CN114062087A - Method for preparing chromosome of melastoma plant based on FISH hybridization - Google Patents
Method for preparing chromosome of melastoma plant based on FISH hybridization Download PDFInfo
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- CN114062087A CN114062087A CN202111309818.8A CN202111309818A CN114062087A CN 114062087 A CN114062087 A CN 114062087A CN 202111309818 A CN202111309818 A CN 202111309818A CN 114062087 A CN114062087 A CN 114062087A
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- 210000000349 chromosome Anatomy 0.000 title claims abstract description 67
- 238000000034 method Methods 0.000 title claims abstract description 32
- 238000009396 hybridization Methods 0.000 title claims abstract description 23
- 240000005338 Melastoma malabathricum Species 0.000 title 1
- 241000266322 Melastoma Species 0.000 claims abstract description 51
- 238000010494 dissociation reaction Methods 0.000 claims abstract description 18
- 230000005593 dissociations Effects 0.000 claims abstract description 18
- 238000005070 sampling Methods 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 23
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 10
- 229960000583 acetic acid Drugs 0.000 claims description 10
- 229960001701 chloroform Drugs 0.000 claims description 10
- 239000012362 glacial acetic acid Substances 0.000 claims description 10
- 239000000725 suspension Substances 0.000 claims description 10
- 229960004756 ethanol Drugs 0.000 claims description 7
- 108010059892 Cellulase Proteins 0.000 claims description 5
- 108010059820 Polygalacturonase Proteins 0.000 claims description 5
- 238000010009 beating Methods 0.000 claims description 5
- 229940106157 cellulase Drugs 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 5
- 238000001035 drying Methods 0.000 claims description 5
- 108010093305 exopolygalacturonase Proteins 0.000 claims description 5
- 239000011521 glass Substances 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 238000005406 washing Methods 0.000 claims description 5
- 210000004027 cell Anatomy 0.000 abstract description 6
- 238000004458 analytical method Methods 0.000 abstract description 3
- 210000002421 cell wall Anatomy 0.000 abstract description 3
- 230000002559 cytogenic effect Effects 0.000 abstract description 3
- 239000006185 dispersion Substances 0.000 abstract description 3
- 230000000394 mitotic effect Effects 0.000 abstract description 3
- 210000000745 plant chromosome Anatomy 0.000 abstract description 3
- 231100000331 toxic Toxicity 0.000 abstract description 2
- 230000002588 toxic effect Effects 0.000 abstract description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
- G01N1/31—Apparatus therefor
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- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a method for preparing a chromosome of a melastoma plant based on FISH hybridization, which relates to the technical field of cytogenetics and specifically comprises the following steps: (1) sampling; (2) fixing; (3) hypotonic; (4) carrying out enzymolysis; (5) and (5) tabletting. The method for preparing the chromosome of the melastoma plant has the advantages of multiple splitting phases, thorough cell wall dissociation, better chromosome dispersion and clean and clear background, is convenient for karyotype analysis research of the melastoma plant chromosome and subsequent FISH hybridization research of the melastoma plant, lays a foundation for development and utilization of germplasm resources of the melastoma plant and research of cytology and molecular cytology, and the fixing solution can stop mitotic cells in the early and middle stages, does not need pretreatment, reduces the toxic action of the pretreatment solution on the cells, combines the pretreatment and the fixation together, and effectively improves the tabletting efficiency.
Description
Technical Field
The invention relates to the technical field of cytogenetics, in particular to a method for preparing a chromosome of a melastoma plant based on FISH hybridization.
Background
The research on chromosomes is the basic research in the genetic field and is a base stone and a catalyst in multiple research fields. The melastoma plant has high ornamental value, high medicinal value and wide development prospect. The molecular cytogenetic mechanism of the melastoma plant is limited by the fact that the melastoma plant has the phenomena of small chromosome, easy overlapping and the like.
The conventional flaking method in the market at present needs to pretreat the chromosome sample of the melastoma plants firstly and then fix the chromosome sample, is complex in operation and low in flaking efficiency, and the phenomena of incomplete cell separation, cell membrane rupture and chromosome overflow are easy to occur in the melastoma plant chromosome flaking obtained by the conventional flaking method, so that the subsequent FISH hybridization research cannot be carried out. Therefore, the skilled person provides a method for preparing chromosome of melastoma plant based on FISH hybridization to solve the problems mentioned in the background art.
Disclosure of Invention
The invention aims to provide a method for flaking the chromosomes of the melastoma genus plant based on FISH hybridization, so as to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a method for preparing a chromosome of a melastoma plant based on FISH hybridization specifically comprises the following steps:
(1) sampling: carrying out water culture on the melastoma plants, and taking 0.5cm root tips as a chromosome sample when root systems of the melastoma plants grow to 1-2 cm;
(2) fixing: fixing the chromosome sample by using a fixing solution;
(3) hypotonic: taking the dye sample out of the fixing solution, and putting the dye sample into 0.075mol/L KCL solution or pure water for hypotonic;
(4) enzymolysis: after the chromosome sample is settled down, clamping the chromosome sample into another culture medium dish to cut off the root crown part, only leaving the root tip, and placing the chromosome sample in the mixed solution of pectinase and cellulase for dissociation;
(5) tabletting: sucking out the dissociation liquid, washing with pure water, fixing with the fixing liquid, beating with a gun head to obtain suspension, dripping the suspension onto a glass slide, and drying to obtain the chromosome slide.
As a further scheme of the invention: the water culture mode in the step (1) is as follows: putting tender stem segments of the melastoma plants into clear water for culturing, wherein the water culture time is 25-45 d.
As a still further scheme of the invention: the stationary liquid in the step (2) is a mixture of ethanol, glacial acetic acid and trichloromethane.
As a still further scheme of the invention: the mass ratio of ethanol, glacial acetic acid and trichloromethane in the stationary liquid is 5:3: 2.
As a still further scheme of the invention: the fixed time in the step (2) is 24 h.
As a still further scheme of the invention: the dissociation time in the enzymolysis in the step (4) is 10-12 h.
As a still further scheme of the invention: the enzymolysis temperature in the step (4) is 37 ℃.
Compared with the prior art, the invention has the beneficial effects that: the invention discloses a method for flaking the chromosome of a melastoma plant based on FISH hybridization, the flaking of the chromosome of the melastoma plant obtained by adopting the chromosome flaking method of the invention has the advantages of multiple splitting phases, thorough cell wall dissociation, better chromosome dispersion and clean and clear background, the karyotype analysis research of the chromosome of the melastoma plant and the subsequent FISH hybridization research of the melastoma plant are convenient to carry out, and the foundation is laid for the development and utilization of germplasm resources of the melastoma plant and the research of cytology and molecular cytology.
Drawings
In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.
FIG. 1 is a drawing showing chromosome production by the chromosome production method of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In the embodiment of the present invention, the first and second substrates,
example 1
A method for preparing a chromosome of a melastoma plant based on FISH hybridization specifically comprises the following steps:
(1) sampling: carrying out water culture on the melastoma plants, and taking 0.5cm root tips as a chromosome sample when root systems of the melastoma plants grow to 1cm, wherein the water culture mode is as follows: putting tender stem segments of the melastoma plant into clear water for culturing, wherein the water culture time is 25d, and the water culture season is early summer;
(2) fixing: fixing the chromosome sample by using a fixing solution, wherein the fixing solution is a mixture of ethanol, glacial acetic acid and trichloromethane, the mass ratio of the ethanol to the glacial acetic acid to the trichloromethane in the fixing solution is 5:3:2, and the fixing time is 24 hours;
(3) hypotonic: taking the dye sample out of the fixing solution, and putting the dye sample into 0.075mol/L KCL solution or pure water for hypotonic;
(4) enzymolysis: after the chromosome sample is settled down, clamping the chromosome sample into another culture medium dish to cut off the root crown part, only leaving the root tip, and placing the chromosome sample into a mixed solution of pectinase and cellulase for dissociation, wherein the dissociation time is 10h, and the enzymolysis temperature is 37 ℃;
(5) tabletting: sucking out the dissociation liquid, washing with pure water, fixing with the fixing liquid, beating with a gun head to obtain suspension, dripping the suspension onto a glass slide, and drying to obtain the chromosome slide.
Example 2
A method for preparing a chromosome of a melastoma plant based on FISH hybridization specifically comprises the following steps:
(1) sampling: carrying out water culture on the melastoma plants, and taking 0.5cm root tips as a chromosome sample when root systems of the melastoma plants grow to 1.5cm, wherein the water culture mode is as follows: putting tender stem segments of the melastoma plant into clear water for culturing, wherein the water culture time is 35d, and the water culture season is early summer;
(2) fixing: fixing the chromosome sample by using a fixing solution, wherein the fixing solution is a mixture of ethanol, glacial acetic acid and trichloromethane, the mass ratio of the ethanol to the glacial acetic acid to the trichloromethane in the fixing solution is 5:3:2, and the fixing time is 24 hours;
(3) hypotonic: taking the dye sample out of the fixing solution, and putting the dye sample into 0.075mol/L KCL solution or pure water for hypotonic;
(4) enzymolysis: after the chromosome sample is settled down, clamping the chromosome sample into another culture medium dish to cut off the root crown part, only leaving the root tip, and placing the chromosome sample into a mixed solution of pectinase and cellulase for dissociation, wherein the dissociation time is 12h, and the enzymolysis temperature is 37 ℃;
(5) tabletting: sucking out the dissociation liquid, washing with pure water, fixing with the fixing liquid, beating with a gun head to obtain suspension, dripping the suspension onto a glass slide, and drying to obtain the chromosome slide.
Example 3
A method for preparing a chromosome of a melastoma plant based on FISH hybridization specifically comprises the following steps:
(1) sampling: carrying out water culture on the melastoma plants, and taking 0.5cm root tips as a chromosome sample when root systems of the melastoma plants grow to 2cm, wherein the water culture mode is as follows: putting tender stem segments of the melastoma plant into clear water for culturing, wherein the water culture time is 45d, and the water culture season is early summer;
(2) fixing: fixing the chromosome sample by using a fixing solution, wherein the fixing solution is a mixture of ethanol, glacial acetic acid and trichloromethane, the mass ratio of the ethanol to the glacial acetic acid to the trichloromethane in the fixing solution is 5:3:2, and the fixing time is 24 hours;
(3) hypotonic: taking the dye sample out of the fixing solution, and putting the dye sample into 0.075mol/L KCL solution or pure water for hypotonic;
(4) enzymolysis: after the chromosome sample is settled down, clamping the chromosome sample into another culture medium dish to cut off the root crown part, only leaving the root tip, and placing the chromosome sample into a mixed solution of pectinase and cellulase for dissociation, wherein the dissociation time is 11h, and the enzymolysis temperature is 37 ℃;
(5) tabletting: sucking out the dissociation liquid, washing with pure water, fixing with the fixing liquid, beating with a gun head to obtain suspension, dripping the suspension onto a glass slide, and drying to obtain the chromosome slide.
In conclusion, the method for flaking the chromosomes of the melastoma plant has the advantages of multiple mitotic phases, thorough cell wall dissociation, better chromosome dispersion and clean and clear background, is convenient for karyotype analysis research of the chromosomes of the melastoma plant and subsequent FISH hybridization research of the melastoma plant, lays a foundation for development and utilization of germplasm resources of the melastoma plant and cytology research, can stay mitotic cells in the early-middle period without pretreatment, reduces the phenomenon of excessively shortened chromosomes caused by toxic action of pretreatment liquid on the cells, combines pretreatment and fixation, effectively improves flaking efficiency, solves the problems of complex operation and low efficiency of the prior flaking method and flaking the melastoma plant chromosomes of the melastoma plant obtained by the prior flaking method, the problems of incomplete cell separation, cell membrane rupture and chromosome overflow and incapability of subsequent FISH hybridization research are easy to occur.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (7)
1. A method for preparing a sheet of a chromosome of a melastoma plant based on FISH hybridization is characterized in that: the chromosome slide making method specifically comprises the following steps:
(1) sampling: carrying out water culture on the melastoma plants, and taking 0.5cm root tips as a chromosome sample when root systems of the melastoma plants grow to 1-2 cm;
(2) fixing: fixing the chromosome sample by using a fixing solution;
(3) hypotonic: taking the dye sample out of the fixing solution, and putting the dye sample into 0.075mol/L KCL solution or pure water for hypotonic;
(4) enzymolysis: after the chromosome sample is settled down, clamping the chromosome sample into another culture medium dish to cut off the root crown part, only leaving the root tip, and placing the chromosome sample in the mixed solution of pectinase and cellulase for dissociation;
(5) tabletting: sucking out the dissociation liquid, washing with pure water, fixing with the fixing liquid, beating with a gun head to obtain suspension, dripping the suspension onto a glass slide, and drying to obtain the chromosome slide.
2. The method for preparing the chromosome of the melastoma plant based on FISH hybridization according to claim 1, wherein the method comprises the following steps: the water culture mode in the step (1) is as follows: putting tender stem segments of the melastoma plants into clear water for culturing, wherein the water culture time is 25-45 d.
3. The method for preparing the chromosome of the melastoma plant based on FISH hybridization according to claim 1, wherein the method comprises the following steps: the stationary liquid is a mixture of ethanol, glacial acetic acid and trichloromethane.
4. The method for preparing the chromosome of the melastoma plant based on FISH hybridization according to claim 3, wherein the method comprises the following steps: the mass ratio of ethanol, glacial acetic acid and trichloromethane in the stationary liquid is 5:3: 2.
5. The method for preparing the chromosome of the melastoma plant based on FISH hybridization according to claim 1, wherein the method comprises the following steps: the fixed time in the step (2) is 24 h.
6. The method for preparing the chromosome of the melastoma plant based on FISH hybridization according to claim 1, wherein the method comprises the following steps: the dissociation time in the enzymolysis in the step (4) is 10-12 h.
7. The method for preparing the chromosome of the melastoma plant based on FISH hybridization according to claim 1, wherein the method comprises the following steps: the enzymolysis temperature in the step (4) is 37 ℃.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102645360A (en) * | 2012-04-16 | 2012-08-22 | 北京林业大学 | Lagerstroemia plant stem tip chromosome tablet preparation method |
| CN106932256A (en) * | 2017-01-22 | 2017-07-07 | 河南师范大学 | A kind of method for preparing asparagus root tip cell chromosome division phases sample |
| CN110926896A (en) * | 2019-12-04 | 2020-03-27 | 怀化学院 | Method for metaphase mitosis phase mounting of rhizosphere chromosome of zingiber plant and application thereof |
| AU2020103466A4 (en) * | 2020-01-16 | 2021-01-28 | Sugarcane Research Institute of Yunnan Academy of Agricultural Sciences | Efficient method for preparing chromosome from shoot tip of sugarcane or sugarcane related species |
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- 2021-11-07 CN CN202111309818.8A patent/CN114062087A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102645360A (en) * | 2012-04-16 | 2012-08-22 | 北京林业大学 | Lagerstroemia plant stem tip chromosome tablet preparation method |
| CN106932256A (en) * | 2017-01-22 | 2017-07-07 | 河南师范大学 | A kind of method for preparing asparagus root tip cell chromosome division phases sample |
| CN110926896A (en) * | 2019-12-04 | 2020-03-27 | 怀化学院 | Method for metaphase mitosis phase mounting of rhizosphere chromosome of zingiber plant and application thereof |
| AU2020103466A4 (en) * | 2020-01-16 | 2021-01-28 | Sugarcane Research Institute of Yunnan Academy of Agricultural Sciences | Efficient method for preparing chromosome from shoot tip of sugarcane or sugarcane related species |
Non-Patent Citations (1)
| Title |
|---|
| 杨亚飞;刘沈徽;黄东益;吴文嫱;: "适于荧光原位杂交的大薯叶片染色体制片技术", 热带生物学报, no. 01, 25 March 2020 (2020-03-25) * |
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