Application of SLC2A9 gene in identification of chicken ovary state and application product

Abstract

The invention relates to the technical field of biotechnology and animal genetic breeding, and aims at providing application of an SLC2A9 gene in identifying the chicken ovary state. The second purpose of the invention is to provide an application of the SLC2A9 gene or the expression product thereof as a marker in identifying or evaluating the follicular atresia degree of chicken. The third purpose of the invention is to provide the application of the SLC2A9 gene or the expression product of the SLC2A9 gene as a marker in identifying or evaluating the senescence state of the chicken ovary. The invention finds that the SLC2A9 gene or the expression product thereof can be used as a specific biomarker for evaluating the chicken ovarian development state, follicular atresia degree and ovarian aging degree. By detecting the expression level, the objective and quantitative evaluation of the ovarian physiological status of the individual chicken can be realized, and the limitation of only depending on external phenotype observation is overcome.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and animal genetic breeding technology, and in particular to the application of the SLC2A9 gene in identifying the ovarian status of chickens and related products. Background Technology

[0002] The ovary is the decisive organ for egg production in poultry. Exploring the developmental status of the ovary at different stages and its regulatory factors is of great significance for improving egg production performance. One of the key factors contributing to ovarian aging, functional decline, and exacerbated follicular atresia is metabolic disorder and redox imbalance. SLC2A9, a member of the solute carrier family, focuses on the transmembrane transport of uric acid. Uric acid has been proven to be closely related to purine metabolism and oxidative stress, and studies have also shown that uric acid can improve ovarian reserve function. Chickens are typical uric acid-excreting animals. Transcriptomic and metabolomic analyses of chicken ovarian tissue have revealed a close association between the purine metabolite adenosine and reproductive function. Existing research indicates that SLC2A9 maintains uric acid balance in the chicken liver, kidneys, and intestines through a feedback regulatory mechanism. Recent studies have confirmed that uric acid has a bidirectional regulatory role in avian tissues: excessive accumulation can induce inflammatory damage, while at physiological concentrations, it can enhance antioxidant enzyme activity by activating the Nrf2-ARE signaling pathway, significantly alleviating oxidative stress damage. Experiments on chicken intestinal epithelial cells have clearly shown that uric acid pretreatment can reduce MDA content and enhance cell viability by upregulating nuclear Nrf2 expression and downstream target gene activity. This suggests that SLC2A9 may participate in the regulation of oxidative stress through precise regulation of local purine metabolites in the ovary.

[0003] Exploring the role of the SLC2A9 gene in ovarian development is beneficial for elucidating the genetic mechanisms of egg production traits in poultry and accelerating the breeding of animal breeds. Therefore, clarifying the role of the SLC2A9 gene in the follicle selection process will not only provide new insights into the regulatory mechanisms of follicle development, but also lay the foundation for further research on the regulatory role of the SLC2A9 gene in poultry egg production traits. Summary of the Invention

[0004] To address the technical problems mentioned in the background section, this invention provides the application of the SLC2A9 gene in identifying chicken ovarian status and related products.

[0005] The present invention is achieved by the following technical solution: One of the objectives of the present invention is to propose the application of the SLC2A9 gene in the identification or evaluation of chicken ovarian development status, and to achieve the identification or evaluation by detecting the expression level of the gene or its expression product.

[0006] The second objective of this invention is to propose the application of the SLC2A9 gene in identifying or assessing the degree of follicular atresia in chickens, by detecting the expression level of the gene or its expression product.

[0007] The third objective of this invention is to propose the application of the SLC2A9 gene in identifying or assessing the aging state of chicken ovaries, by detecting the expression level of the gene or its expression product.

[0008] Furthermore, the identification or assessment is achieved by detecting the expression level of the SLC2A9 gene or its expression product, wherein a higher expression level indicates a better ovarian development status, a milder degree of follicular atresia, or a younger ovarian aging status.

[0009] Furthermore, the higher expression level is specifically manifested in the fact that the expression level in the ovarian tissue of chickens at peak egg production (W30) is significantly higher than that in the ovarian tissue of chickens at late egg production (W90).

[0010] The fourth objective of this invention is to provide a kit for identifying or assessing the developmental status of chicken ovaries, the degree of follicular atresia, or the aging status of ovaries. The kit includes primers and probes for detecting SLC2A9 gene mRNA, or antibodies for detecting SLC2A9 protein.

[0011] Furthermore, the detection of the expression product of the SLC2A9 gene involves detecting its mRNA or protein.

[0012] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0013] This study reveals for the first time the direct link between SLC2A9 and chicken ovarian status: Through multi-omics analysis, this invention, for the first time, demonstrates in chicken ovarian tissue that the expression levels of the SLC2A9 gene at both the transcriptional (mRNA) and translational (protein) levels are significantly negatively correlated with the decline of ovarian function from peak egg production (W30) to late egg production (W90). This provides a novel perspective and target for understanding the molecular mechanisms of chicken ovarian aging.

[0014] This invention provides a clear and detectable biomarker: It clarifies that the SLC2A9 gene or its expression product can serve as a specific biomarker for assessing ovarian development, follicular atresia, and ovarian aging in chickens. By detecting its expression level, an objective and quantitative assessment of the physiological state of an individual chicken's ovary can be achieved, overcoming the limitations of relying solely on external phenotypic observation.

[0015] Strong correlation and forward-looking application value: This discovery is not only a scientific correlation, but also has direct application potential. The detection kit developed based on this biomarker can be widely used in poultry breeding, production management, and reproductive health monitoring, providing important technical means and decision-making basis for early selection of high-fertility breeder chickens and timely adjustment of feeding strategies to delay the decline in egg production performance.

[0016] The detection methods are mature and easy to promote: the detection methods based on this marker are all mature and conventional technologies in the field, which are easy to standardize and promote in laboratory and production practice, and the technology transfer threshold is low. Attached Figure Description

[0017] Figure 1 The histological morphology of the ovary of chickens at different laying stages;

[0018] Figure 2 To investigate the genomic differences in SLC2A9 gene expression in laying hens at different laying stages;

[0019] Figure 3 To investigate the differences in histone expression of the SLC2A9 gene in laying hens at different laying stages. Detailed Implementation

[0020] The present invention will now be further described in conjunction with the accompanying drawings and 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.

[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0022] Example 1:

[0023] Phenotypic and histological analysis of chicken ovary status:

[0024] 1. Experimental grouping and phenotypic data analysis

[0025] The chickens used in this experiment were divided into two groups. The experimental samples were white-feathered hens at 30 weeks of age (W30, n=6) peak laying period and 90 weeks of age (W90, n=6) late laying period, from the Livestock and Poultry Genetic Resources and Breeding Experimental Base of China Agricultural University. Ovarian tissue (composed of small white follicles, large white follicles, small yellow follicles, and ovarian stroma) was collected from 6 chickens in each group, flash-frozen in liquid nitrogen, and stored at -80℃. Any two samples from each group were mixed to form one sample, resulting in three biological replicates for each group. Phenotypic measurements such as body weight, follicle number, and ovarian weight were performed on the two groups of laying hens.

[0026] Body weight, ovarian weight as a percentage of live weight, and follicle count (3-5 mm, 6-8 mm, and >9 mm) (n=6 per group) of W30 and W90 white-feathered hens were analyzed. Data are expressed as mean ± SD. Table 1 shows that compared to W30 hens, W90 hens had a significantly reduced number of small yellow follicles (6-8 mm) (P<0.05), no significant difference in the number of white follicles (3-5 mm) (P>0.05), and a significantly reduced number of follicles larger than 9 mm in diameter (P<0.01). After normalization, ovarian weight showed a significant difference between W30 and W90 hens (P<0.05). However, there was no significant difference in body weight between the two groups (P>0.05).

[0027] Table 1 shows the statistics of live weight, ovarian weight, and number of follicles.

[0028] parameter W30 W90 P-Value Body weight (g) 1436.67±206.56 1584.38±201.07 0.237938812 3-5mm follicles 23±4.05 18.17±4.92 0.092694099 6-8mm follicle 10.67±3.39 6.17±3.31 0.042248414 Follicle >9mm 6.83±0.98 5.33±0.52 0.007899757

[0029] 2. Paraffin sections and HE staining of ovarian tissue

[0030] Follicles of various grades were dissected from ovarian tissue, washed with PBS, and preserved in 4% paraformaldehyde solution for paraffin section preparation. The follicles were then dehydrated using ethanol at different concentrations, followed by clearing with xylene and paraffin embedding. Finally, the embedded paraffin blocks were sectioned to a thickness of 5 μm using a microtome.

[0031] HE staining: First, dewax the sections, bake them at 65°C for 1 hour, treat with xylene and hydrate with graded ethanol, then rinse with distilled water, stain with hematoxylin for 15 minutes, and rinse thoroughly with distilled water. Differentiate with 1% hydrochloric acid ethanol for 10 seconds, then bluing in tap water for 10 seconds, then rinsing in tap water for 10 minutes, dehydrating with graded ethanol, staining with eosin for 1 minute, dehydrating with graded ethanol, then soaking in xylene for 5 minutes, and finally mounting the sections with neutral resin.

[0032] Microscopic examination: The HE-stained paraffin sections of follicles were photographed and observed under a microscope.

[0033] Experimental results are as follows Figure 1 As shown, the follicles at all stages of the W30 ovary are tightly arranged. The ovarian stroma of the W30 White Leghorn laying hen contains a large number of rapidly growing follicles, and the ovarian surface shows swelling and bulging of the ovarian tissue. Simultaneously, some larger secondary follicles undergo atresia. Figure 1 AC). Follicles at all stages of ovarian development were relatively sparse in W90 hens. In the ovarian stroma of W90 White Leghorn laying hens, primordial follicles were more numerous, while rapidly growing follicles were fewer. Atretic follicles were also observed. Figure 1 DF).

[0034] Example 2:

[0035] Transcriptomic validation of the SLC2A9 gene in chicken ovaries:

[0036] Total RNA extraction and quality control

[0037] RNA was extracted from chicken ovarian tissue using TRIZOL. RNA purity was detected using a NanoDrop2000 micro spectrophotometer, and RNA concentration and integrity were detected using an Agilent 2100 spectrophotometer.

[0038] Library construction and sequencing

[0039] Library construction and sequencing were performed by Guangzhou Gediao Biotechnology Co., Ltd.

[0040] Bioinformatics analysis

[0041] Quality control

[0042] Raw reads were subjected to quality control using FASTP to filter out low-quality data and obtain clean reads. The filtered data were then further analyzed for base composition and quality distribution; data that passed quality control were used for subsequent analyses.

[0043] Sequence alignment analysis

[0044] The clean reads were aligned to a chicken ribosome database using the bowtie2 alignment tool. Reads that were aligned to ribosomes were removed, leaving unmapped reads for subsequent transcriptome analysis. The clean reads were then aligned to the reference genome Ensemblrelease110 using HISAT2. Alignment to exon, intron, and intergenic regions was statistically analyzed.

[0045] Expression statistics

[0046] Transcripts were reconstructed using Stringtie, and the expression levels of all genes in each sample were calculated using RSEM (RNA-Seqby Expectation-Maximization). The FPKM value was calculated using the following formula:

[0047] In the formula, for the expression level FPKM value of gene i, C represents the number of segments aligned to gene i, N represents the number of segments aligned to the reference genome, and L represents the number of bases in gene i.

[0048] Intergroup difference analysis

[0049] Differentially expressed genes (DEGs) were analyzed using DESeq2 software. The screening criteria were as follows: genes with FC ≥ 1.7 and FDR < 0.05 were considered significantly upregulated genes; genes with FC ≤ 1 / 1.7 and FDR < 0.05 were considered significantly downregulated genes.

[0050] A total of 139 differentially expressed genes were detected, of which 39 were significantly downregulated and 100 were significantly upregulated. Among them, the differentially expressed gene SLC2A9 was significantly upregulated in the W30 group compared to the W90 group. The results were visualized using a t-test. Figure 2 As shown.

[0051] Example 3:

[0052] Proteomic validation of SLC2A9 protein in chicken ovary:

[0053] Protein extraction:

[0054] An appropriate amount of ovarian tissue was added to 2 ml of lysis buffer and homogenized for 5 min using a tissue homogenizer. The homogenate was then incubated on ice for 2 h, centrifuged at 12,000 rpm for 15 min at 4 °C, and the supernatant was collected to obtain purified protein. Subsequently, the protein was enzymatically digested with trypsin and desalted using an iSTcartridge.

[0055] Establish a spectral database

[0056] High pH reverse phase separation

[0057] After the peptide mixture was redissolved in buffer A, it was separated at high pH using a reverse column connected to an Ultimate3000 system. The separation was performed using a linear gradient, and the fractions were collected and dried in a vacuum concentrator for later use.

[0058] Low pH nano-HPLC-MS / MS analysis (DDA qualitative library preparation)

[0059] The desalted lyophilized peptides were reconstituted in Solvent A and analyzed by LC-MS / MS using an online nanojet ion source. The OrbitrapLumos mass spectrometer operated in data-dependent acquisition mode, automatically switching between MS and MS / MS acquisition.

[0060] DIA Data Acquisition

[0061] DIA data acquisition was performed on the mass spectrometry data obtained from data-dependent acquisition in section 4.2.5.2.

[0062] Qualitative and quantitative analysis of proteins

[0063] Quality control was performed using QuiC (Biognosys) software, and then Pulsar software was used to construct a database for the data obtained in DDA acquisition mode. The DDA data results were analyzed according to the DDA reference database to identify proteins. The false positive rate (FDR) for protein levels was set to 1%.

[0064] Examining transcriptomics results

[0065] In the proteomics results, the t-test was used to validate the important candidate gene SLC2A9 screened from the transcriptome. The experimental results are as follows: Figure 3 As shown, SLC2A9 protein expression was significantly upregulated in the W30 group compared to the W90 group, and the trend was the same as that of the transcriptome results.

[0066] In summary, this invention, through combined transcriptomic and proteomic analysis, has for the first time demonstrated the biological function of the SLC2A9 gene in chicken ovaries, laying an experimental foundation for exploring the mechanism by which the chicken SLC2A9 gene regulates follicle development.

[0067] This invention demonstrates that the SLC2A9 gene can inhibit follicular atresia and regulate redox balance. It clarifies the important regulatory role of the chicken SLC2A9 gene in ovarian development, providing new insights into the expression regulation mechanisms of follicular selection and laying the foundation for further research on the regulatory role of the SLC2A9 gene in poultry egg production traits.

[0068] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

[0069] In conclusion, the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The application of the SLC2A9 gene in identifying or assessing the developmental status of chicken ovaries, characterized in that, The identification or evaluation is achieved by detecting the expression level of the SLC2A9 gene or its expression product.

2. The application of the SLC2A9 gene in identifying or assessing the degree of follicular atresia in chickens, characterized in that... The identification or evaluation is achieved by detecting the expression level of the SLC2A9 gene or its expression product.

3. The application of the SLC2A9 gene in identifying or assessing the aging status of chicken ovaries, characterized in that... The identification or evaluation is achieved by detecting the expression level of the SLC2A9 gene or its expression product.

4. The application according to any one of claims 1 to 3, characterized in that, The identification or assessment is achieved by detecting the expression level of the SLC2A9 gene or its expression product, wherein a higher expression level indicates a better ovarian development status, a milder degree of follicular atresia, or a younger ovarian aging status.

5. The application according to claim 4, characterized in that, The higher expression level is specifically manifested in the fact that the expression level in the ovarian tissue of chickens at peak egg production (W30) is significantly higher than that in the ovarian tissue of chickens at late egg production (W90).

6. A kit for identifying or assessing the developmental status of chicken ovaries, the degree of follicular atresia, or the state of ovarian senescence, characterized in that, The kit includes primers and probes for detecting SLC2A9 gene mRNA, or antibodies for detecting SLC2A9 protein.

7. The reagent kit according to claim 6, characterized in that, The detection of the expression product of the SLC2A9 gene includes detecting its mRNA or protein.

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