A method for assessing chicken aging based on PDCD10 gene transcripts and its application

Abstract

This invention provides a method for assessing chicken aging based on the PDCD10 gene transcript and its application, belonging to the field of biodetection technology. This invention provides the application of the PDCD10 gene transcript as a marker of chicken aging, and the nucleotide sequence of the cDNA corresponding to the PDCD10 gene transcript is shown in SEQ ID No. 1. The PDCD10 gene transcript of this invention exhibits a continuous and linearly decreasing expression characteristic with increasing age in multiple chicken tissues, showing a consistent molecular change pattern across tissues during aging, which can more accurately reflect the overall physiological aging state of chickens, improving the stability and reliability of chicken aging assessment. This invention can be used for the graded assessment and dynamic monitoring of the aging state of chicken flocks, providing a molecular-level reference for flock management, culling timing selection, and adjustment of feeding strategies during the breeding process.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for assessing chicken aging based on PDCD10 gene transcripts and its application. Background Technology

[0002] In chicken farming, aging affects meat quality (such as muscle toughness and fat deposition), egg production, and milk yield. Monitoring aging status can guide timely slaughter or culling, balancing farming costs and output. Existing research and applications of chicken aging assessment often reveal performance and phenotypic changes only in the mid-to-late stages of aging, failing to provide early warning. Disease, malnutrition, management stress, and seasonal changes can all lead to aging-like behaviors, making it difficult to isolate purely aging signals, resulting in delays and subjectivity. Furthermore, current methods for analyzing gene or signaling pathways related to chicken aging focus on changes in expression at a specific time point or in a particular tissue, which can introduce bias due to tissue analysis, failing to accurately and comprehensively reflect the overall aging status of the chicken. Therefore, there is an urgent need to provide a method that can accurately, stably, and comprehensively reflect the aging status of chickens to enhance the reliability of aging assessment. Summary of the Invention

[0003] To address the aforementioned technical problems, the primary objective of this invention is to provide the application of the PDCD10 gene transcript as a marker of aging in chickens. The PDCD10 gene transcript exhibits a sustained, linearly decreasing expression pattern in multiple chicken tissues with increasing age, and can serve as a molecular indicator for assessing the aging state of chickens. Its expression level can reflect the process of transition from juvenile to senescent stages in chickens.

[0004] The second objective of this invention is to provide a reagent for detecting the expression level of the PDCD10 gene transcript in one or more of the following applications: detection of chicken aging status, chicken breeding, chicken flock management, and assessment of chicken stress resistance.

[0005] A third objective of this invention is to provide a detection product for assessing the aging state of chickens.

[0006] The fourth objective of this invention is to provide a method for assessing chicken age based on PDCD10 gene transcripts.

[0007] The fifth objective of this invention is to provide an application of the above-mentioned chicken age assessment method in assessing the aging state of chickens.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of the PDCD10 gene transcript as a marker of aging in chickens, and the nucleotide sequence of the cDNA corresponding to the PDCD10 gene transcript is shown in SEQ ID No. 1.

[0009] As one implementation method, the expression level of the PDCD10 gene transcript in chicken cells or tissues is reduced.

[0010] In one embodiment, the chicken cells or tissues are derived from one or more of the following: adipose tissue, liver, kidney, heart, lung, brain, skeletal muscle, digestive tract, gonads, and endocrine glands.

[0011] This invention also provides the application of reagents for detecting the expression level of PDCD10 gene transcripts in any one or more of the following: (1) Detect the aging status of chickens; (2) Chicken breeding; (3) Chicken flock management; (4) Assessment of chickens' stress resistance; The nucleotide sequence of the cDNA corresponding to the PDCD10 gene transcript is shown in SEQ ID No. 1.

[0012] As one implementation method, the chicken flock management includes group management, breeder chicken renewal, culling timing determination, and / or feeding strategies.

[0013] The present invention also provides a detection product for assessing the aging state of chickens, the detection product comprising a reagent for detecting the expression level of the PDCD10 gene transcript, the nucleotide sequence of the cDNA corresponding to the PDCD10 gene transcript being shown in SEQ ID No. 1.

[0014] As one implementation method, the reagent includes primers that specifically recognize PDCD10 gene transcripts.

[0015] This invention also provides a method for assessing chicken age based on PDCD10 gene transcripts, comprising the following steps: selecting chicken tissue of a determined age from the flock to be assessed as a standard sample; comparing the expression level of PDCD10 gene transcripts in the chicken tissue to be tested with the expression level of PDCD10 gene transcripts in the standard sample to assess the chicken age in the tissue to be tested; the nucleotide sequence of the cDNA corresponding to the PDCD10 gene transcript is shown in SEQ ID No. 1; When the expression level of the PDCD10 gene transcript in the test chicken tissue is greater than that in the standard sample, the test chicken is determined to be younger than the standard sample. When the expression level of the PDCD10 gene transcript in the test chicken tissue is lower than that in the standard sample, the test chicken is determined to be older than the standard sample.

[0016] The present invention also provides the application of the above-mentioned chicken age assessment method in assessing the aging state of chickens.

[0017] As one implementation method, the assessment method for the aging state of chickens includes: when the chickens are 20 to 50 weeks old, they are considered to be in the juvenile stage; when the chickens are 50 to 80 weeks old, they are considered to be in the middle-aged stage; and when the chickens are 80 to 100 weeks old, they are considered to be in the old age stage.

[0018] The advantages of this invention compared to existing technologies are as follows: This invention provides the application of the PDCD10 gene transcript as a marker of aging in chickens. The nucleotide sequence of the cDNA corresponding to the PDCD10 gene transcript is shown in SEQ ID No. 1. The PDCD10 gene transcript exhibits a sustained, linearly decreasing expression pattern across multiple chicken tissues with increasing age, demonstrating consistent molecular changes throughout the aging process. This provides a more accurate reflection of the overall physiological aging state of chickens, improving the stability and reliability of aging assessment. This invention overcomes the shortcomings of existing technologies in terms of accuracy, stability, and applicability, providing a more comprehensive and objective reflection of the aging state of chickens. It can be used for the graded assessment and dynamic monitoring of aging status in chicken flocks, providing molecular-level reference for flock management, culling timing selection, and adjustments to feeding strategies during the breeding process, and has significant practical application value. Attached Figure Description

[0019] Figure 1 This is a time-varying trend graph of the results of clustering transcript expression patterns.

[0020] Figure 2 The results of functional enrichment analysis of the PDCD10 gene transcript set.

[0021] Figure 3 This is a trend diagram showing the expression level of the PDCD10 gene transcript in standard samples of chickens at different ages. Detailed Implementation

[0022] This invention provides the application of the PDCD10 gene transcript as a marker of aging in chickens, and the nucleotide sequence of the cDNA corresponding to the PDCD10 gene transcript is shown in SEQ ID No. 1.

[0023]

[0024] In this invention, the PDCD10 gene transcript is a known transcript, and the sequence shown in SEQ ID No. 1 is the cDNA sequence of the PDCD10 gene transcript annotated in the chicken (Gallus gallus) genome database Ensembl Gallus gallus version 106 as ENSGALT00000015429.

[0025] This invention selects multi-tissue samples from chickens at different age stages, including juvenile, middle-aged, and old-age stages. The juvenile stage is 20-50 weeks; the middle-aged stage is 50-80 weeks; and the old-age stage is 80-100 weeks. In this invention, the chickens include laying hens or broilers, preferably laying hens. This invention is applicable to other chicken breeds; as an optional implementation, the chicken breed includes purebred Loch Ness Red chickens. In this invention, the chicken cells or tissues are derived from one or more of the following: adipose tissue, liver, kidney, heart, lungs, brain, skeletal muscle, digestive tract, gonads, and endocrine glands. In this invention, chicken test samples are obtained from chicken cells or tissues, and the number of chicken test samples can be 5, 10, 15, 20, or 25.

[0026] In this invention, the single sampling amount for each test sample is 10-200 mg, preferably 50 mg, 100 mg, or 150 mg. After sampling, the test sample is added to 0.5-2.0 mL of lysis buffer for lysis. The lysis buffer of this invention is an RNA extraction lysis buffer. As an optional embodiment, the lysis buffer is TRIzol reagent, and the volume of the lysis buffer is preferably 1.0 mL or 1.5 mL.

[0027] Total RNA is then extracted from the sample to be tested using methods including the Trizol method, magnetic bead method, or a combination of the Trizol method and magnetic bead method. The extracted total RNA must meet at least one of the following quality standards: RNA concentration of 100–3000 ng / µL; A260 / A280 ratio of 1.8–2.1; RNA integrity index (RIN) ≥ 6.0, preferably ≥ 7.0. This invention screens total RNA that meets the quality requirements for subsequent full-length transcriptome sequencing analysis.

[0028] In this invention, the total RNA sampling amount for each sample is 0.5–2.0 µg, preferably 1.0 µg or 1.5 µg. A full-length transcriptome sequencing library suitable for the Oxford Nanopore Technologies (ONT) platform is constructed. The construction of the full-length transcriptome sequencing library in this invention is completed according to the ONT full-length transcriptome library construction workflow.

[0029] After the full-length transcriptome sequencing library was constructed, the obtained full-length transcriptome sequencing data was aligned to the Ensembl chicken reference genome version Gallus_gallus-106. Transcript annotation and quantitative analysis were performed on the sequencing data to obtain transcript expression matrices of chickens in different tissues and at different age stages. Then, based on the changing trends of transcript expression levels in chickens at different age stages, expression pattern clustering analysis was performed on the transcripts. The transcript expression cluster Cluster 7 screened in this invention showed a relatively consistent expression decline characteristic in different tissues, suggesting that it may be closely related to the systemic aging process in chickens.

[0030] Then, transcripts with stable and consistent expression levels in multiple tissues were further screened within the transcript expression cluster 7. In this invention, the expression levels are expressed using TPM, CPM, FPKM, or RPKM. Based on the quantitative expression results at the transcript level, the cDNA sequences of the PDCD10 gene transcripts screened in this invention, as shown in SEQ ID No. 1, exhibited a stable decreasing trend in expression levels across different tissues and age stages. This invention, by simultaneously analyzing the age-related gene or transcript expression characteristics in multiple tissues, screens key molecules exhibiting consistent trends in most tissues, effectively avoiding the biases caused by single-tissue analysis and more accurately reflecting the overall aging state of the chicken.

[0031] This invention also provides the application of reagents for detecting the expression level of PDCD10 gene transcripts in any one or more of the following: (1) Detect the aging status of chickens; (2) Chicken breeding; (3) Chicken flock management; (4) Assessment of chicken stress resistance; the nucleotide sequence of the cDNA corresponding to the PDCD10 gene transcript is shown in SEQ ID No. 1. In this invention, chicken flock management includes group management, breeder chicken renewal, culling timing determination and / or feeding strategies.

[0032] This invention also provides a detection product for assessing the aging state of chickens. The detection product includes a reagent for detecting the expression level of the PDCD10 gene transcript, the nucleotide sequence of the cDNA corresponding to the PDCD10 gene transcript being shown in SEQ ID No. 1. In this invention, the reagent includes primers that specifically recognize the PDCD10 gene transcript. The primers in this invention can be designed using conventional methods.

[0033] The present invention also provides a method for assessing chicken age based on PDCD10 gene transcripts, comprising the following steps: selecting chicken tissues of a determined age in the flock to be assessed as standard samples, comparing the expression level of PDCD10 gene transcripts in the chicken tissues to be tested with the expression level of PDCD10 gene transcripts in the standard samples, and assessing the chicken age of the tissues to be tested in weeks. When the expression level of the PDCD10 gene transcript in the test chicken tissue is greater than that in the standard sample, the test chicken is determined to be younger than the standard sample. When the expression level of the PDCD10 gene transcript in the test chicken tissue is lower than that in the standard sample, the test chicken is determined to be older than the standard sample.

[0034] In this invention, the method for detecting the expression level of the PDCD10 gene transcript is any one of the following: (1) The expression level of PDCD10 gene transcript in the chicken tissues to be tested was detected by transcriptome sequencing; (2) The expression level of PDCD10 gene transcript in the chicken tissue to be tested was detected using the above-mentioned detection products.

[0035] The present invention also provides the application of the above-mentioned chicken age assessment method in assessing the aging state of chickens.

[0036] In this invention, the method for assessing the aging state of chickens includes: when the chicken is 20-50 weeks old, it is determined that the chicken is in its youth stage; when the chicken is 50-80 weeks old, it is determined that the chicken is in its middle age stage; and when the chicken is 80-100 weeks old, it is determined that the chicken is in its old age stage.

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of this invention.

[0038] Unless otherwise specified, the materials, reagents, etc. used in the following examples are commercially available. Unless otherwise specified, they are generally used under conventional conditions or under conditions recommended by the company.

[0039] Example 1: Screening and Analysis of PDCD10 Gene Transcripts Associated with Multi-Tissue Senescence in Chickens 1. Acquisition of multi-tissue samples from chickens at different age stages Five purebred Loch Ness Red chickens at different ages (20, 50, 80, and 100 weeks) were selected as the research subjects. Tissue samples from 33 different tissues were collected from each chicken, including adipose tissue (1 sample), liver tissue (1 sample), kidney tissue (1 sample), brain tissue (6 samples), muscle tissue (2 samples), reproductive system tissue (6 samples), digestive system tissue (12 samples), and endocrine system tissue (4 samples) to reflect the synergistic changes among different tissues during aging. Tissue samples were immediately flash-frozen in liquid nitrogen or stored in RNA preservation solution at -80°C for later use.

[0040] 2. Total RNA extraction and quality control Take 100 mg of the above tissue samples and add 1.0 mL of lysis buffer for lysis. Extract total RNA from each tissue sample using the Trizol method. The extracted total RNA must meet at least one of the following quality standards: RNA concentration of 100–3000 ng / µL, A260 / A280 ratio of 1.8–2.1, and RNA integrity index (RIN) ≥7.0. Total RNA meeting the quality requirements is used for subsequent full-length transcriptome sequencing analysis.

[0041] 3. Full-length transcriptome sequencing based on ONT RNA-seq Take 1.0 µg of total RNA extracted from each tissue sample in step 2, construct a full-length transcriptome sequencing library suitable for the Oxford Nanopore Technologies (ONT) platform, and perform sequencing on the ONT sequencing platform to obtain full-length transcriptome sequencing data. Transcript sequences are accurately captured through ONT full-length transcriptome sequencing.

[0042] 4. Cluster analysis of expression trends of aging-related transcripts The full-length transcript sequencing data obtained in step 3 were aligned to the Ensembl chicken reference genome version Gallus_gallus-106. Transcript annotation and quantitative analysis were performed on the sequencing data to obtain transcript expression matrices of chickens in different tissues and at different age stages.

[0043] Based on the changing trends of transcript expression levels in chickens at different age stages, expression pattern cluster analysis was performed on the transcripts. Transcripts were divided into 10 expression trend clusters (Cluster 1~Cluster 10) according to their expression change patterns at different time points, and the overall change trend of each cluster at four time points was displayed as a line graph. By comparing the overall expression change trends of transcripts during aging, 10 transcript expression trend clusters with different expression dynamic characteristics were obtained, as shown in [the figure]. Figure 1The x-axis represents different age-related time points (20 weeks, 50 weeks, 80 weeks, and 100 weeks), and the y-axis represents the average transcript expression value after normalization. Figure 1 It can be seen that during aging, different expression trend clusters exhibit significantly different dynamic change patterns over time, effectively characterizing the overall response characteristics of transcripts as they change with physiological stages. The expression level of transcript expression cluster 7 shows a continuous, almost linear, decreasing trend with increasing age. This indicates that transcript expression cluster 7 exhibits a relatively consistent expression decline characteristic in different tissues, suggesting that it may be closely related to the systemic aging process in chickens.

[0044] 5. Screening of transcripts co-expressed in multiple tissues Within the transcript expression cluster 7, transcripts exhibiting a declining expression trend across multiple tissues were further screened. Specifically, the average expression levels of transcripts in each tissue at different age stages (20 weeks, 50 weeks, 80 weeks, and 100 weeks) were calculated, and their expression trends with age were compared. Transcripts showing a gradual decrease in expression with age in at least two-thirds of the tissues were identified as multi-tissue synergistically declining transcripts. Statistical analysis revealed that within the transcript expression cluster 7, multiple transcripts expressing in over 22 tissues and exhibiting a sustained declining trend with age were identified, demonstrating significant cross-tissue synergistic change characteristics.

[0045] 6. Identification of PDCD10 gene transcripts Among the aforementioned multi-tissue co-expressed transcripts, the PDCD10 gene transcript (whose cDNA sequence is shown in SEQ ID No. 1) from the transcript expression cluster Cluster7 was detected in 25 chicken tissues (ovary, infundibulum, vagina, medulla oblongata, uterus, brain, ileum, hypothalamus, liver, esophagus, kidney, lung, olfactory bulb, pectoral muscle, medulla oblongata, isthmus, cerebellum, pituitary gland, gizzard, thigh muscle, abdominal fat, spleen, proventriculus, pancreas, and rectum). Furthermore, its expression showed a stable decreasing trend across different age groups (20 to 100 weeks), demonstrating a significant cross-tissue synergistic change. The transcript number corresponding to the PDCD10 gene transcript is ENSGALT00000015429.

[0046] This invention performs functional annotation analysis on the obtained PDCD10 gene transcript set, and the results of the functional enrichment analysis of the transcript set are as follows: Figure 2 As shown, the horizontal axis represents the salience level of the functional item (-log). 10(p-value), with the vertical axis representing the corresponding function category. The position and size of different bubbles together reflect the salience of each function item. From Figure 2 The results show that the PDCD10 gene transcript is enriched in biological processes related to cellular responses to reactive oxygen species, oxidative stress, and chemical stress, indicating that this transcript may be involved in molecular regulation processes related to stress and cellular homeostasis during chicken aging. The results also demonstrate that the expression of the PDCD10 gene transcript in multiple chicken tissues shows a sustained decline with aging, stably reflecting the aging state of chickens at different age stages. This demonstrates the feasibility and effectiveness of the PDCD10 gene transcript in real-world data, suggesting its potential use for assessing aging status or progression in chickens.

[0047] Example 2: Application of PDCD10 gene transcripts in the assessment of multi-tissue aging in chickens This embodiment verifies the feasibility of using PDCD10 gene transcripts in assessing aging status in chickens and in production practice. In production practice, the expression level of PDCD10 gene transcripts in chicken flocks shows a negative correlation with age, and combined with the multi-tissue collaborative analysis framework constructed in this invention, accurate assessment of the aging status of chicken flocks can be achieved.

[0048] 1. Standard Sample Collection and Data Acquisition Purebred Loch Ness Red chickens of the same species but different population as those in Example 1 were selected and divided into different age groups according to their actual production cycle: 20 weeks, 50 weeks, 80 weeks, and 100 weeks. Five chickens were randomly selected from each age group, and 33 tissue samples closely related to production performance were collected: fat (1 sample), liver (1 sample), kidney (1 sample), brain (6 samples), muscle (2 samples), reproductive system (6 samples), digestive system (12 samples), and endocrine system-related tissues (4 samples).

[0049] The PDCD10 gene transcripts in the above tissue samples were detected using ONT RNA-seq long-read transcriptome sequencing technology to obtain full-length transcript sequencing data of this chicken population. The sequencing procedure, library construction method, and data processing steps were consistent with those in Example 1. The TPM index was used to represent the expression level of specific PDCD10 transcripts in different tissues and at different age stages. The results are shown in Table 1 and [Table data would be inserted here]. Figure 3 .

[0050] Table 1. Expression levels of specific PDCD10 transcripts in standard samples.

[0051] As shown in Table 1 above, among different flocks of purebred Loch Ness Red chickens, the expression of 25 chicken tissues (ovary, infundibulum, vagina, medulla oblongata, uterus, brain, ileum, hypothalamus, liver, esophagus, kidney, lung, olfactory bulb, pectoral muscle, enlarged part, isthmus, cerebellum, pituitary gland, gizzard, thigh muscle, abdominal fat, spleen, proventriculus, pancreas, and rectum) showed a stable decreasing trend at different age stages (20 weeks to 100 weeks).

[0052] 2. Determination of Aging Status The expression levels of PDCD10 gene transcripts in any of the 25 tissues of chickens at 50, 70, and 100 weeks of age were detected using the method described in step 1. The expression levels of PDCD10 gene transcripts in different chicken tissues, expressed as a TPM index, are shown in Table 2.

[0053] Table 2. Expression levels of specific PDCD10 transcripts in the test samples.

[0054] Comparing the expression levels of PDCD10 gene transcripts in different tissues of the test chickens in Table 2 with the age of chickens in the standard samples in Table 1, it can be seen that the expression levels of PDCD10 gene transcripts in the seven tissues of the test chickens—ovary, uterus, hypothalamus, medulla oblongata, isthmus, pituitary gland, and abdominal fat—show a stable correlation with the chicken's age. This indicates that the expression levels of PDCD10 gene transcripts in different tissues can accurately assess the aging status and aging process of chickens.

[0055] The results above show that the expression changes of PDCD10 gene transcripts in the 25 tissues of this invention are consistent, and the systematic characteristics of chicken aging state are obvious. This indicates that PDCD10 gene transcripts can accurately assess the aging state or aging process of chickens, and improve the stability and reliability of the assessment results.

[0056] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. The application of the PDCD10 gene transcript as a marker of aging in chickens, characterized in that, The nucleotide sequence of the cDNA corresponding to the PDCD10 gene transcript is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, The expression level of the PDCD10 gene transcript in chicken cells or tissues is reduced.

3. The application according to claim 2, characterized in that, The chicken cells or tissues are derived from one or more of the following: adipose tissue, liver, kidney, heart, lung, brain, skeletal muscle, digestive tract, gonads, and endocrine glands.

4. The reagent for detecting the expression level of the PDCD10 gene transcript can be used in one or more of the following applications: (1) Detect the aging status of chickens; (2) Chicken breeding; (3) Chicken flock management; (4) Assessment of chickens' stress resistance; The nucleotide sequence of the cDNA corresponding to the PDCD10 gene transcript is shown in SEQ ID No.

1.

5. The application according to claim 4, characterized in that, The management of chicken flocks includes group management, replacement of breeding chickens, determination of culling time and / or feeding strategies.

6. A detection product for assessing the aging state of chickens, characterized in that, The detection product includes a reagent for detecting the expression level of the PDCD10 gene transcript, and the nucleotide sequence of the cDNA corresponding to the PDCD10 gene transcript is shown in SEQ ID No.

1.

7. The testing product according to claim 6, characterized in that, The reagent includes primers that specifically recognize PDCD10 gene transcripts.

8. A method for assessing chicken age based on PDCD10 gene transcripts, characterized in that, Includes the following steps: Chicken tissues of a certain age were selected from the flock to be evaluated as standard samples. The expression level of the PDCD10 gene transcript in the chicken tissue to be tested was compared with the expression level of the PDCD10 gene transcript in the standard sample to evaluate the age of the chicken tissue to be tested. The nucleotide sequence of the cDNA corresponding to the PDCD10 gene transcript is shown in SEQ ID No.

1. When the expression level of the PDCD10 gene transcript in the test chicken tissue is greater than that in the standard sample, the test chicken is determined to be younger than the standard sample. When the expression level of the PDCD10 gene transcript in the test chicken tissue is lower than that in the standard sample, the test chicken is determined to be older than the standard sample.

9. The application of the chicken age assessment method according to claim 8 in assessing the aging state of chickens.

10. The application according to claim 9, characterized in that, The methods for assessing the aging state of chickens include: when the chickens are 20-50 weeks old, they are considered to be in the juvenile stage; when the chickens are 50-80 weeks old, they are considered to be in the middle age stage; and when the chickens are 80-100 weeks old, they are considered to be in the old age stage.

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