Molecular studies on the formation of calcified hard tissues in marine crustaceans

本研究旨在鉴定海洋甲壳类日本对虾（Penaeus japonicus）外骨骼形成（特别关注钙化过程）的编码蛋白mrna。为此，利用差异显示和Northern分析，在角质层身体部位寻找在蜕皮后阶段（外骨骼在这些阶段变得钙化）特异性或主要表达的mrna。在本研究中，鉴定了具有上述表达谱的6个mRNA物种，并分离了其中4个cDNA克隆。获得并分析了三个mRNA物种（命名为DD-1、4和9）的核苷酸序列。DD-1含有一种富含天冬酰胺的碱性蛋白质的编码序列，这种蛋白质与已知的蛋白质不相似。概念上的DD-9蛋白与构成昆虫角质层的一个小蛋白家族的序列相似。DD-4似乎编码了一个富含脯氨酸的酸性蛋白，其序列与钙结合蛋白calphotin相似。阐明DD-4蛋白在甲壳类动物外骨骼钙化过程中的作用具有重要意义。本研究从日本参表皮部分分离到了一种推测为几丁质酶（内源性几丁质酶）的cDNA。该蛋白与消化器官肝胰腺中表达的几丁质酶不同。

This study was carried out to identify mRNAs encoding proteins which regulate formation of the exoskeleton (with particular intesest in the calcification process) in the marine crustacean Penaeus japonicus. For this purpose, mRNAs that were expressed specifically or predominantly at postmolt stages (the exoskeleton becomes calcified at these stages) in cuticular body parts were sought using the differential display and Northern analyzes.In this study, six mRNA species exhibiting the above expression profile were identified, and for four out of the six, cDNA clones isolated. Nucleotide sequences were obtained and analyzed for three mRNA species (named DD-1,4, and 9). DD-1 contained a coding sequence for an asparagine-rich alkaline protein which resembled no known proteins. The conceptual DD-9 protein exhibited sequence similarities to a family of small proteins constituting insect cuticle. DD-4 seemed to encode a proline-rich acidic protein whose sequence was similar to that of the calcium binding protein calphotin. It is of great interest to elucidate the function of the DD-4 protein in the calcification of the crustacean exoskeleton.In this study, a cDNA encoding a putative chitinase (endo-type chitinolytic enzyme) was isolated from cuticular body parts of P.japonicus. This protein was distinct form the chitinases expressed in the digestive organ hepatopancreas.