CN107142256A - The method of the secondary mutation breeding of Cordyceps militaris ultraviolet - Google Patents
The method of the secondary mutation breeding of Cordyceps militaris ultraviolet Download PDFInfo
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- CN107142256A CN107142256A CN201710328540.6A CN201710328540A CN107142256A CN 107142256 A CN107142256 A CN 107142256A CN 201710328540 A CN201710328540 A CN 201710328540A CN 107142256 A CN107142256 A CN 107142256A
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Abstract
The invention discloses a kind of method of the secondary mutation breeding of Cordyceps militaris ultraviolet, belong to edible mushroom medicinal fungus genetic breeding field.One kind is specifically disclosed on the basis of first time mutagenic treatment, is handled by second of ultraviolet radiation mutagenesis, to improve the breeding method of Cordyceps militaris spawn amount of variability.The amount of variability of Cordyceps militaris spore can be dramatically increased in method disclosed by the invention, so as to be conducive to technical staff to screen strain excellent from the colony after mutagenesis, and then improve the medicinal/edibility and its economic value of Cordyceps militaris.
Description
Technical field
The invention belongs to edible mushroom medicinal fungus genetic breeding field, and in particular to a kind of side of the secondary mutation breeding of ultraviolet
Method, more particularly to a kind of method of the secondary mutation breeding of Cordyceps militaris ultraviolet for the amount of variability for improving strain.
Background technology
Cordyceps sinensis (Classification system Cordyceps sinensis (Berk.) Sacc) also known as cordyceps sinensis, are China's special products
A kind of rare tonic herb, it is equally celebrated for their achievements with ginseng, pilose antler, it is one of traditional three big tonics.Its nutritional ingredient is higher than ginseng, can enter
Medicine, also edible, is superior delicacies, with very high nutritive value.Cordyceps sinensis can strengthen the immunity of body, nourishing
Lung kidney, has obvious inhibitory action to lung cancer, liver cancer etc..Clinically to chronic cough of deficiency lung, asthma, hemoptysis of pulmonary tuberculosis, night sweat, kidney
Red blood cell decreased after virtual waist knee joint soreness, impotence and seminal emission, neurasthenia and chemotherapy, radiotherapy is all effective in cure.
Cordyceps militaris (Classification system Cordyceps militaris), also referred to as northern Chinese caterpillar Fungus, northern worm pupa grass, northern pupa worm
Grass etc., belongs to Clavicipitaceae Cordyceps, and xenogenesis is belonged to together with cordyceps sinensis, and medical value is similar to the cordyceps sinensis having won fame both at home and abroad.
The worldwide distribution of Cordyceps militaris, natural resources quantity seldom, Yunnan (Kunming, peacefulness, river and mountain), Jilin (peace is mainly originated in China
Figure, Yongji), Liaoning (Shenyang), the ground such as the Inner Mongol (Zhelimu League), be born in squama wing in pin, broad-leaf forest or mixed forest surface soil layer
On the pupal cell of mesh insect.
Requirement of the Cordyceps militaris to environment is relatively low, and liquid fermentation can form mycelium, and artificial extensive solid culture can be obtained
Stroma.The composition of Cordyceps militaris such as Cordyceps sinensis polysaccharide and cordycepic acid, the content of some compositions suitable with the content of natural cordyceps sinensis
Even more than cordyceps sinensis, the content of such as cordycepin is more than 35 times of natural cordyceps.The effective active composition of Cordyceps militaris
Cordyceps sinensis polysaccharide is considered as non-specific immunomodulator, can activate the immunocyte of human body.Compared with cordyceps sinensis, Cordyceps militaris
With following advantage：Cordyceps militaris as Cordyceps type sepecies, it is widely distributed, recognized and connect by countries in the world scholar
By；Cordyceps militaris has been bred as complete stroma under artificial condition；Cordyceps militaris contains cordycepin and Cordyceps sinensis polysaccharide, and its unique pharmacology is made
With the great attention for increasingly causing pharmacy circle.Because with above advantage, Cordyceps militaris has become medicinal cordyceps sinensis in Cordyceps
Outstanding person in bacterium.
Due to Cordyceps militaris unique medical value, nutritive value, scientific research value and potential huge economic value, manually
The technology for cultivating Cordyceps militaris receives extensive concern.Cordyceps militaris plantation is the working procedure of a series of complex operation, including bacterial strain
Selection, parent species seed selection and preservation, strain prepare, go out grass management and market sale etc., and each program is essential and extremely closes
It is important.Wherein breeding is the primary content in these programs again, without excellent strain, the preparation or optimization of other working procedures
Just without basis, the Cordyceps militaris underproduction or cultivation failure may finally be directly contributed.
But the artificial culture of existing Cordyceps militaris is paid close attention to improvement or optimization culture based formulas and condition of culture more or totally examined
Consider the culture medium prescription and condition of culture of each step, strain domestication is paid close attention to less.For example, CN1724641A discloses a kind of northern winter
The preparation method and applications of worm summer grass liquid culture nutritine growth medium.The invention is main by choosing different raw materials, including Hu trailing plants
The materials such as fore-telling, glucose, are then sufficiently mixed, and are fermented after adding a certain amount of streptomysin, and northern Chinese caterpillar Fungus is made
Liquid culture nutritine growth medium；Then trip temperature, the different controls of time are entered during northern Chinese caterpillar Fungus is cultivated, to reach preferably
Cultivate effect.CN101463325A discloses a kind of northern Chinese caterpillar Fungus industrial planting method, is prepared through strain, dispensing is filled
Bottle, sterilize, be inoculated with, cultivating, going out grass harvesting step, factory culture northern Chinese caterpillar Fungus, employing common artificial generation material conduct
Culture materials, reduce production cost.The northern Chinese caterpillar Fungus quality stability that the invention is obtained is good, and biological transformation ratio is high.
CN104054513A discloses a kind of northern Chinese caterpillar Fungus that can be used as medicine instead of cordyceps sinensis, can replace natural cordyceps medicine source
Spawn incubation method, the technical scheme comprises the following steps：(1) prepared by parent species；(2) parent species turn original seed and cultigen；(3) suspend
Fluid strain or the preparation of/liquid spawn；(4) artificial cultivation of Coragyceps militaris bacterium.The northern Chinese caterpillar Fungus bacterium of the Invention Announce
Plant cultural method, by increasing capacitance it is possible to increase the yield of cordyceps sinensis.
The content of the invention
To solve the problems, such as that Cordyceps militaris manually cultivates bacterial strain screening, the present invention provides a kind of secondary mutagenesis of Cordyceps militaris ultraviolet and educated
The method planted.The amount of variability of strain can be dramatically increased in the method that the present invention is provided, so as to be conducive to technical staff from mutagenesis
Strain excellent is screened in colony afterwards.
The present invention solves the technical scheme that above-mentioned technical problem is used：
The present invention provides a kind of method of the secondary mutation breeding of Cordyceps militaris ultraviolet, comprises the following steps：
1) Cordyceps militaris spore suspension is prepared；
2) first time mutagenic treatment；
3) second of ultraviolet radiation mutagenesis processing；
4) bacterium screening is chosen.
Preferably, the irradiation time of second of ultraviolet radiation mutagenesis processing is 1~2.5 minute.
In one embodiment of the invention, second of ultraviolet radiation mutagenesis processing, irradiation time is 2 points
Clock.
Preferably, the first time mutagenic treatment is selected from ultraviolet mutagenesis processing, X-ray line mutagenic treatment, gamma ray and sulfuric acid
In diethylester mutagenic treatment, 5-bromouracil mutagenic treatment, mustargen mutagenic treatment, N " wide methyl N " NTG mutant treatment
It is a kind of.
Preferably, the first time mutagenic treatment is ultraviolet radiation mutagenesis processing, and irradiation time is 0.8~1.5 minute.
In one embodiment of the invention, the first time mutagenic treatment is ultraviolet radiation mutagenesis processing, irradiation
Time is 1 minute.
, should be first by ultraviolet before second of ultraviolet radiation mutagenesis processing in the embodiment of the present invention
Light source uviol lamp is opened 20~60 minutes.
In one embodiment of the invention, should be first by ultraviolet before second of ultraviolet radiation mutagenesis processing
Light source uviol lamp is opened 20 minutes.
, should be first by ultraviolet before the processing of first time ultraviolet radiation mutagenesis in the embodiment of the present invention
Light source uviol lamp is opened 20~60 minutes.
In one embodiment of the invention, should be first by ultraviolet before the processing of first time ultraviolet radiation mutagenesis
Light source uviol lamp is opened 20 minutes.
Prepare Cordyceps militaris spore suspension and select sterile saline or phosphate buffer.
Ultraviolet irradiation mutagenesis mutagenic treatment should be carried out at dark, and preferably dark desinfection chamber or superclean bench, case are built-in
15-20 watts of uviol lamp 1, hangs on 30 centimetres of eminences.It should first be turned on light 20~60 minutes during mutagenic treatment, make uviol lamp wavelength steady
It is fixed, then Cordyceps militaris spore suspension is poured into sterile petri dish, ware lid is opened, irradiated several minutes.
When the work spore count contained by every milliliter of Cordyceps militaris spore suspension is about at 106~113, such as people in the art
Member is known, and to obtain single bacterium colony, spore suspension extension rate scope can be ten thousand times of 1000-10.
The present invention achieves following technique effect：
The amount of variability of Cordyceps militaris spore can be dramatically increased in the method that the present invention is provided, thus be conducive to technical staff from
Strain excellent is screened in colony after mutagenesis, and then filters out cordycepin, adenosine, polysaccharide or the high preponderant strainses of Cordyceps militaris yield
Strain, to improve the medicinal/edibility and economic value of Cordyceps militaris.
Embodiment
In order to promote the understanding of the present invention, below with reference to some embodiments, and language-specific will be used to retouch
State the present invention.It is to be understood, however, that these embodiments are not intended to limit the scope of the invention.Described reality
Any change and the further modification in mode are applied, and any of the present invention further applies, and is people in the art
What member generally will recognize that.
The method of the secondary mutation breeding of Cordyceps militaris ultraviolet of embodiment 1
Including four steps：
1) Cordyceps militaris spore suspension is prepared
Starting strain：Cordyceps militaris kind ycc-01.
Fresh sterile spore is moved into 5mL sterile salines, after shaking up spore suspension.Spore suspension
Final concentration of every milliliter contain 108~115, spore.
2) first time mutagenic treatment
Carried out in dark superclean bench, the built-in 15-20 watts of uviol lamp of case 1 hangs on 30 centimetres of eminences, mutagenic treatment
Shi Yingxian is turned on light 20 minutes, makes Wavelength stabilized, and then Cordyceps militaris spore suspension is poured into a diameter of 6 centimetres of sterile petri dish
In, ware lid is opened, is irradiated 1 minute.
After testing, the work spore count contained by every milliliter of Cordyceps militaris spore suspension after irradiation is about at 106~113.
Therefore to obtain single bacterium colony, first with sterilized water by the spore suspension after irradiation dilute 100,000 times, 1,000,000 times,
10000000 times, then take respectively and release liquid 0.3mL and be coated on the culture dish equipped with agar medium, 4~6 are cultivated at 22 DEG C
It on each flat board until obtaining single bacterium colony.
3) second of ultraviolet radiation mutagenesis processing
Carried out in dark superclean bench, the built-in 15-20 watts of uviol lamp of case 1 hangs on 30 centimetres of eminences, mutagenic treatment
Shi Yingxian turns on light 20 minutes, makes Wavelength stabilized, then to obtain first time mutagenic treatment Cordyceps militaris single bacterium colony flat board opening ware
Lid, irradiates 2 minutes.Select pure, healthy and strong single bacterium colony to move into slant tube, bacterium screening is chosen for next step.
4) bacterium screening is chosen
Pure, physically well developed single bacterium colony is selected to move into slant tube, a bacterium colony only connects an inclined-plane, waits to grow
Afterwards, then it is respectively connected in 2~3 bottles of 300mL liquid nutrient solutions, then classification carries out experiment in cultivation, according to this area conventional method
The content of Cordyceps militaris cordycepin is detected to evaluate the amount of variability of Cordyceps militaris.
The detection method of content of the cordycepin of embodiment 2
The method with reference to disclosed in CN102060898B embodiments 5.
The fruiting bodies of cordyceps militaris that 150g drying is manually cultivated is weighed, after firmly crushing, planetary ball mill is added to
During 250mL ball millings are filled, adjustment rotating speed is 500r/min, and alumina balls number is 20 (10 big balls and 10 beads), using reciprocal cross
For grinding, alt time 5min, processing total time is 30min, obtains fruiting bodies of cordyceps militaris powder.150g powder is added to
It is 2.5 with 0.1mol/LHCl adjustment pH, temperature is 45 DEG C, is 30KHZ ultrasonications with frequency in 1500mL distilled water
1h, centrifugation (3000r/min, 10min) after having handled, collects supernatant, and 45g charcoal absorption pigments are added in supernatant,
Refilter, 45g charcoal absorption pigments are added in filtrate, are repeated, untill supernatant does not have color, by this
Clear liquid is first extracted with 300mL ethyl acetate in cavitation suspension extraction device, discards ethyl acetate layer, remaining liq is used again
300mL chloroform is extracted in cavitation suspension extraction device, discards D101 chromatographic columns on chloroform layer, remaining liq, first with distillation
Water elution is colourless to efflux, then the ethanol water for being 50% with the pH volume fractions for being 5.5 is eluted with 1mL/min speed,
Eluent is collected, vacuum drying produces cordycepin crystal, and weighing obtains the fruiting bodies of cordyceps militaris that every 150g drying is manually cultivated
Cordycepin content.
The strain amount of variability of embodiment 3 is detected
The amount of variability of Cordyceps militaris after mutagenic treatment is evaluated by evaluation index of the content of cordycepin.
Detected sample：
Sample 1：Unsuccessful 100 pure, the physically well developed single bacterium colonies of single bacterium obtained after embodiment 1 first time mutagenic treatment
Move into slant tube, a bacterium colony only connects an inclined-plane, after length is good, then be respectively connected to 2~3 bottles of 300mL liquid nutrient solutions
In, classification is cultivated.The cordycepin content for the fructification that each single bacterium colony culture is obtained is determined respectively.
Sample 2：The single bacterium obtained after second of the ultraviolet radiation mutagenesis processing of embodiment 1 is unsuccessful pure, physically well developed
100 single bacterium colonies are moved into slant tube, and a bacterium colony only connects an inclined-plane, after length is good, then are respectively connected to 2~3 bottles of 300mL
In liquid nutrient solution, classification is cultivated.The cordycepin content for the fructification that each single bacterium colony culture is obtained is determined respectively.
Control sample 1：Do not do mutagenic treatment the sterile spores of Cordyceps militaris kind ycc-01 move into 5mL sterile salines or
In phosphate buffer, shake up after spore suspension.Final concentration of every milliliter of spore suspension contains 108~115, spore.
Take and release liquid 0.3mL and be coated on the culture dish equipped with agar medium, 4~6 days are cultivated at 22 DEG C until on each flat board
Obtain single bacterium colony.100 pure, physically well developed single bacterium colonies are selected to move into slant tube, a bacterium colony only connects an inclined-plane, treats
After length is good, then it is respectively connected in 2~3 bottles of 300mL liquid nutrient solutions, classification is cultivated.Each single bacterium colony culture is determined respectively
The cordycepin content of obtained fructification.
Control sample 2：Method according to embodiment 1 first time mutagenic treatment handles Cordyceps militaris spore, and processing time is 3 points
Clock.100 pure, physically well developed single bacterium colonies are selected to move into slant tube, a bacterium colony only connects an inclined-plane, after length is good,
It is respectively connected to again in 2~3 bottles of 300mL liquid nutrient solutions, classification is cultivated.The son that each single bacterium colony culture is obtained is determined respectively
The cordycepin content of entity.
Cordyceps militaris plantation method：
1) potato 200g, glucose 20g, peptone 3g, yeast extract 2g, potassium dihydrogen phosphate 2g, magnesium sulfate 1g, water are used
1000mL formulas routinely breed production and use liquid spawn.
2) 750mL glass preserving jar is used for container, every bottle of 45 grams of wheats, add water 70mL, 1.05 kg/cms
Pressure sterilization 75 minutes, cooling inoculation.
3) bacterium bottle is first put in 20 DEG C of lucifuge cultures 6 days, is then transferred to brightness and intersects culture, daily daytime is with 1000 luxs
Intensity illumination, evening light culture, ambient humidity 85%, daily ventilation 3 times.
4) whole 55 days production cycles, fructification length is controlled at 8~12 centimetres, other routinely to operate, every batch of sampling
Sampling determines cordycepin content afterwards.
Result of the test：
Testing result is shown, compared with control sample 1, and the son for having more than 20% single bacterium colony culture to obtain in sample 1 is real
The cordycepin content changing value of body is more than 50%；There is the worm for the fructification that more than 80% single bacterium colony culture obtains in sample 2
Careless cellulose content changing value is more than 50%；There is the cordyceps sinensis for the fructification that more than 40% single bacterium colony culture obtains in control sample 2
Cellulose content changing value is more than 50%.
Above test result indicates that, the amount of variability of strain can be dramatically increased in the processing of second ultraviolet radiation mutagenesis,
And the Mutagenic Effect (amount of variability) is more than the amount of variability of simple extension ultraviolet treatment with irradiation time.The pupa worm that the present invention is announced
The method of the careless secondary mutation breeding of ultraviolet is beneficial to technical staff and strain excellent is screened from the colony after mutagenesis.
Provided herein is any and all embodiment or the use of exemplary language (for example, " such as, such as ") be only intended to
The present invention is better described, without being construed as limiting to the scope of the present invention, Unless Otherwise Requested.Language in specification should not be by
It is construed to indicate that any element being not claimed is necessary for implementing the present invention.
All publications and patents cited in this specification application is incorporated herein by reference, and individually goes out as each
Version thing or patent application are specifically and individually indicated and are incorporated by reference into.In addition, any theory as described herein, mechanism,
Prove or find to be further intended to strengthen the understanding of the present invention, and be not intended to limit the invention in any way so
Theory, mechanism, proof or find.Whilst there has been described in the foregoing the present invention is illustrated in detail in and described in, but originally
Invention should be considered as illustrative and not restrictive.
Claims (10)
1. the method for the secondary mutation breeding of Cordyceps militaris ultraviolet, it is characterised in that：
1) Cordyceps militaris spore suspension is prepared；
2) first time mutagenic treatment；
3) second of ultraviolet radiation mutagenesis processing；
4) bacterium screening is chosen.
2. the method for the secondary mutation breeding of Cordyceps militaris ultraviolet according to claim 1, it is characterised in that described second
Ultraviolet radiation mutagenesis processing, irradiation time is 1~2.5 minute.
3. the method for the secondary mutation breeding of Cordyceps militaris ultraviolet according to claim 2, it is characterised in that described second
Ultraviolet radiation mutagenesis processing, irradiation time is 2 minutes.
4. the method for the secondary mutation breeding of Cordyceps militaris ultraviolet according to claim 1, it is characterised in that the first time
Mutagenic treatment is selected from ultraviolet mutagenesis processing, X-ray line mutagenic treatment, gamma ray and dithyl sulfate mutagenic treatment, 5-bromouracil
One kind in mutagenic treatment, mustargen mutagenic treatment, N " wide methyl N " NTG mutant treatment.
5. the method for the secondary mutation breeding of Cordyceps militaris ultraviolet according to claim 4, it is characterised in that the first time
Mutagenic treatment is ultraviolet radiation mutagenesis processing, and irradiation time is 0.8~1.5 minute.
6. according to the method for the secondary mutation breeding of Cordyceps militaris ultraviolet according to claim 5, it is characterised in that described
Mutagenic treatment is ultraviolet radiation mutagenesis processing, and irradiation time is 1 minute.
7. the method for the secondary mutation breeding of Cordyceps militaris ultraviolet according to claim 1~6 any one, its feature exists
In before the processing of second ultraviolet radiation mutagenesis, first ultraviolet light source uviol lamp should be opened 20~60 minutes.
8. the method for the secondary mutation breeding of Cordyceps militaris ultraviolet according to claim 7, it is characterised in that purple at second
Before outside line irradiation mutagenic treatment, first ultraviolet light source uviol lamp should be opened 20 minutes.
9. the method for the secondary mutation breeding of Cordyceps militaris ultraviolet according to claim 4~6 any one, its feature exists
In, first time ultraviolet radiation mutagenesis processing before, should first by ultraviolet light source uviol lamp open 20~60 minutes.
10. the method for the secondary mutation breeding of Cordyceps militaris ultraviolet according to claim 9, it is characterised in that for the first time
Before ultraviolet radiation mutagenesis processing, first ultraviolet light source uviol lamp should be opened 20 minutes.
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| CN113179852A (en) * | 2021-03-31 | 2021-07-30 | 山东中泰药业有限公司 | A method for cultivating Ganoderma with Chinese medicinal residue |
| CN113557948A (en) * | 2021-08-13 | 2021-10-29 | 泰山学院 | Method for cultivating novel variety of wisteria |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107739720A (en) * | 2017-11-13 | 2018-02-27 | 大连春天生物菌业孵化基地有限公司 | A kind of poor general culture medium cultivates the application of new varieties in ultraviolet mutagenesis Cordyceps militaris |
| CN113179852A (en) * | 2021-03-31 | 2021-07-30 | 山东中泰药业有限公司 | A method for cultivating Ganoderma with Chinese medicinal residue |
| CN113557948A (en) * | 2021-08-13 | 2021-10-29 | 泰山学院 | Method for cultivating novel variety of wisteria |
| CN113557948B (en) * | 2021-08-13 | 2023-09-08 | 泰山学院 | Cultivation method of novel variety of wisteria variation |
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