共查询到20条相似文献,搜索用时 15 毫秒
1.
2.
Reduced variation in transgene expression from a binary vector with selectable markers at the right and left T-DNA borders 总被引:3,自引:0,他引:3
Madan K. Bhattacharyya Bruce A. Stermer Richard A. Dixon 《The Plant journal : for cell and molecular biology》1994,6(6):957-968
A new binary vector for Agrobacterium-mediated plant transformation was constructed, in which two selectable markers, for kanamycin and hygromycin resistance, were placed next to the right and left T-DNA borders, respectively, and a CaMV 35S promoter-driven β-glucuronidase (GUS) gene was placed between these markers as a reporter gene (transgene). Using double antibiotic selection, all transgenic tobacco plants carrying at least one intact copy of the T-DNA expressed the transgene, and this population exhibited reduced variability in transgene expression as compared with that obtained from the parent vector pBI121. Absence of the intact transgene was the major reason for transgenic plants with little or no transgene expression. Integration of truncated T-DNAs was also observed among transgenic plants that expressed the transgene and carried multiple T-DNA inserts. The copy number of fully integrated T-DNAs was positively associated with transgene expression levels in R0 plants and R1 progeny populations. Variability due to position effect was determined among 17 plants carrying a single T-DNA insert. The coefficient of variability among these plants was only 35.5%, indicating a minor role for position effects in causing transgene variability. The new binary vector reported here can therefore be used to obtain transgenic populations with reduced variability in transgene expression. 相似文献
3.
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
4.
Agrobacterium-mediated transformation of the commercially important citrus cultivar Washington navel orange 总被引:6,自引:0,他引:6
Transgenic Washington navel orange [Citrus sinensis (L.) Osbeck] plants were obtained using Agrobacterium-mediated transformation of seedling epicotyl tissue. An average of 45% (58 out of 128 segments) of the epicotyl segments
produced shoots expressing the β-glucuronidase (GUS)-intron reporter gene when using Agrobacterium strain C58 C1, compared to 29% (38 out of 128 segments) for EHA101-5 and 0% for LBA4404. Co-culture of 21-day-old Washington
navel epicotyl stem segments gave greater transformation efficiency than co-culture of 35- or 56-day-old stem segments. After
6 weeks, regenerated shoots were micro-grafted in vivo onto seedling rootstocks of Carrizo citrange. Stable integration of
the transgene sequence was confirmed by expression of the plant intron-containing GUS gene, PCR and Southern hybridization.
The apomictic (non-zygotic) state of the transgenic plants was confirmed by isoenzyme and random amplified polymorphic DNA
analyses. More than 50 transgenic plants have been obtained and are growing in the greenhouse.
Received: 14 April 1998 / Revision received: 9 June 1998 / Accepted: 8 July 1998 相似文献
5.
S. B. Preuss C.-Z. Jiang H.-K. Baik C. I. Kado A. B. Britt 《Molecular & general genetics : MGG》1999,261(4-5):623-626
Stable transformation of plants by Agrobacterium T-DNAs requires that the transgene insert into the host chromosome. Although most of the Agrobacterium Ti plasmid genes required for this process have been studied in depth, few plant-encoded factors have been identified, although
such factors, presumably DNA repair proteins, are widely presumed to exist. It has previously been suggested that the UVH1 gene product is required for stable T-DNA integration in Arabidopsis. Here we present evidence suggesting that uvh1 mutants are essentially wild type for T-DNA integration following inoculation via the vacuum-infiltration procedure.
Received: 23 June 1998 / Accepted: 21 February 1999 相似文献
6.
S. Mori E. Oka H. Umehara H. Kobayashi Y. Hoshi M. Kondo K. Ogata M. Nakano 《Biologia Plantarum》2008,52(3):513-516
Transgenic plants of Tricyrtis hirta carrying the intron-containing β-glucuronidase (GUS) gene under the control of the CaMV35S promoter have been cultivated
for two years. Four independent transgenic plants produced flowers 1–2 years after acclimatization, and all of them contained
one copy of the transgene as indicated by inverse polymerase chain reaction (PCR) analysis. All the four transgenic plants
showed stable expression of the gus gene in leaves, stems, roots, tepals, stamens and pistils as indicated by histochemical and fluorometric GUS assays, although
differences in the GUS activity were observed among different organs of each transgenic plant. No apparent gus gene silencing was observed in transgenic T. hirta plants even after two years of cultivation. 相似文献
7.
A broad exploration of a transgenic population of citrus: stability of gene expression and phenotype 总被引:12,自引:0,他引:12
M. Cervera J. A. Pina J. Juárez L. Navarro L. Peña 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2000,100(5):670-677
A collection of 70 transgenic citrus plants for the uidA and nptII genes have been maintained under screenhouse conditions over a period of 4–5 years. A detailed scanning of the plants allowed
us to detect four phenotypic off-type plants and a large variation of transgene integration and expression patterns among
the population. Off-type plants were analysed and characterised as nucellar tetraploids, probably originating from tetraploid
starting tissues rather than from somaclonal variation events. Transgene integration and expression analyses revealed that:
(1) a significant negative correlation was found between copy number and GUS activity; (2) rearrangements of the T-DNA inserts
did not imply low expression levels; and (3) stability of integration and expression of the transgenes was confirmed for all
the transformants grown under natural environmental conditions. These combined features validate transformation as a tool
for the genetic improvement of citrus.
Received: 11 January 1999 / Accepted: 19 January 1999 相似文献
8.
9.
M. Fladung 《Molecular & general genetics : MGG》1999,260(6):574-581
The stability of transgenes in the genome of transformed plants depends strongly on their correct physical integration into
the host genome as well as on flanking target DNA sequences. For long-lived species like trees, however, no information is
available so far concerning inactivation or loss of transgenes due to gene silencing or somatic genome rearrangement events.
In this study, four independently transformed 35S-rolC transgenic hybrid aspen plants (Populus tremula L. × tremuloides Michx.), each harbouring one copy of the transgene, were investigated during continuous growth in the greenhouse. In one
of these transgenic lines (Esch5:35S-rolC-##1) individuals frequently show phenotypic reversions, while in the remaining three lines (Esch5:35S-rolC-#3, -#5, -#16) the gene was essentially stable. Molecular analysis including PCR, Southern and Northern assays clearly showed
that the transgene had been lost in the revertant tissue of the unstable line. Sequencing of T-DNA right and left borders,
and flanking DNA regions, in all four transgenic aspen lines revealed no differences either in the type of flanking DNA (G-C
to A-T ratio) or with respect to the presence of enhancers or MAR (matrix associated repeats)-like structures. Primers located
within the left and right flanking regions in the three stable lines could be used to recover the target sites from the untransformed
plants. This was not possible, however, with the unstable line, indicating that at least one flanking sequence does not derive
from the plant target DNA but is of unknown origin. PCR using other primer pairs, and inverse PCR analysis, revealed an additional
truncated T-DNA copy of 1050 nucleotides adjacent to the left border of the complete copy in this line. Sequencing of this
truncated T-DNA revealed that it represented an inverted copy of part of the right half of the original construct. This special
feature would allow the inverted repeat to pair with right border sequences of the complete copy. This would explain the frequently
observed reversion resulting in transgene loss as due to intrachromosomal base-pairing leading to double-stranded loops of
single-stranded DNA during mitotic cell divisions.
Received: 9 June 1998 / Accepted: 6 October 1998 相似文献
10.
Zhai W Chen C Zhu X Chen X Zhang D Li X Zhu L 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2004,109(3):534-542
The genetic loci and phenotypic effects of the transgene Xa21, a bacterial blight (BB) resistance gene cloned from rice, were investigated in transgenic rice produced through an Agrobacterium-mediated transformation system. The flanking sequences of integrated T-DNAs were isolated from Xa21 transgenic rice lines using thermal asymmetric interlaced PCR. Based on the analysis of 24 T-DNA- Xa21 flanking sequences, T-DNA loci in rice could be classified into three types: the typical T-DNA integration with the definite left and right borders, the T-DNA integration linked with the adjacent vector backbone sequences and the T-DNA integration involved in a complicated recombination in the flanking sequences. The T-DNA integration in rice was similar to that in dicotyledonous genomes but was significantly different from the integration produced through direct DNA transformation approaches. All three types of integrated transgene Xa21 could be stably inherited and expressed the BB resistance through derived generations in their respective transgenic lines. The flanking sequences of the typical T-DNA integration consisted of actual rice genomic DNA and could be used as probes to locate the transgene on the rice genetic map. A total of 15 different rice T-DNA flanking sequences were identified. They displayed restriction fragment length polymorphisms (RFLPs) between two rice varieties, ZYQ8 and JX17, and were mapped on rice chromosomes 1, 3, 4, 5, 7, 9, 10, 11 and 12, respectively, by using a double haploid population derived from a cross between ZYQ8 and JX17. The blast search and homology comparison of the rice T-DNA flanking sequences with the rice chromosome-anchored sequence database confirmed the RFLP mapping results. On the basis of genetic mapping of the T-DNA- Xa21 loci, the BB resistance effects of the transgene Xa21 at different chromosome locations were investigated using homozygous transgenic lines with only one copy of the transgene. Among the transgenic lines, no obvious position effects of the transgene Xa21 were observed. In addition, the BB resistance levels of the Xa21 transgenic plants with different transgene copy numbers and on different genetic backgrounds were also investigated. It was observed that genetic background (or genome) effects were more obvious than dosage effects and position effects on the BB resistance level of the transgenic plants. 相似文献
11.
High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures 总被引:6,自引:0,他引:6
K. Rajasekaran R. L. Hudspeth J. W. Cary D. M. Anderson T. E. Cleveland 《Plant cell reports》2000,19(6):539-545
Stable transformation of cotton (Gossypium hirsutum L.) at a high frequency has been obtained by particle bombardment of embryogenic cell suspension cultures. Transient and
stable expression of the β-glucuronidase (GUS) gene was monitored in cell suspension cultures. Transient expression, measured 48 h after bombardment,
was abundant, and stable expression was observed in over 4% of the transiently expressing cells. The high efficiency of stable
expression is due to the multiple bombardment of rapidly dividing cell suspension cultures and the selection for transformed
cells by gradually increasing the concentrations of the antibiotic Geneticin (G418). Southern analysis indicated a minimum
transgene copy number of one to four in randomly selected plants. Fertile plants were obtained from transformed cell cultures
less than 3 months old. However, transgenic and control plants from cell cultures older than 6 months produced plants with
abnormal morphology and a high degree of sterility.
Received: 20 January 1999 / Revision received: 1 October 1999 / Accepted: 11 October 1999 相似文献
12.
Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants, so
transgene copy number analysis is identified as one most important task after obtaining transgenic plants. In this paper,
TaqMan real-time PCR was used to estimate the copy number of exogenous MAC12.2 and NPTII genes in transgenic precocious trifoliate orange (Poncirus trifoliata [L.] Raf) in order to overcome the limitations of Southern blot analysis, which is labor-intensive, time-consuming, in considerable
needs of DNA, etc. We developed a real-time PCR assay which permitted the determination of the copy number of transgene (MAC12.2 and NPTII), relative to a conserved endogenous gene (PtLTP) in transgenic lines. R value is 0.92 by comparing the results to that of Southern blot analysis, indicating a strong correlation
coefficient between TaqMan real-time PCR assay and Southern blot method. 相似文献
13.
14.
We present a simple and rapid method for screening second-generation transgenic rice plants (T1) to identify homozygous plants. The plasmid (pfd11) used for rice transformation contains a partially deleted cytochrome
c gene (cyc) for comparing with the endogenous cyc for copy number. After polymerase chain reaction (PCR) amplification of a segment
of the cyc in transgenic rice DNA followed by agarose gel electrophoresis, two specific bands are obtained. The upper band
represents the endogenous cyc, and the lower band represents the partially deleted cyc in the transgene. The first-generation
plants (T0) that harbor a single copy of the transgene are selected based on the fact that the density of the lower band is half as
dense as the upper band. Next, only plants harboring a single copy of the transgene are advanced to the second generation
(T1). The same PCR procedure is used again, and homozygous T1 plants are easily identified from samples in which the intensity of the two bands is the same. 相似文献
15.
H. Silos-Espino A. Valdez-Ortiz Q. Rascón-Cruz E. Rodríguez-Salazar O. Paredes-López 《Plant Cell, Tissue and Organ Culture》2006,86(3):397-403
A system for genetic transformation of an elite prickly pear cactus (Opuntia ficus-indica L., cultivar Villa Nueva) by Agrobacterium tumefaciens was developed. Beginning with direct bacterial infection by using a hypodermic syringe to the meristematic tissue termed areoles, transgenic plants were obtained by selection with 100 mg l−1 kanamycin. Transient and stable GUS activities were monitored on kanamycin-resistant shoots and regenerated plants, respectively. Genetic transformation of regenerated plants growing under selection was demonstrated by PCR and Southern blot analysis; transgene copy number in the genome of transgenic plants ranged from two to six, while the transformation frequency obtained by the system reported here was of 3.2%. This method may be useful for routine transformation and introduction of several important genes in prickly pear cactus. 相似文献
16.
Marker gene elimination from transgenic barley,using co-transformation with adjacent `twin T-DNAs' on a standard Agrobacterium transformation vector 总被引:14,自引:0,他引:14
Matthews Peter R. Wang Ming-Bo Waterhouse Peter M. Thornton Sarah Fieg Sarah J. Gubler Frank Jacobsen John V. 《Molecular breeding : new strategies in plant improvement》2001,7(3):195-202
We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes. 相似文献
17.
18.
Effect of ploidy and homozygosity on transgene expression in primary tobacco transformants and their androgenetic progenies 总被引:5,自引:0,他引:5
A. Beaujean R. S. Sangwan M. Hodges B. S. Sangwan-Norreel 《Molecular & general genetics : MGG》1998,260(4):362-371
Expression of a transgene is rarely analysed in the androgenetic progenies of the transgenic plants. Here, we report differential
transgene expression in androgenetic haploid and doubled haploid (DH) tobacco plants as compared to the diploid parental lines,
thus demonstrating a gene dosage effect. Using Agrobacterium-mediated transformation, and bacterial reporter genes encoding neomycin phosphotransferase (nptII) and β-glucuronidase (uidA/ GUS), driven respectively by the mas 1′ and mas 2′ promoters, we have generated more than 150 independent transgenic (R0) Nicotiana tabacum plants containing one or more T-DNA copies. Transgene analyses of these R0, their selfed R1 lines and their corresponding haploid progenies showed an obvious position effect (site of T-DNA insertion on chromosome)
on uidA expression. However, transgene (GUS) expression levels were not proportional to transgene copy number. More than 150 haploids
and doubled haploids, induced by treatment with colchicine, were produced from 20 independent transgenic R0 plants containing single and multiple copies of the uidA gene. We observed that homozygous DH plants expressed GUS at approximately 2.9-fold the level of the corresponding parental
haploid plants. This increase in transgene expression may be attributed mainly to the increase (2-fold) in chromosome number.
Based on this observation, we suggest a strong link between chromosome number (ploidy dosage effect) and transgene expression.
In particular, we demonstrate the effect on its expression level of converting the transgene from the heterozygous (in R0 plants) to the homozygous (DH) state: e.g. an increase of 50% was observed in the homozygous DH as compared to the original
heterozygous diploid plants. We propose that ploidy coupled with homozygosity can result in a new type of gene activation,
creating differences in gene expression patterns.
Received: 27 April 1998 / Accepted: 12 August 1998 相似文献
19.
K. M. Doshi F. Eudes A. Laroche D. Gaudet 《In vitro cellular & developmental biology. Plant》2007,43(5):429-435
Wheat and triticale plants were transformed by bombardment of isolated scutella with a genetic construct consisting of the
two anthocyanin biosynthesis regulatory genes, C1 and Bperu, each under the control of the Ltp1 embryo-specific promoter. Transgenic plants were obtained in the absence of selective pressure and selectable marker gene
at a transformation frequency of 0.93% and 1.55% in triticale and wheat, respectively. Initial screening of T0 lines was performed by polymerase chain reaction (PCR), and further confirmation of PCR positives was done using real-time
PCR and by phenotypic observation. In this study, quantitative real-time PCR (qRT-PCR) was developed to determine the transgene
copy number in transgenic wheat and triticale. A conserved wheat housekeeping gene, puroindoline-b, was used as an internal control to calculate the transgene copy number in wheat and the SYBR green detection method with
a standard curve, constructed on the basis of serially diluted plasmid, was used to calculate the transgene copy in triticale.
Estimated transgene copies varied from 3 to 8 in wheat and 4 to 7 in triticale lines. The presence of anthocyanin regulatory
genes, promoter, and termination sequences was detected in six wheat lines and four triticale lines. However, anthocyanin-pigmented
embryos were only observed visually in mature T1 seeds of two transgenic wheat lines and a single triticale line. Multisite insertion and reorganization of transgenes was
likely the explanation for the failure of expression for the anthocyanin genes in the remaining wheat and triticale transgenic
lines. 相似文献
20.
The trait for somatic embryogenesis is being introduced sexually into alfalfa (Medicago sativa) breeding populations to facilitate genetic transformation of this crop. Cocultivation experiments were conducted with an agronomically-improved embryogenic clone from one such population as well as with two other embryogenic clones, one of which was the source of the embryogenic trait in the breeding populations. Transgenic plants were produced from the agronomically-improved clone whereas none were produced from the other two clones. Among the 16 transgenic plants analyzed there was a range in both copy number and number of integration sites for the NPT-II gene; those plants regenerated after a prolonged selection phase in vitro generally had the highest numbers in both respects. There was no evidence of sectoral chimerism of the transgene in a subsample of transgenic plants analyzed by PCR. 相似文献