RNA Interference in the Tobacco Hornworm, Manduca sexta, Using Plastid-Encoded Long Double-Stranded RNA
RNA Interference in the Tobacco Hornworm, Manduca sexta, Using Plastid-Encoded Long Double-Stranded RNA
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DOI：
10.3389/fpls.2019.00313
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发表时间：
2019-03-14
影响因子：
5.6
通讯作者：
Donly, Cam
中科院分区：
文献类型：
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作者：
Burke, William G.;Kaplanoglu, Emine;Donly, Cam
RNA干扰（RNAi）是一种很有前途的控制害虫的方法，它通过沉默重要昆虫基因的表达来干扰发育和生理；然而，某些昆虫目对这一过程有抵抗力。在这项研究中，我们着手测试质体内合成的植物表达的 dsRNA 沉默抗 RNAi 昆虫（鳞翅目烟草天蛾）基因表达的能力。以曼杜卡液泡型H+ ATP酶亚基A（v-ATPaseA）基因为靶标，我们首先通过将体外合成的dsRNA直接喂给M. sexta幼虫来评估两种不同长度的dsRNA产物的RNAi效率。我们发现，当以水滴形式口服递送时，2222 bp 的长 dsRNA 在诱导致死和沉默 v-ATPaseA 基因方面最有效。我们进一步转化了 M. sexta 宿主植物烟草的质体基因组，以在其质体中产生这种长 dsRNA，并用 M. sexta 幼虫对转质体植物进行了生物测定。在测试的昆虫中，质体衍生的 dsRNA 对幼虫的存活没有影响，并且对 v-ATPaseA 基因的表达没有观察到统计上显着的影响。比较转质体叶组织中存在的 v-ATPaseA 和较短尺寸的对照基因 GFP 的 dsRNA 绝对量，发现与短对照产物相比，长 dsRNA 产物的浓度较低。我们认为 dsRNA 的稳定性和长度可能影响了质体中产生的数量，导致测试昆虫中 RNAi 效率低下。我们的结果表明，许多因素决定了植物中 RNAi 的有效性，包括可能的权衡效应，因为增加 dsRNA 产物长度可能会因质体中产生和积累的 dsRNA 量减少而抵消。
RNA interference (RNAi) is a promising method for controlling pest insects by silencing the expression of vital insect genes to interfere with development and physiology; however, certain insect Orders are resistant to this process. In this study, we set out to test the ability of in planta-expressed dsRNA synthesized within the plastids to silence gene expression in an insect recalcitrant to RNAi, the lepidopteran species, Manduca sexta (tobacco hornworm). Using the Manduca vacuolar-type H+ ATPase subunit A (v-ATPaseA) gene as the target, we first evaluated RNAi efficiency of two dsRNA products of different lengths by directly feeding the in vitro-synthesized dsRNAs to M. sexta larvae. We found that a long dsRNA of 2222 bp was the most effective in inducing lethality and silencing the v-ATPaseA gene, when delivered orally in a water droplet. We further transformed the plastid genome of the M. sexta host plant, Nicotiana tabacum, to produce this long dsRNA in its plastids and performed bioassays with M. sexta larvae on the transplastomic plants. In the tested insects, the plastid-derived dsRNA had no effect on larval survival and no statistically significant effect on expression of the v-ATPaseA gene was observed. Comparison of the absolute quantities of the dsRNA present in transplastomic leaf tissue for v-ATPaseA and a control gene, GFP, of a shorter size, revealed a lower concentration for the long dsRNA product compared to the short control product. We suggest that stability and length of the dsRNA may have influenced the quantities produced in the plastids, resulting in inefficient RNAi in the tested insects. Our results imply that many factors dictate the effectiveness of in planta RNAi, including a likely trade-off effect as increasing the dsRNA product length may be countered by a reduction in the amount of dsRNA produced and accumulated in the plastids.