Low temperature stress biological model and construction method and its application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a low temperature stress biological model and a construction method and application thereof. In this study, a low temperature stress mouse model expressing NPAFP protein was successfully constructed by homologous recombination technology, and the stable expression of NPAFP protein in mice was driven by CAG promoter. These transgenic mice will serve as the basis for studying the function of NPAFP protein in mammals, especially the physiological response and mechanism under low temperature stress. By constructing a low temperature stress tilapia model that stably expresses NPAFP protein, the survival rate of tilapia in low temperature environment is improved, providing a new solution for the sustainable development of aquaculture. By constructing a low temperature stress zebrafish model that stably expresses NPAFP protein, the cold resistance function of NPAFP in aquatic organisms is further explored, and its potential in low temperature stress and oxidative stress protection is verified.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a low temperature stress biological model and a construction method and application thereof. Background Art

[0002] During the Cenozoic Era 65 million years ago, the Antarctic continent began to move slowly, and its temperature gradually dropped to below 0°C about 20 million years ago. Temperature is an important factor affecting the survival, reproduction and distribution of organisms. As the sea temperature dropped, most fish became extinct due to their inability to adapt (Verde C, Parisi E, di Prisco G. The evolution of thermal adaptation in polar fish [J]. Gene, 2006, 385: 137-145.). According to statistics, the Southern Ocean, which accounts for about 10% of the total area of ​​the Earth's oceans, has 50 families and 322 species of fish, accounting for only 1.3% of the total number of fish on Earth (Eastman J T. The nature of the diversity of Antarcticfishes [J]. Polar Biology, 2005, 28 (2): 93-107.). In extremely cold environments, some homeothermic vertebrates hibernate, while poikilothermic vertebrates that cannot regulate their body temperature, such as Antarctic icefish, have evolved adaptively, producing antifreeze proteins to prevent their body fluids from freezing, thus adapting to the extremely cold environment and surviving (DeVries AL, Wohlschlag D E. Freezing resistance in some Antarctic fishes[J]. Science, 1969, 163(3871): 1073-1075.). In the 1960s, scientists first discovered antifreeze glycoprotein (AFGP) in the blood of Antarctic notothenioid fish (Nototheniidae) (Chen L, DeVries AL, Cheng CH C. Convergent evolution of antifreeze glycoproteins in Antarctic notothenioid fish and Arctic cod[J]. Proceedings of the National Academy of Sciences of the United States of America, 1997, 94(8): 3817-3822.).

[0003] In the previous study (“Evolution of an antifreeze protein by neofunctionalization under escape from adaptive conflict”, Deng et al. PNAS, 2010.), it was first discovered and proved that the Antarctic fish Lycodichthys dearborni evolved an antifreeze protein AFPIII through neofunctionalization in the process of escaping adaptive conflict, and named it LdAFPII. This protein evolved from SAS-B and has multiple repeated functional domains (such as LdAFPIII-1 with one repeated functional domain, LdAFPIII-4 with four repeated functional domains, LdAFPIII-12 with twelve repeated functional domains, etc.), and each AFPIII domain contains 62 amino acids. The sequence of LdAFPII protein was published in the previous study (“Cloning and evolutionary analysis of the gene of the multimeric type III antifreeze protein of Antarctic eel Pout (Lycodichthys dearborni)”, Yu Jing et al., Acta Genetica Sinica, 2005, 32 (8): 789-794.). Each AFPIII domain contains 62 amino acids. Using alphafold3 to predict and optimize the protein structure, we changed the amino acids at positions 36 and 37 from KL to RI, and the amino acid at position 50 from D to E to increase the flexibility of the protein structure. In order to explore its application in neurological diseases, it was named NeuroProtection AFPIII (such as NPAFP1, NPAFP4, NPAFP12, etc.). NPAFP proteins with different domains have different antifreeze abilities. NPAFP1 contains one AFPIII domain, while NPAFP4 contains four AFPIII domains. According to research, NPAFP protein not only plays an important role in the antifreeze process, but also has the potential to improve the tolerance of cells and tissues to low temperature and oxidative stress.

[0004] Animal experimental models are an indispensable and important tool in medical and biological research. They are used to simulate human diseases to study their pathogenesis, drug efficacy and treatment methods. As a classic mammalian experimental model, mice are widely used in genetics, cell biology and drug development. In addition to mammalian models, the study of low temperature adaptability of aquaculture species is also of great ecological and economic significance. Tilapia (Oreochromis niloticus), as an important aquaculture species, has weak cold tolerance, which limits its cultivation in cold waters. Zebrafish (Danio rerio) has become an ideal model organism for studying low temperature tolerance and oxidative stress protection mechanisms due to its short life cycle, transparent embryos and easy genetic manipulation. Zebrafish is not only convenient for observing embryonic development, but also can be used to study the function of specific genes through genetic manipulation. Different models have their own characteristics and scope of application. Choosing a suitable animal model is crucial to the success of scientific research.

[0005] As global climate change continues to intensify, extreme cold weather poses a severe challenge to the survival and reproduction of animals, especially for those species that are originally adapted to warmer environments. Cold stress caused by cold environments not only affects the growth and reproduction of wild animals and plants, but also has a significant impact on aquaculture, agriculture and animal health. Therefore, exploring and developing molecular mechanisms and protection strategies that can enhance species' tolerance to low temperatures has become an important direction for current biological research and agricultural science and technology development. Although existing studies have revealed the potential of NPAFP proteins in antifreeze, how to effectively apply them to different species, especially mammals, aquaculture species and model organisms to improve their cold tolerance, is still a scientific problem that needs to be solved urgently. Summary of the invention

[0006] The present application found that by constructing an organism model that stably expresses NPAFP protein, it is possible to study the resistance of organisms to low temperature stress. Based on this, the present invention was completed.

[0007] In a first aspect, the present invention provides a low temperature stress biological model, wherein the mammalian model uses gene editing technology to introduce a constructed plasmid expressing NPAFP protein into the mammalian body to obtain an individual that stably inherits and expresses NPAFP protein.

[0008] Furthermore, the NPAFP protein is selected from NPAFP1, NPAFP2, NPAFP3 and / or NPAFP4; the DNA sequences of NPAFP1, NPAFP2, NPAFP3 and / or NPAFP4 are shown in SEQ ID NOs. 1 to 4, respectively.

[0009] Preferably, the NPAFP protein is NPAFP4.

[0010] Furthermore, the individual that stably inherits and expresses the NPAFP protein is a heterozygous individual or a homozygous individual.

[0011] Furthermore, the low temperature stress biological model includes a low temperature stress mammal model and a low temperature stress aquatic organism model.

[0012] Furthermore, the cold stress mammalian model includes the cold stress mouse model, the cold stress rat model and the cold stress monkey model.

[0013] In one embodiment of the present invention, when the cold stress mammal model is a cold stress mouse model, the constructed plasmid for expressing the NPAFP protein is a Donor vector plasmid, and the insertion site in the mouse body is H11.

[0014] Furthermore, the low temperature stress aquatic organism model includes a low temperature stress fish model, a low temperature stress bivalve model, a low temperature stress shrimp model and a low temperature stress phytoplankton model.

[0015] Furthermore, the low temperature stress fish model includes a low temperature stress tilapia model, a low temperature stress zebrafish model and a low temperature stress crucian carp model.

[0016] In one embodiment of the present invention, when the low temperature stress aquatic organism model is a low temperature stress tilapia model, the plasmid expressing the NPAFP protein and the Tol2 transposase are co-injected into the single-cell stage of the tilapia embryo.

[0017] In one embodiment of the present invention, when the low temperature stress aquatic organism model is a low temperature stress zebrafish model, the plasmid expressing the NPAFP protein and the Tol2 transposase are co-injected into the single-cell stage of the zebrafish embryo.

[0018] In a second aspect, the present invention provides a method for preparing a low temperature stress mouse model, wherein the low temperature stress mouse model is prepared by introducing a constructed plasmid expressing NPAFP protein into a mouse body through gene editing technology to obtain heterozygous and homozygous individuals that stably inherit and express NPAFP protein; the preparation method comprises the following steps: (1) Plasmid construction: Use CAG promoter to construct the Donor vector plasmid expressing NPAFP; (2) Construction of specific primers: Design specific primers targeting the NPAFP gene at the 5' and 3' ends of the H11 insertion site, as well as in the upstream and downstream regions of the H11 insertion site; (3) Microinjection: The sample in (1) was microinjected into the fertilized eggs of mice with a C57BL / 6JGpt background. The fertilized eggs that survived the injection were transplanted into pseudopregnant female mice and waited for them to become pregnant and give birth. (4) Genotyping: The F0 pups born to the recipient mice were tail-clipped and toe-clipped at 5-7 days old and numbered. Genomic DNA was extracted for PCR and sequencing to identify the genotype of the mice. (5) Revealing the function of the NPAFP gene: Using positive F1 generation mice for self-fertilization, we obtained 3-month-old heterozygous and WT mice of the same generation to further study and reveal the function of the NPAFP gene; (6) The model with NPAFP function is the cold stress mouse model.

[0019] Furthermore, the NPAFP protein is selected from NPAFP1, NPAFP2, NPAFP3 and / or NPAFP4; the DNA sequences of NPAFP1, NPAFP2, NPAFP3 and / or NPAFP4 are shown in SEQ ID NOs. 1 to 4, respectively.

[0020] Preferably, the NPAFP protein is NPAFP4.

[0021] In a third aspect, the present invention provides a method for preparing a low temperature stress tilapia model, wherein the low temperature stress tilapia model uses gene editing technology to co-inject a constructed plasmid expressing NPAFP protein and Tol2 transposase into tilapia embryos to obtain a heterozygous individual that stably inherits and expresses NPAFP protein; the preparation method comprises the following steps: (1) Construction of plasmid: Use CMV promoter to construct a plasmid expressing NPAFP; (2) Construction of specific primers: Design of primers specific to the NPAFP gene sequence; (3) Microinjection: The expression plasmid and Tol2 transposase were co-injected into the one-cell stage of tilapia embryos; (4) Genotype identification: Extract genomic DNA for PCR and sequencing to identify the genotype of tilapia; (5) Revealing the function of the NPAFP gene: Using the self-fertilized offspring of the positive F1 generation tilapia, the heterozygous siblings of 3-month-old and WT tilapia, further studies revealed the function of the NPAFP gene; (6) The model with NPAFP function is the low temperature stress tilapia model.

[0022] Furthermore, the NPAFP protein is selected from NPAFP1, NPAFP2, NPAFP3 and / or NPAFP4; the DNA sequences of NPAFP1, NPAFP2, NPAFP3 and / or NPAFP4 are shown in SEQ ID NOs. 1 to 4, respectively.

[0023] Preferably, the NPAFP protein is NPAFP4.

[0024] In a fourth aspect, the present invention provides a method for preparing a low temperature stress zebrafish model, wherein the low temperature stress zebrafish model is prepared by injecting a constructed plasmid expressing NPAFP protein and Tol2 transposase into zebrafish embryos through gene editing technology to obtain a heterozygous individual that stably inherits and expresses NPAFP protein; the preparation method comprises the following steps: (1) Construction of plasmid: Use CMV promoter to construct a plasmid expressing NPAFP; (2) Construction of specific primers: Design of primers specific to the NPAFP gene sequence; (3) Microinjection: The expression plasmid and Tol2 transposase were co-injected into zebrafish embryos at the one-cell stage; (4) Genotype identification: Extract genomic DNA for PCR and sequencing identification to identify the genotype of zebrafish; (5) Revealing the function of the NPAFP gene: Using the offspring of self-fertilized positive F1 generation zebrafish, heterozygous siblings of 3-month-old zebrafish and WT zebrafish, further studies revealed the function of the NPAFP gene; (6) The model with NPAFP function is the low temperature stress zebrafish model.

[0025] Furthermore, the NPAFP protein is selected from NPAFP1, NPAFP2, NPAFP3 and / or NPAFP4; the DNA sequences of NPAFP1, NPAFP2, NPAFP3 and / or NPAFP4 are shown in SEQ ID NOs. 1 to 4, respectively.

[0026] Preferably, the NPAFP protein is NPAFP4.

[0027] In a fifth aspect, the present invention provides a use of the low temperature stress biological model as described in the first aspect in studying oxidative stress-related diseases, wherein the oxidative stress-related diseases include but are not limited to oxidative stress caused by low temperature.

[0028] In a sixth aspect, the present invention provides an application of the low temperature stress biological model as described in the first aspect in aquaculture.

[0029] Beneficial Effects This study successfully constructed a mouse model expressing NPAFP protein through homologous recombination technology, and used the CAG promoter to drive the stable expression of NPAFP protein in mice. These transgenic mice will serve as the basis for studying the function of NPAFP protein in mammals, especially the physiological response and mechanism under low temperature stress.

[0030] By constructing a transgenic tilapia model that stably expresses NPAFP protein, the survival rate of tilapia in low temperature environments can be improved, providing a new solution for the sustainable development of aquaculture.

[0031] By constructing a zebrafish model that stably expresses NPAFP protein, we further explored the cold-resistance function of NPAFP in aquatic organisms and verified its potential in protecting against low temperature stress and oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Construction of mice stably expressing and inheriting NPAFP and experimental design.

[0033] Figure 2 Construction and experimental design of tilapia stably expressing and genetically expressing NPAFP.

[0034] Figure 3 Construction and experimental design of zebrafish stably expressing and genetically expressing NPAFP.

[0035] Figure 4 Cold stress experiment on mouse tilapia and zebrafish transfected with NPAFP. DETAILED DESCRIPTION

[0036] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0037] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified. Example 1 NPAFP and its nucleotide sequence NPAFP is a protein composed of multiple tandem repeats of AFPIII domains. NPAFP1 contains one AFPIII domain, NPAFP2 contains two AFPIII domains, NPAFP3 contains three AFPIII domains, and NPAFP4 contains four AFPIII domains; as follows: Nucleotide sequence of NPAFP1: ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA Nucleotide sequence of NPAFP2: ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA Nucleotide sequence of NPAFP3: ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAA Nucleotide sequence of NPAFP4: ATGAAGTCAGTTGTTTTAACTGGTTTGCTGTTCGTCCTCCTTTGTGTCGACCACATGAGTTCAGCCAACAAGGCGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAAGATGTGACGACATGTCCAGGCTTTAAGTCCGTGGTGGCCAACCAGCTGATCCCCATAAATACTGCCCTGACTCTAGTGATGATGAAGGCGGAGGAAGTCAGCCCAAAGGGCATCCCTGCCGAGGAGATCCCCAGAATAGTGGGAATGCAAGTGAACAGGGCAGTGTATCTGGAGCAAACCCTCATGCCAGATATGGTGAAAAACTATGAATAA Example 2 Construction of a mouse model stably expressing and inheriting NPAFP 2.1 Test method Construct plasmid: Construct a Donor vector plasmid expressing NPAFP using the CAG promoter; Construction of specific primers: Design specific primers targeting the NPAFP gene at the 5' and 3' ends of the H11 insertion site, as well as the upstream and downstream regions of the H11 insertion site (Tables 1 and 2); Microinjection: CRISPR / Cas9 and Donor vector were used to microinject the sample into the fertilized eggs of mice with C57BL / 6JGpt background. The fertilized eggs that survived the injection were transplanted into pseudo-pregnant female mice and waited for them to become pregnant and give birth. Genotyping: The F0 pups born to the recipient mice were numbered after their tails and toes were cut off at 5-7 days old, and their genomic DNA was extracted for PCR and sequencing to identify the genotype of the mice. Revealing the function of NPAFP gene: Using positive F1 generation mice to self-cross to obtain WT and positive mice of the same generation, further research revealed the function of NPAFP gene; The model with NPAFP function is the cold stress mouse model.

[0039] Table 1. Specific primers for the construction of homozygous mouse model of low temperature stress 5'arm F 1: AGTCTTTCCCTTGCCTCTGCT R 1: AGGCGGGCCATTTACCGTAAGTTA 3'arm F 2: TCAATCCAGCGGACCTTCCTT R 2: ATATCCCCTTGTTCCCTTTCTGC WT F 3: AGTCTTTCCCTTGCCTCTGCT R 3: GGGTCTTCCACCTTTCTTCAG Table 2. Specific primers for construction of heterozygous mouse model of low temperature stress 5'arm F 1: AGTCTTTCCCTTGCCTCTGCT R 1: AGGCGGGCCATTTACCGTAAGTTA 3'arm F 2: TCAATCCAGCGGACCTTCCTT R 2: GGGTCTTCCACCTTTCTTCAG WT F 3: AGTCTTTCCCTTGCCTCTGCT R 3: GGGTCTTCCACCTTTCTTCAG 2.2 Test results like Figure 1 As shown in Figure A, a Donor vector plasmid expressing NPAFP was constructed using the CAG promoter, and the NPAFP expression plasmid was inserted into the H11 site of C57BL / 6 mice using homologous recombination technology. Figure 1 As shown in B, specific primers targeting the NPAFP gene were designed at the 5' and 3' ends of the H11 insertion site, as well as the upstream and downstream regions of the H11 insertion site. Figure 1 Middle C and Figure 1 D in the middle is the result of genotyping mice by PCR electrophoresis technology, from which we can know that homozygous and heterozygous mice that stably inherit and express NPAFP1 protein were successfully obtained, and heterozygous mice that stably express NPAFP4 protein were also successfully constructed. These transgenic mice can be used to study the role of NPAFP in resistance to cold stress and diseases related to oxidative stress.

[0040] Example 3 Construction of a Tilapia Model Stably Expressing and Genetically Inheriting NPAFP 3.1 Test methods Construction of plasmid: The plasmid expressing NPAFP was constructed using the CMV promoter; Construction of specific primers: Design primers specific for the NPAFP gene sequence (Table 3); Microinjection: The expression plasmid and Tol2 transposase were co-injected into tilapia embryos at the one-cell stage; Genotype identification: Extract genomic DNA for PCR and sequencing identification to identify the genotype of tilapia; The function of NPAFP gene was revealed: The function of NPAFP gene was further revealed by using the offspring of self-pollinated positive F1 tilapia, heterozygous 3-month-old siblings and WT tilapia; The model with NPAFP function is the low temperature stress tilapia model.

[0041] Table 3. Specific primers for constructing the low temperature stress model of tilapia F ATGAAGTCAGTTGTTTTAACTGG R CTCATAGTTTTTCACCATATCTGG 3.2 Test results like Figure 2 As shown in Figure A, the CMV promoter was used to drive the expression of NPAFP protein, and the expression plasmid was co-injected with Tol2 transposase into the one-cell stage of tilapia embryos. Figure 2 As shown in B, the Tol2 transposase randomly inserts the NPAFP gene containing the CMV promoter into the genome of tilapia to form stable transgenic individuals. Due to the randomness of the insertion, the insertion site cannot be predicted in advance. Therefore, PCR amplification of the NPAFP gene sequence was performed using the designed specific primers, and the transgenic individuals were identified by PCR electrophoresis technology ( Figure 2 The test results are as follows Figure 2 As shown in D, heterozygous tilapia individuals that stably inherited and expressed NPAFP1 and NPAFP4 proteins were obtained.

[0042] Example 4 Construction of a zebrafish model with stable expression and genetic expression of NPAFP 4.1 Test methods Construction of plasmid: The plasmid expressing NPAFP was constructed using the CMV promoter; Construction of specific primers: Design primers specific to the NPAFP gene sequence (Table 4); Microinjection: The expression plasmid and Tol2 transposase were co-injected into tilapia embryos at the one-cell stage; Genotype identification: Extract genomic DNA for PCR and sequencing identification to identify the genotype of zebrafish; Revealing the function of NPAFP gene: Using the offspring of self-fertilized positive F1 generation zebrafish, heterozygous and WT zebrafish of 3-month-old siblings, further studies revealed the function of NPAFP gene; The model with NPAFP function is the low temperature stress zebrafish model.

[0043] Table 4. Specific primers for constructing a low temperature stress zebrafish model F ATGAAGTCAGTTGTTTTAACTGG R CTCATAGTTTTTCACCATATCTGG 4.2 Test results like Figure 3 As shown in Figure A, the CMV promoter was used to drive the expression of NPAFP protein, and the expression plasmid was co-injected with Tol2 transposase into zebrafish embryos at the one-cell stage. Figure 3 As shown in B, the Tol2 transposase randomly inserts the NPAFP gene containing the CMV promoter into the genome of zebrafish to form stable transgenic individuals. Due to the randomness of the insertion, the insertion site cannot be predicted in advance. Therefore, PCR amplification of the NPAFP gene sequence was performed using the designed specific primers, and the transgenic individuals were identified by PCR electrophoresis technology ( Figure 3 The test results are as follows Figure 3 As shown in D, zebrafish heterozygous individuals that stably inherit and express NPAFP1 and NPAFP4 proteins were obtained.

[0044] Example 5: Cold tolerance of animal models The obtained transgenic mouse, tilapia and zebrafish models were subjected to low temperature stress experiments.

[0045] 5.1 Mouse cold stress test Figure 4 Middle A and Figure 4 Panel B shows a low temperature stress experiment on mice transfected with NPAFP.

[0046] like Figure 4 As shown in A, transgenic mice and their WT siblings were treated at 4°C, and the tails of the mice were observed after 6 weeks.

[0047] like Figure 4 As shown in B, NPAFP significantly improved the transgenic mice's tolerance to low temperatures. Compared with the tails of WT mice, the tails of NPAFP mice were less frostbitten, and the tails of mice expressing NPAFP4 were more normal than those of mice expressing NPAFP1, and had a stronger ability to resist low temperatures.

[0048] 5.2 Tilapia low temperature stress test Figure 4 Middle C and Figure 4 Figure D shows the low temperature stress experiment of NPAFP-transfected tilapia.

[0049] like Figure 4 As shown in C, the transgenic tilapia and its sibling WT tilapia were gradually cooled and maintained at 10°C for 5 hours before being rewarmed to observe the survival of the fish.

[0050] like Figure 4 As shown in D, the statistical results show that NPAFP significantly improves the cold tolerance of transgenic tilapia, and the cold tolerance of tilapia expressing NPAFP4 is stronger than that of NPAFP1.

[0051] 5.3 Zebrafish low temperature stress test Figure 4 ZhongE and Figure 4 Figure F shows the low temperature stress experiment of zebrafish transfected with NPAFP.

[0052] like Figure 4 As shown in E, the transgenic zebrafish and their sibling WT zebrafish were treated at 12°C for 12 h and then rewarmed to observe the body condition of the fish.

[0053] like Figure 4 As shown in F, the phenotype observation indicated that NPAFP significantly improved the cold tolerance of transgenic zebrafish, and the effect of NPAFP4 was better than that of NPAFP1.

[0054] In summary, NPAFP significantly improves the cold tolerance of transgenic mice, tilapia and zebrafish, and the cold tolerance is proportional to the number of AFPIII domains. These transgenic mice, tilapia and zebrafish can be used for functional verification of resistance to cold stress and their application in oxidative stress-related disease models.

Claims

1. A low temperature stress biological model, wherein the low temperature stress biological model uses gene editing technology to introduce a constructed plasmid expressing NPAFP protein into an organism to obtain an individual that stably inherits and expresses NPAFP protein; the NPAFP protein is selected from NPAFP1, NPAFP2, NPAFP3 and / or NPAFP4; the DNA sequences of NPAFP1, NPAFP2, NPAFP3 and / or NPAFP4 are shown in SEQ ID NO.1~4 respectively; the individual that stably inherits and expresses NPAFP protein is a heterozygous individual or a homozygous individual.

2. A low temperature stress biological model as described in claim 1, wherein the low temperature stress biological model includes a low temperature stress mammal model or a low temperature stress aquatic organism model; the low temperature stress mammal model includes a low temperature stress mouse model, a low temperature stress rat model or a low temperature stress monkey model; the low temperature stress aquatic organism model includes a low temperature stress fish model, a low temperature stress bivalve model, a low temperature stress shrimp model or a low temperature stress phytoplankton model; the low temperature stress fish model includes a low temperature stress tilapia model, a low temperature stress zebrafish model or a low temperature stress crucian carp model.

3. A low temperature stress biological model as described in claim 2, when the low temperature stress mammal model is a low temperature stress mouse model, the constructed plasmid for expressing NPAFP protein is a Donor vector plasmid, and the insertion site in the mouse body is H11.

4. A low temperature stress biological model as claimed in claim 2, when the low temperature stress aquatic biological model is a low temperature stress tilapia model, the plasmid expressing the NPAFP protein and the Tol2 transposase are co-injected into the single-cell stage of the tilapia embryo.

5. A low temperature stress biological model as claimed in claim 2, when the low temperature stress aquatic biological model is a low temperature stress zebrafish model, the plasmid expressing the NPAFP protein and the Tol2 transposase are co-injected into the single-cell stage of the zebrafish embryo.

6. A method for preparing a low temperature stress mouse model, wherein the low temperature stress mouse model is prepared by introducing a constructed plasmid expressing NPAFP protein into a mouse body through gene editing technology to obtain heterozygous and homozygous individuals that stably inherit and express NPAFP protein; the NPAFP protein is selected from NPAFP1, NPAFP2, NPAFP3 and / or NPAFP4; the DNA sequences of NPAFP1, NPAFP2, NPAFP3 and / or NPAFP4 are shown in SEQ ID NO.1-4 respectively; the preparation method comprises the following steps: (1) Plasmid construction: Use CAG promoter to construct the Donor vector plasmid expressing NPAFP; (2) Construction of specific primers: Design specific primers targeting the NPAFP gene at the 5' and 3' ends of the H11 insertion site, as well as in the upstream and downstream regions of the H11 insertion site; (3) Microinjection: The sample in (1) was microinjected into the fertilized eggs of mice with a C57BL / 6JGpt background. The fertilized eggs that survived the injection were transplanted into pseudopregnant female mice and waited for them to become pregnant and give birth. (4) Genotyping: The F0 pups born to the recipient mice were tail-clipped and toe-clipped at 5-7 days old and numbered. Genomic DNA was extracted for PCR and sequencing to identify the genotype of the mice. (5) Revealing the function of the NPAFP gene: Using positive F1 generation mice for self-fertilization, we obtained 3-month-old heterozygous and WT mice of the same generation to further study and reveal the function of the NPAFP gene; (6) The model with NPAFP function is the cold stress mouse model.

7. A method for preparing a low temperature stress tilapia model, wherein the low temperature stress tilapia model is prepared by injecting a constructed plasmid expressing NPAFP protein and Tol2 transposase into tilapia embryos through gene editing technology to obtain heterozygous individuals that stably inherit and express NPAFP protein; the NPAFP protein is selected from NPAFP1, NPAFP2, NPAFP3 and / or NPAFP4; the DNA sequences of NPAFP1, NPAFP2, NPAFP3 and / or NPAFP4 are shown in SEQ ID NO.1-4 respectively; the preparation method comprises the following steps: (1) Construction of plasmid: Use CMV promoter to construct a plasmid expressing NPAFP; (2) Construction of specific primers: Design of primers specific to the NPAFP gene sequence; (3) Microinjection: The expression plasmid and Tol2 transposase were co-injected into tilapia embryos at the one-cell stage; (4) Genotype identification: Extract genomic DNA for PCR and sequencing to identify the genotype of tilapia; (5) Revealing the function of the NPAFP gene: Using the self-fertilized offspring of the positive F1 generation tilapia, the heterozygous siblings of 3-month-old and WT tilapia, further studies revealed the function of the NPAFP gene; (6) The model with NPAFP function is the low temperature stress tilapia model.

8. A method for preparing a low temperature stress zebrafish model, wherein the low temperature stress zebrafish model is injected into zebrafish embryos with a constructed plasmid expressing NPAFP protein and Tol2 transposase by gene editing technology to obtain heterozygous individuals that stably inherit and express NPAFP protein; the preparation method comprises the following steps: (1) Construction of plasmid: Use CMV promoter to construct a plasmid expressing NPAFP; (2) Construction of specific primers: Design of primers specific to the NPAFP gene sequence; (3) Microinjection: The expression plasmid and Tol2 transposase were co-injected into zebrafish embryos at the one-cell stage; (4) Genotype identification: Extract genomic DNA for PCR and sequencing identification to identify the genotype of zebrafish; (5) Revealing the function of the NPAFP gene: Using the offspring of self-fertilized positive F1 generation zebrafish, heterozygous siblings of 3-month-old zebrafish and WT zebrafish, further studies revealed the function of the NPAFP gene; (6) The model with NPAFP function is the low temperature stress zebrafish model.

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