Lymnaea project - elucidation of mechanisms of molluscan shell formation

为了对肺螺lynaea的发育遗传学有一个基本的认识，我们进行了不同发育阶段的组织学观察。我们还准备了cDNA文库，用于从原鳖形成前和形成后的胚胎，以及成年胚套、肌肉和中肠腺组织中提取mRNA。利用SDS-PAGE对壳基质蛋白进行分析，分离出三个突出的条带。以消化腺为参照，对地幔特异性基因进行减法克隆，分离出编码p型atp酶成员的基因片段，以及含有锌指结构域的转录因子片段。为了对这些基因产物的功能进行反向遗传研究，目前正在开发用于该物种的显微注射和RNA干扰的基本技术。关于海洋双壳类的壳基质蛋白，我们从珍珠贝Pinctada fucata中分离出一种酸性极强的蛋白，命名为Aspein，含有60%的Asp残基。Aspein的转录本被证明是特定于壳的方解石棱柱层部分，这表明Aspein参与了棱柱结构的形成和/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.