CN101086017B - Mushroom 507 bacteria molecular specific mark and its obtaining method and uses - Google Patents
Mushroom 507 bacteria molecular specific mark and its obtaining method and uses Download PDFInfo
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- CN101086017B CN101086017B CN2007100403229A CN200710040322A CN101086017B CN 101086017 B CN101086017 B CN 101086017B CN 2007100403229 A CN2007100403229 A CN 2007100403229A CN 200710040322 A CN200710040322 A CN 200710040322A CN 101086017 B CN101086017 B CN 101086017B
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Abstract
The invention relates to a molecular special mark for mushroom 507 bacterial and the preparation method and application. The section of molecular marked DNA of mushroom 507 bacterial is 368 bp, the specific PCR augmentation primer squence is 5'AGGTCTATGTTCAGGAAGT 3'and 5'ATGCTGTATACGTGTTGTG 3'. The method comprises following steps: culturing bacterial filament and extracting genetic DNA; getting specific DNA section of mushroom 507 bacterial; getting specific PCR augmentation primer; SCAR- PCR augmentation; and ionophresis detection. The molecular mark can be used for fast determination and detection for mushroom 507 bacterial.
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 mushroom 507 bacterial classifications.
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.
Along with the foundation of the fast development of Protocols in Molecular Biology, particularly molecule marker and genetic marker technology with ripe, easy for developing, the identification of strains technology provides effective means fast and accurately.SCAR (Sequence CharacterizedAmplified Region) mark is a kind of very stable molecule marker, on using, have rapid, easy, characteristics cheaply, the present invention has set up the SCAR molecule marker of mushroom 507 bacterial classifications, can carry out Rapid identification to mushroom 507 bacterial classifications.
Summary of the invention
The objective of the invention is for can to mushroom 507 bacterial classifications carry out easy, identify fast and accurately and detect, a kind of molecule marker of mushroom 507 bacterial classifications is provided, the preparation method of the molecule marker of this mushroom 507 bacterial classifications is provided simultaneously.
A kind of molecular specific mark of mushroom 507 bacterial classifications is the specific fragment of 368bp size, and its specific PCR amplimer to sequence is: 5 ' AGGTCTATGTTCAGGAAGT 3 ' and 5 ' ATGCTGTATACGTGTTGTG 3 '.
A kind of preparation method of molecular specific mark of mushroom 507 bacterial classifications comprises the following steps:
(1) extraction of mycelium culture and genomic dna.
(2) the pulsating acquisition of the specific DNA of Xianggu mushroom strain 507: adopt round pcr, through a large amount of shaker tests, (primer sequence: AGGGCGTAAG) obtained the specific DNA segment of Xianggu mushroom strain 507, molecular weight is 405bp, with this fragment cloning order-checking to adopt random primer.
(3) acquisition of specific PCR amplimer: based on the dna sequence dna that obtains, design specific PCR amplimer, the right sequence of primer is as follows: 5 ' AGGTCTATGTTCAGGAAGT 3 ' and 5 ' ATGCTGTATACGTGTTGTG 3 '.
(4) increase to 507 bacterial strains being carried out SCAR-PCR with the specific PCR amplimer.
(5) specific fragment (Fig. 1) of the visible 368bp size of electrophoresis detection.And other mushroom strain there is no the specificity segment.
According to adopting this a pair of special primer to carry out pcr amplification, can produce the 368bpDNA fragment as the foundation that mushroom 507 bacterial strains are identified and detected.
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 produced the specificity that has 507 bacterial strains in the bacterial strain at 164 mushrooms of collected China.
Description of drawings
Fig. 1 mushroom 507 bacterial strain special primer for checking carry out the SCAR-PCR amplification to Xianggu mushroom strain
23 is 507 bacterial strains (Sanming City, Fujian Province fungal studies institutes) among the figure, and arrow is depicted as mushroom 507 bacterial strains and amplifies the special DNA band that molecular weight is about 368bp, the negative contrast of ck, and all the other are numbered other Xianggu mushroom strain.
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.
Embodiment 1
(1) the pulsating acquisition of the specific DNA of Xianggu mushroom strain 507: adopt round pcr, through a large amount of shaker tests, (primer sequence: AGGGCGTAAG) obtained the specific DNA segment of Xianggu mushroom strain 507, molecular weight is 368bp, with this fragment cloning order-checking to adopt random primer.
(2) specific PCR amplimer: based on the dna sequence dna that obtains, design specific PCR amplimer, the right sequence of primer is as follows: 5 ' AGGTCTATGTTCAGGAAGT 3 ' and 5 ' ATGCTGTATACGTGTTGTG 3 '.
(3) extraction of mycelium culture and genomic dna: the Xianggu mushroom strain 507 of cryopreservation is transferred on the PDA inclined-plane, cultivates activation for 25 ℃.Receive after two weeks in the 100mL PD liquid nutrient medium, 25 ℃ of two weeks of 100r/min shaking culture, collect mycelia, it is frozen to put into-20 ℃ of refrigerators.Extract genomic dna with improved CTAB method, DNA is diluted to 20ng/ μ L, and-20 ℃ of refrigerator storages are standby.
(4) pcr amplification of SCAR molecule marker: the amplification system cumulative volume is: 25 μ L, 10 * PCR buffer, 2.5 μ L, 25mmol/L MgCL
22 μ L, 10mmol/L dNTP 0.25L, 2.5U/ μ L Taq DNA enzyme 0.5 μ L, 10 μ mol/L, 507 bacterial strains detect primer special to each 1 μ L, 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, 61 ℃ of 15second, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min.
In order to get rid of the false negative situation of other bacterial classification, in the SCAR-PCR reaction system, add ITS (InternalTranscribed Spacer) universal primer to (ITS1, ITS4), verify the integrity of all template DNAs.
(5) 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% fine jade vinegar sugar gel, in 0.5 * tbe buffer liquid, electrophoresis under the 5V/cm voltage is after electrophoresis finishes, with EB dyeing, on the gel imaging instrument, take a picture then.
Embodiment 2
To 114 (for produce with kind) individual collection from mushroom production in all parts of the country with bacterial classification and minority wild species totally 164 bacterial strains carry out SCAR and increase.The single-minded SCAR mark that has had only mushroom 507 bacterial strains to amplify in 164 bacterial classifications.
Claims (2)
1. the molecular specific mark of mushroom 507 bacterial classifications, it is characterized in that: adopt the specific PCR amplimer that 5 ' AGGTCTATGTTCAGGAAGT 3 ' and 5 ' ATGCTGTATACGTGTTGTG 3 ' are increased, obtain size and be described molecular specific mark for the dna segment of 368bp.
2. the molecular specific mark of a kind of mushroom 507 bacterial classifications according to claim 1 is applied to the Rapid identification and the detection of mushroom 507 bacterial classifications.
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| CN2007100403229A CN101086017B (en) | 2007-04-29 | 2007-04-29 | Mushroom 507 bacteria molecular specific mark and its obtaining method and uses |
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| CN101086017B true CN101086017B (en) | 2010-08-25 |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101265494B (en) * | 2008-05-09 | 2011-06-01 | 上海市农业科学院 | Molecular mark, detecting method and application of mushroom Xiangjiu strain |
| 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 |
| CN110714094B (en) * | 2019-11-22 | 2022-06-03 | 福建农林大学 | SCAR molecular marker for specifically identifying tremella strain and identification method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1944677A (en) * | 2006-10-17 | 2007-04-11 | 西北农林科技大学 | Method for identifying Chinese cabbage variety and inspecting seed purity by using SCAR marker |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1944677A (en) * | 2006-10-17 | 2007-04-11 | 西北农林科技大学 | Method for identifying Chinese cabbage variety and inspecting seed purity by using SCAR marker |
Non-Patent Citations (5)
| Title |
|---|
| Lian-Hua Qin et al.Use of intersimple sequence repeats markers to developstrain-specific SCAR markers for Lentinula edodes.FEMS Microbiol Lett257 1.2006,257(1),112-116. |
| Lian-Hua Qin et al.Use of intersimple sequence repeats markers to developstrain-specific SCAR markers for Lentinula edodes.FEMS Microbiol Lett257 1.2006,257(1),112-116. * |
| 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 2.2005,24(2),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 2.2005,24(2),259-266. * |
| 宋春艳等.香菇生产菌株的SCAR鉴定.菌物学报24.2005,24132-138. * |
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