Boltaña Sebastian, Castellana Barbara, Goetz Giles, Tort Lluis, Teles Mariana, Mulero Victor, Novoa Beatriz, Figueras Antonio, Goetz Frederick W, Gallardo-Escarate Cristian, Planas Josep V, Mackenzie Simon
Institute of Biotechnology and Biomedicine, University Autónoma de Barcelona, Barcelona 08193, Spain.
University of Stirling, School of Natural Sciences, Stirling FK9 4LA, Scotland, UK.
Int J Mol Sci. 2017 Feb 3;18(2):317. doi: 10.3390/ijms18020317.
This study describes the development and validation of an enriched oligonucleotide-microarray platform for (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in with an emphasis upon immunity and the immune response.
本研究描述了一种用于金头鲷的富集寡核苷酸微阵列平台的开发与验证，以提供该物种转录组学研究的平台。通过组装源自mRNA公共数据库的金头鲷序列以及来自两个广泛的靶向cDNA文库的大量表达序列标签（EST）读数，构建了一个转录组数据库，这些文库表征了受细菌和病毒攻击调控的mRNA转录本。通过分析用两种革兰氏阴性细菌病原体相关分子模式（PAMP；脂多糖（LPS）和肽聚糖（PGN））攻击后的单核细胞/巨噬细胞激活谱，进一步验证了所开发的微阵列。在大约10,000个测序的EST中，我们总共获得了6837个长度超过100 nt的EST，分别从细菌引发和病毒引发的cDNA文库中获得了3778个和3059个EST。通过基因本体论（GO）对细菌引发和病毒引发的cDNA文库中的重叠群进行功能分类表明，两个文库中排名前五的类别所占比例相同：代谢（约占重叠群总数的24%）、载体蛋白/膜转运（约15%）、效应器/调节剂和细胞通讯（约11%）、核苷、核苷酸和核酸代谢（约7.5%）以及细胞内传感器/信号转导（约5%）。使用这种富集寡核苷酸平台进行的转录组分析确定了巨噬细胞样细胞对PGN和LPS反应中的差异变化，突出了与PAMP宿主识别紧密相关的反应性基因盒。正如在其他鱼类中观察到的那样，PGN是巨噬细胞样细胞中炎症反应的强大激活剂。我们已经开发并验证了一种寡核苷酸微阵列（SAQ），该微阵列提供了一个富集的平台，用于研究金头鲷的基因表达，重点是免疫和免疫反应。
