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WO2002016624A1 - Reduction de la transmission de transgenes dans des plantes - Google Patents

Reduction de la transmission de transgenes dans des plantes Download PDF

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Publication number
WO2002016624A1
WO2002016624A1 PCT/SG2000/000124 SG0000124W WO0216624A1 WO 2002016624 A1 WO2002016624 A1 WO 2002016624A1 SG 0000124 W SG0000124 W SG 0000124W WO 0216624 A1 WO0216624 A1 WO 0216624A1
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Prior art keywords
plant
sequence
promoter
site
recombinase
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PCT/SG2000/000124
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English (en)
Inventor
Venkatesan Sundaresan
Yan Hong
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Institute Of Molecular Agrobiology
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Application filed by Institute Of Molecular Agrobiology filed Critical Institute Of Molecular Agrobiology
Priority to PCT/SG2000/000124 priority Critical patent/WO2002016624A1/fr
Priority to EP00959109A priority patent/EP1313865A1/fr
Priority to CA002419646A priority patent/CA2419646A1/fr
Priority to AU2000270487A priority patent/AU2000270487B2/en
Priority to AU7048700A priority patent/AU7048700A/xx
Priority to BR0017326-6A priority patent/BR0017326A/pt
Priority to CNB008198519A priority patent/CN1296484C/zh
Priority to ARP010104006A priority patent/AR032632A1/es
Publication of WO2002016624A1 publication Critical patent/WO2002016624A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8265—Transgene containment, e.g. gene dispersal

Definitions

  • the present invention relates to methods for site-specific recombination in plant cells, as well as DNA vectors which can be employed in such methods. More particularly, the invention relates to recombinant DNA vectors containing recombinase-specific sequences and methods for creating a transgenic plant that has been modified such that two site-specific recombination systems may be employed to eliminate one or more controlled utility genes present as a transgene(s).
  • the methods and vectors of the present invention can be used to express one or more transgenes with desirable traits and to prevent transmission of the transgenes to progeny of the plant.
  • transgenes can be limited to a particular stage of plant development, a particular plant tissue, particular environmental conditions, or a particular time or location, or a combination of these situations.
  • the two site-specific recombination system may be contained in one transgenic plant or may be contained in two transgenic plants that are to be crossed.
  • Site-specific recombination is the reciprocal genetic exchange between defined sequences present on one or more DNA segments.
  • site-specific recombination strand exchange occurs in a conservative manner by the precise breakage and rejoining of the DNA within the site-specific recombination sequences.
  • reactions are catalyzed by a system-specific recombinase, which in some cases also requires additional factors to facilitate cleavage and excision of insertion of donor DNA sequences.
  • a site specific recombination system has many potential uses for rearranging genetic material in higher eukaryotic cells. Such a system can operate to invert, insert or delete a targeted DNA sequence positioned between defined site-specific recombination sites located on a DNA molecule.
  • bacteriophage PI Cre/lox site-specific recombination system (Hoess et al. 1982; Abremski et al 1983) consists of two components: (i) a recombinase enzyme (Ore) which is the product of the ere gene; and (ii) DNA sequence specific recombination sites (lox) at which the-recombinase acts.
  • the Ore gene encodes a 38 kDa recombinase which, without any other additional factors, catalyzes recombination between two lox sites.
  • the 38 kD "Ore" protein product of the bacteriophage PI ere gene is sufficient to catalyze recombination between lox sites without any additional co- factors.
  • the nucleotide sequence of the lox site consists of two inverted 13 base pair (bp) repeats separated by an asymmetric 8 bp spacer in which each inverted repeat acts as a binding site for the Cre recombinase.
  • Each inverted repeat and the contiguous 4 bp of the spacer comprise a binding domain for Cre (Hoess et al. 1984), and strand exchange occurs at a 6-bp staggered cut within the spacer (Hoess et al. 1985).
  • the asymmetric nature of the 8 bp spacer of the lox site gives a directionality to the lox site and thereby determines the type of recombination event. It has previously been demonstrated that recombination between two directly repeated lox sequences separated by an intervening DNA segment results in the excision of the DNA segment between the sites, whereas recombination between two inverted lox sites produces an inversion of an intervening DNA segment (Abremski et al. 1983). In addition, recombination between unlinked lox sites forms a cointegrate molecule. Due to the fact that the Cre//ox recombination requires only one gene product and a 34 bp sequence, this system provides a simple means to manipulate DNA in eukaryotic cells.
  • the bacteriophage P 1 cre gene product can be expressed as a functional recombinase in tobacco (Dale and Ow 1990).
  • the Cre recombinase recognizes its target lox sites and mediates reciprocal genetic crossovers at these sites.
  • the recombination event within plant chromatin is conservative, i.e. without loss or alteration of the lox sequence or DNA adjacent to the lox sequences.
  • the Cxdlox system thus provides a useful a tool for manipulating DNA in plant cells without disruption of the DNA sequences flanking the lox sites.
  • Bayley et al. (1992) utilized the C ⁇ e/lox system to exchange gene activity in transgenic plants.
  • the system was used to excise a firefly luciferase gene which had previously been incorporated into the tobacco genome between a functional promoter and a distal hybromycin phosphotransferase (hpt) gene.
  • Deletion of the luciferase blocking DNA resulted in the fusion of a promoter with a distally located coding sequence and the excision resulted in the exchange of luciferase activity for hybromycin resistance of the transgenic plant.
  • This experiment proved the feasibility of activating a gene distal from the promoter by site specific deletion of the intervening sequence with the C ⁇ ellox recombinase recombination system.
  • the FLP-FRT recombination system from Saccharomyces cerevisiae is based on site- specific recombination mediated by the FLP recombinase at a site-specific recombination target DNA sequence (FRT).
  • the FLP recombinase is encoded by the yeast plasmid 2 um and catalyzes a site specific recombination reaction that results in inversion of a segment of the 2 um plasmid.
  • DNAse protection experiments have shown that the FRT site consists of three 13 bp symmetry elements surrounding an 8 bp core region (Andrews et al, 1985).
  • the FRT site is characterized by three 13 bp repeat elements surrounding an 8-bp core sequence.
  • Two of the repeats are direct repeats located on one side of the core while an additional copy of the repeat is inverted in relation to these two and is located on the other side of the 8 bp core sequence.
  • the FLP protein binds to the symmetry elements and cleaves opposite strands of the DNA at either end of the core, becoming covalently attached to the 3' phosphate and leaving an 8 base overhang with a 5' OH terminus (Schwartz and Sadowski 1989). Jayaram et al.
  • the "minimal" FLP substrate resides in a 13-bp dyad symmetry element consisting of two flanking repeats surrounding an 8-bp core located within the 65-bp recombination region which contains an additional copy of the 13 bp symmetry element.
  • Kilby et al. (1995) have expressed the FLP recombinase under the regulation of a heat shock promoter using as a target the FRT site containing both the minimal FLP recognition sequence containing two inverted 13 bp repeats separated by the 8 bp core and the additional 13 bp flanking repeat.
  • the presence of the additional 13 bp repeat element enhances the reactivity of the minimal FLP substrate.
  • sequences extraneous to the recombination region can also affect the efficiency of the recombination reaction.
  • the FLP recombinase has been shown to function efficiently in the progeny of crosses made between primary transformed tobacco plants (Lloyd and Davis, 1994). These authors, however, were unsuccessful in their attempts to obtain a transgenic Arabidopsis plant expressing FLP recombinase. By inserting two directionally repeated FRT sites flanking a target gene it is possible, by addition of FLP recombinase, to excise the intervening DNA fragment by site- specific eviction. FLP recombinase mediated excision has also been shown to be reversible, providing means for the introduction of DNA into specific sites in mammalian chromosomes (O'Gorma et ⁇ /., 1991).
  • the present invention is directed to DNA constructs, vectors and methods for using the constructs and vectors to construct a transgenic plant that contains one or more transgenes whose excision can be induced by crossing the plant with plants containing additional factors, by subsequent transformation of the plant with the additional factors, or by providing a single vector containing all of the factors required for controlled excision of the transgene. More particularly, the invention relates to transgenic plants that have been modified such that a transgene that has been introduced into a plant can be excised at various stages of growth and thus limited to a particular stage of plant development, a particular plant tissue, particular environmental conditions, or a particular time or location, or a combination of these situations.
  • a DNA construct which comprises a first and second site-specific recombinase that work in tandem to (1) control activation of the first recombinase and (2) direct the controlled excision of a transgene from a transgenic plant.
  • the DNA construct comprises a first DNA sequence comprising a plant-active first promoter operably linked to one or more transgenes whose expression results in ⁇ an altered plant phenotype and a second DNA sequence comprising a transiently-active second promoter that is active only under specific conditions.
  • the first DNA sequence is flanked by a unique first site-specific recombination sequence that is recognized by a first site-specific recombinase.
  • the transiently active promoter is operably linked to the structural gene for a first site-specific recombinase but the second promoter and first recombinase are separated by a blocking sequence that is in turn bounded at each end by a second and distinct unique site- specific recombination sequence that is recognized by a second site-specific recombinase.
  • the DNA construct comprises the first and second DNA sequences as described in the previous above embodiment and also a third DNA sequence comprising a plant-active third promoter operably linked to the second site-specific recombinase that is specific for the second site-specific recombination sequence.
  • the plant-active third promoter is preferably a regulatable promoter.
  • vectors containing the DNA constructs are provided.
  • transgenic plant tissue containing the DNA constructs are provided.
  • Plant tissue may be plant cells, plant tissue, plant organs, plants, seeds and the like.
  • FIGURES Figure 1 is a Schematic illustration of the system of the present invention wherein the prevention of transmission of a transgene is described.
  • Figure 2 is a schematic representation of the construction of a NOS blocker sequence flanked by two FRT site-specific recombination sites.
  • Figure 3 is a schematic representation of the known restriction map of Arabidopsis Ecotype Lansburg and a clonmg scheme for isolating the atDMCl promoter region on a plasmid vector for use in the practice of the invention.
  • Figure 4 is a schematic representation of the construction of an DNA sequence comprising the DNA sequences of / ⁇ t-35S promoter-GUS-NOS3'-/ox-OCS3"-6 ⁇ r-NOS as a contiguous DNA sequence for use in the practice of the present invention.
  • Figure 5 is a schematic representation showing the assembly of a DNA sequence comprising the a ⁇ MCl-FRT-' t///NOSter"blocker-FRT-Cre-NOS3 'sequences as a contiguous DNA sequence for use in the practice of the present invention.
  • Figure 6 is a schematic representation showing the assembly of a DNA sequence comprising the atDMCl promoter-FRT-nptll/NOS ter blocker-FRT-Cre-NOS3'-NOS promoter- bar-OCS-/ox-NOS 3'-GUS-35 S-lox as a contiguous DNA sequence for use in the practice of the present invention.
  • the present invention involves methods for the construction of a transgenic plant that contains one or more genes whose genetic transmission through reproduction of the plant can be regulated by employing two site-specific recombinase systems. This system achieves a control of gene transmission in subsequent generations that result from the transgenic plant. By controlling the excision of genes that affect the plant phenotype, it is possible to grow transgenic plants where the phenotype is advantageous or desired but the phenotype is not desired in subsequent generations.
  • site-specific recombination sequences are arranged along the DNA molecule to effect excision of the intervening DNA (e.g., as direct repeats of lox separated by the intervening DNA).
  • intervening DNA e.g., as direct repeats of lox separated by the intervening DNA.
  • One skilled in the relevant art can readily appreciate, however, that such a system could alternatively be employed in a way such that the recombination between adjacent site-specific recombination sequences could also cause inversion of the intervening DNA if the sequences were arranged in the necessary orientation along the DNA molecule (e.g., as inverted repeats of lox separated by the intervening DNA).
  • site-specific excision sequence when used in the present specification defines a site-specific recombination sequence that is oriented in such a way as to effect removal of the DNA positioned between two such sequences.
  • a gene that results in an altered plant phenotype is any gene whose expression leads to the plant exhibiting a trait or traits that would distinguish it from a plant of the same species not expressing the gene. Examples of such altered phenotypes include a different growth habit, altered flower or fruit color or quality, premature or late flowering, increased or decreased yield, sterility, mortality, disease susceptibility, altered production of secondary metabolites, or an altered crop quality such as taste or appearance.
  • genes useful for expression in transformed plant cells are known in the art. See for example, U.S. Patent No. 5,659,026.
  • Exemplary genes include, but are not limited to, Bt genes or patatin genes for insect resistance; the Hml gene and chitinase genes for disease resistance; the pat, bar, EPSP synthase gene or ALS genes for herbicide resistance; genes encoding proteins with altered nutritional properties; genes encoding enzymes involved in starch or oil biosynthetic pathways; down-or up-regulatory sequences for metabolic pathway enzymes; and the like.
  • Bt genes or patatin genes for insect resistance include, but are not limited to, Bt genes or patatin genes for insect resistance; the Hml gene and chitinase genes for disease resistance; the pat, bar, EPSP synthase gene or ALS genes for herbicide resistance; genes encoding proteins with altered nutritional properties; genes encoding enzymes involved in starch or oil biosynthetic pathways; down-or up-regulatory
  • Zhao et al. discloses the construction of a prior art superbinary vector pPHP 10525.
  • This vector contains virB, virC and virG genes isolated from superviral strain A281.
  • the vector includes 35Sbar and ubi/GUS plant expression cassettes inserted between the T-DNA borders.
  • Plant expression cassettes preferably comprise a structural gene to which is attached regulatory DNA regions that permit expression of the gene in plant cells.
  • the regulatory regions consist at a minimum of a promoter capable of directing expression of a gene in a plant cell.
  • the promoter is positioned upstream or at the 5' end of the gene to be expressed.
  • a terminator is also provided as a regulatory region in the plant expression cassette and is capable of providing polyadenylation and transcription terminator functions in plant cells.
  • the terminator is attached downstream or at the 3' end of the gene to be expressed. Marker genes, included in the vector, are useful for assessing transfonnation frequencies in this invention.
  • transiently-active promoter any combination of repressible, inducible, and transiently active promoters can be employed with any or all of the DNA sequences contemplated by the present invention (i.e., transgene(s), first, and second recombinases).
  • a transiently-active promoter is any promoter that is active either during a particular phase of plant development or under particular environmental conditions, and is essentially inactive at other times. Any appropriate transiently-active promoter can be used, and selection of an appropriate promoter will be governed by-such considerations as plant type and the time at which excision of the transgene is desired.
  • the transiently-active promoter is preferably not a "leaky” promoter, meaning that it is active substantially only during a well-defined phase of plant growth or under particular environmental conditions, and substantially inactive at all other times. Such requirements will also be useful for the plant-active promoter controlling the expression of the second recombinase that in certain embodiments will be an inducible promoter. This property prevents the premature "triggering" of the system.
  • transiently-active promoters and inducible promoters which can be applied in the present system, as in, for example, the SPL promoter described in Yang et al. (1999).
  • a plant-active promoter is any promoter that is active in cells of a plant of interest. Plant-active promoters can be of viral, bacterial, fungal, animal or plant origin.
  • a gene and a promoter are to be considered to be operably linked if they are on the same strand of DNA, in the same orientation, and are located relative to one another such that the promoter directs transcription of the gene (i.e. in cis).
  • the presence of intervening DNA sequences between the promoter and the gene does not preclude an operable relationship.
  • the blocking sequence can be any sequence that prevents expression of the gene linked to the transiently-active promotor, such as a termination signal.
  • the gene encoding the repressor is responsive to an outside stimulus, or encodes a repressor element that is itself responsive to an outside stimulus, so that repressor function can be controlled by the outside stimulus.
  • the stimulus is preferably one to which the plant is not normally exposed, such as a particular chemical, temperature shock, or osmotic shock. In this way, the simple application of the stimulus will block the repression of the gene it is operably linked to, yet there will be a low probability of the repressor being accidentally or incidentally blocked.
  • the chemical is preferably non-toxic to the crop and to non-pest animals.
  • a preferred system is the TnlO tet repressor system, which is responsive to tetracycline. Gatz, et al. (1992).
  • a modified Cauliflower Mosaic Virus (CaMV) 35S promoter containing one or more, preferably three, tet operons is used; the TnlO tet repressor gene produces a repressor protein thai binds to the tet operon(s) and prevents the expression of the gene to which the promoter is linked.
  • CaMV Cauliflower Mosaic Virus
  • the presence of tetracycline inhibits binding of the TnlO tet repressor to the tet operon(s), allowing free expression of the linked gene.
  • This system is preferred because the stimulus, tetracycline, is not one to which the plant would normally be exposed, so its application can be controlled. Also, since tetracycline has no harmful effects on plants or animals, its presence would not otherwise impede the normal development of the plant, and residual amounts left on the seed or plant after treatment would have no significant environmental impact.
  • the recombinase/excision sequence system can be any one that selectively removes DNA in a plant genome. The excision sequences are preferably unique in the plant, so that unintended cleavage of the plant genome does not occur.
  • a preferred system is the bacteriophage C ⁇ ellox system, wherein the Cre protein performs site- specific recombination of DNA at lox sites.
  • Other systems include the resolvases (Hall, 1993), FLP (Pan, et al., 1993), SSV1 encoded integrase (Muskhekishvili, et al., 1993), and the maize Ac/Ds transposon system (Shen and Hohn, 1992).
  • a site-specific excision sequence is a DNA sequence that is recognized by a site-specific recombinase.
  • a site-recombinase is an enzyme that recognizes a site-specific excision sequence or set of specific excision sequences and effects the removal of, or otherwise alters, DNA between specific excision sequences.
  • the methods used for the actual transformation of the target plant are not critical to this invention.
  • the transformation of the plant is preferably permanent, e.g. by integration of introduced sequences into the plant genome, so that the introduced sequences are passed onto successive plant generations.
  • plant transformation techniques well-known to workers in the art, and new techniques are continually becoming known. Any technique that is suitable for the target plant can be employed with this invention.
  • the sequences can be introduced in a variety of forms, such as a strand of DNA, in a plasmid, or in an artificial chromosome, to name a few.
  • the introduction of the sequences into the target plant cells can be accomplished by a variety of tecliniques, as well, such as calcium phosphate-DNA co- precipitation, electroporation, microinjection, Agrobacterium infection, liposomes or microprojectile transformation.
  • tecliniques such as calcium phosphate-DNA co- precipitation, electroporation, microinjection, Agrobacterium infection, liposomes or microprojectile transformation.
  • Those of ordinary skill in the art can refer to the literature for details, and select suitable techniques without undue experimentation.
  • the methods used to regenerate transformed cells into whole plants are not critical to this invention, and any method suitable for the target plant can be employed.
  • the literature describes numerous techniques for regenerating specific plant types, (e.g., via somatic embryogenesis, Umbeck, et al., 1987) and more are continually becoming known.
  • Those of ordinary skill in the art can refer to the literature for details and select suitable techniques without undue experimentation.
  • site-specific recombination involves eviction of an intervening ⁇ blocking DNA fragment to allow activation of a silent first recombinase and subsequent excision of a transgene. It is also possible to activate genes by site-specific reversion of the intervening DNA fragment when the flanking recombination sites (lox, e.g.) are inverted in their orientation relative to each other.
  • the present invention can be used to make a variety of transgenic plants.
  • the method is particularly suited for use with plants that are planted as a yearly crop from seed.
  • These include, but are not limited to, fiber crops such as cotton and flax; dicotyledonous seed crops such as soybean, sunflower, rapeseeds and peanut; annual ornamental flowers; monocotyledonous grain crops such as maize, wheat and sorghum; leaf crops such as tobacco; vegetable crops such as lettuce, carrot, broccoli, cabbage and cauliflower; and fruit crops such as tomato, zucchini, watermelon, cantaloupe and pumpkin.
  • the transgenic plants of the present invention are prepared by introducing into their genome a series of functionally interrelated DNA sequences, containing several basic elements.
  • a first DNA sequence is provided which comprises a plant-active promoter operably linked to one or more transgenes whose expression results in an altered plant phenotype. This first DNA sequence is flanked by unique first site-specific recombination sequences that are recognized by a first site-specific recombinase.
  • a second DNA sequence is provided which comprises a second plant-active promoter that is active at a particular stage in plant development or under particular environmental conditions ("transiently-active promoter").
  • This transiently-active promoter is operably linked to a blocking sequence and a distal first site- specific recombinase, wherein the blocking sequence separates the promoter and the first recombinase.
  • the blocking sequence is flanked on each side by a unique second site-specific excision sequence.
  • the second site-specific excision sequences which flank the blocking sequence are recognizable by a second site-specific recombinase, which can bind lo the second site-specific excision sequences to direct the removal of the blocking sequence.
  • the blocking sequence prevents transcription of the first recombinase when present and causes expression of the first recombinase when excised.
  • the methods of the present invention also disclose a third DNA sequence encoding a plant-active promoter operably linked to a second site-specific recombinase which, when expressed, directs the precise excision of the blocking sequence that is flanked on either end by the second-site-specific recombination sequence.
  • a first plant contains the first and second DNA sequences which result in an altered plant phenotype due to expression of the transgenes.
  • DNA containing the third DNA sequence is introduced into a second plant. Without crossing the first to the second plant, the structural gene for the first recombinase is not expressed, even in the stage(s) of the plant life cycle during which the transiently-active promoter is activated.
  • the second recombinase is expressed in the resulting hybrid plant and effects removal of the blocking sequence at the second site-specific excision sequences.
  • the structural gene for the first recombinase specific for the first specific excision sequence is expressed from the transiently-active promoter that is active, for example, in the flowers, prior to or during gametophyte development.
  • the transgene remains expressed and intact until the expression of the first recombinase during gametophyte development in the flowers drives the excision of the DNA sequence of the first gene and promoter flanked by the first specific excision sequences. The excision occurs prior to formation of the pollen and embryo sacs, and hence prevents transmission of the transgene to the developing seeds.
  • the third gene that encodes the recombinase specific for the second site-specific excision sequences can also be provided in the same construct with the first and the second DNA sequences of the invention.
  • the recombinase specific for the second site-specific excision sequences is preferably operably linked to an inducible promoter.
  • the inducible promoter When the inducible promoter is expressed, the second recombinase is expressed, followed by excision of the blocker sequence of the second DNA sequence, activation of the first recombinase based on the expression characteristics of the transiently active promoter, and subsequent excision of the transgene(s) of the first DNA sequence.
  • the third gene that encodes the recombinase specific for the second specific excision sequences can be introduced by re- transformation of a plant that has already been transformed with the first and second DNA sequences of the invention.
  • a construct with the third DNA sequence of the invention operably linked to a plant-active promoter is introduced into a background where the first and second sequences of the invention have already been incorporated into the plant genome.
  • the second recombinase When the third DNA sequence is expressed, the second recombinase is activated, followed by excision of the blocker sequence of the second DNA sequence, activation of the first recombinase based on the expression characteristics of the transiently active promoter, and subsequent excision of the transgene(s) of the first DNA sequence.
  • EXAMPLE 1 This example demonstrates the isolation and mutagenesis of the atDMCl promoter.
  • Figure 3 there is shown the known restriction map of Arabidopsis Ecotype Lansburg and a cloning scheme for isolating the atDMCl promoter region.
  • the atDMCl promoter described in the present invention can be generated using standard molecular genetic cloning and Polymerase Chain Reaction (PCR) techniques that are known to those skilled in the art.
  • Genomic DNA is isolated from Arabidopsis Ecotype Lansburg and PCR-amplified with the primers DMC ⁇ S'-gttaacaccgtttatatgagacaaaatcagctatg-S'; Seq ID No.l) and DMC4 (5'- catccccacttgcgaattcactacc-3'; Seq ID No.2).
  • the resulting product is then digested with restriction enzymes RindUI and EcoRI to yield a RinaTW EcoRI DNA fragment.
  • the genomic DNA is then PCR-amplified with primers DMC3 (5'-ggtagtgaattcgcaagtggggatg-3'; Seq ID No.
  • EXAMPLE 2 This example demonstrates the construction of a NOS blocker sequence flanked by two FRT sites.
  • Figure 2 there is shown a schematic representation of the cloning process which can be utilized to obtain the blocking sequence flanked on either side by an FRT site.
  • the FRT sites of this example contain both the minimal FLP recognition sequence and the additional flanking repeat to ensure high efficiency of recombination. Small letters in the FRT sequences represent restriction enzyme specific sequences while capital letters indicate the FRT nucleotides.
  • the plasmid pMUCBS this-is pMUC9 with pBS+(KS) polylinker; Jones, J. et al.
  • FRT-1 , FRT-2, FRT-3, and FRT-4 sequences depicted in the Figures are synthetic oligolucleotides that were synthesized using standard techniques as well known within the art.
  • the resulting DNA is then digested with Xhol, filled in to remove overhangs, digested with Pstl and ligated to the FRT-l/FRT-2 double strand DNA fragment of SEQ ID No. 6.
  • the resulting plasmid is then digested with Pstl and ligated with a Pstl fragment containing the nos terminator and nptll gene from plasmid pB1121(Clontech).
  • the resulting plasmid contains the nptlllNOS ter blocker flanked by FRT sites (represented by solid arrows). This DNA sequence can be removed and operably linked to a plant-active promoter utilizing the remaining restriction enzyme sites as indicated in Figure 2.
  • EXAMPLE 3 This example demonstrates the construction of an DNA sequence containing the transiently-expressed atDMCl promoter and Cre gene separated by a blocking sequence that is flanked on either end by FRT site-specific recombination sequences.
  • Figure 5 there is shown a schematic representation of the cloning strategy utilized to construct a DNA sequence containing the Cre gene under the control of the atDMCl promoter.
  • the plasmid generated in Example 1 is digested with KindHUXbal.
  • Plasmid pED23 (Dale et al, 1990) containing the 35S promoter operably linked to the Cre recombinase gene which is further operably linked at its 3' end to a NOS termination sequence is also digested with HindL ⁇ UXbal. The two digestion products are then ligated, digested with Xbal, and filled in to remove overhangs. The blocker sequence flanked by FRT sites of Example 2 is also digested with Spel, filled in to remove overhangs, and then digested with Kpril.
  • the two resulting DNA fragments are then ligated to yield the following DNA fragments in a 5' to 3' orientation: atDMCl promoter-FRT-n 7t// NOSler blocker-FRT-Cre-NOS3'.
  • the promoter and Cre gene are separated by a blocking sequence flanked by FRT site-specific recombination sequences.
  • EXAMPLE 4 This example demonstrates the construction of a FLP recombinase gene operably linked to the 35S promoter and the NPT II gene, which encodes for kanamycin resistance, operably linked to the NOS promoter. Utilizing standard molecular cloning techniques as in Examples 1- 3, the FLP recombinase is operably linked to a plant-active promoter and inserted on a plant compatible DNA vector along with the gene for kanamycin resistance.
  • EXAMPLE 5 This example demonstrates the construction of a cassette comprising the GUS gene operably linked to the 35S promoter, with the promoter and gene flanked by lox sequences.
  • this DNA sequence also contains the BAR gene operably linked to the "NOS promoter.”
  • NOS and OCS represent 3' transcription termination and processing sequences for the inserted genes that were derived from Agrobacterium tumefaciens nopaline synthase (NOS) and Octopine synthase (OCS) gene sequences.
  • NOS Agrobacterium tumefaciens nopaline synthase
  • OCS Octopine synthase
  • a iox core sequence as shown with Clal/Sacl overhangs is directionally inserted into the polylinker site of plasmid pTML23.
  • a lox core sequence with an Accl overhang and an EcoRV blunt end is inserted into the polylinker site. This results in two directly repeated lox sites within the polylinker that are separated by EcoRI, Pstl and MM sites.
  • Plasmid pSLJ512 containing the NOS promoter operably linked to the bar gene and the OCS site is digested with Pstl, filled in to remove overhangs, digested with Bc/I and ligated with the above lox polylinker that has been digested with C/ ⁇ l, filled in to remove overhangs, and digested with Bg/I.
  • the resulting plasmid is digested with Xhol and EcoRV and the NOS promoter/ ⁇ r/OCS fragment is ligated with EcoRV/XhoI digested plasmid pSP72.
  • the resulting plasmid is digested with Pstl/EcoRI and ligated with the ⁇ stl/EcoIRI fragment from plasmid pB1121(Clontech) containing the GUS gene operably linked to the 35S promoter to yield the DNA sequence containing the GUS gene operably linked to the 35S promoter, with the promoter and gene flanked by lox sequences and the BAR gene operably linked to the NOS promoter.
  • EXAMPLE 6 This example demonstrates the assembly of the DNA sequence comprising the sequence of the atDMCl -FRT-nptllfNOSte ⁇ blocker-FRT-Cre-NOS3'-NOS promoter-BAR-OCS3'-/ox- GUS-35S-/ox.
  • Plasmid pZP200A Hajdukiewicz et al.
  • the resulting plasmid and the plasmid containing the atDMCl promoter-FRT-H/?t/Z/NOSter blocker-FRT-Cre-NOS3' sequences are digested with Sail and HindUI and ligated to yield a plasmid containing the ⁇ tD Ci-FRT-nptH/NOSter blocker-FRT-Cre-NOS3'-NOS promoter-BAR- OCS3'-/ox-GUS-35S-/ox DNA fragment on a single DNA vector.
  • EXAMPLE 7 This example demonstrates a method for the Production of whole plants using the two recombinase system.
  • One kind of transgenic tobacco plant can be made using the techniques disclosed in the present invention that contains the first and second DNA sequences of the disclosed invention on a single DNA fragment in the following orde ⁇ atDMCI promoter-FRT- npt ⁇ NOS ter blocker-FRT-Cr ⁇ -NOS3'-NOS promoter-bar-OCS-Zox-NOS 3'-GUS-35 S-lox.
  • a plant with this construct that actively expresses GUS and BAR proteins is crossed with a plant that contains an DNA sequence with the FLP recombinase gene operably linked to the 35S promoter and the NPTII kanamycin resistance gene operably linked to the NOS promoter.
  • Progeny that contain the complete system will be selected by their resistance to both kanamycin and bastar, expression of GUS, location of Cre under atDMCl promoter as a result of the deletion of blocking sequence and the presence of all of the other components of the systems by PCR with suitable primers.
  • EXAMPLE 8 This example describes methods for the evaluation of transformed plants from Example 7 for the subsequent production of seeds with the transgene deleted.
  • the hybrid progeny are allowed to grow, self-fertilize and produce seeds. Seeds are germinated and progeny that contain the complete system are selected by their resistance to both kanamycin and bastar, loss of expression of GUS, transient expression of Cre under the regulation of the atDMCl promoter, and the loss of the 35S-GUS cassette and the presence of all of the other components of the system. Because all of the aforementioned DNA sequences are known, one skilled in the relevant art will readily appreciate that their presence and expression can readily be monitored using standard PCR techniques with the appropriate primers. Transgenic rapeseeds plants containing the complete system can be generated in the same way as for tobacco.

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Abstract

L'invention concerne un procédé de production d'une plante génétiquement modifiée exprimant un transgène présentant une caractéristique recherchée faiblement transmise, cette caractéristique étant perdue dans les générations suivantes en raison de l'excision du transgène. Ledit procédé consiste à hybrider une première plante régénérée à partir d'une cellule végétale transfectée avec des séquences d'ADN composées d'un premier gène dont l'expression aboutit à un phénotype végétal modifié lié à un promoteur, de la cassette de gène et du promoteur logés sur les deux côtés par les premières séquences d'excision spécifiques, d'un deuxième gène codant une rembinase spécifique aux premières séquences d'excision spécifiques, lié à un promoteur actif de manière transitoire, le gène et le promoteur étant séparés par une séquence de blocage logées sur les deux côtés par les deuxièmes séquences d'excision spécifiques, avec une deuxième plante régénérée à partir d'une deuxième cellule végétale avec des séquences d'ADN composées d'un troisième gène codant une recombinase spécifique aux deuxièmes séquences d'excision spécifiques, lié à un promoteur ; et, à cultiver une plante hybride à partir de la graine hybride. Dans un autre mode de réalisation, une plante individuelle peut être transformée de manière stable avec les séquences citées ci-dessus de manière à obtenir le même résultat. L'invention concerne en outre les cellules végétales, les tissus végétaux, les graines végétales, et les plantes entières contenant les séquences d'ADN selon l'invention.
PCT/SG2000/000124 2000-08-25 2000-08-25 Reduction de la transmission de transgenes dans des plantes WO2002016624A1 (fr)

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PCT/SG2000/000124 WO2002016624A1 (fr) 2000-08-25 2000-08-25 Reduction de la transmission de transgenes dans des plantes
EP00959109A EP1313865A1 (fr) 2000-08-25 2000-08-25 Reduction de la transmission de transgenes dans des plantes
CA002419646A CA2419646A1 (fr) 2000-08-25 2000-08-25 Reduction de la transmission de transgenes dans des plantes
AU2000270487A AU2000270487B2 (en) 2000-08-25 2000-08-25 Reduction of transmission of transgenes in plants
AU7048700A AU7048700A (en) 2000-08-25 2000-08-25 Reduction of transmission of transgenes in plants
BR0017326-6A BR0017326A (pt) 2000-08-25 2000-08-25 Redução de transmissão de transgenes em plantas
CNB008198519A CN1296484C (zh) 2000-08-25 2000-08-25 降低植物中转基因的传递
ARP010104006A AR032632A1 (es) 2000-08-25 2001-08-22 Reduccion de la transmision de transgenes en plantas

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002010415A3 (fr) * 2000-07-28 2002-06-20 Univ Connecticut Methodes d'excision controlees et automatiques d'adn heterologue issu de plantes transgeniques et cassettes de genes d'excision d'adn
WO2006032426A3 (fr) * 2004-09-23 2006-07-20 Basf Plant Science Gmbh Cassettes de recombinaison et technique d'excision de sequence dans des plantes
WO2008043844A1 (fr) * 2006-10-13 2008-04-17 Vrije Universiteit Brussel Préparation de plantes transgéniques

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103215280B (zh) * 2013-05-08 2014-08-20 山东省农业科学院高新技术研究中心 一种花生spl转录因子基因及其编码蛋白与应用

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WO1993001283A1 (fr) * 1991-07-08 1993-01-21 The United States Of America As Represented By The Secretary Of Agriculture Plantes transgeniques sans genes de selection
WO1996004393A2 (fr) * 1994-08-01 1996-02-15 Delta And Pine Land Company Regulation de l'expression d'un gene de plante
WO1999010488A1 (fr) * 1997-08-28 1999-03-04 The Salk Institute For Biological Studies Recombinaison specifique de site chez les eucaryotes et produits de recombinaison utilises a cet effet
WO1999011807A1 (fr) * 1997-09-05 1999-03-11 Purdue Research Foundation Expression selective des genes chez les vegetaux
WO1999023234A1 (fr) * 1997-10-30 1999-05-14 Mogen International N.V. Inhibition de la remobilisation des composes stockes avant et apres recolte
WO1999025841A1 (fr) * 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Nouvelle sequence d'acides nucleiques codant une recombinase flp

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Publication number Priority date Publication date Assignee Title
WO1993001283A1 (fr) * 1991-07-08 1993-01-21 The United States Of America As Represented By The Secretary Of Agriculture Plantes transgeniques sans genes de selection
WO1996004393A2 (fr) * 1994-08-01 1996-02-15 Delta And Pine Land Company Regulation de l'expression d'un gene de plante
WO1999010488A1 (fr) * 1997-08-28 1999-03-04 The Salk Institute For Biological Studies Recombinaison specifique de site chez les eucaryotes et produits de recombinaison utilises a cet effet
WO1999011807A1 (fr) * 1997-09-05 1999-03-11 Purdue Research Foundation Expression selective des genes chez les vegetaux
WO1999023234A1 (fr) * 1997-10-30 1999-05-14 Mogen International N.V. Inhibition de la remobilisation des composes stockes avant et apres recolte
WO1999025841A1 (fr) * 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Nouvelle sequence d'acides nucleiques codant une recombinase flp

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Title
KILBY N ET AL: "FLP recombinase in transgenic plants: constitutive activity in stably transformed tobacco and generation of marked cell clones in Arabidopsis", PLANT JOURNAL,GB,BLACKWELL SCIENTIFIC PUBLICATIONS, OXFORD, vol. 8, no. 5, 1 November 1995 (1995-11-01), pages 637 - 652, XP002089983, ISSN: 0960-7412 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002010415A3 (fr) * 2000-07-28 2002-06-20 Univ Connecticut Methodes d'excision controlees et automatiques d'adn heterologue issu de plantes transgeniques et cassettes de genes d'excision d'adn
WO2006032426A3 (fr) * 2004-09-23 2006-07-20 Basf Plant Science Gmbh Cassettes de recombinaison et technique d'excision de sequence dans des plantes
WO2008043844A1 (fr) * 2006-10-13 2008-04-17 Vrije Universiteit Brussel Préparation de plantes transgéniques

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CN1296484C (zh) 2007-01-24
AR032632A1 (es) 2003-11-19
CA2419646A1 (fr) 2002-02-28

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