DE10035084A1 - Nucleic acid molecule encoding a plant AMP deaminase - Google Patents

Abstract

The isolation and cloning of a nucleic acid sequence coding for the AMP deaminase from Arabidopsis thaliana is described. The isolated nucleic acid molecule containing the nucleic acid sequence enables the expression and isolation of a high-purity vegetable AMP deaminase, which makes it possible to detect herbicidal inhibitors of this AMP deaminase. In addition, this isolated AMP deaminase can be used to produce transgenic plants which have increased resistance to AMP deaminase inhibitors.

Description

Catalyzed the AMP deaminase (adenosine monophosphate deaminase) (EC 3.5.4.6) in eukaryotes the irreversible hydrolytic deamination of the amino group of AMP (adenosine monophosphate) in equimolar amounts IMP (inosine monophosphate) and ammonia. The AMP deaminase (AMP-DA) is found in all eukaryotic organisms to be found (Meyer et al., Biochemistry, 28 (1989), 8734-8743; Thakkar et al., Biochem. J., 290 (1993), 335-341). The enzyme is the key enzyme in the Control of energy balance and purine metabolism in eukaryotic cells considered. The AMP deaminase comes from its function in the breakdown of AMP in plants plays an important role in regulating the energy status of the cell zu (Lowenstein J.M., Int. J. Sports Med., 11 (1990), 3746).

The vegetable AMP deaminase is due to its role in the vegetable Purine metabolism as a herbicidal site of action for the first time by Dancer (Dancer et al., Plant Physiol., 114 (1997), 119-129). A well known inhibitory effective molecule of AMP deaminase is the carbocyclic derivative of Coformycins, the polygocidin. This is a natural product from the Microorganism Saccharothrix spp. (Dancer et al., Plant Physiol., 114 (1997), 119-129). The effectiveness of polygocidine is different on mono- and dicotyledons Weeds have been shown. The application rate required for the control Active ingredient is less than 32 g / ha post-emergence to be a herbicidal Achieve effect (Bush et al., Phytochemistry, 32 (1993), 737-739). Symptoms observed on the plant are after application of coformycin for example the impairment of plant growth as a result of  Bleaching and necrosis on apical meristems of plant tissues. Dancer (Dancer et al., Plant Physiol., 114 (1997), 119-129) could show that in planta the herbicidal effect of carbocyclic coformycin on the inhibition of AMP Deaminase activity by the in vivo phosphorylated form (phosphorylated carbocyclic coformycin) is based.

The result of inhibiting AMP deaminase is believed to be an accumulation of AMP, which by the activity of the adenylate kinase to ADP (adenosine diphosphate) is converted. A measurable consequence of the application of the herbicide is the increase the ATP concentration, since the synthesis of ATP (adenosine triphosphate) by the Availability of ADP and free phosphate is determined (Dancer et al., Plant Physiol., 114: 119-129 (1997). Inhibition of AMP deaminase in planta results consequently due to the role of the enzyme in metabolism to malfunction the adenine nucleotide status of the plant cell, which has a serious impact on almost all energy-dependent metabolic pathways of the plant.

Plant-based AMP deaminases could be partially derived from tissues from different plants be cleaned up (Yoshino & Murakami, Z. Plant Physiology, 99 (1980), 331-338; Lo Floc'h & Lafleuriel, Physiol. Veg., 21, (1982), 15-17, Yabuki & Ashihara, Phytochemistry, 31 (1992) 1905-1909, Dancer et al., Plant Physiol., 114 (1997), 119-129). So far, however, no peptide sequences from partial purified AMP deaminase fractions can be obtained from plants so that of which oligonucleotides for the detection of corresponding gene sequences could have been derived.

So far, no complete vegetable nucleic acid sequences have been found the similarity to other AMP deaminases from pro- and possess eukaryotic organisms and code for a functional protein. Just an "Expressed Sequence Tag" (T21250) from Arabidopsis thaliana (TIGR-  Database Institute for Genome Research) similar to an AMP deaminase has become known without proof or use of a function.

A vegetable protein with AMP deaminase activity can be tested be used to identify potentially herbicidal substances (Dancer et al., Plant Physiol., 114 (1997), 119-129). The probability of Finding new economically attractive crop protection agents increases through the possibility of as many as possible in a simple, manageable method To be able to test substances for their inhibitory potentially herbicidal activity.

The availability of required is advantageous for such a test system Amounts of high purity enzyme. The availability of a recombinant Plant-based AMP deaminase would be the process of finding Ease inhibitors of the enzyme considerably. On the one hand, larger quantities were possible easier and cheaper to manufacture, and on the other hand, would Use of the influence of disruptive side activities that are prevented at the Use of raw vegetable extracts may occur or be considered Need to become. The possibility of producing a recombinant enzyme requires the provision of a DNA molecule or cDNA molecule that can Has nucleic acid sequence for such a plant AMP deaminase coded.

The task now was to provide genetic information about at least one RNA or DNA sequence of an AMP deaminase from a plant organism obtained and then to build an in vitro screening system for herbicides Develop and use active ingredients.

To solve this task, it is necessary to make at least one full-length cDNA to isolate a plant in order to then possibly use the full-length cDNA  Possibility to get more AMP deaminases from others, preferably to identify and possibly isolate plant organisms. Here, a cDNA molecule is a double-stranded DNA molecule which is characterized by Synthesis of a DNA strand complementary to the mRNA, the so-called cDNA first strand, and subsequent synthesis of the first strand complementary second strand of DNA is obtained. This method is one of those A method known to a person skilled in the art to determine an existing one at the mRNA level Sequence information into a more manipulable and clonable nucleic acid that DNA to convict.

The isolated nucleic acid molecules thus obtained and those contained on them Sequence information can then be used to generate an in vitro Realize test system (screening) based on the use of at least proteins encoded and expressed by a cDNA sequence. To do this the cDNA obtained is cloned into a suitable expression system serves, the protein encoded by the cDNA, in this case the AMP deaminase, in to express a suitable host organism. Then this can recombinant protein in a sufficiently pure form from the expression mixture obtained and stands for biochemical analysis or also directly for an in vitro test system is available in which the effectiveness of substances, for example from different chemical libraries, based on exactly this protein can be tested.

The invention relates to an isolated nucleic acid molecule containing a nucleic acid sequence which codes for a protein which has the biological activity of a plant AMP deaminase, the nucleic acid sequence being selected from the group consisting of:

• a) Nucleic acid sequences that given under SEQ ID NO 1 Include nucleic acid sequence and / or that for a protein with the below Encode SEQ ID No 2 given amino acid sequence;  
• b) Nucleic acid sequences with those mentioned under (a) Hybridize nucleic acid sequences;
• c) Nucleic acid sequences, their sequence based on the genetic code is degenerate compared to those mentioned under (a) or (b) nucleic acid sequences; and
• d) nucleic acid sequences, the fragments, derivatives or allelic variants of the represent under (a) to (c) nucleic acid sequences.

The term derivative denotes here and in the overall content of these Description changes that do not affect either Have amino acid sequence, as for example by changing the third base (the so-called wobble base) of a triplet codon one Nucleic acid sequence is the case, or which has an impact on the Amino acid composition, but the total protein then always nor the biochemical characteristics and / or enzymatic properties an AMP deaminase.

All variants of the nucleic acid sequence given under SEQ ID NO 1 can are obtained from the same organism from which the Nucleic acid molecule according to (a), (b) or (c). In general, you can Such allelic variants can be found by either using the entire nucleic acid sequence according to SEQ ID NO 1 or fragments thereof in search for the relevant gene banks, or by using the base probably conserved areas of those shown in SEQ ID NO 2 Amine acid sequence PCR primer (starter molecules for the polymerase chain reaction) developed that are degenerate at multiple positions.

Nucleic acid molecules that match nucleic acid molecules that have the nucleic acid sequence contain according to SEQ ID NO 1 or fragments thereof, can hybridize z. B. isolated from genomic or from cDNA libraries of different organisms  become. The identification and isolation of such nucleic acid molecules Plants or other organisms can be used using the nucleic acid molecules according to the invention take place (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

For example, nucleic acid molecules can be used as the hybridization sample be exactly the or essentially that specified under SEQ ID NO 1 Have sequence or fragments of this sequence. With the as Hybridization samples used nucleic acid molecules can also be synthetic nucleic acid molecules act with the help of common Synthetic techniques were produced and their sequence essentially with the sequence hybridized according to the invention. Are RNA molecules considered Hybridization sample used, these are generally with the help of in vitro transcription of a DNA molecule made into a corresponding and integrated in vitro transcription vector known to the person skilled in the art. A nucleic acid sequence that has significant homology to coding Nucleic acid sequence SEQ ID NO 1 or the sequence complementary to it is characterized in that the coding sequence in general at least 70%, preferably at least 80%, particularly preferably at least 90%, very particularly preferably at least 95%, particularly preferably at least 98%, or most preferably at least 99% sequence identity to the nucleic acid sequence SEQ ID NO 1 or its complement over a length of at least 20, preferably at least 30, particularly preferably at least 40, very particularly preferably at least 60, particularly preferably at least 100 Bases in a continuous sequence, and most preferred over the full length which has SEQ ID NO 1. Any combination of the degrees and Minimum sizes can be used to define the nucleic acid sequence according to the invention are used, with more stringent conditions being the preferred choice represent. Thus, both a nucleic acid sequence is provided by way of example at least a sequence identity of 90% over 20, preferably over 30 nucleotides  represent an object of the invention, as well as in a Nucleic acid sequence with 95% sequence identity over a length of 40 Nucleotides is the case.

Also nucleic acid molecules or fragments, derivatives or alleles Variants thereof the subject of the invention that specifically with the Nucleic acid molecule according to the invention or fragments, derivatives or alleles Hybridize variants thereof. The corresponding fragments, derivatives or alleles Variants can be created by insertion, deletion, addition, substitution or inversion have arisen. The minimum length of a hybridizing nucleic acid molecule is 15 nucleotides. The term "hybridize specifically" means that under conventional hybridization conditions, preferably under stringent conditions, no significant cross hybridization with Nucleic acid sequences that are different for proteins than for the invention Encode protein that can be observed. As specific Hybridization conditions apply, for example, 0.03 M sodium chloride and 0.003 M tri-sodium citrate at approx. 60 ° C. Such nucleic acid molecules have one Total length of at least 20 nucleotides, preferably a length of at least 50 nucleotides and particularly preferably a length of at least 100 nucleotides. Such molecules can be used as PCR primers or as Hybridization samples are used. The coding nucleic acid sequence SEQ ID NO 1 can be changed by nucleotide substitutions, such as by 1, 2, or 3 to 10, 25, 50, 100 or 300 exchanges. Fragments of Nucleic acid sequence as named under (a), (b), or (c) of the invention can be as Primer, for example as a PCR primer (Polymerase Chain Reaction Primer), as Primer for alternative amplification procedures, as a detection molecule with radioactive or non-radioactive labeling or for cloning into suitable Vectors are used. Such primers, detection molecules or others Fragments are preferably at least 10, at least 15, at least 20, at least 25, at least 30 or at least 40 nucleotides in length.  

They are typically 40, 50, 60, 70, 100 or 150 nucleotides long, for example or, for example, 200, 300, 400, 500, 600 or 700 nucleotides long or only a few nucleotides such as 5 or 10 nucleotides shorter than that entire coding sequence according to SEQ ID NO 1.

Another object of the invention is a method for producing a cDNA which codes for a plant AMP deaminase, preferably for an AMP deaminase from Arabidopsis thaliana, and which was isolated from a plant tissue mRNA. For example, the method includes the following steps, but is not limited to them, such as

• a) the isolation of an mRNA population from plant tissue; and
• b) reverse transcription of the mRNA population obtained; and
• c) the second strand synthesis based on the reverse transcribed first strand after (b); and
• d) the PCR amplification of the cDNA obtained according to (a) to (c) Double stranded molecule before or after linking with suitable adapters; and
• e) the enrichment and identification of those coding for the AMP deaminase cDNA or at least fragments thereof.

The invention also relates to vectors which are those according to the invention Contain nucleic acid sequence, the non-homologous host organism for example a bacterium, a yeast cell, an insect cell or a Is mammalian cell. The term vector is a naturally occurring or artificially created nucleic acid molecule, which Propagation, expression or transmission of others such as the Nucleic acid molecules according to the invention with those contained on them Nucleic acid sequences are used.  

An additional object of the invention are host cells that the Nucleic acid molecules or vectors according to the invention, which the contain nucleic acid sequences according to the invention. The host cell can be a prokaryotic, e.g. bacterial, or eukaryotic cell, for example a plant cell, a yeast cell, an insect cell or a Be a mammalian cell.

In particular, the invention relates to a vector that the contains nucleic acid sequence according to the invention, the non-homologous Host organism E. coli or Saccharomyces cerivisiae, or an insect cell especially a Schneider cell of the SL-3 line.

Another object of the invention is the use of one or more Nucleic acid molecules, the nucleic acid sequences thereof according to the invention included, for the transformation of prokaryotic or eukaryotic cells. In this Context means transformation in the case of eurkaryotic cells, and here specifically plant cells, a stable integration of one in a certain place of the genome of the host organism does not occur either in the organism itself in an identical or similar form exists at least once or from another organism was removed.

In the case of transformation of prokaryotic cells or eukaryotic cells in which if there is no chromosomal integration, transformation means Transfer and stable replication of extrachromosomal vectors such as for example of plasmids or viruses. In both cases it contains the transformed one Nucleic acid molecule, the nucleic acid sequence according to the invention, and the appropriate for the start and stop of transcription Regulatory elements. In the case of transformed eurkaryotes and here in particular Plant cells can orient the integrated nucleic acid sequence with respect to the regulatory elements surrounding them in sense orientation, d. i.e., the  Nucleic acid sequence potentially codes for a corresponding polypeptide, or in antisense orientation, d. that is, the nucleic acid sequence does not exactly code for one Polypeptide.

Another object of the invention is the production of a recombinant protein which has the biochemical characteristics of an AMP deaminase, the production being characterized, for example, in that:

• a) a nucleic acid molecule that the nucleic acid sequence according to SEQ ID NO 1 or contains fragments, derivatives or allelic variants thereof in one appropriate expression vector is integrated; and
• b) the nucleic acid molecule that the nucleic acid sequence according to SEQ ID Containing NO 1 or fragments, derivatives or allelic variants thereof Expression vector according to (a) is transferred into a corresponding host cell; and
• c) the expressed protein, if appropriate, specifically from the host organism or is isolated from the medium surrounding it;

The protein is preferably enzymatically active even after its isolation.

The overexpression of foreign proteins occurs increasingly in the form of Fusion proteins. Such fusion proteins are in their possibilities and Details have been described in a variety of publications such as for example in Goedel (ed.), Methods in Enzymology, 185 (1991) "Gene Expression technology; Kost, Current Opinion in Biotechnology, 8 (1997), No. 5; Markides, Microbiol. Reviews, 60 (1996), 512-538; Olins, Current Opinion in Biotechnology, 7 (1996), No. 5; Rudoph et al., In Protein Function, IRL. Press (1997), Creighton ed., 57-99; Shatzman, Current Opinion in Biotechnology, 6 (1995), No. 5th

Such fusion proteins have several advantages. If the foreign protein is linked to a host cell's own protein domain, proteolytic degradation is often slowed down considerably, or the toxicity of the foreign protein to the host organism is reduced, which means that the yield of the desired foreign protein can be significantly increased. In addition, the fusion of a hydrophobic protein, which tends to form inclusion bodies, with a hydrophilic protein often prevents aggregation. A recombinant protein can be purified significantly more efficiently if a protein domain is added through the gene fusion which enables purification by means of affinity chromatography. A distinction is made here between real fusion proteins, which consist of heterologous proteins or protein domains, from proteins which have so-called "tags". These are short terminal amino acid sequences. Such a "tag" can be, for example, a poly-His tag, the fusion protein containing this "tag" then being purified using Ni ²⁺ complexation. In addition, "tags" such as the poly-Arg tag, the poly-Glu tag or the Asp-Tyr-Lys-Asp ₄ -Lys (FLAG) are also known. Possible fusion partner proteins are, for example, β-galactosidase, protein A, streptavidin, glutathione-S-transferase or the maltose binding protein, to name just a few. In addition to the fusion with only one "tag", which can be either amino-terminal or carboxy-terminal, dual fusions can also be carried out, ie different "tags" are located at both the carbox-terminal and the amino-terminal end of the fusion protein. A fusion protein in this form can then be purified in two successive steps.

In the production of recombinant proteins, the based on eukaryotic amino acid sequences, the use of Yeast cells or insect cells as host cells.

Because a yeast cell already has many features of an expression system higher Having eukaryotes, one is largely able to use this Expression system to get authentic eukaryotic proteins in some  All post-translational modifications of the original do not carry recombinant protein.

In addition, one is now able to produce efficient heterologous gene expression perform in insect cells. Here, the recombinant protein can both Cytosol of the insect cells are present or also in the endoplasmic reticulum (ER) to be secreted. When expressing recombinant protein in insect cells the greatest security is that in the end there is an authentic protein that then also contains all modifications that the non-recombinant protein also contains having.

Another object of the invention is the use of the recombinant Protein based on the nucleic acid sequence of the invention and by this is encoded to find new inhibitors. In contrast to the one in U.S. Pat. No. 5,786,165 and by Dancer (Dancer et al., Plant Physiol. 114 (1997), 119-129) described AMP deaminase tests exists when using the nucleic acid sequence according to the invention for the first time the possibility that described test method on the basis of a highly purified and perform enriched protein, increasing the sensitivity of the test system on the one hand, but also the unambiguity of the results obtained on the other hand, is improved.

In this sense, a recombinant protein is a protein that in a heterologous host system, in the case of plant AMP deaminase for example in a bacterial cell or in a yeast cell and either in its amino acid composition according to a sequence SEQ ID NO 2 corresponds, or that in its amino acid composition of deviates from the amino acid composition given in SEQ ID NO 2, but then still the biochemical characteristics and / or enzymatic Features of a vegetable AMP deaminase. Furthermore, can a recombinant protein also at the amino terminal or / and carboxy terminal end changes / additions that  for example, facilitate cleaning up or Increase purification yield and preferably its enzymatic Properties not or if then only marginally influenced.

The invention furthermore relates to the use of the recombinant Protein based on the nucleic acid sequence according to the invention for Discovery of new herbicidal inhibitors.

In addition, another object of the invention is the use of recombinant protein based on the nucleic acid sequence of the invention based, for the biochemical or structural characterization of inhibitors.

By providing the nucleic acid molecules of the invention, it is also possible with the help of genetic engineering to produce plants that at least another gene coding for a homologous or heterologous AMP deaminase contain and also an expression of these homolologous or heterologous AMP deaminase. In the case of a heterologous AMP deaminase means this using a nucleic acid sequence coding for an AMP deaminase genetic engineering methods that are not exactly introduced into the plant previously existed in the plant genome into which it is now integrated. The nucleic acid molecules according to the invention are used with regulatory Linked elements that involve transcription and translation in plant cells ensure, and incorporated into plant cells to ensure optimal and stable Transcription and translation in the plant cells of the target organism guarantee.

For the expression of the various inventive described above In principle, nucleic acid sequences in plants come in a plant Cell functional promoter into consideration. The promoter can be homologous or heterologous in relation to the plant species used. For example, the 35S promoter of the Cauliflower mosaic virus (Odell et al., Nature 313 (1985),  810-812), which ensures constitutive expression in all tissues of a plant. However, promoters can also be used that only lead to one external influences determined time or in a certain tissue the Plant lead to expression of subsequent nucleic acid sequences (Stockhaus et al., 1989, EMBO J. 8: 2245-2251).

In addition to promoters, nucleic acid segments can be used to initiate transcription also contain nucleic acid sequences that further increase the Ensure transcription activity, for example so-called enhancer Elements.

The term “regulatory elements” can also include termination signals include the correct completion of the transcription and the addition of a Poly-A tail serve to the transcript, which has a function in the Stabilization of the transcripts is attributed. Such elements are in the Literature described and are interchangeable. Examples of such Termination sequences are the 3 'untranslated regions that the Polyadenylation signal of the nopaline synthase gene (NOS gene) or the Octopin synthase gene (Gielen et al., EMBO J. 8 (1989) 23-29) from agrobacteria comprise, or the 3'-untranslated regions of the genes of the storage proteins from soybean, as well as the genes of the small subunit from ribulose-1,5- Bisphosphate carboxylase (ssRUBISCO).

The invention furthermore also relates to transgenic plant cells which have a Contain nucleic acid molecule according to the invention, this with regulatory elements that are linked to transcription in plant cells guarantee. The regulatory elements are preferably heterologous in relation to the nucleic acid molecule according to the invention, d. that is, they are not in any natural biological connection with the invention Nucleic acid sequence.  

Another object of the invention are vectors that the invention Contain nucleic acid molecules, especially those in which the described Nucleic acid molecules are linked to regulatory elements that make up the Transcription of the nucleic acid sequences according to the invention in plant cells guarantee.

The introduction of nucleic acid molecules according to the invention into plant cells is preferably carried out using plasmids. Are preferred for this Plasmids used that have stable integration into the plant genome guarantee.

To prepare for the introduction of and on foreign nucleic acid molecules A large number of nucleic acid sequences contained in higher plants are available Cloning vectors are also available from plasmids, among others Replication signal for E.coli and a marker gene for the selection of transformed Contain bacterial cells. Examples of such vectors are pBR322, pUC- Series, M13mp series, pACYC184 etc. The nucleic acid molecule that the Desired nucleic acid sequence, such as for example SEQ ID NO 1, can be inserted into the vector at an appropriate restriction site. The plasmid obtained is used for the transformation of E. coli cells. Transformed E.coli cells are cultivated in a suitable medium, then harvested and lysed. The plasmid is made using standard methods recovered. As an analysis method to characterize the obtained Plasmid DNA is generally used for restriction analysis and sequence analysis used. After each manipulation, the plasmid DNA can be cleaved and the resulting fragmented nucleic acid molecules in selected or not selected form can be linked with other nucleic acid molecules.

In particular, the invention relates to the use of one or more Nucleic acid molecules which contain the nucleic acid sequence according to the invention for the production of transformed plants, the transformed plants being a towards non-transformed, but otherwise genetically identical plants,  increased resistance to one or more AMP deaminase inhibitors exhibit.

Another object of the invention is a transformed cell, preferably one transformed plant cell which contains the nucleic acid sequence according to the invention contains, wherein, in the case of the transformed plant cell, particularly preferably the chromosomally stable integrated nucleic acid sequence is understood. Farther the invention relates to transformed plant tissue and fertile transformed Plants containing the nucleic acid sequence according to the invention. About that In addition, the subject of the invention is the seed, which of the transformed plants according to the invention is obtained.

A particularly preferred object of the invention are transformed Plant cells, plants and plant seeds of crops such as alfalfa, Cotton, barley, oats, potatoes, corn, rice, rye, soybeans, summer rape, Sunflower, tomato, wheat, winter rape or sugar beet.

The transgenic plant cells can be prepared according to those known to the person skilled in the art Techniques to regenerate whole plants. That through regeneration of the transgenic plant cells according to the invention are available plants also subject of the present invention. The subject of Invention plants containing the transgenic plant cells described above contain. In principle, the transgenic plants can be any plants trade any plant species, d. H. both monocot and dicot Plants. They are preferably useful plants, in particular useful plants such as alfalfa, cotton, oats, potatoes, corn, rice, rye, soybeans, summer rape, Sunflower, tomato, wheat, winter rape or sugar beet.

For the introduction of nucleic acid molecules in naked form or integrated in A variety of cloning vectors are available in a plant host cell  Techniques available. These techniques include plant transformation Cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation agent (Hernalsteens et al., Nature, 287: 654-658 (1980); Fraley et al., Crit. Rev. Plant Sci., 4 (1985, 1-46; An et al., EMBO J., 4: 277-287 (1985); Bevan et al., Nucleic Acids Research, 12 (1984), 8711-8721; Chan et al., Plant Mol. Biol., 22: 491-506 (1993); Hiei et al., The Plant J., 6 (1994), 271-282; Guerineau and Mullineaux, in: Plant Molcular Biology Labfax; edited by Croy, Oxford, BIOS Scientific Publishers (1993), 121-148. In addition, the protoplast transformation method can be used (Donn et al., Abstracts VIIth International Congress on Plant Tissue and Cell Culture, (1990), 55; Morocz et al., Theoretical and Applied Genetics, 80 (1990), 721-726; Potrykus et al., Mol. Gen. Genet., 199 (1985), 183-188). One too widespread method is the introduction of nucleic acid molecules by means of biolistic method (particle bombardment technique) (Christou, Curr. Opinion Biotechnol., 4 (1993), 135-143; Christou et al., Bio / Technology, 9: 957-962 (1991); Iglesias et al., 1994, Planta, 192: 84-91; Russell et al., In Vitro Cell Div. Biol, 28P (1992) 97-100; Pereira and Erickson, Plant Cell. Rep., 1995, 14: 290-293; Becker et al., The Plant Journal, 5 (1994), 299-307).

Methods for plant transformation are also known, for example injection or electroporation. With injection and electroporation as well in the protoplast transformation of nucleic acid molecules in plant cells are in themselves no special requirements for the plasmids used posed. Simple plasmids such as e.g. B. pUC derivatives (Vieira and brass, Gene, 19 (1982), 259-268) can be used. But from such transformed cells to regenerate whole plants is the presence a selectable marker gene is necessary.

Depending on the method of introduction desired nucleic acid sequences in the However, plant cells can also have other nucleic acid sequences besides the  nucleic acid sequences according to the invention may be required. Are z. B. for the Transformation of the plant cell, the Ti plasmid (from Agrobacterium tumefaciens) or the Ri plasmid (from Agrobacterium rhizogenes) must be used at least the right boundary, but often the right and left boundary the Ti and Ri plasmid T-DNA as a flank region with the genes to be introduced get connected.

If Agrobacteria are used for the transformation, the one to be introduced must be Nucleic acid molecule can be cloned into special plasmids, either in an intermediate vector or in a so-called binary vector. The intermediate vectors may be due to nucleic acid sequences that are homologous to nucleic acid sequences in the T-DNA, by homologous recombination in the Ti or Ri plasmid of the agrobacteria can be integrated. This contains also the vir region necessary for the transfer of the T-DNA. intermediaries Vectors cannot replicate in agrobacteria. Using a helper plasmid the intermediate vector can be transferred to Agrobaterium tumefaciens (Conjugation). Binary vectors can be found in E.coli as well as in agrobacteria replicate. They contain a selection marker gene and a linker or Polylinker framed by the right and left T-DNA border regions become. They can be transformed directly into the agrobacteria (Holster et al., Mol. Gen. Genet. 163: 181-187 (1978). The one for the transformation of agrobacteria plasmids used also contain a selection marker gene, for example the NPT II gene, which allows the selection of transformed bacteria. The agrobacterium serving as the host cell is said to be a plasmid that contains a vir region bears included. The vir region is for the transfer of T-DNA into the plant cell necessary. Additional T-DNA may be present. That so transformed Agrobacterium is used to transform plant cells.

The use of T-DNA for the transformation of plant cells is intensive examined and sufficient (Hoekema, In: The Binary Vector System Offsetdrukkerrij Kanters B.V., Alblasserdam (1985), Chapter V; Fraley et al., Crit. Plant. Sci., 4: 1-46 and An et al., EMBO J. 4 (1985), 277-287).  

Binary vectors are e.g. T. commercially available, e.g. B. pBIN19 (Clontech Laboratories, Ins., USA).

For the transfer of the nucleic acid molecules into the plant cell, plant Explants with Agrobacterium tumefaciens or Agrobacterium rhizogenes can be cultivated. From the infected plant material (e.g. leaf pieces, stem segments, roots, but also protoplast or suspension-cultivated plant cells) can then in a suitable medium, which may contain antibiotics or biocides for the selection of transformed cells, whole plants are regenerated again. The plants thus obtained can then be examined for the presence of the introduced nucleic acid sequences. Is the nucleic acid sequence introduced once in the genome of the plant cell integrated, it is usually stable there and remains in the descendants of the originally transformed cell obtained. It usually contains one Selection marker that shows resistance to the transformed plant cells a biocide or an antibiotic such as kanamycin, G 418, bleomycin, Hygromycin or phosphinotricin u. a. taught. The individually chosen marker should therefore the selection of transformed cells over cells to which the introduced nucleic acid sequence is missing.

The transformed cells grow within the plant in the usual way (see also McCormick et al., Plant Cell Reports 5 (1986), 81-84). The resulting plants that have the same transformed genetic makeup or others Possess inheritance, to be crossed. The resulting hybrids Individuals have the corresponding phenotypic properties. Two or more generations should be attracted to ensure that the phenotypic trait is stably maintained and inherited. Also should Seeds are harvested to ensure the appropriate phenotype or other idiosyncrasies have been preserved.

All of these methods are known to the person skilled in the art and can be carried out directly as in the described literature can be used because it is established  Standard techniques. The choice of each used Transformation technology essentially depends on the type of plant to be transformed as there are significant differences in efficiency when using the different Systems based on the respective plant species exist. Some types can be transform only according to one of the three methods given.

The following examples serve to illustrate the invention without however, limit their scope in any way.

EXAMPLE 1 RNA isolation from plant tissue from Arabidopsis thaliana

The following is the exact sequence of steps and the amounts used individual components listed in detail.

Green leaf material was frozen in liquid nitrogen and closed in a mortar crushed with a vegetable powder. Plant material was in the GTC buffer resuspended and incubated for 20 min with gentle shaking. The suspension was centrifuged and the aqueous supernatant applied to a CsCI cushion (5.7 M) and centrifuged. The ultracentrifugation took place at 35000 rpm, for 18 h and at 20 ° C. (Method according to Chirgwin et al., Biochem. 18 (1979), 5294-5299).

The RNA pellet obtained was taken up in DEPC H ₂ O (water treated with diethyl pyrocarbonate) and precipitated with 96% ethanol pa The precipitated RNA was dissolved in DEPC (diethyl pyrocarbonate) treated H ₂ O and stored at -80 ° C until further use. 5 µg of the total RNA isolated were used for reverse transcription.

GTC buffer

4 M guanidinium thiocyanate
25 mM Na citrate, pH 5.2
0.5% N-lauroyl sarcosine
0.1 M β-mercaptoethanol

EXAMPLE 2 Creation of full-length cDNA using a marathon cDNA amplification kit (Clontech)

In a gene bank, homologous amino acid and nucleic acid Sequences searched.

AMP deaminase cDNAs of various species were used as starting sequences used (human, mouse, yeast). Homology was possible at the protein level between the AMP deaminase described in mouse and the EST (Expressed Sequence Tag) T21250 can be found from A.thaliana.

A Lambda UNI-ZAP was used for the full length cloning of the EST T21250 (Stratagene) Arabidopsis thaliana cDNA bank using PCR for further homologous Sequences examined.

For this, a PCR (polymerase chain reaction) with different homologs was carried out Primers from the EST T21250 and a T3 primer (poly linker in the vector lambda UNI-ZAP).

A volume of 1 µl was used by the cDNA bank for PCR amplification and for this purpose with 5 µl PCR-Taq buffer (Qiagen), 1 µl T3 primer (25 pmol), 1 µl GA3 / GA4 primer (25 µmol), 1 µl dNTPs (25 mmol), 10 µl 5 * Q solution and 30 µl H ₂ O and 1 µl Taq polymerase [5 U / µl] (Qiagen) were added.

The PCR program carried out comprised the following cycles:
3 min, 94 ° C; 1 cycle;
0.5 min, 94 ° C; 1 min, 55 ° C; 2 min, 72 ° C; 25 cycles;
10 min, 72 ° C

There were approximately 400 bp (pL2) and approximately 500 bp (pL13) PCR fragments be amplified. The two PCR fragments were in the pCRII TOPO (Invitrogen) vector using the topoisomerase according to the manufacturer's instructions (Invitrogen) cloned.

The resulting pCRII TOPO vector, which contained the cloned PCR fragments, was with the commercially available standard primers "T7" and "SP6" from both Directions sequenced. By overlapping the nucleic acid sequences pI2, pI13 and the EST T21250 resulted in the sequence "pL13 full EST.seq". This Nucleic acid sequence contains the postulated 3 'region of the AMP deaminase (Stop codon) and parts of the poly A tail).

T3 primer (SEQ ID NO 3)

5'-AAT TAA CCC TCA CTA AAG GG 3 '

GA3 primer (SEQ ID NO 4)

5'-CCA-GAT-AAA CCT GTT GCA TCT C-3 '

GA4 primer (SEQ ID NO 5)

5'-CGG AAC TCC ACC CTT ATG TGT G-3 '

pL13ful1EST.seq Length: 914 SEQ ID No .: 17

By translating pL13ful1EST.seq, the following peptide partial sequence is obtained Arabidopsis thaliana AMP deaminase. The putatively translated peptide sequence consists of 292 amino acids. SEQ ID No .: 18

With the sequence pL13ful1EST a database search was carried out in the database Bioscout (Lion) performed. There was a nucleic acid sequence with the following  Acession No. AAC27176 of July 22, 1998 found. The gene was called putative Designated amidase. Due to the small transcript size compared to The size of the transcript found in the Northern blot became the marathon cDNA amplification kit (Clontech) for full-length cloning of AMP deaminase used.

The Marathon cDNA amplification kit is used in two parts subdivided.

• a) Reverse transcription (first and second strand synthesis) and ligation of the Adapters, and
• b) 2nd PCR amplifications

All buffers not defined in detail in Example 2 are identical to those from Manufacturer of the buffers supplied with the cDNA amplification kit.

The total RNA isolated according to Example 1 was transformed using the Oligotex mRNA kit (Qiagen) isolated the mRNA.

The Arabidopsis thaliana mRNA thus obtained was used as the starting material for the first-strand synthesis. 4 µl mRNA (1 µg) and 1 µl cDNA Synthesis Primer (10 µM) were incubated for 2 min at 70 ° C and then for 2 min on ice and with 2 µl 5 × first strand buffer; 1 µl dNTP (deoxy nucleotide triphosphate) mix (10 mM); 1 µl H ₂ O; 1 µl AMV reverse transcriptase (20 units / µl) filled up. The subsequent incubation was carried out for 1 hour at a temperature of 42 ° C.

48.4 ul sterile H ₂ O and 16 ul 5 × second strand buffer, 1.6 ul dNTP mix (10 mM) and 4 ul 20 × second strand enzyme cocktail and then a second strand cDNA were added to a volume of 10 ul of the first strand reaction Synthesis carried out. Incubation was carried out for 1.5 hours at a temperature of 42 ° C. This was followed by the addition of 2 μl (10 units) of T4 DNA polymerase.

This was followed by an incubation for 45 minutes at a temperature of 42 ° C executed. The reaction was stopped with a 4 μl EDTA / glycogen mix and with  a phenol / chloroform extraction. The aqueous phase became the nucleic acid is concentrated by means of ethanol precipitation.

The cDNA pellet resulting from the ethanol precipitation was dissolved in 10 μl sterile H ₂ O. 5 µl of this was used for the subsequent adapter ligation. 2 ul marathon cDNA adapter (10 ul), 2 ul 5x DNA ligation buffer, 1 ul T4 DNA ligase (1 ul / ul) were added to the ligation mixture. This was followed by incubation for 12 hours at a temperature of 16 ° C. The ligation mixture was resuspended in 250 μl tricin-EDTA buffer and used as a DNA template for the PCR amplifications.

1. PCR amplification

A volume of 2 μl of the total amount of cDNA was used for PCR amplification and for this purpose with 5 μl PCR buffer, 1 μl I 3'AMP-DA4 primer (10 pmol), 1 μl AP1 primer (10 pmol), 1 µl dNTPs (25 mmol), 2 µl MgCl2 (25 mmol) and 37 µl H ₂ O as well as 1 µl Taq polymerase [5 U / µl] (Boehringer) were added.

The PCR program carried out comprised the following cycles:
1 min, 94 ° C; 1 cycle;
1 min, 94 ° C; 1 min, 54 ° C; 3 min, 72 ° C; 5 cycles;
1 min, 94 ° C; 1 min, 54 ° C; 3 min, 70 ° C; 5 cycles;
1 min, 94 ° C; 1 min, 58 ° C; 3 min, 68 ° C; 25 cycles

1 µl of the 1st PCR amplification was used as the DNA template for the following "nested" PCR (2nd PCR amplification) used.

2. PCR amplification

A volume of 1 μl of DNA was removed from the DNA generated in the first PCR reaction (1st PCR amplification) and with 5 μl of PCR buffer; 1 µl 3'AMP-DA5 primer (10 pmol), 1 µl AP2 primer (10 pmol), 1 µl dNTPs (25 mmol), 2 µl MgCl ₂ (25 mmol) and 37 µl H ₂ O as well as 1 µl Taq -Polymerase [5 U / µl] mixed.

The PCR program carried out comprised the following cycles:
1 min, 94 ° C; 1 cycle;
1 min, 94 ° C; 1 min, 54 ° C; 3 min, 72 ° C; 5 cycles;
1 min, 94 ° C; 1 min, 54 ° C; 3 min, 70 ° C; 5 cycles;
1 min, 94 ° C; 1 min, 58 ° C; 3 min, 68 ° C; 25 cycles.

The PCR batches were separated on a 1% TBE agarose gel (Sambrook et al., (1989) Molecular Cloning - A Laboratory Manual; Cold Spring Harbor Laboratory Press, New York). Corresponding to the coelectrophoretized marker DNA a PCR amplification product of approximately 1200-1300 bp was obtained. The resulting PCR product was transferred via the T / A cloning site into the cloning plasmid pT- Adv (Clontech) introduced.

1 × TBE

100mM tris (hydroxymethyl) amminomethane
83 mM boric acid
1 mM EDTA (ethylenediaminetetraacetate)

Marathon cDNA Synthesis Primer (52-mer) (SEQ ID NO 6)

5'-TTCTAGAATTCAGCGGCCGC (T) ₃₀

N-1 N-3 '
N-1 = G, A or C; N = G, A, C or T

Adapter Primer 1 (AP1; 27 mer) (SEQ ID NO 7)

5'-CCATCCTAATACGACTCACTATAGGGC-3 '

Nested Adapter Primer 2 (AP2; 23 mer) (SEQ ID NO 8)

5'-ACTCACTATAGGGCTCGAGCGGC-3 '

The 3 primers named above come from the Marathon TM cDNA Amplification Kit (Clontech)  

3'AMP-DA4 primer (1261-1246) (SEQ ID NO 9)

5'-GCA-GGT CTA-CTC-AGC-GAA-TAT-TAC-TAC CTC ATC G-3 '

3'AMP-DA5 primer (1169-1152) (SEQ ID NO 10)

5'-GCA-CTA-CTC GGT-GAG-AGC-TGT CGA CTT TCC TAA CG-3 '

The two gene-specific primers (DA4 and DA5) were postulated from the cDNA derived from AMP deminase. The position of the primer on the corresponding DNA sequence is given in parentheses. The primers were synthesized by the cyanoethylphosphoamidite method (Gibco BRL).

The resulting vector pT-AdV, which contained the cloned AMP deaminase cDNA, was with the commercially available standard primers "M13 forward" and "M13 reverse "sequenced from both directions. By overlapping the sequences the following complete sequence (SEQ ID NO 1) was obtained for the cDNA of the Arabidopsis thaliana AMP deaminase.

The full-length cDNA of AMP deaminase was amplified using a polymerase chain reaction (PCR) and cloned into the expression vector PASK IBA3 (IBA). The cDNA produced from Arabidopsis thaliana, as described in Example 2, was used as template. A volume of 2 μl of the total amount of cDNA was used for the PCR amplification and with 5 μm PGR buffer, 1 μl 5 ′ AMP-DA1 primer (25 pmol), 1 μl 3 ′ AMP-DA1 primer (25 μmol ), 1 µl dNTPs (25 mmol), 2 µl MgCl ₂ (25 mmol) and 37 µl H ₂ O as well as 1 µl native Pfu polymerase [2.5 U / pl] (Stratagene).

The PCR program carried out comprised the following cycles:
1 min, 94 ° C; 1 cycle;
1 min, 94 ° C; 1 min, 55 ° C; 5 min, 72 ° C; 30 cycles
10 min, 72 ° C; 1Zyklus

5'AMP-DA1 primer (SEQ ID NO 11)

5'-CTA-GCT-AGC TAA CGA-GGG-CAA AAA ATG GAA CCC AAC CC-3 '

3'AMP-DA1 primer (SEQ ID NO 12)

5'-GCA-GGT CTA-CTC-AGC-GCT-AGG-AGG-TGG-AAC-AAC-TTC-ATC-AGA-G'3 '

The corresponding theoretical size of the nucleic acid sequence for the AMP Arabidopsis thaliana deaminase is 2520 bp (coding reading frame). The resulting PCR fragment had a size of approximately 2500 bp. This Fragment was isolated and inserted into the bacterial expression vector PASK IBA3 (IBA) integrated according to the manufacturer's instructions.

For this, the vector pASK IBA3 with Xba I / Bsa I and the PCR amplificate was used Sew I and Bsa I cut. The vector was generated using a preparative TBE Agarose gel isolated.

The correspondingly amplified PCR amplificate was digested using a PCR Purification Column (Qiagen) purified and in the bacterial expression vector PASK IBA 3 (IBA), integrated. The ligation product was used for transformation competent Escherichia coli DH10B cells (Gibco). The vector was called PASK Designated IBA3 AMP-DA. The resulting vector, which cloned the AMP Deaminase cDNA was used with the standard primers "PASK IBA3 forward" and "PASK IBA3 reverse" sequenced from both directions.

In the bacterial expression vector PASK IBA3 AMP-DA, the stop codon is the AMP deaminase (TAA) replaced by a spacer (CCTCCT). Out the spacer results in an additional pro-pro-amino acid sequence on carboxy terminal end. The spacer is followed by a nucleic acid sequence which is for encodes a 10 amino acid so-called strep-tag (IBA), and which with ends with a stop codon, so that this clearly means the carboxy-terminal end is defined.  

The cDNA clone coding for the Arabidopsis thaliana AMP deaminase, the which corresponds to SEQ ID NO 1, was in accordance with the Budapest Treaty on the international recognition of the deposit of microorganisms for the purposes of patent proceedings in the DSMZ-German collection of microorganisms and Zellkulturen GmbH deposited. The assigned DSMZ catalog number is: DSMZ plasmid PASK IBA3-AMP deaminase - DSM 13440

Example 3

From various plants (Zea mays, Brassica napus, Arabidopsis thaliana) was isolated as described in Example 1, according to standard methods on a Agarose gel separated under denaturing conditions and then on a filter membrane is transferred.

Gel electrophoresis of RNA in formaldehyde gel

2.4 g agarose (1.2%) were boiled up in 5/6 volumes of 1 × gel buffer and after cooling with 1/6 volume 37% formaldehyde. The RNA samples that had previously been subjected to ethanol precipitation, were pelleted and dried. The RNA was resuspended in 30 ul denaturation buffer, 15 minutes heated to 70 ° C and mixed with 3 µl application buffer before application. The Electrophoresis was carried out at 100 V for 5 hours.

10-fold gel buffer (MOPS buffer)

200 mM MOPS, pH 7.0 (3-morpholinopropanesulfonic acid)
50 mM Na acetate
10mM EDTA

denaturation

2.2 M formaldehyde
50% formamide
10% MOPS buffer
1 mg / ml ethidium bromide

job buffers

10% Ficoll 400
0.1% bromophenol blue

This was followed by the transfer of the RNA to a filter membrane and subsequent Hybridization with the Arabidopsis probe according to SEQ ID NO 1.

The nylon membrane Hybond-NX (Amersham Lifescience) used. The transfer was made using the Sambrook method, Fritsch, Maniatis (Molecular Cloning, A Laboratory Manual).

20 × SSC (sodium salt citrate, tri-sodium citrate) was used as the transfer medium. used; the transfer from the gel to the filter membrane took place via a Total time of 24 hours. The membrane was then washed with 4 × SSC washed. The RNA was on the membrane with the Stratalinker II (Stratagene) covalently (UV crosslink) fixed. The membrane was washed with 50 ml Herby buffer Roll hybridization oven prehybridized at 60 ° C for 4 hours. The marking of the Nucleic acid fragments were carried out using the Random Primer Labeling System Kit (Gibco BRL) according to the manufacturer's instructions. The radioactive marked, heat denatured probe was then added to the hybridization mix. Hybridization was carried out at a temperature of 60 ° C for 16 hours. The Nylon membrane was washed with Wash Buffer 1 for 10 minutes at 60 ° C once washed. This was followed by successive further washing steps for every 10 Minutes at 60 ° C with wash buffer 2 and wash buffer 3 until sufficient Stringency was achieved. Sufficient stringency means that non-specific  Foreign hybridizations of the Arabidopsis thaliana hybridization probe on the Filter membrane could no longer be detected. Only then was the exposure of the Filters in the presence of an X-ray film.

Herby buffer

250 mM Na-phosphate buffer, pH 7.2
1mM EDTA
1% BSA (bovine serum albumin)
6.5% SDS (sodium dodecyl sulfate)

Wash buffer 1

3 × SSC (0.45 M NaCl, 0.045 M trisodium citrate; pH 7.0)
0.5% SDS

Wash buffer 2

1 × SSC (0.15 M NaCl, 0.015 M trisodium citrate, pH 7.0)
0.5% SDS

Wash buffer 3

0.1 × SSC (0.015 M NaCl, 0.0015 M tri-sodium citrate, pH 7.0)
0.1% SDS

20 × SSC

3 M NaCl
0.3 M tri-sodium citrate, pH 7

The hybridization patterns clearly showed the presence of an RNA Arabidopsis thaliana, which corresponds to the length of the full-length cDNA clone. Also in the case of total RNA isolates from Zea mays and Brassica napus a weaker hybridizing signal could be isolated  can be demonstrated which in its running distance that of the hybridizing Arabidopsis thaliana RNA corresponded. Because in all cases the same starting quantity the total RNA was applied to the gel, you can see the weaker one Hybridization signal in the case of Zea mays and Brassica napus in two Interpret directions. Either the RNA lies for the AMP deaminase encoded, in Zea mays and Brassica napus in smaller quantities than in Arabidopsis thaliana based on the total amount of RNA, or the sequence of the AMP Deaminase RNA from Zea mays and Brassica napus has a certain heterology compared to the hybridization probe used from Arabidopsis thaliana. A combination of both effects is also possible, so that one expresses less AMP deaminase RNA, which also has a certain sequence heterology to the hybridization probe obtained from Arabidopsis thaliana, in which Sum results in a weaker signal. In any case, however it is assumed that a comparable, because specifically hybridizing, AMP deaminase sequence at least also in agriculturally relevant Cultivated plants such as Zea mays and Brassica napus exist.

Example 4

The present full-length cDNA of the AMP deaminase from Arabidopsis thaliana (as indicated in SEQ ID NO 1) was converted into the vectors pRMHa-N (day on N- terminus) and pRMHa-C (day at C-terminus), which are described in Benting J. et al., Anal Biochem, 278 (2000), 59-68.

For this, the full-length cDNA of the AMP-DA was amplified using PCR and then into the corresponding interfaces of the insect Expression vectors pRMHa-N or pRMHa-C cloned. With the vector pRMHa-N is the His tag (corresponds to 10 histidine residues) at the Nterminus the AMP deaminase, the vector pRMHa-C contains the His tag (corresponds to  10 histidine residues) at the C-terminus of AMP deaminase. The primers were like this selected that the SEQ ID NO 1 after the PCR into the two vectors could be inserted that a complete open reading frame (His-Tag plus of the AMP deaminase encoded by the cDNA).

The plasmid PASK IBA3-AMP-DA was used as template for the cDNA of the AMP deaminase. 1 µl (100 ng / µl) of the plasmid was used and with 5 µl PCR buffer, 1 µl 5'AMP-DA pRN-Ntag (25 pmol), 1 µl 3'AMP-DA pRN-Ntag (25 µmol) , or 1 µl 5'AMP-DA pRN-Ctag (25 pmol), 1 µl 3'AMP-DA pRN-Ctag (25 µmol), 1 µl dNTPs (25 mmol) and 40 µl H ₂ O and 1 µl native Pfu polymerase [2.5 U / µl] (Stratagene) added.

The PCR program carried out comprised the following cycles:
1 min, 94 ° C; 1 cycle;
0.5 min, 94 ° C; 1 min, 55 ° C; 4 min, 72 ° C; 30 cycles
10 min, 72 ° C; 1Zyklus

The PCR reactions were carried out on a 1% TBE agarose gel according to the standard method separated (see e.g. Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). A PCR amplificate of approximately 2500 bp was obtained. The resulting PCR product was directed into the Bam HI and Kpnl restriction sites Expression vectors pRMHa-N or pRMHA-C integrated.

Primers used 5'AMP-DA pRN-Ntag (SEQ ID NO 13)

5'-CGC-GGA-TCC-ATG-GAA CCC AAT-ATT-TAC-3 '

3'AMP-DA pRN-Ntag (SEQ ID NO 14)

5'-CGG GGT ACC TTA-TGG-AAC-AAC-TTC-ATC-3 '

5'AMP-DA pRN-Ctag (SEQ ID NO 15)

5'-GGG GTA CCG TCA TGG AAC CCA ATA TTT ACC-3 '

3'AMP-DA pRN-Ctag (SEQ ID NO 16)

5'-CGC-GGA-TCC-TGG-AAC-AAC-TTC ATC AGA G-3 '

Transfection of the Schneider cells

Cells from the Schneider cell line SL-3 (Schneider, I., J. Embryol. Exp. Morphol. 27 (1972), 353-365) were added at 27 ° C in SF-900II medium (Gibco-BRL) cultivated from 2% fetal calf serum (Gibco-BRL).

SL-3 cells were seeded 1 day before the transfection in a concentration of 2 × 10 ⁶ cells / 3 cm dish. The cells were washed with serum-free SF-900II medium. A transfection was carried out with 1.5 µg pRMHa-N-AMP-DA / pRMHa-C-AMP-Da and 0.5 µg PIZ / V5-His (Invitrogen) and 6 µl FuGene6 (Roche) in SF-900II (without addition of fetal calf serum and antibiotics) in a total volume of 1 ml. Two days after the transfection, the cells were placed under selection (1 mg / ml Zeocin) (Invitrogen). An aliquot was then removed for a gel electrophoretic analysis (Western blot) and for an enzymatic activity test.

Western blot and activity test

The Western blot was carried out according to the usual methods. (see e.g. Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The detection of the  expressed recombinant AMP deaminase was done exactly as by Benting (Benting J. et al., Anal Biochem, 278 (2000), 59-68).

The expected band of approximately 90 KDa was clearly detected in the Western blot, which is the expected size of the recombinant protein to be expressed, the AMP Deaminase.

The cell lysates were resuspended in test buffer (60 mM Na citrate (Merck), 100 mM KCl (Merck), 0.02% Triton X-100 (Merck), pH 7.1). The reaction was started by adding 50 μl of substrate solution (0.6 mM AMP (Sigma), 1 mM ATP (Sigma), 1 μM diadenosine pentaphosphate (Sigma), dissolved in test buffer). After an incubation of 2 h at 25 ° C., the reaction was stopped by adding 50 μl phenol solution. (0.1 M phenol (Merck), 0.17 mM Na nitroprusside (Aldrich) and 50 µl DCA solution (0.125 M NaOH (Merck), 0.38 M Na ₂ HPO ₄ (Merck), 2 mM dichloroisocyanic acid Na (Aldrich)) After two hours, a measurement in a photometer at 650 nm showed that the expressed recombinant AMP deaminase had the desired activity, so that the AMP deaminase is available in a highly pure form that can be used allowed directly in HTS (High Throughput Screening).

SEQUENCE LISTING

Claims (26)

1. An isolated nucleic acid molecule containing a nucleic acid sequence which codes for a protein which has the biological activity of a plant AMP deaminase, said nucleic acid sequence selected from the group consisting of:

• a) nucleic acid sequences which comprise the nucleic acid sequence given under SEQ ID NO 1 and / or which code for a protein with the amino acid sequence given under SEQ ID NO 2;
• b) nucleic acid sequences which hybridize with the nucleic acid sequences mentioned under (a);
• c) nucleic acid sequences whose sequence is degenerate on the basis of the genetic code in comparison to the nucleic acid sequences mentioned under (a) or (b); and
• d) nucleic acid sequences which are fragments, derivatives or allelic variants of the nucleic acid sequences mentioned under (a) to (c).

2. An isolated nucleic acid molecule according to claim 1, wherein the Nucleic acid sequence from Arabidopsis thaliana is obtained and the SEQ ID NO 1 corresponds. 3. One or more nucleic acid molecules according to claim 1 or 2, wherein Fragments or derivatives by insertion, deletion, addition, substitution, and / or Inversions have arisen. 4. A method for producing a cDNA coding for a plant AMP deaminase, comprising the steps:

• a) isolation of an mRNA population from plant tissue;
• b) reverse transcription of the mRNA population obtained;
• c) second strand synthesis based on the reverse transcribed first strand according to (b);
• d) PCR amplification of the cDNA double-stranded molecule obtained according to (a) to (c) before or after linkage with suitable adapters and
• e) Enrichment and identification of the cDNA coding for the AMP deaminase or at least fragments thereof.

5. The method according to claim 4, wherein the vegetable tissue from Arabidopsis thaliana. 6. A recombinant vector containing a nucleic acid molecule after Claims 1 to 3, wherein the vector additionally contains regulatory elements that a Expression in a homologous or non-homologous host organism guarantee. 7. A vector according to claim 6, wherein the non-homologous host organism, is a bacterium, a yeast cell or an insect cell. 8. A vector according to claim 7, wherein the non-homologous host organism E.coli, Saccharomyces cerivisiae or a cell of the Schneider cell line SL3. 9. A vector according to claim 6, wherein the homologous or non-homologous Host organism is a plant. 10. Use of one or more nucleic acid molecules according to the Claims 1 to 3 and 6 to 9 for the transformation of prokaryotic or eukaryotic Cells. 11. Use according to claim 10, wherein the orientation of the integrated Nucleic acid sequence with respect to the surrounding regulatory units in sense or in antisense orientation.   12. Cells containing one or more nucleic acid molecules according to claim 1 heterologous included. 13. Cells according to claim 12, which have an increased resistance to a or more AMP deaminase inhibitors. 14. Plant cells that according to one or more nucleic acid molecules Claim 1 contain heterologous. 15. Plant cells according to claim 14, which have an increased resistance to have one or more AMP deaminase inhibitors. 16. Plant tissue that according to one or more nucleic acid molecules Claim 1 contains heterologously. 17. Plant tissue according to claim 16, which has an increased resistance to one or more AMP deaminase inhibitors. 18. Fertile plants that contain one or more nucleic acid molecules Claim 1 contain heterologous. 19. Fertile plants according to claim 18, which have an increased resistance to have one or more AMP deaminase inhibitors. 20. Seed comprising one or more nucleic acid molecules according to claim 1 heterologous contains. 21. Seed according to claim 20, which has an increased resistance to one or more AMP deaminase inhibitors.   22. Plant according to claim 18, which to the crop plants alfalfa, cotton, Barley, oats, potatoes, corn, rice, rye, soy, summer rape, sunflower, Heard of tomato, wheat, winter rape or sugar beet. 23. A process for the production of a recombinant protein which has the biochemical characteristics of an AMP deaminase, characterized in that:

• a) a nucleic acid molecule according to claim 1 is integrated into a suitable expression vector;
• b) the expression vector containing the nucleic acid molecule according to claim 1 is transferred into a corresponding host cell; and
• c) the expressed protein is optionally isolated from the host organism or the medium surrounding it, the protein being enzymatically active even after its isolation.

24. Recombinant protein, which by a method according to claim 23 has been manufactured. 25. Use of a recombinant protein according to claim 24 for Finding new AMP deaminase inhibitors. 26. Use of a manufactured with a method according to claim 23 recombinant protein for biochemical or structural characterization herbicidal inhibitors.