DE10114063A1 - New DNA sequences from potato, useful for producing plants with altered properties, e.g. tolerance of flooding, also related proteins, antibodies and inhibitory sequences - Google Patents

Abstract

DNA sequences (I) that encode six specific plant proteins, are new. DNA sequences (I) that encode: (i) a protein with mitochondrial carrier protein activity (IIa) of 97 amino acids (aa); (ii) a protein with transferrin binding protein activity (IIb) of 255 aa; (iii) a protein with receptor-like protein kinase activity (IIc) of 186 aa; (iv) a protein with elongation factor EF-2 activity (IId) of 320 aa; (v) a protein with non-long terminal repeat retroelement reverse transcriptase activity (IIe) of 248 aa; or (vi) a protein with helicase activity (IIf) of 228 aa. All are from plants and the specification includes the protein and DNA sequences. Independent claims are also included for the following: (1) DNA sequence (Ia) comprising a coding region of (I); (2) DNA sequence (Ib) that encodes the specified proteins but (i) differs from (Ia) within the degeneracy of the genetic code, (ii) hybridizes to (Ia) or (i), (iii) has at least 70% homology with (Ia), or (iv) is a fragment or allelic, or other, variant of (Ia) or (i)-(iii); (3) ribozyme (III) or antisense RNA (IV) that is complementary to (I)-(Ib), binds specifically to (and cleaves for (III)) mRNA transcribed from (I)-(Ib), so reduces or inhibits synthesis of the protein; (4) expression vector containing (I)-(Ib), (III) or (IV); (5) host cell containing the vector of (4); (6) proteins (IIa)-(IIf); (7) preparation of the proteins of (6) by growing cells of (5); and (8) antibodies (Ab) that bind specifically to (IIa)-(IIf).

Description

The present invention relates to DNA sequences ver encode different plant proteins, u. a. a protein whose biological function of a mitochondrial carrier protein corresponds, a protein whose biological function is one Transferrin binding protein corresponds to a protein whose biological function of a receptor-like protein kinase corresponds, a protein whose biological function is one Elongation factor EF-2 corresponds, a protein whose biolo gische function of a non-LTR retroelement reverse transcript corresponds to a protein and its biological function corresponds to a helicase. The present invention relates also various uses of these DNA sequences preferably for the isolation of promoters (constitutive, aerobic or anaerobically inducible) and terminators.

So far, the foreign gene expression in crops such as Potato only a few own promoters and potato Terminators available, which also has a number of disadvantages len, z. B. in terms of inducibility and / or Strength of gene expression. For foreign gene expression in potatoes So far, however, are mainly heterologous promoters and terminators used, which are also mostly detrimental Have properties. So z. B. the anaerobically inducible Maize GapC4 promoter weaker in anaerobic conditions Comparison to the CaMV 35S promoter under aerobic conditions. In addition, it is also induced by pathogen attack.

Thus, the present invention is the technical problem underlying new proprietary promoters and terminators  to provide, preferably the above disadvantages do not have.

The solution to this technical problem is provided by the Ready position of the marked in the patent claims achieved. It was in the to the present Invention leading experiments, in particular by means of Method of differential display (DD), after new anaerobic Inducible tuber-specific genes sought. Were both new anaerobic and aerobic inducible tuber-specific Found genes as well as new genes that are constitutively expressed become. Based on the new genes found their Promoters and terminators are identified that are present especially for the production of desired proteins in plants, preferably potato plants, where - ever according to the type of promoter and the desired purpose - the Ex constitutive or aerobic or anaerobically inducible can follow. The use of homologous Pro promises engines and terminators in potato a higher functional probability than the use of heterologous promoters and terminators in potato. By using the new homologous promoters and terminators in potato may be one higher foreign gene expression, better tissue specificity or bes sere inducibility can be achieved. Furthermore, the Verwen tion of the promoters and terminators thus obtained also in other organisms, especially in plants, to a higher Foreign gene expression, better tissue specificity or better Inducibility lead. The promoters and terminators can z. B. in potato not only to control the Fremdgenexpres but also to control expression of potato proteins genes are used. In addition, RNA controls, which are not translated, z. Antisense RNA or ribozyme me, be transcribed, and so efficient expression of a desired gene. The transcription can be constitutive, tissue-specific or inducible. Furthermore, transcription in other organisms may be due to These promoters and terminators are controlled. With help the sequence of new genes and their nontranscribed ones  As well as untranslated areas can also Informa to improve or alter other genes, Pro motors and terminators by modifying the same, Modifi cations of others or by combinations with other elements be won. The identification of anaerobically induced In addition, it is possible to use transgenic genes Plants with altered characteristics regarding anaerobiosis, z. As flooding tolerance, by overexpression or Repres sion of the genes found. Finally, z. B. the gene encoding the elongation factor EF-2 changed tion of the translational profile of a plant and thus ge aimed post-translational regulation of plant Gene expression can be used.

The present invention thus relates to a DNA sequence coding for one of the following plant proteins:

• a) a protein whose biological function corresponds to a mitochondrial carrier protein and which has the amino acid sequence shown in Figure 1;
• b) a protein whose biological function corresponds to a trans ferrin binding protein and which has the amino acid sequence shown in Figure 2;
• c) a protein whose biological function corresponds to a receptor protein-like protein kinase and which has the amino acid sequence shown in Figure 3;
• d) a protein whose biological function corresponds to an elon gation factor EF-2 and which has the amino acid sequence shown in Figure 4;
• e) a protein whose biological function corresponds to a non-LTR retroelement reverse transcriptase and which has the amino acid sequence shown in Figure 5; or
• f) a protein whose biological function corresponds to a helicase and which has the amino acid sequence shown in FIG .

Preferably, this DNA sequence comprises the coding region of a DNA sequence shown in Figure 1, 2, 3, 4, 5 or 6.

The present invention further relates to a DNA sequence which encodes a protein having the above-mentioned biological properties and which is different from the above DNA sequence in the codon sequence due to the degeneracy of the genetic code which hybridizes with the above DNA sequences 1, 2, 3, 4, 5 or 6 has a homology of at least 75%, preferably 85%, more preferably 90% and most preferably 95%, of the DNA sequence of the DNA sequence of FIG. 1, 2, 3 or is an allelic variant or another variant of the above DNA sequences.

The term "hybridi." Used in the present invention "refers to conventional hybridization conditions conditions, preferably on hybridization conditions in which 5 × SSPE, 1% SDS, 1 × Denhardts solution is used as the solution and / or the hybridization temperatures between 35 ° C and 70 ° C, preferably at 65 ° C. After hybridization is preferably first with 2 × SSC, 1% SDS and then with 0.2 × SSC at temperatures between 35 ° C and 70 ° C, preferably washed at 65 ° C (to define SSPE, SSC and Denhardts Solution, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989)). Particularly preferred are stringent Hybridization conditions, as described for example in Sambrook et al., supra.

The terms "variant" or "fragment" used in the present invention include DNA sequences which are opposite to the sequences indicated in FIG. 1, 2, 3, 4, 5 or 6 by deletion (s), insertion ( FIG. en), exchange (s) and / or other modifications known in the art, or comprise a fragment of the original DNA sequence, wherein the protein encoded by these DNA sequences still has the biological properties of the original protein and in plants is biologically active. These include allelic variants. Methods for generating the above-mentioned changes in the DNA sequence are known in the art and described ben in standard works of molecular biology, for example in Sambrook et al., Supra. The person skilled in the art is also able to determine whether a protein encoded by such a modified nucleic acid sequence still has the original biological properties, eg. B. by means of the following assays. For these, it is suitable to express the protein as a fusion protein with N-terminal thioredoxin and C-terminal His-tag using the kit pBAD / TOPO® ThioFusion ™ expression system from Invitrogen (Groningen, NL) in E. coli cells and via a Ni-NTA column to clean (QIAexpress, Qiagen, Hilden).

Activity assay for the mitochondrial carrier protein

Functional detection as a mitochondrial carrier protein is carried out by reconstituiton studies on liposomes according to standard conditions taking into account various substrates (see Palmieri et al. (1999) FEBS Letters 462: 472-476). First, 10 ng purified overexpressed protein is reconstituted in liposomes. External substrate of the liposomes are removed from the proteoliposomes via a Sephadex G-75 column (Amersham Pharmacia Biotech, Uppsala, Sweden) with 50mM NaCl, 10mM PIPES, pH 7.0. The transport is then started at 25 ° C by the addition of radioactive substrate [ ³ H] Carnitine (Amersham). In control reactions, inhibitors are added at the same time. After 60 min, the reaction is stopped by addition of 40 mM pyridoxal-5'-phosphate and 15 mM batho-phenanthroline, the excess radioactivity is removed via a Sephadex G-75 column and the radioactivity integrated into the liposomes is determined. The transport activity is then derived from the ratio of radioactivity used to incorporated radioactivity.

Activity assay for the transferrin-binding protein

The binding of transferrin-binding proteins can be carried out by means of a competitive transferrin-binding assay. For this purpose, an Immunolon II microtiter plate (Dynatech Laboratories, Chantilly, VI, USA) is first with transferrin by incubation with 400 ul of 50 ug / ml transferrin (Sig ma, Deisenhofen) in coating buffer (50 mM NaHCO ₃ , pH 9.6 ) at 4 ° C overnight. The plate is then washed in triplicate with in each case 400 μl of coating buffer without transferrin, with 400 μl each of 0.5% gelatin in TBS-Tween (15 mM Tris, 150 mM NaCl, 0.5% Tween 80, pH 8.0). Blocked for one hour at room temperature and washed three times with 400 ul TBS-Tween. Several dilution steps of the E. coli over-expressed and purified transferrin-binding protein in 100 μl each of 0.5% gelatin in TBS-Tween are incubated for one hour in the transferrin-loaded plate. The plate is then washed with 400 μl of 0.5% gelatin in TBS-Tween, 100 μl of biotinylated TfbA (Strutzberg et al. (1995) Infection and Immunity 63, 3846-3850) added for one hour, and again with 400 μl of 0 , 5% gelatin washed in TBS-Tween. The biotinylated TfbA is detected photometrically at 405 nm as described by Strutzberg et al. (1995). For comparison, a calibration series with non-biotinylated TfbA is provided to determine the specific binding activity of the trans ferrin-binding protein.

Activity assay for the receptor protein kinase

10 ng of the overexpressed in E. coli and purified Receptor protein kinase is once with and once without 10 ng GST (glutathione S-transferase, Sigma, Deisenhofen) in 15 ul 50 mM Tris-HCl, pH 7.3, 10 μCi γ- ³² P] ATP (6000 Ci mmol ^-1 ), 20 μM ATP, 1 mM DTT, 10 mM MnCl ₂ incubated at 30 ° C for 60 min. The reaction is stopped by the addition of 35 μl of ice-cold 10% (v / v) TCA. The proteins are sedimented by centrifugation, the pellet is washed with 50 .mu.l ice-cold 10% (v / v) TCA and then resuspended in 4 .mu.l 1 M Tris and 5 .mu.l 2 × SDS sample buffer (Bio-Rad, Munich). The sample is loaded on a 10% polyacrylamide gel and separated at a current of 15 mA. First, the gel is stained by Coomassie to check the order of comparable amounts. Subsequently, the gel is dried and a car radiogram taken with an X-ray film at room temperature. The intensity of the bands is quantified in a Fluor-S-Max imaging system (Bio-Rad). Kinase activity is demonstrated by autophosphorylation of the receptor protein kinase in the sample without GST as well as by phosphorylation of the GST and auto-phosphorylation of the receptor protein kinase in the sample with GST (by Knaap et al. (1999) Plant Physiology 120: 559-569).

Activity assay for elongation factor 2

To show the function as elongation factor-2, be Activity assays after reconstitution of overexpressed EF-2 into functional 80 S ribosomes under standard conditions guided. The GTP binding is via incorporation and hydrolysis determined by GTP as in Rao and Bodley ((1996) Protein Expe Rimental Purification 8: 91-96). As alternative this may be radioactively labeled EF-2 in active 80 S ribosome incorporated and detected (see Nygard et al. (1987) Biochim Biophys Acta 910: 245-253). The production such products are known in the art.

Activity assay for reverse transcriptase

10 ng of Reverse Transcriptase overexpressed and purified in E. coli are dissolved in 50 μl of 100 mM Tris-HCl, pH 7.5, 30 mM NaCl, 10 mM MgCl ₂ in the presence of 100 μM each of dATP, dGTP, dCTP and 5 μCi [α- ³² P] dTTP (3000 Ci mmol ^-1 ) and 5 μM synthetic RNA (5'-GUACGGACCG UUAGCAGAUA GGCGUACAGU CCGCUAGGGC GAUUAGCGCC GGAUAUCGGC AUGUACUCGA UCG-3 ', Metablon, Plagegg Martinsried) and 10 μM DNA primer (5'-CGATCGAGTAC-3 ', Metablon) for 60 min at 30 ° C incubated. The negative control is a sample without reverse transcriptase. In each case 10 .mu.l of the reaction mixtures are separated on a 15% polyacrylamide gel at a current of 30 mA. The gel is dried and an autoradiogram made with an X-ray film at room temperature. The activity of reverse transcriptase is demonstrated in the reverse transcriptase sample by a band of labeled DNA compared to the negative control (Lanford et al., (1999) Journal of Virology 73: 1885-1893).

Activity assay for the helicase

First, oligodeoxyribonucleotides of 10 nt, 30 nt, 100 nt, and 300 nt lengths complementary to the plasmid pUC19 are synthesized (Metablon, Planegg-Martinsried) and then by standard protocol (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) by T4 polynucleotide kinase and [γ- ³² P] ATP at their 5 'ends. 1 μg of pUC19 plasmid DNA are dissolved in 25 μl of 100 mM Hepes, pH 7.6, 2 mM DTT, 0.2 mg / ml BSA and 14 mM MgCl ₂ , denatured for 5 min at 99 ° C and placed on ice. For this purpose, 10 ng of the overexpressed in E. coli and purified helicase, 4 mM ATP, 1 uM of the labeled oligonucleotides and H ₂ O are added to a Gesamtvo lumen of 50 ul. The negative control is a sample without helicase. The reaction is carried out at 30 ° C. for 30 minutes and stopped by adding 5 μl of stop buffer (200 mM EDTA, 1% SDS, 20% glycerol, bromophenol blue saturated). The reaction products are applied to a non-denaturing 15% polyacrylamide gel. As a positive control, a reaction mixture which has been previously denatured by a 5minü term heat treatment at 99 ° C, also applied with. At a current of 30 mA, the fragments are separated. The gel is dried and a car radiogram with an X-ray film at room temperature ago taken. While the positive control lane all and the negative control lane do not show labeled fragments, in the sample lane the fragments released from the plasmid DNA strand by helicase activity become visible (Chong et al (2000) PNAS 97: 1530-1535).

The DNA sequences of the invention are for various Purposes of use. So they can be used for recombinant production  of the respective protein for analysis, for further Cha characterization or to protect plants against harmful effects. You can dar beyond as hybridization probes for identification serve new, related DNA sequences or as information source for the development of PCR primers. Finally they can for the production of anti-protein antibodies, for example by DNA immunization method, and as antigen to Er production of anti-DNA antibodies.

It is conceivable that in certain cases or under be Conditions agreed reduced activity and / or con concentration of the proteins of the invention is desirable. For example, a reduction of the receptor-like Protein kinase to an altered phenotype in a plant to lead. In particular, a reduction of HAESA in Arabi dopsis thaliana for a delayed release of withered flowers Organs (Jinn, T.-L., Stone, H.M., and Walker, J.C. (2000, Gene. Dev. 14: 108-117) and the repression of OsBR11 in rice too a reduced range of internodes (Yamamuro, C. Iha ra, Y., Wu, X., Noguchi, T., Fujioka, S., Takatsuot, S., Ashi kari, M. and Matsuoka, M. (2000), Plant Cell 12: 1591-1606) to lead. Also, a reduction of the elongation factor EF2 under anaerobic conditions, such as. B. in short phases the flooding of fields occurs to a diminished Lead translation. This will under these conditions the Downregulated metabolism, so that the energy consumption ver decreases and the carbohydrate content of the tuber is preserved. The reduction of the proteins of the invention may, for. B. by the lowering or inhibition of the expression of the invention appropriate DNA sequences can be achieved. Therefore, one concerns another preferred embodiment of the present invention an antisense RNA characterized by being too one of the above DNA sequences is complementary and to can specifically bind the translated mRNA and the Synthesis of a protein encoded by these DNA sequences or a protein related to it can, and a ribozyme which is characterized in that it  is complementary to one of the above DNA sequences and specific to the mRNA transcribed from these DNA sequences can bind and cleave them, thereby reducing the synthesis of protein also encoded by these DNA sequences or inhibited. Preferably, these are antisense RNAs and Ribozymes complementary to a coding region of the mRNA. The person skilled in the art is capable of starting from the disclosed DNA sequences to produce and deliver appropriate antisense RNAs turn. Suitable procedures are for example in EB- B1 0 223 399 or EP-A1 0 458. Ribozymes are RNA Enzymes and consist of a single RNA strand. These For example, other RNAs may cleave intermolecularly, such as the mRNAs transcribed from the DNA sequences according to the invention. In principle, these ribozymes must have two domains, (1) a catalytic domain and, (2) a domain belonging to the Target RNA is complementary and can bind to them, which the Prerequisite for a cleavage of the target RNA is. outgoing It is from literature approaches now possible to construct specific ribozymes that a desired RNA at a specific, preselected site see, for example, Tanner et al., in: Antisense Research and Applications, CRC Press, Inc. (1993), 415-426). Preferably, the above ribozymes or hybridize Antisense RNAs over a range of at least 15, more preferably at least 25, and most preferably at least 50 Bases with that of the DNA sequences of the invention trans scribed mRNA.

The DNA sequences of the invention or the above described antisense RNAs or ribozymes encoding DNAs can also be inserted into a vector or expression vector become. Thus, the present invention also encompasses these DNA Sequence-containing vectors or expression vectors. The "Vector" refers to a plasmid (pUC18, pBR322, pBlueScript etc.), on a virus or another suitable vehicle. In a preferred embodiment the DNA sequences of the invention in the vector with reguli functionally linked to elements that express their expression in  allow prokaryotic or eukaryotic host cells. Such vectors contain, in addition to the regulatory elements, for example, a promoter, typically a replica origin and specific phenotypic genes Allow selection of a transformed host cell. To the regulatory elements for expression in prokaryotes, For example, E. coli, include the lac, trp promoter or T7 Promoter, and for expression in eukaryotes of the AOX1 or GAL1 promoter in yeast and the CMV, SV40, RVS-40 promoter, CMV or SV40 enhancer for expression in animal cells len. Further examples of suitable promoters are the Me tallothionein I and polyhedrin promoters. To suitable Expression vectors for E. coli include, for example, pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8, the latter being is awarded. To the vector suitable for expression in yeast These include pY100 and Ycpad1, for expression in mammals cells pMSXND, pKCR, pEFBOS, cDM8 and pCEV4. To the erfin The expression vectors according to the invention also include baculovirus derived vectors for expression in insect cells, for example pAcSGHisNT-A.

General methods known in the art can be used for Construction of expression vectors containing the inventive contain DNA sequences and suitable control sequences, be used. These methods include, for example, in in vitro recombination techniques, synthetic methods, as well as in vivo recombination methods, as described, for example, in Sambrook et al., Supra. These vectors can NEN have more functional units that a stabilization cause the vectors in the host organism, such as a bak terial origin of replication or the 2 micron DNA to Sta bilisation in Saccharomyces cerevisiae. Furthermore, "left border "and" right border "sequences of agrobacterial T-DNA be included, creating a stable integration into the Erb good by plants is possible. Furthermore, a termina be present, the correct termination of the Transcription and the addition of a poly A sequence to the Transcript serves. Such elements are in the literature  (see Gielen et al., EMBO J. 8 (1989), 23-29) and are interchangeable.

To prepare for the introduction of the DNA Sequences in higher plants are a large number of Cloning vectors are available that provide a replication signal for E. coli and a marker gene for selection transformed Contain bacterial cells. Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184, etc. The Foreign gene can be attached to a suitable restriction site in the vector will be introduced. The resulting plasmid is for used the transformation of E. coli cells. transformed E. coli cells are grown in a suitable medium, subsequently harvested and lysed, yielding the plasmid will be. As an analytical method for characterizing the gewon plasmid DNA are generally restriction analyzes, Gel electrophoresis and other biochemical molecular biology used. After each manipulation, the Plasmid DNA cleaved and recovered DNA fragments can with linked to other DNA sequences. Each plasmid DNA Se can be cloned in the same or other plasmids become.

For the introduction of DNA, z. B. in a plant host cell, a variety of techniques are available. These Techniques include the transformation of plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation agent, the fusion of protoplasts, the injection, the electroporation of DNA, the introduction of DNA using the biolistic method as well as other possibilities.

In the injection and electroporation of DNA in plant cells There are no special requirements for the ver used plasmids. There may be simple plasmids, z. B. pUC derivatives. But they should be like that transformed cells whole plants are regenerated, there should be a selectable marker. Depending on the introduction method  desired genes in the plant cell can additional DNA sequences may be required. Are z. B. for the Transformation of the plant cell the Ti or Ri plasmid ver applies, so at least the right border must, but often the right and left borders of the Ti and Ri plasmid T-DNA connected as flank area with the genes to be introduced become.

If Agrobacteria are used for the transformation, it is favorable to loosen the DNA to be introduced into special plasmids kidney, in particular in an intermediate or in a bi nary vector. The intermediate vectors may be due to Sequences that are homologous to sequences in the T-DNA homologous recombination into the Ti or Ri plasmid of Agrobak be integrated. This also contains the for the Transfer of the T-DNA necessary vir region. Intermediate vector can not replicate in agrobacteria. By means of a Helper plasmids may be the intermediate vector on Agrobacterium tumefaciens. Binary vectors can both replicate in E. coli as well as in agrobacteria. They contain a selection marker gene and a linker or polylinker, which is framed by the right and left T-DNA border region become. They can be transformed directly into the agrobacteria become. The host cell serves as an Agrobacterium Plasmid carrying a vir region. The vir region is necessary for the transfer of T-DNA into the plant cell. Additional T-DNA may be present. The trans Formed Agrobacterium is used to transform Pflan used in cells.

For the transfer of the DNA into the plant cell, plant Explants expediently with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From the infected plant material, e.g. B. leaf pieces, Stengelsegmen te, roots, protoplasts or suspension-cultured plants Cells can then be stored in a suitable medium Antibiotics or biocides for selection of transformed cells can contain, whole plants are regenerated again. The  so obtained plants can then be for the presence of led DNA to be studied. Alternative systems for trans formation of plants are the transformation by means of the biolistic approach that induces electrically or chemically DNA uptake into protoplasts, electroporation of par tially permeabilized cells, the macroinjection of DNA into Inflorescences, the microinjection of DNA into microspores and Pro-embryo DNA uptake by germinating pollen and the DNA uptake in embryos by swelling (to overview: Po trykus, Physiol. Plant (1990), 269-273). While the trans formation of dicotyledonous plants via Ti plasmid vector systems Help of Agrobacterium tumefaciens is established recent work indicates that monocotyledonous plants of the Transformation by means of Agrobacterium-based vectors are accessible.

The suitable plants may in principle be Pflan zen of any plant species, d. H. either monocots as well as dicotyledonous plants. It is preferable Gramineae, Chenopodia, Leguminosae, Brassicaceae, Solanaceae, algae, mosses and mushrooms, even more preferred around Wheat, barley, rice, corn, sugar beet, cane, rape, Mustard, turnip rape, flax, pea, bean, lupine, tobacco and potato. Those desired for the expression of the desired protein Plant parts in principle concern any plants In part, propagating material and harvested products of these Plants, e.g. As fruits, seeds, tubers, rhizomes, sämlin ge, cuttings, etc.

The DNA sequences according to the invention can also be used in conjunction with a DNA coding for another protein or peptide be inserted into a vector such that the invention DNA sequences, for example in the form of a fusion protein, z. With a His-tag to facilitate cleaning recombinant production, can be expressed.

The present invention also relates to the above be written vectors containing host cells. To these host cells  count bacteria (for example the E. coli strains HB101, DH1, x1776, JM101, JM109, BL21 and SG13009), yeast, preferably S. cerevisiae, insect cells, preferably sf9- Cells, and animal cells, preferably mammalian cells. preferred Mammalian cells are CHO, VERO, BHK, HeLa, COS, MDCK, 293- and WI38 cells. Method for transforming these hosts cells, for the phenotypic selection of transformants and for the expression of the DNA molecules according to the invention under Ver The use of the vectors described above are on the Fachgebiet known.

The present invention further relates to an inventions Protein or a protein encoded in accordance with the DNA sequence according to the invention with its biological activity and methods for their recombinant production using the invention appropriate expression vectors. The inventive method involves culturing the host described above cells under conditions that control the expression of the protein (or fusion protein) allow (preferably stable Expres sion), and the extraction of the protein from the culture or from the host cells. The skilled person is aware of conditions, trans cultivated or transfected host cells to cultivate. Ge suitable cleaning methods (for example, preparative chro matography, affinity chromatography, immunoaffinity matography, HPLC, etc.) are also well known. On Protein according to the invention or a protein related thereto is not only in the above or below described measures, but can also be used for example in biological assays for activity determination Use, for example in combination with a Variety of other proteins for a high-throughput Screening. In addition, it may, for. B. for the production of anti be useful as a reagent (for example in markier ter form) in competitive assays for quantitative determination of the protein in. plants, as markers for plant parts, in where the corresponding protein is preferably produced (entwe the constitutive or during a certain stage visibly of tissue differentiation, etc.), and also to  Isolation of interacting proteins. Finally, one can Protein according to the invention or a protein related thereto also for the isolation of inhibitors, antagonists or ago nists be used, which is, for example, Pep tide or other small molecules can act.

Another preferred embodiment of the present invention The invention relates to antibodies which are resistant to an above written protein of the invention are directed. These Antibodies can be monoclonal, polyclonal or synthetic Be antibodies or fragments thereof. In this context the term "fragment" means all parts of the monoclonal Antibody (eg Fab, Fv or single chain Fv fragments), which have the same epitopic specificity as the complete anti have body. The preparation of such fragments is the Specialist known. Preferably, the inventions antibodies according to the invention to monoclonal antibodies. The inventor The antibodies according to the invention can be prepared according to standard methods are provided, preferably one of the inventive protein encoded according to DNA sequences or a synthetic one Fragment thereof serve as immunogen. Method of extraction Monoclonal antibodies are known in the art. The inventor The antibodies according to the invention can also be used, for example, for immunoassay precipitation of a protein according to the invention, for isolation related proteins from cDNA expression libraries, etc. used become. The present invention also relates to a hybridoma, that the above-described monoclonal antibody he testifies.

The present invention also relates to the use of a DNA sequence according to the invention, a ribo according to the invention zyms or antisense RNA, an expression of the invention vector, a protein according to the invention or an antibody pers for the production of a plant, preferably potato plant ze with altered properties, preferably under ana eroben conditions become effective.  

Furthermore, the present invention relates to the use of a DNA sequence of the invention or a fragment, preferably with a length of at least 15 nt as a probe or PCR Primer, for screening for preferably constitutive tubers specific, aerobic or anaerobic inducible or anaerobic inducible promoters or terminators. The so isolated Promoters or terminators are for a number of applications useful, as discussed above, z. B. for expression of a desired foreign gene in a Plant, preferably potato plant, under either not inducible or inducible conditions. Procedure for Screening for such controls, e.g. B. in a genome potato bank, and to identify it Professional known and u. a. in Example 2 below described. Method for determining the relevant sequences an isolated promoter or terminator are those skilled in the art also known and include z. B. the generation of Dele mutants in vitro, in which the 5 'and 3' End sequences are removed, so for the activity the promoter / terminator responsible DNA sequence to be able to border.

Finally, the present invention relates to a kit which a DNA sequence of the invention or an above be contains written antibody. Depending on the design of the diagnostic kits, the DNA sequence or the antibody be immobilized. The antibody may be in, for example, Immunoassays in liquid phase or to a solid support ge to be bound. In this case, the antibody in different ways and be marked. Suitable markers and markers Methods are known in the art. Examples of Immu Nassays are ELISA and RIA.  

Brief description of the figures

Figure 1 DNA sequence of fragment 15-1 and deduced amino acid sequence
The biological function of the encoded protein ent speaks that of a mitochondrial carrier protein. Further homologies of the longest reading frame: TC57459 (tomato, unknown protein), TC93200 (Arabidopsis, unknown protein).

Figure 2 DNA sequence of fragment 15-2 and deduced amino acid sequence
The biological function of the encoded protein ent speaks a transferrin binding protein. Highest homologies of longest reading frame found: AI771120 (tomato, hypo thetic protein), AW549368 (tomato, putative protein), TC101033 (Arabidopsis, putative protein), TC94111 (Arabidopsis, hypothetical protein)

Fig. 3 DNA sequence of the fragment 15-3 and derived amino acid sequence
The biological function of the protein encoded by it corresponds to that of a receptor-like mitochondrial carrier protein. Further homologies of the longest reading frame: BE922542 + BE919557 (potato, ESTs), NP00614 (tomato, Hcr2-OA).

Figure 4 DNA sequence of fragment 15-5 and deduced amino acid sequence
The biological function of the encoded protein ent speaks that of the elongation factor EF-2.

Figure 5 DNA sequence of fragment 15-6 and deduced amino acid sequence
The biological. Function of the encoded protein ent speaks of a non-LTR retroelement reverse transcriptase. Further homologies of the longest reading frame: BE450381 (tomato, EST).

Figure 6 DNA sequence of fragment 15-7 and deduced amino acid sequence
The biological function of the encoded protein ent speaks that of a helicase. Further homologies of the longest reading frame: BE924293 (potato, EST), AW218000 (tomato, unknown protein), TC87605 (Arabidopsis, unknown protein).

Fig. 7 Results of RT-PCR
The reactions were carried out with potato RNA (-: without cDNA, B: leaf, C: tuber, aerobic, I: tuber, anaerobic) to detect gene expression of fragment 15-1 (homologous to a mitochondrial carrier protein) fragment 15-2 (homologous to trans ferrin binding protein), 15-3 (homologous to a receptor-like protein kinase), fragment 15-5 (homologous to EF-2), fragment 15-6 (homologous to a non-LTR retroelement reverse Transcriptase) and fragment 15-7 (homologous to a helicase). All bands had the expected size and were excised, cloned and checked by sequencing.
The results indicate that the genes of fragments 15-2, 15-5 and 15-6 are anaerobically induced in potato tubers, and thus their promoters are useful for anaerobic induction in tubers. In contrast, the genes of fragments 15-1, 15-3 and 15-7 are active in potato leaves and aerobic bones. Their promoters are thus constitutive.

Fig. 8 Results of genomic PCR
Reactions to detect genes in potatoes were performed with genomic DNA (-: without genomic DNA, +: with genomic DNA potato cv. Désirée) and gene-specific primers (P: patatin as control, 15-1, 15-2, 15-3 , 15-5, 15-6 and 15-7). The bands were excised, cloned and checked by sequencing. The upper band of 15-6 corresponds to a rearrangement of 15-6. In no case could intron sequences be detected.

Fig. 9 principle of inverse PCR (iPCR)
Genomic DNA is digested with restriction enzyme X. The ends of the 5 'and 3' flanking sequences thus generated are then joined together by ligation, thus circularizing the fragment. Incubation with restriction enzyme Y again gives a linear fragment from which the original flanking sequence is amplified via PCR primers A and B.

Fig. 10 Schematic representation of the transgenic potato line 2-13 construct for iPCR
RB: right boundary; LB: left border of T-DNA. In addition, the XbaI site in the vector and the BclI site in the GUS gene (90-95 nt), the neomycin phospho transferase (NPTII) selection marker, the transcription directions (large arrows) and the primer binding sites (small arrows; L 77-58 nt, GUS-R 357-335 nt).

Fig. 11 Results of the iPCR
Lane 1: 1 kb ladder (Gibco BRL), lanes 2 and 4: 20 μl of a 50 μl iPCR batch of genomic DNA from non-transgenic control plants (1 μg or 2 μg genomic DNA). Lanes 3 and 5: 20 μl of an iPCR mixture with genomic DNA from potato plants with the GapC4-GUS construct (1 or 2 μg genomic DNA).

The following examples illustrate the invention.

example 1 Identification of new z. T. aerobic or anaerobic inducible potato Gene

To identify aerobically or anaerobically induced potatoes tuber genes became potato tubers aerobic and anaerobic inku biert. Then the tubers became the total RNA iso which was subsequently transcribed into cDNA. from that were amplified by PCR gene fragments using  Polyacrylamide gel electrophoresis separated (PAGE) and by means of Silver staining were visualized. Possible differentially expressed gene fragments were excised from the gel and reamplified by PCR. The amplificates were cloned and sequenced. The sequences were homologated by homology analyzed.

(A) Aerobic and anaerobic incubation of potato tubers

For anaerobic incubation, the company's Anaerocult kit was used Merck (Darmstadt, Germany) used. This consists of a gas-tight anaerobic pot and Anaerocult A, a silica gel, the Moisture emits carbon dioxide and binds oxygen. In addition, the kit includes a test strip, the anaerotest, to control the anaerobiosis. Anaerobic conditions become by a color change of the test stick from blue to white shows. The sample material was tubers from the Dési potato variety used. The plants were in the greenhouse in pots ago dressed and the tubers after the harvest about one to two months stored at 4 ° C. Four were washed for incubation whole tubers with an average diameter of 5 cm placed in the anaerobic pot. The samples were added for 96 hours Room temperature and darkness anaerobically incubated. For reference An equal number of tubers were also at room temperature and darkness stored aerobically for 96 hours. After the incubation The tubers were peeled, sliced and fried frozen nitrogen. The samples were either directly further processed or until later use at -80 ° C stored.

(B) Isolation of total RNA from tubers

The frozen tuber material was crushed with a hammer reduced and then ground in liquid nitrogen. Per Sample was mixed with 2 ml of material containing 4 ml of Z6 buffer (8 M guanidium). Hydrochloride, 20 mM MES, 20 mM EDTA. The attitude pH to 7.5 was done with NaOH cookies. Directly before Use was the 26-buffer with 2-mercaptoethanol (final concentrations 50 mM) (Logeman et al., (1987) Anal. Biochem. 163, 16-20). The samples were used to separate the cell debris  Centrifuged at 15,000 × g and 4 ° C. for 20 minutes. The whole Nucleic acids were then treated with 1/10 volume 3M NaAc (pH 4.8). and 0.8 volume of ice-cold isopropanol. After 5 minutes Incubation on ice was centrifuge for 30 min at 15,000 x g and 4 ° C gassed and the supernatant discarded. The pellet was mixed with 2 ml of 80% Ethanol, 15 min at 15 000 × g and 4 ° C centrifu and then dried for 10 min at room temperature. The Further steps to purify the RNA were made according to the protocol and with the solutions from the Qiagen RNA / DNA "Midi Kit" (Hilden, Germany). The resulting RNA pellet was added in 300 μl RNase-free water resuspended. The RNA concentrations of the individual samples were determined photometrically. The RNA was either used immediately or frozen at -20 ° C.

(C) first-strand cDNA synthesis from total RNA

For cDNA synthesis, 2 μg total RNA was used. The ker primer mixture consisted of four different primers, which differed in the last base, z. DT ₁₁ AA, dT ₁₁ AT, dT ₁₁ AG, and dT ₁₁ AC.

The following components were added to the total RNA: 10 μl 10 mM dNTP mix, 5 μl 5 × reverse transcriptase buffer, 10 μl 50 mM anchor primer mix, 1 μl 5 mg / ml BSA, 4 μl 25 U / μl M-MuLV reverse Transcriptase (NEB), ad 50 μl H ₂ O. The reaction was carried out at 37 ° C. for 1 hour.

The following anchor primers and anchor primer mixtures were used:

designation base sequence 1. Roth-dt ₁₁ -AT 5'-TTTTTTTTTTTAT-3 ' 2. Roth dT ₁₁ -AA 5'-TTTTTTTTTTTAA-3 ' 3. Roth-dT ₁₁ -AG 5'-TTTTTTTTTTTAG-3 ' 4. Roth-dT ₁₁ -AC 5'-TTTTTTTTTTTAC-3 ' 5. Roth-dT ₁₁ -GT 5'-TTTTTTTTTTTGT-3 ' 6. Roth-dt ₁₁ -GA 5'-TTTTTTTTTTTGA-3 ' 7. Roth-dT ₁₁ -GG 5'-TTTTTTTTTTTGG-3 ' 8. Roth dT ₁₁ GC 5'-TTTTTTTTTTTGC-3 ' 9. Roth dT ₁₁ -CT 5'-TTTTTTTTTTTCT-3 ' 10. Roth dT ₁₁ -CA 5'-TTTTTTTTTTTCA-3 ' 11. Roth dT ₁₁ -CG 5'-TTTTTTTTTTTCG-3 ' 12. Roth-dT ₁₁ -CC 5'-TTTTTTTTTTTCC-3 ' 13. dT ₂₀ 5'-tttttttttttttttttttt-3 ' 14. DD T ₁₈ AN 5'-GATCGATCTTTTTTTTTTTTTTTTTTAN-3 '

(D) "Differential Display" PCR

CDNA was used as template for the PCR. The PCR reactions were performed in Eppendorf "Master Cycler" devices (Eppen village, Hamburg). By means of the "differential display" (DD) -PCR were Fragments from cDNA amplified. It was among others called random primer, each with a non-specific sequence and a 50% GC content.

Reaction batch using 10mer and 14mer Random Spri numbers: 1 ul cDNA, 2 ul 10 × Taq polymerase buffer, 0.5 ul 50 μM anchor primer, 1 ul 10 μM random primer, 0.6 ul 50 mM MgCl ₂ , 0.5 ul 10 mM dNTPs, 0.1 μl 10 U / μl Taq DNA polymerase (Gibco BRL, Neu-Isenburg, Germany), ad 20 μl with H ₂ O.

Reaction batch using 18mer random primers: 2 μl cDNA, 2 μl 10 × Taq polymerase buffer, 2 μl 10 μM anchor primer, 2 μl 10 μM random primer, 1 μl 50 mM MgCl ₂ , 0.1 μl 10 mM dNTPs, 0.1 μl 10 μg / l Stoffel fragment of the "Ampli Taq" DNA polymerase (Perkin Elmer (Rodgau-Jügelsheim, Germany) ad 20 μl with H ₂ O.

The annealing temperatures for the primers used varied between 42 ° C and 57 ° C. PCR profile modified to Liang and Pardee ((1992) Science 257, 967-971) for 10mer and 14mer primers: (1) denaturation (4 min, 94 ° C), (2) denaturation (1 min, 94 ° C), (3) annealing of the primers (1 min, 42 ° C / 46 ° C), (4) elongation (3 min, 72 ° C); (2) - (4): 40 cycles; (5) End elongation (10 min, 72 ° C).

PCR profile according to Kociok et al. ((1998) Mol. Biotechnol. 9, 25-33) for 18mer primers

• a) denaturation (3 min, 96 ° C);
• b) (1) denaturation (5 min, 95 ° C), (2) annealing of the primers (5 min, 42 ° C), (3) elongation (5 min, 72 ° C);  
• c) (1) denaturation (2 min, 94 ° C), (2) annealing of the primers (5 min, 42 ° C), (3) elongation (5 min, 72 ° C), 2 cycles;
• d) (1) denaturation (1 min, 94 ° C), (2) annealing of the primers (1 min, 57 ° C), (3) elongation (2 min, 72 ° C), 30 cycles;
• e) End elongation (7 min, 72 ° C).

The following random primers were used:

designation base sequence 10-mer random primer Roth-150 02 5'-CAATGCGTCT-3 Roth-150 08 5'-GGAAGACAAC-3 ' Roth-150 10 5'-CCATTTACGC-3 '

The original DD primers from the 10 × 10 nt DD primer kit (Roth, Karlsruhe, Germany) were extended at the 5 'end by the base sequences GATC or GATCGATC to 14mers and 18mers, respectively, to obtain the following primers:

14mer chance primer DD 14.1 5'-GATCGTGCAATGAG-3 ' DD 14.2 5'-GATCGGAAGACAAC-3 '

18mer accident primer DD 18.1.2 5'-GATCGATCGTGCAATGAG-3 ' DD 18.2.2 5'-GATCGATCCAATGCGTCT-3 ' DD 18.2 5'-GATCGATCGGAAGACAAC-3 ' DD 18.6 5'-GATCGATCAGGTTCTAGC-3 ' DD 18.9 5'-GATCGATCAGAAGCGATG-3 ' DD 18.10 5'-GATCGATCCCATTTACGC-3 ' Ap13-18 5'-GATCGATCAGTTAGGCAA-3 '

(E) Polyacrylamide gel electrophoresis of DNA and silver staining

The amplified fragments were either horizontal Polyacrylamide gel electrophoresis on a "Multiphor II" electro Phorese unit (Pharmacia, Freiburg, Germany) separated (9% gel, degree of crosslinking 2%) or via vertical polyacryla mid-gel electrophoresis on a direct blotting sequencer system 1500 "from GATC (Konstanz, Germany) (5% gel, degree of crosslinking  2.6%). The detection of the amplified cDNA bands was carried out by silver staining essentially according to the protocol of Böc kelmann et al. ((1999) Skin, Pharmacol, Appl., Skin, Physiol. 54-63).

(F) Elution of DNA fragments from silver-colored polyacryla midgels and reamplification

The relevant gel bands were cut out of the polyacrylamide gel with a scalpel. About 2 to 3 bands of the same type were combined in reaction vessels and comminuted with a pipette tip. Subsequently, 50 .mu.l of TE was added to the samples, incubated for 6 min at 99.degree. C. and centrifuged for 5 min at 10,000.times.g at RT. 2 μl of the supernatant was used for subsequent reamplification by PCR as a template. In the case of apparently weak cDNA bands, the gel pieces were transferred to 50 μl of 0.5 M NH ₃ Ac / 1 mM EDTA and incubated at 37 ° C. overnight (Böckelmann et al., (1999), supra). Subsequently, the samples were centrifuged at 10,000 × g and 2 μl of the supernatant was used as a template for the PCR. This modified method was also used when the aforementioned method in the Reamplifika tion led to no result. For the reamplification of the eluted cDNA fragments from the silver-stained polyacrylamide gels, a second PCR with the same primer pairs, which were also used for the DD-PCR, was chosen. The reaction mixture used was composed as follows: 2 μl eluate, 5 μl 10 × Taq polymerase buffer, 2 μl 50 mM MgCl ₂ , 0.1 μl 10 mM dNTP, 1 μl 10 pH primer 1, 1 μl 10 pH primer 2, 0.5 μl 10 U / μl Stoffel Fragment Ampli Taq DNA Polymerase (Perkin Elmer), ad 50 μl with H ₂ O.

The following cycle program was used: (1) Denaturation (3 min, 98 ° C), (2) denaturation (1 min, 98 ° C), (3) addition of the Primer (1 min, 57 ° C), (4) elongation (2 min, 72 ° C), (2) - (4) 30 Cycles, (5) end elongation (10 min, 72 °). The PCR samples were in a 1% agarose gel on the size and purity of the fragments examined.  

(G) Cloning of PCR products

For further cloning and sequencing they were excised and reamplified fragments in the linearized pCR2.1 vector from the TA cloning kit from Invitrogen (Groningen, The Netherlands) and then into competent E. coli cell len of the strain INVαF 'transformed. The ligation was after the Information provided by the manufacturer. Deviating from that However, instead of 2 to 3 μl PCR batch 6 μl PCR approach for the Ligation reaction used. The transformation took place as from Manufacturer described. 50 and 250 μl each of the transforma were prepared on LB plates with ampicillin (final concentrations 100 mg / l) and the substrate X-gal (40 mg / l) and incubated at 37 ° C overnight. Positive clones were through Blue / white selection identified.

(H) Sequencing of plasmid DNA

The sequencing of plasmid DNA was carried out after the dideoxy Chain termination method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467) using a DNA sequencingautoma model "373 A strech" by Perkin Elmer. The used System works with a four-color DNA marking technique, which allows the entire DNA sequence in a gel trace too detect. For sequencing, the following primers were used det: M13for (5'-GTAAAACGACGGCCAG-3 ') and M13rev (5'-CAGGAAA CAGCTATGAC-3 ').

(I) Sequence analysis

The program LASERGENE (DNASTAR, Madison, USA) was used for the creation of restriction maps, for the sequence comparison and the execution of virtual mutations. Via the NCBI homepage (http://www.ncbi.nln.nih.gov) and the Expasy homepage (http://www.expasy.ch), the nucleic acid sequences were translated into amino acid sequences and subsequently searched for open reading star. NCBI and TIGR databases (http://www.tigr.org) provided homology comparisons to known sequences. The sequences for the fragments 15-1 ( Fig. 1), 15-2 ( Fig. 2), 15-3 ( Fig. 3), 15-5 ( Fig. 4) 15-6 ( Fig ) and 15-7 ( Fig. 6).

(J) RT-PCR

To examine the expression of genes corresponding to the cDNAs chen, RT-PCRs were performed. The cDNA synthesis was carried out starting from 2 μg RNA from potatoes (leaf, aerobic and tuber aerobic or anaerobic).

Reaction batch reverse transcription: 2 μg RNA, 1 μl oligo dT (400 μg / μl), ad 12 μl H ₂ O, it was cooled on ice for 10 min at 70 ° C. Thereafter, 4 μl 5 × reaction buffer (Gibco BRL), 2 μl 0.1 M DTT, 1 μl dNTPs (10 mM each) were added. This was followed by 10 min incubation at 42 ° C and short centrifugation. Finally ^, 1 μl of Superscript II ™ RNAseH reverse transcriptase (Gibco BRL) was added and it was incubated at 42 ° C. for 50 minutes, then at 70 ° C. for 15 minutes. The reaction mixture for PCR was:
1 μl of cDNA, 5 μl of 10 × buffer, 5 μl of dNTPs (2 mM each), 0.5 μl of primer 1, 0.5 μl of primer 2, 1.5 or 3 μl MgCl ₂ (25 mM), 1 μl Taq. Polymerase (1 μl, MBI-Fermentas (St. Leon Rot, Germany)), ad 50 μl H ₂ O. PCR program: (1) 95 ° C., 2 min, (2) 95 ° C., 30 sec, (3) 53 ° C, 1 min, (4) 72 ° C, 1 min; (2) - (4) 35 cycles, (5) 72 ° C, 5 min.

The following primers were used:

Result

(K) GENOMIC PCR

To detect the genes in potatoes, PCRs were used with genom potato potato (leaf and tuber) from cv. Desiree carried out. The DNA was synthesized using the "RNA / DNA Midi" kit Company QIAGEN isolated.

Reaction batch: 1-2 μl DNA, 5 μl 10 × buffer, 5 μl dNTPs (2 mM each), 0.5 μl primer 1, 0.5 μl primer 2,
1.5 μl MgCl ₂ (25 mM), 1 μl Taq polymerase (1 U / μl, MBI fermentase), ad 50 μl H ₂ O. PCR program: (1) 95 ° C, 2 min, (2 ) 95 ° C, 30 sec, (3) 53 ° C, 1 min, (4) 72 ° C, 2-4 min, (2) - (4) 35 cycles, (5) 72 ° C, 5 min.

The following primers were used:

(L) INVERSE PCR

One technique for isolating missing gene fragments as well as promoters and terminators of genes is inverse PCR (iPCR). The iPCR is a direct method to obtain the sequence information of the 5 'and 3' flanking regions of known genes or gene fragments. This method has already been used successfully in plants to isolate, for example, a seed-specific lipoxygenase promoter from Pisum sativum or a triticum aestivum puroindoline gene promoter. Starting material for the iPCR is genomic DNA containing the known fragment and flanking sequences ( Figure 9). This is digested with a restriction enzyme for which there is no cleavage site in the known gene fragment. Among other things, this results in a fragment consisting of the known sequence flanked by unknown sequence segments at both ends. The linear DNA fragments are treated with DNA ligase. In this case, the fragments can also react with themselves, resulting in, inter alia, circular DNA molecules. This approach is now digested with a second restriction enzyme which cuts within the known sequence. This linearizes the circularized fragment. The result is a sequence section in which the unknown portions at both ends are bounded by the known sequences. With the help of these known sequences, primers can be selected to amplify the unknown fragment ren. Based on the original fragment, the binding sites for the primer are facing away from each other, ie inversely compared to the standard PCR, from which the name of this method is derived. The amplified fragment is cloned and sequenced to obtain the sequence information about the flanking regions. The boundary between the original 5 'and 3' region is defined by the interface of the first restriction erizome. The process is repeated using the new sequence information until the entire desired sequence region is identified.

To perform the iPCR, a transgenic line was used which carries a known T-DNA fragment. Genomic DNA from leaves 2-13 (Bülow et al., 1999, Mol. Plant Microbe Interact., 12, 182-188) carrying the E. coli GUS gene under the control of the maize GapC4 promoter ( Fig. 10) was isolated using Qiagen's "DNeasy Plant Mini Kit". For control also genomic DNA of a non-transgenic line was prepared. 1 or 2 micrograms of genomic DNA from leaves of the transgenic line 2-13 and DNA from leaves of a non-trans gene control plant were digested with XbaI as the first restriction enzyme completely. Subsequently, the DNA fragments were purified with Qiagen's "Nucleotide Removal Kit". The DNA was eluted with 50 μl H ₂ O and then ligated. To test the ligation, the fragments were separated in an agarose gel. For comparison, unligated digested genomic DNA was also plotted ( Figure 11). The track of XbaI-digested genomic DNA was seen to be uniform in smear. In the trace with digested genomic DNA after the Liga tion a striking change in the migration behavior of the fragments was observed. As expected, the fragments grown larger by the ligation were slower, so they were at a higher position in the gel. Subsequently, the partially cultured DNA was linearized. For this purpose, the batch was completely digested with 40 units of the second restriction enzyme BclI in a reaction volume of 200 ul. Thereafter, the DNA was purified with the "nucleotide removal kit" from Qiagen and eluted with 50 μl H ₂ O. After this purification of the DNA, the actual iPCR was performed.

The iPCR was carried out according to a protocol of Digeon et al., Plant Mol. Biol. 39 (1999), 1101-1112. As primers, GUS-L (5'-TCGCGATCCAGACTGAATGC-3 ') and GUS-R (5'-CATTTGAAGCCGATGTCACG-3') were selected. The primer GUS-L hybridizes with nucleotides 77 to 58 of the sense strand of the E. coli GUS gene. The primer GUS-R hybridizes with nucleotides 357 to 335 of the antisense strand of the E. coli GUS gene. Due to the size of the amplicon, the iPCR was performed with a Gibco BRL "High Fidelity Platinum" Taq DNA Polymerase. The reaction mixture was composed as follows: 5 μl of DNA eluate, 1.5 μl of 50 mM MgSO ₄ , 1 μl of 10 mM dNTP, 5 μl of 10 × 10 high fidelity PCR buffer (Gibco BRL), 1.25 μl of 10 μM primer GUS -L, 1.25 μl 10 μM Primer GUS-R, 0.3 μl 5 U / μl High Fidelity Platinum Taq Polymerase (Gibco BRL), ad 50 μl with H ₂ O. The following iPCR program was used in used in an Eppendorf "Master cycler" apparatus: (1) denaturation (4 min, 94 ° C), (2) denaturation (30 sec, 94 ° C), (3) annealing of the primers (30 sec, 60 ° C) , (4) Elongation (3 min, 68 ° C), (2) - (4) 35 cycles, (5) End Elongation (10 min, 72 ° C).

The result was tested in a 1% agarose gel agarose gel ( Figure 11). A vigorous band in lanes 3 and 5 was seen with the expected fragment size of about 3.2 kb. The amplificates showed no difference in starch at 1 and 2 μg starting DNA. In the control reactions (lanes 2 and 4), the approximately 3.2 kb band could not be detected; only nonspecific bands at about 500 bp could be detected. The amplified fragment was excised and cloned into the pCR2.1 vector by Invitrogen's TA cloning kit. Sequence analysis was performed with the oligonucleotides M13 for (5'-GTAAAACGACGGCCAG-3 ') and M13 rev (5'-CAGGAAACAGCTATGAC-3') as sequencing primers. Subsequently, the sequence was compared with the well-known sequence of the GapC4 promoter-GUS construct. The homology comparison performed with the sequence revealed that the fragment was composed on the 5 'side of the GapC4 promoter and on the 3' side of the GUS gene. It was thus possible to demonstrate that the method of the iPCR can be used to identify unknown pro-motors of known genes.

EXAMPLE 2 SCREENING OF GENERAL LIBRARIES FOR ISOLATION OF GENOMIC DNA FRAGMENTS (PROMOTERS / TERMINATORS)

To isolate the genes belonging to the cDNA fragments as well as their promoters and terminators, the genomic DNA library of Solanum tuberosum cv. Saturna in λ-vector EMBL3 (Strata gene, Heidelberg, Germany) by DANISCO (Copenhagen). The screening of the DNA libraries is done according to the manufacturer's protocol. The pre-cultivation of the bacteria is done overnight in 50 ml of LB medium with 0.2% (w / v) maltose and 10 mM MgSO ₄ at 30 ° C. After centrifugation (10 min at 3000 x g), the bacteria are adjusted with 10 mM MgSO ₄ to an OD ₆₀₀ of 0.5. The phage titer is determined by infecting the bacteria (see below) with in each case differently diluted phage suspensions and subsequent counting of the plaques. To isolate the genomic DNA clones 1 × 10 ⁶ pfu (plaque forming units) are used.

In the first round of screening, 400 μl of MRA (P2) cells (genomic DNA library) (Stratagene, Heidelberg, Germany) are infected with 50000 pfu for 20 min at 37 ° C, with 4.5 ml (small plates; Diameter: 9 cm) of warm (45 ° C.) Mg ²⁺ top agarose mixed and each with one NZY plate (1% (w / v) casein hydrolyzate, 0.5% (w / v) yeast extract , 0.2%, (w / v) MgSO ₄ , 0.5% (w / v) NaCl, 1.5% (w / v) agar). After 12-16 h incubation at 37 ° C, the infectious events in the form of plaques in the bacterial lawn are visible. Subsequently, the DNA can be fixed from the phages on an applied nitrocellulose membrane (45 .mu.m pore size, Schleicher & Schull, Dassel, Germany). For this purpose, the membranes are first placed on the 4 ° C cold plates and marked the orientation of the membranes on the plates by three punctures each with a cannula. After transfer of the DNA to the membrane for 10 minutes, the membrane is washed successively for 10 minutes on impregnated filter paper with denaturing solution (0.5 M NaOH, 1.5 M NaCl), neutralization solution (1.5 M NaCl, 0.5 M) Tris / HCl pH 7.0) and equilibration solution (2 x SSPE or 2 x SSC) and then baked at 80 ° C for 1 h. The identifi cation of positive phage clones then takes place via hybridization of the membranes with digoxygenin-labeled (DIG) probes. These can be prepared with the aid of appropriate kits and subsequently detected by means of a color reaction ("DIG DNA Labeling and Detection" kit from Roche, Mannheim, Germany). After color detection on the filters, the areas on the plate that can be assigned to the signals are punched out with an inverted sterile Pasteur pipette and placed in 1 ml of SM buffer plus 20 μl of chloroform. After vigorous vortexing, the phages elute overnight at 4 ° C from the agar block into the solution. Starting from this phage suspension, as described above, bacteria are infected, plated and analyzed, in each case only 10 to 100 pfu serve as a carrier of the DNA sought. By repeating these procedures three times, the signals can be assigned to isolated plaques. The phage DNA is then isolated by standard methods. DNA fragments which contain the desired DNA regions (promoter or terminator) can then be subcloned into E. coli.

Example 3 Verification of inducibility and strength of isolated Promoto Based on the post-harvest production of scFv in transgenic kar Toffeln

The DNA sequence of an inducible potato tuber promoter becomes by linker ligation at its 5 'and 3' ends such modified to have a HincII restriction cut at the 5 'end and at the 3 'end an NcoI restriction cleavage site. The following oligonucleotide pair will be used for the linker ligation det: HincII linker: 5'-GATCGTCGACGATC-3 '; NcoI linker: 5'- GATCCCATGGGATC-3 '. The Artsaenko et al., (1998) Molecular Breeding 4, 313-319, described for an endo plasmatic reticulum localized scFv antibody encoded, is ligated by means of a linker ligation (at the 5 'end with 5' CATGCCATGGCATG-3 ', [5'-phosphorylated oligonucleotide] and am 3'-end with 5'-GCTCTAGAGC-3 '[5'-phosphorylated oligonucleotide]  modified to have an NcoI restriction at the 5 'end interface and at the 3 'end an XbaI restriction site having. From the plasmid pRT100 (Töpfer et al. (1987) Nucleic Acid Research 15, 5890), the CaMV 35S promoter is replaced by Re striktionsverdau with HincII and XbaI removed. Instead, become the two nucleic acid fragments described above, the for the promoter to be examined and the scFv antibody codie ren, advertised. After cleavage with Hindill the expression cassette isolated and in the binary vector pSR 8-30 (Düring et al. (1993), Plant Journal 3, 587-598; Porsch et al. (1998), Plant Molecular Biology 37, 581-585), wherein the expression vector pSR 8-30 / scFv (ox) is obtained.

This is used to transform E. coli SM10 (Koncz and Schell (1986), Molecular and General Genetics 204, 383-296). Transformants are mixed with Agrobacterium GV 3101 (Koncz and Schell (1986), supra) and incubated overnight at 28 ° C (Koncz et al. (1987), Proc. Natl. Acad. Sci. USA 84, 131-135). The agro Bacteria are selected on carbenicillin, the here for necessary bla gene in the above expression vector is present. Selection clones of Agrobacterium tumefaciens on truncated and incised several times on the midrib Leaving the potato plant cv. Désirée upset, and the Leaves incubated for 2 days at 20 ° C in the dark. After that, the Washed off agrobacteria and plant the potato leaves added growth substances, so that preferably regenerate shoots. Further, by the addition of kanamycin (100 mg / L) in the Plant medium untransformed cells in the potato leaves killed. Adolescent sprouts are cut off and in the medium without plant growth substances, but with kanamycin, rooted. The further cultivation of the potato plants takes place in the usual way.

To demonstrate the expression of the scFv antibody is abgeschnit tenes or intact leaf material or intact or cut tuber material by means of the Anaerocult system (Merck, Darm city, Germany) as in Bülow et al. (1999) Mol. Plant-Mi crobe Interact. 12, 182-188). After 40  Hours, the leaf material is removed and mortared. The after The presence of the expressed scFv antibody is shown over the c-myc "tag" using the monoclonal antibody 9E10-IgG (Cam bridge Research Chemicals, Northwich, Cheshire, UK) or protein L (Clontech, Palo Alto, CA, USA) in Western blot or ELISA provided. This is the total protein of the potato material isolated and used in the appropriate detection methods.

Transgenic potato plants determined as expression positive are cultivated in the greenhouse (in a pot or in a strawberry) or in the field under normal horticultural or agricultural conditions. The tubers were harvested after conventional handling and stored. For the post-harvest production of the scFv antibody, the tubers are placed in a steel or plastic reaction vessel having a gas supply valve at the bottom and a gas discharge valve at the top. The room air in the container is quickly displaced (for the detection of an anaerobically inducible promoter) by technical nitrogen or carbon dioxide. Under slow gas flow (1 m ³ per hour per m ² base), a constant composition of the gas phase in the reaction vessel is set. After 40 hours, the tubers are removed from the reaction container, homogenized, centrifuged off the solid portion and the aqueous supernatant of the chromatographic purification of the scFv antibody supplied. By comparing samples before and after oxygen deprivation, it can then be shown (in the presence of an anaerobically inducible promoter) that no scFv antibody is produced prior to displacement of oxygen, but thereafter significant amounts thereof.

Example 4 Production of transgenic potato plants with a gene under Control of a constitutive promoter

The DNA sequence of a potato promoter described above becomes by linker ligation at its 5 'and 3' ends such modified it to have an XbaI restriction cut at the 5 'end and at the 3 'end receives an XhoI restriction cleavage site.  

The following oligonucleotide pair will be used for the linker ligation det: XbaI linker: 5'-GATCTCTAGAGATC-3 '; XhoI linker: 5'- GATCCTCGAGGATC-3 '. By restriction digestion with XbaI and XhoI becomes the promoter of the kanamycin resistance-conferring cassette of the binary vector pLH9000 (Hausmann and Töpfer (1999), lectures Plant Breeding 45, 155-172), and in its place the DNA sequence of the constitutive potato promoter after restriction used with XbaI and XhoI. In the HindIII restriction section This vector is an expression cassette containing the gene for the T4 lysozyme under the control of the CaMV 35S promoter contains, cloned. The fragment to be transferred is from the binary vector pSR8-40 (Porsch et al., (1998), Plant Molecular Biology 37, 581-585) by restriction digestion with Hind cut.

The expression vector is used to transform E. coli SM10 used. Transformants were digested with Agrobacterium GV 3101 mixed and incubated overnight at 28 ° C (see C. Koncz and J. Schell (1986), Mol. Genet. 204, 383-396; C. Koncz. et al. (1987), Proc. Natl. Acad. Sci. USA 84, 131-135). The agrobacts are selected for streptomycin and spectinomycin, wherein the aadA gene necessary for this purpose in the above Ex Pressionsvektor present. Selection clones of Agrobacterium tume Faciens are cut off on and several times on the middle rip pe-cut leaves of the potato plant cv. Désirée up incubated and the leaves incubated for 2 days at 20 ° C in the dark. Thereafter, the agrobacteria are washed off and the potato plant growth substances added, so that preferably shoots regenerated. Further, by the addition of kanamycin in the plant medium untransformed cells in the potato scroll killed. Transgenic cells, on the other hand, express this Kanamycin resistance gene under the control of the promoter, therefore only they multiply. Out of these cells Sprouts are therefore transgenic. These are cut off and on Medium without plant growth substances, but with kanamycin, bewur tent. The further cultivation of the potato plants takes place in usual way.  

To check for incorporation of foreign DNA into the chromosome of the Potato can also all regenerated lines using Sout hybridization to the integrated foreign DNA become. These are genom with methods known in the art isolated from leaves of the individual lines with the DNA Restriction enzyme NotI digested and placed on an agarose gel separates. The fragments are deposited on a positively charged nylon Membrane transferred and labeled DNA of the gene for T4 lyso zym hybridized as a probe. The methods are known to those skilled in the art known. The detection of the expression of T4 lysozyme is by means of a polyclonal antibody (see Düring et al. (1993) Plant Journal 3, 587-598) in Western Blot. To further over testing the efficacy of the DNA transfer method and selec tion mechanism can genomic DNA from leaves of each Lines by PCR of the entire T-DNA with the border-specific Primers 5'-GGC AGG ATA TAT TCA ATT GTA AAT-3 'and 5'-GTA AAC CTA AGA GAA AAG AGC GTT TA-3 '. In the agarose gel is the size of the amplificates with controls from purified plas compared to mid-DNA. The fragments are cloned and se The T-DNA sequence is confirmed.

The following clones were deposited with the DSMZ, Braunschweig, in accordance with the provisions of the Budapest Treaty on February 22, 2001:

Claims (14)

1. DNA sequence encoding one of the following plant proteins:

• a) a protein whose biological function corresponds to a mitochondrial carrier protein and which has the amino acid sequence shown in Figure 1;
• b) a protein whose biological functions correspond to a tranferrin binding protein and which has the amino acid sequence shown in Figure 2;
• c) a protein whose biological function corresponds to a receptor protein-like protein kinase and which has the amino acid sequence shown in Figure 3;
• d) a protein whose biological function corresponds to an elon gation factor EF-2 and which has the amino acid sequence shown in Figure 4;
• e) a protein whose biological function corresponds to a non-LTR retroelement reverse transcriptase and which has the amino acid sequence shown in Figure 5; or
• f) a protein whose biological function corresponds to a helicase and which has the amino acid sequence shown in Fig. 6.

A DNA sequence according to claim 1 comprising the coding region of a DNA sequence shown in Figures 1, 2, 3, 4, 5 or 6. 3. DNA sequence which encodes a protein having the respective biological function defined in claim 1,

• a) which differs from the DNA sequence of claim 2 in the codon sequence due to the degeneracy of the genetic code;
• b) which hybridizes with the DNA sequence of claim 2 or 3 (a);
• c) having at least 75% homology to the DNA sequence of claim 2 or 3 (a); or
• d) which is a fragment, an allelic variant or another variant of the DNA sequence of claim 2 or 3 (a) to 3 (c).

4. ribozyme, characterized in that it is a DNA sequence according to one of claims 1 to 3 is complementary and to the of specifically bind to this DNA sequence transcribed mRNA and this can cleave, thereby increasing the synthesis of this DNA se codon protein is reduced or inhibited. 5. antisense RNA, characterized in that it leads to a DNA Sequence according to one of claims 1 to 3 is complementary and to specifically bind the mRNA transcribed from this DNA sequence may, thereby increasing the synthesis of that encoded by this DNA sequence Protein is reduced or inhibited. 6. expression vector, a DNA sequence according to any one of claims 1 to 3 or a ribozyme according to claim 4 or the antisense Containing RNA according to claim 5 encoding DNA. 7. A host cell containing an expression vector according to claim 6 is transformed. 8. protein with the defined in claim 1 biological radio tion, that of a DNA sequence according to one of claims 1 to 3 is encoded. 9. A process for the preparation of a protein having a claim 1 defined biological function, which is the breeding of the host Cell according to claim 7 under suitable conditions and the Gewin tion of the protein from the cell or the culture medium.   10. An antibody which is specific to a protein according to claim 8 binds. 11. Use of a DNA sequence according to one of claims 1 to 3, a ribozyme according to claim 4, an antisense RNA according to An Claim 5, an expression vector according to claim 6, a Pro A protein according to claim 9 or an antibody according to claim 10 Production of a plant with altered properties. 12. Use of a DNA sequence according to one of claims 1 to 3 or a fragment thereof for screening for terminators or constitutively, aerobic or anaerobically inducible plant promoters. 13. Use of a DNA sequence according to claim 1 (d) for the production a plant with a modified translation profile. 14. Kit comprising a DNA sequence according to one of claims 1 to 3 or an antibody according to claim 10.