Function of a short spacer between rice EPSP synthase and rps20 genes.

我们将水稻愈伤组织中表达的带有序列(S)的片段定位到水稻蜡质位点周围328kb的BAC重叠群的物理图谱上。在杂交片段中，我们试图鉴定位于蜡质下游约45kb区域的强杂交片段。这些片段含有两个看家基因，分别编码5-烯丙酮基-3-磷酸合成酶(Epsps)和核糖体蛋白小亚基20(Rps20)。有趣的是，这些看家基因是相邻的，只有一个303个碱基对的间隔区，并以尾巴对尾巴的方向定向。通过对愈伤组织文库的筛选，获得了这两个基因的近全长cDNA克隆。克隆的EPSPS基因具有1683bp的开放阅读框，编码一个由561个氨基酸(AA)组成的多肽，这是利用该方法首次在水稻中发现的表达基因。EPSPS是莽草酸途径中的一个关键酶，定位于高等植物的质体中。这种酶是已知的…更多的目标是草甘膦，它已经被用作一种非选择性除草剂。之前已经分离到水稻rps20肽的一个基因，我们的rps2O cDNA克隆与该肽的注册序列完全匹配。从水稻品种的根、苗、小穗和花药中提取总RNA，进行Northern杂交分析。在根、幼苗和小穗的RNA样品中很容易检测到2.2kb的EPSPS基因转录本，但在花药RNA中检测不到。在我们检测的所有组织中都检测到0.75kb的rps20转录本，尽管这两个水稻品系的根中表达很弱。我们已经检查了这两种基因的转录本的一定程度。这些转录本在间隔区之外进行；另一个基因的表达可能被来自相反基因的反义RNA修饰，导致双链RNA，这可能在称为转录后基因沉默的机制中触发RNA降解。这里发现的短间隔区在高等植物的核基因组织中并不常见。该片段可能与植物核基因组中间隔区的扩展或收缩有关，并可能在相反基因转录物引起的基因调控中发挥作用。较少

We mapped the fragments with sequence (s) expressed in the rice callus onto a physical map for a 328-kb BAC contig around the rice waxy locus. Among the hybridized fragments, we attempted to characterize strongly hybridized fragments located in a region about 45 kb downstream of waxy. The fragments were found to contain two housekeeping genes encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) and ribosomal protein small subunit 20 (rps20). Interestingly, these housekeeping genes are adjoined, with only a 303-bp spacer, and are directed in a tail-to-tail orientation. Nearly-full-length cDNA clones of the two genes were obtained by screening the callus cDNA library. A clone for the EPSPs gene possessed an open reading frame of 1683 bp encoding a deduced 561-amino-acid (aa) polypeptide that is the first expressed gene identified in rice using this method. EPSPs is a key enzynae on the shikimate pathway and is localized in the plastids of higher plants. This enzyme is known to be … More targeted by glyphosate, which has been employed as a nonselective herbicide. A gene for the rice rps20 peptide had previously been isolated, and our rps2O cDNA clone completely matched the registered sequence for this peptide. Northern blotting analyses were conducted using blots of total RNAs extracted from roots, seedlings, spikelets and anthers of Japonica and Indica rice strains. The 2.2-kb transcript of the EPSPs gene was easily detected in RNA samples from roots, seedlings and spikelets, but not the RNA from anthers. A 0.75-kb rps20 transcript was detected in all the tissues we examined, although the expression in roots from both rice strains was very weak. We have examined an extent of the transcript of either gene. These transcripts proceeded beyond the spacer ; the expression of the other gene might be modified by the antisense RNA from the opposite gene, resulting in double-stranded RNA, which might act as a trigger for RNA degradation in the mechanism called post-transcriptional gene silencing. The short spacer found here is uncommon in the nuclear gene organization in higher plants. This segment may have evolutionary implications about spacer expansion or contraction in the plant nuclear genome, and may play a role in gene regulation caused by the opposite gene transcript. Less