Genome-wide gene expression analysis of amphioxus (Branchiostoma belcheri) following lipopolysaccharide challenge using strand-specific RNA-seq.

Amphioxus is the closest living proxy for exploring the evolutionary origin of the immune system in vertebrates. To understand the immune responses of amphioxus to lipopolysaccharide (LPS), 5 ribosomal RNA (rRNA)-depleted libraries of amphioxus were constructed, including one control (0 h) library and 4 treatment libraries at 6, 12, 24, and 48 h post-injection (hpi) with LPS. The transcriptome of Branchiostoma belcheri was analyzed using strand-specific RNA sequencing technology (RNA-seq). A total of 6161, 6665, 7969, and 6447 differentially expressed genes (DEGs) were detected at 6, 12, 24, and 48 hpi, respectively, compared with expression levels at 0 h. We identified amphioxus genes active during the acute-phase response to LPS at different time points after stimulation. Moreover, to better visualize the resolution phase of the immune process during immune response, we identified 6057 and 5235 DEGs at 48 hpi by comparing with 6 and 24 hpi, respectively. Through real-time quantitative PCR (qRT-PCR) analysis of 12 selected DEGs, we demonstrated the accuracy of the RNA-seq data in this study. Functional enrichment analysis of DEGs demonstrated that most terms were related to defense and immune responses, disease and infection, cell apoptosis, and metabolism and catalysis. Subsequently, we identified 1330, 485, 670, 911, and 1624 time-specific genes (TSGs) at 0, 6, 12, 24, and 48 hpi. Time-specific terms at each of 5 time points were primarily involved in development, immune signaling, signal transduction, DNA repair and stability, and metabolism and catalysis, respectively. As this is the first study to report the transcriptome of an organism with primitive immunity following LPS challenge at multiple time points, it provides gene expression information for further research into the evolution of immunity in vertebrates.