RNA-interference of four embryonically expressed genes in the codling moth Cydia pomonella

The codling moth is a major worldwide pest of apple, other tree fruits and nuts. In this project we are using RNAi to inhibit the expression of specific codling moth genes in order to evaluate their functions and explore the potential use of this approach for future control strategies. We previously cloned four cDNAs from codling moth embryos that are homologous to Drosophila genes with known functions (lin19, Deformed, musashi, and pumilio). I synthesized dsRNAs specific to these four cDNAs for use in RNAi knockdown experiments. I first designed gene-specific primers for the four cDNAs and a green fluorescent protein (gfp) control cDNA incorporating T7 promoter sequences. The primers were commercially synthesized and I used them to make template preparations from the embryonic cDNAs using the polymerase chain reaction (PCR). The specificity of each PCR reaction was evaluated by gel electrophoresis and PCR parameters were optimized as necessary. The templates were then transcribed using T7 RNA polymerase to produce double-stranded RNA (dsRNA). The purity and concentrations of the dsRNAs were evaluated by gel electrophoresis and spectrophotometry. These dsRNA will be used to inject codling moth embryos in order to evaluate their effects on embryonic development and survival.