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  • 1
    Electronic Resource
    Electronic Resource
    The promoter of a cytosolic glutamine synthetase gene from the conifer Pinus sylvestris is active in cotyledons of germinating seeds and light-regulated in transgenic Arabidopsis thaliana (2001)
    Copenhagen : Munksgaard International Publishers
    Physiologia plantarum 112 (2001), S. 0 
    Details
    ISSN: 1399-3054
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: We have isolated and characterized a genomic clone encoding Scots pine (Pinus sylvestris) cytosolic glutamine synthetase (GS). The clone contains the 5′ end half of the gene including part of the coding region and 980 bp upstream of the translation initiation codon. The major transcription start site (+1) was mapped around 180 nucleotides upstream of the translation initiation codon. Sequence analysis of the 5′-upstream region of the gene reveals the presence of putative regulatory elements including a poly-CT consensus sequence, a purine-rich tandem repeat and two AT-rich regions. Fusions of the upstream gene region to uidA were shown to be transiently expressed in the cotyledons of germinating pine seeds transformed by microprojectile bombardment. Stable transformation of Arabidopsis thaliana revealed the shoot apical meristem as the major region of heterologous permanent expression in Arabidopsis, in agreement with the expression of the GS gene in Pinus. Moreover, quantitative data derived from fluorometric β-glucuronidase assays in control and continuous light-grown transgenic Arabidopsis plants indicate that the isolated upstream region of the gene contains regulatory sequences involved in the response to light.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1034/j.1399-3054.2001.1120312.x
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  • 2
    Electronic Resource
    Electronic Resource
    Modifying transient β-glucuronidase expression in pine species using introns (1998)
    Springer
    Plant cell, tissue and organ culture 52 (1998), S. 183-187 
    Details
    ISSN: 1573-5044
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effect of introns on gene expression was evaluated. Several intron-promoter combinations were introduced by microparticle bombardment into two pine species, stone pine (Pinus pinea L.) and salgareño pine (Pinus nigra Arn. ssp. Salzmannii (Dunal) Franco). Gene expression was evaluated by measuring transient GUS expression. Two promoters (CaMV35S and double CaMV35S modified) and two introns (intron 1 from maize genes alcohol dehydrogenase-1 and Shrunken-1) were used in our study. In both pine species tested, the Sh1-int1 increased transient GUS expression from 2 to 6-fold compared to the intron-less construction. On the contrary, the inclusion of the Adh1-int1 associated with the double CaMV35S modified resulted in a dramatic decrease in the expression in both pine species analyzed. Our results suggest that Sh1-int1 may be useful for the acquisition of the required levels of genetic activity of new agronomic traits introduced into pines.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006036704801
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  • 3
    Electronic Resource
    Electronic Resource
    Transient expression of the uidA gene in Pinus pinea cotyledons: A study of heterologous promoter sequences (1999)
    Springer
    Plant cell, tissue and organ culture 56 (1999), S. 69-78 
    Details
    ISSN: 1573-5044
    Keywords: conifer transformation ; uidA expression ; microparticle bombardment ; transient gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transfer and expression of the β-glucuronidase gene (uidA) in cultured cotyledons of stone pine (Pinus pinea L.) was obtained by microprojectile bombardment. Conditions for optimum transient expression were established by using plasmid pBI121 delivered by 1.0 μm-diameter gold particles, into 1-day-old cultured cotyledons. Helium pressure of 6.2 MPa, microcarrier travel distance of 6 cm, and 0.8 μg of plasmid DNA per bombardment, were the best parameters for high levels of transient uidA expression. By using these parameters, 98% of bombarded cotyledons showed β-glucuronidase activity, with a mean of 63 Gus foci per cotyledon. This system was used to study the expression of uidA gene driven by several heterologous promoters. The expression under the control of the sunflower polyubiquitin gene (UbB1) promoter (Δ1 deletion) was higher (99% of GUS positive cotyledons) than under the control of the CaMV35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower uidA expression, as determined histochemically. These results were confirmed by using the GUS fluorometric assay. Use of a deletion of the sunflower polyubiquitin promoter resulted in GUS activity detectable 35 days after bombardment, and significant levels of GUS activity were confirmed at the end of that period. The results will be useful to design protocols for stable transformation and high levels of transgene expression in P. pinea.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006250609262
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  • 4
    Electronic Resource
    Electronic Resource
    Factors involved in Agrobacterium tumefaciens-mediated gene transfer into Pinus nigra Arn. ssp. salzmannii (Dunal) Franco (2000)
    Springer
    Euphytica 114 (2000), S. 195-203 
    Details
    ISSN: 1573-5060
    Keywords: conifers ; salgareño pine ; tissue culture ; transformation ; transient gene expression ; uidA expression ; vir gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Cotyledons from dissected sterile embryos of salgareño pine (Pinus nigra Arn. ssp. salzmannii (Dunal) Franco) were inoculated with different disarmed Agrobacterium tumefaciens strains harbouring the binary vector p35SGUSint. The transient expression of a β-glucuronidase gene (uidA) was studied, using a histochemical staining procedure. Nineteen days after inoculation, the activity of β-glucuronidase was detected in epidermal and subepidermal layers of cotyledonary explants. The EHA105 strain harbouring a disarmed agropine-type Ti-plasmid (pTiBO542) was the most effective for gene transfer of the uidA gene. The effects of exudates and extracts from 0-day-old embryos on induction of vir gene expression in A. tumefaciens were also examined. The results of this study showed that salgarño pine embryo exudates contain a substance(s) that induce vir gene expression, in similar way to that observed with 100 μM acetosyringone (AS).All these findings suggest that T-DNA processing and transfer might take place when Agrobacterium infects suitable tissues of salgareño pine.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1003946131356
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  • 5
    Electronic Resource
    Electronic Resource
    Induction of a virulence response in Agrobacterium tumefaciens by exudates of Pinus pinea cotyledons (1998)
    Springer
    Plant cell, tissue and organ culture 55 (1998), S. 175-181 
    Details
    ISSN: 1573-5044
    Keywords: Conifer ; transformation ; virulence genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract As a preliminary step in efforts to develop a successful protocol for Agrobacterium-mediated transformation of cotyledonary explants of Pinus pinea L. embryos, we tested the ability of embrionary exudates of this species to induce the expression of the virulence genes virA, virB, virC, virD, virE and virG in Agrobacterium tumefaciens containing vir: lacZ fusion constructs. The results obtained in the vir induction assay indicated the absence of bactericidal or bacteriostatic plant compounds affecting A. tumefaciens growth, and showed that cotyledonary and embrionary exudates of P. pinea are able to induce all virulence genes studied, except virG. The data suggest that A. tumefaciens can be used for gene transfer into this important forest and fruit species.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006295622061
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