CN1840704A - Cotton in-situ hybridization method - Google Patents
Cotton in-situ hybridization method Download PDFInfo
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- CN1840704A CN1840704A CN 200610011287 CN200610011287A CN1840704A CN 1840704 A CN1840704 A CN 1840704A CN 200610011287 CN200610011287 CN 200610011287 CN 200610011287 A CN200610011287 A CN 200610011287A CN 1840704 A CN1840704 A CN 1840704A
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Abstract
The related cotton in-situ hybridization method comprises: producing with 2% cellulase and 0.5% pectinase at 37Deg for 1-2.5h, preparing probe marked by multiple colors, pre-processing, preparing hybridization liquid to denaturize 8-10min at 90-97Deg; hybridizing while co-denaturizing the liquid for 8-10min at 80-85Deg, and detecting. This invention can eliminate background interference and improve detection sensitivity.
Description
Technical field
The present invention relates to biology field, relate to the in-situ hybridization method that grows cotton particularly.
Background technology
Utilize the dna probe of labelled with radioisotope to detect after the endonuclear rDNA of Africa xenopus succeeds in the cell film-making from Gall in 1969 and Pardue, Pardue etc. are template with the mouse satellite DNA again, utilize RNA that external synthetic contains 3H for probe has successfully carried out in situ hybridization with the Metaphase Chromosome sample, started the hybridization in situ technique of RNA-DNA.1974, Evans combined chromosome banding technique and hybridization in situ technique for the first time, has improved the accuracy of the assignment of genes gene mapping.1981, the Roumam reported first fluorescein-labeled cDNA do in situ hybridization.In the same year, usefulness biotin labeling Nucleotide such as Langer prepare probe.1986, Cremer and Licher etc. confirmed that respectively fluorescence in situ hybridization (FISH) technology is applied to the feasibility that interphasic nucleus detects chromosome aneuploid.Subsequently, this technology is applied to all respects of Plant Genome Research.
The development of hybridization in situ technique has been passed through the in situ hybridization carried out from the probe that uses labelled with radioisotope to using in situ hybridization---the fluorescence in situ hybridization that nonradioactive labeling's probe carries out, the course of bacterial artificial chromosome-fluorescence in situ hybridization till now (BAC-FISH) again, its accurate positioning and detection sensitivity improve constantly, and range of application more and more widely.Initial in situ hybridization is by position, activity and the expression of specific gene in the radiolabeled probe in detecting cell of tool on karyomit(e).But along with the development of molecular cytogenetics and the raising of detection means, original isotropic substance in situ hybridization shows many shortcomings, all need label probe again as each detection, the probe of mark shows tangible unstable, and the application of radiation autography needs the time shutter (reaching a couple of days even a few weeks longer sometimes) of long period just can detect signal, and signal resolution is low.And the FISH technology has just shown powerful application prospect once appearance.It uses the nucleic acid molecule of different radio-labeling substance markers, as digoxigenin labeled Brdurd triphosphopyridine nucleotide (digoxigenin-dUTP) or biotin labeling Brdurd triphosphopyridine nucleotide (biotin-dUTP) preparation probe, continuous with different fluorescein molecules again, detect different probe molecules by fluorescence excitation, signal is amplified, easy to detect, simple to operate, the susceptibility height, can use a plurality of probes simultaneously and in a section, karyomit(e) be positioned, and to residing period of karyomit(e) and form all can be unrestricted.
At present, monochromatic labeling technique is adopted in the in situ hybridization of cotton usually, causes background interference easily, directly influences sensitivity and accuracy that hybridization signal detects.
Summary of the invention
(1) technical problem that will solve
The purpose of this invention is to provide a kind of effective elimination background interference, improve the cotton in-situ hybridization method of detection sensitivity and accuracy.
(2) technical scheme
Cotton in-situ hybridization method of the present invention, it may further comprise the steps:
(1) film-making: get the cotton tip of a root, pre-treatment, fixing, under 37 ℃, dissociated 1～2.5 hour with 2% cellulase and 0.5% polygalacturonase then, compressing tablet ,-70 ℃ or liquid nitrogen are freezing, take off cover plate, drying;
(2) preparation probe: extract DNA or RNA, purifying is sheared, and electrophoretic separation purpose fragment is carried out multi-color marking with fluorescein, makes probe;
(3) pre-treatment before the hybridization: dry film-making, use RNA (RnaseA) enzyme of 100ug/ml and 0.1%～0.01% stomach en-(Pepsin) to handle successively, fix with 4% Paraformaldehyde 96 again, use 70%, 90%, 100% ethanol dehydration then successively, dry;
(4) preparation hybridization solution:
The hybridization solution cumulative volume is 20ul:
100% deionized formamide 10ul;
50% T 500 4ul;
20 * SSC (standard sodium citrate salt) 2ul;
10%SDS (sodium laurylsulfonate) 1ul;
With different each 1ul of fluorescein-labeled probe;
Supply with the deionized water of sterilization during deficiency 20ul;
With the hybridization solution mixing,, change over to and handle 10～15 minutes in the frozen water in 90～97 ℃ of sex change 8～10 minutes.
(5) hybridization: will hybridize drop in film-making, and add the plastic film cover plate, packing in 80～85 ℃ of co-variations 8～10 minutes, changes over to hybridize in 37 ℃ of water-baths and spends the night.
(6) wash-out, microscopic inspection: use the SSC wash-out, add the antibody of the fluorescein coupling of using with mark then, lining dyes, and with anti-fluorescence decay agent mounting, observes fluorescent signal, gathers and the processing hybrid image.
Cotton in-situ hybridization method of the present invention, wherein, pre-treatment described in the step (1) is with santochlor or a-bromonaphthalene or cycloheximide was at room temperature handled 1.5～2 hours or handled 12～15 hours at 4 ℃, fixedly be to adopt 95% ethanol-glacial acetic acid to handle 2～24 hours, compressing tablet is the acetic acid treatment with 45% or 60%.
Cotton in-situ hybridization method of the present invention, wherein, the DNA of the extraction described in the step (2) or RNA are selected from 51 cotton genomic dnas, the cotton genomic dna fragment, the 45S rRNA of cotton, 26S rRNA, 18S rRNA, 5S rRNA, 5.8S rRNA, the rRNA of soybean, Arabidopis thaliana, paddy rice, any in the endonuclease bamhi of cotton genomic dna, the PCR molecule marker fragment.
Cotton in-situ hybridization method of the present invention, wherein, shearing described in the step (2) is to lash 2000 times or 115 ℃ with disposable syringe to sterilize 5～10 minutes, and described purpose clip size is 300～600bp, and the concentration of described probe is 1～5ng/ul.
Cotton in-situ hybridization method of the present invention, wherein, the fluorescein described in the step (2) is selected from two or more in avidin fluorescent yellow, vitamin H, rhodamine, digoxin, fluorescein isothiocyanate, texas Red, cyanine dyes 3, horseradish peroxidase, the alkaline phosphatase.
Preferably, described fluorescein is selected vitamin H and digoxin for use.
Cotton in-situ hybridization method of the present invention, wherein, drying described in the step (3) is film-making was placed 50～60 ℃ of incubators dry 12 hours or to spend the night, it is 37 ℃ of following incubations 50～60 minutes that the RNA enzyme is handled, pepsin is to handle 30～40 minutes down at 37 ℃, Paraformaldehyde 96 is handled and fix 8～10 minutes under 37 ℃, dewatering time be every grade 2～3 minutes.
Cotton in-situ hybridization method of the present invention, wherein, step (6) is specially: the hybridization film-making is removed cover plate with 2 * SSC rinsing earlier, use 2 * SSC (containing 0.1%SDS) to handle 3 * 5 minutes down again at 37 ℃, use 0.2 * SSC (containing 0.1%SDS) to handle 3 * 5 minutes down then, then with 2 * SSC wash-out 2～3 times under room temperature at 37 ℃.Dry film-making, add 5% bovine serum albumin (BSA) encapsulant, add-on is the 50ul/ sheet, with cover plate, blockades 15～30 minutes under 37 ℃.The drying liquid (film-making can not be seen dried) of blockading adds the antibody of the fluorescein coupling of using with mark, and add-on is the 30ul/ sheet, with cover plate, puts into 37 ℃ of water-baths 1 hour.Use 1 * PBS/Teween20 in 37 ℃ of washings 3 * 5 minutes then.Dry film-making, add 5%BSA, add-on is the 50ul/ sheet, with cover plate, blockades 15～30 minutes under 37 ℃.The drying liquid (film-making can not be seen dried) of blockading adds the antibody that the another kind of fluorescein used with mark is complementary, and add-on is the 30ul/ sheet.With 4,6-diamino-2-phenylindone (DAPI) or iodate third ingot (PI) lining dyed 5～10 minutes.With 1 * PBS washing 3 * 3 minutes, dry back with anti-fluorescence decay agent mounting.Observe fluorescent signal with Leica MRA2 that charge coupled device (CCD) camera is installed or AXiosKoP2 fluorescent microscope (production of Zeiss opticinstrument International Trading Company Ltd) at last, and carry out the collection and the processing of in situ hybridization image with QFISH imaging system software.
(3) beneficial effect
Cotton in-situ hybridization method of the present invention adopts the multi-color marking probe, can effectively eliminate background interference, improves detection sensitivity and accuracy.
Description of drawings
Fig. 1 in situ hybridization figure of probe on cotton of vitamin H and the double-colored mark of digoxin, among the figure: target cell is inferior to cotton, with yellow signal occurring behind the biotin labeled paddy rice 45S rRNA probe hybridization, with danger signal occurring behind the cotton probe hybridization of the Bi Keshi of digoxigenin labeled, the microscope magnification is 912 * 735 * 24;
Fig. 2 in situ hybridization figure of probe on cotton of vitamin H and the double-colored mark of digoxin, among the figure: target cell is No. 7, new sea, green appears behind the 18S rRNA probe hybridization with biotin labeled Arabidopis thaliana, danger signal occurs behind inferior No. 1 probe hybridization of stone system with digoxigenin labeled, the microscope magnification is 912 * 735 * 24.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
The cotton in situ hybridization of embodiment 1 double-colored mark
1. somatic chromosome film-making: soaking seed 12 hours in 58 ℃ warm water than cotton seed (department of agriculture of Agricultural University Of Shanxi provides) in the Asia, is seeded in then in the fine sand that scalding is crossed, and puts under 30 ℃ of conditions and sprout.When treating root length 3～5cm, cut the tip of a root, under room temperature, handled 2 hours with cycloheximide (25ppm).With distillation washing 10 minutes, fix 24 hours then with 95% ethanol-glacial acetic acid (3: 1).After fixedly finishing,, use 2% cellulase (Cellulase R-10, Sigma company produces) and 0.5% polygalacturonase (Pectinase, the production of Sigma company) mixed solution to dissociate 2.5 hours in 37 ℃ again, use 45% acetate compressing tablet at last with distillation washing 10 minutes.Microscopically selects good film-making to take off cover plate with liquid nitrogen, and speed is placed on 70 ℃ the incubator and dries, and deposits standby for-20 ℃.
2. preparation probe: adopt ordinary method to extract the 45S rRNA purifying of paddy rice,, do agarose gel electrophoresis (gum concentration is 0.8%) then, obtain the purpose fragment of 300～600bp, with vitamin H (Roche Holding Ag's production) mark 115 ℃ of sterilizations 5 minutes; Adopt and use the same method, make probe with digoxin (Roche Holding Ag's production) mark Bi Keshi cotton (National Cotton germplasm wild cotton garden provides), concentration and probe concentration is 5ng/ul.
3. pre-treatment before the hybridization: film-making is placed 60 ℃ of incubator dried overnight, 37 ℃ of following incubations 60 minutes, use 2 * SSC to float cover plate then and washed 2 * 5 minutes with the RnaseA (production of Sigma company) of 100ug/ml; Use 0.1% Pepsin (production of Sigma company) to handle 40 minutes down again, use 2 * SSC to float cover plate then and washed 2 * 5 minutes at 37 ℃; Then under 37 ℃, fix 10 minutes, use 2 * SSC washing 2 * 5 minutes then with 4% Paraformaldehyde 96; Last use successively 70%, 90%, 100% ethanol dehydration, dewatering time be every grade 3 minutes, dry.
4. preparation hybridization solution: the hybridization solution cumulative volume is 20ul:
100% deionized formamide 10ul;
50% T 500 4ul;
20×SSC 2ul；
10％SDS 1ul；
Biotin labeled 45S rRNA probe 1ul;
The cotton probe 1ul of the Bi Keshi of digoxigenin labeled;
The deionized water 1ul of sterilization.
With hybridization solution mixing on vortex mixer, then place on the PCR instrument 97 ℃ of sex change 10 minutes, change in the frozen water and handled 10 minutes.
5. 20ul is hybridized drop in film-making, add the plastic film cover plate, packing in 80 ℃ of co-variations 10 minutes, changes over to hybridize in 37 ℃ of water-baths and spends the night.
6. wash-out, microscopic inspection: the hybridization film-making is removed cover plate with 2 * SSC rinsing earlier, use 2 * SSC (containing 0.1%SDS) to handle 3 * 5 minutes down again, use 0.2 * SSC (containing 0.1%SDS) to handle 3 * 5 minutes down then, then with 2 * SSC wash-out 3 times under room temperature at 37 ℃ at 37 ℃.Dry film-making, add 5% BSA, add-on is the 50ul/ sheet, with cover plate, blockades 15 minutes under 37 ℃.The drying liquid (film-making can not be seen dried) of blockading adds Aindin-Fluo antibody (Roche Holding Ag's production), and add-on is the 30ul/ sheet, with cover plate, puts into 37 ℃ of water-baths 1 hour.Use 1 * PBS/Teween20 in 37 ℃ of washings 3 * 5 minutes then.Dry film-making, add 5%BSA, add-on is the 50ul/ sheet, with cover plate, blockades 30 minutes under 37 ℃.The drying liquid (film-making can not be seen dried) of blockading adds Anti-D antibody (Roche Holding Ag's production), and add-on is the 30ul/ sheet.Dyed 10 minutes with the DAPI lining.With 1 * PBS washing 3 * 3 minutes, dry back with anti-fluorescence decay agent mounting.Observe fluorescent signal with the AXiosKoP2 fluorescent microscope (production of Zeiss opticinstrument International Trading Company Ltd) that the CCD camera is installed at last, and carry out the collection and the processing of in situ hybridization image with QFISH imaging system software.
The result adopts hybridizing method of the present invention can effectively eliminate background interference, detection sensitivity and accuracy height as shown in Figure 1.
The cotton in situ hybridization of embodiment 2 double-colored marks
1. somatic chromosome film-making: the new extra large seventh-seeded of upland cotton (National Cotton germplasm wild cotton garden provides) was soaked seed 12 hours in 58 ℃ warm water, was seeded in then in the fine sand that scalding is crossed, and put under 30 ℃ of conditions and sprouted.When treating root length 3～5cm, cut the tip of a root, under room temperature, handled 2 hours with cycloheximide (25ppm).With distillation washing 10 minutes, fix 2 hours then with 95% ethanol-glacial acetic acid (3: 1).After fixedly finishing,, use 2% cellulase (Cellulase R-10, Sigma company produces) and 0.5% polygalacturonase (Pectinase, the production of Sigma company) mixed solution to dissociate 1 hour in 37 ℃ again, use 60% acetate compressing tablet at last with distillation washing 10 minutes.Microscopically selects good film-making to take off cover plate with liquid nitrogen, and speed is placed on 80 ℃ the incubator and dries, and deposits standby for-20 ℃.
2. preparation probe: adopt ordinary method to extract the 18S rRNA purifying of Arabidopis thaliana,, do agarose gel electrophoresis (gum concentration is 0.8%) then, obtain the purpose fragment of 300～600bp, with vitamin H (Roche Holding Ag's production) mark 115 ℃ of sterilizations 10 minutes; Adopt and use the same method, make probe with cotton rock system inferior No. 1 (National Cotton germplasm wild cotton garden provides) in digoxin (Roche Holding Ag's production) mark, concentration and probe concentration is 5ng/ul.
3. pre-treatment before the hybridization: film-making was placed 60 ℃ of incubators dry 12 hours, 37 ℃ of following incubations 60 minutes, use 2 * SSC to float cover plate then and washed 2 * 5 minutes with the RNA enzyme (production of Sigma company) of 100ug/ml; Use 0.1% Pepsin (production of Sigma company) to handle 30 minutes down again, use 2 * SSC to float cover plate then and washed 2 * 5 minutes at 37 ℃; Then under 37 ℃, fix 8 minutes, use 2 * SSC washing 2 * 5 minutes then with 4% Paraformaldehyde 96; Last use successively 70%, 90%, 100% ethanol dehydration, dewatering time be every grade 2 minutes, dry.
4. preparation hybridization solution: the hybridization solution cumulative volume is 20ul:
100% deionized formamide 10ul;
50% T 500 4ul;
20×SSC 2ul；
10％SDS 1ul；
Biotin labeled 18S rRNA probe 1ul;
Inferior No. 1 probe 1ul of stone system of digoxigenin labeled;
The deionized water 1ul of sterilization.
With hybridization solution mixing on vortex mixer, then place on the PCR instrument 90 ℃ of sex change 8 minutes, change in the frozen water and handled 15 minutes.
5. 20ul is hybridized drop in film-making, add the plastic film cover plate, packing in 85 ℃ of co-variations 8 minutes, changes over to hybridize in 37 ℃ of water-baths and spends the night.
6. wash-out, microscopic inspection: the hybridization film-making is removed cover plate with 2 * SSC rinsing earlier, use 2 * SSC (containing 0.1%SDS) to handle 3 * 5 minutes down again, use 0.2 * SSC (containing 0.1%SDS) to handle 3 * 5 minutes down then, then with 2 * SSC wash-out 2 times under room temperature at 37 ℃ at 37 ℃.Dry film-making, add 5% BSA, add-on is the 50ul/ sheet, with cover plate, blockades 30 minutes under 37 ℃.The drying liquid (film-making can not be seen dried) of blockading adds Aindin-Fluo antibody (Roche Holding Ag's production), and add-on is the 30ul/ sheet, with cover plate, puts into 37 ℃ of water-baths 1 hour.Use 1 * PBS/Teween20 in 37 ℃ of washings 3 * 5 minutes then.Dry film-making, add 5%BSA, add-on is the 50ul/ sheet, with cover plate, blockades 30 minutes under 37 ℃.The drying liquid (film-making can not be seen dried) of blockading adds Anti-D antibody (Roche Holding Ag's production), and add-on is the 30ul/ sheet.Dyed 10 minutes with the PI lining.With 1 * PBS washing 3 * 3 minutes, dry back with anti-fluorescence decay agent mounting.Observe fluorescent signal with the AXiosKoP2 fluorescent microscope (production of Zeiss opticinstrument International Trading Company Ltd) that the CCD camera is installed at last, and carry out the collection and the processing of in situ hybridization image with QFISH imaging system software.
Adopt hybridizing method of the present invention can effectively eliminate background interference, detection sensitivity and accuracy height.
Claims (9)
1, the in-situ hybridization method that grows cotton, it may further comprise the steps:
(1) film-making: get the cotton tip of a root, pre-treatment, fixing, under 37 ℃, dissociated 1～2.5 hour with 2% cellulase and 0.5% polygalacturonase then, compressing tablet ,-70 ℃ or liquid nitrogen are freezing, drying;
(2) preparation probe: extract DNA or RNA, purifying is sheared, and electrophoretic separation purpose fragment is carried out multi-color marking with fluorescein, makes probe;
(3) pre-treatment before the hybridization: dry film-making, use the RNA enzyme of 100ug/ml and 0.1%～0.01% pepsin successively, fix with 4% Paraformaldehyde 96 again, use 70%, 90%, 100% ethanol dehydration then successively, dry;
(4) preparation hybridization solution:
The hybridization solution cumulative volume is 20ul:
100% deionized formamide 10ul;
50% T 500 4ul;
20 * standard sodium citrate salt 2ul;
10% sodium laurylsulfonate 1ul;
With different each 1ul of fluorescein-labeled probe;
Supply with the deionized water of sterilization during deficiency 20ul;
With the hybridization solution mixing,, change over to and handle 10～15 minutes in the frozen water in 90～97 ℃ of sex change 8～10 minutes.
(5) hybridization: will hybridize drop in film-making, and add the plastic film cover plate, packing in 80～85 ℃ of co-variations 8～10 minutes, changes over to hybridize in 37 ℃ of water-baths and spends the night.
(6) detect: with standard Trisodium Citrate eluting salt, the antibody lining that adds the fluorescein coupling of using with mark then dyes, and with anti-fluorescence decay agent mounting, observes fluorescent signal, gathers and the processing hybrid image.
2, method according to claim 1, it is characterized in that the pre-treatment described in the step (1) is with santochlor or a-bromonaphthalene or cycloheximide was at room temperature handled 1.5～2 hours or handled 12～15 hours at 4 ℃, fixedly be to adopt 95% ethanol-glacial acetic acid to handle 2～24 hours, compressing tablet is the acetic acid treatment with 45% or 60%.
3, method according to claim 1, the DNA or the RNA that it is characterized in that the extraction described in the step (2) are selected from 51 cotton genomic dnas, the cotton genomic dna fragment, the 45S rRNA of cotton, 26S rRNA, 18S rRNA, 5S rRNA, 5.8S rRNA, the rRNA of soybean, Arabidopis thaliana, paddy rice, any in the endonuclease bamhi of cotton genomic dna, the PCR molecule marker fragment.
4, method according to claim 1 is characterized in that the shearing described in the step (2) is to lash 2000 times or 115 ℃ with disposable syringe to sterilize 5～10 minutes.
5, method according to claim 1 is characterized in that the purpose clip size described in the step (2) is 300～600bp.
6, method according to claim 1 is characterized in that the fluorescein described in the step (2) is selected from two or more in avidin fluorescent yellow, vitamin H, rhodamine, digoxin, fluorescein isothiocyanate, texas Red, cyanine dyes 3, horseradish peroxidase, the alkaline phosphatase.
7, method according to claim 6 is characterized in that the fluorescein described in the step (2) selects vitamin H and digoxin for use.
8, method according to claim 1, the concentration that it is characterized in that the probe described in the step (2) is 1～5ng/ul.
9, method according to claim 1, it is characterized in that the drying described in the step (3) is film-making was placed 50～60 ℃ of incubators dry 12 hours or to spend the night, it is 37 ℃ of following incubations 50～60 minutes that the RNA enzyme is handled, pepsin is to handle 30～40 minutes down at 37 ℃, Paraformaldehyde 96 is handled and fix 8～10 minutes under 37 ℃, the ethanol dehydration time be every grade 2～3 minutes.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559909A (en) * | 2012-02-15 | 2012-07-11 | 四川农业大学 | Fluorescence in-situ hybridization method for Rubus metaphase chromosomes |
| CN102692497A (en) * | 2012-06-11 | 2012-09-26 | 云南农业大学 | Method for observing cell microtubule framework through soybean root tip treatment by immunofluorescence staining |
| CN104099416A (en) * | 2014-07-08 | 2014-10-15 | 河南省农业科学院芝麻研究中心 | FISH (fluorescence in situ hybridization) method of sesame chromosome |
| CN109211644A (en) * | 2018-10-26 | 2019-01-15 | 四川农业大学 | A kind of method of conyza blinii chromosome sectioning |
-
2006
- 2006-01-26 CN CN 200610011287 patent/CN1840704A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559909A (en) * | 2012-02-15 | 2012-07-11 | 四川农业大学 | Fluorescence in-situ hybridization method for Rubus metaphase chromosomes |
| CN102692497A (en) * | 2012-06-11 | 2012-09-26 | 云南农业大学 | Method for observing cell microtubule framework through soybean root tip treatment by immunofluorescence staining |
| CN104099416A (en) * | 2014-07-08 | 2014-10-15 | 河南省农业科学院芝麻研究中心 | FISH (fluorescence in situ hybridization) method of sesame chromosome |
| CN109211644A (en) * | 2018-10-26 | 2019-01-15 | 四川农业大学 | A kind of method of conyza blinii chromosome sectioning |
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