共查询到20条相似文献,搜索用时 31 毫秒
1.
Summary Whole cell transformation ofLactobacillus plantarum CCM 1904 by electroporation was optimized. Pulse duration and electric field strength were shown to be important parameters: the optimum conditions were 12.5 kV/cm, a time-constant of 10 ms for an exponential decay waveform and 6.7 kV/cm applied during 2.5 ms for a square waveform. Transformation efficiency was increased if cells were cultivated on medium containing sorbitol and harvested during their early exponential growth phase: 8 × 10–4 transformants/g pGK12 DNA per viable cell were obtained, with a survival rate of 10%–30% Cryotreatment by several freeze-and-thaw cycles decreased transformant yields. Transformation efficiency with different plasmids was studied and plasmid pGK12 was found to transformL. plantarum the most efficiently. Transformation by electroporation ofL. plantarum is strain dependent. The best results were obtained withL. plantarum NCIB 7220, giving 5 × 106 transformants/gmg plasmid pGK12 DNA. 相似文献
2.
Summary A method was developed for the introduction of plasmids into Clostridium botulinum by electroporation. A 4.4 kb plasmid vector, pGK12, which contains genes for resistance to erythromycin (Emr) and chloramphenicol (Cmr) was electroporated into C. botulinum type A (Hall A). The highest transformation efficiency was obtained using midlog phase cells, 10% PEG 8000 as the electroporation solution, and 2.5 kV field strength. The transformation efficiency was highest (103 transformants/g of DNA) when 1 g of plasmid DNA and 4 × 108 CFU/ml of recipient cells were used. Plasmid DNA recovered from the transformants was indistinguishable from that introduced on the basis of restriction enzyme digestion and agarose gel electrophoresis. 相似文献
3.
Ruirong Yi Takashi Tachikawa Hiroyuki Mukaiyama Yusuke Mochida Mariko Ishikawa Tadanori Aimi 《Mycoscience》2009,50(2):123-129
We cloned a gene encoding the succinate dehydrogenase iron-sulfur protein subunit (sip) from a bipolar mushroom, Pholiota microspora, and introduced a point mutation that confers carboxin resistance into this gene. Using this homologous selective marker
and also a heterologous drug selective marker, the hygromycin B phosphotransferase gene (hph), we successfully constructed a DNA-mediated transformation system in P. microspora. Both these selection markers have high transformation efficiency: the efficiency of carboxin resistance transformation was
about 88.8 transformants/μg pMBsip2 DNA using 5 × 106 protoplasts in regeneration plates containing 1.0 μg/ml carboxin, and the efficiency of hygromycin B resistance transformation
was about 122.4 transformants/μg pMBhph1 DNA using 5 × 106 protoplasts in regeneration plates containing 150 μg/ml hygromycin B. Southern hybridization analysis showed that the introduced
sequence (mutant sip or hph) was integrated into the chromosomal DNA in these transformants with a copy number of one or more. 相似文献
4.
C T Hou W Brown D P Labeda T P Abbott D Weisleder 《Journal of industrial microbiology & biotechnology》1997,19(1):34-38
A bacterium isolated from a dry soil sample collected from McCalla, AL, USA, converted linoleic acid to a novel compound,
12,13,17-trihydroxy-9 (Z)-octadecenoic acid (THOA). The organism is a Gram-positive, non-motile rod (0.5 μ m × 2 μ m). It was identified as a species of Clavibacter ALA2. The product was purified by high pressure liquid chromatography, and its structure was determined by 1H and 13C nuclear magnetic resonance and Fourier transform infrared spectroscopies, and by mass spectrometer. Maximum production
of THOA with 25% conversion of the substrate was reached after 5–6 days of reaction. THOA was not further metabolized by
strain ALA2. This is the first report of a 12,13,17-trihydroxy unsaturated fatty acid and its production by microbial transformation.
Some dihydroxy intermediates were also detected. THOA has a structure similar to those of known plant self-defense substances.
Received 13 January 1997/ Accepted in revised form 05 May 1997 相似文献
5.
Cultures able to dechlorinate cis-1,2-dichloroethene (cDCE) were selected with ethene (3–20%, v/v) as the sole source of carbon and energy. One mixed culture (K20) could degrade
cDCE (400 μmol l–1) or vinyl chloride (100 μmol l–1) in the presence of ethene (≤ 80 μmol l–1 and ≤ 210 μmol l–1, respectively). This culture consists of at least five bacterial strains. All five strains were able to degrade cDCE cometabolically in pure culture. The mixed culture K20 was highly tolerant against cDCE (up to 6 mmol l–1 in the liquid phase). Degradation of cDCE (200 μmol l–1) was not affected by the presence of trichloroethene (100 μmol l–1) or tetrachloroethene (100 μmol l–1). Transformation yields (Ty, defined as unit mass of chloroethene degraded per unit mass of ethene consumed) of the mixed culture K20 were relatively
high (0.51 and 0.61 for cDCE and vinyl chloride, respectively). The yield for cDCE with ethene as auxiliary substrate was ninefold higher than any values reported with methane or methane/formate as auxiliary
substrate. The viability of the cells of the mixed culture K20 (0.3 mg of cells ml–1) was unaffected by the transformation of ≤ 200 μmol l–1
cDCE in 300 min.
Received: 9 March 1999 / Accepted: 21 July 1999 相似文献
6.
Efficient transformation of pBR322 and its derived
plasmids, which have been widely used as cloning vectors in Escherichia
coli, was observed in Pseudomonas avenae (K1), the pathogen of
leaf blight disease in cereals. Moreover, there was a 10- to 50-fold
transformation efficiency (1.3–3.0 × 106/μg DNA) in the
proline-auxotrophic mutant (Pr47), whose virulence to rice seedlings
decreased. Similar enhancement of the frequency of transfer by mobilization
of RSF1010, a broad host range plasmid, was observed in the recipient Pr47
strain in mating with donor Pseudomonas syringae. The plasmids
harbored in these strains were maintained very stably after subcultures.
Thus, a highly efficient transformation system with pBR322-derived plasmids
used as a vector and Pseudomonas as a host bacterium was developed.
Received: 13 July 1996 / Accepted: 26 August 1996 相似文献
7.
For the first time, the phosphomannose isomerase (PMI, EC 5.3.1.8)/mannose-based “positive” selection system has been used
to obtain genetically engineered sugarcane (Saccharum spp. hybrid var. CP72-2086) plants. Transgenic lines of sugarcane were obtained following biolistic transformation of embryogenic
callus with an untranslatable sugarcane mosaic virus (SCMV) strain E coat protein (CP) gene and the Escherichia coli PMI gene manA, as the selectable marker gene. Postbombardment, transgenic callus was selectively proliferated on modified MS medium containing
13.6 μM 2,4-D, 20 g l−1 sucrose and 3 g l−1 mannose. Plant regeneration was obtained on MS basal medium with 2.5 μM TDZ under similar selection conditions, and the regenerants
rooted on MS basal medium with 19.7 μM IBA, 20 g l−1 sucrose, and 1.5 g l−1 mannose. An increase in mannose concentration from permissive (1.5 g l−1) to selective (3 g l−1) conditions after 3 weeks improved the overall transformation efficiency by reducing the number of selection escapes. Thirty-four
vigorously growing putative transgenic plants were successfully transplanted into the greenhouse. PCR and Southern blot analyses
showed that 19 plants were manA-positive and 15 plants were CP-positive, while 13 independent transgenics contained both transgenes. Expression of manA in the transgenic plants was evaluated using a chlorophenol red assay and enzymatic analysis. 相似文献
8.
Plant regeneration via somatic embryogenesis was achieved from leaf petioles of Pelargonium sp. `Frensham' cultured on Murashige and Skoog medium containing 15 μM N6-benzyladenine, and 5 μM α-naphthaleneacetic acid (NAA). More than 80% of these somatic embryos converted into plants when
isolated and cultured on Murashige and Skoog medium supplemented with 15 μM NAA. Stable transgenic plants were obtained by
co-cultivation of the petioles (prior to culture) with Agrobacterium tumefaciens strains LBA4404 (harbouring a binary vector pBI121 carrying the nptII and gus genes) and LBG66 (harbouring a binary plasmid pJQ418 carrying the gus/int:nptII fusion gene). Transformants were selected using kanamycin and transformation was verified by β-glucuronidase histochemical
assay and polymerase chain reaction. Southern analysis further confirmed the integration of these genes into the genome of
transgenic plants. We report here for the first time, an Agrobacterium-mediated model transformation system coupled with regeneration via somatic embryogenesis for production of transgenics in
Pelargonium sp.
Received: 20 September 1996 / Accepted: 13 November 1996 相似文献
9.
In this study, an Agrobacteriurn tumefaciens-mediated transformation (ATMT) protocol was successfully developed for the genetic transformation of a taxol-producing fungus,
Cladosporium cladosporioides MD2, and the co-cultivation conditions affecting the transformation efficiency were optimized. The optimal transformation
conditions were that 1 ml of C. cladosporioides MD2 spore suspension (108 spores/ml) was mixed with an equal volume of A. tumefaciens cultures, which contained 400 μl of A. tumefaciens LBA4404 (OD660 ≈ 0.6) and 600 μl LB medium that were used to make up difference in volume, and the mix cultures were supplemented with 300 μM
acetosyringone (AS) and co-cultivated at 26°C and 50 rpm for 48 h. Stable transformants were obtained through analysis of
the mitotic stability of inserted T-DNA and the presence of hygromycin resistance gene (hpt II). This study laid a fine groundwork for development of transgenic C. cladosporioides MD2 strains. 相似文献
10.
The Alternaria mycotoxin tenuazonic acid (TA) was quantified in fruit juices (n = 50), cereals (n = 12) and spices (n = 38) using a recently developed stable isotope dilution assay (SIDA). [13 C6,15 N]-TA was used as the internal standard. Method validation revealed low limits of detection (LODs) of 0.15 μg/kg (fruit juices),
1.0 μg/kg (cereals) and 17 μg/kg (spices). The respective limits of quantitation were about three times higher. Recovery was
about 100% for all matrices. The precision (relative standard deviation of replicate analyses of naturally contaminated samples)
was 4.2% (grape juice; 1.7 μg/kg), 3.5% (whole wheat flour; 36 μg/kg) and 0.9% (curry powder; 215 μg/kg). The median content
of TA in the analyzed samples was 1.8 μg/kg (fruit juices), 16 μg/kg (cereals) and 500 μg/kg (spices). Positive samples amounted
to 86% (fruit juices), 92% (cereals) and 87% (spices). 相似文献
11.
A protocol for Agrobacterium-mediated transformation was developed for in vitro leaf explants of an elite, mature Prunus serotina tree. Agrobacterium tumefaciens strain EHA105 harboring an RNAi plasmid with the black cherry AGAMOUS (AG) gene was used. Bacteria were induced for 12 h with 200 μM acetosyringone for vir gene induction before leaf explant inoculation. Explants were co-cultured for 3 days, and then cultured on woody plant medium
supplemented with 9.08 μM thidiazuron, 1.07 μM napthaleneacetic acid, 60 μM silver thiosulphate, 3% sucrose, plus 200 mg l−1 timentin in darkness for 3 weeks. Regenerating shoots were selected 27 days after initial co-culture, on Murashige and Skoog
medium with 3% sucrose, 8.88 μM 6-benzylaminopurine, 0.49 μM indole-3-butyric acid, 0.29 μM gibberellic acid, 200 mg l−1 timentin, and 30 mg l−1 kanamycin for five subcultures. After 5–6 months of selection, transformation efficiencies were determined, based on polymerase
chain reaction (PCR) analysis of individual putative transformed shoots relative to the initial number of leaf explants tested.
The transformation efficiency was 1.2%. Southern blot analysis of three out of four PCR-positive shoots confirmed the presence
of the neomycin phosphotransferase and AG genes. Transgenic shoots were rooted (37.5%), but some shoot tips and leaves deteriorated or died, making acclimatization
of rooted transgenic plants difficult. This transformation, regeneration, and rooting protocol for developing transgenic black
cherry will continue to be evaluated in future experiments, in order to optimize the system for several mature black cherry
genotypes. 相似文献
12.
Scott P. Burns Maria Gallo Barry L. Tillman 《In vitro cellular & developmental biology. Plant》2012,48(1):58-66
Agrobacterium-mediated transformation, employing direct shoot organogenesis, allows for mature transgenic plants to be obtained quickly
(3–4 mo). In this study, peanut (Arachis hypogaea L.) cultivars Florida-07, Georgia Green, Georgia Brown, New Mexico Valencia A, and VC-2 were selected to test their shoot
induction response for use in future transformation experiments. Two types of cotyledon explants were examined, those that
previously had an attached embryo axis upon cotyledon separation (explant A) and those that were embryo axis-free upon separation
(explant B). Explants were placed onto a shoot induction medium with N
6-benzyladenine concentrations ranging from 10–80 μM for Florida-07, Georgia Green, and VC-2; 10–20 μM for Georgia Brown; and
10–640 μM for New Mexico Valencia A. Following a 4-wk culture period, explants were visually rated based on a scale of 1–4,
where 1 indicated slight greening, but no growth, and 4 indicated greening, adventitious bud formation, as well as small leaf
expansion. A difference in shoot induction was observed for the cotyledon explants examined (P > t = <0.0001). Explant A had greater shoot induction with a visual rating of 1.8 ± 0.1; explant B had a rating of 1.6 ± 0.1
(P > t = <0.0001). Additionally, cultivars responded to the culture conditions differently (cultivar × N
6-benzyladenine interaction). Georgia Green on 10 μM N
6-benzyladenine produced the most shoot buds (24.6%) and the highest visual rating (2.1), followed by VC-2 on 10 μM N
6-benzyladenine (22.1%, 1.8), New Mexico Valencia A on 640 μM N
6-benzyladenine (21.4%, 1.8), Georgia Brown on 80 μM N
6-benzyladenine (9.0%, 1.7), and Florida-07 on 40 μM N
6-benzyladenine (7.1%, 1.8). Of the tested varieties, Georgia Green, New Mexico Valencia A, and VC-2 were best suited for future
transformation experiments based on their shoot bud production. 相似文献
13.
Vojcic L Despotovic D Martinez R Maurer KH Schwaneberg U 《Applied microbiology and biotechnology》2012,94(2):487-493
Bacillus subtilis strains are used for extracellular expression of enzymes (i.e., proteases, lipases, and cellulases) which are often engineered
by directed evolution for industrial applications. B. subtilis DB104 represents an attractive directed evolution host since it has a low proteolytic activity and efficient secretion. B. subtilis DB104 is hampered like many other Bacillus strains by insufficient transformation efficiencies (≤103 transformants/μg DNA). After investigating five physical and chemical transformation protocols, a novel natural competent
transformation protocol was established for B. subtilis DB104 by optimizing growth conditions and histidine concentration during competence development, implementing an additional
incubation step in the competence development phase and a recovery step during the transformation procedure. In addition,
the influence of the amount and size of the transformed plasmid DNA on transformation efficiency was investigated. The natural
competence protocol is “easy” in handling and allows for the first time to generate large libraries (1.5 × 105 transformants/μg plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA. 相似文献
14.
A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration from
Agrobacterium tumefaciens- inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166
and XP85 and inoculated with A. tumefaciens strain EHA105 containing the binary vector pBISN1. PBISN1 has the neomycin phosphotransferase gene (nptII) and an intron interrupted β-glucuronidase (GUS) reporter gene (gusA). Co-cultivation was carried out for 4 d in the dark on callus induction medium (CIM): Gamborg B5 + 2.79 μM kinetin + 2.26 μM
2,4-dichlorophenoxyacetic acid (2,4-D) supplemented with 100 μM acetosyringone. Embryogenic calluses resistant to kanamycin
(Km) were then recovered on CIM + 25 mg l−1 Km + 250 mg l−1 timentin after 12 weeks. Subsequently, a large number of Km-resistant and GUS-positive transformants, tens to hundreds per
explant were regenerated via somatic embryogenesis on Gamborg B5 + 4.92 μM 6 (γ,γ-dimethylallylamino)-purine (2iP) + 1.93 μM
α-naphthaleneacetic acid (NAA) + 25 mg l−1 Km + 250 mg l−1 timentin after 8 weeks. Using this protocol, the transformation frequency was 5.0% and 5.0% for leaf sections, 17.8% and
18.3% for cotyledons, and 15.9% and 16.7% for hypocotyl explants of cvs. XP85 and XP166, respectively. Stable integration
of the model transgenes with 1–3 copy numbers was confirmed in all ten randomly selected transgenic events by Southern blot
analysis of gusA. Progeny analysis by histochemical GUS assay showed stable Mendelian inheritance of the transgenes. Thus, A. tumefaciens-mediated transformation of cotyledons or hypocotyls provides an effective and reproducible protocol for large-scale production
of transgenic celery plants. 相似文献
15.
Primary production by phytoplankton in the eutrophic Mikawa Bay, Japan, was studied by simultaneous measurements of natural
carbon isotope ratio (δ
13C) and short-term carbon uptake rates (13C tracer study) of size-fractionated nannoplankton (<10 μm) and net plankton (>10 μm) samples. Short-term photosynthetic rates,
which represent the physiological state of algae, were variable regardless of standing stock sizes. Theδ
13C values of particulate organic carbon (POC) in June and July displayed horizontal variations for both the net plankton fraction
(−19.8 to −12.7‰) and the nannoplankton fraction (−22.0 to −12.8‰). For both fractions, low concentrations of POC had more
negativeδ
13C values (−22 to −18‰). Highδ
13C values for the net plankton were found when POC concentrations were much higher, due to red tide. This suggests that the
increase in algal standing crop for the net plankton fraction resulted from accelerated photosynthetic activity. However the
nannoplankton fractions with higher POC values have relatively lowδ
13C values. 相似文献
16.
M Ozkan SG Desai Y Zhang DM Stevenson J Beane EA White ML Guerinot LR Lynd 《Journal of industrial microbiology & biotechnology》2001,27(5):275-280
Characteristics of 13 newly isolated thermophilic, anaerobic, and cellulolytic strains were compared with previously described
strains of Clostridium thermocellum: ATCC 27405 and JW20 (ATCC 31549). Colony morphology, antibiotic sensitivity, fermentation end-products, and cellulose degradation
were documented. All 13 strains were sensitive to erythromycin (5 μg/ml) and chloramphenicol (25 μg/ml), and all strains but
one were sensitive to kanamycin (20 μg/ml). Polymerase chain reaction (PCR) amplification using primers based on gene sequences
from C. thermocellum ATCC 27405 was successful for all 13 strains in the case of the hydrogenase gene and 11 strains in the case of phosphotransacetylase/acetate
kinase genes. Ten strains amplified a product of the expected size with primers developed to be specific for C. thermocellum 16SrRNA primers. Two of the 13 strains did not amplify any product with the PCR primers designed for the phosphotransacetylase/acetate
kinase and 16SrRNA primers. A MboI-like GATC- recognizing restriction activity was present in all of the five strains examined. The results of this study have
several positive implications with respect to future development of a transformation system for cellulolytic thermophiles.
Journal of Industrial Microbiology & Biotechnology (2001) 27, 275–280.
Received 12 September 2000/ Accepted in revised form 20 November 2000 相似文献
17.
A genetic transformation protocol for green ash (Fraxinus pennsylvanica) hypocotyl explants was developed. Green ash hypocotyls were transformed using Agrobacterium tumefaciens strain EHA105 harboring binary vector pq35GR containing the neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) fusion gene, and an enhanced green fluorescent protein gene. Pre-cultured hypocotyl explants were
transformed in the presence of 100 μM acetosyringone using 90 s sonication plus 10 min vacuum-infiltration. Kanamycin at 20 mg l−1 was used for selecting transformed cells. Adventitious shoots regenerated on Murashige and Skoog medium supplemented with
13.3 μM 6-benzylaminopurine, 4.5 μM thidiazuron, 50 mg l−1 adenine sulfate, and 10% coconut water. GUS- and polymerase chain reaction (PCR)-positive shoots from the cut ends of hypocotyls
were produced via an intermediate callus stage. Presence of the GUS and nptII genes in GUS-positive shoots were confirmed by PCR and copy number of the nptII gene in PCR-positive shoots was determined by Southern blotting. Three transgenic plantlets were acclimatized to the greenhouse.
This transformation and regeneration system using hypocotyls provides a foundation for Agrobacterium-mediated transformation of green ash. Studies are underway using a construct containing the Cry8Da protein of Bacillus thuringiensis for genetic transformation of green ash. 相似文献
18.
Summary The generation of transgenic Cucumis sativus cv. Greenlong plants resistant to phosphinothricin (PPT) was obtained using Agrobacterium tumefaciens-mediated gene transfer. The protocol relied on the regeneration of shoots from cotyledon explants. Transformed shoots were
obtained on Murashige and Skoog medium supplemented with 4.4 μM 6-benzylaminopurine 3.8 μM abscisic acid, 108.5 μM adenine sulfate, and 2 mg l−1 phosphinothricin. Cotyledons were inoculated with the strain EHA105 harboring the neomycin phosphotransferase II (npt II), and phosphinothricin resistance (bar) genes conferring resistance to kanamycin and PPT. Transformants were selected by using increasing concentrations of PPT
(2–6 mg l−1). Elongation and rooting of putative transformants were performed on PPT-containing (2 mg l−1) medium with 1.4 μM gibberellic acid and 4.9 μM indolebutyric acid, respectively. Putative transformants were confirmed for transgene insertion through PCR and Southern
analysis. Expression of the bar gene in transformed plants was demonstrated using a leaf painting test with the herbicide Basta. Pre-culture of explants
followed by pricking, addition of 50 μM acetosyringone during infection, and selection using PPT rather than kanamycin were found to enhance transformation frequency
as evidenced by transient β-glucuronidase assay. Out of 431 co-cultivated explants, 7.2% produced shoots that rooted and grew
on PPT, and five different plants (1.1%) were demonstrated to be transgenic following Southern hybridization. 相似文献
19.
D. Licht S. S. Johansen E. Arvin B. K. Ahring 《Applied microbiology and biotechnology》1997,47(2):167-172
Degradation of indole and quinoline by Desulfobacterium␣indolicum was studied in batch cultures. The first step in the degradation pathway of indole and quinoline was a hydroxylation at the
2 position to oxindole and 2-hydroxyquinoline respectively. These hydroxylation reactions followed saturation kinetics. The
kinetic parameters for indole were an apparent maximum specific transformation rate (V
Amax) of 263 μmol mg total protein−1 day−1 and an apparent half-saturation constant (K
Am) of 139 μM. The V
Amax for quinoline was 170 μmol mg total protein−1 day−1 and K
Am was 92 μM. Oxindole inhibited indole hydroxylation whereas 2-hydroxyquinoline stimulated quinoline hydroxylation. An adaptation
period of approximately 20 days was required before transformation of 2-hydroxyquinoline in cultures previously grown on quinoline.
Indole and quinoline were hydroxylated with a lag phase shorter than 4 h in a culture adapted to ethanol. Chloramphenicol
inhibited the hydroxylation of indole and quinoline in ethanol-adapted cells, indicating an inducible enzyme system. Chloramphenicol
had no effect on the hydroxylation of indole in quinoline-adapted cells or on the hydroxylation of quinoline in indole-adapted
cells. This indicated that it was the same inducible enzyme system that hydroxylated indole and quinoline.
Received: 16 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996 相似文献
20.
Diel variation in urea decomposing activity in the euphotic zone of brackish Lake Nakaumi 总被引:1,自引:0,他引:1
Diel variations in urea decomposing activity in the euphotic zone of brackish Lake Nakaumi were measured under fixed light
intensity. The decomposition rate of urea was 17 to 44 μ mol urea m−3 h−1 in the light and 10 to 27 μ mol urea m−3 h−1 in the dark. Higher decomposition rates were obtained in the upper euphotic zone. A clear diel periodicity in the urea decomposition
rate was observed, with high rates from 1200 to 1600 and low rates from 0000 to 0400. Chlorophyll a specific decomposing activity ranged from 12 to 21 μg urea C mg chl.a
−1 h−1 in the light and 7 to 13 μg urea C mg chl.a
−1 h−1 in the dark. In the light, high values were obtained from 1600 to 2000 and low values from 0400 to 0800. The diel change
in specific decomposing activity exhibited a similar pattern to that of the photosynthetic assimilation number, following
the diel change in photosynthetic activity.
Received: March 10, 1999 / Accepted: October 22, 1999 相似文献