转基因植物对有机污染物的吸收、转化和降解

有机污染物是土壤、水体和大气环境的重要污染物.利用和加强植物修复作用是控制环境污染的有效途径.近年来,一些具有修复功能的外源基因被陆续引入到植物中,使转基因植物的生物修复能力大大增强.文章介绍了植物对污染环境中有机污染物,尤其是持久性有机污染物(POPs)的吸收、转化和降解作用,阐述了转基因植物用于被污染环境修复方面的研究进展和应用前景.     

Chemical and Biological Parameters as Tools to Evaluate and Improve Heavy Metal Phytoremediation

In this review, chemical and biological parameters are discussed thatstrongly influence the speciation of heavy metals, their availability tobiological systems and, consequently, the possibilities to usebioremediation as a cleanup tool for heavy metal polluted sites. In orderto assess heavy metal availability, a need exists for rapid, cost-effectivesystems that reliably predict this parameter and, based on this, thefeasibility of using biological remediation techniques for site managementand restoration. Special attention is paid to phytoremediation as anemerging technology for stabilization and remediation of heavy metalpollution. In order to improve phytoremediation of heavy metal pollutedsites, several important points relevant to the process have to beelucidated. These include the speciation and bioavailability of the heavymetals in the soil determined by many chemical and biological parameters,the role of plant-associated soil microorganisms and fungi inphytoremediation, and the plants. Several options are described how plant-associated soil microorganisms canbe used to improve heavy metal phytoremediation.     

Siderophores,NTA, and Citrate: Potential Soil Amendments to Enhance Heavy Metal Mobility in Phytoremediation

Phytoremediation of heavy metal-contaminated sites is often limited by the low bioavailability of the contaminants. Complexing agents can help to improve this technique by enhancing heavy metal solubility. We investigated the effect of three organic chelating agents, that is, the siderophore desferrioxamine B (DFOB), nitrilotriacetate (NTA), and citrate on binding of Cu, Zn, and Cd by either Namontmorillonite, kaolinite, or goethite. The different effects of the complexing agents on metal sorption can mainly be explained by the differences in stability constants and surface charge characteristics. In the presence of clay minerals, NTA was the most efficient ligand with respect to mobilization of heavy metals. In goethite suspensions, the effect of DFOB was more pronounced. In all systems, Cu proved to be the most affected element by the presence of the ligands. In batch experiments with heavy metal-contaminated soils from field sites, NTA was the most efficient metal mobilizer.     

Metal Tolerance and Accumulation Ability of the Ni Hyperaccumulator Streptanthus polygaloides Gray (Brassicaceae)

High concentrations of metals occur in some plant species (termed hyperaccumulators), such as the Ni hyperaccumulator Streptanthus polygaloides. We determined the tolerance of S. polygaloides to, and its accumulation abilities for, six metals (Ni, Zn, Cu, Co, Mn, and Pb). Potting mix concentrations used for all metals ranged from 0 to 1200 μg/g dry weight. For Ni, a treatment of 1600 μg/g was included. For Mn, treatments of 1600, 2000, and 2500 μg/g also were used, and for Pb these concentrations plus 3500 μg/g were included. Germination, plant number per pot, and size at days 30 and 39, number of plants at the end of the experiment (day 49), flower production, and metal concentration in the aboveground biomass were documented. Lead and Ni showed no consistent effects on plant performance, but yielded increased tissue metal concentrations. Streptanthus polygaloides was more sensitive to Co, Cu, and Zn, as ≥ 400 mg/g significantly suppressed plant growth, survival, and flower production. Tissue metal concentrations also were increased to maxima of 1500 μg Co/g, 120 μg Cu/g, and 6000 μg Zn/g. Manganese affected S. polygaloides less markedly, as ≥ 800 mg/kg decreased growth, survival, and flower production. Maximum tissue Mn concentration was 2900 μg/g. We concluded that S. polygaloides would be an appropriate phytoextractor for soils contaminated with Ni or low levels of Co but would not be useful for Cu, Zn, Mn, and Pb.     

Development of Bacterium-Based Heavy Metal Biosorbents: Enhanced Uptake of Cadmium and Mercury by Escherichia coli Expressing a Metal Binding Motif

A gene coding for a de novo peptide sequence containing a metal binding motif was chemically synthesized and expressed in Escherichia coli as a fusion with the maltose binding protein. Bacterial cells expressing the metal binding peptide fusion demonstrated enhanced binding of Cd²⁺ and Hg²⁺ compared to bacterial cells lacking the metal binding peptide. The potential use of genetically engineered bacteria as biosorbents for the removal of heavy metals from wastewaters is discussed.     

毛果杨HMA基因家族的生物信息学分析

重金属转运ATP酶（heavy metal transporting ATPase,HMA）是一种通过水解ATP跨膜运送重金属阳离子的转运蛋白,属于P-ATPase家族中一个亚类。不同HMA蛋白对重金属离子的转运具有选择性,在植物修复重金属污染土壤方面起着重要作用。依据毛果杨全基因组测序的结果,以及HMA基因蛋白的序列和功能特征,从毛果杨基因组中鉴定了13个HMA基因家族成员,分属于Zn亚类（Zn2＋/Co2＋/Cd2＋/Pb2＋P1B-ATPase）和Cu亚类（Cu＋/Ag＋P1B-ATPase）两个亚家族,主要分布于1、3号染色体上。生物信息学分析表明,毛果杨HMA基因的氨基酸序列一致性介于21.3%~89.3%,且具有保守的基序CPC、HP、DKTGT、TGEx、GDG、PxD和CxxC等。蛋白理化特征分析显示,多数毛果杨HMA蛋白稍偏酸性,结构稳定性较好,蛋白脂溶指数高,稍具疏水性。密码子偏好性分析显示,毛果杨HMA蛋白14个氨基酸中存在16个高频密码子,另有1个终止密码子为高频密码子,显示出毛果杨的种属特征。研究结果展示了毛果杨HMA基因家族的基本信息和特点,为深入研究毛果杨HMA基因的功能搭建了基础平台。     

Increased metal tolerance and bioaccumulation of zinc and cadmium in Chlamydomonas reinhardtii expressing a AtHMA4 C-terminal domain protein

The use of microalgal biomass for metal pollutant bioremediation might be improved by genetic engineering to modify the selectivity or capacity of metal biosorption. A plant cadmium (Cd) and zinc (Zn) transporter (AtHMA4) was used as a transgene to increase the ability of Chlamydomonas reinhardtii to tolerate 0.2 mM Cd and 0.3 mM Zn exposure. The transgenic cells showed increased accumulation and internalization of both metals compared to wild-type. AtHMA4 was expressed either as the full-length (FL) protein or just the C-terminal (CT) tail, which is known to have metal-binding sites. Similar Cd and Zn tolerance and accumulation was observed with expression of either the FL protein or CT domain, suggesting that enhanced metal tolerance was mainly due to increased metal binding rather than metal transport. The effectiveness of the transgenic cells was further examined by immobilization in calcium alginate to generate microalgal beads that could be added to a metal contaminated solution. Immobilization maintained metal tolerance, while AtHMA4-expressing cells in alginate showed a concentration-dependent increase in metal biosorption that was significantly greater than alginate beads composed of wild-type cells. This demonstrates that expressing AtHMA4 FL or CT has great potential as a strategy for bioremediation using microalgal biomass.     

伴矿景天植物螯合肽合酶基因的克隆及功能分析

重金属超积累植物由于长期生长在高浓度的重金属环境中,使得经由植物螯合肽（phytochelatins, PCs）解毒途径来应对重金属毒害代价高昂。我们从Zn/Cd超积累植物伴矿景天（Sedum plumbizincicola）中克隆了植物螯合肽合酶（phytochelatin synthase, PCS）基因SepPCS。该基因在裂殖酵母和拟南芥中表达后都具有PCS活性,而且能够互补它们的PCs缺失突变体的Cd敏感表型。SepPCS在伴矿景天中的表达受到高浓度Cd处理的诱导。与其近亲非超积累生态型东南景天（S. alfredii）相比,虽然伴矿景天地上部PCs与Cd的摩尔比远低于东南景天,但是在高浓度Cd处理时PCs含量以及PCs与Cd的摩尔比急剧增加。我们推测在伴矿景天应对Cd毒害的过程中, PCs起到一定的作用,并且在高浓度Cd胁迫时地上部PCs依赖的解毒作用有所加强。     

Seedling Growth and Heavy Metal Accumulation of Candidate Woody Species for Revegetating Korean Mine Spoils

Selecting plant species that can overcome harsh soil and microclimatic conditions and speed the recovery of degraded minelands remains a worldwide restoration challenge. This study evaluated the potential of three woody species and various organic and inorganic fertilization treatments for revegetating abandoned metalliferous mines in Korea. We compared survival, growth, and heavy metal uptake of species common to Korean minelands in two spoil types and a reference forest soil. Substrate type and fertilization both influenced seedling growth and metal concentrations substantially, but they had little effect on seedling survival. Fertilization increased the growth of all three species when grown in mine spoils but influenced the growth of seedlings grown in forest soil only marginally. Initial seedling survival and growth indicate that the study species can tolerate the heavy metal concentrations and other soil constraints of metalliferous spoil types. We estimate that plants can stabilize 2–22% of various heavy metals contained in spoil materials into plant biomass during 20 years of plantation growth. Combined with the erosion control and site amelioration benefits of mineland reforestation, stabilization of heavy metals in forest biomass justifies this treatment on abandoned Korean metalliferous mines.     

镉锌复合污染对龙葵苗期生长和镉锌累积特性的影响

采用营养液培养法研究了苗期龙葵(Solanumnigrum L.)对镉锌复合污染的反应及其对镉锌的吸收和积累特性.结果表明:(1)高浓度镉(200μmol·L-1)、锌(500μmol·L-1)胁迫使苗期龙葵植株生长受到严重抑制,但低浓度镉锌复合处理的部分植株生物量甚至超过了对照.(2)添加锌对苗期龙葵地上部镉积累有显著的影响(P<0.05);当锌浓度为100μmol·L-1时,镉积累量达到峰值,其中的100Cd/100Znμmol·L-1处理组合的镉积累量为所有处理中的最大值(地上部积累量高达171.31μg·plant-1);当锌浓度增加到500μmol·L-1时,植株体内镉积累量则呈现下降趋势.(3)添加镉对苗期龙葵地上部锌积累也有显著的影响(P<0.05);当镉浓度达50μmol·L-1时,根、茎、叶中锌积累量均达到最高值;当镉浓度为200μmol·L-1时,各处理单株地上部锌积累量最低.(4)从植株整体的镉、锌积累量来看,苗期龙葵地上部镉积累量最高达根系的14.67倍,而其锌积累量最高达根系的13.59倍.研究发现,龙葵幼苗根系、茎和叶中镉、锌含量随营养液中镉、锌浓度的增加而显著增加(P<0.05);添加锌在一定程度上缓解了镉对苗期龙葵的毒害效应,低浓度锌还可以提高植株对镉的吸收和积累;添加镉在一定程度上增强了植株对锌的耐受性和吸收富集能力;植株地上部的镉、锌积累量明显高于根系.     

Phytoremediation of pyrene in a Cecil soil under field conditions

We evaluated the effects of annual ryegrass (Lolium multiflorum Lam.) and phosphorus (P) availability on the dissipation of pyrene added at a concentration of approximately 600 mg kg-1 dry soil in the top 7.5 cm of a Cecil loamy sand (fine, kaolinitic, thermic Typic Kanhapludults) in a 10-month experiment under field conditions in Clemson, South Carolina. Plastic canopies were installed to prevent flooding of plots and raindrop dispersion of pyrene. Treatment factors were pyrene, vegetation, and available P levels. Each of the eight treatments had four replicates. The soil was adjusted to low and high P concentrations (an average of 41 and 66 kg extractable P ha-1, respectively). After a 175-d lag period for all treatments, the rate of pyrene removal followed first-order kinetics. The first-order rate constant was significantly higher in nonvegetated (0.098 d-1) than vegetated treatments (0.034 d-1). These data suggest that the presence of easily biodegradable organic matter from plant roots slowed the removal rate of pyrene. The levels of available P did not affect the rate of pyrene dissipation. Pyrene decreased below the detection limit of 6.25 mg kg-1 dry soil in all treatments after 301 d.     

镉中毒大鼠睾丸与肝脏金属硫蛋白表达的时相研究

啮齿目动物睾丸对镉毒性较肝脏更敏感.为阐明睾丸的镉毒性分子机制,比较了肝脏与睾丸金属硫蛋白（MT）表达的时相变化.mRNA采用RT-PCR技术分析并用光密度扫描定量;蛋白质定量用ELISA方法.结果显示,睾丸中存在MT,镉中毒后MT1与MT2 mRNA明显升高,但MT没有相应增加;肝脏镉中毒后MTmRNA与MT均明显升高.结果提示：镉虽然能诱导睾丸MTmRNA的转录,但没有促进其MT的合成,这可能是睾丸对镉毒性与致癌作用较肝脏更敏感的重要原因.     

Integrated Fertilization Regimes Boost Heavy Metals Accumulation and Biomass of <i>Sedum alfredii</i> Hance

The hyperaccumulator Sedum alfredii Hance (S. alfredii) may be employed for zinc (Zn) and cadmium (Cd)-polluted soil remediation. However, the low phytoremediation efficiency, related to the low biomass production, limits its use with that purpose. In this experiment, nitrogen (N), phosphorus (P), and potassium (K) fertilizers, and organic manure were applied to investigate the phytoremediation ability of S. alfredii. Hydroponic and pot experiments were conducted using Zn-Cd polluted soil. The hydroponic experiment indicated that appropriate fertilizer application could increase (p < 0.05) the amount of accumulated Zn and Cd in S. alfredii. When N supply ranged from 0.5 to 2.5 mmol L⁻¹, it could improve growth and accumulation of Zn and Cd in whole plants of S. alfredii. The 1 mmol L^-1 N was an optimal N dosage for shoot biomass production and Cd accumulation in shoots, while the 2.5 mmol L^-1 was an optimal N dosage for Zn accumulation in shoots. Both low (<0.05 mmol L^-1) and high (>0.8 mmol L^-1) P supply decreased growth, and Zn/Cd accumulation in whole plants of the studied species. The 0.1 mmol L^-1 P was an optimal dosage for S. alfredii biomass production and Zn/Cd accumulation in shoots. The supply levels within the range from 0.3 to 1 mmol L^-1 K could significantly improve the biomass production of S. alfredii and its capability to accumulate Zn and Cd in the biomass. The 0.5 mmol L^-1 K was an optimal dosage for the whole biomass production and Zn accumulation in shoots, while the 1 mmol L^-1 was an optimal K dosage for Zn accumulation in shoots, which was 17.2% higher than the control. Moreover, the soil pot experiment showed that the combination of organic (fermented manure) and inorganic fertilizers made significant effects on the Zn and Cd-polluted soil remediation by S. alfredii. These effects varied, however, with the application of different proportions of N, P, K and organic matter. The Zn accumulation by S. alfredii reached the highest efficiency ability under the highest fertilizer mixing rate (N: 50 mg kg^-1, P: 40 mg kg^-1, K: 100 mg kg^-1, organic matter: 1%). Even more, S. alfredii showed the strongest ability to accumulate Cd with a lower fertilizer mixing rate (N: 25mg kg^-1, P: 20mg kg^-1, K: 50 mg kg^-1, organic matter: 0.5%).

     

Characterization of Transgenic Tobacco Plants Containing Bacterial bphc Gene and Study of Their Phytoremediation Ability

Genetically modified plants can serve as an efficient tool for remediation of diverse dangerous pollutants of the environment such as pesticides, heavy metals, explosives and persistent organic compounds. Transgenic lines of Nicotiana tabacum containing bacterial bphC gene from the degradation pathway of polychlorinated biphenyls (PCBs) were tested. The product of the bphC gene – enzyme 2,3-dihydroxybiphenyl-1,2-dioxygenase is responsible for cleaving of the biphenyl ring. The presence of bphC gene in transgenic plants was detected on DNA, RNA and protein level. The expression of the bphC/His gene was verified after purification of the enzyme from plants by affinity chromatography followed by a Western blot and immunochemical assay. The enzyme activity of isolated protein was detected.Efficient transformation of 2,3-DHB by transgenic plants was achieved and the lines also exhibited high production of biomass. The transgenic plants were more tolerant to the commercial PCBs mixture Delor 103 than non-transgenic tobacco. And finally, the higher decrease of total PCB content and especially congener 28 in real contaminated soil from a dumpsite was determined after cultivation of transgenic plant in comparison with non-transgenic tobacco. The substrate specificity of transgenic plants was the same as substrate specificity of BphC enzyme.     

Production and Analysis of Tobacco Transgenic Plants Expressing the Agrobacterial Gene for Tryptophan Monooxygenase

The expression of the agrobacterial iaaM gene for tryptophan monooxygenase, the enzyme catalyzing the first step in the auxin biosynthesis, induced substantial physiological and biochemical changes in transgenic tobacco (Nicotiana tabacum L.) plants. All lines of transgenic plants grown in vitro manifested abnormal phenotypes: enhanced root formation, adventitious roots on stems, and curled leaves. When grown in vivo, plants manifested abnormal, normal, or intermediate phenotype. Under conditions of a greenhouse, the abnormal plants contained the highest amount of auxins in their leaves and manifested an increased number of adventitious roots, poor reproductivity, and the loss in seed germination. Transgenic plants with the normal phenotype did not substantially differ from the wild-type plants in their morphology, and their auxin content was lower than in the abnormal plants. The intermediate-phenotype plants were devoid of some morphological properties characteristic of the abnormal plants. Only the seeds of normal- and intermediate-phenotype transgenic plants germinated at a high rate.     

Utilization of Grasses for Potential Biofuel Production and Phytoremediation of Heavy Metal Contaminated Soils

This research focuses on investigating the use of common biofuel grasses to assess their potential as agents of long-term remediation of contaminated soils using lead as a model heavy metal ion. We present evidence demonstrating that switch grass and Timothy grass may be potentially useful for long-term phytoremediation of heavy metal contaminated soils and describe novel techniques to track and remove contaminants from inception to useful product. Enzymatic digestion and thermochemical approaches are being used to convert this lignocellulosic feedstock into useful product (sugars, ethanol, biocrude oil + biochar). Preliminary studies on enzymatic hydrolysis and fast pyrolysis of the Switchgrass materials that were grown in heavy metal contaminated soil and non-contaminated soils show that the presence of lead in the Switchgrass material feedstock does not adversely affect the outcomes of the conversion processes. These results indicate that the modest levels of contaminant uptake allow these grass species to serve as phytoremediation agents as well as feedstocks for biofuel production in areas degraded by industrial pollution.