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1.
High-resolution structural analysis of biolistic transgene integration into the genome of wheat 总被引:7,自引:0,他引:7
S. A. Jackson P. Zhang W. P. Chen R. L. Phillips B. Friebe S. Muthukrishnan B. S. Gill 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2001,103(1):56-62
Transformation of plant genomes by biolistic methods has become routine over the past decade. However, relatively little is
known about how transgenes are physically integrated into the host genome. Using a high-resolution physical mapping technique,
fluorescence in situ hybridization on extended DNA fibers (fiber-FISH), 13 independent transgenic wheat lines were analyzed
to determine the structural arrangement of stably inherited transgenes in host-plant chromosomes. Twelve transgenic lines
were transformed with a single plasmid and one line was co-transformed with two separate plasmids, which co-segregated genetically.
Three basic integration patterns were observed from the fiber-FISH experiments: Type I, large tandemly repeated integration;
Type II, large tandem integrations interspersed with unknown DNA; and Type III, small insertions, possibly interspersed with
unknown DNA. Metaphase FISH showed that the integration of transgenes was in both hetero- and euchromatic, as well as proximal,
interstitial and distal, regions of the chromosomes. In the transgenic plants, the type of promotor used, rather than the
chromosomal site of transgene integration, was most critical for transgene expression. The integration of the transgenes was
not associated with detectable chromosomal rearrangements.
Received: 25 August 2000 / Accepted: 31 October 2000 相似文献
2.
M. Uzé I. Potrykus C. Sautter 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,99(3-4):487-495
Two non-linked marker genes (gus and bar) were co-introduced by microprojectile bombardment into wheat cells. Four different DNA structures were compared with respect
to ability to integrate into the wheat genome: circular or linear (l) DNA as a single- or double-stranded plasmid (ss and
ds, respectively). In eight independent experiments, linearized DNA integrated in the ds or ss form with a high efficiency
of up to 14% for l-ssDNA. Molecular analyses by Southern blotting showed that all DNA forms gave a similar complicated integration
pattern of the bar gene.
Received: 20 July 1998 / Accepted: 30 January 1999 相似文献
3.
A population of wheat and tritordeum transformants showing a high degree of marker gene stability and heritability 总被引:6,自引:0,他引:6
M. E. Cannell A. Doherty P. A. Lazzeri P. Barcelo 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,99(5):772-784
The stability and heritability of three marker genes was investigated in a population of twelve independent transgenic cereal
lines (six wheat and six tritordeum). Integration patterns, inheritance of structural transgenes and inheritance of expression
were analysed in the T0 and T1 generations for all 12 lines. Transmission and expression were analysed in the T2 generation for 9 lines and in the T3 generation for the six wheat lines. Inheritance of integration patterns was highly stable, and transmission of the transgenes
and inheritance of their expression followed Mendelian ratios in the majority of lines. A gradual reduction in uidA expression was observed over three generations, which was not accompanied by a similar reduction in bar expression. Some unexpected phenomena associated with transgene inheritance were also observed and are discussed.
Received: 9 February 1999 / Accepted: 11 February 1999 相似文献
4.
Green fluorescent protein as a visual marker for wheat transformation 总被引:12,自引:0,他引:12
M. C. Jordan 《Plant cell reports》2000,19(11):1069-1075
Wheat (Triticum aestivum L.) transformation via particle bombardment is now established in many laboratories, but transformation efficiencies are still
largely low and the highest efficiencies can only be obtained with certain genotypes. For rapid optimization and improvement
of wheat transformation protocols, a non-destructive marker which permits early detection of transformed cells is needed.
We have assessed the ability of a modified version of the Aequorea victoria green fluorescent protein (GFP) to act as a marker for detecting transformed cells and tissues of wheat. Multicellular clusters
emitting green fluorescence were observed 14 days after particle bombardment with a sGFPS65T gene construct, and gfp-expressing shoots (often with expressing roots) could be observed as early as 21 days after bombardment. These shoots can
be removed from the callus and grown further until they are ready to transfer to soil. Transgenic wheat plants could be selected
on the basis of gfp expression alone although the inclusion of antibiotic resistance as a selectable marker could improve the efficiency. Using
sgfpS65T as a marker gene in an experiment comparing bombardment parameters allowed the rapid identification of variables that could
be targeted for optimization.
Received: 29 March 2000 / Accepted: 29 March 2000 相似文献
5.
An efficient wheat transformation procedure: transformed calli with long-term morphogenic potential for plant regeneration 总被引:9,自引:0,他引:9
L. Zhang J. J. Rybczynski W. G. Langenberg A. Mitra R. French 《Plant cell reports》2000,19(3):241-250
A method for producing large numbers of transgenic wheat plants has been developed. With this approach, an average of 9.7%
of immature embryo explants were transformed and generated multiple self-fertile, independently transformed plants. No untransformed
plants, or escapes, were regenerated. This transformation procedure uses morphogenic calli derived from scutellum tissue of
immature embryos of Triticum aestivum cv. Bobwhite co-bombarded with separate plasmids carrying a selectable marker gene (bar) and a gene of interest, respectively. Transformed wheat calli with a vigorous growth phenotype were obtained by extended
culture on media containing 5.0 mg/l bialaphos. These calli retained morphogenic potential and were competent for plant regeneration
for as long as 11 months. The bar gene and the gene of interest were co-expressed in T0 progeny plants. This wheat transformation protocol may facilitate quantitative
production of multiple transgenic plants and significantly reduce the cost and labor otherwise required for screening out
untransformed escapes.
Received: 15 June 1998 / Revision received: 6 April 1999 / Accepted: 26 April 1999 相似文献
6.
Genotype screening for proliferative embryogenesis and biolistic transformation of short-season soybean genotypes 总被引:5,自引:0,他引:5
Eighteen of 20 short-season soybean (Glycine max (L.) Merrill) genotypes (maturity group 0 and 00) screened for proliferative embryogenic capacity formed secondary globular
embryos, at rates of 1–70% of cultured immature cotyledons. Five genotypes produced embryogenic cultures which were proliferative
for at least 6 months. Proliferative embryogenic cultures of AC Colibri and X2650–7–2–3 were bombarded using a Bio-Rad PDS-1000/He
particle gun. Co-bombardments with plasmid pairs pHygr (encoding a type IV aminoglycoside phosphotransferase;aphIV) and pRD300pat (encoding a phosphinothricin N-acetyltransferase;pat) or pRD300pat and pFF19G (β-glucuronidase;uidA or gus) resulted, respectively, in 12 hygromycin-selected lines with multiple insertions of aphIV and pat, and two l-phosphinothricin-selected lines plus three β-glucuronidase-positive lines recovered without selection. Although
fertile plants were recovered from young proliferative cultures, transgenic plants, which were derived from cultures 12–14
months of age, were sterile.
Received: 8 January 1998 / Revision received: 12 January 1999 / Accepted: 12 July 1999 相似文献
7.
Association of transgene integration sites with chromosome rearrangements in hexaploid oat 总被引:9,自引:0,他引:9
S. Svitashev E. Ananiev W. P. Pawlowski D. A. Somers 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,100(6):872-880
Transgene loci in 16 transgenic oat (Avena sativa L.) lines produced by microprojectile bombardment were characterized using phenotypic and genotypic segregation, Southern
blot analysis, and fluorescence in situ hybridization (FISH). Twenty-five transgene loci were detected; 8 lines exhibited
single transgene loci and 8 lines had 2 or 3 loci. Double FISH of the transgene and oat C- and A/D-genome-specific dispersed
and clustered repeats showed no preferences in the distribution of transgene loci among the highly heterochromatic C genome
and the A/D genomes of hexaploid oat, nor among chromosomes within the genomes. Transgene integration sites were detected
at different locations along individual chromosomes, although the majority of transformants had transgenes integrated into
subtelomeric and telomeric regions. Transgene integration sites exhibited different levels of structural complexity, ranging
from simple integration structures of two apparently contiguous transgene copies to tightly linked clusters of multiple copies
of transgenes interspersed with oat DNA. The size of the genomic interspersions observed in these transgene clusters was estimated
from FISH results on prometaphase chromosomes to be megabases long, indicating that some transgene loci were significantly
larger than previously determined by Southern blot analysis. Overall, 6 of the 25 transgene loci were associated with rearranged
chromosomes. These results suggest that particle bombardment-mediated transgene integration may result from and cause chromosomal
breakage and rearrangements.
Received: 29 July 1999 / Accepted: 9 November 1999 相似文献
8.
Altered functional properties of tritordeum by transformation with HMW glutenin subunit genes 总被引:4,自引:0,他引:4
L. Rooke F. Barro A. S. Tatham R. Fido S. Steele F. Békés P. Gras A. Martin P. A. Lazzeri P. R. Shewry P. Barcelo 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,99(5):851-858
The high-molecular-weight (HMW) subunits of wheat glutenin are the major determinants of the gluten visco-elasticity that
allows wheat doughs to be used to make bread, pasta and other food products. In order to increase the proportions of the HMW
subunits, and hence improve breadmaking performance, particle bombardment was used to transform tritordeum, a fertile amphiploid
between wild barley and pasta wheat, with genes encoding two HMW glutenin subunits (1Ax1 and 1Dx5). Of the 13 independent
transgenic lines recovered (a transformation frequency of 1.4%) six express the novel HMW subunits at levels similar to, or
higher than, those of the endogenous subunits encoded on chromosome 1B. Small-scale mixograph analysis of T2 seeds from a line expressing the transgene for 1Dx5 indicated that the addition of novel HMW subunits can result in significant
improvements in dough strength and stability, thus demonstrating that transformation can be used to modify the functional
properties of tritordeum for improved breadmaking.
Received: 15 January 1999 / Accepted: 5 February 1999 相似文献
9.
The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants 总被引:10,自引:0,他引:10
James VA Avart C Worland B Snape JW Vain P 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2002,104(4):553-561
Segregating T1, T2 and T3 transgenic rice populations, derived from independent particle-bombardment-mediated transformation events were examined in
order to assess the effect of gene dosage on transgene expression levels and stability. The expression level of the unselected
β-glucuronidase (gusA) reporter gene was quantified in plants from these populations. The gusA gene dosage was determined by segregation analysis of progeny seedlings at the structural level (by PCR) and at the expression
level. For some transformation events a gene dosage effect on transgene expression was observed, leading to higher transgene
expression levels in homozygous progeny than in hemizygous progeny or primary transgenic plants. However, in many other transformation
events, the homozygous state appears to be disadvantageous, being associated with lower transgene expression levels, gene
silencing or counter-selection of homozygous plants across generations. Change of gene dosage is probably one of the key factors
influencing transgene expression levels and stability in transgenic rice. This is particularly important when considering
molecular genetic studies and crop improvement programmes. The possible influence of matrix attachment regions (MARs) in increasing
the likelihood of an additive effect on transgene expression level is discussed.
Received: 21 March 2001 / Accepted: 29 June 2001 相似文献
10.
11.
J. P. Martinant T. Cadalen A. Billot S. Chartier P. Leroy M. Bernard L. Saulnier G. Branlard 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1998,97(7):1069-1075
Two mapping populations were used for the analysis of the water-extractable arabinoxylans. One originated from a cross between
the hexaploid cultivars ‘Courtot’ and ‘Chinese Spring’ and the other from a cross between an amphiploid (Synthetic) and cv
‘Opata’. Arabinose (Ara), and xylose (Xyl) contents were quantified for the 91 and 76 lines obtained from the two crosses,
respectively. Relative viscosity (ηrel) of the wheat flour aqueous extract was evaluated by capillary viscometry. Both crosses gave similar correlation coefficients
between sugar contents and relative viscosity. There were strong positive relationships between arabinose, xylose and arabinoxylan
contents. The relative viscosity was strongly and positively related to the arabinoxylan content and strongly and negatively
related to the Ara/Xyl ratio (arabinose content to xylose content). For one of the two crosses two measurements of relative
viscosity were generated from 2 years of consecutive harvesting. As a strong correlation was observed between these two measurements,
an important genotypic effect can be deduced for the relative viscosity of water-extractable arabinoxylans. QTL (quantitative
trait locus) research did not reveal any chromosomal segments that were strongly implicated in variations in sugar content.
However, a QTL was found for relative viscosity values and the Ara/Xyl ratio on the long arm of the 1B chromosome for the
two crosses considered. This QTL explained 32–37% of the variations in relative viscosity and 35–42% of the variations in
the Ara/Xyl ratio. Genes located at this QTL controlled relative viscosity through modifying the Ara/Xyl ratio. Variations
in the Ara/Xyl ratio were supposedly related to differences in the molecular structure of water-extractable arabinoxylans.
Minor QTLs were also obtained for relative viscosity and Ara/Xyl ratio, but the chromosomes concerned were different for the
two populations evaluated.
Received: 5 January 1998 / Accepted: 15 May 1998 相似文献
12.
Flow cytometric analysis of the chromosomes and stability of a wheat cell-culture line 总被引:3,自引:0,他引:3
T. Schwarzacher M. L. Wang A. R. Leitch G. Moore J. S. Heslop-Harrison N. Miller 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1997,94(1):91-97
A rapidly growing, long-term suspension culture derived from Triticum aestivum L. (wheat) was synchronized using hydroxyurea and colchicine, and a chromosome suspension with chromosomes was made. After staining with the DNA-specific fluorochromes Hoechst 33258 and Chromomycin univariate and bivariate flow-cytometry histograms showed 15 clearly resolved peaks corresponding to individual chromosome
types or groups of chromosomes with similar DNA contents. The flow karyotype was closely similar to a histogram of DNA content
measurements of Feulgen-stained chromosomes made by microdensitometry. We were able to show the stability of the flow karyotype
of the cell line over a year, while a parallel subculture had a slightly different, stable, karyotype following different
growth conditions. The data indicate that flow cytometric analysis of plant karyotypes enables accurate, statistically precise
chromosome classification and karyotyping of cereals. There was little overlap between individual flow-histogram peaks, so
the method is useful for flow sorting and the construction of chromosome specific-recombinant DNA libraries. Using bivariate
analysis, the AT:GC ratio of all the chromosomes was remarkably similar, in striking contrast to mammalian flow karyotypes.
We speculate about a fundamental difference in organization and homogenization of DNA sequences between chromosomes within
mammalian and plant genomes.
Received: 24 April 1996 / Accepted: 24 May 1996 相似文献
13.
The development of a nuclear male sterility system in wheat. Expression of the barnase gene under the control of tapetum specific promoters 总被引:14,自引:0,他引:14
M. De Block D. Debrouwer T. Moens 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1997,95(1-2):125-131
Nuclear male sterility within Triticum aestivum is considered as the ideal basis for the development of a hybridization system for wheat. We engineered nuclear male sterility
in wheat by introducing the barnase gene under the control of tapetum-specific promoters derived from corn and rice. A biolistic-mediated transformation method,
based on the use of the poly(ADP-ribose)polymerase inhibitor niacinamide, was set up which enriched for low-copy integrations
(1–3 copies). Most of these copies were not linked and segregated in the next generation.
Received: 22 January 1997 / 7 February 1997 相似文献
14.
A wheat cDNA coding for a thaumatin-like protein reveals a high level of RFLP in wheat 总被引:1,自引:0,他引:1
D. Mingeot J. M. Jacquemin 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1997,95(5-6):822-827
A cDNA clone that reveals a high level of polymorphism between wheat varieties was isolated from a wheat cDNA library. When
hybridized to DraI-, EcoRV- and HindIII digested DNA this clone, gbx3832, enables us to distinguish 42 different patterns among 48 varieties: 37 varieties are
clearly identified, the remaining 11 are divided into five groups. Base-sequence analysis of the clone reveals 72–74% sequence
identity to mRNAs encoding thaumatin-like proteins from different cereals.
Received: 27 January 1997 / Accepted: 18 April 1997 相似文献
15.
G. Schwarz M. Herz X. Q. Huang W. Michalek A. Jahoor G. Wenzel V. Mohler 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,100(3-4):545-551
Genetic mapping and the selection of closely linked molecular markers for important agronomic traits require efficient, large-scale
genotyping methods. A semi-automated multifluorophore technique was applied for genotyping AFLP marker loci in barley and
wheat. In comparison to conventional 33P-based AFLP analysis the technique showed a higher resolution of amplicons, thus increasing the number of distinguishable
fragments. Automated sizing of the same fragment in different lanes or different gels showed high conformity, allowing subsequent
unambigous allele-typing. Simultaneous electrophoresis of different AFLP samples in one lane (multimixing), as well as simultaneous
amplification of AFLP fragments with different primer combinations in one reaction (multiplexing), displayed consistent results
with respect to fragment number, polymorphic peaks and correct size-calling. The accuracy of semi-automated co-dominant analysis
for hemizygous AFLP markers in an F2 population was too low, proposing the use of dominant allele-typing defaults. Nevertheless, the efficiency of genetic mapping,
especially of complex plant genomes, will be accelerated by combining the presented genotyping procedures.
Received: 10 April 1999 / Accepted: 11 May 1999 相似文献
16.
Embryo survival and transgene integration rates are two major factors that influence the efficiency of transgenic animal production by pronuclear microinjection. Recombinase A protein-coated transgenes were compared for transgene integration and embryo survival with their non-coated counterparts in both single- and double-stranded forms. Murine zygotes were microinjected with a large 30 kb αS1-casein/human lysozyme DNA construct and a small 5.5 kb β-lactoglobulin/desaturase DNA construct using four different construct preparations for each gene. The preparations included recombinase A protein-coated, single- and double-stranded DNA constructs and non-coated, single- and double-stranded DNA constructs. Using conventional non-coated, double-stranded DNA constructs, we obtained a transgene integration efficiency of 1.5% (1352 embryos transferred produced 20 transgenic pups). The same double-stranded DNA constructs coated with recombinase A protein yielded a similar percentage of transgene integration (1.1%, 18/1697). Using single-stranded DNA, non-coated constructs produced a transgene integration rate of 0.5%, while none of the 1040 zygotes injected with recombinase A-coated constructs produced transgenic pups. While recombinase A protein coating produced no effect on embryo survival, litter size or pregnancy rate with double-stranded constructs, a detrimental effect was observed on embryo survival (P < 0.001) and pregnancy rate (P < 0.005) with recombinase A protein coating of single-stranded human lysozyme DNA constructs. A trend toward increased embryo survival (P = 0.054) with no difference in pregnancy rate (P > 0.05) was observed with the recombinase A protein coating of single-stranded desaturase constructs. These results suggest that recombinase A protein coating of single- and double-stranded DNA constructs produced no significant differences (P > 0.05) in the efficiency of generating transgenic mice with respect to the percentage of transgenic animals born. 相似文献
17.
D. Mingeot J. M. Jacquemin 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,98(6-7):1132-1137
Wheat anonymous probes were selected for their efficiency for providing a readable hybridization pattern and revealing RFLP
among wheat varieties. We report the mapping of 132 such probes (20 wheat-leaf cDNA, 28 wheat-root cDNA and 84 genomic DNA)
on the reference population of the International Triticeae Mapping Initiative (ITMI) derived from the cross W-7984 with Opata85.
Each probe has been characterized for its polymorphism information content. The 132 probes allowed us to map 160 loci.
Received: 7 July 1998 / Accepted: 19 October 1998 相似文献
18.
Candidate gene analysis of quantitative disease resistance in wheat 总被引:16,自引:0,他引:16
J. D. Faris W. L. Li D. J. Liu P. D. Chen B. S. Gill 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,98(2):219-225
Knowledge of the biological significance underlying quantitative trait loci (QTLs) for disease resistance is generally limited.
In recent years, advances in plant-microbe interactions and genome mapping have lead to an increased understanding of the
genes involved in plant defense and quantitative disease resistance. Here, we report on the application of the candidate-gene
approach to the mapping of QTLs for disease resistance in a population of wheat recombinant inbreds. Over 50 loci, representing
several classes of defense response (DR) genes, were placed on an existing linkage map and the genome was surveyed for QTLs
associated with resistance to several diseases including tan spot, leaf rust, Karnal bunt, and stem rust. Analysis revealed
QTLs with large effects in regions of putative resistance (R) genes, as previously reported. Several candidate genes, including
oxalate oxidase, peroxidase, superoxide dismutase, chitinase and thaumatin, mapped within previously identified resistance
QTLs and explained a greater amount of the phenotypic variation. A cluster of closely linked DR genes on the long arm of chromosome
7B, which included genes for catalase, chitinase, thaumatins and an ion channel regulator, had major effects for resistance
to leaf rust of adult plants under conditions of natural infestation. The results of this study indicate that many minor resistance
QTLs may be from the action of DR genes, and that the candidate-gene approach can be an efficient method of QTL identification.
Received: 12 June 1998 / Accepted: 24 July 1998 相似文献
19.
We report a large-scale study on the frequency of transgene and T-DNA backbone integration following Agrobacterium-mediated transformation of immature barley embryos. One hundred and ninety-one plant lines were regenerated after hygromycin selection and visual selection for GFP expression at the callus stage. Southern blotting performed on a subset of 53 lines that were PCR positive for the GFP gene documented the integration of the GFP gene in 27 of the lines. Twenty-three of these lines expressed GFP in T1 plantlets. Southern blotting with a vector backbone probe revealed that 13 of the 27 lines possessed one or more vector backbone fragments illustrating the regular occurrence of vector backbone integration following Agrobacterium infection of barley immature embryos. 相似文献
20.
Factors that influence Agrobacterium rhizogenes-mediated transformation of broccoli (Brassica oleracea L. var. italica) 总被引:2,自引:0,他引:2
An improved broccoli transformation system was developed by optimising several factors that affect the rate of effective
Agrobacterium-mediated transformation. Leaf explants of cultivar Shogun were co-cultivated with Agrobacterium rhizogenes strain A4T harbouring the binary vector pART278. The T-DNA of this binary vector contains a neomycin phosphotransferase II
(NOS-NPTII-NOS) gene for kanamycin resistance and a β-glucuronidase (35S-GUS-OCS) gene. Several media and factors were evaluated
including combinations of arginine, mannopine, acetosyringone and the use of feeder cell layers. The new protocol includes
the use of 200 μm acetosyringone in LB medium for bacterial growth, the use of a Brassica campestris feeder cell layer, 10 mm mannopine and 50 μm acetosyringone in the co-cultivation medium and 1 mm arginine in the selection medium. The use of this optimised protocol produced transformation rates of 33% in preliminary
experiments transforming broccoli with the antisense 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene from pTOM13.
Received: 2 July 1998 / Revision received: 9 February 2000 / Accepted: 17 February 2000 相似文献