CN106282396B - Identify the method and primer pair of terraced rib hickory chick mating type - Google Patents
Identify the method and primer pair of terraced rib hickory chick mating type Download PDFInfo
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Abstract
The present invention relates to a kind of methods and primer pair for identifying terraced rib hickory chick mating type, belong to biology techniques field.The primer pair includes detecting the primer pair P6-1F/P6-1R of the primer pair P8-4F/P8-4R and detection mating type MAT1-2 of mating type MAT1-1.This method is to extract bacterial strain DNA, carries out PCR amplification with two pairs of primer pairs respectively, if electrophoresis detection all obtains expected band, it is effective cultivated strains that bacterial strain, which includes two kinds of mating type genes,；If being only able to detect a kind of amplified band, bacterial strain only has a kind of mating type, it is impossible to be used in cultivation.Testing result high specificity of the invention, it is reliable and stable quick, suitable for the research in terms of hickory chick strain improvement, cultivation, mating type gene and systematic growth.
Description
Technical field
The invention belongs to biology techniques fields, and in particular to a kind of terraced rib hickory chick of identification (Morchella importuna) mating type method and primer pair.The technology of the present invention is suitable for hickory chick strain improvement, cultivation, mating type
Research in terms of gene and systematic growth.
Background technique
The zoogamy of sac fungus it is now recognized that controlled by single mating site MAT1, this mating site include with
The MAT1-1 mating type that MAT1-1-1 gene is characterized and the MAT1-2 mating type characterized by MAT1-2-1 gene.It is of the same clan to hand over
The sac fungus self-fertility matched, and the sac fungus self-sterility of different ancestor mating, it is necessary to by being respectively provided with mating type MAT1-1 and friendship
Zoogamy could be completed after the stud mating of distribution type MAT1-2.
Terraced rib hickory chick is a kind of higher delicious edible mushroom of economic value, and current wild dry product price is in 2000 yuan/public affairs
Jin or so.In recent years, the artificial cultivation of terraced rib hickory chick succeeded and rose upsurge, many numerous and confused large area in place in the whole nation
Commercial growth, but not fruiting the case where frequent occurrence, even occur 1 hickory chick of entire greenhouse or field sometimes and do not go out,
It traces it to its cause in addition to climatic environmental factor, most important is exactly strain problem, it is ensured that strain is reliably primary key problem in technology.
We obtain itself MAT1-1-1 and MAT1-2-1 full length gene sequence, pass through to terraced rib hickory chick gene order-checking
List cystospore mating type proportion grading simultaneously combines mating site structure analysis, it was demonstrated that terraced rib hickory chick is that different ancestor's mating type is true
Bacterium, sexual reproduction (fruiting) need two kinds of distribution type individual combinations that could complete.The spore separation and tissue separation of hickory chick
It is likely to have to a kind of bacterial strain of mating type, and also will appear what a kind of mating type was lost in the Subculture of strain
Phenomenon.It therefore, is the key that obtain effective cultivated strains to the detection of strain mating type.
Summary of the invention
It is an object of the present invention to solve the deficiency of the existing technology and provide a kind of sides for identifying terraced rib hickory chick mating type
Method and primer pair, this method are to identify terraced rib sheep tripe by the presence or absence of band after two pairs of primer pair PCR amplifications of detection
The mating type of bacteria strain determines whether as effective cultivated strains, and provides reliable bacterial strain to be selected for crossbreeding.
To achieve the above object, The technical solution adopted by the invention is as follows:
The method for identifying terraced rib hickory chick mating type, including the primer pair P8-4F/ for detecting mating type MAT1-1
P8-4R, for detecting the primer pair P6-1F/P6-1R of mating type MAT1-2, corresponding PCR amplification system and amplification journey
Sequence；
The P8-4F/P8-4R special primer centering:
The nucleotides sequence of P8-4F is classified as 5 '-GCTCTCTTGTGCCCCTTTTGACTAT-3 '；(SEQ ID No.1)
The nucleotides sequence of P8-4R is classified as 5 '-TCTACCAGCCATGTGAAACAAGCAA-3 '；(SEQ ID No.2)
The P6-1F/P6-1R special primer centering:
The nucleotides sequence of P6-1F is classified as 5 '-GAGACTCAAATCTGACTGACTTCCT-3 ' (SEQ ID No.3)
The nucleotides sequence of P6-1R is classified as 5 '-GAAGAACCTCAGATAAGCGTAAAAT-3 '.(SEQ ID No.4)
It is further preferred that identification mating type MAT1-1 and MAT1-2 PCR amplification system it is identical, include 2 ×
Each 1 μ L of forward and reverse primer, DNA profiling 10 ng, ddH of PCRmix 12.5 μ L, 10 μM/L2O complements to 25 μ L.
It is further preferred that the PCR amplification program of identification of M AT1-1 mating type are as follows: 94 DEG C of 3 min of initial denaturation；94℃
30 sec are denaturalized, 60 DEG C of 30 sec of annealing, 72 DEG C of 90 sec of extension, 30 recycle；72 DEG C of 10 min of extension；
The PCR amplification program of identification of M AT1-2 mating type are as follows: 94 DEG C of 3 min of initial denaturation；94 DEG C of 30 sec of denaturation, 58 DEG C are moved back
30 sec of fire, 72 DEG C of 90 sec of extension, 30 recycle；72 DEG C of 10 min of extension.
When detecting the MAT1-1 mating type of terraced rib hickory chick bacterial strain, can obtain size is the unique band of 1837bp, and should
Band contains MAT1-1-1 full-length gene；When detecting the MAT1-2 mating type of terraced rib hickory chick bacterial strain, can obtain size is
The unique band of 1744bp, and the band contains MAT1-2-1 full-length gene.
The present invention provides the PCR primer pair for identifying terraced rib hickory chick mating type, including for detecting mating type MAT1-1's
Primer pair P8-4F/P8-4R and primer pair P6-1F/P6-1R for detecting mating type MAT1-2.
The reagent and kit of the terraced rib hickory chick mating type of identification of above-mentioned PCR primer pair is also claimed in the present invention.
The present invention also provides above-mentioned PCR primer pair, reagent or kits to identify answering in terraced rib hickory chick mating type
With.
Present invention simultaneously provides above-mentioned PCR primer pair, reagent or kits in the application for identifying terraced rib hickory chick breeding.
In the present invention, the unique band of 1837bp or so is amplified with primer pair P8-4F/P8-4R, shows that bacterial strain contains
MAT1-1 mating type；The unique band of 1744bp or so is amplified with primer pair P6-1F/P6-1R, shows that bacterial strain is handed over containing MAT1-2
Distribution type；Two pairs of primer pair PCR amplifications can detect the bacterial strain of expected band, and the mating type containing there are two types of can be used as effective cultivation
Cultivation strain, and only with the bacterial strain of single mating type, it is impossible to be used in cultivation can be used as the alternative bacterial strain of crossbreeding.
The present invention is based on the features for two mating gene orders that gene order-checking obtains, and devise two pairs of special primers,
It is respectively used to the detection of terraced rib hickory chick bacterial strain MAT1-1 and MAT1-2 mating type.Amplified band is single band, and band has
It is as a result reliable and stable without the mating type that can determine bacterial strain.
The mating gene order and detection method of ladder rib hickory chick involved in the present invention are not reported both at home and abroad at present.
Primer through the invention, the amplifiable full length sequence for obtaining MAT1-1-1 and MAT1-2-1 gene, for the sexual of hickory chick
Reproduction and phyletic evolution research are all significant.
Compared with prior art, the present invention has the advantages that:
Terraced rib hickory chick MAT1-1-1 and MAT1-2-1 gene order all has not been reported both at home and abroad at present, using of the invention
Two pairs of primer pairs can expand to obtain the full length sequence of the two genes, not only can identify ladder by the presence or absence of amplified band
The mating type of rib hickory chick bacterial strain, and can be used for the research in terms of further zoogamy and phyletic evolution.Terraced rib sheep tripe
In bacteria cultivation, since strain problem brings direct economic loss per acre at 4000 yuan or more.It, can be with by the application of this technology
Whether be effective cultivated strains, the testing cost of each sample only needs 50 yuan or so, thus bring if being identified in 4 hours
Social benefit is obvious.
Detailed description of the invention
Fig. 1 is that ladder rib hickory chick YPL6 list cystospore mass mating type MAT1-1 detects electrophoretogram: M represents Marker-
Band in DL2000(figure from top to bottom is followed successively by 2000bp, 1000bp, 750bp, 500bp)；1-24 represents terraced rib hickory chick
List cystospore bacterial strain YPL6-1-YPL6-24.
Fig. 2 is that ladder rib hickory chick YPL6 list cystospore mass mating type MAT1-2 detects electrophoretogram: M represents Marker-
Band in DL2000(figure from top to bottom is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp)；1-24 generation
Table ladder rib hickory chick list cystospore bacterial strain YPL6-1-YPL6-24.
Fig. 3 is that the terraced effective cultivated strains of rib hickory chick detect electrophoretogram: M is represented in Marker-DL2000(figure from top to bottom
Band be followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp)；1,2,3,4 be respectively bacterial strain SP1, SP2, YPL6,
YPL7 detects MAT1-1 mating type；5,6,7,8 be bacterial strain SP1, SP2, YPL6, YPL7 detection MAT1-2 mating type respectively.
Specific embodiment
Below with reference to embodiment, the present invention is described in further detail.
It will be understood to those of skill in the art that the following example is merely to illustrate the present invention, and it should not be regarded as limiting this hair
Bright range.In the examples where no specific technique or condition is specified, described technology or conditions according to the literature in the art
Or it is carried out according to product description.Reagents or instruments used without specified manufacturer is that can be obtained by purchase
Conventional products.
The terraced rib hickory chick YPL6 list cystospore mass mating type identification of embodiment 1
Terraced rib hickory chick YPL6 list cystospore group bacterial strain YPL6-1-YPL6-24 is seeded in PDA culture medium, and 23 DEG C
10d, the appropriate mycelium of picking are cultivated, CTAB method extracts DNA, takes 3 μ L in 1%(W/V) agarose gel electrophoresis detection DNA mass
And concentration.
Respectively using the DNA of YPL6-1-YPL6-24 as template, PCR amplification, 25 μ L are carried out with primer pair P8-4F/P8-4R
P8-4F, P8-4R primer each 1 μ of the amplification system including 2 × PCRmix(TSINGKE Bio Inc), 12.5 μ L, 10 μM/L
L, DNA profiling 1 μ L, ddH2O 9.5 µL.PCR amplification program are as follows: 94 DEG C of 3 min of initial denaturation；94 DEG C of denaturation 30 sec, 60 DEG C
Anneal 30 sec, 72 DEG C of 90 sec of extension, 30 circulations；72 DEG C of 10 min of extension.
Respectively using the DNA of YPL6-1-YPL6-24 as template, PCR amplification, 25 μ L are carried out with P6-1F/P6-1R primer pair
P6-1F, P6-1R primer each 1 μ of the amplification system including 2 × PCRmix(TSINGKE Bio Inc), 12.5 μ L, 10 μM/L
L, DNA profiling 1 μ L, ddH2O 9.5µL.PCR amplification program are as follows: 94 DEG C of 3 min of initial denaturation；94 DEG C of denaturation 30 sec, 58 DEG C
Anneal 30 sec, 72 DEG C of 90 sec of extension, 30 circulations；72 DEG C of 10 min of extension.
All amplified productions are in 1.2%(W/V) electrophoresis detection, the result is shown in Figure 1 and Fig. 2 on Ago-Gel, bacterial strain YPL6-
2、YPL6-3、YPL6-5、YPL6-7、YPL6-9、 YPL6-10、YPL6-11、YPL6-17 、YPL6-20、YPL6-21、YPL6-
23 have a unique band of 1.8kb or so when primer pair P8-4F/P8-4R is expanded, and with when P6-1F/P6-1R primer pair amplifies without
Band is accredited as mating type MAT1-1 bacterial strain.Bacterial strain YPL6-1, YPL6-4, YPL6-6, YPL6-8, YPL6-12, YPL6-13,
YPL6-14, YPL6-15, YPL6-16, YPL6-18, YPL6-19, YPL6-22, YPL6-24 expand in primer pair P8-4F/P8-4R
Without band when increasing, and there is the unique band of 1.7kb or so when primer pair P6-1F/P6-1R amplification, is accredited as mating type MAT1-2 bacterium
Strain.
The identification of the effective cultivated strains of the terraced rib hickory chick of embodiment 2
Strain to be tested is terraced rib hickory chick bacterial strain SP1, SP2, YPL6, YPL7, strain culturing, DNA extraction, PCR amplification body
System and amplification program are the same as embodiment 1.
As a result amplified production electrophoresis detection on 1.2% (W/V) Ago-Gel is shown in that Fig. 3, SP2, YPL6, YPL7 bacterial strain are used
P8-4F/P8-4R, P6-1F/P6-1R primer pair PCR amplification have band, and it is effective cultivation bacterium that illustrating tool, there are two types of mating types
Strain.And without band when SP1 bacterial strain P8-4F/P8-4R primer pair amplifies, P6-1F/P6-1R primer pair amplifies have band, are
MAT1-2 mating type bacterial strain, cannot function as cultivated strains.Fruiting after bacterial strain YPL6, YPL7 cultivation.
The basic principles, main features and advantages of the present invention have been shown and described above.The technology of the industry
Personnel are it should be appreciated that the present invention is not limited to the above embodiments, and the above embodiments and description only describe this
The principle of invention, without departing from the spirit and scope of the present invention, various changes and improvements may be made to the invention, these changes
Change and improvement all fall within the protetion scope of the claimed invention.The claimed scope of the invention by appended claims and its
Equivalent thereof.
Sequence table
SEQ ID No.1
GCTCTCTTGT GCCCCTTTTG ACTAT 25
SEQ ID No.2
TCTACCAGCC ATGTGAAACA AGCAA 25
SEQ ID No.3
GAGACTCAAA TCTGACTGAC TTCCT 25
SEQ ID No.4
GAAGAACCTC AGATAAGCGT AAAAT 25
SEQUENCE LISTING
<110>KUNMING INST OF BOTANY CAS
<120>method and primer pair of terraced rib hickory chick mating type are identified
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 25
<212> DNA
<213>artificial sequence
<400> 1
GCTCTCTTGT GCCCCTTTTG ACTAT 25
<210> 2
<211> 25
<212> DNA
<213>artificial sequence
<400> 3
TCTACCAGCC ATGTGAAACA AGCAA 25
<210> 3
<211> 25
<212> DNA
<213>artificial sequence
<400> 3
GAGACTCAAA TCTGACTGAC TTCCT 25
<210> 4
<211> 25
<212> DNA
<213>artificial sequence
<400> 4
GAAGAACCTC AGATAAGCGT AAAAT 25
Claims (9)
1. the method for identifying terraced rib hickory chick mating type, which is characterized in that including for detecting specifically drawing for mating type MAT1-1
Object is to P8-4F/P8-4R, for detecting the primer pair P6-1F/P6-1R of mating type MAT1-2, corresponding PCR amplification system
And amplification program；
The P8-4F/P8-4R special primer centering, the nucleotides sequence of P8-4F are classified as 5 '-
The nucleotides sequence of GCTCTCTTGTGCCCCTTTTGACTAT-3 ', P8-4R are classified as 5 '-
TCTACCAGCCATGTGAAACAAGCAA-3' ；
The P6-1F/P6-1R special primer centering, the nucleotides sequence of P6-1F are classified as 5 '-
The nucleotides sequence of GAGACTCAAATCTGACTGACTTCCT-3 ', P6-1R are classified as 5 '-
GAAGAACCTCAGATAAGCGTAAAAT-3’。
2. the method according to claim 1 for identifying terraced rib hickory chick mating type, which is characterized in that identification mating type
The PCR amplification system of MAT1-1 and MAT1-2 is identical, includes forward and reverse primer of 2 × PCRmix 12.5 μ L, 10 μM/L
Each 1 μ L, DNA profiling 10 ng, ddH2O complements to 25 μ L.
3. the method according to claim 2 for identifying terraced rib hickory chick mating type, which is characterized in that identification of M AT1-1 mating
The PCR amplification program of type are as follows: 94 DEG C of 3 min of initial denaturation；94 DEG C of 30 sec of denaturation, 60 DEG C of 30 sec of annealing, 72 DEG C extend 90
Sec, 30 circulations；72 DEG C of 10 min of extension；
The PCR amplification program of identification of M AT1-2 mating type are as follows: 94 DEG C of 3 min of initial denaturation；94 DEG C of 30 sec of denaturation, 58 DEG C of annealing 30
Sec, 72 DEG C of 90 sec of extension, 30 circulations；72 DEG C of 10 min of extension.
4. the method according to claim 3 for identifying terraced rib hickory chick mating type, which is characterized in that detect terraced rib hickory chick
When the MAT1-1 mating type of bacterial strain, can obtain size is the unique band of 1837bp, and the band contains MAT1-1-1 overall length
Gene；When detecting the MAT1-2 mating type of terraced rib hickory chick bacterial strain, size can be obtained as the unique band of 1744bp, and the band
Contain MAT1-2-1 full-length gene.
5. identifying the PCR primer pair of terraced rib hickory chick mating type, which is characterized in that including the spy for detecting mating type MAT1-1
Different primer pair P8-4F/P8-4R and primer pair P6-1F/P6-1R for detecting mating type MAT1-2；
The P8-4F/P8-4R special primer centering, the nucleotides sequence of P8-4F are classified as 5 '-
The nucleotides sequence of GCTCTCTTGTGCCCCTTTTGACTAT-3 ', P8-4R are classified as 5 '-
TCTACCAGCCATGTGAAACAAGCAA-3' ；
The P6-1F/P6-1R special primer centering, the nucleotides sequence of P6-1F are classified as 5 '-
The nucleotides sequence of GAGACTCAAATCTGACTGACTTCCT-3 ', P6-1R are classified as 5 '-
GAAGAACCTCAGATAAGCGTAAAAT-3’。
6. the reagent of the terraced rib hickory chick mating type of the identification containing the PCR primer pair described in claim 5.
7. the kit of the terraced rib hickory chick mating type of the identification containing the PCR primer pair described in claim 5.
8. PCR primer pair, reagent as claimed in claim 6 described in claim 5 or kit as claimed in claim 7 exist
Identify the application in terraced rib hickory chick mating type.
9. PCR primer pair, reagent as claimed in claim 6 described in claim 5 or kit as claimed in claim 7 exist
Identify the application of terraced rib hickory chick breeding.
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| CN107151698B (en) * | 2017-04-01 | 2020-10-02 | 中国科学院昆明植物研究所 | Method for identifying mating types of eleven black morchella flora |
| CN111500759A (en) * | 2020-05-07 | 2020-08-07 | 云南菌视界生物科技有限公司 | Method and primer pair for identifying mating type genes of commercially-cultured morchella species |
| CN116064749A (en) * | 2022-08-19 | 2023-05-05 | 河南省科学院生物研究所有限责任公司 | Mating type separation method of Morchella mycelium |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101649350A (en) * | 2009-06-01 | 2010-02-17 | 中国农业大学 | Molecular detection method for rapidly distinguishing mating types of Chinese caterpillar fungus |
| CN103184280A (en) * | 2013-02-04 | 2013-07-03 | 北京市农林科学院 | Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-818 thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101649350A (en) * | 2009-06-01 | 2010-02-17 | 中国农业大学 | Molecular detection method for rapidly distinguishing mating types of Chinese caterpillar fungus |
| CN103184280A (en) * | 2013-02-04 | 2013-07-03 | 北京市农林科学院 | Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-818 thereof |
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