Hugu sheep fgf1 gene and application of coded protein thereof

Abstract

The present application relates to the application of Hu sheep FGF1 gene and its coded protein, and belongs to the field of biotechnology. The application of Hu sheep FGF1 gene and its coded protein in regulating the level of oxidative stress, cell apoptosis or cell proliferation of ovary granulosa cells. The present application first systematically identifies the CDS and protein sequence of sheep FGF1 gene, and analyzes its physical and chemical properties, post-translational modification sites, secondary and tertiary structures and systematic evolution relationship, which provides a molecular basis for revealing the role of the gene in oxidative stress and cell fate regulation. In addition, the disclosed sequence information and variation data are helpful for evaluating population genetic diversity, and have important scientific value for the protection, development and evolution research of animal genetic resources.

Description

Technical Field

[0001] This invention relates to the application of the FGF1 gene of Hu sheep and its encoded protein, and belongs to the field of biotechnology. Background Technology

[0002] The FGF1 gene encodes fibroblast growth factor 1 (FGF1), also known as acidic fibroblast growth factor, whose main functions involve cell proliferation, differentiation, repair, and metabolic regulation, especially playing a protective role against oxidative stress. Specific mechanisms include: 1) Scavenging reactive oxygen species and reducing oxidative damage: FGF1 activates downstream signaling pathways, upregulates the expression and activity of antioxidant enzymes (such as SOD2, GPX, CAT), reduces the damage of reactive oxygen species to biomolecules, and maintains the oxidation-antioxidant balance.

[0003] 2) Inhibits oxidative stress-mediated apoptosis: FGF1 regulates apoptosis-related proteins, upregulates anti-apoptotic proteins (such as Bcl-2), downregulates pro-apoptotic proteins (such as Bax, Caspase-3, Caspase-9), blocks apoptosis signals, and reduces apoptosis.

[0004] 3) Regulating the balance between cell proliferation and apoptosis: FGF1 reduces oxidative stress-mediated tissue damage and enhances antioxidant protection by precisely regulating the dynamic balance between proliferation and apoptosis. FGF1 can efficiently activate core signaling pathways such as PI3K / AKT / GSK-3α / β, directly regulating the cell proliferation process: The activated PI3K / AKT / GSK-3α / β signaling pathways can phosphorylate downstream cyclin-dependent kinases (CDKs) and their regulatory factors, promoting the cell cycle transition from G1 phase to S phase and G2 phase to M phase, thus accelerating cell division and proliferation.

[0005] FGF1 is expressed in various tissues (such as liver, kidney, heart, and brain) and plays an important physiological and pathological role in regulating oxidative stress. Oxidative stress is a core mechanism in the development of cardiovascular diseases, neurological diseases, liver diseases, and diabetes. When FGF1 expression is reduced or its function is abnormal, cellular antioxidant capacity decreases, oxidative stress is excessively activated, and disease progression is accelerated. For example, in ischemia-reperfusion injury, supplementing with exogenous FGF1 or upregulating endogenous expression can reduce oxidative damage and improve tissue function. FGF1 has become a potential therapeutic target for oxidative stress-related diseases. Studies have shown that exogenous FGF1, gene-modified vectors, or small molecule compounds can treat diseases such as Alzheimer's disease (improving cognitive impairment) and diabetic nephropathy (delaying kidney failure) by regulating oxidative stress.

[0006] Reproductive performance is a core indicator of sheep production, and oxidative stress significantly inhibits follicle development and early embryonic development in sheep. FGF1, through its antioxidant mechanism, can scavenge excess reactive oxygen species in the reproductive system, protect the structural integrity of oocytes and sperm, inhibit oxidative stress-mediated apoptosis, and regulate the proliferation of reproduction-related cells, thereby controlling the potential of follicle development and early embryonic development, and ultimately improving sheep conception rate, lambing rate, and lamb survival rate, thus optimizing reproductive performance. However, the gene and protein sequences of FGF1 in sheep are currently unknown, and the physicochemical properties, structural domains, functional domains, and secondary and tertiary structures of the protein are not publicly available. In the field of reproductive biology, related research mainly focuses on using model animals such as mice, which have verified the regulatory role of the FGF1 gene in placental development, follicle maturation, and maternal behavior. In large livestock, studies have confirmed that the FGF1 gene is located in bovine ovarian follicles; however, the specific molecular mechanism by which this gene regulates follicle development in large livestock remains unclear. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by proposing the application of the FGF1 gene and its encoded protein in Hu sheep. Using Hu sheep granulosa cells as a model, the regulatory role and specific mechanism of the FGF1 gene in follicle development were thoroughly verified.

[0008] The Hu sheep, belonging to the Bovidae family and the Ovo genus of the order Artiodactyla, is a first-class protected local livestock breed, characterized by its high fertility and high-quality lambskin. Analyzing the key genes regulating the reproductive performance of the Hu sheep and their molecular mechanisms is of great significance for optimizing Hu sheep genetics and breeding, and improving farming efficiency.

[0009] The present invention solves the technical problem through the following technical solution: First, it provides the application of the Hu sheep FGF1 gene and its encoded protein. The nucleotide sequence of the Hu sheep FGF1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the Hu sheep FGF1 gene is shown in SEQ ID NO.2.

[0010] The cloning and sequence analysis of the Hu sheep FGF1 gene involved using total RNA from Hu sheep ovarian tissue as a template. Specific primers (FGF1-F / R) were designed, and the CDS sequence of the Hu sheep FGF1 gene and its encoded protein sequence were obtained by RT-PCR amplification. Bioinformatics analysis revealed the physicochemical properties, phosphorylation sites, and secondary and tertiary structural characteristics of the protein.

[0011] The present invention further provides primer pairs for detecting FGF1 gene expression in Hu sheep, wherein the primer pairs have nucleotide sequences as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0012] This invention further provides the application of the Hu sheep FGF1 gene and its encoded protein in regulating oxidative stress levels, apoptosis, or cell proliferation in Hu sheep ovarian granulosa cells. Specifically, this regulation is achieved by activating the PI3K / AKT / GSK-3α / β signaling pathway. Regulation of oxidative stress levels in Hu sheep ovarian granulosa cells is manifested by reducing intracellular reactive oxygen species (ROS) levels and / or upregulating the expression of antioxidant enzymes SOD2, CAT, and GPX. Regulation of apoptosis in Hu sheep ovarian granulosa cells is manifested by upregulating Bcl-2 expression and / or downregulating Bax and Caspase-9 expression. Regulation of Hu sheep ovarian granulosa cell proliferation is manifested by upregulating proliferating cell nuclear antigen (PCNA) expression.

[0013] This invention further provides the application of the FGF1 gene and its encoded protein in improving the reproductive rate of Hu sheep. Improving the reproductive rate of Hu sheep refers to improving the quality of follicle development in Hu sheep.

[0014] The beneficial effects of the present invention are as follows: (1) The complete coding region (CDS) sequence (SEQ ID NO: 1) of the FGF1 gene and the amino acid sequence of its encoded protein (SEQ ID NO: 2) were cloned from Hu sheep for the first time, filling the gap in the sequence information of the gene in Hu sheep and providing key basic data for related molecular biological research.

[0015] (2) The biological function of the FGF1 gene in the granulosa cells of Hu sheep ovaries was revealed for the first time, confirming that it can improve the functional state of granulosa cells by regulating oxidative stress levels, inhibiting apoptosis and promoting proliferation. As key trophoblast cells in the process of follicle development, the activity of granulosa cells directly affects the fate of follicles. Therefore, this study provides a new theoretical perspective for elucidating the molecular regulatory mechanism of follicle development in Hu sheep.

[0016] (3) The molecular mechanism by which the FGF1 gene exerts the above functions by activating the PI3K / AKT / GSK-3α / β signaling pathway was elucidated for the first time. This discovery provides potential targets for screening candidate drugs or molecular markers that can regulate follicle development.

[0017] (4) This invention provides novel molecular markers for the genetic breeding and reproductive trait improvement of sheep. By detecting the expression level of the FGF1 gene or its protein activity, the ovarian function status can be assessed, assisting in the early selection of individuals with high fertility, and providing theoretical basis and application support for the protection and breeding practices of sheep genetic resources. In summary, this invention systematically identifies the CDS and protein sequence of the sheep FGF1 gene for the first time, and analyzes its physicochemical properties, post-translational modification sites, secondary and tertiary structures, and phylogenetic relationships, providing a molecular basis for revealing the role of this gene in oxidative stress and cell fate regulation. In addition, the disclosed sequence information and variation data help assess population genetic diversity and have important scientific value for the protection, development, and evolutionary research of animal genetic resources. Attached Figure Description

[0018] Figure 1 This is an electrophoresis diagram of PCR in Embodiment 1 of the present invention.

[0019] Figure 2 This is the predicted amino acid percentage of FGF1 protein in Example 2 of the present invention.

[0020] Figure 3 This is the predicted result of the hydrophilicity / hydrophobicity of FGF1 protein in Example 2 of the present invention.

[0021] Figure 4 This is the prediction result of the transmembrane domain of FGF1 protein in Embodiment 2 of the present invention.

[0022] Figure 5 This is the predicted result of the phosphorylation site of FGF1 protein in Example 2 of the present invention.

[0023] Figure 6 This is the predicted result of the secondary structure of FGF1 protein in Example 2 of the present invention.

[0024] Figure 7 This is the predicted result of the tertiary structure of the FGF1 protein in Example 2 of the present invention.

[0025] Figure 8 This is the phylogenetic tree prediction result of the FGF1 protein in Embodiment 2 of the present invention.

[0026] Figure 9 This refers to the relative expression level of FGF1 protein on the HPO axis of Hu sheep in Example 3 of this invention.

[0027] Figure 10 This refers to the expression of FGF1 protein in the ovary of a Hu sheep in Example 3 of the present invention.

[0028] Figure 11 This refers to the relative expression levels of FGF1 protein in different sizes of follicles in Hu sheep in Example 3 of this invention.

[0029] Figure 12 This refers to the expression of FGF1 protein overexpression in Hu sheep granulosa cells in Example 3 of this invention.

[0030] Figure 13 This describes the effect of FGF1 protein overexpression on the level of oxidative stress in Hu sheep granular cells in Example 3 of this invention.

[0031] Figure 14 This describes the effect of FGF1 protein overexpression on the expression level of enzymes related to oxidative stress in Example 3 of this invention.

[0032] Figure 15 This describes the effect of FGF1 protein overexpression on the apoptosis rate of Hu sheep granular cells in Example 3 of the present invention.

[0033] Figure 16 This describes the effect of FGF1 protein overexpression on the expression levels of apoptosis-related genes in Example 3 of this invention.

[0034] Figure 17 This describes the effect of FGF1 protein overexpression on the proliferation of granulosa cells in Hu sheep in Example 3 of this invention.

[0035] Figure 18 This describes the effect of FGF1 protein overexpression on the expression level of proliferating cell nuclear antigen (PCNA) in Example 3 of this invention.

[0036] Figure 19 This describes the effect of FGF1 protein overexpression on the phosphorylation levels of key genes in the PI3K / AKT / GSK-3α / β pathway in Example 3 of this invention.

[0037] Figure 20 This invention relates to the effect of adding pathway inhibitors on the phosphorylation levels of key genes in the PI3K / AKT / GSK-3α / β pathway in Example 3 of this invention.

[0038] Figure 21 This invention relates to the effect of FGF1 protein overexpression on the phosphorylation levels of key genes in the PI3K / AKT / GSK-3α / β pathway after inhibition, as described in Example 3 of this invention.

[0039] Figure 22 This is a graph showing the functional analysis results of the FGF1 protein in Example 3 of the present invention. Detailed Implementation

[0040] The implementation plan is mainly carried out according to the following steps: 1) PCR primers for cloning the FGF1 gene of Hu sheep: FGF1-F and FGF1-R.

[0041] 2) The PCR system for cloning the FGF1 gene of Hu sheep was a 50 µL system, including 2 µL OneStepEnzymeMix, 2 µL of 10 µM upstream primer, 2 µL of 10 µM downstream primer, 1 µL of total RNA, 25 µL of 2×One-StepReactionSolutionA, and RNase-free water to make up to 50 µL.

[0042] 3) The PCR amplification conditions for cloning the FGF1 gene of Hu sheep were as follows: reverse transcription 50℃ for 30 min; pre-denaturation 94℃ for 2 min; denaturation 94℃ for 30 s, annealing 58℃ for 30 s, extension 72℃ for 1 min, 33 cycles; final extension 72℃ for 5 min.

[0043] 4) Use ProtParamtool to analyze the physicochemical properties of FGF1 protein.

[0044] 5) Protein hydrophilicity and hydrophobicity analysis was performed using ProtScale.

[0045] 6) TMHMM was used to analyze transmembrane domains of proteins.

[0046] 7) Phosphorylation sites were determined using NetPhos-3.1.

[0047] 8) SOPMA was used to predict the secondary structure of proteins.

[0048] 9) Use SWISS-MODEL to predict the tertiary structure of proteins.

[0049] 10) Construct a protein evolutionary tree using MEGA.

[0050] 11) The immunohistochemical procedure was as follows: Paraffin sections of sheep ovaries were dewaxed with xylene, hydrated stepwise with graded ethanol, and then rinsed with distilled water. The dewaxed sections were incubated in 3% H2O2 at 37°C for 10 min to inactivate endogenous peroxidase; subsequently, they were placed in citrate buffer and incubated at 100°C for antigen retrieval for 15 min. After naturally cooling to room temperature in citrate buffer, the sections were blocked with 5% bovine serum albumin (BSA) at 37°C for 30 min, followed by incubation with primary antibody anti-FGF1 (1:500, Abclonal, A23167) at 4°C overnight. The negative control group used PBS instead of the primary antibody. After washing with PBS, the corresponding secondary antibody was added and incubated at 37°C for 30 min. All sections were developed with DAB and observed and images were acquired under an optical microscope (Nikon, Japan).

[0051] 12) Intracellular oxidative stress levels were measured using a ROS kit (catalog number S0033S; Beyotime, Shanghai, China). Images were acquired using a confocal microscope (Zeiss LSM900). The mean fluorescence intensity of randomly selected fields of view was quantitatively analyzed using ImageJ software.

[0052] 13) Cell apoptosis was detected using the Annexin V-fluorescein isothiocyanate apoptosis detection kit (catalog number C1052; Vinozan, Nanjing). Experimental procedures were strictly performed according to the kit instructions. Data were collected using a flow cytometer (FACSCalibur, BD, USA). The apoptosis rate was analyzed using FlowJo software.

[0053] 14) Cell proliferation was detected using the Alexa Fluor 555-Click-iTEdU assay kit (catalog number C0075S; Beyotime, Shanghai, China), and cell nuclei were stained with Hoechst 33342. Cell fluorescence was detected using confocal microscopy (Zeiss LSM900). The average fluorescence intensity of randomly selected fields of view was quantitatively analyzed using ImageJ software.

[0054] 15) The qRT-PCR procedure was as follows: Total RNA was extracted from Hu sheep tissues, ovarian follicles, and granulosa cells (GCs) using TRIzol reagent (15996-026; Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using a reverse transcription kit containing gDNA removal agent (Vazyme, Nanjing, China) for qRT-PCR analysis. Subsequently, qRT-PCR was performed using SYBR Master Mix (Vazyme, Nanjing, China) on an ABI 7500 real-time quantitative PCR instrument (Applied BioSystems, Carlsbad, CA, USA). β-actin was used as an internal reference gene, and 2... –ΔΔCT The relative expression level of mRNA was calculated using the qRT-PCR method. Primers for qRT-PCR were designed using Primer 5.0 software, and their sequences are shown in Table 4.

[0055] 16) The Western blot procedure was as follows: RIPA lysis buffer (89900; Thermo Pierce, Waltham, MA, USA) containing protease and phosphatase inhibitors (P1045; Beyotime, Shanghai, China) was used to lyse sheep tissue, ovarian follicles, and granulosa cells at 4°C for 30 min to extract total protein. Protein concentration was determined using a BCA protein quantification kit (P0010; Beyotime, Shanghai, China). 10 μg of total protein was separated by electrophoresis on a 12% SDS-PAGE gel and transferred using a wet transfer method at 100 V for 60 min to a polyvinylidene fluoride (PVDF) membrane. The transferred membrane was blocked with 5% skim milk at room temperature for 2 h, incubated with primary antibody, and then incubated with the corresponding secondary antibody at 37°C for 1 h. Development was performed using ECL Plus chemiluminescence detection reagent (BL523A; Biosharp, China). ImageJ software was used to analyze the grayscale values ​​of each band, and α-tubulin was used as an internal reference for standardization correction. Detailed antibody information is shown in Table 5. Specific methods are described in the following examples.

[0056] Example 1 The sequence of the FGF1 gene was obtained through the following steps: Ovarian tissue was collected from freshly slaughtered Hu sheep (the slaughtered Hu sheep were high-fertility ewes that had given birth to triplets in three consecutive litters, purchased from Qianbao Livestock Co., Ltd.), totaling 3 samples. These samples were preserved in liquid nitrogen and brought back to the laboratory. RNA was extracted from the ovarian tissue using a Trizol extraction kit. 5 μL of RNA solution was aspirated and subjected to 1.5% agarose gel electrophoresis with 1×TAE as the electrophoresis buffer. The electrophoresis results were photographed under ultraviolet transmitted light. The results are shown below. Figure 1 .

[0057] Primers were designed based on the sheep homologous gene sequence of the FGF1 gene published in NCBI, as shown in Table 1. PCR was performed using the primers shown in Table 1, the system shown in Table 2, and the amplification procedure shown in Table 3.

[0058] Table 1

[0059] Table 2

[0060] Table 3

[0061] The PCR products were subjected to 1.5% agarose gel electrophoresis with 1×TAE as the electrophoresis buffer. After electrophoresis, the images were taken under UV transmitted light. The results are shown below. Figure 1 .

[0062] The target band was excised, and the target DNA was recovered via gel extraction and sent to the company for sequencing. The sequencing results are as follows: the CDS sequence of sheep FGF1 is shown in SEQ ID NO.1.

[0063] SEQ ID NO.1 ATGGCTGAAGGAGAAACCACAACCTTCAGGCCCTGACTGAGAAGTTTAACCTGCCTCTAGGCAATTACAAGAAGCCCAAGCTCCTCTATTGCAGCAACGGGGGCTACTTCCTGAGAATCCTCCCAGATGGCAGAGTGGATGGGACGAAGGACAGGAGCGACCAGCACATTCAGCTGCAGCTCTATGCGGAAAGCATAGGGGAGGTGTATATTAAGAGTACGGAGACTGGCCAG TTCTTGGCCATGGACACCAACGGGCTTTTGTACGGCTCACAAACACCCAGTGAGGAATGTTTGTTCCTGGAAAGGCTGGAGGAAAACCATTATAACACCTACATATCCAAGAAGCATGCAGAGAAGAATTGGTTCATTGGTCTCAAGAAGAACGGAAGCTCCAAACTCGGTCCTCGGACTCACTTCGGCCAGAAAGCCATCTTGTTTCTCCCCCTGCCAGTTTCCTCTGATTAA

[0064] Example 2 The obtained FGF1 CDS sequence was translated into a protein using SnapGene software to obtain the protein sequence, as shown in SEQ ID NO.2.

[0065] SEQ ID NO.2 MAEGETTTFRALTEKFNLPLGNYKKPKLLYCSNGGYFLRILPDGRVDGTKDRSDQHIQLQLYAESIGEVYIKSTETGQFLAMDTGLLYGSQTPSEECLFLERLEENHYNTYISKKHAEKNWFIGLKKNGSSKLGPRTHFGQKAILFLPLPVSSD The physicochemical properties of the FGF1 protein were analyzed using ProtParamtool. Results: Molecular weight: 38337.32; Isoelectric point: 5.19; Instability index: 56.91. Structural formula: C1384H23O2N468O568S117; Total number of atoms: 4839; Amino acid composition as shown in [reference needed]. Figure 2 .

[0066] Protein hydrophobicity analysis was performed using ProtScale. Alanine at position 19 showed the highest hydrophobicity (2.189), while threonine at position 294 showed the strongest hydrophilicity (-0.356). The results of the hydrophobicity analysis are as follows: Figure 3 As shown.

[0067] Transmembrane domain analysis of proteins was performed using TMHMM. The domain analysis results are as follows: Figure 4 As shown.

[0068] Phosphorylation sites were determined using NetPhos-3.1 (prediction results are shown in [link]). Figure 5 A total of 29 phosphorylation sites were obtained.

[0069] SOPMA was used to predict the secondary structure of proteins, and the results are as follows: Figure 6 As shown, Alphahelix (Hh): 10.32%; Extendedstrand (Ee): 26.45%; Randomcoil (Cc): 63.23%.

[0070] The tertiary structure of proteins was predicted using SWISS-MODEL, and the results are as follows: Figure 7 As shown.

[0071] A protein phylogenetic tree was constructed using MEGA, and the results are as follows: Figure 8 As shown.

[0072] Example 3 FGF1 is an important antioxidant regulator in the body, mitigating oxidative stress damage through multi-step signal transduction and molecular regulation. After being secreted extracellularly, FGF1 specifically binds to the fibroblast growth factor receptor (FGFR) on the surface of target cells. This leads to phosphorylation of serine / threonine kinase (AKT) within the target cell, generating phosphorylated AKT (P-AKT). P-AKT can then act on glycogen synthesis kinase 3α / β (GSK-3α / β) via intracytoplasmic signal transduction, causing it to phosphorylate as well. Under the regulation of P-GSK-3α / β, a series of antioxidant genes are activated and expressed in large quantities, including SOD2, GPX, and CAT, ultimately reducing intracellular oxidative stress levels. Simultaneously, FGF1 can upregulate the expression of anti-apoptotic proteins (such as Bcl-2) and downregulate the expression of pro-apoptotic proteins (such as Bax and Caspase-9), blocking apoptosis signal transduction, reducing apoptosis in damaged cells, and promoting cell proliferation and protecting cell survival by upregulating cell cycle-related genes (such as PCNA). See [link to relevant documentation] Figure 22 .

[0073] Three healthy, high-fertility female Hu sheep under identical feeding and management conditions were selected as experimental subjects. Hypothalamic, pituitary, and ovarian tissues were collected, and FGF1 mRNA and protein localization and expression levels were detected using quantitative real-time PCR (primer sequences were designed based on sheep FGF1 and β-actin mRNA sequences published by NCBI, see Table 4), immunohistochemistry, and Western blotting (antibody sources are shown in Table 5). Granulosa cells of different sizes were isolated and collected for subsequent detection of oxidative stress levels, apoptosis rate, cell proliferation rate, and related gene expression levels. The results showed that FGF1 may affect oxidative stress, proliferation, and apoptosis of granulosa cells by regulating the expression levels of antioxidant stress-related enzymes, apoptosis, and cell proliferation-related genes, thereby regulating female reproduction. FGF1 protein was expressed in the hypothalamus, pituitary gland, and ovary, and was localized in the granulosa cells of Hu sheep ovarian follicles. Furthermore, the expression levels of FGF1 mRNA and protein increased with increasing follicle diameter. Overexpression of FGF1 reduces oxidative stress levels in granulosa cells, inhibits apoptosis, and promotes cell proliferation. Simultaneously, it upregulates the expression of antioxidant stress-related enzymes, anti-apoptotic proteins (Bcl-2), and cell proliferation antigens (PCNA), while downregulating the expression of pro-apoptotic proteins (such as Bax and Caspase-9). Overexpression of FGF1 activates the PI3K / AKT / GSK-3α / β pathway, increasing AKT and GSK-3α / β phosphorylation levels, and FGF1 can restore the inhibited AKT and GSK-3α / β phosphorylation levels. See [link to relevant documentation]. Figures 9-21 .

[0074] Detecting the expression levels of the FGF1 gene in the hypothalamus, pituitary gland, and ovary, such as... Figure 9As shown, the FGF1 gene exhibits differential expression in the hypothalamus, pituitary gland, and ovary.

[0075] Immunohistochemistry was used to locate the expression of FGF1 protein in ovarian follicles, such as Figure 10 As shown, FGF1 protein is highly expressed mainly in the granulosa cells of ovarian follicles of Hu sheep; Detecting the expression level of the FGF1 gene in follicles of different sizes, such as Figure 11 As shown, the expression levels of FGF1 gene mRNA and protein increase with the increase of follicle diameter; Design FGF1 overexpression vectors and verify their efficiency, such as Figure 12 As shown, the expression levels of both FGF1 gene mRNA and protein significantly increased after transfection with the FGF1 overexpression vector; The changes in reactive oxygen species (ROS) levels after FGF1 gene overexpression were detected using a ROS kit. Figure 13 As shown, overexpression of the FGF1 gene can significantly reduce the level of reactive oxygen species in granulocytes. The effect of FGF1 gene overexpression on the expression levels of related antioxidant enzymes was examined, such as... Figure 14 As shown, overexpression of the FGF1 gene upregulates the mRNA expression levels of antioxidant enzymes SOD2, CAT, and GPX, as well as the protein expression level of SOD2. The change in cell apoptosis rate after FGF1 gene overexpression was detected using an apoptosis kit, such as... Figure 15 As shown, overexpression of the FGF1 gene can significantly reduce the apoptosis rate of granulocytes; The effect of FGF1 gene overexpression on the expression levels of related apoptosis genes was examined, such as... Figure 16 As shown, overexpression of the FGF1 gene upregulates Bcl-2, downregulates Bax expression, and downregulates Caspase-9 mRNA expression. Changes in cell proliferation after FGF1 gene overexpression were detected using an EdU kit, such as... Figure 17 As shown, overexpression of the FGF1 gene promotes granulosa cell proliferation; The effect of FGF1 gene overexpression on the expression level of cell proliferation antigen (PCNA) was examined, such as... Figure 18 As shown, overexpression of the FGF1 gene upregulates the expression level of PCNA; The effect of FGF1 gene overexpression on the phosphorylation levels of AKT and GSK-3α / β was examined, such as... Figure 19 As shown, overexpression of the FGF1 gene significantly upregulated the phosphorylation levels of AKT and GSK-3α / β; The effect of the PI3K / AKT pathway inhibitor LY294002 on the phosphorylation levels of AKT and GSK-3α / β was examined, such as... Figure 20As shown, the phosphorylation levels of AKT and GSK-3α / β decreased in a dose-dependent manner after the addition of LY294002; The effect of the FGF1 gene on the restoration of phosphorylation levels of AKT and GSK-3α / β was examined, such as... Figure 21 As shown, FGF1 can partially restore the suppressed phosphorylation levels of AKT and GSK-3α / β.

[0076] The above results indicate that the FGF1 gene plays multiple regulatory roles in the granulosa cells of Hu sheep ovaries: on the one hand, it upregulates the expression of antioxidant enzymes and reduces intracellular oxidative stress levels by activating the PI3K / AKT / GSK-3α / β pathway; on the other hand, it inhibits apoptosis and promotes cell proliferation by regulating the expression of apoptosis-related genes (Bcl-2 / Bax / Caspase-9) and proliferation-related genes (PCNA). The functional state of granulosa cells is a key factor determining follicle development, maturation, and ovulation. Therefore, the FGF1 gene can serve as an important molecular target for regulating follicle development in Hu sheep and can be used to assess and improve ovarian function in Hu sheep.

[0077] Table 4

[0078] Table 5

[0079] In addition to the above-described embodiments, the present invention may have other implementations. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention.

Claims

1. The application of the FGF1 gene and its encoded protein in Hu sheep, characterized by: The nucleotide sequence of the FGF1 gene of the Hu sheep is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the FGF1 gene of the Hu sheep is shown in SEQ ID NO.

2.

2. The application of the Hu sheep FGF1 gene and its encoded protein according to claim 1, characterized in that: The invention contains primer pairs for detecting FGF1 gene expression in Hu sheep, the nucleotide sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.

4.

3. The application of the Hu sheep FGF1 gene and its encoded protein according to claim 1, characterized in that: The application of the FGF1 gene and its encoded protein in regulating oxidative stress levels, apoptosis, or cell proliferation in Hu sheep ovarian granulosa cells.

4. The application of the Hu sheep FGF1 gene and its encoded protein according to claim 3, characterized in that: Regulation is achieved by activating the PI3K / AKT / GSK-3α / β signaling pathway.

5. The application of the Hu sheep FGF1 gene and its encoded protein according to claim 3, characterized in that: The regulation of oxidative stress levels in ovarian granulosa cells of Hu sheep is manifested by reducing the level of reactive oxygen species (ROS) in granulosa cells and / or upregulating the expression of antioxidant enzymes SOD2, CAT, and GPX.

6. The application of the Hu sheep FGF1 gene and its encoded protein according to claim 3, characterized in that: The regulation of apoptosis in ovarian granulosa cells of Hu sheep was manifested by upregulating Bcl-2 expression and / or downregulating Bax and Caspase-9 expression.

7. The application of the Hu sheep FGF1 gene and its encoded protein according to claim 3, characterized in that: The regulation of granulosa cell proliferation in the ovaries of Hu sheep was manifested by upregulating the expression of proliferating cell nuclear antigen (PCNA).

8. The application of the Hu sheep FGF1 gene and its encoded protein according to claim 1, characterized in that: The application of the FGF1 gene and its encoded protein in improving the reproductive rate of Hu sheep.

9. The application of the Hu sheep FGF1 gene and its encoded protein according to claim 8, characterized in that: Improving the reproductive rate of Hu sheep is to improve the quality of follicle development in Hu sheep.

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