基于滚环转录技术的siRNA递送策略对粘虫的干扰效果

Assessing the interference effect of the siRNA delivery strategy on Mythimna separata using rolling circle transcription

【目的】评估基于滚环转录技术的siRNA递送策略对粘虫Mythimna separata的RNA干扰效果。【方法】采用荧光定量PCR,测定靶标基因Msα-tubulin和Msβ-tubulin的沉默效率;通过饲喂方法进行生物测定,观察经滚环转录制备的RNA微球处理粘虫3龄幼虫后其体重、体长和致死表型特征的变化。【结果】靶标基因沉默效率测定结果表明,RNA微球处理24 h后,Msα-tubulin和Msβ-tubulin的基因表达量分别下降51.6%和44.3%;Msα-tubulin在48 h的表达量下降32.0%,在72 h的表达量上调134.4%;处理48和72 h后,Msβ-tubulin的表达量分别上调120.5%和129.0%。生物学干扰效应结果表明,与对照组相比,处理7 d后,Msα-tubulin处理组幼虫体重、体长分别下降51.1%和34.9%,而Msβ-tubulin处理组的幼虫分别下降49.0%和32.4%;处理14 d后,这两种靶标基因处理组的幼虫死亡率均为42.2%。【结论】滚环转录制备的RNA微球能够触发粘虫的RNAi响应,干扰靶标基因的表达,抑制幼虫体重、体长并引起粘虫明显的死亡。

[Aim]To assess the effect of RNA interference of the siRNA delivery strategy on Mythimna separata using rolling circle transcription.[Methods]We used qPCR to determine the silencing efficiency of the two target genes,Msα-tubulin,and Msβ-tubulin.The RNAi Feeding Bioassay was used to observe changes in M.separata body weight,body length,and lethal phenotypes.Additionally,the 3rd instar larvae were treated with RNA microspheres,which were synthesized using the rolling circle transcription method.[Results]After 24 h of RNA microsphere treatment,Msα-tubulin and Msβ-tubulin expression decreased by 51.6%and 44.3%,respectively,demonstrating the effectiveness of the target gene silencing.After 48 h there was a further 32%decrease in the expression of Msα-tubulin,which then increased by 134.4%at 72 h.After 48 and 72 h of treatment,the expression level of Msβ-tubulin was upregulated by 120.5%and 129.0%,respectively.The RNAi feeding bioassay results showed that after 7 days,larval body weight and body length decreased by 51.1%and 34.9%,respectively in the Msα-tubulin treatment group,and decreased by 49.0%and 32.4%in the Msβ-tubulin treatment group,respectively,compared to the control.After 14 days,the mortality rate of larvae reached 42.2%for both the Msα-tubulin and Msβ-tubulin treatment groups.[Conclusion]RNA microspheres,created via the rolling circle transcription method,can trigger an RNAi response in M.separata,interfere with the expression of target genes,significantly increase M.separata mortality,and reduce larval weight and body length.The siRNA delivery strategy,utilizing rolling circle transcription,offers a novel approach to controlling M.separata.