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1.
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
2.
3.
Sergei F. Krasnyanski Jagdeep Sandhu Leslie L. Domier Dennis E. Buetow Schuyler S. Korban 《In vitro cellular & developmental biology. Plant》2001,37(4):427-433
Summary Two different promoters, a cauliflower mosaic virus (CaMV) 35S promoter with a 5′-untranslated leader sequence from alfalfa
mosaic virus RNA4 (designated as CaMV 35S/AMV) and an E-8 fruit-ripening-specific promoter, were compared to evaluate their
effects on expression of the uidA reporter gene in transgenic tomato plants. In order to generate sufficient numbers of transgenic tomato plants, both a reliable
regeneration system and an efficient Agrobacterium transformation protocol were developed using 8-d-old cotyledons of tomato (Lycopersicon ecsulentum Mill. cv. Swifty Belle). Two sets of constructs, both derivatives of the binary vector pBI121, were used in transformation
of tomato whereby the uidA gene was driven either by the CaMV 35S/AMV or the E-8 fruit-ripening-specific promoter. Southern blot hybridization confirmed
the stable integration of the chimeric uidA gene into the tomato genome. Fruit and leaf tissues were collected from T0 and T1 plants, and assayed for β-glucuronidase (GUS) enzyme activity. As expected, both vegetative and fruit tissues of transgenic
plants carrying the uidA gene under the control of CaMV 35S/AMV showed varying levels of GUS activity, while no expression was observed in vegetative
tissues of transgenic plants carrying the uidA gene driven by the E-8 promoter. All fruits from transgenic plants produced with both sets of constructs displayed expression
of the uidA gene. However, when this reporter gene was driven by the CaMV 35S/AMV, GUS activity levels were significantly higher than
when it was driven by the E-8 fruit-specific promoter. The presence/absence of the uidA gene in T1 plants segregated in a 3∶1 Mendelian ratio. 相似文献
4.
Summary Electroporation conditions were optimized for the transfection of protoplasts isolated from an embryogenic cell line of sweet
organe [Citrus sinensis (L.) Osbeck ev. Hamlin]. Electric field strength (375–450 V cm−1) vector DNA concentration (100 μgml−1), carrier DNA concentration (100 μgml−1), electroporation buffer (pH 8), and preelectroporation heat shock of protoplasts (5 min at 45°C) were optimized. The plasmid
vector pBI221 containing the β-glucuronidase (GUS) coding sequence under the control of the CaMV 35S promoter was used and
GUS activity was measured 24h after electroporation. All variables significantly affected transfection efficiency and when
optimal conditions for each were combined. GUS activity was 7714 pmol 4-methylumbelliferone (MU) mg−1 (protein) min−1. Protoplasts were then electroporated in the presence of green fluorescent protein (GFP) expression vectors pARS101 or pARS108.
Green fluorescent embryos were selected, plants regenerated, and integration of the transgene was confirmed by Southern blot
analysis. Both plasmids were constructed using EGFP, a GFP variant 35 times brighter than wtGFP, having a single, red-shifted
excitation peak, and optimized for human codon-usage. pARS101 was constructed by placing EGFP under the control of a 35S–35S
promoter containing 33 bp of the untranslated leader sequence from alfalfa mosaic virus. pARS108 was constructed similarly
except sequences were added for transport and retention of EGFP in the lumen of the endoplasmic reticulum.
Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the US Department of Agriculture
and does not imply its approval to the exclusion of other products or veudors that may also be suitable. 相似文献
5.
Scaffold/matrix attachment regions (S/MARs) are defined as genomic DNA sequences, located at the physical boundaries of chromatin
loops. Previous reports suggest that S/MARs elements may increase and stabilize the expression of transgene. In this study,
DNA sequence with MAR characteristics has been isolated from B. napus
. The BnMARs sequence was used to flank the CaMV35S-GUS-NOS expression cassette within the T-DNA of the plant expression vector
pPZP212. These constructs were introduced into tobacco plants, respectively and the GUS reporter gene expression was investigated in stably transformed plants. When the forward BnMARs sequence was inserted into
the upstream of CaMV35S promoter, the average GUS activities were much higher than those without BnMARs in transgenic tobacco.
The GUS expression of M(+)35S:GUS, M(+)35S:GUSM(+) and M(+)35S:GUSM(−) constructs increased average 1.0-fold, with or without
BnMARs located downstream of NOS. The GUS expression would not be affected when reverse BnMARs sequence inserted whether upstream
of CaMV35S promoter or downstream of NOS. The GUS expression was affected a little when reverse BnMARs sequence was inserted
the downstream of NOS and BnMARs could not act by serving as of promoter. The results showed that the presence of forward
BnMARs sequence does have an obvious impact on enhancing downstream gene expression and its effect is unidirectional. 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.
Isabel M.G. Padilla Agnieszka Golis Adele Gentile Carmine Damiano Ralph Scorza 《Plant Cell, Tissue and Organ Culture》2006,84(3):309-314
To determine the optimum conditions for Agrobacterium-mediated gene transfer, peach explants including cotyledons, embryonic axes and hypocotyl slices from non-germinated seeds
and epicotyl internode slices from germinating seeds were exposed to Agrobacterium-mediated transformation treatments. The GUS (uidA) marker gene was tested using two different A. tumefaciens strains, three plasmids and four promoters [CaMV35s, (Aocs)3AmasPmas (“super-promoter”), mas-CaMV35s, and CAB]. GFP was tested with six A.␣tumefaciens strains, one plasmid (pLC101) and the doubleCaMV35s (dCaMV35s) promoter. The CaMV35s promoter produced more GUS expression
than the CAB promoter. A. tumefaciens strains EHA105 and LBA4404 harboring the same plasmid (pBIN19) differed in their effects on GUS expression suggesting an
interaction between A. tumefaciens strain and plasmid. A combination of A. tumefaciens EHA105, plasmid pBIN19 and the CaMV35s promoter produced the highest rates of transformation in peach epicotyl internodes
(56.8%), cotyledons (52.7%), leaves (20%), and embryonic axes (46.7%) as evaluated by the percentage of explants expressing
GUS 14 days after co-cultivation. GFP expression under the control of the dCaMV35s promoter was highest for internode explants
but only reached levels of 18–19%. When GFP-containing plasmid pCL101 was combined with each of five A. tumefaciens strains the highest levels of transformation were 20–21% (internode and cotyledons, respectively). When nine peach genotypes
were co-cultivated with A. tumefaciens strain EHA105 and GFP-containing plasmid pCL101 the highest levels of transformation were 26–28% (cotyledons and internodes,
respectively). While GFP represents a potentially useful transformation marker that allows the non-destructive evaluation
of transformation, rates of GFP transformation under the conditions of this study were low. It will be necessary to optimize
expression of this marker gene in peach. 相似文献
8.
Akutsu Masako Ishizaki Takuma Sato Hiroji 《Molecular breeding : new strategies in plant improvement》2004,13(1):69-78
An efficient procedure is described for transformation of calli of the monocotyledonous plant Alstroemeria by Agrobacterium rhizogenes. Calli were co-cultivated with A. rhizogenes strain A13 that harbored both a wild-type Ri-plasmid and the binary vector plasmid pIG121Hm, which included a gene for neomycin phosphotransferase II (NPTII) under the control of the nopaline synthase (NOS) promoter, a gene for hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a gene for -glucuronidase (GUS) with an intron fused to the CaMV 35S promoter. Inoculated calli were plated on medium that contained cefotaxime to eliminate bacteria. Four weeks later, transformed cells were selected on medium that contained 20 mg L–1 hygromycin. A histochemical assay for GUS activity revealed that selection by hygromycin was complete after eight weeks. The integration of the T-DNA of the Ri-plasmid and pIG121Hm into the plant genome was confirmed by PCR. Plants derived from transformed calli were produced on half-strength MS medium supplemented with 0.1 mg L–1 GA3 after about 5 months of culture. The presence of the gusA, nptII, and rol genes in the genomic DNA of regenerated plants was detected by PCR and Southern hybridization, and the expression of these transgenes was verified by RT-PCR. 相似文献
9.
10.
CaMV 35S promoter directs β-glucuronidase expression in Ganoderma lucidum and Pleurotus citrinopileatus 总被引:1,自引:0,他引:1
The cauliflower mosaic virus (CaMV) 35S promoter has been most commonly used in plant transformation studies, but its activity
in mushrooms has not been reported. p301-b is a binary vector containing a bialaphos resistance gene driven by the promoter
of Lentinus edodes glyceraldehyde-3-phosphate dehydrogenase (GPD) gene. CaMV 35S-GUS was inserted into p301-b, and the resulting construct p301-bG
was transformed to protoplasts of Ganoderma lucidum and basidiospores of Pleurotus citrinopileatus. GUS activity was observed in the transformants, indicating that CaMV 35S promoter can direct expression of exogenous gene
in the mushrooms. This is the first report on the application of CaMV 35S promoter in genetic modification of mushrooms. 相似文献
11.
Isolde Saalbach Thomas Pickardt Frank Machemehl Gerhard Saalbach Otto Schieder Klaus Müntz 《Molecular & general genetics : MGG》1994,242(2):226-236
The coding region of the 2S albumin gene of Brazil nut (Bertholletia excelsa H.B.K.) was completely synthesized, placed under control of the cauliflower mosaic virus (CaMV) 35S promoter and inserted into the binary vector plasmid pGSGLUC1, thus giving rise to pGSGLUC1-2S. This was used for transformation of tobacco (Nicotiana tabacum L. cv. Petit Havanna) and of the grain legume Vicia narbonensis L., mediated by the supervirulent Agrobacterium tumefaciens strain EHA 101. Putative transformants were selected by screening for neomycin phosphotransferase (NPT II) and -glucuronidase (GUS) activities. Transgenic plants were grown until flowering and fruiting occurred. The presence of the foreign gene was confirmed by Southern analysis. GUS activity was found in all organs of the regenerated transgenic tobacco and legume plants, including the seeds. In the legume, the highest expression levels of the CaMV 35S promoter-controlled 2S albumin gene were observed in leaves and roots. 2S albumin was localized in the vacuoles of leaf mesophyll cells of transgenic tobacco. The Brazil nut protein was present in the 2S fraction after gel filtration chromatography of the legume seed proteins and could be clearly identified by immunoblotting. Analysis of seeds from the R2 progenies of the legume and of transgenic tobacco plants revealed Mendelian inheritance of the foreign gene. Agrobacterium rhizogenes strain RifR 15834 harbouring the binary vector pGSGLUCl2S was also used to transform Pisum sativum L. and Vicia faba L. Hairy roots expressed the 2S albumin-specific gene. Several shoots were raised but they never completely rooted and no fertile plants were obtained from these transformants. 相似文献
12.
For establishing a transformation system of rice (Oryza sativa), after three days of culture embryogenic suspension-cultured cell clusters were enzymatically macerated for 2 hours in electroporation
buffer containing 2% cellulase and filtered through 550, 400, 250 and 100 μm stainless mesh. Filtered embryogenic microcolonies
of 100–250 μm with pBI121 were electroporated at 400 V/cm for 1.2 ms. Four weeks after the electroporation, stable transformed
calli were obtained at a frequency of 72% on the selection medium containing 100 mg/L kanamycin. GUS gene in the genomic DNA
among 20 out of 22 putative transformed calli lines were detected by PCR analysis. The expression of GUS gene into the kanamycin-resistance
calli was confirmed by spectrophotometric assay and histochemical assay of GUS activity. In a histochemical study of the transgenic
rice regenerants, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in leaf vein and
root apex. 相似文献
13.
伪狂犬病毒gD基因在转基因烟草中的表达 总被引:6,自引:0,他引:6
将猪伪狂犬病毒 (pseudorabiesvirus ,PRV)最主要的保护性抗原基因gD完整编码区亚克隆到修饰的植物双元表达载体pBI 35SL中 ,使其置于强启动子CaMV 35S doubleenhancer TEV 5′UTR下游 ,构建的转基因植物双元表达质粒经农杆菌介导转化烟草 .PCR检测叶片筛选阳性植株 ,Southern杂交进一步证实gD已整合到转基因烟草基因组中 .固相酶联斑点试验和Western印迹表明 ,gD在烟草获得正确表达并具有抗原性 相似文献
14.
Summary A sugar beet transformation method was developed using particle bombardment of short-term suspension cultures of a breeding
line FC607. Highly embryogenic suspension cultures derived from leaf callus were bombarded with the uidA (gusA) reporter gene under the control of either the osmotin or proteinase inhibitor II gene promoter, and the npt II selectable marker gene. Transient uidA expression was visualized as 500–4000 blue units per 200 mg of bombarded cells 2 d after bombardment. Stably-transformed
calluses were recovered on both kanamycin and paromomycin media. The greatest number of GUS (+) calluses was obtained when
50 or 100 mgl−1 of kanamycin was applied 2 d after transformation for 3–5 wk, followed by either no selection or reduced levels of the antibiotic.
PCR analyses of the GUS (+) callus lines revealed the expected size fragment for uidA and npt II genes. Stable incorporation of the uidA gene into the genome was confirmed by Southern blot analyses. Several transformed embryos were detected by histochemical
β-glucuronidase (GUS) staining. 相似文献
15.
K. B. McIntosh J. L. Hulm L. W. Young P. C. Bonham-Smith 《Plant Molecular Biology Reporter》2004,22(1):53-61
Stable transformation ofArabidopsis thaliana is a lengthy process that involves up to 3 mo of plant growth and seed selection. We have developed a rapid, 3-wk transient
assay system to test the functionality ofcis-regulatory regions controlling expression of a reporter gene in plants before undertaking stable transformation. Two-week-oldArabidopsis seedlings were vacuum-infiltrated withAgrobacterium tumefaciens cultures carrying various upstream regulatory regions controllinguidA (β-glucuronidase [GUS]) expression. Seedlings were fixed and stained for GUS activity 3–5 d following infiltration. Regulatory
regions tested in this system include the cauliflower mosaic virus (CaMV)35S promoter, the upstream regulatory region of ribosomal protein geneL23A-1, and a temperature-inducible regulatory region (HSP101B) also fromArabidopsis. The percentage of seedlings positive for GUS activity varied depending on the construct used, with the CaMV35S promoter producing the highest number of GUS-positive seedlings. Temperature induction treatments elicited increased GUS
expression in seedlings transformed with theHSP101B regulatory region. Regardless of construct, GUS expression levels were higher in seedlings collected 5 d followingAgrobacterium infiltration than those collected 3–4 d postinfiltration. 相似文献
16.
Physical,chemical and physiological parameters for electroporation-mediated gene delivery into rice protoplasts 总被引:1,自引:0,他引:1
Transformation of cereal protoplasts has been reported using several methods; however, the efficiencies of transformations are still very low. We have evaluated a number of parameters that influence electroporation-mediated DNA uptake and have also compared the efficiency of transient GUS activity and stable transformation obtained using an optimized electroporation method with that of the PEG method. The electroporation conditions tested were ionic composition of buffer, ionic strength, resistivity of buffer, type of anions, voltage, and capacitance.Protoplasts isolated from suspension cultures derived from immature embryos of rice (cvs Radon and IR-54) were used for this study. Stable transformation or transient GUS expression experiments were carried out using a plasmid construct containing the CaMV 35S promoter driving thebar gene and a rice actin promoter driving thegus A (uid A) gene (pAG35bar). Electroporation under optimized conditions resulted in about 13-fold higher GUS activities compared to the PEG method. Protoplast survival following optimized electroporation conditions was 55–60%, compared to 35–40% with the PEG treatment. Protoplasts isolated from a suspension culture at different ages gave substantially different levels of transient GUS expression following electroporation-mediated DNA uptake. In contrast, the age of the suspension culture did not influence PEG-mediated DNA uptake and transient GUS activities, which remained low throughout the culture period examined (21 months). Putatively transformed calluses were selected after three to four weeks on medium containing phosphinothricin as the selection agent. The transformation frequencies ranged from 6.2×10–5 to 5.4×10–4 with the electroporation method compared to 1.3×10–5 to 5.3×10–5 with the PEG method. Southern blot analysis of PPT-resistant calluses obtained by the electroporation-mediated transformation showed simple intergration patterns of integrated DNA in most of the transformants. 相似文献
17.
TheCaMV 35S promoter is highly active on floral organs and pollen of transgenic strawberry plants 总被引:5,自引:0,他引:5
de Mesa MC Santiago-Doménech N Pliego-Alfaro F Quesada MA Mercado JA 《Plant cell reports》2004,23(1-2):32-38
We have evaluated the expression of the reporter -glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter in flowers and pollen from 14 independent transgenic strawberry lines. Of the 14 lines evaluated, 13 (92.8%) showed GUS activity—as estimated by the histochemical GUS assay—in some floral organs, with expression being most common in the flower stem, sepals, petals, ovary and stigma. Ten of these thirteen transgenic lines (77%) showed GUS activity in pollen, although the percentages of positive pollen per flower varied greatly among the different lines. A study of the GUS expression during pollen maturation showed that the (CaMV 35S) promoter showed low expression in pollen from flower buds before anthesis but was activated in mature pollen following anther dehiscence. The percentages of pollen grains that showed GUS activity ranged from 2.1% to 46.3%. These percentages were similar or even higher when mature pollen was stored dry at room temperature for 2 weeks. After 5 weeks of storage, the percentages of GUS-positive pollen decreased in two of the six lines analysed but remained at similar values in the other four lines. GUS activity was also measured in protein extracts of mature pollen by means of the fluorometric GUS assay, with the values obtained ranging from 3.8 mol MU mg protein–1 h–1 to 0.26 mol MU mg protein–1 h–1. Contrary to the generally held view that the CaMV 35S promoter is virtually silent in pollen, we conclude that it is highly expressed in transgenic strawberry pollen.Abbreviations CaMV 35S Cauliflower mosaic virus promoter - GUS -Glucuronidase (EC 3.2.1.31) - MU 4-Methyl umbelliferone - nos Nopaline synthase promoter - nptII Neomycin phosphotransferase - X-Gluc 5-Bromo-4-chloro-3-indolyl--d-glucuronic acid 相似文献
18.
Genetic transformation system of Dendrobium Sonia 17 was optimized using green fluorescent protein (GFP) and -glucuronidase (GUS) gene as the reporter systems. The 35S-sgfp-TYG-nos (p35S) and pSMDFR, carrying sgfp and gusA gene, respectively, were co-bombarded into the calluses. Parameters optimized were acceleration pressure, target distance, gold particle size, pre-bombardment cultured time, plasmid DNA precipitation, total plasmid DNA and the ratio of the plasmids co-bombarded. Both reporter systems responded similarly to the bombardment parameters investigated. Based on the GUS/GFP spot counts, the GFP expression rate was higher than that for GUS under the control of the same promoter, CaMV 35S. GFP could be used as the reporter system for the co-bombardment as it was rapid and non-destructive system to monitor the transformed tissues. A combination of GFP and antibiotic resistance gene was used to select stable putative transformants. 相似文献
19.
SBgLR (Solanum tuberosum genomic lysine-rich) gene was isolated from a potato genomic library using SB401 (S.
berthaultii 401) cDNA as probe. RT-PCR analysis of SBgLR gene expression profile and microscopic analysis of green fluorescent protein (GFP) expression in tobacco plants transformed
with SBgLR promoter-GFP reporters indicate that SBgLR is a pollen-specific gene. A series of 5′deletions of SBgLR promoter were fused to the β-glucuronidase (GUS) gene and stably introduced into tobacco plants. Histochemical and quantitative assays of GUS expression in transgenic
plants allowed us to localize an enhancer of SBgLR promoter to the region −345 to −269 relative to the translation start site. This 76 bp (−345 to −269) fragment enhanced GUS
expression in leaves, stems and roots when fused to −90/+6 CaMV 35S minimal promoter. Deletion analysis showed that a cis-element, which can repress gene expression in root hairs, was located in the region −345 to −311. Further study indicated
that the −269 to −9 region was sufficient to confer pollen-specific expression of GFP when fused to CaMV 35S enhancer.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Authors Zhihong Lang and Peng Zhou contributed equally to this work. 相似文献
20.
Marek Hraška Slavomír Rakouský Vladislav Čurn 《Plant Cell, Tissue and Organ Culture》2008,94(3):239-251
Constitutive promoters are the most common promoters used to drive the expression of various genes in monocots and dicots.
Therefore, it is of intense interest to ascertain their expression patterns in various plant species, organs and during their
ontogenic development. In this study, the activity of the CaMV 35S promoter in transgenic tobacco plants was assessed. In
contrast to other studies, performed rather on the primary transformants (T0 generation), here, individuals of T1 and T2 generations were used. The expression profiles of the CaMV 35S promoter were tracked within various plant organs and tissues
using the GFP marker. Special attention was given to floral tissues for which the original data regarding the CaMV 35S expression
were obtained. As expected, distinct developmental and organ/tissue specific expression patterns in a plant body were observed.
CaMV 35S activity was detected in most of the plant tissues and during different developmental stages. The GFP signal was
not visible in dry seeds only, but it became clearly apparent within 24–48 h after sowing onto the medium, what, among other
things, enables the discrimination of transgenic and non-transgenic seeds/seedlings. Afterwards, the most pronounced GFP fluorescence
intensity was usually visible in various vascular tissues of both, T1 and T2 plants, indicating the high promoter activity. A stable manifestation of the promoter was retained in the next T2 generation without any evident changes or losses of activity, showing the expression stability of the CaMV 35S. 相似文献