EP1220894A1

EP1220894A1 - Prpp-amidotransferase from plants

Info

Publication number: EP1220894A1
Application number: EP00969449A
Authority: EP
Inventors: Jens Lerchl; Thomas Ehrhardt; Uwe Sonnewald; Ralf Boldt
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 1999-10-11
Filing date: 2000-10-07
Publication date: 2002-07-10
Also published as: WO2001027248A1; DE19949000A1; AU7915900A; WO2001027248A8; CA2387159A1

Abstract

The invention relates to DNA sequences coding for a polypeptide sequence with PRPP-amidotransferase (EC 2.4.2.14) activity. The invention relates furthermore to the use of said nucleic acids for production of a test system.

Description

PRPP amidotranaferase from plants

description

The present invention relates to the identification of plant PRPP amidotransferase (phosphoribosyl pyrophosphate A idotransferase, E.C. 2.4.2.14) as a new target for herbicidal active compounds. The present invention further relates to DNA sequences coding for a polypeptide with PRPP amidotransferase activity. In addition, the invention relates to the use of a nucleic acid coding for a protein with PRPP amidotransferase activity of plant origin for the production of a test system for identifying inhibitors of PRPP amidotransferase with herbicidal activity and inhibitors of plant PRPP amidotransferase identified using this test system. The invention further relates to the use of the nucleic acid SEQ-ID No. 1 or SEQ-ID No. 3 coding for plant PRPP amidotransferase for the production of plants with increased resistance to inhibitors of PRPP amidotransferase, and for the production of plants with a modified purine nucleotide content. In addition, the invention relates to a method for eliminating undesirable plant growth, the plants to be removed being treated with a compound which is specifically PRPP amidotransferase, coded by a DNA sequence SEQ-ID No. 1 or a DNA sequence hybridizing with this DNA sequence, binds and inhibits their function.

Plants are able to synthesize their cell components from carbon dioxide, water and inorganic salts.

This process is only possible by using biochemical reactions to build up organic substances. 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, which are involved in many cellular reactions. Coupled hydrolysis of guanosine 5 ^x triphosphate (GTP) defines vesicular for various cellular processes such as protein translation, microtubule assembly Transport, signal transduction and cell division in one direction of reaction. Nucleotides are also the starting metabolites for the biosynthesis of methylxanthines such as caffeine and theobromine in plant families of the Rubiaceae and Theaceae.

Genes encoding PRPP amidotransferase have been isolated from various organisms.

CDNAs coding for PRPP amidotransferase enzymes could be isolated and characterized from various bacterial, animal and plant organisms. Plant PRPP amidotransferase cDNAs were isolated from Glycine max, Vigna aconitifolia and from Arabidσpsis thaliana by complementation of E. coli purF mutants and by DNA hybridization techniques (Ito et al., Plant Molecular Biology 26 (1994), 529-533; Kim et al., The Plant Journal 7 (1995), 77-86). Sequence homologies indicate that the encoded enzymes, like PRPP amidotransferase from E. coli, contain 4Fe-4S clusters. The PRPP amidotransferase amino acid sequences from plants, which are elongated in comparison to E. coli at the N terminal, are similar to plastid signal sequences.

There are several PRPP amidotransferase isoenzymes in plants that are differentially expressed. For example, the RNA for AtATasel from Arabidopsis thaliana preferentially accumulates in the roots, while the AtATase2 transcripts are found more strongly in young leaves and flowers (Ito et al., Plant Molecular Biology 26 (1994), 529-533). In Vigna aconi tifolia, a PRPP amidotransferase RNA accumulates mainly in root nodules and is induced in root tissues by L-glutamine (Kim et al., The Plant Journal 7 (1995), 77-86).

Since plants rely on an effective nucleotide metabolism, it can be assumed that the enzymes involved are suitable targets for herbicides. Active substances that inhibit plant de novo purine biosynthesis have already been described. An example is the natural product hydanthocidin, which inhibits adenylosuccinate synthetase (ASA) after phosphorylation in planta (Siehl et al., Plant Physiol. 110 (1996), 753-758).

Inhibitors for enzymes in purine biosynthesis are also known for their pharmacological activity in animals and microorganisms: Folate analogs inhibit, among other things, the enzyme GAR transformylase and have an antiproliferative, anti-inflammatory and immunosuppressive effect. Mycophenolic acid (MPA) acts as an inhibitor of IMP dehydrogenase in the GMP synthesis pathway, antimicrobial, antivi- ral and immunosuppressive (Kitchin et al., Journal of the American Acade y of Dermatology 37 (1997), 445-449).

Bacterial PRPP amidotransferase can be inhibited, for example, by glutamine antagonists such as azaserine, 6-diazo-5-oxo-L-norleucine (DON) or L-2-amino-4-oxo-5-chloropentanoic acid as well as by mercaptopurine and thioguanosine. Glutamine antagonists are not specific for PRPP amidotransferase and also act on other enzymes of purine biosynthesis, e.g. the formylglycine amide ribotide synthase. A proof of the effectiveness of glutamine antagonists on plant PRPP amidotransferase is still pending.

The object of the present invention was to demonstrate that PRPP amidotransferase is a suitable herbicidal target in plants, the isolation of a complete plant cDNA coding for the enzyme PRPP amidotransferase and its functional expression in bacterial or eukaryotic cells, and the production of an efficient and simple PRPP amidotransferase test system for performing inhibitor-enzyme binding studies.

The object was achieved by isolating genes which code for the plant enzyme PRPP amidotransferase, the production of antisense constructs of PRPP amidotransferase, and the functional expression of PRPP amidotransferase in bacterial or eukaryotic cells.

One object of the present invention relates to the isolation of full-length cDNAs coding for functional PRPP amidotransferase (E.C.2.4.2.14) from tobacco (Nicotiana tabacum).

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 PRPP amidotransferase from tobacco, see Example 1.

The invention further relates to DNA sequences which are derived 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 PRPP amidotransferase.

Nicotiana tabacum cv. Samsun NN, which carry an antisense construct of PRPP amidotransferase, were characterized in more detail. The plants show in different ways Measurements of growth retardation and fading of the leaves. The transgenic lines as well as the descendants of the 1st and 2nd generation showed a reduced growth in soil. In plants with reduced growth, a reduced amount of PRPP amidotransferase RNA compared to the wild type could be detected in the Northern hybridization. Furthermore, by measuring the enzyme activity, a reduced amount of PRPP amidotransferase activity in the transgenic lines could be detected in comparison with wild type plants, see Example 7. A correlation between growth retardation and

Determine reduction in PRPP amidotransferase activity. This clear connection clearly shows PRPP amidotransferase for the first time as a suitable target protein for herbicidal active ingredients.

In order to be able to find efficient inhibitors of plant PRPP amidotransferase, it is necessary to provide suitable test systems with which inhibitor-enzyme binding studies can be carried out. For this purpose, for example, the complete cDNA sequence of the PRPP amidotransferase from tobacco is cloned into an expression vector (pQE, Qiagen) and overexpressed in E. coli, see Example 2.

Alternatively, however, the expression cassette containing a DNA sequence SEQ-ID No. 1 or SEQ ID NO. 3 are expressed, for example, in other bacteria, in yeasts, fungi, algae, plant cells, insect cells or mammalian cells, see Example 4.

The PRPP amidotransferase protein expressed with the aid of the expression cassette according to the invention is particularly suitable for the detection of inhibitors specific for PRPP amidotransferase.

For this purpose, the plant PRPP amidotransferase can be used, for example, in an enzyme test in which the activity of the PRPP amidotransferase is determined in the presence and absence of the active substance to be tested. A comparison of the two activity determinations can be used to make a qualitative and quantitative statement about the inhibitory behavior of the active substance to be tested, see Example 3.

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 allows reproducible from a large number of

Select substances with great potency, in order to subsequently carry out further in-depth tests familiar to the person skilled in the art with these substances.

The invention further relates to a method for identifying substances having a herbicidal action which inhibit PRPP amidotransferase activity in plants, consisting of

a) the production of transgenic plants, plant tissues or plant cells which contain an additional DNA sequence coding for an enzyme with PRPP amidotransferase activity and are able to overexpress an enzymatically active PRPP amidotransferase;

b) applying a substance to transgenic plants,

Plant cells, plant tissue or parts of plants and on non-transformed plants, plant cells, plant tissue or parts of plants;

c) determining the growth or survivability of the transgenic and non-transformed plants, plant cells, plant tissue or plant parts after the application of the chemical substance; and

d) comparing the growth or survivability of the transgenic and non-transformed plants, plant cells, plant tissue or plant parts after the application of the chemical substance;

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 inhibits PRPP amidotransferase enzyme activity in plants.

Another object of the invention is a method for the identification of inhibitors of plant PRPP amidotransferases, with potential herbicidal activity by cloning the gene of a plant PRPP amidotransferase, overexpression in a suitable expression cassette - for example in insect cells - which opens and opens the cells Cell extract is used directly or after enrichment or isolation of the enzyme PRPP amidotransferase 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 herbicidal activity which can be identified using the test system described above.

Another object of the invention is a method for eliminating undesirable plant growth, wherein the plants to be removed are treated with a compound which specifically binds to plant PRPP amidotransferase and inhibits their function.

PRPP amidotransferase inhibitors with herbicidal activity 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.

PRPP amidotransferase inhibitors with herbicidal activity can be used, for example, against the following weeds:

Dicot weeds of the genera:

Sinapis, Lepidium, Galium, Stellaria, Matricaria, Anthemis, Galinsoga, Chenopodium, Urtica, Senecio, Amaranthus, Portulaca, Xanthium, Convolvulus, Ipomoea, Polygonum, Sesbania, Ambrosia, Cirsium, Carduus, Sonchus, Solanum, Rorippernia, Rorippa, Rorippa, Rorippa Lamium, Veronica, Abutilon, Emex, Datura, Viola, Galeopsis, Papaver, Centaurea, Trifolium, Ranunculus, Taraxacum.

Monocot weeds of the genera:

Echinochloa, Setaria, Panicum, Digitaria, Phleum, Poa, Festuca, Eleusine, Brachiaria, Lolium, Bromus, Avena, Cyperus, Sorghum, Agropyron, Cynodon, Monochoria, Fimbristylis, Sagittaria, Eleo-charis, Scirpus, Paspalum, Sphenumum, Ischaemum Dactyloctenium, Agrostis, Alopecurus, Apera.

The invention also relates to expression cassettes, the sequence of which codes for a PRPP amidotransferase from tobacco or its functional equivalent. The nucleic acid sequence can e.g. be a DNA or a cDNA sequence.

The expression cassettes according to the invention also contain regulatory nucleic acid sequences which control the expression of the coding sequence in the host cell. According to a preferred embodiment, an expression cassette according to the invention comprises upstream, ie at the 5 'end of the coding sequence, a promoter and downstream, ie at the 3 'end, a polyadenylation signal and, if appropriate, further regulatory elements which are operatively linked to the intermediate coding sequence for the PRPP amidotransferase gene. An operational link is the sequential one

Arrangement of promoter, coding sequence, terminator and possibly other regulatory elements in such a way that each of the regulatory elements can perform its function as intended in the expression of the coding sequence.

An expression cassette according to the invention is produced by fusing a suitable promoter with a suitable PRPP amidotransferase 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) and in T.J. Silhavy, M.L. Berman 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).

The invention also relates to functionally equivalent DNA sequences which code for a PRPP amidotransferase gene and which, based on the total length of the DNA sequence, have sequence homology with the DNA sequence SEQ-ID NO. 1 or SEQ ID NO. 3 have from 40 to 100%.

Preferred objects of the invention are functionally equivalent DNA sequences which code for a PRPP amidotransferase gene and which, based on the total length of the DNA sequence, have sequence homology with the DNA sequence SEQ-ID NO. 1 or SEQ ID NO. 3 have from 60 to 100%.

A particularly preferred object of the invention are functionally equivalent DNA sequences which code for a PRPP amidotransferase gene and which, based on the total length of the DNA sequence, have sequence homology with the DNA sequence SEQ-ID NO. 1 or SEQ-ID No. 3 have from 80 to 100%.

Functionally equivalent sequences which code for a PRPP amidotransferase 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 ones, for example by chemical synthesis Nucleotide sequences obtained which are 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 PRPP amidotransferase, which furthermore shows the desired function. Mutations include substitutions, additions, deletions, exchanges or insertions of one or more nucleotide residues. Thus, for example, 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 to transform bacteria, cyanobacteria, yeasts, filamentous fungi and algae with the aim of producing sufficient quantities of the enzyme PRPP amidotransferase.

Another object of the invention is a protein from tobacco characterized by the amino acid sequence SEQ-ID NO: 2 or SEQ-ID No. 4 or derivatives or parts of this protein with PRPP amidotransferase activity.

The invention also relates to plant proteins with PRPP amidotransferase activity with an amino acid sequence homology to the tobacco PRPP amidotransferase with SEQ-ID NO: 2 or SEQ-ID NO. 4 of 20 - 100% identity.

Vegetable proteins with PRPP amidotransferase activity with an amino acid sequence homology to the tobacco PRPP amidotransferases with the sequences SEQ-ID NO: 2 or SEQ-ID NO are preferred. 4 out of 50 - 100% identity.

Plant proteins with PRPP amidotransferase activity with an amino acid sequence homology to the tobacco PRPP amidotransferases with the sequences SEQ-ID NO: 2 or SEQ-ID NO are particularly preferred. 4 of 80 - 100% identity. Another object of the invention was the overexpression of the PRPP amidotransferase gene in plants for the production of plants which are tolerant of inhibitors of PRPP amidotransferase.

By overexpressing the gene sequence coding for a PRPP amidotransferase SEQ-ID NO. 1 or SEQ ID NO. 3 In a plant, increased resistance to inhibitors of PRPP amidotransferase is achieved. The transgenic plants produced in this way are also the subject of the invention.

The effectiveness of the expression of the transgenically expressed PRPP amidotransferase gene can be determined, for example, in vitro by proliferation or by a germination test. In addition, a change in the type and level of expression of the PRPP amidotransferase gene and its effect on the resistance to inhibitors of PRPP amidotransferase on test plants can be tested in greenhouse experiments.

The invention also relates to transgenic plants, transformed with an expression cassette according to the invention, containing the DNA sequence 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 PRPP amidotransferase, 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, 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 ,

A change in the nucleotide content in plants can be useful in various cases. Plant-based baby food products are added with nucleotides, for example, in order to achieve a nutritional composition that corresponds to breast milk. Furthermore, an optimized nucleotide content would be useful in the case of enteral feeding of patients. A reduced purine nucleotide content in nutritionally relevant plants is relevant for the dietary nutrition of patients with gout. Nucleotides also have a taste-forming and taste-enhancing effect, so that a changed nucleotide content affects the taste properties of plants.

The invention therefore furthermore relates to plants which, after expression of the DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 have a modified purine nucleotide content in the plant. The content of the purine nucleotides IMP, AMP is preferred and / or GMP or their di- or trinucleotides ADP, ATP or GDP, GTP increased.

A plant with a modified purine nucleotide content is, for example, expressed by expressing an additional DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 produced in the plant in sense or antisense orientation. Modified content of purine nucleotides means that both plants with an increased content of purine nucleotides in sense orientation and also plants with a reduced content in guanosine nucleotides with sense orientation (cosuppression) or antisense orientation can be produced.

In the context of the present invention, increasing the purine nucleotide content means, for example, the artificially acquired one

Ability of an increased biosynthesis for purine nucleotides by functional overexpression of the PRPP amidotransferase gene in the plant compared to the non-genetically modified plant for at least one plant generation.

Another object of the invention is the use of plant PRPP amidotransferase to change the concentrations of methylxanthines in plants.

Sequences are particularly preferred which ensure targeting in the apoplasts, in plastids, the vacuole, the mitochondrium, the endoplasmic reticulum (ER) or, due to the lack of corresponding operative sequences, ensuring that they remain in the compartment of formation, the cytosol (Kermode, Crit. Rev. Plant Sci. 15, 4 (1996), 285-423).

For example, the plant expression cassette can be installed in the plant transformation vector pBinAR, see Example 5.

In principle, any promoter which can control the expression of foreign genes in plants is suitable as promoters of the expression cassette according to the invention. In particular, a plant promoter or a plant virus-derived promoter is preferably used. The is particularly preferred

CaMV 35S promoter from the cauliflower mosaic virus (Franck et al., Cell 21 (1980), 285-294). This promoter contains different recognition sequences for transcriptional effectors, all of which 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 PRPP amidotransferase gene in the plant can be controlled at a specific point in time. Such promoters as for example the PRPl promoter (Ward et al., Plant Mol Biol. (1993) 22, 361-366), a promoter induced by salicylic acid (WO 95/19443), a promoter induced by benzenesulfonamide (EP 388186 ), one that can be induced by tetracycline (Gatz et al., Plant J. (1992) 2, 397-404), one that can be induced by abscisic acid (EP0335528) or one that can be induced by ethanol or cyclohexanone (WO 93 / 21334) Promoters are described in the literature and can be used inter alia.

Furthermore, 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 potato or the ST-LSI promoter from potato (Stockhaus et al., EMBO J., (1989) 8, 2445-245) are to be mentioned.

With the help of a seed-specific promoter, 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 (1995) 10, 1090-1094). 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.

The inserted nucleotide sequence coding for a PRPP amidotransferase can be produced synthetically or obtained naturally or contain a mixture of synthetic and natural DNA components. In general, 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. When preparing an expression cassette, 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. To connect the DNA fragments to one another, adapters or linkers can be attached to the fragments. In addition, artificial DNA sequences are suitable as long as, as described above, for example, they impart the desired property of increasing the level of purine nucleotides in the plant by overexpressing the PRPP amidotransferase gene in crop plants. Such artificial DNA sequences can be determined, for example, by back-translating proteins constructed using molecular modeling, which have PRPP amidotransferase activity, or by using viüro 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. 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.

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 PRPP amidotransferase 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 PRPP amidotransferase expression (e.g. myc-tag or his-tag). However, this is preferably a regulatory protein sequence, such as e.g. a signal or transit peptide that directs the PRPP amidotransferase protein to the desired site of action.

The promoter and terminator regions according to the invention 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. As a rule, the linker has 1 to 10, usually 1 to 8, preferably 2 to 6, restriction sites. In general, 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. Manipulations which provide suitable restriction sites or which remove superfluous DNA or restriction sites can also be used. Where insertions, deletions or substitutions such as transitions and trans versions are possible, viüro mutagenesis, "primer pair", restriction or ligation can be used. With suitable manipulations, such as restriction, "chewing-back" or filling in overhangs for "blunt ends", complementary ends of the fragments can be made available for the ligation.

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.

To transform a host plant with a DNA coding for a PRPP amidotransferase, an expression cassette according to the invention is inserted as an insert into a recombinant vector whose vector DNA contains additional functional regulatory signals, for example sequences for replication or integration. Suitable vectors are described, inter alia, in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press, Chapter 6/7, 71-119).

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, Engineering and Utilization, edited by SD Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Molec. Biol. 42: 205-225 (1991). The construct to be expressed is preferably 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 according to the invention 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 , 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 suspension and then cultivating them in suitable media.

The biosythesis site of purines is generally the leaf tissue, so that leaf-specific expression of the PRPP amidotransferase gene makes sense. However, it is obvious that the purine biosynthesis need not be limited to the leaf tissue, but can also be tissue-specific in all other parts of the plant - for example in fatty seeds.

In addition, constitutive expression of the exogenous PRPP amidotransferase gene is advantageous. On the other hand, inducible expression may also appear desirable.

Using the recombination and cloning techniques cited above, 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, M13mp series and pA-CYC184. 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 the transformation of plants, plant cells, plant tissues or parts of plants. The aim of the use is preferably to increase the PRPP amidotransferase content in the plant.

Depending on the choice of the promoter, 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 object of the present invention.

The invention is illustrated by the following examples, but is not limited to these: Examples

Genetic engineering processes on which the exemplary embodiments are based:

General cloning procedures

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 cells, 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).

Sequence analysis of recombinant DNA

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 to avoid polymerase errors in constructs to be expressed.

Analysis of total RNA from plant tissues:

Total RNA from plant tissues was, as in Logemann et al. (Anal. Biochem. 163 (1987), 21). For the analysis, 20 μg 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 marked with a Random Pri ed 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.

Unless otherwise stated, the chemicals used were purchased in pa quality from Fluka (Neu-Ulm), Merck (Darmstadt), Roth (Karlsruhe), Serva (Heidelberg) and Sig a (Deisenhofen). Solutions were prepared with treated, pyrogen-free water, hereinafter referred to as H0, from a Milli-Q Water System water treatment plant (Millipore, Eschborn). Restriction endonucleases, 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.

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). Alternatively, the agrobacterial strain LBA4404 (Clontech) or other suitable strains can be used. For cloning, the vectors pUC19 (anish-Perron, Gene 33 (1985), 103-119) pBluescript SK- (Stratagene), pGEM-T (Promega), pZerO (Invitrogen), pBinl9 (Bevan et al., Nucl. Acids Res. 12 (1984), 8711-8720) and pBinAR (Höfgen and Willmitzer, Plant Science 66 (1990), 221-230).

example 1

Isolation of cDNAs coding for a functional PRPP amidotransferase from tobacco.

To isolate cDNAs from Nicotiana tabacum coding for PRPP amidotransferase, a cDNA clone from Arabidopsis coding for PRPP amidotransferase (AtATasel; Ito et al., Plant Molecular Biology 26 (1994), 529-533; GenBank Accession nu via D28868) used as a template for generating a hybridization probe by means of PCR.

The reaction mixtures contained approx. 1 ng / μl template DNA, 0.5 UM of oligonucleotides 5 ⁽ -cgc tct aga act agt gga tc-3 'and 5 * -tcg agg tcg acg gta tc-3', 200 μM deoxy nucleotides (Pharmacia), 50 mM KC1, 10 mM Tris-HCl (pH 8.3 at 25 ° C, 1.5 mM MgCl ₂ ) and 0.02 U / ul Taq polymerase (Perkin Elmer). The amplification conditions were set as follows:

Annealing temperature: 50 ° C, 1 min

Denaturation temperature: 94 ° C, 1 min. Elongation temperature: 72 ° C, 2 min

Number of cycles: 30

The resulting fragment of 1.9 kb was used for a heterologous screening of a cDNA library from Nicotiana tabacum var. SR-1 (Stratagene). 3.0 x 10 ⁵ lambda phages from the cDNA library 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. The PCR fragment described above was used as the hybridization probe and was radioactively labeled with the aid of the “Multiprime DNA labeling System” (Amersham Buchler) in the presence of α- 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 approx. 12 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 and isolated using standard techniques (Sambrook et al. (1989); Cold Spring Harbor Laboratory Press: ISBN 0-87969-309-6) and converted into plasmids (strategists).

After restriction and sequence analysis, two distinguishable clones Ntpurl.l (clone 7.2) containing the DNA sequence SEQ-ID No. 1 and Ntpurl .2 (clone 9.2) containing the DNA sequence SEQ-ID No. 3 are identified which reading frames code with homology to AtATAsel from Arabidopsis thaliana. The amino acid sequences of Ntpurl.l (SEQ-ID No. 2 - length: 573 amino acids) and Ntpurl.2 (SEQ-ID No. 4 - length: 573 amino acids) are 97% identical, see Table 1. The homology at the amino acid level to AtATasel is 81% for Ntpurl.l and 85% for Ntpurl.2. The continuous reading frames begin with nucleotide base 49 (Ntpurl.l) or 25 (Ntpurl.2) and are translated into polypeptides with a length of 573 amino acids. Table 1

Amino acid comparison Ntpurl .1 x Ntpurl .2:

1 MAATVSTASAAATNKSPLSQPLDKPFCSPSQKLLSLSPKTLPKPYRTLVT 50 MIMIMMM IMIIMIMI

1 MAATVSTASAAATNKYPLSQPLDKPFCSLSQKLLSLSPKTHPKPYRTLIT ASSKNPLNDWSFKKSADNTLDSYFDDEDKPREECGWGIYGDSEASRLC 50 51 100 51 100 101 ASSKNPLNDVISFKKSADNTLDSYFDDDDKPREECGWGIYGDSEASRLC YLALHALLHRGQEGAGIVAVNDDVLKSITGVGLVSDVFNESKLDQLPGDM 150

MIM

101 YLALHALQHRGQEGAGIVAVNDDVLKSITGVGLVSDVFNESKLDQLPGDM 150 151 AIGHVYSTAGSSMLKNVQPFVANYKFGSVGVAHNGNLVNYKLLRGELEE 200

MIM MMMI MIMMMMMIMMMIMMMIMI MM 151 AIGHVRYSTAGSSMLKNVQPFVASYKFGSVGVAHNGNLVNYKLLRSELEE 200

201 NGSIFNTSSDTΞWLHLIAISKARPFLLRIVEACEKIEGAYSMVFVTEDK 250

MMMMMMMMMMMMMMMMMMMMMMMIMI

201 NGSIFNTSSDTEWLHLIAISKARPFLLRIVEACEKIEGAYSMVFVTEDK 250 251 LVAVRDPHGFRPLVMGRRSNGAWFASETCALDLIEATYEREVNPGE W 300

MMMMMMMMMMMMMMMMMMMMMMMMM

251 LVAVRDPHGFRPLVMGRRSNGAWFASETCALDLIEATYEREVNPGEVW 300 301 VDKDGVHSIYLMPHPEHKSCIFEHIYFALPNSWFGRSVYESRRAFGEIL 350

MMM I! MMM II M I M M M I M M M M M M M M M M I

301 VDKDGVQSICLMPHPERKSCIFEHIYFALPNSWFGRSVYESRRAFGEIL 350 351 ATEAPVECDVGIAVPDSGIVAALGYAAKAGVPFQQGLIRSHYVGRTFIEP 400

IMMIIMI MMIMMMMMMMMMMMIMMMIMM

351 ATEAPVECDWIAVPDSGWAALGYAAKAGVPFQQGLIRSHYVGRTFIEP 400 401 SQKIRDFGVKLKLSPVRALLEGKRVVVVDDSIVRGTTSSKIVRLLKEAGA 450

MMMMMMMMM-MMMMMMMMMMMMMIMM

401 SQKIRDFGVKLKLSPVRAVLEGKRVWVDDSIVRGTTSSKIVRLLKEAGA 450 451 KEVHMRIASPPIIASCYYGVDTPSSDELIS RMSVEEIKEFIGSDSLAFL 500

MMMMMMMMMMMMMMMMMMMMMMMIMI

451 KEVHMRIASPPIIASCYYGVDTPSSDELIS RMSVEEIKEFIGSDSLAFL 500 501 PMDSLNKLLGNDSKSFCYACFSGYPVEPTGKVKRIGDFMDDGLSGDMDS 550

1111111111 i 1111111 II 1111111! 111111111111111 M 11111

501 PMDSLNKLLGNDSKSFCYACFSGNYPVEPTGKVKRIGDFMDDGLSGDMDS 550 551 IDGGLPGSSRVQKTILNEVRTG 573

11111111! 1111111111111 551 IDGGWLPGSSRVQKTILNEVRTS 573

The vegetable proteins (Ntpurl.l, Ntpurl.2, AtATasel) show sequences of bacteria and PRPP amidotransferase

Human has an elongated N-terminus with a large proportion of basic amino acids (Table 2), which indicates the function of a

Indicates transit peptides for plastid import (von Heijne et al., Eur. J. Biochem. 180 (1989), 535-545). Table 2

Sequence comparison of PRPP amidotransferase proteins from Arabidopsis thaliana (AtATasel), Bacillus subtilis (BacSu__purF) human (purl_hum) and Nicotiana tabaccum (Ntpurl.l, Ntpurl.2)

1 50

AtATasel SLN QTILLTPINL SLSSPNPSLN

BacSu_purF

Ntpurl LAPHLLFLLS SFFPPPMAAT VSTASAAATN KSPLSQPLDK PFCSPSQKL.

Ntpurl-2 LS SFFPPP AAT VSTASAAATN KYPLSQPLDK PFCSLSQKL. purl_hum

51 100

AtATasel LHISLS.FLL PSPLLLLHSS MESPPTSPLL HHPKNNSHAP FDYHNDEDDE

BacSu_purF MLAEIK

Ntpurl ..LSLSP TL PKPYRTLVTA SSKPLNDW SFKKSADNTL DSYFDDED .. Ntpurl-2 ..LSLSPKTH PKPYRTLITA SSKNPL DVI SFKKSADNTL DSYFDDDD .. purl_hum MELEEL

101 150

AtATasel KPREECGWG IYGDPE. .ASRLFYLA LHALQHRGQE GAGIVTVSPE

BacSu_purF GLNEECGVFG I GHEE. .APQITYYG LHSLQHRGQE GAGIVATDGE

Ntpurl KPREECGWG IYGDSE. .ASRLCYLA LHALLHRGQE GAGIVAVN.D

Ntpurl-2 KPREECGWG IYGDSE. .ASRLCYLA LHALQHRGQE GAGIVAVN.D purl_hum GIREECGVFG CIASGEWPTQ LDVPHVITLG LVGLQHRGQE SAGIVTSDGS 151 200

AtATasel KV..LQTITG VGLVSEVFNE SKLDQL. PGE FAIAHVRYST AGASMLKNVQ

BacSu_purF K ... LTAHKG QGLITEVFQN GELSKV.KGK GAIGHVRYAT AGGGGYENVQ

Ntpurl DV..LKSITG VGLVSDVFNE SKLDQL. PGD MAIGHV YST AGSSMLKNVQ

Ntpurl-2 DV..LKSITG VGLVSDVFNE SKLDQL. PGD MAIGHVRYST AGSSMLKNVQ purl_hum SVPTFKSHKG MGLVNHVFTE DNLKKLYVSN LGIGHTRYAT TGKCELENCQ

201 250

AtATasel PFV.AGYRFG SIGVAHNGNL VNYKTLRAML EENGSIFNTS SDTEWLHLI

BacSu_purF PLLFRSQNNG SLALAHNGNL VNATQLKQQL ENQGSIFQTS SDTEVLAHLI

Ntpurl PFV.ANYKFG SVGVAHNGNL VNYKLLRGEL EENGSIFNTS SDTEWLHLI

Ntpurl-2 PFV.ASYKFG SVGVAHNGNL VNYKLLRSEL EENGSIFNTS SDTEWLHLI purl_hum PFWETLH.G KIAVAHNGEL VNAARLRKKL LRHGIGLSTS SDSEMITQLL

251 300

AtATasel AISKAR. .PFFMRIID ACEKLQGAYS MVFVTEDKLV AVRDPYGFRP BacSu_purF KRSGHF. .TLKDQIKN SLSMLKGAYA FLIMTETEMI VALDPNGLRP

Ntpurl AISKAR. .PFLLRIVE ACEKIEGAYS MVFVTEDKLV AVRDPHGFRP

Ntpurl-2 AISKAR. .PFLLRIVΞ ACEKIEGAYS MVFVTEDKLV AVRDPHGFRP purl_hum AYTPPQEQDD TPDWVARIKN LMKEAPTAYS LLIMHRDVIY AVRDPYGNRP 301 350

AtATasel LVMGR R SNGAWFASE TCALDLIEAT YEREVYPGEV

BacSu_purF LSIGM M GD.AYWASE TCAFDWGAT YLREVEPGEM

Ntpurl LVMGR R SNGAWFASE TCALDLIEAT YEREVNPGEV

Ntpurl-2 LVMGR R SNGAWFASE TCALDLIEAT YEREVNPGEV purl_hum LCIGRLIPVS DINDKEKKTS ETEGWWSSE SCSFLSIGAR YYREVLPGEI 351 400 AtATasel LWDKDGVKS QCLMPKFEPK Q.. CIFEHI YFSLPNSIVF GRSVYESRHV

BacSu_purF LIINDEGMKS ERFSMNINRS I. .CSMEYI YFSRPDSNID GINVHSARKN

Ntpurl VWDKDGVHS IYLMPHPEHK S. .CIFEHI YFALPNSWF GRSVYESRRA

Ntpurl-2 WVDKDGVQS ICLMPHPERK S. .CIFEHI YFALPNSWF GRSVYESRRA purl_hum VEISRHNVQT LDIISRSEGN PVAFCIFEYV YFARPDSMFE DQMVYTVRYR 401 450

AtATasel FGEILATESP VECDWIAVP DSGWAALGY AAKSGVPFQQ GLIRSHYVGR

BacSu_purF LGKMLAQESA VEADWTGVP DSSISAAIGY AEATGIPYEL GLIKNRYVGR

Ntpurl FGEILATEAP VECDVGIAVP DSGIVAALGY AAKAGVPFQQ GLIRSHYVGR

Ntpurl-2 FGEILATEAP VECDWIAVP DSGWAALGY AAKAGVPFQQ GLIRSHYVGR purl_hum CGQQLAIEAP VDADLVSTVP ESATPAALAY AGKCGLPYVE VLCKNRYVGR

451 500

AtATasel TFIEPSQKIR DFGVKLKLSP VRGVLEGKRV VWDDSIVRG TTSSKIVRLL

BacSu_purF TFIQPSQALR EQGVRMKLSA VRGWEGKRV VMVDDSIVRG TTSRRIVTML

Ntpurl TFIEPSQKIR DFGVKLKLSP VRALLEGKRV VWDDSIVRG TTSSKIVRLL

Ntpurl-2 TFIEPSQKIR DFGVKLKLSP VRAVLEGKRV VWDDSIVRG TTSSKIVRLL purl_hum TFIQPNMRLR QLGVAKKFGV LSDNFKGKRI VLVDDSIVRG NTISPIIKLL

501 550

AtATasel REAGAKEVHM RIASPPIVAS CYYGVDTPSS EELISNRLSV EEINEFIGSD

BacSu_purF REAGATEVHV KISSPPIAHP CFYGIDTSTH EELIASSHSV GEIRQEIGAD

Ntpurl KEAGAKEVHM RIASPPIIAS CYYGVDTPSS DELISNRMSV EEIKEFIGSD Ntpurl-2 KEAGAKEVHM RIASPPIIAS CYYGVDTPSS DELISNRMSV EEIKEFIGSD purl_hum KESGAKEVHI CFMELEINPAN

551 600

AtATasel SLAFLSFDTL KKHL GK ... .DSK. SFCYA

BacSu_purF TLSFLSVEGL LKGI GRKYD. DSNCGQCLA

Ntpurl SLAFLPMDSL NKLL GN ... .DSK. SFCYA

Ntpurl-2 SLAFLPMDSL NKLL GN ... .DSK. SFCYA purl_hum SWYLSVEGL VSSVQEGIKF KKQKEKKHDI MIQENGNGLE CFEKSGHCTA

601 650

AtATasel CFTGDYPVKP TEVKVKRGGG DFIDDGLVGS FENIEAGVR

BacSu_purF CFTGKYPTEI YQDTVLPHVK EAVLTK Ntpurl CFSGNYPVEP TG.KVKR.IG DFMDDGLSGD MDSIDGGLP GSSRVQKTIL Ntpurl-2 CFSGNYPVEP TG.KVKR.IG DFMDDGLSGWGTLDKG

651

AtATasel

BacSu_purF

Ntpurl NEVRTG Ntpurl-2 NEVRTS purl_hum

Example 2

Expression of PRPP amidotransferase from tobacco in E. coli

With the aim of detecting the activity of the PRPP amidotransferase enzyme encoded by Ntpurl.2, Ntpurl.2 was expressed in E. coli. For this, a fragment of 1523 bp was amplified in a PCR with Pfu polymerase using the oligonucleotides Jle336: 5 '-ttttgctagcgactcgtattttgacg-3' and Jle337: 5 '-aaaaagatctcaggttctaacttcat -3 ^λ and Ntpurl .2-DNA as template. The generated DNA Fragment codes for an N-terminally shortened PRPP amidotransferase enzyme by 86 amino acids, which no longer contains the assumed transit peptide. This shortened form of the PRPP amidotransferase enzyme begins N-terminally with the amino acids MDSYFDDDD. An Nhel cleavage site and a BglII cut line were inserted by means of the oligonucleotides, via which the fragment generated was ligated into the expression vector pETlla (Novagen) cleaved with Nhel and BamHI.

For expression, the E. coli strain BL21 (DE3) LysS (Novagen) was transformed with the construct pETNtpurl.2 generated in this way. After overnight culture one day culture at OD OO _E = 0.1 was inoculated and induced after reaching an OD ₆ oo = 0.7 with mM IPTG. A whole cell extract was extracted using the pressure digestion method ("French Press") in 50 mM Tris-HCl, pH 7.4; 150mM NaCl generated. An overexpressed protein of approx. 65 kDa was cut out of the gel after SDS polyacrylamide gel electrophoresis. The protein was injected into rabbits to generate antisera (commissioned by Eurogentec, Herstal, Belgium).

Example 3

Test system for measuring the activity of plant PRPP amidotransferase activity

The method described above for measuring plant PRPP amidotransferase activity according to Reynolds et al. (Archieves of Biochemistry and Biophysics 229 (1984), 623-631) is not suitable for high-throughput testing due to the use of radioactive substrates. Based on the method described in Shid and Ishii (Journal of Biological Chemistry 66 (1969), 175-181) for PRPP amidotransferase from E. coli, an alternative test system was developed with which the plant PRPP amidotransferase activity in Protein extract is detected on the basis of the formation of the reaction product glutamate. The concentration of the resulting glutamate is measured by reaction with glutamate dehydrogenase (GluDH) and photometric monitoring of the formation of APADH at 363 nm.

PRPP + L-glutamine ^PRAT 1 »PRA + L-glutamate + PPi

GluDH _ L-glutamate + APAD + H ₂ 0 ► α-oxoglutarate + APADH + NH ₄ ⁺ (PRPP = phosphoribosyl pyrophosphate, PRA = phosphoribosylamine, APAD = 3-acetylpyridine adenine dinucleotide, PRAT = PRPP amidotransferase)

5 For this purpose, the reaction mixture (see below) was incubated for up to 60 minutes at 37 ° C and the reaction was stopped by incubating at 95 ° C for 5 minutes.

Reaction approach:

10

375 µL 100 mM Tris / HCl buffer pH 8.0

75 µL 100 mM MgCl ₂

75 µL 30 mM phosphoribosyl pyrophosphate

75 μL 100 mM L-glutamine 15 5500 μμLL H ₂ 0

100 _U - protein extract 750 μL

The detection of the glutamate formed was carried out in detection batch 20 (see below) by photometric measurement of the APADH increase at 363 nm after the addition of the glutamate dehydrogenase.

Verification approach:

25 375 µL 100 mM Tris / HCl buffer pH 8.0

75 µL 500 mM KC1

125 µL H ₂ 0

75 µL 3mM APAD

100 μL of the reaction mixture

30 750 μL

Start the detection reaction with 2 μl (approx. 4 units) glutamate dehydrogenase (Sigma).

35 The test system is particularly suitable for measuring the PRPP amidotransferase activity from plant material and in expression extracts, for example from baculovirus-infected insect cells.

40 Example 4

Functional expression of PRPP amidotransferase from tobacco in insect cells

45 The Bac-to-Bac expression system from GibcoBRL was used to express Ntpurl.l in baculovirus-infected insect cells. Ntpurl.l was used for a PCR. The Reak tion mixtures contained approx. 1 ng / μl Ntpurl.l DNA, 0.5 μM of the oligonucleotides 5 ^λ -tat agg atc cat gga ctc cta ttt tga cg-3 'and 5 ^v -atg aat tct agc tgg ttc taa ctt c-3 ', 200 μM deoxy nucleotides (Pharmacia), 0.04 U / μl Pfu polymerase (Stratagene) and was adjusted to buffer conditions according to the manufacturer.

The amplification conditions were set as follows:

Step 1:

Denaturation temperature: 95 ° C, 0.5 min annealing temperature: 40 ° C, 0.5 min elongation temperature: 72 ° C, 2 min number of cycles for Step 1: 2

Step 2:

Denaturation temperature: 95 ° C, 0.5 min annealing temperature: 50 ° C, 0.5 min elongation temperature: 72 ° C, 3 min

Number of cycles for Step 2: 25

The PCR product was ligated into the Vector pFast-Bacl (GibcoBRL) cut with StuI. The correct orientation of the insert was ensured by control digestion with Kpnl. The transfer vector pFastBacNtpurl.2 obtained was used according to the manufacturer's instructions to generate recombinant baculoviruses using Sf21 insect cells (Invitrogen). Sf21 insect cells were infected with the recombinant baculovirus (BvNtpurl.2). The cells were harvested by centrifugation after 2-4 days. A protein of approx. 54 kDal was identified in the total extract by SDS-polyacrylamide gel electrophoresis in accordance with the expected size of the PRPP amidotransferase. A whole cell extract was produced by the pressure digestion method ("French Press") in extraction buffer (100 mM HEPES pH 8.0; 2.5 mM EDTA; 10% glycerol; 20 mM DTE; 0.2 mM PEFA block) and after desalting on a PDIO column (Pharmacia) for measuring the PRPP amidotransferase activity in the described assay (see example 3).

Example 5

Generation of vectors for plant transformation

To generate binary vectors for the plant transformation, the clone Ntpurl.l was cleaved with Smal and EcoRV and a 1482 bp fragment was isolated, which was converted into the vector with Smal cleaved pBinAR (Höfgen and Willmitzer, Plant Science 66 (1990), 221-230). The antisense or sense constructs obtained in this way were designated pBinAR-NtpurlA or pBinAR-Ntpurl, see Figure 1.

Example 6

Generation of transgenic tobacco plants

The plasmids pBinAR-NtpurlA and pBinAR-Ntpurl were transformed in Agrobacterium tumefaciens C58Cl: pGV2260 (Deblaere et al., Nucl. Acids. Res. 13 (1984), 4777-4788). To transform tobacco plants (Nicotiana tabacum cv. Samsun NN), a 1:50 dilution of an overnight culture of a positively transformed agrobacterial colony in Murashige-Skoog medium (Murashige and Skoog Physiol. Plant. 15 (1962), 473) with 2% sucrose (2MS -Medium) is used. Leaf disks of sterile plants (each about 1 cm ² ) 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 on a weekly basis on MS medium with 500 mg / 1 claforan (cefotaxime sodium), 50 mg / 1 kamanycin, 1 mg / 1 benzylaminopurine (BAP ), 0.2 mg / 1 naphthylacetic acid and 1.6 g / 1 glucose. 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 after cultivation for two weeks in a climatic chamber in a 16 hour light / 8 hour dark rhythm at 60% humidity for PRPP amidotransferase expression and - Activity as well as changes in metabolite levels and phenotypic growth characteristics were examined. Altered nucleotide contents can e.g. according to the method of Stitt et al., FEBS Letters 145 (1982), 217-222.

Example 7

Analysis of transgenic plants

Transgenic plants that have been transformed with the construct with pBinAR-Ntpurl are characterized by different degrees of reduced growth and large-area bleaching of the leaves compared to untransformed control plants (Fig. 2). The RNA analysis by the Northern blot technique showed a reduced amount of Ntpurl.1-RNA in transgenic lines with the described phenotype (Fig. 3). This Effects were also observed in subsequent generations of the transgenic lines.

In order to test the growth reduction correlation, the 5 PRPP amidotransferase activity in the transgenic lines was measured and compared with that in untransformed controls. For this purpose, approx. 30 g leaves of approx. 20 cm high plants were homogenized with 50 ml extraction buffer at + 4 ° C.

10 extraction buffers:

100 mM HEPES pH 8.0

2.5 mM EDTA

10% glycerol

15 20 mM DTE

0.2 M PEFA block (40mM)

The digestion extract was filtered through Miracloth (Calbiochem, Bad Soden) and centrifuged at 16000 rpm in the Sorval centrifuge.

20 fugated. The resulting supernatant was precipitated with ammonium sulfate at 4 ° C. The 30% - 60% step was solubilized in the extraction buffer and desalted on a PD-10 column (Pharmacia, Sweden). The extract obtained in this way is stable for at least 24 hours and can be stored at -20 ° C after the addition of glycerol (50% final

25 tration) can be stored for a long time. The extract can be used directly to determine activity. The PRPP amidotransferase activity was significantly reduced in the transgenic lines compared to wild type plants, see Fig. 4. Fig. 4A shows the PRPP amidotransferase activity based on the pro-

30 tons. Fig. 4B shows the PRPP amidotransferase activity based on the fresh weight.

These data establish a direct connection between reduced PRPP amidotransferase activity and reduced growth 35 of the tobacco plants and therefore show PRPP amidotransferase as a suitable target protein for herbicidal active substances for the first time.

Example 8 0 Search for inhibitors of PRPP amidotransferase activity

To search for inhibitors of PRPP amidotransferase activity, the in vitro assay described in Example 3 with high-throughput methods can be used. The PRPP amidotransferase 5 activity can be prepared from plant tissues, see example 7. Alternatively, a plant PRPP amidotransferase can be found in E. coli, insect cells or another suitable expression system can be expressed. In this way, known PRPP amidotransferase inhibitors - such as glutamine antagonists - were identified.

Example 9

Analysis of adenine and guanine nucleotide levels in transgenic plants.

Of wild type plants and transgenic plants, which with the

Construct pBinAR-Ntpurl were transformed and their successor generation (lines 3.1, 3.2, 3.9., 25.1 and 38.8.), Leaf material (5 disks each with a diameter of 6 mm) were harvested and immediately frozen in liquid nitrogen. TCA extracts were then prepared using standard methods and used to determine the nucleotide contents.

AMP is strong in the transgenic lines with the exception of line 38.8 in the green leaf area and less in yellow leaf areas compared to the wild type (WT) (see Fig. 5).

No change was found for the guanosine nucleotides GTP, GDP and GMP compared to the wild type.

Claims

claims

1. DNA sequence containing the coding region of a plant PRPP amidotransferase, characterized in that this DNA sequence contains the nucleotide sequence SEQ-ID No. 1 or SEQ-ID No. 3 has.

2. DNA sequences that match the DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 according to claim 1 or parts thereof or

Derivatives derived from these sequences by insertion, deletion or substitution hybridize and code for a protein which has the biological activity of a PRPP amidotransferase.

3. Protein with PRPP amidotransferase activity, containing an amino acid sequence which is a partial sequence of at least 100 amino acids from SEQ-ID No. 2 or SEQ-ID No. 4.

4. Protein according to claim 3, characterized in that it contains the partial sequence 100 - 450 from SEQ-ID No. as amino acid sequence. 2 or SEQ-ID No. 4 contains.

5. Protein according to claim 4, characterized in that it as the amino acid sequence in SEQ-ID No. 2 or SEQ-ID No. 4 shown sequence contains.

6. Use of a DNA sequence according to claim 1 or 2 for introduction into pro- or eukaryotic cells, this sequence optionally being linked to control elements which ensure transcription and translation in the cells and for expression of a translatable mRNA which carries out the synthesis a plant PRPP amidotransferase causes leads.

7. Use of a DNA sequence according to claim 1 or 2 for the production of a test system for the identification of inhibitors of plant PRPP amidotransferase with herbicidal activity.

Sign.

8. A method for finding substances that inhibit the activity of plant PRPP amidotransferase, characterized in that in a first step using a DNA sequence according to claim 1 or 2 PRPP amidotransferase is produced and in a second step Activity of the plant PRPP amidotransferase is measured in the presence of a test substance.

9. The method according to claim 9, characterized in that the measurement of the plant PRPP amidotransferase is carried out in a high-throughput screening (HTS).

10. A method for identifying substances with herbicidal activity which inhibit PRPP amidotransferase activity in plants, consisting of

b) the application of a substance to transgenic plants, plant cells, plant tissue or parts of plants and to non-transformed plants, plant cells, plant tissue or parts of plants;

d) comparing the growth or survivability of the transgenic and the non-transformed plants, plant cells, plant tissue or plant parts after the ringing on the chemical substance;

11. Test system based on the expression of a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 according to claim 1 or 2 for the identification of inhibitors of plant PRPP amidotransferase with herbicidal activity.

5

12. Test system according to claim 11 for the identification of inhibitors of plant PRPP amidotransferase, characterized in that the enzyme is incubated with a test substrate to be examined and after a suitable reaction

10 time the enzymatic activity of the enzyme is determined in comparison to the activity of the uninhibited enzyme.

13. Plant PRPP amidotransferase inhibitors.

14. Inhibitors of plant PRPP amidotransferase, identified using a test system according to claim 11 or 12.

15. Inhibitors according to one of claims 13 or 14 for use as a herbicide. 20

16. A method for eliminating undesirable plant growth, characterized in that the plants to be removed are treated with a compound which is specifically coded to PRPP amidotransferase by a DNA sequence

25 Claim 1 or 2, binds and inhibits their function.

17. Plant with modified purine nucleotide content produced by additional expression of a DNA sequence SEQ-ID No. 1 or SEQ-ID No. 3 according to claim 1 or 2 in

30 Sense or antisense orientation.

5

0

5