Zebra fish gene marker for detecting influence of urban particulate matters on heart development and application of zebra fish gene marker

Abstract

The invention relates to a zebrafish gene marker for detecting the influence of urban particulate matters on heart development and application. The gene marker is a g2e3 gene. Female zebra fish is exposed in urban particulate matter to be detected, after the female zebra fish is mated, whether the g2e3 gene in a filial generation of the zebra fish is remarkably reduced or not is detected, and the heart development condition of the zebra fish is evaluated, so that whether the urban particulate matter affects the heart development of the zebra fish or not is judged; the development condition of the zebra fish heart can be reflected more intuitively and conveniently, and the application prospect is wide.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection of environmental pollution, and particularly relates to a zebrafish gene marker for detecting the influence of urban particulate matter on heart development and application. BACKGROUND

[0002] Air pollution is a global environmental problem that has a profound impact on human health, ecosystems, and climate. Urban particulate matter (UPM) is an important cause of air pollution. These particulate matters provide a reaction site for chemical reactions in the atmosphere, and some gaseous pollutants will adsorb on the surface of particulate matter and generate secondary pollutants under suitable conditions, thereby aggravating air pollution. Toxic and harmful substances such as heavy metals and polycyclic aromatic hydrocarbons in urban particulate matter enter the city and are inhaled into the human body. For larger particles, such as PM 10 , they are blocked by the nasal cavity and throat, but long-term inhalation may also cause respiratory diseases such as rhinitis and pharyngitis. Fine particulate matter, such as PM 2.5 , can penetrate deep into the lungs and even pass through the alveoli into the blood circulation, thereby causing diseases such as asthma, bronchitis, and pneumonia, and is also related to the occurrence of cardiovascular diseases.

[0003] Genes are functional fragments of DNA that carry genetic information. As the basic unit of controlling biological traits, they play a role by guiding protein synthesis. They follow the central dogma: transcription forms mRNA, which is then translated into protein, which in turn performs life activities. Gene expression has precise spatiotemporal specificity and regulates almost all life processes such as growth and development, immune response, etc. For example, during embryonic development, gene temporal expression ensures orderly cell differentiation and organ formation; and when its expression is abnormal, such as activation of proto-oncogenes or silencing of tumor suppressor genes, it may lead to the occurrence of diseases such as tumors. Genetic variation is the basis of evolution and genetic diversity, and is also closely related to many genetic diseases. In-depth study of genes is the core of understanding life and combating diseases.

[0004] Using animal models to study pathogenesis is a very effective research method. Suitable animal models include mice, rats, or zebrafish, etc. Taking mice as an example, its genome has a high similarity to that of humans. Researchers can use gene editing techniques (such as CRISPR-Cas9) to introduce gene mutations related to human diseases into the mouse genome, thereby constructing an animal model that simulates human diseases. However, as an animal model, mice also have some shortcomings, such as long breeding cycle, high cost, complex drug administration method, and inability to directly observe the details of early embryonic development. SUMMARY

[0005] The present application aims to provide a zebrafish gene marker for detecting the influence of urban particulate matter on heart development and application, which can reflect the influence of urban particulate matter on heart development.

[0006] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is:

[0007] A zebrafish gene marker for detecting the influence of urban particulate matter on heart development, characterized in that the gene marker is a g2e3 gene.

[0008] Another application of the present application provides a kit for detecting the influence of urban particulate matter on heart development, comprising reagents for detecting the zebrafish gene marker of claim 1.

[0009] Another application of the present application provides a method using the kit according to the above-mentioned kit, comprising the following steps:

[0010] S1, divide female zebrafish into a control group and a detection group, use culture water to culture the control group, and use culture water containing urban particulate matter to be detected to culture the detection group;

[0011] S2, mate the female zebrafish in the two groups with male zebrafish respectively, collect fertilized eggs, culture embryos, and detect the expression level of the g2e3 gene in each group of embryos; compared with the control group, if the expression of the g2e3 gene in the embryos of the detection group is significantly decreased, it is judged that the urban particulate matter in the culture water has an influence on heart development.

[0012] Preferably, in step S2, the expression level of the g2e3 gene in each group of embryos includes the following steps: extracting RNA of each group of embryos, determining the RNA concentration, reverse transcribing the RNA into cDNA, quantitatively analyzing the g2e3 gene in the zebrafish embryos, taking the beta-actin gene in the zebrafish embryos as an internal reference, adding corresponding primers of the g2e3 gene and corresponding primers of the beta-actin gene, and performing fluorescent quantitative PCR.

[0013] Preferably, in step S2, the primer sequence of the g2e3 gene is shown in SEQ ID NO. 2 and SEQ ID NO. 3.

[0014] Another application of the present application provides the application of a reagent for detecting a zebrafish gene marker in the preparation of a kit for detecting the influence of urban particulate matter on heart development, wherein the gene marker is a g2e3 gene.

[0015] Preferably, the primer sequence of the g2e3 gene is shown in SEQ ID NO. 2 and SEQ ID NO. 3.

[0016] Preferably, the kit uses steps include:

[0017] S1, divide female zebra fish into a control group and a detection group, the control group is cultured using aquaculture water, and the detection group is cultured using aquaculture water containing urban particulate matter to be detected;

[0018] S2, the two groups of female zebra fish are mated with male zebra fish respectively, fertilized eggs are collected, embryos are cultured, and the expression level of the g2e3 gene in each group of embryos is detected.

[0019] Preferably, in step S2, the expression level of the g2e3 gene in each group of embryos is detected, including the following steps: extracting RNA of each group of embryos, determining the RNA concentration, reverse transcribing the RNA into cDNA, quantitatively analyzing the g2e3 gene in zebra fish embryos, taking the beta-actin gene in zebra fish embryos as an internal reference, adding corresponding primers of the g2e3 gene and corresponding primers of the beta-actin gene, and performing fluorescence quantitative PCR.

[0020] The present application provides a method for detecting the influence of urban particulate matter on heart development, characterized in that it comprises the following steps:

[0021] S1, divide female zebra fish into a control group and a detection group, the control group is cultured using aquaculture water, and the detection group is cultured using aquaculture water containing urban particulate matter to be detected;

[0022] S2, the two groups of female zebra fish are mated with male zebra fish respectively, fertilized eggs are collected, embryos are cultured, and the expression level of the g2e3 gene in each group of embryos is detected.

[0023] The present application provides a method for detecting the influence of urban particulate matter on heart development, characterized in that it comprises the following steps: BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The solubility curve of the internal reference gene beta-actin and the target gene g2e3 qPCR reaction is shown in Figure A: beta-actin; B: g2e3.

[0025] Figure 2Figure 1 shows the relative expression level of g2e3 in the control group and the UPM group. D21-Control represents the relative expression level of g2e3 in the zebrafish embryos obtained by mating the parent fish that were cultured in water for 21 days. D21-UPM represents the relative expression level of g2e3 in the zebrafish embryos obtained by mating the parent fish that were exposed to UPM for 21 days. * indicates P<0.05. DETAILED DESCRIPTION

[0026] The present application is further illustrated by the following figures and specific examples.

[0027] In the following examples, equipment and materials were obtained from the following companies:

[0028] Wild-type zebrafish were purchased from Nanjing Yishuliuhua Biotechnology Co., Ltd. (Nanjing, China);

[0029] Zebrafish breeding system was purchased from Nanjing Yishuliuhua Biotechnology Co., Ltd. (Nanjing, China);

[0030] UPM was purchased from National Institute of Standards and Technology, item number: 1648a;

[0031] Ultra-micro spectrophotometer was purchased from DeNovix, model number: DS-11FX;

[0032] PCR instrument was purchased from BIO-RAD, model number: T100;

[0033] Microplate centrifuge was purchased from TIANGEN, model number: OSE-MP25;

[0034] Fluorescent quantitative PCR instrument was purchased from BIO-RAD, model number: CFX 96 Touch;

[0035] Eastep RT Master Mix Kit was purchased from promega, item number: LS2052;

[0036] BlasTaq Green 2× qPCR Master Mix was purchased from abm, item number: G891;

[0037] Quantitative sealing film was purchased from BIO-RAD, item number: MSB1001;

[0038] 1.8 mL cryogenic storage tube was purchased from Thermo, item number: 375418;

[0039] 1.5 mL EP tube was purchased from Borys Technology, item number: BHY02S1CA-S.

[0040] UPM is urban particulate matter, which is black solid, and is a mixture of various urban particulate matters, including PM 2.5 , PM 10 , inorganic elements and organic pollutants, wherein the inorganic elements include aluminum, iron, lead, etc., and the organic pollutants include polycyclic aromatic hydrocarbons, polychlorinated biphenyls, DDT, etc.

[0041] The specific experimental methods and results are as follows:

[0042] I. Embryo sample collection and processing

[0043] 1. Preparation of UPM

[0044] Take 0.6 g of UPM with a 15 mL centrifuge tube, and dissolve it in 15 mL of ultrapure water to prepare a UPM mother liquor with a concentration of 40 mg / mL. After 6 h of ultraviolet disinfection, use an ultrasonic mixer to treat for 1 h to ensure that it is fully dispersed. After treatment, seal the centrifuge tube with sealing film, and store it in a 4°C refrigerator.

[0045] 2. Zebrafish breeding and exposure

[0046] Wild-type zebrafish were purchased from Nanjing Yushuliuhua Biotechnology Co., Ltd. New zebrafish were acclimated at room temperature for a period of time, and then were bred in a circulating water breeding system at a density of 5 per tank, with water temperature maintained at 28 ± 0.5°C, pH value controlled between 7.0 and 8.0, and light cycle set to 14 h light and 10 h darkness. After a week of acclimation, when the fish were stable, subsequent experiments were performed.

[0047] Select 10 female fish, and divide them into a control group and an experimental group, 5 in each group, and breed them in separate fish tanks, with 1 L of breeding water added to each fish tank. The fish tank of the experimental group needs to additionally add 2.5 mL of UPM mother liquor to prepare a UPM treatment solution with a final concentration of 100 μg / mL. Replace the water every other day, and culture for 21 days.

[0048] 3. Zebrafish embryo collection and processing

[0049] Adult male and female fish were placed on both sides of the partition in the spawning tank according to a 1:1 gender ratio, and they were placed in a constant temperature incubator at 28°C overnight. The next day, the partition was removed within 0.5 h after the start of light, and the fish were allowed to mate for 2 h before returning the adult fish to the original tank, and then the fertilized eggs were collected. The collected fertilized eggs were washed with system water, and dead embryos and other impurities were removed, and transparent and normally shaped zebrafish embryos were selected for subsequent experiments. The cleaned embryos were transferred to culture dishes at n = 50 per group, and control and experimental groups were set up, with 3 replicates per group, and were placed in a constant temperature incubator at 28°C, and the culture water was changed every 24 h and the dead embryos were removed. The embryos were cultured to 3 dpf, and the embryos were transferred to cryogenic tubes at n = 50 per group, the liquid in the cryogenic tubes was aspirated as much as possible, then placed in liquid nitrogen for rapid freezing, and finally the samples were stored in a -80°C refrigerator.

[0050] II. RNA extraction from embryo samples

[0051] Add 1 mL Trizol and 2 grinding beads to a 1.5 mL EP tube, and then add the sample. After balancing, place it in the grinder for grinding: 65 Hz, 60 s, 10 s, 6 cycles; grind twice, and stand at room temperature for 5 min. Centrifuge at 12,000 rpm for 10 min at 4°C, and transfer the supernatant to a new EP tube; add 200 μL chloroform to the tube, shake vigorously for 15 s, and stand at room temperature for 3 min. Centrifuge at 12,000 rpm for 15 min at 4°C, and the upper aqueous phase is RNA; transfer the upper aqueous phase to a new 1.5 mL EP tube, add an equal volume of isopropanol, mix thoroughly, and stand at -20°C overnight; centrifuge at 12,000 rpm for 10 min at 4°C, and the gel at the bottom of the tube is RNA, discard the supernatant, and wash the precipitate with 1 mL of 75% ethanol; centrifuge at 7,500 rpm for 5 min at 4°C, discard the supernatant; centrifuge at 7,500 rpm for 2 min at 4°C, aspirate the liquid as much as possible, stand at room temperature for 5-10 min, and dry the RNA; add 20 μL RNase-Free H2O and mix.

[0052] III. RNA quantification and reverse transcription

[0053] Refer to the Eastep RT Master Mix Kit kit, and follow the instructions. After repeated improvements, the specific operation process is as follows:

[0054] 1. Use a ultramicro spectrophotometer to measure the RNA concentration in the zebrafish embryo sample:

[0055] Open the sampling arm, wipe the mirror paper clean the measurement base; suction 1 μL DEPC water as a blank sample added to the measurement base surface, click "blank" in the software to zero; after zero, wipe the droplets on the base and sampling arm with a lens paper, suction 1 μL of the RNA sample to be tested, drop on the measurement base surface, click "test" in the software to determine the sample RNA concentration, and record as c (ng / μL). After determining the concentration, if the concentration is too high, you can add an appropriate amount of RNase-Free H2O to adjust the concentration between 500-600 ng / μL. Record the final concentration.

[0056] 2. Reverse transcription reaction system configuration, RNA template volume x = 2000 / c:

[0057] Eastep RT Master Mix, 4 μL;

[0058] RNase-Free ddH2O, 16-x μL; Total RNA, x μL;

[0059] Total volume, 20 μL.

[0060] 3. The reaction procedure is as follows:

[0061] Use the PCR instrument to perform the following heating incubation procedure on the reverse transcription reaction tube: reverse transcription, 42℃, 60 min; enzyme inactivation, 98℃, 5 min; 4℃, keep.

[0062] 4. After the reaction is completed, the reverse transcription product (cDNA) is 20 μL, and 180 μL of ddH2O is added to dilute it 10 times for subsequent Real-time PCR experiments.

[0063] Four, gene quantification and analysis in zebrafish embryos

[0064] Use Real-time PCR method to quantitatively analyze the genes in zebrafish embryos. Refer to the BlasTaq Green2× qPCR Master Mix kit, and follow the instructions. After repeated improvement, the specific operation process is as follows:

[0065] 1. Primer: β-actin is used as an internal reference in zebrafish embryos. The primers of the target gene and β-actin are synthesized in Shengong Bioengineering Co., Ltd., including sequence-specific forward primer (Forward primer) and reverse primer (Reverse primer).

[0066] 2. PCR reaction system configuration (μL)

[0067] PCR system was prepared according to Table 1, and the whole process was carried out on ice. Three replicates were set for each reaction well of the PCR plate. The primer sequence is shown in Table 2. The nucleotide sequence of g2e3 is shown in SEQ ID No. 1.

[0068] Table 1

[0069] Table 2

[0070]

[0071] 3. PCR reaction

[0072] After sealing with a quantitative sealing film, it was placed in a microplate centrifuge for centrifugation for 10 s, and the side wall liquid was centrifuged to the bottom. Fluorescent quantitative PCR was performed using a BIO-RAD fluorescent quantitative PCR instrument. The program is as follows (Table 3):

[0073] Table 3

[0074]

[0075] 4. Data analysis

[0076] CT value reading was performed using Bio-Rad CFX Manager software, and the formula was:

[0077] Expression = 2 Ct(β-a )-Ct(g2e3)

[0078] The expression level of each sample in the control group and the experimental group was calculated respectively.

Claims

1. A zebrafish gene marker for detecting the influence of urban particulate matter on heart development, characterized in that, The genetic marker is a g2e3 gene.

2. A kit for detecting the effect of urban particulate matter on heart development, characterized in that, The reagent comprises the zebrafish genetic marker of claim 1.

3. A method of using the kit according to claim 2, characterized in that, The method comprises the following steps: S1, female zebrafish are divided into a control group and a detection group, the control group is cultured using aquaculture water, and the detection group is cultured using aquaculture water containing urban particulate matter to be detected; S2, the two groups of female zebrafish are mated with male zebrafish respectively, fertilized eggs are collected, embryos are cultured, and the expression levels of the g2e3 gene in the two groups of embryos are detected; compared with the control group, if the expression of the g2e3 gene in the embryos of the detection group is significantly decreased, it is determined that the urban particulate matter in the aquaculture water will affect heart development.

4. The method of claim 3, wherein, In step S2, the expression levels of the g2e3 gene in the two groups of embryos are detected, including the following steps: extracting RNA of the two groups of embryos, determining the RNA concentration, reverse transcribing the RNA into cDNA, quantitatively analyzing the g2e3 gene in the zebrafish embryos, taking the β-actin gene in the zebrafish embryos as an internal reference, adding corresponding primers of the g2e3 gene and corresponding primers of the β-actin gene, and performing fluorescent quantitative PCR.

5. The method of claim 4, wherein, In step S2, the primer sequence of the g2e3 gene is shown in SEQ ID NO. 2 and SEQ ID NO.

3.

6. Use of a reagent for detecting a zebrafish genetic marker in the manufacture of a kit for detecting the effect of urban particulate matter on heart development, characterized in that, The genetic marker is a g2e3 gene.

7. Use according to claim 6, characterized in that, The primer sequence of the g2e3 gene is shown in SEQ ID NO. 2 and SEQ ID NO.

3.

8. Use according to claim 6, characterized in that, The use steps of the kit comprise: S1, female zebrafish are divided into a control group and a detection group, the control group is cultured using aquaculture water, and the detection group is cultured using aquaculture water containing urban particulate matter to be detected; S2, the two groups of female zebrafish are mated with male zebrafish respectively, fertilized eggs are collected, embryos are cultured, and the expression levels of the g2e3 gene in the two groups of embryos are detected; compared with the control group, if the expression of the g2e3 gene in the embryos of the detection group is significantly decreased, it is determined that the urban particulate matter in the aquaculture water will affect heart development.

9. Use according to claim 8, characterized in that, In step S2, the expression levels of the g2e3 gene in the two groups of embryos are detected, including the following steps: extracting RNA of the two groups of embryos, determining the RNA concentration, reverse transcribing the RNA into cDNA, quantitatively analyzing the g2e3 gene in the zebrafish embryos, taking the β-actin gene in the zebrafish embryos as an internal reference, adding corresponding primers of the g2e3 gene and corresponding primers of the β-actin gene, and performing fluorescent quantitative PCR.

10. A method of detecting the effect of urban particulate matter on heart development, characterized in that, The method comprises the following steps: S1, female zebrafish are divided into a control group and a detection group, the control group is cultured using aquaculture water, and the detection group is cultured using aquaculture water containing urban particulate matter to be detected; S2, the two groups of female zebrafish are mated with male zebrafish respectively, fertilized eggs are collected, embryos are cultured, and the expression levels of the g2e3 gene in the two groups of embryos are detected; compared with the control group, if the expression of the g2e3 gene in the embryos of the detection group is significantly decreased, it is determined that the urban particulate matter in the aquaculture water will affect heart development.

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