CN109988716B - Method for protecting ganoderma lucidum strain by uracil auxotrophy - Google Patents
Method for protecting ganoderma lucidum strain by uracil auxotrophy Download PDFInfo
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Abstract
The invention belongs to the technical field of strain protection, and relates to a method for protecting ganoderma lucidum strains by utilizing uracil auxotrophy, which comprises the following steps: hybridizing the ganoderma uracil auxotroph monokaryon 80-3 with a ganoderma monokaryon wild type strain, and separating the monosporium after fruiting to obtain a monokaryon strain; screening the obtained mononucleon strain to obtain uracil auxotroph mononucleon strain; and (3) singly hybridizing the obtained uracil auxotroph monokaryon strain, and culturing to obtain the ganoderma lucidum uracil auxotroph dinucleotide strain. The growth speed of the obtained ganoderma lucidum uracil auxotroph binuclear strain on a PDA culture medium added with uracil is improved by 97.5 percent compared with that on the PDA culture medium, the ganoderma lucidum auxotroph binuclear strain cannot grow normally in a basic culture medium without uracil additives, and ganoderma lucidum strains can be effectively protected.
Description
Technical Field
The invention belongs to the technical field of strain protection, relates to a ganoderma lucidum strain protection method, and in particular relates to a ganoderma lucidum strain protection method by utilizing uracil auxotrophy.
Background
Ganoderma lucidum is a traditional edible and medicinal fungus in China, and contains various bioactive components such as ganoderan, triterpene compounds, nucleosides, amino acids, ergosterol, alkaloids, etc. Proved by many-sided researches on pharmacology, clinical application and the like of ganoderma lucidum, the ganoderma lucidum polysaccharide has the effects of reducing blood sugar, promoting the immune function of intestinal mucosa immune system, delaying aging, protecting liver, promoting sleep and the like. The development of ganoderma lucidum cultivation and production depends on strains, the breeding of ganoderma lucidum strains depends on long-term accumulation, and a great deal of labor, physical and time cost is required to be input, but ganoderma lucidum is asexually propagated, and the strain production process has replicability, so that ganoderma lucidum strains are easy to imitate.
Auxotrophs lose the ability to synthesize a substance due to mutation of the gene, do not grow normally in minimal medium, and must be supplemented with certain nutrients to produce growth. If it is not known exactly what kind of nutritional deficiency is, its production is extremely limited, so that the intellectual property rights of breeders are fully protected.
The ganoderma lucidum is used as edible and medicinal fungi and has higher safety by adopting an auxotroph mark. Uracil auxotrophs are a common marker in microorganisms. 5-fluoroorotic acid is a substrate analogue of orotidine nucleotides in the uracil synthetic pathway, which can be converted into 5-fluorouracil nucleotides having strong cytotoxicity, thereby inhibiting growth of the wild-type strain. Whereas uracil auxotrophs cannot be metabolized to produce toxic 5-fluorouracil nucleotides due to disruption of the uracil synthetic pathway, and thus 5-fluoroorotic acid resistance cannot be obtained.
CN 107557354A discloses a method for protecting pleurotus eryngii strain by uracil auxotroph, CN 105802952A discloses a method for protecting crystal form mushroom strain by uracil auxotroph, CN 106978413A discloses a method for protecting flammulina velutipes strain by uracil auxotroph, and although the methods for protecting pleurotus eryngii, mushrooms and flammulina velutipes strain are disclosed, no study is made on the method for protecting ganoderma lucidum strain in the prior art.
Disclosure of Invention
The invention aims to provide a method for protecting ganoderma lucidum strains by utilizing uracil auxotrophs, which is simple and low in cost, and the obtained ganoderma lucidum uracil auxotroph binuclear strain cannot normally grow in a culture medium without uracil additives, so that the ganoderma lucidum strains are protected.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
the invention provides a method for protecting ganoderma lucidum strains by utilizing uracil auxotrophy, which comprises the following steps:
(1) Hybridizing the ganoderma uracil auxotroph monokaryon 80-3 with a ganoderma monokaryon wild type strain, and separating the monosporium after fruiting to obtain a monokaryon strain;
(2) Screening the mononucleosome strain obtained in the step (1) to obtain uracil auxotroph mononucleosome strain;
(3) And (3) singly hybridizing the uracil auxotroph mononucleon strain obtained in the step (2), and culturing to obtain the ganoderma lucidum uracil auxotroph dinuclear strain.
The ganoderma lucidum uracil auxotroph mononucleoid 80-3 has a preservation number of: CGMCC No.12879, the preservation date is: the classification name of the ganoderma lucidum (Ganoderma lucidum) is 2016, 9 months and 19 days, the preservation unit is China general microbiological culture Collection center, and the address is 1 th or 3 rd of North West Lu in the Korean region of Beijing city.
The uracil auxotroph binuclear strain has a deposit number of: CGMCC No.16098, the preservation date is: the classification name of the ganoderma lucidum (Ganoderma lucidum) is 24 days 8.2018, the preservation unit is the China general microbiological culture Collection center, and the address is North Silu No.1 and No. 3 in the North Yangyang area of Beijing city.
Preferably, the fruiting of step (1) is carried out on wood chip medium.
The wood chip culture medium consists of 78wt.% of wood chips, 20wt.% of wheat bran, 1wt.% of gypsum and 1wt.% of sucrose, the water content of the mixed wood chip culture medium is 60-65%, and the wood chip culture medium is packaged and sterilized for fruiting.
Preferably, the step of monospore separation in step (1) is:
(a) Collecting spores which are ejected by fruiting body single plants naturally after fruiting, soaking the spores in sterile water, and diluting to obtain a dispersion;
(b) The dispersion liquid is coated on a protoplast regeneration screening culture medium, after culturing, germinating spores are transferred to a PDA culture medium added with uracil, and after culturing, edge hyphae are selected, and the edge hyphae are cultured on a new PDA culture medium added with uracil, and after repeating culturing for 4-6 times, the mononuclear body strain is obtained.
The protoplast regeneration screening culture medium is a culture medium obtained by adding uracil and 5-fluoroorotic acid into a PDA culture medium, wherein the concentration of uracil in the protoplast regeneration screening culture medium is 0.05mmol/L, and the concentration of 5-fluoroorotic acid is 0.5g/L.
The PDA culture medium added with uracil is a culture medium obtained by adding uracil into the PDA culture medium, and the concentration of uracil in the PDA culture medium added with uracil is 0.1mmol/L.
The temperature of the culture in the step (b) is 20-28 ℃, the culture time is 14-16d, the temperature of the culture is 20-28 ℃, and the culture time is 6-8d.
Preferably, the PDA medium is a PDA medium prepared by the steps of: 39g of PDA powder was mixed with sterile water to a volume of 1L, sterilized and poured into a plate to obtain the PDA culture medium.
The PDA powder is potato dextrose agar with the model number of CND07-F0185 produced by BD company.
Preferably, the dilution in step (a) is performed using sterile water, the concentration of spores in the resulting spore liquid being 1X 10 4 And each mL.
Preferably, the step of screening in step (2) is: and (3) inoculating the mononucleon strain obtained in the step (1) into a basic culture medium, a basic culture medium added with uracil and a screening culture medium respectively and independently, wherein the mononucleon strain which can grow in the basic culture medium added with uracil and the screening culture medium but does not grow in the basic culture medium is the uracil auxotroph mononucleon strain.
Preferably, the hybridization of step (3) is performed on minimal medium supplemented with uracil.
Preferably, the minimal medium is a minimal medium prepared by the steps of: mixing glucose and KH 2 PO 3 、(NH 4 ) 2 HPO 3 、MgSO 4 ·7H 2 O, thiamine HCl, agar powder and water are added with water to 1L, and the basic culture medium is obtained by pouring a flat plate after sterilization.
The basic culture medium is prepared from glucose and KH 2 PO 3 、(NH 4 ) 2 HPO 3 、MgSO 4 ·7H 2 A culture medium consisting of O, thiamine HCl, agar powder and water, wherein each 1L of the basic culture medium contains 20g of glucose and 1g of KH 2 PO 3 ，1.5g(NH 4 ) 2 HPO 3 ，0.3g MgSO 4 ·7H 2 O,500 mug Thiamine HCl, 15g agar powder and the balance water.
The basic culture medium added with uracil is a culture medium obtained by adding uracil into the basic culture medium, and the concentration of uracil in the basic culture medium added with uracil is 0.05mmol/L.
The screening culture medium is obtained by adding uracil and 5-fluoroorotic acid into a basic culture medium, wherein the concentration of uracil in the screening culture medium is 0.05mmol/L, and the concentration of 5-fluoroorotic acid is 0.5g/L.
As a preferred technical scheme of the method provided by the invention, the method comprises the following steps:
(1) Hybridizing Ganoderma uracil auxotroph monokaryon 80-3 with wild strain of Ganoderma monokaryon, fruiting on wood chip culture medium, collecting spores ejected from fruiting body, soaking in sterile water, and diluting to 1×10 4 Performing culture on the mycelia, namely obtaining dispersion liquid, coating the dispersion liquid on a protoplast regeneration screening culture medium, transferring germination spores to a uracil-added PDA culture medium after culture, selecting edge mycelia after culture, and performing culture on the edge mycelia on a new uracil-added PDA culture medium again, and repeatedly culturing for 4-6 times to obtain a mononuclear body strain;
(2) Inoculating the mononuclear body strain obtained in the step (1) into a basic culture medium, a basic culture medium added with uracil and a screening culture medium respectively and independently, wherein the mononuclear body strain which can grow in the basic culture medium added with uracil and the screening culture medium but does not grow in the basic culture medium is the uracil auxotroph mononuclear body strain;
(3) And (3) singly hybridizing the uracil auxotroph mononucleon strain obtained in the step (2) on a basic culture medium added with uracil, and culturing to obtain the ganoderma lucidum uracil auxotroph dinuclear strain.
Compared with the prior art, the invention has the following beneficial effects:
the method provided by the invention is simple, has low cost, the growth speed of the ganoderma lucidum uracil auxotroph binuclear strain on the PDA culture medium added with uracil is improved by 97.5% compared with that on the PDA culture medium, the ganoderma lucidum strain can not normally grow in the basic culture medium without uracil additives, and the ganoderma lucidum strain can be effectively protected.
Drawings
FIG. 1 is a microscopic image of a ganoderma lucidum uracil auxotroph binuclear strain obtained in example 1;
FIG. 2 is a graph showing growth rates of the ganoderma lucidum uracil auxotroph binuclear strain obtained in example 1 in PDA medium and PDA medium supplemented with uracil.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof. Wherein the Ganoderma wild type mononuclear body strain 241 and the binuclear body strain "Hunong Ganoderma lucidum No. one" are all preserved by edible fungus division center of China center for agricultural microbiological culture collection center.
Example 1
The embodiment provides a method for protecting ganoderma lucidum strains by utilizing uracil auxotrophs, which comprises the following steps:
(1) Hybridizing Ganoderma uracil auxotroph monomer 80-3 with Ganoderma monomer strain 241, fruiting on wood chip culture medium, collecting fruiting body single plant naturally ejected spore, soaking in sterile water, and diluting to 1×10 4 Coating dispersion liquid on a protoplast regeneration screening culture medium, culturing in a biochemical incubator at 25 ℃ for 14d, transferring germination spores to a PDA culture medium added with uracil, culturing in the biochemical incubator at 25 ℃ for 7d, selecting edge mycelia, culturing the edge mycelia on a new PDA culture medium added with uracil again, and repeatedly culturing for 5 times to obtain a mononuclear body strain;
(2) Inoculating the mononucleon strain obtained in the step (1) into a basic culture medium, a basic culture medium added with uracil and a screening culture medium independently, wherein the mononucleon strain which can grow in the basic culture medium added with uracil and the screening culture medium but does not grow in the basic culture medium is the uracil auxotroph mononucleon strain;
(3) And (3) singly hybridizing the uracil auxotroph mononucleon strain obtained in the step (2) on a basic culture medium added with uracil, and culturing to obtain the ganoderma lucidum uracil auxotroph dinuclear strain.
The microscopic images of the obtained ganoderma lucidum uracil auxotroph binuclear strain are shown in fig. 1, the growth rates of the obtained ganoderma lucidum uracil auxotroph binuclear strain in the PDA culture medium and the uracil-added PDA culture medium are shown in fig. 2, and the average growth rate of the obtained ganoderma lucidum uracil auxotroph strain in the PDA culture medium is 1.97mm/d, and the growth rate of the obtained ganoderma lucidum uracil auxotroph strain in the uracil-added PDA culture medium is 3.89mm/d.
As is clear from example 1, the average growth rate of the ganoderma lucidum uracil auxotroph dual-nucleus strain obtained in example 1 was 1.97mm/d on PDA medium, the average growth rate was 3.89mm/d on uracil-added PDA medium, and the growth rate was improved by 97.5%.
In conclusion, the method provided by the invention is simple and low in cost, the growth speed of the ganoderma lucidum uracil auxotroph dual-nucleus strain on the PDA culture medium added with uracil is improved by 97.5% compared with that of the ganoderma lucidum uracil auxotroph dual-nucleus strain on the PDA culture medium, the ganoderma lucidum strain cannot normally grow in a basic culture medium without uracil additives, and the ganoderma lucidum strain can be effectively protected.
The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.
Claims (1)
1. The ganoderma uracil auxotroph binuclear strain is characterized in that the strain is ganoderma lucidum (Ganoderma lucidum) UL26 strain, the preservation number is CGMCC NO.16098, the preservation date is 2018, 8 months and 24 days, the preservation unit is China general microbiological culture Collection center, and the preservation address is North Xielu No.1, 3 in the Korean region of Beijing city.
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| CN105802952A (en) * | 2016-03-29 | 2016-07-27 | 上海市农业科学院 | Method for protecting lentinus edodes strain through uracil auxotroph |
| CN106978413A (en) * | 2016-12-26 | 2017-07-25 | 上海市农业科学院 | A kind of method that Needle mushroom strain protection is carried out using uracil auxotrophy |
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| CN104642142A (en) * | 2015-03-18 | 2015-05-27 | 云南明侍达生物科技有限责任公司 | Mutation breeding method of lucid ganoderma strains |
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| CN105802952A (en) * | 2016-03-29 | 2016-07-27 | 上海市农业科学院 | Method for protecting lentinus edodes strain through uracil auxotroph |
| CN106978413A (en) * | 2016-12-26 | 2017-07-25 | 上海市农业科学院 | A kind of method that Needle mushroom strain protection is carried out using uracil auxotrophy |
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