CN-122016441-A - Improved method for preparing yellow stone climbing-sword-like dyeing body

Abstract

The invention provides an improved preparation method of a yellow stone climbing-type Japanese sea-ball (PHA) chromosome, which comprises the steps of gradually heating and injecting PHA twice, wherein the water temperature is increased from 18 ℃ to 20 ℃ in a process of temporary culture of Huang Danpa hours, after injecting PHA into a first needle, the temperature is increased to 22.5-23 ℃ after 12 hours, injecting PHA into a second needle, the water temperature is kept unchanged at 22.5-23 ℃, after injecting PHA for 3 hours into the second needle, sampling, washing head and kidney in physiological saline to collect cells, 0.5% KCl hypotonic, fixing by a Carnot fixing solution, centrifuging, dripping tablets by a cold dripping method, and naturally drying, and dyeing the tablets by Giemsa. The method promotes the proliferation of the yellow stone climbing somatic cells by heating and injecting PHA twice, so that the metaphase division phase is increased, the morphology is clear and dispersed, and the quality and the preparation success rate of chromosome nuclei are obviously improved.

Inventors

• YANG RUIBIN
• TANG MAOGANG
• ZHAO HENG
• XIA YONG
• XIONG HAO
• LU JIAYI
• ZHOU XIAOYUN
• SU XIAOJING
• YANG XUEFEN

Assignees

• 华中农业大学

Dates

Publication Date

20260512

Application Date

20251217

Claims (4)

1. 1. An improved method for preparing a yellow stone gladiolus chromosome is characterized in that the method for preparing yellow stone gladiolus chromosome is carried out according to the following steps: 1. In the process of carrying out Huang Danpa-up culture on the yellow stone, raising the water temperature from 18 ℃ to 20 ℃ within 12h, injecting a first needle PHA by the yellow stone, raising the water temperature to 22.5-23 ℃ after 12h, injecting a second needle PHA by the yellow stone, and maintaining the water temperature at 22.5-23 ℃ after 3 h; 2.3 h later, bleeding Huang Danpa of the red-tail of the fresh water fish in water, dissecting the fish body, taking out the head and the kidney, placing the fish in a plate filled with normal saline for freshwater fish for cleaning, placing the fish in a culture plate filled with normal saline after blood clot removal, grinding the head and the kidney tissues by forceps, sucking the fish into a 10mL centrifuge tube, repeatedly blowing the fish into the centrifuge tube by using a suction tube, adding the normal saline, uniformly mixing, centrifuging the fish at a speed of 1200r/min for 12min, and discarding the supernatant; 3. Adding 5mL of KCl hypotonic solution with mass fraction of 0.5% into the product of the step two, blowing uniformly, standing at room temperature, hypotonic treatment for 30min, adding 1mL of Carnot's stationary liquid prepared according to the ratio of methanol to glacial acetic acid=3:1, blowing uniformly, centrifuging at 1200r/min for 12min, discarding supernatant, and mixing uniformly; 4. Adding 5ml of Carnot's fixing liquid into the product obtained in the step three, blowing the product and the Carnot's fixing liquid uniformly by using a rubber head dropper, standing and fixing for 20min at room temperature, centrifuging for 10min at a speed of 1200r/min, and discarding the supernatant; 5. repeating the steps for 1 time, and discarding the supernatant; 6. adding 5ml of Carnot's fixing solution into the product obtained in the step five, blowing uniformly, standing and fixing at room temperature for 10min, centrifuging at 1200r/min for 10min, and discarding the supernatant; 7. Adding 0.5ml of Carnot's stationary liquid into the cell sediment after the centrifugation in the step six, uniformly mixing to prepare a cell suspension, sucking the cell suspension into a rubber head dropper, dripping the cell suspension onto a pre-frozen glass slide, then putting the glass slide above the flame of an alcohol lamp for 2-3 times, and naturally drying at room temperature; 8. And (3) dyeing the chromosome slide glass dried in the step seven with Giemsa staining solution with the volume fraction of 10% for 10min, flushing with tap water, and naturally drying to finish the preparation of chromosome samples, thus obtaining the chromosome of the glabrous sarcandra.
2. 2. The method for preparing the improved yellow stone climbing Japanese sea weed herb chromosome according to claim 1, wherein in the first step, the temperature of water is raised from 18 ℃ to 20 ℃ in 12h, the first needle PHA is injected into the yellow stone climbing according to the proportion of PHA to fish weight of 1g, the temperature of water is raised to 22.5-23 ℃ after 12h, and the second needle PHA is injected into the yellow stone climbing according to the proportion of PHA to fish weight of 6-8 μg of 1g, and the temperature of water is maintained at 22.5-23 ℃.
3. 3. The method of claim 1, wherein in the first step, after injecting the second needle PHA for 3 hours, the colchicine solution is injected in a ratio of 4. Mu.g to 1g of the fish body weight.
4. 4. The method for preparing modified yellow stone-climbing-me-dium chromosome as set forth in claim 1, wherein the Carnot's fixative is prepared from methanol and glacial acetic acid at a volume ratio of 3:1.

Description

Improved method for preparing yellow stone climbing-sword-like dyeing body Technical Field The invention relates to a preparation method of fish chromosomes, in particular to an improved preparation method of a yellow stone gliptis screen. Background Huang Danpa also known as Shi-Pang Zi and Huangshi-Pang belong to the genus of Leptodermia, pazuidae, and Shi-Pang. The species is a special small benthic fish at the upstream of the Yangtze river, and is mainly distributed in the dry tributaries of Jinshajiang and Minjiang river systems, and covers the land of Qinghai, sichuan, yunnan, tibet and the like. Typical habitats are mountain stream in remote mountain areas, streams and rushes in small rivers. The yellow stone glabrous sartan belongs to low-temperature cold water fish, and the suitable survival temperature is 14-18 ℃. The Huang Danpa is regarded as precious economic fish in the production area because of tender and delicious meat quality, and the market price is very high. Because of excessive fishing and changes in habitat, the amount of natural resources is small and difficult to catch. Chromosome is used as a main carrier of genetic information, the number and karyotype analysis are the core content of cytogenetic research, and the chromosome has key significance for explaining species evolution rules, classification relations and fish genetic breeding. Chromosome preparation is the basis of karyotype analysis, and metaphase chromosomes with high division indexes and clear morphology are required to be obtained. The quantity and quality of split phases of different fishes are uneven due to the differences of tissue structures, physiological states and treatment conditions. Therefore, a stable and efficient species-specific chromosome preparation system is established, and a reliable cytological basis can be provided for subsequent research by systematic condition optimization and repeated verification. The types of the Pachyrhizus that have been studied on the present day are very limited, mainly including the black macula proto-macula, huang Danpa macula, qingshi Pangzhu, fujian Maxiao and Zhonghua Maxiao et al (Li Shushen et al 1981, yu Xian et al 1989, ji Xihai 1992). The method of "PHA living injection-colchicine treatment" is generally adopted by combing the prior literature (Wu Yunfei 1999, zhu Chuankun and Pan Zhengjun 2022) and is generally characterized by comprising the steps of injecting PHA once into the chest or abdominal cavity with a dosage ranging from 5 to 15 mug per gram of fish body weight, treating the PHA for about 2 to 24 hours after injecting the PHA with colchicine 1 to 6 mug per gram, and treating the PHA for 2 to 6 hours to accumulate metaphase division phases, and obtaining materials and preparing chromosome specimens by the steps of hypotonic, fixing, dripping and giemsa staining, and the like, wherein the PHA living injection-colchicine treatment method is generally adopted for most fishes in the Uygur family or the like (Wu Yunfei to 15 mug per gram of fish body weight). Although the conventional method described above has been successful in many fish species, it is a significant challenge to apply it to the species specific to the species of the family of the species Leptodermaceae, in particular the plateau such as Huang Danpa, and it is difficult to obtain the ideal result Zhang Lin (2023) points out that the sensitivity and response pattern of the different species to PHA are different when summarizing the experience of 7 difficult aquatic animals. Experiments are carried out by referring to the existing traditional chromosome karyotype manufacturing method, and the obtained yellow stone climbing gliptin chromosome has the advantages of small number of metaphase phases, uneven dispersion and unclear morphology, as shown in figure 1. Therefore, a stable and reliable chromosome preparation method optimized for biological characteristics of the yellow-shaped glyptohave been needed. Disclosure of Invention The invention provides an improved method for preparing a yellow stone climbing chromosome, which aims to solve the problems of small quantity of split phases, uneven dispersion and unclear morphology of low-temperature perching fishes such as Huang Danpa macula, and the like. On the basis of fully referencing the classical method, the invention systematically optimizes and innovates key steps. By adopting a method of gradually heating treatment and injecting PHA twice, the proliferation of somatic cells of the yellow stone is effectively promoted, so that the number of split phases in the middle of the prepared chromosome is more, and the chromosome is clear, well dispersed and complete in number. By improving the preparation method, the quality of the prepared chromosome karyotype and the success rate of the preparation are effectively improved. The invention provides an improved method for preparing a yellow stone glabra miq chromosome, which comprises the followi