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

基因复制和随后的亚功能化被认为在适应新的环境挑战中起着决定性作用。对于暴露于环境毒素的生物体，如植食性昆虫遇到有毒植物的次级化合物，这似乎尤其如此。众所周知，强心苷可阻断主要且普遍存在的动物酶Na，K-ATP酶，该酶负责维持膜电位、驱动次级转运和恢复轴突静息电位。暴露于其宿主植物中的强心苷的几种昆虫，如马利筋虫Oncopeltus fasciatus，在Na，K-ATP酶α-亚基的强心苷结合位点中显示氨基酸取代，这强烈降低了酶对这些毒素的敏感性。号手术fasciatus中，靶位点不敏感性与α-亚基的两轮重复有关，产生了对强心苷具有不同敏感性的三种基因旁系同源物，并且推测还具有强烈不同的动力学特征。三个α-亚基可以与四个β-亚基二聚化以产生功能性Na，K-ATP酶。该项目通过a）α-β-组合的体外表达和功能表征，B）在蛋白质水平鉴定组织特异性Na，K-ATP酶组合，和c）RNAi介导的β-亚基敲低，随后进行表型筛选，探索所得酶的组织特异性功能作用和酶功能与强心苷抗性之间的权衡。 除了其基本的离子泵送功能之外，Na，K-ATP酶还在信号转导中起重要作用，因此参与形态发生过程，如上皮中紧密连接或分隔连接的形成。这些额外的功能也需要进行调查，以阐明潜在的机制的Na，K-ATP酶基因的靶位点不敏感性和亚功能化的演变。对其中不同β-亚基已被敲低的新鲜蜕皮昆虫的气管和表皮进行形态学筛选将作为阐明这种功能是否也受适应毒性强心苷影响的第一步。

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.