Laboratoire des Sciences de l'Environnement Marin (LEMAR), UMR CNRS 6539, IUEM, Technopole Brest-Iroise, 29280 Plouzané, France.
Dev Comp Immunol. 2012 Oct;38(2):368-76. doi: 10.1016/j.dci.2012.03.003. Epub 2012 Mar 23.
The Manila clam, Ruditapes philippinarum, is an economically-important, commercial shellfish; harvests are diminished in some European waters by a pathogenic bacterium, Vibrio tapetis, that causes Brown Ring disease. To identify molecular characteristics associated with susceptibility or resistance to Brown Ring disease, Suppression Subtractive Hybridization (SSH) analyzes were performed to construct cDNA libraries enriched in up- or down-regulated transcripts from clam immune cells, hemocytes, after a 3-h in vitro challenge with cultured V. tapetis. Nine hundred and ninety eight sequences from the two libraries were sequenced, and an in silico analysis identified 235 unique genes. BLAST and "Gene ontology" classification analyzes revealed that 60.4% of the Expressed Sequence Tags (ESTs) have high similarities with genes involved in various physiological functions, such as immunity, apoptosis and cytoskeleton organization; whereas, 39.6% remain unidentified. From the 235 unique genes, we selected 22 candidates based upon physiological function and redundancy in the libraries. Then, Real-Time PCR analysis identified 3 genes related to cytoskeleton organization showing significant variation in expression attributable to V. tapetis exposure. Disruption in regulation of these genes is consistent with the etiologic agent of Brown Ring disease in Manila clams.
菲律宾蛤仔（Ruditapes philippinarum）是一种具有重要经济价值的商业贝类；在一些欧洲水域，一种致病性细菌——鳗弧菌（Vibrio tapetis）导致的“棕环病”降低了其产量。为了鉴定与棕环病易感性或抗性相关的分子特征，采用抑制性消减杂交（SSH）分析方法，从经体外培养的鳗弧菌（V. tapetis）攻击 3 小时后的菲律宾蛤仔免疫细胞（血细胞）中构建了差异表达的 cDNA 文库。对这两个文库的 998 个序列进行了测序，并通过计算机分析鉴定了 235 个独特的基因。BLAST 和“基因本体论”分类分析表明，60.4%的表达序列标签（ESTs）与参与各种生理功能（如免疫、细胞凋亡和细胞骨架组织）的基因具有高度相似性；而 39.6%的基因仍然未知。从 235 个独特基因中，我们根据生理功能和文库中的冗余性选择了 22 个候选基因。然后，实时 PCR 分析鉴定了 3 个与细胞骨架组织相关的基因，它们的表达在鳗弧菌暴露后发生了显著变化。这些基因的调控中断与菲律宾蛤仔棕环病的病原体一致。
