CN103194535B - Method for identification or auxiliary identification of mating type of protoplast monokaryon of Lepista sordid and special primer pair SR-5*16 used therein - Google Patents

Method for identification or auxiliary identification of mating type of protoplast monokaryon of Lepista sordid and special primer pair SR-5*16 used therein Download PDF

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CN103194535B
CN103194535B CN201310043763.XA CN201310043763A CN103194535B CN 103194535 B CN103194535 B CN 103194535B CN 201310043763 A CN201310043763 A CN 201310043763A CN 103194535 B CN103194535 B CN 103194535B
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paint face
face mushroom
mating type
identified
strain
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CN103194535A (en
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许峰
刘宇
李登进
赵爽
王守现
王兰青
耿小丽
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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 identification or auxiliary identification of the mating type of the protoplast monokaryon of Lepista sordid and a special primer pair SR-5*16 used therein. The method comprises the following steps: with genome DNAs of the protoplast monokaryon of two to-be-identified Lepista sordid strains as templates respectively, carrying out PCR amplification by using a PCR primer pair as represented by SEQ ID No. 1 and 2; and detecting the size of obtained PCR products, determining that the mating types of the protoplast monokaryon of the two to-be-identified Lepista sordid strains are identical if both PCR products of the protoplast monokaryon of the two Lepista sordid strains contain or do not contain 500 bp to 800 bp DNA fragments, and determining that the mating types of the protoplast monokaryon of the two to-be-identified Lepista sordid strains are different if the PCR product of the protoplast monokaryon of one of the two Lepista sordid strains contains 500 bp to 800 bp DNA fragments while the PCR product of the protoplast monokaryon of the other of the two Lepista sordid strains does not contain 500 bp to 800 bp DNA fragments.

Description

The method of discriminating or auxiliary discriminating paint face mushroom protoplastis monocaryon mating type and primer special thereof are to SR-5 × 16
Technical field
The present invention relates to differentiate or the method for the auxiliary paint face of discriminating mushroom protoplastis monocaryon mating type and primer special thereof to SR-5 × 16.
Background technology
The genetic system that system that nutrition is not affine is a kind of recognition of nonself, in most fungies, this system can cause that the upper visibly different individuality of heredity produces distinctive somatic incompatibility (Qi Yuancheng etc. at cross-connecting area, the comparison of the experiment of Chinese cultivated oyster cap fungus kind somatic incompatibility and RAPD analytical results. fungus journal, 2010,29(3)) reaction, the intensity of somatic incompatibility reaction is because of individual different different.In fungi, nutrition is not affine is called again that somatocyte is not affine or heterokaryon is not affine, and its effect is to prevent the fusion between the different individuality of gene on individuality that in same, mating type is different or the not affine site of nutrition, to keep the stability in idiogenetics.Mating type be according between the individuality of mating factor, can complete mating in conjunction with and definite bond type (China Standards Press's the first editing cubicle, Chinese agriculture standard compilation (edible fungus rolls). China Standards Press, 2010).In the syngenesis of fungi and the process of nourishing and growing, all there is mechanism or a system own or dissident identifies.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 being prevalent in fungi, in the fungies such as ascomycetes, basidiomycetes, zygomycetes, is a kind of ubiquitous phenomenon.In heterokaryon forming process, non-own recognition system finally causes the core that genetic background is different can not form stable heterokaryon by the interaction of a series of protein factors, namely so-called heteronuclear incompatibility.Heterokaryon incompatibility is one and is subject to the process of gene regulating and the often mycelium meeting death of Heterokaryon incompatibility.
Heterocaryotic mycelium prepares at protoplastis the phenomenon that occurs monokaryon protoplastis in process.These monokaryon protoplastiss cultivate through regeneration and genetic screening becomes protoplastis monocaryon, because protoplastis monocaryon is to be directed to vegetative hyphae, do not live through maiotic vegetative progeny, the heredity that it is edible mushrooms and breeding research provide a kind of very important base mateiral.The cross-breeding mode of protoplastis monocaryon is to access between two same PDA flat board from double-core parent's protoplastis monocaryon respectively two, and face-off is cultivated and 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, be difficult in protoplastis monocaryon, disperseing and dilution according to parental trait, in the procedure of breeding, can in a smaller range of variation, go selection to there is the sterile monocaryon of parental trait, effectively overcome that the parental trait that caused by spore monocaryon genetic diversity in traditional breeding method process disperses and the obstacle of selection difficulty, reduced the workload of screening filial generation.Meanwhile, because protoplastis monocaryon obtains under laboratory condition, without seasonal restriction, therefore shortened breeding time.
Paint face mushroom Lepista sordida(Fr) Sing belongs to Basidiomycotina (Basidiomycomycotina), Hymenomycetes (Hymenomycetes), Agaricales (Agaricales), Tricholomataceae (Tricholomataceae), Lepista lentinus (Lepista).This bacterium is nutritious, has certain medicinal efficacy.Mainly be distributed in the ground such as Guizhou, Heilungkiang, Liaoning, Hebei, Henan, Gansu, Qinghai, Sichuan, Xinjiang, Shanxi, the Inner Mongol and Fujian in China, a kind of famous and precious edible mushrooms and medicinal fungus, Lepista sordida is also in the bench-scale testing stage, wild yielding poorly and rareness, its price is extremely expensive, is a kind of wild-type strain that has very much potentiality to be exploited.But exist and yield poorly in actual production, insect resistance capacity is poor, adaptive temperature narrow range, sporophore is frangible, is difficult for the difficult problems urgently to be resolved hurrily such as transport, preservation.This just requires the relevant fundamental research of genetic breeding of paint face mushroom to go into overdrive, for traditional genetic breeding is established solid theoretical basis and technical support.
Summary of the invention
Technical problem to be solved by this invention be to provide a kind of differentiate or the method for the auxiliary paint face of discriminating mushroom protoplastis monocaryon mating type and primer special thereof to SR-5 × 16.
Discriminating provided by the present invention or the auxiliary PCR primer pair of differentiating paint face mushroom protoplastis monocaryon mating type, name is called SR-5 × 16, 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 20 deoxynucleotides, and SEQ ID No.2 is made up of 19 deoxynucleotides.
The discriminating that contains PCR primer pair SR-5 × 16 or auxiliary reagent or the test kit of differentiating paint face mushroom protoplastis monocaryon mating type also belong to protection scope of the present invention.
Described reagent or test kit, except PCR primer pair SR-5 × 16, also contain other the conventional reagent that carries out PCR and DNA sequencing.
The PCR of experimental results show that primer pair of the present invention SR-5 × 16, the reagent that contains PCR primer pair SR-5 × 16 or test kit can be used for differentiating or the auxiliary paint face of discriminating mushroom protoplastis monocaryon mating type.
Discriminating provided by the present invention or the auxiliary method of differentiating paint face mushroom protoplastis monocaryon mating type, comprise the steps: that respectively genomic dna taking two strain paint face mushroom protoplastis monocaryons to be identified is as template, use PCR primer pair SR-5 × 16 that formed by two single stranded DNAs of SEQ ID No.1 and SEQ ID No.2 to carry out pcr amplification, detect the size of the PCR product obtaining, determine by the following method the mating type of described two strain paint face mushroom protoplastis monocaryons to be identified according to the size of pcr amplification product:
If the PCR product of described two strain paint face mushroom protoplastis monocaryons to be identified all contains the DNA fragmentation (or the PCR product of described two strain paint face mushroom protoplastis monocaryons to be identified is the DNA fragmentation of 500bp-800bp) of 500bp-800bp, identical or the candidate of the mating type of described two strain paint face mushroom protoplastis monocaryons to be identified is that mating type is identical, if the PCR product of described two strain paint face mushroom protoplastis monocaryons to be identified does not all contain the DNA fragmentation (or the PCR product of described two strain paint face mushroom protoplastis monocaryons to be identified is all for size is the DNA fragmentation of 500bp-800bp) of 500bp-800bp, identical or the candidate of the mating type of described two strain paint face mushroom protoplastis monocaryons to be identified is that mating type is identical, if the DNA fragmentation that the PCR product of the paint face mushroom protoplastis monocaryon to be identified of the strain in described two strain paint face mushroom protoplastis monocaryons to be identified contains 500bp-800bp (or the PCR product of a strain in described two strain paint face mushroom protoplastis monocaryons to be identified paint face mushroom protoplastis monocaryon to be identified be 500bp-800bp DNA fragmentation), the PCR product of another strain paint face mushroom protoplastis monocaryon to be identified does not contain the DNA fragmentation (or the PCR product of another strain paint face mushroom protoplastis monocaryon to be identified is not the DNA fragmentation of 500bp-800bp) of 500bp-800bp, and mating type difference or the candidate of described two strain paint face mushroom protoplastis monocaryons to be identified are mating type difference.
In aforesaid method, the DNA fragmentation of described 500bp-800bp is specially the DNA fragmentation of 641bp.
In one embodiment of the invention, the primer annealing condition that described pcr amplification adopts is 56 DEG C of annealing 30s.The PCR temperature programming adopting in described pcr amplification is as follows: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C are extended 40s, 30 circulations; 72 DEG C are extended 7min.
In aforesaid method, the mating type of two strain paint face mushroom protoplastis monocaryons is identical refers to that the Mycelial interaction place of two strain paint face mushroom protoplastis monocaryons, without antagonism line, can not produce clamp connexion by the cultivation that stands facing each other of two strain paint face mushroom protoplastis monocaryons together; The mating type difference of two strain paint face mushroom protoplastis monocaryons refers to that there is antagonism line at the Mycelial interaction place of two strain paint face mushroom protoplastis monocaryons produces clamp connexion, cross fertile by the cultivation that stands facing each other of two strain paint face mushroom protoplastis monocaryons together.
Experimental results show that, Auele Specific Primer of the present invention has specificity to SR-5 × 16 pair paint face mushroom protoplastis monocaryon, and it is 100% that the present invention utilizes the consistence of the method whether method that PCR primer pair SR-5 × 16 differentiate that whether mating type between paint face mushroom protoplastis monocaryon is identical is identical with mating type between existing somatic incompatibility experimental technique discriminating paint face mushroom protoplastis monocaryon.Present method has shortened the discriminating time of paint face mushroom protoplastis monocaryon mating type and has simplified the step that mating type is differentiated, its accuracy and reliability are high.The monocaryon obtaining by protoplastis monokaryonization is called Protoplasted monokaryon, different from spore monocaryon, protoplastis monocaryon prepares at protoplastis the haploid vegetative progeny of monokaryon directly obtaining from paint face mushroom heterocaryotic mycelium in process, and therefore the Protoplasted monokaryon of paint face mushroom only has the mating type A of two kinds of forms xb xand A yb ytwo pairs.In conventional cross-breeding, only having the monokaryon bacterial strain that mating type is 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 genetic breeding and the production practice of research paint face mushroom.The present invention is applicable to the research of the aspect such as discriminating and genetic breeding of the relevant speciality such as Edible Fungi, strain improvement field to paint face mushroom protoplastis Protoplasted monokaryon.
Brief description of the drawings
Fig. 1 is the pcr amplification product electrophorogram with primer pair of the present invention SR-5 × 16 pair part paint face mushroom protoplastis monocaryon.
In figure, 1 to No. 9 corresponding bacterial strain is followed successively by: No. 110, and No. 67, No. 55, No. 137; No. 179, No. 139, No. 100, No. 65, double-core bacterial strain; M:DNA Marker.
Fig. 2 is the electrophorogram that with primer SRAP-me5 and SRAP-em16, paint face mushroom protoplastis monocaryon is carried out PCR.
In figure, the bacterial strain of 1 to 7 correspondence is followed successively by: No. 110, and No. 58, No. 99, No. 179, No. 69, No. 104, the genomic dna of double-core bacterial strain; M:DNA Marker.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.
Paint face mushroom Lepista sordida(Fr) Sing(swallow continues bright etc. paint face mushroom protein crude extract control pepper virus disease and growth-promoting functions research. Chinese biological control journal 28 (2) 269-274) public can be from field acquisition, also can obtain from commercial channels, also can obtain from Beijing City Agriculture and Forestry Institute, to repeat the present invention's experiment.Hereinafter, be all called for short paint face mushroom.
1, the paint face mushroom protoplastis monocaryon in following embodiment all obtains by protoplastis monokaryonization, and concrete grammar is as follows:
1.1 substratum
MM damping fluid is by solute and solvent composition; Described solvent is 50mM toxilic acid damping fluid (pH5.5); Described solute and the concentration in MM damping fluid thereof are as follows: 0.5M N.F,USP MANNITOL.
Liquid MCM substratum: 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 water are settled to 1L.
Solid MCM flat board: add agar in liquid MCM substratum, making its concentration is 20g/L.
The preparation method of solid regenerated flat board (RM): add sorbyl alcohol and agar in liquid MCM substratum, make its concentration be respectively 1M and 20g/L.
1.2 protoplastis preparations
1) get paint face mushroom mycelia in liquid MCM substratum, 160rpm, 25 DEG C cultivate 4 days (3-5 days all can), filter and collect mycelia with 3 layers of aseptic lens wiping paper, and then use 0.6M Osmitrol washs 2-3 time.
2) by step 1) paint face mushroom mycelia (about 1g) be suspended in 1.5% lywallzyme solution and (1.5g lywallzyme 0.6M Osmitrol dissolved and be settled to 100mL; Lywallzyme is purchased from Guangdong Bide Biotechnology Co., Ltd., catalog number: Bd_8110001023), in shaking bath (32 DEG C, 60rpm), temperature is bathed 2 hours, filters and collect filtrate with 3 layers of aseptic lens wiping paper.
3) by step 2) the centrifugal 10min of filtrate 3000rpm collecting precipitation.
4) by step 3) precipitation with after MM damping fluid washing three times, be suspended in 100 μ l MM damping fluids, be protoplastis solution.
The acquisition of 1.3 Protoplasted monokaryons
It is 10 that the protoplastis of acquisition is diluted to concentration with MM damping fluid 3-10 4individual/mL, evenly coats RM upper, and 28 DEG C leave standstill cultivation 10-14 days; After single bacterium colony that grows out, be forwarded on solid MCM substratum with aseptic inoculation pin, cultivate 5-7 days for 28 DEG C.
By the Protoplasted monokaryon number consecutively obtaining, and in the lower observation of the microscope (Olympus BX51) that amplifies 400 times, if do not find clamp connexion, this bacterial strain is paint face mushroom protoplastis monocaryon.Obtain altogether 30 strain paint face mushroom protoplastis monocaryons, 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, the mating type of the paint face mushroom protoplastis monocaryon in following embodiment is differentiated according to following antagonistic experiment method
PDA substratum: 200g potato, clean peeling is cut into small pieces, and the 1000ml that adds water boils half hour, and filtered through gauze is got filtrate and is added 20g glucose and 20g agar again, 121 DEG C of sterilizings 20 minutes.
The paint face mushroom protoplastis monocaryon of two strains mating type to be identified is inoculated on PDA substratum together, opens 2cm between between the two, cultivate 7-10 days for 25 DEG C.If the mycelia of the paint face mushroom protoplastis monocaryon of two strains mating type to be identified grows together, the mycelia of examining under a microscope junction does not have clamp connexion, and the mating type of the paint face mushroom 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 paint face mushroom protoplastis monocaryon of two strains mating type to be identified crosses, the mycelia on picking antagonism line, to on PDA substratum 25 DEG C cultivate after 3-5 days, examine under a microscope and whether have clamp connexion, if without clamp connexion, the mating type of the paint face mushroom protoplastis monocaryon of this two strain mating type to be identified is identical; If there is clamp connexion, the mating type difference of the paint face mushroom protoplastis monocaryon of this two strain mating type to be identified.
The mating type of differentiating the 1.3 30 strain paint face mushroom protoplastis monocaryons that obtain 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 experiment has antagonism line, the mating type difference of two bacterial strains; "×" represents that between two bacterial strains, somatic incompatibility is tested without antagonism line, and the mating type of two bacterial strains is identical.
Table 1. somatic incompatibility experimental technique is differentiated the whether identical result of mating type between paint face mushroom protoplastis monocaryon
? 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 is differentiated the whether identical result of mating type between paint face mushroom protoplastis monocaryon
? 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, utilize PCR primer pair SR-5 × 16 to differentiate the mating type of paint face mushroom protoplastis monocaryon
1, differentiate or assist the PCR reagent of differentiating paint face mushroom protoplastis monocaryon mating type
The discriminating of the present embodiment or the auxiliary reagent of differentiating paint face mushroom protoplastis monocaryon mating type are by PCR primer pair SR-5 × 16,10 × Taq damping fluid, dNTP mix, Taq archaeal dna polymerase and ddH 2o composition.
Wherein, PCR primer pair SR-5 × 16 are made up of SR-5 × 16-pre-F and these two single stranded DNAs of SR-5 × 16-pre-R, and its sequence is as follows:
SR-5×16-pre-F:5’-ATCAGGTAGGTAGGCGTAAC-3’（SEQ?ID?No.1），
SR-5×16-pre-R:5’-TGCGTACGAATTCGGGTAA-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 and differentiate paint face mushroom protoplastis monocaryon mating type
The above-mentioned 1.3 30 strain paint face mushroom protoplastis monocaryons that obtain are inoculated into respectively on PDA substratum, cultivate 7-10 days for 25 DEG C, the about 0.1g of mycelia on scraping PDA substratum, with the DNeasy Plant Mini Kit extraction DNA of QIAGEN.
Measure the concentration of gained DNA solution with extinction photometer, dilution stoste, making its OD value is 1, concentration is 50ng/ μ L.
Every strain paint face mushroom protoplastis monocaryon all adopts following PCR system and following PCR condition, taking paint face mushroom protoplastis monocaryon genomic dna as template, utilize the discriminating of step 1 or the reagent of the auxiliary paint face of discriminating mushroom protoplastis monocaryon mating type to carry out pcr amplification respectively.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, 56 DEG C of annealing 30s, 72 DEG C are extended 40s, 30 circulations; 72 DEG C are extended 7min, 12 DEG C of preservations.
By pcr amplification product electrophoresis on 1.3% sepharose of every strain paint face mushroom protoplastis monocaryon, gel imaging, result shows that the PCR product of 36,42,55,58,67,81,82,86,87,99,110,113,117,119, No. 137 paint face mushroom protoplastis monocaryons is the DNA fragmentation of a 500bp-800bp; 50, in the PCR product of 53,61,65,66,69,71,72,75,88,100,104,111,139, No. 179 paint face mushroom protoplastis monocaryons, there is no DNA band.Fig. 1 has shown the pcr amplification product of part paint face mushroom protoplastis monocaryon.
The DNA fragmentation that reclaims respectively the 500bp-800bp of every strain paint face mushroom protoplastis monocaryon, checks order, and result shows that the size of the DNA fragmentation of the 500bp-800bp of 15 all strain paint face mushroom protoplastis monocaryons is 641bp.
Determine according to the size of pcr amplification product that by the following method whether the mating type between every two strain bacterial strains of the above-mentioned 1.3 30 strain paint face mushroom protoplastis monocaryons that obtain is identical: if the PCR product of two strain paint face mushroom protoplastis monocaryons to be identified all contains the DNA fragmentation of 500bp-800bp, described two strain paint face mushroom protoplastis monocaryons to be identified are the bacterial strain that mating type is identical, if the PCR product of two strain paint face mushroom protoplastis monocaryons to be identified does not all contain the DNA fragmentation of 500bp-800bp, described two strain paint face mushroom protoplastis monocaryons to be identified are the bacterial strain that mating type is identical, if the DNA fragmentation that the PCR product of the paint face mushroom protoplastis monocaryon to be identified of the strain in two strain paint face mushroom protoplastis monocaryons to be identified contains 500bp-800bp, the PCR product of another strain paint face mushroom protoplastis monocaryon to be identified does not contain the DNA fragmentation of 500bp-800bp, and described two strain paint face mushroom protoplastis monocaryons to be identified are the bacterial strain that mating type is different.
The present invention utilizes result that PCR primer pair SR-5 × 16 differentiate that whether the mating type of every two strain paint face mushroom protoplastis monocaryons is identical as shown in table 4 and 5.Show that it is 100% that the present invention utilizes the consistence of the method (table 1 and 2) whether method that PCR primer pair SR-5 × 16 differentiate that whether the mating type of every two strain paint face mushroom protoplastis monocaryons is identical is identical with mating type between existing somatic incompatibility experimental technique discriminating paint face mushroom protoplastis monocaryon.
The mating type of differentiating the 1.3 30 strain paint face mushroom protoplastis monocaryons that obtain according to the method, result is as shown in table 4 and 5.In table 4 and table 5, " √ " represent the present invention utilize PCR primer pair SR-5 × 16 differentiate two paint face mushroom protoplastis monocaryons between mating type difference, "×" represent the present invention utilize PCR primer pair SR-5 × 16 differentiate two paint face mushroom protoplastis monocaryons between mating type identical.
Table 4, the present invention utilize PCR primer pair SR-5 × 16 to differentiate the whether identical result of mating type between paint face mushroom protoplastis monocaryon.
? 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 PCR primer pair SR-5 × 16 to differentiate the whether identical result of mating type between paint face mushroom protoplastis monocaryon.
? 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 SR-5 × 16 are screened by the following method and to be obtained:
1. molecule marker experiment
By the above-mentioned 1.3 30 strain paint face mushroom protoplastis monocaryons that obtain, and the double-core bacterial strain of paint face mushroom, totally 31 kinds of bacterial strains, are inoculated on PDA substratum, 25 DEG C of cultivations.After for some time, the about 0.1g of mycelia on scraping PDA substratum, with the DNeasy Plant Mini Kit extraction DNA of QIAGEN.
Measure the concentration of gained DNA solution with extinction photometer, dilution stoste, making its OD value is 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, upstream primer SRAP-me5:5 '-TGAGTCCAAACCGGAAG-3 ', downstream primer SRAP-em16:5 '-GACTGCGTACGAATTCGG-3 '.
PCR temperature programming is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 1min, 35 DEG C of annealing 1min, 72 DEG C are extended 1min, 5 circulations; 94 DEG C of sex change 1min, 50 DEG C of annealing 1min, 72 DEG C are extended 1min, 35 circulations; 72 DEG C are extended 10min, and 12 DEG C of preservations are above-mentioned amplified production, electrophoresis on 1.3% sepharose, and gel imaging, analyzes mating type specific band.Find by analysis: in the fragment obtaining with primer SRAP-me5 and SRAP-em16 amplification, a part of Protoplasted monokaryon has the fragment of a length in 700bp left and right, 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: the specific band of cutting No. 110 Protoplasted monokaryons of glue recovery---700bp left and right.Cut glue and reclaim the sepharose DNA recovery test kit that used kit provides for middle Ke Ruitai.Press the specification sheets operation in test kit, reclaim the DNA of specific fragment, for ligation, build recombinant vectors.
2.2 connect: linked system is that 4 μ L DNA reclaim liquid, 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 on ice after this system, be placed in the water-bath of 16 DEG C, spend the night.
2.3 transform: the intestinal bacteria TOP10 competent cell (Tiangen) of-70 DEG C of preservations is placed in to 4 DEG C and thaws.Get 5 μ L connecting fluids and 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, adds liquid LB substratum (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 hickie screenings: the good nutrient solution of rejuvenation is uniformly coated on and is added on sodium ampicillin, the IPTG of 0.024mg/mL and the solid LB substratum of 0.04mg/mL (adding the agar powder of 20g/L on liquid LB substratum) that final concentration is 0.1mg/mL, 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, be seeded to and contain in the LB liquid nutrient medium that final concentration is 0.1mg/mL sodium ampicillin, at 37 DEG C, 180rpm cultivates 12-16 hour.
2.5PCR checking changing effect 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 are extended 2min, 30 circulations; 72 DEG C are extended 7min, and after PCR reaction finishes, the sepharose with 1% carries out electrophoresis, ultraviolet imagery.Entrust the calm and peaceful biotechnology of Sino-U.S. (Beijing) company limited to carry out sequencing the bacterium liquid of the positive colony with object band.
The sequencing result of this 700bp left and right specific band is as SEQ ID No.3, and its size is 727bp.Design PCR primer pair SR-5 × 16 that are made up of SR-5 × 16-pre-F and these two single stranded DNAs of SR-5 × 16-pre-R according to SEQ ID No.3, its sequence is as follows:
SR-5×16-pre-F:5’-ATCAGGTAGGTAGGCGTAAC-3’（SEQ?ID?No.1），
SR-5×16-pre-R:5’-TGCGTACGAATTCGGGTAA-3’（SEQ?ID?No.2）。

Claims (9)

1. the application of the method for discriminating or auxiliary discriminating paint face mushroom protoplastis monocaryon mating type in the breeding of paint face mushroom; Described discriminating or the auxiliary method of differentiating paint face mushroom protoplastis monocaryon mating type, comprise the steps: that respectively genomic dna taking two strain paint face mushroom protoplastis monocaryons to be identified is as template, use the PCR primer pair being formed by two single stranded DNAs shown in SEQ ID No.1 and SEQ ID No.2 to carry out pcr amplification, detect the size of the PCR product obtaining, determine by the following method the mating type of described two strain paint face mushroom protoplastis monocaryons to be identified according to the size of pcr amplification product:
If the PCR product of described two strain paint face mushroom protoplastis monocaryons to be identified all contains the DNA fragmentation of 500bp-800bp, identical or the candidate of the mating type of described two strain paint face mushroom protoplastis monocaryons to be identified is that mating type is identical, if the PCR product of described two strain paint face mushroom protoplastis monocaryons to be identified does not all contain the DNA fragmentation of 500bp-800bp, the identical or candidate of the mating type of described two strain paint face mushroom protoplastis monocaryons to be identified is that mating type is identical; If the DNA fragmentation that the PCR product of the paint face mushroom protoplastis monocaryon to be identified of the strain in described two strain paint face mushroom protoplastis monocaryons to be identified contains 500bp-800bp, the PCR product of another strain paint face mushroom protoplastis monocaryon to be identified does not contain the DNA fragmentation of 500bp-800bp, and mating type difference or the candidate of described two strain paint face mushroom protoplastis monocaryons to be identified are mating type difference; Described paint face mushroom is paint face mushroom Lepista sordida (Fr) Sing.
2. application according to claim 1, is characterized in that: the DNA fragmentation that the DNA fragmentation of described 500bp-800bp is 641bp.
3. application according to claim 1 and 2, is characterized in that: the primer annealing condition that described pcr amplification adopts is 56 DEG C of annealing 30s.
4. application according to claim 1 and 2, is characterized in that: the PCR temperature programming adopting in described pcr amplification: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, 72 DEG C are extended 40s, 30 circulations; 72 DEG C are extended 7min, 12 DEG C of preservations.
5. differentiate or assist the PCR primer pair of differentiating paint face mushroom protoplastis monocaryon mating type, it is characterized in that: the name of described primer pair is called SR-5 × 16, is made up of two single stranded DNAs shown in SEQ ID No.1 and SEQ ID No.2; Described paint face mushroom is paint face mushroom Lepista sordida (Fr) Sing.
6. contain the discriminating of PCR primer pair claimed in claim 5 or the reagent of the auxiliary paint face of discriminating mushroom protoplastis monocaryon mating type; Described paint face mushroom is paint face mushroom Lepista sordida (Fr) Sing.
7. contain the discriminating of PCR primer pair claimed in claim 5 or the test kit of the auxiliary paint face of discriminating mushroom protoplastis monocaryon mating type; Described paint face mushroom is paint face mushroom Lepista sordida (Fr) Sing.
8. PCR primer pair claimed in claim 5, reagent claimed in claim 6 or test kit claimed in claim 7 are in discriminating or the auxiliary application of differentiating in paint face mushroom protoplastis monocaryon mating type; Described paint face mushroom is paint face mushroom Lepista sordida (Fr) Sing.
9. PCR primer pair claimed in claim 5, reagent claimed in claim 6 or test kit claimed in claim 7 application in the breeding of paint face mushroom; Described paint face mushroom is paint face mushroom Lepista sordida (Fr) Sing.
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