|Walker Iii, William|
|Allen, Margaret - Meg|
Submitted to: Entomological Society of America Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 11/25/2009
Publication Date: 12/15/2009
Citation: Walker III, W. B., Allen, M. L. 2009. Gene Expression Studies in Lygus lineolaris. Entomological Society of America Annual Meeting.
Technical Abstract: Genes are expressed in insect cells, as in all living organisms, by transcription of DNA into RNA followed by translation of RNA into proteins. The intricate patterns of differential gene expression in time and space directly influence the development and function of every aspect of the organism. While some genes are expressed in all cells at all times (constitutive expression), many are linked to specific functions that require timing, such as molting; or circumstance, such as feeding or mating. We have used reverse-transcription quantitative polymerase chain reaction (qRT-PCR) to study various genes in the pest insect, Lygus lineolaris. 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. Another 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 qRT-PCR and/or may produce one or more specific changes in function, or even gross morphological or developmental changes. RNAi gene knock-down 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, L. lineolaris (Allen 2007). 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. We described the three PG sequences in some detail; PG transcripts are present only in feeding insect stages, not eggs (Allen and Mertens 2008), and have been linked to Lygus feeding damage (Celorio-Mancera et al. 2008). 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 (Walker and Allen (in press)). 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 (manuscript in preparation). Additionally, RNAi treatment targeting some of the aforementioned cuticle sequences results in incomplete molting and subsequent death (manuscript in preparation). Allen, M. L. 2007. Expressed sequenced tags from Lygus lineolaris (Hemiptera: Miridae), the tarnished plant bug. Genet Mol Res 6: 206-13. Allen, M. L., and J. A. Mertens. 2008. Molecular cloning and expression of three polygalacturonase cDNAs from the tarnished plant bug, Lygus lineolaris. Journal of Insect Science 8.27: 1-14. Celorio-Mancera, M. D., M. L. Allen, A. L. Powell, H. Ahmadi, M. R. Salemi, B. S. Phinney, K. A. Shackel, L. C. Greve, L. R. Teuber, and J. M. Labavitch. 2008. Polygalacturonase causes lygus-like damage on plants: cloning and identification of western tarnished plant bug (Lygus hesperus) polygalacturonases secreted during feeding. Arthropod-Plant Interactions 2: 215-225. Walker, W. B., and M. L. Allen. (in press). Expression and RNA interference of salivary polygalacturonase genes in the tarnished plant bug, Lygus lineolaris. Journal of Insect Science.