Construction method and application of zebra fish circadian rhythm disorder model

Abstract

The invention discloses a construction method and application of a zebra fish circadian rhythm disorder model, and belongs to the technical field of zebra fish screening / evaluation model construction. The method comprises the following steps: culturing zebrafish in an embryonic period in a dynamic photoperiod environment for 7 days to construct a model; adopting fixed 12-hour light / dark circulation (8: 00-20: 00 illumination) in a normal control group; the first-day illumination of the model group is consistent with that of the control group, the dark period is advanced by 6 hours every day from the second day to the fourth day, the dark period is delayed by 6 hours every day from the fifth day to the seventh day, and finally the model group is synchronized with the normal control group. The light / dark duration in every 24 hours is 12 hours, and obtaining a zebra fish circadian rhythm disorder model with consistent illumination duration and consistent development stages on the seventh day. The method can establish the circadian rhythm disorder model which has the advantages of short modeling period, low time and economic cost, high similarity with human rhythm, avoidance of other interference factors and the like, and can be used for evaluating the circadian rhythm disorder improving effects of medicines, functional foods, health care products and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of zebrafish screening / evaluation model construction, and particularly relates to a construction method and application of a zebrafish circadian rhythm disorder model. Background Art

[0002] The circadian rhythm refers to a physiological rhythm oscillation system with a period of approximately 24 hours, regulated by the endogenous biological clock. It is precisely regulated through a transcription-translation negative feedback loop composed of core clock genes (Clock, Bmal1, Per, Cry, etc.). It is involved in the regulation of multiple physiological processes, and the accuracy and stability of the biological clock are closely related to the health of the body. However, due to many factors such as work demands, the use of smart electronic devices, and increased stress in life, more and more people are experiencing disrupted work and rest schedules. Among them, working conditions such as shift work and frequent flights across time zones require people to frequently adjust their biological rhythms. Therefore, circadian rhythm disorders have now become a major public health issue.

[0003] The zebrafish is the third most popular model organism after rats and mice. The zebrafish model offers advantages such as rapid, efficient, low-cost, and low-drug in vitro experiments. Furthermore, it shares a high degree of similarity with humans in terms of genomics, nervous system development, and function. Like humans, it also exhibits a typical diurnal rhythm pattern, making it an excellent model animal for developing circadian rhythm disorder models. However, reports on zebrafish models of circadian rhythm disorders are currently scarce.

[0004] Existing mouse or rat models of circadian rhythm disorders mostly utilize methods such as sleep deprivation, continuous light or darkness, and knockout of core clock genes. These methods have the advantage of relatively conserving sleep and rhythm regulation pathways between mammalian models and humans. However, they also have challenges such as long experimental periods, a lack of dynamic models that mimic human social rhythm disorders, and discrepancies that may arise from differences in light exposure or feeding schedules. Furthermore, rodents are primarily nocturnal, and their rhythmic physiology differs significantly from that of diurnal humans. Summary of the Invention

[0005] In order to overcome the shortcomings of the above-mentioned existing methods and technologies, the purpose of the present invention is to provide a method for constructing and applying a zebrafish circadian rhythm disorder model, so as to solve the technical problems that the existing circadian rhythm disorder model has a long existence time, is not conducive to high-throughput screening, and has large differences from human physiological rhythms.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention discloses a method for constructing a zebrafish circadian rhythm disorder model, wherein zebrafish embryos are placed in a culture medium and cultured under different light-dark alternating environments for 7 days to obtain a zebrafish circadian rhythm disorder model;

[0008] The different light-dark alternation environments are as follows: starting at 8:00 on the first day, there is a 12-hour light period and a 12-hour dark period. Starting from the second day, the dark period begins 6 hours earlier every day for three consecutive days, and then the dark period is delayed by 6 hours every day for three consecutive days. Until the seventh day of the experiment, the light-dark cycle is exactly the same as that of the first day (and the normal rhythm control group); every day, the light and dark time within each 24 hours is guaranteed to be 12 hours.

[0009] Preferably, the zebrafish embryos are quality-screened zebrafish embryos less than 2 hpf.

[0010] Preferably, the light source during the illumination period is cool white light with a light intensity of 1000 lux.

[0011] Preferably, the light intensity during the dark period is 0 lux.

[0012] Preferably, the culture medium is zebrafish E3 embryo culture medium.

[0013] Preferably, the zebrafish is an AB strain or a specifically marked transgenic zebrafish strain.

[0014] Preferably, the zebrafish circadian rhythm disorder model has a reduced swimming distance, a chaotic swimming trajectory, and a decreased sensitivity to environmental stimuli.

[0015] The second aspect of the present invention discloses the application of a method for constructing a zebrafish circadian rhythm disorder model in the evaluation of products related to the efficacy of improving circadian rhythm disorders.

[0016] Preferably, the product is a medicine, food or health product.

[0017] A third aspect of the present invention discloses a method for evaluating the efficacy of a product for improving circadian rhythm disorders, comprising the following steps:

[0018] 1) Construct a zebrafish circadian rhythm disorder model according to the above method;

[0019] 2) Zebrafish embryos were placed in culture medium and cultured with a 12-hour light period and a 12-hour dark period starting at 8:00 AM daily for 7 days to obtain a normal control zebrafish circadian rhythm model;

[0020] 3) Administer a functional product for improving circadian rhythm disorders to a zebrafish circadian rhythm disorder model and a zebrafish circadian rhythm regularity model, respectively, and analyze the statistical differences in the developmental phenotypes and photoperiodic behavioral changes of the two groups of zebrafish to evaluate the efficacy of the product for improving circadian rhythm disorders.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention provides a method for constructing a zebrafish circadian rhythm disorder model, which includes: 1) selecting a 7-day modeling cycle, during which zebrafish larvae are in a biological window period most suitable for neuroethological and developmental studies, and their nutrition is completely dependent on yolk supply, avoiding interference factors caused by feeding differences; 2) mathematical modeling is used to ensure that the cumulative light duration over 7 days is consistent (84 hours), and the light cycle changes so that the light and dark durations are 12 hours per 24 hours, without changing the light or dark durations. The purpose of disrupting the zebrafish circadian rhythm is achieved only by changing the phases of the light and dark periods, eliminating confounding effects such as sleep deprivation; the method precisely focuses on the interference of "light cycle changes" on the rhythm to avoid nonspecific effects; 3) the characteristics of zebrafish that are photosensitive throughout the body and have obvious diurnal biological rhythms are used to design a 7-day stepped dynamic light cycle to simulate complex rhythm disorder scenarios such as human shift work and cross-time zone travel, with strong physiological or pathological correlations. Compared with existing static illumination simulation methods, the "bidirectional dynamic regulation" model design is closer to actual physiological states and pathological triggers, and can significantly improve the ecological validity of the model. Multi-dimensional experimental results from behavioral studies (including spontaneous activity, light and dark stimulation, and vibration stimulation), transcriptomics (including enrichment of circadian rhythm-related GO terms and KEGG pathways), and molecular biology (including qPCR verification of core clock gene expression) show that the zebrafish circadian rhythm disorder model constructed by this method not only has the general advantages of the zebrafish model, but also has a shorter modeling time and lower cost than rat or mouse models. Evaluation can be completed within 7 days, which complies with the substitution and optimization principles of the 3R principle of animal protection and welfare. At the same time, the experimental results are visualized, easy to understand, highly stable, and reproducible. They can be used not only to explore the effects and mechanisms of circadian rhythm disorders, but also to evaluate the efficacy of drugs, foods, or health products in improving circadian rhythm disorders.

[0023] Furthermore, before the experiment, zebrafish embryos less than 2hpf were strictly quality screened. After quality screening, the zebrafish embryos had good fertilization quality, were in the cleavage stage, had highly synchronized cell division, and had not yet undergone tissue differentiation. Photoperiod phase shift intervention was initiated in the early stage of zebrafish embryos, utilizing their light sensitivity and developmental synchronization. At this time, grouping ensured that all embryos were at the same developmental starting point, avoiding individual differences after tissue differentiation, eliminating the impact of subsequent developmental differences on experimental results, and reducing the differences between the normal control group and the disordered group caused by non-light factors. In addition, the developmental stage of the zebrafish was completely consistent when the relevant experiments were conducted on the 7th day.

[0024] Furthermore, the light intensity during the light period is 1000 lux, and the light intensity during the dark period is 0 lux, which can avoid interference caused by external light. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the illumination of two groups of zebrafish in the zebrafish circadian rhythm disorder model of the present invention; wherein A is the normal control group (i.e., the circadian rhythm regular group) and B is the circadian rhythm disorder group;

[0026] Figure 2 This is a statistical graph showing the survival rates of two groups of zebrafish in the zebrafish circadian rhythm disorder model of the present invention;

[0027] Figure 3 Statistical graphs of neurobehavioral tests of two groups of zebrafish in the zebrafish circadian rhythm disorder model of the present invention; wherein A is the total swimming distance of the spontaneous activity test, B is the swimming distance per minute of the spontaneous activity test, C is a schematic diagram of the swimming trajectory of the spontaneous activity test, D is the total swimming distance of the light and dark stimulation test, E is the swimming distance per minute of the light and dark stimulation test, F is the total swimming distance of the light and dark stimulation test under different lighting conditions, G is the total swimming distance of the vibration stimulation test, H is the swimming distance per second of the vibration stimulation test, and I is the swimming distance amplitude of the vibration stimulation test. **p<0.01, ****p<0.0001, all of which are statistically significantly different;

[0028] Figure 4 This is the transcriptome sequencing enrichment result diagram of the present invention; wherein A is the GO term enrichment result, and B is the KEGG pathway enrichment result;

[0029] Figure 5 3 is a histogram of the mRNA expression of the zebrafish transcriptome of the present invention; wherein A represents per1b, B represents cry1b, and **p<0.01 indicates a statistically significant difference. DETAILED DESCRIPTION

[0030] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0031] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0032] Herein, all features such as values, amounts, amounts, and concentrations defined in numerical ranges or percentage ranges are for brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to include and specifically disclose all possible subranges and individual values within the range (including integers and fractions).

[0033] In this document, unless otherwise specified, “include,” “including,” “contains,” “has” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0034] In this article, light / dark cycle (L / D cycle) refers to a periodic environmental pattern in which light periods and dark periods alternate, characterized by the length of the period (Period) and the phase (Phase).

[0035] In this article, light-phase refers to the period of time during the light / dark cycle when the organism receives light, while dark-phase refers to the period of time during the light / dark cycle when the organism receives no light.

[0036] In this article, the light source during the light period is cool white light with a light intensity of 1000 lux; the light intensity during the dark period is 0 lux.

[0037] In this article, unless otherwise specified, “hpf” refers to hours post-fertilization.

[0038] In this article, unless otherwise specified, “dpf” refers to days post-fertilization.

[0039] Herein, zebrafish include but are not limited to AB strain zebrafish, and may also be transgenic zebrafish, such as transgenic Tg (huc:eGFP), Tg (mpeg1:eGFP) and other zebrafish strains.

[0040] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0041] The present invention provides a method for constructing a zebrafish circadian rhythm disorder model, comprising the following steps:

[0042] 1) Perform rigorous embryo quality screening on zebrafish embryos younger than 2 hpf to avoid potential problems affecting experimental results due to unfertilized embryos or poor embryo quality (e.g., adhesions, abnormal morphology, developmental delay, etc.);

[0043] 2) The zebrafish embryos screened in step 1) were randomly divided into groups and placed in E3 embryo culture medium; the normal control group adopted a fixed light cycle of 12 hours of light / 12 hours of darkness (8:00-20:00 for the light period); the circadian rhythm disorder group adopted a light cycle of 12 hours of light / 12 hours of darkness (8:00-20:00 for the light period) on the first day, and the dark period was advanced by 6 hours every day for 3 consecutive days starting from the next day, and then the dark period was delayed by 6 hours every day for 3 days. By the 7th day, the light timing of the circadian rhythm disorder group was restored to be completely synchronized with that of the normal control group, ensuring that the light and dark duration of the zebrafish in every 24 hours was 12 hours, and the total light duration was completely consistent.

[0044] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0045] The following examples utilize conventional instruments and equipment in the art. Experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or as recommended by the manufacturer. The various raw materials used in the following examples, unless otherwise specified, were conventional commercially available products, with specifications conventional in the art.

[0046] Example 1 Construction of a zebrafish circadian rhythm disorder model

[0047] 1. Experimental Preparation

[0048] 1. Prepare zebrafish embryos: The night before, place adult AB strain zebrafish in a 1:1 ratio of male to female in a dedicated zebrafish spawning tank, using a baffle to separate the males and females. The next morning, at the start of the light cycle (8:00 AM), remove the baffle to allow the males and females to begin breeding and spawning. Adult zebrafish are maintained in a water system maintained at 28.5 ± 1°C and a pH of 7.2-7.6. The light cycle in the fish room is 14 hours light / 10 hours dark (14L:10D).

[0049] 2. Prepare 60×E3 Embryo Culture Medium: Add 8.7g NaCl, 0.4g KCl, 1.1g CaCl2, and 2.445g MgCl2·6H2O to a beaker. Dissolve in ultrapure water and pour into a 500mL volumetric flask. Bring to volume with ultrapure water. Sterilize by autoclaving and cool to obtain 60×E3 Embryo Culture Medium. Store at 4°C. Dilute 60×E3 Embryo Culture Medium to 1×: Add 16.5mL 60×E3 Embryo Culture Medium and 33.33μL 3mg / mL methylene blue solution to a 1L volumetric flask. Bring to 1L with ultrapure water to obtain 1×E3 Embryo Culture Medium. Set aside.

[0050] 2. Construction of a zebrafish circadian rhythm disorder model

[0051] like Figure 1 As shown, several 0-2 hpf zebrafish embryos were selected, quality screened, and randomly divided into a circadian rhythm disorder group and a normal control group (i.e., a circadian rhythm regularity group). Zebrafish embryos in each group were placed in six-well plates, with 30-40 embryos per well, and 5 mL of 1×E3 embryo culture medium was added. The six-well plates were then placed in corresponding experimental chambers for a seven-day light experiment. Every day during the light-on period of the normal control group, the zebrafish were fed a medium change and dead embryos were aspirated. After completing the seven-day dynamic light cycle experiment, the zebrafish in the circadian rhythm disorder group were obtained as the zebrafish circadian rhythm disorder model.

[0052] Among them, the lighting time of the experimental box of the circadian rhythm disorder group was set as follows: Day 1: 8:00-20:00; Day 2: 8:00-14:00; Day 3: 2:00-8:00, 20:00-24:00; Day 4: 0:00-8:00, 14:00-24:00; Day 5: 0:00-2:00, 20:00-24:00; Day 6: 0:00-14:00; Day 7: 2:00-20:00. The light source was cold white light (1000 lux) to simulate daylight.

[0053] The lighting time of the experimental box of the normal control group was set as follows: from the first day to the seventh day, lighting was on from 8:00 to 20:00 every day, that is, 12L:12D, and the light source was cool white light (1000 lux) to simulate daylight.

[0054] 3. Verification of the Safety of the Zebrafish Circadian Rhythm Disruption Model

[0055] While changing the medium and aspirating dead embryos every day, the survival rate of each group of zebrafish was recorded and the developmental status was observed.

[0056] Record the results as Figure 2 As shown in the figure, there was no significant difference in the survival rate of the two groups of zebrafish after 7 days of light treatment, which to a certain extent shows that this experimental method has no significant effect on the development of zebrafish, indicating that this method is safe and harmless to zebrafish.

[0057] 4. Behavioral Verification of the Zebrafish Circadian Rhythm Disruption Model

[0058] After seven days of light exposure, normally moving zebrafish larvae from each group were selected under a stereomicroscope at the same time (10:00 AM) and placed into a 48-well plate, with one larvae and an equal volume of 1× E3 embryo culture medium placed in each well. Various swimming parameters of the zebrafish in each group were measured using the DanioVision Tracking System (Noldus IT). Zebrafish were allowed to acclimate for 5 minutes before each test.

[0059] First, a spontaneous activity test was conducted, measuring the distance the fish swam spontaneously under normal light (1000 lux) over 30 minutes and plotting the behavioral trajectory. Next, a vibration stimulation test was conducted, with the zebrafish receiving vibration stimulation for 30 seconds, repeated 10 times, under normal light. Finally, a light-dark stimulation test was conducted, with the zebrafish placed in darkness (0 lux) for 5 minutes, then immediately illuminated to maximum light (5450 lux) for 5 minutes, repeated five times. By comparing these behavioral parameters, we determined whether the zebrafish's neurobehavioral activity was affected.

[0060] The experimental results are as follows Figure 3 As shown in Figure 2, the spontaneous activity of zebrafish in the circadian rhythm disorder group was significantly different from that in the normal control group, specifically in terms of a significant decrease in their swimming distance ( Figure 3 A and B), the swimming trajectory is relatively chaotic ( Figure 3 Middle C); The zebrafish in the circadian rhythm disorder group also showed significant abnormalities under light and dark stimulation compared with the normal control group, as shown by a significant decrease in swimming distance during the light and dark periods, indicating that the zebrafish in the circadian rhythm disorder group were less sensitive to light and dark stimulation ( Figure 3 (D-F in the middle); Vibration stimulation test is often used to detect the zebrafish's ability to perceive external stimuli. The results showed that the zebrafish in the circadian rhythm disorder treatment group had a significant decrease in sensitivity to the external environment, indicating that the circadian rhythm disorder model had a significant impact on the behavior of zebrafish ( Figure 3 Medium G~I).

[0061] 5. Transcriptome Sequencing Validation of the Zebrafish Circadian Rhythm Disruption Model

[0062] At the same time (12:00 PM) after completing the seven-day light treatment, zebrafish larvae with normal behavior from each group were collected under a stereomicroscope. After the culture medium was drained as much as possible, they were immediately snap-frozen in liquid nitrogen. RNA sequencing was performed on both groups of zebrafish, and gene expression was quantitatively analyzed by comparing the raw data to high-quality sequencing data.

[0063] The experimental results are as follows Figure 4 As shown in the figure, differentially expressed genes can be obtained by screening with |log2FC|≥0.58 and padj≤0.05. Enrichment analysis of these differentially expressed genes shows that terms such as rhythm process and circadian rhythm are significantly enriched, and the phototransduction signaling pathway is significantly enriched, which shows that this method has a significant effect on the circadian rhythm system of zebrafish and can successfully construct a zebrafish circadian rhythm disorder model.

[0064] 6. Detection of mRNA Expression in Zebrafish Circadian Rhythm Disorder Model

[0065] At the same time after the seven-day light experiment, two groups of zebrafish were collected and the expression of core clock gene mRNA in the two groups of zebrafish was detected by qPCR. The specific steps are as follows:

[0066] Add 1 mL of Trizol reagent to an EP tube containing 30 zebrafish larvae. Grind using a tissue grinder. After grinding, let it rest on ice for 5 minutes. Centrifuge at 12,000 × g at 4°C for 10 minutes, and remove the supernatant. Add 200 μL of chloroform to the supernatant, cap the tube, shake on ice for 2 minutes, let it rest for 3-5 minutes, and centrifuge at 12,000 × g at 4°C for 10-15 minutes. Transfer the aqueous phase to a new centrifuge tube, add 500 μL of isopropanol, mix thoroughly, and let it rest at room temperature for 20-30 minutes. Centrifuge at 12,000 × g at 4°C for 10 minutes, and discard the supernatant. Slowly add 1 mL of 75% ethanol to the tube, cap it, and gently swirl to rinse the sides. Discard the supernatant. Add 500 μL of 75% ethanol to the tube, blow up the precipitate, shake gently 10-15 times, centrifuge at 12,000 × g at 4°C for 5 minutes, discard the supernatant; centrifuge for 5 minutes, and let it dry at room temperature; add an appropriate amount of ddH2O, dissolve it, and then measure the RNA concentration.

[0067] RNA was reverse transcribed to obtain cDNA. The reverse transcription conditions were as follows: Step 1: 42°C, 15 min; Step 2: 85°C, 5 min; Step 3: 4°C, and then the end. Real-time PCR reaction was performed on the obtained cDNA. The reaction conditions were as follows: Step 1: pre-denaturation at 95°C, 30 s, and one cycle; Step 2: PCR reaction at 95°C, 5 s, and then at 60°C, 30 s, and 40 cycles; Step 3: melting curve at 95°C, 15 s, and then at 65°C, 60 s, and then at 95°C, 15 s, 50°C, 30 s, and one cycle, and then placed at 4°C. -ΔΔC The expression of the zebrafish core clock genes per1b and cry1b was obtained by relative quantitative analysis using the primer sequences shown in Table 1 , compared with the expression level of the internal control actin (β-actin).

[0068] Table 1 Primer sequences for zebrafish embryo experiments

[0069]

[0070] The experimental results are as follows Figure 5 As shown in the figure, there was a statistically significant difference in the expression levels of core clock genes (per1b and cry1b) between the circadian rhythm disorder group and the normal control group (p < 0.01), indicating that the use of this circadian rhythm disorder model did produce a circadian rhythm disorder effect on zebrafish, and the model construction was effective at this time.

[0071] Example 2 Application of zebrafish circadian rhythm disorder model

[0072] The zebrafish circadian rhythm disorder model is used to evaluate the efficacy of drugs, foods, or health products in improving circadian rhythm disorders, including the following steps:

[0073] Step 1: construct a zebrafish circadian rhythm disorder model and a normal control group of the same size according to the scheme of Example 1;

[0074] Step 2: administer the drug, food, or health product to be evaluated to each group of zebrafish, analyze the statistical differences in the photoperiodic behavior changes between the sample group and the circadian rhythm disorder group, and evaluate the efficacy of the drug, food, or health product.

[0075] The above content is only for explaining the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A method for constructing a zebrafish circadian rhythm disorder model, characterized in that: Zebrafish embryos were placed in culture medium and cultured under different light-dark alternating conditions for 7 days to obtain a zebrafish circadian rhythm disorder model; The different light-dark alternation environments are as follows: starting at 8:00 on the first day, there is a 12-hour light period and a 12-hour dark period. Starting from the second day, the dark period begins 6 hours earlier every day for three consecutive days, and then the dark period begins 6 hours later every day for three consecutive days. Until the seventh day of the experiment, the light-dark cycle is consistent with the first day; every day, the light and dark time within each 24 hours is guaranteed to be 12 hours.

2. The method for constructing a zebrafish circadian rhythm disorder model according to claim 1, wherein: The zebrafish embryos are quality screened zebrafish embryos less than 2 hpf.

3. The method for constructing a zebrafish circadian rhythm disorder model according to claim 1, wherein: The light source during the photoperiod was cool white light with a light intensity of 1000 lux.

4. The method for constructing a zebrafish circadian rhythm disorder model according to claim 1, wherein: The light intensity during the dark period was 0 lux.

5. The method for constructing a zebrafish circadian rhythm disorder model according to claim 1, wherein: The culture medium was zebrafish E3 embryo culture medium.

6. The method for constructing a zebrafish circadian rhythm disorder model according to claim 1, wherein: The zebrafish is an AB strain or a specifically marked transgenic zebrafish strain.

7. The method for constructing a zebrafish circadian rhythm disorder model according to claim 1, characterized in that: The zebrafish circadian rhythm disorder model has a reduced swimming distance, a chaotic swimming trajectory, and a decreased sensitivity to environmental stimuli.

8. Use of the method for constructing a zebrafish circadian rhythm disorder model according to any one of claims 1 to 7 in evaluating products that improve circadian rhythm disorders.

9. The use according to claim 8, characterized in that The product is a medicine, food or health product.

10. A method for evaluating the efficacy of a product for improving circadian rhythm disorders, characterized in that: The following steps are involved: 1) constructing a zebrafish circadian rhythm disorder model according to the method of any one of claims 1 to 7; 2) Placing zebrafish embryos in culture medium, starting at 8:00 AM daily, with a 12-hour light period and a 12-hour dark period, alternating light and dark for 7 days to establish a zebrafish circadian rhythm model; 3) Administer a functional product for improving circadian rhythm disorders to a zebrafish circadian rhythm disorder model and a zebrafish circadian rhythm regularity model, respectively, and analyze the statistical differences in the developmental phenotypes and photoperiodic behavioral changes of the two groups of zebrafish to evaluate the efficacy of the product for improving circadian rhythm disorders.

Images