WO1998010074A2

WO1998010074A2 - Adenylosuccinate synthetase

Info

Publication number: WO1998010074A2
Application number: PCT/EP1997/004812
Authority: WO
Inventors: Jens Lerchl; Ralf-Michael Schmidt; Helmut Schiffer; Uwe Sonnewald; Ralf Badur
Original assignee: Basf Aktiengesellschaft
Priority date: 1996-09-04
Filing date: 1997-09-04
Publication date: 1998-03-12
Also published as: JP2001500008A; BR9711658A; CA2264677A1; AU4553097A; AU741624B2; EP0927246A2; IL128618A0; WO1998010074A3

Abstract

Expression cartridges are disclosed which confer to plants, plant cells, tissues or parts a resistance against inhibitors of the vegetable adenylosuccinate synthetases. Also disclosed is the use of the expression cartridges in appropriate vectors to transform plants, plant cells, tissues or parts.

Description

Adenylosuccinate synthetase

description

The present invention relates to expression cassettes, coding for non-vegetable adenylosuccinate synthetases (ADSS), which impart resistance to plant ADSS inhibitors to plants; Vectors and microorganisms containing such expression cassettes; transgenic plants transformed therewith; the corresponding expression products and nucleic acid sequences; and an expression kit for use in transforming a plant host.

As photoautotrophic organisms, 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. In particular, plants must also synthesize the nucleotides as components of the nucleic acids de novo.

It can be assumed that the efficient formation, use and distribution of the nucleotides strongly influence the cell division and the growth of a plant. Since plants rely on a functioning de novo nucleotide biosynthesis, this biosynthetic path offers itself as a target for the use of herbicides. The complex reactions that ensure nucleotide biosynthesis are divided into purine and pyridine idiosynthesis.

The enzyme reactions of purine biosynthesis can start from

Pliosphoribosyl pyrophosphate (PRPP) can be divided into the following steps:

a) Synthesis of the pyrimidine ring b) Synthesis of the purine ring c) Branching from the IMP to the AMP or GMP

The reaction sequence of step c) is shown schematically in the attached FIG. 1.

Some of the enzymes involved in purine biosynthesis represent potential targets for herbicidal agents. The ADSS occupies a special position. The enzyme catalyzes the following reaction:

IMP + L-Aspartate + GTP ± + Adenylosuccinate + GDP + Pi The ADSS was purified from E. coli for homogeneity (Bass et al., 1987, Aren. Biochem. Biophys., 256, 335-342), the crystal structure was determined (Poland et al., 1993, J. Biol. Che. , 268, 25334-25342) and kinetic properties of the enzyme 5 were characterized (Kang and Fromm, 1995, J. Biol. Chem., 270,

15539-15544). The enzyme was also purified from Dictyostelium diseoideum (Jahngen and Rossomando, 1984, Arch. Biochem. Biophys., 229, 145-154) and rabbit muscle (J. Biol. Chem., 1974, 249 (2), 459-464). Vegetable adenylosuccinate synthetase 10 was obtained from wheat seedlings (Hatch, 1967, Phytochemistry, 6,

115-119) and from maize (Siehl et al., 1996, Plant Physiol., 110, 753-758) for a determination of the enzyme activity.

The adenylosuccinate synthetase is present in E. coli as a dimer of two 15 48 kD polypeptides. No data are available for herbal ADSS.

Genes coding for ADSS have so far been derived from Escherichia coli (EMB access number J04199), Bacillus subtilis (J02732),

20 Haemophilus influenzae (46263), Saccharomyces pombe (L22185), Schizosaccharomyces pombe (M98805), Dictyostelium diseoidem (M58471), Homo sapiens (X65503) and Mus musculus (M74495) were isolated. By comparing data, "expressed-sequence tags", so-called est sequences, from Arabidopsis thaliana can be found

25 (T42641) and Oryza sativa (D15352) with clear similarity to the mentioned ADSS.

Complete cDNA sequences of plant adenylosuccinate synthetases have also been described from Arabidopsis thaliana and maize (US-A-5519125) 30 and from wheat (WO96 / 19576).

No cofactors are required to measure ADSS activity. However, the active substances hadacidin and alanosine (Stayton et al., 1983, Curr. Top. Cell. Regul., 22, 103-141) as well as hydantoine or its metabolite 5 '-phosphohydantocidine have been described (Siehl et al., 1996, Plant Physiol., 110, 753-758), which inhibit the enzyme activity of plant ADSS.

The phytotoxic active ingredient hydantocidin was isolated from Streptomyces 40 hygroscopicus (strain SANK 63584) (Nakajima et al., 1991, J. Antibiot., 44, 293-300). It has been shown that 5'-phosphohydantoeidine as the plant metabolite is the actual inhibitor of ADSS (Siehl et al., 1996, Plant Physiol., 110, 753-758). Hydantocidin exerts its toxic effect only in plants, has a low mammalian toxicity and does not act on various microorganisms investigated (Nakajima et al., 1991, J. Antibiot., 44, 293-300). It would be desirable, if ways could be found to specifically change the response of plants to ADSS inhibitors.

The invention is therefore based on the object of providing means by means of which the response of plants to ADSS inhibitors can be specifically modified. In particular, this modification should be feasible by genetic engineering.

This object is achieved by providing an expression cassette containing regulatory under genetic control

Nucleic acid sequences the coding nucleic acid sequence for a protein which imparts resistance to inhibitors of plant adenylosuccinate synthetase to a plant host.

The invention will now be explained in more detail with reference to the following figures. It shows

Figure 1 shows the de novo purine biosynthesis in plants; FIG. 2 shows the nucleic acid sequence of the adenylosuccinate synthase from Escherichia coli, including the 5 'and 3' terminal Ba HI cleavage sites; FIG. 3 shows the amino acid sequence of the adenylosuccinate synthetase from Escherichia coli; FIG. 4 oligonucleotide sequences for isolating the nucleic acid sequence of the adenylosuccinate synthetase from Escherichia coli; FIG. 5 shows the nucleic acid sequence of the transit peptide of chloroplast plastid transketolase in three reading frames; FIG. 6 shows the structure of the expression cassettes and transformation vectors pTP09, pTPlO and pTPll;

FIG. 7 shows the structure of the expression cassettes and the transformation vector pTP09-ASS.

A first object of the present invention relates to expression cassettes which are suitable for transforming a plant host and which contain a coding sequence which give the host resistance to inhibitors of plant ADSS.

In the context of the present invention, resistance means the artificially acquired resistance to the action of plant ADSS inhibitors. It includes the partial and, in particular, the complete insensitivity to these inhibitors for at least one generation of plants.

According to a preferred embodiment of the invention, expression cassettes for a plant host are selected from whole plants, plant cells, plant tissues or plant parts, such as leaves, roots, fruits. In particular, the plant host is selected from crop plants, cells, tissues or parts derived therefrom. As non-limiting examples of suitable crop plants can be mentioned: cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, potato, tobacco, tomato, rapeseed, alfalfa, lettuce and the various tree, nut and Wine species.

In particular in the case of crop plants, the present invention offers the advantage that, after induction of a selective resistance of the crop plant to plant ADSS inhibitors, these inhibitors can be used as specific herbicides against non-resistant plants. Non-limiting examples of inhibitors of this type include alanosine, hadacidin, hydantocidin and metabolites and functionally equivalent derivatives thereof. Functionally equivalent derivatives of plant ADSS inhibitors have a comparable spectrum of action as the specifically named substances, with lower, equal or higher inhibitory activity (e.g. expressed in g inhibitor per hectare of cultivated area, required to completely suppress the growth of non-resistant plants).

The invention relates in particular to expression cassettes whose coding sequence contains a non-vegetable ADSS nucleic acid sequence which is tolerant to plant ADSS inhibitors or a functional equivalent thereof. The ADSS nucleic acid sequence can e.g. be a DNA or a cDNA sequence.

Coding sequences suitable for insertion into an expression cassette according to the invention are, for example, those which essentially comprise a microbial DNA sequence which are suitable for an adenylosuccinate synthetase from microorganisms of the genera Escherichia, Bacillus, Haemophilus, Dictyostelium, Saccharomyces or Schizosaccharomyces and in particular from Escherichia coli subtilis, Haemophilus influenzae, Dictyostelium discoideum, Saccharomyces pombe or Schizosaccharomyces pombe. Particularly suitable is an ADSS sequence which essentially corresponds to the DNA sequence from E. coli shown in FIG. 2 (SEQ ID N0: 1).

In addition, artificial DNA sequences are suitable as long as they induce the desired resistance as described above. Such artificial DNA sequences can be determined, for example, by back-translation of proteins constructed by means of molecular modeling which have adenylosuccinate synthetase activity or by in vitro selection. Are particularly suitable for ADSS coding DNA sequences obtained by back-translating an ADSS polypeptide sequence 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.

The invention also relates to the sequences which are functionally equivalent to the above nucleic acid sequences and to the amino acid sequences derived therefrom. According to the invention, functional equivalents are sequences which essentially correspond to one another. This means in particular sequence variants which have arisen as a result of natural or artificial mutations in an ADSS sequence, provided that the desired ADSS activity, which is necessary to maintain plant metabolism, is retained. The exogenous ADSS activity transformed in the plant host can therefore be higher, comparable or somewhat lower than that of the endogenous plant ADSS. Mutations include substitutions, deletions, exchanges or insertions of one or more nucleotide or amino acid residues.

Substitutions mean exchanges of nucleotides or amino acids. So-called silent or conservative exchanges are preferred. A silent nucleotide exchange does not change the amino acid sequence. A conservative exchange results in an amino acid substitution by an amino acid residue with comparable properties, e.g. Size, charge, polarity, solubility. Examples of amino acid pairs with similar properties are Glu and Asp, Val and Ile, Ser and Thr.

According to the invention, a deletion means the removal of at least one nucleotide or at least one amino acid residue. Preferred positions for deletions are the DNA regions which code for the termini of the polypeptide and the links between the individual protein domains. According to the invention, particular preference is given to DNA sequences which code for ADSS proteins and which are reduced by N-terminal truncations by up to about 100, e.g. by 20 to 100 amino acids, from the sequence shown in FIG. 3 (SEQ ID N0: 2).

According to the invention, insertions comprise insertions of at least one nucleotide or amino acid residue into one of the sequences according to the invention. Sequences which code for fusion proteins are to be mentioned as further functionally equivalent nucleic acid sequences according to the invention, part of the fusion protein being a non-vegetable ADSS polypeptide or a functionally equivalent part thereof. The second part of the fusion protein can be, for example, another polypeptide with the same or different enzymatic activity. However, this is preferably a regulatory protein sequence, such as a signal or transit peptide, which directs ADSS to the desired site of action.

The invention thus also relates to expression cassettes whose coding sequence codes for an ADSS fusion protein, part of the fusion protein being a transit peptide which controls the translocation of the ADSS sequence. Chloroplast-specific transit peptides are particularly preferred which, after translocation of the ADSS sequence into the plant chloroplasts (main site of purine biosynthesis in plants), are enzymatically split off from the ADSS part. The transit peptide is particularly preferably derived from plastid transketolase (TK) or a functional equivalent of this transit peptide. An expression cassette which contains one of the transit peptide DNA sequences shown in FIG. 5 (SEQ ID NOs: 3,4,5) is particularly preferred.

The expression cassettes according to the invention also comprise 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, i.e. at the 5 'end, the coding sequence a promoter and downstream, i.e. at the 3 'end a polyadenylation signal, and optionally further regulatory elements which are operatively linked to the intervening coding sequence for ADSS and / or transit peptide. An operative link is understood to mean the sequential arrangement of the said regulatory elements in such a way that each of the regulatory elements can fulfill its function in the expression of the coding sequence.

In principle, any promoter which can control the expression of foreign genes in plants is suitable as the promoter of the expression cassette according to the invention. A plant promoter is preferably used in particular. The 35S CaMV promoter from the Cauliflower mosaic virus (Franck et al. (1980) Cell 21, 285-294) is particularly preferred. This promoter contains different recognition sequences for transcriptional effectors, which in their entirety lead to a constitutive expression of the introduced gene (Benfey et al. (1989) EMBO J. 8, 2195-2202).

Preferred polyadenylation signals are plant polyadenylation signals, preferably those which essentially correspond to T-DNA polyadenylation signals from Agrobacterium tumefaciens, in particular the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5.

An expression cassette according to the invention is produced by fusing a suitable promoter with a suitable ADSS-DNA and preferably a DNA coding for a chloroplast-specific transit peptide inserted between the promoter and ADSS-DNA, and a polyadenylation signal according to common recombination and cloning techniques, as described, for example, in T. Maniatis, EF 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-Interscience (1987).

The ADSS-DNA or cDNA required for the production of expression cassettes according to the invention is preferably obtained with the aid of the

Polymerase chain reaction (PCR) amplified. Methods for DNA amplification by means of PCR are known, for example from Innis et al. , PCR Protocols, A Guide to Methods and Applications, Academic Press (1990). The PCR-generated DNA fragments can expediently be checked by sequence analysis to avoid polymerase errors in constructs to be expressed.

The invention also relates to an expression kit which comprises at least three expression cassettes, at least two of which contain a variable region or frame shift sequence which differ from one another by insertion or deletion, preferably insertion, of a number of nucleotides which cannot be divided by 3 and thus shift the reading frame for a sequence inserted downstream by one or two nucleotides. The 5 'end of the frame shift sequence is linked to the transit peptide sequence. The frame shift sequence includes a restriction site at the 3 'end into which, for example, the ADSS-DNA sequence is inserted. The provision of at least three vectors, each containing an expression cassette of the kit with different reading frames for the inserted gene, ensures that the fusion constructs consist of any DNA sequence and a DNA sequence coding for a transit peptide. can be expressed in three different reading frames, with at least one of the constructs leading to the expression of functional gene product (such as ADSS). The construction diagrams for an expression kit according to the invention containing three expression cassettes and which is particularly suitable for the transformation of plants are shown in the attached FIG. 6.

The invention also relates to the use of such kits for transforming a plant host.

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 pACYC184. Binary vectors which can replicate both in E. coli and in agrobacteria, such as e.g. pBin19 (Bevan et al. (1980) Nucl. Acids Res. 12, 8711).

Another object of the invention therefore relates to recombinant vectors, such as e.g. Plasmids or viruses containing at least one expression cassette according to the invention. A particularly preferred recombinant plasmid called pTP09-ASS contains the gene construct shown in FIG. 7 and gives the plant host transformed therewith resistance to plant ADSS inhibitors, such as e.g. Hydantocidin.

To transfect a host plant with an ADSS-DNA, an expression cassette according to the invention is inserted as an insert in a recombinant vector, the vector DNA of which contains additional functional regulatory signals, for example sequences for replication or integration. Suitable vectors are inter alia in "Methods in Plant Molecular Biology and Biotechnology" (CRC Press), Chap. 6/7, p.71-119.

The vectors according to the invention can be used to transform plants, plant cells, plant tissues or parts.

The DNA constructs fused according to the invention can also be transferred into plant genomes by various other known methods. Suitable methods are, for example, protoplast transformation by polyethylene glycol-induced DNA uptake, electroporation, sonication or microinjection, and the transformation of intact cells or tissue by micro or macro injection into tissue or embryos, tissue electroporation, incubation of dry embryos in DNA-containing solution, biolistic shear gene transfer and particularly preferably Agrobacterium transform ation. 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, pp. 128-143 and in Potrykus (1991) Annu. Rev. Plant Physiol. Plant Molec. Biol. 42, 205-225).

The fused construct is preferably cloned into a vector, for example pBin19, which is suitable for transforming Agrobacterium tumefaciens. Agrobacteria transformed with such a vector can then be used in a known manner to transform plants, in particular crop plants, such as e.g. of tobacco plants can be used, for example, by wounding leaves or leaf pieces in an agrobacterial solution and then cultivating them in suitable media. The transformation of plants by agrobacteria is known, among other things, from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1, Engineering and Utilization, edited by S-d Kung and R. Wu, Academic Press, 1993, pp. 15-38 and from S.B. Gelvin, Molecular Genetics of T-DNA Transfer from Agrobacterium to Plants, also in Transgenic Plants, pp. 49-78. Transgenic plants which express ADSS-DNA integrated into the expression cassette according to the invention can be regenerated in a known manner from the transformed cells of the wounded leaves or leaf pieces.

The effectiveness of the transgenically expressed ADSS can be tested, for example, in vitro by an enzyme assay, as described in Baugher et al. , Biochem. Res. Commun., 94: 123-129 (1980); Stayton et al., Curr. Top. Cell. Regul. , 22: 103-141 (1983); and Bass et al., Arch. Biochem. Biophys., 256: 335-342 (1987).

The invention also relates to microorganisms, such as Bacteria or fungi which contain a recombinant vector according to the invention.

The invention also relates to transgenic plants transformed with a vector or microorganism according to the invention, and to transgenic cells, tissues, parts and propagation material of such plants. Transgenic crop plants, such as e.g. Cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, potato, tobacco, tomato, rapeseed, alfalfa, lettuce and the various tree, nut and wine species.

The transgenic plants, plant cells, tissue or parts can be treated with an active ingredient which inhibits the plant ADSS, as a result of which those which have not been successfully transformed Plants, cells, tissues or parts of plants die. Examples of suitable active substances are alanosine, hadacidin and in particular hydantocidin as well as metabolites and functional derivatives of these compounds. The ADSS-DNA inserted into the expression cassette according to the invention can thus be used as a selection marker.

Another object of the invention thus relates to the use of vectors or microorganisms transformed therewith for the transformation of plants, plant cells, plant tissues or plant parts, in particular for the expression of exogenous proteins, glycoproteins or fusion proteins. The aim of the use is preferably to impart resistance to inhibitors of plant ADSS.

The invention also relates to the expression products produced according to the invention, in particular the fusion proteins from transit peptide and protein part with non-plant ADSS activity.

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

Example 1: PCR amplification of Escherichia coli adenylosuccinate synthetase using synthetic oligonucleotides

The PCR amplification of the Escherichia coli ADSS was carried out in a DNA thermal cycler from Perkin Elmer. The oligonucleotides used (SEQ ID NO: 6, 7) are shown in FIG. 4 and were taken from the published sequence. The reaction mixtures contained 8ng / μl genomic DNA from Escherichia coli, 0.5 μM of the corresponding oligonucleotides, 200 μM nucleotides (Pharmacia), 50 mM KCl, 10 M Tris-HCl (pH 8.3 at 25 ° C.), 1 , 5 mM MgCl) and 0.02 U / μl Taq polymerase (Perkin Elmer). The amplification conditions were set as follows:

Annealing temperature: 45 ° C, 1 min

Denaturation temperature: 92 ° C, 1 min. Elongation temperature: 72 ° C, 1.5 min

Number of cycles: 40

The result was a fragment of 1300 base pairs which was ligated into the vector pGEM-T (Promega). E. coli XL-I Blue was transformed with the ligation mixture and the plasmid pGEM-ASS was obtained. Example 2: Generation of plant expression cassettes

In the plasmid pBinl9 (Bevan et al. (1980) Nucl. Acids Res. 12, 8711) (commercially available from Clontech, Palo Alto, CA, USA), a 35S CaMV promoter was developed as an EcoRI-Kpnl-Frag (corresponding the nucleotides 6909-7437 of the Cauliflower mosaic virus (Franck et al. (1980) Cell 21, 285) The polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., (1984) EMBO J. 3, 835), nucleotides 11749-11939, derived from the octopine synthase (OCS) gene, was isolated as a PvuII-Hindlll fragment and after addition of Sphl linkers to the PvuII interface between the Sphl-Hindlll- Interfaces of the vector were cloned, resulting in the plasmid pBinAR (Höfgen and Willmitzer (1990) Plant Science 66, 221-230).

The cDNA sequence of the transit peptide (TP) of the transketolase (TK) was taken from the plasmid pBluescript TK-26 (DE-A-19501906) and with the aid of synthetic oligonucleotides as a Kpnl-BamHI fragment via a polymerase chain reaction into the plasmid pBinAR - riert. By varying the 3 '-specific oligonucleotide used, three vectors were obtained as plant expression cassettes (pTP09, pTPlO, pTPll, see FIGS. 5 and 6), which allow chimeric gene constructs with the cDNA transit sequence of the plastid transketolase in three different reading frames to create.

Example 3: Creation of a plant expression cassette for the adenylosuccinate synthetase

The DNA fragment coding for the adenylosuccinate synthetase was cloned into the vector pTP09 as a BamHI fragment. The plasmid pTP09-ASS resulted (see FIG. 7). Fusion of the transit peptide (TP / TK) with the adenylosuccinate synthetase ensured the import of the protein into the chloroplasts.

Example 4: Sequence analysis of recombinant DNA

The sequencing of recombinant DNA molecules was carried out with a laser fluorescence DNA sequencer from Pharmacia according to the method of Sanger (Sanger et al. (1977) Proc. Natl. Acad. Sei. USA 74, 5463-5467). Fragments resulting from a polymerase chain reaction were sequenced and checked to avoid polymerase errors in constructs to be expressed. Example 5: Production of transgenic tobacco plants which contain a microbial adenylosuccinate synthetase in chloroplast

The plasmid pTP09-ASS was in Agrobacterium tumefaciens

C58C1: pGV2260 transformed. The transformation of Agrobacterium tumefaciens was carried out according to the method of Höfgen and Willmitzer (Nucl. Acid Res. (1988) 16, 9877). The agrobacteria were grown in YEB medium (Vervliet et al., J. Gen. Virol. (1975) 26, 33). For the transformation of 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 ((1962) Physiol. Plant. 15, 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 to 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 kanamycin, 1 mg / 1 benzylaminopurine (BAP), 0.2 mg / 1 naphthylacetic acid and 1.6 g / 1 glucose, and growing rungs were transferred to MS medium with 2% sucrose, 250 mg / 1 claforan and 0.8% Bacto agar.

The same procedure was followed in a second transformation, except that 220 mg / l of hydantocidine was added as an antibiotic.

Example 6: Analysis of total RNA from plant tissues

For a more detailed investigation of the expression, the total RNA of tobacco plants, as in Logemann et al. (Anal. Biochem. (1987) 163,21). For the analysis, 20 μg RNA were separated in a 1.5% agarose gel containing formaldehyde. After electrophoretic separation of the RNA molecules, the RNA was transferred to a nylon membrane by capillary transfer. The detection of specific transcripts was carried out as described in Amasino (Anal. Biochem. (1986) 152, 304). The cDNA fragments used as a probe were radioactively labeled with a random primed DNA labeling kit (Boehringer, Mannheim). Example 7: Enzyme assay of adenylosuccinate synthetase isolated from transgenic tobacco plants

The test mixture contained 14 mM Tris-HCl pH 8.3; 6 mM MgCl ₂ ; 0.4 mM IMP; 0.1 mM GTP; 0.5 mM phosphoenol pyruvate; 0.1 mM ATP; 2 U / ml pyruvate kinase; 3 mM aspartate and in a 1 ml test batch 10 to 100 μl enzyme preparation. There was an incubation of 5 to 20 min at 25 ° C. in the presence or absence of the inhibitor hydantocidin and subsequent measurement at 280 nm (absorption of adenylosuccinate) on a two-beam photometer against a batch without aspartate as a blank. The recombinant ADSS showed no inhibition by hydantocidin.

Example 8: Testing of hydantocidine-resistant tobacco plants

Tobacco plants transformed with the plasmid pTP09-ASS were grown in tissue culture on 2MS medium with 0.8% Bacto agar, 250 mg / 1 claforan, 50 mg / 1 kanamycin, and axial shoots were transferred to corresponding medium with 220 mg / 1 hydantocidin. Untransformed plants died within a few weeks, while resistant plants continued to grow.

SEQUENCE LOG

(1. GENERAL INFORMATION:

(i) LOGIN!

(A) NAME: BASF Aktiengesellschaft

(B) ROAD: - - <C) LOCATION: Ludwigshafen

(E) COUNTRY: Germany

<F) POSTAL CODE: D-67056

(ii) DESCRIPTION OF THE INVENTION: Adenylosuccinate Synthetase

(iii) NUMBER OF SEQUENCES: 7

(iv) COMPUTER READABLE VERSION:

(A) DISK: Floppy disk (B) COMPUTER: IBM? C compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patentin Release # 1.0, Version tfl.30 (EPA)

(2) INFORMATION ABOUT SEQ ID NO: 1: (i) SEQ IDENTIFICATION:

(A) LENGTH: 1311 base pairs

(B) TYPE: nucleotide

(C) STRANDFORM: Eir.zelstrang

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Genomic DNA

(iii) HYPOTHETICAL: NO

(iv) ANTISENSE: NO

(vi) ORIGINAL ORIGIN:

(A) ORGANISM: Escherichia coli

(ix) FEATURE:

(A) NAME / KEY: misc_feature

(B) LOCATION:! .. 6

(D) OTHER INFORMATION: / function = "BamHI interface"

(ix) NOTE:

(A) NAME / KEY: CDS (ß) LOCATION: 7..1302

(D) OTHER INFORMATION: / product »" Adenylosυccinate Synthetase "

(ix) FEATURE:

(A) NAME / KEY: misc_fea ure

(B) LOCATION: 1303..1305 (D) OTHER INFORMATION: / function- "Stop codon"

(ix) FEATURE:

(A) NAME / KEY: misc_f ea ure

(3) LOCATION: 1306..1311

(D) OTHER INFORMATION -./function- "BamHI interface"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GGATCC ATG GGT AAC AAC GTC GTC GTA CTG GGC ACC CAA TGG GGT GAC 48

Met Oly Asn Asn Val Val Val Leu Gly Thr Gin Trp Gly Asp

I S 10

GAA GGT AAA GGT AAG ATC GTC GAT CTT CTG ACT GAA CGG GCT AAA TAT 96

Glu Gly Lys Gly Lys Ilβ Val Asp Leu Leu Thr Glu Arg Ala Lys Tyr 15 20 25 30

GTT GTA CGC TAC CAG GGC GGT CAC AAC GCA GGC CAT ACT CTC GTA ATC 144

Val Val Arg Tyr Gin Gly Gly His Asn Ala Gly His Thr Leu Val Ile 35 40 45

AAC GGT GAA AAA ACC GTT CTC CAT CTT ATT CCA TCA GGT ATT CTC CGC 192

Asn Gly Glu Lys Thr Val Leu His Leu Ile Pro Ser Gly Ile Leu Arg 50 55 60

GAG AAT GTA ACC AGC ATC ATC GGT AAC GGT GTT GTG CTG TCT CCG GCC 240 Glu Asn Val Thr Ser Ile Ile Gly Asn Gly Val Val Leu Ser Pro Ala 65 70 75

GCG CTG ATG AAA GAG ATG AAA GAA CTG GAA GAC CGT GGC ATC CCC GTT 288

Ala Leu Met Lys Glu Met Lys Glu Leu Glu Asp Arg Gly Ile Pro Val

80 85 90

CGT GAG CGT CTG CTG CTG TCT GAA GCA TGT CCG CTG ATC CTT GAT TAT 36 Arg Glu Arg Leu Leu Leu Ser Glu Ala Cys Pro Leu Ile Leu Asp Tyr 9S 100 105 110

CAC GTT GCG CTG GAT AAC GCG CGT GAG AAA GCG CGT GGC GCG AAA GCG 384 His Val Ala Leu Asp Asn Ala Arg Glu Lys Ala Arg Gly Ala Lys Ala 115 120 125

ATC GGC ACC ACC GGT CGT GGT ATC GGG CCT GCT TAT GAA GAT AAA GTA 32 Ile Gly Thr Thr Gly Arg Gly Ile Gly Pro Ala Tyr Glu Asp Lys Val 130 135 140

GCA CGT CGC GGT CTG CGT GTT GGC GAC CTT TTC GAC AAA GAA ACC TTC 480 Ala Arg Arg Gly Leu Arg Val Gly Asp Leu Phe Asp Lys Glu Thr Phe 145 150 155

GCT GAA AAA CTG AAA GAA CTG ATG GAA TAT CAC AAC TTC CAG TTG GTT 528 Ala Glu Lys Leu Lyε Glu Val Met Glu Tyr His Asn Fhe Glr. Leu Val 160 165 170

AAC TAC TAC AAA GCT GAA GCG GTT GAT TAC CAG AAA GTT CTG CAT GAT 576 Asn Tyr Tyr Lys Ala Glu Ala Val Asp Tyr Gin Lys Val Leu Asp Asp 175 180 185 190

ACG ATG GCT GTT GCC GAC ATC CTG ACT TCT ATG GTG GTT GAC GTT TCT 624 Thr Met Ala Val Ala Asp Ile Leu Thr Ser Met Val Val Asp Val Ser 195 200 205

GAC CTG CTC GAC CAG GCG CGT CAG CGT GGC GAT TTC GTC ATG TTT GAA 672 Asp Leu Leu Asp Gin Ala Arg Gin Arg Gly Asp Phe Val Met Fhe Glu 210 215 220

GGT GCG CAG GGT ACG CTG CTG GAT ATC GAC CAC GGT ACT TAT CCG TAC 720 Gly Ala Gin Gly Thr Leu Leu Asp Ile Asp Hie Gly Thr Tyr Pro Tyr 225 230 235 GTA ACT TCT TCC AAC ACC ACT GCT GGT GGC GTG GCG ACC GGT TCC GGC 76θ Val Thr Ser Ser Asn Thr Thr Ala Gly Gly Val Ala Thr Gly Ser Gly 240 245 250 CTG GGC CCG CGT TAT GTT GAT TAC GTT CTG GGT ATC CTC AAA GCT TAC 816

Leu Gly Pro Arg Tyr Val Asp Tyr Val Leu Gly Ile Leu Lys Ala Tyr

255 260 265 270

TCC ACT CGT GTA GGT GCA GGT CCG TTC CCG ACC GAA CTG TTT GAT GAA 864

Ser Thr Arg Val Gly Ala Gly Pro Phe Pro Thr Glu Leu Phe Asp Glu 275 280 285

ACT GGC GAG TTC CTC TGC AAG CAG GGT AAC GAA TTC GGC GCA ACT ACO 912

Thr Gly Glu P ^~ he Leu ^' Cys Lys Gin Gly Asn Glu Phe Gly Ala Thr Thr 290 295 300

GGG CGT CGT CGT CGT ACC GGC TGG CTG GAC ACC GTT GCC GTT CGT CGT 960

Gly Arg Arg Arg Arg Thr Gly Trp Leu Asp Thr Val Ala Val Arg Arg 305 310 315

GCG GTA CAG CTG AAC TCC CTG TCT GGC TTC TGC CTG ACT AAA CTG GAC 1008

Ala Val Gin Leu Asn Ser Leu Ser Gly Phe Cys Leu Thr Lys Leu Asp 320 325 330

GTT CTG GAT GGC CTG AAA GAG GTT AAA CTC TGC GTG GCT TAC CGT ATG 1056

Val Leu Asp Gly Leu Lys Glu Val Lys Leu Cys Val Ala Tyr Arg Met

335 340 345 350

CCG GAT GGT CGC GAA GTG ACT ACC ACT CCG CTG GCA GCT GAC GAC TGG 1104 Pro Asp Gly Arg Glu Val Thr Thr Thr Pro Leu Ala Ala Aso Asp Trp

355 360 365

AAA GGT GTA GAG CCG ATT TAC GAA ACC ATG CCG GGC TGG TCT GAA TCC 1152

Lys Gly Val Glu Pro Ile Tyr Glu Thr Met Pro Gly Trp Ser Glu Ser 370 375 380

ACC TTC GGC GTG AAA GAT CGT AGC GGC CTG CCG CAG GCG GCG CTG AAC 1200 Thr Phe Gly Val Lys Asp Arg Ser Gly Leu Pro Gin Ala Ala Leu Asn 385 390 395

TAT ATC AAG CGT ATT GAA GAG CTG ACT GGT GTG CCG ATC GAT ATC ATC 1248

Tyr Ile Lys Arg Ile Glu Glu Leu Thr Gly Val Pro Ile Asp Ile Ile 400 405 41C TCT ACC GAT CCG GAT CGT ACT GAA ACC ATG ATT CTG CGC GAC CCG TTC 1296

Ser Thr Asp Pro Asp Arg Thr Glu Thr Met Ile Leu Arg Asp Pro Phe

415 420 425 430

GAC GCG TAAGGATCC 1 11

Asp Ala

(2) INFORMATION ON SEQ ID NO: 2;

(i) S EQUENCE XENNZ E I CHEN:

(A) LONG: 432 amino acids

(B) TYPE: amino acid <D> TOPOLOGY: l inea r

(i i) TYPE OF MOLECULE; Prote in

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2

Met Gly Asn Asn Val Val Val Leu Gly Thr Gin Trσ Gly Aso Glu Gly 1 5 10 ^'' 15

Lys Gly Lys Ile Val Asp Leu Leu Thr Glu Arg Ala Lys Tyr Val Val 20 25 30

Lys Leu Lyε Glu Val Met Glu Tyr His Asn Phe Gin Leu Val Asn Tyr

165 170 175

Tyr Lys Ala Glu Ala Val Asp Tyr Gin Lys Val Leu Asp Asp Thr Met

180 185 190

Ala Val Ala Asp Ile Leu Thr Ser Met Val Val Asp Val Ser Asp Leu 195 200 205

Leu Asp Gin Ala Arg Gin Arg Gly Asp Phe Val Met Phe Glu Gly Ala 210 215 ^* 220

Gin Gly Thr Leu Leu Asp Ile Asp His Gly Thr Tyr Pro Tyr Val Thr

225 230 ^" 235 240

Ser Ser Asr. Thr Thr Ala Gly Gly Val Ala Thr Gly Ser Gly Leu Gly 245 250 255

Pro Arg Tyr Val Asp Tyr Val Leu Gly Ile Leu Lys Ala Tyr Ser Thr 260 265 270

Arg Val Gly Ala Gly Pro Phe Pro Thr Glu Leu Phe Asp Glu Thr Gly 275 280 285

Glu Phe Leu Cys Lys Gin Gly Asn Glu Phe Gly Ala Thr Thr Gly era 290 295 300

Arg Arg Arg Thr Gly Trp Leu Asp Thr Val Ala Val Arg Arg Ala Val 305 310 315 320 Gin Leu Asr. Ser Leu Ser Gly Phe Cys Leu Thr Lys Leu Asp Val Leu

325 330 335

Asp Gly Leu Lys Glu Val Lys Leu Cys Val Ala Tyr Arg Met Pro Asp 340 345 350

Gly Arg Glu Val Thr Thr Thr Pro Leu Ala Ala Asp Aso Tro Lys Gly 355 360 36 ^* 5 Val Glu Pro Ile Tyr Glu Thr Met Pro Gly Trp Ser Glu Ser Thr Phe 370 375 380

Gly Val Lys Asp Arg Ser Gly Leu Pro Gin Ala Ala Leu Asn Tyr Ile

385 390 395 400

Ly3 Arg Ile Glu Glu Leu Thr Gly Val Pro Ile Asp Ile Ile Ser Thr 405 410 415

Asp Pro Asp Arg Thr Glu Thr Met Ile Leu Arg Asp Pro Phe Asp Ala 420 425 430

(2) INFORMATION ON SEQ ID NO: 3:

(i) ZICK SEQUENCE KEYS:

(A) LENGTH: 258 base pairs

(B) TYPE: nucleotide (C) STRAX3F0RM: single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (iv) ANTISENSE: NO

(vi) ORIGINAL ORIGIN:

(I) CRGANELLE: chloroplast

(ix) FEATURE:

(A) NAME / KEY: misc_feature

(B) LOCATION: 1..S (D) OTHER INFORMATION: / function = "Knpl interface ^»

(ix) FEATURE:

(A) NAME / KEY: transit_peptide

(B) LOCATION: 7..252

(ix) FEATURE: (A) NAME / KEY: misc_fea: ure

(B) LOCATION: 253..253

(D) OTHER INFORMATION: / function- "BamHI interface"

(xi) SEQUENCE DESCRIPTION: SEQ ID MO: 3: GGTACCATGG CGTCTTCTTC TTCTCTCACT CTCTCTCAAG CTATCCTCTC ICGTTCTGTC 60

CCTCGCCATG GCTCTGCCTC TTCTTCTCAA CTTTCCCCTT CTTCTCTCAC TTTTTCCGGC 120

CTTAAATCCA ATCCCAATAT CACCACCTCC CGCCGCCGTA CTCCTTCCTC CGCCGCCGCC 180

GCCGCCGTCG TAAGGTCACC GGCGATTCCT GCCTCAGCTG CAACCGAAAC CATAGAGAAA 240

ACTGAGACTG CGGGATCC 258

(2) INFORMATION ON SEQ ID NO: 4:

(i) SEQUENCE KEY OAK:

(A) LENGTH: 260 base pairs (B) TYPE: nucleotide

(C) STRAND FORM; Single strand (0) TOPOLOGY: lir.ear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTISENSE: NO

(vi) ORIGINAL ORIGIN:

(I) ORGANELLE: chloroplast

(ix) FEATURE: (A) NAME / KEY: misc_feature

(B) LOCATION: 1..6 (D) OTHER INFORMATION: / function = * KpnI interface "

(ix) FEATURE:

(A) NAME / KEY: transi _peptide

(B) LOCATION: 7..2S2 (ix) FEATURE:

(A) NAME / KEY: misc feature

(B) LOCATION: 2S3..254

(D) OTHER INFORMATION: / function = "frame shifr. Insert"

(ix) FEATURE:

(A) NAME / KEY: misc_feature (B) LOCATION: 255..260

(D) OTHER INFORMATION: / function- "BamHI interface"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

GGTACCATGG CGTCTTCTTC TTCTCTCACT CTCTCTCAAG CTATCCTCTC TCGTTCTGTC 60

CCTCGCCATG GCTCTGCCTC TTCTTCTCAA CTTTCCCCTT CTTCTCTCAC TTTTTCCGGC 120

CTTAAATCCA ATCCCAATAT CACCACCTCC CGCCGCCGTA CTCCTTCCTC CGCCGCCGCC 180

GCCGCCGTCG TAAGGTCACC GGCGATTCGT GCCTCAGCTG CAACCGAAAC CATAGAGAAA 240 ACTGAGACTG CGCTGGATCC 260

(2) INFORMATION ON SEQ ID NO: 5:

(i) SEQUENCE IDENTIFIER:

(A) LENGTH: 259 base pairs

(B) AST: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

<ii) MOLECULE TYPE: cDMA

(iii) HYPOTHETICAL: NO

(iv) ANTISΞNSΞ: NO

(vi) ORIGINAL ORIGIN:

(I) ORGANΞLLE: chloroolast

(ix) FEATURE:

(A) NAME / KEY: misc_feature

(B) LOCATION: 1..6 (D) OTHER INFORMATION: / fur.ct ion = "Koni Schni tts te lle ' (ix) FEATURE:

(A) NAME / KEY: transit peptide

(B) LOCATION: 7..252

(ix) FEATURE: (A) NAME / KEY: misc feature

(B) LOCATION: 253 (D) OTHER INFORMATION: / product- "Fraπe shift insert"

(ix) FEATURE:

(A) NAME / KEY: misc feature

(B) LOCATION -.254..259 (D) OTHER INFORMATION: / function- "BanHI interface"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

GGTACCATGG CGTCTTCTTC TTCTCTCACT CTCTCTCAAG CTATCCTCTC TCGTTCTGTC 60

CCTCGCCATG GCTCTGCCTC TTCTTCTCAA CTTTCCCCTT CTTCTCTCAC TTTTTCCGGC 120

CTTAAATCCA ATCCCAATAT CACCACCTCC CGCCGCCGTA CTCCTTCCTC CGCCGCCGCC 180

GCCGCCGTCG TAAGGTCACC GGCGATTCGT GCCTCAGCTG CAACCGAAAC CATAGAGAAA 240

ACTGAGACTG CGGGGATCC 259 (2) INFORMATION ABOUT SEQ ID NO: 6:

(i) SEQUENCE LABEL:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc «" syr.the ic "

(iii) HYPOTHETICAL: NO

(iv) ANTISENSE: NO

(xi) SEQUENZ3ES FLASH: SEQ ID NO: 6: AAGGATCCAT GGGTAACAAC GTCGTCGTAC TGG 33

(2) INFORMATION ON SEQ ID NO: 7:

(i) SEQUENCE CHARACTERS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleotide

(C) STRANDFORM: Ξiπzelstrang

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc «" synthetic "

(iii) HYPOTHETICAL: NO

(iv) ANTISENSE: NO (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: AAGGATCCCG TACCAGAATT ACG 23

Claims

claims

1. Expression cassette containing, under genetic control of regulatory nucleic acid sequences, the coding nucleic acid sequence for a protein which imparts resistance to inhibitors of plant adenylosuccinate synthetase to a plant host.

2. Expression cassette according to claim 1, characterized in that the inhibitor is selected from alanosine, hadacidin, hydantocidin and metabolites and functionally equivalent derivatives thereof.

3. Expression cassette according to claim 1 or 2, characterized in that the coding nucleic acid sequence codes for a protein which comprises non-vegetable adenylosuccinate synthetase or a functional equivalent thereof.

4. Expression cassette according to claim 3, characterized in that the non-vegetable adenylosuccinate synthetase is derived from a microbial adenylosuccinate synthetase.

5. Expression cassette according to claim 4, characterized in that the microbial adenylosuccinate synthetase is derived from Escherichia coli, Bacillus subtilis, Haemophilus influencae, Dictyostelium discoideum, Saccharomyces pombe or Schizosaccharomyces pombe.

6. Expression cassette according to one of the preceding claims, characterized in that the coding nucleic acid sequence for a protein containing an amino acid sequence according to SEQ ID NO: 2 or a functional equivalent thereof or a nucleic acid sequence from residue + 7 to + 1305 according to SEQ ID NO-. l or a functional equivalent thereof.

7. Expression cassette according to one of the preceding claims, characterized in that the coding nucleic acid sequence also codes for a chloroplast-specific transit peptide.

8. Expression cassette according to claim 7, characterized in that the coding nucleic acid sequence for the transit peptide encodes plastid transketolase or a functional equivalent thereof.

9. Recombinant vector comprising an expression cassette according to one of claims 1 to 8.

10. The vector of claim 9, which corresponds substantially to the vector 5 pTP09-ASS.

11. Microorganism containing a recombinant vector according to claim 9 or 10.

10 12. Microorganism according to claim 11 from the genus Agrobacterium and in particular the species Agrobacterium tumefaciens.

13. Use of a vector according to one of claims 9 and 10 or a microorganism according to one of claims 11 and 12

15 for the transformation of plants, plant cells, tissues or parts.

14. Use according to claim 13, wherein the plants, plant cells, tissues or parts resistance to inhibitors

20 vegetable adenylosuccinate synthetase is mediated.

15. Use of a coding nucleic acid sequence as defined in one of claims 1 to 6 as a selection or marker gene in plants, plant cells, tissues or

25 parts.

16. Transgenic plant transformed with a vector according to one of claims 9 and 10 or with a microorganism according to one of claims 11 and 12, or transgenic cells, tissue,

30 parts or transgenic material.

17. Transgenic plant according to claim 16, selected from crops such as cereals, corn, soybeans, rice, cotton, sugar beet, canola, sunflower, flax, potato, tobacco, tomato,

35 rapeseed, alfalfa, lettuce and the various tree, nut and wine species.

18. A process for the production of transgenic plants according to one of claims 16 and 17, characterized in that

40 plant cells, tissue or parts or protoplasts transformed with a vector according to one of claims 9 and 10 or with a microorganism according to one of claims 11 and 12, which cultivated transformed cells, tissues, parts of plants or protoplasts in a growth medium

45 and possibly regenerated plants from the culture.

19. Expression product of an expression cassette defined in claims 1 to 8.

20. Expression product according to claim 19, characterized in that it is essentially an amino acid sequence of amino acid

+1 to + 432 according to SEQ ID NO: 2 or a functional equivalent thereof.

21. Expression kit for the expression of a foreign gene in a plant host, which comprises at least three expression cassettes, at least two expression cassettes of which, under the genetic control of regulatory nucleic acid sequences, each contain a frame shift sequence which differ from one another by insertion or deletion of one do not differ by 3 15 divisible number of nucleotides and shift the reading frame for a downstream coding nucleic acid sequence by one or two nucleotides.

22. Expression kit according to claim 21, characterized in that 20 each expression cassette a coding sequence for

Contains chloroplast-specific transit peptide and the frame shift sequence optionally contained follows its 3 'end.

23. Expression kit according to claim 22, containing expression cassettes, each of the following nucleotide sequences: SEQ ID NO: 3 from nucleic acid residue +7 to +252 SEQ ID NO: 4 from nucleic acid residue +7 to +254 SEQ ID NO: 5 from Nucleic acid residue +7 to +253

30 or a functional equivalent thereof.

24. Use of an expression kit according to one of claims 21 to 23 for transforming a plant host.

35

40

45