CAS Key Laboratory of Tropical Marine Bio-resources and Ecology (LMB), Guangdong Provincial Key Laboratory of Applied Marine Biology (LAMB), South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou, 510301, PR China.
University of Chinese Academy of Sciences, Beijing, 100049, PR China.
BMC Genomics. 2020 Apr 16;21(1):306. doi: 10.1186/s12864-020-6698-6.
The sea cucumber Holothuria leucospilota belongs to echinoderm, which is evolutionally the most primitive group of deuterostomes. Sea cucumber has a cavity between its digestive tract and the body wall that is filled with fluid and suspended coelomic cells similar to blood cells. The humoral immune response of the sea cucumber is based on the secretion of various immune factors from coelomocytes into the coelomic cavity. The aim of this study is to lay out a foundation for the immune mechanisms in echinoderms and their origins in chordates by using RNA-seq.
Sea cucumber primary coelomocytes were isolated from healthy H. leucospilota and incubated with lipopolysaccharide (LPS, 10 μg/ml), polyinosinic-polycytidylic acid [Poly (I:C), 10 μg/ml] and heat-inactived Vibrio harveyi (10 cell/ml) for 24 h, respectively. After high-throughput mRNA sequencing on an Illumina HiSeq2500, a de novo transcriptome was assembled and the Unigenes were annotated. Thirteen differentially expressed genes (DEGs) were selected randomly from our data and subsequently verified by using RT-qPCR. The results of RT-qPCR were consistent with those of the RNA-seq (R = 0.61). The top 10 significantly enriched signaling pathways and immune-related pathways of the common and unique DEGs were screened from the transcriptome data. Twenty-one cytokine candidate DEGs were identified, which belong to 4 cytokine families, namely, BCL/CLL, EPRF1, IL-17 and TSP/TPO. Gene expression in response to LPS dose-increased treatment (0, 10, 20 and 50 μg/ml) showed that IL-17 family cytokines were significantly upregulated after 10 μg/ml LPS challenge for 24 h.
A de novo transcriptome was sequenced and assembled to generate the gene expression profiling across the sea cucumber coelomocytes treated with LPS, Poly (I:C) and V. harveyi. The cytokine genes identified in DEGs could be classified into 4 cytokine families, in which the expression of IL-17 family cytokines was most significantly induced after 10 μg/ml LPS challenge for 24 h. Our findings have laid the foundation not only for the research of molecular mechanisms related to the immune response in echinoderms but also for their origins in chordates, particularly in higher vertebrates.
海参属于棘皮动物，在进化上是最原始的后口动物群。海参的消化道和体壁之间有一个充满液体和悬浮体腔细胞的腔，类似于血细胞。海参的体液免疫反应基于从体腔细胞分泌到体腔中的各种免疫因子。本研究旨在通过 RNA-seq 为棘皮动物的免疫机制及其在脊索动物中的起源奠定基础。
从健康的糙海参中分离出海参原代体腔细胞，分别用脂多糖（LPS，10μg/ml）、聚肌苷酸-聚胞苷酸[Poly（I:C），10μg/ml]和热灭活哈维弧菌（10 个细胞/ml）孵育 24h。在 Illumina HiSeq2500 上进行高通量 mRNA 测序后，组装了一个从头转录组，并对 Unigenes 进行了注释。从我们的数据中随机选择了 13 个差异表达基因（DEGs），并通过 RT-qPCR 进行了后续验证。RT-qPCR 的结果与 RNA-seq 的结果一致（R=0.61）。从转录组数据中筛选出共同和独特 DEGs 的前 10 个显著富集的信号通路和免疫相关通路。鉴定出 21 个细胞因子候选 DEGs，它们属于 4 个细胞因子家族，即 BCL/CLL、EPRF1、IL-17 和 TSP/TPO。对 LPS 剂量递增处理（0、10、20 和 50μg/ml）的基因表达进行分析表明，在 LPS 刺激 24h 后，IL-17 家族细胞因子显著上调。
对 LPS、Poly（I:C）和 V. harveyi 处理的海参体腔细胞进行了从头转录组测序和组装，生成了基因表达谱。DEGs 中的细胞因子基因可分为 4 个细胞因子家族，其中 10μg/ml LPS 刺激 24h 后，IL-17 家族细胞因子的表达最为显著。我们的研究结果不仅为棘皮动物免疫反应相关的分子机制研究奠定了基础，也为它们在脊索动物中的起源，特别是在高等脊椎动物中的起源奠定了基础。
