稻胚發育早期表現之DNA結合蛋白基因ose2之研究

Abstract

在植物體中，basic leucine zipper proteins（簡稱bZIP）是DNA binding protein family的一類，用以在各種發育，生理及環境時調控相關基因的表現。由水稻台農67號之授粉後3-5天幼胚及其週圍組織所製備的cDNA基因庫（cDNA library）篩選一選殖株，稱為pOSE2，經核?酸定序分析，序列全長1,180 bp，可轉譯出217個胺基酸之蛋白質，此OSE2蛋白質經比對得知為bZIP蛋白，具有保守的鹼性胺基酸區（basic amino acid domain）和leucine zipper domain。首先以ose2 cDNA探針對水稻IR36之染色體基因庫（genomic library）進行染色體基因選殖株之篩選及定序分析，已完成gOSE2IR中2,833 bp的序列分析，並經與pOSE2之序列比對，發現其中有三個片段與cDNA有較高的相似性，但平均的相似性只有75.78％，再將其核?酸序列推演出胺基酸序列，發現具有高保守性的鹼性胺基酸區及leucine zipper功能區，因此推測gOSE2IR可能為其他的bZIP蛋白。又利用ose2 cDNA探針對水稻台農67號之部份染色體基因庫（partial genomic library）進行篩選，得一選殖株，稱為gOSE2Ta，所攜帶的染色體DNA約7.6 kb，其中之4,834 bp已定序完成，經與pOSE2之序列比較分析，發現gOSE2Ta具有四個exons和三個introns。並且經由primer extension的方法得知染色體基因選殖株gOSE2Ta的轉錄起始點位於起始密碼ATG上游-97 bp處。除此之外還對水稻不同組織之total RNA作RNA slot blot分析，並利用OSE2抗血清對不同組織之蛋白質進行Western blot分析，結果顯示OSE2蛋白及其RNA具有些許的組織特異性，主要表現於水稻的胚及花器官組織中，因此推測OSE2在水稻胚胎發育時，可能扮演調節基因表現之角色。

The basic leucine zipper (bZIP) class of DNA binding protein contains a basic amino acid domain and a leucine zipper dimerization domain. Several plant bZIP proteins have been shown to play an essential role in the regulation genes expressed during seed development and various environmental stimuli. A cDNA clone-pOSE2 screened from a cDNA library of rice (Oryza sativa L. japonica cv. Tainung 67) young embryos and their adjacent tissues of 3-5 DAP seeds. The cDNA insert has a length of 1180 bp, encodes a bZIP protein which corresponds to 217 amino acids. We reported two genomic clones, designated as gOSE2IR and gOSE2Ta, respectively screened from O. sativa L. indica cv. IR36 and japonica cv. Tainung 67 genomic libraries. The gOSE2IR, a fragment of 2833 bp has been sequened and compared with the sequence of pOSE2, shared homology around 75%. Deduced amino acid sequences of gOSE2IR found a conserved basic domain and leucine zipper domain. So, we suggested that gOSE2IR may encode another bZIP protein. On the other hand, the gOSE2Ta containing 4834 bp in size, has 99.9% identity to the ose2 cDNA in coding region and there are four exons in gOSE2Ta. The transcription start site of ose2 was located 97 bp upstream from the ATG codon on gOSE2Ta. RNA slot blot and Western blot studies have shown that the ose2 gene expressed in embryos and panicle tissues. Results indicate that OSE2 protein may play a role in regulation genes expressed in rice during embryogenesis.