CN112280892B - Primer and method for rapidly screening high-temperature-resistant strain of agaricus bisporus W192 strain - Google Patents
Primer and method for rapidly screening high-temperature-resistant strain of agaricus bisporus W192 strain Download PDFInfo
- Publication number
- CN112280892B CN112280892B CN202011383787.6A CN202011383787A CN112280892B CN 112280892 B CN112280892 B CN 112280892B CN 202011383787 A CN202011383787 A CN 202011383787A CN 112280892 B CN112280892 B CN 112280892B
- Authority
- CN
- China
- Prior art keywords
- strain
- agaricus bisporus
- pcr amplification
- primer
- temperature resistant
- 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.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
A primer and a method for rapidly screening a high-temperature resistant strain of an agaricus bisporus W192 strain are disclosed, wherein the sequence of the primer is as follows: 4140-1F: TTCCGTTCAGTGCTACC; 4140-1R TGCAAAGAGAGCTGGTACA. The method can quickly screen the high-temperature resistant strain of the agaricus bisporus W192 strain, can prejudge the high-temperature resistant characteristic of the W192 spore strain in advance for fruiting experiments, and greatly saves the time for breeding the high-temperature resistant strain and promotes the breeding process.
Description
Technical Field
The invention belongs to the technical field of edible fungus breeding, and relates to a primer and a method for quickly screening a high-temperature-resistant strain of an agaricus bisporus W192 strain.
Background
Agaricus bisporus (Agaricus bisporus) with Chinese names mushroom, Agaricus campestris, and white mushroom. It contains rich protein, polysaccharide, vitamins, nucleotide and unsaturated fatty acid, and has rich nutrients, rich meat quality, delicious taste, low heat energy and high health function. In foreign countries, agaricus bisporus still accounts for the dominant position of edible mushroom consumption so far, and taking the second major world of edible mushroom production-the united states (accounting for about 16% of the total world production) as an example, the agaricus bisporus accounts for more than 90% of the total world production. In China, the agaricus bisporus consumption market develops rapidly, and the fresh mushrooms are increasingly sold in the market.
The main cultivated species of the agaricus bisporus are A15, S512, S608 and S130 of Sylvan company, U1, U3 of Horst mushroom test station in the Netherlands, and the like. These varieties are suitable for domestic facility cultivation. In China, germplasm resources of agaricus bisporus are very lacking, and great difficulty is brought to the work of improving and breeding agaricus bisporus strains. At present, the main cultivated varieties of domestic agaricus bisporus comprise As2796, 2000 and 192, Zhenong No.1 and the like, but only the W192 strain is an industrial strain which is independently bred and suitable for industrial production in China, and other domestic industrial varieties of agaricus bisporus are imported abroad. Therefore, the lack of domestic agaricus bisporus industrialized strains limits the development of domestic agaricus bisporus industrialization.
Temperature is a major factor affecting the growth of agaricus bisporus. The temperature requirement of the agaricus bisporus is divided into two stages, and the development temperature range of the mycelium is 4-32 ℃. The optimum temperature of the agaricus bisporus mycelium in the growth stage is kept at 22-25 ℃. The temperature range of the growth and development of the agaricus bisporus sporocarp is 4-25 ℃, and the temperature regulation of the cultivation environment is one of the main regulation factors for the industrial production of the agaricus bisporus. Therefore, it is necessary to design a primer and a method for rapidly screening a high temperature resistant strain of Agaricus bisporus W192 strain.
Disclosure of Invention
The invention aims to provide a method for judging the optimal rice straw usage amount for planting stropharia rugoso-annulata.
In order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a primer for rapidly screening a high-temperature-resistant strain of an agaricus bisporus W192 strain is disclosed, and the sequence of the primer is as follows:
4140-1F:TTCCGTTCAGTGCTACC；
4140-1R:TGCAAAGAGAGCTGGTACA。
in order to achieve the above objects and other related objects, the present invention provides the following technical solutions: a method for rapidly screening a high-temperature resistant strain of Agaricus bisporus W192 strain comprises the following steps:
step 1: extraction of W192 Strain DNA
Culturing a W192 hybrid strain or spore strain to be detected on a PDA (personal digital assistant) flat plate, then picking up hyphae, and cracking the picked hyphae by using a DNA (deoxyribonucleic acid) lysate to obtain a hypha lysate;
step 2: PCR amplification
Performing PCR amplification using the hyphal lysate as a template for PCR amplification using the primers 4140-1F and 4140-1R of claim 1;
and step 3: if the band with the product size of 781bp can be amplified, the strain is a high temperature resistant strain, otherwise, the strain is a non-high temperature resistant strain.
The preferable technical scheme is as follows: the PCR amplification procedure was: pre-denaturation at 94 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 48 deg.C for 30s, extension at 72 deg.C for 1min, 35 cycles, preservation at 72 deg.C for 10min, and preservation at 4 deg.C.
The preferable technical scheme is as follows: the PCR amplification system is as follows: the total volume was 20. mu.l, and included 10. mu.l of PCR mix, 7ul of double distilled water, 1ul of primer 4140-1F, 1ul of primer 4140-1R, and 1ul of hyphal lysate.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
the method can quickly screen the high-temperature resistant strain of the agaricus bisporus W192 strain, can prejudge the high-temperature resistant characteristic of the W192 spore strain in advance for fruiting experiments, and greatly saves the time for breeding the high-temperature resistant strain and promotes the breeding process.
Drawings
FIG. 1 shows the screening electrophoresis of 4140 molecular marker. Note: m: d2000 marker; CK is the amplification result of the W192 starting strain; 1-6: amplification results of 6 thermostable strains of W192.
FIG. 2 is a PCR amplification electrophoretogram of the W192 spore strain. Note: m: marker; CK is an original strain; 1-23: temperature was used to screen the spore strains.
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
Please refer to fig. 1-2. It should be understood that the structures, ratios, sizes, and the like shown in the drawings and described in the specification are only used for matching with the disclosure of the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions under which the present invention can be implemented, so that the present invention has no technical significance, and any structural modification, ratio relationship change, or size adjustment should still fall within the scope of the present invention without affecting the efficacy and the achievable purpose of the present invention. In addition, the terms "upper", "lower", "left", "right", "middle" and "one" used in the present specification are for clarity of description, and are not intended to limit the scope of the present invention, and the relative relationship between the terms and the terms is not to be construed as a scope of the present invention.
Example 1: primer and method for rapidly screening high-temperature-resistant strain of agaricus bisporus W192 strain
Primers 4140-1F and 4140-1R, seq.No.1 and seq.No.2, respectively, were designed based on gene 4140 and used as primers for screening a thermostable strain of Agaricus bisporus W192 strain. The sequence of gene 4140 is seq.No. 3. According to the primer, the detection method comprises the following specific steps:
(1) extraction of W192 Strain DNA
Culturing the collected W192 hybrid strain or spore strain on a PDA (personal digital assistant) plate, picking hyphae when the diameter of a bacterial colony on the plate is about 2cm, cracking the picked hyphae by using a DNA (deoxyribonucleic acid) cracking solution, and using the cracked liquid as a PCR (polymerase chain reaction) template. The DNA lysate was purchased from Shanghai Biotech engineering Co., Ltd.
(2) PCR amplification
The hyphal lysate was used as a PCR template for ordinary PCR amplification using primers 4140-1F and 4140-1R.
The two primer sequences were:
4140-1F:TTCCGTTCAGTGCTACC；
4140-1R:TGCAAAGAGAGCTGGTACA。
(3) PCR amplification conditions
The PCR amplification procedure was as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 95 deg.C for 30S, annealing at 48 deg.C for 30S, extension at 72 deg.C for 1min, 35 cycles, preservation at 72 deg.C for 10min, and preservation at 4 deg.C.
The PCR amplification system is as follows: the total volume was 20. mu.l, including 10ul of PCR mix, 7ul of double distilled water, 1ul of primer 4141-1F, 1ul of primer 4140-1R and 1ul of DNA template. PCR mix was purchased from Dalibobao corporation.
(4) Screening of high temperature resistant strains
The product size of ordinary PCR amplification by primers 4140-1F and 4140-1R is 781bp, if DNA of hybrid strain or spore strain of W192 strain is used as template, PCR amplification is carried out by primers 4140-1F and 4140-1R, if band with product size of 781bp can be amplified, as shown in FIG. 1, it is considered as high temperature resistant strain, and vice versa.
The invention carries out genome re-sequencing and comparative analysis on 5 screened high-temperature strains of W192 and an original strain, selects genes with different base sequences for verification, screens to obtain a gene with the number of 4140, designs a primer on a mutation site, takes DNA of the W192 strain as a template, can obtain a specific product with the length of 781bp by the high-temperature resistant strain, and can distinguish the high-temperature resistant strain from the original strain if a normal strain does not have the specific product. The method can quickly, accurately and simply identify the W192 high-temperature resistant strain by a PCR amplification method, thereby not only greatly shortening the process of breeding the W192 strain, but also improving the accuracy of breeding the high-temperature resistant strain.
Example 2: primer and method for rapidly screening high-temperature-resistant strain of agaricus bisporus W192 strain
Collection of spore strains of the W192 Strain
Collecting spore in spore collector, coating spore on PDA plate with spore concentration of 10 months 3 Perml, each plate 9cm in diameter was coated with 100ul of spore suspension and incubated in an incubator at 25 ℃.
Temperature treatment of spore strain of W192 Strain
After the germinated spore strain is treated at 40 ℃ for 24 hours, the germinated spore strain is transferred to 25 ℃ for continuous culture, and the spore strain which can continuously grow is used as an alternative of a high-temperature resistant strain.
DNA extraction of the spore strain W192
50mg of hypha of the spore strain is picked and put into a centrifugal tube with the volume of 1.5mL, 50ul of DNA lysate is added, and the mixture is incubated at 80 ℃ for 5min, so that the preparation of the DNA extract is finished.
PCR amplification
The DNA extract of spore strain is used as template, 4140-1F and 4140-1R are used as primers, and PCR amplification is carried out.
PCR amplification conditions
The PCR amplification procedure was as follows: pre-denaturation at 94 deg.C for 5min, denaturation at 95 deg.C for 30S, annealing at 48 deg.C for 30S, extension at 72 deg.C for 1min, 35, preservation at 72 deg.C for 10min, and preservation at 4 deg.C.
The PCR amplification system is as follows: the total volume was 20. mu.l, including 10ul PCR mix, 7ul double distilled water, 1ul primer 4140-1F, 1ul primer 4140-1R and 1ul DNA template.
6. Screening of high temperature resistant strains
The product size of the ordinary PCR amplification by using the primers 4140-1F and 4140-1R is 781bp, if the DNA of the spore strain of the W192 strain is used as a template, the PCR amplification is carried out by using the primers 4140-1F and 4140-1R, if a band with the product size of 781bp (as shown in figure 2) can be amplified, the strain is considered as a high temperature resistant strain, and vice versa.
The foregoing is illustrative of the preferred embodiment of the present invention and is not to be construed as limiting thereof in any way, and any modifications or variations thereof that fall within the spirit of the invention are intended to be included within the scope thereof.
Sequence listing
<110> Shanghai city academy of agricultural sciences
<120> primer and method for rapidly screening high-temperature-resistant strain of agaricus bisporus W192 strain
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 17
<212> DNA
<213> Artificial sequence
<400> 1
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence
<400> 2
<210> 3
<211> 781
<212> DNA
<213> Gene 4140
<400> 3
ttccgttcag tgctaccgtg acacacacgt gccattcaac caaatcacac ccgagtctct 60
cacgctagag gctatacact gcttaaacac tgcttaactt ctcttcgacc agacaagctg 120
gacaaacacg gcttgggaat ttgactgaca tcagtcatgt ggacattcgg ctgaatcaaa 180
catgataact tgttcggcag ctcgggataa gtggatgcag cctcaaacct gcggtggcat 240
ttcgatcaat tgtgtatatt tgaaaaccac aggtccattc ggtcagtata taccatgaat 300
gattgcaata gtttctactt gctgatccgt gccagacgca ataggaaacc cgagtctagc 360
aaagcccatg tcaggggagt aggtatactc gaccccatgg actcccaatc aacggccttt 420
ttacctcgaa taaaacgtct acccactgga gggacaatgc tcatcggagc tgtcctactt 480
atcgcctgtt aatcgtcgat gttgcagctc caccgaccat atcaattgtt actgcaaacg 540
gagtatggta tacctgcgaa cgttatctag agcgaccgag atacactgat tagtactatg 600
tatgcatcgc ccattcagtt gtgtgctcgc gttcacgcgc acgcttcatc cacgcttcaa 660
ttcgccttac tttatcgttc cgggagggag ttcccctttc ctctgactgc aactccttcc 720
ttcgaaatat atgtcccgag gcttcgagcc tagttgacgg cctgtaccag ctctctttgc 780
a 781
Claims (3)
1. A method for rapidly screening a high-temperature-resistant strain of an agaricus bisporus W192 strain is characterized by comprising the following steps: comprises the following steps:
step 1: extraction of W192 Strain DNA
Culturing a W192 hybrid strain or a spore strain to be detected on a PDA (personal digital assistant) flat plate, picking hyphae, and cracking the picked hyphae by using a DNA (deoxyribonucleic acid) cracking solution to obtain a hypha cracking solution;
step 2: PCR amplification
Performing PCR amplification by using the hypha lysate as a template for PCR amplification and using primers 4140-1F and 4140-1R;
4140-1F: TTCCGTTCAGTGCTACC；
4140-1R:TGCAAAGAGAGCTGGTACA；
and step 3: if the band with the product size of 781bp can be amplified, the strain is a high temperature resistant strain, otherwise, the strain is a non-high temperature resistant strain.
2. The method for rapidly screening the thermostable strain of Agaricus bisporus W192 strain according to claim 1, wherein: the PCR amplification procedure was: pre-denaturation at 94 deg.C for 5min, denaturation at 95 deg.C for 30s, annealing at 48 deg.C for 30s, extension at 72 deg.C for 1min, 35 cycles, preservation at 72 deg.C for 10min, and preservation at 4 deg.C.
3. The method for rapidly screening the thermostable strain of Agaricus bisporus W192 strain according to claim 1, wherein: the PCR amplification system is as follows: the total volume was 20. mu.l, and included 10. mu.l of PCR mix, 7. mu.l of double distilled water, 1. mu.l of primer 4140-1F, 1. mu.l of primer 4140-1R and 1. mu.l of hyphal lysate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011383787.6A CN112280892B (en) | 2020-12-01 | 2020-12-01 | Primer and method for rapidly screening high-temperature-resistant strain of agaricus bisporus W192 strain |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011383787.6A CN112280892B (en) | 2020-12-01 | 2020-12-01 | Primer and method for rapidly screening high-temperature-resistant strain of agaricus bisporus W192 strain |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112280892A CN112280892A (en) | 2021-01-29 |
| CN112280892B true CN112280892B (en) | 2022-08-02 |
Family
ID=74426181
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011383787.6A Active CN112280892B (en) | 2020-12-01 | 2020-12-01 | Primer and method for rapidly screening high-temperature-resistant strain of agaricus bisporus W192 strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112280892B (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110257264A (en) * | 2019-07-29 | 2019-09-20 | 上海市农业科学院 | A method of screening agaricus bisporus high-temperature resistant strain |
-
2020
- 2020-12-01 CN CN202011383787.6A patent/CN112280892B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN112280892A (en) | 2021-01-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110423694B (en) | Artificially domesticated bolete HZ18006 and SSR marker fingerprint spectrum thereof | |
| CN111876333B (en) | Lentinus edodes strain XG-3 capable of producing polysaccharide at high yield and application thereof | |
| Abe et al. | rDNA ITS sequence of Rhizopus oryzae: its application to classification and identification of lactic acid producers | |
| CN110423693B (en) | Boletus aereus strain YL1701-2 and SSR marker fingerprint spectrum thereof | |
| CN113088455B (en) | Lentinus edodes strain suitable for industrial cultivation and molecular identification method thereof | |
| CN112266878B (en) | Flammulina velutipes suitable for industrial cultivation and molecular identification method thereof | |
| CN112280892B (en) | Primer and method for rapidly screening high-temperature-resistant strain of agaricus bisporus W192 strain | |
| CN107475384A (en) | A kind of screening technique of manganese/Cd uptake efficient ecological type dyers' grapes | |
| CN112481405B (en) | Primer and method for rapidly screening high-temperature-resistant strain of agaricus bisporus A15 strain | |
| CN1702176A (en) | One-step dual PCR method for detecting fire blight of pear | |
| CN113337408B (en) | Lentinus edodes strain JXB5 and application thereof | |
| CN106434381B (en) | Pure white true pleurotus cornucopiae bacterial strain and its molecular labeling, specific primer to and application | |
| WO2006024961A2 (en) | Production of artificial hybrid lingzhi and uses thereof | |
| CN111996128B (en) | Flammulina velutipes suitable for industrial cultivation and molecular identification method thereof | |
| CN112063535B (en) | Hypsizigus marmoreus strain | |
| CN112280891A (en) | Primer and method for screening high-temperature-resistant strain of agaricus bisporus A15 strain | |
| CN109777884B (en) | Aegilops sharonensis 1Ssh chromosome specific molecular marker and application thereof | |
| CN103131755A (en) | Method for rapidly detecting brassica oleracea var. italic hybrid purity by using SRAP mark | |
| CN106755541B (en) | Molecular marker, primer and probe for identifying lyophyllum decastes | |
| CN108531641B (en) | Method and primer for identifying same-nucleus sterile single spore strain of agaricus bisporus and mating type of agaricus bisporus based on SNP locus difference | |
| CN114015585B (en) | Needle mushroom variety 'nong Wan jin 10' and specific molecular marker thereof | |
| Aminah et al. | Influence of pH and temperature on in vitro mycelial growth performance of wild edible Schizophyllum commune of northern Malaysia | |
| JP5595453B2 (en) | New species of Pleurotus sp. And method for producing the same | |
| CN113005220B (en) | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes J1011 strain, construction method and application thereof | |
| CN113186328B (en) | Identification method of microsatellite DNA marker fingerprint of flammulina velutipes xujin 18 strain and construction method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |