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At least 163 records · Page 9Linked to original sources

PubMed1994

Functional expression of the yeast FLP/FRT site-specific recombination system in Nicotiana tabacum.

The FLP/FRT site-specific recombination system of Saccharomyces cerevisiae was expressed in stably transformed tobacco plants. The FLP protein efficiently catalyzes recombination between two directly repeated FLP recombination target (FRT) sites, deleting the sequence between them. In the constructs tested here, this deletion places the CaMV 35S promoter adjacent to a hygromycin resistance marker; transcriptional activation of the marker allows direct selection of recombination events. After crossing plants containing an integrated FLP expression construct with plants containing a FLP substrate, F1 seedlings can be selected directly for hygromycin resistance, indicating that recombination occurs at, or very early after zygote formation. Molecular analysis confirmed the expected recombination product.

Agrobacterium tumefaciens↗

PubMed1994

Molecular analysis of transgenic plants generated by microprojectile bombardment: effect of petunia transformation booster sequence.

Supercoiled plasmid expression vectors containing the petunia transformation booster sequence (TBS) were introduced by microprojectile bombardment into dicotyledenous (tobacco) and monocotyledonous (maize) cells. TBS effected a 7.8- to 16-fold increase in transformation frequencies in tobacco, and a 1.7- to 2.4-fold increase in maize. Although TBS contains a well-defined transcription enhancer element, no increases in plasmid gene expression were observed. TBS did not alter integration patterns in transformants, and did not affect segregation of linkage in R1 progeny. Computer analyses of the TBS sequence revealed numerous modular elements previously shown to be associated with putative chromosomal replication origin regions in eukaryotes, including DNA unwinding elements, scaffold-associated regions and pyrimidine tracts.

Aminobutyrates↗

PubMed1997

Gene targeting approaches using positive-negative selection and large flanking regions.

We report here on strategies aimed at improving the frequency of detectable recombination in plants by increasing the efficiency of selecting double-recombinants in transgenic calli. Gene targeting was approached on the Gln1 and the Pzfloci of Lotus japonicus, using Agrobacterium tumefaciens T-DNA replacement vectors. Large flanking regions, up to 22.9 kb, surrounding a positive selection marker were presented as substrates for homologous recombination. For easier detection of putative recombinants the negative selectable marker cytosine deaminase was inserted at the outside borders of the flanking regions offered for cross-over. A combination of positive and negative selection allowing double-recombinants to grow, while counter-selecting random insertions, was used to select putative targeting events. The more than 1000-fold enrichment observed with replacement vectors designed to minimize gene silencing demonstrated the efficiency of the negative selection. Using five different replacement vectors an estimated total of 18,974 transformation events were taken through the positive-negative selection procedure and 185 resistant calli obtained. Targeting events could not be verified in the survivors by PCR screening and Southern blot analysis. With this approach the frequency of detectable gene targeting in L. japonicus was below 5.3 x 10(-5), despite the large flanking sequences offered for recombination.

Antimetabolites↗

PubMed1998

Construction of a new vector conferring methotrexate resistance in Nicotiana tabacum plants.

A new binary vector encoding for Candida albicans dihydrofolate reductase (DFR1) has been constructed and used as a dominant selectable marker for plant transformation. Transgenic tobacco plants with an increased resistance to methotrexate (Mtx) were obtained by co-transformation of tobacco leaf discs with Agrobacterium tumefaciens strains carrying two new binary vectors: pTI20 and pTI18. Co-transformants of Nicotiana tabacum were directly selected for and rooted on medium containing both kanamycin (kan) and Mtx. Leaf discs of transgenic plants were assayed for capacity of regeneration at different Mtx concentrations. Analysis of transcripts was performed on total RNA extracted from two Mtx-resistant plants. The transgenic plants increased resistance to Mtx can be explained by the exceptionally low capacity of Mtx to bind C. albicans dihydrofolate reductase, accountable by the presence of two amino acid residues strategically important in Mtx binding.

Agrobacterium tumefaciens↗

PubMed1993

Correlation between arrested secondary plasmodesmal development and onset of accelerated leaf senescence in yeast acid invertase transgenic tobacco plants.

Mature leaves of a transgenic tobacco plant (Nicotiana tabacum L. var. Samsun, line A41-10) that constitutively express a yeast-derived acid invertase gene develop symptoms which are characterized by the presence of greenish-yellow and green sectors in the same leaf, and onset of early leaf senescence. Previous studies indicated that invertase activity was two- to threefold higher in the greenish-yellow sectors than in the green sectors. Our structural analyses revealed that development of secondary plasmodesmata, via modification of existing primary plasmodesmata, between mesophyll cells was inhibited severely in the greenish-yellow sectors, but only marginally in the green sectors. In contrast, the structure and function of primary plasmodesmata in the same symptomatic sectors remained unaltered as determined by structural and dye coupling studies. It is hypothesized that secondary plasmodesmata differ from primary plasmodesmata in having special abilities to traffic information molecules to coordinate leaf development and physiological function(s). Arrest of secondary plasmodesmal development by high invertase activity in the transgenic tobacco leaf may have prevented this type of trafficking and hence resulted in early leaf senescence. The results also indicate that the yeast acid invertase-expressing tobacco may provide an effective experimental system for the molecular characterization of cellular mechanisms that regulate the development, function, and possible turnover of secondary plasmodesmata.

Gene Expression↗

PubMed1995

FLP recombinase in transgenic plants: constitutive activity in stably transformed tobacco and generation of marked cell clones in Arabidopsis.

FLP site-specific recombinase was expressed in stably transformed tobacco and Arabidopsis. FLP-expressing tobacco lines were crossed with other transformed tobacco lines that contained a stably integrated FLP recognition target construct(s). The target construct consisted of two directly-oriented FLP recognition targets (FRTs), flanking a hygromycin resistance cassette located between a GUS coding region and an upstream 35S CaMV promoter. Excision of the hygromycin resistance cassette by FLP-mediated recombination between FRTs brings the GUS coding region under the transcriptional control of the CaMV 35S promoter. In the absence of FLP-mediated recombination, the GUS gene is transcriptionally silent. GUS activity was observed in the progeny of all crosses made between FLP recombinase-expressing and target-containing tobacco lines, but not in the selfs of parents. The predicted recombination product remaining after excision was confirmed by PCR and Southern analysis. In Arabidopsis, inducible expression of FLP recombinase was achieved from the soybean Gmhsp 17.6L heat-shock promoter. Heat-shock induction of FLP expression in plants containing the target construct led to activation of constitutive GUS expression in a subset of cells, whose progeny, therefore, were GUS-positive. A variety of clonal sectors were produced in plants derived from seed that was heat-shocked during germination. The ability to control the timing of GUS activation was demonstrated by heat-shock of unopened flower heads which produced large sectors. It was concluded that heat-shock-induced expression of FLP recombinase provides a readily controllable method for generating marked clonal sectors in Arabidopsis, the size and distribution of which reflects the timing of applied heat-shock.

Arabidopsis↗

PubMed1994

Increased disease susceptibility of transgenic tobacco plants with suppressed levels of preformed phenylpropanoid products.

It has been proposed that natural products synthesized by plants contribute to their resistance to pests and pathogens. We show here that transgenic tobacco plants with suppressed levels of the phenylpropanoid biosynthetic enzyme phenylalanine ammonia-lyase (L-phenylalanine ammonia-lyase, EC 4.3.1.5) and correspondingly low levels of chlorogenic acid, the major soluble leaf phenylpropanoid product, exhibit more rapid and extensive lesion development than wild-type plants after infection by the virulent fungal pathogen Cercospora nicotianae. These observations provide direct evidence that phenylpropanoid products contribute to disease limitation. No induction of transcripts encoding phenylalanine ammonia-lyase or the lignin branch pathway enzyme caffeic acid O-methyltransferase was observed during the infection and there was no perturbation in the pattern of soluble phenylpropanoids. Hence, increased disease susceptibility does not involve inhibition of a pathogen-induced response but likely reflects inhibition of the developmental accumulation of chlorogenic acid. Demonstration of the contribution of such preformed protectants to plant health identifies attractive targets for manipulation by breeding or gene transfer to reduce the quantitative impact of disease.

Disease Susceptibility↗

PubMed1992

Somatic and germinal recombination of a direct repeat in Arabidopsis.

Homologous recombination between a pair of directly repeated transgenes was studied in Arabidopsis. The test construct included two different internal, non-overlapping deletion alleles of npt (neomycin phosphotransferase) flanking an active HPT (hygromycin phosphotransferase) gene. This construct was introduced into Arabidopsis by agrobacterium-mediated transformation with selection for resistance to hygromycin, and two independent single-insert lines were analyzed. Selection for active NPT by resistance to kanamycin gave both fully and partly (chimeric) recombinant seedlings. Rates for one transgenic line were estimated at less than 2 x 10(-5) events per division for germinal and greater than 10(-6) events per division for somatic recombination, a much smaller difference than between meiotic and mitotic recombination in yeast. Southern analysis showed that recombinants could be formed by either crossing over or gene conversion. A surprisingly high fraction (at least 2/17) of the recombinants, however, appeared to result from the concerted action of two or more independent simple events. Some evolutionary implications are discussed.

Alleles↗

PubMed1999

Double-strand break-induced recombination between ectopic homologous sequences in somatic plant cells.

Homologous recombination between ectopic sites is rare in higher eukaryotes. To test whether double-strand breaks (DSBs) can induce ectopic recombination, transgenic tobacco plants harboring two unlinked, nonfunctional homologous parts of a kanamycin resistance gene were produced. To induce homologous recombination between the recipient locus (containing an I-SceI site within homologous sequences) and the donor locus, the rare cutting restriction enzyme I-SceI was transiently expressed via Agrobacterium in these plants. Whereas without I-SceI expression no recombination events were detectable, four independent recombinants could be isolated after transient I-SceI expression, corresponding to approximately one event in 10(5) transformations. After regeneration, the F1 generation of all recombinants showed Mendelian segregation of kanamycin resistance. Molecular analysis of the recombinants revealed that the resistance gene was indeed restored via homologous recombination. Three different kinds of reaction products could be identified. In one recombinant a classical gene conversion without exchange of flanking markers occurred. In the three other cases homologous sequences were transferred only to one end of the break. Whereas in three cases the ectopic donor sequence remained unchanged, in one case rearrangements were found in recipient and donor loci. Thus, ectopic homologous recombination, which seems to be a minor repair pathway for DSBs in plants, is described best by recombination models that postulate independent roles for the break ends during the repair process.

Base Sequence↗

PubMed2000

Genetic engineering on shikonin biosynthesis: expression of the bacterial ubiA gene in Lithospermum erythrorhizon.

The naphthoquinone pigment shikonin from Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) was the first plant secondary metabolite produced in industrial scale from plant cell cultures. We have now manipulated the biosynthetic pathway leading to shikonin in L. erythrorhizon by introduction of the bacterial gene ubiA. This gene of Escherichia coli encodes 4-hydroxybenzoate-3-polyprenyltransferase, a membrane-bound enzyme that catalyzes a key step in ubiquinone biosynthesis. Using geranyl diphosphate (GPP) as substrate, it is able to catalyze the formation of 3-geranyl-4-hydroxybenzoate (GBA), a principal step of shikonin biosynthesis. The prokaryotic ubiA gene was fused to two signal sequences for targeting of the resulting peptide to the endoplasmic reticulum (ER). Constructs with different constitutive promoters were introduced into L. erythrorhizon using Agrobacterium rhizogenes-mediated transformation. In the resulting hairy root lines, high UbiA enzyme activities could be observed, reaching 133 pkat mg(-1). Expression of ubiA resulted in an accumulation of GBA in an amount exceeding that of the control culture by a factor of 50. However, the ubiA-transformed lines showed only a marginal (average 22%) increase of shikonin production in comparison to the control lines, and there was no significant correlation of UbiA enzyme activity and shikonin accumulation. This suggests that overexpression of ubiA alone is not sufficient to increase shikonin formation, and that further enzymes are involved in the regulation of this pathway.

Alkyl and Aryl Transferases↗

PubMed1999

Restoration of wild-type virus by double recombination of tombusvirus mutants with a host transgene.

Nicotiana benthamiana plants transformed with the coat protein gene of tomato bushy stunt virus (TBSV) failed to elicit effective virus resistance when inoculated with wildtype virus. Subsequently, R1 and R2 progeny from 13 transgenic lines were inoculated with a TBSV mutant containing a defective coat protein gene. Mild symptoms typical of those elicited in nontransformed plants infected with the TBSV mutant initially appeared. However, within 2 to 4 weeks, up to 20% of the transgenic plants sporadically began to develop the lethal syndrome characteristic of wild-type virus infections. RNA hybridization and immunoblot analyses of these plants and nontransformed N. benthamiana inoculated with virus from the transgenic lines indicated that wild-type virus had been regenerated by a double recombination event between the defective virus and the coat protein transgene. Similar results were obtained with a TBSV deletion mutant containing a nucleotide sequence marker, and with a chimeric cucumber necrosis virus (CNV) containing the defective TBSV coat protein gene. In both cases, purified virions contained wild-type TBSV RNA or CNV chimeric RNA derived by recombination with the transgenic coat protein mRNA. These results thus demonstrate that recombinant tombus-viruses can arise frequently from viral genes expressed in transgenic plants.

Capsid↗

PubMed1999

A plant-derived edible vaccine against hepatitis B virus.

The infectious hepatitis B virus represents 42 nm spherical double-shelled particles. However, analysis of blood from hepatitis B virus carriers revealed the presence of smaller 22 nm particles consisting of a viral envelope surface protein. These particles are highly immunogenic and have been used in the design of hepatitis B virus vaccine produced in yeast. Upon expression in yeast, these proteins form virus-like particles that are used for parenteral immunization. Therefore, the DNA fragment encoding hepatitis B virus surface antigen was introduced into Agrobacterium tumerifacience LBA4404 and used to obtain transgenic lupin (Lupinus luteus L.) and lettuce (Lactuca sativa L.) cv. Burpee Bibb expressing envelope surface protein. Mice that were fed the transgenic lupin tissue developed significant levels of hepatitis B virus-specific antibodies. Human volunteers, fed with transgenic lettuce plants expressing hepatitis B virus surface antigen, developed specific serum-IgG response to plant produced protein.

Administration, Oral↗

PubMed1998

Recombination between viral DNA and the transgenic coat protein gene of African cassava mosaic geminivirus.

Nicotiana benthamiana was transformed with three different constructs (pCRA1, pCRA2 and pJC1) containing the coat protein coding sequence of African cassava mosaic virus (ACMV). Transformed plants were inoculated with a coat protein deletion mutant of ACMV that induces mild systemic symptoms in control plants. Several inoculated plants of transgenic lines CRA1/3, CRA1/4, CRA2/1 and CRA2/2 developed severe systemic symptoms typical of ACMV. DNA analysis revealed that, in these plants, recombination had occurred between the mutant viral DNA and the integrated construct DNA, resulting in the production of recombinant virus progeny with 'wild-type' characteristics. No reversion of mutant to 'wild-type' virus was observed in pJC1-transformed plants. Recombinant virus from several transgenic plants was analysed by PCR and parts of DNA A of virus progeny were cloned. Sequence analysis revealed that only a few nucleotides were changed from the published sequence.

Capsid↗

PubMed1990

Properties of the maize transposable element Activator in transgenic tobacco plants: a versatile inter-species genetic tool.

The maize controlling element Activator (Ac) transposes autonomously from an integrated T-DNA vector to new sites in the genomes of tobacco and other heterologous plant species. Here we demonstrate that critical functions required for transposition of Ac in maize are conserved in tobacco and that Ac transposes at high frequency for at least five generations. Ac structure and terminal sequences are conserved upon transposition and a characteristic 8-bp duplication of target sequences is generated upon integration. Ac remains unmethylated, transcriptionally active, and capable to trans-activate transposition of the nonautonomous Dissociation (Ds) element throughout several generations. In tobacco, as in maize, Ac transposes adjacent to low copy or unique DNA, and transcriptional analysis of unique target DNA provides evidence that an Ac element transposed into a gene. In maize, increasing copies of Ac delay the timing and reduce the frequency of early transposition of Ac and Ds. In tobacco, increasing copies of Ac correlate with an increased frequency of Ds trans-activation. These data firmly establish that the Ac and Ds transposable elements are versatile genetic tools well suited for use as insertional mutagens and demonstrate that thorough investigation of mechanism and regulation of transposition is facilitated in heterologous settings.

Base Sequence↗

PubMed2002

[In vitro culture and the Agrobacterium-mediated genetic transformation of Morinda officinalis].

OBJECTIVE: To establish an effective system for the Agrobacterium-mediated genetic transformation of M. officinalis, for laying a foundation for the improvement of breeds and introduction of foreign objective genes. METHOD: The explants used for culture were the nodular stem segments from M. officinalis. Agrobacterium tumefaciens strain was EHA101, containing vector plasmid pGA482GG. The GUS gene and NPT II gene were introduced into the plasmid. RESULT: MT basal medium with BA 1 mg.L-1 was effective to inducing the direct shoot formation, and the frequency of shoot formation was 97.8%. As BA concentrations increased, the ability of shoot formation decreased. The explants oriented with their apical ends protruding from the medium produced more shoots than when they were placed with their basal end upright or were placed horizontally. The optimal rooting medium for regenerating shoots was MT basal medium supplemented with 0.2 to 0.5 mg.L-1 NAA, and a root induction rate over 80.0% was observed. The selection pressure for kanamycin was 50 mg.L-1. Cefotaxime was used as antibiotics, and the concentration was 300 mg.L-1. After 1.5 months, 14.8% resistant shoots were emerged from the explants. Histochemical GUS assay showed that 22.2% of the resistant plants were GUS-positive. CONCLUSION: Plant regeneration system and Agrobacterium-mediated genetic transformation have been established for M. officinalis in vitro.

Agrobacterium tumefaciens↗

PubMed1991

Regeneration of transgenic Lycium barbarum L.

A simple and effective system for the transformation and regeneration of Lycium barbarum L. has been developed. Young stem segments from Lycium barbarum L. were infected by Agrobacterium tumefaciens harboring a vector containing neomycin phosphotransferase II (npt-II) gene derived from non-oncogenic Ti plasmid. Calli originating from young stem segments on selective induction medium could differentiate into buds on selective differentiation medium rapidly and finally developed into whole plants. NPT-II enzyme activity assay and DNA hybridization indicated that the foreign gene had been integrated into the genome of Lycium barbarum L. and could be expressed in plants.

Kanamycin↗

PubMed2003

Comparison of hCMV immediate early and CaMV 35S promoters in both plant and human cells.

Cauliflower mosaic virus 35S promoter, widely used in transgenic crop plants, is known to be recognized in widely differing kinds of cells. Its activity in human cells may have impact on the risk assessment for the environmental release of genetically modified plants. In this study, transient expression of several constructs containing beta-glucuronidase (GUS) gene driven by cauliflower mosaic virus 35S promoter or by immediate early promoter of human cytomegalovirus (pCMV) was tested in both potato leaf protoplasts and cultured human cells. The results showed very low but measurable activity of 35S promoter in human 293T-cells (0.01% of that revealed when using pCMV) and in 293 cells that do not produce SV40 T antigen this activity was even lower. On the other hand, in potato protoplasts, pCMV displayed nearly 1% activity seen with p35S.

Bacterial Outer Membrane Proteins↗

PubMed1998

Towards efficient conversion of electricity into edible biomass in crop production systems: a transgenic approach.

A major problem of controlled environment crop production is the procurement and distribution of adequate light for plant growth. Some electrically efficient light sources lack specific wavelengths required for photomorphogenesis yet provide a light spectrum with a high potential photosynthetic yield. To enable use of these lamps, we describe a concept to genetically modify plants to change their morphogenic responses to light. Photoreceptors in the red and far-red light sensing phytochrome family and the blue light sensing cryptochrome family may be useful to increase or decrease plant sensitivity to regions of the photomorphogenic spectrum. Uses of developmental masterswitches such as COP and DET, and downstream developmental controls, such as the cytokinin biosynthesis enzyme isopentenyl transferase, are also detailed. If photomorphogenic needs can be altered, then lamp selection can be based solely on electrical efficiency of light production and on potential photosynthetic yield from light produced. The combination of lamp electrical efficiency and photosynthetic yield, termed photosynthetic efficacy, was calculated for plant lighting sources. The low-pressure sodium (LPS) and high-pressure sodium (HPS) lamps provide the highest photosynthetic efficacies relative to microwave, metal halide, red and blue light-emitting diodes (LED), and fluorescent lamps. Typically plants grow tall and spindly under LPS or red LED irradiation and in some cases under the broader spectrum of HPS. The strategy of genetic modification of plant light requirements may enable use of electrically efficient and photosynthetically useful red-biased light sources without supplementation from less electrically efficient blue or less photosynthetically useful far-red light sources.

Biomass↗