Abstract: The purpose of this study was to screen candidate genes related to tibial bone metabolism and reveal the genetic mechanism of bone metabolism changes in laying hens by analyzing the transcriptional expression profile of the tibia of laying hens at the early and late stages of laying. Three Hy-line Grey laying hens aged 19 weeks old (early laying) and 79 weeks old (late laying) were selected to construct a tibia transcriptome library, and transcriptome data were used to screen differentially expressed genes and predict protein interactions; Five differentially expressed genes were randomly selected and their expression levels was verified using real-time fluorescence quantitative PCR. The effective reads of 19 034 718-22 668 315 were obtained from the 6 cDNA libraries of the tibia of Hy-line Grey laying hens with Q30 value of at least 93.68% and comparison rate of 83.78%-85.72%. A total of 1 211 differentially expressed genes were screened from the tibia of laying hens at the early and late stages of laying, of which 645 differentially expressed genes were down regulated and 566 differentially expressed genes were up regulated. GO analysis results showed that most of the differential genes were enriched in GO terms related to cell differentiation and cell development. KEGG analysis results showed that the differentially expressed genes were significantly enriched in 11 KEGG pathways (P<0.05). There were 5 pathways related to bone metabolism, namely MAPK signaling pathway, TGF-β signaling pathway, Toll-like receptor signaling pathway, glycosaminoglycan biosynthesis-chondroitin sulfate/dermatan sulfate and glycosaminoglycan degradation. Among the above 5 pathways, 59 differentially expressed genes were involved, of which 29 were up regulated and 30 were down regulated. In addition, protein interaction analysis was conducted on 59 differentially expressed genes related to bone metabolism pathways, identifying a total of 53 coding proteins and screening out 5 hub genes (IL6, IL1B, FOS, TGFB2 and BMP4). Five differentially expressed genes were randomly selected from 1 211 differentially expressed genes for real-time fluorescent quantitative PCR verification, and the results were consistent with the results of RNA-Seq sequencing. This study revealed the differential gene expression in the tibia tissue of laying hens during the early and late stages of egg laying, and screened multiple differentially expressed genes and signaling pathways that affect tibia bone metabolism in laying hens, providing a theoretical basis for studying the molecular mechanism of tibia tissue regulation of bone metabolism in laying hens.

Key words: laying hen, tibia, differentially expressed genes, bone metabolism