Preparation method of PPAR (peroxisome proliferator activated receptor) gamma gene deleted zebrafish

Abstract

The invention relates to a preparation method of PPAR gamma gene deleted zebrafish, belongs to the field of molecular biology, and particularly relates to preparation of PPAR gamma gene deleted mutant zebrafish. The method comprises the following steps: 1, synthesizing two sgRNA chains; 2, combining the two sgRNA chains with Cas9 protein, and microinjecting the combined sgRNA chains and Cas9 protein into fertilized eggs in a single cell period of zebra fish; 3, detecting mutation efficiency, and feeding the F0 generation zebrafish to adult fish; 4, backcrossing the F0-generation adult zebrafish and the wild type zebrafish; 5, selecting individuals with consistent filial generation deletion positions after backcross for selfing to obtain F1-generation PPAR gamma mutation zebrafish; and 6, carrying out selfing on the obtained F1 generation PPAR gamma deletion homozygous individuals. According to the invention, a PPAR gamma gene deletion mutant strain is obtained in zebrafish by using a gene editing technology, the designed double-acting target primer can realize accurate and efficient knockout of a target gene in a zebrafish genome, the knockout rate reaches 85%, and embryo development after microinjection does not have malformation. The obtained PPAR gamma gene deletion mutant zebrafish can be used for research on lipid metabolism and deposition.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology, and particularly relates to preparation of a PPARγ gene deletion mutant zebrafish. BACKGROUND

[0002] Fat deposition is a complex physiological process in animal body, and lipid metabolism regulation is a core issue of concern in animal physiology and agricultural production. When the decomposition metabolism of lipids in the body is not as fast as the synthesis metabolism, fat begins to deposit. Fat cells in different parts form and deposit in sequence, and in mammals, visceral fat cells basically stop growing after birth, while intramuscular fat (IMF) cells form relatively late, and the marble pattern of meat quality is deposited after sexual maturity . IMF, as a key indicator of determining meat flavor, tenderness and juiciness , its deposition process is precisely regulated by a multi-level molecular network. Fat deposition in bony fish mainly occurs in the abdominal cavity, visceral mass, intramuscular and intermuscular, as one of the main energy sources of fish, it participates in the growth and development and metabolism of fish body and plays an important physiological role . Appropriate IMF content in animal muscle can meet the dietary needs of consumers with low-fat nutrition, and also provide better sensory experience when eating , the positive effect of IMF on the sensory quality of animal meat has been confirmed in beef , broiler , pork and salmon . However, excessive deposition of lipids endangers the health of economic animals, and reduces the feed conversion rate in breeding, also causes difficulty in handling during the slaughter process and pollutes the environment .

[0003] PPARγ is peroxisome proliferator-activated receptor gamma, as a ligand-activated transcription factor nuclear receptor (NR) superfamily member, is a core transcription factor for regulating adipogenesis and lipid storage, and is also essential for regulating insulin sensitivity and adipogenesis . PPARγ drives fat cell differentiation, survival and triglyceride (TG) deposition by activating downstream genes such as lipoprotein lipase (LPL), fatty acid binding protein (FABP) and perilipin (PLIN) , and can also affect IMF deposition efficiency by regulating the balance of fatty acid oxidation / storage in muscle tissue .

[0004] Gene editing can realize the insertion or knockout of specific sites at the genome level, thereby realizing the directional modification of economic traits such as growth, reproduction and meat quality Zebrafish (Danio rerio) is the third largest vertebrate model after mammals, and can also be used as an ideal vertebrate model for studying lipid metabolism regulation due to its transparent embryo, rapid reproduction and visible development process, and has unique advantages in analyzing the development and energy allocation mechanism of fish adipose tissue. At present, the gene editing model of PPARgamma has revealed the complexity of lipid metabolism regulation in mammals , but the systematic function research of fish PPARgamma is still in its infancy. SUMMARY

[0005] The application provides a preparation method of PPARgamma gene deletion zebrafish, which is expected to provide innovative strategies for precise fat deposition regulation and genetic improvement of aquatic animals, and to provide cross-species comparison basis for vertebrate fat metabolism evolution research.

[0006] The preparation method of the PPARgamma gene deletion zebrafish according to the application is carried out according to the following steps:

[0007] I. Two target points at similar positions of the same exon are selected, CRISPR probe primers for knockout are designed according to the target point position and zebrafish PPARgamma gene sequence, and two sgRNA chains are synthesized in vitro;

[0008] II. Combine the two sgRNA chains with Cas9 protein and microinject them into zebrafish single-cell stage zygotes;

[0009] III. Test the mutation efficiency of the zygotes 48 hours after injection by PCR, and breed F0 generation zebrafish into adult fish;

[0010] IV. Backcross the F0 generation zebrafish adult fish with wild type to obtain mutant heterozygous zebrafish, and breed the zebrafish to be more than 1 month old, and sequence to detect the frameshift after knocking out the gene;

[0011] V. Select female and male PPARgamma mutant heterozygous zebrafish with consistent frameshift and knockout position for self-crossing to obtain F1 generation PPARgamma mutant homozygous zebrafish, which is PPARgamma gene deletion mutant zebrafish homozygote, and breed to adult fish;

[0012] VI. Cross the obtained F1 generation PPARgamma deletion homozygous individuals to obtain PPARgamma gene deletion mutant homozygous zebrafish.

[0013] Further, the sequence of the core target site in step one is designed as follows: two core target sites are located in the same exon, and the sequence of the core target site is PPARγ-1 5'-GCGTCAGGTCCATCATGTGCGGG-3', and PPARγ-2 5'-AGCGCTCAGGCCGAAGCCCACGG-3'.

[0014] Further, the CRISPR probe primer in step one is composed of a forward primer and a reverse primer, wherein the forward primer is composed of a T7 promoter sequence, a modified core target site sequence and an sgRNA scaffold, and the reverse primer is composed of a universal pT7 scaffold;

[0015] The sequence of the modified core target site of the core target site PPARγ-1 is 5'-GCGTCAGGTCCATCATGTGC-3';

[0016] The sequence of the modified core target site of the core target site PPARγ-2 is 5'-AGCGCTCAGGCCGAAGCCCA-3'.

[0017] Further, the base numbers of the T7 promoter sequence and the sgRNA scaffold in the forward primer are 20 and 14 bp respectively, the T7 promoter sequence is 5'-TTCTAATACGACTCACTATA-3', the sgRNA scaffold is 5'-GTTTTAGAGCTAGA-3', the sequence of the PPARγ-1 forward primer is a, TTCTAATACGACTCACTATA GCGTCAGGTCCATCATGTGCGTTTTAGAGCTAGA, the sequence of the PPARγ-2 forward primer is b, TTCTAATACGACTCACTATA GAGCGCTCAGGCCGAAGCCCA GTTTTAGAGCTAGA, and the modified core target site sequence is 5' end G base, and the "NGG" base of the target site PAM region is removed; wherein the 5' end of the core target site should be G base, which is not a requirement of the Cas9 target site itself, but because the T7 promoter is used in the sgRNA in vitro transcription vector, the former requires the first two positions of the transcription start site to be GG; the three bases next to the 3' end of the target site constitute the protospacer adjacent motif (PAM) region, and the sequence should be "NGG", wherein N is any base.

[0018] Further, the pT7 scaffold sequence in the reverse primer is: 5'-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'.

[0019] Further, the PPAR gamma, 1 sgRNA chain and the PPAR gamma, 2 sgRNA chain are mixed in a concentration ratio of 1:1 in step two.

[0020] Further, the microinjection system in step two is: Cas9 protein 2 muL, 10x NEBuffer 0.8 muL, sgRNA 1600 ng, and ddH2O is supplemented to 6 muL.

[0021] Further, the test primers for PCR test of mutation efficiency in step three are: PPAR gamma-E2-SF 5'-TCGATTTTCTCTGTGTTGTTCG-3'; PPAR gamma-E2-SR 5'-CGTGTGTCCTGTAGTTCTCCTG-3'.

[0022] Further, in step three, 20 eggs are taken for digestion and DNA extraction, and the knockout efficiency is detected by polyacrylamide gel electrophoresis.

[0023] Further, in step four, when backcrossing with wild type zebrafish, zebrafish with different genders from the fish are selected for 1:1 backcrossing.

[0024] The zebrafish PPAR gamma gene deletion primer of the present application is composed of two groups of primer pairs of PPAR gamma-1 primer pair and PPAR gamma-2 primer pair;

[0025] The sequence of the forward primer of PPAR gamma-1 is: a, TTCTAATACGACTCACTATA GCGTCAGGTCCATCATGTGCGTTTTAGAGCTAGA; and the sequence of the forward primer of PPAR gamma, 2 is: b, TTCTAATACGACTCACTATA GAGCGCTCAGGCCGAAGCCCA GTTTTAGAGCTAGA;

[0026] The reverse primer of PPAR gamma-1 and PPAR gamma-2 is the sequence of pT7 scaffold: 5'-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'.

[0027] Advantages and beneficial effects of the present application:

[0028] The present application selects two suitable target site sequences at the second exon of the zebrafish PPAR gamma gene, and designs two PPAR gamma gene deletion probes, so that efficient and accurate knockout of the target gene in the zebrafish genome can be realized, and the embryonic development after microinjection does not appear to be deformed.

[0029] The present application uses two sgRNA chains designed to act on different target points, which are mixed 1:1 and combined with Cas9 protein to inject into one fertilized egg, which can cut the longer sequence of the target gene, has high knockout efficiency, simple process, low gene mutation cost and is easy to popularize.

[0030] The PPAR gamma gene deletion mutant zebrafish constructed by the present application can provide a basic model for fish fat deposition and metabolic regulation.

[0031] The gene knockout efficiency of a single target point in the prior art is low, and the knockout efficiency in the zebrafish model is about 65% On average; and designing multiple action sites for simultaneous knockout can greatly increase the gene knockout efficiency, and the gene knockout efficiency of multiple action sites designed for the same exon in the zebrafish model can reach 77.8% Even 83.3% By double-target knockout of zebrafish PPAR gamma gene, the knockout efficiency reaches 85%, which provides an efficient operation model for fish fat regulation.

[0032] The present application uses gene editing technology to obtain a PPAR gamma gene deletion mutant strain in zebrafish, and the double-action target primer designed can realize accurate and efficient knockout of the target gene in the zebrafish genome, with a knockout rate of 85%, and the embryonic development after microinjection does not appear to be deformed. The obtained PPAR gamma gene deletion mutant zebrafish can be used for the research of fish lipid metabolism and deposition. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The agarose gel electrophoresis test result for preparing the sgRNA in vitro transcription template by PCR amplification;

[0034] Figure 2 Mutation rate detection of PPAR gamma gene knockout zebrafish eggs at 48 h;

[0035] Figure 3 Mutation of PPAR gamma gene knockout zebrafish F0 generation up to 2 months old;

[0036] Figure 4 Mutation of PPAR gamma gene knockout zebrafish mutant F0 generation after backcrossing of heterozygous offspring;

[0037] Figure 5 The mutation sequencing alignment diagram of the PPARγ gene knockout zebrafish hybrid offspring;

[0038] Figure 6 The polypropylene gel electrophoresis test results of the F1 generation of the PPARγ gene knockout zebrafish;

[0039] Figure 7 The relative expression amount results of the PPARγ gene in the muscle of the wild type (WT) and mutant lines (PPARγ - / - The polypropylene gel electrophoresis test results of the homozygous line;

[0040] Figure 8 The relative expression amount results of the PPARγ gene in the muscle of the wild type (WT) and mutant lines (PPARγ - / - );

[0041] Figure 9 The difference results of the T-CHO content, a key index of fat deposition in the muscle of the two genotypes of zebrafish;

[0042] Figure 10 The difference results of the TG content, a key index of fat deposition in the muscle of the two genotypes of zebrafish. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0044] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0045] Embodiment 1: Preparation of PPARγ gene knockout zebrafish

[0046] 1. Two target points at similar positions of the same exon are selected, CRISPR probe primers for knockout are designed according to the target point positions and the sequence of the zebrafish PPARγ gene, and two sgRNA chains are synthesized in vitro.

[0047] The core target point sequence is designed, and the two designed core target points are located on the same exon. In this embodiment, the design of the primer sgRNA chain of the two zebrafish PPARγ gene knockout CRISPR probes is given. The CRISPR probe primer is composed of a forward primer and a reverse primer, wherein the forward primer is composed of a T7 promoter sequence, a modified core target point sequence and an sgRNA scaffold, and the reverse primer is composed of a universal pT7 scaffold.

[0048] The forward primer and the reverse primer are combined to amplify and replicate the sgRNA in vitro transcription template containing the T7 transcription promoter, the core target sequence. The T7 promoter sequence is 5'-TTCTAATACGACTCACTATA-3'; the sgRNA scaffold is 5'-GTTTTAGAGCTAGA-3'; and the universal reverse primer pT7 scaffold sequence is 5'-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'.

[0049] The genomic data of the target gene PPARy was downloaded from the zebrafish genome database of NCBI (https: / / www.ncbi.nlm.nih.gov / ), the core target primer was designed online for the CDS region by using the CHOPCHOP (https: / / chopchop.cbu.uib.no / ) website, and the suitable action target was selected as the core target sequence. In this embodiment, two action targets were finally selected, which were located on the positive strand of the second exon of the same exon: PPARy-1 5'-GCGTCAGGTCCATCATGTGCGGG-3', and PPARy-2 5'-AGCGCTCAGGCCGAAGCCCACGG-3'.

[0050] The T7 promoter sequence, the core target sequence (the 5' end should be a G base, and the "NGG" base of the target PAM region is removed) and the sgRNA scaffold constitute the forward primer. The 5' end of the core target should be a G base, which is not a requirement of the Cas9 target itself, but because the sgRNA in vitro transcription vector used in this embodiment uses a T7 promoter, the latter requires the first two positions of the transcription start site to be GG; the three bases next to the 3' end of the target constitute the protospacer adjacent motif (PAM) region, which requires the sequence to be "NGG", wherein N is any base.

[0051] The forward primer sequence of the final two kinds of zebrafish PPARy gene deletion primer sgRNA strands in this embodiment is: a, TTCTAATACGACTCACTATA GCGTCAGGTCCATCATGTGC GTTTTAGAGCTAGA; b, TTCTAATACGACTCACTATA G AGCGCTCAGGCCGAAGCCCA GTTTTAGAGCTAGA.

[0052] sgRNA strand preparation: two sgRNA forward primers were combined with a universal reverse primer pT7 scaffold to obtain sgRNA in vitro transcription templates by PCR. Among them, the PCR system of sgRNA in vitro transcription template was: 10 μM sgRNA 10 μL, 10 μM pT7 scaffold 10 μL, Dream Taq master mix 100 μL, ddH2O to 180 μL. The PCR reaction conditions were: 95°C for 3 min; 95°C for 30 s, 58°C for 30 s, 72°C for 30 s, 33 cycles; 72°C for 5 min; 4°C for ∞.

[0053] The above PCR product was purified and recovered with a kit (Gene JET Gel Extraction Kit, K0692), and the mass and concentration were detected by Nanodrop 8000 spectrophotometer. 300 ng of PCR product was taken for agarose gel electrophoresis, Figure 1 The agarose gel electrophoresis test results of PCR amplification for preparing sgRNA in vitro transcription templates are shown in FIG. 2, and the fragment size is about 120 bp, and the sgRNA in vitro transcription template is successfully amplified. Figure 1

[0054] The sgRNA was synthesized by in vitro transcription at 37°C for 6 h using an sgRNA synthesis kit (HiScribe™ T7 Quick High Yield RNA Synthesis Kit (E2050S)). The sgRNA synthesis reaction system was: T7 RNA Polymerase Mix 1 μL, NTP Buffer Mix 5 μL, Template DNA 400 ~ 700 ng, ddH2O to 15 μL. To eliminate residual DNA, 1 μL of DNase I and preheated 37°C 9 μL of ddH2O were added after incubation for 15 min. The synthesized sgRNA was purified and recovered with an RNA purification kit (MACHEREY-NAGEL), and its concentration and mass were detected, and then aliquoted at -80°C for storage for standby.

[0055] ​2. Two sgRNA strands were combined with Cas9 protein and microinjected into zebrafish single-cell stage fertilized eggs; that is, two sgRNAs were mixed 1:1, combined with Cas9 protein (NEB Company, item number M0386S), microinjected into single-cell stage zebrafish embryos, and about 80 eggs were injected. Among them, the microinjection system was: sgRNA final concentration 5 pmol / μL (1600 ng), 10x NEBuffer 0.8 μL, Cas9 protein 2 μL, ddH2O to 6 μL. The injection system was prepared and used immediately, first incubated at 37℃ for 5 min, and finally added 2 μL of phenol red, and the control group was added with the same amount of 25% phenol red.

[0056] 3. PCR test step two mutant efficiency of fertilized eggs microinjected 48 h, and F0 generation zebrafish were raised to adult fish;

[0057] 3.1 Detection of knockout efficiency, F0 generation zebrafish were raised: 20 48 hpf (48 hours past fertilization, 48 h after fertilization) injected embryos of each gene and control group were randomly taken, and genomic DNA was extracted by alkaline lysis method. The alkaline lysis method was to add 10 μL of 50 mM NaOH to each sample at 95℃ for 40 min, then add 1 μL of 1M Tris-HCl (pH8.0) vortex to mix to terminate digestion, centrifuge for 10 s, and take the supernatant to obtain the sample genomic DNA.

[0058] 3.2 PCR test mutation: the sample genomic DNA was used as a template, and the corresponding detection primers were used for PCR to test the sgRNA knockout efficiency. Among them, the corresponding upstream and downstream detection primers were: PPARγ-E2-SF 5'-TCGATTTTCTCTGTGTTGTTCG-3'; PPARγ-E2-SR 5'-CGTGTGTCCTGTAGTTCTCCTG-3'. The PCR reaction system for testing sgRNA knockout efficiency was: zebrafish genomic DNA 1 μL, upstream and downstream detection primers (0.4*2) μL, Taq enzyme 5 μL, ddH2O to 3.2 μL. The PCR reaction conditions were: 95℃ for 3 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, 35 cycles; 72℃ for 5 min; 4℃ for ∞. The PCR products were subjected to polyacrylamide gel electrophoresis test. Figure 2 For PPARγ gene knockout zebrafish egg 48 h mutation rate detection, the mutation rate detection was about 85%.

[0059] 3.3 The surviving embryos after injection were raised to 60 days, and the tail fins were digested by alkaline lysis to obtain genomic DNA, which was subjected to PCR to test the mutation and screen the PPARy gene deletion F0 generation zebrafish. The alkaline lysis method is as described in step 3.1, and the PCR test of the mutation is as described in step 3.2. Figure 3 The mutation of the PPARy gene knockout zebrafish F0 generation was observed to 2 months old. Figure 3 As a result, PPARy gene deletion F0 generation zebrafish with larger mutant fragments were screened out.

[0060] Four, the F0 generation zebrafish was backcrossed with wild type to obtain mutant heterozygous zebrafish, and the zebrafish of this generation was raised to 1 month old or more, and sequencing was performed to detect the frameshift after the gene was knocked out;

[0061] 4.1, the mutant heterozygous zebrafish obtained by backcrossing the F0 generation zebrafish with wild type was raised to 1 month old or more;

[0062] 4.2, the tail was cut and the fin bar was digested by alkaline lysis, and PCR amplification was performed using detection primers to test the gene mutation. The alkaline lysis method is as described in step 3.1, and the PCR test of the mutation is as described in step 3.2. Figure 4 The mutation of the PPARy gene knockout zebrafish mutant F0 generation backcrossed in this embodiment is shown in the figure, and one band is about 32 bp deletion.

[0063] 4.3, gene sequencing was performed on the heterozygous zebrafish individuals successfully inherited mutation, and the sequencing results were compared. Figure 5 The sequencing comparison diagram of the PPARy gene knockout zebrafish mutant in this embodiment (WT is wild type) is shown. The PPARy knockout individual is 12 bp and 20 bp less than the wild type at target points 1 and 2, respectively, a total of 32 bp, and the number of bases deleted is not a multiple of 3, so that the gene appears to be a frameshift mutation. The wild type zebrafish encodes a 115 aa amino acid chain, and the protein sequence is: HIYSSAVMLHRRFSVQKASLSADMVDTQTFGWPVGFGLSALELEELEDDTHSLDIKPFSTLDYSSISGIDYENNPTQNDPTPHMMDLTHMYSLQDTGELQDTRAHLQARTQQLQP; after the predicted frameshift mutation, the gene encodes a 103 aa amino acid chain, and the protein sequence is: HIYSSAVMLHRRFSVQKASLSADMVDTQTFGFGLSALELEELEDDTHSLDIKPFSTLDYSSISGIDYENNPTHDGPDAHVQLTGHRRTTGHTSPSTGQNTAATA.

[0064] 5. Select and self-pollinate female and male PPARγ mutant heterozygous zebrafish with consistent frameshift and knockout positions to obtain homozygous zebrafish containing F1 generation PPARγ mutation, which are PPARγ gene deletion mutant zebrafish homozygous, and raise them to adulthood.

[0065] Zebrafish DNA was extracted by tail cutting and alkaline digestion, and homozygous mutants were detected by polypropylene gel electrophoresis. The fish were then raised to adulthood. The specific procedures are as described in step 4.2. Figure 6 The polypropylene gel electrophoresis results of the F1 generation of PPARγ gene knockout zebrafish are used to examine the mutation status of the PPARγ gene knockout zebrafish after self-crossing in this embodiment (M is Marker DL 1000). Figure 6 It can be seen that nearly 1 / 4 of the zebrafish mutants have a 32bp deletion in both PPARγ bands compared to the wild type, which conforms to Mendel's laws of inheritance. This 1 / 4 is the F1 generation of homozygous mutants, which can be used to construct subsequent homozygous mutant lines with PPARγ gene deletion.

[0066] 6. Self-cross the obtained F1 generation PPARγ deletion homozygous individuals to obtain a PPARγ gene deletion mutation homozygous zebrafish line.

[0067] The obtained progeny were tailed and DNA was extracted by alkaline digestion. Mutations were examined by polypropylene gel electrophoresis. The specific procedures are as described in step 4.2. Figure 7 The polypropylene gel electrophoresis results of this embodiment show that the PPARγ gene in this zebrafish strain is deleted by 32 bp (M represents Marker DL 1000, and W represents the wild-type control). From Figure 7 As can be seen, this embodiment successfully established a homozygous mutant line of zebrafish with PPARγ gene deletion.

[0068] In this embodiment, the relative expression level of the PPARγ gene in the homozygous mutant line with gene deletion was analyzed, and the gene deletion type was significantly lower than that in the wild type. Figure 8 The PPARγ gene in zebrafish wild-type (WT) and mutant lines (PPARγ) - / - The relative expression levels in muscle, with a decrease in PPARγ expression by % in mutant muscle (* indicates a significant difference, P<0.05). Figure 8 As shown, PPARγ expression decreased by approximately 76% in muscle and approximately 60% in liver in the mutant strain. After PPARγ knockout, PPARγ... - / - In zebrafish mutant lines, the expression level of this gene was significantly decreased in key tissues such as the liver and muscle fat deposition, and PPARγ expression was also significantly reduced. - / - The zebrafish mutant line has been successfully constructed.

[0069] The total cholesterol (T-CHO) and total triglyceride (TG) contents in the muscles of the PPARγ gene deletion homozygous mutant line and wild type zebrafish were determined. The T-CHO and TG contents in the muscles of the gene deletion type were significantly lower than those of the wild type (P<0.001); Figure 9 The T-CHO content in the muscles of the PPARγ knockout zebrafish homozygous line was significantly decreased (WT is the wild type, *** indicates a significant difference P<0.001) due to the difference in the key index of fat deposition T-CHO content in the muscles of the two genotypes of zebrafish. Figure 10 The T-CHO content in the muscles of the PPARγ knockout zebrafish homozygous line was significantly decreased (WT is the wild type, *** indicates a significant difference P<0.001) due to the difference in the key index of fat deposition TG content in the muscles of the two genotypes of zebrafish. Figure 9 and Figure 10 It can be seen that the T-CHO and TG contents in the muscles of the example are about 0.015 and 0.335 mmol / gprot, respectively, indicating that knocking out the PPARγ gene significantly reduces the lipid level in the muscles of zebrafish. The PPARγ gene deletion homozygous mutant line of zebrafish can be used as an effective model for fish lipid regulation.

Claims

1. A method for preparing a PPARy gene deleted zebrafish, characterized by The method for preparing the PPARγ gene deletion zebrafish is carried out according to the following steps: I. Two target points at similar positions of the same exon are selected, and CRISPR probe primers for knocking out are designed according to the positions of the target points and the sequence of the PPARγ gene of the zebrafish, and two sgRNA chains are synthesized in vitro; II. The two sgRNA chains are combined with Cas9 protein and microinjected into zebrafish single-cell stage fertilized eggs; III. The mutation efficiency of the fertilized eggs 48 hours after injection is tested by PCR, and the F0 generation zebrafish is bred to adult fish; IV. The F0 generation zebrafish is backcrossed with wild type to obtain mutant heterozygous zebrafish, and the zebrafish of this generation is bred to be more than one month old, and the frameshift after knocking out the gene is detected by sequencing; V. Female and male PPARγ mutant heterozygous zebrafish with consistent knockout positions are screened for self-crossing to obtain F1 generation PPARγ mutant homozygous zebrafish, which is PPARγ gene deletion mutant zebrafish homozygote, and is bred to adult fish; VI. The F1 generation PPARγ deletion homozygous individual is self-crossed to obtain PPARγ gene deletion mutant homozygous zebrafish.

2. The method for preparing a PPARy gene deleted zebrafish according to claim 1, characterized in that Step I core target sequence design: two core target points are located on the same exon, and the core target sequence is: PPARγ-1 5'-GCGTCAGGTCCATCATGTGCGGG-3', PPARγ-2 5'-AGCGCTCAGGCCGAAGCCCACGG-3'.

3. The method for preparing a PPARγ gene-deleted zebrafish according to claim 1, characterized in that, The CRISPR probe primers in step I are composed of forward primers and reverse primers, wherein the forward primers are composed of T7 promoter sequence, modified core target sequence and sgRNA scaffold, and the reverse primers are composed of universal pT7 scaffold; The modified core target sequence of the core target PPARγ-1 is 5'-GCGTCAGGTCCATCATGTGC-3'; The modified core target sequence of the core target PPARγ-2 is 5'-AGCGCTCAGGCCGAAGCCCA-3'.

4. The method for preparing PPARγ gene-deleted zebrafish according to claim 3, characterized in that, The base numbers of T7 promoter sequence and sgRNA scaffold in the forward primer are 20 and 14 bp respectively, the T7 promoter sequence is 5'-TTCTAATACGACTCACTATA-3', and the sgRNA scaffold is 5'-GTTTTAGAGCTAGA-3'; the forward primer sequence of PPARγ-1 is: a, TTCTAATACGACTCACTATA GCGTCAGGTCCATCATGTGC GTTTTAGAGCTAGA; the forward primer sequence of PPARγ-2 is: b, TTCTAATACGACTCACTATA G AGCGCTCAGGCCGAAGCCCAGTTTTAGAGCTAGA.

5. The method for preparing a PPARγ gene-deleted zebrafish according to claim 3, characterized in that, The sequence of pT7 scaffold in the reverse primer is: 5'-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'.

6. The method for preparing a PPARγ gene-deleted zebrafish according to claim 1, characterized in that... The PPARγ-1 sgRNA strand and the PPARγ-2 sgRNA strand in step two are mixed at a concentration of 1:1; the microinjection system is: Cas9 protein 2 μL, 10×NEBuffer 0.8 μL, sgRNA 1600 ng, and ddH2O is supplemented to 6 μL.

7. The method for preparing PPARγ gene-deleted zebrafish according to claim 1, characterized in that, The test primers for PCR test of mutation efficiency in step three are: PPARγ-E2-SF 5'-TCGATTTTCTCTGTGTTGTTCG-3'; PPARγ-E2-SR 5'-CGTGTGTCCTGTAGTTCTCCTG-3'.

8. The method for preparing PPARγ gene-deleted zebrafish according to claim 1, characterized in that, In step three, when PCR test of mutation efficiency is performed, 20 eggs are digested to extract DNA, and the knockout efficiency is detected by polyacrylamide gel electrophoresis.

9. The method for preparing PPARγ gene-deleted zebrafish according to claim 1, characterized in that, In step four, when backcrossed with wild-type zebrafish, zebrafish of different genders are selected for 1:1 backcrossing.

10. The primer for deleting PPARy gene of zebra fish in the preparation of PPARy gene deletion zebra fish according to claim 1, wherein, The zebrafish PPARγ gene deletion primers are composed of two groups of primer pairs, PPARγ-1 primer pair and PPARγ-2 primer pair; ​ The sequence of the forward primer of PPARγ-1 is: a, TTCTAATACGACTCACTATA GCGTCAGGTCCATCATGTGCGTTTTAGAGCTAGA; The sequence of the forward primer of PPARγ-2 is: b, TTCTAATACGACTCACTATA G AGCGCTCAGGCCGAAGCCCAGTTTTAGAGCTAGA; The sequence of the reverse primer of PPARγ-1 and PPARγ-2 is the sequence of pT7 scaffold: 5'-GATCCGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'.

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