Institute of Parasitology, BFS, Justus-Liebig-University, Giessen, Germany.
Institute of Parasitology, BFS, Justus-Liebig-University, Giessen, Germany; Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom.
Int J Parasitol. 2019 Jul;49(8):615-624. doi: 10.1016/j.ijpara.2019.01.006. Epub 2019 May 25.
Facilitated by the Schistosoma mansoni genome project, multiple transcriptomic studies were performed over the last decade to elucidate gene expression patterns among different developmental stages of the complex schistosome life cycle. While these analyses enable the identification of candidate genes with key functions in schistosome biology, a diverse molecular tool set is needed that allows comprehensive functional characterization at the single gene level. This includes the availability of reliable reference genes to confirm changes in the transcription of genes of interest over different biological samples and experimental conditions. In particular, the investigation of one key aspect of schistosome biology, the pairing-dependent gene expression in females and males, requires knowledge on reference genes that are expressed independently of both pairing and of in vitro culture effects. Therefore, the present study focused on the identification of quantitative reverse transcription (qRT)-PCR reference genes suitable for the investigation of pairing-dependent gene expression in the S. mansoni male. The "pipeline" we present here is based on qRT-PCR analyses of high biological replication combined with three different statistical analysis tools, BestKeeper, geNorm, and NormFinder. Our approach resulted in a statistically robust ranking of 15 selected reference genes with respect to their transcription stability between pairing-unexperienced and -experienced males. We further tested the top seven candidate genes for their transcription stability during invitro culture of adult S. mansoni. Of these, the two most suitable reference genes were used to investigate the influence of the pairing contact on the transcription of genes of interest, comprising a tyrosine decarboxylase gene Smtdc1, an ebony ortholog Smebony, and the follistatin ortholog Smfst in S. mansoni males. Performing pairing, separation and re-pairing experiments with adult S. mansoni in vitro, our results indicate for the first time that pairing can act as a molecular on/off-switch of specific genes to strictly control their expression in schistosome males.
曼氏血吸虫基因组项目的推动下，过去十年进行了多项转录组研究，以阐明复杂血吸虫生活史不同发育阶段的基因表达模式。虽然这些分析使能够鉴定在血吸虫生物学中具有关键功能的候选基因，但需要多样化的分子工具集，以便在单个基因水平上进行全面的功能表征。这包括提供可靠的参考基因，以确认不同生物样本和实验条件下感兴趣基因的转录变化。特别是，调查血吸虫生物学的一个关键方面，即雌雄虫配对依赖性基因表达，需要了解独立于配对和体外培养效应表达的参考基因。因此，本研究集中于鉴定适合曼氏血吸虫雄虫配对依赖性基因表达研究的定量逆转录（qRT）-PCR 参考基因。我们在这里提出的“流水线”基于高生物学复制的 qRT-PCR 分析，并结合了三种不同的统计分析工具，BestKeeper、geNorm 和 NormFinder。我们的方法对 15 个选定的参考基因进行了统计上稳健的排序，这些基因在配对未经验证和经验证的雄虫之间的转录稳定性。我们进一步测试了前 7 个候选基因在曼氏血吸虫成虫体外培养过程中的转录稳定性。其中，使用两个最适合的参考基因来研究配对接触对感兴趣基因转录的影响，包括一个酪氨酸脱羧酶基因 Smtdc1、一个 ebony 同源物 Smebony 和一个 follistatin 同源物 Smfst 在曼氏血吸虫雄虫中的转录。通过在体外进行成年曼氏血吸虫的配对、分离和重新配对实验，我们的结果首次表明，配对可以作为特定基因的分子开/关开关，严格控制它们在血吸虫雄虫中的表达。
