CN103184280B - 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
- Publication number
- CN103184280B CN103184280B CN201310042607.1A CN201310042607A CN103184280B CN 103184280 B CN103184280 B CN 103184280B CN 201310042607 A CN201310042607 A CN 201310042607A CN 103184280 B CN103184280 B CN 103184280B
- Authority
- CN
- China
- Prior art keywords
- paint face
- face mushroom
- mating type
- identified
- strain
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 230000013011 mating Effects 0.000 title claims abstract description 78
- 238000000034 method Methods 0.000 title claims abstract description 34
- 241000908176 Lepista Species 0.000 title abstract description 11
- 210000001938 protoplast Anatomy 0.000 title abstract 9
- 235000001674 Agaricus brunnescens Nutrition 0.000 claims description 123
- 239000003973 paint Substances 0.000 claims description 121
- 108020004414 DNA Proteins 0.000 claims description 42
- 238000013467 fragmentation Methods 0.000 claims description 23
- 238000006062 fragmentation reaction Methods 0.000 claims description 23
- 238000012408 PCR amplification Methods 0.000 claims description 13
- 239000003153 chemical reaction reagent Substances 0.000 claims description 10
- 241000547652 Lepista sordida Species 0.000 claims description 9
- 238000000137 annealing Methods 0.000 claims description 9
- 238000012360 testing method Methods 0.000 claims description 9
- 102000053602 DNA Human genes 0.000 claims description 6
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 6
- 238000009395 breeding Methods 0.000 claims description 6
- 230000001488 breeding effect Effects 0.000 claims description 5
- 230000008859 change Effects 0.000 claims description 5
- 230000004087 circulation Effects 0.000 claims description 5
- 238000004925 denaturation Methods 0.000 claims description 5
- 230000036425 denaturation Effects 0.000 claims description 5
- 230000003321 amplification Effects 0.000 abstract description 2
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 2
- 238000003752 polymerase chain reaction Methods 0.000 abstract 7
- 239000000047 product Substances 0.000 description 20
- 230000001580 bacterial effect Effects 0.000 description 16
- 238000002474 experimental method Methods 0.000 description 10
- 239000012530 fluid Substances 0.000 description 10
- 239000007788 liquid Substances 0.000 description 10
- 241000233866 Fungi Species 0.000 description 9
- 238000013016 damping Methods 0.000 description 9
- 230000000392 somatic effect Effects 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 6
- 230000008485 antagonism Effects 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 229920001817 Agar Polymers 0.000 description 4
- 229920002684 Sepharose Polymers 0.000 description 4
- 239000008272 agar Substances 0.000 description 4
- 238000012214 genetic breeding Methods 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 3
- 238000009402 cross-breeding Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 230000003993 interaction Effects 0.000 description 3
- 235000010355 mannitol Nutrition 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 241000222485 Agaricales Species 0.000 description 2
- 241000222382 Agaricomycotina Species 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241000222433 Tricholomataceae Species 0.000 description 2
- KLOHDWPABZXLGI-YWUHCJSESA-M ampicillin sodium Chemical compound [Na+].C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C([O-])=O)(C)C)=CC=CC=C1 KLOHDWPABZXLGI-YWUHCJSESA-M 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 229940064457 osmitrol Drugs 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000003716 rejuvenation Effects 0.000 description 2
- 238000007790 scraping Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- MEIRRNXMZYDVDW-MQQKCMAXSA-N (2E,4E)-2,4-hexadien-1-ol Chemical compound C\C=C\C=C\CO MEIRRNXMZYDVDW-MQQKCMAXSA-N 0.000 description 1
- 241000235349 Ascomycota Species 0.000 description 1
- 241000221198 Basidiomycota Species 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000222418 Lentinus Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010038049 Mating Factor Proteins 0.000 description 1
- 239000012807 PCR reagent Substances 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 240000005373 Panax quinquefolius Species 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 239000006002 Pepper Substances 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 235000016761 Piper aduncum Nutrition 0.000 description 1
- 235000017804 Piper guineense Nutrition 0.000 description 1
- 244000203593 Piper nigrum Species 0.000 description 1
- 235000008184 Piper nigrum Nutrition 0.000 description 1
- 240000001462 Pleurotus ostreatus Species 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- CWHJIJJSDGEHNS-MYLFLSLOSA-N Senegenin Chemical compound C1[C@H](O)[C@H](O)[C@@](C)(C(O)=O)[C@@H]2CC[C@@]3(C)C(CC[C@]4(CCC(C[C@H]44)(C)C)C(O)=O)=C4[C@@H](CCl)C[C@@H]3[C@]21C CWHJIJJSDGEHNS-MYLFLSLOSA-N 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- DPDMMXDBJGCCQC-UHFFFAOYSA-N [Na].[Cl] Chemical compound [Na].[Cl] DPDMMXDBJGCCQC-UHFFFAOYSA-N 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 238000011021 bench scale process Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000010448 genetic screening Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- SKEFKEOTNIPLCQ-LWIQTABASA-N mating hormone Chemical compound C([C@@H](C(=O)NC(CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCS(C)=O)C(=O)NC(CC=1C=CC(O)=CC=1)C(O)=O)NC(=O)[C@@H](N)CC=1C2=CC=CC=C2NC=1)C1=CN=CN1 SKEFKEOTNIPLCQ-LWIQTABASA-N 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000009871 tenuigenin Substances 0.000 description 1
- 108010046845 tryptones Proteins 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000009105 vegetative growth Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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 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 IS-818.
Discriminating provided by the present invention or the auxiliary PCR primer pair of differentiating paint face mushroom protoplastis monocaryon mating type, name is called IS-818, 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 22 deoxynucleotides, and SEQ ID No.2 is made up of 21 deoxynucleotides.
The discriminating that contains PCR primer pair IS-818 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 IS-818, also contain other the conventional reagent that carries out PCR and DNA sequencing.
The PCR of experimental results show that primer pair IS-818 of the present invention, the reagent that contains PCR primer pair 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 genomic dna taking two strain paint face mushroom protoplastis monocaryons to be identified is as template, use the PCR primer pair IS-818 being formed by two single stranded DNAs of SEQ IDNo.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 800bp-1000bp) 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 (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 800bp-1000bp) 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 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 800bp-1000bp (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 800bp-1000bp 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 800bp-1000bp) of 800bp-1000bp, 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 800bp-1000bp is specially the DNA fragmentation of 861bp.
In one embodiment of the invention, the primer annealing condition that described pcr amplification adopts is 62 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, 62 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 IS-818 to 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 IS-818 differentiates 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 to part paint face mushroom protoplastis monocaryon with primer pair IS-818 of the present invention.
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 I SSR-818, 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:DNAMarker.
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 the limited public affairs of the green moral biotechnology in Guangdong
Department, catalog number: Bd_8110001023), temperature bath 2 hours in shaking bath (32 DEG C, 60rpm), filters and collects 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.Obtained 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 IS-818 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 IS-818,10 × Taq damping fluid, dNTP mix, Taq archaeal dna polymerase and ddH
2o composition.
Wherein, PCR primer pair IS-818 is made up of IS-818-pre-F and these two single stranded DNAs of IS-818-pre-R, 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 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, 62 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 800bp-1000bp; 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 800bp-1000bp of every strain paint face mushroom protoplastis monocaryon, checks order, and result shows that the size of the DNA fragmentation of the 800bp-1000bp of 15 all strain paint face mushroom protoplastis monocaryons is 861bp.
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 800bp-1000bp, 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 800bp-1000bp, 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 800bp-1000bp, the PCR product of another strain paint face mushroom protoplastis monocaryon 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 bacterial strain that mating type is different.
The present invention utilizes result that PCR primer pair IS-818 differentiates 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 the present invention utilizes method that PCR primer pair IS-818 differentiates that whether the mating type of every two strain paint face mushroom protoplastis monocaryons is identical and existing somatic incompatibility experimental technique to differentiate that the consistence of the method (table 1 and 2) whether mating type between paint face mushroom protoplastis monocaryon is identical is 100%.
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 IS-818 differentiate two paint face mushroom protoplastis monocaryons between mating type difference, "×" represent the present invention utilize PCR primer pair IS-818 differentiate two paint face mushroom protoplastis monocaryons between mating type identical.
Table 4, the present invention utilize PCR primer pair IS-818 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 IS-818 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 IS-818 screens by the following method and to obtain:
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 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.
PCR temperature programming is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 41 DEG C of annealing 45s, 72 DEG C are extended 2min, 34 circulations; 72 DEG C are extended 7min, 12 DEG C of preservations.
By above-mentioned amplified production, electrophoresis on 1.3% sepharose, gel imaging, analyzes mating type specific band.Find by analysis: in the fragment obtaining with primer I SSR-818 amplification, a part of Protoplasted monokaryon has the fragment of a length in 900bp 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---900bp 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 900bp left and right specific band is as SEQ ID No.3, and its size is 943bp.Design the PCR primer pair IS-818 being made up of IS-818-pre-F and these two single stranded DNAs of IS-818-pre-R 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 (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 IDNo.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 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 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 800bp-1000bp, the PCR product of another strain paint face mushroom protoplastis monocaryon 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 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 800bp-1000bp is 861bp.
3. application according to claim 1 and 2, is characterized in that: the primer annealing condition that described pcr amplification adopts is 62 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, 62 DEG C of annealing 30s, 72 DEG C are extended 40s, 30 circulations; 72 DEG C are extended 7min.
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 IS-818, 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310042607.1A CN103184280B (en) | 2013-02-04 | 2013-02-04 | Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-818 thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310042607.1A CN103184280B (en) | 2013-02-04 | 2013-02-04 | Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-818 thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103184280A CN103184280A (en) | 2013-07-03 |
| CN103184280B true CN103184280B (en) | 2014-12-10 |
Family
ID=48675726
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310042607.1A Expired - Fee Related CN103184280B (en) | 2013-02-04 | 2013-02-04 | Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-818 thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103184280B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282396B (en) * | 2016-11-07 | 2019-05-28 | 云南省农业科学院生物技术与种质资源研究所 | Identify the method and primer pair of terraced rib hickory chick mating type |
| CN108486269B (en) * | 2018-03-02 | 2021-08-20 | 昆明理工大学 | Method for identifying homokaryon strains in trametes hirsuta S0301 |
| CN111394491B (en) * | 2019-01-03 | 2021-12-28 | 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 |
| 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 |
| CN111304091A (en) * | 2019-11-28 | 2020-06-19 | 昆明理工大学 | Method for improving ganoderic acid content in ganoderma lucidum cell culture |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101649350B (en) * | 2009-06-01 | 2012-08-22 | 中国农业大学 | Molecular detection method for rapidly distinguishing mating types of Chinese caterpillar fungus |
-
2013
- 2013-02-04 CN CN201310042607.1A patent/CN103184280B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| Development of SCAR markers to determine the mating types of Lepista nuda protoplast monokaryons;Li Dengjin et al;《Curr Microbiol》;20131219;第1-7页 * |
| Li Dengjin et al.Development of SCAR markers to determine the mating types of Lepista nuda protoplast monokaryons.《Curr Microbiol》.2013,第1-7页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103184280A (en) | 2013-07-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103184280B (en) | Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-818 thereof | |
| Nikam et al. | Pathogenic, cultural, morphological and molecular variability among eight isolates of Alternaria solani, causing early blight of tomato | |
| CN104611414B (en) | Utilize method and the application of ssr primer qualification pomegranate kind | |
| Talukdar et al. | Genetics of Yellow Mosaic Virus (YMV) resistance in cultivated soybean [Glycine max (L) Merr.] | |
| CN103276070B (en) | Method for identifying mating types of lepista sordida protoplasted monokaryons and special primer pair IS-879b thereof | |
| CN103184281B (en) | Method for identifying or assisting in identifying mating types of Lepista sordid protoplast monokaryons and special primer pairs IS-873 thereof | |
| CN103194535B (en) | Method for identification or auxiliary identification of mating type of protoplast monokaryon of Lepista sordid and special primer pair SR-5*16 used therein | |
| CN103184282B (en) | Method and special primer SR-5*13 for differentiating and auxiliarily differentiating mating types of bioplast monokaryons of lepista sordida (Fr) sings | |
| CN106434641A (en) | Molecular marker linked with Chinese cabbage purple head gene BrPur | |
| CN103305606B (en) | Method for identifying mating type of protoplasted monokaryon of lepista sordida and special primer pair SR-6*4 thereof | |
| CN103224990B (en) | Method for identifying mating types of Lepista sordida protoplast monokaryons and special primer pair SR-4*7 therefor | |
| CN103276073B (en) | Method for identifying mating types of lepista sordida protoplasted monokaryons and special primer pair SR-1x8b thereof | |
| CN103233079B (en) | Method for identifying mating types of Lepista sordida protoplast monokaryons and special primer pair SR-5*1 therefor | |
| CN103224992B (en) | Method for identifying mating types of Lepista sordida protoplast monokaryons and special primer pair SR-1*10 therefor | |
| CN103276072B (en) | Method for identifying mating types of lepista sordida protoplasted monokaryons and special primer pair SR-2x8 thereof | |
| CN103266173B (en) | Method for identifying lepista sordida protoplast monokaryon mating types and special primer pair SR-6*14 therefor | |
| CN103290117B (en) | Method for identifying protoplast monokaryon mating types of lepista sordida and special primer pair SR-4*2 thereof | |
| CN101838705A (en) | Method for detecting burkholderia cepacia flora on fruits or vegetables and identifying seeds | |
| CN103224991B (en) | Method for identifying mating types of Lepista sordida protoplast monokaryons and special primer pair IS-857c therefor | |
| CN103233080B (en) | Method for identifying mating types of Lepista sordida protoplast monokaryons and special primer pair SR-1*3 therefor | |
| CN104278028A (en) | Sequence of dasypyrum villosum 6VS DNA permeating into powdery mildew resistant near-isogenic line of wheat and application | |
| CN103233081B (en) | Method for identifying mating types of Lepista sordida protoplast monokaryons and special primer pair SR-6*6 therefor | |
| CN106636173B (en) | The method and its bacterial strain of enhanced green fluorescence protein label maize head dust-brand bacterial strain and application | |
| CN105861640B (en) | A kind of method of Rapid identification edible sunflower cenospecies SH338 authenticity and purity | |
| CN106978505A (en) | Detect primer pair and its application of eggplant stemphylium botryosum |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20141210 |