Method for creating fluorescent zebra fish based on enhancer of muscle specific gene

Abstract

The invention discloses a method for creating fluorescent zebra fish based on an enhancer of a muscle specific gene. The invention provides a method for creating fluorescent zebrafish, which comprises the following steps: by taking an upstream promoter and an enhancer of a fmyhc2.2 gene as target sequences, amplifying the upstream promoter and the enhancer and seamlessly connecting the upstream promoter and the enhancer with a fluorescent protein carrier to form a recombinant plasmid, carrying out microinjection on a zebrafish embryo, selecting F0 generation individuals capable of driving fluorescent protein expression, and culturing until sexual maturity, thereby obtaining the fluorescent zebrafish. Selecting male and female individuals for selfing to obtain F1 generation, observing and selecting individuals with strong fluorescence from F1 generation embryos through a fluorescence microscope, and culturing the individuals with strong fluorescence to sexual maturity to obtain the transgenic zebrafish with remarkably discolored skin. The fluorescent zebra fish is created by utilizing the enhancer of the muscle specific gene, an innovative method is provided for creating a zebra fish ornamental strain, a support is provided for researching a regulation mechanism of zebra fish muscle related genes, and the fluorescent zebra fish has important scientific value and industrial application value.

Description

Technical Field

[0001] This invention relates to the field of aquatic organism breeding technology, and more specifically, to a method for creating fluorescent zebrafish based on enhancers of muscle-specific genes. Background Technology

[0002] With the development of genetic engineering technology, transgenic model organisms have become important tools for functional gene screening, developmental biology research, and drug screening. Among them, zebrafish (Danio rerio) has become a widely used model organism due to its advantages such as rapid embryonic development, high transparency, and ease of gene manipulation. To achieve stable expression of target genes, constructing transgenic zebrafish has become a routine technique in zebrafish research.

[0003] In recent years, with the advancement of gene editing and transgenic technologies, the methods for constructing zebrafish models have been continuously optimized. The main methods for constructing transgenic zebrafish include the Tol2 transposon system, CRISPR / Cas9-mediated gene editing technology, electroporation transfection technology, and the pIGLET safe harbor landing site system. Among these, the Tol2 transposon system is a highly efficient tool widely used in zebrafish gene manipulation. Originally derived from killifish, the Tol2 transposon's "cutting-insertion" characteristic allows DNA carrying the target sequence to be stably integrated into the host genome, offering advantages such as high integration efficiency of the target fragment, fewer insertion sites, and more stable gene expression. To achieve tissue-specific expression, the selection of promoters and enhancers is crucial. Promoters are key elements regulating gene transcription, while enhancers improve expression efficiency. In zebrafish, several muscle-specific promoters have been identified and used for muscle development, disease research, and the construction of transgenic models, such as... mylz2 , mylpfa , α- actin These elements can drive the specific expression of fluorescent proteins (such as EGFP, mCherry, etc.) in skeletal muscle, thereby enabling the visualization of muscle tissue and having significant research value.

[0004] However, most fluorescent zebrafish based on muscle-specific expression are currently used for research purposes, with related studies focusing primarily on functional validation or disease modeling, such as for cardiac regeneration research, neuroscience research, and disease model construction. Their potential for creating ornamental fish has not been fully explored. Few studies have utilized muscle-specific enhancers to construct stably heritable, clearly fluorescent transgenic zebrafish strains. Therefore, how to construct a fluorescently colored zebrafish strain with high ornamental value and stable heritability has become an urgent problem to be solved in this field. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of existing transgenic zebrafish strains that can be stably inherited and have clear fluorescence expression. The present invention provides a method for creating fluorescent zebrafish based on enhancers of muscle-specific genes.

[0006] The first objective of this invention is to provide a method for creating fluorescent zebrafish based on enhancers of muscle-specific genes.

[0007] A second objective of this invention is to provide an application of the method.

[0008] The third objective of this invention is to provide zebrafish fmyhc2.2 Application of gene promoters and muscle-specific enhancers in the preparation of fluorescent transgenic zebrafish.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a method for creating fluorescent zebrafish based on enhancers of muscle-specific genes, comprising the following steps: S1. Amplification of the target fragment: [The following text appears to be incomplete and requires further context: "to ampl fmyhc2.2 Gene promoter and muscle-specific enhancer fragments are amplified to obtain the target fragment containing the enhancer or promoter; fmyhc2.2 The gene promoter sequence is shown in SEQ ID NO: 1, and the muscle-specific enhancer sequence is shown in SEQ ID NO: 2; S2. Constructing the plasmid expression cassette: The target fragment containing the enhancer or promoter is seamlessly ligated into an empty vector backbone containing a fluorescent protein fragment for homologous recombination to obtain pminiTol2-enhancer- fmyhc2.2 -p4100-Fluorescent protein particle expression cassette; S3. Microinjection: The plasmid expression cassette was mixed with the Tol2 transposon to obtain a recombinant plasmid system, which was then microinjected into zebrafish embryos; S4. Zebrafish breeding and screening: After injection, fluorescent embryos are selected, and after culture, F0 generation male and female individuals that can drive fluorescent protein expression are selected for mating to obtain F1 generation fluorescent zebrafish with heritable skin coloration.

[0010] This invention utilizes Tol2 transposase to convert zebrafish... fmyhc2.2 Fluorescent protein particles containing promoter fragments and muscle-specific enhancer fragments were injected into zebrafish single-cell stage fertilized eggs, and cultured and screened to obtain transgenic zebrafish strains with visible fluorescence, exhibiting strong muscle specificity and high expression efficiency. Studies have shown that using specific... fmyhc2.2Only promoter fragments and muscle-specific enhancer fragments can drive the stable and efficient expression of fluorescent proteins in skeletal muscle, achieving a clearly visible muscle coloration effect, avoiding problems such as non-specific expression or irregular expression patterns, and improving visual appeal. The fluorescent zebrafish constructed using the method of this invention exhibits good genetic stability, and the target trait can be stably inherited to offspring, making them suitable for long-term breeding, display, and further breeding.

[0011] Preferably, the fluorescent protein in S2 is selected from one or more of EGFP, tdTomato, BFP, and YFP. In particular, based on the method of the present invention, various fluorescent transgenic zebrafish can be created using fluorescent proteins of different colors.

[0012] Preferably, the homologous recombination method in S2 is as follows: amplify the homologous arms of the target fragment and the linearized vector, perform seamless ligation, transform, and select bacteria to obtain the plasmid expression cassette.

[0013] Preferably, the hollow carrier in S2 is selected from one of pminiTol2, pT2KXIGΔin, and pT2AL200R150G.

[0014] More preferably, the hollow carrier in S2 is pminiTol2.

[0015] More preferably, the fluorescent protein empty vector in S2 is pminiTol2-EGFP / tdTomato.

[0016] Preferably, homologous recombination in S2 is seamlessly ligated using the ClonExpress Ultra One Step Cloning Kit V2.

[0017] More preferably, the final concentration of the plasmid expression cassette used in S3 is 50-100 ng / µL; and the final concentration of the Tol2 transposon is 200-400 ng / µL.

[0018] Preferably, S3 is microinjected into zebrafish I-cell stage fertilized eggs.

[0019] Preferably, the microinjection volume in S3 is 1-2 nL.

[0020] This invention provides the application of the above method in the preparation of fluorescently colored transgenic zebrafish.

[0021] This invention also provides zebrafish fmyhc2.2 Application of gene promoters and muscle-specific enhancers in the preparation of fluorescent transgenic zebrafish.

[0022] The present invention has the following beneficial effects: This invention provides a method for creating fluorescent zebrafish based on enhancers of muscle-specific genes, in order to... fmyhc2.2 The upstream promoter and enhancer of the gene are the target sequences. These sequences are amplified and combined with a fluorescent protein vector to form a recombinant plasmid. The fluorescent protein gene fragment is integrated into the zebrafish genome using the Tol2 transposon system. Zebrafish embryos are microinjected with the fluorescent protein, and F0 generation individuals capable of driving fluorescent protein expression are selected and cultured to sexual maturity. Male and female individuals are self-crossed to obtain F1 generation. In the F1 generation embryos, individuals with strong fluorescence are observed under a fluorescence microscope and selected. These individuals with strong fluorescence are then cultured to sexual maturity to obtain transgenic zebrafish with significantly changed skin color. This invention utilizes the enhancer of a specific muscle-specific gene to create fluorescent zebrafish, driving stable and efficient expression of the fluorescent protein in skeletal muscle, achieving a clearly visible muscle coloration effect. This avoids problems such as non-specific expression or irregular expression patterns, improving ornamental value. It possesses good genetic stability, and the target trait can be stably inherited to offspring, making it suitable for long-term breeding, display, and further breeding. This invention provides an innovative method for creating ornamental zebrafish strains and supports research on the regulatory mechanisms of zebrafish muscle-related genes, possessing significant scientific and industrial application value. Attached Figure Description

[0023] Figure 1 The results of H3K27ac and H3K4me1 signal analysis for ChIP-seq and ATAC-seq data are shown in the figure.

[0024] Figure 2 for fmyhc2.2 A schematic diagram of promoter nested PCR amplification (a) and a schematic diagram of the recombinant plasmid constructed in this invention (b).

[0025] Figure 3 The F0 generation fluorescent zebrafish embryo (a) and adult fish (b) constructed for this invention.

[0026] Figure 4 The F1 generation fluorescent zebrafish embryo (a) and adult fluorescent zebrafish (b) constructed for this invention are shown.

[0027] Figure 5 For no enhancer fmyhc2.2 Transgenic zebrafish F0 generation embryos (a) and adult fish (b), and cFos F0 generation embryos (c) and adult fish (d) with minimal promoter + enhancer. Detailed Implementation

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0029] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0030] The wild-type TUB strain zebrafish used in the following examples were purchased from the China Zebrafish Resource Center (CZRC). The embryos used for microinjection were obtained from the natural mating and spawning of sexually mature male and female parents.

[0031] Example 1: Construction of a recombinant plasmid for a muscle-specific gene 1. fmyhc2.2 Selection and amplification of upstream promoter and enhancer regions Based on ChIP-seq and ATAC-seq data, it was found that the H3K27ac and H3K4me1 signals are in fmyhc2.2 The ATAC-seq peak region upstream of the gene is highly enriched, suggesting it may be an enhancer, such as... Figure 1 As shown. Verification has shown that it can enhance... fmyhc2.2 The expression of this signal is used as a muscle-specific enhancer, with its sequence position being Chr5 32187149-32189255, and the specific sequence is shown in SEQ ID NO: 2.

[0032] Amplify separately fmyhc2.2 Upstream gene promoters and muscle-specific enhancers fmyhc2.2 The sequence of the gene promoter is shown in SEQ ID NO: 1, and the sequence position is: Chr5 32218197-32222297. fmyhc2.2 The gene promoter was amplified using nested PCR; the target fragment was 4.4 kb. Figure 2 As shown in Figure a, the primer sequences used for amplification are shown in Table 1. The PCR system consisted of: 10 μL of 5× PrimeSTAR GXL Buffer, 4 μL of dNTP Mixture, 1 μL of upstream primer, 1 μL of downstream primer, 1 μL of template DNA, 2 μL of PrimeSTAR GXL DNA Polymerase, and sterile deionized water to a final volume of 50 μL. fmyhc2.2 The gene promoter nested PCR reaction conditions were as follows: 94℃ pre-denaturation for 1 min, 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 68℃ extension (first round extension for 10 min, second round extension for 70 s, third round extension for 40 s), 35 cycles; and a final extension at 72℃ for 5 min. The first-round product was amplified using the wild-type zebrafish genome as a template, the second-round product was amplified using the first-round product as a template, and the third-round product was amplified using the second-round product as a template.

[0033] Table 1 PCR amplification primers

[0034] The enhancer was directly amplified using wild-type zebrafish DNA as a template. The target fragment was 2kb, and the PCR reaction system was the same as above. The PCR reaction conditions for enhancer amplification were: 94℃ pre-denaturation for 1 min, 98℃ denaturation for 10 s, 58℃ annealing for 15 s, 68℃ extension for 2 min, for 35 cycles; and a final extension at 72℃ for 5 min.

[0035] enhancer PCR products and fmyhc2.2 After verifying the correct band size by agarose gel electrophoresis of the three gene promoter products, the products were purified and recovered to obtain the target fragments containing enhancers and promoters. After measuring the concentration, the recovered products were sent to Qingke Company, where bidirectional sequencing was performed using selected upstream and downstream primers. After confirming that the sequences were correctly aligned with existing sequences on the NCBI website, they were subsequently ligated to blunt-end vectors for homologous arm amplification.

[0036] 2. Construction of recombinant plasmids (1) Construction of plasmid expression cassette The fluorescent proteins used are EGFP (green fluorescent protein) and tdTomato (red fluorescent protein), which are characterized by strong luminescence, low toxicity, and clear imaging. Similarly, other different types of fluorescent proteins can be used in the art to construct various transgenic lines in this invention.

[0037] The basic structure of the plasmid expression cassette is: [muscle-specific enhancer]-[muscle-specific promoter]-[fluorescent protein coding sequence]-[polyA signal]. After amplifying the fragments of the enhancer and promoter, they are homologously recombined with the empty vector of pminitol2-EGFP / tdTomato fluorescent protein through seamless ligation.

[0038] First, homologous arm primers were designed based on the plasmid map. The primer sequences used for homologous arm amplification are shown in Table 2. PCR amplification was performed using the plasmid with blunt ends as a template. The reaction system was as follows: 25 μL of Ex Premier, 1 μL each of forward and reverse primers F and R, 1 μL of plasmid template, and sterile water to a final volume of 50 μL.

[0039] Table 2. Primers for homologous arm amplification

[0040] The PCR program was as follows: 94℃ pre-denaturation for 1 min, 98℃ denaturation for 10 s, 60℃ annealing for 15 s, 68℃ extension (30 s / kb), 35 cycles; final extension at 68℃ for 5 min. After amplification, 1 μL of Speedy Cut DpnI fast digester (BBI, China) was added, and the mixture was digested at 37℃ for 1 h to remove residual template plasmid. The PCR products were then recovered using a DNA purification kit (Omega, USA), and the concentration was determined before use.

[0041] The homologous recombination reaction system was then prepared using the ClonExpress Ultra One Step Cloning Kit V2 (Vazyme, China): linearized vector (volume calculated as 0.02 × vector base pairs / concentration), insert fragment (0.04 × fragment base pairs / concentration), 2 × ClonExpress Mix 5 μL, and ddH2O to a final volume of 10 μL. After mixing and brief centrifugation, the mixture was incubated at 50°C for 5 min in a PCR instrument. Immediately after incubation, the mixture was placed on ice to cool, followed by competent cell transformation, plate culture, and overnight incubation at 37°C. The next day, single colonies were selected and amplified in 2 mL of ampicillin broth. Plasmids were extracted using the alkaline cleavage method, and enzyme digestion was performed to verify the band size. Plasmids suspected of successful recombination were sent to KINK Technology for sequencing. The plasmid expression cassette was located in the center of the Tol2 transposon element, as shown in the image. Figure 2 As shown in b, enhancer and promoter fragments in the expression cassette were measured and compared. After correct comparison, 500 μL of positive bacterial culture was inoculated into 10 mL of ampicillin liquid medium and amplified overnight in a shaker at 37 °C. Finally, plasmid was extracted using an endotoxin-free plasmid mini-extraction kit (TIANGEN, China), and the concentration was determined before storage at -20 °C for later use.

[0042] (2) Synthesis of Tol2 transposon mRNA Tol2 transposon mRNA was synthesized via in vitro transcription: The T7pase plasmid was linearized using NotI and BamHI restriction endonucleases, followed by incubation with proteinase K at 50°C for 30 min to remove residual RNase and other transcriptional repressors. The mRNA was then purified and recovered using the phenol-chloroform-isoamyl alcohol extraction method, and its concentration was measured and electrophoresis performed. Transcription was performed at 37°C for 4 h using an in vitro transcription kit (Thermofisher, USA), followed by sample loading to verify transcription efficiency. RNA was purified using lithium chloride precipitation, and after concentration measurement, it was aliquoted into 10 μL tubes and stored at -80°C.

[0043] Subsequently, a plasmid expression cassette was inserted between the left and right arms of the Tol2 transposon to construct a recombinant plasmid. The constructed recombinant plasmid was finally confirmed by the company's sequencing verification.

[0044] Example 2: Construction of transgenic zebrafish Adult wild-type zebrafish (WT / TUB) were fed for about an hour the night before mating. They were then removed from the rearing tank at a 2:2 female-to-male ratio and placed in the breeding tank, separated by a partition and covered with a light-blocking plate to isolate them from light. At 9:00 AM the next morning, the light-blocking plate was removed, and the partition was taken off. The male and female fish then freely mated in the tank, with the female laying eggs and the male releasing sperm. The sperm and eggs combined in the water to form fertilized eggs. Single-celled fertilized eggs (within 30 minutes of ovulation) were selected for microinjection. The transient expression of fluorescent protein was observed after injecting the recombinant plasmid prepared in Example 1. The specific procedure was as follows: the total volume for microinjection was 4 μL, containing: Tol2 enzyme 400 ng / μL, recombinant plasmid 50 ng / μL, phenol red 0.2 μL, and finally, enzyme-free water was added to bring the volume to 4 μL. Using the PUL-1000 needle puller, place the 1B100-4 capillary tube into the puller and secure both ends; set the program: Heat-574, Force-250, Distance-5.00, Delay-0. Click START to complete the needle pull. The needle is ready for use after the bevel is formed.

[0045] Subsequently, injection was performed using a PV860 pneumatic picoliter perfusion system. An embryo fixation mold was prepared beforehand using 3% agarose gel, with a volume of 40 mL pure water and 1.2 g agarose. The mixture was heated until the agarose was completely dissolved, poured onto a plate, and the mold was placed inside. After cooling, the mold was carefully removed. Zebrafish fertilized eggs were placed in the mold, and a small amount of E3 medium-methylene blue culture medium was added to prevent dehydration. Under a microscope, the needle was cut open, and a capillary tube was installed at the tip of the syringe. The fertilized egg was located under the microscope and injected from the vegetal pole to the animal pole, with an injection volume of 1-2 nL.

[0046] Twenty-four hours after fertilization, dead eggs were removed under high magnification, and the culture medium was replaced with fresh medium. Forty-eight hours after fertilization, unruptured embryos were demembraned and observed under a fluorescence microscope. The expression of fluorescent protein in the embryonic trunk proved the recombinant plasmid's effectiveness. Subsequently, embryos with strong fluorescence were selected, such as... Figure 3 As shown in a, the zebrafish were placed in a recirculating aquaculture system for culture 7 days after fertilization.

[0047] Example 3: Screening of transgenic fish lines Following microinjection as described in Example 2, F0 generation zebrafish cultured at approximately two months of age exhibited irregular skin coloration, such as... Figure 3 As shown in b, the inserted fragment has already been integrated into its genome, suggesting that the discolored individuals may pass this trait on to the next generation. These discolored individuals were selected and fed individually to accelerate growth. After reaching sexual maturity at 3 months, male and female individuals were selected for self-fertilization. Similarly, fluorescent embryos were selected 48 hours after fertilization. Figure 4As shown in figure a, the fluorescent protein is expressed uniformly throughout the trunk and exhibits strong muscle specificity.

[0048] The fluorescent F1 embryos were further cultured, and at around two months of age, the F1 zebrafish showed a significant and uniform skin color change. Figure 4 As shown in b, the F1 generation of transgenic fish is a stably heritable line. The skin coloration rate of the F1 generation adult fish is 2 / 3 (n=30), which shows high expression efficiency.

[0049] Example 4: Transgenic zebrafish constructed with different muscle-specific genes 1. Alone fmyhc2.2 Promoter transgenesis Based on the construction method of Example 1, the enhancer fragment in the recombinant plasmid is removed, i.e., a separate... fmyhc2.2 After the promoter fragment was used to construct a plasmid expression cassette and mixed with Tol2 transposon mRNA, it was microinjected according to the method in Example 2, and zebrafish were screened and cultured. The results showed that using the promoter fragment alone... fmyhc2.2 The promoter cannot effectively drive the expression of fluorescent proteins, such as Figure 5 As shown in a; its adult fish will not exhibit coloration, such as Figure 5 As shown in b, the proof is... fmyhc2.2 The promoter needs to work in conjunction with the upstream enhancer to drive the expression of fluorescent proteins.

[0050] 2. cFos Promoter transgenesis Based on the construction method of Example 1, fmyhc2.2 Gene promoter replacement with mouse cFos Experiments were conducted using the smallest promoter. cFos The specific sequence is shown in SEQ ID NO: 3. cFos The minimal promoter contains only the TATA box and some core promoter elements required for basic transcription. It has a weak ability to drive gene expression on its own and produces only a very low fluorescence signal in the absence of enhancers, which is used to verify enhancer activity.

[0051] Insert the enhancer from Example 1 cFos Upstream of the promoter, it can significantly drive the expression of fluorescent proteins in the trunk, demonstrating that this enhancer is muscle-specific. Figure 5 As shown in c; however, the adult fish did not show any color development after cultivation, such as Figure 5 As shown in d, it indicates fmyhc2.2 The promoter + enhancer combination produces better results than fluorescent zebrafish. cFos Minimal promoter + enhancer.

[0052] Meanwhile, the present invention also employs other... fmyhcThe gene promoter was validated in experiments, which showed that the offspring emitted light, but the fluorescence was only visible under a fluorescence microscope and could not be observed directly with the naked eye. Only by using... fmyhc2.2 Promoter + enhancer enable offspring to emit light and the color rendering effect is visible to the naked eye. cFos The promoter plus enhancer makes the offspring glow, but the color-changing effect is not visible to the naked eye; only the promoter plus enhancer is used. fmyhc2.2 The promoter prevents the offspring from emitting light.

[0053] Example 5: A method for creating fluorescent zebrafish based on enhancers of muscle-specific genes This embodiment provides a method for creating fluorescent zebrafish based on enhancers of muscle-specific genes, including the following steps: S1. Amplification of the target fragment: amplified by nested PCR. fmyhc2.2 Upstream promoter of the gene, muscle-specific enhancer was directly amplified using wild-type zebrafish DNA as a template to obtain the target fragment containing enhancer and promoter respectively; S2. Construction of recombinant plasmids: The target fragment containing an enhancer or promoter is homologously recombined with the empty pminiTol2 vector containing fluorescent protein through seamless ligation to obtain a plasmid expression cassette; the plasmid expression cassette is then inserted between the left and right arms of the Tol2 transposon to construct the recombinant plasmid. S3. Microinjection: The recombinant plasmid was mixed with Tol2 transposon mRNA and then microinjected into zebrafish embryos at the I-cell stage. The final concentration of the plasmid was 50 ng / µL, and the final concentration of Tol2 enzyme was 200 ng / µL to drive fluorescent protein expression and gene fragment integration. S4. After injection, F0 generation male and female individuals that can significantly drive fluorescent protein expression are observed under a fluorescence microscope and mated to obtain F1 generation. Individuals with uniform distribution of fluorescent protein under a fluorescence microscope are heritable F1 generation fluorescent zebrafish. The skin of F1 generation fluorescent zebrafish changes significantly after sexual maturity.

[0054] In summary, this invention utilizes the Tol2 transposon system to construct a transgenic zebrafish strain with high expression efficiency and strong muscle specificity. By screening and optimizing the combination of muscle-specific enhancers and promoters, the fluorescent protein is driven to be expressed stably and efficiently in skeletal muscle, achieving a clearly visible muscle coloration effect. This avoids problems such as non-specific expression or irregular expression patterns, thus improving the aesthetic appeal. The constructed fluorescent zebrafish exhibits good genetic stability, and the target trait can be stably inherited by offspring, making it suitable for long-term rearing, display, and further breeding.

[0055] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for creating fluorescent zebrafish based on enhancers of muscle-specific genes, characterized in that, Includes the following steps: S1. Amplification of the target fragment: [The following text appears to be incomplete and requires further context: "to amplify the target fragment respectively..."] fmyhc2.2 Gene promoter and muscle-specific enhancer fragments are amplified to obtain the target fragment containing the enhancer or promoter; fmyhc2.2 The gene promoter sequence is shown in SEQ ID NO: 1, and the muscle-specific enhancer sequence is shown in SEQ ID NO: 2; S2. Constructing the plasmid expression cassette: The target fragment containing the enhancer or promoter is seamlessly ligated into an empty vector backbone containing a fluorescent protein fragment for homologous recombination to obtain pminiTol2-enhancer- fmyhc2.2 -p4100-Fluorescent protein particle expression cassette; S3. Microinjection: The plasmid expression cassette was mixed with the Tol2 transposon to obtain a recombinant plasmid system, which was then microinjected into zebrafish embryos; S4. Zebrafish breeding and screening: After injection, fluorescent embryos are selected, cultured, and F0 generation male and female individuals that can drive fluorescent protein expression are selected for mating to obtain F1 generation fluorescent zebrafish with heritable skin coloration.

2. The method according to claim 1, characterized in that, The fluorescent protein in S2 is selected from one or more of the following: green fluorescent protein (EGFP), red fluorescent protein (tdTomato), blue fluorescent protein (BFP), and yellow fluorescent protein (YFP).

3. The method according to claim 2, characterized in that, The hollow carrier in S2 is selected from one of pminiTol2, pT2KXIGΔin, and pT2AL200R150G.

4. The method according to claim 3, characterized in that, The empty vector for the fluorescent protein in S2 was pminiTol2-EGFP / tdTomato.

5. The method according to claim 1, characterized in that, The homologous recombination method in S2 is as follows: amplify the homologous arms of the target fragment and the linearized vector, perform seamless ligation, transform, and select bacteria to obtain the plasmid expression cassette.

6. The method according to claim 1, characterized in that, The final concentration of the plasmid expression cassette used in S3 is 50-100 ng / µL; the final concentration of the Tol2 transposon is 200-400 ng / µL.

7. The method according to claim 1, characterized in that, S3 was injected via microinjection into zebrafish I-cell stage fertilized eggs.

8. The method according to claim 1, characterized in that, The microinjection volume in S3 is 1-2 nL.

9. The application of the method according to any one of claims 1 to 5 in the preparation of fluorescently colored transgenic zebrafish.

10. Zebrafish fmyhc2.2 The application of gene promoters and muscle-specific enhancers in the preparation of fluorescently colored transgenic zebrafish is characterized by, The fmyhc2.2 The gene promoter sequence is shown in SEQ ID NO: 1; the muscle-specific enhancer sequence is shown in SEQ ID NO: 2.

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