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Use of I-Sce I to induce DNA double-strand breaks in Nicotiana.
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[Plant regeneration from Agobacterium-mediated CTV-cp gene transformation of Poncirus trifoliata Raf].
OBJECTIVE: To lay a foundation for the resistant breeding, the anti-virus CTV-cp gene was transformed into the epicotyles mediated by Agrobacterium tumefaciens in Poncirus trifoliata. METHOD: The explants used for the genetic transformation were the epicotyls from P. trifoliata. The Agrobacterum tumefaciens strain was EHA101 containing vector plasmid pGA482GG. The coat protein gene (CTV-cp gene), GUS gene and NPT II gene were introduced into the transformation plasmid. RESULTS: Ceftaxime used as antibiotics was better than carbenicillin. The concentration was 300 mg.L-1; The selection pressure for kanamycin was 50 mg.L-1; 70.0% of the resistant plants were GUS-positive; extra gene was proved to be in P. trifoliata plant by southern blot examination. CONCLUSION: An effective genetic transformation mediated by Agrobacterium tumefacines, which harbours a CTV-cp gene, has been developed in P. trifoliata. Transgenic CTV-cp plants were obtained.
A Brassica napus gene family which shows sequence similarity to ascorbate oxidase is expressed in developing pollen. Molecular characterization and analysis of promoter activity in transgenic tobacco plants.
The genomic clone named Bp10 contains a member of a small pollen-specific gene family of B. napus. The expression of the Bp10 gene family is maximal in early binucleate microspores and declines considerably in mature trinucleate pollen. Homologues of the Bp10 genes are expressed in the pollen of other plant species. The pollen-specific expression of the gene contained in the genomic clone was confirmed in tobacco plants transformed with a chimeric Bp10 promoter/GUS construct. A promoter fragment of 396 bp is sufficient to direct a strong and correct spatial and temporal expression in transgenic plants. The Bp10 gene family codes for proteins of 62 kDa showing approximately 30% sequence identify to cucumber and pumpkin ascorbate oxidases (AAOs). However, the AAO active centres are not conserved in the Bp10 products, suggesting an evolutionary relationship but a different enzymatic activity for these proteins. Expression of a recombinant Bp10 protein in E. coli inhibits bacterial growth on minimal medium, suggesting the production of an enzymatically active polypeptide in bacteria. No AAO activity could be correlated with the expression of the recombinant protein. Moreover, substances affecting AAO activity do not appear to influence the inhibitory activity of the protein produced in bacteria. However, as indicated by the rescue of bacterial growth in the presence of sodium bicarbonate or gaseous CO2, the Bp10 protein activity could be modulated by CO2 levels.
Engineered male sterility in plants.
We constructed two chimaeric ribonuclease genes that are specifically expressed in anthers of tobacco and rapeseed plants. The expression of these genes affects the production of functional and viable pollen yielding plants that are male sterile. This dominant gene for nuclear male sterility should facilitate the production of hybrid seed in various crops.
[The import of DNA topoisomerase type II from Drosophila melanogaster into the chloroplasts of transgenic Nicotiana tabacum plants].
The construct containing the structural gene of the type II DNA topoisomerase of Drosophila melanogaster fused to the sequence of the chloroplast transit peptide directed by the 35 S promoter was produced on the basis of the binary vector Bin 19. The construct was introduced into Nicotiana tabacum plants by Agrobacterium-mediated transformation. The integration of the construct into genomic DNA was demonstrated by PCR and Southern blot hybridization. Expression of the chimeric gene at the transcription level was studied by Northern blotting. The protein produce of the expression was identified in chloroplasts by Western blot hybridization with polyclonal anti-type II Drosophila topoisomerase antibodies.
Promoter sequences of two homologous pectin esterase genes from Chinese cabbage (Brassica campestris L. ssp. pekinensis) and pollen-specific expression of the GUS gene driven by a promoter in tobacco plants.
The promoter regions of two genomic clones, GBAN215-6 and GBAN215-12 from Chinese cabbage (Brassica campestris L. ssp. pekinensis), were sequenced. The nucleotide sequences of their promoter regions were compared with that of the Bp19 pollen-specific gene of Brassca napus. High nucleotide sequence homologies were observed among these three genes in the region between 210 bp upstream and the putative transcription start site. A sequence motif TGTGGTG, which is similar to that of the PB core motif (TGTGGTT) of two tomato pollen-specific genes, LAT52 and LAT56, was present in these two cloned genes. To determine regulatory sequences responsible for the anther-specific expression of the gene BAN215-6, two recombinant plasmids, pBPE3 (-274- + 109) and pBPE4 (-816- + 109) containing different lengths of the promoter fused with the GUS gene, were constructed and introduced into tobacco plants by Agrobacterium-mediated transformation. The result showed that the 383 bp (-274- + 109) of the BAN215-6 promoter region was sufficient for the anther-specific expression of the GUS gene. The GUS expression in a tobacco plants transformed with these constructs was first detected in uninucleate microspores and persisted at in vitro germinated pollen tubes. The expression level was increased during anther development, reaching the highest level in mature pollens.
In vitro organogenesis and transgenosis aspects in globe artichoke (Cynara scolymus L.).
The genetic transformation of globe artichoke (Cynara scolymus L.) cells is possible. However, the percentage of transformed cells is still low and the regeneration process appeared to be the critical step towards the obtention of transgenic plants. The present work reports the organogenesis potentialities from new vegetal materials: cotyledons and leaves from in vitro artichoke plants. The results showed that cotyledons gave better rates of neoformation than leaves and this was reached in a shorter time. Regarding the transfer of genetic resistance to globe artichoke, a model system was developed using Nicotiana benthamiana as systemic host for artichoke viral diseases. Mutagenized sequences of the replicase gene of artichoke motteled crinckle virus (AMCV) as transferable genetic material for resistance induction were performed. Transgenic lines of Nicotiana benthamiana were obtained and some of them presented a considerable attenuation of symptoms when challenged with AMCV.
Pollen ablation of transgenic tobacco plants by expression of the diphtheria toxin A-chain gene under the control of a putative pectin esterase promoter from Chinese cabbage.
We previously showed that a 383 bp (-274 to approximately +109) promoter of a pollen-specific gene, GBAN215-6, had a property of a late gene in pollen development in transgenic tobacco plants. It drove GUS gene expression from uninucleate microspores to pollen tube growth of trinucleated cells. To more precisely characterize the specificity of the promoter, we placed the diphtheria toxin A-chain (DTx-A) coding region under the control of the GBAN215-6 promoter. Transgenic tobacco plants containing the GBAN215-6/DTx-1 were phenotypically normal until an early stage of flowering. The dehisced anthers do not contain pollen grains and the filament length of stamen was shorter than that of normal plants. Microscopic examination showed that ablation of pollen by the expression of DTx-A was variable. The transgenic tobacco plants containing one copy of the DTx-A gene show 50% aborted and 50% normal pollen, which suggests that this gene acts gametophytically. However, most of the transgenic plants with high copy number were male-sterile. When these male-sterile tobacco plants were backcrossed as female with pollen from wild-type tobacco plants, the fruit capsule sizes and seed yields of the next generation (BC1 lines) were severely reduced and the segregation of male-sterile to fertile plants in BC1 seeds was not Mendelian.
Defective interfering RNA-mediated resistance against cymbidium ringspot tombusvirus in transgenic plants.
Defective interfering (DI) RNA of cymbidium ringspot tombusvirus was cloned downstream from the bacteriophage T7 RNA polymerase promoter. In vitro synthesized RNA was biologically active when coinoculated with parental genomic RNA onto Nicotiana benthamiana plants and prevented the occurrence of apical necrosis. N. benthamiana plants were transformed with the DI RNA sequences in both the positive and negative orientations relative to the cauliflower mosaic virus 35S promoter. Integration of DI RNA sequences in the plant genome was verified using PCR amplification of DNA extracts and Northern blot analysis of RNA extracts. DI RNA-related transcripts were detected in uninfected transgenic plants, but inoculation with the parental virus induced replication of the DI RNA only in transgenic plants expressing DI RNA in the positive orientation. Transgenic plants in which DI RNA accumulated were protected from apical necrosis and death.
Deletions in the 3' untranslated region of cowpea chlorotic mottle virus transgene reduce recovery of recombinant viruses in transgenic plants.
The 3' untranslated region (UTR) adjacent to the capsid gene is frequently included with the transgene in the construction of capsid protein mediated virus resistant transgenic plants. Since ribonucleotide sequences within the 3' UTR are involved in the initiation of viral replication, the presence of this sequence may encourage the participation of the transgene in RNA recombination. Experiments were designed to explore the involvement of the 3' UTR of cowpea chlorotic mottle virus (CCMV) in RNA recombination events between transgenes and challenging viruses. Nicotiana benthamiana was transformed with CCMV transgenes consisting of the 3' two-thirds of the capsid gene and fragments of the associated 3' UTR lacking the terminal 69, 83, or 214 nucleotides. Plants were inoculated with wild-type CCMV transcripts for RNAs 1 and 2 and a movement defective RNA 3 transcript lacking the 3' third of the capsid gene. While no recombinant virus was detected in plants expressing 3' deletion constructs, 3% of control transformants containing an identical segment of the capsid gene with the complete 3' UTR yielded viable recombinant virus. These results suggest that RNA recombination between transgenic RNA and viral RNA can be reduced significantly by omitting or disrupting the 3' UTR in the transgene.
Cotton (Gossypium hirsutum L.) pollen-specific polygalacturonase mRNA: tissue and temporal specificity of its promoter in transgenic tobacco.
A gene (G9) expressed during late microsporogenesis in cotton (Gossypium hirsutum L.) was isolated. Sequence analysis of the cDNA (1.3 kb) as well as the gene (2.6 kb) revealed an open reading frame of 1233 bases encoding a protein of 43.9 kDa. The coding region of the gene is interrupted by three introns. Northern analysis of the RNA from developing anthers showed that the transcripts appear 12 days before anthesis and that the maximal concentration of RNA occurs in pollen on the day of anthesis. This pattern of gene expression suggests functions in post-anthesis events. Sequence comparisons with other known plant genes indicated that G9 is homologous to polygalacturonases. The G9 promoter conferred tissue and temporal specificity of beta-glucuronidase (GUS) expression in transgenic tobacco plants. Thus, the G9 promoter can be used to drive gene expression in homologous as well as heterologous plants in a tissue-specific manner.
Characterization of a tobacco gene encoding a pollen-specific polygalacturonase.
We report here the isolation and characterization of a gene which is specifically expressed during late pollen development in Nicotiana tabacum L. cv. Havana and which exhibits homology to bacterial, fungal and plant polygalacturonases. This gene is ca. 4.3 kb, from the transcription start-site to the 3' polyadenylation-site sequences. It contains three introns of 620, 706 and 1400 bp and encodes a 1.5 kb message that contains an A-rich 5'-untranslated-leader sequence of 81 bases and a variable-length 3'-untranslated sequence of between 180 and 320 bases. Located within intron 3 is a 414 bp sequence which exhibits 79% homology to a sequence within the endochitinase gene; both sequences share the same internal repeat structure and exhibit features consistent with them being defective transposable elements. The predicted protein sequence coded for by Npg1 shows, in addition to a number of highly conserved cysteines, four conserved domains with the bacterial and fungal polygalacturonase genes. The pollen-specific polygalacturonases as a group can be distinguished from the fruit-ripening polygalacturonases by a number of criteria. It is suggested that these differences reflect the functional differences between plant endo- and exo-polygalacturonases. Npg1 is one of a two-member gene family expressed predominantly in the male gametophyte upon first microspore mitosis. From expression studies of promoter::GUS transgenes it is clear that the -744 bp to +74/+85 bp of Npg1 sequence (with respect to the transcription start site) is sufficient to drive the expression of the GUS reporter gene in a manner that reflects the spatial and temporal expression of Npg1 as determined by dot-blot and northern analysis.
The maize transposable element Ac excises in progeny of transformed tobacco.
To assess the potential of the maize transposable element Ac for gene tagging in heterologous plant species we monitored transcription, excision and transposition of the element in transgenic tobacco plants and their selfed progeny. Ac excised in the majority of primary regenerants and continued to excise in the first-generation progeny plants. In one primary regenerant Ac was transcribed but did not excise. Fourteen of eighteen kanamycin-resistant progeny from this plant showed Ac excision, suggesting that excision of Ac may have been activated during meiosis or in embryo development. This finding, together with the more general observation of continued Ac mobility in the progeny of transformed plants in which Ac had excised, suggests that Ac will be useful for gene tagging.
A chimaeric tryptophan decarboxylase gene as a novel selectable marker in plant cells.
A novel selection system for plant genetic transformation was developed based on the enzyme tryptophan decarboxylase (TDC; EC 4.1.1.28) from Catharanthus roseus. This enzyme converts the toxic tryptophan analogue 4-methyl tryptophan (4-mT) into the non-toxic compound 4-methyl tryptamine. Expression of tdc in transgenic plants that have no endogenous TDC-activity allows selection on 4-mT. A vector was constructed containing a tdc cDNA clone under control of the constitutively expressed cauliflower mosaic virus 35S promoter. This vector was used in Agrobacterium-mediated tobacco leaf disc transformation experiments. The optimal concentration for selection with 4-mT was found to be 0.1 mM. The transformed nature of shoots obtained after tdc gene transfer and subsequent selection on 0.1 mM 4-mT was confirmed by northern blot analysis.
Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site-specific recombination system.
The Cre-lox site-specific recombination system of bacteriophage P1 was used to excise a firefly luciferase (luc) gene which had previously been incorporated into the tobacco genome. The excision event was due to site-specific DNA recombination between two lox sequences flanking the luc gene and was catalyzed by the Cre recombinase introduced by cross-fertilization. Recombination resulted in the fusion of a promoter with a distally located hygromycin phosphotransferase (hpt) coding sequence and the excision event was monitored as a phenotypic change from expression of luc to expression of hpt. The efficiency of recombination was estimated from the exchange of gene activity and confirmed by molecular analysis. The relevance to potential applications of site-specific deletion-fusion events for chromosome engineering are discussed.
Expression of the CMS-associated urfS sequence in transgenic petunia and tobacco.
The expression of a 25 kDa protein, encoded by the fused mitochondrial pcf gene, is associated with cytoplasmic male sterility (CMS) in petunia. To investigate the role of the 25 kDa protein in CMS we have transformed petunia and tobacco plants with constructs expressing a portion of the urfS sequence of the pcf cDNA which encodes the 25 kDa protein. The urfS sequence was fused with two different mitochondrial targeting sequences. The chimeric gene coding region was placed under the control of the CaMV 35S promoter or a tapetum-specific promoter. Expression of the PCF protein was obtained in mitochondria of transgenic petunia and tobacco plants, yet fertility of the plants was not affected. Analysis of the location of the urfS-encoded protein revealed that it fractionates primarily into the soluble fraction in the transgenic plants whereas the genuine 25 kDa protein is found primarily in the soluble fraction but also in the membrane portion of immature buds from CMS petunia plants. Fertile transgenic plants were obtained which expressed the 25 kDa protein in the tapetal layer of post-meiotic anthers, while CMS plants express the endogenous 25 kDa protein in both the tapetal layer and sporogenous tissue of pre-meiotic anthers.
A GCC element and a G-box motif participate in ethylene-induced expression of the PRB-1b gene.
The PRB-1b gene codes for a basic-type pathogenesis-related protein and is activated at the transcriptional level by the plant hormone ethylene. To identify cis-acting DNA elements essential for ethylene induction, deleted and mutant forms of the PRB-1b promoter, fused to the beta-glucuronidase (GUS) coding region, were introduced in transgenic tobacco plants. A 73 bp fragment (X1 region) of the PRB-1b promoter, located between positions -213 and -141, was sufficient to confer ethylene responsiveness to the reporter gene. The X1 region contains a TAAGAGCCGCC motif (GCC-box) well conserved in several ethylene-inducible genes. A substitution mutation in this sequence, in the context of a 213 bp PRB-1b promoter, completely abolished ethylene induction in transgenic tobacco, defining this conserved motif as part of a cis-acting element responsive to ethylene. Three other mutations in the X1 region caused a pronounced decrease in the PRB-1b promoter activity in transgenic plants, but did not affect ethylene inducibility. One of them, localized in a G-box like motif (CACGTG), disrupted the binding site for a nuclear factor, as observed in gel-shift analysis. Interestingly, the mobility of the complex formed on the G-box element was dependent on its phosphorylation state. These results suggest that a cis-acting element involved in the perception of the ethylene signal resides in a GCC motif and acts in concert with additional elements in the regulation of ethylene-induced PRB-1b expression.