CN103224991B - Method for identifying mating types of Lepista sordida protoplast monokaryons and special primer pair IS-857c therefor - Google Patents

Method for identifying mating types of Lepista sordida protoplast monokaryons and special primer pair IS-857c therefor Download PDF

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CN103224991B
CN103224991B CN201310188032.4A CN201310188032A CN103224991B CN 103224991 B CN103224991 B CN 103224991B CN 201310188032 A CN201310188032 A CN 201310188032A CN 103224991 B CN103224991 B CN 103224991B
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lepista sordida
protoplastis
mating type
identified
strain
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CN103224991A (en
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许峰
刘宇
耿小丽
王守现
赵爽
李登进
王鹏
王兰青
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Beijing Academy of Agriculture and Forestry Sciences
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Beijing Academy of Agriculture and Forestry Sciences
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Abstract

The invention discloses a method for identifying the mating types of Lepista sordida protoplast monokaryons and a special primer pair IS-857c therefor. The method comprises the following steps: respectively taking genome DNAs (deoxyribonucleic acid) of two Lepista sordida protoplast monokaryons to be identified as templates, performing PCR (polymerase chain reaction) amplification by using a PCR primer pair composed of two single-chain DNAs as shown in SEQ ID NO.1 and SEQ ID NO.2, and detecting the sizes of the obtained PCR products; if both or neither of the PCR products of the two Lepista sordida protoplast monokaryons to be identified contains a DNA segment of 500-800bp, indicating that the mating types of the two Lepista sordida protoplast monokaryons to be identified are the same; and if one of the two contains a DNA segment of 500-800bp and the other does not contain a DNA segment of 500-800bp, indicating that the mating types of the two Lepista sordida protoplast monokaryons to be identified are different.

Description

The method of discriminating Lepista sordida protoplastis monocaryon mating type and primer special thereof are to IS-857c
Technical field
The present invention relates to differentiate Lepista sordida protoplastis monocaryon mating type method and primer special thereof to IS-857c.
Background technology
System that nutrition is not affine is a kind of genetic system of recognition of nonself, in most fungi, this system can cause the upper visibly different individuality of heredity to produce distinctive somatic incompatibility (Qi Yuancheng etc. at cross-connecting area, the experiment of Chinese cultivated oyster cap fungus kind somatic incompatibility is compared with RAPD analytical results. fungus journal, 2010,29(3)) react, the intensity of somatic incompatibility reaction is different because of individual difference.In fungi, nutrition is not affine is also called that somatocyte is not affine or heterokaryon is not affine, and its effect is to prevent the fusion between individuality that on individuality that mating type in same is different or the not affine site of nutrition, gene is different, to keep the stability in idiogenetics.Mating type be can complete between the individuality according to mating factor mating combine and determine bond type (China Standards Press first editing cubicle, Chinese agriculture standard compendium (edible fungus rolls). China Standards Press, 2010).Mechanism or system that oneself or a dissident identifies all is there is in the syngenesis and process of nourishing and growing of fungi.The syngenesis stage is by mating type factor identification; And the identification of vegetative growth phase is undertaken by the not affine system of heterokaryon.Heterokaryon incompatibility is a kind of biological phenomena be prevalent in fungi, is a kind of ubiquitous phenomenon in the fungies such as ascomycetes, basidiomycetes, zygomycetes.In heterokaryon forming process, non-own recognition system finally causes the different core of genetic background can not form stable heterokaryon by the interaction of a series of protein factor, namely so-called heteronuclear incompatibility.Heterokaryon incompatibility be one by the process of gene regulating and often the mycelium of Heterokaryon incompatibility can be dead.
Heterocaryotic mycelium prepares in process the phenomenon occurring monokaryon protoplastis at protoplastis.These monokaryon protoplastiss become protoplastis monocaryon through regeneration cultivation and genetic screening, because protoplastis monocaryon is directed to vegetative hyphae, do not live through maiotic vegetative progeny, it is that the heredity of edible mushrooms and breeding research provide a kind of very important base mateiral.The cross-breeding mode of protoplastis monocaryon accesses same PDA flat board between two from the protoplastis monocaryon of double-core parent respectively two, and opposite culture is hybridized.In crossover process, the tenuigenin of two protoplastis monocaryons is recombinated, and forms the filial generation of a heterogeneous heteronuclear.The principal feature of this method is, according to parental trait not easily dispersion and dilution in protoplastis monocaryon, the sterile monocaryon selecting to have parental trait can be removed in a smaller range of variation in the procedure of breeding, effectively overcome the obstacle of parental trait dispersion and the selection difficulty caused by Spore monokaryons genetic diversity in traditional breeding method process, decrease the workload of screening filial generation.Meanwhile, because protoplastis monocaryon obtains in laboratory conditions, without seasonal restriction, therefore shorten breeding time.
Lepista sordida (Lepista sordida) belongs to Basidiomycotina (Basidiomycomycotina), Hymenomycetes (Hymenomycetes), Agaricales (Agaricales), Tricholomataceae (Tricholomataceae), Lepista lentinus (Lepista).This bacterium is nutritious, has certain medicinal efficacy.The ground such as Guizhou, Heilungkiang, Liaoning, Hebei, Henan, Gansu, Qinghai, Sichuan, Xinjiang, Shanxi, the Inner Mongol and Fujian are mainly distributed in China, a kind of famous and precious edible mushrooms and medicinal fungus, Lepista sordida is also in the bench-scale testing stage, wildly to yield poorly and rare, its price is extremely expensive, is a kind of wild-type strain having very much potentiality to be exploited.But the difficult problem that exist in actual production and yield poorly, insect resistance capacity is poor, adaptive temperature narrow range, and sporophore is frangible, not easily transports, preservation etc. is urgently to be resolved hurrily.This just requires that the relevant fundamental research of the genetic breeding of Lepista sordida is gone into overdrive, for traditional genetic breeding establishes solid theoretical basis and technical support.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of discriminating or assists the method and primer special thereof of differentiating Lepista sordida protoplastis monocaryon mating type to IS-857c.
Discriminating provided by the present invention or the auxiliary PCR primer pair differentiating Lepista sordida protoplastis monocaryon mating type, name is called IS-857c, is made up of two single stranded DNAs shown in SEQ ID No.1 and SEQ ID No.2.
Wherein, SEQ ID No.1 is made up of 24 deoxynucleotides, and SEQ ID No.2 is made up of 20 deoxynucleotides.
Reagent or the test kit of the discriminating containing PCR primer pair IS-857c or auxiliary discriminating Lepista sordida protoplastis monocaryon mating type also belong to protection scope of the present invention.
Described reagent or test kit except PCR primer pair IS-857c, also containing other conventional reagent carrying out PCR and DNA sequencing.
Experiment of the present invention proves that PCR primer pair IS-857c, reagent containing PCR primer pair IS-857c or test kit can be used for differentiating or auxiliaryly differentiate Lepista sordida protoplastis monocaryon mating type.
Discriminating provided by the present invention or the auxiliary method differentiating Lepista sordida protoplastis monocaryon mating type, comprise the steps: respectively with the genomic dna of two strain Lepista sordida protoplastis monocaryons to be identified for template, pcr amplification is carried out with the PCR primer pair IS-857c that two single stranded DNAs by SEQ ID No.1 and SEQ ID No.2 form, detect the size of the PCR primer obtained, determine the mating type of described two strain Lepista sordida protoplastis monocaryons to be identified according to the size of pcr amplification product by the following method:
If the DNA fragmentation of PCR primer all containing 500bp-800bp of described two strain Lepista sordida protoplastis monocaryons to be identified (or the PCR primer of described two strain Lepista sordida protoplastis monocaryons to be identified is the DNA fragmentation of 500bp-800bp), identical or the candidate of the mating type of described two strain Lepista sordida protoplastis monocaryons to be identified is that mating type is identical, if the DNA fragmentation of the PCR primer of described two strain Lepista sordida protoplastis monocaryons to be identified all not containing 500bp-800bp (or the PCR primer of described two strain Lepista sordida protoplastis monocaryons to be identified all not sized by be the DNA fragmentation of 500bp-800bp), identical or the candidate of the mating type of described two strain Lepista sordida protoplastis monocaryons to be identified is that mating type is identical, if the PCR primer of the Lepista sordida protoplastis monocaryon that the strain in described two strain Lepista sordida protoplastis monocaryons to be identified is to be identified contains the DNA fragmentation (or the PCR primer of a strain in described two strain Lepista sordida protoplastis monocaryons to be identified Lepista sordida protoplastis monocaryon to be identified is the DNA fragmentation of 500bp-800bp) of 500bp-800bp, the PCR primer of the Lepista sordida protoplastis monocaryon that another strain the is to be identified DNA fragmentation containing 500bp-800bp (or the PCR primer of another strain Lepista sordida protoplastis monocaryon to be identified be the DNA fragmentation of 500bp-800bp), the different or candidate of the mating type of described two strain Lepista sordida protoplastis monocaryons to be identified is mating type difference.
In aforesaid method, the DNA fragmentation of described 500bp-800bp is specially the DNA fragmentation of 765bp.
In one embodiment of the invention, the primer annealing condition that described pcr amplification adopts is 57 DEG C of annealing 30s.The PCR temperature programming adopted in described pcr amplification is as follows: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 40s, 30 circulations; 72 DEG C extend 7min.
In aforesaid method, described two strain Lepista sordida protoplastis monocaryons to be identified are cultivated by identical the referring to of the mating type of described two strain Lepista sordida protoplastis monocaryons to be identified together, do not produce clamp connexion between the mycelia of described two strain Lepista sordida protoplastis monocaryons to be identified; The mating type difference of described two strain Lepista sordida protoplastis monocaryons to be identified refers to cultivates described two strain Lepista sordida protoplastis monocaryons to be identified together, produces clamp connexion between the mycelia of described two strain Lepista sordida protoplastis monocaryons to be identified.
In aforesaid method, described Lepista sordida specifically can be Lepista sordida (Lepista sordida) CFCC89562.
Experiment proves, Auele Specific Primer of the present invention has specificity to IS-857c to Lepista sordida protoplastis monocaryon, the present invention utilize PCR primer pair IS-857c to differentiate the method whether mating type between Lepista sordida protoplastis monocaryon identical and existing somatic incompatibility experimental technique differentiate that the consistence of the method whether mating type between Lepista sordida protoplastis monocaryon identical is 100%.Present method shorten Lepista sordida protoplastis monocaryon mating type the discriminating time and simplify mating type differentiate step, its accuracy and reliability high.The monocaryon obtained by Protoplasting preparation is called Protoplasted monokaryon, different from Spore monokaryons, protoplastis monocaryon prepares the haploid vegetative progeny of the monokaryon directly obtained from Lepista sordida heterocaryotic mycelium in process at protoplastis, and therefore the Protoplasted monokaryon of Lepista sordida only has the mating type A of two kinds of forms xb xand A yb ytwo pairs.In the cross-breeding of routine, the mononuclear bacterial strain only having mating type different is cross fertile, and therefore the qualification of mating type is prerequisite and the prerequisite of cross-breeding.The present invention is significant to the genetic breeding and production practice of studying Lepista sordida.The present invention is applicable to the research of the relevant art such as Edible Fungi, strain improvement to aspects such as the discriminating of Lepista sordida protoplastis Protoplasted monokaryon and genetic breedings.
Accompanying drawing explanation
Fig. 1 is to the pcr amplification product electrophorogram of part Lepista sordida protoplastis monocaryon with primer pair IS-857c of the present invention.
In figure, the bacterial strain of 1 to 12 correspondence is followed successively by: No. 110, No. 58, No. 99, No. 67, No. 55, No. 137, No. 179, No. 69, No. 104, No. 139, No. 100, double-core bacterial strain; M:DNA Marker.
Fig. 2 is the electrophorogram with primer I SSR primer I SSR-UBC857, Lepista sordida protoplastis monocaryon being carried out to PCR.
In figure, the bacterial strain of 1 to 12 correspondence is followed successively by: No. 110, No. 58, No. 99, No. 67, No. 55, No. 137, No. 179, No. 69, No. 104, No. 139, No. 100, double-core bacterial strain; M:DNA Marker.
Fig. 3 is the clamp connexion photo produced between the mycelia of the two strain Lepista sordida protoplastis monocaryons that mating type is different.
In figure, arrow shows clamp connexion.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Lepista sordida in following embodiment is Lepista sordida (Lepista sordida) CFCC89562, the public can from China Committee for Culture Collection of Microorganisms forestry microorganism center (China Forest Microbiological Culture Collection administrative center, China Forestry Culture Collection Center english abbreviation CFCC, is called for short forestry microorganism center) obtain.Hereinafter, all Lepista sordida is called for short.
1, the Lepista sordida protoplastis monocaryon in following embodiment is all obtained by Protoplasting preparation, and concrete grammar is as follows:
1.1 substratum
MM damping fluid is by solute and solvent composition; Described solvent is 50mM maleate buffer (pH5.5); Described solute and the concentration in MM damping fluid as follows: 0.5M N.F,USP MANNITOL.
Liquid MCM substratum: by water-soluble to yeast extract 2g, Tryptones 2g, glucose 20g, magnesium sulfate 0.5g, potassium primary phosphate 0.5g and dipotassium hydrogen phosphate 1g and be settled to 1L with water.
Solid MCM is dull and stereotyped: in liquid MCM substratum, add agar, make its concentration be 20g/L.
The preparation method of solid regenerated flat board (RM): add sorbyl alcohol and agar in liquid MCM substratum, makes its concentration be respectively 1M and 20g/L.
1.2 protoplastis preparations
1) get Lepista sordida mycelia in liquid MCM substratum, 160rpm, 25 DEG C of cultivations, 4 days (3-5 days), filter with 3 layers of aseptic lens wiping paper and collect mycelia, then with 0.6M Osmitrol washing 2-3 time.
2) by step 1) Lepista sordida mycelia (about 1g) be suspended in 1.5% lywallzyme solution and (1.5g lywallzyme 0.6M Osmitrol dissolved and is settled to 100mL; Lywallzyme purchased from Guangdong Bide Biotechnology Co., Ltd., catalog number: Bd_8110001023), temperature bath 2 hours, filters with 3 layers of aseptic lens wiping paper and collects filtrate in the shaking bath (32 DEG C, 60rpm).
3) by step 2) the centrifugal 10min of filtrate 3000rpm and collecting precipitation.
4) by step 3) precipitation with after MM buffer solution three times, be suspended in 100 μ l MM damping fluids, be protoplast solution.
The acquisition of 1.3 Protoplasted monokaryons
It is 10 that the protoplastis MM damping fluid of acquisition is diluted to concentration 3-10 4individual/mL, is spread evenly across on RM, 28 DEG C of quiescent culture 10-14 days; Be forwarded on solid MCM substratum with aseptic inoculation pin after single bacterium colony that grows out, cultivate 5-7 days for 28 DEG C.
By the Protoplasted monokaryon number consecutively obtained, and observe under the microscope (Olympus BX51) of amplification 1000 times, if do not find clamp connexion, then this bacterial strain is Lepista sordida protoplastis monocaryon.Obtain 30 strain Lepista sordida protoplastis monocaryons altogether, their numbering is respectively 36,42,50,53,55,58,61,65,66,67,69,71,72,75,81,82,86,87,88,99,100,104,110,111,113,117,119,137,139,179.
2, differentiate that whether the mating type of Lepista sordida protoplastis monocaryon is identical
Two strain Lepista sordida protoplastis monocaryons to be identified are cultivated by identical the referring to of the mating type of two strain Lepista sordida protoplastis monocaryons to be identified together, do not produce clamp connexion between the mycelia of these two strain Lepista sordida protoplastis monocaryons to be identified; The mating type difference of two strain Lepista sordida protoplastis monocaryons to be identified refers to cultivates two strain Lepista sordida protoplastis monocaryons to be identified together, produces clamp connexion between the mycelia of these two strain Lepista sordida protoplastis monocaryons to be identified.Mating type between 30 strain Lepista sordida protoplastis monocaryons in 1.3 is differentiated according to following somatic incompatibility experimental technique:
PDA substratum: 200g potato, clean peeling is cut into small pieces, and the 1000ml that adds water boils half hour, filtered through gauze, gets filtrate and adds 20g glucose and 20g agar again, 121 DEG C of sterilizings 20 minutes.
Be inoculated into together on PDA substratum by the Lepista sordida protoplastis monocaryon of two strains mating type to be identified, open 2cm between the two, 25 DEG C of cultivations are observed for 10 days.If the mycelia of the Lepista sordida protoplastis monocaryon of visual inspection two strain mating type to be identified grows together, confirm that the mycelia of junction does not have clamp connexion further by microscopic examination, the mating type of the Lepista sordida protoplastis monocaryon of this two strain mating type to be identified is identical.If there is antagonism line in the place that the mycelia of the Lepista sordida protoplastis monocaryon of visual inspection two strain mating type to be identified crosses, mycelia then on picking antagonism line, being seeded on PDA substratum 25 DEG C cultivates after 5 days, whether clamp connexion is had again by microscopic examination, if without clamp connexion, the mating type of the Lepista sordida protoplastis monocaryon of this two strain mating type to be identified is identical; If there is clamp connexion, the mating type of the Lepista sordida protoplastis monocaryon of this two strain mating type to be identified is different.Fig. 3 shows the clamp connexion produced between the mycelia of two different strain Lepista sordida protoplastis monocaryons of mating type.
Differentiate the mating type of the 1.3 30 strain Lepista sordida protoplastis monocaryons obtained according to the method, result as shown in Tables 1 and 2.In table 1 and table 2, " √ " represents that between two bacterial strains, somatic incompatibility experimental result shows that the mating type of two bacterial strains is different; "×" represents that between two bacterial strains, somatic incompatibility experimental result shows that the mating type of two bacterial strains is identical.
Table 1. somatic incompatibility experimental technique differentiates the result whether mating type between Lepista sordida protoplastis monocaryon is identical
? 36 42 50 53 55 58 61 65 66 67 69 71 72 75 81
36 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
42 × ? ? ? ? ? ? ? ? ? ? ? ? ? ?
50 √ √ ? ? ? ? ? ? ? ? ? ? ? ? ?
53 √ √ × ? ? ? ? ? ? ? ? ? ? ? ?
55 × × √ √ ? ? ? ? ? ? ? ? ? ? ?
58 × × √ √ × ? ? ? ? ? ? ? ? ? ?
61 √ √ × × √ √ ? ? ? ? ? ? ? ? ?
65 √ √ × × √ √ × ? ? ? ? ? ? ? ?
66 √ √ × × √ √ × × ? ? ? ? ? ? ?
67 × × √ √ × × √ √ √ ? ? ? ? ? ?
69 √ √ × × √ √ × × × √ ? ? ? ? ?
71 √ √ × × √ √ × × × √ × ? ? ? ?
72 √ √ × × √ √ × × × √ × × ? ? ?
75 √ √ × × √ √ × × × √ × × × ? ?
81 × × √ √ × × √ √ √ × √ √ √ √ ?
82 × × √ √ × × √ √ √ × √ √ √ √ ×
86 × × √ √ × × √ √ √ × √ √ √ √ ×
87 × × √ √ × × √ √ √ × √ √ √ √ ×
88 √ √ × × √ √ × × × √ × × × × √
99 × × √ √ × × √ √ √ × √ √ √ √ ×
100 √ √ × × √ √ × × × √ × × × × √
104 √ √ × × √ √ × × × √ × × × × √
110 × × √ √ × × √ √ √ × √ √ √ √ ×
111 √ √ × × √ √ × × × √ × × × × √
113 × × √ √ × × √ √ √ × √ √ √ √ ×
117 × × √ √ × × √ √ √ × √ √ √ √ ×
119 × × √ √ × × √ √ √ × √ √ √ √ ×
137 × × √ √ × × √ √ √ × √ √ √ √ ×
139 √ √ × × √ √ × × × √ × × × × √
179 √ √ × × √ √ × × × √ × × × × √
Table 2. somatic incompatibility experimental technique differentiates the result whether mating type between Lepista sordida protoplastis monocaryon is identical
? 82 86 87 88 99 100 104 110 111 113 117 119 137 139 179
36 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
42 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
50 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
53 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
55 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
58 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
61 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
65 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
66 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
67 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
69 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
71 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
72 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
75 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
81 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
82 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
86 × ? ? ? ? ? ? ? ? ? ? ? ? ? ?
87 × × ? ? ? ? ? ? ? ? ? ? ? ? ?
88 √ √ √ ? ? ? ? ? ? ? ? ? ? ? ?
99 × × × √ ? ? ? ? ? ? ? ? ? ? ?
100 √ √ √ × √ ? ? ? ? ? ? ? ? ? ?
104 √ √ √ × √ × ? ? ? ? ? ? ? ? ?
110 × × × √ × √ √ ? ? ? ? ? ? ? ?
111 √ √ √ × √ × × √ ? ? ? ? ? ? ?
113 × × × √ × √ √ × √ ? ? ? ? ? ?
117 × × × √ × √ √ × √ × ? ? ? ? ?
119 × × × √ × √ √ × √ × × ? ? ? ?
137 × × × √ × √ √ × √ × × × ? ? ?
139 √ √ √ × √ × × √ × √ √ √ √ ? ?
179 √ √ √ × √ × × √ × √ √ √ √ × ?
Embodiment 1, PCR primer pair IS-857c is utilized to differentiate the mating type of Lepista sordida protoplastis monocaryon
1, differentiate or assist the PCR reagent differentiating Lepista sordida protoplastis monocaryon mating type
The discriminating of the present embodiment or auxiliaryly differentiate that the reagent of Lepista sordida protoplastis monocaryon mating type is by PCR primer pair IS-857c, 10 × Taq damping fluid, dNTP mix, Taq archaeal dna polymerase and ddH 2o forms.
Wherein, PCR primer pair IS-857c is made up of these two single stranded DNAs of IS-857c-F and IS-857c-R, and its sequence is as follows:
IS-857c-F:5’-TGAGATATAATCACGAGTGCAAAT-3’（SEQ?ID?No.1）
IS-857c-R:5’-TAGGACAAAATGGTAGGGGA-3’（SEQ?ID?No.2）。
10 × Taq damping fluid, dNTP mix and Taq archaeal dna polymerase are all purchased from Sheng Xubai river, Beijing company (CNS).
2, differentiate or assist discriminating Lepista sordida protoplastis monocaryon mating type
The 30 strain Lepista sordida protoplastis monocaryons obtained above-mentioned 1.3 are inoculated on PDA substratum respectively, and 25 DEG C of mycelia cultivated on 7-10 days, scraping PDA substratum are about 0.1g, extract DNA with the DNeasy Plant Mini Kit of QIAGEN.
Measure the concentration of gained DNA solution with extinction photometer, dilution stoste, make its OD value be 1, concentration is 50ng/ μ L.
Every strain Lepista sordida protoplastis monocaryon all adopts following PCR system and following PCR condition, respectively with Lepista sordida protoplastis monocaryon genomic dna for template, utilize the discriminating of step 1 or auxiliary differentiate that the reagent of Lepista sordida protoplastis monocaryon mating type carries out pcr amplification.PCR reaction system is as shown in table 3:
The PCR reaction system of table 3,20 μ L
PCR temperature programming: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 40s, 30 circulations; 72 DEG C extend 7min, 12 DEG C of preservations.
By pcr amplification product electrophoresis on the sepharose of 1.3% of every strain Lepista sordida protoplastis monocaryon, gel imaging, result shows that the PCR primer of 36,42,55,58,67,81,82,86,87,99,110,113,117,119, No. 137 Lepista sordida protoplastis monocaryons is the DNA band (DNA fragmentation) of a 500bp-800bp; DNA band is not had in the PCR primer of 50,53,61,65,66,69,71,72,75,88,100,104,111,139, No. 179 Lepista sordida protoplastis monocaryons.Fig. 1 shows the pcr amplification product of part Lepista sordida protoplastis monocaryon.
Reclaim the DNA band (DNA fragmentation) of the 500bp-800bp of every strain Lepista sordida protoplastis monocaryon respectively, check order, result shows that the size of the DNA band (DNA fragmentation) of the 500bp-800bp of 15 all strain Lepista sordida protoplastis monocaryons is 765bp.
Whether the mating type between the every two strain bacterial strains determining the above-mentioned 1.3 30 strain Lepista sordida protoplastis monocaryons obtained according to the size of pcr amplification product is by the following method identical: if the DNA fragmentation of PCR primer all containing 500bp-800bp of two strain Lepista sordida protoplastis monocaryons to be identified, described two strain Lepista sordida protoplastis monocaryons to be identified are the bacterial strain that mating type is identical, if the DNA fragmentation of the PCR primer of two strain Lepista sordida protoplastis monocaryons to be identified all not containing 500bp-800bp, described two strain Lepista sordida protoplastis monocaryons to be identified are the bacterial strain that mating type is identical, if the PCR primer of the Lepista sordida protoplastis monocaryon that the strain in two strain Lepista sordida protoplastis monocaryons to be identified is to be identified contains the DNA fragmentation of 500bp-800bp, the DNA fragmentation of PCR primer not containing 500bp-800bp of the Lepista sordida protoplastis monocaryon that another strain is to be identified, described two strain Lepista sordida protoplastis monocaryons to be identified are the bacterial strain that mating type is different.
The result that the present invention utilizes PCR primer pair IS-857c to differentiate that whether the mating type of every two strain Lepista sordida protoplastis monocaryons is identical is as shown in table 4 and 5.The method whether mating type of every two strain Lepista sordida protoplastis monocaryons is identical and existing somatic incompatibility experimental technique differentiate that the consistence of the method (table 1 and 2) whether mating type between Lepista sordida protoplastis monocaryon is identical is 100% to show the present invention to utilize PCR primer pair IS-857c to differentiate.
Differentiate the mating type of the 1.3 30 strain Lepista sordida protoplastis monocaryons obtained according to the method, result is as shown in table 4 and 5.In table 4 and table 5, " √ " represents that the mating type between the two strain Lepista sordida protoplastis monocaryons that the present invention utilizes PCR primer pair IS-857c to differentiate is different, and "×" represents that the mating type between the two strain Lepista sordida protoplastis monocaryons that the present invention utilizes PCR primer pair IS-857c to differentiate is identical.
Table 4, the present invention utilize the result whether mating type between PCR primer pair IS-857c discriminating Lepista sordida protoplastis monocaryon is identical.
? 36 42 50 53 55 58 61 65 66 67 69 71 72 75 81
36 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
42 × ? ? ? ? ? ? ? ? ? ? ? ? ? ?
50 √ √ ? ? ? ? ? ? ? ? ? ? ? ? ?
53 √ √ × ? ? ? ? ? ? ? ? ? ? ? ?
55 × × √ √ ? ? ? ? ? ? ? ? ? ? ?
58 × × √ √ × ? ? ? ? ? ? ? ? ? ?
61 √ √ × × √ √ ? ? ? ? ? ? ? ? ?
65 √ √ × × √ √ × ? ? ? ? ? ? ? ?
66 √ √ × × √ √ × × ? ? ? ? ? ? ?
67 × × √ √ × × √ √ √ ? ? ? ? ? ?
69 √ √ × × √ √ × × × √ ? ? ? ? ?
71 √ √ × × √ √ × × × √ × ? ? ? ?
72 √ √ × × √ √ × × × √ × × ? ? ?
75 √ √ × × √ √ × × × √ × × × ? ?
81 × × √ √ × × √ √ √ × √ √ √ √ ?
82 × × √ √ × × √ √ √ × √ √ √ √ ×
86 × × √ √ × × √ √ √ × √ √ √ √ ×
87 × × √ √ × × √ √ √ × √ √ √ √ ×
88 √ √ × × √ √ × × × √ × × × × √
99 × × √ √ × × √ √ √ × √ √ √ √ ×
100 √ √ × × √ √ × × × √ × × × × √
104 √ √ × × √ √ × × × √ × × × × √
110 × × √ √ × × √ √ √ × √ √ √ √ ×
111 √ √ × × √ √ × × × √ × × × × √
113 × × √ √ × × √ √ √ × √ √ √ √ ×
117 × × √ √ × × √ √ √ × √ √ √ √ ×
119 × × √ √ × × √ √ √ × √ √ √ √ ×
137 × × √ √ × × √ √ √ × √ √ √ √ ×
139 √ √ × × √ √ × × × √ × × × × √
179 √ √ × × √ √ × × × √ × × × × √
Table 5. the present invention utilizes the result whether mating type between PCR primer pair IS-857c discriminating Lepista sordida protoplastis monocaryon is identical.
? 82 86 87 88 99 100 104 110 111 113 117 119 137 139 179
36 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
42 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
50 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
53 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
55 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
58 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
61 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
65 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
66 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
67 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
69 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
71 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
72 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
75 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
81 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
82 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?
86 × ? ? ? ? ? ? ? ? ? ? ? ? ? ?
87 × × ? ? ? ? ? ? ? ? ? ? ? ? ?
88 √ √ √ ? ? ? ? ? ? ? ? ? ? ? ?
99 × × × √ ? ? ? ? ? ? ? ? ? ? ?
100 √ √ √ × √ ? ? ? ? ? ? ? ? ? ?
104 √ √ √ × √ × ? ? ? ? ? ? ? ? ?
110 × × × √ × √ √ ? ? ? ? ? ? ? ?
111 √ √ √ × √ × × √ ? ? ? ? ? ? ?
113 × × × √ × √ √ × √ ? ? ? ? ? ?
117 × × × √ × √ √ × √ × ? ? ? ? ?
119 × × × √ × √ √ × √ × × ? ? ? ?
137 × × × √ × √ √ × √ × × × ? ? ?
139 √ √ √ × √ × × √ × √ √ √ √ ? ?
179 √ √ √ × √ × × √ × √ √ √ √ × ?
Wherein, PCR primer pair IS-857c to screen by the following method and obtains:
1. molecule marker experiment
The 30 strain Lepista sordida protoplastis monocaryons obtained above-mentioned 1.3, and the double-core bacterial strain of Lepista sordida, totally 31 strain bacterial strains, be inoculated on PDA substratum, 25 DEG C of cultivations.After for some time, the mycelia on scraping PDA substratum is about 0.1g, extracts DNA with the DNeasy Plant Mini Kit of QIAGEN.
Measure the concentration of gained DNA solution with extinction photometer, dilution stoste, make its OD value be 1, concentration is about 50ng/ μ L.PCR reaction system is as shown in table 6:
The PCR reaction system of table 6:20 μ L
Wherein, the sequence of upstream and downstream primer is 5 '-acacacacacacacacyg-3 ', and name is called ISSR-UBC857.
PCR temperature programming is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 41 DEG C of annealing 45s, 72 DEG C extend 2min, 34 circulations; 72 DEG C extend 7min, 12 DEG C of preservations.
By above-mentioned amplified production, electrophoresis on the sepharose of 1.3%, gel imaging, analyzes mating type specific band.Find by analysis: in the fragment obtained with primer I SSR-UBC857 amplification, a part of Protoplasted monokaryon has a length in the fragment of about 800bp, and another part Protoplasted monokaryon is without this band (Fig. 2).
2. the recovery of specific band and cloning and sequencing
2.1 obtain object band: cut the specific band that glue reclaims No. 110 Protoplasted monokaryons---about 800bp.The sepharose DNA that cutting glue recovery used kit provides for middle Ke Ruitai reclaims test kit.By the specification sheets operation in test kit, reclaim the DNA of specific fragment, for ligation, build recombinant vectors.
2.2 connect: linked system is 4 μ L DNA Ethylene recov, the ddH of 4 μ L 2o, 0.5 μ L pMD19-T Vector(TaKaRa), 1 μ L T 4dNA ligase damping fluid (New England BioLabs), 0.5 μ L T 4dNA ligase (BioLabs).Configure this system on ice after, be placed in the water-bath of 16 DEG C, spend the night.
2.3 transform: the intestinal bacteria TOP10 competent cell (Tiangen) that-70 DEG C are preserved is placed in 4 DEG C and thaws.Get 5 μ L connecting fluids to mix with the competent cell of 50 μ L, place 30min on ice, then ice bath 2min after 42 DEG C of water-bath 90s, add LB liquid medium (sodium-chlor, the 10g/L of 1m L; Peptone, 10g/L; Yeast powder, 5g/L), 37 DEG C, rejuvenation 1 hour under 90rpm condition.
2.4 blue hickies screenings: the nutrient solution that rejuvenation is good is uniformly coated on and adds that final concentration is the sodium ampicillin of 0.1mg/mL, on the solid LB media of IPTG and 0.04mg/mL of 0.024mg/mL (LB liquid medium adding the agar powder of 20g/L), after coating evenly, the constant temperature culture 12-16 hour of 37 DEG C.After treating to grow blue hickie in flat board, draw hickie with the pipettor of 10 μ L, being seeded to containing final concentration is in the LB liquid nutrient medium of 0.1mg/mL sodium ampicillin, and at 37 DEG C, 180rpm cultivates 12-16 hour.
2.5PCR verifies changing effect and sample presentation order-checking: use primer pair RV-M (5 '-GAGCGGATAACAATTTCACACAGG-3 ') and M13-47 (5 '-CGCCAG GGTTTTCCCAGTCACGAC-3 '), be PCR with cultured bacterium liquid, PCR system is prepared by table 2.Response procedures is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 50s, 57.8 DEG C of annealing 1min, 72 DEG C extend 2min, 30 circulations; After 72 DEG C of extension 7min, PCR reactions terminate, the sepharose with 1% carries out electrophoresis, ultraviolet imagery.Calm and peaceful biotechnology (Beijing) company limited of Sino-U.S. is entrusted to carry out sequencing the bacterium liquid with the positive colony of object band.
The sequencing results of this about 800bp specific band is as SEQ ID No.3, and its size is 822bp.Design the PCR primer pair IS-857c be made up of this two single stranded DNAs of IS-857c-F and IS-857c-R according to SEQ ID No.3, its sequence is as follows:
IS-857c-F:5’-TGAGATATAATCACGAGTGCAAAT-3’（SEQ?ID?No.1）
IS-857c-R:5’-TAGGACAAAATGGTAGGGGA-3’（SEQ?ID?No.2）。

Claims (9)

1. differentiate or the auxiliary application of method in Lepista sordida breeding differentiating Lepista sordida protoplastis monocaryon mating type; Described discriminating or the auxiliary method differentiating Lepista sordida protoplastis monocaryon mating type, comprise the steps: respectively with the genomic dna of two strain Lepista sordida protoplastis monocaryons to be identified for template, pcr amplification is carried out by the PCR primer pair be made up of two single stranded DNAs shown in SEQ ID No.1 and SEQ ID No.2, detect the size of the PCR primer obtained, determine the mating type of described two strain Lepista sordida protoplastis monocaryons to be identified according to the size of pcr amplification product by the following method:
If the DNA fragmentation of PCR primer all containing 500bp-800bp of described two strain Lepista sordida protoplastis monocaryons to be identified, identical or the candidate of the mating type of described two strain Lepista sordida protoplastis monocaryons to be identified is that mating type is identical, if the PCR primer of described two strain Lepista sordida protoplastis monocaryons to be identified is not all containing the DNA fragmentation of 500bp-800bp, the identical or candidate of the mating type of described two strain Lepista sordida protoplastis monocaryons to be identified is that mating type is identical; If the PCR primer of the Lepista sordida protoplastis monocaryon that the strain in described two strain Lepista sordida protoplastis monocaryons to be identified is to be identified contains the DNA fragmentation of 500bp-800bp, the DNA fragmentation of PCR primer not containing 500bp-800bp of the Lepista sordida protoplastis monocaryon that another strain is to be identified, the mating type difference of described two strain Lepista sordida protoplastis monocaryons to be identified or candidate are that mating type is different;
Described Lepista sordida is numbered CFCC89562 China Committee for Culture Collection of Microorganisms forestry microorganism center.
2. application according to claim 1, is characterized in that: the DNA fragmentation of described 500bp-800bp is the DNA fragmentation of 765bp.
3. application according to claim 1 and 2, is characterized in that: the primer annealing condition that described pcr amplification adopts is 57 DEG C of annealing 30s.
4. application according to claim 1 and 2, is characterized in that: the PCR temperature programming adopted in described pcr amplification: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 57 DEG C of annealing 30s, 72 DEG C extend 40s, 30 circulations; 72 DEG C extend 7min.
5. differentiate or the auxiliary PCR primer pair differentiating Lepista sordida protoplastis monocaryon mating type, it is characterized in that: the name of described primer pair is called IS-857c, be made up of two single stranded DNAs shown in SEQ ID No.1 and SEQ ID No.2; Described Lepista sordida is numbered CFCC89562 China Committee for Culture Collection of Microorganisms forestry microorganism center.
6. the discriminating containing PCR primer pair according to claim 5 or the auxiliary reagent differentiating Lepista sordida protoplastis monocaryon mating type; Described Lepista sordida is numbered CFCC89562 China Committee for Culture Collection of Microorganisms forestry microorganism center.
7. the discriminating containing PCR primer pair according to claim 5 or the auxiliary test kit differentiating Lepista sordida protoplastis monocaryon mating type; Described Lepista sordida is numbered CFCC89562 China Committee for Culture Collection of Microorganisms forestry microorganism center.
8. PCR primer pair according to claim 5, reagent according to claim 6 or test kit according to claim 7 are being differentiated or the auxiliary application differentiated in Lepista sordida protoplastis monocaryon mating type; Described Lepista sordida is numbered CFCC89562 China Committee for Culture Collection of Microorganisms forestry microorganism center.
9. PCR primer pair according to claim 5, reagent according to claim 6 or the application of test kit according to claim 7 in Lepista sordida breeding; Described Lepista sordida is numbered CFCC89562 China Committee for Culture Collection of Microorganisms forestry microorganism center.
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