Method for constructing homozygous line of fast-growing red crucian carp and primer

Abstract

The application belongs to the field of fish breeding, and discloses a construction method and primers of a homozygous line of fast-growing red crucian carp. acvr2b The construction method comprises the following steps: preparing gRNA of a red crucian carp gene, microinjecting a mixture of the gRNA and a Cas9 protein into a one-cell fertilized egg of the red crucian carp, screening to obtain F0 generation gene knockout mutants, self-crossing the F0 generation gene knockout mutants to obtain F1 generation mutants, screening to obtain homozygous mutants, and self-crossing the homozygous mutants to propagate. acvr2b The application successfully establishes a homozygous knockout line in the red crucian carp for the first time through a CRISPR / Cas9 gene editing technology. acvr2b The mutant red crucian carp obtained in the application has a body weight increased by 30.68% and a muscle protein content increased by 13.81% compared with a wild type at 12 months of age, and has a significantly improved intestinal digestion and absorption capacity, thereby providing high-quality germplasm resources for fish genetic breeding and having a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of fish breeding technology and discloses a method for constructing a homozygous strain of fast-growing red crucian carp and primers. Background Technology

[0002] Fish muscle tissue is an important source of high-quality protein for humans, and its growth rate directly determines the profitability of aquaculture. In fish farming, the key to improving growth rate lies in promoting the proliferation and differentiation of myofibril cells, thereby accelerating muscle tissue development. This process not only shortens the farming cycle and reduces production costs but also reduces the risk of disease during farming, which is of great significance to the sustainable development of aquaculture. Red crucian carp, as an important germplasm resource of the genus Carapax in the Cyprinidae family, has attracted widespread attention due to its strong resistance and delicious flesh. However, compared with common crucian carp, red crucian carp have a slower growth rate, limiting its value for widespread aquaculture.

[0003] Acvr2 (Activin receptor type II) is a member of the TGF-β receptor family and plays a negative regulatory role in muscle growth and development. Studies have found that in acvr2 Significant increases in muscle mass were observed in both humans and mice with active natural variants; [the text abruptly ends here, likely due to an incomplete sentence or missing information.] acvr2 Soluble receptor with outer membrane domain fused to immunoglobulin Fc fragment acvr2 / Fc Two weeks later, it can increase the muscle mass of mice by 60%; because activin A can also bind to acvr2 To regulate muscle growth, causing mstn Re-injection into (myosin) gene knockout mice acvr2 / Fc Afterwards, it can also promote muscle growth by 15-25%. These studies suggest that... acvr2 It plays an important role in muscle growth, but its potential has not yet been realized. acvr2 Breeding research in economically important fish species, especially red crucian carp. Summary of the Invention

[0004] The technical problem to be solved by this invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a method and primers for constructing a rapid-growing homozygous red crucian carp strain. This method is based on CRISPR / Cas9 gene editing technology to target activin receptor type II B (β2B) in red crucian carp. acvr2b The gene was knocked out, and the resulting acvr2b The red crucian carp mutant had a 30.68% higher body weight, a 13.81% higher muscle protein content, and significantly improved intestinal digestion and absorption capacity at 12 months of age compared to the wild type.

[0005] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0006] A method for constructing a homozygous strain of fast-growing red crucian carp includes the following steps:

[0007] (1) For red carp acvr2b Specific targets were designed based on gene exon sequences, and corresponding gRNAs were synthesized based on these targets. The mixture of gRNA and Cas9 protein was injected into one-cell stage fertilized eggs of red crucian carp via microinjection, and F0 generation gene knockout mutants were obtained through screening.

[0008] The preparation process of the gRNA includes: designing specific forward primers based on the target sequence; performing PCR using the F primer and universal reverse primers with the gRNA plasmid as a template; purifying and recovering the PCR product to obtain the gRNA DNA template; and preparing the gRNA DNA template into a... acvr2b gRNA of genes;

[0009] The nucleotide sequence of the target site is as follows:

[0010] 5'-GGCTGCACTGTTACGCCTCC-3' (as shown in SEQ ID NO:1);

[0011] The specific forward primer is:

[0012] 5'-TGTAATACGACTCACTATAGGCTGCACTGTTACGCCTCCGTTTTAGAGCTAGAAATAGC-3' (as shown in SEQ ID NO: 2);

[0013] The universal reverse primer is:

[0014] 5'-AGCACCGACTCGGTGCCACT-3' (as shown in SEQ ID NO:3)

[0015] (2) Self-cross the sexually mature F0 generation gene knockout mutants to obtain F1 generation mutants, and screen them to obtain homozygous mutants;

[0016] (3) Self-cross the homozygous mutant to obtain the fast-growing red crucian carp homozygous strain.

[0017] The preferred method described above uses the following PCR amplification reaction system and procedure: 0.2 µL of 5 U / µL high-fidelity DNA polymerase, 2 µL of 50 mM MgSO4, 1 µL of 10 mM dNTP mix, 5 µL of 10×High Fidelity PCR Buffer, 2 µL of gRNA plasmid DR274, 1 µL of specific forward primer, 1 µL of universal reverse primer, and nuclease-free water to a total volume of 50 µL; the amplification program parameters are: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 15 s, 66℃ annealing for 30 s, 68℃ extension for 20 s, cycling 30-35 times, and 68℃ for 10 min.

[0018] Preferably, the in vitro transcription system is as follows: 4.0 μL of 5×TranscriptAid Reaction Buffer, 2 μL of 10 mM ATP Tris buffered, 2 μL of 10 mM CTP Tris buffered, 2 μL of 10 mM GTP Tris buffered, 2 μL of 10 mM UTP Tris buffered, 2.0 μL of TranscriptAidEnzyme Mix, 2 μg of template, and nuclease-free water to a final volume of 20 μL; after thorough mixing, incubate at 37°C for 3.0 h, add 1 μL of TuRBo DNase, mix well, incubate at 37°C for 20 min, and then purify using lithium chloride precipitation.

[0019] The Cas9 protein is mainly composed of an α-helix recognition region (REC) and a nuclease-active region (NUC). The REC region is responsible for binding gRNA, recognizing and binding gRNA by regulating the conformation of its domains. The NUC region can recognize and bind to the PAM region and perform DNA cleavage. Compared with plasmid- and RNA-based CRISPR / Cas9, Cas9-RNP can directly enter the nucleus for gene editing with the help of the nuclear localization sequence (NLS) at the end of the Cas9 protein, without the need for protein translation and expression, thus improving gene editing efficiency.

[0020] Preferably, in the microinjection mixing system of step (1), red crucian carp acvr2b The concentration of gRNA used for the gene was 80 ng / μL, and the concentration of Cas9 protein used was 100 ng / μL.

[0021] Preferably, in step (1), the injection volume of each fertilized egg is 3.0 μL; the fertilized eggs after injection are placed in water at a temperature of 25-28℃ for incubation.

[0022] Preferably, the screening and identification method for F0 generation gene knockout mutants in step (1) includes the following steps: extracting genomic DNA from F0 generation individuals and wild-type control individuals respectively; using the genomic DNA of both as templates; designing specific detection primers based on the target site for PCR; performing Sanger sequencing on the PCR products; and confirming the gene knockout effect by comparing the sequencing peak diagrams. acvr2b When overlapping peaks appear in the terminal region of the gene target site, it indicates that the gene has been successfully knocked out, and F0 generation gene knockout mutants can be obtained by screening accordingly.

[0023] Preferably, the specific detection primer sequences are as follows:

[0024] The forward primer is:

[0025] 5'-AATCTGCTTCTATTCTGTGTTT-3' (as shown in SEQ ID NO:4);

[0026] The reverse primer is:

[0027] 5'-TGAAGGAGGTTCTGATGTTAC-3' (as shown in SEQ ID NO:5).

[0028] Based on a general inventive concept, the present invention also provides a method for preparing red crucian carp. acvr2b Amplification-specific primers for gene gRNA include:

[0029] The specific forward primer is:

[0030] 5'-TGTAATACGACTCACTATAGGCTGCACTGTTACGCCTCCGTTTTAGAGCTAGAAATAGC-3' (as shown in SEQ ID NO: 2);

[0031] The universal reverse primer is:

[0032] 5'-AGCACCGACTCGGTGCCACT-3' (as shown in SEQ ID NO:3).

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] 1. This invention is the first to successfully establish a gene editing system in red crucian carp using CRISPR / Cas9 gene editing technology. acvr2b Homozygous knockout strains are important for further understanding acvr2b The mechanism by which genes play a role in the negative regulation of muscle growth is of great significance.

[0035] 2. The invention obtained acvr2bThe mutant red crucian carp showed a 30.68% increase in body weight and a 13.81% increase in muscle protein content at 12 months of age compared to the wild type, and a significant improvement in intestinal digestion and absorption capacity. It provides high-quality germplasm resources for fish genetic breeding and has good application prospects. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings and tables used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings and tables described below are some embodiments of the present invention. For those skilled in the art, other accompanying drawings and tables can be obtained based on these accompanying drawings and tables without creative effort.

[0037] Figure 1 The red carp provided in the embodiments of the present invention acvr2b Schematic diagram of gene target sites;

[0038] Figure 2 Provided for embodiments of the present invention acvr2b Images of mutant and wild-type red crucian carp;

[0039] Figure 3 Provided for embodiments of the present invention acvr2b A diagram illustrating that the mutant red crucian carp has a significantly higher weight than the wild-type red crucian carp;

[0040] Figure 4 Provided for embodiments of the present invention acvr2b A comparative illustration of paraffin sections of muscle tissue from mutant red crucian carp and wild-type red crucian carp.

[0041] Figure 5 Provided for embodiments of the present invention acvr2b Mutant Red Carp acvr2b A schematic diagram of gene mRNA levels, * indicates significant differences ( p <0.05);

[0042] Figure 6 Provided for embodiments of the present invention acvr2b A schematic diagram of the protein content in the muscle tissue of mutant red crucian carp. * indicates a significant difference. p <0.05);

[0043] Figure 7 This is provided by the embodiments of the present invention. acvr2b A schematic diagram of the intestinal morphology of a mutant red crucian carp. Detailed Implementation

[0044] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0045] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0046] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0047] TranscriptAid Reaction Buffer: TranscriptAid reaction buffer, ThermoFisher (manufacturer), K0441 (model).

[0048] ATP Tris buffered: ATP solution, ThermoFisher (manufacturer), K0441 (model);

[0049] CTP Tris buffered: CTP solution, ThermoFisher (manufacturer), K0441 (model);

[0050] GTP Tris buffered: GTP solution, ThermoFisher (manufacturer), K0441 (model).

[0051] UTP Tris buffered: UTP solution, ThermoFisher (manufacturer), K0441 (model);

[0052] TranscriptAid Enzyme Mix: TranscriptAid enzyme mixture, ThermoFisher (manufacturer), K0441 (model).

[0053] The high-fidelity DNA polymerase product used in this invention is Platinum®. Taq DNA High Fidelity Polymerase.

[0054] Example:

[0055] A method for constructing a homozygous strain of fast-growing red crucian carp includes the following steps:

[0056] 1. Design and synthesis of the CRISPR system

[0057] (1) Design and synthesis of gRNA

[0058] Red Carp acvr2bThe gene's Gene ID is 113112785. Designed using the ZiFiT website (http: / / zifit.partners.org / zifit / Disclaimer.aspx). acvr2b Gene targets; when designing targets, the following should be noted: acvr2b Target sites are designed from conserved sequence regions of genes. The target sequence length for gRNA is generally 18-22 bp. The target sequence should avoid four consecutive Ts, and the fourth-to-last base should ideally be G. The PAM of the target sequence should be NGG. The final designed target fragment is:

[0059] 5'-GGCTGCACTGTTACGCCTCC-3' (as shown in SEQ ID NO:1), as Figure 1 As shown.

[0060] Fragments containing the target and gRNA sequences were amplified using target-specific forward primers (F primers) and universal reverse primers (R primers). The amplification system was as follows: 0.2 µL of high-fidelity DNA polymerase (5 U / µL), 2 µL of 50 mM MgSO4, 1 µL of 10 mM dNTP mix, 5 µL of 10× High Fidelity PCR Buffer, 2 µL of DR274 plasmid, 1 µL of gRNA-specific forward primer (F primer), 1 µL of gRNA universal reverse primer (R primer), and nuclease-free water to a total volume of 50 µL.

[0061] Specific forward primers:

[0062] 5'-TGTAATACGACTCACTATAGGCTGCACTGTTACGCCTCCGTTTTAGAGCTAGAAATAG-3' (as shown in SEQ ID NO: 2);

[0063] Universal reverse primer:

[0064] 5'-AGCACCGACTCGGTGCCACT-3' (as shown in SEQ ID NO:3).

[0065] The amplification program was as follows: pre-denaturation at 94℃ for 5 min, followed by 30-35 cycles of denaturation at 94℃ for 15 s, annealing at 66℃ for 30 s, extension at 68℃ for 20 s, and final extension at 68℃ for 10 min. The purified fragment was used as a template for gRNA synthesis. In vitro transcription was performed using an in vitro transcription kit. The transcription system consisted of: 4.0 μL of 5×TranscriptAid Reaction Buffer, 2 μL (10 mM) of ATP Tris buffered, 2 μL (10 mM) of CTP Tris buffered, 2 μL (10 mM) of GTP Tris buffered, 2 μL (10 mM) of UTP Tris buffered, 2.0 μL of TranscriptAid Enzyme Mix, 2 μg of template, and nuclease-free water to a final volume of 20 μL.

[0066] After thorough mixing, incubate at 37°C for 3.0 h, add 1 μL of TuRBo DNase, mix well, incubate at 37°C for 20 min, then purify using lithium chloride precipitation method, and store at -80°C.

[0067] (1) Purchase of Cas9 protein

[0068] The Cas9 protein used for the injection was purchased directly from Thermo Fisher Scientific (A36497, 1 mg / mL).

[0069] 2. Microinjection and culture of red crucian carp embryos

[0070] (1) Obtaining red crucian carp embryos

[0071] The red crucian carp used in the experiment were obtained from the Engineering Research Center for Polyploid Reproduction and Breeding Technology of the Ministry of Education. During the breeding season, several mature male and female red crucian carp were selected and spawning was induced by injecting a mixed spawning stimulant consisting of luteinizing hormone-releasing hormone analogue (LRH-A), human chorionic gonadotropin (HCG), and domperidone (DOM) into the base of their pectoral fins. The dosage of LRH-A was 6 μg / kg, the dosage of HCG was 300 IU / kg, and the dosage of DOM was 1 mg / kg. The female broodstock was injected first, and the male broodstock was injected 4-5 hours later, with the dosage of the male broodstock halved. After about 10-12 hours, the abdominal surface of the fish was dried with a towel. The female red crucian carp was taken out, and the abdomen was gently pressed to allow the eggs to flow into a stainless steel basin. The same method was used to gently press the abdomen of the male fish, and a small amount of sperm was squeezed into the basin. The fish were then quickly shaken to ensure that the eggs and sperm were fully fertilized before being evenly spread in a glass culture dish.

[0072] (2) Microinjection

[0073] Prepared red carp acvr2bThe gRNA and Cas9 protein of the gene were mixed and incubated at room temperature to obtain a mixture. When the fertilized eggs of red crucian carp in the glass culture dish developed to the one-cell stage (the fertilized egg membrane was raised), microinjection was performed using a microinjector. The injection concentration was Cas9 protein (100 ng / μL) and gRNA (80 ng / μL), and the injection volume for each fertilized egg was 3.0 μL.

[0074] (3) Embryo culture and knockout detection

[0075] The injected embryos and wild-type control embryos were transferred to a constant temperature water incubation system at 25-28℃ for hatching. After hatching, the fry were initially fed egg yolk, and after 10 days, they were fed brine shrimp. Half of the water was changed daily. Once the fry were able to feed normally, they were stocked at a size of 3m. 2 Aquarium. Two months later, the tail fins of the fish were harvested to extract the genome. Specific primers containing the knockout site were designed for detection. Mutation rate was detected using forward and reverse primers, and F0 generation gene knockout mutants were obtained through screening. acvr2b Mutant red crucian carp and wild red crucian carp are shown in the morphology. Figure 2 ;

[0076] Forward primer:

[0077] 5'-AATCTGCTTCTATTCTGTGTTT-3' (as shown in SEQ ID NO:4);

[0078] Reverse primer:

[0079] 5'-TGAAGGAGGTTCTGATGTTAC-3' (as shown in SEQ ID NO:5).

[0080] 3. Preparation of mutant homozygotes

[0081] F0 generation gene knockout mutant experimental fish were cultured in dedicated ponds for one year. During the breeding season (late March to early May), when the water temperature stabilized at 20-25℃, the F0 generation gene knockout mutants were self-crossed to obtain F1 generation mutants. F1 generation mutants with 100% mutation efficiency were selected as parents and cultured in dedicated ponds for self-crossing propagation to obtain red crucian carp. acvr2b Mutant homozygous strains are fast-growing homozygous red crucian carp strains.

[0082] like Figure 3-4 As shown, the invention obtained acvr2b The mutant red crucian carp had a 30.68% higher body weight at 12 months of age compared to the wild type, and there were significant changes in muscle cells.

[0083] Table 1 lists the embodiments of the present invention. acvr2b Statistical table of total number, average area and total area of ​​myofibrils in mutant red crucian carp and wild red crucian carp.

[0084] Table 1 acvr2b Muscle fiber indices of knockout red crucian carp and control red crucian carp

[0085]

[0086] Note: a The representative showed a significant difference from the red carp.

[0087] like Figure 5-6 As shown in Table 1, the present invention obtains acvr2b Mutant Red Carp acvr2b The mRNA level of the gene was significantly reduced, muscle protein content increased by 13.81%, and the total number of myofibrils (145±6.5) was significantly higher than that of wild-type red crucian carp (121±4.2). p <0.05, mean area of ​​myofibroblasts (1222.2±89.1 μm) 2 The concentration was significantly higher than that of wild-type red crucian carp (572.6±60.1 μm). 2 () p <0.05, total area of ​​myofibroblasts (177214.9±7982 μm) 2 The concentration of the red crucian carp was significantly higher than that of wild-type red crucian carp (69298.1±899 μm). 2 () p <0.05).

[0088] Table 2 lists the embodiments of the present invention. acvr2b Statistical table of intestinal morphological parameters of knockout red crucian carp and control red crucian carp.

[0089] Table 2 acvr2b Intestinal morphological parameters of knockout red crucian carp and control red crucian carp

[0090]

[0091] Note: a The representative showed a significant difference from the red carp.

[0092] like Figure 7 As shown in Table 2, the present invention obtains acvr2b The intestinal tissue of the mutant red crucian carp exhibited better digestive and absorptive capacity, with the intestinal fold length (152.52±15.12 μm) significantly higher than that of the wild-type red crucian carp (112.31±10.35 μm). p <0.05, the intestinal fold width (39.38±2.98 μm) was significantly higher than that of wild-type red crucian carp (33.62±2.51 μm). p <0.05, and the intestinal muscle layer thickness (50.47±2.85 μm) was significantly higher than that of wild-type red crucian carp (41.11±1.03 μm). p<0.05).

[0093] In summary, this invention provides a method for knocking out gene defects using CRISPR / Cas9 gene editing technology. acvr2b The method of achieving rapid growth of red crucian carp through gene technology is of great significance for the improvement of germplasm resources and has promising application prospects. Furthermore, it can shorten the time to market for red crucian carp, and is simple to operate and highly efficient, making it important in fish genetics and breeding.

Claims

1. A method for constructing a homozygous strain of fast-growing red crucian carp, characterized in that, Includes the following steps: (1) For red carp acvr2b Specific targets were designed based on gene exon sequences, and corresponding gRNAs were synthesized based on these targets. The mixture of gRNA and Cas9 protein was injected into one-cell stage fertilized eggs of red crucian carp via microinjection, and F0 generation gene knockout mutants were obtained through screening. The preparation process of the gRNA includes: designing specific forward primers based on the target sequence; performing PCR using the specific forward primers and universal reverse primers with the gRNA plasmid as a template; purifying and recovering the PCR product to obtain the gRNA DNA template; and preparing the gRNA DNA template into a... acvr2b gRNA of genes; red crucian carp acvr2b The concentration of gRNA used was 80 ng / μL, and the concentration of Cas9 protein used was 100 ng / μL; the injection volume for each fertilized egg was 3.0 μL. The nucleotide sequence of the target site is as follows: 5'-GGCTGCACTGTTACGCCTCC-3'; The specific forward primer is: 5'-TGTAATACGACTCACTATAGGCTGCACTGTTACGCCTCCGTTTTAGAGCTAGAAATAGC-3'; The universal reverse primer is: 5'-AGCACCGACTCGGTGCCACT-3'; (2) Self-cross the sexually mature F0 generation gene knockout mutants to obtain F1 generation mutants, and screen them to obtain homozygous mutants; (3) Self-cross the homozygous mutant to obtain the fast-growing red crucian carp homozygous strain; The specific PCR amplification reaction system and procedure are as follows: 0.2 µL of 5 U / µL high-fidelity DNA polymerase, 2 µL of 50 mM MgSO4, 1 µL of 10 mM dNTP mix, 5 µL of 10×High Fidelity PCR Buffer, 2 µL of gRNA plasmid DR274, 1 µL of specific forward primer, 1 µL of universal reverse primer, and nuclease-free water to a total volume of 50 µL; the amplification program parameters are: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 15 s, 66℃ annealing for 30 s, 68℃ extension for 20 s, cycle 30-35, 68℃ for 10 min; The in vitro transcription system was as follows: 4.0 μL of 5×TranscriptAid Reaction Buffer, 2 μL of 10 mM ATP Tris buffer, 2 μL of 10 mM CTP Tris buffer, 2 μL of 10 mM GTP Tris buffer, 2 μL of 10 mM UTP Tris buffer, 2.0 μL of TranscriptAid Enzyme Mix, 2 μg of template, and nuclease-free water to a final volume of 20 μL. After thorough mixing, the mixture was incubated at 37°C for 3.0 h. Then, 1 μL of TuRBoDNase was added, mixed, and incubated at 37°C for 20 min. Finally, the mixture was purified using lithium chloride precipitation.

2. The construction method according to claim 1, characterized in that, In step (1), the injected fertilized eggs are placed in water at a temperature of 25-28℃ for incubation.

3. The construction method according to claim 1, characterized in that, The screening and identification method for F0 generation gene knockout mutants in step (1) includes the following steps: extracting genomic DNA from F0 generation individuals and wild-type control individuals respectively; using the genomic DNA of both as templates; designing specific detection primers based on the target site for PCR; performing Sanger sequencing on the PCR products; and confirming the gene knockout effect by comparing the sequencing peak diagrams. acvr2b When overlapping peaks appear in the terminal region of the gene target site, it indicates that the gene has been successfully knocked out, and F0 generation gene knockout mutants can be obtained by screening accordingly.

4. The construction method according to claim 3, characterized in that, The specific detection primer sequences are as follows: The forward primer is: 5'-AATCTGCTTCTATTCTGTGTTT-3'; The reverse primer is: 5'-TGAAGGAGGTTCTGATGTTAC-3'.

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