Application of transcription factor HOXA13 as poultry abdominal fat percentage detection marker

Abstract

The invention belongs to the technical field of genes, and particularly relates to application of a transcription factor HOXA13 serving as a poultry abdominal fat rate detection marker. In the invention, compared with broiler chickens with high abdominal fat percentage (greater than 1.4%), the expression of FPKM of the HOXA13 gene in an abdominal fat tissue transcriptome of broiler chickens with low abdominal fat percentage (less than 0.6%) is remarkably improved, and AUC in an ROC curve drawn for the expression of the HOXA13 gene of a sample is equal to 0.815, which indicates that the HOXA13 gene can better represent the abdominal fat content of the broiler chickens.

Description

Technical Field

[0001] The invention belongs to the field of gene technology, and specifically relates to an application of a transcription factor HOXA13 as a marker for detecting abdominal fat rate of poultry. Background Art

[0002] As an efficient production system, broilers provide humans with economical and high-quality animal protein. The efficient use of broiler feed plays an important role in food security in my country and even the world. However, genetic selection for commercial purposes has led to an unprecedented increase in the growth rate of broilers, which has also led to excessive deposition of abdominal adipose tissue. Excessive abdominal adipose tissue deposition reduces feed efficiency, impairs animal health, and has a negative impact on the slaughter process and the environment. Excessive abdominal fat deposition is a common industry problem associated with the selection of broilers for high body weight and high growth rate. It is a core factor that limits broiler meat production efficiency and efficient nutrient utilization, and has caused huge economic losses to the broiler industry. Due to the positive genetic and phenotypic correlation between abdominal fat weight and broiler body weight, reducing abdominal fat content through traditional breeding methods faces great challenges. New strategies are needed to reveal the mechanisms of abdominal adipose tissue development.

[0003] The exponential growth of omics data and the rapid development of high-throughput sequencing technology have provided unprecedented opportunities for the above potential mechanisms. Many studies have attempted to elucidate the mechanisms and regulatory targets of abdominal adipose tissue formation through methods such as proteomics, 16s, and LC / MS-based lipidomics. Chromatin accessibility provides valuable information for identifying regulatory elements and mechanisms, and can be used to determine various cis-regulatory elements and predict transcription factor binding sites. Combining transcriptome with ATAC-seq technology has become a valuable strategy to identify the underlying mechanisms of complex animal phenotypes. For example, studies have used ATAC-seq and RNA-seq technologies to map the chromatin accessibility and developmental transcriptome of pig skeletal muscle at different developmental stages, as well as the epigenetic mechanism of fertility differences between Meishan pigs and Duroc pigs. In the growth cycle of chickens, the proliferation and hypertrophy of adipocytes are closely related to abdominal fat deposition, and it is of great significance to determine the key physiological stages of adipocyte differentiation.

[0004] At present, most of the attention paid to abdominal fat tissue deposition in broilers at home and abroad is to regard it as a slaughter performance indicator, and there are few studies on the specific molecular mechanisms of abdominal fat deposition. There is a positive genetic and phenotypic correlation between abdominal fat weight and broiler body weight. It is still very challenging to reduce abdominal fat content through traditional breeding methods. New strategies are needed to reveal the internal mechanism of abdominal fat deposition, which is of great significance to the broiler breeder industry. Summary of the invention

[0005] In the present invention, the key transcription factor HOXA13 that affects abdominal fat deposition is screened out at a relatively upstream level of the three-dimensional genome. By identifying the transcription factor HOXA13, poultry individuals with low abdominal fat content are quickly and accurately screened out, thereby improving the accuracy and efficiency of abdominal fat content identification and providing a reference basis for breeding low-fat broiler varieties.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On one hand, the present invention provides an application of a reagent for detecting transcription factor HOXA13 in preparing a detection product for detecting abdominal fat rate of poultry.

[0008] The present invention also provides an application of a reagent for detecting transcription factor HOXA13 in poultry genetic breeding.

[0009] Preferably, in the above application, the transcription factor HOXA13 is overexpressed, and the abdominal fat rate of poultry is reduced.

[0010] Preferably, in the above application, the reagent for detecting the transcription factor HOXA13 includes a reagent for detecting the expression of the transcription factor HOXA13, a reagent for detecting the mRNA synthesized with the participation of the transcription factor HOXA13, or a reagent for detecting the protein synthesized with the participation of the transcription factor HOXA13.

[0011] More preferably, in the above application, the reagent for detecting the expression of transcription factor HOXA13 includes a detection reagent based on a dual luciferase reporter gene detection system, a detection reagent based on a gel shift assay, a detection reagent based on a yeast detection system, a detection reagent based on a DNA footprinting method, or a detection reagent based on a yeast single hybrid assay; the reagent for detecting mRNA synthesized by the transcription factor HOXA13 or the reagent for detecting protein synthesized by the transcription factor HOXA13 includes a detection reagent based on western blotting detection.

[0012] Preferably, in the above application, the poultry is chicken.

[0013] Preferably, in the above application, the detection product includes a detection reagent or a detection kit.

[0014] The beneficial effects of the present invention include: in the present invention, compared with broilers with high abdominal fat rate (>1.4%), the FPKM expression of the HOXA13 gene in the abdominal adipose tissue transcriptome of broilers with low abdominal fat rate (<0.6%) is significantly increased, and the AUC in the ROC curve drawn for the HOXA13 gene expression of the sample is 0.815, indicating that the HOXA13 gene can better characterize the abdominal fat content of broilers. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1APCA results of transcriptomes at different ages between D3 and D14 of abdominal fat dynamic development;

[0016] Figure 1B Heat map of differentially expressed genes in the transcriptome between D3 and D14 of abdominal fat dynamic development;

[0017] Figure 1C The number of up-regulated and down-regulated differentially expressed genes in the transcriptome between D3 and D14 of abdominal fat dynamic development;

[0018] Figure 1D This is the clustering heat map of ATAC-seq samples between D3 and D14 of abdominal fat dynamic development;

[0019] Figure 1E Heat map of ATAC-seq differential motifs between D3 and D14 of abdominal fat dynamic development;

[0020] Figure 1F The number of up-regulated and down-regulated differential motifs by ATAC-seq between D3 and D14 of abdominal fat dynamic development;

[0021] Figure 1G Combined RNA-seq and ATAC-seq analysis of abdominal fat dynamics between D3 and D14;

[0022] Figure 1H The expression of HOXA13, FABP4, and CPT-1A proteins at different time points of abdominal fat dynamic development;

[0023] Fig. 1I The quantitative results of HOXA13 protein expression at different time points of abdominal fat dynamic development;

[0024] Figure 1J The quantitative results of FABP4 protein expression at different time points of abdominal fat dynamic development;

[0025] Figure 1K The quantitative results of CPT-1A protein expression at different time points of abdominal fat dynamic development;

[0026] Figure 2A The expression of transcription factor HOXA13 and proliferation and differentiation-related proteins in the ICP2 differentiation model;

[0027] Figure 2B Quantification of transcription factor HOXA13 and proliferation and differentiation-related protein bands in the ICP2 differentiation model;

[0028] Figure 3A The changes in the degree of chromatin opening in the promoter regions of FASN, ACACA, and ELOVL6 genes in ATAC-seq after ICP2 cell differentiation;

[0029] Figure 3B The HOXA13 binding motif identified in ATAC-seq after ICP2 cell differentiation;

[0030] Figure 3C The TG and TC contents of ICP2 cells transfected with pcDNA3.1 and Flag-HOXA13 after differentiation;

[0031] Figure 3D Oil Red O staining of ICP2 cells transfected with pcDNA3.1 and Flag-HOXA13 after differentiation induction;

[0032] Figure 3E Semi-quantitative analysis of Oil Red O staining after differentiation induction in ICP2 cells transfected with pcDNA3.1 and Flag-HOXA13, respectively;

[0033] Figure 3F Effects of interfering with HOXA13 on TG and TC content in ICP2 cells;

[0034] Figure 3G Interference with Oil Red O staining of HOXA13 in ICP2 cells;

[0035] Figure 3H Semi-quantification of Oil Red O staining for HOXA13 interference in ICP2 cells;

[0036] Fig. 3I Prediction of the binding of HOXA13 to the promoter region of genes related to lipid metabolism;

[0037] Figure 4A CUT&Tag for Flag-HOXA13. Distribution of HOXA13 binding in the whole genome;

[0038] Figure 4B Changes in the binding of HOXA13 to genes related to lipid catabolism;

[0039] Figure 5A The expression of HOXA13 gene in white-feathered broilers with high and low abdominal fat rates;

[0040] Figure 5B Shown is the ROC curve of HOXA13 gene expression in white-feather broilers with high and low abdominal fat rates. DETAILED DESCRIPTION

[0041] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0042] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0043] An embodiment of the present invention provides an application of a reagent for detecting transcription factor HOXA13 in preparing a detection product for detecting abdominal fat rate of poultry.

[0044] In some specific examples, in the above application, when the transcription factor HOXA13 is overexpressed, the abdominal fat rate of poultry is reduced.

[0045] In some specific examples, in the above application, the reagent for detecting the transcription factor HOXA13 includes a reagent for detecting the expression of the transcription factor HOXA13, a reagent for detecting the mRNA synthesized with the participation of the transcription factor HOXA13, or a reagent for detecting the protein synthesized with the participation of the transcription factor HOXA13.

[0046] In some specific examples, in the above applications, the reagents for detecting the expression of the transcription factor HOXA13 include detection reagents based on a dual luciferase reporter gene detection system, detection reagents based on a gel shift assay, detection reagents based on a yeast detection system, detection reagents based on a DNA footprinting method, or detection reagents based on a yeast single hybrid assay; the reagents for detecting mRNA in which the transcription factor HOXA13 participates in the synthesis or the reagents for detecting proteins in which the transcription factor HOXA13 participates in the synthesis include detection reagents based on western blotting detection.

[0047] In some specific examples, in the above applications, the poultry is chicken.

[0048] In some specific examples, in the above applications, the detection product includes a detection reagent or a detection kit.

[0049] The embodiment of the present invention also provides an application of a reagent for detecting transcription factor HOXA13 in poultry genetic breeding.

[0050] In some specific examples, in the above application, when the transcription factor HOXA13 is overexpressed, the abdominal fat rate of poultry is reduced.

[0051] In some specific examples, in the above application, the reagent for detecting the transcription factor HOXA13 includes a reagent for detecting the expression of the transcription factor HOXA13, a reagent for detecting the mRNA synthesized with the participation of the transcription factor HOXA13, or a reagent for detecting the protein synthesized with the participation of the transcription factor HOXA13.

[0052] In some specific examples, in the above applications, the reagents for detecting the expression of the transcription factor HOXA13 include detection reagents based on a dual luciferase reporter gene detection system, detection reagents based on a gel shift assay, detection reagents based on a yeast detection system, detection reagents based on a DNA footprinting method, or detection reagents based on a yeast single hybrid assay; the reagents for detecting mRNA in which the transcription factor HOXA13 participates in the synthesis or the reagents for detecting proteins in which the transcription factor HOXA13 participates in the synthesis include detection reagents based on western blotting detection.

[0053] In some specific examples, in the above applications, the poultry is chicken.

[0054] In some specific examples, in the above applications, the detection product includes a detection reagent or a detection kit.

[0055] It should be noted that in the present invention, ATAC-seq and RNA-seq were jointly analyzed between D3 and D14 of abdominal fat dynamic development, and a total of 4 genes were found between up-regulated motifs and up-regulated DEGs, among which HOXA13 had the largest difference fold. The expression changes of the selected transcription factors in the dynamic development of abdominal fat were verified by Western blotting, indicating that HOXA13 is involved in the regulation of abdominal adipose tissue formation.

[0056] It should also be noted that in the present invention, chicken adipocytes ICP2 were induced to differentiate using 200 μM oleic acid, and protein blotting verified that the expression of adipogenic differentiation-related proteins was upregulated, such as C / EBPα, FABP4, IGF2, and SREBP-1; while the expression of cell cycle-related factors PCNA, CDK1, and the key rate-limiting enzyme of β-oxidation CPT1 was significantly reduced. The above results indicate that the adipocyte differentiation model was successfully constructed. In addition, the protein expression of the transcription factor HOXA13 was significantly reduced after differentiation, suggesting its potential role in regulating adipogenic differentiation.

[0057] It should also be noted that in the present invention, overexpression and interference experiments were carried out in ICP2 cells, and it was found that overexpression of HOXA13 can effectively inhibit lipid deposition in ICP2 cells, and the TG and TC contents in the cells were significantly reduced. ICP2 cells in which HOXA13 was interfered showed more obvious lipid deposition and increased the TG and TC contents in the cells. Therefore, it can be shown that HOXA13 can bind to a series of gene promoter regions involved in adipocyte differentiation and maturation to regulate their expression; in addition, the JASPAR website was further used to predict the binding of HOXA13 to the promoter regions of lipid metabolism-related genes, and it was found that HOXA13 can bind to the promoter regions of CPT-1A and PPARα genes.

[0058] It should also be noted that, in the present invention, the CUT&Tag technology is used to detect the interaction binding sites of HOXA13 and lipid metabolism-related genes, in order to clarify the intrinsic mechanism of HOXA13 regulating the proliferation and differentiation of chicken adipocytes, and to provide a reference for breeding new broiler varieties with high meat production efficiency. The results showed that the binding of transcription factor HOXA13 in the whole genome is mainly concentrated in the intron and promoter regions. In addition, HOXA13 has higher binding peaks in the CPT1A, CD36, ACSL4 and ACSL6 genes; and the binding peaks at the CPT1A and CD36 genes are upregulated after differentiation. The above results show that HOXA13 may bind to the promoter regions of genes related to fat decomposition and transport such as CPT1A and CD36, and positively regulate lipid catabolism.

[0059] It should also be noted that in the present invention, IP-MS was used to further identify the protein pool interacting with HOXA13. It is intended to clarify the mechanism by which transcription factor HOXA13 regulates adipogenesis. The results showed that HOXA13 can interact with lipid metabolism-related or chromatin structure regulatory proteins such as CPT1A, USP1, VTG2, IKZF2, and YES1 after ICP2 cell differentiation.

[0060] It should also be noted that in the present invention, compared with broilers with high abdominal fat rate (>1.4%), the FPKM expression of the HOXA13 gene in the abdominal adipose tissue transcriptome of broilers with low abdominal fat rate (<0.6%) was significantly increased, and the AUC=0.815 in the ROC curve drawn for the HOXA13 gene expression of the sample indicated that the gene could better characterize the abdominal fat content of broilers.

[0061] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0062] 1. Screening of transcription factors regulating abdominal fat deposition based on the epigenome of dynamic changes in broiler abdominal fat

[0063] 300 white-feathered broilers were selected and slaughtered to calculate the abdominal fat percentage of broilers. On this basis, RNA-seq was used to analyze the differentially expressed genes at D3 and D14 during the dynamic development of abdominal fat. Total RNA was extracted from abdominal adipose tissue using the TRIZOL kit protocol provided by Agbio (China). Illumina Kit (Illumina, USA) was used to construct RNA-seq libraries and sequenced by Shanghai Personal Biotechnology Co., Ltd. The raw data were filtered, quality controlled, and aligned with the chicken genome (GRCg7b) using HISAT2. DESeq2 software was further used to identify differentially expressed genes based on the threshold of log2 FoldChange>1 and P value <0.05. ATAC-seq was further used to analyze the changes in chromatin accessibility during the dynamic development of abdominal fat.

[0064] Among them, ATAC-seq was performed by Wuhan Frasergen Bioinformatics Co., Ltd.; the specific steps are as follows: the preprocessing steps mainly include nucleic acid extraction, Tn5 transposase cleavage, introduction of adapters and purification using AMpure beads; after sequencing on the Illumina platform, SOAPnuke was used to filter the raw data, and then the reads were aligned with the chicken reference genome (GRCg7b) using the Burrows-Wheeler Alignment algorithm; MACS2 (v2.1.1) software was used to identify the distribution of open chromatin regions throughout the genome; the annotatePeak function of ChIPseeker was used to annotate the peaks, and the promoter region was defined as <3kb from the transcription start site; DESeq2 was used to screen differential peaks under the conditions of log2Fold Change>1 and P-value<0.05; DiffBind was used for accessibility analysis with parameters of |log2FoldChange|>1 and P-value<0.05, and the MEME Suite function in the JASPAR database was used to perform DNA motif enrichment analysis on the differential peaks.

[0065] In addition, the expression changes of HOXA13 protein during the dynamic development of abdominal fat were detected by Western blotting. The specific steps were referred to "Liu Y, Shen J, Yang X, Sun Q, Yang X. Folic Acid Reduced Triglycerides Depositionin Primary Chicken Hepatocytes. J Agric Food Chem. 2018, 19; 66(50): 13162-13172."; specifically as follows: The abdominal adipose tissue was fully lysed using RIPA lysis buffer containing 1% PMSF from Zhonghui Hecai Biological Company, and the supernatant was taken after centrifugation at 4°C and 12000rpm for 5 minutes. The supernatant was then purified by using AccuRefScientific The protein concentration was standardized using a commercial BCA kit provided by Biotin (Xi'an, China); the protein was boiled at 95°C for 15 min, and equal amounts of protein (25 μg) were electrophoresed through SDS-polyacrylamide gel to detect the expression of the target protein; the primary antibody was diluted 1:1000 with 5% BSA, and the secondary antibody was diluted 1:2000 (DIYIBio, Shanghai) for incubation; the detailed information of the antibodies used is listed in Table 1; images were captured using the iBright FL1500 system (Thermo Fisher, USA), and quantitative analysis was performed using Image J (National Institutes of Health, USA) with GAPDH or β-actin as an internal reference.

[0066] Table 1 Antibody information used in Western blotting

[0067] protein company Molecular size (kDa) Dilution ratio β-actin PTMbio(PTM-5028) 42 1:1000 IGF2 Wanleibio(WL02665) 20 1:1000 PPARγ Abways(CY6675) 57 1:1000 SREBP-1 Wanleibio(WL02093) 68；125 1:1000 GAPDH Abways(AB0037) 36 1:1000 CPT-1A abcam(ab220789) 88 1:1000 FABP4 Abways(CY6768) 15 1:1000 HOXA13 abcam(ERP10375) 39 1:1000 PCNA Abways(AB0051) 29 1:1000 C\EBPα Abways(CY5723) 43 1:1000 CDK-1 Abways(CY5176) 34 1:1000

[0068] Test results such as Figures 1A to 1K As shown, Figures 1A to 1K The results showed that the transcription factor HOXA13, which mediates abdominal fat deposition in broilers, was mined at the upstream three-gene level and its expression changes in the dynamic development of abdominal fat were verified; among them, a total of 4 genes were crossed between the up-regulated motif and the up-regulated DEGs, and HOXA13 had the largest difference fold; the expression changes of the selected transcription factors in the dynamic development of abdominal fat were verified by protein blotting, indicating that HOXA13 may be involved in the regulation of abdominal adipose tissue formation.

[0069] 2. Exploring the expression changes of transcription factor HOXA13 in chicken adipocytes ICP2

[0070] Immortalized chicken preadipocytes (ICP2) were purchased from the Key Laboratory of Chicken Genetics and Breeding, Ministry of Agriculture and Rural Affairs, Northeast Agricultural University (Harbin); ICP2 cells were cultured in DMEM / F12 medium (Gibco, USA, the same below), supplemented with 10% fetal bovine serum (BI, Germany), 100 unit / mL penicillin and 100 μg / mL streptomycin, and cultured in a 37°C incubator containing 5% CO2; when the density reached about 80%, complete medium (DMEM / F12+10% FBS) containing 200 μM oleic acid was used to induce differentiation for 1 day; the adipogenic differentiation and cell cycle-related protein expressions of ICP2 cells were detected by the above-mentioned protein blotting detection method to verify that the model was successfully constructed.

[0071] The results are as follows Figure 2A and Figure 2B As shown in the figure, the expression of proteins related to adipogenic differentiation was upregulated, such as C / EBPα, FABP4, IGF2, and SREBP-1; while the expression of cell cycle-related factors PCNA, CDK1, and the key rate-limiting enzyme of β-oxidation CPT1 was significantly reduced. The above results indicate that the adipocyte differentiation model was successfully constructed. In addition, the protein expression of the transcription factor HOXA13 was significantly reduced after differentiation, suggesting its potential role in regulating adipogenic differentiation.

[0072] In addition, the above-mentioned ATAC-seq method was used to further detect the changes in the chromatin openness of lipid metabolism-related genes before and after ICP2 cell differentiation. Figures 3A to 3B As shown, the results showed that HOXA13 was predicted in the up-regulated motifs, further suggesting that HOXA13 may regulate gene transcription by binding to the promoter regions of lipid metabolism-related genes during adipogenic differentiation.

[0073] In addition, to explore the function of the HOXA13 gene, overexpression and interference experiments were performed in ICP2 cells. Both the overexpression vector and small interfering RNA were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. When the cells were seeded into a 6-well plate and cultured to a density of about 70%, ICP2 cells were transfected using serum-free and double-antibody-free culture medium and Thermo Fisher's Lipofectamine2000 transfection reagent. After 6 hours of transfection, the normal culture medium was replaced, and the differentiation medium was replaced one day later. After the treatment, the cells were digested with trypsin to collect the cell precipitate. The protein concentration was determined by the BCA kit, and the intracellular TG and TC contents were detected according to the instructions of the commercial kit of Nanjing Jiancheng Company. The results showed that overexpression of HOXA13 can effectively inhibit lipid deposition in ICP2 cells, while interference performance promotes cell adipogenic differentiation (see Appendix). FIG. 3C to FIG. 3H ).

[0074] Based on the above results, we further used the JASPAR website to predict the binding of HOXA13 to the promoter regions of lipid metabolism-related genes, and found that HOXA13 can bind to the promoter regions of CPT-1A and PPARα genes ( Fig. 3I ).

[0075] 3. Identification of HOXA13 binding sites to lipid metabolism genes based on Flag-tagged CUT&Tag

[0076] ICP2 overexpressing Flag-tagged HOXA13 was subjected to CUT&Tag sequencing to detect the interaction between HOXA13 and lipid metabolism-related genes; specifically, concanavalin A-coated magnetic beads were added to the cell suspension; next, incubation was performed with Flag primary antibody, followed by incubation with secondary antibody; pA-Tn5 transposase (ABclonal) that can recognize the antibody was added to the above system, and the Tn5 transposase cut the genome and inserted the adapter sequence near the target protein; library construction and sequencing were performed on the Illumina NovaSeq platform by Fraser Gene Information Co., Ltd. (Wuhan, China); the Burrows-Wheeler Alignment program was used to obtain clear reads and align with the chicken reference genome GRCg7b; peaks were called by MACS2, and differential peaks were evaluated using DESeq2 when P-value < 0.05 and log2 Fold Change absolute value > 1. Peaks were annotated by the annotatePeak function of ChIPseeker, and the promoter region was set to be <3 kb from TSS. Target genes can also be predicted based on differential peaks in the promoter region.

[0077] The results are as follows Figures 4A to 4B As shown, the results showed that HOXA13 binding in the whole genome was mainly concentrated in introns and promoter regions. In addition, HOXA13 had higher binding peaks in CPT1A, CD36, ACSL4 and ACSL6 genes; and the binding peaks at CPT1A and CD36 genes were upregulated after differentiation; that is, HOXA13 can bind to the promoter regions of genes related to fat decomposition and transport such as CPT1A and CD36, and positively regulate lipid catabolism.

[0078] 4. Identification of HOXA13-interacting protein library based on IP-MS

[0079] Combining the above three results, immunoprecipitation-mass spectrometry (IP-MS) was used to identify the set of proteins interacting with HOXA13. The steps were as follows: After the preparation of the antigen-antibody-magnetic bead complex, the separated immunoprecipitated substances were detected by mass spectrometry (Thermo Fisher Scientific, USA); about 320 μl of SDT (4% SDS, 100 mM Tris-HCl, pH = 7.6) buffer was used for sample lysis and protein extraction; the sample was incubated with trypsin at 37°C overnight to obtain a peptide sample. Then, the peptide was desalted on a C18 column (Empore TM SPE column C18, layer diameter 7mm, Sigma), vacuum centrifugation concentration, reconstituted in 40μl 0.1% formic acid. The peptide content was estimated using UV spectral density with an extinction coefficient of 1.1 at 280nm. Then 1μg of peptide sample was prepared for LC-MS / MS analysis; LC-MS / MS analysis was performed on a Q Exactive mass spectrometer (ThermoFisher Scientific, USA), which was coupled to Easy nLC (Thermo Fisher Scientific, USA) for 120min, and the peptide recognition mode was enabled when the instrument was running. The MS raw data of each sample were merged and searched using MaxQuant 1.5.3.17 software for identification and quantitative analysis.

[0080] The results showed that HOXA13 could interact with lipid metabolism-related or chromatin structure regulatory proteins such as CPT1A, USP1, VTG2, IKZF2 and YES1 after ICP2 cell differentiation, as shown in Table 2.

[0081] Table 2 Proteins related to lipid metabolism or chromatin structure regulation that interact with HOXA13 in IP-MS

[0082] Protein name -10lgP CPT1A 31.52 USP1 24.6 VTG2 60.45 IKZF2 29.99 YES1 26.72

[0083] 5. Changes in HOXA13 gene expression in abdominal fat transcriptome of white-feathered broiler chickens with high and low abdominal fat rates

[0084] In a large group of 1000 white-feathered broiler chickens with the same genetic background and feeding conditions, sampling was carried out at the 42nd day of age. Combined with the general situation of abdominal fat rate of this breed and the probability of its extreme value, the abdominal fat rate of broiler chickens was calculated by slaughtering. 30 broilers with high abdominal fat rate (>1.4%) and 30 broilers with low abdominal fat rate (<0.6%) were selected, and the expression changes of HOXA13 gene in high and low abdominal fat rate groups were identified according to the above-mentioned transcriptome determination method; ROC curve was drawn using the Micro-Informatics website (https: / / www.bioinformatics.com.cn / ).

[0085] The results are as follows FIG. 5A to FIG. 5B As shown, the results showed that compared with broilers with high abdominal fat rate (>1.4%), the FPKM expression of HOXA13 gene in the abdominal adipose tissue transcriptome of broilers with low abdominal fat rate (<0.6%) was significantly increased, and the AUC=0.815 in the ROC curve drawn for the HOXA13 gene expression of the sample indicated that the gene could better characterize the abdominal fat content of broilers.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. Application of reagents for detecting transcription factor HOXA13 in the preparation of detection products for detecting abdominal fat rate of poultry.

2. Application of reagents for detecting transcription factor HOXA13 in poultry genetic breeding.

3. The use according to claim 1 or 2, characterized in that: Overexpression of the transcription factor HOXA13 reduces the abdominal fat rate of poultry.

4. The use according to claim 1 or 2, characterized in that: The reagents for detecting the transcription factor HOXA13 include reagents for detecting the expression of the transcription factor HOXA13, reagents for detecting the mRNA synthesized with the participation of the transcription factor HOXA13, or reagents for detecting the protein synthesized with the participation of the transcription factor HOXA13.

5. The use according to claim 4, characterized in that: Reagents for detecting the expression of transcription factor HOXA13 include detection reagents based on a dual luciferase reporter gene detection system, detection reagents based on a gel shift assay, detection reagents based on a yeast detection system, detection reagents based on a DNA footprinting method, or detection reagents based on a yeast single hybridization assay; reagents for detecting mRNA synthesized by the transcription factor HOXA13 or reagents for detecting proteins synthesized by the transcription factor HOXA13 include detection reagents based on western blot detection.

6. The use according to claim 1, 2 or 5, characterized in that: The poultry is chicken.

7. The use according to claim 4, characterized in that: The poultry is chicken.

8. The use according to claim 1, 2, 5 or 7, characterized in that: Testing products include testing reagents or testing kits.

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