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1.
A protocol has been developed to produce a cholera toxin B subunit (CTB) in tobacco tolerant to the herbicide phosphinothricin
(PPT) by means of in vitro selection. The synthetic CTB subunit gene was altered to modify the codon usage to that of tobacco
plant genes. The gene was then cloned into a plant expression vector and was under the control of the ubiquitin promoter and
transformed into tobacco plants by Agrobacterium-mediated transformation. Transgenic plantlets were selected in a medium supplemented with 5 mg/L PPT. Polymerase chain reaction
analysis confirmed stable integration of the synthetic CTB gene into a chromosomal DNA. A high level of CTB (1.8% of total
soluble protein) was expressed in transgenic plants, which was 18-fold higher than that under the control of the expressed
CaMV 35S promoter with native gene. The transgenic plants when transferred to a greenhouse proved to be resistant to 2% PPT. 相似文献
2.
Hye Jin Choi Thummala Chandrasekhar Hyo-Yeon Lee Kyung-Moon Kim 《Plant Cell, Tissue and Organ Culture》2007,91(3):235-242
Transgenic herbicide-resistant sweet potato plants [Ipomoea batatas (L.) Lam.] were produced through Agrobacterium-mediated transformation system. Embryogenic calli derived from shoot apical meristems were infected with Agrobacterium tumefaciens strain EHA105 harboring the pCAMBIA3301 vector containing the bar gene encoding phosphinothricin N-acetyltransferase (PAT) and the gusA gene encoding β-glucuronidase (GUS). The PPT-resistant calli and plants were selected with 5 and 2.5 mg l−1 PPT, respectively. Soil-grown plants were obtained 28–36 weeks after Agrobacterium-mediated transformation. Genetic transformation of the regenerated plants growing under selection was demonstrated by PCR,
and Southern blot analysis revealed that one to three copies of the transgene were integrated into the plant genome of each
transgenic plant. Expression of the bar gene in transgenic plants was confirmed by RT-PCR and application of herbicide. Transgenic plants sprayed with Basta containing
900 mg l−1 of glufosinate ammonium remained green and healthy. The transformation frequency was 2.8% determined by herbicide application
which was high when compared to our previous biolistic method. In addition, possible problems with multiple copies of transgene
were also discussed. We therefore report here a successful and reliable Agrobacterium-mediated transformation of the bar gene conferring herbicide-resistance and this method may be useful for routine transformation and has the potential to develop
new varieties of sweet potato with several important genes for value-added traits such as enhanced tolerance to the herbicide
Basta. 相似文献
3.
Nguyen Hoang Loc Nguyen Van Song Nguyen Quang Duc Tien Tang Thuy Minh Phan Thi Quynh Nga Tae-Geum Kim Moon-Sik Yang 《Plant Cell, Tissue and Organ Culture》2011,105(1):39-45
A gene encoding the B subunit of the enterotoxigenic Escherichia coli heat-labile enterotoxin (LTB) was adapted to the optimized plant coding sequence, and fused to the endoplasmic reticulum
retention signal SEKDEL in order to enhance its expression level and protein assembly in plants. The synthetic LTB (sLTB)
gene was placed into a plant expression vector under the control of the CaMV 35S promoter, and subsequently introduced into
the watercress (Nasturtium officinale L.) plant by the Agrobacterium-mediated transformation method. The integration of the sLTB gene into the genomic DNA of transgenic plants was confirmed
by genomic DNA PCR amplification. The assembly of plant-produced LTB protein was detected by western blot analysis. The highest
amount of LTB protein produced in transgenic watercress leaf tissue was approximately 1.3% of the total soluble plant protein.
GM1-ganglioside enzyme-linked immunosorbent assay indicated that plant-synthesized LTB protein bound specifically to GM1-ganglioside, which is the receptor for biologically active LTB on the cell surface, suggesting that the plant-synthesized
LTB subunits formed biologically active pentamers. 相似文献
4.
Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment 总被引:5,自引:0,他引:5
Dai Shunhong Zheng Ping Marmey Philippe Zhang Shiping Tian Wenzhong Chen Shouyi Beachy Roger N. Fauquet Claude 《Molecular breeding : new strategies in plant improvement》2001,7(1):25-33
We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T0, T1 and T2 transgenic plants. Oryza
sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hygr), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T1 and T2 generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T1 lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression. 相似文献
5.
Overexpression of antifungal pathogenesis-related (PR) proteins in crop plants has the potential for enhancing resistance
against fungal pathogens. Thaumatin-like proteins (TLPs) are one group (PR-5, permatins) of antifungal PR-proteins isolated
from various plants. In the present study, a plasmid containing a cDNA of rice tlp (D34) under the control of the CaMV-35S promoter was introduced into tobacco plants through Agrobacterium-mediated transformation system. A considerable overproduction of TLP was observed in transformed tobacco plants by Western
blot analysis. There was a large accumulation of tlp mRNA in transgenic plants as revealed by Northern blot analysis. Southern blot analysis of the DNA from transgenic tobacco
plants confirmed the presence of the rice tlp gene in the genomic DNA of transgenic tobacco plants. Immunoblot analysis of intracellular and extracellular proteins of
transgenic tobacco leaves using a Pinto bean TLP antibody demonstrated that the 23-kDa TLP was secreted into the extracellular
matrix. T2 progeny of regenerated plants transformed with TLP gene were tested for their disease reaction to Alternaria alternata, the brown spot pathogen. Transgenic tobacco plants expressing TLP at high levels showed enhanced tolerance to necrotization
caused by the pathogen.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
6.
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
7.
Three types of transgenic tobacco plants were acquired by separate transformation or co-transformation of a vacuolar Na+/H+ antiporter gene, SeNHX1, and a betaine synthesis gene, BADH. When exposed to 200 mM NaCl, the dual gene-transformed plants displayed greater accumulation of betaine and Na+ than their wild-type counterparts. Photosynthetic rate and photosystem II activity in the transgenic plants were less affected
by salt stress than wild-type plants. Transgenic plants exhibited a greater increase in osmotic pressure than wild-type plants
when exposed to NaCl. More importantly, the dual gene transformed plants accumulated higher biomass than either of the single
transgenic plants under salt stress. Taken together, these findings indicate that simultaneous transformation of BADH and SeNHX1 genes into tobacco plants can enable plants to accumulate betaine and Na+, thus conferring them more tolerance to salinity than either of the single gene transformed plants or wild-type tobacco plants.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
8.
Fathi Hassan Jochen Meens Hans-Jrg Jacobsen Heiko Kiesecker 《Journal of biotechnology》2009,143(4):302-308
Embryo axes excised from mature seeds of pea (Pisum sativum L.) cv. ‘Sponsor’ were used as explants for Agrobacterium-mediated transformation using pGreenII 0229 binary vectors. The vectors harbored a chimeric chitinase gene (chit30), driven by the constitutive 35S promoter or the elicitor inducible stilbene synthase (vst) promoter from grape (Vitis vinifera L.). The secretion signal of the bacterial chitinase gene from Streptomyces olivaceoviridis ATCC 11238 (DSM 41433) was replaced by the A. thaliana basic chitinase leader sequence. Functional properties of the recombinant gene were tested in tobacco as a model system before the long process of pea transformation was undertaken. Several transgenic pea clones were obtained and the transgenic nature confirmed by different molecular methods. The accumulation and activity of chitinase in stably transformed plants were examined by Western blot analysis and in-gel assays, which showed the presence of an additional 3 isoform bands. Using in vitro bioassays with Trichoderma harzanium as a model, we found an inhibition or delay of hyphal extension, which might indicate enhanced antifungal activity compared with non-transformed pea plants. Up to the 4th generation, the transgenic plants did not show any phenotypic alterations compared with non-transgenic control plants. 相似文献
9.
Nacyra Assad-García Neftali Ochoa-Alejo Enrique García-Hernández Luis Herrera-Estrella June Simpson 《Plant cell reports》1992,11(11):558-562
A protocol for the Agrobacterium-mediated transformation of tomatillo was developed. Up to 40 transgenic plants could be obtained in experiments using 60 cotyledon expiants. The transformed nature of the regenerated plants was confirmed by NPT II and Southern blot hybridization analysis. Using the b-glucuronidase system the tissue specific and developmental patterns of expression of the Cauliflower Mosaic Virus 35S promoter were determined in transgenic tomatillo plants. It was found that this promoter is developmentally regulated during fruit and seed formation. 相似文献
10.
Hui-Juan Li Ai-Fang Yang Xue-Cheng Zhang Feng Gao Ju-Ren Zhang 《Plant Cell, Tissue and Organ Culture》2007,89(1):37-48
Sucrose: sucrose 1-fructosyltransferase (1-SST) cDNA from Lactuca sativa, coding the enzyme responsible for lower degree polymers fructan biosynthesis, was cloned by RT-PCR and RACE methods. The
1-SST cDNA under the control of CaMV 35S promoter was introduced into tobacco by Agrobacterium-mediated leaf disc transformation protocol. Fructan synthesis in vitro and carbohydrate analysis showed that sense transgenic
tobacco plant displayed sucrose: sucrose 1-fructosyltransferse activity. After freezing stress, significant increases in electrolyte
leakage and malondialdehyde were found in the wild type and anti-sense transgenic plants, while no apparent differences were
observed in sense transgenic plants. Meanwhile, water soluble carbohydrate, fructan and fructose of sense transgenic plants
remarkably increased, compared with those of wild type and anti-sense plants. No significant difference was detected in superoxide
dismutase activity between transgenic and wild type plants. The above results demonstrated that the expression of 1-SST gene improved the freezing resistance of transgenic tobacco plants. 相似文献
11.
R.L.R. Canche-Moo A. Ku-Gonzalez C. Burgeff V.M. Loyola-Vargas L.C. Rodríguez-Zapata E. Castaño 《Plant Cell, Tissue and Organ Culture》2006,84(3):373-377
Agrobacterium-mediated plant transformation protocol was evaluated as a fast method to obtain genetically modified Coffea canephora plantlets. Leaf explants were used as source material for Agrobacterium tumefaciens-mediated transformation involving a vacuum infiltration protocol, followed by a step of somatic embryogenesis induction and
a final selection of the transformed plants. A. tumefaciens strain C58CI containing the binary vector pER10W-35SRed was used. PCR amplification of DsRFP gene and visual detection of
the red fluorescent protein demonstrated 33% transformed embryos. The protocol presented here produces reliable transgenic
coffee embryos in two months. 相似文献
12.
Z. T. Li S. Dhekney M. Dutt M. van Aman J. Tattersall K. T. Kelley D. J. Gray 《In vitro cellular & developmental biology. Plant》2006,42(3):220-227
Summary A translational fusion between the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTH) genes
was used to optimize parameters influencing Agrobacterium-mediated transformation of Vitis vinifera L. cv. Thompson Seedless. The corresponding bifunctional protein produced from this EGFP/NPTH fusion gene allowed for a single
promoter to drive expression of both green fluorescence and kanamycin resistance, thus conserving promoter resources and climinating
potential promoter-promoter interactions. The fusion gene, driven by either a double cauliflower mosaic virus 35S (CaMV 35S)
promoter or a double cassava vein mosaic virus (CsVMV) promoter, was immobilized into Agrobacterium strain EHA 105. Somatic embryos capable of direct secondary embryogenesis were used as target tissues to recover transgenic
plants. Simultaneous visualization of GFP fluorescence and kanamycin selection of transgenic cells, tissues, somatic embryos,
and plants were achieved. GFP expression and recovery of embryogenic culture lines were used as indicators to optimize transformation
parameters. Preculturing of somatic embryos for 7 d on fresh medium prior to transformation minimized Agrobacterium-induced tissue browning/necrosis. Alternatively, browning/necrosis was reduced by adding 1 gl−1 of the antioxidant dithiothreitol (DTT) to post co-cultivation wash media. While combining preculture with antioxidant treatments
did not result in a synergistic improvement in response, either treatment resulted in recovery of more stable embryogenic
lines than did the control. A 48h co-cultivation period combined with 75 mgl−1 kanamycin in selection medium was optimal. DNA analysis confirmed stable integration of transgenes into the grape genome:
63% had single gene insertions, 27% had two inserts, and 7 and 3% had three and four inserts, respectively. Utilizing optimized
procedures, over 1400 stable independent transgenic embryogenic culture lines were obtained, of which 795 developed into whole
plants. Transgenic grapevines have exhibited normal vegetative morphology and stable transgene expression for over 5 yr. 相似文献
13.
14.
Based on the Cre/loxP system, we have developed a novel marker-free system mediating a direct auto-excision of loxP-flanked marker genes from T1 transgenic rice without any treatment or further offspring crossing. To achieve this, the floral-specific promoter OsMADS45 was isolated from rice and the expression patterns of OsMADS45 promoter was characterised by using the pOs45:GUS transgenic plants. Furthermore, the binary vector with Cre recombinase under the control of OsMADS45 promoter was constructed and introduced into rice by Agrobacterium-mediated transformation and transgenic rice plants were generated. Southern blot analysis showed that auto-excision of the
selective markers occurred in some T1 progeny of the transgenic plants, suggesting that a high auto-excision frequency can be achieved with our Cre/loxP system. This auto-excision strategy provides an efficient way of removing the selectable marker gene from transgenic rice.
Xianquan Bai and Qiuyun Wang contributed equally to the work. 相似文献
15.
In the present study, an efficient Agrobacterium-mediated gene transformation system was developed for soybean [Glycine max (L.) Merrill] based on the examinations of several factors affecting plant transformation efficiency. Increased transformation
efficiencies were obtained when the soybean cotyledonary node were inoculated with the Agrobacterium inoculum added with 0.02% (v/v) surfactant (Silwet L-77). The applications of Silwet L-77 (0.02%) during infection and l-cysteine (600 mg l−1) during co-cultivation resulted in more significantly improved transformation efficiency than each of the two factors alone.
The optimized temperature for infected explant co-cultivation was 22°C. Regenerated transgenic shoots were selected and produced
more efficiently with the modified selection scheme (initiation on shoot induction medium without hygromycin for 7 days, with
3 mg l−1 hygromycin for 10 days, 5 mg l−1 hygromycin for another 10 days, and elongation on shoot elongation medium with 8 mg l−1 hygromycin). Using the optimized system, we obtained 145 morphologically normal and fertile independent transgenic plants
in five important Chinese soybean varieties. The transformation efficacies ranged from 3.8 to 11.7%. Stable integration, expression
and inheritance of the transgenes were confirmed by molecular and genetic analysis. T1 plants were analyzed and transmission of transgenes to the T1 generation in a Mendelian fashion was verified. This optimized transformation system should be employed for efficient Agrobacterium-mediated soybean gene transformation. 相似文献
16.
Marcel J. A. de Groot Remko Offringa Jürgen Groet Mirjam P. Does Paul J. J. Hooykaas Peter J. M. van den Elzen 《Plant molecular biology》1994,25(4):721-733
Previously we have demonstrated gene targeting in plants after Agrobacterium-mediated transformation. In these initial experiments a transgenic tobacco line 104 containing a T-DNA insertion with a defective neomycin phosphotransferase (nptII) gene was transformed with a repair construct containing an otherwise defective nptII gene. Homologous recombination between the chromosomally located target and the incoming complementary defective nptII construct generated an intact nptII gene and led to a kanamycin-resistant (Kmr) phenotype. The gene targeting frequency was 1×10–5. In order to compare direct gene transfer and Agrobacterium-mediated transformation with respect to gene targeting we transformed the same transgenic tobacco line 104 via electroporation. A total of 1.35×108 protoplasts were transformed with the repair construct. Out of nearly 221 000 transformed cells 477 Kmr calli were selected. Screening the Kmr calli via PCR for recombination events revealed that in none of these calli gene targeting had occurred. To establish the origin of the high number of Kmr calli in which gene targeting had not occurred we analysed plants regenerated from 24 Kmr calli via PCR and sequence analysis. This revealed that in 21 out of 24 plants analysed the 5-deleted nptII gene was fused to the hygromycin phosphotransferase (hpt) gene that was also present on the repair construct. Sequence analysis of 7 hpt/nptII gene fusions showed that they all contained a continuous open reading frame. The absence of significant homology at the fusion site indicated that fusion occurred via a process of illegitimate recombination. Therefore, illegitimate recombination between an introduced defective gene and another gene present on the repair construct or the chromosome has to be taken into account as a standard byproduct in gene targeting experiments. 相似文献
17.
Meadow fescue (Festuca pratensis Huds.) is an important cool-season forage grass in Europe and Asia. We developed a protocol for producing meadow fescue transgenic
plants mediated by Agrobacterium tumefaciens transformation. Embryogenic calli derived from mature embryos were transformed with A. tumefaciens strain AGL1 carrying the binary vector pDM805, coding for the phosphinothricin acetyltransferase (bar) and β-glucuronidase (uidA) genes. Bialaphos was used as the selective agent throughout all phases of tissue culture. In total, 40 independent transgenic
plants were recovered from 45 bialaphos-resistant callus lines and an average transformation efficiency of 2% was achieved.
The time frame from infection of embryogenic calli with Agrobacterium to transferring the transgenic plants to the greenhouse was 18 weeks. In a study of 11 BASTA-resistant transgenic lines,
the uidA gene was expressed in 82% of the transgenic lines. Southern blot analysis revealed that 82% of the tested lines integrated
one or two copies of the uidA gene.
C. Gao and J. Liu contributed equally to the work. 相似文献
18.
In this study, floral spray and floral dip were used to replace the vacuum step in the Agrobacterium-mediated transformation of a superoxide dismutase (SOD) gene into Arabidopsis. The transgene was constructed by using a CaMV 35S promoter to drive a rice cytosolic CuZnSOD coding sequence in Arabidopsis. The transgene construct was developed in binary vectors and mobilized into Agrobacterium. When Arabidopsis plants started to initiate flower buds, the primary inflorescence shoots were removed and then transformed by floral spray or floral dip. More than 300 transgenic plants were generated to assess the feasibility of floral spray used in the in planta transformation. The result indicates that the floral spray method of Agrobacterium can achieve rates of in planta transformation comparable to the vacuum-infiltration and floral dip methods. The floral spray method opens up the possibility of in planta transformation of plant species which are too large for dipping or vacuum infiltration. 相似文献
19.
20.
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