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1.
Proteolytic enzymes existing in plant cell cultured media are the major reason for the loss of secreted human granulocyte-macrophage
colony-stimulating factor (hGM-CSF). The addition of pepstatin, aprotinin and PMSF relatively decreased the proteolytic degradation
of hGM-CSF in a conditioned medium, but sufficient prevention against the proteolytic activity could not be obtained with
chemical protease inhibitors. Gelatin, as a competitive substrate for protease, showed a stabilizing effect in a conditioned
medium. Compared to the initial hGM-CSF concentration in a conditioned medium, with 10 g/L of gelatin, 68% of the hGM-CSF
remained after 5 days. In a cell culture experiment, 5 g/L of gelatin significantly stimulated the hGM-CSF production and
accumulation in culture media, with no growth inhibition. Compared to the controls (4.72 μg/L), the extracellular hGM-CSF
level could be increased to 39.78 μg/L with the addition of 5 g/L of gelatin. 相似文献
2.
Production and secretion of biologically active human granulocyte-macrophage colony stimulating factor in transgenic tomato suspension cultures 总被引:4,自引:0,他引:4
A complementary DNA encoding human granulocyte-macrophage colony stimulating factor (hGM-CSF) was cloned and introduced into tomato (Lycopersicon esculentum cv. Seokwang) using Agrobacterium-mediated transformation. Genomic PCR and Northern blot analysis demonstrated the integration of the construction into the plant nuclear genome and expression of the hGM-CSF in transgenic tomato. The cell suspension culture was established from leaf-derived calli of the transgenic tomato plants transformed with the hGM-CSF gene. Recombinant hGM-CSF was synthesized by the transgenic cell culture and secreted into the growth medium at 45 g l–1 after 10 d' cultivation. 相似文献
3.
Interleukin-18 (IL-18), otherwise known as interferon-gamma-inducing factor (IGIF), is one of several well characterized and
important cytokines that contribute to host defenses. The complementary DNA (cDNA) of mature human interleukin-18 gene (hIL-18)
was fused with the signal peptide of the rice amylase 1A gene (Ramy1A) and introduced into the plant expression vector under
the control of a duplicated CaMV 355 promoter. The recombinant plasmid was transformed into tobacco (Nicotiana tabacum L. cv Havana) using theAgrobacterium-mediated transformation method. The integration of the hIL-18 gene into the genome of transgenic tobacco plants was confirmed
by polymerase chain reaction (PCR) amplification and its expression was observed in the suspension cells that were derived
from the transgenic plant callus by using Northern blot analysis. The hIL-18 protein was detected in the extracts of the transgenic
callus and in the medium of the transgenic tobacco suspension culture by using immunoblot analysis. Based upon enzyme-linked
immunosorbant assay (ELISA) results, the expression level of the hIL-18 protein approximated 166 μg/L in the suspension culture
medium. Bioassay results from the induction of interferon-γ from a KG-1 cell line indicated that the hIL-18 secreted into
the suspension culture medium was bioactive. 相似文献
4.
5.
Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome this problem, we sought an expression system in which heterologous gene expression could be induced at high levels. We selected a rice amylase expression system in which the promoter Ramy3D is induced to express recombinant protein by sucrose starvation. This induction system was found to give good yield of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system. 相似文献
6.
Bimal Kumar Ghimire Eun Soo Seong Eun Jung Goh Eun Young Kang Joung Kuk Ahn Chang Yeon Yu Ill Min Chung 《Plant Growth Regulation》2011,63(1):1-6
Codonopsis lanceolata Trautv (Companulaceae) is a folk medicine in Korea. To shift the content of tocopherol and enhance its antioxidant properties,
we overexpressed the γ-tocopherol methyltransferase (γ-tmt) gene in C. lanceolata. The antioxidant activity of methanolic crude extracts of the transgenic plants was compared to that of control plants using
the 1,1-diphenyl-2-picrylhydrazyl radical scavenging method, with α-tocopherol and butylated hydroxy toluene as standard antioxidants.
The antioxidant activity of the leaf and root extracts of transgenic plants was higher (IC 50 12–17.33 and 408–524 μg/ml, respectively) than that of control plant leaf and root extracts (18 and 529 μg/ml, respectively).
High-performance liquid chromatography analysis of phenolic compounds confirmed an increase in the levels of 12 major phenolic
acids and flavonoids in the leaf and root extracts of transgenic plants compared to control plants. We also found that the
rate of photosynthesis was 48% higher in transgenic plants than in control plants. Based on these results, we suggest that
increases in the α-tocopherol level in transgenic C. lanceolata plants may result in increases in the photosynthetic performance and antioxidant metabolism of these plants. 相似文献
7.
Characterization of auxin-binding protein 1 from tobacco: content, localization and auxin-binding activity 总被引:1,自引:0,他引:1
There is evidence that auxin-binding protein 1 (ABP1) is an auxin receptor on the plasma membrane. Maize (Zea mays L.) possesses a high level of auxin-binding activity due to ABP1, but no other plant source has been shown to possess such
an activity. We have analyzed the ABP1 content of tobacco (Nicotiana tabacum L.) to examine whether or not the ABP1 content of maize is exceptionally high among plants. The ABP1 content of tobacco leaves
was shown by quantitative immunoblot analysis to be between 0.7 and 1.2 μg ABP1 per gram of fresh leaf. This value is comparable
to the reported value in maize shoots, indicating that ABP1 is present at a similar level in both monocot and dicot plants.
The ABP1 content of tobacco leaves was increased up to 20-fold by expression of a recombinant ABP1 gene, and decreased to
half of the original value by expression of the antisense gene. Although ABP1 was found mainly in the endoplasmic reticulum
fraction, a secreted protein showing a molecular size and epitopes similar to intracellular ABP1 was also detected in the
culture medium of tobacco leaf disks. The secretion of this protein was dependent on the expression level of the ABP1 gene.
Received: 24 February 1999 / Accepted: 25 March 1999 相似文献
8.
Qi-Feng Wang Yue Zhao Qiong Yi Kun-Zhi Li Yong-Xiong Yu Li-Mei Chen 《Acta Physiologiae Plantarum》2010,32(6):1209-1220
Numerous studies with transgenic plants have demonstrated that overexpression of enzymes related to organic acid metabolism
under the control of CaMV 35S promoter increased organic acid exudation and Al-resistance. The synthesis of organic acids
requires a large carbon skeleton supply from leaf photosynthesis. Thus, we produced transgenic tobacco overexpressing cytosolic
malate dehydrogenase (MDH) cDNA from Arabidopsis thaliana (amdh) and the MDH gene from Escherichia coli (emdh), respectively, under the control of a leaf-specific light-inducible promoter (Rubisco small subunit promoter, PrbcS) in
the present study. Our data indicated that an increase (120–130%) in MDH-specific activity in leaves led to an increase in
malate content in the transgenic tobacco leaves and roots as well as a significant increase in root malate exudation compared
with the WT plants under the acidic (pH 4.5) conditions irrespective of 300 μM Al3+ stress absence or presence. After being exposed to 25 μM Al3+ in a hydroponic solution, the transgenic plants exhibited stronger Al-tolerance than WT plants and the degree of A1 tolerance
in the transgenic plants corresponded with the amount of malate secretion. When grown in an Al-stress perlite medium, the
transgenic tobacco lines showed better growth than the WT plants. The results suggested that overexpression of MDH driven
by the PrbcS promoter in transgenic plant leaves enhanced malate synthesis and improved Al-resistance. 相似文献
9.
Transgenic tobacco (Nicotiana tabacum L. cv. Wisconsin 38) lines expressing a mutant calmodulin (VU-3) that hyperactivates NAD kinase exhibit an enhanced elicitor-stimulated
oxidative-burst reaction (S.A. Harding et al., 1997, EMBO J. 16: 1137–1144). VU-3 transgenic tobacco was used in the present
study to investigate the relationship between calmodulin signalling, the production of active oxygen species and cell death
in response to infection with an incompatible pathogen. Following P. syringae pv. syringae 61 infection, suspension cells derived from VU-3 transgenic plants exhibited a stronger oxidative burst (3- to 4-fold higher
primary and secondary burst reactions), greater media alkalinization (3-fold) and more rapid cell death (4-fold greater mortality
at 20 h post infection) than did infected control tobacco cells. Infection of leaf tissues with P. syringae pv. syringae 61 also resulted in an enhanced cell death response compared to control tobacco tissues. This cell death response of VU-3
leaf tissues, but not control leaf tissues, was further enhanced by the presence of 50 μM salicylic acid, suggesting that
this transgenic line is more sensitive to the effects of this agent. Overall, the data support the model that calmodulin signalling
pathways are involved in the plant oxidative burst and contribute to the regulation of cell death in infected plant tissues
undergoing the hypersensitive response.
Received: 6 January 1998 / Accepted: 7 March 1998 相似文献
10.
Vaclav Diopan Violetta Shestivska Vojtech Adam Tomas Macek Martina Mackova Ladislav Havel Rene Kizek 《Plant Cell, Tissue and Organ Culture》2008,94(3):291-298
Phytoremediation is a process that utilizes plants to remove, transfer, stabilize, or destroy pollutants in soil, sediment,
and groundwater. Plants used for such purposes have several requirements. Genetic engineering these plants could be an effective
tool used to acquire features needed for such purposes within a substantial amount of time. This paper aims to utilize electrochemical
techniques to analyze transgenic tobacco and, thus, to reveal their heavy metals phytoremediation potential. Total thiol and
metallothionein (MT) quantities were determined in the control and transgenic tobacco plants. The total content of thiols
in transgenic plants varied within the range of 561 to 1,671 μg g−1. Furthermore, the determination of MT was done on transgenic tobacco plants. The level of human MT in transgenic tobacco
plants varied between 25 and 95 μg g−1. However, a plant cell protects itself by synthesizing low molecular mass thiols such as reduced glutathione and phytochelatins
to protect itself against heavy metals toxicity. The most important thiols, cysteine (Cys), glutathione (GSH), oxidised glutathione
(GSSG) and phytochelatin 2 (PC2), were determined in the non-transgenic and transgenic tobacco plants by high performance
liquid chromatography with electrochemical detection. Tobacco plants synthesizing the highest amount of metallothionein have
the highest basal level of phytochelatin 2 as well as reduced glutathione and free cysteine. It clearly follows from the results
obtained that the biosynthesis of particular thiols is mutually linked, which contributes to a better protection of a transgenic
plant against heavy metals effects. 相似文献
11.
E. A. Trifonova A. V. Romanova S. S. Sangaev M. V. Sapotsky V. I. Malinovsky A. V. Kochetov 《Biologia Plantarum》2012,56(3):571-574
The gene of Zinnia elegans L. coding for S-like extracellular ribonuclease (ZRNase II) was used to produce transgenic tobacco plants with an increased ribonuclease activity. The protein-coding part of ZRNase
II included the signal peptide sequence so the transgenic protein was located extracellularly. The cDNA of ZRNase II was cloned under the control of 2′-promoter of the mannopine synthase (MAS 2′) gene from Ti-plasmid of Agrobacterium tumefaciens. It was shown that the resultant transgenic plants had an increased ribonuclease activity of the crude extracts and the induction
of MAS 2′ promoter by wounding additionally increased the activity. The plants of two transforming lines characterized by
different ribonuclease activities were used to analyze the transgene influence on plant resistance to tobacco mosaic virus.
The plants demonstrated either absence of disease symptoms or a significant delay in their appearance, depending on the virus
content in the inoculum and ribonuclease activity. 相似文献
12.
13.
Cysteine serves as a precursor for the synthesis of various sulfur-containing metabolites, and the cysteine synthase (CS)
gene plays a central role in the sulfur cycle in nature. In the present study, rcs1, a cytosolic CS gene of rice, was introduced into the genome of tobacco (Nicotiana tabacum). The tolerance of wild-type tobacco plants as well as of the resulting transgenic tobacco plants overexpressing the rcs1 gene to toxic levels of ozone (O3, 0.15 μ mol−1) was measured after various lengths of exposure. Leaf lesions in plants exposed for 2 weeks to O3 were more prevalent in the leaves of the wild-type plants than in those of the transgenic tobacco plants. Transgenic tobacco
plants showed a higher growth rate and a higher chlorophyll content than the wild-type plants. Cysteine synthase activity
and cysteine and glutathione contents were higher in transgenic plants than in wild-type plants irrespective of the length
of the O3 treatment. Our results indicate that the CS gene plays a role in the protection of the plant against toxic O3 gas, probably through the mechanism of an over-accumulation of such sulfur-rich antioxidants as cysteine and glutathione. 相似文献
14.
Atrazine Tolerance of Grass Species with Potential for Use in Vegetated Filters in Australia 总被引:2,自引:0,他引:2
The response of introduced (Pennisetum clandestinum Hochst.) and native (Stipa aristiglumis F. Muell., Themeda australis (R. Br.) Stapf, Danthonia spp. (L.) grasses to the herbicide atrazine was studied in plants with the potential for use in vegetated filters (biofilters)
designed to reduce chemical loads in agricultural runoff. The response was detected by photosynthetic inhibition using leaf
chlorophyll fluorescence. With continuous short-term (14 days) dosing with atrazine in sand culture, P. clandestinum showed the greatest tolerance, regardless of the dose (20–500 μg/L). In a clay vertosol soil in the glasshouse, the four
species were tolerant to longer-term (84 days) application of three successive doses of simulated run-on, each dose containing
100 μg/L atrazine, a concentration which is comparable to the highest reported in runoff from agricultural land in Australia.
Even with a subsequent single atrazine dose at ∼
∼5000 μg/L, established plants of the four species showed signs of quick recovery (7–21 days) to normal photosynthetic activity.
In a field experiment with simulated run-on, applied to P. clandestinum pasture in sandy loam soil, repeated doses at concentrations up to 1000 μg/L gave no significant response; only a subsequent
single dose of 5000 μg/L had significant effects, from which plants soon recovered. Although damage from atrazine can be demonstrated
with continuous dosing in non-adsorbing media, in soil culture the tolerance of the four selected species to repeat doses
of atrazine shows they may be used confidently for biofiltering purposes. P. clandestinum was especially tolerant. 相似文献
15.
Chao Ma Binggang Ma Juan He Qingnan Hao Xiaoyan Lu Lei Wang 《Plant Molecular Biology Reporter》2011,29(1):117-124
To conduct RNAi interference of Lyc-β and Lyc-ε genes, two plant expression vectors were constructed by inserting the intron fragments of the gusA gene into the two target gene fragments, which were designed in anti-sense directions. After the Agrobacterium tumefaciens-mediated transformation, 13 transgenic tomato plants (seven and six for Lyc-β and Lyc-ε, respectively) were obtained, which was further validated by PCR. Real-time PCR revealed that the messenger RNA abundance
of Lyc-β gene and Lyc-ε gene in transgenic tomato plants was significantly reduced to 8.95% and 13.16%, respectively, of the level of the wild-type
plant. Subsequent high-performance liquid chromatography analysis found that transgenic tomato plant had significantly increased
lycopene content, with the highest value of 13.8 μg/g leaf dry weight, which was about 4.2-fold that of wild-type plant. Moreover,
Lyc-β and Lyc-ε interference gene effects were observed on downstream products as well. β-Carotene and lutein contents decreased in Lyc-β RNAi lines, ranging from 40.7 to 117.3 μg/g and 4.9 to 23.5 μg/g leaf dry weight, respectively. In Lyc-ε RNAi lines, β-carotene content increased, ranging from 195.8 to 290.2 μg/g, while lutein content decreased, ranging from
3.7 to 11.3 μg/g. For total carotenoids, Lyc-β RNAi lines resulted in 2.9-fold decrease, while Lyc-ε RNAi lines yielded 1.7-fold increase in contents when compared to wild-type control. This study demonstrated that RNAi gene
technology is an effective method for enhancing lycopene content in plants. 相似文献
16.
Xiangbin Xu Sai Guo Kai Chen Hongmiao Song Junjun Liu Longbiao Guo Qian Qian Huizhong Wang 《Biotechnology letters》2010,32(10):1533-1539
A 1681 bp PsPR10 promoter was isolated from Pinus strobus and a series of 5′-deletions were fused to the β-glucuronidase (GUS) reporter gene and introduced into tobacco. GUS activity in P796 (−796 to +69) construct transgenic plant roots was similar
with that of P1681 and higher than those of the P513 (−513 to +69) and P323 (−323 to +69) transgenic plants. Moreover, the
abiotic stresses of NaCl, PEG 6000 and mannitol, and salicylic acid (SA), abscisic acid (ABA) and jasmonic acid (JA) induced
higher GUS activity in the roots of P796 transgenic tobacco. This study provides a potential inducible root-specific promoter
for transgenic plants. 相似文献
17.
In this study we have utilized Nicotiana tabacum with a molecular farming purpose in attempt of producing transgenic plants expressing the human tissue transglutaminase (htTG).
Three plant expression constructs were used enabling targeting and accumulation of the recombinant protein into the plant
cell cytosol (cyto), the chloroplasts (chl) and the apoplastic space (apo). Analysis of transgenic T0 plants revealed that recombinant htTG was detectable in all three transgenic lines and the accumulation levels were in a
range of 18–75 μg/g of leaf material. In the T1 generation, the recombinant htTG was still expressed at high level and a significant catalytic activity was detected into
the leaf protein extracts. Southern blot analyses revealed that apo and chl plants of T1 generation possess a high copy number of the recombinant htTG in their genome, while the cyto plants carry a single copy. 相似文献
18.
Stanley C. K. Cheung Samuel S. M. Sun Juliana C. N. Chan Peter C. Y. Tong 《Transgenic research》2009,18(6):943-951
Human insulin-like growth factor binding protein-3 (hIGFBP-3) is a multifunctional protein which has high affinity for insulin-like
growth factor-I (IGF-I). It combines with IGF-I to form a tertiary complex in circulation, thus regulating the activity of
IGF-I. Furthermore, recombinant hIGFBP-3 (rhIGFBP-3) has been found to negatively regulate cell proliferation and induce apoptosis.
In this study, we have established an efficient plant bioreactor platform for mass production of rhIGFBP-3. Different expression
constructs, driven by the seed-specific phaseolin promoter, were designed and transformed into tobacco plant via Agrobacterium. To enhance protein expression level, the signal peptide (SP) and the C-terminal tetrapeptide AFVY of phaseolin were used
to direct rhIGFBP-3 to protein storage vacuole (PSV) in tobacco seed for stable accumulation. Western blot analysis showed
that rhIGFBP-3 was successfully synthesized in transgenic tobacco seeds, with the highest protein expression of 800 μg/g dry
weight. The localization of rhIGFBP-3 in PSV was also evident by confocal immunofluorescence microscopy. Our results indicated
that protein sorting sequences could benefit the expression level of rhIGFBP-3 and it is feasible to use plant as “bio-factory”
to produce therapeutic recombinant proteins in large quantity. 相似文献
19.
X. Niu X. Li P. Veronese R. A. Bressan S. C. Weller P. M. Hasegawa 《Plant cell reports》2000,19(3):304-310
Substantial improvement in peppermint (Mentha x piperita L. var. Black Mitcham) genetic transformation has been achieved so that the frequency of transgenic plants regenerated (percent
of leaf explants that produced transformed plants) was 20-fold greater than with the original protocol. Essential modifications
were made to conditions for Agrobacterium tumefaciens co-cultivation that enhanced infection, and for selection of transformed cells and propagules during regeneration. A systematic
evaluation of co-cultivation parameters established that deletion of coconut water from the co-cultivation medium resulted
in substantially increased transient β-Glucuronidase (GUS) activity, in both the frequency of explants expressing gusA and the number of GUS foci per explant (>700 explants). Co-cultivation on a tobacco cell feeder layer also enhanced A. tumefaciens infection. Enhanced transformation efficiencies were further facilitated by increased selection pressure mediated by higher
concentrations of kanamycin in the medium during shoot induction, regeneration, and rooting: from 20 to 50 mg/l in shoot induction/regeneration
medium and from 15 to 30 mg/l in rooting medium. Raising the concentration of kanamycin in media substantially lowered the
number of "escapes" without significant reduction in plant regeneration. These modifications to the protocol yielded an average
transformation frequency of about 20% (>2000 explants) based on expression of GUS activity or the tobacco antifungal protein,
osmotin, in transgenic plants. Genetic transformation of peppermint has been enhanced to the extent that biotechnology is
a viable alternative to plant breeding and clonal selection for improvement of this crop.
Received: 7 December 1998 / Revision received: 27 April 1999 / Accepted: 14 May 1999 相似文献
20.
The Lhcb2 gene from hyperaccumulator Sedum alfredii was up-regulated more than three-fold while the non-hyperaccumulator accumulated one or two-fold higher amount of the mRNA
than control plants under different concentrations of Cd2+ for 24 h. Lhcb2 expression was up-regulated more than five-fold in a non-hyperaccumulator S. alfredii when exposed to 2 μM Cd2+ or 50 μM Zn2+ for 8 d and the hyperaccumulator had over two-fold more mRNA abundance than the control plants. Over-expression of SaLhcb2 increased the shoot biomass by 14–41% and the root biomass by 21–57% without Cd2+ treatment. Four transgenic tobacco lines (L5, L7, L10 and L11) possessed higher shoot biomass than WT plants with Cd2+. Four transgenic lines (L7, L8, L10 and L11) accumulated 6–35% higher Cd2+ amounts in shoots than the wild type plants. 相似文献