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In this study Red Star potato plants were genetically modified to resist insect attack by Colorado potato beetle, thr ough insertion of synthetic version of such a hybrid gene, SN19. Drawbacks of expression of a single resistance gene are the limited target spectrum and the potential for rap id adaptation of the pest.
In this study Red Star potato plants were genetically modified to resist insect attack by Colorado potato beetle, thr ough insertion of synthetic version of such a hybrid gene, SN19. Drawbacks of expression of a single resistance gene are the limited target spectrum and the potential for rap id adaptation of the pest.
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In this study Red Star potato plants were genetically modified to resist insect attack by Colorado potato beetle, thr ough insertion of synthetic version of such a hybrid gene, SN19. Drawbacks of expression of a single resistance gene are the limited target spectrum and the potential for rap id adaptation of the pest.
Droits d'auteur :
Attribution Non-Commercial (BY-NC)
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Téléchargez comme TXT, PDF, TXT ou lisez en ligne sur Scribd
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 expression of SN19 in transgenic potato 3. Ferré J., Van Rie J. (2002) Ann. Rev. Entomol., plants could provide excellent protection 47, 501-533. against several major potato pests in the 4. Forgash D.A. (1985) Proceedings of the 17th Congress of Entomology, vol. 740 (D.N. Ferro, R.H. field. Whereas an expanded host range has Voss, Eds), USA: Massachusetts Agricultural Ex- economic advantages, it may also have dis
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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 D., Boersma W.J.A., Hilgers L.A.T. (2001) Vacto be included in the safety assessment ofcine, 19, 2749-2755. any such crop, as indeed it was for already6. Lazo G.R., Stein P.A., Ladwig R.A. (1991) commercialized insect-resistant transgenic Biotechnology (NY), 9, 963-967. crops. 7. Loseva O.I., Ibrahim M., Candas M., Koller Changes in toxin binding sites is the mostC., Bauer L., La B.J. (2002) Insect Bi ochem. commonly occurring resistance mechanismMolec. Biol., 32, 567-577. 8. Mersereau M., Gregory J., Anath D. (1990) against Cry proteins in insects (3), and oc- Gene, 90, 149-151. cur in Cry3Aa-resistant CPB (7). For this 9. Naimov S., Weemen-Hendriks M., Dukianreason pyramiding or stacking of two djiev S., de Maagd R.A. (2001) Appl. Environ. genes encoding proteins with different re- Microbiol., 67, 5328-5330. ceptor recognition properties (12) or de
10. Perlak F.J., Fuchs R.L., Dean D.A., McPherploying
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