Classifications

• • 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
  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1235—Diphosphotransferases (2.7.6)
• • 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
  • C12N15/8261—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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
• • 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
  • G01N2333/91—Transferases (2.)
  • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • G01N2333/91205—Phosphotransferases in general
  • G01N2333/9124—Diphosphotransferases (2.7.6)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2430/00—Assays, e.g. immunoassays or enzyme assays, involving synthetic organic compounds as analytes
  • G01N2430/20—Herbicides, e.g. DDT
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

• Phosphoribosyl pyrophosphate synthetase 1 as a herbicidal target
• the present invention relates to the identification of plant phosphoribosyl pyrophosphate synthetase 1 (E.C. 2.7.6.1) as a new target for herbicidal active compounds.
• the present invention further relates to the use of the DNA sequence SEQ-ID No. 1, SEQ ID No. 3 or SEQ ID No. 5 or parts or derivatives thereof coding for a polypeptide with phosphoribosyl pyrophosphate synthetase 1 Activity for the preparation of a test system for identifying inhibitors of phosphoribosyl pyrophosphate synthetase 1 with herbicidal activity.
• the invention also relates to a method or a test system for finding substances which inhibit the activity of the plant phosphoribosyl pyrophosphate synthetase 1, and inhibitors of plant phosphoribosyl pyrophosphate synthetase 1 identified using these methods or this test system.
• 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 are also a component of essential factors such as coenzyme A, as well as nicotinamide and flavin coenzymes that are involved in many cellular responses.
• GTP guanosine 5'-triphosphate
• ASA adenylosuccinate synthetase
• PRPP Phosphoribosyl pyrophosphate
• the phosphobosyl pyrophosphate synthetase of some organisms also require Mg 2+ .
• Eukaryotes often have more than one phosphoribosyl pyrophosphate synthetase. For example, people have three isoforms with redundant functions.
• Phosphoribosyl pyrophosphate synthetase 2 is imported into the chloroplasts (Krath and Hove-Jensen, Plant Physiology 119 (1999), 497-505). This is consistent with results that have been demonstrated by several enzymes of purine de novo biosynthesis in the chloroplasts of Arabidopsis (Senecoff and Meager, Plant Physiology 102 (1993), 387-399; Ito et al., Plant Mol Biol 26 (1994) , 529-533; Schnorr et al., Plant Journal 6 (1994), 113-121).
• the suitability of an enzyme as a target for herbicides can be demonstrated by reducing the enzyme activity, for example using antisense technology in transgenic plants. If reduced growth is brought about by introducing an antisense DNA for a specific gene into a plant, this indicates the suitability of the enzyme whose activity is reduced as a site of action for herbicidal active ingredients.
• the antisense inhibition of acetolactate synthase (ALS) in transgenic potato plants leads to comparable phenotypes, such as the treatment of control plants with ALS-inhibiting herbicides (Höfgen et al., Plant Physiology 107 (1995), 469-477).
• the object of the present invention was to demonstrate that
• Phosphoribosyl pyrophosphate synthetase 1 is a suitable herbicidal target in plants, and the preparation of an efficient and simple phosphoribosyl pyrophosphate synthetase 1 test system for carrying out inhibitor-enzyme binding studies.
• the object was achieved by isolating genes which code for the plant enzyme phosphoribosyl pyrophosphate synthetase 1, the production of antisense constructs of the plant phosphoribosyl pyrophosphate synthetase 1 and their expression in plants, and the functional expression of the plant phosphoribosyl pyrophosphate synthetase 1 in bacterial or eukaryotic cells.
• cDNAs coding for plant phosphoribosyl pyrophosphate synthetase 1 from Nicotiana tabacum and Arabidopsis thaliana and a partial cDNA from Physcomitrella patens were first isolated and sequenced, see Example 1 and Example 6, sequence listing SEQ-ID No. 1, SEQ ID No. 3 and SEQ-ID No. 5th
• Another object of the invention relates to methods for identifying inhibitors of plant phosphoribosyl pyrophosphate synthetase 1 by high-throughput methods.
• the invention therefore relates to the functional expression of plant phosphoribosyl pyrophosphate synthetase 1, in particular phosphoribosyl pyrophosphate synthetase 1 from tobacco and Arabidopsis thaliana in suitable expression systems, and to the use of the enzymes thus produced in an in vitro test system for measuring the phosphoribosyl pyrophosphate synthetase 1 activity ,
• the cDNA sequence of the phosphoribosyl pyrophosphate synthetase 1 or suitable fragments of the cDNA sequence of the phosphoribosyl pyrophosphate synthetase 1 from tobacco or Arabidopsis thaliana is cloned into an expression vector (pQE, Qiagen) and overexpressed in E. coli.
• the expression cassette containing a DNA sequence or partial DNA sequence of SEQ-ID No. 1 or SEQ ID NO. 3 or derivatives of these sequences can be expressed, for example, in other bacteria, in yeasts, fungi, algae, plant cells, insect cells or mammalian cells.
• Another object of the invention is the use of DNA sequences from SEQ ID NO. 1 or SEQ ID NO. 3 are derived or hybridize with one of these sequences and which code for a protein which has the biological activity of a phosphoribosyl pyrophosphate synthetase 1.
• the vegetable phosphoribosyl pyrophosphate synthetase 1 protein expressed with the aid of an expression cassette is particularly suitable for the detection of inhibitors specific for phosphoribosyl pyrophosphate synthetase 1.
• the plant phosphoribosyl pyrophosphate synthetase 1 can be used, for example, in an enzyme test in which the activity of the phosphoribosyl pyrophosphate synthetase 1 is determined in the presence and absence of the active substance 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 7.
• a large number of chemical compounds can be checked quickly and easily for herbicidal properties. The method makes it possible to selectively reproducibly select those with great 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.
• Another object of the invention is a method for the identification of inhibitors of plant phosphoribosyl pyrophosphate synthetase 1, with potentially herbicidal activity by cloning the gene of a plant phosphoribosyl pyrophosphate synthetase 1, in a suitable expression cassette - for example in insect cells - to overexpress the cells opens and the cell extract is used directly or after enrichment or isolation of the enzyme phosphoribosyl pyrophosphate synthetase 1 in a test system for measuring the enzyme activity in the presence of low molecular weight chemical compounds.
• Another object of the invention is a method for identifying substances with herbicidal activity which inhibit the phosphoribosyl pyrophosphate synthetase 1 activity in plants, consisting of
• transgenic plants, plant tissues or plant cells which contain an additional DNA sequence coding for an enzyme with phosphoribosyl pyrophosphate synthetase 1 activity and are able to overexpress an enzymatically active phosphoribosyl pyrophosphate synthetase 1;
• the suppression of the growth or survivability of the non-transformed plants, plant cells, plant tissues or plant parts without, however, strongly suppressing the growth or survivability of the transgenic plants, plant cells, plant tissues or plant parts shows that the substance from b) shows herbicidal activity and the phosphoribosyl pyrophosphate synthetase 1 inhibits enzyme activity in plants.
• the invention further relates to compounds with herbicidal activity which can be identified using the test system described above.
• Inhibitors of phosphoribosyl pyrophosphate synthetase 1 with herbicidal activity can be used as defoliants, desiccants, herbicides 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 one used Amount off.
• Inhibitors of phosphoribosyl pyrophosphate synthetase 1 with herbicidal activity can be used, for example, against the following weeds:
• the invention also relates to the use of expression cassettes, the sequence of which codes for a phosphoribosyl pyrophosphate synthetase 1 from Arabidopsis thaliana or Nicotiana tabacum or the functional equivalent thereof for the production of a test assay for finding compounds with herbicidal activity.
• the nucleic acid sequence can e.g. be a DNA or a cDNA sequence.
• an expression cassette according to the invention comprises a promoter upstream, ie at the 5 'end of the coding sequence, and a polyadenylation signal and downstream, ie at the 3' end, and, if appropriate, further regulatory elements which are associated with the coding sequence for the phosphoribosyl intermediate Pyrophosphate synthetase 1 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.
• Such an expression cassette is produced by fusing a suitable promoter with a suitable phosphoribosyl pyrophosphate synthetase 1 DNA sequence and a polyadenylation signal according to common recombination and
• the invention also relates to the use of functionally equivalent DNA sequences which code for a phosphoribosyl pyrophosphate synthetase 1 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%.
• the preferred object of the invention is the use of functionally equivalent DNA sequences which code for a phosphoribosyl pyrophosphate synthetase 1 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 60 to 100%.
• a particularly preferred object of the invention is the use of functionally equivalent DNA sequences which code for a phosphoribosyl pyrophosphate synthetase 1 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 80 to 100%.
• Functionally equivalent sequences which code for a phosphoribosyl pyrophosphate synthetase 1 gene are those sequences which, despite a different nucleotide sequence, still have the desired functions. Functional equivalents thus include naturally occurring variants of the sequences described herein as well as artificial, e.g. Artificial 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 phosphoribosyl pyrophosphate synthetase 1, 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, for example, further narrow down the coding sequence contained therein or, for example, also insert further ones 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 can also be used to transform bacteria, cyanobacteria, yeast, filamentous fungi and algae and eukaryotic cells (e.g. insect cells) with the aim of producing sufficient amounts of the enzyme phosphoribosyl pyrophosphate synthetase 1.
• Another object of the invention is the use of a protein from Nicotiana tabacum or Arabidopsis thaliana characterized by the amino acid sequence SEQ-ID NO. 2, SEQ ID No. 4 or derivatives or parts of this protein with phosphoribosyl pyrophosphate synthetase 1 activity for the preparation of a test system for finding compounds with herbicidal activity.
• the invention also relates to the use of plant proteins with phosphoribosyl pyrophosphate synthetase 1 activity with an amino acid sequence homology to the phosphoribosyl pyrophosphate synthetase 1 from Nicotiana tabacum, Arabidopsis thaliana or Physcomitrella patens with the SEQ-ID NO. 2, SEQ ID NO. 4 or SEQ ID No. 6 of 20 - 100% identity for the production of a test system for finding compounds with herbicidal activity.
• plant proteins with phosphoribosyl pyrophosphate synthetase 1 activity with an amino acid sequence homology to the phosphoribosyl pyrophosphate synthetase 1 from Nicotiana tabacum, Arabidopsis thaliana or Physcomitrella patens with the sequences SEQ-ID NO. 2, SEQ ID NO. 4 or SEQ-ID No. 6 of 50 - 100% identity for the production of a test system for the discovery of compounds with herbicidal activity.
• the effectiveness of the expression of the transgenically expressed phosphoribosyl pyrophosphate synthetase 1 gene can be determined, for example, in vitro by proliferation or by a germination test.
• a change in the type and level of expression of the phosphoribosyl pyrophosphate synthetase 1 gene and its effect on the resistance to inhibitors of phosphoribosyl pyrophosphate synthetase 1 can be tested on test plants in greenhouse experiments.
• the invention also relates to transgenic plants transformed with an expression cassette containing the DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3, by additional expression of the DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 have become tolerant of inhibitors of phosphoribosyl pyrophosphate synthetase 1, as well as transgenic cells, tissues, parts and propagation material of such plants.
• Transgenic crop plants such as barley, wheat, rye, corn, soybean, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rape, are particularly preferred.
• Endoplasmic reticulum ER or, due to the lack of corresponding operative sequences, ensure that it remains in the compartment of formation, the cytosol (Kermode, Crit. Rev. Plant Sei. 15, 4 (1996), 285-423).
• the plant expression cassette can be built into the plant transformation vector pBinAR.
• any promoter which can control the expression of foreign genes in plants is suitable as promoters of the expression cassette.
• 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 can also contain a chemically inducible promoter which can be used to control the expression of the exogenous phosphoribosyl pyrophosphate synthetase 1 gene in the plant at a particular point in time.
• a chemically inducible promoter which can be used to control the expression of the exogenous phosphoribosyl pyrophosphate synthetase 1 gene in the plant at a particular point in time.
• promoters such as the PRP1 promoter (Ward et al., Plant Mol. Biol. 22 (1993), 361-366), a salicylic acid-inducible promoter (WO 95/19443), a benzene-sulfonamide-inducible (EP 388186 ), one that can be induced by tetracycline (Gatz et al., Plant J.
• promoters are particularly preferred which ensure expression in tissues or parts of plants in which the biosynthesis of purines or their precursors takes place. Promoters that ensure leaf-specific expression should be mentioned in particular.
• the promoter of the cytosolic FBPase from potatoes or the ST-LSI promoter from potatoes should be mentioned (Stockhaus et al., EMBO J., 8 (1989) 2445-245).
• a foreign protein can be stably expressed up to a proportion of 0.67% of the total soluble seed protein in the seeds of transgenic tobacco plants (Fiedler and Conrad, Bio / Technology 10 (1995), 1090-1094).
• the expression cassette according to the invention can therefore, for example, be a seed-specific promoter (preferably the phaseolin promoter, the USP or LEB4-
• the inserted nucleotide sequence coding for a phosphoribosyl pyrophosphate synthetase 1 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. These codons preferred by plants can be determined from codons with the highest protein frequency, which are expressed in 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, as described above, for example, they impart the desired property of expression of the phosphoribosyl pyrophosphate synthetase 1 gene.
• Such artificial DNA sequences can be determined, for example, by back-translation of proteins constructed using molecular modeling, which have phosphoribosyl pyrophosphate synthetase 1 activity, or by in-v / o selection. Coding DNA sequences which are particularly suitable are those obtained by back-translating a
• Polypeptide sequence was obtained according to the codon usage specific for the host plant.
• the specific codon usage can easily be determined by a person skilled in plant genetic methods by computer evaluations of other, known genes of the plant to be transformed.
• Suitable equivalent nucleic acid sequences are sequences which code for fusion proteins, part of the fusion protein being a plant phosphoribosyl pyrophosphate synthetase 1 polypeptide or a functionally equivalent one Is part of it.
• the second part of the fusion protein can be, for example, a further polypeptide with enzymatic activity or an antigenic polypeptide sequence with the aid of which detection of phosphoribosyl pyrophosphate synthetase 1 expression is possible (for example myc-tag or histag).
• it is preferably a regulatory protein sequence, such as a signal or transit peptide, that the
• Phosphoribosyl pyrophosphate synthetase 1 protein to the desired site.
• the promoter and terminator regions should expediently be provided in the transcription direction with a linker 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, 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 expression cassette according to the invention contains, in the 5'-3 'transcription direction, the promoter according to the invention, any sequence and a region for the transcriptional termination. 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 pTiACH ⁇ (Gielen et al., EMBO J., 3rd (1984), 835) or functional equivalents.
• Expression cassette inserted as an insert in a recombinant vector whose vector DNA contains additional functional regulation signals, for example sequences for replication or integration.
• Suitable vectors are described, inter alia, in “Methods in Plant Molecular Biology and Biotechnology” (CRC Press, chapters 6/7, 71-119).
• 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 gun, 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, Engineering and Utilization, published by SD Kung and R. Wu, Academic Press (1993), 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec.Biol.
• the construct to be expressed is cloned into a vector which is suitable for transforming Agrobacterium tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711).
• Agrobacteria transformed with an expression cassette can also be used in a known manner to transform plants, in particular crop plants, such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa, Salad and the various tree, nut and wine species and legumes can be used, for example by bathing wounded leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media.
• crop plants such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, hemp, potato, tobacco, tomato, rapeseed, alfalfa, Salad and the various tree, nut and wine species and legumes can be 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 phosphoribosyl pyrophosphate synthetase 1 gene is useful.
• 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 phosphoribosyl pyrophosphate synthetase 1 gene is advantageous.
• inducible expression may also appear desirable.
• Cloning techniques allow the expression cassettes to be cloned into suitable vectors that allow them to multiply, for example in E. coli.
• suitable cloning vectors include pBR332, pUC series, M13mp series and pACYC184.
• Binary vectors which replicate both in E. coli and in agrobacteria are particularly suitable can.
• Another object of the invention relates to the use of an expression cassette according to the invention for the transformation of plants, plant cells, plant tissues or parts of plants.
• the aim of the use is preferably to increase the phosphoribosyl pyrophosphate synthetase 1 content in the plant.
• 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.
• Cloning methods such as: restriction cleavage, DNA isolation, agarose gel electrophoresis, purification of DNA fragments, transfer of nucleic acids to nitrocellulose and nylon membranes, linking of DNA fragments, transformation of E. coli cells, cultivation of bacteria, sequence analysis of recombinant DNA were carried out according to Sambrook et al., Cold Spring Harbor Laboratory Press (1989); ISBN 0-87969-309-6.
• the transformation of Agrobacterium tumefaciens was carried out according to the method of Höfgen and Willmitzer (Nucl. Acid Res. 16 (1988), 9877).
• the cultivation of the Agrobacteria occurred in YEB medium (Vervliet et al., Gen. Virol. 26 (1975), 33).
• Total plant RNA was determined according to the method of Logemann et al., Analytical Biochem. 163 (1987), 16) isolated and separated in formaldehyde-containing agarose gels (Lehrach et al., Biochem. 16 (1977), 4743). Capillary transfer to nylon membranes (Gene Screen, NEN) was carried out in 20 ⁇ SSC (1.5 M NaCl, 150 mM sodium citrate) overnight. After two hours of prehybridization in hybridization buffer (500 mM sodium phosphate (pH 7.2), 7% SDS, 0.5% bovine serum albumin, 1 mM EDTA), the hybridization was carried out with a radiolabelled NtPrsI probe at 65 ° C. for 16 hours. The filters were then washed under the following conditions: 10 minutes at 65 ° C in 6 x SSC, 0.1% SDS and 5 minutes at 65C in 4 x SSC, 0.1% SDS.
• hybridization buffer 500 mM sodium phosphate (pH
• 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 used according to the manufacturer's instructions.
• E. coli (XL-1 Blue) bacteria were purchased from Stratagene.
• the Agrobacterium strain used for plant transformation (C58C1 with the plasmid pGV 3850kan) was developed by Debleare et al., Nucl. Acid Res. 13 (1985), 4777).
• RBPRPP3 5'-aag aat tcg gat cca cca tgg tct tga agt tgt tct ctg gta ctg c-3 'RBPRPP4 5'-aag aat tcg gat cct caa agg aa atg cta ctg ac-3'
• a cDNA fragment from Arabidopsis thaliana (Var. Landsberg erecta) leaf cDNA was amplified using these oligonucleotides (35 Cycles, 30sec 94DC, 45 sec 45DC, 2 min 72DC) and then bluntly cloned into a pGEM-T vector (Promega) using EcoRV.
• the identity of the Arabidopsis Prs1 cDNA clone was confirmed by sequencing, see SEQ-ID No. Third
• This clone was used to screen a source leaf cDNA bank from Nicotiana tabacum variety Samsun NN in DDZAPII.
• the cDNA bank was plated with a titer of 2.5D10 5 plaque-forming units and analyzed using the plaque screening method (T. Maniatis, EF Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor , NY 1989).
• 12 phage populations were isolated, with which a second screening was carried out, whereby genetically uniform phage populations could be isolated which were used for in vivo excision. Restriction analysis showed no differences between the cDNA clones and four clones with the largest insertions were selected for sequencing.
• the sequence data show that cDNA clones are coding for the 5-phosphoribosyl-1-pyrophosphate synthetase.
• the cDNA clone NtPrsI - see SEQ-ID No. 1 - is 1251 bp long and has a start codon at position 21 and a termination codon TAG at position 1089.
• the open reading frame codes for a 356 amino acid protein with a molecular weight of 38.3 KDa.
• the 152 bp 3'-untranslated region contains no polyadenylation signal.
• the binary vector pBinAR (Höfgen and Willmitzer, Plant Science 66 (1990), 221-230) was cleaved in the polylinker with BamHI.
• the orientation of the ligated NtPrsI cDNA fragment was checked by restriction with HindIII.
• the resulting Hindlll fragment of 300 bp confirmed the antisense orientation.
• the plasmid pBinAR-NtPrsI antisense ( Figure 2) consists of 3 fragments A, B and C.
• fragment A contains the 35S promoter of the Cauliflower Mosaic Virus (CaMV), which is a constitutive expression in transgenic Plants. This fragment comprises nucleotides 6909 to 7437 of the CaMV (Franck, Cell 21 (1980), 285).
• CaMV Cauliflower Mosaic Virus
• Fragment B contains a 1226 bp NtPrsI cDNA fragment which was isolated as a BamHI fragment from the plasmid pBluescriptSK- ⁇ / fPrs7 and was cloned into the BamHI site of the polylinker of the vector pBinAR.
• Fragment C contains the polyadenylation signal of gene 3 (octopine synthase, OCS) of the T-DNA of the Ti plasmid pTiACH ⁇ (Gielen et al., EMBO J. 3 (1984), 835), which comprises nucleotides 11749-11939.
• the individual fragments were provided with EcoRI / Notl linkers during the generation of the cDNA bank and then cloned into the EcoRI restriction sites of the polylinker of pBluescript. Accordingly, the Notl interfaces are not Original part of the pBluescript polylinker.
• Figure 2 shows the expression cassette for the expression of the NtPrsI cDNA (1226 bp) in antisense orientation in transgenic tobacco plants
• A 35S promoter of the Cauliflower Mosaic Virus (CaMV), 529 bp nucleotides 6909 to 7437 of the CaMV.
• CaMV Cauliflower Mosaic Virus
• B NtPrsI cDNA (1226 bp) in antisense orientation, which was isolated as a BamHI fragment from the plasmid pBluescript SK-NtPrs1 and comprises the bases 1-1208 of the coding region of NtPrsI.
• C Polyadenylation signal of gene 3 (octopine synthase, OCS) of the T-DNA of the Ti plasmid pTiACH ⁇ (192 bp).
• Tobacco plants (Nicotiana tabacum cv.Samsun NN) were used 10 ml of an overnight culture of a positively transformed agrobacterial colony in YEB medium (Vervliet et al., Gen. Virol. 26 (1975), 33).
• Leaf disks of sterile plants (each about 1 cm 2 ) were incubated in a Petri dish with this solution for 5-10 min. There was a two day incubation in Darkness at 25 ° C on MS medium (Murashige-Skoog medium,
• Transgenic plants that are transformed with the construct pBinAR-NtPrsI Antisense are characterized by large chlorotic areas in the leaves and by a strong fading of the leaves compared to untransformed wild-type plants.
• transgenic pBinAR-NtPrsl antisense plants are those that are severely impaired in their growth.
• RNA analysis was carried out by strand-specific labeling of an NtPrsI cDNA fragment.
• the plasmid pBluescript SK-NtPrs1 was digested with EcoRI and a 1251 bp fragment, which has the coding region of the NtPrsI gene, was isolated.
• a 3 'oligonucleotide with the following sequence was used for the reaction: 5' CTT CAA GTT CCA GAC AAC AGT GTC-3 '.
• a kit from the company came for the reaction
• the reaction mixture contained approx. 10 ng fragment DNA, 0.1 mM of the oligonucleotide, 5 ml of 10 ⁇ buffer, 1.5 mM MgCl 2 , 5 mM deoxy nucleotides (dGTP, dATP, dTTP), 50 mCi a- 32 P-dCTP (Amersham ) and 1 ml DyNazyme polymerase.
• the amplification conditions were chosen as follows:
• Denaturation temperature 95 ° C, 5 sec annealing temperature: 45 ° C, 30 sec elongation temperature: 72 ° C, 1 min number of cycles: 40
• RNA was isolated from about 0.5-1.0 cm large sink leaves, 20 Dg RNA were separated in a formaldehyde-containing agarose gel and transferred to a nylon membrane by capillary blotting. This was hybridized with the strand-specifically labeled NtPrsI gene probe.
• Figure 3 shows the hybridization signals for wild-type (WT) and transgenic plants. The hybridization signals were then quantified using a phosphoimager. According to the phenotype of the plants, the transcript accumulation of the ⁇ / fPrs7 synthetase mRNA in line 45-1 is dramatically reduced. Plants 33.4, 14.5 and 30.4 only show a reduced mRNA accumulation and, analogously, a less pronounced growth phenotype.
• the strain 16/14 used was by H.L.K. Whitehouse collected in Gransden Wood, Huntingdonshire (England) and subcultured by Engel spores (Am. J. Bot. 55 (1968), 438-446).
• the moss plants were proliferated via spores and regeneration of gametophores.
• the protonema tissue developing from the haploid spore consists of chloroplast-rich chloronema and chloroplast-poor caulonema, on which buds form after approx. 12 days. These buds grow into gametophores and contain antheridia and archegonia. After fertilization, the diploid sporophyte, short Seta and spore capsule result, in which the maturing spores develop.
• RNAse H digest 12D C (2h), 16D C (1 h) and 22D C (1 h). The reaction was stopped by incubation at 65DC (10 min) and transferred to ice.
• Double-stranded DNA was filled in using T4-DNA polymerase (Röche, Mannheim) at 37DC (30 min) and nucleotides were separated off by phenol / chloroform extraction and Sephadex G50 chromatography. EcoRI adapters (Pharmacia, Freiburg, Germany) were ligated to the cDNA ends using T4-DNA ligase (Röche, 12D C, 16h) and phosphorylated using polynucleotide kinase (Röche, 37DC, 30 min). The DNA was separated by means of gel electrophoresis and those of more than 300 base pairs were isolated by means of phenol extraction and concentrated by means of Elutip-D columns (Schleicher and Schuell, Dassel, Germany).
• the double-stranded DNA obtained was ligated in lambda ZAPII and proceeded using the Gigapack Gold Kit (Stratagene, Amsterdam, Netherlands) according to the manufacturer's instructions. Plasmid DNA was obtained by in vivo excision and was used for the transformation of E. coli XLI blue bacteria. Clones were grown in liquid culture from individual colonies, plasmid DNA was isolated and these were used for sequence reactions. An EST coding for 5-phosphoribosyl-1-pyrophosphate synthesis could be identified by homology comparison, see SEQ-ID No. 5th
• the fragment of Prs1 from Arabidopsis thaliana is obtained from the vector pGEM-T AtPrsI ( Figure 2) with the restriction enzymes BamHI and EcoRI (interfaces inserted via primers RBPRPP3 and RBPRPP4) and cut identically Transfer vector pFastBacHTb (GibcoBRL) cloned.
• the construct pFASTBAC Tb-AtPrs1 obtained is used according to the manufacturer's instructions (GibcoBRL) to generate recombinant baculovirus.
• this virus is used to infect Sf21 insect cells in order to generate active 5-phosphoribosyl-1-pyrophosphate synthetase 1 enzyme.
• the specific enzymatic phosphoribosyl pyrophosphate synthesis 1 activity is measured photometrically after ultrasound digestion of the cells.
• the following components are used for each 90DI approach:
• cordycepin-5'-triphosphate an inhibitor of 5-phosphoribosyl-1-pyrophosphate synthetase from other higher eukaryotes - also inhibits plant-based 5-phosphoribosyl-1-pyrophosphate synthetase 1 in its enzymatic activity.

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