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Double recoverable block of function – a molecular controlof transgene flow with enhanced reliability

Published online by Cambridge University Press:  15 November 2005

Viktor Kuvshinov ,
Andrey Anisimov ,
Bukhari M. Yahya  and
Anne Kanerva
Viktor Kuvshinov
Affiliation:
UniCrop Ltd, Helsinki Business & Science Park, Viikinkaari 4, FIN-00790, Helsinki, Finland
Andrey Anisimov
Affiliation:
UniCrop Ltd, Helsinki Business & Science Park, Viikinkaari 4, FIN-00790, Helsinki, Finland
Bukhari M. Yahya
Affiliation:
UniCrop Ltd, Helsinki Business & Science Park, Viikinkaari 4, FIN-00790, Helsinki, Finland
Anne Kanerva
Affiliation:
UniCrop Ltd, Helsinki Business & Science Park, Viikinkaari 4, FIN-00790, Helsinki, Finland

Abstract

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Despite all the achieved benefits and potential promises from recombinant DNA technology of plants, the potential of transgene spread to wild relatives and to non-transgenic crops is still of a wide-spread concern. We continue to develop recoverable block of function (RBF) technology for gene flow control in transgenic plants. RBF consists of two elements: blocking construct (BC) and recovering construct (RC). Natural expression of the BC (barnase) in embryos and sprouts blocks a physiological function essential for survival or reproduction of the transgenic plant (mRNA synthesis and germination). Artificially induced (heat shock treatment) RC (barstar) recovers the blocked function enabling transgenic plant to reproduce. In natural conditions without artificial induction of RC the transgenic plant can not reproduce itself. However, a single RBF may still fail because of the potential for mutations and gene silencing of the inserted constructs. To minimize the frequency of such an inactivation, we developed a double RBF, in which a single insert comprising two BC, flanking a transgene of interest, was constructed and transferred into tobacco (Nicotiana tabacum (L.)). We used a barstar gene driven by a heat shock or 35S promoter as a RC, and two different promoters were used for barnase genes in the BC. One BC contained a seed germination specific cysteine endopeptidase promoter (BC1) and the other contained the cruciferin promoter (BC2), which is active during fruit development and embryogenesis. Three alternative constructs of double RBF are described, and a segregating two-insert as well as a one-insert cassettes, were compared. One-insert system comprising two BC with different nucleotide sequences but degenerate codons that expressed the same Barnase protein appeared to be the most reliable choice. The biological and molecular data obtained suggest that double RBF is a potent transgene containment technique that can safely be applied in agriculture.

Keywords

barnasebarstarcontrol of gene flowgene containmentGMO risk managementtobacco (Nicotiana tabacum (L.))recoverable block of function
Type
Research Article
Information
Environmental Biosafety Research , Volume 4 , Issue 2 , April 2005 , pp. 103 - 112
Copyright
© ISBR, EDP Sciences, 2005

References

Akasofu, H, Yamauchi, D, Minamikawa, T (1990) Nucleotide sequence of the gene for the Vigna mungo sulfhydryl-endopeptidase (SH-EP). Nucl. Acid Res. 18: 1892 CrossRef
Becker, D, Elke, K, Schell, J, Masterson, R (1992) New plant binary vectors with selectable markers located proximal to the left T-DNA border. Plant Mol. Biol. 20: 1195-1197 CrossRef
Bright SWJ, Greenland AJ, Jepson I, Paine JAM, inventors. 1994. Improved Plant Germplasm. PCT application WO 9403619
Czarnecka, E, Key, JL, Gurley, WB (1989) Regulatory domains of the Gmhsp 17.5-E heat shock promoter of soybean. Mol. Cell Biol. 9: 3457–3463 CrossRef
Fabijanski SF, Arnison PG, Robert L, Schernthaner J, inventors. 2004. Methods and genetic compositions to limit outcrossing and undesired gene flow in crop plants. US patent No. 6,753,460
Gallagher SR, ed. (1992) GUS protocols. Using the GUS gene as a reporter of gene expression. Academic Press Inc., San Diego, London, New York.
Gatz C, Frohberg C, Wendenburg R (1992) Stringent repression and homogeneous de-repression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants. Plant J. 2: 397-404
Gray AJ (2000) The transfer of traits to wild relatives. Predicting Field Performance in Crop Protection (Copping L.G., ed.). BCPC Symposium Proceedings No. 74. British Crop Protection Council, Farnham, Surrey, UK, pp 165-174
Gressel, J (1999) Tandem constructs: preventing the rise of superweeds. Trends Biotech. 17: 361–366 CrossRef
Hartley, RW (1989) Barnase and Barstar: two small proteins to fold and fit together. Trends Biochem. Sci. 14: 450–454 CrossRef
Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir and T-region of the A. tumefaciens Ti-plasmid. Nature (London) 303: 179-180
Horvath, H, Jensen, L, Wong, O, Kohl, E, Ullrich, S, Cochran, J, Kannangara, C, von Wettstein D (2001) Stability of transgene expression, field performance and recombination breeding of transformed barley lines. Theor. Appl. Genet. 102: 1-11
Kling, J (1996) Could transgenic supercrops one day breed superweeds? Science 274: 180-181 CrossRef
Kuvshinov, V, Koivu, K, Kanerva, A, Pehu, E (2001) Molecular control of transgene escape from genetically modified plants. Plant Sci. 160: 517-522 CrossRef
Kuvshinov V, Koivu K, Kanerva A, Pehu E, inventors. 2002. Molecular control of transgene escape by a repressible excision system. PCT application WO 02064801
Kuvshinov, V, Anissimov, A, Yahya, BM (2004) Barnase gene inserted in the intron of GUS – a model for controlling transgene flow in host plants. Plant Sci. 167: 173-182 CrossRef
Kuvshinov V, Koivu K, Kanerva A, Pehu E, inventors. 2005. Molecular control of transgene segregation and its escape by a recoverable block of function (RBF) system. US patent No. 6,849,776
Lazzeri, PA, Shewry, PR (1993) Biotechnology of cereals. Biotechnology and Genetic Engineering Review 11: 79-146 CrossRef
Mette, MF, Aufsatz, W, van der Winden, J, Matzke, MA, Matzke, AJ (2000) Transcriptional silencing and promoter methylation triggered by double-stranded RNA. EMBO J. 19: 5194-5201 CrossRef
Morel, JB, Mourrain, P, Beclin, C, Vaucheret, H (2000) DNA methylation and chromatin structure affect transcriptional and post-transcriptional transgene silencing in Arabidopsis. Curr. Biol. 10: 1591-1594 CrossRef
Noguchi, T, Fujioka, S, Choe, S, Takatsuto, S, Yoshida, S, Yuan, H, Feldmann, KA, Tax, FE (1999) Brassinosteroid-insensitive dwarf mutants of Arabidopsis accumulate brassinosteroids. Plant Physiol. 121: 743-752 CrossRef
Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810–812
Rodin, J, Sjodahl, S, Josefsson, LG, Rask, L (1992) Characterization of a Brassica napus gene encoding a cruciferin subunit: estimation of sizes of cruciferin gene families. Plant Mol. Biol. 20: 559–563 CrossRef
Schernthaner JP, Fabijanski SF, Arnison PG, Racicot M, Obert LS (2003) Control of seed germination in transgenic plants based on the segregation of two-component genetic system. Proc. Natl. Acad. Sci. (USA) 100: 6855- 6859
Tax, FE, Vernon, DM (2001) T-DNA-associated duplication/translocations in Arabidopsis. Implications for mutant analysis and functional genomics. Plant Physiol. 126: 1527-1538 CrossRef
Vancanneyt, G, Schmid, R, O’Connor-Sanchez, A, Willmitzer, L, Rocha-Sosa M (1990) Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol. Gen. Genet. 220: 245-250 CrossRef
Vaucheret, H, Beclin, C, Fagard, M (2001) Post-transcriptional gene silencing in plants. J. Cell Sci. 114: 3083-3091
Windels, P, Taverniers, L, Depicker, A, Van Bockstaele, E, De Loose M (2001) Characterisation of the roundup ready soybean insert. Eur. Food Res. Technol. 213: 107–112 CrossRef
Yamauchi, D, Terasaki, Y, Okamoto, T, Minamikawa, T (1996) Promoter regions of cysteine endopeptidase genes from legumes confer germination-specific expression in transgenic tobacco seeds. Plant Mol. Biol. 30: 321-329 CrossRef