CN107653194A - A kind of culture medium and cultural method for being used to cultivate preeminent hickory chick - Google Patents
A kind of culture medium and cultural method for being used to cultivate preeminent hickory chick Download PDFInfo
- Publication number
- CN107653194A CN107653194A CN201711126323.5A CN201711126323A CN107653194A CN 107653194 A CN107653194 A CN 107653194A CN 201711126323 A CN201711126323 A CN 201711126323A CN 107653194 A CN107653194 A CN 107653194A
- Authority
- CN
- China
- Prior art keywords
- preeminent
- hickory chick
- morchella
- culture medium
- extract
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- 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
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
Abstract
The invention discloses a kind of culture medium and cultural method for being used to cultivate preeminent hickory chick, it is related to edible mushroom field.Contain willow leaf extract in culture medium disclosed by the invention for cultivating preeminent hickory chick, it is applicable to what the present invention was separated and identified, and it is CCTCC NO to cultivate deposit number:M 2016032 new preeminent hickory chick, using the medium culture provided by the invention preeminent hickory chick, there is the features such as mycelial growth rate is fast, and biomass height, anti-miscellaneous bacteria ability is strong, and pollution rate is few, and the sclerotium generation time is short.
Description
Technical field
The present invention relates to edible mushroom field, in particular to a kind of culture medium for being used to cultivate preeminent hickory chick and
Cultural method.
Background technology
Hickory chick (Morchella) is a kind of Rare edible fungus kind, because of its cap surface irregularity, shape such as sheep tripe
Gain the name.Hickory chick (Morchella) is Ascomycetes (Ascomycetes), Pezizale (Pezizales), Morchellaceae
(Morohellaceae), the dual-purpose fungi of rare food, the medicine of morchella (Morchella), also known as morel, sheep tripe dish,
Sheep mushroom, sheep tripe mushroom.Hickory chick species known to China has had much at present, it is relatively common have hickory chick, it is small push up hickory chick,
Morchellaconica, Morchella crassipes, small hickory chick etc..
Hickory chick is that foremost delicious food bacterium, its cap part contain isoleucine, leucine, rely ammonia in sac fungus
7 kinds of acid, methionine, phenylalanine, threonine and valine amino acid needed by human.The nutrition of hickory chick is quite abundant, according to
Measure, hickory chick also contain several amino acids, particularly paddy containing crude protein 20%, crude fat 26%, carbohydrate 38.1%
Histidine content is up to 1.76%.Therefore, it is believed that being " very good protein source ", and there is the laudatory title of " meat or fish in element ".
In addition, hickory chick at least contains 8 kinds of vitamins according to surveying and determination：Vitamin B1, vitamin B2, vitamin B12, nicotinic acid,
Pantothenic acid, pyrrole sound of vomiting alcohol, biotin, folic acid etc..The nutrition of hickory chick, can be suitable with cow's milk, meat and fish meal.Therefore, in the world
Often it is referred to as one of " healthy food ".
Wild toadstool is distributed in China Shaanxi, Gansu, Qinghai, Tibet, Xinjiang, Sichuan, Shanxi, Jilin, Jiangsu, cloud
The areas such as south, Henan, Hebei, Beijing, Hunan, Guizhou.Being grown under natural environment, monomer is up to more than 200 grams, at present, China
The hickory chick having found has kind more than 20.
It is therefore desirable to provide the application of more hickory chick new varieties and hickory chick new varieties, authentication method and
Cultural method etc..
In consideration of it, special propose the present invention.
The content of the invention
It is an object of the invention to provide a kind of new hickory chick bacterial strain, has and improves immunity, suppresses tumour cell increasing
The effect of growing etc..
Another object of the present invention is to provide a kind of extract, it is extracted from from above-mentioned preeminent hickory chick.
Another object of the present invention is to provide a kind of medicine or health products for improving immunity.
Another object of the present invention is to provide the medicine of prevention or treatment tumour.
Another object of the present invention is to provide a kind of authentication method for identifying above-mentioned preeminent hickory chick.
Another object of the present invention is to provide the application of above-mentioned preeminent hickory chick or its extract.
Another object of the present invention is to provide a kind of culture medium for being used to cultivate above-mentioned preeminent hickory chick.
Another object of the present invention is to provide a kind of method for cultivating above-mentioned preeminent hickory chick.
What the present invention was realized in：
On the one hand, the present invention provides a kind of preeminent hickory chick, and its deposit number is CCTCC NO:M2016032.
The present inventor is separated from a kind of fructification of hickory chick in Yunnan, obtains mycelial samples after purification,
By observing the form of mycelial samples, it is one of kind of Morchellaceae to judge it, passes through further ITS sequence
Analysis, the rDNA-ITS sector sizes that the DNA extracted with mycelial samples enters performing PCR amplification gained are about 750bp, by NCBI's
BLAST is compared, and the sequence similarity of the mycelial samples and Morchella eximia voucher strains is 99%, is thus inferred
The hickory chick bacterial strain is a kind of Morchella eximia voucher new hickory chick bacterial strain, is preeminent hickory chick
(Morchella eximia voucher)。
The bacterial strain was preserved in China typical culture collection center (CCTCC), address on January 13rd, 2016：It is Chinese military
Chinese Wuhan University, Classification And Nomenclature are：Morchella eximia voucher SAAS-1, deposit number are：CCTCC NO:M
2016032。
On the other hand, the present invention provides a kind of extract, and it extracts from above-mentioned preeminent hickory chick and obtained.
Further, in some embodiments of the invention, the extract is water extract or alcohol extract.
Further, in some embodiments of the invention, the extract is polyoses extract.
Further, in some embodiments of the invention, the water extract or alcohol extract can be prepared via a method which：
Take the preeminent new fresh sporophore of hickory chick as described above, after cleaning, freeze；
By lyophilized fructification, ethanol mixing is added, ultrasonication, obtains crude extract；
Crude extract is centrifuged, supernatant (precipitation is standby) is taken, dries, obtain alcohol extract,
Above-mentioned precipitation is taken, is dried, water is added, is heated, is filtered, collects filtrate, concentration filtrate obtains water extract.
Experimental verification in the embodiment of the present invention, extract provided by the invention, which has, to be promoted T cell propagation and suppresses swollen
The effect of tumor cell proliferation.
On the other hand, the present invention provides a kind of medicine or health products for improving immunity, and it includes：Above-mentioned preeminent sheep tripe
Bacterium, or above-mentioned extract.
Experimental verification in the embodiment of the present invention, extract provided by the invention have the function that to promote T cell propagation.Cause
This, the extract or the preeminent hickory chick can be used for preparing the medicine or health products for improving immunity.Correspondingly, containing the extraction
The medicine or health products of thing or the preeminent hickory chick, which also have, promotes T cell propagation, improves the action effect of immunity.
On the other hand, the present invention provides a kind of medicine for preventing or treating tumour, and it includes：Preeminent sheep tripe as described above
Bacterium, or extract as described above.
Experimental verification in the embodiment of the present invention, extract provided by the invention have the work for suppressing tumor cell proliferation
With.Therefore, the extract or the preeminent hickory chick can be used for the medicine for preparing prevention or treatment tumour.Correspondingly, carried containing this
The medicine or health products for taking thing or the preeminent hickory chick also have suppression tumor cell proliferation, reach the purpose of prevention or treatment.
On the other hand, the present invention provides a kind of authentication method of preeminent hickory chick as described above, and it includes：
The ITS region sequences of bacteria to be tested are expanded, obtain sequence to be checked；
The sequence to be checked is compared with reference sequences, if the sequence to be checked contains the reference sequences,
It is the preeminent hickory chick to indicate the bacteria to be tested；
Wherein, the reference sequences are as shown in SEQ ID NO.1.
Further, in some embodiments of invention, the ITS areas of the bacteria to be tested are expanded with fungi universal primer
Sequence.
Further, in some embodiments of invention, treated described in fungi universal primer ITS1 and the ITS4 amplification
Survey the ITS region sequences of strain；ITS1 base sequence is as shown in SEQ ID NO.2, ITS4 base sequence such as SEQ ID NO.3
It is shown.
On taxology, from traditional form on to divide hickory chick is divided into 28 kinds, but in molecular biology
Angle be but only divided into three or four kinds, illustrate to lack and the reference frame of strain identified by hypha form.Because these are asked
Topic causes the chaotic phenomenon of strain occurs in the production of hickory chick, therefore establishes a set of science, effective strain idenfication system
It is a major issue of urgent need to resolve.So the mode being combined using traditional form and molecular biology, while from shape
State feature and molecular level identification hickory chick strain are a relatively good approach.
ITS(Internal Transcribed Spacer)：The Internal Transcribed Spacer, it is eucaryote ribosomes RNA
(rRNA) part for gene nontranscribed domain.Being generally used for the ITS sequence of Fungal identification includes ITS1,5.8S and ITS2.Ribose
Vivo transcription spacer region includes 2 different non-coding regions, i.e. ITS1 and ITS4.Fungi ITS zone lengths typically 650~
750bp (base-pair).ITS sequence identification refers to carry out DNA sequencing to ITS sequence, by will be sequenced obtained ITS sequence with
Know that the ITS sequence of fungi is compared, so as to obtain a kind of method of unknown fungi kind information.
Traditional taxology authentication method is to distinguish different strain according to sporophore shape, but because present environment becomes
Change greatly, seriously polluted, sporophore shape can produce change due to being influenceed by growing environment and different developmental phases, so it is difficult to
Accurately make a distinction.
Authentication method application rDNA provided by the invention ITS sequence analysis method bacteria to be tested is identified, with
SEQ ID NO.1 are CCTCC NO from deposit number as reference sequences, the reference sequences:M2016032 preeminent hickory chick
RDNA ITS sequence, therefore, there is the characteristics of qualification result is accurate, reliable using the authentication method.
On the other hand, it is CCTCC NO that the present invention, which provides deposit number as described above,:M 2016032 preeminent hickory chick
Or the preeminent Morchella esculenta extract is preparing the application in improving immunity medicine or health products.
On the other hand, it is CCTCC NO that the present invention, which provides deposit number as described above,:M 2016032 preeminent hickory chick
Or application of the preeminent Morchella esculenta extract in prevention or tumor is prepared.
Through the experimental verification in the embodiment of the present invention, as a result show, the water extract that is extracted from above-mentioned preeminent hickory chick or
Alcohol extract has the function that to promote T cell propagation and suppresses tumor cell proliferation.Illustrate, preeminent hickory chick as described above or super
Group's Morchella esculenta extract, which can be used for preparing, improves immunity medicine or health products, and the neck such as prevention or tumor
Domain.
On the other hand, a kind of culture medium for being used to cultivate preeminent hickory chick, it is suitable to cultivate deposit number as described above
For CCTCC NO:M 2016032 preeminent hickory chick, contain willow leaf extract in the culture medium.
Further, in some embodiments of invention, the willow leaf extract is Poplar leaves water extract.
Further, in some embodiments of invention, the Poplar leaves water extract is prepared by the following method to obtain：
Poplar leaves are boiled with decocting, filters, takes filtrate to produce the Poplar leaves water extract.
Further, in some embodiments of invention, the culture medium also contains：Glucose and KH2PO4。
Further, in some embodiments of invention, based on every L, the culture medium contains：18-22g glucose and
0.8-1.1g KH2PO4。
Preeminent Morchella esculenta (L.) Pers sporophore provided by the invention comes from Qujing of Yunnan, inventor according to the living environment of fructification and
The seeds of surrounding, continuous improvement has been carried out to its mycelial culture medium prescription and creative thinking, its result are shown, phase
To ordinary culture medium, culture medium provided by the invention can make the preeminent hickory chick well by adding willow leaf extract
Growth, show the characteristics of mycelia is sturdy, the speed of growth is fast, sclerotium quantity is more.
Another further aspect, it is CCTCC NO that the present invention, which provides a kind of culture deposit number as described above,:M2016032 is preeminent
The method of hickory chick, this method use medium culture as described above.
The invention has the advantages that：
Preeminent hickory chick provided by the invention, deposit number are CCTCC NO:M 2016032.It is identified, the preeminent sheep tripe
Bacterium is a kind of new preeminent hickory chick bacterial strain, and its extract promotes T cell propagation and suppresses the effect of tumor cell proliferation, and this is super
Group hickory chick or its extract can be used for improve immunity and prevent or treatment tumour, correspondingly, the preeminent hickory chick or its
Extract can be used for preparing the medicine or health products for improving immunity, or prepare in the medicine of prevention or treatment tumour, tool
Have broad application prospects；
In addition, the method for authentication method application rDNA provided by the invention ITS sequence analysis is reflected to bacteria to be tested
Fixed, using SEQ ID NO.1 as reference sequences, the reference sequences are CCTCC NO from deposit number:M's 2016032 is preeminent
The rDNA of hickory chick ITS sequence, therefore, using the authentication method, can accurately identify whether bacteria to be tested is preservation
Numbering is CCTCC NO:M 2016032 preeminent hickory chick, it has the characteristics of qualification result is accurate, reliable.
Further, the culture medium and corresponding cultural method of preeminent hickory chick provided by the invention, it is according to deposit number
For CCTCC NO:The initial growth environmental quality of M 2016032 preeminent hickory chick, is carried by adding Poplar leaves in the medium
Thing is taken, the preeminent hickory chick can be made to grow well, shows the characteristics of mycelia is long, sclerotium quantity is more.
Brief description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below by embodiment it is required use it is attached
Figure is briefly described, it will be appreciated that the following drawings illustrate only certain embodiments of the present invention, therefore be not construed as pair
The restriction of scope, for those of ordinary skill in the art, on the premise of not paying creative work, can also be according to this
A little accompanying drawings obtain other related accompanying drawings.
Fig. 1 is the DNA electrophoresis detection figures of the preeminent hickory chick extraction in the embodiment of the present invention (in figure：M：Marker；1、2、
3rd, 4,5 be mycelia DNA of the same race)；
Fig. 2 is PCR electrophoresis of the DNA of the preeminent hickory chick extraction in the embodiment of the present invention under 52-62 DEG C of thermograde
Figure is (in figure：M：Marker；13rd, 14 be 58 DEG C；23rd, 24 be blank control group)；
Fig. 3 is PCR electrophoretograms of the DNA of the preeminent hickory chick extraction in the embodiment of the present invention at a temperature of 58 DEG C (in figure：
M：Marker；16 be blank control group)；
Fig. 4 is the glue reclaim electrophoretogram in the embodiment of the present invention (in figure：M：Marker)；
Fig. 5 is the extraction electrophoretogram of the bacterium solution plasmid in the embodiment of the present invention (in figure：M：Marker)；
Fig. 6 is the restriction enzyme digestion and electrophoresis figure (M in the embodiment of the present invention：Marker；1st, 2,3,4, No. 6 are successfully transferred to carrier)；
Fig. 7 is the Blast comparison results in the embodiment of the present invention；
Fig. 8 is the chadogram constructed with different hickory chick ITS sequences in the embodiment of the present invention；
Fig. 9 is that the preeminent hickory chick alcohol extracting thing of various concentrations of the flow cytomery in the embodiment of the present invention is thin to T
The influence result of born of the same parents' propagation；
Figure 10 be the embodiment of the present invention in flow cytomery different volumes than preeminent hickory chick water extract to T
The influence result of cell propagation；
Figure 11 is the preeminent hickory chick alcohol extracting thing of various concentrations in the embodiment of the present invention to three kinds of tumor cell proliferations
Influence result；
Figure 12 is for the different volumes in the embodiment of the present invention than preeminent hickory chick water extract to three kinds of tumor cell proliferations
Influence result；
Figure 13 is the chadogram constructed with different preeminent hickory chick ITS sequences in the embodiment of the present invention；
Figure 14 is Morchella eximia the voucher SAAS-1 and Morchella in the embodiment of the present invention
eximia voucher LIP:Ph253 ITS sequence contrast partial results.
Embodiment
, below will be in the embodiment of the present invention to make the purpose, technical scheme and advantage of the embodiment of the present invention clearer
Technical scheme be clearly and completely described.Unreceipted actual conditions person, builds according to normal condition or manufacturer in embodiment
The condition of view is carried out.Agents useful for same or the unreceipted production firm person of instrument, it is the conventional production that can be obtained by commercially available purchase
Product.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
The separation and identification of preeminent hickory chick
The separation of 1 preeminent hickory chick
Original hickory chick collection is separated, obtains the mycelial samples of experiment after purification from Qujing of Yunnan.
The identification of 2 preeminent hickory chicks
2.1 cultural hypha
Take a small amount of strains tested with culture medium to access PDA culture medium, constant temperature and humidity incubator is put into after sticking sealed membrane
In cultivated.Condition of culture is constant to be：25 DEG C of temperature, relative humidity 40%, and no light.
The mycelia of access grows aerial hyphae after 1 day or so, and the speed of growth is accelerated, and mycelia typically covers with flat board at 3 days,
Mycelia color is white, mycelia browning in 5 days or so, is changed into brown, granular brown material is grown among culture medium.Due to bacterium
Silk extraction DNA dosage is bigger, and general scraping is inoculated with the white hypha of 4 days or so as the material subsequently tested.
2.2CTAB methods extract DNA
(1) after the completion of cultural hypha, picking part mycelia is in 2ml centrifuge tube.
(2) take the μ l of CTAB 800 of 65 DEG C of preheating, add in centrifuge tube, 65 DEG C of water-bath 1h, or shake up mixed liquor.
(3) 12000rpm, 4 DEG C of centrifugation 20min, takes supernatant to be transferred in another 2ml centrifuge tube.
(4) (4 DEG C) of precooling isometric phenol is added：Chloroform (1:1), fully mix, 12000rpm, 4 DEG C of centrifugations
10min, supernatant is taken to be transferred in 2ml centrifuge tube.
(5) with (4 DEG C) of precooling isometric chloroform：Isoamyl alcohol (24:1) extract 2 times, fully mix, 12000rpm, 4
DEG C centrifugation 10min, take supernatant to be transferred in 1.5ml centrifuge tube.
(6) 60 μ l NaAc (3M, pH5.2) is added, and adds (- 20 DEG C) isopropanol of 500 μ l precoolings in -20 DEG C of standings
More than 20min, precipitate DNA.
(7) 12000rpm, 4 DEG C of centrifugation 10min, abandons supernatant.
(8) ethanol of (4 DEG C) 70% of 1ml precoolings is added, is gently turned upside down, 12000rpm, 4 DEG C of centrifugation 10min, is abandoned
Ethanol, spontaneously dry, stand more than 15min.
(9) 50 μ l ddH2O is added, gently beaing dissolves precipitation.
(10) DNA extracts are in -20 DEG C of preservations.
After Morciiella Esculeuta Mycelia genomic DNA takes 5 μ l to be well mixed with 2 μ 6 × Loading of l Buffer, with 2% agarose
Then Ago-Gel is placed in gel imaging system in carrying out electrophoresis on gel-electrophoretic apparatus and is detected and observed figure by gel
As result.Experimental result is shown in Fig. 1.Fig. 1 shows that with CTAB methods extraction Morciiella Esculeuta Mycelia be effective, and the DNA is subsequent reality
The basis tested.
The PCR amplifications of 2.3ITS region sequences
2.3.1 annealing temperature is screened
Using fungi universal primer ITS1 (5 '-TCCGTAGGTGAACCTGCG-3 ', SEQ ID NO.2) and ITS4 (5 '-
TCCTCCGCTTATTGATATGC-3 ', SEQ ID NO.3) carry out ITS region sequences amplification.
25 μ lPCR reaction systems：10 × PCR Buffer, 2.5 μ l, rTaq enzymes (5U/ μ l) 0.2 μ l, MgCl2(25mmol/
μ l) 2 0.3 μ l, primer ITS4 of μ l, dNTPs (10mmol/ μ l) 0.5 μ l, primerITS5 0.5 μ l, the μ l of DNA profiling 2.5,
ddH2O 16.5μl。
Response procedures：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 45s；52-62 DEG C of thermograde annealing 45s；72 DEG C of extensions
45s；35 circulations；72 DEG C of filling-in 10min.
In order to filter out mycelia PCR optimum annealing temperature, the annealing temperature in response procedures is arranged to 52-62 DEG C,
Other conditions are constant, then enter performing PCR amplification, after PCR primer is well mixed with 2 μ 6 × Loading of l Buffer, with 2%
Then Ago-Gel is placed in gel imaging system and detected simultaneously in carrying out electrophoresis on gel-electrophoretic apparatus by Ago-Gel
Observe image result.Experimental result is shown in Fig. 2.
Band is most clear when Fig. 2 shows 58 DEG C, and brighter, so 58 DEG C are comparatively proper annealing temperature
Degree.
After the optimum annealing temperature for filtering out mycelia DNA PCR is 58 DEG C, regular-PCR is carried out as reaction condition, its
His condition is constant.After PCR primer is well mixed with 2 μ 6 × Loading of l Buffer, with 2% Ago-Gel in gel electricity
Electrophoresis is carried out on swimming instrument, then Ago-Gel is placed in gel imaging system and is detected and observed image result.As a result
See Fig. 3.Fig. 3 shows that when annealing temperature is 58 DEG C, band is all very limpid in sight.
2.4 enter performing PCR amplification by the annealing temperature filtered out, after the completion of, take 5 μ lPCR products and 2 μ l6 × Loading
After Buffer is well mixed, with 2% Ago-Gel in carrying out electrophoresis on gel-electrophoretic apparatus, then Ago-Gel is placed in solidifying
Detected in glue imaging system and observe result.
2.5 glue reclaim
(1) after cutting glue, smashed to pieces as far as possible in 1.5ml centrifuge tube with 1ml pipette tips.
(2) 300 μ l (being no more than 600 μ l) Binding B, 55 DEG C of water-bath 10min is added.
(3) it is transferred to after melting in sleeve pipe, makes film contact liq, after being sufficiently humidified so as to 2min, centrifugation.
(4) 600 μ l Wash D are added, centrifugation, abandon liquid.
(5) 400 μ l Wash D are added, centrifugation, abandon liquid.
(6) blank pipe centrifugation after, standing allow alcohol volatilize more than 15min.
(7) ddH is added2The μ l of O 30, place more than half an hour, allow glue reclaim product to be substantially dissolved in water.
(8) after taking 5 μ l glue reclaims products to be well mixed with 2 μ 6 × Loading of l Buffer, with 2% Ago-Gel in
Electrophoresis is carried out on gel-electrophoretic apparatus, then Ago-Gel is placed in gel imaging system and is detected and observed result.Knot
Fruit sees Fig. 4.Fig. 4 shows that glue reclaim effect is fine, and band is limpid in sight.
2.6 clone
(1) the μ l of glue reclaim product 4 are sequentially added in micro centrifugal pipe (can suitably increase and decrease, at most according to glue reclaim concentration
No more than 4 μ l), the μ l of pEASY-T3Cloning Vector 1, it is gently mixed, 25 DEG C of reaction 10min., will be micro- after reaction terminates
Type centrifuge tube is positioned on ice.
(2) connection product is added in 50 μ l Trans1-T1 competent cells and (added when competent cell just thaws
Enter connection product, and be placed on all the time on ice chest.), flick mixing, ice bath 20-30 minutes.
(3) 42 DEG C of water-bath heat shocks 50 seconds, are immediately placed on 5 minutes on ice.
(4) add having balanced to the LB fluid nutrient mediums of room temperature for 250 μ l, 200rpm, 37 DEG C cultivate 1 hour.
(5) take 20 μ l 500mM IPTG and 40 μ l 20mg/ml X-gal to drop on flat board and mix, be equably coated in standard
(contain millesimal ammonia benzyl in culture medium) on the flat board got ready, placed 30 minutes in 37 DEG C of incubators.
(6) after IPTG and X-gal is absorbed after waiting until 30 minutes, 200 μ l bacterium solutions are taken equably to be coated on flat board, at 37 DEG C
(to obtain more clone, 12000rpm winks are from discarding 100 μ l supernatants, retain 200 μ l or so, gently for overnight incubation in incubator
Precipitation is beaten in featheriness, it is fully dissolved, and is drawn remaining whole bacterium solutions and is carried out coated plate).
(do not exceeded 16 hours) after (7) 16 hours and the culture dish being incubated overnight is taken out, observed and select and (use lancet
Head) different zones bacterium (be preferably selected a region and there was only single bacterium), be put into together with small pipette tips in 1.5ml centrifuge tubes,
Then toward adding 1ml LB liquid mediums in centrifuge tube, by centrifuge tube be positioned over 200rpm on shaking table, 37 DEG C cultivate 3 hours.3
Enter performing PCR amplification in this, as DNA profiling after hour to be verified (PCR system and reaction condition are all same as above).
(8) select white monoclonal to be inoculated in LB fluid nutrient mediums, 200rpm, 37 DEG C of overnight incubations, extract within second day
Plasmid, take 17 μ l plasmids to carry out digestion identification with restriction enzyme EcoR I, and run glue observation result.As a result it is as shown in Figure 5.
Fig. 5 shows that plasmid band becomes clear, and concentration is higher, may be used as subsequent experimental and carries out digestion.
(6) part plasmid is taken, digestion identification is carried out with restriction enzyme EcoR I, with 2% Ago-Gel in gel
Electrophoresis is carried out on electrophoresis apparatus, then Ago-Gel is placed in gel imaging system and is detected and observed image result.It is real
Test result and see Fig. 6.Electrophoretogram shows that the plasmid of 1,2,3,4, No. 6 has all been transferred to carrier, and No. 5 do not have then.
(10) by digestion successfully remaining plasmid deliver to Shanghai life work be sequenced.
Sequencing result is following (SEQ ID NO.4)：
5’-GGATCGGCAGTGATTGTATACGACTCACTATAGGGCGAATTGGGCCCGACGTCGCATGCTCCCGGC
CGCCATGGCGGCCGCGGGAATTCGATTGGATCGCCCTTGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCT GCGGAAGGATCATTACCAAGAACCACACAGAAAAGGGCAGCCGAGGGGCCAACCAGGGCTAGTAGCTTTACGTTGTT GAACGTCCTGGAACGGACCCGGAGCCGCCCCCATCTAAACACTCTGCGTACCCATCCCACCTTGCTTCCCCCGGCCA TCCGCTGGGGGGAGGAACAACAACCAAAACTCTTTGTGAAGAAACAGACGTCAGAATCATAACCAAAAAAAAGTTAA AACTTTCAACAACGGATCTCTTGGTTCCCACATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAG AATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGTATTCCGGGGGGCATGCCTGTTCGAGCGTCA TAAAAACCTCCTCCCCCTTCGGGTTTGATTACTATCGTTGGGGGGGTATTGGCCTACTGGGAAAGCGATTTGGCAAT TGCCTTCCCACTGTCCTAAATACACTTAGACCCGCCTCCAGATGCGACAGCACCGAGGCCATCAACCGTGGAGTTAT GGAATACCGTTCTCCACACGCCGATGGCAAACCGGTCGCAGTTGCGGGCGTAAATTGGAGCCCTCTTCAGGACCCTC GTGGCCTAGCATCCACCATACATATTTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAAG CGGAGGAAAAGGGCGATCCCAATCACTAGTGAATTCGCGGCCGCCTGCAGGTCGACCATATTGGGAGAGCTCCCAAC
GCGTTGGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCTGGGTCATAGCTGTTTCCTG
GGGTGAAATT-3’。
2.7BLAST compare
After above-mentioned sequencing sequence is removed into primer sequence, submit and (be as above used as progress by the use of the partial sequence that underscore marks
The sequence formally contrasted, SEQ ID NO.1) arrive NCBI (National Center for Biotechnology
Information), http://www.ncbi.nlm.nih.gov/, carry out Blast comparisons.
For Blast comparison results as shown in Fig. 7 and Figure 14, Figure 14 shows Morchella eximia voucher SAAS-
1 ITS sequence and preeminent hickory chick (Morchella eximia voucher LIP:Ph253 ITS sequence part) compares knot
(Query represents Morchella eximia voucher LIP to fruit:Ph253；Sbjct represents Morchella eximia
voucher SAAS-1).Comparison result shows, sequence (SEQ ID NO.1) and the Morchella eximia of submission
Voucher ITS sequence comparative analysis, there is the difference (shown in arrow as shown in figure 14) of 3 bases, sequence similarity in both
Up to 99%.Thus it is Morchella eximia voucher to infer sample, entitled preeminent hickory chick, is a kind of new preeminent sheep
Tripe bacteria strain.It is named as：Morchella eximia voucher SAAS-1.
2.8 constructing system chadograms
2.8.1 chadogram is built with software PHYML 3.0.Downloaded from NCBI official websites (www.ncbi.nlm.nih.gov)
The ITS sequence for the hickory chick kind being currently known, after handling each ITS sequence using software MUSCLE v3.7, according to maximum likelihood
Method carries out Genetic relationship to it), by Morchella eximia voucher SAAS-1 ITS sequence (i.e. SEQ ID
NO.1) with other hickory chick species:Morchella septentr, Morchella delicious (small hickory chick),
Morchella brunnea, Morchella elata (Morchella elata), Morchella purpuras (heteropleural silk purple sheep tripe
Bacterium), Morchella norvegie, Morchella disparil, Morchella snyderi, Morchella
Angustic, Morchella eximioid, Morchella costata (costae hickory chick), Morchella capitata,
Morchella exuberan, Morchella sextelat, Morchella eximia (preeminent hickory chick), Morchella
Septimel, Morchella conica (Morchellaconica), Morchella dunalii, Morchella gigas,
Morchella semilibe (half-open hickory chick), Morchella Pakistan, Morchella populiph,
Morchella punctipe,Morchella tridenti,Morchella quercusi,Morchella elatoide,
Morchella frustrat, Morchella tomentos, Morchella rufobrun (reddish brown hickory chick),
Morchella anatolic, Morchella steppico, Morchella spongiol (small sponge hickory chick),
Morchella dunensis,Morchella prava,Morchella diminuti,Morchella ulmaria,
Morchella cryptica, Morchella castanea, Morchella vulgaris (common hickory chick),
Morchella esculent,Morchella American,Morchella Virginia,Morchella sceptrif,
Morchella palazoni, Morchella crassipe (Morchella crassipes), Morchella fluviali and
Aspergillus flavus (aspergillus flavus) ITS sequence builds chadogram together, as a result as shown in Figure 8.
Fig. 8 shows, preeminent hickory chick Morchella eximia voucher SAAS-1 provided in an embodiment of the present invention its
He compares the affiliation of 46 hickory chick kinds, be as a result found that while Morchella eximia voucher SAAS-1 with
The homologous similitude of preeminent hickory chick (Morchella eximia) is higher, but certain difference in affiliation still be present.
2.8.2 by the Morchella eximia voucher SAAS-1 of the present embodiment ITS sequence (SEQ ID
NO.1) ITS sequence with other 14 kinds preeminent hickory chick (Morchella eximia) subspecies uses and above-mentioned steps identical
Method builds chadogram, determines status of the bacterial strain in preeminent hickory chick, as a result as shown at 13.
It can be seen that from Figure 13 result, Morchella eximia voucher SAAS-1 and Morchella eximia
Voucher FR0410 affiliations are nearer.
The above results, absolutely prove preeminent hickory chick (Morchella eximia provided in an embodiment of the present invention
Voucher SAAS-1) it is a kind of new preeminent hickory chick bacterial strain, never recorded by prior art.
To sum up result, above-mentioned isolated hickory chick bacterial strain are a kind of new preeminent hickory chick bacterial strains, the bacterial strain in
On January 13rd, 2016 is preserved in China typical culture collection center (CCTCC), address：Wuhan, China Wuhan University, classification life
It is entitled：Morchella eximia voucher SAAS-1, deposit number are：CCTCC NO:M 2016032.
Embodiment 2
What the present embodiment provided identifies that deposit number is CCTCC NO:The authentication method of M 2016032 preeminent hickory chick,
It includes：
1 expands the ITS region sequences of bacteria to be tested using fungi universal primer ITS1 and ITS4, obtains sequence to be checked；
Wherein, ITS1 base sequence is as shown in SEQ ID NO.2, and ITS4 base sequence is as shown in SEQ ID NO.3.
Wherein, PCR reaction systems (25 μ l)：10 × PCR Buffer, 2.5 μ l, rTaq enzymes (5U/ μ l) 0.2 μ l, MgCl2
μ l, primerITS50.5 μ l, the DNA moulds of (25mmol/ μ l) 2 0.3 μ l, primer ITS4 of μ l, dNTPs (10mmol/ μ l) 0.5
Plate (bacteria to be tested DNA) 2.5 μ l, ddH2O 16.5μl。
PCR response procedures：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 45s；58 DEG C of thermogrades annealing 45s；72 DEG C of extensions
45s；35 circulations；72 DEG C of filling-in 10min.
2 sequence to be checked is compared with reference sequences, if sequence to be checked contains reference sequences, it indicates that described to be measured
Strain is that deposit number is CCTCC NO:M 2016032 preeminent hickory chick.
Wherein, reference sequences are as shown in SEQ ID NO.1.The reference sequences are CCTCC NO from deposit number:M
The rDNA of 2016032 preeminent hickory chick ITS sequence.
Embodiment 3
Present embodiments provide and extract from preeminent hickory chick (CCTCC NO:M 2016032) preeminent Morchella esculenta extract,
The preeminent Morchella esculenta extract is preeminent hickory chick water extract or preeminent hickory chick alcohol extracting thing.
The extracting method of the preeminent Morchella esculenta extract is as follows：
1 takes preeminent hickory chick (CCTCC NO:M 2016032) new fresh sporophore 200g, it is real with 75% alcohol flushing
Body twice, place -80 DEG C of refrigerators and freeze 4h by fructification.
2 are transferred to lyophilized fructification in 50mL centrifuge tubes, and by 1:10 (M/V) ratio adds 95% (v/v) ethanol,
40 DEG C, (400W) ultrasonication three times, crushes 30min every time.Broken interval time is 30min.
3 in 10000rpm centrifugations (16000 × G) 15 minutes, and (precipitation is standby, for preparing preeminent sheep tripe for transfer supernatant
Bacterium water extract) it is evaporated to rotary vacuum evaporator, 60 DEG C of conditions, produce preeminent hickory chick alcohol extracting thing.
Precipitation in 4 steps 3 is using (60 DEG C) the processing 2h extractions of heated-air drying instrument, according to 1:20 (w/v) ratios, which add, steams
Distilled water, in 100 DEG C of water-baths three times, 2 hours every time.Filtering, collect filtrate and be concentrated into the 1/ of original volume in freeze concentration instrument
10, that is, obtain preeminent hickory chick water extract.
The extracting method step is simple, fast, it is not necessary to complex instrument, and it is more disposably can largely to extract hickory chick
Sugar.
The preeminent Morchella esculenta extract that the present embodiment provides has the work for promoting T cell propagation and suppressing tumor cell proliferation
With, immunity medicine or health products are improved available for preparing, and in the field such as preparation prevention or tumor.
Embodiment 4
The facilitation that the preeminent Morchella esculenta extract that checking embodiment 3 obtains is bred to T cell, experimental method are as follows：
1 every group of experiment takes 10 mouse, daily gavage 1 time, continues 30 days,
2 by separating spleen after mouse anesthesia, grinding, and the suspension after grinding is utilized into 70 μm of membrane filtrations,
3 filtrate 1500rpm centrifuge 5min, and cell liquid is put into warm bath 10 minutes in ACK lysis buffers,
4 add anti-mouse CD4 magnetic-particles (BD Bioscience, San Jose, CA, USA), are separated from splenocyte
Go out CD4+T cells.
5 insert T cell in the hydroxyl fluorescein diacetate succinimide lipoprotein solution that concentration is 5mM, 37 DEG C of warm bath
10min, it is transferred in the cold culture mediums of RPMI 1640 containing 10%FBS, 1000rpm centrifugation 3min, is repeated once.
6 respectively using preeminent hickory chick water extract (0 μ g/mL (as control), 0.25 μ g/mL, 2.5 μ containing various concentrations
G/mL, 5 μ g/mL) the medium cultures of RPMI 1640 and addition have various concentrations (volume ratio) (0% (as control),
0.1%, 1%, 10%, 50%) the medium culture T cells of RPMI 1640 of preeminent hickory chick alcohol extracting thing, 37 DEG C, 72h,
Cell is collected, and with flow cytometry analysis T cell proliferative conditions.As a result (figure is flow cytometer pair as shown in Figure 9 and Figure 10
The detection collection of illustrative plates of T cell, abscissa represent fluorescence intensity during detection cell, and ordinate represents that cell count is count).
Fig. 9 shows various concentrations (0 μ g/mL, 0.25 μ g/mL, 2.5 μ g/mL, 5 μ g/mL) preeminent hickory chick water extract
Influence to T cell propagation, as shown in figure 9, preeminent hickory chick water extract can promote T cell to breed, and concentration is bigger, and T is thin
Born of the same parents' quantity is more, illustrates that preeminent hickory chick water extract promotes T cell proliferation function more obvious.
Figure 10 shows the preeminent hickory chick alcohol extract of various concentrations (0%, 0.1%, 1%, 10%, 50%) to T cell
The influence of propagation, as shown in Figure 10, preeminent hickory chick alcohol extract can promote T cell to breed, and the preeminent hickory chick alcohol added
Extract amount is more, and T cell quantity is more, promotes cultivation effect more obvious.
It is indicated above that the preeminent Morchella esculenta extract that embodiment 3 obtains T cell is bred there is facilitation, it can
For preparing the medicine or health products that improve immunity.
Embodiment 5
The inhibitory action of the tumor cell proliferation for the preeminent Morchella esculenta extract that checking embodiment 3 obtains, experimental method is such as
Under：
1 has been respectively placed in tumour cell (Daudi, NAMALWA, SU-DHL-4, three kinds of different human lymphoma cells)
In full culture medium, 37 DEG C, 5%CO2Incubator in cultivated, change liquid once within every 2 days, every bottle plus 5mL culture mediums.
2 are rinsed twice, at 25 DEG C, with the second two containing 0.28% trypsase with 20mL 0.01%M kaliumphosphate buffers
Amine tetraacethyl (1X) digests 5 minutes, takes the logarithmic proliferation phase cell that grows fine.
Cell concentration is adjusted to 2.5*104/mL by 3 using blood counting chamber counting, and 150 μ L cell suspensions are added into 96
In orifice plate, after cultivating 1 day, culture medium is drawn, addition contains various concentrations (0 μ g/mL (as a control group), 1 μ g/mL, 10 μ g/
ML, 100 μ g/mL, 1000 μ g/mL) preeminent hickory chick alcohol extract culture medium and containing various concentrations, (volume ratio, 0 ‰ (as right
According to group), the culture medium of 0.01 ‰, 0.1 ‰, 1 ‰, 10 ‰) preeminent hickory chick water extracts, cultivate 24 hours.
Under 4 dark conditions, 30 μ L 5mM MTT solution, CO are added per hole2Culture 5h is carried out in incubator, is sucked in hole
Culture medium, add 150 μ L dimethyl sulfoxide (DMSO), lucifuge stand 20 minutes after ELIASA detection cell growth status.Result figure
Shown in 11 and Figure 12.
Figure 11 shows the shadow of the preeminent hickory chick alcohol extract of the various concentrations human lymphoma cell propagation different to three kinds
Result is rung, as shown in figure 11, preeminent hickory chick alcohol extract has the function that suppressing tumour cell such as lymphoma cell breeds.It is dense
Degree is bigger more obvious to human lymphoma cell's inhibitory action.
Figure 12 shows the shadow of the preeminent hickory chick water extract of the different volumes human lymphoma cell propagation different to three kinds
Result is rung, as shown in figure 12, preeminent hickory chick water extract has the function that suppressing tumour cell such as lymphoma cell breeds.It is dense
Degree is bigger more obvious to human lymphoma cell's inhibitory action.
It these results suggest that, the preeminent Morchella esculenta extract that embodiment 3 provides is to tumour cell such as human lymphoma cell
There is Inhibit proliferaton, the preeminent Morchella esculenta extract for being indicated above the offer of embodiment 3 can be used for preparing prevention or control
Treat the medicine of tumour.
Embodiment 6
It is CCTCC NO that what the present embodiment provided, which is used to cultivate deposit number,:The solid of M 2016032 preeminent hickory chick
Culture medium, contain：Murphy juice, willow leaf extract, glucose, KH2PO4And agar.
The culture medium that the present embodiment provides is prepared via a method which：
1 fetches earth beans 200g, peeling, is cut into the fritter of 2cm length, is cooked, filtered through gauze, leaves and takes potato filtrate.
2 choose the wild growth willow old leaf of Qujing of Yunnan or fallen leaves (brown or yellow), dry.Take and dry Poplar leaves
200g, 1cm*0.5cm strips are cut into, add 500mL water, boiling 20min, filtered through gauze, leave and take filtrate, be i.e. Poplar leaves are extracted
Thing.
3 mix above two filtrate, add 20g glucose, KH thereto2PO41g, agar 15g, adds water to 1L, matches somebody with somebody
It is CCTCC NO to be made for cultivating deposit number:The culture medium of M 2016032 preeminent hickory chick, also is understood as improveing
PDA culture medium.
Embodiment 7
The culture medium that embodiment 6 provides is CCTCC NO to deposit number:The culture effect of M 2016032 preeminent hickory chick
Fruit, method are as follows.
Preeminent hickory chick is seeded on the culture medium that embodiment 6 provides, as experimental group, made with common PDA culture medium
To compare, the growing state of preeminent hickory chick is observed.As a result it see the table below 1.
Table 1
In table：++ represent that mycelium growth vigor is general, ++++representing that mycelium growth vigor is fine, * * represent that sclerotium quantity is general, * * * *
Represent that sclerotium quantity is a lot.
As seen from the results in Table 1, cultivated using the culture medium of embodiment 6, preeminent hickory chick (CCTCC NO:M
2016032) growth such as the dense degree of growing way, the per day speed of growth, mycelia, full plate time, Sclerotia forming time, sclerotium quantity
Index is superior to common PDA culture medium.
Morphologic observation result shows that on the culture medium that embodiment 6 provides, 6h starts to sprout after inoculation, early growth period bacterium
Silk is close to culture medium, is grown to culture medium outer rim direction, and mycelia color is glossy to be translucent, generally unbranched thicker
Mycelium, slow-growing after 2 days, mycelia darkens, aerial hyphae increase, and mycelia is white and has " Y " or branching shape point
Branch, after 4 days, mycelia by it is faint yellow be in gradually sepia, mycelia is twisted together more, and slabbing is assembled in many places；After 6 days, sclerotium is presented, and is in
Brown, between a diameter of 1.2-4.3mm of sclerotium, quality is hard, in dark brown.
Embodiment 8
Present embodiments provide the present embodiment offer is CCTCC NO for cultivating deposit number:M2016032's is preeminent
The fluid nutrient medium of hickory chick, contains：Analysis for soybean powder, willow leaf extract, glucose, KH2PO4And agar.
The preparation method of the fluid nutrient medium is as follows：
Common willow old leaf or fallen leaves (brown or yellow) are chosen, are dried；
The Poplar leaves 200g dried is taken, is cut into 1cm*0.5cm strips, adds 500mL water, boiling 20min, filtered through gauze,
Leave and take filtrate；
5g analysis for soybean powder is added into filtrate, 20g glucose, 1L is added water to, is CCTCCNO for cultivating deposit number:
The fluid nutrient medium of M2016032 preeminent hickory chick.
Embodiment 9
The fluid nutrient medium that embodiment 8 provides is CCTCC NO to deposit number:The training of M 2016032 preeminent hickory chick
Effect is supported, method is as follows.
By eugonic inoculation in solid slope culture medium to the Liquid Culture for filling 100mL embodiments 8 and providing
In the 500mL of base triangular flask, cultivated 4 days under the conditions of 25 DEG C, during culture, 150rpm is with quiescent culture according to 24h/24h weeks
Phase is carried out, and prevents from forming bacterium ball, observes mycelial growth situation；Control is used as using common PDA liquid medium.As a result such as following table
Shown in 2.
Table 2
In table：++ represent that mycelium growth vigor is general, ++++represent that mycelium growth vigor is fine.
As shown in Table 2, compared with common PDA liquid medium, using embodiment 8 provide fluid nutrient medium culture,
Mycelial growth rate is fast, and biomass is high, and anti-miscellaneous bacteria ability is strong, and pollution rate is few, and the sclerotium generation time is short.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.Within the spirit and principles of the invention, that is made any repaiies
Change, equivalent substitution, improvement etc., should be included in the scope of the protection.
SEQUENCE LISTING
<110>Academy of Agricultural Sciences, Shanghai City
<120>A kind of culture medium and cultural method for being used to cultivate preeminent hickory chick
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 739
<212> DNA
<213>Artificial sequence
<400> 1
ggaagtaaaa gtcgtaacaa ggtttccgta ggtgaacctg cggaaggatc attaccaaga 60
accacacaga aaagggcagc cgaggggcca accagggcta gtagctttac gttgttgaac 120
gtcctggaac ggacccggag ccgcccccat ctaaacactc tgcgtaccca tcccaccttg 180
cttcccccgg ccatccgctg gggggaggaa caacaaccaa aactctttgt gaagaaacag 240
acgtcagaat cataaccaaa aaaaagttaa aactttcaac aacggatctc ttggttccca 300
catcgatgaa gaacgcagcg aaatgcgata agtaatgtga attgcagaat tcagtgaatc 360
atcgaatctt tgaacgcaca ttgcgccctc tggtattccg gggggcatgc ctgttcgagc 420
gtcataaaaa cctcctcccc cttcgggttt gattactatc gttggggggg tattggccta 480
ctgggaaagc gatttggcaa ttgccttccc actgtcctaa atacacttag acccgcctcc 540
agatgcgaca gcaccgaggc catcaaccgt ggagttatgg aataccgttc tccacacgcc 600
gatggcaaac cggtcgcagt tgcgggcgta aattggagcc ctcttcagga ccctcgtggc 660
ctagcatcca ccatacatat tttgacctcg gatcaggtag ggatacccgc tgaacttaag 720
catatcaata agcggagga 739
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence
<400> 2
tccgtaggtg aacctgcg 18
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
tcctccgctt attgatatgc 20
<210> 4
<211> 1000
<212> DNA
<213>Artificial sequence
<400> 4
ggatcggcag tgattgtata cgactcacta tagggcgaat tgggcccgac gtcgcatgct 60
cccggccgcc atggcggccg cgggaattcg attggatcgc ccttggaagt aaaagtcgta 120
acaaggtttc cgtaggtgaa cctgcggaag gatcattacc aagaaccaca cagaaaaggg 180
cagccgaggg gccaaccagg gctagtagct ttacgttgtt gaacgtcctg gaacggaccc 240
ggagccgccc ccatctaaac actctgcgta cccatcccac cttgcttccc ccggccatcc 300
gctgggggga ggaacaacaa ccaaaactct ttgtgaagaa acagacgtca gaatcataac 360
caaaaaaaag ttaaaacttt caacaacgga tctcttggtt cccacatcga tgaagaacgc 420
agcgaaatgc gataagtaat gtgaattgca gaattcagtg aatcatcgaa tctttgaacg 480
cacattgcgc cctctggtat tccggggggc atgcctgttc gagcgtcata aaaacctcct 540
cccccttcgg gtttgattac tatcgttggg ggggtattgg cctactggga aagcgatttg 600
gcaattgcct tcccactgtc ctaaatacac ttagacccgc ctccagatgc gacagcaccg 660
aggccatcaa ccgtggagtt atggaatacc gttctccaca cgccgatggc aaaccggtcg 720
cagttgcggg cgtaaattgg agccctcttc aggaccctcg tggcctagca tccaccatac 780
atattttgac ctcggatcag gtagggatac ccgctgaact taagcatatc aataagcgga 840
ggaaaagggc gatcccaatc actagtgaat tcgcggccgc ctgcaggtcg accatattgg 900
gagagctccc aacgcgttgg atgcatagct tgagtattct atagtgtcac ctaaatagct 960
tggcgtaatc tgggtcatag ctgtttcctg gggtgaaatt 1000
Claims (6)
1. a kind of culture medium for being used to cultivate preeminent hickory chick, it is CCTCC NO that it, which is suitable to culture deposit number,:M2016032's
Preeminent hickory chick, it is characterised in that contain willow leaf extract in the culture medium.
2. culture medium according to claim 1, it is characterised in that the willow leaf extract is Poplar leaves water extract.
3. culture medium according to claim 2, it is characterised in that the Poplar leaves water extract is prepared by the following method
Arrive：
Poplar leaves are boiled with decocting, filters, takes filtrate to produce the Poplar leaves water extract.
4. according to the culture medium described in claim any one of 1-3, it is characterised in that the culture medium also contains：Glucose and
KH2PO4。
5. culture medium according to claim 4, it is characterised in that based on every L, the culture medium contains：18-22g grapes
Sugar and 0.8-1.1g KH2PO4。
6. a kind of method for cultivating preeminent hickory chick, the deposit number of the preeminent hickory chick is CCTCC NO:M2016032, its
It is characterised by, it uses the medium culture any one of claim 1-5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711126323.5A CN107653194B (en) | 2017-11-14 | 2017-11-14 | It is a kind of for cultivating the culture medium and cultural method of preeminent hickory chick |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711126323.5A CN107653194B (en) | 2017-11-14 | 2017-11-14 | It is a kind of for cultivating the culture medium and cultural method of preeminent hickory chick |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107653194A true CN107653194A (en) | 2018-02-02 |
| CN107653194B CN107653194B (en) | 2019-09-27 |
Family
ID=61121618
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711126323.5A Active CN107653194B (en) | 2017-11-14 | 2017-11-14 | It is a kind of for cultivating the culture medium and cultural method of preeminent hickory chick |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107653194B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108901589A (en) * | 2018-04-18 | 2018-11-30 | 上海沃施生物科技有限公司 | A kind of separation method of wild delicious lactarius strain |
| CN109504613A (en) * | 2019-01-23 | 2019-03-22 | 上海市农业科学院 | A kind of cultural method and its culture medium of Morchella esculenta (L.) Pers mycelium |
| CN109504612A (en) * | 2019-01-23 | 2019-03-22 | 上海市农业科学院 | A kind of cultural method and its culture medium of Morchella esculenta (L.) Pers mycelium |
| CN109566272A (en) * | 2019-01-23 | 2019-04-05 | 上海市农业科学院 | A kind of cultural method and its culture medium of Morchella esculenta (L.) Pers mycelium |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101946634A (en) * | 2010-09-13 | 2011-01-19 | 云南省农业科学院高山经济植物研究所 | Manufacturing method for morchella mother culture |
-
2017
- 2017-11-14 CN CN201711126323.5A patent/CN107653194B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101946634A (en) * | 2010-09-13 | 2011-01-19 | 云南省农业科学院高山经济植物研究所 | Manufacturing method for morchella mother culture |
Non-Patent Citations (2)
| Title |
|---|
| 刘伟等: "我国羊肚菌产业发展的现状及趋势", 《食药用菌》 * |
| 熊川等: "羊肚菌生活史周期、人工栽培及功效研究进展", 《中国食用菌》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108901589A (en) * | 2018-04-18 | 2018-11-30 | 上海沃施生物科技有限公司 | A kind of separation method of wild delicious lactarius strain |
| CN109504613A (en) * | 2019-01-23 | 2019-03-22 | 上海市农业科学院 | A kind of cultural method and its culture medium of Morchella esculenta (L.) Pers mycelium |
| CN109504612A (en) * | 2019-01-23 | 2019-03-22 | 上海市农业科学院 | A kind of cultural method and its culture medium of Morchella esculenta (L.) Pers mycelium |
| CN109566272A (en) * | 2019-01-23 | 2019-04-05 | 上海市农业科学院 | A kind of cultural method and its culture medium of Morchella esculenta (L.) Pers mycelium |
| CN109566272B (en) * | 2019-01-23 | 2022-09-20 | 上海市农业科学院 | Method for culturing morchella mycelium and culture medium thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107653194B (en) | 2019-09-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107653194B (en) | It is a kind of for cultivating the culture medium and cultural method of preeminent hickory chick | |
| CN106613350B (en) | Schizophyllum commune and domestication and cultivation method and application thereof | |
| CN103430855B (en) | A kind of low temperature resistance straw mushroom bacterial and selection thereof | |
| CN105219654B (en) | One plant of aspergillus flavus strain for not producing aflatoxin and its application in aflatoxin pollution of peanuts biological control | |
| Kammerer et al. | Identification of a new Waitea circinata variety causing basal leaf blight of seashore paspalum | |
| CN107312815A (en) | A kind of Resistance Identification method in pepper anthracnose room | |
| CN109452088A (en) | Needle mushroom X18 and its cultural method | |
| CN109082382A (en) | Cordyceps sinensis Hirsutella sinensis ZJB18002 and its application | |
| CN103614411A (en) | Research method for American ginseng hairy root induction and plant regeneration | |
| CN106434371A (en) | Neopestalotiopsis zerumbet and method of using same to identify Alpinia zerumbet leaf diseases | |
| CN104651319B (en) | Fusarium graminearum low-toxicity virus FgHV2/JS16 and application thereof | |
| CN104072593A (en) | Hericium erinaceus glycoprotein with anti-tumor and agglutination activity and preparation method thereof | |
| CN109735457B (en) | Mutant eurotium cristatum and application thereof in preventing and controlling barnyard grass | |
| CN109452089B (en) | White beech mushroom H7 and cultivation method thereof | |
| CN108315266B (en) | Paecilomyces cicadae and application thereof | |
| CN107711296A (en) | A kind of preeminent hickory chick and its authentication method | |
| CN107823225A (en) | The application of preeminent hickory chick | |
| CN109619207A (en) | A kind of fresh adding oil preserved bean curd and preparation method thereof | |
| Zohmangaiha et al. | Ganoderma Species of Mizoram, India | |
| CN109220514B (en) | Separation and artificial domestication cultivation method of new wild edible fungi | |
| CN109055248A (en) | A kind of bio-control yeast bacterium activated freeze dried powder and preparation method thereof | |
| CN113462586A (en) | Method for biologically preventing and treating soft rot of picked strawberry fruits and storing and refreshing strawberry fruits by Debaryomyces hansenii Y3 | |
| CN107325973A (en) | One plant there is High pathogenicity to hazel stinkbug muscardine bacterial strain and its application | |
| CN106635822B (en) | A kind of elm mushroom bacterial strain and its cross breeding method | |
| Kozar | The pathway of infection of Ustilago hordei |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |