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1.
Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression
of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene
expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene ( uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why
chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs
on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs
do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results
show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have
an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene
expression in A. thaliana wild-type plants and suppressed gene silencing ( sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression
in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing.
Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.
Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work 相似文献
2.
The rice (Oryza sativa L.) BAHD acyltransferase gene OsAt10 affects growth and metabolism of cells and regulates cell response to environmental stress. However, influence of the OsAt10 gene on low-temperature stress tolerance has not been evaluated in plant cells. Here, cell suspension cultures of plant species Arabidopsis (Arabidopsis thaliana L.), cotton (Gossypium hirsutum L.), white pine (Pinus strobus L.), and rice (Oryza sativa L.) were used to generate transgenic cell lines via Agrobacterium tumefaciens-mediated genetic transformation to examine the effects of OsAt10 on cold stress tolerance. OsAt10 transgenic cell lines of A. thaliana, G. hirsutum, P. strobus, and O. sativa were confirmed by molecular analyses including Southern blotting ND northern blotting, following by physiological and biochemical analyses under cold stress. The experimental results demonstrated that growth rate, cell viability, lipid peroxidation, ion leakage, antioxidative enzyme activity, polyamines level, and cell morphology were changed in transgenic cells under cold stress, compared to the controls. In transgenic A. thaliana cells, overexpression of the OsAt10 gene increases expression of polyamines biosynthesis genes under cold stress. In transgenic A. thaliana plants, overexpression of the OsAt10 gene increased cold stress tolerance by regulating expression of stress marker genes, TBARS content, ion leakage level, antioxidant enzymes activity, and polyamines content, indicating that the OsAt10 gene could be economically important for improving low-temperature stress tolerance in plants. 相似文献
3.
Molecular genetic studies on the model plant Arabidopsis thaliana often involve multiple rounds of Agrobacterium-mediated transformation. Such procedures require multiple marker genes that would allow for efficient selection of transgenic
plants in each cycle of transformation. Here, we report on a selection marker cassette based on a codon-modified glyphosate
N-acetyltransferase ( GAT) gene whose expression is driven by a powerful EL2Ω promoter. After introduction of the GAT expression cassette into A. thaliana via Agrobacterium-mediated transformation, glyphosate-resistant primary transformants are efficiently selected by glyphosate, either added to
the culture medium or by spraying a glyphosate solution onto seedlings grown in soil. Robust glyphosate-resistant phenotypes
are always associated with the presence of the GAT cassette. In addition, RT-PCR analysis of T 2 transformants has demonstrated that resistance to glyphosate is associated with higher levels of GAT expression. Resistance conferred by GAT is specific to glyphosate and not to other commonly used selection chemical compounds. These results demonstrate the versatility
of the GAT cassette suitable for both large-scale, soil-based selection system of transgenic plants as well as their characterization
in vitro. 相似文献
4.
Expansins are non-enzymatic plant proteins breaking hydrogen bonds between cellulose microfibrils and hemicellulose polymer
matrix. Each plant has many expansin genes, whose protein products participate in the regulation of plant growth and development
mainly by regulating cell expansion. To analyze the effects of elevated expansin expression on the plant organ sizes, we cloned
the AtEXPA10 gene from Arabidopsis thaliana and PnEXPA1 gene from Populus nigra. Transgenic tobacco plants expressing the target genes were obtained. The obtained transgenic tobacco plants were shown to
have significantly larger leaves and longer stems compared to control plants. The flowers were quite insignificantly larger,
but at the same time transgenic plants had more flowers. The microscopic studies showed that the organs of AtEXPA10-carrying plants were larger mainly due to stimulated cell proliferation, whereas the overexpression of the PnEXPA1 gene activated cell expansion. 相似文献
5.
以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础. 相似文献
6.
Human beta-defensin-2 (hBD-2) is a small antimicrobial peptide with potent activity against different Gram-negative bacteria
and fungal/yeast species. Since human beta-defensins and plant defensins share structural homology, we set out to analyse
whether there also exists a functional homology between these defensins of different eukaryotic kingdoms. To this end, we
constructed a plant transformation vector harbouring the hBD-2 coding sequence, which we transformed to Arabidopsis thaliana plants, giving rise to A. thaliana plants indeed expressing hBD-2. Furthermore, we could demonstrate that this heterologously produced hBD-2 possesses antifungal activity in vitro. Finally,
we could show that hBD-2 expressing A. thaliana plants are more resistant against the broad-spectrum fungal pathogen Botrytis cinerea as compared to untransformed A. thaliana plants, and that this resistance is correlated with the level of active hBD-2 produced in these transgenic plants. Hence,
we demonstrated a functional homology, next to the already known structural homology, between defensins originating from different
eukaryotic kingdoms. To our knowledge, this is the first time that this is specifically demonstrated for plant and mammalian
defensins. 相似文献
7.
The potential use of human P450-transgenic plants for phytoremediation of pesticide contaminated soils was tested in laboratory and greenhouse experiments. The transgenic P450 CYP1A2 gene Arabidopsis thaliana plants metabolize number of herbicides, insecticides and industrial chemicals. The P450 isozymes CYP1A2 expressed in A. thaliana were examined regarding the herbicide simazine (SIM). Transgenic A. thaliana plants expressing CYP1A2 gene showed significant resistance to SIM supplemented either in plant growth medium or sprayed on foliar parts. The results showed that SIM produces harmful effect on both rosette diameter and primary root length of the wild type (WT) plants. In transgenic A. thaliana lines, the rosette diameter and primary root length were not affected by SIM concentrations used in this experiment. The results indicate that CYP1A2 can be used as a selectable marker for plant transformation, allowing efficient selection of transgenic lines in growth medium and/or in soil-grown plants. The transgenic A. thaliana plants exhibited a healthy growth using doses of up to 250 μmol SIM treatments, while the non-transgenic A. thaliana plants were severely damaged with doses above 50 μmol SIM treatments. The transgenic A. thaliana plants can be used as phytoremediator of environmental SIM contaminants. 相似文献
8.
A gene encoding staphylokinase from Staphylococcus aureus was cloned into the plant transformation binary vector pCAMBIA 1304. The transgene was introduced into the genome of A. thaliana via in planta
Agrobacterium tumefaciens–mediated genetic transformation. The presence of the staphylokinase gene was confirmed by PCR in 60% of the investigated
plants. The presence of the fusion protein (119 kDa) was confirmed by SDS–PAGE and Western blot analysis in protein extracts
from putative transgenics. Furthermore, the amidolytic assay confirmed the activity of SAK in protein extracts in 23 out of
45 transgenic lines of A. thaliana plants. 相似文献
9.
An efficient gene transfer system without tissue culture steps was developed for kidney bean by using sonication and vacuum
infiltration assisted, Agrobacterium-mediated transformation. Transgenic kidney bean with a group 3 lea (late embryogenesis abundant) protein gene from Brassica napus was produced through this approach. Among 18 combinations of transformation methods, Agrobacterium-mediated transformation combined with 5 min sonication and 5 min vacuum infiltration turned to be optimal, resulting in the
highest transformation efficiency. Transgenic kidney bean plants demonstrated enhanced growth ability under salt and water
deficit stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by
drought stress. Transgenic lines with high level of lea gene expression showed higher stress tolerance than lines with lower expression level. Stress tolerance of transgenic kidney
bean correlated much better with lea gene expression levels than with gene integration results. There is no prior report on the production of transgenic kidney
bean using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation. 相似文献
10.
Gibberellins (GAs) are endogenous hormones that play an important role in regulating plant stature by increasing cell division
and promoting seed germination. The GA2-oxidase gene from Arabidopsis thaliana ( AtGA2ox8) was introduced into Brassica napus L. by Agrobacterium-mediated floral-dip transformation with the aim of decreasing the amount of bioactive GA and hence reduced the plant height.
As anticipated, the transgenic plant exhibited dwarf phenotype. Importantly, compared with the wild type, the transgenic plants
had delayed the seed germination, increased the chlorophyll content (28.7–36.3%) and photosynthesis capacity (14.3–18.7%)
in a single leaf. At the same time, the photosynthesis capacity of the whole plants was significantly enhanced (35.7–48.6%)
due to the extra leaves and branches. 相似文献
11.
该研究利用海岛棉‘新海21’和陆地棉ND203以及模式植物拟南芥,通过转基因及荧光定量检测等方法探究海岛棉 GbHCT13基因(GenBank 登录号MW048849)在纤维发育中的功能。结果显示:(1)成功构建重组载体pCAMBIA3301 GbHCT13,经农杆菌介导法转化、除草剂抗性基因筛选、荧光定量检测方法鉴定获得转 GbHCT13基因拟南芥T 3代植株4株;qRT PCR检测表明,转基因植株中 GbHCT13基因表达量较野生型极显著增加。(2)转基因拟南芥过表达 GbHCT13基因使植株同一时期的生长较野生型旺盛,株形、叶片数、抽薹数和茎秆表皮毛数量均与野生型存在差异;组织化学分析发现,转 GbHCT13基因的拟南芥较野生型茎秆初生木质部生长活跃,导管增粗,次生木质部导管细胞壁横截面积变大,但髓质细胞无明显变化;过表达 GbHCT13使拟南芥中木质素合成途径基因发生不同程度改变,其中 CAD、 CCoAOMT、 PAL和4 CL与 GbHCT13基因的表达呈正相关。(3)经大田筛选、分子鉴定,成功获得转 GbHCT13基因棉花植株3株;转 GbHCT13基因棉花的棉纤维伸长率增加,纤维强度增大;沉默 GbHCT13基因使棉花植株木质素含量降低,茎秆表皮毛数量减少,木质部导管细胞数量减少,导管细胞壁中木质素沉积量降低,而棉株并未发生株高上的明显矮化现象,且木质素合成通路中的 CAD、 CCoAOMT、 CCR、 PAL 4个基因的表达均呈降低趋势,说明抑制 GbHCT13使得棉花生长代谢受阻,影响纤维发育起始。研究表明, GbHCT13基因能影响棉花植株中木质素合成从而调控纤维的生长发育,其功能与 GbHCT13基因在模式植物拟南芥中的基本一致。 相似文献
12.
Christolea crassifolia HARDY: gene ( CcHRD) belongs to the AP2/ERF-like tanscritpion factor family, and overexpression of HRD gene has been proved to result in improved water use efficiency and enhanced drought resistance in multiple plant species. In the present study, we cloned the CcHRD gene from Christolea crassifolia, which shares 99.1% sequence similarity with the HRD gene from Arabidopsis thaliana. We generated transgenic tomato plants expressing CcHRD gene by agrobacterium-mediated genetic transformation. Our results revealed that the transgenic tomato plants showed a more developed root system and higher fruit yield than the wild-type plants. Furthermore, the leaf relative water content, chlorophyll content and Fv/Fm value in transgenic plants were significantly higher than the wild type, while the relative conductivity and MDA content of transgenic plant leaves were markedly lower than those of wild type under drought stress. We also observed that the major agronomic traits of transgenic tomato plants were improved under natural drought stress compared with those of the wild type. In summary, results in this transgenic study showed that the CcHRD gene could enhance the drought resistance in tomato, and also provided important information for the application of drought-responsive genes in improving crop plant resistance to abiotic stresses. 相似文献
13.
Chromosomal integration of multicopy transgene inserts in higher plants is often followed by loss of expression. We have analysed whether this inactivation can trigger repeat-induced point mutations (RIP) as has been observed in Neurospora crassa. We have previously characterized transgenic lines of Arabidopsis thaliana containing the hygromycin phosphotransferase ( hpt) gene either as a unique sequence in plants expressing the gene, or as multimeric, closely linked repeats in clones that were resistant to hygromycin directly after transformation but exhibited gene inactivation in the subsequent generation. At the sequence level, we have determined the mutation frequencies in the promoter and coding regions of active and inactive copies of transgene inserts after passage through three sexual generations. No RIP-like mutations were found in inactivated genes. Comparison of our data with those from Neurospora suggest that sequence divergence within plant repetitive DNA is either much slower than in Neurospora or is generated by a different mechanism. 相似文献
14.
A reproducible and efficient transformation system utilizing the nodal regions of embryonal axis of blackgram ( Vigna mungo L. Hepper) has been established via Agrobacterium tumefaciens. This is a report of genetic transformation of Vigna mungo for value addition of an agronomic trait, wherein the gene of interest, the glyoxalase I driven by a novel constitutive Cestrum yellow leaf curling viral promoter has been transferred for alleviating salt stress. The overexpression of this gene under
the constitutive CaMV 35S promoter had earlier been shown to impart salt, heavy metal and drought stress tolerance in the
model plant, tobacco. Molecular analyses of four independent transgenic lines performed by PCR, Southern and western blot
revealed the stable integration of the transgene in the progeny. The transformation frequency was ca. 2.25% and the time required
for the generation of transgenic plants was 10–11 weeks. Exposure of T1 transgenic plants as well as untransformed control
plants to salt stress (100 mM NaCl) revealed that the transgenic plants survived under salt stress and set seed whereas the
untransformed control plants failed to survive. The higher level of Glyoxalase I activity in transgenic lines was directly
correlated with their ability to withstand salt stress. To the best of our knowledge this is the only report of engineering
abiotic stress tolerance in blackgram.
Prasanna Bhomkar, Chandrama P. Upadhyay are contributed equally.
An erratum to this article can be found at 相似文献
15.
Two cultivars of potato ( Solanum tuberosum L.) were transformed with a barley antiporter gene HvNHX2 driven by the CaMV 35S promoter. The expressed transgene conferred a higher NaCl tolerance to one of the cultivars. Under
salt stress, the more salt-tolerant transgenic plants had longer roots, higher dry weight, and suppressed cell expansion as
compared to wild-type plants. The salt tolerance of the plants grown in vitro was not accompanied by elevated total sodium
in any plant organs tested. Instead, higher potassium was found in roots of transgenic plants. Possible mechanisms of plant
salt tolerance are discussed. 相似文献
16.
A vector was constructed for the isolation of gene fusions to the lacZ reporter gene following T-DNA integration into the genome of Arabidopsis thaliana. To facilitate the generation of tagged A. thaliana plants, we established a modified method for high-frequency transformation of A. thaliana by Agrobacterium tumefaciens. The main modification required was to inhibit the methylation of T-DNA in the transformed calli. Apparently, cytosine residues of the nos-nptII gene used as a selectable marker were methylated, and the expression of this gene was suppressed. Treatment of the calli with the cytosine methylation inhibitor 5-azacytidine led to a dramatic increase (from 3% to 96%) in the regeneration of transformed (kanamycin-resistant) shoots. A total of 150 transgenic plants were isolated, and in 17 of these expression of the lacZ reporter was detected by in situ staining. The T-DNA insert together with flanking plant DNA sequences was cloned into Escherichia coli by plasmid rescue from some of the T 3 transformants that harbored one copy of the integrated T-DNA. Comparison of the rescued DNA with the corresponding DNA of the transgenic plant showed that most of the rescued plasmids had undergone rearrangements. These rearrangements could be totally avoided if an mcrAB (modified cytosine restriction) mutant of E. coli was used as the recipient in plasmid rescue. 相似文献
17.
Since past three decades new discoveries in plant genetic engineering have shown remarkable potentials for crop improvement.
Agrobacterium Ti plasmid based DNA transfer is no longer the only efficient way of introducing agronomically important genes into plants.
Recent studies have explored a novel plant genetic engineering tool, Rhizobia sp ., as an alternative to Agrobacterium, thereby expanding the choice of bacterial species in agricultural plant biotechnology. Rhizobia sp. serve as an open license source with no major restrictions in plant biotechnology and help broaden the spectrum for plant
biotechnologists with respect to the use of gene transfer vehicles in plants. New efficient transgenic plants can be produced
by transferring genes of interest using binary vector carrying Rhizobia sp. Studies focusing on the interactions of Rhizobia sp. with their hosts, for stable and transient transformation and expression of genes, could help in the development of an
adequate gene transfer vehicle. Along with being biologically beneficial, it may also bring a new means for fast economic
development of transgenic plants, thus giving rise to a new era in plant biotechnology, viz. “ Rhizobia mediated transformation technology.” 相似文献
18.
The auxin-inducible gene ARGOS from Arabidopsis thaliana is expressed in growing tissues and controls the plant organ size by regulating cell proliferation and meristematic competence.
The promoter of the dahlia ( Dahlia pinnata Cav.) mosaic virus (DMV) resembles the well-known cauliflower mosaic virus 35S promoter but shows a higher activity in transgenic
tobacco plants ( Nicotiana tabacum L.). We obtained transgenic tobacco plants expressing the Arabidopsis ARGOS gene under the control of the DMV promoter. Several of the T0 generation plants exhibited an accelerated transition to flowering,
a slight increase in flower size, and a significant increase in the leaf size. The T1 transgenic plants were characterized
by faster growth, the increased leaf size, and somewhat enlarged flowers as compared with control plants. These phenotypic
traits, as well as stability and inheritance of the transgene were demonstrated also in T2 transgenic plants. 相似文献
19.
Plant secondary metabolites, including pharmaceuticals, flavorings and aromas, are often produced in response to stress. We
used chemical inducers of the pathogen defense response (jasmonic acid, salicylate, killed fungi, oligosaccharides and the
fungal elicitor protein, cryptogein) to increase metabolite and biomass production in transformed root cultures of the medicinal
plant, Withania somnifera, and the weed, Convolvulus sepium. In an effort to genetically mimic the observed effects of cryptogein, we employed Agrobacterium rhizogenes to insert a synthetic gene encoding cryptogein into the roots of C. sepium, W. somnifera and Tylophora tanakae. This genetic transformation was associated with stimulation in both secondary metabolite production and growth in the first
two species, and in growth in the third. In whole plants of Convolvulus arvensis and Arabidopsis thaliana, transformation with the cryptogein gene led, respectively, to increases in the calystegines and certain flavonoids. A similar
transgenic mimicry of pathogen attack was previously employed to stimulate resistance to the pathogen and abiotic stress.
In the present study of biochemical phenotype, we show that transgenic mimicry is correlated with increased secondary metabolite
production in transformed root cultures and whole plants. We propose that natural transformation with genes encoding the production
of microbial elicitors could influence interactions between plants and other organisms. 相似文献
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