Tobacco rattle virus vector: A rapid and transient means of silencing manduca sexta genes by plant mediated RNA interference.
Tobacco rattle virus vector: A rapid and transient means of silencing manduca sexta genes by plant mediated RNA interference.
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DOI：
10.1371/journal.pone.0031347
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Baldwin IT
中科院分区：
文献类型：
--
作者：
Kumar P;Pandit SS;Baldwin IT
在食草性昆虫中，可以通过喂养稳定转化为表达选定中肠表达基因的双链RNA(DsRNA)的宿主植物来实现RNAi。然而，稳定转化植株的发展是一个缓慢而费力的过程，在这里，我们开发了一种快速、可靠和瞬时的方法。我们使用病毒载体在寄主植物烟草中产生dsRNA，以暂时沉默该植物的鳞翅目专业食草动物Manduca sexta的中肠基因。为了比较更长的、未切碎的dsRNA对昆虫基因沉默的有效性，我们在一种稳定转化为表达昆虫基因的dsRNA的植物中，沉默了弱小核糖核酸的所有组合(NaDCL1-4)。获得了稳定表达312个碱基的反向重复序列(ir-CyP6B46)片段的弱小核糖核酸(N.tenuata)植株。在摄食这些植物后，六纹夜蛾幼虫中肠中细胞色素P6B46的转录本显著减少。将相同的312个碱基的cDNA反义克隆到TRV载体中，并将其农业渗透到弱小氮藻的植株中。当幼虫摄入这些植物时，观察到CYP6B46转录本的类似减少，而最密切相关的MsCYP6B45的转录本没有减少。我们使用这种瞬时方法快速沉默了另外两个中肠表达的MsCyp的表达。当取食ir-CyP6B46植物的幼虫对两种NaDCL组合(DCL1/3/4和DCL2/3/4)瞬时沉默并含有更高浓度的更长的未切块的CYP6B46 dsRNA时，中肠中的CYP6B46转录本进一步减少。CYP6B46 dsRNA在寄主植物中的稳定和瞬时表达为在六分枝杆菌幼虫中沉默该基因提供了一种特异和可靠的手段，但瞬时系统更适合于高通量分析。在ir-CyP6B46植物中瞬时沉默NaDCL增加了MsCYP6B46的沉默，这表明昆虫的RNAi机制更有效，摄入的dsRNA长度更长。
RNAi can be achieved in insect herbivores by feeding them host plants stably transformed to express double stranded RNA (dsRNA) of selected midgut-expressed genes. However, the development of stably transformed plants is a slow and laborious process and here we developed a rapid, reliable and transient method. We used viral vectors to produce dsRNA in the host plant Nicotiana attenuata to transiently silence midgut genes of the plant's lepidopteran specialist herbivore, Manduca sexta. To compare the efficacy of longer, undiced dsRNA for insect gene silencing, we silenced N. attenuata's dicer genes (NaDCL1- 4) in all combinations in a plant stably transformed to express dsRNA targeting an insect gene. Stable transgenic N. attenuata plants harboring a 312 bp fragment of MsCYP6B46 in an inverted repeat orientation (ir-CYP6B46) were generated to produce CYP6B46 dsRNA. After consuming these plants, transcripts of CYP6B46 were significantly reduced in M. sexta larval midguts. The same 312 bp cDNA was cloned in an antisense orientation into a TRV vector and Agro-infiltrated into N. attenuata plants. When larvae ingested these plants, similar reductions in CYP6B46 transcripts were observed without reducing transcripts of the most closely related MsCYP6B45. We used this transient method to rapidly silence the expression of two additional midgut-expressed MsCYPs. CYP6B46 transcripts were further reduced in midguts, when the larvae fed on ir-CYP6B46 plants transiently silenced for two combinations of NaDCLs (DCL1/3/4 and DCL2/3/4) and contained higher concentrations of longer, undiced CYP6B46 dsRNA. Both stable and transient expression of CYP6B46 dsRNA in host plants provides a specific and robust means of silencing this gene in M. sexta larvae, but the transient system is better suited for high throughput analyses. Transiently silencing NaDCLs in ir-CYP6B46 plants increased the silencing of MsCYP6B46, suggested that insect's RNAi machinery is more efficient with longer lengths of ingested dsRNA.
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