CN110408717A - The specific amplification primer of Ganoderma mitochondria rns gene and its application - Google Patents
The specific amplification primer of Ganoderma mitochondria rns gene and its application Download PDFInfo
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- CN110408717A CN110408717A CN201910663597.0A CN201910663597A CN110408717A CN 110408717 A CN110408717 A CN 110408717A CN 201910663597 A CN201910663597 A CN 201910663597A CN 110408717 A CN110408717 A CN 110408717A
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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
- C12Q1/6869—Methods for sequencing
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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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- G—PHYSICS
- G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
- G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
- G16B10/00—ICT specially adapted for evolutionary bioinformatics, e.g. phylogenetic tree construction or analysis
Abstract
The invention discloses a kind of specificity amplification primer of Ganoderma mitochondria rns gene and its applications, are related to genetic evolution field.The primer includes upstream primer and downstream primer, and upstream primer nucleotide sequence is as shown in SEQ ID NO.1, and downstream primer nucleotide sequence is as shown in SEQ ID NO.2.The primer disposably, in large quantity can carry out specific amplified to ganoderma lucidum species, the gene interference of animal in sample or environment, plant, microorganism is effectively excluded, provides great convenience for the monitoring of Ganoderma species identification, breed breeding, Phylogenetic Analysis and strain.Specific amplification primer amplified production size provided by the invention is 650-750bp, existing enough genetic locus are identified for species differentiation, ensure that a sequencing reaction can survey full sequence, and does not have to be separately separated and extract species mitochondria, with easy to operate, save the cost, advantage easy to use.
Description
Technical field
The present invention relates to genetic evolution fields, specifically relate to, and the specific amplified for being related to Ganoderma mitochondria rns gene draws
Object and its application.
Background technique
Mitochondria is Eukaryotic energy plants, provides the source energy (ATP) for major part eucaryote.Mitochondria contains
Have the genome of oneself, it was reported that it is derived by Endosymbiotic bacterium.Chondriogen is due to relatively small size, list
Close heredity, independently of the evolutionary rate of karyogene, and a large amount of advantages such as homologous gene and molecular labeling have become grind at present
Study carefully the powerful of systematic growth and species taxonomy.
Ganoderma Lucidum (Amanita) be mycota, Basidiomycota, agaric guiding principle, Agaricales, ganoderma lucidum Cordycepps, one in ganoderma lucidum Pseudomonas
The general name of class fungi is a kind of important medicinal fungi.It is reported that the fructification of ganoderma lucidum have invigorating qi for tranquilization, it is relieving cough and asthma, prolong
The effect of year lengthens one's life, can be used for treating dizziness egersis, shortness of breath and palpitation, neurasthenia, consumptive disease cough and asthma.Ganoderma lucidum is medicinal existing in China
More than 2000 years history, the magical treasure that strengthening by means of tonics is considered as by successive dynasties medicine man, is strengthened the body resistance to consolidate the constitution.Modern medicine study shows
Ganoderma lucidum polysaccharide is one of the main active constituent in ganoderma lucidum, is lost to neurasthenia, hyperlipidemia, coronary disease and angina pectoris, the rhythm of the heart
Often, hepatitis, indigestion, tracheitis etc. have different degrees of curative effect.The species diversity of Ganoderma is very rich, existing at present
Tens species are described in Ganoderma.And with the expansion of cultivation scale, lucidum strain name in the market is more chaotic,
The phenomenon that homonym, synonym, is very prominent, and which has limited the further development and utilization to ganoderma lucidum resource.
Summary of the invention
First invention purpose of the invention is: in view of the above problems, providing the special expansion of mitochondria rns gene
Increase primer, which disposably, in large quantity can carry out specific amplified to ganoderma lucidum species, effectively exclude sample or environment
The gene interference of middle animal, plant, microorganism provides pole for the monitoring of Ganoderma species identification, Phylogenetic Analysis and strain
Big convenience.
Second goal of the invention of the invention is, provides application of the above-mentioned primer in identification ganoderma lucidum species.
The technical solution adopted by the invention is as follows:
The specific amplification primer of Ganoderma mitochondria rns gene comprising upstream primer and downstream primer, upstream primer core
Nucleotide sequence is as shown in SEQ ID NO.1, and downstream primer nucleotide sequence is as shown in SEQ ID NO.2.
Application of the specific amplification primer of above-mentioned Ganoderma mitochondria rns gene in identification ganoderma lucidum species.
Application of the specific amplification primer of Ganoderma mitochondria rns gene of the invention in identification ganoderma lucidum species,
Include the following steps:
According to the mitochondria rns gene of known ganoderma lucidum species, determine in the conserved sequence in gene order and design
Swim primer and downstream primer；
With upstream primer and downstream primer PCR amplification Ganoderma mitochondria rns gene, if without non-specificity in PCR product
Amplification, then primer specificity is good；
With the mitochondria rns gene of upstream primer and downstream primer PCR amplification species to be measured, if special nothing but in PCR product
Specific amplification, then the species are ganoderma lucidum species.
Application of the specific amplification primer of Ganoderma mitochondria rns gene of the invention in identification ganoderma lucidum species, on
The amplified fragments between primer and downstream primer are swum in 400-800bp.
Application of the specific amplification primer of Ganoderma mitochondria rns gene of the invention in identification ganoderma lucidum species, PCR
When reaction, 30 μ L systems include:
Application of the specific amplification primer of Ganoderma mitochondria rns gene of the invention in identification ganoderma lucidum species, PCR
Program are as follows: 95 DEG C of 5min；94 DEG C of 45s, 56 DEG C of 45min, 72 DEG C of 30s, 35 circulations；72 DEG C of extension 10min.
Application of the specific amplification primer of Ganoderma mitochondria rns gene of the invention in identification ganoderma lucidum species, PCR
Product carries out electrophoresis detection with 1% Ago-Gel, if having on PCR product swimming lane and an only band, without non-specific expansion
Volume increase life.
Application of the specific amplification primer of Ganoderma mitochondria rns gene of the invention in identification ganoderma lucidum species, electricity
After swimming detection, if recycling PCR product without non-specific amplification, being sequenced after purification, sequencing result and known ganoderma lucidum species
Sequence in rns gene database is compared, phylogenetic tree construction, examines PCR product sequence and known Ganoderma object
The species of Sequence clustering together in kind rns gene database, if the species in species to be measured and database constitute single offspring,
Then species to be measured are accredited as the species.
In conclusion by adopting the above-described technical solution, the beneficial effects of the present invention are:
The present invention provides the specific amplification primers of Ganoderma mitochondria rns gene and its application, the primer can be primary
Property, in large quantity specific amplified is carried out to ganoderma lucidum species, effectively exclude animal in sample or environment, plant, microorganism
Gene interference provides great convenience for the monitoring of Ganoderma species identification, breed breeding, Phylogenetic Analysis and strain.This
The specific amplification primer amplified production size that invention provides is 650-750bp, and existing enough genetic locus are used for species differentiation
Identification ensures that a sequencing reaction can survey full sequence, and without from being wherein separately separated after extraction species total DNA
Species mitochondrial DNA is extracted, there is easy to operate, save the cost, advantage easy to use.
Detailed description of the invention
Examples of the present invention will be described by way of reference to the accompanying drawings, in which:
Fig. 1 is the electrophoresis result figure that primer specificity detects in the embodiment of the present invention 2；
Fig. 2 is the electrophoresis detection result figure of the pcr amplification product of 15 ganoderma lucidum species DNA in the embodiment of the present invention 3；
Fig. 3 is the phylogenetic tree constructed in the embodiment of the present invention 3.
Specific embodiment
All features disclosed in this specification or disclosed all methods or in the process the step of, in addition to mutually exclusive
Feature and/or step other than, can combine in any way.
Any feature disclosed in this specification (including any accessory claim, abstract), unless specifically stated,
It is replaced by other equivalent or with similar purpose alternative features.That is, unless specifically stated, each feature is a series of
An example in equivalent or similar characteristics.
Embodiment 1
The foundation and design of primers in Ganoderma rns gene order fragment data library.Download all Ganodermas delivered
Species mitochondria rns gene order establishes Ganoderma rns gene order fragment data library.Then 6.06 software pair of MEGA is utilized
Ganoderma rns sequence is compared, and obtains the conservative section of Ganoderma rns gene.Ganoderma rns base is designed according to conservative section
The universal primer of cause.The primer of design should meet: the expanding fragment length between upstream and downstream primer between 400-800bp,
In order to have enough genetic locus for species differentiation identify, ensure that a sequencing reaction can survey full sequence,
Extract species total DNA after do not have to from be wherein separately separated extract species mitochondrial DNA, easy to operate, save the cost.
In the present embodiment, 5 pairs of primers are constructed altogether for further screening.By analyzing primer sequence, arrange
Except inside is likely to occur the primer of secondary structure；It is compared simultaneously, guarantees primer sequence and Ganoderma rns gene order
Other sequences obtain 1 pair of optimal primer sequence, forward primer to finally screen without obvious homology in fragment data library
For SEQ ID NO.1:5 '-GGGGATAAGGTGTAGAGGTGAG-3 ', reverse primer is SEQ ID NO.2:5 '-
ATGAATTCACTAAATCCAATA-3’。
Embodiment 2
Primer specificity and amplification method validation.The present embodiment is by the optimization experiment of multiple single factor tests, finally
The PCR amplification condition and reaction system of Ganoderma rns gene has been determined.
Wherein, best PCR amplification condition are as follows: 95 DEG C of 5min；94 DEG C of 45s, 56 DEG C of 45s, 72 DEG C of 30s, 35 circulations；72℃
Extend 10min.
30 μ L systems include: 2 × PCR Mix, 15 μ L, 1 μ L of DNA profiling, upstream and downstream primer each 1 μ L, ddH2O polishing is extremely
30μL。
Carry out PCR amplification with best PCR amplification condition provided in this embodiment and system, with verify primer specificity and
The validity of method.Experimental method is as follows:
Known 5 kinds of ganoderma lucidum species: red sesame, Ganoderma tropicum, Ganoderma tsugae, plain boiled pork ganoderma lucidum, purple sesame are taken, first by each object
The fructification of kind is fully ground with liquid nitrogen, then utilizes OMEGA fungal DNA extraction kits (E.Z.N.A.TM Fungal DNA
Kit, the U.S.) total DNA is extracted, extracting method repeats no more herein as shown in the kit specification.
The total DNA of extraction is purified (D1300, Beijing Suo Laibao Science and Technology Ltd) using DNA purification kit, pure
Change method is as described in the kit specification.Then using total DNA as template (1 μ L), above-mentioned PCR reaction condition and reaction are utilized
System is expanded, and amplified production detects (110v, 30min) with 1% agarose gel electrophoresis, as a result as shown in Fig. 1.By Fig. 1
It is found that the test sample of 1-5 swimming lane is respectively the PCR product of ganoderma lucidum species DNA in above-mentioned 5 in Fig. 1.As shown in Figure 1,5 kinds
Ganoderma species all obtain single band.It can be seen that the primer specificity that embodiment 1 provides is good, the present embodiment provides
PCR amplification condition and system it is scientific and effective.
Embodiment 3
The PCR amplification condition and system that the primer and embodiment 2 that the present embodiment is provided using embodiment 1 provide identify object
Kind, further foundation is provided for primer provided by the invention and its application.
In the present embodiment, for the specificity of further verifying primer, to 15 ganoderma lucidum species, 3 sides in the present embodiment
Ear species, 4 russule species and 9 ascus fungus kinds have carried out rns gene magnification.Specifically comprise the following steps:
First to 15 ganoderma lucidum species (be known as Ganoderma, but do not know it is specific why species) and 16 non-Ganoderma objects
Kind carries out the extraction of DNA respectively.First each species fructification is fully ground respectively using liquid nitrogen, it is then true using OMEGA
Bacterium DNA extraction kit (E.Z.N.A.TM Fungal DNA Kit, the U.S.) extracts total DNA.Using total DNA as template (1 μ L),
It is expanded using the PCR reaction condition and reaction system that are provided in embodiment 2,1% agarose gel electrophoresis of amplified production
It detects (110v, 25min).
Electrophoresis result is as shown in Fig. 2, the test sample of 1-15 swimming lane is respectively that the PCR of 15 ganoderma lucidum species is produced in Fig. 2
Object.As shown in Figure 2, the swimming lane of 15 ganoderma lucidum species obtains single band, and size is between 400-800bp.And it picks up the ears
Category, Russula and sac fungus species species do not obtain amplified band (not shown), have further demonstrated that the primer has object
Species specificity.
Ganoderma pcr amplification product is purified using PCR purification kit (D0033, the green skies), purified product into
Row sequencing, using MEGA software to the sequence construct phylogenetic tree of acquisition, constructed development tree is as shown in Figure 3.It can by Fig. 3
Know, 15 in the present embodiment ganoderma lucidum species are respectively: red sesame, Ganoderma tropicum, ganoderma lucidum, Ganoderma tsugae, Sichuan ganoderma lucidum, dim light
Ganoderma lucidum, curved handle ganoderma lucidum, rubber ganoderma lucidum, plain boiled pork ganoderma lucidum, purple light ganoderma lucidum, band-like ganoderma lucidum, weber ganoderma lucidum, ganoderma tenue, ganoderma lipsiense,
Purple sesame.The close species of Phylogenetic Relationships have closer affiliation, the result and Ganoderma morphology and molecule of this research
The result of classification is almost the same, shows that rns gene can be used as the effective molecule mark for analyzing species Phylogenetic Relationships of picking up the ears
Note.
To sum up, the specific amplification primer of Ganoderma mitochondria rns gene provided by the invention, can disposably, it is large quantities of
Amount ground carries out specific amplified to environmental samples or ganoderma lucidum species, is Ganoderma species identification, breed breeding, Phylogenetic Analysis
And strain monitoring provides great convenience, can effectively exclude the gene interference of animal in sample or environment, plant, microorganism；
The specific amplification primer amplified production size is 650-750bp, and existing enough genetic locus are identified for species differentiation, and can
Guarantee that a sequencing reaction can survey full sequence, and do not have to be separately separated and extract species mitochondria, there is easy to operate, section
About cost, advantage easy to use.
The invention is not limited to specific embodiments above-mentioned.The present invention, which expands to, any in the present specification to be disclosed
New feature or any new combination, and disclose any new method or process the step of or any new combination.
SEQUENCE LISTING
<110>Institute of Nuclear and Biotechnology, Sichuan Academy of Agriculture Science
<120>specific amplification primer of Ganoderma mitochondria rns gene and its application
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 22
<212> DNA
<213>artificial sequence
<400> 1
ggggataagg tgtagaggtg ag 22
<210> 2
<211> 21
<212> DNA
<213>artificial sequence
<400> 2
atgaattcac taaatccaat a 21
Claims (8)
1. the specific amplification primer of Ganoderma mitochondria rns gene, which is characterized in that it includes upstream primer and downstream primer,
Upstream primer nucleotide sequence is as shown in SEQ ID NO.1, and downstream primer nucleotide sequence is as shown in SEQ ID NO.2.
2. the specific amplification primer of Ganoderma mitochondria rns gene described in claim 1 answering in identification ganoderma lucidum species
With.
3. application according to claim 2, which is characterized in that it includes the following steps:
According to the mitochondria rns gene of known ganoderma lucidum species, determines the conserved sequence in gene order and design upstream and draw
Object and downstream primer；
With upstream primer and downstream primer PCR amplification Ganoderma mitochondria rns gene, if without non-specific amplification in PCR product,
Then primer specificity is good；
With the mitochondria rns gene of upstream primer and downstream primer PCR amplification species to be measured, if without non-specificity in PCR product
Amplification, then the species are ganoderma lucidum species.
4. application according to claim 3, which is characterized in that the amplified fragments between upstream primer and downstream primer exist
400-800bp。
5. application according to claim 4, which is characterized in that when PCR reacts, 30 μ L systems include:
6. application according to claim 5, which is characterized in that PCR program are as follows: 95 DEG C of 5min；94 DEG C of 45s, 56 DEG C of 45s；
72 DEG C of 30s, 35 circulations；72 DEG C of extension 10min.
7. application according to claim 6, which is characterized in that PCR product carries out electrophoresis inspection with 1% Ago-Gel
It surveys, if having on PCR product swimming lane and an only band, no non-specific amplification is generated.
8. application according to claim 7, which is characterized in that after electrophoresis detection, if recycling PCR without non-specific amplification
Product is sequenced after purification, and sequencing result is compared with the sequence in known ganoderma lucidum species rns gene database, building
Phylogenetic tree examines object of the PCR product sequence together with Sequence clustering in known ganoderma lucidum species rns gene database
Kind, if the species in species to be measured and database constitute single offspring, species to be measured are accredited as the species.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110358856A (en) * | 2019-07-23 | 2019-10-22 | 四川省农业科学院生物技术核技术研究所 | It the amplimer of Pleurotus mitochondria nad4L gene and its picks up the ears the application in species in identification |
| CN112980998A (en) * | 2021-04-27 | 2021-06-18 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Molecular marker, specific primer and identification method of high-quality ganoderma leucocontextum strain I140033 |
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| CN112980998A (en) * | 2021-04-27 | 2021-06-18 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Molecular marker, specific primer and identification method of high-quality ganoderma leucocontextum strain I140033 |
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