Nanotechnology, new fascinating field of science, is bringing many application's options. However, it is necessary to understand their potential environmental risk and toxicity. Zinc selenide quantum dots (ZnSe QDs) are getting valuable due to wide industrial usage, mainly as cadmium free diodes or stabilizing ligand. Thanks to unique properties, they could also open the possibilities of application in the agriculture. Their effects on living organisms, including plants, are still unknown. Therefore, the attention of this work was given to antioxidant response of Arabidopsis thaliana to 100 and 250 μM ZnSe QDs foliar feeding. ZnSe QDs treatment had no statistically significant differences in morphology but led to increased antioxidant response in the leaves at the level of gene expression and production secondary antioxidant metabolites. Concurrently, analysis of growth properties of Agrobacterium tumefaciens was done. 250 μM ZnSe solution inhibited the Agrobacterium tumefaciens viability by 60%. This is the first mention about effect ZnSe QDs on the plants. Although QDs induced oxidative stress, the apply treatment dose of ZnSe QDs did not have significant toxic effect on the plants and even no morphological changes were observed. However, the same amount of ZnSe QD induced an inhibitory effect on Agrobacterium tumefaciens.
- MeSH
- Agrobacterium tumefaciens účinky léků růst a vývoj MeSH
- antioxidancia metabolismus MeSH
- Arabidopsis genetika růst a vývoj metabolismus MeSH
- exprese genu * MeSH
- kvantové tečky toxicita MeSH
- metabolomika * MeSH
- sloučeniny selenu aplikace a dávkování toxicita MeSH
- sloučeniny zinku aplikace a dávkování toxicita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Many Gram-negative bacteria use N-acyl-homoserine lactones (N-acyl-HSL) as quorum sensing signal molecules. N-acyl-HSL are the subject of investigation by many research groups. Simple methods have been designed to detect N-acyl-HSL using bacterial strains that doesn't produce its own signal molecules, but the reporter gene is expressed when exogenous N-acyl-HSL are present. This article reviews and discusses the most common and most currently obtainable systems using bacteria such as Agrobacterium tumefaciens, Chromobacterium violaceum, Escherichia coli or Vibrio fischeri. These biological sensing elements can be used to detect and study a wide range of N-acyl-HSL and related molecules interfering with quorum sensing systems.
Organophosphorus compounds have been widely employed to the development of warfare nerve agents and pesticides, resulting in a huge number of people intoxicated annually, being a serious problem of public health. Efforts worldwide have been done in order to design new technologies that are capable of combating or even reversing the poisoning caused by these OP nerve agents. In this line, the bioremediation arises as a promising and efficient alternative for this purpose. As an example of degrading enzymes, there is the organophosphate-degrading (OpdA) enzyme from Agrobacterium radiobacter, which has been quite investigated experimentally due to its high performance in the degradation of neurotoxic nerve agents. This work aims to look into the structural and electronic details that govern the interaction modes of these compounds in the OpdA active site, with the posterior hydrolysis reaction prediction. Our findings have brought about data about the OpdA performance towards different nerve agents, and among them, we may realize that the degradation efficiency strongly depends on the nerve agent structure and its stereochemistry, being in this case the compound Tabun the one more effectively hydrolyzed. By means of the chemical bonds (AIM) and orbitals (FERMO) analysis, it is suggested that the initial reactivity of the OP nerve agents in the OpdA active site does not necessarily dictate the reactivity and interaction modes over the reaction coordinate.
- MeSH
- Agrobacterium tumefaciens enzymologie MeSH
- bakteriální proteiny chemie metabolismus MeSH
- biodegradace * MeSH
- biokatalýza MeSH
- fosfatasy chemie metabolismus MeSH
- katalytická doména MeSH
- kvantová teorie MeSH
- lidé MeSH
- nervová bojová látka chemie metabolismus MeSH
- sarin chemie metabolismus MeSH
- simulace molekulového dockingu MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Plant transformation via Agrobacterium tumefaciens is characterized by integration of commonly low number of T-DNAs at random positions in the genome. When integrated into an active gene region, promoterless reporter genes placed near the T-DNA border sequence are frequently transcribed and even translated to reporter proteins, which is the principle of promoter- and gene-trap lines. RESULTS: Here we show that even internal promotorless regions of T-DNAs are often transcribed. Such spontaneous transcription was observed in the majority of independently transformed tobacco BY-2 lines (over 65%) and it could effectively induce silencing if an inverted repeat was present within the T-DNA. We documented that the transcription often occurred in both directions. It was not directly connected with any regulatory elements present within the T-DNAs and at least some of the transcripts were initiated outside of the T-DNA. The likeliness of this read-through transcription seemed to increase in lines with higher T-DNA copy number. Splicing and presence of a polyA tail in the transcripts indicated involvement of Pol II, but surprisingly, the transcription was able to run across two transcription terminators present within the T-DNA. Such pervasive transcription was observed with three different T-DNAs in BY-2 cells and with lower frequency was also detected in Arabidopsis thaliana. CONCLUSIONS: Our results demonstrate unexpected pervasive read-through transcription of T-DNAs. We hypothesize that it was connected with a specific chromatin state of newly integrated DNA, possibly affected by the adjacent genomic region. Although this phenomenon can be easily overlooked, it can have significant consequences when working with highly sensitive systems like RNAi induction using an inverted repeat construct, so it should be generally considered when interpreting results obtained with the transgenic technology.
- MeSH
- Agrobacterium tumefaciens genetika MeSH
- Arabidopsis genetika MeSH
- buněčné linie MeSH
- DNA bakterií genetika MeSH
- genetická transkripce * MeSH
- geneticky modifikované rostliny MeSH
- messenger RNA genetika MeSH
- obrácené repetice genetika MeSH
- promotorové oblasti (genetika) genetika MeSH
- reportérové geny MeSH
- tabák genetika MeSH
- Publikační typ
- časopisecké články MeSH
Intact, growing cells of strongly acidophilic fungi Acidea extrema and Acidothrix acidophila have been successfully transformed by introduction of heterologous DNA fragment (composed of the glyceraldehyde-phosphate-dehydrogenase gene promoter from Emericella nidulans, a metallothionein-coding gene AsMt1 from Amanita strobiliformis and glyceraldehyde-phosphate-dehydrogenase gene terminator from Colletotrichum gloeosporioides) with the length of 1690 bp. The transformation procedure was based on the DNA transfer mediated by Agrobacterium tumefaciens bearing disarmed helper plasmid pMP90 and binary vector pCambia1300 with inserted DNA fragment of interest. The transformants proved to be mitotically stable, and the introduced gene was expressed at least at the level of transcription. Our work confirms that metabolic adaptations of strongly acidophilic fungi do not represent an obstacle for genetic transformation using conventional methods and can be potentially used for production of heterologous proteins. A promising role of the fast growing A. acidophila as active biomass in biotechnological processes is suggested not only by the low susceptibility of the culture grown at low pH to contaminations but also by reduced risk of accidental leaks of genetically modified microorganisms into the environment because highly specialized extremophilic fungi can poorly compete with common microflora under moderate conditions.
- MeSH
- Agrobacterium tumefaciens genetika MeSH
- Amanita genetika MeSH
- Ascomycota genetika MeSH
- exprese genu MeSH
- genetické vektory MeSH
- koncentrace vodíkových iontů MeSH
- metalothionein genetika MeSH
- plazmidy MeSH
- promotorové oblasti (genetika) MeSH
- terminace genetické transkripce MeSH
- transformace genetická * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Agrobacterium tumefaciens is widely used as a versatile tool for development of stably transformed model plants and crops. However, the development of Agrobacterium based transient plant transformation methods attracted substantial attention in recent years. Transient transformation methods offer several applications advancing stable transformations such as rapid and scalable recombinant protein production and in planta functional genomics studies. Herein, we highlight Agrobacterium and plant genetics factors affecting transfer of T-DNA from Agrobacterium into the plant cell nucleus and subsequent transient transgene expression. We also review recent methods concerning Agrobacterium mediated transient transformation of model plants and crops and outline key physical, physiological and genetic factors leading to their successful establishment. Of interest are especially Agrobacterium based reverse genetics studies in economically important crops relying on use of RNA interference (RNAi) or virus-induced gene silencing (VIGS) technology. The applications of Agrobacterium based transient plant transformation technology in biotech industry are presented in thorough detail. These involve production of recombinant proteins (plantibodies, vaccines and therapeutics) and effectoromics-assisted breeding of late blight resistance in potato. In addition, we also discuss biotechnological potential of recombinant GFP technology and present own examples of successful Agrobacterium mediated transient plant transformations.
In this study, the Agrobacterium tumefaciens-mediated transformation method for Ganderma weberianum has been established. Driven by the cauliflower mosaic virus (CaMV) 35S promoter, the hygromycin phosphotransferase (hpt), β-glucuronidase (uidA), and enhanced green fluorescent protein (egfp) genes have been efficiently expressed in transgenic mycelia and spores. The transformation system was composed of the growing mycelia, A. tumefaciens strain GV3101, and the expression vector pBI-H1, harboring the CaMV 35S promoter and selective hpt marker. The genetic transformation of G. weberianum was achieved through co-cultivation of Agrobacterium lawn and fungal mycelia at 28 °C on yeast extract agar (YEA) medium. Stable genetic transformants were obtained through successive hygromycin B selections and single spore isolation. Over 80 % of transformants showed genetic stability even after ten rounds of subculturing. The simple and efficient genetic transformation method is a useful tool for molecular genetics analyses and gene manipulation of G. weberianum.
- MeSH
- Agrobacterium tumefaciens genetika metabolismus MeSH
- Ganoderma genetika MeSH
- genetické techniky * MeSH
- genetické vektory genetika metabolismus MeSH
- transformace genetická * MeSH
- zelené fluorescenční proteiny MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
Barley is one of the most important cereal crops grown worldwide. It has numerous applications, but its utility could potentially be extended by genetically manipulating its hormonal balances. To explore some of this potential we identified gene families of cytokinin dehydrogenases (CKX) and isopentenyl transferases, enzymes that respectively irreversibly degrade and synthesize cytokinin (CK) plant hormones, in the raw sequenced barley genome. We then examined their spatial and temporal expression patterns by immunostaining and qPCR. Two CKX-specific antibodies, anti-HvCKX1 and anti-HvCKX9, predominantly detect proteins in the aleurone layer of maturing grains and leaf vasculature, respectively. In addition, two selected CKX genes were used for stable, Agrobacterium tumefaciens-mediated transformation of the barley cultivar Golden Promise. The results show that constitutive overexpression of CKX causes morphological changes in barley plants and prevents their transition to flowering. In all independent transgenic lines roots proliferated more rapidly and root-to-shoot ratios were higher than in wild-type plants. Only one transgenic line, overexpressing CKX under the control of a promoter from a phosphate transporter gene, which is expressed more strongly in root tissue than in aerial parts, yielded progeny. Analysis of several T1-generation plants indicates that plants tend to compensate for effects of the transgene and restore CK homeostasis later during development. Depleted CK levels during early phases of development are restored by down-regulation of endogenous CKX genes and reinforced de novo biosynthesis of CKs.
- MeSH
- Agrobacterium tumefaciens MeSH
- cytokininy biosyntéza genetika MeSH
- exprese genu * MeSH
- fertilita genetika MeSH
- geneticky modifikované rostliny enzymologie genetika MeSH
- ječmen (rod) enzymologie genetika MeSH
- kořeny rostlin embryologie genetika MeSH
- oxidoreduktasy biosyntéza genetika MeSH
- rostlinné proteiny biosyntéza genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Haloalkane dehalogenases catalyze the hydrolysis of carbon-halogen bonds in various chlorinated, brominated and iodinated compounds. These enzymes have a conserved pair of halide-stabilizing residues that are important in substrate binding and stabilization of the transition state and the halide ion product via hydrogen bonding. In all previously known haloalkane dehalogenases, these residues are either a pair of tryptophans or a tryptophan-asparagine pair. The newly-isolated haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 (EC 3.8.1.5) possesses a unique halide-stabilizing tyrosine residue, Y109, in place of the conventional tryptophan. A variant of DatA with the Y109W mutation was created and the effects of this mutation on the structure and catalytic properties of the enzyme were studied using spectroscopy and pre-steady-state kinetic experiments. Quantum mechanical and molecular dynamics calculations were used to obtain a detailed analysis of the hydrogen-bonding patterns within the active sites of the wild-type and the mutant, as well as of the stabilization of the ligands as the reaction proceeds. Fluorescence quenching experiments suggested that replacing the tyrosine with tryptophan improves halide binding by 3.7-fold, presumably as a result of the introduction of an additional hydrogen bond. Kinetic analysis revealed that the mutation affected the substrate specificity of the enzyme and reduced its K(0.5) for selected halogenated substrates by a factor of 2-4, without impacting the rate-determining hydrolytic step. We conclude that DatA is the first natural haloalkane dehalogenase that stabilizes its substrate in the active site using only a single hydrogen bond, which is a new paradigm in catalysis by this enzyme family.
- MeSH
- Agrobacterium tumefaciens enzymologie metabolismus MeSH
- analýza hlavních komponent MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- biokatalýza MeSH
- halogenované uhlovodíky chemie metabolismus MeSH
- halogeny chemie metabolismus MeSH
- hydrolasy chemie genetika metabolismus MeSH
- hydrolýza MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- kvantová teorie MeSH
- molekulární modely MeSH
- mutageneze cílená MeSH
- mutantní proteiny chemie metabolismus MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- stabilita enzymů MeSH
- substituce aminokyselin MeSH
- substrátová specifita MeSH
- tyrosin chemie MeSH
- vodíková vazba MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Modified versions of the Cry3A gene of Bacillus thuringiensis (Bt) were transferred into Norway spruce (Picea abies). Both the biolistic approach and Agrobacterium tumefaciens mediated procedure were employed for transformation of embryogenic tissue (ET) cultures. The latter method proved to be more efficient yielding 70 transgenic embryogenic tissue lines compared with 18 lines obtained by biolistics. The modified Cry3A genes were driven by a 35S promoter and the nptII screenable selection marker gene was used in all vectors. The transgenic ETs were molecularly characterized and converted into mature somatic embryos. Germinating embryos formed plantlets which were finally planted into perlite and their Cry3A gene transcription activities were demonstrated by RT-PCR.
- MeSH
- Agrobacterium tumefaciens genetika MeSH
- Bacillus thuringiensis genetika MeSH
- bakteriální proteiny genetika MeSH
- endotoxiny genetika MeSH
- genetická transkripce MeSH
- genetické vektory MeSH
- geneticky modifikované rostliny * MeSH
- hemolyziny genetika MeSH
- jedle embryologie genetika MeSH
- promotorové oblasti (genetika) MeSH
- semena rostlinná genetika růst a vývoj MeSH
- somatická embryogeneze rostlin MeSH
- transformace genetická MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
