CN-121983118-A - Method for analyzing influence of salmonella infection on chicken liver transcriptome RNA m6A modification

Abstract

The invention discloses a method for analyzing the influence of salmonella infection on chicken liver transcriptome RNA m6A modification, which relates to the technical field of bioengineering and comprises the following steps of animal model establishment and sample acquisition, RNA extraction, sequencing and data quality control, bioinformatics analysis and difference m6A peaks identification, data integration and function enrichment analysis; according to the invention, through integrating MeRIP-seq and transcriptome sequencing technology, a dynamic regulation mechanism of RNA m6A modification in the liver process of salmonella infection chicken is disclosed, an m6A modification and gene expression association analysis framework under an infection model is constructed, the analysis depth of an avian immune response epigenetic regulation mechanism is remarkably improved, and through establishing a standardized experimental flow and a bioinformatics analysis system, accurate recognition of differential modification sites and functional association thereof from the transcriptome level is realized, so that a new technical path is provided for poultry disease resistance research.

Inventors

• CHEN BIAO
• HUANG CHAO
• CHEN ZHIWU
• SU YONGCHUN
• Liao Zurong
• WU DONGXIAO
• Ding Zhenxuan

Assignees

• 广西金陵农牧集团有限公司
• 江西农业大学

Dates

Publication Date

20260505

Application Date

20251125

Claims (8)

1. 1. A method of analyzing the effect of salmonella infection on chicken liver transcriptome RNA m6A modification comprising the steps of: Firstly, breeding chicks and dividing the chicks into a salmonella infection group and a control group, obtaining chicken liver tissue samples of the salmonella infection group and the control group, wherein the infection group is inoculated with 3X 10 7 CFUs salmonella suspension by lavage, the control group is filled with phosphate buffer solution with the same dosage, and liver tissue is collected 14 days after infection; extracting total RNA of the chicken liver tissues of the salmonella infection group and the control group collected in the first step, carrying out MeRIP-seq sequencing to enrich the RNA fragment modified by m6A, and simultaneously carrying out input RNA-seq sequencing to serve as transcriptome control; Analyzing the sequencing data obtained in the step two by using a bioinformatics tool, identifying m6A peaks by MACS software, performing genome-wide peak scanning by exomePeak2, and screening the difference m6A peaks to obtain a gene list with the modification degree changed; and step four, based on the difference m6A peaks data obtained in the step three, integrating the input RNA-seq data of the step two, identifying genes with obviously changed m6A modification degree and gene expression quantity, and carrying out functional enrichment analysis to reveal the regulation and control relation between modification and expression.
2. 2. The method of claim 1, wherein in the first step, the bred chicks are commercial white feather broilers and are subjected to Salmonella inoculation at 3 days of age.
3. 3. The method of claim 1, wherein in the second step, the total RNA is extracted by Trizol method and is broken into fragments of about 100nt after quality inspection for use in MeRIP-seq and input RNA-seq library establishment.
4. 4. The method of claim 1, wherein in the second step, the MeRIP-seq sequencing is performed using Illumina NovaSeq X Plus platform, and the sequencing data is aligned to the chicken reference genome GRCg b after quality control by fastp using PE150 mode.
5. 5. The method of claim 1, wherein in the third step, the screening of the difference m6A peaks is performed by diffReps software and is related to a difference modification gene.
6. 6. The method of claim 1, wherein in the third step, sequencing data analysis comprises screening for differentially expressed genes using edgel R software under conditions of FDR <0.05 and |log2FC| >1.
7. 7. The method of claim 1, wherein in the fourth step, the functional enrichment analysis comprises GO and KEGG enrichment, wherein the genes associated with the difference m6A peaks are significantly enriched in carbon metabolism, tricarboxylic acid cycle, bacterial invasion of epithelial cell pathways, and the genes expressed differentially are significantly enriched in NF-. Kappa.B signaling pathway, JAK-STAT signaling pathway, and PPAR signaling pathway.
8. 8. The method of claim 1, wherein in the fourth step, the genes with significantly changed m6A modification degree and gene expression level comprise NFKBIA, IL16, CD5, IL21R and STAT4.

Description

Method for analyzing influence of salmonella infection on chicken liver transcriptome RNA m6A modification Technical Field The invention relates to the technical field of bioengineering, in particular to a method for analyzing the influence of salmonella infection on chicken liver transcriptome RNA m6A modification. Background Salmonella infection is widely present in various poultry production systems. The mechanism of salmonella infection in chickens is complex, and pathogen invasion into the host can trigger infection and stimulate the immune response of the host. Salmonella invades intestinal epithelial cells through the gastrointestinal tract of the host. During the invasion phase, salmonella relies on its genes encoding virulence factors (lipopolysaccharides) to adhere and enter host cells, triggering the pathological response of the body. Salmonella can cause systemic infections leading to serious diseases such as fowl typhoid, with concomitant high mortality. The host immune response has an important influence on the salmonella infection result, and the immune performance of the chicken flock is greatly influenced by the age and health condition of the chicken flock. In general, chicks are more susceptible to acute systemic infections, manifested by severe intestinal symptoms and high mortality, and adult chickens are mostly Salmonella carriers, resulting in long-term transmission of pathogens. Cytokines such as IL-6 play an important role in the immunomodulation process and help regulate the processes of inflammatory response and pathogen clearance. Salmonella evades the host's immune defenses through a variety of mechanisms, such as the ability of pathogens to survive and multiply within macrophages, resulting in the spread of infection throughout the host. The long-term persistence of pathogens in the cecum and other tissues increases the risk of repeated outbreaks within the poultry population. In recent years, the role of epigenetic activity has been of interest in the study of disease resistance in poultry, particularly chickens. DNA methylation, histone modification, RNA modification (e.g., N6-methyladenosine, m 6A), non-coding RNA regulation, etc., which regulate gene expression without altering the DNA sequence. The m6A modification has a more complex interaction relationship with the immune response. Studies have shown that m6A modifications are capable of modulating the expression of immune-related genes during viral infections (e.g., avian leukemia virus). RNA m6A modification is involved in the regulation of NF- κB signaling pathways during viral infection in the body, which pathways play an important role in the inflammatory response. The above studies indicate that RNA m6A modification affects the expression of immune-related important genes possibly playing a role in Salmonella-infected chickens and in the pathogenic process, but no study has been reported to report changes in transcriptome m6A modification after Salmonella infection in chickens. Therefore, the invention provides a method for analyzing the influence of salmonella infection on chicken liver transcriptome RNA m6A modification so as to solve the problems in the prior art. Disclosure of Invention Aiming at the problems, the invention aims to provide a method for analyzing the influence of salmonella infection on chicken liver transcriptome RNA m6A modification, which takes commercial white feather broilers as research objects, uses salmonella to infect chicks in a mode of gavage at the age of 3 days, collects chicken liver tissues of an experimental group and a control group after 14 days, performs MeRIP-seq and subsequent analysis, compares dynamic changes of chicken liver transcriptome m6A modification after salmonella infection, identifies m6A modification characteristics of chicken liver transcriptome infected with salmonella, lays a foundation for discussing how m6A modification affects immune response, and provides theoretical basis for deeply disclosing that m6A modification affects poultry to cope with salmonella infection. In order to achieve the aim, the invention is realized by the following technical scheme that the method for analyzing the influence of salmonella infection on chicken liver transcriptome RNA m6A modification comprises the following steps: Firstly, breeding chicks and dividing the chicks into a salmonella infection group and a control group, obtaining chicken liver tissue samples of the salmonella infection group and the control group, wherein the infection group is inoculated with 3X 10 7 CFUs salmonella suspension by gastric lavage, the control group is filled with Phosphate Buffer (PBS) with the same dosage, and the tissue is collected 14 days after infection; Extracting total RNA of chicken liver tissues of a salmonella infection group and a control group, carrying out MeRIP-seq sequencing to enrich the RNA fragments modified by m6A, and simultaneously carrying out input RNA-seq sequencing