CN106591213A - Method for rapidly separating and cultivating single spores from cordyceps militaris fruiting bodies - Google Patents
Method for rapidly separating and cultivating single spores from cordyceps militaris fruiting bodies Download PDFInfo
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- CN106591213A CN106591213A CN201710071453.7A CN201710071453A CN106591213A CN 106591213 A CN106591213 A CN 106591213A CN 201710071453 A CN201710071453 A CN 201710071453A CN 106591213 A CN106591213 A CN 106591213A
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- culture
- sporophore
- cordyceps militaris
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- monospore
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N3/00—Spore forming or isolating processes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
Abstract
The invention relates to a method for rapidly separating and cultivating single spores from cordyceps militaris fruiting bodies. The method includes the steps that parts in areas 1/2 to 1/3 away from the tops of the cordyceps militaris fruiting bodies are taken and cut, then a section obtained after cutting is taken and pasted on a dry sealing cover, inversely hung to the upper portion of sterilization water and cultivated in a sealed mode, and spore suspension liquid is obtained; the spore suspension liquid is absorbed and transferred to a plate medium, smeared to be even to be inverted and cultivated for 12 hours or above in the dark; single germinated spores are sought and selected, transferred to a sterilization cultivating tube and cultivated in a sealed mode at the temperature of 18 DEG C to 22 DEG C; single-spore cultures are obtained. The method is high in practicability and convenient to operate; the spores of the ordyceps militaris fruiting bodies can be simply and rapidly separated and cultivated, experiment operating is simplified, and the cultivating success rate is increased. The separated cultures can be used for single cell cross experiments, genetic diversity analysis, germplasm resource identification, genetic map establishing and functional gene researching; compared with a traditional method, the analyzing period can be greatly shortened, and the efficiency is improved.
Description
Technical field
The invention belongs to bio-science field, more particularly to a kind of sharp separation culture monospore from Cordyceps militaris (L.) Link.sporophore
Method.
Background technology
Cordyceps militaris (L.) Link. Cordyceps militaris have various medicinal efficacies as a kind of entomogenous fungi, in health care
Field has broad application prospects.Although at aspects such as artificial culture, strain improvement, molecular biology, active component
Numerous studies work has been done to Cordyceps militaris (L.) Link., but also there is obvious technical barrier in terms of artificial culture in spawn degeneration.
In Cordyceps militaris (L.) Link. culture studies, the selection-breeding of excellent species and the research of high yield condition of culture are still Cordyceps militaris (L.) Link. research
Emphasis.There are some researches show, many spore bacterial strains of Cordyceps militaris (L.) Link. fungus (including many ascospore bacterial strains and many conidium bacterial strains) or tissue divide
Under the same conditions carpogenic ability Jing often changes bacterial strain from source；Many spore bacterial strains or tissue isolation strains are than single
Easily there is spawn degeneration in spore bacterial strain.
During existing Cordyceps militaris spawn selection-breeding, after obtaining spore, the general method by plate streaking with being coated with, by spore
Son is diluted to suitable degree, after bacterium colony is formed, further selects.This selection is although simple to operate, but
Due to the restriction of technology itself, it is difficult to obtain monospore and grow the bacterium colony to be formed, the bacterium colony of acquisition needs further to be separated
With select.This method needs to carry out substantial amounts of repeated labor, low separation efficiency, and time-consuming for selection-breeding, it is difficult to meets production
Need.
Therefore by technological innovation, sharp separation culture obtains a large amount of single-ascospore strains, has to the cultivation of Cordyceps militaris (L.) Link. important
Theory and practice meaning.
The content of the invention
For the deficiencies in the prior art, the invention provides a kind of sharp separation culture monospore from Cordyceps militaris (L.) Link.sporophore
Method.
The present invention is achieved through the following technical solutions：
A kind of method of the sharp separation culture monospore from Cordyceps militaris (L.) Link.sporophore, including step is as follows：
(1) add water, sterilize to the culture tube with closure, obtain aquesterilisa, be dried closure, it is standby；
(2) take away from 1/2～1/3 intra-zone position of Cordyceps militaris (L.) Link.sporophore top, be divided into several sections, every section of Cordyceps sporophore
Internal diameter of the length less than closure；
(3) wherein one section Cordyceps sporophore is taken, is pasted onto and is dried on closure, hung by the feet above aquesterilisa, lid is closed, in 20
24～48h is cultivated under the conditions of～25 DEG C, is treated that the spore on Cordyceps sporophore is kicked down and formed in aquesterilisa spore suspension；
(4) PDA culture medium is poured in culture dish, plating medium is obtained, drawn spore suspension and be transferred to flat board training
On foster base, smear uniform, be placed at 20～25 DEG C of temperature dark inversion and cultivate more than 12 hours；
(5) take PDA culture medium in addition to be fitted in clean culture tube, it is standby after sterilizing；
(6) culture dish of step (4) is placed under body formula mirror, the spore of single sprouting, the spore that picking is sprouted are found in observation
Son, is transferred in the culture tube in step (5), and lid culture is closed under the conditions of 18～22 DEG C；Obtain single cell culture thing.
Currently preferred, step (1), the culture tube of step (5) are centrifuge tube with cover, and centrifuge tube specification is 1.5ml
Or 2ml, aquesterilisa is 1ml.
Currently preferred, sterilising temp is 120～125 DEG C in step (1), step (5), 10～30min of sterilization time,
Preferably, sterilising temp is 121 DEG C, sterilization time 20min.
Currently preferred, step (2) takes to be split away from 2/5～1/3 intra-zone position of Cordyceps militaris (L.) Link.sporophore top, most
For preferred, take and split away from 1/3 intra-zone position of Cordyceps militaris (L.) Link.sporophore top.
Currently preferred, the length of step (2) every section of Cordyceps sporophore is 5～7mm.
Currently preferred, the bonding method in step (3) is that Cordyceps sporophore is pasted onto into closure by vaseline
On, it is preferred that vaseline is applied in into the end of Cordycepses, is so hung upside down on closure.
Currently preferred, step (3) Cordyceps sporophore is pasted onto the inner central position for being dried closure.
Currently preferred, PDA culture medium in step (4) adds chloromycetin or oxytetracycline, and addition is 0.1～
0.3g/L, it is preferred that addition is 0.2g/L.Suppress the growth of antibacterial, reduce interference.
It is currently preferred, per 1cm in step (4)20.1～0.3 μ l spore suspension is smeared in culture medium.
Currently preferred, the culture dish of a diameter of 8cm adds 10 μ l spore suspension in step (4), is smeared with painting rod
Uniformly.
Currently preferred, the dark cultivation temperature of being inverted of step (4) is 22～24 DEG C, and incubation time is 18～24h.
It is currently preferred, it is the credibility of the single cell culture thing for further checking the method to be obtained, the method is also wrapped
Include, the single cell culture thing that step (6) is obtained is carried out extracting DNA after expanding propagation, MAT gene tests are then carried out, if only amplifying
A kind of fragment of copulation gene, the culture is single cell culture thing, is not otherwise single cell culture thing.
MAT gene tests concrete steps and nucleotide sequence are referring to " mating type gene is used as molecular markers for identification Cordyceps militaris (L.) Link.
The nuclear phase preliminary study of degenerative strain ", Wang Hong, Wei waits quietly, edible fungi journal 2010.17 (4)：1～4, text page 2.
Above-mentioned expanding propagation is carried out by the ordinary skill in the art.
The spore suspension transfer of the present invention, the transfer of the spore of single sprouting use the liquid-transfering gun and 10 μ l pipette tips for sterilizing
Carry out.
The invention has the advantages that：
1st, present invention centrifuge tube hangs Cordyceps sporophore acquisition spore suspension, the traditional triangular flask of replacement or culture by the feet
Ware, compact and flexible, convenient operation.
2nd, present invention liquid-transfering gun is inserted and takes 10 μ l pipette tips picking unit cells from plating medium, relative to traditional fire repeatedly
Inoculating needle sterilizing is burnt, pipette tips is changed quickly, operation is simple.
3rd, the present invention fills PDA culture medium with centrifuge tube, and the unit cell of picking is cultivated, and replaces traditional culture dish training
Support, simple to operate, pollution is few.
4th, detected with copulation gene pairss culture, to confirm that gained culture, as single cell culture thing, is follow-up study
The accuracy of experiment material used provides foundation.
5th, the present invention can the quick isolated single ascospore from Cordyceps militaris (L.) Link. fungus sporophore, and it cultivated obtain
Obtain single cell culture thing.
Description of the drawings
Fig. 1 is the MAT gene amplification results of the single cell culture thing after culture；
Fig. 2. it is that the comparison diagram that the unit cell obtained in culture medium is applied to spore suspension is collected using agar；
Wherein a figures are the spore collected with agar, and drop point compares concentration, it is difficult to picking unit cell；B figures are by spore suspension
The unit cell obtained in culture medium is applied to than relatively decentralized, it is easy to picking unit cell.
Fig. 3 a are the length schematic diagram of Cordyceps militaris (L.) Link. Stroma；
Fig. 3 b are to hang smearing culture base after acquisition spore suspension using the Cordyceps militaris (L.) Link.sporophore of different footpaths section to obtain
Unit cell culture comparison diagram；
Wherein, from left to right, respectively with 0～1cm of Cordyceps militaris (L.) Link., 1～2cm, 2～3cm footpath section hangs and obtains spore culture dish
The unit cell culture that smearing culture base is obtained after suspension.Unit cell culture in the section of 0～1cm footpaths is most, and 1～2cm and 2～
Unit cell culture is not obtained in the section of 3cm footpaths；
Specific embodiment
The embodiment of the invention discloses a kind of method that monospore sharp separation culture is carried out from Cordyceps militaris (L.) Link.sporophore, ability
Field technique personnel can use for reference present disclosure, be suitably modified technological parameter realization.Specifically, it is all similar to replace
Change and change apparent to those skilled in the art, they are considered as being included in the present invention.The side of the present invention
Method is described by preferred embodiment, and related personnel substantially can be without departing from present invention, spirit and scope
It is interior the method for the invention to be modified or suitably changes with combining to realize and apply the technology of the present invention.
For a further understanding of the present invention, the one kind provided the present invention with reference to embodiment is from Cordyceps militaris (L.) Link.sporophore
The method for carrying out monospore sharp separation culture is described in detail.
All operations of the present invention are both needed to operate in an aseptic environment.
Embodiment 1
(1) prepare 1.5ml centrifuge tubes, 1ml distilled water is added in pipe, it is standby after sterilizing 20min at 121 DEG C.Using front, high speed
Centrifugation, the water on centrifugation cap wall is dried.
(2) in 1/3rd regions of Cordyceps militaris (L.) Link.sporophore top will there is the position for producing spore function to cut into several segments,
Length per segment is 6mm, to be advisable less than centrifuge tube inner cap diameter.
(3) a bit of Cordyceps sporophore is gripped with tweezers, smears some vaseline, be pasted onto centrifugation lid inner side, hung by the feet
Above aquesterilisa, close to cover and 36h is cultivated under the conditions of 24 DEG C, wait the spore on Cordyceps sporophore to kick down formation spore in water and hang
Supernatant liquid.
(4) PDA culture medium (200g Rhizoma Solani tuber osis, 20g glucoses, 18g agar powders, 1000ml water), 121 DEG C of sterilizings are prepared
20min, pours into standby in the culture dish of a diameter of 8cm, and culture medium can also add chloromycetin or the oxytetracycline, addition to be
0.2g/L, primarily to suppressing the growth of antibacterial, reduces interference.
(5) draw 10 μ l spore suspension with the liquid-transfering gun and pipette tips after sterilizing to be transferred on plating medium, with painting rod
Smear uniform, 23 DEG C of dark inversions cultivate 20h.
(6) PDA culture medium of 100 μ l is filled with centrifuge tube, it is standby after sterilizing.
(7) culture dish in step 5 is placed under body formula mirror, the spore of the single sprouting of observation searching, 24 unit cells of picking,
The spore for taking the sprouting of 10 μ l pipette tips pickings is inserted with the liquid-transfering gun of sterilizing, picking thing is transferred in the centrifuge tube in step 6,20
Lid culture is closed under the conditions of DEG C.And finally obtain single cell culture thing.
(8) it is the credibility of single cell culture thing of further checking the method to be obtained.Culture is carried out to carry after expanding propagation
DNA is taken, is detected with copulation gene.In theory, each spore is monoploid, only carries a kind of mating type gene.If
A kind of fragment of copulation gene is only amplified, illustrates that the culture obtained by us is single cell culture thing, be not otherwise.After culture
Single cell culture thing MAT gene amplifications result as shown in Figure 1.
Embodiment 2
(1) prepare the centrifuge tube of 2ml, 1ml distilled water is added in pipe, it is standby after sterilizing 20min at 121 DEG C.Using front, high speed
Centrifugation, the water on centrifugation cap wall is dried.
(2) in 1/3rd regions of Cordyceps militaris (L.) Link.sporophore top will there is the position for producing spore function to cut into several segments,
Length per segment is 5mm, to be advisable less than centrifuge tube inner cap diameter.
(3) vaseline is smeared into centrifugation lid inside bottom after sterilization, with tweezers a bit of Cordyceps sporophore is gripped,
Centrifugation lid inner side is pasted onto, is hung by the feet above aquesterilisa, closed to cover and 48h is cultivated under the conditions of 20 DEG C, waited on Cordyceps sporophore
Spore maturation after kick down and formed in water spore suspension.
(4) PDA culture medium (200g Rhizoma Solani tuber osis, 20g glucoses, 18g agar powders, 1000ml water), 121 DEG C of sterilizings are prepared
20min, pours into standby in the culture dish of a diameter of 8cm, and culture medium can also add chloromycetin or the oxytetracycline, addition to be
0.2g/L, primarily to suppressing the growth of antibacterial, reduces interference.
(5) draw 10 μ l spore suspension with the liquid-transfering gun and pipette tips after sterilizing to be transferred on plating medium, with painting rod
After smearing uniformly, 20～25 DEG C of dark inversions cultivate 24h.
(6) PDA culture medium of 200 μ l is filled with centrifuge tube, it is standby after sterilizing.
(7) culture dish in step 5 is placed under body formula mirror, the spore of single sprouting is found in observation, with the liquid relief of sterilizing
Rifle inserts the spore for taking the sprouting of 10 μ l pipette tips pickings, and picking thing is transferred in the centrifuge tube in step 6, and lid is closed under the conditions of 18 DEG C
Culture.And finally obtain single cell culture thing.
(8) it is the credibility of single cell culture thing of further checking the method to be obtained.We carry out expanding propagation to culture
After extract DNA, detected with copulation gene.In theory, each spore is monoploid, only carries a kind of mating type gene.
If only amplifying a kind of fragment of copulation gene, illustrate that the culture obtained by us is single cell culture thing, be not otherwise.
Claims (10)
1. a kind of method of the sharp separation culture monospore from Cordyceps militaris (L.) Link.sporophore, including step is as follows：
(1) add water, sterilize to the culture tube with closure, obtain aquesterilisa, be dried closure, it is standby；
(2) take away from 1/2～1/3 intra-zone position of Cordyceps militaris (L.) Link.sporophore top, be divided into several sections, the length of every section of Cordyceps sporophore
Less than the internal diameter of closure；
(3) wherein one section Cordyceps sporophore is taken, is pasted onto and is dried on closure, hung by the feet above aquesterilisa, lid is closed, in 20～25
24～48h is cultivated under the conditions of DEG C, is treated that the spore on Cordyceps sporophore is kicked down and formed in aquesterilisa spore suspension；
(4) PDA culture medium is poured in culture dish, plating medium is obtained, drawn spore suspension and be transferred to plating medium
On, smear uniform, it is placed at 20～25 DEG C of temperature dark inversion and cultivates more than 12 hours；
(5) take PDA culture medium in addition to be fitted in clean culture tube, it is standby after sterilizing；
(6) culture dish of step (4) is placed under body formula mirror, the spore of single sprouting is found in observation, the spore that picking is sprouted turns
In moving to the culture tube in step (5), lid culture is closed under the conditions of 18～22 DEG C；Obtain single cell culture thing.
2. the method for the sharp separation culture monospore from Cordyceps militaris (L.) Link.sporophore according to claim 1, it is characterised in that step
Suddenly (1), the culture tube of step (5) are centrifuge tube with cover, and centrifuge tube specification is 1.5ml or 2ml, and aquesterilisa is 1ml；Step
(1), sterilising temp is 120～125 DEG C in step (5), 10～30min of sterilization time, it is preferred that sterilising temp is 121 DEG C, is gone out
Bacterium time 20min.
3. the method for the sharp separation culture monospore from Cordyceps militaris (L.) Link.sporophore according to claim 1, it is characterised in that step
Suddenly (2) take and are split away from 2/5～1/3 intra-zone position of Cordyceps militaris (L.) Link.sporophore top, highly preferred, take real away from Cordyceps militaris (L.) Link.
Split 1/3 intra-zone position of body top.
4. the method for the sharp separation culture monospore from Cordyceps militaris (L.) Link.sporophore according to claim 1, it is characterised in that step
Suddenly the length of (2) every section of Cordyceps sporophore is 5～7mm.
5. the method for the sharp separation culture monospore from Cordyceps militaris (L.) Link.sporophore according to claim 1, it is characterised in that step
Suddenly the bonding method in (3) is that Cordyceps sporophore is pasted onto on closure by vaseline, it is preferred that be applied in vaseline
The end of Cordycepses, is so hung upside down on closure；Preferably, step (3) Cordyceps sporophore is pasted onto and is dried the interior of closure
Side middle position.
6. the method for the sharp separation culture monospore from Cordyceps militaris (L.) Link.sporophore according to claim 1, it is characterised in that step
Suddenly the PDA culture medium in (4) adds chloromycetin or oxytetracycline, and addition is 0.1～0.3g/L, it is preferred that addition is 0.2g/
L。
7. the method for the sharp separation culture monospore from Cordyceps militaris (L.) Link.sporophore according to claim 1, it is characterised in that step
Suddenly per 1cm in (4)20.1～0.3 μ l spore suspension is smeared in culture medium.
8. the method for the sharp separation culture monospore from Cordyceps militaris (L.) Link.sporophore according to claim 1, it is characterised in that step
Suddenly the culture dish of a diameter of 8cm adds 10 μ l spore suspension in (4), is smeared uniformly with painting rod.
9. the method for the sharp separation culture monospore from Cordyceps militaris (L.) Link.sporophore according to claim 1, it is characterised in that step
Suddenly (4) dark cultivation temperature of being inverted is 22～24 DEG C, and incubation time is 18～24h.
10. the method for the sharp separation culture monospore from Cordyceps militaris (L.) Link.sporophore according to claim 1, it is characterised in that
For the credibility of the further single cell culture thing that inspection the method is obtained, the method also includes, the monospore obtained to step (6)
Culture carries out extracting DNA after expanding propagation, then carries out MAT gene tests, if only amplifying a kind of fragment of copulation gene, the training
Foster thing is single cell culture thing, is not otherwise single cell culture thing.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710071453.7A CN106591213A (en) | 2017-02-09 | 2017-02-09 | Method for rapidly separating and cultivating single spores from cordyceps militaris fruiting bodies |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710071453.7A CN106591213A (en) | 2017-02-09 | 2017-02-09 | Method for rapidly separating and cultivating single spores from cordyceps militaris fruiting bodies |
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ID=58586983
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| CN201710071453.7A Pending CN106591213A (en) | 2017-02-09 | 2017-02-09 | Method for rapidly separating and cultivating single spores from cordyceps militaris fruiting bodies |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107493979A (en) * | 2017-10-16 | 2017-12-22 | 常德炎帝生物科技有限公司 | Cordyceps militaris ascospore cross breeding method based on Protocols in Molecular Biology |
| CN109197379A (en) * | 2018-11-12 | 2019-01-15 | 贵州大学 | A kind of efficient monospore expanding propagation method of arbuscular mycorrhizal fungi |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107493979A (en) * | 2017-10-16 | 2017-12-22 | 常德炎帝生物科技有限公司 | Cordyceps militaris ascospore cross breeding method based on Protocols in Molecular Biology |
| CN109197379A (en) * | 2018-11-12 | 2019-01-15 | 贵州大学 | A kind of efficient monospore expanding propagation method of arbuscular mycorrhizal fungi |
| CN109197379B (en) * | 2018-11-12 | 2020-11-13 | 贵州大学 | Efficient single-spore propagation method for arbuscular mycorrhizal fungi |
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