Brown M, Davies I M, Moffat C F, Craft J A
Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow, Scotland, UK.
Mar Environ Res. 2006 Jul;62 Suppl:S128-35. doi: 10.1016/j.marenvres.2006.04.057. Epub 2006 Apr 18.
The lack of genomic resources for aquatic invertebrates restricts their use as sentinel species in coastal environments. It is known that where genomic data are not available, suppression subtractive hybridisation (SSH) can generate cDNA libraries representative of pollutant-responsive gene transcription in aquatic vertebrates. To assess whether the approach was equally suited to aquatic invertebrates, altered gene expression in digestive gland of the mussel, Mytilus edulis, in response to exposure to benzo[a]pyrene (BaP) (1 mg/l) was investigated with SSH and a nylon macroarray. Screening of the subtracted libraries showed 112/250 up-regulated and 25/55 down-regulated clones were positive for differential expression and characterisation of these identified 87 with unique sequence suitable for array on a nylon membrane. The transcripts isolated were from a diverse range of genes involved in general stress, oxidative stress, cell adhesion, transcriptional and translational regulation, transport mechanisms, energy metabolism, cell metabolism, lipid metabolism, protein turnover and activation, lysosomal activity and 22 cryptic clones. Subsequent use of the clones in macroarray format to analyse expression of BaP-responsive genes (0 vs 4 day exposed) showed 0-100-fold increased levels of the forward-subtracted probes and between 0 and 0.1-fold down-regulation of the reverse-subtracted probes. Only 15% of the clones showed less than 2-fold change in expression. The gene ontology of the transcripts isolated demonstrates that BaP elicits a multitude of responses with a major feature being disruption of cellular redox status. The results indicate that the use of SSH and a macroarray is a robust method to discover novel pollutant-responsive genes in aquatic invertebrates.
水生无脊椎动物缺乏基因组资源，这限制了它们在沿海环境中作为指示物种的应用。众所周知，在没有基因组数据的情况下，抑制性消减杂交（SSH）可以生成代表水生脊椎动物中污染物响应基因转录的cDNA文库。为了评估该方法是否同样适用于水生无脊椎动物，利用SSH和尼龙宏阵列研究了紫贻贝（Mytilus edulis）消化腺中基因表达的变化，该变化是对暴露于苯并[a]芘（BaP）（1 mg/l）的响应。对消减文库的筛选显示，112/250个上调克隆和25/55个下调克隆在差异表达方面呈阳性，对这些克隆的鉴定确定了87个具有适合尼龙膜阵列的独特序列。分离出的转录本来自多种基因，包括一般应激、氧化应激、细胞粘附、转录和翻译调控、转运机制、能量代谢、细胞代谢、脂质代谢、蛋白质周转与激活、溶酶体活性以及22个未知克隆。随后以宏阵列形式使用这些克隆来分析BaP响应基因的表达（暴露0天与4天），结果显示正向消减探针的水平增加了0 - 100倍，反向消减探针下调了0至0.1倍。只有15%的克隆表达变化小于2倍。所分离转录本的基因本体分析表明，BaP引发了多种反应，主要特征是细胞氧化还原状态的破坏。结果表明，使用SSH和宏阵列是在水生无脊椎动物中发现新型污染物响应基因的一种可靠方法。
