Molecular control of transgene segregation and its escape by a recoverable block of funtion (rbf) system

US20040025200A1Inactive Publication Date: 2004-02-05UNICROP LTD
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  • Molecular control of transgene segregation and its escape by a recoverable block of funtion (rbf) system
  • Molecular control of transgene segregation and its escape by a recoverable block of funtion (rbf) system
  • Molecular control of transgene segregation and its escape by a recoverable block of funtion (rbf) system

Examples

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Effect test

example 1

[0180] Continuous Compensation of a Metabolite Eliminated by Constitutively Expressing the Blocking Construct (BC) of L-Asparaginase / L-Glutaminase Enzyme;

[0181] Recovery with Externally Applied L-Asparagine / L-Glutamine

[0182] The schematic positions of the genes are shown in FIG. 1A. The Recoverable Block of Function (RBF) system is an example of how the system works at the metabolite level--in this case amino acid or nitrogen metabolism level. It is the most controlled Recoverable Block of Function (RBF) system. A block of the nitrogen metabolism leads to lethal effects resulting from the lack of amino acids, especially during seed germination. The L-asparaginase is active in sink tissues rich in asparagine. Expression of the enzyme in physiologically active tissues can inhibit the normal nitrogen metabolism. The Blocking Construct (BC) is placed close to the gene of interest or between genes of interest. The L-asparaginase from Lupinus angustifolius (Dickson, et al., 1992, Plant Mo...

example 2

[0183] Antisense mRNA of Ent-Kaurene Synthase A Temporally Expressed in Germinating Seeds of Turnip Rape

[0184] Recovery with Externally Applied Gibberellins

[0185] The schematic positions of the genes are shown in FIG. 1B. The Recoverable Block of Function (RBF) system works at RNA silencing level with the recovery of the block working on hormone level. A mutation in a single copy gene encoding for Ent-kaurene synthase A leads to dwarf phenotype of Arabidopsis thaliana. Expression of the antisense RNA of the enzyme leads to the same phenotype alteration due to a block of the gibberellin synthesis in the turnip rape plant. This is a temporal block of function because the enzyme is situated under the SH-EP (EP cysteine endopeptidase) promoter from Vigna mungo. The expression of the antisense ent-kaurene synthase A starts during late embryogenesis and rises at the stage of seed germination. The host plant carrying the Recoverable Block of Function (RBF) develops a dwarf phenotype and is...

example 3

[0186] Recoverable Block of Function (RBF) with Barnase Controlled by SH-EP Endopeptidase Promoter;

[0187] Recovery by Barstar Gene Controlled by HS Heat Shock Promoter

[0188] The schematic positions of the genes are shown in FIG. 2. The Recoverable Block of Function (RBF) system works at the protein interaction level. It consists of bamase and barstar genes originating from Bacillus amyloliquefaciens (Hartley, 1989, Trends Biochem. Sci. 14: 450-454). Barnase is the best known lethal gene since Mariani, et al. (1990, Nature 347: 737-741) used it for engineering fertility control in transgenic plants. When expressed in the plant Barnase blocks the expression of all the genes through destroying RNA molecules. Acting as Blocking Construct (BC) the barnase gene was expressed under cysteine endopeptidase promoter (SH-EPp) cloned from Vigna mungo and, previously named sulfhydryl-endopeptidase promoter (Akasofu et al., 1990, Nucl. Acid Res. 18: 1892; Yamauchi et al., 1996, Plant Mol. Biol. 3...

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Abstract

The invention is related to a method and a complex of DNA constructs for providing an increased level of control of transgene segregation in Sexually Reproducing Multicellular Organisms (SRMOs), which are prone to out-crossing with their wild-type or cultivated relatives. The method and constructs allow the farmer to reuse the transgenic crop. without risk. The RBF system comprises one or more Transgenes of Interest (TGIs) encoding desired gene products, one or more Blocking Constructs (BC), and one or more user-controlled means for recovering the blocked functions. The BC has the capacity of blocking at least one function essential for the survival and / or reproduction of the SRMO. Preferably more than one BC flanking the TGIs are used. The BC may be located in close proximity to the TGI, preferably in an intron or flanking the TGIs. The blocked function is recoverable by user-controlled interventions preferably applicable under confined conditions. The intervention is combined with one or more Recovering Constructs (RC). Different types of RBF systems are disclosed. The Double and Triple and Segregating RBF systems and intron-inserted BC are the preferred embodiments of the present invention. The Transgenic Inserts (TI) of the Triple RBF system and their interactions are shown in FIG. 10.

Description

THE TECHNICAL FIELD OF THE INVENTION[0001] The present invention is related to an improved method and complex of DNA constructs, herein called Recoverable Block of Function (RBF) systems, for obtaining an increased security level in the control of transgene segregation and transgene escape including introgression. The method and system also allows farmers to reuse their transgenic crop without risking leakage of transgenes into the environment. The increased security level is achieved by Recoverable Block of Function (RBF) systems comprising one or more blocking nucleotide constructs (BC) closely linked to a Transgene of Interest (TGI), wherein the Blocking Construct (BC) is inserted into an intron of the Transgene of Interest (TGI), if one Blocking Construct (BC) is used or between two Blocking Constructs (BC), if more than one are used, as well as one or more Recovering Constructs (RC), i.e. nucleotide constructs for recovering the blocked function under a user-controlled chemical...

Claims

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Application Information

Patent Timeline
05 Feb 2004
Publication
US20040025200A1
IPC
A01H1/02; A01H1/04; C12N15/31; C12N15/55; C12N15/82
CPC
A01H1/02; A01H1/04; C12N15/8213; C12N15/8241; C12N15/8287; C12N15/8261; C12N15/8263; C12N15/8265
Inventors
KUVSHINOV, VIKTOR; KOIVU, KIMMO