Study on the transcriptome for breast muscle of chickens and the function of key gene RAC2 on fibroblasts proliferation

Abstract

Abstract Background Growth performance is significant in broiler production. In the growth process of broilers, gene expression varies at different growth stages. However, limited research has been conducted on the molecular mechanisms of muscle growth and development in yellow-feathered male chickens. Results In the study, we used RNA-seq to study the transcriptome of the breast muscle of male Jinghai yellow chickens at 4 (M4F), 8 (M8F) and 12 weeks (M12F) of age. The results showed that 4608 differentially expressed genes (DEGs) were obtained by comparison in pairs of the three groups with Fold Change (FC) ≥ 2 and False Discovery Rate (FDR) ≤ 0.05, and 83, 3445 and 3903 DEGs were obtained separately from M4FvsM8F, M4FvsM12F and M8FvsM12F. Six genes were found as co-differentially expressed in the three age groups, namely SNCG, MYH1A, ARHGDIB, ENSGALG00000031598, ENSGALG00000035660 and ENSGALG00000030559. The GO analysis showed that 0, 304 and 408 biological process (BP) were significantly enriched in M4FvsM8F, M4FvsM12F and M8FvsM12F groups, respectively. KEGG pathway enrichment showed that 1, 2, 4 and 4 pathways were significantly enriched in M4FvsM8F, M4FvsM12F, M8FvsM12F and all DEGs, respectively. They were steroid biosynthesis, carbon metabolism, focal adhesion, cytokine-cytokine receptor interaction, biosynthesis of amino acids and salmonella infection. We constructed short hairpin RNA (shRNA) to interfere the differentially expressed gene RAC2 in DF-1 cells and detected mRNA and protein expression of the downstream genes PAK1 and MAPK8. Results of qPCR showed that RAC2, PAK1 and MAPK8 mRNA expression significantly decreased in the shRAC2–2 group compared with the negative control (NC) group. Western Blot (WB) results showed that the proteins of RAC2, PAK1 and MAPK8 also decreased in the shRAC2–2 group. Cell Counting Kit-8 (CCK-8) and 5-Ethynyl-2′-deoxyuridine (EdU) assay both showed that the proliferation of DF-1 cells was significantly inhibited after transfection of shRAC2–2. Conclusions The results of RNA-seq revealed genes, BP terms and KEGG pathways related to growth and development of male Jinghai yellow chickens, and they would have important guiding significance to our production practice. Further research suggested that RAC2 might regulate cell proliferation by regulating PAKs/MAPK8 pathway and affect growth of chickens.

Keywords

• Jinghai yellow chicken
• Growth and development
• RNA-seq
• qPCR
• RNAi