Degree Type

Dissertation

Date of Award

2018

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

Major

Biochemistry

First Advisor

Dipali G. Sashital

Abstract

RNA interference (RNAi) is a highly conserved cellular process whereby small regulatory RNAs bound to argonaute proteins produce sequence-specific silencing of longer complementary RNAs. The agricultural biotechnology industry has taken advantage of RNAi to control insect pests through the use of transgenic crops expressing insecticidal RNAs. Upon introduction of double-stranded RNA into a pest, the complementary target messenger RNA is depleted and results in a lethal phenotype. For reasons that are not fully defined, certain insects respond differently to orally introduced RNAs, leaving holes in the manageability of all agricultural pests through this promising new technology. Furthermore, there are indications that insects may be able to develop resistance to crop-mediated RNAi through natural downregulation of RNAi pathway genes, among other proposed mechanisms. Using bioinformatics, next-generation sequencing, and insect bioassays, eight genes essential for RNAi were examined in three important agricultural insect pests for their potential involvement both in the differing responses to exogenous RNAs observed across these insects, and in development of resistance to insecticidal RNAs. These genes include drosha, dicer-1, dicer-2, pasha, loquacious, r2d2, argonaute 1, and argonaute 2.

Putative homologues of the well-characterized Drosophila melanogaster genes were identified in the western corn rootworm (Diabrotica virgifera virgifera), fall armyworm (Spodoptera frugiperda), and southern green stink bug (Nezara viridula) and compared using translated gene products. All genes were present in each insect and most showed conservation of basic protein domain structure, but differences in the number of isoforms and expression level of pasha, loquacious, r2d2, argonaute 1, and argonaute 2 were found. Sequencing experiments in each insect revealed the presence of small RNAs typical of the products of RNAi pathways, including conserved microRNAs. Abundance and distribution of these RNAs varied across life stage and insect. Finally, transcript depletion experiments were conducted in rootworm, and adverse phenotypic effects for each gene were observed. Taken together, these results suggest that while differences in these eight genes could contribute to variation in the RNAi pathways of these insects and therefore to variation in response to exogenous RNAs, they are unlikely to promote development of resistance to RNAi-based technology through expression pattern changes.

Copyright Owner

Courtney Anne Davis-Vogel

Language

en

File Format

application/pdf

File Size

214 pages

Included in

Biochemistry Commons

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