Title: Gene knockdown through RNAi in Lygus lineolaris Authors
|Walker Iii, William|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: October 21, 2010
Publication Date: N/A
Technical Abstract: A powerful method for discovering gene function is RNA interference (RNAi). When double-stranded RNA that corresponds to an insect gene transcript is introduced into the insect, cellular mechanisms are activated that degrade that specific transcript. The decreased transcript levels can then be measured by reverse-transcription quantitative polymerase chain reaction (qRT-PCR) and/or may produce one or more specific changes in function, or even gross morphological or developmental changes. RNAi gene silencing may result in death of the insect if the target gene is both vital and sufficiently eliminated by the treatment. Our laboratory has sequenced and analyzed a small set of transcribed genes (mRNA) from our target insect, Lygus lineolaris. We have used qRT-PCR to study various genes in this pest insect. Comparison of the transcript quantity in an insect at a specific life stage, in a specific tissue, or under specific conditions provides evidence for the relevance of differential gene expression in the insect. Validation of transcription levels is made by comparison with presumed constitutive genes. We targeted three putative polygalacturonase (PG) sequences that appeared to be associated with salivation and pectin degradation, one apoptosis-associated sequence (IAP) and several cuticle-associated sequences. PG sequences have been described in some detail: PG transcripts are present only in feeding insect stages, not eggs, and have been linked to Lygus feeding damage. Our expression studies have clearly shown that all three PGs are expressed primarily in salivary gland tissue, and we have been able to measure transcriptional changes based on diet and following RNAi treatment, using qRT-PCR. Our PG RNAi treatments did not result in any obvious phenotypic or functional change. However, RNAi treatment using the IAP sequence target results in death. Additionally, RNAi treatment targeting some of the aforementioned cuticle sequences results in incomplete molting and subsequent death.