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1.
Wang H  Chen X  Xing X  Hao X  Chen D 《Plant cell reports》2010,29(12):1391-1399
Atrazine chlorohydrolase (AtzA) catalyzes hydrolytic dechlorination and can be used in detoxification of atrazine, a herbicide widely employed in the control of broadleaf weeds. In this study, to investigate the potential use of transgenic tobacco plants for phytoremediation of atrazine, atzA genes from Pseudomonas sp. strain ADP and Arthrobacter strain AD1 were transferred into tobacco. Three and four transgenic lines, expressing atzA-ADP and atzA-AD1, respectively, were produced by Agrobacterium-mediated transformation. Molecular characterization including PCR, RT-PCR and Southern blot revealed that atzA was inserted into the tobacco genome and stably inherited by and expressed in the progenies. Seeds of the T1 transgenic lines had a higher germination percentage and longer roots than the untransformed plants in the presence of 40–150 mg/l atrazine. The T2 transgenic lines grew taller, gained more dry biomass, and had higher total chlorophyll content than the untransformed plants after growing in soil containing 1 or 2 mg/kg atrazine for 90 days. No atrazine residue remained in the soil in which the T2 transgenic lines were grown (except 401), while, in the case of the untransformed plants, 0.91 mg (81.3%) and 1.66 mg (74.1%) of the atrazine still remained in the soil containing 1 and 2 mg/kg of atrazine, respectively, indicating that the transgenic lines could degrade atrazine effectively. The transgenic tobacco lines developed could be useful for phytoremediation of atrazine-contaminated soil and water.  相似文献   

2.
A mixed enrichment culture of microorganisms capable of accelerated mineralization of atrazine was isolated from soil treated with successive applications of the herbicide. Liquid cultures of this consortium, in the presence of simple carbon sources, mineralized 96% of the applied atrazine (0.56 mM) within 7 days. Atrazine mineralization in culture is initiated with the formation of the metabolite hydroxyatrazine. In soil treated with atrazine at a concentration of 0.14 mM (concentration is based on total soil mass), and then inoculated with the microbial consortium, the parent compound was completely transformed in 25 days. After 30 days of incubation, 60% of the applied atrazine was accounted for as14CO2. As was found with the liquid cultures, hydroxyatrazine was the major metabolite. After 145 days, soil extractable hydroxyatrazine declined to zero and 86% of the applied atrazine was accounted for as14CO2. No metabolites, other than hydroxyatrazine, were recovered from either the liquid culture or soil inoculated with the consortium. The use of the mixed microbial culture enhanced mineralization more than 20 fold as compared to uninoculated soil.  相似文献   

3.
AIMS: To isolate and characterize atrazine-degrading bacteria in order to identify suitable candidates for potential use in bioremediation of atrazine contamination. METHODS AND RESULTS: A high efficiency atrazine-degrading bacterium, strain AD1, which was capable of utilizing atrazine as a sole nitrogen source for growth, was isolated from industrial wastewater. 16S rDNA sequencing identified AD1 as an Arthrobacter sp. The atrazine chlorohydrolase gene (atzA) isolated from strain AD1 differed from that found in the Pseudomonas sp. ADP by only one nucleotide. However, it was found located on the bacterial chromosome rather than on plasmids as previously reported for other bacteria. CONCLUSIONS: Atrazine chlorohydrolase gene, atzA, either encoded by chromosome or plasmid, is highly conserved. SIGNIFICANCE AND IMPACT OF THE STUDY: Comparison analysis of atrazine degradation gene structure and arrangement in this and other bacteria provides insight into our understanding of the ecology and evolution of atrazine-degrading bacteria.  相似文献   

4.
Bacterial communities that cooperatively degrade atrazine commonly consist of diverse species in which the genes for atrazine dechlorination and dealkylation are variously distributed among different species. Normally, the first step in degradation of atrazine involves dechlorination mediated by atzA, followed by stepwise dealkylation to yield either N-ethylammelide or N-isopropylammelide. As the liberated alkylamine moieties are constituents of many organic molecules other than atrazine, it is possible that a large number of alkylamine-degrading bacteria other than those previously described might contribute to this key step in atrazine degradation. To examine this hypothesis, we isolated 82 bacterial strains from soil by plating soil water extracts on agar media with ethylamine as a sole carbon source. Among the relatively large number of isolates, only 3 were able to degrade N-ethylammelide, and in each case were shown to carry the atzB gene and atzC genes. The isolates, identified as Rhizobium leguminosarum, Flavobacterium sp., and Arthrobacter sp., were all readily substituted into an atrazine-degrading consortium to carry out N-ethylammelide degradation. The distribution of these genes among many different species in the soil microbial population suggests that these genes are highly mobile and over time may lead to generation of various atrazine-degrading consortia.  相似文献   

5.
One of the various ecosystemic services sustained by soil is pollutant degradation mediated by adapted soil bacteria. The pathways of atrazine biodegradation have been elucidated but in situ expression of the genes involved in atrazine degradation has yet to be demonstrated in soil. Expression of the atzA and atzD genes involved in atrazine dechlorination and s‐triazine ring cleavage, respectively, was investigated during in situ degradation of atrazine in the soil drilosphere and bulked samples from two agricultural soils that differed in their ability to mineralize atrazine. Interestingly, expression of the atzA gene, although present in both soils, was not detected. Atrazine mineralization was greatest in Epoisses soil, where a larger pool of atzD mRNA was consistently measured 7 days after atrazine treatment, compared with Vezin soil (146 vs. 49 mRNA per 10 6 16S rRNA, respectively). Expression of the atzD gene varied along the degradation time course and was profoundly modified in soil bioturbated by earthworms. The atzD mRNA pool was the highest in the soil drilosphere (casts and burrow‐linings) and it was significantly different in burrow‐linings compared with bulk soil (e.g. 363 vs. 146 mRNA per 10 6 16S rRNA, 7 days after atrazine treatment in Epoisses soil). Thus, consistent differences in atrazine mineralization were demonstrated between the soil drilosphere and bulk soil. However, the impact of bioturbation on atrazine mineralization depended on soil type. Mineralization was enhanced in casts, compared with bulk soil, from Epoisses soil but in burrow‐linings from Vezin soil. This study is the first to report the effects of soil bioturbation by earthworms on s‐triazine ring cleavage and its spatial variability in soil.  相似文献   

6.
阿特拉津氯水解酶定向改造的关键是开发一种廉价的、表型改变明显的高通量筛选方法。利用高错误倾向PCR和DNA洗牌相结合的突变方法,对来源于假单胞菌ADP和节杆菌AD1的阿特拉津氯水解酶基因进行随机突变,以雨生红球藻为受体、以阿特拉津为选择压力对突变文库进行高通量筛选。筛选到的12个突变子序列分析显示,突变均为点替换,位点分散在全基因上,是在高错误倾向PCR及DNA洗牌过程中逐渐累积形成的。酶活力分析显示,突变子的酶活力均高于野生株,在添加1.0 mg/L阿特拉津培养液中的活力是野生株的1.9~3.6倍,在添  相似文献   

7.
Atrazine belongs to the widely used herbicides blocking the electron transport chain in chloroplasts, thus resulting in the generation of active oxygen species. In the present work, we demonstrated that, at low concentrations mimicking residual amounts, atrazine enhanced the susceptibility of Arabidopsis plants to further treatments with the same herbicide applied at the recommended field rate. Arabidopsis thaliana plants were treated three times (at five-day intervals) with 1 µM atrazine. Five days after the last treatment, the plants were sprayed with 5 mM atrazine. Atrazine increased the levels of lipid peroxidation products, hydrogen peroxide, and ion leakage, and caused changes in the activities of antioxidant enzymes, such as superoxide dismutase, guaiacol peroxidase, and catalase.From Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 243–249.Original English Text Copyright © 2005 by Ivanov, Alexieva, Karanov.This article was submitted by the authors in English.This revised version was published online in April 2005 with a corrected cover date.  相似文献   

8.
Bacterial atrazine catabolism is initiated by the enzyme atrazine chlorohydrolase (AtzA) in Pseudomonas sp. strain ADP. Other triazine herbicides are metabolized by bacteria, but the enzymological basis of this is unclear. Here we begin to address this by investigating the catalytic activity of AtzA by using substrate analogs. Purified AtzA from Pseudomonas sp. strain ADP catalyzed the hydrolysis of an atrazine analog that was substituted at the chlorine substituent by fluorine. AtzA did not catalyze the hydrolysis of atrazine analogs containing the pseudohalide azido, methoxy, and cyano groups or thiomethyl and amino groups. Atrazine analogs with a chlorine substituent at carbon 2 and N-alkyl groups, ranging in size from methyl to t-butyl, all underwent dechlorination by AtzA. AtzA catalyzed hydrolytic dechlorination when one nitrogen substituent was alkylated and the other was a free amino group. However, when both amino groups were unalkylated, no reaction occurred. Cell extracts were prepared from five strains capable of atrazine dechlorination and known to contain atzA or closely homologous gene sequences: Pseudomonas sp. strain ADP, Rhizobium strain PATR, Alcaligenes strain SG1, Agrobacterium radiobacter J14a, and Ralstonia picketti D. All showed identical substrate specificity to purified AtzA from Pseudomonas sp. strain ADP. Cell extracts from Clavibacter michiganensis ATZ1, which also contains a gene homologous to atzA, were able to transform atrazine analogs containing pseudohalide and thiomethyl groups, in addition to the substrates used by AtzA from Pseudomonas sp. strain ADP. This suggests that either (i) another enzyme(s) is present which confers the broader substrate range or (ii) the AtzA itself has a broader substrate range.  相似文献   

9.
Atrazine sensitive leguminous plants were grown in a soil spiked with atrazine and augmented with an atrazine-degrading bacterium, Arthrobacter sp. strain MCM B-436, to ascertain its degradative efficiency. Germination and survival of plants was correlated with atrazine removal from soil. This experiment was carried out at laboratory as well as field level, showing consistent results. This bioindicator approach serves as an efficient measure for atrazine removal and could be easily adapted to determine atrazine degradation efficiency of other microbial strains.  相似文献   

10.
Atrazine degradation previously has been shown to be carried out by individual bacterial species or by relatively simple consortia that have been isolated using enrichment cultures. Here, the degradative pathway for atrazine was examined for a complex 8-membered enrichment culture. The species composition of the culture was determined by PCR-DGGE. The bacterial species included Agrobacterium tumefaciens, Caulobacter crescentus, Pseudomonas putida, Sphingomonas yaniokuyae, Nocardia sp., Rhizobium sp., Flavobacterium oryzihabitans, and Variovorax paradoxus. All of the isolates were screened for the presence of known genes that function for atrazine degradation including atzA,-B,-C,-D,-E,-F and trzD,-N. Dechlorination of atrazine, which was obligatory for complete mineralization, was carried out exclusively by Nocardia sp., which contained the trzN gene. Following dechlorination, the resulting product, hydroxyatrazine was further degraded via two separate pathways. In one pathway Nocardia converted hydroxyatrazine to N-ethylammelide via an unidentified gene product. In the second pathway, hydroxyatrazine generated by Nocardia sp. was hydrolyzed to N-isopropylammelide by Rhizobium sp., which contained the atzB gene. Each member of the enrichment culture contained atzC, which is responsible for ring cleavage, but none of the isolates carried the atzD,-E, or -F genes. Each member further contained either trzD or exhibited urease activity. The enrichment culture was destabilized by loss of Nocardia sp. when grown on ethylamine, ethylammelide, and cyanuric acid, after which the consortium was no longer able to degrade atrazine. The analysis of this enrichment culture highlights the broad level bacterial community interactions that may be involved in atrazine degradation in nature.  相似文献   

11.
小麦几丁质酶基因的异种表达及其功能鉴定   总被引:3,自引:0,他引:3  
几丁质酶参与植物的发育及防卫反应,并与人类疾病发生有关.文章研究了小麦几丁质酶基因Wch2经根癌农杆菌介导的烟草瞬间表达和转基因拟南芥的稳定表达,Western杂交及酶活测定证实,瞬间表达的小麦几丁质酶分子量约30 kD,具有降解几丁质多聚物的功能;Wch2在转入拟南芥后表达量高,尖孢镰刀菌接种的鉴定表明,表达Wch2的转基因植株的抗病性显著高于表达绿色荧光蛋白的对照植株.这些结果说明Wch2的异种表达,可用于植物抗病基因工程,以增强植物的抗病性.  相似文献   

12.
The potential of soil microorganisms to mineralize atrazine was studied in soil samples collected from fields with various histories of atrazine application. In contrast to many previous studies, which showed no atrazine mineralization activity, all the tested soils mineralized atrazine regardless of their atrazine application history. However, the delay before mineralization and the variation in the subsequent mineralization rate were in agreement with the initial copy number of the atrazine dechlorinaze gene, and the proliferation rate of the degraders. Soils from corn fields, which had up to 100 copies of the atzA gene per gram of soil, had a lag period of 4-5 days before atrazine mineralization started, and final mineralization percentages ranged from 40% to 54%. However, soils from fields that were never amended with atrazine had much longer lag periods (more than 17 days), which decreased after enrichment of the degrader population with high concentrations of atrazine for 15 days. Generally the mineralization rate and the atzA gene copy number increased after the enrichment period. The atrazine mineralization potential was measured by PCR of genes from the atrazine mineralization pathway. Magnetic capture hybridization was the most efficient of the two tested methods for purifying target DNA of PCR inhibitors, without reducing the copy number of the required fragment. Nested PCR proved to be the most effective method for predicting the exact potential of the soil to mineralize the pollutant even without enrichment of a small population with the target genes. This method can complement microcosm studies and eliminate futile efforts when the potential to mineralize the pollutant does not exist in the soil.  相似文献   

13.
Alfalfa is very sensitive to soil acidity and its yield and stand duration are compromised due to inhibited root growth and reduced nitrogen fixation caused by Al toxicity. Soil improvement by liming is expensive and only partially effective, and conventional plant breeding for Al tolerance has had limited success. Because tobacco and papaya plants overexpressing Pseudomonas aeruginosa citrate synthase (CS) have been reported to exhibit enhanced tolerance to Al, alfalfa was engineered by introducing the CS gene controlled by the Arabidopsis Act2 constitutive promoter or the tobacco RB7 root-specific promoter. Fifteen transgenic plants were assayed for exclusion of Al from the root tip, for internal citrate content, for growth in in vitro assays, or for shoot and root growth in either hydroponics or in soil assays. Overall, only the soil assays yielded consistent results. Based on the soil assays, two transgenic events were identified that were more aluminum-tolerant than the non-transgenic control, confirming that citrate synthase overexpression can be a useful tool to help achieve aluminum tolerance. Pierluigi Barone and Daniele Rosellini contributed equally to this work.  相似文献   

14.
Green fluorescent protein (GFP) has emerged as a powerful new tool in a variety of organisms. An engineered sGFP(S65T) sequence containing optimized codons of highly expressed eukaryotic proteins has provided up to 100-fold brighter fluorescence signals than the original jellyfish GFP sequence in plant and mammalian cells. It would be useful to establish a non-invasive, quantitative detection system which is optimized for S65T-type GFP, one of the brightest chromophore mutants among the various GFPs. We demonstrate here that highly fluorescent transgenic Arabidopsis can be generated, and the fluorescence intensity of whole plants can be measured under non-disruptive, sterile conditions using a quantitative fluorescent imaging system with blue laser excitation. Homozygous plants can be distinguished from heterozygous plants and fully fertile progenies can be obtained from the analyzed plants. In the case of cultured tobacco cells, GFP-positive cells can be quantitatively distinguished from non-transformed cells under non-selective conditions. This system will be useful in applications such as mutant screening, analysis of whole-body phenomena, including gene silencing and quantitative assessments of colonies from microorganisms to cultured eukaryotic cells. To facilitate the elucidation of protein targeting and organelle biogenesis in planta, we also generated transgenic Arabidopsis that stably express the plastid- or mitochondria-targeted sGFP(S65T). Etioplasts in dark-grown cotyledons and mitochondria in dry seed embryos could be visualized for the first time in transgenic Arabidopsis plants under normal growing conditions.  相似文献   

15.
The potential of a microbial consortium for treating waters contaminated with atrazine was considered. In conventional liquid culture, atrazine and its two dealkylated by-products were equally metabolised by the microbial consortium. Transient production of hydroxyatrazine was observed during atrazine catabolism, indicating that the catabolic pathway was similar to the one reported for isolates capable of atrazine mineralisation. This consortium was then inoculated to sediments sampled from an artificial recharge site. These sediments were contaminated by atrazine and diuron and exhibited only a slow endogenous herbicide dissipation. Inoculated microorganisms led to extensive atrazine degradation and survived for more than 10 weeks in the sediments. A rudimentary bioreactor was then setup using a soil core originating from the same recharge site. Degrading microorganisms rapidly colonised the core and expressed their degrading activity. The efficiency of the bioreactor was improved in the presence of spiked environmental surface waters. Atrazine degraders thus possibly benefited from the other organic sources in developing and expressing their activity. The microbial consortium did not initially exhibit the capacity to degrade diuron, which was used as reference compound. No change in this characteristic was detected throughout the study. Received: 13 December 1999 / Received revision: 26 April 2000 / Accepted: 5 May 2000  相似文献   

16.
We previously identified a Pseudomonas sp. strain, ADP, which rapidly metabolized atrazine in liquid culture, agar plates, and soils (R. T. Mandelbaum, D. L. Allan, L. P. Wackett, Appl. Environ. Microbiol. 61:1451-1457, 1995). In this study, we report the cloning and partial characterization of a gene region from Pseudomonas sp. strain ADP that encodes atrazine degradation activity. A 22-kb EcoRI genomic DNA fragment, designated pMD1, was shown to encode atrazine dechlorination activity in Escherichia coli DH5 alpha. Atrazine degradation was demonstrated by a zone-clearing assay on agar medium containing crystalline atrazine and by chromatographic methods. A gene conferring the atrazine-clearing phenotype was subsequently subcloned as a 1.9-kb AvaI fragment in pACYC184, designated pMD4, and was expressed in E. coli. This result and random Tn5 mutagenesis established that the 1.9-kb AvaI fragment was essential for atrazine dechlorination. High-pressure liquid and thin-layer chromatographic analyses were used to rigorously establish that E. coli containing pMD4 degraded atrazine and accumulated hydroxyatrazine. Hydroxyatrazine was detected only transiently in E. coli containing pMD1. This is consistent with the idea that hydroxyatrazine is the first metabolite in atrazine degradation by Pseudomonas sp. strain ADP. A 0.6-kb ApaI-PstI fragment from pMD4, containing the putative atrazine chlorohydrolase gene, hybridized to DNA from atrazine-degrading bacteria isolated in Switzerland and Louisiana.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

17.
Atrazine-degrading microorganisms designated EAA-3 and EAA-4, belonging to the genus Nocardioides, were obtained from an agricultural soil in Nigeria. The degradation kinetics of the two strains revealed total disappearance of 25 mg l?1 of atrazine in less than 72 h of incubation at the rate of 0.42 mg l?1 h?1 and 0.35 mg l?1 h?1, respectively. Screening for atrazine catabolic genes in these organisms revealed the presence of trzN, atzB, and atzC. Other genes, specifically atzA, atzD, and trzD, were not detected. Potential intermediates of atrazine catabolic route such as hydroxyatrazine, desethylatrazine, and desisopropylatrazine were utilized as sources of carbon and energy, while desisopropyl desethyl-2-hydroxyatrazine and desisopropyl-2-hydroxyatrazine were attacked but in the presence of glucose. A soil microcosm study showed that degradation was faster in microcosms contaminated with 13 mg of atrazine per g?1 of soil compared with 480 mg g?1 of soil. In the former, degradation was 10% higher in the inoculated soil than the non-inoculated control (natural attenuation) over the 28-day study period. Corresponding value obtained for the latter was nearly 70% higher. This study has demonstrated that the bacterial strains isolated enhanced atrazine degradation and the catabolic activities of these strains were not affected with increasing soil atrazine concentration.  相似文献   

18.
The level of expression of highly conserved, plasmid-borne, and widely dispersed atrazine catabolic genes (atz) was studied by RT-qPCR in two telluric atrazine-degrading microbes. RT-qPCR assays, based on the use of real-time PCR, were developed in order to quantify atzABCDEF mRNAs in Pseudomonas sp. ADP and atzABC mRNAs in Chelatobacter heintzii. atz gene expression was expressed as mRNA copy number per 10(6) 16S rRNA. In Pseudomonas sp. ADP, atz genes were basally expressed. It confirmed atrazine-degrading kinetics indicating that catabolic activity starts immediately after adding the herbicide. atz gene expression increased transitorily in response to atrazine treatment. This increase was only observed while low amount of atrazine remained in the medium. In C. heintzii, only atzA was basally expressed. atzA and atzB expression levels were similarly and significantly increased in response to atrazine treatment. atzC was not expressed even in the presence of high amounts of atrazine. This study showed that atz genes are basally expressed and up-regulated in response to atrazine treatment. atz gene expression patterns are different in Pseudomonas ADP and C. heintzii suggesting that the host may influence the expression of plasmid-borne atrazine-catabolic potential.  相似文献   

19.
Aims:  The aim of this work was to enrich stable mixed cultures from atrazine-contaminated soil. The cultures were examined for their atrazine biodegradation efficiencies in comparison with J14a, a known atrazine-degrading strain of Agrobacterium radiobacter . The cultures were also characterized to identify community structure and bacterial species present.
Methods and Results:  The cultures were enriched and then stabilized in bacterial media. The stable mixed cultures and J14a were tested in a medium containing 100 μg l−1 of atrazine. For all cultures, atrazine was removed 33–51% within 7 days and the cell optical density increased from 0·05 to between 0·50 and 0·70. Four isolates designated ND1, ND2, ND3 and ND4 were purified from the mixed cultures and identified based on sequence analysis of the 16 S rRNA gene as Alcaligenes faecalis , Klebsiella ornithinolytica , Bacillus megaterium and Agrobacterium tumefaciens , respectively. An atrazine-degrading gene, atzA , was present in ND2 and ND4.
Conclusions:  The stable mixed cultures obtained could degrade atrazine. Klebsiella ornithinolytica ND2 and Ag. tumefaciens ND4 are atrazine degraders.
Significance and Impact of the Study:  The novel stable mixed cultures could be used for bioremediating crop fields contaminated with atrazine. This is the first report of the atzA gene in Kl. ornithinolytica .  相似文献   

20.
ABSTRACT

Sugarcane top-derived biochar was added to an alluvial soil, a moist soil and a paddy soil at the rate of 0.2% and 0.5% (w/w). After the addition of 0.2% and 0.5% biochar, the sorption coefficients (Kd) of atrazine (Ce = 10 mg L?1) were increased by 26.97% and 79.58%, respectively, in the moist soil with a low level of total organic carbon (TOC), while it increased by 31.43% and 60.06%, respectively, in the paddy soil with a high TOC content. The half-time persistence values of atrazine in the alluvial soil, moist soil and paddy soil were 28.18, 23.74 and 39.84 d, respectively. In the 0.2% biochar amended soils, the corresponding half-times of atrazine for the alluvial soil, moist soil and paddy soil were extended by 10.33, 11.81 and 1.42 d, and they were prolonged by 16.83, 17.52 and 14.74 d, respectively, in the 0.5% biochar amended soils. Atrazine degradation products (deisopropylatrazine and desethylatrazine) decreased after they accumulated to 3.2 and 1 mg kg?1, respectively. Generally, increasing sorption was accompanied by decreasing degradation of atrazine which is found in biochar-amended soils.  相似文献   

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