School of Biotechnology and Environmental Engineering, Myongji University, Yongin 449-728, Korea.
Planta. 2013 Jan;237(1):211-24. doi: 10.1007/s00425-012-1764-1. Epub 2012 Sep 25.
To be effective in crop biotechnology applications, gene promoters need to be stably active over sequential generations in a population of single-copy transgenic lines. Most of the stress-inducible promoters characterized in plants thus far have been analyzed at early (T₀, T₁ or T₂) generations and/or by testing only a small number of transgenic lines. In our current study, we report our analysis of OsNCED3, a stress-inducible rice promoter involved in ABA biosynthesis, in various organs and tissues of transgenic rice plants over the T(2-4) homozygous generations. The transgene copy numbers in the lines harboring the OsNCED3:gfp construct were determined and six single- and two double-copy transgenic lines were analyzed for promoter activity in comparison with the Wsi18, a stress-inducible promoter previously characterized. The exogenous promoter activities were found to be significantly enhanced in the roots and leaves, whereas zero or low levels of activity were evident in grains and flowers, under drought and high-salinity conditions. The highest induction levels of gfp transcripts in the OsNCED3:gfp plants upon drought treatments were 161- and 93-fold in leaves and roots, respectively, and these levels were comparable with those of gfp transcripts in the Wsi18:gfp plants. A comparison of the promoter activities between the T₂-T₄ plants revealed that comparable activity levels were maintained over these three homozygous generations with no evidence of silencing. Thus, our results provide the OsNCED3 promoter that is stress-inducible in a whole rice plant except for in the aleurones and endosperm and stably active over three generations.
为了在作物生物技术应用中取得成效，基因启动子需要在单拷贝转基因系的群体中连续几代保持稳定的活性。迄今为止，在植物中鉴定的大多数应激诱导启动子都在早期（T0、T1 或 T2）世代进行了分析，或者仅测试了少数几个转基因系。在我们目前的研究中，我们报告了我们对 OsNCED3 的分析，OsNCED3 是一种参与 ABA 生物合成的应激诱导水稻启动子，在 T（2-4）纯合世代的各种器官和组织中的分析。在含有 OsNCED3:gfp 构建体的系中确定了转基因拷贝数，并分析了六个单拷贝和两个双拷贝转基因系与先前表征的应激诱导启动子 Wsi18 的启动子活性。在外源启动子活性在干旱和高盐条件下，在根和叶中显著增强，而在谷物和花朵中则没有或低水平的活性。在 OsNCED3:gfp 植物受到干旱处理后，gfp 转录物的最高诱导水平分别在叶片和根中达到 161 倍和 93 倍，这些水平与 Wsi18:gfp 植物中的 gfp 转录物水平相当。对 T₂-T₄ 植物之间的启动子活性进行比较表明，在这三个纯合世代中，在没有沉默证据的情况下，维持了可比的活性水平。因此，我们的结果提供了一个除了糊粉层和胚乳之外，在整个水稻植株中都是应激诱导的 OsNCED3 启动子，并且在三代中稳定活跃。
