CN101805730A - Convenient and rapid DNA extracting method of peanut health tissues and diseased tissues - Google Patents
Convenient and rapid DNA extracting method of peanut health tissues and diseased tissues Download PDFInfo
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Abstract
The invention relates to a convenient and rapid DNA extracting method of peanut health tissues and diseased tissues. The method comprises two steps of: sample materials preparation: testing the leaf and stem tissues, shell tissues or peanut cotyledons, the weight of which is 3-10 mg, adding the sample materials into a 1.5ml centrifugal tube preset with an alkaline lysate or enzymolysis liquid, and grinding by a grinding rod till the visible particles do not exist; DNA extracting liquid preparation: treating the ground sample materials by an alkaline decoction method or a enzymolysis method to obtain the DNA extracting liquid. The method is also suitable for peanut health tissues and diseased tissues. Compared with the DNA extracting method, the invention has short required time, the required time of single sample by the alkaline decoction method does not exceed 20min, and the required time of single sample by the enzymolysis method does not exceed 0.5h; the dosage of the sample is little, and each sample only needs 3-10mg; the method has no need of liquid nitrogen to grind, therefore the cost is low, and the requirements of peanut molecule marker auxiliary breeding and transgene breeding are satisfied. The invention has no influence on the plant growth or seed germination, and has significant meaning on the practicality of peanut molecule breeding technology.
Description
Technical field
The present invention relates to the DNA extraction category of plant tissue, be specifically related to the easy rapid DNA extracting method of peanut health tissues and diseased tissues.
Background technology
In the prior art, generally adopting etiolated seedling blade, whole grain or half granule seed is the DNA that parent material, liquid nitrogen grinding are extracted the peanut tissue, and it comprises 10 steps at least, above (the Kochert et al.1991 of 2h how consuming time; Choi et al.1999; Burrow et al.2001; .2002 such as Wang Chuantang; The old .2008 that waits quietly).When handling a large amount of sample, also be very inconvenient even utilize the ball milling instrument, can not satisfy the requirement of high-throughput researchs such as molecular mark, map construction and transformant screening.Cut the hybridization with Southern if not being directly used in enzyme, the DNA extraction step can be simplified greatly.
Round pcr has become the indispensable important means of modern molecular biology research.Carry out the whole year for ease of test, and it is very necessary setting up the techniqueflow that a cover utilizes different parent material rapid extraction peanut DNA to be used for PCR.Directly take a sample in the peanut season of growth, can save germination, dark trouble of cultivating from the plant of outdoor growth; Utilize the seed plumule directly to extract DNA, can satisfy the need of gene clone, Seed Inspection, analysis of genetic diversity; In researchs such as transgenosis and molecular mark, if with the seed is that parent material extracts DNA, do not have under the greenhouse available situation in northern peanut producing region, can do sth. in advance six months at least carries out, thereby save manpower and land resources, but such DNA extraction method must be nondestructive to seed.Utilize the report of peanut seed extraction DNA less, consuming time longer so far.The sub-benevolence DNA extraction of a kind of nondestructive peanut method has been set up in Chenault etc. (2007) research, needs the 20mg peanut sample can prepare pcr template, and 20 steps are arranged, and needs 2.5h.Hu Xiaohui etc. (2009) have improved the method for Kang (1998) etc., and by 10 steps, 1.5h consuming time extracts peanut DNA from half sheet peanut cotyledon, influences seed germination through testing this method.
Similar with the traditional research method that is adopted on the other plant disease, the research of peanut fungal disease generally adopts the way of separation and Culture pathogenic bacteria to carry out.From diseased tissues, directly extract DNA, can be used for the Molecular Identification of known cause of disease or unknown cause of disease, infer its classification position, for the separation and Culture cause of disease provides reference.
Summary of the invention
The application's goal of the invention is the deficiency that remedies existing DNA extraction method, provides a kind of peanut health tissues and diseased tissues easy rapid DNA extracting method.This method is suitable equally for peanut health tissues and peanut diseased tissues.
The application's DNA extraction method is a cover proven technique system, peanut leaf, stem tissue, sophisticated shell tissue, seed plumule, cotyledon with field growing are that parent material all can carry out DNA extraction, be not subjected to the peanut restriction in vegetative period, carry out DNA extraction at any time, but Study on Acceleration process fast and effectively.
The present invention realizes by following technical solution:
Study the easy rapid DNA extracting method of a kind of peanut health tissues and diseased tissues, comprise and prepare sample material and preparation DNA extraction liquid step, it is characterized in that:
Described preparation sample material is that the sample material that finger to finger test is got is blade, stem tissue, shell tissue or the peanut cotyledon of weight at 3～10mg; Sample material adds the centrifuge tube that presets alkaline lysis liquid or enzymolysis solution, is ground to no visual particulate matter with grinding rod, and is standby;
Described preparation DNA extraction liquid is meant and uses alkaline-heating method or enzymolysis process that above-mentioned ground sample material is handled, and obtains the DNA extraction liquid of sample, wherein:
Described alkaline-heating method is that boiling water boiled 10～40sec after each sample ground in the alkaline lysis liquid of 20～60 μ l, the Tris-HCl that adds 4 times of volumes, boil 1～2min after, the centrifugal 5～10min of 10000rpm, get supernatant liquor, add equal-volume TE damping fluid and be DNA extraction liquid;
Described enzymolysis process, be after sample grinds in 100～200 μ l enzymolysis solutions, put 55～58 ℃ of water-bath 20min, add volume ratio with volume and be 25: 24: 1 phenol: chloroform: primary isoamyl alcohol, the centrifugal 5min of 10000rpm, supernatant liquor changes in another centrifuge tube, adding is with the volume Virahol, and the centrifugal 2～5min of 10000rpm abandons supernatant liquor, be deposited in room temperature and dry, add 100～150 μ l TE damping fluids and be DNA extraction liquid.
The DNA extraction method of above-mentioned peanut tissue is characterized in that described alkaline lysis liquid is 0.1～0.5M NaOH solution.
The DNA extraction method of above-mentioned peanut tissue is characterized in that described Tris-HCl damping fluid contains 5～8mg/ml PVP40.
The DNA extraction method of above-mentioned peanut tissue is characterized in that described enzymolysis solution is the solution that contains 10mM Tris-HCl, 5mMEDTA, 5～8mg/ml PVP, 40,50～100 μ g/ml Proteinase Ks.
The DNA extraction method of above-mentioned peanut tissue is characterized in that described TE damping fluid is that 20ml 0.1M Tris-HCI and 0.4ml 5mM EDTA constant volume 200ml are formulated.
The application of the DNA extraction method of above-mentioned peanut tissue is characterized in that this method is suitable equally for peanut health tissues and peanut diseased tissues.
Two kinds of resulting DNA extraction liquid of method add 2 μ l templates with 15 μ l reaction systems and calculate enough 50 PCR reaction uses.
Sampling method uniqueness of the present invention is being parent material when sampling only to need push blade with the pipe (for example, the rear end of common gel ink pen pen core) of corresponding internal diameter with the blade, makes it separate the leaf dish that promptly obtains the about 1.12mm of diameter with leaf tissue on every side; Be instrument with common shaver blade then when being parent material with the cotyledon, fix peanut, heavily about 5mg size a slice gets final product under the end-grain cutting away from embryo.
Advantage of the present invention:
Compare with existing DNA extraction method, the present invention's weak point consuming time, amount of samples is few, and expense is low, can satisfy the requirement of peanut molecular mark and transgenic breeding.The alkaline-heating method single sample time spent is no more than 20min, and the enzymolysis process single sample is no more than 0.5h, and method in the past needs 1.5h at least and must use liquid nitrogen when grinding.Each sample of DNA extraction of the present invention only needs 3～10mg to get final product; Need not liquid nitrogen grinding, to plant strain growth or not influence of seed germination,, significant for the peanut molecule breeding technical applicationization.
The present invention not only can be used for the DNA extraction of peanut health tissues, and is applicable to the DNA extraction of peanut diseased tissues.
Description of drawings
Fig. 1 is that the DNA that extracts with peanut leaf is the peanut ITS electrophorogram that template amplifies with primer rDNAa/rDNAb;
Fig. 2 is a wild species A.duranensis ITS sequencer map.
Fig. 3 is to be the PCR product electrophorogram of template employing primer TBPfex1/TBPrex1 acquisition with the DNA that the peanut plumule extracts;
Fig. 4 is that the DNA that extracts with the peanut diseased tissues is the scab cause of disease 18s rDNA electrophorogram that template amplification goes out;
Fig. 5 is to be template with the peanut fruit rot DNA that shell extracts that is injured, and makes the product electrophorogram of pcr amplification acquisition with primer I TS1/ITS4;
Fig. 6 is that primer is to IT-ISJ29R/IT-ISJ3F amplified production 6% denaturing polyacrylamide gel electrophoresis collection of illustrative plates.
Fig. 7 is that the DNA that extracts with the peanut cotyledon is a template, with the peanut FAD2 gene electrophorogram of primers F L1/FR1 amplification acquisition;
Fig. 8 is that the DNA that extracts with the peanut cotyledon is the peanut ITS electrophorogram that template amplifies with primer rDNA a/rDNA b;
1-8 is that the DNA that extracts with peanut material cotyledon to be detected is that template is made pcr amplification product electrophorogram (the negative contrast of P) with EGFP primer EGPF-F1/EGPF-R1 among Fig. 9.
Embodiment
Below in conjunction with drawings and Examples the present invention is further set forth.
Among Fig. 1, M is a molecular weight standard, and 1 is cultivar and wild species filial generation, and 2 is the chemical mutation body.
Among Fig. 3, M is a molecular weight standard, and 1,2,3 is different Cultivars, and 4 is A.rigonii hybrid generation, and 5 is the hybrid generation of A.glabrata.
Among Fig. 4, M is a molecular weight standard, and L is for to prepare the 18s rDNA that DNA cloning goes out from sick leaf, and S prepares the 18S rDNA that DNA cloning goes out for stem's scab.
Among Fig. 5, M is a Takara DL2000DNA molecular weight standard, and 1～3 is 3 different fruit rot pods.
Among Fig. 6, M is a molecular weight standard, 1～20 be respectively that B4, Valencia 101,08-29,08-30,08-31, flower educate that 16,08 surveys-A2, A6, A7, A8, A10, A30, chimera enterprise, volt peanut, evergreen are grown thickly, a brood of monkey in north, the Yihe River, Wenshang climb overgrow, three pod public affairs, Suining two nests, red precipice, Xingcheng volt greatly.
Among Fig. 7, M is a molecular weight standard, 1～7 is respectively that spring spends 646, spring spends 10, Guangdong oil 256, JYH1, CTWE, FB4, assorted 9307 PCR product far away.
Among Fig. 8, M is a molecular weight standard, 1～7 is respectively that spring spends 646, spring spends 10, Guangdong oil 256, JYH1, CTWE, FB4, assorted 9307 PCR product far away.
Among Fig. 9, M is a molecular weight standard, and 1～8 is respectively 8 in the seed to be detected that obtains by transgenic technology.
Embodiment one: extract DNA from peanut leaf
One, test materials and reagent
Material: 3 genotype are adopted in this test, comprise the filial generation of wild species (Arachis.duranensis), 1 cultivar and wild species A.glabrata, 1 chemical mutation body material.
Reagent: alkaline lysis liquid contains the Tris-HCl damping fluid of 5～8mg/ml PVP40.
Two, test method
1. template preparation
A, on peanut plant, choose a slice leaflet, at master pulse one side perforating, obtain the about 1.12mm leaf of diameter dish with Neutral ball-point for ball-point pen.When the peanut material relatively preciousness or peanut grow early stage lobe numbers more after a little while for reduce as far as possible to its influence of growing, can directly punching sampling on plant.
B, the leaf dish is placed the 1.5ml centrifuge tube that is added with 60 μ l alkaline lysis liquid (can freezing preservation standby), grind no macroscopic particle with grinding rod.
C, boil 30sec, add 4 times of volume Tris-HCl damping fluids.
D, boil 2min, the centrifugal 5min of 12000rpm.
E, get supernatant liquor, add isopyknic TE damping fluid and be DNA extraction liquid.
2.PCR
PCR system: 12.5 μ l, 2 * Tiangen Taq PCR MasterMix, 10 μ M primers
Primer sequence finds disclosed Auele Specific Primer on the net by us, then by Genscript (Jin Site) company synthetic, generally is a pipe 2OD.
(rDNAa:GGAAGTAAAAGTCGTAACAAGG/rDNAb:CCTCCTCCGCTTATTGATATG C) each 0.5 μ l, 2 μ lDNA masterplates, 9.5 μ l aseptic deionized waters.
The PCR condition: 94 ℃ of pre-sex change 3min, 94 ℃ of 50sec, 55 ℃ of 1min, 72 ℃ of 1.5min, 35 circulations, 72 ℃ are extended 7min.
3.PCR product reclaims purifying, order-checking
The PCR product is through 1% agarose gel electrophoresis, and 100V, 30min, electrophoresis picture show and obtain the purpose fragment, further the PCR product are reclaimed (Tiangen, common DNA product purification test kit), operate according to the test kit specification sheets.Purpose fragment after the recovery is connected in the pBS-T carrier, transform (Tiangen, pBS-T clones test kit), operate according to clone's test kit specification sheets, the positive single colony inoculation of picking white contains in the LB liquid nutrient medium of penbritin in 1ml, be inverted overnight incubation for 37 ℃, send the order-checking of Genscript company.
Three, result
Electrophoresis picture (Figure of description Fig. 1) shows that target DNA fragment is clear, even show that in another research the very thick wild species peanut of wax layer also is suitable for this method and extracts DNA, its sequencing result good (Figure of description Fig. 2), 3 materials have all obtained the purpose fragment, confirmation this method is simple and reliable, be applicable to the extraction of dissimilar peanut cultivation kind material blade DNA, and do not need painstakingly to select young leaflet tablet.
Embodiment two: extract DNA from the peanut plumule
One, test materials and reagent
Material: 5 genotype materials are adopted in this experiment, comprise the hybrid generation of 1 cultivar and wild species A.rigonii, 1 chemical mutation body, 3 peanut cultivating varieties.
Reagent: alkaline lysis liquid contains the Tris-HCl damping fluid of 5～8mg/ml PVP40.
Two, test method
1. template preparation
Get a peanut seed, two cotyledons of strip off cut with a knife and get the plumule part, be i.e. several prematurity blades.Following steps are with scheme one B, C, D, E.
2.PCR
PCR system: 12.5 μ l, 2 * Taq PCR MasterMix, each 0.5 μ L of 10nM primer (TBPfex1/TBPrex1), 2 μ LDNA masterplates, 9.5 μ L aseptic deionized waters.
The PCR condition: 94 ℃ of pre-sex change 3min, 94 ℃ of 50sec, 55 ℃ of 1min, 72 ℃ of 1.5min, 35 circulations, 72 ℃ are extended 7min.
3.PCR product electrophoretic separation
The PCR product is through 1% agarose gel electrophoresis, 100V, 30min.
Three, result
PCR product electrophoresis picture (Figure of description 3) shows that the target DNA extraction effect is better, and 5 materials have all obtained target gene fragment, confirms that this method is simple and reliable, applicable to different kind of material plumule DNA extraction.
Embodiment three: clone fungi 18S rDNA from the shot hole diseased tissues
One, test materials and reagent
Material: Peanut Scab diseased tissues.
Reagent: alkaline lysis liquid contains the Tris-HCl damping fluid of 5～8mg/ml PVP40.
Two, test method
1. template preparation
On peanut plant, choose the sick leaf of a slice shot hole, at master pulse one side perforating, obtain the about 1.12mm leaf of diameter dish with Neutral ball-point for ball-point pen; Stem's disease is organized the thin slice that then cuts the about 4mm of diameter with razor blade.Following steps are with B, C, D, the E step of " template preparation " among the embodiment one.
2.PCR
PCR system: each 2 μ l of 25 μ l, 2 * Tiangen Taq PCR MasterMix, 10 μ M fungi Auele Specific Primers (NS26:5 '-CTGCCCTATCAACTTTCGA-3 '/R518:5 '-ATTACCGCGGCTGCTGG-3 '), 1 μ LDNA masterplate, 20 μ l aseptic deionized waters.
The PCR condition: 95 ℃ of pre-sex change 6min, 94 ℃ of 1min, 55 ℃ of 50sec, 72 ℃ of 1min, 32 circulations, 72 ℃ are extended 6min.
3.PCR product reclaims purifying, order-checking
The PCR product is through 1% agarose gel electrophoresis, 100V, and 30min, following steps are with embodiment one.
Three, result
PCR reaction back gel electrophoresis (in the Figure of description 4) confirms from the pathogenic bacteria 18sDNA of sick leaf and the extraction of sick stem kind in full accord; Shows that this method is simple and reliable, the 18S rDNA sequence of the Peanut Scab pathogen that the further order-checking of and repeatability is strong. obtains is as follows: TGC CCT ATC AAC TTT CGA TGG TAG GAT AGT GGC CTA CCA TGG TGG TGACGG GTG ACG GAG AAT TAG GGT TCG ATT CCG GAG AGG GAG CCT GAG AAA CGGCTA CCA CAT CCA AGG AAG GCA GCA GGC GCG CAAATT ACC CAA TCC TG ACA CGGGGA GGT AGT GAC AAT AAA TAA CAA TAC CGG GCT CAT AGA GTC TGG TAA TTGGAA TGA GTA CAA TCT AAA TCC CTT AAC GAG GAT CCA TTG GAG GGC AAG TCTGGT GCC AGC AGC CGC GGT AAT. Show that alkaline lysis is applicable to the research of peanut fungal pathogen fully.
Embodiment four: extract fungal DNA, pathogen identification in the rotten diseased tissues of peanut
One, test materials and reagent
Material: the peanut fruit rot shell of being injured.
Reagent: alkaline lysis liquid contains the Tris-HCl damping fluid of 5～8mg/ml PVP40.
Two, test method
1. template preparation
Get the about 6.5mm in middle layer of the shell of being injured
2Thin slice, following steps are with embodiment one.
2.PCR
PCR system: each 1 μ l of 12.5 μ l, 2 * Tiangen Taq PCR MasterMix, the general fungi Auele Specific Primer of 10 μ M (ITS1:5 '-tccgtaggtgaacctgcgg-3 '/ITS4:5 '-tcctccgcttattgatatgc-3 '), 1 μ l DNA masterplate, 4.5 μ l aseptic deionized waters.
The PCR condition: 95 ℃ of pre-sex change 5min, 95 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 1min, 35 circulations, 72 ℃ are extended 5min.
3.PCR product reclaims purifying, order-checking
The PCR product is through 1.5% agarose gel electrophoresis, 100V, and 30min reclaims test method such as order-checking with embodiment one.
Three, result
PCR product gel electrophoresis (in the Figure of description 5) confirms to have the purpose fragment, the sickle spore bacterium sequence height homology of including among pathogenic bacteria ITS sequence that further order-checking obtains and the GenBank.Show that this method is applicable to the research of fruit rot fungal pathogen.Concrete sequence is as follows:
TCCGTAGGTGAACCTGCGGAGGGATCATTACCGAGTTATACAACTCATCAACCCTGTGAACATACCTAAAACGTTGCTTCGGCGGGAACAGACGGCCCTGTAACAACGGGCCGCCCCCGCCAGAGGACCCCTAACTCTGTTTTTATAATGTTTTTCTGAGTAAACAAGCAAATAAATTAAAACTTTCAACAACGGATCTCTTGGCTCTGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCAGTGAATCATCGAATCTTTGAACGCACATTGCGCCCGCCAGTATTCTGGCGGGCATGCCTGTTCGAGCGTCATTACAACCCTCAGGCCCCCGGGCCTGGCGTTGGGGATCGGCGGAAGCCCCCTGTGGGCACACGCCGTCCCTCAAATACAGTGGCGGTCCCGCCGCAGCTTCCATTGCGTAGTAGCTAACACCTCGCAACTGGAGAGCGGCGCGGCCATGCCGTAAAACACCCAACTTCTGAATGTTGACCTCGAATCAGGTAGGAATACCCGCTGAACTTAAGCATATCAATAAGCGGAGGA。
Embodiment five: extract DNA from blade, carry out the IT-ISJ marker research
One, test materials and reagent
Material: as following table.
Table 2. peanut material source
Reagent: alkaline lysis liquid contains the Tris-HCl damping fluid of 5～8mg/ml PVP40.
Two, test method
1. template preparation
With embodiment one.
2.PCR
PCR system: 7.5 μ l, 2 * Taq PCR MasterMix, each 0.75 μ l of 10nM primer each (IT-ISJ29R:GGAATTCCACCTGCACTA/IT-ISJ3F:GCATGCCAGGTAAGTAAG), 2 μ lDNA masterplates, 6 μ l aseptic deionized waters.
The PCR condition: 94 ℃ of pre-sex change 5min, 94 ℃ of 45sec, 50 ℃ of 45sec, 72 ℃ of 1min, 40 circulations, 72 ℃ are extended 10min.。
3.PCR product polyacrylamide gel electrophoresis
6% polyacrylamide gel electrophoresis 30min, argentation dyeing fast.
Three, result
PCR reaction back polyacrylamide gel electrophoresis (electrophorogram is in Figure of description 6) is added up its polymorphism situation according to gel figure and is seen Table 3, and display material band polymorphism is better.This test has been carried out 3 times and has been repeated, and banding pattern does not change, and illustrates that this method is simple and reliable, and the DNA that is extracted can be used for Study on Molecular Marker.
Table 3. primer is to the expanding effect of IT-ISJ29R/IT-ISJ3F to 20 parts of peanut samples
Embodiment six: extract DNA from cotyledon
One, test materials and reagent
Table 1 peanut material source
Reagent: the enzymolysis solution, the TE damping fluid that contain 10mMTris-HCl, 5mM EDTA, 5～8mg/ml PVP, 40,50～100 μ g/ml Proteinase Ks.
Two, test method
1. template preparation
A, get a peanut seed, cut the thick thin slice cotyledon of about 0.40mm, reduce damage as far as possible cotyledon with razor blade.
B, be placed in the 1.5ml centrifuge tube that is added with 200 μ l enzymolysis solutions (can freezing preservation standby), grind no macroscopic particle with grinding rod.
C, 55 ℃ of water-bath 20min add equal-volume phenol/chloroform/primary isoamyl alcohol.
The centrifugal 5min of D, 10000rpm.
E, get supernatant liquor, add the equivalance Virahol, the centrifugal 2-5min of 10000rpm.
F, abandon supernatant liquor, be deposited in room temperature and dry, add with the isopyknic TE damping fluid of supernatant and be DNA extraction liquid.
2.PCR
The PCR system: 7.5 μ l, 2 * Taq PCR MasterMix, 10nM primer each (FL1:5 '-AAG GGT TCC ACA TTCAAA CCC TCC ATT-3 ', FR1:5 '-CAA TGC TTT GTA AAC TGG GGT GCC ATC-3 ' and rDNAa/rDNA b) each 0.6 μ l, 2 μ lDNA masterplates, 4.3 μ l aseptic deionized waters.
PCR condition: primers F L1/FR1 respective conditions: 94 ℃ of pre-sex change 3min, 94 ℃ of 1min, 67 ℃ of 30sec, 72 ℃ of 1min, 35 circulations, 72 ℃ are extended 2min; Primer rDNA a/rDNA b respective conditions: 94 ℃ of pre-sex change 3min, 94 ℃ of 50sec, 55 ℃ of 1min, 72 ℃ of 1min 30sec, 35 circulations, 72 ℃ are extended 7min.
3.PCR product electrophoresis
The PCR product of FL1/FR1, rDNA a/rDNA b primer amplification is respectively through 2%, 0.8% agarose gel electrophoresis, 30min.
Three, result
PCR product gel electrophoresis (electrophorogram is in Figure of description 7,8) shows that this method is applicable to the extraction of different genotype peanut material cotyledon DNA.
Embodiment seven: extract DNA and identify transgenic seed from cotyledon
One, test materials and reagent
Material: 8 parts in the screening seed to be detected that obtains by transgenic technology.
Reagent: the enzymolysis solution, the TE damping fluid that contain 10mM Tris-HCl, 5mM EDTA, 5～8mg/ml PVP, 40,50～100 μ g/ml Proteinase Ks.
Two, test method
1. template preparation
Template preparation with embodiment six.
2.PCR
PCR system: 12.5 μ l, 2 * Taq PCR MasterMix, each 0.5 μ l of 10nM primer each (EGPF-F1:ACCCTCGTGACCACCCTGACCTAC/EGPF-R1:GACCATGTGATCGCG CTTCTCGTT), 1 μ lDNA masterplate, 10.5 μ l aseptic deionized waters.
The PCR condition: 94 ℃ of pre-sex change 3min, 94 ℃ of 1min, 61.6 ℃ of 40sec, 72 ℃ of 1min, 35 circulations, 72 ℃ are extended 5min.
3.PCR product gel electrophoresis
1.0% agarose gel electrophoresis, 30min.
Three, result
PCR product gel electrophoresis (electrophorogram is in Figure of description 9) shows 6 parts of positive materials.Illustrate that this method is applicable to the Screening and Identification research of transgenic line.
Reference:
Wang Chuantang, Huang Yue, Yang Xin road etc. the low pH value method of modified CTAB method and high salt is extracted the effect [J] of peanut DNA. peanut journal, 2002,31 (3): 20-23.
Chen Jing, Hu Xiaohui .CTAB methods such as Miao Huarong are extracted the expanding effect [J] of the total DNA of peanut in SSR and SRAP. peanut journal, 2008,37 (1): 29-31.
Hu Xiaohui, seedling Hua Rong, Shi Yunqing etc. the sub-DNA extraction research of peanut dry peanut [J]. both sides of the Straits peanut scientific seminar collection of thesis/Chinese agriculture science and technology press, 2009:293-296
BUROW?M.D.；SIMPSON?C.E.；STARR?J.L.and?PATERSON?A.H.Transmission?genetics?ofchromatin?from?a?synthetic?amphidiploid?to?cultivated?peanut(Arachis?hypogaea?L.)：broadeningthe?gene?pool?of?a?monophyletic?polyploid?species[J].Genetics?2001，159：823-837.
CHOI?K.；BUROW?M.D.；CHURCH?G.；BUROW?G.；PATERSON?A.M.；SIMPSON?C.E.andSTARR?J.L.Genetics?and?mechanisim?of?resistance?to?Meloidogyne?arenaria?in?peanut[J].Journal?ofNematology?1999，31：283-290.
CHU，Y.；RAMOS，L.；HOLBROOK，C.C.and?OZIAS-AKINS，P.Frequency?of?a?loss-of-functionmutation?in?oleoyl-PC?desaturase(ahFAD2A)in?the?mini-core?of?the?US?peanut?germplasmcollection[J].Crop?Science，2007，47：2372-2378.
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Claims (6)
1. the easy rapid DNA extracting method of peanut health tissues and diseased tissues comprises and prepares sample material and preparation DNA extraction
The liquid step is characterized in that:
Described preparation sample material is that the sample material that finger to finger test is got is blade, stem tissue, shell tissue or the peanut cotyledon of weight at 3～10mg; Sample material adds the 1.5ml centrifuge tube that presets alkaline lysis liquid or enzymolysis solution, is ground to no visual particulate matter with grinding rod, and is standby;
Described preparation DNA extraction liquid is meant and uses alkaline-heating method or enzymolysis process that above-mentioned ground sample material is handled, and obtains the DNA extraction liquid of sample, wherein:
Described alkaline-heating method is that boiling water boiled 10～40sec after each sample ground in the alkaline lysis liquid of 20～60 μ l, the Tris-HCl that adds 4 times of volumes, boil 1～2min after, the centrifugal 5～10min of 10000rpm, get supernatant liquor, add equal-volume TE damping fluid and be DNA extraction liquid;
Described enzymolysis process, be after sample grinds in 100～200 μ l enzymolysis solutions, put 55～58 ℃ of water-bath 20min, add volume ratio with volume and be 25: 24: 1 phenol: chloroform: primary isoamyl alcohol, the centrifugal 5min of 10000rpm, supernatant liquor changes in another centrifuge tube, adding is with the volume Virahol, and the centrifugal 2～5min of 10000rpm abandons supernatant liquor, be deposited in room temperature and dry, add 100～150 μ l TE damping fluids and be DNA extraction liquid.
2. the DNA extraction method of peanut tissue according to claim 1 is characterized in that described alkaline lysis liquid is 0.1～0.5M NaOH solution.
3. the DNA extraction method of peanut tissue according to claim 1 is characterized in that described Tris-HCl damping fluid contains 5～8mg/ml PVP40.
4. the DNA extraction method of peanut tissue according to claim 1 is characterized in that described enzymolysis solution is the solution that contains 10mMTris-HCl, 5mMEDTA, 5～8mg/ml PVP, 40,50～100 μ g/ml Proteinase Ks.
5. the DNA extraction method of peanut tissue according to claim 1, it is formulated to it is characterized in that described TE damping fluid is that 20ml0.1M Tris-HCl and 0.4ml 5mM EDTA are settled to 200ml.
6. the application of the DNA extraction method of peanut tissue according to claim 1 is characterized in that this method is suitable equally for peanut health tissues and peanut diseased tissues.
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| CN103528914A (en) * | 2013-10-16 | 2014-01-22 | 山东省花生研究所 | Method for extracting and measuring peanut colesterolo totale |
| CN103882010A (en) * | 2014-03-25 | 2014-06-25 | 北京市农林科学院 | Method for rapidly extracting DNA (Desoxvribose Nucleic Acid) of plant genome with high flux |
| CN103882011A (en) * | 2014-03-26 | 2014-06-25 | 中国计量学院 | Method for high-throughput rapid extraction of total DNA of plant and kit thereof |
| CN106676100A (en) * | 2017-01-17 | 2017-05-17 | 安徽江淮园艺种业股份有限公司 | Extraction method of plant genome deoxyribonucleic acid (DNA) |
| CN107034213A (en) * | 2017-06-15 | 2017-08-11 | 商丘市农林科学院 | The one DNA rapid extracting methods for cultivating peanut tissue |
| CN107254468A (en) * | 2017-08-11 | 2017-10-17 | 山东省花生研究所 | A kind of peanut leaf tissue grinding methods and device for being used to extract DNA |
| CN107287104A (en) * | 2017-08-11 | 2017-10-24 | 山东省花生研究所 | A kind of peanut leaf tissue grinder system for being used to extract DNA |
| CN107287191A (en) * | 2017-07-13 | 2017-10-24 | 重庆市畜牧科学院 | The extractant extracted for minim DNA and its application method and application |
| CN108918510A (en) * | 2018-03-29 | 2018-11-30 | 江苏金运农业科技发展有限公司 | A kind of high throughput assay method of plant tissue's content of inorganic phosphorus |
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| CN101372703A (en) * | 2007-08-21 | 2009-02-25 | 中国科学院生态环境研究中心 | Method for extracting plant phyllospheric microorganism genome DNA |
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| CN103528914A (en) * | 2013-10-16 | 2014-01-22 | 山东省花生研究所 | Method for extracting and measuring peanut colesterolo totale |
| CN103528914B (en) * | 2013-10-16 | 2016-08-17 | 山东省花生研究所 | A kind of method for extracting and measuring peanut colesterolo totale |
| CN103882010A (en) * | 2014-03-25 | 2014-06-25 | 北京市农林科学院 | Method for rapidly extracting DNA (Desoxvribose Nucleic Acid) of plant genome with high flux |
| CN103882011A (en) * | 2014-03-26 | 2014-06-25 | 中国计量学院 | Method for high-throughput rapid extraction of total DNA of plant and kit thereof |
| CN106676100A (en) * | 2017-01-17 | 2017-05-17 | 安徽江淮园艺种业股份有限公司 | Extraction method of plant genome deoxyribonucleic acid (DNA) |
| CN107034213A (en) * | 2017-06-15 | 2017-08-11 | 商丘市农林科学院 | The one DNA rapid extracting methods for cultivating peanut tissue |
| CN107287191A (en) * | 2017-07-13 | 2017-10-24 | 重庆市畜牧科学院 | The extractant extracted for minim DNA and its application method and application |
| CN107254468A (en) * | 2017-08-11 | 2017-10-17 | 山东省花生研究所 | A kind of peanut leaf tissue grinding methods and device for being used to extract DNA |
| CN107287104A (en) * | 2017-08-11 | 2017-10-24 | 山东省花生研究所 | A kind of peanut leaf tissue grinder system for being used to extract DNA |
| CN108918510A (en) * | 2018-03-29 | 2018-11-30 | 江苏金运农业科技发展有限公司 | A kind of high throughput assay method of plant tissue's content of inorganic phosphorus |
| CN111961662A (en) * | 2020-08-31 | 2020-11-20 | 江西农业大学 | DNA extracting solution and method for extracting sesame leaf genome DNA |
| CN111961662B (en) * | 2020-08-31 | 2021-11-02 | 江西农业大学 | DNA extracting solution and method for extracting sesame leaf genome DNA |
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