Lymnaea project - elucidation of mechanisms of molluscan shell formation

为了对肺状蜗牛的发育遗传学有基本的了解，我们在不同发育阶段进行了组织学观察。我们还从原球囊形成前和形成后的胚胎以及成体外套膜、肌肉和中肠腺组织中提取了mRNA的cDNA文库。对壳层基质蛋白进行了SDS-PAGE分析，分离出3条显著条带。以消化腺为参照，对外套膜特异基因进行消减克隆，分离到编码P型ATPase成员的基因片段和含有锌指结构域的转录因子基因片段。为了逆转这些基因产物的遗传学功能，目前正在开发针对该物种的显微注射和RNA干扰的基本技术。针对海洋双壳类贝壳基质蛋白，我们从珠母贝中分离到一种酸性极强的蛋白，命名为Aspin，其天冬氨酸残基含量为60%。ASpein的转录本显示只在壳层的钙化棱柱层部分，这表明ASpein参与了棱柱状结构的形成和/或方解石的沉淀。

In order to obtain basic insight into developmental genetics of the pulmonate snail Lymnaea, we have conducted histological observations at various developmental stages. We have also prepared cDNA libraries for mRNA extracted from embryos of both before and after formation of the protochonch, and from adult mantle, muscle, and midgut gland tissues. Shell matrix proteins were analyzed using SDS-PAGE, and three prominent bands were separated. Subtraction cloning for the mantle specific genes using the digestive gland as a reference, resulted in isolation of a gene fragment encoding a member of P-type ATPases, as well as a fragment for a transcription factor containing a Zn-finger domain. So as to reverse genetically study functions of those gene products, basic techniques for microinjections and RNA interference for this species are now being developed. Concerning the shell matrix proteins of marine bivalves, we have isolated an extremely acidic protein, named Aspein, with 60% Asp residues, from the pearl oyster Pinctada fucata. The transcript for Aspein is shown to be specific to the calcitic prismatic layer part of the shell, suggesting that Aspein is involved in prismatic architecture formation and/or calcite precipitation.