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Rayssa Almeida Garcia

Federal University of Brasilia, Brazil

Title: Strategy to overcome nucleic acid degrading enzymes in insect pests for the RNAi application

Biography

Rayssa Almeida Garcia is a PhD student in the Molecular Biology Program of the Federal University of Brasilia – Brazil,  at the age of 23. She does her research work at in the Plant-Pest Interaction laboratory at Embrapa Genetic Resources and Biotechnology- Brasilia/ Brazil, under supervising of Dr Maria Fatima Grossi-de-Sa, have published  a large number of papers in reputed journals and is a fellow member of the World Academy of Science.

Abstract

Gene silencing through RNA interference as a biotechnological approach for the control of crop insect-pests have been intensively applied in the last few years. dsRNA microinjection and in vitro feeding are the most wildly used approaches for administering RNAi in insects. However, RNAi efficiency appears to be variable among different insect groups, especially when applied by feeding - for some insect groups the oral delivery of the dsRNAs has been reported highly ineffective. In initial studies, our gene silencing data for cotton boll weevil (Anthonomus grandis)  were unclear when dsRNA administration was done by feeding. The purpose of this work was to assess the possibilities of RNAi as a tool for the control of this insect pest using oral delivery of dsRNAs and to investigate the reason for the low efficiency in gene silencing,  aiming to develop a strategy to deal with the efficiency and usage of dsRNA by feeding. Data showed an optimal nucleasic activity of the A. grandis gut nucleases at acid pH, ranging from 5.5 to 6.5, and the A. grandis' gut homogenate significative degraded both dsRNA and dsDNA. Three nuclease sequences were found in A. grandis transcriptome, named AgNuc1, AgNuc2, and AgNuc3; in which AgNuc2 and AgNuc3  showed to be highly expressed in the insect gut. The silencing of the three nuclease genes strongly diminished dsRNA degradation when dsRNA were incubated with homogenate from silenced insects. On the other hand, when dsRNAs were protected with a Cell Penetrating Peptide (CPP) fused with a dsRNA Binding Domain (DRBD), no dsRNA degradation was found. Furthermore, dsRNAs complexed with CPP-DRBD were found to enter into A. grandis gut cells. The dsRNA complexed administered in the diet caused a greater gene silencing, compared to naked dsRNA. All data point out to the relevance for overcoming the gut nucleases with/or in parallel with the RNAi applications for the control of crop insect-pests.