EP1224292A2 - Synthase de formylglycinamidinribotide d'origine vegetale - Google Patents

Synthase de formylglycinamidinribotide d'origine vegetale

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Publication number
EP1224292A2
EP1224292A2 EP00981201A EP00981201A EP1224292A2 EP 1224292 A2 EP1224292 A2 EP 1224292A2 EP 00981201 A EP00981201 A EP 00981201A EP 00981201 A EP00981201 A EP 00981201A EP 1224292 A2 EP1224292 A2 EP 1224292A2
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EP
European Patent Office
Prior art keywords
synthase
plant
ribotide
purl
formylglycinamidine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00981201A
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German (de)
English (en)
Inventor
Jens Lerchl
Thomas Ehrhardt
Uwe Sonnewald
Ralf Boldt
Gotthard Kunze
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BASF SE
Original Assignee
BASF SE
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Application filed by BASF SE filed Critical BASF SE
Publication of EP1224292A2 publication Critical patent/EP1224292A2/fr
Withdrawn legal-status Critical Current

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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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • 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
    • C12N15/8274—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 for herbicide resistance
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/9015—Ligases (6)

Definitions

  • the present invention relates to the identification of plant formylglycinamidine ribotide synthase (EC 6.3.5.3.) As a new target for herbicidal active compounds.
  • the present invention further relates to DNA sequences SEQ-ID No. 1, SEQ ID No. 3 or SEQ-ID No. 5 coding for a polypeptide with formylglycinamidine- 0 ribotide synthase activity.
  • the invention relates to the use of a nucleic acid coding for a protein with formylglycinamidine ribotide synthase activity of plant origin for the production of a test system for identifying inhibitors of formylglycinamidine ribotide synthase with herbicidal activity. 5 Furthermore, the invention relates to the use of the nucleic acid SEQ-ID No. 1, SEQ ID No. 3 or SEQ-ID No. 5 coding for plant formylglycinamidine ribotide synthase for the production of plants with increased resistance to inhibitors of formylglycinamidine ribotide synthase.
  • the invention relates to a method for eliminating undesired plant growth, characterized in that the plants to be removed are treated with a compound which is coded specifically for formylglycinamidine ribotide synthase by a DNA sequence SEQ-ID No. 1, SEQ ID No. 3 or SEQ-ID No. 5 or a DNA sequence hybridizing with this DNA sequence, binds and inhibits their 5 function.
  • Plants are able to synthesize their cell components from carbon dioxide, water and inorganic salts.
  • Nucleotides are synthesized de novo in plants. They are of particular importance as part of the nucleic acids. In a covalent bond, nucleotides activate carbohydrates for the biosynthesis of polysaccharides. They also activate head groups for the biosynthesis of lipids. Nucleotides are involved in almost all metabolic pathways. Nucleoside triphosphates, especially ATP, drive most of the cell's energy-intensive reactions. Adenine nucleotides can also be found as a component in essential factors such as coenzyme A, as well as nicotinamide and flavin coenzymes,
  • nucleotides are the starting metabolites for the biosynthesis of methylxanthines such as Cof-5 fein and theobromine in plant families of the Rubiaceae and Theaceae. Since plants rely on an effective nucleotide metabolism, it can be assumed that enzymes which are involved in nucleotide biosynthesis are suitable as target proteins for herbicides. Active substances that inhibit the 5 plant de novo purine biosynthesis have already been described.
  • ASA adenylosuccinate synthetase
  • Bacterial PRPP amidotransferase can be caused by glutamine antagonists, e.g. Acivicin (L- (alpha S, 5S) -alpha-amino-3-chloro-
  • glutamine antagonists e.g. Acivicin (L- (alpha S, 5S) -alpha-amino-3-chloro-
  • Glutamine antagonists are not specific for PRPP amidotransferase but also inhibit mammalian formylglycinamidine ribotide synthase (EC 6.3.5.3.) And have an antiproliferative effect on tumor tissue (Elliot and Weber, Biochemical Pharmacology 34 (1985),
  • formylglycinamidine ribotide synthase converts the carboxa id oxygen of the formylglycinamide ribonucleotide (FGAR) into an imino group.
  • FGAR formylglycinamidine ribotide
  • CDNAs coding for formylglycinamidine ribotide synthase could be isolated and characterized from various bacterial and animal organisms.
  • the coding genes have already been isolated for most enzymes of the purine biosynthetic pathway in plants.
  • formylglycine amide ribotide synthase only two partial EST (expressed sequence tag) sequences from Arabidopsis thaliana (Gen.
  • formylglycinamidine ribotide synthase is a suitable herbicidal target in plants, the isolation of a complete plant cDNA coding for the enzyme formylglycinamidine ribotide synthase and its functional expression in bacterial or eukaryotic cells, as well as the production of an efficient and simple formylglycinamidine ribotide synthase test system for carrying out inhibitor-enzyme binding studies.
  • One object of the present invention accordingly relates to the isolation of cDNAs coding for Fonnylglycinamidinribotid synthase from Arabidopsis thaliana, Nicotiana tabacum and Chilopsis linearis.
  • Another object of the invention relates to methods for identifying inhibitors of formylglycinamidine ribotide synthase in plants by high-throughput methods.
  • the invention therefore relates to the functional expression of plant formylglycine amide ribotide synthase, in particular of formylglycine amide ribotide synthase from Arabidopsis thaliana and tobacco in suitable expression systems, and to the use of the enzymes thus produced in an in vitro test system for measuring the formyl glycine amide ribotide synthase activity ,
  • the object was achieved by isolating genes which code for the plant enzyme formylglycinamidine ribotide synthase, the production of antisense constructs of formylglycinamidine ribotide synthase, and the functional expression of formylglycinai ⁇ idinribotide synthase in bacterial or eukaryotic cells.
  • One object of the present invention relates to the isolation of full-length cDNAs coding for functional formylglycine amidribotide synthase (EC6.3.5.3.) From Arabidopsis thaliana and Nicotiana tabacum and a partial cDNA sequence from Chilopsis linearis.
  • a first object of the present invention is a DNA sequence SEQ-ID NO. 1 or SEQ ID NO. 3 containing the coding region of a plant formylglycinamidine ribotide synthase from Nicotiana tabacum and Arabidopsis thaliana, see Examples 1 and 2.
  • Another object of the invention are DNA sequences which of
  • SEQ ID NO. 1 or SEQ ID NO. 3 are derived or hybridize to one of these sequences and which code for a protein which has the biological activity of a formylglycinamidine ribotide
  • the cDNA sequence of the formylglycinamidine ribotid synthase or suitable fragments of the cDNA sequence of the formylglycinamidine ribotid synthase from Arabidopsis thaliana is cloned into an expression vector (pQE, Qiagen) and overexpressed in E. coli.
  • the expression cassette containing a partial DNA sequence of SEQ-ID No. 1 or SEQ ID NO. 3 can be expressed, for example, in other bacteria, in yeasts, fungi, algae, plant cells, insect cells or mammalian cells.
  • the formylglycaminididine ribotide synthase protein expressed with the aid of an expression cassette is particularly suitable for the detection of inhibitors specific for formylglycinamidine ribotide synthase. 5
  • the vegetable formylglycina idinribotid synthase can be used, for example, in an enzyme test in which the ac Activity of formylglycinamidine ribotide synthase is determined in the presence and absence of the active ingredient to be tested. By comparing the two activity determinations, a qualitative and quantitative statement can be made about the inhibitory behavior of the active substance to be tested, see Example 8.
  • test system With the help of the test system according to the invention, a large number of chemical compounds can be checked quickly and easily for herbicidal properties.
  • the method makes it possible to selectively select those with a high potency from a large number of substances in order to subsequently carry out further in-depth tests known to the person skilled in the art with these substances.
  • Another object of the invention is a method for identifying substances with herbicidal activity which inhibit the formylglycine amide ribotide synthase activity in plants, consisting of
  • transgenic plants, plant tissues, or plant cells which contain an additional DNA sequence coding for an enzyme with formylglycinamidine ribotide synthase activity and are capable of overexpressing an enzymatically active formylglycinamidine ribotide synthase;
  • Another object of the invention is a method for identifying inhibitors of plant formylglycinamidine ribotide synthase, with potentially herbicidal activity by cloning the gene of a plant formylglycinamidine ribotide synthase, for overexpression in a suitable expression cassette - for example in insect cells - which opens and opens the cells uses the cell extract directly or after enrichment or isolation of the enzyme formylglycinamidine ribotide synthase in a test system for measuring the enzyme activity in the presence of low molecular weight chemical compounds.
  • the invention further relates to compounds with a herbicidal action which can be identified using the test system described above.
  • Another object of the invention is a method for eliminating undesirable plant growth, characterized in that the plants to be removed are treated with a compound which is coded specifically for formylglycine amide ribotide synthesis by a DNA sequence SEQ-ID No. 1, SEQ ID No. 3 or SEQ-ID No. 5 or a DNA sequence hybridizing with this DNA sequence, binds and inhibits their function.
  • Effects can be used as defoliants, desiccants, haulm killers and in particular as weed killers. Weeds in the broadest sense are understood to mean all plants that grow up in places where they are undesirable. Whether the active ingredients found with the aid of the test system according to the invention act as total or selective herbicides depends, inter alia, on the amount used.
  • Inhibitors of formylglycinamidine ribotide synthase with herbicidal activity can be used, for example, against the following weeds:
  • Echinochloa Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristylis, Sagittaria, Eleocharis, Scirpus, Paspalum, Ischaemum, Sphenoclea, Dactyloctenium, Agrostis, Alopecurus, Apera.
  • the invention also relates to expression cassettes whose encoding sequence for a Formylglycinamidinribotid synthase from Arabidopsis thaliana, Nicotiana tabacum or chilopsis or their functional equivalent.
  • the nucleic acid sequence can be, for example, a DNA or a cDNA sequence.
  • the expression cassettes of the invention also include regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell.
  • an expression cassette according to the invention comprises upstream, ie at the 5 'end of the coding sequence, 5 a promoter and downstream, ie at the 3' end, a polyadenylation signal and optionally further regulatory elements which are associated with the coding sequence for the Formylglycinamidinribotid synthase gene are operatively linked.
  • An operative link is understood to mean the sequential arrangement of promoter, coding sequence, terminator and, if appropriate, further regulatory elements in such a way that each of the regulatory elements can fulfill its function as intended when expressing the coding sequence.
  • An expression cassette according to the invention is produced by fusing a suitable promoter with a suitable formylglycinamidine ribotide synthase DNA sequence and a polyadenylation signal according to common recombination and cloning techniques, as described, for example, in T. Maniatis, E.F. Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989) as in T.J. Silhavy, M.L. Ber an and L.W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and in Ausubel, F.M. et al. , Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Inter-science (1987). 5
  • the invention also relates to functionally equivalent DNA sequences which code for a formylglycine amide ribotide synthase gene and which, based on the total length of the DNA sequence, have sequence homology with the DNA sequence SEQ-ID NO. 1, SEQ ID NO. 3 or SEQ-ID No. 5 have from 40 to 100%. 0
  • a preferred object of the invention are functionally equivalent DNA sequences which code for a formylglycinamidine ribotide synthase gene and which, based on the total length of the DNA sequence, have sequence homology with the DNA sequence SEQ-ID NO. 1, SEQ ID NO. 3 5 or SEQ-ID No. 5 have from 60 to 100%.
  • a particularly preferred object of the invention are functionally equivalent DNA sequences which code for a formylglycinamidine ribotide synthase gene and which, based on the total length of the DNA sequence, have sequence homology with the DNA sequence SEQ-ID NO. 5 1, SEQ-ID No. 3 or SEQ-ID No. 5 have from 80 to 100%.
  • Functions equivalent sequences which code for a formylglycine amidribotide synthase gene are, according to the invention, those sequences which, despite a different nucleotide sequence, still have the desired functions.
  • Functional equivalents thus include naturally occurring variants of the sequences described here as well as artificial, e.g. nucleotide sequences obtained by chemical synthesis and adapted to the codon use of a plant.
  • a functional equivalent is also understood to mean, in particular, natural or artificial mutations of an originally isolated sequence coding for a formylglycine amide ribotide synthase, which furthermore shows the desired function. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues.
  • the present invention also encompasses those nucleotide sequences which are obtained by modifying this nucleotide sequence.
  • the aim of such a modification can e.g. further narrowing down the coding sequence contained therein or e.g. also the insertion of further restriction enzyme interfaces.
  • Functional equivalents are also those variants whose function is weakened or enhanced compared to the original gene or gene fragment.
  • the expression cassette according to the invention can also be used for the transformation of bacteria, cyanobacteria, yeast, filamentous fungi and algae and eukaryotic cells (e.g. insect cells) with the aim of producing sufficient quantities of the enzyme for ylglycinamidine ribotide synthase. 5
  • Another object of the invention is a protein from Arabidopsis thaliana, Nicotiana tabacum or Chilopsis linearis characterized by the amino acid sequence SEQ-ID NO. 2, SEQ ID No. 4 or SEQ-ID No. 6 or derivatives or parts of this protein with formyl glycinamidine ribotide synthase activity.
  • the invention also relates to vegetable proteins with formylglycinamidine ribotid synthase activity with an amino acid sequence homology to formylglycinamidine ribotide synthase from Arabidopsis thaliana, Nicotiana tabacum or Chilopsis linearis with 5 SEQ ID NO. 2, SEQ ID NO. 4 or SEQ-ID No. 6 of 20 - 100% identity.
  • Vegetable proteins with formylglycinamidine ribotide synthase activity with an amino acid sequence homology to the formylglycinamidine ribotide synthase from Arabidopsis thaliana, Nicotiana tabacum or Chilopsis linearis with the sequences 5 SEQ-ID NO are preferred. 2, SEQ ID NO. 4 or SEQ-ID No. 6 out of 50 - 100% identity.
  • Vegetable proteins with formylglycaminamide ribotide synthase activity with an amino acid sequence homology to formylglycinamidine ribotide synthase from Arabidopsis thaliana, Nicotiana tabacum or Chilopsis linearis are particularly preferred with the
  • Another object of the invention was the overexpression of the formyl-5-glycinamidine ribotide synthase gene in plants for the production of plants which are tolerant of inhibitors of the formylglycine amide ribotide synthase.
  • transgenic plants produced in this way are also the subject of the invention. 5
  • the effectiveness of the expression of the transgenically expressed formylglycine amide ribotide synthase gene can be determined, for example, in vitro by proliferation or by a germination test.
  • the invention also relates to transgenic plants transformed with an expression cassette containing the DNA sequence 5 SEQ-ID No. 1 or SEQ-ID No. 3, which by additional expression of the DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 have become tolerant of inhibitors of formylglycinamidine ribotide synthase, as well as transgenic cells, tissues, parts and propagation material of such plants.
  • Transgenic crop plants such as e.g. Barley, wheat, rye, corn, soy, 0 rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa, lettuce and the various tree, nut and wine species, as well as legumes.
  • Sequences are particularly preferred which target in the 5 apoplasts, in plastids, the vacuole, the mitochondrium, the endoplasmic reticulum (ER) or, owing to the lack of corresponding operative sequences, remain in the compartment of the ent Stand, the cytosol, ensure (Kermode, Crit. Rev. Plant Sei. 15, 4 (1996), 285-423).
  • the plant expression cassette can be installed in the plant transformation vector pBinAR, see Example 5.
  • any promoter which can control the expression of foreign genes in plants is suitable as promoters of the expression cassette according to the invention.
  • a plant promoter or a plant virus-derived promoter is preferably used.
  • the CaMV 35S promoter from the cauliflower mosaic virus (Franck et al., Cell 21 (1980), 285-294) is particularly preferred.
  • This promoter contains different recognition sequences for transcriptional effectors, which in their entirety lead to permanent and constitutive expression of the introduced gene (Benfey et al., EMBO J., 8 (1989), 2195-2202).
  • the expression cassette according to the invention can also contain a chemically inducible promoter, by means of which the expression of the exogenous formylglycinamidine ribotide synthase gene in the plant can be controlled at a specific point in time.
  • a chemically inducible promoter by means of which the expression of the exogenous formylglycinamidine ribotide synthase gene in the plant can be controlled at a specific point in time.
  • promoters as e.g. the PRPl promoter (Ward et al., Plant. Mol. Biol. (1993) 22, 361-366), a salicylic acid-inducible promoter (WO 95/19443), a benzenesufonamide-inducible (EP 388186), a by Tetracycline-inducible (Gatz et al., Plant J. (1992) 2, 397-404), a scisic acid-inducible (EP0335528) or ethanol- or cycl
  • the expression cassette according to the invention can therefore contain, for example, a seed-specific promoter (preferably the phaseolin promoter, the USP or LEB4 promoter), the LEB4 signal peptide, the gene to be expressed and an ER retention signal.
  • a seed-specific promoter preferably the phaseolin promoter, the USP or LEB4 promoter
  • the LEB4 signal peptide the gene to be expressed and an ER retention signal.
  • the inserted nucleotide sequence coding for a formylglycine namidine ribotide synthase can be produced synthetically or naturally be won or contain a mixture of synthetic and natural DNA components.
  • synthetic nucleotide sequences with codons are generated which are preferred by plants. This plant preferred codons kön- g nen be determined from codons with the highest protein frequency which are expressed in the most interesting plant species.
  • various DNA fragments can be manipulated in order to obtain a nucleotide sequence which expediently reads in the correct direction and which is equipped with a correct reading frame.
  • adapters or linkers can be attached to the fragments.
  • artificial DNA sequences are suitable as long as they impart the desired property of increasing the level of purine nucleotides in the plant by overexpressing the formylglycine amidine ribotide synthase gene.
  • Such artificial DNA sequences can be determined, for example, by back-translation of proteins constructed using molecular modeling, which have formylglycine amidribotide synthase activity, or by in vitro selection. Coding DNA sequences which are obtained by back-translating a polypeptide sequence according to the codon usage specific for the host plant are particularly suitable.
  • a person skilled in the art familiar with plant genetic methods, can easily determine the specific codon usage by computer evaluations of other, known genes of the plant 5 to be transformed.
  • Sequences which code for fusion proteins are to be mentioned as further suitable equivalent nucleic acid sequences according to the invention, part of the fusion protein being a plant formylglycinamidine ribotide synthase polypeptide or a functionally equivalent part thereof.
  • the second part of the fusion protein can e.g. be another polypeptide with enzymatic activity or an antigenic polypeptide sequence that can be used to detect formylglycinamidine ribotide synthase expression (e.g. mye-tag or his-tag).
  • this is preferably a regulatory protein sequence, such as e.g. a signal or transit peptide that directs the formylglycine amide ribotide protein to the desired site of action.
  • Terminator regions in the direction of transcription are provided with a linker 0 or polylinker which contains one or more restriction sites for the insertion of this sequence.
  • the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites.
  • the linker has a size of less than 100 bp, 5 often less than 60 bp, but at least 5 bp within the regulatory ranges.
  • the promoter according to the invention can be both native or homologous and foreign or heterologous to the host plant.
  • the invention Expression cassette contains the promoter according to the invention, any sequence and a region for the transcriptional termination in the 5 '-3' transcription direction. Different termination areas are interchangeable.
  • Preferred polyadenylation signals are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular gene 3 of T-DNA (octopine synthase) of the Ti plasmid pTiACH5 (Gielen et al., EMBO J. , 3 (1984), 835) or functional equivalents.
  • an expression cassette according to the invention is inserted as an insert into a recombinant vector whose vector DNA contains additional functional regulation signals, for example sequences for replication or integration.
  • additional functional regulation signals for example sequences for replication or integration.
  • transformation The transfer of foreign genes into the genome of a plant is called transformation.
  • the methods described for the transformation and regeneration of plants from plant tissues or plant cells for transient or stable transformation are used. Suitable methods are the protoplast transformation by polyethylene glycol-induced DNA uptake, the biolistic approach with the gene cannon, the electroporation, the incubation of dry embryos in DNA-containing solution, the microinjection and the gene transfer mediated by Agrobacterium. The methods mentioned are described, for example, in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,
  • the construct to be expressed is cloned into a vector that is suitable, Agro-bacteriuu. transform tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711).
  • Agrobacteria transformed with an expression cassette according to the invention can also be used in a known manner to transform plants, in particular crop plants, such as cereals, maize, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rape, Alfalfa, lettuce and the various tree, nut and wine species as well as legumes are used, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
  • the biosythesis site of purines is generally the leaf tissue, so that leaf-specific expression of the formylglycine amide ribotide synthase gene makes sense.
  • the purine biosynthesis need not be restricted to the leaf tissue, but can also be tissue-specific in all other parts of the plant - for example in fatty seeds.
  • constitutive expression of the exogenous formylglycinamidine ribotide synthase gene is advantageous.
  • inducible expression may also appear desirable.
  • the expression cassettes according to the invention can be cloned into suitable vectors which enable their multiplication, for example in E. coli.
  • suitable cloning vectors include pBR332, pUC series, Ml3mp series and pACYC184.
  • Binary vectors which can replicate both in E. coli and in agrobacteria are particularly suitable.
  • Another object of the invention relates to the use of an expression cassette according to the invention for transforming plants, plant cells, plant tissues or parts of plants.
  • the aim of the use is preferably to increase the formylglycine amidribotide synthase content in the plant. 5
  • the expression can take place specifically in the leaves, in the seeds or in other parts of the plant.
  • Such transgenic plants, their reproductive material and their plant cells, tissue or parts are a further subject of the present invention.
  • the invention is illustrated by the following examples, but is not limited to these.
  • Cloning methods such as Restriction cleavages, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of Escherichia coli lines, cultivation of bacteria and sequence analysis of recombinant DNA were carried out as in Sambrook et al. (1989) (Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6). Screening of cDNA libraries
  • ⁇ phages from the corresponding cDNA libraries were plated on agar plates with E. coli XLl-Blue as a bacterial strain.
  • the phage DNA was transferred to nitrocellulose filters (Gelman Sciences) using standard methods (Sambrook et al. (1989), Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) and fixed on the filters.
  • PCR fragments or DNA fragments obtained by restriction cleavage were used as hybridization probes and were radioactively labeled with the aid of a "Multiprime DNA labeling System” (Amersham Buchler) in the presence of 32 P-dCTP (specific activity 3000 Ci / mmol) according to the manufacturer's instructions.
  • the membranes were hybridized after prehybridization at 60 ° C. in 3 ⁇ SSPE, 0.1% sodium dodecyl sulfate (w / v), 0.02% polyvinylpyrrolidone (w / v), 0.02% Ficoll 400 (w / v ) and 50 mg / ml calf thymus DNA for 12-16 hours.
  • the filters were then washed in 2 x SSPE, 0.1% sodium dodecyl sulfate (w / v) at 60 ° C. for 60 minutes. Positive hybridizing phages were visualized by autoradiography, isolated using standard techniques (Sambrook et al. (1989); Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) and converted into plasmids (Stratagene).
  • the sequencing of recombinant DNA molecules was carried out with a laser fluorescence DNA sequencer from ABI according to the method of Sanger (Sanger et al., Proc. Natl. Acad. Sci. USA, 74 (1977), 5463-5467). Fragments resulting from a polymerase chain reaction were sequenced and checked in order to avoid polymerase errors in constructs to be expressed.
  • RNA from plant tissues was isolated as in Logemann et al. (Anal. Biochem. 163 (1987), 21). For the analysis, 20 ⁇ g RNA were separated in a 1.5% agarose gel containing formaldehyde and transferred to nylon membranes (Hybond, Amersham). The detection of specific transcripts was carried out as described for Amasino (Anal. Biochem. 152 (1986), 304). The DNA fragments used as a probe were radioactively labeled with a random primed DNA labeling kit (Röche, Mannheim) and hybridized according to standard methods, see Hybond user instructions, Amersham. Hyridization signals were visualized by autoradiography using X-OMAT AR films from Kodak.
  • DNA-modifying enzymes and molecular biological kits were developed by the companies AGS (Heidelberg), Amersham (Braunschweig), Biometra (Göttingen), Röche (Mannheim), Genomed (Bad Oeynnhausen), New England Biolabs (Schwalbach / Taunus), Novagen ( Madison, Wisconsin, USA), Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg) Qiagen (Hilden) and Stratagene (Heidelberg). Unless otherwise stated, they were named after AGS (Heidelberg), Amersham (Braunschweig), Biometra (Göttingen), Röche (Mannheim), Genomed (Bad Oeynnhausen), New England Biolabs (Schwalbach / Taunus), Novagen ( Madison, Wisconsin, USA), Perkin-Elmer (Weiterstadt), Pharmacia (Freiburg) Qiagen (Hilden) and Stratagene (Heidelberg). Unless otherwise stated, they were named after
  • E. coli, XL-1 Blue The bacterial strains used below (E. coli, XL-1 Blue) were obtained from Stratagene.
  • E. coli AT 2465 was obtained from the coli genetic stock center (Yale University, New Haven).
  • the Agrobacterium strain used for plant transformation (Agrobacterium tumefaciens, C58C1 with the plasmid pGV2260 or pGV3850kan) was developed by Deblaere et al. (Nucl. Acids Res. 13 (1985), 4777).
  • the LBA4404 agrobacterial strain (Clontech) or other suitable strains can be used.
  • the vectors pUC19 Yamaphilid, XL-1 Blue
  • an expressed sequence tag (est) partially coding for formylglycinamidine ribotide synthase was obtained from Arabidopsis thaliana (GenBank Accession number AA042492), sequenced and as a template for generating a hybridization probe by means of a poly erase chain reaction (PCR) uses t.
  • the reaction mixtures contained approx.
  • the authorization conditions were set as follows:
  • Annealing temperature 50 ° C, 1 min denaturation temperature: 94 ° C, 1 min
  • the resulting fragment of 465 bp was used for a heterologous screening of 6.5 * 10 5 pfu of a UniZAP XR cDNA bank (Stratagene) created from RNA from Arabidopsis thaliana (whole plant). After restriction and sequence analysis, two distinguishable clones (purL-19 and purL-23) were identified, which encode reading frames with homology to formylglycinamidine ribotide synthase from Escherichia coli, Bacillus subtilis, Saccharomyces ce-revisiae and Drosophila melanogaster.
  • purL-48.1 was identified by means of restriction analysis and then sequenced, see SEQ-ID No. 1.
  • the purL-48.1 cDNA has a length of 4570 base pairs and is identical in the overlapping area (nucleotide 3328-4570) with the shorter purL-19 cDNA.
  • a continuous reading frame codes in purL-4 ⁇ .l for a polypeptide of 1407 amino acids and a calculated mass of 153.952 kda, see SEQ-ID No. 2.
  • the amino acid sequence is similar to formylglycinamidine ribotide synthase from other organisms, see Table 1. There is a relationship to formylglycinamidine ribotide synthase from humans (GenBank number
  • purL-48.1 has an extension of 53 up to 88 amino acids compared to formylglycina idinribotid synthase from other organisms, which indicates its importance as a transit peptide.
  • the computer-aided evaluation of the sequence predicts a possible function as a signal peptide for an import into the plastids for amino acids 1-53. Localization of the enzyme in plastids is consistent with its function in purine biosynthesis. A stop codon is found in the reading frame upstream of the start methionine.
  • PurL-48.1 is the first full-length plant cDNA for a formylglycinamidine ribotide synthase.
  • purL_yeast kdi — ytnst svinelrsc- ihyvngiaq- -seqdt-1— v-lt-dsa-d purL_Drome sa-ee-sv— rlreedg.av -s-rm-r — h -e.ys-qaeh —aldel-v- purL_human -ga-ghtr- kl-gklp.-l qg-e v-.
  • Consensus -DLDYYTD-F EL-KNPT- VELFDFAQSN SEHSRHWFFN GD-VIDG-KQ
  • 351 400 purL_e. coli k - kn -f-ttpdhvl say - aavm e-s - gryfa dhe-g. r. y- purL_yeast qft rn -hklnpeyti says - aavl dsenda-ffa pns-t.
  • Consensus -QQE LF TAETHN-PTA V-PFPGA-TG -GGRIRD - A TGRG-K - AG 451 500 purL_e.coli lv-fs-s — rf. ed-gk-eriv ta-d-mt-gp 1-gaafn-e- purL_yeast ls-fs-sd-1 n.
  • 701 750 purL_e.coli -ea-eelh-s -h- rh fdnq-i— - dvl 1 — m purL_yeast -ha-aeqk-i ve- -1 lkt — im pilf — p — m purL_Drome -w — d — v - -l.ekp d leqa-n-fnr sevs-f ky dr purL_hu ⁇ .an - i - dr-iv -v-drec-vr rngqgd-pt v - rk purL-ara-48- - in-g - c- -i-sta-ac skeg-. , , .p pav -k d k
  • Consensus PD KTVTPD LK-PTG-GDD - LLVDLS- -KG — RLGGS ALAQVF-QLG 1001 1050 purL_e.coli dk-a-vr q fy-ai- —vqk..l —y — ra purL_yeast -ks-tvy-n - i flesli q-hqqkediv —y — r —i purL_Drome kdt-n-trsd v-gka-av— s — gdg ..- iqv cv i purL_human eh lpe n-vra-si— g — kd -..- l es v -vtc purL-ara-48- -de py — nv — gv- —i-en ..- vsi -v —
  • 1101 1150 purL_e.coli r sv-aq h-1-.dcvhy —q-vsgd-f -ita — q- ... -f — s-tt- purL_yeast -skf-ki-ne nk — isi —kpsfq-qe ikii-st-nd viyans purL_Drome —r-rstyek pny-lv tegf-ld -1-ngkse ..
  • SEQ ID No. 3 - a polypeptide of 1017 amino acids in length, see SEQ ID No. 4.
  • the polypeptide shows similarity to the formylglycinamidine ribotide synthase from Arabidopsis thaliana (purL-48.1) (80.1% identity).
  • Double-stranded cDNA was generated from mRNA from Chilopsis linearis and used to produce a cDNA library in the vector pBluescript SKII (la bda ZAP II RI library construction kit, Stratagene). Individual clones from this bank were sequenced. The 3'-sided sequence of the clone 74_chi_005_el0 was similar to purL-Ntl.l from tobacco and purL-48.1 from Arabidopsis thaliana. The clone 74_chi_005_el0 was fully sequenced. It encodes a 478bp partial cDNA for a polypeptide of 97 amino acids. At the amino acid level, 74_chi_005_el0 is 82.4% identical to purL-48.1 and 75.2% identical to purL-Ntl.l.
  • cDNA coded by purL-48.1 it was cloned into suitable expression vectors (for example of the pQE, Qiagen or pET, Novagen series).
  • suitable expression vectors for example of the pQE, Qiagen or pET, Novagen series.
  • fragments of different lengths of the purL-48.1 cDNA can be generated, for example, by PCR and ligated into the vectors prepared by treatment with restriction endonucleases.
  • the expression constructs obtained can be used to convert the E.
  • coli strain CGSC # 4537 (genotype: fhuA2, lacYl, glnV44 (AS), gal-6, ⁇ ⁇ , nadB4, purL66, rpsL9, malTl ( ⁇ R ), xylA7, mtlA2, ⁇ argHl; E. coli genetic stock center, Yale University, New Haven).
  • AS lacYl
  • glnV44 AS
  • gal-6 ⁇ ⁇ , nadB4
  • xylA7, mtlA2, ⁇ argHl E. coli genetic stock center, Yale University, New Haven
  • the minimal media should contain, for example, 0.4% glucose, 0.2% casaminoacids, 100 ⁇ g / ml thiamine, 100 ⁇ g / ml inosine, 100 ⁇ g / ml biotin, 100 ⁇ g / ml nicotinate, 100 ⁇ M IPTG and 50-100 ⁇ g / ml of the antibiotic in question, to which the expression vector mediates resistance.
  • the cloning vector can be transformed into CGSC # 4537 without a purL-48.1 insert as a negative control. It was shown that only the bacteria transformed with purL-48.1 expression constructs are able to grow on minimal media without adenine.
  • the enzyme encoded by purL-48.1 is therefore functional and the first functional formylglycinamidine ribotide synthase isolated from plants.
  • Yeast Sacharomyces cerevisiae
  • Ade6 gene which codes for the yeast-specific formylglycinamidine ribotide synthase, is rendered inoperative.
  • a yeast mutant suitable for complementation the method of Güldener et al. , Nucleic Acids Research 24 (1996), 2519-2524 in application to a suitable starting strain, such as, for example, SEY6210 (Robinson et al., Protoplasma 150 (1989), 79-82).
  • the mutant yeast generated accordingly should not show growth on minimal media without adenine.
  • cDNA encoded by purL-48.1 it is cloned into suitable yeast expression vectors (for example pYEBH2, Riesmeier et al., EMBO J. 11 (1992), 4705-4713 or pYES2, Invitrogen).
  • suitable yeast expression vectors for example pYEBH2, Riesmeier et al., EMBO J. 11 (1992), 4705-4713 or pYES2, Invitrogen.
  • cDNAs of different lengths can be generated, for example, by PCR and ligated into the vectors prepared by treatment with restriction enducleases.
  • the expression constructs obtained are used to complement the yeast mutant generated. To do this, it is transformed with the expression constructs and plated on minimal media (eg SDG media, Clontech, Matchmaker system) without adenine.
  • the cloning vector without purL-48.1 insert is transformed into the yeast mutant as a negative control. It is shown that only the yeasts transformed with purL-48.1 expression constructs are capable of growing on minimal media without adenine.
  • the enzyme encoded by purL-48.1 thus represents the first functional formylglycinamidine ribotide syn- thase isolated from plants.
  • binary vectors were first generated for the plant transformation.
  • the clone purL-19 was cleaved with BamHI and SalI and the fragment of 1127 kb obtained was cleaved in the vector pBinAR (Höfgen and Willmitzer, Plant Science 66 (1990), 221-230), 1990. ligated.
  • the clone purL-19 was cleaved with BamHI and Kpnl and the generated fragment of 964 bp was ligated into the likewise cleaved vector pBinAR.
  • the Binary constructs AtSpurL and AtASpurL Figure 2 obtained in this way were transformed into Arabidopsis thaliana.
  • the binary vectors AtSpurL and AtASpurL were transformed into Agrobacterium tumefaciens C58C1: pGV2260 (Deblaere et al.,
  • Greenhouse 22 ° C day / 18 ° C night temperature, 16 h photoperiod 35, 450 ⁇ E * m- 2 * s " 1 , 68% humidity.
  • the plasmids NtSpurL and NtASpurL were transformed into Agrobacterium tumefaciens C58C1: pGV2260 (Deblaere et al., Nucl. Acids. Res. 13 (1984), 4777-4788).
  • pGV2260 Agrobacterium tumefaciens C58C1: pGV2260 (Deblaere et al., Nucl. Acids. Res. 13 (1984), 4777-4788).
  • Leaf disks of sterile plants were incubated in a Petri dish with a 1:50 agrobacterial dilution for 5-10 minutes. This was followed by a 2-day incubation in the dark at 25 ° C. on 2MS medium with 0.8% Bacto agar. The cultivation was continued after 2 days with 16 hours of light / 8 hours of darkness and in a weekly rhythm on MS medium with 500 mg / 1 claforan (cefotaxime sodium), 50 mg / 1 kanamycin, 1 mg / 1 benzylaminopurine (BAP ), 0.2 mg / 1 naphthylacetic acid and 1.6 g / 1 glucose continued.
  • Growing shoots were transferred to MS medium with 2% sucrose, 250 mg / 1 Claforan and 0.8% Bacto agar. Regenerated shoots were obtained on 2MS medium with kanamycin and claforan, transferred to soil after rooting and analyzed after cultivation in a climatic chamber in a 16 hour light / 8 hour dark rhythm at 60% humidity.
  • Transgenic plants were examined for formylglycinamidine ribotide synthase expression and activity as well as for altered metabolite levels and phenotypic growth characteristics. Altered nucleotide levels can e.g. using the method of Stitt et al. , FEBS Letters 145 (1982), 217-222.
  • Lines of transgenic plants that have been transformed with the constructs AtSpurL, AtASpurL, NtSpurL or NtASpurL are characterized by a different degree of reduced growth compared to untransformed control plants.
  • the RNA analysis by Northernblot-Teehnik showed a reduced amount of purL-48.1 or purL-Ntl.l in transgenic lines with the described phenotype.
  • the formylglycinamidine ribotide synthetic activity can be determined by a method as described in Example 8.
  • the measurement of the formylglycinamidine ribotide synthase activity is carried out with modifications according to the methods described (Methods in Enzymology. 51 (1978), 193-201). After adapting the method to high-throughput methods, it is possible to search for inhibitors of formylglycine amidribotide synthase activity using one of the described in vitro assays. For this purpose, the formylglycinamidine ribotide synthetic activity can be prepared from plant tissues.
  • a complete or shortened plant formylglycinamidine ribotide synthase (preferably formylglycinamidine ribotide synthase from Arabidopsis thaliana) can be generated in a suitable prokaryotic (e.g. E. coli) or eukaryotic (e.g. yeast, insect cell) expression system. After digestion of the plant tissues or cells in suitable buffers and, if necessary, further purification steps e.g. The formylglycinamidine ribotide synthase activity can be quantified using chromatographic methods. In this way it is possible to correlate the growth phenotype with the formylglycine amide ribotide synthase activity in transgenic lines.
  • a suitable prokaryotic e.g. E. coli
  • eukaryotic e.g. yeast, insect cell

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Abstract

L'invention concerne des séquences d'ADN codant un polypeptide à activité de formylglycinamidinribotide (E.C.6.3.5.3). L'invention concerne en outre l'utilisation de ces acides nucléiques pour produire un système de test.
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