Jiangsu Key Laboratory for Biodiversity & Biotechnology and Jiangsu Key Laboratory for Aquatic Crustacean Diseases, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, China.
Fish Shellfish Immunol. 2013 Aug;35(2):607-17. doi: 10.1016/j.fsi.2013.05.013. Epub 2013 Jun 4.
We used the Illumina/Solexa deep sequencing technology to sequence two small RNA libraries prepared from hemocytes of Procambarus clarkii under normal and infection conditions. The high-throughput sequencing approach resulted in approximately 12,801,827 and 8,410,455 raw reads corresponding to 10,949,754 and 6,648,161 high-quality mappable reads for the normal and infected hemocyte samples, respectively. Bioinformatic analyses identified 195 unique miRNAs, including 30 that are conserved in crustaceans, 48 that are novel to crayfish but are present in other arthropods (PN-type), and 117 that are completely new (PC-type). Thirty-three miRNAs displayed significant differential expressions between the two hemocyte samples (p < 0.0001). Of these, 15 (45.5%) were significantly up-regulated and 18 (54.5%) were significantly down-regulated upon challenge with Spiroplasma eriocheiris. Integrating comparative genomic and bibliomic analysis, of the 33 significant miRNAs identified, 19 were conserved and immune-related in P. clarkii and Eriocheir sinensis infected with S. eriocheiris infection; 24 were conserved and immune-related in P. clarkii and Marsupenaeus japonicus immune response to S. eriocheiris or white spot syndrome virus (WSSV) infection. Function annotation of target genes revealed a broad range of biological processes and signal transduction pathways that regulated by crayfish miRNAs. Thereinto, pcl-miR-34, pcl-miR-7, PN-pcl-let-7, pcl-miR-1, and pcl-miR-2b are highly conserved in vertebrates and invertebrates and function in the similar pathways. To our knowledge, this is the first report of comprehensive identification of P. clarkii miRNAs and of expression analysis of P. clarkii miRNAs after exposure to S. eriocheiris in crayfish, and many miRNAs were differentially regulated under normal and infection conditions. Our results should help develop new control strategies for efficient immune protection against S. eriocheiris infections in crustaceans.
我们使用 Illumina/Solexa 深度测序技术对正常和感染条件下克氏原螯虾血细胞中两个小 RNA 文库进行测序。高通量测序方法分别得到约 12801827 和 8410455 个原始读数，对应正常和感染血细胞样本的 10949754 和 6648161 个高质量可映射读数。生物信息学分析鉴定出 195 个独特的 miRNA，其中 30 个在甲壳动物中保守，48 个在螯虾中是新的，但在其他节肢动物中存在（PN 型），117 个是完全新的（PC 型）。在两个血细胞样本之间，有 33 个 miRNA 的表达差异显著（p < 0.0001）。其中，15 个（45.5%）显著上调，18 个（54.5%）显著下调。在感染螺原体后，螯虾和中华绒螯蟹的 33 个显著 miRNA 中有 19 个保守且与免疫相关；在螯虾和日本沼虾对螺原体或白斑综合征病毒（WSSV）感染的免疫反应中，有 24 个保守且与免疫相关。靶基因的功能注释显示，调控螯虾 miRNA 的生物过程和信号转导途径广泛。其中，pcl-miR-34、pcl-miR-7、PN-pcl-let-7、pcl-miR-1 和 pcl-miR-2b 在脊椎动物和无脊椎动物中高度保守，在相似的途径中发挥作用。据我们所知，这是首次全面鉴定克氏原螯虾 miRNA 并分析克氏原螯虾 miRNA 在感染螺原体后的表达情况，许多 miRNA 在正常和感染条件下差异表达。我们的研究结果有助于开发针对甲壳动物螺原体感染的有效免疫保护的新控制策略。
