Analysis method of differential metabolite and hub gene common enrichment pathway

Abstract

The invention relates to the technical field of poultry molecular breeding, in particular to an analysis method of a differential metabolite and hub gene common enrichment pathway. Selecting differential metabolites of laying ducks and hub genes to enrich metabolic pathways; dividing the laying ducks into laying ducks with high residual feed intake and laying ducks with low residual feed intake; screening out significant difference pathways by comparing metabolic pathways of the laying ducks with high residual feed intake and the laying ducks with low residual feed intake; obtaining significant differential genes in the significant differential pathway, and inputting the significant differential genes into the STRING protein interaction database to establish a PPI protein interaction network diagram; based on the PPI protein interaction network diagram, establishing a gene network diagram of significantly differential genes by adopting a Cytoscape network analysis method; and analyzing according to the gene network diagram to obtain the key differential genes of the laying ducks. Analysis is carried out from the perspective of a differential metabolite and hub gene common enrichment analysis pathway, and breeding for optimizing the animal feed utilization rate is facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of poultry molecular breeding, and in particular to a method for analyzing a common enrichment pathway of differential metabolites and hub genes. Background Art

[0002] Metabolomics studies metabolites in organisms and can perform efficient, comprehensive and accurate quantitative analysis of biological samples. It combines modern mathematical statistics and machine learning to more comprehensively screen out metabolites with significant differences. Metabolomics is an important omics strategy in animal husbandry research and has multiple characteristics, the most notable of which is the ability to detect biological molecules other than genomes, transcriptomes, proteomes and metallomics. In recent years, transcriptomics technology has developed rapidly and can comprehensively and systematically understand the gene expression patterns of cells, tissues or organs at the RNA level, explain their physiological properties, and gain an in-depth understanding of their growth, development, differentiation, aging and other mechanisms.

[0003] Feed cost is the main factor determining profitability in most domestic animal production, accounting for about 70% to 80% of the total production cost, and has been used as a core indicator to measure the economic benefits of the breeding industry. Egg duck breeding is a dominant and characteristic industry in my country, with a breeding volume of about 80% of the world. Duck eggs and their products (preserved eggs, salted eggs) are traditional delicacies in my country. In recent years, my country's large-scale and systematic breeding of high-yield egg ducks has made good progress, especially in terms of egg production, reaching more than 300 eggs at 72 weeks of age; but in terms of feed utilization efficiency, the average feed-egg ratio of egg ducks is 2.7 to 2.8:1, which is low compared with other poultry (chickens and meat ducks), and there are large differences between and within breeds, resulting in low overall breeding benefits of the industry. Therefore, a method for identifying animals with high feed utilization efficiency is urgently needed, so as to provide a reference for the screening of egg duck varieties with high feed utilization efficiency in the future. Summary of the invention

[0004] In order to solve the problems existing in the prior art, the present invention provides an analysis method for the common enrichment pathway of differential metabolites and hub genes. The method comprises: selecting differential metabolites and hub genes of laying ducks to enrich metabolic pathways; dividing laying ducks into high residual feed intake laying ducks and low residual feed intake laying ducks; screening out significantly different pathways by comparing the metabolic pathways of high residual feed intake laying ducks and low residual feed intake laying ducks; obtaining significantly different genes in the significantly different pathways, and inputting the significantly different genes into the STRING protein interaction database to establish a PPI protein interaction network diagram; using the Cytoscape network analysis method based on the PPI protein interaction network diagram to establish a gene network diagram of significantly different genes; analyzing according to the gene network diagram to obtain the key differential genes of laying ducks. The present application analyzes from the perspective of the common enrichment analysis pathway of differential metabolites and hub genes, which is helpful to optimize the breeding of animal feed utilization.

[0005] The present invention adopts the following technical scheme, a method for analyzing the common enrichment pathway of differential metabolites and hub genes, comprising:

[0006] Differential metabolites and hub genes of laying ducks were selected to enrich metabolic pathways;

[0007] According to the residual feed intake of laying ducks, laying ducks are divided into high residual feed intake laying ducks and low residual feed intake laying ducks.

[0008] By comparing the metabolic pathways of laying ducks with high residual feed intake and laying ducks with low residual feed intake, 9 significantly different pathways were screened out;

[0009] Obtaining significantly differentially expressed genes in the significantly differentially expressed pathways, inputting the significantly differentially expressed genes into the STRING protein interaction database, and establishing a PPI protein interaction network diagram of the significantly differentially expressed genes;

[0010] Based on the PPI protein interaction network diagram, a Cytoscape network analysis method is used to establish a gene network diagram of the significantly differentially expressed genes;

[0011] According to the gene network diagram, analysis is performed to obtain key differential genes of laying ducks.

[0012] Furthermore, the differential metabolites include: 15-deoxy-Δ12, 14-prostaglandin J2, L-saccharomyces cerevisiae, L-glutamine, methyl palmitate and deoxycholic acid.

[0013] Furthermore, the hub genes include: IL6, IL10, CSF3, VCAM1, CD44, PTGS2, CXCL8-3, SOCS3, TNFSF11 and IL-2.

[0014] Furthermore, the significantly different pathways include: neuroactive ligand-receptor interaction pathway, GABAergic synaptic signaling pathway, cAMP signaling pathway, cAMP signaling pathway, calcium signaling pathway, nitrogen metabolism, tyrosine metabolism pathway, ovarian steroidogenesis pathway and gluconeogenesis pathway.

[0015] Furthermore, the differential metabolites are obtained by:

[0016] The differential metabolites were obtained by the VIP value of the OPLS-DA model and combined with the pvalue of the independent sample T test. The screening threshold for obtaining the differential metabolites was: |log2FC|≥0&OPLS-DA_VIP≥1&P-value≤0.05; which was expressed as: the absolute value of the log2 difference multiple ≥0, the VIP value of the orthogonal partial least squares discriminant analysis ≥1 and the P value was less than 0.05, and the above three conditions were met at the same time.

[0017] Further, after obtaining the differential metabolites:

[0018] The metabolites were grouped in pairs according to the pathways they participated in or the functions they performed through the KEGG PATHWAY database. For the differential metabolites in the pairwise groups, one of the samples was used as the control, and the differential metabolites were displayed on the KEGG pathway map to establish a KEGG annotated pathway map of differential metabolites.

[0019] Furthermore, the key differential genes of laying ducks were obtained by analyzing the gene network diagram, specifically:

[0020] The gene network diagram is compared with the differential metabolite KEGG annotation pathway diagram of laying ducks, and the differential genes that exist in the differential metabolite KEGG annotation pathway diagram and are the same as those in the gene network diagram are obtained as key differential genes.

[0021] The beneficial effects of the present invention are as follows: the present invention proposes a relevant analytical method for identifying metabolic pathways for identifying high and low residual feed intake, and for the problem of analyzing pathways for the co-enrichment of differential metabolites and hub genes, an analytical method suitable for identifying high and low residual feed intake of animals is established. The application of these metabolites and enrichment pathways is helpful to optimize the breeding of animal feed utilization, and can provide some valuable clues for subsequent actual production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 This is a schematic flow chart of a method for analyzing a pathway for common enrichment of differential metabolites and hub genes according to an embodiment of the present invention;

[0024] Figure 2 A PPI protein interaction network diagram of a significantly differentially expressed gene according to an embodiment of the present invention;

[0025] Figure 3 for Figure 2 Schematic diagram of key differentially expressed genes obtained by calculating DEGREE scores;

[0026] Figure 4 A volcano plot of differential metabolites according to an embodiment of the present invention;

[0027] Figure 5This is a diagram of the arachidonic acid metabolic pathway in which the differential metabolite 15d-PJG2 and the hub gene PTGS2 are co-enriched according to an embodiment of the present invention. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] A schematic flow chart of a method for analyzing a common enrichment pathway of differential metabolites and hub genes according to an embodiment of the present invention is shown in FIG. Figure 1 As shown, including:

[0030] Differential metabolites and hub genes of laying ducks were selected to enrich metabolic pathways;

[0031] According to the residual feed intake of laying ducks, laying ducks are divided into high residual feed intake laying ducks and low residual feed intake laying ducks.

[0032] By comparing the metabolic pathways of laying ducks with high residual feed intake and laying ducks with low residual feed intake, 9 significantly different pathways were screened out;

[0033] Obtain the significantly differentially expressed genes in the significantly differentially expressed pathways, input the significantly differentially expressed genes into the STRING protein interaction database, and establish a PPI protein interaction network diagram of the significantly differentially expressed genes;

[0034] Based on the PPI protein interaction network diagram, the Cytoscape network analysis method was used to establish a gene network diagram of significantly differentially expressed genes;

[0035] The key differential genes of laying ducks were obtained through analysis based on the gene network diagram.

[0036] In a specific embodiment of the present invention, the selected differential metabolites include 15-deoxy-Δ12, 14-prostaglandin J2, L-saccharomyces cerevisiae, L-glutamine, methyl palmitate and deoxycholic acid; the selected hub genes include: IL6, IL10, CSF3, VCAM1, CD44, PTGS2, CXCL8-3, SOCS3, TNFSF11 and IL-2.

[0037] In the embodiment of the present invention, the VIP value of the OPLS-DA model is used, and the pvalue of the independent sample T test is combined to find the differential metabolites. The differential metabolite screening threshold is: |log2FC|≥0&OPLS-DA_VIP≥1&P-value≤0.05, which is expressed as: the absolute value of the log2 difference multiple is ≥0, the VIP value of the orthogonal partial least squares discriminant analysis is ≥1, and the P value is less than 0.05. The above three conditions need to be met at the same time; after obtaining the differential metabolites, the identified differential metabolites are further annotated for metabolic pathways. The metabolites use the KEGG PATHWAY database to group the metabolites in pairs according to the functions they participate in or perform, and for the differential metabolites in the two groups, one of the samples is used as a control to display the differential metabolites on the KEGG pathway diagram, thereby obtaining a KEGG annotated pathway diagram of the differential metabolites.

[0038] In a specific embodiment of the present invention, the 9 significantly different pathways screened out include: neuroactive ligand-receptor interaction pathway, GABAergic synaptic signaling pathway, cAMP signaling pathway, cAMP signaling pathway, calcium signaling pathway, nitrogen metabolism, tyrosine metabolism pathway, ovarian steroidogenesis pathway and gluconeogenesis pathway.

[0039] like Figure 2 As shown, an embodiment of the present invention provides a PPI protein interaction network diagram of significantly differentially expressed genes, in which nodes represent proteins, the color of the nodes represents the FDR (q-value) level, the size and color of the nodes represent the average abundance of gene expression, and the connecting lines represent the association between proteins; the correlation between two proteins is expressed by the thickness of the line, the thicker the line, the stronger the correlation, and the depth of the line color represents the comprehensive score, which is calculated in Cytoscape.

[0040] The embodiment of the present invention further obtains the key hub genes in the significantly differentially expressed genes by calculating the DEGREE score, such as Figure 3 As shown, the darker the color, the more important the gene. Based on the function and importance of the gene, PTGS2 is the gene most likely to be related to residual feed intake in the liver.

[0041] The enrichment analysis of the metabolic pathways of differential metabolites, the embodiment of the present invention maps the differential metabolites to metabolite databases such as KEGG and PubChem, and then searches and analyzes the metabolic pathways of the pathway databases of the corresponding species, and then enriches the differential metabolites in the KEGG database, and displays the KEGG annotated pathway diagram of the differential metabolites. Figure 4As shown in the figure, according to the enriched metabolite KEGG pathway diagram, 15d-PGJ2, one of the differential metabolites in the arachidonic acid pathway, was found. At the same time, the hub gene PTGS2 analyzed in the liver transcriptome regulated the process of arachidonic acid metabolism to 15d-PGJ2, thus jointly enriching the arachidonic acid metabolic pathway diagram as shown in the figure Figure 5 As shown, circles represent metabolites and rectangles represent genes.

[0042] In summary, by finding the key differentially expressed genes in the arachidonic acid metabolic pathway map obtained through co-enrichment, a reference can be provided for the subsequent screening of laying ducks.

[0043] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for analyzing the common enrichment pathway of differential metabolites and hub genes, characterized in that: include: Differential metabolites and hub genes of laying ducks were selected to enrich metabolic pathways; According to the residual feed intake of laying ducks, laying ducks are divided into high residual feed intake laying ducks and low residual feed intake laying ducks. By comparing the metabolic pathways of laying ducks with high residual feed intake and laying ducks with low residual feed intake, 9 significantly different pathways were screened out; Obtaining significantly differentially expressed genes in the significantly differentially expressed pathways, inputting the significantly differentially expressed genes into the STRING protein interaction database, and establishing a PPI protein interaction network diagram of the significantly differentially expressed genes; Based on the PPI protein interaction network diagram, a Cytoscape network analysis method is used to establish a gene network diagram of the significantly differentially expressed genes; The key differential genes of laying ducks are obtained by analyzing the gene network diagram, and laying ducks are screened according to the key differential genes.

2. The method for analyzing a common enrichment pathway of differential metabolites and hub genes according to claim 1, characterized in that: The differential metabolites include: 15-deoxy-Δ12, 14-prostaglandin J2, L-saccharomyces cerevisiae, L-glutamine, methyl palmitate and deoxycholic acid.

3. The method for analyzing the common enrichment pathway of differential metabolites and hub genes according to claim 1, characterized in that: The hub genes include: IL6, IL10, CSF3, VCAM1, CD44, PTGS2, CXCL8-3, SOCS3, TNFSF11 and IL-2.

4. The method for analyzing a common enrichment pathway of differential metabolites and hub genes according to claim 1, characterized in that: The significantly different pathways include: neuroactive ligand-receptor interaction pathway, GABAergic synaptic signaling pathway, cAMP signaling pathway, cAMP signaling pathway, calcium signaling pathway, nitrogen metabolism, tyrosine metabolism pathway, ovarian steroidogenesis pathway and gluconeogenesis pathway.

5. The method for analyzing the common enrichment pathway of differential metabolites and hub genes according to claim 1, characterized in that: The differential metabolites are obtained by: The differential metabolites were obtained by the VIP value of the OPLS-DA model and combined with the pvalue of the independent sample T test. The screening threshold for obtaining the differential metabolites was: |log2FC|≥0&OPLS-DA_VIP≥1&P-value≤0.05; which was expressed as: the absolute value of the log2 difference multiple ≥0, the VIP value of the orthogonal partial least squares discriminant analysis ≥1 and the P value was less than 0.05, and the above three conditions were met at the same time.

6. The method for analyzing the common enrichment pathway of differential metabolites and hub genes according to claim 1, characterized in that: After obtaining the differential metabolites: The metabolites were grouped in pairs according to the pathways they participated in or the functions they performed through the KEGG PATHWAY database. For the differential metabolites in the pairwise groups, one of the samples was used as the control, and the differential metabolites were displayed on the KEGG pathway map to establish a KEGG annotated pathway map of differential metabolites.

7. The method for analyzing the common enrichment pathway of differential metabolites and hub genes according to claim 6, characterized in that: According to the gene network diagram, the key differential genes of laying ducks are analyzed and obtained, specifically: The gene network diagram is compared with the differential metabolite KEGG annotation pathway diagram of laying ducks, and the differential genes that exist in the differential metabolite KEGG annotation pathway diagram and are the same as those in the gene network diagram are obtained as key differential genes.

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