Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/35900
標題: 水稻與三種代表性裸子植物的油體與油體膜蛋白異構型之特性分析
Characterization of Oil Bodies and Oleosin Isoforms in Rice and Three Representative Gymnosperm Species
作者: 吳世欣
Wu, Lawrence Shih-Hsin
關鍵字: oil bodies
油體
oleosin
rice
gymnosperm
油體膜蛋白
水稻
裸子植物
出版社: 農業生物科技學研究所
摘要: 水稻種子的油體存在於胚與糊粉層(aleurone layer)這兩種組織細胞內, 兩處的油體大小相似(0.5-1μm), 均以TAGs(triacylglycerols)為主要成份, 而且兩種oleosin isoforms的含量比例亦相似, 並沒有觀察到兩處油體有何不同。TAGs分析顯示34%的TAGs在胚, 含油量明顯低於糊粉層。 水稻受粉後七天TAGs開始累積, 而16kDa與18kDa兩種型式的油體膜蛋白則在受粉後九天出現。配合西方墨點法的結果發現在油體形成早期16kDa比18kDa較早出現或是表現較強, 但是到種子成熟後期, 兩種isoform的量就沒有明顯差異。 在發芽實驗部份, 發芽過程中18kDa消失的速率略大於16kDa的消失速率。TAGs的消耗速率緩慢, 在浸種後五天內只消耗約38%的TAGs, 留下約62%的TAGs, 而五天後水稻種子已長出綠色葉片, 剩油量在往後數天已沒有明顯變化。藉由穿透式電子顯微鏡觀察發芽各階段中胚與糊粉層細胞中油體的變化, 發現胚細胞的油體在浸種四天後明顯減少, 但是糊粉層細胞內的油體十天後卻仍沒有減少的跡象。這樣的結果似乎指示糊粉層中的油體扮演的不是能量儲存的角色。 在不同的被子植物種子油體上均有兩個以上的油體膜蛋白, 這些油體膜蛋白可以依分子量劃分為兩類, 分別稱為high與low Mr isoform, 並且可以用西方墨點法加以區分。 在海松 (Pinus koraiensis), 銀杏 (Ginkgo biloba)與蘇鐵 (Cycas revoluta)三種裸子植物中 (分別代表三類在分類上不同綱的裸子植物)發現這些裸子植物的油體膜蛋白只有一個, 使用水稻與上述三種植物的油體膜蛋白所製作出的抗體進行免疫交叉確認實驗, 得知此單一的油體膜蛋白是屬於low Mr isoform, 而且與被子植物的油體膜蛋白一樣僅分佈在油體上。 裸子植物的油體分佈在種子的胚與大配子體部份, 純化的油體表面具負電斥力與立體障礙防止油體聚集與融合。測量油體的三種主要成份, neutral lipid, phospholipid與protein, 並將其換算成比率, 發現與由被子植物油體所推測的模式 (model)運算出來的預測比率相近。由上述結果推論裸子植物與被子植物的油體具有相同的表面特性與組成結構。 以水稻16kDa oleosin基因全長為探針篩選出芝麻的L-form oleosin 15kDa (accession no. AF091840)。cDNA全長共730個nucleotide, 包括5''端非轉錄區56個, open reading frame 435個, 3''端非轉錄區236個。轉錄出的胺基酸序列分子量為15194.60 Da。 在釋迦 (Annona squamosa) 與草麻黃 (Ephedra sinica) 的種子油體上有兩個可能是oleosin的蛋白質。經過與水稻和芝麻兩個isoforms的抗體作免疫交叉互認後發現, 這兩個蛋白質應是 oleosin, 而且依分子量大小分屬L-form 與 H-form oleosin。證明較原始的被子植物與較高等的裸子植物均具有兩個 oleosin, 有別於其他三類的裸子植物僅具有一個oleosin。 將41個從EMBL data bank (包括芝麻15 kDa oleosin) 找尋出的oleosin 蛋白質序列作譜系關係分析, 推算出兩個isoforms分歧的年代並與化石紀錄相比。發現基因複製(gene duplication)應該發生在被子植物的祖先。結合免疫互認的結果, 我們推測在尼藤目(Gnetales, 裸子植物, 麻黃屬為該目下的一屬)與被子植物分歧之前oleosin 基因已完成複製形成兩種isoforms。這樣的推測同時也支持尼藤目與被子植物是姊妹群(sister group)的論點。
Oleosins are hydrophobic abundant proteins localized in the oil bodies of plant seeds. Two distinct oleosin isoforms of molecular masses 18 and 16 kDa are present in rice oil bodies. Both oleosin isoforms of similar ratio were found in rice embryos and aleurone layers. Triacylglycerols and oleosins were accumulated concomitantly in maturing rice seeds in accord with the active assembly of oil bodies, and partly mobilized in postgerminative seedlings. Approximately, 60% of the stored triacylglycerols in rice were not utilized as a result that the majority of oil bodies in embryos were mobilized in five days after imbibition while those in aleurone layers were left intact in postgerminative seedlings. . Two immunologically distinct oleosins are present in seed oil bodies of diverse angiosperms, and classified as high and low Mr isoforms according to their relative molecular masses in each species. Only one putative oleosin was found in seed oil bodies of three representative gymnosperm species, Pinus koraiensis, Ginkgo biloba, and Cycas revoluta. Immunological cross-recognition using antibodies against the three putative gymnosperm oleosins and those against the two (high and low Mr) rice oleosin isoforms suggested that a singular low Mr oleosin isoform, but no high Mr isoform, was present in seed oil bodies of these gymnosperms. Oil bodies were found in embryos and megagametophytes of these three species. Seed oil bodies purified from these three gymnosperms maintained their integrity via electronegative repulsion and steric hindrance on the surface of the organelles. The compositions of the three essential constituents (neutral lipid, phospholipid and protein) in seed oil bodies from these three species were determined and compared with those calculated from the oil body model proposed in angiosperms. The results suggested that seed oil bodies of gymnosperms and angiosperms possess similar surface properties and structural organization. A full-length cDNA clone (accession no. AF091840) with an open reading frame encoding a L-form oleosin of sesame was obtained using a DNA probe containing the full length of rice 16kDa oleosin. The cDNA fragment comprises 730 nucleotides, consisting of a 56-nucleotide 5' untranslated region, an open reading frame of 435 nucleotides, and a 236-nucleotide 3' untranslated region. The deduced sequence comprises 145 amino acid residues of Mr 15194.60. The sesame oleosin 15kDa and other oleosin amino acid sequences available from the EMBL data library were included in phylogenetic analysis. Two putative oleosin was found in seed oil bodies of Annona squamosa (representative primitive angiosperm) and Ephedra sinica (representative advanced gymnosperm). Immunological cross-recognition using antibodies against the putative oleosins and those against the two (high and low Mr) rice and sesame oleosin isoforms suggested that two oleosin isoforms were present in seed oil bodies of primitive angiosperm and advanced gymnosperm. Our amino acid sequence comparisons and previous immunological study indicate that there are two distinct oleosin isoforms present in angiosperms, and that the divergence of these two isoforms occurred before the splitting of the Gnetales and angiosperms. The result should be supposed that angiosperm and ephedra had common ancient, that is ephedra (one genus of Gnetales) been sister group of angiosperms.
URI: http://hdl.handle.net/11455/35900
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