EXPRESSION OF SYNTHETIC SN 19 HYBRID

DELTA-ENDOTOXIN ENCODING GENE IN

TRANSGENIC POTATO
S. Naimov1, G. Zahmanova1, R. Boncehva1, M. Kostova1, I. Minkov1, S. Dukiandjiev
1,
R. de Maagd2
University of Plovdiv, Department of Plant Physiology and Molecular Biology, Plo
vdiv,
Bulgaria1
Business Unit Cell Cybernetics, Plant Research International B.V, Wageningen, Th
e
Netherlands2
ABSTRACT
Expression of Bacillus thuringiensis delta-endotoxins has proven to be a success
ful strategy
for obtaining insect resistance in transgenic plants. Drawbacks of expression of
a
single resistance gene are the limited target spectrum and the potential for rap
id adaptation
of the pest. Hybrid toxins with a wider target spectrum in combination with exis
ting
toxins may be used as tool to mitigate these problems. In this study Red Star po
tato plants
were genetically modified to resist insect attack by Colorado potato beetle, thr
ough insertion
of synthetic version of such a hybrid gene, SN19. This makes it a useful tool fo
r resistance
management strategies.
Introduction
for CPB is much lower than that of
Cry3Aa, the most active natural toxin (1).
The coleopteran Colorado potato beetle is
Somewhat higher activity against CPB was
one of the most destructive pests of culti

reported for Cry1Ia (14). A cry1Ba/cry1Ia

vated potato. It s life cycle, feeding habits,
hybrid gene (SN19) encoding a protein
and demonstrated ability to develop resis

consisting of domains I and III of Cry1Ba

tance to chemical insecticides have made
and domain II of Cry1Ia, with high activity
control of CPB an increasing agricultural
against CPB was constructed and described
problem (4, 15). Presently CPB control is
earlier by us (9).
accomplished primarily by the use of
chemical insecticides, through the use of Cry toxins have been expressed in a
different insecticidal Cry proteins origi-number of plant species. Expression of
one
nating from Bacillus thuringiensis in member of this family usually results insp
rays, or by the expression of Cry proteins resistance against a single pest inse
ct or
in transgenic plants.against a few relatively closely related in-
The cry gene family is a large, still sect species within one order. Cry3Aa-exgr
owing
family of homologous genes, withpressing potatoes with resistance to CPB
each gene encoding a protein active on in-(11) is an examples of this.
sect larvae of a subset of species usuallyIn this manuscript we describe producb
elonging
to the same order (13). Cry1tion of synthetic , codon optimized Cry1proteins are
generally active against lepi-gene and it s expression in transgenic podopterans
(larvae of moths and butterflies). tato plants under the control of a chrysanCry
1Ba
also has some activity against co-themum ribulose-1,5-bisphosphate carleopterans
(beetles), although its toxicity boxylase/oxygenase small subunit (Rubisco
Biotechnol. & Biotechnol. Eq. 20/2006/3 38
SSU) promoter and terminator.
Materials and Methods
SN19 synthesis. In order to obtain a synthetic
SN19 encoding DNA sequence a
rapid PCR method was used. Two thousand
and eighty nucleotide sequence coding
for a truncated version of SN19 gene
was designed in order to meet specific requirements
for high level expression in
plants. The full length sequence was broken
down to 85 overlapping oligonucleotides,
80 bp each. For sequence optimization
and poly A signals elimination web
based tools were used (for details see:
http://www.entelechon.com, http://gcua.
schoede.de, and http://www.kazuso.or.jp).
All oligonucletides used for this research
were produced and kindly provided bySynGen Inc, Canada. In order to assemblefull
length sequence all primers were mixed
together in equal molar ratios, and elongation
of the overlapping primer areas was
performed by Pfu-Turbo DNA polymerase(Stratagene). The PCR product from thisstag
e was subsequently used as a template
for second PCR reaction with two primers
flanking 5 and 3 ends of the gene. Additionally
Nco I and BamH I restriction sites
were given at 5 and respectively at the
3 end of the sequence. The resulted PCR
product were separated on 0.8% agarose
gel and purified using a QIAEX II agarose
gel extraction kit (Qiagen), cloned inpGemT-easy (Promega) giving pSN66, and
sequenced in both directions.
Construction of binary vectors. A Nco I-
BamH I fragment of PSN66 containing the
truncated, synthetic SN19 gene (2080 bp)
was cloned into a Nco I-BamH I sites of
pIV 1.1 (Plant Research International BV,
The Netherlands) and subsequently sub-
cloned in pBinPlus vector (16) by AscI-
PacI insertion. Resulted plasmid pMH65
was transferred into A. tumefaciens Agl0
(6) by electroporation (8). A. tumefaciens
mediated potato transformation was performed
following the protocol described
39
previously (5). The obtained transgeniclines were subsequently multiplied and
adapted to greenhouse conditions: 25 ºCand a 16h-light/8h-dark-cycle.
Protein quantification. Leaf tissue (0.2 g)
was ground with 400 µl extraction buffer
(50mM NaOH, 20mM NaS2O5, 5 mM
EDTA, and 10% Polyvinylpoly-pyrrolidone),
subsequently neutralized with 80 µl
1M Tris-HCl, pH 5.5, and centrifuged at14,000 rpm for 10 min. The supernatantwas
transferred into a new eppendorf tube
and additionally centrifuged at 14,000 rpmfor 10 min. Protein concentrations in
supernatant
were determined by the Bradford
method (Bio-Rad Laboratories). The
amount of Cry protein of interest was estimated
by dot blot analysis as follows.
Equal amounts of soluble leaf protein (20
µg) were transferred to a nitrocellulose
membrane using a S&S Minifold Dot
blotter (Schleicher & Schuell). The immunological
detection was performed bytreating the membrane with blocking solution
containing Tris buffered-saline (TBS:
10 mM TrisHCl, pH7.6, 150 mM NaCl),
5% (w/v) non-fat dry milk, and 3% (w/v)
Bovine serum albumin for 1h, washed three
times with TBST buffer (TBS buffer, with
0.2% Tween-20). 1:1000 diluted anti-SN19
serum was applied and the membrane was
incubated for 1h at room temperature. After
three washing steps with TBST, alkaline
phosphatase conjugated anti-rabbit IgG(Sigma-Aldrich) was added (1:1000) and
incubated for 1h. The membranes were
washed three times with TBST buffer, and
once with carbonate buffer (0.1 M
NaHCO3, 1.0 mM MgCl2, pH 9.8). After
15 minutes incubation with 50 ml carbonate
buffer, the membranes were developed
with 0.015% (w/v) 5-Bromo-4-chloro-3indolyl
phosphate (Sigma-Aldrich) and
0.03% (w/v) Nitro Blue Tetrazolium
(Sigma-Aldrich) in carbonate buffer. Serialdilutions of trypsin activated SN19 i
n
phosphate buffed saline (10 mM
Na2HPO4/KH2PO4, 0.8% (w/v) NaCl)
Biotechnol. & Biotechnol. Eq. 20/2006/3
Expression(% of total soluble protein)
0,16
0,14
0,12
0,1
0,08
0,06
0,04
0,02
0
tr1 tr2 tr3 tr5 tr7 tr8 tr11 tr13 tr14 tr16 tr17 tr19 tr23 tr24
Line

Figure. SN19 expression levels, estimated by dot blotting analyses.

added to negative control plant extracts
were used for comparison and estimation
of SN19 content in leaf tissues.
Results and Discussion
Although current transgenic plants expressing
a Cry protein are effectively protected
against one or a few relatively related
pests, their activity spectrum is limited.

Expression of the SN19 gene in potato

was an approach to prove our hypothesis
that an effective expression of a single hybrid
delta-endotoxin gene could provide
effective resistance against a coleopteran
pest.
For the reconstruction of the gene encoding
the toxic fragment of SN19, a synthetic
oligonucletides were designed and
subsequently assembled. Sequence analysisof the bacterial gene identified a numb
er of
potential RNA instability elements and
polyadenylation sites.
By combination of PCR with primers designed
for an codon optimized SN 19 sequence
and amplification of the full length
Biotechnol. & Biotechnol. Eq. 20/2006/3 40
product a completely synthetic SN19 gene
was reconstructed, eliminating all putative
polyadenylation sites, mRNA instabilitysequences, and consecutive C+G and A+T
stretches. This DNA sequence was clonedbetween a promoter and terminator fragmen
t
derived from the chrysanthemum Rubisco
SSU gene in the binary transformation
vector pBINplus. This resulted in
transformation vectors pMH65 and. The
described expression cassette was introduced
in potato cultivar RedStar by
Agrobacterium tumefaciens mediated
transformation. 14 transgenic potato lines,
were obtained and successfully adapted togreenhouse conditions and protein expre
ssion
levels were estimated.
In agreement with results reported in
previous studies (10) we found that a significant
modification of the Bacillus
thuringiensis delta-endotoxin encoding
hybrid gene SN19 was necessary for successful
expression in plants. In our hands
expression levels up 0,15% of total soluble
protein were achieved (Figure). According
our previous experience we conclude that
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Voss, Eds), USA: Massachusetts Agricultural Ex-
economic advantages, it may also have dis

perimental Station Research Bulletin, 33-52.

advantages in the form of increased effects
5. Lauterslager T.G.M., Florack D.E.A., van der
on non-target and/or beneficial insects. Pre-
Wal T.J., Molthoff J.W., Langeveld J.P.M., Bosch
release testing for these effects would have
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9. Naimov S., Weemen-Hendriks M., Dukianreason
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We would like to thank SynGen Inc. Can-
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for providing oligonucleotides used in1217.
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