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来说，这似乎尤其正确，比如食植物昆虫遇到有毒植物的次生化合物。众所周知，心脏糖苷可以阻断Na， k - atp酶这一重要且普遍存在的动物酶，而Na， k - atp酶负责维持膜电位、驱动二次转运和恢复轴突静息电位。一些暴露于宿主植物心糖苷的昆虫，如乳草虫Oncopeltus fasciatus，在Na， k - atp酶α亚基的心糖苷结合位点上显示出氨基酸取代，这大大降低了酶对这些毒素的敏感性。在筋膜棘鱼中，靶位不敏感与α -亚基的两轮复制有关，产生了对心糖苷敏感性不同的三个基因相似物，可能也有强烈不同的动力学特征。这3个α亚基可以与4个β亚基二聚产生功能性的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)在蛋白质水平上鉴定组织特异性Na， k - atp酶组合，以及c) rnai介导的β亚基敲低，然后进行表型筛选，探索所产生的酶的组织特异性功能作用以及酶功能与心脏糖苷抗性之间的权衡。除了其必需的离子泵送功能外，Na， k - atp酶还在信号转导中发挥重要作用，因此参与了上皮细胞紧密或分隔连接的形成等形态发生过程。这些额外的功能也需要研究，以阐明Na， k - atp酶基因靶位点不敏感和亚功能化进化的机制。对不同β -亚基被敲除的刚蜕皮臭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到毒性心苷适应的影响的第一步。

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.