共查询到20条相似文献,搜索用时 15 毫秒
1.
Selma Gulbitti-Onarici Mohsin Abbas Zaidi Ibrahim Taga Sebahattin Ozcan Illimar Altosaar 《Molecular biotechnology》2009,42(3):341-349
Expression of cry1Ac gene from Bacillus
thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1
promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic
plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of
Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance
against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they
were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded
leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without
pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay. 相似文献
2.
Abranches R Arcalis E Marcel S Altmann F Ribeiro-Pedro M Rodriguez J Stoger E 《Planta》2008,227(3):649-658
A number of recent reports suggest that the functional specialization of plant cells in storage organs can influence subcellular
protein sorting, so that the fate of a recombinant protein tends to differ between seeds and leaves. In order to test the
general applicability of this hypothesis, we investigated the fate of a model recombinant glycoprotein in the leaves and seeds
of a leguminous plant, Medicago truncatula. Detailed analysis of immature seeds by immunofluorescence and electron microscopy showed that recombinant phytase carrying
a signal peptide for entry into the endoplasmic reticulum was efficiently secreted from storage cotyledon cells. A second
version of the protein carrying a C-terminal KDEL tag for retention in the endoplasmic reticulum was predominantly retained
in the ER of seed cotyledon cells, but some of the protein was secreted to the apoplast and some was deposited in storage
vacuoles. Importantly, the fate of the recombinant protein in the leaves was nearly identical to that in the seeds from the
same plant. This shows that in M. truncatula, the unanticipated partial vacuolar delivery and secretion is not a special feature of seed cotyledon tissue, but are conserved
in different specialized tissues. Further investigation revealed that the unexpected fate of the tagged variant of phytase
likely resulted from partial loss of the KDEL tag in both leaves and seeds. Our results indicate that the previously observed
aberrant deposition of recombinant proteins into storage organelles of seed tissue is not a general reflection of functional
specialization, but also depends on the species of plant under investigation. This discovery will have an impact on the production
of recombinant pharmaceutical proteins in plants.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
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The B subunit of Escherichia coli heat-labile enterotoxin (LTB) has been transformed to plants for use as an edible vaccine. We have developed a simple and reliable Agrobacterium-mediated transformation method to express synthetic LTB gene in N. tabacum using a phosphinothricin acetyltransferase (bar) gene as a selectable marker. The synthetic LTB gene adapted to the coding sequence of tobacco plants was cloned to a plant expression vector under the control of the ubiquitin promoter and transformed to tobacco by Agrobacterium-mediated transformation. Transgenic plants were selected in the medium supplemented with 5 mg l-1 phosphinothricin (PPT). The amount of LTB protein detected in the transgenic tobacco was approximately 3.3% of the total soluble protein, approximately 300-fold higher than in the plants generated using the native LTB gene under the control of the CaMV 35S promoter. The transgenic plants that were transferred to a greenhouse had harvested seeds that proved to be resistant to herbicide. Thus, the described protocol could provide a useful tool for the transformation of tobacco plants. 相似文献
5.
Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity. 相似文献
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Fernando Gallardo Myroslawa Miginiac-Maslow Rajbir S. Sangwan Paulette Decottignies Eliane Keryer François Dubois Evelyne Bismuth Susana Galvez Brigitte Sangwan-Norreel Pierre Gadal Claude Crétin 《Planta》1995,197(2):324-332
Chloroplastic NADP+-malate dehydrogenase (cpMDH, EC 1.1.1.82) is a key enzyme in the carbonfixation pathway of some C4 plants such as the monocotyledons maize or Sorghum. We have expressed cpMDH from Sorghum vulgare Pers. in transgenic tobacco (Nicotiana tabacum L.) (a dicotyledonous C3 plant) by using a gene composed of the Sorghum cpMDH cDNA under the control of cauliflower mosaic virus 35S promoter. High steady-state levels of cpMDH mRNA were observed in isogenic dihaploid transgenic tobacco lines. Sorghum cpMDH protein was detected in transgenic leaf extracts, where a threefold higher cpMDH activity could be measured, compared with control tobacco leaves. The recombinant protein was identical in molecular mass and in N-terminal sequence to Sorghum cpMDH. The tobacco cpMDH protein which has a distinct N-terminal sequence, could not be detected in transgenic plants. Immunocytochemical analyses showed that Sorghum cpMDH was specifically localized in transgenic tobacco chloroplasts. These data indicate that Sorghum cpMDH preprotein was efficiently synthesized, transported into and processed in tobacco chloroplasts. Thus, C3-C4 photosynthesis specialization or monocotyledon-dicotyledon evolution did not affect the chloroplastic proteinimport machinery. The higher levels of cpMDH in transgenic leaves resulted in an increase of l-malate content, suggesting that carbon metabolism was altered by the expression of the Sorghum enzyme. 相似文献
8.
Cao Jun Shelton Anthony M. Earle Elizabeth D. 《Molecular breeding : new strategies in plant improvement》2001,8(3):207-216
We produced 49 broccoli plants (Brassica oleracea L. ssp. italica) containing a Bacillus thuringiensis cry1Ab gene under control of the chemically inducible PR-1a promoter from tobacco. Most of them showed substantial or complete control of neonate diamondback moth larvae, regardless of whether the transgene was induced or not. Ten plants were selected for detailed study via northern and western analysis and insect bioassays. They expressed the cry1Ab gene and gave complete insect control when treated with the chemical inducers INA (2,6-dichloroiso-nicotinic acid) or BTH (1,2,3-benzothiadiazole-7-carbothioic acid S-methyl ester); however, leaves treated with water alone were also partially or completely protected from insect damage. Transgenic progeny plants showed greater inducibility than primary transformants at the molecular level. Two progeny lines produced cry1Ab mRNA and Cry1Ab protein and gave insect control only after induction, both when detached leaves and intact plants were tested. The relevance of these results to resistance management strategies is discussed. 相似文献
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Isolde Saalbach Thomas Pickardt Frank Machemehl Gerhard Saalbach Otto Schieder Klaus Müntz 《Molecular & general genetics : MGG》1994,242(2):226-236
The coding region of the 2S albumin gene of Brazil nut (Bertholletia excelsa H.B.K.) was completely synthesized, placed under control of the cauliflower mosaic virus (CaMV) 35S promoter and inserted into the binary vector plasmid pGSGLUC1, thus giving rise to pGSGLUC1-2S. This was used for transformation of tobacco (Nicotiana tabacum L. cv. Petit Havanna) and of the grain legume Vicia narbonensis L., mediated by the supervirulent Agrobacterium tumefaciens strain EHA 101. Putative transformants were selected by screening for neomycin phosphotransferase (NPT II) and -glucuronidase (GUS) activities. Transgenic plants were grown until flowering and fruiting occurred. The presence of the foreign gene was confirmed by Southern analysis. GUS activity was found in all organs of the regenerated transgenic tobacco and legume plants, including the seeds. In the legume, the highest expression levels of the CaMV 35S promoter-controlled 2S albumin gene were observed in leaves and roots. 2S albumin was localized in the vacuoles of leaf mesophyll cells of transgenic tobacco. The Brazil nut protein was present in the 2S fraction after gel filtration chromatography of the legume seed proteins and could be clearly identified by immunoblotting. Analysis of seeds from the R2 progenies of the legume and of transgenic tobacco plants revealed Mendelian inheritance of the foreign gene. Agrobacterium rhizogenes strain RifR 15834 harbouring the binary vector pGSGLUCl2S was also used to transform Pisum sativum L. and Vicia faba L. Hairy roots expressed the 2S albumin-specific gene. Several shoots were raised but they never completely rooted and no fertile plants were obtained from these transformants. 相似文献
11.
Daisuke Yamauchi Yoko Terasaki Takashi Okamoto Takao Minamikawa 《Plant molecular biology》1996,30(2):321-329
Cysteine endopeptidases, SH-EP from Vigna mungo and EP-C1 from Phaseolus vulgaris, act to degrade seed storage protein during seed germination. Using transgenic tobacco plants, expression of SH-EP and promoter activity of the EP-C1 gene were analyzed in transgenic tobacco plants. The promoters of the two genes in tobacco seeds showed germination-specific activation, although post-translational processing of SH-EP and regulatory regions of promoter of the gene for EP-C1 were found to differ between leguminous seeds and transgenic tobacco seeds. 相似文献
12.
Targeting and glycosylation of patatin the major potato tuber protein in leaves of transgenic tobacco 总被引:4,自引:0,他引:4
Patatin, the most abundant protein in the storage parenchyma cells of potato (Solanum tuberosum L.) tubers, is a vacuolar glycoprotein that consists of a number of closely related polypeptides and is encoded by a large gene family. To analyse the glycosylation pattern and the nature of the glycans on a single patatin polypeptide in a heterologous tissue we introduced a single chimaeric patatin gene into tobacco (Nicotiana tabacum L.) and studied its product in leaves. Patatin isolated from the leaves of transgenic tobacco plants is glycosylated at asparagine (Asn)60, and Asn90, but the third glycosylation site (Asn202) has no glycan. The two glycans are typical small complex glycans with xylose, fucose, mannose and N-acetylglucosamine in a ratio 1:1:3:2, the same ratio as found on patatin isolated from potato tubers. Expression of patatin in tobacco leaves was accompanied by the correct processing of the signal peptide, and the proper targeting of the glyco-protein to the vacuoles of mesophyll cells.Abbreviations Asn
asparagine
- ConA
concanavalin A
- EndoH
endoglycosidase H
- Fuc
fucose
- GlcNAc
N-acetylglucosamine
- HPLC
high-performance liquid chromatography
- Man
mannose
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecyl-sulfate
- Ser
serine
- TFMS
trifluoromethanesulfonic acid
- Thr
threonine
- Xyl
xylose 相似文献
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Genetic manipulation in cultivars of oilseed rape (Brassica napus) using Agrobacterium 总被引:1,自引:0,他引:1
G. Ooms A. Bains M. Burrell A. Karp D. Twell E. Wilcox 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1985,71(2):325-329
Summary The response of oilseed rape cultivars to infection with Agrobacterium tumefaciens and A. rhizogenes and the possibility of regenerating genetically transformed oilseed rape plants were examined. The frequency at which Agrobacterium induced galls or hairy-roots on in vitro cultured plants ranged from 10% to 70%, depending on the cultivar. From galls induced by the tumorigenic strain T37, known to be strongly shoot inducing on tobacco, roots developed frequently. Occasionally, shoots formed and some of these produced tumour cell specific nopaline. Attempts to grow the transformed shoots into plants have so far been unsuccessful. Whole plants transformed with Ri-T-DNA, however, were regenerated. These had crinkled leaves and abundant, frequently branching roots that showed reduced geotropism, similar to previously isolated Ri T-DNA transformed tobacco and potato plants. The transformed oilseed rape plants flowered, but failed to form seeds. 相似文献
16.
The resurrection plant Craterostigma plantagineum Hochst. is used as an experimental system to investigate desiccation tolerance in higher plants. A search for genes activated during early stages of dehydration identified the gene CpEdi-9, which is expressed in mature seeds and in response to dehydration in the phloem cells of vascular tissues of leaves. Elements for the tissue-specific expression pattern reside in the isolated promoter of the CpEdi-9 gene, as shown through the analysis of transgenic plants. The
CpEdi-9 promoter could be a suitable tool for expressing genes in the vascular system of dehydrated plants.
CpEdi-9 encodes a small (10 kDa) hydrophilic protein, which does not have significant sequence homologies to known genes. The predicted protein CpEDI-9 shares some physicochemical features with LEA proteins from plants and a nematode. Based on the unique expression pattern and on the nucleotide sequence we propose that CpEdi-9 defines a new class of hydrophilic proteins that are supposed to contribute to cellular protection during dehydration. This group of proteins may have evolved because desiccation tolerance requires the abundant expression of protective proteins during early stages of dehydration in all tissues.Abbreviations ABA
Abscisic acid
- ABRE
ABA-responsive element
- Edi
Early dehydration induced
- GUS
Glucuronidase
- LEA
Late embryogenesis abundant
- MU
Methylumbelliferone
This article is dedicated to Prof. Dr. Francesco Salamini on the occasion of his 65th birthday and his departure from the Max Planck Institute in Köln 相似文献
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Kushnir SG Shlumukov LR Pogrebnyak NJ Berger S Gleba Y 《Molecular & general genetics : MGG》1987,209(1):159-163
Summary Mesophyll protoplasts of plastome chlorophyll-deficient, streptomycin-resistant Nicotiana tabacum were fused with those of wild type Atropa belladonna using the polyethylene-glycol/high pH/high Ca++/dimethylsulfoxide method. Protoplasts were cultured in nutrient media suitable for regeneration of tobacco but not Atropa cells. In two experiments, a total of 41 cell lines have been selected as green colonies. Cytogenetic (chromosomal number and morphology) and biochemical (isozyme analyses of esterase, amylase and peroxidase) studies were used to evaluate the nuclear genetic constitution of regenerated plants. To study plastid genetic constitution, restriction endonuclease analysis of chloroplast DNA was performed. Three groups of regenerants have been identified: (a) nuclear hybrids (4 cell lines); (b) Atropa plants, most probably arising from rare surviving parental protoplasts (4 lines) and (c) Nicotiana/Atropa cybrids possessing a tobacco genome and an Atropa plastome (33 lines). Most of cybrids obtained were diploid, morphologically normal plants phenotypically similar to tobacco. Some plants flowered and yielded viable seeds. Part of cybrid regenerants were variegated, variegation being transmitted to sexual progeny. Electron microscopic analysis of the mesophyll cells of variegated leaves revealed the presence of heteroplastidic cells. Analysis of thylakoid membrane polypeptides shows that in the cybrids the content of at least one of the major polypeptides, presumably a chlorophyll a/b binding protein is drastically reduced. 相似文献
19.
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
20.
The expression of a bacterial cytokinin biosynthesis gene (PI-II-ipt) in Nicotiana plumbaginifolia Viviani plants has been correlated with enhanced resistance to Manduca sexta and Myzus persicae. We expressed the PI-II-ipt gene in N. tabacum and Lycopersicon esculentum and observed similar antifeedent effects with the transgenic tobacco but not tomato. A 30 to 50 % reduction in larval weight
gain was observed with some of the tomato plants but these results could not be repeated consistently. Leaf surface extracts
from transgenic N. plumbaginifolia leaves killed 100 % of M. sexta second instars at concentrations of 0.05 % (w/v) whereas the N. tabacum extracts were at least 20 times less active. Extract suspensions were stable for up to 2 days at ambient temperatures below
42 °C and for at least 3 months at 4 °C when stored in the dark. HPLC analysis of the N. plumbaginifolia extracts yielded an active fraction that reduced hatching of M. sexta eggs by 30 % and killed first, second and third instars within 24, 48 and 72 hours of exposure, respectively. The activity
appears to be associated with oxygen-containing aliphatic compounds, possibly diterpenes, as analyzed by TLC, UV absorption
and fragmentation with EIMS. Based on the partial characterization of this activity, the production, secretion or accumulation
of secondary metabolites in leaves of cytokinin producing PI-II-ipt N. plumbagini-folia plants appears to be responsible for the observed insect resistance. 相似文献