Preparation and regeneration method and application of phellinus igniarius protoplast
Preparation and regeneration of mulberry yellow protoplasts through protoplast technology, and screening of excellent strains using mutagenesis technology, solving the problems of difficult artificial cultivation of mulberry yellow and long growth cycle, and achieving rapid breeding of mulberry yellow species and biological effectors Increase in output.
Patent Information
- Application Number
- CN202411409236.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-10
- Publication Date
- 2025-02-25
AI Technical Summary
The artificial cultivation of wild mulberry yellow is difficult and the growth cycle is long, which limits the further development and utilization of mulberry yellow. A fast and effective method is needed to breed mulberry yellow species with stable traits and short growth cycles.
By using protoplast technology, mulberry yellow protoplasts were prepared and regenerated, and the complex mulberry yellow protoplasts were used to screen out the mulberry yellow dominant mutant strains with improved mycelium growth rate, biomass, flavonoid yield and antioxidant activity.
It has achieved rapid breeding of mulberry chlorophyllium species, shortened the growth cycle, improved the bioeffect product yield of mycelium, enhanced antioxidant activity, and met the market's growing demand for mulberry chlorophyllium products.
Smart Images
Figure FT_1 
Figure FT_2 
Figure FT_3
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of plant cell biology, and in particular relates to a preparation and regeneration method of mulberry linterus protoplasts and an application thereof. Background Art
[0002] Phellinus linteus Sanghuangporus spp. ) is a rare large medicinal fungus, belonging to the phylum Basidiomycota, class Agaricomycetes, order Hymenochaetales, family Hymenochaetaceae, genus Moriformis Sanghuangporus . Phellinus igniarius is also known as mulberry, mulberry mushroom, mulberry ear and mulberry parasite. Phellinus igniarius is slightly cold in nature, sweet and spicy in taste. The general method of taking mulberry igniarius is to boil water and take it. Phellinus igniarius is rich in nutrients and bioactive substances, including polysaccharides, terpenes, phenols, flavonoids, pyrones, amino acids and aromatic acids, as well as more than ten trace elements such as iron, calcium, zinc and magnesium. The main chemical components are larch acid, verrucine, mushroom acid, meta-dimethoxyphthalic acid, ergobinol, peroxidase and amino acids. The main medicinal ingredients include polysaccharides such as β-glucan (β-Glucans), proteoglycans and acidic polysaccharides, which have extremely high medicinal value. Phellinus igniarius is currently recognized as the most effective medicinal fungus in the world's biological anticancer agents. Its anti-tumor, antioxidant, anti-inflammatory, anti-mutation, liver protection, hypoglycemic and immune enhancement effects have been significant in clinical trials. It can prevent or treat a variety of diseases and is widely used around the world.
[0003] The artificial cultivation of wild Phellinus linteus is difficult and the fruiting body growth cycle is long, generally 3 to 4 years, which limits the further development and utilization of Phellinus linteus. In order to meet the growing market demand, it has become an important task to breed Phellinus linteus strains with stable traits, short fruiting body growth cycle and low difficulty in artificial cultivation. Protoplast technology is an effective means for breeding excellent strains, such as protoplast fusion, protoplast mutagenesis and protoplast gene transformation. Among them, the preparation and regeneration of protoplasts are the premise and basis of protoplast technology. For this reason, the present invention provides a preparation and regeneration method of Phellinus linteus protoplasts, which lays the foundation for breeding excellent Phellinus linteus strains using protoplast technology. Summary of the invention
[0004] The purpose of the present invention is to provide a preparation and regeneration method of Phellinus igniarius protoplasts and application thereof.
[0005] To achieve the above object, the present invention adopts the following technical solution: A preparation and regeneration method of Phellinus linteus protoplasts, comprising the following steps: S1: Inoculate the 4℃ low-temperature preserved Phellinus linteus onto the modified PDA medium plate, incubate at 26℃ in the dark for activation, and then take the 10×10mm 2 5 pieces of mycelium blocks were inoculated into a 250 mL Erlenmeyer flask containing 100 mL of modified PDB medium, and cultured at 26°C in the dark, with shaking once in the morning and evening every day during the culture process; the mycelium cultured in the dark for 8 to 16 days was washed with sterile water, and then the surface moisture was absorbed with sterile filter paper to obtain Phellinus igniarius wet mycelium; S2: Add 1.0wt% to 2.0wt% of lytic enzyme and 0.5wt% of collapsing enzyme to a 0.6mol / L mannitol solution to obtain a mixed enzyme solution; filter the mixed enzyme solution through a 0.2μm microporous filter membrane for sterilization and set aside; take 0.3g of Phellinus igniarius wet mycelium and add it to a 1.5mL centrifuge tube containing 1.0mL of the mixed enzyme solution, seal the centrifuge tube and shake the centrifuge tube up and down for 10s, then enzymolyze at 20~40℃ and 120r / min for 1~5h to obtain Phellinus igniarius mycelium enzymolysis solution; S3: Filter the enzymatic hydrolyzate of Phellinus linteus hyphae with a sterilized G3 sand core funnel, collect the filtrate into a 1.5 mL centrifuge tube, centrifuge at 4°C and 3000 g for 10 min, discard the supernatant, and wash the precipitate with 0.6 mol / L mannitol solution for 3 times to obtain purified Phellinus linteus protoplasts; disperse the purified Phellinus linteus protoplasts in 0.6 mol / L mannitol solution to obtain a Phellinus linteus protoplast suspension; S4: dilute the phellinus linus protoplast suspension with 0.6 mol / L mannitol solution to obtain a phellinus linus protoplast dilution solution; take 1.0 mL of the phellinus linus protoplast dilution solution and spread it on a modified PDA regeneration medium plate, and place it at 26° C. in the dark for regeneration culture.
[0006] The above step S1 is: inoculate the Phellinus linteus strains stored at 4°C onto a modified PDA medium plate, place it in a dark place at 26°C for activation, and then take out the 10×10 mm 2 Five mycelium blocks were inoculated into a 250-mL Erlenmeyer flask containing 100 mL of modified PDB medium and cultured at 26°C in the dark. The culture was shaken once in the morning and once in the evening every day during the culture process. The mycelium that had been cultured in the dark for 10 days was washed with sterile water, and then the surface moisture was absorbed with sterilized filter paper to obtain the wet mycelium of Phellinus igniarius.
[0007] The above-mentioned step S2 is: adding a final concentration of 1.5wt% of lytic enzyme and a final concentration of 0.5wt% of disintegrating enzyme to a 0.6mol / L mannitol solution to obtain a mixed enzyme solution; filtering the mixed enzyme solution through a 0.2μm microporous filter membrane for sterilization and then setting it aside; taking 0.3g of Phellinus igniarius wet mycelium and adding it to a 1.5mL centrifuge tube containing 1.0mL of the mixed enzyme solution, sealing the centrifuge tube and shaking the centrifuge tube up and down for 10s, and then shaking the enzymolysis in a constant temperature water bath at 30°C and 120r / min for 3h to obtain Phellinus igniarius mycelium enzymolysis solution.
[0008] The formula of the above-mentioned improved PDB culture medium is: dissolve 24g of PDB culture medium powder in plant water extract, make up to 1L with distilled water, and the pH is natural; the preparation method of the plant water extract is: add 10g of mulberry branch slice powder and 10g of Oasis No. 1 powder to 1L of distilled water, extract at 100℃ for 1h, filter with sterile filter paper and collect the filtrate to obtain the plant water extract.
[0009] The improved PDB culture medium formula is: dissolve 24g of PDB culture medium powder in plant water extract, dilute to 1L with distilled water, and the pH is natural; the preparation method of the plant water extract is: add 10g of mulberry branch slice powder and 10g of Oasis No. 1 powder to 1L of distilled water, extract at 100°C for 1h, filter with sterile filter paper and collect the filtrate to obtain the plant water extract.
[0010] The formula of the above-mentioned improved PDA regeneration medium is: dissolve 109.2g of mannitol and 37g of PDA medium powder in plant water extract, dilute to 1L with distilled water, and the pH is natural; the preparation method of the plant water extract is: add 10g of mulberry branch slice powder and 10g of Oasis No. 1 powder to 1L of distilled water, extract at 100°C for 1h, filter with sterile filter paper and collect the filtrate to obtain the plant water extract.
[0011] The above-mentioned method for preparing and regenerating mulberry linterus protoplasts is applied in breeding excellent mulberry linterus strains using protoplast technology.
[0012] The above application is specifically: subjecting the protoplast suspension of Phellinus igniarius to combined mutagenesis with ultraviolet light and lithium chloride, regenerating and culturing the protoplasts after mutagenesis, and obtaining, through screening, superior mutant strains of Phellinus igniarius with improved mycelium growth rate, mycelium biomass, flavonoid production and antioxidant activity of mycelium ethanol extract. BRIEF DESCRIPTION OF THE DRAWINGS
[0013] Figure 1 :Effects of PDB medium and improved PDB medium on the growth rate of Phellinus linterus mycelium.
[0014] Figure 2 :Effects of enzymatic hydrolysis temperature and time on protoplast yield of Phellinus linteus.
[0015] Figure 3 :Effect of the content of lytic enzyme in mixed enzyme solution on the yield of protoplasts of Phellinus igniarius.
[0016] Figure 4 :Effect of cell age on preparation of protoplasts from Phellinus linteus.
[0017] Figure 5 :Analysis of protoplast regeneration results of Phellinus igniarius under different enzymatic hydrolysis times.
[0018] Figure 6 :Preliminary screening results of mutant strains with excellent growth conditions. U represents ultraviolet mutagenesis, L represents lithium chloride mutagenesis, and UL represents ultraviolet-lithium chloride combined mutagenesis.
[0019] Figure 7 : The antagonistic phenomenon produced by the starting strain G1 of Phellinus igniarius and the mutants UL27, U8 and L13 respectively.
[0020] Figure 8 : RAPD amplification electrophoresis patterns of mutant strains UL27, U8, L13 and the starting strain G1 of Phellinus igniarius.
[0021] Fig. 9 :Ethanol extracts from mycelium of different strains remove ABTS + The ability of free radicals.
[0022] Fig.10 :Reduction of Fe by ethanol extracts of mycelium from different strains 3+ ability. DETAILED DESCRIPTION
[0023] The present invention is described below by specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.
[0024] The Phellinus igniarius strain G1 used in the present invention is provided by the National Juncao Engineering Technology Research Center of Fujian Agriculture and Forestry University.
[0025] The mulberry branch slice powder used in the present invention is obtained by slicing and crushing the dried young branches of the mulberry plant of the Moraceae family, and is purchased from Duojuntang Biotechnology Co., Ltd. The Oasis No. 1 powder used in the present invention is obtained by crushing the hay of the perennial Gramineae plant Oasis No. 1 of the genus Arundo donax, and is provided by the National Juncao Engineering Technology Research Center of Fujian Agriculture and Forestry University.
[0026] The PDA culture medium powder used in the present invention was purchased from Beijing Hongrun Baoshun Technology Co., Ltd. The PDB culture medium powder used in the present invention was purchased from Shanghai Yuanye Biotechnology Co., Ltd.
[0027] The lysozyme used in the present invention was purchased from Fuzhou Luoyin Biotechnology Co., Ltd. The lysozyme used in the present invention was purchased from Beijing Wokai Biotechnology Co., Ltd.
[0028] Embodiment 1: S1: Protoplast preparation material preparation The Phellinus igniarius strain G1 stored at 4°C was inoculated onto a modified PDA medium plate and incubated at 26°C in the dark for activation. 2 Five mycelium blocks were inoculated into a 250-mL Erlenmeyer flask containing 100 mL of modified PDB medium and cultured at 26°C in the dark. The culture was shaken once in the morning and once in the evening every day during the culture process. The mycelium that had been cultured in the dark for 10 days was washed with sterile water, and then the surface moisture was absorbed with sterilized filter paper to obtain the wet mycelium of Phellinus igniarius.
[0029] Among them, the formula of the improved PDA culture medium is: dissolve 37g of PDA culture medium powder in plant water extract (add 10g of mulberry branch slice powder and 10g of Oasis No. 1 powder to 1L of distilled water, extract at 100℃ for 1h, filter with sterile filter paper and collect the filtrate to obtain plant water extract), make up to 1L with distilled water, and the pH is natural; the culture medium sterilization conditions are: high pressure sterilization pot 121℃, sterilization time 20min.
[0030] Among them, the formula of the improved PDB culture medium is: dissolve 24g of PDB culture medium powder in plant water extract (add 10g of mulberry branch slice powder and 10g of Oasis No. 1 powder to 1L of distilled water, extract at 100℃ for 1h, filter with sterile filter paper and collect the filtrate to obtain plant water extract), make up to 1L with distilled water, and the pH is natural; the culture medium sterilization conditions are: high pressure sterilization pot 121℃, sterilization time 20min.
[0031] S2: Protoplast acquisition To a 0.6 mol / L mannitol solution, add 1.5 wt% of lytic enzyme and 0.5 wt% of disintegrating enzyme to obtain a mixed enzyme solution; filter the mixed enzyme solution through a 0.2 μm microporous filter membrane for sterilization and set aside; take 0.3 g of Phellinus igniarius wet mycelium and add it to a 1.5 mL centrifuge tube containing 1.0 mL of the mixed enzyme solution, seal the centrifuge tube and oscillate the centrifuge tube up and down for 10 seconds, and then oscillate the enzymolysis in a constant temperature water bath at 30°C and 120 r / min for 3 hours to obtain Phellinus igniarius mycelium enzymolysis solution.
[0032] S3: Protoplast purification The enzymatic hydrolyzate of Phellinus igniarius mycelium was filtered through a sterilized G3 sand core funnel, and the filtrate was collected into a 1.5 mL centrifuge tube. The filtrate was centrifuged at 4°C and 3000 g for 10 min, the supernatant was discarded, and the precipitate was washed three times with a 0.6 mol / L mannitol solution to obtain purified Phellinus igniarius protoplasts; the purified Phellinus igniarius protoplasts were dispersed in a 0.6 mol / L mannitol solution to obtain a Phellinus igniarius protoplast suspension for later use.
[0033] S4: Protoplast regeneration The phellinus linteus protoplast suspension was diluted to 10 6 / mL to obtain the mulberry linterus protoplast dilution; take 1.0mL of the mulberry linterus protoplast dilution and spread it on the improved PDA regeneration medium plate, incubate it at 26℃ in the dark for 5 days, then count the colonies and calculate the regeneration rate, regeneration rate / %=(number of regenerated colonies in group A-number of regenerated colonies in group B) / total number of protoplasts×100.
[0034] Among them, the formula of the improved PDA regeneration medium in Group A is: dissolve 109.2g mannitol and 37g PDA medium powder in plant water extract (add 10g mulberry branch slice powder and 10g Oasis No. 1 powder to 1L distilled water, extract at 100℃ for 1h, filter with sterile filter paper and collect the filtrate to obtain plant water extract), make up to 1L with distilled water, and the pH is natural; the medium sterilization conditions are: high pressure sterilization pot 121℃, sterilization time 20min.
[0035] Among them, the formula of the improved PDA regeneration medium in group B is: dissolve 37g of PDA medium powder in distilled water, make up to 1L with distilled water, and the pH is natural; the medium sterilization conditions are: high pressure sterilization pot at 121℃, sterilization time 20min.
[0036] Comparative Example 1: (1) Inoculate the Phellinus igniarius strain G1 stored at 4°C onto a PDA culture medium plate and incubate it at 26°C in the dark for activation. Regularly observe the changes in colony morphology and mycelial growth and measure the colony diameter. Take the most vigorous growth of 10×10mm 2 Five mycelium blocks were inoculated into a 250-mL Erlenmeyer flask containing 100 mL of PDB culture medium and cultured at 26°C in the dark. The mycelium was shaken once in the morning and evening every day during the culture process. The mycelium that had been cultured in the dark for 10 days was washed with sterile water, and then the surface moisture was absorbed with sterilized filter paper. The mycelium was dried in an oven at 60°C to constant weight and weighed to obtain the dry weight of Phellinus igniarius mycelium.
[0037] Among them, the formula of PDA culture medium is: dissolve 37g PDA culture medium powder in distilled water, make up to 1L with distilled water, and the pH is natural; the culture medium sterilization conditions are: high pressure sterilization pot 121℃, sterilization time 20min.
[0038] Among them, the formula of PDB culture medium is: dissolve 24g PDB culture medium powder in distilled water, make up to 1L with distilled water, and the pH is natural; the culture medium sterilization conditions are: high pressure sterilization pot 121℃, sterilization time 20min.
[0039] (2) Inoculate the 4°C low-temperature preserved Phellinus igniarius strain G1 onto a modified PDA medium plate and incubate at 26°C in the dark for activation. Regularly observe the changes in colony morphology and mycelial growth and measure the colony diameter. Take the most vigorous growth of 10×10 mm 2 Five mycelium blocks were inoculated into a 250-mL Erlenmeyer flask containing 100 mL of modified PDB medium and cultured at 26°C in the dark. The mycelium was shaken once in the morning and evening every day during the culture process. The mycelium that had been cultured in the dark for 10 days was washed with sterile water, and then the surface moisture was absorbed with sterilized filter paper. The mycelium was dried in an oven at 60°C to constant weight and weighed to obtain the dry weight of Phellinus igniarius mycelium.
[0040] Among them, the formulas of the improved PDA medium and the improved PDB medium are the same as those in Example 1.
[0041] When the diameter of the culture dish is 120 mm, the mycelial growth rate of Phellinus igniarius in PDA medium and modified PDA medium is shown in Table 1. As can be seen from the table, the additional addition of mulberry branches and water extracts of Oasis No. 1 in PDA medium has a promoting effect on the mycelial growth rate of Phellinus igniarius, and the increase is obvious. It can be inferred that there are some special ingredients in the additives that can stimulate the growth and development of Phellinus igniarius mycelium.
[0042] Table 1 Effects of PDA medium and improved PDA medium on the growth of mulberry mycelium The growth of Phellinus linterus hyphae in the modified PDB medium is shown in Figure 1 As shown, A is the growth state of Phellinus linteus hyphae in PDB liquid medium, and B is the growth state of Phellinus linteus hyphae in improved PDB medium. After static culture at 26°C in the dark for the same time, it can be seen that the Phellinus linteus hyphae in B grow evenly, densely, thickly and in larger volume. The dry weight of the mycelium is measured as shown in Table 2.
[0043] Table 2 Effects of PDB medium and improved PDB medium on the growth of Phellinus linterus hyphae Comparative Example 2: S1: Same as S1 in Example 1.
[0044] S2: Add 1.5wt% lytic enzyme and 0.5wt% disintegrating enzyme to a 0.6mol / L mannitol solution to obtain a mixed enzyme solution; filter the mixed enzyme solution through a 0.2μm microporous filter membrane for sterilization and set aside; take 0.3g of Phellinus igniarius wet mycelium and add it to a 1.5mL centrifuge tube containing 1.0mL of the mixed enzyme solution, seal the centrifuge tube and oscillate the centrifuge tube up and down for 10s, then oscillate in a constant temperature water bath at 20~40℃ and 120r / min for 1~5h to obtain Phellinus igniarius mycelium hydrolyzate; aspirate the Phellinus igniarius mycelium hydrolyzate and count it with a hemocytometer and calculate the protoplast concentration (pieces / mL), and the protoplast concentration is used to represent the protoplast yield.
[0045] The results are as follows Figure 2 As shown. Under the light-proof environment at 120 r / min, as the enzymatic hydrolysis time increases, the protoplast yield has a trend of increasing and then decreasing. This is because a large number of hyphae overlap and intersect in the early stage of enzymatic hydrolysis, and the contact area between the hyphae and the enzymatic hydrolysis solution is not large. The enzymatic hydrolysis solution can only dissolve the accessible mycelial cell wall first. Under the conditions of 25-35℃, the protoplast yield increases first and then decreases, reaching a peak at about 3h of enzymatic hydrolysis, with the maximum yield of 3.75×10 6 / mL, and the protoplast yield decreased after 3h, mainly because the enzymatic hydrolysis time was too long, and the over-reaction of the enzymatic hydrolysate may cause damage to some protoplasts. At 20℃, the protoplast yield was always low and did not change much. At 40℃, the protoplast yield was very high at first, and began to decrease after 2h of enzymatic hydrolysis, because the high temperature easily led to enzyme inactivation. Therefore, the optimal enzymatic hydrolysis conditions were 30℃ and 3h.
[0046] Comparative Example 3: S1: Same as S1 in Example 1.
[0047] S2: Add 1.0wt% to 2.0wt% of lytic enzyme and 0.5wt% of disintegrating enzyme to a 0.6mol / L mannitol solution to obtain a mixed enzyme solution; filter the mixed enzyme solution through a 0.2μm microporous filter membrane for sterilization and set aside; take 0.3g of wet mycelium of Phellinus igniarius and add it to a 1.5mL centrifuge tube containing 1.0mL of the mixed enzyme solution, seal the centrifuge tube and oscillate the centrifuge tube up and down for 10s, then oscillate in a constant temperature water bath at 30℃ and 120r / min for 3h to obtain the enzymatic hydrolyzate of Phellinus igniarius mycelium; aspirate the enzymatic hydrolyzate of Phellinus igniarius mycelium and count it with a hemocytometer and calculate the protoplast concentration (pieces / mL), and the protoplast concentration is used to represent the protoplast yield.
[0048] The results are as follows Figure 3As shown. The concentration of lytic enzyme plays a key role in the enzymatic reaction and is the key to promoting the dissolution of the cell wall. Too little lytic enzyme will lead to insufficient enzymatic reaction, resulting in some cells with small contact area or relatively thick cell walls not completely undergoing enzymatic reaction, resulting in the inability to release protoplasts. Too high concentration will lead to excessive enzymatic reaction, which may cause damage to the cell structure and damage or even death of the released protoplasts. The highest protoplast yield was obtained when the mixed enzyme solution contained a final concentration of 1.5wt% lytic enzyme. Therefore, the optimal final concentration of lytic enzyme is 1.5wt%.
[0049] Comparative Example 4: S1: Inoculate the 4℃ low-temperature preserved Phellinus igniarius strain G1 onto a modified PDA medium plate, place it in a dark place at 26℃ for activation, and then take the most vigorous growth of 10×10mm 2 Five mycelium blocks were inoculated into a 250-mL Erlenmeyer flask containing 100 mL of modified PDB medium and cultured at 26°C in the dark. The culture was shaken once in the morning and once in the evening every day during the culture process. The mycelium that had been cultured in the dark for 8 to 16 days was washed with sterile water, and then the surface moisture was absorbed with sterilized filter paper to obtain the wet mycelium of Phellinus igniarius.
[0050] Among them, the formula of the improved PDA culture medium is: dissolve 37g of PDA culture medium powder in plant water extract (add 10g of mulberry branch slice powder and 10g of Oasis No. 1 powder to 1L of distilled water, extract at 100℃ for 1h, filter with sterile filter paper and collect the filtrate to obtain plant water extract), make up to 1L with distilled water, and the pH is natural; the culture medium sterilization conditions are: high pressure sterilization pot 121℃, sterilization time 20min.
[0051] Among them, the formula of the improved PDB culture medium is: dissolve 24g of PDB culture medium powder in plant water extract (add 10g of mulberry branch slice powder and 10g of Oasis No. 1 powder to 1L of distilled water, extract at 100℃ for 1h, filter with sterile filter paper and collect the filtrate to obtain plant water extract), make up to 1L with distilled water, and the pH is natural; the culture medium sterilization conditions are: high pressure sterilization pot 121℃, sterilization time 20min.
[0052] S2: Add 1.5wt% lytic enzyme and 0.5wt% disintegrating enzyme to 0.6mol / L mannitol solution to obtain a mixed enzyme solution; filter the mixed enzyme solution through a 0.2μm microporous filter membrane for sterilization and set aside; take 0.3g of Phellinus igniarius wet mycelium and add it to a 1.5mL centrifuge tube containing 1.0mL of the mixed enzyme solution, seal the centrifuge tube and shake the centrifuge tube up and down for 10s, then shake the enzyme in a constant temperature water bath at 30℃ and 120r / min for 3h to obtain Phellinus igniarius mycelium hydrolyzate; aspirate the Phellinus igniarius mycelium hydrolyzate and count it with a hemocytometer and calculate the protoplast concentration (pieces / mL), and the protoplast concentration is used to represent the protoplast yield.
[0053] The results are as follows Figure 4 The age of the fungus affects the preparation of protoplasts from Phellinus igniarius. When 8-day-old mycelium of Phellinus igniarius was used to prepare protoplasts, the protoplast yield reached a peak after 2 hours of enzymatic hydrolysis, with a yield of 3.82×10 6 / mL, because the cell wall of young mycelium is easier to be hydrolyzed by enzymes. The cell wall of mycelium with a culture age of 16 days is obviously difficult to hydrolyze, and the yield is not high. The protoplast yield of 10-day-old Phellinus igniarius mycelium when hydrolyzed for 3 hours is not much different from the protoplast yield of 8-day-old Phellinus igniarius mycelium when hydrolyzed for 2 hours, but the total mass of mycelium cultured for 10 days is greater than that of mycelium cultured for 8 days. Therefore, considering all factors, the best choice is the 10-day-old Phellinus igniarius mycelium.
[0054] Comparative Example 5: S1: Same as S1 in Example 1.
[0055] S2: Add 1.5wt% lytic enzyme and 0.5wt% lytic enzyme to a 0.6mol / L mannitol solution to obtain a mixed enzyme solution; filter the mixed enzyme solution through a 0.2μm microporous filter membrane for sterilization and set aside; take 0.3g of Phellinus igniarius wet mycelium and add it to a 1.5mL centrifuge tube containing 1.0mL of the mixed enzyme solution, seal the centrifuge tube and shake the centrifuge tube up and down for 10s, then shake and enzymolyze in a constant temperature water bath at 30℃ and 120r / min for 1~5h to obtain Phellinus igniarius mycelium enzymolysis solution; S3: Same as S3 in Example 1.
[0056] S4: Same as S4 in Example 1.
[0057] The results are as follows Figure 5 As shown. With the extension of enzymatic hydrolysis time, the regeneration rate of Phellinus igniarius protoplasts showed a downward trend, because the enzymatic hydrolyzate was reacting all the time, and some cell structures were severely damaged, resulting in inability to regenerate. In addition, with the extension of enzymatic hydrolysis time, before 3h, it may be because more and more cells with plasmolysis occurred, and the cell wall on the surface of the cells became less and less, but they had not been completely hydrolyzed into protoplasts, so the regeneration of these cells would become easier. Although different enzymatic hydrolysis times can lead to changes in the regeneration rate of Phellinus igniarius protoplasts, the regeneration results of protoplasts shown on the modified PDA regeneration medium fluctuated little, and the regeneration rate was about 0.80%.
[0058] Embodiment 2: S1: Phellinus linteus protoplast mutagenesis S1-1: Dilute the phellinus linterus protoplast suspension prepared in Example 1 with 0.6 mol / L mannitol solution to 10 6 / mL, and get the Phellinus igniarius protoplast dilution. Take 21 sterilized modified PDA regeneration medium plates containing 0.1wt% lithium chloride, and add 1mL of Phellinus igniarius protoplast dilution to each plate. Turn on the ultraviolet light tube (15W) for 20 minutes before irradiation to stabilize the light wave. The mutagenesis process is carried out under dark conditions, and the illumination is completed by a red light bulb. After preheating, the plate is irradiated at a distance of 30cm from the ultraviolet lamp for 20s, and then an appropriate volume of each dish is diluted and spread on the regeneration medium, wrapped tightly with tin foil and placed at 26℃ in the dark for static culture.
[0059] Among them, the formula of the improved PDA regeneration medium is: dissolve 109.2g mannitol and 37g PDA medium powder in plant water extract (add 10g mulberry branch slice powder and 10g Oasis No. 1 powder to 1L distilled water, extract at 100℃ for 1h, filter with sterile filter paper and collect the filtrate to obtain plant water extract), make up to 1L with distilled water, and the pH is natural; the medium sterilization conditions are: high pressure sterilization pot 121℃, sterilization time 20min.
[0060] S1-2: Dilute the phellinus linterus protoplast suspension prepared in Example 1 with 0.6 mol / L mannitol solution to 10 6 / mL, and get the Phellinus igniarius protoplast dilution. Take 21 sterilized plates containing modified PDA regeneration medium, and add 1mL of Phellinus igniarius protoplast dilution to each plate. Turn on the ultraviolet light tube (15W) and preheat for 20 minutes before irradiation to stabilize the light wave. The mutagenesis process is carried out under dark conditions, and the lighting is completed by a red light bulb. After preheating, irradiate the plate at a distance of 30cm from the ultraviolet lamp for 20s, then take an appropriate volume of each dish, dilute it, and spread it on the regeneration medium, wrap it tightly with tin foil, and place it at 26℃ to avoid light for static culture.
[0061] Among them, the formula of the improved PDA regeneration medium is: dissolve 109.2g mannitol and 37g PDA medium powder in plant water extract (add 10g mulberry branch slice powder and 10g Oasis No. 1 powder to 1L distilled water, extract at 100℃ for 1h, filter with sterile filter paper and collect the filtrate to obtain plant water extract), make up to 1L with distilled water, and the pH is natural; the medium sterilization conditions are: high pressure sterilization pot 121℃, sterilization time 20min.
[0062] S1-3: Dilute the phellinus linterus protoplast suspension prepared in Example 1 with 0.6 mol / L mannitol solution to 10 6Take 21 sterilized modified PDA regeneration medium plates containing 0.1wt% lithium chloride, add 1mL of Phellinus linteus protoplast dilution to each plate, induce in dark conditions for 10min, then take an appropriate volume of each plate, dilute and spread on the regeneration medium, wrap tightly with tin foil and place at 26℃ in dark for static culture.
[0063] Among them, the formula of the improved PDA regeneration medium is: dissolve 109.2g mannitol and 37g PDA medium powder in plant water extract (add 10g mulberry branch slice powder and 10g Oasis No. 1 powder to 1L distilled water, extract at 100℃ for 1h, filter with sterile filter paper and collect the filtrate to obtain plant water extract), make up to 1L with distilled water, and the pH is natural; the medium sterilization conditions are: high pressure sterilization pot 121℃, sterilization time 20min.
[0064] S2: The initial screening of dominant strains is mainly a comprehensive screening of the growth characteristics of the induced strains visible to the naked eye. Observe and record the growth of the induced protoplasts cultured at 26°C in the dark every day, and use the growth characteristics of the starting strains that have not been induced as a control. It is generally believed that colonies with fast mycelium growth rate have more potential in the later mycelium fermentation stage, and strains with weak growth on flat culture media are unlikely to have outstanding performance even in the later mycelium fermentation stage. A comprehensive investigation of mycelium growth rate, colony thickness, density, etc. is used as an important indicator for initial screening.
[0065] S3: The purpose of identifying dominant strains is to identify the strains as new strains from both macroscopic and microscopic aspects, which are different from the starting strains. Macroscopic refers to antagonism experiments. When two strains with different genetic materials are cultured in the same culture dish, there will be obvious antagonism between the two colonies, that is, there will be no intersection between the two, and there is a clear separation line in the middle, which inhibits the development of hyphae. Microscopic means that from the perspective of molecular biology, two strains with different genetic materials must be different. This study used random amplified polymorphic DNA (RAPD) technology to identify that there are differences in genetic information between the new strain and the starting strain. S3-1: When hyphae of different strains are grown in the same space, obvious antagonistic lines can be seen at the junction of the hyphae of the two strains, which will restrict the growth of each other's hyphae. This is the antagonistic phenomenon. The strength of the antagonistic phenomenon can also reflect the size of the genetic differences between strains. The starting strain of Phellinus igniarius and the single colony of Phellinus igniarius that was initially screened after mutagenesis were inoculated on the modified PDA flat culture medium. The two strains were cultured at 26°C in the dark with a distance of 1.5 cm, and observed whether there were obvious antagonistic lines between the two colonies. If the hyphae of the two colonies grow together and no antagonistic lines are produced, it means that the strains of the two colonies are the same strain, not new individuals that have mutated. On the contrary, if antagonistic lines are produced, it can be said that the two strains are different individuals, the genetic material has mutated, and new traits are likely to be produced.
[0066] S3-2: Mycelial fermentation was performed on the dominant strains that were initially screened and antagonistic to the starting strains. Five 1 cm × 1 cm bacterial blocks were taken from the modified PDA regeneration medium and inoculated into the modified seed medium. They were cultured at 26°C, 140 r / min and away from light for 8 days. After shaking, they were inoculated into the fermentation medium for fermentation. The inoculation amount was 10% of the liquid strain. The culture was cultured at 26°C, 140 r / min and away from light for 8 days. The fermentation product was filtered, the mycelium was collected, and it was rinsed with distilled water 3 times. The fermentation liquid on the surface of the mycelium after fermentation was washed off, centrifuged, and then freeze-dried. The mycelial DNA was extracted from the obtained dry mycelium using the CTAB method. The arbitrarily primed PCR method (also known as random amplified polymorphic DNA (RAPD) and random primer PCR (RP-PCR) was used. The principle is to use a single-stranded artificially synthesized random oligonucleotide sequence (usually 10 base pairs) as a primer, and the cDNA produced by reverse transcription of the genomic DNA or RNA under study as a template to amplify the PCR product. The amplified products are separated by agarose electrophoresis, run on the gel, and their polymorphism is observed and analyzed. The polymorphism of these amplified DNA fragments reflects the polymorphism of the corresponding region of the genomic DNA. Theoretically, there are certain differences in the amplification patterns obtained using the same primers for different genomic DNAs, and the number of amplified bands depends on the complexity of the genome. RAPD-PCR amplification system: 1μL (100ng) DNA template, 2.5μL 10×PCR buffer (500mmol / KCl, 15mmol / LMgCl 2 ,100mmol / L Tris·HCl, pH 8.3), 2μL 0.2mmol / L dNTPs, 0.5U Taq DNA polymerase, 0.5μL 10mmol / L effective primer sequence, and finally ddH2 O was added to 25 μL. The amplification conditions were: 93°C pre-denaturation for 2 min, followed by 45 cycles of the following: 93°C denaturation reaction for 1 min, 36°C annealing reaction for 1 min, 72°C extension reaction for 1.5 min, and the last cycle of 72°C extension reaction for 10 min. After the cycle was completed, the reaction product was stored at 4°C. The reaction product was subjected to 1.2% agarose gel electrophoresis, and the spectrum was finally analyzed to see if there were significant differences.
[0067] S4: Flavonoid compounds in Phellinus igniarius are very important active substances. The total flavonoids yield after fermentation of Phellinus igniarius mycelium is used as an important indicator for rescreening. The mutant strains that were initially screened and confirmed to have genetic material mutations from antagonistic tests and molecular levels were fermented with mycelium, and their mycelial biomass, flavonoid content in mycelium, and total intracellular flavonoids yield were determined. The dominant strains that were antagonistic to the starting strains were fermented with mycelium after identification. Five 1cm×1cm blocks on the improved PDA regeneration medium were inoculated into the improved seed medium, and cultured at 26℃, 140r / min in the dark for 8 days to obtain liquid strains. After shaking, they were inoculated into a 250mL fermentation culture bottle containing 100mL of improved fermentation medium for fermentation. The inoculation amount was 10% of the liquid strains, and cultured at 26℃, 140r / min in the dark for 8 days. Filter the fermentation product, collect the mycelium, rinse it with distilled water three times, wash off the fermentation liquid on the surface of the mycelium after fermentation, centrifuge, and then freeze-dry. Weigh the obtained dry mycelium and calculate its biomass. Take 1g of dry mycelium, put it into a 25mL volumetric flask, dilute it with 80% ethanol, ultrasonically extract it for 2h (40kHz), centrifuge, take the supernatant, freeze-dry it, and set it aside. Refer to NaNO 2 -Al(NO 3 ) 3 Colorimetric method, the experiment uses NaNO 2 -Al(NO 3 ) 3 The total flavonoid content was determined by the colorimetric method, that is, in an alkaline nitrite solution, flavonoid compounds and aluminum ions formed a red complex with a characteristic absorption peak at 510 nm. The absorbance of the reaction product at 510 nm was measured to calculate the total flavonoid content in the sample. The standard substance was rutin, and a standard curve was prepared. According to the standard substance concentration curve equation, the content of flavonoid compounds in the three selected mycelia was calculated, the total intracellular flavonoid production was calculated, and the strain with the highest flavonoid production was selected.
[0068] S5: Determination of the antioxidant capacity of mycelial ethanol extract in vitro The ethanol extract was collected by centrifugation after ultrasonic extraction with 80% ethanol (liquid-to-solid ratio 10:1, m:v, 40kHz, 2h), and then the mycelium after fermentation of Phellinus igniarius was treated by freeze-drying to obtain the extract powder for evaluating its antioxidant capacity. The TEAC test (Trolox equivalent antioxidant capacity) was used to evaluate the ability of the compounds in the ethanol extract of Phellinus igniarius mycelium to scavenge ABTS free radicals; the FRAP (Ferric reducing antioxidant power) test was used to detect the antioxidant reduction of Fe under acidic conditions. 3+ to evaluate its antioxidant activity.
[0069] S5-1: Prepare 1mmol / L Trolox standard solution (prepared with 80% ethanol) and 7.4mmol / L ABTS solution; dissolve a certain amount of ethanol extract of Phellinus igniarius and Trolox in PBS phosphate buffer (20mmol / L, pH 7.4) respectively for later use. Mix 13.2mg potassium persulfate with 20mL of the prepared ABTS solution, shake well and place for 12~16h (room temperature, dark). When it changes to dark blue-green, it means that ABTS has been oxidized to blue-green ABTS cation free radicals. Store away from light for later use. Adjust the absorption wavelength of the UV spectrophotometer to 734nm, and dilute the mixed solution with PBS (20mmol / L, pH7.4) phosphate buffer until its absorbance value at the absorption wavelength of 734nm is about 0.7. After preparation, the antioxidant capacity of the ethanol extract of Phellinus igniarius needs to be determined within 4h. Mix 3.9 mL of ABTS cation mixed solution with 100 μL of mycelium ethanol extract solution, shake gently, react for 25 minutes, and measure the absorbance value at 734 nm absorption wavelength. In order to more intuitively present the antioxidant capacity, the ABTS free radical scavenging ability of the sample is expressed as Trolox equivalent (TEAC value), and different concentrations of Trolox standard solution are used as the control group system to draw the TEAC curve of the dominant strain.
[0070] S5-2: First, prepare the FRAP (Ferric reducing antioxidant power) reaction solution. The preparation method is as follows: 10mmol / L TPTZ solution, 20mmol / L ferric chloride solution and 300mmol / L acetate buffer with a pH value of 3.5 are mixed in a ratio of 1:1:10; then the powder extracted from the ethanol of Phellinus igniarius mycelium and dried is dissolved in 80% ethanol to adjust the appropriate concentration. After mixing, take 0.5mL and then take 4.0mL of FRAP reaction solution, mix and shake them well, and then keep them away from light. After reacting for 15 minutes, adjust the absorption wavelength of the UV spectrophotometer to 593nm and measure its absorbance value. In order to more intuitively present the antioxidant capacity of the sample to Fe 3+ The reducing ability was expressed as ferrous sulfate equivalent (FRAP value). Different concentrations of ferrous sulfate standard solution were used as the control group system to draw the FRAP curve of the dominant strain.
[0071] The initial screening can only be analyzed based on the comprehensive growth of mycelium. Most of the strains that have been induced are negative mutations. Compared with the original strains, they grow slower and have sparser mycelium. Only a few have improved mycelium growth rate. From the 90 strains that can be regenerated after mutagenesis, one strain was selected from each of the three different mutagenesis groups, and three dominant strains from different mutagenesis treatments were obtained ( Figure 6 ), the three strains were the best in each group in terms of growth rate and mycelium density. The three strains initially screened provided a guarantee for the subsequent determination of flavonoid production and the determination of the in vitro antioxidant activity of mycelium after fermentation, the re-screening and identification of new strains, and the comparison of the advantages and disadvantages of different mutagenesis methods.
[0072] Table 3 Growth of mutant strains UL27, U8, and L13 like Figure 7 As shown, the starting strain G1 and the mutant strains UL27, U8 and L13 each had obvious antagonistic lines. From a macroscopic perspective, the mutagenic strains were identified as new strains with different traits. The three dominant strains were different from the starting strain G1 of Phellinus igniarius.
[0073] RAPD amplification electrophoresis pattern Figure 8 As shown, compared with the starting strain G1, the mutant strains UL27, U8 and L13 all had slight differences at different bp, proving that their genetic materials had mutated and the strains were identified as new strains.
[0074] The results of flavonoid yield analysis are shown in Table 4. Compared with the starting strain G1, the flavonoid content of the mutant strains UL27, U8 and L13 all changed significantly, and the total flavonoids increased by 64.8%, 24.3% and 56.7%, respectively. The results showed that the strains that underwent compound mutagenesis, the strain UL27 obtained after primary screening and rescreening, had the highest increase in flavonoid yield, and the strains U8 and L13 that underwent single mutagenesis had a smaller increase in flavonoid yield. This result can infer to a certain extent that compound mutagenesis may be more conducive to positive mutations and more likely to help people cultivate new varieties with excellent traits.
[0075] Table 4 Analysis of flavonoid production of mutant strains and starting strains The results of in vitro antioxidant capacity analysis of mycelium ethanol extracts are shown in Fig. 9 As shown in the figure, the TEAC values of mutant strains UL27, U8, and L13 increased by 38.5%, 14.9%, and 32.2% respectively compared with the starting strains, and the antioxidant capacity was significantly improved. The results showed that the strain UL27 obtained after primary screening and rescreening from the strains that underwent compound mutagenesis had the highest improvement in the antioxidant capacity of the strains measured, while the antioxidant capacity of the strains with single mutagenesis increased less. Therefore, compound mutagenesis may be more conducive to positive mutations and more likely to help people cultivate new varieties with excellent traits.
[0076] Fe 3+ The results of reducing capacity determination are as follows Fig.10 As shown in the figure, the FRAP values of mutant strains UL27, U8, and L13 increased by 48.9%, 24.4%, and 32.7% respectively compared with the starting strains, and the antioxidant capacity was significantly improved. The results showed that the strain UL27 obtained after primary screening and rescreening from the strains that underwent compound mutagenesis had the highest improvement in the antioxidant capacity of the strains measured, while the antioxidant capacity of the strains with single mutagenesis increased less. Therefore, compound mutagenesis may be more conducive to positive mutations and more likely to help people cultivate new varieties with excellent traits.
Claims
1. A method for preparing and regenerating protoplasts of Phellinus linteus, characterized in that: The following steps are involved: S1: Inoculate the 4℃ low-temperature preserved Phellinus linteus onto the modified PDA medium plate, incubate at 26℃ in the dark for activation, and then take the 10×10mm 2 5 pieces of mycelium blocks were inoculated into a 250 mL Erlenmeyer flask containing 100 mL of modified PDB medium, and cultured at 26°C in the dark, with shaking once in the morning and evening every day during the culture process; the mycelium cultured in the dark for 8 to 16 days was washed with sterile water, and then the surface moisture was absorbed with sterile filter paper to obtain Phellinus igniarius wet mycelium; S2: Add 1.0wt% to 2.0wt% of lytic enzyme and 0.5wt% of collapsing enzyme to a 0.6mol / L mannitol solution to obtain a mixed enzyme solution; filter the mixed enzyme solution through a 0.2μm microporous filter membrane for sterilization and set aside; take 0.3g of Phellinus igniarius wet mycelium and add it to a 1.5mL centrifuge tube containing 1.0mL of the mixed enzyme solution, seal the centrifuge tube and shake the centrifuge tube up and down for 10s, then enzymolyze at 20~40℃ and 120r / min for 1~5h to obtain Phellinus igniarius mycelium enzymolysis solution; S3: Filter the enzymatic hydrolyzate of Phellinus linteus hyphae with a sterilized G3 sand core funnel, collect the filtrate into a 1.5 mL centrifuge tube, centrifuge at 4°C and 3000 g for 10 min, discard the supernatant, and wash the precipitate with 0.6 mol / L mannitol solution for 3 times to obtain purified Phellinus linteus protoplasts; disperse the purified Phellinus linteus protoplasts in 0.6 mol / L mannitol solution to obtain a Phellinus linteus protoplast suspension; S4: dilute the phellinus linus protoplast suspension with 0.6 mol / L mannitol solution to obtain a phellinus linus protoplast dilution solution; take 1.0 mL of the phellinus linus protoplast dilution solution and spread it on a modified PDA regeneration medium plate, and place it at 26° C. in the dark for regeneration culture.
2. The method according to claim 1, characterized in that: The step S1 is: inoculate the Phellinus linteus strains stored at 4°C onto a modified PDA culture medium plate, place it in a dark place at 26°C for activation, and then take the 10×10 mm thick, vigorous growth 2 Five mycelium blocks were inoculated into a 250-mL Erlenmeyer flask containing 100 mL of modified PDB medium and cultured at 26°C in the dark. The culture was shaken once in the morning and once in the evening every day during the culture process. The mycelium that had been cultured in the dark for 10 days was washed with sterile water, and then the surface moisture was absorbed with sterilized filter paper to obtain the wet mycelium of Phellinus igniarius.
3. The method according to claim 1, characterized in that: The step S2 comprises: adding a lytic enzyme with a final concentration of 1.5 wt% and a disintegrating enzyme with a final concentration of 0.5 wt% to a 0.6 mol / L mannitol solution to obtain a mixed enzyme solution; filtering the mixed enzyme solution through a 0.2 μm microporous filter membrane for sterilization and setting aside for later use; taking 0.3 g of wet mycelium of Phellinus igniarius and adding it to a 1.5 mL centrifuge tube containing 1.0 mL of the mixed enzyme solution, sealing the centrifuge tube and shaking the centrifuge tube up and down for 10 seconds, and then shaking and enzymolyzing in a constant temperature water bath at 30° C. and 120 r / min for 3 hours to obtain an enzymatic hydrolyzate of Phellinus igniarius mycelium.
4. The method according to claim 1, characterized in that: The formula of the improved PDB culture medium is: dissolve 24g of PDB culture medium powder in plant water extract, dilute to 1L with distilled water, and the pH is natural; the preparation method of the plant water extract is: add 10g of mulberry branch slice powder and 10g of Oasis No. 1 powder to 1L of distilled water, extract at 100°C for 1h, filter with sterile filter paper and collect the filtrate to obtain the plant water extract.
5. The method according to claim 1, characterized in that: The formula of the improved PDB culture medium is: dissolve 24g of PDB culture medium powder in plant water extract, dilute to 1L with distilled water, and the pH is natural; the preparation method of the plant water extract is: add 10g of mulberry branch slice powder and 10g of Oasis No. 1 powder to 1L of distilled water, extract at 100°C for 1h, filter with sterile filter paper and collect the filtrate to obtain the plant water extract.
6. The method according to claim 1, characterized in that: The formula of the improved PDA regeneration medium is: dissolve 109.2g of mannitol and 37g of PDA medium powder in plant water extract, dilute to 1L with distilled water, and the pH is natural; the preparation method of the plant water extract is: add 10g of mulberry branch slice powder and 10g of Oasis No. 1 powder to 1L of distilled water, extract at 100°C for 1h, filter with sterile filter paper and collect the filtrate to obtain the plant water extract.
7. Use of the method as claimed in claim 1 in breeding excellent Phellinus linterus strains using protoplast technology.
8. The use according to claim 7, characterized in that: The mulberry linterus protoplast suspension described in claim 1 is subjected to ultraviolet and lithium chloride combined mutagenesis, and the protoplasts after mutagenesis are regenerated and cultured, and the mulberry linterus dominant mutant strains with improved mycelium growth rate, mycelium biomass, flavonoids production and antioxidant activity of mycelium ethanol extract are obtained through screening.