Gene duplications and subfunctionalization in response to challenges by dietary cardiac glycosides in phytophagous insects

点击翻译按钮获取中文摘要

Gene duplication and ensuing subfunctionalization are believed to play a decisive role in adaptation to new environmental challenges. This seems to be especially true for organisms exposed to environmental toxins like phytophagous insects encountering secondary compounds from toxic plants. Cardiac glycosides are well known for blocking the prominent and ubiquitous animal enzyme, Na,K-ATPase, which is responsible for maintaining membrane potentials, driving secondary transports, and restoring axonal resting potentials. Several insects exposed to cardiac glycosides in their host plants, like the milkweed bug Oncopeltus fasciatus, show amino acid substitutions in the cardiac glycoside binding site of the Na,K-ATPase alpha-subunit that strongly reduce the sensitivity of the enzyme for these toxins. In O. fasciatus, target-site insensitivity is associated with two rounds of duplications of the alpha-subunit, yielding three gene paralogs with differing sensitivities to cardiac glycosides and presumably also strongly differing kinetic characteristics. The three alpha-subunits may dimerize with four beta-subunits to yield functional Na,K-ATPases. This project explores the tissue-specific functional role of the resulting enzymes and the trade-offs between enzyme function and cardiac glycoside resistance by a) in vitro expression and functional characterization of the alpha-beta-combinations, b) identification of tissue specific Na,K-ATPase combinations at the protein level, and c) RNAi-mediated knockdown of the beta-subunits followed by phenotypic screening. In addition to its essential ion pumping function, the Na,K-ATPase also plays an important role in signal transduction and as such is involved in morphogenetic processes like the formation of tight or septate junctions in epithelia. These additional functions also need to be investigated in order to elucidate the mechanisms underlying the evolution of target-site insensitivity and subfunctionalization of the Na,K-ATPase genes. Morphological screening of tracheal tubes and epidermis of freshly molted bugs in which different beta-subunits have been knocked down will serve as a first step to elucidate whether this function is also affected by adaptation to toxic cardiac glycosides.