CN110408724A - The primer and molecule labelling method of the molecular labeling of fragrant Na needle mushroom new strains - Google Patents
The primer and molecule labelling method of the molecular labeling of fragrant Na needle mushroom new strains Download PDFInfo
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Classifications
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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
- C12N1/145—Fungal isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- 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 present invention relates to the primers and molecule labelling method of a kind of molecular labeling of fragrant Na needle mushroom new strains.The primer of the molecular labeling of fragrant Na needle mushroom Flammulina fennae HMGIM-A151357, including FAA-F-3 and FAA-R-4 or FAA-F-5 and FAA-R-7；FAA-F-3:GCATTGGCAAGAGTTTCA, FAA-R-4:AGGCAAGTCATCCGTTTT；FAA-F-5:GCATTTGGACTATTGCCTCA, FAA-R-7:CGTCCTATGTCGTAGAGTAGCG.Two pairs of specific nucle primer sequence combination Standard PCRs of the present invention, electrophoresis method can quick and precisely identify this fragrant Na Needle mushroom strain, distinguish common Needle mushroom strain in the market, discrimination method is simple and specificity is high.
Description
Technical field
The present invention relates to a kind of molecular labeling primer of new strains and molecule labelling method more particularly to a kind of sweet smell Na Jinzhen
The primer and molecule labelling method of the molecular labeling of mushroom new strains.
Background technique
Currently, the industry development of edible mushroom is swift and violent, the annual yield of China edible mushroom reaches 30,000,000 tons or more, Zhan Quanqiu
75% or more, practitioner is more than 20,000,000 people, mushroom industry come in planting industry in addition to grain, dish fruit, oil after the
Five, be more than cotton, tealeaves and silkworm and mulberry.
In today that mushroom industry flourishes, more and more Rare edible fungus kinds progress into the view of people
Open country, many original rare kinds are gradually tamed, such as dictyophora phalloidea, Agrocybe chaxingu, from pleat umbrella.But also there is large quantities of wild food medicines
It is not studied due to failing by human knowledge with bacterium.In 150,000 kinds of macro fungis having at present, only 80 kinds or so
Wild edible and medical fungi tamed by the mankind, and the kind of large-scale planting more only have more than 20 kinds.With people's living standard
Gradually rise, the requirement for quality of the life is higher, and macro fungi due to its be rich in it is various with nutrition and function
Ingredient, including fungi polysaccharide, triterpenes, sterol etc. have very good effect for human health, are increasingly subject to people's
Pay attention to.
Needle mushroom (Flammulina velutipes) is one of edible mushroom manually cultivated extensively at present and a kind of warp
Ji is worth very high edible and medicinal fungi.Needle mushroom contains multiple nutritional components, wherein 8 kinds of amino acid contents needed for growth in humans
It is higher, it is especially very rich with lysine and arginine content, the healthy growth and intellectual development of children can be promoted, therefore often claimed
For " increasing intelligence mushroom ".Needle mushroom contains Flammulin, has significant anti-cancer function, and often edible needle mushroom can also prevent high blood
Pressure treats the diseases such as hepatitis and intestine gastric ulcer.
Based on different Needle mushroom strains, need to carry out identification differentiation.However, it is easy to operate and identify specificity it is high
Discrimination method be there is an urgent need to.
Summary of the invention
Against the above deficiency, the present invention provides the primer and molecular labeling of a kind of molecular labeling of fragrant Na needle mushroom new strains
Method.
The present invention provides a kind of primer of the molecular labeling of fragrant Na needle mushroom new strains, which is isolated from Liaoning Province's sheet
On the old bald sub- mountain broad leaf tree dead wood in the county Huan Ren, small stream city, original strain is obtained by the way that fructification is carried out tissue separation and purified,
It is fragrant Na needle mushroom Flammulina fennae new strains through morphology and molecular biology identification, is sweet smell by the Strain Designation
Na needle mushroom Flammulina fennae HMGIM-A151357, in preservation on June 20 in 2019 to Chinese Typical Representative culture
Collection (abbreviation CCTCC, Wuhan, China), deposit number are as follows: CCTCC NO:M 2019478.
The isolated bacterial strain mycelia of inventor flushes, and currently obtains son in fact by artificial domesticating cultivation
Body, commodity property are excellent.And the sequencing and assembling that its full-length genome is completed to the bacterial strain, by with edible mushroom genome ratio
It is right, belong to fragrant Na needle mushroom (Flammulina fennae Bas) strain, and in particular to come from mycota (Fungi) basidiomycetes
Door (Basidiomycota) agaric guiding principle (Agaricomycetes) Agaricales (Agaricales) swollen coral Cordycepps
(Physalacriaceae) genus flammulina (Flammulina).
Fragrant Na acupuncture needle massee fruiting bodies of the invention are yellow, and cap is light yellow, hemispherical, larger, are not easy parachute-opening, surface
There is the thin skin of colloid；Stem is elongated, and light yellow to brown, stem base portion is deeper than color at the top of stem；Bacterial context white, it is relatively thin；
Lamella is close cream-colored or light yellow, recessed life or prolongs life.Growth cycle is -70 days 60 days, and the management of producing mushroom time is -13 days 10 days.
22-24 DEG C of mycelium optimum temperature, former base form 15-16 DEG C, and sporophore growth optimum temperature is 16-18 DEG C.Mycelium is general
The speed of growth is moderate in logical PDA culture medium, and mycelia is slightly dense, and initial stage is white to canescence, and the later period shows faint yellow, and produces
Fecula spore.
Compared with common needle mushroom, fragrant Na needle mushroom of the invention has more abundant calcium content, fruiting tolerable temperature
It is higher, it is beneficial to energy conservation, and cap is not easy parachute-opening, commodity property is excellent, and content of cellulose is low, it is not easy to get stuck between the teeth when edible, mouthfeel
It is more excellent.
The molecular labeling of fragrant Na needle mushroom new strains (fragrant Na needle mushroom Flammulina fennae HMGIM-A151357)
Primer, including FAA-F-3 and FAA-R-4 or FAA-F-5 and FAA-R-7；
Wherein,
FAA-F-3:5 ' --- GCATTGGCAAGAGTTTCA---3 '
FAA-R-4:5 ' --- AGGCAAGTCATCCGTTTT---3 '
FAA-F-5:5 ' --- GCATTTGGACTATTGCCTCA---3 '
FAA-R-7:5 ' --- CGTCCTATGTCGTAGAGTAGCG---3 '.
Two pairs of specific nucle primer sequence combination Standard PCRs, electrophoresis methods of the invention can quick and precisely identify this
Fragrant Na Needle mushroom strain, to distinguish common Needle mushroom strain in the market, discrimination method is simple and specificity is high.
Detailed description of the invention
Fig. 1 is the ITS sequencing result of the new strains of the invention of embodiment 1.
Fig. 2 is an otherness site of embodiment 2.
Fig. 3 is another otherness site of embodiment 2.
Fig. 4 is the PCR amplification electropherogram spectrum of the primer pair of embodiment 3.
Fig. 5 is the PCR amplification electropherogram spectrum of another primer pair of embodiment 3.
Specific embodiment
The present invention provides one plant of fragrant Na needle mushroom new strains isolated by field acquisition, pass through molecular biology
Identify and character identify be new strains, measure its full-length genome data and with widely used Needle mushroom strain gene in the market
It is compared analysis, design specific primer completes its molecular labeling, realizes the protection of own bacterial strain, provides for commercial applications
Property right guarantee.
A kind of sweet smell Na needle mushroom new strains are isolated from the old bald sub- mountain broad leaf tree dead wood in Liaoning Province, the county Huan Ren, Benxi,
Original strain is obtained by the way that fructification is carried out tissue separation and purified, is sweet smell Na Jinzhen through morphology and molecular biology identification
The Strain Designation is sweet smell Na needle mushroom Flammulina fennae HMGIM- by mushroom Flammulina fennae new strains
A151357 is protected in preservation on June 20 in 2019 to China typical culture collection center (abbreviation CCTCC, Wuhan, China)
Hiding number are as follows: CCTCC NO:M 2019478.
The sequencing and assembling that its full-length genome is completed to the bacterial strain, by being compared with common needle mushroom gene in the market
Analysis, screening obtain differential gene collection, design specific primer and PCR amplification, by amplified production electrophoresis detection and are sequenced.As a result
Show that two pairs of nucleic acid probe primers of the invention can amplify specific band for the sweet smell Na Strains of Flammulina velutipes, and for
Common needle mushroom is then without band, and the band that Successful amplification comes out is after inspection is sequenced, and fragrant Na needle mushroom genome alignment,
Similitude reaches 98% or more, shows that these two pair nucleic acid probe primer can be with specific amplification sweet smell Na Strains of Flammulina velutipes, to area
The Strains of Flammulina velutipes not circulated in the market.
The molecular labeling of fragrant Na needle mushroom new strains (fragrant Na needle mushroom Flammulina fennae HMGIM-A151357)
Primer, including FAA-F-3 and FAA-R-4 or FAA-F-5 and FAA-R-7；
Wherein,
FAA-F-3:5 ' --- GCATTGGCAAGAGTTTCA---3 '
FAA-R-4:5 ' --- AGGCAAGTCATCCGTTTT---3 '
FAA-F-5:5 ' --- GCATTTGGACTATTGCCTCA---3 '
FAA-R-7:5 ' --- CGTCCTATGTCGTAGAGTAGCG---3 '
The molecular labeling of fragrant Na needle mushroom new strains (fragrant Na needle mushroom Flammulina fennae HMGIM-A151357)
Method carries out PCR amplification using primer pair FAA-F-3 and FAA-R-4 or FAA-F-5 and FAA-R-7, obtains specific amplification
Band is accredited as fragrant Na needle mushroom new strains (fragrant Na needle mushroom Flammulina fennae HMGIM-A151357).
The molecule labelling method further comprises using primer pair FAA-F-3 and FAA-R-4 or FAA-F-5 and FAA-
R-7 carries out the PCR amplification of common needle mushroom, shows without amplified band, shows fragrant Na needle mushroom new strains (fragrant Na needle mushroom
Flammulina fennae HMGIM-A151357) identification specificity.
The molecule labelling method further comprises that ITS1/ITS4 is used to carry out control amplification for primer pair, expands shaping
Band shows the validity of PCR amplification system.
Below in conjunction with specific embodiment, invention is further explained.
1 molecular biology identification of embodiment
The fragrant Na needle mushroom new strains sweet smell Na being isolated from the old bald sub- mountain broad leaf tree dead wood in Liaoning Province, the county Huan Ren, Benxi
Needle mushroom Flammulina fennae HMGIM-A151357, using Magen (Mei Ji) biology HiPure Fungal DNA
Mini Kit II fungal genomic DNA extraction agent box extract mycelium DNA, with ITS1 (TCCGTAGGTGAACCTGCGG),
ITS4 (TCCTCCGCTTATTGATATGC) is that primer carries out genome ITS region PCR test, and amplified production is sent to Shanghai beauty
Lucky biological order-checking, obtains the sequence that base number is 760bp, and sequence information is as shown in Figure 1.
In the website NCBI using blast program by this sequence alignment analysis, qualification result is Flammulina fennae.
Embodiment 2
Artificial cultivation method
The fragrant Na Needle mushroom strain of purifies and separates is seeded in 13cm × 25cm Polypropylene Bag equipped with Primary spawn material,
Primary spawn material ingredient be 98% sorghum grain, 2% calcium carbonate, 24-25 DEG C be protected from light culture cover with bacterium bag to mycelia after obtain original seed.
Original seed is seeded in the polypropylene fruiting bag of cultivar compost, cultivar compost ingredient is 50% cotton seed hulls, 38% wood
Bits, 10% wheat bran, 2% calcium carbonate are placed in 24-25 DEG C, are protected from light culture to bacterium in the culturing room of relative air humidity 70% or so
Filament length expires bacterium bag.Then a bag flower bud is opened, and increases humidity to 95% or so, after water droplet occurs in compost surface, changes wind 2 daily
Secondary culture 4 days or so.Then it is cooled to 6 DEG C, relative air humidity 85% or so, (daily) the progress cold stimulation of illumination 2h, is handled
Time is 4 days or so.Then temperature is risen to 18 DEG C, relative air humidity is 85% or so, no light, and controls carbon dioxide
Concentration waits the long up to 10cm or more of young mushroom, cap that can harvest when starting open and flat less than 5%.
3 specific primer design of embodiment
By by the coded protein sequence of this fragrant Na Strains of Flammulina velutipes (Coding sequence, abbreviation CDs sequence, under
It is compared with common needle mushroom CDs sequence together), it is determined that differential gene determines the gene02699 of the bacterial strain by screening
Gene and common needle mushroom gene similitude are only 75%, and specific differential position is as shown in Figure 2.
By screening, determine that the gene11357 gene of the bacterial strain and common needle mushroom gene similitude are only 76%, specifically
Differential position figure is as shown in Figure 3.
According to fig. 2 with the difference site of Fig. 3, devise 2 pairs of specific primers pair, respectively primer pair FAA-F-3 and
FAA-R-4, primer pair FAA-F-5 and FAA-R-7, specific sequence information are as follows:
FAA-F-3:5 ' --- GCATTGGCAAGAGTTTCA---3 '
FAA-R-4:5 ' --- AGGCAAGTCATCCGTTTT---3 '
FAA-F-5:5 ' --- GCATTTGGACTATTGCCTCA---3 '
FAA-R-7:5 ' --- CGTCCTATGTCGTAGAGTAGCG---3 '
The verifying of 4 specific primer of embodiment
It is identified using above-mentioned primer pair and PCT system, PCR reaction system is 50 μ L, archaeal dna polymerase mixed liquor
(Premix TaqTM, RR003A) and 25 μ L, fragrant 5 μ L of Na needle mushroom genomic DNA template, primer each 5 μ L, ddH2O10 μ L, control
Using common needle mushroom genomic DNA as template, shown in response procedures table 1.
Table 1: response procedures
After reaction, by product and DNA Marker, loading is placed in 2% Ago-Gel PCR respectively, 120V, electrophoresis
25min then takes out gel, and observed under fluorescent light is taken pictures, as shown in Figure 4 and Figure 5.
As can be seen from Figure 4 and Figure 5, fragrant Na needle mushroom and common needle mushroom be when using ITS1/ITS4 as primer, can be with
Amplify band, therefore the product of the primer pair amplifies cannot directly distinguish two kinds of needle mushrooms, but specific primer FAA-F-
3/FAA-R-4, FAA-F-5/FAA-R-7 can amplify in specific manner fragrant Na needle mushroom sequence, and PCR product electricity by PCR
Swimming figure can quickly distinguish two kinds of needle mushrooms.These two pair primer amplified primer size is respectively 620-630bp, 560-
570bp。
Therefore, two pairs of specific nucle primer sequence combination Standard PCRs, electrophoresis methods of the invention can quick and precisely reflect
This fixed fragrant Na Needle mushroom strain, to distinguish common Needle mushroom strain in the market.
The above, preferable specific embodiment only of the invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Design is subject to equivalent substitution or change, should be covered by the scope of protection of the present invention.
Sequence table
<110>Guangdong Microbes Inst (microbiological analysis inspection center, Guangdong Province), the limited public affairs of Guangdong Guangdong microorganism science and technology
Department
<120>primer and molecule labelling method of the molecular labeling of fragrant Na needle mushroom new strains
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
Claims (4)
1. the primer of the molecular labeling of fragrant Na needle mushroom new strains, the sweet smell Na needle mushroom new strains are fragrant Na needle mushroom
Flammulina fennae HMGIM-A151357, deposit number are as follows: CCTCC NO:M 2019478, which is characterized in that described
The primer of the molecular labeling of fragrant Na needle mushroom new strains (fragrant Na needle mushroom Flammulina fennae HMGIM-A151357),
Including FAA-F-3 and FAA-R-4 or FAA-F-5 and FAA-R-7；
Wherein,
FAA-F-3:5 ' --- GCATTGGCAAGAGTTTCA---3 '
FAA-R-4:5 ' --- AGGCAAGTCATCCGTTTT---3 '
FAA-F-5:5 ' --- GCATTTGGACTATTGCCTCA---3 '
FAA-R-7:5 ' --- CGTCCTATGTCGTAGAGTAGCG---3 '.
2. the molecular labeling side of fragrant Na needle mushroom new strains (fragrant Na needle mushroom Flammulina fennae HMGIM-A151357)
Method, which is characterized in that PCR amplification is carried out using primer pair FAA-F-3 and FAA-R-4 or FAA-F-5 and FAA-R-7, obtains spy
Specific amplification band is accredited as fragrant Na needle mushroom new strains (fragrant Na needle mushroom Flammulina fennae HMGIM-
A151357)。
3. molecule labelling method according to claim 2, which is characterized in that the molecule labelling method further comprises adopting
The PCR amplification of common needle mushroom is carried out with primer pair FAA-F-3 and FAA-R-4 or FAA-F-5 and FAA-R-7, is shown without amplification
Band shows that the identification of fragrant Na needle mushroom new strains (fragrant Na needle mushroom Flammulina fennae HMGIM-A151357) is special
It is anisotropic.
4. molecule labelling method according to claim 2 or 3, which is characterized in that the molecule labelling method further wraps
It includes and ITS1/ITS4 is used to carry out control amplification for primer pair, amplify the validity that band shows PCR amplification system.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910856311.0A CN110408724A (en) | 2019-09-11 | 2019-09-11 | The primer and molecule labelling method of the molecular labeling of fragrant Na needle mushroom new strains |
| PCT/CN2019/119579 WO2021047032A1 (en) | 2019-09-11 | 2019-11-20 | Primers for molecularly labeling new strain of flammulina fennae and molecular labeling method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910856311.0A CN110408724A (en) | 2019-09-11 | 2019-09-11 | The primer and molecule labelling method of the molecular labeling of fragrant Na needle mushroom new strains |
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| Publication Number | Publication Date |
|---|---|
| CN110408724A true CN110408724A (en) | 2019-11-05 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111518708A (en) * | 2020-01-03 | 2020-08-11 | 福建农林大学 | Yellow needle mushroom strain nong jin 49 not easy to open and molecular marker identification method thereof |
| WO2021047031A1 (en) * | 2019-09-11 | 2021-03-18 | 广东省微生物研究所（广东省微生物分析检测中心） | Novel flammulina fennae strain as well as molecular marker primer and molecular marking method thereof |
| WO2021047032A1 (en) * | 2019-09-11 | 2021-03-18 | 广东省微生物研究所（广东省微生物分析检测中心） | Primers for molecularly labeling new strain of flammulina fennae and molecular labeling method |
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| CN111518708A (en) * | 2020-01-03 | 2020-08-11 | 福建农林大学 | Yellow needle mushroom strain nong jin 49 not easy to open and molecular marker identification method thereof |
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