CN102492771A - Method for constructing chromosome physical map of rose plant - Google Patents
Method for constructing chromosome physical map of rose plant Download PDFInfo
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- CN102492771A CN102492771A CN2011103904606A CN201110390460A CN102492771A CN 102492771 A CN102492771 A CN 102492771A CN 2011103904606 A CN2011103904606 A CN 2011103904606A CN 201110390460 A CN201110390460 A CN 201110390460A CN 102492771 A CN102492771 A CN 102492771A
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Abstract
The invention relates to a method for constructing a chromosome physical map of a rose plant, which comprises the following steps: preparing a mitosis metaphase chromosome slide through an improved enzymolysis and deplasmolysis low-permeability method, wherein in the probe marking process, a nick translation method is adopted, and digoxin-modified nucleotide is used to mark plasmid DNA (deoxyribonucleic acid) carrying target genes and enable the DNA to become a hybridization probe; and then, directly hybridizing a chromosome-denatured DNA single chain with the DNA probe, and carrying out specific immune reaction on anti-digoxin antibody coupled with rhodamine and the digoxin marked on the probe to detect the positions and distribution of the target genes on the chromosome, thus obtaining the chromosome physical map. The invention can carry out fluorescent in-situ hybridization on a rose plant which has a small chromosome and many cell inclusions, and can stably obtain a clear chromosome physical map. Besides, the method is universally applicable and easy to operate with respect to a rose plant.
Description
Technical field
The present invention relates to plant molecular cytogenetics field, specifically is a kind of method of utilizing laser confocal microscope to make up gul physical chromosomal map spectrum.
Background technology
Make up the method that the plant chromosome physical map mainly adopts chromosome fluorescence in-situ hybridization.Chromosome fluorescence in-situ hybridization (fluorescence in situ hybrisization; FISH) be meant with the dna single chain that carries after the dna probe of special modified nucleotide mark directly unwinds with karyomit(e) and hybridize; With coupling the monoclonal antibody of fluorescein molecule and the specific immune response of probe marked material are arranged again; Come to detect intuitively position and the distribution of dna molecular on karyomit(e), thereby make up the physical chromosomal map spectrum of goal gene.One of effective means that it is that donor gene infiltrate to be analyzed in molecular cytogenetic and the plant breeding.
Conventional fluorescence in-situ hybridization method can be in a lot of plants widespread usage.But because most plants seed germination difficulty in the rose, engrafting method commonly used is bred in the production, and plant root is real to be the root system of stock, and therefore can only select stem apex for use is the film-making material.And the rose stem apex is more with respect to its cell content of the tip of a root, cell walls is thicker; And the karyomit(e) of gul is that minisome (mean length 3 μ m) waits this a series of problems; Cause general fluorescence in-situ hybridization method to be applied to gul; Can't stably make up physical chromosomal map spectrum clearly, limit the application of molecular cytogenetics in gul research.
Summary of the invention
The objective of the invention is characteristics, a kind of method of utilizing laser confocal microscope to make up gul physical chromosomal map spectrum is provided to gul.
Gul among the present invention refer to Rosaceae rose (
RosaLinnaeus) each wild taxonomic species (species) and Cultivar (cultivar) in.
The method of structure gul physical chromosomal map of the present invention spectrum is: go the hypotonic legal system of wall to have chromosome sectioning thread metaphase with the enzymolysis of improvement; Probe mark adopts nick-translation, uses the Nucleotide mark of modifying through digoxin (Digoxigenin-11-dUTP) to carry the DNA of goal gene, makes it to become hybridization probe.Then with dna probe directly with the karyomit(e) sex change after the dna single chain hybridize; Have the anti digoxin antibody (Anti-digoxigenin) of rhodamine (Rhodamine) and the digoxin (Digoxigenin-11-dUTP) of probe marked to carry out specific immune response with coupling again, position and the distribution of testing goal gene on karyomit(e) obtains the physical chromosomal map spectrum.Concrete grammar is following:
1, Chromosome Preparation
1) collection of material, pre-treatment and preservation: in the fine morning in spring, choose firm sprouting and do not have the vegetative bud of powder month in and month out of disease and pest, gather its stem apex 2-3cm; Peel off the outside spire of stem apex with tweezers, cut the about 1-2cm of stem apex, stem apex was put in the pretreatment fluid of oxine and 0.02% NST-757 of the 0.002mol/l that 1:1 disposes 3-4 hour; After stem apex taken out from pretreatment fluid,, be put in then in the Klorvess Liquid of 0.075mol/l 30 minutes with aseptic water washing 3 times; After from Klorvess Liquid, taking out stem apex,, be put in Kano stationary liquid (absolute ethyl alcohol: in the glacial acetic acid=3v:1v) then with aseptic water washing 3 times; Fixed overnight is taken out stem apex from stationary liquid, with aseptic water washing 3 times; Be put in then in 70% the ethanolic soln, place 4 ℃ of refrigerators, (can for a long time) preserves subsequent use;
2) chromosome sectioning: the stem apex of powder takes out from preserve liquid month in and month out, behind aseptic water washing 3 times, under body formula anatomical lens; Isolate shoot tip meristem in the gnotobasis condition; Cut 1-2mm and be put in the 0.25-0.1mol/l hydrochloric acid soln 10-15 minute, the shoot tip meristem after the acidolysis is clean with aseptic water washing, be put in enzymolysis in the mixed solution of 2%-5% cellulase and 1%-3% polygalacturonase; In 37 ℃ of enzymolysis 3-4 hours; Enzymolysis completely shoot tip meristem wash gently 2 times with sterilized water, be put in 37 ℃ the sterilized water hypotonic 90 minutes; Hypotonic medium is removed, and (absolute ethyl alcohol: in the glacial acetic acid=3v:1v), the vortex mixing makes it to become the protoplastis suspension to add the Kano stationary liquid; 3000rpm is centrifugal with suspension, removes supernatant, and it is resuspended to add the Kano stationary liquid once more, the vortex mixing; Draw suspension, drip on the also freezing anticreep slide glass of prior cleaning, suspension is evenly blown open, and place rapidly spirit lamp flame envelope baking 2-3 second; Dry air then, the film-making for preparing are observed with 40 * object lens under ordinary optical microscope, chosen more metaphase mutually and karyomit(e) disperse film-making preferably; Be put in the slide box, and place-20 ℃ of refrigerators, (can for a long time) preserves;
2, probe mark
1) probe purity and concentration detect: use the absorption spectrum nucleic acid detection method---remove background with distilled water, and then after being connected with the segmental plasmid of 45SrDNA and diluting, the light absorption value A when measuring wavelength respectively and being 260nm and 280nm
260And A
280, according to the double-stranded DNA method of calculation, calculate the purity and the concentration of plasmid double-stranded DNA, choose purity greater than 1.8, concentration is carried out following experiment greater than the plasmid of 62.5 μ g/ml;
2) probe mark: adopt nick-translation; With digoxin (Digoxigenin-11-dUTP; Luo Shi Roche company) this plasmid of mark, promptly each reaction system contains 1 μ g DNA and digoxigenin labeled reagent (Digoxigenin Nick Translation), reacts 90 minutes down in 15 ℃; After the termination reaction, subsequent use in-20 ℃ of preservations;
3, chromosome fluorescence in-situ hybridization
The chromosome sectioning of powder month in and month out that step 1 is preserved drips 10 μ g/ml pepsin solutions in the film-making upper surface in 60 ℃ of oven dry 1-2 hour, is put in 37 ℃ of constant incubators 20-40 minute; With 2 * SSC rinsing 5 minutes, dewatered 3 times each 5 minutes dry air successively with 75%-95%-100% (volumn concentration) ethanol; Under 70 ℃-80 ℃ in 70% methane amide (volumn concentration) after the sex change; Dewatered successively 3 times each 5 minutes at the cold ethanol of 75%-95%-100% (volumn concentration) immediately; Dry air; Configuration contains the hybridization solution of 50% methane amide (volumn concentration), 10% T 500,23% 2 * SSC, 3% salmon sperm dna, 14% digoxin labelled probe, and hybridization solution after 80 ℃ of-90 ℃ of sex change, was put in rapidly in the frozen water 10-20 minute; To hybridize mixed solution and be added drop-wise to equably on the chromosome sectioning, spend the night in 37 ℃ of hybridization;
4, signal detection
37 ℃ down with 2 * SSC wash-out 2 times, each 5 minutes, at room temperature use 2 * SSC, each wash-out of 1 * PBS 1 time then respectively, each 5 minutes; The drip-dry slide glass, signal detection adopts anti digoxin antibody (Anti-digoxigenin, the Luo Shi Roche company) test kit that combines rhodamine (Rhodamine), after accomplishing with antibodies; With 1 * PBS wash-out 3 times, each 5 minutes, the drip-dry slide glass was with DAPI (4; 6-diamidino-2-phenylindole) contaminated 5 minutes, use 1 * PBS wash-out once more 3 times, each 5 minutes, the drip-dry slide glass; On slide glass, drip anti-fluorescent quenching mountant, extrude unnecessary liquid, mounting, dry air; Under laser confocal microscope, find karyomit(e) with ordinary light source earlier, use laser scanning again, taking pictures after the removal background obtains the physical chromosomal map spectrum.
Plant according to the invention can be each wild taxonomic species (species) and the Cultivar (cultivar) in the rose.
The present invention is on the basis of general fluorescence in-situ hybridization method; The improvement enzymolysis goes the hypotonic legal system of wall to have chromosome sectioning thread metaphase; The karyomit(e) that when film-making, better divides the acellular film that sheds to wrap up through the mechanical force of whizzer and vortex appearance fully spreads on the plane karyomit(e).To karyomit(e) is the more gul chromosome sectioning of minisome and cell content, and enough metaphase in cell division phases of acquisition that can be stable and karyomit(e) disperse the high-quality chromosome sectioning of better, acellular film parcel.Then, before probe mark, detect the concentration and the purity of DNA earlier, guarantee the quality of probe; Use suitable pepsin in when hybridization, make probe more be prone to permeates cell membranes and combine with target chromosome; In testing process, adopt the Laser Scanning Confocal Microscope of laser scanning, to compare with general fluorescent microscope, it detects sensitiveer, and do not need 3 antibody to carry out signal and amplify, and can remove background automatically, and the power between the detectable signal, be used for quantitative analysis.Utilize method of the present invention to have following advantage: to karyomit(e) is the more gul fluorescence in situ hybridization of minisome and cell content, can stably obtain physical chromosomal map spectrum clearly.The present invention has generally suitable, easy-operating characteristics to the plant of rose.
Description of drawings
Fig. 1 be before the chromosome fluorescence in-situ hybridization powder month in and month out (
Rosa chinensis' Pallida ', 2n=2x) karyostasis chromosome map.
Fig. 2 for carry out with the 45SrDNA probe behind the chromosome fluorescence in-situ hybridization powder month in and month out (
Rosa chinensis' Pallida ', 2n=2x) karyostasis chromosome map, red point is hybridization site.The 45SrDNA assignment of genes gene mapping is in the nucleus outside.
Fig. 3 be before the chromosome fluorescence in-situ hybridization powder month in and month out (
Rosa chinensis' Pallida ', 2n=2x) metaphase chromosome map.
Fig. 4 for carry out with the 45SrDNA probe behind the chromosome fluorescence in-situ hybridization powder month in and month out (
Rosa chinensis' Pallida ', 2n=2x) metaphase chromosome map, red point is hybridization site.The 45SrDNA assignment of genes gene mapping is in the karyomit(e) close end.
Embodiment
Following being embodied as is convenient to better understand the present invention, but do not limit the present invention.Experimental technique among the following embodiment like no specified otherwise, is ordinary method.Used experiment reagent among the following embodiment like no specified otherwise, is to buy from the routine biochemistry reagent suppliers and obtains.Following percentage composition like no specified otherwise, is the quality percentage composition.
Among the embodiment used gul material be Cultivar month in and month out powder (
Rosa chinensis' Pallida ' 2n=2x), collected by Flower Research Institute(FRI) of Yunnan Woody flower center, is stored in Flower Research Institute(FRI) of Yunnan experiment base.
Embodiment 1:
1, Chromosome Preparation
1) collection of material, pre-treatment and preservation
In the fine morning in spring, choose firm sprouting and do not have the vegetative bud of powder month in and month out of disease and pest, gather its stem apex 2-3cm, be put in the collection bag that sterilized water is housed.Collection bag is placed in the ice chest, in 1 hour, be transported to the laboratory and carry out pre-treatment.Carefully peel off the outside spire of stem apex in the laboratory with tweezers, cut the about 1-2cm of stem apex.Stem apex was put in the pretreatment fluid of 1:1 configuration of oxine and 0.02% NST-757 of 0.002mol/l 3-4 hour.After stem apex taken out from pretreatment fluid,, be put in then in the Klorvess Liquid of 0.075mol/l 30 minutes with aseptic water washing 3 times.After from Klorvess Liquid, taking out stem apex, with aseptic water washing 3 times, be put in then the Kano stationary liquid (absolute ethyl alcohol: in the glacial acetic acid=3v:1v), fixed overnight.Stem apex is taken out from stationary liquid,, be put in then in 70% the ethanolic soln, place 4 ℃ of refrigerators, but prolonged preservation is subsequent use with aseptic water washing 3 times.
2) chromosome sectioning
The stem apex of powder is month in and month out taken out from preserve liquid, with aseptic water washing 3 times.Then, under body formula anatomical lens, in the gnotobasis condition, isolate shoot tip meristem, cut 1-2mm with scalpel with tweezers and dissecting needle.The shoot tip meristem that cuts was put in the 0.25mol/l hydrochloric acid soln 10 minutes.Shoot tip meristem after the acidolysis is clean with aseptic water washing, be put in enzymolysis in the mixed solution of 2% cellulase and 1% polygalacturonase, in 37 ℃ of enzymolysis 3-4 hours.Enzymolysis completely shoot tip meristem wash gently 2 times with sterilized water, be put in 37 ℃ the sterilized water hypotonic 90 minutes.Hypotonic medium is removed, and (absolute ethyl alcohol: in the glacial acetic acid=3v:1v), the vortex mixing makes it to become the protoplastis suspension to add the Kano stationary liquid.3000rpm is centrifugal with suspension, removes supernatant.It is resuspended to add the Kano stationary liquid once more, the vortex mixing.Draw suspension, drip on the also freezing anticreep slide glass of prior cleaning, suspension is evenly blown open, and place spirit lamp flame envelope baking 2-3 second, dry air then rapidly.The film-making for preparing is observed down with 40 * object lens under ordinary optical microscope.Chosen more metaphase mutually and karyomit(e) disperse film-making preferably, be put in the slide box, and place-20 ℃ of refrigerators, but prolonged preservation.
2, probe mark
1) probe purity and concentration detect: use the absorption spectrum nucleic acid detection method---remove background with distilled water, and then after being connected with the segmental plasmid of 45SrDNA and diluting, the light absorption value A when measuring wavelength respectively and being 260nm and 280nm
260And A
280According to the double-stranded DNA method of calculation, calculate the purity and the concentration of plasmid double-stranded DNA.Choose purity greater than 1.8, concentration is carried out following experiment greater than the plasmid of 62.5 μ g/ml.
2) probe mark: adopt nick-translation, with digoxin (Digoxigenin-11-dUTP, Luo Shi Roche company) this plasmid of mark.Be that each reaction system contains 1 μ g DNA and digoxigenin labeled reagent (Digoxigenin Nick Translation), reacted 90 minutes down in 15 ℃.After the termination reaction, subsequent use in-20 ℃ of preservations.
3, chromosome fluorescence in-situ hybridization
The chromosome sectioning of powder month in and month out that step 1 is preserved drips 10 μ g/ml pepsin solutions in the film-making upper surface in 60 ℃ of oven dry 1-2 hour, is put in 37 ℃ of constant incubators 20-40 minute; With 2 * SSC rinsing 5 minutes, dewatered 3 times each 5 minutes dry air successively with 75%-95%-100% (volumn concentration) ethanol.Under 70 ℃-80 ℃, in 70% methane amide (volumn concentration), after the sex change, dewatered 3 times each 5 minutes dry air immediately successively at the cold ethanol of 75%-95%-100% (volumn concentration); Configuration contains the hybridization solution of 50% methane amide (volumn concentration), 10% T 500,23% 2 * SSC, 3% salmon sperm dna, 14% digoxin labelled probe, and hybridization solution after 80 ℃ of-90 ℃ of sex change, was put in rapidly in the frozen water 10-20 minute.To hybridize mixed solution and be added drop-wise to equably on the chromosome sectioning, spend the night in 37 ℃ of hybridization.
4, signal detection
37 ℃ down with 2 * SSC wash-out 2 times, each 5 minutes, at room temperature use 2 * SSC, each wash-out of 1 * PBS 1 time then respectively, each 5 minutes, the drip-dry slide glass.Signal detection adopts anti digoxin antibody (Anti-digoxigenin, the Luo Shi Roche company) test kit that combines rhodamine (Rhodamine).After accomplishing with antibodies, with 1 * PBS wash-out 3 times, each 5 minutes.The drip-dry slide glass, (4,6-diamidino-2-phenylindole) dip-dye is 5 minutes, uses 1 * PBS wash-out once more 3 times, each 5 minutes with DAPI.The drip-dry slide glass drips anti-fluorescent quenching mountant on slide glass, extrude unnecessary liquid, mounting, dry air.Under laser confocal microscope, find karyomit(e) with ordinary light source earlier, use laser scanning again, taking pictures after the removal background obtains the physical chromosomal map spectrum.
Embodiment 2:
1, Chromosome Preparation
1) collection of material, pre-treatment and preservation
In the fine morning in spring, choose firm sprouting and do not have the vegetative bud of powder month in and month out of disease and pest, gather its stem apex 2-3cm, be put in the collection bag that sterilized water is housed.Collection bag is placed in the ice chest, in 1 hour, be transported to the laboratory and carry out pre-treatment.Carefully peel off the outside spire of stem apex in the laboratory with tweezers, cut the about 1-2cm of stem apex.Stem apex was put in the pretreatment fluid of 1:1 configuration of oxine and 0.02% NST-757 of 0.002mol/l 3-4 hour.After stem apex taken out from pretreatment fluid,, be put in then in the Klorvess Liquid of 0.075mol/l 30 minutes with aseptic water washing 3 times.After from Klorvess Liquid, taking out stem apex, with aseptic water washing 3 times, be put in then the Kano stationary liquid (absolute ethyl alcohol: in the glacial acetic acid=3v:1v), fixed overnight.Stem apex is taken out from stationary liquid,, be put in then in 70% the ethanolic soln, place 4 ℃ of refrigerators, but prolonged preservation is subsequent use with aseptic water washing 3 times.
2) chromosome sectioning
The stem apex of powder is month in and month out taken out from preserve liquid, with aseptic water washing 3 times.Then, under body formula anatomical lens, in the gnotobasis condition, isolate shoot tip meristem, cut 1-2mm with scalpel with tweezers and dissecting needle.The shoot tip meristem that cuts was put in the 0.1mol/l hydrochloric acid soln 15 minutes.Shoot tip meristem after the acidolysis is clean with aseptic water washing, be put in enzymolysis in the mixed solution of 5% cellulase and 3% polygalacturonase, in 37 ℃ of enzymolysis 3-4 hours.Enzymolysis completely shoot tip meristem wash gently 2 times with sterilized water, be put in 37 ℃ the sterilized water hypotonic 90 minutes.Hypotonic medium is removed, and (absolute ethyl alcohol: in the glacial acetic acid=3v:1v), the vortex mixing makes it to become the protoplastis suspension to add the Kano stationary liquid.3000rpm is centrifugal with suspension, removes supernatant.It is resuspended to add the Kano stationary liquid once more, the vortex mixing.Draw suspension, drip on the also freezing anticreep slide glass of prior cleaning, suspension is evenly blown open, and place spirit lamp flame envelope baking 2-3 second, dry air then rapidly.The film-making for preparing is observed down with 40 * object lens under ordinary optical microscope.Chosen more metaphase mutually and karyomit(e) disperse film-making preferably, be put in the slide box, and place-20 ℃ of refrigerators, but prolonged preservation.
2,3,4 steps are identical with embodiment 1.
Claims (1)
1. one kind makes up the method that the gul physical chromosomal map is composed, and it is characterized in that carrying out according to the following steps:
1) Chromosome Preparation
(1) collection of material, pre-treatment and preservation: in the fine morning in spring, choose firm sprouting and do not have the vegetative bud of powder month in and month out of disease and pest, gather its stem apex 2-3cm; Peel off the outside spire of stem apex with tweezers, cut the about 1-2cm of stem apex, stem apex was put in the pretreatment fluid of oxine and 0.02% NST-757 of the 0.002mol/l that 1:1 disposes 3-4 hour; After stem apex taken out from pretreatment fluid,, be put in then in the Klorvess Liquid of 0.075mol/l 30 minutes with aseptic water washing 3 times; After from Klorvess Liquid, taking out stem apex,, be put in then by absolute ethyl alcohol: in the Kano stationary liquid of glacial acetic acid=3v:1v preparation with aseptic water washing 3 times; Fixed overnight is taken out stem apex from stationary liquid, with aseptic water washing 3 times; Be put in then in 70% the ethanolic soln, place 4 ℃ of refrigerators, preserve subsequent use;
(2) chromosome sectioning: the stem apex of powder takes out from preserve liquid month in and month out, behind aseptic water washing 3 times, under body formula anatomical lens; Isolate shoot tip meristem in the gnotobasis condition; Cut 1-2mm and be put in the 0.25-0.1mol/l hydrochloric acid soln 10-15 minute, the shoot tip meristem after the acidolysis is clean with aseptic water washing, be put in enzymolysis in the mixed solution of 2%-5% cellulase and 1%-3% polygalacturonase; In 37 ℃ of enzymolysis 3-4 hours; Enzymolysis completely shoot tip meristem wash gently 2 times with sterilized water, be put in 37 ℃ the sterilized water hypotonic 90 minutes; Hypotonic medium is removed, added by absolute ethyl alcohol: in the Kano stationary liquid of glacial acetic acid=3v:1v preparation, the vortex mixing makes it to become the protoplastis suspension; 3000rpm is centrifugal with suspension, removes supernatant, and it is resuspended to add the Kano stationary liquid once more, the vortex mixing; Draw suspension, drip on the also freezing anticreep slide glass of prior cleaning, suspension is evenly blown open, and place rapidly spirit lamp flame envelope baking 2-3 second; Dry air then, the film-making for preparing are observed with 40 * object lens under ordinary optical microscope, chosen more metaphase mutually and karyomit(e) disperse film-making preferably; Be put in the slide box, and place-20 ℃ of refrigerators, preserve;
2) probe mark
(1) probe purity and concentration detect: remove background with distilled water, and then after being connected with the segmental plasmid dilution of 45SrDNA, the light absorption value A when measuring wavelength respectively and being 260nm and 280nm
260And A
280, according to the double-stranded DNA method of calculation, calculate the purity and the concentration of plasmid double-stranded DNA, choose purity greater than 1.8, concentration is carried out following experiment greater than the plasmid of 62.5 μ g/ml;
(2) probe mark: adopt nick-translation, with this plasmid of digoxigenin labeled, promptly each reaction system contains 1 μ g DNA and digoxigenin labeled reagent, reacts 90 minutes down in 15 ℃, and is after the termination reaction, subsequent use in-20 ℃ of preservations;
3) chromosome fluorescence in-situ hybridization
The chromosome sectioning of powder month in and month out that step 1) is preserved drips 10 μ g/ml pepsin solutions in the film-making upper surface in 60 ℃ of oven dry 1-2 hour, is put in 37 ℃ of constant incubators 20-40 minute; With 2 * SSC rinsing 5 minutes, dewatered 3 times each 5 minutes dry air successively with volumn concentration 75%-95%-100% ethanol; Under 70 ℃-80 ℃ in volumn concentration 70% methane amide after the sex change; Dewatered successively 3 times each 5 minutes at the cold ethanol of volumn concentration 75%-95%-100% immediately; Dry air; Configuration contains the hybridization solution of volumn concentration 50% methane amide, 10% T 500,23% 2 * SSC, 3% salmon sperm dna, 14% digoxin labelled probe, and hybridization solution after 80 ℃ of-90 ℃ of sex change, was put in rapidly in the frozen water 10-20 minute; To hybridize mixed solution and be added drop-wise to equably on the chromosome sectioning, spend the night in 37 ℃ of hybridization;
4) signal detection
37 ℃ down with 2 * SSC wash-out 2 times, each 5 minutes, at room temperature use 2 * SSC, each wash-out of 1 * PBS 1 time then respectively, each 5 minutes; Drip-dry slide glass, signal detection adopt to combine the anti digoxin antibody test kit of rhodamine, after accomplishing with antibodies, with 1 * PBS wash-out 3 times; Each 5 minutes, the drip-dry slide glass, (4,6-diamidino-2-phenylindole) dip-dye is 5 minutes with DAPI; Use 1 * PBS wash-out once more 3 times, each 5 minutes, the drip-dry slide glass dripped anti-fluorescent quenching mountant on slide glass; Extrude unnecessary liquid, mounting, dry air; Under laser confocal microscope, find karyomit(e) with ordinary light source earlier, use laser scanning again, taking pictures after the removal background obtains the physical chromosomal map spectrum.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073568A (en) * | 2014-07-18 | 2014-10-01 | 西南大学 | Fluorescence in situ hybridization method for metaphase chromosome of mulberry |
| CN111323288A (en) * | 2020-04-03 | 2020-06-23 | 贵州省烟草科学研究院 | Winged tobacco monosome identification method |
-
2011
- 2011-12-01 CN CN 201110390460 patent/CN102492771B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 余朝文等: "植物45S rDNA的染色体位置的CPD染色和FISH分析", 《广西植物》 * |
| 刘源: "树霉荧光原位杂交体系的建立及优化", 《中国优秀硕士论文全文数据库》 * |
| 葛台明等: "对植物染色体标本去壁低渗制片法的探讨", 《鄂西大学学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104073568A (en) * | 2014-07-18 | 2014-10-01 | 西南大学 | Fluorescence in situ hybridization method for metaphase chromosome of mulberry |
| CN104073568B (en) * | 2014-07-18 | 2016-01-06 | 西南大学 | The fluorescence in-situ hybridization method of mulberry tree Metaphase Chromosome |
| CN111323288A (en) * | 2020-04-03 | 2020-06-23 | 贵州省烟草科学研究院 | Winged tobacco monosome identification method |
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