Screening and activity evaluation of lethal RNAi target genes inPlutella xylostella based on RCT technology

To screen for key RNA interference (RNAi) target genes for controlling Plutella xylostella, trehalase (PxTRE), vacuolar ATP synthase subunit E (PxATPE), ribosomal protein S13 (PxRPS13), and chitinase (PxCHT) were selected. Multiple RNA microspheres (RMS) were designed and synthesized using rolling circle transcription (RCT) technology. After administering the RMS to third-instar larvae via oral feeding, larval development and mortality were assessed. The results showed that gene silencing was significant after 48 hours of treatment. Compared to the RMS-EGFP control group, mRNA expression levels of PxTRE, PxATPE, PxRPS13, and PxCHT were reduced by 74.84%, 73.36%, 62.90%, and 38.10%, respectively (P < 0.05). In addition, multi-RMS RMS-PxTA (RMS-PxTRE/ATPE) and RMS-PxTC (RMS-PxTRE/CHT) significantly downregulated the expression of the corresponding genes. In the RMS-PxTA treatment, the expression levels of PxTRE and PxATPE were reduced by 86.16% and 96.38%, respectively (P < 0.05). Similarly, in the RMS-PxTC treatment, the expression levels of PxCHT were decreased by 45.22% and 28.02%, respectively (P < 0.05) . After 72 hours, the larval mortality rates in the RMS-PxTRE、RMS-PxATPE、RMS-PxCHT和RMS-PxRPS13、RMS-PxTA and RMS-PxTC treatments were 62.22%, 86.67%, 60.00%, 57.78%, 35.56%, and 66.67%, respectively. Surviving larvae in the treatment groups exhibited reduced feeding, shortened body length, and molting failure. These findings suggest that RCT technology is a viable method for rapid screening of RNAi target genes in P. xylostella. Among the tested target genes, PxATPE demonstrates potential as a highly effective target for RNA-based biopesticide, offering promising prospects for environmentally friendly control of P. xylostella.