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In vitro Study of Herves Transposable Element of Anopheles gambiae and Use of RNA Interference (RNAi) in Culex quinquefasciatus

Abstract

Transposable elements (TEs) and RNA interference (RNAi) are excellent genetic tools that could help control the incidence and spread of mosquito-borne diseases such as malaria, dengue, yellow fever, etc. This study aimed to 1) understand the RNAi mechanism in Culex quinquefasciatus, which will be used to study genes involved in pathogen transmission and will help reveal role of RNAi in antiviral immunity in this species; and 2) characterize the DNA sequences that regulate Herves transposase binding, which will help us understand its pre- and post-integration behavior within the host.

By using the white eye-pigmentation gene as a marker for RNAi function we demonstrated that introducing dsRNA into embryos of Cx. quinquefasciatus induces a specific functional RNAi response, which silenced the white gene, consequently allowing a white-eye phenotype to be produced in the hatched larvae and adults. Sequence-specific knockdown of key RNAi components was achieved by introducing the homologous dsRNA into embryos of Cx. quinquefasciatus. We found ago2-mediated slicing to be more critical than dcr2-mediated dicing for the functional RNAi response in Cx. quinquefasciatus. Our phylogenetic analysis confirmed the previously reported results that RNAi components for small RNA biogenesis are present in Cx. quinquefasciatus. In addition, we found important differences in the number and the expression of ago genes, the predicted domain architecture of Dcr, and the RNAi response between Cx. quinquefasciatus and the model organism D. melanogaster.

Transposition of Class II TEs is regulated by both cis-acting sequences and trans-acting host factors. In this study, we used purified Herves transposase to characterize the specific DNA-binding sites of the Herves transposase. The purified active Herves transposase showed site-specific binding to the subterminal and terminal sequences of the L- and R- ends of the element, respectively. Furthermore, the transposase bound strongly with the R-TIR but failed to bind to the L-TIR. We identified an 8bp sequence repeat as the transposase binding motif that is conserved on both the L- and R-end sequences and is critical and sufficient for Herves transposase binding.

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