose 10g / L, glucose 10g / L, yeast extract 2g / L, peptone 2g / L, KH2PO4 4.6g / L, magnesium sulfate 0.5g / L, agar 20g / L, regeneration stabilizer (sucrose) 0.8mol / L, and growth promoters (20mg / L VB1, 500mg / L bovine serum albumin, 20mg / L VB1 + 500mg / L bovine serum albumin).

[0084] The formula for calculating the protoplast regeneration rate is: C(%) = B*10 / A×100%. Where: A represents the number of protoplasts; B represents the number of colonies regenerated on the regeneration medium; and C represents the protoplast regeneration rate.

[0085] The result is Figure 7 It can be seen that when VB1 and bovine serum albumin are added to the regeneration medium as growth promoters, VB1 and BSA have a significant promoting effect on the regeneration of Tremella fuciformis spores, and the protoplast regeneration rate of Tremella fuciformis spores is further increased from 32.64% to 43.52%.

[0086] This invention provides a method for the preparation and regeneration of *Tremella fuciformis* spore protoplasts. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.

Claims

1. A method for preparing and regenerating protoplasts of Tremella fuciformis spores, characterized by, It comprises the following steps: (1) Activated culture of tremella spore: picking tremella spore colony on PDA solid medium for activated culture to obtain activated tremella spore; (2) Seed liquid culture of tremella spore: inoculating the activated tremella spore obtained in step (1) into seed liquid medium for fermentation culture to obtain tremella spore suspension, centrifuging the tremella spore suspension to obtain the precipitate, which is the tremella spore mycelium; (3) Preparation of tremella spore protoplast enzyme solution: washing and centrifuging the tremella spore mycelium obtained in step (2) to remove the supernatant, resuspending in a complex enzyme solution for enzyme hydrolysis to obtain the tremella spore protoplast enzyme solution; (4) Regeneration of tremella spore protoplast: centrifuging the tremella spore protoplast enzyme solution obtained in step (3) to remove the supernatant, separating out the protoplast, washing and diluting with a stabilizing agent to prepare a protoplast suspension, and coating the protoplast suspension on a regeneration medium for culture to form regenerated colonies; In step (3), the complex enzyme solution comprises lyticase, lyticase and stabilizing agent, and the total enzyme concentration is 10-30 mg / mL, pH=5-8. In step (4), the regeneration medium has the following formula: maltose 5-20 g / L, glucose 1-10 g / L, yeast powder 1-5 g / L, peptone 1-5 g / L, KH2PO4 1-10 g / L, magnesium sulfate 0.1-2 g / L, agar 15-30 g / L, regeneration stabilizing agent, growth promoter, and water as solvent; specifically, the regeneration stabilizing agent is 0.4-1.2 mol / L sucrose, and the growth promoter is 10-50 mg / L VB1+100-1000 mg / L bovine serum albumin. The tremella spore is derived from a tremella strain with the preservation number of CGMCC No. 40060 and the strain number of S-41. In step (3) or step (4), the stabilizing agent is a 0.4-1.2 mol / L potassium chloride solution prepared with 0.5-1.0 mol / L sodium phosphate buffer as solvent, pH=5-8. In the step (3), the resuspension is performed with an initial concentration of 3.6 x 10 8 / mL.

2. The method of claim 1, wherein, In step (1), the activated culture has the following culture conditions: 24-30 ℃, and culture for 5-8 days.

3. The method of claim 1, wherein, In step (2), the fermentation culture has the following culture conditions: 20-32 ℃, 150-200 rpm, and culture for 3-5 days.

4. The method of claim 1, wherein, In step (2), the seed culture medium has the following formula: glucose 5-30 g / L, yeast powder 1-5 g / L, peptone 1-5 g / L, magnesium sulfate 0.1-2 g / L, potassium dihydrogen phosphate 1-10 g / L, dipotassium hydrogen phosphate 0.1-2 g / L, and the rest is water.

5. The method of claim 1, wherein, In step (3), the complex enzyme solution is prepared as follows: weighing lyticase and lyticase in a mass ratio of 1:1-9:1 in a stabilizing agent, dissolving, and sterilizing through a 0.22 μm water system membrane for standby use.

6. The method of claim 1, wherein, The total enzyme concentration of the complex enzyme solution is 30 mg / mL, and pH=5.

7. The method of claim 1, wherein, In step (3), the enzymolysis condition is 26-35 ℃, 200 rpm for 2-8 h.

8. The method of claim 7, wherein, The enzymolysis condition is 26 ℃, 200 rpm for 4 h.

9. The method of claim 1, wherein, In step (4), the culture condition is 20-32 ℃, inverted culture for 10-15 days.

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