فعالیت تجزیه کننده dsRNA در میدگات سن گندم

Sunn pest Eurygaster integriceps Puton (Hemiptera: Scutelleridae) is a serious pest of cereals which causes severe quantitative and qualitative damage to crops. The post-transcriptional gene silencing mechanism, RNA interference (RNAi), has a potential to be used in crop protection strategy against important insect pests by targeting mRNA via double-stranded RNA (dsRNA). The presence of nucleases in the insect midget that results in the decomposition of dsRNA can be linked to the RNAi technique success or to the concentration of dsRNA used in this technique. The aim of the current study is to evaluate the Sunn pest midgut dsRNA degrading activity. To do this, dsRNA was synthesized by using T7 RNA polymerase. Sequence-specific primers were designed with a T7 promoter at the 5ˈend to synthesize cDNA fragments which contained the T7 promoter region in the 5ˈend of both sense and antisense strands. The amplified dsDNA was used as template for dsRNA synthesis with the MEGAscript® RNAi kit (Ambion, Life Technologies). Fifth instar larvae midgut was dissected and homogenized in 100 µl of 2-distilled water then the sample was centrifuged for 3 min at 3000 rpm and the resulting supernatants were separated as midgut juice. Ten and 150 ng of dsRNA was added to 10 µl midgut juice which was serially diluted (i.e. 100, 50, 20, 10, and 2% of original extract). As a control, 10 and 150 ng of dsRNA was added to 10 µl 2-distilled water. As well as a second control consisting of 10 µl of midgut juice, without adding dsRNA was considered. After 5 minutes incubation time, the samples were analysed using 1% agarose electrophoresis. By incubating 10 ng of dsRNA in 100% and 50% midgut juice, Sunn pest midgut juice degraded dsRNA and its band disappeared in the agarose gel. The experimental data showed the presence of a dsRNA degrading activity in the midgut juice of the Sunn pest.