Method for preparing procambarus clarkii embryo chromosomes

Abstract

The present application belongs to the field of animal cytology experimental technology, and specifically discloses a preparation method of crayfish embryo chromosomes, comprising the following steps: taking crayfish embryos in the nauplius stage, immersing in a colchicine solution, culturing in a constant-temperature incubator, discarding supernatant after centrifugation, and obtaining cell mass precipitate; then, low penetration, fixation, sheet preparation, sheet spraying, staining and microscope examination are performed, and the crayfish embryo chromosomes are obtained. The present application first uses crayfish embryos in the nauplius stage to prepare chromosomes, overcomes the limitation of sample quantity, does not need to dissect parent crayfish, and has the advantages of clean division phase, more division phase quantity, clear chromosome quantity and morphology, which is beneficial to the analysis of chromosome type and chromosome heredity; the operation is simple, the preparation period is short, and the cost is low. Finally, the present application can obtain clear somatic cell chromosomes of 188 in the crayfish.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of animal cell experiment, and provides a preparation method of crayfish embryo chromosome. BACKGROUND

[0002] Procambarus clarkia, the scientific name of which is Procambarus clarkia, belongs to Arthropoda, Crustacea, Decapoda, Reptantia, Astacidae and Cambarus in taxonomy. The crayfish is a wide-temperature crayfish with strong adaptability, high breeding rate, mixed diet, fast growth and non-death within several hours away from water. The crayfish meat is tender and nutritious, rich in eight kinds of amino acids necessary for human body, and low in fat content, and contains more tropomyosin and paramyosin. It is deeply loved by consumers, and the export volume is also increasing day by day. The sales and purchase prices are rising continuously, and the breeding prospects and benefits are good. At present, the crayfish on the market has the problems of self-breeding, close cross and the like, resulting in more and more small crayfish and the problems of small head and less meat. In order to further study the genetic breeding and variety improvement of the crayfish, it is necessary to study the chromosome of the crayfish. Through the study of the chromosome of the crayfish, the crayfish can be improved at the level of chromosome operation.

[0003] Since the chromosome of Procambarus clarkii is small in shape and large in number, the previous method for preparing the chromosome of Procambarus clarkii mainly uses sperm or ovary of killed crayfish to prepare chromosome or uses antennal gland of killed crayfish to prepare somatic chromosome, a large number of crayfish need to be killed, and the chromosome split phase has impurities, so that the number and shape of the chromosome are not clear, which is not conducive to the analysis of the chromosome type and the genetic analysis of the chromosome. SUMMARY

[0004] The technical problem to be solved by the present application is to overcome the deficiencies and defects mentioned in the background, and to provide a preparation method of crayfish embryo chromosome, which uses embryo to prepare chromosome, does not need to kill a large number of crayfish, and the chromosome split phase is clean, the number and shape of the chromosome are clear, which is conducive to the analysis of the chromosome type and the genetic analysis of the chromosome.

[0005] To solve the above technical problems, the technical solution provided by the present application is as follows:

[0006] A preparation method of crayfish embryo chromosome, comprising the following steps:

[0007] (1) preliminary treatment: taking crayfish embryo at the nauplius stage, immersing in colchicine solution, culturing in a constant temperature incubator, centrifuging and discarding the supernatant to obtain cell mass precipitate;

[0008] (2) low penetration: removing the colchicine solution, performing low penetration treatment, and removing the low penetration liquid;

[0009] (3) Fixing: the cell mass obtained after step (2) is pre-fixed and post-fixed;

[0010] (4) Preparing slice: the embryo obtained after step (3) is taken into a centrifugal tube, and the cell is digested by adding chain protease, and then is softened by adding acetic acid solution, and then the fertilization membrane of the embryo is torn off, and the embryo cell is broken up to be dispersed, and then the upper liquid is poured off after centrifugation, and then the cell suspension is obtained by blowing and beating with a pipette after adding fixing solution;

[0011] (5) Spraying slice: the pre-cooled glass slide is placed obliquely, and the cell suspension obtained in step (4) is taken by a dropper and sprayed vertically on the glass slide, and then the glass slide is baked on the outer flame of an alcohol lamp and dried in air;

[0012] (6) Staining: the glass slide on which the chromosomes are attached obtained in step (5) is stained by Giemsa staining solution;

[0013] (7) Microscopic examination: observation and photography are carried out under a microscope.

[0014] In the above preparation method, preferably, in step (1), the concentration of the colchicine solution is 500 mg / mL.

[0015] Preferably, in step (1), the temperature of the constant temperature incubator is 22-25℃, and the incubation time is 3-4 h; the speed of centrifugation is 1000-1200 r / min, and the time is 5-10 min.

[0016] Preferably, in step (2), the hypotonic treatment is hypotonic treatment with 0.75% KCl at 37℃ for 20-30 min.

[0017] Preferably, in step (3), the pre-fixing and post-fixing are that the cell obtained after removing the hypotonic solution is pre-fixed in Carnoy's fixing solution pre-cooled to 4℃ for 1.5-3 h, and the pre-fixing solution is replaced twice, and then the cell is post-fixed in post-fixing solution pre-cooled to 4℃.

[0018] More preferably, the Carnoy's fixing solution is obtained by mixing methanol and glacial acetic acid at a volume ratio of 3:1, and the post-fixing solution is obtained by mixing methanol and glacial acetic acid at a volume ratio of 1:1.

[0019] Preferably, in step (4), the concentration of the chain protease is 2 mg / mL, and the digestion time is 20 min; the volume concentration of the acetic acid is 50-60%, and the softening time is 15-20 min.

[0020] Preferably, in step (4), the centrifugation speed is 1000-1200r / min, and the time is 5-10min; the post-fixing solution is obtained by mixing methanol and glacial acetic acid at a volume ratio of 1:1.

[0021] Preferably, in step (5), the baking time is 2-3s.

[0022] Preferably, in step (6), the Giemsa staining solution is prepared as follows: 0.5g of Giemsa particles is weighed into a mortar, a little of glycerol is added, the Giemsa particles are crushed, and glycerol is added to make the total amount 33mL, and the mixture is mixed to obtain a mixed solution; the mixed solution is heated at 60℃ for 90min in a water bath, 33mL of methanol is added and mixed, and then filtered into a brown glass bottle while hot, and placed in a cool and dry place for aging for at least half a month, and then diluted with a phosphate buffer at a volume ratio of 9:1 for use. The staining concentration obtained by this method is uniform, and there are basically no large staining particles.

[0023] Compared with the prior art, the present application has the following beneficial effects:

[0024] The present application first uses the nauplius stage of the crayfish embryos to prepare chromosomes, overcomes the limitation of sample quantity, does not need to dissect the parent crayfish, and has clean mitotic phases, more mitotic phase numbers, and clear chromosome numbers and shapes, which is beneficial to the karyotype and genetic analysis of the chromosomes; the operation is simple, the preparation period is short, and the cost is low. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0026] Figure 1 The self-made sharp forceps used for peeling the egg envelope of the crayfish.

[0027] Figure 2 The peeling comb used for peeling the egg envelope of the crayfish.

[0028] Figure 3 It is a photo of the crayfish in the process of carrying eggs.

[0029] Figure 4 It is a diagram of the embryonic development of the crayfish at different stages.

[0030] Figure 5 It is a picture of the embryonic chromosome mitotic phase of the crayfish prepared by the present application. DETAILED DESCRIPTION

[0031] For the convenience of understanding the present application, the present application will be described more fully below with reference to the accompanying drawings and preferred embodiments of the application, but the scope of the present application is not limited to the following specific embodiments.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The materials used in the present application are only for the purpose of describing specific embodiments, and are not intended to limit the scope of the present application.

[0033] Unless otherwise specifically indicated, various materials, reagents, instruments and equipment used in the present application can be purchased on the market or can be prepared by existing methods.

[0034] The present application uses self-made sharp tweezers and stripping comb to strip the outer membrane of shrimp eggs.

[0035] As shown in Figure 1 The biggest difference between the tweezers and conventional tweezers is that the head of the tweezers is very sharp, which is polished multiple times by artificial sanding, and the sharp tip is powerful (indicated by the two arrows on the left), which can easily penetrate the embryo membrane without damaging the embryo. In addition, the self-made tweezers have a very suitable arc (60 degrees) (indicated by the rightmost arrow), which is beneficial to hook the embryo membrane and facilitate the tearing off of the degraded or acidified embryo membrane. The head of the ordinary tweezers is relatively rough and has a certain thickness, which is easy to touch the embryo when clamping the embryo membrane.

[0036] As shown in Figure 2 The self-made stripping comb of the present application has uniform spacing between the comb gears (indicated by the left arrow), and the spacing between the comb gears can be adjusted according to the diameter of the egg (indicated by the right arrow), which is not easy to scratch the egg when stripping the egg, and is beneficial to the greatest extent to protect the embryo of the crayfish.

[0037] Example 1:

[0038] A preparation method of crayfish embryo chromosomes according to the present application, comprising the following steps:

[0039] 1. Preparation of reagents and related materials:

[0040] (1) Preparation of physiological saline: 0.8 g of NaCl is dissolved in deionized water to make up to 100 mL;

[0041] (2) Phytohemagglutinin (PHA): concentration 2 mg / mL, solvent is sterile physiological saline;

[0042] (3) Colchicine: 500 mg / mL, solvent is sterile physiological saline;

[0043] (4) 0.75% KCl solution: 0.75 g KCl was dissolved in 100 mL sterilized ddH2O;

[0044] (5) Pre-fixing solution: prepared by mixing methanol and glacial acetic acid at a volume ratio of 3:1, and used immediately;

[0045] (6) Post-fixing solution: prepared by mixing methanol and glacial acetic acid at a volume ratio of 1:1, and used immediately;

[0046] (7) Giemsa staining solution: 0.5 g of Giemsa particles was placed in a mortar, a small amount of glycerol was added, and the Giemsa particles were crushed, then 33 mL of glycerol was added, and the mixture was mixed well. The mixture was poured into a small beaker and heated in a 60°C water bath for 90 min, then 33 mL of methanol was added and mixed well, and then filtered into a brown glass bottle while hot. It is placed in a cool and dry place and matures for half a month before use. When used, it is diluted with phosphate buffer at a ratio of 9:1 to prepare the staining working solution.

[0047] (8) Glass slide treatment: soak in 2% hydrochloric acid alcohol (95% alcohol 100 parts, add concentrated hydrochloric acid 2 parts) for several hours, then rinse with running water and let the water drip dry. After taking out, dry in the clean bench, seal the dried slides and store in the 4°C refrigerator.

[0048] 2. Select healthy and well-developed small crayfish embryos (as shown in Figure 3 ), and follow the steps below to prepare chromosomes:

[0049] (1) Pre-treatment: take the embryos at the nauplius stage (as shown in Figure 4 ) and incubate them in a 22-25°C constant temperature incubator with 500 mg / mL colchicine solution for 3.5 h; centrifuge at 1000 r / min for 5 min, discard the supernatant, and obtain the cell mass;

[0050] (2) Hypotonic treatment: remove the colchicine solution and use 0.75% KCl hypotonic solution for hypotonic treatment at 37°C for 25 min, and carefully remove the hypotonic solution;

[0051] (3) Fixation: add pre-cooled Carnoy's fixative (methanol: glacial acetic acid = 3:1) to 4°C, fix at 4°C for 1.5-3 h, replace the Carnoy's fixative twice in the middle, and then perform post-fixation in pre-cooled post-fixing solution (methanol: glacial acetic acid = 1:1) at 4°C;

[0052] (4) Slide preparation: take the embryos obtained after step (3) fixation in a centrifuge tube, and add 2 mg / mL concentration of chainase protease for digestion for 20 min, then use 55% volume concentration of acetic acid for softening for 20 min, and then use self-made forceps and stripping comb (as shown in Figure 1 and Figure 2) tearing the fertilization membrane of the embryo, then quickly crushing to dissociate and disperse the embryo cells, centrifuging at 1000r / min for 5min, then pouring away the upper liquid, adding fixing liquid and uniformly blowing with a pipette to obtain a cell suspension;

[0053] (5) spraying the slide: placing a pre-cooled slide obliquely, using a dropper to suck an appropriate amount of cell suspension, spraying it vertically and quickly on the slide, quickly baking on the outer flame of an alcohol lamp for 2-3s, and drying in air;

[0054] (6) staining: staining for 30min using the Giemsa staining solution prepared by the application, slowly washing the staining solution, and drying in air;

[0055] (7) microscopic examination: observing and photographing under a microscope.

[0056] Generally, a small crayfish lays about 200-300 eggs at a time, and the embryo cell mass in the nauplius stage is very large, and the yolk is very small, which is very beneficial to the preparation of chromosomes. In addition, using embryos to prepare chromosomes does not require killing a large number of small crayfishes, and the use of embryos to prepare chromosome division phases is clean, the number and shape of chromosomes are clear, which is beneficial to the analysis of karyotype and chromosome inheritance. Generally, the embryo membrane of fish can be dissolved by trypsin, and the outer membrane of the embryo can be easily removed by a sharp forceps. However, the egg membrane of shrimps does not react with trypsin, making it difficult to remove the outer membrane of the small crayfish egg, which is difficult to use for chromosome preparation. The application uses 2mg / mL of chain protease to dissolve the outer membrane of the shrimp egg and 50-60% acetic acid for softening, and then uses a self-made sharp forceps to remove the outer membrane of the shrimp egg, thereby ensuring the integrity of the embryo (Table 1).

[0057] Table 1: Experimental results of different concentrations of chain protease and acetic acid under different treatment times

[0058]

[0059] The main function of the colchicine solution is to destroy the spindle in the metaphase of cell division, so that the cell stays in the metaphase of division, at which time the shape and number of chromosomes are clearest. The key factor for the success of the experiment is to control the concentration of the chromosome solution for treating the embryo cells. The embryo is a living individual, and its cell development is still ongoing when it is immersed in the colchicine solution. If the concentration of colchicine is too low, it cannot destroy the spindle, and if the concentration is too high, it will be toxic and kill the embryo. Therefore, it is particularly important to control the reasonable concentration (Table 2).

[0060] Table 2: Experimental results of different concentrations of colchicine solution

[0061]

[0062]

[0063] As Figure 5 shown in the figure are the embryonic chromosome division phase pictures of the crayfish prepared by the present application. As can be seen from the figure, through the method, the number of clear somatic chromosomes of the crayfish can be obtained, which is 188, and the number and shape of the crayfish chromosomes are clear, which lays a good experimental foundation for subsequent karyotype identification and ploidy identification. The method is not only practical for Procambarus clarkii, but also applicable to other shrimps such as Northeast Procambarus and Macrobrachium rosenbergii.

Claims

1. A method for preparing chromosomes from crayfish embryos, characterized in that, Includes the following steps: (1) Pretreatment: Take crayfish embryos in the nauplius stage, immerse them in a colchicine solution with a concentration of 500 mg / mL, and culture them in a constant temperature incubator at 22-25℃ for 3-4 h. After centrifugation at 1000-1100 r / min for 5-10 min, discard the supernatant to obtain cell clumps. (2) Hypotonic treatment: Remove colchicine solution, treat with 0.75% KCl at 37℃ for 20-30 min to induce hypotonicity, and then remove the hypotonic solution; (3) Fixation: The cell clusters obtained after step (2) are pre-fixed and then fixed; (4) Slide preparation: Take the embryo obtained after fixation in step (3) into a centrifuge tube, add streptolysin at a concentration of 2 mg / mL for digestion for 20 min, then add acetic acid solution at a volume concentration of 50-60% for softening for 15-20 min, tear off the fertilization membrane of the embryo, then crush it to dissociate and disperse the embryonic cells, centrifuge, pour off the liquid on the top layer, add fixative and blow evenly with a pipette to obtain a cell suspension; the centrifugation speed is 1000-1100 r / min and the time is 5-10 min; the fixative is obtained by mixing methanol and glacial acetic acid at a volume ratio of 1:1; (5) Spraying: Place the pre-cooled glass slide at an angle, use a dropper to draw up the cell suspension obtained in step (4), spray it vertically onto the glass slide, bake it over the outer flame of an alcohol lamp, and then dry it in the air. (6) Staining: The slides with chromosomes attached obtained in step (5) were stained with Giemsa stain. The Giemsa stain was prepared as follows: 0.5g of Giemsa particles were weighed and placed in a mortar, a little glycerol was added, the Giemsa particles were crushed, and then glycerol was added to make a total volume of 33mL. The mixture was mixed well to obtain a solution. The solution was heated in a water bath at 60℃ for 90min, 33mL of methanol was added and mixed well, and the mixture was filtered into a brown glass bottle while hot. It was placed in a cool and dry place to mature for at least half a month. It was then diluted with phosphate buffer at a volume ratio of 9:1 before use. (7) Microscopic examination: Observe and photograph under a microscope.

2. The preparation method according to claim 1, characterized in that, In step (3), the pre-fixation and post-fixation are performed by adding the cells obtained after removing the hypotonic solution to Carnoy fixative solution pre-cooled to 4°C for 1.5-3 hours, changing the Carnoy fixative solution twice in between, and then performing post-fixation in post-fixative solution pre-cooled to 4°C.

3. The preparation method according to claim 2, characterized in that, The Carnoy fixative is obtained by mixing methanol and glacial acetic acid in a volume ratio of 3:1, and the post-fixative is obtained by mixing methanol and glacial acetic acid in a volume ratio of 1:

1.

4. The preparation method according to claim 1, characterized in that, In step (5), the baking time is 2-3 seconds.

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