Schwob E, Choi S Y, Simmons C, Migliaccio F, Ilag L, Hesse T, Palme K, Söll D
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511.
Plant J. 1993 Sep;4(3):423-32. doi: 10.1046/j.1365-313x.1993.04030423.x.
The site I 22 kDa auxin-binding proteins from maize are encoded by a small gene family comprising at least five members. Here the cloning and molecular analysis of the Zm-ERabp1, Zm-ERabp4, and Zm-ERabp5 genes is presented. All three encode 22-23 kDa proteins displaying a transit peptide, a C-terminal KDEL sequence, as well as glycosylation and auxin-binding sites. The Zm-ERabp4 and Zm-ERabp5 genes are very similar. The Zm-ERabp1 gene encodes a related protein, but its promoter, leader and signal peptide are very different. Northern analysis using gene-specific oligonucleotide probes indicates that Zm-ERabp4 is expressed in leaves and coleoptiles but weakly in roots, whereas Zm-ERabp5 expression is barely detectable in these tissues. RNA-PCR indicated that all three genes are none the less expressed in many tissues. Primer-extension analysis revealed an unusually long (320 bases) Zm-ERabp1 leader containing an 80 codon ORF which, if expressed, would encode a positively charged protein with some similarity to transcription factors. In a transient promoter-reporter gene expression system using maize leaf protoplasts the Zm-ERabp1 promoter is more active than the Zm-ERabp4 and Zm-ERabp5 promoters. Promoter deletion analysis of Zm-ERabp1 has identified a negative regulatory sequence in a region from -364 bp and -130 bp, deletion of which results in about twofold higher expression. This region contains both enhancer- and G-box-related sequences. Deletion of -126 bp to +64 bp, which contains the TATA box and transcription start, results in a large decrease in expression.
玉米中22 kDa生长素结合蛋白的位点I由一个至少包含五个成员的小基因家族编码。本文介绍了Zm-ERabp1、Zm-ERabp4和Zm-ERabp5基因的克隆及分子分析。这三个基因均编码22 - 23 kDa的蛋白质，这些蛋白质具有转运肽、C端KDEL序列以及糖基化和生长素结合位点。Zm-ERabp4和Zm-ERabp5基因非常相似。Zm-ERabp1基因编码一种相关蛋白，但其启动子、前导序列和信号肽有很大差异。使用基因特异性寡核苷酸探针进行的Northern分析表明，Zm-ERabp4在叶片和胚芽鞘中表达，但在根中表达较弱，而Zm-ERabp5在这些组织中几乎检测不到表达。RNA-PCR表明这三个基因在许多组织中均有表达。引物延伸分析揭示了一个异常长（320个碱基）的Zm-ERabp1前导序列，其中包含一个80个密码子的开放阅读框，如果该开放阅读框表达，将编码一种与转录因子有一定相似性的带正电荷的蛋白质。在使用玉米叶片原生质体的瞬时启动子 - 报告基因表达系统中，Zm-ERabp1启动子比Zm-ERabp4和Zm-ERabp5启动子更活跃。对Zm-ERabp1进行的启动子缺失分析在 - 364 bp至 - 130 bp区域鉴定出一个负调控序列，缺失该序列会导致表达量提高约两倍。该区域同时包含增强子相关序列和G盒相关序列。缺失包含TATA盒和转录起始位点的 - 126 bp至 + 64 bp区域会导致表达量大幅下降。
