WO2000047754A1 - Procede d'inhibition de l'expression de genes cibles chez les plantes - Google Patents

Procede d'inhibition de l'expression de genes cibles chez les plantes Download PDF

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WO2000047754A1
WO2000047754A1 PCT/EP2000/001524 EP0001524W WO0047754A1 WO 2000047754 A1 WO2000047754 A1 WO 2000047754A1 EP 0001524 W EP0001524 W EP 0001524W WO 0047754 A1 WO0047754 A1 WO 0047754A1
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plant
dna construct
sequence
chimeric
plants
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Wolfgang Werr
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Rhobio
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Priority to CA002359749A priority patent/CA2359749A1/fr
Priority to EP00915151A priority patent/EP1151119A1/fr
Publication of WO2000047754A1 publication Critical patent/WO2000047754A1/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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]

Definitions

  • the present invention relates to a method for inhibiting the expression of target genes in plants and to DNA constructs used to implement this method.
  • Plant transcription factors mostly belong to gene families containing numerous members.
  • An example how large such families can grow is the myMamily comprised of at least 97 genes in Arabidopsis.
  • homeobox and MADS box genes are found in multiple copies comprising several subfamilies in all plant species analyzed so far. In genetic screens for loss of function alleles only few of these genes have been associated with a mutant phenotype.
  • One explanation for numerous silent family members may be genetic redundancy; each gene specific contributions may be subtle or dependent on special conditions. This fraction of silent genes in plant as others genomes provides a major challenge in molecular biology because these are foreseen to oppose a functional analysis also in reverse genetic approaches.
  • the authors of the present invention now propose an alternative approach for blocking the expression of genes in plants which meets the above requirements and presents other advantages.
  • the method of the invention distinguishes from previous technologies in that this is, as further described, a dominant approach with high penetrance. This method is independent from any DNA sequence homology with the targeted gene, which is crucial for antisense and cosuppression approaches, and moreover circumvents the problem of redundancy in genes and/or functions.
  • the method of the invention involves the use of a chimeric DNA construct comprising at least one repressor sequence in transcriptional fusion with at least one plant-specific sequence that codes for a protein or fragment thereof that bind to DNA or that activates transcription either by binding to DNA itself or by interacting with a DNA-binding protein ; said repressor sequence being operably linked to elements allowing the transcription of said fused sequences.
  • plant-specific sequence means a sequence that originates from plants or has been modified (or shown) to function in plants, i.e. to actually bind to DNA in plants or to activate transcription either by binding to DNA itself or by interacting with a DNA-binding protein.
  • the repressor sequence of said chimeric DNA construct preferably does not contain a sequence that codes for a second other DNA-binding domain, so that no undesirable interaction could occur.
  • said repressor sequence is at least the repressor domain of the Drosophila engrailed gene (eng).
  • the product of the Drosophila engrailed (eng) locus was shown to be involved in the regulatory interactions that govern early embryogenesis (Poole et al, 1985). Eng is a homeodomain-containing transcription factor that is required for cell fate specification through out development of the fly. A pioneering experiment was performed in Drosophila where the DNA-binding homeodomain of the engrailed (eng) gene was replaced by its Fushi tarazu (Ftz) counterpart, and expression of the chimeric gene controlled by heat shock promoter.
  • the sequence specificity of the Ftz homeodomain directed the strong transcriptional eng repressor function to Ftz target genes resulting in phenocopies of Ftz loss of function alleles in transgenic progeny (John et al., 1995).
  • the Eng protein was shown to be an active repressor in Drosophilia (John et al, 1995) and cultured animal cells (Han & Manley, 1993).
  • Conlon et al use an approach in which the DNA-binding domain of a transcription activator is fused to the engrailed repressor domain to assist in the analysis of Xenopus and Zebrafish transcription factors (Conlon et al, 1996). But no stable transformation has been carried out until now in plants, in order to obtain transgenic plants with heritable mutant phenotype.
  • the term "repression domain of the Drosophila engrailed gene (eng)” means a fragment of the Drosophila engrailed gene or of a derivative sequence thereof, said fragment comprising a nucleotide sequence that encodes a polypeptide interfering with the general transcription machinery and transcriptional activators. Said fragment advantageously comprises a minimal repression sequence coding for a polypeptide of 55 residues (Poole et al., 1985, Han K. et al., 1993). The whole engrailed gene may also be used but most preferably without its homeodomain.
  • a derivative sequence is understood as meaning a sequence which differs from the sequence of the Drosophila engrailed gene by mutation, insertion, deletion or substitution of one or more bases, or by the degeneracy of the genetic code so long as it codes for a polypeptide which is substantially the same as the Engrailed product.
  • Engrailed related domains which share the same repression activity, may also be used, such as those described in Smith et al. (1996).
  • the repressor sequence that is used may be a sequence that codes for at least the Kruppel-associated box-A (KRAB-A) domain of zinc finger proteins (Witzgall et al., 1994) for at least the RE-1- silencing transcription factor (REST) (Thiel et al., 1998), or for at least the BTB (for Broad-complex Tramtrac and Brie) domain, also known as POZ-domain (Ahmad et al., 1998 ; Huynh & Bardwell, 1998).
  • KRAB-A Kruppel-associated box-A domain of zinc finger proteins
  • REST RE-1- silencing transcription factor
  • BTB for Broad-complex Tramtrac and Brie domain
  • Chimeric DNA constructs further comprising corepressor sequences associated with said repressor sequence are also encompassed.
  • corepressors of interest one may cite KAP-1 (Friedman et al.,
  • said repressor sequence is fused to a plant- specific sequence that encodes a DNA-binding protein domain.
  • DNA binding protein domain refers to the whole protein that contains such a DNA- binding domain but also to parts of it independently of their ability to bind to DNA.
  • a plant transcription factor or a DNA-binding fragment thereof, is used as the DNA-binding domain containing protein.
  • a complete transcription factor is preferably used, as it preserves interactions between proteins involved in regulation of transcription.
  • the repressor sequence is fused to a sequence that codes for a protein or parts thereof that activates transcription by binding to a DNA-binding protein such as a transcription factor.
  • a DNA-binding protein such as a transcription factor.
  • proteins may either increase the affinity of DNA-binding proteins to their target sites or mediate signalling to the transcriptional initiation complex and are generally called coactivators.
  • said transcription factor may preferably be selected from the group consisting of : STM ("Shootmeristemless"), member of the Knotted class of homeodomain proteins, which is an essential gene for development and function of the shoot apical meristem ;
  • AP3 member of the MADS box proteins, which is a floral organ identity B function gene essential for the development of petals and stamen.
  • AP3 protein is known to form heterodimers with the PISTILLATA (PI) gene product.
  • STM and AP3 encode plant transcription factors with well-characterized loss of function alleles. Both genes are not only members of different classes of plant transcription factors but deficiencies in both genes cause phenotypic effects at different developmental stages, early in the vegetative phase or rather late in the floral development.
  • ZmHox - member of the homeobox proteins more particularly ZmHox 1a/1 b and 2a/2b, which are expressed in maize meristems and proliferating cells from the early embryo to late reproductive organs. This expression pattern suggests a contribution to plant growth and morphogenesis;
  • Ms-41-A and Zm-41-A are associated with male fertility. Recent results assertain that Ms41-A protein from
  • Arabidopsis is a transcription factor belonging the family recently described as ARF1 family for Auxin Response Factor 1 (Ulmasov et al., 1997). Ms41-A analogues may also be used, such as those described in WO 97/23618 or the Arabidopsis gene Monopteros which encodes a transcription factor mediating embryo axis formation and vascular development (Hardtke et al., 1998), and the Arabidopsis gene ETTIN involved in floral development (Sessions, 1997).
  • the plants transformed with a chimeric DNA construct of the invention comprising such Ms-41-A factor are expected to be male sterile.
  • the chimeric DNA construct of the invention may further comprise a sequence that encodes at least the hormone-binding domain of a steroid hormone receptor, said sequence being in frame with the fusion construct consisting of at least one repressor sequence in transcriptional fusion with at least one plant-specific sequence that codes for a protein or fragment thereof that activates transcription either by binding to DNA itself or by interacting with a DNA-binding protein ; whereby the nuclear localization of the chimeric protein, that is the product of the translation of said fusion construct is dependent on the application of said steroid hormone or analogues thereof.
  • the steroid hormone receptor that is used may be for example the glycocorticoid receptor (GR).
  • GR glycocorticoid receptor
  • dexamethasone may be used to induce the nuclear translocation of the chimeric protein.
  • NLS nuclear localization signals
  • the repression domain of the Drosophila engrailed gene (eng) is operably linked to elements allowing its expression.
  • Such elements may more particularly comprise a promoter and polyadenylation signals.
  • the promoter that is used is a constitutive promoter.
  • the promoter that is used is a tissue- specific promoter or a developmentally regulated promoter. This allows a conditional loss of functions that may be desired, for example to design new plant varieties.
  • the promoter that is used is an inducible promoter.
  • the polyadenylation that can be used may be for example the 35S polyA terminator of cauliflower mosaic virus (CaMV), as disclosed in
  • 35S promoter or advantageously the double 35S constitutive promoter of CaMV as described in Kay et al. (1987) ; rice actin promoter followed by the rice actin intron (PAR- IAR) included in plasmid pAct1-F4 as described in Mc Elroy et al. (1991); the constitutive promoter EF-1 ⁇ of the gene encoding for plant elongation factor described in WO 90/02172 or in Axelos et al.
  • PCRU promoter of radish cruciferin gene allowing the expression specifically in seeds, as described in Depigny-This et al. (1992) ;
  • HMWG promoter High Molecular Weight Glutenin from barley (Anderson et al., 1989) ; the promoter of maize ⁇ zein (P ⁇ zein) included in p ⁇ 63 plasmid in Reina et al. (1990) allowing the expression in albumen of maize seeds ;
  • PGEA1 and PGEA6 promoters corresponding to the non coding 5' region of the genes GEA1 and GEA6, expressed in the grains in Arabidopsis thaliana (Gaubier et al., 1993) ; and ⁇ -phaseolin promoter (Riggs et al., 1989). • specific promoters which drive expression in particular plant tissues which are involved in the control of fertility :
  • the promoters of interest one may cite the Brassicaceae A3 or A9 promoter described in WO 92 1 1379, the A6 promoter described in WO 93 02197, or TA29, TA26, TA13 promoters described in WO 89 10396 ; the Ms41-A anther-specific promoter described in WO 97/23618, which may also be used in male sterility systems ; a dehiscence-zone specific promoter such as the one described in EP 692 030.
  • inducible promoters a promoter inducible in stress conditions, for example heat shock, wound or interaction with pathogens (Kuhlemeier et al., 1987, WO 94/21793) ; and an ethanol-inducible promoter (Salter et al., 1998) ; and - the PR1a promoter inducible by salicylic acid for example
  • the DNA construct of the invention is advantageously inserted in a vector, e.g. a plasmid, for use in plant cell transformation.
  • a vector e.g. a plasmid
  • the transformation of plant cells may be effected by transferring the above vectors in protoplasts, in particular after incubating those protoplasts in a solution of polyethyleneglycol (PEG) in the presence of divalent cations (Ca 2+ ) as described in Krens et al. (1982).
  • the transformation of plant cells may also be effected by electroporation as described in Fromm et al. (1986).
  • a gene gun may also be used allowing the projection of metal particles coated with a DNA construct of the invention, whereby genes are delivered into cell nucleus (Sanford et al., 1988).
  • Another method for transforming plant cells is cytoplasmic or nuclear micro-injection.
  • plant cells are transformed with a DNA construct of the invention, by means of a host cell infecting said plant cells.
  • a further subject of the present invention is thus a host cell transformed with a chimeric DNA construct as previously described.
  • the above host cell is Agrobacterium tumefaciens, as used in particular in the methods of Bevan et al. (1984) and An (1986), or Agrobacterium rhizogenes, in particular as used in the method of Jouanin et al. (1987).
  • Plant cell transformation is preferably effected by transferring the Agrobacterium tumefaciens T region of an extra-chromosomal circular plasmid that induces tumors (Ti) e.g by using a binary system.
  • helper Ti plasmid is a modified plasmid that has no T-DNA longer but still contains the virulence genes vir, necessary for transforming a plant cell. This plasmid is maintained in Agrobacterium.
  • the present invention also provides a transgenic plant or parts thereof, said plant being transformed with a DNA construct of the invention, or deriving from a plant initially transformed with a DNA construct of the invention.
  • Such transgenic plants exhibit heritable phenotypes.
  • the term "deriving" refers to plants of the following generations, as long as the parent plant is fertile.
  • parts of transgenic plants refer in particular to leaves, fruits, seeds, roots or cells that have been genetically transformed.
  • the preferred plants that are used for transformation may be for example selected from the group consisting of Arabidopsis thaliana, rice, tobacco, maize, Brassica, wheat, tomato and flowers (Petunia, rose, carnation).
  • the present invention thus provides a method for obtaining a transgenic plant, wherein a DNA construct of the invention is transferred and expressed in a plant cell and said cell is cultured under conditions for regenerating a whole transgenic plant.
  • a chimeric DNA construct of the invention may be used for inhibiting the expression of a target gene in the genome of a plant, the transcription of which involves a DNA-binding protein domain as previously defined. Inhibiting the expression of a target gene in the genome of a plant may be desired in many purposes. Many transcription factors known by the man skilled in the art play a role in the control of metabolic pathways (starch, lipids, amino acids...) or are involved in the plant development, or in the plant sensibility to pathogen. For example, blocking a gene whose expression is necessary for pollen or another formation (e.g. the Ms-41-A transcription factor, as above described) produces male sterility. Blocking the gene controlled by the AP-3 transcription factor also leads to male sterility. As another example, blocking the gene which codes for the enzyme which catalyses the conversion of sugars to starch can be used to produced sweet corn (see EP 475 584).
  • transgenic plants with enriched content in lysine by using, according to the invention, the opaque 2 transcription factor (Schmidt et al., 1990), involved in the control of the expression of certain zeins.
  • Use of other members of Myb- related transcription factors playing a role in the regulation of phenylpropanoid and lignin biosynthesis (Tamagnone et al., 1998) could also be interesting. Some others could be involved in cellular development and senescence.
  • the chimeric construct, fusion of the repressor domain to the plant transcription factor or part of it, can be advantageously used to identify essential protein-protein interaction domains and interacting protein partners in planta in transgenic plants.
  • expression of a chimeric protein comprising the repressor domain in fusion to a plant transcription factor is known to cause a dominant phenocopy.
  • the method according to the invention is therefore also suitable to study protein-protein interactions in planta.
  • the chimeric DNA construct of the invention can be used to produce phenocopies of loss of function mutants in genes involved in transcriptional control, by reversing the biological function from activation to repression and by providing chimeric fusion proteins in excess over the endogenous function.
  • Phenocopy is an artificial (transgenic) situation mimicking a mutant phenotype.
  • the term phenocopy is used by Smith et al. (1996), referring to the original description of John et al. (1995), describing a Ftz loss of function phenotype mimicked by the Eng Ftz chimeric protein.
  • the phenocopy caused by the expression of the chimeric fusion protein allows to associate a biological function to a given transcription factor or gene involved in transcriptional control.
  • a chimeric DNA construct of the invention may more particularly be used in a method for determining the function of a transcription factor in plants, comprising the steps of : i) fusing a sequence encoding said transcription factor to a repressor sequence to form a DNA construct as previously defined; ii) transforming plant cells with said DNA construct; iii) culturing the plants obtained from the transformed cells and observing a phenocopy of a mutation correlated with the loss of expression of genes controlled by said transcription factor.
  • the chimeric DNA construct of the invention allows the production of a cDNA library, said library being useful to isolate new genes controlled by the transcriptional activator as above described.
  • the present invention thus encompasses a method for identifying new genes in plants comprising the steps of : i) obtaining transgenic plants transformed with a chimeric DNA construct of the invention ; ii) comparing the RNA population from said transgenic plants with the RNA population of a plant that has not been transformed with a chimeric DNA construct of the invention, by amplifying (for example by Polymerase Chain Reaction in a differential display approach (Liang et al., 1993) the RNAs repressed by expression of the chimeric DNA construct, identified as genes inactive in said transgenic plants but active in the plant that has not been transformed with a chimeric DNA construct of the invention.
  • the plants to be compared should genotypically be as identical as possible.
  • the plants that have not been transformed with a chimeric DNA construct of the invention are transgenic plants transformed with a blank vector.
  • the method for identifying new genes in plants comprises the steps of : i) obtaining transgenic plants transformed with a chimeric DNA construct comprising a sequence that encodes at least the hormone- binding domain of a steroid hormone receptor and/or comprising an inducible promoter; ii) submitting said transgenic plants to an induction by means of a steroid hormone or analogues thereof and/or promoter inducer, whereby a phenocopy is created due to the loss of expression of target genes; iii) comparing the RNA populations from said transgenic plants before and shortly after induction, by amplifying (for example by Polymerase
  • RNAs repressed by expression of the chimeric DNA construct identified as genes active before but inactive after induction.
  • a differential display approach associated with the AFLP technique described by Vos et al. (1995) or the PCR- Select System (Clontech) is used.
  • the preferred embodiment of the method of the invention wherein an inducible system is used advantageously allows to identify direct target genes controlled by the transcriptional factor, at any time during the plant development.
  • Phenocopies are hardly sensitive to genetic redundancy. In contrast to mutagenesis approaches like gene machines where knock outs of single genes only give phenotypes in non-redundant situations, the phenocopy approach is expected to be informative in redundant situations. This may be important for species which are not or not truly diploid (e.g. wheat or maize respectively). Also in many Brassica crops the genome is triplicated relative to Arabidopsis.
  • the advantage in redundant situations is related to the fact that the phenocopy is caused by the chimeric protein making the method independent from DNA sequence homology, crucial for antisense and cosuppression approaches, which focuses effects to single or few genes (depending on the degree of sequence similarity).
  • the newly created chimeric repressor protein may perform and compete with all kinds of interactions and functions related to the transcription factor fused with the repressor. It thus uses functional protein domains and redundant protein functions that partly or fully compensate for the loss of a single gene product (reduced by antisense or cosuppression) therefore should be also effected by the chimeric repressor protein.
  • Phenocopies support results obtained in gene machines, where many additional elements in the genome frequently interfere with the conclusive association of a phenotype to a specific insertion.
  • the phenocopy approach is also helpful in transferring information between species, e.g. from Arabidopsis to Brassica crops or between grass crops (rice, maize, wheat, barley) and in comparing regulatory networks between species. By this way, it is possible to detect similarities and differences between groups of genes regulated by the same transcription factor within several species.
  • Figure 1 represents a top view into the shoot apical meristem of a young wild-type Arabidopsis seedling. In addition to both cotyledons, it has developed four elaborated leaves plus two additional leaf primordia in the center covering the functional shoot apical meristem.
  • Figure 2 represents a top view into the shoot apical meristem of
  • a functional shoot apical meristem is missing and has presumably been consumed by initiation of the single leaf primordium.
  • Figure 3 represents a flower of wild-type Arabidopsis thaliana with outermost sepals (green), white petals, yellow stamen and central carpel.
  • Figure 4 represents a flower of Arabidopsis thaliana phenocopy eng-AP3. A single sepal has been removed frontally, two neighbouring normal sepals are slightly displaced left and right to allow insight into the second and third floral whorls. Petals and stamen are obviously replaced by sepaloid organs close to the central carpel and small filamentous structures, one visible at the bottom between the two sepaloid petals.
  • Figure 5 represents a eng-STM construct.
  • Figure 6 represents a eng-AP3 construct.
  • the cDNA clone that is used is D 2 B clone as described in Poole et al, 1985. This clone was obtained by inserting a 2 kb EcoRI fragment of the engrailed cDNA in a pEMBL vector.
  • the engrailed cDNA sequence is identical to the data base entry (Genbank Access. M 10017) except for the lack of the 3' end downstream the EcoRI site gaattc, i.e the lack of the 3' end from nucleotide 2014 included.
  • the construct obtained was pRT ⁇ eng.
  • the pRT ⁇ Not-Asc is described in ⁇ berlacker et al. (1996). It is a derivative of pRT100 (T ⁇ pfer et al., 1987) and contains the CaMV 35S promoter and a polyA signal of Cabb B.
  • the homeodomain of STM was removed in an independent experiment.
  • the plasmid pRT ⁇ eng-STM was first partially digested with Hindll, secondly to completion with BamHI and ends were ligated after fill in reaction with Klenow enzyme.
  • the sequence of the AP3 coding region that is used is disclosed in Jack et al., (1992) and is identical to the data base entry (Genbank - Access - D21125).
  • the AP3 cDNA clone coding region is fused to a myc epitope at its carboxy-terminus.
  • the protein coding region including the myc epitope was amplified by PCR with primers adding terminal BamHI sites. The resulting BamHI fragment was inserted into the BamHI site of pRT ⁇ eng. This construct was designated as pRT ⁇ eng-AP3.
  • tumefaciens colony was grown at small scale (5 ml) in liquid YEB medium (5 g beef extract, 5 g saccharose, 1 g yeast extract, 1 g bacto-tryptone, 2 mM MgS0 per liter) at 28° C for 48 hours. From this preculture 0.1 ml each are used to inoculate 4 x 500 ml LB medium (5 g yeast extract, 10 g bacto-tryptone, 10 g NaCI per liter) which are grown with vigorous shaking for 16 hours at 28°C.
  • BASTA eng/stm-GR; kanamycin
  • trans-dominant alterations phenocopy weak stm alleles and have been reproduced in various independent large-scale experiments ( ⁇ 1000 transgenic T1 progeny). Generally stm phenocopies are found in 75 % to 90 % of the primary transformants. Alterations may be subtle early in seedling development but are manifested with increasing numbers of leave primordia until the primary SAM ultimately looses its activity in the vast majority of transgenic eng-STM plants.
  • both chimeric repressors eng-STM and eng-AP3 are obviously recruited by either DNA sequence specificity, protein-protein interactions or both to their natural target genes which are repressed resulting in the phenocopy of the respective loss of function mutant phenotype.
  • embryogenic callus of type II
  • Such callus is obtained from immature embryos having the genotype HI II or A188 x B73 according to the method and the media described in Dennehey et al, 1994, and may be multiplied and maintained by successive prickings every two-weeks on the initiation medium.
  • Plants are regenerated from these callus by modifying the hormonal and osmotic equilibrium of the cells according to the method described by Vain et al, 1989.
  • a method of genetic transformation leading to a stable integration of the modified genes in the genome of the plant involves the use of a gene gun.
  • the target cells are fragments of callus having a surface area of 10 to 20 mm 2 .
  • the fragments are laid in the center of a Petri dish containing a culture medium identical to the initiation medium, further containing 0.2 M of mannitol + 0.2 M of sorbitol (16 fragments per dish). Tissues are then bombarded as previously described.
  • the dishes are then sealed using Scellofrais® and cultivated in dark at 27°C.
  • the first planting is effected 24 hours afterwards, then every two-weeks during three months in a medium identical to the initiation medium but containing a selective agent.
  • the selective agents may be for example active ingredients of herbicidal agents (Basta®, Round up®) or antibiotics (hygromycin, kanamycin).
  • callus that have not been inhibited by the selected agents develop. These are usually composed of cells resulting from the division of a cell having integrated in its genome one or more copies of gene of interest. The frequency of obtaining such callus is about 0.8 callus per bombarded dish. Callus are identified, individualized, amplified and cultivated so as to regenerate plants. In order to avoid any interference with non transformed cells, all these steps are effected in culture media that contain the selective agent. The regenerated plants are acclimated and cultivated in the greenhouse were they can be crossed or self-pollinated.
  • the transformation of plants may be carried out with Agrobacterium tumefaciens and immature embryos, as described by Ishida et al., (1996).
  • Example 5 Expression of the endogenous STM and AP3 genes
  • transgenic eng-STM or eng-AP3 phenocopy plants were subjected to RNA gel blot and RT-PCR analysis.
  • the chimeric transcripts derived from the CaMV 35S promoter were easily detectable in total or poly(A) + RNA from pooled seedlings or flowers (12 individual progeny) probed with the STM or AP3 coding regions.
  • the high abundance of chimeric transcripts interfered with the detection of the shorter native mRNA in these gel blot experiments.
  • Discrimination was achieved by probing with the natural 3'UTR sequences that are lacking in the transgenes. The result obtained for the STM 3'UTR confirms transcription of the native STM gene although at a significantly reduced level.
  • Example 6 Phenocopies rely on the incorporation of the chimeric eng-STM protein into the nuclear compartment Although the changes in size or identity of the expressing organs might explain the lower transcript levels observed in stm and ap3 phenocopy plants we aimed to elaborate further evidence against homology based gene silencing.
  • the chimeric eng-STM polypeptide therefore was expressed in C- terminal fusion with the hormone-binding domain of the glucocorticoid receptor in transgenic Arabidopsis plants. Due to this addition the resulting eng-STM-GR polypeptide should be cytoplasmatic and incorporated into the nuclear compartment only after hormone application. A linkage between the dexamethasone treatment and alterations in SAM activity thus should depend on the nuclear import of the chimeric eng-STM-GR protein and be incompatible with cosuppression.
  • To test for dexamethasone inducibility we focused on one T 2 family with phenotypically normal kanamycine resistant seedlings. After confirming their normal development to the 4-5 leaf stage 10 transgenic seedlings were sprayed with dexamethasone and photographed daily to document the developmental progress. As controls either wild-type plants treated with dexamethasone or untreated transgenic progeny were analysed over the same time interval. In the control plants, 4-6 leaf primordia newly appear within the depicted 6-day interval in these.
  • Example 7 Expression of the C-terminal deletion polypeptide eng-STM ( ⁇ HD)
  • transgenic plants were raised expressing the C-terminal deletion polypeptide eng-STM ( ⁇ HD).
  • This deletion protein still spans 296 aa of STM protein sequence including the conserved KNOX (B ⁇ rglin, 1997) and ELK domains (Kerstetter et al., 1994) but lacks the DNA-binding homeodomain.
  • Transcription of the eng-STM ( ⁇ HD) transgene was confirmed in BASTA resistant progeny by RT-PCR resulting in the predicted 192 bp amplicon (compared to 374 bp for eng-STM).
  • the obtained primary transformants exhibited stm phenocopies in frequency and strength indistinguishable from those observed with the full-length protein.
  • the trans-dominant negative function provided by the chimeric eng-STM protein is therefore independent of the DNA-binding activity contributed by the STM homeodomain.

Abstract

La présente invention concerne un procédé d'inhibition de l'expression de gènes cibles chez les plantes. Un produit de recombinaison d'ADN chimère comprend au moins une séquence de répresseur en fusion transcriptionnelle avec au moins une séquence spécifique aux plantes qui code une protéine, ou un fragment de celle-ci, se liant à l'ADN, ou qui active la transcription soit en se liant à l'ADN lui-même, soit en interagissant avec une protéine liée à l'ADN.
PCT/EP2000/001524 1999-02-09 2000-02-09 Procede d'inhibition de l'expression de genes cibles chez les plantes WO2000047754A1 (fr)

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AU36563/00A AU770893B2 (en) 1999-02-09 2000-02-09 A method for inhibiting the expression of target genes in plants
CA002359749A CA2359749A1 (fr) 1999-02-09 2000-02-09 Procede d'inhibition de l'expression de genes cibles chez les plantes
EP00915151A EP1151119A1 (fr) 1999-02-09 2000-02-09 Procede d'inhibition de l'expression de genes cibles chez les plantes

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WO2004018687A2 (fr) 2002-08-07 2004-03-04 Basf Plant Science Gmbh Sequences d'acide nucleique codant des proteines associees a une reaction de stress abiotique
WO2004092398A2 (fr) 2003-04-15 2004-10-28 Basf Plant Science Gmbh Sequence d'acides nucleiques codant pour des proteines associees a la reponse de stress abiotique et cellules vegetales et plantes a tolerance accrue au stress environnemental
JP2005295879A (ja) * 2004-04-09 2005-10-27 Japan Science & Technology Agency 花の形態が改変された植物体の生産方法およびこれを用いて得られる植物体、並びにその利用
JP2005295878A (ja) * 2004-04-09 2005-10-27 Japan Science & Technology Agency 花芽形成遅延植物体の生産方法、及びこれを用いて得られる植物体、並びにその利用
JP2006006248A (ja) * 2004-06-28 2006-01-12 Japan Science & Technology Agency 葉の形態形成が制御された植物体の生産方法およびこれを用いて得られる植物体、並びにその利用
JP2006020607A (ja) * 2004-07-09 2006-01-26 Japan Science & Technology Agency 葉の形態が改変された植物体の生産方法およびこれを用いて得られる植物体、並びにその利用
JP2006042730A (ja) * 2004-08-06 2006-02-16 Japan Science & Technology Agency 単子葉植物の雄性不稔体の生産方法およびこれを用いて得られる植物体、並びにその利用
JP2006101827A (ja) * 2004-10-08 2006-04-20 Japan Science & Technology Agency 雄性不稔形質転換植物体の生産方法およびこれを用いて得られる植物体、並びにその利用
JP2006280242A (ja) * 2005-03-31 2006-10-19 Japan Science & Technology Agency 完全不稔性植物体の生産方法およびこれを用いて得られる植物体、並びにその利用
US7342148B2 (en) 2001-12-26 2008-03-11 National Institute Of Advanced Industrial Science And Technology Gene and peptide for transcriptional repressor
WO2008142034A2 (fr) 2007-05-22 2008-11-27 Basf Plant Science Gmbh Plantes présentant une tolérance et/ou une résistance accrues au stress environnemental et une production de biomasse accrue
EP2053057A2 (fr) 2004-09-24 2009-04-29 BASF Plant Science GmbH Séquences d'acide nucléique codant pour des protéines associées à la réponse au stress abiotique et cellules végétales et plantes dotées d'une tolérance accrue au stress environnemental
DE112008002435T5 (de) 2007-09-18 2010-07-22 Basf Plant Science Gmbh Pflanzen mit erhöhtem Ertrag
DE112008002456T5 (de) 2007-09-21 2011-01-13 Basf Plant Science Gmbh Pflanzen mit erhöhtem Ertrag
WO2011009801A1 (fr) 2009-07-23 2011-01-27 Basf Plant Science Company Gmbh Plantes à rendement amélioré
DE112008003318T5 (de) 2007-12-19 2011-04-21 Basf Plant Science Gmbh Pflanzen mit erhöhtem Ertrag und erhöhter Toleranz gegenüber Umweltstress (IY-B)
DE112009000313T5 (de) 2008-02-27 2011-04-28 Basf Plant Science Gmbh Pflanzen mit erhöhtem Ertrag
WO2011061656A1 (fr) 2009-11-17 2011-05-26 Basf Plant Science Company Gmbh Plantes à rendement accru
EP2333078A2 (fr) 2006-03-24 2011-06-15 BASF Plant Science GmbH Protéines associées à une réponse au stress abiotique et homologues
DE112009002267T5 (de) 2008-09-23 2012-01-19 Basf Plant Science Gmbh Pflanzen mit erhöhtem Ertrag (LT)
DE112009001994T5 (de) 2008-08-19 2012-01-19 Basf Plant Science Gmbh Pflanzen mit erhöhtem Ertrag durch Erhöhen oder Erzeugen einer oder mehrerer Aktivitäten in einer Pflanze oder einem Teil davon
EP2455472A2 (fr) 2006-10-13 2012-05-23 BASF Plant Science GmbH Plantes ayant un rendement amélioré
DE112009002342T5 (de) 2008-10-23 2012-06-14 Basf Plant Science Gmbh Verfahren zum Herstellen einer transgenen Zelle mit erhöhtem Gehalt an gamma-Aminobuttersäure (GABA)
DE112009002577T5 (de) 2008-10-23 2012-06-21 Basf Plant Science Gmbh Pflanzen mit erhöhtem Ertrag (NUE)
WO2014134567A1 (fr) * 2013-03-01 2014-09-04 The Regents Of The University Of California Procédés et compositions pour cibler des arn-polymérases et biogenèse de l'arn non codant pour des loci spécifiques
WO2016128470A1 (fr) 2015-02-11 2016-08-18 Basf Se Hydroxyphénylpyruvate dioxygénases résistantes aux herbicides

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EP2278018A2 (fr) 2002-08-07 2011-01-26 BASF Plant Science GmbH Gènes codant des protéines associées au stress abiotique
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EP2674496A2 (fr) 2003-04-15 2013-12-18 BASF Plant Science GmbH Protéines de codage de séquences dýacide nucléique associées à une réponse au stress abiotique et cellules végétales avec une augmentation de la tolérance au stress environnemental
JP2005295879A (ja) * 2004-04-09 2005-10-27 Japan Science & Technology Agency 花の形態が改変された植物体の生産方法およびこれを用いて得られる植物体、並びにその利用
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JP2006006248A (ja) * 2004-06-28 2006-01-12 Japan Science & Technology Agency 葉の形態形成が制御された植物体の生産方法およびこれを用いて得られる植物体、並びにその利用
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