CN114561427B - Cultivation method of transgenic zebra fish for detecting organic pollutants in water body - Google Patents

Abstract

The invention discloses a cultivation method of transgenic zebra fish for detecting organic pollutants in water. The invention prepares the transgenic zebra fish by using a plasmid which expresses a mosquito-eating fish cyp1a gene promoter and simultaneously expresses green fluorescent protein and luciferase and the non-body-spot horse fish. In the actual water quality monitoring work, the pollution condition of the organic matters in the water body can be qualitatively estimated by directly observing the fluorescence of the transgenic zebra fish, and the pollutants in the water body can be quantitatively analyzed by measuring the luciferase activity of the transgenic zebra fish.

Description

Cultivation method of transgenic zebra fish for detecting organic pollutants in water body

Technical Field

The invention relates to the field of biological monitoring, in particular to a cultivation method of transgenic zebra fish for detecting organic pollutants in water.

Background

Biological monitoring refers to the process of evaluating environmental quality from a biological perspective, and further clarifies the nature, degree and range of environmental pollution in which biological individuals, populations or communities are located by observing the reaction and influence of the biological individuals, populations or communities on the environmental quality and the change of the environmental quality, including biological population and community investigation, acute and chronic toxicity tests, water body microorganism tests and fish tissue pollutant analysis. Germany is the earliest country for biological monitoring, and as early as the beginning of the 20 th century, the work of monitoring water quality by aquatic organisms has been carried out, and up to date, developed countries such as the european union and the united states have established relatively complete environmental biological monitoring systems. The China starts relatively late, and a national aquatic organism monitoring network is established for the first time along with the publication of an aquatic organism monitoring manual in 1993, so that aquatic organism monitoring work starts to be carried out nationwide. Biological monitoring work was not paid attention again until the beginning of the 21 st century.

At present, the traditional biological monitoring method for monitoring the persistent organic pollutants in the water body is generally to collect biological samples in polluted environments including aquatic organisms such as fishes, shellfishes and the like, and reflect the concentration level of the pollutants in the environment by analyzing the expression level of persistent organic pollutant metabolism-related enzymes such as dioxins and the like in the body, so that the method is time-consuming and labor-consuming and cannot be used for visual monitoring. The prior art has disclosed zebra fish strain and green strain (doi: 10.1016/j.aquatox.2013.03.010; doi:10.1016/j.aquatox.2018.10.022; doi: 10.1371/journ.fine.fine.0064334; doi: 10.1016/j.jhazmat.2020.122192) which can be applied to detection of dioxin-like persistent organic pollutants in water, respectively, utilizing green and zebra fish cyp1a promoters or modified zebra fish cyp1a promoters to drive fluorescent expression for environmental monitoring, but still has the problem of low detection sensitivity, and no report of practical application of the strain in environmental monitoring has been seen so far.

How to improve the sensitivity of the detection of the fluorescent fish and how to make the detection more convenient and faster is a key problem facing the practical application of pushing the fluorescent fish in the environment detection.

Disclosure of Invention

In order to solve the technical problems that biological monitoring is time-consuming and labor-consuming and cannot be used for visual monitoring in the prior art, the invention provides a method for cultivating transgenic zebra fish for detecting organic pollutants in a water body.

It is a first object of the present invention to provide a plasmid for detecting organic pollutants in a body of water.

A second object of the present invention is to provide a gene editing plasmid for constructing a bleachless horse fish.

It is a third object of the present invention to provide a gRNA for constructing a horse fish free of body spots.

A fourth object of the present invention is to provide a method of constructing a non-body spot horse fish.

The fifth object of the invention is to provide a cultivation method of the transgenic zebra fish for detecting organic pollutants in water.

A sixth object of the present invention is to provide the use of the above plasmid, the above gene editing plasmid and/or the above cultivation method in one or more of water environment, water quality monitoring and/or detection of organic pollutants in water.

In order to achieve the above object, the present invention is realized by the following means:

a plasmid for detecting organic contaminants in a body of water, the plasmid comprising an expression cassette; the expression cassette contains a promoter and a reporter gene of the mosquito-eating fish cyp1a gene with the nucleotide sequence shown as SEQ ID NO.1, wherein the promoter of the mosquito-eating fish cyp1a gene with the nucleotide sequence shown as SEQ ID NO.1 is upstream of the reporter gene.

Preferably, the reporter gene is a gene encoding a fluorescent protein and/or a gene encoding a luciferase.

The promoter of the mosquito-eating fish cyp1a gene with the nucleotide sequence shown as SEQ ID NO.1 can simultaneously start the expression of fluorescent protein and luciferase.

More preferably, the fluorescent protein is one of an orange fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, a blue fluorescent protein, a cyan fluorescent protein, and/or a green fluorescent protein.

More preferably, the fluorescent protein is green fluorescent protein and Gene ID is LT726875.1.

Further, the Gene ID of the luciferase is LT726875.1.

More preferably, the nucleotide sequence of the plasmid is shown in SEQ ID NO. 4.

The plasmid for detecting the organic pollutants in the water body provided by the invention contains the fusion expression protein of the green fluorescence and the fluorescent dye enzyme, and can be used for directly observing the green fluorescence and detecting and analyzing the activity of the fluorescent dye enzyme.

A gene editing plasmid for constructing the fumosoroseus, which has PAM region and the sequence of SEQ ID NO.9 from 5 'end to 3' end.

Preferably, the sequence shown in SEQ ID NO.9 is a reverse complementary sequence of the sequence shown in SEQ ID NO.5, a sequence shown in SEQ ID NO.6, a sequence shown in SEQ ID NO.8, a sequence shown in SEQ ID NO.7 and a reverse complementary sequence of the sequence shown in SEQ ID NO.5 in sequence from the 5 'end to the 3' end.

Preferably, the PAM region has a nucleotide sequence of 5'-CCC-3'.

A gRNA for constructing a pseudomackerel with a nucleotide sequence shown in SEQ ID No. 10.

A method of constructing a somatic stain-free horse fish comprising the steps of: silencing the mitfa gene of the fertilized eggs of the zebra fish; screening and identifying fertilized eggs; hybridization and purification; the homozygote zebra fish is the zebra fish without body color spots.

Preferably, the method for silencing the mitfa gene of the fertilized eggs of the zebra fish comprises the following steps: and injecting the gene editing plasmid, the Cas9 protein and the gRNA with the nucleotide sequence shown as SEQ ID NO.10 into the fertilized eggs of the zebra fish, so that the sequence with the nucleotide sequence shown as SEQ ID NO.8 in the gene editing plasmid is integrated into the genome of the fertilized eggs of the zebra fish.

More preferably, the sequence of the nucleotide sequence shown in SEQ ID NO.8 in the gene editing plasmid is integrated between the promoter and the transcription initiation site of the mitfa gene.

The sequence with the nucleotide sequence shown as SEQ ID NO.5 is a targeting site, the 5 'end sequence of the targeting site, namely the sequence with the nucleotide sequence shown as SEQ ID NO.6, and the 3' end sequence of the targeting site, namely the sequence 7 with the nucleotide sequence shown as SEQ ID NO.6, are taken as homologous arm sequences, and the sequence with the nucleotide sequence shown as SEQ ID NO.8 on a gene editing plasmid and the corresponding zebra fish genome sequence of the targeting site are subjected to cross exchange by utilizing the homologous recombination principle of the zebra fish, so that the sequence with the nucleotide sequence shown as SEQ ID NO.8 is inserted between a promoter and a transcription initiation site of a mitfa gene to cause insertion mutation, thereby causing the mitfa gene to be incapable of transcription and losing functions.

Preferably, the method of integrating fertilized eggs into zebra fish is microinjection.

More preferably, the fertilized egg of the zebra fish is in a cell phase.

The zebra fish without body color spot has no interference of the zebra fish body color melanin, and is more beneficial to fluorescent observation.

A cultivation method of transgenic zebra fish for detecting organic pollutants in water body comprises the following steps: preparing the horse fish without body color spots; introducing the plasmid into fertilized eggs of the somatic stain-free horse fish prepared by the method; screening and identifying fertilized eggs; hybridization and purification; the obtained homozygote zebra fish is the transgenic zebra fish for detecting the organic pollutants in the water body.

Preferably, the above plasmid is introduced into a fertilized egg in a cell phase by microinjection.

More preferably, the plasmid injection amount of a single fertilized egg is controlled to 20 to 50pg.

Preferably, the screening is a dioxin screening.

More preferably, the screening is to treat fertilized eggs with dioxin and/or DMSO, select fertilized eggs that express green fluorescence in dioxin and do not express green fluorescence in DMSO for subsequent hatching.

Preferably, the incubation condition is a constant temperature culture at 28 ℃.

The application of the plasmid, the gene editing plasmid and/or the cultivation method in one or more of water environment, water quality monitoring and/or detection of organic pollutants in water is also within the protection scope of the invention.

Preferably, the organic pollutants of the water body comprise dioxin-like substances and/or polychlorinated biphenyl-like substances.

More preferably, the dioxin-like substance contains dioxin and/or benzopyrene.

More preferably, the polychlorinated biphenyls comprise 3,3', 4' -tetrachlorobiphenyl, indeno [ cd ] pyrene, and/or benzo [ k ] fluoranthene.

Compared with the prior art, the invention has the following beneficial effects:

the invention discovers for the first time that the promoter of the mosquito-eating fish cyp1a gene has high sensitivity to dioxin-like persistent organic matters in the environment, and compared with the promoter of the zebra fish cyp1a gene, the promoter of the mosquito-eating fish cyp1a gene has stronger green fluorescence intensity and higher luciferase activity under the exposure of pollutants with the same concentration.

The invention prepares the transgenic zebra fish by using a plasmid which expresses a mosquito-eating fish cyp1a gene promoter and simultaneously expresses green fluorescent protein and luciferase and the non-body-spot horse fish. In the actual water quality monitoring work, the pollution condition of the organic matters in the water body can be qualitatively estimated by directly observing the fluorescence of the transgenic zebra fish, and the pollutants in the water body can be quantitatively analyzed by measuring the luciferase activity of the transgenic zebra fish. The invention provides a more convenient and more sensitive biological detection means, which can rapidly detect persistent organic pollutants in water.

Drawings

FIG. 1 is a plasmid map; a is pICSEI-cyp 1 acromoter-eGFP/Luc plasmid; b is a map of the pICSEI-GAcyp 1 acromoter-eGFP/Luc plasmid.

FIG. 2 shows the result of the pICSceI-GAcyp 1apromoter-eGFP/Luc plasmid luciferase assay; a is the luciferase activity of the pICREI-cyp 1 apomoter-eGFP/Luc plasmid and the pICREI-GAcyp 1 apomoter-eGFP/Luc plasmid in the same concentration (100. Mu.g/L) of benzopyrene treatment; b is the luciferase activity of pICSEI-GAcyp 1apromoter-eGFP/Luc plasmid at different concentrations (benzopyrene treatment).

FIG. 3 shows the identification results of F2 transgenic zebra fish homozygotes.

FIG. 4 is the effect of dioxin on luciferase activity of transgenic zebra fish.

FIG. 5 is a graph showing the response of transgenic zebra fish to different concentrations of dioxin.

FIG. 6 is a graph showing the response of transgenic zebra fish to dioxin treatment for various durations.

FIG. 7 is a response of transgenic zebra fish to other organic pollutants.

Detailed Description

The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

Example 1 preparation of pICSEI-GAcyp 1apromoter-eGFP/Luc plasmid

(1) Extraction of genomic DNA of mosquito-eating fish

Taking mosquito-eating fish, rinsing 20-30 mg of tail fin in distilled water for several times, sucking water with water-absorbing paper to show water, placing in a 1.5mL centrifuge tube, shearing, and extracting according to the operation instruction of tissue DNA extraction kit (purchased from Tiangen Biochemical technology (Beijing) Co., ltd.) to obtain the genome DNA of the mosquito-eating fish.

(2) Amplification primer design

According to the coding region sequence of the mosquito-eating fish cyp1a Gene (Gene ID: MK 286594.1) obtained by inquiring at NCBI, the cyp1a promoter sequence is obtained by calling in a mosquito-eating fish genome database (GCA_ 019740435.1), and an upstream amplification primer GAcyp1a-F is designed: 5'-GAACCATAGGGAAGATTGCTGACAT-3', downstream amplification primer GAcyp1a-R:5'-AAATAAAGTCAAGCATCATAGAATCATC-3'.

(3) PCR amplification of mosquito-feeding fish cyp1a gene promoter

Using the mosquito-feeding fish genomic DNA as a template, PCR amplification was performed using KOD Plus Neo high-fidelity polymerase (available from Toyobo) using the above primers GAcyp1a-F and GAcyp1 a-R.

The reaction system (50. Mu.L) was: 10 XPCR Buffer, 5. Mu.L; dNTPs mix (2 mM each), 5. Mu.L; 25mM MgSO ₄ ，3μL；GAcyp1a-F，1.5μL；GAcyp1a-R，1.5μL；KOD Plus Neo DNA Polymerase，1 μl; mosquito-eating fish genomic DNA (200 ng), 1. Mu.L; ddH ₂ O，32μL。

The reaction procedure is: pre-denaturation at 94℃for 2min; denaturation at 98℃for 10s, annealing at 55℃for 30s, extension at 68℃for 2min for 40 cycles; extending at 72 ℃ for 5min; preserving at 25 ℃.

After the PCR reaction, 1% agarose gel electrophoresis is carried out, and a PCR product is recovered by using an agarose gel recovery kit (purchased from Tiangen Biochemical technology (Beijing)) to obtain the instant mosquito fish cyp1a gene promoter, which is marked as GAcyp1a promoter, and the nucleotide sequence of the instant mosquito fish cyp1a promoter is shown as SEQ ID NO. 1.

The PCR product was cloned into pEASY-Blunt Cloning vector vector. The reaction system is as follows: PCR product, 2. Mu.L; pEASY-Blunt Cloning vector,1 μL; adding ddH ₂ O was made up to 5. Mu.L. The reaction was carried out at 25℃for 18min. Ligation product 1 was obtained.

After the reaction was completed, 5. Mu.L of the ligation product 1 was added to 50. Mu.L of Trans1-T1 competent cells, gently flicked, mixed well, ice-bathed for 30min, heat-shocked at 42℃for 30s, and immediately placed on ice for 2min. 250 μLLB medium was added and incubated at 200rpm and 37℃for 1 hour. Centrifuging at 5000rpm for 1min, discarding part of supernatant, and mixing the rest. All bacterial solutions were plated onto LA plates and incubated overnight at 37℃with inversion.

The following day, colony PCR was performed using M13 universal primers to pick positive clones, LA liquid medium was added and incubated overnight at 200rpm and 37 ℃. The plasmid was extracted and submitted to sequencing by biological engineering (Shanghai) Inc., and the sequencing results were aligned with NCBI sequences. The sequence alignment shows that the correct plasmid is pEASY-GAcyp1apromoter plasmid carrying GAcyp1a promoter (SEQ ID NO. 1).

(4) Integration of the mosquito-eating fish cyp1a Gene promoter into the pICSEI-cyp 1 acromoter-eGFP/Luc plasmid

The pISCEI-cyp1apromoter-eGFP/Luc plasmid is a plasmid carrying a zebra fish cyp1A gene promoter, the pISCEI-CMV-eGFP/Luc is taken as a framework, the zebra fish cyp1A gene promoter is inserted between ApaI and AgeI sites, the nucleotide sequence of the plasmid is shown as SEQ ID NO.2, and the structure of the plasmid is shown as figure 1A.

The pEASY-GAcyp1 apromiter plasmid and the pICEI-cyp 1 apromiter-eGFP/Luc plasmid (SEQ ID NO. 2) were digested simultaneously with the fast cutting enzymes ApaI and AgeI (both purchased from Semer Feicher technologies Co.) respectively, and digested in a water bath at 37℃for 30min.

The GAcyp1a promoter (SEQ ID NO. 1) and the pICeI-eGFP/Luc backbone (SEQ ID NO. 3) from the pICeI-cyp 1 apromiter-eGFP/Luc plasmid (SEQ ID NO. 2) were recovered by agarose gel electrophoresis.

GAcyp1a promoter (SEQ ID NO. 1) was ligated to the pICeI-eGFP/Luc backbone (SEQ ID NO. 3) using T4DNA ligase (available from Semer Feicher technologies). The reaction system (20. Mu.L) was: 10 Xbuffer, 2. Mu.L; GAcyp1 acromoter (SEQ ID NO. 1), 50ng; pISCEI-eGFP/Luc backbone (SEQ ID NO. 3), 100ng; t4DNA ligase, 0.2. Mu.L; adding ddH ₂ O was made up to 20. Mu.L. Ligation was performed at 22℃for 1 hour in a PCR apparatus to obtain ligation product 2.

10. Mu.L of the ligation product 2 was added to 100. Mu.LDH 5. Alpha. Competent cells (purchased from Beijing Bomaide Gene technology Co., ltd.), gently flicked, mixed well, ice-cooled for 30min, heat-shocked at 42℃for 60s, immediately placed on ice for 2min, and incubated at 37℃for 60min at 180rpm with 500. Mu.LLB medium. Centrifuging at 5000rpm for 1min, removing part of supernatant, mixing the rest, coating all bacterial liquid on LA plate, and culturing at 37deg.C overnight.

The following day, positive clones were picked after colony PCR identification and incubated overnight at 200rpm and 37 ℃. Extracting plasmid, enzyme cutting, identifying, and sequencing positive plasmid in Shanghai Biotechnology Co. The sequence alignment shows that the correct plasmid is the pICSceI-GAcyp 1 apromiter-eGFP/Luc plasmid, the nucleotide sequence of the plasmid is shown as SEQ ID NO.4, and the structure is shown as figure 1B.

Example 2 functional identification of pICSEI-GAcyp 1apromoter-eGFP/Luc plasmid

1. Experimental method

(1) Cell culture

Fluorescence induction effect verification tests were performed using human hepatoma cell HepG 2.

Human hepatoma cell HepG2 was used at 1X 10 ⁴ The density of each/well was inoculated into 96-well cell culture plates and cultured in high-sugar DMEM medium with serum free of antibiotics.

(2) Cell transfection

When HepG2 cells in the 96-well plate were grown to a confluence of 70 to 80%, pICEI-cyp 1 apromiter-eGFP/Luc plasmid (SEQ ID NO. 2) and pICEI-GAcyp 1 apromiter-eGFP/Luc plasmid (SEQ ID NO. 4) were transfected with Promega ViaFectTM transfection reagent, respectively, and a Renilla luciferase control plasmid (from Promega double luciferase assay kit)Luciferase Assay System) into HepG2 cells.

HepG2 cells transfected with the pICSceI-cyp 1 acromoter-eGFP/Luc plasmid (SEQ ID NO. 2) were designated as group D.rerio; hepG2 cells transfected with the pICSEI-GAcyp 1apromoter-eGFP/Luc plasmid (SEQ ID NO. 4) were designated as group G.affinis.

Culturing is continued for 6-8 hours.

(3) Benzopyrene exposure

Benzopyrene was added to the cell culture medium in a concentration gradient (0. Mu.g/L, 1. Mu.g/L, 10. Mu.g/L, 100. Mu.g/L and 1000. Mu.g/L) and the culture was continued for 24 hours.

(4) Dual luciferase activity assay

Kit for dual luciferase assay according to PromegaLuciferase Assay System the operation shows that the activity of bifluorescein enzyme in cells was measured using an enzyme-labeled instrument.

2. Experimental results

As shown in FIG. 2A, the activity of the G.affilm group bifluorescence enzyme was higher than that of the D.reio group, also exposed to benzopyrene at a concentration of 100. Mu.g/L. As shown in FIG. 2B, when the G.afinis group was exposed to different concentrations of benzopyrene, the cellular bifluorescence activity increased with increasing benzopyrene concentration.

The above results indicate that the sensitivity of the mosquito-eating fish cyp1a gene promoter to the response to dioxin-like pollutants is higher than the zebra fish cyp1a gene promoter, and the activity of the mosquito-eating fish cyp1a gene promoter is positively correlated with the concentration of the dioxin-like pollutants.

Example 3 preparation and screening of transgenic Zebra fish

1. Experimental method

(1) Preparation method of horse fish without body color spot

The targeting sites of 6 CRISPR/CAS9 Gene editing systems are designed according to the 5' UTR region of the zebra fish mitfa Gene (Gene ID: ENSDARG 00000003732), and are named as mitfa-1-6, and the specific sequences are as follows:

mitfa-1：5’-CTTCAGCTGGCCAAGACGAC-3’；

mitfa-2：5’-CAAGAACTGACCAGTCGTCT-3’；

mitfa-3：5’-GCTCGAGTACAGTCACTACC-3’；

mitfa-4：5’-CAGTCACTACCAGGTGAGAT-3’；

mitfa-5(SEQ ID NO.5)：5’-GACACAAAATGTATTTAAGG-3’；

mitfa-6：5’-ACAAAATGTATTTAAGGGGG-3’。

wherein the targeting efficiency of mitfa-5 (SEQ ID NO. 5) is highest as the targeting site for the final insertion mutant sequence.

By utilizing a CRISPR/CAS9 gene editing system, a random sequence (SEQ ID NO. 8) is inserted between a promoter and a transcription start site of the zebra fish mitfa gene through homologous recombination, so that insertion mutation is caused, and the mitfa gene cannot be transcribed and is disabled.

The method for constructing the gene editing plasmid is as follows:

amplifying to obtain a 5 'end sequence (SEQ ID NO. 6) and a 3' end sequence (SEQ ID NO. 7) of a zebra fish mitfa gene targeting site mitfa-5 (SEQ ID NO. 5) as homologous arm sequences; ligating the 5 'end sequence (SEQ ID NO. 6) and the 3' end sequence (SEQ ID NO. 7) of mitfa-5 (SEQ ID NO. 5) to both ends of the random sequence (SEQ ID NO. 8), respectively; respectively connecting reverse complementary sequences of a mitfa gene targeting site mitfa-5 (SEQ ID NO. 5) to two ends of a homologous arm sequence to obtain a homologous insert with a nucleotide sequence shown as SEQ ID NO. 9; finally, a PAM region sequence (5 ' -CCC-3 ') is added in front of the 5' end of the homologous insert (SEQ ID NO. 9) to obtain the connecting fragment.

Integration of the resulting ligated fragments into blunt-ended vectorAnd (3) Cloning vector (Beijing full gold) to obtain the gene editing plasmid.

Co-injecting Cas9 protein, gRNA (SEQ ID NO. 10) and a gene editing plasmid into a cell-phase embryo of zebra fish, culturing the embryo to 2 months after injection, cutting tail to extract zebra fish genome DNA, and detecting exogenous random sequence fragments by PCR (polymerase chain reaction) with the following PCR primers:

MitfaKI-JC-F：5’-CAAATGTAATCACCTGGCTCACCTT-3’；

MitfaKI-JC-R：5’-AGGTTAACACACAAAATCCCATCTC-3’。

and (3) continuously culturing the zebra fish with the random fragment inserted into the zebra fish to sexual maturity, hybridizing the zebra fish with the wild zebra fish, selecting offspring with the random fragment inserted into the zebra fish, continuously culturing the zebra fish to sexual maturity, performing selfing, and then screening homozygotes without body colors from the zebra fish to sexual maturity. Thus obtaining the horse fish without body color spots.

(2) Microinjection

And before microinjection, picking female and male fishes of the sexually mature non-body-spot zebra fish, wherein the ratio of the female to the male fishes is 2:2, separating the female fishes from the male fishes by using a partition board, and starting light for stimulating the mating and spawning of the zebra fish the next day, wherein the average spawning of each female zebra fish is 100-200. According to the injection requirement, the partition plates are sequentially opened, so that the male zebra fish and the female zebra fish can be mated freely. After the partition plate is opened for a few minutes, the female zebra fish can discharge fertilized eggs.

The fertilized eggs are collected and washed simply with clear water, the fertilized eggs in a cell stage are sucked by a suction tube and sequentially discharged into a groove of an injection disc of a Eppendorf FemtoJet X4 microinjection instrument.

The system of injection (10. Mu.L) is: pICSceI-GAcyp 1 acromoter-eGFP/Luc plasmid (SEQ ID NO. 4) at a final concentration of 50 ng/. Mu.L; ISceI enzyme, 0.5 μl;10 Xbuffer, 1. Mu.L; phenol red, 1 μl; adding ddH ₂ O was made up to 10. Mu.L.

And adjusting proper injection pressure and injection time according to the size of the needle tip of the capillary glass tube. The injection needle passes through the plant pole of the fertilized ovum to reach the boundary of the animal pole, the injection pedal is lightly stepped to inject the injection into the animal pole, and the injection needle is lightly pulled out to complete the injection. The plasmid injection amount of single fertilized egg is controlled at 20-50 pg.

After injection, fertilized eggs are washed from an injection tray into a clean culture dish by filtered water, and cultured at constant temperature of 28 ℃ to timely remove abnormal eggs and dead eggs which cannot normally develop. After hatching, the transgenic zebra fish is obtained and is continuously fed to 2 months old.

(3) Identification of transgenes

2 months old transgenic zebra fish is cut off from the tail, and PCR is carried out by using a one-step PCR trace genotype identification kit (purchased from Nanjing yao Yu Biotechnology Co., ltd.) under the following reaction conditions: 56 ℃ for 2h;95 ℃ for 5min;16 ℃ for 1min; preserving at 12 ℃. Thus obtaining the genome DNA of the transgenic zebra fish.

The upstream detection primer GAcyp1a-JC-F was designed based on the pICSEI-GAcyp 1 acromoter-eGFP/Luc plasmid (SEQ ID NO. 4): 5'-AGCCAGTTCTGTGTCTCATACGC-3' downstream detection primer GAcyp1a-JC-R:5'-TTGAAGTTCACCTTGATGCCGTT-3'.

PCR amplification was performed using the above primers GAcyp1a-JC-F and GAcyp1a-JC-R using the transgenic zebra fish genomic DNA as a template and 2 XSan Taq PCR Mix (available from Shanghai Biotechnology Co., ltd.).

The reaction system (20. Mu.L) was: 2 Xsan Taq PCR Mix, 10. Mu.L; GAcyp1a-JC-F (10 uM), 0.8. Mu.L; GAcyp1a-JC-R (10 uM), 0.8. Mu.L; 1 mu L of transgenic zebra fish genome DNA; ddH ₂ O，7.4μL。

The reaction procedure is: 94 ℃ for 5min;94 ℃,30s,55 ℃,30s,72 ℃ and 30s for 35 cycles; extending at 72 ℃ for 10min; preserving at 16 ℃.

And after the PCR reaction is finished, agarose gel electrophoresis detection is carried out. The positive bands were recovered and sent to the biological engineering (Shanghai) Co.Ltd for sequencing. The sequencing result is compared with the plasmid sequence, and if the sequence comparison result is correct, the zebra fish is successfully transferred into the pICSEI-GAcyp 1 acromoter-eGFP/Luc plasmid (SEQ ID NO. 4). Namely, the P0 generation transgenic zebra fish is successfully obtained.

(4) Screening of transgenic zebra fish homozygotes

And carrying out one-to-one pairing hybridization on the P0 generation transgenic zebra fish and the wild zebra fish, and collecting F1 generation fertilized eggs.

Randomly picking 30F 1 generation fertilized eggs to extract genome DNA, and the specific method is as follows:

preparing a lysate: to CTAB (hexadecyltrimethylammonium bromide, cetyltrimethylammonium bromide) was added 100mM Tris-HCl (pH 8.5), 0.5M EDTA, v/v=10% SDS, 5M NaCl; mixing 100 mu L of the lysate, 1 mu L of proteinase K with 20mg/mL of fertilized eggs by vortex oscillation, and carrying out water bath pyrolysis for 2-3 h at 55 ℃ and mixing by vortex oscillation for several times until the tissue blocks are completely cracked;

150. Mu.L of chloroform was added, mixed upside down, centrifuged at 12000rpm for 4min, and the supernatant (about 80. Mu.L) was carefully aspirated into a new 0.6mL centrifuge tube; adding equal volume of isopropanol, reversing and uniformly mixing to obtain flocculent precipitate, and centrifuging at 12000rpm for 4min; discarding the supernatant, adding 300 mu L of 75% ethanol, reversely suspending, washing and precipitating, and centrifuging at 10000rpm for 4min; discarding supernatant, inverting, air drying, adding 20 μLddH ₂ O is dissolved and precipitated, and F1 generation fertilized egg genome DNA is obtained.

The concentration and purity of F1 generation fertilized egg genomic DNA were measured using a micro-spectrophotometer.

PCR identification was performed according to step (3) of this example. Fertilized eggs that were selected for PCR amplification to obtain positive bands were incubated for 2 days with 1nM final concentration of dioxin (TCDD).

And observing the fluorescence expression condition of the fish larvae by using a fluorescence microscope. And selecting the fries expressing green fluorescence, namely, the F1 generation positive transgenic zebra fish homozygotes, and feeding the fries to sexual maturity.

The sexually mature F1 generation transgenic zebra fish homozygote is hybridized with wild zebra fish, and F2 generation fertilized eggs are collected.

Randomly picking 30F 2 fertilized eggs of 72hpf, placing the fertilized eggs into a 90mm glass culture dish, and adding 30mL ddH ₂ O, TCDD in a final concentration of 1nM or DMSO in an equivalent volume to that of TCDD is added. After 24h incubation at 28 ℃, the fluorescence expression of the fish larvae was observed under a fluorescent microscope. As shown in FIG. 3, zebra fish which do not express fluorescence in DMSO and green fluorescence in TCDD are screened out, namely F2 generation transgenic zebra fish homozygote.

And (3) the F2 generation transgenic zebra fish homozygote is fed to sexual maturity, and selfing is carried out to obtain the F3 generation transgenic zebra fish.

And hybridizing the F3 generation transgenic zebra fish with the wild zebra fish to obtain the F4 generation transgenic zebra fish. If the F4 transgenic zebra fish expresses green fluorescence in TCDD with the final concentration of 1nM, the F3 transgenic zebra fish is shown to be homozygote. Thus obtaining the F3 generation transgenic zebra fish homozygote.

Example 4 functional identification of transgenic zebra fish

1. Experimental method

30mL ddH was added to each of two 90mm glass petri dishes ₂ O and 150 fertilized eggs of 72 hpf. To one of the dishes was added TCDD at a final concentration of 1 nM; to another dish, DMSO was added in an equivalent volume to TCDD as a control group.

After 24h of culture at 28 ℃, fertilized eggs are placed in liquid nitrogen for quick freezing.

Kit for luciferase assay according to PromegaLuciferase Assay System the operation shows that the activity of firefly luciferases in fertilized eggs was measured by an enzyme-labeled instrument.

2. Experimental results

As shown in fig. 4, the luciferase activity of the transgenic zebra fish was significantly increased after the transgenic zebra fish was treated with dioxin, as compared with the control group. The method shows that the dioxin can obviously induce the expression of the transgenic zebra fish luciferase, so that the concentration of dioxin pollutants in water can be reacted by measuring the activity of the luciferase.

Example 5 responsiveness of transgenic zebra fish to dioxins

1. Experimental method

(1) Response test of transgenic zebra fish to different concentrations of dioxin

30mL ddH was added to each of 6 90mm glass petri dishes ₂ O and 30F 4 fertilized eggs of 72 hpf.

TCDD with five concentration gradients of 0.005nM, 0.01nM, 0.05nM, 0.1nM and 0.5nM were added to each of the 5 dishes; DMSO was added to the last dish in the same volume as TCDD as control group.

After incubation at 28℃for 36h, the fluorescent expression of the transgenic zebra fish was observed under a fluorescent microscope.

(2) Response test of transgenic zebra fish to dioxin with different treatment time lengths

30mL ddH was added to each of 3 90mm glass petri dishes ₂ O and 30F 4 fertilized eggs of 72 hpf.

TCDD was added to each of the 2 dishes at a final concentration of 0.005nM, 0.01 nM; DMSO was added to the last dish in the same volume as TCDD as control group.

Culturing at 28deg.C for 120 hr, and observing fluorescent expression of transgenic zebra fish under a fluorescent microscope at 72 hr, 96 hr and 120 hr.

2. Experimental results

As shown in fig. 5, compared with the control group, the transgenic zebra fish treated with dioxin at different concentrations can express green fluorescence, the lower limit value of the detection concentration of the transgenic zebra fish to the dioxin is 0.005nM, and the fluorescence intensity of the transgenic zebra fish is gradually increased along with the increase of the dioxin concentration.

As shown in fig. 6, the fluorescence intensity of the transgenic zebra fish was also gradually increased as the treatment time of dioxin was prolonged, as compared with the control group.

The fluorescent intensity of the transgenic zebra fish is directly proportional to the concentration of the dioxin and the treatment time, and the content of the dioxin in the water body can be intuitively reflected.

Example 6 responsiveness of transgenic zebra fish to other organic pollutants

1. Experimental method

30mL ddH was added to each of 6 90mm glass petri dishes ₂ O and 30F 4 fertilized eggs of 72 hpf.

TCDD with a final concentration of 0.05nM was added to each of the 1 dishes as a positive control; 3,3', 4' -tetrachlorobiphenyl (PCB 77) at a final concentration of 10. Mu.g/L, indeno [ cd ] pyrene (IcdP) at a final concentration of 10. Mu.g/L, and benzo [ k ] fluoranthene (BkF) at a final concentration of 10. Mu.g/L were added to the remaining 3 dishes, respectively; DMSO was added to the last dish in equal volume to dioxin as a negative control group.

Culturing at 28 ℃ for 72 hours, and observing the fluorescent expression condition of the transgenic zebra fish under a fluorescent microscope.

2. Experimental results

As shown in FIG. 7, the transgenic zebra fish can express green fluorescence when 3,3', 4' -tetrachlorobiphenyl, indeno [ cd ] pyrene and benzo [ k ] fluoranthene exist in the water body. The transgenic zebra fish cultivated by the method can respond to dioxin and polychlorinated biphenyl substances, and can be used for detecting different types of organic pollutants.

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Sequence listing

<110> agricultural university of south China

<120> a method for breeding transgenic zebra fish for detecting organic pollutants in water

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2780

<212> DNA

<213> Gambusia affinis

<400> 1

ttattagttg cactccttac cagcgggctc aactcttgga gcttgtaaaa gaccgggagg 60

gttgacgagg tcatcttgtt cagagttttc gtgattcgtt ttgccctata gcgacctcag 120

cgtgttgcat gactgcaaac agtgaagcct gggtggcaga ttgcatgact ccaaagctac 180

aagatcctct catttacagc gcgcgcgcga atacacgtgc accttcgtgc gggcaatgag 240

tggggggcgt aaaaaaaaaa gaaaaaaaaa agatttgagc ttttttttct ctttgttcta 300

cgagtttaaa acctttgtgt cttttccagg ggctacagcc ttcgcgtgac ttaaccaagc 360

agatttagga ttgtccagaa gacggagacc ctcccctctg gacggcagct ccgcttctca 420

cgcaacctgc tgcaccagag gcgtagcgcc ggagcgcacg gagaaagaaa ccctgcagat 480

tcagcacatg cagcccccca ggggatcggg acagcgctgt tcagcgcgga ccgaagggaa 540

ttggcgaaca aattaagtta tgactttggg accttattca agagaaaagt caaattttcc 600

ttgagagctg tgtccaaggc cggtggatct tggtgcacaa actgtctatt aagggggaaa 660

aaaggtcaca atattattat taagaatatg aatggaagta aaagacataa acgagatata 720

acgccctccg tcggcaaaaa cctattttag cagtctttta aaataaccaa aataaatatt 780

cccatctgat ggcttgtgtt tttatttgaa taaacattgt tttgacagca tttcactatt 840

atgggtgctc tgatttttaa cattcaatag cacacgcgca gaataaccgt caaagtcacg 900

aatacaacat tgctgcattg tttgtttgta ttatatattt tctcactata tatcacctaa 960

aagattacct atatcagtaa ataatttcag aaaaagttaa atgaacactc atatatccat 1020

tacatacaag gtgaaacatt ctaaatgttt atttgtgaat gtgatttaca gttaatgatg 1080

accagcaaat tatttactta ggaaaaaaat aacaaaaata aaagatttta aagtagaaat 1140

aacagccaac ggacatgtac attatactta gtctgaactt ggtctgtgtc tctcttctct 1200

cttgattttt acatgaaagt atttctagta gatttttttc ttccaataaa ctttccatca 1260

taatgctttg atacagcatt gtgtgaacat cttctagcaa taatattttg tggttaattc 1320

tcccggtagt aggtgtaggt ggatcttcag acaaaaagtt agatatctgg tccataacca 1380

tatatggctt tacatgctca ttacgtctac ataaaaacaa tatttaatac tcatccaaac 1440

catttattgt cgtaagaaac ttttttccct gagaaacaaa gttctaactt tttgcttgaa 1500

ctatatgctt tataatgact ttgttttagc tatttgagac aagatttcta aattgttcaa 1560

tgttctattt tatgaagatg catgagctct gtcagaacat gtaaacaaaa acaactgcag 1620

atatctgcaa tcttttcctt ccttcttgtc cagcattaat ccaacagatt agaaaacaac 1680

gtgggcatgg catgagcgaa ggggacaaaa atgtttaaat tcacaaaaac aaaagtgtag 1740

ctgagaacca gcttagtgct cttaataatg tgttattgtt gctgtttttt gtttgtttat 1800

ttatgaattt tttttccttt aacatagtgg atttttgttt caaagttgta acggtgcatt 1860

agttaagcca ctcattacgc agcactttat ttaagtgtat gaactcgtct ggccttttgc 1920

tccggcagca gcgccgcctg ttcctcctcc ctccctctaa ctctgcccag catcctcctc 1980

gaaggggagg ggaaggtttg atctctgcgc tctcacgcaa ctggtcaatc tttaactcct 2040

gcggagtgca tacaagtaca agcacgcaat ggcatctgtt tttatcagca ctgtgtaacc 2100

atgcctggaa aaggctggca tggtcagcag tcttgttctt acttccaaat atggaaggta 2160

agatggctga atctttgttt ggcatcacaa tcacaagcaa ataacccatc tcaattgctt 2220

ccaattaagt ccgcttcttg cgagaaatcc ttacagtatc aagaagctcc ttcaccaggc 2280

atgaactctt ctgcggttat cctgacagac cgcactcgca gaaggtctct gcatcagcaa 2340

atatcctacc tgcacagacc aagccgaagt gcattcaagt gtattaatcg taccccatgt 2400

cctgcattag aaaactgttg cactgaaaat acatagcaga tactgcaaag tttgaaacac 2460

ctgcaaaaca agtgcatgat gacagtgatt tgaactgtct tatctcagat cacacactga 2520

tttacagcca gttctgtgtc tcatacgcag ctaaacttga agaaagcagt ggagcatttg 2580

caatacacac gaattgtgaa atgtaagaac cacacacaga cacgcccacc aactttttta 2640

tgttccaaac ttcattcatg gcagagaatt agaaaggaga gccgcagaaa tttggaggag 2700

gggagatgat gtcacccaca gcagccaatc agattgtgca gtgctctata aattataagt 2760

ccactctcgg ttttgaagac 2780

<210> 2

<211> 8239

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60

attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120

gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180

caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240

ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300

cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360

agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420

cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480

caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540

gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600

taaaacgacg gccagtgagc gcgtagggat aacagggtaa tgcgcgcgta atacgactca 660

ctatagggcg aattgggtac cgggcccaac aggagggtaa atagagcaga actagtgaac 720

ctcttctcct cctccagctc acgcaacgtg gccaatcttt aacccgcgct acaggtgcgc 780

gcacgcgatg ctgtttgatc agtttatcgt agcgtgtttc acacagcgat aacagtctga 840

ggtcgcagga actcttccca taaacccacc gcagaacaaa cactccggct ttaacactcc 900

tcgtgctttt gtgcatgaac cgctgacatg cacgctctcc gacggccacg cgcgtctacc 960

ccattctgcc agctcttcct gttgacagtc aatgagatgc atgaaaaatg tgtgaaggaa 1020

tctgcagcag cggttcacaa acgcacgcac acactctcac acacacacac ctttgcacgc 1080

gatgctttac ctgttgctta atgagttacg agcgcgtgcc agatcagcag agactcaaac 1140

atgcaggcaa ttatcggatg tgttgcaaca aacaatttat ttagttcaca taattgccta 1200

aaccatcaca ctgatttatg acactttagc ttagacagct ttaaaagata aataaacatc 1260

tcgagcatgc tgtttaactt ttcatgattt atatattctg attttattgg gcttatttat 1320

ttctcacata attattatcc gcattgagtt tgctgtatta agagttgtga tgaaatgtgg 1380

gattgatttc tcagttaatg cacgtcgctt ttgtctacaa actgttctgt aaatattaac 1440

attacattac ataacatcaa aaaacactga taagcccagt tctgccttaa ttataaaggc 1500

taattaagcg tctcatttat taatttattt catttattta tgttttaaat atatatttgt 1560

ttacatattg caaatttagt tggaaatgca tgttaaaaat attagtgctg tataattatt 1620

acccaatcaa aaatgttgca tttgtgttaa atattgacat atatatgatc acattacata 1680

aatattgaca tatatatgat tacaatacat aaatattgac atatatatat ataataatgt 1740

tacataaata ttgacatata tgattacgtt acataaatat tgacatatat ataattactt 1800

tacataaata ttgatatata tataattaca ttacataaat atgagcatat atatataatt 1860

acattacata aatattgaca tatatattta attacaatac ataaatattg acatatatat 1920

aattacatta cataaatatt gacatatatg attacattac ataaatattg acatatataa 1980

gtacgttaca taaatattga gatatatata tataattaca atacataaat attggcatat 2040

atataattac gttacataaa tattgacata tatatatata tatatatata tatatatata 2100

tatatatata tatatatata tataattaca atacataaat attgacatat atgattacgt 2160

tacataaata ttgacacata tataattacg ttacataaat attgacatat atataattac 2220

gtttcataaa tattgacata tatataataa cgatacataa atattgacat atatatgatt 2280

acaatacata aatattggca taattaaaat gacattacat aaatattgac atatatatga 2340

ctatattaca aaaatattga catatatata tatacacaca cacacacata tatatataca 2400

attacgttga atcaatattg acatatatat gaatgaatta caaaaatatt gacatatata 2460

taataacgtt acataaatat tgacatatat ttgattttat taaataaata ttggcatata 2520

tataattaca ttacataaat attgacatat atatgattac attacataaa tattgacata 2580

tatataataa cgttacataa atattgacat atatttgatt ttattaaata aatattggca 2640

tatatatgat tacattacat aaatattgac atatatatat atatatatat atatatatat 2700

atatatatat atatatatat atttacttta cataaatatt gacatatttg acttagtccc 2760

tttaatcagg ggtcgccact gcggaatgaa ccgccaactt atccagcata gtttttacgc 2820

agcagatgcc cttccagctg caacccaaca ctgggaaaca cacataccct ctcattcaca 2880

cgcacactca tacactacgg ccaatttagt tcatcagttc ccctaaagtg catgtgtttg 2940

gactgtggag aaaaccggag cacccggagg aaacccacgc catgcaaact ccacacagaa 3000

atgccagctg acccagctcg aaccagcgat cgtgctactc actgcacttt ataaatatat 3060

atttttcatt cataactttt gtatacattt tacatagtct tttgtaccat gtatgtgtgc 3120

gtgtgttaca tacatcaatc tccttcccac agtttagata tgtgtgaggt gagtgtgtgt 3180

aattactcag ggagtttact cagtgcaatc gatcagcctg taataaaatc tcagcccttc 3240

tcagcatcaa agcctcctgc gctcggtgac gtccgcggag gacagccaat cacggcgagc 3300

tctgcgctat aaaagattta ccgctggaat agtgcagcac cggtcgccac catggtgagc 3360

aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta 3420

aacggccaca agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg 3480

accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc 3540

accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac 3600

ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac 3660

gacggcaact acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc 3720

atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag 3780

tacaactaca acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag 3840

gtgaacttca agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac 3900

cagcagaaca cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc 3960

acccagtccg ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag 4020

ttcgtgaccg ccgccgggat cactctcggc atggacgagc tgtacaagtc cggccggact 4080

cagatctcga gctcaagctt cgaattcgaa gacgccaaaa acataaagaa aggcccggcg 4140

ccattctatc cgctggaaga tggaaccgct ggagagcaac tgcataaggc tatgaagaga 4200

tacgccctgg ttcctggaac aattgctttt acagatgcac atatcgaggt ggacatcact 4260

tacgctgagt acttcgaaat gtccgttcgg ttggcagaag ctatgaaacg atatgggctg 4320

aatacaaatc acagaatcgt cgtatgcagt gaaaactctc ttcaattctt tatgccggtg 4380

ttgggcgcgt tatttatcgg agttgcagtt gcgcccgcga acgacattta taatgaacgt 4440

gaattgctca acagtatggg catttcgcag cctaccgtgg tgttcgtttc caaaaagggg 4500

ttgcaaaaaa ttttgaacgt gcaaaaaaag ctcccaatca tccaaaaaat tattatcatg 4560

gattctaaaa cggattacca gggatttcag tcgatgtaca cgttcgtcac atctcatcta 4620

cctcccggtt ttaatgaata cgattttgtg ccagagtcct tcgataggga caagacaatt 4680

gcactgatca tgaactcctc tggatctact ggtctgccta aaggtgtcgc tctgcctcat 4740

agaactgcct gcgtgagatt ctcgcatgcc agagatccta tttttggcaa tcaaatcatt 4800

ccggatactg cgattttaag tgttgttcca ttccatcacg gttttggaat gtttactaca 4860

ctcggatatt tgatatgtgg atttcgagtc gtcttaatgt atagatttga agaagagctg 4920

tttctgagga gccttcagga ttacaagatt caaagtgcgc tgctggtgcc aaccctattc 4980

tccttcttcg ccaaaagcac tctgattgac aaatacgatt tatctaattt acacgaaatt 5040

gcttctggtg gcgctcccct ctctaaggaa gtcggggaag cggttgccaa gaggttccat 5100

ctgccaggta tcaggcaagg atatgggctc actgagacta catcagctat tctgattaca 5160

cccgaggggg atgataaacc gggcgcggtc ggtaaagttg ttccattttt tgaagcgaag 5220

gttgtggatc tggataccgg gaaaacgctg ggcgttaatc aaagaggcga actgtgtgtg 5280

agaggtccta tgattatgtc cggttatgta aacaatccgg aagcgaccaa cgccttgatt 5340

gacaaggatg gatggctaca ttctggagac atagcttact gggacgaaga cgaacacttc 5400

ttcatcgttg accgcctgaa gtctctgatt aagtacaaag gctatcaggt ggctcccgct 5460

gaattggaat ccatcttgct ccaacacccc aacatcttcg acgcaggtgt cgcaggtctt 5520

cccgacgatg acgccggtga acttcccgcc gccgttgttg ttttggagca cggaaagacg 5580

atgacggaaa aagagatcgt ggattacgtc gccagtcaag taacaaccgc gaaaaagttg 5640

cgcggaggag ttgtgtttgt ggacgaagta ccgaaaggtc ttaccggaaa actcgacgca 5700

agaaaaatca gagagatcct cataaaggcc aagaagggcg gaaagatcgc cgtggcggcc 5760

ctagagcggc ctatgatgaa ctctcgagta gatccagaca tgataagata cattgatgag 5820

tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat 5880

gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc 5940

attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaatt cgcggccggg 6000

ggatcggtgg agctccagct tttgttccct ttagtgaggg ttaattgcgc gcattaccct 6060

gttatcccta cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 6120

ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 6180

taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 6240

aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 6300

attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 6360

cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 6420

gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 6480

ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 6540

agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 6600

tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 6660

ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 6720

gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 6780

ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 6840

gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 6900

aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 6960

aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 7020

ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 7080

gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 7140

gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 7200

tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 7260

ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 7320

ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 7380

atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 7440

ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 7500

tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 7560

attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 7620

tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 7680

ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 7740

gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 7800

gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 7860

gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 7920

aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 7980

taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 8040

tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 8100

tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 8160

atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca 8220

tttccccgaa aagtgccac 8239

<210> 3

<211> 5612

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ataagtccac tctcggtttt gaagacaccg gtcgccacca tggtgagcaa gggcgaggag 60

ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag 120

ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc 180

atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac 240

ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc 300

gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac 360

aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag 420

ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac 480

agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag 540

atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc 600

cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc 660

ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc 720

gccgggatca ctctcggcat ggacgagctg tacaagtccg gccggactca gatctcgagc 780

tcaagcttcg aattcgaaga cgccaaaaac ataaagaaag gcccggcgcc attctatccg 840

ctggaagatg gaaccgctgg agagcaactg cataaggcta tgaagagata cgccctggtt 900

cctggaacaa ttgcttttac agatgcacat atcgaggtgg acatcactta cgctgagtac 960

ttcgaaatgt ccgttcggtt ggcagaagct atgaaacgat atgggctgaa tacaaatcac 1020

agaatcgtcg tatgcagtga aaactctctt caattcttta tgccggtgtt gggcgcgtta 1080

tttatcggag ttgcagttgc gcccgcgaac gacatttata atgaacgtga attgctcaac 1140

agtatgggca tttcgcagcc taccgtggtg ttcgtttcca aaaaggggtt gcaaaaaatt 1200

ttgaacgtgc aaaaaaagct cccaatcatc caaaaaatta ttatcatgga ttctaaaacg 1260

gattaccagg gatttcagtc gatgtacacg ttcgtcacat ctcatctacc tcccggtttt 1320

aatgaatacg attttgtgcc agagtccttc gatagggaca agacaattgc actgatcatg 1380

aactcctctg gatctactgg tctgcctaaa ggtgtcgctc tgcctcatag aactgcctgc 1440

gtgagattct cgcatgccag agatcctatt tttggcaatc aaatcattcc ggatactgcg 1500

attttaagtg ttgttccatt ccatcacggt tttggaatgt ttactacact cggatatttg 1560

atatgtggat ttcgagtcgt cttaatgtat agatttgaag aagagctgtt tctgaggagc 1620

cttcaggatt acaagattca aagtgcgctg ctggtgccaa ccctattctc cttcttcgcc 1680

aaaagcactc tgattgacaa atacgattta tctaatttac acgaaattgc ttctggtggc 1740

gctcccctct ctaaggaagt cggggaagcg gttgccaaga ggttccatct gccaggtatc 1800

aggcaaggat atgggctcac tgagactaca tcagctattc tgattacacc cgagggggat 1860

gataaaccgg gcgcggtcgg taaagttgtt ccattttttg aagcgaaggt tgtggatctg 1920

gataccggga aaacgctggg cgttaatcaa agaggcgaac tgtgtgtgag aggtcctatg 1980

attatgtccg gttatgtaaa caatccggaa gcgaccaacg ccttgattga caaggatgga 2040

tggctacatt ctggagacat agcttactgg gacgaagacg aacacttctt catcgttgac 2100

cgcctgaagt ctctgattaa gtacaaaggc tatcaggtgg ctcccgctga attggaatcc 2160

atcttgctcc aacaccccaa catcttcgac gcaggtgtcg caggtcttcc cgacgatgac 2220

gccggtgaac ttcccgccgc cgttgttgtt ttggagcacg gaaagacgat gacggaaaaa 2280

gagatcgtgg attacgtcgc cagtcaagta acaaccgcga aaaagttgcg cggaggagtt 2340

gtgtttgtgg acgaagtacc gaaaggtctt accggaaaac tcgacgcaag aaaaatcaga 2400

gagatcctca taaaggccaa gaagggcgga aagatcgccg tggcggccct agagcggcct 2460

atgatgaact ctcgagtaga tccagacatg ataagataca ttgatgagtt tggacaaacc 2520

acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 2580

tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg 2640

tttcaggttc agggggaggt gtgggaggtt ttttaattcg cggccggggg atcggtggag 2700

ctccagcttt tgttcccttt agtgagggtt aattgcgcgc attaccctgt tatccctacg 2760

cgcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 2820

ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 2880

taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 2940

cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 3000

tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 3060

gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 3120

atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 3180

ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 3240

cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 3300

tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 3360

gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 3420

aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 3480

tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 3540

aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 3600

aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 3660

ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 3720

ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 3780

atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 3840

atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 3900

tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 3960

gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 4020

tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 4080

gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 4140

cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 4200

gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 4260

atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 4320

aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 4380

atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 4440

aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 4500

aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 4560

gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 4620

gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 4680

gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 4740

ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 4800

ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 4860

atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 4920

gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa 4980

tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat 5040

agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg 5100

tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac 5160

catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta 5220

aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag 5280

ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg 5340

taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat tcgccattca 5400

ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg 5460

cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac 5520

gacgttgtaa aacgacggcc agtgagcgcg tagggataac agggtaatgc gcgcgtaata 5580

cgactcacta tagggcgaat tgggtaccgg gc 5612

<210> 4

<211> 8368

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60

attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120

gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180

caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240

ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300

cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360

agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420

cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480

caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540

gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg 600

taaaacgacg gccagtgagc gcgtagggat aacagggtaa tgcgcgcgta atacgactca 660

ctatagggcg aattgggtac cgggccctta ttagttgcac tccttaccag cgggctcaac 720

tcttggagct tgtaaaagac cgggagggtt gacgaggtca tcttgttcag agttttcgtg 780

attcgttttg ccctatagcg acctcagcgt gttgcatgac tgcaaacagt gaagcctggg 840

tggcagattg catgactcca aagctacaag atcctctcat ttacagcgcg cgcgcgaata 900

cacgtgcacc ttcgtgcggg caatgagtgg ggggcgtaaa aaaaaaagaa aaaaaaaaga 960

tttgagcttt tttttctctt tgttctacga gtttaaaacc tttgtgtctt ttccaggggc 1020

tacagccttc gcgtgactta accaagcaga tttaggattg tccagaagac ggagaccctc 1080

ccctctggac ggcagctccg cttctcacgc aacctgctgc accagaggcg tagcgccgga 1140

gcgcacggag aaagaaaccc tgcagattca gcacatgcag ccccccaggg gatcgggaca 1200

gcgctgttca gcgcggaccg aagggaattg gcgaacaaat taagttatga ctttgggacc 1260

ttattcaaga gaaaagtcaa attttccttg agagctgtgt ccaaggccgg tggatcttgg 1320

tgcacaaact gtctattaag ggggaaaaaa ggtcacaata ttattattaa gaatatgaat 1380

ggaagtaaaa gacataaacg agatataacg ccctccgtcg gcaaaaacct attttagcag 1440

tcttttaaaa taaccaaaat aaatattccc atctgatggc ttgtgttttt atttgaataa 1500

acattgtttt gacagcattt cactattatg ggtgctctga tttttaacat tcaatagcac 1560

acgcgcagaa taaccgtcaa agtcacgaat acaacattgc tgcattgttt gtttgtatta 1620

tatattttct cactatatat cacctaaaag attacctata tcagtaaata atttcagaaa 1680

aagttaaatg aacactcata tatccattac atacaaggtg aaacattcta aatgtttatt 1740

tgtgaatgtg atttacagtt aatgatgacc agcaaattat ttacttagga aaaaaataac 1800

aaaaataaaa gattttaaag tagaaataac agccaacgga catgtacatt atacttagtc 1860

tgaacttggt ctgtgtctct cttctctctt gatttttaca tgaaagtatt tctagtagat 1920

ttttttcttc caataaactt tccatcataa tgctttgata cagcattgtg tgaacatctt 1980

ctagcaataa tattttgtgg ttaattctcc cggtagtagg tgtaggtgga tcttcagaca 2040

aaaagttaga tatctggtcc ataaccatat atggctttac atgctcatta cgtctacata 2100

aaaacaatat ttaatactca tccaaaccat ttattgtcgt aagaaacttt tttccctgag 2160

aaacaaagtt ctaacttttt gcttgaacta tatgctttat aatgactttg ttttagctat 2220

ttgagacaag atttctaaat tgttcaatgt tctattttat gaagatgcat gagctctgtc 2280

agaacatgta aacaaaaaca actgcagata tctgcaatct tttccttcct tcttgtccag 2340

cattaatcca acagattaga aaacaacgtg ggcatggcat gagcgaaggg gacaaaaatg 2400

tttaaattca caaaaacaaa agtgtagctg agaaccagct tagtgctctt aataatgtgt 2460

tattgttgct gttttttgtt tgtttattta tgaatttttt ttcctttaac atagtggatt 2520

tttgtttcaa agttgtaacg gtgcattagt taagccactc attacgcagc actttattta 2580

agtgtatgaa ctcgtctggc cttttgctcc ggcagcagcg ccgcctgttc ctcctccctc 2640

cctctaactc tgcccagcat cctcctcgaa ggggagggga aggtttgatc tctgcgctct 2700

cacgcaactg gtcaatcttt aactcctgcg gagtgcatac aagtacaagc acgcaatggc 2760

atctgttttt atcagcactg tgtaaccatg cctggaaaag gctggcatgg tcagcagtct 2820

tgttcttact tccaaatatg gaaggtaaga tggctgaatc tttgtttggc atcacaatca 2880

caagcaaata acccatctca attgcttcca attaagtccg cttcttgcga gaaatcctta 2940

cagtatcaag aagctccttc accaggcatg aactcttctg cggttatcct gacagaccgc 3000

actcgcagaa ggtctctgca tcagcaaata tcctacctgc acagaccaag ccgaagtgca 3060

ttcaagtgta ttaatcgtac cccatgtcct gcattagaaa actgttgcac tgaaaataca 3120

tagcagatac tgcaaagttt gaaacacctg caaaacaagt gcatgatgac agtgatttga 3180

actgtcttat ctcagatcac acactgattt acagccagtt ctgtgtctca tacgcagcta 3240

aacttgaaga aagcagtgga gcatttgcaa tacacacgaa ttgtgaaatg taagaaccac 3300

acacagacac gcccaccaac ttttttatgt tccaaacttc attcatggca gagaattaga 3360

aaggagagcc gcagaaattt ggaggagggg agatgatgtc acccacagca gccaatcaga 3420

ttgtgcagtg ctctataaat tataagtcca ctctcggttt tgaagacacc ggtcgccacc 3480

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 3540

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 3600

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 3660

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 3720

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 3780

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 3840

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 3900

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 3960

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 4020

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 4080

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 4140

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtcc 4200

ggccggactc agatctcgag ctcaagcttc gaattcgaag acgccaaaaa cataaagaaa 4260

ggcccggcgc cattctatcc gctggaagat ggaaccgctg gagagcaact gcataaggct 4320

atgaagagat acgccctggt tcctggaaca attgctttta cagatgcaca tatcgaggtg 4380

gacatcactt acgctgagta cttcgaaatg tccgttcggt tggcagaagc tatgaaacga 4440

tatgggctga atacaaatca cagaatcgtc gtatgcagtg aaaactctct tcaattcttt 4500

atgccggtgt tgggcgcgtt atttatcgga gttgcagttg cgcccgcgaa cgacatttat 4560

aatgaacgtg aattgctcaa cagtatgggc atttcgcagc ctaccgtggt gttcgtttcc 4620

aaaaaggggt tgcaaaaaat tttgaacgtg caaaaaaagc tcccaatcat ccaaaaaatt 4680

attatcatgg attctaaaac ggattaccag ggatttcagt cgatgtacac gttcgtcaca 4740

tctcatctac ctcccggttt taatgaatac gattttgtgc cagagtcctt cgatagggac 4800

aagacaattg cactgatcat gaactcctct ggatctactg gtctgcctaa aggtgtcgct 4860

ctgcctcata gaactgcctg cgtgagattc tcgcatgcca gagatcctat ttttggcaat 4920

caaatcattc cggatactgc gattttaagt gttgttccat tccatcacgg ttttggaatg 4980

tttactacac tcggatattt gatatgtgga tttcgagtcg tcttaatgta tagatttgaa 5040

gaagagctgt ttctgaggag ccttcaggat tacaagattc aaagtgcgct gctggtgcca 5100

accctattct ccttcttcgc caaaagcact ctgattgaca aatacgattt atctaattta 5160

cacgaaattg cttctggtgg cgctcccctc tctaaggaag tcggggaagc ggttgccaag 5220

aggttccatc tgccaggtat caggcaagga tatgggctca ctgagactac atcagctatt 5280

ctgattacac ccgaggggga tgataaaccg ggcgcggtcg gtaaagttgt tccatttttt 5340

gaagcgaagg ttgtggatct ggataccggg aaaacgctgg gcgttaatca aagaggcgaa 5400

ctgtgtgtga gaggtcctat gattatgtcc ggttatgtaa acaatccgga agcgaccaac 5460

gccttgattg acaaggatgg atggctacat tctggagaca tagcttactg ggacgaagac 5520

gaacacttct tcatcgttga ccgcctgaag tctctgatta agtacaaagg ctatcaggtg 5580

gctcccgctg aattggaatc catcttgctc caacacccca acatcttcga cgcaggtgtc 5640

gcaggtcttc ccgacgatga cgccggtgaa cttcccgccg ccgttgttgt tttggagcac 5700

ggaaagacga tgacggaaaa agagatcgtg gattacgtcg ccagtcaagt aacaaccgcg 5760

aaaaagttgc gcggaggagt tgtgtttgtg gacgaagtac cgaaaggtct taccggaaaa 5820

ctcgacgcaa gaaaaatcag agagatcctc ataaaggcca agaagggcgg aaagatcgcc 5880

gtggcggccc tagagcggcc tatgatgaac tctcgagtag atccagacat gataagatac 5940

attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt tatttgtgaa 6000

atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca agttaacaac 6060

aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaattc 6120

gcggccgggg gatcggtgga gctccagctt ttgttccctt tagtgagggt taattgcgcg 6180

cattaccctg ttatccctac gcgcttggcg taatcatggt catagctgtt tcctgtgtga 6240

aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc 6300

tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc 6360

cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 6420

ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt 6480

cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca 6540

ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa 6600

aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat 6660

cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc 6720

cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc 6780

gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt 6840

tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac 6900

cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg 6960

ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca 7020

gagttcttga agtggtggcc taactacggc tacactagaa ggacagtatt tggtatctgc 7080

gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa 7140

accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa 7200

ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac 7260

tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta 7320

aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt 7380

taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata 7440

gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc 7500

agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac 7560

cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag 7620

tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac 7680

gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc 7740

agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg 7800

gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc 7860

atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct 7920

gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc 7980

tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc 8040

atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc 8100

agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc 8160

gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca 8220

cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt 8280

tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 8340

ccgcgcacat ttccccgaaa agtgccac 8368

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gacacaaaat gtatttaagg 20

<210> 6

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

cgtttgggta aaaaaaacaa tatga 25

<210> 7

<211> 26

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ttaaatacat tttgtgtctt ttcttt 26

<210> 8

<211> 1551

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

gcccttaagg gcgacacgcg aagtcgatgt cgcgtctgcc tgaagtcaat actgacgatg 60

gtcatagctg tttcctgtcc atagcagaaa gtcaaaagcc tccgaccgga ggcttttgac 120

ttgatcggca cgtaagaggt tccaactttc accataatga aataagatca ctaccgggcg 180

tattttttga gttatcgaga ttttcaggag ctaaggaagc taaaatgagt attcaacatt 240

tccgtgtcgc acttattccg ttttttgcgg cattttgcct tcctgttttt gctcacccag 300

aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 360

aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 420

tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 480

aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 540

tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 600

ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 660

taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 720

agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 780

caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 840

tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 900

gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 960

cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 1020

caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 1080

ggtaatgagg gcccaaatgt aatcacctgg ctcaccttcg ggtgggcctt tctgcgttgc 1140

tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga tgctcaagtc 1200

agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 1260

tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 1320

cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 1380

ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 1440

ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 1500

ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag t 1551

<210> 9

<211> 1648

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

cccccttaaa tacattttgt gtccgtttgg gtaaaaaaaa caatatgagc ccttaagggc 60

gacacgcgaa gtcgatgtcg cgtctgcctg aagtcaatac tgacgatggt catagctgtt 120

tcctgtccat agcagaaagt caaaagcctc cgaccggagg cttttgactt gatcggcacg 180

taagaggttc caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt 240

tatcgagatt ttcaggagct aaggaagcta aaatgagtat tcaacatttc cgtgtcgcac 300

ttattccgtt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga 360

aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca 420

acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt 480

ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg 540

gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc 600

atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata 660

acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt 720

tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag 780

ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca 840

aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg 900

aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg 960

ctgataaatc tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag 1020

atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg 1080

aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taatgagggc 1140

ccaaatgtaa tcacctggct caccttcggg tgggcctttc tgcgttgctg gcgtttttcc 1200

ataggctccg cccccctgac gagcatcaca aaaatcgatg ctcaagtcag aggtggcgaa 1260

acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 1320

ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 1380

cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 1440

tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 1500

gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 1560

ggattagcag agcgaggtat gtaggcggtg ctacagagtt taaatacatt ttgtgtcttt 1620

tctttccccc ttaaatacat tttgtgtc 1648

<210> 10

<211> 96

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

gacacaaaat gtatttaagg gttttagagc tagaaatagc aagttaaaat aaggctagtc 60

cgttatcaac ttgaaaaagt ggcaccgagt cggtgc 96

Claims (10)

1. A plasmid for detecting organic contaminants in a body of water, the plasmid comprising an expression cassette; the expression cassette contains a promoter and a reporter gene of a mosquito-eating fish cyp1a gene with nucleotide sequences shown as SEQ ID NO.1, wherein the promoter of the mosquito-eating fish cyp1a gene with the nucleotide sequences shown as SEQ ID NO.1 is upstream of the reporter gene; the organic pollutants in the water body comprise dioxin, benzopyrene, 3', 4' -tetrachlorobiphenyl, indeno [ cd ] pyrene and/or benzo [ k ] fluoranthene.

2. The plasmid according to claim 1, wherein the reporter gene is a gene encoding a fluorescent protein and/or a gene encoding a luciferase.

3. The plasmid according to claim 2, wherein the nucleotide sequence of the plasmid is shown in SEQ ID No. 4.

4. The cultivation method of the transgenic zebra fish for detecting the organic pollutants in the water body is characterized by comprising the following steps of: introducing the plasmid of any one of claims 1 to 3 into fertilized eggs of a somatic zebra fish; screening and identifying fertilized eggs; hybridization and purification; the homozygote zebra fish is the transgenic zebra fish for detecting the organic pollutants in the water body;

the preparation method of the somatic stain-free horse fish comprises the following steps of:

injecting a gene editing plasmid, cas9 protein and gRNA with a nucleotide sequence shown as SEQ ID NO.10 into the fertilized eggs of the zebra fish, wherein the gene editing plasmid sequentially comprises a PAM region and a sequence with a nucleotide sequence shown as SEQ ID NO.9 from the 5 'end to the 3' end, so that the sequence with a nucleotide sequence shown as SEQ ID NO.8 in the gene editing plasmid is integrated into the genome of the fertilized eggs of the zebra fish;

screening and identifying fertilized eggs; hybridization and purification; the homozygote zebra fish is the zebra fish without body color spots.

5. The cultivation method according to claim 4, wherein said PAM region has a nucleotide sequence of 5'-CCC-3'.

6. The cultivation method as claimed in claim 4, wherein the nucleotide sequence shown in SEQ ID NO.8 of the gene editing plasmid is integrated between the promoter and the transcription initiation site of the mitfa gene.

7. The cultivation method as claimed in claim 4, wherein the plasmid according to any one of claims 1 to 3 is introduced into fertilized eggs in a cell phase by microinjection.

8. The method according to claim 4, wherein the method for screening and identifying fertilized eggs is dioxin screening.

9. The method of claim 8, wherein the method of screening and identifying fertilized eggs is to treat fertilized eggs with dioxin and/or DMSO, select fertilized eggs that express green fluorescence in dioxin and do not express green fluorescence in DMSO, and use them for subsequent hatching.

10. Use of a plasmid according to any one of claims 1 to 3 or a cultivation method according to any one of claims 4 to 9 in water environment, water quality monitoring and/or detection of one or more of organic pollutants of a water body, characterized in that the organic pollutants of a water body comprise dioxins, benzopyrenes, 3', 4' -tetrachlorobiphenyls, indeno [ cd ] pyrenes and/or benzo [ k ] fluoranthenes.