Project 5: Phytorermediation of Pollutants Using Transgenic Plants

接触有毒挥发性有机化合物(VOCs)继续对人类健康构成重大威胁。氯仿、四氯化碳(CT)和三氯乙烯(TCE)等VOCs以及苯和其他单环芳烃是水和空气中最常见的污染物。这些化合物都对哺乳动物肝脏细胞色素P450 2E1的氧化敏感。在过去由NIEHS资助的威斯康星大学的工作中，我们已经证明了TCE和CT通过与哺乳动物相似的途径被无菌杨树细胞培养氧化，并且野生型杨树能够吸收和降解TCE和CT。 我们的工作使人们认识到野生型植物对TCE和CT的吸收太弱，无法显著降低根区水中的TCE和CT浓度，这为对VOCs具有更高降解活性的植物的发展提供了动力。为此，我们在植物中表达了哺乳动物细胞色素P450 2E1(CYP2E1)，在转基因杨树中实现了对TCE的更大数量级的氧化。我们目前正在进行野生型杨树和转基因杨树对三氯乙烯的田间降解试验 能够进行质量平衡测量的试验台设备中的刻度。在这一应用中，我们提出了以下具体目标：比较野生型和现有的转基因杨树品系在田间试验中降解TCE、CT和PCE的能力；确定受污染物存在上调的基因；克隆和分析参与TCE及其代谢产物降解的植物基因；确定转CYP2E1基因杨树中TCE和苯的代谢物；通过增加叶表面开放气孔的数量来增加对VOCs的吸收；在植物中表达降解有机磷神经毒素的有机磷羟基酶PON1；并测定了VOCs对转基因植物根际物质、叶凋落物和根际土壤的生态毒性。

Exposure to toxic volatile organic compounds (VOCs) continues to pose a significant risk to human health. VOCs such as chloroform, carbon tetrachloride (CT) and trichloroethylene (TCE), along with benzene and other single-ring aromatics are among the most common contaminants in water and air. These compounds share a susceptibility to oxidation by the mammalian hepatic cytochrome P450 2E1. In past NIEHS-funded work at UW, we have demonstrated that TCE and CT are oxidized by axenic poplar cell cultures using a pathway similar to that in mammals, and that wild-type poplar are able to take up and degrade TCE and CT. Our work has led to the realization that TCE and CT uptake by wild-type plants is too weak to significantly reduce their concentration in root zone water, providing motivation for the development of plants with increased degradative activity toward VOCs. Toward this end we have expressed mammalian cytochrome P450 2E1 (CYP2E1) in plants, achieving orders of magnitude greater oxidation of TCE in transgenic poplar. We are presently engaged in testing TCE degradation by wild-type and CYP2E1 transgenic poplar at field scale in a test bed facility capable of mass balance measurements. In this application we propose the following specific aims: compare the abilities of wild-type and existing CYP2E1 transgenic poplar lines to degrade TCE, CT, and PCE in field tests; identify genes that are up-regulated by the presence of pollutants; clone and analyze the plant genes involved in the degradation of TCE and its metabolites; determine the metabolites of TCE and benzene in CYP2E1 transgenic poplar; increase the uptake of VOCs from the air by increasing the number of open stomata on leaf surfaces; express the organophosphorus hydroxylase, paraoxonase PON1, in plants for the degradation of organophosphate neurotoxins; and determine the ecotoxicity to earthworms of root materials, leaf litter and rhizosphere soil of CYP2E1 transgenic plants exposed to VOCs.