CN103289997A - Molecular marker of pure white Hypsizygus marmoreus Finc-W-62 strain, its acquisition method and application - Google Patents
Molecular marker of pure white Hypsizygus marmoreus Finc-W-62 strain, its acquisition method and application Download PDFInfo
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- CN103289997A CN103289997A CN2013100305518A CN201310030551A CN103289997A CN 103289997 A CN103289997 A CN 103289997A CN 2013100305518 A CN2013100305518 A CN 2013100305518A CN 201310030551 A CN201310030551 A CN 201310030551A CN 103289997 A CN103289997 A CN 103289997A
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Abstract
The invention relates to a molecular marker of a pure white Hypsizygus marmoreus Finc-W-62 strain. Its specific PCR amplification primer pair sequences are 5'-ATAAGGCGTTCCAAACTCATCTC-3' and 5'-TAAGAGACCAACTGCCCTTATCTC-3'; and the size of its DNA fragment is 1082bp. The invention also discloses an acquisition method and application of the molecular marker of the pure white Hypsizygus marmoreus Finc-W-62 strain.
Description
[technical field]
The invention belongs to biological technical field, be specifically related to a kind of molecule marker and preparation method and application of pure white Hypsizygus marmoreus Finc-W-62 bacterial classification.
[background technology]
Hypsizygus marmoreus (Latin title Hypsizygus marmoreus) is subordinate to Basidiomycotina, Hymenomycetes, Agaricales, white mushroom section, beautiful gill fungus genus, because it has unique crab delicate flavour, so claim that again it is crab mushroom.Initial artificial culture started from Japan before and after 1978, its delicious flavour, sense of food are tender and crisp, nutritious, and it is distinguished the flavor of than flat mushroom aquatic foods, and its meat is thicker than sliding mushroom, and its matter is more tough than mushroom, and mouthfeel is excellent, and its dry product also thick flavor overflows.Hypsizygus marmoreus contains abundant VITAMIN and 17 seed amino acids, and the beta-glucan that extracts in the sporophore has very high anti-tumor activity, is a kind of protective foods low in calories, low-fat.
The discriminating of tradition edible fungus species, especially for Hypsizygus marmoreus, the mode of appearance that its main foundation is sporophore, antagonistic effect, fruiting contrast etc.Mode of appearance is subject to planting environment (temperature, humidity, CO
2Concentration, intensity of illumination and ventilation etc.) and the influence of prescription, the difficulty that causes mode of appearance to identify.Between the nearer bacterial strain of sibship (between some parent and filial generation), antagonism is not obvious, differentiates that by antagonistic effect bacterial classification also has difficulties.At least 4 months consuming time of fruiting simultaneous test is unfavorable for evaluation and the detection of bacterial classification.Along with development of molecular biology, molecular labeling method is used for the discriminating of bacterial classification more and more widely.SCAR(Sequence-characterized Amplified Region) mark is a kind of very stable molecule marker, can be easy, quick, stable carry out strain identification, but at present pair SCAR molecule marker of pure white Hypsizygus marmoreus Finc-W-62 is not arranged as yet, so that Rapid identification pure white Hypsizygus marmoreus Finc-W-62.
[summary of the invention]
The technical problem that the present invention solves is to overcome the problem that prior art exists, and a kind of molecule marker and preparation method thereof of pure white Hypsizygus marmoreus Finc-W-62 bacterial classification is provided.
The present invention adopts round pcr to carry out a large amount of shaker tests, wherein, (sequence is: the specific DNA fragment that GGCGGATAAG) has obtained Hypsizygus marmoreus bacterial strain Finc-W-62 to adopt random primer, molecular weight is 1093bp, with this fragment cloning order-checking, based on the dna sequence dna of this fragment, design specific PCR amplimer, its special primer to sequence is: 5'-ATAAGGCGTTCCAAACTCATCTC-3' and 5'-TAAGAGACCAACTGCCCTTATCTC-3'.This primer can obtain the molecular weight size and be the specific fragment of 1082bp Hypsizygus marmoreus Finc-W-62 bacterial strain is carried out pcr amplification, is the molecule marker of Hypsizygus marmoreus bacterial strain Finc-W-62.
The present invention also discloses a kind of preparation method of molecule marker of pure white Hypsizygus marmoreus Finc-W-62 bacterial classification, comprises the steps:
1) obtains mycelia or sporophore;
2) extracting genome DNA;
3) specific DNA fragment is analyzed and obtained to the RAPD method: adopt the used random primer sequence of specific DNA fragment of RAPD method analysis and acquisition Hypsizygus marmoreus bacterial strain Finc-W-62 to be: GGCGGATAAG.
4) special primer design: based on the specific DNA fragment sequence of above-mentioned acquisition, design specific PCR amplimer, primer is 5'-ATAAGGCGTTCCAAACTCATCTC-3' and 5'-TAAGAGACCAACTGCCCTTATCTC-3' to sequence.
5) pcr amplification of SCAR molecule marker: with the specific PCR amplimer genome of Hypsizygus marmoreus Finc-W-62 bacterial strain is carried out the SCAR-PCR amplification, obtain the specific molecular marker of 1082bp.
6) electrophoresis detection.
The pcr amplification reaction system of RAPD method is in the described step 3): cumulative volume 25 μ L: genomic templates 20-30ng/ μ L 1.0 μ L, random primer 0.5pmol/ μ L 1.0 μ L, 10mM dNTP mix 0.5 μ L, 10 * Taq damping fluid, 2.5 μ L, 25mM MgCl
22.0 μ L, Taq enzyme 5U/ μ L 0.25 μ L adds water to 25 μ L; PCR reaction conditions: pre-95 ℃ of 3min of sex change; 94 ℃ of 45s, 36 ℃ of 60s, 72 ℃ of 2min, 35 circulations; 72 ℃ are extended 10min; Pure white Hypsizygus marmoreus Finc-W-62 bacterial strain specific DNA fragment size is 1082bp.
The pcr amplification of SCAR molecule marker in the described step 5), its system is: cumulative volume 25 μ L, template 20～30ng/ μ L 1 μ L, Hypsizygus marmoreus Finc-W-62 special primer is to each 0.5 μ L of 0.5pmol/ μ L, 10mM dNTP mix 0.5 μ L, 10 * Taq Buffer, 2.5 μ L, 25mM MgCl
22.0 μ L, Taq enzyme 5U/ μ L 0.25 μ L adds water to 25 μ L; PCR reaction conditions: pre-95 ℃ of 3min of sex change; 94 ℃ of 30s, 62 ℃ of 30s, 72 ℃ of 1min, 35 circulations; 72 ℃ are extended 5min.
The molecule marker that the present invention discloses a kind of pure white Hypsizygus marmoreus Finc-W-62 bacterial strain again is used for the Rapid identification of pure white Hypsizygus marmoreus Finc-W-62 bacterial strain and the application of detection, be specially and adopt the special detection primer of Hypsizygus marmoreus Finc-W-62 bacterial strain that the Hypsizygus marmoreus bacterial strain is carried out pcr amplification, according to the dna fragmentation that can produce the 1082bp size, as the foundation that Hypsizygus marmoreus Finc-W-62 bacterial strain is identified and detected.
Detection method of the present invention is compared with the authentication methods such as morphologic detection, antagonistic effect and the contrast of cultivation fruiting of routine, has short, advantage of high accuracy detection time.It can overcome morphologic detection, the antagonistic effect tolerance range is poor, can overcome the shortcoming that the cultivation fruiting contrasts length consuming time again.Present method finds that it has the specificity of Finc-W-62 bacterial strain in being applied to existing white Hypsizygus marmoreus bacterial strain detection.
[description of drawings]
Fig. 1 be random primer S265 to the electrophoretogram of the pcr amplification product of 4 Hypsizygus marmoreus bacterial strains, wherein 1 is H-W, 2 is Finc-W-62,3 is G-W, 4 is GC-W, M is 10kbp DNA ladder.
Fig. 2 is special primer to the electrophoretogram to the pcr amplification product of 4 Hypsizygus marmoreus bacterial strains, and wherein 1 is H-W, and 2 is Finc-W-62, and 3 is G-W, and 4 is GC-W, and M is 10kbp DNA ladder.
[embodiment]
Described " H-W " in following examples is the pure white Hypsizygus marmoreus kind that Japanese Big Dipper Co., Ltd. in 2002 seed selection is released, snow-white, the no bitter taste of its entire body; Described " G-W " bacterial strain is a kind of Hypsizygus marmoreus bacterial strain of buying from the market, and its original hase is grey, and along with the g and D of sporophore bleaches gradually, collection period, stem was canescence, and cap is pure white; Described " GC-W " bacterial strain is the white jade mushroom bacterial strain that Japanese Ge Cheng newly breeds.
The preparation method of the molecule marker of a kind of pure white Hypsizygus marmoreus Finc-W-62 comprises the steps:
1) mycelium culture and aerial hyphae obtain
The Finc-W-62 of cryopreservation and H-W, G-W and GC-W are inoculated on the PDA substratum, cultivated 20 days under the condition of 22 ℃ of temperature, humidity 75%, scrape and get aerial hyphae ,-20 ℃ frozen.
2) strain gene group DNA extracts
Extract according to giving birth to worker's biotechnology (Shanghai) limited-liability company (hereinafter to be referred as " giving birth to the worker ") SK1375 fungal gene group DNA extraction agent box specification sheets, with resulting dna solution place-20 ℃ standby.
3) the RAPD method is analyzed
See also table 1, select for use 14 random primers to carry out RAPD and analyze.Pcr amplification and system are set up (25 μ L): genomic templates (20-30ng/ μ L) 1.0 μ L, random primer (0.5pmol/ μ L) 1.0 μ L, dNTP mix (10mM each) 0.5 μ L, 10 * Taq damping fluid, 2.5 μ L, MgCl
2(25mM) 2.0 μ L, Taq enzyme (5U/ μ L) 0.25 μ L adds water to 25 μ L.PCR reaction conditions: pre-95 ℃ of 3mim of sex change; 94 ℃ of 45s, 36 ℃ of 60s, 72 ℃ of 2min, 35 circulations; 72 ℃ are extended 10min.
Table 1 random primer
Through screening, random primer S265 can increase and obtain the peculiar DNA band of Finc-W-62 bacterial strain, sees also Fig. 1.As can be seen from Figure 1, the band of a frame position is peculiar by Finc-W-62, does not occur and all there is this band on H-W, G-W and the GC-W place swimming lane correspondence position.
The distinctive DNA band of a frame position Finc-W-62 in the cutting drawing 1 reclaims DNA.Recovery method reclaims the test kit explanation according to giving birth to worker UNIQ-10 pillar DNA glue.
The DNA that reclaims adopts conventional order-checking, the results are shown in sequence table, and it is the dna fragmentation of 1093bp.
4) acquisition of special primer
Dna fragmentation two ends base sequence according to above-mentioned 1093bp, with Primer premier5.0 software design special primer sequence, several bases are respectively given up at described dna fragmentation two ends, and the amplimer of final design is 5'-ATAAGGCGTTCCAAACTCATCTC-3' and 5'-TAAGAGACCAACTGCCCTTATCTC-3' to sequence.
5) pcr amplification of SCAR molecule marker
With the specific PCR amplimer Finc-W-62 and H-W, G-W and GC-W are carried out the SCAR-PCR amplification.Amplification system: cumulative volume 25 μ L, template (20-30ng/ μ L) 1 μ L, Hypsizygus marmoreus Finc-W-62 bacterial strain detect primer special to (0.5pmol/ μ L) each 0.5 μ L, dNTPmix (10mM each) 0.5 μ L, 10 * Taq Buffer2.5 μ L, MgCl
2(25mM) 2.0 μ L, Taq enzyme (5U/ μ L) 0.25 μ L adds water to 25 μ L; PCR reaction conditions: pre-95 ℃ of 3min of sex change; 94 ℃ of 30s, 62 ℃ of 30s, 72 ℃ 1min35 circulation; 72 ℃ are extended 5min.
6) electrophoresis detection
Get above-mentioned pcr amplification product 6 μ L, with 1 μ L sample loading buffer mixing, point sample is on 1.5% sepharose, in 0.5 * tbe buffer liquid, electrophoresis under the 5V/cm voltage is after electrophoresis finishes, dye with EB, carry out agarose gel electrophoresis, take a picture at the gel imaging instrument then, its result sees also Fig. 2 electrophoretogram.
As can be seen from Figure 2, have only the new bacterial strain Finc-W-62 of Hypsizygus marmoreus of the present invention that the specific amplified band is arranged, and other three kinds existing Hypsizygus marmoreus bacterial strain H-W, G-W and GC-W all do not have amplified band, illustrate that thus 5'-ATAAGGCGTTCCAAACTCATCTC-3' and 5'-TAAGAGACCAACTGCCCTTATCTC-3' are the SCAR molecule marker primers of Finc-W-62.Be 1082bp with this primer to the dna fragmentation molecular weight that amplifies, described size is that the specific DNA fragment of 1082bp is the SCAR molecule marker of the new bacterial strain Finc-W-62 of Hypsizygus marmoreus of the present invention.
In the present embodiment, adopt mycelia to carry out the extraction of genomic dna, obviously, adopt sporophore can carry out the extraction of genomic dna too.The method of extracting has been that prior art is disclosed, is not giving unnecessary details at this.
More than describing is embodiments of the invention only, forgives and can understand, and under the prerequisite that does not depart from the present invention's design, all should be included within the technical conceive of the present invention simple modification of the present invention and replacement.
Claims (5)
1. the molecule marker of a pure white Hypsizygus marmoreus Finc-W-62 bacterial classification, it is characterized in that: the specific PCR amplimer is 5'-ATAAGGCGTTCCAAACTCATCTC-3' and 5'-TAAGAGACCAACTGCCCTTATCTC-3' to sequence; The dna fragmentation size is 1082bp.
2. the preparation method of the molecule marker of a pure white Hypsizygus marmoreus Finc-W-62 bacterial classification is characterized in that: comprise the steps:
1) obtains mycelia or sporophore;
2) extracting genome DNA;
3) specific DNA fragment is analyzed and obtained to the RAPD method: adopt the used random primer sequence of specific DNA fragment of RAPD method analysis and acquisition Hypsizygus marmoreus bacterial strain Finc-W-62 to be: GGCGGATAAG.
4) special primer design: based on the specific DNA fragment sequence of above-mentioned acquisition, design specific PCR amplimer, primer is 5'-ATAAGGCGTTCCAAACTCATCTC-3' and 5'-TAAGAGACCAACTGCCCTTATCTC-3' to sequence.
5) pcr amplification of SCAR molecule marker: with the specific PCR amplimer genome of Hypsizygus marmoreus Finc-W-62 bacterial strain is carried out the SCAR-PCR amplification, obtain the specific molecular marker of 1082bp.
6) electrophoresis detection.
3. the preparation method of the molecule marker of a kind of pure white Hypsizygus marmoreus Finc-W-62 bacterial strain according to claim 2, it is characterized in that: the pcr amplification reaction system of RAPD method is in the described step 3): cumulative volume 25 μ L: genomic templates 20-30ng/ μ L 1.0 μ L, random primer 0.5pmol/ μ L 1.0 μ L, 10mM dNTP mix0.5 μ L, 10 * Taq damping fluid, 2.5 μ L, 25mM MgCl
22.0 μ L, Taq enzyme 5U/ μ L 0.25 μ L adds water to 25 μ L; PCR reaction conditions: pre-95 ℃ of 3min of sex change; 94 ℃ of 45s, 36 ℃ of 60s, 72 ℃ of 2min, 35 circulations; 72 ℃ are extended 10min; Pure white Hypsizygus marmoreus Finc-W-62 bacterial strain specific DNA fragment size is 1082bp.
4. according to the preparation method of the molecule marker of claim 2 or 3 described a kind of pure white Hypsizygus marmoreus Finc-W-62 bacterial strains, it is characterized in that: the pcr amplification of SCAR molecule marker in the described step 5), its system is: cumulative volume 25 μ L, template 20～30ng/ μ L 1 μ L, Hypsizygus marmoreus Finc-W-62 special primer is to each 0.5 μ L of 0.5pmol/ μ L, 10mM dNTP mix0.5 μ L, 10 * Taq Buffer, 2.5 μ L, 25mM MgCl
22.0 μ L, Taq enzyme 5U/ μ L 0.25 μ L adds water to 25 μ L; PCR reaction conditions: pre-95 ℃ of 3min of sex change; 94 ℃ of 30s, 62 ℃ of 30s, 72 ℃ of 1min, 35 circulations; 72 ℃ are extended 5min.
5. the molecule marker of a kind of pure white Hypsizygus marmoreus Finc-W-62 bacterial strain according to claim 1 is applied to Rapid identification and the detection of pure white Hypsizygus marmoreus Finc-W-62 bacterial strain.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434381A (en) * | 2016-10-31 | 2017-02-22 | 上海丰科生物科技股份有限公司 | Pure white hypsizigus marmoreus strain, and molecular marker, specific primer pair and application thereof |
| CN106434382A (en) * | 2016-10-31 | 2017-02-22 | 上海丰科生物科技股份有限公司 | Pure white hypsizigus marmoreus strain, and molecular marker, specific primer pair and application thereof |
| CN112126702A (en) * | 2020-09-21 | 2020-12-25 | 东营市菇健生物科技有限公司 | White beech mushroom GJ7 strain and SSR marker primer and application thereof |
| CN112280687A (en) * | 2020-09-21 | 2021-01-29 | 东营市菇健生物科技有限公司 | Mushroom-Jian-Hypsizygus marmoreus GJ5 strain, SSR marker primer and application thereof |
-
2013
- 2013-01-25 CN CN201310030551.8A patent/CN103289997B/en active Active
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434381A (en) * | 2016-10-31 | 2017-02-22 | 上海丰科生物科技股份有限公司 | Pure white hypsizigus marmoreus strain, and molecular marker, specific primer pair and application thereof |
| CN106434382A (en) * | 2016-10-31 | 2017-02-22 | 上海丰科生物科技股份有限公司 | Pure white hypsizigus marmoreus strain, and molecular marker, specific primer pair and application thereof |
| CN106434382B (en) * | 2016-10-31 | 2018-12-21 | 上海丰科生物科技股份有限公司 | Pure white true pleurotus cornucopiae bacterial strain and its molecular labeling, specific primer to and application |
| CN106434381B (en) * | 2016-10-31 | 2018-12-21 | 上海丰科生物科技股份有限公司 | Pure white true pleurotus cornucopiae bacterial strain and its molecular labeling, specific primer to and application |
| CN112126702A (en) * | 2020-09-21 | 2020-12-25 | 东营市菇健生物科技有限公司 | White beech mushroom GJ7 strain and SSR marker primer and application thereof |
| CN112280687A (en) * | 2020-09-21 | 2021-01-29 | 东营市菇健生物科技有限公司 | Mushroom-Jian-Hypsizygus marmoreus GJ5 strain, SSR marker primer and application thereof |
| CN112126702B (en) * | 2020-09-21 | 2022-06-10 | 东营市菇健生物科技有限公司 | White beech mushroom GJ7 strain and SSR marker primer and application thereof |
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