CN117265080B - Zebra fish gene markers and application thereof in assessing risk of nerve injury caused by environmental pollutants - Google Patents
Zebra fish gene markers and application thereof in assessing risk of nerve injury caused by environmental pollutants Download PDFInfo
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Abstract
The invention provides a group of zebra fish gene markers and application thereof in evaluating the risk of nerve injury caused by environmental pollutants, belonging to the technical field of pollutant detection; a set of zebra fish gene markers for assessing the risk of nerve damage by environmental contaminants, comprising: c6ast3, endou and tspan34 genes; the application of the zebra fish gene marker in evaluating the risk of nerve injury caused by environmental pollutants; and a method for assessing the risk of nerve damage caused by environmental contaminants; the invention discovers three genes of c6ast3, endou and tspan34 of zebra fish for the first time, and the three genes are cultured in a sample containing a nerve injury causing substance, and compared with a blank control group, the expression level of the three genes is obviously increased.
Description
Technical Field
The invention belongs to the technical field of pollutant detection, and relates to a group of zebra fish gene markers and application thereof in assessing the risk of nerve injury caused by environmental pollutants.
Background
Pollution is an unavoidable problem in modern society, and various types of pollutants are produced by any industry or household. Contaminants have a great impact on the environment and health and must therefore be detected and monitored in order to take the necessary measures to reduce or eliminate the contaminants. Methods for detecting contaminants are involved in many areas, including chemistry, physics, engineering, and data analysis, among others. Common contaminant detection methods include gas chromatography, liquid chromatography, mass spectrometry, atomic absorption spectrometry, nuclear magnetic resonance, marine mass index, and the like.
Of course, there are also many common biological detection methods for monitoring and studying water pollution conditions, including biocenosis, productivity assays, residual toxicity assays, acute toxicity tests, and the like. The existing method for detecting the neurotoxicity of pollutants is to add a target compound into a growth medium of zebra fish, and observe whether the behavior response of the zebra fish is affected, such as swimming speed rotation behavior and the like.
In addition, zebra fish has 87% homology with human genes, and a large number of new functions of the genes in zebra fish are not developed and utilized. At present, a scientific, accurate and rapid environmental pollutant detection method is urgently needed.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a group of zebra fish gene markers and application thereof in evaluating the risk of nerve injury caused by environmental pollutants.
The technical scheme of the invention is as follows:
a set of zebra fish gene markers for assessing the risk of nerve damage by environmental contaminants, comprising: c6ast3, endou and tspan34 genes.
Use of a set of zebra fish gene markers for assessing the risk of nerve damage by environmental contaminants, the zebra fish gene markers comprising: c6ast3, endou and tspan34 genes.
According to a preferred embodiment of the invention, the primer sequences for the c6ast3, endou and tspan34 genes are as follows:
c6ast3-RT-F：GTCTGTGTCTGAACTGCTGC SEQ ID NO.1；
c6ast3-RT-R：GAGGAGTAGTGGTTGGCGAT SEQ ID NO.2；
endou-RT-F：TTGGCCAATGTGTCAGATGC SEQ ID NO.3；
endou-RT-R：GCCGGTCTGAGAAGAAGGAASEQ ID NO.4；
tspan34-RT-F：ATCAGCGGTGGACAGGTAAC SEQ ID NO.5；
tspan34-RT-R：CCAGGAAGATGATGCCGTTG SEQ ID NO.6。
a method of assessing the risk of nerve damage from environmental contaminants comprising the steps of:
(1) Dividing 6dpf zebra fish into a blank control group and a detection group, wherein the blank control group is cultivated by using fish-farming water, and the detection group is cultivated by using fish-farming water containing environmental pollutants;
(2) After culturing for 24-25h, respectively collecting each group of zebra fish, and detecting the expression levels of marker genes c6ast3, endou and tspan34 in each group of zebra fish; compared with a blank control group, the three genes c6ast3, endou and tspan34 of the zebra fish in the detection group are obviously increased; the risk of nerve damage by environmental contaminants is judged.
According to a preferred embodiment of the invention, in step (1), the zebra fish strain is wild-type AB zebra fish.
According to a preferred embodiment of the invention, in step (1), 3 zebra fish are present in parallel, and 20-25 zebra fish are present in parallel.
According to the present invention, in the step (1), the method for preparing the fish culture water comprises: firstly preparing E3 fish culturing water, wherein each liter of E3 fish culturing water comprises 14.62-15g NaCl, 0.63-1.00g KCl and CaCl 2 2.42-2.9g、MgSO 4 ·7H 2 4.06-4.98g of O and the balance of deionized water, and then diluting the prepared E3 fish culture water by more than 50 times to obtain the fish culture water.
Further preferably, the prepared E3 fish farming water is diluted 50 times to obtain fish farming water.
According to a preferred embodiment of the invention, in step (1), the environmental contaminant is wastewater.
Further preferably, the environmental pollutants include chromium-containing wastewater, arsenic-containing wastewater, lead-containing wastewater, mercury-containing wastewater, copper-containing wastewater.
According to the preferred embodiment of the present invention, in the step (2), the zebra fish is cultured in a constant temperature incubator at 28℃for 24-25 hours.
According to a preferred embodiment of the invention, in step (2), the primer sequences for the c6ast3, endou and tspan34 genes are as follows:
c6ast3-RT-F：GTCTGTGTCTGAACTGCTGC SEQ ID NO.1；
c6ast3-RT-R：GAGGAGTAGTGGTTGGCGAT SEQ ID NO.2；
endou-RT-F：TTGGCCAATGTGTCAGATGC SEQ ID NO.3；
endou-RT-R：GCCGGTCTGAGAAGAAGGAASEQ ID NO.4；
tspan34-RT-F：ATCAGCGGTGGACAGGTAAC SEQ ID NO.5；
tspan34-RT-R：CCAGGAAGATGATGCCGTTG SEQ ID NO.6。
according to the preferred embodiment of the present invention, in the step (2), total RNAs of different groups of zebra fish are extracted using an RNA extraction kit, RNA concentration is measured using an ultra-micro spectrophotometer, the RNA is reverse transcribed into cdnas using a reverse transcription kit, and the expression levels of marker genes c6ast3, endou and tspan34 in each group of zebra fish are detected by adding corresponding primers of three genes c6ast3, endou and tspan34 respectively into a qRTPCR instrument using each group of reverse transcribed cdnas as templates according to a real-time fluorescent quantitative PCR specification, and three parallel tests are set for each group.
The beneficial effects of the invention are as follows:
1. the invention discovers for the first time that three genes of c6ast3, endou and tspan34 of zebra fish are cultured in a sample containing a substance causing nerve injury, and compared with a blank control group, the expression level of the three genes is obviously increased.
2. The gene panel consisting of three genes of c6ast3, endou and tspan34 of zebra fish can be used for evaluating the risk of nerve injury caused by environmental pollutants.
3. The invention further provides a method for evaluating the risk of nerve damage caused by the environmental pollutants, and the method provided by the invention has the advantages of short operation flow, easiness in operation, accurate and efficient detection result and provides a new method for detecting and evaluating the risk of nerve damage caused by the environmental pollutants.
Drawings
Fig. 1 is a graphical representation of the behavioral swimming speed and distance of zebra fish for five environmental pollutants from each group (< 0.001 vs. blank).
Fig. 2 is a schematic representation of the expression levels of the environmental pollutant-induced nerve injury marker genes c6ast3, endou and tspan34 in each group (P < 0.05 vs. blank, P < 0.01 vs. blank, P < 0.001 vs. blank).
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments.
The wild type AB strain zebra fish is provided by a drug screening key laboratory of Shandong national academy of sciences of biological sciences, and can also be purchased directly from the national zebra fish resource center. And is executed strictly according to the relevant processing criteria of the international experimental animal protection certification and evaluation institution. The animal use license number was SWS20211117.
The preparation process of the fish culture water comprises the following steps: firstly, preparing E3 fish-raising water, the mixture ratio of which is 14.658g NaCl, 0.633g KCl and CaCl 2 2.425g, mgSO4.7H2O 4.06g and purified water 1L. 20mL of E3 fish-farming water was taken, 980mL of deionized water was added, and the fish-farming water was prepared.
Composition of environmental pollutants: chromium-containing wastewater (chromium chloride hexahydrate, 4 mg/L), arsenic-containing wastewater (arsenic pentoxide, 4.5 mg/L), lead-containing wastewater (lead acetate, 3 mg/L), mercury-containing wastewater (mercurous chloride, 2 mg/L), copper-containing wastewater (copper sulfate, 3 mg/L); the solvent for preparing the environmental pollutants is the fish-farming water.
Example 1
A method of assessing the risk of nerve damage caused by environmental contaminants, comprising the steps of:
(1) Acquisition of nerve injury marker Gene
Adult zebra fish are cultured in a constant temperature zebra fish culture box at 28 ℃ according to the proportion of 14: feeding in a dark and light circulating environment for 10 hours according to the following ratio of 2:2, the male and female are put into a spawning cylinder for isolation in the afternoon 1 day before the experiment, 8:30, extracting a baffle plate, collecting fertilized eggs of zebra fish after about 2 hours, transferring the fertilized eggs into culture water containing 2mg/L of methylene blue, placing the culture water into a constant-temperature illumination incubator at 28 ℃ for light control incubation, placing embryos under a microscope for observation after 4 hours, selecting normal-development embryos to hatch to 6dpf after fertilization, and timely removing dead eggs every day during the period.
Zebra fish of 6dpf were transferred into 6-well plates, 20 per well, three per group, 20 per group, and divided into a blank group and a nerve damage environmental pollutant group (lead acetate, 50mg/L, solvent is fish-farming water). 5mL of fish-farming water was added to the blank control group, and 5mL of nerve-damaging environmental pollutants was added to the nerve-damaging environmental pollutants group.
After 24 hours of treatment, respectively collecting each group of zebra fish, washing the zebra fish for two to three times by using fish-farming water, transferring the zebra fish into a centrifuge tube, and sucking the upper liquid to leave the tissue of the bottom zebra fish; extracting total RNA of zebra fish, performing transcriptome study on the extracted total RNA of zebra fish in a blank control group and a nerve injury environment pollutant group, performing significance analysis on gene expression differences of the two groups of zebra fish, performing information analysis by using a statistical method, comparing the gene expression differences of the two groups of zebra fish, and finding out specific genes related to conditions. As a result of analysis of transcriptome sequencing, c6ast3, endou and tspan34 are pollutant nerve injury marker genes. Primer sequences were designed for the nerve injury environmental pollutant marker genes c6ast3, endou and tspan 34:
the Gene sequences are derived from NCBI, gene ID 541399, 791216, 751731 respectively
c6ast3-RT-F：GTCTGTGTCTGAACTGCTGC SEQ ID NO.1
c6ast3-RT-R：GAGGAGTAGTGGTTGGCGAT SEQ ID NO.2
endou-RT-F：TTGGCCAATGTGTCAGATGC SEQ ID NO.3
endou-RT-R：GCCGGTCTGAGAAGAAGGAA SEQ ID NO.4
tspan34-RT-F：ATCAGCGGTGGACAGGTAAC SEQ ID NO.5
tspan34-RT-R：CCAGGAAGATGATGCCGTTG SEQ ID NO.6
(2) Assessment of environmental pollutant-induced nerve loss risk using behavioural experiments
5 kinds of environmental pollution waste water are artificially simulated, namely chromium-containing waste water, arsenic-containing waste water, lead-containing waste water, mercury-containing waste water and copper-containing waste water. The influence of nerve injury environmental pollutants on the behavior of zebra fish is observed firstly based on behaviours. Zebra fish eggs are obtained and incubated according to the steps, the zebra fish with the length of 6dpf are transferred into a 6-hole plate and are divided into a blank control group and an environmental pollutant group, 5mL of fish raising water is added into the blank group, and 5mL of environmental pollutant is added into each environmental pollutant group.
After 24h treatment, transferring each group of zebra fish larvae with the length of 6dpf into a 48-hole plate, placing the 48-hole plate into a zebra ox zebra fish behavioral analyzer camera bellows to adapt for 10 minutes, starting to detect the influence of environmental pollutants on the zebra fish larvae, wherein the detection time is 20 minutes, recording data once per minute, carrying out data treatment by using Zeblab software, and calculating the total swimming distance and the swimming speed of the zebra fish. As shown in fig. 1, the environmental pollutants of mercury and copper do not have a significant effect on the behavior of the zebra fish, and the other three pollutants reduce the swimming speed and the total swimming distance of the zebra fish.
(3) Assessment of environmental pollutant-induced nerve injury risk using gene markers
After the behavioural experiment is completed, respectively collecting each group of zebra fish, washing the zebra fish for two to three times by using fish-farming water, transferring the zebra fish into a centrifuge tube, and sucking the upper liquid to leave the tissue of the bottom zebra fish; extracting total RNA of different groups of zebra fish by using an RNA extraction kit, measuring the concentration of the RNA by using an ultra-micro spectrophotometer, reversely transcribing the RNA into cDNA by using a reverse transcription kit, referring to a real-time fluorescent quantitative PCR instruction, taking the cDNA after each group of reverse transcription as a template, adding a primer to be detected for amplification in a qRTPCR instrument, setting three parallel tests in each group, and detecting the expression levels of nerve injury environmental pollutant marker genes c6ast3, endou and tspan34, wherein the reaction system and the conditions are shown in the following tables 1 and 2:
RT-PCR reaction system:
TABLE 1
Reaction conditions:
TABLE 2
Rpl13a was used as an internal control in the experiment and 3 duplicate wells were made. Removing the error is larger, the dissolution curve is not single and the amplification curve is not singleLine abnormal data, reuse 2 ΔΔCT The method performs analysis statistics. The results are shown in FIG. 2, which shows that the expression of the nerve injury marker genes c6ast3, endou and tspan34 can be significantly increased after the treatment of 5 environmental pollutants compared with the blank control group.
In summary, the invention discovers for the first time that three genes c6ast3, endou and tspan34 of zebra fish are cultured in samples containing nerve-damaging substances, and the expression levels of the three genes are obviously increased compared with a blank control group. The gene panel consisting of three genes of c6ast3, endou and tspan34 of zebra fish can be used for evaluating the risk of nerve injury caused by environmental pollutants. The method provided by the invention has the advantages of short operation flow, easiness in operation and accurate and efficient detection result, and provides a new method for detecting and evaluating the nerve loss risk caused by the environmental pollutants.
Claims (9)
1. A set of zebra fish gene markers for assessing the risk of nerve damage by environmental contaminants, comprising:c6ast3、endouandtspan34and (3) a gene.
2. The application of a primer for detecting a zebra fish gene marker in preparing a kit for evaluating the risk of nerve injury caused by environmental pollutants is characterized in that the zebra fish gene marker comprises:c6ast3、endouandtspan34a gene;
c6ast3、endouandtspan34the primer sequences of the genes are as follows:
c6ast3-RT-F：GTCTGTGTCTGAACTGCTGCSEQ ID NO.1；
c6ast3-RT-R：GAGGAGTAGTGGTTGGCGATSEQ ID NO.2；
endou-RT-F：TTGGCCAATGTGTCAGATGCSEQ ID NO.3；
endou-RT-R：GCCGGTCTGAGAAGAAGGAASEQ ID NO.4；
tspan34-RT-F：ATCAGCGGTGGACAGGTAACSEQ ID NO.5；
tspan34-RT-R：CCAGGAAGATGATGCCGTTGSEQ ID NO.6。
3. the use of claim 2, wherein the kit-of-parts step comprises:
(1) Dividing 6dpf zebra fish into a blank control group and a detection group, wherein the blank control group is cultivated by using fish-farming water, and the detection group is cultivated by using fish-farming water containing environmental pollutants;
(2) After culturing 24-25 and h, collecting each group of zebra fish, and detecting marker genes in each group of zebra fishc6ast3、endouAndtspan34expression level of (2); compared with the blank control group, the detection group zebra fishc6ast3、endouAndtspan34the expression of the three genes is obviously increased; the risk of nerve damage by environmental contaminants is judged.
4. The use according to claim 3, wherein in step (1) the zebra fish strain is wild-type AB zebra fish.
5. The use according to claim 3, wherein in step (1) 3 zebra fish of each group are parallel and 20-25 zebra fish of each group are parallel.
6. The use according to claim 3, wherein in step (1), the method of preparing the fish farming water comprises: firstly preparing E3 fish culturing water, wherein each liter of E3 fish culturing water comprises NaCl 14.62-15g, KCl 0.63-1.00g and CaCl 2 2.42-2.9 g、MgSO 4 ·7H 2 O4.06-4.98 g and the balance deionized water, and then diluting the prepared E3 fish culture water by more than 50 times to obtain the fish culture water;
and diluting the prepared E3 fish culture water by 50 times to obtain the fish culture water.
7. The use according to claim 3, wherein in step (1) the environmental contaminant is chromium-containing wastewater, arsenic-containing wastewater, lead-containing wastewater, mercury-containing wastewater, copper-containing wastewater.
8. The use according to claim 3, wherein in step (2) zebra fish are incubated in a constant temperature incubator at 28 ℃ for 24-25h.
9. The use according to claim 3, wherein in step (2), total RNA of different groups of zebra fish is extracted by using an RNA extraction kit, the concentration of RNA is measured by using an ultra-micro spectrophotometer, RNA is reverse transcribed into cDNA by using a reverse transcription kit, and the cDNA after completion of the reverse transcription of each group is used as a template and added respectively by referring to real-time fluorescent quantitative PCR instructionsc6ast3、endouAndtspan34corresponding primers of the three genes are amplified in a qRT-PCR instrument, each group is provided with three parallel tests, and marker genes in each group of zebra fish are detectedc6ast3、endouAndtspan34is a target expression level.
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| CN109633140A (en) * | 2018-12-19 | 2019-04-16 | 中国环境科学研究院 | A method of perfluorochemical neurodevelopment toxicity is evaluated using zebra fish |
| CN109971864A (en) * | 2019-02-28 | 2019-07-05 | 南华大学 | A method of it is damaged using the expression quantity evaluation low dosage radiated by gamma-ray of zebrafish embryo brain ywhah gene |
| CN115656435A (en) * | 2022-10-25 | 2023-01-31 | 中国农业科学院农业质量标准与检测技术研究所 | Method for evaluating influence of bisphenol pollutants on nervous system of zebra fish based on social behaviors of zebra fish |
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| CN109633140A (en) * | 2018-12-19 | 2019-04-16 | 中国环境科学研究院 | A method of perfluorochemical neurodevelopment toxicity is evaluated using zebra fish |
| CN109971864A (en) * | 2019-02-28 | 2019-07-05 | 南华大学 | A method of it is damaged using the expression quantity evaluation low dosage radiated by gamma-ray of zebrafish embryo brain ywhah gene |
| CN115656435A (en) * | 2022-10-25 | 2023-01-31 | 中国农业科学院农业质量标准与检测技术研究所 | Method for evaluating influence of bisphenol pollutants on nervous system of zebra fish based on social behaviors of zebra fish |
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