In plants the alc gene expression system responds more rapidly following induction with acetaldehyde than with ethanol

It has recently been proposed that acetaldehyde is the physiological inducer of the alc gene system and hence indirectly the activator of the AlcA promoter in Aspergillus nidulans. Here we show that this chemical induces expression of a GUS (beta-D-glucuronidase) reporter under the control of the alc gene system in transgenic potato tubers more rapidly than ethanol allowing tighter control of transgene expression. Furthermore by analysis of metabolite levels we demonstrate that the application of inducer has few effects on metabolism. We propose that this system is therefore ideal for the temporal regulation of important metabolic enzyme activities.     

Local induction of the alc gene switch in transgenic tobacco plants by acetaldehyde

The alc promoter system, derived from the filamentous fungi Aspergillus nidulans, allows chemically regulated gene expression in plants and thereby the study of gene function as well as metabolic and developmental processes. In addition to ethanol, this system can be activated by acetaldehyde, described as the physiological inducer in A. nidulans. Here, we show that in contrast to ethanol, acetaldehyde allows tissue-specific activation of the alc promoter in transgenic tobacco plants. Soil drenching with aqueous acetaldehyde solutions at a concentration of 0.05% (v/v) resulted in the rapid and temporary induction of the alc gene expression system exclusively in roots. In addition, the split root system allows activation to be restricted to the treated part of the root. The temporary activation of the alc system by soil drenching with acetaldehyde could be prolonged over several weeks by subsequent applications at intervals of 7 d. This effect was demonstrated for the root-specific induction of a yeast-derived apoplast-located invertase under the control of the alcohol-inducible promoter system. In leaves, which exhibit a lower responsiveness to acetaldehyde than roots, the alc system was induced in the directly treated tissue only. Thus, acetaldehyde can be used as a local inducer of the alc gene expression system in tobacco plants.     

Ethanol vapor is an efficient inducer of the alc gene expression system in model and crop plant species

We have demonstrated that low concentrations of ethanol vapor efficiently induce the alc gene expression system in tobacco (Nicotiana tabacum cv Samsun NN), potato (Solanum tuberosum cv Solara), and oilseed rape (Brassica napus cv Westar). For many situations, this may be the preferred method of induction because it avoids direct application of comparatively high concentrations of an ethanol solution. Although induction was seen with less than 0.4 microM ethanol vapor, maximal induction of the chloramphenicol acetyl transferase gene was achieved after 48 h in leaves of tobacco plants enclosed with 4.5 microM ethanol vapor. In the absence of ethanol, there is no detectable gene expression. Treatment of potato tubers with ethanol vapor results in uniform beta-glucoronidase (GUS) expression. Vapor treatment of a single oilseed rape leaf resulted in induction of GUS in the treated leaf only and (14)C-ethanol labeling in tobacco confirmed that the inducer was not translocated. In contrast, enclosure of the roots, aerial parts, or whole plant with ethanol vapor resulted in induction of GUS activity in leaves and roots. The data reported here broaden the utility of the alc system for research and crop biotechnology.     

Marker-free site-specific gene integration in rice based on the use of two recombination systems

Transgene integration mediated by heterologous site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. This approach of plant transformation generates a precise site-specific integration (SSI) structure consisting of a single copy of the transgene construct. As a result, stable transgene expression correlated with promoter strength and gene copy number is observed among independent transgenic lines and faithfully transmitted through subsequent generations. Site-specific integration approaches use selectable marker genes, removal of which is necessary for the implementation of this approach as a biotechnology application. As SSR systems are also excellent tools for excising marker genes from transgene locus, a molecular strategy involving gene integration followed by marker excision, each mediated by a distinct recombination system, was earlier proposed. Experimental validation of this approach is the focus of this work. Using FLPe-FRT system for site-specific gene integration and heat-inducible Cre-lox for marker gene excision, marker-free SSI lines were developed in the first generation itself. More importantly, progeny derived from these lines inherited the marker-free locus, indicating efficient germinal transmission. Finally, as the transgene expression from SSI locus was not altered upon marker excision, this method is suitable for streamlining the production of marker-free SSI lines.     

A reporter gene system used to study developmental expressionof alternative oxidase and isolate mitochondrial retrograde regulationmutants in Arabidopsis

Perturbation of mitochondrial function causes altered nuclear gene expression in plants. To study this response, called mitochondrial retrograde regulation, and developmental gene expression, a transgenic Arabidopsis thaliana (Col-0) line containing a firefly luciferase gene controlled by a promoter region of the Arabidopsis alternative oxidase 1a gene (AtAOX1a) was created. The transgene and the endogenous gene were developmentally induced in young cotyledons to a level higher than in older cotyledons and leaves. Analysis of transgene expression suggests that this is true for emerging leaves as well. Antimycin A (AA), a mitochondrial electron transport chain inhibitor, and monofluroacetate (MFA), a TCA cycle inhibitor, induced expression of the transgene and the endogenous gene in parallel. The following comparative responses of the transgene to inhibitors were observed: (a) the response in cotyledons to AA treatment differed greatly in magnitude from the response in leaves; (b) the induction kinetics in cotyledons following MFA treatment differed greatly from the kinetics in leaves; and (c) the induction kinetics following MFA treatment differed from the kinetics of AA in both leaves and cotyledons. The transgenic line was used in a genetic screen to isolate mutants with greatly decreased transgene and AtAOX1a induction in response to AA. Some of these mutant lines showed greatly decreased induction by MFA, but one did not. Taken altogether, the data provide genetic evidence that suggests that induction of the AtAOX1a gene by distinct mitochondrial perturbations are via distinct, but overlapping signaling pathways that are tissue specific.     

The expression of tomato prosystemin gene in tobacco plants highly affects host proteomic repertoire

Systemin, an octadecapeptide isolated from tomato, is a primary signal molecule involved in the local and systemic responses to pest attack, elicited by activation of a set of defence genes. It derives from processing of prosystemin, a prohormone of almost 200 amino acids. Prosystemin orthologues have been found in other Solanaceae species but not in tobacco, where are present hydroxyproline-rich peptides functionally but not structurally related to tomato systemin. Molecular events leading to the release of signalling peptides from protein precursors are unknown in plants; the occurrence of a family of signal molecules suggests that initiation of wound response may involve different processing mechanisms. It has been previously shown that the protein product from an engineered tomato prosystemin gene is processed in tobacco, thus suggesting that the components responsible for its post-translational modifications are present in this species. By analyzing analysing the proteome repertoire of transformed tobacco plant leaves with 2-DE, here we demonstrate that the constitutive expression of the tomato prosystemin gene highly affected host protein synthesis. In particular, engineered plants showed a number of differentially synthesized proteins that were identified by PMF MALDI-TOF and microLC-ESI-IT-MS/MS experiments as polypeptide species involved in protection from pathogens and oxidative stress, or in carbon/energy metabolism. Significant differences in over-produced proteins were observed with respect to previous data reported on systemin-engineered tomato plants. Our results strongly support the need of using proteomic approaches during systematic analysis of plant tissues to investigate the principle of substantial equivalence in transgenic plants expressing a transgene coding for a signalling molecule.     

Site-specific excisional recombination strategies for elimination of undesirable transgenes from crop plants

A major limitation of crop biotechnology and breeding is the lack of efficient molecular technologies for precise engineering of target genomic loci. While transformation procedures have become routine for a growing number of plant species, the random introduction of complex transgenenic DNA into the plant genome by current methods generates unpredictable effects on both transgene and homologous native gene expression. The risk of transgene transfer into related plant species and consumers is another concern associated with the conventional transformation technologies. Various approaches to avoid or eliminate undesirable transgenes, most notably selectable marker genes used in plant transformation, have recently been developed. These approaches include cotransformation with two independent T-DNAs or plasmid DNAs followed by their subsequent segregation, transposon-mediated DNA elimination, and most recently, attempts to replace bacterial T-DNA borders and selectable marker genes with functional equivalents of plant origin. The use of site-specific recombination to remove undesired DNA from the plant genome and concomitantly, via excision-mediated DNA rearrangement, switch-activate by choice transgenes of agronomical, food or feed quality traits provides a versatile “transgene maintenance and control” strategy that can significantly contribute to the transfer of transgenic laboratory developments into farming practice. This review focuses on recent reports demonstrating the elimination of undesirable transgenes (essentially selectable marker and recombinase genes) from the plant genome and concomitant activation of a silent transgene (e.g., a reporter gene) mediated by different site-specific recombinases driven by constitutive or chemically, environmentally or developmentally regulated promoters. These reports indicate major progress in excision strategies which extends application of the technology from annual, sexually propagated plants towards perennial, woody and vegetatively propagated plants. Current trends and future prospects for optimization of excision-activation machinery and its practical implementation for the generation of transgenic plants and plant products free of undesired genes are discussed.     

CMV抑制PVYN CP基因沉默及其介导的抗病性

以前曾报道用RNA介导的抗病毒策略,获得了高度抗病的表达马铃薯Y病毒坏死株系外壳蛋白基因(PVY^N CP)的转基因烟草,并对T1、T2代转基因植株进行了遗传和抗病性分析。此次以T,代转基因植株为试验材料,在筛选高度抗病植株并证明其抗病性是基于转基因沉默的基础上,采用Northern杂交的方法,证明CMV侵染抑制了转基因植株中PVY^N CP基因的沉默,而且CMV对PVY^N CP基因沉默的抑制部位是发生在接种后的新生叶上,接种叶及其下部叶片中PVY^N CP基因沉默则未受到影响。采用ELISA方法对CMV PVY^N复合接种的转基因植株进行PVY^N检测,结果表明,接种叶及下部叶没有检测到PVY^N,植株叶片对PVY^N表现为抗病。而在CMV接种后植株新生叶中则检测出了高滴度的PVY^N,植株叶片对PVY^N表现为感病。该文报道了在表达PVY^N CP基因的RNA介导抗性转基因植株中,异源病毒侵染抑制了转基因的沉默,并导致转基因植株的抗病性丧失。     

Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitive response and nonspecific disease resistance.

The rapid and effective activation of disease resistance responses is essential for plant defense against pathogen attack. These responses are initiated when pathogen-derived molecules (elicitors) are recognized by the host. We have developed a strategy for creating novel disease resistance traits whereby transgenic plants respond to infection by a virulent pathogen with the production of an elicitor. To this end, we generated transgenic tobacco plants harboring a fusion between the pathogen-inducible tobacco hsr 203J gene promoter and a Phytophthora cryptogea gene encoding the highly active elicitor cryptogein. Under noninduced conditions, the transgene was silent, and no cryptogein could be detected in the transgenic plants. In contrast, infection by the virulent fungus P. parasitica var nicotianae stimulated cryptogein production that coincided with the fast induction of several defense genes at and around the infection sites. Induced elicitor production resulted in a localized necrosis that resembled a P. cryptogea-induced hypersensitive response and that restricted further growth of the pathogen. The transgenic plants displayed enhanced resistance to fungal pathogens that were unrelated to Phytophthora species, such as Thielaviopsis basicola, Erysiphe cichoracearum, and Botrytis cinerea. Thus, broad-spectrum disease resistance of a plant can be generated without the constitutive synthesis of a transgene product.     

Wound-inducible and organ-specific expression of ORF13 from Agrobacterium rhizogenes 8196 T-DNA in transgenic tobacco plants

Tissue-specific expression of the ORF13 promoter from Agrobacterium rhizogenes 8196 was assessed throughout the development of transgenic tobacco plants using a GUS reporter gene. ORF13 exhibited high activity in roots but with different patterns of expression. The activity of the ORF13 promoter in vascular tissues increased from the base to the tip of the stem. The ORF13 promoter is wound inducible in a limited area adjacent to the wound site. The time course of wound induction of ORF13 in transgenic tobacco containing an ORF13 promoter-GUS translational fusion was similar to that previously described for genes involved in plant defense responses. A series of 5′ deletions of the ORF13 promoter fused to the β-glucuronidase gene was examined for expression in roots and leaves of transgenic plants. Cis-acting elements that modulate quantitative expression of the transgene after wounding were detected.     

Experimental models for creation of transgenic plants resistant to stress factors

Experimental models of the potato primary transgenic plants which express the hybrid gene cry3aM-licBM2 have been created. Modecular analysis and the biotests of the experimental models allow proposing a new system of cry genes expression in plants. The system is based on the expression of hybrid genes possessing the sequence of reporter lichenase gene and the use as a regulator element of a light-induced promoter providing preferential expression of the controled genes only in green plant tissues (leaves)--the target tissues for pests. The lichanase presence in hybrid proteins facilitates selection and analysis of the expression level of the hybris proteins in transgenic organisms. Basing on the lichenase properties in hybrid proteins it seems possible to use this reporter system for transgene monitoring in agrocoenosis as this system is rather simple and precise and does not need large material and time expenses.     

In vivo gene transfer by electroporation allows expression of a fluorescent transgene in hamster testis and epididymal sperm and has no adverse effects upon testicular integrity or sperm quality

The study of gene function in testis and sperm has been greatly assisted by transgenic mouse models. Recently, an alternative way of expressing transgenes in mouse testis has been developed that uses electroporation to introduce transgenes into the male germ cells. This approach has been successfully used to transiently express reporter genes driven by constitutive and testis-specific promoters. It has been proposed as an alternative method for studying gene function in testis and sperm, and as a novel way to create transgenic animals. However, the low levels and transient nature of transgene expression that can be achieved using this technique have raised concerns about its practical usefulness. It has also not been demonstrated in mammals other than mice. In this study, we show for the first time that in vivo gene transfer using electroporation can be used to express a fluorescent transgene in the testis of a mammal other than mice, the Syrian golden hamster. Significantly, for the first time we demonstrate expression of a transgene in epididymal sperm using this approach. We show that expression of the transgene can be detected in sperm for as long as 60 days following gene transfer. Finally, we provide the first systematic demonstration that this technique does not lead to any significant long-term adverse effects on testicular integrity and sperm quality. This technique therefore offers a novel way to study gene function during fertilization in hamsters and may also have potential as a way of creating transgenic versions of this important model species.     

Ethanol-inducible gene expression: first step towards a new green revolution?

The introduction of dwarf varieties of cereals was fundamental to the green revolution, but in the post-genomic era, the manipulation of plant morphology could be more sophisticated. A recent publication by Tahar Ait-ali et al. describes the use of the ethanol-inducible transgene expression system to re-examine plant architecture, and the genes that determine it. Their findings have implications for the manipulation of plant height and yield, and demonstrate the efficacy of regulated transgene expression for functional genomics.     

Characterization of an ethanol-inducible promoter system in Catharanthus roseus hairy roots

Efforts to engineer Catharanthus roseus hairy roots to produce commercially significant amounts of valuable compounds, such as the terpenoid indole alkaloids vinblastine and vincristine, require the development of tools to study the effects of overexpressing key metabolic and regulatory genes. The use of inducible promoters allows researchers to control the timing and level of expression of genes of interest. In addition, use of inducible promoters allows researchers to use a single transgenic line as both the control and experimental line, minimizing the problems associated with clonal variation. We have previously characterized the use of a glucocorticoid-inducible promoter system to study the effects of gene overexpression within the terpenoid indole alkaloid pathway on metabolite production. Here the feasibility of using an ethanol-inducible promoter within C. roseus hairy roots is reported. This ethanol-inducible promoter is highly sensitive to ethanol concentration with a concentration of 0.005% ethanol causing a 6-fold increase in CAT reporter activity after 24 h of induction. The ethanol-inducible CAT activity increased 24-fold over a 72-h induction period with 0.5% ethanol.     

Recent advances in transgenic technology

Techniques that allow modification of the mammalian genome have made a considerable contribution to many areas of biological science. Despite these achievements, challenges remain in two principal areas of transgenic technology, namely gene regulation and efficient transgenic livestock production. Obtaining reliable and sophisticated expression that rivals that of endogenous genes is frequently problematic. Transgenic science has played an important part in increasing understanding of the complex processes that underlie gene regulation, and this in turn has assisted in the design of transgene constructs expressed in a tightly regulated and faithful manner. The production of transgenic livestock is an inefficient process compared to that of laboratory models, and the lack of totipotential embryonic stem (ES) cell lins in farm animal species hampers the development of this area of work. This article highlights recent progress in efficient transgene expression systems, and the current efforts being made to find alternative means of generating transgenic livestock.