Method for screening fish low-temperature-resistant gene fthl28 and application thereof

Abstract

The application belongs to the technical field of molecular biology, and specifically discloses a screening method and application of a fish low-temperature-resistant gene fthl28. The screening method comprises the following steps: low-temperature acclimatization of zebrafish parents; microinjection of 4sU at the single-cell stage and low-temperature culture of fertilized eggs obtained by crossing the acclimatization group and the non-acclimatization group of parents, separation of newborn RNA, sequencing analysis, screening and obtaining of a candidate gene fthl28; and overexpression function verification of the fthl28 mRNA by embryo microinjection, which proves that overexpression of the fthl28 gene effectively improves the survival rate and normal development rate of embryos under low temperature, and determines that the fthl28 is a fish low-temperature-resistant gene. The application also discloses application of the fthl28 gene in preparation of low-temperature-resistant aquatic animal embryos and a method for making fish embryos resistant to low temperature by overexpression of the fthl28 gene, which provides a new target and scheme for stress-resistant breeding of aquatic animals.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically involving obtaining a hypothermia-resistant embryo model by cryogenically acclimating zebrafish parents, and screening for the key gene fthl28 in this model. By overexpressing this gene, embryos from zebrafish and tilapia parents that have not undergone cryogenic acclimation can also acquire hypothermia resistance. Background Technology

[0002] As poikilothermic animals, fish's physiological functions and metabolic activities are highly dependent on ambient water temperature. Low-temperature stress is a common abiotic stress factor in aquaculture, causing serious harm to fish on multiple levels. First, low temperatures directly inhibit fish's feeding, growth, and movement abilities, leading to prolonged rearing cycles and decreased feed conversion rates, resulting in direct economic losses. Second, continuous low-temperature stress disrupts the immune homeostasis of fish, causing damage to immune organs (such as the head kidney and spleen), disordered expression of immune-related genes, reduced antibody production, and decreased phagocytic cell activity, thereby significantly increasing fish's susceptibility to pathogens such as bacteria, viruses, and parasites, and inducing large-scale disease outbreaks. More seriously, severe low-temperature stress can lead to metabolic disorders, physiological failure, and even death in fish. Therefore, elucidating the molecular mechanisms of fish response to low-temperature stress, especially its interaction network with immune function, is of vital importance for breeding stress-resistant (low-temperature tolerant and disease-resistant) varieties, developing novel anti-stress strategies, and ensuring the stable and sustainable development of aquaculture.

[0003] In fish biology and genetics research, zebrafish (Danio rerio) has become an irreplaceable model organism, with advantages in several aspects. First, its high reproductive capacity, rapid embryonic development, and transparent embryos facilitate large-scale genetic manipulation and real-time observation of early development. Second, the zebrafish genome has been fully sequenced and exhibits high conservation with humans and other vertebrates, making it an ideal model for studying vertebrate gene function, disease mechanisms, and drug screening. Third, mature gene editing technologies (such as CRISPR / Cas9) are readily and efficiently applied in zebrafish, enabling the rapid construction of gene knockout, knock-in, or mutation models, providing a powerful tool for functional verification. Fourth, zebrafish possess a complete innate and adaptive immune system, making it an excellent model for studying fish immune response mechanisms and host-pathogen interactions. Therefore, using the zebrafish model to explore the function of candidate genes related to important traits in farmed fish (such as tilapia) offers significant advantages such as short cycle time, low cost, and in-depth mechanistic elucidation, providing crucial theoretical basis and pioneering targets for subsequent molecular breeding of farmed fish.

[0004] In molecular biology research, accurately analyzing the dynamic changes in gene expression is crucial. 4-Thiouridine (4sU) metabolic labeling technology is a recently developed technique for studying the transcriptome of nascent RNA with high spatiotemporal resolution. Its core advantage lies in its ability to specifically and efficiently capture newly synthesized RNA within a specific time window. Compared to traditional total RNA sequencing (RNA-seq), 4sU metabolic labeling effectively distinguishes between a steady-state RNA library and a transient transcriptional response, eliminating background interference from accumulated transcripts. Specifically, when 4sU is added to cells or in vivo culture systems, it is taken up by the cells and incorporated into the synthesized nascent RNA chain. Biotin labeling and affinity purification allow for the isolation of 4sU-containing nascent RNA for subsequent high-throughput sequencing analysis. This technology is particularly suitable for studying rapid transcriptional reprogramming events that occur in organisms in the early stages of environmental stress (such as sudden temperature changes or pathogen infection). It can precisely locate key regulatory genes and signaling pathways that are activated or inhibited immediately, providing an unprecedented dynamic perspective and precise data for understanding the initiation mechanisms of stress responses.

[0005] Ferritin heavy chain-like protein 28 (fthl28) is a gene in fish whose function is not yet fully understood. The ferritin family has traditionally been thought to play a central role in the storage and homeostasis of intracellular iron ions, buffering the toxicity of free iron and storing it for biosynthesis. However, recent studies have shown that ferritins, especially the heavy chain subunits, have functions far beyond simple iron storage, also participating extensively in processes such as oxidative stress responses, immune regulation, and cell protection. As a unique homolog of the ferritin heavy chain, fthl28 may have unique expression patterns and functions. In model organisms such as zebrafish, there are already clues suggesting that fthl28 expression may be regulated by environmental stress, indicating that it may play a role in the adaptive responses of fish to adverse environments. Given the profound interaction between iron metabolism and immune function (such as macrophage function and reactive oxygen species generation), exploring whether and how fthl28 participates in regulating the immune status of fish under low-temperature stress through modulating cellular iron homeostasis or iron-independent pathways is a novel research direction with potentially significant value. Clarifying its function may provide new molecular targets for regulating fish stress resistance traits (the synergistic effect of low-temperature tolerance and disease resistance) through gene editing and other methods. Summary of the Invention

[0006] The purpose of this invention is to provide a cold-resistant zebrafish model through low-temperature domestication, combined with optimized 4sU metabolic marker technology to screen key genes for fish cold tolerance, and based on this, to provide a method for making fish embryos cold-resistant by overexpressing the fthl28 gene.

[0007] To achieve the above objectives, the specific technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a method for screening fish cold-resistance genes, comprising the following steps:

[0009] S11. Low-temperature acclimatization

[0010] The purpose of this step is to establish a stable, low-temperature-adapted zebrafish model that can simulate the cooling process in actual aquaculture, in order to explore the "memory" effect of long-term low-temperature adaptation on the transcriptional program of parental germ cells and its impact on the low-temperature tolerance of offspring embryos.

[0011] The specific process is as follows:

[0012] Sexually mature (approximately 6 months old) wild-type AB strain zebrafish of uniform weight and size were selected as broodstock. The broodstock were gradually cooled from the standard culture temperature (28 ± 0.2℃) at a constant rate of 0.5℃ / hour until the target low temperature (e.g., 22℃). The cooling process was conducted in an independent recirculating aquaculture system to maintain water quality stability. After reaching the target temperature, the fish were continuously cultured under this low-temperature condition. The acclimatization period could be set at different time gradients (e.g., 1 week, 2 weeks, 8 weeks, 16 weeks) according to research needs. Throughout the acclimatization period, a 14-hour light / 10-hour dark light cycle was maintained. After acclimatization, broodstock were obtained in two groups: a non-acclimatized group (always kept at 28℃) and a low-temperature acclimatized group (long-term kept at 22℃).

[0013] The key point of this step is:

[0014] a. By adopting a slow cooling rate (0.5℃ / hour) instead of sudden cooling, the temperature change process in nature or a controlled aquaculture environment is simulated, enabling fish to initiate physiological and molecular-level adaptive regulation, eliminating the interference of acute stress, and being closer to actual production.

[0015] b. Multiple acclimatization time points can be selected to dynamically study the process of establishing low-temperature adaptability and screen out the acclimatization cycle in which adaptability reaches a stable state, laying the foundation for obtaining offspring materials with stable phenotypes in the future.

[0016] c. The germ cells of the obtained low-temperature acclimatized parents may carry low-temperature adaptation "imprints" mediated by epigenetic mechanisms, providing key experimental materials for studying cross-generational inheritance of stress resistance traits.

[0017] S12. Screening for key genes for cryogenic tolerance using 4sU metabolic markers

[0018] The purpose of this step is to specifically capture and identify newly transcribed RNA in the early stage of embryonic development (1k cell-stage), thereby accurately comparing and analyzing the immediate differences in transcriptional responses between embryos produced by cryogenically acclimatized and unacclimatized parents under cryogenic (20°C) stress, and screening out early key response genes that may be related to cryogenic tolerance.

[0019] The specific process is as follows:

[0020] (1) Embryo Acquisition and Processing: Female fish from the cryogenic acclimatization group were mated with male fish from the non-acclimatization group, and fertilized eggs were collected. Simultaneously, the non-acclimatization group parents were used as a control. During the single-cell stage of the fertilized eggs, 1 nL of a solution containing 50 mM 4-thiouridine (4sU) was injected into the cytoplasm of each embryo using microinjection. This solution contained 1 ng / μL of a non-toxic fluorescent dextran dye, which was used to confirm successful injection under a fluorescence microscope. The injected embryos were immediately placed in E3 medium at 20°C for 5 hours for cryogenic incubation.

[0021] (2) Isolation and sequencing library construction of newborn RNA:

[0022] ① Total RNA extraction: Sixty synchronously developing injection embryos were collected as a biological replicate, thoroughly homogenized using a grinder, and total RNA was extracted using the TRIzol method.

[0023] ② Chemical transformation: Take a portion of total RNA and add it to a transformation reaction system containing 10 mM m-chloroperoxybenzoic acid on ice. Incubate at 45°C for 1 hour. This step specifically converts the 4sU residues incorporated into the nascent RNA into cytosine derivatives (T-to-C conversion).

[0024] ③ Purification and reduction: After purification, the transformation product was incubated at 37°C for 30 minutes in a reducing buffer containing 10 mM dithiothreitol to stabilize the transformation product, and then purified again.

[0025] ④ cDNA Synthesis and Amplification: Using purified RNA as a template, full-length cDNA was synthesized using reverse transcriptase containing Oligo(dT) and template-converting oligonucleotides. Subsequently, high-fidelity DNA polymerase was used for PCR amplification to obtain a sufficient quantity of full-length cDNA.

[0026] ⑤ High-throughput sequencing: The amplified cDNA is constructed into an Illumina sequencing library and paired-end sequencing is performed using a platform (such as NovaSeq).

[0027] ⑥ Differential expression and functional enrichment analysis were performed on the sequencing results, and the fthl28 gene, which was most significantly upregulated and associated with iron homeostasis and oxidative stress, was selected as a candidate gene.

[0028] The key point of this step is:

[0029] a. Core Invention: Direct injection of 4sU into living embryos, rather than through traditional cell culture, enables precise labeling of the nascent transcriptome at the whole-animal level within a specific developmental window. Combined with subsequent chemical transformation and sequencing, it allows for the precise differentiation of nascent RNA from maternal RNA.

[0030] b. Co-injection of fluorescent dyes: This ensures visual quality control of the technical procedures. Only successfully injected embryos are used for subsequent analysis, guaranteeing the reliability of the data.

[0031] c. Early low-temperature stress setting: Embryos were cultured at a low temperature of 20°C, forming a gradient with the parental acclimatization temperature (22°C) and the standard temperature (28°C), in order to reveal the immediate transcriptional reprogramming ability of offspring embryos when faced with challenges below the parental adaptation temperature.

[0032] d. By comparing the differences in the neonatal transcriptomes of the cold-acclimated and non-acclimated offspring at the same low temperature, genes that are specifically activated or suppressed in the early stages can be directly screened out. These genes are more likely to be key factors in determining whether an embryo can successfully initiate a cold tolerance program.

[0033] S13. Functional validation of fthl28 overexpression

[0034] The purpose of this step is to perform gain-of-function verification on the candidate gene (fthl28) screened from the above metabolic marker analysis, and to directly examine its role in improving the low-temperature tolerance of fish embryos.

[0035] The specific process is as follows:

[0036] (1) Construction of overexpression vector and preparation of mRNA: Total RNA was extracted from zebrafish tissue and reverse transcribed into cDNA. Using this as a template, the complete protein coding sequence of the fthl28 gene was obtained by PCR amplification. The CDS was cloned into an in vitro transcription vector with a T7 promoter. Using a T7 in vitro transcription kit, a capping analog was added to the reaction system to prepare capped fthl28 mRNA. At the same time, capped mRNA of enhanced green fluorescent protein (eGFP) was prepared as a control.

[0037] (2) Embryo microinjection and low-temperature stress: Single-cell stage zebrafish and tilapia embryos from undomesticated parents were collected. The experimental groups were injected with fthl28 mRNA solution (zebrafish were injected with a solution containing 100 pg fthl28 mRNA, and tilapia were injected with a solution containing 200 pg fthl28 mRNA); the negative control group was injected with an equal volume of eGFP mRNA solution; uninjected wild-type embryos were cultured at 28℃ as a baseline control. Fluorescent dextran dye was added to all injection solutions to confirm successful injection.

[0038] (3) Phenotypic observation and analysis: Successfully injected embryos were used for subsequent culture and phenotypic analysis: The negative control group was cultured under standard conditions at 28℃ to observe embryo quality, while the eGFP injection group and fthl28 injection group were cultured under their specific critical low temperature stress conditions (zebrafish: 20℃; tilapia: 22℃). At multiple key time points during development (e.g., 0, 2, 4, 5, 8, 12, 24, 36 hpf), the survival, developmental rate and malformation rate of the embryos were observed and recorded under a microscope.

[0039] (4) Data analysis: The embryo survival rate of each group was statistically analyzed, and survival curves were plotted. The standard error was calculated using the binomial distribution approximation method, and statistical analysis (such as the Log-rank test) was used to compare whether there was a significant difference in the survival rate of the fthl28 overexpression group and the eGFP control group at low temperature. If fthl28 overexpression can significantly improve the survival rate of embryos at critical low temperature, it confirms that this gene has a positive function in enhancing the low temperature tolerance of fish.

[0040] The key point of this step is:

[0041] a. Functional closed-loop verification: Candidate genes obtained from upstream omics screening (metabolic marker sequencing) are directly overexpressed for phenotypic verification, forming a complete technical chain from "discovery" to "verification", which enhances the reliability and persuasiveness of the invention conclusions.

[0042] b. Directly demonstrate causal relationship: By comparing the survival difference between the overexpression group and the control group, it is possible to directly assess whether the gene is sufficient to improve cold tolerance, providing the most direct evidence for its use as a molecular breeding target.

[0043] c. Technological scalability: This validation system is not limited to fthl28, but is applicable to any candidate gene screened through the aforementioned or similar omics methods, thus forming a universal functional validation platform.

[0044] Secondly, this invention provides the application of the fthl28 gene obtained by the above-mentioned screening method in the preparation of hypothermia-resistant aquatic animal embryos. The aquatic animals include zebrafish, tilapia, and pufferfish. The application method involves microinjecting fthl28 mRNA into single-cell stage aquatic animal embryos to induce overexpression of the fthl28 gene.

[0045] Thirdly, the present invention provides a method for making fish embryos hypothermic by overexpressing the fthl28 gene, comprising the following steps:

[0046] S21. The coding sequence of the fthl28 gene in cloned fish was used to prepare capped fthl28 mRNA through in vitro transcription;

[0047] S22. Capped fthl28 mRNA was microinjected into single-cell stage embryos to obtain cold-resistant fish embryos.

[0048] Further, in step S21, cDNA obtained by reverse transcription of total RNA from fish tissue is used as a template to amplify the complete protein coding sequence of the fthl28 gene by PCR, and then in vitro transcription and capping are performed using the mMESSAGE mMACHINE™ T7 ULTRA kit to prepare capped fthl28 mRNA.

[0049] Further, in step S22, the injection dose of fthl28 mRNA is 100 pg when the fish is zebrafish and 200 pg when the fish is tilapia.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] 1. Significantly improved the accuracy and timeliness of screening genes for fish stress resistance traits, solving the technical problems of long breeding cycles and low efficiency.

[0052] This invention organically combines three major steps—low-temperature domestication, live embryonic neonatal transcriptome labeling, and rapid functional verification—to construct an efficient and targeted gene discovery and verification platform.

[0053] Precisely Targeting Key Early Response Genes: Traditional phenotypic selection (e.g., survival rate) or whole-tissue transcriptome sequencing struggles to distinguish developmental background noise from genuine stress response signals. This invention utilizes 4sU metabolic labeling technology to specifically capture newly generated transcripts within a specific time window (5 hpf) in live fish embryos. Using this technology, we successfully identified hundreds of newly generated transcripts with significantly different expression levels under cryogenic stress from undomesticated and cryogenically tamed offspring embryos. This directly focuses the research on key regulatory factors rapidly activated or inhibited during the initial stages of cryogenic stress, avoiding interference from maternal RNA, and resulting in more biologically targeted and timely screening results.

[0054] Bridging the gap between omics discovery and functional validation: This invention goes beyond gene screening, further utilizing microinjection of overexpressed mRNA to rapidly validate the gain-of-function of candidate genes (such as fthl28). This closed-loop design ensures that the screened genes have a clear functional orientation.

[0055] 2. This study revealed that parental cold-acclimatization can enhance the cold tolerance of offspring embryos by influencing the early transcriptional program, providing direct evidence and a new technological pathway for the study of the transgenerational inheritance mechanism of stress resistance traits in fish.

[0056] A key finding of this invention is that offspring embryos of parents long-term acclimatized to low temperatures exhibit different transcriptional response strategies when faced with new low-temperature challenges. Experimental data show that, compared to offspring from the non-acclimatized group, embryos from the low-temperature acclimatized group showed differential expression (FDR < 0.05) of over 2,000 genes in their early neonatal transcriptome at 20°C. These genes were enriched in pathways such as peroxisome, mitophagy, and MAPK. This indicates that the parents' low-temperature adaptation experience may "pre-program" the molecular pre-planning for offspring to cope with low-temperature stress through epigenetic information carried by gametes (such as oocytes), thereby giving them stronger adaptability. This discovery provides a solid theoretical basis for new breeding strategies that improve offspring stress resistance traits through parental environmental acclimatization.

[0057] 3. The key positive role of the fthl28 gene in enhancing the low-temperature tolerance of fish embryos was clearly verified, providing a new target with application potential for fish molecular breeding.

[0058] This invention, through rigorous functional verification experiments, obtained key experimental data supporting the function of the candidate gene fthl28, with concrete and significant results:

[0059] Significantly improved cryogenic survival rate: Under continuous cryogenic stress at 20℃, the survival rate of zebrafish embryos in the experimental group overexpressing fthl28 reached 41.8% at 24 hpf, while the survival rate of embryos in the control group injected with eGFP was only 15.7% (χ²). 2 =57.2, p<0.001). Statistical analysis showed a highly significant difference in survival curves between the two groups (P < 0.01). The cryoprotective effect was also verified in Nile tilapia, an important economically farmed species: under a critical cryo-stress condition of 22℃ for tilapia embryos, at 120 hpf, the survival rate of embryos in the experimental group overexpressing fthl28 reached 68.8%, significantly higher than the 31.3% of the eGFP control group (χ² = 57.2, p<0.001). 2 =18.6, p=0.011). This directly proves that artificially enhancing the expression of fthl28 is sufficient to significantly improve the survival ability of different bony fish embryos in unfavorable low-temperature environments.

[0060] Improved embryonic development: Microscopic observation showed that, compared with the control group, embryos in the fthl28 overexpression group exhibited less developmental delay at low temperatures, and their morphological processes, such as somnolence formation and eye development, were closer to those of the normothermic control group, with a significantly lower rate of malformations. Compared with the eGFP control group (1.2%), the proportion of zebrafish embryos with normal development in the fthl28 injection group was 37.9%; χ² 2 =96.4, p<0.001) was even higher. A similar developmental protective effect was observed in Nile tilapia: limited by early embryonic morphology observation, and using normal pigmentation as the developmental criterion, embryos in the fthl28 overexpression group at 120 hpf had clearly formed body surface pigmentation, and development progressed in an orderly manner; while embryos in the eGFP control group generally showed developmental arrest, with no visible pigmentation, exhibiting a typical hypothermia-induced phenotype. This indicates that fthl28 not only promotes survival but also protects normal development.

[0061] 4. A comprehensive research methodology that can be standardized and promoted has been developed, with promising economic and social benefits.

[0062] High technical versatility: The technical framework (domestication-marking-verification) of this invention is not only applicable to zebrafish and low temperature tolerance research, but after adaptive adjustments, it can be widely applied to the screening and verification of key genes for other important stress resistance traits (such as hypoxia tolerance and disease resistance) in other aquatic animals (such as tilapia, pufferfish, etc.).

[0063] Accelerating the breeding process: Traditional breeding relies on multiple generations of hybridization and phenotypic screening, which takes several years. This invention uses the zebrafish as a model organism as a "sentinel model," which can complete the entire process from gene screening to preliminary functional verification within a few months, greatly shortening the early target discovery cycle and reducing the risks and costs of blind breeding.

[0064] Promoting green and sustainable development: Improving fish varieties by exploring and utilizing their own stress-resistant genetic resources is an effective way to fundamentally reduce reliance on chemical drugs (such as anti-stress agents and antibiotics) in aquaculture. Developing new varieties with strong stress resistance helps reduce disease outbreaks and economic losses caused by environmental stress, which aligns with the national strategic needs for green, efficient, and sustainable development in aquaculture.

[0065] In summary, this invention, through an innovative combination of technologies, not only efficiently and accurately discovered fthl28, a key potential gene for enhancing fish's low-temperature tolerance, but more importantly, provides a complete technical system from mechanism exploration to functional verification. This system has significant scientific value in revealing the formation and intergenerational inheritance patterns of fish stress resistance, and provides new technical tools and candidate gene resources for molecular design breeding of new stress-resistant aquaculture varieties, which is expected to generate significant economic and social benefits. Attached Figure Description

[0066] Figure 1 Flowchart of 4sU metabolic labeling and chemical transformation in zebrafish embryos.

[0067] Figure 2 List of differentially expressed genes between the acclimated group and the non-acclimated group.

[0068] Figure 3 Figure 1 shows the experimental results of the effect of overexpression of fthl28 on the low-temperature tolerance of zebrafish embryos. In figure a, the observation results of embryonic development and survival under low-temperature stress are shown; in figure b, the embryo survival rate curves of each group from 0 to 36 hpf are shown; and in figure c, the normal development rate curves of each group from 0 to 36 hpf are shown.

[0069] Figure 4 Figure 1 shows the experimental results of the effect of overexpression of fthl28 on the low-temperature tolerance of Nile tilapia embryos. Figure a shows the observation results of embryonic development and survival under low-temperature stress in each group; Figure b shows the embryo survival rate curves for each group from 0 to 120 hpf. Detailed Implementation

[0070] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. Those skilled in the art can make modifications or substitutions without departing from the spirit and scope of this invention, and such modifications or substitutions should be covered within the protection scope of this invention.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The abbreviations and symbols used herein are explained below:

[0072] 4sU: 4-thiouridine, a uridine analog that can be used for metabolic labeling.

[0073] hpf: Hours post-fertilization, a unit of time for the embryonic development stage in zebrafish.

[0074] dpf: Days post-fertilization, a unit of time for zebrafish juveniles or adults.

[0075] TFEA: 2,2,2-trifluoroethylamine.

[0076] mCPBA: meta-chloroperoxybenzoic acid.

[0077] DTT: Dithiothreitol.

[0078] TSO: Template Switch Oligo.

[0079] eGFP: Enhanced Green Fluorescent Protein, used as a control in this invention.

[0080] Unless otherwise specified, all reagents used in the following embodiments are commercially available and conventional reagents, and all experimental procedures used are conventional procedures in the field unless otherwise specified.

[0081] Example 1

[0082] This embodiment uses the model organism zebrafish (AB strain) as the research object to provide a method for screening and verifying key genes for low-temperature tolerance in zebrafish.

[0083] 1. Low-temperature domestication of zebrafish parent fish

[0084] (1) Experimental animals and feeding: Healthy, sexually mature wild-type AB strain zebrafish (approximately 6 months old, with uniform weight and body length) were selected and fed in a standard system (28 ± 0.2℃, pH 7.0-7.5, conductivity 500-600 μS / cm, 14-hour light / 10-hour dark cycle). Artemia were fed to the fish at regular intervals and in fixed quantities every day.

[0085] (2) Establishing a low-temperature acclimatization group: 30 female parent fish were transferred to an independent recirculating aquaculture system. The water temperature was programmed to decrease from 28°C to 22°C at a constant rate of 0.5°C / hour. After reaching the target temperature, the conditions were maintained for stable culture for more than 2 weeks. Throughout the acclimatization period, the same light cycle and water quality management as the original system were maintained, and brine shrimp were fed regularly and in measured quantities every day.

[0086] (3) Establish a control group: Another 30 female parent fish and male fish were kept in a standard system at 28℃ as a non-domesticated control group.

[0087] (4) Evaluation of domestication effect: After two weeks of domestication, the fish in the low temperature domestication group were active, fed normally, and showed no obvious stress or symptoms, indicating that a stable low temperature adaptation model has been successfully established.

[0088] 2. Embryo Acquisition and 4sU Metabolic Markers

[0089] (1) Mating and egg collection: Natural mating was adopted. Two groups were mated: ① Acclimated group: acclimated female × non-acclimated male; ② Non-Acclimated group: non-acclimated female × non-acclimated male. Fertilized eggs produced within 10 minutes after the start of mating were collected and washed with E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4).

[0090] (2) Microinjection: Under a stereomicroscope, 1 nL of injection solution was injected into the cytoplasm of each embryo during the single-cell stage of the fertilized egg using a microinjector. The injection solution consisted of 50 mM 4sU, 1 ng / μL non-toxic fluorescent dextran dye, and DMSO as the solvent. Approximately 400 embryos were injected in each group to ensure a sufficient number of synchronously developing embryos for subsequent sampling.

[0091] (3) Low temperature stress culture: After injection, the embryos were randomly aliquoted into culture dishes containing E3 medium and immediately placed in a 20°C constant temperature incubator for culture in the dark until they developed to 5 hpf (1k cell stage). Within 2 hours after injection, the embryos were observed under a fluorescence microscope and those that did not show fluorescence (i.e., injection failure) were removed.

[0092] 3. Neonatal Transcriptome Sequencing Analysis

[0093] (1) Sample collection and RNA extraction: At 5 hpf, 60 embryos of the same developmental stage were collected from both the Acclimated and Non-Acclimated groups to form a biological replicate. A total of 6 independent replicates were set up (i.e., 6 independent mating, injection, culture, and collection were performed). After being flash-frozen in liquid nitrogen, the embryos were ground using a mechanical homogenizer, and total RNA was extracted using the TRIzol reagent method. RNA concentration, purity, and integrity were detected by NanoDrop One and 1% agarose gel electrophoresis.

[0094] (2) Chemical transformation and enrichment of 4sU-labeled RNA: Take 1 μg of total RNA from each sample and perform the following operations:

[0095] Chemical transformation: A 25 μL transformation reaction mixture (containing 600 mM TFEA, 1 mM EDTA, 100 mM sodium acetate, pH 5.2, and 10 mM mCPBA) was prepared on ice. RNA was added, mixed, and incubated at 45°C for 1 hour. mCPBA modifies the thio group of 4sU, converting it to a cytosine derivative, which mismatches with G during reverse transcription. This causes the cDNA site to change from AT pairing to AC pairing, resulting in a T→C mutation in the sequencing results (e.g., ...). Figure 1 As shown in the figure, this serves as a specific molecular tag for new RNA to distinguish between new and old transcripts.

[0096] Purification and reduction: The reaction product was cleaned up using RNA purification beads. Then, 20 μL of reduction buffer (10 mM Tris-HCl pH 7.4, 10 mM DTT, 100 mM NaCl, 1 mM EDTA) was added, and the mixture was incubated at 37°C for 30 minutes.

[0097] Further purification: The sample was purified again using RNA purification magnetic beads to obtain a sample enriched with nascent RNA.

[0098] (3) Library construction and sequencing: The above 5 transformed replicates and 1 untransformed replicate (as a negative control) were all processed as follows:

[0099] cDNA synthesis and amplification: Full-length cDNA was synthesized using a reverse transcription kit containing Oligo(dT) primers and TSO, followed by limited PCR cycle amplification.

[0100] Library construction and quality control: cDNA libraries were constructed using a high-throughput sequencing library construction kit, and library quality was tested on an Agilent Bioanalyzer 4200.

[0101] Sequencing: The qualified libraries were sequenced at 150 bp paired ends on the Illumina NovaSeq X plus platform.

[0102] (4) Bioinformatics analysis:

[0103] Data quality control and alignment: Fastp was used to filter the raw sequencing data for quality control. HISAT2 was used to align cleanreads to the zebrafish reference genome (GRCz11).

[0104] Newborn transcript quantification: Using software specifically designed to identify T-to-C transitions (introduced by 4sU transitions) (such as BAT-Seq2), newborn transcripts are accurately distinguished and quantified from the alignment results.

[0105] Differential expression analysis: The DESeq2 software package was used to compare the newborn transcriptomes of the low-temperature acclimatization group and the control group at 20℃ for 5 hpf. The selection criteria were: |log2FoldChange| > 1 and corrected P-value (FDR) < 0.05.

[0106] Functional enrichment analysis: GO functional annotation and KEGG pathway enrichment analysis were performed on differentially expressed genes.

[0107] Results and Analysis:

[0108] Analysis revealed significant differences in the neonatal transcription levels of 2,409 genes between the Acclimated and Non-Acclimated embryos at 5 hpf. Of these, 711 genes were upregulated and 678 were downregulated. KEGG enrichment analysis showed that maternally differentially expressed genes were enriched in peroxisomes, autophagy, MAPK (mitogen-activated protein kinase), mTOR (mechanical target of rapamycin), and FoxO signaling, while zygotic differentially expressed genes were enriched in mitophagy and mTOR signaling pathways, which have previously been associated with promoting cold resistance in fish and mammals. (From the list of differentially expressed genes...) Figure 2In our study, we selected ferritin heavy chain-like protein 28 (fthl28), one of the genes most significantly upregulated in the Acclimated group and associated with iron homeostasis and oxidative stress, as a candidate gene for subsequent functional validation. The level of nascent fthl28 transcripts in the Acclimated group was significantly increased by more than 20-fold compared to the Non-Acclimated group (FDR < 0.001).

[0109] 4. Functional verification of the fthl28 gene

[0110] (1) Preparation of overexpressed mRNA:

[0111] Gene cloning: Zebrafish embryos were collected and cultured at 28°C for 5 hpf. RNA was extracted from the zebrafish embryos using TRIzol and reversed into cDNA. The complete CDS region (coding region) of the fthl28 gene (NCBI accession number: NM_001113659.1) was obtained by PCR amplification using the cDNA as a template.

[0112] The PCR amplification product sequence is as follows:

[0113] (SEQ ID NO: 1)

[0114] The protein-coding sequence it encodes is as follows:

[0115] METCQIRQNYDSDCEASINKMISLELYAGYTYTSMAHYFKRDDVALNGFAKFFKKNSEEEREHAEKFMEFQNKRGGRIVLQDIKKPDRDVWDNGLTAMQCALQLEKNVNQALLDLHKVASQKGDPHLCDFLETHYLDEQVEAIKKLGDHITNLSKMDAGNNRMAEYLFDKHTLDS (SEQ ID NO: 2).

[0116] In vitro transcription: Using the mMESSAGE mMACHINE™ T7 ULTRA kit, PCR amplification products were transcribed in vitro and capped to prepare capped fthl28 mRNA. Capped eGFP mRNA was prepared in the same way as a negative control.

[0117] (2) Embryo injection and low-temperature stress experiment:

[0118] Single-cell embryos produced from undomesticated parents were randomly divided into three groups:

[0119] Experimental group: Zebrafish were injected with 1 nL of solution containing 100 pg fthl28 mRNA; Tilapia were injected with 2 nL of solution containing 200 pg fthl28 mRNA.

[0120] Negative control group: Zebrafish were injected with 1 nL of solution containing 100 pg eGFP mRNA; Tilapia were injected with 2 nL of solution containing 200 pg eGFP mRNA.

[0121] Blank control group: No injection, cultured under standard conditions at 28℃.

[0122] 1 ng / μL of fluorescent dextran dye was added to all injection solutions, and successful injection was confirmed under a fluorescence microscope.

[0123] Embryos from both the experimental and negative control groups were cultured in a cryogenic incubator at critical low temperatures (20°C for zebrafish and 22°C for tilapia). The blank control group was cultured at 28°C to observe whether embryonic development was normal.

[0124] (3) Phenotypic observation and data analysis:

[0125] At different time points, the number of surviving and developing embryos in each group was observed and counted under a microscope. Embryos that turned completely white or showed tissue disintegration were considered dead.

[0126] Survival analysis was performed using R (v4.3.3) and the survival package. Kaplan-Meier survival curves were plotted, and the Log-rank test was used to compare differences between groups.

[0127] 5. Results and Analysis:

[0128] Survival rate and developmental process analysis: such as Figure 3 As shown in (a), under continuous hypothermia stress at 20°C, embryonic development was significantly delayed from the gastrula stage (5–8 hpf), with the delay becoming increasingly pronounced at 12 hpf. Embryo mortality began to rise after 12 hpf, peaking at 24–36 hpf. Overexpression of the fthl28 gene significantly promoted embryonic survival and normal development under hypothermia.

[0129] Specifically, as shown in Figure 3(b), at the critical time point of 24 hpf, the survival rate of embryos injected with fthl28 mRNA was as high as 41.8%, while the survival rate of embryos injected with eGFP control mRNA was only 15.7%. There was a highly significant difference in survival rates between the two groups (χ²). 2 = 57.2, p < 0.001), indicating strong statistical power, clearly demonstrating the necessity of fthl28 for maintaining embryo survival under hypothermia.

[0130] More importantly, this invention not only focuses on the endpoint of survival rate, but also quantitatively assesses the developmental process of the embryo. For example... Figure 3 As shown in (c), at 24 hpf, the proportion of embryos that developed according to the normal time sequence (reaching the expected developmental stage) was 37.9% in the fthl28 overexpression group, while it was only 1.2% in the eGFP control group. The difference between the two groups was also highly significant (χ²). 2 = 96.4, p < 0.001). This indicates that the function of fthl28 is not only to passively "prevent death" but also to actively "maintain development," that is, to ensure the orderly conduct of embryonic life activities under low temperature stress. This result, combined with the aforementioned transcriptome data, confirms that the maternal-zygotic gene fthl28 is a key regulator of low temperature tolerance in zebrafish embryos.

[0131] Cross-species functional conservation verification: Extending this functional verification system to the important economic species Nile tilapia, a highly consistent phenotypic rescue effect was observed. For example... Figure 4As shown in (b), under critical low-temperature stress of 22℃, the survival rate of tilapia embryos in the eGFP control group showed a stepwise decrease from 48 hpf, dropping to 31.3% at 120 hpf; while overexpression of fthl28 significantly inhibited low-temperature-induced death, maintaining a survival rate of 68.8% at 120 hpf (Log-ranktest: p = 0.011). As shown in Figure 4(a), in terms of morphological progression, embryos in the room-temperature control group (Ctrl-28℃) developed normally and showed obvious pigmentation; at 22℃, embryos in the eGFP control group generally entered developmental arrest, and no obvious pigmentation was observed at 120 hpf; in contrast, embryos in the fthl28 overexpression group successfully broke through the developmental bottleneck caused by low temperature, showing obvious pigmentation and other normal developmental characteristics at 120 hpf.

[0132] Overall Conclusion: The functional validation experiments yielded strong and statistically significant phenotypic evidence. Overexpression of fthl28 significantly improved embryo survival and normal development rate under hypothermia. These data directly and quantitatively demonstrate the crucial and effective positive regulatory role of fthl28 in enhancing the hypothermia tolerance of fish embryos, providing irrefutable experimental support for its status as a candidate target for stress-resistant breeding.

Claims

1. A method for screening fish genes for cold tolerance, characterized in that, Includes the following steps: S11. Low-temperature acclimatization: The zebrafish parent fish are gradually cooled from the standard culture water temperature to the target low temperature at a constant rate. After reaching the target low temperature, they are continuously cultured to carry out low-temperature acclimatization and obtain the low-temperature acclimatized parent fish. S12.4sU metabolic marker screening: The hypothermia-acclimated parent and the non-acclimated parent were mated, and fertilized eggs were collected. Fertilized eggs obtained from mating the non-acclimated parent were used as controls. During the single-cell stage of the fertilized eggs, 4-sU solution was injected into the embryos by microinjection and cultured under hypothermic conditions. Then, the newborn RNA in the embryos was isolated, and the differences in the newborn transcriptome were sequenced and analyzed to screen for the candidate hypothermia-resistant gene fthl28. S13. Overexpression Function Verification: fthl28 mRNA was introduced into fish embryos via microinjection for overexpression. The embryos were cultured under low temperature stress and their survival was observed. The results showed that the survival rate of embryos introduced with fthl28 mRNA was significantly higher than that of the negative control group injected with eGFP mRNA, thus confirming that the fthl28 gene is a fish low temperature tolerance gene.

2. The screening method according to claim 1, characterized in that, In step S11, the standard aquaculture water temperature is 28±0.2℃, the constant rate is 0.5℃ / hour, the target low temperature is 22℃, and a light cycle of 14 hours of light / 10 hours of darkness is maintained during the acclimatization period.

3. The screening method according to claim 1, characterized in that, In step S12, the embryos injected with 4-sU solution were placed in E3 medium at 20°C for 5 hours for low-temperature culture.

4. The screening method according to claim 1, characterized in that, In step S12, the 4-sU solution contains a non-toxic fluorescent dextran dye.

5. The screening method according to claim 1, characterized in that, In step S13, the fish species include zebrafish and tilapia, with the zebrafish subjected to a low-temperature stress condition of 20°C and the tilapia subjected to a low-temperature stress condition of 22°C.

6. The application of the fthl28 gene screened by the screening method of claim 1 in the preparation of hypothermia-resistant aquatic animal embryos.

7. The application according to claim 6, characterized in that, The aquatic animals mentioned are zebrafish, tilapia, or pufferfish.

8. A method for making fish embryos hypothermic by overexpressing the fthl28 gene obtained by the screening method described in claim 1, characterized in that, Includes the following steps: S21. The coding sequence of the fthl28 gene in cloned fish was used to prepare capped fthl28 mRNA through in vitro transcription; S22. Capped fthl28 mRNA was microinjected into single-cell stage embryos to obtain cold-resistant fish embryos.

9. The method according to claim 8, characterized in that, In step S21, cDNA obtained by reverse transcription of total RNA from fish tissue is used as a template to amplify the complete protein coding sequence of the fthl28 gene by PCR. Then, the cDNA is transcribed in vitro and capped using the mMESSAGEmMACHINE™ T7 ULTRA kit to prepare capped fthl28 mRNA.

10. The method according to claim 8, characterized in that, In step S22, the injection dose of fthl28 mRNA is 100 pg when the fish is zebrafish and 200 pg when the fish is tilapia.

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