Genomic-Bioinformatic Analysis of Transcripts Enriched in the Third-Stage Larva of the Parasitic Nematode Ascaris suum

Differential transcription in Ascaris suum was investigated using a genomic-bioinformatic approach. A cDNA archive enriched for molecules in the infective third-stage larva (L3) of A. suum was constructed by suppressive-subtractive hybridization (SSH), and a subset of cDNAs from 3075 clones subjected to microarray analysis using cDNA probes derived from RNA from different developmental stages of A. suum. The cDNAs (n = 498) shown by microarray analysis to be enriched in the L3 were sequenced and subjected to bioinformatic analyses using a semi-automated pipeline (ESTExplorer). Using gene ontology (GO), 235 of these molecules were assigned to ��biological process�� (n = 68), ��cellular component�� (n = 50), or ��molecular function�� (n = 117). Of the 91 clusters assembled, 56 molecules (61.5%) had homologues/orthologues in the free-living nematodes Caenorhabditis elegans and C. briggsae and/or other organisms, whereas 35 (38.5%) had no significant similarity to any sequences available in current gene databases. Transcripts encoding protein kinases, protein phosphatases (and their precursors), and enolases were abundantly represented in the L3 of A. suum, as were molecules involved in cellular processes, such as ubiquitination and proteasome function, gene transcription, protein�Cprotein interactions, and function. In silico analyses inferred the C. elegans orthologues/homologues (n = 50) to be involved in apoptosis and insulin signaling (2%), ATP synthesis (2%), carbon metabolism (6%), fatty acid biosynthesis (2%), gap junction (2%), glucose metabolism (6%), or porphyrin metabolism (2%), although 34 (68%) of them could not be mapped to a specific metabolic pathway. Small numbers of these 50 molecules were predicted to be secreted (10%), anchored (2%), and/or transmembrane (12%) proteins. Functionally, 17 (34%) of them were predicted to be associated with (non-wild-type) RNAi phenotypes in C. elegans, the majority being embryonic lethality (Emb) (13 types; 58.8%), larval arrest (Lva) (23.5%) and larval lethality (Lvl) (47%). A genetic interaction network was predicted for these 17 C. elegans orthologues, revealing highly significant interactions for nine molecules associated with embryonic and larval development (66.9%), information storage and processing (5.1%), cellular processing and signaling (15.2%), metabolism (6.1%), and unknown function (6.7%). The potential roles of these molecules in development are discussed in relation to the known roles of their homologues/orthologues in C. elegans and some other nematodes. The results of the present study provide a basis for future functional genomic studies to elucidate molecular aspects governing larval developmental processes in A. suum and/or the transition to parasitism.

本研究借助基因组生物信息学手段，对猪蛔虫（*Ascaris suum*）的差异转录水平展开研究。通过抑制性消减杂交（SSH）技术，构建了富集猪蛔虫感染性第三期幼虫（L3）相关分子的互补DNA（cDNA）文库；随后从3075个克隆中选取部分cDNA，利用源自猪蛔虫不同发育阶段总RNA的cDNA探针开展基因芯片分析。经基因芯片验证在L3中富集的498条cDNA（n=498）被测序，并通过半自动化分析流程ESTExplorer进行生物信息学注释。借助基因本体论（GO），其中235个分子被划分为生物过程（n=68）、细胞组分（n=50）及分子功能（n=117）三大类别。在组装得到的91个基因簇中，56个分子（占比61.5%）在自由生活线虫秀丽隐杆线虫（*Caenorhabditis elegans*）、布氏线虫（*C. briggsae*）及其他物种中存在同源/直系同源蛋白，而剩余35个分子（占比38.5%）与当前基因数据库收录的所有序列均无显著相似性。猪蛔虫L3中高丰度表达的转录本涵盖蛋白激酶、蛋白磷酸酶（及其前体）与烯醇化酶，同时还包括参与泛素化、蛋白酶体功能、基因转录、蛋白质-蛋白质相互作用等细胞过程的相关分子。计算机模拟分析（in silico）预测，50个秀丽隐杆线虫的同源/直系同源分子分别参与细胞凋亡与胰岛素信号通路（2%）、ATP合成（2%）、碳代谢（6%）、脂肪酸生物合成（2%）、间隙连接（2%）、葡萄糖代谢（6%）及卟啉代谢（2%）；但其中34个分子（占比68%）无法被定位到特定代谢通路中。该50个分子中仅少数被预测为分泌型蛋白（10%）、锚定蛋白（2%）和/或跨膜蛋白（12%）。功能层面，其中17个分子（占比34%）被预测与秀丽隐杆线虫的（非野生型）RNA干扰（RNAi）表型相关，多数表现为胚胎致死（Emb，共13种类型，占比58.8%）、幼虫停滞（Lva，占比23.5%）与幼虫致死（Lvl，占比47%）。研究人员针对这17个秀丽隐杆线虫直系同源分子构建了遗传互作网络，结果显示其中9个与胚胎发育及幼虫发育（66.9%）、信息存储与加工（5.1%）、细胞加工与信号转导（15.2%）、代谢（6.1%）及功能未知（6.7%）相关的分子存在极显著互作关系。本研究结合其同源/直系同源分子在秀丽隐杆线虫及其他部分线虫中的已知功能，探讨了这些分子在猪蛔虫发育过程中的潜在作用。本研究结果为后续功能基因组学研究奠定了基础，以阐明调控猪蛔虫幼虫发育过程及/或向寄生状态转变的分子机制。