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A minimal gene cassette comprised of the ubiquitin (Ubi) promoter + green fluorescent protein (Gfp) gene + Nos terminator DNA sequences, derived from the plasmid vector pPZP201-Gfp was utilized for transformation of creeping bentgrass using particle bombardment. Bentgrass calli bombarded individually
with equivalent amounts of the cassette or whole plasmid DNA were compared for Gfp expression and the GFP-positive calli were subsequently regenerated into plants. Percentage of GFP expressing calli and the
number of GFP spots/calli were significantly higher in calli that were bombarded with the minimal gene cassette when compared
to the whole plasmid. The Gfp expression was stable up to the T2 generation in minimal gene cassette transformants and there was a lower degree of gene silencing. Southern blot analysis
of transgenic plants derived from minimum gene cassette bombardment revealed the presence of single or few copy of the transgene
and fairly simple integration patterns. In comparison, whole plasmid transformants had multiple copies and complex integration
patterns of the transgene. These results illustrate the advantages of using simple gene cassette for stable plant transformation
in bentgrass with possible applications to other plant species. 相似文献
3.
Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.) 总被引:1,自引:0,他引:1
V. Srivastava V. Vasil I. K. Vasil 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1996,92(8):1031-1037
Molecular analysis of the transgenes bar and gus was carried out over successive generations in six independent transgenic lines of wheat, until the plants attained homozygosity. Data on expression and integration of the transgenes is presented. Five of the lines were found to be stably transformed, duly transferring the transgenes to the next generation. The copy number of the transgenes varied from one to five in the different lines. One line was unstable, first losing expression of and then eliminating both the transgenes in R3 plants. Although the gus gene was detected in all the lines, GUS expression had been lost in R2 plants of all but one line. Rearrangement of transgene sequences was observed, but it had no effect on gene expression. All the stable lines were found to segregate for transgene activity in a Mendelian fashion. 相似文献
4.
Influence of plant development and environment on transgene expression in potato and consequences for insect resistance 总被引:8,自引:0,他引:8
Down Rachel E. Ford Louise Bedford Simon J. Gatehouse Laurence N. Newell Christine Gatehouse John A. Gatehouse Angharad M.R. 《Transgenic research》2001,10(3):223-236
Clonal replicates of different transformed potato plants expressing transgene constructs containing the constitutive Cauliflower Mosaic Virus (CaMV) 35S promoter, and sequences encoding the plant defensive proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA), and bean chitinase (BCH) were propagated in tissue culture. Plants were grown to maturity, at first under controlled environmental conditions, and later in the glasshouse. For a given transgene product, protein accumulation was found to vary between the different lines of clonal replicates (where each line was derived from a single primary transformant plant), as expected. However, variability was also found to exist within each line of clonal replicates, comparable to the variation of mean expression levels observed between the different clonal lines. Levels of GNA, accumulated in different parts of a transgenic potato plant, also showed variation but to a lesser extent than plant–plant variation in expression. With the majority of the clonal lines investigated, accumulation of the transgene product was found to increase as the potato plant developed, with maximum levels found in mature plants. The variation in accumulation of GNA among transgenic plants within a line of clonal replicates was exploited to demonstrate that the enhanced resistance towards larvae of the tomato moth, Lacanobia oleracea L., caused by expression of this protein in potato, was directly correlated with the level of GNA present in the plants, and that conditions under which the plants were grown affect the levels of GNA expression and subsequent levels of insect resistance. 相似文献
5.
Variation in GUS activity in vegetatively propagated Hevea brasiliensis transgenic plants 总被引:1,自引:0,他引:1
Lardet L Leclercq J Bénistan E Dessailly F Oliver G Martin F Montoro P 《Plant cell reports》2011,30(10):1847-1856
Hevea brasiliensis transgenic plants are regenerated from transgenic callus lines by somatic embryogenesis. Somatic embryogenesis is not yet
available for commercial propagation of Hevea clones, which requires conventional grafting of buds on rootstock seedlings (budding). The stability of transgene expression
in budded plants is therefore necessary for further development of genetic engineering in rubber trees. Transgene expression
was assessed by fluorimetric beta-glucuronidase (GUS) activity in fully developed leaves of in vitro plants from transgenic
lines and their sub-lines obtained by budding. A large variation in GUS activity was found in self-rooted in vitro plants
of five transgenic lines, and the absence of activity in one line suggested transgene silencing. Beyond confirming transmissibility
of the reporter gene by budding and long-term expression, a quantification of GUS activity revealed that greater variability
existed in budded plants compared to self-rooted mother in vitro plants for three transgenic lines. Although somatic embryogenesis
provided more stable GUS activity, budding remained an efficient way of propagating transgenic plants but transgene expression
in budded plants should be verified for functional analysis and further development. 相似文献
6.
Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants 总被引:5,自引:0,他引:5
The utility of green fluorescent protein (GFP) for biological research is evident. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. Fluorescence intensity was linear with increasing levels of GFP over a range that encompasses transgene expression in plants by the cauliflower mosaic virus 35S promoter. Standard curves were used to estimate GFP concentration in planta and in protein extracts. These values were consistent with ELISA measurements of GFP in protein extracts from transgenic plants, indicating that the technique is a reliable measure of recombinant GFP expression. The levels of in planta GFP expression in both homozygous and hemizygous plants was then estimated. Homozygous transgenic plants expressed twice the amount of GFP than hemizygous plants, suggesting additive transgene expression. This methodology may be useful to simplify the characterization of transgene expression in plants.Abbreviations
ELISA
Enzyme-linked immunosorbent assay
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HRP
Horseradish peroxidase
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GFP
Green fluorescent protein
Communicated by M.C. Jordan 相似文献
7.
We developed a site-directed integration (SDI) system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined target locus by recombinase-mediated
cassette exchange (RMCE). We produced site-specific transgenic tobacco plants from four target lines and examined expression
of the transgene in T1 site-specific transgenic tobacco plants, which were obtained by backcrossing. We found that site-specific
transgenic plants from the same target lines showed approximately the same level of expression of the transgene. Moreover,
we demonstrated that site-specific transgenic plants showed much less variability of transgene expression than random-integration
transgenic plants. Interestingly, transgenes in the same direction at the same target locus showed the same level of activity,
but transgenes in different directions showed different levels of activity. The expression levels of transgene did not correlate
with those of the target gene. Our results showed that the SDI system could benefit the precise comparisons between different
gene constructs, the characterization of different chromosomal regions and the cost-effective screening of reliable transgenic
plants. 相似文献
8.
9.
D. Gahakwa S. B. Maqbool X. Fu D. Sudhakar P. Christou A. Kohli 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,101(3):388-399
The success of contemporary breeding programmes involving genetic engineering depends on the stability of transgene expression
over many generations. We studied the stability of transgene expression in 40 independent rice plant lines representing 11
diverse cultivated varieties. Each line contained three or four different transgenes delivered by particle bombardment, either
by cotransformation or in the form of a cointegrate vector. Approximately 75% of the lines (29/40) demonstrated Mendelian
inheritance of all transgenes, suggesting integration at a single locus. We found that levels of transgene expression varied
among different lines, but primary transformants showing high-level expression of the gna, gusA, hpt and bar transgenes faithfully transmitted these traits to progeny. Furthermore, we found that cry1Ac and cry2A transgene expression was stably inherited when primary transformants showed moderate or low-level expression. Our results
show that six transgenes (three markers and three insect-resistance genes) were stably expressed over four generations of
transgenic rice plants. We showed that transgene expression was stable in lines of all the rice genotypes we analysed. Our
data represent a step forward in the transfer of rice genetic engineering technology from model varieties to elite breeding
lines grown in different parts of the world.
Received: 22 March 1999 / Accepted: 6 December 1999 相似文献
10.
Wahlroos Tony Susi Petri Solovyev Andrej Dorokhov Yurii Morozov Sergeyi Atabekov Josif Korpela Timo 《Molecular breeding : new strategies in plant improvement》2005,14(4):455-462
An approach that enables the increase of the quantity of a specific amino acid in crop plants is reported. Oleosin gene from Arabidopsis thaliana or 30K movement protein gene of Tobacco mosaic virus (TMV; genus Tobamovirus) were cloned under the control of napin or hybrid promoters, and in fusion to synthetic poly-histidine (poly-His) sequences for transformation into spring turnip rape (Brassica rapa subsp. oleifera; synonym to B. campestris). The most stable expression cassettes for the poly-His production prior to the plant transformation were selected by analyzing the protein expression in in vitro translation and in transient plant expression systems using GFP as marker. Expression of the poly-His-constructs in transgenic Brassica rapa plants was analyzed using dot and western blotting and PCR. The constructs were stably expressed in the third generation of the transgenic plant lines. Histidine content was measured from the seeds of the transgenic plants, and some plant lines had more than 20% increase in histidine content compared to wild type. The methodology may be widely applicable to increase the content of any amino acid in crop plants including those encoded by rare codons. 相似文献
11.
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
12.
J. Leclercq L. Lardet F. Martin T. Chapuset G. Oliver P. Montoro 《Plant cell reports》2010,29(5):513-522
An efficient genetic transformation procedure using a recombinant green fluorescent protein (GFP) has been developed in Hevea brasiliensis clone PB260. Transformation experiments have been performed using an Agrobacterium tumefaciens binary vector harbouring both uidA and S65T-GFP reporter genes in order to compare selection methods using glucuronidase assay (GUS activity) and paromomycin
resistance, GFP activity and paromomycin resistance, or GFP activity only. At transient level, the number of spots showing
GUS or GFP activities was similar for 4 and 5 days after coculture. After selection, stable transformation events were observed
and led to the establishment of transgenic callus lines. A higher number of lines were generated with GFP selection compared
to the GUS one. GFP selection is less time-consuming in terms of callus subculturing, and offers the possibility of producing
antibiotic resistance marker-free transgenic plants. 相似文献
13.
14.
A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations 总被引:15,自引:0,他引:15
Afolabi AS Worland B Snape JW Vain P 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2004,109(4):815-826
Transgenic locus composition and T-DNA linkage configuration were assessed in a population of rice plants transformed using the dual-binary vector system pGreen (T-DNA containing the bar and gus
genes)/pSoup (T-DNA containing the aphIV and gfp genes). Transgene structure, expression and inheritance were analysed in 62 independently transformed plant lines and in around 4,000 progeny plants. The plant lines exhibited a wide variety of transgenic locus number and composition. The most frequent form of integration was where both T-DNAs integrated at the same locus (56% of loci). When single-type T-DNA integration occurred (44% of loci), pGreen T-DNA was preferentially integrated. In around half of the plant lines (52%), the T-DNAs integrated at two independent loci or more. In these plants, both mixed and single-type T-DNA integration often occurred concurrently at different loci during the transformation process. Non-intact T-DNAs were present in 70–78% of the plant lines causing 14–21% of the loci to contain only the mid to right border part of a T-DNA. In 53–66% of the loci, T-DNA integrated with vector backbone sequences. Comparison of transgene presence and expression in progeny plants showed that segregation of the transgene phenotype was not a reliable indicator of either transgene inheritance or T-DNA linkage, as only 60–80% of the transgenic loci were detected by the expression study. Co-expression (28% of lines) and backbone transfer (53–66% of loci) were generally a greater limitation to the production of marker-free T1 plants expressing the gene of interest than co-transformation (71% of lines) and unlinked integration (44% of loci). 相似文献
15.
The Saccharomyces cerevisiae chitinase,encoded by the CTS1-2 gene,confers antifungal activity against Botrytis cinerea to Transgenic tobacco 总被引:2,自引:0,他引:2
The Saccharomyces cerevisiae chitinase, encoded by the CTS1-2 gene has recently been confirmed by in vitro tests to possess antifungal abilities. In this study, the CTS1-2 gene has been evaluated for its in planta antifungal activity by constitutive overexpression in tobacco plants to assess its potential to increase the plant's defence against fungal pathogens. Transgenic tobacco plants, generated by Agrobacterium-mediated transformation, showed stable integration and inheritance of the transgene. Northern blot analyses conducted on the transgenic tobacco plants confirmed transgene expression. Leaf extracts from the transgenic lines inhibited Botrytis cinerea spore germination and hyphal growth by up to 70% in a quantitative in vitro assay, leading to severe physical damage on the hyphae. Several of the F1 progeny lines were challenged with the fungal pathogen, B. cinerea, in a detached leaf infection assay, showing a decrease in susceptibility ranging from 50 to 70%. The plant lines that showed increased disease tolerance were also shown to have higher chitinase activities. 相似文献
16.
Background
Phenotypic characterization of transgenic cell lines, frequently used in plant biology studies, is complicated because transgene expression in individual cells is often heterogeneous and unstable. To identify the sources and to reduce this heterogeneity, we transformed tobacco (Nicotiana tabacum L.) BY-2 cells with a gene encoding green fluorescent protein (GFP) using Agrobacterium tumefaciens, and then introduced a simple cloning procedure to generate cell lines derived from the individual transformed cells. Expression of the transgene was monitored by analysing GFP fluorescence in the cloned lines and also in lines obtained directly after transformation. 相似文献17.
Isolation and Characterization of Chloroplast DNA from the Duckweed Spirodela oligorrhiza 总被引:2,自引:1,他引:1
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Chloroplast DNA of the duckweed Spirodela oligorrhiza, isolated by CsCl gradient centrifugation, was characterized by its buoyant density, guanine + cytosine content, melting behavior, circularity, and contour length. In all these characteristics, chloroplast DNA of S. oligorrhiza is similar to the chloroplast genomes of other higher plants, except that it has a significantly larger size. 相似文献
18.
Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny 总被引:17,自引:1,他引:16
Philippe Vain Barbara Worland Ajay Kohli John W. Snape Paul Christou George C. Allen William F. Thompson 《The Plant journal : for cell and molecular biology》1999,18(3):233-242
To investigate the effect of matrix attachment regions (MARs) on transgene expression levels and stability in cereal crops, we generated 83 independent transgenic rice callus lines containing a gusA expression cassette either as a simple expression unit, or flanked with MARs from tobacco (Rb7) or yeast (ARS1). Transgenic rice plants were regenerated from these callus lines and analysed at the structural and expression levels over two generations. In the first generation (T0), both Rb7 and ARS1 MARs significantly increased transgene expression levels. In the populations of plants containing MARs, we observed a significant reduction in the number of non-expressing lines compared to the population of plants without MARs. However, variation in β-glucuronidase (GUS) expression levels between independent lines was similar both in the presence and absence of flanking MARs. In the presence of MARs, GUS activity increased in proportion to transgene copy number up to 20 copies, but was generally reduced in lines carrying a higher copy number. In the population of plants without MARs, there was no correlation between expression level and transgene copy number. In the second generation (T1), transgene expression levels were significantly correlated with those of the T0 parents. The Rb7 MARs significantly improved the stability of transgene expression levels over two generations, and therefore appear to offer protection against transgene silencing. Our study shows that the exploitation of MARs may be an important strategy for stabilising transgene expression levels in genetically engineered cereals. 相似文献
19.
A red fluorescent protein, DsRed2, as a visual reporter for transient expression and stable transformation in soybean 总被引:6,自引:0,他引:6
Fluorescent proteins such as green fluorescent protein (GFP) from Aequorea victoria are often used as markers for transient expression and stable transformation in plants, given that their detection does not require a substrate and they can be monitored in a nondestructive manner. We have now evaluated the red fluorescent protein DsRed2 (a mutant form of DsRed from Discosoma sp.) for its suitability as a visual marker in combination with antibiotic selection for genetic transformation of soybean [Glycine max (L.) Merrill]. Transient and stable expression of DsRed2 in somatic embryos was readily detected by fluorescence microscopy, allowing easy confirmation of gene introduction. We obtained several fertile transgenic lines, including homozygous lines, that grew and produced seeds in an apparently normal manner. The red fluorescence of DsRed2 was detected by fluorescence microscopy without background fluorescence in both leaves and seeds of the transgenic plants. Furthermore, in contrast to seeds expressing GFP, those expressing DsRed2 were readily identifiable even under white light by the color conferred by the transgene product. The protein composition of seeds was not affected by the introduction of DsRed2, with the exception of the accumulation of DsRed2 itself, which was detectable as an additional band on electrophoresis. These results indicate that DsRed2 is a suitable reporter (even more suitable than GFP) for genetic transformation of soybean. 相似文献
20.
To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T8 plant, having uidA (or gus) driven by the barley endosperm-specific B1-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T4 plant, having sgfp(S65T) driven by the barley endosperm-specific D-hordein promoter (localized on the subtelomeric region of chromosome 2H).
Both lines stably expressed the two transgenes in the generations prior to the cross. Three independently crossed F1 progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F2 seeds followed a 3:1 Mendelian segregation ratio and transgenes were localized by FISH to the same location as in the parental
plants. FISH was used to screen F2 plants for homozygosity of both transgenes; four homozygous plants were identified from the two crossed lines tested. FISH
results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating
that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F3 and F4 generations. Thus, even after crossing independently transformed, homozygous parental plants containing a single, stably
expressed transgene, progeny were obtained that continued to express multiple transgenes through generation advance. Such
stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies. 相似文献