Stable oil bodies sheltered by a unique caleosin in cycad megagametophytes.
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.
Jiang
Pei-Luen
PL
Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung 402, Taiwan, ROC.
Chen
Jeff C F
JC
Chiu
Shau-Ting
ST
Tzen
Jason T C
JT
D000595:Amino Acid Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D032462:Cycadopsida / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015388:Organelles / Q000378:metabolism*; D010743:Phospholipids; D010938:Plant Oils / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D018749:RNA, Plant / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014280:Triglycerides
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D010743:Phospholipids; D010938:Plant Oils; D010940:Plant Proteins; D018749:RNA, Plant; D011994:Recombinant Proteins; D014280:Triglycerides; C402836:caleosin protein, Sesamum indicum