Table_9_Full-length transcriptome sequencing and comparative transcriptome analysis of Eriocheir sinensis in response to infection by the microsporidian Hepatospora eriocheir.xls

As a new generation of high-throughput sequencing technology, PacBio Iso-Seq technology (Iso-Seq) provides a better alternative sequencing method for the acquisition of full-length unigenes. In this study, a total of 22.27 gigabyte (Gb) subread bases and 128,614 non-redundant unigenes (mean length: 2,324 bp) were obtained from six main tissues of Eriocheir sinensis including the heart, nerve, intestine, muscle, gills and hepatopancreas. In addition, 74,732 unigenes were mapped to at least one of the following databases: Non-Redundant Protein Sequence Database (NR), Gene Ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG), KEGG Orthology (KO) and Protein family (Pfam). In addition, 6696 transcription factors (TFs), 28,458 long non-coding RNAs (lncRNAs) and 94,230 mRNA-miRNA pairs were identified. Hepatospora eriocheir is the primary pathogen of E. sinensis and can cause hepatopancreatic necrosis disease (HPND); the intestine is the main target tissue. Here, we attempted to identify the key genes related to H. eriocheir infection in the intestines of E. sinensis. By combining Iso-Seq and Illumina RNA-seq analysis, we identified a total of 12,708 differentially expressed unigenes (DEUs; 6,696 upregulated and 6,012 downregulated) in the crab intestine following infection with H. eriocheir. Based on the biological analysis of these DEUs, several key processes were identified, including energy metabolism-related pathways, cell apoptosis and innate immune-related pathways. Twelve selected genes from these DEUs were subsequently verified by quantitative real-time PCR (qRT-PCR) analysis. Our findings enhance our understanding of the E. sinensis transcriptome and the specific association between E. sinensis and H. eriocheir infection.

作为新一代高通量测序技术，PacBio Iso-Seq测序技术（Iso-Seq）为全长单基因簇的获取提供了更具优势的测序方案。本研究从中华绒螯蟹（Eriocheir sinensis）的六大主要组织——心脏、神经组织、肠道、肌肉、鳃及肝胰腺中，共计获得22.27吉字节（Gb）的子读段碱基数据与128614个非冗余单基因簇（平均长度：2324 bp）。此外，共有74732个单基因簇可比对至以下至少一个数据库：非冗余蛋白质序列数据库（NR）、基因本体论（GO）、京都基因与基因组百科全书（KEGG）、KEGG同源基因（KO）及蛋白质家族数据库（Pfam）。本研究还鉴定得到6696个转录因子（TFs）、28458个长链非编码RNA（lncRNAs）以及94230对mRNA-miRNA调控对。中华绒螯蟹肝孢虫（Hepatospora eriocheir）是中华绒螯蟹的主要致病菌，可引发肝胰腺坏死病（HPND），肠道为其主要侵染靶组织。本研究旨在筛选中华绒螯蟹肠道中与肝孢虫侵染相关的关键基因。通过联合Iso-Seq与Illumina RNA测序（RNA-seq）分析，我们在被肝孢虫侵染的中华绒螯蟹肠道中共鉴定得到12708个差异表达单基因簇（DEUs），其中6696个上调、6012个下调。通过对这些DEUs的生物学功能富集分析，我们筛选得到多个关键调控通路，涵盖能量代谢相关通路、细胞凋亡通路以及先天免疫相关通路。随后，我们从这些DEUs中选取12个基因，通过实时荧光定量PCR（qRT-PCR）完成了实验验证。本研究结果深化了我们对中华绒螯蟹转录组以及中华绒螯蟹与肝孢虫侵染间特异性互作关系的认知。