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A 6.5 kb cucumber genomic DNA fragment containing the icl gene was introduced into Nicotiana plumbaginifolia and shown to direct isocitrate lyase (ICL) mRNA synthesis in transgenic seedlings upon germination, in a temporally regulated manner. Two putative icl promoter fragments, of 2900 and 572 bp, were subsequently linked to the GUS reporter gene and introduced into N. plumbaginifolia. Both constructs directed GUS expression after transgenic seed germination, and although the 572 bp fragment gave only 1% of the activity of the 2900 bp fragment, it directed expression in the same cotyledon-specific and temporally regulated pattern. Seedlings were transferred to darkness after 18 days growth in the light, to induce a starvation response. The 2900 bp construct was activated by starvation and repressed by exogenous sucrose, whereas the 572 bp construct was not starvation-responsive. To localize the region of the 2900 bp promoter fragment which is responsible for regulation by sucrose, further deletions were make, linked to GUS, and assayed in a cucumber protoplast transient assay system. Constructs with promoters of 2900, 2142 and 1663 bp were activated by starvation and repressed by sucrose, but promoters of 1142 and 572 bp showed no such response. We conclude that the icl gene promoter contains at least two distinct cis-acting elements, one required for the response to sucrose and the other which participates in expression upon seed germination. 相似文献
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Investigation of the endosperm-specific sucrose synthase promoter from rice using transient expression of reporter genes in guar seed tissue 总被引:1,自引:0,他引:1
We report the investigation of an endosperm-specific promoter from the rsus3 gene from rice (Oryza sativa). The promoter was characterized by deletion analysis and transient expression in guar (Cyamopsis tetragonoloba) seed-tissue. Transient expression was monitored by histochemical GUS assay, and quantitative dual reporter assays comprising firefly luciferase as a test reporter, and Renilla luciferase and GUS as reference reporters. These revealed high expression levels of the reporter genes directed by the rsus3 promoter in guar endosperm. Specificity for this tissue in seeds was apparent from a virtual absence of reporter activity in guar cotyledons. Removal of a putative intron region only slightly raised the expression level, whereas duplication of the minimal promoter region, in a tandem-repeat rsus3 promoter construct, retained endosperm specificity in guar, and displayed three times the reporter activity observed with the single copy construct. 相似文献
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Jason A. Able Carl Rathus Ian D. Godwin 《In vitro cellular & developmental biology. Plant》2001,37(3):341-348
Summary This report outlines the development of optimized particle inflow gun (PIG) parameters for producing transgenic sorghum (Sorghum bicolor (L.) Moench). Both transient and stable expression were examined when determining these parameters. The uidA reporter gene (GUS) encoding β-glucuronidase was used in transient experiments and the green fluorescent protein (GFP) used
to monitor stable expression. Initially, optimization was conducted using leaf segments, as the generation of sorghum callus
in sufficiently large quantities is time-consuming. Following leaf optimization, experiments were conducted using callus,
identifying a high similarity between the two tissue types (r
s=0.83). High levels of GUS expression were observed in both leaf and callus material when most distant from the DNA expulsion
point, and using a pressure greater than 1800 kPa. A higher level of expression was also observed when the aperture of the
helium inlet valve was constricted. Using the optimized conditions (pressure of 2200 kPa, distance to target tissue of 15
cm from the expulsion point, and the aperture of the helium inlet valve at one full turn), three promoters (Ubiquitin, Actinl and CaMV 35S) were evaluated over a 72-h period using GUS as the reporter gene. A significantly higher number of GUS foci were counted
with the Ubiquitin construct over this period, compared to the Actinl and CaMV 35S constructs. Stable callus sectors (on 2 mg 1−1 bialaphos) with GFP expression were visualized for as long as 6 wk post-bombardment. Using this optimized protocol, several
plants were regenerated after having been bombarded with the pAHC20 construct (containing the bar gene), with molecular evidence confirming integration. 相似文献
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Inducible promoters are important in regulating the expression of resistance genes when plants are attacked by insects or pathogens. Evaluation of the Shpx6b peroxidase promoter from the tropical forage legume Stylosanthes humilis[ Curtis MD, Rae AL, Rusu AG, Harrison SJ & Manners JM (1997) A peroxidase gene promoter induced by phytopathogens and methyl jasmonates in transgenic plants. Molecular Plant Microbial Interactions 10: 326–338] in transgenic tobacco plants Nicotiana tabacum L. (Solanaceae) demonstrated that this promoter could drive expression of both the β‐glucuronidase (GUS uidA gene of E. coli) and green fluorescent protein (GFP) reporter genes in leaf tissues during attack by chewing insects – larvae of potato tuber moth (PTM) Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae) and sucking insects – green peach aphids Myzus persicae Sulzer (Homoptera: Aphididae). Strong GUS expression was present in tissues next to cells damaged by PTM larvae 24 h after infestation. With aphid infestation, GUS expression was limited to sites of feeding, and was observed 48 h after infestation. The expression of GFP mirrored that of GUS expression for both treatments, but was normally detected 48 h after infestation. Similarly, the exogenous application of methyl jasmonate (MeJa) induced GUS uniformly across leaf tissue, and mechanical wounding activated GUS expression at wound sites, similar to PTM larvae. GFP expression was observed 48 h after treatment, and for mechanical wounding GFP was localised in a manner similar to PTM damage. For MeJa treatment, GFP expression was more pronounced in cells around the midrib, and it was not uniformly induced across the leaf tissue. GUS reporter gene levels were also assayed to quantify expression, and the results were consistent with the observed histological patterns of expression. The results presented here show that the Shpx6b promoter switches on the expression of linked genes after damage by insect herbivores, and could be useful in regulating the expression of heterologous genes for insect and/or pathogen resistance in transgenic plants. 相似文献
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Effects of the polyubiquitin gene Ubi.U4 leader intron and first ubiquitin monomer on reporter gene expression in Nicotiana tabacum 总被引:5,自引:0,他引:5
Plesse Bertrand Criqui Marie-Claire Durr Andrée Parmentier Yves Fleck Jacqueline Genschik Pascal 《Plant molecular biology》2001,45(6):655-667
We have previously shown by RNA gel blot analyses that the tobacco polyubiquitin-encoding gene Ubi.U4 is expressed in a complex pattern during plant development (Genschik et al., 1994). In order to study its tissue-specific expression, we cloned the fragment containing the –263 bp proximal promoter of the gene, the leader intron and the first ubiquitin monomer in front of the reporter GUS gene. Histochemical analyses for GUS activity during tobacco plant development revealed that the gene is expressed at variable amounts in many plant tissues with high levels in metabolically active and/or dividing cells and in the vascular tissues of the plant. We also analysed the expression pattern of constructs in which either the intron or the intron together with the first ubiquitin monomer were deleted. Our results indicate that the ubiquitin leader intron is not only a quantitative determinant of gene expression but may also influence the tissue-specific expression pattern. 相似文献
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Joseph F. Petolino Andrew Worden Krisi Curlee James Connell Tonya L. Strange Moynahan Cory Larsen Sean Russell 《Plant molecular biology》2010,73(6):617-628
A transgene, flanked by zinc finger nuclease (ZFN) cleavage sites, was deleted from a stably transformed plant by crossing it with a second plant expressing a corresponding ZFN gene. A target construct, containing a GUS reporter gene flanked by ZFN cleavage sites, a GFP reporter gene and a PAT selectable marker gene, was transformed into tobacco. Basta®-resistant plants were regenerated and screened for GUS and GFP expression. A second construct, containing a ZFN gene driven by the constitutive CsVMV promoter and an HPT selectable marker gene, was also transformed into tobacco. Selected T0 plants were grown to maturity and allowed to self-pollinate. Homozygous target plants, which expressed GUS and GFP, were crossed with homozygous ZFN plants, which expressed the ZFN gene. Numerous GUS-negative plants were observed among the hybrids with one particular cross displaying ~35% GUS-negative plants. Evidence for complete deletion of a 4.3 kb sequence comprising the GUS gene was obtained and sequence confirmed. Co-segregation in F2 progenies of ‘truncated’ and ‘intact’ target sequences with expected reporter gene phenotypes were observed. Since ZFNs can be designed to bind and cleave a wide range of DNA sequences, these results constitute a general strategy for creating targeted gene deletions. 相似文献
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Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells 总被引:7,自引:0,他引:7
This report describes the delivery of plasmid DNA containing either the β-glucuronidase (GUS) or the green fluorescent protein (GFP) reporter gene into intact plant cells of bamboo callus, lilium
scales, and Nicotiana benthamiana suspension culture cells. By first plasmolyzing the tissues or cells with 0.4 m sucrose in the presence of plasmid DNA, electroporation effectively delivers plasmid DNA into the intact plant cells. Transient
expression of the GUS gene, as revealed by histochemical assays, showed the presence of blue-staining areas in the electroporated
tissues. A short exposure of cells to 2% DMSO (dimethyl sulfoxide) prior to plasmolysis elevated the level of transient GUS
activity. When plasmid DNA containing a synthetic GFP gene was used, a strong green fluorescence was observed in N. benthamiana suspension culture cells that were subjected to plasmolysis and electroporation. These results suggest that plasmolysis brings
the plasmid DNA into the void space that is in close vicinity to the plasmalemma, allowing electroporation to efficiently
deliver the plasmid DNA into intact plant cells.
Received: 15 June 1998 / Revision received: 18 August 1998 / Accepted: 28 August 1998 相似文献
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Sergei F. Krasnyanski Jagdeep Sandhu Leslie L. Domier Dennis E. Buetow Schuyler S. Korban 《In vitro cellular & developmental biology. Plant》2001,37(4):427-433
Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa
mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their
effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable
regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation
of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed
the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T0 and T1 plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic
plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative
tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression
of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than
when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T1 plants segregated in a 3∶1 Mendelian ratio. 相似文献
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The effect of dithiothreitol (DTT) on the expression of the β-glucuronidase (GUS) reporter gene under the control of the CaMV-35 S promoter has been investigated by radioactive labelling
and immunoprecipitation of the enzyme in protoplasts from stably transformed tobacco plants and compared with that observed
in protoplasts transiently expressing the same gene construct. An increase in net accumulation of GUS during the culture period
in response to externally added DTT (2 mm) was observed both in protoplasts from transformed tobacco plants and in electroporated protoplasts. DTT had no effect on
rate of degradation of the mature GUS protein, as shown in a pulse-chase experiment. Relevant aspects of protoplast physiology,
such as viability, synthesis of 35S-labelled cellular proteins, or synthesis and export of pathogenesis-related proteins (one putative chitinase and two 1,3-β-glucanases) were not affected by the reducing reagent.
Received: 15 December 1997 / Revision received: 14 April 1998 / Accepted: 1 May 1998 相似文献
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In silico analysis showed that the differentially expressed type 3 oil palm metallothionein-like genes MT3-A and MT3-B share at least 11 common putative promoter regulatory elements. The identified motifs include W-boxes, TATCCA element, binding
element for cytokinin response regulators and pollen-specific elements. A high degree of conservation was observed in their
genomic organisation where the coding regions are divided at two identical positions in both genes by two AT-rich introns.
Promoter activity of the MT3-B gene was analysed using a transient assay by bombarding oil palm tissue slices with a β-glucuronidase (GUS) gene construct
and a stable reporter assay by analysing GUS expression in transformed Arabidopsis thaliana plants. Transient expression analysis revealed MT3-B promoter activity in oil palm root tissues but not in fruit mesocarp at 12 weeks after anthesis and spear leaves. The T3
homozygous transgenic Arabidopsis plants, harbouring the MT3-B promoter/GUS construct, showed reporter activity in cotyledons and mature leaves with lower expression levels in root tissues.
The expression levels in the roots of the T3 homozygous transgenic plants increased five- and 2.5-folds when treated with
80 μM of Zn2+ and Fe2+, respectively. Altogether, these results indicate that the MT3-A and MT3-B promoter activities may be regulated by a variety of abiotic factors and MT3-B promoter may potentially be manipulated for use in plant genetic engineering for induced synthesis of gene product. 相似文献
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Characterization of the regulatory elements of the maize P-rr gene by transient expression assays 总被引:2,自引:0,他引:2
The maize P-rr gene conditions floral-specific flavonoid pigmentation, especially in the kernel pericarp and cob. We analyzed the P-rr promoter by transient expression assays, in which segments of the P-rr promoter were fused to the GUS reporter gene and introduced into maize cells by particle bombardment. A basal P-rr promoter fragment (–235 to +326) gave low, but significant, levels of GUS reporter gene expression. Interestingly, two widely spaced segments containing enhancer-like activity were found. When tested individually, both the proximal (–1252 to –236) and distal (–6110 to –4842) segments boosted expression of the basal P-rr promoter::GUS construct about five-fold. A 1.6 kb segment of the P-rr promoter (–1252 to +326) containing the proximal enhancer and the 5-untranslated leader driving the GUS reporter gene showed preferential expression in BMS and embryogenic suspension cell cultures vs. endosperm-derived suspension cell cultures. These results demonstrate the application of transient assay techniques for the identification of regulatory elements responsible for floral-specific regulation of the complex P-rr gene promoter in maize. 相似文献