共查询到20条相似文献,搜索用时 15 毫秒
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Gregorio Godoy-Hernández Elide Avilés Berzunza Lizbeth Castro Concha María de Lourdes Miranda-Ham 《Plant Cell, Tissue and Organ Culture》2006,84(3):365-368
Hypocotyls, roots, leaf sections and shoot tips from Tagetes erecta plantlets were inoculated with Agrobacterium tumefaciens, harboring the binary vector pCAMBIA2301, containing the β-glucuronidase gene. Histochemical GUS assays of infected tissues
showed transient gus gene expression after 3 days. 相似文献
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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
4.
Recently, a new gene encoding β-glucuronidase from Streptococcus equi subsp. zooepidemicus (SEZ) was identified and expressed in Escherichia coli. In this paper, the characterization of the enzyme is described. Specific enzyme activity was 120,000 U/mg purified protein
at 37°C and pH = 7.0. The temperature and pH value, at which the enzyme has the highest specific activity, were determined
and were found to be approximately 52°C and 5.6, respectively. The mutant strain SEZ glcHis was designed for the efficient isolation of β-glucuronidase from S. equi subsp. zooepidemicus. It was observed that the specific activity of β-glucuronidase in the cytoplasmic extract of a mutated strain was about 45%
lower than in the cytoplasmic extract of a wild-type strain. The specific activity of purified β-glucuronidase from SEZ glcHis was four times as low as β-glucuronidase purified from E. coli. Comparing the specific activity of purified streptococcal β-glucuronidase from E. coli with E. coli β-glucuronidase (the enzyme with the highest specific activity was supplied by Sigma), the former is 1.8 higher than the
latter. 相似文献
5.
Factors that influence Agrobacterium rhizogenes-mediated transformation of broccoli (Brassica oleracea L. var. italica) 总被引:2,自引:0,他引:2
An improved broccoli transformation system was developed by optimising several factors that affect the rate of effective
Agrobacterium-mediated transformation. Leaf explants of cultivar Shogun were co-cultivated with Agrobacterium rhizogenes strain A4T harbouring the binary vector pART278. The T-DNA of this binary vector contains a neomycin phosphotransferase II
(NOS-NPTII-NOS) gene for kanamycin resistance and a β-glucuronidase (35S-GUS-OCS) gene. Several media and factors were evaluated
including combinations of arginine, mannopine, acetosyringone and the use of feeder cell layers. The new protocol includes
the use of 200 μm acetosyringone in LB medium for bacterial growth, the use of a Brassica campestris feeder cell layer, 10 mm mannopine and 50 μm acetosyringone in the co-cultivation medium and 1 mm arginine in the selection medium. The use of this optimised protocol produced transformation rates of 33% in preliminary
experiments transforming broccoli with the antisense 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene from pTOM13.
Received: 2 July 1998 / Revision received: 9 February 2000 / Accepted: 17 February 2000 相似文献
6.
Transformation of tomato (Lycopersicon esculentum Mill.) was carried out using disarmed Agrobacterium tumefaciens strain EHA 105 harboring a binary vector pBIG-HYG-bspA. The plasmid contains the bspA (boiling stable protein of aspen) gene under the control of a CaMV35S promoter and nopaline synthase (NOS) terminator, hygromycin phosphotransferase gene (hpt) driven by nopaline synthase promoter and polyadenylation signal of Agrobacterium gene7 as terminator and a promoterless gus gene. Very strong β-glucuronidase (GUS) expression was observed in transformed tomato plants but never in non-transformed
(control). Since GUS expression was observed only in transformed plants, the possibility of the presence of endogenous GUS
enzymes was ruled out. Possibility of false GUS positives was also ruled out because the GUS positive explants reacted positively
to polymerase chain reaction (PCR) and PCR-Southern tests carried out for the presence of bspA gene, which indicated the integration of T-DNA in tomato genome. The promoterless GUS expression was hypothesized either
due to leaky NOS termination signal of bspA gene or due to different cryptic promoters of plant origin. It was concluded that GUS expression was observed in the putative
transgenics either due to the read through mechanism by the strong CaMV35S promoter or due to several cryptic promoters driving
the gus gene in different transgenic lines. 相似文献
7.
Q. Zheng Y. P. Zheng G. D. Wang W. M. Guo W. F. Fan C. Wang 《Russian Journal of Plant Physiology》2012,59(2):266-274
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I. S. Curtis J. B. Power P. Hedden D. A. Ward A. Phillips K. C. Lowe M. R. Davey 《Plant cell reports》1999,18(7-8):554-560
A transformation system is described for Datura meteloides using the supervirulent Agrobacterium tumefaciens strain 1065, carrying both the β-glucuronidase (gusA) and neomycin phosphotransferase II (nptII) genes between the T-DNA border sequences of the binary vector. The importance of conditions such as the preculture period
of the plant tissues, wounding, bacterial dilution and incubation time were evaluated in terms of transgenic plant production.
A preculture period of 2–3 days, using a 1:20 or 1:10 (vol:vol) dilution of an overnight bacterial culture, resulted in optimum
shoot regeneration, with 48% from a total of 576 explants regenerating transformed shoots. Expression of the gusA and nptII genes was confirmed by a GUS fluorometric assay and by NPTII ELISA. Southern analysis revealed the integration of both
transgenes, which segregated as dominant Mendelian traits in seed progeny.
Received: 7 September 1998 / Revision received: 16 November 1998 / Accepted: 16 November 1998 相似文献
11.
Identification of T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium 总被引:11,自引:0,他引:11
J. Nam K. S. Mysore C. Zheng M. K. Knue A. G. Matthysse S. B. Gelvin 《Molecular & general genetics : MGG》1999,261(3):429-438
We have identified T-DNA tagged Arabidopsis mutants that are resistant to transformation by Agrobacterium tumefaciens (rat mutants). These mutants are highly recalcitrant to the induction of both crown gall tumors and phosphinothricin-resistant
calli. The results of transient GUS (β-glucuronidase) assays suggest that some of these mutants are blocked at an early step
in the Agrobacterium-mediated transformation process, whereas others are blocked at a step subsequent to translocation of T-DNA into the nucleus.
Attachment of Agrobacterium to roots of the mutants rat1 and rat3 was decreased under various incubation conditions. In most mutants, the transformation-deficient phenotype co-segregated
with the kanamycin resistance encoded by the mutagenizing T-DNA. In crosses with susceptible wild-type plants, the resistance
phenotype of many of these mutants segregated either as a semi-dominant or dominant trait.
Received: 26 October 1998 / Accepted: 8 January 1999 相似文献
12.
Two important marker proteins used in plant gene expression studies are green fluorescent protein (GFP) and β-glucuronidase
(GUS). We have compared the utility of each in the analysis of a relatively weakArabidopsis thaliana promoter. The background green fluorescence of arabidopsis tissues and organs has been catalogued. This background fluorescence
makes it difficult to detect weak promoter activity driving GFP, a problem compounded by the lack of amplification of the
GFP signal. In the case of β-glucuronidase, due to diffusion of the enzymatic product, GUS may over-report promoter activity.
However, because of the enzymatic amplification of the signal and the low β-glucuronidase activity of untransformed arabidopsis
tissues, weak promoter activity is more easily and more accurately detected using GUS. 相似文献
13.
Xanthomonas campestris pv. campestris, the causal agent of black-rot disease of cruciferous plants, and an important industrial microbe, was able to express the
Escherichia coliβ-glucuronidase reporter gene (uidA) when fused to the E. coli lactose operon promoter on a wide-host-range plasmid vector. The gene fusion is expressed constitutively at high levels in
both complex and defined media using a wide range of carbon sources, and is not repressible by glucose or inducible by the
gratuitous lac inducer isopropyl β-d-thiogalactoside. An X. campestris campestris strain with a lesion in the clp (catabolite-repressor-like protein) locus, and containing the plac/uidA fusion, was tested for β-glucuronidase activity. We found that the expression of the plac/uidA fusion gene is dependent on the presence of catabolite-repressor-like protein, with an approximately 75% reduction of expression
in the clp -deficient mutant.
Received: 1 April 1996 / Received revision: 21 June 1996 / Accepted: 15 July 1996 相似文献
14.
Zaldívar-Cruz J.M. Ballina-Gómez H. Guerrero-Rodríguez C. Avilés-Berzunza E. Godoy-Hernández G.C. 《Plant Cell, Tissue and Organ Culture》2003,73(3):281-284
Hypocotyls from annatto seedlings, were inoculated with Agrobacterium tumefaciens harboring a binary vector, pBI.121 or pCAMBIA2301, containing the -glucuronidase (gus) gene. Histochemical GUS assay of infected hypocotyls from two annatto varieties showed transient gus gene expression between 3 and 12 days after inoculation.These authors contributed equally to this work.) 相似文献
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Genetically transformed plants of Cymbidium were regenerated after cocultivating protocorm-like bodies (PLB) with Agrobacterium tumefaciens strain EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (gus), hygromycin phosphotransferase (hpt) and neomycin phosphotransferase II (nptII). PLB of three genotypes maintained in liquid new Dogashima medium (NDM), were subjected to transformation experiments. The
PLB inoculated with Agrobacterium produced secondary PLB, 4 weeks after transfer onto 2.5 g L−1 gellan gum-solidified NDM containing 10 g L−1 sucrose, 20 mg L−1 hygromycin and 40 mg L−1 meropenem. Transformation efficiency was affected by genotype and the presence of acetosyringone during cocultivation. The
highest transformation efficiency was obtained when PLB from the genotype L4 were infected and cocultivated with Agrobacterium on medium containing 100 μM acetosyringone. Transformation of the hygromycin-resistant plantlets regenerated from different
sites of inoculated PLB was confirmed by histochemical GUS assay, PCR analysis and Southern blot hybridization. 相似文献
16.
Agrobacterium-mediated transformation of Asparagus officinalis L.: molecular and genetic analysis of transgenic plants 总被引:3,自引:0,他引:3
Limanton-Grevet A. Jullien M. 《Molecular breeding : new strategies in plant improvement》2001,7(2):141-150
Four long-term embryogenic lines of Asparagus officinalis were co-cultured with the hypervirulent Agrobacterium tumefaciens strain AGL1Gin carrying a uidA gene and an nptII gene. 233 embryogenic lines showing kanamycin resistance and -glucuronidase (GUS) activity were obtained. Transformation frequencies ranged from 0.8 to 12.8 transformants per gram of inoculated somatic embryos, depending on the line. Southern analysis showed that usually 1 to 4 T-DNA copies were integrated. Regenerated plants generally exhibited the same insertion pattern as the corresponding transformed embryogenic line. T1 progeny were obtained from crosses between 6 transformed plants containing 3 or 4 T-DNA copies and untransformed plants. They were analysed for GUS activity and kanamycin resistance. In three progenies, Mendelian 1:1 segregations were observed, corresponding to one functional locus in the parent transgenic plants. Southern analysis confirmed that T-DNA copies were inserted at the same locus. Non-Mendelian segregations were observed in the other three progenies. T2 progeny also exhibited non-Mendelian segregations. Southern analysis showed that GUS-negative and kanamycin-sensitive plants did not contain any T-DNA, and therefore inactivation of transgene expression could not be responsible for the abnormal segregations. 相似文献
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Transgenic carnations obtained byAgrobacterium tumefciens-mediated transformation of leaf explants 总被引:1,自引:0,他引:1
Anne-Claire van Altvorst Tjitske Riksen Herma Koehorst Hans J. M. Dons 《Transgenic research》1995,4(2):105-113
For the development of anAgrobacterium-mediated transformation procedure of carnation (Dianthus caryophyllus L.), an intron-containing -glucuronidase (gus) gene was used to monitor the frequency of transformation events soon after infection of leaf explants. The efficiency of gene transfer was dependent on the carnation genotype, explant age and cocultivation time. Leaf explants from the youngest leaves showed the highest number of GUS-positive spots. After selection on a kanamycin-containing medium, transgenic shoots were generated among a relatively high number of untransformed shoots. The selection procedure was modified in such a way that the contact between explant and medium was more intense. This improved the selection and decreased the number of escapes. Kanamycin-resistant and GUS-positive plants were obtained from five cultivars after infection of leaf explants with the supervirulentAgrobacterium strain AGLO. A higher transformation frequency was observed with the binary vector pCGN7001 than with the p35SGUSint vector. Integration of the genes into the carnation genome was demonstrated by Southern blot hybridization. The number of incorporated T-DNA insertions varied between independent transformants from one to eight. Transformants were morphologically identical to untransformed plants. Segregation of the genes occurred in a Mendelian way. 相似文献
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Optimization of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation conditions in mature embryos of elite wheat 总被引:1,自引:0,他引:1
Immature embryos have been used frequently as target tissues in the genetical transformation of wheat. However, obtaining
a large number of high quality immature embryos throughout the year is a laborious and delicate process, because of the need
to cultivate the plants under controlled conditions. To circumvent this, we have employed mature embryos rather than immature
ones as starter explants for Agrobacterium-mediated transformation of an elite wheat (Triticum aestivum L.) cultivar EM12. The neomycin phosphotransferase ІІ (npt ІІ) and β-glucuronidase (gus) genes were used as selectable and screenable marker genes, respectively, to assess and optimize the performance of T-DNA
delivery. With the aid of an orthogonal design, the effect of four factors in combination on transfer DNA (T-DNA) delivery
was studied. These factors were preculture duration, different kinds of inoculation, length of inoculation and co-culture
condition. Optimal conditions for T-DNA delivery were obtained for mature embryos precultured for 14 days, followed by immersing
in inoculation suspension with full strength Murashige and Skoog (MS) salts in darkness at 23–25°C for 3 h, and then co-culturing
with Agrobacterium under desiccating condition in the dark at 23–24°C for 2–3 days. Complete analysis of transgene insertion demonstrated that
the optimized method for Agrobacterium-mediated transformation of mature embryos of wheat was efficient and practicable. 相似文献
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Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants 总被引:18,自引:0,他引:18
Keith Lindsey Wenbin Wei Michael C. Clarke Hayley F. McArdle Leonie M. Rooke Jennifer F. Topping 《Transgenic research》1993,2(1):33-47
As part of a gene tagging strategy to study the developmental regulation of patterns of plant gene expression, a promoterlessuidA (gus A) gene, encoding the -glucuronidase (GUS) reporter, was introduced into populations of tobacco,Arbidopsis and potato byAgrobacterium-mediated gene transfer. The objective was to generate random functional fusions following integration of thegusA gene downstream of native gene promoters. We describe here a detailed analysis of levels and patterns ofgusA activation in diverse organs and cell types in those populations.gusA activation occurred at high frequency in all three species, and unique patterns of fusion gene expression were found in each transgenic line. The frequency ofgusA activation was differentially blased in different organs in the three species. Fusion gene activity was identified in a wide range of cell types in all organs studied, and expression patterns were stably transmissible to the T2 and T3 progeny. Developmentally-regulated and environmentally-inducible expression ofgusA is described for one transgenic line. Phenotypic variants were detected in the transgenic population. These results demonstrate the potential of T-DNA insertion as a means of creating functional tags of genes expressed in a wide spectrum of cell types, and the value of the approach as a complement to standard T-DNA insertional mutagenesis and transposon tagging for developmental studies is discussed. 相似文献
20.
Agrobacterium-mediated transformation and plant regeneration from hypocotyl segments of Japanese persimmon (Diospyros kaki Thunb) 总被引:1,自引:0,他引:1
Hypocotyl segments from the seeds of Japanese persimmon (Diospyros kaki Thunb) were cultured on a modified Murashige and Skoog medium supplemented with N-(2-chloro-4-pyridyl)-N′-phenylurea, zeatin
or 6-benzylaminopurine. The highest frequency of shoot regeneration was observed when the segments were cultured on medium
containing 2 mg/l of zeatin. This culture system was adapted to Agrobacterium-mediated transformation. The hypocotyl segments were inoculated with Agrobacterium tumefaciens strains harboring binary vectors, which contained the neomycin phosphotransferase II gene and the β-glucuronidase gene. Regenerated shoots were selected on a medium containing kanamycin. Histochemical GUS assay showed that
the shoots regenerated from the segments inoculated with EHA101/pSMAK251 expressed the gus gene. The presence and integration of the gus gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. The regeneration frequency of transformed
shoot was 11.1%. The transgenic shoots were rooted and developed into whole plants within 4–5 months.
Received: 18 August 1997 / Revision received: 8 October 1997 / Accepted: 11 November 1997 相似文献