DE10138091A1 - Process for influencing mineral intake in transgenic plants - Google Patents

Abstract

The invention relates to a method for influencing mineral absorption and in particular iron absorption in transgenic plants. Furthermore, the invention relates to DNA sequences which code for FER proteins, in particular for the FER protein from tomato. Furthermore, the invention relates to transgenic plants and plant cells which contain a nucleic acid molecule comprising a DNA sequence according to the invention and, as a result thereof, have an altered mineral intake, in particular iron intake, compared to wild type plants or cells, as well as harvest products and propagation material of the transgenic plants.

Description

The invention relates to a method for influencing the mineral intake and especially iron absorption in transgenic plants. The invention further relates to DNA sequences which are required for FER proteins, in particular for the FER protein from tomato, encode. Furthermore, the invention relates to transgenic plants and plant cells, which a Contain and due to the inventive DNA sequence comprising nucleic acid molecule in comparison to wild type plants or cells, a changed mineral intake, in particular have iron absorption, as well as crop products and propagation material of transgenic plants.

Iron is a necessary component of many important enzymes and proteins Nutrient element for all organisms. The efficient iron absorption of the plants from the soil is a complex and strictly regulated process induced by iron deficiency stress. Two active iron uptake mechanisms are distinguished in higher plants: strategy I (for higher plants with the exception of the Gramineae) and Strategy II (Gramineae). Next Numerous physiological and morphological changes is the central core of the Strategy I the reduction of Fe (III) to Fe (II) at the root surface (Briat and Lobreaux (1997) Trans. Plant Sci. 2: 187-193). This reduced iron comes from the plant added. For this purpose, Strategy I plants activate the iron chelate reductase in Roots, which has been detected by a color reaction in many plants, and Iron transporter. To a large extent, these reactions become more transcriptional Level switched on at the iron deficiency in the root, like studies in the model system Arabidopsis thaliana. In Arabidopsis, induction is due to iron deficiency the FeIII chelate reductase gene, fro2 (Robinson et al. (1999) Nature 397: 694-697), and that Gene for a transporter of divalent metal ions, irt1 (Koshunova et al. (1999) Plant Mol. Biol. 40: 37-44). The genes for NRAMP proteins with transport capacities for iron, such as ATNRAMP3 and ATNRAMP4, are rooted in Arabidopsis thaliana Iron deficiency increasingly expressed (Thomine et al. (2000) Proc. Natl. Acad. Sci. USA 97: 4991-4996), while on the other hand the expression of the gene of ATNRAMP1, which is for a protein encoded with lower iron transport capacities than ATNRAMP3 and ATNRAMP4, not is influenced by iron deficiency (Curie et al. (2000) Biochem. J. 347: 749-755). On an important point, however, is that the transport mechanisms induced by iron deficiency are not are specific for iron, but also reinforced by other divalent metal ions can be used, for example zinc, manganese, cadmium (Nelson (1999) EMBO J., 18: 4361-4371; Thomine (2000) Proc. Natl. Acad. Sci. USA, 97: 4991-4996; Koshunova et al. (1999) Plant Mol. Biol. 40: 37-44). The central core of Strategy II is increased production and Secretion of phytosiderophores and the subsequent uptake of complexes Phytosiderophores with divalent iron via induced transport mechanisms (Mori (1999) Curr. Opin. Plant Biol. 2: 250-253).

Because a positive influence on the mineral intake leads to an increased vitality of the Plants, higher biomass production and thus better yields are new and efficient approaches to influencing the mineral balance for agriculture of great interest. So far, the mineral intake in plants has been influenced by Minerals are added to the nutrient medium, to the soil or directly to the plants were (fertilization). Plants were used, for example, to remedy iron deficiency treated or sprayed with a solution containing iron chelate complexes. genetic engineering Approaches have so far not led to the desired success or have not so far have been described.

The previously used to remedy mineral deficiency or to influence the Mineral nutrient approaches to fertilization are very expensive, complex and u. U. harmful to the environment.

Surprisingly, it has now succeeded, for the first time, in coding an FER protein To provide DNA sequence. The DNA sequence disclosed in this application is suitable for a positive influence on the mineral balance and the enrichment and Storage of minerals in transgenic plants.

The fer genes coding for FER proteins control iron absorption and in roots the reactions of iron deficiency stress. The FER protein is a DNA binding regulator, which in the root of strategy I plants on the iron uptake controlled transcriptional level.

For example, this is done by inducing genes for iron and transporters other metal ions, such as nramp or irt, or also of genes that are responsible for the Encode iron reductase.

The targeted control of fer expression in transgenic plants offers diverse and promising options for influencing the plant's mineral metabolism. By providing the fer gene for transmission to plants, there is now Possibility to influence the activity of the FER protein and thus have positive effects the mineral metabolism and on the enrichment and storage of minerals cause. The activity of the FER protein is influenced, for. B. by a altered expression of the fer gene in the transgenic plants.

This makes it possible, in contrast to the prior art, the mineral balance predominantly to be influenced internally in the plant, without any significant external addition of minerals is necessary. The internal influence takes place by changing the FER protein content in transgenic plants.

The changed activation of FER in transgenic plants has several positive effects Effects. On the one hand, this can increase the vitality of the plants, a higher one Biomass production and ultimately better yields can be achieved. Cultivars like z. B. cereals, potatoes can thus be better grown on mineral deficient soils, so that the expensive and environmentally harmful fertilization with minerals is eliminated.

On the other hand, thanks to the provision of the fer gene, plants with increased Expression are generated which are better due to an increased mineral intake Have competition for minerals and therefore against pests and Weed plants are preferred so that these plants are more resistant to pests and Weed plants are wild type plants without increased fer expression. This would make the expensive or environmentally harmful pest and weed control may or may not be necessary at least be reduced.

In addition, an altered tissue-specific expression of fer genes achieved that the plants minerals in certain parts of the plant such as For example, enrich leaves, roots, tubers, fruits or seeds and thereby for the human nutrition become more valuable.

Finally, the present invention provides the ability to produce plants that can withdraw heavy metal ions from the soil due to their altered fer expression and can therefore be used for the remediation of floors contaminated with heavy metals (Phytoremediation) because usually heavy metals like cadmium are similar or the same Use uptake mechanisms like iron.

It has now been possible for the first time to isolate and characterize a fer gene of the tomato. The DNA sequences according to the invention open up a fundamental new possibility to influence vegetable mineral metabolism. You can use suitable vectors in the genome of plants are transmitted, causing transgenic plants with altered Synthesis of FER protein can be generated. Have these altered levels of FER protein Effect on regulation of fer target genes, such as genes for Metal transporters and iron reductases, and lead to a changed efficiency of the Mineral absorption from the soil, especially iron, zinc, manganese, copper, but also from Heavy metal ions such as cadmium and / or a changed distribution and storage of these minerals in the various plant organs and Cell compartments.

Basically, the new DNA sequences are for transfer to any plants suitable. However, preference is given to using the fer gene from dicotyledonous plants for the same or for other dicotyledonous plants, e.g. B. tomato, potato, sugar beet, etc. The The use of fer DNA sequences obtained from monocotyledonous plants is therefore for other monocot plants preferred, e.g. B. the use of the fer gene from barley in gesture, Rice or other cereals.

The transfer of additional fer gene copies into the genome of plants as well as the organ and ontogenesis-specific expression of the fer gene result from altered FER Production in the transgenic plant to an altered expression of the potential Target genes, such as genes for iron reductase and mineral transporters. This will Minerals are transported in a different way to target organs, in which fer in is expressed in a changed manner.

It is therefore an object of the present invention to provide DNA Sequences coding for FER regulator proteins, as well as in the provision of transgenic plants, which have a changed content compared to their wild type FER regulator protein and, due to this, an altered mineral balance, especially have a changed iron intake.

Another task is to develop methods for changing the mineral intake, in particular to provide iron absorption in transgenic plants. Further tasks result from the above or the following description. The above and other objects of the invention are set forth in the in the independent Claims defined objects solved. Preferred embodiments are in the dependent claims.

In the context of the present invention, a DNA sequence is disclosed for the first time FER protein encoded. The sequence preferably originates from Lycopersicon esculentum. A preferred DNA sequence coding for an FER protein from tomato is in SEQ ID No. 1 and 2, with SEQ ID No. 1 shows the fer gene and SEQ ID No. 2 from the fer Indicates mRNA-derived cDNA sequence. SEQ ID No. 3 shows the amino acid sequence of the FER protein from tomato.

In the context of the present invention, the term “fer gene” or “for an FER protein coding DNA sequence "for a nucleic acid sequence used for an active DNA-binding regulatory protein encoded, the regulatory protein in the root of Strategy I plants control the iron uptake at the transcriptional level and where the coding regions of the nucleic acid sequence with the coding sequence in SEQ ID No. 1 or 2 a sequence identity degree of at least 45%, 50%, 55%, 60%, preferably at least 70%, 80%, particularly preferably at least 90% and most preferably of at least 92%, 94%, 96%, 98%.

By providing the DNA sequences according to the invention, the iron uptake in transgenic plants can be positively influenced, this being particularly preferred by Overexpression of the fer gene takes place in the transgenic plants or cells.

The observation that the mineral balance through transmission and expression exogenous fer DNA sequences can be positively influenced, can be ideal for use the production of plants based on wild-type plants improved mineral balance are more vital, more productive and more resistant.

The basic requirement for the production of such useful plants is the availability of more suitable ones Transformation systems. Here has been a broad one for the past two decades Developed and established a range of transformation methods. These techniques include transforming plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation agent, the fusion of Protoplasts, the direct gene transfer of isolated DNA into protoplasts, injection and Electroporation of DNA into plant cells, the introduction of DNA using biolistic Methods and other options. These transformation techniques are known to the person skilled in the art known or you can in overview articles or corresponding reference works being found.

In addition to using the fer-DNA sequences disclosed here for the first time, the Specialist other DNA sequences coding for FER proteins from other organisms as a tomato using conventional molecular biological techniques and in the Use within the scope of the present invention. For example, the person skilled in the art can derive suitable hybridization probes from the fer sequences according to the invention and for the screening of cDNA and / or genomic banks of the desired one Organism, e.g. B. potato or cereals from which a new fer gene is to be isolated, deploy. Here, the person skilled in the art can familiarize with hybridization, cloning and Sequencing methods fall back in any bio or genetic engineering laboratory are well known and established (see also e.g. Sambrook et al. (1989) Molecular Cloning: Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). The person skilled in the art can of course also use the sequences according to the invention synthesize suitable oligonucliotide primers for PCR amplification of fer sequences and deploy. Of course, there is also the option of the whole, below as SEQ ID NO: 1 or SEQ ID No. 2 indicated DNA sequence as hybridization probe in use conventional hybridization techniques.

In general, the person skilled in the art can compare genes with a high level by comparing the fer gene sequence or FER amino acid sequence with sequences from different sequence databases (genomic sequences, BAC sequences, EST sequences, cDNA sequences) from different organisms (e.g. from various genome projects) Identify similarity. These genes can then be processed using conventional methods of PCR and gene cloning. In order to identify fer-like genes from organisms that are not in databases, oligonucleotides for PCR or RT-PCR can be derived from the fer sequence. Regions that are largely identical between the tomato fer gene and the gene similar to this gene in Arabidopsis are suitable for the design of such oligonucleotides (see FIG. 3).

In the recombinant nucleic acid molecules according to the invention, which are used for an FER Containing protein coding DNA sequence, the coding nucleic acid sequence can be like in the native gene, i.e. in the 5'-3 'direction in operative association with one in plants active promoter. In this way, an overexpression of the coding Sequence can be achieved in the transgenic plants, and thus an increase in FER Activity. Increased activity can also be achieved with partial sequences of the entire cDNA if the partial fragments have the necessary properties for regulation.

On the other hand, downregulation of endogenous FER activity can also occur be desirable, e.g. For example, it can be useful in certain cells Switch off mineral intake and mineral transport to do this on another to be able to control the appropriate place better. To do this you would have to have the fer activity in the Switch off cells where it is undesirable, e.g. B. by the antisense approach, the phenomenon co-suppression or double hybrid RNA formation. In all cases it is not necessary imperatively the entire nucleic acid sequence coding naturally for an FER protein can be transmitted and transcribed, but rather fragments of the coding Region are sufficient, insofar as these fragments achieve the desired effects, i. H. co- Ensure suppression on the one hand or antisense inhibition on the other. The expert can e.g. B. produce suitable deletion constructs and by transfer to transgenic Plant cells check whether a certain fragment of the coding region effect according to the invention, namely the inhibition of endogenous FER activity or not. Like in Used within the scope of this application includes the phrase "DNA sequence for FER protein "encodes" DNA sequences and fragments that are not code for an active protein, its presence and transcription in sense or Antisense orientation but a co-suppression or antisense inhibition in transgenic plant cells. One can see such fragments related to the Also refer to the invention as "antisense or suppression active" FER DNA fragments. The complete genomic clone of an FER protein or parts thereof can also are transferred to achieve the desired inhibition of endogenous FER.

In any case, the term "antisense" is to be understood to mean that the for an FER Protein coding sequence or an "antisense-active" fragment thereof in antisense Orientation, ie "upside down" in 3 '→ 5' direction, in operative connection with a there is an active promoter region in plant cells and is transcribed. The RNA that the transcription of such an antisense gene is complementary to the RNA of the endogenous gene and can prevent the synthesis of the FER protein product by Hybridization comes between the native and the antisense RNA.

In the case of the alternatively usable co-suppression construct, this is for an FER protein coding sequence or a "co-suppression active" fragment thereof in sense Orientation (i.e. in 5 '→ 3' direction) in operative connection with one in plant cells active promoter region. With the phenomenon of co-suppression, the endogenous gene inactivated by additionally introduced, identical (partial) copies of this gene. This Inactivation is probably due to an RNA-dependent mechanism which is involved in RNA-directed RNA polymerase.

In the case of double hybrid RNA, the sequence coding for an FER protein is both in sense (5 '→ 3') - as well as antisense (3 '→ 5') - orientation directly or through an intron or other sequence operatively linked.

Furthermore, all that is required to carry out the method according to the invention is more suitable regulatory sequences that require the transcription of an operably linked fer DNA Control the sequence in the transformed plant or plant cell. Here too Sequences suitable for a person skilled in the art can be taken from the prior art without any problems or by yourself isolate particularly suitable promoter sequences. Suitable for various plants Promoters can be found in the literature. In addition, new promoters can be created using current genome projects can be identified. For example, transgenic plant lines can follow new promoter activities are screened, which integrates promoterless GUS constructs (Springer (2000) plans Cell 12: 1007-1020). Explosion analysis of numerous or the entirety of all EST sequences (RNA profiling, Affymetrix) can draw conclusions allow suitable promoters. This allows promoters to be identified that organ-specific (e.g. root, seeds, flowers, leaf etc.), tissue-specific (e.g. Sliding vessels, cortex, epidermis) are development-specific or inducible.

In a particularly preferred embodiment, the coding for the FER protein DNA sequence under the control of the 35S promoter.

Although tissue-specific expression is preferred, the expression of DNA sequence according to the invention in plant cells of any kind of regulatory Sequences in question that ensure transcription in plant cells. Everyone is here active promoter included in plant cells. The promoter can be chosen in this way be that the expression is constitutive or only in specific tissues to one certain point in time of plant development and / or at a time due to external influences, biotic or abiotic stimuli specific point in time (induced gene expression). In relation the promoter can be homologous or heterologous to the plant to be transformed. at Use of a constitutive promoter can be a cell or tissue specific Expression can also be achieved in that the gene expression in the cells or Tissues in which it is not desired is inhibited, for example by expression of antibodies which bind the gene product and thus inhibit its activity, or with suitable inhibitors.

Constitutive or tissue- or development-specific or inducible genes or The skilled person can promote the prior art, in particular the relevant ones take scientific journals and databases. In addition, the One of ordinary skill in the art is able to use routine methods to find other suitable promoters isolate. So the expert can with the help of common molecular biological methods Identify regulatory nucleic acid elements, using examples here as methods names are transgenic for the identification of genes with interesting promoters Plant lines with reporter gene constructs (see above), RNA profiling, Affymetrix, in genes or promoters described in the literature, subtractive screening of banks specifically expressed cDNA, and for cloning the promoters. The cloning over PCR products, genome walking, isolation of genomic clones, cosmids, BACs, Lambda, inverse PCR or the like to obtain insertion points for transgenic lines, Cloning: hybridization with probes.

Furthermore, there are optional transcription or termination sequences that the serve correct completion of the transcription, and the addition of a PolyA tail to the The transcript can serve a function in stabilizing the transcripts is attributed. Such elements are described in the literature and are arbitrary interchangeable.

• a) regulatorische Sequenzen eines in Pflanzengeweben, insbesondere in Wurzeln, aktiven Promotors;
• b) operativ daran gebunden eine DNA-Sequenz, die für ein FER-Protein kodiert; und
• c) ggf. operativ daran gebunden regulatorische Sequenzen, die als Transkriptions-, Terminations- und/oder Polyadenylierungssignale in Pflanzenzellen dienen können.

Finally, chimeric gene constructs in which fer-coding DNA sequences are under the control of regulatory sequences which ensure expression in plant cells are produced by means of conventional cloning methods (see, for example, Sambrook et al. (1989), supra). The present invention thus relates to a recombinant nucleic acid molecule comprising:

• a) regulatory sequences of a promoter active in plant tissues, in particular in roots;
• b) operatively linked to a DNA sequence encoding an FER protein; and
• c) if necessary, operatively linked regulatory sequences which can serve as transcription, termination and / or polyadenylation signals in plant cells.

The DNA sequence encoding an FER protein can be isolated from natural sources or can be synthesized by known methods. The invention preferably comes from DNA sequence from Lycopersicon esculentum.

Using common molecular biological techniques (see for example Sambrook et al. (1989) supra), it is possible to construct desired constructs for the transformation of plants prepare or manufacture. The for genetic engineering manipulation in prokaryotic cells usually used cloning, mutagenizing, Sequence analysis, restriction analysis and other biochemical-molecular biological Methods are well known to those of ordinary skill in the art. So not only can suitable ones chimeric gene constructs with the desired fusion of promoter and fer DNA sequence rather, the skilled artisan can use routine techniques, if desired, different types of mutations or deletions in the FER coding DNA sequence introduce.

• a) DNA-Sequenzen, die die in SEQ ID No. 1 oder SEQ ID No. 2 angegebene kodierende Nukleotidsequenz oder Teile davon umfassen;
• b) DNA-Sequenzen, die eine Nukleotidsequenz umfassen, die die in SEQ ID No. 2 angegebene Aminosäuresequenz oder Fragmente davon kodieren;
• c) DNA-Sequenzen, die eine Nukleotidsequenz umfassen, die zu einer Nukleotidsequenz von a) oder b) degeneriert ist, oder Teile dieser Nukleotidsequenz umfassen;
• d) DNA-Sequenzen, die zu der in SEQ ID No. 1 bzw. SEQ ID No. 2 angegebenen Nukleotidsequenz eine Sequenzidentität von mindestens 60%, vorzugsweise mindestens 80%, besonders bevorzugt mindestens 90% und am meisten bevorzugt . mindestens 92, 94, 96, 98% aufweist;
• e) DNA-Sequenzen, die ein Derivat, Analog oder Fragment einer Nukleotidsequenz von a), b), c) oder d) darstellen.

In a preferred embodiment, the DNA sequence encoding an active FER protein is selected from the group consisting of:

• a) DNA sequences that the in SEQ ID No. 1 or SEQ ID No. 2 comprise specified coding nucleotide sequence or parts thereof;
• b) DNA sequences which comprise a nucleotide sequence which the in SEQ ID No. 2 encode specified amino acid sequence or fragments thereof;
• c) DNA sequences which comprise a nucleotide sequence which is degenerate to a nucleotide sequence of a) or b), or comprise parts of this nucleotide sequence;
• d) DNA sequences which correspond to the sequence shown in SEQ ID No. 1 or SEQ ID No. 2 indicated nucleotide sequence a sequence identity of at least 60%, preferably at least 80%, particularly preferably at least 90% and most preferred. has at least 92, 94, 96, 98%;
• e) DNA sequences which represent a derivative, analog or fragment of a nucleotide sequence of a), b), c) or d).

The DNA sequences according to the invention also include fragments, derivatives and allelic Variants of the DNA sequences described above, which encode an FER protein. The The term "derivative" in this context means that the sequences differ from the DNA sequences described above differ at one or more positions, however have a high degree of similarity to these sequences. The deviations from the DNA sequences described above can, for example, by deletion, Substitution, insertion or recombination have arisen. Similarity or homology means a sequence identity of at least 45%, 50%, 55%, in particular one Identity of at least 60%, preferably an identity of at least 70%, 80%, particularly preferred of at least 90%, 92%, 94% and most preferred of at least 96% and at least 98%. The DNA sequences show through these coding proteins a sequence identity to that in SEQ ID No. 2 or 3 specified Amino acid sequence of at least 45%, 50%, 55%, in particular an identity of at least 60%, preferably an identity of at least 70%, 80%, particularly preferred of at least 90%, 92%, 94% and most preferably at least 96%, 98%.

For the DNA sequences that are similar to the sequences described above and Represent derivatives of these sequences, there are usually variations of these Sequences that represent modifications that perform the same biological function. It can be both naturally occurring variations, for example to sequences from other organisms, or to mutations, these mutations being based on may have occurred naturally or have been introduced by targeted mutagenesis.

Furthermore, the variations can be synthetically produced sequences. at the allelic variants can be both naturally occurring variants as also synthetically produced or produced by recombinant DNA techniques Variants.

In a particularly preferred embodiment, the described one for an FER Protein-coding DNA sequence from Lycopersicon esculentum (as in SEQ ID No. 1 or SEQ ID No. 2 specified).

The invention further relates to vectors and microorganisms, the invention Contain nucleic acid molecules and their use in the production of plant cells and plants, their mineral balance, especially their iron intake compared to wild-type plant cells or plants. These are the Vectors especially around plasmids, cosmids, viruses, bacteriophages and others in the Genetic engineering common vectors. The microorganisms are primarily Bacteria, viruses, fungi, yeast and algae.

Furthermore, in the present invention, a recombinant FER protein Lycopersicon esculentum, in particular a recombinant protein with the in SEQ ID No. 2 and No. 3 amino acid sequence provided.

• a) Herstellung eines rekombinanten Nukleinsäuremoleküls, das folgende Sequenzen umfasst:
  • - regulatorische Sequenzen eines in Pflanzen aktiven Promotors;
  • - operativ daran gebunden eine DNA-Sequenz, die ein FER-Protein kodiert; und
  • - optional operativ daran gebunden regulatorische Sequenzen, die als Transkriptions-, Terminations- und/oder Polyadenylierungssignale in Pflanzenzellen dienen können;
• b) Übertragung des Nukleinsäuremoleküls aus a) auf pflanzliche Zellen; und
• c) gegebenenfalls die Regeneration intakter Pflanzen aus den transformierten Pflanzenzellen.

The invention further relates to a method for producing plants or plant cells with a mineral balance, in particular changed iron absorption, which has changed compared to wild-type plants or plant cells, comprising the following steps:

• a) Production of a recombinant nucleic acid molecule which comprises the following sequences:
  • - regulatory sequences of a promoter active in plants;
  • operatively linked to a DNA sequence encoding an FER protein; and
  • optionally operatively linked to it regulatory sequences that can serve as transcription, termination and / or polyadenylation signals in plant cells;
• b) transfer of the nucleic acid molecule from a) to plant cells; and
• c) optionally the regeneration of intact plants from the transformed plant cells.

The invention further relates to plant cells that the invention Nucleic acid molecules that encode an FER protein. The invention also relates Harvest products and propagation material of transgenic plants as well as the transgenic plants themselves, which contain a nucleic acid molecule according to the invention. Transgenic plants of the present invention have due to the introduction of an FER-coding DNA Sequence of an iron uptake changed compared to wild type plants.

Prepare for the introduction of foreign genes into higher plants or their cells a large number of cloning vectors are available that provide a replication signal for E. coli and a marker gene for the selection of transformed bacterial cells. Examples for such vectors are pBR322, pUC series, M13mp series, pACYC184 etc. The The desired sequence can be inserted into the vector at a suitable restriction site be introduced. The plasmid obtained is then used for the transformation of E. coli Cells used. Transformed E. coli cells are in a suitable medium grown and then harvested and lysed, and the plasmid is recovered. As Analysis method for the characterization of the plasmid DNA obtained are in general restriction analyzes, gel electrophoresis and other biochemical-molecular biological Methods used. After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained are linked to other DNA sequences.

As already mentioned, DNA is introduced into a plant host cell A variety of techniques are available, with the skilled person choosing the most appropriate method can easily determine. These techniques include transformation plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation medium, the fusion of protoplasts that Injection, electroporation, direct gene transfer of isolated DNA into protoplasts Introduction of DNA using biolistic methods as well as other possibilities have been well established for several years and are part of the usual repertoire of the specialist in belong to plant molecular biology or plant biotechnology.

In the injection and electroporation of DNA into plant cells, none per se made special demands on the plasmids used. The same applies to the direct one Gene transfer. Simple plasmids such as e.g. B. pUC derivatives can be used. Should but whole plants are regenerated from such transformed cells Presence of a selectable marker gene is recommended. The experts are familiar Known selection marker, and it is not a problem for him, a suitable marker select.

Depending on the method of introducing the desired genes into the plant cell, others can DNA sequences may be required. Are z. B. for the transformation of the plant cell Ti or Ri plasmid used, at least the right limit, but often the right and left delimitation of the T-DNA contained in the Ti and Ri plasmid as Flank area with the genes to be introduced. Be for transformation When using agrobacteria, the DNA to be introduced must be cloned into special plasmids either in an intermediate or in a binary vector. The intermediate vectors may be due to sequences homologous to sequences in the T-DNA are, by homologous recombination in the Ti or Ri plasmid of the agrobacteria to get integrated. This also contains the necessary for the transfer of the T-DNA Region. However, intermediate vectors cannot replicate in agrobacteria. through of a helper plasmid, the intermediate vector on Agrobacterium tumefaciens transferred (conjugation). Binary vectors, however, can be used in E. coli as well also replicate in agrobacteria. They contain a selection marker gene and a linker or polylinkers, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria. That serving as the host cell Agrobacterium is said to contain a plasmid which has a fer sequence within the T-DNA carries, which is transferred into the plant cell. Additional T-DNA may be present. The agrobacterium transformed in this way becomes the transformation of plant cells used. The use of T-DNA for the transformation of plant cells is intensively examined and sufficient in well-known overview articles and manuals for plant transformation have been described. For the transfer of the DNA into the Plant cells can advantageously 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 protoplasts or Suspension-cultivated plant cells) can then in a suitable medium, which Antibiotics or biocides for the selection of transformed cells can contain whole Plants are regenerated.

Once the inserted DNA is integrated into the genome of the plant cell, it is there in the Usually stable and remains in the progeny of the originally transformed cell receive. It usually contains a selection marker, which is the transformed one Plant cells resistance to a biocide or an antibiotic such as kanamycin, G 418, Bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea, Gentamycin or Phosphinotricin u. a. taught. The individually selected marker should therefore be the Selection of transformed cells against cells that lack the inserted DNA, allow. Alternative markers are also suitable for this, such as nutritive markers, Screening markers (such as GFP, green fluorescent protein). Of course, it can also be completely open Selection markers are dispensed with, but with a rather high one Screening needs goes hand in hand. If marker-free transgenic plants are desired, please Experts also have strategies available for subsequent removal of the marker gene allow e.g. B. cotransformation, sequence-specific recombinases.

The regeneration of the transgenic plants from transgenic plant cells takes place after usual regeneration methods using known nutrient media. The so Plants obtained can then be subjected to conventional procedures including molecular biological methods, such as PCR, blot analysis, for the presence of the introduced Nucleic acid encoding an FER protein or for the presence of the gene product, i.e. the FER protein to be examined.

The transgenic plant or the transgenic plant cells can be any act any monocot or dicot plant or plant cell in which the Mineral balance, especially the iron absorption changed, should preferably be improved. They are preferably useful plants or cells of useful plants. Especially it is preferably solanaceae such as potato, tobacco, tomato, paprika, but also other dicotyledons (e.g. sugar beet) or monocotyledonous plants (e.g. rice, corn, wheat). In principle, any crop is desirable for the implementation of the invention for Nutritional purposes directly or indirectly.

The invention also relates to propagation material and crop products of the plants according to the invention, for example fruits, seeds, tubers, rhizomes, seedlings, Cuttings etc.

The specific expression of the FER protein in the plants according to the invention or in the Plant cells according to the invention can be prepared using conventional molecular biological and biochemical methods can be demonstrated and tracked. These are the expert Techniques known and he is easily able to find a suitable detection method choose, for example a Northern blot analysis for the detection of FER-specific RNA or to determine the amount of accumulation of FER-specific RNA or a Southern blot analysis for the detection of DNA sequences coding for FER.

• a) die Übertragung eines rekombinanten Nukleinsäuremoleküls oder Vektors, der eine DNA-Sequenz enthält, die für ein FER-Protein kodiert, auf Pflanzenzellen;
• b) die Regeneration von Pflanzen aus den transformierten Pflanzenzellen.

The invention further relates to a method for changing the mineral intake, in particular the iron intake in plants, comprising the following steps:

• a) the transfer of a recombinant nucleic acid molecule or vector, which contains a DNA sequence which codes for an FER protein, to plant cells;
• b) the regeneration of plants from the transformed plant cells.

The present invention is illustrated in the following examples, which are only the Serve to illustrate the invention and are not to be understood in any way as a limitation are explained.

Examples

The T3238fer tomato line, derived from the boron-inefficient T3238 tomato line as a mutant is not in a position to undertake the strategy I To activate plant-typical iron deficiency stress reactions (Brown et al. (1971) Physiol. Plant. 25: 48-53; Brown et al. (1974) Physiol. Plant. 31: 221-224). The mutated plants show Iron deficiency neither increased reductase activity nor other physiological or morphological changes necessary for the efficient absorption of iron in the case of iron deficiency be turned on. Since the fer mutation has numerous effects, fer probably acts centrally at the transcriptional level. When growing in soil, fer plants die with Mutation continues during the vegetative phase. Plants with fer mutation can be partial or be completely saved by being in the form of iron all the time in earth Iron hydroxyethylene diamine triacetic acid (Fe-HEDTA) fertilized or on MS medium get dressed by. The fer mutant is a recessive mutant in which a only gene affected is that on chromosome 6 of the tomato 0.33 cM from TG590 and 2.0 cM was mapped away from TG118 (Ling et al. (1996) Mol. Gen. Genet. 252: 87-92).

Marker-assisted isolation of the fer gene

The marker-assisted gene cloning technique was used to isolate the fer gene (Chromosome Walk). This technique has been successfully used in the tomato several times applied. In the case of the cloning of fer, the method was carried out analogously to that of chloronerva, which was successfully completed two years ago and in Ling et al. (PNAS Prod. Natl. Acad. Sci. USA (1999) 96: 7098-7103) with all references. The Techniques for working with YAC, BAC and Cosmid clones are Birren et al. (Green et al., Eds, Genome Analysis, A laboratory Manual, CSHL Press, USA, 1997).

The beginning of gene mapping and chromosome walking in fer was described in Ling et al. (1996) Mol. Gen. Gent. 252: 87-92). In this publication the mapping described by fer on chromosome 6 between the two markers TG590 and TG118.

For the marker-assisted gene cloning technique, the method described in Ling et al. (1996) supra described mapping population expanded from 1244 F2 plants to 1815 F2 plants of the crossbreed L. esculentum T3238fer x L. pennellii. 6 recombinant plants were found between TG590 and fer, and 35 recombinant plants between fer and TG118. The one described in Ling et al. (1996) supra started chromosome walk was continued as shown in Fig. 1. First, based on the in Ling et al. (1996) supra described YAC end 149AR isolated two further YAC clones and mapped their end (267L and 328N1). With 267L and 328N1, the YAC 337 was isolated, spanning the entire fer region and ending on both sides of the fer gene after two recombination events. With the end of Y337D, BAC clones were identified, two of which were characterized in more detail. The BAC clones were identified by filter hybridization. Filters and BAC clones were purchased from Research Genetics, Inc. USA. The end of BAC 53M23 co-existed with fer. Starting from 53M23 and 337D, a cosmid contour was developed as in Ling et al. (1999) described supra. Two BAC clones, BAC 56B23 and BAC 53M23, were fully sequenced using the shot gun method. For this purpose, the sheared and fractionated BAC-DNA in 1 kb was cloned and sequenced using the Zero Blunt TOPO PCR Cloning Kit, Invitrogen. With the help of the cosmids and corresponding sequence analysis programs, the sequence of the fer region was compiled completely. At various points in the sequence, mapping probes were derived so that the fer gene could finally be located in an area of 18 kb. This 18 kb area contains two open reading frames, one of which codes for a transposase and is therefore out of the question as a fer gene. The second open reading frame codes for a 34 kD protein and is the fer gene.

Molecular analysis of the fer gene in T3238FER wild-type and T3238fer mutant lines

The fer gene co-regulates with the fer mutation and has an RFLP polymorphism between the mutant line T3238fer and the mother line T3238FER. While the mother line T3238FER has an EcoRV fragment of 4.8 kb in an RFLP analysis, as can also be seen from the sequence analysis, the mutant line T3238fer has two fragments of 4.5 kb and 4.0 kb (see FIG . 2). This polymorphism indicates an insertion or a rearrangement, which affect the fer region in the mutant.

Description of fer DNA and FER amino acid sequences

A comparison of the sequence of the fer gene with sequences from the gene database showed that the fer gene for a protein with a conserved basic helix-loop-helix DNA binding domain coded. The fer gene shows the highest sequence similarity with a gene from Arabidopsis thaliana located on chromosome II (DNA Accession No. AC005851 coding sequence from 72093 to 73201 bp, gene = At2g28160, here in the following Atfer called protein accession no. AAC984501.1). So far, none has been found for the Arabidopsis gene Functional analysis described. It can therefore not be said whether the sequences are only similar between fer and atfer or whether the genes are actually homologous, d. H. the functions are homologous.

Outside the conserved helix-loop-helix domain, there are sequence similarities between fer and atfer in the N-terminal and C-terminal region, both on the DNA and on the protein level (see FIG. 3a). In contrast, other helix-loop helix proteins have no sequence similarities outside of the conserved motif. A comparison of the cDNA and genomic sequence shows that the fer gene has three introns. The positions of the first two introns are conserved in fer and atfer (see Fig. 3b), which may mean that fer and atfer are closely related.

Expression analysis of the fer gene

Northern blot analyzes of total RNA from roots, leaves and stems show that the fer gene is only expressed in root tissues ( FIG. 4a). With the help of reverse transcription PCR, no or only very small amounts of transcripts could be detected in cotyledons, leaves or flowers (see FIG. 4b). The expression of the fer gene in the root depends on the addition of trivalent iron. Plants grown in Hoagland solution (Scholz et al. (1987) Bioch. Physiol. Plant. (1987) 63: 99-104) with 100 μM Fe-HEDTA showed a strong expression of fer in roots (4a). For wild-type and fer plants that were grown up to three weeks of age in Hoagland solution with 10 µM Fe-NaEDTA, but then in Hoagland-Lösurg with 0.1 µM, 10 µM and 50 µM Fe-NaEDTA respectively the expression of the fer gene was constitutive in the roots ( FIG. 4b). In contrast, no transcripts were detectable by Northern blot analysis in roots of wild-type plants which were grown in Hoagland solution with 100 μM Fe-HEDTA.

Expression of target genes in fer mutants

An analysis of the expression of transporter genes in the example "Expression Analysis of the Fer- Gens "described wild-type fer plants, each in Hoagland solution with 0.1 µM, 10 µM and 50 µM Fe-NaEDTA were grown, revealed that nramp genes in the roots of remote Plants were not expressed. In contrast, these nramp genes are used in wild-type Roots expressed constitutively.

FER regulates nramp genes either directly by binding to nramp promoters or indirectly after switching on a signal cascade. FER is therefore a direct or indirect one Regulator of nramp genes in the root.

Generation of transgenic plants with expression of the fer gene from tomato

The coding sequence of the fer gene was inserted behind the 35S promoter Plant transformation vector (pBINAR, Höfgen et al. (1990) Plant Science 66: 221-230) cloned and transferred to mutant fer tomato plants. Transformation and regeneration of fer plants presented no problem in vitro because the fer phenotype in vitro on MS Medium is saved.

When transferred to soil, it was observed that non-transgenic control plants soon Leaf chlorosis and necrosis developed, while those with the 35S-fer construct transformed plants were saved. The fer gene can thus be used to correct Mineral chloroses can be used. The transformation and regeneration was after usual protocols performed.

Description of the figures

Figure 1 illustrates the marker-assisted isolation of the fer gene.

(top) The coverage of the fer region with YAC and BAC clones is shown. mapped Ends of genomic BAC and YAC clones are shown and their spacing of fer gene based on the number of recombination events.

(middle) Enlarged view of the fer region with the cosmids shown.

(below) Representation of open reading frames in the 18 kb fer region.

Fig. 2 shows the result of Southern blot hybridization of the fer gene in Tfer and different wild type lines

Genomic DNA from the mutant line T3238fer, the wild type lines T3238FER, Moneymaker, LA483 and TA56 were cut with the restriction enzyme EcoRV and hybridized in a Southern blot analysis with the fer cDNA as a probe.

Fig. 3a shows the comparison of cDNA sequences from tomato and Arabidopsis thaliana.

FIG. 3b shows a comparison of amino acid sequences of FER from tomato and Arabidopsis thaliana.

The coding sequences of Lefer (Le stands for Lycopersicon esculentum) and Atfer (At stands for Arabidopsis thaliana) begins with the ATG start codon and ends with the TAA Stop codons were created using the DNASTAR Inc. Clustal Program (Meg Align 4.0) - Sequence analysis packages compared. Identical base positions have a black background. The Amino acid sequences FER (tomato) and ATFER were created using the Clustal program compared. Identical amino acid positions are highlighted in black.

• a) Northern-Blot-Analyse:
  fer Transkripte wurden auf Gesamt RNA von Wurzeln, Blättern und Stengeln von Wildtyppflanzen (Mm, Moneymaker) und fer-Pflanezn (Tfer) in einer Northern-Blot-Analyse mit radioaktiv markierter fer cDNA nachgewiesen. Die Pflanzen wurden jeweils in Hoagland- Lösung mit 100 µM Fe-NaEDTA oder Fe-HEDTA angezogen.
• b) Reverse Transkription-PCR-Analyse:
  mRNA wurde revers mit oligodT-Primern in cDNA transkribiert. Mit spezifischen Oligonukleotidprimern wurden DNA-Fragmente von fer und als Positivkontrolle vom Elongationsfaktorgen ef-1αamplifiziert. Die PCR-Produkte wurden im Agarosegel aufgetrennt und durch Southern-Blot-Hybridisierung nachgewiesen. Für die Expressionsanalyse in verschiedenen Geweben wurde RNA von zwei Wochen alten Keimlingen isoliert (r = Wurzel, rt = Wurzelspitzen, hy = Hypokotyl, cot = Kotyledonen, 1 = erste Blatt). Zwei Wochen alte Pflanzen wurden in Hoagland-Lösung mit entweder 0,1, 10 oder 50 µM Fe- EDTA überführt. Wurzelproben zur RNA-Präparation wurden vor Beginn des Experiments (0) nach sechs Stunden (6 h), acht Stunden (8 H), zwei, vier und acht Tagen (2-8d) entnommen.

Figure 4 shows expression analyzes of the fer gene

• a) Northern blot analysis:
  Fer transcripts were detected on total RNA from roots, leaves and stems of wild-type plants (Mm, Moneymaker) and fer plants (Tfer) in a Northern blot analysis with radiolabelled fer cDNA. The plants were each grown in Hoagland solution with 100 µM Fe-NaEDTA or Fe-HEDTA.
• b) Reverse transcription PCR analysis:
  mRNA was reverse transcribed with oligodT primers in cDNA. DNA fragments of fer and as a positive control of the elongation factor gene ef-1α were amplified with specific oligonucleotide primers. The PCR products were separated in an agarose gel and detected by Southern blot hybridization. For expression analysis in different tissues, RNA was isolated from two-week-old seedlings (r = root, rt = root tips, hy = hypocotyl, cot = cotyledons, 1 = first leaf). Plants two weeks old were transferred to Hoagland solution with either 0.1, 10 or 50 µM Fe-EDTA. Root samples for RNA preparation were taken before the start of the experiment (0) after six hours (6 h), eight hours (8 H), two, four and eight days (2-8d).

FIG. 5 shows the expression of nramp genes in wild-type and fer plants. The reverse transcription-PCR analysis was carried out as in connection with FIG. 4b, but with nramp-specific oligonucleotides. SEQUENCE LISTING

Claims (11)

1. DNA sequence coding for an FER protein selected from the group consisting of a) DNA sequences that the in SEQ ID No. 1 or SEQ ID No. 2 comprise specified coding nucleotide sequence or parts thereof, b) DNA sequences which comprise a nucleotide sequence which the in SEQ ID No. 2 or SEQ ID No. 3 encode specified amino acid sequence or fragments thereof; c) DNA sequences which comprise a nucleotide sequence which is degenerate to a nucleotide sequence of a) or b) or parts of this nucleotide sequence; d) DNA sequences which correspond to the sequence shown in SEQ ID No. 1 or SEQ ID No. 2 indicated nucleotide sequence has a sequence identity of at least 45%, 50%, 55%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% and most preferably at least 92, 94, 96, 98%; e) DNA sequences which represent a derivative, analog or fragment of a nucleotide sequence of a), b), c) or d). 2. DNA sequence according to claim 1, which is derived from tomato. 3. Recombinant nucleic acid molecule, comprising the following elements: regulatory sequences of a promoter active in plant cells, operatively linked to a DNA sequence according to claim 1 or 2, - If necessary, operationally linked regulatory sequences which can serve as transcription, termination and / or polyadenylation signals in the plant cell. 4. Recombinant nucleic acid molecule according to claim 1, wherein the DNA sequence can be in sense and / or antisense orientation. 5. Recombinant FER protein which is encoded by a DNA sequence according to claim 1 or 2 or which has an amino acid sequence selected from the group consisting of: a) Amino acid sequence as in SEQ ID No. 2 or 3 indicated or fragments thereof, and b) amino acid sequence which corresponds to the sequence shown in SEQ ID No. 2 or 3 given amino acid sequence has a sequence identity of at least 45%, 50%, 55%, preferably at least 60%, 70%, 80%, particularly preferably at least 90% and most preferably at least 92, 94, 96, 98%. 6. Recombinant FER protein according to claim 5, which is derived from tomato. 7. A method for changing the mineral balance, in particular for changing the mineral intake and for changing the iron intake in transgenic plants, comprising the following steps: a) production of a recombinant nucleic acid molecule according to claim 3 or 4, b) transfer of the nucleic acid molecule from a) to transgenic plants or plant cells; and a) if necessary regeneration of plants from the transformed plant cells. 8. Plant cell containing a DNA sequence according to claim 1 or 2 or one recombinant nucleic acid molecule according to claim 3 or 4, or prepared according to claim 7th 9. The transgenic plant cell according to claim 8, which is compared to wild-type cells has increased FER protein content. 10. Transgenic plant cell according to claim 8 or 9, one opposite Wild type cells have increased mineral intake, especially iron intake. 11. Transgenic plant containing a plant cell according to one of claims 8 to 10 or produced by a method according to claim 7, and parts of these plants, transgenic crop products and transgenic propagation material of these plants, such as Protoplasts, plant cells, calli, seeds, tubers, cuttings, as well as the transgenic Offspring of these plants.