Application of transcription factor HOXA13 in detection of abdominal fat rate of poultry as a marker

Abstract

The application belongs to the technical field of genes and particularly relates to application of a transcription factor HOXA13 as an abdominal fat rate detection marker of poultry. In the application, compared with high abdominal fat rate (>1.4%) broilers, the FPKM expression of the HOXA13 gene in the abdominal fat tissue transcriptome of low abdominal fat rate (<0.6%) broilers is significantly increased, and the AUC in the ROC curve drawn according to the HOXA13 gene expression of the sample is 0.815, indicating that the HOXA13 gene can preferably represent the abdominal fat content of broilers.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genes, and particularly relates to application of a transcription factor HOXA13 to detection of an abdominal fat rate of poultry as a marker. BACKGROUND

[0002] As an efficient production system, broiler chickens provide humans with economic and high-quality animal protein. Efficient utilization of broiler chicken feed plays an important role in food security in China and even the world. However, due to genetic selection for commercial purposes, the growth rate of broiler chickens has never been faster, which has also led to excessive deposition of abdominal adipose tissue. Excessive deposition of abdominal adipose tissue reduces feed efficiency, damages animal health, and has a negative impact on the slaughtering process and the environment. Excessive abdominal fat deposition is an industry problem that commonly occurs with high body weight and high growth rate selection of broiler chickens, and is a core factor limiting the meat production efficiency and nutrient utilization of broiler chickens, causing huge economic losses to the broiler chicken industry. Since there is a positive correlation between abdominal fat weight and broiler chicken body weight in terms of genetics and phenotype, it is a huge challenge to reduce the abdominal fat content through traditional breeding methods. New strategies are needed to reveal the mechanism of abdominal adipose tissue development.

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

[0004] Currently, the focus of attention on abdominal adipose tissue deposition in broiler chickens is mainly on slaughter performance indicators, and there are few studies on the specific molecular mechanisms of abdominal fat deposition regulation. There is a positive correlation between abdominal fat weight and broiler chicken body weight in terms of genetics and phenotype, and it is still a great challenge to reduce the 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 chicken industry. SUMMARY

[0005] In the present invention, HOXA13, a key transcription factor affecting abdominal fat deposition, was screened at a relatively upstream level of the three-dimensional genome. By identifying the transcription factor HOXA13, poultry individuals with low abdominal fat content were quickly and accurately screened. This not only improves the accuracy and efficiency of abdominal fat content identification, but also provides a reference basis for breeding low-fat broiler breeds.

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

[0007] In one aspect, the present invention provides a use of a reagent for detecting the transcription factor HOXA13 in preparing a detection product for detecting the 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 in which the transcription factor HOXA13 participates in the synthesis, or a reagent for detecting the protein in which the transcription factor HOXA13 participates in the synthesis.

[0011] More preferably, in the above application, the reagent for detecting the expression of the 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 one-hybrid assay; the reagent for detecting the mRNA synthesized by the transcription factor HOXA13 or the reagent for detecting the protein synthesized by the transcription factor HOXA13 includes a detection reagent based on western blot 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 a high abdominal fat rate (>1.4%), the FPKM expression of the HOXA13 gene in the abdominal adipose tissue transcriptome of broilers with a low abdominal fat rate (<0.6%) is significantly increased, and the AUC of 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 1AFigure 6. PCA plot of transcriptome of abdominal adipose tissue dynamic development between D3 and D14 of different age;

[0016] Figure 1B Figure 7. Heatmap of differentially expressed genes of transcriptome of abdominal adipose tissue dynamic development between D3 and D14;

[0017] Figure 1C Figure 8. Number of up-regulated and down-regulated differentially expressed genes of transcriptome of abdominal adipose tissue dynamic development between D3 and D14;

[0018] Figure 1D Figure 9. Cluster plot of ATAC-seq of abdominal adipose tissue dynamic development between D3 and D14;

[0019] Figure 1E Figure 10. Heatmap of differentially expressed motifs of ATAC-seq of abdominal adipose tissue dynamic development between D3 and D14;

[0020] Figure 1F Figure 11. Number of up-regulated and down-regulated differentially expressed motifs of ATAC-seq of abdominal adipose tissue dynamic development between D3 and D14;

[0021] Figure 1G Figure 12. Combined analysis of RNA-seq and ATAC-seq of abdominal adipose tissue dynamic development between D3 and D14;

[0022] Figure 1H Figure 13. Protein expression of HOXA13, FABP4 and CPT-1A at different time points of abdominal adipose tissue dynamic development;

[0023] Figure 1I Figure 14. Quantitative results of protein expression of HOXA13 at different time points of abdominal adipose tissue dynamic development;

[0024] Figure 1J Figure 15. Quantitative results of protein expression of FABP4 at different time points of abdominal adipose tissue dynamic development;

[0025] Figure 1K Figure 16. Quantitative results of protein expression of CPT-1A at different time points of abdominal adipose tissue dynamic development;

[0026] Figure 2A Figure 17. Protein expression of transcription factor HOXA13 and proliferation and differentiation related proteins in ICP2 differentiation model;

[0027] Figure 2B Figure 18. Quantitative results of protein bands of transcription factor HOXA13 and proliferation and differentiation related proteins in ICP2 differentiation model;

[0028] Figure 3A Figure 19. Changes in chromatin opening degree of FASN, ACACA and ELOVL6 gene promoter regions in ATAC-seq of ICP2 cells after differentiation;

[0029] Figure 3B HOXA13 binding motifs identified in ATAC-seq of differentiated ICP2 cells;

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

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

[0032] Figure 3E Oil red O staining semi-quantification of ICP2 cells transfected with pcDNA3.1 and Flag-HOXA13 respectively after induction of differentiation;

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

[0034] Figure 3G Oil red O staining of ICP2 cells transfected with HOXA13 after induction of differentiation;

[0035] Figure 3H Oil red O staining semi-quantification of ICP2 cells transfected with HOXA13 after induction of differentiation;

[0036] Figure 3I Prediction of HOXA13 binding to promoter regions of lipid metabolism related genes;

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

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

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

[0040] Figure 5B ROC curve of HOXA13 gene expression in white-feathered broilers with high and low abdominal fat rate. DETAILED DESCRIPTION

[0041] The examples are provided to better illustrate the present application but are not intended to limit the present application to their details. Therefore, the skilled in the art can make non-essential improvements and modifications to the embodiments according to the above disclosure, which still fall within the scope of the present application.

[0042] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. Unless otherwise defined, all terms of art used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of the disclosure. As used herein, it is also to be understood that the use of "a" or "an", which means "one or more", "at least one", and "one or more than one" is open-ended, and for that reason, specifies the inclusion of one or more of something which is compatible with that specific use. As used herein, the term "or" as used herein, unless otherwise indicated, is generally intended to mean "and / or", but it is also possible that "or" means "and" or "or" means "exactly one" in various instances, and it is intended to cover each of the different options as long as the description is consistent with such options.

[0043] The application provides a use of a reagent for detecting a transcription factor HOXA13 in preparation of a detection product for detecting an abdominal fat rate of an avian.

[0044] In some specific examples, in the use, the transcription factor HOXA13 is overexpressed, and the abdominal fat rate of the avian is reduced.

[0045] In some specific examples, in the use, the reagent for detecting the transcription factor HOXA13 comprises a reagent for detecting expression of the transcription factor HOXA13, a reagent for detecting mRNA synthesized by the transcription factor HOXA13, or a reagent for detecting a protein synthesized by the transcription factor HOXA13.

[0046] In some specific examples, in the use, the reagent for detecting expression of the transcription factor HOXA13 comprises 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 one-hybrid assay; and the reagent for detecting mRNA synthesized by the transcription factor HOXA13 or the reagent for detecting a protein synthesized by the transcription factor HOXA13 comprises a detection reagent based on a Western blot detection.

[0047] In some specific examples, in the use, the avian is a chicken.

[0048] In some specific examples, in the use, the detection product comprises a detection reagent or a detection kit.

[0049] The application also provides a use of a reagent for detecting a transcription factor HOXA13 in genetic breeding of an avian.

[0050] In some specific examples, in the use, the transcription factor HOXA13 is overexpressed, and the abdominal fat rate of the avian 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 mRNA synthesized by the transcription factor HOXA13, or a reagent for detecting protein synthesized by the transcription factor HOXA13.

[0052] In some specific examples, in the above application, the reagent for detecting the expression of the transcription factor HOXA13 includes a reagent for detecting the expression of the transcription factor HOXA13, a reagent for detecting mRNA synthesized by the transcription factor HOXA13, or a reagent for detecting protein synthesized by the transcription factor HOXA13.

[0053] In some specific examples, in the above application, the avian is a chicken.

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

[0055] It should be noted that, in the present application, the ATAC-seq and RNA-seq between D3 and D14 of the dynamic development of abdominal fat are jointly analyzed, and 4 genes are co-interacted between the up-regulated motif and the up-regulated DEGs, of which HOXA13 is the largest in the difference multiple. The expression change of the screened transcription factor in the dynamic development of abdominal fat is 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 application, the chicken fat cells ICP2 are induced to differentiate by 200 μM oleic acid, and Western blotting verification shows that the expression of adipogenic differentiation related proteins is up-regulated, for example: C / EBPα, FABP4, IGF2, SREBP-1; while the cell cycle related factors PCNA, CDK1 and the key rate-limiting enzyme CPT1 of beta oxidation are significantly reduced, which indicates that the fat cell differentiation model is successfully constructed. In addition, the protein expression of the transcription factor HOXA13 is significantly reduced after differentiation, which suggests its potential role in regulating adipogenic differentiation.

[0057] It also needs to be explained that in the present application, 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 are significantly reduced. The ICP2 cells in which HOXA13 is interfered show more obvious lipid deposition, and the TG and TC contents in the cells are increased. Therefore, it can be explained that HOXA13 can bind to a series of gene promoter regions involved in adipocyte differentiation and maturation to regulate their expression; in addition, further use of the JASPAR website for HOXA13 and lipid metabolism-related gene promoter region binding prediction found that HOXA13 can bind to the CPT-1A and PPARα gene promoter regions.

[0058] It also needs to be explained that in the present application, the CUT&Tag technology is used to detect the interaction binding sites of HOXA13 and lipid metabolism-related genes, and to clarify the internal mechanism of HOXA13 regulating chicken adipocyte proliferation and differentiation, and to provide a reference for breeding new varieties of high-meat-yield efficiency broilers. The results show that the binding of transcription factor HOXA13 in the whole genome range is mainly concentrated in the intron and promoter region. In addition, HOXA13 has a higher binding peak in CPT1A, CD36, ACSL4 and ACSL6 genes; and the binding peak at CPT1A and CD36 genes is up-regulated after differentiation. The above results show that HOXA13 may bind to the promoter regions of CPT1A and CD36 and other genes related to fat decomposition and transport, and positively regulate lipid decomposition and metabolism.

[0059] It also needs to be explained that in the present application, IP-MS is used to further identify the HOXA13 interacting protein library. The mechanism of transcription factor HOXA13 regulating fat formation is clarified. The results show that HOXA13 can interact with CPT1A, USP1, VTG2, IKZF2, YES1 and other lipid metabolism-related or chromatin structure regulating proteins after ICP2 cell differentiation.

[0060] It also needs to be explained that in the present application, compared with high abdominal fat rate (>1.4%) broilers, the FPKM expression of HOXA13 gene in the abdominal fat tissue transcriptome of low abdominal fat rate (<0.6%) broilers is significantly increased, and the ROC curve drawn for the expression of HOXA13 gene in the sample shows that the AUC=0.815 indicates that the gene can better represent the abdominal fat content of broilers.

[0061] In order to better understand the present application, the content of the present application will be further explained in combination with specific examples below, but the content of the present application is not limited to the examples below.

[0062] I. Screening of transcription factors regulating abdominal fat deposition based on the dynamic changes of abdominal fat of broilers

[0063] Select 300 white-feathered broilers, slaughter to calculate the abdominal fat rate of broilers; on this basis, use RNA-seq to analyze the differentially expressed genes of D3 and D14 in the dynamic development process of abdominal fat; use the TRIZOL kit protocol provided by Agbio (China) to extract total RNA from abdominal fat tissue; use Illumina Kit (Illumina, USA) to construct RNA-seq library, and Shanghai Personal Biotechnology Co., Ltd. to sequence; filter and quality control the original data, and use HISAT2 to align with chicken genome (GRCg7b); DESeq2 software is further used to identify differentially expressed genes according to the threshold of log2 FoldChange>1 and P value<0.05; further analyze the changes of chromatin accessibility in the dynamic development process of abdominal fat by ATAC-seq.

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

[0065] In addition, the changes in HOXA13 protein expression during the dynamic development of abdominal fat were detected by Western blotting. The specific steps were as follows: the abdominal adipose tissue was fully lysed using RIPA lysis buffer containing 1% PMSF produced by Zhonghui Hecai Biological Company, and the supernatant was collected after centrifugation at 4°C and 12000 rpm for 5 min. The supernatant was collected using AccuRefScientific Protein concentrations were normalized using a commercial BCA kit provided by Biotin (Xi'an, China). Proteins were boiled at 95°C for 15 min, and equal amounts of protein (25 μg) were electrophoresed on SDS-polyacrylamide gels to detect target protein expression. Primary antibodies were diluted 1:1000 in 5% BSA, and secondary antibodies (DIYIBio, Shanghai) were diluted 1:2000 for incubation. Details of the antibodies used are listed in Table 1. Images were captured using the iBright FL1500 system (Thermo Fisher Scientific, USA), and quantitative analysis was performed using Image J (National Institutes of Health, USA) with GAPDH or β-actin as an internal control.

[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 Figure 1A to Figure 1K As shown, Figure 1A to Figure 1K The results showed that HOXA13, a transcription factor that mediates abdominal fat deposition in broiler chickens, was mined at the upstream three-gene level and its expression changes during the dynamic development of abdominal fat were verified. Among them, 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 during 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 the transcription factor HOXA13 in chicken adipocytes ICP2

[0070] Immortalized chicken preadipocytes (ICP2) were purchased from the Key Laboratory of Poultry Genetics and Breeding of Ministry of Agriculture and Rural Affairs, Northeast Agricultural University (Harbin); ICP2 cells were cultured in DMEM / F12 medium (Gibco, USA, 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%, the cells were induced to differentiate for 1 day using complete medium (DMEM / F12 + 10% FBS) containing 200 μM oleic acid; the expression of lipogenic differentiation-related proteins and cell cycle-related proteins in ICP2 cells was detected by the Western blotting method described above to verify the successful construction of the model.

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

[0072] In addition, the ATAC-seq method described above was further used to detect the changes in chromatin openness of lipid metabolism-related genes before and after differentiation of ICP2 cells. The results are shown in Figure 3A to 3-B The results show that HOXA13 is predicted in the up-regulated motif, further suggesting that HOXA13 may regulate gene transcription by binding to the promoter region of lipid metabolism-related genes during adipogenic differentiation.

[0073] In addition, to explore the function of HOXA13 gene, overexpression and interference experiments were performed in ICP2 cells. The overexpression vector and small interfering RNA were synthesized by Shanghai Shengong Bioengineering Technology Service Co., Ltd. When the cells were seeded into 6-well plates and cultured to a density of about 70%, ICP2 cells were transfected using serum-free and double-antibiotic-free medium and Lipofectamine 2000 transfection reagent from Thermo Fisher. After 6 h of transfection, the cells were cultured in normal medium, and the medium was changed after one day. After the treatment, the cells were collected by trypsin digestion. The protein concentration was determined by BCA kit, and the TG and TC contents in the cells were detected according to the commercial kit instructions of Nanjing Jiancheng Bioengineering Co., Ltd. The results show that overexpression of HOXA13 can effectively inhibit lipid deposition in ICP2 cells, while interference promotes adipogenic differentiation of the cells (see Figure 3C to Figure 3H ).

[0074] Based on the above results, further HOXA13 and lipid metabolism-related gene promoter region binding prediction was performed using the JASPAR website, and it was found that HOXA13 can bind to CPT-1A and PPARa gene promoter regions Figure 3I ).

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

[0076] ICP2 overexpressing HOXA13 containing Flag tag was subjected to CUT&Tag sequencing to detect HOXA13 interaction with lipid metabolism-related genes; specifically including: adding concanavalin A-coated magnetic beads to the cell suspension; Next, incubate with Flag primary antibody, followed by secondary antibody; pA-Tn5 transposase (ABclonal company) that can recognize antibodies is added to the above system, Tn5 transposase cuts the genome and inserts a linker sequence near the target protein; Library construction and sequencing were performed on the Illumina NovaSeq platform of Fosangene Information Co., Ltd. (Wuhan, China); Clear reads were obtained using the Burrows-Wheeler Alignment program and aligned to the chicken reference genome GRCg7b; Peaks were called by MACS2, and differential peaks were assessed using DESeq2 under the conditions of P-value < 0.05 and log2 Fold Change absolute value > 1. Peak values were annotated by the annotatePeak function of ChIPseeker, with the promoter region set to < 3 kb from the TSS, and target genes can also be predicted according to differential peaks in the promoter region.

[0077] The results are shown in Figure 4A to 4-B , which show that HOXA13 binding in the whole genome range is mainly concentrated in introns and promoter regions. In addition, HOXA13 has higher binding peaks in CPT1A, CD36, ACSL4 and ACSL6 genes; and the binding peaks at CPT1A and CD36 genes are up-regulated after differentiation; that is, HOXA13 can bind to the promoter regions of CPT1A and CD36 and other genes related to fat decomposition and transport, positively regulating lipid decomposition metabolism.

[0078] IV. Identification of HOXA13 interaction protein library based on IP-MS

[0079] Based on the above three results, the IP-MS method was used to identify the HOXA13 interacting protein set. After the preparation of the antigen-antibody-magnetic bead complex, the separated immunoprecipitated material was 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 polypeptide sample. Then desalination was performed on a C18 chromatographic column (Empore TM SPE chromatographic column C18, layer diameter 7 mm, Sigma), vacuum centrifugation concentration, reconstitution in 40 μl of 0.1% formic acid. The peptide content was estimated by ultraviolet spectral density at 280 nm with an extinction coefficient of 1.1. Then 1 μg of peptide sample was prepared for LC-MS / MS analysis; LC-MS / MS analysis was performed on a Q Exactive mass spectrometer (Thermo Fisher Scientific, USA) coupled with Easy nLC (Thermo Fisher Scientific, USA) for 120 min. The MS raw data of each sample were combined and searched using MaxQuant 1.5.3.17 software for identification and quantitative analysis.

[0080] The results showed that HOXA13 could interact with CPT1A, USP1, VTG2, IKZF2 and YES1, etc. lipid metabolism related or chromatin structure regulation proteins after ICP2 cell differentiation, as shown in the following table 2.

[0081] Table 2 Lipid metabolism or chromatin structure regulation related proteins interacting with HOXA13 in IP-MS

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

[0083] Five, the expression change of HOXA13 gene in the abdominal fat transcriptome of high and low abdominal fat rate populations of white-feathered broilers

[0084] In a large population of 1000 white-feathered broilers with the same genetic background and feeding conditions, sampling was performed at 42 days 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 broilers was calculated by slaughter, and 30 broilers with high abdominal fat rate (>1.4%) and 30 broilers with low abdominal fat rate (<0.6%) were selected. The expression change of HOXA13 gene in high and low abdominal fat rate populations was identified according to the above transcriptome determination method; the ROC curve was drawn using the micro bioinformatics website (https: / / www.bioinformatics.com.cn / ).

[0085] The results are as follows Figure 5A to Figure 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 solutions of the present invention and are not limiting. 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 solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. Application of a reagent for detecting the expression of the transcription factor HOXA13 in the preparation of a detection product for detecting the abdominal fat rate of white-feathered broiler chickens.

2. The use according to claim 1, characterized in that Overexpression of the transcription factor HOXA13 reduces the abdominal fat rate of white-feathered broiler chickens.

3. The use according to claim 1 or 2, characterized in that Reagents for detecting the expression of the transcription factor HOXA13 include detection reagents based on a dual-luciferase reporter gene assay system, detection reagents based on a gel shift assay, detection reagents based on a DNA footprinting method, or detection reagents based on a yeast one-hybrid assay.

4. The use according to claim 1 or 2, characterized in that Testing products include testing reagents or testing kits.

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