Piece preparation method of diptera insect chromosomes
By using semen materials and colchicine injection technology, combined with HCl, KCl and glacial acetic acid treatment, the problem of unclear chromosomes of the salivary glands of Diptera insects was solved, and chromosomal products with clear morphology were achieved, which promoted the development of research.
Patent Information
- Application Number
- CN202411909306.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-24
- Publication Date
- 2025-04-08
AI Technical Summary
In the prior art, the salivary gland chromosomes of Diptera insects are difficult to clearly observe, which leads to difficulties in research.
The semen was used as the observation material, and chromosome division was stagnated in the meso-meiosis through colchicin injection, and HCl and KCl solutions were used for dissociation and hypotonic treatment. Finally, clear chromosomes were obtained by glacial acetic acid separation and tableting technology.
A chromosome with clear morphology is obtained, which is easy to observe and count, solves the problem of unclear observation of traditional salivary gland chromosomes, and provides a chromosome production method that is more suitable for Diptera insects.
Smart Images
Figure CN119779786A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of cytogenetics, and in particular to a method for preparing chromosomes of dipteran insects. Background Art
[0002] Diptera is an order in the phylum Arthropoda, subphylum Mandibulata, class Insecta, and subclass Pterygota. It is the fourth largest order in the class Insecta, after Coleoptera, Lepidoptera, and Hymenoptera. There are 85,000 known species in the world, distributed globally. More than 4,000 species are known in China. Some of these species are vectors that spread diseases to humans or other animals and plants. In agricultural production, the larvae of some groups of Diptera, such as seed flies, leaf miners, fruit flies, and wheat gall midges, are important agricultural pests. This patent takes the Tephritidae as an example. This type of insect flies flexibly and has a bee-like body shape, so it is called "needle bee", also known as fruit borer, yellow fly, melon maggot, etc. Fruit flies are important pests of horticultural crops in the Asia-Pacific region. They have strong reproductive capacity and a wide range of hosts. Their harm can be seen all year round, causing significant economic losses. They are also important pests in the international trade of agricultural products. Understanding the karyotype characteristics of Diptera insects can lay a certain theoretical foundation for further research on the genetic composition, evolutionary relationships, species identification, genome structure and control of Diptera.
[0003] Chromosome preparation can be observed under a microscope. Since the salivary gland cells of dipteran insects no longer undergo mitosis after developing to a certain stage, but remain in the interphase permanently, as the larvae grow, the salivary gland chromosomes continue to replicate themselves without separating. After many replications, chromosome threads are formed, which are about 100-150 times larger than ordinary cell metaphase chromosomes. They are giant chromosomes. Therefore, the current preparation of chromosomes of dipteran insects mainly uses salivary glands as materials. However, salivary gland chromosomes are linear, with one end connected to the same centromere. Chromosomes are easily entangled with each other, making it difficult to find clearly distributed chromosomes and difficult to observe. Therefore, it is necessary to find a chromosome preparation method that is more suitable for dipteran insects.
[0004] Based on the above problems, the purpose of this application is to provide a method for preparing chromosomes of Diptera insects. Summary of the invention
[0005] In view of the above shortcomings, the present invention provides a method for preparing chromosomes of dipteran insects, which solves the problems that the chromosomes of dipteran insects are unclear and not conducive to observation and research when using salivary glands as materials for chromosome preparation. The method of the present invention can obtain chromosomes with clear morphology. The specific technical scheme is as follows:
[0006] A method for preparing chromosomes of dipteran insects comprises the following steps:
[0007] (1) Pretreatment: After the newly emerged male fruit flies are anesthetized, colchicine is microinjected into the abdomen, and the fruit flies that have recovered their vitality are placed in a centrifuge tube for later use;
[0008] (2) Obtaining testes: dissecting the fruit flies injected with colchicine, taking out the testes, placing them on a glass slide, and dripping physiological saline into them for later use;
[0009] (3) Staining: First, add 0.5-1.5 mol / L HCl solution to the testis treated with physiological saline and dissociate for 2-3 min. Then, add 0.05-0.07 mol / L KCl solution to the dissociated testis for hypotonic treatment for 8-10 min. After absorbing the KCl solution with filter paper, add dye solution for staining for 5-10 min and set aside.
[0010] (4) Color separation, sheet pressing and microscopic examination: Glacial acetic acid is dripped onto the stained testis for color separation and then directly covered with a cover slip. After sheet pressing, microscopic examination is performed to observe the morphology and number of chromosomes under an optical microscope.
[0011] Furthermore, in step (1), the abdominal microinjection is performed at the third intersegment of the abdomen.
[0012] Furthermore, in step (1), the concentration of colchicine is 8-15 ng / μL, and the injection volume is 350-450 nL.
[0013] Furthermore, in step (2), the dissection is performed 2-4 hours after colchicine injection.
[0014] Furthermore, in step (2), the dissection operation steps are as follows: crush the thorax of the fruit fly with forceps, pull out the abdomen together with the contents, tear open the abdominal shell with forceps, and find and clamp the light yellow testis in the contents.
[0015] Furthermore, in step (3), the dye solution is a modified phenol fuchsin dye solution.
[0016] Furthermore, in step (4), the mass concentration of the glacial acetic acid is 40-50%.
[0017] Furthermore, in step (4), the pressing step is to place the slide on toilet paper, fold the toilet paper back to cover the cover glass, press lightly with fingers to keep the cover glass from shifting, absorb excess liquid with the toilet paper, and tap the cover glass to evenly disperse the chromosomes.
[0018] Compared with the prior art, the present invention has the following beneficial effects:
[0019] The present invention selects testis as observation material, and uses colchicine to inject abdomen 2-4h and then takes out testis for film making, and finally obtains chromosome with clear morphology. Wherein, select testis as observation material, compared with the salivary gland selected by tradition, the color is obvious, it is easier to dissect, and the testis chromosome is short rod-shaped, easy to observe and count; and using colchicine to inject fruit fly abdomen, chromosome division can be stagnated in meiotic metaphase, chromosome morphology is clear, easy to disperse evenly, easy to observe, especially using microinjection instrument to inject colchicine, it is easier to work than immersion method, and microinjection wound is small, does not affect insect vitality; secondly, using 0.5-1.5mol / L HCl dissociation treatment, after increasing cell membrane permeability, then using 0.05-0.07mol / L KCL hypotonic treatment, cells are more likely to burst, shortening the processing time; finally using 40-50% glacial acetic acid color separation, it is helpful to fix the chromosome structure, and the passive staining of cytoplasm can be removed, avoiding the background color being too dark, affecting the observation of chromosome. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for describing the embodiments are briefly introduced below.
[0021] Figure 1 : Chromosome observation of pepper fruit fly in Example 1;
[0022] Figure 2 : The chromosome observation of Bactrocera fasciatus in Example 2;
[0023] Figure 3 : Chromosome observation of Bactrocera melonensis in Example 3;
[0024] Figure 4 : This is the chromosome observation of Bactrocera dorsalis in Example 4;
[0025] Figure 5 : This is the chromosome observation of South Asian fruit fly in Example 5. DETAILED DESCRIPTION
[0026] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.
[0027] The instruments used in the following examples are: a dissecting microscope, a microinjection instrument, a common optical microscope, dissecting tweezers, a glass slide, a cover slip, cotton wool, and filter paper.
[0028] Example 1
[0029] In this embodiment, the chromosome preparation is carried out using pepper fruit fly as the material, and the specific steps are as follows:
[0030] (1) Pretreatment: Take the newly emerged male fruit flies and place them on ice for freezing anesthesia. Use tweezers to pick up the fully anesthetized male fruit flies and place them on the surface of a water agar plate (agar 40 g / L), with the abdomen facing upward and the wings attached to the surface of the plate. Use a cotton ball containing 75% alcohol to gently wipe the abdomen of the male fruit flies. Wait for the alcohol to air-dry, select the third intersegment of the abdomen as the injection site, and use a microinjector to inject 350 nL of colchicine (concentration of 15 ng / μL). After the injection, the adult insects that have recovered their vitality are placed back into a 50 mL clean centrifuge tube for later use.
[0031] (2) Obtaining testes: Take the above-mentioned fruit flies injected 2 hours after injection and place them in a clean culture dish with a drop of physiological saline (0.9% NaCl solution), and dissect them under a dissecting microscope. The operation steps are as follows: Use forceps to crush the thorax of the male insect to make it unable to move, pull out the abdomen together with the contents, tear open the abdominal shell with forceps, and find the pale yellow testes among the contents. Clip the testes and place them in another drop of physiological saline, remove excess fat tissue as much as possible, and the processed testes are ready for staining.
[0032] (3) Staining: The dissected testes were placed on a clean glass slide for staining. Specifically, 0.5 mol / L HCl solution was added to the peeled testes for 3 min of dissociation to decompose excess adipose tissue and increase cell membrane permeability. After the dissociation was completed, the HCl solution was removed with filter paper. Then, 0.05 mol / L KCl solution was added to the dissociated testes for hypotonic treatment for 10 min. After the KCl solution was removed with filter paper, a drop of modified phenol fuchsin dye was added and dyed for 5 min for later use.
[0033] (4) Color separation, pressing and microscopic examination: Add glacial acetic acid with a mass concentration of 40% to the stained testes for color separation and directly cover with a coverslip. Place the slide on toilet paper folded into long strips. Fold the toilet paper back to cover the coverslip. Press lightly with fingers to keep the coverslip from shifting. At the same time, use the toilet paper to absorb excess liquid. Use a stylus pen to tap the coverslip moderately to disperse the chromosomes more evenly. Observe the morphology and number of chromosomes under an optical microscope (100× objective lens).
[0034] Example 2
[0035] In this example, Bactrocera tabacum is used as material for chromosome preparation, and the specific steps are as follows:
[0036] (1) Pretreatment: Take the newly emerged male fruit flies and place them on ice for freezing anesthesia. Use tweezers to pick up the fully anesthetized male fruit flies and place them on the surface of a water agar plate (agar 40 g / L), with the abdomen facing upward and the wings attached to the surface of the plate. Use a cotton ball containing 75% alcohol to gently wipe the abdomen of the male fruit flies. After the alcohol is air-dried, select the third intersegment of the abdomen as the injection site and inject 450 nL of colchicine (concentration of 8 ng / μL) using a microinjector. After the injection, the adult insects that have recovered their vitality are placed back into a 50 mL clean centrifuge tube for later use.
[0037] (2) Obtaining testes: Take the above-mentioned fruit flies injected 4 hours after injection and place them in a clean culture dish with a drop of physiological saline (0.9% NaCl solution), and dissect them under a dissecting microscope. The operation steps are as follows: Use forceps to crush the thorax of the male insect to make it unable to move, pull out the abdomen together with the contents, tear open the abdominal shell with forceps, and find the pale yellow testes among the contents. Clip the testes and place them in another drop of physiological saline, remove excess fat tissue as much as possible, and the processed testes are ready for staining.
[0038] (3) Staining: Place the dissected testis on a clean glass slide and perform staining. Specifically, add 1.5 mol / L HCl solution to the peeled testis and dissociate for 2 min to decompose excess adipose tissue and increase cell membrane permeability. After dissociation, remove the HCl solution with filter paper. Then, add 0.07 mol / L KCl solution to the dissociated testis for hypotonic treatment for 8 min. After removing the KCl solution with filter paper, add a drop of modified phenol fuchsin dye and stain for 10 min for later use.
[0039] (4) Color separation, pressing and microscopic examination: Add 50% glacial acetic acid to the stained testes for color separation and directly cover with a coverslip. Place the slide on toilet paper folded into long strips. Fold the toilet paper back to cover the coverslip. Press lightly with fingers to keep the coverslip from shifting. At the same time, use the toilet paper to absorb excess liquid. Use a stylus pen to tap the coverslip moderately to disperse the chromosomes more evenly. Observe the morphology and number of chromosomes under an optical microscope (100× objective lens).
[0040] Example 3
[0041] In this embodiment, chromosome preparation is carried out using Bactrocera melonensis as material, and the specific steps are as follows:
[0042] (1) Pretreatment: Take the newly emerged male fruit flies and place them on ice for freezing anesthesia. Use tweezers to pick up the fully anesthetized male fruit flies and place them on the surface of a water agar plate (agar 40 g / L), with the abdomen facing upward and the wings attached to the surface of the plate. Use a cotton ball containing 75% alcohol to gently wipe the abdomen of the male fruit flies. Wait for the alcohol to air-dry, select the third intersegment of the abdomen as the injection site, and use a microinjector to inject 400 nL of colchicine (concentration of 10 ng / μL). After the injection, the adult insects that have recovered their vitality are placed back into a 50 mL clean centrifuge tube for later use.
[0043] (2) Obtaining testes: Take the above-mentioned fruit flies injected 3 hours after injection and place them in a clean culture dish with a drop of physiological saline (0.9% NaCl solution), and dissect them under a dissecting microscope. The operation steps are as follows: Use forceps to crush the thorax of the male insect to make it unable to move, pull out the abdomen together with the contents, tear open the abdominal shell with forceps, and look for the pale yellow testes in the contents. Clip the testes and place them in another drop of physiological saline, remove excess fat tissue as much as possible, and the processed testes are ready for staining.
[0044] (3) Staining: The dissected testes were placed on a clean glass slide for staining. Specifically, 1 mol / L HCl solution was added to the peeled testes for 3 min of dissociation to decompose excess adipose tissue and increase cell membrane permeability. After the dissociation was completed, the HCl solution was removed with filter paper. Then, 0.06 mol / L KCl solution was added to the dissociated testes for hypotonic treatment for 9 min. After the KCl solution was removed with filter paper, a drop of modified phenol fuchsin dye was added and dyed for 8 min for later use.
[0045] (4) Color separation, pressing and microscopic examination: Add glacial acetic acid with a mass concentration of 45% to the stained testes for color separation and directly cover with a coverslip. Place the slide on toilet paper folded into long strips. Fold the toilet paper back to cover the coverslip. Press lightly with fingers to keep the coverslip from shifting. At the same time, use the toilet paper to absorb excess liquid. Use a stylus pen to tap the coverslip moderately to disperse the chromosomes more evenly. Observe the morphology and number of chromosomes under an optical microscope (100× objective lens).
[0046] Example 4
[0047] In this example, chromosome preparation was performed using Bactrocera dorsalis as material, and the specific steps were as follows:
[0048] (1) Pretreatment: Take the newly emerged male fruit flies and place them on ice for freezing anesthesia. Use tweezers to pick up the fully anesthetized male fruit flies and place them on the surface of a water agar plate (agar 40 g / L), with the abdomen facing upward and the wings attached to the surface of the plate. Use a cotton ball containing 75% alcohol to gently wipe the abdomen of the male fruit flies. Wait for the alcohol to air-dry, select the third intersegment of the abdomen as the injection site, and inject 400 nL of colchicine (concentration of 10 ng / μL) using a microinjector. After the injection, the adult insects that have recovered their vitality are placed back into a 50 mL clean centrifuge tube for later use.
[0049] (2) Obtaining testes: Take the above-mentioned fruit flies injected 3 hours after injection and place them in a clean culture dish with a drop of physiological saline (0.9% NaCl solution), and dissect them under a dissecting microscope. The operation steps are as follows: Use forceps to crush the thorax of the male insect to make it unable to move, pull out the abdomen together with the contents, tear open the abdominal shell with forceps, and look for the pale yellow testes in the contents. Clip the testes and place them in another drop of physiological saline, remove excess fat tissue as much as possible, and the processed testes are ready for staining.
[0050] (3) Staining: Place the dissected testis on a clean glass slide and perform staining. Specifically, add 1 mol / L HCl solution to the peeled testis and dissociate for 3 min to decompose excess adipose tissue and increase cell membrane permeability. After the dissociation is complete, remove the HCl solution with filter paper. Then, add 0.07 mol / L KCl solution to the dissociated testis for hypotonic treatment for 10 min. After removing the KCl solution with filter paper, add a drop of modified phenol fuchsin dye and stain for 10 min for later use.
[0051] (4) Color separation, pressing and microscopic examination: Add glacial acetic acid with a mass concentration of 45% to the stained testes for color separation and directly cover with a coverslip. Place the slide on toilet paper folded into long strips. Fold the toilet paper back to cover the coverslip. Press lightly with fingers to keep the coverslip from shifting. At the same time, use the toilet paper to absorb excess liquid. Use a stylus pen to tap the coverslip moderately to disperse the chromosomes more evenly. Observe the morphology and number of chromosomes under an optical microscope (100× objective lens).
[0052] Example 5
[0053] In this example, the chromosome preparation was carried out using Bactrocera australis as the material, and the specific steps were as follows:
[0054] (1) Pretreatment: Take the newly emerged male fruit flies and place them on ice for freezing anesthesia. Use tweezers to pick up the fully anesthetized male fruit flies and place them on the surface of a water agar plate (agar 40 g / L), with the abdomen facing upward and the wings attached to the surface of the plate. Use a cotton ball containing 75% alcohol to gently wipe the abdomen of the male fruit flies. Wait for the alcohol to air-dry, select the third intersegment of the abdomen as the injection site, and inject 400 nL of colchicine (concentration of 10 ng / μL) using a microinjector. After the injection, the adult insects that have recovered their vitality are placed back into a 50 mL clean centrifuge tube for later use.
[0055] (2) Obtaining testes: Take the above-mentioned fruit flies injected 3 hours after injection and place them in a clean culture dish with a drop of physiological saline (0.9% NaCl solution), and dissect them under a dissecting microscope. The operation steps are as follows: Use forceps to crush the thorax of the male insect to make it unable to move, pull out the abdomen together with the contents, tear open the abdominal shell with forceps, and look for the pale yellow testes in the contents. Clip the testes and place them in another drop of physiological saline, remove excess fat tissue as much as possible, and the processed testes are ready for staining.
[0056] (3) Staining: Place the dissected testis on a clean glass slide and perform staining. Specifically, add 1 mol / L HCl solution to the peeled testis and dissociate for 3 min to decompose excess adipose tissue and increase cell membrane permeability. After the dissociation is complete, remove the HCl solution with filter paper. Then, add 0.07 mol / L KCl solution to the dissociated testis for hypotonic treatment for 10 min. After removing the KCl solution with filter paper, add a drop of modified phenol fuchsin dye and stain for 10 min for later use.
[0057] (4) Color separation, pressing and microscopic examination: Add glacial acetic acid with a mass concentration of 40-50% to the stained testes for color separation and directly cover with a coverslip. Place the slide on toilet paper folded into long strips. Fold the toilet paper back to cover the coverslip. Press lightly with fingers to keep the coverslip from shifting. At the same time, use the toilet paper to absorb excess liquid. Use a stylus pen to tap the coverslip moderately to disperse the chromosomes more evenly. Observe the morphology and number of chromosomes under an optical microscope (100× objective lens).
[0058] The chromosome morphology of the testicular cells of the five fruit flies in the metaphase of meiosis was observed, and the dividing cells with fully dispersed chromosomes, clear outlines and consistent numbers were selected for observation and counting. It was determined that the chromosomes of the five fruit flies were all complete diploid (2n=12), of which there were 5 pairs of pairable autosomes, and the sex chromosomes were XY type, which could be as small as dots, with a total of 6 pairs of chromosomes. The results are as follows Figures 1 to 5 As shown, each example obtained a chromosome with a clear structure.
[0059] In summary, the present invention selects testis as observation material, and uses colchicine to inject abdomen 2-4h and then takes out testis for film making, and finally obtains chromosome with clear morphology. Wherein, select testis as observation material, compared with the salivary gland selected by tradition, color is obvious, easier to dissect, and testis chromosome is short rod-shaped, easy to observe and count; and using colchicine to inject fruit fly abdomen, chromosome division can be stagnant in meiotic metaphase, chromosome morphology is clear, easy to disperse evenly, easy to observe, especially using microinjection instrument to inject colchicine, it is easier to work than immersion method, and microinjection wound is small, does not affect insect vitality; secondly, using 0.5-1.5mol / L HCl dissociation treatment, after increasing cell membrane permeability, then using 0.05-0.07mol / L KCL hypotonic treatment, cells are more likely to burst, shortening the processing time; finally using 40-50% glacial acetic acid color separation, it is helpful to fix chromosome structure, and passive staining of cytoplasm can be removed, avoiding background color too dark, affecting the observation of chromosome.
[0060] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.
Claims
1. A method for preparing chromosomes of Diptera insects, characterized in that: The following steps are involved: (1) Pretreatment: After the newly emerged male fruit flies are anesthetized, colchicine is microinjected into the abdomen, and the fruit flies that have recovered their vitality are placed in a centrifuge tube for later use; (2) Obtaining testes: dissect the fruit flies injected with colchicine, take out the testes, place them on a glass slide, and drip physiological saline into them for later use; (3) Staining: First, add 0.5-1.5 mol / L HCl solution to the testis treated with physiological saline and dissociate for 2-3 min. Then, add 0.05-0.07 mol / L KCl solution to the dissociated testis for hypotonic treatment for 8-10 min. After absorbing the KCl solution with filter paper, add dye solution for staining for 5-10 min and set aside. (4) Color separation, sheet pressing and microscopic examination: Glacial acetic acid is dripped onto the stained testis for color separation and then directly covered with a cover slip. After sheet pressing, microscopic examination is performed to observe the morphology and number of chromosomes under an optical microscope.
2. The method for preparing chromosomes of a dipteran insect according to claim 1, characterized in that: In step (1), the abdominal microinjection is performed at the third intersegment of the abdomen.
3. The method for preparing chromosomes of Diptera insects according to claim 1, characterized in that: In step (1), the concentration of colchicine is 8-15 ng / μL, and the injection volume is 350-450 nL.
4. The method for preparing chromosomes of Diptera insects according to claim 1, characterized in that: In step (2), the dissection is performed 2-4 hours after colchicine injection.
5. The method for preparing chromosomes of Diptera insects according to claim 1, characterized in that: In step (2), the dissection operation steps are as follows: crush the thorax of the fruit fly with forceps, pull out the abdomen together with the contents, tear open the abdominal shell with forceps, and find and clamp the light yellow testis in the contents.
6. The method for preparing chromosomes of Diptera insects according to claim 1, characterized in that: In step (3), the dye solution is a modified phenol fuchsin dye solution.
7. The method for preparing chromosomes of Diptera insects according to claim 1, characterized in that: In step (4), the mass concentration of the glacial acetic acid is 40-50%.
8. The method for preparing chromosomes of dipteran insects according to claim 1, characterized in that: In step (4), the pressing step is to place the slide on toilet paper, fold the toilet paper back to cover the cover glass, press lightly with fingers to keep the cover glass from shifting, absorb excess liquid with the toilet paper, and tap the cover glass to evenly disperse the chromosomes.