CN112782144B - Method for detecting toxicity of organic pollutants in water body based on DNA damage repair defect living cell comet - Google Patents
Method for detecting toxicity of organic pollutants in water body based on DNA damage repair defect living cell comet Download PDFInfo
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Abstract
The invention discloses a method for detecting toxicity of organic pollutants in a water body based on DNA damage repair defect living cells comet, which comprises the steps of firstly collecting water sample enrichment, treating the water sample enrichment with DNA damage repair defect living cells (TDP1KO and TDP2KO), simultaneously using the DNA damage repair normal living cells (WT) treated by the water sample enrichment as a contrast, combining a single cell gel electrophoresis technology, analyzing and measuring DNA comet migration tail moment values of the DNA damage repair defect living cells and the normal cells, and evaluating the toxicity of the organic pollutants in the water body. The method improves the sensitivity of genetic toxicity detection of the organic pollutants in the water body, can detect the ultra-micro organic pollutants in the water, has important significance for early warning of harm and health of environmental pollution, and can explore the molecular mechanism of biological toxicity of the organic pollutants in the water from the aspect of DNA damage repair.
Description
Technical Field
The invention belongs to the field of environmental health genetic toxicology, and particularly relates to a method for detecting toxicity of organic pollutants in water by using DNA damage repair defect living cells in combination with a single cell electrophoresis experiment technology.
Background
With the increase of population and the rapid development of modern industry and agriculture, the environmental pollution problem becomes more serious, and the results of epidemiological investigation at home and abroad show that organic pollutants exist in tap water and source water, and the organic matters can enter human bodies through water and food chains to cause the DNA damage of genetic materials of the human bodies, influence the genetic stability of cells, and cause the abortion, the stillbirth, the teratogenesis and certain hereditary diseases and cancers of pregnant women. Therefore, it is important to detect the toxicity of organic pollutants in water to cellular DNA and to know whether the damage can cause the change of cell genetic materials.
Many developed countries are typically based on bacterial or mammalian cell testing systems that detect the genotoxicity of contaminants in aqueous samples using the Ames method (Ames test), chromosome aberration analysis, or Single Cell Gel Electrophoresis (SCGE). The single cell gel electrophoresis (comet assay) is a quantitative detection means widely used for eukaryotic single cell DNA damage. The working principle is that DNA in cells is damaged, and the superhelix high-level structure of the DNA is changed, so that the structure of the DNA is loose. If cells are embedded into low-melting-point agarose gel, the gel is fixed on a glass slide, then cell membranes and nuclear membranes are damaged by lysis solution, so that nuclear DNA and other components enter the gel due to membrane damage, then electrophoresis is carried out under alkaline conditions, macromolecular nuclear DNA in the cells can still remain in situ, if the cellular DNA is damaged, the DNA is changed from double-stranded uncoiling into single-stranded DNA under the action of alkaline electrophoresis solution (pH 13), the DNA of the damaged cells can migrate to the anode under the action of an electric current field, through dyeing gel cellular DNA, comet tailing can be observed under a fluorescence microscope, the comet tail length is positively correlated with the DNA damage degree, the higher the damage degree of the cellular DNA is, the more chain breakage and DNA fragments are generated, and the longer and brighter comet tail moment is detected by fluorescence. If the cells have no DNA damage, no broken chain and fragment DNA are generated, and the fluorescent microscope observation shows that the circular fluorophore is more regular and has no tailing phenomenon. Compared with other common genetic ecotoxicology biomarkers, the SCGE has the characteristics of simplicity, convenience, rapidness, sensitivity, low cost, high efficiency and the like.
Over the last half century, various laboratory scientists have discovered and elucidated some of the key genes for DNA damage repair and their biological roles, such as TDP1, TDP2, XRCC1, PARP1, and others. Among them, TDP1 and TDP2 have the enzymatic activities of degrading the 3 'and 5' DNA ends of protein tyrosine-DNA complex, and participate in various DNA repair signal transduction and repair paths. TDP1 cleaves DNA breaks caused by topoisomerase I-mediated and oxidative damage-induced 3' phosphate and alkylation damage, while TDP2 repairs topoisomerase II-mediated DNA damage.
However, at home and abroad, no technical method for detecting the potential genetic toxicity of the organic pollutants in the water body by combining DNA damage repairing defective living cells with a single-cell gel electrophoresis experimental technology is provided so far. Therefore, the method is efficient, rapid and sensitive, can provide information about the genotoxicity mechanism of the pollutants, is used for evaluating the genetic toxicity of the organic matters in the water, and has important health significance and social and economic benefits.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings in the background art and provide a method for efficiently, quickly and sensitively detecting the toxicity of organic pollutants in water.
In order to solve the technical problems, the technical scheme provided by the invention is as follows:
a method for detecting toxicity of organic pollutants in water based on DNA damage repair defect living cell comet includes the following steps:
(1) collecting a water sample enrichment, treating DNA damage repair defect living cells by the water sample enrichment, and meanwhile, using the DNA damage repair normal living cells treated by the water sample enrichment as a control; respectively centrifuging and precipitating the treated DNA damage repair defect living cells and normal living cells, then resuspending the cells by PBS, and centrifuging to obtain a first cell suspension for later use;
(2) preparing the first cell suspension obtained after the treatment of the step (1) into a comet electrophoresis glass slide cell sample;
(3) removing a cover glass of the comet-star electrophoresis glass slide cell sample obtained in the step (2), immersing the glass slide into an alkaline lysis solution for cell lysis, taking out the lysed glass slide, placing the glass slide into an electrophoresis tank for electrophoresis, and neutralizing the glass slide cell sample after the electrophoresis is finished;
(4) after neutralization, dripping DNA fluorescent dye on a glass slide cell sample, covering a cover glass, observing change of comet (Olive) by using a fluorescence microscope, measuring comet tail moment values of DNA damage repairing defect living cells and normal living cells treated by the water sample enrichment, and obtaining a DNA comet migration tail moment value.
In the above method, preferably, the living cell deficient in DNA repair is obtained by knocking out DNA repair gene TDP1 and/or TDP2 from chicken B lymphocyte (DT 40).
The research proves that cells with TDP1 and/or TDP2 gene defects are allergic to poison causing DNA damage, and the change of comet tail moment of cells with DNA damage repair defects can be caused by the ultra-micro poison concentration if a comet analysis method is also adopted, so that the sensitivity of organic pollutant toxicity detection in a water body can be improved by applying the DNA damage repair defects cells and the comet analysis, and information about the genotoxicity mechanism of pollutants can be provided. That is, after the TDP1 and/or TDP2 are knocked out, the DNA repair function of chicken B lymphocytes is lost, and the chicken B lymphocytes are sensitized to the genetic toxicity of organic pollutants in a water body.
The reason for using the chicken B lymphocyte cell line (DT40) was: firstly, DT40 is the only somatic cell that can efficiently realize gene knockout except embryonic stem cells; secondly, due to efficient gene targeting, gene mutant cloning of known DNA damage repair pathways has been established (Zeng nucleic acids res.2012); thirdly, the epigenetics and karyotype of the knockout cell are stable; and fourthly, the DT40 cells grow in a suspension mode, the cells proliferate quickly, can be split three times per day, and are easy to culture.
More preferably, the DNA damage repair deficient living cell is a TDP1KO cell line or a TPD 2KO cell line, the TDP1KO cell line is obtained by knocking out a gene segment shown as SEQ ID NO:1 on a TDP1 gene of a chicken B lymphocyte (DT40), and the TPD 2KO cell line is obtained by knocking out a gene segment shown as SEQ ID NO:2 on a TDP2 gene of the chicken B lymphocyte DT 40. TDP1 and/or TDP2 genes are knocked out in a chicken B lymphocyte cell line (DT40), compared with wild cells, TDP1 and/or TDP2 gene knockout cells obviously delay DNA repair, sensitize water body pollutants and detect trace organic pollutants in water.
Preferably, in the step (1), the method for collecting the water sample enrichment specifically comprises the following steps: collecting a water sample, standing for a period of time, filtering to remove suspended particles, enriching by a macroporous resin adsorption column, removing water by nitrogen, eluting by methanol, acetone and dichloromethane in sequence, concentrating and drying the obtained eluent, and finally dissolving by dimethyl sulfoxide to obtain a water sample enrichment substance; the first cell suspension is fineThe cell concentration is 0.5-1.0x105Individual cells/mL.
More preferably, the macroporous resin is XAD-2, XAD-4, XAD-7, XAD-8 or XAD-2/XAD-8 resin; the standing time is 24-36 h; the concentration temperature is 45-50 ℃; and storing the water sample enrichment in a dark place at the temperature of between 18 ℃ below zero and 70 ℃ below zero for later use.
Preferably, in the step (2), the method for preparing the comet electrophoresis slide specifically comprises the following steps: dripping Agarose (Agarose) with normal melting point on a frosted glass slide, quickly covering a cover glass, and solidifying to obtain a first layer of Agarose; and (2) adding an isometric Low-Melting-point Agarose (Low Melting point Agarose) solution into the first cell suspension obtained after the treatment in the step (1), blowing and beating uniformly to form a second cell suspension, spreading and dripping the second cell suspension onto the first layer of glue, covering a cover glass, solidifying to obtain a second layer of cell glue, and completing the preparation of the comet-star electrophoresis slide.
More preferably, the normal melting point agarose has a melting point of more than 90 ℃ and a mass concentration of 0.6%; the melting point of the low-melting-point agarose is 42-65 ℃, and the mass concentration of the solution is 1.2%; the curing temperature is 0-4 ℃, and the curing time is 30-120 min. Gelatinizing normal melting point agarose at 35-40 deg.C, and melting at more than 90 deg.C; the low melting point agarose is gelatinized at 26-30 deg.C and melted at 42-65 deg.C. The volume of the first cell suspension and the low melting point agarose solution was 200. mu.L, and the volume of the second cell suspension added to the first layer of gel was 150. mu.L.
Preferably, in the step (3), the alkaline lysis solution comprises 0.5M EDTA and 5M NaCL, the pH of the alkaline lysis solution is adjusted to 13 by using 5M NaOH, and 1% by volume of Triton X-100 and 1% by volume of dimethyl sulfoxide are added before use; the temperature of the alkaline lysis solution is 4 ℃, the pyrolysis temperature is 4 ℃, and the pyrolysis time is 10-120 min (more preferably 60 min).
Preferably, in the step (3), the specific operation of electrophoresis includes the following steps: injecting an alkaline electrophoresis solution with the pH value of 13.0 into an electrophoresis tank, standing for 30-45min, and then starting electrophoresis, wherein the current is 200-300 mA, the voltage is 12-25V, and the electrophoresis time is 20-30 min; and after electrophoresis, placing the glass slide in a Tris-HCL buffer solution for neutralization rinsing for 5-10 minutes, wherein the concentration of the Tris-HCL buffer solution is 0.4M, the rinsing times are 3 times, and the rinsing time is 15min each time.
Preferably, in the step (4), the DNA fluorescent dye is 1xSYBR Green I solution, and the dropping amount is 50-100 μ L; observing comets by a fluorescence microscope, obtaining comet images, and measuring comet tail moment values of DNA damage repairing defective living cells and normal living cells treated by the water sample concentrate by using CASP software to obtain DNA comet migration tail moment values.
The method of the invention firstly uses macroporous resin to enrich the water sample, and then uses DNA damage to repair the defective living cells and combines the Single Cell Gel Electrophoresis (SCGE) experimental technology to research the genetic toxicity of the organic pollutants in the water. The key technology is that DNA damage repairing defect living cells are selected, and the defect that normal living cells selected by the traditional SCGE method cannot detect ultra-micro pollutants is overcome. An ideal result is obtained through the optimization research of the experimental conditions of the single cell gel electrophoresis. Compared with the traditional comet analysis method, the method established by the invention can detect ultra-micro organic pollutants in water, has important significance for early warning of environmental pollution and health hazards, and can explore the molecular mechanism of the biotoxicity of the organic pollutants in water from the aspect of DNA damage repair.
Compared with the prior art, the invention has the beneficial effects that:
1. the method improves the sensitivity of genetic toxicity detection of the organic pollutants in the water body, can detect the ultra-micro organic pollutants in the water, and has important significance for early warning of environmental pollution and health hazards.
2. The method fills the blank about applying the DNA damage repair defect living cells to combine with the single cell gel electrophoresis experiment technology to detect the potential genetic toxicity of the organic pollutants in the water body, applies the DNA damage repair defect living cells to the SCGE, also expands the research objects of the SCGE, and can also explore the molecular mechanism of the biological toxicity of the organic pollutants in the water from the aspect of DNA damage repair and analyze the biological genetic toxicity of the pollutants.
3. Compared with the traditional comet analysis method, the chicken B lymphocyte cell line DT40 adopted by the method is simple to operate and low in cost by using other animal cells, and particularly, a Gene knockout-out (Gene Knock-out) defective cloned cell line of a DNA damage repair pathway is used as a toxicity test object of organic matters in water, so that the defective cell line is more sensitive to the epigenetic inheritance of the toxicity of the organic matters, and the influence of ultra-micro organic poisons on the cell phenotype can be detected.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a schematic diagram of Southern-blot analysis of gene knockdown in TDP1 and TPD2 cells; wherein, FIG. 1A shows the gene knockout of TDP1, and FIG. 1B shows the gene knockout of TDP 2;
FIG. 2 is a graph showing the data of the damage of single cell electrophoresis analysis of TDP1KO cell DNA by different concentrations of water samples (concentration ratio: 1-fold, 2-fold and 4-fold);
FIG. 3 shows the data of the damage of single cell electrophoresis analysis to TDP2KO cell DNA by different concentration water samples (concentration ratio: 1-fold, 5-fold and 10-fold).
Detailed Description
In order to facilitate understanding of the invention, the invention will be described more fully and in detail with reference to the accompanying drawings and preferred embodiments, but the scope of the invention is not limited to the specific embodiments below.
Unless otherwise defined, all terms of art used hereinafter have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the present invention are commercially available or can be prepared by existing methods.
Example (b):
the invention relates to a method for detecting toxicity of organic pollutants in water, which comprises the following steps:
1. pretreatment of water samples
In 6 months in 2019, 20L of water sample is collected in the Hunan river drinking water source section (Wangchuan) of Changsha, Hunan province according to the method specified by the State environmental protection agency. After standing for 24h, suspended particles were removed by filtration and enriched by means of an XAD-2 resin (Sigma) adsorption column. Removing water by using nitrogen at the flow rate of 30-40 mL/min, sequentially eluting by using methanol, acetone and dichloromethane, concentrating and drying eluent at 50 ℃, finally dissolving by using dimethyl sulfoxide (DMSO), and keeping constant volume to 2.0mL at minus 18 ℃ in a dark place for later use.
2. Cell culture and preparation
Constructing a gene homologous recombination targeting vector, transfecting DT40 cells, knocking out TDP1 gene (the gene sequence of which is shown as SEQ ID NO: 1) and TDP2 gene (the gene sequence of which is shown as SEQ ID NO: 2) of DT40 cells in different DT40 cells respectively, screening and identifying to obtain TDP1 and TPD2 gene Knock-out cell lines (Knock out, KO) which are named as TDP1KO cell line and TDP2KO cell line respectively, and storing the DNA damage repair defect cell lines in a cell bank of the institute of biological and environmental engineering of Changsha institute. Complete medium with RPMI1640 at 5% CO2And culturing at 39 ℃ for 1:3 passages.
Sequencing analysis shows that compared with a normal DT40 Wild-type cell (Wild type, WT), the TDP1KO cell line deletes the 485-2868 gene segment shown as SEQ ID NO:1 in the TDP1 gene, and the TDP2KO cell line deletes the 82-8725 gene segment shown as SEQ ID NO:2 in the TDP2 gene.
The Southern-blotting analysis was used to analyze the gene knockout of TDP1 and TPD2, and FIG. 1A shows the following steps from right to left: two alleles of TDP1 (TDP 1)+/+) One allele knock-out (TDP 1)+/-) Two allele knock-outs (TDP 1)-/-) (ii) a FIG. 1B shows, from right to left: three alleles of TDP2 (TDP 2)+/+/+) One allele knock-out (TDP 2)+/+/-) Two allele knock-outs(TDP2+/-/-) Three allele knockouts (TDP 2)-/-/-)。
The detection effects of TDP1KO and TDP2KO cells and WT cells are compared by performing a contamination experiment with TDP1KO, TDP2KO and normal DT40 wild-type cells (WT), respectively. The drinking water from Xiangjiang river in Changsha city is treated by infecting cells with XAD-2 resin adsorption column enriched matter, and three concentration treatment groups and non-infected blank control group CK (water sample without organic pollutant is used to treat normal DT40 wild cell) are set. Each concentration was 3 replicates. The cell concentration was 5X 105The cells/mL were treated with ice for 30 minutes, centrifuged at 2000rpm for 5min at 4 ℃ to remove the supernatant, resuspended in precooled PBS, centrifuged under the same centrifugation conditions to remove the supernatant, resuspended in PBS and adjusted to a cell concentration of 1X 105Individual cells/mL, to obtain a first cell suspension for use.
3. Detection of DNA damage degree by single cell gel electrophoresis
3.1 spreading glue
First, 150. mu.L of 0.6% (g/mL) of conventional Agarose was dropped onto a frosted glass slide, quickly covered with a cover slip, and cured at 4 ℃ for 1-2 hours. Then, 200. mu.L of the prepared cell suspension was put into a 1.5mL Eppendorf tube, 200. mu.L of 1.2% (g/mL) low melting point Agarose was added thereto, the mixture was blown out uniformly to form a second cell suspension, 150. mu.L of the mixture was applied to the first layer, and the mixture was covered with a cover slip and solidified at 4 ℃ for 60 minutes.
3.2 cleavage
And (3) removing a cover glass of the prepared glass slide, immersing the glass slide into alkaline lysis solution at 4 ℃, and lysing for 60 min.
3.3 electrophoresis
And taking out the cracked glass slide, putting the glass slide into an electrophoresis tank, slowly injecting an alkaline electrophoresis solution with the pH value of 13.0 into the tank, standing for 45min, and then starting electrophoresis at the current of 200mA and the voltage of 20V for 20 min.
3.4 neutralization
Slides were rinsed by immersion in 0.4mmol/L Tris-HCL buffer (pH 7.5) for 15min each and neutralized 3 times.
3.5 dyeing
70. mu.L of 1 XSSYBR Green I solution was added dropwise to the gel, followed by coverslipping and microscopic examination. Or storing the film under the conditions of 4 ℃, humidity and light-cut, dyeing again when observing, and performing microscopic examination for 24 h.
3.6DNA Damage level analysis
The observation was done with a fluorescence microscope (BX41, Olympus) under green excitation and photographed with a cold CCD under auto-exposure conditions using a random software. After obtaining the comet image, analyzing and measuring the parameters of DNA migration of the infected DNA damage repair defect living cells and normal living cells by using CASP software: the higher the transferred comet tail moment value is, the greater the potential genetic toxicity of organic pollutants in the water body is. ANOVA statistical analysis is carried out by using Origin 8.0 software, significance t test is carried out on an experimental group and a control group, and P <0.05 is used as a significance basis. And C, analyzing the comet image by using CASP software, wherein in evaluation parameters, an Olive tail moment value (OTM) simultaneously reflects the content of DNA in comet tails and the shape characteristics of the comet tails, and is a common index for quantifying the DNA damage degree. At least 100 cells were analyzed per slide.
The solution formulations described in the examples are as follows:
is Ca-free2+、Mg2+Phosphate Buffer (PBS)
Mother liquor 1: NaH2PO4·2H2O0.15M (23.4g/L), added 2.34g to 100mL deionized water, and dissolved completely, taking 96 mL.
Mother liquor 2: na (Na)2HPO4·12H2O0.15M (53.72g/L), added 26.86g to 500mL deionized water, and dissolved completely, 404 mL.
Mother liquor 3: NaCI 0.145M (8.5g/L), added 4.25g to 500mL of deionized water, and dissolved completely, 500mL was taken.
The mother liquors 1, 2 and 3 were mixed together in a total volume of 1000mL, and the pH was adjusted to 7.4.
② cell complete culture solution
DT40 cell culture medium consisted of 90% RIMP1640, 10% fetal bovine serum, 1% chicken serum and 50. mu.M beta-mercaptoethanol, plus 1 fold of penicillin mixed solution (100X). At 5% CO2And culturing at 39 deg.CCell, passage 1: 3.
③ agarose gel solution
0.6% (g/mL) of ordinary agarose gel and 1.2% (g/mL) of low-melting agarose gel were prepared using PBS as a solvent, and the low-melting agarose gel was boiled in a microwave oven and then placed in a 42 ℃ water bath for use.
Alkaline cracking liquid
Accurately measuring 150mL of 5M NaCl, 60mL of 0.5M EDTA and 3mL of 1M Tris pH10, adjusting the pH to 10 with 10M NaOH, metering to 294mL, and refrigerating at 4 ℃ for later use. When in use, 3ml of DMMSO and 3ml of TRITON X-10 are added, and the mixture is fully mixed by a stirrer.
Stock solution: 5M NaCl, 0.5M EDTA, 1M Tris pH10, 10M NaOH, DMSO.
Alkaline electrophoresis buffer solution
Accurately measuring 2mL of 0.5M EDTA and 5mL of 10M NaOH, diluting to 990mL with deionized water, refrigerating at 4 deg.C for use, adding 10mL of LDMSO 20min in advance, and mixing.
Neutral buffer solution
0.4M Tris buffer was prepared, pH adjusted to 7.0 with 10M HCl and stored at room temperature.
Seventhly, DNA staining solution
SYBR Green I (10000X) was diluted 1:10000 with PBS and used as received.
FIG. 2 shows the data of the damage of single cell electrophoresis to cellular DNA from different concentration water samples, which were treated with a sample without contamination (control) and with different concentration ratios (1-fold, 2-fold and 4-fold concentration, respectively) for 1 hour with DT40 wild-type cells (WT) and TDP1 knock-out cells (TDP1 KO), and analyzed by single cell electrophoresis under alkaline conditions for cellular DNA damage.
FIG. 3 shows the data of the damage of single cell electrophoresis to cellular DNA from different concentrations of water samples, which were treated with DT40 wild-type cells (WT) and TDP2 knock-out cells (TDP 2KO) for 1 hour using a sample without a sample (control) and with different concentration ratios (1-fold, 5-fold and 10-fold, respectively), and analyzed for cellular DNA damage by single cell electrophoresis under alkaline conditions.
As can be seen from fig. 2 and 3, compared to DT40 wild-type cells (WT), TDP1 and TDP2 knockout cells are more sensitive to epigenetic inheritance of organic toxicity, and can detect ultra-small amounts of organic pollutants in water.
Sequence listing
<110> Changsha college
<120> method for detecting toxicity of organic pollutants in water body based on DNA damage repair defect living cell comet
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caattgatca gttctgtaag attaccttgt ttcagactta agtattctaa catgagttga 660
acttcacgca gttttcagtg gggctgacag acttcctgag ctgctcattc tgcaaactga 720
agaacatgaa gtagtccaaa gcagatttac tccgataaag tttgggttct acttacatag 780
tttaattttt gtgttttgtg tagaacacat ttaggttatt tttaagtttg aagttttgaa 840
tcattcttgt atctggataa attatcgctt cagtatacat acatatcatt tttggcattc 900
ttatttgtaa tattgagttc catttgaatc tcttatgcaa atatgttgaa ttgatcaaca 960
tctagttttt aaacatatgt tttaaagaaa tgaaaaaaac tgaatcagta acttgacaga 1020
gctggtctta agtgatagag atgcaaagtc agtctgcgtt aaatgtttgc ttcagaaaat 1080
tactggtaga agagtctgag ttacgttata attcctcttg tatgaggaat atatatgtga 1140
aggatattat aatgaggatt gtgttaagaa taggcatttt tactttaaga cagagcacta 1200
tatgcacgta taatgtttct gaagctaata tagggcatgg ctttctgcta tgaagtgcac 1260
aaccttctac ttcctggtca caaagtagct tttctcattg atttgatttc cactgttggg 1320
tggtaaagct acaaatattt ctttctttct gtagagtata tctgcttggt tctactcctg 1380
gacgatatca aggtattgat aaagagaagt ggggtcatct taagctcaga aaggtactct 1440
aacttttttt actgatatca ttttaactgt aaatgtaatt tgagagcatt cgttagttgc 1500
acctaatttt gcagctacac aacaatattg taatacattg atacagctta agctagcaaa 1560
agcataagct tacaacagtg cagatcttgc acatcatatt cgtcagccct attacccagg 1620
tcagcttgca ttctgaagtg ttatttgctt acagatatgt gaagattcac catcattcag 1680
gtggcttcct atattaaaaa attggacact ataaaaatcc agtcttctgt gactaggagt 1740
ccaatatctg ctcaggtttt cttgccagta agacaatgag taaaaacaaa ataaaacaga 1800
aagctgtaaa ggataaaaat gttcttatgg cagatgtttc tgtgattttg aaaagattaa 1860
tgaataacaa tgtagatata tctgataaca ctacactgac taaagccttt tagtcataaa 1920
ccccccccac acacagagga gaggaaaaag gtttctacta cacacaagtt agtatgtgct 1980
tatatccatc tgtttttaat gtgacattta aattccgttc acagcttaac aacaacttaa 2040
aaaaaaaccg tttagttttc ttttcaattg ttctgttttc ctctcattat agcttgggct 2100
ttggggtatg aaaagaattt taagatcagc tatcatcaaa tgtaaaaagg cttgtggtta 2160
caacttctga aactcttgtt tcatggttat aggaaaagat cctggcattc tttttactag 2220
ttacttaaag gttggcttta atgctttagg ataaaatttt gatgtttcat cttctcttaa 2280
agagtagtat gtttatttgg agatcataga atcatagaat ggcttaggtt ggaaggggcc 2340
tcatggatca tcaggttcca accccctgcc acaggtaggg ttgccagcca ctagatcaag 2400
tactagatca gataacctat ggggcctcat ccagcctggc cttaaacatc accagggaca 2460
agacatccac aacctctcca ggcaacctgt tccagcgcct caccagtctc tcagttaaaa 2520
acaaacaaca acaacaaaaa accacgaaca aacaaaaaac tccttccccc taacatctaa 2580
ttaaatcccc ctttctttaa tttaaaacca ttcccccttg ttctgtcaca atctactcat 2640
gtaaaaagtt gatttccctc aagtttataa actcccttta aatactggga ggccacaatt 2700
aggtctctcc atagcacttc tctttttcca ggctgagcaa gcccaagatc ctaatgggtc 2760
ttaattgcca ggttgttgtc agaaatgact atagaggata ttggaagcaa tattaaactc 2820
ttacttaagg ttttctttgt ttgttgaatg tcttaatgtt ttaattttcc tcaatagctt 2880
ttgaaagacc atgcttcatc aataccagca caggagtctt ggcctgttgt gggacagttc 2940
tcaagcattg gttcactggg agctgatggg tccaaatggc tgtgctcaga gttccaggag 3000
agcctggtgg ctgcaggcag tggtgtggca gctcttctta aatgtgatgt tccgattcat 3060
ttggtatgtc ctttaggtac tagttttaaa ctccagtgtg attttattca ttctgaaaaa 3120
aatgaagctt ttaatggttt attagtttga taacaagttt tctttaaggt gatttttttg 3180
ttgtttgaaa attgatctca tgtacattta aagctacatt attaaagaca actttttttt 3240
ttctccatga aggtttatcc tactgtgagc aatgtgaggc aaagtctgga aggctatcct 3300
ggtaagcaga tgagcaaaag gaaatccatt tattagtttt ttgtggtaac agtgtcttgc 3360
aaaatcattt cagtggttgc tgttgaatga agctgaagtt gccaaattgc tgacaacttg 3420
gctgagtagg tggcagcaga attctattat ttgtattagt tggattctga cttccagttc 3480
tattatgttt gaatataaaa 3500
<210> 2
<211> 8880
<212> DNA
<213> Chicken (Gallus Gallus domesticus)
<400> 2
aaagatcttc atgtccaaat tatcctcaaa ttagacgtaa taatgcagtc gtcgtacaaa 60
tcctaatact gattaaagga ggtgtgagcc tgtgcagcat ttcaggggtt aaagactttg 120
cagtgactaa aaatcttttg gtaatgaaca gtattgctta catcaggttt tatgcctatg 180
tcaggagcat gcttcacact tccatcctgt acaggaaaag agggtgaaaa tgcaagaagc 240
ttgtttgctt cattctggtg gtatgagtaa cttcattctg gagcaaactg tttggagggc 300
caccagcatt ctttgtttgt ttgttatttg aaaaatggat cccttgccat tacagttatt 360
tgtctataaa ggatgataat gtgcaagcta atatgaaagc attagtatct gtgattgcct 420
tttatacttg taccatttga agcttcctgt taatacactt aatgattaat ttaataatta 480
cataaattgg tgcgtctgaa tacggtaatt tatcaatgta aattaaactg gtttaaacaa 540
atgttaattg gttcttctga atccaacaga aagccttaac taaattgatt tttctcttga 600
tttgtaataa tatgcgttga ttttggtttt ttttctgtaa aagtgaactt ataagcaaga 660
ggacgacttt ttacacagtc taatagtgat agggcaaggg ggaatggctt taaactgaaa 720
gagaatgaga tttaggtcag atgttaggaa gaaattctca gagtgcagtg aggcgctgac 780
acagctcccc agagaagctg tggtgcccca tccctggagg tgctcaaggc caggttggat 840
ggggccctgg gcagctgagc tggtgggggg cagccctgcc catggcacgg ggtggggctg 900
ggtgggcttt gagggccctt cctacccaaa ccattctgtg attctatgaa gtttgtcctt 960
tgactgctta gagaaggtct actcaacagt gtaacaaaaa aggacagcta taaagttgtg 1020
cctaaaagag cactttcaga actcctaccc cacacgtgcc attacttgtg gcatagctct 1080
aaggagcttg gcattgatga atggcagatt agctgagaag gatctattgt aggggaatga 1140
gatggagggt agaatttcct agaaatctta tctattgcat attatgatcc gaagctgctt 1200
tccaaattcc ttgcatatgt aataattgag aactgtgcaa atgttttcaa gacaaaagga 1260
agtcgtgcgt tttattgcca tgttggtact gtgcccataa tagtcattgt tttctgcctt 1320
cagcatcagt ggtatgtcta cgtgctactt aatatcatta gagaggatct taaaatatct 1380
aggaagataa ggaaaaaaaa tgcacctgaa atctccgacc ctgttctgtt tgacatcaga 1440
gtgtaataat gttacatttc agtagataac ttctaaactt gttttgcttt cagatacagc 1500
ccagatgtgg tgtttttaca ggaggttatc ccaccgtatc tctgtattct acagaggaga 1560
gcaggcggtt acaccattat tccaggtacg aagtaagcct tgatttctta agacagaaac 1620
tggaaaaact aagtacagtt ttgttctgtc ttccttgcca aaagctccaa cctcctgtag 1680
caactgaaaa gagtaagatg aagaaggaaa cgctcaaatt acttcctcta ttttactacc 1740
agcatatagt tggatgcaaa ttactcccgt gaaaggtttc ttgactgatg gcctttgttt 1800
tgtttttttt ccccacgttt tgaaaaactg atagcaaaat tatcccagaa agcaccgtag 1860
atgagtagtt gcttaaacat ttgtattaaa aacatattcg caccactgct agggaacttc 1920
tgaataatca ttttgtacat tttgcaaatt aaaagttttc tcccagatta taataaggag 1980
atctgtggaa gagctttaac tttatcctgt agcagaagat gtccttgacc ttttatgtgc 2040
ctattcctct ccaggtaacg tagatggcta tttcactgcc atgctgttga agaaaccaag 2100
agtgaaagta ctaaaacagg agataatacg ttttccaaca acttccatga tgaggaacct 2160
tttggttgtg catgtgagtc agtctgtcag tactacagct gtgtgtctgt cttcagaaca 2220
atagagtaca ggttggcctt actcagtggc agtcataaac agagcataaa catttcttca 2280
gacatatgaa tcgagttgca tttggtatta atacaattaa gtctcttatg ttactggctg 2340
tactttgttt tcatatctga taacctctgg aaacaaatta gaaacccgct atcctcccat 2400
aaagcacttc tccaatttaa aagacatatt aaccagagct ctgtcattca ttaattgatt 2460
ttctgattag tttttgtcta tgatcttcat atcacagtgt gaaaaatagc atacctgttt 2520
tgtttcacgt gcaggtgaac atatctggta atgaactttg ccttatgact tctcatctgg 2580
agagcactag agatcactca aaggagcgta tgaagcaact gcaaatagtg ttaaacaaaa 2640
tgcaggagga gtctcagtcc accactgtta tatttggggg ggatacaaac ctcagagaca 2700
gcgaggtaaa tagaaataag caacctgatt atttggggaa ggtattctgg taaaatcttg 2760
tcagactttt ctgttgtctt cacgagaaac aaactgcaca gcagaaatct gcaacttctt 2820
cttaatcata tatccccctt ttaagagtta gaagctgtgg tattgctacg ttgaggcttt 2880
gttacagctg agatgttgct atatgttatg agacgtaaaa ggtatgtttt tttccccata 2940
actaaaaatg cataattctc aaaaagtatt tctgagaagt ttcagctttc agagcatctt 3000
ctgattttga gaacttgatc ttttattcat tcagctctct gattctaggg atttaatgct 3060
aaaaaaaaat gaacgaaaga agaaggtgct ctggagaaat acagtttaaa atctgtcccc 3120
catcatggag acattcagga ttagaagcca tttttcgctg ctcatgttaa aagtgaaatt 3180
tctgcattat tagtaaaaat ttgtttttcc ctaatagtga aattccactg ctatgagcag 3240
gaattacttt atttcagaaa acatggcctt gacccctatg cctgaagatt tcagagctga 3300
actctgtgtt ttgtagcatt gaaggccaat ggcatcctgg cttgtatcag gaatggtgtg 3360
gtgagcagga ctagggaagt aatcctgcca ctgtactcag cactggtgag ggcccacctc 3420
aagtaccgtg cacagttttg ggcacctcag tacagaaagg acatggaggt gctggagcag 3480
gtccaaagaa gggcaacaag gcttgtgaag ggcttggaga atatgcccta tgaggagcaa 3540
ctaaaggaac tggggctgtt cggtctgggg aagaggaggc tgaggggaga ccctattgct 3600
ctcttcaaat acttgaaatg tgattgcagt gagagcaaag atggtttctt ctcattggtg 3660
acgggtgaca ggatgagggg aaatggcctc aagttgcacc aggggaggtt taggttgaat 3720
atcaggaaga acttcattac agaaagggtt gttaagcact ggaataggct ccccagggag 3780
gtggctgagt caccatccct ggatgtgatt aaaaactgtt tggatgtggt gctcagggtc 3840
atgatttagc agagggctgt tagagttagg gtagtatggt tgggttgcag ttggacttga 3900
tgatctttaa ggtcttttcc aacctgagca attctatgat tctatgcttt ggatttagga 3960
agtatgacaa gaattggtca caccgtagag tctactgaga agtaacacaa tttagaaatt 4020
cctctagtag ctttgttctt cacaactcat gttttacagg ggaaaaaaag caatattttg 4080
acagctcagt tctgttttct cccttcaggt ggctaagcta ggtggcttac ctaaaaacat 4140
cacggatatc tgggagtttc tgggcaaacc ccagcactgc cgctatacgt gggacacaag 4200
ctccaacacc aacctgcgca tagagagtaa atgcaagttg aggtttgatc gcctttactt 4260
tcggcctgca gcagaagggg gacatatcat tccacgcaat atggacttga tcggattaga 4320
aaagctggac tgtggcagat tcccaagtga tcactggggt cttctgtgtc gctttgatgt 4380
gatattataa cgaatagggc ttgtgctgtt tggtcttaag ggttctgttc ttgtttacac 4440
tcccagttta agtagggcat gttgacagtt tcagccatag caaattccct actttgactc 4500
ctgctggaat gtcttctcta attccaaggc tccttgttcc tcactgtatg catctgctgt 4560
tgaatcgtga ctttaaatat atatttttaa taaaaccaaa gttcagctct aaaacaggag 4620
agaggaataa atagaatcca taaatgtact aggtgatttt aaagatgctt ttattttgtc 4680
ctgtattata agctcataat aaatgctgct tcttttttaa aatgctcttc tgatgactgc 4740
tttgttttgt aactgttttc atcatccttt acagcagtgt cttttgtgag gtgtattgtg 4800
tcagtttctg aagctcgtat tgtaaataca tacaggcctc ctcagtcagg acaataaact 4860
gtggaaaaaa tggctaggtt ctcacaaact aaaagtttct atgtaagaat taagccaaca 4920
ttgaacagat ttaagaatga tgtgcctttc acgtgatgtg aaaaggctat agtacatgta 4980
gtacatggaa gttgtacgct tggggtgtta aataccctgg tttatattat taatataaag 5040
ctgctttatt tatgtttata ttaatgtaaa gctggatagg agtttcaggc ttaagatttc 5100
atcattctga tgagcttata gaagctgaat actcttacaa aagcagctca gggtttagct 5160
gactttgcaa aagataaaca ggcgttctta ctcaagaact gtgtaatcag ccttgtgaaa 5220
gctatcattt tatagctgtg caaagtacta agtggtgtca ttaaaaagga attcttgctt 5280
ctgaatttca agcatgaagt tcagtgcaca ggcatggaga ggcagcgcag acaacccgtg 5340
tcagtggtac aaagtgcacc aaacgtctgt actaatgcac agcgttcttt cttccaggca 5400
ctgacaaact aatgcttttt ttatcttggg tcttaagcca aaagctttaa aatgcctcgt 5460
gttttcctgg gaatttgtgg caggtcaggc ccagctggcc caaatagcaa acaaataaag 5520
ctcacggttt aaggagatat gcagggaaat cacttgtcaa ttactctcat gggcaaaata 5580
ggctcgattt ccagaaatta acttaaattc attactaatt aacatatgtc cttaattact 5640
aattcaggaa gcaagataac gaacaccttt cctcccactt tcccaggctc cacatcactc 5700
caggcatttt tgaggagttg tttgtgtact gattgactta agactgcaag atgggttatc 5760
tctatcacta acatggttgg aggaagtttt ctctgtcctt ctgtaacagt ctgtgaacaa 5820
cgctgaaggg caaagccagt gtaaataagt actggggaca gtgtgactgg atagatacag 5880
aatgtcatca taaggcccca tcagacccat gttaggccag cagaaatgag ctatccagca 5940
ctgagaaaag ccaaaattag ctcttgtggc attatgtggc ttagcctaag atgctccaat 6000
cagccaagca tcaaggaaga tgaaaggtgt gtgaatgtta cgttttccat ctccatcttg 6060
cagtgctgct tgtagcccag aaaaggagaa cttggcccaa tatagggcag tggaaggtga 6120
gtttgtggat ccttacccta aaatagtcag gatctttaac cagttcctca caaggataga 6180
gaggcccgag gtacatgtgt ttttcacagg aggcaacctt actaaacaag gttatatggc 6240
atgatggcta ttttttgctt atgtggatgc atcctctgca ctgttgcgga tgcagatgtt 6300
ctaggttttt aatttgagaa agaaagaagg aaaaaatgat tcagagcagt gtgtgactta 6360
actcagtgct tcacttccag attcatctct ggtattgact atgagtaccc tgctttgatc 6420
agctgtcctt cttaacctct gaagtagtga gtgggcacag ggcattttta ttaaggaaca 6480
agcagagaca tgacactgta agagccattt catttttcta attataaagg taggcaagag 6540
gaagcccata tttagtgtaa ttagctgtta atcattctct cagaaagagg aaattatgct 6600
ttacagcctt taaaaatctc cttgaactac attaaaagca atcatactta aaaaattaag 6660
atgatgtaaa tgctgcatgt ttcactgact attgaatgtg aagcactaca tctttaataa 6720
ttaaatacac tttgaaccac ttagtaagta cccaaattac tgacaatcaa gagcagcaag 6780
agtcctaaca ttaatttgcc tcttaattta atcagtgatg taaatggtaa aatatcggct 6840
gaatatgtca actaaaacct gtgagataac tgaatttaaa ggagtagaaa cagcaaagta 6900
aaatcaagta aaggatttct ggggctacaa cttaacaact tgatttcttc tcctgtttca 6960
tcaatgtaat actatggcat ctatacacca ggtgacagtg agttcattta acttcatttg 7020
ggtaaatttc ccatgttttg cattcactaa atgtatacgg aagattaatt aatgaagact 7080
tgtaaataac atctacttat ttattggaga gaaggcatgt cgtggagcat cagctcaaag 7140
agaagaatcc cagagactgc aaagccatca gtttaatgct gccattttgg taacaaggaa 7200
gaaaacagga ggcttaagtg gatgctggag atcataggga ctctcagtgt gtgataatgc 7260
aagttgagga aatgtcagat catggtgaga gcacacgtgg atagcatctc ttctctgctc 7320
ccctgttctc tgtacaacgg ttgctacact acaaaaaatt catacagttt ctgcagtaac 7380
tcacagtaaa atcctgtgca cctgtggcaa taaatccacc tactgacata aagagataca 7440
gcaaaactac agctttgact aagaaaaagg atgtgagtcg gggcttcccc accaccagcg 7500
ctcagctgct ctgtgggaac atttggcact gatggggcgc aggtcaggag cagcctccaa 7560
aacacctctg tctgtccagc aatgagttga cacgcctcct actgttaaaa taaaaagaag 7620
cctactgtta aaaaatatgg gaaaggtggg aatcggaggg aaagtaaaat aaatgagtaa 7680
aaaaaaaacc agctgtgtgc tacagagatg acatttttac atcttcagaa gaactttttt 7740
ctccctaatt ctgtagcatt tgcaaaatac cattttcctg agttaagtgc tggcaatttg 7800
ttagagatga ggcaggcccc agtgacacag cagagcagtg gaggagagga tgtgggactt 7860
tgacaagctg gtgttgcaga ttagcatgaa atcttcacaa ttaaagctcg aagttcatag 7920
ctctctgtgt tttatttcac tggaaaacga aactttgtgc ttgaatagtt gccaagatga 7980
acaaacttaa gatactcctt ttctatggaa cgtggacttt ggcgttgagt agcggaaggt 8040
gtatttattt ccaattattt tgtactgcaa ttgtaaaaat caaagaaatg cacaaaccat 8100
ttcagcatgt tctacctgca aatgcaccac ggacactgag cagaaagttg tgactggagc 8160
agcgaggaca gcacaaaatc agatactgca atggctaaga ttttattgaa tagctgtgct 8220
gaataagcta tgggtacttt tgtggtggct tcagatggag atactggggg cttcagatgg 8280
aggatggggg actttttcag ccaaacagca tggggtctcc aaccccatat ttgtatgaaa 8340
gtcggcatgt agaaaccaat aggaaagtgc aaagagaaaa gtgtagatcc agatgttcac 8400
ccaggagcac ctcctgcaag agagatgttg gccgcacgtt ccttttccag tacctatgca 8460
gctggttagg atagaaattc cccactaaaa gcaacactga ccccaaaaca ttaagcttgg 8520
gaccaggtca ctcacagctc atgtgtacct caagaacttc acacagcgtg gtggcacaga 8580
ggaggtagga tccctccctt atggcccctt ggaagcctac tgggaactct aagtttgttg 8640
tctgagaaac attatacaaa gcctcatgtg tctgaagaca ttaagcattc tgggatgagc 8700
atggacaatg aatacaagtg caaagttcca ggctccgttg cgaggaaggg aggtttatca 8760
ggactgtaag gtcagcctag cagggaaaac aactttctat ggtagacttt cactaataaa 8820
gtttctgtgt attcttccct aggcaagaaa gaggtaaaaa aaagatgggg ctctgaacaa 8880
Claims (8)
1. A method for detecting toxicity of organic pollutants in a water body based on DNA damage repair defect living cell comet is characterized by comprising the following steps:
(1) collecting a water sample enrichment, treating DNA damage repairing defective living cells by the water sample enrichment, and simultaneously using the DNA damage repairing normal living cells treated by the water sample enrichment as a control; respectively centrifuging and precipitating the treated DNA damage repair defect living cells and normal living cells, then resuspending the cells by PBS, and centrifuging to obtain a first cell suspension for later use;
the DNA damage repair defect living cell is obtained by knocking out a DNA repair gene TDP1 and/or TDP2 by a chicken B lymphocyte DT 40; the DNA damage repair defect living cell is a TDP1KO cell line or a TPD 2KO cell line, the TDP1KO cell line is obtained by knocking out a gene segment shown as SEQ ID NO. 1 on a TDP1 gene by a chicken B lymphocyte DT40, and the TPD 2KO cell line is obtained by knocking out a gene segment shown as SEQ ID NO. 2 on the TDP2 gene by the chicken B lymphocyte DT 40;
(2) preparing the first cell suspension obtained after the treatment of the step (1) into a comet electrophoresis glass slide cell sample;
(3) removing a cover glass of the cell sample of the comet-star electrophoresis glass slide obtained in the step (2), immersing the cell sample into alkaline lysis solution for cell lysis, taking out the lysed glass slide, placing the lysed glass slide into an electrophoresis tank for electrophoresis, and neutralizing the cell sample of the glass slide after the electrophoresis is finished;
(4) and after the neutralization is finished, dripping DNA fluorescent dye on a glass slide cell sample, covering a cover glass, observing comet change by using a fluorescence microscope, measuring comet tail moment values of DNA damage repairing defect living cells and normal living cells treated by the water sample enrichment, and obtaining a DNA comet migration tail moment value.
2. The method according to claim 1, wherein in the step (1), the method for collecting the water sample concentrate specifically comprises the following steps: collecting a water sample, standing for a period of time, filtering to remove suspended particles, enriching by a macroporous resin adsorption column, removing water by nitrogen, eluting by methanol, acetone and dichloromethane in sequence, concentrating and drying the obtained eluent, and finally dissolving by dimethyl sulfoxide to obtain a water sample enrichment substance; the cell concentration of the first cell suspension is 0.5-1.0x105Individual cells/mL.
3. The process of claim 2, wherein the macroporous resin is XAD-2, XAD-4, XAD-7, XAD-8 or XAD-2/XAD-8 resin; the standing time is 24-36 h; the concentration temperature is 45-50 ℃; and storing the water sample enrichment in the dark at a temperature of between 18 ℃ below zero and 70 ℃ below zero for later use.
4. The method according to claim 1, wherein in the step (2), the preparation method of the comet electrophoresis slide cell sample specifically comprises the following steps: dripping agarose with normal melting point on a frosted glass slide, quickly covering a cover glass, and solidifying to obtain a first layer of agarose; and (2) adding an isovolumetric low-melting-point agarose solution into the first cell suspension obtained after the treatment in the step (1), uniformly blowing and beating to form a second cell suspension, spreading and dripping the second cell suspension onto the first layer of gel, covering a cover glass, and curing to obtain a second layer of cell gel, thereby completing the preparation of the comet-electrophoresis glass slide cell sample.
5. The method of claim 4, wherein the normal melting agarose has a melting point greater than 90 ℃ and a mass concentration of 0.6%; the melting point of the low-melting-point agarose is 42-65 ℃, and the mass concentration of the solution is 1.2%; the curing temperature is 0-4 ℃, and the curing time is 30-120 min.
6. The method according to claim 1, wherein in the step (3), the alkaline lysate comprises 0.5M EDTA and 5M NaCL, the alkaline lysate has pH =13, 1% by volume of Triton X-100 and 1% by volume of dimethylsulfoxide are added before use; the temperature of the alkaline lysis solution is 4 ℃, the cell lysis temperature is 4 ℃, and the lysis time is 10-120 min.
7. The method according to claim 1, wherein in the step (3), the specific operation of electrophoresis comprises the following steps: injecting an alkaline electrophoresis solution with the pH =13.0 into an electrophoresis tank, standing for 30-45min, and then starting electrophoresis, wherein the current is 200-300 mA, the voltage is 12-25V, and the electrophoresis time is 20-30 min; and after electrophoresis, placing the glass slide in a Tris-HCL buffer solution for neutralization and rinsing for 5-10 minutes, wherein the concentration of the Tris-HCL buffer solution is 0.4M, the rinsing times are 3 times, and the rinsing time is 15min each time.
8. The method of any one of claims 1 to 7, wherein in the step (4), the DNA fluorescent dye is 1xSYBR Green I solution, comets are observed by a fluorescence microscope, after comet images are obtained, the comet tail moment values of the DNA damage repair defect living cells and the normal living cells treated by the water sample enrichment are measured respectively by CASP software, and the DNA comet migration tail moment value is obtained.
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