The efficiency of RNA interference (RNAi) has always limited the research on the phenotype innovation of Lepidoptera insects. Previous studies have found that double‐stranded RNA‐degrading enzyme (dsRNase) is an important factor in RNAi efficiency, but there have been no relevant reports in butterflies (Papilionoidea). Papilio xuthus is one of the important models in butterflies with an extensive experimental application value. To explore the effect of dsRNase in the RNAi efficiency on butterflies, six dsRNase genes (PxdsRNase 1–6) were identified in P. xuthus genome, and their dsRNA‐degrading activities were subsequently detected by ex vivo assays. The result shows that the dsRNA‐degrading ability of gut content (<1 h) was higher than hemolymph content (>12 h). We then investigated the expression patterns of these PxdsRNase genes during different tissues and developmental stages, and related RNAi experiments were carried out. Our results show that different PxdsRNase genes had different expression levels at different developmental stages and tissues. The expression of PxdsRNase2, PxdsRNase3, and PxdsRNase6 were upregulated significantly through dsGFP injection, and PxdsRNase genes can be silenced effectively by injecting their corresponding dsRNA. RNAi‐of‐RNAi studies with PxEbony, which acts as a reporter gene, observed that silencing PxdsRNase genes can increase RNAi efficiency significantly. These results confirm that silencing dsRNase genes can improve RNAi efficiency in P. xuthus significantly, providing a reference for the functional study of insects such as butterflies with low RNAi efficiency.

中文翻译：

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通过敲除蝴蝶 Papilio xuthus 中的双链 RNA 降解酶提高 RNAi 效率

RNA干扰（RNAi）的效率一直限制着鳞翅目昆虫表型创新的研究。此前的研究发现双链RNA降解酶（dsRNase）是RNAi效率的重要因素，但在蝴蝶（Papilionoidea）中尚未见相关报道。凤蝶是蝴蝶的重要模型之一，具有广泛的实验应用价值。为了探讨 dsRNase 对蝴蝶 RNAi 效率的影响，六双链RNA酶基因（PxdsRNase 1–6）被识别于徐苏斯随后通过离体测定检测了它们的 dsRNA 降解活性。结果表明，肠道内容物（<1 h）的 dsRNA 降解能力高于血淋巴内容物（>12 h）。然后我们研究了这些的表达模式核糖核酸酶并对不同组织和发育阶段的基因进行了研究，并进行了相关的RNAi实验。我们的结果表明，不同核糖核酸酶基因在不同的发育阶段和组织中有不同的表达水平。的表达式为PxdsRNA酶2,PxdsRNA酶3， 和PxdsRNA酶6通过 dsGFP 注射显着上调，并且核糖核酸酶通过注射相应的 dsRNA 可以有效地沉默基因。 RNAi-of-RNAi 研究黑檀木，作为报告基因，观察到沉默核糖核酸酶基因可以显着提高RNAi效率。这些结果证实沉默双链RNA酶基因可以提高RNAi效率徐苏斯显着，为低RNAi效率的蝴蝶等昆虫的功能研究提供参考。