Cosuppression and RNAi induced by Arabidopsis ortholog gene sequences in tobacco
Cosuppression and RNAi induced by Arabidopsis ortholog gene sequences in tobacco
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烟草中拟南芥直系同源基因序列诱导的共抑制和 RNAi
DOI：
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发表时间：
2010
影响因子：
2.4
通讯作者：
H. Kodama
中科院分区：
文献类型：
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作者：
S. Oka;K. Midorikawa;H. Kodama
拟南芥 ω-3 脂肪酸去饱和酶 (AtFAD7) 催化三烯脂肪酸 (TA) 的合成。转基因烟草品系 T15 由有义 AtFAD7 构建体产生，并显示出类似共抑制的表型，即极低的 TA 水平。 AtFAD7 与烟草直系同源基因 NtFAD7 的编码序列具有中等相似性（约 69%）。 AtFAD7 siRNA 以高水平积累，AtFAD7 和 NtFAD7 mRNA 在 T15 植物中均被降解。 T15 中的低 TA 表型依赖于烟草 RNA 依赖性 RNA 聚合酶 6 (NtRDR6)。我们还生产了针对 AtFAD7 基因序列的烟草 RNAi 植物。 AtFAD7 siRNA 水平为微量，这与叶 TA 水平的轻微降低有关。出乎意料的是，这种 RNAi 植物表现出 NtFAD7 转录水平的增加。为了研究翻译抑制对 NtFAD7 mRNA 稳定性的影响，用翻译抑制剂放线菌酮处理野生型烟草植物的叶子。放线菌酮治疗后 NtFAD7 mRNA 水平显着增加。这些结果表明，低水平的 AtFAD7 siRNA 或放线菌酮的翻译抑制增加了 NtFAD7 mRNA 的稳定性。 AtFAD7转基因的共存增加了靶向AtFAD7基因的RNAi构建体的沉默程度，其中发生了NtRDR6依赖性的siRNA扩增。这些结果表明 NtRDR6 可以增强共抑制和 RNAi 中的沉默效应。
The Arabidopsis ω-3 fatty acid desaturase (AtFAD7) catalyzes the synthesis of trienoic fatty acids (TA). A transgenic tobacco line, T15, was produced by a sense AtFAD7 construct and showed a cosuppression-like phenotype, namely extremely low TA levels. The sequence similarity between AtFAD7 and a tobacco ortholog gene, NtFAD7, was moderate (about 69%) in the coding sequences. AtFAD7 siRNAs accumulated at a high level, and both AtFAD7 and NtFAD7 mRNAs are degraded in T15 plants. The low-TA phenotype in T15 was dependent on a tobacco RNA-dependent RNA polymerase6 (NtRDR6). We also produced tobacco RNAi plants targeting AtFAD7 gene sequences. The AtFAD7 siRNA level was trace, which was associated with a slight reduction in leaf TA level. Unexpectedly, this RNAi plant showed an increased NtFAD7 transcript level. To investigate the effect of translational inhibition on stability of the NtFAD7 mRNAs, leaves of the wild-type tobacco plants were treated with a translational inhibitor, cycloheximide. The level of NtFAD7 mRNAs significantly increased after cycloheximde treatment. These results suggest that the translational inhibition by low levels of AtFAD7 siRNAs or by cycloheximide increased stability of NtFAD7 mRNA. The degree of silencing by an RNAi construct targeting the AtFAD7 gene was increased by co-existence of the AtFAD7 transgene, where NtRDR6-dependent amplification of siRNAs occurred. These results indicate that NtRDR6 can emphasize silencing effects in both cosuppression and RNAi.