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PubMed1998

Flank matrix attachment regions (MARs) from chicken, bean, yeast or tobacco do not prevent homology-dependent trans-silencing in transgenic tobacco plants.

The effect of flanking matrix attachment regions (MARs) on homology-dependent trans-silencing was tested using two strong trans-silencing loci. The transgenic tobacco line 271 carries at a single locus a p35S-RiN-tNos transgene which is able to silence, in trans and at the transcriptional level, the expression of any p35S-driven transgene irrespective of its position. The transgenic tobacco line 6b8 carries at a single locus a p35S-uidA-tRbcS transgene which is able to silence in trans, at the post-transcriptional level, the expression of any uidA-expressing transgene irrespective of its position. Various transgenic tobacco lines carrying a target p35S-uidA-tNos transgene, flanked on each side by MARs from chicken, bean, yeast or tobacco, were crossed with lines carrying the 271 and 6b8 loci. Expression of the target transgene was silenced in all hybrids, irrespective of the presence or absence of MAR sequences. These results therefore demonstrate that MARs are not able to protect transgene expression from strong silencing loci that act in trans.

Animals↗

PubMed1993

Efficient pathogen-derived resistance induced by integrated potato virus Y coat protein gene in tobacco.

The coat protein (CP) gene from potato virus Y (Hungarian isolate, PVY-H) was engineered into Agrobacterium tumefaciens binary vector for expression in different tobacco lines. Three different Nicotiana tabacum breeding lines were transformed and the integration of the CP gene was confirmed by PCR technique using genomic DNA preparations. The transcription and expression of the integrated CP gene was detected by Northern and Western blots. Pathogen-derived resistance was demonstrated by inoculation of the R1 progeny of the transformed lines with purified PVY-H. The efficiency of protection varied between different transgenic plants ranging from almost complete to no protection. Five CP expressing tobacco lines were resistant to challenge infection with PVY-H as indicated by attenuation or absence of symptom development associated with reduction or lack of detectable virus accumulation. Data from Western blots showed that there is no correlation between the level of the expressed CP and the extent of protection. This suggests that the mechanism of the observed resistance is independent of the level of CP accumulation in the transgenic tobacco plants.

Agrobacterium tumefaciens↗

PubMed1998

Characterization and expression of a heptaubiquitin gene from tomato.

Ubiquitin is highly conserved 76 amino acid protein involved, among other functions, in the selective degradation of proteins in the cell. From a tomato (Lycopersicon esculentum Mill. cv. Craigella) genomic library, we have isolated a clone encoding a polyubiquitin gene, designated ubq1-1 comprising seven repeats of ubiquitin and two C-terminal extension amino acids. The ubq1-1 gene contains an intron of 1128bp immediately upstream of the translation start codon. DNA sequence comparison revealed that the 5' and 3' non-coding regions of the tomato ubq1-1 gene are nearly identical to the sequence of a polyubiquitin cDNA clone isolated from potato (Garbarino et al., 1992; Plant Mol. Biol. 20, 235-244). The ubq1-1 gene is expressed in leaves to rather low levels in tomato, and the abundance of ubq1-1 transcripts is increased under heat shock conditions. For functional analyses, a chimeric gene construct containing the intron and 1.6kb of ubq1-1 sequence 5' to the intron fused to the gus reporter gene was introduced into the tobacco genome. In leaves of transgenic tobacco plants, reporter gene expression was generally lower from the ubq1-1 promoter than from the cauliflower mosaic virus 35S RNA promoter. In addition, the tomato ubq1-1 promoter was not found to respond to heat shock in transgenic tobacco plants. Histochemical analysis of the plants demonstrated localization of gus reporter gene activity in the vascular systems of the leaves and the roots. Deletion of the intron from the reporter gene construct markedly reduced reporter gene expression in transformed tobacco plants, thus suggesting that the intron may influence transcript levels deriving from the ubq1-1 promoter.

Base Sequence↗

PubMed2001

Effects of a mustard trypsin inhibitor expressed in different plants on three lepidopteran pests.

The effects of mustard trypsin inhibitor MTI-2 expressed at different levels in transgenic tobacco, arabidopsis and oilseed rape lines have been evaluated against three different lepidopteran insect pests. 1. Plutella xylostella (L.) larvae were the most sensitive to the ingestion of MTI-2. The inhibitor expressed at high levels in arabidopsis plants caused rapid and complete mortality. High mortality and significantly delayed larval development were also detectable in oilseed rape expressing MTI-2 at lower levels. 2. Mamestra brassicae (L.) larvae were sensitive only at high MTI-2 expression level, as obtained in transgenic tobacco and arabidopsis, whereas no effects were observed for larvae fed on plants showing relatively low expression levels such as those of oilseed rape lines. 3. Feeding bioassays with Spodoptera littoralis (Boisduval) larvae were carried out using the same oilseed rape lines, showing that at these low expression levels no mortality was observed although a delay in larval development did occur. The levels of insect gut proteolytic activities of the larvae still alive at the end of a 7 day feeding bioassay were usually higher than in the controls, but no new proteinases were expressed in any case. The combined results described in this paper demonstrate altogether the relevance of a case-by-case analysis [target insects and proteinase inhibitor (PI) level of expression in planta] in a PI-based strategy for plant protection.

Animals↗

PubMed1997

Expression of de novo high-lysine alpha-helical coiled-coil proteins may significantly increase the accumulated levels of lysine in mature seeds of transgenic tobacco plants.

We have designed protein molecules based on an alpha-helical coiled-coil structure. These proteins can be tailored to complement nutritionally unbalanced seed meals. In particular, these proteins may contain up to 43% mol/mol of the essential amino acid lysine. Genes encoding such proteins were constructed using synthetic oligonucleotides and the protein stability was tested for in vivo by expression in an Escherichia coli model system. A protein containing 31% lysine and 20% methionine (CP 3-5) was expressed in transgenic tobacco seeds utilizing the seed specific bean phaseolin and soybean beta-conglycinin promoters. Both promoters provided a level of expression in the mature transgenic tobacco seeds which resulted in a significant increase in the total lysine content of the seeds. Several of these transgenic lines were analyzed for three generations to determine the stability of gene expression. Plants transformed with the soybean beta-conglycinin promoter/CP 3-5 gene consistently expressed the high-lysine phenotype through three generations. However, expression of the high-lysine phenotype in plants transformed with the bean phaseolin/CP 3-5 was variable. This is the first report of a significant increase in seed lysine content due to the seed-specific expression of a de novo protein sequence.

Amino Acid Sequence↗

PubMed1997

The RY sequence is necessary but not sufficient for the transcription activation of a winged bean chymotrypsin inhibitor gene in developing seeds.

A winged bean Kunitz-type chymotrypsin inhibitor (WCI) is expressed in seeds and tuberous roots. In seeds, the expression of WCI is restricted to the period between the mid- and late-maturation stage. To understand the mechanisms that regulate the expression of WCI genes, we analyzed the promoter activity of the upstream region of the WCI-3b gene, which encodes a major WCI protein, in transgenic tobacco plants. By using a series of constructs with 5' deletions in the upstream sequences, the region between -882 and -623, relative to the transcription start site, was shown to contain multiple sequences which are responsible for high level expression in mid-maturation stage seeds. However, when this region was fused to the cauliflower mosaic virus 35S core promoter in both orientations, the chimeric promoters showed only a weak transcription activity in transgenic tobacco plants. Further analyses using internal deletion constructs revealed that the region between -882 and -174 is required for the transcription activation. Disruption of the RY sequence at -517, which is conserved in many seed protein genes, resulted in a drastic reduction of the transcription activity in seeds. These results suggest that sequences necessary for high level induction of the WCI-3b gene transcription in developing seeds are dispersed in the region between -882 and -174, and that the RY sequence is one of these sequences.

Chymotrypsin↗

PubMed1998

A 42 bp fragment of the pmas1' promoter containing an ocs-like element confers a developmental, wound- and chemically inducible expression pattern.

Synthesis of mannopine in plant tissues infected with Agrobacterium tumefaciens is controlled by a divergent promoter (pmas2' and pmas1') that in 479 bp contains all the cis-acting elements necessary to direct tissue-specific and wound-inducible expression. In this report, using transgenic tobacco plants harboring a pmas1'-beta-glucuronidase (GUS) gene fusion, we investigated the developmental expression pattern directed by pmas1' in the early stages of development and the responses of pmas1' to different chemical inducers. It was found that this promoter can respond to auxins, cytokinins, methyl jasmonate (MJ), salicylic acid (SA) and its analogue 2,6-dichloroisonicotinic acid (iNA). Treatment with chemical inducers also showed that the effects of iNA are organ-dependent, that wound-induction is a complex response mediated by at least two different chemical signals, and that MJ stimulates changes in the tissue-specific and developmental expression pattern directed by the ptmas1' promoter. Using chimeric promoters we demonstrate that an ocs-like element (ocs+1) directs MJ responses in an orientation-dependent manner and that sequences around the ocs+1 are important to maintain the inducible and developmental properties of this cis-regulatory element.

Acetates↗

PubMed1998

Fusions between green fluorescent protein and beta-glucuronidase as sensitive and vital bifunctional reporters in plants.

By fusing the genes encoding green fluorescent protein (GFP) and beta-glucuronidase (GUS) we have created a set of bifunctional reporter constructs which are optimized for use in transient and stable expression studies in plants. This approach makes it possible to combine the advantage of GUS, its high sensitivity in histochemical staining, with the advantages of GFP as a vital marker. The fusion proteins were functional in transient expression studies in tobacco using either DNA bombardment or potato virus X as a vector, and in stably transformed Arabidopsis thaliana and Lotus japonicus plants. The results show that high level of expression does not interfere with efficient stable transformation in A. thaliana and L. japonicus. Using confocal laser scanning microscopy we show that the fusion constructs are very suitable for promoter expression studies in all organs of living plants, including root nodules. The use of these reporter constructs in the model legume L. japonicus offers exciting new possibilities for the study of the root nodulation process.

Arabidopsis↗

PubMed1998

Functional architecture of a late pollen promoter: pollen-specific transcription is developmentally regulated by multiple stage-specific and co-dependent activator elements.

The tomato lat52 gene encodes an essential cysteine-rich protein preferentially transcribed in the vegetative cell during pollen maturation. Detailed analyses of the identity, organization and role of cis-regulatory elements in controlling the precise developmental and tissue-specific expression of lat52 during pollen development were performed. Analysis of a series of 5' promoter deletion mutants stably introduced into tobacco demonstrated differential developmental activation of deletion mutants during pollen development. All major cis-regulatory elements required for pollen-specific transcription were located within the upstream region -492 to -52. This region was shown to comprise three independent activator domains A, B and C, each sufficient to activate the minimal CaMV 35S promoter in a pollen-specific manner. 5' deletion and gain of function approaches were used to show that domain A and the previously defined motif PBII (sub-domain B1) were largely redundant in the presence of downstream sequences in mature pollen. Within domain B two novel pollen-specific sub-domains B2 and B3 were identified. Within domain C, the activity of the PBI motif (sub-domain C1) was shown to be strictly dependent upon a downstream 20 bp pollen-specific activator unit -72 to -52 (sub-domain C2), containing two novel co-dependent regulatory elements AGAAA and TCCACCATA. These results demonstrate that transcriptional activation of lat52 is controlled by a complex of pollen-specific cis-regulatory elements which cooperate to achieve maximum levels of gene expression throughout pollen maturation. Alternative models of the interaction of identified cis-regulatory elements with putative trans-acting factors within the lat52 promoter and their developmental utilization are presented.

Enhancer Elements, Genetic↗

PubMed1999

Auxin and brassinosteroid differentially regulate the expression of three members of the 1-aminocyclopropane-1-carboxylate synthase gene family in mung bean (Vigna radiata L.).

Indole-3-acetic acid (IAA) markedly increased ethylene production by inducing the expression of three 1aminocyclopropane-1-carboxylate (ACC) synthase cDNAs (pVR-ACS1, pVR-ACS6 and pVR-ACS7) in mung bean hypocotyls. Results from nuclear run-on transcription assay and RNA gel blot studies revealed that all three genes were transcriptionally active displaying unique patterns of induction by IAA and various hormones in etiolated hypocotyls. Particularly, 24-epibrassinolide (BR), an active brassinosteroid, specifically enhanced the expression of VR-ACS7 by a distinct temporal induction mechanism compared to that of IAA. In addition, BR synergistically increased the IAA-induced VR-ACS6 and VR-ACS7 transcript levels, while it effectively abolished both the IAA- and kinetin-induced accumulation of VR-ACS1 mRNA. In light-grown plants, VR-ACS1 was induced by IAA in roots, and VR-ACS6 in epicotyls. IAA- and BR-treatments were not able to increase the VR-ACS7 transcript in the light-grown tissues. These results indicate that the expression of ACC synthase multigene family is regulated by complex hormonal and developmental networks in a gene- and tissue-specific manner in mung bean plants. The VR-ACS7 gene was isolated, and chimeric fusion between the 2.4 kb 5'-upstream region and the beta-glucuronidase (GUS) reporter gene was constructed and introduced into Nicotiana tabacum. Analysis of transgenic tobacco plants revealed the VR-ACS7 promoter-driven GUS activity at a highly localized region of the hypocotyl-root junction of control seedlings, while a marked induction of GUS activity was detected only in the hypocotyl region of the IAA-treated transgenic seedlings where rapid cell elongation occurs. Although there was a modest synergistic effect of BR on the IAA-induced GUS activity, BR alone failed to increase the GUS activity, suggesting that induction of VR-ACS7 occurs via separate signaling pathways in response to IAA and BR. A scheme of the multiple regulatory pathways for the expression of ACC synthase multigene family by auxin and BR is presented.

Brassinosteroids↗

PubMed1999

Green fluorescent protein (GFP) as a marker during pollen development.

The transient expression of three mutant forms of green fluorescent protein (GFP) genes, GFP4, GFP5ER, and GFP4S65C, under several constitutive and pollen-specific promoters throughout pollen development in Nicotiana tabacum, Arabidopsis thaliana and Antirrhinum majus is described. Immature pollen of tobacco, Arabidopsis and snapdragon, isolated at different developmental stages, were bombarded with plasmids containing the GFP and cultured in vitro for several days until maturity. The expression of GFP was monitored every day during in vitro maturation, germination and pollination, as well as after in situ pollination. The expression pattern of each GFP construct was compared in parallel experiments to that of beta-glucuronidase (GUS) constructs expressed by the same promoters. The results show that the expression level of all three GFP mutant forms was dependent on the strength of the promoter used. The strongest promoter was the DC3 promoter, and no notable differences in the intensity and brightness of all three versions of GFP were observed. GFP-expressing pollen from tobacco and snapdragon developed in vitro for several days until maturity and germinated in vitro as well as on the surface of stigmata, strongly suggesting that all three GFPs are not toxic for the development of functional pollen. Furthermore, stably transformed tobacco plants expressing GFP under the control of the strong pollen-expressed DC3 and LAT52 promoters were not impaired in reproductive function, confirming that GFP can be used as a non-destructive marker for plant reproductive biology and development.

Arabidopsis↗

PubMed1999

Identification of cis-regulatory elements required for endosperm expression of the rice storage protein glutelin gene GluB-1.

Rice storage protein glutelin genes are coordinately regulated during seed development. A previous 5' deletion analysis using transgenic tobacco revealed that the minimum 5' region necessary for endosperm specificity was within -245 bp of the transcription start site, and included the AACA and GCN4 motifs that are highly conserved in the 5'-flanking regions of all glutelin genes. In this paper, the sequence elements essential for endosperm-specific expression are characterized in stable transgenic tobacco plants by both loss-of-function and gain-of-function experiments using this minimum promoter. Base substitution analysis shows that the proximal AACA motif between -73 and -61, and the GCN4 motif between -165 and -158 act as critical elements. An ACGT motif between -81 and -75, and Skn-I-like elements between -173 and -169 also play important roles in controlling the seed-specific expression. When the distal region between -245 and -145 containing the AACA and the GCN4 motifs or the proximal region between -113 and -46 containing the ACGT and AACA motifs is fused to a truncated promoter (-90 to +9) of the CaMV 35S gene fused to the beta-glucuronidase (GUS) reporter gene, high levels of seed-specific expression are observed in these fusions, thereby indicating that either pair of motifs is sufficient to confer seed expression in these fusions. However, when substituted for by the CaMV 35S core promoter (-46 to +1), seed expression is abolished, suggesting that the sequence between -90 and -46 of the CaMV 35S promoter containing G-box-like motif (as-1 element) is required for such specific expression in addition to AACA and GCN4 motifs. Therefore, we conclude that at least three cis-regulatory elements, the AACA motif, GCN4 motif and ACGT motif, are necessary to mediate endosperm expression of the GluB-1 glutelin gene.

Base Sequence↗

PubMed2003

Expression of the B subunit of E. coli heat-labile enterotoxin in the chloroplasts of plants and its characterization.

Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1-ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.

Bacterial Toxins↗

PubMed1993

Transgenic plants expressing a functional single-chain Fv antibody are specifically protected from virus attack.

Expression of viral genes in transgenic plants is a very effective tool for attenuating plant viral infection. Nevertheless, the lack of generality and risk issues related to the expression of viral genes in plants might limit the exploitation of this strategy. Expression in plants of antibodies against essential viral proteins could provide an alternative approach to engineer viral resistance. Recently, expression of complete or engineered antibodies has been successfully achieved in plants. The engineered single-chain Fv antibody scFv (refs 10, 11) is particularly suitable for expression in plants because of its small size and the lack of assembly requirements. Here we present evidence that constitutive expression in transgenic plants of a scFv antibody, directed against the plant icosahedral tombusvirus artichoke mottled crinkle virus, causes reduction of infection incidence and delay in symptom development.

Antibodies, Viral↗

PubMed1992

Production of active Bacillus licheniformis alpha-amylase in tobacco and its application in starch liquefaction.

As a first example of the feasibility of producing industrial bulk enzymes in plants, we have expressed Bacillus licheniformis alpha-amylase in transgenic tobacco, and applied the seeds directly in starch liquification. The enzyme was properly secreted into the intercellular space, and maximum expression levels of about 0.3% of total soluble protein were obtained. No apparent effect of the presence of the enzyme on plant phenotype was observed. The molecular weight of the enzyme produced in tobacco was around 64 kD. The difference, compared to 55.2 kD for the bacterial enzyme, was found to result from complex-type carbohydrate chains attached to the protein. Application studies on the liquefaction of starch were done with transgenic seeds containing the recombinant alpha-amylase. The resulting hydrolysis products were virtually identical with those obtained from degradation with alpha-amylase from Bacillus licheniformis.

Bacillus↗

PubMed1992

Potato virus X as a vector for gene expression in plants.

The suitability of potato virus X (PVX) as a gene vector in plants was tested by analysis of two viral constructs. In the first, the GUS gene of Escherichia coli was substituted for the viral coat protein gene. In the second, GUS was added into the viral genome coupled to a duplicated copy of the viral promoter for the coat protein mRNA. The viral construct with the substituted coat protein gene accumulated poorly in inoculated protoplasts and failed to spread from the site of infection in plants. These results suggest a role for the viral coat protein in key stages of the viral infection cycle and show that gene replacement constructs are not suitable for the production of PVX-based gene vector. The construct with GUS coupled to the duplicated promoter for coat protein mRNA also accumulated less well in protoplasts than the unmodified PVX, but did infect systemically and directed high level synthesis of GUS in inoculated and systemically infected tissue. Although there was some genome instability in the PVX construct, much of the viral RNA in the systemically infected tissue had retained the foreign gene insertion, especially in infected Nicotiana clevelandii plants. These data point to a general utility of PVX as a vector for unregulated gene expression in plants.

Base Sequence↗

PubMed1993

The ubiquitous presence of exopolygalacturonase in maize suggests a fundamental cellular function for this enzyme.

Exopolygalacturonase (exoPG) is a pectin-degrading enzyme abundant in maize pollen. Using immunochemistry and in situ hybridization it is shown that in addition to its presence in pollen, exoPG is also present in sporophytic tissues, such as the tapetum and mesophyll cells. The enzyme is located in the cytoplasm of pollen and of some mesophyll cells. In other mesophyll cells, the tapetum and the pollen tube, exoPG is located in the cell wall. The measurement of enzyme activity shows that exoPG is ubiquitous in the vegetative organs. These results suggest a general function for exoPG in cell wall edification or degradation. ExoPG is encoded by a closely related multigene family. The regulation of the expression of one of the exoPG genes was analyzed in transgenic tobacco. Reporter GUS activity was detected in anthers, seeds and stems but not in leaves or roots of transgenic plants. This strongly suggests that the ubiquitous presence of exoPG in maize is the result of the expression of different exoPG genes.

Base Sequence↗

PubMed1993

The promoter of the Arabidopsis thaliana plastocyanin gene contains a far upstream enhancer-like element involved in chloroplast-dependent expression.

Plastocyanin is part of the photosynthetic electron transport chain in the chloroplast and is encoded in the nucleus. Expression of the Arabidopsis thaliana plastocyanin gene is organ specific: high mRNA levels are observed in young green parts of the plant. Furthermore, expression is dependent on the presence of light and functional chloroplasts. When grown in the presence of norflurazon under white light conditions, resulting in the photo-oxidative destruction of the chloroplast, plastocyanin mRNA levels are strongly reduced. A -1579 to -9 promoter fragment confers light-regulated and chloroplast-dependent expression to the beta-glucuronidase reporter gene in transgenic tobacco plants. This suggests that regulation takes place at the level of transcription. A plastocyanin promoter deletion series ranging from -1579 to -121 which was also tested in tobacco, revealed the presence of a strong positive regulating element (PRE) in the -1579 to -705 region. Deletion of this part of the promoter resulted in a approximately 100-fold reduction of GUS expression as measured in mature leaves. Surprisingly, this enhancer-like element was capable of stimulating transcription from a position downstream of its reporter. Moreover, it could also activate a truncated CaMV 35S promoter. Deletion of this element coincides with the loss of chloroplast-dependency of reporter gene expression, as judged by norflurazon treatment of transgenic seedlings. So, the activity of the PRE itself might depend on the presence of functional chloroplasts.

Arabidopsis↗