PxAdoR Gene Silencing

A double-stranded RNA (348 bp) corresponding to a portion of the PxAdoR was synthesized by using a method that eliminates the cloning step [16]. Here, templates for in vitro transcription were produced by adding T7 promoter sequences to each 5’ end of cDNA template prepared by SMART-RACE cDNA Amplification Kit (Clontech) through polymerase chain reaction (PCR). The primers Pxds-F and Pxds-R (Table 1) used to generate a cDNA with T7 promoter sequences, were designed based on the PxAdoR sequence at positions 1314–1661. The PCR products were examined on agarose gel prior to in vitro transcription to verify that the products show a single band and the expected sizes. All dsRNA preparations were quantified spectrophotometrically and stored at −20°C until use.

Second instar larvae of P. xylostella were starved for 24 h and fed for 48 h with cabbage dishes which were cut into 10 cm diameter to fit the size of a petri dish [17], and drenched in 500 ng/mL [17]of Pxds or green fluorescent protein (GFP) -dsRNA 5 mins, and aired. Being a nontoxic reporter gene, GFP-dsRNA (dsGFP) was used as a control.