CN106282370A - Morchella esculenta (L.) Pers mating type determines gene identification primer special and fruiting energy force prediction method - Google Patents
Morchella esculenta (L.) Pers mating type determines gene identification primer special and fruiting energy force prediction method Download PDFInfo
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Abstract
The invention belongs to Molecular Identification technical field, be specifically related to Morchella esculenta (L.) Pers mating type and determine gene identification primer special and fruiting energy force prediction method.The technical problem to be solved in the present invention is that Morchella esculenta (L.) Pers strain is difficult to judge whether it has fruiting ability before the planting.It is to have invented the primer identifying that two mating type genes of Morchella esculenta (L.) Pers are special first that the present invention solves the technical scheme of above-mentioned technical problem.The present invention can make Morchella esculenta (L.) Pers confirm fruiting ability before cultivation, it is to avoid cannot bring unnecessary loss by fruiting.
Description
Technical field
The invention belongs to Molecular Identification technical field, the Morchella esculenta (L.) Pers mating type being specifically related to Morchella esculenta (L.) Pers determines gene, copulation
Type identifies primer special and fruiting energy force prediction method.
Background technology
Morchella esculenta (L.) Pers is a kind of delicious edible fungi in ascomycetes, and its sporophore contains the aminoacid of 7 kinds of needed by human, Gan Han
Nontoxic, useful the intestines and stomach, phlegm-eliminiating and qi-regulating drug effect.The nutrition of Morchella esculenta (L.) Pers is the abundantest, and according to surveying and determination, Morchella esculenta (L.) Pers is containing crude protein 20%, thick
Fat 26%, carbohydrate 38.1%, possibly together with several amino acids, particularly content of glutamic acid is up to 1.76%.Therefore, have
People is considered " the best protein source ", and has the laudatory title of " meat or fish in element ".Owing to Morchella esculenta (L.) Pers extensively gets consumer reception, supply
Should not ask, cannot meet the demand of Morchella esculenta (L.) Pers consumption market by gathering wild toadstool.Therefore, a group of people is occurred in that in recent years
The technology of work cultivation Morchella esculenta (L.) Pers, the such as patent of Authorization Notice No. CN103583232B, CN103202177B, CN101628834B
With the cultural method described in application publication number CN103782802A, CN103583240A, CN101053302.But, real producing
In trampling, there is the phenomenon that fruiting is unstable in tame Morchella esculenta (L.) Pers.It is currently used for the Morchella esculenta (L.) Pers bacterium that artificial culture mode produces
Strain is numerous, and for replacing the old bacterial strain degenerated, the annual bacterial strain also having a large amount of new selection-breeding puts into Morchella esculenta (L.) Pers and produces.Have quite a lot of
In the case of, the Morchella esculenta (L.) Pers bacterial strain planted finally cannot fruiting, cause planting that almost No kernels or seeds are gathered, as in a year of scarcity at family.Cause Morchella esculenta (L.) Pers without
The reason of method fruiting, outside removing field management is not good at causing the factors such as illumination, temperature, moisture, ventilation to be not suitable for, Morchella esculenta (L.) Pers bacterium
The mating type disappearance planted also occupies important reason.
Most of funguses, including Morchella esculenta (L.) Pers, there are the mycelia of two kinds of mating types, in sexual reproduction respectively
Play the part of and be equivalent to female and male role.And in Morchella esculenta (L.) Pers, two kinds of mating types are respectively MAT1-1-1 and MAT1-2-1 type.
Only existing when MAT1-1-1 and MAT1-2-1 type mycelia, Morchella esculenta (L.) Pers just can complete the syngenesis of similar male and female copulation simultaneously
Journey, and successfully grow sporophore (fruiting).Accordingly, it is capable to success fruiting Morchella esculenta (L.) Pers strain must simultaneously have MAT1-1-1 and
Two kinds of mycelia of MAT1-2-1 type.After Morchella esculenta (L.) Pers strain, is repeatedly passed at long-term preservation, it is susceptible to degenerate, two kinds will be caused
Possible result: any of which in two kinds of mycelia of (1) MAT1-1-1 and MAT1-2-1 type is degenerated and disappeared, (2) gene
Sudden change causes MAT1-1-1 type to be changed into MAT1-2-1 type or MAT1-2-1 type is transformed into MAT1-1-1 type.Both change all incite somebody to action
Cause the Morchella esculenta (L.) Pers strain only remaining a kind of copulation originally simultaneously having the mycelia of two kinds of mating types of MAT1-1-1 and MAT1-2-1
Type, it is impossible to complete sexual reproduction, thus cause fruiting failure.The present invention is badly in need of developing the most just differentiating sheep
Whether tripe bacterium strain has the technology of fruiting ability.
Summary of the invention
The technical problem to be solved in the present invention is that Morchella esculenta (L.) Pers strain is difficult to judge whether it has fruiting energy before the planting
Power.It is to find first and isolated two kinds of mating types of Morchella esculenta (L.) Pers that the present invention solves the technical scheme of above-mentioned technical problem
Gene on the basis of, it is provided that amplification Morchella esculenta (L.) Pers mating type gene primer pair.
Further, the nucleotides sequence of above-mentioned primer pair is classified as SEQ ID No.7 and SEQ ID No.8, and SEQ
Shown in ID No.9 and SEQ ID No.10.
The present invention also provides above-mentioned primer in identifying Morchella esculenta (L.) Pers strain mating type on the basis of such scheme
Purposes.
Meanwhile, the invention provides a kind of method identifying Morchella esculenta (L.) Pers strain mating type.The method is in comprising the following steps:
A, the STb gene of extraction Morchella esculenta (L.) Pers. Mycelium；
B, use above-mentioned primer to carrying out PCR amplification；
C, according to amplification products therefrom judge be Morchella esculenta (L.) Pers. Mycelium be MAT1-1-1 type mycelia, MAT1-2-1 type mycelia, also
It it is the mycelia having MAT1-1-1 and MAT1-2-1 type concurrently.
Wherein, the disconnected mode described in said method step c is: if being classified as SEQ ID No.7 and SEQ with nucleotides sequence
Primer pair amplifies shown in ID No.8 goes out the fragment of an about 1.2-1.5kb size, illustrates that sample has MAT1-1-1 copulation
Type, on the contrary the most no；If going out one greatly with nucleotide sequence primer pair amplifies as shown in SEQ ID No.9 and SEQ ID No.10
The fragment of about 0.4-0.6kb size, illustrates that sample has MAT1-2-1 mating type, otherwise the most no.
Enter one, present invention also offers and a kind of predict that can Morchella esculenta (L.) Pers the method for fruiting, it is characterised in that include following
Step:
A, the STb gene of extraction Morchella esculenta (L.) Pers. Mycelium；
B, use above-mentioned primer to carrying out PCR amplification；
C, according to amplification products therefrom judge be Morchella esculenta (L.) Pers. Mycelium be MAT1-1-1 type mycelia, MAT1-2-1 type mycelia, also
It it is the mycelia having MAT1-1-1 and MAT1-2-1 type concurrently；
If d Morchella esculenta (L.) Pers. Mycelium is double be tool MAT1-1-1 and MAT1-2-1 type mycelia, it determines for can fruiting, otherwise then sentence
Wei can not.
Wherein, the judgment mode described in said method step c is: if being classified as SEQ ID No.7 and SEQ with nucleotides sequence
Primer pair amplifies shown in ID No.8 goes out the fragment of an about 1.2-1.5kb size, illustrates that sample has MAT1-1-1 copulation
Type, on the contrary the most no；If going out one greatly with nucleotide sequence primer pair amplifies as shown in SEQ ID No.9 and SEQ ID No.10
The fragment of about 0.4-0.6kb size, illustrates that sample has MAT1-2-1 mating type, otherwise the most no.
The beneficial effects of the present invention is: the present invention provides the method for detection Morchella esculenta (L.) Pers mating type first, and is planting
Can predict before training Morchella esculenta (L.) Pers bacterial strain can the method for fruiting, experiment proof can extremely accurate predict that can Morchella esculenta (L.) Pers bacterial strain fruiting
With, contribute to that agricultural production is eliminated mating type as early as possible and have defective bacterial strain, it is to avoid blindly going into operation causes damage, aborning
There is good application prospect.
Explanation of nouns:
" Morchella esculenta (L.) Pers " of the present invention: refer to fungus belongs to all edible fungi of morchella (Morchella genus),
Include but not limited to ladder rib Morchella esculenta (L.) Pers (Morchella importuna), six younger sister's Morchella esculenta (L.) Perss (Morchella sextelata),
Seven younger sister's Morchella esculenta (L.) Perss (Morchella septimelata), Morchellaconica (Morchella conica), yellow Morchella esculenta (L.) Pers
(Morchella esculenta), Morchella elata (Morchella elata), big legs Morchella esculenta (L.) Pers (Morchella
Crassipes) etc..Most common of which cultivation kind is ladder rib Morchella esculenta (L.) Pers, six younger sister's Morchella esculenta (L.) Perss, seven younger sister's Morchella esculenta (L.) Perss.
Heretofore described " fruiting ", refers to that Morchella esculenta (L.) Pers produces sporophore.
Detailed description of the invention
Owing to, in Morchella esculenta (L.) Pers, two kinds of mating types are respectively MAT1-1-1 and MAT1-2-1 type.Only as MAT1-1-1 and
MAT1-2-1 type mycelia exists simultaneously, and Morchella esculenta (L.) Pers just can complete the sexual reproduction of similar male and female copulation, and successfully grows son
Entity (fruiting).Accordingly, it is capable to the Morchella esculenta (L.) Pers strain of success fruiting must simultaneously have two kinds of bacterium of MAT1-1-1 and MAT1-2-1 type
Silk.After Morchella esculenta (L.) Pers strain long-term is preserved, repeatedly passed on, it is susceptible to degenerate, will cause two kinds of possible results: (1)
Any of which in two kinds of mycelia of MAT1-1-1 and MAT1-2-1 type is degenerated and is disappeared, and (2) gene mutation causes MAT1-1-
1 type is changed into MAT1-2-1 type or MAT1-2-1 type is transformed into MAT1-1-1 type.Both changes all will cause originally gathering around simultaneously
There is Morchella esculenta (L.) Pers strain only remaining a kind of mating type of the mycelia of two kinds of mating types of MAT1-1-1 and MAT1-2-1, it is impossible to complete sexual
Reproductive process, thus cause fruiting failure.
In Morchella esculenta (L.) Pers, mating type MAT1-1-1 of mycelia and MAT1-2-1 respectively by gene mrlMAT1-1-1,
MrlMAT1-2-1 controls.The mycelia with mrlMAT1-1-1 gene is MAT1-1-1 mating type, has mrlMAT1-2-1 base
The mycelia of cause is mrlMAT1-2-1 mating type.
Present invention firstly discovers that the sequence of mrlMAT1-1-1, mrlMAT1-2-1 gene of morchella.mrlMAT1-
The global DNA sequence of 1-1, mrlMAT1-2-1 gene comprises upstream and downstream non-express sequence and intron, sees SEQ ID No.1
With SEQ ID No.2.In sequence listed below, upstream and downstream non-express sequence underscore labelling, intron square frame mark
Note (in the present invention, the order of all nucleotide sequences is from 5 ' ends to 3 ' ends if no special instructions).
The global DNA sequence (SEQ ID No.1) of mrlMAT1-1-1 gene:
ATCACCGACGAGAGCACGCTGCTCACTGGGTTTTCCCTTACTTTACATGCATTTCCTGACACATACAACCTTTTATC AACGGTACACTTCAATAAAGTAGAGTCTACTAAGGCTCTCTTGTGCCCCTTTTGACTATTATTTCTATCCTTCTATT TTATCTTCATGTCACTCCGTCCGGTTTACCTTACTGGACTGGTTCGTGAGAACCCGCCTTCTGAGTCCGTTATGCTC
ATGGACGCTATAAAGCGGACTCTTTGTCTTCGTAACGCCACTTTGGGTTGCGCTCTCTATCTCTCGAAAACCCATCA
ATACCTTTTAATGCAAGACCGAGGCGGTGGAATCTGGGTTCAACACAAAGACTGTCGATATATTGTGCCCCCACCAG
GAGGGTCGGTATTTCAGGTGCAGGGCAGGCCCCTCCCGTTCCAGAGATGGCTCACTCTTATGCATCGACTTGTACGT
CAGGATGGAAGGGTTGGGCTAGACCCTAAGATCCCATACAAGTGCGCAGTCCAAAGGCTTTATGGTGAACACTACAA
GAGTAATAGGCCGATTGTTGATGAACCGTTGACTAAACGGTCTGTTTTGAAAGCCCTAAACCCTTATATAGCACATA
GAA
CTTGGATATCAAAATTCCTAGGAGGGCTAGGATTTACCCAGATGCTGATTTCAGCACTCACCAAAGGTGTATGGCAA
CGTGAGAAGAGGAAAGCTATGTGGTCCAATATAGCAAGGTTGTACACCTACCACCGGGATCAAGGGACGCTCACATC
TACTCTCGAAGATTTTATCAAAGCGCAACTTATCGAGAATAAACAAGATCCTTCCCCTAGCTCATTCCTACACAACT
GCGGTATAAATCTCGACGATGTGAGAAAACGAATGACACCCACTTGCATTCCACCTGATGCTCAAATCTTTGCTATT
GGGACTGTGGCACCCATCGAGGAGACTTATTCTGGGATTCAAGATATTCCAAATGTTCACTTTCAAGCGCCTACAGT
TCCTGACTCTACCGCCATGGAATCTGCCAAAACGGAGCCCATTATAATGTCGCCCATATCAACTATATGTACGGTAT
CGAAGGATTATATTTCGTCTCGAGCTCGGCAGAGAAGCAGGAAAACAATTGCACGCTCGGAATTAACAGGAATTACC
CCTCAAGACGCATGTCAAAACGCTCTATCGCCACCAGCTCTCCCAACTTCTCAACAATCCATCTCCAATCAAATGCT
TGTAGATCCTCCTCAGGACTGGAATTCTATGACCCCAGTCGCTTACAATACGAATCAGCTCGATCCAAGATTGAACT
TTTTACCCATTAAACCGCTTCCTGGGAGGCTGCCCGCTTTTGATAAGACCCTCCGAGAGCCAGGGACATACCCAGAG
CCAGAGATATACCCAGAACCAGATACATATTCAGAATCACGACATTTCCAGGAGTCACAGTTTTATCCACAACAAAT
TTTTTCTAGCGGGTTAAAATTCTGTATAAGCCCAAAGTCTGATGTAGAACCACGGACATATACAGCTCCCAACATTT
ATCCAGAACCTTATACTCTTAGACCTGAAAACGCAACTTCGCAGGTATTTCATGGGATATATAATCCCCAAAATATG
GAAATTGAAGAGCCTTTCGATTTTACAAAAG AAAATACATATTTCGTTTCGGAAACCTACACCTCCAAAGACTT
TCCACAGCCTCAATCACAGACATTCCAGGAGTTCGTAAACTAGGACCACACTACAGTACAAGGACATTAAATTGAAG TAAACGTATCTCATGTTTTATAATTACTTTGTTTTGCTTGTTTCACATGGCTGGTAGATATATCGTCATTTTAAAAC GTTTGAGCGGCCATTACGCTCAATCTGATTTGTCAAACCAGCTACTTATATATATGTGCTATATAACTA
The global DNA sequence (SEQ ID No.2) of mrlMAT1-2-1 gene:
ATTTTCTGCCATTTATAACATATATATATAAATCAAACCTATTTCGACGTATATCAACCACGCCCGTAATTTCTTCG ATCTTTACTATTCCGGTCACCTCTTTGAAACATCTTATTCTGTTAGCCGCCCATCTCTGAGCTTTTTCTACTCTTAC CACCCTATCAGTTCATCGATAAAGAGGTTCCATGTTTCCAGGCCTCCAAAAGCCTGGGTTGCGTCCGAAGAATAGCT
CTACTCTTCGGCCAGAACAGATGCTCGAAGAAGCTATGGGTCATATCAATCAGAGCTTTGCCAATTCAAATGAAAAG
ACCGCTTTAGATAGATTGGCAGGACTTAAACCTCGCCGTGACCCTCCTTCTTTCGATTTCACTGGATCAGACGGCCA
TATCCTGAGAGTTAAGGTTTTATCTCTGGACCAACGAGGGCCAGGGAAAGAAAGTGATGACCATGGGCCCCAAACAT
TTACTGTTTTAGAAAAAACCAACCATATAAACGGTTATCCTTGTGTTAAGACCAATGAGGGTGATCTGGTCTTCGTT
TATGACTCTACTGTCACTTCTGCAGAAACAATTTACCAAGGCCATGGAAATTTTGTGGATCCACAGCTGTCAGCGCA
AGGCGTGAGTCGAGTTACCAAGAAGAATTTATTAGAGCATGTTCCTCGTCCTATGAATGC
TTTTATAATCTATCGCAACACAGAGATGAAGGCTGTTCGCCAGAAATACCCGGGGATTGGGAACAACGATGTTTGTA
GGTTTTTTGTTGCTCATTTATCCTTCAGAATCTGATAATATGTGTTCGTTTTGGGCCTTTTGATTATTTTATTGACA ATTTACGTTGTAGCGCGGATTGTCGGGCAACGGTGGCGAGAGCTTGACCCTAAGATTAGAGAGCATTACAAGGTGAA TTAAAACAATCACTTCTTCAAGTAAATATGCTCATATTGCTAATCTAGAAAAGGATTTGGCCGCAAAATCTGCACGA GAGCACCACGTAA
CDS sequence is there is in the most also research in being found that this mrlMAT1-1-1 gene of Morchella esculenta (L.) Pers and mrlMAT1-2-1 gene
Row.The CDS sequence of mrlMAT1-1-1 gene is as shown in SEQ ID No.3.The CDS sequence such as SEQ ID of mrlMAT1-2-1 gene
Shown in No.4.
The most also it is found that this mrlMAT1-1-1 gene of Morchella esculenta (L.) Pers and the mating type of mrlMAT1-2-1 gene code are determined
The peptide sequence of determining cause.The aminoacid sequence of the protein of mrlMAT1-1-1 gene code such as SEQ ID No.5 institute
Show.
The aminoacid sequence of the protein of mrlMAT1-2-1 gene code is as shown in SEQ ID No.6.
On the basis of above-mentioned creative work, applicant further obtains and investigated the multiple of morchella can
MrlMAT1-1-1, mrlMAT1-2-1 gene of edible kind, finds its total conserved sequence.Wherein, shown in SEQ ID No.1
MrlMAT1-2-1 gene order shown in mrlMAT1-1-1 and SEQ ID No.2 is all belonging to ladder rib Morchella esculenta (L.) Pers and six younger sister's Gaster caprae seu Ovis
Bacterium, the two gene order of the two kind is identical.Also find mrlMAT1-1-1, mrlMAT1-2-1 base that morchella is various
Because of sequence high conservative, the most of the same race between similarity mrlMAT1-1-1 between 98.1%-100%, mrlMAT1-2-1 is
99.4-100%.The present invention studies on this basis and obtains a set of primer pair, and this set primer is selected as at multistage conserved sequence
On the premise of design of primers site, for amplifying the primer pair of full length gene as far as possible.This primer that the present invention obtains is to not only
Can detect whether target gene exists, moreover it is possible to detect its length the most normal, cause base to facilitate detection to be likely to be due to sudden change
Because Partial Fragment lacks, mrlMAT1-1-1 or mrlMAT1-2-1 gene outcome is made to lose the situation of normal function.This set primer
To the primer included for specific amplification mrlMAT1-1-1 gene to MrlMAT1-1-1F, MrlMAT1-1-1-R, and it is used for
The primer of specific amplification mrlMAT1-2-1 gene is to MrlMAT1-2-1-F, MrlMAT1-2-1-R.
MrlMAT1-1-1F (SEQ ID No.7): ATGGCTCACTCTTATGCATCGACTTGTAC
MrlMAT1-1-1-R (SEQ ID No.8): GGCTGTGGAAAGTCTTTGGAGGTGTAG
MrlMAT1-2-1-F (SEQ ID No.9): AAGACCGCTTTAGATAGATTGGCAGGAC
MrlMAT1-2-1-R (SEQ ID No.10): AACAGCCTTCATCTCTGTGTTGCGATAG
Utilize above-mentioned primer can be identified the mating type of Morchella esculenta (L.) Pers. Mycelium by PCR method: to be MAT1-1-1 type mycelia,
MAT1-2-1 type mycelia, still has the mycelia of MAT1-1-1 and MAT1-2-1 type concurrently.
And when using PCR method to identify Morchella esculenta (L.) Pers mating type, carrying of wherein PCR concrete steps and details, such as DNA profiling
Take, the setting of PCR program, the selection etc. of primer annealing temperature, according to the reagent manufacture of used different manufacturers and difference,
But it is all the groundworks that are easily accomplished under the guidance of prior art of those skilled in the art, as with reference to various textbooks, examination
These details are no longer repeated in the present invention by the description of agent box, molecular biology manual etc..
Inventor, by identifying the mating type of Morchella esculenta (L.) Pers strain, checks that it has two kinds of mating types the most simultaneously,
Prediction Morchella esculenta (L.) Pers strain success fruiting, thus be used for identifying self-control or the quality of commercially available Morchella esculenta (L.) Pers strain.
When specifically using at the scene, when with primer, MrlMAT1-1-1F, MrlMAT1-1-1-R being expanded Morchella esculenta (L.) Pers sample,
Amplify the fragment of an about 1.2-1.5kb size, can illustrate that sample has MAT1-1-1 mating type.If without band, say
Bright sample does not have MAT1-1-1 mating type.
When MrlMAT1-2-1-F, MrlMAT1-2-1-R being expanded Morchella esculenta (L.) Pers sample with primer, amplify one about
The fragment of 0.4-0.6kb size, can illustrate that sample has MAT1-2-1 mating type.If without band, illustrate that sample does not has
MAT1-2-1 mating type.
And when this Morchella esculenta (L.) Pers strain has two kinds of mating types of MAT1-1-1 and MAT1-2-1 simultaneously, then judge that this strain can become
Merit fruiting, i.e. strain are qualified.When this strain only has a kind of mating type in MAT1-1-1 or MAT1-2-1, then judge this bacterium
Kind can not success fruiting, i.e. strain defective.
In Morchella esculenta (L.) Pers cultivation practices, fruiting ability prediction and result verification is carried out below by way of applicable the inventive method
Embodiment, to be described in further detail the present invention.
Embodiment one, use the inventive method predict that can Morchella esculenta (L.) Pers strain fruiting
Purpose: 5 strain Morchella esculenta (L.) Perss are carried out the qualification of mating type, and the Morchella esculenta (L.) Pers strain being respectively prepared these 5 bacterial strains produces
Can product be predicted by fruiting.
Time: the time being predicted according to PCR result is in JIUYUE, 2014, in March, 2015 actual fruiting time.
Place: Xindu District, Chengdu, Sichuan Province
Wherein A, B, C are inventor's unit one's own Morchella esculenta (L.) Pers bacterial strain, and D, E are from two kinds of commercially available Morchella esculenta (L.) Pers strains
Bacterial strain.A, B are ladder rib Morchella esculenta (L.) Pers (Morchella importuna), and C, D are six younger sister Morchella esculenta (L.) Pers (Morchella
Sextelata), E is seven younger sister's Morchella esculenta (L.) Perss (Morchella septimelata).
In result form, pcr amplification product has, and inserts " √ ", without then inserting "×".Result sees table 1.
Sampling process: the mycelia of the appropriate Morchella esculenta (L.) Pers bacterial strain to be measured of sterile working's picking, accesses and fills 25ml murphy juice Fructus Vitis viniferae
Sugar culture fluid (the preparation method of every 1000 milliliters of culture fluid: weigh 200g Rhizoma Solani tuber osi and clean peeling and be cut into small pieces, add water well-done
(boiling 20～30 minutes, can be poked by Glass rod), with eight layers of filtered through gauze, heating, adds 20g glucose, and stirring is all
Even, slightly supply moisture again to 1000 milliliters after cooling) 250 milliliters of triangular flasks in, 20 degrees Celsius of lower quiescent culture 7 days, to obtain final product
Mycelial samples.
Extraction process: use the fungal genomic DNA Rapid extraction test kit (production number SK8229) of the raw work in Shanghai to extract
STb gene, as pcr template.
The PCR process of identification of M AT1-1-1:
Use the Q5 of NEB companyTMHot Start High-Fidelity 2X Master Mix expands.Every 20 micro-
Rise reaction system and contain 1 microlitre Morchella esculenta (L.) Pers STb gene, the primer shown in 1 microlitre Seq NO.7, drawing shown in 1 microlitre Seq NO.8
Thing, 7 microlitre distilled water, 10 microlitre Q5TM Hot Start High-Fidelity 2X Master Mix。
PCR program:
98 degree 2 minutes,
98 degree 30 seconds, 60 degree 20 seconds, 72 degree 45 seconds (circulating 30 times),
72 degree 2 minutes,
The PCR process of identification of M AT1-2-1:
Use the Q5 of NEB companyTMHot Start High-Fidelity 2X Master Mix expands.Every 20 micro-
Rise reaction system and contain 1 microlitre Morchella esculenta (L.) Pers STb gene, the primer shown in 1 microlitre Seq NO.9, drawing shown in 1 microlitre Seq NO.10
Thing, 7 microlitre distilled water, 10 microlitre Q5TM Hot Start High-Fidelity 2X Master Mix。
PCR program:
98 degree 2 minutes,
98 degree 30 seconds, 64 degree 20 seconds, 72 degree 20 seconds (circulating 30 times)
72 degree 2 minutes.
PCR primer is electrophoresis detection on the gel containing 1% agarose.The positive is appeared as with respective strap.
The mating type qualification result of table one .5 strain Morchella esculenta (L.) Pers
By above-mentioned strain then according to disclosed " Sichuan Morchella esculenta (L.) Pers Efficient Cultivation pattern and technology " (" edible and medical fungi " delivered
The 3rd phase of volume 24 in 2016, page 151～154) described in flow process cultivate, results to sporophore be then fruiting, 0 son reality
It is then non-fruiting that body produces.Experimental result sees table 2.
Table two. to 5 Morchella esculenta (L.) Pers strain products can the prediction of fruiting, and actual fruiting situation.
| Bacterial strain number | It is accredited as mating type | Can prediction fruiting | Reality whether fruiting |
| A | Only MAT1-1-1 | Estimating can not fruiting | Non-fruiting |
| B | MAT1-1-1 and MAT1-2-1 | It is expected to fruiting | Fruiting |
| C | MAT1-1-1 and MAT1-2-1 | It is expected to fruiting | Fruiting |
| D | Only MAT1-2-1 | Estimating can not fruiting | Non-fruiting |
| E | MAT1-1-1 and MAT1-2-1 | It is expected to fruiting | Fruiting |
As can be seen here, by identifying that can mating type prediction Morchella esculenta (L.) Pers strain the rate of accuracy reached 100% of fruiting.If according to biography
System pattern without identifying that mating type is blindly gone into operation, then will run in 5 strains 2 cannot fruiting, risk probability 40%, can
Bring the biggest economic loss can to plantation family.
Embodiment two, use the inventive method predict that can Morchella esculenta (L.) Pers strain fruiting
Purpose: 12 strain Morchella esculenta (L.) Perss are carried out the qualification of mating type, and the Morchella esculenta (L.) Pers strain product making these 12 bacterial strains
Can be predicted by fruiting.
Time: the time being predicted according to PCR result is in October, 2015, in April, 2016 actual fruiting time.
Place: Sichuan Province's Aba Prefecture
Wherein M1-M8 is the one's own bacterial strain of our unit, and S1-S4 is the bacterial strain from four kinds of commercially available Morchella esculenta (L.) Pers strains.
M1～M4 is six younger sister's Morchella esculenta (L.) Perss, M5～M8 is ladder rib Morchella esculenta (L.) Pers, and S1 is seven younger sister's Morchella esculenta (L.) Perss, and S2 is ladder rib Morchella esculenta (L.) Pers, and S3 is big legs
Morchella esculenta (L.) Pers (Morchella crassipes), S4 is six younger sister's Morchella esculenta (L.) Perss
Concrete test method sees embodiment one.
Pcr amplification product has, and inserts " √ ", without then inserting "×".In result form, pcr amplification product has, and inserts
" √ ", without then inserting "×".Result sees table 2.
The mating type qualification result of table three .12 strain Morchella esculenta (L.) Pers
By above-mentioned strain then according to disclosed " Sichuan Morchella esculenta (L.) Pers Efficient Cultivation pattern and technology " (" edible and medical fungi " delivered
The 3rd phase of volume 24 in 2016, page 151～154) described in flow process cultivate, results to sporophore be then fruiting, 0 son reality
It is then non-fruiting that body produces.Experimental result sees table 4.
Table four. to 12 Morchella esculenta (L.) Pers strain products can the prediction of fruiting, and actual fruiting situation
As can be seen here, by identifying that can mating type prediction Morchella esculenta (L.) Pers strain the rate of accuracy reached 100% of fruiting.If according to biography
System pattern without identifying that mating type is blindly gone into operation, then will run in 12 strains 5 cannot fruiting, risk probability 42%,
Bring the biggest loss may to plantation family.
Claims (8)
1. expand the primer pair of Morchella esculenta (L.) Pers mating type gene.
Primer pair the most according to claim 1, it is characterised in that:
The nucleotides sequence of described primer pair is classified as SEQ ID No.7 and SEQ ID No.8, and SEQ ID No.9 and SEQ ID
Shown in No.10.
3. the primer described in claim 1 or 2 is to the purposes in identifying Morchella esculenta (L.) Pers strain mating type.
4. the method identifying Morchella esculenta (L.) Pers strain mating type, it is characterised in that comprise the following steps:
A, the STb gene of extraction Morchella esculenta (L.) Pers. Mycelium；
B, use primer described in claim 1 or 2 are to carrying out PCR amplification；
C, according to amplification products therefrom judge be Morchella esculenta (L.) Pers. Mycelium be MAT1-1-1 type mycelia, MAT1-2-1 type mycelia, or hold concurrently
The mycelia of tool MAT1-1-1 and MAT1-2-1 type.
Method the most according to claim 4, it is characterised in that: described judgment mode is: if being classified as SEQ with nucleotides sequence
Primer pair amplifies shown in ID No.7 and SEQ ID No.8 goes out the fragment of an about 1.2-1.5kb size, illustrates that sample has
There is MAT1-1-1 mating type, on the contrary the most no；If with nucleotide sequence primer as shown in SEQ ID No.9 and SEQ ID No.10
To amplifying the fragment of an about 0.4-0.6kb size, illustrate that sample has MAT1-2-1 mating type, on the contrary the most no.
6. can prediction Morchella esculenta (L.) Pers the method for fruiting, it is characterised in that comprise the following steps:
A, the STb gene of extraction Morchella esculenta (L.) Pers. Mycelium；
B, use primer described in claim 1 or 2 are to carrying out PCR amplification；
C, according to amplification products therefrom judge be Morchella esculenta (L.) Pers. Mycelium be MAT1-1-1 type mycelia, MAT1-2-1 type mycelia, or hold concurrently
The mycelia of tool MAT1-1-1 and MAT1-2-1 type.
If Morchella esculenta (L.) Pers. Mycelium double be tool MAT1-1-1 and MAT1-2-1 type mycelia, it determines for can fruiting, otherwise be then determined as
Can not.
Method the most according to claim 6, it is characterised in that: described judgment mode is: if being classified as SEQ with nucleotides sequence
Primer pair amplifies shown in ID No.7 and SEQ ID No.8 goes out the fragment of an about 1.2-1.5kb size, illustrates that sample has
There is MAT1-1-1 mating type, on the contrary the most no；If with nucleotide sequence primer as shown in SEQ ID No.9 and SEQ ID No.10
To amplifying the fragment of an about 0.4-0.6kb size, illustrate that sample has MAT1-2-1 mating type, on the contrary the most no.
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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 |
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| 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 |
| CN113317124A (en) * | 2021-05-17 | 2021-08-31 | 中国科学院昆明植物研究所 | Rapid identification method for fruiting rate of morchella esculenta |
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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 |
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| CN109628626A (en) * | 2018-12-07 | 2019-04-16 | 广东东阳光药业有限公司 | Identify specific primer, the kit, method and its application of terraced rib hickory chick |
| CN109628626B (en) * | 2018-12-07 | 2022-07-26 | 东莞东阳光保健品研发有限公司 | Specific primer, kit and method for identifying morchella ladder and application of specific primer, kit and method |
| CN109628625B (en) * | 2018-12-07 | 2022-08-12 | 东莞东阳光保健品研发有限公司 | Specific primer, kit and method for identifying morchella esculenta and application of specific primer, kit and method |
| 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 |
| CN113317124A (en) * | 2021-05-17 | 2021-08-31 | 中国科学院昆明植物研究所 | Rapid identification method for fruiting rate of morchella esculenta |
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