EP0805865A1 - Deoxyribonucleic acid coding for glutathion-s-transferase and its use - Google Patents

Abstract

The present invention relates to a novel deoxyribonucleic acid (DNA) and its use for the transformation of vectors, host organisms and plants and for the breeding of plants with a high resistance to herbicides.

Description

FOR DESOXYRIBONUCLEIC ACID ENCODING GLUTATHION-S-TRANSFERASE AND THEIR USE

The present invention relates to a new deoxyribonucleic acid (DNA) and its use for the transformation of vectors, host organisms and plants and for the production of plants which have increased resistance to herbicides.

It is already known to genetically modify plants in such a way that they have increased resistance to certain herbicides. This makes it possible to use herbicides which, with a low selectivity, would otherwise have advantageous properties, in the crops of those plants which in the original non-transgenic form would be damaged by these herbicides. The provision of herbicide-resistant plants thus increases the selection of herbicides which can be used, and it is also possible in many cases to manage with relatively small amounts of herbicide, e.g. if the undesired plants can only be combated after they have emerged when the respective damage threshold has been reached. There is therefore a considerable need to produce new crop plants which are more resistant to other herbicides.

Glutathione-S-transferases are multifunctional proteins that play an essential role in the detoxification of cytotoxic substances. The enzymes catalyze the conjugation of reduced glutathione to electrophilic, hydrophobic

Substrates that can be of natural or synthetic origin.

The physiological substrates of plant glutathione S transferases and their role in the metabolism of plants are not known in detail. However, the participation of these enzymes in the detoxification and thus in the selectivity mechanism for a number of important herbicides from the group of

Thiocarbamates, chloroacetanilides and S-triacines: Mozer TJ., Tiemeier DC, Jaworski EG, Biochemistry 22: 1068-1072 (1983); Moore RE, Davies M S., O'Connell KM, Harding EJ, Wiegand RC, Tiemeier DC, Nucleic Acids Res. 14 7227-7235 (1983), Grove G, Zarlengo RP, Timmermann KP, Li N, Tarn MF, Tue CPD, Nucleic Acids Res 16 425-438 (1988)

The new deoxyribonucleic acid has now been found, which codes for the new protein glutathione-S-transferase IIIc (hereinafter "GSTIIIc"), which has the amino acid sequence listed in SEQ ID NO 2 (the new DNA according to the invention in the following) is referred to as "GSTIIIc-DNA")

It has furthermore been found that plants in whose genome the new GSTIIIc DNA was incorporated have an increased resistance to herbicides, preferably heteroaryloxyacetamide herbicides, in comparison with the corresponding “starting plants”

The new GSTIIIc DNA was isolated from mutin maize (Zea mais). This DNA codes for the protein GSΗIIc of the amino acid sequence according to SEQ ID NO 2. In plant cells, 2 molecules of the protein GSΗIIc spontaneously form the dimeric active enzyme (hereinafter referred to as "GSTIIIc enzyme"), which ensures the detoxification of the herbicide used and thus the plants against that

Herbicide resistant

The GSTIIIc DNA preferred according to the invention has the sequence listed in SEQ ID NO: 1

Also preferred according to the invention is the GSTIIIc DNA as contained on the vector plasmids pET3a-GSTIIIc and pSS-GSTIIIc described below

The new GSTIIIc DNA can be in the form of a single strand or in the form of the double strand which additionally contains a strand which is complementary to the respective single strand

In a preferred embodiment of the present invention, the

GSTIIIc-DNA at the 5'-end preceded by a promoter which is effective in plants. The usual promoters which are active in plants can be used. The promoter of the gene of the small subunit of ribulose-1,5-b-phosphate-carboxylase (rbsc) is exemplary. see EMBO Journal, vol 5 No. 9, 2063-2071 (1986)) Promoters of plant viruses are preferably used, where in the CaMN 35S RΝA promoter should be mentioned as an example. The known construct from the CaMN 35S enhancer and the CaMN 35S promoter ("CaMV 35S double promoter") which follows in 5'-3 'order is particularly preferably used. A corresponding preferred construct from the GSTIIIc-DΝA and the CaMN double promoter is contained on the vector plasmid pSS-GSTMc explained below. However, it is also possible to use the natural promoter which regulates the expression of the GSTIIIc-DΝA in maize, variety mutin.

The type of the 3'-termination sequence following the GSTTIIc-DΝA in 5'-3 'order is largely variable and is not of critical importance for the present invention. Plant 3'-termination sequences are preferably used. The natural termination sequence of the GSTÜIc gene from maize, variety Mutin, can also be used as an example.

Also part of the present invention is the new protein GSΗIIc with the amino acid sequence according to SEQ ID ΝO: 2 and its likewise new dimer

(GSTIIIc-Enzy), which, as already explained above, spontaneously arises from 2 molecules of the protein GSTIIIc after their formation in plant cells.

The DNA sequences or protein according to the invention (optionally in the dimeric form) also include the DNA sequences or protein sequences derived from this DNA or from this protein. Under DNA or

Proteins with derived sequences are to be understood as DNA or protein which still have the essential features of the GSTIIIc-DNA or the protein GSTIIIc (possibly as a GSTIIIc enzyme in the dimeric form), which are therefore essentially equivalent, i.e. produce the herbicide resistance according to the invention to a sufficient extent in plants. Derived in such

Sequences may be missing individual DNA's, codons, and / or partial DNA sequences or individual amino acids or partial amino acid sequences (in the case of DNA, for example, by using restriction enzymes) and / or by other, essentially equivalent DNA, codons and DNA Partial sequences or amino acids or partial amino acid sequences can be replaced. Such modifications can be due to the degeneration of the genetic code or result from the manipulation of the GSTIIIc DNA or the protein GSTIIIc or the GSTIIIc enzyme. The DNA according to the invention can also contain DNA's, codons or DNA sequences which facilitate its handling, for example so-called Linkers or which of such linkers remain after manipulations (eg after cuts with restriction enzymes). The GSTIIIc DNA or the protein GSTIIIc or the GSTIIIc enzyme can be of natural origin or can be partially or completely in synthesized form.

Part of the present invention is also a recombinant prokaryotic or eukaryotic DNA which contains the new GSTIIIc DNA or a DNA derived therefrom as "foreign" or "additional" DNA. Examples include viral DNA, microbial DNA (in particular bacterial DNA, such as DNA from Escherichia coli or Agrobacterium tumefaciens) and vegetable DNA.

The GSTHIc DNA according to the invention and the DNA sequences derived therefrom and the recombinant prokaryotic or eukaryotic DNA and the DNA sequences derived therefrom can be used as "foreign" or "additional" DNA in vectors (in particular plasmids, cosmids or phages), in transformed or transgenic microorganisms (preferably bacteria, such as Escherichia coli or Agrobacterium tumefaciens) as well as in transformed or transgenic plant cells and plants or in their DNA. These vectors, microorganisms, plant cells and plants and their DNA are part of the present invention. Like the recombinant prokaryotic and eukaryotic DNA, they can be obtained by the person skilled in the art with knowledge of the present description, in particular SEQ ID NO: 1 and SEQ ID NO: 2, according to the generally known and / or customary methods and methods.

The vector plasmids pET3a-GSTIIIc and pSS-GSTIIIc may be mentioned as particularly preferred vectors. Both vectors contain the GSTIIIc DNA according to the invention as shown in SEQ ID NO: 1.

The plasmid pET3a-GSTIIIc contains the GSTIIIc DNA on a Ndel / BamHI-

Fragment. To construct this plasmid, the GSTIIIc DNA according to SEQ ID NO: 1 was cloned into the Ndel and BamHI interfaces of the vector pET-3a (Novagen / Madison) (Studier FW, Moffatt BA, J. Mol. Biol. 189: 1 13- 130; Studier FW, J. Mol. Biol. 219: 37-44 (1991)). This plasmid (5306 bps) is shown in Fig. 1. The direction of the arrow shows the direction of the promoter and the gene or the GSTIIIc DNA with the start codon ATG. "Amp" stands for the ampicillin resistance gene. To construct the plasmid pSS-GSTIIIc, the GSTIIIc DNA as Xbal / BamHI fragment was purified from the vector pET3a-GSTIIIc and cloned into the plasmid Bluescript-SKII (Xbal / BamHI-linearized; Stratagene). The GSTIIIc DNA was then isolated from the plasmid Bluescript-SKII-GSTIIIc obtained in this way via an Sstl / Smal restriction cleavage and ligated into the vector pRTlOl (Sstl / Smal-linerized; Töpfer et al. 1987). The GSTIIIc DNA was then purified as an EcoRI / Smal fragment from the vector pRTlOl-GSTTIIc thus obtained and cloned into the binary vector pSS (EcoRI / Smal-linearized; Voß et al. 1994). In the resulting pSS-GSTIIIc vector, the coding GSTIIIc DNA is under the control of a duplicated 35S RNA promoter from CaMV. The plasmid pSS-GSTIIIc (10000 bps) is shown as Fig. 2.

If necessary, the person skilled in the art can isolate the GSTIIIc DNA according to the invention from the plasmids mentioned by the generally customary methods and methods.

The Escherichia coli strains DS pET3a-GSTfflc and DS pSS-GSΗIIc and their mutants may be mentioned as particularly preferred transformed or transgenic microorganisms according to the invention. The strain DS pET3a-GSTIIIc contains the plasmid pET3a-GSΗÜc and the strain DS pSS-GSTIIIc contains the plasmid pSS-GSTIIIc. Mutants according to the invention are those microorganisms which still contain the features which are essential for the implementation of the invention, that is to say in particular also contain the plasmids pET3a-GSTIIIc and / or pSS-GSTIIIc or DNA sequences derived therefrom. These strains can be propagated according to the generally accepted methods. The plamides pET3a-GSTIIIc and pSS-GSTIIIc can also be isolated from these microorganisms by the generally customary methods.

The Escherichia coli strain pET3a-GSTIIIc was obtained from the German Collection of Microorganisms (DSM), Mascheroder Weg lb, D-38124 Braunschweig, Federal Republic of Germany in accordance with the provisions of the Treaty of Budapest on the international recognition of the deposit of microorganisms for the Purposes of patent procedures deposited (Filing date: January 17, 1995). The strain received the deposit number DSM 9677.

As already explained above, the present invention also includes transgenic plants and plant cells (including protoplasts) and plant Parts (such as callus, leaves, stems, bleeds, parts of blood, roots, tubers, seeds and other propagation material) of such transgenic plants which contain in their genome the GSTIIIc DNA or DNA sequences derived therefrom and / or a recombinant prokaryotic or eukaryotic DNA according to the invention contained as "foreign" or "additional" DNA

The transgenic plants according to the invention also include the progeny of the transgenic plants obtainable according to the invention and the cross-breeding results with other plants and their progeny as long as these transgenic plants contain the GSTIIIc DNA or DNA sequences derived therefrom as "foreign" or "additional" DNA

Preferred transgenic plants are those plants which contain the GSTIIIc DNA according to SEQ ID NO 1 as "foreign" or "additional" DNA in their genome

Particularly preferred transgenic plants are those plants which have in their genome the construct of the CaMV 35S double promoter and the GSTIIIc

Contain DNA as SEQ ID NO 1 as "foreign" or "additional" DNA

Very particularly preferred transgenic plants are those plants which contain in their genome the GSTIIIc DNA according to SEQ ID NO: 1 and / or the construct from the CaMV 35S double promoter and the GSTIIIc DNA according to SEQ ID NO 1 as "foreign" DNA. So far, no plants have become known, apart from the maize variety Mutin, which contain a DNA corresponding to GSTIIIc DNA in their genome

In connection with the present invention, “foreign” DNA is to be understood as such DNA which is not naturally contained in a certain prokaryotic or eukaryotic (including plant) genome, but is only incorporated into this genome by human intervention (transformation) "Additional" DNA is a DNA that is already present in the respective prokaryotic or eukaryotic genome, but is added to this genome by human intervention (transformation). The "foreign" or "to" ¬ sentence "DNA can be incorporated in one or more copies depending on the need and type of the present case As has already been explained, the main objective of the present invention is that new plants are produced which have an increased resistance to herbicides, preferably to heteroaryloxyacetamide herbicides.

Thus, novel transgenic plants according to the invention are preferred which, in addition to the properties stated above (content of “foreign” or “additional” GSTIIIc DNA), have an increased resistance to herbicides, in particular, in comparison to the corresponding non-transgenic plants against heteroaryloxyacetamide herbicides. Cultures of these transgenic plants can thus be treated with the herbicides to control unwanted plants without damaging the crop plants.

The increased herbicide resistance of the transgenic plant cells and plants according to the invention is important for agriculture and forestry, for ornamental plant cultivation, medicinal plant cultivation and plant cultivation.

The present invention thus also relates to a process for the production of transgenic plant cells (including protoplasts) and plants (including plant parts and seeds) with increased resistance to herbicides, which is characterized in that

(a) one or more copies of the GSTIIIc-DNA and / or recombinant DNA according to the invention, which contain the GSTIIIc-DNA, which code for the protein GScIIc, are inserted into the genome of plant cells (including protoplasts) and optionally

(b) completely transformed plants are regenerated from the transformed plant cells (including protoplasts) and, if appropriate, propagated and if appropriate

(c) the desired plant parts (including seeds) are obtained from the transgenic plants of the parent generation or further generations obtained therefrom.

Process steps (a), (b) and (c) can be carried out in a conventional manner by known processes and methods. Transgenic plant cells (including protoplasts) and plants (including plant parts and seeds) which contain the GSTIIIc DNA one or more times as “foreign” or “additional” DNA, and also such transformed plant cells and plants which can be obtained by the above processes, also belong to the present invention.

Parts of the present invention are also:

(a) use of the GSTIIIc DNA and / or the recombinant DNA according to the invention and / or the recombinant vectors according to the invention and / or the transformed microorganisms according to the invention for the transformation of plant cells (including protoplasts) and plants (including plant parts and seeds), the

(b) Use of the transgenic plant cells according to the invention (including protoplasts) and plants (including plant parts and seeds) for the production of propagation material and for the production of new plants and their propagation material, the

c) Use of the cDNA or its parts contained on the plasmid pET3a-GSTIIIc and of the DNA sequences corresponding to the sequence information according to the sequence listing SEQ ID NOJ for the determination of DNA which code for GSTIIIc protein or GSTIIIc enzyme in plants and (in general) in the generation of transgenic plant cells (including protoplasts) and plants (including plant parts and seeds) and the use of the protein sequence encoded by the GSTIIIc DNA of pET3a-GSTIIIc and the protein according to SEQ ID NO: 2 in the isolation and the Detection of the GSTIIIc-DNA (eg by the usual antibody technique).

A number of different methods are available for using the GSTIIIc DNA, optionally as a construct with the CaMV35S double promoter, as "foreign" or "additional" DNA in the genetic material of plants or plant cells. The gene transfer can be carried out according to the generally known methods Methods are carried out, the person skilled in the art having the appropriate method without

Can identify difficulties The Ti plasmid from Agrobacterium tumefaciens is available as a particularly inexpensive and widely applicable vector for the transfer of "foreign" or "additional" DNA into genomes of dicotyledonous and monocotyledonous plants. The genetic material which codes for the protein GSTIIIc is optionally used together with regulatory DNA sequences in the T-DNA of suitable Ti plasmids (for example Zambryski et al., 1983) and by infection of the plant, infection of parts of plants or Plant tissues, such as, for example, from leaf disks, stems, hypocotyls, cotyledons, meristems and tissues derived therefrom, such as, for example, secondary embryos and calli, or by coculturing protoplasts with Agrobacterium tumefaciens.

An alternative is the incubation of purified DNA, which contains the desired gene or the desired DNA, in plant protoplasts (eg Hain et al., 1985; Krens et al., 1982; Paszkowski et al., 1984) in the presence of polycations or Calcium salts and polyethylene glycol.

The DNA uptake can also be favored by an electric field (electroporation) (e.g. Fromm et al., 1986).

The DNA can also be introduced in a known manner via plant pollen by "bombarding" pollen or other plant parts with physically accelerated particles which carry the DNA (cf. EP-A 0 270 356).

The regeneration of the plants takes place in a known manner with the help of suitable ones

Culture media (e.g. Nagy and Maliga 1976).

In a preferred embodiment of the method according to the invention, the GSTIIIc-DNA from the plasmid pET3a-GSTIIIc is cloned into a binary expression vector (e.g. Voss et al. (1994)). The chimeric gene construct is then transferred to Agrobacterium tumefaciens (Koncz and Schell 1986).

Alternatively, the chimeric gene construct on the vector pSS-GSTIIIc is transferred in the usual way by direct gene transfer to plant protoplasts (for example Hain et.al. 1985). The plasmid can be in circular, but preferably in linear form. When using this plasmid with reporter gene, kanamycin-resistant protoplasts are then checked for expression of GSTIIIc

Transformed (transgenic) plants or plant cells are produced using the known methods, e.g. by leaf disc transformation (e.g. Horsch et al. 1985), by coculture regenerating plant protoplasts or cell cultures with Agrobacterium tumefaciens (e.g. Marton et al. 1979, Hain et al. 1985) or by direct DNA transfection. Resulting transformed plants are either selected by selection for the expression of the reporter gene, e.g. by phosphorylation of kanamycin sulfate in vitro (Reiss et al. 1984; Schreier et al. 1985) or by the expression of nopaline synthase (according to Aerts et al. 1983) or by GSΗIIc by Northern blot analysis and Western blot analysis grasslands. The protein GSTIIIc can also be detected in a known manner in Western blot analyzes with the aid of specific antibodies in transformed plants.

The cultivation of the transformed plant cells and the regeneration to complete plants is carried out according to the generally customary methods with the aid of the appropriate nutrient media.

Both the transformed plant cells and the transformed plants which contain the GSTIIIc DNA according to the invention and which are constituents of the present invention show a considerably higher resistance to

Herbicides, especially against heteroaryloxyacetamide herbicides.

In connection with the present invention, the term "plants" means both complete plants and parts of plants, such as leaves, seeds, tubers, cuttings, etc. "Plant cells" include protoplasts, cell lines, plant calli, etc. "Propagation material" means plants and plant cells which can be used to multiply the transformed plants and plant cells and is therefore also part of the present invention

Plants which can be given increased resistance to herbicides by incorporating (transforming) the GSTIIIc DNA according to the invention include practically all plants. A particular need for

Resistance is naturally produced in crops such as forest plants, such as spruce, fir, Douglas fir, pine, larch, beech and oak, and Plants supplying food and raw materials, e.g. cereals (in particular wheat, rye, barley, oats, millet, rice and corn), potatoes, legumes (such as legumes and in particular alfalfa, soybeans), vegetables (in particular cabbages and tomatoes), fruit (in particular apples , Pears, cherries, grapes, citrus, pineapple and bananas), oil palms, tea, cocoa and coffee bushes, tobacco, sisal and cotton as well as medicinal plants such as rauwolfia and digitalis. Potatoes, sugar beets, sugar cane, cereals such as wheat and barley and sorghum, and rice are particularly preferred. The GSTIIIc DNA according to the invention is preferably incorporated into the genome of plants as "foreign" DNA.

Preferred herbicides, against which an increased herbicide resistance can be generated according to the invention, belong to the group of heteroaryloxyacetamides. The heteroaryloxyacetamides of the general formula (I) are particularly preferred.

Het-O-CH ₂ -CO-NR ¹ R ² (I)

in which

Het stands for an optionally substituted heteroaromatic radical with preferably 5 ring members, which preferably contains at least one nitrogen atom and one sulfur or oxygen atom, the particularly preferred radical being 5-trifluoromethyl-1,3,4-thiadiazol-2-yl Rest is called;

R ¹ for optionally substituted alkyl or alkoxy (each with preference

1-4 carbon atoms); and

R stands for an optionally substituted aryl radical (preferably phenyl radical, which is preferably substituted by halogen).

Such herbicides are already known (see e.g. EP-A-18 497 and the corresponding US Pat. No. 4,645,525, EP-A-94 541 and the corresponding one

U.S. Patent No. 4,585,471 and EP-A-348,737 and corresponding U.S. Patent No. 4,968,342). The herbicides mentioned in these patent applications or patent documents are particularly preferred in connection with the present invention. Resistance to the herbicide mentioned in EP-A-348 737 and in the corresponding US Pat. No. 4,968,342 with the chemical name (5-trifluoromethyl-1,3,4-thiadiazol-2-yl - oxy) -acetic acid-N-isopropyl-N- (4-fluorophenyl) -amιd (proposed common name thiafluamide) This resistance allows the use of the herbicide mentioned also in those crops which are not in the non-resistant form in a acceptable damage from the herbicide

The present invention will be explained in more detail using the following exemplary embodiments

I. Isolation of GSTIIIc DNA from Maize

The known methods and methods of molecular biology are used to isolate the GSTIIIc DNA, as described, for example, in the following manual: Maniatis T., Fritsch E.F., Sambrook J Molecular Cloning, A Laboratory Manual, Cold Spring Habor Laboratory, Second Edition 1989

To isolate the GSTIIIc DNA, the protein from etiolated maize seedlings (zea mais), mutin variety, is first purified (1) and the amino acid sequence is determined completely by protein sequence analysis (2). Also from maize seedlings, mRNA is isolated (3) and by Reverse transcriptase enzymatically rewritten into cDNA (4) The cDNA obtained in this way is used as a template in the polymerase chain creatone (Mullis KB, Faloona FA, (1987) Methods Enzymol 155 335-350) for the isolation of the GSTIIIc DNA

1. Isolation of the GSTIHc protein from maize, variety, mutin

To purify the GSTIIIc protein, maize seedlings are broken down and mixed with 0.2 M Tπs / HCl pH 7.8, 1 M EDTA (2 ml / g filter weight). The suspension is centrifuged and the protein fraction in the supernatant is saturated with fractionated ammonium sulfate 30% and 70% obtained. The GSTIIIc protein is isolated by chromatography on the following columns with the buffer conditions listed A) Sephadex G-100 (separation medium based on dextran), v = 500 ml (Pharmacia)

Buffer A: 50 mM potassium phosphate pH 7.3

B) DEAE-Sepharose (cross-linked agarose matrix with covalently bound diethylamino-ethyl group), v = 50 ml (Pharmacia)

Buffer AJ0 mM potassium phosphate pH 7.3 Buffer B: 1.0 M potassium phosphate pH 7.3 Gradient: 0-100% B in 500 ml

C) Glutathione-sulfobromophthalein agarose, v = 20 ml (Sigma) buffer A: 50 mM potassium phosphate pH 7.3

Buffer B: 50 mM potassium phosphate pH 8.0, 5 mM glutathione

D) Mono Q HR 5/5 (anion exchange material based on cross-linked agarose with charged -CH ₂ N (CH ₃ ) ₃ + groups, particle size of 10 + 5 μm), v = 1 ml (Pharmacia) buffer A: 20 mM Tris / HCl pH 7.5

Buffer B: 20 mM Tris HCl pH 8.0, 1.0 M NaCl gradient: 0-25% B in 20 ml

2. Protein sequence analysis of the GSTIIIc protein

The GSTIIIc protein isolated from maize (variety mutin) is reduced, carboxymethylated and dialyzed against 0.2 M ammonium hydrogen carbonate

(Glazer A.N., Delange RJ., Sigman D.S., (1975) Chemical Modification of Proteins, Elsevier Biomedical Press, Amsterdam). The subsequent cleavage of the protein with the endoproteases Asp-N (sequencing grade, Boehringer Mannheim) or trypsin (TPCK-treated, Worthington) takes place at 23 ° C. for 16 hours with a protein protease ratio of 1: 100.

The cleavage reactions are terminated by adding 0J% trifluoroacetic acid. Insoluble peptides are centrifuged off, the soluble peptides are separated from one another by reversed-phase HPLC under the following conditions:

Column: Vydac C 18 (silica-based separating gel with aliphatic chains (C ₁₈ )), 0.46 cm x 25 cm Eluent A OJ% trifluoroacetic acid

Eluent B OJ% trifluoroacetic acid, 90% acetonitrile

Gradient 0 - 60% B in 40 min

Flow rate 0.25 ml / min

The amino acid sequence of the isolated peptides is applied using the

Biosystems protein sequencers 470A and 473A determined The complete protein sequence of the GSTIIIc protein is shown in SEQ ID No 2

3. Isolation of poly (A) + mRNA from maize (variety Mutin)

The GSTIIIc DNA, the mRNA and the corresponding cDNA contain nucleotide sequences that can be derived from one another. To isolate the GSTIIIc DNA, polyadenyherized mRNA from localized maize seedlings is first prepared. The isolation is carried out using Dynabeads Oligo (dT) _{25 in} accordance with the manufacturer's protocol (Dynal, Oslo / Norway; Jakobsen KS, Breivold E, (1990 ) Nucleic Acids Res 18.3669) This method of purifying poly (A) + RNA is based on the base pair bond between the poly (A) + residues at the 3 'end of mRNA and oligo (dT) residues that are covalent on the surface are bound by magnetic metal balls (Dynabeads) 0.2 g of plant material are used per milligram of Dynabeads. The mRNA isolated with this method is used without further purification steps for the enzymatic synthesis of cDNA

4. Enzy atic synthesis of cDNA

The preparation of cDNA is carried out using the "First Strand cDNA Synthesis Kit" (Pharmacia PL Biochemicals Ine). This method is based on the enzymatic activity of viral DNA polymerases (reverse transcriptases), which synthesize DNA according to an RNA matrix ( Maniatis T, Fπtsch EF, Sambrook j, (1982) Molecular Cloning A laboratory manual, Cold Spring Harbor Laboratory)

According to the manufacturer's instructions, the reaction is carried out as follows 5 μm poly (A) + mRNA from maize, type Mutin were mixed with:

1 μm 0.5 M dithioeritrite

I μm d (T) ₁₆ _ ₁₈ primer

I I μm reaction mixture (bulk reaction mix) consisting of:

Murine reverse transcriptase, 135 mM Tris / HCl, pH 8.3, 204 mM KCl,

27 mM MgCl ₂ , 0.24 mg / ml BSA, 5.4 mM dATP, dCTP, dGTP, dTTP.

After a reaction time of one hour at 37 ° C, the mRNA is removed by alkaline hydrolysis. The synthesized cDNA is precipitated and used as a template for the polymerase chain reaction.

Amplification, cloning and sequencing of the GSTIIIc DNA

To isolate the GSTIIIc DNA, the nucleotide sequence coding for GSTIIIc is first amplified with the aid of the polymerase chain reaction (Mullis K.B., Faloona F.A., (1987) Methods Enzymol. 155: 335-350). The cDNA produced from maize mRNA serves as the template for the reaction. For specific enrichment of the GSTHIc DNA, the primers 1

(forward) according to SEQ ID NO: 3 and primer 2 (reverse) according to SEQ ID No: 4.

Reaction batches with the following composition are produced:

5 μm cDNA (200 ng / μl) 1 μm 50 μM primer 1

1 μm 50 μM primer 2

4 µl 250 µM dATP, dCTP, dGTP, dTTP

5 ul 200 mM Tris HCl, pH 8.8, 100 mM KCl, 60 mM (NH ₄ ) ₂ SO ₄ , 15 mM Mg Cl ₂ , 1% Triton X-100 34 ul H, O

The polymerase chain reaction takes place in a GeneAmp PCR System 9600 thermal cycler (Perkin Elmer). 1 μl of Pfu-Dna polymerase (Stratagene, 2500 U / ml) is added as the heat-stable polymerase. Overall 35 reaction cycles with the following temperatures and reaction times were carried out 45 sec at 94 ° C, 45 sec at 58 ° C, 45 sec at 72 ° C. The amplified DNA fragment, which contains the nucleotide sequence coding for GSTIIIc, is obtained by agarose gel electrophoresis purified and, as already described, cloned into the vector pET 3a. The complete DNA

Sequence of GSTIIIc DNA (SEQ ID NO-1) is determined using the Sequenase DNA Sequencing Kit (United States Biochemical / Cleveland) (Sanger F., Coulson R, (1975) J Mol Biol 94 441-448).

Transformation of rice

The transformation of rice (Oryza sativa) can be carried out in accordance with the methods described in the following references

Zhang H M., Yang H., Rech E.L, Golds T J, Davis A.S, Mulligan BJ., (1988) Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts. Plant Cell Rep 7 379-384

Zhang W., Wu R., (1988) Efficient regeneration of transgenic plants from rice protoplasats and correctly regulated expression of the foreign gene in the plant. Theor. Appl. Gen 76 835-840

Shimamoto K, Terada R, Izawa T., Fujimoto H, (1989) Fertile transgenic rice plants regenerated from transformed protoplasts Nature 338.274-276

Datta S K, Peterhans A, Datta K, Potrykus I, (1990) Genetically engineered fertile indica-πce recovered from protoplasts Biotechnol 6 736-740

Hayashimoto A., Li Z, Murai N, (1990) A polyethylene glycolmediated transformation system for production of fertile transgenic rice plants Plant

Physiol 93 857-863

The method of Hayashimoto et al (1990) was used without changes for the transfer of the vector pSS-GSTIIIc into rice Kanamycin-resistant transformants were checked in Northern blot experiments for the expression of GSTIIIc. The formation of the GSTIIIc protein was detected by specific antibodies. The enzymatic activity of the protein could be shown in the crude extract of transformed rice plants by detecting the enzyme-catalyzed modification of the herbicide (5-trifluoromethyl-l, 3,4-thiadiazol-2-yl-oxy) -acetic acid-N -isoproypl-N- (4-fluoφhenyl) amide.

In comparison to non-transformed controls, transgenic rice plants "show resistance to the herbicide mentioned.

ID. Transformation of tobacco

a) Culture of tobacco sprouts and isolation of tobacco protoplasts

Nicotiana tabacum (Petit Havana SRI) is propagated as a sterile sprout culture on hormone-free LS medium (Linsmaier and Skoog 1965). At intervals of approx. 6-8 weeks, shoot sections are placed on fresh LS

Medium implemented. The shoot cultures are kept in a culture room at 24-26 ° C under 12 h light (1000-3000 lux).

To isolate leaf protoplasts, approx. 2 g of leaves (approx. 3-5 cm long) are cut into small pieces (0.5 cm x 1 cm) with a fresh razor blade. The leaf material is in 20 ml enzyme solution consisting of K3

Medium (Nagy and Maliga 1976), 0.4 M sucrose, pH 5.6, 2% cellulase RIO (Serva), 0.5% Macerozym RIO (Serva) for 14-16 h at room temperature. The protoplasts are then separated from cell remnants by filtration through 0.30 mm and 0J mm steel sieves. The filtrate is centrifuged at 100 xg for 10 minutes. During this centrifugation, intact protoplasts float and collect in a band at the top of the enzyme solution. The pellet from cell residues and the enzyme solution are aspirated with a glass capillary. The pre-cleaned protoplasts are made up to 10 ml with fresh K3 medium (0.4 M sucrose as an osmotic agent) and floated again. The washing medium is suctioned off and the protoplasts are diluted to 1-2 × 10 ⁵ / ml for culture or subsequent infection with agrobacteria (coculture). The protoplast concentration is determined in a counting chamber.

b) Construction of the vector pSS-GSTIIIc and transfer into Agrobacterium tumefaciens

The GSTIIIc DNA and the Shine-Delgarno sequence were cut out as an Xbal BamHI fragment from the vector pET3a-GSTIIIc, purified and into the plasmid Bluescript II Sk +/- (Stratagene, La Jolla, California), hereinafter referred to as pBS -SKII, cloned The interfaces Xbal and BamHl were then used

GSTIIIc-DNA cut out of the vector pBS-SKII-GSTIIIc via the interfaces Sstl and Smal, isolated and into the vector pRTl O l (Sstl / Smal linearized) ligated (Töpfer R, Matzeit V., Gronenborn B., Schell J., Steinbiß HH, (1987) Nucleic Acids Res. 14: 5890).

The GSTIIIc DNA was then purified as an EcoRI / Smal fragment from the vector pRTl01 -GSTIIIc and linearized into the expression vector pSS (EcoRI / Smal, cloned (Voss A et al, Molec. Breeding 1: 15-26 (1995)) .

Any other expression vectors and intermediate vectors which have appropriate interfaces can be used instead of the vectors mentioned, it being easy for a person skilled in the art to make a suitable selection based on the above information. The resulting intermediate vector pSS-GSTIIIc, which contains the GSTIIIc DNA, is transferred to Agrobacterium tumefaciens, which contains a functional vir region (Koncz and Schell 1986, van Haute et al. 1983).

c) Transformation of regenerating tobacco protoplasts by co-culture with Agrobacterium tumefaciens

The method of Marton et al. 1979 with little ones

Changes used. The protoplasts are isolated as described and at a density of l-2xl0 ⁵ / ml in K3 medium (0.4 M sucrose, 0.1 mg / ml NAA, 0.2 mg kinetin) for two days in the dark and one or two days incubated under low light (500 lux) at 26 ° C.

As soon as the first division of the protoplasts occurs, 30 μl of an agrobacterium suspension according to b) are added to 3 ml of regenerating protoplasts in minimally A (Am) medium (density approx. 10 ⁹ agrobacteria / ml). The coculture time is 3-4 days at 20 ° C in the dark. The tobacco cells are then filled into 12 ml centrifuge tubes, diluted to 10 ml with sea water (600 mOsm / kg) and pelleted at 60 xg for 10 minutes. This washing process is repeated 1-2 more times to remove most of the agrobacteria. The Zeil suspension is at a density of 5 x 10 ⁴ / ml in K3 medium (0.3 m sucrose) with 1 mg / 1 NAA (naphthyl-1-acetic acid), 0.2 mg / 1 kinetin and 500 mg / 1 of the cephalosporin antibiotic cefotaxime. The cell suspension is diluted every week with fresh K3 medium and the osmotic value of the medium is gradually increased by 0.05 M sucrose (approx. 60 mOsm / kg) per Week reduced. The selection with kanamycin (100 mg / 1 kanamycin sulfate (Sigma), 660 mg / g active km) is started 2-3 weeks after the coculture in agarose "bead type culture" (Shillito et al 1983). Colonies resistant to kanamycin can be distinguished from the background of remaining colonies 3-4 weeks after the start of the selection.

d) Direct transformation of tobacco protoplasts with DNA. Calcium nitrate-PEG transformation.

In a Petri dish, approximately 10 ⁶ protoplasts in 180 μl K3 medium are carefully mixed with 20 μl aqueous DNA solution containing 20 μg plasmid pSS-GSTIIIc. The plasmid pSS-GSTIIIc can be obtained by known methods from the plasmids pET3a-GSTIIIc, pRTlOl, pBS-SKII and pSS. Then 200 μl of fusion solution (0J M calcium nitrate, 0.45 M mannitol, 25% polyethylene glycol (PEG 6000), pH 9) are carefully added. After 15 minutes, 5 ml of washing solution (0.275 M

Calcium nitrate pH 6) added and after a further 5 minutes the protoplasts are transferred into a centrifuge tube and pelleted at 60 x g. The pellet is taken up in a small amount of K3 medium and cultivated as described in the next section. Alternatively, the protoplasts according to Hain et al. 1985 are transformed.

e) Culture of protoplasts incubated with DNA and selection of kanamycin-resistant calli:

A modified "bead type culture" technique (Shillito et al. 1983) is used for the culture and selection of kanamycin-resistant colonies described below. One week after treatment of the protoplasts with DNA (cf. d), 3 ml of the Zeil suspension are mixed with 3 ml of K3 medium (0.3 M sucrose + hormones; 1.2% (Seaplaque) LMT agarose (low melting agarose, marine Colloids) are mixed in 5 cm Petri dishes, for this purpose agarose is dry autoclaved and briefly boiled in the microwave after adding K3 medium. After the agarose has solidified, the agarose disks ("beads") with the embedded tobacco microcalli are added in 10 for further culture and selection cm Petri dishes transferred and each 10 ml K3 medium (OJ M sucrose, 1 mg / 1 NAA, 0.2 mg / 1 Kinetin) and 100 mg / 1 kanamycin sulfate (Sigma) added. The liquid medium is changed every week. The osmotic value of the medium is gradually reduced.

The exchange medium (K3 + Km) is reduced by 0.05 M of saccharide (approx. 60 mOsm) per week.

Scheme of selection of kanamycin-resistant tobacco colonies after DNA transformation:

0.4 M 0J M 0.25 M 0.20 M 0.15 M 0.10 sucrose in the liquid medium

E S K

Weeks after DNA uptake

(K3 medium 1 mg / 1 NAA, 0.2 mg / 1 kinetin)

A = DNA uptake E = embedding in agarose

S = selection with kanamycin (100 mg / 1 kanamycin sulfate) K = kanamycin-resistant colonies can be clearly distinguished from the background

e) Regeneration of kanamycin-resistant plants:

As soon as the kanamycin-resistant colonies have reached a diameter of approx. 0.5 cm, half is placed on regeneration medium (LS medium, 2% sucrose, 0.5 mg / 1 benzylaminopurine BAP) and exposed to 12 h light (3000- 5000 lux) and 24 ° C in the culture room. The other half is propagated as callus culture on LS medium with 1 mg / 1 NAA, 0.2 mg / 1 kinetin, 0.1 mg / 1 BAP and 100 mg / 1 kanamycin sulfate. When the regenerated shoots are about 1 cm in size, they are cut off and placed on 1/2 LS medium (1% sucrose, 0.8% agar) without growth regulators for rooting. The shoots are rooted on 1/2 MS medium with 100 mg / 1 kanamycin sulfate and later converted into soil. g) Transformation of leaf disks by Agrobacterium tumefaciens

For the transformation of leaf disks (Horsch et al. 1985) - approx. 2-3 cm long leaves of sterile shoot cultures are punched into 1 cm diameter discs and with a suspension of appropriate agrobacteria (approx. 10 ⁹ / ml) (cf. c) in Am medium, see below) for approx.

Incubated for 5 minutes. The infected leaf pieces are kept on MS medium (see below) without hormones for 3-4 days at approx. 24 ° C. During this time, Agrobacterium grows over the leaf pieces. The leaf pieces are then washed in MS medium (0.5 mg / ml BAP, OJ mg / ml NAA) and on the same medium (0.8% agar) with 500 μg / ml cefotaxim and 100 μg / ml kanamycin sulfate. The medium should be replaced after two weeks. Transformed kanamycin-resistant shoots become visible after another 2-3 weeks.

Biochemical detection method of transformation

Neomycin phosphotransferase (NPT II) enzyme test:

NPT II activity in plant tissue is determined by in situ phosphorylation of kanamycin, as described by Reiss et al. (1984) and by Schreier et al. (1985) modified as follows. 50 mg of plant tissue are homogenized in 50 μl extraction buffer (10% glycerol, 5% 2-mercaptoethanol, 0.1% SDS, 0.025% bromophenol blue, 62.5 mM Tris pH 6.8) with the addition of glass powder on ice and for 10 minutes centrifuged in an Eppendorf centrifuge at 4 ° C. 50 μl of the supernatant are placed on a native polyacrylamide gel (145 x 110 x 1.2 mm; separating gel: 10% acrylamide, 0.33% bisacrylamide, 0.375 M Tris pH 8.8, stacking gel: 5% acrylamide, 0J65% bisacrylamide, 0J25 M Tris pH 6.8) and electrophoresed overnight at 4 ° C and 60 V. As soon as the bromophenol blue marker runs out of the gel, the gel is washed twice with distilled water for 10 minutes and once for 30 minutes with reaction buffer (67 mM Tris-maleate, pH 7J, 42 mM MgC, 400 mM ammonium chloride ). The gel is placed on a glass plate of the same size and covered with 40 ml of 1% agarose in reaction buffer which contains the substrates kanamycin sulfate (20 μg / ml) and 20-200 μCi ³² P ATP (Amersha). The sandwich gel is 30 min. incubated at room temperature and then a sheet of phosphocellulose paper P81 (Whatman) is placed on the agarose. About four Filtrieφapierlagen 3 MM, (Whatman) and some paper towels stacked. The transfer of in situ phosphorylated radioactive kanamycin phosphate to the P81 paper is stopped after 3-4 hours. The P81 paper is for 30 min. incubated in a solution of proteinase K and 1% sodium dodecyl sulfate (SDS) at 60 ° C and then washed 3-4 times in 250 ml of 10 mM phosphate buffer pH 7.5 at 80 ° C, dried and for 1-12 h at - 70 ° C autoradiographed (XAR5 film Kodak).

All percentages in the examples above and in the examples below relate to percentages by weight, unless stated otherwise.

In the plant cells and plants obtained according to the above and examples, the presence of the GSTIIIc DNA was confirmed by Southern blot analysis. The expression of the GSTIIIc-DNA was detected by Northern blot analysis and Wester blots with the help of specific antibodies.

Some of the media used in the transformation of plants or plant cells are described below:

Am medium

3.5 g K ₂ HPO ₄

1.5 g KH ₂ PO ₄

0.5 g Na ₃ citrate

0J g MgSO ₄ x 7H ₂ O 1 g (NH ₄ ⁾ 2 ^S0 4

2 g glucose ad 1 1

Medium for sterile shoot culture of tobacco

Macro elements 1/2 of the concentration of MS salts Macro elements 1/2 of the concentration of MS salts

Fe-EDTA Murashige and Skoog (MS)

Myo-inositol 100 mg / 1

Sucrose 10 mg / 1

Agar 8 mg / 1

Vitamins Ca-panthotenate 1 mg / 1

Biotin 10 mg / 1

Nicotinic acid 1 mg / 1

Pyridoxine 1 mg / 1

Thiamine 1 mg / 1 pH 5.7 before autoclaving

K3 medium

On the culture of Nicotiana tabacum petit Havana SRI, Nicotiana tabacum Wiscon¬ sin 38, and Nicotiana plumaginifolia protoplasts (Nagy and Maliga, 1976)

Macro elements NH ₄ N0 ₃ 250 mg / 1

KNO ₃ 2500 mg / 1

CaCl ₂ -2H ₂ O 900 mg / 1

MgSO ₄ -7H ₂ O 250 mg / 1

NaH ₂ PO ₄ JH ₂ O 150 mg / 1

(NH ₄ ) ₂ SO ₄ 134 mg / 1

CaHPO ₄ JH ₂ 0 50 mg / 1

Micro elements H ₃ BO ₃ 3 mg / 1

MnSO ₄ JH ₂ O 10 mg / 1

ZnS0 ₄ -4H ₂ 0 2 mg / 1

KI 0.75 mg / 1

Na ₂ MoO ₄ -2H ₂ O 0.25 mg / 1

CuSO ₄ -5H ₂ O 0.025 mg / 1

CoCl ₂ -6H ₂ O 0.025 mg / 1

Fe-EDTA Na ₂ EDTA 37.2 mg / 1

FeS0 ₄ -7H ₂ 0 27.8 mg / 1

Inositol 100 mg / 1

Sucrose 137 g / i

(= 0.4 M)

Xylose 250 mg / 1

Vitamins nicotinic acid 1 mg / 1

Pyridoxine 1 mg / 1

Thiamine 10 mg / 1

Hormones NAA 1.0 mg / 1

Kinetin 0.2 mg / 1 pH 5.6

Sterilize the filter Linsmaier and Skoog Medium (Linsmaier and Skoog 1965)

Murashige and Skoog Medium (Murashige and Skoog, 1962) are for the culture of regenerating protoplasts and for tissue culture of tobacco tumors and callus Linsmaier and Skoog (LS) Medium with the following modifications

Thiamine is weighed in a higher concentration 0.4 mg / 1 instead of OJ mg / 1,

Glycine, pyridoxine and nicotinic acid are missing.

Macro elements NH ₄ NO ₃ 1650 mg / 1

KN0 ₃ 1900 mg / 1

CaCl ₂ -2H ₂ 0 440 mg / 1

MgS0 ₄ -7H ₂ 0 370 mg / 1

KH ₂ P0 ₄ 170 mg / 1

Micro elements H ₃ B0 ₃ 6.2 mg / 1

MnS0 ₄ JH ₂ 0 22.3 mg / 1

ZnS0 ₄ -4H ₂ 0 8.6 mg / 1

CI 0.83 mg / 1

Na ₂ Mo0 ₄ -2H ₂ 0 0.25 mg / 1

CuS0 ₄ -5H ₂ 0 0.025 mg / 1

CoCl ₂ -6H ₂ 0 0.025 mg / 1

Fe-EDTA Na ₂ EDTA 37.2 mg / 1

FeS0 ₄ -7H ₂ 0 27.8 mg / 1

Inositol 100 mg / 1

Sucrose 30 g i

Agar 8 mg / 1

Vitamins thiamine 0.4 mg / 1

Hormones NAA 1 mg / 1

Kinetin 0.2 mg / 1 pH 5.7 The following literature can be cited for the transformation of plants or plant cells:

Fraley R.T., Rogers S.G., Horsch R.B., Sanders P.R., Flick J.S., Adams S.P., Bittner M.L., Brand L.A., Fink C.L., Fry J.S., Fallupi G R., Goldberg S.B., Hoffmann N.L., Woo S.C. (1983). Expression of bacterial genes in plant cells.

Proc. Natl. Acad. Be. USA 80: 4803-4807.

Fromm M.E., Taylor L.P., Walbot V. (1986) Stable transformation of maize after gene transfer by electroporation. Nature 319: 791-793

Hain, R., Stabel, P., Czerailofsky, A.P., Steinbiß, H.H., Herrera-Estrella, L., Schell, J. (1985) Uptake, Integration, expression and genetic transmission of a selectable chimeric gene by plant protoplasts. Mol. Gen. Genet. 199: 161-168

Hernalsteens J.P., Thia-Tong L., Schell J., Van Montagu M. (1984) An Agrobacterium-transformed cell eulture from the monocot Asparagus officinalis. EMBO J. 3: 3039-3041

Herrera-Estrella L., De Block M., Messens E., Hernalsteens J.P., van Montagu M., Schell J. (1983) EMBO J. 2: 987-995.

Horsch R.B., Fry J.E. Hoffmann N.L., Eichholtz D., Rogers S.G., Fraley R.T. (1985) A simple and general method for transferring genes into plants. Science 277: 1229-1231

Krens F.H., Molendijk L., Wullems G.J., Schilperoort R.A. (1982) In vitro transformation of plant protoplasts with Ti-plasmid DNA. Nature 296: 72-74

Koncz C, Schell J. (1986) The promotor of T _L -DNA gene 5 controls the tissue- specific expression of chimaeric genes carried by a noval type of Agrobacterium linary vector. Mol. Gen. Genet. (1986) 204: 338-396

Linsmaier DM, Skoog F. (1965) Organic growth factor requirements of tobaeco tissue eultures. Physiol. Plant 18: 100-127 Marton L., Wullems GJ, Molendijk L., Schilperoort PR (1979) In vitro transformation of cultured cells from Nicotiana tabacum by Agrobacterium tumefaciens. Nature 277: 1229-131

Nagy J.I., Maliga P. (1976) Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana sylvestris. Z. plant physiol. 78: 453-455

Paszkowski I., Shillito R.D., Saul M, Mandak V., Hohn T., Hohn B., Potrykus I. (1984) Direct gene transfer to plants. EMBO J. 3: 2717-2722

Shillito R.D., Paszkowski J. Potrykus I. (1983) Agarose plating and bead type culture technique enable and stimulate development of protoplast-derived colonies in an number of plant species. Pl. Cell. Rep. 2: 244-247 Van den Elzen P.J.M.,

Townsend J., Lee K.Y., Bedbrook J.R.

Van den Elzen P.J.M., Townsend J., Lee K.Y., Bedbrook J.R. (1985) A chimaeric resistance gene as a selectable marker in plant cells. Plant Mol. Biol. 5: 299-302.

Veiten J., Veiten L., Hain R, Schell J. (1984) Isolation of a dual plant promotor fragment from the Ti plasmid of Agrobacterium tumefaciens. EMBO J. 12: 2723-

2730

Van Haute E., Joos H, Maes M., Warren G, Van Montagu M., Schell J. (1983) Intergenic transfer and exchange recombination of restriction fragments clones in pBR 322: a novel strategy for the reversed genetics of Ti plasmids of Agrobacterium tumefacines. EMBO J. 2: 41 1-418.

Zambryski P., Joos H, Genetello C, van Montagu M., Schell J. (1983) Ti-plasmid vector for the introduction of DNA into plant cells without altering their normal regeneration capacity. EMBO J. 12: 2143-2150.

Reiss, B., Sprengel, Will H., and Schaller H. (1984) A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell tracts, GENE 1081: 21 1-217 Schreier PH, Seftor EA, Schell J. and Bohnert HJ. (1985) The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplasts. EMBO J. 1: 25-32

The following published patent applications can also be listed:

EP-A 116 718 EP-A-126 546

EP-A 159 418 EP-A-164 597

EP-A 120 515 EP-A-175 966

EP-A-120 516 WO 84/02913 EP-A-172 112 WO 84/02919

EP-A-140 556 WO 84/02920

EP-A-174 166 WO 83/01176 EP-A-122 791

The increased resistance of the transformed plants according to the invention is explained using the following example:

Evidence of the increased resistance of transformed plants:

To test the increased resistance to herbicidal herterooxyacetamides, the damage to the plants transformed according to the invention is assessed in the

Determination compared to control plants. The compound (5- trifluoromethyl-1,3,4-thiadiazol-2-yl-oxy-acetic acid-N-isopropyl-N- (4-fluoro phhenyl) amide is used as test herbicide.

Seeds of the Fl generation of the transformed plants are placed in pots (d = 11 cm) in the greenhouse on land with 3% humus. The plants are grown at a temperature of 23 ° C and a relative humidity of 70 - 80%. The treatment with the herbicidal compound mentioned above, formulated as 70 WP (wettable powder), takes place 24 hours after laying out the seeds at concentrations which correspond to an application rate of 2,000-4,000 g of active ingredient / ha.

Evaluation takes place 20 days after treatment with herbicide. The percentage of total damage to transgenic plants in comparison to corresponding control plants which were treated with the same concentration of active ingredient is rated.

The transformed tobacco plants, in which the GSTIHc DNA according to the invention was used, show a markedly increased resistance to high amounts of herbicide (2,000-4,000 g ha) compared to corresponding non-transformed corresponding control plants.

SEQUENCE LOG

(1. GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Bayer AG

(B) ROAD: Bayerwerk

(C) LOCATION: Leverkusen

(E) COUNTRY: Germany

(F) POSTCODE: D-51368

(G) TELEPHONE: 0214/30 66400 (H) TELEFAX: 0214/30 3482 (I) TELEX: 85 101-265 by d

(ii) DESCRIPTION OF THE INVENTION: Deoxyribonucleic Acid and its Use

(iii) NUMBER OF SEQUENCES: 4

(iv) COMPUTER READABLE VERSION:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPA)

(2) INFORMATION ON SEQ ID NO: 1:

(i) SEQUENCE LABEL:

(A) LENGTH: 666 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO

(iv) ANTISENSE: NO

(ix) FEATURE:

(A) NAME / KEY: CDS

(B) LOCATION: 1..663

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATG GCG CCT CTG AAG CTG TAC GGG ATG CCG CTG TCC CCC AAC GTG GTG 48

Met Ala Pro Leu Lys Leu Tyr Gly Met Pro Leu Ser Pro Asn Val Val 1 5 10 15

CGC GTG GCC ACC GTG CTC AAC GAG AAG GGC CTC GAC TTC GAG ATC GTC 96

Arg Val Ala Thr Val Leu Asn Glu Lys Gly Leu Aεp Phe Glu Ile Val 20 25 30

CCC GTC GAC CTC ACC ACC GGC GCC CAC AAG CAG CCC GAC TTC CTC GCC 144 Pro Val Asp Leu Thr Thr Gly Ala His Lys Gin Pro Asp Phe Leu Ala 35 40 45

CTC AAC CCT TTC GGC CAG ATC CCG GCT CTC GTC GAC GGA GAC GAA GTC 192 Leu Asn Pro Phe Gly Gin Ile Pro Ala Leu Val Asp Gly Asp Glu Val 50 55 60

CTC TTC GAG TCC CGT GCG ATC AAC CGG TAC ATC GCC AGC AAG TAC GCG 240 Leu Phe Glu Ser Arg Ala Ile Asn Arg Tyr Ile Ala Ser Lys Tyr Ala 65 70 75 80

TCG GAG GGC ACG GAC CTG CTC CCC GCG ACG GCG TCG GCG GCG AAG CTG 288 Ser Glu Gly Thr Asp Leu Leu Pro Ala Thr Ala Ser Ala Ala Lys Leu 85 90 95 GAG GTG TGG CTG GAG GTG GAG TCG CAC CAC TTC CAC CCG AAC GCG TCG 336 Glu Val Trp Leu Glu Val Glu Ser His His Phe His Pro Asn Ala Ser 100 105 110

CCG CTG GTG TTC CAG CTG CTC GTG AGG CCG CTC CTG GGC GGC GCC CCC 384 Pro Leu Val Phe Gin Leu Leu Val Arg Pro Leu Leu Gly Gly Ala Pro 115 120 125

GAC GCG GCG GTG GTG GAG AAG CAC GCG GAG CAG CTC GCC AAG GTG CTC 432 Asp Ala Ala Val Val Glu Lys His Ala Glu Gin Leu Ala Lys Val Leu 130 135 140

GAC GTG TAC GAG GCG CAC CTG GCC CGC AAC AAG TAC CTC GCC GGG GAC 480 Asp Val Tyr Glu Ala His Leu Ala Arg Asn Lys Tyr Leu Ala Gly Asp 145 150 155 160

GAG TTC ACG CTC GCC GAC GCC AAC CAC GCG TCC TAC CTG CTC TAC CTC 528 Glu Phe Thr Leu Ala Asp Ala Asn His Ala Ser Tyr Leu Leu Tyr Leu 165 170 175

AGC AAG ACC CCC AAG GCC GGG CTC GTC GCC GCC CGC CCC CAC GTC AAG 576 Ser Lys Thr Pro Lys Ala Gly Leu Val Ala Ala Arg Pro His Val Lys 180 185 190

GCC TGG TGG GAG GCC ATC GCC GCC CGC CCC GCG TTC CAG AAG ACC GTC 624 Ala Trp Trp Glu Ala Ile Ala Ala Arg Pro Ala Phe Gin Lys Thr Val 195 200 205

GCC GCC ATC CCC TTG CCC CCG CCG CCC TCC TCC TCG GCT TGA 666

Ala Ala Ile Pro Leu Pro Pro Pro Pro Ser Ser Ser Ala 210 215 220 (2) INFORMATION ON SEQ ID NO: 2:

(i) SEQUENCE LABEL:

(A) LENGTH: 221 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: Protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Met Ala Pro Leu Lys Leu Tyr Gly Met Pro Leu Ser Pro Asn Val Val 1 5 10 15

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

Pro Val Asp Leu Thr Thr Gly Ala His Lys Gin Pro Asp Phe Leu Ala 35 40 45

Leu Asn Pro Phe Gly Gin Ile Pro Ala Leu Val Asp Gly Aεp Glu Val 50 55 60

Leu Phe Glu Ser Arg Ala Ile Asn Arg Tyr Ile Ala Ser Lys Tyr Ala 65 70 75 80

Ser Glu Gly Thr Asp Leu Leu Pro Ala Thr Ala Ser Ala Ala Lys Leu 85 90 95

Glu Val Trp Leu Glu Val Glu Ser His His Phe His Pro Asn Ala Ser 100 105 110

Pro Leu Val Phe Gin Leu Leu Val Arg Pro Leu Leu Gly Gly Ala Pro 115 120 125 sp Ala Ala Val Val Glu Lys His Ala Glu Gin Leu Ala Lys Val Leu

130 135 140

Asp Val Tyr Glu Ala His Leu Ala Arg Asn Lys Tyr Leu Ala Gly Asp 145 150 155 160

Glu Phe Thr Leu Ala Asp Ala Asn His Ala Ser Tyr Leu Leu Tyr Leu 165 170 175

Ser Lys Thr Pro Lys Ala Gly Leu Val Ala Ala Arg Pro His Val Lys 180 185 190

Ala Trp Trp Glu Ala Ile Ala Ala Arg Pro Ala Phe Gin Lys Thr Val 195 200 205

Ala Ala Ile Pro Leu Pro Pro Pro Pro Ser Ser Ser Ala 210 215 220

(2) INFORMATION ON SEQ ID NO: 3:

(i) SEQUENCE LABEL:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTISENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

AGCGCATATG GCGGCTCTGA AGCTGTACGG GAT ₃ 3

(2) INFORMATION ON SEQ ID NO: 4:

(i) SEQUENCE LABEL:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iv) ANTISENSE: NO

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

GACGGATCCT CAAGCCGAGG AGGAGGGCGG 30

INTERNATIONAL FORM

Bayer AG

Plant protection business area

Research / biotechnology

Plant protection center Monhei

RECEIPT CONFIRMATION AT THE FIRST DEPOSIT,

51368 Leverkusen issued in accordance with Rule 7.1 by the INTERNATIONAL DEPOSIT AGENT specified below

I. LABELING OF THE MICROORGANISM

Reference number assigned by the DEPOSIT: INPUT NUMBER assigned by the INTERNATIONAL DEPOSIT: BB pET3a-GSTIIIc

DSM 9677

II. SCIENTIFIC DESCRIPTION AND / OR PROPOSED TAXONOMIC NAME

With the microorganism designated under I.

(X) a scientific description

(X) submitted a proposed taxonomic name. (Tick the appropriate).

III. ENTRANCE AND ACCEPTANCE

This international depository accepts the microorganism designated under I, which it received on 19 95 - 01 - 17 (date of first deposit) ¹ .

IV. RECEIPT FOR CONVERSION

The microorganism under 1 bczςichnctc was received by this international depository on (date of first deposit) and an application for the conversion of this initial deposit into a deposit under the Budapest Treaty was received on (date of receipt of the application for conversion).

V. INTERNATIONAL DEPOSIT

Name: DSM-GERMAN COLLECTION OF signatures to represent the international depository

MIKROORGANISMEN UND ZELLKULTUREN GmbH authorized person (s) or the authorized person (s):

Address: Maschcrodcr Weg lb D-38124 Üraunschweig U. u? _* r Ό

Daium 1 995-01-18

^{1 from} an international un cnvorbcπ.

Claims

claims

1 DNA (deoxyribonucleic acid) which codes for the protein glutathione-S-transferase IIIc (GSTIIIc) according to SEQ ED NO 2 or a protein sequence derived therefrom (GSTIIIc-DNA).

2. DNA according to claim 1, which the sequence according to SEQ ID NO. 1 or a sequence derived therefrom

3. DNA according to claim 1, which has the sequence of the GSTIIIc-DNA which is contained on the vector plasmid pET3a-GSTIIIc or a sequence derived therefrom.

4. DNA according to claims 1 to 3, which is in the form of a DNA double strand containing the DNA according to claims 1 to 3 and a complementary DNA strand

5 DNA according to claims 1 to 4, in which a promoter effective in plants is connected upstream at the 5 'end of the coding sequence.

6 DNA according to claim 5, wherein the CaMN 35S promoter or the CaMV 35S double promoter composed of the CaMV 35S enhancer and the CaMV 35S promoter is used as the promoter

7. DNA according to claim 5, which is a construct of the GSTIIIc-DNA and the CaMV 35S double promoter, as it is on the vector plasmid pSS-GSTIIIc

8 Recombinant prokaryotic or eukaryotic DNA, which contains the DNA according to claims 1 to 7

9 recombinant DNA, which is contained in plants or plant cells and contains the DNA according to claims 1 to 7

10 vectors containing the DNA according to claims 1 to 7 or the recombinant DNA according to claims 8 and 9

1 1. Vector plasmids pET3a-GSTIIIc and pSS-GSTIIIc.

12. Transformed microorganisms which contain the DNA according to claims 1 to 7 or the recombinant DNA according to claims 8 and 9.

13. Escherichia coli strain DS pET3a-GSTIIIC (according to DSM 9677) and its mutants, which still have the features essential for carrying out the invention.

14. Transgenic plants (including parts of these plants and their propagation material, such as protoplasts, plant cells, calli, seeds, tubers or cuttings, etc.) which in their genome have the DNA according to claims 1 to 7 in addition to the DNA of the corresponding not contain transgenic plants.

15. Transgenic plants according to claim 14, which contain the DNA according to claim 6 in their genome.

16. Transgenic plants according to claim 14, which contain the DNA according to claim 7 in their genome.

17. Transgenic plants according to claims 14 to 16, which have a content of glutathione-S-transferase IIIc (GSΗIIc) optionally in the dimeric form.

18. Transgenic plants according to claims 14 to 17 which, compared to the corresponding non-transgenic plants, have an increased resistance to herbicides, in particular to heterooxyacetamide herbicides.

19. Use of the DNA according to claims 1 to 7 and / or the recombinant DNA according to claims 8 and 9 and / or the vectors according to claims 10 and 11 and / or the transformed microorganisms according to claims 12 and 13 for Generation of transgenic plant cells (including protoplasts) and plants (including plant parts and seeds).

0 A process for the production of the transgenic plants according to claim 14, characterized in that

(a) the DNA according to claim 1 and / or the recombinant DNA according to claim 8 is used in the genome of plant cells (including protoplasts) and optionally

(b) from the transgenic plant cells (including protoplasts) completely transformed plants are regenerated and, if appropriate, multiplied and, if appropriate

(c) the desired plant parts (including propagation material) are obtained from the transgenic plants of the parent generation or further generations obtained therefrom.

21. Use of the transgenic plants according to claim 14 for the production of propagation material and for the production of new plants which contain the DNA according to claim 1 or the recombinant DNA according to claim 8 and their propagation material.

22. Use of DNA which corresponds wholly or partly to the GSTIIIc-DNA which is contained on the plasmid pET3a-GSTIIIc or which wholly or partly corresponds to SEQ ID NO: 1 as a probe for detecting the content of GSTIIIc-DNA or their constituents in a DNA to be examined for this content.

23 Use of the DNA coding for the protein GSTIIIc according to SEQ ID NO: 2 for the production of transgenic plants which, in comparison with the corresponding non-transgenic plants, have an increased resistance to herbicides, in particular heterooxyacetamide herbicides

24 protein with an amino acid sequence according to SEQ ED NO 2 or a sequence derived therefrom

25 Protein according to claim 24 in the dimeric form