CN103266173A - Method for identifying lepista sordida protoplast monokaryon mating types and special primer pair SR-6*14 therefor - Google Patents
Method for identifying lepista sordida protoplast monokaryon mating types and special primer pair SR-6*14 therefor Download PDFInfo
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Abstract
The invention discloses a method for identifying lepista sordida protoplast monokaryon mating types and a special primer pair SR-6*14 therefor. The method comprises the following steps of: with genomic DNA (deoxyribonucleic acid) of two lepista sordida protoplast monokaryon to be identified as templates, carrying out PCR (polymerase chain reaction) amplification by using a PCR primer pair consisting of two single-stranded DNA shown by SEQ ID NO.1 and SEQ ID No.2; and detecting the sizes of the obtained PRC products, wherein if the PCR products of the two lepista sordida protoplast monokaryon to be identified contain or do not contain the DNA fragment of 500bp-800bp, the mating types of the two lepista sordida protoplast monokaryon to be identified are same, if the PCR product of one of the two lepista sordida protoplast monokaryon to be identified contains the DNA fragment of 500bp-800bp, and the PRC product of the other of the two lepista sordida protoplast monokaryon to be identified does not contain the DNA fragment of 500bp-800bp, the mating types of the two lepista sordida protoplast monokaryon to be identified are different.
Description
Technical field
The present invention relates to differentiate that the method for the fragrant mushroom protoplastis of paint face monocaryon mating type and primer special thereof are to SR-6 * 14.
Background technology
The genetic system that the not affine system of nutrition is a kind of recognition of nonself, in most fungies, this system can cause that hereditary visibly different individuality produces distinctive somatocyte incompatibility (Qi Yuancheng etc. at cross-connecting area, the comparison of the experiment of Chinese cultivated oyster cap fungus kind somatocyte incompatibility and RAPD analytical results. the fungus journal, 2010,29(3)) reaction, the intensity of somatocyte incompatibility reaction is because of individual different different.In fungi, nutrition is not affine, and to be called somatocyte again affine or heterokaryon is not affine, and its effect is to prevent the fusion between the different individuality of gene on individuality that same intrabreeding type is different or the not affine site of nutrition, to keep the stability in the idiogenetics.Mating type be according to can finish between the individuality of mating factor mating in conjunction with the bond type determined (China Standard Press's first editing cubicle, the Chinese agriculture standard collects (edible mushrooms volume). China Standard 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.The heterokaryon incompatibility is a kind of biological phenomena that is prevalent in the fungi, is a kind of ubiquitous phenomenon in fungies such as ascomycetes, basidiomycetes, zygomycetes.In the heterokaryon forming process, non-own recognition system finally causes the different nuclear of genetic background can not form stable heterokaryon by the interaction of a series of protein factors, just so-called heteronuclear incompatibility.The heterokaryon incompatibility is one and is subjected to the process of gene regulating and the often mycelium meeting death of heterokaryon incompatibility.
Heterocaryotic mycelium prepares the phenomenon that occurs the monokaryon protoplastis in the process at protoplastis.These monokaryon protoplastiss cultivate through regeneration and genetic screening becomes the protoplastis monocaryon, because the protoplastis monocaryon is to be directed to vegetative hyphae, do not live through maiotic vegetative progeny, it provides a kind of very important base mateiral for heredity and the breeding research of edible mushrooms.The cross-breeding mode of protoplastis monocaryon is to insert same PDA flat board to two in twos from double-core parent's protoplastis monocaryon respectively, 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 the protoplastis monocaryon, disperseing and dilution according to parent's proterties, the sterile monocaryon that in the procedure of breeding, can go to select to have parent's proterties in the smaller range of variation, overcome effectively that parent's proterties of being caused by spore monocaryon genetic diversity in the traditional breeding method process is disperseed and the obstacle of selection difficulty, reduced the workload of screening filial generation.Simultaneously, because the protoplastis monocaryon obtains, do not have seasonal restriction under laboratory condition, therefore shortened breeding time.
The fragrant mushroom of paint face (Lepista sordida) 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 ground such as Guizhou, Heilungkiang, Liaoning, Hebei, Henan, Gansu, Qinghai, Sichuan, Xinjiang, Shanxi, the Inner Mongol and Fujian in China, be a kind of famous and precious edible mushrooms and medicinal fungus, the fragrant mushroom of artificial culture paint face also is in the bench-scale testing stage, wild yielding poorly and rareness, its price is extremely expensive, is a kind of wild-type strain that potentiality to be exploited is arranged very much.Yield poorly but exist in the actual production, insect resistance capacity is poor, the adaptive temperature narrow range, and sporophore is frangible, is difficult for the difficult problem that transportation, preservation etc. need to be resolved hurrily.This just requires the relevant fundamental research of genetic breeding of the fragrant mushroom of paint face to go into overdrive, for traditional genetic breeding is established solid theory and technical support.
Summary of the invention
Technical problem to be solved by this invention provide a kind of differentiate or the method for the fragrant mushroom protoplastis of the auxiliary paint face of discriminating monocaryon mating type and primer special thereof to SR-6 * 14.
The PCR primer of discriminating provided by the present invention or the fragrant mushroom protoplastis of auxiliary discriminating paint face monocaryon mating type is right, and name is called SR-6 * 14, is made up of two single stranded DNAs shown in SEQ ID No.1 and the SEQ ID No.2.
Wherein, SEQ ID No.1 is made up of 20 deoxynucleotides, and SEQ ID No.2 is made up of 20 deoxynucleotides.
Contain the PCR primer to the discriminating of SR-6 * 14 or assist reagent or the test kit of differentiating the fragrant mushroom protoplastis of paint face monocaryon mating type also to belong to protection scope of the present invention.
Described reagent or test kit except the PCR primer to SR-6 * 14, also contain other the conventional reagent that carries out PCR and dna sequencing.
The PCR of experimental results show that primer of the present invention to SR-6 * 14, contain the PCR primer to the reagent of SR-6 * 14 or test kit can be used for differentiating or the fragrant mushroom protoplastis of the auxiliary paint face of discriminating monocaryon mating type.
Discriminating provided by the present invention or the auxiliary method of differentiating the fragrant mushroom protoplastis of paint face monocaryon mating type, comprise the steps: that respectively the genomic dna with the fragrant mushroom protoplastis of two strain paint face to be identified monocaryon is template, use the PCR primer of being formed by two single stranded DNAs of SEQ ID No.1 and SEQ ID No.2 that pcr amplification is carried out in SR-6 * 14, detect the size of resulting PCR product, determine the mating type of the described fragrant mushroom protoplastis of two strain paint face monocaryon to be identified according to the size of pcr amplification product according to following method:
If, for the two to be the identification of Lepista sordida protoplast monokaryons PCR products contain 500bp-800bp DNA fragment ( or the identification of the two strains to be Lepista sordida protoplast monokaryons PCR products were of 500bp-800bp DNA fragment ) , the to-be identified two Lepista sordida protoplast monokaryons same mating type , or candidate for the same mating type , if , pending identification of the two Lepista sordida protoplast monokaryons PCR product , not containing 500bp-800bp DNA fragment ( or the identification of the two strains to be Lepista sordida protoplast monokaryons not the size of the PCR product is 500bp-800bp DNA fragment ) , the identification of the two to be described Lepista sordida protoplast monokaryons same mating type , or candidate for the same mating type ; if , pending identification of the two Lepista sordida protoplast monokaryons be identified in a Lepista sordida protoplast monokaryons a PCR product contains 500bp-800bp DNA fragment ( or the identification of the two strains to be Lepista sordida monokaryons protoplast to be identified in a strain of Lepista sordida mononuclear protoplast the PCR product was 500bp-800bp DNA fragment ) , the identification of other strains to be Lepista sordida monokaryons protoplast does not contain a PCR product of 500bp-800bp DNA fragment ( or the identification of other strains to be protoplasts Lepista sordida monokaryons PCR product is not 500bp-800bp DNA fragment ) , said to be the identification of two Lepista sordida protoplast monokaryons different mating type , or candidate for the different mating type .
In the aforesaid method, the dna fragmentation of described 500bp-800bp is specially the dna fragmentation of 636bp.
In one embodiment of the invention, the primer annealing condition of described pcr amplification employing is 57 ℃ of annealing 30s.The PCR temperature programming that adopts in the described pcr amplification is as follows: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 40s, 30 circulations; 72 ℃ are extended 7min.
In the aforesaid method, identical the referring to of the mating type of the fragrant mushroom protoplastis of described two strain paint face to be identified monocaryon cultivated the fragrant mushroom protoplastis of described two strain paint face to be identified monocaryon together, do not produce clamp connexion between the mycelia of the described fragrant mushroom protoplastis of two strain paint face monocaryon to be identified; The mating type difference of the fragrant mushroom protoplastis of described two strain paint face to be identified monocaryon refers to the fragrant mushroom protoplastis of described two strain paint face to be identified monocaryon is cultivated together, produces clamp connexion between the mycelia of the described fragrant mushroom protoplastis of two strain paint face monocaryon to be identified.
In the aforesaid method, the fragrant mushroom of described paint face specifically can be fragrant mushroom (Lepista sordida) CFCC89562 of paint face.Experimental results show that, Auele Specific Primer of the present invention has specificity to the fragrant mushroom protoplastis of SR-6 * 14 couple paint face monocaryon, the present invention utilize the PCR primer to SR-6 * 14 differentiate between the fragrant mushroom protoplastis of paint face monocaryons mating type whether identical method and existing somatocyte incompatibility experimental technique differentiate between paint face's perfume mushroom protoplastis monocaryon mating type whether the consistence of identical method be 100%.Present method has shortened the discriminating time of the fragrant mushroom protoplastis of paint face monocaryon mating type and has simplified step, its accuracy and reliability height that mating type is differentiated.Monocaryon by the acquisition of protoplastis monokaryon is called the protoplasma monocaryon, different with the spore monocaryon, the protoplastis monocaryon is to prepare the haploid vegetative progeny of monokaryon that directly obtains the fragrant mushroom heterocaryotic mycelium from the paint face in the process at protoplastis, so the protoplasma monocaryon of the fragrant mushroom of paint face has only the mating type A of two kinds of forms
xB
xAnd A
yB
yTwo pairs.In the cross-breeding of routine, having only the different monokaryon bacterial strain of mating type is cross fertile, so the evaluation of mating type is prerequisite and the prerequisite of cross-breeding.The present invention is significant to genetic breeding and the production practice of the fragrant mushroom of research paint face.The present invention is applicable to that relevant speciality fields such as Edible Fungi, strain improvement are to the research of aspects such as the discriminating of the fragrant mushroom protoplastis of paint face protoplasma monocaryon and genetic breeding.
Description of drawings
Fig. 1 is for using primer of the present invention to the pcr amplification product electrophorogram of the fragrant mushroom protoplastis of SR-6 * 14 couple part paint face monocaryon.
Among the figure, the bacterial strain of 1 to 12 correspondence is followed successively by: No. 110, and No. 58, No. 99, No. 67, No. 55, No. 137, No. 179, No. 69, No. 104, No. 139, No. 100, the double-core bacterial strain; M:DNA Marker.
Fig. 2 is for carrying out the electrophorogram of PCR with primer SRAP-me6 and the fragrant mushroom protoplastis of the paint face of SRAP-em14 monocaryon.
Among the figure, the bacterial strain of 1 to 12 correspondence is followed successively by: No. 110, and No. 58, No. 99, No. 67, No. 55, No. 137, No. 179, No. 69, No. 104, No. 139, No. 100, the genomic dna of double-core bacterial strain; M:DNA Marker.
The clamp connexion photo that produces between the mycelia of Fig. 3 for the different fragrant mushroom protoplastis of the two strain paint face monocaryon of mating type.
Arrow shows clamp connexion among the figure.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The fragrant mushroom of paint face among the following embodiment is fragrant mushroom (Lepista sordida) CFCC89562 of paint face, the public can be from China Committee for Culture Collection of Microorganisms forestry microorganism center (China Forest microbial strains preservation administrative center, China Forestry Culture Collection Center english abbreviation CFCC is called for short forestry microorganism center) obtain.Hereinafter, all be called for short the fragrant mushroom of paint face.
1, the fragrant mushroom protoplastis of the paint face among following embodiment monocaryon is all by the acquisition of protoplastis monokaryon, and concrete grammar is as follows:
1.1 substratum
The 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 the MM damping fluid thereof are as follows: 0.5M N.F,USP MANNITOL.
Liquid MCM substratum: water-soluble and water is settled to 1L with yeast extract 2g, Tryptones 2g, glucose 20g, sal epsom 0.5g, potassium primary phosphate 0.5g and dipotassium hydrogen phosphate 1g.
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): in liquid MCM substratum, add sorbyl alcohol and agar, make its concentration be respectively 1M and 20g/L.
1.2 protoplastis preparation
1) get the fragrant mushroom mycelia of paint face in liquid MCM substratum, 160rpm, 25 ℃ cultivate 4 days (3-5 days all can), filter and collect mycelia with 3 layers of aseptic lens wiping paper, wash 2-3 time with the 0.6M Osmitrol then.
2) the fragrant mushroom mycelia of the paint face of step 1) (about 1g) being suspended in 1.5% lywallzyme solution (dissolves the 1.5g lywallzyme and is settled to 100mL with the 0.6M Osmitrol; Lywallzyme is available from Guangdong Bide Biotechnology Co., Ltd., and catalog number: Bd_8110001023), temperature was bathed 2 hours in shaking bath (32 ℃, 60rpm), filtered and collect filtrate with 3 layers of aseptic lens wiping paper.
3) with step 2) the centrifugal 10min of filtrate 3000rpm and collecting precipitation.
4) with the precipitation of step 3) with MM damping fluid washing three times after, be suspended in the 100 μ l MM damping fluids, be protoplastis solution.
1.3 the acquisition of protoplasma monocaryon
It is 10 that the protoplastis that obtains is diluted to concentration with the MM damping fluid
3-10
4Individual/mL, evenly coat on the RM, 28 ℃ leave standstill cultivation 10-14 days; Waiting to grow out is forwarded on the solid MCM substratum with the aseptic inoculation pin behind single bacterium colony, cultivates 5-7 days for 28 ℃.
With the protoplasma monocaryon number consecutively that obtains, and in microscope (Olympus BX51) observation down of amplifying 1000 times, if do not find clamp connexion, then this bacterial strain is the fragrant mushroom protoplastis of paint face monocaryon.Obtain the fragrant mushroom protoplastis of 30 strain paint face monocaryon 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, whether the mating type of the fragrant mushroom protoplastis of discriminating paint face monocaryon is identical
Identical the referring to of the mating type of the fragrant mushroom protoplastis of two strain paint face to be identified monocaryon cultivated the fragrant mushroom protoplastis of two strain paint face to be identified monocaryon together, do not produce clamp connexion between the mycelia of the fragrant mushroom protoplastis of these two strain paint face to be identified monocaryon; The mating type difference of the fragrant mushroom protoplastis of two strain paint face to be identified monocaryon refers to the fragrant mushroom protoplastis of two strain paint face to be identified monocaryon is cultivated together, produces clamp connexion between the mycelia of the fragrant mushroom protoplastis of these two strain paint face to be identified monocaryon.1.3 in the fragrant mushroom protoplastis of 30 strain paint face monocaryon between mating type differentiate according to following somatocyte incompatibility experimental technique:
The PDA substratum: the 200g potato, clean peeling is cut into small pieces, and adds water 1000ml and boils half hour, and filtered through gauze is got filtrate and is added 20g glucose and 20g agar again, sterilizes 20 minutes for 121 ℃.
The fragrant mushroom protoplastis of the paint face of two strains mating type to be identified monocaryon is inoculated on the PDA substratum together, opens 2cm between between the two, cultivate for 25 ℃ and observed in 10 days.If the mycelia of the fragrant mushroom protoplastis of the paint face of visual inspection two strains mating type to be identified monocaryon grows together, the mycelia of further confirming the junction by microscopic examination does not have clamp connexion, and the mating type of the fragrant mushroom protoplastis of the paint face of this two strain mating type to be identified monocaryon is identical.If there is the antagonism line in the place that the mycelia of the fragrant mushroom protoplastis of the paint face of visual inspection two strains mating type to be identified monocaryon crosses, the mycelia on the picking antagonism line then, being seeded on the PDA substratum 25 ℃ cultivated after 5 days, by microscopic examination whether clamp connexion is arranged again, if no clamp connexion, the mating type of the fragrant mushroom protoplastis of the paint face of this two strain mating type to be identified monocaryon is identical; If clamp connexion is arranged, the mating type difference of the fragrant mushroom protoplastis of the paint face of this two strain mating type to be identified monocaryon.Fig. 3 has shown the clamp connexion that produces between the mycelia of the fragrant mushroom protoplastis of two different strain paint face of mating type monocaryon.
Differentiate the mating type of the fragrant mushroom protoplastis of the 1.3 30 strain paint face that obtain monocaryons according to this method, the result is shown in table 1 and 2.In table 1 and the table 2, somatocyte incompatibility experimental result shows the mating type difference of two bacterial strains between two bacterial strains of " √ " expression; Somatocyte incompatibility experimental result shows that the mating type of two bacterial strains is identical between two bacterial strains of " * " expression.
Table 1. somatocyte incompatibility experimental technique is differentiated the mating type identical result whether between the fragrant mushroom protoplastis of paint face 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. somatocyte incompatibility experimental technique is differentiated the mating type identical result whether between the fragrant mushroom protoplastis of paint face 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 | √ | √ | √ | × | √ | × | × | √ | × | √ | √ | √ | √ | × | ? |
1, differentiates or assists the PCR reagent of differentiating the fragrant mushroom protoplastis of paint face monocaryon mating type
The discriminating of present embodiment or the auxiliary reagent of differentiating the fragrant mushroom protoplastis of paint face monocaryon mating type by the PCR primer to SR-6 * 14,10 * Taq damping fluid, dNTP mix, Taq archaeal dna polymerase and ddH
2O forms.
Wherein, the PCR primer is made up of SR-6 * 14-F and these two single stranded DNAs of SR-6 * 14-R SR-6 * 14, and its sequence is as follows:
SR-6×14-F:5’-TCCAAACCGGTAGGTAACAG-3’（SEQ?ID?No.1），
SR-6×14-R:5’-ATCGTCACATTCGGAGTCAA-3’（SEQ?ID?No.2）。
10 * Taq damping fluid, dNTP mix and Taq archaeal dna polymerase are all contained the rising sun hundred river companies (CNS) available from Beijing.
2, differentiate or assist the fragrant mushroom protoplastis of discriminating paint face monocaryon mating type
The fragrant mushroom protoplastis of the above-mentioned 1.3 30 strain paint face that obtain monocaryon is inoculated into respectively on the PDA substratum, cultivated 7-10 days for 25 ℃, scrape the about 0.1g of the mycelia of getting on the PDA substratum, with the DNeasy Plant Mini Kit extraction DNA of QIAGEN.
With the concentration of extinction photometer mensuration gained dna solution, dilution stoste, making its OD value is 1, and concentration is 50ng/ μ L.
The fragrant mushroom protoplastis of every strain paint face monocaryon all adopts following PCR system and following PCR condition, be template with the fragrant mushroom protoplastis of paint face monocaryon genomic dna respectively, utilize the discriminating of step 1 or the reagent of the fragrant mushroom protoplastis of the auxiliary paint face of discriminating monocaryon mating type to carry out pcr amplification.The PCR reaction system is as shown in table 3:
The PCR reaction system of table 3,20 μ L
PCR temperature programming: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 40s, 30 circulations; 72 ℃ are extended 7min, 12 ℃ of preservations.
Pcr amplification product electrophoresis on 1.3% sepharose with the fragrant mushroom protoplastis of every strain paint face monocaryon, gel imaging, the result shows that the PCR product of 50,53,61,65,66,69,71,72,75,88,100,104,111,139, No. 179 fragrant mushroom protoplastis of paint face monocaryons is the DNA band (dna fragmentation) of a 500bp-800bp; 36, there is not the DNA band in the PCR product of 42,55,58,67,81,82,86,87,99,110,113,117,119, No. 137 fragrant mushroom protoplastis of paint face monocaryons.Fig. 1 has shown the pcr amplification product of the fragrant mushroom protoplastis of part paint face monocaryon.
Reclaim the DNA band (dna fragmentation) of the 500bp-800bp of the fragrant mushroom protoplastis of every strain paint face monocaryon respectively, check order, the result shows that the size of DNA band (dna fragmentation) of the 500bp-800bp of the fragrant mushroom protoplastis of all 15 strain paint face monocaryon is 636bp.
Determine according to following method according to the size of pcr amplification product whether the mating type between per two strain bacterial strains of the fragrant mushroom protoplastis of the above-mentioned 1.3 30 strain paint face that obtain monocaryons is identical: if the PCR product of the fragrant mushroom protoplastis of two strain paint face to be identified monocaryon all contains the dna fragmentation of 500bp-800bp, the fragrant mushroom protoplastis of described two strain paint face to be identified monocaryon is the identical bacterial strain of mating type, if the PCR product of the fragrant mushroom protoplastis of two strain paint face to be identified monocaryon does not all contain the dna fragmentation of 500bp-800bp, the fragrant mushroom protoplastis of described two strain paint face to be identified monocaryon is the identical bacterial strain of mating type; If the PCR product of the fragrant mushroom protoplastis of the paint face monocaryon that the strain in the fragrant mushroom protoplastis of the two strain paint face to be identified monocaryon is to be identified contains the dna fragmentation of 500bp-800bp, the PCR product of the fragrant mushroom protoplastis of the paint face monocaryon that another strain is to be identified does not contain the dna fragmentation of 500bp-800bp, and the fragrant mushroom protoplastis of described two strain paint face to be identified monocaryon is the different bacterial strain of mating type.
The present invention utilize mating type that the PCR primer differentiates the fragrant mushroom protoplastis of per two strain paint face monocaryon to SR-6 * 14 whether identical result shown in table 4 and 5.Show the present invention utilize mating type that the PCR primer differentiates the fragrant mushroom protoplastis of per two strain paint face monocaryon to SR-6 * 14 whether identical method and existing somatocyte incompatibility experimental technique differentiate between paint face's perfume mushroom protoplastis monocaryon mating type whether the consistence of identical method (table 1 and table 2) be 100%.In table 4 and the table 5, " √ " expression the present invention utilizes the PCR primer to the mating type difference between the fragrant mushroom protoplastis of the two strain paint face monocaryon of SR-6 * 14 discriminatings, and " * " expression the present invention utilizes the PCR primer identical to the mating type between the fragrant mushroom protoplastis of the two strain paint face monocaryon of SR-6 * 14 discriminatings.
Table 4, the present invention utilize the PCR primer that SR-6 * 14 is differentiated mating type identical result whether between the fragrant mushroom protoplastis of paint face monocaryons.
| ? | 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 utilize the PCR primer that SR-6 * 14 is differentiated mating type identical result whether between the fragrant mushroom protoplastis of paint face monocaryons.
| ? | 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, the PCR primer obtains according to following method screening SR-6 * 14:
1. molecule marker experiment
With the fragrant mushroom protoplastis of the above-mentioned 1.3 30 strain paint face that obtain monocaryon, and the double-core bacterial strain of the fragrant mushroom of paint face, totally 31 strain bacterial strains are inoculated on the PDA substratum, 25 ℃ of cultivations.After for some time, scrape the about 0.1g of the mycelia of getting on the PDA substratum, with the DNeasy Plant Mini Kit extraction DNA of QIAGEN.
With the concentration of extinction photometer mensuration gained dna solution, dilution stoste, making its OD value is 1, and concentration is about 50ng/ μ L.The PCR reaction system is as shown in table 4:
The PCR reaction system of table 4:20 μ L
Wherein, upstream primer is SRAP-me6:5 '-tgagtccaaaccggtag-3 ',
Downstream primer is SRAP-em14:5 '-gactgcgtacgaattcag-3 '.
The PCR temperature programming is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 1min, 35 ℃ of annealing 1min, 72 ℃ are extended 1min, 5 circulations; 94 ℃ of sex change 1min, 50 ℃ of annealing 1min, 72 ℃ are extended 1min, 35 circulations; 72 ℃ are extended 10min, 12 ℃ of preservations.
With above-mentioned amplified production, electrophoresis on 1.3% sepharose, gel imaging is analyzed the mating type specific band.Find by analysis: in the fragment with primer SRAP-me6 and SRAP-em14 amplification acquisition, a part of protoplasma monocaryon has a length at the band of 600bp-700bp, and another part protoplasma monocaryon does not have this band (Fig. 2).
2. the recovery of specific band and cloning and sequencing
2.1 obtain the purpose band: cut specific band---the 600bp-700bp that glue reclaims No. 179 protoplasma monocaryons.Cut glue and reclaim the sepharose DNA recovery test kit that used kit provides for middle Ke Ruitai.Press the specification sheets operation in the test kit, reclaim the DNA of specific fragment, be used for ligation, make up 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).Dispose this system on ice after, be placed in 16 ℃ the water-bath, spend the night.
2.3 transform: place 4 ℃ to thaw the intestinal bacteria TOP10 competent cell (Tiangen) of-70 ℃ of preservations.Get the competent cell that 5 μ L connect liquid and 50 μ L and mix, place 30min on ice, ice bath 2min behind 42 ℃ of water-bath 90s then, liquid LB substratum (sodium-chlor, the 10g/L of adding 1mL; Peptone, 10g/L; Yeast powder, 5g/L), 37 ℃, rejuvenation is 1 hour under the 90rpm condition.
2.4 blue hickie screening: the nutrient solution that rejuvenation is good is uniformly coated on the solid LB substratum (adding the agar powder of 20g/L on the liquid LB substratum) of the IPTG that adds sodium ampicillin that final concentration is 0.1mg/mL, 0.024mg/mL and 0.04mg/mL, after the coating evenly, 37 ℃ constant temperature culture 12-16 hour.Treat to grow in the flat board after the blue hickie, draw hickie with the pipettor of 10 μ L, be seeded to and contain in the LB liquid nutrient medium that final concentration is the 0.1mg/mL sodium ampicillin, at 37 ℃, 180rpm cultivated 12-16 hour.
2.5PCR checking changing effect and sample presentation order-checking: with primer to RV-M (5 '-GAGCGGATAACAATTTCACACAGG-3 ') and M13-47 (5 '-CGCCAG GGTTTTCCCAGTCACGAC-3 '), be PCR with cultured bacterium liquid, the PCR system is pressed table 2 preparation.Response procedures is: 95 ℃ of pre-sex change 5min; 95 ℃ of sex change 50s, 57.8 ℃ of annealing 1min, 72 ℃ are extended 2min, 30 circulations; 72 ℃ are extended 7min, and after the PCR reaction finished, the sepharose with 1% carried out electrophoresis, ultraviolet imagery.The bacterium liquid that will have the positive colony of purpose band entrusts the calm and peaceful biotechnology of Sino-U.S. (Beijing) company limited to carry out sequencing.
The sequencing result of this 600bp-700bp specific band such as SEQ ID No.3, its size is 674bp.Design the PCR primer be made up of SR-6 * 14-F and these two single stranded DNAs of SR-6 * 14-R to SR-6 * 14 according to SEQ ID No.3, its sequence is as follows:
SR-6×14-F:5’-TCCAAACCGGTAGGTAACAG-3’（SEQ?ID?No.1），
SR-6×14-R:5’-ATCGTCACATTCGGAGTCAA-3’（SEQ?ID?No.2）。
Claims (10)
1. differentiate or assist the method for differentiating the fragrant mushroom protoplastis of paint face monocaryon mating type, comprise the steps: that respectively the genomic dna with the fragrant mushroom protoplastis of two strain paint face to be identified monocaryon is template, use the PCR primer of being formed by two single stranded DNAs shown in SEQ ID No.1 and the SEQ ID No.2 to carrying out pcr amplification, detect the size of resulting PCR product, determine the mating type of the described fragrant mushroom protoplastis of two strain paint face monocaryon to be identified according to the size of pcr amplification product according to following method:
If the PCR product of the fragrant mushroom protoplastis of described two strain paint face to be identified monocaryon all contains the dna fragmentation of 500bp-800bp, identical or the candidate of the mating type of the fragrant mushroom protoplastis of described two strain paint face to be identified monocaryon is that mating type is identical, if the PCR product of the fragrant mushroom protoplastis of described two strain paint face to be identified monocaryon does not all contain the dna fragmentation of 500bp-800bp, the identical or candidate of the mating type of the described fragrant mushroom protoplastis of two strain paint face monocaryon to be identified is that mating type is identical; If the PCR product of the fragrant mushroom protoplastis of the paint face monocaryon that the strain in the fragrant mushroom protoplastis of the described two strain paint face to be identified monocaryon is to be identified contains the dna fragmentation of 500bp-800bp, the PCR product of the fragrant mushroom protoplastis of another strain paint face to be identified monocaryon does not contain the dna fragmentation of 500bp-800bp, and mating type difference or the candidate of the described fragrant mushroom protoplastis of two strain paint face monocaryon to be identified are the mating type difference.
2. method according to claim 1, it is characterized in that: the dna fragmentation of described 500bp-800bp is the dna fragmentation of 636bp.
3. method according to claim 1 and 2 is characterized in that: the primer annealing condition that described pcr amplification adopts is 57 ℃ of annealing 30s.
4. according to claim 1 or 2 or 3 described methods, it is characterized in that: the PCR temperature programming that adopts in the described pcr amplification: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 57 ℃ of annealing 30s, 72 ℃ are extended 40s, 30 circulations; 72 ℃ are extended 7min.
5. the application of arbitrary described method in the fragrant mushroom breeding of paint face among the claim 1-4.
6. the PCR primer of discriminating or the fragrant mushroom protoplastis of auxiliary discriminating paint face monocaryon mating type is right, and it is characterized in that: the right name of described primer is called SR-6 * 14, is made up of two single stranded DNAs shown in SEQ ID No.1 and the SEQ ID No.2.
7. the reagent that contains the right discriminating of the described PCR primer of claim 6 or the fragrant mushroom protoplastis of the auxiliary paint face of discriminating monocaryon mating type.
8. the test kit that contains the right discriminating of the described PCR primer of claim 6 or the fragrant mushroom protoplastis of the auxiliary paint face of discriminating monocaryon mating type.
The described PCR primer of claim 6 to, the described reagent of claim 7 or the described test kit of claim 8 differentiate or the fragrant mushroom protoplastis of the auxiliary paint face of discriminating monocaryon mating type in application.
10. the described PCR primer of claim 6 application in the fragrant mushroom breeding of paint face to, the described reagent of claim 7 or the described test kit of claim 8.
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