Generation of secondary small interfering RNA in cell-autonomous and non-cell autonomous RNA silencing in tobacco
Generation of secondary small interfering RNA in cell-autonomous and non-cell autonomous RNA silencing in tobacco
复制标题
DOI：
10.1007/s11103-006-9124-9
复制
发表时间：
2007-01
影响因子：
5.1
通讯作者：
K. Shimamura;S. Oka;Yumi Shimotori;T. Ohmori;H. Kodama
中科院分区：
文献类型：
--
作者：
K. Shimamura;S. Oka;Yumi Shimotori;T. Ohmori;H. Kodama
长度为21-25个核苷酸的小干扰RNA（siRNA）种类在RNA干扰（RNAi）中引导mRNA切割、翻译停滞和异染色质形成。为了描绘RNAi的靶区域，将包含靶mRNA的部分与β-葡萄糖醛酸糖苷酶基因之间的转录融合的构建体生物射弹递送到显示RNAi表型的烟草叶中，并瞬时表达测定序列。通过扩增该嵌合转录物来监测RNAi效应。利用该方法，我们研究了烟草内质网ω-3脂肪酸去饱和酶基因（NtFAD 3）的传递性RNA沉默。在NtFAD 3 RNAi植物中，RNAi的靶区域被限制在对应于发夹双链RNA的茎序列的诱导物区域中，表明内源NtFAD 3 mRNA不是RNA依赖性RNA聚合酶的模板。在NtFAD 3过表达的植物和NtFAD 3 RNAi植物之间的杂交植物中产生次级NtFAD 3 siRNA。类似地，在系统性沉默的接穗中产生次级siRNA。虽然这些次级siRNA优先起源于诱导物区域下游的3′区域，但是在沉默的接穗中产生的次级siRNA（非细胞自主的次级siRNA）导致靶mRNA的强烈降解，但是在杂交植物中的次级siRNA（细胞自主的次级siRNA）显示有限的RNA降解活性。这些结果表明，这种用于确定RNAi效率的体内测定是描述RNAi机制的有用工具。
Small interfering RNA (siRNA) species with 21–25 nucleotides in length guide mRNA cleavage, translational arrest, and heterochromatin formation in RNA interference (RNAi). To delineate the target region of RNAi, a construct harboring a transcriptional fusion between parts of the target mRNA and the β-glucuronidase gene was biolistically delivered into tobacco leaves showing an RNAi phenotype and the assay sequence was transiently expressed. The RNAi effect was monitored by amplification of this chimeric transcript. By using this assay method, we addressed the transitive RNA silencing of a tobacco endoplasmic reticulum ω-3 fatty acid desaturase gene (NtFAD3). In theNtFAD3RNAi plants, the target region of RNAi was restricted in the inducer region corresponding to a stem sequence of the hairpin double-stranded RNA, indicating that endogenousNtFAD3mRNA was not a template for an RNA-dependent RNA polymerase. The secondaryNtFAD3siRNAs were produced in the crossbred plants between theNtFAD3overexpressed plant and theNtFAD3RNAi plant. Similarly, the secondary siRNAs were generated in the systemically silenced scion. Although these secondary siRNAs originated preferentially from the 3′ region downstream of the inducer region, the secondary siRNAs produced in the silenced scion (non-cell autonomous secondary siRNAs) resulted in the strong degradation of the target mRNA, but the secondary siRNAs in the crossbred plants (cell-autonomous secondary siRNAs) showed limited RNA degradation activity. These results showed that this in vivo assay for determination of RNAi efficiency is a useful tool to delineate RNAi mechanisms.