Phytoremediation 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)等卤代VOCs是水和空气中最常见的污染物。这些化合物都对哺乳动物细胞色素P450 2E1的氧化敏感。在过去NIEHS资助的工作中，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，并且杨树能够吸收和降解VOCs。我们的工作使人们认识到野生型的TCE和CT摄取 植物太弱，不能显著降低根区水中的浓度，这促使人们寻找对VOCs具有更高降解活性的植物。我们在植物中表达了哺乳动物细胞色素P450 2E1，在包括杨树在内的几种植物中实现了对TCE的更大氧化和对VOCs的更大去除。与对照杨树相比，我们最新的r2e1杨木克隆具有高达132倍的TCE新陈代谢。我们目前正在测试转基因杨树对VOC的降解，以确定适合大田研究的优良无性系。在拟议的工作中，我们将把转基因杨树带到试验台规模，通过全尺寸树木的质量平衡研究，测试卤化VOC吸收与野生类型的VOC吸收。我们还将测试转基因植物从空气中去除VOCs的能力。我们还将利用最近完成的杨树基因组序列继续分析野生型植物在杨树中卤代挥发性有机化合物的代谢。根据基因组序列，我们将克隆与已知参与污染物降解的哺乳动物基因最相似的杨树基因。随着TCE和其他VOCs降解途径中所有基因的过度表达，我们将获得具有增强修复水和空气中VOCs能力的转基因植物。我们还将使用基因组序列来分析因此而上调的基因 利用杨树微阵列对污染物进行检测。这些实验有望提供有关哪些基因参与污染物降解的重要线索。我们期望这些实验将导致发现有用的杨树启动子，只有在底物存在的情况下才能表达解毒转基因。新的工作将集中在将降解有机磷神经毒素的基因引入植物中。哺乳动物细胞色素P450亚型已被证明能降解有机磷化合物。我们将使用我们现有的含有CYP3A4的杨树结构来确定毒死蜱是否被吸收和转化。为了优化降解有机磷化合物的体系，我们将表达细胞色素P3A4和有机磷水解酶PON1。随着我们对降解污染物的基因的新了解，我们将能够设计出更好的植物来进行植物修复。

Exposure to toxic volatile organic compounds (VOCs) continues to pose a significant risk to human health. Halogenated VOCs such as chloroform, carbon tetrachloride (CT) and trichloroethylene (TCE) are among the most common contaminants in water and air. These compounds share a susceptibility to oxidation by mammalian cytochrome P450 2E1. In past NIEHS-funded work, we have demonstrated that TCE and CT are oxidized by axenic poplar cell cultures using a pathway similar to that in mammals, and that poplar trees are able to take up and degrade VOCs. 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, motivating a search for plants with increased degradative activity toward VOCs. We have expressed mammalian cytochrome P450 2E1 in plants, achieving greater oxidation of TCE and greater removal of VOCs in several plants, including poplar. Our latest r2E1 aspen clones have up to 132-fold greater metabolism of TCE compared to control aspens. We are presently testing VOC degradation by transgenic poplar to identify superior clones for field-scale study. In the proposed work we will take transgenic poplar to test-bed scale, testing halogenated VOC uptake compared to wild-type by mass balance studies with full size trees. We will also test the ability of transgenic plants to remove VOCs from air. We will also continue analysis of wild-type plant metabolism of halogenated VOCs in poplar using the recently completed poplar genome sequence. Informed by the genome sequence, we will clone the poplar genes that are most similar to the mammalian genes known to be involved in degradation of pollutants. With overexpression of all genes in the pathway for degradation of TCE and other VOCs, we will produce transgenic plants with enhanced ability for remediation of VOCs in water and air. We will also use the genome sequence for analysis of genes that are upregulated in response to pollutants using poplar microarrays. These experiments are expected to yield important clues about which genes are involved in the degradation of pollutants. We expect these experiments to lead to the discovery of useful poplar promoters that will enable expression of detoxifying transgenes only when the substrates are present. New work will focus on the introduction into plants of genes for the degradation of organophosphorus neurotoxins. Mammalian cytochrome P450 isoforms have been shown to degrade organophosphorus compounds. We will use our existing poplar constructs containing CYP3A4 to determine whether chlorpyrifos is taken up and transformed. To optimize the system for degradation of organophosphorous compounds, we will express CYP3A4 and organophosphorus hydrolase, PON1. With our new knowledge of the genes involved in degradation of pollutants, we will be able to design superior plants for phytoremediation.