Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation
Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon b...
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| Published in: | Цитология и генетика |
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| Date: | 2013 |
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Інститут клітинної біології та генетичної інженерії НАН України
2013
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| Cite this: | Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation / N. Shcherbak, O. Kishchenko, L. Sakhno, I. Komarnytsky, M. Kuchuk // Цитология и генетика. — 2013. — Т. 47, № 3. — С. 21-32. — Бібліогр.: 48 назв. — англ. |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860057441316634624 |
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| author | Shcherbak, N. Kishchenko, O. Sakhno, L. Komarnytsky, I. Kuchuk, M. |
| author_facet | Shcherbak, N. Kishchenko, O. Sakhno, L. Komarnytsky, I. Kuchuk, M. |
| citation_txt | Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation / N. Shcherbak, O. Kishchenko, L. Sakhno, I. Komarnytsky, M. Kuchuk // Цитология и генетика. — 2013. — Т. 47, № 3. — С. 21-32. — Бібліогр.: 48 назв. — англ. |
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| description | Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.
Проанализирована способность lox-сайтов Cre/lox системы рекомбинации бактериофага Р1 влиять на экспрессию трансгенов при расположении этой последовательности непосредственно возле правого бордера (RB) перед кодирующей последовательностью гена. Нативная и мутированная последовательность lox-сайта были размещены в векторах для трансформации возле гена bar и проведена генетическая трансформация растений с помощью агробактерии и биолистическим методом. Lox-опосредованная экспрессия гена bar, обусловливающая устойчивость растений к фосфинотрицину, наблюдалась только у растений, которые получены с помощью агробактериальной трансформации. Методом РТ-ПЦР анализа подтверждено, что в трансгенных растениях, устойчивых к фосфинотрицину, происходит транскрипция гена bar. Сконструирован вектор, в котором ген gus и предшествующий ему lox-сайт размещены вблизи правого бордера, и проведена трансформация табака этим вектором. Экспрессия гена gus задетектирована в листьях трансгенных растений. Векторы, у которых последовательность lox-сайта предшествует гену bar возле правого бордера (RB-lox-bar), успешно использованы для получения устойчивых к фосфинотрицину трансгенных растений таких видов, как Beta vulgaris, Brassica napus, Lactuca sativa и Solanum tuberosum. Наши результаты подтверждают возможность использования последовательности lox-сайта возле правого бордера для контроля экспрессии гена bar в трансгенных растениях.
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21ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 3
Lox sites of the Cre/lox recombination system from
bacteriophage P1 were analyzed for their ability to affect
on transgene expression when inserted upstream from a
gene coding sequence adjacent to the right border (RB)
of T-DNA. Wild and mutated types of lox sites were
tested for their effect upon bar gene expression in plants
obtained via Agrobacterium-mediated and biolistic trans-
formation methods. Lox-mediated expression of bar gene,
recognized by resistance of transgenic plants to PPT,
occurred only in plants obtained via Agrobacterium-me-
diated transformation. RT-PCR analysis confirms that
PPT-resistant phenotype of transgenic plants obtained
via Agrobacterium-mediated transformation was caused
by activation of bar gene. The plasmid with promoterless
gus gene together with the lox site adjacent to the RB was
constructed and transferred to Nicotiana tabacum as well.
Transgenic plants exhibited GUS activity and expression
of gus gene was detected in plant leaves. Expression of bar
gene from the vectors containing lox site near RB allowed
recovery of numerous PPT-resistant transformants of such
important crops as Beta vulgaris, Brassica napus, Lactuca
sativa and Solanum tuberosum. Our results demonstrate
that the lox site sequence adjacent to the RB can be used
to control bar gene expression in transgenic plants.
Introduction. Designing plants with a required
expression pattern of transgenes remains one of the
major problems of plant biotechnology. A variety
of regulatory elements is necessary in all areas of
plant genetic engineering, from basic researches
to development of economically valuable crops.
Development efforts ever more have focused on
the use of tissue-specific or inducible promoters to
control expression of the gene of interest [1, 2].
Nevertheless, the successful selection of transgenic
plants requires strong constitutive promoters and
traditionally regulatory elements derived from non-
plant sources (actually in most cases from plant
pathogens) are often used. For a number of vectors
used for plant transformation nowadays such as
pPZP [3] and pCAMBIA derivatives (http://www.
cambia.org/daisy/cambia/materials/vectors.html),
as well as the pCGN [4] 35S promoter from cauli-
flower mosaic virus (CaMV) has been used to drive
the selective marker genes. Though, it has been
found that expression of transgenes under control
of 35S promoter was unstable under field condition
because of naturally occurring CaMV infection and
homology depended gene silencing resulting from
their interaction [5]. CaMV 35S enhanñers can
influence the expression of nearby genes [6] that
could lead to the loss of tissue-specific expression
of transgenes controlled by appropriate promoter
[7, 8]. In addition, the repetitive use of the same
promoter is known to induce transgene inactivation
due to homology of the sequences [9–11]. One of
the approach to escape these problems is to create
a synthetic promoter [12, 13]. Thereby, alternative
regulatory sequences with little sequence similar-
ity either to the plant pathogens or to extensively
used promoters, are going to be available sources as
building blocks for the promoter engineering.
Lox site sequences used in our experiments were
initially selected for recombination events in trans-
genic plant. Since the introduction of the Cre/lox
system from bacteriophage P1 into plant genome
[14] it has become the best characterized and the
most widely used recombination system in both
commercial plant biotechnology and basic plant re-
search [15]. The Cre/lox recombination system has
been used to induce chromosomal rearrangements
[16, 17], to insert foreign genes precisely into a
pre-existing sites [18] and it has also been used as
the method for producing marker free transgenic
crops [19–23]. Though, to our knowledge, none
of recombination sites has been functionally tested
in plants for their ability to influence transgene ex-
pression. In our research we found that DNA se-
quence of lox site from bacteriophage P1 promoted
gene expression when placed adjacent to the right
border (RB) in the proximal upstream region of
gene coding sequence. Genome DNA sequences
which normally do not function as gene regulatory
elements and become activated as promoter when
УДК 577.21:582.926.2
N. SHCHERBAK, O. KISHCHENKO, L. SAKHNO, I. KOMARNYTSKY, M. KUCHUK
Institute of Cell Biology and Genetic Engineering NAS of Ukraine, Kyiv
E-mail: natasha@iicb.kiev.ua
Lox-DEPENDENT GENE EXPRESSION IN TRANSGENIC PLANTS
OBTAINED VIA AGROBACTERIUM-MEDIATED TRANSFORMATION
© N. SHCHERBAK, O. KISHCHENKO, L. SAKHNO,
I. KOMARNYTSKY, M. KUCHUK, 2013
22 ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 3
N. Shcherbak, O. Kishchenko, L. Sakhno et al.
placed in a new context adjacent to gene referred
to as cryptic [24–26]. By analogy with cryptic gene
regulatory elements lox site sequence confers strong
constitutive expression of a transgene under cer-
tain conditions only, notably, placed adjacent to
the RB in the proximal upstream region of gene
coding sequence. Previously we described the use
of vectors containing lox site between the RB of
T-DNA and promoterless bar gene for obtaining
PPT-resistant transgenic plants [27, 28]. After the
initial demonstration of the feasibility of lox-medi-
ated expression of bar gene we used set of vectors
for the further investigations. The vectors with pro-
moterless gus gene were constructed too. The po-
tential benefit of lox-mediated expression for crop
species transformation has been studied by example
of Beta vulgaris, Solanum tuberosum, Lactuca sativa
and Brassica napus. We have finally demonstrated
that lox-mediated expression provides an effective
alternative for at least bar gene expression in trans-
genic plants from a wide range of plant species.
Materials and methods. Vector constructions.
Plasmid vectors pICH3737, pCBV19, pICH3744,
pICH9393, pICH9414, pICH9702 and pICH3831
were generously donated by «Icon Genetics
GmbH» (Germany). Vector pICH3737 includes bar
gene coding region without promoter near RB bet-
ween loxA and loxM sites. Construct pICH9393 is
the pICH3737 derived vector that contained wild
type of lox site loxP instead of loxA and nos termi-
nator between lox site and RB. Plasmids pICH3744
and pICH9414 were the same as pICH337 and
pICH9393, respectively, except that the additional
lox site in a direct orientation was introduced in
the constructs. All plasmid vectors held nptII gene
under control of nos promoter as well. Constructs
pICH9702 and pICH1567 had -lox-bar- located
internally in the T-DNA and differed with the wild
type of loxP sites in pICH9702 and mutative loxA
sites in plasmid pICH1567. Also we used the vector
pICH3831 containing promoterless bar gene adja-
cent to the RB (without lox site).
Plasmid pCB100 contained loxP site between RB
promoterless gus gene coding sequence. The plasmid
was derived from pICH9414 by substitution of bar
gene coding region for gus gene (RB-lox-gus-). The gus
gene fragment had previously been digested with SacI
and PstI and inserted into the vector pICH9414 which
was incurred a treatment of same restriction enzymes.
Plasmid pCB108 was the pICH3737-derived
vector and contained bar gene coding sequence
with a loxA site located upstream without nptII
gene (deletion in XhoI/Ehe fragment). Plasmid
pCB148 contained AT rich region between RB and
promoterless bar gene. The plasmid was derived
from pCB108 by substituting loxA site (BamHI/
Ecl136II restriction) for the spacer between to-
bacco plastome genes: petB (gene of cytochrome
b6/f complex) and psbB (gene of photosystem II).
Plasmid pCB164 contained bar gene coding region
near LB with the loxA between bar gene and LB
(LB-lox-bar). This vector also contained -lox-gus-
gene located in the middle of T-DNA.
Obtained plasmids were transformed into Agro-
bacterim tumefaciens strain GV3101 and resulting
bacteria were used for plant transformation.
Plant transformation and growth condition. For
Agrobacterium-mediated plant transformation ex-
periments aseptic plant or seedlings were used.
Aseptic plants were grown at 25 ºC (potato at
23 ºC), 16 h photoperiod under 4000 lx light inten-
sity. Transformation was accomplished using Agro-
bacterim tumefaciens strain GV3101 containing the
indicated construct. Agrobacterium cultures were
grown 48 h in LB medium with appropriate anti-
biotics (100 mg/l carbenicillin, 50 mg/l rifampicin,
25 mg/l gentamicin) at 28 ºC in dark. The resulting
cultures were independently centrifuged at 4000 g
for 15 min and resuspended in the equal volume
of cocultivation media supplemented with 0.2 mM
acetosyringone. Obtained bacterial suspensions
were cultivated for 1 h on rotary shaker and than
used for plant genetic transformation.
Agrobacterium-mediated transformation of Nico-
tiana tabacum (cv. Petit Havanna Ottawa) was car-
ried out using the conventional leaf disc method. To-
bacco leaf discs were incubated with A.tumefaciens
containing binary vector for 24 h at 26 ºC in dark.
Infected discs were transferred to regeneration
medium (MS medium [29] supplemented with
1 mg/l BA, 0.1 mg/l NAA, 7 g/l agar, 25 g/l su-
crose) containing 400 mg/l cefotaxime and 100 mg/l
kanamycin for selection. Kanamycin-resistant shoots
were rooted on selective MS medium with 100 mg/l
kanamycin. In the groups of about thirty transfor-
mants transgenic plants were tested for PPT-resis-
tance on the selective medium containing 5 mg/l PPT.
For biolistic transformation of tobacco the plas-
mids were prepared from amplified E. coli XL-blue
cultures using the QIAGEN Plasmid Maxi Kit.
23ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 3
Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation
To absorb DNA to the microprojectiles (Tung-
sten M-17 Microcarriers, 1.1 m, BioRad) 10 l
of DNA (1 g/ l) was added to 50 l of a sus-
pension of tungsten particles (0.06 mg/ml in 50 %
glycerol) in a 1.5-ml Eppendorf tube. After DNA
addition of the DNA, 10 l of PEG/MgCl2 solu-
tion (50 % PEG 2000, 5 M MgCl2) was added to
the suspension. After 20 min of incubation at room
temperature the particles were pelleted by centrifu-
gation in a Microfuge for 30 s, and the supernatant
was removed. The final microprojectile prepara-
tion was resuspended in 60 l of absolute ethanol.
Leaves were placed abaxial side up on MS medium
supplemented with 1 mg/l BA and 0.1 mg/l NAA
and bombarded in 14 cm from the end of the bar-
rel of the particle gun. The pressure in the sample
chamber was reduced to 0.05 atmospheres prior to
bombardment. In all experiments, ten Petri plates
were bombarded per construction.
Seeds of diploid O-type sugar beet (Beta vulgaris
breeding lines KS 3, KS 7, SÑ 01733, SÑ 023-
2 and SÑ 03441) were provided by Institute for
Sugar Beet Research of the Ukrainian Academy of
Agrarian Sciences (Kyiv, Ukraine). Prior to sur-
face sterilization sugar beet seeds were incubated
at +4 ºC for a week then soaked in water at the
room temperature overnight. Seeds were surface
sterilized in 40 % (v/v) formalin for 2 min, trans-
ferred to 70 % ethanol for 30 s, treated with 30 %
(v/v) bleach (1.5 % sodium hypochlorite) for
20 min and washed 3 times for 10 min in auto-
claved distilled water. Seeds were germinated on
MS medium containing 15 g/l sucrose and 2 mg/l
BA at 22 ºC in the dark. Genetic transformation of
sugar beet was carried out as previously reported
[30]. Agrobacterium culture resuspended in MS
medium supplemented with 20 g/l sucrose, 2 g/l
glucose, 2 mg/l BA and 0.2 mM acetosyringone
was used for vacuum infiltration of sugar beet etio-
lated derooted seedlings. After vacuum-infiltration,
seedlings were transferred to sterile filter paper and
incubated in the dark at 22–24 ºC for 3 days. Then
the seedlings were cut into 0.7–1.0 cm pieces, in-
cised and placed on MS medium containing 15 g/l
sucrose and 2 mg/l BA. Within 6–8 weeks a friable
callus has arisen from cotyledons and hypocotyls.
Kanamycin-resistant callus was isolated and further
cultivated on regeneration medium composed of
MS basal salts, 30 g/l sucrose, 29 mM silver thio-
sulfate, 0.5 g/l polyvinil pirrolidone, 1 mg/l BA,
0.3 mg/l IAA and 0.4 mg/l gibberellic acid,
300 mg/l cefotaxime and 100 mg/l kanamycin.
Callus cultures were cultivated at 24 ºC under
scattered light and 16h photoperiod with 3 weeks
subcultivation period. Shoot regeneration occurred
within 4–10 weeks. Selected shoots were trans-
ferred to MS medium with 100 mg/l kanamycin
for root formation.
Aseptic plants of potato (Solanum tuberosum L.
cv. Lugavskoj) used for genetic transformation were
provided by Institute for Potato Research of the
Ukrainian Academy of Agrarian Sciences (Neme-
shaevo, Ukraine). Internodes of propagated in vitro
potato plants were cut into about 0.8 cm fragment
and pre-cultivated on MS medium supplemented
with 40 mg/l adenine hemisulphate, 2 mg/l Dicam-
ba and 0.5 mg/l BA for a week. The explants were
vacuum-infiltrated with bacterial suspension. After
three-day cocultivation the explants were washed,
blotted with sterile filter paper and transferred on
MS medium supplemented with 25 g/l sucrose,
29 mM silver thiosulfate, 0.5 g/l polyvinil pirroli-
done, 40 mg/l adenine hemisulphate, 200 mg/l,
1 mg/l zeatin, 2 mg/l gibberellic acid, 500 mg/l
cefotaxime and 100 mg/l kanamycin. Shoots re-
generated within 6–8 weeks.
For canola genetic transformation (Brassica na-
pus varieties Kalinovskij, VNIS-100) the leaves of
3–4 weeks aseptic plants were used. The explants
were placed on MS medium containing 2 mg/l
2,4-D, 1 mg/l NAA, 0.1 mg/l BA, 0.1 mg/l ki-
netin and 1 g/l sodium thiosulfate as an agent in-
creasing the susceptibility of the plant tissues to
Agrobacterium and positively affected transforma-
tion efficiency [31]. After 3-day pre-cultivation ex-
plants were soaked in the Agrobacterium culture for
20 min and placed on the same medium for cal-
lus formation. The explants with callus were then
transferred to MS-basal medium supplemented with
2 mg/l BA, 1 mg/l zeatine, 1 mg/l NAA, 1 mg/l
gibberellic acid, 1 mg/l abscisic acid and 5 mg/l
PPT for shoot development. Green shoots formed
in 3–4 weeks were replaced on a hormone-free MS
medium complemented with 5 mg/l PPT. Regene-
rated plants were grown on MS medium with 10 mg/l
PPT and root development occurred under these
conditions without any additional initiation factors.
Cotyledon explants of lettuce (Lactuca sativa
cv. Odeskij kucheriavij) were obtained after seed
surface sterilization seeds in 50 % Domestos (com-
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 324
N. Shcherbak, O. Kishchenko, L. Sakhno et al.
mercial bleach ~ 5 % sodium hypochlorite) for
15 min and germination on MS medium under 16 h
day length illumination at 25 ºC for 6–7 day. Fully-
expanded cotyledons were cut at the base of the
petiole and placed in Agrobacterial solution, which
preparing was described above. In 20 min coty-
ledon explants were moved to sterile filter paper
and incubated at room temperature for 2 day in
darkness. After that the explants were placed on the
B5 based [32] regeneration medium (B5 medium
salts with 2.5 % sucrose, 3 mg/l kinetin, 0.5 mg/l
NAA) supplemented with 100 mg/l kanamycin
for selection. As shoot appeared, each one was
tested for PPT resistant by removing and placing
on the selective medium with 5 mg/l PPT. Well
established plants were transferred into the soil in
the greenhouse.
PCR analysis. Genomic DNA was isolated from
young leaves as describd [33]. PCR was carried out
in a reaction volume of 20 l containing 50 ng
DNA, 200 M each of forward and reverse prim-
ers, 200 M dNTPs and 1 U Taq DNA polymerase
(«Fermentas»). Thermal cycling (=amplification)
was performed on a Mastercycler® personal («Ep-
pendorf») with an initial denaturation step at 94 ºC
for 3 min, followed by 35 cycles of denaturation at
94 ºC for 30 s; primer annealing at 65 ºC for 30 s,
and elongation at 72 ºC for 30 or 45 s. Sequences
of the primers used:
for bar gene amplification were
barpr1 5 -ATGAGCCCAGAACGACGCCCGGCC-3
barpr2 5 -GCATGCGCACGGTCGGGTGTTGG-3
barpr3 5 -CCGTACCGAGCCGCAGGAAC-3
barpr4 5 -CAGATCTCGGTGACGGGCAGGAC-3
for nptII gene amplification
kanpr1 5 -CCTGAATGAACTCCAGGACGAGCA-3
kanpr2 5 -GCTCTAGATCCAGAGTCCCGCTCAG-
AAG-3
for gus gene amplification
guspr1 5 -TGGGTGGACGATATCACCGTGGTGA-3
guspr2 5 -GGCCCCAATCCAGTCCATTAATGCG-3
The products of the amplification were sepa-
rated on a 1 % (w/v) agarose gels.
RNA isolation and RT-PCR. For reverse tran-
scription polymerase chain reactions (RT-PCR)
1 g of total RNA isolated from young leaf tissues
of tobacco and beet plants was used. The RNA ex-
traction protocol was followed as Logemann et al.
[34]. DNase I (RNase-free) treatment was used to
eliminate DNA contamination from RNA samples.
First strand cDNA was synthesized from 1 g to-
tal RNA using M-MLV reverse transcriptase and
random hexamer primers («Fermentas») at 37 ºC
for 1 h. Then 2 l of the reaction mixture was used
as a template for PCR with specific primers for
transgenes.
Histochemical assay of GUS activity. Histoche-
mical staining for -glucuronidase (GUS) activi-
ty was performed as described by Jefferson [35]
with modifications. Tissues were stained for 12 h
at 37 ºC in 0.1 M phosphate buffer pH 7.0 con-
taining 1 mM X-Gluc (5-bromo-4-chloro-3-indo-
lyl- -D-glucuronide), 10 mM EDTA, 2.5 mM
K4[Fe(CN)6], 2.5 mM K3[Fe(CN)6], 2 mM dithio-
treitol, 0.1 % (v/v) Triton X-100 and 20 % (v/v)
methanol. After staining, chlorophyll from green tis-
sues was removed by washing in 70 % (v/v) ethanol.
GUS quantification. Protein was extracted from
in vitro plant leaves by homogenization in GUS ex-
traction buffer (50 mM NaH2PO4, 10 mM EDTA,
0.1 % Triton X-100, 0.1 % Sarcosyl). Aliquots of
the extract (50 l) were added to 250 l of assay
buffer (extraction buffer containing 1 mM MUG)
and incubated at 37 ºC. After 1 h incubation 50 l
samples were removed and placed in 2.95 ml
stop buffer (200 M sodium carbonate). Specific
activity of GUS expression was determined with
fluorometric assay using MUG as a substrate and
quantified by the PerkinElmer LS 55 Fluorescence
spectrometer with 365 nm excitation and 455 nm
emission wavelengths. Total soluble protein was
determined as described by Bradford method [36]
using bovine serum albumin (BSA) as a standard.
GUS activity was expressed as picomole 4-MU per
minute per milligram protein.
Results. Genetic transformation experiments with
vectors contain lox site and promoterless bar gene
sequences. In our experiments a set of vectors based
on lox site disposition adjacent to promoterless bar
gene sequence were used (Fig. 1). Most of plasmid
vectors held nptII gene under control of nos pro-
moter as well. In experiments with the vectors con-
taining nptII gene transgenic plants were selected
according to their growth capacity on the medium
with kanamycin and then tested on the selective
medium containing PPT. The presence of bar gene
in selected tobacco plants was confirmed with PCR
analysis (Fig. 2). Herbicide resistance/sensitiv-
25ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 3
Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation
ity studies of transformants harboring RB-lox-bar
sequence reproducibly resulted in approximately
80 % of PPT-resistant transgenic plants. The re-
sults from a series of transformation experiments
are represented at Fig. 3.
Neither the localization of nos terminator be-
tween lox site and the RB nor the presence of ad-
ditional lox site within the T-DNA had an effect on
bar gene expression: the number of ÐÐÒ-resistant
transgenic plants obtained using pICH3737 and
pICH3744 vectors was similar to that with the
vectors pICH9414 and pICH9393. The frequency
of PPT-resistant transgenic plants obtained using
these 4 vectors did not differ from each other at
>95 % confides using Students T-test. We can con-
clude that additional lox site placed in the direct
orientation and therefore can recombine with lox
near RB (pICH3744, pICH9414) and create fa-
vourable conditions for putative recombination had
no effect of the lox-mediated expression.
In contrast, disposition -lox-bar- internally
in the T-DNA thoroughly changed the result
of PPT tests. Transformation experiments with
plasmid vectors pICH1567 and pICH9702 resulted
without obtaining any PPT-resistant transgenic
plants. Another strategy of experiments with direct
selection on the regeneration medium with PPT
immediately after co-cultivation also was used for
these constructs. Invariable result was obtained in
the numerous transformation experiments: no PPT-
resistant transgenic plants were obtained after co-
cultivation with the Agrobacterium strain containing
pICH1567 and pICH9702. Based on these data we
suggest that lox-mediated expression occurred only
when lox site was associated with RB.
The results of biolistic transformation experi-
ments have been estimated using a scheme such
Fig. 1. Schematic representation of the T-DNA region of transformation vectors used in this study: RB – right border, LB –
left border, Pnos – promoter of the nopaline synthase gene, n3 – terminator of the nopaline synthase gene, ocs3 – terminator
of the octopine synthase gene, P35 – CaMV 35S promoter, AT – AT rich region of intergenic spacer of chloroplast DNA
Fig. 2. PCR analysis of DNA isolated from transgenic
tobacco plants using primers for bar gene: 1 – negative
control with DNA from non-transformed plant; 2 –
positive control, plasmid pICH3737 DNA; 3–10 – DNA
from different transgenic plants; Ì – molecular weight
marker 1 kb Plus DNA Ladder (Gibco BRL)
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N. Shcherbak, O. Kishchenko, L. Sakhno et al.
as Agrobacterium-mediated transformation: plants
were regenerated on the medium with kanamycin
and then tested on the selective medium containing
PPT. Only 3 % of obtained kanamycin resistant
plants were tolerant to PPT. The result of direct
transformation led us to conclude that lox-
mediated expression of bar gene depended on the
method of DNA delivery and occurred mainly
in plants obtained via Agrobacterium-mediated
transformation.
A set of vectors was also used for transforma-
tion experiments with direct selection of transgenic
plants on the medium with PPT. Vector pCB108
was constructed to investigate lox-mediated exp-
ression without presence of additional promoter
or selective gene sequence in the construct. In
order to determine if combining of lox site and
LB sequence produces the same result as that
seen with RB-lox, vector pCB164 was designed.
Transgenic tobacco lines were selected immedia-
tely after cocultivation on the medium with 5 mg/l
PPT. We noted a general decrease of transforma-
tion efficiency by comparing these vectors with
ones characterized above (Fig. 4). Substitution of
the lox site by AT rich region of intergenic spacer
of chloroplast DNA in construct pCB148 resulted
in loss of ability to obtain PPT resistant plants
and therefore to indicate bar gene expression.
Constructions and activities of RB-lox-gus-fusion.
Translational fusion was constructed between gus
reporter gene and RB-lox upstream regions. We used
plasmid pICH9414 for designing a new vector –
pCB100 which contains promoterless gus gene
instead of bar gene. Transgenic tobacco plants were
raised and GUS activity was assayed using standard
histochemical analysis (Fig. 5). The GUS activity
varied between individual transgenic plants though
most of them exhibited the absence of gus gene
expression. Fluorometric assay of GUS activity was
carried out to quantify distinguishes between gus
gene expression controlled by 35S promoter and
RB-lox- sequences. GUS activity directed by RB-
lox- sequences in some transgenic plants obtained
with vector pCB100 was fairly high but less that
activity of gus gene under control of 35S promoter.
Crop species transformation. To investigate the
possibilities of lox-mediated expression in crop
species RB-lox-bar vectors were inserted into
sugar beet, lettuce, canola and potato plants via
Agrobacterium-mediated transformation. In general,
the data obtained from stable transgenic crop
species showed good agreement with the discussed
above data of tobacco transformation.
Twenty one transgenic callus clones of sugar beet
were selected on the medium supplemented with
100 mg/l kanamycin after Agrobacterium-mediated
transformation with vector pICH3744. Eight of
them were morphogenic and were able to regenerate
shoots on selective medium. Herbicide resistance/
sensitivity studies of sugar beet were carried out by
100
80
60
40
20
0
3737 3744 9393 9414 3831 9702 1567
Fig. 3. Quantification of PPT resistant transgenic plants
(%). Each bar represents quantity of PPT resistant
transgenic tobacco plants obtained in experiments with
appropriate constructs. Averages and standard deviations
(±SD, n = 3) of three independent transformation ex-
periments for each constructs are shown. Approximately
thirty kanamycin resistant transgenic lines were analyzed
in one experiment for each constructs
Fig. 4. Efficiency of tobacco plants regeneration (%)
on the selective medium with 5 mg/l PPT, after Agro-
bacterium-mediated transformation. Regeneration effi-
ciency was estimated as ratio of explants number produ-
cing green shoots on selective medium to total number
of explants analyzed in experiment. Regeneration effi-
ciency represents as averages (±SD) of three independent
transformation experiments for each constructs
27ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 3
Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation
cultivation of obtained transgenic plant petioles on
regeneration medium supplemented with 10 mg/l
ÐÐÒ (Fig. 6, i, k).
Lettuce and potato transgenic plants were rege-
nerated on the selective medium with kanamycin
and then screened by herbicide selection. Lettuce
green shoots were transferred on the selective
medium (with 5 mg/l ÐÐÒ) for rooting and resistant
rooted plants were identified for the further analysis.
Internodes of transgenic kanamycin resistant potato
plants were cultivated on regeneration medium
with 5 mg/l ÐÐÒ and hereby transgenic potato
clones tolerant to PPT were selected (Fig. 6, g, h).
An alternative method with direct selection on
the regeneration medium containing 5 mg/l PPT
for Brassica napus was used. It was shown that
kanamycin resistant marker could not be used suc-
cessfully as reliable marker for transgenic Brassica
napus plant selection [37, 38]. The same is true for
canola selection on the regeneration medium with
kanamycin in our experiments. Therefore Brassica
napus transgenic plants with lox-depended bar gene
expression were obtained using direct selection on the
regeneration medium with PPT (Fig. 6, a, b). Eighty
eight independent transgenic lines of Brasica napus
resistant to PPT were obtained in our experiments.
PPT-resistant primary transformants of all ex-
amined species were transferred to soil and treated
with BASTA herbicide. The results of herbicide ap-
plication of transgenic plants in greenhouse entirely
agreed with in vitro test results for all plant species
used in this report (Fig. 6, l, m). Self-fertilization
seeds (R1) from the primary transformants of to-
bacco and canola were harvested and germinated
on the medium containing 5mg/l phosphinotricin.
The 3:1 segregation of the selectable marker gene
in progeny of tobacco and canola transgenic plants
was observed (Fig. 6, d).
RT-PCR analysis. The effect of the lox site
sequence on expression level of bar gene in obtained
transgenic plants has been further confirmed with
reverse transcription PCR (RT-PCR) technique.
No expression of bar gene was observed in the leaves
of wild-type plants. In the case of transgenic plants
that hosted RB-lox-bar and 35S-bar gene transcript
was detected (Fig. 7). Results of RT-PCR analysis
corroborated lox-mediated expression of bar gene
in transgenic plants.
In silico analysis. In silico analysis of RB-lox
region using Plat CARE and PLACE database
(http://bioinformatics.psb.ugent.be/webtools/
plantcare/html) revealed TATA motifs within loxA
site (vectors pICH3737 and pICH3744) sequen-
ces located 94, 82 and 72 bp upstream of the
Fig. 5. Expression analysis of LB-lox-gus sequence in
transgenic tobacco plant: a – histochemical staining of
GUS activity of transgenic tobacco plants transformed
with vector pCB100. GUS activity was detected in
primary transformants (in vitro plant leaves), b – the
bar diagram denotes GUS activities of in vitro transgenic
plant generated for the GUS vectors: pCB100 and
pICBV19. The data present average (±SD) of five
independent GUS-positive lines
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N. Shcherbak, O. Kishchenko, L. Sakhno et al.
transcription start site (Fig. 8, b). The same
TATA motif was found in loxP sequence (vectors
pICH9393 and pICH9414). Several sequences that
resemble previously identified plant cis-acting re-
gulatory elements were identified in the region
adjacent to lox site, mainly in RB sequence: CAAT
boxes, ATGCAAAT motif, as well as TGA-element
and ATCT-motif that have partial overlap with the
CAAT box (Fig. 8, a).
Discussion. Teeri et al. [39] demonstrated that
activation of promoterless nptII coding sequence
adjacent to the RB of T-DNA is possible after
plant transformation. The establishment of this
protocol has opened up the possibility to identify
new regulatory sequences needed to drive trans-
gene expression. In the studies presented here,
we report that lox site located near RB and
upstream of the transcription start site positively
affected expression of bar gene coding sequence
situated in vectors without control of any known
promoter sequence. Whereas bar gene coding se-
quence was placed in close to the RB we com-
pared our result with published data of gene exp-
ression in promoter tagging experiments. If the
transcriptional activation of introduced bar gene
in our experiments occurred by plant promoter
tagging we would expect 4–5 % of transformants
resistant to PPT. Foster et al. [25] reported that one
from thousand transgenic tobacco line created by
T-DNA tagging demonstrated constitutive gus gene
expression. According to the published data 4 % is a
usual result for strong gene expression in promoter
tagging experiments [40, 41]. The frequency of gus
gene transcription fusion and gus gene expression
in different plant tissues was higher [42]. Koo et
al. [43] revealed 13 % of gene trapping efficiency
Fig. 6. Regeneration and selection of PPT resistant transgenic plants: a, b – regeneration of canola plants after
cocultivation with Agrobacterium containing vector pICH9393 on the selective medium with 5 mg/l PPT; c –
regeneration of tobacco plants transformed with vector pCB164 on the selective medium with 5 mg/l PPT; d –
selection of F1 tobacco plants (pCB108) on the medium with 5 mg/l PPT; e, f – callus formation and regeneration
of sugar beet plants transformed with vector pICH3737; g, h – transgenic (g) and non-transgenic control (h) potato
explants at the stage of regeneration on the selective medium; k, i – sugar beet transgenic (k) and non-transgenic (i) explants
at the stage of regeneration on the selective medium; l, m – sugar beet transgenic (l) and non-transgenic control (m) plants
herbicide application in greenhouse
29ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 3
Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation
and Yamamoto et al. [44] reported the same result
for promoterless trapping vectors and 41 % of gene
trapping efficiency for IRES-type trapping vector.
We note that in our studies, however, enormous
for promoter tagging experiments number of
transgenic plants was resistant to PPT. The further
investigation confirmed that localization of the lox
site and bar gene near RB created a strong gene
expression system that is more effective in case
of Agrobacterium-mediated transformation but for
a wide range of plant species, including several
important crops.
To gain insight into the mechanisms of lox-
mediated expression, we studied the sequence of
lox site and adjacent region in binary vector
and compared it with several known promoters.
Several TATA motifs within lox site sequence were
revealed. Furthermore, the minimal 35S promoter
Fig. 7. RT-PCR analysis of PPT-resistant transgenic plants: a – PCR analysis of cDNA synthesized with reverse
transcriptase from tobacco RNA using primers for bar gene (barpr3 and barpr4); 1 – negative control with cDNA
from non-transformed plant; 4 – positive control with cDNA of tobacco transformed with plasmid pICBV19 (bar
gene under35S promoter); 2, 6, 8 – cDNA of transgenic tobacco plants (pICH3737); 3, 5, 7 – control: the same
samples without using of reverse transcriptase; M – molecular weight marker 1 kb Plus DNA Ladder (Gibco BRL);
b – PCR analyses of cDNA synthesized with reverse transcriptase from RNA isolated from sugar beet plants using
primers for bar gene (barpr1 and barpr2); 1 – negative control with cDNA from non-transformed plant; 3 – positive
control with cDNA of sugar beet transformed with plasmid pICBV19; 2 – cDNA of transgenic sugar beet (pICH3744)
with primers for bar gene; 4 – control: the same sample without using of reverse transcriptase; 5 – control: DNA
of transgenic sugar beet (pICH3744) with primers for bar gene; M – molecular weight marker (Promega); c – PCR
analyses of cDNA synthesized with reverse transcriptase from RNA isolated from sugar beet plants using primers
for nptII gene (kanpr1 and kanpr2); 1 – negative control with cDNA from non-transformed plant; 2 – cDNA of
transgenic sugar beet (pICH3744) with primers for nptII gene; 3 – control: the same sample without using of reverse
transcriptase; 4 – positive control with DNA of transgenic sugar beet (pICH3744); M – molecular weight marker
(Promega)
Fig. 8. Sequence of RB-lox region: a – putative regulated sequences reviled by Plat CARE and PLACE database are
underlined, TATA box are in bold, lox site sequences are underlined with dashed lines; b – sequence comparison of
loxA and loxP sites with the min 35S promoter
ISSN 0564–3783. Öèòîëîãèÿ è ãåíåòèêà. 2013. Ò. 47. ¹ 330
N. Shcherbak, O. Kishchenko, L. Sakhno et al.
is associated with very low basal transcription in the
absence of additional upstream regulatory motifs
[45] and it is highly improbable that minimal
promoter can support expression level sufficient for
strong herbicide tolerance of transgenic plants. In
addition, we constructed the vector with arbitrary
AT rich sequence (spacer between tobacco plastome
genes) placed instead of lox site. Transformation
experiments with such vector resulted without
obtaining any PPT-resistant plant.
Only several regulatory sequences were found
in the region adjacent to lox site, including RB
sequence. Neither G-box nor as-1 element present
in 35S promoter [46] and in other plant, viral and
Agrobacteria promoter were found in this sequences.
As-1 element associated with expression in roots
has been reported to act synergistically with other
subdomains to confer expression in different tissues.
The elements like as-1 element are present in other
viral and Agrobacteria promoter sequences [47, 48].
We can conclude that the DNA context surrounding
several regulatory sequences which were found in
RB-lox region differs markedly from the previously
examined promoter elements. We suppose that using
the lox site in genetic vectors will help to express
transgenes with reduced possibility of homology
dependent gene silencing as this element have reduced
sequence homology with widely used promoters.
We note that gene expression mediated by these
two elements (RB-lox-) being critically dependent on
their spacing in the vector and method of T-DNA
delivery. Though separating of the lox sequence and
the RB by 282 kb of nos terminator sequence did
not influence bar gene expression, no occurrence
of bar gene expression was observed when lox-bar
sequence was replaced in the middle of the construct.
Experiments with vector pCB164 containing one lox
site placed near LB upstream of bar gene demonstrated
possibilities of lox-depended expression as well.
Constructs based on lox-mediated expression
constructs have been used effectively for obtaining
herbicide resistant transgenic plant of sugar beet,
canola, lettuce and potato. It seems to be completely
real to involve new crop species in transformation
experiments with vectors containing lox-site near
the RB instead of conventional promoters.
One of the goals of this research was to elu-
cidate the main principles of lox-mediated ex-
pression. This goal is far from complete, but our re-
sults do confirm that lox site provides an interesting
alternative to promoters derived from plant pathogens
in that lox sites are not associated with them and can
be used to control gene expression in transgenic plant.
Í.Ë. Ùåðáàê, Å.Ì. Êèùåíêî, Ë.À. Ñàõíî,
È.Ê. Êîìàðíèöêèé, Í.Â. Êó÷óê
Lox-ÎÏÎÑÐÅÄÎÂÀÍÍÀß ÝÊÑÏÐÅÑÑÈß
ÃÅÍÎÂ Â ÒÐÀÍÑÃÅÍÍÛÕ ÐÀÑÒÅÍÈßÕ,
ÏÎËÓ×ÅÍÍÛÕ Ñ ÏÎÌÎÙÜÞ
ÀÃÐÎÁÀÊÒÅÐÈÀËÜÍÎÉ ÒÐÀÍÑÔÎÐÌÀÖÈÈ
Ïðîàíàëèçèðîâàíà ñïîñîáíîñòü lox-ñàéòîâ Cre/lox
ñèñòåìû ðåêîìáèíàöèè áàêòåðèîôàãà Ð1 âëèÿòü íà
ýêñïðåññèþ òðàíñãåíîâ ïðè ðàñïîëîæåíèè ýòîé ïîñ-
ëåäîâàòåëüíîñòè íåïîñðåäñòâåííî âîçëå ïðàâîãî áîð-
äåðà (RB) ïåðåä êîäèðóþùåé ïîñëåäîâàòåëüíîñòüþ
ãåíà. Íàòèâíàÿ è ìóòèðîâàííàÿ ïîñëåäîâàòåëüíîñòü
lox-ñàéòà áûëè ðàçìåùåíû â âåêòîðàõ äëÿ òðàíñ-
ôîðìàöèè âîçëå ãåíà bar è ïðîâåäåíà ãåíåòè÷åñêàÿ
òðàíñôîðìàöèÿ ðàñòåíèé ñ ïîìîùüþ àãðîáàêòåðèè è
áèîëèñòè÷åñêèì ìåòîäîì. Lox-îïîñðåäîâàííàÿ ýêñ-
ïðåññèÿ ãåíà bar, îáóñëîâëèâàþùàÿ óñòîé÷èâîñòü
ðàñòåíèé ê ôîñôèíîòðèöèíó, íàáëþäàëàñü òîëüêî ó
ðàñòåíèé, êîòîðûå ïîëó÷åíû ñ ïîìîùüþ àãðîáàê-
òåðèàëüíîé òðàíñôîðìàöèè. Ìåòîäîì ÐÒ-ÏÖÐ àíà-
ëèçà ïîäòâåðæäåíî, ÷òî â òðàíñãåííûõ ðàñòåíèÿõ,
óñòîé÷èâûõ ê ôîñôèíîòðèöèíó, ïðîèñõîäèò òðàíñ-
êðèïöèÿ ãåíà bar. Ñêîíñòðóèðîâàí âåêòîð, â êîòî-
ðîì ãåí gus è ïðåäøåñòâóþùèé åìó lox-ñàéò ðàç-
ìåùåíû âáëèçè ïðàâîãî áîðäåðà, è ïðîâåäåíà òðàíñ-
ôîðìàöèÿ òàáàêà ýòèì âåêòîðîì. Ýêñïðåññèÿ ãåíà
gus çàäåòåêòèðîâàíà â ëèñòüÿõ òðàíñãåííûõ ðàñòå-
íèé. Âåêòîðû, ó êîòîðûõ ïîñëåäîâàòåëüíîñòü lox-
ñàéòà ïðåäøåñòâóåò ãåíó bar âîçëå ïðàâîãî áîðäåðà
(RB-lox-bar), óñïåøíî èñïîëüçîâàíû äëÿ ïîëó÷åíèÿ
óñòîé÷èâûõ ê ôîñôèíîòðèöèíó òðàíñãåííûõ ðàñòå-
íèé òàêèõ âèäîâ, êàê Beta vulgaris, Brassica napus,
Lactuca sativa è Solanum tuberosum. Íàøè ðåçóëüòàòû
ïîäòâåðæäàþò âîçìîæíîñòü èñïîëüçîâàíèÿ ïîñëåäî-
âàòåëüíîñòè lox-ñàéòà âîçëå ïðàâîãî áîðäåðà äëÿ êîíò-
ðîëÿ ýêñïðåññèè ãåíà bar â òðàíñãåííûõ ðàñòåíèÿõ.
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Received 26.11.12
ABBREVIATIONS
RB – T-DNA right border
LB – T-DNA left border
PPT – phosphinotricin
GUS – -Glucuronidase
NAA – naphthalene-acetic acid
BA – 6-benzylaminopurine
MUG – 4-methylumbelliferyl -d-glucuronidase
MU – 4-methylumbelliferone
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| id | nasplib_isofts_kiev_ua-123456789-126571 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0564-3783 |
| language | English |
| last_indexed | 2025-12-07T17:02:04Z |
| publishDate | 2013 |
| publisher | Інститут клітинної біології та генетичної інженерії НАН України |
| record_format | dspace |
| spelling | Shcherbak, N. Kishchenko, O. Sakhno, L. Komarnytsky, I. Kuchuk, M. 2017-11-26T17:53:47Z 2017-11-26T17:53:47Z 2013 Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation / N. Shcherbak, O. Kishchenko, L. Sakhno, I. Komarnytsky, M. Kuchuk // Цитология и генетика. — 2013. — Т. 47, № 3. — С. 21-32. — Бібліогр.: 48 назв. — англ. 0564-3783 DOI: 10.3103/S0095452713030079 https://nasplib.isofts.kiev.ua/handle/123456789/126571 577.21:582.926.2 Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants. Проанализирована способность lox-сайтов Cre/lox системы рекомбинации бактериофага Р1 влиять на экспрессию трансгенов при расположении этой последовательности непосредственно возле правого бордера (RB) перед кодирующей последовательностью гена. Нативная и мутированная последовательность lox-сайта были размещены в векторах для трансформации возле гена bar и проведена генетическая трансформация растений с помощью агробактерии и биолистическим методом. Lox-опосредованная экспрессия гена bar, обусловливающая устойчивость растений к фосфинотрицину, наблюдалась только у растений, которые получены с помощью агробактериальной трансформации. Методом РТ-ПЦР анализа подтверждено, что в трансгенных растениях, устойчивых к фосфинотрицину, происходит транскрипция гена bar. Сконструирован вектор, в котором ген gus и предшествующий ему lox-сайт размещены вблизи правого бордера, и проведена трансформация табака этим вектором. Экспрессия гена gus задетектирована в листьях трансгенных растений. Векторы, у которых последовательность lox-сайта предшествует гену bar возле правого бордера (RB-lox-bar), успешно использованы для получения устойчивых к фосфинотрицину трансгенных растений таких видов, как Beta vulgaris, Brassica napus, Lactuca sativa и Solanum tuberosum. Наши результаты подтверждают возможность использования последовательности lox-сайта возле правого бордера для контроля экспрессии гена bar в трансгенных растениях. en Інститут клітинної біології та генетичної інженерії НАН України Цитология и генетика Оригинальные работы Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation Lox-опосредованная экспрессия генов в трансгенных растениях, полученных с помощью агробактериальной трансформации Article published earlier |
| spellingShingle | Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation Shcherbak, N. Kishchenko, O. Sakhno, L. Komarnytsky, I. Kuchuk, M. Оригинальные работы |
| title | Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation |
| title_alt | Lox-опосредованная экспрессия генов в трансгенных растениях, полученных с помощью агробактериальной трансформации |
| title_full | Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation |
| title_fullStr | Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation |
| title_full_unstemmed | Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation |
| title_short | Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation |
| title_sort | lox-dependent gene expression in transgenic plants obtained via agrobacterium-mediated transformation |
| topic | Оригинальные работы |
| topic_facet | Оригинальные работы |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/126571 |
| work_keys_str_mv | AT shcherbakn loxdependentgeneexpressionintransgenicplantsobtainedviaagrobacteriummediatedtransformation AT kishchenkoo loxdependentgeneexpressionintransgenicplantsobtainedviaagrobacteriummediatedtransformation AT sakhnol loxdependentgeneexpressionintransgenicplantsobtainedviaagrobacteriummediatedtransformation AT komarnytskyi loxdependentgeneexpressionintransgenicplantsobtainedviaagrobacteriummediatedtransformation AT kuchukm loxdependentgeneexpressionintransgenicplantsobtainedviaagrobacteriummediatedtransformation AT shcherbakn loxoposredovannaâékspressiâgenovvtransgennyhrasteniâhpolučennyhspomoŝʹûagrobakterialʹnoitransformacii AT kishchenkoo loxoposredovannaâékspressiâgenovvtransgennyhrasteniâhpolučennyhspomoŝʹûagrobakterialʹnoitransformacii AT sakhnol loxoposredovannaâékspressiâgenovvtransgennyhrasteniâhpolučennyhspomoŝʹûagrobakterialʹnoitransformacii AT komarnytskyi loxoposredovannaâékspressiâgenovvtransgennyhrasteniâhpolučennyhspomoŝʹûagrobakterialʹnoitransformacii AT kuchukm loxoposredovannaâékspressiâgenovvtransgennyhrasteniâhpolučennyhspomoŝʹûagrobakterialʹnoitransformacii |