CN108731994A - A kind of production method of climbing groundsel root tip chromosomes sample slice - Google Patents

Abstract

This proposal discloses a method for making a Senecio root tip chromosome specimen sheet in the technical field of chromosome production, including the following steps: 1: Select the intact root tip of the Senecio plant; 2: Place the root tip in the ice-water mixture Soaking in medium pretreatment for 15 to 17 hours; Three: Hypotonic treatment of the root tip with KCl solution; Fourth: Wash the root tip after the hypotonic treatment with double distilled water, and then fix it with Carnoy’s fixative; Fifth: Use double distilled water Wash the fixed root tip with water, and then use a mixed enzyme solution to enzymolyze it; 6: Wash the enzymolyzed root tip with double distilled water and treat it with double distilled water for 40-60 minutes; 7: Cut off the front of the root tip 2-3mm Put it on an ice slide, then spread out the root tip tissue, drip Carnoy's fixative solution, drip modified phenol fuchsin solution, stain for 15-20 minutes, cover with a cover glass, then tap, bake, and press. The root tip chromosome specimen sheet of Senecio was prepared. The Senecio chromosome specimen sheet prepared by this protocol has the characteristics of good dispersion, clear shape and high recognition.

Description

A kind of making method of Senecio root tip chromosome specimen sheet

technical field

The invention belongs to the technical field of chromosome preparation, and in particular relates to a method for making a Senecio root tip chromosome specimen sheet.

Background technique

Senecio is a plant of the genus Senecio in the family Asteraceae, also known as Jiuliming. It is widely distributed in my country, with rich wild resources and complex chemical components. The light water extract has certain inhibitory effect on HIV-1 virus), anti-leptohelix, anti-trichomonas and other pharmacological effects. It is mainly used to treat influenza, pneumonia, mumps, acute enteritis, acute urinary tract infection, erysipelas, eczema, dry and wet ringworm sores, trichomonal vaginitis, burns and scalds, etc. With the continuous development of science and technology, the continuous improvement of its chemical composition, pharmacological effects and toxicology research makes Senecio have broad prospects for development and utilization.

Senecio is clinically used as a broad-spectrum antibacterial drug, which has obvious curative effect in the treatment of bacterial infection and is not easy to produce bacterial resistance. Qian Gang et al. found that the antibacterial pharmacological effect of Senecio is a quantitative trait controlled by genes, but each The distribution of genes on the chromosome and the way of gene interaction are not clear. Therefore, it is necessary to precisely locate the genes that control antibacterial traits and conduct cytological genetic analysis on them, so as to apply them to the research of antibacterial aspects and make better use of Senecio in the medical field. The most commonly used method for gene mapping is the combination of chromosome banding technology and fluorescence in situ hybridization technology. The preparation of high-quality chromosome specimen slices is the key step and prerequisite for banding in FISH (fluorescence in situ hybridization) technology. In FISH of mitotic metaphase, the efficiency of hybridization depends on the proximity of the probe to the chromosome, and a large number of metaphases with clear background, dispersed chromosomes and good morphology must be obtained. At the same time, some studies have found that the pyrrolizidine alkaloids in the plant components of Senecio have certain hepatotoxicity, mutagenic toxicity, embryotoxicity, etc., but did not point out the specific species. How to identify whether the poisoned species is similar to certain species in my country It is very important for the development and utilization of Senecio that Chinese herbal medicines of the genus Senecio are of the same species. Karyotype analysis and banding techniques can clarify the composition of biological genomes, identify the morphological characteristics of each chromosome, and provide cytological basis for the identification of species resources. Therefore, it is of great scientific significance to make Senecio-stained specimen slices for the mechanism research and medicinal development of Senecio.

A search of prior art literature found that Li Derong only carried out simple karyotype analysis in the article "Karyon Morphology of Senecio (Asteraceae: Senecio Family) and Its Systematic Significance), while other techniques for chromosome analysis, such as chromosome Separation, chromosome engineering, various banding techniques and in situ hybridization techniques all require high-quality chromosome specimens. Among them, chromosome microdissection in chromosome separation technology has high requirements for chromosome specimens. Therefore, it is of great significance for the follow-up research of Senecio to produce a chromosome specimen with less impurities, good chromosome dispersion, clear shape, and high recognition.

Contents of the invention

The present invention aims at the deficiencies of the prior art and aims to provide a method for preparing Senecio chromosome specimen slices with good dispersion, clear shape and high identification.

A kind of making method of Senecio root tip chromosome specimen sheet in this scheme, comprises the following steps:

Step 1: Select the root tip of the intact white new root on the Senecio plant;

Step 2: Soak the root tip in the ice-water mixture for 15-17 hours;

Step 3: using 0.075mol/L KCl solution to perform hypotonic treatment on the pretreated root tip for 30 minutes;

Step 4: Wash the root tip after the hypotonic treatment in Step 3 with double distilled water, and then fix it with Carnoy's fixative for 24-72 hours;

Step 5: Wash the root tip fixed in Step 4 with double distilled water, and then use a mixed enzyme solution of cellulase and pectinase to enzymatically hydrolyze for 60-90 minutes;

Step 6: wash the enzymatically hydrolyzed root tip with double distilled water and treat it with double distilled water for 40-60 minutes;

Step 7: Cut the front end of the root tip 2-3 mm after the hypotonic treatment in step 6 and put it on the ice slide, then spread the root tip tissue, drip Carnoy's fixative to cover the dispersed area of the tissue, and wait for Carnoy's fixative to cover the scattered area. After the volatilization is complete, drop the modified phenol fuchsin solution to cover the dispersed area of the tissue, cover with a cover glass after staining for 15-20 minutes, absorb the staining solution from the edge of the cover glass, tap the cover glass, then bake and press the slices. Senecio root tip chromosome specimens were obtained.

Beneficial effects of this scheme: 1. The Senecio chromosome sheet production method provided by the present invention does not consume expensive reagents, the pretreatment method is non-toxic, can increase the number of metaphase mitotic figures, and make chromosomes disperse and spread, which is environmentally friendly and economical. 2. The root tip of Senecio is relatively small. The method of making slices used in the present invention can better control the range of root tip tissue migration on the glass slide. On the one hand, it reduces material loss and improves experimental efficiency. On the one hand, it prevents tissue overlap, which is beneficial to The cells are dispersed and the tissue is dispersed as much as possible to reduce the possibility of the cover glass breaking when knocking the slices; in addition, the root tip is placed in the ice-water mixture for 15-17 hours to slow down the speed of cell division and facilitate more metaphase mitosis. In addition, the mixed enzyme solution of cellulase and pectinase was used to enzymatically hydrolyze for 60-90 minutes to remove the cell wall of Senecio. Compared with the acid hydrolysis method, the cell wall was more fully dissociated and the root tip tissue was easier to disperse. 3. Cells with good chromosome shape can be found in the phase contrast microscope without staining in the process of the preparation of the present invention, which can be used in chromosome banding and fluorescence in situ hybridization experiments, and can also be sealed with resin after peeling off for permanent preservation .

In this program, fresh root tips with more mitotic cells are selected, and then soaked in ice-water mixture to slow down their mitotic speed, so as to obtain more metaphase mitotic cells; then use 0.075mol/L KCl solution to hypotonically expand the root tip cells , which is conducive to the dispersion of chromosomes, and then fixed by Carnot’s fixative for 24 to 72 hours to control the dispersion speed of chromosomes; after fixation, the cell wall and other cell structures are further destroyed by enzymatic hydrolysis, and the protoplasts are released, which is easier to compress and get dispersed. Chromosome slices; after enzymatic hydrolysis, low-osmotic treatment with double distilled water is used to reduce the cytoplasmic concentration, clean the chromosome specimen, and disperse the chromosomes outside the membrane; finally, control the dispersion speed of the chromosomes through the combination of borneol and Carnot’s fixative, and control the The distribution area makes the prepared Senecio root tip chromosome specimens have the characteristics of good dispersion, clear shape and high recognition.

Further, in step 4, the raw materials and volume ratios included in Carnot's fixative solution are: absolute alcohol: glacial acetic acid = 3:1.

Further, the mixed enzymolysis solution described in step five is obtained by dissolving cellulase and pectinase in PBS solution; each 10ml of mixed enzymolysis solution contains 0.20-0.25g cellulase and 0.20-0.25g pectinase; enzymolysis , the enzymatic hydrolysis temperature is 35°C to 37°C.

Further, in step 7, use the needle tip to scatter the tissue on the ice slide before tapping the coverslip. The area of the dispersed area of the tissue is smaller than the area of the coverslip. The raw materials and volume ratio included in the fixative are: absolute alcohol: glacial acetic acid equal to 3:1. Adding Carnoy's fixative to chromosomes before staining is beneficial to control the dispersion speed of chromosomes and phenol fuchsin staining.

Description of drawings

Fig. 1 is the effect diagram of microscopic examination of Senecio root tip chromosome specimen sheet in Example 1 of the present invention;

Fig. 2 is the microscopic examination effect diagram of Senecio root tip chromosome specimen sheet in the embodiment 2 of the present invention;

Figure 3 is an enlarged view of A in Figure 2;

Fig. 4 is a microscopic examination effect diagram of the Senecio root tip chromosome specimen slice in Example 3 of the present invention.

Detailed ways

The following is further described in detail through specific implementation methods:

Example 1

A method for making a Senecio root tip chromosome specimen sheet: the material comes from the F1 generation obtained by crossing Zunyi wild high antibacterial Senecio and low antibacterial Senecio, and hydroponics For the young shoots of the F1 generation, when the root length is about 2-3cm, cut off the 0.5cm-long root tip, then put the root tip into the ice-water mixture for 17 hours, take it out after soaking, and use 0.075mol/L KCL solution for hypotonicity Treat for 30min. The root tips after hypotonic treatment were washed with double distilled water, and then fixed with Carnoy's fixative solution (absolute ethanol: glacial acetic acid = 3:1, V/V) for 36 h. The fixed root tips were taken out, washed with water for 30 minutes, and then enzymatically hydrolyzed with a mixed enzyme solution of 2.5% cellulase and 2.5% pectinase at 37°C for 90 minutes; after enzymatic hydrolysis, the root tips were hypotonic in double distilled water for 40 minutes. Production: Cut out the root tip meristematic zone, place it on an ice slide, add a small amount of double distilled water dropwise to keep the root tip in a liquid environment, peel off the root tip with a needle, add an appropriate amount of Carnot’s fixative dropwise, and wait until When the Carnoy's fixative solution was dry, the dye was added dropwise and stained for 20 minutes. Then use tweezers to pick up the coverslip for coverslipping. Use one side of the coverslip to touch the dye solution and then slowly cover the coverslip. No air bubbles will be generated during the coverslip. After covering, gently suck from the edge of the coverslip. To remove the staining solution, try not to move the tissue and liquid between the cover glass and the slide glass, and knock the cover glass after it is out of the floating state, and knock the whole piece evenly and vertically with a blunt object For the cover glass, relative movement between the cover glass and the slide glass is prohibited. The method of knocking on the slide is to cover the slide evenly with force, perpendicular to the plane of the slide, and then bake the slide on the outer flame of the alcohol lamp. The method of baking is that the slide passes After the flame of the alcohol lamp, a thin water mist is produced on the surface and the dye solution does not boil. Finally, wrap the whole slide with a paper towel, and press the slide vertically with your thumb on the paper towel. The maximum force required to destroy the coverslip.

Microscopic examination effect (as shown in Figure 1): Observe the prepared chromosome specimen sheet under the magnification of 1000 times (10×100). It can be used for other chromosome techniques such as karyotype analysis and banding.

Example 2

A method for making a chromosomal specimen sheet of Senecio root tip: hydroponic young young branches of wild Senecio in Zunyi, Guizhou, and when the root length is about 2 to 3 cm, cut the root tip with a length of 0.5 cm, and then put the root tip in ice Soak in the water mixture for 16 hours, take it out after soaking, and treat it with 0.075mol/L KCL solution for 30 minutes. The root tips after hypotonic treatment were washed with double distilled water, and then fixed with Carnoy's fixative solution (absolute ethanol: glacial acetic acid = 3:1, V/V) for 48 hours. The fixed root tip was taken out, washed with water for 30 minutes, and then hydrolyzed with a mixed enzyme solution of 2.5% cellulase and 2.5% pectinase at 37°C for 70 minutes, and then hypotonic in double distilled water for 40 minutes. Production: Cut out the root tip meristematic zone, place it on an ice slide, add a small amount of double distilled water dropwise to keep the root tip in a liquid environment, peel off the root tip with a needle, add an appropriate amount of Carnot’s fixative dropwise, and wait until When the Carnoy's fixative solution was dry, the dye was added dropwise and stained for 20 minutes. Then use tweezers to pick up the coverslip for coverslipping. Use one side of the coverslip to touch the dye solution and then slowly cover the coverslip. No air bubbles will be generated during the coverslip. After covering, gently suck from the edge of the coverslip. To remove the staining solution, try not to move the tissue and liquid between the cover glass and the slide glass, and knock the cover glass after it is out of the floating state, and knock the whole piece evenly and vertically with a blunt object For the cover glass, relative movement between the cover glass and the slide glass is prohibited. The method of knocking on the slide is to cover the slide evenly with force, perpendicular to the plane of the slide, and then bake the slide on the outer flame of the alcohol lamp. The method of baking is that the slide passes After the flame of the alcohol lamp, a thin water mist is produced on the surface and the dye solution does not boil. Finally, wrap the whole slide with a paper towel, and press the slide vertically with your thumb on the paper towel. The maximum force required to destroy the coverslip.

Microscopic examination results (as shown in Figures 2-3): Observe the prepared chromosome specimens under the magnification of 1000 times (10 times eyepiece × 100 times objective lens), the background of the cells under the microscope is very light, the chromosome shape is clear, well dispersed, suitable for Counting can be used for other chromosome techniques such as karyotype analysis and banding without staining.

Example 3

A method for making a Senecio root tip chromosome specimen sheet: the material comes from the F1 generation obtained by crossing Zunyi wild high antibacterial Senecio and low antibacterial Senecio, and hydroponics For the young shoots of the F1 generation, when the root length is about 2-3cm, cut off the 0.5cm-long root tip, then put the root tip into the ice-water mixture for 17 hours, take it out after soaking, and use 0.075mol/L KCL solution for hypotonicity Treat for 30min. The root tips after hypotonic treatment were washed with double distilled water, and then fixed with Carnoy's fixative solution (absolute ethanol: glacial acetic acid = 3:1, V/V) for 58 hours. Take out the fixed root tip, wash it with double distilled water for 3-5 times, and use a mixed enzyme solution of 2.5% cellulase and 2.5% pectinase to enzymolyze it at 37°C for 90 minutes; 40min. Production: cut the root tip meristematic zone, place it on an ice slide, add a small amount of double-distilled water dropwise to keep the root tip in a liquid environment, peel off the root tip with a needle, add the dye solution dropwise, and stain for 20 minutes. Then use tweezers to pick up the coverslip for coverslipping. Use one side of the coverslip to touch the dye solution and then slowly cover the coverslip. No air bubbles will be generated during the coverslip. After covering, gently suck from the edge of the coverslip. To remove the staining solution, try not to move the tissue and liquid between the cover glass and the slide glass, and knock the cover glass after it is out of the floating state, and knock the whole piece evenly and vertically with a blunt object For the cover glass, relative movement between the cover glass and the slide glass is prohibited. The method of knocking on the slide is to cover the slide evenly with force, perpendicular to the plane of the slide, and then bake the slide on the outer flame of the alcohol lamp. The method of baking is that the slide passes After the flame of the alcohol lamp, a thin water mist is produced on the surface and the dye solution does not boil. Finally, wrap the whole slide with a paper towel, and press the slide vertically with your thumb on the paper towel. The maximum force required to destroy the coverslip.

Microscopic examination effect (as shown in Figure 4): Observing the obtained chromosome specimen sheet under the magnification of 1000 times (10 times eyepiece × 100 times objective lens), the background of the cells under the microscope is light, the shape of the chromosome is not clear, the dispersion is not good, and it is not suitable for Counting (mainly due to the lack of fixation of Carnoy's fixative in the film-making process).

What is described above is only an embodiment of the present invention, and common knowledge such as specific structures and characteristics known in the scheme are not described here too much. It should be pointed out that for those skilled in the art, under the premise of not departing from the structure of the present invention, several modifications and improvements can also be made, and these should also be regarded as the protection scope of the present invention, and these will not affect the implementation of the present invention. Effects and utility of patents. The scope of protection required by this application shall be based on the content of the claims, and the specific implementation methods and other records in the specification may be used to interpret the content of the claims.

Claims (4)

1. a kind of preparation method of Senecio root tip chromosome specimen sheet, is characterized in that: comprise the following steps: Step 1: Select the root tip of the intact white new root on the Senecio plant; Step 2: Soak the root tip in the ice-water mixture for 15-17 hours; Step 3: using 0.075mol/L KCl solution to perform hypotonic treatment on the pretreated root tip for 30 minutes; Step 4: Wash the root tip after the hypotonic treatment in Step 3 with double distilled water, and then fix it with Carnoy's fixative for 24-72 hours; Step 5: Wash the root tip fixed in Step 4 with double distilled water, and then use a mixed enzyme solution of cellulase and pectinase to enzymatically hydrolyze for 60-90 minutes; Step 6: wash the enzymatically hydrolyzed root tip with double distilled water and treat it with double distilled water for 40-60 minutes; Step 7: Cut the front end of the root tip 2-3 mm after the hypotonic treatment in step 6 and put it on the ice slide, then spread the root tip tissue, drip Carnoy's fixative to cover the dispersed area of the tissue, and wait for Carnoy's fixative to cover the scattered area. After the volatilization is complete, drop the modified phenol fuchsin solution to cover the dispersed area of the tissue, cover with a cover glass after staining for 15-20 minutes, absorb the staining solution from the edge of the cover glass, tap the cover glass, then bake and press the slices. Senecio root tip chromosome specimens were obtained. 2. the preparation method of Senecio root tip chromosome specimen sheet according to claim 1 is characterized in that: in step 4, the raw material and volume ratio that Carnot's fixative comprises are: dehydrated alcohol: glacial acetic acid=3: 1. 3. The method for making the Senecio root tip chromosome specimen sheet according to claim 1 or 2, characterized in that: the mixed enzymolysis solution described in step 5 is obtained by dissolving cellulase and pectinase in PBS solution; 10ml of mixed enzymatic hydrolysis solution contains 0.20-0.25g cellulase and 0.20-0.25g pectinase; during enzymatic hydrolysis, the enzymatic hydrolysis temperature is 35°C-37°C. 4. the preparation method of Senecio root tip chromosome specimen sheet according to claim 3, is characterized in that: in step 7, before knocking cover glass, earlier scatters tissue on ice slide with needle point, tissue disperse area The area is smaller than the area of the cover glass, and the dripping Carnot's fixative covers the scattered area of the tissue. The raw materials and volume ratio included in the dripping Carnot's fixative are: absolute alcohol: glacial acetic acid is equal to 3:1.