中华绒螯蟹围食膜结构观察及其主要蛋白质种类的鉴定

为揭示中华绒螯蟹围食膜的分泌、形态和功能,实验采用组织切片技术和电镜技术观察了中华绒螯蟹消化道及围食膜的基本形态,采用几丁质酶溶解法和SDS-PAGE电泳法研究了围食膜的基本组成成分,采用液相色谱电离串联质谱(LC-ESI-MS/MS)分析了围食膜蛋白的种类。显微切片结果显示,中华绒螯蟹整个消化道表面都有围食膜存在,除中肠外围食膜区室化作用明显;围食膜与上皮细胞分离时,柱状细胞变成不规则圆形,核消失。电镜结果显示,围食膜形成时上皮细胞内含有大量线粒体和分泌泡;中肠上皮细胞表面密生微绒毛。围食膜能被几丁质酶溶解,围食膜蛋白质SDS-PAGE图谱显示有7条比较清晰的电泳条带,蛋白质分子量主要分布在40 ku以上。LC-ESI-MS/MS分析结果发现,中华绒螯蟹围食膜蛋白主要包括钠、钾-ATP酶、ATP合成酶、肌动蛋白、微管蛋白、硫氧还原蛋白、加氧酶等。研究表明,中华绒螯蟹的围食膜由上皮细胞分泌形成,可能参与免疫、渗透调节和营养物质转运等生理过程。

Observation of the peritrophic membrane structure and identification of major protein species of Eriocheir sinensis

The aim of this study was to investigate the secretion, structure and function of peritrophic membrane (PM) in digestive tract of Eriocheir sinensis. Histology and electron microscopy techniques were employed to observe the intestinal and PM morphology of E. sinensis. Chitinase dissolution test and SDS-PAGE gel electrophoresis were used to research the composition of the PM and then the function of PM protein was indentified by LC-ESI-MS/MS. Microscopic examination showed the surfaces of gastrointestinal tract of E. sinensis were all covered with PM. Compartmentalization of PM was observed in intestine except midgut. When PM was detached from the epithelial cells, the columnar epithelial cells were becoming irregular round with the nucleus disappeared. A large number of mitochondria and secretory vesicles were observed in cytoplasm under the electron microscope. PM was noticed to be dissolved by chitinase. SDA-PAGE map showed that there were 7 clearer electrophoresis bands and their molecular weight was mainly distributed in more than 40 ku. Results from LC-ESI-MS/MS analysis showed that the PM proteins included sodium-potassium ATPase, ATP synthetase, actin and tubulin, thioredoxin, oxygenase, etc. These results indicated that the PM of E. sinensis is secreted by epithelial cells, improves absorption by forming compartment, and may participate in physiological processes of immune, osmotic regulation and nutrients transportation.