CN110261206B - Method for flaking karyotype analysis of large-leaf chrysosporium - Google Patents
Method for flaking karyotype analysis of large-leaf chrysosporium Download PDFInfo
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- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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Abstract
The invention relates to a method for preparing a slide for analyzing karyotype of a Chrysosplenium macrocarpum, which is a short evergreen herb and has important medicinal development value due to flavonoid. The invention discloses a flaking method suitable for karyotype analysis of a large-leaf chrysosporium, which comprises the processes of obtaining root tips from fresh large-leaf chrysosporium plants, preprocessing, fixing, front hypotonic treatment, enzymolysis wall removal, rear hypotonic treatment, smearing and dyeing, microscopic examination and observation. The invention provides a simple and applicable method for analyzing and flaking the karyotype of the chrysosporium grandiflorum, which provides help for the subsequent scientific research of the chrysosporium grandiflorum and lays a foundation for the classification, development, application and research of chrysosporium plants.
Description
Technical Field
The invention belongs to the fields of cytogenetics and cytobiology, and particularly relates to a chromosome flaking method of a Jinyao.
Background
The Chrysosplenium grandiflorum (Chrysosplenium macrocarpum Oliv.) is a perennial herb of Chrysosplenium genus (Chrysosplenium) of Saxifragaceae family (Saxfragaceae), and has alternate leaves with stems, nearly circular to broad-egg-shaped leaves, 11-13 round teeth at the edges, brown and soft hair on the ventral surface, no hair on the back and brown and soft hair on the leaf stems. The flower stem is sparse to grow brown long and soft hair. A plurality of basal leaves with handles, leaf leathery texture, inverted egg shape, blunt tip, full edge or unobvious microwave-shaped small teeth, wedge-shaped base, fluffy brown and soft hair on the ventral surface and no hair on the back; the cauline leaves are usually 1, the leaves are narrow and oval, the edges are usually provided with 13 round teeth, the back is hairless, and the ventral surface and the edges are fluffy and brownish and soft. Multiple gore parasol inflorescences; inflorescence branching sparse brown soft hair or nearly hairless; from bract oval to broad oval, blunt pointed at the tip, generally with 9-15 scallops (sometimes less pronounced), wedge-shaped at the base; the sepals are nearly oval to broad oval, the tips are slightly concave, and no hairs exist; stamen are elevated to sepals; the ovary is half lower, the flower column is about 5mm long and is nearly straight; without a flower disc. A capsule; the seeds are dark brown, nearly egg-shaped and closely protruded by micro-emulsion heads. The flower and fruit period is 4-6 months.
The golden waist plants are distributed in Asia, Europe, Africa and America in 70 types all around the world, and the Asia temperate zone distribution is taken as the main distribution. China currently finds that about 36 species of Chrysosplenium plants are widely distributed in more than twenty provinces such as Yunnan, Tibet, Sichuan, Guizhou, Hubei, Hunan, northeast and the like.
The plants of the genus Chrysosplenium have high medicinal value due to the fact that the plants are rich in flavonoids, and Chinese plant records, Chinese medicine plant atlas references and Chinese herbal medicine compilation records relate to the medicinal effect records of the plants, and are used for clearing away heat and toxic materials, treating liver and gallbladder diseases and the like. The medicinal history of Jinyao is widely applied and recorded in traditional Chinese medicine, for example, the Tibetan medicine is called as Shijima, and the Di Ma Er Dan Zeng Ping Med of Tibetan medicine works Jingzhu Bencao carries that the Shijima is born in the mountain stonewall, has bitter taste and cool property, is slow in vomiting and diarrhea and treats biliary diseases. "besides, the plants of the genus Chrysosplenium are also recorded in the Mongolian medicine book" No-error Mongolian medicine reference ". In recent years, researches show that the plant of the genus Chrysosplenium generally contains higher flavonoids and triterpenoids, has good antitumor and antiviral activities, the pentacyclic triterpene separated from the C.nudae has strong inhibition effects on malignant brown tumor (A375), 4 kinds of gastric cancer (ST-KM, Kato-III, NKPS, KKLS) and bladder cancer (KK-47), and the specific and ubiquitous Chrysosplenone B and Chrysosplenone C (Chrysosplenol B, Chrysosplenol C) in the plant have obvious antiviral activities. The physiological activity shows that the Chrysosplenium plant is worthy of further research and development.
Chromosomes are the basis of genetic material inherent in an organism and are the primary carriers of genetic material. The size, number and form (i.e. karyotype characteristics) of the chromosome are relatively stable in the growth, development and generation and multiplication processes of plants, are not easily affected by environmental condition changes to generate variation, and can reflect the characteristics and genetic differences of species to a great extent. Therefore, the karyotypic characteristics and even the number of chromosomes of different species can provide cytological evidence for classification, phylogeny and identification of genetic relationship of the plants of the genus. The chrysosplenium plants are numerous, and the differences of different flavonoid components are large, so that the clear identification and the screening of the chrysosplenium species with high medicinal value are the primary tasks of the development and the application of the chrysosplenium plants. When the number, morphology, karyotype, etc. of chromosomes in a specific group of cells are compared and studied, the karyotype characteristics of chromosome specimens for karyotyping must be complete and clear.
According to data, the fact that no method for preparing the chromosome of the plant of the genus Chrysosporium exists at present is found, and the conventional chromosome flaking method, namely the flaking method and the wall-removing hypotonic method have many problems for flaking the chromosome of the great-leaf Chrysosporium, such as difficulty in chromosome dispersion, insufficient stretching, more cytoplasm residues, insufficient dyeing, unstable flaking result and the like. Therefore, obtaining a method suitable for karyotyping of the great-leaf chrysosporium is an urgent problem to be solved at present.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a method suitable for the karyotype analysis and flaking of the chrysosporium grandiflorum, which provides help for the subsequent scientific research of the chrysosporium grandiflorum and lays a foundation for the classification, development and application research of chrysosporium plants.
In one embodiment, the present invention provides a simple and useful method for preparing a karyotype of the great-leaf chrysosporium. The method comprises the following specific steps:
(1) obtaining a root tip: taking the root tip of the gorgeous plant;
(2) pretreatment: pre-treating the root tip in a refrigerator at 4 deg.c for 2-3 hr;
(3) fixing: putting the pretreated root tip into a fixing solution for fixing and storing;
(4) front hypotonic: taking out the root tip, and treating in redistilled water for 40 min;
(5) enzymatic de-walling: adding 0.2mL of 2% mixed enzyme, and putting the mixture into a water bath kettle at 28 ℃ for 4.5 hours in water bath, wherein the mixed enzyme is formed by equal volumes of 4% cellulase and 4% pectinase.
(6) And (3) low-permeability after treatment: absorbing enzyme solution, washing root tip with redistilled water, and placing in redistilled water for hypotonic water bath at 28 deg.C for about 40 min;
(7) knocking-out and dyeing: taking root tips after hypotonic, carrying out knocking treatment, and putting the root tips into a dye vat with an improved giemsa dye liquor for dyeing for 40min, wherein the formula of the improved giemsa dye liquor is as follows: grinding and mixing Giemsa powder and glycerol according to the mass-volume ratio of 2:1 to prepare a mother solution, and mixing the Giemsa mother solution and a phosphate buffer solution according to the volume ratio of 1: 20, performing;
(8) and (6) microscopic observation.
In one embodiment, the golden waist is a big leaf golden waist.
In one embodiment, the step 1) is about 9 am in a sunny day, selecting a luxuriant big-leaf plant with a golden waist growing in a pot plant, taking about 3cm of the root tip part of the plant, and then cleaning soil attached to the surface of the root by using distilled water.
In one embodiment, the step 3) is to suck water from the pretreated root tips, put the root tips into a small bottle containing a fixing solution, and perform fixed storage, or transfer the root tips into a 75% ethanol solution for long-term storage after fixing for 24 hours, and store the root tips in a refrigerator at 4 ℃.
In one embodiment, the step 4) is to take out the root tip with tweezers, then to re-steam and wash, cut the white part of the tip about 1mm from the petri dish, and place the cut tip in an EP tube containing hypotonic solution (double-distilled water) to soak for 40min, during which the hypotonic solution can be changed for 3 times.
In one embodiment, the step 6) is to suck out the enzyme solution, wash the root tip with redistilled water for 2-3 times, and then place the root tip in redistilled water for hypotonic treatment, and water bath is carried out at 28 ℃ for about 40 min.
In one embodiment, said step 7) is. 2-3 root tips after hypotonic are taken to be placed on a glass slide on which a fixing solution (ethanol: glacial acetic acid is 3:1) is dripped, the root tips are crushed as much as possible by a glass rod, cells are dispersed, the crushed histiocytes are spread on the glass slide as uniformly as possible after crushing, the fixing solution is added during the root tip crushing to keep the water environment, and the tissue of the root tips is uniformly dispersed on the glass slide and then is dried by inner flame of an alcohol lamp. Directly placing the dried glass slide into a dye vat with an improved giemsa dye solution, immersing and dyeing for 40min, and after dyeing, washing the glass slide clean with redistilled water for later use, wherein the formula of the improved giemsa dye solution is as follows: grinding and mixing Giemsa powder and glycerol according to the mass-volume ratio of 2:1 to prepare a mother solution, and mixing the Giemsa mother solution and a phosphate buffer solution according to the volume ratio of 1: 20, and (3) performing.
Preferably, the formula of the fixing liquid is as follows: the volume ratio of ethanol to glacial acetic acid is 3: 1;
preferably, the mixed enzyme comprises the following components: the Cellulase is Cellulase R-10, and the pectinase is PECTOLYASE Y-23;
preferably, the improved giemsa dyeing solution comprises the following components: giemsa powder, glycerol, methanol, disodium hydrogen phosphate, potassium dihydrogen phosphate and redistilled water, wherein the dye liquor is prepared by grinding and mixing Giemsa powder and glycerol to prepare mother liquor, and the Giemsa mother liquor and a phosphate buffer solution are mixed according to the volume ratio of 1: 20 for dilution.
In one embodiment, the present invention provides a method for preparing a karyotype of a chrysosporium grandiflorum by parameter adjustment, comprising the following steps:
(1) obtaining a root tip: selecting a luxuriantly growing big leaf plant with a tip part of about 3cm from a pot plant at about 9 am in sunny days, and cleaning soil attached to the surface of the root by using distilled water;
(2) pretreatment: putting the cleaned root tips into a small bottle filled with redistilled water, and putting the small bottle in a refrigerator at 4 ℃ for pretreatment for 2.5 hours;
(3) fixing: removing water from the pretreated root tip, placing into a small bottle containing fixing solution (ethanol: glacial acetic acid: 3:1), fixing and storing, or fixing for 24 hr, transferring into 75% ethanol solution, storing for a long time, and storing in 4 deg.C refrigerator;
(4) front hypotonic: taking out root tip with tweezers, washing with redistilled water, cutting white part of the tip about 1mm from a culture dish, soaking in EP tube containing hypotonic solution (double distilled water) for 40min, wherein the hypotonic solution can be replaced for 3 times;
(5) enzymatic de-walling: after hypotonic reaction, using a liquid transfer machine to suck off hypotonic solution, adding about 0.2mL of 2% mixed enzyme (4% Cellulase R-10, 4% PECTOLYASE Y-23 is prepared into mixed enzyme according to a ratio of 1: 1), then putting into a water bath kettle at 28 ℃ for water bath for 4.5h, and slightly shaking an EP tube for about 3 times in the enzymolysis process to enable the enzyme solution to fully act on the material.
(6) And (3) low-permeability after treatment: absorbing enzyme solution, washing root tip with redistilled water for 2-3 times, and performing hypotonic treatment in redistilled water at 28 deg.C for about 40 min;
(7) knocking-out and dyeing: 2-3 root tips after hypotonic are taken to be placed on a glass slide on which a fixing solution (ethanol: glacial acetic acid is 3:1) is dripped, the root tips are crushed as much as possible by a glass rod, cells are dispersed, the crushed histiocytes are spread on the glass slide as uniformly as possible after crushing, the fixing solution is added during the root tip crushing to keep the water environment, and the tissue of the root tips is uniformly dispersed on the glass slide and then is dried by inner flame of an alcohol lamp. Directly placing the dried glass slide into a dye vat with an improved giemsa dye solution, immersing and dyeing for 40min, and after dyeing, washing the glass slide clean with redistilled water for later use, wherein the formula of the improved giemsa dye solution is as follows: grinding and mixing Giemsa powder and glycerol according to the mass-volume ratio of 2:1 to prepare a mother solution, and mixing the Giemsa mother solution and a phosphate buffer solution according to the volume ratio of 1: 20, performing;
(8) microscopic observation: the ideal, dispersed and clear chromosome was found using an OLYMPUS BX51 microscope, 10 Xeyepiece, 100 Xoleoscope, then photographed with an OLYMPUS DP73 camera and stored.
In one embodiment, the invention provides a slide glass product containing a Goldwaist chromosome Giemsa staining karyotype prepared by the method for preparing a Goldwaist chromosome.
In one embodiment, the invention provides a use of the method for preparing a golden waist chromosome karyotype.
Corresponding to the conventional preparation method suitable for other plant chromosome karyotypes in the prior art, the method suitable for analyzing and preparing the golden waist chromosome karyotype has the following advantages:
in the pretreatment step, the low-temperature treatment can disperse the chromosome of the mallotus macrophyllus better than the use of colchicine and 8-hydroxyquinoline, so that a clearer split phase is obtained, and the observation is convenient.
In the enzymolysis step, experiments show that 2% mixed enzymolysis effect obtained by mixing 4% of cellulase and 4% of pectinase according to equal volume is optimal, and a well-dispersed and clear chromosome map can be obtained. The test compares the treatment results of 1%, 2% and 2.5% of mixed enzyme after equal volume of cellulase and pectinase is mixed; experiments show that the cell wall of the waist of the mallotus macrophyllus can be better broken when the enzymolysis time is 4.5 hours, and the experiments compare the treatment time of 4 hours, 4.5 hours and 5 hours.
In the step of flaking, the method of directly breaking the root tips by using the glass rod is simpler than the method of flaking by using a cover glass, and the cell dispersion effect is better, thereby facilitating microscopic examination.
In the dyeing step, compared with the dyeing liquid of phenol fuchsin and gentian violet, the improved giemsa dyeing liquid can obtain better dyeing effect, and the belt type of the big-leaf golden waist chromosome is clearer.
In the hypotonic step, finding the appropriate hypotonic time may allow the chromosomes to spread out for easy observation.
Correspondingly, the invention can achieve the following beneficial effects:
1. the invention has the advantages of convenient material acquisition and simple and easy operation.
2. The invention can achieve the effects of good and clear chromosome dispersion and good dyeing.
3. The invention lays a foundation for the subsequent chromosome-based research of the broad-leaf chrysosporium.
4. The invention provides help for the follow-up research on chromosomes of other species of the Chrysosplenium.
Drawings
FIG. 1 is a flow chart of the method of the present invention;
FIG. 2 is a diagram showing the karyotype effect of the low-temperature pretreated great-leaf chrysosporium;
FIG. 3 is a graph showing the effect of pretreatment with colchicine, 8-hydroxyquinoline;
FIG. 4 is a graph showing the effect of treatments with different concentrations of mixed enzyme, wherein a is a 1% mixed enzyme treatment group; panel b is 2% mixed enzyme treatment group; FIG. c is a 2.5% mixed enzyme treatment group;
FIG. 5 is a graph showing the effect of karyotype on the thorny leaf.
Detailed Description
In order to better understand the technical scheme of the invention, the technical scheme provided by the invention is described in detail by combining the embodiment.
Example 1 method for preparing karyotype of Rheum palmatum
A method for preparing a chromosome karyotype of a large leaf chrysosporium comprises the following specific experimental steps:
(1) obtaining a root tip: selecting a luxuriantly growing big leaf plant with a tip part of about 3cm from a pot plant at about 9 am in sunny days, and cleaning soil attached to the surface of the root by using distilled water;
(2) pretreatment: putting the cleaned root tips into a small bottle filled with redistilled water, and putting the small bottle in a refrigerator at 4 ℃ for pretreatment for 2 to 3 hours, or adopting a mixed solution of 0.02 percent of colchicine and 2mmol of 8-hydroxyquinoline for pretreatment;
(3) fixing: removing water from the pretreated root tip, placing into a small bottle containing fixing solution (ethanol: glacial acetic acid: 3:1), fixing and storing, or fixing for 24 hr, transferring into 75% ethanol solution, storing for a long time, and storing in 4 deg.C refrigerator;
(4) front hypotonic: taking out root tip with tweezers, washing with redistilled water, cutting white part of the tip about 1mm from a culture dish, soaking in EP tube containing hypotonic solution (double distilled water) for 40min, wherein the hypotonic solution can be replaced for 1-3 times;
(5) enzymatic de-walling: after hypotonic, using a pipette to suck the hypotonic solution, adding about 0.2mL of 1% -2.5% mixed enzyme (Cellulase and pectinase, wherein the Cellulase is Cellulase R-10, and the pectinase is PECTOLYASE Y-23), then putting into a water bath kettle at 28 ℃ for 4-4.5h in a water bath, and slightly shaking an EP tube for about 3 times in the enzymolysis process to enable the enzyme solution to fully act on the material.
(6) And (3) low-permeability after treatment: absorbing enzyme solution, washing root tip with redistilled water for 2-3 times, and performing hypotonic treatment in redistilled water at 28 deg.C for about 40 min;
(7) knocking-out and dyeing: 2-3 root tips after hypotonic are taken to be placed on a glass slide on which a fixing solution (ethanol: glacial acetic acid is 3:1) is dripped, the root tips are crushed as much as possible by a glass rod, cells are dispersed, the crushed histiocytes are spread on the glass slide as uniformly as possible after crushing, the fixing solution is added during the root tip crushing to keep the water environment, and the tissue of the root tips is uniformly dispersed on the glass slide and then is dried by inner flame of an alcohol lamp. Directly placing the dried glass slide into a dye vat with an improved giemsa dye solution, immersing and dyeing for 40min, and after dyeing, washing the glass slide clean with redistilled water for later use, wherein the formula of the improved giemsa dye solution is as follows: firstly, grinding and mixing Giemsa powder and glycerol according to the mass-volume ratio of 2:1-1:2 to prepare a mother solution, and mixing the Giemsa mother solution and a phosphate buffer solution according to the volume ratio of 1: 20, performing;
(8) microscopic observation: the ideal, dispersed and clear chromosome was found by observation under an OLYMPUS BX51 microscope with 10 Xeyepiece and 100 Xoleoscope, and then photographed with an OLYMPUS DP73 camera and the photograph was saved.
Example 2 Effect of different pretreatment regimens on the karyotype of the Chrysosplenium megatherium
In order to explore the influence of different treatment modes on the karyotype of the chrysosporium grandiflorum, and seek to find the most suitable pretreatment mode for preparing the karyotype of the chrysosporium grandiflorum, the implementation adopts the following experimental design:
comparing the 4 ℃ low-temperature treatment group with the colchicine and 8-hydroxyquinoline mixed solution treatment group, and setting 3 parallel experiments for each group on the influence of the chromosome karyotype of the leaf-golden waist, wherein the specific steps are as follows:
1. experimental methods
(1) Obtaining a root tip: selecting a luxuriantly growing big leaf plant with a tip part of about 3cm from a pot plant at about 9 am in sunny days, and cleaning soil attached to the surface of the root by using distilled water;
(2) pretreatment: putting the cleaned root tips into a small bottle containing redistilled water, putting the small bottle in a refrigerator at 4 ℃, and performing pretreatment for 2.5h (low-temperature treatment group), or performing pretreatment by adopting a mixed solution of 0.02% of colchicine and 2mmol of 8-hydroxyquinoline 1:1 (mixed solution treatment group);
(3) fixing: removing water from the pretreated root tip, placing into a small bottle containing fixing solution (ethanol: glacial acetic acid: 3:1), fixing and storing, or fixing for 24 hr, transferring into 75% ethanol solution, storing for a long time, and storing in 4 deg.C refrigerator;
(4) front hypotonic: taking out root tip with tweezers, washing with redistilled water, cutting white part of the tip about 1mm from a culture dish, soaking in EP tube containing hypotonic solution (double distilled water) for 40min, wherein the hypotonic solution can be replaced for 3 times;
(5) enzymatic de-walling: after hypotonic, using a pipette to suck the hypotonic solution, adding about 0.2mL of 1% -2.5% mixed enzyme (Cellulase and pectinase, wherein the Cellulase is Cellulase R-10, and the pectinase is PECTOLYASE Y-23), then putting into a water bath kettle at 28 ℃ for 4-4.5h in a water bath, and slightly shaking an EP tube for about 3 times in the enzymolysis process to enable the enzyme solution to fully act on the material.
(6) And (3) low-permeability after treatment: absorbing enzyme solution, washing root tip with redistilled water for 2-3 times, and performing hypotonic treatment in redistilled water at 28 deg.C for about 40 min;
(7) knocking-out and dyeing: 2-3 root tips after hypotonic are taken to be placed on a glass slide on which a fixing solution (ethanol: glacial acetic acid is 3:1) is dripped, the root tips are crushed as much as possible by a glass rod, cells are dispersed, the crushed histiocytes are spread on the glass slide as uniformly as possible after crushing, the fixing solution is added during the root tip crushing to keep the water environment, and the tissue of the root tips is uniformly dispersed on the glass slide and then is dried by inner flame of an alcohol lamp. Directly placing the dried glass slide into a dye vat with an improved giemsa dye solution, immersing and dyeing for 40min, and after dyeing, washing the glass slide clean with redistilled water for later use, wherein the formula of the improved giemsa dye solution is as follows: firstly, grinding and mixing Giemsa powder and glycerol according to the mass-volume ratio of 2:1-1:2 to prepare a mother solution, and mixing the Giemsa mother solution and a phosphate buffer solution according to the volume ratio of 1: 20, performing;
(8) microscopic observation: the ideal, dispersed and clear chromosome was found by observation under an OLYMPUS BX51 microscope with 10 Xeyepiece and 100 Xoleoscope, and then photographed with an OLYMPUS DP73 camera and the photograph was saved.
2. Results of the experiment
As can be seen from microscopic examination results, the number of the metaphase karyotype of the megakaryoid chromosome obtained by pretreating for 2.5h in a refrigerator at 4 ℃ is obviously more than that obtained by pretreating for 2.5h by using a mixed solution of 0.02% of colchicine and 2mmol of 8-hydroxyquinoline 1: 1. As can be seen from the results of FIGS. 2-3, the chromosome shrinkage of the cryogenically treated chromosome is appropriate in length and size, while the chromosome of the golden loin of the lobule pretreated with a mixture of 0.02% colchicine and 2mmol of 8-hydroxyquinoline 1:1 is excessively shrunk, has a short length and a fuzzy chromosome shape, and is not suitable for the test operations such as the karyotype analysis and the staining hybridization at the later stage. Therefore, the pretreatment is carried out for 2.5h in a refrigerator at 4 ℃, which is the best pretreatment step for preparing the karyotype of the chrysosporium grandiflorum.
Example 3 Effect of different enzymatic parameters on the karyotype of the Chrysosplenium megalobium
In order to explore the influence of different enzymolysis parameters on the karyotype of the chrysosporium grandiflorum, and seek to find the most suitable enzymolysis parameter mode for preparing the karyotype of the chrysosporium grandiflorum, the following experimental design is adopted in the implementation:
1% mixed enzyme treatment group: preparing mixed enzyme from 4% Cellulase R-10 and 4% PECTOLYASE Y-23 at a ratio of 1:1, and adding 1% of the mixed enzyme during enzymolysis;
2% mixed enzyme treatment group: preparing mixed enzyme from 4% Cellulase R-10 and 4% PECTOLYASE Y-23 at a ratio of 1:1, and adding 2% of the mixed enzyme during enzymolysis;
2.5% mixed enzyme treatment group: preparing mixed enzyme from 4% Cellulase R-10 and 4% PECTOLYASE Y-23 at a ratio of 1:1, and adding 2.5% of the mixed enzyme during enzymolysis;
each group is provided with 3 parallel experiments, and the specific steps are as follows:
1. experimental methods
(1) Obtaining a root tip: selecting a luxuriantly growing big leaf plant with a tip part of about 3cm from a pot plant at about 9 am in sunny days, and cleaning soil attached to the surface of the root by using distilled water;
(2) pretreatment: putting the cleaned root tips into a small bottle filled with redistilled water, and putting the small bottle in a refrigerator at 4 ℃ for pretreatment for 2.5 hours;
(3) fixing: removing water from the pretreated root tip, placing into a small bottle containing fixing solution (ethanol: glacial acetic acid: 3:1), fixing and storing, or fixing for 24 hr, transferring into 75% ethanol solution, storing for a long time, and storing in 4 deg.C refrigerator;
(4) front hypotonic: taking out root tip with tweezers, washing with redistilled water, cutting white part of the tip about 1mm from a culture dish, soaking in EP tube containing hypotonic solution (double distilled water) for 40min, wherein the hypotonic solution can be replaced for 3 times;
(5) enzymatic de-walling: after hypotonic, using a pipette to suck the hypotonic solution, adding corresponding mixed enzyme according to the steps of the 1% mixed enzyme treatment group, the 2% mixed enzyme treatment group and the 2.5% mixed enzyme treatment group, then putting the mixture into a water bath kettle at 28 ℃ for water bath for 4.5h, and slightly shaking the EP tube for about 3 times in the enzymolysis process to ensure that the enzyme solution fully acts on the material.
(6) And (3) low-permeability after treatment: absorbing enzyme solution, washing root tip with redistilled water for 2-3 times, and performing hypotonic treatment in redistilled water at 28 deg.C for about 40 min;
(7) knocking-out and dyeing: 2-3 root tips after hypotonic are taken to be placed on a glass slide on which a fixing solution (ethanol: glacial acetic acid is 3:1) is dripped, the root tips are crushed as much as possible by a glass rod, cells are dispersed, the crushed histiocytes are spread on the glass slide as uniformly as possible after crushing, the fixing solution is added during the root tip crushing to keep the water environment, and the tissue of the root tips is uniformly dispersed on the glass slide and then is dried by inner flame of an alcohol lamp. Directly placing the dried glass slide into a dye vat with an improved giemsa dye solution, immersing and dyeing for 40min, and after dyeing, washing the glass slide clean with redistilled water for later use, wherein the formula of the improved giemsa dye solution is as follows: firstly, grinding and mixing Giemsa powder and glycerol according to the mass-volume ratio of 2:1-1:2 to prepare a mother solution, and mixing the Giemsa mother solution and a phosphate buffer solution according to the volume ratio of 1: 20, performing;
(8) microscopic observation: the ideal, dispersed and clear chromosome was found using an OLYMPUS BX51 microscope, 10 Xeyepiece, 100 Xoleoscope, then photographed with an OLYMPUS DP73 camera and stored.
2. Results of the experiment
As can be seen from FIG. 4, the different concentrations of the mixed enzymes have a great influence on the definition of the chromosome karyotype, 1% of the mixed enzymes have too weak enzymolysis effect, the cell wall is not completely removed, the chromosome karyotype is wrapped in a thick film, karyotype analysis cannot be performed, 2.5% of the mixed enzymes have too large enzymolysis effect, the chromosome karyotype becomes fuzzy, 2% of the mixed enzymes can obtain the clear chromosome karyotype, and thus, 4% of Cellulase R-10 and 4% of PECTOLYASE Y-23 are prepared into the mixed enzymes according to the ratio of 1:1, and 2% of the mixed enzymes are added during enzymolysis to obtain the optimal enzymolysis concentration for preparing the chromosome karyotype of the golden waist.
Further, in order to explore the influence of different enzymolysis times on the chromosome karyotype of the chrysosporium grandiflorum, the most suitable enzymolysis time for preparing the chromosome karyotype of the chrysosporium grandiflorum is sought, and the following experiment is further designed:
4h group: putting the 2% mixed enzyme into a water bath kettle at 28 ℃ for 4 h;
4.5h group: putting the 2% mixed enzyme into a water bath kettle at 28 ℃ for water bath for 4.5 hours;
and 5h group: putting the 2% mixed enzyme into a water bath kettle at 28 ℃ for water bath for 5 hours;
each group is provided with 3 parallel experiments, and the specific steps are as follows:
1. experimental methods
The same experimental procedure as described above was used, wherein in step (5) the enzymatic wall removal, comparative experiments were performed for 4h, 4.5h and 5h, respectively.
2. Results of the experiment
From the final microscopic examination result, it is known that, compared with other enzymolysis time, the clear chromosome karyotype can be obtained by enzymolysis for 4.5h, and therefore, the enzymolysis for 4.5h is the best enzymolysis time for preparing the great-leaf chrysosporium chromosome karyotype.
EXAMPLE 4 Effect of different staining patterns on the preparation of the karyotype of the Chrysosporium grandiflorum
In order to explore the influence of different staining modes on the karyotype of the chrysosporium grandiflorum, and seek to find out the most suitable staining mode for preparing the karyotype of the chrysosporium grandiflorum, the following experimental design is adopted in the implementation:
giemsa staining group: staining with conventional Giemsa solution
Modified giemsa staining group 1: grinding and mixing Giemsa powder and glycerol according to the mass-volume ratio of 2:1 to prepare a mother solution, and mixing the Giemsa mother solution and a phosphate buffer solution according to the volume ratio of 1: 20 proceed with
Modified gimsa staining group 2: grinding and mixing Giemsa powder and glycerol according to the mass-to-volume ratio of 1:2 to prepare a mother solution, and mixing the Giemsa mother solution and a phosphate buffer solution according to the volume ratio of 1: 20, performing;
phenol magenta dye set: dyeing with phenol fuchsin;
gentian violet staining group: dyeing with gentian violet;
each group is provided with 3 parallel experiments, and the specific steps are as follows:
1. experimental methods
(1) Obtaining a root tip: selecting a luxuriantly growing big leaf plant with a tip part of about 3cm from a pot plant at about 9 am in sunny days, and cleaning soil attached to the surface of the root by using distilled water;
(2) pretreatment: putting the cleaned root tips into a small bottle filled with redistilled water, and putting the small bottle in a refrigerator at 4 ℃ for pretreatment for 2.5 hours;
(3) fixing: removing water from the pretreated root tip, placing into a small bottle containing fixing solution (ethanol: glacial acetic acid: 3:1), fixing and storing, or fixing for 24 hr, transferring into 75% ethanol solution, storing for a long time, and storing in 4 deg.C refrigerator;
(4) front hypotonic: taking out root tip with tweezers, washing with redistilled water, cutting white part of the tip about 1mm from a culture dish, soaking in EP tube containing hypotonic solution (double distilled water) for 40min, wherein the hypotonic solution can be replaced for 3 times;
(5) enzymatic de-walling: after hypotonic, using a pipette to suck the hypotonic solution, adding about 0.2mL of 2% mixed enzyme (Cellulase and pectinase, wherein the Cellulase is 4% Cellulase R-10, and the pectinase is 4% PECTOLYASE Y-23), then putting into a water bath kettle at 28 ℃ for water bath for 4.5h, and slightly shaking an EP tube for about 3 times in the enzymolysis process to enable the enzyme solution to fully act on the material.
(6) And (3) low-permeability after treatment: absorbing enzyme solution, washing root tip with redistilled water for 2-3 times, and performing hypotonic treatment in redistilled water at 28 deg.C for about 40 min;
(7) knocking-out and dyeing: 2-3 root tips after hypotonic are taken to be placed on a glass slide on which a fixing solution (ethanol: glacial acetic acid is 3:1) is dripped, the root tips are crushed as much as possible by a glass rod, cells are dispersed, the crushed histiocytes are spread on the glass slide as uniformly as possible after crushing, the fixing solution is added during the root tip crushing to keep the water environment, and the tissue of the root tips is uniformly dispersed on the glass slide and then is dried by inner flame of an alcohol lamp. The dried slide glass was stained by the aforementioned Giemsa staining group, modified Giemsa staining group 1, modified Giemsa staining group 2, phenol magenta staining group, and gentian violet staining group
(8) Microscopic observation: the ideal, dispersed and clear chromosome was found by observation under an OLYMPUS BX51 microscope with 10 Xeyepiece and 100 Xoleoscope, and then photographed with an OLYMPUS DP73 camera and the photograph was saved.
2. Results of the experiment
As can be seen from the microscopic examination result, the karyotype obtained by improving the Giemsa chromosome group 1 is the clearest, and clear banding patterns appear in the chromosome, so that the chromosome identification and the subsequent karyotype analysis experiment are facilitated. Therefore, the Giemsa powder and the glycerol are ground and mixed according to the mass volume ratio of 2:1 to prepare a mother solution, and the Giemsa mother solution and a phosphate buffer solution are mixed according to the volume ratio of 1: 20 is the best staining mode for the preparation of the karyotype of the great-leaf chrysosporium.
Example 5 preparation of a karyotype of the Chrysosporium grandiflorum obtained by combining the optimal parameters of examples 2-3
A method for preparing the karyotype of the great-leaf chrysosporium by parameter adjustment comprises the following specific steps:
(1) obtaining a root tip: selecting a luxuriantly growing big leaf plant with a tip part of about 3cm from a pot plant at about 9 am in sunny days, and cleaning soil attached to the surface of the root by using distilled water;
(2) pretreatment: putting the cleaned root tips into a small bottle filled with redistilled water, and putting the small bottle in a refrigerator at 4 ℃ for pretreatment for 2.5 hours;
(3) fixing: removing water from the pretreated root tip, placing into a small bottle containing fixing solution (ethanol: glacial acetic acid: 3:1), fixing and storing, or fixing for 24 hr, transferring into 75% ethanol solution, storing for a long time, and storing in 4 deg.C refrigerator;
(4) front hypotonic: taking out root tip with tweezers, washing with redistilled water, cutting white part of the tip about 1mm from a culture dish, soaking in EP tube containing hypotonic solution (double distilled water) for 40min, wherein the hypotonic solution can be replaced for 3 times;
(5) enzymatic de-walling: after hypotonic reaction, using a liquid transfer machine to suck off hypotonic solution, adding about 0.2mL of 2% mixed enzyme (4% Cellulase R-10, 4% PECTOLYASE Y-23 is prepared into mixed enzyme according to a ratio of 1: 1), then putting into a water bath kettle at 28 ℃ for water bath for 4.5h, and slightly shaking an EP tube for about 3 times in the enzymolysis process to enable the enzyme solution to fully act on the material.
(6) And (3) low-permeability after treatment: absorbing enzyme solution, washing root tip with redistilled water for 2-3 times, and performing hypotonic treatment in redistilled water at 28 deg.C for about 40 min;
(7) knocking-out and dyeing: 2-3 root tips after hypotonic are taken to be placed on a glass slide on which a fixing solution (ethanol: glacial acetic acid is 3:1) is dripped, the root tips are crushed as much as possible by a glass rod, cells are dispersed, the crushed histiocytes are spread on the glass slide as uniformly as possible after crushing, the fixing solution is added during the root tip crushing to keep the water environment, and the tissue of the root tips is uniformly dispersed on the glass slide and then is dried by inner flame of an alcohol lamp. Directly placing the dried glass slide into a dye vat with an improved giemsa dye solution, immersing and dyeing for 40min, and after dyeing, washing the glass slide clean with redistilled water for later use, wherein the formula of the improved giemsa dye solution is as follows: grinding and mixing Giemsa powder and glycerol according to the mass-volume ratio of 2:1 to prepare a mother solution, and mixing the Giemsa mother solution and a phosphate buffer solution according to the volume ratio of 1: 20, performing;
(8) microscopic observation: the ideal, dispersed and clear chromosome was found by observation under an OLYMPUS BX51 microscope with 10 Xeyepiece and 100 Xoleoscope, and then photographed with an OLYMPUS DP73 camera and the photograph was saved.
As can be seen from the effect of FIG. 5, the method of example 5 can obtain a karyotype of the Chrysosporium grandiflorum with good dispersity, suitable chromosome length and clear banding pattern
The invention has been described in detail with respect to a general description and specific embodiments thereof, but it will be apparent to those skilled in the art that modifications and improvements can be made based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (1)
1. A method for preparing a golden waist chromosome slide comprises the following specific steps of (1) obtaining root tips: selecting a luxuriantly growing big leaf plant with a tip part of about 3cm from a pot plant at about 9 am in sunny days, and cleaning soil attached to the surface of the root by using distilled water;
(2) pretreatment: putting the cleaned root tips into a small bottle filled with redistilled water, and putting the small bottle in a refrigerator at 4 ℃ for pretreatment for 2.5 hours;
(3) fixing: removing water from the pretreated root tip, and putting the root tip into a small bottle filled with a fixing solution, wherein the fixing solution is ethanol: the glacial acetic acid is 3:1, and is fixedly stored;
(4) front hypotonic: taking out root tip with tweezers, washing with redistilled water, cutting white part of the tip about 1mm from a culture dish, soaking in EP tube containing double distilled water for 40min, and replacing hypotonic solution for 3 times;
(5) enzymatic de-walling: after hypotonic reaction, sucking off hypotonic solution by using a liquid transfer device, adding 0.2mL of 2% mixed enzyme, then putting the mixture into a water bath kettle at 28 ℃ for water bath for 4.5h, and slightly shaking an EP (ethylene propylene glycol) tube for 3 times in the enzymolysis process to enable the enzyme solution to fully act on the material, wherein the mixed enzyme is 4% cellulase and 4% pectinase, and the ratio of the mixed enzyme to the pectinase is 1: 1;
(6) and (3) low-permeability after treatment: absorbing enzyme solution, washing root tip with redistilled water for 2-3 times, and performing hypotonic treatment in redistilled water at 28 deg.C for about 40 min;
(7) knocking-out and dyeing: 2-3 root tips after hypotonic are taken and placed on a glass slide on which a fixing solution is dripped, wherein the fixing solution is ethanol: 1, crushing a root tip with a glass rod as much as possible, dispersing cells, uniformly spreading the crushed histiocytes on a glass slide as much as possible after crushing, adding a fixing solution during crushing the root tip to keep a water environment, uniformly dispersing the tissue of the root tip on the glass slide, drying the tissue of the root tip by using an alcohol lamp inner flame, directly putting the dried glass slide into a dye vat with an improved Giemsa dye solution, immersing and dyeing for 40min, and washing the glass slide clean with redistilled water for later use, wherein the formula of the improved Giemsa dye solution is as follows: grinding and mixing Giemsa powder and glycerol according to the mass-volume ratio of 2:1 to prepare a mother solution, and mixing the Giemsa mother solution and a phosphate buffer solution according to the volume ratio of 1: 20, performing;
(8) microscopic observation: the ideal dispersed and clear chromosome is found by using an OLYMPUS BX51 bright field microscope, a 10-fold ocular lens and a 100-fold oil lens for observation, then the chromosome is photographed by using an OLYMPUS DP73 camera, and the picture is stored.
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