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標題: 水稻一似核糖體結合蛋白基因 Ose725之選殖與分析
Molecular Cloning and Characterization of a Ribosome-Associated Protein Gene Ose725 from Rice
作者: 李佳鳳
Lee, Chia-Feng
關鍵字: Rice;水稻;Ribosome-Associated Protein;核糖體結合蛋白
出版社: 分子生物學研究所
The cDNA Ose725 is one of the 38 cDNA clones isolated by differential screening of a cDNA library made with poly(A) RNA from embryos at the stage of 10 days after pollination. The cDNA Ose725 is 1336-bp in length, consisting of a 213-bp 5'-end untranslated region, a 942-bp coding region and a 163-bp 3'-end untranslated region. Within the 213-bp 5''-end region, a DNA fragment of 60 GA repeats was observed. The deduced translation product of the coding region is 313 amino acids with an estimated molecular mass of 33582.7 dalton with an isoelectric point of 4.8. Amino acid sequence of Ose725 shows 79 % identity to that of ribosome-associated protein from soybean and chickpea. The Ose725 is thus thought to be a gene coding for a ribosome-associated protein gene in rice. A His-Ose725 fusion protein was constructed in the pET30c expression system and the protein product was purified by passing through “His-Bind” nickel affinity column. Polyclonal antiserum against the His-Ose725 fusion protein was raised from rabbit. Western blot analysis indicated that the accumulation of Ose725 protein is not only in the embryos, but also in young roots ﹑mature leaves ﹑endosperms and seedlings. These results imply that the Ose725 protein might play an important role in the tissues where cell growed fast. Genomic Southern assay incicated that Ose725 is a single copy gene.

本實驗室早期以水稻授粉後十天之胚建立一個 cDNA 基因庫。經由差異性篩選得到38個水稻胚特有表現選殖群組, Ose725 為其中一個選殖系。本論文即針對此基因進行選殖與分析。 Ose725 基因之 cDNA 全長為 1336-bp ,含有 213-bp 之 5'' 端非轉譯區, 942-bp 之轉譯區以及 163-bp 之 3'' 端非轉譯區。在 5'' 端非轉譯區之 213-bp 中包含了一段 60 個 GA repeats 的區域,轉譯區之 942-bp 可供轉譯出一個含有 313 個胺基酸的蛋白,其分子量為 33582.70 ,等電點為 4.82 。根據 Ose725 之胺基酸序列,上網比對發現與已發表的大豆和雞豆核糖體結合蛋白有極高的相同度 (79%) ,推論 Ose725 屬於核糖體結合蛋白的基因。根據南方墨點法分析結果, Ose725 在水稻染色體中屬於單一基因。將 Ose725 之轉譯區構築於 pET30c 的表現載體上,於 E. coli 中以 IPTG 誘導其大量表現蛋白後,收集蛋白製備 Ose725 的抗血清,再針對水稻胚發育各時期及各組織,對 Ose725 蛋白的累積情形作探討。經由西方轉漬分析的結果發現, Ose725 蛋白在水稻胚發育的各時期呈現穩定性的存在,而在幼葉﹑幼根﹑胚﹑胚乳以及幼苗可偵測到其存在,在成熟葉片及小支梗則呈現較微弱的存在,然而在老根及內外穎則偵測不到其存在,由此結果及已報導之文獻推論 Ose725 蛋白在細胞分裂旺盛的組織中扮演重要角色。
Appears in Collections:分子生物學研究所

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