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標題: | 水稻胚發育早期相關基因-OSE4之研究 Characterization of Rice Early Embryogenesis Related Gene - OSE4 |
作者: | 徐久逵 |
出版年 : | 1995 |
學位: | 碩士 |
摘要: | 胚胎發育過程在植物生活史中占了十分重要的地位,我們相信研究參與其中的因數將有助於對胚胎發育機制的瞭解。 由授粉後3-5天的水稻幼胚及週圍組織所製備之cDNA基因庫(cDNA library)中所選殖之選殖株-OSE4,經核?酸序列分析,序列全長1801 bp,可轉譯出一479個胺基酸之蛋白質,分子量約為52 KDa。該蛋白質經比對得知分別與phosphodiesterase I及nucleotide pyrophosphatase有40%的相似性。且由胺基酸序列分析推測此蛋白質等電點為4.88,靠近胺基端具有三段membrane associated helices。 將OSE4 cDNA殖入表現型載體(expression vector)pET-30,誘導其蛋白質表現。但表現結果並不如預期,推測可能因為此蛋白質與寄主的細胞膜結合,對寄主的生理造成影響。若將OSE4 cDNA自5'端切除298 bp,即相對於該蛋白質的前兩段membrane associated helices,再殖入表現型載體誘導,即可得到大量表現。 利用pET His•Tag純化系統以及SDS-PAGE電泳純化此大量表現之蛋白質,並進行抗體之製備,以期利用免疫抗體分析進一步瞭解此蛋白質在生體中之合成情形。 Embryogenesis plays an important role in plant life cycle. We want to understant this, mechanism through reseaches on facfors involved in embryogenesis. A cDNA clone-OSE4 screened from a cDNA library of rice young embryo is 1801 bp long, and encodes a 52 KD polypeptide which corresponds to 479 amino acids compared with protein sequences of phosphodiesterase I and nucleotide pyrophosphatase, OSE4 protein shares homology around 40% with them. Data from amino acid sequence analysis reveals that this protein has 3 membrane-associated helics near N-terminal with a pI value at 4.88. To avoid binding interference from ose4 protein and cell membrane, 298bp at 5' end of OSE4 which encodes the first two membrane-associated helics were deleted. Then the truncated OSE4 DNA were cloned into an expression vector pET-30 to induce producfion of fusion protein. This protein was purified by pET His•Taq purification system and SDS-PAGE and has been used to prepare polyclonal antibody. We hope to understand the biosynthetic process and cellular localization of OSE4 protein through immunocytochemistry studies in the future. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76127 |
全文授權: | 未授權 |
顯示於系所單位: | 植物科學研究所 |
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