CN101265493B - Molecular mark, detecting method and application of mushroom cultivation strain - Google Patents
Molecular mark, detecting method and application of mushroom cultivation strain Download PDFInfo
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- CN101265493B CN101265493B CN2008100372600A CN200810037260A CN101265493B CN 101265493 B CN101265493 B CN 101265493B CN 2008100372600 A CN2008100372600 A CN 2008100372600A CN 200810037260 A CN200810037260 A CN 200810037260A CN 101265493 B CN101265493 B CN 101265493B
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Abstract
The invention relates to a molecular mark of mushroom cultivation strains 241-4, a detecting method and the application thereof. The molecular mark is a molecular specific detecting mark of gene SCAR, and a PCR special primer pair thereof has the sequence that 241-4F:5<1>CTCTGGTCATTGTTACTGTG 3<1> and 241-4R:5<1>CTCTGGCAAGGCTTAT 3<1>. The detecting method comprises the steps that are (1) mycelium culture, (2) the extraction of genome DNA, (3) the detection of the SCAR molecular mark, and (4) electrophoresis detection. The method has the application that the gene SCAR in mushroom strains is expanded, and the mushroom strains with SCAR marks are the mushroom cultivation strains 241-4. Compared with the conventional morphological detection, an antagonism test and a fruiting test, the molecular mark, the detecting method and the application thereof have the advantages of short detecting time and high accuracy; the detecting method has specificity of 241-4 strains in the thirty collected major mushroom strains in China.
Description
Technical field
The invention belongs to the detection range of cultivating champignon bacterial classification, particularly relate to molecule marker, its detection method and the application of mushroom cultivation strain-4.
Background technology
China's mushroom production is from 1.95 ten thousand tons of 2,420,000 tons of developing 2005 rapidly of nineteen eighty-three, account for more than 70% of global mushroom ultimate production, rewritten the ranking list of world's edible mushrooms output, and constantly dwindle and the Twospore Mushroom poor distance of ranking first, scholarly forecast is arranged, because the fast development of Chinese mushroom industry, it will become the highest edible mushrooms of world wide production in nearly 10 years.China's mushroom is with its alarming development speed, and fine quality and cheap cost are that world mushroom industry personage attractes attention the fashionable world of Chinese mushroom.
The contribution rate of good quality strain in mushroom per unit area yield and quality is very important, and this has determined the critical role of mushroom strain in the mushroom industry.China had signed " international new variety of plant protection method " in 1999; this not only requires us to respect other national kind intellecture property; also to strengthen simultaneously protecting the kind intellecture property of our country oneself; really protect the kind property right of China in order to set up edible mushrooms new variety resignation system; must at first set up sophisticated cultivar identification technology, for the new variety registration lays the foundation.Especially Japan on April 1st, 2004 came into effect " seedling method amendment ", to the export mixes of China edible mushrooms especially mushroom great threat.With regard to domestic, the phenomenon of cultivating champignon bacterial classification " different name of the same race, xenogenesis is of the same name " has brought great loss not only for the mushroom farming, has influenced their cultivation enthusiasm, has also greatly influenced the fast development of Chinese mushroom; And, more and more higher along with the appearance of large-scale factory culture mode to the requirement of cultivating champignon bacterial strain quality, need more easy, the identification of strains technology fast and accurately of development, with guarantee every batch with kind all accurate.
In the face of this situation, just need us further to accelerate the strain identification Study on Technology, in the research of mushroom, introduce more efficiently strain identification system.
Summary of the invention
The purpose of this invention is to provide molecule marker, its detection method and the application of a kind of mushroom cultivation strain-4, by adopting round pcr, through shaker test, obtained the specific DNA segment of Xianggu mushroom strain 241-4, based on this segmental dna sequence dna, design specific PCR amplimer, thereby by the specific fragment detection of 241-4 bacterial strain being identified mushroom 241-4 bacterial strain and being detected.
The molecule marker of a kind of mushroom cultivation strain-4 of the present invention, it is the molecular specific certification mark of gene SCAR, the primer special of its pcr amplification is 241-4F/R, 241-4F is 5 ' CTCTGGTCATTGTTACTGTG 3 ', 241-4R is 5 ' CTCTGGCAAGGCTTAT 3 ', and PCR product size is 555bp.
The segmental gene order of the SCAR that amplifies is as follows:
5′-CTCTGGTCATTGTTACTGTGTTTATTGGAGTACTGAGCAACTACAAGACTCTGTAGCACAACCTACTGAGCTTGTATTAAGGCATTGACATTCTCTGCACTGAGCTGGGTGGCATACTGCTTGGGTTAGCTGAAGGGCGCTCCAGTGTGTTTCAACTTCAACAAGTGCAGTCTCCTTCAACACGGAATAGGAATCAATGGGTAATGTTTGAGATCTTCAGACGACTGAAAATCAATACACGATGAATAAAACTAATTAAACAAGAAAAGAAAAAGACCGAATACATGTACTAAAAACGAGGAACGATCTTCAAGGCTAACTCCCCATTGTTGGTTCAAGCTATCATGAAAGAAAGCCGGGAGTTTTTGGGATAAGAATGCTGCAGTGCAAGGTACATGTTCGGCAAACAAAGGGCAGTTAGCCCGACCGATCCAGAAATCGCGAATATAAATCTAAAATCAAAATCAATTAACAAGTTCGGTTGGAAAACCAGACAAACAAATACATACCAAGAATGGTGCCGCCTCCTCAAATAGAGCATAAGCCTTGCCAGAG-3′。
The detection method of the molecule marker of mushroom cultivation strain of the present invention-4 comprises the following steps:
(1) mycelium culture
The mushroom strain of 4 ℃ of preservations is transferred on the PDA flat board, 23 ℃～25 ℃ lucifuges are cultivated, and receive in the 100mL PDY substratum 100rpm～150rpm behind 10d～14d, collect mycelia behind 23 ℃～25 ℃ shake-flask culture 10d～14d, put into-20 ℃ of refrigerators guarantors and freeze standby;
(2) extraction of genomic dna
Extract the genomic dna of mycelia with improved CTAB (cetyl trimethylammonium bromide) method:
1) with-20 ℃ of cryodesiccated mushroom mycelium grind into powders, add 2 * CTAB extract of 65 ℃ of preheatings, more than 65 ℃ of insulation 45min, or the jog mixing;
2) 12000rpm, 4 ℃ of centrifugal 20min get supernatant liquor;
3) add isopyknic phenol, chloroform and primary isoamyl alcohol mixed solution (the mixed volume ratio of mixed solution is 25: 24: 1), mixing 10min gently, 12000rpm, 4 ℃ of centrifugal 10min get supernatant liquor and move in the new centrifuge tube;
4) add isopyknic chloroform and primary isoamyl alcohol mixed solution (the mixed volume ratio of mixed solution is 24: 1), mixing 10min gently, 12000rpm, 4 ℃ of centrifugal 10min get supernatant liquor and move in the new centrifuge tube;
5) add the Virahol of-20 ℃ of precoolings of 2/3 volume (with respect to previous step rapid in supernatant liquor), shake 5min gently, 8000rpm, 4 ℃ of centrifugal 10min remove supernatant;
6) precipitation is washed 2～3 times with 75% (percent by volume) ethanol, 10mmol/L Potassium ethanoate (KAC), each 8000rpm, the centrifugal 5min of room temperature;
7) 95% (percent by volume) ethanol of adding precooling turns upside down gently, and the centrifugal 10min of 8000rpm room temperature abandons ethanol, and vacuum is drained or dried naturally;
8) add 100 μ L, 10 * TE (Tris/EDTA) damping fluid, beat gently and make resolution of precipitate;
9) add 37 ℃ of water-baths of 1 μ L 10mg/mL RNaseA, 1hr removes RNA;
Ultraviolet spectrophotometry detects total genomic dna concentration and purity, adjusts the concentration unanimity of sample DNA, refrigerates standby;
(3) detection of SCAR molecule marker
Amplification system (cumulative volume 25 μ L): 10 * PCR buffer, 2.5 μ L, 25mmol/L MgCL
22 μ L, 10mmol/LdNTP 0.25L, 2.5U/ μ L Taq DNA enzyme 0.5 μ L, each 1 μ L of 10 μ mol/L 241-4F/R primers, the template DNA 1 μ L of extraction (concentration 1ng～10ng/ μ L), ddH
2O 18.6 μ L;
PCR reaction conditions: 94 ℃ of 1min; 94 ℃ of 15second, 58 ℃ of 15second, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min;
(4) electrophoresis detection
Get above-mentioned pcr amplification product 8 μ L, with 1 μ L sample loading buffer mixing, point sample is on 1.5% sepharose, in 0.5 * tbe buffer liquid (Tris-borate buffer), electrophoresis under the 5V/cm voltage is after electrophoresis finishes, with EB (ethidium bromide) dyeing, on the gel imaging instrument, take a picture analytical results then.
The application of the molecule marker of a kind of mushroom cultivation strain-4 of the present invention is the pcr amplification special primer for checking that utilize the SCAR of mushroom strain 241-4, carries out the SCAR amplification, and what have the SCAR mark promptly is mushroom cultivation strain-4.
Beneficial effect of the present invention:
This detection method is compared with conventional morphologic detection, antagonistic effect, fruiting experiment, has weak point detection time, advantage of high accuracy.This detection required time only needs 2-3 days, and conventional antagonistic effect required time needs two time-of-weeks at least, and fruiting experiment then needs at least 3 months time; This method is planted the specificity that has the 241-4 bacterial strain in the bacterial classification 30 mushroom masters of collected China.
Description of drawings
Fig. 1 is the SCAR amplification collection of illustrative plates of 241-4, and wherein M is 100bpDNAladder, and NC is contrast, and the arrow indication is the 241-4 specific marker.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
1, mycelium culture
The mushroom strain of 4 ℃ of preservations is transferred on the PDA flat board, and 25 ℃ of lucifuges are cultivated.Receive in the 100mL PDY substratum 150rpm, 25 ℃ of shake-flask culture behind the 14d.When treating that mycelial growth reaches a certain amount of, collect mycelia, put into-20 ℃ of refrigerators guarantors and freeze standby.
2, the extraction of genomic dna
Extract genomic dna with improved CTAB method.Ultraviolet spectrophotometry detects total genomic dna concentration and purity, adjusts the concentration unanimity of sample DNA, refrigerates standby.
3, the detection of SCAR molecule marker
Amplification system (cumulative volume 25 μ L): 10 * PCR buffer, 2.5 μ L, 25mmol/L MgCL
22 μ L, 10mmol/LdNTP 0.25L, 2.5U/ μ L Taq DNA enzyme 0.5 μ L, each 1 μ L of 10 μ mol/L 241-4F/R, template DNA 1 μ L (concentration 1ng～10ng/ μ L), ddH
2O 18.6 μ L.PCR reaction conditions: 94 ℃ of 1min; 94 ℃ of 15second, 58 ℃ of 15second, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min.
4, electrophoresis detection
Get above-mentioned pcr amplification product 8 μ L, with 1 μ L sample loading buffer mixing, point sample is on 1.5% sepharose, and in 0.5 * tbe buffer liquid, electrophoresis under the 5V/cm voltage after electrophoresis finishes, with EB dyeing, is taken a picture on the gel imaging instrument then.
Adopt special-purpose 241-4 to detect primer Xianggu mushroom strain is carried out pcr amplification, mushroom 241-4 bacterial strain can amplify the special DNA band that molecular weight is 555bp.Shown in arrow mark among Fig. 1.
Utilize the Auele Specific Primer of 241-4 that 114 collections are carried out the SCAR amplification from mushroom production in all parts of the country with bacterial classification and minority wild species.There are 6 bacterial classifications to amplify the SCAR mark of 241-4 in 114 bacterial classifications, wherein four is the 241-4 bacterial strain of collecting in different places, and two other is respectively shake rich No. 1 of the teacher of the Wang You 12 of state edible mushrooms institute and Xinyang, Henan Province agrotechnical station of Qingyuan County, Zhejiang Province.
Claims (6)
1. the molecule marker of a mushroom cultivation strain-4, it is characterized in that: this molecule marker is the molecular specific certification mark of gene SCAR, the primer special of its pcr amplification is 241-4F/R, 2414F is 5 ' CTCTGGTCATTGTTACTGTG3 ', 241-4R is 5 ' CTCTGGCAAGGCTTAT 3 ', and PCR product size is 555bp;
The segmental gene order of the SCAR that amplifies is as follows:
5′-CTCTGGTCATTGTTACTGTGTTTATTGGAGTACTGAGCAACTACAAGACTCTGTAGCACAACCTACTGAGCTTGTATTAAGGCATTGACATTCTCTGCACTGAGCTGGGTGGCATACTGCTTGGGTTAGCTGAAGGGCGCTCCAGTGTGTTTCAACTTCAACAAGTGCAGTCTCCTTCAACACGGAATAGGAATCAATGGGTAATGTTTGAGATCTTCAGACGACTGAAAATCAATACACGATGAATAAAACTAATTAAACAAGAAAAGAAAAAGACCGAATACATGTACTAAAAACGAGGAACGATCTTCAAGGCTAACTCCCCATTGTTGGTTCAAGCTATCATGAAAGAAAGCCGGGAGTTTTTGGGATAAGAATGCTGCAGTGCAAGGTACATGTTCGGCAAACAAAGGGCAGTTAGCCCGACCGATCCAGAAATCGCGAATATAAATCTAAAATCAAAATCAATTAACAAGTTCGGTTGGAAAACCAGACAAACAAATACATACCAAGAATGGTGCCGCCTCCTCAAATAGAGCATAAGCCTTGCCAGAG-3′。
2. the detection method of the molecule marker of mushroom cultivation strain-4 according to claim 1 comprises the following steps:
(1) mycelium culture
Mushroom strain is transferred on the PDA flat board, and 23 ℃～25 ℃ lucifuges are cultivated, and receive behind 10d～14d in the 100mL PDY substratum, collect mycelia behind 100rpm～150rpm, 23 ℃～25 ℃ shake-flask culture 10d～14d;
(2) extraction of genomic dna
Extract the genomic dna of mycelia with improved CTAB method, ultraviolet spectrophotometry detects total genomic dna concentration and purity, adjusts the concentration unanimity of sample DNA;
(3) detection of SCAR molecule marker
The DNA that extracts is carried out the pcr amplification of gene SCAR;
(4) electrophoresis detection
Above-mentioned pcr amplification product and sample loading buffer mixing, point sample are on sepharose, and electrophoresis with EB dyeing, is taken a picture analytical results on the gel imaging instrument.
3. the detection method of the molecule marker of mushroom cultivation strain-4 according to claim 2 is characterized in that: the improved CTAB method in the described step (2):
1) with-20 ℃ of cryodesiccated mushroom mycelium grind into powders, add 2 * CTAB extract of 65 ℃ of preheatings, more than 65 ℃ of insulation 45min, or the jog mixing;
2) 12000rpm, 4 ℃ of centrifugal 20min get supernatant liquor;
3) add the mixed solution of isopyknic phenol, chloroform and primary isoamyl alcohol, the mixed volume ratio of its mixed solution is 25: 24: 1, mixing 10min gently, and 12000rpm, 4 ℃ of centrifugal 10min get supernatant liquor and move in the new centrifuge tube;
4) add the mixed solution of isopyknic chloroform and primary isoamyl alcohol, the mixed volume ratio of its mixed solution is 24: 1, mixing 10min gently, and 12000rpm, 4 ℃ of centrifugal 10min get supernatant liquor and move in the new centrifuge tube;
5) Virahol of-20 ℃ of precoolings of adding 2/3 volume shakes 5min gently, 8000rpm, and 4 ℃ of centrifugal 10min remove supernatant;
6) precipitation is 75% ethanol, 10mmol/L Potassium ethanoate KAC washing 2～3 times with percent by volume, 8000rpm at every turn, the centrifugal 5min of room temperature;
7) percent by volume of adding precooling is 95% ethanol, turns upside down gently, and the centrifugal 10min of 8000rpm room temperature abandons ethanol, and vacuum is drained or dried naturally;
8) add 100 μ L, 10 * TE damping fluid, beat gently and make resolution of precipitate;
9) add 37 ℃ of water-baths of 1 μ L 10mg/mL RNaseA, 1hr removes RNA;
10) the DNA extraction thing is standby in-20 ℃ of refrigerator storages.
4. the detection method of the molecule marker of mushroom cultivation strain-4 according to claim 2 is characterized in that: the cumulative volume of the pcr amplification in the described step (3) is 25 μ L, comprising: 10 * PCR buffer, 2.5 μ L, 25mmol/LMgCl
22 μ L, 10mmol/L dNTP 0.25L, 2.SU/ μ L Taq DNA enzyme 0.5 μ L, 10 μ mol/L 241-4 bacterial strains detect primer special to each 1 μ L, the template DNA 1 μ L of concentration 1ng～10ng/ μ L, ddH
2O 18.6 μ L;
PCR reaction conditions: 94 ℃ of 1min; 94 ℃ of 15second, 58 ℃ of 15second, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min;
5. the detection method of the molecule marker of mushroom cultivation strain-4 according to claim 2, it is characterized in that: the pcr amplification product in the described step (4) is 8 μ L, sample loading buffer is 1 μ L, sepharose is 1.5%, electrophoresis is in 0.5 * tbe buffer liquid, electrophoresis under the 5V/cm voltage.
6. the application of the molecule marker of a kind of mushroom cultivation strain-4 according to claim 1 is gene SCAR amplifications of mushroom strain being carried out described molecule marker, and what have the SCAR mark promptly is mushroom cultivation strain-4.
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| CN101712984B (en) * | 2009-11-30 | 2011-12-21 | 浙江省林业科学研究院 | Molecular specificity mark primer for authenticating mushroom strains 241 and 241-4 and detecting method |
| CN101962687B (en) * | 2010-11-15 | 2012-10-17 | 上海百茸食用菌有限公司 | Method for evaluating agrocybe cylindracea Ag..c0003 by molecule marker technology |
| CN106801093B (en) * | 2017-02-07 | 2020-07-24 | 上海市农业科学院 | SSR marker fingerprint spectrum of No. 1 Ganxiang mushroom strain and construction method and application thereof |
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| CN101041857A (en) * | 2007-04-29 | 2007-09-26 | 上海市农业科学院食用菌研究所 | Molecule differential mark for mushroom 9015 and Qingke 20 bacterial and method and application thereof |
| CN101086018A (en) * | 2007-04-29 | 2007-12-12 | 上海市农业科学院食用菌研究所 | Mushroom 7402 bacteria molecular specific mark method and uses |
| CN101086019A (en) * | 2007-04-29 | 2007-12-12 | 上海市农业科学院食用菌研究所 | Mushroom 'Shenxiang'93 bacteria molecular specific mark, and method and uses |
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| CN101041857A (en) * | 2007-04-29 | 2007-09-26 | 上海市农业科学院食用菌研究所 | Molecule differential mark for mushroom 9015 and Qingke 20 bacterial and method and application thereof |
| CN101086018A (en) * | 2007-04-29 | 2007-12-12 | 上海市农业科学院食用菌研究所 | Mushroom 7402 bacteria molecular specific mark method and uses |
| CN101086019A (en) * | 2007-04-29 | 2007-12-12 | 上海市农业科学院食用菌研究所 | Mushroom 'Shenxiang'93 bacteria molecular specific mark, and method and uses |
Non-Patent Citations (2)
| Title |
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| 吴学谦等.SCAR分子标记技术在香菇菌株鉴定上的应用研究.菌物学报24 02.2005,24(02),259-266. |
| 吴学谦等.SCAR分子标记技术在香菇菌株鉴定上的应用研究.菌物学报24 02.2005,24(02),259-266. * |
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