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1.
Human growth hormone (hGH), a pituitary-derived polypeptide, evidences a wide range of biological functions, including protein
synthesis, cell proliferation, and metabolism. A synthetic hGH gene (shGH) has been synthesized on the basis of plant-optimized
codon usage via an overlap PCR strategy and located in a plant expression vector under the control of the rice amylase 3D
(Ramy3D) promoter, which is induced by sugar starvation. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Donjin) via particle bombardment transformation methods. The integration of the shGH gene into the chromosome of the
transgenic rice callus was verified via genomic DNA PCR amplification and shGH expression in transgenic rice suspension cells
was confirmed via Northern blot analysis. The shGH protein was detected in the transgenic rice cell suspension culture medium
following induction with sugar starvation, using Western blot analysis. The quantity of shGH that accumulated in the transgenic
rice cell suspension medium was 57 mg/l. The shGH accumulated in the transgenic rice cell suspension culture medium evidenced
a biological activity similar to that of Escherichia coli-derived recombinant hGH. These results indicate that the shGH was generated and accumulated in the transgenic rice cell suspension
culture medium, and manifested biological activity. 相似文献
2.
It has been suggested that plant cell culture is the most suitable system for producing small-to-medium quantities of specialized, expensive, and high-purity proteins. Here, we report that a heterodimeric protein, human interleukin-12 (hIL-12), was expressed and secreted into culture medium in a biologically active form. A transgenic plant expressing hIL-12 was constructed by sexual crossing of plants that expressed each subunit of the protein. From a piece of transgenic plant, callus was induced and cell suspension culture was established. The biological activity and amount of hIL-12 secreted into culture medium were analyzed using bioassays and ELISA. Analysis of cellular localization demonstrated that the protein was secreted into the culture medium together with its intrinsic signal peptide. 相似文献
3.
Tae-Ho Kwon Young-Mi Shin Young-Sook Kim Yong-Suk Jang Moon-Sik Yang 《Biotechnology and Bioprocess Engineering》2003,8(2):135-141
The human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene was introduced into tobacco plants. The cell suspension
culture was established from leaf-derived calli of the transgenic tobacco plants in order to express and secrete a biologically
active hGM-CSF. The recombinant hGM-CSF from the transgenic plant cell culture (prhGM-CSF) was identified as a yield of about
180 μg/L in the culture filtrate, as determined by ELISA. The addition of 0.5 g/L polyvinylpyrrolidone (PVP) to the plant
cell culture medium both stabilized the secreted prhGM-CSF and increased the level of production approximately 1.5-fold to
270 μg/L. The biological activity of the prhGM-CSF was confirmed by measuring the proliferation of the hGM-CSF-dependent cell
line, TF-1. Interestingly, the specific activity of the prhGM-CSF was estimated to be approximately 2.7 times higher than
that of a commercially available preparation fromE. coli. 相似文献
4.
Kim NS Yu HY Chung ND Shin YJ Kwon TH Yang MS 《Protein expression and purification》2011,76(1):121-126
A synthetic bovine trypsinogen (sbTrypsinogen) was synthesized on the basis of rice-optimized codon usage via an overlap PCR strategy, prior to being expressed under the control of the sucrose starvation-inducible rice α-amylase 3D (RAmy3D) promoter. Secretion of trypsin into the culture medium was achieved by using the existing signal peptide. The plant expression vector was introduced into rice calli (Oryza sativa L. cv. Dongjin), mediated by Agrobacterium tumefaciens. The integration of the sbTrypsinogen gene into the chromosome of the transgenic rice callus was verified via genomic DNA PCR amplification, and sbTrypsin expression in transgenic rice suspension cells was confirmed via Northern blot analysis. Western blot analysis detected glycosylated proteins in the culture medium, having masses from 24 to 26 kDa, following induction by sugar starvation. Proteolytic activity of the rice-derived trypsin was confirmed by gelatin zymogram, and was similar to that of the commercial bovine-produced trypsin. The yields of sbTrypsin that accumulated in the transgenic rice cell suspension medium were 15 mg/L at 5 days after sugar starvation. 相似文献
5.
Expression and production of bioactive human interleukin-18 in transgenic tobacco plants 总被引:4,自引:0,他引:4
The cDNA of human interleukin-18 (hIL-18) was successfully inserted into the genome of tobacco plant, Nicotiana tabacum cv. NC-89, using Agrobacterium tumefaciens-mediated transformation. Insertion and translation of hIL-18 in transformants were confirmed by PCR, ELISA, and Western blot, respectively. The transformed extracts contained the recombinant hIL-18 protein up to 0.05% of total soluble protein. Activity of the recombinant hIL-18 in plant cells was confirmed by the induction of IFN- on IL-18-responsive J6-1 cells by the extracts obtained from the transformants. The expression level of hIL-18 (351 ng g–1 tobacco tissue) obtained in the present study may be sufficient to induce responses/effects in vivo. 相似文献
6.
G. H. Fleming O. Olivares-Fuster S. Fatta Del-Bosco J. W. Grosser 《In vitro cellular & developmental biology. Plant》2000,36(6):450-455
Summary An alternative method for transforming sweet organe [Citrus sinensis (L.) Osbeck] has been developed. Plasmid DNA encoding the non-destructive selectable marker enhanced green fluorescent protein
gene was introduced using polyethylene glycol into protoplasts of ‘Itaborai’ sweet organe isolated from an embryogenic nucellar-derived
suspension culture. Following protoplast culture in liquid medium and transfer to solid medium, transformed calluses were
identified via expression of the green fluorescent protein, physically separated from non-transformed tissue, and cultured on somatic embryogenesis
induction medium. Transgenic plantlets were recovered from germinating somatic embryos and by in vitro rooting of shoots. To expedite transgenic plant recovery, regenerated shoots were also micrografted onto sour orange seedling
rootstocks. Presence of the transgene in calluses and regenerated sweet organe plants was verified by gene amplification and
Southern analyses. Potential advantages of this transformation system over the commonly used Agrobacterium methods for citrus are discussed. 相似文献
7.
Kim TG Lee HJ Jang YS Shin YJ Kwon TH Yang MS 《Protein expression and purification》2008,61(2):117-121
The synthetic gene (sPI-II) harboring the chymotrypsin (C1) and trypsin (T1) inhibitor domains of the Nicotiana alata serine proteinase inhibitor II gene has been previously expressed, and extracellular protease activity was shown to be reduced in the suspension culture medium. In this study, the sPI-II gene was introduced into transgenic rice cells expressing rhGM-CSF (recombinant human granulocyte–macrophage colony-stimulating factor), in an effort to reduce protease activity and increase rhGM-CSF accumulation in the suspension culture medium. The integration and expression of the introduced sPI-II gene in the transgenic rice cells were verified via genomic DNA PCR amplification and Northern blot analysis, respectively. Relative protease activity was found to have been reduced and rhGM-CSF production was increased 2-fold in the co-transformed cell suspension culture with rhGM-CSF and the sPI-II gene, as compared with that observed in the transformed cell suspension culture expressing rhGM-CSF only. These results indicate that a transformed plant cell suspension culture system expressing the proteinase inhibitor can be a useful tool for increasing recombinant protein production. 相似文献
8.
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
9.
10.
Hai Ping Hong Hongyi Zhang Paula Olhoft Steve Hill Hunt Wiley Effie Toren Helke Hillebrand Todd Jones Ming Cheng 《In vitro cellular & developmental biology. Plant》2007,43(6):558-568
A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes
as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced
on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with
various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect
on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a
low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside,
and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction
medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected
calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been
regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including
leaf, stem, and roots. Southern and T1 plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy
number ranging from 1–5 copies. 相似文献
11.
H. Brittain-Loucas S. R. Bowley B. D. McKersie 《In vitro cellular & developmental biology. Plant》1998,34(4):281-284
Summary The effects of callus inoculation concentration and culture duration on somatic embryogenesis of orchardgrass,Dactylis glomerata L., were evaluated in suspension cultures of an embryogenic genotype Embryogen-P. Somatic embryo formation was induced in
liquid SH medium containing 30 μM dicamba (SH-30 and 1.5% casein hydrolysate; embryo development was in liquid SH medium without plant growth regulators (SH-0);
and embryo maturation and germination occurred on solid SH-0 medium. Callus proliferation in SH-30 suspension cultures was
greatest when callus was inoculated into the liquid medium at a relatively high concentration of 4% (4 g callus/100 ml medium),
but the induction of somatic embryos was highest in this medium if the callus was inoculated at a lower concentration (<2%).
In a second experiment, somatic embryo yield was highest when SH-0 development medium was inoculated with suspension culture
callus at 0.1% concentration and declined markedly as inoculation concentration increased. Cell concentration is a critical
factor in regulating the somatic embryogenesis response in orchardgrass suspension cultures. 相似文献
12.
13.
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
14.
Mami Arie Kiyoshi Hikichi Kiyoshi Takahashi Muneharu Esaka 《Physiologia plantarum》2000,110(2):232-239
15.
Heleen M. van der Maas Eliza R. de Jong Saskia Rueb Lambert A. M. Hensgens Frans A. Krens 《Plant molecular biology》1994,24(2):401-405
Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4×106 cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture. 相似文献
16.
Embryogenic cell suspension cultures of Santalum album were transformed with Agrobacterium tumefaciens harboring pD35SHER plant expression vector having hepatitis B small surface antigen (HBsAg) with a C-terminal ER retention
signal. The transformed colonies were selected on culture medium supplemented with kanamycin and subsequently the transgenic
nature of these colonies was confirmed by PCR analysis. The expression of HBsAg was confirmed by RT-PCR analysis and Western
blot analysis and the expression was quantified using monoclonal antibody-based ELISA. Cell suspension cultures were initiated
from the colony with expression of 11.09 μg(HBsAg) g−1(f.m.). To further increase the expression of HBsAg, transgenic S. album suspensions were cultured on media with various medium additives and cells growing in medium with 30 mM trehalose showed
the expression of 19.95 μg(HBsAg) g−1(f.m.). 相似文献
17.
Delivery of plasmid DNA into intact plant cells by electroporation of plasmolyzed cells 总被引:7,自引:0,他引:7
This report describes the delivery of plasmid DNA containing either the β-glucuronidase (GUS) or the green fluorescent protein (GFP) reporter gene into intact plant cells of bamboo callus, lilium
scales, and Nicotiana benthamiana suspension culture cells. By first plasmolyzing the tissues or cells with 0.4 m sucrose in the presence of plasmid DNA, electroporation effectively delivers plasmid DNA into the intact plant cells. Transient
expression of the GUS gene, as revealed by histochemical assays, showed the presence of blue-staining areas in the electroporated
tissues. A short exposure of cells to 2% DMSO (dimethyl sulfoxide) prior to plasmolysis elevated the level of transient GUS
activity. When plasmid DNA containing a synthetic GFP gene was used, a strong green fluorescence was observed in N. benthamiana suspension culture cells that were subjected to plasmolysis and electroporation. These results suggest that plasmolysis brings
the plasmid DNA into the void space that is in close vicinity to the plasmalemma, allowing electroporation to efficiently
deliver the plasmid DNA into intact plant cells.
Received: 15 June 1998 / Revision received: 18 August 1998 / Accepted: 28 August 1998 相似文献
18.
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
19.
Annegret Tewes Renate Manteuffel Klaus Adler Ernst Weber Ulrich Wobus 《Plant cell reports》1991,10(9):467-470
Summary Long-term cultures of four different cultivars of barley (Hordeum vulgare L.) have been established. Both callus and suspension cultures formed embryogenic structures at high frequency even after more than 18 months of culture. These compact proembryogenic cell clusters synthesize seed storage globulins whereas loose cell aggregates in callus culture and suspension cultures of fine dispersed consistency were free of globulins. Globulin synthesis was especially intense in compact structures of callus cultures established from suspension culture-derived protoplasts. Within the cells storage globulins are deposited in the vacuolar compartment as in zygotic embryos. The molecular data provided recommend the system for studies on factors determining seed protein gene expression and intracellular protein transport.Abbreviations MS
Murashige and Skoog (1962)
- 2,4-D
2,4-dichlorophenoxyacetic acid 相似文献
20.
Transfer and Expression of an Artificial Storage Protein (ASP1) Gene in Cassava (Manihot Esculenta Crantz) 总被引:4,自引:0,他引:4
In order to increase the nutritional quality of cassava storage roots, which contain up to 85% starch of their dry weight, but are deficient in protein, a synthetic ASP1 gene encoding a storage protein rich in essential amino acids (80%) was introduced into embryogenic suspensions of cassava via Agrobacterium-mediated gene transfer. Transgenic plants were regenerated from suspension lines derived from hygromycin-resistant friable embryogenic callus lines. Molecular analysis showed the stable integration of asp1 in cassava genome and its expression at RNA level in transformed suspension lines. PCR and Southern analyses proved the transgenic nature of the regenerated plant lines. The expression of asp1 at RNA level was demonstrated by RT-PCR. The ASP1 tetramer could be detected in leaves as well as in primary roots of cultured transgenic plants by western blots. These results indicate that the nutritional improvement of cassava storage roots may be achieved by constitutive expression of asp1 in transgenic plants. 相似文献