共查询到20条相似文献,搜索用时 31 毫秒
1.
High-efficiency induction of soybean hairy roots and propagation of the soybean cyst nematode 总被引:8,自引:0,他引:8
Cotyledon explants of 10 soybean [Glycine max (L.) Merr.] cultivars were inoculated with Agrobacterium rhizogenes strain K599 with and without binary vectors pBI121 or pBINm-gfp5-ER possessing both neomycin phosphotransferase II (nptII) and β-glucuronidase (gus) or nptII and green fluorescent protein (gfp) genes, respectively. Hairy roots were produced from the wounded surface of 54–95% of the cotyledon explants on MXB selective
medium containing 200 μg ml−1 kanamycin and 500 μg ml−1 carbenicillin. Putative individual transformed hairy roots were identified by cucumopine analysis and were screened for transgene
incorporation using polymerase chain reaction. All of the roots tested were found to be co-transformed with T-DNA from the
Ri-plasmid and the transgene from the binary vectors. Southern blot analysis confirmed the presence of the 35S-gfp5 gene in the plant genomes. Transgene expression was also confirmed by histochemical GUS assay and Western blot analysis for
the GFP. Attempts to induce shoot formation from the hairy roots failed. Infection of hairy roots of the soybean cyst nematode
(Heterodera glycines Ichinohe)-susceptible cultivar, Williams 82, with eggs of H. glycines race 1, resulted in the development of mature cysts about 4–5 weeks after inoculation. Thus the soybean cyst nematode could
complete its entire life cycle in transformed soybean hairy-root cultures expressing GFP. This system should be ideal for
testing genes that might impart resistance to soybean cyst nematode.
Received: 13 July 1999 / Accepted: 8 August 1999 相似文献
2.
High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures 总被引:6,自引:0,他引:6
K. Rajasekaran R. L. Hudspeth J. W. Cary D. M. Anderson T. E. Cleveland 《Plant cell reports》2000,19(6):539-545
Stable transformation of cotton (Gossypium hirsutum L.) at a high frequency has been obtained by particle bombardment of embryogenic cell suspension cultures. Transient and
stable expression of the β-glucuronidase (GUS) gene was monitored in cell suspension cultures. Transient expression, measured 48 h after bombardment,
was abundant, and stable expression was observed in over 4% of the transiently expressing cells. The high efficiency of stable
expression is due to the multiple bombardment of rapidly dividing cell suspension cultures and the selection for transformed
cells by gradually increasing the concentrations of the antibiotic Geneticin (G418). Southern analysis indicated a minimum
transgene copy number of one to four in randomly selected plants. Fertile plants were obtained from transformed cell cultures
less than 3 months old. However, transgenic and control plants from cell cultures older than 6 months produced plants with
abnormal morphology and a high degree of sterility.
Received: 20 January 1999 / Revision received: 1 October 1999 / Accepted: 11 October 1999 相似文献
3.
Parshikov IA Freeman JP Williams AJ Moody JD Sutherland JB 《Applied microbiology and biotechnology》1999,52(4):553-557
Cultures of the fungi Aspergillus niger, Cunninghamella verticillata, and Penicillium simplicissimum, grown in a sucrose/peptone medium, transformed N-acetylphenothiazine to N-acetylphenothiazine sulfoxide (from 13% to 28% of the total) and phenothiazine sulfoxide (from 5% to 27%). Phenothiazin-3-one
(4%) and phenothiazine N-glucoside (4%) were also produced by C. verticillata. The probable intermediate, phenothiazine, was detected only in cultures of P. simplicissimum (6%).
Received: 15 January 1999 / Received revision: 7 May 1999 / Accepted: 21 May 1999 相似文献
4.
The role of the alternative respiratory pathway in the stimulation of cephalosporin C formation by soybean oil in Acremonium chrysogenum 总被引:1,自引:0,他引:1
L. Karaffa E. Sándor J. Kozma C. P. Kubicek A. Szentirmai 《Applied microbiology and biotechnology》1999,51(5):633-638
Addition of soybean oil to Acremonium chrysogenum cultures growing on sugars doubled the specific production of cephalosporin C during the idiophase of growth. While the addition
of soybean oil had no effect on the total rate of respiration, the respiration that proceeded via the alternative, cyanide-insensitive
pathway exhibited a more than twofold increase. Addition of soybean oil also stimulated the formation of isocitrate lyase
activities. Inhibition of oxidative metabolism of one of the products of isocitrate lyase (succinate) by thenoyltrifluoroacetone
completely inhibited the alternative respiratory pathway. The role of soybean-oil-stimulated alternative respiration in the
stimulation of cephalosporin C production and the role of isocitrate lyase are discussed.
Received: 13 October 1998 / Revised revision: 14 January 1999 / Accepted: 22 January 1999 相似文献
5.
Ye K Shibasaki S Ueda M Murai T Kamasawa N Osumi M Shimizu K Tanaka A 《Applied microbiology and biotechnology》2000,54(1):90-96
An engineered yeast with emission of fluorescence from the cell surface was constructed. Cell surface engineering was applied
to display a visible reporter molecule, green fluorescent protein (GFP). A glucose-inducible promoter GAPDH as a model promoter was selected to control the expression of the reporter gene in response to environmental changes. The
GFP gene was fused with the gene encoding the C-terminal half of α-agglutinin of Saccharomyces cerevisiae having a glycosylphosphatidylinositol anchor attachment signal sequence. A secretion signal sequence of the fungal glucoamylase
precursor protein was connected to the N-terminal of GFP. This designed gene was integrated into the TRP1 locus of the chromosome of S. cerevisiae with homologous recombination. Fluorescence microscopy demonstrated that the transformant cells emitted green fluorescence
derived from functionally expressed GFP involved in the fusion molecule. The surface display of GFP was further verified by
immunofluorescence labeling with a polyclonal antibody (raised in rabbits) against GFP as the first antibody and Rhodamine
Red-X-conjugated goat anti-rabbit IgG as the second antibody which cannot penetrate into the cell membrane. The display of
GFP on the cell surface was confirmed using a confocal laser scanning microscope and by measuring fluorescence in each cell
fraction obtained after the subcellular fractionation. As GFP was proved to be displayed as an active form on the cell surface,
selection of promoters will endow yeast cells with abilities to respond to changes in environmental conditions, including
nutrient concentrations in the media, through the emission of fluorescence.
Received: 23 August 1999 / Received revision: 16 November 1999 / Accepted: 29 November 1999 相似文献
6.
Regeneration of garlic callus as affected by clonal variation, plant growth regulators and culture conditions over time 总被引:3,自引:0,他引:3
A long-term regeneration system for garlic (Allium sativum L.) clones of diverse origin was developed. Callus was initiated on a modified Gamborg's B-5 medium supplemented with 4.5 μM 2,4-D and maintained on the same basal medium with 4.7 μM picloram+0.49 μM 2iP. Regeneration potential of callus after 5, 12 and 16 months on maintenance medium was measured using several plant growth
regulator treatments. The 1.4 μM picloram+13.3 μM BA treatment stimulated the highest rate of shoot production. Regeneration rate decreased as callus age increased, but healthy
plantlets from callus cultures up to 16-months-old were produced for all clones. Regeneration of long-term garlic callus cultures
could be useful for clonal propagation and transformation.
Received: 24 September 1998 / Revision received: 27 January 1999 / Accepted: 26 February 1999 相似文献
7.
Organophosphorus hydrolase (OPH) is capable of degrading a variety of pesticides and nerve agents. We have developed a versatile
monitoring technique for detecting the amount of OPH during the expression and purification steps. This involves fusion of
the gene for green fluorescent protein (GFP) to the 5′ end of the OPH gene and subsequent expression in Escherichia coli. The synthesized fusion protein was directly visualized due to the optical properties of GFP. Western blot analyses showed
that the correct fusion protein was expressed after IPTG-induction. Also, the in vivo GFP fluorescence intensity was proportional
to the OPH enzyme activity. Moreover, the OPH, which forms a dimer in its active state, retained activity while fused to GFP.
Enterokinase digestion experiments showed that OPH was separated from the GFP reporter after purification via immobilized
metal affinity chromatography, which in turn was monitored by fluorescence. The strategy of linking GFP to OPH has enormous
potential for improving enzyme production efficiency, as well as enhancing field use, as it can be monitored at low concentrations
with inexpensive instrumentation based on detecting green fluorescence.
Received: 27 April 1999 / Received last revision: 18 October 1999 / Accepted: 1 November 1999 相似文献
8.
Production of (R)-3-pentyn-2-ol through stereoinversion of racemic 3-pentyn-2-ol by Nocardia fusca AKU 2123 总被引:1,自引:0,他引:1
Wet cells of Nocardia fusca AKU 2123 are good catalysts for the production of (R)-3-pentyn-2-ol (PYOH) from (RS)-PYOH through a stereoinversion reaction. Under optimal conditions (350 mM potassium phosphate buffer, pH 8.0, 30% (w/v)
wet cells, 0.12% NADPH, 10% glucose, and 30 U/ml glucose dehydrogenase) (R)-PYOH of high optical purity (98.7% e.e.) was produced from 2% (v/v) (RS)-PYOH with a yield of 70.4% by 140 h incubation.
Received: 22 January 1999 / Received revision: 23 April 1999 / Accepted: 1 May 1999 相似文献
9.
Genotype screening for proliferative embryogenesis and biolistic transformation of short-season soybean genotypes 总被引:5,自引:0,他引:5
Eighteen of 20 short-season soybean (Glycine max (L.) Merrill) genotypes (maturity group 0 and 00) screened for proliferative embryogenic capacity formed secondary globular
embryos, at rates of 1–70% of cultured immature cotyledons. Five genotypes produced embryogenic cultures which were proliferative
for at least 6 months. Proliferative embryogenic cultures of AC Colibri and X2650–7–2–3 were bombarded using a Bio-Rad PDS-1000/He
particle gun. Co-bombardments with plasmid pairs pHygr (encoding a type IV aminoglycoside phosphotransferase;aphIV) and pRD300pat (encoding a phosphinothricin N-acetyltransferase;pat) or pRD300pat and pFF19G (β-glucuronidase;uidA or gus) resulted, respectively, in 12 hygromycin-selected lines with multiple insertions of aphIV and pat, and two l-phosphinothricin-selected lines plus three β-glucuronidase-positive lines recovered without selection. Although
fertile plants were recovered from young proliferative cultures, transgenic plants, which were derived from cultures 12–14
months of age, were sterile.
Received: 8 January 1998 / Revision received: 12 January 1999 / Accepted: 12 July 1999 相似文献
10.
Terashima M Murai Y Kawamura M Nakanishi S Stoltz T Chen L Drohan W Rodriguez RL Katoh S 《Applied microbiology and biotechnology》1999,52(4):516-523
Recombinant human α1-antitrypsin (rAAT) was expressed and secreted from transgenic rice cell suspension cultures in its biologically active form.
This was accomplished by transforming rice callus tissues with an expression vector, p3D-AAT, containing the cDNA for mature
human AAT protein. Regulated expression and secretion of rAAT from this vector was achieved using the promoter, signal peptide,
and terminator from a rice α-amylase gene Amy3D. The Amy3D gene of rice is tightly controlled by simple sugars such as sucrose. It was possible, therefore, to induce the expression
of the rAAT by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically.
Although transgenic rice cell produced a heterogeneous population of the rAAT molecules, they had the same N-terminal amino
acids as those found in serum-derived (native) AAT from humans. This result indicates that the rice signal peptidase recognizes
and cleaves the novel sequence between the Amy3D signal peptide and the first amino acid of the mature human AAT. The highest molecular weight band seen on Western blots
(AAT top band) was found to have the correct C-terminal amino acid sequence and normal elastase binding activity. Staining
with biotin-concanavalin A and avidin horseradish peroxidase confirmed the glycosylation of the rAAT, albeit to a lesser extent
than that observed with native AAT. The rAAT, purified by immunoaffinity chromatography, had the same association rate constant
for porcine pancreatic elastase as the native AAT. Thermostability studies revealed that the rAAT and native AAT decayed at
the same rate, suggesting that the rAAT is correctly folded. The productivity of rice suspension cells expressing rAAT was
4.6–5.7 mg/g dry cell. Taken together, these results support the use of rice cell culture as a promising new expression system
for production of biologically active recombinant proteins.
Received: 18 January 1999 / Received revision: 26 April 1999 / Accepted: 1 May 1999 相似文献
11.
M. Walfridsson M. Anderlund X. Bao B. Hahn-Hägerdal 《Applied microbiology and biotechnology》1997,48(2):218-224
Saccharomyces cerevisiae was transformed with the Pichia stipitis CBS 6054 XYL1 and XYL2 genes encoding xylose reductase (XR) and xylitol dehydrogenase (XDH) respectively. The XYL1 and XYL2 genes were placed under the control of the alcohol dehydrogenase 1 (ADH1) and phosphoglycerate kinase (PGK1) promoters in the yeast vector YEp24. Different vector constructions were made resulting in different specific activities
of XR and XDH. The XR:XDH ratio (ratio of specific enzyme activities) of the transformed S. cerevisiae strains varied from 17.5 to 0.06. In order to enhance xylose utilisation in the XYL1-, XYL2-containing S. cerevisiae strains, the native genes encoding transketolase and transaldolase were also overexpressed. A strain with an XR:XDH ratio
of 17.5 formed 0.82 g xylitol/g consumed xylose, whereas a strain with an XR:XDH ratio of 5.0 formed 0.58 g xylitol/g xylose.
The strain with an XR:XDH ratio of 0.06, on the other hand, formed no xylitol and less glycerol and acetic acid compared with
strains with the higher XR:XDH ratios. In addition, the strain with an XR:XDH ratio of 0.06 produced more ethanol than the
other strains.
Received: 12 March 1997 / Received revision: 17 April 1997 / Accepted: 27 April 1997 相似文献
12.
Biotechnology of succinic acid production and markets for derived industrial products 总被引:29,自引:4,他引:25
Succinic acid, derived from fermentation of agricultural carbohydrates, has a specialty chemical market in industries producing
food and pharmaceutical products, surfactants and detergents, green solvents and biodegradable plastics, and ingredients to
stimulate animal and plant growth. As a carbon-intermediate chemical, fermentation-derived succinate has the potential to
supply over 2.7 × 108 kg industrial products/year including: 1,4-butanediol, tetrahydrofuran, γ-butyrolactone, adipic acid, n-methylpyrrolidone and linear aliphatic esters. Succinate yields as high as 110 g/l have been achieved from glucose by the
newly discovered rumen organism Actinobacillus succinogenes. Succinate fermentation is a novel process because the greenhouse gas CO2 is fixed into succinate during glucose fermentation. New developments in end-product recovery technology, including water-splitting
electrodialysis and liquid/liquid extraction have lowered the cost of succinic acid production to U.S. $ 0.55/kg at the 75 000
tonne/year level and to $ 2.20/kg at the 5000 tonne/year level. Research directions aimed at further improving the succinate
fermentation economics are discussed.
Received: 27 October 1998 / Received revision: 22 January 1999 / Accepted: 22 January 1999 相似文献
13.
Genetically transformed plants of a phalaenopsis orchid [Doritaenopsis Coral Fantasy×Phalaenopsis (Baby Hat×Ann Jessica)] were regenerated after cocultivation of cell clumps with Agrobacterium tumefaciens strains LBA4404 (pTOK233) and EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (GUS) and hygromycin resistance.
The efficiency of transformation was markedly increased by 10 h cocultivation of cell clumps with A. tumefaciens that had been induced with 200 μm acetosyringone, and by inclusion of 500 μm acetosyringone in the cocultivation medium. Hygromycin-resistant cell clusters (0.5–3 mm in diameter) were selected from
the infected cell clumps after 4–6 weeks of culture on agar (8 g/l)-solidified new Dogashima medium (NDM) containing 20 g/l
sucrose, 0.1 mg/l naphthaleneacetic acid, 1.0 mg/l benzyladenine (BA), 50 mg/l hygromycin and 300 mg/l cefotaxime. The cell
clusters proliferated 4 weeks after transfer onto the same medium. To induce callus greening, the carbon source was changed
from sucrose to maltose. The green calli obtained produced protocorm-like bodies (PLBs) after 4 weeks of culture on phytohormone-free
NDM medium. Regeneration of transgenic plantlets was enhanced by incubating PLBs on NDM medium supplemented with 0.1 mg/l
abscisic acid, followed by partial desiccation for 10–30 min. Successful transformation was confirmed by histochemical GUS
assay, PCR analysis and Southern hybridization of transformants. With this transformation system, more than 100 hygromycin-resistant
phalaenopsis plantlets were produced about 7 months following infection of the cell aggregates.
Received: 10 November 1998 / Revision received: 4 June 1999 / Accepted: 22 June 1999 相似文献
14.
Babu KR Swaminathan S Marten S Khanna N Rinas U 《Applied microbiology and biotechnology》2000,53(6):655-660
Escherichia coli TG1 transformed with a temperature-regulated interferon-α expression vector was grown to high cell density in defined medium
containing glucose as the sole carbon and energy source, utilizing a simple fed-batch process. Feeding was carried out to
achieve an exponential increase in biomass at growth rates which minimized acetate production. Thermal induction of such high
cell density cultures resulted in the production of ∼4 g interferon-α/l culture broth. Interferon-α was produced exclusively
in the form of insoluble inclusion bodies and was solubilized under denaturing conditions, refolded in the presence of arginine
and purified to near homogeneity, utilizing single-step ion-exchange chromatography on Q-Sepharose. The yield of purified
interferon-α was ∼300 mg/l with respect to the original high cell density culture broth (overall yield of ∼7.5% active interferon-α).
The purified recombinant interferon-α was found by different criteria to be predominantly monomeric and possessed a specific
bioactivity of ∼2.5 × 108 IU/mg based on viral cytopathic assay.
Received: 8 October 1999 / Received revision: 8 December 1999 / Accepted: 12 December 1999 相似文献
15.
A method employing isolated microspore culture was established for the androgenic embryogenesis of timothy (Phleum pratense L). Embryos/calli were obtained and green plants regenerated. The induction medium was PG-96 (1.0 mg l−1 2,4-D, 0.1 mg l−1 Kinetin) supplemented with 6% maltose monohydrate. Timothy microspore culture was genotype-dependent, among 12 genotypes,
6 produced embryos/calli and 4 produced green plants. Macerating the spikes with a blender and purifying the microspores at
a mannitol/maltose monohydrate interface gave a relatively high percentage of cell vitality. The optimum microspore developmental
stage was from the very late uninucleate stage to the binucleate stage. Heat shock promoted the initiation of microspore culture.
Over 150 regenerated green plants were obtained; in a random sample of 32 of these 65.6% were doubled haploids (6n=42). Albinism
was a problem in plant regeneration (9.3–22%). This paper is the first to describe timothy androgenic embryogenesis by isolated
microspore culture.
Received: 9 September 1999 / Revision received: 6 December 1999 / Accepted: 13 December 1999 相似文献
16.
Torres R Ramón F de la Mata I Acebal C Castillón MP 《Applied microbiology and biotechnology》1999,53(1):81-84
At 28 °C, Streptomyces lavendulae produced high levels of penicillin V acylase (178 IU/l of culture) when grown on skim milk as the sole nutrient source for
275 h. The enzyme showed catabolite repression by glucose and was produced in the stationary phase of growth. Penicillin V
was a good inducer of penicillin V acylase formation, while phenoxyacetic acid, the side-chain moiety of penicillin V, did
not alter enzyme production significantly. The enzyme was stable between pH 6 and 11 and at temperatures from 20 °C to 55 °C.
This extracellular enzyme was able to hydrolyse natural penicillins and unable to hydrolyse penicillin G.
Received: 22 March 1999 / Received revision: 16 June 1999 / Accepted: 20 June 1999 相似文献
17.
Production of transgenic gentian plants by particle bombardment of suspension-culture cells 总被引:3,自引:0,他引:3
Cell suspension cultures were established from leaf explants of gentian (Gentiana triflora×G. scabra) for the generation of transgenic plants by particle bombardment. The parameters for the bombardment of suspension culture
cells with a particle gun were examined by monitoring the transient expression of a gene for β-glucuronidase driven by the
cauliflower mosaic virus (CaMV) 35S promoter. We found that prior culture of suspension culture cells for 5 days on solid
medium was optimum for successful particle bombardment. Putative transformed calli were obtained from bombarded cells after
a two-step selection procedure. Cells were cultured first with 30 mg l–1 hygromycin in liquid MS medium that contained 10 mg l–1
N-phenyl-N′-1,2,3-thiadiazol-5-yl urea, 1 mg/l 1-naphthaleneacetic acid and 30 g l–1 sucrose and then on solid medium prepared from the same liquid medium plus 2 g l–1 gellan gum. After 12 weeks of selection on solid medium that contained 30 mg l–1 hygromycin, two transgenic gentian plants were regenerated from each selected callus. Analysis by the polymerase chain reaction
and Southern blotting revealed the stable integration of transferred DNA.
Received: 3 June 1999 / Revision received: 21 September 1999 / Accepted: 20 September 1999 相似文献
18.
Barley microspore-derived doubled-haploid embryos have been produced in vitro. The development of embryo desiccation technology will allow long-term storage, germplasm preservation and low delivery cost.
Treatment of the microspore-derived embryos was essential to induce desiccation tolerance and to arrest further development
and plant regeneration. At the concentrations used, a treatment with trehalose was more efficient than with sucrose, and mannitol
was harmful to the embryos. Up to 80% of the desiccated embryos produced complete green plants when transferred to regeneration
medium, by the application of a 0.6 m trehalose or a 10–5 m abscisic acid treatment to the embryos in the culture induction medium. The morphology of these plants was similar to plants
produced directly from non-desiccated embryos.
Received: 28 September 1998 / Revision received: 27 November 1998 / Accepted: 5 January 1999 相似文献
19.
A range of biomimetic analogues of the nicotinamide nucleotide coenzymes NAD(P)(H) have been developed based on the structure
of a triazine dye template. These biomimetic redox coenzymes are relatively straightforward and inexpensive to synthesise
and display NAD+-like activity with different dehydrogenases, despite their apparently minimal structural similarity to the native coenzyme
NAD+. Horse liver alcohol dehydrogenase oxidises butan-1-ol, using the most active biomimetic coenzyme (Nap 1), with a k
cat value an order of magnitude lower and a K
m for the coenzyme two orders of magnitude higher than those using native NAD+. The enzymatically reduced biomimetic coenzymes may be reoxidised by phenazine methosulfate. We believe that these coenzymes
may find applications in biotransformations and biosensors, and in the development of biomimetic catalysts where the redox
enzyme itself is replaced by a synthetic binding site.
Received: 26 October 1998 / Received revision: 25 January 1999 / Accepted: 31 January 1999 相似文献
20.
Pi Nyvall Jerome Pelloux Howard V. Davies Marianne Pedersén Roberto Viola 《Planta》1999,209(1):143-152
Red algae (Rhodophyceae) are photosynthetic eukaryotes that accumulate starch granules in the cytosol. Starch synthase activity
in crude extracts of Gracilaria tenuistipitata Chang et Xia was almost 9-fold higher with UDP[U-14C]glucose than with ADP[U-14C]glucose. The activity with UDP[U-14C]glucose was sensitive to proteolytic and oxidative inhibition during extraction whilst the activity with ADP[U-14C]glucose appeared unaffected. This indicates the presence of separate starch synthases with different substrate specificities
in G. tenuistipitata. The UDPglucose: starch synthase was purified and characterised. The enzyme appears to be a homotetramer with a native Mr of 580 kDa and displays kinetic properties similar to other α-glucan synthases such as stimulation by citrate, product (UDP)
inhibition and broad primer specificity. We propose that this enzyme is involved in cytosolic starch synthesis in red algae
and thus is the first starch synthase described that utilises UDPglucose in vivo. The biochemical implications of the different
compartmentalisation of starch synthesis in red algae and green algae/plants are also discussed.
Received: 29 January 1999 / Accepted: 11 March 1999 相似文献