环境重金属污染的植物修复及基因工程在其中的应用

随着工业技术的发展,重金属在土壤和水体中的含量越来越高,重金属污染已日益成为威胁人类健康和人类生活质量的严重的社会问题和环境问题。植物修复可部分解决这一问题且正引起人们的普遍关注。但现在发现许多用于修复的超量积累植物生长缓慢、植株矮小、地上部生物量小,成了实际应用中的最大限制。利用基因工程手段改变植物对重金属吸收、转运、积累和忍耐的机制,从而提高植物对重金属的富集能力,将成为今后植物修复领域研究的一个重要方向。     

Identification and characterization of PAH-degrading endophytes isolated from plants growing around a sludge dam

Abstract

This study involved the isolation of bacteria endophytes with PAH-degrading ability from plants growing around a sludge dam. A total of 19 distinct isolates that were morphologically identified were isolated from 4 species of plant with a follow-up confirmatory identification using the molecular technique. Polymerase chain reaction (PCR) of the 16S rRNA gene with specific primers (16S-27F PCR and 16S-1491R PCR) was carried out. The sequence of the PCR products was carried out, compared with similar nucleotides available in GenBank. Results of the phylogenetic analysis of the isolates indicated their belonging to 4 different clades including Proteobacteria, Actinobacteria, Cyanobacteria, and Firmicutes. These were related to the genera Bacillus, Pseudomonas, Terribacillus, Virgibacillus, Stenotrophomonas, Paenibacillus, Brevibacterium, Geobacillus, Acinetobacter. From the result, Pseudomonas demonstrated a high incidence in the plants sampled. The in-vitro degradation study and the presence of dioxygenase genes indicated that these lists of endophytes are able to use the list of PAHs tested as their source of food and energy leading to their breakdown. This means that the bacterial endophytes contributed to the remediation of petroleum hydrocarbons in planta, a situation that may have been phytotoxic to plant alone. Therefore, these bacteria endophytes could be potential organisms for enhanced phytoremediation of PAHs.     

持久性有机污染土壤的植物修复及其机理研究进展

随着人类对化学品的依赖程度越来越高,环境的有机污染状况也越来越严重.有机污染土壤的植物修复是指利用植物在生长过程中,吸收、降解、钝化有机污染物的一种原位处理污染土壤的方法,具有应用成本低、生态风险小、对环境副作用小等特点.本文综述了近年来国内外有机污染土壤的植物修复研究进展情况,重点介绍了多氯联苯、多环芳烃、农药和硝基芳香化合物等持久性有机污染物的植物修复,阐述了有机污染土壤植物修复的关键机制,并分析了该技术在实际工程应用中的局限性及应考虑的因素.最后,指出了今后该领域的重点研究方向.     

汞污染土壤植物修复中转基因技术的应用

汞是一种全球性污染物.随着工业的发展、汞矿开采的加速,土壤汞污染日益严峻,给生态环境和人类生活带来了严重的威胁.因此,汞污染土壤的修复问题受到了广大学者的关注.针对受污染的土壤修复问题,植物修复技术具有成本低和不会给环境带来新的危害等特点.本文对目前国内外汞污染土壤修复植物修复技术的研究进行了综述,介绍了转基因技术在汞污染土壤中的应用,探讨了转基因技术在环境治理和环境修复中潜在的应用价值.     

Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of PCB phytoremediation

The aim of this work is to increase the efficiency of the biodegradation of polychlorinated biphenyls (PCBs) by the introduction of bacterial genes into the plant genome. For this purpose, we selected the bphC gene encoding 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas testosteroni B-356 to be cloned into tobacco plants. The dihydroxybiphenyldioxygenase enzyme is the third enzyme in the biphenyl degradation pathway, and its unique function is the cleavage of biphenyl. Three different constructs were designed and prepared in E. coli: the bphC gene being fused with the beta-glucuronidase (GUS) gene, with the luciferase (LUC) gene, and with histidine tail in three separate plant cloning vectors. The GUS and LUC genes were chosen because they can be used as markers for the easy detection of transgenic plants, while histidine tail better enables the isolation of protein expressed in plant tissue. The prepared vectors were then introduced into cells of Agrobacterium tumefaciens. The transient expression of the prepared genes was first studied in cells of Nicotiana tabacum. Once this ability had been established, model tobacco plants were transformed by agrobacterial infection with the bphC/GUS, bphC/LUC, and bphC/His genes. The transformed regenerants were selected on media using a selective antibiotic, and the presence of transgenes and mRNA was determined by PCR and RT-PCR. The expression of the fused proteins BphC/GUS and BphC/LUC was confirmed histochemically by analysis of the expression of their detection markers. Western blot analysis was performed to detect the presence of the BphC/His protein immunochemically using a mouse anti-His antibody. Growth and viability of transgenic plants in the presence of PCBs was compared with control plants.     

Exploitation of endophytes for sustainable agricultural intensification

Intensive agriculture, which depends on unsustainable levels of agrochemical inputs, is environmentally harmful, and the expansion of these practices to meet future needs is not economically feasible. Other options should be considered to meet the global food security challenge. The plant microbiome has been linked to improved plant productivity and, in this microreview, we consider the endosphere – a subdivision of the plant microbiome. We suggest a new definition of microbial endophyte status, the need for synergy between fungal and bacterial endophyte research efforts, as well as potential strategies for endophyte application to agricultural systems.     

Endophyte-assisted phytoremediation: mechanisms and current application strategies for soil mixed pollutants

Abstract

Phytoremediation uses plants and associated microbes to remove pollutants from the environment and is considered a promising bioremediation method. Compared with well-described single contaminant treatments, the number of studies reporting phytoremediation of soil mixed pollutants has increased recently. Endophytes, including bacteria and fungi, exhibit beneficial traits for the promotion of plant growth, stress alleviation, and biodegradation. Moreover, endophytes either directly or indirectly assist host plants to survive high concentrations of organic and inorganic pollutants in the soil. Endophytic microorganisms can also regulate the plant metabolism in different ways, exhibiting a variety of physiological characteristics. This review summarizes the taxa and physiological properties of endophytic microorganisms that may participate in the detoxification of contaminant mixtures. Furthermore, potential biomolecules that may enhance endophyte mediated phytoremediation are discussed. The practical applications of pollutant-degrading endophytes and current strategies for applying this valuable bio-resource to soil phytoremediation are summarized.     

Heavy metal pollution in aquatic ecosystems and its phytoremediation using wetland plants: an ecosustainable approach

This review addresses the global problem of heavymetal pollution originating from increased industrialization and urbanization and its amelioration by using wetland plants both in a microcosm as well as natural/field condition. Heavymetal contamination in aquatic ecosystems due to discharge of industrial effluents may pose a serious threat to human health. Alkaline precipitation, ion exchange columns, electrochemical removal, filtration, and membrane technologies are the currently available technologies for heavy metal removal. These conventional technologies are not economical and may produce adverse impacts on aquatic ecosystems. Phytoremediation of metals is a cost-effective "green" technology based on the use of specially selected metal-accumulating plants to remove toxic metals from soils and water. Wetland plants are important tools for heavy metal removal. The Ramsar convention, one of the earlier modern global conservation treaties, was adopted at Ramsar, Iran, in 1971 and became effective in 1975. This convention emphasized the wise use of wetlands and their resources. This review mentions salient features of wetland ecosystems, their vegetation component, and the pros and cons involved in heavy metal removal. Wetland plants are preferred over other bio-agents due to their low cost, frequent abundance in aquatic ecosystems, and easy handling. The extensive rhizosphere of wetland plants provides an enriched culture zone for the microbes involved in degradation. The wetland sediment zone provides reducing conditions that are conducive to the metal removal pathway. Constructed wetlands proved to be effective for the abatement of heavymetal pollution from acid mine drainage; landfill leachate; thermal power; and municipal, agricultural, refinery, and chlor-alkali effluent. the physicochemical properties of wetlands provide many positive attributes for remediating heavy metals. Typha, Phragmites, Eichhornia, Azolla, Lemna, and other aquatic macrophytes are some of the potent wetland plants for heavy metal removal. Biomass disposal problem and seasonal growth of aquatic macrophytes are some limitations in the transfer of phytoremediation technology from the laboratory to the field. However, the disposed biomass of macrophytes may be used for various fruitful applications. An ecosustainable model has been developed through the author's various works, which may ameliorate some of the limitations. The creation of more areas for phytoremediation may also aid in wetlands conservation. Genetic engineering and biodiversity prospecting of endangered wetland plants are important future prospects in this regard.     

The use of halophytic plants for salt phytoremediation in constructed wetlands

This research studied the use of constructed wetlands (CWs) to reduce water salinity. For this purpose, three halophytic species of the Chenopodiaceae family (Salicornia europaea, Salsola crassa, and Bienertia cycloptera) that are resistant to saline conditions were planted in the CWs, and experiments were conducted at three different salinity levels [electrical conductivity (EC)～2, 6, 10 dS/m]. EC and concentrations of calcium (Ca), magnesium (Mg), sodium (Na), and chlorine (Cl) were measured before and after phytoremediation with a retention time of 1 week. The results suggested that these plants were able to grow well and complete their life cycles at all the salinity levels within this study. Moreover, these plants reduced the measured parameters to acceptable levels. Therefore, these plants can be considered good options for salt phytoremediation.     

Enhancing ascorbate in fruits and tubers through over-expression of the L-galactose pathway gene GDP-L-galactose phosphorylase

Ascorbate, or vitamin C, is obtained by humans mostly from plant sources. Various approaches have been made to increase ascorbate in plants by transgenic means. Most of these attempts have involved leaf material from model plants, with little success reported using genes from the generally accepted l-galactose pathway of ascorbate biosynthesis. We focused on increasing ascorbate in commercially significant edible plant organs using a gene, GDP-l-galactose phosphorylase (GGP or VTC2), that we had previously shown to increase ascorbate concentration in tobacco and Arabidopsis thaliana. The coding sequence of Actinidia chinensis GGP, under the control of the 35S promoter, was expressed in tomato and strawberry. Potato was transformed with potato or Arabidopsis GGP genes under the control of the 35S promoter or a polyubiquitin promoter (potato only). Five lines of tomato, up to nine lines of potato, and eight lines of strawberry were regenerated for each construct. Three lines of tomato had a threefold to sixfold increase in fruit ascorbate, and all lines of strawberry showed a twofold increase. All but one line of each potato construct also showed an increase in tuber ascorbate of up to threefold. Interestingly, in tomato fruit, increased ascorbate was associated with loss of seed and the jelly of locular tissue surrounding the seed which was not seen in strawberry. In both strawberry and tomato, an increase in polyphenolic content was associated with increased ascorbate. These results show that GGP can be used to raise significantly ascorbate concentration in commercially significant edible crops.     

Identification and characterization of bacterial endophytes of rice

We isolated seven different bacteria from rice seedlings grown from surface sterilized seeds. Three were associated with the rice seed husk and the other four were growing endophytically within the seed. Microscopic studies revealed that the endophytes were concentrated in the root stele region. Some of the bacteria exhibited strong anti-fungal activity against Rhizoctonia solani, Pythium myriotylum, Guamannomyces graminis and Heterobasidium annosum.     

Dark septate endophytes: a review of facultative biotrophic root-colonizing fungi

Dark septate root endophytes (DSE) are conidial or sterile fungi (Deuteromycotina, Fungi Imperfecti) likely to be ascomycetous and colonizing plant roots. They have been reported for nearly 600 plant species representing about 320 genera and 100 families. DSE fungi occur from the tropics to arctic and alpine habitats and comprise a heterogeneous group that functionally and ecologically overlaps with soil fungi, saprotrophic rhizoplane-inhabiting fungi, obligately and facultatively pathogenic fungi and mycorrhizal fungi. Numerous species of undescribed sterile and anamorphic taxa may also await discovery. Although DSE are abundant in washed root and soil samples from various habitats, and are easily isolated from surface-sterilized roots of ecto-, ectendo-, endo- and non-mycorrhizal host species, their ecological functions are little understood. Studies of DSE thus far have yielded inconsistent results and only poorly illustrate the role of DSE in their natural habitats. These inconsistencies are largely due to the uncertain taxonomic affinities of the strains of DSE used. In addition, because different strains of a single anamorph taxon seem to vary greatly in function, no clear generalizations on their ecological role have been drawn. This paper reviews the current literature on DSE and the ecology and discusses the need for and direction of future research.     

Presence of Neotyphodium-like endophytes in European grasses

In order to improve knowledge of Neotyphodium‐likc fungi in European grasses, microscopic detection of endophytic mycelium was achieved in living grasses and in seeds. Samples of grasses were gathered near permanent pastures or along paths in France (93 samples, 13 genera, 22 species). Typical convoluted mycelium was found inside leaf sheaths of four genera, especially in Festuca and Lolium spp. The results suggest that endophytes were present most often in the southern regions where plants may suffer from summer drought. Endophytes were looked for in 489 seed samples (63 genera, 237 species) received from 24 European botanical gardens. Neotyphodium was found in seeds of only six genera (22 species), especially in Festuca (15 species) and Lolium (three species). No endophytic mycelium was found in the other 215 species, although other work had demonstrated the presence of the fungus in 39 of the species analysed. Neotyphodium‐likc mycelium was detected for the first time in Festuca juncifolia, F. trachyphylla, F. vaginata, F. pseudodalmatica, F. rupicaprina, F. arenaria, Vulpia ciliata and Micropyrum tenellum. Further studies are needed for the correct identification of the Neotyphodium species observed.     

Enhancing the growth of Vicia faba plants by microbial inoculation to improve their phytoremediation potential for oily desert areas

Two experiments were conducted to investigate the effect of inoculating Vicia faba plants (broad beens) raised in clean and oily sand with nodule-forming rhizobia and plant-growth-promoting rhizobacteria (PGPR) on growth of these plants in sand and to test whether this can improve the phytoremediation potential of this crop for oily desert areas. It was found that crude oil in sand at concentrations < 1.0% (w/w) enhanced the plant heights, their fresh and dry weights, the total nodule weights per plant, and the nitrogen contents of shoots and fruits. Similar enhancing effects were recorded when roots of the young plants were inoculated with nodule bacteria alone, PGPR alone, or a mixture of one strain of nodule bacteria and one of the PGPR. Such plant growth effects were associated with a better phytoremediation potential of V. faba plants for oily sand. The total numbers of oil-utilizing bacteria increased in the rhizosphere and more hydrocarbons were eliminated in sand close to the roots. The nodule bacteria tested were two strains of Rhizobium leguminosarum and the PGPR were Pseudomonas aeruginosa and Serratia liquefaciens. The four strains were found to use crude oil, n-octadecane, and phenanthrene as sole sources of carbon and energy. It was concluded that coinoculation of V. faba plant roots in oily sand with nodule bacteria and PGPR enhances the phytoremediation potential of this plant for oily desert sand through improving plant growth and nitrogen fixation.     

Fungal endophytes from Dioscorea zingiberensis rhizomes and their antibacterial activity

AIMS: The aim of the study was to isolate and characterize the endophytic fungi from the rhizomes of the Chinese traditional medicinal plant Dioscorea zingiberensis and to detect their antibacterial activities. METHODS AND RESULTS: After strict sterile sample preparation, nine fungal endophytes were isolated from rhizomes of the Chinese traditional medicinal plant D. zingiberensis. The endophytes were classified by morphological traits and internal transcribed spacer (ITS) rRNA gene sequence analysis. Their ITS rDNA sequences were 99-100% identical to Nectria, Fusarium, Rhizopycnis, Acremonium and Penicillium spp. respectively. Of these, the most frequent genera were Fusarium and Nectria. One isolate, Dzf7, was unclassified on the basis of its low sequence similarity. The next closest species was Alternaria longissima (c. 92.4% sequence similarity). Endophyte isolate Dzf5 showed the closest sequence similarity (c. 99.5%) to an uncultured soil fungus (DQ420800) obtained from Cedar Creek, USA. Bioassays using a modified broth dilution test were used to detect the antibacterial activity of n-butanol extracts of both mycelia and culture filtrates of D. zingiberensis showed biological activity against Bacillus subtilis, Staphylococcus haemolyticus, Escherichia coli and Xanthomonas vesicatoria. Minimal inhibitory concentration (MIC) values of the extracts were between 31 x 25 microg ml(-1) and 125 microg ml(-1). CONCLUSIONS: Endophytic fungus Dzf2 (c. 99 x 8% sequence similarity to Fusarium redolens) isolated from D. zingiberensis rhizome showed the most potent antibacterial activities. SIGNIFICANCE AND IMPACT OF THE STUDY: Endophytic fungi isolated from D. zingiberensis may be used as potential producers of antibacterial natural products.     

内生真菌对草坪植物病原真菌抑制作用的比较

分别从野生牧草羽茅(Achnatherum sibiricum (L.) Keng)、栽培种高羊茅(Festuca arundinacea Schreb.)(品种Millennium)、栽培种黑麦草(Lolium perenne L.) (品种Justus)中分离出内生真菌Neotyphodium sp.、N. coenophialum和N. lolli,通过体外培养法比较了这3种内生真菌对草坪植物病原真菌的抑制作用.结果表明,从羽茅中分离的内生真菌Neotyphodium sp.在两菌相交前对所有供试的病原真菌都有一定的抑制作用,其中对枝孢霉属(Cladosporium sp.)、弯孢霉属(Curvularia sp.)和拟茎点属(Phomopsis sp.)病原真菌的抑制效果尤为显著,对峙培养3d后的抑菌率分别达70.1%、52.3%和30.9%,营养竞争作用、重寄生作用是其主要的拮抗机制;从高羊茅中分离的内生真菌N. coenophialum对枝孢霉属病原真菌存在一定的抑制作用;而从黑麦草中分离的内生真菌N. lolli与病原真菌对峙培养时,病原真菌菌落慢慢侵占整个营养空间,内生真菌停止生长并逐渐褐变死亡.体外培养结果说明Neotyphodium sp.对供试病原真菌的拮抗效果优于N. coenophialum和N. lolli,由此推测Neotyphodium sp.与宿主植物羽茅的共生可能有利于宿主植物抵抗病原真菌的侵扰.     

The use of dialdehyde starch derivatives in the phytoremediation of soils contaminated with heavy metals

Products of the reaction between dialdehyde starch and Y-NH₂ compounds (e.g. semicarbazide or hydrazine) are effective ligands for metal ions. The usefulness of these derivatives was tested in the experiment, both in terms of the immobilization of heavy metal ions in soil and the potential application in phytoextraction processes. The experimental model comprised maize and the ions of such metals as: Zn(II), Pb(II), Cu(II), Cd(II), and Ni(II). The amount of maize yield, as well as heavy metal content and uptake by the aboveground parts and roots of maize, were studied during a three-year pot experiment. The results of the study indicate the significant impact of heavy metals on reduced yield and increased heavy metal content in maize. Soil-applied dialdehyde starch derivatives resulted in lower yields, particularly disemicarbazone (DASS), but in heavy metal-contaminated soils they largely limited the negative impact of these metals both on yielding and heavy metal content in plants, particularly dihydrazone (DASH). It was demonstrated that the application of dihydrazone (DASH) to a soil polluted with heavy metals boosted the uptake of Zn, Pb, Cu, and Cd from the soil, hence there is a possibility to use this compound in the phytoextraction of these metals from the soil. Decreased Ni uptake was also determined, hence the possibility of using this compound in the immobilization of this metal. The study showed that dialdehyde starch disemicarbazone was ineffective in the discussed processes.