Huang Xuena, Gao Yangchun, Jiang Bei, Zhou Zunchun, Zhan Aibin
Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, China.
Liaoning Key Lab of Marine Fishery Molecular Biology, Liaoning Ocean and Fishery Science Research Institute, Dalian, Liaoning, China.
Gene. 2016 Jan 15;576(1 Pt 1):79-87. doi: 10.1016/j.gene.2015.09.066. Epub 2015 Sep 30.
As invasive species have successfully colonized a wide range of dramatically different local environments, they offer a good opportunity to study interactions between species and rapidly changing environments. Gene expression represents one of the primary and crucial mechanisms for rapid adaptation to local environments. Here, we aim to select reference genes for quantitative gene expression analysis based on quantitative Real-Time PCR (qRT-PCR) for a model invasive ascidian, Ciona savignyi. We analyzed the stability of ten candidate reference genes in three tissues (siphon, pharynx and intestine) under two key environmental stresses (temperature and salinity) in the marine realm based on three programs (geNorm, NormFinder and delta Ct method). Our results demonstrated only minor difference for stability rankings among the three methods. The use of different single reference gene might influence the data interpretation, while multiple reference genes could minimize possible errors. Therefore, reference gene combinations were recommended for different tissues - the optimal reference gene combination for siphon was RPS15 and RPL17 under temperature stress, and RPL17, UBQ and TubA under salinity treatment; for pharynx, TubB, TubA and RPL17 were the most stable genes under temperature stress, while TubB, TubA and UBQ were the best under salinity stress; for intestine, UBQ, RPS15 and RPL17 were the most reliable reference genes under both treatments. Our results suggest that the necessity of selection and test of reference genes for different tissues under varying environmental stresses. The results obtained here are expected to reveal mechanisms of gene expression-mediated invasion success using C. savignyi as a model species.
由于入侵物种已成功在各种截然不同的当地环境中定殖，它们为研究物种间相互作用以及快速变化的环境提供了一个很好的机会。基因表达是快速适应当地环境的主要关键机制之一。在此，我们旨在基于定量实时聚合酶链反应（qRT-PCR）为模式入侵海鞘萨氏海鞘（Ciona savignyi）选择用于定量基因表达分析的内参基因。我们基于geNorm、NormFinder和ΔCt法这三个程序，分析了10个候选内参基因在海洋领域的两种关键环境胁迫（温度和盐度）下在三个组织（虹吸管、咽和肠）中的稳定性。我们的结果表明这三种方法在稳定性排名上仅有微小差异。使用不同的单个内参基因可能会影响数据解读，而多个内参基因可将可能的误差降至最低。因此，建议针对不同组织使用内参基因组合——在温度胁迫下，虹吸管的最佳内参基因组合是RPS15和RPL17，在盐度处理下是RPL17、UBQ和TubA；对于咽，在温度胁迫下最稳定的基因是TubB、TubA和RPL17，而在盐度胁迫下最佳的是TubB、TubA和UBQ；对于肠，在两种处理下最可靠的内参基因是UBQ、RPS15和RPL17。我们的结果表明在不同环境胁迫下针对不同组织选择和测试内参基因的必要性。预期此处获得的结果将揭示以萨氏海鞘作为模式物种的基因表达介导的入侵成功机制。
