ADO gene deleted zebrafish model as well as construction method and application thereof

Abstract

The invention discloses an ADO (adenosine dioxygenase) gene deleted zebrafish model as well as a construction method and application thereof. According to the invention, ADOa gene knockout is carried out on a model organism (zebrafish) to successfully construct an ADO gene deleted zebrafish model. Based on the model, the function of the ADO gene in fish taurine metabolism can be researched more deeply, so that a new scientific basis is provided for understanding the ADO function of zebra fish and even other fishes, and meanwhile, the model can be used for drug screening and provides a theoretical basis and technical support for breeding and genetic improvement of aquaculture industry.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of animal model construction, and particularly relates to an ADO gene deletion zebrafish model and a construction method and application thereof. BACKGROUND

[0002] With the rapid development of molecular biology and genetic engineering technology, gene editing technology, especially the CRISPR-Cas9 system, has become an important tool in current biological scientific research. This technology has been widely used in many biological fields due to its high efficiency, precision and easy operation, including the construction of disease models, the study of gene function, and the treatment of genetic diseases.

[0003] Zebrafish (Danio rerio) as a classic model organism in modern life science research, with its unique biological characteristics (transparent embryo, fast growth cycle) and highly conserved genome with humans, has become an irreplaceable platform for gene function research and human disease modeling, and is widely used in biomedical research. In particular, in the fields of gene function research, genetic disease mechanism analysis and drug efficient screening, zebrafish models provide researchers with a window to understand gene function and disease mechanism in depth.

[0004] Cysteamine dioxygenase (ADO) as a key oxygen sensor can oxidize two different types of substrates at the same time, and plays an important role in maintaining cellular oxygen homeostasis and cysteine metabolism. In human cells, ADO uses O2 molecules as a cofactor to catalyze the oxidation of the N-terminal cysteine residues of G protein signaling regulator 4 / 5 (RGS4 / 5) and interleukin-32 (IL-32), thereby causing them to be degraded through the Arg / N-terminal degradation pathway, and then exerting regulatory functions. Although the function of ADO in mammalian physiological processes has been studied and understood to some extent, the function research and regulatory mechanism of ADO gene in non-mammalian models, especially in model organisms such as zebrafish, are still not clear enough.

[0005] With the rapid development of global aquaculture industry, the demand for high-quality and sustainable feed is more urgent than ever, and taurine, as a conditional essential amino acid for aquatic animals, is widely used in aquatic feed as an immune enhancer and attractant. How to improve the absorption and metabolism of farmed fish through genetic improvement and biotechnology has become a research hotspot. However, the lack of effective models for gene function research in aquatic animals has limited the progress of scientific research and application in this field.

[0006] Therefore, it is urgent to develop a zebrafish ADO gene mutation model based on gene editing technology for studying the role of ADO gene in taurine metabolism in fish, thereby providing new scientific basis for understanding the function of ADO in zebrafish and even other fish, and also can be used for drug screening, providing theoretical basis and technical support for breeding and genetic improvement of aquaculture industry. SUMMARY

[0007] The present application aims at solving at least one of the above technical problems in the prior art. To this end, the present application aims to provide an ADO gene deletion zebrafish model and a construction method and application thereof. The present application successfully constructs an ADO gene deletion animal model by using a model organism (zebrafish), and it is also found that knocking out a specific ADOa gene can effectively improve the response to pathogen stimulation. Based on this model, the role of ADO gene in taurine metabolism in fish can be further studied, thereby providing new scientific basis for understanding the function of ADO in zebrafish and even other fish, and also can be used for drug screening, providing theoretical basis and technical support for breeding and genetic improvement of aquaculture industry.

[0008] In a first aspect, the present application provides a construction method of an ADO gene deletion model, comprising the following steps: knocking out or silencing the ADO gene of an organism.

[0009] In the present application, the term "ADO" refers to cysteamine (2-aminoethanethiol) dioxygenase, which is an important member of the thiol dioxygenase family and plays an extremely important role in cells. It can maintain thiol metabolism balance by catalyzing small molecule cysteamine, and promote the ordered degradation of key signal proteins by modifying the N-terminal cysteine residues thereof. At the same time, ADO is an oxygen-responsive protein, and its activity is limited under low oxygen. It is pointed out in the prior art that by comparing the sequences of fish ADO proteins, it is found that the sequence homology of fish ADO is high, and it is speculated that the function of ADO in fish is relatively conservative.

[0010] In some embodiments of the present application, the ADO gene is an ADOa gene.

[0011] In the present application, the ADOa gene is located on chromosome 12 of zebrafish.

[0012] In some embodiments of the present application, the knockout site corresponding to the ADOa gene is as shown in Figure 1 .

[0013] In some embodiments of the present application, the knocking out or silencing comprises using at least one gene editing technology or physical-chemical mutagenesis technology selected from the group consisting of RNA interference, plasmid-mediated gene knockout, embryonic stem cell knockout, CRISPR system, transcription activator-like effector nucleases (TALENs) and zinc finger nucleases (ZFNs).

[0014] Of course, other technologies capable of achieving the specified gene silencing or knocking out can also be used by those skilled in the art, including but not limited to the above-mentioned RNA interference, plasmid-mediated gene knockout, embryonic stem cell knockout, CRISPR system, TALENs and ZFNs.

[0015] In some embodiments of the present application, the construction method uses the CRISPR system for knocking out.

[0016] In some embodiments of the present application, the CRISPR system comprises CRISPR-Cas9.

[0017] Of course, other CRISPR systems can also be selected by those skilled in the art according to the use requirements, including but not limited to Cas9, Cas12a, Cas13a and Cas13b.

[0018] In some embodiments of the present application, the construction method uses the CRISPR system for knocking out, and the transcript of the gRNA in the CRISPR system is obtained by annealing the following oligonucleotide chains:

[0019] 5'-GGTGTTTCTCCTGAAATC-3' (SEQ ID NO: 7) and 5'-GATTTCAGGAGAAACACC-3' (SEQ ID NO: 8).

[0020] Of course, other gRNAs or their transcripts with equivalent effects can also be designed by those skilled in the art according to the use requirements, for use with the corresponding CRISPR system and the corresponding target site.

[0021] In some embodiments of the present application, the transcript is further connected with a sticky end.

[0022] In some embodiments of the present application, the sticky end comprises at least one of 5'-TAGG-3' and 5'-AAAC-3'.

[0023] In some embodiments of the present application, the sticky end is 5'-TAGG-3' and 5'-AAAC-3'.

[0024] In some embodiments of the present application, 5'-TAGG-3' and 5'-AAAC-3' are connected on different chains.

[0025] In some embodiments of the present application, the sticky ends are obtained by at least one of the following ways:

[0026] (1) by end-repairing the annealing product of the oligonucleotide strands; or

[0027] (2) by annealing after connecting the sticky ends on at least one of the oligonucleotide strands.

[0028] In some embodiments of the present application, the sticky ends are obtained by annealing after connecting the sticky ends on both of the oligonucleotide strands.

[0029] In some embodiments of the present application, the two oligonucleotide strands are SEQ ID NO: 7 and 8.

[0030] In some embodiments of the present application, the two oligonucleotide strands after connecting the sticky ends are shown in SEQ ID NO: 4 and 5.

[0031] In some embodiments of the present application, the organism comprises aquatic animals.

[0032] In some embodiments of the present application, the organism is fish.

[0033] In some embodiments of the present application, the organism is zebrafish.

[0034] In the present application, the organism refers to any species that can survive to adulthood after knocking out or silencing the ADOa gene, including but not limited to the above-mentioned zebrafish. As a model organism well known in the art, zebrafish can represent its generality and feasibility in a wide range of species.

[0035] In some embodiments of the present application, the construction method specifically comprises: synthesizing sgRNA, mixing it with Cas9 system, injecting into embryos, and obtaining after typing by sequencing screening.

[0036] In a second aspect of the present application, the use of the ADO gene deletion model constructed by the construction method of the above-mentioned aspect in drug screening is provided.

[0037] In some embodiments of the present application, the drug comprises a cysteamine (2-aminoethanethiol) dioxygenase (ADO) targeted drug or a taurine metabolic pathway targeted drug.

[0038] In some embodiments of the present application, the taurine metabolic pathway comprises:

[0039] (1) taurine forms conjugated bile acids such as taurocholic acid, taurochenodeoxycholic acid, and the like with free bile acids such as cholic acid, chenodeoxycholic acid, and the like;

[0040] (2) catalyzing the reaction of hydrogen peroxide and chloride under the action of neutrophil and monocyte myeloperoxidase to generate hypochlorous acid (HClO), and then HClO reacts with taurine to generate stable and less toxic taurine chloramine (RNHCl);

[0041] (3) forming amidine taurine through the amidine transfer of taurine and arginine;

[0042] (4) generating taurine urea acid;

[0043] (5) generating hydroxyethyl taurine.

[0044] In some embodiments of the present application, the drug has at least one of the following functions:

[0045] (1) improving the growth rate and / or weight gain rate of fish;

[0046] (2) improving the immunity of fish;

[0047] (3) improving the environmental adaptability of fish; and

[0048] (4) improving the feeding amount of fish.

[0049] In some embodiments of the present application, the drug is a veterinary drug.

[0050] In some embodiments of the present application, preferably, the veterinary drug is an aquatic animal drug.

[0051] The beneficial effects of the present application are:

[0052] 1. The present application successfully constructs an ADO deletion zebrafish model that can be stably inherited, does not affect the normal growth and survival of zebrafish, and can be used as an effective model for double oxygenase drug screening and taurine metabolism research, thereby reflecting the function of ADO, providing a powerful tool for in-depth study of gene function.

[0053] 2. The ADO deletion zebrafish model in the present application responds more obviously to pathogen stimulation. The experimental period of the entire construction process is short, the model itself has fast reproduction and development speed, good repeatability, strong reliability, and can effectively reduce experimental costs.

[0054] 3. The ADO deletion zebrafish model in the present application can be effectively used for drug screening and functional research in taurine metabolism, and can realize the function of fish participating in metabolism and protein regulation, signal pathway regulation, and immune and pathogen stimulation response, thereby providing guidance significance for actual production and genetic breeding. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1Figure 1 is a diagram of CRISPR / Cas9 target analysis of zebrafish ADOa gene in the embodiments of the present application, wherein the underlined part represents the sgRNA targeting site, the red labeled part represents the mutant sequence, and the "-" represents the edited base deleted after editing.

[0056] Figure 2 Figure 2 is an electrophoresis identification diagram (A) and a juvenile fish morphology diagram (B) of ADOa gene knockout F0 generation zebrafish.

[0057] Figure 3 Figure 3 is an electrophoresis identification diagram (A) and a juvenile fish morphology diagram (B) of ADOa gene knockout F1 generation zebrafish.

[0058] Figure 4 Figure 4 is an electrophoresis identification diagram (A) and a juvenile fish morphology diagram (B) of ADOa gene knockout F2 generation homozygous mutant zebrafish.

[0059] Figure 5 Figure 5 is a high-resolution melting curve of different zebrafish. The black curve represents wild type (+ / +) zebrafish, the red curve represents ADOa knockout (- / -) zebrafish, and the blue curve represents ADOa heterozygous (+ / -) zebrafish. DETAILED DESCRIPTION

[0060] The content of the present application is further described in detail through specific examples. The raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial channels or can be obtained by existing technical methods, unless otherwise specified. Unless otherwise specified, the test or test method is a conventional method in the art.

[0061] Example 1

[0062] This example provides a method for constructing an ADO gene deletion zebrafish model. The specific construction steps include:

[0063] (1) Design of zebrafish ADO gene targeting knockout system sgRNA:

[0064] ADO gene in zebrafish is a protein coding gene, and there are two subtypes of ADOa and ADOb. Among them, ADOa is located on chromosome 12 of zebrafish, and there is 1 reference transcript. In this embodiment, the target for knockout is located in the protein coding region (Coding sequence, CDS) of the ADO gene.

[0065] The target sequence of the sgRNA is: 5'-GGGGTGTTTCTCCTGAAATC-3' (SEQ ID NO: 1).

[0066] The PCR amplification primers of the sgRNA (for amplifying it by PCR to connect it to the plasmid vector) are:

[0067] The forward amplification primer of the sgRNA is: 5'-TTCGGTCCCTCCAGTCACTT-3' (SEQ ID NO: 2).

[0068] The reverse amplification primer of the sgRNA is: 5'-GCTGAAGGTCCTGTACGGTA-3' (SEQ ID NO: 3).

[0069] The following oligonucleotide chains Oligo 1-2 are synthesized according to the nucleotide sequence composition by using the conventional means in the art (such as solid-phase synthesis).

[0070] Oligo 1: 5'-TAGGGGTGTTTCTCCTGAAATC-3' (SEQ ID NO: 4).

[0071] Oligo 2: 5'-AAACGATTTCAGGAGAAACACC-3' (SEQ ID NO: 5).

[0072] 10 μL of 10 μM Oligo 1 and Oligo 2 are respectively taken by a pipette into a 1.5 mL centrifuge tube, 80 μL of ddH2O is added, and it is mixed by vortexing gently, centrifuged briefly to make all the liquid at the bottom of the centrifuge tube, and then the centrifuge tube is placed in a 95℃ water bath for heating for 10 min, and annealed naturally to obtain double-stranded DNA containing sticky ends TAGG and AAAC, which is the transcript of the sgRNA.

[0073] (2) Preparation of the sgRNA expression cassette:

[0074] The commercially available pDR274 plasmid is subjected to restriction cleavage by using Bsa I enzyme according to the system shown in the following table.

[0075] Table 1 Enzyme cleavage system

[0076]

[0077]

[0078] The reaction temperature is 37℃, and the reaction time is 60 min, to obtain the cleavage product.

[0079] The double-stranded DNA obtained in the above step was connected with the linear pDR274 plasmid digested by Bsal enzyme using T4 ligase, and then PCR amplification was performed in the system shown in the following table, with the digested product obtained in the above step as a template, to obtain a recombinant pDR274-sgRNA expression vector (i.e., sgRNA expression cassette).

[0080] Table 2 PCR amplification system

[0081] Component Amount 2x Accurate Taq Master Mix (dye plus) 12.5 μL Template 1 μL Upstream primer (Oligo 1 in the above step) 1 μL Downstream primer (1861_m13) 1 μL RNase-free water 9.5 μL Total volume 25 μL

[0082] The downstream primer 1861_m13 is 5'-GTAAAACGACGGCCAGT-3' (SEQ ID NO: 6).

[0083] The PCR amplification reaction program is shown in the following table.

[0084] Table 3 PCR amplification reaction program

[0085]

[0086] The recombinant pDR274-sgRNA expression vector was transformed into E. coli cells DH5a, and a single positive clone containing the recombinant plasmid was screened by colony PCR. After sequencing verification, it was ready for use.

[0087] The plasmid was extracted from the positive clone using the E.Z.N.A. Endo-free Plasmid Mini Kit II kit, and the extraction method was referred to the instruction or performed according to the following steps:

[0088] The ETR solution, N3 buffer and solution I in the kit were pre-placed on ice. The recombinant E. coli positive clone obtained in the above step was inoculated into 20 mL of resistant medium at a volume ratio of 1:1000, 37°C, 170 rpm shaking for 12-16 h, then centrifuged at 5000g for 10 min at room temperature, and the supernatant was discarded.

[0089] 500 μL of solution I (4°C, pre-added with RNase A) was added to the precipitated bacteria, and the bacterial solution was blown and resuspended thoroughly, then transferred to a 2 mL centrifuge tube. 500 μL of solution II (from the kit) was added, and the solution was mixed gently until it became clear, and then it was placed at room temperature for 3 min. 250 μL of N3 buffer was added, and the tube was gently inverted several times until a white flocculent precipitate appeared, and then it was centrifuged at 13000g for 10 min at 4°C under pre-cooling conditions.

[0090] The supernatant after centrifugation was carefully aspirated and transferred to a new 1.5 mL centrifuge tube, and 0.1 times the volume of ETR solution was added to each tube. After mixing by blowing, it was placed on ice for 10 min and heat shocked at 42°C for 5 min.

[0091] Centrifuge at 13000g for 5 min at room temperature, take the supernatant to a new 2 mL centrifuge tube, add 0.5 times volume of ethanol, invert 6-7 times to mix. Add the mixed solution to the collection tube of the column in the kit, add 700 μL of mixed solution each time, centrifuge at 13000g for 1 min, discard the filtrate. Add 500 μL of HBC buffer to the column, centrifuge at 13000g for 1 min, discard the filtrate. Add 700 μL of DNA cleaning buffer to the column, centrifuge at 13000g for 1 min, discard the filtrate. Repeat the cleaning step once. Centrifuge at 13000g for 1 min again, place the column in a 1.5 mL centrifuge tube, add 80-100 μL of enzyme-free water, stand at room temperature for 1 min, centrifuge at 13000g for 3 min to obtain the plasmid solution.

[0092] Use an ultraviolet spectrophotometer to determine the concentration and purity of the plasmid in the solution, and store it in a -20°C refrigerator for standby use.

[0093] According to the system shown in the following table, use the obtained plasmid as a template, and use high-fidelity DNA polymerase KOD FX to perform PCR amplification of the template.

[0094] Table 4 KOD FX-based amplification system

[0095] Component Amount 2x PCR buffer for KOD FX 25 μL 2 mM dNTPs 10 μL Primer 3111_gRNA 1.5 μL Primer 2134_M13 1.5 μL Template About 50 ng KOD FX 1 μL Autoclaved water Added to 50 μL

[0096] The sequence of primer 3111_gRNA is 5'-TTTAAAAGCACCGACTCGGTGCCAC-3' (SEQ ID NO: 9); the sequence of primer 2134_M13 is 5'-CTCAGGAAACAGCTATGACA-3' (SEQ ID NO: 10). The PCR amplification reaction program is shown in the following table.

[0097] Table 5 PCR amplification reaction program

[0098]

[0099]

[0100] The PCR amplification product is the target fragment. The target fragment is cut under ultraviolet light, and the E.Z.N.A. Gel Extraction Kit is used for gel recovery. The recovery method is referred to the instruction for use, or the following steps are used:

[0101] According to 1 g / mL gel block density per 1.5 mL centrifuge tube, add 1 volume of binding buffer (XP2) to each tube, incubate at 50-60°C for 7 min until the gel block completely disappears, during which, vortex / shake the centrifuge tube every 2-3 min, and after the gel block completely disappears, cool the solution to room temperature. Transfer the solution to the assembled HiBind DNA Mini collection tube, centrifuge at 13000 g for 1 min at room temperature, and discard the filtrate. Add 300 μL of binding buffer (XP2) to the precipitate, centrifuge at 13000 g for 1 min at room temperature, and discard the filtrate. Add 700 μL of SPW washing buffer to the precipitate, centrifuge at 13000 g for 1 min at room temperature, discard the filtrate, and repeat once. Empty spin the centrifuge column at 13000 g for 2 min at room temperature. Perform a 56°C metal bath. Transfer the centrifuge column to a new 1.5 mL tube, add 30-50 μL of elution buffer or deionized water to the center of the column membrane, stand at room temperature for 2 min, and centrifuge at 13000 g for 1 min to elute again, and the solution of the target fragment is obtained.

[0102] Again, use KOD FX to amplify according to the above procedure, and then use the E.Z.N.A. Cycle Pure Kit kit to recover, or use the following procedure:

[0103] Transfer the amplification product to a 1.5 mL centrifuge tube, add 4-5 volumes of CP buffer (if the amplification product is <200 bp, add 5 volumes of CP buffer and 0.4 volumes of 100% isopropanol). Vortex thoroughly to mix the liquid, and centrifuge briefly to spin all the liquid to the bottom of the tube. Transfer it to the assembled HiBind DNA Mini collection tube, centrifuge at 13000 g for 1 min at room temperature, and discard the filtrate. Add 700 μL of DNA washing buffer to the precipitate, centrifuge at 13000 g for 1 min, and discard the filtrate. Repeat the washing step once. Empty spin at 13000 g for 2 min, and perform a 56°C metal bath for 2 min. Transfer the centrifuge column to a new 1.5 mL tube, add 30-50 μL of elution buffer (TE buffer or sterilized deionized water can also be used) to the center of the column membrane, stand at room temperature for 2 min (a 56°C metal bath for 2 min can also be used to speed up elution). Centrifuge at 13000 g for 1 min to elute again, and the solution of the target fragment is obtained.

[0104] (3) In vitro transcription:

[0105] Use the MEGAscript T7 Transcription Kit kit, and perform in vitro transcription according to the system shown in the following table, using the target fragment obtained in step (2) as a template.

[0106] Table 6 In vitro transcription system

[0107]

[0108]

[0109] Specifically, RNAPolymerase Enzyme Mix was placed on ice in advance, and 10x reaction buffer and NTP (ATP, CTP, GTP and UTP) were vortexed respectively until completely dissolved. The vortexed 10x reaction buffer was placed at room temperature to raise the temperature to room temperature, while the NTP solution was placed on ice. Before use, all the liquid of each reagent above was spun to the bottom of the tube. After mixing the components according to the table above, the PCR tube was flicked or gently mixed with a pipette gun, and the whole liquid was spun to the bottom of the tube after a short centrifugation. Incubate at 37℃ for 4-6h. After incubation, add 50μL LiCl precipitation solution to terminate the reaction and precipitate the RNA, vortex to mix, spin the whole liquid to the bottom of the tube, and incubate at -20℃ overnight. Centrifuge at 12000rpm for 15min at 4℃ to form a pellet. Remove the supernatant, wash the pellet once with 1mL of 70% ethanol (75% can also be used). Centrifuge at 12000rpm for 15min at 4℃ to remove unbound nucleotides. Remove the ethanol and air dry the RNA pellet. Add 20-30μL of nuclease-free water to resuspend, vortex quickly, and spin the whole liquid to the bottom of the tube after a short centrifugation. The sgRNA is obtained, and after quantification, it is stored at -20℃ or -80℃ for future use.

[0110] (4) Microinjection:

[0111] The sgRNA (45ng / μL) obtained in the above steps and commercial Cas9 protein (New England Biolabs, USA) were co-microinjected into wild-type zebrafish embryos at the 1-cell stage. After successful injection, genomic DNA was extracted by alkaline lysis method to confirm whether the editing was successful. The specific extraction steps are as follows: select several injected wild-type zebrafish embryos, add 30-50μL NaOH (50nM) solution, and react at 95℃ for 10min until the tissue is completely dissolved. After cooling at room temperature, 3-5μL Tris-HCl (pH 8.0) is added to neutralize the reaction system. The neutralized reaction system is centrifuged at 12000rpm for 1min, and the supernatant (i.e. zebrafish genomic DNA) is used as the PCR template for genotype identification. Use SEQ ID NO: 2 and 3 as PCR forward and reverse primers to amplify the template by PCR to confirm the editing, and the PCR amplification system is shown in the table below.

[0112] Table 7 PCR amplification system

[0113] Component Amount Zebrafish genomic DNA 1 μL 2x T5 Super PCR Mix (Nanjing Keygen Biotech Co., Ltd.) 10 μL SEQ ID NO: 2 forward primer (10 μM) 1 μL SEQ ID NO: 3 reverse primer (10 μM) 1 μL ddH2O To 20 μL

[0114] The PCR amplification reaction program is shown in the table below.

[0115] Table 8 PCR amplification reaction program

[0116]

[0117] The same batch of other embryos showing a messy peak in the melting curve were raised to sexual maturity as parents to breed the next generation. The next generation of embryos were screened by the above PCR method to detect and screen zebrafish of effective mutation types, thereby obtaining ADO gene mutant zebrafish. The obtained ADO gene mutant zebrafish were crossed with wild-type zebrafish to produce heterozygous mutant (+ / -), and then the mutation type was determined by sequencing. Then based on the sequencing results, the heterozygous zebrafish (+ / -) of the same promoter fragment deletion mutation type were selected for self-crossing to produce homozygous offspring (- / -), thereby obtaining homozygous ADO gene mutant zebrafish with known mutation type.

[0118] (5) Genotype identification:

[0119] In order to further clarify the genotype of the obtained homozygous ADO gene mutant zebrafish, the inventors extracted genomic DNA from the tail fin samples of wild-type and homozygous ADO gene mutant zebrafish using the above alkaline lysis method, and performed PCR amplification detection using SEQ ID NO: 2 and 3 as PCR forward and reverse primers based on the above PCR amplification system. High resolution melting curve analysis (HRMA) was used to genotype the amplification products. According to the melting curve differences of wild-type zebrafish, heterozygous mutant zebrafish and homozygous mutant zebrafish obtained in the above steps, different mutation types were distinguished. For genotypes that are difficult to distinguish by HRMA, non-denaturing PAGE gel electrophoresis was used to process the amplification products, and then heteroduplex mobility analysis (HMA) was performed, and further distinguished according to the bands.

[0120] The results are shown in Figure 1-5 .

[0121] Figure 1 Figure 1 is a diagram of CRISPR / Cas9 target analysis of zebrafish ADOa gene. The underlined part represents the sgRNA targeting site, the red labeled part represents the mutant sequence, and the "-" represents the deleted base after editing. From the results, it can be known that the F1 generation heterozygous (+ / -) mutant of ADOa gene knockout was successfully constructed.

[0122] Figure 2The electrophoresis identification diagram and adult fish morphology diagram of the F0 generation of ADOa gene knockout zebrafish are shown in the figure, and it can be found that the amplification band of the ADOa+ / - type zebrafish (i.e. heterozygote) is consistent with the expectation, which indicates that the ADOa+ / - type zebrafish is successfully constructed, and the F0 adult fish morphology of the ADOa+ / - type zebrafish is consistent with the wild type without obvious difference.

[0123] Figure 3 The electrophoresis identification diagram and adult fish morphology diagram of the F0 generation of ADOa gene knockout zebrafish are shown in the figure, and it can be found that the amplification band of the ADOa+ / - type zebrafish (i.e. heterozygote) is consistent with the expectation, which indicates that the ADOa+ / - type zebrafish is successfully constructed, and the F0 adult fish morphology of the ADOa+ / - type zebrafish is consistent with the wild type without obvious difference.

[0124] Figure 4 The electrophoresis identification diagram and adult fish morphology diagram of the F0 generation of ADOa gene knockout zebrafish are shown in the figure, and it can be found that the amplification band of the ADOa+ / - type zebrafish (i.e. heterozygote) is consistent with the expectation, which indicates that the ADOa+ / - type zebrafish is successfully constructed, and the F0 adult fish morphology of the ADOa+ / - type zebrafish is consistent with the wild type without obvious difference.

[0125] Figure 5 The high-resolution melting curve of different zebrafish is shown in the figure, wherein the black curve represents the wild type (+ / +) zebrafish, the red curve represents the ADOa knockout type (- / -) zebrafish, and the blue curve represents the ADOa heterozygote (+ / -) zebrafish.

[0126] In summary, the ADOa knockout type (- / -) zebrafish is successfully constructed, which can be used as an effective animal model to provide support for the research on the double oxygenase drug screening and taurine metabolism model.

[0127] Example 2

[0128] In this embodiment, the ADOa knockout type (- / -) zebrafish is constructed according to the above-mentioned steps in Example 1, and is used together with the wild type for ADO related pathway research, and it is found that the difference in response to pathogenic stimulation can be exhibited based on the difference in the regulation of the related gene pathways of the wild type zebrafish, so as to be used as an effective zebrafish research model.

[0129] The above-mentioned embodiments are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above-mentioned embodiments, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A method for constructing an ADO gene deletion model, comprising the following steps: Knockout or silence of the ADO gene in an organism; Preferably, the ADO gene is the ADOa gene; Preferably, the target sequence of the ADO gene is shown in SEQ ID NO:

1.

2. The construction method according to claim 1, characterized in that, The knockout or silencing includes knockout or silencing using at least one of the following gene editing techniques or physicochemical mutagenesis techniques: RNA interference, plasmid-mediated gene knockout, embryonic stem cell knockout, CRISPR system, transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs).

3. The construction method according to claim 2, characterized in that, The construction method uses a CRISPR system for knockout, where the transcript of the gRNA in the CRISPR system is obtained by annealing the following oligonucleotide chains: 5'-GGTGTTTCTCCTGAAATC-3' (SEQ ID NO: 7) and 5'-GATTTCAGGAGAAACACC-3' (SEQ ID NO: 8).

4. The construction method according to claim 3, characterized in that, The transcript is also attached to a sticky end; Preferably, the adhesive end comprises at least one of 5'-TAGG-3' and 5'-AAAC-3'.

5. The construction method according to claim 4, characterized in that, The viscous ends are obtained by at least one of the following methods: (1) Obtained by end-repair of oligonucleotide chain annealing products; or (2) Annealing after attaching sticky ends to at least one oligonucleotide chain; Preferably, the sticky ends are obtained by annealing after attaching sticky ends to two oligonucleotide chains.

6. The construction method according to claim 1, characterized in that, The organisms include aquatic animals; Preferably, the organism is a fish; Preferably, the organism is a zebrafish.

7. The application of the ADO gene deletion model constructed by the construction method according to any one of claims 1-6 in drug screening.

8. The application according to claim 7, characterized in that, The drugs include cysteine ​​(2-aminoethanethiol) dioxygenase (ADO) targeted drugs or taurine metabolism pathway targeted drugs.

9. The application according to claim 7, characterized in that, The drug has at least one of the following functions: (1) Improve fish growth rate and / or weight gain rate; (2) Enhance fish immunity; (3) Improve the environmental adaptability of fish; and (4) Increase the amount of food consumed by fish.

10. The application according to claim 7, characterized in that, The drug is a veterinary drug; preferably, the veterinary drug is a drug for aquatic animals.

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