CN106282396A - Identify method and the special primer pair of ladder rib Morchella esculenta (L.) Pers mating type - Google Patents
Identify method and the special primer pair of ladder rib Morchella esculenta (L.) Pers mating type Download PDFInfo
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- CN106282396A CN106282396A CN201610975616.XA CN201610975616A CN106282396A CN 106282396 A CN106282396 A CN 106282396A CN 201610975616 A CN201610975616 A CN 201610975616A CN 106282396 A CN106282396 A CN 106282396A
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Abstract
The present invention relates to a kind of method identifying ladder rib Morchella esculenta (L.) Pers mating type and special primer pair, belong to biology techniques field.Described primer is to including that the special primer detecting mating type MAT1 1 special primer to P8 4F/P8 4R and detection mating type MAT1 2 is to P6 1F/P6 1R.The method for extract bacterial strain DNA, respectively with two to primer to carrying out PCR amplification, if electrophoresis detection all obtain expection band, then bacterial strain comprises two kinds of mating type genes, is effective cultivated strains；If being only able to detect a kind of amplified band, then a kind of mating type of bacterial strain, it is impossible to be used for cultivating.The testing result high specificity of the present invention, reliable and stable quickly, it is adaptable to the research in terms of Morchella esculenta (L.) Pers strain improvement, cultivation, mating type gene and phylogeny.
Description
Technical field
The invention belongs to biology techniques field, be specifically related to a kind of identify ladder rib Morchella esculenta (L.) Pers (Morchella importuna) method of mating type and special primer pair.The technology of the present invention is applicable to Morchella esculenta (L.) Pers strain improvement, cultivation, mating type
Research in terms of gene and phylogeny.
Background technology
Ascomycetous syngenesis it is now recognized that by single copulation site MAT1 control, this copulation site include with
MAT1-1 mating type that MAT1-1-1 gene is characterized and the MAT1-2 mating type being characterized with MAT1-2-1 gene.Friendship of the same clan
The ascomycetes self-fertility joined, and the ascomycetes self-sterility of different ancestor's copulation, it is necessary to by being respectively provided with mating type MAT1-1 and friendship
Syngenesis just can be completed after the stud mating of distribution type MAT1-2.
Ladder rib Morchella esculenta (L.) Pers is the delicious edible fungi that a kind of economic worth is higher, and current wild dry product price is 2000 yuan/public affairs
About Jin.In recent years, the artificial culture of ladder rib Morchella esculenta (L.) Pers succeeded and rose upsurge, a lot of local numerous and confused large area in the whole nation
Commercial growth, but the situation of fruiting does not often occur, and the most even occurs that whole booth or 1 Morchella esculenta (L.) Pers of field do not go out,
Tracing it to its cause in addition to climatic environmental factor, topmost is exactly strain problem, it is ensured that strain is reliably primary key problem in technology.
We are to ladder rib Morchella esculenta (L.) Pers gene order-checking, it is thus achieved that itself MAT1-1-1 and MAT1-2-1 full length gene sequence, pass through
List cystospore mating type proportion grading also combines copulation site structure analysis, it was demonstrated that ladder rib Morchella esculenta (L.) Pers is that different ancestor's mating type is true
Bacterium, sexual reproduction (fruiting) needs two kinds of distribution type individualities combinations just can complete.The spore separation of Morchella esculenta (L.) Pers and separate tissue
It is likely to have to the bacterial strain of a kind of mating type, and the Subculture of strain also there will be what a kind of mating type was lost
Phenomenon.Therefore, the detection to strain mating type is the key obtaining effective cultivated strains.
Summary of the invention
The invention aims to solve the deficiencies in the prior art, it is provided that a kind of side identifying ladder rib Morchella esculenta (L.) Pers mating type
Method and special primer pair, the method is by detecting two to special primer to the presence or absence of band after PCR amplification, identifying ladder rib Gaster caprae seu Ovis
The mating type of bacteria strain, determines whether as effective cultivated strains, and provides reliable bacterial strain to be selected for cross-breeding.
For achieving the above object, the technical solution used in the present invention is as follows:
The method identifying ladder rib Morchella esculenta (L.) Pers mating type, including the special primer for detecting mating type MAT1-1 to P8-4F/P8-
4R, for detecting the special primer of mating type MAT1-2 to P6-1F/P6-1R, corresponding PCR amplification system and amplification program；
Described 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)
Described 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 identify that mating type MAT1-1 is identical with the PCR amplification system of MAT1-2, all include 2 × PCRmix
Each 1 L of forward and reverse primer of 12.5 L, 10 M/L, DNA profiling 10 ng, ddH2O complements to 25 L.
It is further preferred that the PCR amplification program of identification of M AT1-1 mating type is: 94 DEG C of denaturation 3 min；94℃
Degeneration 30 sec, 60 DEG C of annealing 30 sec, 72 DEG C extend 90 sec, 30 circulations；72 DEG C extend 10 min；
The PCR amplification program of identification of M AT1-2 mating type is: 94 DEG C of denaturation 3 min；94 DEG C of degeneration 30 sec, 58 DEG C of annealing 30
Sec, 72 DEG C extend 90 sec, 30 circulations；72 DEG C extend 10 min.
When detecting the MAT1-1 mating type of ladder rib Morchella esculenta (L.) Pers bacterial strain, it is possible to 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 ladder rib Morchella esculenta (L.) Pers bacterial strain, it is possible to obtain size is
The unique band of 1744bp, and this band contains MAT1-2-1 full-length gene.
The present invention provides the PCR primer pair identifying ladder rib Morchella esculenta (L.) Pers mating type, including for detecting mating type MAT1-1
Special primer is to P8-4F/P8-4R with for detecting the special primer of mating type MAT1-2 to P6-1F/P6-1R.
The present invention is also claimed above-mentioned PCR primer reagent and test kit to identifying ladder rib Morchella esculenta (L.) Pers mating type.
The present invention also provide for above-mentioned PCR primer to, reagent or test kit in identifying ladder rib Morchella esculenta (L.) Pers mating type should
With.
Present invention simultaneously provides above-mentioned PCR primer to, reagent or test kit in the application identifying ladder rib Morchella esculenta (L.) Pers breeding.
In the present invention, with primer, P8-4F/P8-4R is amplified the unique band of about 1837bp, show that bacterial strain contains
MAT1-1 mating type；With primer, P6-1F/P6-1R is amplified the unique band of about 1744bp, show that bacterial strain contains MAT1-2 and hands over
Distribution type；Two pairs of primers can detect the bacterial strain of expection band to PCR amplification, containing two kinds of mating types, and can be as effectively planting
Cultivation strain, and only there is the bacterial strain of single mating type, it is impossible to it is used for cultivating, can be as the alternative bacterial strain of cross-breeding.
The feature of two copulation gene orders that the present invention obtains based on gene order-checking, devises two pairs of special primers,
It is respectively used to ladder rib Morchella esculenta (L.) Pers bacterial strain MAT1-1 and the detection of MAT1-2 mating type.Amplified band is single band, having of band
Without i.e. can determine that the mating type of bacterial strain, result is reliable and stable.
Copulation gene order and the detection method of terraced rib Morchella esculenta (L.) Pers involved in the present invention are not all reported at present both at home and abroad.
By the primer of the present invention, the amplifiable full length sequence obtaining MAT1-1-1 and MAT1-2-1 gene, sexual for Morchella esculenta (L.) Pers
Reproduction and phyletic evolution research are the most significant.
Compared with prior art, it has the beneficial effect that the present invention
Ladder rib Morchella esculenta (L.) Pers MAT1-1-1 and MAT1-2-1 gene order all have no report at present both at home and abroad, utilize two couple of the present invention
Primer pair, can expand the full length sequence obtaining the two gene, and the presence or absence not only by amplified band identifies ladder rib sheep
The mating type of tripe bacteria strain, and may be used for the research in terms of further syngenesis and phyletic evolution.Ladder rib Morchella esculenta (L.) Pers is planted
In training, owing to strain problem brings the direct economic loss of every mu more than 4000 yuan.By the application of this technology, can be 4
Whether be effective cultivated strains, the testing cost of each sample only needs about 50 yuan, the society thus brought if identifying in hour
Meeting benefit is obvious.
Accompanying drawing explanation
Fig. 1 is for ladder rib Morchella esculenta (L.) Pers YPL6 list cystospore mass mating type MAT1-1 detection electrophoretogram: M represents Marker-
In DL2000(figure, band from top to bottom is followed successively by 2000bp, 1000bp, 750bp, 500bp)；1-24 represents ladder rib Morchella esculenta (L.) Pers
List cystospore bacterial strain YPL6-1 YPL6-24.
Fig. 2 is for ladder rib Morchella esculenta (L.) Pers YPL6 list cystospore mass mating type MAT1-2 detection electrophoretogram: M represents Marker-
In DL2000(figure, band from top to bottom is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp)；1-24 generation
Table ladder rib Morchella esculenta (L.) Pers list cystospore bacterial strain YPL6-1 YPL6-24.
Fig. 3 represents in Marker-DL2000(figure from top to bottom for ladder rib Morchella esculenta (L.) Pers effective cultivated strains detection electrophoretogram: M
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 is that bacterial strain SP1, SP2, YPL6, YPL7 detect MAT1-2 mating type respectively.
Detailed description of the invention
Below in conjunction with 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 should not be regarded as limiting this
Bright scope.Unreceipted concrete technology or condition person in embodiment, according to the technology described by the document in this area or condition
Or carry out according to product description.Agents useful for same or instrument unreceipted production firm person, be and can be obtained by purchase
Conventional products.
Embodiment 1 ladder rib Morchella esculenta (L.) Pers YPL6 list cystospore mass mating type is identified
Ladder rib Morchella esculenta (L.) Pers YPL6 list cystospore colony bacterial strain YPL6-1 YPL6-24 is seeded in PDA culture medium, 23 DEG C of cultivations
10d, the appropriate mycelium of picking, CTAB method is extracted DNA, is taken 3 L at 1%(W/V) agarose gel electrophoresis detection DNA mass and dense
Degree.
Respectively with the DNA of YPL6-1 YPL6-24 as template, with primer, P8-4F/P8-4R carried out PCR amplification, 25 L
Amplification system includes 2 × PCRmix(TSINGKE Bio Inc) P8-4F, P8-4R primer each 1 of 12.5 L, 10 M/L
L, DNA profiling 1 L, ddH2O 9.5 µL.PCR amplification program is: 94 DEG C of denaturation 3 min；94 DEG C of degeneration 30 sec, 60 DEG C
Anneal 30 sec, and 72 DEG C extend 90 sec, 30 circulations；72 DEG C extend 10 min.
Respectively with the DNA of YPL6-1 YPL6-24 as template, with P6-1F/P6-1R primer to carrying out PCR amplification, 25 L
Amplification system includes 2 × PCRmix(TSINGKE Bio Inc) P6-1F, P6-1R primer each 1 of 12.5 L, 10 M/L
L, DNA profiling 1 L, ddH2O 9.5µL.PCR amplification program is: 94 DEG C of denaturation 3 min；94 DEG C of degeneration 30 sec, 58 DEG C
Anneal 30 sec, and 72 DEG C extend 90 sec, 30 circulations；72 DEG C extend 10 min.
All amplified productions are at 1.2%(W/V) electrophoresis detection on agarose gel, result is shown in Fig. 1 and Fig. 2, 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 about 1.8kb when P8-4F/P8-4R is expanded by primer, and with during 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,
P8-4F/P8-4R is expanded by YPL6-14, YPL6-15, YPL6-16, YPL6-18, YPL6-19, YPL6-22, YPL6-24 at primer
Without band during increasing, and when P6-1F/P6-1R is expanded by primer, there is the unique band of about 1.7kb, be accredited as mating type MAT1-2 bacterium
Strain.
The qualification of the embodiment 2 ladder effective cultivated strains of rib Morchella esculenta (L.) Pers
Test strains be ladder rib Morchella esculenta (L.) Pers bacterial strain SP1, SP2, YPL6, YPL7, strain culturing, DNA extraction, PCR amplification system and
Amplification program is with embodiment 1.
Amplified production is electrophoresis detection on 1.2% (W/V) agarose gel, and result is shown in Fig. 3, and SP2, YPL6, YPL7 bacterial strain is used
P8-4F/P8-4R, P6-1F/P6-1R primer has band to PCR amplification, illustrates have two kinds of mating types, is effectively to cultivate bacterium
Strain.And SP1 bacterial strain with during P8-4F/P8-4R primer pair amplifies without band, P6-1F/P6-1R primer pair amplifies has band, for
MAT1-2 mating type bacterial strain, it is impossible to as cultivated strains.Fruiting after bacterial strain YPL6, YPL7 cultivation.
The ultimate principle of the present invention, principal character and advantages of the present invention have more than been shown and described.The technology of the industry
Personnel, it should be appreciated that the present invention is not restricted to the described embodiments, simply illustrating this described in above-described embodiment and description
The principle of invention, without departing from the spirit and scope of the present invention, the present invention also has various changes and modifications, and these become
Change and improvement both falls within scope of the claimed invention.Claimed scope by appending claims and
Equivalent defines.
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 the special primer pair of ladder rib Morchella esculenta (L.) Pers 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 identifying ladder rib Morchella esculenta (L.) Pers mating type, it is characterised in that include drawing for detecting the special of mating type MAT1-1
Thing is to P8-4F/P8-4R, for detecting the special primer of mating type MAT1-2 to P6-1F/P6-1R, and corresponding PCR amplification system
And amplification program；
Described P8-4F/P8-4R special primer centering, the nucleotides sequence of P8-4F is classified as 5 '-GCTCTCTTGTGCCCCTTTTGA
The nucleotides sequence of CTAT-3 ', P8-4R is classified as 5 '-TCTACCAGCCATGTGAAACAAGCAA-3 '；
Described P6-1F/P6-1R special primer centering, the nucleotides sequence of P6-1F is classified as 5 '-GAGACTCAAATCTGACTGACT
The nucleotides sequence of TCCT-3 ', P6-1R is classified as 5 '-GAAGAACCTCAGATAAGCGTAAAAT-3 '.
The method identifying ladder rib Morchella esculenta (L.) Pers mating type the most according to claim 1, it is characterised in that identify mating type
The PCR amplification system of MAT1-1 with MAT1-2 is identical, all includes forward and reverse primer of 2 × PCRmix 12.5 L, 10 M/L
Each 1 L, DNA profiling 10 ng, ddH2O complements to 25 L.
The method identifying ladder rib Morchella esculenta (L.) Pers mating type the most according to claim 2, it is characterised in that identification of M AT1-1 copulation
The PCR amplification program of type is: 94 DEG C of denaturation 3 min；94 DEG C of degeneration 30 sec, 60 DEG C of annealing 30 sec, 72 DEG C extend 90
Sec, 30 circulations；72 DEG C extend 10 min；
The PCR amplification program of identification of M AT1-2 mating type is: 94 DEG C of denaturation 3 min；94 DEG C of degeneration 30 sec, 58 DEG C of annealing 30
Sec, 72 DEG C extend 90 sec, 30 circulations；72 DEG C extend 10 min.
The method identifying ladder rib Morchella esculenta (L.) Pers mating type the most according to claim 3, it is characterised in that detection ladder rib Morchella esculenta (L.) Pers
During the MAT1-1 mating type of bacterial strain, it is possible to obtain size is the unique band of 1837bp, and this band contains MAT1-1-1 total length
Gene；When detecting the MAT1-2 mating type of ladder rib Morchella esculenta (L.) Pers bacterial strain, it is possible to obtain size is the unique band of 1744bp, and this band
Contain MAT1-2-1 full-length gene.
5. identify the PCR primer pair of ladder rib Morchella esculenta (L.) Pers mating type, it is characterised in that include the spy for detecting mating type MAT1-1
Different primer is to P8-4F/P8-4R with for detecting the special primer of mating type MAT1-2 to P6-1F/P6-1R.
6. contain the reagent to identifying ladder rib Morchella esculenta (L.) Pers mating type of the PCR primer described in claim 5.
7. contain the test kit to identifying ladder rib Morchella esculenta (L.) Pers mating type of the PCR primer described in claim 5.
8. the reagent described in, claim 6 or the test kit described in claim 7 are existed by the PCR primer described in claim 5
Identify the application in ladder rib Morchella esculenta (L.) Pers mating type.
9. the reagent described in, claim 6 or the test kit described in claim 7 are existed by the PCR primer described in claim 5
Identify the application of ladder rib Morchella esculenta (L.) Pers breeding.
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| CN107151698A (en) * | 2017-04-01 | 2017-09-12 | 中国科学院昆明植物研究所 | Identify the mating type method of 11 kinds in black hickory chick monoid |
| 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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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107151698A (en) * | 2017-04-01 | 2017-09-12 | 中国科学院昆明植物研究所 | Identify the mating type method of 11 kinds in black hickory chick monoid |
| 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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