Method and system for regulating and controlling attack behavior of snakehead based on prolactin PRL signal channel

Abstract

The invention belongs to the technical field of biology, discloses a method for regulating the attack behavior of channa argus based on a prolactin PRL signal channel, provides a transcriptional regulation channel of an olfactory prolactin receptor (PRLR) for regulating the attack behavior of channa argus, and finds that a prl gene and caor24 olfactory receptor differential expression are screened from attack difference individuals before and after channa argus breeding. The transcriptional regulation and control effect of the snakehead PRL / PRLR / STAT5 signal channel on caor24 is verified. The method for regulating and controlling the attack behavior of the snakeheaded fish based on the PRL signal channel is established, and a new thought is provided for regulation and control of the attack behavior of the fish. The method provides a practical solution thought for aquaculture attack behavior regulation and control.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a method and system for regulating the attacking behavior of snakehead based on prolactin PRL signal pathway. BACKGROUND

[0002] Snakehead (Channa argus) has a special reproductive behavior. During the breeding period, male fish chases and bites female fish, and completes mating by crossing the body. After mating, the male and female parents do not leave the nest, and after spawning, they hide in the nest bottom or nearby, guard the fish eggs and fry, and attack the organisms approaching the nest to prevent them from being preyed on by other predators. This parental care behavior significantly improves the survival rate of offspring. However, the parental aggression increases during the breeding period, which can easily cause the parents to be injured and die, resulting in serious economic losses. At present, there is still no effective artificial intervention and prevention method, and there is a lack of targeted regulation means. Therefore, it is necessary to conduct in-depth research on the mechanism of the attacking behavior of snakehead during the breeding period to find new regulation methods and provide new ideas for the regulation of the attacking behavior.

[0003] Fish live in a complex water environment and need to perceive chemical clues in the surrounding environment to respond. Pheromones play an important role in the life processes of animals such as mating and reproduction, showing social status and warning threats, and are an important medium for animal social communication. Fish perceive pheromones through the action of specific pheromone receptors, and many pheromone receptors are olfactory receptors. After the olfactory receptor binds to the ligand, it activates a series of downstream signal pathways, causing the excitation of neurons. Prolactin (PRL) is a polypeptide hormone synthesized and secreted by a group of lactating cells distributed in the anterior pituitary. It mediates downstream signal pathways through its receptor prolactin receptor (PRLR) on the cell membrane to exert biological functions. However, although prolactin has been proven to affect the parental care behavior of terrestrial mammals, there is less research on fish. Due to the unique physiological environment of fish and the complexity of water exposure, further research challenges are added.

[0004] Through the above analysis, the problems and defects of the prior art are:

[0005] The parental aggression increases during the breeding period, which can easily cause the parents to be injured and die, resulting in serious economic losses. At present, there is still no effective artificial intervention and prevention method, and there is a lack of targeted regulation means. SUMMARY

[0006] In view of the problems in the prior art, the application provides a method for regulating the attacking behavior of snakehead based on the prolactin (PRL) signal pathway.

[0007] The application is achieved, a method for regulating the attacking behavior of snakehead based on prolactin (PRL) signal pathway comprises:

[0008] Step 1, using transcriptome sequencing (RNA-seq) technology, screening prl gene and caor24 olfactory receptor differential expression in attacking differential individuals before and after snakehead reproduction;

[0009] Step 2, using E. coli expression system to express snakehead PRL recombinant protein rPRL in prokaryotic, verifying the interaction of snakehead PRL recombinant protein rPRL with PRLRa and PRLRb and the activation of downstream transcription factor STAT5;

[0010] Step 3, locating mRNA of prlr, stat5 and caor24 on olfactory capsule and proving co-expression in olfactory neurons, verifying the transcriptional regulation of snakehead PRL / PRLR / STAT5 signal pathway on caor24.

[0011] Further, the coding sequence of the caor24 gene is SEQ ID NO: 1.

[0012] Further, the rPRL protein is the protein encoded by the PRL gene of claim 1, and the amino acid sequence is shown as SEQ ID NO: 2;

[0013] The PRL recombinant protein rPRL is expressed in prokaryotic using E. coli expression system transformed by prokaryotic expression vector pET-PRL-His;

[0014] The coding sequence of the prlra gene is SEQ ID NO: 3;

[0015] The coding sequence of the prlrb gene is SEQ ID NO: 4;

[0016] The coding sequence of the stat5a gene is SEQ ID NO: 5;

[0017] The coding sequence of the stat5b gene is SEQ ID NO: 6.

[0018] Further, the recombinant plasmid pcDNA3.1-PRLRa, pcDNA3.1-PRLRb, pcDNDA3.1 empty eukaryotic expression plasmid and pSTAT5 activity report plasmid are transfected with pRL-TK renilla luciferase report plasmid as an internal reference, and the dual luciferase report gene detection system is used to analyze the difference of firefly luciferase activity between different groups; then the rPRL protein and solvent are used to stimulate cells, and the change of firefly luciferase activity is analyzed.

[0019] Further, the recombinant plasmid pcDNA3.1-PRLRa, pcDNA3.1-PRLRb, pcDNDA3.1 empty eukaryotic expression plasmid and pGL3-Pcaor24 reporter plasmid are transfected with the sea renilla luciferase reporter gene plasmid pRL-TK as an internal reference to measure the relative luciferase activity; the recombinant plasmid pcDNA3.1-STAT5a, pcDNA3.1-STAT5b, pcDNDA3.1(+) empty eukaryotic expression plasmid and pGL3-Pcaor24 reporter plasmid are transfected with the pRL-TK sea renilla luciferase reporter gene plasmid as an internal reference to measure the relative luciferase activity; the recombinant plasmid pcDNA3.1-STAT5a, pcDNA3.1-STAT5b or pcDNDA3.1(+) empty eukaryotic expression plasmid and pGL3-Pcaprlra or pGL3-Pcaprlrb reporter plasmid are transfected with the pRL-TK sea renilla luciferase reporter gene plasmid as an internal reference to measure the relative luciferase activity;

[0020] The transcriptional regulation of caor24 by the PRL / PRLR / STAT5 signal pathway is inhibited after treatment with 3nM of the JAK2 inhibitor TG101348.

[0021] Further, the regulation method is based on the physiological mechanism of the increased aggressiveness of the snakehead during the breeding period to reduce the damage of the parent fish during breeding.

[0022] The regulation method is based on the chemical communication mode and signal transduction of pheromones.

[0023] The species is Channa argus.

[0024] Another object of the present application is to provide a system for regulating the aggressiveness of Channa argus based on the prolactin (PRL) signal pathway, which comprises:

[0025] The screening module is used for screening the differential expression of prl gene and caor24 olfactory receptor in the different individuals before and after the breeding of Channa argus by using the transcriptome sequencing (RNA-seq) technology.

[0026] The verification module is used for verifying the interaction of Channa argus PRL recombinant protein rPRL with PRLRa and PRLRb and the activation of downstream transcription factor STAT5 by using the E. coli expression system to express the Channa argus PRL recombinant protein rPRL in prokaryotes.

[0027] The positioning module is used for positioning the mRNA of prlr, stat5 and caor24 on the olfactory capsule and proving co-expression in olfactory neurons, thereby verifying the transcriptional regulation effect of the PRL / PRLR / STAT5 signal pathway of the snakehead on caor24.

[0028] Another object of the present application is to provide a computer device comprising a memory and a processor, wherein the memory stores a computer program, and the computer program is executed by the processor to enable the processor to perform the steps of the method for regulating the aggression behavior of snakehead based on the prolactin (PRL) signal pathway.

[0029] Another object of the present application is to provide a computer readable storage medium storing a computer program, wherein the computer program is executed by a processor to enable the processor to perform the steps of the method for regulating the aggression behavior of snakehead based on the prolactin (PRL) signal pathway.

[0030] Another object of the present application is to provide an information data processing terminal for implementing the system for regulating the aggression behavior of snakehead based on the prolactin (PRL) signal pathway.

[0031] In combination with the above technical solutions and solved technical problems, the technical solution to be protected by the present application has the following advantages and positive effects:

[0032] Firstly, the present application proposes a behavior regulation strategy taking the prolactin (PRL) signal as the core for the aggression behavior of snakehead and other parent fish induced by low transparency of aquaculture water and dominant chemical communication. The scheme controls the excitability and threshold of the olfactory related pathway through the closed loop design around "PRL—receptor (PRLR)—downstream effector gene", reduces the territoriality and pairing conflict triggered by odor cues, thereby significantly reducing the body surface tearing and fin damage of parent fish, stabilizing the pairing and group structure, improving the continuity of the spawning and fertilization process, and finally improving the fry efficiency and survival consistency. Compared with the traditional method of relying only on isolation and separation of tanks, shielding or changing stocking density, the present method directly acts on the endocrine—sensory pathway of behavior occurrence, belongs to the "upstream—key link" regulation on the causal path, and has predictable and repeatable effects, and is more adaptable to production conditions.

[0033] At the molecular level, the present application first establishes a "PRL-PRLR-STAT5-caor24" regulation model, locks caor24 as the effector of PRL pathway downstream attack behavior, and clearly links the chain from hormone signal to sensory receptor expression to behavior output. The technical advantages of this model are: first, the target is clear, which is convenient for constructing a monitoring and feedback regulation framework around PRLR activation, STAT5 transcriptional activity and caor24 expression level; second, the path is explainable, which can be used as an intervention point to reduce interference with non-related neuroendocrine branches and reduce "non-target effects"; third, it has good migration, PRL-STAT5 is a commonly conserved signal axis in hard fish, which provides a standardized reference and evaluation index for subsequent horizontal verification and working condition adaptation in related economic fish.

[0034] Based on the above model, the present application further illustrates the transcriptional regulation of olfactory receptor genes (including caor24) by PRLR via STAT5, and establishes an operational methodology for "regulating the attack behavior of snakehead through PRL pathway": by controlling the process of pathway activity and target gene expression, the amplification gain of olfactory input and the behavior triggering threshold are kept in the "low conflict interval", and the olfactory-driven attack reflex triggered by low transparency is weakened from the source. This mechanism-level regulation replaces simple physical isolation and frequent human intervention, reduces stress and the risk of secondary infection, and balances output indicators and fish welfare; in key links such as high-density breeding of brood fish, breeding induction and fry transition, it can obtain more stable breeding performance and fry quality with less management cost, providing a feasible technical route and new ideas for the engineering management of attack behavior in aquaculture.

[0035] Second, as the creative evidence of the claims of the present application, it is also reflected in the following important aspects:

[0036] (1) The expected income and commercial value of the technical solution of the present application after transformation are:

[0037] The present application discloses a method and system for regulating the attack behavior of snakehead based on prolactin (PRL) signal pathway. After transformation, the present application can significantly reduce the attack behavior of snakehead brood fish during the breeding period, directly reduce the casualties of brood fish caused by fighting, and reduce the loss of valuable brood fish and economic cost. At the same time, a stable and low-stress breeding environment can improve the fertilization rate, hatching rate and healthy fry yield, directly increasing the breeding income. This targeted regulation method is more efficient and labor-saving than traditional manual intervention, which can reduce management cost and drug use, and meets the trend of green aquaculture. Facing snakehead and other potential high-aggressive fish species, this patent technology provides a possibility of chemical communication induction scheme, which has great commercialization potential and industrial value.

[0038] (2) The technical solution of the present application fills the technical gap in the industry at home and abroad:

[0039] The present application fills the technical blank in the field of aquaculture at home and abroad to engineer the attack behavior of fish by targeting specific physiological signal pathways. The core lies in that the prolactin (PRL) signal pathway is first used as a key target for regulating the attack behavior triggered by olfactory chemical communication during the breeding period of snakehead. Unlike traditional extensive behavior management methods, the present application starts from the source of attack behavior triggered by olfaction, and builds a clear regulation model of 'olfactory perception-PRL signal activation-attack behavior expression'. This provides a feasible engineering technology route based on clear molecular mechanism for the long-term lack of effective means to deal with the problem of snakehead aggressiveness in the aquaculture industry, and provides the possibility from basic theory discovery to practical application scheme.

[0040] (3) The technical scheme of the present application solves the technical problems that people have been eager to solve but have always failed to succeed:

[0041] The present application successfully solves the problem of attack behavior triggered by chemical communication during the breeding period of snakehead in aquaculture. The key regulation target of the prolactin (PRL) signal pathway is precisely locked to connect the external chemical communication signal of olfactory perception and the internal attack behavior expression. The method and system proposed by the present application can actively and accurately block the occurrence chain of attack behavior by targeting intervention of the pathway from the source of olfactory perception, and provide an efficient and controllable attack behavior solution for the industry. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 is a method flowchart for regulating the attack behavior of snakehead based on the prolactin (PRL) signal pathway provided by the embodiment of the present application.

[0043] Figure 2 is a system structure block diagram for regulating the attack behavior of snakehead based on the prolactin (PRL) signal pathway provided by the embodiment of the present application.

[0044] Figure 3 is an effect diagram provided by the embodiment of the present application.

[0045] Figure 4 is a comparison diagram of the number of olfactory receptor genes of different fish provided by the embodiment of the present application.

[0046] Figure 5 is a phylogenetic tree diagram of snakehead olfactory receptors provided by the embodiment of the present application.

[0047] Figure 6 is a phylogenetic tree diagram of snakehead OR provided by the embodiment of the present application.

[0048] Figure 7 is a principal component analysis (PCA) diagram of differentially expressed genes (DEG) of snakehead during the breeding period provided by the embodiment of the present application.

[0049] Figure 8 This refers to the number of differentially expressed genes in the olfactory sac and brain provided in the embodiments of the present invention. A. Bar chart of differentially expressed genes in the olfactory sac and brain; B. Venn diagram of co-expressed genes in each group of the olfactory sac and brain; C. Volcano diagram of differentially expressed genes in the brain; D. Volcano diagram of differentially expressed genes in the olfactory sac.

[0050] Figure 9 This is a heatmap of differentially expressed genes in the brain transcriptome of snakehead fish provided in an embodiment of the present invention.

[0051] Figure 10 This is a heatmap of differentially expressed genes in the olfactory sac transcriptome of snakehead fish provided in an embodiment of the present invention.

[0052] Figure 11 This is a predicted binding site map of STAT5 on the caor24 promoter provided in an embodiment of the present invention.

[0053] Figure 12 This is a qPCR verification diagram of the transcriptome results provided in the embodiments of the present invention.

[0054] Figure 13 This is the prokaryotic expression and purification of rPRL provided in the embodiments of the present invention. M: protein marker; (1): bacterial lysate before induction; (2): bacterial lysate after induction; (3): supernatant after induction; (4): precipitate after induction; (5): flow-through solution; (6): washing solution; (7): elution solution containing protein.

[0055] Figure 14 This illustrates the localization of prlra and prlrb mRNA in the olfactory sac of the snakehead fish, as provided in this embodiment of the invention. AD: Sections treated with antisense probes; E, F: Sections treated with sense probes; EG: HE staining of the olfactory sac.

[0056] Figure 15 This is a colocalization diagram of prlra, prlrb, and ompa in the olfactory sac of snakehead fish, provided in an embodiment of the present invention.

[0057] Figure 16 This is an rPRL activity verification diagram provided in an embodiment of the present invention.

[0058] Figure 17 This is a localization diagram of caor24 in the olfactory sac of snakehead fish provided in an embodiment of the present invention.

[0059] Figure 18 This invention provides examples of the co-localization of caor24, stat5, and prlr in the olfactory sac via fluorescence in situ hybridization. A: Co-localization of prlra, prlrb, and stat5; B: Co-localization of prlra, prlrb, and caor24.

[0060] Figure 19 This is a verification diagram of the transcriptional regulation of caor24 by PRLR and STAT5 provided in the embodiments of the present invention.

[0061] Figure 20 This is a verification diagram of STAT5's transcriptional regulation of prlr provided in an embodiment of the present invention. Detailed Implementation

[0062] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0063] like Figure 1 As shown in the figure, a method for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway provided by an embodiment of the present invention includes the following steps:

[0064] S101, using transcriptome sequencing (RNA-seq) technology, screened for differential expression of the prl gene and caor24 olfactory receptor in individuals with different attack patterns before and after breeding in snakehead fish;

[0065] S102, the recombinant protein rPRL of snakehead pRL was expressed in prokaryotes using the Escherichia coli expression system to verify the interaction between the recombinant protein rPRL and its receptors PLRRa and PLRB and its activation of the downstream transcription factor STAT5.

[0066] S103 was used to locate the mRNAs of prlr, stat5, and caor24 on the olfactory sac and demonstrate their co-expression in olfactory neurons, thus verifying the transcriptional regulatory role of the PRL / PRLR / STAT5 signaling pathway on caor24 in snakehead.

[0067] The coding sequence of the caor24 gene provided in this embodiment of the invention is SEQ ID NO: 1.

[0068] The rPRL protein provided in this embodiment of the invention is the protein encoded by the PRL gene as described in claim 1, and its amino acid sequence is shown in SEQ ID NO: 2;

[0069] The recombinant PRL protein rPRL was expressed in prokaryotes using an E. coli expression system transformed with the prokaryotic expression vector pET-PRL-His.

[0070] The coding sequence of the prlra gene is SEQ ID NO: 3;

[0071] The coding sequence of the prlrb gene is SEQ ID NO: 4;

[0072] The coding sequence of the stat5a gene is SEQ ID NO: 5;

[0073] The coding sequence of the stat5b gene is SEQ ID NO: 6.

[0074] The embodiments of this invention provide that recombinant plasmids pcDNA3.1-PRLRa, pcDNA3.1-PRLRb, pcDNDA3.1 empty eukaryotic expression plasmid, and pSTAT5 activity reporter plasmid, as well as pRL-TK Renilla luciferase reporter gene plasmid as an internal control, were transfected. The differences in firefly luciferase activity among different groups were analyzed using a dual luciferase reporter gene detection system. Then, rPRL protein and solvent were used to stimulate cells to analyze changes in firefly luciferase activity.

[0075] This invention provides examples of transfection with recombinant plasmids pcDNA3.1-PRLRa, pcDNA3.1-PRLRb, pcDNDA3.1 empty eukaryotic expression plasmid, and pGL3-Pcaor24 reporter plasmid, as well as with the René luciferase reporter gene plasmid pRL-TK as an internal control, to measure relative luciferase activity; and the recombinant plasmids pcDNA3.1-STAT5a, pcDNA3.1-STAT5b, pcDNDA3.1(+) empty eukaryotic expression plasmid, and pGL3- The Pcaor24 reporter plasmid and the pRL-TK Renilla luciferase reporter gene plasmid were transfected, and the relative luciferase activity was measured. The recombinant plasmids pcDNA3.1-STAT5a, pcDNA3.1-STAT5b or pcDNDA3.1(+) empty vector eukaryotic expression plasmids and pGL3-Pcaprlra or pGL3-Pcaprlrb reporter plasmids, as well as the pRL-TK Renilla luciferase reporter gene plasmid as an internal control, were transfected, and the relative luciferase activity was measured.

[0076] The transcriptional regulation of Caor24 by the PRL / PRLR / STAT5 signaling pathway was inhibited by treatment with 3 nM of the JAK2 inhibitor TG101348, and the transcriptional regulation of Caor24 induced by STAT5a and STAT5b was suppressed.

[0077] The regulation method provided in this invention is based on understanding the physiological mechanism of increased aggression in snakehead fish during the breeding season in order to reduce damage to parent fish during breeding.

[0078] The aforementioned regulation method is based on the chemical communication mechanism and signal transduction of pheromones;

[0079] The species mentioned is the snakehead (Channa argus).

[0080] likeFigure 2 As shown, an embodiment of the present invention provides a system for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway, comprising:

[0081] The screening module is used to screen for differential expression of the prl gene and caor24 olfactory receptor in individuals with different expression before and after breeding by using transcriptome sequencing (RNA-seq) technology.

[0082] The validation module is used to express the recombinant snakehead PRL protein rPRL in prokaryotes using the E. coli expression system, and to verify the interaction between the recombinant snakehead PRL protein rPRL and PLRRa and PLRB and its activation of the downstream transcription factor STAT5.

[0083] The localization module was used to locate the mRNAs of prlr, stat5, and caor24 on the olfactory sac and demonstrate their co-expression in olfactory neurons, verifying the transcriptional regulation of caor24 by the PRL / PRLR / STAT5 signaling pathway in snakehead.

[0084] The working principle of this invention is primarily manifested in the discovery and screening of differentially expressed targets. By conducting RNA-seq analysis on individuals exhibiting significant differences in aggressive behavior before and after breeding, genes with significantly different expression levels related to aggressive behavior were screened, particularly the endocrine hormone-related prl group (prl) and the olfactory receptor gene caor24. This process not only establishes a preliminary molecular link between aggressive behavior and the PRL signaling axis but also provides a gene-level entry point for subsequent functional validation and pathway construction, achieving a targeted transformation from phenotypic differences to molecular candidate targets.

[0085] In the target function validation phase, the system efficiently prepared recombinant snakehead pRL protein (rPRL) using an E. coli prokaryotic expression system. Protein-protein interaction analysis confirmed that rPRL specifically binds to two receptor homologs, PRLRa and PRLRab, triggering phosphorylation activation of the downstream transcription factor STAT5. The key significance of this step lies in verifying the integrity and function of the pRL signaling pathway in snakehead, demonstrating the molecular-level coupling between pRL and its receptor and downstream transcription factors, thus laying the theoretical and experimental foundation for pRL signaling to serve as an upstream node in the regulation of attack behavior.

[0086] In the spatial localization and regulatory mechanism analysis phase, the system performed in situ hybridization to locate the mRNAs of prlr, stat5, and caor24 in the olfactory sac tissue. The results showed that the three were co-expressed in olfactory neurons. Combined with transcriptional activity detection, it was demonstrated that the PRL / PRLR / STAT5 signaling pathway can directly regulate the transcriptional level of caor24, thereby affecting the reception and amplification of olfactory signals. This mechanism reveals the molecular pathway of aggressive behavior from endocrine signaling pathways through sensory organ input to behavioral output, realizing a causal closed loop from signal source to behavioral phenotype, and providing an operable technical principle for precise intervention of snakehead aggression behavior using the PRL pathway.

[0087] The purpose of this invention is to find new technical means to regulate the aggressive behavior of fish during the breeding season. It proposes a transcriptional regulatory pathway of olfactory prolactin receptor (PRLR) to regulate the aggressive behavior of snakehead fish, and establishes a method for regulating the aggressive behavior of snakehead fish based on the prolactin (PRL) signaling pathway, providing a new idea for the regulation of fish aggressive behavior.

[0088] To achieve the above objectives, the present invention adopts the following technical solution:

[0089] All olfactory receptor genes were identified from the whole genome. Differentially expressed genes (DEGs) in the olfactory sac and brain of individuals with different reproductive and attack patterns were obtained using RNA-seq technology. Increased expression of the prl gene was found in the brain of snakehead during the breeding season. The caor24 olfactory receptor gene was upregulated in the olfactory sac. The promoter of caor24 has multiple transcription factor signaling sites downstream of PRL and binding sites for transcription activator protein 5 (STAT5), indicating that caor24 may be a sex pheromone receptor regulated by PRL.

[0090] Preferably, the expression level of caor24 in the olfactory sac of male snakehead during the breeding season is significantly higher than that of females, which enhances the sensitivity of male snakehead to female pheromones and triggers male nest-protecting attack behavior.

[0091] In situ hybridization of the mRNAs of PRL receptors prlra and prlrb on the olfactory sac revealed that prlra and prlrb are expressed in the sensory epithelium of the olfactory epithelium and in olfactory neurons. Dual-luciferase reporter assays showed that rPRL can initiate signal transduction of the downstream JAK2-STAT5 pathway by interacting with PRLRa or PRLRb, activating STAT5 in HEK293T cells, and the activity of pSTAT5 showed a concentration-dependent effect with the concentration of rPRL protein added.

[0092] Preferably, the recombinant protein rPRL from snakehead pRL is expressed in prokaryotic E. coli using the prokaryotic expression vector pET-PRL-His transformed E. coli expression system.

[0093] In situ hybridization of caor24, prlr, and stat5 revealed that caor24 is expressed in microvilli neurons (mORNs) of the olfactory sac, and that caor24, prlr, and stat5 co-localize in activated olfactory neurons (ORNs) of the olfactory sac, indicating that PRLR in ORNs exerts transcriptional regulation on caor24 through STAT5. Dual luciferase reporter assays showed that both PRLR and STAT5 significantly increased the expression of the caor24 reporter gene plasmid. Treatment with the JAK2 inhibitor TG101348 at 3 nM inhibited the transcriptional regulation of caor24 induced by STAT5a and STAT5b, demonstrating that PRLR regulates caor24 expression through the JAK2-STAT5 signaling pathway. The PRL-PRLR-JAK2-STAT5-Caor24 signaling pathway increases the sensitivity of males to sex pheromones emitted by the opposite sex during the reproductive period, promoting aggressive behavior during this time. The positive feedback loop of STAT5a-PRLRa-STAT5a can amplify the effect of PLRa.

[0094] Experimental animals: Two-year-old male snakehead fish (40.25±7.80cm in length) with strong aggressive behavior and protecting their young after spawning were used in the experiment, while two-year-old male snakehead fish (38.75±4.33cm in length) with weak aggressive behavior and not in the breeding season were used.

[0095] The working principle of this invention is primarily manifested in the discovery and screening of differentially expressed targets. By conducting RNA-seq analysis on individuals exhibiting significant differences in aggressive behavior before and after breeding, genes with significantly different expression levels related to aggressive behavior were screened, particularly the endocrine hormone-related prl group (prl) and the olfactory receptor gene caor24. This process not only establishes a preliminary molecular link between aggressive behavior and the PRL signaling axis but also provides a gene-level entry point for subsequent functional validation and pathway construction, achieving a targeted transformation from phenotypic differences to molecular candidate targets.

[0096] In the target function validation phase, the system efficiently prepared recombinant snakehead pRL protein (rPRL) using an E. coli prokaryotic expression system. Protein-protein interaction analysis confirmed that rPRL specifically binds to two receptor homologs, PRLRa and PRLRab, triggering phosphorylation activation of the downstream transcription factor STAT5. The key significance of this step lies in verifying the integrity and function of the pRL signaling pathway in snakehead, demonstrating the molecular-level coupling between pRL and its receptor and downstream transcription factors, thus laying the theoretical and experimental foundation for pRL signaling to serve as an upstream node in the regulation of attack behavior.

[0097] In the spatial localization and regulatory mechanism analysis phase, the system performed in situ hybridization to locate the mRNAs of prlr, stat5, and caor24 in the olfactory sac tissue. The results showed that the three were co-expressed in olfactory neurons. Combined with transcriptional activity detection, it was demonstrated that the PRL / PRLR / STAT5 signaling pathway can directly regulate the transcriptional level of caor24, thereby affecting the reception and amplification of olfactory signals. This mechanism reveals the molecular pathway of aggressive behavior from endocrine signaling pathways through sensory organ input to behavioral output, realizing a causal closed loop from signal source to behavioral phenotype, and providing an operable technical principle for precise intervention of snakehead aggression behavior using the PRL pathway.

[0098] SEQ ID NO: 1

[0099] >caor24

[0100] ATGGAAAATAACTCTGAGATTATCTTTGTGCTTCAGGGTCTCAATGACTCCCTGACAAACCGACAGATTTACTTCACCTTGGCTCTGATGTTGTACGTCTTCACCATCCTTGTCAACCTGATTCTGATCGTTACTATCGGTCTGGAGACAACGCTCCATGAGCCCATGTACGTCTTCGTGTGCAACCTGTGTGTTAACGGCATCTGTGGAGCGTCAAGCTTCTACCCCAAGTTTCTTCGTGACCTTCTGGCCGACTCTCAAACAATCACATACTCTGGCTGTTTGACTCAGATCTTTGTGGCTTATTGTTATGTTTTCTGTGAATTCACCAATCTCACTGTTATGGCGTACGACCGCTACCTGGCCATCTGCAAGCCGCTGCAGTACCAGGCGCTAATGTCGATGCACAAAGTGGCAAAGCTGCTGCTGCTCACCTGGTGCTTTTCGCTGTTGGAGACATTTGTTGGTGTTCTCCTGACAGCCAGGCTCACCATCTGCAGCCGCCATGTCGGCAAGATATTCTGCACCAACTGGGAGGTGGTGAAACTGTCGTGCTCCAACACGGCCGGCAACAACATCTATACCTTCGTTCTCACATTCTCACACTTATCTCAAACAGGGCTCATTCTGGTGTCGTACACCCGCCTAGTCAGAACGTCGCTGCAGTCACGCTCCAGCCACAGAAAGTTCTTTCAGACATGTCTGCCACACCTTATCGCTCTGCTCGTCTTTGCCACTTCGCTGTTATTCGACGTCATGTACTCCCGTTATGGTGGTGGCACGCTGCAGACGCTGCAGAATGTGCTAGCTGTAGAGTTTCTGGTTATCCCACCTCTCATCAACCCCATCATCTATGGCATTAAACTGCAGCAGATCAGGTCCAGGATTGTCCACAACTTTACCCACAAACCAGAGCTTCTAAGATTATTCTAA

[0101] SEQ ID NO:2

[0102] >prl

[0103] MAHRGTKGSKLFMTLLYMVASCSAVPINDLLDRASQRSDKLHSLSTMLTQELDSHFPPIGRVLMPRPSMCHTSSLQTPNDKEQALQVSESDLLSLARSLLQAWVDPLEVLSTNANTLPHPAQNSIFNKIQELQEHSKNLEDGLDILSGKMGPAAQTISSLPYSGGNDIGQDKISKLTNFHFLLSCFRRDSHKIDSFLKVLRCRAAKLQPE

[0104] MCSEQ ID NO:3

[0105] >Channa_chr19_0353[mRNA]prlra

[0106]

[0107] >Channa_chr12_0725[mRNA]prlrb

[0108]

[0109] SEQ ID NO:5

[0110] >STAT5A

[0111]

[0112] SEQ ID NO:6

[0113] >STAT5B

[0114]

[0115] I. Specific application areas or related products of this invention.

[0116] Example 1

[0117] Identification of gene family members of olfactory receptors in snakehead fish and screening of hormone and sex pheromone receptors that play a role in breeding-season aggressive behavior.

[0118] Protein sequences of olfactory receptors (OR), trace amine-associated olfactory receptors (TAAR), type I vomeronasal receptors (V1R), and type II vomeronasal receptors (V2R) were obtained from various fish species, including zebrafish (Danio rerio), medaka (Oryzias latipes), stickleback (Gasterosteusaculeatus), red-finned pufferfish (Takifugu rubripe), and green pufferfish (Tetraodon nigroviridis). Protein-coding sequences obtained from the *Channa argus* genome (GCA_027943205.1) in NCBI were retrieved using TBLASTN. The obtained nucleic acid sequences were translated into protein sequences using TBtools. BLASTP was used to search the NCBI non-redundant protein database, and the sequences with the highest scores were identified. GPCRs for non-olfactory genes were removed. A phylogenetic tree was constructed from the obtained sequences, and the resulting phylogenetic tree was then refined. After the RNA samples passed quality control, cDNA sequencing libraries were constructed and transcriptome sequencing was performed. gDNA was removed from the RNA, and the first-strand cDNA was synthesized via reverse transcription. The resulting product was detected by qPCR. The expression detection primers are as follows:

[0119] Table 1. Primers used for qPCR validation of transcriptome results.

[0120]

[0121] Based on OR sequences from other fish species, 116 ORs, 65 TAARs, 6 V1Rs, and 65 V2Rs were identified from the snakehead genome (see [link to relevant data]). Figure 4 , Figure 5 Snakehead fish possesses 5 β-class ORs, 2 γ-class ORs, 63 δ-class ORs, 9 ε-class ORs, 26 ζ-class ORs, and 11 η-class ORs. The δ-class ORs are the most numerous, accounting for 50% of the total ORs (see...). Figure 6 Transcriptome sequencing and principal component analysis were performed on the brains and olfactory sacs of snakehead fish exhibiting aggressive behavior during both the non-breeding and breeding seasons. The results showed that the brains of snakehead fish at different time periods exhibited good separation and clustering (see...). Figure 75656 DEGs were identified in the brain of snakehead fish, of which 3567 genes were upregulated and 2089 genes were downregulated. 848 differentially expressed genes were identified in the olfactory sac, of which 431 genes were upregulated and 417 genes were downregulated. 12891 genes were co-expressed in the brain tissue of breeding snakehead fish compared to non-breeding snakehead fish, and 12946 genes were co-expressed in the olfactory sac of breeding snakehead fish compared to non-breeding snakehead fish (see...). Figure 8 Fifteen differentially expressed genes associated with parental care and aggression were identified from the transcriptome of snakehead brain. Genes associated with parental care and aggression during the breeding season include prl, c-fos, oxt, gal, bndf, nts, and cartpt; genes associated with aggression include ar, c-fos, hrh2, crh, npy2, npy2r, slc6a3, drd5, and sstr4 (see [link to relevant documentation]). Figure 9 Three olfactory receptors showed significant upregulation in aggressive and maternal behaviors after breeding in the olfactory sac of the snakehead (see [link to relevant documentation]). Figure 10 The downstream pathway of PRL primarily activates the JAK2-STAT5 signaling pathway via the receptor PRLR to transduce signals and regulate gene expression. By selecting all STAT5-recognized motifs from mice in the Jaspar database and scanning the promoters of the three olfactory receptor genes up to the start codon (2000 bp), multiple STAT5 binding sites were found only at the caor24 promoter (see...). Figure 11 Eight genes were selected from the DEG of the brain and olfactory sac for qPCR validation. As shown in the figure, the differences in gene expression levels during transcription were almost identical to the differences in expression levels obtained from qPCR (see figure). Figure 12 ).

[0122] Example 2

[0123] Prokaryotic expression of recombinant PRL protein (rPRL) from snakehead fish

[0124] Escherichia coli Rosetta-gami B(DE3) strain transformed with the prokaryotic expression vector pET-PRL-His was inoculated into 200 mL of LB liquid medium containing ampicillin and chloramphenicol at a ratio of 1:100. The culture was shaken at 37°C and 180-200 rpm until OD600≈0.4. 10 mL of the bacterial culture was retained as the sample before induction (1). The remaining bacterial culture was inoculated with IPTG at a final concentration of 0.5 mM and induced at 16°C and 120 rpm for 12-16 h. After centrifugation at 3500 rpm for 10 min and discarding the supernatant, 20 mL of lysis buffer was added. The culture was lysed using an ultrasonic homogenizer at 60 W power for 10 s, stopped for 10 s, and then lysed for 30 min. A small amount of the lysed liquid was taken as the sample after IPTG induction (2). The remaining liquid was centrifuged at 4°C and 12000 rpm for 20 min. A small portion of the supernatant was taken as the sample (3). The precipitate was resuspended in lysis buffer and taken as the sample (4). NTA-Ni packing material (Beyotime, Shanghai) was loaded into a gravity column. After equilibration with PBS, the remaining liquid from the previous step was filtered through a 0.45 μm filter and poured into the gravity column. The liquid was repeatedly flowed through the gravity column, and the liquid was left in the gravity column. The packing material was mixed, and the column was placed on ice and shaken for 1 hour. The liquid in the gravity column was released (5). The column was washed with 6 gravity column volumes of washing buffer (6), and 3 packing material volumes of elution buffer (7) were added to elute the protein. The elution buffer was dialyzed overnight with PBS containing 10% glycerol. The protein concentration was determined using a BCA protein concentration assay kit (Beyotime, Shanghai) and an ELISA reader (Agilent, USA). The protein was filtered through a 0.45 μm filter in a clean bench and aliquoted into 1.5 mL molecular tubes. The tubes were then flash-frozen in liquid nitrogen and stored at -80°C. The seven components obtained above were added to protein loading buffer and denatured at 95°C for 5 min. The effects of protein induction, expression and purification were observed by SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue G250 staining.

[0125] Example 3

[0126] Localized expression of prolactin and its receptor in snakehead fish

[0127] To maintain good tissue morphology, the olfactory sac of the anesthetized snakehead fish, along with the surrounding bone tissue, was removed. The olfactory sac tissue was fixed in 1×PBS containing 4% paraformaldehyde for 18 hours. The olfactory sac was then separated from the bone tissue with forceps. The olfactory sac was dehydrated by a gradient of alcohol, cleared with xylene, and embedded in paraffin. The tissue was cut into 7μm thick paraffin strips using a tissue slicer. The strips were then spread and baked in DEPC water containing 30% alcohol and DEPC water at 38℃, respectively. The slides were then transferred to anti-detachment glass slides and dried overnight in a 37℃ oven.

[0128] RNA probe synthesis: The *ompa* gene of *Channa argus* was cloned. Primers were designed for a specific region of 300-700 bp on the CDS of the gene. A protective base (CGC) and the SP6 polymerase promoter (ATTTAGGTGACACTATAGAAGCG) were added to the 5' end of the upstream primer, and a protective base (CCG) and the T7 polymerase promoter (TAATACGACTCACTATAGGGAGACA) were added to the 5' end of the downstream primer (see Table 2). Using a single-clone bacterial culture as a template, DNA was amplified by PCR. After purification by agarose gel electrophoresis and gel extraction, in vitro transcription was performed to obtain RNA probes labeled with digoxigenin (DIG) or biotin. The DNA template was digested with DNase I, and the RNA was purified using the lithium chloride / ethanol method. RNA concentration was detected using an OTC (Optical Stability Test Cell) analyzer, and probe integrity was checked by 1.5% agarose gel electrophoresis. The obtained RNA probes were stored at -80°C.

[0129] Table 2 Primers used for in situ hybridization

[0130]

[0131] In situ hybridization using the NBT-BICP chromogenic system: Sections were dewaxed and rehydrated with xylene and 100%, 95%, 85%, and 70% ethanol and 1×PBS. They were then washed with 0.2M HCl to inactivate endogenous alkaline phosphatase. After PBS washing, the tissue was immersed in PBS containing 10 μg / mL proteinase K at 37°C for 5 min. PBS washing removed the proteinase K. The tissue was then washed with triethanolamine (TEA) solution (pH = 8.0) containing 0.25% acetic anhydride for 15 min to acetylate positively charged proteins and prevent them from adsorbing negatively charged RNA probes. After washing with 5×SSC, the tissue was pre-hybridized with hybridization buffer at the hybridization temperature for 1 h. The RNA probe-containing hybridization buffer was then denatured at 95°C for 5 min, placed on ice for 2 min to unravel the higher-order structure of the RNA, and used to cover the tissue. Hybridization was then performed in a molecular hybridization furnace at 55°C for 16–20 h. The following day, the sections were washed successively with SSC and MABT buffers at concentrations of 5×, 2×, 1×, and 0.2×. After blocking with in situ hybridization blocking solution for 30 min, the tissues were covered with anti-DIG Fab fragment monoclonal antibody diluted 1:400 in blocking solution and incubated at room temperature for 2 h. After incubation, the sections were washed four times with MABT to remove unbound antibody. The sections were then developed using the BCIP / NBT alkaline phosphatase chromogenic kit (Beyotime, Shanghai) for 30-60 min. After development, the sections were passed through 70%, 85%, 95%, and 100% ethanol and xylene, mounted with neutral resin, and observed and photographed using an optical microscope.

[0132] Fluorescence in situ hybridization co-localization: Endogenous horseradish peroxidase (HRP) in the tissue was inactivated with 3% H2O2 before washing with 0.2M HCl, and an RNA probe labeled with digoxigenin and biotin was added simultaneously during hybridization. After blocking, the antibody was incubated with HRP-labeled streptavidin for 2 hours, followed by MABT washing and Tyramide SuperBoost. TM Kits with Alexa Fluor TM Develop with 647 Tyramides for 20 min. Cover tissue with 3% H2O2 for 20 min to inactivate HRP-containing antibodies. Block with blocking solution for 30 min, then incubate with HRP-labeled DIG antibody Anti-DigPOD(Poly)fab fragments for 2 h. Wash 4 times with MABT and then use Tyramide SuperBoost. TM Kits with Alexa Fluor TM After developing the cells with 647 Tyramides for 20 min, the cell nuclei were counterstained with Dapi, mounted with anti-fluorescence quenching mounting medium, and observed and photographed under a fluorescence microscope.

[0133] HE staining results showed that the olfactory plate of the snakehead olfactory sac can be divided into sensory epithelium (SE) and central medulla (CC). Olfactory neurons, namely ciliated neurons (CO) and microvilli neurons (MO), exist in the SE; ciliated neurons are located deep within the SE, while microvilli neurons are located superficially. In situ hybridization results showed that the mRNAs of prlra and prlrb are widely expressed in the sensory epithelium of the snakehead olfactory sac, and may be expressed in both ciliated and microvilli olfactory neurons (see...). Figure 14 ).

[0134] Fluorescence in situ hybridization results showed that prlra and prlrb co-localized with the olfactory marker protein ompa, which is specifically expressed in ORNs, and also demonstrated that prlra and prlrb are expressed on olfactory neurons, and may affect the expression of olfactory neuron genes through downstream signal transduction (see...). Figure 15 ).

[0135] Example 4

[0136] Verification of the interaction between prolactin and its receptor in snakehead fish

[0137] Construction of the prokaryotic expression vector pET-PRL-His: The pET-HTH vector, modified from the pET-32a(+) (Novagen, Germany) plasmid, was double-digested with BamHI and KpnI. After 1% agarose gel electrophoresis, the linearized vector was purified and recovered by gel excision. Using correctly sequenced pRL-positive bacterial clones as templates, primers with homologous arms were designed to amplify the target DNA fragment by PCR. After gel excision and recovery, homologous recombination was performed using the Seamless Cloning Kit (Beyotime, Shanghai). The homologous recombination product was transformed into Rosetta-gami B(DE3) competent cells. Positive colonies were obtained by screening with ampicillin-containing plates, and the sequence of single colonies was verified by Sanger sequencing.

[0138] Construction of the 5×pSTAT5-TA-luc reporter gene plasmid: Using the pSTAT3-TA-luc reporter gene plasmid (Beyotime, Shanghai) as a template, a linearized vector was obtained by PCR. A DNA sequence with five STAT5 binding sites (ATTTCCAGGAAAT) and homologous arms was synthesized by a certain company and homologously recombined with the linearized vector. The product was transformed into DH5α competent cells, screened by ampicillin plates, and verified by Sanger sequencing.

[0139] Construction of eukaryotic expression vectors: Using positive clones of prlra and prlrb as templates, DNA fragments of prlra and prlrb ORFs with homologous arms were obtained. These fragments were then homologously recombinated with linearized vectors obtained by double digestion of pcDNA3.1(+) (Invitrogen, USA) with Hind III and BamHI. The resulting vectors were then transformed into competent cells to verify sequence correctness.

[0140] Plasmid extraction: One day before plasmid extraction, 10 μL of bacterial culture was inoculated into 30 mL of LB liquid medium containing ampicillin and chloramphenicol. The endotoxin-free plasmid mini-extraction kit (Tiangen, Beijing) was used to extract the endotoxin-free plasmid using the overnight cultured bacterial culture according to the kit instructions. The concentration and purity of the plasmid were detected using a nucleic acid concentration analyzer (OSTC).

[0141] Prepare the complete culture medium according to the following formula: DMEM high-glucose medium (Biosharp, Bengbu), 10% fetal bovine serum (Absin, Shanghai), and 1% 100× triple antibody stock solution (Kingclone, Beijing). Resuscitate HEK293T cells from liquid nitrogen, centrifuge to discard the cell cryopreservation solution, resuspend the cells in the complete culture medium, and evenly seed them into 25 mm² culture flasks. Incubate at 37°C in a 5% CO₂ incubator. When the cells reach a density of 95%, digest them with trypsin-EDTA cell digestion solution (Biosharp, Bengbu), add three times the volume of complete culture medium to stop digestion, centrifuge, discard the supernatant, add complete culture medium, resuspend the cells, and seed them into twice the volume of 25 mm² flasks. 2 In the culture flask.

[0142] After passage 3, the cells were digested, centrifuged, the supernatant was discarded, and the cells were resuspended and plated in 24-well cell culture dishes. After 24 hours, when the cell density reached 60%-70%, the cells were transfected using Lipo8000™ transfection reagent (Beyotime, Shanghai). Each well was transfected with 250 ng of pcDNA3.1-PRLRa, pcDNA3.1-PRLRb, or pcDNDA3.1 empty eukaryotic expression plasmid, 225 ng of pSTAT5 activity reporter plasmid, and 25 ng of Renina luciferase pRL-TK plasmid. 24 hours after transfection, the culture medium in the dishes was replaced with rPRL protein and solvent at final concentrations of 50, 300, and 1000 ng / mL, respectively, to stimulate the cells. 24 hours later, cells were lysed using cell lysis buffer from a dual luciferase reporter gene assay kit (Beyotime, Shanghai). The lysate was centrifuged at 12000g for 3 min, and the supernatant was collected. The luminescence values ​​of firefly luciferase and senna luciferase in 20 μL of sample were detected using an ELISA reader (Agilent, USA). The relative luciferase activity was calculated based on the ratio of firefly luciferase to senna luciferase.

[0143] Example 5

[0144] PRLR validates transcriptional regulation of caor24 by STAT5.

[0145] Construction of eukaryotic expression vectors and reporter plasmids: pcDNA3.1-STAT5a and pcDNA3.1-STAT5b plasmids were constructed. The pGL3-Basic plasmid was linearized by double digestion with Kpn I and Hind III. Primers with homologous arms were designed for the promoters of caor24, caprlra, and caprlrb (see Table 3). The promoter DNA of caor24, caprlra, and caprlrb with homologous arms was obtained by PCR. This DNA was then homologously recombinated with the linearized vector obtained from the pGL3-Basic plasmid, transformed into E. coli, and sequenced for verification. Plasmid extraction was then performed.

[0146] Table 3 Primers used for vector construction

[0147]

[0148] In situ hybridization: DIG and biotin-labeled probes for caor24 and biotin-labeled probes for stat5a were synthesized. Fluorescent in situ hybridization co-localization was performed using DIG-labeled prlra and prlrb with biotin-labeled caor24 cRNA probes.

[0149] Verification of the transcriptional regulatory effect of PRLR on caor24: HEK293T cells in 24-well cell culture dishes were transfected with 250 ng of the eukaryotic expression vector pcDNA3.1-PRLRa, pcDNA3.1-PRLRb, or the empty eukaryotic expression plasmid pcDNDA3.1, 225 ng of the reporter plasmid pGL3-Pcaor24, and 25 ng of the Renilla luciferase pRL-TK plasmid using Lipo8000™ transfection reagent. Before transfection, 1000 ng / mL of rPRL was added to the culture medium, and the activity of the two luciferases was measured after 48 h.

[0150] Verification of the transcriptional regulatory effect of STAT5 on caor24: HEK293T cells in 24-well cell culture dishes were transfected with eukaryotic expression vectors pcDNA3.1-STAT5a, pcDNA3.1-STAT5b, or pcDNDA3.1(+) empty eukaryotic expression plasmid, 225 ng of reporter plasmid pGL3-Pcaor24, and 25 ng of Renilla luciferase pRL-TK plasmid. Another group of cells was transfected with 3 nM of the JAK inhibitor TG101348, following the same method. Cells were lysed after 48 h, and the relative luciferase activity of 20 μL of cell lysis products was measured.

[0151] Verification of the transcriptional regulatory effect of STAT5 on caprlra and caprlrb: HEK293T cells in 24-well cell culture dishes were transfected with eukaryotic expression vectors pcDNA3.1-STAT5a, pcDNA3.1-STAT5b, or pcDNDA3.1(+) empty eukaryotic expression plasmids, 225 ng of pGL3-Pcaprlra or pGL3-Pcaprlrb reporter plasmids, and 25 ng of Renilla luciferase pRL-TK plasmid. Relative luciferase activity was measured after 48 h.

[0152] II. Evidence related to the technical effects obtained by the embodiments of the present invention.

[0153] Prokaryotic expression and NTA-Ni affinity chromatography were performed on *E. coli* Rosetta-gami B(DE3) strain transformed with the pET-PRL-His plasmid to obtain the recombinant snakehead pRL protein (rPRL), which has 6 His tags at the C-terminus. Figure 13 SDS-PAGE gel electrophoresis results showed that rPRL was expressed in a soluble form, mainly present in the supernatant, with only a small amount present in the precipitate. Figure 13 ).

[0154] To demonstrate the potential role of the PRL / PRLR / STAT5 system in the olfactory system of snakehead fish, a dual-luciferase reporter assay was performed on the eukaryotic expression vectors pcDNA3.1-PRLRa and pcDNA3.1-PRLRb, and the pSTAT5 activity reporter plasmid pGL6-5×pSTAT5 TA-luc. The results showed that rPRL stimulation dose-dependently increased pSTAT5 activity in HEK293T cells transfected with the PRLR eukaryotic expression plasmid. In HEK293T cells transfected with PRLRb, pSTAT5 activity gradually increased with gradually increasing rPRL concentrations, but was not as high as in the group transfected with PRLRa at the same rPRL concentration. Even without rPRL stimulation, pSTAT5 activity was significantly increased in the PRLRa transfected group. There was no difference in pSTAT5 activity in the untransfected PRLR control group (see [link to study].) Figure 16 ).

[0155] To verify the transcriptional regulation of caor24 by PRLR through STAT5, fluorescence in situ hybridization (FISH) was performed to co-localize prlra, prlrb, stat5, and caor24. A dual luciferase reporter assay was then used to verify the transcriptional regulation of caor24 by PRLR, the transcriptional regulation of caor24 by STAT5, and the transcriptional regulation of caprlra and caprlrb by STAT5. FISH results showed that caor24 was located in the ORN of the olfactory sac of the snakehead fish, and its expression level was high in microvilli neurons (see...). Figure 17 Fluorescence in situ hybridization co-localization results showed that caor24, stat5, prlra, and prlrb all had positive signals in the ORN of the snakehead olfactory sac and exhibited co-localization (see [link to original text]). Figure 18Dual-luciferase reporter assays showed that transfected pcDNA3.1-PRLRa and pcDNA3.1-PRLRb significantly increased luciferase expression compared to the control group, with relative luciferase activity increasing by 104.88% and 43.90%, respectively. PRLRa had a stronger transcriptional regulatory effect on caor24 than PRLRb. This indicates that PRLR can activate downstream signaling pathways in cells and promote caor24 expression (see...). Figure 19 The relative luciferase activities of the groups transfected with pcDNA3.1-STAT5a and pcDNA3.1-STAT5b plasmids were significantly higher than those of the control group. After treatment with the JAK inhibitor TG101348 targeting JAK2, the relative luciferase activities of the groups transfected with pcDNA3.1-STAT5a and pcDNA3.1-STAT5b plasmids were not different from those of the control group (see [link to relevant documentation]). Figure 19 The results indicate that STAT5 plays a transcriptional regulatory role in caor24. It demonstrates that PRL binding to PRLR activates the downstream JAK2-STAT5 signaling pathway, increasing STAT5 phosphorylation and regulating caor24 transcription via STAT5. The relative luciferase activity in the groups transfected with pGL3-Pcaprlra and pcDNA3.1-STAT5a plasmids was significantly higher than that in the control group, while no significant differences were observed in other groups (see...). Figure 20 This demonstrates that STAT5a can directly regulate the transcription of prlra.

[0156] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway, characterized in that, The method for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway includes the following steps: Step 1: Using transcriptome sequencing (RNA-seq) technology, differential expression of the prl gene and caor24 olfactory receptor was screened in individuals with different expression before and after breeding of snakehead fish. Step 2: Prokaryotic expression of snakehead pRL recombinant protein rPRL using the E. coli expression system to verify the interaction between snakehead pRL recombinant protein rPRL and PRLRa and PRLRab and its activation of downstream transcription factor STAT5; Step 3: Locate the mRNAs of prlr, stat5, and caor24 on the olfactory sac and demonstrate their co-expression in olfactory neurons, verifying the transcriptional regulatory role of the PRL / PRLR / STAT5 signaling pathway on caor24 in snakehead.

2. The method for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway as described in claim 1, characterized in that, The coding sequence of the caor24 gene is SEQ ID NO:

1.

3. The method for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway as described in claim 1, characterized in that, The rPRL protein is the protein encoded by the PRL gene according to claim 1, and its amino acid sequence is shown in SEQ ID NO: 2; The recombinant PRL protein rPRL was expressed in prokaryotes using an E. coli expression system transformed with the prokaryotic expression vector pET-PRL-His. The coding sequence of the prlra gene is SEQ ID NO: 3; The coding sequence of the prlrb gene is SEQ ID NO: 4; The coding sequence of the stat5a gene is SEQ ID NO: 5; The coding sequence of the stat5b gene is SEQ ID NO:

6.

4. The method for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway as described in claim 1, characterized in that, Recombinant plasmids pcDNA3.1-PRLRa, pcDNA3.1-PRLRb, pcDNDA3.1 empty eukaryotic expression plasmid, and pSTAT5 activity reporter plasmid, as well as pRL-TK Renilla luciferase reporter gene plasmid as an internal control, were transfected. The differences in firefly luciferase activity among different groups were analyzed using a dual luciferase reporter gene detection system. Then, the cells were stimulated with rPRL protein and solvent to analyze the changes in firefly luciferase activity.

5. The method for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway as described in claim 1, characterized in that, Relative luciferase activity was measured by transfecting recombinant plasmids pcDNA3.1-PRLRa, pcDNA3.1-PRLRb, pcDNDA3.1 empty eukaryotic expression plasmid, and pGL3-Pcaor24 reporter plasmid, as well as pRL-TK luciferase reporter gene plasmid as an internal control. The recombinant plasmids pcDNA3.1-STAT5a, pcDNA3.1-STAT5b, pcDNDA3.1(+) empty eukaryotic expression plasmid, and pGL3-Pcaor24 reporter plasmid were also transfected. The r24 reporter plasmid and the pRL-TK Renilla luciferase reporter gene plasmid were transfected, and the relative luciferase activity was measured. The recombinant plasmids pcDNA3.1-STAT5a, pcDNA3.1-STAT5b or pcDNDA3.1(+) empty vector eukaryotic expression plasmids and pGL3-Pcaprlra or pGL3-Pcaprlrb reporter plasmids, as well as the pRL-TK Renilla luciferase reporter gene plasmid as an internal control, were transfected, and the relative luciferase activity was measured. The transcriptional regulation of Caor24 by the PRL / PRLR / STAT5 signaling pathway was inhibited by treatment with 3 nM of the JAK2 inhibitor TG101348, and the transcriptional regulation of Caor24 induced by STAT5a and STAT5b was suppressed.

6. The method for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway as described in claim 1, characterized in that, The aforementioned regulation method is based on understanding the physiological mechanisms of increased aggression in snakehead fish during the breeding season in order to reduce damage to parent fish during breeding. The aforementioned regulation method is based on the chemical communication mechanism and signal transduction of pheromones; The species mentioned is the snakehead (Channa argus).

7. A system for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway, implementing the method for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway as described in any one of claims 1-6, characterized in that, The system for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway includes: The screening module is used to screen for differential expression of the prl gene and caor24 olfactory receptor in individuals with different expression before and after breeding by using transcriptome sequencing (RNA-seq) technology. The validation module is used to express the recombinant snakehead PRL protein rPRL in prokaryotes using the E. coli expression system, and to verify the interaction between the recombinant snakehead PRL protein rPRL and P RLRa and PRLRb and its activation of the downstream transcription factor STAT5. The localization module was used to locate the mRNAs of prlr, stat5, and caor24 on the olfactory sac and demonstrate their co-expression in olfactory neurons, verifying the transcriptional regulation of caor24 by the PRL / PRLR / STAT5 signaling pathway in snakehead.

8. A computer device, characterized in that, The computer device includes a memory and a processor. The memory stores a computer program that, when executed by the processor, causes the processor to perform the steps of the method for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway as described in any one of claims 1-6.

9. A computer-readable storage medium storing a computer program, which, when executed by a processor, causes the processor to perform the steps of the method for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway as described in any one of claims 1-6.

10. An information data processing terminal, characterized in that, The information data processing terminal is used to implement the system described in claim 7 for regulating snakehead attack behavior based on the prolactin (PRL) signaling pathway.

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