CN103215263A - SSR (simple sequence repeat) molecular marker of agaricus blazei murrill, preparation method thereof and method of applying SSR molecular marker to identification of agaricus blazei murrill strains - Google Patents
SSR (simple sequence repeat) molecular marker of agaricus blazei murrill, preparation method thereof and method of applying SSR molecular marker to identification of agaricus blazei murrill strains Download PDFInfo
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Abstract
The invention discloses an SSR (simple sequence repeat) molecular marker of agaricus blazei murrill and a method of applying the SSR molecular marker to identification of agaricus blazei murrill strains. The SSR molecular marker is (1) primers A5'-CTCTCTCTTCCATCATCCTCT-3' and 5'-CGCCAGTTGTGCACAT-3'; (2) primers B5'-CGCATCTTGGGCTCCTT-3' and 5'-GGGGAAGCACGATCGTT-3'; (3) primers C5'-CTCCCTATGACCAACAGCA-3' and 5'-CCCCACGGATTGCC-3'; and (4) primers D5'-CCGCACTCTCCTCCAGTT-3' and 5'-GTGAGTCTGTGTGTGTGTGAGT-3'. The SSR molecular marker solves the problems that the developed and used SSR molecular markers of agaricus blazei murrill are not available in the prior art and the SSR molecular markers can not be utilized to quickly identify the agaricus blazei murrill strains, and has the characteristics of stable polymorphism, quickness in detection and convenience in use.
Description
Technical field
The present invention relates to Agaricus blazei Murrill SSR molecule marker, its preparation method and the application method on discriminating Agaricus blazei Murrill bacterial strain.
Background technology
Agaricus blazei Murrill Latin formal name used at school
Agaricus blazei Murrill,Be a kind of rare edible medicinal fungus, it belongs on classification of fungi is learned has carried on a shoulder pole bacterium door, Agaricales, Agaricus edibilis, Agaricus, originates in ground such as the U.S., Brazil, and artificial culture starts from Japan.1992, agricultural academy of sciences in Fujian Province's introduced this bacterial classification in the edible mushrooms center, and cultivates successfully (river branch and 1993) at home first, then is generalized to all parts of the country.This mushroom is delicious flavour, nutritious not only, (wears and kindly helps secure the success of and Yang Zhu good 2008 and order has hypotensive and anticancer dietotherapy effect; Dai
Et al.2009).The value of considering this mushroom is high, and from calendar year 2001, we utilize Co
60Radioinduction screening Agaricus blazei Murrill mutant strain and obtain some have bacterial strains that proterties to a certain degree changes (the river branch and etc. 2003; River branch and grade 2004; Weng Baiqi etc. 2007), and give popularization.
Little satellite (microsatellite) sequence is called simple sequence again and repeats that (simple sequence repeat SSR), is to be the series connection reiterated DNA sequences of repeating unit with a few Nucleotide (general 2-4).It is distributed in the eukaryotic gene group evenly, at random, widely, and the both sides order of microsatellite sequence is often more conservative in the biology of the same race, and its primer amplified has good repeatability and fidelity.Because there is nontranscribed domain in most of microsatellite sequences, variation frequency height, thereby tool extensive site variation between kind.Therefore, little satellite has divided the development and utilization of mark to become a focus in the eukaryotic gene group.Research to the little satellite of fungi at present mainly concentrates on the animals and plants pathogenic bacteria, the development and application of edible mushrooms SSR molecule marker also considerably less (Zhang Ruiying etc. 2010), be used on mushroom, straw mushroom, the Ganoderma more, as 200710040319,201110146770,200810204544,200810204545,200810204550 etc., also develop the SSR molecule marker that uses at present at Agaricus blazei Murrill, can't utilize the SSR molecule marker to carry out bacterial strain discriminating fast.
The approach of the main exploitation edible mushrooms SSR primer of having reported at present has 3: one is based on dna sequence data designs and develops the SSR primer.This is approach very efficiently for oneself through having the edible mushrooms of enriching EST or genomic dna sequence data.Two are based on the method exploitation microsatellite marker that ISSR suppresses PCR.It had successful report, reported the SSR site that utilizes this method exploitation needle mushroom and black fungus but also have, and the result is very undesirable.May be since in the fungal gene group distribution density of SSR relatively low, so the ISSR amplified production is not to be sequence between the SSR fully, this causes in the sequence that this method obtains false positive than higher (Zhang Ruiying etc. 2010).The 3rd, that develops in recent years utilizes biotin labeling SSR probe, after genomic DNA fragment hybridization, contains the method for the genomic fragment of SSR sequence with enrichment with avidin absorption hybridized fragment.
Summary of the invention
The purpose of this invention is to provide Agaricus blazei Murrill SSR molecule marker, its preparation method and the application method on discriminating Agaricus blazei Murrill bacterial strain, it has overcome does not have the Agaricus blazei Murrill SSR molecule marker that exploitation is used temporarily in the prior art, can't utilize the SSR molecule marker to its problem of carrying out bacterial strain discriminating fast, have the advantages that polymorphism is stable, detection is quick, easy to use.
The following 1. primer of sequence A 5 '-CTCTCTCTTCCATCATCCTCT-3 ' and 5 '-CGCCAGTTGT GCACAT-3 ' of Agaricus blazei Murrill SSR molecule marker primer; 2. primer B 5 '-CGCATCTTGGGCTCCTT-3 ' and 5 '-GGGGAAGCAC GATCGTT-3 '; 3. primer C 5 '-CTCCCTATGACCAACAGCA-3 ' and 5 '-CCCCACGGAT TGCC-3 '; 4. primer D 5 '-CCGCACTCTCCTCCAGTT-3 ' and 5 '-GTGAGTCTGT GTGTGTGTGAGT-3 '.The above-mentioned Agaricus blazei Murrill SSR molecule marker that goes out newly developed has been filled up the blank of present Agaricus blazei Murrill SSR molecule marker, makes that utilizing the SSR molecule marker that Agaricus blazei Murrill is carried out quick bacterial strain differentiates and become possibility.
The preparation method of above-mentioned Agaricus blazei Murrill SSR molecule marker may further comprise the steps:
1., take by weighing 0.1g Agaricus blazei Murrill Co ⑴, CTAB method extract the Agaricus blazei Murrill genomic dna:
60Mycelia or the sporophore of radioinduction bacterial strain J2～J66 after rinsing well with deionized water, change over to rapidly in the 1.5ml centrifuge tube; 2., add liquid nitrogen, smash to pieces with electric drill rapidly, the back adds 600mlCTAB and in 65 ℃ of water-bath 1h, and every 20min counter-rotating shakes up once; 3., add the synthetic chloroform-primary isoamyl alcohol of equal-volume, solution and chloroform-primary isoamyl alcohol volume ratio 24:1 that step (1) is 2. obtained, put upside down mixing after, in the centrifugal 6min of 12000rpm, get supernatant liquor in new pipe; 4., in supernatant liquor, add the dehydrated alcohol of its 2 times of volumes or the Virahol of its 2/3 volume, in-20 ℃ of placement 20min, in order to deposit D NA; 5., step (1) solution 4. is in the centrifugal 6min of 12000rpm, remove supernatant liquor after, add 75% ethanol and clean DNA behind the centrifugal 2min of 12000rpm, remove supernatant liquor once more; 6., the TE damping fluid of the air-dry back adding of the DNA that step (1) is 5. made 50 μ l is standby with dissolving DNA; Because the CTAB method is slightly different for the extracting method of Different Crop, aforesaid method is to the genome DNA extracting method behind the traditional CT AB method improvement, and the Agaricus blazei Murrill genomic dna yield of using this method to extract is higher.
⑵, utilize the technical point of Streptomycin sulphate enrichment with magnetic bead and biotin labeled probe hybridization from the SSR of Agaricus blazei Murrill genomic dna microsatellite sequence and to its order-checking; Agaricus blazei Murrill genetic background know little about it and genomic data limited, it is inapplicable to Agaricus blazei Murrill directly to utilize dna sequence data to design and develop the method for SSR primer.Utilize the technical point of Streptomycin sulphate enrichment with magnetic bead and biotin labeled probe hybridization to be an approach that efficient is higher from the SSR of Agaricus blazei Murrill genomic dna microsatellite sequence and at its sequences Design SSR molecule marker primer, the most suitable to Agaricus blazei Murrill.And since the SSR molecule marker of this method preparation to have polymorphism stable, also overcome the high shortcoming of method exploitation microsatellite marker false positive that suppresses PCR based on ISSR.
⑶, carry out design of primers at the SSR microsatellite sequence of Agaricus blazei Murrill genomic dna, and carry out pcr amplification with primer Agaricus blazei Murrill genomic dna, the electrophorogram that obtains by polyacrylamide gel electrophoresis detects the specific band that bacterial strain amplifies, and can carry out the SSR molecule marker primer that the Agaricus blazei Murrill bacterial strain is differentiated by the specific band that amplifies thereby filter out: 1. primer A 5 '-CTCTCTCTTCCATCATCCTCT-3 ' and 5 '-CGCCAGTTGTGCACAT-3 '; 2. primer B 5 '-CGCATCTTGGGCTCCTT-3 ' and 5 '-GGGGAAGCACGATCGTT-3 '; 3. primer C 5 '-CTCCCTATGACCAACAGCA-3 ' and 5 '-CCCCACGGATTGCC-3 '; 4. primer D 5 '-CCGCACTCTCCTCCAGTT-3 ' and 5 '-GTGAGTCTGT GTGTGTGTGAGT-3 '.
Above-mentioned Agaricus blazei Murrill SSR molecule marker is at the application method of differentiating on the Agaricus blazei Murrill bacterial strain, and this method may further comprise the steps:
⑴, the special SSR molecule marker of usefulness Agaricus blazei Murrill primer be primer A 5 '-CTCTCTCTTCCATCATCCTCT-3 ' and 5 '-CGCCAGTTGTGCACAT-3 ' 1.; 2. primer B 5 '-CGCATCTTGGGCTCCTT-3 ' and 5 '-GGGGAAGCACGATCGTT-3 '; 3. primer C 5 '-CTCCCTATGACCAACAGCA-3 ' and 5 '-CCCCACGGATTGCC-3 '; 4. primer D 5 '-CCGCACTCTCCTCCAGTT-3 ' and 5 '-GTGAGTCTGT GTGTGTGTGAGT-3 ' carry out the PCR reaction to the Agaricus blazei Murrill genomic dna respectively: the PCR reaction system is: the dna solution 1 μ l that comprises extraction in the PCR reaction system of 25 μ l, 10nmol/L primer 2 μ l, each 1 μ l of upstream and downstream primer wherein, 2.5mmol/L dNTP 2 μ l, 10 * PCR buffer, 2.5 μ l
TaqArchaeal dna polymerase 1U; The PCR response procedures is: 94 ℃ of pre-sex change 7min; 30 circulations subsequently, each 94 ℃ of sex change 30s that circulate, 55 ℃ of annealing 30s, 72 ℃ are extended 30s; Last 72 ℃ are extended 10min;
⑵, step ⑴ gained PCR product is carried out polyacrylamide gel electrophoresis, silver dye colour developing and take pictures and obtain the electrophoresis picture;
⑶, PCR product electrophoresis picture is carried out the band polymorphism analysis, the bacterial strain kind is differentiated: 1. primer A: only lack the 243bp band and identify bacterial strain J22, J26, J29, J30 by the specific band of following bacterial strain; Lack 224 simultaneously, the 243bp band identifies bacterial strain J3, J19, J38, J54; The 197bp band identifies bacterial strain J29, J31; 2. primer B:318bp band identifies bacterial strain J19; The 372bp band identifies bacterial strain J45; The 389bp band identifies bacterial strain J65; The 426bp band identifies bacterial strain J51; 3. primer C:244bp band identifies bacterial strain J14; 4. primer D:106bp band identifies bacterial strain J19; The 201bp band identifies bacterial strain J45.
Because the band that goes out of Agaricus blazei Murrill SSR primer amplification has very high specificity, its reaction result can be divided into: do not react, indistinction and having any different.Carry out the purpose that specific band that pcr amplification obtains reaches quick strain identification by the Agaricus blazei Murrill bacterial strain that need differentiate with the special SSR molecule marker of Agaricus blazei Murrill primer.
Figure of description
Fig. 1 is that the special SSR molecule marker of Agaricus blazei Murrill primer A is to Agaricus blazei Murrill J1 and Co
60The pcr amplification figure of radioinduction J2～J66 bacterial strain;
Fig. 2 is that the special SSR molecule marker of Agaricus blazei Murrill primer B is to Agaricus blazei Murrill J1 and Co
60The pcr amplification figure of radioinduction J2～J66 bacterial strain;
Fig. 3 is that the special SSR molecule marker of Agaricus blazei Murrill primer C is to Agaricus blazei Murrill J1 and Co
60The pcr amplification figure of radioinduction J2～J66 bacterial strain;
Fig. 4 is that the special SSR molecule marker of Agaricus blazei Murrill primer D is to Agaricus blazei Murrill J1 and Co
60The pcr amplification figure of radioinduction J2～J66 bacterial strain.
Embodiment
One, material and instrument:
Agaricus blazei Murrill is the original strain J1 of mutagenesis not: in the preservation of existing Fujian Province academy of agricultural sciences's soil and fertilizer institute.
Agaricus blazei Murrill Co
60Radiomutation bacterial strain J2～J66 is in the preservation of existing Fujian Province academy of agricultural sciences's soil and fertilizer institute.Its mutagenic condition is: cobalt source radiation activity 1.12*10
15Bq, dosage are 0.2-1.5kGy, and absorbed dose rate is 11.36Gy/min.
CTAB: Chinese name: hexadecyl-trimethylammonium-brometo de amonio, originate from BBI company, analytical pure;
Chloroform, primary isoamyl alcohol, Virahol, dehydrated alcohol, stationary liquid (100 ml/L ethanol): Beijing Chemical Plant, analytical pure;
Agaricus blazei Murrill SSR molecule marker primer A, B, C, D: it is synthetic that the worker is given birth in Shanghai;
DNTP, 10 * PCR buffer,
TaqThe biological GenStar of DNA polysaccharase: Kang Run;
Staining fluid (20 g/L AgNO
3): the Beijing Chemical Plant, industrial benchmark content is more than 99%;
Colour developing liquid (30 g/L Na
2CO
3): the Beijing Chemical Plant, top grade is pure;
Stop bath (100 mL/L HAc): the Beijing Chemical Plant, top grade is pure;
Eppendorf whizzer: model 5418;
PCR thermal cycler: Bio-Rad company, model C 1000;
Electrophoresis apparatus: JUNYI company; Model JY-SCZ6 or JY-SCZ7.
Two, now provide embodiment to describe Agaricus blazei Murrill SSR molecule marker of the present invention, its preparation method and the application method on discriminating Agaricus blazei Murrill bacterial strain in detail.
1. the preparation method of Agaricus blazei Murrill SSR molecule marker is as follows:
1., take by weighing 0.1g Agaricus blazei Murrill Co ⑴, CTAB method extract the Agaricus blazei Murrill genomic dna:
60Mycelia or the sporophore of radioinduction bacterial strain J2～J66 after rinsing well with deionized water, change over to rapidly in the 1.5ml centrifuge tube; 2., add liquid nitrogen, after smashing to pieces with electric drill rapidly, add 600mlCTAB, and every 20min counter-rotating shakes up once in 65 ℃ of water-bath 1h; 3., add the synthetic chloroform-primary isoamyl alcohol of equal-volume, solution and its volume ratio 24:1 in making 2., put upside down mixing after, in the centrifugal 6min of 12000rpm, get supernatant liquor in new pipe; 4., in supernatant liquor, add the dehydrated alcohol of its 2 times of volumes or the Virahol of its 2/3 volume, in-20 ℃ of placement 20min, with deposit D NA; 5., in the centrifugal 6min of 12000rpm, remove supernatant liquor after, add 75% ethanol and clean DNA, behind the centrifugal 2min of 12000rpm, remove supernatant liquor; 6., the TE damping fluid of the air-dry back adding of the DNA that will 5. make 50 μ l is standby with dissolving DNA;
, utilize Streptomycin sulphate enrichment with magnetic bead and biotin labeled probe hybridization technical point from the SSR of Agaricus blazei Murrill genomic dna microsatellite sequence and to its order-checking:
1., adjust to determine gained Agaricus blazei Murrill genomic dna work quality concentration to be 25mg/L;
2., digestion with restriction enzyme: use
MseI and
Eco2 kinds of restriction enzymes of RI carry out prozyme to the Agaricus blazei Murrill genomic dna to be cut, and contains 2 μ l in the 40 μ l endonuclease reaction systems
MseI(12U/ μ l), 2 μ l
EcoRI (10U/ μ l), lO * damping fluid of 2 μ g DNA and 4 μ l.Enzyme is cut the afterreaction system in 85 ℃ of water-bath 1Omin deactivation restriction enzymes;
3., enzyme cuts product and is connected with joint: contain 5 μ l, 10 * T4 ligase buffer in the 50 μ l reaction systems, 2 μ l dna ligases, 35 μ l genome enzymes are cut product, 2 μ l joints (0.01mmol/L), joint by
EcoThe Oligo A of few fine and soft sour 5'-CTCGTAGACTGCGTACC-3 ' of nuclear of the terminal strand of RI and 5'-AATTGGTACGCAGTCTAC-3 ' annealed combination and
MseThe Oligo B of few fine and soft sour 5'-GACGATGAGTCCTGAG-3 ' of nuclear of the terminal strand of I and 5'-TACTCAGGACTCAT-3 ' annealed combination forms.Ligation is spent the night for 16 ℃, takes out and is heated to 70 ℃, insulation 10min, deactivation ligase enzyme activity;
4., connect product P CR amplification: be that primer connection product carries out the inhibition pcr amplification with the single stranded oligonucleotide of forming Oligo A, Oligo B respectively, with 2% cool lipolysaccharide detected through gel electrophoresis expanding effect.Contain 10 μ l, 10 * Ex in the 100 μ l pcr amplification reaction systems
TaqDamping fluid, 4 kinds of dNTP (2.5mmol/L) of 8 μ l, 6 μ lMgCl2 (25mmo1/L), 8 μ l primers (0.01mmol/L), 4 μ l connect product and 0.5 μ l Ex
Taq(5U/ μ l).Response procedures: 94 ℃ of pre-sex change 5min; 30 circulations subsequently, each 94 ℃ of sex change 30s that circulate, 60 ℃ of annealing 30s, 72 ℃ are extended 30s; Last 72 ℃ are prolonged secondary 7min;
5., the little satellite fragment of enrichment with magnetic bead: operate according to the Streptomycin sulphate magnetic bead operation instruction that Invitrogen provides, it is as follows wherein to improve step: ⑴ washs magnetic bead: get activated magnetic bead, with 0.5 * SSC damping fluid of 500 μ l washing 3 times, then with 100 μ l O.5 * SSC damping fluid suspension magnetic bead.⑵ magnetic bead combines with probe: add 10 μ l probes (1 μ mol/L) in bead suspension, shake up the back room temperature and place 10min.Siphon away supernatant in the magnetic field, wash magnetic bead 4 times, behind the unconjugated probe of wash-out, form biotin labeled (AG) that be combined on the Streptomycin sulphate magnetic bead with O.l * SSC damping fluid of 300 μ l
15, (CT)
15, (AC)
15, (GT)
15Probe.⑶ hybridization: step amplified production is 4. added in above-mentioned magnetic bead and the little satellite probe binding substances 68 ℃ of hybridization lh.⑷ wash-out: after hybridization finishes, mixture is placed room temperature cooling 5min, in magnetic field, siphon away supernatant liquor, wash magnetic bead 4 times with 400 μ l, 0.1 * SSC damping fluid, the dna fragmentation that wash-out is not hybridized.⑸ collect the dna fragmentation of enrichment.By specification is suspended in magnetic bead in 95% the first phthalein amine (it is 8.2 that the lOmmol/L EDTA that adding accounts for volume about 5% regulates pH), 5min comes out in 65 ℃, in magnetic field, draw the supernatant liquor that contains the dna fragmentation that discharges, with chloroform-isopropanol method deposit D NA fragment, be dissolved in the 100 μ lTE damping fluids that to place refrigerator standby;
6., PCR product cloning and conversion: draw above-mentioned dna fragmentation pregnant solution 1 μ l as template, respectively with form Oligo A,
The single stranded oligonucleotide of Oligo B is that primer carries out the inhibition pcr amplification.Utilize pMD-18T that pcr amplification product is transformed among the competence DH5 α.Through cultivating the picking positive colony in the LB liquid nutrient medium that contains the microbiotic ampicillin, the concussion overnight incubation.With primer (AG)
15, (CT)
15, (AC)
15, (GT)
15With universal primer M13 positive colony bacterium liquid being carried out pcr amplification detects.Getting 5 μ l PCR products detects with 2% agarose gel electrophoresis.The clone who chooses the PCR product checks order.
⑶, carry out design of primers at the SSR microsatellite sequence of Agaricus blazei Murrill genomic dna, promptly little satellite SSR sequence that separation is obtained is analyzed, remove repetition or invalid microsatellite sequence, choose the base sequence at the two ends of little satellite tumor-necrosis factor glycoproteins and design Agaricus blazei Murrill SSR molecule marker primer.And carry out pcr amplification with this primer Agaricus blazei Murrill genomic dna, the electrophorogram that obtains by polyacrylamide gel electrophoresis detects the specific band that bacterial strain amplifies, and can carry out the SSR molecule marker primer that the Agaricus blazei Murrill bacterial strain is differentiated by the specific band that amplifies thereby filter out: 1. primer A 5 '-CTCTCTCTTCCATCATCCTCT-3 ' and 5 '-CGCCAGTTGTGCACAT-3 '; 2. primer B 5 '-CGCATCTTGGGCTCCTT-3 ' and 5 '-GGGGAAGCACGATCGTT-3 '; 3. primer C 5 '-CTCCCTATGACCAACAGCA-3 ' and 5 '-CCCCACGGATTGCC-3 '; 4. primer D 5 '-CCGCACTCTCCTCCAGTT-3 ' and 5 '-GTGAGTCTGT GTGTGTGTGAGT-3 '.
2. Agaricus blazei Murrill SSR molecule marker is differentiating that the application method on the Agaricus blazei Murrill bacterial strain is as follows:
⑴, the special SSR molecule marker of usefulness Agaricus blazei Murrill primer be primer A 5 '-CTCTCTCTTCCATCATCCTCT-3 ' and 5 '-CGCCAGTTGTGCACAT-3 ' 1.; 2. primer B 5 '-CGCATCTTGGGCTCCTT-3 ' and 5 '-GGGGAAGCACGATCGTT-3 '; 3. primer C 5 '-CTCCCTATGACCAACAGCA-3 ' and 5 '-CCCCACGGATTGCC-3 '; 4. primer D 5 '-CCGCACTCTCCTCCAGTT-3 ' and 5 '-GTGAGTCTGT GTGTGTGTGAGT-3 ' carry out PCR reaction to the Agaricus blazei Murrill genomic dna respectively: the PCR reaction system is the dna solution 1 μ l that comprises extraction in the PCR reaction system of 25 μ l, 10nmol/L primer 2 μ l, each 1 μ l of upstream and downstream primer wherein, 2.5mmol/L dNTP 2 μ l, 10 * PCR buffer, 2.5 μ l
TaqArchaeal dna polymerase 1U; The PCR response procedures is: 94 ℃ of pre-sex change 7min; 30 circulations subsequently, each 94 ℃ of sex change 30s that circulate, 55 ℃ of annealing 30s, 72 ℃ are extended 30s; Last 72 ℃ are extended 10min;
, to step ⑴ gained PCR product carry out polyacrylamide gel electrophoresis, silver dyes colour developing, obtains the electrophoresis picture.1., the preparation polyacrylamide gel specified operational procedure is:, draw the PCR product of 7 μ l and the sample-loading buffer of 1 μ l, fully sample on the mixing.After finishing, last sample uses 140V voltage electrophoresis.When tetrabromophenol sulfonphthalein indicator during, finish electrophoresis apart from the following 1cm of gel～2cm.2., after electrophoresis finishes, close electrophoresis apparatus, take out sheet glass and the medication the shank of the key is carefully pried open, remove upper glass plate, to face down with gel with the sheet glass of gel and immerse in the off-the-shelf clean porcelain dish that is placed with distilled water, gel promptly falls, or the medication the shank of the key is one jiao of glue strip off gently, and gel can fall in the dish; 3., gel is fixing: outwell the water in the porcelain dish, add stationary liquid (100 ml/L ethanol), slowly shake 15 min on shaking table; Outwell the stationary liquid in the porcelain dish, wash 2 times with distilled water again; 4., the dyeing of gel: outwell the water in the porcelain dish, add staining fluid (20g/L AgNO
3), on shaking table, slowly shake dyeing 20 min; 5., wash glue: outwell the staining fluid in the porcelain dish, wash 2 times, amount to 1min with distilled water; 6., the development of gel: add a small amount of colour developing liquid (30g/L Na
2CO
3) flushing 15 s, adding certain volume colour developing liquid again, the jog porcelain dish is generally 5～6min up to seeing band clearly, and the time is not long, otherwise background can be dark excessively; 7., stop developing: outwell the developing solution in the porcelain dish, pour stop bath (100 ml/L HAc) rapidly into, slowly shake 2～3min; 8., the acquisition of electrophorogram: outwell the stop bath in the porcelain dish, wash 2 times with distilled water, each 2min is placed on air drying with gel, and available digital camera or the shooting of gel imaging instrument obtain electrophorogram.
⑶, PCR product electrophoresis picture is carried out strip analysis, carry out the discriminating of bacterial strain kind by the specific band of Agaricus blazei Murrill bacterial strain, specific as follows:
The special SSR molecule marker of Agaricus blazei Murrill primer: 1. primer A 5 '-CTCTCTCTTCCATCATCCTCT-3 ' and 5 '-CGCCAGTTGTGCACAT-3 '; 2. primer B 5 '-CGCATCTTGGGCTCCTT-3 ' and 5 '-GGGGAAGCACGATCGTT-3 '; 3. primer C 5 '-CTCCCTATGACCAACAGCA-3 ' and 5 '-CCCCACGGATTGCC-3 '; 4. primer D 5 '-CCGCACTCTCCTCCAGTT-3 ' and 5 '-GTGAGTCTGT GTGTGTGTGAGT-3 ' are to Agaricus blazei Murrill J1 and Co
60The pcr amplification figure of radioinduction J2～J66 bacterial strain asks for an interview accompanying drawing 1～4, and concrete application of sample order please see the following form:
| The electrophoresis | Bacterium source | |
| 0 | |
|
| 2 | Agaricus blazei Murrill is the original strain J1 of mutagenesis not | |
| 1、3～66 | Be followed successively by Agaricus blazei Murrill Co 60Radiomutation bacterial strain J2～J66 |
By the strip analysis of electrophoresis picture, identify the Agaricus blazei Murrill bacterial strain
The summary that primer detects dissociant:
| Detection case | Detect dissociant |
| Only detected by 1 pair of primer | J3、J14、J22、J26、J29、J30、J31、J38、J51、J54、J65 |
| Can be detected by 2 pairs of primers | J45 |
| Can be detected by 3 pairs of primers | J19 |
As seen, the special SSR molecule marker of Agaricus blazei Murrill primer A, B, C, D can be fast to Agaricus blazei Murrill Co
60Radioinduction J2～J66 bacterial strain carries out bacterial strain and differentiates.
SEQUENCE LISTING
＜110〉Fujian Province Agriculture InstituteSoil and Fertilizer Institute
＜120〉Agaricus blazei Murrill SSR molecule marker, its preparation method and the application method on discriminating Agaricus blazei Murrill bacterial strain
<130> 2013
<160> 8
<170> PatentIn version 3.3
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Claims (3)
1. Agaricus blazei Murrill SSR molecule marker is characterized in that: the following 1. primer of the sequence of this molecule marker primer A 5 '-CTCTCTCTTCCATCATCCTCT-3 ' and 5 '-CGCCAGTTGTGCACAT-3 '; 2. primer B 5 '-CGCATCTTGGGCTCCTT-3 ' and 5 '-GGGGAAGCACGATCGTT-3 '; 3. primer C 5 '-CTCCCTATGACCAACAGCA-3 ' and 5 '-CCCCACGGATTGCC-3 '; 4. primer D 5 '-CCGCACTCTCCTCCAGTT-3 ' and 5 '-GTGAGTCTGT GTGTGTGTGAGT-3 '.
2. the preparation method of Agaricus blazei Murrill SSR molecule marker according to claim 1, it is characterized in that: it may further comprise the steps,
1., take by weighing 0.1g Agaricus blazei Murrill Co ⑴, CTAB method extract the Agaricus blazei Murrill genomic dna:
60Mycelia or the sporophore of radioinduction bacterial strain J2～J66 after rinsing well with deionized water, change over to rapidly in the 1.5ml centrifuge tube; 2., add liquid nitrogen, smash to pieces with electric drill rapidly, the back adds 600mlCTAB and in 65 ℃ of water-bath 1h, and every 20min counter-rotating shakes up once; 3., add the synthetic chloroform-primary isoamyl alcohol of equal-volume, solution and chloroform-primary isoamyl alcohol volume ratio 24:1 that step (1) is 2. obtained, put upside down mixing after, in the centrifugal 6min of 12000rpm, get supernatant liquor in new pipe; 4., in supernatant liquor, add the dehydrated alcohol of its 2 times of volumes or the Virahol of its 2/3 volume, in-20 ℃ of placement 20min; 5., step (1) solution 4. is in the centrifugal 6min of 12000rpm, remove supernatant liquor after, add 75% ethanol and clean DNA behind the centrifugal 2min of 12000rpm, remove supernatant liquor once more; 6., the air-dry back of the DNA that step (1) is 5. made adds the TE damping fluid of 50 μ l;
⑵, utilize the technical point of Streptomycin sulphate enrichment with magnetic bead and biotin labeled probe hybridization from the SSR of Agaricus blazei Murrill genomic dna microsatellite sequence and to its order-checking;
⑶, carry out design of primers at the SSR microsatellite sequence of Agaricus blazei Murrill genomic dna, and carry out pcr amplification with primer Agaricus blazei Murrill genomic dna, the electrophorogram that obtains by polyacrylamide gel electrophoresis detects the specific band that bacterial strain amplifies, and can carry out the SSR molecule marker primer that the Agaricus blazei Murrill bacterial strain is differentiated by the specific band that amplifies thereby filter out: 1. primer A 5 '-CTCTCTCTTCCATCATCCTCT-3 ' and 5 '-CGCCAGTTGTGCACAT-3 '; 2. primer B 5 '-CGCATCTTGGGCTCCTT-3 ' and 5 '-GGGGAAGCACGATCGTT-3 '; 3. primer C 5 '-CTCCCTATGACCAACAGCA-3 ' and 5 '-CCCCACGGATTGCC-3 '; 4. primer D 5 '-CCGCACTCTCCTCCAGTT-3 ' and 5 '-GTGAGTCTGT GTGTGTGTGAGT-3 '.
3. Agaricus blazei Murrill SSR molecule marker according to claim 1 is at the application method of differentiating on the Agaricus blazei Murrill bacterial strain, and it is characterized in that: it may further comprise the steps,
, with the special SSR molecule marker of described Agaricus blazei Murrill primer 1. primer A 5 '-CTCTCTCTTCCATCATCCTCT-3 ' and 5 '-CGCCAGTTGTGCACAT-3 '; 2. primer B 5 '-CGCATCTTGGGCTCCTT-3 ' and 5 '-GGGGAAGCACGATCGTT-3 '; 3. primer C 5 '-CTCCCTATGACCAACAGCA-3 ' and 5 '-CCCCACGGATTGCC-3 '; 4. primer D 5 '-CCGCACTCTCCTCCAGTT-3 ' and 5 '-GTGAGTCTGT GTGTGTGTGAGT-3 ' carry out the PCR reaction to the Agaricus blazei Murrill genomic dna respectively: the PCR reaction system is: the dna solution 1 μ l that comprises extraction in the PCR reaction system of 25 μ l, 10nmol/L primer 2 μ l, each 1 μ l of upstream and downstream primer wherein, 12.5mmol/L dNTP 2 μ l, 10 * PCR buffer, 2.5 μ l
TaqArchaeal dna polymerase 1U; The PCR response procedures is: 94 ℃ of pre-sex change 7min; 30 circulations subsequently, each 94 ℃ of sex change 30s that circulate, 55 ℃ of annealing 30s, 72 ℃ are extended 30s; Last 72 ℃ are extended 10min;
, to step ⑴ gained PCR product carry out polyacrylamide gel electrophoresis, silver dyes colour developing, obtains the electrophoresis picture;
⑶, PCR product electrophoresis picture is carried out strip analysis, the bacterial strain kind is differentiated: 1. primer A: only lack the 243bp band and identify bacterial strain J22, J26, J29, J30 by the specific band of following bacterial strain; Lack 224 simultaneously, the 243bp band identifies bacterial strain J3, J19, J38, J54; The 197bp band identifies bacterial strain J29, J31; 2. primer B:318bp band identifies bacterial strain J19; The 372bp band identifies bacterial strain J45; The 389bp band identifies bacterial strain J65; The 426bp band identifies bacterial strain J51; 3. primer C:244bp band identifies bacterial strain J14; 4. primer D:106bp band identifies bacterial strain J19; The 201bp band identifies bacterial strain J45.
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| CN115088557A (en) * | 2022-06-20 | 2022-09-23 | 中华全国供销合作总社昆明食用菌研究所 | Agaricus blazei murill strain ZJJJSR 001 and cultivation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105255882A (en) * | 2015-11-22 | 2016-01-20 | 吉林农业大学 | Agaricus bisporus ssr molecular marker specific primer system and application thereof |
| CN105255882B (en) * | 2015-11-22 | 2018-06-19 | 吉林农业大学 | Agaricus bisporus SSR molecular marker special primer system and its application |
| CN115088557A (en) * | 2022-06-20 | 2022-09-23 | 中华全国供销合作总社昆明食用菌研究所 | Agaricus blazei murill strain ZJJJSR 001 and cultivation method thereof |
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