AU2021105469A4 - Method for preparing chromosome karyotype of coleoptera larva - Google Patents
Method for preparing chromosome karyotype of coleoptera larva Download PDFInfo
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- AU2021105469A4 AU2021105469A4 AU2021105469A AU2021105469A AU2021105469A4 AU 2021105469 A4 AU2021105469 A4 AU 2021105469A4 AU 2021105469 A AU2021105469 A AU 2021105469A AU 2021105469 A AU2021105469 A AU 2021105469A AU 2021105469 A4 AU2021105469 A4 AU 2021105469A4
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- 210000000349 chromosome Anatomy 0.000 title claims abstract description 32
- 241000254173 Coleoptera Species 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 23
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 30
- 229960000583 acetic acid Drugs 0.000 claims abstract description 15
- 239000012362 glacial acetic acid Substances 0.000 claims abstract description 15
- NNJPGOLRFBJNIW-HNNXBMFYSA-N (-)-demecolcine Chemical compound C1=C(OC)C(=O)C=C2[C@@H](NC)CCC3=CC(OC)=C(OC)C(OC)=C3C2=C1 NNJPGOLRFBJNIW-HNNXBMFYSA-N 0.000 claims abstract description 10
- NNJPGOLRFBJNIW-UHFFFAOYSA-N Demecolcine Natural products C1=C(OC)C(=O)C=C2C(NC)CCC3=CC(OC)=C(OC)C(OC)=C3C2=C1 NNJPGOLRFBJNIW-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000007788 liquid Substances 0.000 claims abstract description 10
- HZLHRDBTVSZCBS-UVJJDBRNSA-N 4-[(e)-(4-aminophenyl)-(4-imino-3-methylcyclohexa-2,5-dien-1-ylidene)methyl]-2-methylaniline;hydrochloride Chemical compound Cl.C1=CC(=N)C(C)=C\C1=C(C=1C=C(C)C(N)=CC=1)/C1=CC=C(N)C=C1 HZLHRDBTVSZCBS-UVJJDBRNSA-N 0.000 claims abstract description 8
- 238000003825 pressing Methods 0.000 claims abstract description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 15
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 8
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 8
- 210000001519 tissue Anatomy 0.000 claims description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 4
- 239000012153 distilled water Substances 0.000 claims description 4
- 239000008103 glucose Substances 0.000 claims description 4
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 235000011148 calcium chloride Nutrition 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 238000004043 dyeing Methods 0.000 abstract description 8
- 238000002224 dissection Methods 0.000 abstract description 4
- 238000002474 experimental method Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 10
- 239000000463 material Substances 0.000 description 4
- 241000346653 Holotrichia parallela Species 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 229910014813 CaC2 Inorganic materials 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 230000016507 interphase Effects 0.000 description 2
- 230000031864 metaphase Effects 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 210000003079 salivary gland Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 210000001550 testis Anatomy 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000006727 cell loss Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000007614 genetic variation Effects 0.000 description 1
- 210000002149 gonad Anatomy 0.000 description 1
- 230000021121 meiosis Effects 0.000 description 1
- 230000017346 meiosis I Effects 0.000 description 1
- 230000023439 meiosis II Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000011197 physicochemical method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 230000015909 regulation of biological process Effects 0.000 description 1
- 230000020509 sex determination Effects 0.000 description 1
- 210000001082 somatic cell Anatomy 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
-
- 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
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- Health & Medical Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
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- Biochemistry (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Biomedical Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
The present disclosure belongs to the technical field of genetics, and particularly relates to a
method for preparing a chromosome karyotype of coleoptera larva. placing an antennal lobe of a
three-age mature larva of coleoptera into 0.05% colcemid solution to be treated for 25-45
minutes; then putting same into 0.075 mol/L KCl solution for hypotonic treatment for 20-30
minutes; using a carbol fuchsin dye liquid to dye the same on a slide for 20-30 minutes after
treatment is completed, using 40%-50% (volume ratio) glacial acetic acid to separate color, and
then pressing the slide to obtain a chromosome karyotype of coleoptera larva. The present
disclosure performs dyeing directly after hypotonic treatment without fixing, thereby simplifying
an experimental procedure, making steps simpler and more convenient, and saving time.
1/2
DRAWINGS:
Seiectamaterial Select a three-age mature larva of a coleoptera
larva
Take an antennal lobe out by means of dissection
Carry out
pretreatment under a stereoscopic microscope, and treat same
with 0.05% colcemid solution for 25-45 minutes
carry out hypotonic
treatment Carry out hypotonic treatment with 0.075 mol/L
KCI for 20-30 minutes
Dye Carry out dyeing with a carbol fuchsin dye
ye liquid for 20-30 minutes
[ Press a slide Use 40%-50% glacial acetic acid to separate
color and press the slide
Carry out microscopic
examination
FIG. 1
Description
1/2
Seiectamaterial Select a three-age mature larva of a coleoptera larva
Take an antennal lobe out by means of dissection Carry out pretreatment under a stereoscopic microscope, and treat same with 0.05% colcemid solution for 25-45 minutes
carry out hypotonic treatment Carry out hypotonic treatment with 0.075 mol/L KCI for 20-30 minutes
Dye Carry out dyeing with a carbol fuchsin dye ye liquid for 20-30 minutes
[ Press a slide Use 40%-50% glacial acetic acid to separate color and press the slide
Carry out microscopic examination
FIG. 1
[0001] 1. Technical Field
[0002] The present disclosure belongs to the technical field of genetics, and particularly relates to a method for preparing a chromosome karyotype of coleoptera larva.
[0003] 2. Description of Related Art
[0004] The chromosome is the carrier of genetic material. Research on the chromosome not only has great significance to understand the genetic variation, sex determination, individual development, regulation of physiological processes, etc., but also provides an important basis for determining the status of biological groups and clarifying the history and evolution of species, and further plays a role in solving the problem of classification among sibling species. Moreover, developing genomics imposes increasing requirements on the analysis of biological chromosome karyotypes.
[0005] For dispersing cells, the liquid nitrogen refrigeration slide peeling method after slide pressing, the slide tearing method and the method for preparing a cell suspension by centrifugation are generally used. When the material to be prepared has a small size and a limited quantity, the centrifugation method should not be used for preparing the cell suspension. When the gonad of the insect is small, it is difficult to use the dissecting needle to tear the slide, resulting in insufficient tearing of the slide. During microscopic examination, it is observed that most tissues are not separated, and moreover, scratches will be engraved, by the dissecting needle, on the slide due to the improper force, influencing the observation. The slide is peeled after the slide is pressed, such that some cells will stick to the cover slip, resulting in cell loss, which requires to be improved.
[0006] The testicle is generally selected for the karyotype analysis of the adult insect. The salivary gland of the larva has been a common tissue for chromosome karyotype research of the larva due to its giant chromosome. The coleoptera does not have the salivary gland, and accordingly, adult testicles are generally selected for analysis of chromosome karyotypes in the existing research. However, the whole process of meiosis of spermatogonia is divided into four stages, including interphase I, meiosis I, interphase II and meiosis II, which cause much interference in chromosome counting, and accordingly, it is better to directly select somatic cells for counting.
[0007] Aiming at the problem existing in the prior art, the objective of the present disclosure is to provide a method for preparing a chromosome karyotype of a coleoptera larva.
[0008] In order to achieve the above objective, the present disclosure uses the following technical solution:
[0009] the method for preparing a chromosome karyotype of a coleoptera larva includes: placing an antennal lobe of a three-age mature larva of a coleoptera into 0.05% colcemid solution to be treated for 25-45 minutes; then putting same into 0.075 mol/L KCl solution for hypotonic treatment for 20-30 minutes; using a carbol fuchsin dye liquid to dye the same on a slide for 20 minutes after treatment is completed, using 40%-50% (volume ratio) glacial acetic acid to separate color, and then pressing the slide to obtain a chromosome karyotype of a coleoptera larva.
[0010] On the basis of the above solution, a tissue is the antennal lobe of the mature larva.
[0011] On the basis of the above solution, the colcemid solution is prepared from normal saline, where the normal saline consists of 6.8 g/L NaCl, 0.2 g/L CaCl2, 0.2 g/L KCl, 0.2 g/L MgCl2•6H20, 0.15 g/L NaHCO3, 7.7 g/L glucose and 1000 mL of distilled water.
[0012] On the basis of the above solution, when the glacial acetic acid is used for separating the color, color of a background dye is made faded to the greatest extent, and filter paper is used for absorbing excess dye and glacial acetic acid.
[0013] When the slide is pressed, a cover slip is gently put down, so as to avoid too many bubbles between the cover slip and the slide. After the slide is covered with the cover slip, double-layer filter paper covers the slide, and is pressed by an index finger, and the index finger gradually applies a force from left to right without vertical pressing or moving the cover slip.
[0014] The method of the present disclosure is suitable for all three-age mature larvae of coleoptera, including but not limited to three-age mature larvae of Holotrichia parallela.
[0015] The technical solution of the present disclosure has the advantages:
[0016] the present disclosure provides the simple method for preparing a chromosome karyotype of a coleoptera larva. The method has the following beneficial effects: a material is easy to acquire, preparation time is short, preparation steps are few, operation is simple, and the minimum time required to prepare the chromosome karyotype of the coleoptera larva is only 1.5 2 hours, thereby improving efficiency of preparing the chromosome karyotype of the coleoptera larva; and moreover, the method described in the present disclosure does not use a centrifuge to separate cells, thereby reducing dependence of an experiment on an instrument, avoiding an influence of centrifugation on the cells, and improving a success rate of preparing a karyotype slide specimen.
[0017] Fixation is to rapidly kill a tissue cell by means of a physico-chemical method, and maintains a morphological structure and a real-life state of an inclusion of the tissue cell to the greatest extent. Moreover, it is better to use a fixing liquid right after it is prepared since a fixing effect will be influenced by long-time storage, and fixing time is generally 15-1440 minutes, which is too long. In the present disclosure, without a cell wall, an animal cell is easier to treat. The carbol fuchsin dye liquid used in the present disclosure contains alcohol, the alcohol also may achieve a fixing function, and accordingly, the present disclosure performs dyeing directly after hypotonic treatment without fixing, thereby simplifying an experimental procedure, making steps simpler and more convenient, and saving time.
[0018] The present disclosure may clearly count a somatic chromosome by selecting the antennal lobe of the three-age mature larva, and the antennal lobe is easy to identify and simple to dissect, and may be easy to acquire.
[0019] FIG. 1 is a schematic diagram of steps of a method of the present disclosure; and
[0020] FIG. 2 is a chromosome image of an antennal lobe of a three-age mature larva of Holotrichia parallela observed under a 100x objective lens through the method of the present disclosure.
[0021] The embodiments of the present invention are described in detail below, and the examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end, the same or similar reference numbers represent the same or similar elements or elements having the same or similar function. The embodiments described below with reference to the accompanying drawings are exemplary only for the purpose of explaining the present invention, and should not be construed as limiting the present invention.
[0022] Embodiment 1
[0023] As shown in FIG. 1, a method for preparing a chromosome karyotype of a coleoptera larva includes:
[0024] (1) a material was selected: a three-age mature larva of coleoptera was selected;
[0025] (2) pretreatment was carried out: an antennal lobe was taken out by means of dissection under a stereoscopic microscope and was treated with 0.05% colcemid solution for 25-45 minutes;
[0026] (3) hypotonic treatment was carried out: hypotonic treatment was carried out with 0.075 mol/L KCl for 20-30 minutes;
[0027] (4) dyeing was carried out: dyeing was carried out with a carbol fuchsin dye liquid for -30 minutes;
[0028] (5) a slide was pressed: 40%-50% glacial acetic acid was used for separating color, and then the slide was pressed, where when the glacial acetic acid was used for separating the color, color of a background dye was made faded to the greatest extent, and filter paper was used for absorbing excess dye and glacial acetic acid; when the slide was pressed, a cover slip was gently put down, so as to avoid too many bubbles between the cover slip and the slide; and after the slide was covered with the cover slip, double-layer filter paper covered the slide, and was pressed by an index finger, and the index finger gradually applied a force from left to right without vertical pressing or moving the cover slip; and
[0029] (6) microscopic examination was carried out: an OLYMPUS BX53 upright fluorescence microscope was used for observation, an OLYMPUS DP74 microscopy imaging system was used for photographing, cells in a metaphase were found under a 40x objective lens, cells having an excellent chromosome dispersion effect and excellent morphology were selected to be photographed under a 100x objective lens, and Photoshop image software was used for analyzing a karyotype.
[0030] The colcemid solution was prepared from normal saline, where the normal saline consisted of 6.8 g/L NaCl, 0.2 g/L CaC2, 0.2 g/L KCl, 0.2 g/L MgCl2•6H20, 0.15 g/L NaHCO3, 7.7 g/L glucose and 1000 mL of distilled water.
[0031] Embodiment 2
[0032] The method for preparing a chromosome karyotype of a coleoptera larva includes:
[0033] (1) a material was selected: a three-age mature larva of coleoptera was selected;
[0034] (2) pretreatment was carried out: an antennal lobe was taken out by means of dissection under a stereoscopic microscope and was treated with 0.05% colcemid solution for 30 minutes;
[0035] (3) hypotonic treatment was carried out: hypotonic treatment was carried out with 0.075 mol/L KCl for 20 minutes;
[0036] (4) dyeing was carried out: dyeing was carried out with a carbol fuchsin dye liquid for 28 minutes;
[0037] (5) a slide was pressed: 50% glacial acetic acid was used for separating color, and then the slide was pressed, where when the glacial acetic acid was used for separating the color, color of a background dye was made faded to the greatest extent, and filter paper was used for absorbing excess dye and glacial acetic acid; when the slide was pressed, a cover slip was gently put down, so as to avoid too many bubbles between the cover slip and the slide; and after the slide was covered with the cover slip, double-layer filter paper covered the slide, and was pressed by an index finger, and the index finger gradually applied a force from left to right without vertical pressing or moving the cover slip; and
[0038] (6) microscopic examination was carried out: an OLYMPUS BX53 upright fluorescence microscope was used for observation, an OLYMPUS DP74 microscopy imaging system was used for photographing, cells in a metaphase were found under a 40x objective lens, cells having an excellent chromosome dispersion effect and excellent morphology were selected to be photographed under a 100x objective lens, and Photoshop image software was used for analyzing a karyotype.
[0039] The colcemid solution was prepared from normal saline, where the normal saline consisted of 6.8 g/L NaCl, 0.2 g/L CaC2, 0.2 g/L KCl, 0.2 g/L MgCl2•6H20, 0.15 g/L NaHCO3, 7.7 g/L glucose and 1000 mL of distilled water.
[0040] The karyotypes of the prepared chromosomes were shown in FIG. 2. The number of chromosomes of an antennal lobe of a three-age mature larva of Holotrichia parallela observed under a 100x objective lens was 2n=22.
[0041] It should be understood that although the present description is described in terms of the implementation, not every implementation includes only one separate technical solution, and such a description mode of the description is merely for the sake of clarity. A person skilled in the art should take the description as a whole, and the technical solutions in all the embodiments may be appropriately combined to form other implementations that can be understood by a person skilled in the art.
Claims (5)
1. A method for preparing a chromosome karyotype of coleoptera larva, the following steps: placing an antennal lobe of a three-age mature larva of coleoptera into 0.05% colcemid solution to be treated for 25-45 minutes; then putting same into 0.075 mol/L KC solution for hypotonic treatment for 20-30 minutes; using a carbol fuchsin dye liquid to dye the same on a slide for 20 minutes after treatment is completed, using 40%-50% (volume ratio) glacial acetic acid to separate color, and then pressing the slide to obtain a chromosome karyotype of coleoptera larva.
2. The method for preparing a chromosome karyotype of coleoptera larva according to claim 1, a tissue is the antennal lobe of the mature larva.
3. The method for preparing a chromosome karyotype of coleoptera larva according to claim 1, the normal saline consists of 6.8 g/L NaCl, 0.2 g/L CaCl2, 0.2 g/L KCl, 0.2 g/L MgCl2•6H20, 0.15 g/L NaHCO3, 7.7 g/L glucose and 1000 mL of distilled water.
4. The method for preparing a chromosome karyotype of coleoptera larva according to claim 1,when the glacial acetic acid is used for separating the color, color of a background dye is made faded to the greatest extent, and filter paper is used for absorbing excess dye and glacial acetic acid.
5. The method for preparing a chromosome karyotype of coleoptera larva according to claim 1, a cover slip is gently put down, so as to avoid too many bubbles between the cover slip and the slide; after the slide is covered with the cover slip, double-layer filter paper covers the slide, and is pressed by an index finger, and the index finger gradually applies a force from left to right without vertical pressing or moving the cover slip.
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| AU2021105469A AU2021105469A4 (en) | 2021-08-13 | 2021-08-13 | Method for preparing chromosome karyotype of coleoptera larva |
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