CN101358238B - Molecular specificity labeled primers of mushroom Cr02 strain and test method - Google Patents
Molecular specificity labeled primers of mushroom Cr02 strain and test method Download PDFInfo
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- CN101358238B CN101358238B CN200810120314XA CN200810120314A CN101358238B CN 101358238 B CN101358238 B CN 101358238B CN 200810120314X A CN200810120314X A CN 200810120314XA CN 200810120314 A CN200810120314 A CN 200810120314A CN 101358238 B CN101358238 B CN 101358238B
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Abstract
The invention provides a molecular specificity labeled primer for shiitake mushroom Cr02 strains and a method for identifying and testing the shiitake mushroom Cr02 strains by utilizing the primer. The primer sequence is: the upstream primer: 5'-AGGCAAACTGGTTCCATGATA-3' and the downstream primer: 5'-CTGTGAAGTCA T TACAGTCGC-3'. The beneficial effect of the molecular specificity labeled primer liesin that the testing method of the invention has the advantages of short testing time and high accuracy by comparing with the conventional morphological test, the antagonistic test and the fruiting test. The testing time of the invention is required only one to three days, while the conventional antagonistic test requires at least two weeks and the fruiting test requires at least three months.
Description
(1) technical field
The present invention relates to the molecular specificity labeled primers of mushroom Cr 02 bacterial, and the method for utilizing this primer that mushroom Cr 02 bacterial is differentiated and detected.
(2) background technology
Mushroom (Lentinula edodes (Berk.) Pegler) originates from the temperate zone-subtropical zone in Asia and Australia.Mushroom is the well-known whole world with its nutrition, medical value and unique taste, particularly in China and other country in Southeast Asia.China is the country of mushroom culture the earliest, and mushroom production reaches global 70% at present.Mushroom strain is the most important production means during mushroom is produced, kind surplus the mushroom strain that uses in the China Business cultivation has surpassed 100 at present.In mushroom was produced, some mushroom strains with high yield and high quality cultivation characteristic were be evaluated as excellent species in industrial community usually, for example the Cr02 that always cultivates on producing, celebrating 20, Shen 8, fragrant 9 bacterial classifications etc.These excellent species obtain commerial growing in China, even have introduced a fine variety some other Asian countries and regional as Japan, Korea S, TaiWan, China etc., and therefore the grower has also obtained huge economic interests.Yet for a long time, the bacterial classification on China mushroom main product ground is produced and sold comparatively chaotic always, " xenogenesis different name of the same name, of the same race " phenomenon ubiquity, particularly some excellent species frequently palmed off and appeared on the bacterial classification market, greatly damaged the mushroom breeder and the producer's interests.Thereby for mushroom industry, it is particularly urgent to set up a kind of easy, quick, reliable excellent species authenticate technology.
The foundation of the fast development of Protocols in Molecular Biology, particularly molecular marking technique is with ripe, and is easy for developing, the strain identification technology provides effective means fast and accurately.The molecular marking technique of some PCR-based such as RAPD (Random Amplified Polymorphic DNA, randomly amplified polymorphic DNA), AFLP (Amplified Fragment Length Polymorphism, amplified fragment length polymorphism) classification that has been used for edible fungus species the nineties in eighties of last century is identified, but that these marks are used for the evaluation of mushroom strain is all not ideal enough.SCAR (SequenceCharacterizedAmplified Region, characteristic fragment amplification zone) mark is to be proposed on the RAPD basis by Paran and Michelmore in 1993, it is based on to the segmental order-checking of special RAPD, primer according to a pair of 18～24 bases of two ends sequences Design, carry out under higher annealing temperature that specific amplified realizes, the competition between the random primer binding site has been got rid of in the employing of its specificity primer, thereby it is a kind of very stable molecule marker, has rapid, easy, characteristics cheaply on using.
(3) summary of the invention
The present invention seeks to set up the SCAR molecule marker of mushroom Cr 02 bacterial, the molecular specificity labeled primers of mushroom Cr 02 bacterial is provided, and a kind of method that can carry out Rapid identification to mushroom Cr 02 bacterial.
The technical solution used in the present invention is:
The molecular specificity labeled primers of mushroom Cr 02 bacterial, described primer sequence is:
Upstream primer 5 '-AGGCAAACTGGTTCCATGATA-3 ';
Upstream primer 5 '-CTGTGAAGTCATTACAGTCGC-3 '.
This primer is to being the employing round pcr, through a large amount of shaker tests, adopt RAPD to be labeled as the DNA fragment specific that primer obtains mushroom Cr 02 bacterial, with this fragment cloning order-checking, based on the dna sequence dna that obtains, designed Auele Specific Primer carries out pcr amplification with this primer to mushroom Cr 02 bacterial, can obtain the specific fragment of 1084bp size.
The invention still further relates to the method that a kind of described molecular specificity labeled primers is identified and detected mushroom Cr 02 bacterial, described method is: extract mushroom strain genomic dna to be measured as template, with described molecular specificity labeled primers as amplimer, carry out pcr amplification, amplified production is carried out electrophoresis detection, if the DNA band of 1084bp appears in electrophoresis result, bacterial classification then to be measured is a mushroom Cr 02 bacterial; Described molecular specificity labeled primers sequence is:
Upstream primer 5 '-AGGCAAACTGGTTCCATGATA-3 ';
Downstream primer 5 '-CTGTGAAGTCATTACAGTCGC-3 '.
Described method key is the selection of amplimer, and DNA extraction, PCR reaction system and reaction conditions are determined, and electrophoresis detection, all can carry out according to this area ordinary method.
Preferably, PCR reaction system of the present invention is composed as follows:
PCR Buffer final concentration is 1 *
dNTPs 1mmol/L
MgCl
2 2.5mmol/L
Taq DNA enzyme 2.5U
Each 1.25 μ M of upstream and downstream primer
Template DNA 60ng
Surplus is ddH
2O;
The PCR reaction conditions is as follows:
Behind 94 ℃ of pre-sex change 6min; 92 ℃ of sex change 40s, 54.5 ℃ of annealing 40s, 72 ℃ are extended 2min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last.
PCR Buffer final concentration is 1 *, be meant that the concentration of each component in reaction system is identical with 1 * PCR Buffer among the PCR Buffer, selecting volume usually for use is 10 * PCR Buffer of reaction system volume 1/10.10 * PCR Buffer composition is: 100mMTris-HCl (pH8.5), 500mMKCl, 25mM MgCl
2And 1.0%Triton-X-100, solvent is ddH
2O.
Concrete, the method for the invention is as follows:
(1) get mushroom strain to be measured, be seeded to the PDA inclined-plane, cultivate 14d for 25 ℃, be forwarded in the PD liquid nutrient medium, 25 ℃ of following shaking culture 14d collect mycelia, extract mushroom strain genomic dna to be measured with the SDS-CTAB method;
(2) genomic dna that extracts with step (1) is a template, as amplimer, carries out pcr amplification with described molecular specificity labeled primers:
Per 20 μ L are composed as follows for the PCR reaction system:
10×PCR?Buffer 2μL
10mmol/L?dNTPs 2μL
25mmol/L?MgCl
2 2μL
5U/ μ L Taq DNA enzyme 0.5 μ L
Each 1 μ L of 25 μ M upstream and downstream primers
20ng/ μ L template DNA 3 μ L
DdH
2O complements to 20 μ L;
The PCR reaction conditions is as follows:
Behind 94 ℃ of pre-sex change 6min; 92 ℃ of sex change 40s, 54.5 ℃ of annealing 40s, 72 ℃ are extended 2min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last;
(3) get step (2) amplified production 5 μ L, with 1 μ L0.25% bromjophenol blue damping fluid mixing, point sample is on 1.5% sepharose, electrophoresis in 1 * TAB damping fluid, under the 5V/cm voltage, the EB dyeing of electrophoresis end back, EB dyeing was dyeed 30 minutes in the aqueous solution that contains 0.5 μ g/ml EB usually.Take a picture on automatic gel images analyser, if the DNA band of 1084bp appears in electrophoresis result, bacterial classification then to be measured is a mushroom Cr 02 bacterial.
Beneficial effect of the present invention is mainly reflected in: detection method of the present invention is compared with conventional morphologic detection, antagonistic effect, fruiting experiment, has weak point detection time, advantage of high accuracy; The inventive method detects required time and needed only 1～3 day, and conventional time-of-week of needed times at least two of antagonistic effect, fruiting experiment then needs at least 3 months time.
(4) description of drawings
Fig. 1 is for carrying out the result of pcr amplification to mushroom strain; M is a dna molecular amount standard; The negative contrast of C; The arrow indication is for being the special DNA band of 1084bp from being numbered the molecular weight that 17 mushroom Cr 02 bacterial amplifies; All the other are numbered other mushroom commonly used and produce bacterial classification.
(5) embodiment
The present invention is described further below in conjunction with specific embodiment, but protection scope of the present invention is not limited in this:
Embodiment 1:
(1) extraction of mycelium culture and genomic dna: the mushroom strain of cryopreservation is transferred to PDA inclined-plane (PDA slant medium: remove skin potato 200 grams, be cut into small pieces, add 1000 milliliters in water and boiled 30 minutes, the elimination potato ball adds water with filtrate and complements to 1000 milliliters, add glucose 20 grams, agar 15 grams dissolve the back packing, sterilize 30 minutes for 121 ℃, cool off the inclined-plane) on, cultivate down for 25 ℃.Receive 100ml PD liquid nutrient medium (PD liquid nutrient medium: remove skin potato 200 grams behind the 14d, be cut into small pieces, add 1000 milliliters in water and boiled 30 minutes, the elimination potato ball adds water with filtrate and complements to 1000 milliliters, add glucose 20 grams, sterilized 30 minutes for 121 ℃, cooling promptly gets the PD liquid nutrient medium) in, 25 ℃ of following 100r/min shaking culture 14d, collect mycelia, it is standby to put into-20 ℃ of refrigerator preservations.The SDS-CTAB method is adopted in the extraction of genomic dna, and uses DNA purification kit (Hangzhou BIOER Technology Co., Ltd) that the DNA that extracts is carried out purifying.The genomic dna of purifying with 1.5% agarose gel electrophoresis qualitative detection, is used DNA/RNA ultraviolet spectrophotometer detection by quantitative earlier again.The DNA extraction thing is standby in-20 ℃ of refrigerator storages.
(2) design specific PCR amplimer, the right sequence of primer be upstream primer 5 '-AGGCAAACTGGTTCCATGATA-3 ' and downstream primer 5 '-CTGTGAAGTCATTA C AGTCGC-3 ', synthetic by Shanghai biotechnology company limited.
(3) pcr amplification of SCAR molecule marker: 10 * PCR Buffer2 μ l, 10mmol/LdNTPs2 μ l, 25mmol/L MgCl
22 μ l, 5U/ μ l Taq DNA enzyme 0.5 μ l, 25mM special primer are to each 1 μ l, 20ng/ μ l template DNA 3 μ l, ddH
2O complements to 20 μ l.Amplified reaction carries out on TC-XP type amplification instrument.Behind 94 ℃ of pre-sex change 6min; 92 ℃ of sex change 40s, 54.5 ℃ of annealing 40s, 72 ℃ are extended 2min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last.
(4) electrophoresis detection: get step (3) pcr amplification product 5ul, with 1ul0.25% bromjophenol blue damping fluid mixing, point sample is on 1.5% sepharose, in 1 * TAB damping fluid, electrophoresis under the 5V/cm voltage, after electrophoresis finished, dyeing was 30 minutes in the aqueous solution that contains 0.5 μ g/ml EB, took a picture on the automatic gel images analyser of the clear JS-380A of training then.
According to the method described above, (on behalf of mushroom strain, numbering 1～23 be followed successively by: 1: No. 2, Shen Xiang to multiple mushroom strain respectively; 2: No. 6, Shen Xiang No. 43: Shen Xiang; 4: No. 9, Shen Xiang; 5: No. 7, Shen Xiang; 6: No. 8, Shen Xiang; 7: No. 10, Shen Xiang; 8: Shen Xiang 12; 9: Su Xiang; 10: Wu Xiang; 11:L26; 12:L03; 13:L66; 14:241; 15:241-4; 16: celebrating section 20; 17:Cr02; 18:Cr04; 19: rich No. 1 of Fujian; 20: No. 1, Shanghai farming; 21: the fragrant 939-9 in Zhejiang; 22: the fragrant 939-6 in Zhejiang; 23: fragrant nine) detect, with the negative contrast of sterilized water, electrophoresis result is seen Fig. 1.Wherein number 17 and be mushroom Cr 02 bacterial, amplifying molecular weight is the special DNA band of 1084bp, and all the other are numbered other mushrooms commonly used and produce bacterial classification, and not seeing has the special DNA band of 1084bp to produce.
Sequence table _ ST25
SEQUENCE?LISTING
＜110〉Zhejiang Prov. Forest Science Inst
＜120〉molecular specificity labeled primers of mushroom Cr 02 bacterial and detection method
<130>
<160>2
<170>PatentIn?version3.4
<210>1
<211>21
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>1
<210>2
<211>21
<212>DNA
<213>Unknown
<220>
＜223〉artificial sequence
<400>2
Claims (4)
1. the molecular specificity labeled primers of mushroom Cr 02 bacterial, described primer sequence is:
Upstream primer 5 '-AGGCAAACTGGTTCCATGATA-3 ';
Downstream primer 5 '-CTGTGAAGTCATTACAGTCGC-3 '.
2. method of mushroom Cr 02 bacterial being identified and being detected with the described molecular specificity labeled primers of claim 1, described method is: extract mushroom strain genomic dna to be measured as template, with described molecular specificity labeled primers as amplimer, carry out pcr amplification, amplified production is carried out electrophoresis detection, if the DNA band of 1084bp appears in electrophoresis result, bacterial classification then to be measured is a mushroom Cr 02 bacterial.
3. method as claimed in claim 2 is characterized in that described method is as follows:
(1) get mushroom strain to be measured, be seeded to the PDA inclined-plane, cultivate 14d for 25 ℃, be forwarded in the PD liquid nutrient medium, 25 ℃ of following shaking culture 14d collect mycelia, extract mushroom strain genomic dna to be measured with the SDS-CTAB method;
(2) genomic dna that extracts with step (1) is a template, as amplimer, carries out pcr amplification with described molecular specificity labeled primers:
Per 20 μ L are composed as follows for the PCR reaction system:
10×PCR?Buffer 2μL
10mmol/L?dNTPs 2μL
25mmol/L?MgCl
2 2μL
5U/ μ L Taq DNA enzyme 0.5 μ L
Each 1 μ L of 25 μ M upstream and downstream primers
20ng/ μ L template DNA 3 μ L
DdH
2O complements to 20 μ L;
The PCR reaction conditions is as follows:
Behind 94 ℃ of pre-sex change 6min; 92 ℃ of sex change 40s, 54.5 ℃ of annealing 40s, 72 ℃ are extended 2min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last;
(3) get step (2) amplified production 5 μ L, with 1 μ L, 0.25% bromjophenol blue damping fluid mixing, point sample is on 1.5% sepharose, electrophoresis in 1 * TAB damping fluid, under the 5V/cm voltage, electrophoresis finishes the back and dyes with EB, take a picture on automatic gel images analyser, if the DNA band of 1084bp appears in electrophoresis result, bacterial classification then to be measured is a mushroom Cr 02 bacterial.
4. method as claimed in claim 3 is characterized in that described EB dyeing is dyeing in the aqueous solution that contains 0.5 μ g/ml EB 30 minutes.
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|---|---|---|---|
| CN200810120314XA CN101358238B (en) | 2008-08-25 | 2008-08-25 | Molecular specificity labeled primers of mushroom Cr02 strain and test method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810120314XA CN101358238B (en) | 2008-08-25 | 2008-08-25 | Molecular specificity labeled primers of mushroom Cr02 strain and test method |
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| CN101358238A CN101358238A (en) | 2009-02-04 |
| CN101358238B true CN101358238B (en) | 2010-12-15 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000325084A (en) * | 1999-05-20 | 2000-11-28 | Mori Sangyo Kk | Differentiation of variety |
| CN101086015A (en) * | 2007-04-29 | 2007-12-12 | 上海市农业科学院食用菌研究所 | Mushroom 45 bacteria molecular specific mark and its obtaining method and uses |
-
2008
- 2008-08-25 CN CN200810120314XA patent/CN101358238B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000325084A (en) * | 1999-05-20 | 2000-11-28 | Mori Sangyo Kk | Differentiation of variety |
| CN101086015A (en) * | 2007-04-29 | 2007-12-12 | 上海市农业科学院食用菌研究所 | Mushroom 45 bacteria molecular specific mark and its obtaining method and uses |
Non-Patent Citations (1)
| Title |
|---|
| 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》.2005,第24卷(第2期),259-266. * |
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Granted publication date: 20101215 Termination date: 20120825 |