CN113174339A

CN113174339A - Oomycopora ovales high-yield strain HMGIM-T43 and breeding method thereof

Info

Publication number: CN113174339A
Application number: CN202110646322.3A
Authority: CN
Inventors: 杜娜; 谢意珍; 胡惠萍; 吴清平; 刘远超; 张智; 陈建; 蔡曼君; 肖春; 李向敏; 黄龙花
Original assignee: Guangdong Yuewei Biotechnology Co ltd; Institute of Microbiology of Guangdong Academy of Sciences
Current assignee: Guangdong Yuewei Biotechnology Co ltd; Institute of Microbiology of Guangdong Academy of Sciences
Priority date: 2021-06-10
Filing date: 2021-06-10
Publication date: 2021-07-27
Anticipated expiration: 2041-06-10
Also published as: CN113174339B

Abstract

The invention belongs to the technical field of edible fungus breeding, and discloses a high-yield oospore oudemansiella mucida strain HMGIM-T43 and a breeding method thereof. The oospore oudemansiella mucida high-yield strain HMGIM-T43 is preserved in Guangdong province microbial strain collection center at 4 month and 7 days 2021, and the preservation number is GDMCC No. 61599. The breeding method comprises the following steps: activating and culturing original strains of oospore oudemansiella mucida to obtain mycelia; separating and washing the obtained mycelium, performing enzymolysis by using muramidase, and centrifuging to remove supernatant to obtain a protoplast; the protoplast is subjected to ARTP mutagenesis and regeneration, primary screening and secondary screening to obtain the oospore oudemansiella mucida high-yield strain HMGIM-T43. According to the invention, an excellent Oudemansiella oothecoides strain is bred by utilizing an ARTP mutagenesis technology, and the problems of long cultivation period, low conversion efficiency and the like caused by poor quality of the Oudemansiella oothecoides strain are solved.

Description

Oomycopora ovales high-yield strain HMGIM-T43 and breeding method thereof

Technical Field

The invention belongs to the technical field of edible fungus breeding, and particularly relates to a high-yield oospore oudemansiella mucida strain HMGIM-T43 and a breeding method thereof.

Background

Oomyces aeolorinus Hymenopellis rapanipes, which is known under the trade name "Collybia nigricans", also known as Bispodophytora bisporus, belongs to the kingdom Fungi (Fungi), Basidiomycota (Basidiomycota), Agaricales (Agaricales), Hymenophilaceae (Physalceae), and Oesophagostoma (Hymenopellis). A rare edible fungus, namely black-skin Termitomyces albuminosus, which is popular in the domestic market in recent years, is similar to Termitomyces albuminosus (Termitiomyces sp.) in appearance, black in whole body and connected with a slender false root below a stipe. Except that the rhizoid of "black skin termitomyces albuminosus" was attached to ground sapropel, and the rhizoid of termitomyces albuminosus was attached to a fungus bed of termites. In addition, oospore oudemansiella mucida belongs to the asian species, and previously "black skin termitomyces albuminosus" was identified as long-rooted oudemansiella mucida (h.radiacata) or pholioma lepista (h.furfurfurfurmoracea), both of which are derived from north america and europe, and has not been found in china at present.

The research shows that the contents of crude protein and soluble sugar of oospore oudemansiella mucida are 32.12% and 24.25% respectively, which are higher than the protein content of most edible fungi, and the total content of 17 amino acids is 18.14%. The total dietary fiber content of oospore oudemansiella mucida is 41.0% in the early research of the team, which is beneficial to improving the intestinal function of the human body; the agaricus bisporus also contains abundant mineral elements of phosphorus (679mg/100g), potassium (3230mg/100g), calcium (9.92mg/100g), magnesium (103mg/100g), iron (23.2mg/100g) and zinc (5.99 mg/100g), and shows that the agaricus bisporus is an edible fungus with abundant and balanced nutritional ingredients. Oospore oudemansiella mucida is also one of traditional medicinal fungi, and also has various effects of lowering blood pressure, resisting oxidation, resisting tumors and the like. Because the taste and the appearance of the fungus are similar to those of wild collybia albuminosa, and the fungus is rich in nutrition and delicious in taste, the fungus is more and more popular with people, and the market demand is increasingly sharply increased. At present, the first-grade product of the black skin termitomyces albuminosus in the wholesale market is 26-28 yuan per kilogram, the second-grade product is 24-26 yuan per kilogram, and the third-grade product is 15-20 yuan per kilogram. If the edible mushroom is sold in retail market or bought in a restaurant, the price of the edible mushroom can be as high as 60 yuan per kilogram, which is much higher than that of common mushrooms, golden mushrooms and oyster mushrooms, and the edible mushroom is a high-grade mushroom product in edible mushroom varieties and has wide market prospect.

The artificial cultivation of oospore oudemansiella mucida is late in China. In 2007, Sunxia was the first time to artificially domesticate and cultivate Oudemansiella oothecoides. In 2012, the Huhui duckweed of the team firstly provides a cultivation method for directly fruiting oospore oudemansiella mucida fungi bags. In 2013, the success of artificial large-scale annual cultivation is reported continuously. In 2019, Yanzhelan and the like research the industrialized facility cultivation technology of oospore oudemansiella mucida, the cultivation is successful, the yield of each bag of fresh mushrooms is about 0.3 kg, and the supply is short of the demand. The oospore oudemansiella mucida is artificially cultivated in Shandong, Sichuan, Guangdong, Hunan, Jiangsu, Shanxi and the like in a certain scale, but has low transformation efficiency, unstable quality and high artificial cultivation cost, and limits further popularization and cultivation. At present, the problems of long period and low transformation efficiency caused by poor strain quality generally exist in the cultivation of oospore oudemansiella mucida. The invention aims to breed the excellent Oudemansiella oothecoides strain with short cultivation period and high transformation efficiency.

Atmospheric Room Temperature Plasma (ARTP) is a new method for jet mutation breeding of Atmospheric frequency glow discharge Plasma (RF APGD) proposed in recent years. The RF APGD adopts a bare metal electrode structure, the dielectric covering layer is removed, and the breakdown voltage is obviously reduced. Compared with the traditional mutagenesis, the ARTP mutagenesis technology has higher high efficiency and convenience, and simultaneously has high safety which cannot be replaced by physical radiation or chemical reagents, and the application of the ARTP mutagenesis technology in the aspect of edible fungus breeding has great prospect.

The ARTP mutagenesis technique has been successfully applied to mutagenesis breeding of 60 kinds of microorganisms such as bacteria, fungi (macrofungi, yeasts and molds), algae, plants and the like, and plays an important role in the field of microorganism breeding. At present, some progress has been made in the application of ARTP mutagenesis technology to edible fungi. 2014, Ohwei Hua firstly utilizes an ARTP mutagenesis technology to breed the straw mushroom, and the obtained straw mushroom has the growth speed improved by 57 percent at low temperature compared with the original strain and the low temperature stress resistance improved by 24 hours. In 2017, the Agrocybe aegerita is bred by the moderate technique of ARTP mutagenesis, and a strain with biological efficiency improved by 9.03% is obtained. In 2017, the white flammulina velutipes is bred by the poplar mushroom through an ARTP mutagenesis technology, and an excellent strain with the disease resistance improved by 15.49% is obtained. In 2018, Zhang Henan utilizes ARTP mutagenesis technology to breed phellinus igniarius, the yield of the obtained flavone is improved by 86.7%, and the mutagenized strain has higher in-vitro antioxidant activity than the original strain. In 2019, Hericium erinaceus is bred by the Zyguyi method through an ARTP mutagenesis technology, and the yield of the obtained fruiting bodies and the content of polysaccharides are respectively increased by 22% and 16%. However, there is no report of breeding the high spawning spore oudemansiella mucida strain by adopting an ARTP mutagenesis technology at present.

Disclosure of Invention

Aiming at the defects and shortcomings of the prior art, the primary object of the invention is to provide a high-yield strain HMGIM-T43 of Oomyelia ovalis.

The invention also aims to provide a breeding method of the oospore oudemansiella mucida high-yield strain HMGIM-T43. The purpose of the invention is realized by the following technical scheme:

a high-yield strain HMGIM-T43 of oospore oudemansiella rapanipes is deposited in Guangdong province microorganism culture collection center (GDMCC) at 7 days 4 and 2021, and the deposit number is GDMCC No. 61599. The address of the preservation unit is No. 59 building No. 5 building of No. 100 college of Jifura Zhonglu, Guangzhou city.

The breeding method of the oospore oudemansiella mucida high-yield strain HMGIM-T43 comprises the following steps:

(1) activating and culturing original strains of oospore oudemansiella mucida to obtain mycelia;

(2) separating and washing the mycelium obtained in the step (1), performing enzymolysis by using muramidase, and centrifuging to remove supernatant to obtain protoplast;

(3) and (3) carrying out induced mutation and regeneration on the protoplast obtained in the step (2) by adopting ARTP, carrying out primary screening and secondary screening to obtain the oospore oudemansiella mucida high-yield strain HMGIM-T43.

Further, the oospore oudemansiella starting strain in the step (1) is the strain HMGIM-W160136. The strain HMGIM-W160136 is disclosed in patent 202010554107.6, and is preserved in Guangdong province microorganism culture collection center with the preservation number of GDMCC NO: 61004.

Further, the culturing step in the step (1) is as follows: inoculating the activated starting strain to a PDA culture medium plate, and placing the plate in an incubator at 25 +/-1 ℃ for dark culture for 7 days; punching the edges of the colonies with a punch with a diameter of 5mm, selecting the bacterial blocks of the original strains, inoculating the bacterial blocks into a PD liquid culture medium, standing and culturing for 1d, and culturing for 7d in a shaking table at 25 +/-1 ℃ and at a speed of 140r/min in a dark place to obtain mycelia.

Further, the separation and washing steps in the step (2) are as follows: placing the cultured mycelium in a sterile centrifuge tube, centrifuging at 8000r/min for 10min at 4 deg.C, removing supernatant, washing mycelium with sterile 0.6mol/L mannitol for 3 times, centrifuging, and removing supernatant.

Further, the lywallzyme in the step (2) is prepared by the following method: dissolving a proper amount of solid enzyme in 0.6mol/L mannitol solution, and filtering and sterilizing by using a 0.22um microporous membrane to obtain the lywallzyme with the mass concentration of 2%; the amount of the muramidase added was 1mL per 250-300mg of fresh mycelium.

Further, the enzymolysis conditions in the step (2) are as follows: performing enzymolysis for 2.5h in a constant-temperature water bath at 30 ℃.

Further, the centrifugation in the step (2) means centrifugation at 4000r/min for 10min at a temperature of 4 ℃.

Further, the protoplast in the step (2) is further washed 3 times with sterile 0.6mol/L mannitol, centrifuged and the supernatant removed, and the protoplast is diluted with sterile 0.6mol/L mannitol to form a suspension, which is then counted by a cell counter (automatic cell counter) and adjusted to 1X 10⁶-10⁷one/mL.

Further, the ARTP mutagenesis conditions in the step (3) are as follows: the helium gas supply pressure is 0.15-0.20MPa, the helium gas injection flow is 10L/min, the radio frequency power is 100W, the working distance is 2mm, and the mutagenesis irradiation time is 10-80 s.

Further, the regeneration step in the step (3) is: putting the mutagenized protoplast into a MYG regeneration culture medium, and performing vortex oscillation to form a new bacterial suspension; and (3) coating the bacterial suspension on a cellobiose regeneration culture medium plate, and culturing at 25 +/-1 ℃ in a dark place for 7 days.

Further, the primary screening in the step (3) comprises the following steps: selecting strains with hypha growth rate higher than 5% of that of original strains as control group, subculturing the selected strains for 5 generations, and screening for 5 generations to obtain strains with excellent shape and stable heredity.

Further, the re-screening in the step (3) comprises the following steps: inoculating the primary screened strain into a sterile stock culture material for culturing, then inoculating the primary screened strain into a culture bag for culturing, carrying out fruiting management, and screening the strains with short culture period, good commodity characters and high fruiting body yield.

Compared with the prior art, the invention has the beneficial effects that:

according to the invention, an excellent Oudemansiella oothecoides strain is bred by utilizing an ARTP mutagenesis technology, and the problems of long cultivation period, low conversion efficiency and the like caused by poor quality of the Oudemansiella oothecoides strain are solved.

Drawings

FIG. 1 shows the lethality (a) and positive mutation rate (b) of the starting strain at different ARTP mutagenesis times in the examples.

FIG. 2 is a diagram showing the domesticated cultivated fruit body of all the strains in the examples.

FIG. 3 is a graph showing a standard classification of fruiting bodies of oospore oudemansiella mucida (Collybia nigrescens).

FIG. 4 is an ISSR electrophoretogram of CK and HMGIM-T43 strains in examples.

FIG. 5 is a scanning electron micrograph of the CK and HMGIM-T43 mycelia of Oomyces oosporus Aoshima in example.

FIG. 6 is a scanning electron micrograph of fruiting bodies of Olympic Mushroom CK and HMGIM-T43 in example.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The culture formulations used in the following examples:

1. enriching the comprehensive PDA: 200g/L of potato, 20g/L of glucose, 20g/L of agar and 10g/L, KH of peptone₂PO₄3g/L、MgSO₄1.5g/L, vitamin B₁10mg/L, pH is natural.

2. Enrichment of integrated PD: 200g/L of potato, 20g/L of glucose and 10g/L, KH g of peptone₂PO₄3g/L, MgSO41.5g/L, vitamin B₁10mg/L, pH is natural.

3. MYG regeneration medium: maltose 10g/L, glucose 4g/L, yeast powder 4g/L, agar 15g/L, pH is natural, and the components are dissolved in 0.6mol/L osmotic pressure stabilizer.

4. Cellobiose regeneration medium: 15g/L cellobiose, 2g/L peptone, 4g/L yeast powder and 15g/L agar, has natural pH value, and is dissolved in 0.6mol/L osmotic pressure stabilizer.

5. Stock culture medium: 98-99% of sorghum, 1-2% of calcium carbonate, 55% of water content and natural pH.

6. Production of seed culture medium: 38-42% of cottonseed hulls, 35-40% of sawdust, 18-20% of bran and 1-2% of calcium carbonate; water content is 60%, and pH is natural.

7. Cultivation medium: 45% of cottonseed hulls, 30% of sawdust, 20% of bran, 3% of corn flour, 1% of calcium superphosphate and 1% of gypsum, 65% of water content and natural pH.

Example 1

This example is the preparation of protoplast suspensions and ARTP mutagenesis.

(1) The starting strain HMGIM-W160136(HMGIM-W160136 is collected from Fujian plum blossom mountain and preserved in Guangdong province microbial culture Collection center with the preservation number GDMCC NO:61004 and disclosed in patent 202010554107.6) was activated, and the pure culture was inoculated onto a 90mm PDA medium plate and cultured in an incubator at 25. + -. 1 ℃ for 7 days in the dark. Punching the edges of the bacterial colonies by a puncher with the diameter of 5mm, selecting bacterial blocks of the original strains, inoculating the bacterial blocks into a 250mL conical flask filled with 100mL PD liquid culture medium, standing and culturing for 1d, and culturing for 7d in a shaking table at the temperature of 25 +/-1 ℃ in the dark at the speed of 140r/min to obtain mycelia prepared from the bacterial suspension.

(2) Protoplast suspension is prepared by placing cultured mycelia in a sterile centrifuge tube in a clean bench, centrifuging at 8000r/min for 10min at 4 deg.C, and removing supernatant. The mycelia were washed 3 times with sterile 0.6mol/L mannitol, centrifuged and the supernatant removed. Preparing 2% muramidase, weighing a proper amount of solid enzyme, dissolving the solid enzyme in 0.6mol/L mannitol solution, and filtering and sterilizing by using a 0.22um microporous membrane. Adding 1mL of muramidase into fresh mycelium of 250-300mg per mycelium, performing constant-temperature water bath at 30 ℃, and performing enzymolysis for 2.5 h. The enzyme solution was collected in a 2mL EP tube, centrifuged at 4000r/min at 4 ℃ for 10min, and the supernatant was removed. The purified protoplasts were obtained by washing 3 times with sterile 0.6mol/L mannitol, centrifuging and removing the supernatant. Diluting the protoplast with sterile 0.6mol/L mannitol to obtain suspension,counting with a cell counter (automatic cell counter), adjusting to 1 × 10⁶-10⁷one/mL.

(3) The protoplast ARTP mutation and regeneration are carried out, an ARTP mutation system is started, the helium gas supply pressure is adjusted to 0.15-0.20MPa, and the operation parameters are as follows: the helium gas injection flow rate is 10L/min, the radio frequency power is 100W, the working distance is 2mm, the mutagenesis irradiation time is respectively set to 10s, 20s, 30s, 40s, 50s, 60s, 70s and 80s, and the treatment time is 0s as a control. First, 10. mu.L of a protoplast suspension was uniformly applied to the surface of a sterile metal slide, and the metal slide was placed on a sterile stage of ARTP with tweezers and plasma-irradiated for the above-mentioned different mutagenesis times, respectively. And (3) placing the mutagenized metal slides in 2mL MYG regeneration culture medium EP tubes respectively, taking the slides treated by 0s as a control group, and performing vortex oscillation for 2min to form new bacterial suspension. And (3) coating 100 mu L of suspension on a cellobiose regeneration culture medium plate, placing the cellobiose regeneration culture medium plate at 25 +/-1 ℃ for dark culture, paralleling each gradient by 3, and counting the number of colonies after 7d culture. Calculating the lethality curve under different treatment time, determining the optimal mutagenesis irradiation time and carrying out multiple rounds of ARTP mutagenesis breeding. Lethality ═ number of (non-mutagenized colonies)/non-mutagenized colonies × 100%.

(4) HMGIM-W160136 was subjected to ARTP mutagenesis as a starting strain (CK) for 10s, 20s, 30s, 40s, 50s, 60s, 70s and 80s, respectively, to obtain lethality at various mutagenesis times (a in FIG. 1). Along with the change of mutagenesis time, the lethality of the strain continuously rises, and due to the influence of high fault tolerance rate in the SOS repair process, the lethality rate drops in the middle and then rises, and when the maximum lethality rate is reached, the lethality rate begins to drop continuously. Because the strain is affected by high fault tolerance rate at the treatment time of 50s, the fatality rate is about 88%; the lethality of the strain at 60s treatment time was about 96%. The forward mutation was obtained by increasing the hyphal growth rate by 5% or more, and the forward mutation rates at different mutagenesis times were obtained (b in FIG. 1). When the treatment time is 60s, the positive mutation rate reaches about 53 percent at most. Because the mutation effect is best when the strain has the lethality of more than 90%, the ARTP mutagenesis is carried out by selecting 60s processing time according to positive mutation rate data and comprehensive consideration.

The results of the above examples are combined to show that the optimum mutagenesis conditions for screening Oudemansiella ovospora are selected by using ARTP mutagenesis technology and using the lethality rate and the positive mutation rate as indexes, the mutagenesis time is set as 60s, the air flow is 10L/min, and the power is 100W, the lethality rate of the Oudemansiella ovospora is 96.00%, and the highest positive mutation rate is 53.00%.

Example 2

This example is a preliminary screening of the growth rate of hyphae of the mutagenized strain.

(1) Selecting a mutagenic strain with better growth vigor by taking the original strain as a control group as in the step (1) in the above example 1, and subculturing the mutagenic strain for 5 generations; after 5 generations of screening, strains with excellent shapes and stable heredity are obtained for cultivation test.

(2) Using the growth rate of CK hyphae (0.81cm/d) as a reference, 958 mutant strains were obtained by 4 rounds of ARTP mutagenesis, strains having a hyphae growth rate of 5% or more higher than that of the original strain were selected, and 10 dominant strains having a stable growth rate were obtained by 5 generations of selection (see Table 1 below).

TABLE 1 determination of genetic stability of mutagenized strains

Note: the growth speed of the hyphae is improved after the CK is domesticated and cultivated and separated and purified; data are expressed as mean ± sem (n ═ 3), with different letters indicating significant differences between strains (P < 0.05).

Example 3

This example is a rescreening of mutagenized strain acclimatization and cultivation.

(1) Preparing stock seed, mixing jowar and calcium carbonate, adjusting water content to 65%, placing into high temperature resistant polypropylene bags (17cm × 35cm), each bag containing 80-100g of dry material, and sterilizing at 126 deg.C under 0.147MPa for 90 min. Inoculating 10 Olympic acid oudemansiella strains into sterile stock culture material, culturing in 25 + -1 deg.C incubator in dark for 7-14 days, and allowing mycelia to grow over the bag.

(2) Preparing a cultivation fungus bag, namely prewetting carbon source raw materials such as wood chips, cottonseed hulls and the like one day in advance; adding bran, corn flour and other nitrogen sources on the dosing day, fully mixing, adjusting the water content to 65%, and putting into high temperature resistant polypropylene bags (17cm multiplied by 35cm), wherein each bag is equivalent to 400g of dry materials. A plastic rod (1.5cm multiplied by 8cm) is used for punching a hole in the center of the high-temperature-resistant polypropylene cultivation material, a plastic ring is sleeved on a bag opening, and a matched cover is buckled. Sterilizing at 126 deg.C under 0.147MPa for 90 min. Inoculating about 20g of the above 1.5.1 stock to about 1100g of high temperature resistant polypropylene cultivation material, culturing in dark in a culture room with temperature of 24 + -1 deg.C, air relative humidity of 65-75%, carbon dioxide concentration of 2500-.

(3) And (4) fruiting management, namely transferring the completely cultured fungus bags to a fruiting room, removing a cover, scratching the surface of the fungus bags, and vertically placing the fungus bags with upward openings (gaps should be reserved between the bags). The room temperature is 26 +/-1 ℃, the relative air humidity is 85-90%, the illumination intensity is 600- & lt800 lux & gt, the illumination is carried out for 12h every day, and the carbon dioxide content in the space is kept between 350- & lt1000 mg/L & gt. Spraying water mist to young mushroom 1-2 times every day after primordium is formed, ventilating for 2-3 times (10-15 min each time) with relative space humidity of 90-95%, and harvesting until fruiting body stipe grows to 5.0-7.0 cm. Weighing the collected fresh sporocarp, calculating biological conversion rate, screening mutagenic strain with excellent agronomic character, short cultivation period and high sporocarp yield, and measuring nutrient components. Biological conversion rate is the average fresh weight of fruit body (g)/dry weight of cultivation material (g) × 100%.

(4) The cultivation period of the mutant strain is statistically recorded (see table 2 below), 12 mutant strains are co-domesticated and cultivated, the hypha cultivation period of CK and 12 mutant strains is 60d, the primordium appearing time is 7-12d, and the primordium time of the mutant strain HMGIM-T43 is respectively shortened by 5d compared with CK. The harvesting time of 13 sporophores is 3-10 days, and compared with CK, the harvesting time of the sporophores of the mutant strain HMGIM-T43 is respectively shortened by 1 day. The cultivation period comprises hypha cultivation, primordium time and fruiting body harvesting time, the cultivation period of the mutagenic strain HMGIM-T43 is 72d as short as possible, and is 6d shorter than CK. In conclusion, the mutagenized strain performed better in the cultivation period is the HMGIM-T43 strain.

TABLE 2 growth period survey of mutagenized strains

(5) The commodity characteristics, the yield of fruiting bodies and the appearance quality of the edible fungi have important influence on the commercialization of the edible fungi. FIG. 2, wherein (a) CK, (b) HMGIM-T14, (c) HMGIM-T19, (d) HMGIM-T22, (e) HMGIM-T42, and (f) HMGIM-T43. From the appearance, it was noted that the mutagenized strain fruiting bodies were clustered, showing brown or dark brown fruiting bodies. The quality of edible fungi is very important, and according to the edible fungi standard of edible and edible congeneric Collybia albuminosa, part 3: black skin termitomyces grading criteria (FIG. 3 and Table 3), and we performed statistics on the appearance of the mutagenized strains (Table 4). Meanwhile, CK and 12 mutant strains were subjected to classification. The length of the stipe of the mutant strain CK and 12 strains is between 5.66 and 6.93cm, wherein the stipe of the HMGIM-T512 strain (6.93 +/-0.23 cm) is the longest; the stipe length of HMGIM-T494 strain (6.63. + -. 0.17 cm); the HMGIM-T520 strain (5.66. + -. 0.31cm) had the shortest stipe, and all strains had stipe lengths within the range of the classification standard. The thicknesses of the stipes of the original strain and the 12 mutant strains are between 1.08 and 1.77cm, wherein the stipe of the HMGIM-T42 strain (1.77 +/-0.10 cm) is the thickest; secondly, the thickness of the stipe of HMGIM-T19(1.70 +/-0.15 cm) and HMGIM-T512 strain (1.75 +/-0.16 cm); the stipe of the HMGIM-T199 strain (1.09. + -. 0.13cm) was the finest. In addition, the thicknesses of the stipes of CK (1.51 + -0.10 cm), HMGIM-T19(1.70 + -0.15 cm), HMGIM-T42(1.77 + -0.10 cm), HMGIM-T43 (1.53 + -0.08 cm), HMGIM-T481(1.53 + -0.22 cm), HMGIM-T490(1.66 + -0.10 cm), HMGIM-T494 (1.57 + -0.10 cm) and HMGIM-T512 strains (1.75 + -0.16 cm) were between 1.5 and 2.0 cm. The cap diameter of CK and 12 mutant strains is between 1.77 and 3.36cm, wherein the cap diameter of HMGIM-T494 strain (3.36 +/-0.41 cm) is the largest; the second largest diameter of the pileus of HMGIM-T512 (3.11. + -. 0.25cm) and HMGIM-T490 strains (2.80. + -. 0.24 cm); the strain HMGIM-T23 (1.77. + -. 0.13cm) had the smallest pili diameter. In addition, the diameters of the pileus of HMGIM-T42 (2.30. + -. 0.13cm), HMGIM-T43 (2.39. + -. 0.10cm), and HMGIM-T481 strain (2.24. + -. 0.84cm) were between 2.0 and 2.4 cm.

TABLE 3 Black Collybia albuminosa grading Standard

(6) According to the above, CK and 12 mutant strains were graded as follows, and 3 strains with higher first-grade products were HMGIM-T42, HMGIM-T43 and HMGIM-T481 strains, respectively; the 2 strains with higher tertiary product accounts are respectively HMGIM-T14 and HMGIM-T23; grades four and five are the worst grades, and 8 strains which account for the higher grades are CK, HMGIM-T19, HMGIM-T199, HMGIM-T490, HMGIM-T494, HMGIM-T512, HMGIM-T520 and HMGIM-T527 respectively. As described above, the HMGIM-T42, HMGIM-T43 and HMGIM-T481 strains showed the most advantageous morphological appearance.

(7) We performed statistics on the yield of the mutagenized strains (see Table 4 below). The yield of the CK and 12 mutant strain fruiting bodies is 52.17-88.20 g/bag, and the biological efficiency is 13.04-22.05%. The yields of the mutant strains HMGIM-T43 and HMGIM-T490 are respectively 83.12 +/-7.30 g/bag and 83.22 +/-3.48 g/bag, which are obviously higher than those of other strains. The yields of the mutagenized strains HMGIM-T520, HMGIM-T494 and HMGIM-T14 were 78.13 + -5.22 g/bag, 76.71 + -5.22 g/bag and 74.25 + -3.00 g/bag, respectively, which were significantly higher than those of the other strains. In addition, the biological efficiencies of the mutant strains HMGIM-T43 and HMGIM-T490 are respectively 20.70% and 20.80%, and are improved by more than 50.00% compared with the biological efficiency of CK.

TABLE 4 commercial fruiting body traits and yields of mutagenized strains

Note: data are expressed as mean ± sem (n ═ 15), with different letters indicating significant differences between strains (P < 0.05).

The results of the above examples are combined to show that the regenerated colonies of 958 strain were obtained by performing ARTP mutagenesis on HMGIM-W160136 protoplast suspension and performing 4 rounds of mutagenesis under the condition of selecting the optimal mutagenesis time of 60 s. And (4) carrying out primary screening by increasing the growth speed of hypha by 5%, and then carrying out fruiting body fruiting test. The total cultivation period of HMGIM-T43 strain was 72d and that of HMGIM-T490 strain was 79d, which were 6d and 1d shorter than CK, respectively. The biological efficiencies of the fruit bodies of HMGIM-T43 and HMGIM-T490 strains were 20.7% and 20.80%, respectively, which were improved by 59.33% and 59.50%, respectively, compared to CK. In addition, the minimal time for the primordium of the HMGIM-T43 strain was 7 days, and it was also shown to be the most excellent in morphological appearance.

Example 4

This example describes the determination of the nutrient content of the mutagenized strain.

(1) An amino acid content determination reference (GB 5009.124-2016); a crude polysaccharide content determination reference (NY/T1676-2008/7); protein content determination reference (GB 5009.5-2016/first method); fat content determination reference (GB 5009.6-2016/first method); ash (GB 5009.4-2016/first process); total dietary fiber (GB 5009.88-2014); a crude fiber content determination reference (GB/T5009.10-2003); a phosphorus content determination reference (GB 5009.87-2016 third method); a potassium content determination reference (GB 5009.91-2017 third method); a calcium content determination reference (GB 5009.92-2016 third method); a magnesium content determination reference (GB 5009.241-2017 second method); an iron content measurement reference (second method of GB 5009.90-2016); the zinc content is determined by reference (GB 5009.14-2017 second method), and the determination is finished by Guangdong institute of microbiology (Guangdong center for microbiological analysis and detection).

(2) The HMGIM-T43 and HMGIM-T490 strains obtained from the primary screening and secondary screening of the mutagenized strains were superior to other mutagenized strains by determining the fruiting body protein, crude polysaccharide, amino acids, potassium and calcium contents of the CK and 2 mutagenized strains (see Table 5 below). CK. The protein content of HMGIM-T43 and HMGIM-T490 strains were 22.40g/100g, 26.10g/100g, and 25.20g/100g, respectively; compared with CK, the protein content of HMGIM-T43 and HMGIM-T490 strains is increased by 16.52% and 12.50%, respectively. CK. The crude polysaccharide content of HMGIM-T43 and HMGIM-T490 strains were 4.06g/100g, 5.08g/100g, and 2.91g/100g, respectively; compared with CK, the crude polysaccharide content of the HMGIM-T43 strain is increased by 25.12%; in addition, crude polysaccharide content of HMGIM-T490 strain was reduced by 28.33%. CK. The calcium content of HMGIM-T43 and HMGIM-T490 strains were 9.92g/100g, 17.40g/100g, and 16.40g/100g, respectively; compared with CK, the calcium content of HMGIM-T43 and HMGIM-T490 strains is increased by 75.40% and 65.32%, respectively.

(3) In terms of amino acids, the co-assay of the CK and 2 mutant strains contained 16 amino acids (see Table 6 below). The flavor characteristics of amino acids are mainly classified into fresh, sweet, bitter and tasteless. CK. The total amino acid content of HMGIM-T43 and HMGIM-T490 strains was 15.41-19.05g/100g, with the amino acid content of HMGIM-T43 strain being up to 19.05g/100g, an increase of 24.21% compared to CK, followed by HMGIM-T490 strain. The content of glutamic acid (Glu) in one of the umami amino acids is 2.89-4.64g/100g at most, and accounts for 18.80-24.20% of the total amino acid content, wherein the content of glutamic acid (Glu) in HMGIM-T43 is 4.64g/100g at most. The total content of umami amino acids (glutamic acid (Glu) and aspartic acid (Asp)) is 4.21-6.48g/100g, and accounts for 27.30-33.90% of the total amino acid content, wherein the content of umami amino acids in the HMGIM-T43 strain is 6.48g/100g at most. The total content of sweet amino acids (threonine (Thr), serine (Ser), proline (Pro), glycine (Gly), and alanine (Ala)) is 2.96-4.22g/100g, and the total content is only glutamic acid (Glu). In addition, alanine (Ala) interacts with umami substances to produce a umami enhancing effect.

(4) The E/T ratio of essential amino acids to total amino acids of strains CK, HMGIM-T43 and HMGIM-T490 is 0.29-0.32, and the E/N ratio of essential amino acids to nonessential amino acids is 0.41-0.46. The ideal protein pattern proposed by FAO/WHO is that E/T reaches 0.40 and E/N is more than 0.60, which shows that the amino acids of 2 mutant strains are close to the ideal protein pattern proposed by FAO/WHO and belong to better protein.

TABLE 5 partial nutrient content of mutagenized strain fruiting body

Note: the institute of microbiology (Guangdong institute of science and technology) of Guangdong province was entrusted to complete the detection.

TABLE 6 amino acid content of mutagenized bacterial fruiting bodies (g/100gDW)

Note: essential amino acids for human body; t is the total amount of amino acids; e is the total amount of essential amino acids; n is the mass fraction of non-essential amino acids.

(5) The first limiting amino acid of each of the CK, HMGIM-T43 and HMGIM-T490 strains was methionine (Met) as calculated by the human essential Amino Acid Score (AAS) (see Table 7 below). In addition, the HMGIM-T43 and HMGIM-T490 strains had lysine (Lys) lower than that of the standard model protein, and other essential amino acids higher than that of the standard model protein.

TABLE 7 scoring of essential amino acids in humans in the fruiting bodies of the mutagenized strains

(6) By comparing the above analysis of protein, crude polysaccharide, amino acid, potassium and calcium contents of CK, HMGIM-T43 and HMGIM-T490 strains, it is more advantageous to consider the HMGIM-T43 strain in combination. The water, ash, fat, crude fiber, total dietary fiber, carbohydrate, phosphorus, magnesium, iron and zinc contents of the fruit bodies of the CK and HMGIM-T43 strains were further determined (see Table 8 below). The detection of the HMGIM-T43 strain fruiting body in a dry state shows that the crude fiber and the total dietary fiber of the HMGIM-T43 strain are 4.70g/100g and 33.25g/100g respectively, which are in line with the daily intake range of human bodies and have little difference with the content of CK. In addition, the phosphorus, magnesium and zinc contents of HMGIM-T43 strain were 622.00mg/100g, 111.00 mg/100g and 5.83mg/100g, respectively, with no significant difference compared to CK. The iron content of HMGIM-T43 strain was 3.82mg/100g, 1/6 times that of CK. In conclusion, the HMGIM-T43 strain with short primordium time, high yield and more first-grade products in commodity shapes is obtained through primary screening and secondary screening.

TABLE 8 nutrient content of mutagenized strain fruiting bodies

The results of the above examples were combined to show that the protein, crude polysaccharide and calcium contents of HMGIM-T43 strain were 26.10g/100g, 5.08g/100g and 17.40mg/100g, respectively, in terms of nutrients, and were 16.52%, 25.12% and 75.40% higher than CK, respectively. The comprehensively screened excellent strain HMGIM-T43 with short cultivation period, high yield and rich nutrition.

Example 5

This example was an ISSR analysis of the mutagenized strain.

(1) Inoculating the mutant strain and the activated strain of the original strain onto a PDA culture medium plate (a plate stuck with cellophane membrane), placing in an incubator at 25 + -1 deg.C, culturing in the dark for 8 days, collecting mycelia with sterile forceps, and oven drying in an oven for use. DNA extraction of the mutant strain and the original strain refers to a CTAB method, and primer sequences are specifically shown in a table 9; the ISSR reaction system is shown in Table 10; the PCR amplification reaction procedure is shown in Table 11; after the PCR reaction was completed, the product was stored at 4 ℃ or the product was checked using 1.5% agar gel.

TABLE 9 primer sequence information

TABLE 10 ISSR reaction System

TABLE 11 PCR extension reaction procedure

(2) ISSR-PCR polymorphic amplification of CK and HMGIM-T43 strains, ISSR electropherograms (see FIG. 4) showing that CK and HMGIM-T43 differ at about 950bp under amplification with

primers

890 and 855; CK and HMGIM-T43 differed at about 1500bp under amplification of primer 846; CK and HMGIM-T43 differed at about 650bp under amplification of primer 812. The ISSR polymorphism amplification result proves that the genetic material of the HMGIM-T43 strain is changed on a molecular level, which indicates that a new strain is obtained by ARTP mutagenesis. And through ISSR molecular identification, the CK and HMGIM-T43 have obvious difference in amplification under 890, 855, 846 and 812 four pairs of primers, and the fact that the HMGIM-T43 gene of the strain is mutated into a variant strain is shown.

Example 5

The SEM of the present embodiment.

(1) A small amount of fresh 5X 5mm clumps of the original strain and the mutagenized strain were taken for sample pretreatment. Firstly, fixing for 5 hours by using 3% glutaraldehyde; the fixative was aspirated and washed 6 times with 6mmol/L PBS for 20min each. Dehydrating with 30% and 50% ethanol for 2 times, each for 10 min; dehydrating with 70% and 90% ethanol for 15min for 1 time; dehydrating with anhydrous ethanol for 3 times, each for 15 min; absorbing ethanol and replacing with tert-butanol for 2 times, each for 20 min; and finally freeze drying. And (3) putting the fruiting bodies of the original strain and the mutagenic strain into a 60 ℃ oven for drying, and taking a small amount of samples of 5 multiplied by 5mm respectively. And (4) drying the sample, spraying gold, preparing the sample, placing the sample in a sample chamber, and observing the appearance of the sample under an electron microscope.

(2) The appearance structures of mycelium and fruiting body of oospore oudemansiella mucida CK and HMGIM-T43 were observed and compared by Scanning Electron Microscopy (SEM), and the results are shown in fig. 5 and 6. From FIG. 5, no lock-like association of the CK and HMGIM-T43 mycelia was obtained. In FIG. 5, (a) and (b) are appearance microstructures of the CK mycelia when they are enlarged 2000 times and 5000 times under an electron microscope, respectively; in FIG. 5, (c) and (d) are apparent microstructures of HMGIM-T43 mycelium at 2000-fold and 5000-fold enlargement under an electron microscope, respectively. We can find that under the same times, the thickness of HMGIM-T43 hyphae is more uniform and the surface is smooth; the CK hyphae have uneven thickness, most of the surface is smooth, and the very small part has lighter ornamentation. The result shows that the morphological structure of the mycelium is changed while the high-yield strain is screened by the ARTP mutagenesis method, and compared with the appearance structure of CK, the HMGIM-T43 has uniform mycelium thickness and smooth surface; there is no lock-like union. From FIG. 6, it is shown that both the CK and HMGIM-T43 fruiting bodies have only two basidiospores. FIG. 6 (a) and (b) show appearance microstructures of CK fruit body when it is enlarged 1000 times and 5000 times under an electron microscope, respectively; in FIG. 6, (c) and (d) are appearance microstructures of the HMGIM-T43 fruiting body when it is enlarged 1000 times and 5000 times under an electron microscope, respectively. The appearance and the shape of the sporocarp have no obvious difference. From the observation of the mycelia and the fruiting bodies, we know that both CK and HMGIM-T43 belong to the Tricholoma oothecoides Oudemansiei.

The results of the above examples were combined to show that the morphological structure of HMGIM-T43 hyphae was explored by SEM scanning. The results show that the HMGIM-T43 mycelium has no locked combination, uniform mycelium thickness and smooth surface. The fruiting bodies of both CK and HMGIM-T43 strains have only 2 basidiospores and belong to the species Oudemansiella bisporus.

The experimental data show that the ARTP mutagenesis technology is utilized to breed oospore oudemansiella mucida, a better experimental result is obtained, and the same experimental result is obtained after the cultivation experiment is repeated twice. By integrating various factors such as hypha growth speed, genetic stability, fruiting period, yield, fruiting body quality, nutrient components and the like, the excellent strain obtained by the method obviously improves the quality of the oospore oudemansiella mucida strain, and lays a certain foundation for the cultivation of the oospore oudemansiella mucida.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

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Claims

1. A high-yield strain HMGIM-T43 of oospore oudemansiella rapanipes is preserved in Guangdong province collection center of microorganism strains at 7/4/2021 with the preservation number being GDMCC No. 61599.

2. The breeding method of the oospore oudemansiella mucida high-producing strain HMGIM-T43 as claimed in claim 1, characterized by comprising the following steps:

3. The breeding method of the oospore oudemansiella mucida high-producing strain HMGIM-T43 according to claim 2, characterized in that: the oospore oudemansiella mucida starting strain in the step (1) is a strain HMGIM-W160136.

4. The method for breeding the high-producing strain HMGIM-T43 of oospore oudemansiella mucida according to claim 2 or 3, wherein the culturing step in step (1) is: inoculating the activated starting strain to a PDA culture medium plate, and placing the plate in an incubator at 25 +/-1 ℃ for dark culture for 7 days; punching the edges of the colonies with a punch with a diameter of 5mm, selecting the bacterial blocks of the original strains, inoculating the bacterial blocks into a PD liquid culture medium, standing and culturing for 1d, and culturing for 7d in a shaking table at 25 +/-1 ℃ and at a speed of 140r/min in a dark place to obtain mycelia.

5. The method for breeding the high-producing strain HMGIM-T43 of oospore oudemansiella mucida according to claim 2 or 3, wherein the separation and washing steps in step (2) are as follows: placing the cultured mycelium in a sterile centrifuge tube, centrifuging at 8000r/min for 10min at 4 deg.C, removing supernatant, washing mycelium with sterile 0.6mol/L mannitol for 3 times, centrifuging, and removing supernatant.

6. The method for breeding a high-producing strain HMGIM-T43 of oospore oudemansiella mucida according to claim 2 or 3, wherein the lywallzyme in step (2) is formulated by the following method: dissolving a proper amount of solid enzyme in 0.6mol/L mannitol solution, and filtering and sterilizing by using a 0.22um microporous membrane to obtain the lywallzyme with the mass concentration of 2%; the addition amount of the muramidase is 1mL of the muramidase added to each 250-300mg of fresh mycelium; the enzymolysis conditions are as follows: performing enzymolysis for 2.5h in a constant-temperature water bath at 30 ℃.

7. The breeding method of the oospore oudemansiella mucida high-producing strain HMGIM-T43 according to claim 2 or 3, characterized in that: the centrifugation in the step (2) refers to centrifugation at the temperature of 4 ℃ and the speed of 4000r/min for 10 min.

8. The breeding method of the oospore oudemansiella mucida high-producing strain HMGIM-T43 according to claim 2 or 3, characterized in that: washing the protoplast in the step (2) with sterile 0.6mol/L mannitol for 3 times, centrifuging, removing supernatant, diluting the protoplast with sterile 0.6mol/L mannitol to obtain suspension, counting with a cell counter, and adjusting to 1 × 10⁶-10⁷one/mL.

9. The method for breeding the high-producing strain HMGIM-T43 of oospore oudemansiella mucida according to claim 2 or 3, wherein the ARTP mutagenesis conditions in step (3) are as follows: the helium gas supply pressure is 0.15-0.20MPa, the helium gas injection flow is 10L/min, the radio frequency power is 100W, the working distance is 2mm, and the mutagenesis irradiation time is 10-80 s; the regeneration step is as follows: putting the mutagenized protoplast into a MYG regeneration culture medium, and performing vortex oscillation to form a new bacterial suspension; and (3) coating the bacterial suspension on a cellobiose regeneration culture medium plate, and culturing at 25 +/-1 ℃ in a dark place for 7 days.

10. The method for breeding the high-producing strain HMGIM-T43 of Onospora oodoea according to claim 2 or 3, wherein the preliminary screening in step (3) comprises the steps of: selecting strains with hypha growth rate higher than 5% of that of original strains by taking the original strains as a control group, subculturing the selected strains for 5 generations, and screening for 5 generations to obtain strains with excellent shapes and stable heredity; the secondary screening comprises the following steps: inoculating the primary screened strain into a sterile stock culture material for culturing, then inoculating the primary screened strain into a culture bag for culturing, carrying out fruiting management, and screening the strains with short culture period, good commodity characters and high fruiting body yield.