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Functional analysis of rice oleosin and ribosomal protein p40
|關鍵字:||rice;水稻;RNA interference;oleosin;ribosomal protein p40;transgenic plant;RNA干擾;油體膜蛋白;核醣體蛋白p40;轉殖植物||出版社:||分子生物學研究所||引用:||源起 吳妤憶 (2003) 利用RNA干擾法探討水稻穀粒發育相關基因之功能，中興大學分子生物學研究所碩士論文 Demianova, M., Formosa, T.G. and Ellis, S.R. (1996) Yeast proteins related to the p40/laminin receptor precursor are essential components of the 40 S ribosomal subunit. J Biol Chem 271: 11383-11391. Siloto, R.M., Findlay, K., Lopez-Villalobos, A., Yeung, E.C., Nykiforuk, C.L. and Moloney, M.M. (2006) The accumulation of oleosins determines the size of seed oilbodies in Arabidopsis. Plant Cell 18: 1961-1974. Susantad, T. and Smith, D.R. (2008) siRNA-mediated silencing of the 37/67-kDa high affinity laminin receptor in Hep3B cells induces apoptosis. Cell Mol Biol Lett 13: 452-464. Tai, S.S., Chen, M.C., Peng, C.C. and Tzen, J.T. (2002) Gene family of oleosin isoforms and their structural stabilization in sesame seed oil bodies. Biosci Biotechnol Biochem 66: 2146-2153. 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The functions of rice oleosin and ribosomal protein Osp40 were analyzed by knocking-out or knocking-down their expression using RNA interference (RNAi) approache. Transgenic rice plants 35S::ole16i, 35S::ole18i and 705::p40i containing RNAi constructs of ole16, ole18 and Osp40a were generated seperately.
There are two oleosin isoforms OLE16 and OLE18 in rice oil bodies. Different effects on triacylglycerol packaging to oil bodies were observed in transgenic rice seeds that eliminating one of their two oleosin isoforms. Oil bodies isolated from both 35S::ole16i and 35S::ole18i transgenic seeds were found to be of comparable size and stability to those isolated from wild-type rice seeds, although they were merely sheltered by a single oleosin isoform. Electron microscopy revealed a few large, irregular oil clusters in 35S::ole16i transgenic seed cells, whereas accumulated oil bodies in 35S::ole18i transgenic seed cells were comparable to or slightly larger than those in wild-type seed cells. The drastic difference between the triacylglycerol contents of crude seed extracts and isolated oil bodies from 35S::ole16i transgenic plants could be attributed to the presence of large, unstable oil clusters that were sheltered by insufficient amounts of oleosin and therefore could not be isolated together with stable oil bodies.
Three ribosomal protein p40 genes Osp40a, Osp40b and Osp40c are identified from the rice genome. In addition to the major 40 kD protein band, this study also observed a minor 33 kD protein band in most of the rice tissues. The 705::p40i transgenic plants with the suppression of Osp40s revealed retarded growth and showed smaller in size or shorter in length in all vegetative and reproductive organs. The longitudinal section of stem showed much smaller cell sizes in p40i plants suggesting that the reduction of Osp40 protein inhibited cell elongation which could be the cause of the shorter stem and peduncle length. The cross section of stem and leaf of p40i plants also revealed smaller cell sizes which resulted in a smaller stem and leaf and also reduced the sizes of vascular bundles. However, the epidermis cells of leaf in p40i plants showed no different from those of the wild type. No stable homozygous p40i transgenic line was survived and the progenies of survived p40i lines would segregate into various sized seedlings. The protein analyses of these various sized seedlings revealed that the greater the suppression of Osp40s, the less the total proteins synthesized and that resulted in the smaller seedlings. Although the size of each part of the p40i plant has been reduced as a result of the limited amount of the available Osp40s proteins, all vegetative and reproductive organs remain functional. Taken together, Osp40s are essential proteins for rice growth and development and function as a key component in the translation machinery.
水稻基因組中共有三個核醣體蛋白p40基因，分別命名為Osp40a、Osp40b與Osp40c。除了40 kDa位置的主要訊號外，水稻p40蛋白質另以33 kDa形式存在於各組織中。705::p40i植株中因Osp40蛋白質表現量受抑制，造成植株生長遲緩，各營養或生殖組織變小或變短。由稻稈縱切發現細胞變小，故推測Osp40蛋白缺失造成細胞伸長能力受抑制，進一步造成稻稈與總花柄長度縮減。稻稈與葉片橫切顯示細胞尺寸縮小，進一步造成微管束變小。然而葉片表皮細胞大小不變。目前尚未獲得p40i同質品系之植株，由異質品系p40i植株所採收的種子播種後的幼苗生長速度不一致，Osp40蛋白質含量越低，幼苗生長受抑制越明顯。縱然p40i轉植株各部位變小係由於可用的Osp40蛋白量有限，各營養或生殖組織仍保有其功能性。總而言之，Osp40蛋白為水稻生長與發育之必要性蛋白質，並且於蛋白質轉譯工作扮演一重要角色。
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