Application of chicken PDYN gene in regulating expression of gonadotropin-releasing hormone

Abstract

This invention relates to the application of the chicken PDYN gene in regulating gonadotropin-releasing hormone (GnRH) expression. The nucleotide sequence of the PDYN gene is shown in SEQ ID NO: 1; the regulation is positive. This invention is the first to clone the PDYN gene in chickens using PCR technology. The sequence matched the predicted sequence on the NCBI website, indicating the gene's actual presence in chickens. By constructing overexpressed and interfered primary hypothalamic neuronal cell models in chickens, it was demonstrated that the PDYN gene positively regulates GnRH expression. This is of great significance for improving the poultry reproductive regulation system and rapidly and effectively enhancing conventional breeding techniques.

Description

Technical Field

[0001] This invention relates to the application of the chicken PDYN gene in regulating the expression of gonadotropin-releasing hormone (GnRH), and belongs to the field of biotechnology. Background Technology

[0002] Dynorphin (PDYN) is a member of the opioid peptide family, which includes enkephalins, endorphins, orphanonephrines, and endorphins. Opioid peptides are widely distributed in the brain and play significant regulatory roles in immunity, developmental behavior, respiration, analgesia, and the cardiovascular system. In mammals, neurokinin B (NKB) and PDYN neurons have been reported to participate in mediating the feedback regulation of GnRH by gonadotropins.

[0003] The hypothalamus-pituitary-gonadal (HPG) axis is a universal mechanism for reproductive regulation in vertebrates. The hypothalamus, as the initiator of the HPG axis, regulates the activity of downstream components and maintains the normal reproductive cycle by releasing GnRH hormones through GnRH neurons. However, due to their evolutionary independence, birds exhibit significant differences from mammals and other vertebrates in physiological characteristics and gene expression regulation. It is evident that the PDYN gene, which clearly regulates GnRH expression in mammals, remains a blank in birds, and its regulatory mechanism is currently unclear. Therefore, investigating whether the chicken PDYN gene participates in regulating GnRH expression is crucial for improving poultry reproductive regulation systems, rapidly and effectively enhancing conventional breeding techniques, and protecting and protecting rare birds. Summary of the Invention

[0004] The purpose of this invention is to provide the application of the chicken PDYN gene in regulating the expression of gonadotropin-releasing hormone. Experiments have shown that the PDYN gene can effectively regulate the expression of GnRH, laying the foundation for a sound poultry reproductive regulation system.

[0005] To achieve the above objectives, the technical solution for the application of the chicken PDYN gene in regulating gonadotropin-releasing hormone expression in this invention is as follows:

[0006] The application of the chicken PDYN gene in regulating the expression of gonadotropin-releasing hormone, wherein the nucleotide sequence of the CDS region of the PDYN gene is shown in SEQ ID NO: 1; the regulation is positive regulation.

[0007] The beneficial effects of the above technical solution are as follows: This invention is the first to clone the PDYN gene in chickens using PCR technology. The sequence matched the predicted sequence on the NCBI website, indicating that the gene truly exists in chickens. By constructing chicken primary hypothalamic neuron cell models with overexpression and interference, it was demonstrated that the PDYN gene positively regulates GnRH expression. This is of great significance for improving the poultry reproductive regulation system and rapidly and effectively enhancing conventional breeding techniques.

[0008] As a further improvement, the PDYN gene was overexpressed in primary chicken hypothalamic neurons through genetic transformation, which increased the secretion level of gonadotropin-releasing hormone; the PDYN gene was interfered with in primary chicken hypothalamic neurons, which decreased the secretion level of gonadotropin-releasing hormone.

[0009] The beneficial effects of the above technical solution are as follows: After proving that the PDYN gene truly exists in chickens, this invention fully demonstrates that the PDYN gene can regulate the expression of GnRH by examining the relationship between the PDYN gene and GnRH expression from both positive and negative perspectives.

[0010] As a further improvement, the overexpression of the PDYN gene in primary chicken hypothalamic neurons is achieved by constructing a PDYN recombinant expression vector to overexpress the PDYN gene.

[0011] The beneficial effects of the above technical solution are as follows: through genetic transformation, the PDYN gene can be efficiently expressed in primary hypothalamic neurons of laying hens using recombinant expression vectors, laying the foundation for studying the relationship between PDYN gene and GnRH expression.

[0012] As a further improvement, the recombinant expression vector is constructed by ligating the cloned PDYN gene into the pcDNA3.1 vector.

[0013] The beneficial effects of the above technical solution are as follows: pcDNA3.1 vector is a eukaryotic vector that can be expressed efficiently and stably after transfection into cells.

[0014] As a further improvement, the PDYN gene interference in the primary hypothalamic neurons of chickens is a synthetic interference fragment that, after transfection of the cells, inhibits the expression of the PDYN gene.

[0015] The beneficial effects of the above technical solution are as follows: the use of gene interference is simple to operate, requires no vector construction, and can significantly inhibit the expression of the PDYN gene. Attached Figure Description

[0016] Figure 1 This is an electrophoresis diagram of the PCR product of the CDS region of the chicken PDYN gene in Example 1 of the present invention;

[0017] Figure 2 The CDS sequence and encoded amino acid sequence of the chicken PDYN gene in Example 1 of this invention;

[0018] Figure 3 The relative expression levels of the chicken PDYN gene in different tissues of 15-week-old and 30-week-old chickens in Example 1 of this invention;

[0019] Figure 4 The gene structure and phylogenetic tree of the chicken PDYN gene in Example 1 of this invention;

[0020] Figure 5 This invention relates to the efficiency detection of PDYN overexpression vector and interfering fragment in primary chicken hypothalamic neurons in Example 2 of this invention.

[0021] Figure 6 This illustrates the effect of PDYN gene overexpression on GnRH expression in Example 2 of the present invention. Detailed Implementation

[0022] The present invention will now be further described in conjunction with specific embodiments. It should be noted that, without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. The equipment and raw materials used are all commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0023] In the following embodiments:

[0024] Gene sequencing, primer synthesis, and vector construction were all provided by Shanghai Sangon Biotech.

[0025] The gene interference fragment was provided by Guangzhou Ruibo Biotechnology Co., Ltd.

[0026] Example 1: Cloning and Sequence Analysis of Chicken PDYN Gene

[0027] Six healthy Hy-Line Brown chickens aged 15 weeks and 30 weeks were selected. After euthanasia, tissue samples from each chicken, including the hypothalamus, pituitary gland, ovary, heart, liver, spleen, lungs, and kidneys, were rapidly frozen in liquid nitrogen and then transferred to a -80°C freezer for later use.

[0028] 1.1 Extraction of total RNA from various tissues of Hy-Line Brown chickens

[0029] (1) Take an appropriate amount (the size of a soybean) of hypothalamic tissue sample into a 2 mL sterile enzyme-free tube (the sterile enzyme-free tube has been pre-filled with 1 mL of Trizol solution and two steel balls with diameters of 2-3 mm and 4-6 mm respectively), and then grind it in a grinder at 60 Hz for 60-90 seconds until the tissue block is no longer visible to the naked eye.

[0030] (2) Place the ground sample on ice and let it stand for 5 minutes to allow it to fully lyse. Then add 200 μL of chloroform and shake the sample rapidly. Continue to place it on ice until the sample shows obvious stratification. At this time, use a low-temperature high-speed centrifuge and adjust the centrifuge parameters to 4℃, 12000 r / min, and 15 min.

[0031] (3) Try to draw the supernatant into a new 1.5mL sterile enzyme-free tube, add 600μL isopropanol, and place it in a -80℃ refrigerator for 30min.

[0032] (4) Use a low-temperature high-speed centrifuge at 4℃ and 12000r / min for 15min.

[0033] (5) Discard the supernatant, add 1 mL of freshly prepared 75% anhydrous ethanol, mix by inverting, and centrifuge at 8000 r / min for 15 min at 4°C. Repeat this step twice.

[0034] (6) Discard the liquid in the tube and let the remaining liquid on the tube wall dry in a fume hood. Add 20-40 μL of sterile, enzyme-free water and store in a -80°C refrigerator for later use.

[0035] 1.2 Total RNA quality detection

[0036] (1) Concentration and purity detection

[0037] The concentration and purity of total RNA were determined using a Nanodrop2000 UV spectrophotometer, and RNA samples with an OD260 / OD280 ratio between 1.8 and 2.0 were retained.

[0038] (2) Integrity Detection

[0039] The integrity of total RNA was detected using 1% gel electrophoresis. The total RNA concentration was diluted to 100 ng / μL and mixed thoroughly with 2 μL loading buffer. After electrophoresis, RNA samples with intact quality were retained based on the bands and brightness.

[0040] 1.3 cDNA Synthesis

[0041] cDNA synthesis from various chicken tissues was performed using the Vazyme reverse transcription kit, strictly following the manufacturer's instructions. Specific details are shown in Tables 1-1 and 1-2.

[0042] Table 1-1 Genomic DNA Removal Reaction System

[0043] 4×gDNA wiper 4μL Total RNA 1ng RNase Free H2O Up to 16μL total 16μL

[0044] Table 1-2 Reverse Transcription Reaction System

[0045] 5×HiScript III qRT SuperMix 4μl Step 1 reaction solution 16μL total 20μL

[0046] The reaction system in Table 1-1 was tested in a PCR instrument at 42℃ for 2 min.

[0047] The reaction systems in Table 1-2 were subjected to 37℃ for 15 min and 85℃ for 5 s in a PCR instrument.

[0048] 1.4 Amplification of the PDYN gene CDS sequence

[0049] Using Hy-Line Brown hypothalamic cDNA samples as templates, PCR amplification of the PDYN gene was performed using KOD One™ PCR Master Mix-Blue. The designed primers contained the entire CDS region of the PDYN gene. Primer information is shown in Table 1-3, amplification buffer system is shown in Table 1-4, and reaction procedure is shown in Table 1-5.

[0050] Table 1-3 PCR Primer Information

[0051]

[0052] Table 1-4 PCR reaction system

[0053] KOD OneTM PCR Master Mix-Blue- 5μL upstream primer 0.5μL Downstream primer 0.5μL template cDNA 1μL RNase water 3μL total 10μL

[0054] Table 1-5 PCR reaction procedures

[0055]

[0056] 1.5 Purification and Recovery of PCR Products

[0057] The PCR product of the PDYN gene was recovered and then sequenced. The PCR product recovery procedure was performed according to the Tiangen kit instructions below.

[0058] (1) Place the adsorption column into the collection tube and add 500 μL of equilibration solution BL. After centrifuging at 12000 rpm for one minute, discard the liquid in the collection tube and put the adsorption column that has completed column equilibration back into the collection tube for later use.

[0059] (2) After cutting the correct strip from the agarose gel, cut it into small pieces as much as possible, immediately put it into a new 2mL EP tube, and weigh the cut strip.

[0060] (3) Add an appropriate amount of PN solution at a ratio of 0.1g:100μL, place it in a 50℃ water bath to fully dissolve the gel block, and gently turn it upside down until the gel block is completely dissolved.

[0061] (4) Centrifuge at 4°C and 12000 rpm for 15 minutes using a low-temperature high-speed centrifuge. Transfer the dissolved gel to the above adsorption column, let it stand at room temperature for 2 minutes, then centrifuge at 12000 rpm for 1 minute and discard the waste liquid;

[0062] (5) Repeat step (4);

[0063] (6) After placing the adsorption column into the collection tube, centrifuge the empty column at 12,000 rpm for 2 min to remove as much PW solution as possible. Then let it stand at room temperature for 3 min to dry completely;

[0064] (7) Place the adsorption column into a clean centrifuge tube, add 50 μL of EB elution buffer, let stand at room temperature for two minutes, then centrifuge at 12000 rpm for 2 minutes to collect DNA.

[0065] 1.6 Quantitative Real-Time PCR

[0066] Using cDNA from the hypothalamus of Hy-Line Brown chickens as a template and GADPH as an internal control gene, SYBR Green dye was used, and each sample was subjected to three technical replicates. The real-time PCR reaction system is shown in Table 1-6, the reaction procedure is shown in Table 1-7, and the real-time PCR primer information is shown in Table 1-8.

[0067] Table 1-6 Real-time quantitative PCR amplification reaction system

[0068] 2xSYBR Green Mix 5μL upstream primer 0.5μL Downstream primer 0.5μL template cDNA 1μL RNase water 3μL total 10μL

[0069] Table 1-7 Real-time quantitative PCR reaction procedures

[0070]

[0071]

[0072] Table 1-8 Primer Information for Quantitative Real-Time PCR

[0073]

[0074] 1.7 Sequence Analysis

[0075] Chicken PDYN nucleic acid and amino acid sequence analysis was performed using NCBI online BLAST, PDYN protein physicochemical parameters were analyzed using ProtParm online software, gene structure analysis was performed using GSDS online software, and a PDYN gene phylogenetic tree was constructed using Mega 7.0.

[0076] 1.8 Experimental Results

[0077] The PDYN gene was subjected to PCR reaction followed by gel electrophoresis with 1% agarose gel. The results are shown below. Figure 1Our designed primers have the upstream end at the 5' end and the downstream end at the 3' end, producing a PCR product of 1349 bp. The CDS region of the PDYN gene is completely contained within this PCR product. The fragment, consistent with the expected size, was purified and sequenced. Sequence alignment revealed a complete match with the sequence XM_040650978.1 annotated on the NCBI website. Its full-length CDS sequence is 948 bp, encoding 315 amino acids. (See [link to relevant documentation]). Figure 2 .

[0078] Spatiotemporal expression profiling of the PDYN gene revealed its widespread expression in various tissues. High expression levels were observed in reproductive organs (hypothalamus and pituitary gland) and the lungs, while lower expression levels were found in the heart, liver, spleen, kidneys, and ovaries. Figure 3 .

[0079] Physicochemical analysis of chicken PDYN protein showed that its molecular weight is 33357.68, its theoretical isoelectric point (PI) is 6.6, and it is an acidic protein. The molecular formula of this protein is C0. 1441 H 2272 N 426 O 447 S 20 The protein has a total of 4606 atoms and is composed of 315 amino acids. Among them, there are 34 negatively charged residues (Asp+Glu) and 33 positively charged residues (Arg+Lys). The instability index is 55.13, indicating that the protein is unstable (see Table 1-9).

[0080] Table 1-9 Physicochemical Properties Analysis of PDYN Protein

[0081]

[0082]

[0083] The exon-intron structure of the chicken PDYN gene was analyzed using the online website GSDS (http: / / gsds.cbi.pku.edu.cn / ). A phylogenetic tree of the PDYN gene in different species was constructed using MEGA 7.0 neighbor-joining. (See attached image.) Figure 4 .from Figure 4 The results showed that the PDYN gene consists of two exons and one intron. Phylogenetic results showed that it clustered with birds (ducks, zebra finches, and robins), with clawed frogs and zebrafish, and with rats, mice, pigs, and humans.

[0084] Example 2: PDYN gene regulates gonadotropin-releasing hormone expression

[0085] 2.1 Construction of PDYN gene overexpression vector and plasmid extraction

[0086] The CDS region sequence of the PDYN gene containing the 3×flag tag sequence was synthesized and cloned into the expression vector pcDNA3.1(+) to construct an overexpression plasmid for chicken PDYN. The following procedures were performed according to the instructions of the Tiangen Biotech Endotoxin-Free Plasmid Kit:

[0087] (1) Column equilibration step: After placing the adsorption column CP4 into the collection tube, add 500 μl of equilibration solution BL to the adsorption column CP4, centrifuge at 12,000 rpm for 1 min, and discard the liquid in the collection tube for later use.

[0088] (2) Centrifuge 15 mL of overnight culture at 12,000 rpm for 1 min and discard the supernatant;

[0089] (3) Add 500 μL of P1 solution containing RNase A to the centrifuge tube containing the precipitate, suspend the precipitate completely using a vortex mixer, then add 500 μL of P2 solution and gently invert the tube 6-8 times.

[0090] (4) Add 500 μl of P4 solution to the centrifuge tube, mix well, let stand at room temperature for 10 min, and centrifuge at 12,000 rpm for 10 min.

[0091] (5) Transfer the collected supernatant into the CS filter column in batches, centrifuge at 12,000 rpm for 1 min, and collect the filtrate into a clean 15 mL centrifuge tube.

[0092] (6) Add 0.3 times the volume of isopropanol to the above filtrate, mix well, and then add it to the treated adsorption column in portions. Centrifuge at 12,000 rpm for 1 min and discard the supernatant.

[0093] (7) Add 500 μL of PD solution to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the supernatant;

[0094] (8) Add 600 μL of washing solution PW to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid.

[0095] (9) Repeat step 8;

[0096] (10) Centrifuge the empty column at 12,000 rpm for 2 min, discard the waste liquid, and let it dry completely at room temperature for 5 minutes; residual PW solution;

[0097] (11) Place the adsorption column into a clean centrifuge tube, add 100 μL of RNase-free water, let stand at room temperature for 2 min, centrifuge at 12,000 rpm for 1 min, collect the plasmid, and store at -20℃.

[0098] 2.2 Isolation and Culture of Primary Chicken Hypothalamic Neurons

[0099] (1) Coat the cell plate with poly-L-lysine for 12 hours, then aspirate the waste liquid and wash the cell plate twice with PBS buffer, and put it into the cell culture incubator to dry for later use.

[0100] (2) Place the hatching eggs in an egg tray that has undergone strict ultraviolet disinfection and then place them between cells;

[0101] (3) Wipe the hatching eggs with alcohol swabs, use the handle of large scissors to break the blunt end of the hatching eggs, then use tweezers to remove the chicken embryo and immerse it in alcohol for 30 seconds for disinfection.

[0102] (4) After cutting off the chicken embryo’s head from the base with large scissors, use ophthalmic scissors to peel off the skin on the head and cut off the top of the head to remove the skull and expose the entire brain tissue. Use small tweezers to remove the hypothalamic tissue and wash it twice in a mixture of 98% D-Hanks and 2% penicillin and streptomycin. Then transfer it to a mixture of 98% DMEM high glucose medium and 2% penicillin and streptomycin.

[0103] (5) After removing the hypothalamus from 40 embryos, transfer the tissue to a clean 1.5 mL EP tube and crush the cells using a cell grinder. Then transfer the cells to a 15 mL centrifuge tube and add 1.5 times the volume of trypsin. Digest the cells in a 37°C cell culture incubator for about 10 minutes. During this period, take the tube out every 3 minutes to observe and gently shake it. After digestion, there should be no tissue clumps, but the tissue should be aggregated in a flocculent manner.

[0104] (6) After digestion, add twice the volume of complete culture medium to stop the digestion process, and remove impurities using 200-mesh and 500-mesh cell filters. After filtration, centrifuge at 1200 rpm for 7 min. After centrifugation, discard the supernatant and add 5 mL of complete culture medium to resuspend, and centrifuge again at 1000 rpm for 6 min (this process is repeated three times).

[0105] (7) The final cell concentration was adjusted to 1×10⁶ using the trypan blue staining method for cell counting. 6 After the number of cells / mL is increased, plate culture is performed;

[0106] (8) After culturing the cells for 24 hours, replace them with Neurobasal serum-free medium containing 2% B27 and cytarabine. Continue culturing for another 24 hours, then replace them with Neurobasal serum-free medium containing 2% B27 and cytarabine.

[0107] (9) When the cells are about 75% adherent, conduct subsequent experiments.

[0108] 2.3 Primary hypothalamic neuronal cell culture and transfection

[0109] Hypothalamic neurons were seeded and cultured in 6-well plates. When the cell density reached 70% or higher, transfection was performed. The transfection groups were divided into PDYN gene overexpression group (pcDNA3.1-PDYN), overexpression control group (pcDNA3.1 empty vector group), PDYN gene interference group (NCR-PDYN), and interference control group (NCR). The steps are as follows:

[0110] (1) Starve hypothalamic neurons for 30 min using 1.5 mL of serum-free and antibiotic-free culture medium;

[0111] (2) Prepare the transfection reagent solution for the overexpression transfection group according to Table 2-1. After preparing reagent 1 and reagent 2, let them stand and incubate for 5 minutes. Then, gently mix reagent 1 and reagent 2 evenly and incubate for 15 minutes. Prepare the transfection reagent solution for the gene interference group according to Table 2-2. After preparation, the cells can be directly transfected.

[0112] (3) Add the prepared transfection reagents to each well according to the groups shown below, and gently shake well;

[0113] (4) After 4 hours of transfection, the medium was replaced with a medium containing serum and antibiotics, and cultured for 24 hours.

[0114] Table 2-1 Preparation of Transfection Reagent Solutions

[0115]

[0116] Table 2-2 Proportions of Interference Transfection Reagent Solutions

[0117] riboFECT™ CP reagent 6μl riboFECT™ CP Buffer (x1) 60μl Interference fragment / NC 5μl

[0118] 2.4 PDYN overexpression, interference efficiency identification, and GnRH expression detection

[0119] 24 hours after transfection, RNA was extracted from the cells, reverse transcribed, and then subjected to quantitative real-time PCR to detect PDYN overexpression and interference efficiency in primary hypothalamic neurons. The RNA extraction, reverse transcription, and quantitative real-time PCR process are described in Example 1.

[0120] 2.5 Detection of GnRH hormone secretion in cell culture medium

[0121] After transfection with primary chicken hypothalamic neurons, the cells were incubated at 37°C for 24 hours in a 5% CO2 incubator. The cell supernatant was then collected, centrifuged at 3000 rpm for 20 minutes at 4°C, and transferred to a new 1.5 mL centrifuge tube. The procedure was performed using the instructions for the chicken GnRH enzyme-linked immunosorbent assay kit provided by Jiangsu Enzyme Immunoassay Co., Ltd.

[0122] 2.6 Experimental Results

[0123] The effect of PDYN overexpression vector on PDYN mRNA expression level was examined, and it was found that PDYN overexpression significantly upregulated PDYN mRNA level (P<0.01). Figure 5 (5A). The effect of PDYN interference fragments on PDYN mRNA expression levels was examined, and it was found that interfering with PDYN significantly upregulated PDYN mRNA levels (P<0.01). (See [reference needed]). Figure 5 (5B). The results showed that PDYN overexpression and interference were successful.

[0124] To evaluate the effect of PDYN on GnRH expression in primary hypothalamic neurons of laying hens, we used qRT-PCR and ELISA to determine the effect of co-treatment with low and high concentrations of estrogen after PDYN overexpression on GnRH expression in primary hypothalamic neurons of laying hens. qRT-PCR results showed that, compared with the control group, PDYN overexpression significantly promoted GnRH mRNA expression (P<0.05). Figure 6 (6A) ELISA results showed that the GnRH secretion level and gene expression level trended in the same direction. Figure 6 (6B); Compared with the control group, interference with PDYN significantly inhibited the expression of GnRH mRNA (P<0.05), see Figure 6 (6C), ELISA results showed that the GnRH secretion level and gene expression level trended in the same direction. Figure 6 (6D).

[0125] In summary, this invention is the first to clone the CDS region sequence of the chicken PDYN gene using PCR, obtaining a 948bp coding region sequence. Sequencing confirmed its consistency with the sequence predicted on the NCBI website, indicating the actual existence of this gene in chickens. Overexpression of the PDYN gene in primary chicken hypothalamic neurons significantly increased GnRH gene expression and hormone secretion; conversely, interference with PDYN gene expression significantly decreased both GnRH gene expression and hormone secretion, demonstrating that the PDYN gene regulates GnRH gene expression and hormone secretion, and that PDYN promotes GnRH expression. This invention contributes to enriching the theory of poultry reproductive regulation, thereby enriching the system of poultry reproductive performance, rapidly and effectively improving conventional breeding techniques, and enhancing the economic benefits of animal husbandry.

[0126] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the description and drawings of the present invention shall also be included within the scope of protection of the present invention.

Claims

1. The application of the chicken PDYN gene in regulating gonadotropin-releasing hormone expression, characterized by: The nucleotide sequence of the CDS region of the PDYN gene is shown in SEQ ID NO: 1; the regulation is positive regulation.

2. The application of the chicken PDYN gene according to claim 1 in regulating gonadotropin-releasing hormone expression, characterized in that: Overexpression of the PDYN gene in primary chicken hypothalamic neurons via genetic transformation increases gonadotropin-releasing hormone (GnRH) secretion levels; interference with the PDYN gene in primary chicken hypothalamic neurons decreases GnRH secretion levels.

3. The application of the chicken PDYN gene in regulating gonadotropin-releasing hormone expression according to claim 2, characterized in that: The overexpression of the PDYN gene in primary chicken hypothalamic neurons was achieved by constructing a PDYN recombinant expression vector and overexpressing the PDYN gene.

4. The application of the chicken PDYN gene according to claim 3 in regulating gonadotropin-releasing hormone expression, characterized in that: The recombinant expression vector is constructed by ligating the cloned PDYN gene into the pcDNA3.1 vector.

5. The application of the chicken PDYN gene according to claim 2 in regulating gonadotropin-releasing hormone expression, characterized in that: The PDYN gene interference in chicken primary hypothalamic neurons is a synthetic interference fragment that, after transfection of cells, inhibits the expression of the PDYN gene.

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