合浦珠母贝外套膜细胞engrailed、BMP2和Smad3的mRNA表达水平的相关性

软体动物engrailed蛋白和骨形成相关蛋白对胚胎贝壳区域边界形成可能具有重要作用，engrailed还被推测为调节基质蛋白在外套膜组织区域化表达的重要调控因子．因此，弄清调控engrailed在软体动物中特征表达的分子机制有着重要的研究意义．但是，由于贝类基因组测序尚不完整，目前也没有建立获得贝类细胞系，以致于许多预测可能参与调控的基因需要通过克隆来鉴定，而且经典的研究细胞信号通路的方法也很难得到应用．目前，在中国南海广泛养殖的合浦珠母贝中，已获知其BMP2和Smad3的cDNA全长，以该贝的基因组为模板，PCR扩增获得了一段engrailed编码区片段．经软件分析，该片段含有EH4结构域，且与其他物种engrailed蛋白具有很高的同源性．研究的贝中，特别是外套膜组织中，engrailed、BMP2和Smad3三者表达之间的相关性，将有助于我们理解贝壳形成的分子机制．贝壳缺刻后半定量PCR试验结果表明，三者均参与贝壳修复，且在贝壳缺刻后的修复过程中，engrailed和Smad3的mRNA表达变化规律非常相似，提示它们之间可能存在相互影响的联系．用地塞米松(DXM)和过氧化氢(H₂O₂)分别处理原代培养的贝外套膜组织迁出细胞，实时相对定量PCR检测engrailed、BMP2和Smad3的mRNA表达水平，统计分析结果表明，三者具有显著的相关性．上述所有结果为进一步研究贝类生物矿化的发育和信号转导机制提供了新的思路和基础．

Correlations Among mRNA Expression Levels of Engrailed, BMP2 and Smad3 in Mantle Cells of Pearl Oyster Pinctada fucata

Molluscan engrailed proteins and bone morphogenetic proteins have attracted lots of interests in their potential important roles during embryonic shell compartment boundary formation. Engrailed is also proposed to be one of the important regulators of matrix proteins regional expressions in oyster mantle tissue, therefore determination of the regulation mechanisms on the particular expression patterns of engrailed in mollusks would be of great scientific value. However the study has been bogged down because the whole genomes of the oysters have not been sequenced completely and no oyster cell lines are available at present so that many genes of the regulators and mediators need to be cloned and classic signal pathways assays are hard to be applied. In previous study, the bone morphogenetic protein 2 (BMP2) and the oyster mothers against decapentaplegic 3 homolog (Smad3) have been identified from the pearl oyster Pinctada fucata. It is interesting to investigate the relationships among the oyster engrailed, BMP2 and Smad3 expressions in the oyster. Thereby a partial fragment of an engrailed homolog has been amplified from the genome template of the pearl oyster Pinctada fucata. Alignments with the deduced amino acids sequence show that the DNA-binding homeobox (EH4) domain of the oyster engrailed shares extremely high similarities with other engrailed proteins. Shell notching experiment and semi quantitative polymerase chain reaction results suggest that the change pattern of the oyster engrailed mRNA level is similar to that of the oyster mothers against decapentaplegic 3 homolog (Smad3) in shell repair process. Then the Smad3, the engrailed and the BMP2 mRNA expression levels in primary cultured oyster mantle tissue cells that are treated with dexamethasone (DXM) and hydrogen peroxide (H₂O₂) respectively have been examined. Real time quantitative polymerase chain reaction results show that the engrailed, the Smad3 and the BMP2 mRNA levels in the oyster mantle cells exhibit significant correlations with each other. All the results provide important clues and basis for further study on developmental and signal transduction mechanisms on oyster biomineralisation.