T7 ribomax system

The full-length Of-Relish cDNA in pMD-18T was used as template to amplify with T7- Relish-F and T7-Relish-R (Table 1) that contain the binding site of T7 RNA polymerase. The purified PCR product (1.0 μg) was transcribed to RNA strands using RiboMax™ T7 system (Promega, USA). After annealing, the dsRNA was then digested with RNase A and DNase1, precipitated with 2.2 μL 3 M sodium acetate and 55 μL 95% ethanol, washed with 75% ethanol, and resuspended in nuclease-free water. Integrity of the dsRNA was checked by gel electrophoresis, and its concentration was measured in an Eppendorf BioPhotometer D30. Finally, the dsRNA was adjusted to 5 μg/μL for injection and 2 μL was injected into each day 1, 3^rd instar larva. There were three injected larvae in each group with three biological replicates. The injected larvae were treated with 500 μL of Trizol solution at 84, 96, 108, and 120 h post injection for total RNA preparation. The larvae injected with dsGFP were used as the control group. To assess the RNAi effect on AMP expression, the bacteria were injected into dsRNA-treated larvae at 84 h post dsRNA injection and the larvae were treated with 500 μL Trizol at 0, 6, 12, and 24 h post bacterial infection. All reactions were completed in triplicate using independently extracted RNA samples.

Double-stranded (ds) RNAs of OfPAP and green fluorescent protein (GFP) were prepared by following the protocol of RiboMax^™ T7 system (Promega, USA). A template for in vitro transcription reactions was prepared by PCR amplification of a clone containing the OfPAP ORF using gene-specific primers (Table 1) with a T7 polymerase binding sequence at 5’ end. After phenol-chloroform extraction and heat treatment, the dsRNA was diluted with nuclease-free water to a final concentration of 5 μg/μL for injection into 4^th instar larvae. GFP dsRNA and nuclease-free DEPC water were served as negative controls. Fourth instar larvae were injected intra-abdominally with 20 μg of OfPAP dsRNA (RNAi group). The GFP control group received a 4 μl injection with 20 μg of GFP dsRNA. The third group of larvae received a 4 μL injection of nuclease-free DEPC water to mimic the handling effect. Sixty 4^th instar larvae were treated in three independent experiments, respectively. The puncture was sealed immediately with liquid paraffin. Larvae were removed from the slide after injection and raised at 25°C until analysis. qRT-PCR was performed to confirm the efficacy of RNAi as described above. All reactions were completed in triplicate using independently extracted RNA samples.