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Functional genomics of nuclear tRNAs in plants

植物核 tRNA 的功能基因组学

基本信息

批准号：
16510147
负责人：
AKAMA Kazuhito
金额：
$ 2.18万
依托单位：
Shimane University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16510147/
关键词：
tRNA Arabidopsis genomics amber suppressor GFP Agrobacterium seed maturation amino acid usage GUS 形質転換パーティクルガン

项目摘要

Functional analysis of entire tRNA genes in Arabidopsis whose genome had been determined recently. First, expression patterns of 23 different kinds of isoacceptor tRNA gene families were investigated with corresponding gene probes. Northern blot analysis indicated that tRNA^<Leu> (CAA) was tissue-specifically expressed in root and flower-bud. During seed-maturation after fertilization, several tRNA species such as tRNA^<Lys>(CUU),tRNA^<Pro>(UGG),tRNA^<Glu>(CUC) and tRNA^<Gln>(UUG) were abundant in mature seeds, while the level of remaining tRNAs examined were decreased in the same stage. Analysis of tRNAs that were sampled from various stages in the seed maturation with denatured polyacrylamide gel electrophoresis showed degradation of tRNAs in the late stage of the maturation. This result suggests that most of tRNA species were target of ribonuclease, but some of tRNAs were escaped from this degradation via an unknown mechanism.Second, in order to explore the functional expression of … More tRNAs in plant cells, reporter genes such as a GUS and luciferase has been widely used so far. In this study, we have evaluated a GFP marker gene : GFP/amber, in which a premature amber codon was artificially introduced in the fourth codon (Lys) from an initiation codon, was constructed by an in vitro site-directed mutagenesis. When the GFP/amber was introduced in onion epidermal cells with a particle gun, no fluorescence was observed as expected, but when introduced and co-expressed with a gene coding for suppressor tRNA^<Ser> (NtS2-am), weak fluorescence was detected. Therefore, GFP/amber would be used alternatively to explore functional expression of the specific tRNA in plant cells. In parallel, on the basis of the NtS2-am, which is known as the most active suppressor tRNA known so far, a novel in vivo system for analysis of effect of 5'-flanking region of tRNA on its transcription, i.e., substitution of the 5'-flanking sequence of the NtS2 for that of a target gene. We examined five RNA polymerase III genes in this system. Interestingly, relative activity of in vivo transcription of these genes was well correlated to that of transcription of the same genes in the cell-free system of tobacco BY-2. Less

拟南芥全tRNA基因的功能分析，其基因组已被确定。首先，用相应的基因探针研究了23种不同类型的异受体tRNA基因家族的表达模式。北方印迹分析表明，tRNA^<Leu>（CAA）在根和花芽中特异表达。在受精后的种子成熟过程中，成熟种子中富含tRNA^<Lys>（CUU）、tRNA^<Pro>（UGG）、tRNA^<Glu>（CUC）和tRNA^<Gln>（UUG）等几种tRNA，而在同一时期检测到的其余tRNA的水平下降。用变性聚丙烯酰胺凝胶电泳分析种子成熟过程中不同阶段的tRNA，结果表明，成熟后期tRNA发生降解。这一结果表明，大多数tRNA是核糖核酸酶的靶标，但也有一些tRNA通过未知的机制逃脱了核糖核酸酶的降解。 ...更多信息迄今为止，植物细胞中的tRNA、GUS和荧光素酶等报道基因已被广泛使用。在这项研究中，我们已经评估了一个绿色荧光蛋白标记基因：绿色荧光蛋白/琥珀，其中一个过早的琥珀密码子是人工引入的第四个密码子（赖氨酸）从一个起始密码子，是通过体外定点突变构建的。当用粒子枪将GFP/amber导入洋葱表皮细胞时，如预期的那样没有观察到荧光，但是当与编码抑制性tRNA γ（NtS 2-am）的基因一起导入并共表达<Ser>时，检测到弱荧光。因此，GFP/琥珀将交替用于探索特异性tRNA在植物细胞中的功能表达。与此同时，在已知最具活性的抑制性tRNA NtS 2-am的基础上，建立了一种新的分析tRNA 5 ′侧翼区对其转录影响的体内系统，NtS 2的5 '侧翼序列被靶基因的5'侧翼序列取代。我们研究了五个RNA聚合酶III基因在这个系统中。有趣的是，这些基因在体内转录的相对活性与烟草BY-2的无细胞系统中相同基因的转录活性有很好的相关性。少