2.6 ∣ RNA interference
DNA fragments of candidate genes were amplified with Taq PCR Master Mix
(2X, with Blue Dye) (Sangon Biotech, Shanghai, China) and inserted into
the RNAi vector L4440 plamsid (Addgene, USA) following the Gateway
procedure. The resulting constructs were verified by complete sequencing,
then recombinant plasmids were transfected into DH5α (TianGen Biotech
Co. LTD, Beijing, China). DH5α cells were cultured in SOC solid medium
containing 2mg/L Amp+ (pH=7.0) for 12 hours. The cells were then
transferred to a MS liquid medium for another 12 hours. Plasmid DNA for
dsRNA production was isolated using E.Z.N.A.TM Plasmid Mini Kit I
(Sangon Biotech, Shanghai, China), and was then transfected into the
HT115 cells through Thermal Activation. HT115 cells with a dsRNA
production vector were cultured in SOC solid medium containing 2mg/L
Amp+ and 10ml/L Tet (PH=7.0) for 12 hours, and then were transferred
into 2YT liquid medium containing 1.2 ml/L Amp and 2 ml/L Tet for
obtaining large amounts of dsRNAs. After purification, dsRNA was sprayed
onto early second male nymphs. Silencing of dsRNA was examined using
qRT-PCR at 12, 24, 48, and 72h after RNAi treatments.