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

本研究旨在鉴定海洋甲壳动物日本对虾外骨骼(尤其是钙化过程)形成调控蛋白的编码蛋白。为此，利用差异显示和Northern分析，寻找在蜕皮后阶段(外骨骼在这些阶段变得钙化)角质层身体部分特异或主要表达的mRNAs。在本研究中，鉴定了6个具有上述表达谱的mRNA物种，并从6个物种中分离到4个cDNAs克隆。获得了3个mRNA物种(命名为DD-1、4和9)的核苷酸序列并进行了分析。DD-1含有一个富含天冬酰胺的碱性蛋白的编码序列，与已知的蛋白质相似。概念性的DD-9蛋白与组成昆虫角质层的一组小蛋白具有序列相似性。DD-4似乎编码一种富含脯氨酸的酸性蛋白，其序列与钙结合蛋白calPhotin相似。研究DD-4蛋白在甲壳类外骨骼钙化过程中的作用是非常有意义的。本研究从日本对虾的角质层中分离到一个编码几丁质酶(几丁质酶)的基因。该蛋白不同于消化器官肝胰腺中表达的几丁质酶。

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.