Plant Transgenesis Research Articles

TGP – база данных промоторов для трансгенеза растений

TGP – база данных промоторов для трансгенеза растений

Simple database to select promoters for plant transgenesis

The experiments with transgenic plants frequently demand selection of promoters providing appropriate transcription patterns. The set of promoters commonly used in vectors and genetic constructs is very limited, and these promoters provide only a few variants of gene expression patterns. Moreover, identical promoters in a complex construct can induce transgene silencing. This problem can be solved using a variety of plant gene promoters with experimentally verified characteristics. However, this requires a time-consuming analysis of literature data. Here, we describe a database of plant promoters (TransGene Promoters, TGP; http://wwwmgs.bionet.nsc.ru/mgs/dbases/tgp/home.html ). TGP contains the information on genomic DNA segments providing certain expression patterns of reporter genes in experiments with transgenic plants. TGP was constructed on the SRS platform, and its interface allows users to search for the promoters with particular characteristics.

An improved particle bombardment for the generation of transgenic plants by direct immobilization of relleasable Tn5 transposases onto gold particles

We have developed a modified particle bombardment method for plant transgenesis. An intein-tag and a 6×Cys-tag were successively fused to the N-terminus of a hyperactive Tn5 transposase. The modified transposase was immobilized on bare gold microscopic particles via covalent binding of a 6×Cys-tag sulfydryl groups to the gold surface. The tethered transposase can bind the transposon DNA in vitro to form the transposome in the absence of Mg²⁺ ions. After bombardment of the gold particles carrying the transposomes into the plant cells, the transposomes will be released from the carrier due to the activated self-cleavage function of intein-tag. Our data showed this procedure integrated foreign DNA into the plant genome with an increased transformation frequency as compared to the conventional particle bombardment method. A single copy insertion can also be obtained by decreasing of the assembled transposon DNA amount in relation to plant cell biomass.

Advances and perspectives in artificial chromosomes

Artificial chromosomes (ACs) are genetic-engineered vector systems with defined native chromosomal elements. ACs have large carrying capacity and genetic stability without integration into host genome, thus avoiding random insertion and positional effects. ACs were first successfully developed in yeast (Yeast artificial chromosome, YAC), and then in bacterium (Bacterial artificial chromosome, BAC), human (Human artificial chromosome, HAC), and plant (Plant artificial chromosome, PAC). Here, we summarized recent progress on ACs, especially, on PAC. To date, YAC and BAC have been widely applied in genome sequencing and gene isolation, while HAC and PAC have been subjected to gene therapy, protein production, and plant transgenesis, respectively. Recently, American scientists reported a man-made genome of prokaryote Mycoplasma mycoides. However, like ACs, this man-made genome was also genetic-engineered product and can't survive as an independent life without a cellular environment.

Potassium chloride and rare earth elements improve plant growth and increase the frequency of the Agrobacterium tumefaciens-mediated plant transformation

Plant transformation efficiency depends on the ability of the transgene to successfully interact with plant host factors. Our previous work and the work of others showed that manipulation of the activity of host factors allows for increased frequency of transformation. Recently we reported that exposure of tobacco plants to increased concentrations of ammonium nitrate increases the frequency of both homologous recombination and plant transgenesis. Here we tested the influence of KCl and salts of rare earth elements, Ce and La on the efficiency of Agrobacterium-mediated plant transformation. We found that exposure to KCl, CeCl(3) and LaCl(3) leads to an increase in recombination frequency in Arabidopsis and tobacco. Plants grown in the presence of CeCl(3) and LaCl(3) had higher biomass, longer roots and greater root number. Analysis of transformation efficiency showed that exposure of tobacco plants to 50 mM KCl resulted in ~6.0-fold increase in the number of regenerated calli and transgenic plants as compared to control plants. Exposure to various concentrations of CeCl(3) showed a maximum increase of ~3.0-fold in both the number of calli and transgenic plants. Segregation analysis showed that exposure to KCl and cerium (III) chloride leads to more frequent integrations of the transgene at a single locus. Analysis of transgene intactness showed better preservation of right T-DNA border during transgene integration. Our data suggest that KCl and CeCl(3) can be effectively used to improve quantity and quality of transgene integrations.

Light intensity affects RNA silencing of a transgene in Nicotiana benthamiana plants.

BackgroundExpression of exogenous sequences in plants is often suppressed through one of the earliest described RNA silencing pathways, sense post-transcriptional gene silencing (S-PTGS). This type of suppression has made significant contributions to our knowledge of the biology of RNA silencing pathways and has important consequences in plant transgenesis applications. Although significant progress has been made in recent years, factors affecting the stability of transgene expression are still not well understood. It has been shown before that the efficiency of RNA silencing in plants is influenced by various environmental factors.ResultsHere we report that a major environmental factor, light intensity, significantly affects the induction and systemic spread of S-PTGS. Moreover, we show that photoadaptation to high or low light intensity conditions differentially affects mRNA levels of major components of the RNA silencing machinery.ConclusionsLight intensity is one of the previously unknown factors that affect transgene stability at the post-transcriptional level. Our findings demonstrate an example of how environmental conditions could affect RNA silencing.

Binding and Glutathione Conjugation of Porphyrinogens by Plant Glutathione Transferases

Overexpression in Escherichia coli of a tau (U) class glutathione transferase (GST) from maize (Zea mays L.), termed ZmGSTU1, caused a reduction in heme levels and an accumulation of porphyrin precursors. This disruption was highly specific, with the expression of the closely related ZmGSTU2 or other maize GSTs having little effect. Expression in E. coli of a series of chimeric ZmGSTU1/ZmGSTU2 proteins identified domains responsible for disrupting porphyrin metabolism. In addition to known heme precursors, expression of ZmGSTU1 led to the accumulation of a novel glutathione conjugate of harderoporphyrin(ogen) (2,7,12,18-tetramethyl-3-vinylporphyrin-8,13,17-tripropionic acid). Using the related protoporphyrinogen as a substrate, conjugation could be shown to occur on one vinyl group and was actively catalyzed by the ZmGSTU. In plant transgenesis studies, the ZmGSTUs did not perturb porphyrin metabolism when expressed in the cytosol of Arabidopsis or tobacco. However, expression of a ZmGSTU1-ZmGSTU2 chimera in the chloroplasts of tobacco resulted in the accumulation of the harderoporphyrin(ogen)-glutathione conjugate observed in the expression studies in bacteria. Our results show that the well known ability of GSTs to act as ligand binding (ligandin) proteins of porphyrins in vitro results in highly specific interactions with porphyrinogen intermediates, which can be demonstrated in both plants and bacteria in vivo.

Methods and Advances in Biotech

AbstractCover illustration: This special issue on “Methods and Advances in Biotech” consists mainly of technical reports on PCR‐based methods, focussing on multiplexing PCR, single cell PCR, real‐time PCR and other molecular biology methods. We also offer reviews on biomolecular computing, transgenesis in plants, and a method to discover tunnels in molecular structures. Picture DNA helix © David Martyn – FOTOLIA.

A farewell to bacterial ARMs?

A plant gene that confers antibiotic resistance provides a 'cleaner' selectable marker for plant transgenesis.

Agrobacterium proteins VirD2 and VirE2 mediate precise integration of synthetic T-DNA complexes in mammalian cells.

Agrobacterium tumefaciens-mediated plant transformation, a unique example of interkingdom gene transfer, has been widely adopted for the generation of transgenic plants. In vitro synthesized transferred DNA (T-DNA) complexes comprising single-stranded DNA and Agrobacterium virulence proteins VirD2 and VirE2, essential for plant transformation, were used to stably transfect HeLa cells. Both proteins positively influenced efficiency and precision of transgene integration by increasing overall transformation rates and by promoting full-length single-copy integration events. These findings demonstrate that the virulence proteins are sufficient for the integration of a T-DNA into a eukaryotic genome in the absence of other bacterial or plant factors. Synthetic T-DNA complexes are therefore unique protein:DNA delivery vectors with potential applications in the field of mammalian transgenesis.

RNA silencing: no mercy for viruses?

'RNA silencing' is a highly conserved mechanism leading to suppression of gene expression through nucleotide sequence-specific interactions that are mediated by 21-24 nucleotide-long RNAs. This process was first discovered as an unexpected consequence of transgenesis in plants, and similarly, it was subsequently identified in animals as an anomaly of antisense gene inhibition. We summarize the progressive steps that paved the way to our current understanding of the molecular basis and fundamental biological roles of RNA silencing in both plants and animals. In particular, we describe the general antiviral function of this mechanism in higher plants where it forms the basis of a highly elaborate immune system. All defense systems show some level of fallibility, and RNA silencing is no exception to this rule, as plant viruses have developed sophisticated ways to counteract various steps of the process. Recent work indicates that viruses are also engaged into a similar arms race in insects, but it remains unclear if RNA silencing plays a defensive role against virus infection of higher vertebrates. We also discuss some biotechnological applications of RNA silencing in mammalian cells that have fueled optimism that this mechanism may hold a promising future in antiviral human therapy.

Tourte Y. Genetically modified organisms: transgenesis in plants.

This is one of many recently published books describing the production and application of GMOs. It is pitched at ‘students and anyone concerned or curious about GMOs’ and, although a attempt has been made to keep the jargon to a minimum, detailed explanations of, for example ‘haplodiploidization to create homozygotes’ are unnecessary and set to trip the non‐specialist reader. The first part of the book gives a brief history of genetic engineering and summarizes the techniques used to integrate recombinant DNA into the plant genome. The latter part uses a dispassionate and generally positive tone to describe potential applications, and the concerns and questions raised by GMOs. The author draws specific examples from his own interests in modifying isoprene biosynthesis and describes other specialized potential applications such as making pharmaceuticals in plants, improving quality or nitrogen uptake, but fails to discuss the impact of the few GM traits currently grown in increasing area around the world. Specific points are well illustrated with examples, some of which hint at the author’s nationality and the fact that the book is a translation from its original 2001 title Les OGM in French. Particularly parochial is the section on regulatory and legal issues which describes, in too much detail, the independent workings of the commission du genie genetique and the commission du genie biomoleculaire in the release of GMOs in France. There is no colour except for the pink inset boxes used to exemplify or add detail throughout the chapters. The figures and photographs are generally helpful but it is a pity that the publishers chose to reproduce them at such a low image quality. It is an interesting little book that provides an insight into the underpinning science, the research applications and the concerns of genetic modification. If you want basic information covering the whole gamut of the GMO field under one cover, then this is a good start but it is less well presented and more parochial than others in the same genre (e.g. Halford, 200?)

Genetically modified organisms: transgenesis in plants

Genetically modified organisms: transgenesis in plants

Efficient male germ line transformation for transgenic tobacco production without selection.

Microspores at late uninucleate/early binucleate stages were isolated from flower buds of tobacco (Nicotiana tabacum L.) and in vitro culture methods optimised for their maturation to fully functional viable pollen which, after application to the stigma of emasculated plants in situ, led to the generation of large numbers of seed. Efficient protocols were established for the biolistic introduction of a construct containing a reporter gene and selectable marker into these microspores and hence, after in vitro maturation and in situ fertilisation, for the generation of transgenic plants. Stable transformants of low copy number were generated by this procedure. The efficiency of transformation achieved allows the production of large numbers of transgenic plants without selection, dispensing with the requirement for a selectable marker in plant transgenesis.