Application of SLC26A2 gene as a marker of tibia chondrodysplasia in broilers

Abstract

This invention discloses the application of the SLC26A2 gene as a biomarker for broiler tibial chondrodysplasia (TD), belonging to the field of molecular biology. This application includes the use of SLC26A2-detecting products in the preparation of diagnostic tools for broiler TD, and the use of SLC26A2 activators in the preparation of drugs for treating broiler TD. Experiments in the examples demonstrate that there are significant differences in SLC26A2 gene expression between normal and TD tissues in broilers, indicating that the SLC26A2 gene can be used as a novel diagnostic and prognostic biomarker for broiler TD.

Description

Technical Field

[0001] This invention belongs to the field of biological technology, specifically relating to the application of the SLC26A2 gene as a marker of tibia chondrogenesis imperfecta in broilers. Background Technology

[0002] Tibial dyschondrosis (TD) is a metabolic skeletal disease caused by metabolic disorders during the rapid growth period of poultry. This condition not only weakens poultry production performance but also causes significant economic losses to the poultry farming industry. The main characteristics of TD include cartilage degeneration in the tibia, increased growth plate thickness, obstructed blood supply, and the formation of opaque cartilage plugs. With the shift from traditional free-range farming to more intensive farming, the incidence of TD has increased. In my country, the incidence of TD has exceeded 10%, accounting for approximately 30% of all poultry skeletal diseases, posing a challenge to my country's rapidly developing poultry farming industry. Therefore, the pathogenesis and prevention of TD are gradually becoming a focus of research.

[0003] Currently, the diagnosis of tibial dyschonoplasia (TD) in broilers mainly relies on the observation of morphological changes in the tibial growth plate, although imaging diagnostic tools such as X-ray examination can also be used as an auxiliary means to assess the condition of TD. However, these traditional diagnostic methods are not cost-effective in actual poultry farming operations, especially in large-scale intensive farms or free-range environments by individual farmers, where they are both complex and time-consuming. In addition, in clinical observation, most TD in broilers presents with subclinical symptoms. When broilers show obvious lameness, it indicates a late stage of the disease, and the condition is generally more severe, often causing irreversible damage to the broilers. Some studies have begun to explore new diagnostic approaches. Publication number CN114088855 A discloses a fecal biomarker for the early diagnosis of tibial dyschonoplasia in broilers and its application. This application proposes an early diagnostic method based on fecal sample metabolomics analysis, which aims to achieve early detection of TD by screening specific biomarkers. Therefore, developing an efficient clinical diagnostic method for TD in broilers and corresponding targeted drugs is of great clinical value and socio-economic benefit for improving poultry health management, reducing economic losses, and promoting the sustainable development of the poultry farming industry. Summary of the Invention

[0004] One of the objectives of this invention is to provide a method for diagnosing tibia chondrogenesis imperfecta in broilers by detecting differences in the expression of the SLC26A2 gene or protein.

[0005] The second objective of this invention is to provide a method for predicting the prognosis of tibial chondropathy by detecting differences in the expression of the SLC26A2 gene or protein.

[0006] The third objective of this invention is to provide a method for treating tibia chondrogenesis imperfecta in broilers by activating the SLC26A2 gene or SLC26A2 protein.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides the application of the SLC26A2 gene as a marker of tibia chondrogenesis imperfecta in broilers, including the application of products that detect SLC26A2 in the preparation of diagnostic tools for tibia chondrogenesis imperfecta in broilers.

[0009] Furthermore, the product for detecting SLC26A2 includes products for detecting the expression level of the SLC26A2 gene.

[0010] Furthermore, the products for detecting SLC26A2 gene expression include products capable of quantifying SLC26A2 gene mRNA or products capable of quantifying SLC26A2 protein.

[0011] Furthermore, the reagents for quantifying SLC26A2 gene mRNA include primers specifically amplifying the SLC26A2 gene used in real-time quantitative PCR, the primer sequences of which are shown in SEQ ID NO.6 and SEQ ID NO.7; the reagents for quantifying SLC26A2 protein include antibodies that specifically bind to SLC26A2 protein.

[0012] The number of amino acids recognized by the anti-SLC26A2 antibody or other fragments in the detection products and diagnostic tools based on the SLC26A2 gene provided by this invention is not particularly limited, as long as the antibody can bind to SLC26A2.

[0013] This invention provides a method for diagnosing tibia chondrogenesis imperfecta in broilers, the method being as follows:

[0014] (1) Obtain broiler chicken samples;

[0015] (2) Detect the expression level of SLC26A2 gene or protein in broiler samples;

[0016] (3) Associate the detected SLC26A2 gene or protein expression level with whether the broiler chickens being tested are diseased;

[0017] (4) If the expression level of the SLC26A2 gene or protein is reduced, it indicates that the broiler is diagnosed with tibial chondrodysplasia or the broiler is determined to have a poor prognosis.

[0018] This invention also provides the use of an activator of SLC26A2 in the preparation of a drug for treating tibia chondropathy in broilers.

[0019] Furthermore, the activator can promote or enhance the expression or activity of SLC26A2 or substances involved in upstream or downstream pathways of SLC26A2.

[0020] Furthermore, the activators of the SLC26A2 gene or SLC26A2 protein mentioned above are not limited, as long as they can promote or enhance the expression activity of SLC26A2 or substances involved in the upstream or downstream pathways of SLC26A2, and are effective in treating tibia chondrogenesis imperfecta in broilers.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention discloses the application of the SLC26A2 gene as a biomarker for tibial chondrodysplasia (TD) in broilers. By detecting the SLC26A2 gene content in broilers, it is possible to determine whether broilers are at high risk of TD. This discovery is of great significance for the clinical diagnosis and prevention strategy development of TD, especially given the current lack of diagnostic methods targeting broiler TD genotypes. Furthermore, applying the SLC26A2 gene to the diagnosis, treatment, and prognostic assessment of broiler TD provides a new direction for using differentially expressed genes of TD in broilers as biomarkers for clinical diagnosis. It also has the potential to become a target for developing novel treatment strategies, promoting the development of broiler TD prevention and control technologies, improving broiler health, and increasing breeding efficiency. Attached Figure Description

[0023] Figure 1 This is a diagram showing the gene and protein expression levels of SLC26A2 chondrocytes after silencing in an embodiment of the present invention; wherein: Figure 1 (A) is a graph showing the gene expression level of SLC26A2 gene after silencing in chondrocytes. Figure 1 (B) shows the protein expression level after SLC26A2 silencing in chondrocytes. Figure 1 (C) shows the bands of SLC26A2 and GAPDH internal reference proteins.

[0024] Figure 2 This is a diagram showing the gene and protein expression levels after SLC26A2 gene overexpression in chondrocytes in an embodiment of the present invention; wherein, Figure 2 (A) is a graph showing the gene expression level after SLC26A2 gene overexpression in chondrocytes. Figure 2 (B) is a band diagram of SLC26A2 and GAPDH internal reference proteins. Figure 2 (C) shows the protein expression level after overexpression of the SLC26A2 gene in chondrocytes.

[0025] Figure 3 This is a diagram showing the expression level of the SLC26A2 gene in chondrocytes in an embodiment of the present invention; wherein: Figure 3 (A) is a graph showing the expression level of the SLC26A2 gene after chondrocytes were attacked with 10 μM thiamethoxam. Figure 3 (B) is a graph showing the expression level of the SLC26A2 gene in TD tissue of broiler chickens; Figure 3 (C) shows the expression levels of chondrocyte development-related genes after SLC26A2 silencing in chondrocytes.

[0026] Figure 4 This is a graph showing the expression levels of chondrocyte development-related genes after overexpression of the SLC26A2 gene in chondrocytes in an embodiment of the present invention.

[0027] Figure 5 This is a graph showing the protein expression level of SLC26A2 in TD tibia tissue of broiler chickens and after thiamethoxam-treated chondrocytes in an embodiment of the present invention.

[0028] Figure 6 This is a graph showing the expression levels of chondrocyte development-related proteins after SLC26A2 silencing in chondrocytes in an embodiment of the present invention.

[0029] Figure 7 This is a graph showing the expression levels of chondrocyte development-related proteins after SLC26A2 overexpression in chondrocytes in an embodiment of the present invention. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments:

[0034] Example

[0035] 1. Sample Acquisition

[0036] Twenty healthy AA broiler chickens of similar weight, aged one day, were purchased from Ludong Breeding Professional Cooperative. They were acclimatized to three days of age and then randomly divided into two groups of 10 chickens each: a control group (CON group) and a TD group (Thiram group). The CON group was fed a standard basal diet, while the TD group was fed a diet supplemented with 50 mg / kg of thiram from day 4 to day 21. Tibial bone samples were collected from the broilers at day 21 and rapidly frozen in liquid nitrogen for subsequent quantitative PCR and Western blotting experiments.

[0037] 2. Obtaining primary broiler cells and establishing a TD chondrocyte model induced by thiram.

[0038] Select 15-day-old chicken embryos and place them on a laminar flow hood after alcohol sterilization. Use forceps to gently tap open the air cell of the embryo. Use ophthalmic forceps to cut the membrane and extract the tissue, placing it into a sterile culture dish. Then, use ophthalmic scissors to remove the tibia along the thigh root of the chicken embryo and wash three times with PBS. Separate the articular cartilage using cytokines. In a sterile culture dish containing PBS, use ophthalmic scissors to cut the cartilage into 1-3 mm pieces, then transfer them to new centrifuge tubes and wash twice with PBS. Afterward, digest the tissue with 0.2% collagenase IV (catalog number: 2091 mg 100, Biofrox, Germany) at 37°C for 12 hours. Subsequently, place the tissue digest through a 400-mesh filter, collect the isolated primary chondrocytes, and wash the cells twice with PBS. Finally, chondrocytes were diluted with DMEM / F12 medium (C11330500BT, Gibco, USA) supplemented with 15% fetal bovine serum (FBS, Yilang, China) and 1% penicillin-streptomycin (Catalog No.: G4003, Servicebio, Wuhan) and seeded at a density of 5 × 10⁶ cells / year. 5 Cells / mL culture plates were incubated in a 5% CO2, 37°C incubator. Chondrocytes were challenged with 10 μM thiram-2 medium for 24 hours to detect changes in gene levels, and for 48 hours to detect changes in protein levels.

[0039] 3. Chondrocyte transfection

[0040] When the chondrocyte density reached 70-80%, the cells were washed three times with PBS. Transfection reagents and siRNA or pEGFP-SLC26A2 plasmid DNA were diluted using opti-MEM (catalog number: 31985-070, Gibco, USA) and DMEM / F12 medium (catalog number: C11330500BT, Gibco, USA) containing 10% FBS (Yilang, China) but without double antibodies, and transfected according to the instructions of Lipofectamine 3000 (catalog number: L3000008, Thermo, USA). siRNA was synthesized by GenePharma (GenePharma, China). After transfection, the culture plates were placed in an incubator for further culture. After 24-72 hours, cells were collected to assess transfection efficiency. The si-SLC26A2 RNA oligo sequences are shown in Table 1 (in accordance with WIPOST.26 standards, all "U" (representing uracil in the nucleotide sequence) in the sequence listing were replaced with "T").

[0041] Table 1

[0042]

[0043] ① Primer Design: Based on the SLC26A2 gene sequence CDS region found on NCBI, a suitable primer was designed at each end to amplify the complete SLC26A2 CDS region. After BLAST testing of primer specificity, the primers were synthesized by Qingke Biotechnology Co., Ltd. Then, the sequences of the vector pEGFP-N1 and SLC26A2 were compared, and suitable restriction enzyme sites were selected. Restriction enzyme sites and protective bases were added to the designed primers. After BLAST testing of primer specificity, the primers were synthesized by Qingke Biotechnology Co., Ltd. The sequences of the two pairs of primers used are shown in Table 2 ((According to the WIPOST.26 standard, in the sequence listing, all "U" (representing uracil in the nucleotide sequence) are replaced with "T")).

[0044] Table 2

[0045]

[0046] ②cDNA synthesis: Total RNA was extracted from broiler tibia cartilage tissue according to the experimental method in point 4. After the concentration was measured by a micro spectrophotometer, it was quantified to 500 ng / μL. Then, the following systems were prepared according to the reverse transcription kit instructions, as shown in Tables 3, 4 and 5.

[0047] Table 3. Reverse Transcription First Step System

[0048] Components Volume (μL) <![CDATA[RNase-free ddH2O]]> 6 Total RNA 2

[0049] Heat at 65℃ for 5 minutes, then quickly place on ice to cool rapidly, and let stand on ice for 2 minutes.

[0050] Table 4. Reverse Transcription Second Step System

[0051] Components Volume (μL) The mixture from the previous step 8 5×gDNA wiper Mix 2

[0052] Gently blow and mix well, react at 42℃ for 2 minutes.

[0053] Table 5. Reverse Transcription Third Step System

[0054] Components Volume (μL) The mixture from the previous step 10 10×RT Mix 2 HiScript III Enzyme Mix 2 Oligo(dT)20VN 1 <![CDATA[RNase-free ddH2O]]> 5

[0055] After mixing thoroughly by pipetting, the mixture was placed in a PCR instrument and reacted at 25°C for 5 min, 37°C for 45 min, and 85°C for 5 s. The product was stored at -80°C.

[0056] ③ Product amplification: Dilute the cDNA product obtained in the previous step 10-fold. Prepare the following reaction system according to the Taq enzyme instructions, as shown in Table 6.

[0057] Table 6 PCR reaction system

[0058] Components Volume (μL) Template DNA 2 Upstream primer (10 μM) 2.5 Downstream primer (10 μM) 2.5 2×Magic Green Taq SuperMix 25 <![CDATA[ddH2O]]> 18

[0059] The reaction procedure is shown in Table 7.

[0060] Table 7 PCR reaction procedure

[0061]

[0062] ④ Product Recovery: Prepare the gel and add the amplification product and DNA Marker obtained in the previous step to the wells. Perform electrophoresis at 110V for 30 minutes. After electrophoresis, image the gel using a multi-functional imaging system. Observe a bright band slightly larger than 1000bp, and cut the product fragment completely.

[0063] ⑤ Product purification: Recover the DNA using the gel extraction purification kit instructions and measure the concentration.

[0064] ⑥ Secondary amplification: Using the gel-recovered product as template DNA, primers with added restriction enzyme sites and homologous arms were used for a second amplification according to the reaction system and procedure in step ③, followed by gel recovery. The gel-recovered product was sequenced by Sangon Biotech Co., Ltd.

[0065] ⑦ Enzyme digestion: The pEGFP-N1 vector strain was plated on an agar plate and incubated overnight at 37°C. The next day, a single colony was picked and placed in 10 mL of broth medium, and cultured at 37°C with shaking for 12 h. Then, the plasmid was extracted according to the instructions of the plasmid extraction kit, and the concentration was measured to 500 ng / μL. The vector plasmid was digested with a rapid digestion enzyme, and the system is shown in Table 8.

[0066] Table 8

[0067] Components Volume (μL) QuickCut green Buffer 5 Empty plasmid 2 Hind III rapid cleavage enzyme 1 XheⅠ rapid cleavage enzyme 1 <![CDATA[ddH2O]]> 41

[0068] The reaction was carried out at 37°C for 5 hours. The reaction product was subjected to nucleic acid electrophoresis, and then recovered using a kit to obtain the purified linearized vector, and the concentration was measured.

[0069] ⑧ Homologous recombination: The DNA fragment and the linearized vector are carried out in the proportions specified in the instructions for recombination. The recombination reaction system is shown in Table 9.

[0070] Table 9

[0071] Components Volume (μL) DNA fragment (20 ng / μL) 3 Linearized vector (20 ng / μL) 1 2×Sealess Master Mix 5 <![CDATA[ddH2O]]> 1

[0072] React at 50°C for 30 minutes, then place the centrifuge tubes on ice.

[0073] ⑨ Transformation: Add 5 μL of ligation solution to 50 μL of freshly thawed DH5α competent cells, mix gently, incubate on ice for 30 min, heat shock in a 42°C water bath for 30 s, immediately place on ice for 2 min, add 500 μL of broth (antibiotic-free), and incubate at 37°C with shaking for 60 min. Then centrifuge at 3000g for 2 min, discard part of the supernatant, and gently pipette to thoroughly mix the cells. Spread 200 μL of the bacterial culture onto a plate and incubate overnight at 37°C.

[0074] ⑩ Identification: After colonies grow, pick a single colony and culture it in 500 μL of broth medium with shaking for 3 hours. Then, use the bacterial culture as a template to amplify DNA. Perform nucleic acid electrophoresis on the amplification product. After confirming that it is a positive colony, add the remaining medium to 10 mL of broth medium and culture at 37°C with shaking for 14-16 hours.

[0075] Plasmid extraction: The overexpression plasmid was extracted according to the instructions of the plasmid extraction kit. After collection, the concentration was measured and quantified to 500 ng / μL. The SLC26A2 overexpression plasmid transfection system is shown in Table 10.

[0076] Table 10

[0077]

[0078] Experimental results:

[0079] like Figure 1 As shown: After si-SLC26A2 was transfected into broiler tibia chondrocytes, the expression of the corresponding genes and proteins decreased, indicating that the si-SLC26A2 transfection model of broiler tibia chondrocytes was successfully established; Figure 2 As shown, after transfecting broiler tibia chondrocytes with the constructed pEGFP-SLC26A2 overexpression vector, SLC26A2 gene expression significantly increased after 24 hours, and protein expression also significantly increased after 48 hours. This indicates that the SLC26A2 overexpression vector was successfully constructed. The above experiments demonstrate that the expression levels of the corresponding SLC26A2 gene and protein changed significantly after appropriate treatment, indicating that the SLC26A2 interference and overexpression model was successfully constructed.

[0080] 4. Total RNA extraction from tibial growth plate tissue and cells and quantitative real-time PCR (qRT-PCR) reaction.

[0081] When the cells reached approximately 80% confluence in the culture dish, a TD in vitro cell model was constructed using 10 μM thiophanate-methyl virus. Total RNA was then extracted from the tibial growth plate and cells using a total RNA extraction kit (FastPure Cell / Tissue Total RNA Isolation Kit V2, Vazyme, China, catalog number: RC112-01). gDNA was removed from the total RNA, and the mRNA was reverse transcribed into cDNA using a reverse transcription kit (HiScript IIQ RT SuperMix for qPCR (+gDNA wiper), Vazyme, China, catalog number R223-01) according to the kit specifications. RT-qPCR was performed using a universal SYBR Green Fast qPCRMix (ABclonal, China, catalog number RK21203). The RT-qPCR instrument (qTOWER3; analytikjena, Germany) was used, and the program was set according to the qPCR Mix specifications. Primer sequences for RT-qPCR were designed using a primer design tool (National Center for Biotechnology Information, USA), and the primers were synthesized by Qingke Technology Co., Ltd., China. Using 2... -△△CT The method normalizes the expression level of the target gene to GAPDH, and the primer sequences are shown in Table 11 ((in accordance with the WIPOST.26 standard, all "U" (representing uracil in the nucleotide sequence) in the sequence listing are changed to "T")).

[0082]

[0083] Experimental results:

[0084] Figure 3A graph showing the expression level of the SLC26A2 gene in chondrocytes, from... Figure 3 As shown in (A), after attacking chondrocytes with 10 μM fumarate, the expression of the SLC26A2 gene decreased significantly; from Figure 3 As shown in (B), in TD tissue of broilers, the expression level of the SLC26A2 gene in the tibial cartilage tissue of TD-type broilers was significantly lower than that of the CON gene in the control group. This indicates that the in vivo and in vitro experimental results in broilers are consistent, suggesting that the expression level of the SLC26A2 gene significantly decreases when TD occurs in broilers, thus demonstrating that SLC26A2 can serve as an important indicator for detecting TD in broilers. Figure 3 As shown in (C), after transfecting si-SLC26A2 into chondrocytes, the cartilage development-related genes Runx2 and ALP were significantly downregulated, while the MMP13 gene was significantly upregulated. This indicates that interfering with the expression of the SLC26A2 gene significantly affects chondrocyte differentiation and development.

[0085] Figure 4 This image shows the expression levels of chondrocyte development-related genes after SLC26A2 overexpression. The pEGFP-SLC26A2 plasmid was transfected into chondrocytes, and gene expression levels were detected by qRT-PCR. The image shows that overexpression of pEGFP-SLC26A2 significantly increased the expression levels of chondrocyte development-related genes Runx2 and ALP, while significantly downregulated MMP13. This indicates that SLC26A2 gene overexpression promotes chondrocyte differentiation.

[0086] The above experiments show that the SLC26A2 gene is an important gene related to cartilage development. Furthermore, after TD occurs in broilers, detecting the expression of the SLC26A2 gene can help determine whether broilers are at high risk of developing TD.

[0087] 5. Extraction of proteins from tibial growth plate tissue and cells, and Western blotting of proteins.

[0088] Tissue samples were homogenized on ice using RIPA lysis buffer (Beyotime, China, catalog number P0013B) supplemented with 1% PMSF protease inhibitor (Beyotime, China, catalog number ST505). Protein concentration was quantified to 1 μg / μL using a BCA protein assay kit (NCM, China, catalog number WB6501). The sample was then mixed with SDS-PAGE loading buffer (NCM, China, catalog number P0015) and heated at 100°C for 10 min to denature the protein and expose the primary antibody binding sites. PAGE gels were prepared using a one-step PAGE gel rapid preparation kit (12%) (Vazyme, China, catalog number E304-01). 10 μL aliquots of the lysate mixture were loaded onto SDS-PAGE gels and electrophoresed at 80 V for 2 h. Proteins from the PAGE gels were transferred to a 0.45 μm pore size polyvinylidene fluoride (PVDF) membrane. PVDF membrane (Millipore, USA, catalog no. IPVH00010) was transferred using a wet transfer method at 150 mA for 2 hours, during which the instrument was cooled to 0-4°C. Non-specific antigens on PVDF were blocked by incubating the membrane with 5% skim milk powder (Servicebio, China, catalog no. GC310001). The membrane was dissolved in TBS (Tris-buffered saline) with 0.1% Tween-20 on a horizontal shaker at room temperature for 1.5 hours. The membrane was then incubated with primary antibody at 4°C for 16 hours, followed by incubation with horseradish peroxidase (HRP)-labeled secondary antibody at room temperature for 1 hour. Blots were developed using the NcmECL Ultra-Enhanced Chemiluminescence (ECL) Kit (NCM, China, catalog number P10100), and the chemiluminescence signals of the blots were captured using UVP ChemStudio (Analytik Jena, Germany). The grayscale values ​​of each blot were analyzed using ImageJ software (National Institutes of Health, USA) and normalized using GAPDH.

[0089] Experimental results:

[0090] Figure 5 This image shows the protein expression levels of SLC26A2 in broiler tibial tissue and chondrocytes after challenged with 10 μM thiram. The protein expression level of SLC26A2 in broiler tibial chondrocytes was detected 48 hours after challenge with 10 μM thiram. Figure 5 As shown in (B), SLC26A2 protein expression was significantly downregulated after thiamethoxam-treated chondrocytes. Figure 5As shown in (A), in the tibial cartilage tissue of TD broiler chickens, the SLC26A2 protein was significantly downregulated in the TD group compared to the control group CON. This indicates that the protein expression in vivo and in vitro experiments showed consistency, suggesting that SLC26A2 was significantly downregulated in broiler chickens with TD, and considering the above in this embodiment... Figure 3 The experimental results show that the expression levels of the SLC26A2 gene and protein are consistent in the tibial cartilage tissue and cells of TD broiler chickens.

[0091] Figure 6 This image shows the expression levels of chondrocyte development-related proteins after SLC26A2 silencing in chondrocytes. Chondrocytes were transfected with si-SLC26A2 for 48 hours, and the expression levels of these proteins were detected by Western blotting. Figure 6 As shown in the figure, compared with the NC group, the expression levels of cartilage development-related proteins COL2α1, BMP2, Runx2, ACAN and ALP in the SLC26A2 gene interference group were significantly downregulated, indicating that interference with the expression of SLC26A2 protein inhibits chondrocyte development.

[0092] Figure 7 This image shows the expression levels of chondrocyte development-related proteins after SLC26A2 overexpression in chondrocytes. The expression levels of these proteins were detected 48 hours after pEGFP-SLC26A2 transfection into chondrocytes. The results are shown below. Figure 7 As shown in the figure, the protein expression levels of cartilage development-related proteins ACAN, BMP2, COL2α1, SOX9, and Runx2 were significantly upregulated compared to the empty vector group (pEGFP group), indicating that overexpression of the SLC26A2 gene is beneficial to chondrocyte development.

[0093] In summary, the SLC26A2 gene was significantly downregulated in the tibia cartilage tissue and cells of TD broilers, with consistent expression levels of both the gene and protein. The consistency between in vivo and in vitro experiments indicates that SLC26A2 is an important indicator gene for TD in broilers. Furthermore, the expression levels of cartilage development-related genes and proteins remained consistent after interfering with and overexpressing the SLC26A2 gene in broiler tibia cartilage cells using quantitative real-time PCR and Western blotting. Therefore, the experimental results of this embodiment demonstrate that the SLC26A2 gene can be used as a detection indicator for TD in broilers.

[0094] The application of the SLC26A2 gene as a marker of tibia chondrogenesis imperfecta in broilers has been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and core ideas of this invention. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims of this invention.

Claims

1. Application of products that detect SLC26A2 gene expression levels in the preparation of diagnostic tools for tibia chondrodysplasia in broilers.

2. The application according to claim 1, characterized in that, The products used to detect SLC26A2 gene expression include products that can quantify SLC26A2 gene mRNA or products that can quantify SLC26A2 protein.

3. The application according to claim 2, characterized in that, The reagents for quantifying SLC26A2 gene mRNA include primers specifically amplifying the SLC26A2 gene used in real-time quantitative PCR, the primer sequences of which are shown in SEQ ID NO. 6 and SEQ ID NO. 7; the reagents for quantifying SLC26A2 protein include antibodies that specifically bind to SLC26A2 protein.

4. Application of SLC26A2 overexpression plasmid in the preparation of a drug for treating tibia chondrogenesis imperfecta in broilers.

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