Long dsRNA but not siRNA initiates RNAi in western corn rootworm larvae and adults
Long dsRNA but not siRNA initiates RNAi in western corn rootworm larvae and adults
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DOI：
10.1111/jen.12224
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发表时间：
2015-07-01
影响因子：
1.9
通讯作者：
Narva, K. E.
中科院分区：
文献类型：
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作者：
Li, H.;Khajuria, C.;Narva, K. E.
在温室测定中，与阴性对照相比，表达靶向西方玉米根虫（WCR，玉米根萤叶甲）v-ATP酶亚基C mRNA用于RNAi的dsRNA的转基因玉米植物提供了针对WCR幼虫摄食损害的显著的根保护。WCR抗性玉米植物中转录的发夹dsRNA以完整的发夹衍生的dsRNA和植物加工的siRNA两者存在。因此，在WCR幼虫和成虫中研究了靶向Dv v-ATPase CmRNA的dsRNA和siRNA引起RNAi应答的能力。在9天的基于饲料的摄食测定中，长度至少60 bp的dsRNA导致高水平的幼虫死亡率。相比之下，15-，25-或27-bp的dsRNA或合并的21-bp的siRNA不会导致暴露的幼虫死亡。当幼虫喂食覆盖有siRNA的饲料时，Dv v-ATPase C转录水平没有变化。相反，当WCR幼虫用覆盖有184-bp dsRNA的饮食喂养时，mRNA水平相对于yfp dsRNA阴性对照降低>20倍。类似地，184-bp dsRNA导致WCR成虫的100%死亡率，而用siRNA处理的饮食喂养的成虫的死亡率与阴性对照相似。在饮食中用siRNA喂养成年人并不影响Dv v-ATPase CmRNA转录物的水平，而用184 bp dsRNA喂养的成年人显示靶mRNA水平降低约35倍。用184-bp dsRNA或21-bp siRNA注射的WCR成虫获得了类似的结果。这些结果表明，只有长的dsRNA或发夹衍生的dsRNA在引起Dv v-ATPase CmRNA的致死性敲低中是有效的。这些结果对于有效的植物递送的dsRNA用于保护转基因玉米免受WCR摄食损害和表达杀虫dsRNA的转基因玉米的风险评估具有意义。
Transgenic maize plants expressing dsRNA targeting western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) v-ATPase subunit C mRNA for RNAi provided significant root protection from WCR larval feeding damage in greenhouse assays compared to negative controls. Transcribed hairpin dsRNA in WCR-resistant maize plants was present as both intact hairpin-derived dsRNA and plant-processed siRNA. Therefore, the ability of dsRNA and siRNA targeting Dv v-ATPase CmRNA to cause an RNAi response was studied in both WCR larvae and adults. In 9-day diet-based feeding assays, dsRNA of at least 60bp in length resulted in high levels of larval mortality. In contrast, 15-, 25- or 27-bp dsRNAs or pooled 21-bp siRNAs did not cause mortality of exposed larvae. When larvae were fed with diet overlaid with siRNAs, Dv v-ATPase C transcript levels did not change. Conversely, when WCR larvae were fed with diet overlaid with 184-bp dsRNA, the mRNA level was reduced by >20-fold relative to yfp dsRNA negative control. Similarly, 184-bp dsRNA caused 100% mortality of WCR adults, whereas the mortality of adults fed on diet treated with siRNAs was similar to the negative control. Feeding adults with siRNAs on diet did not affect the level of Dv v-ATPase CmRNA transcripts, whereas adults fed with the 184-bp dsRNA showed approximately 35-fold reduction in the target mRNA level. Similar results were obtained with the WCR adults injected with 184-bp dsRNA or 21-bp siRNA. These results suggest that only long dsRNA or hairpin-derived dsRNA is effective in causing lethal knock-down of Dv v-ATPase CmRNA. These results have implications for efficacious plant-delivered dsRNA for the protection of transgenic maize from WCR feeding damage and for the risk assessment of transgenic maize expressing insecticidal dsRNA.