Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung 402, Taiwan, ROC.
Plant Physiol Biochem. 2009 Nov-Dec;47(11-12):1009-16. doi: 10.1016/j.plaphy.2009.07.004. Epub 2009 Jul 15.
Stable oil bodies of smaller sizes and higher thermostability were isolated from mature cycad (Cycas revoluta) megagametophytes compared with those isolated from sesame seeds. Immunological cross-recognition revealed that cycad oil bodies contained a major protein of 27 kDa, tentatively identified as caleosin, while oleosin, the well-known structural protein, was apparently absent. Mass spectrometric analysis showed that the putative cycad caleosin possessed a tryptic fragment of 15 residues matching to that of a theoretical moss caleosin. A complete cDNA fragment encoding this putative caleosin was obtained by PCR cloning using a primer designed according to the tryptic peptide and another one designed according to a highly conservative region among diverse caleosins. The identification of this clone was subsequently confirmed by immunodetection and MALDI-MS analyses of its recombinant fusion protein over-expressed in Escherichia coli and the native form from cycad oil bodies. Stable artificial oil bodies were successfully constituted with triacylglycerol, phospholipid and the recombinant fusion protein containing the cycad caleosin. These results suggest that stable oil bodies in cycad megagametophytes are mainly sheltered by a unique structural protein caleosin.
与芝麻种子相比，从成熟苏铁（Cycas revoluta）大配子体中分离出了更小尺寸和更高热稳定性的稳定油体。免疫交叉识别表明，苏铁油体含有一种 27kDa 的主要蛋白，暂定为钙卫蛋白，而众所周知的结构蛋白油体蛋白显然不存在。质谱分析表明，拟南芥钙卫蛋白具有与理论上的苔藓钙卫蛋白相匹配的 15 个残基的胰蛋白酶片段。根据胰蛋白酶肽设计了一个引物，根据不同钙卫蛋白之间高度保守的区域设计了另一个引物，通过 PCR 克隆获得了编码该假定钙卫蛋白的完整 cDNA 片段。随后通过在大肠杆菌中表达重组融合蛋白的免疫检测和 MALDI-MS 分析以及从苏铁油体中分离的天然形式证实了该克隆的鉴定。成功地用三酰基甘油、磷脂和含有苏铁钙卫蛋白的重组融合蛋白构成了稳定的人工油体。这些结果表明，苏铁大配子体中的稳定油体主要由一种独特的结构蛋白钙卫蛋白保护。
