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標題: Cloning and Characterization of Rice Genomic DNAs of RE1M and RE31D
水稻 RE1M 與 RE31D 之染色體 DNA 之選殖與分析
作者: Hou, Chien-Wei
關鍵字: 水稻
染色體組 DNA
genomic DNA
出版社: 分子生物學研究所
摘要: The objectives of the current research are to clone the genomic clones of RE1M and RE31D from 10-day embryos of rice, to analyze these two genes, and to study the regulation of RE31D gene expression. The genomic clones of RE1M and RE31DG were isolated from genomic library of 5-day etiolated shoot of rice selected by plaque hybridization with the cDNA probe of RE1M and RE31D, and named RE1MG and RE31DG, respectively. The RE1MG is 1,853 bp long, and contains a 1,590-bp ORF, a 262-bp 5' untranslated region, and a 334-bp 3' untranslated region, but no intron. The molecular weight of deduced amino acid of RE1MG is 43.7 kDa with 401 amino acids and a pI of 11.34. The identity of RE1MG is not known at this moment. The RE31DG is 3,877 bp long, and contains a 1,775-bp ORF, a 1666-bp 5' untranslated region, a 138-bp 3' untranslated region, and two introns. The molecular weight of deduced amino acid of RE31DG is 60.5 kDa with 541 amino acids and a pI of 11.99. Either the nucleotide sequence or the deduced amino acid sequence of RE31DG is highly homologous with the plant globulin gene. Two chimeric constructs contained 5' promoter region of RE31DG (-367~47 and -890~47) and GUS coding region were transferred to 10-day embryos, mature embryos and calli of rice via gene gun mediated transformation. The results of GUS histochemical staining after 2 days of transformation show that the transient expression of GUS in both constructs were high in 10-day and mature embryos, but low in the calli. The promoter of RE31DG has embryo-specific characters.
本研究之目的在於篩選水稻十天胚特有基因,RE1M 與 RE31D 之染色體組 DNA, 並分析RE31D 其啟動子調節。 本實驗以水稻5 天黃化苗之基因庫為材料,分別以RE1M 及RE31D 之cDNA為探針。篩選出RE1M 與RE31D 之染色體組 DNA,分別命名為RE1MG 及RE31DG。所選殖之RE1MG全長為1,853 bp具有1,590 bp之ORF,包含 5' 端上游啟動子有 262 bp, 3' 端非轉譯區共有 334 bp,沒有 intron。RE1MG 可轉譯出 401 個胺基酸,蛋白質分子量為 43.7 kDa,等電點為 11.34 。RE1MG 之 DNA 核酸序列,經由網路基因資料庫比對結果與其他發表的基因之相似性都很低,因此目前對其屬性仍未知。 RE31DG 全長有 3,877 bp,具有 1,775 bp 之 ORF,包含 5' 端上游啟動子 1,666 bp,3' 端下游區域 2,211 bp,有 2 個 intron。RE31DG 可轉譯出541 個胺基酸,蛋白質分子量為 60.5 kDa,等電點為11.99。將RE31DG 之 DNA 核酸列與推演出的胺基酸序列,經由網路基因資料庫比對之結果顯示與水稻的 globulin 基因有很高的相似性。 分別將 RE31DG 基因啟動子之 bp -368~47 (pBIA-4) 與 bp -890~47 (pBIA-9) 片段構築到 GUS 報導基因上,再利用基因槍轉移法分別傳送至台農67號水稻的十天胚、成熟胚與癒傷組織中。兩天後,經過 GUS 染色分析之結果顯示,此二段啟動子調節之 GUS 基因在十天胚與成熟胚的表現很明顯,在癒傷組織表現微弱,試驗結果顯示RE31DG 之啟動子具有胚獨特性之表現特性。
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