共查询到20条相似文献,搜索用时 9 毫秒
1.
Use of a simple semiquantitative method for appraisal of green fluorescent protein gene expression in transgenic tobacco plants 总被引:1,自引:0,他引:1
We have applied a simple method for evaluation of gfp gene expression in plants using a CCD camera and computerized processing of images. Transgenic tobacco plants were obtained by Agrobacterium tumefaciens-mediated transfer of plasmid T-DNA bearing a m-gfp5-ER sequence governed by the 35S promoter together with the nptII selectable marker gene. Presence of the gfp gene in plants was confirmed by a polymerase chain reaction method. Mean brightness values measured using image analysis software showed differences between transgenic and control plants and suggest the possibility of rapid selection of transgenic individuals among regenerants and their progenies. 相似文献
2.
Sugey Ramona Sinagawa-García Tarinee Tungsuchat-Huang Octavio Paredes-López Pal Maliga 《Plant molecular biology》2009,70(5):487-498
Plastid transformation vectors are E. coli plasmids carrying a plastid marker gene for selection, adjacent cloning sites and flanking plastid DNA to target insertions
in the plastid genome by homologous recombination. We report here on a family of next generation plastid vectors carrying
synthetic DNA vector arms targeting insertions in the rbcL-accD intergenic region of the tobacco (Nicotiana tabacum) plastid genome. The pSS22 plasmid carries only synthetic vector arms from which the undesirable restriction sites have been
removed by point mutations. The pSS24 vector carries a c-Myc tagged spectinomycin resistance (aadA) marker gene whereas in vector pSS30 aadA is flanked with loxP sequences for post-transformation marker excision. The synthetic vectors will enable direct manipulation of passenger genes
in the transformation vector targeting insertions in the rbcL-accD intergenic region that contains many commonly used restriction sites.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
3.
We recently reported an 868-bp plastid DNA minicircle, NICE1, that formed during transformation in a transplastomic Nicotiana tabacum line. Shuttle plasmids containing NICEI sequences were maintained extrachromosomally in plastids and shown to undergo recombination with NICE1 sequences on the plastid genome. To prove the general utility of the shuttle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the CoIE1 ori for maintenance in E. coli and a spectinomcyin resistance gene (aadA) for selection in both systems. In addition, pNICERl carries a defective kanamycin resistance gene, kan*, to target the rescue of a functional kanamycin resistance gene, kan, from the recipient plastid genome. pNICERl was introduced into plastids where recombination could occur between the homologous kan/kan* sequences, and subsequently rescued in E. coli to recover the products of recombination. Based on the expression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recovered at a high frequency. Efficient rescue of kan from the plastid genome in E. coli indicates that NICE 1-based plasmids are suitable for rescuing mutations from any part of the plastid genome, expanding the repertoire of genetic tools available for plastid biology. 相似文献
4.
Lenzi P Scotti N Alagna F Tornesello ML Pompa A Vitale A De Stradis A Monti L Grillo S Buonaguro FM Maliga P Cardi T 《Transgenic research》2008,17(6):1091-1102
Human Papillomavirus (HPV) is the causal agent of cervical cancer, one of the most common causes of death for women. The major
capsid L1 protein self-assembles in Virus Like Particles (VLPs), which are highly immunogenic and suitable for vaccine production.
In this study, a plastid transformation approach was assessed in order to produce a plant-based HPV-16 L1 vaccine. Transplastomic
plants were obtained after transformation with vectors carrying a chimeric gene encoding the L1 protein either as the native
viral (L1v gene) or a synthetic sequence optimized for expression in plant plastids (L1pt gene) under control of plastid expression signals. The L1 mRNA was detected in plastids and the L1 antigen accumulated up
to 1.5% total leaf proteins only when vectors included the 5′-UTR and a short N-terminal coding segment (Downstream Box) of
a plastid gene. The half-life of the engineered L1 protein, determined by pulse-chase experiments, is at least 8 h. Formation
of immunogenic VLPs in chloroplasts was confirmed by capture ELISA assay using antibodies recognizing conformational epitopes
and by electron microscopy.
Contribution No. 129 from CNR-IGV, Portici. 相似文献
5.
D. S. Andreyuk N. P. Matveeva M. I. Tukeeva I. P. Ermakov 《Russian Journal of Developmental Biology》2000,31(2):89-94
We studied the dynamics of mobile potassium, chloride, and nitrate ions during development of the microspore and differentiation
of the pollen grain inNicotiana tabacum L. by measuring their concentration in aqueous extracts from cells destroyed by freezing-thawing using ion-selective electrodes.
Stage-specific changes in the ion content and intracellular concentration in the male gametophyte were found. A relationship
of the dynamics of ions to growth processes and changes in metabolic activity during gametophytogenesis has been discussed.
The changes in the potassium and chloride ion concentrations have been interpreted as regulatory changes controlling protein
synthesis in the pollen grain vegetative cell.
Deceased. 相似文献
6.
Genetically engineered pollen with a visible marker gene could be useful to monitor the movement of transgenic pollen provided there are no negative physiological or fitness effects of expressing such a gene. In this study, we measured the fitness of Nicotiana tabacum cv. Xanthi pollen expressing the marker gene green fluorescent protein (GFP). Average pollen tube germination frequencies and pollen tube growth rates in vitro were measured in three different types of plants: (1) plants producing GFP in pollen cells only (LAT59-GFP), (2) plants synthesizing GFP under the control of a constitutive promoter (CaMV 35S) in which no GFP was produced in pollen, and (3) non-transgenic plants. Pollen synthesizing the GFP protein did not differ significantly in average pollen germination frequencies from pollen without GFP (P=0.65). Average pollen tube growth rates over a 5-h period did not differ significantly between transgenic and non-transgenic types (R2=0.89, 0.98, and 0.95, respectively, for GFP-tagged, 35S-GFP, and wild type). Overall, GFP expression in pollen grains of tobacco was not found to have an effect on pollen fitness under the controlled experimental conditions of this study. 相似文献
7.
Plants protect themselves against pathogens using a range of response mechanisms. There are two categories of nonhost resistance:
Type I, which does not result in visible cell death; and Type II, which entails localized programmed cell death (or hypersensitive
response) in response to nonhost pathogens. The genes responsible for these two systems have not yet been intensively investigated
at the molecular level. Using tobacco plants (Nicotiana tabacum), we compared expression of 12 defense-related genes between a Type I (Xanthomonas axonopodis pv. glycines 8ra) nonhost interaction, and two Type II (Pseudomonas syringae pv. syringae 61 and P. syringae pv. phaseolicola NPS3121) nonhost interactions, as well as those expressed during R gene-mediated resistance to Tobacco mosaic virus. In general, expression of most defense-related genes during R gene-mediated resistance was activated 48 h after challenge by TMV; the same genes were upregulated as early as 9 h after
infiltration by nonhost pathogens. Surprisingly, X. axonopodis pv. glycines (Type I) elicited the same set of defense-related genes as did two pathovars of P. syringae, despite the absence of visible cell death. In two examples of Type II nonhost interactions, P. syringae pv. phaseolicola NPS3121 produced an expression profile more closely resembling that of X. axonopodis pv. glycines 8ra, than that of P. syringae pv. syringae 61. These results suggest that Type I nonhost resistance may act as a mechanism providing a more specific and
active defense response against a broad range of potential pathogens. 相似文献
8.
Summary
Nicotiana tabacum lines carrying maternally inherited resistance to spectinomycin were obtained by selection for green callus in cultures bleached by spectinomycin. Two levels of resistance was found. SPC1 and SPC2 seedlings are resistant to high levels (500 g/ml), SPC23 seedlings are resistant to low levels (50 g/ml) of spectinomycin. Lines SPC2 and SPC23 are derivatives of the SR1 streptomycin-resistant plastome mutant. Spectinomycin resistance is due to mutations in the plastid 16S ribosomal RNA: SPC1, an A to C change at position 1138; SPC2, a C to U change at position 1139; SPC23, a G to A change at position 1333. Mutations similar to those in the SPC1 and SPC2 lines have been previously described, and disrupt a conserved 16S ribosomal RNA stem structure. The mutation in the SPC23 line is the first reported case of a mutation close to the region of the 16S rRNA involved in the formation of the initiation complex. The new mutants provide markers for selecting plastid transformants. 相似文献
9.
To develop an efficient protocol for the transformation of the legume Astragalus sinicus (Chinese milk vetch), cotyledon segments were infected with Agrobacterium tumefaciens strain EHA105 harboring the binary vector pBINm-gfp5-ER which carries the gfp5 gene encoding green fluorescent protein and the kanamycin (Km) resistance gene nptII. The infected explants were cultured on shoot regeneration (SR) medium containing 1.0 mg l–1 -naphthaleneacetic acid (NAA) and 1.0 mg l–1 thidiazuron (TDZ). Putative transformed shoots were selected on SR medium containing 75 g ml–1 Km, 200 g ml–1 Timentin, and transformation was monitored by observation of GFP expression under a dissecting fluorescence microscope with appropriate filters. The identification of GFP-expressing shoots or callus in combination with Km selection allowed the visual selection of growing transgenic cells and shoots with no escapes. Plants were regenerated from seven independent transgenic events and five plants have set seed. GFP expression segregated in the T1 seedlings of the two lines tested in a 3 – 1 ratio. In addition to the GFP expression of the transgenic plants, the transgenic nature of individual plants was confirmed by Southern and Western blot analyses. 相似文献
10.
Promoters of auxin-induced genes from tobacco can lead to auxin-inducible and root tip-specific expression 总被引:13,自引:0,他引:13
E. J. van der Zaal F. N. J. Droog C. J. M. Boot L. A. M. Hensgens J. H. C. Hoge R. A. Schilperoort K. R. Libbenga 《Plant molecular biology》1991,16(6):983-998
In previous studies we have identified several mRNAs which accumulate after addition of 2,4-dichlorophenoxyacetic-acid (2,4-D) to auxin-starved tobacco cells [45, 46]. The mRNAs corresponding to cDNA clone pCNT103 were found to accumulate transiently prior to the cell division response due to auxin treatment. In this study we determined the sequences of three 103-like cDNAs and two 103-like genes, GNT1 and GNT35. To further study the regulation of the expression of these genes their 5 regions were translationally fused with the -D-glucuronidase reporter gene (GUS). The GNT1 5 region led to GUS expression only in the root tips of transgenic plants. By using transgenic hairy-root cultures and transformed cell suspension cultures it was shown that the 5 regions of both GNT1 and GNT35 lead to 2,4-D-inducible expression of GUS activity. The homology of the 103-like genes with other auxin-regulated genes is evaluated.Department of Plant Molecular Biology, Leiden University 相似文献
11.
Summary A chimeric gene under the control of the hsp70 promoter of Drosophila is heat regulated in roots, stems and leaves, but not in pollen of transgenic tobacco plants. For these and other parameters, it behaves similarly to plant heat-shock genes. 相似文献
12.
Ergosterol is the main sterol of most fungi. Production of reactive oxygen species after the treatment of tobacco and tomato cells by nano-molar concentrations of ergosterol was previously observed as well as the activation of some stress activated mitogen-activated protein kinases on alfalfa cells. In this paper, the expression of some defence-related genes after the ergosterol treatment of tobacco Nicotiana tabacum plants is reported. The gene expression of pathogenesis related proteins of families PR1, PR3, PR5 and proteinase inhibitors of class I and II together with enzymes participating in the defence response, such as phenylalanine-ammonia lyase and sesquiterpene cyclase, were monitored by RT-qPCR. In addition, the concentrations of salicylic acid, an important signalling molecule, increased in time due to the enzyme activation. On the other hand, ergosterol did not provoke tissue necrosis and the possible cross-talk between the signalling pathways of salicylate and jasmonate was observed. Collected data shows that ergosterol is able to activate the expression of a number of defence genes and could increase resistance against pathogens. 相似文献
13.
14.
15.
16.
G. L. Creason R. S. Chaleff 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1988,76(2):177-182
Summary Cultures of Nicotiana tabacum cells homozgous for a mutation (S4) at the SuRB locus that confers resistance to the sulfonylurea herbicides chlorsulfuron and sulfometuron methyl (Chaleff and Ray 1984; Chaleff and Bascomb 1987) were used to isolate a doubly mutant cell line (S4 Hra/S4+) resistant to even higher herbicide concentrations. Growth of cells homozygous for both the S4 and Hra mutations (S4 Hra/S4 Hra) was uninhibited by a herbicide concentration 500-fold higher than a concentration by which growth of S4+/S4+ callus was inhibited by 75%. Plants homozygous for both mutations were at least five-fold more resistant to foliar applications of chlorsulfuron than were singly mutant S4+/S4+ plants. This enhanced resistance was inherited as a single, semidominant, nuclear trait that is genetically linked to the S4 mutation. Acetolactate synthase (ALS) activity in extracts of leaves of doubly mutant (S4 Hra/S4 Hra) plants was approximately 20-fold more resistant to inhibition by chlorsulfuron and sulfometuron methyl than was ALS activity in singly mutant (S4+/ S4+) leaf extracts, which was in turn more resistant to inhibition by these compounds than was the normal enzyme. Extracts prepared from plants of these three genotypes possessed the same ALS specific activities. Therefore, Hra represents a second independent mutation at or near the SuRB locus that reduces the sensitivity of tobacco ALS activity to inhibition by sulfonylurea herbicides. 相似文献
17.
18.
We report here a new selectable marker for tobacco immature pollen transformation based on the expression of dihydrofolate
reductase (dhfr) gene which confers resistance to methotrexate (Mtx). Two immature pollen transformation approaches, i.e., male germ line
transformation and particle bombardment of embryogenic mid-bicellular pollen have been used for the production of stable transgenic
tobacco plants. In the first method, two methotrexate-resistant plants were selected from a total of 7161 seeds recovered
after transformation experiments. In the second method, four methotrexate-resistant plants were obtained from 29 bombardments
using 3.7×105 pollen grains per bombardment. Southern analysis confirmed the transgenic nature of T0 and T1 candidate transgenic plants, and a genetic analysis showed that the transgenes are transmitted to subsequent generations. 相似文献
19.
Green fluorescent protein (GFP) was successfully used as a visual reporter at various stages of carrot (Daucus carota L.) transformation. GFP-fluorescence was non-invasively observed in protoplasts, callus and plants after the delivery of
mgfp5-er gene using two transformation methods: direct DNA transfer into polyethylene glycol (PEG) -treated protoplasts and inoculation
of root discs with Agrobacterium rhizogenes. Transient GFP-expression was detected in the treated protoplasts and monitored during the first week of the cell culture
until the stable level of expression was observed. It was useful for the comparison of protoplast susceptibility to DNA uptake
and the transgene expression as the fluorescence declined with various rates depending on the used carrot genotype and PEG-concentration.
GFP-monitoring in callus enabled the selection of stably expressing lines. It also allowed verification of the homogeneous
tissue composition with regard to the expression of the transgene. In plants, GFP-performance depended on the assayed tissue
and organ despite of the constitutive 35S promoter. The expression was visually detected in both vegetative and generative
parts, but particularly strong fluorescence was observed in leaf marginal meristems, petioles, stems, and styles. Those tissues
can be convenient for examination of the transgenic plants during their growth. The results encourage that GFP is a valuable
reporter and can be routinely used for optimization of transformation protocol, selection of transformants and monitoring
transgenic carrot. 相似文献