Transcriptome Profiling Reveals Higher Vertebrate Orthologous of Intra-Cytoplasmic Pattern Recognition Receptors in Grey Bamboo Shark

From an immunologist perspective, sharks are an important group of jawed cartilaginous fishes and survey of the public database revealed a great gap in availability of large-scale sequence data for the group of Chondrichthyans the elasmobranchs. In an attempt to bridge this deficit we generated the transcriptome from the spleen and kidney tissues (a total of 1,606,172 transcripts) of the shark, Chiloscyllium griseum using the Illumina HiSeq2000 platform. With a cut off of > = 300 bp and an expression value of >1RPKM we used 43,385 transcripts for BLASTX analysis which revealed 17,548 transcripts matching to the NCBI nr database with an E-value of < = 10−5 and similarity score of 40%. The longest transcript was 16,974 bases with matched to HECT domain containing E3 ubiqutin protein ligase. MEGAN4 annotation pipeline revealed immune and signalling pathways including cell adhesion molecules, cytokine-cytokine receptor interaction, T-cell receptor signalling pathway and chemokine signaling pathway to be highly expressed in spleen, while different metabolism pathways such as amino acid metabolism, carbohydrate metabolism, lipid metabolism and xenobiotic biodegradation were highly expressed in kidney. Few of the candidate genes were selected to analyze their expression levels in various tissues by real-time PCR and also localization of a receptor by in-situ PCR to validate the prediction. We also predicted the domains structures of some of the identified pattern recognition receptors, their phylogenetic relationship with lower and higher vertebrates and the complete downstream signaling mediators of classical dsRNA signaling pathway. The generated transcriptome will be a valuable resource to further genetic and genomic research in elasmobranchs.

从免疫学家的视角来看，鲨鱼是一类重要的有颌软骨鱼类。对公共数据库的调研显示，软骨鱼纲（Chondrichthyes）下的板鳃亚纲（Elasmobranchii）类群的大规模序列数据存在显著的可获得性缺口。为弥补这一不足，本研究依托Illumina HiSeq2000测序平台，对灰斑竹鲨（Chiloscyllium griseum）的脾脏与肾脏组织开展转录组（transcriptome）测序，共获得1,606,172条转录本。
我们以≥300 bp作为序列长度筛选阈值、≥1RPKM作为表达量阈值，最终筛选得到43,385条转录本用于BLASTX分析。分析结果显示，其中17,548条转录本可匹配至NCBI非冗余（nr）数据库，匹配的期望值（E-value）≤10⁻⁵，相似性得分≥40%。最长的转录本长度为16,974 bp，其匹配到含HECT结构域的E3泛素蛋白连接酶。
通过MEGAN4注释流程分析发现，脾脏中高表达免疫与信号通路相关基因，涵盖细胞黏附分子（cell adhesion molecules）、细胞因子-细胞因子受体相互作用通路、T细胞受体信号通路以及趋化因子信号通路；而肾脏中则高表达多种代谢通路相关基因，包括氨基酸代谢、糖代谢、脂代谢以及外源性物质生物降解通路。
本研究筛选了部分候选基因，通过实时荧光定量PCR（real-time PCR）分析其在不同组织中的表达水平，并借助原位PCR（in-situ PCR）定位某一受体以验证转录组预测结果。此外，我们还对部分已鉴定的模式识别受体（pattern recognition receptors）的结构域进行预测，分析其与低等及高等脊椎动物的系统发育关系，并解析了经典双链RNA（dsRNA）信号通路的完整下游信号介质。
本研究构建的转录组将为板鳃亚纲的后续遗传与基因组学研究提供宝贵的资源。