RNAi-mediated knockdown of candidate genes in Emerald Ash Borer

Candidate genes were selected based on our previous work (Shen et al., 2021 (link)), the selected candidates were expressed significantly different between newly emerged and sexually mature A. planipennis (OBP5, OBP7, OBP10, LW opsin 1 and UV opsin 2) or highly expressed in sexually mature A. planipennis (UV opsin 3). The control consisted injections of dsEGFP (dsRNA against green fluorescent protein), H₂O and nontreated. Double-stranded RNA (dsRNA) was synthesized by using T7 RNA polymerase and gene-specific primers. The primers were designed with a T7 polymerase promoter sequence (TAA​TAC​GAC​TCA​CTA​TAG​G) at the 5’ end (Supplementary Table S1) to generate PCR templates for in vitro transcription of dsRNA. PCR conditions were 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min, finishing with an extension step at 72°C for 10 min. The PCR templates were purified using a universal DNA Purification Kit (Tiangen, Beijing, China). After purification, dsRNA was synthesized and purified using the T7 RiboMAX™ Express RNAi System (Promega, United States) following instructions provided. The synthesized dsRNA was then quantified using NanoDrop ND-2000 (Thermo Scientific, Wilmington, DE, United States), and its quality was examined by agarose gel electrophoresis. The final concentration of dsRNA was diluted to 2 μg/μl.