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

Free access article

Issue		Environ. Biosafety Res. Volume 4, Number 2, April-June 2005


Page(s)		103 - 112
DOI		10.1051/ebr:2005015

Environ. Biosafety Res. 4 (2005) 103-112
DOI: 10.1051/ebr:2005015

Double recoverable block of function - a molecular control of transgene flow with enhanced reliability

Viktor Kuvshinov, Andrey Anisimov, Bukhari M. Yahya and Anne Kanerva

UniCrop Ltd, Helsinki Business & Science Park, Viikinkaari 4, FIN-00790, Helsinki, Finland

(Received February 15, 2005; accepted September 24, 2005)

Abstract
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.

Key words: barnase / barstar / control of gene flow / gene containment / GMO risk management / tobacco (Nicotiana tabacum (L.)) / recoverable block of function

Corresponding author: Viktor Kuvshinov Viktor.Kuvshinov@unicrop.fi

© ISBR, EDP Sciences 2005

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