CN103397083B - Specific probes for identification of asparagus 981 fine variety and its application - Google Patents
Specific probes for identification of asparagus 981 fine variety and its application Download PDFInfo
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Abstract
The invention relates to specific probes for identification of an asparagus 981 fine variety. The specific probe sequences are nucleotide sequences shown as SEQ ID NO:1 or SEQ ID NO:2. The two specific probes obtained in the invention can be used for rapid and accurate identification of the asparagus 981 fine variety.
Description
Technical field
The present invention relates to the excellent strain qualification of marine algae, particularly identify specific probe and the application thereof of Thallus Gracilariae 981 breeding.
Background technology
Red algae Thallus Gracilariae is important product agar-agar marine alga, at present, Thallus Gracilariae 981 breeding come through the wild stocks seed selection of Zhan Shan gulf, Qingdao has growth fast, branch is many, high temperature resistant, fast-growing is degeneration-resistant, agar content is high and good, in ground cultivations such as south Fujian sea area, Shantou City, Guangdong Province Nan ' ao Islands for many years, and in Guangdong, Fujian, Zhejiang, Shandong and Liaoning Coast establish Thallus Gracilariae cultivation industry, but itself and wild Thallus Gracilariae are difficult to distinguish on mode of appearance, be all before by measure both growth traits and physiological and biochemical index distinguished.The invention provides two sections of nucleotide sequences and application thereof, adopt this technology can fast, precise Identification 981 breeding, solve the seedling that produces in the course of cultivation of Thallus Gracilariae and mix phenomenon, avoid causing heavy losses to Thallus Gracilariae cultivation industry.
By measuring and measuring growth traits and physiological and biochemical index is distinguished, the test period is long, complex operation.The invention provides two sections of nucleotide sequences, can fast, precise Identification 981 breeding, solve the seedling that produces in the course of cultivation of Thallus Gracilariae and mix phenomenon, avoid causing heavy losses to Thallus Gracilariae cultivation industry.
Summary of the invention
For the defect existed in prior art, the object of the present invention is to provide two probes, can fast, precise Identification Thallus Gracilariae 981 breeding, solve the seedling that produces in the course of cultivation of Thallus Gracilariae and mix phenomenon, avoid causing heavy losses to Thallus Gracilariae cultivation industry.
The specific probe of qualification Thallus Gracilariae 981 breeding provided by the invention, described specific probe sequence is:
There is the nucleotide sequence that sequence is SEQ ID NO:1 or SEQ ID NO:2;
On the basis of such scheme, described probe is marked by digoxigenin labeled test kit.
The specific probe of above-mentioned qualification Thallus Gracilariae 981 breeding is used for the qualification of Thallus Gracilariae 981 breeding.
Utilize above-mentioned specific probe to identify the method for Thallus Gracilariae 981 breeding, use the method for southern blot hybridization to identify.
The concrete steps of the method for above-mentioned qualification Thallus Gracilariae 981 breeding are as follows:
(1) Thallus Gracilariae genomic dna is extracted;
CTAB method is used to extract Thallus Gracilariae genomic dna;
(2) restriction enzyme of genomic dna cuts;
The Thallus Gracilariae genomic dna that employing EcoR I, EcoR V and EcoR V, Hind III two pairs of enzymes combinations obtain step (1) respectively carries out enzyme and cuts;
(3)Southern Blot；
Gel is put into culture dish, adds 20ml sex change liquid, shake sex change 1h, incline sex change liquid, with distilled water rinsing once; Change neutralizer 20ml, shake 1h, between change a neutralizer; Appropriate transfer liquid 20 × SSC is added in electrophoresis chamber, intermediate support puts a filter paper bar, filter paper two ends are immersed in 20 × SSC, take out the gel of neutralization, loading wells down back-off, on filter paper, is rolled with glass stick and is removed the bubble of glue and filter paper, the nylon membrane identical with glue size is placed on sterile distilled water surface, make it nature water suction to sink, film is proceeded in 20 × SSC and soaks 5min, cover on gel; Two filter paper identical with film size are put in 2 × SSC solution moistening, covers on film, get rid of bubble, filter paper is put folded identical with a film size thieving paper, thieving paper is pressed 0.5-1kg weight, shift 12h.After transfer, film makes marks, clear membrane positive and negative, ultraviolet detection gel transfer effect, film dries naturally, infiltrates in 2 × SSC solution, 120 DEG C of baking 30min;
(4) prehybridization of film, hybridization;
Add the DigEasyHyb5ml of 37-42 DEG C of preheating, sealer, prehybridization 30min, be placed in rapidly 0 DEG C after probe 100 DEG C of 5min sex change, incline prehybridization solution, again adds 2ml prehybridization solution and 10ng sex change label probe, sealer, 42 DEG C of hybridized overnight, pour out hybridization solution after having hybridized, film is put into the 2XSSC that small beaker adds 8ml, 0.1%SDS, room temperature washing 5min, repeat once, to pour out film washing liquid, add the 0.5XSSC of 8ml, 0.1%SDS, 65 DEG C of washing 15min, repeat once;
(5) develop the color;
Film adds 5ml damping fluid I in small beaker, and washing 5min, removes damping fluid I, add 10ml damping fluid II, room temperature places 30min, then removes damping fluid II, film is placed in culture dish, add 10ml damping fluid II, room temperature 30min, film is taken out and is placed in small beaker, add 50ml damping fluid I, washing 15min, repeats once, removes damping fluid I, with damping fluid III10ml, place 5min, incline damping fluid III, adds 5ml nitrite ion, be placed in static waiting in the dark to develop the color, colour developing terminates to take out film and adds 3ml distilled water, washing 5min, dry air;
(6) result is observed;
The enzyme of Thallus Gracilariae sample cuts genomic DNA fragment and probe hybridization result shows: Thallus Gracilariae 981 breeding has occurred band at 600bp, 500bp right position, and all occurs without band in other strain individualities.
Two specific probes of the present invention, can identify Thallus Gracilariae 981 breeding quickly and accurately.
Accompanying drawing illustrates:
The enzyme of Fig. 1 Thallus Gracilariae sample cuts genomic DNA fragment and SEQ ID NO:1 probe hybridization result shows;
The enzyme of Fig. 2 Thallus Gracilariae sample cuts genomic DNA fragment and SEQ ID NO:2 probe hybridization result shows;
In figure, M is Marker, and size is followed successively by from top to bottom: 1000bp, 750bp, 500bp, 250bp.1-3 is the test strip of 981 samples; 4-6 is the detected result of 07-2 sample, and 7-36 is the detected result of the wild tetrasporophyte in gulf, profound mountain, can find out, all occurs without band in the wild tetrasporophyte of 07-2 and gulf, profound mountain;
The combination of primers E-AG/M-CCG AFLP system of step 4 in Fig. 3 embodiment 2;
Note: 1-2 is 981; 3-4 is 07-2; 5-14 is the wild tetrasporophyte in gulf, profound mountain.Black arrow is depicted as 981 distinctive bands;
The combination of primers E-AG/M-CAC AFLP system of step 4 in Fig. 4 embodiment 2;
Note: 1-2 is 981; 3-4 is 07-2; 5-14 is the wild tetrasporophyte in gulf, profound mountain.Black arrow is depicted as 981 distinctive bands;
The electrophorogram of two specific bands after sepharose reclaims of step 5.2 in Fig. 5 embodiment 2;
Note: 1 obtains 981 specific bands for E-AG/M-CCG primer amplification; 2 obtain 981 specific bands for E-AG/M-CAC primer amplification;
M is Marker, and size is followed successively by from top to bottom: 1000bp, 750bp, 500bp, 250bp;
Combination of primers E-AG/M-CCG amplification acquisition 981 specific band positive test symbol in step 5.5 in Fig. 6 embodiment 2;
Note: 1 is the universal primer test strip of pMD18T carrier; 2 is special primer E-AG/M-CCG test strip; M is Marker, and size is followed successively by from top to bottom: 1000bp, 750bp, 500bp, 250bp;
Combination of primers E-AG/M-CAC amplification acquisition 981 specific band positive test symbol in step 5.5 in Fig. 7 embodiment 2;
Note: 1 is the universal primer test strip of pMD18T carrier; 2 is special primer E-AG/M-CAC test strip; M is Marker, and size is followed successively by from top to bottom: 1000bp, 750bp, 500bp, 250bp.
Embodiment
The experimental technique of unreceipted actual conditions in the embodiment of the present invention, usually can condition routinely, and the condition as described in " Molecular Cloning: A Laboratory guide " that J. Pehanorm Brooker (Sambrook) etc. is write operates.
Embodiment 1
The specific probe of qualification Thallus Gracilariae 981 breeding provided by the invention, has sequence for the nucleotide sequence described in SEQ ID NO:1 or SEQ ID NO:2.
Embodiment 2
Thallus Gracilariae extracting genome DNA
Adopt CTAB method to extract, take the fresh Thallus Gracilariae of 2.5g, add 5ml CTAB damping fluid and 2g PVPP solid, add liquid nitrogen and be ground into powder, move into rapidly in 10ml plastic centrifuge tube.65 DEG C of temperature baths, every 5min shakes several times gently, the centrifugal 15min of 12000r/min after 30min, careful Aspirate supernatant, and add Proteinase K to final concentration 100 μ g/ml, 50 DEG C, 3h, period puts upside down mixing frequently.With equal-volume phenol/centrifugal 5min of chloroform 10min, 12000rpm, carefully draw supernatant, use chloroform 5min, the centrifugal 5min of 12000rpm, gets supernatant liquor, add the Virahol of 2/3 volume, put upside down mixing, at-20 DEG C, precipitate 15min, the centrifugal 15min of room temperature 12000rpm, dispel supernatant, wash precipitation with 1ml70% ethanol, the centrifugal 15min of 12000rpm, dispel supernatant, dry air precipitates.Add 200 μ l TE to dissolve, 37 DEG C of standing 10min, take out and mix a little, the centrifugal 5min of 14000rpm.Transfer supernatant, in 1.5ml Eppendorf pipe, adds RNase to final concentration 100 μ g/ml, 37 DEG C of digestion 30min.With equal-volume phenol/centrifugal 5min of chloroform 10min, 12000rpm, get supernatant, use chloroform 5min, the centrifugal 5min of 12000rpm, gets supernatant, add-20 DEG C of dehydrated alcohols of 1/10 volume 3M sodium-acetate and 2 times of volumes, place the centrifugal 15min of 12000rpm after 20min, outwell supernatant for-20 DEG C ,-20 DEG C of washing with alcohol with 70% sink DNA, the centrifugal 15min of 12000rpm, dispel supernatant, dry air precipitates, and DNA is dissolved in 50 μ l TE.Ultraviolet spectrometer measures concentration, and 1% agarose electrophoresis detects the integrity of DNA molecular.
The genomic dna of two 981 samples, two 07-2 samples and wild tetrasporophyte 10 strain of gulf, profound mountain is extracted according to the method.
AFLP (amplified fragment length polymorphism) analyzes the acquisition with the specific fragment of 981 breedings
1, digestion with restriction enzyme
EcoR I and Mse I two kinds of restriction enzymes are utilized to carry out double digestion, system 20 μ L:0.5 μ L DNA (500ng/ μ L), 4 μ L10 × buffer (Tango to the above-mentioned genomic dna amounting to 14 samples
tM), 1.0 μ LEcoR I (10U/ μ L), 1.0 μ LMse I (10U/ μ L).By (system is configuring on ice) after mixture mixing, 37 DEG C are being reacted 6 hours, and 65 DEG C are reacted 6 hours.Get 4 μ L digestion products and cut effect through 1% agarose electrophoresis detection enzyme.
2, ligation
The present invention's joint sequence used is as follows:
The joint sequence of Mse I is:
Mse I-adapter I(M-ad1)5’-GAC GAT GAG TCC TGA-3’；SEQ ID NO：3；
Mse I-adapter II(M-ad1)5’-TAC TCA GGA CTC AT-3’；SEQ ID NO：4；
The joint sequence of EcoR I is:
EcoR I-adapter I(E-ad1)5’-CTC GTA GAC TGC GTA CC-3’；SEQ ID NO：5；
EcoR I-adapter II(E-ad2)5’-AAT TGG TAC GCA GTC-3’；SEQ ID NO：6；
Above-mentioned double digestion product is carried out ligation, linked system 40 μ l: 16 μ l double digestion product, 4 μ L10 × ligation buffer, 1.0 μ l EcoR I joint (5 μMs), 1.0 μ LMse I joints (50 μMs), 1.0 μ LT4DNA Ligase.By (system is configuring on ice) after mixture mixing, 16 DEG C of connections are being spent the night.
3, pre-amplified reaction
The present invention's pre-amplimer used end adds 1 selectivity base, another one indiscriminate property base, and pre-amplification primer sequence is as follows:
EcoR I Primer(E-A)：5’-GAC TGC GTA CCA ATT CA-3’；SEQ ID NO：7；
Mse I Primer(M-C)：5’-GAT GAG TCC TGA GTAAC-3’；SEQ ID NO：8；
Known by preliminary experiment, after connecting product dilution 10 times, expanding effect is best, therefore, connection product is carried out 10 times of dilutions as pre-amplification PCR reaction template, 20 μ L reaction systems: 2.0 μ l connect product (diluting 10 times), 2.0 μ l10 × Taq Buffer, 2.0 μ l dNTPs (2mM), 1.2 μ l MgCl
2(25mM), 1.0 μ l E-A (10 μMs), 1.0 μ l M-C (10 μMs), 0.2 μ l Taq archaeal dna polymerase (10U/ μ l).
Above-mentioned reaction system liquid mixing, reaction conditions: 94 DEG C of denaturation 5min; 94 DEG C of 30s, 56 DEG C of 60s, 72 DEG C of 60s, 28 circulations; 72 DEG C of 10min., 4 DEG C of preservations.Pre-amplified production detects through the agarose gel electrophoresis of 1.5%.
4, selective amplification reaction
The selective amplification primer that the present invention adopts extends two bases again on the basis of pre-amplimer, and EcoR I selective amplification primer selects 2 primers and Mse I selective amplification primer to select 9, amounts to 18 pairs of combination of primers.Primer sequence is as follows:
EcoR I Primers：
E-AA：5’-GAC TGC GTA CCA ATT CAA-3’SEQ ID NO：9；
E-AG：5’-GAC TGC GTA CCA ATT CAG-3’SEQ ID NO：10；
Mse I Primers：
M-CCG：5’-GAT GAG TCC TGA GTA ACC G-3’SEQ ID NO：11；
M-CCT：5’-GAT GAG TCC TGA GTA ACC T-3’SEQ ID NO：12；
M-CGG：5’-GAT GAG TCC TGA GTA ACG G-3’SEQ ID NO：13；
M-CGT：5’-GAT GAG TCC TGA GTA ACG T-3’SEQ ID NO：14；
M-CGA：5’-GAT GAG TCC TGA GTA ACG A-3’SEQ ID NO：15；
M-CAC：5’-GAT GAG TCC TGA GTA ACA C-3SEQ ID NO：16；
M-CCA：5’-GAT GAG TCC TGA GTA ACC A-3’SEQ ID NO：17；
M-CTG：5’-GAT GAG TCC TGA GTA ACT G-3’SEQ ID NO：18；
M-CGC：5’-GAT GAG TCC TGA GTA ACG C-3’SEQ ID NO：19；
Known by preliminary experiment, after pre-expansion product dilution 100 times, expanding effect is best, therefore, pre-expansion product is carried out 100 times of dilutions as selective amplification PCR reaction template, 20 μ L reaction systems: 2.0 μ l pre-expansion products (diluting 100 times), 2.0 μ l10 × Taq Buffer, 2.0 μ ldNTPs (2mM), 1.2 μ l MgCl
2(25mM), 0.4 μ l E-AN (10 μMs), 1.2 μ l M-CNN (10 μMs), 0.2 μ l Taq archaeal dna polymerase (10U/ μ l).
Above-mentioned reaction system mixing, reaction conditions is: 94 DEG C of sex change 5min; 94 DEG C of 30s, 65 DEG C of 60s, 72 DEG C of 60s, totally 12 circulations, each cycle annealing temperature reduces by 0.7 DEG C to 56 DEG C.Then 94 DEG C of 30s, 56 DEG C of 60s, 72 DEG C of 60s, then carry out 28 circulations.Last 72 DEG C extend 10min, 4 DEG C of preservations.Selective amplification product detects expanding effect through 1% agarose electrophoresis, amplifies the polyacrylamide gel electrophoresis (PAGE) of after desirable band 6%, silver dye.
5, object band recovery, connect transform with order-checking
5.1 object band polyacrylamide gels reclaim
The specific band of 981 breedings that Analysis and Screening PAGE plate occurs.Illustrate with reference to Poly-gelDNA extraction kit and carry out the recovery of polyacrylamide gel glue.
(1) cut by the glue blade containing specific band, be placed on sterilized slide glass, covered is pulverized, and proceeds in centrifuge tube, adds 250ul Poly gel DNA elution buffer (2mm × 8mm × 0.8mm).Amass equivalent according to colloid and amplify add-on, do not have glue.
Hatch 1-4h for (2) 65 DEG C.Incubation time depends on the volume of glue, DNA size and incubation temperature.100bpDNA fragment 65 DEG C hatches 4h.
(3) transferred to by solute in Poly-Gel pillar, the supporting collection tube of pillar uses.The centrifugal 10min of room temperature 10000g.
(4) elutriant adds 5 times of volume HBbuffer, vortex mixing (fragment of 100bp at least adds the Hbbuffer of 6 times of volumes).
(5) 750ul mixed solution is added in clean HiBind pillar, the centrifugal 1min of room temperature 10000g.Abandon waste liquid.
(6) remaining mixed solution continues to be added to HiBind, with 5 process.Abandon waste liquid, pillar is reinstalled collection tube.
(7) add 700ulSPW and clean pillar, the centrifugal 1min of room temperature 10000g.(the necessary alcohol dilution of SPW)
(8) abandon waste liquid, the empty centrifugal 2min of pillar 10000g, dries.
(9) pillar is put in 1.5ml centrifuge tube, adds 15-30ul ddH
2o.The centrifugal 1min of 10000g room temperature, eluted dna.
(10) repeating step (9) repeats the target DNA fragment that wash-out can obtain more.
The amplification of 5.2 object fragment amounts
Object fragment is after the recovery that glue cut by polyacrylamide gel, due to amount lacking very, be not suitable for directly doing connecting and transform, therefore, with the DNA fragmentation of returning for template, the PCR (with reference to corresponding selective amplification system) that first time amplifies 20ul system is carried out by original corresponding selective amplification primer, first time amplifies PCR primer as template, the second time of carrying out 50ul × 5 pipe amplifies PCR, 50 μ l reaction systems: 2.0 μ l template DNAs, 5.0 μ l10 × PCR Buffer, 3.0 μ l MgCl
2(25mM), 3.0 μ l EcoRI Primers (10 μMs), 3.0 μ l Mse I Primers (10 μMs), 3.0 μ l dNTPs (2.0mM), Taq archaeal dna polymerase 0.5 μ l.By all amplification PCR primer after 1.2% agarose gel electrophoresis detects, carry out the recovery of object fragment.
5.3 object fragment sepharoses reclaim
There is the PCR primer that 200ul amplifies altogether, after agarose gel electrophoresis, EB dyes 20min, the cutting of object fragment is carried out in gel imaging system, the band cut down is put into a new 1.5ml centrifuge tube, then carry out sepharose recovery according to TaKaRa Agarose Gel DNA Purification Kit specification sheets.
The connection of 5.4 object fragments transforms
Object fragment is according to pMD
tM18-T Vector specification sheets connects, and carries out the preparation of competent cell, and its step is as follows:
1. bacterium liquid is activated: in 1.5mL centrifuge tube, add 1ml LB liquid nutrient medium, add the DH5 α bacterium liquid that 10 μ l glycerine are preserved, 100rpm shaken overnight in 37 DEG C of constant-temperature tables, getting 10 μ l bacterium liquid afterwards new is equipped with in the centrifuge tube of 1ml LB liquid nutrient medium to one, and 37 DEG C of constant-temperature table 100rpm vibrate 3-4h.
2. after activation, bacterium liquid places 10min on ice, makes it cool.
3. 4000rpm4 DEG C of centrifugal 10min, sedimentation cell, removes supernatant, adds ice-cold 0.1M CaCl
2200 μ l, blow and beat suspension cell gently, place 30min on ice.
4. 4000rpm4 DEG C of centrifugal 10min, reclaims cell, and supernatant, adds ice-cold 0.1M CaCl
2100 μ l, blow and beat suspension cell gently.
5. 2-3 days is preserved for 4 DEG C, for subsequent use.
(4) transform:
1. 10 μ l connecting fluids joined in the competent cell that 100 μ l prepare, piping and druming mixing several times gently, places 30min on ice.
2. put into by the solution of above-mentioned mixing and be preheated to 42 DEG C of water-baths, heat shock is 90s (being sure not shake) exactly, is quickly transferred to cooled on ice 3-5min.
3. add not containing the LB liquid nutrient medium 400 μ l of AMP, 100rpm incubation 40min in 37 DEG C of constant-temperature tables.
4. taking out 50-100 μ l bacterium liquid transfers on the agar plate solid medium containing AMP, with aseptic triangle rod even spread.
5. be inverted flat board, continue in 37 DEG C of constant incubators to cultivate 12-16h, during obvious single bacterium colony to appear, take out flat board.
6. 4 DEG C of preservations, for subsequent use.
The detection of 5.5 positive colonies and order-checking
Random picking 10 mono-clonals, put into the centrifuge tube of 1.5ml containing the 1ml LB liquid nutrient medium of AMP, in 37 DEG C of constant-temperature tables, 100rpm cultivates 5-7h, the bacterium liquid after spending the night is carried out positive detection by following system (25ul): 2.0ul template DNA, 2.5 μ l PCR Buffer, 2.0 μ l dNTPs, 2.0 μ l MgCl
2, l μ l M
13forward primer, 1 μ l M
13reverse primer, 0.2 μ l Taq archaeal dna polymerase.PCR response procedures: 94 DEG C of denaturation 5min; 94 DEG C of 45s, 55 DEG C of 45s, 72 DEG C 1min35 circulation; Last 72 DEG C extend 5min.Product 4 DEG C preservation.PCR primer carries out 1.5% agarose electrophoresis, uses Dongsheng DL2000DNAmarker as standard calibration.
M
13forward primer: TGTAAAACGACGGCCAGT SEQ ID NO:22
M
13reverse primer: CAGGAAACAGCTATGACC SEQ ID NO:23
The bacterium liquid being positive colony is drawn 10 μ l to be placed in 1ml and to cultivate 4h containing the LB liquid nutrient medium of Amp, glycerol adding is to final concentration 15%, send to the raw work in Shanghai and carry out unidirectional order-checking through 3730 sequenators, obtain the fragment that two Thallus Gracilariae 981 strains are special, be respectively SEQ ID NO:20 and SEQ ID NO:21.
Embodiment 3
With two the specific fragment designing probes obtained in embodiment 2, probe sequence is respectively: SEQ IDNO:1 and SEQ ID NO:2.By digoxigenin labeled test kit, label probe and certification mark rate are described, prepare label probe and contrast by test kit operation instructions, each 1 μ l point of pipe 2-9 is on film, and film dries naturally, 80 DEG C of baking 2h.Film adds 5ml damping fluid I[and lavation buffer solution in small beaker: 0.1M toxilic acid, 0.15MNaCl, pH7.5 (20 DEG C), 0.3% (v/v) Tween-20], washing 5min, remove damping fluid I, add 10ml damping fluid II[and lock solution, with maleate buffer (0.1M toxilic acid, 0.15M NaCl, pH7.5) according to the ratio of 1: 10,10 × Blocking Solution (pipe 6) is diluted to 1 × working solution], room temperature places 30min, remove damping fluid II, film is placed in culture dish, 10ml damping fluid II is added in culture dish, room temperature 30min, take out film and be placed in small beaker, add 50ml damping fluid I, washing 15min, repeat once, remove damping fluid I, namely damping fluid is detected with damping fluid III[, 0.1M Tris-HCl, 0.1M NaCl, pH9.5 (20 DEG C)] 10ml, place 5min, incline damping fluid III, add 5ml nitrite ion [get 200 μ lNBT/BCIP liquid storages (pipe 5) add 10ml detect in damping fluid mix], be placed in static waiting in the dark to develop the color, colour developing terminates to take out film and adds 3ml distilled water, washing 5min, dry air.Probe dilution is to final concentration 10ng/ μ l the most at last.
According to following table, serial dilutions is carried out to the probe of mark and DIG-labeled control DNA.
For the probe that contrast DNA and mark obtain, compare the colour development difference of the sample point of both dilutions, calculate the output of Digoxigenin labeled DNA probe thus.If probe is all visible with the 0.1pg dilution point contrasting DNA, shows that the probe marked reaches the labeling effciency of expection, the concentration and probe concentration needed for hybridization can be met.
Embodiment 4
With probe in detecting Thallus Gracilariae 981 breeding of preparation in embodiment 3, concrete grammar is as follows:
The restriction enzyme of genomic dna cuts
Method with reference to embodiment 2 extracts the genomic dna of the wild tetrasporophyte in the profound gulf, mountain of three 981 samples, three 07-2 samples and 30 strains, employing EcoR I, EcoR V and EcoR V, Hind III two pairs of enzymes combinations carry out enzyme to Thallus Gracilariae sample gene group respectively and cut, system 100 μ l: 30 μ l DNA (1 μ g/ μ l), 5 μ l EcoR I/Hind III (10U/ μ l), 5 μ l EcoR V (10U/ μ l), 20 μ l10 × Buffer.Enzyme cuts through night, adds 2 times of volume dehydrated alcohols, mixing, and be placed in-20 DEG C of precipitations more than 2 hours, the centrifugal 15min of 12000rpm makes DNA precipitate, and dispels supernatant, and dry air precipitates, 40 μ lTE dissolving DNAs.After agarose gel electrophoresis terminates, coated with each band of preservative film record Marker and sample position.
Gel obtained above is put into culture dish, adds 20ml sex change liquid [1.5M NaCl, 0.5MNaOH], shake sex change 1h, incline sex change liquid, with distilled water rinsing once; Change neutralizer [0.5M Tris (pH7.4), 1.5M NaCl] 20ml, shake 1h, between change a neutralizer; Appropriate transfer liquid 20 × SSC[3M NaCl is added in electrophoresis chamber, 0.3M Trisodium Citrate, pH7.0], intermediate support puts a filter paper bar, filter paper two ends are immersed in 20 × SSC, take out the gel of neutralization, loading wells down back-off, on filter paper, is rolled with glass stick and is removed the bubble of glue and filter paper, the nylon membrane identical with glue size is placed on sterile distilled water surface, make it nature water suction to sink, film is proceeded in 20 × SSC and soaks 5min, cover on gel; Two filter paper identical with film size are put in 2 × SSC solution moistening, covers on film, get rid of bubble, filter paper is put folded identical with a film size thieving paper (high 8-10cm), thieving paper is pressed 0.5-1kg weight, shift 12h.After transfer, film makes marks, clear membrane positive and negative, ultraviolet detection gel transfer effect, film dries naturally, infiltrates in 2 × SSC solution, 120 DEG C of baking 30min.
The prehybridization of film, hybridization
Add DigEasyHyb[and the hybridization buffer of 37-42 DEG C of preheating, bottle 7] 5ml, sealer, prehybridization 30min.Be placed in rapidly 0 DEG C after probe 100 DEG C of 5min sex change, incline prehybridization solution, again adds 2ml prehybridization solution and 10ng sex change label probe, sealer, 42 DEG C of hybridized overnight.Pour out hybridization solution after having hybridized, film put into small beaker and add 8ml film washing liquid I[2XSSC, 0.1%SDS], room temperature washing 5min, repeats once, to pour out film washing liquid, adds 8ml film washing liquid II[0.5XSSC, 0.1%SDS], 65 DEG C of washing 15min, repeat once.
Colour developing:
Film adds 5ml damping fluid I in small beaker, and washing 5min, removes damping fluid I, add 10ml damping fluid II, room temperature places 30min, then removes damping fluid II, film is placed in culture dish, add 10ml damping fluid II, room temperature 30min, film is taken out and is placed in small beaker, add 50ml damping fluid I, washing 15min, repeats once, removes damping fluid I, with damping fluid III10ml, place 5min, incline damping fluid III, adds 5ml nitrite ion, be placed in static waiting in the dark to develop the color, colour developing terminates to take out film and adds 3ml distilled water, washing 5min, dry air.
Result is observed:
The enzyme of Thallus Gracilariae sample cuts genomic DNA fragment and probe hybridization result shows: Thallus Gracilariae 981 sample has occurred band at 600bp, 500bp right position, and all occurs without band in cultivar 07-2 and wild individuality, and result as shown in Figure 1 and Figure 2.
Two probes of the present invention, can identify Thallus Gracilariae 981 breeding quickly and accurately.
Note: the present invention's digoxigenin labeled test kit used is produced by Roche (Roche), the date manufactured: 2011.12;
Other primers of the present invention produce synthesis by Shanghai Sheng Gong bio-engineering corporation;
EcoR I, EcoR V, Hind III are produced by Fermentas company.
Claims (4)
1. identify the specific probe of Thallus Gracilariae 981 breeding, it is characterized in that described specific probe sequence is as follows:
There is the nucleotide sequence that sequence is SEQ ID NO:1 or SEQ ID NO:2.
2. specific probe according to claim 1 is used for the qualification of Thallus Gracilariae 981 breeding.
3. utilize the method for specific probe qualification Thallus Gracilariae 981 breeding described in claim 1, it is characterized in that using the method for southern blot hybridization to identify.
4. utilize the method for specific probe qualification Thallus Gracilariae 981 breeding described in claim 1, it is characterized in that concrete steps are as follows:
(1) Thallus Gracilariae genomic dna is extracted;
CTAB method is used to extract Thallus Gracilariae genomic dna;
(2) restriction enzyme of genomic dna cuts;
The Thallus Gracilariae genomic dna that employing EcoR I, EcoR V and EcoR V, Hind III two pairs of enzymes combinations obtain step (1) respectively carries out enzyme and cuts;
(3)Southern Blot；
Gel is put into culture dish, adds 20ml sex change liquid, shake sex change 1h, incline sex change liquid, with distilled water rinsing once; Change neutralizer 20ml, shake 1h, between change a neutralizer; Appropriate transfer liquid 20 × SSC is added in electrophoresis chamber, intermediate support puts a filter paper bar, filter paper two ends are immersed in 20 × SSC, take out the gel of neutralization, loading wells down back-off, on filter paper, is rolled with glass stick and is removed the bubble of glue and filter paper, the nylon membrane identical with glue size is placed on sterile distilled water surface, make it nature water suction to sink, film is proceeded in 20 × SSC and soaks 5min, cover on gel; Two filter paper identical with film size are put in 2 × SSC solution moistening, covers on film, get rid of bubble, filter paper is put folded identical with a film size thieving paper, thieving paper is pressed 0.5-1kg weight, shift 12h; After transfer, film makes marks, clear membrane positive and negative, ultraviolet detection gel transfer effect, film dries naturally, infiltrates in 2 × SSC solution, 120 DEG C of baking 30min;
(4) prehybridization of film, hybridization;
Add the DigEasyHyb5ml of 37-42 DEG C of preheating, sealer, prehybridization 30min, be placed in rapidly 0 DEG C after probe 100 DEG C of 5min sex change, incline prehybridization solution, again adds 2ml prehybridization solution and 10ng sex change label probe, sealer, 42 DEG C of hybridized overnight, pour out hybridization solution after having hybridized, film is put into 2 × SSC that small beaker adds 8ml, 0.1%SDS, room temperature washing 5min, repeat once, to pour out film washing liquid, add the 0.5 × SSC of 8ml, 0.1%SDS, 65 DEG C of washing 15min, repeat once;
(5) develop the color;
Film adds 5ml damping fluid I in small beaker, and washing 5min, removes damping fluid I, add 10ml damping fluid II, room temperature places 30min, then removes damping fluid II, film is placed in culture dish, add 10ml damping fluid II, room temperature 30min, film is taken out and is placed in small beaker, add 50ml damping fluid I, washing 15min, repeats once, removes damping fluid I, with damping fluid III10ml, place 5min, incline damping fluid III, adds 5ml nitrite ion, be placed in static waiting in the dark to develop the color, colour developing terminates to take out film and adds 3ml distilled water, washing 5min, dry air;
(6) result is observed;
The enzyme of Thallus Gracilariae sample cuts genomic DNA fragment and probe hybridization result shows: Thallus Gracilariae 981 breeding has occurred band at 600bp, 500bp right position, and all occurs without band in other strain individualities.
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