WO2003014364A1

WO2003014364A1 - Method for influencing the acceptance of minerals in transgenic plants

Info

Publication number: WO2003014364A1
Application number: PCT/EP2002/008725
Authority: WO
Inventors: Petra Bauer; Martin Ganal; Beat Keller; Hong-Qing Ling
Original assignee: Ipk - Institut Für Pflanzengenetik Und Kulturpflanzenforschung; Universität Zürich
Priority date: 2001-08-03
Filing date: 2002-08-05
Publication date: 2003-02-20
Also published as: DE10138091A1

Abstract

The invention relates to a method for influencing the acceptance of minerals and especially the acceptance of iron in transgenic plants. The invention also relates to DNA-sequences coding for FER-proteins, especially the FER-protein in tomatoes. The invention further relates to transgenic plants and plant cells which contain a nucleic acid molecule comprising an inventive DNA-sequence and exhibiting an altered acceptance of minerals, especially acceptance of iron, in comparison with wild plants or cells. The invention also relates to harvest crops and multiplication material of transgenic plants.

Description

Process for influencing mineral intake in transgenic plants

The invention relates to a method for influencing the absorption of minerals and in particular the absorption of iron 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 a changed mineral intake, in particular iron intake, compared to wild type plants or cells, as well as crop products and propagation material of the transgenic plants.

As a component of many important enzymes and proteins, iron is a necessary nutrient element for all organisms. The efficient iron uptake of plants from the soil is a complex and strictly regulated process, which is induced in the event of 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). In addition to numerous physiological and morphological changes, the central core of Strategy I is

Reduction of Fe (III) to Fe (II) at the root surface (Briat and Lobreaux (1997) Trans. Plant Sei. 2: 187-193). This reduced iron is absorbed by the plant. For this purpose, Strategy I plants activate iron chelate reductase in roots, which has been detected by a color reaction in many plants, and iron transporters. To a large extent, these reactions are switched on at the transcriptional level in the case of iron deficiency in the root, as studies in the model system Arabidopsis thaliana have shown. In Arabidopsis there is an iron deficiency induction for the Felll chelate reductase gene. fio2 (Robinson et al. (1999) Nature 397: 694-697), and the gene for a transporter of divalent metal ions, irtl (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 increasingly expressed in Arabidopsis thaliana-Wxxrzein in the case of iron deficiency (Thomine et al. (2000) Proc. Natl. Acad. Sei. USA 97: 4991-4996), while on the other hand the expression of the gene of ATNRAMPl, which codes for a protein with lower iron transport capacities than ATNRAMP3 and ATNRAMP4, is not influenced by iron deficiency (Curie et al. (2000) Biochem. J. 347: 749- 755). An important point, however, is that the transport mechanisms induced by iron deficiency are not specific for iron, but are also increasingly used by other divalent metal ions, 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 the increased production and secretion of phytosiderophores and the subsequent uptake of complexes of the phytosiderophores with divalent iron via induced transport mechanisms (Mori (1999) Curr. Opin. Plant Biol. 2: 250-253).

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

The previously used approaches to remedy mineral deficiency or to influence the mineral balance via fertilization are very expensive, complex and possibly. harmful to the environment.

Surprisingly, it has now been possible for the first time to provide a DNA sequence coding for an FER protein. The one disclosed in this application DNA sequence is suitable for a positive influence on the mineral balance and the accumulation and storage of minerals in transgenic plants.

The fer genes coding for FER proteins control the iron uptake and the reactions of iron deficiency stress in roots. The FER protein is a DNA-binding regulator that controls the iron uptake at the transcriptional level in the root of Strategy I plants.

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

In the context of the present invention, FER protein also means that the protein is encoded by a gene, namely the fer gene, which is located at the fer locus in the tomato line T3238fer (see Ling et al. (1996) Mol. Gen. Genet. 252: 87-92) is mutated. This mutation leads to that of Ling et al. (1996, vide supra) described fer phenotype, which is expressed in that, in contrast to the wild type, the mutant is unable to activate the responses of strategy I plants to iron deficiency, ie the plants are, for example, unable to respond to iron deficiency stress by increasing Fe ³⁺ reductase activity.

FER protein or fer gene in the sense of the invention also means that the fer phenotype (as described, for example, by Ling et al. (1996, vide supra) for the tomato line T3238fer) by transforming plants which show this phenotype , with the fer gene according to the present invention, ie a functional sequence according to SEQ ID No. 1 or 2, can be complemented. In other words, a fer gene in the sense of the present invention is a DNA sequence which, when planted with the fer phenotype and is expressed there, restores the fer wild type, that is to say functionally complements the fer phenotype. The complemented plants, like the wild type, can be used for strategy I plants show typical iron deficiency stress reactions and therefore, for example, in contrast to the fer mutant on Fe ^3+, survive in the soil or in the growth medium.

The targeted control of fer expression in transgenic plants offers diverse and promising possibilities for influencing the plant's mineral metabolism. By making the fer gene available for transmission to plants, it is now possible to influence the activity of the FER protein and thus to have positive effects on the mineral metabolism and on the accumulation and storage of minerals. The activity of the FER protein is influenced e.g. by an altered expression of the fer gene in the transgenic plants.

In this way, in contrast to the prior art, it is possible to influence the mineral balance predominantly internally in the plant without the need for a substantial external addition of minerals. The internal influence takes place by changing the FER protein content in transgenic plants.

The changed activation of FER in transgenic plants has various positive effects. On the one hand, this can increase the vitality of the plants, higher biomass production and ultimately better yields. Cultivars such as Cereals, potatoes can thus be better grown on mineral deficient soils, so that the expensive and environmentally harmful fertilization with minerals is no longer necessary.

On the other hand, thanks to the provision of the fer gene, plants with increased fer expression can be produced which, owing to an increased absorption of minerals, have better competition for minerals and are therefore preferred to pests and weed plants, so that these plants are more resistant to pests and weed plants are as wild type plants without increased fer expression. This would eliminate the costly and environmentally harmful pest and weed control, or could at least be reduced.

In addition, by changing the tissue-specific expression of fer genes, the plants can enrich minerals in certain parts of the plant, such as leaves, roots, tubers, fruits or seeds, and thus become more valuable for human nutrition.

Finally, the present invention provides the possibility of producing plants which, owing to their altered fer expression, can extract heavy metal ions from the soil and can therefore be used for the remediation of soils contaminated with heavy metals (phytoremediation) because, as a rule, heavy metals such as cadmium are similar or the same Use 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 of influencing the vegetable mineral metabolism. They can be transferred into the genome of plants by means of suitable vectors, whereby transgenic plants with modified synthesis of FER protein are produced. These changed amounts of FER protein have an effect on the regulation of fer target genes, such as genes for metal transporters and iron reductases, and lead to a changed efficiency of mineral absorption from the soil, in particular iron, zinc, manganese, copper, but also heavy metal ions such as for example cadmium and / or for a changed distribution and storage of these minerals in the various plant organs and cell compartments.

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

The transfer of additional copies of fer genes into the genome of plants and the organ and ontogenesis-specific expression of the fer gene lead to an altered expression of the potential target genes, such as genes for iron reductase and mineral transporters, due to changed FER production in the transgenic plant. As a result, minerals are transported in a different way to target organs, in which fer is expressed in a different way.

It is therefore an object of the present invention to provide DNA sequences which code for FER regulator proteins, and to provide transgenic plants which have a modified FER regulator protein content compared to their wild-type form and, due to this, a changed mineral balance, in particular have a changed iron absorption.

Another object is to provide methods for changing the mineral uptake, in particular the iron uptake in transgenic plants. Further tasks result from the above or the following description. The above and other objects of the invention are achieved by the subjects defined in the independent claims. Preferred embodiments are set out in the dependent claims.

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

In the context of the present invention, the term “fer gene” or “DNA sequence coding for an FER protein” is used for a nuclear acid sequence which codes for an active DNA-binding regulator protein, the regulator protein being in the root of strategy I- Plants control the iron uptake at the transcriptional level and 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 of at least 70%, 75%, 80%, 85%, particularly preferably of 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 done particularly preferably by overexpression of the fer gene in the transgenic plants or cells.

The observation that the mineral balance can be positively influenced by the transfer and expression of exogenous fer DNA sequences can be ideally used for the production of plants that are more vital, more productive and more resistant due to an improved mineral balance compared to wild type plants.

The basic requirement for the production of such crops is the availability of suitable transformation systems. A wide range of transformation methods have been developed and established here over the past two decades. 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 direct gene transfer isolated DNA in protoplasts, the injection and electroporation of DNA into plant cells, the introduction of DNA using biolistic methods and other possibilities. These transformation techniques are known to the person skilled in the art or they can be found in overview articles or corresponding reference works.

In addition to the use of the fer DNA sequences disclosed here for the first time, the person skilled in the art can find further DNA sequences coding for FER proteins from organisms other than tomato by means of conventional molecular biological techniques and can use them in the context 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 use them for the screening of cDNA and / or genomic banks of the desired organism, e.g. Use potatoes or cereals from which a new fer gene is to be isolated. Here, the person skilled in the art can familiarize with hybridization, cloning and

Use sequencing methods that are well known and established in any bio or genetic engineering laboratory (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 to synthesize and use suitable oligonucliotide primers for PCR amplification of fer sequences. Of course, there is also the possibility to use the entire, below as SEQ ID NO: 1 or SEQ ID No. 2 use specified DNA sequence as a hybridization probe in 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 the 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 used with conventional Methods of PCR and gene cloning are processed. 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 fer gene from tomato 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 contain a DNA sequence coding for an FER protein, the coding nucleic acid sequence can be operated as in the native gene, ie in the 5 '-3' direction

Link to a promoter active in plants. In this way, an overexpression of the coding sequence in the transgenic plants can be achieved, 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 properties required for regulation.

On the other hand, downregulation of endogenous FER activity may also be desirable, e.g. For example, it may be useful to turn off mineral intake and transport in certain cells in order to better control these processes at another suitable location. To do this, one would have to switch off the fer activity in the cells where it is undesirable, e.g. B. by the antisense approach, the phenomenon of co-suppression or double hybrid RNA formation. In all cases, the entire nucleic acid sequence that naturally encodes an FER protein does not necessarily have to be transmitted and transcribed, but rather fragments of the encoding can

Region are sufficient, as far as these fragments ensure the achievement of the desired effects, ie co-suppression on the one hand or antisense inhibition on the other. The person skilled in the art can, for example, produce suitable deletion constructs and, by transferring them to transgenic plant cells, check whether a particular fragment of the coding region has the effect according to the invention, namely that Has or not inhibits endogenous FER activity. As used in the context of this application, the formulation “DNA sequence which codes for an FER protein” thus also includes those DNA sequences and fragments which do not code for an active protein, their presence and transcription in sense or Antisense orientation but causes a co-suppression or antisense inhibition in transgenic plant cells. Such fragments can also be referred to in the context of the invention as “antisense or suppression active” FER-DNA fragments. The entire genomic clone of an FER protein or parts thereof can also be transferred in order to achieve the desired inhibition of the endogenous FER.

A further possibility of achieving endogenous FER activity is the method known as RNA interference, in which the formation of a specific protein, in the present case the FER protein, is formed by specifically switching off mRNA molecules. is prevented (Elbashir et al. (2001) Nature 411, 494-498; Waterhouse et al. (2001) Nature 411, 834-842).

In the case of RNA interference, too, the specific RNA supplied from outside leads to the destruction of the corresponding target mRNA and thus to the absence of the corresponding protein. The method therefore does not eliminate the gene, but only its functional product (mRNA and protein). It is therefore a method similar to the anti-sense method, with the anti-sense method using an RNA sequence which is complementary to the mRNA, while with the RNA interference, the anti-sense RNA is in the form a double-stranded RNA is added. It is currently believed that the RNA sequences form a complex with proteins in the cell that specifically recognizes and cleaves the target RNA. The synthesis of certain RNA double-strand chains can thus determine which target mRNA is to be destroyed in the cell. In the case of the RNA interference method, the literature shows which fragment lengths are suitable for inhibiting the FER protein. Short, about 21 base pairs long double-stranded RNA molecules are often advantageous (Elbashir et al. (2001) vide supra). Here too, the person skilled in the art is able to select suitable fragments and test their suitability in the RNA interference method.

In any case, the term “antisense” should be understood to mean that the sequence coding for an FER protein or an “antisense-active” fragment thereof in an antisense orientation, ie “upside down” in 3 ′ -> 5 ′ - Direction that is operatively linked to a promoter region that is active in plant cells and is transcribed. The RNA that is produced when such an antisense gene is transcribed is complementary to the RNA of the endogenous gene and prevents the synthesis of the FER protein product by Hybridization comes between the native and the antisense RNA.

In the case of the co-suppression construct which can be used as an alternative, the sequence coding for an FER protein or a “co-suppression-active” fragment thereof is in the sense orientation (ie in the 5 → 3 direction) in operative association with one in plant cells In the case of the co-suppression phenomenon, the endogenous gene is inactivated by additionally introduced identical (partial) copies of this gene, which is probably due to an RNA-dependent mechanism in which the RNA-directed RNA polymerase is involved is.

In the case of the double hybrid RNA coding for a FER protein sequence lies both in sense (5 ^Λ -> 3 ') - and antisense (3 ^Λ -> 5 ^Λ) - orientation linked directly or operatively by an intron, or other sequence in front. Furthermore, the execution of the method according to the invention only requires suitable regulatory sequences which control the transcription of an operatively linked fer-DNA sequence in the transformed plant or plant cell. Here, too, the person skilled in the art can easily find suitable sequences from the prior art or even isolate particularly suitable promoter sequences. Promoters suitable for various plants can be found in the literature. In addition, new promoters can be identified using current genome projects. For example, transgenic plant lines can be screened for new promoter activities that have integrated promoterless GUS constructs (Springer (2000) plans Cell 12: 1007-1020). Explosion analyzes of numerous or all of all EST sequences (RNA profiling, Affymetrix) can allow conclusions to be drawn about suitable promoters. In this way, promoters can be identified which are organ-specific (e.g. root, seeds, flowers, leaf, etc.), tissue-specific (e.g. gliding vessels, cortex, epidermis) or are development-specific or inducible.

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

Although tissue-specific expression is preferred, for

Expression of the DNA sequence according to the invention in plant cells in question any kind of regulatory sequences which ensure the transcription in plant cells. Every promoter active in plant cells is included here. The promoter can be selected so that the expression is constitutive or only in specific tissues at a certain time

Plant development and / or at a time determined by external influences, biotic or abiotic stimuli (induced gene expression). The promoter can be homologous or heterologous with respect to the plant to be transformed. If a constirutive promoter is used, cell-specific or tissue-specific expression can also be achieved in that the gene expression in the cells or Tissues in which it is not desired are inhibited, for example by expressing antibodies that bind the gene product and thus inhibit its activity, or by suitable inhibitors.

The person skilled in the art can find constitutive or tissue-specific or development-specific or inducible genes or promoters from the prior art, in particular from the relevant scientific journals and databases. In addition, the average specialist is able to isolate other suitable promoters using routine methods. The person skilled in the art can thus identify regulatory nucleic acid elements with the aid of common molecular biological methods, examples of which here are methods for the identification of genes with interesting promoters, transgenic plant lines with reporter gene contracts (see above), RNA profiling, affymetrix, in which Genes or promoters described in the literature, subtractive screening of banks for specifically expressed cDNA, and for cloning the promoters. Cloning via 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, optional transcription or termination sequences are available, which serve to correctly end the transcription, and can also be used to add a PolyA tail to the transcript, which is assigned a function in stabilizing the transcripts. Such elements are described in the literature and are interchangeable.

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 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 that codes for an FER protein; and c) optionally 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 synthesized by known methods. The DNA sequence according to the invention preferably originates from Lycopersicon esculentum.

Using common molecular biological techniques (see, for example, Sambrook et al. (1989) supra), it is possible to prepare or produce desired constructs for the transformation of plants. The cloning, mutagenization, sequence analysis, usually used for genetic engineering manipulation in prokaryotic cells

Restriction analysis and other biochemical-molecular biological methods are well known to those of ordinary skill in the art. Not only can suitable chimeric gene constructs be produced with the desired fusion of promoter and fer-DNA sequence, but the person skilled in the art can, if desired, introduce various mutations or deletions into the FER-coding DNA sequence by means of routine techniques.

In a preferred embodiment, the DNA sequence encoding an active FER protein is selected from the group consisting of: a) DNA sequences that the SEQ ID No. 1 or SEQ ID No. 2 comprise the specified coding nucleotide sequence or fragments thereof, the length of the fragments being sufficient to encode a protein with FER protein activity; b) DNA sequences comprising a nucleotide sequence that the in SEQ ID

No. Encode 2 given amino acid sequence or fragments thereof, the length of the fragments being sufficient to have FER protein activity; c) DNA sequences which comprise a nucleotide sequence which is degenerate to a nucleotide sequence of a) or b) or hybridizes with a complementary strand of the nucleotide sequence of a) or b), or comprise parts of this nucleotide sequence, the length of these parts being sufficient to encode a protein with FER protein activity; 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 60%, preferably at least 80%, particularly preferably at least 90% and most preferably at least 92, 94, 96, 98%; ^" e) DNA sequences that are a derivative, analogue or fragment of a

Represent nucleotide sequence of a), b), c) or d), the length of the fragment being sufficient to encode a protein with FER protein activity.

In the context of this invention, the term “hybridization” means hybridization under conventional hybridization conditions, preferably under stringent conditions, as described, for example, in Sambrook et al. (1989, vide supra).

According to the invention, hybridization in vitro will always be carried out under conditions that are stringent enough to ensure specific hybridization. Such stringent hybridization conditions are known to the person skilled in the art and can be found in the literature (Sambrook et al., 2001, Molecular cloning: A laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press).

In general, "specifically hybridize" means that a molecule binds preferentially to a certain nucleotide sequence under stringent conditions if this sequence is present in a complex mixture of (for example total) DNA or RNA. The term “stringent conditions” generally stands for conditions under which a nucleic acid sequence will preferentially hybridize to its target sequence and to a significantly lesser extent or not at all to other sequences. Stringent conditions are in some cases sequence-dependent and will be different in different circumstances. longer sequences hybridize specifically at higher temperatures. in general, stringent conditions are selected so that the temperature is about 5 ° C lower than the thermal melting point (T _m) for the specific sequence at a defined ionic strength and a defined pH. the T _m is the Temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the molecules complementary to the target sequence hybridize to the target sequence in an equilibrium state, typically stringent conditions are those at which the salt concentration is at least approximately 0.01 to 1.0 M Na trium ion concentration (or another salt) at a pH between 7.0 and 8.3 and the temperature is at least about 30 ° C for short molecules (for example 10-50 nucleotides). In addition, as already mentioned above, stringent conditions can be achieved by adding destabilizing agents such as formamide.

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 term “derivative” in this context means that the sequences differ from the DNA sequences described above in one or more positions, but a high degree of similarity to these Have sequences. The deviations from the DNA sequences described above may have arisen, for example, through deletion, substitution, hisertion or recombination.

Similarity or homology means a sequence identity of at least 45%, 50%, 55%, in particular an identity of at least 60%, 64%, 68%. preferably an identity of at least 70%, 72%, 74%, 76%, 78% "particularly preferably of at least 80%, 82%, 84%, 86%, 88%, even more preferably of at least 90%, 92%, 94% and most preferably at least 96% and at least 98%.

The proteins encoded by these DNA sequences have a sequence identity to that in SEQ ID No. 2 or 3 given amino acid sequence of at least 45%, 50%, 55%, in particular an identity of at least 60%, 64%, 66%, 68%, preferably an identity of at least 70%, 74%, 76%, 78% , 80%, particularly preferably of at least 82%, 86%, 90%, 92%, 94% and most preferably of at least 96%, 98%.

Degrees of homology or sequence identities are usually determined using various alignment programs, such as, for. B. CLUSTAL found. Generally stand that

Suitable algorithms for determining sequence identity / similarity are available to those skilled in the art, e.g. also the program, which is available at http://www.ncbi.nlm.nih.gov/BLAST (e.g. the link "Standard nucleotide-nucleotide BLAST [blastn]").

The DNA sequences, which are similar to the sequences described above and which are derivatives of these sequences, are usually variations of these sequences which represent modifications which have the same biological function. It can be both naturally occurring variations, for example sequences from others Organisms, or around mutations, these mutations may have occurred naturally or have been introduced by targeted mutagenesis.

The variations can also be synthetically produced sequences. The allelic variants can be both naturally occurring variants and also synthetically produced variants or those produced by recombinant DNA techniques.

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

The invention further relates to vectors and microorganisms which contain nucleic acid molecules according to the invention and the use of which enables the production of plant cells and plants whose mineral content, in particular their iron absorption, is changed compared to wild-type plant cells or plants. The vectors are in particular plasmids, cosmids, viruses, bacteriophages and other vectors which are common in genetic engineering. The microorganisms are primarily bacteria, viruses, fungi, yeasts 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.

The invention further relates to a method for producing plants or plant cells with a mineral balance, in particular changed iron absorption, which is different from that of 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 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) if appropriate, the regeneration of intact plants from the transformed plants

Plant cells.

The invention further relates to plant cells which contain the nucleic acid molecules according to the invention which encode an FER protein. The invention also relates to crop products and propagation material of transgenic plants and the transgenic plants themselves which contain a nucleic acid molecule according to the invention. Because of the introduction of an FER-coding DNA sequence, transgenic plants of the present invention have an iron uptake which is different from that of wild-type plants.

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

As already mentioned, a large number of techniques are available for introducing DNA into a plant host cell, and the person skilled in the art can determine the appropriate method in each case without difficulty. These techniques include transforming plant cells with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as

Transformation medium, the fusion of protoplasts, the injection, the electroporation, the direct gene transfer of isolated DNA into protoplasts, the introduction of DNA using biolistic methods as well as other possibilities that have been well established for several years and become the usual repertoire of the specialist in plant molecular biology or plant biotechnology.

When injecting and electroporation of DNA into plant cells, there are no special requirements per se for the plasmids used. The same applies to direct gene transfer. Simple plasmids such as e.g. pUC derivatives can be used. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable marker gene is recommended. The usual selection markers are known to the person skilled in the art and it is not a problem for him to select a suitable marker.

Depending on the method of introducing the desired genes into the plant cell, additional DNA sequences may be required. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, at least the right boundary, but often the right and left boundary of the T-DNA contained in the Ti or Ri plasmid, must be used as a flank region with the genes to be introduced get connected. If agrobacteria are used for the transformation, the DNA to be introduced must be cloned into special plasmids, either in an intermediate or in a binary vector. The intermediate vectors can be integrated into the Ti or Ri plasmid of the agrobacteria on the basis of sequences which are homologous to sequences in the T-DNA by homologous recombination. This also contains the fer region necessary for the transfer of the T-DNA. However, intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors, on the other hand, can replicate in E. coli as well as in Agrobacteria. They contain a selection marker gene and a linker or polylinker, which are framed by the right and left T-DNA border region. They can be transformed directly into the agrobacteria. The agrobacterium serving as the host cell is said to contain a plasmid which carries a fer sequence within the T-DNA which is transferred into the plant cell. Additional T-DNA may be present. The agrobacterium transformed in this way is used to transform plant cells. The use of T-DNA for the transformation of plant cells has been intensively investigated and has been sufficiently described in well-known overview articles and manuals for plant transformation. For the transfer of the DNA into the plant cell, plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. Whole plants can then be regenerated from the infected plant material (for example leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) in a suitable medium, which can contain antibiotics or biocides for the selection of transformed cells.

Once the introduced DNA is integrated in the genome of the plant cell, it is generally stable there and is also retained in the progeny of the originally transformed cell. It usually contains a selection marker, which imparts resistance to a biocide or an antibiotic such as kanamycin, G 418, bleomycin, hygromycin, methotrexate, glyphosate, streptomycin, sulfonylurea, gentamycin or phosphinotricin, among others, to the transformed plant cells. The individually selected marker should therefore allow the selection of transformed cells from cells that lack the inserted DNA. Alternative markers are also suitable for this, such as nutritive markers and screening markers (such as GFP, green fluorescent protein). Of course, selection markers can also be completely dispensed with, but this is associated with a fairly high need for screening. If marker-free transgenic plants are desired, strategies are available to the person skilled in the art which make a subsequent

Allow removal of the marker gene, e.g. Cotransformation, sequence-specific recombinases.

The regeneration of the transgenic plants from transgenic plant cells is carried out according to customary regeneration methods using known nutrient media. The plants obtained in this way can then be examined for the presence of the nucleic acid which codes for an FER protein or for the presence of the gene product, that is to say the FER protein, using customary methods, including molecular biological methods, such as PCR, blot analyzes.

The transgenic plant or the transgenic plant cells can be any monocotyledon or dicotyledonous plant or plant cell in which the mineral balance, in particular the iron absorption, should preferably be improved. They are preferably useful plants or cells of useful plants. Solanaceae such as are particularly preferred

Potatoes, tobacco, tomatoes, peppers, but also other dicotyledonous plants (e.g. sugar beet) or monocotyledonous plants (e.g. rice, corn, wheat). In principle, any useful plant that is directly or indirectly suitable for nutritional purposes is desirable for the implementation of the invention. The invention also relates to propagation material and harvest 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 detected and tracked using conventional molecular biological and biochemical methods. These techniques are known to the person skilled in the art and he is easily able to choose a suitable detection method, for example a Northern blot analysis for the detection of FER-specific RNA or for determining the level of accumulation of FER-specific RNA or a Southem Blot analysis for the detection of DNA sequences coding for FER.

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 coding 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 serve only to illustrate the invention and are in no way to be understood as a limitation. Examples

The tomato line T3238fer, which is derived from the boron-inefficient tomato line T3238 as a mutant, is not able to activate the iron deficiency stress reactions typical of plant I plants under iron deficiency conditions (Brown et al. (1971) Physiol. Plant. 25: 48- 53; Brown et al. (1974) Physiol. Plant. 31: 221-224). The mutated plants show neither increased reductase activity nor other physiological or morphological changes in the case of iron deficiency, which are switched on for the efficient absorption of iron in the case of iron deficiency. Since the fer mutation has numerous effects, fer probably acts centrally at the transcriptional level. When growing in soil, fer plants with fer mutations die during the vegetative phase. Plants with fer mutations can be partially or completely saved by continuously fertilizing them in soil with iron in the form of iron hydroxyethylenediamine triacetic acid (Fe-HEDTA) or by attracting them to MS medium. The fer mutant is a recessive mutant that affects a single gene that was mapped to chromosome 6 of the tomato 0.33 cM from TG590 and 2.0 cM 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 (chromosome walk) was used to isolate the fer gene. This technique has been used successfully several times in the tomato. In the case of the cloning of fer, the method was analogous to that of chloronerva, which was successfully completed two years ago and in Ling et al. (PNAS Prod. Natl. Acad. Sei. 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 of fer on chromosome 6 between the two markers TG590 and TGl 18 was described.

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 crossing L. esculentum T3238fer x L. pennellii. 6 recombinant plants were found between TG590 and fer, and 35 recombinant plants between fer and TGl 18. The 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 two further YAC clones isolated and their end mapped (267L and 328N1). With 267L and 328N1, the YAC 337, which spans the entire fer region and on both sides of the fer gene, was isolated after two each

Recombination events end. 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, Ine, 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) supra. Two BAC clones, BAC 56B23 and BAC 53M23 were completely sequenced using the shot gun method. For this purpose, the sheared and fractionated in lkb BAC-DNA 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 completely put together. Probes were mapped at various points in the sequence so that the fer gene could finally be located in an area of 18 kb. This 18kb area contains two open reading frames, one of which codes for a transposase and therefore as a fer gene is out of the question. 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 EcoRN 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 the fer-DΝA 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 codes for a protein with a conserved basic helix-loop-helix-DΝA binding domain. The fer-gen has the highest sequence similarity with a gene from Arabidopsis thaliana, which is located on chromosome II (DΝA accession Νo. AC005851 coding sequence from 72093 to 73201bp, gene = At2g28160, hereinafter referred to as Atfer, protein accession Νo AAC984501.1). No functional analysis has been described for the Arabidopsis gene. It cannot therefore be said whether the sequences between fer and Atfer are only similar or whether the genes are actually homologous, ie 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 show no sequence similarities outside of the conserved motif. A comparison of the cDNA and genomic sequence shows that the fer gene has three hitrons. 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 were received

Transcripts can be detected in cotyledons, leaves or flowers (see Figure 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 strong expression of fer in roots (4a). For wild-type and fer plants that are up to three weeks old in Hoagland solution

10 μM Fe-NaEDTA, but then grown in Hoagland solution with 0.1 μM, 10 μM and 50 μM Fe-NaEDTA, 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 analyzes of the fer gene” described wild-type fer plants which were grown in Hoagland solution with 0.1 μM, 10 μM and 50 μM Fe-NaEDTA, respectively, showed that nramp- Genes in the roots of fer plants were not expressed. In contrast, these nramp genes are constitutively expressed in wild-type roots.

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 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 converted into mutant fer tomato plants. Transformation and regeneration of fer plants were not a problem in vitro because the fer phenotype is saved in vitro on MS medium.

When transferred to soil, it was observed that non-transgenic control plants soon developed leaf chlorosis and necrosis, while the plants transformed with the 35S-fer construct were saved. The fer gene can thus be used to remedy mineral chloroses. The transformation and regeneration was carried out according to the usual protocols.

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 distance from the 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.

2 shows the result of a Southern blot hybridization of the fer gene in Tfer and various wild type lines. Genomic DNA from the mutant line T3238fer, the wild type lines T3238FER, Moneymaker, LA483 and TA56 was cut with the restriction enzyme EcoRV and cut in a Southem blot Analysis hybridized with the fer cDNA as a probe.

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

3b shows a comparison of the FER amino acid sequences from tomato and Arabidopsis thalianä. The coding sequences of Lefer (Le stands for Lycopersicon esculentum) and Atfer (At stands for Arabidopsis thalianä) began with the ATG start codon and ended with the TAA stop codon using the Clustal program of DNASTAR Inc. (Meg Align 4.0) - Sequence analysis packages compared. Identical base positions have a black background. The amino acid sequences FER (tomato) and ATFER were compared using the Clustal program. Identical amino acid positions are highlighted in black.

4 shows expression analyzes of the fer gene a) Northem blot analysis: fer transcripts were carried out on total RNA from roots, leaves and stems of wild-type plants (Mm, Moneymaker) and fer plants (Tfer) in a Northem blot Analysis with radioactively labeled fer cDNA demonstrated. 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. With specific oligonucleotide primers, DNA fragments of fer and as a positive control of the elongation factor gene were ef-lo. amplified. 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 (6h), eight hours (8H), two, four and eight days (2-8d).

Figure 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.

Claims

PATENT CLAIMS

1. DNA sequence coding for an FER protein selected from the

Group consisting of a) DNA sequences that the SEQ ID No. 1 or SEQ ID No. 2 encoding nucleotide sequence specified or parts thereof, b) DNA sequences comprising a nucleotide sequence which the 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 are 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 or fragments thereof, and b) amino acid sequence which corresponds to that 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 spoke 5, which comes 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 spoke 3 or 4, b) transfer of the nucleic acid molecule from a) to transgenic plants or plant cells; and c) optionally regeneration of plants from the transformed plant cells.

8. Plant cell containing a DNA sequence according to spoke 1 or 2 or a recombinant nucleic acid molecule according to speech 3 or 4, or produced according to claim 7.

9. Transgenic plant cell according to claim 8, which has an increased FER protein content compared to wild-type cells.

10. The transgenic plant cell according to claim 8 or 9, which has an increased mineral intake, in particular iron intake, compared to wild-type cells.

11. Transgenic plant, containing a plant cell according to one of claims 8 to 10 or produced by a method according to spoke 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 progeny of these plants.