CN109988716A - A method of lucidum strain protection is carried out using uracil auxotrophy - Google Patents
A method of lucidum strain protection is carried out using uracil auxotrophy Download PDFInfo
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Abstract
The invention belongs to strain protection technique fields; it is related to a kind of method for carrying out lucidum strain protection using uracil auxotrophy; described method includes following steps: ganoderma lucidum uracil auxotrophy monocaryon 80-3 being hybridized with ganoderma lucidum monocaryon wild-type strain, single spore separation obtains monocaryon bacterial strain after fruiting；Screening gained monocaryon bacterial strain, obtains uracil auxotrophy monocaryon bacterial strain；Gained uracil auxotrophy monocaryon bacterial strain is only hybridized, ganoderma lucidum uracil auxotrophy dicaryon bacterial strain is obtained after culture.Gained ganoderma lucidum uracil auxotrophy dicaryon bacterial strain addition uracil PDA culture medium on the speed of growth relative in PDA culture medium the speed of growth improve 97.5%; without uracil additive minimal medium in can not normal growth, lucidum strain can be effectively protected.
Description
Technical field
The invention belongs to strain protection technique fields, are related to a kind of method of lucidum strain protection, and in particular to Yi Zhongli
The method for carrying out lucidum strain protection with uracil auxotrophy.
Background technique
Ganoderma lucidum is the edible and medical fungi of Chinese tradition, contains ganoderma lucidum polysaccharide, triterpene compound, ucleosides, amino acid, ergot
The various bioactive components such as sterol, alkaloid.Various researchs confirmations such as pharmacology and clinical application to ganoderma lucidum, ganoderma lucidum
Polysaccharide has immune function that is hypoglycemic, promoting Intestinal Mucosal Immunization system, anti-aging, liver protection, promotion sleep and other effects.Spirit
The development of sesame cultivation and production relies on strain, and the breeding of lucidum strain needs to need to put into a large amount of people by long-term accumulation
Power, physics and time cost, but ganoderma lucidum is vegetative propagation, and strain production technology has reproducibility, so that Ganoderma Lucidum
Kind is easy counterfeit.
Auxotroph strain loses the ability for synthesizing certain substance since gene mutates, in basic medium
It can not normal growth, it is necessary to which supplementing certain nutriments just can just generate length.If can not clearly know it is which kind of battalion actually
Defect is supported, production will receive very big limitation, so that the intellectual property of breeder be made adequately to be protected.
Ganoderma lucidum has higher safety using citrinipileatus as edible and medicinal fungi.Uracil auxotrophy is
One kind commonly marks in microorganism.5- fluororotic acid is that the substrate of orotidylic acid in uracil route of synthesis is similar
Object, can be converted into the 5 FU 5 fluorouracil nucleotide of very strong cytotoxicity, to inhibit the growth of wild-type strain.And uracil
Auxotrophic strain is because the route of synthesis of uracil is interrupted, and energy metabolism does not generate toxic 5 FU 5 fluorouracil nucleotide, because
This cannot obtain 5- fluororotic acid resistance.
107557354 A of CN discloses a kind of method for carrying out pleurotus eryngii quel strains protection using uracil auxotrophy,
105802952 A of CN discloses a kind of method protected using uracil auxotrophy crystal form mushroom strain, CN
106978413 A disclose a kind of method for carrying out Needle mushroom strain protection using uracil auxotrophy, though these methods
Strain protection so has been carried out to Pleurotus eryngii, mushroom and needle mushroom, but in the prior art not to lucidum strain protection method into
Row research.
Summary of the invention
The purpose of the present invention is to provide a kind of method for carrying out lucidum strain protection using uracil auxotrophy, the party
Method is simple, at low cost, and obtained ganoderma lucidum uracil auxotrophy dicaryon bacterial strain is in the culture for not containing uracil additive
In base can not normal growth, to realize the protection to lucidum strain.
In order to achieve that object of the invention, the invention adopts the following technical scheme:
The present invention provides a kind of method for carrying out lucidum strain protection using uracil auxotrophy, the method packets
Include following steps:
(1) ganoderma lucidum uracil auxotrophy monocaryon 80-3 is hybridized with ganoderma lucidum monocaryon wild-type strain, after fruiting
Single spore separation obtains monocaryon bacterial strain；
(2) monocaryon bacterial strain obtained by screening step (1), obtains uracil auxotrophy monocaryon bacterial strain；
(3) uracil auxotrophy monocaryon bacterial strain obtained by step (2) is only hybridized, ganoderma lucidum is obtained after culture
Uracil auxotrophy dicaryon bacterial strain.
The deposit number of the ganoderma lucidum uracil auxotrophy monocaryon 80-3 are as follows: CGMCC NO.12879, preservation day
Phase are as follows: on September 19th, 2016, specific name are ganoderma lucidum (Ganoderma lucidum), and depositary institution is Chinese microorganism strain
Preservation administration committee common micro-organisms center, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The deposit number of the uracil auxotrophy dicaryon bacterial strain are as follows: CGMCC NO.16098, preservation date are as follows:
On August 24th, 2018, specific name are ganoderma lucidum (Ganoderma lucidum), and depositary institution is Chinese microorganism strain preservation
Administration committee's common micro-organisms center, address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Preferably, step (1) described fruiting carries out on sawdust medium.
Sawdust medium of the present invention by the sawdust of 78wt.%, the wheat bran of 20wt.%, 1wt.% gypsum and
The sucrose of 1wt.% forms, and the water content of the sawdust medium mixed is 60-65%, supplies after sawdust medium pack sterilizing
Fruiting uses.
Preferably, the step of step (1) described single spore separation are as follows:
(a) spore that fructification single plant launches naturally after collection fruiting, dilutes after spore is impregnated in sterile water, obtains
Dispersion liquid；
(b) dispersion liquid is coated on protoplast regeneration screening and culturing medium, after culture by sprout spore be transferred to addition urine it is phonetic
In the PDA culture medium of pyridine, edge mycelia is selected after being further cultured for, and by edge mycelia in the PDA culture medium of new addition uracil
On cultivated, repetition be further cultured for 4-6 times after obtain monocaryon bacterial strain.
Protoplast regeneration screening and culturing medium of the present invention is that uracil and 5- fluororotic acid are added in PDA culture medium
Obtained culture medium, wherein the concentration of uracil is 0.05mmol/L, 5- fluorine whey in protoplast regeneration screening and culturing medium
The concentration of acid is 0.5g/L.
The PDA culture medium of addition uracil of the present invention is the culture medium for adding uracil in PDA culture medium and obtaining,
The concentration for adding uracil in the PDA culture medium of uracil is 0.1mmol/L.
The temperature of culture described in step (b) of the present invention is 20-28 DEG C, and the time of culture is 14-16d, described to cultivate
Temperature be 20-28 DEG C, the time being further cultured for be 6-8d.
Preferably, the PDA culture medium is the PDA culture medium being prepared by following steps: 39g PDA powder and nothing
It is settled to 1L after the mixing of bacterium water, inverted plate after sterilizing obtains the PDA culture medium.
PDA powder of the present invention is the potato dextrose agar of the model CND07-F0185 of BD company production.
Preferably, step (a) is described is diluted to dilute using sterile water, and the concentration of gained spore liquid miospore is 1 × 104
A/mL.
Preferably, the step of step (2) described screening are as follows: be separately inoculated with monocaryon bacterial strain obtained by step (1)
In minimal medium, addition uracil base basal culture medium and screening and culturing medium in, can addition uracil base basal culture medium and
It is grown in screening and culturing medium, but non-growing monocaryon bacterial strain is the uracil auxotrophy list in basic medium
Nucleome bacterial strain.
Preferably, step (3) hybridization carries out on the minimal medium of addition uracil.
Preferably, the minimal medium is the minimal medium being prepared by following steps: mixed glucose,
KH2PO3、(NH4)2HPO3、MgSO4·7H2O, Thiamine HCl, agar powder and water add water to 1L, and inverted plate obtains after sterilizing
To the minimal medium.
Minimal medium of the present invention is by glucose, KH2PO3、(NH4)2HPO3、MgSO4·7H2O、 Thiamine
The culture medium of HCl, agar powder and water composition, wherein containing 20g glucose, 1g KH in every 1L minimal medium2PO3, 1.5g
(NH4)2HPO3, 0.3g MgSO4·7H2O, 500 μ g Thiamine HCl, 15g agar powders, surplus is water.
The minimal medium of addition uracil of the present invention is the culture that addition uracil obtains in basic medium
Base, it is described addition uracil minimal medium in uracil concentration be 0.05mmol/L.
Screening and culturing medium of the present invention is the culture that addition uracil and 5- fluororotic acid obtain in basic medium
Base, wherein the concentration of uracil is 0.05mmol/L in screening and culturing medium, and the concentration of 5- fluororotic acid is 0.5g/L.
As the optimal technical scheme of the method provided by the present invention, described method includes following steps:
(1) ganoderma lucidum uracil auxotrophy monocaryon 80-3 is hybridized with ganoderma lucidum monocaryon wild-type strain, sawdust training
Fruiting on base is supported, the spore that launches naturally of fructification single plant after fruiting is collected, it is diluted to 1 after spore is impregnated in sterile water ×
104A/mL obtains dispersion liquid, and dispersion liquid is coated on protoplast regeneration screening and culturing medium, and spore will be sprouted after culture and is transferred to
In the PDA culture medium for adding uracil, edge mycelia is selected after being further cultured for, and by edge mycelia in new addition uracil
It is further cultured in PDA culture medium, repetition obtains monocaryon bacterial strain after being further cultured for 4-6 times；
(2) monocaryon bacterial strain obtained by step (1) is separately inoculated in minimal medium, addition uracil is trained substantially
It supports in base and screening and culturing medium, can be grown in the minimal medium and screening and culturing medium of addition uracil, but training substantially
Supporting non-growing monocaryon bacterial strain in base is the uracil auxotrophy monocaryon bacterial strain；
(3) uracil auxotrophy monocaryon bacterial strain obtained by step (2) is enterprising in addition uracil base basal culture medium
Row only hybridizes, and ganoderma lucidum uracil auxotrophy dicaryon bacterial strain is obtained after culture.
Compared with the existing technology, the invention has the following advantages:
Method provided by the invention is simple, at low cost, and ganoderma lucidum uracil auxotrophy dicaryon bacterial strain is phonetic in addition urine
In the PDA culture medium of pyridine the speed of growth relative in PDA culture medium the speed of growth improve 97.5%, without containing urinate it is phonetic
In the minimal medium of pyridine additive can not normal growth, lucidum strain can be effectively protected.
Detailed description of the invention
Fig. 1 is the microscopy picture for the ganoderma lucidum uracil auxotrophy dicaryon bacterial strain that embodiment 1 obtains；
Fig. 2 is that the ganoderma lucidum uracil auxotrophy dicaryon bacterial strain that embodiment 1 obtains is phonetic in PDA culture medium and addition urine
Speed of growth figure in the PDA culture medium of pyridine.
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright
, the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention.Wherein ganoderma lucidum used is wild
Type monocaryon bacterial strain 241, dicaryon bacterial strain " Shanghai agriculture ganoderma lucidum No.1 " are by Chinese agriculture Organism Depositary edible mushroom point
Heart preservation.
Embodiment 1
Present embodiments provide a kind of method for carrying out lucidum strain protection using uracil auxotrophy, the method
Include the following steps:
(1) ganoderma lucidum uracil auxotrophy monocaryon 80-3 is hybridized with ganoderma lucidum monocaryon bacterial strain 241, sawdust medium
Upper fruiting collects the spore that launches naturally of fructification single plant after fruiting, is diluted to 1 × 10 after spore is impregnated in sterile water4
A/mL, obtains dispersion liquid, and dispersion liquid is coated on protoplast regeneration screening and culturing medium, and 25 DEG C in cultivating 14d in biochemical cultivation case
Spore will be sprouted afterwards to be transferred in the PDA culture medium of addition uracil, 25 DEG C are selected edge after cultivating 7d in biochemical cultivation case
Mycelia, and edge mycelia is further cultured in the PDA culture medium of new addition uracil, repetition obtains after being further cultured for 5 times
Monocaryon bacterial strain；
(2) monocaryon bacterial strain obtained by step (1) is separately inoculated in minimal medium, addition uracil is trained substantially
It supports in base and screening and culturing medium, can be grown in addition uracil base basal culture medium and screening and culturing medium, but cultivating substantially
Non-growing monocaryon bacterial strain is the uracil auxotrophy monocaryon bacterial strain in base；
(3) uracil auxotrophy monocaryon bacterial strain obtained by step (2) is enterprising in addition uracil base basal culture medium
Row only hybridizes, and ganoderma lucidum uracil auxotrophy dicaryon bacterial strain is obtained after culture.
The microscopy picture of gained ganoderma lucidum uracil auxotrophy dicaryon bacterial strain is as shown in Figure 1, gained ganoderma lucidum uracil
Auxotroph dicaryon bacterial strain PDA culture medium and addition uracil PDA culture medium in the speed of growth as shown in Fig. 2,
It knows that average growth rate of the gained ganoderma lucidum uracil auxotrophy bacterial strain in PDA culture medium is 1.97mm/d, is adding
The speed of growth in the PDA culture medium of uracil is 3.89mm/d.
By embodiment 1 it is found that 1 gained ganoderma lucidum uracil auxotrophy dicaryon bacterial strain of embodiment is on PDA culture medium
Average growth rate be 1.97mm/d, addition uracil PDA culture medium on average growth rate be 3.89mm/d, growth
Speed improves 97.5%.
In conclusion method provided by the invention is simple, at low cost, ganoderma lucidum uracil auxotrophy dicaryon bacterial strain exists
The speed of growth in the PDA culture medium of uracil is added to cultivate relative to ganoderma lucidum uracil auxotrophy dicaryon bacterial strain in PDA
The speed of growth improves 97.5% on base, without uracil additive minimal medium in can not normal growth, can be right
Lucidum strain effectively protects.
The Applicant declares that the foregoing is merely a specific embodiment of the invention, but protection scope of the present invention not office
It is limited to this, it should be clear to those skilled in the art, any to belong to those skilled in the art and take off in the present invention
In the technical scope of dew, any changes or substitutions that can be easily thought of, and all of which fall within the scope of protection and disclosure of the present invention.
Claims (8)
1. a kind of method for carrying out lucidum strain protection using uracil auxotrophy, which is characterized in that the method includes
Following steps:
(1) ganoderma lucidum uracil auxotrophy monocaryon 80-3 is hybridized with ganoderma lucidum monocaryon wild-type strain, monospore after fruiting
Separation obtains monocaryon bacterial strain；
(2) monocaryon bacterial strain obtained by screening step (1), obtains uracil auxotrophy monocaryon bacterial strain；
(3) uracil auxotrophy monocaryon bacterial strain obtained by step (2) is only hybridized, it is phonetic that ganoderma lucidum urine is obtained after culture
Pyridine auxotroph dicaryon bacterial strain；
The deposit number of the ganoderma lucidum uracil auxotrophy monocaryon 80-3 are as follows: CGMCC NO.12879, preservation date are as follows:
On September 19th, 2016；
The deposit number of the uracil auxotrophy dicaryon bacterial strain are as follows: CGMCC NO.16098, preservation date are as follows: 2018
On August 24,.
2. the method according to claim 1, wherein step (1) described fruiting carries out on sawdust medium.
3. method according to claim 1 or 2, which is characterized in that the step of step (1) described single spore separation are as follows:
(a) spore that fructification single plant launches naturally after collection fruiting, dilutes after spore is impregnated in sterile water, is dispersed
Liquid；
(b) dispersion liquid is coated on protoplast regeneration screening and culturing medium, and spore will be sprouted after culture and is transferred to addition uracil
In PDA culture medium, edge mycelia is selected after being further cultured for, and edge mycelia is enterprising in the PDA culture medium of new addition uracil
Row is further cultured for, and repetition obtains monocaryon bacterial strain after being further cultured for 4-6 times.
4. according to the method described in claim 3, it is characterized in that, the PDA culture medium is prepared by following steps
PDA culture medium: 39g PDA powder is settled to 1L after mixing with sterile water, and inverted plate after sterilizing obtains the PDA culture medium.
5. method according to claim 1-4, which is characterized in that step (a) is described to be diluted to using sterile water
Dilution, the concentration of gained spore liquid miospore are 1 × 104A/mL.
6. method according to claim 1-5, which is characterized in that the step of step (2) described screening are as follows: will walk
Suddenly monocaryon bacterial strain obtained by (1) is separately inoculated in minimal medium, addition uracil base basal culture medium and screening and culturing
In base, it can be grown in addition uracil base basal culture medium and screening and culturing medium, but non-growing list in basic medium
Nucleome bacterial strain is the uracil auxotrophy monocaryon bacterial strain.
7. method according to claim 1-6, which is characterized in that step (3) hybridization is in addition uracil
Minimal medium on carry out.
8. method according to claim 1-7, which is characterized in that described method includes following steps:
(1) ganoderma lucidum uracil auxotrophy monocaryon 80-3 is hybridized with ganoderma lucidum monocaryon wild-type strain, sawdust medium
Upper fruiting collects the spore that launches naturally of fructification single plant after fruiting, is diluted to 1 × 10 after spore is impregnated in sterile water4
A/mL obtains dispersion liquid, and dispersion liquid is coated on protoplast regeneration screening and culturing medium, is transferred to sprouting spore after culture and adds
Add in the PDA culture medium of uracil, edge mycelia is selected after being further cultured for, and by edge mycelia in the PDA of new addition uracil
It is further cultured on culture medium, repetition obtains monocaryon bacterial strain after being further cultured for 4-6 times；
(2) monocaryon bacterial strain obtained by step (1) is separately inoculated in minimal medium, addition uracil base basal culture medium
In screening and culturing medium, it can be grown in the minimal medium and screening and culturing medium of addition uracil, but in minimal medium
In non-growing monocaryon bacterial strain be the uracil auxotrophy monocaryon bacterial strain；
(3) uracil auxotrophy monocaryon bacterial strain obtained by step (2) is carried out on addition uracil base basal culture medium single
Single crosses obtains ganoderma lucidum uracil auxotrophy dicaryon bacterial strain after culture.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114395488A (en) * | 2022-01-26 | 2022-04-26 | 辽宁省农业科学院 | Culture medium for promoting growth of mushroom mononuclear hyphae and preparation method thereof |
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| US20160355831A1 (en) * | 2013-08-09 | 2016-12-08 | Jiangna University | Mortierella alpine uracil auxotroph with ura5 gene knocked out through homologous recombination, and construction method thereof |
| 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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| US20160355831A1 (en) * | 2013-08-09 | 2016-12-08 | Jiangna University | Mortierella alpine uracil auxotroph with ura5 gene knocked out through homologous recombination, and construction method thereof |
| US20160270359A1 (en) * | 2015-03-18 | 2016-09-22 | Yunnan Mingshida-Science-Tech Co., Ltd. | Ganoderma lucidum strain suitable for large-scale liquid fermentation culture, method of mutation breeding the same, and use of the strain |
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114395488A (en) * | 2022-01-26 | 2022-04-26 | 辽宁省农业科学院 | Culture medium for promoting growth of mushroom mononuclear hyphae and preparation method thereof |
| CN114395488B (en) * | 2022-01-26 | 2024-05-17 | 辽宁省农业科学院 | Culture medium for promoting growth of single-core mycelia of lentinus edodes and preparation method thereof |
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