The maize Emb5 is an abscisic acid–responsive gene which is specifically expressed in the late embryo during seed maturity. To further dissect and identify the elements specific for its embryo expression pattern, we investigated the activity of the − 1653 bp upstream of the “full-length” promoter region of this gene in transgenic Arabidopsis plants. We first confirmed that the “full-length” promoter could indeed drive the expression of β-glucuronidase reporter gene (GUS) in the transgenic Arabidopsis seed embryo. Subsequently, DNA fragments of ~ 500 bp in length were generated after a series of progressive deletions from positions − 1653 bp to − 1 bp relative to the transcriptional initiation site. These fragments were fused with GUS and introduced into Arabidopsis. Measurement of the GUS activity in the immature seeds isolated from the transgenic plants revealed that the region between positions − 523 bp and − 1 bp, namely ProEm-D, is absolutely required and sufficient for the temporal and embryo-specific expression of GUS with an activity comparable with the full-length Emb5 promoter in Arabidopsis. Therefore, our results clearly demonstrated that the 523 bp ProEm-D can replace the − 1653 bp Emb5 promoter to drive embryo-specific expression in Arabidopsis seed. Because of its small size and strong embryo-specific activity, it could become the promoter of choice in metabolic pathway engineering to transfer multiple genes for the production of valuable pharmaceutical products in seeds, such as polyunsaturated fatty acids found in fish oils, or pro-vitamin A where at least three transgenes are required to assemble the entire metabolic pathways.

中文翻译：

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玉米 Emb5 启动子的分析确定了负责其胚胎特异性活性的 523 bp 区域

玉米 Emb5 是一种脱落酸反应基因，在种子成熟的晚期胚中特异性表达。为了进一步剖析和鉴定其胚胎表达模式的特异性元件，我们研究了转基因拟南芥植物中该基因“全长”启动子区域上游 - 1653 bp 的活性。我们首先证实“全长”启动子确实可以驱动 β-葡萄糖醛酸酶报告基因 (GUS) 在转基因拟南芥种子胚中的表达。随后，在从相对于转录起始位点 - 1653 bp 到 - 1 bp 的位置进行一系列渐进删除后，产生了约 500 bp 长度的 DNA 片段。这些片段与 GUS 融合并引入拟南芥。对从转基因植物中分离的未成熟种子中 GUS 活性的测量表明，位置 - 523 bp 和 - 1 bp 之间的区域，即 ProEm-D，对于 GUS 的时间和胚胎特异性表达是绝对需要和足够的与拟南芥中全长 Emb5 启动子的活性相当。因此，我们的结果清楚地表明，523 bp ProEm-D 可以替代 - 1653 bp Emb5 启动子以驱动拟南芥种子中的胚胎特异性表达。由于其体积小和胚胎特异性活性强，它可以成为代谢途径工程中的首选启动子，以转移多种基因，用于在种子中生产有价值的药物产品，例如鱼油中发现的多不饱和脂肪酸，