An attempt at application of tRNA genes as promoters of protein-coding genes in transgenic plants.
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Agrobacterium tumefaciens↗
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5'-flanking region responsible for endosperm-specific expression of rice prolamine chimeric gene in transgenic tobacco.
The 5' upstream region (-680-(+)40) containing the complete signal peptide coding sequence of the rice seed storage prolamine gene was amplified in vitro with polymerase chain reaction from the genome of Chinese super rice cultivar Zhonghua 8. Physical map and DNA sequence analysis showed strong homology with the 5'-flanking region of rice prolamine gene reported by Kim in 1988. No changes in the signal peptide coding sequence and a long leader sequence with several small ORFs were found. Chimeric gene containing 5'-flanking region of the prolamine gene has been transcriptionally fused with the beta-glucuronidase reporter gene. The fusion junction was confirmed by both physical map and DNA sequence analysis. The resultant chimeric gene was used to transform the tobacco explants by Ti binary system of Agrobacterium tumefaciens LBA4404. With dot and Southern blotting hybridization, three transgenic tobacco plants with the copies of chimeric GUS genes as many as 20 were obtained. Histochemical analysis revealed that the GUS activity in the endosperm tissues of tobacco seeds at the developmental stage was about 20 DAP. No GUS activity was found in leaves, stems, roots and flowers of the transgenic tobacco plants. Therefore, we concluded that the 5'-upstream cis-elements from -680 to -18 were enough to confer the endosperm-specific and temporal expression of rice prolamine gene.
Base Sequence↗
S1F binding site is related to but different from the light-responsive GT-1 binding site and differentially represses the spinach rps1 promoter in transgenic tobacco.
Nuclear genes encoding plastid ribosomal proteins are more highly expressed in leaves than in roots. This leaf-specific induction seems to be light-independent. We have previously characterized a spinach nuclear factor S1F binding to a cis-element within the rps1 promoter, which negatively regulates both the rps1 and the cauliflower mosaic virus 35S promoters in transient expression assays. Here, we show that the S1F binding site is related to but different from the light-responsive Box II of the pea rbcS-3A promoter, which is recognized by the nuclear factor GT-1. Transgenic plant analyses showed that the S1F site tissue-specifically represses the rps1 promoter in roots as well as in etiolated seedlings. We suggest that the GT-1-related S1F binding site is responsible, at least in part, for the transcriptional repression of rps1 in nonphotosynthetic tissues such as roots.
Base Sequence↗
Establishment of a transgenic tobacco cell suspension culture system for producing murine granulocyte-macrophage colony stimulating factor.
We tested if murine granulocyte-macrophage colony stimulating factor (mGM-CSF) is produced as a biologically active form through plant cell culture. The mGM-CSF gene was cloned into the plant expression vector, pBI121, and Ti-plasmid mediated transformation of tobacco leaves was conducted using Agrobacterium tumefaciens harboring the recombinant mGM-CSF (rmGM-CSF) gene. Cell suspension culture was established from the leaf-derived calli of transgenic tobacco plants. Northern blot analysis indicated the expression of the introduced mGM-CSF gene in both transgenic plant and cell suspension cultures. In addition, the biological activity of rmGM-CSF from plant cell culture was confirmed by measuring the proliferation of GM-CSF dependent FDC-P1 cells.
Agrobacterium tumefaciens↗
Cadmium resistance in transgenic tobacco plants expressing the Nicotiana glutinosa L. metallothionein-like gene.
To understand the function of metallothioneins (MTs) in plants, we introduced the Nicotiana glutinosa MT gene into tobacco (N. tabacum) plants via an Agrobacterium mediated transformation. Full-length MT cDNA was fused between the cauliflower mosaic virus 35S (CaMV 35S) promoter and the nopaline synthase (nos) terminator of the pMBP1 binary vector in sense orientation. Tobacco leaf discs which were cocultivated with Agrobacterium carrying the chimeric MT gene, formed kanamycin-resistant shoots on medium containing kanamycin. The kanamycin-resistant shoots were subsequently rooted on medium containing 200 microM CdSO4. Approximately 30% of individual transgenic plants developed normally. Nontransgenic plants promptly underwent leaf chlorosis, and their growth and development were inhibited on MS medium containing 50 microM CdSO4. Genomic Southern blot analysis showed that the MT gene was stably integrated into the nuclear genome of transgenic tobacco plants. The expression level of MT transcripts was analyzed by RNA gel blot analysis. Self-pollinated seeds obtained from transgenic tobacco plants showing cadmium tolerance were germinated on a medium containing 100 microM CdSO4. PCR analysis from sensitive and stably resistant T2 seedlings for cadmium sulfate confirmed a high correlation between the phenotypic expression of the MT gene and the transgenic genotype, indicating that the MT gene is inherited in the next generation.
Cadmium↗
Transgenic Trifolium repens with foliage accumulating the high sulphur protein, sunflower seed albumin.
With the aim of increasing the rumen-protected level of the sulphur amino acids cysteine and methionine in Trifolium repens, we introduced the coding sequence of the sunflower seed albumin (SSA) into T. repens by Agrobacterium tumefaciens-mediated transformation. The SSA gene was modified such that the protein would be localised to the endoplasmic reticulum (ER). Four different T-DNA constructions all containing the SSA gene driven by either the promoter of a gene encoding the small subunit of ribulose bisphosphate carboxylase (Rubisco) from Arabidopsis thaliana (Assu), the promoter of the gene encoding the small subunit of Rubisco of Medicago sativa (Lssu), or the Cauliflower Mosaic Virus 35S promoter (CaMV35S), were transferred to T. repens cv. Haifa. Transgenic T0-plants and inter-transgenic hybrids were analysed for the level of SSA accumulation in the leaves by western blotting. The highest observed level of SSA accumulation was 0.1% of total extractable leaf protein. We observed that the promoter had a substantive effect on the level of SSA accumulation with Assu > CaMV35S > Lssu. Results from the inter-transgenic hybrids showed that the capacity to synthesise SSA was inherited. However the level of SSA accumulation in the leaves generally appears not to be additive with extra transgenic loci. During this work, we attempted to improve the efficiency of A. tumefaciens-mediated transformation of T. repens using the SAAT-method (Sonication Assisted Agrobacterium-mediated Transformation) on cotyledons of T. repens. T-DNA transfer was in general not enhanced by sonication compared to traditional A. tumefaciens-mediated transformation. Furthermore, Southern blot analyses of plants regenerated from the same cotyledon after A. tumefaciens treatment and under selection, indicated that multiple shoots were usually derived from the same transformation event. We concluded from these results that only one plant from each A. tumefaciens-treated cotyledon should be taken to avoid transgenic clones.
2S Albumins, Plant↗
Synthesis and secretion of tobacco neutral PR-5 protein by transgenic tobacco and yeast.
Tobacco neutral osmotin-like protein was expressed in both homologous and heterologous expression systems. Osmotin-like protein was retained in tobacco cells when expressed as the prepro-form, whereas deletion of the carboxyl-terminal propeptide of 22 amino acids induced its secretion into the extracellular fluid. Without the amino-terminal signal peptide, there was no accumulation of osmotin-like protein. When the construct with both the amino- and carboxyl-terminal peptides was introduced to methylotrophic yeast, divergent osmotin-like protein forms were found in the cell and medium. Results suggest that the amino-terminal signal of the osmotin-like protein is essential for the transport of protein to the endoplasmic reticulum both in tobacco and yeasts but that the carboxyl-terminal propeptide signal is effective only for intracellular retention in tobacco.
Amino Acid Sequence↗
Expression of giant silkmoth cecropin B genes in tobacco.
Cecropin B is a small antibacterial peptide from the giant silkmoth Hyalophora cecropia. To reveal the potential of this peptide for engineering bacterial disease resistance into crops, several cecropin B gene constructs were made either for expression in the cytosol or for secretion. All constructs were cloned in a plant expression vector and introduced in tobacco via Agrobacterium tumefaciens. A cDNA-derived cecropin B gene construct lacking the amino-terminal signal peptide was poorly expressed in transgenic plants at the mRNA level, whereas plants harbouring a full-length cDNA-derived construct containing the insect signal peptide, showed increased cecropin B-mRNA levels. Highest expression was found in plants harbouring a construct with a plant-gene-derived signal peptide. In none of the transgenic plants could the cecropin B peptide be detected. This is most likely caused by breakdown of the peptide by plant endogenous proteases, since a chemically synthesized cecropin B peptide was degraded within seconds in various plant cell extracts. This degradation could be prevented by the addition of specific protease inhibitors and by boiling the extract prior to adding the peptide. In addition, anionic detergents, in contrast to cationic, zwitter-ionic or non-ionic detergents, could prevent this degradation. Nevertheless, transgenic tobacco plants were evaluated for resistance to Pseudomonas solanacearum, the causal agent of bacterial wilt of many crops, and P. syringae pv. tabaci, the causal agent of bacterial wildfire, which are highly susceptible to cecropin B in vitro. No resistance was found. These experiments indicate that introduction and expression of cecropin B genes in tobacco does not result in detectable cecropin B protein levels and resistance to bacterial infections, most likely due to degradation of the protein by endogenous proteases.
Amino Acid Sequence↗
XsiAMT1.1a was identified as a novel ammonium uptake functional gene and its overexpression combined with GA4 application significantly increased yield in Arabidopsis thaliana.
Nitrogen (N) is a key limiting factor for plant yield. Ammonium is one of the main N forms absorbed by plants. Overexpression of ammonium uptake functional genes, such as ammonium transporter (AMT), can increase yield. However, the AMTs reported to enhance yield significantly is still limited. No researches have focused on the effect of overexpressing AMT combined with hormone application on yield improvement. In this study, we first investigated the role of XsiAMT1.1a, a potential ammonium uptake functional gene in an ammonium preference plant Xanthium sibiricum, in ammonium uptake by the analysis of bioinformatics, gene expression and subcellular localization, and the determination of ammonium uptake rate in endogenous silencing and heterologous overexpression plants. Subsequently, the effect of XsiAMT1.1a overexpression combined with hormone application on yield increase was further investigated in model plant Arabidopsis thaliana. Our results showed that XsiAMT1.1a shared the same conserved domains with AtAMT1 subfamily members and localized on the plasma membrane. XsiAMT1.1a was induced by N deficiency and highly expressed during the reproductive period. XsiAMT1.1a endogenous silencing and heterologous overexpression significantly decreased and increased ammonium uptake rates in X. sibiricum and A. thaliana, respectively. Overexpression of XsiAMT1.1a significantly improved total N accumulation, biomass and yield in A. thaliana, while XsiAMT1.1a overexpression combined with GA4 application had a stronger promoting effect on the above indicators. Our research identified a novel ammonium uptake functional gene, XsiAMT1.1a, and provided a new yield-increasing strategy which was verified in A. thaliana.
Arabidopsis↗
Activation of systemic acquired silencing by localised introduction of DNA.
BACKGROUND: In plants, post-transcriptional gene silencing results in RNA degradation after transcription. Among tobacco transformants carrying a nitrate reductase (Nia) construct under the control of the cauliflower mosaic virus 35S promoter (35S-Nia2), one class of transformants spontaneously triggers Nia post-transcriptional gene silencing (class II) whereas another class does not (class I). Non-silenced plants of both classes become silenced when grafted onto silenced stocks, indicating the existence of a systemic silencing signal. Graft-transmitted silencing is maintained in class II but not in class I plants when removed from silenced stocks, indicating similar requirements for spontaneous triggering and maintenance. RESULTS: Introduction of 35S-Nia2 DNA by the gene transfer method called biolistics led to localised acquired silencing (LAS) in bombarded leaves of wild-type, class I and class II plants, and to systemic acquired silencing (SAS) in class II plants. SAS occurred even if the targeted leaf was removed 2 days after bombardment, indicating that the systemic signal is produced, transmitted and amplified rapidly. SAS was activated by sense, antisense and promoterless Nia2 DNA constructs, indicating that transcription is not required although it does stimulate SAS. CONCLUSIONS: SAS was activated by biolistic introduction of promoterless constructs, indicating that the DNA itself is a potent activator of post-transcriptional gene silencing. The systemic silencing signal invaded the whole plant by cell-to-cell and long-distance propagation, and reamplification of the signal.
Base Sequence↗
The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent.
The xylose isomerase gene (xylA) from Thermoanaerobacterium thermosulfurogenes (formerly Clostridium thermosulfurogenes) has been expressed in three plant species (potato, tobacco, and tomato) and transgenic plants have been selected on xylose-containing medium. The xylose isomerase gene was transferred to the target plant by Agrobacterium-mediated transformation. The xylose isomerase gene was expressed using the enhanced cauliflower mosaic virus (CaMV) 35S promoter and the omega' translation enhancer sequence from tobacco mosaic virus. Unoptimized selection studies showed that, in potato and tomato, the xylose isomerase selection was more efficient than the established kanamycin resistance selection, whereas in tobacco the opposite was observed. Efficiency may be increased by optimization. The xylose isomerase system enables the transgenic cells to utilize xylose as a carbohydrate source. It is an example of a positive selection system because transgenic cells proliferate while non-transgenic cells are starved but still survive. This contrasts to antibiotic or herbicide resistance where transgenic cells survive on a selective medium but non-transgenic cells are killed. The results give access to a new selection method which is devoid of the disadvantages of antibiotic or herbicide selection.
Aldose-Ketose Isomerases↗
Expression and sequence requirements for nitrite reductase co-suppression.
We have previously reported that the introduction of a full-length tobacco nitrite reductase Nii1 cDNA under the control of the 35S promoter triggers co-suppression of endogenous Nii genes in 25% of tobacco transformants. Here we show that introduction of chimeric Nii1-uidA, uidA-Nii1 and Nii1-uidA-Nii1 transgenes carrying 186 bp of the 5' end and/or 241 bp of the 3' end of the Nii1 cDNA do not trigger co-suppression of endogenous Nii genes. In addition, we show that when introduced by crossing or transformation into co-suppressed transgenic tobacco lines carrying full-length Nii1 transgenes, these chimeric transgenes are not silenced. These results therefore suggest that the 5' and 3' ends of the Nii1 cDNA are not sufficient to trigger co-suppression and are not targets for homology-dependent RNA degradation. Surprisingly, co-suppression was released in a double transformant obtained by introduction of one of these constructs into the co-suppressed transgenic tobacco line 461-2.1 homozygous for a full-length Nii1 transgene, and in one plant regenerated from untransformed leaf discs (plant 461-2.1*). The reappearance of co-suppression at very low frequency (less than 10(-3)) in the F2 progeny of plant 461-2.1* and the apparent absence of structural modification of the transgene locus suggest a metastable epigenetic modification. The steady-state level of Nii mRNAs in the plant 461-2-.1* was higher than in wild-type plants but lower than in hemizygous plants 461-2.1 which never trigger silencing. These results therefore confirm that transcription of the transgene above a particular threshold is required to trigger co-suppression.
DNA Methylation↗
Apoplastic and cytosolic expression of full-size antibodies and antibody fragments in Nicotiana tabacum.
We compared the expression of a functional recombinant TMV-specific full-size antibody (rAb29) in both the apoplast and cytosol of tobacco plants and a single chain antibody fragment (scFv29), derived from rAb29, was expressed in the cytosol. Cloned heavy and light chain cDNAs of full-size rAb29, which binds to TMV coat protein monomers, were integrated into the plant expression vector pSS. The full-size rAb29 was expressed in the cytosol and targeted to the apoplast by including the original murine antibody leader sequences. Levels of functional full-size rAb29 expression were high in the apoplast (up to 8.5 micrograms per gram leaf tissue), whereas cytosolic expression was low or at the ELISA detection limit. Sequences of the variable domains of rAb29 light and heavy chain were used to generate the single chain antibody of scFv29, which was expressed in the periplasmic space of E. coli and showed the same binding specificity as full-size rAb29. In addition, scFv29 was functionally expressed in the cytosol of tobacco plants and plant derived scFv29 maintained same binding specificity to TMV-coat protein monomers as rAb29.
Antibodies, Viral↗
Overexpression of arginine decarboxylase in transgenic plants.
The activity of arginine decarboxylase (ADC), a key enzyme in plant polyamine biosynthesis, was manipulated in two generations of transgenic tobacco plants. Second-generation transgenic plants overexpressing an oat ADC cDNA contained high levels of oat ADC transcript relative to tobacco ADC, possessed elevated ADC enzyme activity and accumulated 10-20-fold more agmatine, the direct product of ADC. In the presence of high levels of the precursor agmatine, no increase in the levels of the polyamines putrescine, spermidine and spermine was detected in the transgenic plants. Similarly, the activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase were unchanged. No diversion of polyamine metabolism into the hydroxycinnamic acid-polyamine conjugate pool or into the tobacco alkaloid nicotine was detected. Activity of the catabolic enzyme diamine oxidase was the same in transgenic and control plants. The elevated ADC activity and agmatine production were subjected to a metabolic/physical block preventing increased, i.e. deregulated, polyamine accumulation. Overaccumulation of agmatine in the transgenic plants did not affect morphological development.
Adenosylmethionine Decarboxylase↗
Antisense-inhibition of ADP-glucose pyrophosphorylase in developing seeds of Vicia narbonensis moderately decreases starch but increases protein content and affects seed maturation.
The small subunit of a Vicia faba ADP-glucose pyrophosphorylase (AGP) cDNA was expressed in antisense orientation in Vicia narbonensis under the control of the seed-specific legumin B4 promoter. From several independent transgenic lines both ADP-glucose pyrophosphorylase AGP-mRNA and AGP enzyme activity were reduced by up to 95% in the cotyledons during the mid- to late-maturation phase. Starch was moderately decreased and sucrose was increased. In two of three lines, transcripts encoding the large subunit of AGP and the storage protein vicilin were increased, whereas legumin B-mRNA was decreased. Transcripts of other storage-associated genes were not altered. The cotyledons contained more protein and total nitrogen. Despite the reduction in starch, total carbon was not decreased and dry weight was unchanged. Compared to the wild type, transgenic seeds contained more water and accumulated dry weight during a longer period, and therefore had a prolonged seed-filling period. Transgenic cotyledon cells of comparable age to the wild type were more highly vacuolated and contained smaller starch grains, indicating a delay in cellular differentiation. We conclude that a specific alteration in carbon metabolism can have pleiotropic effects on water and nitrogen content and induces temporal changes in seed development.
Cotyledon↗
A cytoplasmic male sterility-associated mitochondrial protein causes pollen disruption in transgenic tobacco.
In higher plants, dominant mitochondrial mutations are associated with pollen sterility. This phenomenon is known as cytoplasmic male sterility (CMS). It is thought that the disruption in pollen development is a consequence of mitochondrial dysfunction. To provide definitive evidence that expression of an abnormal mitochondrial gene can interrupt pollen development, a CMS-associated mitochondrial DNA sequence from common bean, orf239, was introduced into the tobacco nuclear genome. Several transformants containing the orf239 gene constructs, with or without a mitochondrial targeting sequence, exhibited a semi sterile or male-sterile phenotype. Expression of the gene fusions in transformed anthers was confirmed using RNA gel blotting, ELISA, and light and electron microscopic immunocytochemistry. Immunocytological analysis showed that the ORF239 protein could associate with the cell wall of aberrant developing microspores. This pattern of extracellular localization was earlier observed in the CMS common bean line containing orf239 in the mitochondrial genome. Results presented here demonstrate that ORF239 causes pollen disruption in transgenic tobacco plants and may do so without targeting of the protein to the mitochondrion.
Chimera↗
RecA protein stimulates homologous recombination in plants.
A number of RecA-like proteins have been found in eukaryotic organisms. We demonstrate that the prokaryotic recombination protein RecA itself is capable of interacting with genomic homologous DNA in somatic plant cells. Resistance to the DNA crosslinking agent mitomycin C requires homologous recombination as well as excision repair activity. Tobacco protoplasts expressing a nucleus-targeted RecA protein were at least three times as efficient as wild-type cells in repairing mitomycin C-induced damage. Moreover, homologous recombination at a defined locus carrying an endogenous nuclear marker gene was stimulated at least 10-fold in transgenic plant cells expressing nucleus-targeted RecA. The increase in resistance to mitomycin C and the stimulation of intrachromosomal recombination demonstrate that Escherichia coli RecA protein is functional in genomic homologous recombination in plants, especially when targeted to the plant nucleus.
Agrobacterium tumefaciens↗
A gene coding for a monomeric form of cucumber mosaic virus satellite RNA confers tolerance to CMV.
A gene whose transcript bears a monomeric form of cucumber mosaic virus (CMV) satellite RNA was introduced into tobacco (Nicotiana tabacum 'Xanthi' nc) plants by using an Ri plasmid-based vector system. On CMV infection, the transcript of the satellite RNA gene was used as a template to yield unit-length satellite RNA, which was efficiently amplified by the virus. Plants bearing the satellite RNA gene displayed long-term tolerance to CMV infection and were also tolerant to CMV infection by aphids, the natural vector of CMV. Implications of these results concerning the mechanism of satellite RNA replication are discussed.
Animals↗