CN101509032A - Method for identifying pleurotus citrinopilestus by using molecular marker technology - Google Patents

Abstract

A method which uses a molecular marker technology for identifying Pleurotus citrinopileatus comprises the cultivation and collection of mycelium, the extraction of genome DNA, the establishment of the detection of a SCAR-PCR molecular marker, and the detection of a SCAR-PCR product. The method has the advantages of high sensitivity, less usage of needed DNA, and short detection time, high accuracy, easy judgment, good reproducibility, and the like, compared with conventional morphology detection, antagonistic test, and fruiting test. The method provides an effective strain identification method for the use of Pleurotus citrinopileatus germplasm resources and the registration work of new varieties.

Description

A kind of method of utilizing molecular marking technique to identify Pleurotus Citrinopileatus Sing

Technical field

The present invention relates to the authentication method of a kind of microorganism, be specifically related to utilize molecular marking technique to identify the method for Jiayu Pleurotus Citrinopileatus Sing, belong to biological technical field.

Background technology

Pleurotus Citrinopileatus Sing (Pleurotus citrinopileatus Singer) has another name called YUHUANG MUSHROOM, is the Northeast distinctive food medicine dual-purpose bacterium, belongs to Basidiomycotina, Hymenomycetes, Agaricales, Tricholomataceae, pleurotus.Pleurotus Citrinopileatus Sing had both been had delicious flavour, aromatic flavour, a nutritious edibleness, had the pharmaceutical use of nourishing and fit keeping function again, can be used for treating impotence due to deficiency of the kidney and dysentery.Pleurotus Citrinopileatus Sing contains polysaccharose substance and rich in amino acid, protein, also contain multiple nutritional components such as VITAMIN and mineral substance, belong to high nutrition, low calorific food, the title of " plain meat " is arranged, the polysaccharide that extracts from the Pleurotus Citrinopileatus Sing mycelium has restraining effect to people's colon poorly differentiated adenocarcinoma cell.By to its chemical composition analysis, find wherein to contain materials such as lovastatin, D-N.F,USP MANNITOL, ergosterol, 1-decanol, the necessary aminoacids content of high-quality Pleurotus Citrinopileatus Sing accounts for 40.8% of himself total amino acid content.

Yet the bacterial classification of China edible mushrooms is relatively more chaotic, and the phenomenon of homonym, synonym is serious.Traditional authentication method has according to morphological feature, as 7 proterties such as bacteria cover diameter, cap thickness, stem diameter, stem length, cap color, a bacterium time and output such as Yang Yanjie by comparing the sporophore stage, method with cluster analysis is handled testing data, proposition is with authentication method (" Liaoning agricultural sciences ", 2005 (5): 21～22) of genetic distance to bacterium classification.The workload of using this method is big, is subject to the environmental factors shadow and the normal instability that occurs, and can cause bigger error to judgement.

Other have the scholar from the difference of isozyme zymogram to identifying between bacterial strain.Li Ximei etc. to Henan Province performance preferably the mycelium of 38 flat mushroom bacterial strains carry out esterase isozyme and measure, according to the difference of zymogram, 38 bacterial strains of test are divided into 12 types, and point out PL ₇₉Though the white bacterial strain in bacterial strain and river belongs to strong door mountain, Sichuan Russula delica, they do not belong to a bacterial strain (" Agricultural University Of He'nan's journal ", 1989,23 (1): 73～77).Yet can be subjected to the influence of the heredity, culture condition, sampling point (mycelia, cap, stem), specimen preparation, deposition condition, dyeing process (comprising dyeing time) etc. of thalline with this method.

Along with development of molecular biology, for the evaluation between bacterial strain provides new method.Chinese scholar Zhang Jinxia etc. use little inter-satellite district (inter simple sequence repeat, ISSR) technology, 30 bacterial strains of Pleurotus cornucopiae (paul.Pers.) Rolland Pleurotus cornucopiae of cultivation in the period of China nineteen eighty-two to 2004 years 22 have been carried out grappling ISSR to be analyzed, by cluster analysis, under the level of genetic similarity 61%, 30 strains testeds are divided into 15 monoids, i.e. the bacterial strain of 15 certain hereditary differences of tool (" fungus journal ", 2007,26 (1): 115～121).

Though scholars are more to the aspect researchs such as cultivation, nutrition and effect of Pleurotus Citrinopileatus Sing, but the identification research to the Pleurotus Citrinopileatus Sing bacterial classification is very few, therefore be necessary the authenticate technology of Pleurotus Citrinopileatus Sing bacterial classification is studied, develop easy, strain identification technology fast and accurately.

Summary of the invention

The purpose of this invention is to provide a kind of method of utilizing molecular marking technique to identify the Jiayu Pleurotus Citrinopileatus Sing.The Pleurotus Citrinopileatus Sing bacterial classification that provided by the global edible mushrooms in Jiayu County, Hubei institute is provided described Jiayu Pleurotus Citrinopileatus Sing.

A kind of method of utilizing molecular marking technique to identify the Jiayu Pleurotus Citrinopileatus Sing of the present invention comprises the extraction of mycelium culture and collection, genomic dna, the detection foundation of SCAR-PCR molecule marker and the detection of SCAR-PCR product; It is characterized in that

1, the detection of SCAR-PCR molecule marker, the detection primer of use are Jiayu Pleurotus Citrinopileatus Sing F/R, wherein, Jiayu Pleurotus Citrinopileatus Sing F is 5 '-ATGAACTAATGACCGCTTG-3 ', Jiayu Pleurotus Citrinopileatus Sing R is 5 '-CCAATAGTAAGGGTCCTCC-3 ';

2, the detection of SCAR-PCR molecule marker is set up: the SCAR-PCR amplification system is 10 * PCRbuffer, 2.5 μ l, 25mmol/L MgCL ₂1 μ l, 2.5mmol/L dNTP 2 μ l, 5U/ul TaqDNA Polymerase 0.3 μ l, 10 μ mol/L detect each 1 μ l of primer, 1ng～10ng/ μ l template DNA 1 μ l, ddH ₂O 16.2 μ l;

The SCAR-PCR reaction conditions is 94 ℃ of 1min; 94 ℃ of 45second, 64 ℃ of 45second, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min;

3, the detected result of SCAR-PCR product: show that in the DNA of electrophoresis detection collection of illustrative plates the molecular weight that amplifies is the DNA band of 470bp, is the sign of Jiayu Pleurotus Citrinopileatus Sing.

The full name of described SCAR is sequence characterized amplified regions.

The method of utilizing molecular marking technique to identify the Jiayu Pleurotus Citrinopileatus Sing provided by the invention, have highly sensitive, needed DNA consumption is few, compares with conventional morphologic detection, antagonistic effect, fruiting experiment, has short, advantages such as accuracy is high, easy judgement, good reproducibility detection time.Specific as follows:

1, detection time is short: this detection method, from the sporophore position or from obtaining to be not less than the mycelial sample detection of 0.5g generally as long as 2～3 days, and conventional antagonistic effect required time needs two time-of-weeks at least, and conventional morphologic detection and fruiting experiment then need at least 2 months time.

2, accuracy height, judgement easily, and good reproducibility: this detection method is material with the genomic dna, DNA is stable genetic material, be not subject to the influence of extraneous factor etc., the dominant marker of its 470bp judges very clear simultaneously, reduce the artificial deviation of judging, improved accuracy greatly; In the conventional antagonistic effect, the antagonism manifestation has 3 kinds at least, even more, sometimes manifestation is not obvious, also is subjected to the influence of culture environment, and the antagonism performance not only can occur between the different varieties in planting, also can show between the bacterial strain between planting simultaneously, giving accurately, judgement causes difficulty; The easier influence that is subjected to each side's factors such as environment, time of fruiting experiment, poor repeatability has strengthened detection difficult; Diacritic feature is few between the different varieties of morphologic detection in same genus kind, some in addition do not have what difference, also need judgement person to have rich knowledge and experience simultaneously, can make accurately and to judge.

3, the dominant marker that obtains of this method can reduce the workload that Jiayu Pleurotus Citrinopileatus Sing kind is assert in addition, this method only needs 1 positive strain and 1 negative strain contrast just can compare fast during detection, and assert new variety with conventional morphology, antagonistic effect, fruiting experiment method, need all interior different varietiess of the same race are taken out of relatively together, just can make correct identification, this will very big man power and material, may make identification to carry out when wide in variety.

The present invention is the utilization of Pleurotus Citrinopileatus Sing germ plasm resource and the registration work of new variety, and more effective strain identification method is provided, and strengthens all significant to the protection work of China's Pleurotus Citrinopileatus Sing germ plasm resource.

Description of drawings

1 for using primer Jiayu Pleurotus Citrinopileatus Sing F/R to detect the DNA collection of illustrative plates of Pleurotus Citrinopileatus Sing bacterial classification amplification.Wherein M～16 are the swimming lane numbering; The molecular weight of the numeral dna fragmentation in left side, the arrow indication is a Jiayu Pleurotus Citrinopileatus Sing mark.

Embodiment

In order fully to disclose the method for utilizing molecular marking technique to identify the Jiayu Pleurotus Citrinopileatus Sing of the present invention, be illustrated below in conjunction with embodiment.

Embodiment 1: a kind of method of utilizing molecular marking technique to identify the Jiayu Pleurotus Citrinopileatus Sing

A kind of method of utilizing molecular marking technique to identify the Jiayu Pleurotus Citrinopileatus Sing may further comprise the steps:

One, mycelium culture and collection

(1) if confession was tested the preservation time of Pleurotus Citrinopileatus Sing bacterial strain less than 6 months, then mycelium culture is carried out as follows;

1, with the broken PDA substratum that contains the Pleurotus Citrinopileatus Sing mycelia of inoculation rake rake, the 250ml triangular flask is gone in switching, contains in the 100ml PDA liquid nutrient medium;

2, be placed on 25 ℃ of shaking tables, rotating speed 90～130r/min cultivated 7～10 days;

3, with the cultured mycelia of filtered through gauze, distilled water flushing, filter paper blots;

4, take by weighing 0.5～1.3g mycelia, wrap with filter paper, be stored in-20 ℃ standby.

(2) if supply the test Pleurotus Citrinopileatus Sing bacterial strain preservation time to be not less than 6 months, then mycelium culture adopts following method:

1, gets Pleurotus Citrinopileatus Sing bacterial classification beans piece size and be transferred on the PDA inclined-plane, cultivated 7～10 days for 25 ℃;

2, with the broken PDA substratum that contains the Pleurotus Citrinopileatus Sing mycelia of inoculation rake rake, the 250ml triangular flask is gone in switching, contains in the 100ml PDA liquid nutrient medium;

3, be placed on 25 ℃ of shaking tables, rotating speed 90～130r/min cultivated 7～10 days;

4, with the cultured mycelia of filtered through gauze, distilled water flushing, filter paper blots;

5, take by weighing 0.5～1.3g mycelia, wrap with filter paper, be stored in-20 ℃ standby.

Two, the extraction of genomic dna, described genomic dna can extract from the mycelium of bacterial strain or sporophore.

1, go bail for and deposit standby Pleurotus Citrinopileatus Sing mycelia 0.5～1.3g, or sporophore 0.5～1.3g, put into mortar, add the rapid grind into powder of liquid nitrogen, change the centrifuge tube of 7ml over to;

2, the extract that adds 2.5mL65 ℃ of preheating adds 50 μ l mercaptoethanols simultaneously, and concussion makes protein denaturation precipitation up and down;

3,65 ℃ of water-bath 1h are every 10min vibration 1 time;

4, the chloroform isoamyl alcohol mixing that adds 2.5ml removes deproteinize;

5,8000r/min, 4 ℃ of centrifugal 10min get supernatant to the 7ml centrifuge tube;

6, the CTAB/NaCl mixing that adds 65 ℃ of preheatings of 1/5 volume, the chloroform isoamyl alcohol mixing of adding 2.5ml;

7,10000r/min, 4 ℃ of centrifugal 10min get supernatant;

8, add the CTAB precipitated liquid of 2.5ml65 ℃ of preheating, put upside down mixing, as seen 65 ℃ of water-bath 1h maybe can spend the night to precipitating;

9,12000r/min behind 4 ℃ of centrifugal 10min, carefully removes supernatant liquor;

10, with 0.5ml TE solution or sterilized water dissolution precipitation 10min;

11, add saturated phenol of 0.25ml and 0.25mL chloroform isoamyl alcohol, mixing;

12,12000r/min, 4 ℃ of centrifugal 10min get supernatant, add the dehydrated alcohol of 2 times of volume precoolings, mixing;

13, place-20 ℃ of refrigerator 1h or spend the night;

14,12000r/min, 4 ℃ of centrifugal 10min abandon supernatant;

15, natural air drying precipitation, with 30 μ l TE solution or aseptic deionized water dissolution precipitations ,-20 ℃ of preservations are standby.

Three, the detection of SCAR-PCR molecule marker is set up

SCAR-PCR amplification system: 10 * PCR buffer, 2.5 μ l, 25mmol/L MgCL ₂1 μ l, 2.5m mol/L dNTP 2 μ l, 5U/ul Taq DNA Polymerase 0.3 μ l, each 1 μ l of 10 μ mol/L special primer for checking Jiayu Pleurotus Citrinopileatus Sing F/R, 1ng～10ng/ μ l template DNA 1 μ l, ddH ₂O16.2 μ l.

SCAR-PCR reaction conditions: 94 ℃ of 1min; 94 ℃ of 45second, 64 ℃ of 45second, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min.

Described special primer for checking Jiayu Pleurotus Citrinopileatus Sing F/R is to adopt round pcr, through a large amount of shaker tests, has obtained the dna segment of Jiayu Pleurotus Citrinopileatus Sing bacterial strain, based on this segmental dna sequence dna, and the detection primer of design Jiayu Pleurotus Citrinopileatus Sing bacterial classification.Described special primer for checking Jiayu Pleurotus Citrinopileatus Sing F/R particular content is: Jiayu Pleurotus Citrinopileatus Sing F is 5 '-ATGAACTAATGACCGCTTG-3 ', Jiayu Pleurotus Citrinopileatus Sing R is 5 '-CCAATAGTAAGGGTCCTCC-3 '.

Four, pcr amplification product detects

Specifically detection method is, gets pcr amplification product 8 μ l and adds 1.5 μ l, 6 * tetrabromophenol sulfonphthalein sample-loading buffer, and mixing at 1.0% sepharose, contains Goldview ^TMThe DNA dyestuff carries out electrophoresis, and 5V/cm constant voltage electrophoresis 50min observes and takes pictures by the ultraviolet gel imaging system.Perhaps, get pcr amplification product 8 μ l, with 1.5 μ l, 6 * tetrabromophenol sulfonphthalein sample-loading buffer mixing, on the fine jade vinegar sugar gel of point sample in 1%, in 0.5 X tbe buffer liquid, 5V/cm constant voltage electrophoresis 0.5～1h, after electrophoresis finishes,, on the gel imaging instrument, take a picture then with EB dyeing.

The pcr amplification product detected result adopts detection primer Jiayu Pleurotus Citrinopileatus Sing F/R that the Pleurotus Citrinopileatus Sing bacterial strain is carried out SCAR-pcr amplification, has only Jiayu Pleurotus Citrinopileatus Sing bacterial strain can amplify the DNA band that molecular weight is 470bp.

Main agents used in the present invention is as follows: (all chemical reagent are analytical pure)

1, CTAB extract: 100mmol/L Tris-HCl, 2.0% CTAB, 20mmol/LEDTA, 1.4mol/L NaCl, pH 8.0;

2, CTAB precipitated liquid: 50mmol/L Tris-HCl, 1.0% CTAB, 10mmol/LEDTA, pH8.0;

3、CTAB/NaCl：0.7mol/L?NaCl，10％?CTAB；

4, TE damping fluid: 10mmol/L Tris HCl, 1mmol/L EDTA;

5、0.5×TBE：44.5mmol/L?Tris，50mmol/L?HBO ₃、1mmol/LEDTA；

6, sample-loading buffer: 0.1% tetrabromophenol sulfonphthalein, 40% sucrose;

7, EB:10mg/ml ethidium bromide;

8, pcr amplification reagent is available from the precious biotech firm in Dalian.

Key instrument used in the present invention is as follows: the dual-purpose clean work station of the single horizontal vertical of SW-CJ-1FB type: Purifying Equipment Co., Ltd., Suzhou;

Portable Autoclave: three Shens, Shanghai;

The full temperature of HYG-A is shaken a bottle cabinet: the laboratory, Taicang;

Refrigerated centrifuge: Sigma 3K30;

Pcr amplification instrument: Eppendorf AG22331 Hamburg;

Palm type whizzer: the kylin medical apparatus Lx-100 of factory of Haimen City, Jiangsu palm type whizzer;

Gel imaging system: TANON-2008.

Untitled.ST25

SEQUENCE?LISTING

＜110〉University Of Agriculture and Forestry In Fujian

＜120〉a kind of method of utilizing molecular marking technique to identify Pleurotus Citrinopileatus Sing

<130>

<160>2

<170>PatentIn?version?3.1

<210>1

<211>19

<212>DNA

＜213〉Pleurotus Citrinopileatus Sing

<400>1

<210>2

<211>19

<212>DNA

＜213〉Pleurotus Citrinopileatus Sing

<400>2

Claims (1)

1, a kind of method of utilizing molecular marking technique to identify Pleurotus Citrinopileatus Sing comprises the extraction of mycelium culture and collection, genomic dna, the detection foundation of SCAR-PCR molecule marker and the detection of SCAR-PCR product; It is characterized in that

(1) detection of SCAR-PCR molecule marker, the detection primer of use are Jiayu Pleurotus Citrinopileatus Sing F/R, wherein, Jiayu Pleurotus Citrinopileatus Sing F is 5 '-ATGAACTAATGACCGCTTG-3 ' ', Jiayu Pleurotus Citrinopileatus Sing R is 5 '-CCAATAGTAAGGGTCCTCC-3 ';

(2) detection of SCAR-PCR molecule marker is set up: the SCAR-PCR amplification system is 10 * PCR buffer, 2.5 μ l, 25mmol/L MgCL ₂1 μ l, 2.5m mol/L dNTP 2 μ l, 5U/ulTaq DNA Polymerase 0.3 μ l, 10 μ mol/L detect each 1 μ l of primer, 1ng～10ng/ μ l template DNA 1 μ l, ddH ₂O 16.2 μ l;

The SCAR-PCR reaction conditions is 94 ℃ of 1min; 94 ℃ of 45second, 64 ℃ of 45second, 72 ℃ of 1min, 30 circulations; 72 ℃ of 5min;

(3) detected result of SCAR-PCR product: show that in the DNA of electrophoresis detection collection of illustrative plates the molecular weight that amplifies is the DNA band of 470bp, is the sign of Jiayu Pleurotus Citrinopileatus Sing.

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