CN101712984B

CN101712984B - Molecular specificity mark primer for authenticating mushroom strains 241 and 241-4 and detecting method

Info

Publication number: CN101712984B
Application number: CN2009101549231A
Authority: CN
Inventors: 李海波; 吴学谦; 付立忠; 魏海龙; 吴庆其; 贺亮; 程俊文
Original assignee: Zhejiang Academy of Forestry
Current assignee: Zhejiang Academy of Forestry
Priority date: 2009-11-30
Filing date: 2009-11-30
Publication date: 2011-12-21
Anticipated expiration: 2029-11-30
Also published as: CN101712984A

Abstract

The invention provides a molecular specificity mark primer for authenticating mushroom strains 241 and 241-4 and a detecting method for differentiating and authenticating the mushroom strains 241 and 241-4 by using same. The sequence of an upstream primer is 5'-GGTTCTGCTCCTTGTGACTG-3', and the sequence of a downstream primer is 5'-TCTGCTCTTCAGATGCAAGCT-3'. Compared with convectional morphology detection, antagonism tests and fruiting tests, the invention has the advantages of short detection time and high accuracy. The detection time in the method is only 1 to 2 days, but the time of the convectional antagonism tests is at least two weeks, and the time of the fruiting tests is at least three months.

Description

Differentiate molecular specificity labeled primers and the detection method of mushroom strain 241 and 241-4

(1) technical field

The present invention relates to a kind ofly distinguish the molecular specificity labeled primers that mushroom is produced sibling species 241 and 241-4, and utilize this primer that mushroom is produced the method that bacterial classification 241 and 241-4 distinguish and identify.

(2) background technology

Mushroom Lentinula edodes (Berk.) Pegler is delicious and have important food, pharmaceutical use liking of human consumer extremely both at home and abroad because of it, be China commercially produce largest and in the world output be only second to the edible mushrooms of Twospore Mushroom.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.Different mushroom strains has the different production traitss and commercial exploitation and is worth, thus bacterial classification quick and precisely identify for the development of mushroom industry most important.The classical way that mushroom strain is identified mainly is by the antagonism reaction of the macroscopic view of mushroom mycelium, bacterium colony and sporophore shape now being examined, being taken place between growth characteristics, biochemical reactions and different strains etc.These methods are owing to be subject to the influence of environmental factors and physiological situation, to the less bacterial strain of difference on the morphological specificity especially some mushroom sibling specieses for example 241 and 241-4, Cr02 and Cr04, fragrant No. 6 of No. 4, No. 2, Shen Xiang, Shen Xiang and Shen, 939-9 and 939-6 etc. is difficult to realize differentiating fast and accurately.

In addition, because mushroom strain itself has vegetative hereditary property, make different name of the same race on bacterial classification market, xenogenesis kind of the same name, false, inferior strain phenomenon ubiquity, add the degradation phenomena of strain quality, not only greatly damage the mushroom producer and breeder's interests, also greatly influenced the sustainable and healthy development of China's mushroom industry.Therefore, set up a cover science reliably, mushroom strain identification system quickly and easily, not only help the excavation and the utilization of China's mushroom germ plasm resource, have the good mushroom strain service of China's independent intellectual property right better for seed selection, and be standard present stage confusion mushroom strain market, promote pressing for of mushroom industry sustainable and healthy development.

The foundation of the fast development of Protocols in Molecular Biology, particularly molecule marker and genetic marker technology is with ripe, and is easy for developing, the identification of strains technology provides effective means fast and accurately.SCAR (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 object of the invention provides a kind ofly distinguishes the molecular specificity labeled primers that mushroom is produced sibling species 241 and 241-4, and a kind of this primer that utilizes is produced the method that bacterial classification 241 and 241-4 distinguish fast to mushroom.

The technical solution used in the present invention is:

A kind of mushroom of differentiating is produced the molecular specificity labeled primers of bacterial classification 241 and 241-4, and described primer sequence is:

Upstream primer: 5 '-GGTTCTGCTCCTTGTGACTG-3 '

Downstream primer: 5 '-TCTGCTCTTCAGATGCAAGCT-3 '

This primer through a large amount of shaker tests, adopts the RAPD mark to obtain the differential DNA fragment of mushroom 241 and 241-4 bacterial classification to being to adopt round pcr, with this fragment cloning order-checking, is the basic design Auele Specific Primer to obtain dna sequence dna.To mushroom 241 and 241-4 bacterial classification are carried out pcr amplification, can from 241 bacterial classifications, obtain the specific fragment of 1609bp size with this primer.

The invention still further relates to and a kind ofly utilize described molecular specificity labeled primers that mushroom is produced the method that bacterial classification 241 and 241-4 differentiate fast, described method comprises: 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 1609bp appears in electrophoresis result, bacterial classification then to be measured is mushroom 241 bacterial classifications, if the DNA band of 1609bp does not appear in electrophoresis result, bacterial classification then to be measured is a mushroom 241-4 bacterial classification; Described molecular specificity labeled primers sequence is:

Upstream primer 5 '-GGTTCTGCTCCTTGTGACTG-3 '

Downstream primer 5 '-TCTGCTCTTCAGATGCAAGCT-3 '

Described method key is that selection, DNA extraction, PCR reaction system and the reaction conditions of amplimer is definite, and electrophoresis detection, all can carry out according to this area ordinary method.

Need to prove that molecular specificity labeled primers of the present invention and detection method are only applicable to the differentiation that mushroom is produced sibling species 241 and 241-4, bacterial strain promptly to be measured is to be defined as in the scope of mushroom 241 and 241-4.Mushroom 241-4 is to be that starting strain is gathered specific strain with section banksia rose mushroom strains 241, experiment sieving such as compares through antagonism, product and breeds, and its institute's mushroom that produces type rounding, lid are greatly, meat is thick, handle is short, meat bacteria organization's densification, water content is low, and very suitable oven dry is a present dried mushroom kind best in quality.Because mushroom 241 and 241-4 bacterial classification morphological specificity difference are very little, situation with these two kinds of strain name marked erroneous on the bacterial classification market happens occasionally, and employing the inventive method, can distinguish fast and identify the mushroom on the bacterial classification market 241 and 241-4 bacterial strain, method be simple, quick, accurately.

Described pcr amplification reaction system is composed as follows:

The PCRBuffer final concentration is 1 *

dNTPs 1mM

MgCl ₂ 2.5mM

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, 60 ℃ of annealing 40s, 72 ℃ are extended 2min, totally 30 circulations; In 72 ℃ of flat 7min of benefit, final temperature is 4 ℃ at last.

Concrete, 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

10mM?dNTPs 2μL

25mM?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, 60 ℃ 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 * TAE damping fluid, under the 5V/cm voltage, electrophoresis finishes back EB dyeing (normally dyeing 30 minutes) in the aqueous solution that contains 0.5 μ g/ml EB, on automatic gel images analyser, take a picture, if the DNA band of 1609bp appears in electrophoresis result, bacterial classification then to be measured is mushroom 241 bacterial classifications, if the DNA band of 1609bp does not appear in electrophoresis result, bacterial classification then to be measured is a mushroom 241-4 bacterial classification.

The PCRBuffer final concentration is 1 *, be meant that the concentration of each component in reaction system is identical with 1 * PCR Buffer among the PCRBuffer, selecting volume usually for use is 10 * PCR Buffer of reaction system volume 1/10.10 * PCR Buffer composition is: 100mM Tris-HCl (pH8.5), 500mMKCl, 25mM MgCl ₂And 1.0%Triton-X-100, solvent is ddH ₂O.

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 1～2 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:Marker, i.e. dna molecular amount standard; C: negative control; 1:241; 2:241-4; The arrow indication is that to be numbered the molecular weight that 1 mushroom 241 bacterial classifications amplify be the special DNA band of 1609bp, does not amplify this band and be numbered 2 mushroom 241-4 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 (mushroom 241 and 241-4 produce bacterial classification and provided by Qingyuan County edible mushrooms scientific research center) 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 ℃, cooling back bevel) on, cultivates 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 100rpm/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 (GeneQuant Pro, GE Healthcare company) 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 '-GGTTCTGCTCCTTGTGACTG-3 ' and downstream primer 5 '-TCTGCTCTTCAGATGCAAGCT-3 ', synthetic by Shanghai biotechnology company limited.

(3) pcr amplification of SCAR molecule marker: 10 * PCR Buffer, 2 μ l, 10mM dNTPs2 μ l, 25mM MgCl ₂2 μ l, 5U/ μ l Taq DNA enzyme (Hangzhou BIOER Technology Co., Ltd) 0.5 μ l, 25 μ M special primers 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 (Hangzhou BIOER Technology Co., Ltd).Behind 94 ℃ of pre-sex change 6min; 92 ℃ of sex change 40s, 60 ℃ 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 1ul 0.25% bromjophenol blue damping fluid mixing, point sample is on 1.5% sepharose, in 1 * TAE 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, trains on the automatic gel images analyser of clear JS-380A in Shanghai then and takes a picture.

According to the method described above, respectively to mushroom 241 and 241-4 bacterial classification (1:241; 2:241-4) detect, with the negative contrast of sterilized water, electrophoresis result is seen Fig. 1.Wherein being numbered 1 mushroom 241 bacterial classifications, to have amplified molecular weight be the special DNA band of 1609bp, do not amplify this band and be numbered 2 mushroom 241-4 bacterial classification.

SEQUENCE?LISTING

＜110〉Zhejiang Prov. Forest Science Inst

＜120〉molecular specificity labeled primers and the detection method of discriminating mushroom strain 241 and 241-4

<130>

<160>2

<170>PatentIn?version?3.4

<210>1

<211>20

<212>DNA

<213>Unknown

<220>

＜223〉artificial sequence

<400>1

ggttctgctc?cttgtgactg 20

<210>2

<211>21

<212>DNA

<213>Unknown

<220>

＜223〉artificial sequence

<400>2

tctgctcttc?agatgcaagc?t 21

Claims

1. differentiate that mushroom produces the molecular specificity labeled primers of bacterial classification 241 and 241-4 for one kind, described primer sequence is:

Upstream primer: 5 '-GGTTCTGCTCCTTGTGACTG-3 '

Downstream primer: 5 '-TCTGCTCTTCAGATGCAAGCT-3 '.

2. one kind is utilized the described molecular specificity labeled primers of claim 1 that mushroom is produced the method that bacterial classification 241 and 241-4 differentiate fast, described method comprises: 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 1609bp appears in electrophoresis result, bacterial classification then to be measured is mushroom 241 bacterial classifications, if the DNA band of 1609bp does not appear in electrophoresis result, bacterial classification then to be measured is a mushroom 241-4 bacterial classification; Described molecular specificity labeled primers sequence is:

Upstream primer 5 '-GGTTCTGCTCCTTGTGACTG-3 '

Downstream primer 5 '-TCTGCTCTTCAGATGCAAGCT-3 '.

3. method as claimed in claim 2 is characterized in that:

Described pcr amplification reaction system is composed as follows:

Surplus is ddH ₂O;

The PCR reaction conditions is as follows:

4. 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 slant medium, 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;

Per 20 μ L are composed as follows for the PCR reaction system:

DdH ₂O complements to 20 μ L;

The PCR reaction conditions is as follows:

(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 * TAE damping fluid, under the 5V/cm voltage, electrophoresis finish back EB dyeing, take a picture on automatic gel images analyser, if the DNA band of 1609bp appears in electrophoresis result, bacterial classification then to be measured is mushroom 241 bacterial classifications, if the DNA band of 1609bp does not appear in electrophoresis result, bacterial classification then to be measured is a mushroom 241-4 bacterial classification.

5. method as claimed in claim 4 is characterized in that described EB dyeing is dyeing in the aqueous solution that contains 0.5 μ g/ml EB 30 minutes.