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

基因复制和随之而来的亚功能化被认为在适应新的环境挑战方面起着决定性的作用。对于暴露在环境毒素中的有机体来说，情况似乎尤其如此，比如植食性昆虫遇到有毒植物的次生化合物。众所周知，心脏糖苷可以阻断重要的和普遍存在的动物酶Na，K-ATPase，该酶负责维持膜电位，驱动二次转运，并恢复轴突静息电位。几种昆虫暴露在寄主植物中的心脏糖苷类化合物中，如乳草虫OnCopeltus Fasciatus，在Na，K-ATPaseα亚单位的心脏糖苷结合部位显示出氨基酸替换，大大降低了酶对这些毒素的敏感性。在扁平线虫中，靶点不敏感与两轮阿尔法亚基的复制有关，产生了三个对心脏糖苷类药物敏感性不同的基因同源基因，推测也有非常不同的动力学特征。这三个α-亚基可能与四个β-亚基二聚，从而产生功能性的Na，K-ATPase。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定的Na，K-ATPase组合，以及c)RNAi介导的β-亚基的敲除和表型筛选，探索所产生的酶的组织特异性功能作用和酶功能与心脏糖苷耐药性之间的权衡。除了其基本的离子泵功能，Na，K-ATPase还在信号转导中发挥重要作用，因此参与了形态发生过程，如在上皮细胞中形成紧密或分隔的连接。为了阐明Na，K-ATPase基因靶点不敏感性和亚功能化的进化机制，还需要研究这些额外的功能。对不同β-亚基被击倒的新鲜蜕皮臭虫的气管和表皮进行形态筛选，将是阐明这种功能是否也受到对有毒心脏糖苷的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.