Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, 650500, China; State Key Laboratory of Pharmaceutical Biotechnology, School of Life Science, Nanjing University, Nanjing, 210023, China.
BGI Genomics, BGI-Shenzhen, Shenzhen, 518083, China.
Fish Shellfish Immunol. 2018 May;76:196-205. doi: 10.1016/j.fsi.2018.03.007. Epub 2018 Mar 3.
Amphioxus, a basal chordate, is widely considered to be an existing proxy of the invertebrate ancestor of vertebrates, and it exhibits susceptibility to various pathogen infections and pathogenic mimic challenges. Here, in order to understand more clearly its antibacterial mechanisms, we analyzed the ribosomal RNA (rRNA)-depleted transcriptome of Chinese amphioxus (Branchiostoma belcheri) infected with Vibrio parahaemolyticus (V. p.) via next-generation deep sequencing technology (RNA-seq). We identified a total of 3214 differentially expressed genes (DEGs) by comparing V. p.-infected and control transcriptome libraries, including 2219 significantly up-regulated and 995 significantly down-regulated DEGs in V. p.-infected amphioxus. The DEGs with the top 10 most dramatic expression fold changes after V. p. infection, as well as 53 immune-related DEGs (IRDs) belonging to four primary categories of innate immunity were analyzed further. Through gene ontology (GO) and pathway enrichment analysis, DEGs were found to be primarily related to immune processes, apoptosis, catabolic and metabolic processes, binding and enzyme activity, while pathways involving bacterial infection, immune signaling, immune response, cancer, and apoptosis were overrepresented. We validated the RNA-seq results by detecting the expression levels of 10 IRDs using qRT-PCR, and we surveyed the dynamic variation in gene expression for these IRDs at 0, 6, 12, 24, and 48 h after V. p.
Subsequently, according to the RNA-seq results, the presence of a primitive Toll-like receptor (TLR)-mediated antibacterial immune signaling pathway was predicted in B. belcheri. This study provides valuable information regarding antibacterial immunity for further research into the evolution of immunity in vertebrates and broadens our understanding of the innate immune response against bacterial invasion in amphioxus.
文昌鱼，一种基干脊索动物，被广泛认为是脊椎动物无脊椎动物祖先的现存代表，它易受各种病原体感染和致病模拟挑战。在这里，为了更清楚地了解其抗菌机制，我们通过下一代深度测序技术（RNA-seq）分析了感染副溶血弧菌（V. p.）的中国文昌鱼（Branchiostoma belcheri）的核糖体 RNA（rRNA）耗尽转录组。我们通过比较 V. p.感染和对照转录组文库，共鉴定出 3214 个差异表达基因（DEGs），包括 V. p.感染文昌鱼中 2219 个显著上调和 995 个显著下调的 DEGs。进一步分析了感染副溶血弧菌后 DEGs 表达变化最剧烈的前 10 个基因，以及属于先天免疫四大类的 53 个免疫相关 DEGs（IRDs）。通过基因本体论（GO）和通路富集分析，发现 DEGs 主要与免疫过程、细胞凋亡、分解代谢和代谢过程、结合和酶活性有关，而涉及细菌感染、免疫信号、免疫反应、癌症和细胞凋亡的通路则被过度代表。我们通过 qRT-PCR 检测 10 个 IRD 的表达水平验证了 RNA-seq 的结果，并调查了这些 IRD 在感染副溶血弧菌后 0、6、12、24 和 48 小时的基因表达动态变化。随后，根据 RNA-seq 结果，预测文昌鱼中存在原始 Toll 样受体（TLR）介导的抗菌免疫信号通路。这项研究为进一步研究脊椎动物免疫进化提供了抗菌免疫方面的有价值信息，并拓宽了我们对文昌鱼对抗细菌入侵的先天免疫反应的理解。
