CN103184280A - Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-818 thereof - Google Patents
Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-818 thereof Download PDFInfo
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Abstract
The invention discloses a method for identifying or assisting in identifying the mating type of Lepista sordid protoplast monokaryons and special primer pairs IS-818 thereof. The method for identifying or assisting in identifying the mating types of the Lepista sordid protoplast monokaryons comprises the following steps: respectively taking the genome DNA of two Lepista sordid protoplast monokaryons to be identified as templates; performing polymerase chain reaction (PCR) amplification by using PCR primer pairs shown in SEQ ID No. 1 and 2; detecting the size of the obtained PCR products; if the PCR products of the two Lepista sordid protoplast monokaryons to be identified comprise 800 to 1,000 bp of DNA segments or do not comprise 800 to 1,000 bp of DNA segments, the mating types of two Lepista sordid protoplast monokaryons are identical; and if the PCR product of one of the two Lepista sordid protoplast monokaryons to be identified comprises 800 to 1,000 bp of DNA segments and the PCR product of the other one of the two Lepista sordid protoplast monokaryons to be identified does not comprise 800 to 1,000 bp of DNA segments, the mating types of two Lepista sordid protoplast monokaryons are different.
Description
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 IS-818.
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.
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 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 paint face mushroom 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 auxiliary paint face of discriminating mushroom protoplastis monocaryon mating type and primer special thereof to IS-818.
The PCR primer of discriminating provided by the present invention or auxiliary discriminating paint face mushroom protoplastis monocaryon mating type is right, and name is called IS-818, 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 22 deoxynucleotides, and SEQ ID No.2 is made up of 21 deoxynucleotides.
Contain the PCR primer to the discriminating of IS-818 or assist reagent or the test kit of differentiating paint face mushroom protoplastis monocaryon mating type also to belong to protection scope of the present invention.
Described reagent or test kit except the PCR primer to the IS-818, 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 IS-818, contain the PCR primer to the reagent of IS-818 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 the genomic dna with two strain paint face mushroom protoplastis monocaryons to be identified is template, use the PCR primer of being formed by two single stranded DNAs of SEQ IDNo.1 and SEQ ID No.2 that IS-818 is carried out pcr amplification, detect the size of resulting PCR product, determine the mating type of described two strain paint face mushroom protoplastis monocaryons to be identified according to the size of pcr amplification product according to following method:
If the two to-be- identified and painted face mushroom protoplast monokaryons PCR products contain 800bp-1000bp DNA fragment ( or the identification of the two strains to be painted face mushroom protoplast monokaryons PCR products are 800bp- 1000bp DNA ) , said to be the identification of two Baikal mushroom protoplast monokaryons same mating type , or a candidate for the same mating type , if the identification of the two to be painted face mushroom protoplast monokaryons PCR products , not containing 800bp-1000bp DNA fragment ( or the identification of the two strains to be painted face mushroom protoplast monokaryons not the size of the PCR product is 800bp-1000bp DNA fragment ) , the identification of the two to be painted face protoplast mushroom mating monokaryons candidate of the same type or the same mating type ; if the two strains to be painted face identification protoplast monokaryons mushroom in a mushroom to be painted face identification protoplast monokaryons PCR products , containing 800bp-1000bp DNA fragment ( or the identification of the two strains to be painted face protoplast monokaryons mushroom in a mushroom to be painted face identification protoplast monokaryons PCR product of 800bp-1000bp DNA fragment ) , and the other a mushroom to be painted face identification monokaryons protoplast does not contain a PCR product of 800bp-1000bp DNA fragment ( or the identification of other strains to be painted face mushroom protoplast monokaryons PCR product is not a DNA fragment of 800bp-1000bp ) said to be the identification of two Baikal mushroom protoplast monokaryons different mating type or candidate for the different mating type .
In the aforesaid method, the dna fragmentation of described 800bp-1000bp is specially the dna fragmentation of 861bp.
In one embodiment of the invention, the primer annealing condition of described pcr amplification employing is 62 ℃ 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, 62 ℃ of annealing 30s, 72 ℃ are extended 40s, 30 circulations; 72 ℃ are extended 7min.
In the aforesaid method, the mating type of two strain paint face mushroom protoplastis monocaryons is identical to refer to that the mycelia junction of two strain paint face mushroom protoplastis monocaryons does not have the antagonism line, can not produce clamp connexion with 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 the antagonism line mycelia junction of two strain paint face mushroom protoplastis monocaryons and produces clamp connexion, cross fertile with 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 the paint face mushroom of IS-818 protoplastis monocaryon, the present invention utilize the PCR primer to IS-818 differentiate between paint face mushroom protoplastis monocaryon mating type whether the mating type between identical method and existing somatocyte incompatibility experimental technique discriminating paint face mushroom protoplastis monocaryon whether the consistence of identical method be 100%.Present method has shortened the discriminating time of paint face mushroom protoplastis 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 prepares the haploid vegetative progeny of monokaryon that directly obtains in the process at protoplastis from paint face mushroom heterocaryotic mycelium, so the protoplasma monocaryon of paint face mushroom 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 research paint face mushroom.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 paint face mushroom protoplastis 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 part paint face mushroom of IS-818 protoplastis monocaryon.
1 to No. 9 corresponding bacterial strain is followed successively by among the figure: No. 110, and No. 67, No. 55, No. 137; No. 179, No. 139, No. 100, No. 65, the double-core bacterial strain; M:DNA Marker.
Fig. 2 is for carrying out the electrophorogram of PCR with the paint face mushroom of primer I SSR-818 protoplastis monocaryon.
Among the 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:DNAMarker.
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.
Paint face mushroom Lepista sordida(Fr) the Sing(swallow is continued bright etc. paint face mushroom protein crude extract control pepper virus disease and growth-promoting functions research. and Chinese biological control journal 28 (2) 269-274) public can be from field acquisition, also can obtain from commercial channels, also can obtain from the Beijing City Agriculture and Forestry Institute, to repeat the present invention's experiment.Hereinafter, all be called for short the paint face mushroom.
1, the paint face mushroom protoplastis monocaryon among the following embodiment 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 paint face mushroom mycelia 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 paint face mushroom mycelia (about 1g) of step 1) is suspended in 1.5% lywallzyme solution (with the 1.5g lywallzyme with the dissolving of 0.6M Osmitrol and be settled to 100mL; Lywallzyme is available from the limited public affairs of the green moral biotechnology in Guangdong
Department, 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 400 times, if do not find clamp connexion, then this bacterial strain is paint face mushroom protoplastis monocaryon.Obtained 30 strain paint face mushroom 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, the mating type of the paint face mushroom protoplastis monocaryon among the following embodiment is differentiated according to following antagonistic experiment method
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 paint face mushroom protoplastis monocaryon of two strains mating type to be identified is inoculated on the PDA substratum together, opens 2cm between between the two, cultivated 7-10 days for 25 ℃.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 the 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 the 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 the picking antagonism line then, cultivate after 3-5 days for 25 ℃ to the PDA substratum, examine under a microscope whether clamp connexion is arranged, if no clamp connexion, the mating type of the paint face mushroom protoplastis monocaryon of this two strain mating type to be identified is identical; If clamp connexion, the mating type difference of the paint face mushroom protoplastis monocaryon of this two strain mating type to be identified are arranged.
According to the mating type of these method discriminating 1.3 30 strain paint face mushroom protoplastis monocaryons that obtain, the result is shown in table 1 and 2.In table 1 and the table 2, the experiment of somatocyte incompatibility has antagonism line, the mating type difference of two bacterial strains between two bacterial strains of " √ " expression; The no antagonism line of somatocyte incompatibility experiment between two bacterial strains of " * " expression, the mating type of two bacterial strains is identical.
Table 1. somatocyte incompatibility experimental technique is differentiated the mating type identical result whether 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. somatocyte incompatibility experimental technique is differentiated the mating type identical result whether 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 | √ | √ | √ | × | √ | × | × | √ | × | √ | √ | √ | √ | × | ? |
1, differentiates or assists the PCR reagent of differentiating paint face mushroom protoplastis monocaryon mating type
The discriminating of present embodiment or the auxiliary reagent of differentiating paint face mushroom protoplastis monocaryon mating type by the PCR primer to IS-818,10 * Taq damping fluid, dNTP mix, Taq archaeal dna polymerase and ddH
2O forms.
Wherein, the PCR primer is made up of IS-818-pre-F and these two single stranded DNAs of IS-818-pre-R IS-818, and its sequence is as follows:
IS-818-pre-F:5’-CAGAGAAAGAATTGGCAAAGGT-3’（SEQ?ID?No.1）
IS-818-pre-R:5’-CAATCAACAGGAGGTCTACGA-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 discriminating 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 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.
Every strain paint face mushroom protoplastis monocaryon all adopts following PCR system and following PCR condition, be template with paint face mushroom protoplastis monocaryon genomic dna respectively, 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.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, 62 ℃ 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 every strain paint face mushroom protoplastis monocaryon, gel imaging, the 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 800bp-1000bp; 50, there is not the DNA band 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.Fig. 1 has shown the pcr amplification product of part paint face mushroom protoplastis monocaryon.
Reclaim the dna fragmentation of the 800bp-1000bp of every strain paint face mushroom protoplastis monocaryon respectively, check order, the result shows that the size of dna fragmentation of the 800bp-1000bp of 15 all strain paint face mushroom protoplastis monocaryons is 861bp.
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 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 800bp-1000bp, described two strain paint face mushroom protoplastis monocaryons to be identified are the identical bacterial strain of mating type, if the PCR product of two strain paint face mushroom protoplastis monocaryons to be identified does not all contain the dna fragmentation of 800bp-1000bp, described two strain paint face mushroom protoplastis monocaryons to be identified are the identical bacterial strain of mating type; If the PCR product of the paint face mushroom protoplastis monocaryon that the strain in the two strain paint face mushroom protoplastis monocaryons to be identified is to be identified contains the dna fragmentation of 800bp-1000bp, the PCR product of the paint face mushroom protoplastis monocaryon that another strain is to be identified does not contain the dna fragmentation of 800bp-1000bp, and described two strain paint face mushroom protoplastis monocaryons to be identified are the different bacterial strain of mating type.
The present invention utilize mating type that the PCR primer differentiates per two strain paint face mushroom protoplastis monocaryons to IS-818 whether identical result shown in table 4 and 5.Show the present invention utilize mating type that the PCR primer differentiates per two strain paint face mushroom protoplastis monocaryons to IS-818 whether identical method and existing somatocyte incompatibility experimental technique differentiate between paint face mushroom protoplastis monocaryon mating type whether the consistence of identical method (table 1 and 2) be 100%.
According to the mating type of these method discriminating 1.3 30 strain paint face mushroom protoplastis monocaryons that obtain, the result is shown in table 4 and 5.In table 4 and the table 5, " √ " expression the present invention utilizes the PCR primer to the mating type difference between two paint face mushroom protoplastis monocaryons of IS-818 discriminating, and " * " expression the present invention utilizes the PCR primer identical to the mating type between two paint face mushroom protoplastis monocaryons of IS-818 discriminating.
Table 4, the present invention utilize the PCR primer that IS-818 is differentiated mating type identical result whether 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 the PCR primer that IS-818 is differentiated mating type identical result whether 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, the PCR primer obtains according to following method screening IS-818:
1. molecule marker experiment
With 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 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, the sequence of upstream and downstream primer is 5 '-CACACACACACACACAG-3 ', and name is called ISSR-818.
The PCR temperature programming is: 94 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 41 ℃ of annealing 45s, 72 ℃ are extended 2min, 34 circulations; 72 ℃ are extended 7min, 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 I SSR-818 amplification acquisition, a part of protoplasma monocaryon has the fragment of a length about 900bp, 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 the specific band that glue reclaims No. 110 protoplasma monocaryons---about 900bp.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 1m L; 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: right with primer
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 900bp left and right sides specific band such as SEQ ID No.3, its size is 943bp.Design the PCR primer be made up of IS-818-pre-F and these two single stranded DNAs of IS-818-pre-R to IS-818 according to SEQ ID No.3, its sequence is as follows:
IS-818-pre-F:5’-CAGAGAAAGAATTGGCAAAGGT-3’（SEQ?ID?No.1）；
IS-818-pre-R:5’-CAATCAACAGGAGGTCTACGA-3’（SEQ?ID?No.2）。
Claims (10)
1. differentiate or assist the method for differentiating paint face mushroom protoplastis monocaryon mating type, comprise the steps: that respectively the genomic dna with two strain paint face mushroom protoplastis monocaryons to be identified 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 described two strain paint face mushroom protoplastis monocaryons to be identified according to the size of pcr amplification product according to following method:
If the PCR product of described two strain paint face mushroom protoplastis monocaryons to be identified all contains the dna fragmentation of 800bp-1000bp, 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 800bp-1000bp, 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 PCR product of the paint face mushroom protoplastis monocaryon that the strain in the described two strain paint face mushroom protoplastis monocaryons to be identified is to be identified contains the dna fragmentation of 800bp-1000bp, the PCR product of the paint face mushroom protoplastis monocaryon that another strain is to be identified does not contain the dna fragmentation of 800bp-1000bp, and mating type difference or the candidate of described two strain paint face mushroom protoplastis monocaryons to be identified are the mating type difference.
2. method according to claim 1, it is characterized in that: the dna fragmentation of described 800bp-1000bp is the dna fragmentation of 861bp.
3. method according to claim 1 and 2 is characterized in that: the primer annealing condition that described pcr amplification adopts is 62 ℃ 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, 62 ℃ of annealing 30s, 72 ℃ are extended 40s, 30 circulations; 72 ℃ are extended 7min.
5. the application of arbitrary described method in the breeding of paint face mushroom among the claim 1-4.
6. the PCR primer of discriminating or auxiliary discriminating paint face mushroom protoplastis monocaryon mating type is right, and it is characterized in that: the right name of described primer is called IS-818, 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 auxiliary paint face of discriminating mushroom protoplastis monocaryon mating type.
8. the test kit that contains the right discriminating of the described PCR primer of claim 6 or the auxiliary paint face of discriminating mushroom protoplastis 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 auxiliary paint face of discriminating mushroom protoplastis monocaryon mating type in application.
10. the described PCR primer of claim 6 is to, the described reagent of claim 7 or the application of the described test kit of claim 8 in the breeding of paint face mushroom.
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| CN111394491A (en) * | 2019-01-03 | 2020-07-10 | 中国科学院微生物研究所 | Mushroom mating type molecular marker and application thereof in identification of mushroom mating type |
| CN110029191A (en) * | 2019-05-31 | 2019-07-19 | 上海市农业科学院 | The primer sets of the monocaryon mating type of a kind of identification mushroom Shen 215 strains of perfume and its Essentially derived variety, identification method and application |
| CN110029191B (en) * | 2019-05-31 | 2022-09-06 | 上海市农业科学院 | Primer group for identifying mating types of monokaryons of shiitake mushroom Shenxiang 215 strain and substantive derivative variety thereof, identification method and application |
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