An ARTP mutagenesis protective agent and its application

Abstract

The present invention belongs to the technical field of microbial breeding, and specifically relates to an ARTP mutagenesis protectant and its application. The present invention discloses an ARTP mutagenesis protectant, which comprises the following components in mass percentage: 1-3% of water-soluble acid, 1-2% of repair enzyme, 3-10% of lipid-soluble substances, and the balance is a stabilizer. By screening and adjusting the formula of the mutagenesis protectant, an ARTP mutagenesis protectant formulation 1 is obtained, which reduces the mutagenesis lethality rate of strains and increases the regeneration rate, promotes the SOS repair and the ability of the oxidative damage repair system of gene mutation strains, significantly improves the positive mutation rate, and greatly improves the mutation efficiency. The present invention also explores the technical conditions of ARTP mutagenesis, and screens out conditions such as the optimal addition ratio, the optimal incubation time, and the mutagenesis time, thereby improving the breeding efficiency of target strains.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial breeding, and particularly relates to an ARTP mutagenesis protectant and its application. Background Art

[0002] Atmospheric and room temperature plasma mutagenesis (ARTP) is a method that, under normal temperature and pressure, can generate a large amount of reactive oxygen species, reactive nitrogen species, ultraviolet photons, high-energy ions, charged particles, etc. through plasma energy, thereby inducing gene mutations in organisms, including point mutations and translocations, exchanges, or deletions of large gene fragments. Its damage mechanism to genetic material is diverse, so the possibility of obtaining mutant diversity increases, thus causing mutant biological effects. It can greatly accelerate the breeding cycle of natural domestication and shows its unique advantages in the mutagenesis breeding of microorganisms with complex metabolic networks. Although biological means such as genetic engineering and cell engineering have begun to be used in the transformation of industrial microbial strains and have very attractive application prospects, physical mutagenesis techniques such as ARTP mutagenesis are still the main application means of mutagenesis breeding, and this technique is widely used in bacteria, fungi (including yeasts, molds, and macrofungi), actinomycetes, algae, plant cells, and animal cells.

[0003] However, the biological effects caused by ARTP breeding are diverse and have dual results: it can not only cause gene mutations but also induce organisms to accelerate the repair of DNA damage induced by low-temperature plasma, such as the SOS repair system, oxidative damage repair system, etc. If base mismatches occur during the repair process and the strain survives, mutant strains will be generated. At the same time, ARTP can also stimulate the body to promote the increase in the activity of antioxidant enzymes, promote the growth of individuals, extend the accumulated lifespan, and increase the content of metabolites. If ARTP destroys the structures of nucleic acids, proteins, lipids, and polysaccharides, causes membrane damage and enzyme inactivation, and makes the cells unable to repair the damage, it will cause cell apoptosis; thus resulting in a large loss of positive mutants.

[0004] A large number of studies have shown that the repair of cell damage has become a key step restricting mutant strains, and the addition of free radical scavengers can effectively slow down this destructive effect. The ARTP protective solution disclosed in Patent CN 114317517 A is 10% glycerol as the protective solution after mutagenesis; the ARTP protective solution disclosed in CN 114292838 A is 20% glycerol as the protective solution after mutagenesis. The above-mentioned mutagenesis protective solutions have simple compositions and no significant ability to promote the repair of SOS genes, resulting in the death of a large number of positive mutant strains. There are few reports on ARTP mutagenesis protective solutions at home and abroad. According to literature research, free radical scavengers mainly include superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), astaxanthin, vitamin C, vitamin E, salvianolic acid A, salvianolic acid B, thioredoxin, etc. They eliminate oxidative damage by reducing the content of cellular reactive oxygen free radical molecular intermediates. Patent CN201010146991 invented a high-efficiency composite food antioxidant of tea polyphenols, rosemary, leaf flavonoids, citric acid, and vitamin C; the cell repair promoting solution invented in Patent CN202111359461, whose main components are notoginsenoside, salicin, allantoin, etc., provides sufficient theoretical basis for the present invention.

[0005] In this case, by adding an ARTP mutagenesis protective agent to improve the ARTP mutagenesis and breeding method, the SOS repair and oxidative damage repair systems are enhanced, thereby generating more positive mutant strains. Through the detection of the individual survival rate of the strains and the vitality of the cells, and combined with the verification of the indicator method plate screening and shake flask fermentation experiments, more positive mutant strains are screened out, greatly improving the success rate of breeding. Summary of the Invention

[0006] The technical problem to be solved by the present invention is aimed at the deficiencies of the prior art. Specifically, ARTP overly damages the structures of nucleic acids, proteins, lipids, and polysaccharides, causes membrane damage and enzyme inactivation, resulting in apoptosis of cells that are difficult to repair themselves, and a low resurrection rate of mutants. The present invention provides an ARTP mutagenesis protective agent.

[0007] Another problem to be solved by the present invention is to provide the application of the above ARTP mutagenesis protective agent in ARTP mutagenesis and breeding.

[0008] To solve the above technical problems, the present invention discloses an ARTP mutagenesis protective agent to improve the repair capabilities of the SOS repair and oxidative damage repair systems, thereby generating more positive mutant strains.

[0009] An ARTP mutagenesis protective agent comprises the following components in mass percentages: 1-3% of water-soluble acid, 1-2% of repair enzyme, 3-10% of lipid-soluble substances, and the balance is stabilizer.

[0010] Among them, the water-soluble acids include any one or a combination of several of lauric acid, maleic acid, tea polyphenols, citric acid, vitamin C, glutathione, anthocyanin, chlorogenic acid, glycyrrhizic acid, ferulic acid, hyaluronic acid, glycine, schisandra peptide, astaxanthin, chromium picolinate, rosmarinic acid; the repair enzymes include any one or a combination of several of superoxide dismutase, catalase, glutathione peroxidase, thioredoxin reductase, glucose oxidase, coenzyme Q10; the fat-soluble substances include any one or a combination of several of glycerol, retinol, rosmarinic ether, vitamin E, lecithin, sucrose ester, high acid value camellia seed oil, leaf flavonoids, vitamin A, β-lactoglobulin powder, monoglyceride oleate, dibutylhydroxytoluene; the stabilizers include any one or a combination of several of buffer salts, osmotic pressure solutions, ultrapure water.

[0011] Specifically, the buffer salt is PBS 10-20 mM pH6.0; the osmotic pressure solution is any one of 0.6-0.8 M NaCl, 0.6-0.8 M MgSO4, 0.6-0.8 M KCl.

[0012] Studies have shown that tocopherol (vitamin E), rosemary extract, and high acid value camellia seed oil have high antioxidant capacities; tea polyphenols, rosmarinic ether, leaf flavonoids, and vitamin C have high antioxidant adsorption capacities; coenzyme Q10 can promote cell energy metabolism; lauric acid and astaxanthin are good phenolic antioxidants; glutathione can be used as a multi-organism antioxidant for animals; superoxide dismutase, catalase, and thioredoxin reductase are common, inexpensive, and easily available antioxidant enzymes for damage repair, and their prices are 20-2000 yuan / mg.

[0013] Therefore, in the present invention, by improving the antioxidant capacity, preferably, the water-soluble acids include any one of vitamin C, glutathione, tea polyphenols, astaxanthin, lauric acid; the repair enzymes include any one of superoxide dismutase, catalase, thioredoxin reductase, coenzyme Q10; the fat-soluble substances include any one of glycerol, vitamin E, high acid value camellia seed oil, leaf flavonoids, rosmarinic ether; the stabilizer is any one of PBS 10 mM pH6.0, 0.8 M NaCl to assist in cell damage repair. Considering price cost, availability, and efficacy comprehensively, the above ARTP mutagenesis protectant is preferably one of the following 5 formulations in specific embodiments, but is not limited to these 5 formulations: (by mass percentage)

[0014] Formulation 1: 2% vitamin C, 1% superoxide dismutase, 5% glycerol, and the balance is stabilizer;

[0015] Formula 2: 2% glutathione, 1% catalase, 5% vitamin E, and the balance is a stabilizer;

[0016] Formula 3: 2% tea polyphenols, 1% thioredoxin reductase, 5% high acid value camellia seed oil, and the balance is a stabilizer;

[0017] Formula 4: 2% astaxanthin, 1% thioredoxin reductase, 5% leaf flavonoids, and the balance is a stabilizer;

[0018] Formula 5: 2% lauric acid, 1% coenzyme Q10, 5% rosmarinic ether, and the balance is a stabilizer;

[0019] More preferably, it is Formula 1: 2% vitamin C, 1% superoxide dismutase, 5% glycerol, and the balance is a stabilizer.

[0020] The preparation method of the above ARTP mutagenesis protective agent is specifically to mix the water-soluble acid, repair enzyme and lipid-soluble substances in the formula evenly, dissolve them with a stabilizer, and obtain a sterile protective solution by passing through a yellow aqueous filter membrane with a pore size of 100 μm after centrifugation for standby.

[0021] The application of the above ARTP mutagenesis protective agent in ARTP mutagenesis breeding is also within the scope protected by the present invention.

[0022] Among them, for the above ARTP mutagenesis breeding, the object is any one of bacteria, fungi (including yeasts, molds and macrofungi), actinomycetes, algae, plant cells and animal cells.

[0023] Preferably, they are bacteria and fungi, and more preferably, they are lactic acid bacteria among bacteria, yeasts among fungi and the edible mushroom Pleurotus djamor among edible fungi.

[0024] The ARTP mutagenesis breeding described in the present invention is not limited to the specific breeding objects under the above classification, and the breeding objects satisfying the above classification in the prior art are all applicable to the present invention.

[0025] The above application in ARTP mutagenesis breeding is specifically to mix the object to be mutagenized and bred with the above ARTP mutagenesis protective agent and then perform ARTP mutagenesis, and after the ARTP mutagenesis is completed, place it in the above ARTP mutagenesis protective agent for incubation.

[0026] Among them, the mixing of the object to be mutagenized and bred with the above ARTP mutagenesis protective agent is specifically to prepare the object to be mutagenized and bred into a suspension of 10 5 -10 6 CFU / mL and then mix it with the ARTP mutagenesis protective agent according to a volume ratio of 1-5:1-10, and the preferred volume ratio is 1:1.

[0027] Among them, for the ARTP mutagenesis, the mutagenesis conditions are as follows: under the conditions of a working power of 100 - 120 W, a working gas flow of 10 - 12 SLM, and an irradiation distance of 2 - 4 mm, the irradiation time is set to 0 - 240 s.

[0028] The preferred mutagenesis conditions are as follows: under the conditions of a working power of 100 W, a working gas flow of 10 SLM, and an irradiation distance of 2 mm, the irradiation time is set to 120 s.

[0029] Among them, for the incubation, the conditions are as follows: incubate at 20 - 30 °C for 30 - 120 min.

[0030] The preferred incubation conditions are as follows: incubate at 26 °C for 90 min.

[0031] Beneficial effects:

[0032] (1) The present invention discloses an ARTP mutagenesis protectant, which is composed of the following components in mass percentages: 1 - 3% of water-soluble acid, 1 - 2% of repair enzyme, 3 - 10% of lipid-soluble substances, and the balance is a stabilizer. Through component screening, a preferred ARTP mutagenesis protectant is obtained, which promotes the ability of the SOS repair and oxidative damage repair systems of gene mutation strains, generates more positive mutation strains, and greatly improves the mutation efficiency.

[0033] (2) During the ARTP breeding process of bacteria, yeast, and large edible fungi (including mycelia, protoplasts, spore suspensions), the mutagenesis lethal rate of the ARTP mutagenesis protectant is reduced from more than 90% to less than 55%, greatly improving the regeneration rate of mutant strains; at the same time, the positive mutation rate is increased from 14.28% to 46.67%, greatly increasing the breeding probability of positive mutations.

[0034] (3) The present invention explores the technical conditions of ARTP mutagenesis, screens out the optimal addition ratio, the most suitable incubation time, mutagenesis time, etc., and improves the breeding efficiency of target strains. Description of the drawings

[0035] The following further specifically describes the present invention in conjunction with the drawings and specific embodiments, and the above and / or other advantages of the present invention will become clearer.

[0036] Figure 1 It is the mutagenesis lethal rate of strain GXGD-EF-13-JD-1 (left without mutagenesis protectant, right with mutagenesis protectant formula 1).

[0037] Figure 2 It is the regeneration growth situation of strain GXGD-EF-13-JD-1 after mutagenesis (left without mutagenesis protectant, right with mutagenesis protectant formula 1).

[0038] Figure 3It is the fruiting body and spore suspension of strain GXGD-EF-13-JD-1.

[0039] Figure 4 This is the regeneration growth of strain JM-1 after mutagenesis (left is without mutagenesis protectant, right is with mutagenesis protectant formula 1).

[0040] Figure 5 This is the regeneration growth of strain RSJ-1 after mutagenesis (left is without mutagenesis protectant, right is with mutagenesis protectant formula 1). DETAILED DESCRIPTION

[0041] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0042] In the following embodiments, the strain is Pleurotus djamor, strain number GXGD-EF-13-JD-1, deposited in Guangdong Microbiological Culture Collection Center on November 9, 2022, with a deposit number of GDMCC No: 62953, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0043] In the following examples, the yeast strain is Saccharomyces cerevisiae, the strain number of which is JM-1, and the source of which is any one of CICC 32883, CICC 1369, and CICC 1299.

[0044] In the following examples, the lactic acid strain is Lactobacillus plantarum, the strain number of which is RSJ-1, and the source of which is any one of CICC 24936, CICC 6043, and CICC 25125.

[0045] In the following examples, tea polyphenols, astaxanthin, lauric acid, superoxide dismutase (SOD), catalase, thioredoxin reductase, coenzyme Q10, and glycerol used in the enzyme system were all purchased from Aladdin; vitamin E, high acid value camellia seed oil, vitamin C, and glutathione were all purchased from Yuanye.

[0046] In the following examples, the preparation method of the mutagenesis protective agent is as follows: After uniformly mixing the water-soluble acid, repair enzyme and lipid-soluble substances in the formula, dissolve them with a stabilizer, centrifuge, and then filter and sterilize through a yellow aqueous filter membrane with a pore size of 100 μm to obtain a sterile protective solution for standby. Among them, the stabilizer includes any one of buffer salts, osmotic pressure solutions, and ultrapure water. Specifically, the buffer salt is PBS 10 - 20 mM pH 6.0; the osmotic pressure solution is any one of 0.6 - 0.8 M NaCl, 0.6 - 0.8 M MgSO4, and 0.6 - 0.8 M KCl; the preferred stabilizer is PBS 10 mM pH 6.0 or 0.8 M NaCl.

[0047] Example 1: Exploration of the lethal rate of ARTP mutagenesis

[0048] (1) Strain activation: Transfer the strain GXGD-EF-13-JD-1 to a PDA medium with an inoculation shovel, and place it in a constant temperature incubator at 26 °C for 6 - 7 d. Wait until the mycelium covers the plate for standby.

[0049] Among them, the formula of the PDA medium is: potato juice 200 g / L, glucose 20 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate 1.5 g / L, VB1 0.1 g / L;

[0050] (2) Preparation of the seed liquid: Transfer the well-grown plate in step (1) to the seed medium by inoculating 4 - 5 mycelial blocks with a diameter of 0.5 cm per bottle. There are two parallels in each group, and culture at 26 °C and 150 rpm for 5 - 8 d to prepare the seed liquid. Observe that it is in good condition for standby.

[0051] Among them, the formula of the seed medium is: soybean powder 30 g / L, glucose 30 g / L, yeast extract 3 g / L, potassium dihydrogen phosphate 0.5 g / L, anhydrous magnesium sulfate 0.5 g / L, ferrous sulfate heptahydrate 0.18 g / L, VB1 0.01 g / L, pH 6.0, and the liquid loading amount is 150 mL / 500 mL.

[0052] (3) Dissolve the enzyme system M2 with a stabilizer, centrifuge, and then filter and sterilize through a yellow aqueous filter membrane with a pore size of 100 μm to obtain an enzymolysis solution for standby.

[0053] Among them, the enzyme system M2 is: bovine serum albumin 50 mg, snail enzyme 50 mg, lytic enzyme 50 mg, Lysingenzymes 50 mg, Yatalase 50 mg, β-glucuronidase 100 μL, pectinase 100 mg, cellulase 200 μL, stabilizer (PBS 10 mM pH 6.0) 30 mL, an enzyme concentration of 0.67 g / 100 mL, pH 5.0 - 6.0, and dissolve it with a stabilizer.

[0054] (4) Preparation of mycelium suspension: Shake and disperse the seed liquid cultured in step (2) with glass beads, filter it with a magical filter cloth to obtain 100 - 200 mL of mycelium suspension, centrifuge it at 10000 rpm for 10 min, pour off the supernatant, dilute the precipitate with 0.85% normal saline to 1 mL, concentrate it 100 - 200 times, and reserve it for use.

[0055] (5) Preparation of protoplasts: Mix the enzymolysis solution prepared with enzyme system M2 in step (3) with the mycelium suspension prepared in step (4) to prepare mycelium protoplasts. After purification, adjust the number of protoplasts to 10 5 -10 6 CFU / mL, and reserve it for use.

[0056] (6) ARTP mutagenesis: Respectively pipette 10 - 15 μL of the mycelium protoplast suspension prepared in step (5) and evenly coat it on a small iron sheet (coat more for backup), control the liquid layer thickness to be 0.2 - 0.4 cm, cover it with a glass plate, and reserve it for use. Set the conditions of the ARTP mutagenizer as follows: under the conditions of working power 100 W, working gas flow 10 SLM, and irradiation distance 2 mm, set the irradiation time to 0 - 180 s. After the mutagenesis is completed, place the sample in a 2 mL centrifuge tube containing 150 μL of 0.85% normal saline, incubate it at 26°C in the dark for 2 h, then elute and shake it with a magnetic levitation shaker for no less than 1 min, and reserve it for use.

[0057] (7) Plate coating: Spread the plate mutagenized in step (6) on a regeneration PDA plate, culture it statically at 26°C, and after single colonies grow on the plate, calculate its mutagenic lethality rate as shown in Table 1.

[0058] Among them, the formula of the regeneration PDA plate is: 2 g / L potassium penicillin, 205 g / L sucrose, 200 g / L potato juice, 20 g / L glucose, 3 g / L potassium dihydrogen phosphate, 1.5 g / L magnesium sulfate, 0.1 g / L VB1.

[0059] Table 1 Mutagenic lethality rate of its protoplasts without mutagenic protectant

[0060]

[0061]

[0062] Result analysis: According to the analysis of the mutagenic lethality rate without mutagenic protectant in Table 1, the results show that when the mutagenesis time is 120 s, the lethality rate is high and it has the potential to produce better mutant strains. Therefore, this condition is used as the best condition for further screening of mutagenic protectants.

[0063] Example 2: Exploration of the ARTP mutagenesis protective agent formula

[0064] (1) The activation of the strain, preparation of the seed solution, preparation of the enzyme system, preparation of the mycelium suspension, and preparation of the protoplasts were the same as steps (1)-(5) of Example 1.

[0065] (2) ARTP mutagenesis: Respectively pipette 10 μL of the protoplasts prepared in step (1) and 5 mutagenesis protective agents, and coat them on a small iron sheet in a ratio of 1:1 (coat more for backup), control the liquid layer thickness to be 2-4 mm, cover it with a glass plate, and set aside. Set the conditions of the ARTP mutagenesis instrument as follows: working power 100 W, working gas flow 10 SLM, irradiation distance 2 mm, and set the irradiation time to 120 s. After the mutagenesis is completed, place the samples in 2 mL centrifuge tubes containing 150 μL of the corresponding mutagenesis protective agent respectively. After incubating at 26 °C in the dark for 2 h, elute and shake with a magnetic suspension shaker, and the elution and shaking time is not less than 1 min, and set aside.

[0066] Among them, the 5 mutagenesis protective agents and their formula contents are shown in Table 2.

[0067] Table 2 5 protective agents and their formula contents (by mass fraction)

[0068]

[0069] (3) Plate coating: Coat the plates mutagenized in step (2) on the regenerated PDA plates and incubate them statically at 26 °C. After single colonies grow on the plates, calculate their mutagenesis lethality rates. The results are shown in Table 3.

[0070] Among them, the regenerated PDA plate is: potassium penicillin 2 g / L, sucrose 205 g / L, potato juice 200 g / L, glucose 20 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate 1.5 g / L, VB1 0.1 g / L.

[0071] Table 3 Statistics of the mutagenesis lethality rates corresponding to 5 mutagenesis protective agents

[0072]

[0073] Result analysis: According to the exploration of the 5 mutagenesis protective agents in Table 3, it was found that by analyzing their mutagenesis lethality rates, it was found that these 5 mutagenesis protective agents could effectively reduce their mutagenesis lethality rates, greatly improving the regeneration rate of their mutant strains, providing a sufficient mutant strain regeneration library for the screening of high-yield strains in the later stage. Among them, the mutagenesis lethality rate of formula 1 was 20.65%, and the regeneration rate was the highest. Further research will be carried out on it in the later stage.

[0074] Example 3: Exploration of the usage ratio of the ARTP mutagenesis protective agent

[0075] (1) The activation of the strain, the preparation of the seed liquid, the preparation of the enzyme system, the preparation of the mycelium suspension, and the preparation of the protoplasts are the same as steps (1)-(5) of Example 1.

[0076] (2) ARTP mutagenesis: Respectively pipette 10 μL of the protoplasts prepared in step (1) and mutagenesis protective agent formulation 1, and coat them on a small iron sheet according to the ratios of 1:1, 1:2, 1:5, and 1:10 (coat more for backup). Control the liquid layer thickness to be 0.2 - 0.4 cm, cover it with a glass plate, and set aside. Set the conditions of the ARTP mutagenizer as follows: under the conditions of a working power of 100 W, a working gas flow of 10 SLM, and an irradiation distance of 2 mm, set the irradiation time to 120 s. After the mutagenesis is completed, place the sample in a 2 mL centrifuge tube containing 150 μL of mutagenesis protective agent formulation 1, incubate it at 26 °C in the dark for 2 h, then wash and shake it with a magnetic levitation shaker for no less than 1 min, and set aside.

[0077] (3) Plate coating: Coat the plate mutagenized in step (2) on a regeneration PDA plate and incubate it statically at 26 °C. After single colonies grow on the plate, calculate its mutagenesis lethality rate, as shown in Table 4.

[0078] Table 4 Applicable ratios of mutagenesis protective agent formulation 1 (by volume fraction)

[0079]

[0080] Result analysis: According to the exploration of the applicable ratios of mutagenesis protective agent formulation 1 in Table 4, by analyzing its mutagenesis lethality rate, it is found that the optimal ratio of the bacterial liquid volume to the applicable ratio of the mutagenesis protective agent formulation is 1:1 (volume ratio). Among them, the mutagenesis lethality rate of formulation 1 is 22.47%, and the regeneration rate is the highest; its operation is simple and convenient, and further research will be carried out on it later.

[0081] Example 4: Exploration of the incubation time of the ARTP mutagenesis protective agent

[0082] (1) The activation of the strain, the preparation of the seed liquid, the preparation of the enzyme system, the preparation of the mycelium suspension, and the preparation of the protoplasts are the same as steps (1)-(5) of Example 1.

[0083] (2) ARTP mutagenesis: Respectively pipette 10 μL of the protoplasts prepared in step (1) and mutagenesis protective agent formula 1, coat them on a small iron sheet according to a ratio of 1:1 (coat more for backup), control the liquid layer thickness to be 0.2 - 0.4 cm, cover it with a glass plate, and set aside. Set the conditions of the ARTP mutagenesis instrument as follows: under the conditions of a working power of 100 W, a working gas flow of 10 SLM, and an irradiation distance of 2 mm, set the irradiation time to 120 s. After mutagenesis, place the sample in a 2 mL centrifuge tube containing 150 μL of mutagenesis protective agent formula 1, incubate it at 26 °C in the dark for 30 min, 60 min, 90 min, and 120 min, then use a magnetic levitation shaker to elute and shake, and the elution and shaking time is not less than 1 min, and set aside.

[0084] (3) Plate coating: Spread the plate mutagenized in step (2) on a regeneration PDA plate and incubate it statically at 26 °C. After single colonies grow on the plate, calculate its mutagenic lethality rate. The results are shown in Table 5.

[0085] Table 5 Statistics of lethality rate under different incubation times of mutagenesis protective agent

[0086]

[0087] Result analysis: According to the exploration of the incubation time of the mutagenesis protective agent in Table 5, by analyzing its mutagenic lethality rate, it is found that as the incubation time of the mutagenesis protective agent prolongs, its lethality rate becomes lower, indicating that the mutagenesis protective agent has a good repair effect on mutant damaged strains. Among them, between 90 min and 120 min, the change in its lethality rate is not significant. Considering saving time costs, the optimal incubation time is determined to be 90 min.

[0088] Example 5: Application of ARTP mutagenesis protective agent in protoplasts 1

[0089] (1) The activation of the strain, the preparation of the seed liquid, the preparation of the enzyme system, the preparation of the mycelium suspension, and the preparation of the protoplasts are the same as steps (1) - (5) of Example 1.

[0090] (2) ARTP mutagenesis: Respectively pipette 10 μL of the protoplasts in step (1) and mutagenesis protective agent formula 1, coat them on a small iron sheet according to a ratio of 1:1 (coat more for backup), control the liquid layer thickness to be 0.2 - 0.4 cm, cover it with a glass plate, and set aside. Set the conditions of the ARTP mutagenesis instrument as follows: under the conditions of a working power of 100 W, a working gas flow of 10 SLM, and an irradiation distance of 2 mm, set the irradiation time to 120 s. After mutagenesis, place the sample in a 2 mL centrifuge tube containing 150 μL of mutagenesis protective agent formula 1, incubate it at 26 °C in the dark for 90 min, then use a magnetic levitation shaker to elute and shake, and the elution and shaking time is not less than 1 min, and set aside.

[0091] (3) Coating the plate: Spread the plate mutagenized in step (2) on the regenerated PDA plate and incubate it statically at 26°C. After single colonies grow on the plate, calculate the mutagenic lethality rate, as shown in Figure 1 and Table 6.

[0092] Table 6 Mutagenic lethality rate of strain GXGD-EF-13-JD-1

[0093]

[0094] (4) Measurement of the growth rate of mutant strains: Select 10 - 20 regenerated strains mutagenized in step (3) and incubate them statically on the regenerated PDA plate at 26°C. Observe and measure their growth rates, and calculate their positive mutation rates, as shown in Figure 2 and Tables 7 and 8.

[0095] Table 7 Regenerated growth of strain GXGD-EF-13-JD-1 without mutagenic protectant

[0096]

[0097]

[0098] Table 8 Regenerated growth of strain GXGD-EF-13-JD-1 with mutagenic protectant

[0099]

[0100] Result analysis: According to the results in Table 6, it can be seen that the regeneration rate of the formula without mutagenic protectant and formula 1 with mutagenic protectant increased from 7.56% to 77.86%, and the regeneration rate increased by 9.30 times; according to Figure 2 the pictures and data in Tables 7 and 8, it can be seen that the positive mutation rate of the formula without mutagenic protectant and formula 1 with mutagenic protectant increased from 14.28% to 46.67%, greatly improving the positive mutation rate.

[0101] Example 6: Application of ARTP mutagenic protectant in spore suspension 2

[0102] (1) Collect the spores of strain GXGD-EF-13-JD-1 during fruiting, and adjust the number of the spore suspension to 10 5 -10 6 CFU / mL with 0.8M NaCl for standby, as shown in Figure 3 .

[0103] (2) ARTP mutagenesis: Pipette 10 - 15 μL of the spore suspension in step (1) and mutagenesis protective agent formula 1 respectively, coat them on a small iron sheet at a ratio of 1:1 (coat more for backup), control the liquid layer thickness to be 2 - 4 mm, cover it with a glass plate and set aside. Set the conditions of the ARTP mutagenesis instrument as follows: under the conditions of working power of 100 W, working air flow of 10 SLM, and irradiation distance of 2 mm, set the irradiation time from 0 - 240 s. After mutagenesis, place the sample in a 2 mL centrifuge tube containing 150 μL of mutagenesis protective agent, incubate it at 26 °C in the dark for 90 min, then wash and shake it with a magnetic levitation shaker for no less than 1 min, and set aside.

[0104] (3) Plate coating: Spread the plate mutagenized in step (2) on a regenerated PDA plate and incubate it statically at 26 °C. After single colonies grow on the plate, calculate its mutagenesis lethality rate. The results are shown in Table 9.

[0105] Table 9 Mutagenesis lethality rate of the spore suspension of strain GXGD - EF - 13 - JD - 1

[0106] Mutation time / s Without mutagenesis protectant Formulation 1 0S 0 0 60S 64.06 18.18 120S 85.31 30.3 180S 93.75 48.5 240S 100 78.79 300S 100 93.94 360S 100 90.91

[0107] Result analysis: According to the comparison and exploration of the spore suspension without mutagenesis protective agent and formula 1 in Table 9, by analyzing its mutagenesis lethality rate, its mutagenesis time is increased from 0 - 180 s to 0 - 360 s; at 180 s, its mutagenesis lethality rate drops from 93.75% to 48.5%, and the regeneration rate almost doubles, that is, the amount of mutant strains obtained increases by 1 fold; it shows that the mutagenesis protective agent can well repair the mutant damaged strains.

[0108] Example 7: Application of ARTP mutagenesis protective agent in yeast 3

[0109] (1) Activate strain JM - 1 on a PDA plate and prepare a seed solution; adjust the cell number to 10 5 -10 6 CFU / mL and set aside.

[0110] (2) ARTP mutagenesis: Pipette 10 μL of the bacterial suspension in step (1) and mutagenesis protective agent formula 1 respectively, coat them on a small iron sheet at a ratio of 1:1 (coat more for backup), control the liquid layer thickness to be 0.2 - 0.4 cm, cover it with a glass plate and set aside. Set the conditions of the ARTP mutagenesis instrument as follows: under the conditions of working power of 100 W, working air flow of 10 SLM, and irradiation distance of 2 mm, set the irradiation time to 120 s. After mutagenesis, place the sample in a 2 mL centrifuge tube containing 150 μL of mutagenesis protective agent formula 1, incubate it at 26 °C in the dark for 90 min, then wash and shake it with a magnetic levitation shaker for no less than 1 min, and set aside.

[0111] (3) Coating the plate: Spread the plate mutagenized in step (2) on a regenerated PDA plate and incubate it statically at 26°C. After single colonies grow on the plate, calculate the mutagenic lethality rate, as shown in Figure 4 Table 10.

[0112] Table 10 Mutagenic lethality rate of strain JM-1

[0113]

[0114] Result analysis: According to the results in Table 10, it can be seen that the lethality rate of the non-mutagenic protective agent and Formula 1 decreased from 98.5% to 66.49%, and the regeneration rate increased by 32.01%; the regeneration rate of mutagenic mutant strains was significantly improved, and the potential positive mutation rate was also increased.

[0115] Example 8: Application of ARTP mutagenic protective agent in lactic acid bacteria 4

[0116] (1) Activate strain RSJ-1 on an LB plate and prepare a seed solution; adjust the cell number to 10 5 -10 6 CFU / mL with 0.6M NaCl and set aside.

[0117] Among them, the formula of LB is: Tryptone 10 g / L; Yeast extract 5 g / L; Sodium chloride (NaCl) 10 g / L.

[0118] (2) ARTP mutagenesis: Pipette 10 μL of the spore suspension in step (1) and mutagenic protective agent Formula 1 respectively, and coat them on a small iron sheet according to a ratio of 1:1 (coat more for backup), control the liquid layer thickness to be 0.2 - 0.4 cm, cover it with a glass plate and set aside. Set the conditions of the ARTP mutagenesis instrument as follows: working power 100 W, working gas flow 10 SLM, irradiation distance 2 mm, and set the irradiation time to 120 s. After mutagenesis, place the sample in a 2 mL centrifuge tube containing 150 μL / mutagenic protective agent Formula 1, incubate it at 26°C in the dark for 90 min, then elute and shake it with a magnetic levitation shaker for no less than 1 min and set aside.

[0119] (3) Coating the plate: Spread the plate mutagenized in step (2) on a regenerated PDA plate and incubate it statically at 26°C. After single colonies grow on the plate, calculate the mutagenic lethality rate, as shown in Figure 5 Table 11.

[0120] Table 11 Mutagenic lethality rate of strain RSJ-1

[0121]

[0122] It can be seen from the results in Table 11 that the lethality rate of the non-mutagenesis protective agent and Formula 1 decreased from 99.21% to 27.78%, and the regeneration rate increased by 71.43%; the regeneration rate of mutagenic mutants was greatly increased, and the potential positive mutation rate was also increased.

[0123] The present invention provides an idea and method for an ARTP mutagenesis protective agent and its application. There are many methods and ways to specifically implement this technical solution. The above description is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. An ARTP mutagenesis protective agent, characterized in that, Components in the following mass percentages: 2% vitamin C, 1% superoxide dismutase, 5% glycerol, the balance being stabilizer; wherein, the stabilizer is any one of PBS 10 mM pH6.0 and 0.8 M NaCl.

2. The preparation method of the ARTP mutagenesis protective agent according to claim 1, characterized in that, After mixing vitamin C, superoxide dismutase and glycerol in the formula evenly, dissolve them with the stabilizer, and obtain a sterile mutagenesis protective agent through centrifugal filtration.

3. Application of the ARTP mutagenesis protective agent described in claim 1 in ARTP mutagenesis breeding.

4. The application according to claim 3, characterized in that The object of the ARTP mutagenesis breeding is bacteria and / or fungi.

5. The application according to claim 4, characterized in that, The object of the ARTP mutagenesis breeding is lactic acid bacteria among bacteria, yeast among fungi and / or the edible mushroom Pleurotus djamor among edible fungi.

6. The application according to claim 3 or 4, characterized in that, Mix the object to be mutagenized and bred with the ARTP mutagenesis protective agent described above, and then perform ARTP mutagenesis. After the ARTP mutagenesis is completed, place it in the ARTP mutagenesis protective agent for incubation.

7. The application according to claim 6, wherein The object to be induced to be bred is mixed with the ARTP mutagenesis protective agent, specifically, the object to be induced to be bred is prepared into 10 5 -10 6 The suspension of CFU / mL was then mixed with the ARTP mutagenic protective agent in a volume ratio of 1-5:1-10.

8. The application according to claim 6, wherein The mutagenesis conditions of the ARTP mutagenesis are as follows: under the conditions of working power 100 - 120 W, working gas flow 10 - 12 SLM, irradiation distance 2 - 4 mm, set the irradiation time to 0 - 240 s.

9. The application according to claim 6, wherein The incubation conditions are as follows: incubate at 20 - 30 °C for 30 - 120 min.

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