CN113717865B - Ganoderma lucidum mutant strain and application thereof - Google Patents

Ganoderma lucidum mutant strain and application thereof Download PDF

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CN113717865B
CN113717865B CN202111093569.3A CN202111093569A CN113717865B CN 113717865 B CN113717865 B CN 113717865B CN 202111093569 A CN202111093569 A CN 202111093569A CN 113717865 B CN113717865 B CN 113717865B
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庞欣
齐文武
陈晓云
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BEIJING DAWN AEROSPACE BIO-TECH CO LTD
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Abstract

The invention discloses a ganoderma lucidum mutant strain and application thereof, and ganoderma lucidum Ganoderma lucidum SBL-LZ-KJYB-5 with the preservation number of CGMCC NO.23055. The application of the ganoderma lucidum mutant strain is that the ganoderma lucidum mutant strain is fermented to produce extracellular crude polysaccharide and extracellular polysaccharide. The ganoderma lucidum colony is round, the hypha is dense, neat, white, fast in growth vigor, dry and easy to pick. The physiological and biochemical characteristics of the strain are as follows: the growth temperature range is wider than that of the original strain, the strain is high-temperature resistant, can still grow at 45 ℃, and the optimal growth temperature is 28-32 ℃; the optimal rotation speed of liquid culture is 180rpm; the natural pH, salinity tolerance was stronger than the strain before mutagenesis, and still grew well in PDA medium containing 1% NaCl.

Description

Ganoderma lucidum mutant strain and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to a ganoderma lucidum mutant strain and a mutagenesis and screening method thereof.
Background
Ganoderma is fungus of Ganoderma genus of Polyporaceae of Agaricales of Basidiomycetes. The Chinese dynasty 'Shennong Ben Cao Jing' considers that the ganoderma lucidum can tonify heart qi, calm essence, nourish liver, tonify qi and strengthen bones and muscles, and has high medicinal value. Ganoderma contains various chemical components such as polysaccharide, triterpene, nucleoside, protein, fatty acid, etc., wherein polysaccharide is considered as one of the main active ingredients of Ganoderma. Modern researches have proved that ganoderan has various biological activities such as anti-tumor, immunity enhancing and antiaging.
Therefore, the polysaccharide produced by the ganoderma lucidum mutant strain obtained by the mutagenesis technology in the present time in fermentation is significantly improved.
Disclosure of Invention
In view of the foregoing drawbacks or shortcomings of the prior art, it would be desirable to provide a mutant strain of ganoderma lucidum and methods of mutagenesis and screening thereof.
According to the technical scheme provided by the embodiment of the application, the ganoderma lucidum mutant strain has the preservation number of CGMCC NO.23055, namely ganoderma lucidum Ganoderma lucidum SBL-LZ-KJYB-5.
The application of the ganoderma lucidum mutant strain is that the ganoderma lucidum mutant strain is fermented to produce extracellular crude polysaccharide and extracellular polysaccharide, the ganoderma lucidum mutant strain is fermented for 10 days in a potato glucose broth culture medium of a conventional ganoderma lucidum fermentation medium, and the content of the extracellular crude polysaccharide is 3.10 g/L-3.56 g/L, and is improved by 49% -52.79% compared with a starting strain. The ganoderma lucidum mutant strain is subjected to liquid fermentation in a potato glucose broth culture medium of a conventional ganoderma lucidum fermentation culture medium for 10 days, the content of extracellular polysaccharide of the ganoderma lucidum mutant strain reaches 18g/L-20.77g/L, and the content of extracellular polysaccharide of the ganoderma lucidum mutant strain is improved by 55% -59.52% compared with that of a starting strain. The ganoderma lucidum mutant strain belongs to a mutant variety of ganoderma lucidum, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms (address: north Xiylu No. 1, 3 of the North West way of the Korean area of Beijing, post code: 100101 of the institute of microorganisms of the national academy of sciences of China) in the year 2021 and is classified as ganoderma lucidum (Ganoderma lucidum) with a preservation number of CGMCC No.23055.
The mutagenesis of the ganoderma lucidum mutant strain comprises the following steps:
1) Ground simulated mutagenesis
Strain a preparation:
inoculating the bacterial colony with the size of 0.2cm x 0.2cm of the original bacterial strain on a PDA flat plate culture medium, culturing at 25-35 ℃, and standing by when the diameter of the bacterial colony on the flat plate exceeds 3 cm;
b, ground simulation space mutagenesis test:
the following three groups of combined mutagenesis experiments were performed on the strains on the plates:
vibration test when simulating aircraft flight: fixing a strain flat plate with the diameter exceeding 3cm on a double vibration table, and vibrating for 1h under the condition of 0-400kN, wherein the first 0.5 hour is a low-frequency sinusoidal vibration test, and the second 0.5 hour is a random vibration test;
vacuum low-energy particle irradiation test: placing strain flat plate with diameter exceeding 3cm into space low-energy comprehensive irradiation experimental equipment, at 10 -3 -10 -6 Recovering after irradiation under Pa vacuum for 30min-180min, and irradiating with 1×10 radiation dose 14 eV-4×10 14 eV；
Gravity acceleration test: selecting a gravity acceleration of 4g, and performing an overweight test for 30min;
c, culturing:
taking a ground simulation space mutation sample under a sterile condition for culturing, wherein the steps are as follows: taking a simulated space mutagenesis sample with the culture area of 0.2cm x 0.2cm under the aseptic condition, eluting thalli into an aseptic centrifuge tube by using 5 milliliters of aseptic water, sucking uniform thalli eluent by using a pipettor, taking 0.5 milliliter of the thalli eluent into 4.5 milliliters of aseptic water, sucking uniformly, and so on, performing multiple dilution, taking 10-4 and 10-5 dilutions, coating 50 microliter to 250 microliter of diluted thalli on each plate, coating a plurality of plates on each dilution gradient, inversely culturing for 5 days to 20 days at the temperature of 25 ℃ to 35 ℃ in a culture box after the thalli is absorbed by the culture medium, selecting strains with rapid growth and large single colony, continuously culturing, and preserving, and performing a next screening test;
2) Bacterial strain screening after ground simulated mutagenesis
A, adding high-concentration metabolic intermediate product beta-glucan into a PDA solid culture medium to form a screening culture medium, wherein the preparation method comprises the following steps: weighing 39g of potato dextrose agar culture medium Powder (PDA) and 50-150g of beta-glucan, fixing the volume to 1L by distilled water, sterilizing for 30min at the natural pH value of 121 ℃, pouring into a sterile culture dish, and cooling and solidifying to obtain a solid culture medium;
b, sterilizing the screening culture medium, pouring the culture medium into a plate, and condensing to prepare a screening plate;
c, uniformly coating, scribing and dibbling the strain subjected to the space mutagenesis on the ground simulation on a screening flat plate;
d, placing the inoculated flat plate in a mould incubator, culturing for 3-10 days at a constant temperature of 25-35 ℃, screening strains which grow faster and have larger colony morphology change as candidate strains according to two indexes of growth speed and colony size, carrying out space mutagenesis test when a return aircraft is available,
the screened strain is subjected to passage work of 3-10 generations, the strain is inoculated on a PDA slant culture medium, the strain is preserved in a refrigerator at the temperature of 4 ℃, and after the emission plan is determined, rejuvenation is carried out, and space mutagenesis is carried out;
3) Space mutagenesis test
The space mutagenesis is to transfer the strains screened in the ground simulated mutagenesis into a carrying tube, put the carrying tube into a space capsule, and then send the space capsule into a space mutagenesis environment, wherein the space environment is as follows: microgravity 10 -3 -10 -6 g, vibration in the emission and recovery stages is 0-400kN; the radiation dose in the space capsule is 0.1mGy-0.9mGy.
The space mutation test method comprises the following steps:
a space mutagenesis
Inoculating the mutant strain which grows more rapidly and is selected from the ground simulation experiment on a solid culture medium, putting the inoculated inclined plane into an incubator, and culturing for 3-7 days at 25-35 ℃ so as to facilitate the space mutation experiment;
under the aseptic condition, scraping a cultured inclined plane, adding the inclined plane into a 2mL carrying small pipe, sealing by a sealing film, then putting the carrying small pipe into a carrying large pipe with the solvent of 30-50mL, delivering the carrying large pipe to a person related to a transmitting base, installing the carrying large pipe on a Shenzhou-eleventh-shaped aircraft, carrying out a space mutagenesis test along with the aircraft, putting the carrying pipe into a Shenzhou-eleventh-shaped aircraft, flying along with the aircraft in space for 33 days, and then recovering the carrying pipe;
b recycling and carrying pipe
Taking a carrying recovery sample with a culture area of 0.2cm x 0.2cm under a sterile condition, eluting thalli in a sterile centrifuge tube by using 5 milliliters of sterile water, sucking and beating uniform thalli eluent by using a liquid-transfering device, taking 0.5 milliliter of the thalli eluent into 4.5 milliliters of sterile water, sucking and beating uniformly, and so on, performing multiple ratio dilution, and taking 10 -4 And 10 -5 Coating the diluted solution of multiple times to a plate culture medium, coating 50-250 microlitres of diluted bacteria solution on each plate, coating a plurality of plates on each dilution gradient, and inversely culturing for 3-10 days at 25-35 ℃ in an incubator after the bacteria solution is absorbed by the culture medium;
4) Strain screening after spatial mutagenesis
Selecting single colony which grows rapidly after space mutagenesis, inoculating the single colony on a pre-prepared screening plate by a streaking method, placing the inoculated plate in a mould incubator, culturing for 3-10 days at a constant temperature of 25-35 ℃, and picking the single colony which grows rapidly on the plate.
In the invention, the preparation method of the PDB culture medium comprises the following steps: 35 g of potato dextrose broth is weighed, added into 1000ml of distilled water, naturally pH is uniform, and the mixture is sterilized at 121 ℃ for 30 minutes for later use.
In the invention, the strain is activated on a flat PDA culture medium, cultured for 5-10d at 25-35 ℃, after mycelium grows on a flat plate, bacterial blocks with the size of about 4mm < 2 > are selected and put into 250mL PDB culture medium, and after culturing for 5d-10d at 120r/min-200r/min and 25-35 ℃ in a shaking table, fermentation broth is collected to measure mycelium biomass, extracellular crude polysaccharide content and extracellular polysaccharide content.
In the invention, the original strain ganoderma lucidum (Ganoderma lucidum) SBL-LZ-6 (laboratory number) is obtained by natural collection of the inventor, and compared with a wild strain, the original strain has higher mycelium biomass and the capability of producing extracellular crude polysaccharide and extracellular polysaccharide after culture, but still does not reach an ideal state.
In the invention, after a ground simulated space mutagenesis test is carried out on an original strain, namely ganoderma lucidum (Ganoderma lucidum) SBL-LZ-6, a mutagenesis material is recovered, a screening culture medium plate is utilized to screen a plurality of strains (laboratory number SBL-LZ-MNYB 9-SBL-LZ-MNYB 21) with higher growth speed from a recovered sample, space mutagenesis is carried out on the mutagenesis material by utilizing Shenzhou undecane, and after the mutagenesis material is recovered, a strain capable of producing more mycelia, extracellular crude polysaccharide and extracellular polysaccharide, namely ganoderma lucidum (Ganoderma lucidum) SBL-LZ-KJYB-5 is determined through a screening and final fermentation method to be preserved, so that the purpose of the invention is achieved.
To sum up, the beneficial effects of this application:
(1) The ganoderma lucidum colony is round, the hypha is dense, neat, white, fast in growth vigor, dry and easy to pick. The physiological and biochemical characteristics of the strain are as follows: the growth temperature range is wider than that of the original strain, the strain is high-temperature resistant, can still grow at 45 ℃, and the optimal growth temperature is 28-32 ℃; the optimal rotation speed of liquid culture is 180rpm; the natural pH, salinity tolerance was stronger than the strain before mutagenesis, and still grew well in PDA medium containing 1% NaCl.
(2) The invention adopts a method combining ground simulation and space mutagenesis as a new mutagenesis means of ganoderma lucidum, and has the remarkable characteristic of high mutagenesis beneficial mutation rate;
(3) The screening method of the invention is based on the principle of metabolite feedback inhibition in microbial metabolic pathways, and the high-concentration metabolic end product beta-glucan is added into a PDA culture plate, so that the strains with good growth conditions in the plate are generally strains with faster growth and higher metabolic efficiency, so that only single bacterial colonies with good growth on the plate are selected, and the strains are generally the strains required in production, thus the screening method is an efficient, simple and convenient excellent strain plate screening method;
(4) Compared with the traditional screening method, the method provided by the invention reduces the workload by more than several times, and greatly reduces the blindness of screening.
(4) The method has simple operation process, and the required reagents and materials are common reagents commonly used in laboratories.
Drawings
Other features, objects and advantages of the present application will become more apparent upon reading of the detailed description of non-limiting embodiments, made with reference to the following drawings, in which:
FIG. 1 shows the growth of strains on 1% NaCl PDA plates before and after mutagenesis;
FIG. 2 is a histogram of differential protein subcellular localization;
reference numerals in the drawings:
Detailed Description
The present application is described in further detail below with reference to the drawings and examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention. It should be noted that, for convenience of description, only the portions related to the invention are shown in the drawings.
It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The present application will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.
Example 1
(1) Ground simulation space mutagenesis test
1) Strain preparation
The original strain ganoderma lucidum (Ganoderma lucidum) SBL-LZ-KJYB-5 (laboratory number) is inoculated on a plate containing PDA culture medium for culture, and when the diameter of the strain exceeds 3cm, the ground simulated space mutagenesis test is prepared. The plates of PDA medium were made as follows: 1) Weighing 39g of potato dextrose agar culture medium Powder (PDA), fixing the volume to 1L by distilled water, naturally p H, sterilizing at 121 ℃ for 30min, pouring into a sterile culture dish, and cooling and solidifying to obtain a solid culture medium; 2) 20-30ml of each plate is poured into the culture medium, and the culture can be used for 24 hours of aseptic culture at 33-37 ℃; 4) After inoculation of the starting strain, the strain was incubated at a constant temperature of 30℃for 5 days.
2) The following mutagenesis experiments were performed sequentially on the strains on the plates:
vacuum low energy particle mutagenesis: placing the cultured Ganoderma strain flat plate (phi 9 cm) into space low energy comprehensive irradiation experimental equipment for vacuum low energy particle irradiation test at 10 -5 Recovering after irradiation under Pa vacuum for 120 min, the radiation dose is 2.6X10 14 eV。
Vibration test when simulating aircraft flight: the cultured ganoderma lucidum strain flat plate (phi 9 cm) is fixed on a 400kN double vibration table, the test is carried out for 1h, the low-frequency sinusoidal vibration test is carried out for 0.5 hour, and the random vibration test is carried out for 0.5 hour. This test simulates the vibration experienced by an aircraft during launch and return.
Gravity acceleration test: the cultured strain is put into a 10mL centrifuge tube, and the overweight test is carried out for 30min by selecting the gravity acceleration of 4 g.
3) Culturing
The mutagenic material is recovered. Taking a simulated space mutagenesis sample with a culture area of 0.2cm by 0.2cm under aseptic conditions, eluting thalli into an aseptic centrifuge tube by using 5 milliliters of aseptic water, sucking and beating uniform thalli eluent by using a pipettor, taking 0.5 milliliters of the thalli eluent into 4.5 milliliters of aseptic water, sucking and beating uniformly, and so on, and performing multiple ratio dilution. 10-4 and 10-5 dilutions were applied to PDA medium plates (which were made in the same manner as PDA solid medium plates in strain preparation in step (1)), 100. Mu.l of diluted bacteria were applied to each plate, and multiple plates were applied per dilution gradient. After the bacterial liquid is absorbed by the culture medium, the bacterial liquid is inversely cultured for 5 days at 30 ℃ in an incubator. Selecting strains with rapid growth and large single colony, continuously culturing and preserving, and performing a next screening test.
4) Screening
(1) Production of screening plates
a, weighing 39g of potato dextrose agar culture medium Powder (PDA) and 100g of beta-glucan, fixing the volume to 1L by distilled water, sterilizing for 30min at the natural pH value of 121 ℃, pouring into a sterile culture dish, and cooling and solidifying to obtain a solid culture medium;
b, taking sterilized culture dishes with the diameter of 90mm, pouring 30ml of culture medium into each culture dish, culturing for 24 hours at the temperature of 32 ℃, selecting a sterile flat plate to enter the next working link, and sterilizing and destroying the polluted flat plate.
(2) Inoculation of
Single colonies which grow rapidly after the space mutagenesis is simulated on the ground are selected, and inoculated on a pre-prepared screening plate by a streaking method.
(3) Culturing
The inoculated plates were incubated in a mold incubator at 30℃for 5 days.
(4) Colony picking
Single colonies growing rapidly on the plates were picked.
(2) Space mutagenesis
1) Space mutagenesis. The more rapid growth (laboratory number SBL-LZ-MN 21) selected from the ground simulation experiment was inoculated on a solid medium, and the inoculated slant was placed in an incubator and cultured at 30℃for 3 days to facilitate the space mutagenesis test. Under the aseptic condition, scraping a cultured inclined plane, adding the inclined plane into a 2mL carrying small pipe, sealing the carrying small pipe by a sealing film, and then filling the carrying small pipe into a carrying large pipe with 30-50mL of solvent. And delivering the carrying large pipe to related personnel of the launching base, installing the carrying large pipe on a Shenzhou-eleven-sized flyer, and carrying out a space mutation test along with the flyer. The carrying pipe is put into a Shenzhou eleventh flying boat, and is recovered after flying for 33 days along with the flying boat in space.
2) And (5) recovering the carrying pipe. Taking a carrying recovery sample with a culture area of about 0.2cm & lt 0.2 & gt cm under a sterile condition, eluting thalli into a sterile centrifuge tube by using 5 milliliters of sterile water, sucking and beating uniform thalli eluent by using a pipette, taking 0.5 milliliters of the thalli eluent into 4.5 milliliters of sterile water, sucking and beating uniformly, and so on, and performing multiple ratio dilution. 10-4 and 10-5 times of the dilution liquid is coated on the plate culture medium, 50-250 microlitres of the dilution bacteria liquid is coated on each plate, and a plurality of plates are coated on each dilution gradient. After the bacterial liquid is absorbed by the culture medium, the bacterial liquid is inversely cultured for 5 days at 30 ℃ in an incubator.
3) Screening
(1) 39g of potato dextrose agar culture medium Powder (PDA) and 50-150g of beta-glucan are weighed, distilled water is used for fixing the volume to 1L, natural pH is carried out, sterilization is carried out for 30min at 121 ℃, and the mixture is poured into a sterile culture dish to be cooled and solidified into a solid culture medium. Taking sterilized culture dishes with the diameter of 90mm, pouring 30ml of culture medium into each culture dish, culturing for 24 hours at 37 ℃, selecting a sterile flat plate to enter the next working link, and sterilizing and destroying the polluted flat plate. Single colonies which grow rapidly after space mutagenesis are selected and streaked onto pre-prepared screening plates.
(2) Culturing: the inoculated plates were incubated in a mold incubator at 30℃for 5 days.
(3) Colony selection: single colonies growing rapidly on the plates were picked.
4) Fermentation
Potato dextrose broth (PDB medium) was prepared: 35 g of potato dextrose broth is weighed, added into 1000ml of distilled water, naturally pH is uniform, and the mixture is sterilized at 121 ℃ for 30 minutes for later use.
Activating the strain on a plate PDA culture medium, culturing at 30deg.C for 10d, and picking about 4mm 2 The bacterial blocks with the size are inoculated into 250mL of PDB culture medium and subjected to shaking culture at the temperature of 28 ℃ for 10d at the speed of 120 r/min.
5) Measurement
Mycelium biomass and extracellular crude polysaccharide content determination: culturing for corresponding days according to the test method, collecting fermentation liquor, centrifuging for 20min at 8000r/min, collecting precipitate to obtain mycelium, washing with distilled water for 3 times, freeze drying, and weighing to obtain mycelium biomass. Units are calculated in g/L.
Centrifuging the fermentation liquor, taking supernatant, measuring the volume of the supernatant, slowly adding 4 times of absolute ethyl alcohol, stirring uniformly, standing at 4 ℃ for alcohol precipitation for 12h, centrifuging at 8000r/min for 20min, collecting precipitate, washing the precipitate with ethanol with the same concentration for 2 times, adding distilled water into the precipitate to fully dissolve, heating to volatilize the ethanol, and freeze-drying to obtain the extracellular crude polysaccharide. The measurement results of the extracellular crude polysaccharide of the mutant strain and the starting strain are shown in Table 1.
Determination of extracellular polysaccharide content: and (3) taking a certain amount of supernatant after alcohol precipitation of the fermentation liquor, and measuring extracellular polysaccharide by using a phenol sulfuric acid method. Units are calculated in g/L. The measurement results of extracellular polysaccharide of the mutant strain and the starting strain are shown in Table 1.
6) Results
TABLE 1 Effect of space mutagenesis on growth of mycelium and exopolysaccharide production of Ganoderma lucidum strains
Figure RE-GDA0003325382630000081
Example 2
(1) Ground simulation space mutagenesis test
1) Strain preparation
The original strain ganoderma lucidum (Ganoderma lucidum) SBL-LZ-6 (laboratory number) is inoculated on a plate containing PDA culture medium for culture, and when the diameter of the strain exceeds 3cm, the ground simulated space mutagenesis test is prepared. The plates of PDA medium were made as follows: 1) Weighing 39g of potato dextrose agar culture medium Powder (PDA), fixing the volume to 1L with distilled water, sterilizing for 30min at the natural pH of 121 ℃, pouring into a sterile culture dish, and cooling and solidifying to obtain a solid culture medium; 2) 20-30ml of each plate is poured into the culture medium, and the culture can be used for 24 hours of aseptic culture at 33-37 ℃; 4) After inoculation of the starting strain, the strain was incubated at a constant temperature of 30℃for 5 days.
2) The following mutagenesis experiments were performed sequentially on the strains on the plates:
vacuum low energy particle mutagenesis: placing the cultured Ganoderma strain flat plate (phi 9 cm) into space low energy comprehensive irradiation experimental equipment for vacuum low energy particle irradiation test at 10 -5 Recovering after irradiation under Pa vacuum for 120 min, the radiation dose is 2.6X10 14 eV。
Vibration test when simulating aircraft flight: the cultured ganoderma lucidum strain flat plate (phi 9 cm) is fixed on a 400kN double vibration table, the test is carried out for 1h, the low-frequency sinusoidal vibration test is carried out for 0.5 hour, and the random vibration test is carried out for 0.5 hour. This test simulates the vibration experienced by an aircraft during launch and return.
Gravity acceleration test: the cultured strain is put into a 10mL centrifuge tube, and the overweight test is carried out for 30min by selecting the gravity acceleration of 4 g.
3) Culturing
The mutagenic material is recovered. Taking a simulated space mutagenesis sample with a culture area of 0.2cm by 0.2cm under aseptic conditions, eluting thalli into an aseptic centrifuge tube by using 5 milliliters of aseptic water, sucking and beating uniform thalli eluent by using a pipettor, taking 0.5 milliliters of the thalli eluent into 4.5 milliliters of aseptic water, sucking and beating uniformly, and so on, and performing multiple ratio dilution. 10-4 and 10-5 dilutions were applied to PDA medium plates (which were made in the same manner as PDA solid medium plates in strain preparation in step (1)), 100. Mu.l of diluted bacteria were applied to each plate, and multiple plates were applied per dilution gradient. After the bacterial liquid is absorbed by the culture medium, the bacterial liquid is inversely cultured for 5 days at 30 ℃ in an incubator. Selecting strains with rapid growth and large single colony, continuously culturing and preserving, and performing a next screening test.
4) Screening
(1) Production of screening plates
a, weighing 39g of potato dextrose agar culture medium Powder (PDA) and 100g of beta-glucan, fixing the volume to 1L by distilled water, sterilizing for 30min at the natural pH value of 121 ℃, pouring into a sterile culture dish, and cooling and solidifying to obtain a solid culture medium;
b, taking sterilized culture dishes with the diameter of 90mm, pouring 30ml of culture medium into each culture dish, culturing for 24 hours at the temperature of 32 ℃, selecting a sterile flat plate to enter the next working link, and sterilizing and destroying the polluted flat plate.
(2) Inoculation of
Single colonies which grow rapidly after the space mutagenesis is simulated on the ground are selected, and inoculated on a pre-prepared screening plate by a streaking method.
(3) Culturing
The inoculated plates were incubated in a mold incubator at 30℃for 5 days.
(4) Colony picking
Single colonies growing rapidly on the plates were picked.
(2) Space mutagenesis
1) Space mutagenesis. The solid culture medium is inoculated with the strain selected from the ground simulation experiment (laboratory number SBL-LZ-MN 21) more rapidly, and the inoculated inclined surface is placed in an incubator and cultured for 3 days at 30 ℃ so as to facilitate the space mutagenesis test. Under the aseptic condition, scraping a cultured inclined plane, adding the inclined plane into a 2mL carrying small pipe, sealing the carrying small pipe by a sealing film, and then filling the carrying small pipe into a carrying large pipe with 30-50mL of solvent. And delivering the carrying large pipe to related personnel of the launching base, installing the carrying large pipe on a Shenzhou-eleven-sized flyer, and carrying out a space mutation test along with the flyer. The carrying pipe is put into a Shenzhou eleventh flying boat, and is recovered after flying for 33 days along with the flying boat in space.
2) And (5) recovering the carrying pipe. Taking a carrying recovery sample with a culture area of about 0.2cm & lt 0.2 & gt cm under a sterile condition, eluting thalli into a sterile centrifuge tube by using 5 milliliters of sterile water, sucking and beating uniform thalli eluent by using a pipette, taking 0.5 milliliters of the thalli eluent into 4.5 milliliters of sterile water, sucking and beating uniformly, and so on, and performing multiple ratio dilution. 10-4 and 10-5 times of the dilution liquid is coated on the plate culture medium, 50-250 microlitres of the dilution bacteria liquid is coated on each plate, and a plurality of plates are coated on each dilution gradient. After the bacterial liquid is absorbed by the culture medium, the bacterial liquid is inversely cultured for 5 days at 30 ℃ in an incubator.
3) Screening
(1) 39g of potato dextrose agar culture medium Powder (PDA) and 50-150g of beta-glucan are weighed, distilled water is used for fixing the volume to 1L, natural pH is carried out, sterilization is carried out for 30min at 121 ℃, and the mixture is poured into a sterile culture dish to be cooled and solidified into a solid culture medium. Taking sterilized culture dishes with the diameter of 90mm, pouring 30ml of culture medium into each culture dish, culturing for 24 hours at 37 ℃, selecting a sterile flat plate to enter the next working link, and sterilizing and destroying the polluted flat plate. Single colonies which grow rapidly after space mutagenesis are selected and streaked onto pre-prepared screening plates.
(2) Culturing: the inoculated plates were incubated in a mold incubator at 30℃for 5 days.
(3) Colony selection: single colonies growing rapidly on the plates were picked.
4) Fermentation
Potato dextrose broth (PDB medium) was prepared: 35 g of potato dextrose broth is weighed, added into 1000ml of distilled water, naturally pH is uniform, and the mixture is sterilized at 121 ℃ for 30 minutes for later use.
Activating the strain on a plate PDA culture medium, culturing at 30deg.C for 10d, and picking about 4mm 2 The bacterial blocks with the size are inoculated into 250mL of PDB culture medium and subjected to shaking culture for 10d at the temperature of 30 ℃ at 180 r/min.
5) Measurement
Mycelium biomass and extracellular crude polysaccharide content determination: culturing for corresponding days according to the test method, collecting fermentation broth, centrifuging for 20min at 8000r/min, collecting precipitate to obtain mycelium, washing with distilled water for 3 times, freeze drying, and weighing to obtain mycelium biomass. Units are calculated in g/L.
Centrifuging the fermentation liquor, taking supernatant, measuring the volume of the supernatant, slowly adding 4 times of absolute ethyl alcohol, stirring uniformly, standing at 4 ℃ for alcohol precipitation for 12h, centrifuging at 8000r/min for 20min, collecting precipitate, washing the precipitate with ethanol with the same concentration for 2 times, adding distilled water into the precipitate to fully dissolve, heating to volatilize the ethanol, and freeze-drying to obtain the extracellular crude polysaccharide. The measurement results of the extracellular crude polysaccharide of the mutant strain and the starting strain are shown in Table 2.
Determination of extracellular polysaccharide content: and (3) taking a certain amount of supernatant after alcohol precipitation of the fermentation liquor, and measuring extracellular polysaccharide by using a phenol sulfuric acid method. Units are calculated in g/L. The measurement results of extracellular polysaccharide of the mutant strain and the starting strain are shown in Table 2.
6) Results
TABLE 2 Effect of space mutagenesis on growth of mycelium and extracellular polysaccharide production content of Ganoderma lucidum strains
Figure RE-GDA0003325382630000111
From the test results, the amounts of mycelium and extracellular polysaccharide produced by the ganoderma lucidum strain are related to the strain, and are also related to the culture method greatly. Meanwhile, the effect of the mutant strain on producing extracellular crude polysaccharide and extracellular polysaccharide is more obvious than that of the original strain.
Salt resistance test of mutagenized Strain
As shown in FIG. 1, colonies of approximately 0.2 cm.times.0.2 cm in size of cultivated Ganoderma lucidum were inoculated on PDA plate medium containing 1% NaCl before and after mutagenesis, and cultivated at 30℃for 5d. The strain after mutagenesis obviously grows more densely than the original strain on a flat plate with the salinity of 1%, and the mycelium content is more abundant.
Genetic study of mutagenized Strain
In order to further determine the genetic characteristics of the mutagenized strain, quantitative proteomic analysis and non-targeted metabonomic analysis of ganoderma lucidum strain proteins before and after simulated space mutagenesis treatment were performed using isotope labeled relative and absolute quantification (iTRAQ) techniques and LC-MS/MS techniques.
(1) Proteomics research
As shown in FIG. 2, a total of 14893 peptide fragments and 3783 proteins were identified in proteomic analysis, 982 of the differential proteins, with 490 up-regulated and 492 down-regulated protein expression. Among them, 268 proteins located in cytoplasm, 224 of mitochondria, 195 extracellular, 174 nuclei, 54 plasma membranes, 34 of cytoplasm and nucleus, 26 cytoskeletal, 5 of cytoplasm and fine particle, and 2 peroxisomes.
(2) Metabonomics study
As shown in table 3, the metabonomic analysis results showed that inosine, indole-3-acetic acid, biotin, which was significantly associated with growth, were significantly upregulated, whereas jasmonic acid, which was downregulated, was also upregulated by ribitol associated with the pentose phosphate pathway. The results of proteomics and metabonomics mechanistically explain the reason for the increased growth of the mutant strain and the increased extracellular crude polysaccharide content.
TABLE 3 Table 3
Figure RE-GDA0003325382630000121
Figure RE-GDA0003325382630000131
The above description is only illustrative of the preferred embodiments of the present application and of the principles of the technology employed. Meanwhile, the scope of the invention referred to in this application is not limited to the technical solutions of the specific combination of the above technical features, but also covers other technical solutions formed by any combination of the above technical features or their equivalents without departing from the inventive concept. Such as the above-described features and technical features having similar functions (but not limited to) disclosed in the present application are replaced with each other.

Claims (1)

1. A mutant strain of ganoderma lucidum, characterized by: ganoderma Ganodermatudursbl-LZ-KJYB-5 with the preservation number of CGMCC No.23055.
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