CN107557491A - The fluorescence in-situ hybridization method of zanthoxylum armatum metaphase chromosome - Google Patents
The fluorescence in-situ hybridization method of zanthoxylum armatum metaphase chromosome Download PDFInfo
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Abstract
The invention provides the fluorescence in-situ hybridization method of zanthoxylum armatum metaphase chromosome.Preparation, FISH, signal detection and slide recycling step including zanthoxylum armatum metaphase chromosome slide sample.The present invention helps to establish stable easily metaphase chromosome FISH reaction system, substantially increases the detector efficiency of zanthoxylum armatum metaphase chromosome, and the detection for zanthoxylum armatum chromosome or even Zanthoxylum species chromosome provides new method.
Description
Technical field
The invention belongs to cytogenetics and biology field, and in particular to the fluorescence of zanthoxylum armatum metaphase chromosome
In-situ hybridization method.
Background technology
The method of existing detection plant chromosome generally divides band using plant chromosome, is divided into two major classes, i.e. fluorescence divides band
Divide band with Giemsa.But two methods did not use in xanthoxylum.Main cause is：First, xanthoxylum
Germination is not easy, and time length, seed hull is harder, is not easy water swelling, difficult using the germination clip tip of a root.Second, root
Point tissue is rich in grease, has a strong impact on chromosome observation under microscope, is unfavorable for finding chromosome.3rd, xanthoxylum dye
Colour solid is more, small, is not easy to disperse, and is unfavorable for finding a complete split coil method of cell metaphase chromosome number.Although Zanthoxylum two
Individual species more than hundred, but clear and definite chromosome number purpose species only have 9 species at present：Z. dimorphyllum 2n=36,68, zanthoxylum acanthopodium 2n
=64, zanthoxylum armatum 2n=66, Radix zanthoxyli 2n=68, Chinese prickly ash strangle 2n=68, felt hair Chinese prickly ash 2n=72, sharp leaf Chinese prickly ash 2n=72,
Zanthoxylum simulans 2n=ca.132, Chinese prickly ash 2n=136.The chromosome number of these species, some are slightly identical, and some are close in 2 times
With 4 times of relation.Chinese prickly ash species are at present also in the determination chromosome number purpose stage.Xanthoxylum ploidy is not also bright
Really.According to the Zanthoxylum species chromosome number reported for work some close to 2 times and 4 times of relations, it appears that Chinese prickly ash species exist again
Polyploid, for example, the species that chromosome number is 36 are probably diploid, the species of chromosome number 64,68,72 are probably
Tetraploid, the species of chromosome number 132,136 are probably octoploid.But xanthoxylum is usually single-female generation, institute
To be typically not easy to occur polyploid automatically in nature.And the vegetative manner such as artificially breeding use grafting wide in variety,
Species ploidy will not be increased.Therefore speculate that Chinese prickly ash species should not be polyploid, but diploid.Zanthoxylum more several species contaminate
The determination of colour solid number and species ploidy, the clearly species basic background knowledge is beneficial to, beneficial to seed selection improved seeds.
Fluorescence in situ hybridization technique (fluorescence in situ hybridization, FISH) is in 20th century 80
A kind of on-radiation molecular cytogenetics technology that age end grows up in radioactive in situ hybridization technical foundation, with fluorescence
A kind of new in-situ hybridization method that mark substitutes isotope marks and formed, the technology use fluorescein label probe, detection
The chromosome of metaphase or intermitotic chromatinic hybridization.In general, FISH has safe, easy to operate fast
The advantages that prompt, stability and strong repeatability.At present, this method has been widely used for detection GMOs and positioning, heterologous dyeing
The many aspects such as body identification, genome structure parsing.But enzymolysis tip of a root film-making process, the dye of FISH technology in operation
The factors such as colour solid degenerative process, probe specificity, probes label methods and efficiency be all FISH can hybridize successfully it is crucial because
Element.FISH technology is used successfully on many plants, and yet there are no includes zanthoxylum armatum in any Chinese prickly ash species
In have application report.The present invention provides a kind of fluorescence in-situ hybridization method of zanthoxylum armatum chromosome, establishes stabilization and easily contaminates
Colour solid FISH reaction system, the problems of existing zanthoxylum armatum chromosome detection are overcome, are contaminated for zanthoxylum armatum
The detection of colour solid or even Zanthoxylum species chromosome provides new method.
The content of the invention
The problem of existing for prior art, the present invention provide a kind of FISH of zanthoxylum armatum metaphase chromosome
Method.The technical scheme is that：
A kind of fluorescence in-situ hybridization method of zanthoxylum armatum metaphase chromosome, comprises the following steps：
1) preparation of zanthoxylum armatum metaphase chromosome slide sample：Zanthoxylum armatum seedling is used into Nutrition Soil culture, treated
When tip of a root length is to 1.5~2.0cm, clip 1~1.5cm organizations of root tips, N is used successively2O processing 3h, glacial acetic acid processing 5min, then
It is placed in 75% alcohol in -20 DEG C of preservations；
1~2mm of Root apical meristem is cut after the organization of root tips of Cord blood is cleaned with distilled water, is placed in enzymolysis liquid
55min is digested in 37 DEG C, removes enzymolysis liquid, successively with distilled water, 75% alcohol, 95% alcohol, the 100% alcohol washes tip of a root point
Room temperature is dried after raw tissue；Glacial acetic acid is added in organization of root tips suspension is made；Hanging drop is dried in the air in room temperature on slide
Microscopy after dry, the good slide of microscopy is in -20 DEG C of preservations；
2) FISH：Slide is placed in 4% paraformaldehyde and handles 10min, then with 2 × SSC buffer solutions
Handle twice, each 5min, then handled twice with distilled water, each 5min, then successively with 75% alcohol, 95% alcohol, 100%
The processing of alcohol gradient, each 5min, room temperature are added dropwise 70%FA liquid, covered, 2min are denatured in 80 DEG C after drying；
Slide denaturation is completed after 75% alcohol, 95% alcohol, 100% alcohol gradient that -20 DEG C are sequentially placed into 5s
Processing, each 5min, room temperature are dried；The hybridization solution for being placed with probe, covered, in dark are added dropwise on the slide dried
Under the conditions of in hybridizing box in 37 DEG C of 1.5~2h of Constant temperature hatch；The probe is using 5S rDNA and (GAA)6, wherein 5S rDNA
The base sequence of probe is：5 '-TCAGAACTCCGAAGTTAAGCGTGCTTGGGCGAGAGTAGTAC-3 ', marked using FAM
5 ' ends of its sequence；(GAA)6The base sequence of probe is：5 '-GAAGAAGAAGAAGAAGAA-3 ', it is marked using TAMRM
5 ' ends of sequence；
The slide completed will be hatched under the conditions of lucifuge successively with 2 × SSC buffer solution for cleaning 3min, distilled water processing two
Secondary, each 5min, room temperature is dried；
3) signal detection：DAPI, covered, using fluorescence microscope to slide are added on slide after drying
Detected, acquisition testing image；
4) slide reclaims：Film-making is reclaimed afterwards and is put into 2 × SSC buffer solutions in 60 DEG C of water-bath 5min, is existed successively
Gradient is handled in 75% alcohol, 95% alcohol, 100% alcohol, each 5min, and film-making will be reclaimed after the completion of processing under fluorescent lamp
Place 12h；- 20 DEG C of preservations.
Further, the compound method of the enzymolysis liquid is：0.04g celluloses are added in citrate buffer solution per 1mL
Enzyme and 0.02g pectases；The compound method of wherein citrate buffer solution is：DdH per 50mL2O adds 0.5707g trisodium citrates
With 0.4324g citric acids.
Further, the compound method of the 4% paraformaldehyde liquid is：It is more that 1 × PBS per 100mL adds 4g
Polyformaldehyde, 2~3h of water-bath in 60 DEG C of water-baths, wherein the compound method of the 1 × PBS is：Per 1000mL
ddH2O adds 8g sodium chloride, 0.2g potassium chloride, 1.42g disodium hydrogen phosphates and 0.27g potassium dihydrogen phosphates.
Further, the compound method of 2 × SSC buffer solutions is：DdH per 1000mL2O adds 8.823g citric acids
Trisodium and 17.532g sodium chloride.
Further, the 70%FA liquid by deionized formamide (FA) and 2 × SSC buffer solutions according to volume ratio 7:3 groups
Into.
Further, the hybridization buffer is made up of 1 isometric × TE and 2 × SSC buffer solutions；Wherein 1 × TE's
Compound method is：DdH per 800mL21.211g Tris and 0.372g EDTANa are added in O2·2H2O, pH is adjusted to arrive with HCl
8.0, constant volume to 1000mL, produced after sterilizing.
The beneficial effects of the present invention are：The present invention is based on chromosome fluorescence in-situ hybridization technology, there is provided a kind of leaf of bamboo
The fluorescence in-situ hybridization method of Chinese prickly ash metaphase chromosome, this method help to establish stable easily metaphase chromosome fluorescent in situ
Hybridization reaction system, this method is reproducible, can Reusability film-making, and probe prepare easily, quick, high sensitivity, need
Ask DNA probe amount few, good and cheap, tip of a root film-making is easy, is easily obtained zanthoxylum armatum metaphase chromosome, relative to lucifuge requirement
Relatively low, the time of whole experiment flow shortens, easy to operate.The most important, the present invention are substantially increased in zanthoxylum armatum
The detector efficiency of phase chromosome, the detection for zanthoxylum armatum chromosome or even Zanthoxylum species chromosome provide new method.
Brief description of the drawings
Fig. 1 is that the probe of the embodiment of the present invention 1 marks 5S rDNA and (GAA)6Signal position on zanthoxylum armatum chromosome
Point distributed image, wherein figure a is (GAA)6Signal, figure b is 5S rDNA signals, and figure c is a and b composite diagrams.
Embodiment
Probe groups are synthesized by Beijing Sheng Gong companies used by the present invention is implemented, and the probe of synthesis is diluted to 1 × TE
100M concentration is stored in -20 DEG C, and probe face concentration needs to be diluted to 10M again, also in -20 DEG C of preservations.
The present invention is described in further details with specific embodiment below in conjunction with the accompanying drawings, described is the solution to the present invention
Release rather than limit.
The specific embodiment of the invention provides a kind of fluorescence in-situ hybridization method of zanthoxylum armatum metaphase chromosome, including following
Step：
1) preparation of zanthoxylum armatum metaphase chromosome slide sample：Annual zanthoxylum armatum from field production is planted
Strain, balled transplanting to the basin alms bowl for filling Nutrition Soil, incubated at room temperature two months, when tip of a root length is to 1.5~2.0cm, in the morning 10:
30 and afternoon 4:30 clip 1~1.5cm organizations of root tips, use N successively2O processing 3h, glacial acetic acid processing 5min, are subsequently placed in 75%
In -20 DEG C of preservations in alcohol；
1~2mm of Root apical meristem is cut after the organization of root tips of Cord blood is cleaned with distilled water, is placed in enzymolysis liquid
55min is digested in 37 DEG C, removes enzymolysis liquid, successively with distilled water, 75% alcohol, 95% alcohol, the 100% alcohol washes tip of a root point
Room temperature is dried after raw tissue；Glacial acetic acid is added in organization of root tips suspension is made；Hanging drop is dried in the air in room temperature on slide
Microscopy after dry, the good slide of microscopy is in -20 DEG C of preservations；The compound method of the enzymolysis liquid is：Citrate buffer solution per 1mL
Middle addition 0.04g cellulases and 0.02g pectases；The compound method of wherein citrate buffer solution is：DdH per 50mL2O adds
0.5707g trisodium citrates and 0.4324g citric acids；
2) FISH：Slide is placed in 4% paraformaldehyde and handles 10min, then with 2 × SSC buffer solutions
Handle twice, each 5min, then handled twice with distilled water, each 5min, then successively with 75% alcohol, 95% alcohol, 100%
The processing of alcohol gradient, each 5min, room temperature are added dropwise 70%FA liquid, covered, 2min are denatured in 80 DEG C after drying；It is described
The compound method of 4% paraformaldehyde liquid is：1 × PBS per 100mL adds 4g paraformaldehydes, in 60 DEG C of water-baths
2~3h of water-bath, wherein the compound method of the 1 × PBS is：DdH per 1000mL2O adds 8g sodium chloride, 0.2g chlorine
Change potassium, 1.42g disodium hydrogen phosphates and 0.27g potassium dihydrogen phosphates；The compound method of 2 × SSC buffer solutions is：Per 1000mL
ddH2O adds 8.823g trisodium citrates and 17.532g sodium chloride；The 70%FA liquid by deionized formamide (FA) and 2 ×
SSC buffer solutions are according to volume ratio 7:3 compositions；
Slide denaturation is completed after 75% alcohol, 95% alcohol, 100% alcohol gradient that -20 DEG C are sequentially placed into 5s
Processing, each 5min, room temperature are dried；The hybridization solution for being placed with probe is added dropwise on the slide dried, 10 μ are added dropwise in every slide
L is placed with the hybridization solution of probe, wherein every kind of probe contains 0.5 μ L, remaining as hybridization solution；Covered, under dark condition
In 37 DEG C of 1.5~2h of Constant temperature hatch in hybridizing box；The probe is using 5S rDNA and (GAA)6, wherein 5S rDNA probes
Base sequence is：5 '-TCAGAACTCCGAAGTTAAGCGTGCTTGGGCGAGAGTAGTAC-3 ', its sequence is marked using FAM
5 ' end；(GAA)6The base sequence of probe is：5 '-GAAGAAGAAGAAGAAGAA-3 ', its sequence is marked using TAMRM
5 ' ends；The hybridization buffer is made up of 1 isometric × TE and 2 × SSC buffer solutions；Wherein 1 × TE compound method is：Often
800mL ddH21.211g Tris and 0.372g EDTANa are added in O2·2H2O, pH to 8.0 is adjusted with HCl, constant volume arrives
1000mL, produced after sterilizing；
The slide completed will be hatched under the conditions of lucifuge successively with 2 × SSC buffer solution for cleaning 3min, distilled water processing two
Secondary, each 5min, room temperature is dried；
3) signal detection：DAPI, covered, using fluorescence microscope to slide are added on slide after drying
Detected, acquisition testing image；Can be with the hybridization signal of clear view to zanthoxylum armatum, such as Fig. 1 institutes by fluorescence microscope
Show, probe signals as indicated with an arrow, are respectively positioned on chromosome intermediate region, do not appear in end of chromosome.Chromosome uses
DAPI is dyed, and display blueness, scale size is 5 μm；
4) slide reclaims：Film-making is reclaimed afterwards and is put into 2 × SSC buffer solutions in 60 DEG C of water-bath 5min, is existed successively
Gradient is handled in 75% alcohol, 95% alcohol, 100% alcohol, each 5min, and film-making will be reclaimed after the completion of processing under fluorescent lamp
Place 12h；- 20 DEG C of preservations.
Although embodiments of the invention have been shown and described above, it is to be understood that above-described embodiment is example
Property, it is impossible to limitation of the present invention is interpreted as, one of ordinary skill in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, changed, replacing and modification.
Sequence table
<110>Sichuan Agricultural University
<120>The fluorescence in-situ hybridization method of zanthoxylum armatum metaphase chromosome
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 41
<212> DNA
<213>Artificial sequence ()
<400> 1
tcagaactcc gaagttaagc gtgcttgggc gagagtagta c 41
<210> 2
<211> 18
<212> DNA
<213>Artificial sequence ()
<400> 2
gaagaagaag aagaagaa 18
Claims (6)
1. the fluorescence in-situ hybridization method of zanthoxylum armatum metaphase chromosome, it is characterised in that comprise the following steps：
1) preparation of zanthoxylum armatum metaphase chromosome slide sample：Zanthoxylum armatum seedling is used into Nutrition Soil culture, treats the tip of a root
When length is to 1.5~2.0cm, clip 1~1.5cm organizations of root tips, N is used successively2O processing 3h, glacial acetic acid processing 5min, are subsequently placed in
In -20 DEG C of preservations in 75% alcohol；
1~2mm of Root apical meristem is cut after the organization of root tips of Cord blood is cleaned with distilled water, is placed in enzymolysis liquid in 37
DEG C enzymolysis 55min, remove enzymolysis liquid, successively with distilled water, 75% alcohol, 95% alcohol, mitogenetic group of the 100% alcohol washes tip of a root
Rear room temperature is knitted to dry；Glacial acetic acid is added in organization of root tips suspension is made；By hanging drop after room temperature is dried on slide
Microscopy, the good slide of microscopy is in -20 DEG C of preservations；
2) FISH：Slide is placed in 4% paraformaldehyde and handles 10min, is then handled with 2 × SSC buffer solutions
Twice, each 5min, then handled twice with distilled water, each 5min, then successively with 75% alcohol, 95% alcohol, 100% alcohol
Gradient processing, each 5min, room temperature are added dropwise 70%FA liquid, covered, 2min are denatured in 80 DEG C after drying；
Slide denaturation is completed to handle after 75% alcohol, 95% alcohol, 100% alcohol gradient that are sequentially placed into -20 DEG C in 5s,
Each 5min, room temperature are dried；The hybridization solution for being placed with probe, covered, in dark condition are added dropwise on the slide dried
Under in hybridizing box in 37 DEG C of 1.5~2h of Constant temperature hatch；The probe is using 5S rDNA and (GAA)6, wherein 5S rDNA probes
Base sequence be：5 '-TCAGAACTCCGAAGTTAAGCGTGCTTGGGCGAGAGTAGTAC-3 ', its sequence is marked using FAM
5 ' ends of row；(GAA)6The base sequence of probe is：5 '-GAAGAAGAAGAAGAAGAA-3 ', its sequence is marked using TAMRM
5 ' end；
The slide completed will be hatched to be handled twice with 2 × SSC buffer solution for cleaning 3min, distilled water successively under the conditions of lucifuge,
Each 5min, room temperature are dried；
3) signal detection：DAPI is added on slide after drying, covered, slide is carried out using fluorescence microscope
Detection, acquisition testing image；
4) slide reclaims：Film-making is reclaimed afterwards and is put into 2 × SSC buffer solutions in 60 DEG C of water-bath 5min, successively in 75% wine
Gradient is handled in essence, 95% alcohol, 100% alcohol, each 5min, places recovery film-making under fluorescent lamp after the completion of processing
12h；- 20 DEG C of preservations.
2. the fluorescence in-situ hybridization method of zanthoxylum armatum metaphase chromosome according to claim 1, it is characterised in that described
The compound method of enzymolysis liquid is：0.04g cellulases and 0.02g pectases are added in citrate buffer solution per 1mL；Wherein lemon
The compound method of lemon acid buffer is：DdH per 50mL2O adds 0.5707g trisodium citrates and 0.4324g citric acids.
3. the fluorescence in-situ hybridization method of zanthoxylum armatum metaphase chromosome according to claim 1, it is characterised in that described
The compound method of 4% paraformaldehyde liquid is：1 × PBS per 100mL adds 4g paraformaldehydes, in 60 DEG C of water-baths
2~3h of water-bath, wherein the compound method of the 1 × PBS is：DdH per 1000mL2O adds 8g sodium chloride, 0.2g chlorine
Change potassium, 1.42g disodium hydrogen phosphates and 0.27g potassium dihydrogen phosphates.
4. the fluorescence in-situ hybridization method of zanthoxylum armatum metaphase chromosome according to claim 1, it is characterised in that described
The compound method of 2 × SSC buffer solutions is：DdH per 1000mL2O adds 8.823g trisodium citrates and 17.532g sodium chloride.
5. the fluorescence in-situ hybridization method of zanthoxylum armatum metaphase chromosome according to claim 1, it is characterised in that described
70%FA liquid is by deionized formamide (FA) and 2 × SSC buffer solutions according to volume ratio 7:3 compositions.
6. the fluorescence in-situ hybridization method of zanthoxylum armatum metaphase chromosome according to claim 1, it is characterised in that described
Hybridization buffer is made up of 1 isometric × TE and 2 × SSC buffer solutions；Wherein 1 × TE compound method is：Per 800mL
ddH21.211g Tris and 0.372g EDTANa are added in O2·2H2O, pH to 8.0, constant volume to 1000mL, sterilizing are adjusted with HCl
After produce.
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| CN201710985222.7A CN107557491B (en) | 2017-10-20 | 2017-10-20 | Fluorescence in situ hybridization method for zanthoxylum bungeanum intermediate chromosome |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108441539A (en) * | 2018-04-11 | 2018-08-24 | 四川农业大学 | A method of using few sequence detection forest chromosome end |
| CN109825553A (en) * | 2019-03-06 | 2019-05-31 | 南京林业大学 | A method of poplar whole chromosome is identified using oligonucleotide probe |
| CN114540534A (en) * | 2021-03-17 | 2022-05-27 | 浙江农林大学 | Mao bamboo oligonucleotide probe |
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| CN103409523A (en) * | 2013-08-12 | 2013-11-27 | 南开大学 | Fluorescence in situ hybridization method of 5S rDNA on plant chromosome |
| CN105039542A (en) * | 2015-07-17 | 2015-11-11 | 南京农业大学 | Novel method for painting chromosomes by adopting oligonucleotide probe dye liquor |
| CN106399499A (en) * | 2016-09-18 | 2017-02-15 | 新乡医学院 | Fluorescence in-situ hybridization method for asparagus fern medium-term chromosomes |
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2017
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| CN103409523A (en) * | 2013-08-12 | 2013-11-27 | 南开大学 | Fluorescence in situ hybridization method of 5S rDNA on plant chromosome |
| CN105039542A (en) * | 2015-07-17 | 2015-11-11 | 南京农业大学 | Novel method for painting chromosomes by adopting oligonucleotide probe dye liquor |
| CN106399499A (en) * | 2016-09-18 | 2017-02-15 | 新乡医学院 | Fluorescence in-situ hybridization method for asparagus fern medium-term chromosomes |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108441539A (en) * | 2018-04-11 | 2018-08-24 | 四川农业大学 | A method of using few sequence detection forest chromosome end |
| CN108441539B (en) * | 2018-04-11 | 2022-01-28 | 四川农业大学 | Method for detecting end of forest chromosome by using oligonucleotide sequence |
| CN109825553A (en) * | 2019-03-06 | 2019-05-31 | 南京林业大学 | A method of poplar whole chromosome is identified using oligonucleotide probe |
| CN114540534A (en) * | 2021-03-17 | 2022-05-27 | 浙江农林大学 | Mao bamboo oligonucleotide probe |
| CN114540534B (en) * | 2021-03-17 | 2023-08-25 | 浙江农林大学 | Moso bamboo oligonucleotide probe |
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