CN103409524A - Fluorescence in situ hybridization method for positioning 45S rDNA on plant chromosome - Google Patents
Fluorescence in situ hybridization method for positioning 45S rDNA on plant chromosome Download PDFInfo
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Abstract
The invention discloses a fluorescence in situ hybridization method for positioning the 45S rDNA on a plant chromosome. The fluorescence in situ hybridization method mainly comprises the following steps of preparation of a chromosome specimen, preparation of 45S rDNA probes, fluorescence in situ hybridization of the chromosome and hybridization signal detection. The provided method for probe marking and hybridization is simple, convenient, quick and accurate, and mainly solves the problem about complex and cumbersome marking of the existing probes for fluorescence in situ hybridization. The probes provided by the invention are oligonucleotide probes which are not marked any more, and a fluorophore for modification can be added during synthesis of the probes, and compared with the prior art, the method is simple and the cost is low. By utilizing the technology, FISH positioning of the 45S rDNA on the chromosome can be completed within 3 hours.
Description
Technical field
The invention belongs to cytogenetics and technical field of molecular biology, relate to a kind of easy, quick fluorescence in-situ hybridization method of locating 45S rDNA on plant chromosome.
Background technology
Fluorescence in situ hybridization technique is the comparatively ripe molecule of a development and the biological study means of cell levels, and the probe be labeled is by carrying out in conjunction with realizing the location to target sequence, qualitative and relative quantitative assay with targets such as karyomit(e) and interphasic nucleus or DNA fibers.Ribosomal RNA gene (rDNA) is a kind of multiple copied sequence of the repetition of connecting, mainly comprise 18S-5.8S-25SrDNA(45S) and two parts of 5S rDNA, they are respectively different transcriptional units, form separately tandem repetitive sequence and are positioned at specific site on karyomit(e).18S-5.8S-25SrDNA(45S) and 5S rDNA being widely distributed in plant, have and highly repeat and the high conservative characteristic, can be used as a kind of mark of molecular cytogenetics accurately, this mark is different from general sign mark, be a kind of chromosomal marker that can be deep into molecular level, can partly reflect chromosome structure and genome constitutions information.
In classical 45S rDNA in-situ hybridization method, plasmid probe will carry out probe mark, probe sex change, hybridization solution complicated component, and hybridization time is long, and after hybridization, wash-out is bothersome, hybridization signal needs the processes such as antibody test, operate more loaded down with trivial details, waste time and energy.Existing hybridizing method can not meet the demand of chromosomal localization far away.Development along with fluorescence in situ hybridization, the multicolor fluorescence mark has become possibility, utilize multicolor fluorescence in situ hybridization can be simultaneously by the probe positioning of multiple not isolabeling to karyomit(e), just can analyze the karyomit(e) that is difficult to distinguish on some forms, but due to complex steps, time-consuming, effort and each experimental result are unstable etc., and factors makes this technical development far lag behind the development of other technologies.Therefore, improve the method for existing probe mark method and hybridization, the efficiency that improves hybridization is to be badly in need of the key issue solved.
Summary of the invention
The objective of the invention is to overcome the complicated loaded down with trivial details method of mark of existing fluorescence in situ hybridization probe, a kind of easy, probe mark and hybridizing method fast and accurately are provided, utilize this technology can in three hours, complete the FISH location of 45S rDNA on karyomit(e).
For achieving the above object, the invention discloses following technology contents:
The fluorescence in-situ hybridization method of a kind of 45S rDNA on plant chromosome is characterized in that being undertaken by following step:
1. chromosome specimen preparation
(1) vegetable material is cultivated routinely, treats that seed or the plant tip of a root grow to 0.5-1cm;
(2) cut the eugonic tip of a root, with pre-treatment 3-5 hour under saturated santochlor solution room temperature;
(3) suck pretreatment fluid, the tip of a root with distilled water or the hypotonic 30min of 0.075mol/L Repone K, is then used 3:1(methyl alcohol: Glacial acetic acid) the fixing 20min-1 hour of stationary liquid;
(4) with distillation washing three times, each 5min, fully wash away stationary liquid;
(5) with polygalacturonase and the cellulase enzyme 2.5%(w/w that mixes than 1:1 of mark by weight), at 37 ℃ of tips of a root 1 hour of dissociating;
(6) distilled water washes away enzyme liquid, and with hypotonic 15-30min after 0.075mol/L Repone K, use 3:1 methyl alcohol: the Glacial acetic acid stationary liquid is 20min fixedly;
(7) get the tip of a root in 4 ℃ of distilled water, on freezing clean glass slide, dripping stationary liquid in advance, with tweezers, the tip of a root is fully smashed to pieces, then added 1 stationary liquid, cell is dispelled to dry air;
(8) with the Giemsa staining fluid dyeing 10min of 1:30 dilution, tap water rinses airing;
(9) microscopy, select finely disseminated chromosome specimen to be placed in-20 ℃ of refrigerators to preserve stand-by;
2. 45S rDNA probe preparation
From Arabidopis thaliana 45S rDNA sequence, choosing the highly conserved sequence of one section 20 bases left and right, 5 ' or 3 ' end at probe when synthetic connects a fluorophor:
5 '-TCGTAACAAGGTTTCCGTAG-3 ' (SEQ NO:1), 5 ' end band green fluorescence flag F AM(Fluoresceincarboxylic acid);
3. chromosome fluorescence in-situ hybridization:
A: chromosome specimen pre-treatment
(1) on fluorescent microscope, write down the coordinate of mark division phase, and with glass cutter, divide the position of phase in the slide glass rear indicia;
(2) slide glass is soaked to 5min in 45% acetic acid and fade, dry air;
B: chromosome specimen sex change
(1) in mark, add 30 μ L 70% deionized formamides/2 * SSC, covered, process 2min for 70 ℃ in PCR instrument or baking oven;
(2) remove cover plate, 70%, 85%, the 100% cold ethanol series dehydration of-20 ℃, every grade of 3min, dry air;
C: hybridization
(1) with 2 * SSC, dilute probe to 5ng/ μ L;
(2) every sample adds 10 μ L probes, and covers the cover glass of 18 * 18mm;
(3) 37 ℃ of hybridization 1-2 hour in wet box;
D: wash-out after hybridization
(1) at 4 * SSC, lucifuge washing 10min under room temperature in 0.2%Tween 20;
(2) distilled water flushing a moment, the lucifuge dry air;
E: hybridization signal detects
(1) drip 3 μ L and contain DAPI(4', 6-diamidino-2-phenylindone) anti-fluorescence quenching, covered;
(2) Nikon 80i fluorescence microscope, can be observed blue division phase according to the sample coordinate recorded under ultraviolet excitation, under the blue excitation optical excitation, can be observed green 45S rDNA hybridization signal;
(3) the cold CCD of SPOT RT KE carries out IMAQ, and it is synthetic that SPOT 4.1 softwares carry out image.
The positively effect that the fluorescence in-situ hybridization method of 45S rDNA disclosed by the invention on plant chromosome compared with prior art has is:
(1) probe of the present invention is that oligonucleotide probe can add fluorophor to modify when synthetic, need not carry out mark again, and compared with prior art method is simple, with low cost.
(2) oligonucleotide probe is easy to penetrate into karyomit(e), so the chromosome specimen pre-treatment step can be simplified.
(3) oligonucleotide probe does not need sex change, and the composition of hybridization solution is only 2xSSC, simply more a lot of than classical way composition.
(4) hybridization time only needs 1 hour, does not need to spend the night.
(5) after hybridization, elution step is easy, does not need methane amide
(6) hybridization signal detects and can directly observe, and does not need the signal detection step such as antibody incubation.
The accompanying drawing explanation
Fig. 1 is the FISH positioning result of 45S rDNA on the rye Metaphase Chromosome, and arrow is depicted as 45S rDNA hybridization signal site;
Fig. 2 is the FISH positioning result of 45S rDNA on the willow Metaphase Chromosome, and arrow is depicted as 45S rDNA hybridization signal site;
Fig. 3 is the FISH positioning result of 45S rDNA on the rye Metaphase Chromosome, and arrow is depicted as 45S rDNA hybridization signal site;
Fig. 4 is the FISH positioning result of 45S rDNA on the paddy rice Metaphase Chromosome, and arrow is depicted as 45S rDNA hybridization signal site;
Fig. 5 is the FISH positioning result of 45S rDNA on the green onion Metaphase Chromosome, and arrow is depicted as 45S rDNA hybridization signal site.
Embodiment
Below in conjunction with embodiment, the present invention is described, the scheme of embodiment described here, do not limit the present invention, one of skill in the art can make improvements and change according to spirit of the present invention, described these improvement and variation all should be considered as within the scope of the invention, and scope of the present invention and essence are limited by claim.The model of reagent source used, instrument sees the following form:
| Reagent, instrument title | Source, model |
| Santochlor | Shanghai Tian Lian Fine Chemical Co., Ltd |
| Methyl alcohol | Tianjin chemical reagent six factories |
| Ethanol | Tianjin chemical reagent six factories |
| Glacial acetic acid | Tianjin Chemical Reagents Factory No.1 |
| KCl | Tianjin Chemical Reagents Factory No.1 |
| NaCl | Tianjin Chemical Reagents Factory No.1 |
| HCl | Tianjin Chemical Reagents Factory No.1 |
| MgCl 2 | Tianjin Chemical Reagents Factory No.1 |
| Trisodium citrate | Tianjin good fortune chemical reagent factory in morning |
| Na 2HPO 4 | Tianjin Chemical Reagents Factory No.1 |
| KH 2PO 4 | Tianjin Chemical Reagents Factory No.1 |
| Polygalacturonase | SEVER |
| Cellulase | Yakult |
| Giemsa | Beijing chemical reagents corporation |
| Tween 20 | Beijing Ding Guo Bioisystech Co., Ltd |
| Paraformaldehyde | Beijing Ding Guo Bioisystech Co., Ltd |
| Deionized formamide | Sigma |
| Anti-fluorescence quenching | Vectashield |
| DAPI(4', 6-diamidino-2-phenylindone) | Roche |
| DIG High Prime | Roche |
| RNase A | Roche |
| Anti-digoxigenin-fluorescein | Roche |
| The PCR instrument | Eppendorf 5331 |
| Fluorescent microscope | Nikon 80i |
| Cold CCD | SPOT RT KE |
Embodiment 1
The FISH location of 45S rDNA on the rye Metaphase Chromosome
1. chromosome specimen preparation
(1) after the rye seed germination, treat that the tip of a root grows to 0.5cm.
(2) cut off root tips, with pre-treatment under saturated p-dichlorobenzene aqueous solution room temperature 5 hours.
(3) tip of a root with 0.075mol/L Repone K hypotonic solution hypotonic 30min, is then used 3:1(methyl alcohol: Glacial acetic acid) the fixing 20min of stationary liquid.
(4) with distillation washing three times, each 5min, fully wash away stationary liquid.
(5) with polygalacturonase and the cellulase enzyme 2.5%(w/w that mixes than 1:1 of mark by weight), at 37 ℃ of tips of a root 1 hour of dissociating.
(6) distilled water washes away enzyme liquid, and with the hypotonic 30min of 0.075mol/L Repone K hypotonic solution, use 3:1 methyl alcohol: the Glacial acetic acid stationary liquid is 20min fixedly.
(7) with smear method, prepare chromosome specimen, get the tip of a root in 4 ℃ of distilled water, on freezing clean glass slide, dripping stationary liquid in advance, with tweezers, the tip of a root is fully smashed to pieces, then added 1 stationary liquid, cell is dispelled to dry air.
(8) with Giemsa staining fluid dyeing (Beijing chemical reagents corporation) tap water of 1:30 dilution, rinse airing.
(9) microscopy, select finely disseminated karyomit(e) with the fluorescent microscope scale, write down the position of coordinate and with marking pen in the position of reverse side marker chromosomes, chromosome specimen is placed in-20 ℃ of refrigerators preserve stand-by.
2. 45S rDNA probe preparation
45S rDNA conserved sequence 5 '-TCGTAACAAGGTTTCCGTAG-3 ' (SEQ NO:1) gives birth to work by Shanghai and synthesizes, 5 ' end band green fluorescence flag F AM(Fluoresceincarboxylic acid, work is given birth in Shanghai).The probe distilled water diluting is to save backup in 50ng/ μ L storage liquid-20 ℃ refrigerator.
3. chromosome fluorescence in-situ hybridization (FISH) method
A. chromosome specimen pre-treatment
(1) on fluorescent microscope, write down the coordinate of sample division phase, and regional in the hybridization of slide glass rear indicia with glass cutter.
(2) slide glass is soaked to 5min in 45% acetic acid and fade, dry air;
B. chromosome specimen sex change
(1) in mark, add 30 μ L 70% deionized formamides/2 * SSC, covered, process 2min for 70 ℃ in PCR instrument or baking oven.
(2) get rid of rapidly cover plate, the cold ethanol series of-20 ℃ (70%, 85%, 100%) dehydration, every grade of 3min, dry air.
C. hybridization
(1) with 2 * SSC, dilute probe to 5ng/ μ l.
(2) every sample adds 10 μ L probes, and covers the cover glass of 18 * 18mm.
(3) 37 ℃ of hybridization 1 hour in wet box.
D. wash-out after hybridizing
(1) at 4 * SSC, lucifuge washing 10min under room temperature in 0.2%Tween 20.
(2) distilled water flushing a moment, dry air (lucifuge).
E. hybridization signal is observed
(1) drip the anti-fluorescence quenching (Vectashield) that 3 μ l contain 1.5 μ g/mL DAPI, covered.
(2) Nikon 80i fluorescence microscope, can be observed blue karyomit(e) under ultraviolet excitation, under the blue excitation optical excitation, can be observed green 45S rDNA hybridization signal.
(3) the cold CCD of SPOT RT KE carries out IMAQ, and it is synthetic that SPOT 4.1 softwares carry out image.
Embodiment 2
The FISH location of 45S rDNA on the willow Metaphase Chromosome
1. chromosome specimen preparation
(1) the clip poplar-branch is soaked in tap water, changes water every day, treats that the tip of a root grows to 1cm.
(2) cut off root tips, with pre-treatment under saturated p-dichlorobenzene aqueous solution room temperature 3 hours.
(3) tip of a root with 0.075mol/L Repone K hypotonic solution hypotonic 30min, is then used 3:1(methyl alcohol: Glacial acetic acid) fixing 40min.
(4) tip of a root is washed three times with distillation, and each 5min, fully wash away stationary liquid.
(5) with polygalacturonase and the cellulase enzyme 2.5%(w/w that mixes than 1:1 of mark by weight), at 37 ℃ of tips of a root 1 hour of dissociating.
(6) distilled water washes away enzyme liquid, and with the hypotonic 30min of 0.075mol/L Repone K hypotonic solution, use 3:1 methyl alcohol: the Glacial acetic acid stationary liquid is 20min fixedly.
(7) with smear method, prepare chromosome specimen, get the tip of a root in 4 ℃ of distilled water, on freezing clean glass slide, dripping stationary liquid in advance, with tweezers, the tip of a root is fully smashed to pieces, then added 1 stationary liquid, cell is dispelled to dry air.
(8) with Giemsa staining fluid dyeing (Beijing chemical reagents corporation) tap water of 1:30 dilution, rinse airing.
(9) microscopy, select finely disseminated karyomit(e) with the fluorescent microscope scale, write down the position of coordinate and with marking pen in the position of reverse side marker chromosomes, chromosome specimen is placed in-20 ℃ of refrigerators preserve stand-by.
2. 45S rDNA probe preparation
45S rDNA conserved sequence 5 '-TCGTAACAAGGTTTCCGTAG-3 ' (SEQ NO:1) gives birth to work by Shanghai and synthesizes, 5 ' end band green fluorescence flag F AM(Fluoresceincarboxylic acid, work is given birth in Shanghai).The probe distilled water diluting is to save backup in 50ng/ μ L storage liquid-20 ℃ refrigerator.
3. chromosome fluorescence in-situ hybridization (FISH) method
A. chromosome specimen pre-treatment
(1) on fluorescent microscope, write down the coordinate of sample division phase, and regional in the hybridization of slide glass rear indicia with glass cutter.
(2) slide glass is soaked to 5min in 45% acetic acid and fade, dry air;
B. chromosome specimen sex change
(1) in mark, add 30 μ L 70% deionized formamides/2 * SSC, covered, process 2min for 70 ℃ in PCR instrument or baking oven.
(2) get rid of rapidly cover plate, the cold ethanol series of-20 ℃ (70%, 85%, 100%) dehydration, every grade of 3min, dry air.
C. hybridization
(1) with 2 * SSC, dilute probe to 5ng/ μ L.
(2) every sample adds 10 μ L probes, and covers the cover glass of 18 * 18mm.
(3) 37 ℃ of hybridization 1.5 hours in wet box.
D. wash-out after hybridizing
(1) at 4 * SSC, lucifuge washing 10min under room temperature in 0.2%Tween 20.
(2) distilled water flushing a moment, dry air (lucifuge).
E. hybridization signal is observed
(1) drip the anti-fluorescence quenching (Vectashield) that 3 μ L contain 1.5 μ g/mL DAPI, covered.
(2) Nikon 80i fluorescence microscope, can be observed blue karyomit(e) under ultraviolet excitation, under the blue excitation optical excitation, can be observed green 45S rDNA hybridization signal.
(3) the cold CCD of SPOT RT KE carries out IMAQ, and SPOT 4.1 softwares carry out the synthetic (see figure 2) of image.
Embodiment 3: comparing embodiment
Classical in-situ hybridization method
The FISH positioning step of 45S rDNA on the rye Metaphase Chromosome:
1. chromosome specimen preparation
(1) after the rye seed germination, treat that the tip of a root grows to 0.5cm.
(2) cut off root tips, with pre-treatment under saturated p-dichlorobenzene aqueous solution room temperature 5 hours.
(3) tip of a root with 0.075mol/L Repone K hypotonic solution hypotonic 30min, is then used 3:1(methyl alcohol: Glacial acetic acid) fixing 20min.
(4) tip of a root is washed three times with distillation, and each 5min, fully wash away stationary liquid.
(5) with polygalacturonase and the cellulase enzyme 2.5%(w/w that mixes than 1:1 of mark by weight), at 37 ℃ of tips of a root 1 hour of dissociating.
(6) distilled water washes away enzyme liquid, and with the hypotonic 30min of 0.075mol/L Repone K hypotonic solution, use 3:1 methyl alcohol: the Glacial acetic acid stationary liquid is 20min fixedly.
(7) with smear method, prepare chromosome specimen, get the tip of a root in 4 ℃ of distilled water, on freezing clean glass slide, dripping stationary liquid in advance, with tweezers, the tip of a root is fully smashed to pieces, then added 1 stationary liquid, cell is dispelled to dry air.
(8) with Giemsa staining fluid dyeing (Beijing chemical reagents corporation) tap water of 1:30 dilution, rinse airing.
(9) microscopy, select finely disseminated chromosome specimen to be placed in-20 ℃ of refrigerators to preserve stand-by.
2.45S rDNA probe preparation
Contain the plasmid (being so kind as to give by professor Song Yunchun of Wuhan University) of 45S rDNA from tomato, adopt the random priming mark, the digoxigenin labeled thing is Digoxigenin-11-dUTP(Roche).1 μ g template DNA is diluted in 16 μ L aseptic double-distilled waters, sex change 10min in boiling water bath, put into rapidly the cooling 10min of frozen water, add 4 μ L DIG High Prime(Roche) 4 μ L, 37 ℃ of temperature were bathed 20 hours, 65 ℃ of temperature are bathed 10 minutes termination reactions, save backup in-20 ℃ of refrigerators.
3. chromosome fluorescence in-situ hybridization (FISH) method
A. chromosome specimen pre-treatment:
Slide glass being soaked to 5min in 45% acetic acid fades, dry air, drip 100 μ L 100 μ g/mL RNase A(2xSSC:0.3 mol/L NaCl, 0.03 the preparation of mol/L trisodium citrate) cover cover plate, 37 ℃ of temperature were bathed 1 hour, with 2xSSC washing 3x5min(washing 3 times, each 5min), 70%, 85%, 100% ethanol series dehydration, every grade 5 minutes, air-dry rear dropping 100 μ L 0.01% pepsin(stomach en-s) (10 mM HCl preparation), cover cover plate, 37 ℃ of temperature are bathed 10 min, 1xPBS(0.137M NaCl, 0.0027 M KCl, 0.01M Na
2HPO
4, 0.002 M KH
2PO
4) washing 2x5min, contain 50 mM MgCl
21xPBS washing 5 minutes, 1% paraformaldehyde is (with containing 50 mM MgCl
21xPBS preparation) process 10min under room temperature, 1xPBS washing 5 minutes, 70%, 85%, 100% ethanol series dehydration, every grade 5 minutes, air-dry.
B. chromosome specimen sex change:
Drip 30 μ L 70% deionized formamides/2 * SSC, covered, process 2min for 70 ℃ in PCR instrument or baking oven.Get rid of rapidly cover plate, the cold ethanol series of-20 ℃ (70%, 85%, 100%) dehydration, every grade of 3min, dry air.
C. hybridization:
Probe is diluted to 10 times with hybridization solution (60% deionized formamide, 2xSSC, 10% T 500), in 95 ℃ of sex change 10 minutes, to insert rapidly in frozen water 10 minutes, every sample drips 10 μ L probes, cover 18 * 18mm cover glass, 37 ℃ of hybridization are spent the night in wet box.
D. wash-out after hybridizing:
37 ℃ of washing 3x5min of 60% methane amide (diluting with 2xSSC), TNT damping fluid (100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20) washing 5 minutes.
E. hybridization signal detects:
Drip 100 μ L TNB damping fluid (100 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% blocking reagent), cover cover plate, in 37 ℃ of wet boxes, temperature is bathed 30min, and the TNT damping fluid washes away cover plate, dripping 30 μ L is the Anti-digoxigenin-fluorescein(Roche of 2 μ g/ μ L with the TNB dilution), cover cover plate, in 37 ℃ of wet boxes, temperature is bathed 30min, and TNT washs 3x5min.
F. hybridization signal is observed:
(1) drip the anti-fluorescence quenching (Vectashield) that 3 μ L contain 1.5 μ g/ml DAPI, covered.
(2) Nikon 80i fluorescence microscope, can be observed blue karyomit(e) under ultraviolet excitation, under the blue excitation optical excitation, can be observed green 45S rDNA hybridization signal.
(3) the cold CCD of SPOT RT KE carries out IMAQ, and SPOT 4.1 softwares carry out the synthetic (see figure 3) of image.
Method of the present invention
Step:
With classical in situ hybridization, compare
(1) probe prepares aspect, and probe of the present invention is that oligonucleotide probe can add fluorophor to modify when synthetic, need not carry out mark again, and compared with prior art method is simple, with low cost.
(2) oligonucleotide probe is easy to penetrate into karyomit(e), so the chromosome specimen pre-treatment step can be simplified.
(3) oligonucleotide probe does not need sex change, and the composition of hybridization solution is only 2xSSC, simply more a lot of than classical way composition.
(4) hybridization time only needs 1 hour, does not need to spend the night.
(5) after hybridization, elution step is easy, does not need methane amide
(6) hybridization signal detects and can directly observe, and does not need the signal detection step such as antibody incubation
Conclusion:
The inventive method easy steps is terse, and required cost is low, consuming timely shortly from hybridizing to result, observes and can within 3 hours, complete.
Embodiment 4
Apply method of the present invention as paddy rice (see figure 4), green onion (see figure 5), etc. on various plants, test and achieve success on 30 various plants.
Method: with embodiment 1
Step: with embodiment 1
Conclusion: the inventive method can be applied to the 45S rDNA FISH location of various plants.
SEQUENCE LISTING
<110 > Nankai University
<120 > fluorescence in-situ hybridization method of a kind of 45S rDNA on plant chromosome
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213 > artificial sequence
<400> 1
tcgtaacaag gtttccgtag 20
Claims (1)
1. the fluorescence in-situ hybridization method of a 45S rDNA on plant chromosome is characterized in that being undertaken by following step:
The chromosome specimen preparation
(1) vegetable material is cultivated routinely, treats that seed or the plant tip of a root grow to 0.5-1cm;
(2) cut the eugonic tip of a root, with pre-treatment 3-5 hour under saturated santochlor solution room temperature;
(3) suck pretreatment fluid, the tip of a root with distilled water or the hypotonic 30min of 0.075mol/L Repone K, is then used 3:1(methyl alcohol: Glacial acetic acid) the fixing 20min-1 hour of stationary liquid;
(4) with distillation washing three times, each 5min, fully wash away stationary liquid;
(5) with polygalacturonase and the cellulase enzyme 2.5%(w/w that mixes than 1:1 of mark by weight), at 37 ℃ of tips of a root 1 hour of dissociating;
(6) distilled water washes away enzyme liquid, and with hypotonic 15-30min after 0.075mol/L Repone K, use 3:1 methyl alcohol: the Glacial acetic acid stationary liquid is 20min fixedly;
(7) get the tip of a root in 4 ℃ of distilled water, on freezing clean glass slide, dripping stationary liquid in advance, with tweezers, the tip of a root is fully smashed to pieces, then added 1 stationary liquid, cell is dispelled to dry air;
(8) with the Giemsa staining fluid dyeing 10min of 1:30 dilution, tap water rinses airing;
(9) microscopy, select finely disseminated chromosome specimen to be placed in-20 ℃ of refrigerators to preserve stand-by;
The preparation of 45S rDNA probe
From Arabidopis thaliana 45S rDNA sequence, choosing the highly conserved sequence of one section 20 bases left and right, 5 ' or 3 ' end at probe when synthetic connects a fluorophor:
5 '-TCGTAACAAGGTTTCCGTAG-3 ', 5 ' end band green fluorescence flag F AM;
Chromosome fluorescence in-situ hybridization:
A: chromosome specimen pre-treatment
(1) on fluorescent microscope, write down the coordinate of mark division phase, and with glass cutter, divide the position of phase in the slide glass rear indicia;
(2) slide glass is soaked to 5min in 45% acetic acid and fade, dry air;
B: chromosome specimen sex change
(1) in mark, add 30 μ L 70% deionized formamides/2 * SSC, covered, process 2min for 70 ℃ in PCR instrument or baking oven;
(2) remove cover plate, 70%, 85%, the 100% cold ethanol series dehydration of-20 ℃, every grade of 3min, dry air;
C: hybridization
(1) with 2 * SSC, dilute probe to 5ng/ μ L;
(2) every sample adds 10 μ L probes, and covers the cover glass of 18 * 18mm;
(3) 37 ℃ of hybridization 1-2 hour in wet box;
D: wash-out after hybridization
(1) at 4 * SSC, lucifuge washing 10min under room temperature in 0.2%Tween 20;
(2) distilled water flushing a moment, the lucifuge dry air;
E: hybridization signal detects
(1) drip the anti-fluorescence quenching that 3 μ L contain DAPI, covered;
(2) Nikon 80i fluorescence microscope, can be observed blue division phase according to the sample coordinate recorded under ultraviolet excitation, under the blue excitation optical excitation, can be observed green 45S rDNA hybridization signal;
(3) the cold CCD of SPOT RT KE carries out IMAQ, and it is synthetic that SPOT 4.1 softwares carry out image.
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| CN106755507A (en) * | 2017-02-07 | 2017-05-31 | 电子科技大学 | Differentiate the molecular detecting method of cultivation rye and wild chromosomes of rye |
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| KR101863820B1 (en) * | 2017-07-28 | 2018-06-01 | 삼육대학교산학협력단 | Novel 45S rDNA Nucleic Acid Probes for Advansed FISH and Novel FISH method Using the Same |
| CN108760706A (en) * | 2018-06-08 | 2018-11-06 | 农业部环境保护科研监测所 | A kind of method of quick screening low cadmium-accumulation rice varieties |
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| KR101863820B1 (en) * | 2017-07-28 | 2018-06-01 | 삼육대학교산학협력단 | Novel 45S rDNA Nucleic Acid Probes for Advansed FISH and Novel FISH method Using the Same |
| CN107541553A (en) * | 2017-09-08 | 2018-01-05 | 河南省农业科学院芝麻研究中心 | One group is used to distinguish BAC mark of the sesame 13 to chromosome |
| CN108760706A (en) * | 2018-06-08 | 2018-11-06 | 农业部环境保护科研监测所 | A kind of method of quick screening low cadmium-accumulation rice varieties |
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| CN108977565A (en) * | 2018-08-14 | 2018-12-11 | 南京林业大学 | A kind of probe combinations and method suitable for plant chromosome rDNA physical positioning |
| CN109161581A (en) * | 2018-09-06 | 2019-01-08 | 南京晓庄学院 | A kind of Allium mongolicum Regel chromosome fluorescence in-situ hybridization method |
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