SGER: Role of Cytochrome P450 in Chemosensory Mediation of Oviposition in Papilio polyxenes

了解鳞翅目、蝴蝶和飞蛾等以植物为食的昆虫如何识别和利用它们赖以为生的植物，是了解这一物种丰富的生物群体多样化的关键。无法确定宿主植物如何被识别和加工的特定基因一直是一个主要障碍；尽管基因组计划提供了大量的清单，但为影响行为和生理的基因分配功能仍然是一项挑战，而且大多数模式生物（如遗传学上著名的“果蝇”——黑腹果蝇）的生态学特征存在大量的知识空白。介导植物-昆虫相互作用的关键基因是细胞色素P450单加氧酶，这种酶不仅负责解毒食叶毛虫消耗的杀虫植物化合物，而且还负责分解寻找植物产卵的雌性蝴蝶的触角和腿探测到的植物气味。黑燕尾凤蝶（Papilio polyxenes）提供了一个在单一物种中表征p450的绝佳机会，该物种影响毛虫对寄主植物的利用和雌性蝴蝶对寄主植物的识别。它的化学生态具有独特的特征，众所周知，产卵的兴奋剂以及影响毛虫生长的有毒化学物质。此外，作为一种非模式物种，其分子生物学的记录异常充分，毛虫肠道中的P450酶CYP6B1和CYP6B3有助于对寄主植物中的杀虫化合物进行解毒，而雌性蝴蝶腿上的CYP4G28可能参与了雌性蝴蝶对寄主植物的识别。因此，本SGER的目标是通过1确定CYP4G28功能。确定该基因是否在雌性蝴蝶产卵前用于“品尝”和识别寄主植物的感觉结构中特异性表达（使用免疫定位和原位杂交技术）；2. 通过分子模型来确定刺激产卵的化学物质是否“适合”进入CYP4G28蛋白的活性位点；3. 进行行为生物测定，以确定抑制蝴蝶前腿中的p450是否会阻止雌性蝴蝶识别它们的寄主植物。拟议活动产生的更广泛影响包括包括植物化学、分子生物学和昆虫学在内的跨学科培训；学生们将接受基础广泛的训练，这对他们在21世纪的竞争力至关重要。研究结果将发表在科学期刊、分子生物学和化学生态学结合的教科书、通俗著作和公开演讲中。对整个社会的其他潜在好处之一是对细胞基因组p450的进化过程有了更深入的了解。这些酶有助于杀虫剂抗性的发展和获得新的寄主植物，包括作物，具有巨大的生态和经济重要性的现象。

SGER: Role of cytochrome P450 in chemosensory mediation of oviposition in Papilio polyxenesMay Berenbaum, University of Illinois at Urbana-ChampaignUnderstanding how plant-feeding insects such as the Lepidoptera, the butterflies and moths, recognize and utilize the plants on which they feed is key to understanding the diversification of this exceptionally species-rich group of organisms. The inability to identify specific genes underlying how hostplants are identified and processed has been a major obstacle; although genome projects provide enormous inventories the challenge remains to assign function to genes affecting behavior and physiology and substantial knowledge gaps characterize the ecology of most model organisms such as Drosophila melanogaster, the "fruit fly" of genetics fame. Key genes mediating plant-insect interactions are the cytochrome P450 monooxygenases, enzymes responsible not only for detoxifying insecticidal plant compounds consumed by leaf-feeding caterpillars but also breaking down plant odorants detected by the antennae and legs of female butterflies searching for plants on which to lay eggs . An extraordinary opportunity for characterizing P450s within a single species that influence hostplant use by caterpillars and hostplant recognition by female butterflies is presented by the black swallowtail Papilio polyxenes. Its chemical ecology is uniquely well-characterized, stimulants for egg-laying as well as toxic chemicals that affect caterpillar growth are known. As well, for a non-model species, its molecular biology is unusually well-documented, with P450 enzymes CYP6B1 and CYP6B3 in caterpillar guts contributing to detoxification of insecticidal compounds in hostplants, and CYP4G28 characterized from the legs of female butterflies possibly involved in hostplant recognition by female butterflies. Thus, the objectives of this SGER are to determine CYP4G28 function by 1. determining whether this gene is expressed specifically in the sensory structures used by female butterflies for "tasting" and recognizing their hostplants prior to laying eggs (using the techniques of immunolocalization and in situ hybridization); 2. molecular modeling to determine whether chemicals known to stimulate egg-laying "fit" into the active site of the CYP4G28 protein; 3. conducting behavioral bioassays to determine if inhibiting the P450s in butterfly forelegs prohibits female butterflies from recognizing their hostplants. Broader impacts resulting from the proposed activities include interdisciplinary training encompassing elements of phytochemistry, molecular biology, and entomology; students will receive broad-based training essential for competitiveness in the 21st century. Findings will be published in scientific journals, in a textbook integrating molecular biology and chemical ecology, and in popular writings and public lectures. Among other potential benefits to society at large will be a greater understanding of the process by which cytochome P450s evolve. These enzymes contribute to the development of insecticide resistance and the acquisition of new hostplants, including crops, phenomena of tremendous ecological and economic importance.