WO2002014521A2 - Plantes a proprietes de croissance modifiees - Google Patents

Plantes a proprietes de croissance modifiees Download PDF

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Publication number
WO2002014521A2
WO2002014521A2 PCT/DE2001/003216 DE0103216W WO0214521A2 WO 2002014521 A2 WO2002014521 A2 WO 2002014521A2 DE 0103216 W DE0103216 W DE 0103216W WO 0214521 A2 WO0214521 A2 WO 0214521A2
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nucleic acid
acid sequence
promoter
seq
plant
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PCT/DE2001/003216
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German (de)
English (en)
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WO2002014521A3 (fr
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Jeff Schell
Bernd Reiss
Elke Kemper
Ellen Mahlow
Kerstin KÖPCHEN
Ernesto Olmos
Sabine Steiner-Lange
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Priority to DE10193390T priority Critical patent/DE10193390D2/de
Priority to AU2001289571A priority patent/AU2001289571A1/en
Publication of WO2002014521A2 publication Critical patent/WO2002014521A2/fr
Publication of WO2002014521A3 publication Critical patent/WO2002014521A3/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)
    • C12N15/8241—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
    • 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
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • 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
    • C12N15/8291—Hormone-influenced development
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to transgenic plants with a regulatory nucleic acid sequence according to SEQ ID NO: 1 which is stably integrated into the genome, or its fragment or derivative or homolog with the biological function of a promoter, and a nucleic acid sequence which is functionally linked to a nucleic acid coding for a gene product, in particular the nucleic acid sequence according to SEQ ID NO: 2, or its fragment or derivative or homologue with the biological activity of the growth regulation.
  • a change in the growth properties of plants can thus be used, for example, to increase yield or to improve the resistance to mechanical environmental influences, such as wind or rain, an approach which has already been followed, for example, in WO 9729123 - by manipulating the expression of GAI genes. Also aesthetic new creations, for example the development of dwarf forms of different ornamental or useful plants, are possible with it.
  • the present invention is therefore based on the object of providing plants with modified growth properties and a process for their production.
  • Figure 1 shows the genomic nucleic acid sequence of the promoter region of Sul 4.1.
  • the core area of the promoter is printed in bold, a TATA box is underlined twice. This region was predicted using a cutoff value of 0.8 using the "Promoter Prediction by Neural Network", available at http://whitefly.lbl.gov/seq_tools/promoter.html. The percentage of the cutoff value is wrong positively recognized eukaryotic promoters below 0.4%.
  • the transcription start point is underlined and indicates the start of full-length EST clones.
  • Figure 2 shows the nucleic acid sequence of an EST clone (cDNA) from Sul 4.1. The numbering continues the numbering from FIG. 1, the last 6 nucleotides from FIG. 1 overlapping with the first 6 nucleotides from FIG. 2. The start ATG and stop codon of the longest open reading frame of this EST clone are shown in bold.
  • FIG. 3 shows the amino acid sequence derived from the open reading frame according to FIG. 2 from Sul 4.1. The numbering begins with the start ATG.
  • FIG. 4a shows the developmental differences between Arabidopsis thaliana Sul 4.1 mutants and wild type plants at the time the wild type bloomed.
  • Figure 4b shows the final size of Arabidopsis thaliana Sul 4.1 mutants compared to wild type plants.
  • Figure 5a shows Sul 4.1 retransformants with slow development / dwarfism compared to wild-type plants and Sul 4.1 mutants.
  • FIG. 5b shows Sul 4.1 retransformants with accelerated development / giant growth in comparison to wild-type plants and Sul 4.1 mutants.
  • FIG. 6 schematically shows the restriction map of the vector pAC102 used.
  • FIG. 7 schematically shows the restriction map of the vector pSEXOOl-VS used.
  • FIG. 8 shows the result of a Northern analysis of Sul 4.1 overexpressing plants in comparison with wild type plants (C24).
  • homologous sequence or “homolog” used here denotes a nucleic acid or amino acid sequence with significant similarity to the comparison sequence or parts thereof.
  • the homologous sequences are therefore nucleic acid sequences which are compatible with the comparison sequences or parts of these sequences under conditions which are not very stringent, particularly preferably under hybridize stringent conditions (for stringent and less stringent conditions see Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory (1989), ISBN 0-87969-309-6)
  • An example of stringent hybridization conditions is: Hybridization in 4 x SSC at 65 ° C (alternatively in 50% formamide and 4 X SSC at 42 ° C), followed by several washing steps in 0.1 x SSC at 65 ° C for a total of about one hour.
  • An example of less stringent hybridization conditions is hybridization in 4 x SSC at 37 ° C., followed by several washing steps in 1 x SSC at room temperature ollen furthermore nucleic acid or Amino
  • derivatives denotes nucleic acid sequences which have modifications in the form of one or more deletions, substitutions, additions, insertions and / or inversions. Genomic equivalents of EST or cDNA sequences containing introns are also to be regarded as derivatives. Derivative also means that a nucleic acid sequence is composed of one or more nucleic acid fragments of a gene and one or more nucleic acid fragments of one or more other genes. The individual domains can be both unmodified and modified.
  • fragment denotes parts of the original nucleic acid sequence with a length of at least 100 nucleotides.
  • the term "functionally linked" used here means that a regulatory sequence such as a promoter controls the expression of a gene or that a nucleic acid sequence is expressed starting from the promoter.
  • vector denotes naturally occurring or artificially created constructs for the uptake, multiplication, expression or transfer of nucleic acids, e.g. Plasmids, phagemids, cosmids, artificial chromosomes, bacteriophages, viruses, retroviruses.
  • expression system means any combination of vectors, restriction enzymes, transformation methods, cell extracts, living cells e.g. prokaryotic or eukaryotic cells or organisms for the purpose of endogenous or exogenous expression of genes.
  • transgenic plant used here refers to plants which were produced by means of recombinant genetic engineering and / or microbiological processes and not by means of conventional breeding processes.
  • the present invention relates to the regulatory nucleic acid sequence (hereinafter also referred to as promoter) which naturally controls the transcription of the Sul 4.1 gene in Arabidopsis thaliana.
  • This nucleic acid sequence according to the invention is listed in SEQ ID NO: 1.
  • the invention further relates to fragments or homologs or derivatives of the nucleic acid sequence according to SEQ ID NO: 1, which have the biological function of a promoter.
  • the invention further relates to nucleic acid sequences which hybridize with the nucleic acid sequence according to SEQ ID NO: 1 and which have the biological function of a promoter.
  • nucleic acid sequences which hybridize under stringent conditions with the nucleic acid sequence according to SEQ ID NO: 1 and which have the biological function of a promoter.
  • Preferred homologues of SEQ ID NO: 1 are regulatory nucleic acid sequences of the following plants: soy, rice, cotton, sugar beet, sunflower, flax, hemp, potato, tobacco, tomato, summer rape, winter rape, alfalfa (lucerne), lettuce, pea, bean, Carrot, onion, the various tree, nut and wine species, also cereals such as barley, wheat, rye, oats, maize, feed grasses, and other types of fruit such as mango, apple, peach, gooseberry, currant, banana, melon, pumpkin and various citrus fruits, for example Lemon, orange, grapefruit or tangerine. Ornamental plants such as cyclamen are also preferred,
  • nucleic acid sequence according to the invention according to SEQ ID NO: 1 or its fragment or homolog or derivative is suitable for example for the identification and isolation of genes homologous to the Sul 4.1 gene in other organisms or of related regulatory sequences in e.g. Arabidopsis thaliana using special hybridization or screening procedures, e.g. as a probe for screening in DNA libraries using hybridization to single-stranded nucleic acids.
  • the nucleic acid sequence according to the invention according to SEQ ID NO: 1 or its fragment or homolog or derivative is furthermore suitable for the specific control of the expression of genes in organisms or cells, preferably for the specific control of the expression of genes which influence growth or interfere with hormone metabolism.
  • the invention thus relates to transgenic plants which have been newly produced by a regulatory nucleic acid sequence according to SEQ ID NO: 1 which is stably integrated into the genome, or its fragment or derivative or homologue with the biological function of a promoter, and a functionally linked to this nucleic acid sequence for a Gene product coding nucleic acid sequence.
  • the present invention relates to a method for producing a plant with modified gene expression, comprising the stable integration of a regulatory nucleic acid sequence according to SEQ ID NO: 1, or its fragment or homolog or derivative with the biological function of a promoter, and a functionally linked to this nucleic acid sequence for a nucleic acid sequence coding for a gene product into the genome of plant cells or plant tissues and regeneration of the plant cells or plant tissues obtained to plants, preferably to fertile plants.
  • the nucleic acids functionally linked to the nucleic acid sequence according to the invention according to SEQ ID NO: 1 or its fragment or homolog or derivative can be endogenous, exogenous genomic DNA segments or cDNAs or their fragments or derivatives.
  • Endogenous means that the nucleic acid sequence originates from the same organism in which it is integrated using the method according to the invention, e.g. a nucleic acid sequence from Arabidopsis thaliana is integrated into Arabidopsis thaliana using the method according to the invention.
  • Exogenous means that the nucleic acid sequence comes from another organism, e.g. a nucleic acid sequence from Arabidopsis thaliana is obtained with the method according to the invention in e.g. Wheat integrated.
  • the nucleic acid sequences can have deletions, substitutions, additions, insertions and / or inversions compared to the naturally occurring nucleic acid sequences.
  • the nucleic acid sequence according to SEQ ID NO: 1 or its fragment or homolog or derivative can be used for regulating the expression of the Sul 4.1 gene which is naturally connected to it.
  • the nucleic acid sequence according to the invention is also suitable for the regulation of the expression of other gene sequences, in particular for the regulation of other genes influencing plant growth, as a result of which plants with a different growth rate and final size can also be achieved.
  • This is primarily the genes of the phytohormone metabolism of auxins, gibberellins, cytokines, abscisic acid and ethylene, which can be regulated by the promoter. Genes for enzymes from the anabolic and catabolic phytohormone metabolism and genes for phytohormone receptors are particularly suitable for this.
  • nucleic acid sequence according to the invention as shown in SEQ ID NO: 1 or its fragment or homolog or derivative can be used for regulating the expression of any gene for any application, both from Arabidopsis thaliana and from other organisms.
  • the promoter can be present in combination with any genes, both in a vector and in transgenic organisms, in particular plants.
  • Sul 4.1 gene Plants with a mutation in the Sul 4.1 gene have a reduced size growth compared to the wild type plants.
  • Sul 4.1 cDNA or Sul 4.1 EST The nucleic acid sequence of the Sul 4.1 gene (hereinafter also referred to as Sul 4.1 cDNA or Sul 4.1 EST) is listed as SEQ ID NO: 2.
  • Sul 4.1 cDNA or Sul 4.1 EST The nucleic acid sequence of the Sul 4.1 gene (hereinafter also referred to as Sul 4.1 cDNA or Sul 4.1 EST) is listed as SEQ ID NO: 2.
  • An EST clone (Genbank Accession number H76408) is available for the Arabidopsis thaliana Sul 4.1 gene, but no function has been known to date.
  • a BAC clone with the corresponding gene region is registered under the gene bank accession number ALI 62508. There are no similarities between Sul 4.1 and other growth-regulating genes. A gene with distant similarity to Sul 4.1, but also unknown function, is located on chromosome 3 of Arabidopsis thaliana (Genbank Accession number AL 096859).
  • the present invention thus also relates to transgenic plants which have been newly produced by a nucleic acid sequence according to SEQ ID NO: 2 which is stably integrated into the genome, or its fragment or derivative with the biological activity of growth regulation, and a regulatory nucleic acid sequence which is functionally linked to this nucleic acid sequence.
  • the present invention relates to transgenic plants which have been newly produced by a nucleic acid sequence stably integrated into the genome, which hybridizes with the nucleic acid sequence according to SEQ ID NO: 2 and has the biological activity of growth regulation, and a regulatory functionally linked to this nucleic acid sequence Nucleic acid sequence.
  • transgenic plants with a nucleic acid sequence stably integrated into the genome, which hybridizes with the nucleic acid sequence according to SEQ ID NO: 2 under stringent conditions and has the biological activity of growth regulation, and a regulatory nucleic acid sequence which is functionally linked to this nucleic acid sequence.
  • the nucleic acid sequence according to SEQ ID NO: 2 or its fragment or derivative or homolog can be any Sul 4.1 sequence or its fragment or derivative or homolog, for example one from plants, algae or cyanobacteria.
  • the nucleic acid sequence according to SEQ ID NO: 2 or its fragment or homolog or derivative is further suitable, for example, for the identification and isolation of genes homologous to the Sul 4.1 gene in other organisms or of related sequences in, for example, Arabidopsis thaliana with the aid of special hybridization or screening Methods, for example as a probe for screening in DNA libraries with the aid of hybridization to single-stranded nucleic acids.
  • the regulatory DNA sequence functionally linked to the nucleic acid sequence according to SEQ ID NO: 2 or its fragment or derivative or homolog, for example a promoter can be the regulatory DNA sequence according to SEQ ID NO: 1 endogenously present with the nucleic acid sequence as well as any other regulatory DNA sequence.
  • the regulatory DNA sequence can be either an endogenous promoter of the organism to be transformed or an exogenous promoter which is located, for example, on the vector used.
  • any regulatory sequence that can control the expression of foreign genes in organisms, for example plants is suitable as a promoter, for example the CaMV 35S promoter from the cauliflower mosaic virus; see. Franck et al., Cell 21: 285-294 (1980).
  • the expression of the nucleic acid sequences can also be achieved by a chemically inducible promoter.
  • promoters examples include the PRPI promoter (Ward et al, Plant Molecular Biology 22, 361-366 (1993)), a promoter induced by salicylic acid (WO 95/19443), a promoter inducible by benzenesulfonamide (EP-A 388186 ), a promoter inducible by tetracycline (Gatz et al., Plant Journal 2, 397-404 (1992)), a promoter inducible by abscisic acid (EP-A 335528) or a promoter inducible by ethanol or cyclohexanone (WO 93/21334) , Depending on the desired expression site, promoters can also be used, for example in certain plant tissues or Plant parts are active.
  • phaseolin promoter US 5504200
  • isoflavone reductase promoter US 5750399
  • a seed-specific promoter for example from tobacco (US 5824863) or the ST-LSI promoter from potato (Stockhaus et al., (1989) EMBO JS, 2445-2452).
  • nucleic acid sequences according to SEQ ID NO: 1 and SEQ ID NO: 2 or their fragments or homologs or derivatives can be of natural origin or have been produced artificially.
  • the nucleic acid sequence according to SEQ ID NO: 1 is present in a sense orientation
  • the nucleic acid sequence according to SEQ ID NO: 2 can be present both in a sense and in an antisense orientation.
  • nucleic acid sequences according to SEQ ID NO: 1 or SEQ ID NO: 2 or their fragments or homologs or derivatives can be used in vectors, expression systems or plants, plant tissues or plant cells or animal cells or microorganisms to change the expression patterns of a wide variety of gene products.
  • the expression of the gene products can be both increased and reduced compared to their natural expression.
  • the present invention relates to a method for producing plants with modified growth properties, comprising the stable integration of a
  • the invention further relates to a method for
  • nucleic acid sequences which according to stringent conditions with the nucleic acid sequence
  • SEQ ID NO: 2 or its fragment or derivative with the biological activity of growth regulation, functional with a regulatory nucleic acid sequence e.g. the
  • Nucleic acid sequence according to SEQ ID NO: 1, or its fragment or derivative or homologue with the biological function of a promoter Another aspect of the invention relates to a method for producing plants with modified growth properties, comprising the stable integration of a nucleic acid sequence according to SEQ ID NO: 1 and / or SEQ ID NO: 2 or its fragment or derivative or homologue into the genome of plant cells and regeneration the plant cells or plant tissues obtained to plants, preferably to fertile plants. Due to the changed growth, increased biomass production and thus an increase in plant yield can be achieved. Experiments suggest that Sul4.1 is involved in phloem transport and thus in sink-source metabolism.
  • the method according to the invention can be used, for example, to increase the productivity of useful plants: an improved distribution of the assimilates in the phloem from their production sites (source) to the buyers (sink), such as seeds, for example from oats, rice or peas; Leaves, eg spinach or cabbage; Stems, eg sugar cane, bamboo or asparagus; Tubers, eg potatoes; Beets, such as fodder beets or sugar beets; and fruits, such as cucumbers lead to a higher yield of the plant. It is also possible to produce short-range varieties with increased resistance to mechanical environmental influences such as wind or rain, for example by introducing the appropriate genes in an antisense orientation. Aesthetically appealing growth forms, such as mini plants, can also be produced.
  • Endogenous or exogenous nucleic acid sequences according to SEQ ID NO: 1 and / or SEQ HD NO: 2 or their fragments or derivatives or homologs can be used for the method according to the invention.
  • Endogenously means that the nucleic acid sequence comes from the same organism into which it is integrated using the method according to the invention, for example a Sul 4.1 gene from Arabidopsis thaliana is integrated using the method according to the invention in Arabidopsis thaliana.
  • Exogenous means that the nucleic acid sequence comes from another organism, for example a nucleic acid sequence from Arabidopsis thaliana is integrated into, for example, wheat using the method according to the invention.
  • nucleic acid sequences After the stable integration of the transformed nucleic acid sequence, one, two or more Sul 4.1 genes or Promoters are present in the genome, which leads to an increased expression of the corresponding genes.
  • the nucleic acid sequences have deletions, substitutions, additions, hisertions and / or inversions compared to the naturally occurring nucleic acid sequences.
  • the present invention further relates to a transformed cell, in particular a transformed plant cell or a transformed plant tissue, in which the nucleic acid sequences according to the invention according to SEQ ID NO: 1 and / or SEQ ID NO: 2 or their fragments or homologs or derivatives are stably integrated. Furthermore, the present invention relates to a plant cell or plant tissue transformed with the nucleic acid sequences according to the invention according to SEQ ID NO: 1 and / or SEQ ID NO: 2 or their fragments or homologs or derivatives, or a transformed plant tissue, which or to a plant, especially a fertile plant is regenerable.
  • the present invention relates to a plant which can be obtained by the process according to the invention.
  • the present invention further relates to seed obtained from plants obtained by the process according to the invention.
  • the invention further relates to the fruits produced by the plants, e.g. Fruit, berries, potatoes and grapes.
  • a nucleic acid sequence is used which is composed of fragments of different Sul 4.1 genes.
  • This composite nucleic acid sequence can also contain modifications such as deletions, additions or substitutions.
  • the methods for producing such nucleic acid sequences are state of the art, familiar to the person skilled in the art under the term “chimeras” and can be carried out easily by him.
  • the method according to the invention can be applied to any organisms.
  • the method according to the invention can be used to increase the yield and to regulate the growth of mono- and dicotyledonous plants as well as algae and cyanobacteria.
  • Particularly preferred plants are cultivated plants, for example, soybean, rice, cotton, sugar beet, sunflower, flax, hemp, potato, tobacco, tomato, summer rape, winter rape, alfalfa (lucerne), lettuce, pea, bean, carrot, onion, the various trees , Nut and wine species, furthermore cereals eg barley, wheat, rye, oats, corn, furthermore fruit types eg mango, apple, peach, gooseberry, currant, banana, melon, pumpkin and citrus fruits such as lemon, orange, grapefruit or tangerine.
  • cultivated plants for example, soybean, rice, cotton, sugar beet, sunflower, flax, hemp, potato, tobacco, tomato, summer rape, winter rape, alfalfa (lucerne),
  • Ornamental plants such as cyclamen, aster, bougainvillea, chrysanthemum, petunia, marguerite, narcissus, snowdrop, delphinium, sunflower, geranium, geese cress, gerbera, gladiolus, brass, hyacinth, lily, magnolia, orchid, rhododendron, rose are also particularly preferred Heart, tulip, poinsettia.
  • the plants transformed with the nucleic acid sequence can be wild type plants, plants obtained by breeding or transgenic plants.
  • the method according to the invention can also be used not only in plants or plant tissues but also in plant cells, for example in a cell culture or in expression systems for changing the expression pattern of Sul 4.1 genes or derivatives or homologues of these genes, or for expression of genes which are related to the regulatory Sequence according to SEQ ID NO: 1 are functionally linked.
  • the method according to the invention is suitable for temporary growth regulation in that the Sul 4.1 -specific promoter is exchanged for an inducible promoter.
  • an inducible promoter By appropriate induction of growth e.g. to special environmental conditions, e.g. extreme dry periods or long periods of cold are reacted to.
  • the present invention further relates to vectors comprising a nucleic acid sequence according to SEQ ID NO: 1, or its fragment or derivative or homolog, and a nucleic acid sequence which is functionally linked to this sequence and which codes for a gene product.
  • the present invention further relates to vectors comprising a nucleic acid sequence according to SEQ ID NO: 2, or its fragment or derivative or homolog and a regulatory DNA sequence.
  • Suitable vectors for the uptake and transfer of the nucleic acid sequences can ensure the multiplication and / or the expression of the uptake nucleic acids in single cells such as Escherichia coli or Agrobacterium tumefaciens or in plant cells, plant tissues or plants. Corresponding vectors can of course occur or can be produced artificially.
  • the vectors can include selection markers, terminator sequences, polylinkers, promoter elements, enhancers, polyadenylation sites and other genetic elements.
  • Vectors suitable for cloning are, for example, pBluescript, plasmids of the pUC series, plasmids of the pGEM series or vectors based on the bacteriophage ⁇ .
  • a plasmid vector used for use in Agrobacterium is, for example, pBinl9 cf. Bevan et al., (1984), Nucleic Acids Research 12, 8711-8721.
  • For transformation and expression in plants represent usable vectors on the Ti plasmid of Agrobacterium species or constructs based on plant viruses and are known to the person skilled in the art.
  • the abovementioned cloning vectors or linearized DNA are also used instead of Ti plasmids.
  • a summary description of vectors that have been used frequently and by default can be found in Guerineau and Mullineaux, Plant Transformation and Expression Vectors, in: Plant Molecular Biology Labfax, published by Croy, Oxford, BIOS Scientific Publishers, 121-148 (1993).
  • Transformation methods used to date in plants are e.g. gene transfer by means of Agrobacterium tumefaciens (e.g. by bathing seeds or leaf pieces in an Agrobacterial solution), by means of plant viruses, by electroporation, by injection (microprojectile bombardment) or injection (microinjection) as well as the incubation of dry embryos in DNA-containing liquids and the transformation of protoplasts with the help of polyethylene glycol. More detailed descriptions of the methods mentioned can be found e.g. in Jens et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by Kung and Wu, Academic Press 128-143 (1993).
  • Example 1 General Clomeration Processes
  • the cloning steps carried out in the context of the present invention such as restriction cleavages, agarose gel electrophoresis, purification of nucleic acid fragments, transfer of nucleic acids to filter materials, transformation and cultivation of bacterial cells, sequencing, PCR etc. were carried out analogously to those in Sambrook et al. (1989) Molecular Cloning, Cold Spring Harbor Laboratory, ISBN 0-87969-309-6 Procedure carried out.
  • Example 2 Production of a T-DNA population of Arabidopsis thaliana Arabidopsis plants of the ecotype C24 were by vacuum infiltration according to Bechtold et al. (1993), N. Bechtold, J. Ellis, G. Pelletier, In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants, C.R. Acad. Sei., Paris, Sciences de la vie, 1993, 316, 1184-9, transformed with the Agrobacterium strain GV3101 ⁇ MP90RK :: pAC102.
  • the strain GV3101pMP90RK is in Koncz & Schell, (1986), Mol. Gen. Genet. 204, 383-396.
  • the plasmid pAC102 is shown in FIG. 6.
  • the pAC102 contained in the Agrobacteria mediates resistance to the antibiotic sulfadiazine, among other things, and also has the property of over-expressing genomic sequence segments located on the right border fragment after integration.
  • a population of approximately 500 individuals comprising transformants was obtained by selection for resistance to sulfadiazine.
  • the Sul 4.1 mutant was detected by screening the population of Arabidopsis thaliana plants transformed with Ti plasmid pAC102 after visual selection of individuals with changed growth properties.
  • the seeds of the Arabidopsis plants treated by vacuum infiltration were applied to a pearlite / sand mixture containing sulfadiazine in order to select transformants.
  • the seedlings were grown in the climatic chamber on short day (8 hours light, 16 hours dark, 22 ° C).
  • Transformants, ie in this case seedlings, which reached the four-leaf stage under selection, were then picked in plastic containers on "mini-tray" earth (Gebr.
  • the genomic sequences flanking the Sul 4.1 T-D ⁇ A were isolated by the so-called plasmid rescue technique.
  • DNA from Sul 4.1 plants was prepared using the CTAB method, cut with EcoRI, the resulting ends ligated with T4 ligase, and the ligase batch was transformed into DH10B cells according to the manufacturer's instructions (Gibco / Brl).
  • the genomic sequences obtained in this way were analyzed by DNA sequencing using the Sanger dideoxy method.
  • the oligonucleotides 1046 and pucli3 were used for this.
  • pucli3 CCGGGTACCGAGCTCGAATTC (SEQ ID NO: 5)
  • DNA sequencing was performed by the MPIZ ADIS service group with PE Biosystems Abi Prism 377 and 3700 devices and the BigDye terminator chemistry. The DNA sequences were compared using the "University of Wisconsin Genetic Computer Group” sequence analysis package or using FASTA. This homology search revealed that the genomic sequence obtained from Sul 4.1 has significant homology to EST 194D3T7 (Genbank Acc. Number H76408).
  • Example 8 Growth measurements
  • Sul4.1 plants and wild type C24 plants were cultivated in parallel rows in a planting bowl under different growing conditions in the greenhouse and after several points in time the key parameters stem length and leaf rosette size were determined by measuring with a ruler. The growth of Sul 4.1 plants has slowed significantly compared to the wild type from the time the flowers begin to bloom. At seed maturity, the size of Sul 4.1 plants is on average only half the size of wild-type plants.
  • Sul4.1 gene is expressed depending on development - only from the flowering stage of the plant.
  • Elementary molecular biological methods such as Southern and Northem blotting were performed as described in Sambrook et al., (1989) Molecular Cloning, Cold Spring Harbor Laboratory, ISBN 0-87969-309-6.
  • EST clone 194D3T7 was inserted between the CaMV 35S promoter and terminator of the plasmid pSEXOOl-VS (see FIG. 7 and SEQ ID NO: 4) and the resulting plasmid pSEX146 was transformed into Agrobacterium strain GV3101pMP90RK by electroporation.
  • This Agrobacterium strain was used for transformation by vacuum infiltration of Arabidopsis wild type C24.
  • Transformants were tested for resistance to sulfadiazine selected (primary transformants). The progeny of these primary transformants were characterized as for example 8 by growth tests in direct comparison with wild type C24 and Sul4.1.

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Abstract

L'invention concerne des plantes transgéniques comportant une séquence d'acide nucléique régulatrice intégrée dans le génome selon SEQ n° ID :1, ou un fragment, un dérivé ou un homologue de ladite séquence ayant la fonction biologique d'un promoteur, ainsi qu'une séquence d'acide nucléique codant un produit génique, lié de manière fonctionnelle à ladite séquence d'acide nucléique, notamment la séquence d'acide nucléique selon SEQ n°ID :2, ou un fragment, un dérivé ou d'un homologue ayant l'activité biologique de régulation de croissance.
PCT/DE2001/003216 2000-08-18 2001-08-17 Plantes a proprietes de croissance modifiees WO2002014521A2 (fr)

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DE10193390T DE10193390D2 (de) 2000-08-18 2001-08-17 Pflanzen mit veränderten Wachstumseigenschaften
AU2001289571A AU2001289571A1 (en) 2000-08-18 2001-08-17 Plants having modified growth properties

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DE10040379A DE10040379A1 (de) 2000-08-18 2000-08-18 Pflanzen mit veränderten Wachstumseigenschaften

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8053184B2 (en) 2005-05-27 2011-11-08 Monsanto Technology Llc Soybean event MON89788 and methods for detection thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] Accession Number AL162508, 28. März 2000 (2000-03-28) BEVAN M, ET AL.: "Arabidopsis thaliana DNA chromosome 5" XP002189243 *
MATHUR JAIDEEP ET AL: "Gene identification with sequenced T-DNA tags generated by transformation of Arabidopsis cell suspension." PLANT JOURNAL, Bd. 13, Nr. 5, M{rz 1998 (1998-03), Seiten 707-716, XP002189242 ISSN: 0960-7412 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8053184B2 (en) 2005-05-27 2011-11-08 Monsanto Technology Llc Soybean event MON89788 and methods for detection thereof
US9017947B2 (en) 2005-05-27 2015-04-28 Monsanto Technology Llc Soybean event MON89788 and methods for detection thereof
US9944945B2 (en) 2005-05-27 2018-04-17 Monsanto Technology Llc Soybean event MON89788 and methods for detection thereof
US10738320B2 (en) 2005-05-27 2020-08-11 Monsanto Technology Llc Soybean event MON89788 and methods for detection thereof
US11390881B2 (en) 2005-05-27 2022-07-19 Monsanto Technology, Llc Soybean event MON89788 and methods for detection thereof

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