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標題: 水稻 ATML1 同源基因之選殖與分析
Molecular Cloning and Characterization of the ATML1 Gene Homologues in Rice
作者: 涂景瑜
Tu, Ching-Yu
關鍵字: Homeobox gene
出版社: 農業生物科技學研究所
摘要: Homeobox gene泛指真核生物中的一轉錄因子蛋白,其共同特色為具有一段 60 個胺基酸的homeodomain,可與特定的DNA序列結合,經常扮演調控生物體分化發育的角色。例如擬南芥的ATML1 (Arabidopsis thaliana meristem L1 layer) 基因,不但具有HD-GL2型的homeodomain,且表現位置極具專一性,從胚胎分化初期到成株均僅侷限於各種分生組織的最表層細胞 (L1 layer),暗示其可能具有界定細胞與組織分化的能力。本實驗假設水稻中也有ATML1同源基因存在,擬選殖之並研究其特性。首先以ATML1的蛋白序列為藍圖,使用tblastn程式搜查找到兩個水稻的EST (expressed sequence tags) 與之具高度相似性,以其EST 片段作為探針,經過水稻幼嫩花穗 cDNA 基因庫的篩選,degenerate PCR 與5' RACE (Rapid Amplification of cDNA Ends) 等三種策略選殖後,得到其相對的基因序列,分別命名為 OSML1A (1699 bp) 與 OSML1B (2567 bp),由基因組雜合分析結果顯示,不論是 OSML1A 及 OSML1B 均為單套 (single-copy) 的基因。OSML1A 殖系尚不完整,可能的轉譯產物未含括 homeodomain,但與 ATML1 蛋白序列相似度達 65﹪,以 OSML1A 為探針多次嘗試北方雜合分析,均未偵測到任何訊號,推測可能是 OSML1A 基因的表現區域十分狹窄且表現量低之故。而 OSML1B 基因應可轉譯出一完整長達 709 個胺基酸的蛋白,分類上屬於 HD-GL2 型的轉錄因子,其基因表現時間始於穎果受粉後第三天,大量表現後逐漸降低至第八天完全消失,然而其表現位置並非侷限於胚體中,於胚乳組織中亦有頗高的含量,由於 OSML1B 蛋白與 ATML1 蛋白相似性僅為 50﹪,故推測 OSML1B 並非 ATML1 的同源基因,其生理與生化功能尚待深入研究。
Homeobox genes are master regulatory genes that specify the body plans and control development of many eukaryotic organisms. They share a common homeodomain (HD) which consists of 60 amino acids and is responsible for sequence-specific binding to target genes. HD proteins act as transcription factors that control gene expression in the precise spatial and temporal patterns. One HD-containing protein, ATML1 (Arabidopsis thaliana meristem L1 layer), was found to be expressed only in the L1 layer of apical meristem during embryogenesis and throughout plant development. Such observations indicated that ATML1 protein might provide positional information necessary for controlling cell specification. By analogy, we proposed that monocotyledonous rice might contain the ATML1 gene homologues. Where using tblastn program obtained by homology search of rice EST (expressed sequence tags) databank two ESTs, C91737 and C74009, which encode polypeptide with high sequence similarity (59% and 65%) to ATML1. The EST fragments were used as probes and various strategies were employed for isolation of their corresponding cDNA clones, designated as OSML1A (1699 bp) and OSML1B (2567 bp) for C91737 and C74009, respectively. Genomic Southern blot analyses indicated that both OSML1A and OSML1B are single-copy genes in rice. The cDNA of OSML1A is truncated and encode a polypeptide of 306 amino acid residues which shows 65% sequence similarity to ATML1. However, no OSML1A transcripts could be detected in northern blot analysis even by using 2 mg of poly(A)+ RNA isolated from various tissues. We reasoned that OSML1A might be expressed only in a very restricted area and only to a limited level. OSML1B encodes a putative transcription factor that can be classified as HD-GL2 homeobox protein. OSML1B was preferentially expressed in the spikelets after pollination, and was undetectable at early embryogenesis stages. Transcripts of OSML1B could also be detected in the upper parts of the spikelets (5DAP), indicated that OSML1B is not an embryo-specific gene. The amino acid sequence similarity between OSML1B and ATML1 is only 50% which suggested that OSML1B might not be the ATML1 gene homologue in rice.
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