DE19904754A1 - Plant guard cell-specific regulatory cis-active element useful for promoter constructs and for identification of components of guard cell signal pathways comprises specific sequences - Google Patents

Abstract

A regulatory cis-active element, which comprises at least a nucleic acid sequence (I), (II) and/or a complementary sequence in the context of a promoter for mediating stomatal guard cell-specific gene expression is new. A new regulatory cis-active element comprises at least a nucleic acid sequence (I), (II) and/or a complementary sequence in the context of a promoter for mediating stomatal guard cell-specific gene expression. Independent claims are also included for: (1) producing a recombinant promoter for guard cell-specific or -additive gene expression, comprising adding a regulatory cis-active element to a foreign promoter, which has no guard cell-specific activity; (2) a recombinant promoter produced by (1); (3) a DNA construct, especially an expression construct, comprising a recombinant promoter as in (2); (4) a vector, plasmid, cosmid, bacmid, virus or phage genome comprising (3); (5) a transformed microorganism comprising (3); and (6) a transgenic plant, plant part, plant cell or seed comprising (3). TAAAG (I) AAAAG (II)

Description

The invention relates to short regulatory cis-active Elements representing the nucleic acid sequence (s) TAAAG and / or AAAAG and / or at least one of the nucleic acid sequences mentioned include complementary sequence and that in the context of a promo mediate a lock cell specificity of gene expression, the use of such short regulatory cis-active Elements in a method of manufacturing promoter con structures for a lock cell-specific or lock cell additi ve gene expression, process for the production of recombinant Promoters based on a lock cell-specific gene expression sion effect output promoters, which one compared to the Output promoter changed activity in lock cells sen, recombi obtainable by the inventive method nante promoters, DNA constructs, in particular expression cas set, vectors and other carrier systems, transformed micro organisms and transgenic plants, parts of plants and seeds, the the recombinant promoter according to the invention in the form of a DNA Constructs included, especially for executing the kits mentioned method according to the invention and a method for Identification of expressed in plant lock cells Genes based on nucleic acid sequence data.

Due to the constantly increasing need for food resources and raw materials resulting from the constant growth of World population results, and considering the medium to long It is the task to limit the cultivation area in good time of biotechnological research to develop it  sustainable and ecologically harmless crops hen. To this end, development and metabolism of plants and the reactivity of plants to bio table and abiotic stress factors are manipulated. This can, among other things, by changing the cell nucleus Plants contain genetic material, deoxyribonucleic acid (DNA) can be achieved.

Locking cells are highly differentiated, often bean-shaped cells in the epiderms of the aerial green parts of higher plants surrounded by air, which are arranged in pairs and leave a gap (gap) between them. The gap under interrupts the otherwise gapless layer of the epidermal cells and thus creates the connection between the outside air and the intercellular system. With most plants, the pore area takes up about 0.5 to 1.5% of the leaf area when the gaps are open. As a result of their special construction, locking cells can regulate their shape through active changes in the turgor so that the gap between them closes or opens. The locking cells are therefore regulators of gas exchange, especially perspiration. The locking cells have the task of regulating the diffusion resistance in such a way that the water loss through transpiration and the CO ₂ uptake for photosynthetic or dark CO ₂ fixation are in a ratio that is adapted to the respective needs. In addition, the locking cells also allow other gaseous substances produced by plants, such as methane, to be released into the atmosphere surrounding the plants. There are indications that interactions or communication between plants or between plants and other types of organisms surrounding them, such as insects, take place at least in part through the mediation of such gaseous substances. There is also evidence of inter-plant communication such. B. by methyl jasmonate and methyl salicylate, which are also released with high probability in the form of gases through the stomata of the plants to the atmosphere surrounding the plants.

The opening and closing of the gap opening is on a ver Change of the turgor in the lock cells themselves and on the Establishment of a difference between the turgor and the lock cells adjacent epidermal cells (neighboring cells).

This can increase the yield of crops be achieved that the photosynthetic performance of the plants ver is changed or water consumption is reduced. Therefor are the guard cells in the epidermis of plant leaf tissue a good starting point. Furthermore, the lock cells a preferred entry point for a variety of pathogens represented by the selective expression of defense genes in the sen cells can be counteracted. A selective leg Flow and control of the expression of certain genes in the Lock cells accordingly represents a promising one Methodology to optimize crop productivity ren. It also appears possible by modulating the stomatal opening width or duration interactions between Plants and / or between plants and other types of organ men, such as insects, which are produced by the plant graced and released through the stomata sub punching are conveyed to influence. Therefore there is a great interest in identification and economic Usability of the specific expression in the lock cells responsible regulatory DNA sequences of the vegetable genome.

So far, several promoters have been published, one preferential gene expression in guard cells of plants ver means (see generally for expressions specific to guard cells Genes and promoters e.g. B. Kaldenhoff, R., et al. (1995); Miller- Röber, B., et al. (1994); Müller-Röber, B., et al. (1995); Naka mura, R.L. et al. (1995); Sarda, X., et al. (1997); Terryn, N., et al. (1993); Thoma, S., et al. (1994); Xia, Y., et al. (1997)). The published patent application DE 42 07 358 A1 also relates a lock cell-specific starter region. It is about  however in each case naturally occurring in their entirety Starter or promoter regions for mediating a gene expression sion.

In contrast, the task of the present Er exists in finding the basic cis-active regulatory Identify elements that derar for the guard cell specificity promoters. This makes it possible to provide recombinant promoters containing a lock cell specific or possibly also lock cell-additive gene expression possible due to the integration of such short regulations scher cis-active elements in the context of different, too the cis-active elements of heterologous promoters (starter region nen) or promoter components a lock cell-specific Ex mediate pression.

The present application solves the problem of the invention lying task by means of the Ge defined in the claims objects and procedures based on the provision of regulatory cis-active elements that have at least one, preferably two or more of the nucleic acid sequences TAAAG and / or AAAAG and / or to the nucleic acid sequences mentioned zen complementary sequences include and in the context of a pro motors (a starter region) a lock cell-specific Genex mediate pression.

In specific embodiments of the invention, it comprises regulatory cis-active element according to the invention essentially chen at least one of the in SEQ. ID. NO. 1, 2, 3 or 4 specified benen nucleotide sequences, for example a dimer, trimer or Tetramer, but possibly also a multimer of up to twenty or more of that in SEQ. ID. NO. 1, 2, 3 or 4 specified nucleotides quence. Some or all of the SEQ. ID. NO. 1 to 4 specified nucleotide sequences combined in a cis active element.  

In a further embodiment, a ßes cis-active element a nucleic acid sequence that of the Nucleic acid sequence of one of the regulators explained above cis-active elements that are essentially at least one the one in SEQ. ID. NO. 1, 2, 3 or 4 specified nucleotide sequences zen include, by mutation, especially deletion, substitutes on, addition and / or insertion of individual nucleotides or smaller group of nucleotides is derived. In particular here is thought of cis-active elements, the derived nu small acid sequence with the starting nucleic acid sequence, d. H. the Nucleic acid sequence of a cis-active element which essentially chen at least one of the in SEQ. ID. NO. 1, 2, 3 or 4 specified benen nucleotide sequences, under stringent conditions hybridizes.

Hybridization under stringent conditions in the sense of the invention is understood to be a hybridization in the procedure according to one or more of the methods specified below. Hybridize: Up to 20 hrs in Church and Gilbert PEG buffer (0.25 M Na ₂ HPO ₄ , 1 mM EDTA, 1% (w / v) BSA, 7% (w / v) SDS, pH 7, 5 with phosphoric acid; Church, GM and Gilbert, W. (1984) Genomic sequencing, Proc. Acad. Sci. USA, 81, 1991-1995) at 42 ° C or in standard hybridization buffers with formamide at 42 ° C or without formamide at 68 ° C (see Sambrook, Fritsch & Maniatis (1989) Molecular cloning: a laboratory manual (2nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Wash: 3 times 30 min at 65 ° C in 3-fold SSC buffer (see Sam brook, Fritsch & Maniatis (1989), loc. Cit.), 0.1% SDS.

The above-mentioned nucleic acid sequences TAAAG and AAAAG or the complementary sequences, the individual, more fold and also in combination in the cis according to the invention active elements can be included, constitute the core sequences zen or core elements of the binding site for a particular Class of transcription factors, in particular the Guard cell specificity of a promoter comprising these elements  condition. In the context of the present documents, Ver points to the nucleic acid sequences mentioned TAAAG and / or AAAAG always the complementary nucleic acid sequences as to be understood with includes.

For steric reasons as well as for specificity reasons these are binding sites for transcription factors and thus such cis-active elements often larger than 5-6 base pairs where however, the environment around the core sequences or core elements compared to this a subordinate role for the transcription factor binding plays. The crucial requirement for that Binding this particular class of transcription factors represents the presence of the above core sequences or core elements TAAAG and / or AAAAG and / or core elements ten with complementary to the listed nucleic acid sequences Nucleic acid sequence.

In addition to the use of the he explained in more detail below regulatory cis-active elements according to the invention Position of recombinant promoters for a lock cell-specific ces or additive gene expression can also be used for identi fication of transcription factors attached to this cis-active Bind element, be used. This identification of specifically binding to cis-active elements according to the invention Transcription factors can in particular by means of the yeast one Hybrid systems (Wang & Reed (1993)) or the Southwestern Screening (Singh et al. (1993)).

In particular, the determination of the lock cell spec opens cis-active elements conveying the possibility with the help of these regulatory elements through Kombinati a variety of other heterologous promoter components combined promoters or starter regions for a gene expression sion in lock cells. These can then be one Gene expression in guard cells of variable strength and given also in combination with an expression in other Ge weave, d. H. a so-called "lock cell-additive gene expression", convey.  

The terms "recombinant promoter" and "recombinant Starter region ", which ver are used to denote a promoter in which at least least the cis-active element imparting lock cell specificity, if it has a sequence that in its entirety in the Nature occurs, not within the genetic environment in which it is naturally present. Alternatively, the cis active element apart from at least one of the core sequences TAAAG and AAAAG or the complementary sequences which must remain unchanged, including nucleotide sections have, which lead to the fact that the entire nucleic acid ab must be regarded as synthetic, that is, in nature is not found.

The other promoter surrounding the cis-active element elements or components can be from the same plant as the cis-active element mentioned or from another type of Or ganisms, especially eukaryotic, but possibly also come from prokaryotic organisms, viruses or phages. Al Alternatively, other promoter elements can also be purely synthetic So to be of origin does not come in this form in nature in front. Other promoter elements are, for example, the TATA box and the transcription initiation body, which together form the not agile and sufficient elements of a so-called "minimal promotors ", which is a transcription of one under his Control of the standing gene - as a rule, however, only in very small scale - enables. Still other promoter elements that may be present, for example, the CCAAT box, the GC box and other cis-active elements that bind mediate transcription factors. Such a mediated bin The addition of transcription factors in turn brings about a union professional and therefore more frequent formation of the initiation comm plexes for transcription at the transcription initiation and thus an increased transcription. This cis Active elements can also be used in special cases additionally a tissue- or cell-specific gene expression  teln, and can in the present context if necessary in addition to the cis found by the invention active elements mediated lock cell specificity. In the latter case are recombi according to the invention nante promoters obtained in addition to a certain original tissue or cell specificity an additional gene expression enable in the locking cells, ie a "locking cell addi tive "gene expression.

Any other cis present in the recombinant promoter active elements enable inducibility or repression Availability of the promoter when certain events occur, e.g. B. a wound or dry stress, and especially in property certain substances, such as phytohormones. Such cis active elements are also called activation or repression designated elements. By integrating such cis-active Elements in a recombinant promoter according to the invention can combination with stronger or weaker versions the inventive cis mediating lock cell specificity active elements with a recombinant promoter locally Lock cells limited induction or repressive ability getting produced. A lock cell can be used here as an example specific promoter repression and thus repression of the genex pression in dry stress or a lock cell-specific act Defense genes when events occur that occur in Connection with the penetration of pathogens into a plant occur.

The other promoter elements can be in the finished promo may also be available in multiple copies. In a minimal ver sion comprises a recombinant promoter according to the invention or possibly also several cis-active elements, the lock cell spec mediate zifity, in combination with a minimal promoter, e.g. B. the 35S minimal promoter.

The invention is based on the identification of two in such cis elements contain five nucleotides  the core sequences or sequence motifs with the nucleic acid sequences TAAAG and AAAAG or the complementary nucleic acid resequences, each in the context of a cis-active Elements mediate a lock cell-specific gene expression can. The described cis-active regulato mediate elements specific expression in closing alone cells of the plants and no expression in other tissues. The core sequences or sequence motifs explained above can individually or within such a cis element occur simultaneously and possibly also in several copies. By targeted installation of the provided according to the invention cis-active elements or sequence motifs or by targeted Er Generation of such cis-active elements within a given benen promoter by mutation including insertion (s) and Deletion (s), in particular the nucleic acid sequence of the previous core sequence (s) TAAAG and / or AAAAG and / or a nucleic acid sequence complementary to the said sequences ge generated, regulatory starter regions that are up to for a long time no expression was possible in the plant's guard cells ten, convert into starter regions that an expression in Ensure the plant's guard cells. In the context of this "Insertion" and "Deletion" of the documents always attacked "mutation" or "mutagenesis" as included will stand.

Accordingly, the invention includes among others Aspect of a method for producing a recombinant promo tors for a lock cell-specific or lock cell additive Gene expression, which is characterized in that in an out gang promoter that has no lock cell specific or lock causes cell-additive gene expression, or to components of a such output promoter at least one regulatory, one lock cell-specific gene expression mediating cis-active Element which contains the nucleic acid sequence (s) TAAAG and / or AAAAG and / or at least one of the nucleic acids mentioned sequences include, added or added by Mu  tation is generated. Various suitable cis-active elements are exemplary in the figures and in the accompanying sequence protocol specified.

In a special embodiment of the invention Processes are used to produce the recombinant promoter two to twenty or more, especially two to 18, e.g. B. two up to 8 cis-active elements, comprising the nucleic acid sequence (s) TAAAG and / or AAAAG or at least one to the named Nu small acid sequences complementary sequence used.

The integration of one or more copies of the described DNA sequences TAAAG and AAAAG or the complementary Se sequences in the form of one or more cis-active elements in the promoter according to the invention or the generation of such copies mutation allows the expression to be varied strength in lock cells. The more of these elements or sequence mo tive contained in the promoter, the higher the Ex usually be strong.

In addition, knowledge of the cis-active el elements or sequence motifs, the activity of transcriptional To modulate starter regions (promoters) in guard cells. By adding at least one of the cis active elements, which the sequence motifs TAAAG and / or AAAAG and / or those gene with complementary sequence include as z. B. in the figures and the attached sequence listing (SEQ. ID. NO. 1-4) are listed for a lock cell-specific or closing cell additive effective promoter sequence (or by Generation of at least one of the cis-active elements in the Output promoter sequence by mutation) can reduce the activity of the Gene expression can be increased specifically in guard cells.

Accordingly, the invention includes among others Aspect of a method for producing a recombinant promo tors with a modified compared to an output promoter, in particular increased activity in guard cells, this is characterized in that in an output promoter, the one  Lock cell-specific or lock cell-additive gene expression causes at least one of the cis-active elements comprising at least one sequence motif TAAAG and / or AAAAG and / or minde least a sequence motif with the nucleic acid sequences mentioned zen complementary sequence, added or by mutation is fathered.

In return, through deletion at least one can the sequence motifs TAAAG or AAAAG or the sequence motifs with complementary sequence within an output promoter, which is a lock cell-specific or additive gene expression acts, and / or change at least one of these sequence motifs ve by block substitution or mutation and / or deletion, In sertion and / or substitution of one or more nucleotides the lock cell-specific activity of the promoter is reduced become. Accordingly, the invention also includes using a method for producing a recombinant promoter one modified compared to an output promoter, in particular reduced activity in guard cells, in which in the out gang promoter, which is a lock cell specific or additive Gene expression causes at least one of the sequence motifs TAAAG and AAAAG or the sequence motifs with the specified nucleus acid sequences of complementary sequence deleted or by Block substitution or by deletion, insertion and / or sub stitution of one or more nucleotides is changed. Un A block substitution or block mutation is understood in the present context the complete replacement of the five nu sequence motifs comprising kleotides through a different, sequence motif also comprising five nucleotides. An example the examples show such a block mutation men.

In a further variant, one can have an off gang promoter also changed promoter activity by "Moving" inventive cis-active elements or at least at least the Se comprising five nucleotides explained above sequence motifs within the nucleic acid sequence of an initial pro  motors. Such a shift or change of position based on at least one further element of the promoter in the output promoter by clones familiar to the person skilled in the art measures, multiple nucleotide deletions, insertions and / or substitutions combined in a suitable manner will, include, be effected.

Another central subject of the invention are recom binant promoters by the inventive method are available, i.e. at least one regulatory, one lock cell-specific gene expression mediating cis-active Element comprising the nucleic acid sequence (s) TAAAG and / or AAAAG and / or at least one of the nucleic acids mentioned sequences complementary sequence. For the rest, is on the foregoing in connection with the terms "recombinant promoter" and "recombinant starter region" ver grasslands. The promoters according to the invention can also additional non-coding nucleic acid in the 5 'and 3' direction real sections, for example linker sequences with interfaces for restriction enzymes.

Otherwise, the invention relates to DNA constructs, in particular expression cassettes that a recombi according to the invention include promoter.

Other possibly in the DNA construct according to the invention besides the present recombinant promoter geneti elements are relatively short, in vivo in part as repetitions special cis-active elements with a length of approximately 70 bp, which are referred to as "enhancers" the. In vivo enhancers usually amplify in different ways chem level of expression of some genes, d. H. they improve that Recognition of the promoter by the RNA polymerase, for which it is described in usually about 100 to in the 5'-flanking region of a gene 500 bp before the "cap" site of the gene, the activity of which they can troll, are arranged. But you can also in the 5 'direction further away, within the transcribed areas or  also lie in the 3 'flanking region. The transcription activating effect of enhancers is independent of their ab stood by the promoter and their orientation regarding the promo tors. In addition, enhancers are usually not promoters specifically, so that enhancers of different origins each other can functionally replace. Many enhancers bind to cells Protein factors that are believed to be themselves interact with DNA binding proteins and the formation of DNA loops, through which distant DNA areas spatially close can be brought together. A lot of cellular enhan cer elements are adjustable and only under certain cells conditions are active. So some enhancers through Substan regulated with hormone action. Often the tissue or cell-specific expression of genes by enhancer elements Right.

In a special embodiment, the DNA construct comprises also a nucleic acid encoding a gene product cut operationally or functionally with the recombinant Pro motor is linked. The necessary are for the expert Measures for the operative or functional addition of a code nucleic acid segment to a promoter as part of its Skills. Such a DNA construct and in particular one Such expression cassettes enable a lock cell speci fish or - if there are certain, explained above ter conditions - lock cell additive expression of the above Gene product in plants. The coding nucleic acid segment can become a host plant in which its expression takes place should be homologous or heterologous. If the gene product in one to be transformed by the DNA construct according to the invention The locking cell has already been extracted, is undergoing a transformation this and the subsequent expression of its coding Area of overexpression of this gene product in the Lock cells. If the gene product is in a lock cell in front of the Introduction of the DNA construct not yet expressed, can a first-time ex by means of the DNA constructs according to the invention  pression of a gene product in this lock cell the.

Alternatively, such a DNA construct can be used instead of one coding nucleic acid section also an "antisense" DNA, So a DNA sequence that z. B. a coding nucleic acid contains section in "antisense" orientation. The this enables expression of an "antisense" transcript tion product in plants can be a reduction or even eli mination of expression, and more precisely, as is suspected, the Translation of one normally expressed in the guard cell Gene product due to hybridization of the "antisense" - Transcription product with the mRNA of the "normal" Bedin genes expressed gene.

An equal effect, reducing or even eliminating expression of a gene product, that of lock cells a transformation with the DNA construct ex is primed, can by a cosuppression effect on smuggling of a DNA construct, which is this special Gene product coding gene under control of the invention Promoters contains can be achieved. A cosuppression effect will regularly with some organisms after transformation or Transfection with DNA constructs that express gene expression enable that already exist in the organism cells, ne ben a variety of transformed organisms, which, as in usually intends to overexpress the target gene due to the additional construct present, observed.

In addition to the "antisense" nucleic acid mentioned above DNA constructs according to the invention can also be used still include further nucleic acid sections, the transcripti on to transcription products other than mRNA, for example leads to the production of ribozymes.

By means of the recombinant lock cells according to the invention specific promoters and the constructs comprising them can be re-inserted into the lock cells by expression more suitable  guided genes or by varying the concentration of endogenous Gene products a change in stomatal properties, at for example a permanent opening or closing, an extension reducing or shortening the opening periods of locking cells, a change in the opening and closing kinetics (i.e. the Speed of stomatal opening and closing) or a Change in opening and closing dependencies, e.g. B. in Ab dependence on phytohormones, environmental factors or pathogens, be brought about. This makes it possible to influence the Gas exchange, such as water vapor and carbon dioxide. About that In addition, there is also the possibility of modulating the stomatal opening width or duration interactions or the com communication between plants and / or between plants and an organisms such as insects, which are determined by certain produced by the plant and released through the stomata to influence gaseous substances. How As already explained, the locking cells also allow the delivery other gaseous substances produced by plants, such as Methane, methyl jasmonate and methyl salicylate to which the plants surrounding atmosphere. According to the current state of knowledge ver are highly likely to average these substances interac tion or communication between plants or between plants zen and other kinds of surrounding organisms, such as insects.

As also mentioned above, but can also targeted expression of defense genes against pathogens in Lock cells can be reached. Patho genes preferentially penetrate plants through the stomata. Therefore is of a lock cell-specific constitutive or esp especially depending on the pathogen detection inductively ven expression of defense genes or of components or proteins that cause closure of a guard cell, a butt potential for pathogen defense and thus for plant protection expected.

A permanent keeping of the lock cells should be due to egg increased perspiration leads to high water loss  Plants lead, but can, for example, in late phases of Growth cycle to accelerate the maturation of culture plants may be desirable. In addition, the product tion of highly perspiration-active plants for the purpose of a con controlled drainage of soils. Surface Plants with roots can lower the Heat capacity of the soil due to the strong water drainage due to the increasingly perspiring plants a seasonal cause earlier heating of the usable areas and thus an earlier one Sowing temperature-sensitive crops, such as. B. the Allow sugar beet. Because the genetics initially introduced changed auxiliary plants in the further phases of the wax are disadvantaged, they do not lead to a hindrance development of crops, but on the contrary through an expansion of the vegetation phase to a yield point training.

The stomata would be kept largely closed slower growth due to reduced gas exchange and consequently a temporal extension of the vegetation phase cause. This can be of interest for ornamental plants. Next agricultural auxiliary plants can be produced which, as poorly transpiring ground cover, erodes the Usable areas at rest or at the beginning of the vegetation phase prevent crops.

A change in the opening period of the column proves to be beneficial for crops. For example, in green harvested species, especially fodder plants, water can prevent older leaf material from drying out and thus increase the yield. An adaptation to locations with a higher atmospheric carbon dioxide concentration (e.g. CO ₂ fumigation in greenhouses) should also be considered. Artificially extending the opening period can compensate for effects such as mineral deficiency with reduced transpiration current due to high carbon dioxide partial pressures in the plant.

By combining lock cell-specific regulatory elements Other control loops can be used for other applications arises. For example, by coupling to chemi sensors an externally induced closing of the gap opening effects and so z. B. a growth stop of certain Useful plants, the so-called "agricultural auxiliary crop zen "can be triggered at any desired time.

As explained, the lock cells can be modified affecting the opening condition of the stomata on two Reaching through: by modulating the content of endogenous, Proteins, peptides present in the guard cells, in particular those with hormone activity, enzymes, "carriers" or "Pum pen "which transport metabolites through membranes; or through Transfer and expression of homologous or heterologous genes or of synthetic origin, the proteins, peptides, enzymes encode me, "carrier" or "pumps" that are not normal Equipment of a lock cell belongs, but its activity or presence of the state of the cell with respect to the opening affect the stomata.

The DNA constructs according to the invention and in particular expression cassettes for lock cell-specific or guard cell additive expression of genes that regulate of gas exchange and perspiration change. Three sensor systems are involved in the regulation of perspiration involved: sensors for measuring the carbon dioxide concentration, Phytohormone sensors and a measuring system for turgor gradients between lock cells and adjacent epidermal cells. Turgora Changes are caused, for example, by influencing the activity of enzymes or transport systems of carbohydrate metabolism possible. In addition to a direct change in the concentration os Motively active carbohydrates, e.g. B. by means of cytosolic or vacuolar invertases or chloroplastic triose phosphate translocators, are consequences for osmoregulation too wait by enriching or reducing precursors os motively active substances. For example, using a modes  fication of ADP-glucose pyrophosphorylase activity the starch influenced synthesis and so the amount of substrates for glycolysis and the citrate cycle can be changed, ultimately reducing the concentration tration of malate, one particularly with regard to the cell turgor important substance that is affected. An increase in apopla static invertase activity in guard cells could have a increased intake of hexoses increases the starch concentration. Such an intervention must be specific to the lock cell, since an increase in apoplastic invertase activity in the strong photosynthetic mesophyll cells to reduce the As similate transport and thus to a high osmotic load of the tissue (v. Schaewen et al. (1990), EMBO J. 9: 3033-3044). Furthermore, an increase in the expression of a Phosphoenolpyruvatcarboxylase or the expression of a change other phosphoenolpyruvate carboxylase. The enzyme catalyzes the reaction of phosphoenol pyruvate to oxaloacetate, which in turn is a precursor to Malat. Proto internal pumps (proton ATPases), which have elec trochemical gradients enable ion transport processes, too contribute to a change in the osmotic potential.

There are other possibilities to influence the activity the carbonate dehydratase, which strikes the balance between dissolved system adjusts carbon dioxide and carbonate and in this respect the cell Lär carbon dioxide concentration affected, but for example also with fructose bisphosphate, which is an enzyme from the active nerative section of the Calvin cycle at the fixation of the Koh lendioxides is involved.

The constructs according to the invention and in particular Expressi on cassettes can also be used for the lock cell-specific Ex pression of genes that use the phytohormone levels of Pflan regulate zen, i.e. a local change in the phytohormone game effect gels.

Local changes in phytohormone levels are at a premium flow of the properties of lock cells particularly interesting sant: during z. B. Auxins cause the column to open  an application of abscisic acid to rapid cleavage Enough. Numerous genes, their products in the hormonal balance intervene have already been cloned (see, inter alia, Klee, H. & Estelle, M. (1991) Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 529-551). Genes 1 and 2 of T-DNA from Agrobacterium tume faciens code for auxin synthesis enzymes, while the indolace tat-lysine synthetase (Romano et al. (1991) Genes & Development 5: 438-446) catalyzed the inactivation of auxins. Cytokini ne by one of the rol c gene of the T-DNA from Agrobacterium rhizogenic encoded glucosidase released (Estruch et al. (1991) EMBO J. 10: 3125-3128); the gene product of the T-DNA gene 6b of A. tumefaciens reduces cytokinin activity (Spanier et al. (1989) Mol. Gen. Genet. 219: 209-216). Ethylene production can be expressed in plants by expression of the aminocyclopropane carboxy lat synthase (Huang et al. (1991) Proc. Nat. Acad. Science 88: 7021-7025); an inhibition of aminocyclopro pancarboxylate oxidase lowers by expression of antisense RNA in contrast, the release of ethylene.

Because of the far-reaching consequences of hormonal effects the plant influences the lock cell speed in the described way only by using the inventions DNA constructs and expression vectors for a lock cell-specific gene expression feasible.

In addition, a change in stomatal properties can also be brought about by modulating regulatory components in the guard cells. Possible regulatory components include: transcription factors, protein kinases and protein phosphatases, farnesyl transferases, enzymes of the phosphatidylinositol metabolism, enzymes of the metabolism of cyclic ADP-ribose or proteins whose activity and specificity are regulated by calcium, for example proteins, the EF - Wear hands or C ₂ domains. Modulation and, in particular, a change in the concentration of regulatory components in the guard cells makes it possible to change complexly inherited properties. For example, constitutive overexpression of the transcription activator CBF1 in transgenic Arabidopsis plants has recently resulted in the production of plants with increased cold tolerance. Arabidopsis plants that express CBF1 to a greater extent show an increased transcription compared to unchanged plants of several genes regulated by CBF (Jaglo-Ottosen et al., 1998). By means of the recombinant promoters according to the invention, an increased expression of CBF1 in the guard cells of plants can be brought about. The targeted changes of stomatal properties explained above, which are made possible by the present invention, are not only of importance for wild-type plants, but also for plants which have already been genetically modified or modified by classical mutation with respect to other parameters. These other mutation changes can, among other things, affect changes in the metabolism of the plant, for example those changes which lead to a reduced or increased synthesis of gaseous substances or their precursors. A modified transpirati on (water release in the form of water vapor) of a resulting transgenic plant can e.g. B. by combining a modified stomatal opening with a suitably modified Wassertrans portability can be achieved via the xylem. In this way, an increased stomatal opening could be coupled with an improved xylem water transport capacity for an increased water release. An increased drought tolerance of plants could, for example, be combined with a reduced stomatal opening with roots that go deeper into the ground. Modified xylem or root parameters could be the result of such a further genetic modification.

By means of the recombinant lock cell sp specific promoters and of these comprehensive constructs but also by expression more appropriate in the lock cells reporter genes introduced for the first time, the Sta cellular concentrations of ions or metabolites  agree, i.e. information about cellular conditions within to get from lock cells. As an example of a reporter gene, which is a determination of the cellular concentration of ions or Metabolites, is called Equorin, which informatio provides information about the content of calcium ions in a cell. Such constructs can accordingly for example Determination of cellular concentrations of ions or metabolites in lock cells of plants under certain environmental or Stress conditions such as dry stress or pathogen infestation are used become.

Another application of the recom binant lock cell-specific promoters and which includes them send constructs lies in the lock cell-specific expressi on other proteins, such as antibodies, or of ribozymes that if necessary, about their immediate effect within the locking cells indirectly also influence the gap opening characteristics of the Can exercise lock cells.

Another application for the invention DNA constructs consists in the use of such a DNA Construct in which the coding nucleic acid section contains a Reporter protein encoded in a process for producing lock cell-specific promoters with a compared to that in activity contained in the construct promotes activity in Locking cells through mutation.

In an embodiment of such a procedure mutations into an inventive DNA construct in vitro managed, which preserve the lock cell specificity as possible, however, the level of expression contained in the DNA construct nen. Promotors should change. To maintain the Ensuring guard cell specificity is especially important for this Care should be taken that the nucleic acid sequence motifs of the Core elements TAAAG and / or AAAAG or the core elements with too the said sequences of complementary nucleic acid sequence  the mutation is not changed or that its number in the Promoter is at least not decreased. It can be assumed that in particular the Se flanking the core elements mentioned sequences within the DNA constructs according to the invention by Mu tation (exchange / insertion / deletion of individual nucleotides or smaller groups of nucleotides) can be changed that the resulting constructs in particular have a stronger Ex pression of coding Se contained in these constructs enable sequences. It can abge in this by mutation constructs directed the mutant cis-active element as a single element, but possibly also in several copies, d. H. as Multimer available. Aside from evidence of an intensified gene expression by the mutant DNA constructs in Pflan zen, what as coding sequences in the mutated DNA con preferentially construct genes for suitable reporter proteins, e.g. B. GUS, luciferase, GFP ("green fluorescence protein"), conditional lethal gene products can also be used as alternatives after cloning the transcription factors attached to the in the cis contained in the mutant starting construct according to the invention bind active element, in vitro using the mutated DNA Constructs are searched for the optimal binding site. On cis-active element that improves in this in vitro test te, d. H. stronger binding of the specific transcription factors, should also increase activity in vivo and consequently increased gene expression over that of this cis mediate active element comprehensive promoter.

Alternatively, a plant with a ß DNA construct in which the coding nucleic acid ab cut a reporter protein to be encoded, transformed and a transgenic plant line of a mutagen produced in this way se, z. B. by chemical reagents such as EMS, high-energy Radiation or by means of a T-DNA or transposon insertion, be subjected. The resulting mutants can then owing to a change in the expression level of the reporter pro teins are analyzed. The change in expression level  ke can indicate a mutation of the closure according to the invention cell-specificity-imparting cis element itself or else to a mutation of the transcript binding to this cis element tion factor or several such factors. It can also be used to change the level of expression responsible mutation in others present in the promoter NEN cis elements or in transcription factors and / or other factors that specifically bind to these cis elements. In addition, a relevant mutation can also, for example in the promoter region of the genes for the transcription factors or the other binding factors are that lead to a changed Transcription of these genes and thus to a higher or lower rigorous concentration of these transcription factors and others Factors in the cell leads. The relevant mutations within of the promoter contained in the DNA construct according to the invention areas that are of particular interest here are at easily determined by sequencing, for example.

An additional area of application of the invention DNA constructs lies in the use of a DNA construct, in which is controlled by the promoter according to the invention coding nucleic acid section encodes a reporter protein, in a method to detect the presence of mutants in the Signaling pathway for lock cell specificity in a lock cell after mutation of a parent cell that contains the DNA construct comprises, or a plant or a part of a plant, the or whose cells comprise the DNA construct, and optionally for Identify one for a change in signaling Pathway mutation responsible for guard cell specificity.

This is done first through transformation or transfection with a DNA construct according to the invention, in which the by the nucleotide coding for the promoter according to the invention acid section encodes a reporter protein, a transformed Guard cell, a transgenic plant or a transgenic plant part, which are explained in more detail below  is also objects of the invention. In the transformed guard cell or the transgenic plant or the transgenic part of the plant is then the lock cell speci fish or lock cell additive expression of the reporter gene proven. These biological starting systems then become egg ner mutation, e.g. B. by means of chemical substances such as EMS, ener greedy radiation, such as X-rays, or T-DNA or Subject to transposon insertion. The mutant cells obtained len, plants or parts of plants are then in terms of Expression level and cell specificity of expression of Re portergens examined. The results obtained are with NEN of the original cells, plants or parts of plants in front of the Mu tion compared. A change, d. H. Increase or decrease expression level of the reporter gene and / or a ver change in the cell specificity of the expression of the reporter gene, d. H. a loss of expression of the reporter gene in closing cell len, which may also occur with a first after the mutation tendency of expression of the reporter gene in another plant cell type or plant tissue can be connected, allow egg ne first statement that the mutated cells, plants or parts of plants potentially mutants in the signaling pathway for Could represent guard cell specificity.

As components of the signaling pathway for lock cells zifität, their change by mutation to the Ver Changes in the expression level and / or the cell specificity of the Expression of the reporter gene can all be cellular Elements to be mentioned in the mediation of the lock cell spec gene expression are involved, d. H. for example that special cis-active element itself and that to this cis-active Element-binding transcription factors, but also the promo Gate regions of the genes of these transcription factors. One poses only a change in the expression level of the closing cell-specific expression of the reporter gene must first be excluded that these are due to mutations of components of the promoter / reporter gene contained in the DNA construct  are to be traced back to the lock cell specificity of the Genex pression are not relevant. This can e.g. B. by sequencing the promoter and possibly also the reporter gene area and / or in particular in particular also easily proven by suitable backcrossing become. In this way, i.e. H. sequencing can also be used Modification / mutation of the responsible for the lock cell specificity literal cis-active element, which in the here described Procedures of central interest can be easily identified. The determination of others on the signaling pathway for locking cells specificity of components involved, which may ver changes and thereby the expression level and / or cell spectrum the expression of the report gene against one is not mutated starting system can change by means of the expert known methods, such as "chromosomal walking" or recloning of T-DNA or transposon-tagged genes.

Another area of application of DNA Constructs in which the coding nucleic acid section Coded reporter protein lies in their use in a ver drive to identify chemical compounds that the Signaling pathway for lock cell specificity in a lock affect cell. In such a procedure again transgenic plants or parts of plants which have an inventive DNA construct included, with chemical compounds to be tested gene, e.g. B. herbicides, treated and then with regard to Expression level and cell specificity of expression of Re portergens examined. The results obtained are with who, when examining the plants or Plant parts before treatment with the chemical substances have been compared. A change, d. H. Increase hung or decrease the level of expression of the reporter gene and / or a change in the cell specificity of the expression of the Reporter gene, d. H. a loss of the guard cell specificity of the Re portergen expression, again allow conclusions to be drawn about egg  ne potential influence on the signaling pathway for locking cell specificity due to the chemical substance.

Regarding possibly through the chemical substance influenced components of the signaling pathway for locking cells specificity is referred to the above explanations. For a final clarification of the effects of chemical ver Binding must also be excluded in a further step be that for the changes in the signaling pathway for Locking cell specificity responsible interaction (s) of chemical compound with components of the in the DNA Construct contained promoter and reporter gene for the Locking cell specificity of gene expression are not relevant, he said follows or take place.

In the process mentioned, the chemical compound also in the form of compositions of two or more chemical Compounds, in liquid, solid or optionally also gas shaped for the investigation.

The method is particularly suitable for egg screening ner variety of substances to be tested, for example in Form of a substance library ("synthetic combinatorial library "), as for example in EP 0 734 530, EP 0 816 309 and EP 0 818 431 can be provided. On this way z. B. who identified new herbicide targets the.

In addition, special DNA Constructs in which the coding nucleic acid section Encoded reporter protein that allows the status to be more cellular Determine concentrations of certain ions or metabolites use in a procedure for the detection of the presence of mutants in the signaling pathway for generation and / or regulation of intracellular concentration ions or metabolites in a guard cell after a muta tion of a parent cell comprising the DNA construct, or egg  ner plant or a part of a plant, its cells comprise the DNA construct and, if necessary, for identification to change the signaling pathway responsible mutation.

As with the immediately preceding procedure, here for transformed by a DNA construct according to the invention Guard cells, transgenic plants or transgenic parts of plants manufactured. In the present method, the code in the contained ko construct used for the transformation area a special reporter protein that allows the status of cellular concentrations of certain ions or To determine metabolites. An example of such a special The reporter protein is an equorine that contains information about the Calcium content in a cell provides. In the transformed Guard cell, the transgenic plant or the transgenic plant The concentration on the one or the other then becomes part Ion (s) or metabolite (s) by means of the lock cell-specific or closing cell additive expressed reporter protein - in the Rule in lock cells - determined. The biological output sy Then, as described above, systems become mutagenesis subject. In the mutant cells, plants or Plant parts will now again the concentration on the or certain ion (s) or metabolite (s) using the reporter pro teins determined and the results obtained are with those of the original cells, plants or parts of plants before the mutation compared. A change, d. H. Increase or decrease the ion or metabo determined by means of the reporter protein lit concentration enables a first statement that that the mutant cells, plants or plant parts potentially Mutants in the signaling pathway for generation and / or Regulation of the intracellular concentration of these ions or Could represent metabolites.

In order to confirm this presumption, however, in this case a mutation within the Si gnalling pathway for guard cell specificity, d. H. u. a. of the promo  Gate section of the DNA construct, or the binding to it Transcription factors such as the promoter sections of these ko genes are excluded. Appropriate measures here have been described above or are known to the person skilled in the art. If this can be ruled out, the determination of the Si gnalling pathway for the generation and / or regulation of intracellular concentration of these ions or metabolites be involved components that have been changed by mutation and thereby to a changed concentration on the or certain ion (s) or metabolite (s) to one not lead mutated starting system, again by means of the expert known methods, such as "chromosomal walking" or recloning of T-DNA or transposon-tagged genes.

DNA constructs according to the invention in which the coding Nucleic acid section encodes a reporter protein, which it does leaves the status of cellular concentrations of certain ions or to determine metabolites, also find Ver use in a process for the identification of chemical Substances that have the status of cellular concentrations at these Change ions or metabolites. In such a process are transgenic plants and parts of plants that the Invention contain appropriate DNA constructs, with chemical to be tested Connections, e.g. B. herbicides treated, then the Konzentra tion on the specific ion (s) or metabolite (s) in the lock cells of the plants or parts of plants by means of Reporter protein determined and the results obtained with ent speaking results by the plants or plant part len before treatment with the chemical compounds have been compared. Will there be a change in ion or Metabolite concentration after chemical treatment must, however, still be excluded for a final clarification sen that a for the measured change in concentration Interaction of the chemical compound to be tested with the DNA construct and in particular with the promo contained therein  gate area, the expression of the reporter protein, by means of of which the ion or metabolite concentration is determined controls, is responsible. Suitable measures for this are Known specialist.

The chemical compound to be examined can also be in Form of compositions of two or more chemical Compounds, in liquid, semi-liquid, solid or given also used in gaseous form for the investigation become.

The method is particularly suitable for egg screening ner variety of substances to be tested, for example in Form of a substance library ("synthetic combinatorial library "), as for example in EP 0 734 530, EP 0 816 309 and EP 0 818 431 can be provided. On this way z. B. who identified new herbicide targets the.

DNA constructs according to the invention in which the coding Nucleic acid section encodes a reporter protein, which it does leaves the status of cellular concentrations of certain ions or to determine metabolites are also used in a procedure for investigating an influence on the Status of cellular concentrations at these ions or Metaboli due to environmental influences such as dry stress or pathogen infestation. In such a process, transgenic plants and plants parts that contain the DNA constructs according to the invention, subject to the environmental impact to be examined and then the Concentration on the specific ion (s) or metabo lit (en) in the guard cells of the plants or parts of plants determined by means of the reporter protein. The results obtained will be back with appropriate results from the Pflan zen or parts of plants before exposure to the environment have been compared. If a change in the ion or metabolite concentration after exposure to the environment This should also be final clarification  be excluded that for the measured concentration ver change that may be due to the environmental impact in its activity changed DNA construct and in particular the contained in the promoter region, the expression of the report terproteins, by means of which the ion or metabolite concentrations tration is determined, controls, is responsible. For this too Suitable measures are known to the person skilled in the art.

In addition, the invention also relates to vectors, plasmi de, cosmids, bacmids, virus and phage genomes, which invent a DNA construct according to the invention, as explained above, integrated in their nucleic acid molecules and trans formed microorganisms which are treated with a DNA Construct were transformed. Regarding the output vectors, plasmids, cosmids, bacmids, viruses or phage genomes, in which a DNA construct according to the invention for generating the inventions vectors, plasmids, cosmids, bacmids, virus or Phage genome is integrated, there is no restriction. The term "microorganism" means in this context u. a. Bacteria, bacteriophages, viruses such as also eukaryotic organisms and cells, such as fungi, yeasts, pro tozoen, algae and human, animal and plant cells. A variety of techniques and methods of performing the Process according to the invention and generally for the production of recombinant promoters and constructs according to the invention starting from an output promoter or promoter elements such as Cloning, transformation of microorganisms, DNA preparation and analysis, are well known to those skilled in the art and can be found in for example from standard works such as Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor New York, USA, and a huge number of in the one relevant journals published individual protocols be removed. Likewise, for the execution of this techni Reagents such as buffers, vectors and  similar carrier systems for foreign DNA, such as plasmids, the esp special additional so-called marker genes for the detection of an er consequent transformation of a host organism with this Trä gene system, host organisms such as viruses, bacteria, He fen, fungi or eukaryotic cells known to those skilled in the art and available to them from a variety of sources or can even use generally available and established ones Protocols are made. The same applies to the manufacturer treatment or generation of the vectors, plasmids according to the invention, Cosmids, virus or phage genomes and transformed microor mechanisms.

The invention further relates to transgenic plants, plants parts, plant cells or seeds which contain a DNA Construct included. The DNA construct can be inserted into the chromo som of the host plant or host plant cell integrated or ex trachromosomal. The terms "plants, parts of plants and plant cells "include any propagation material from Plants, such as plant protoplasts, plant cells, plants zen tissue, e.g. B. callus tissue, developing plantlets, Leaves, unripe whole plants and ripe whole plants. He Plants according to the invention include all monocotyledons and dicotyledons Plants, and in particular crops and crops, such as Kartof fel, tobacco, tomato, sugar beet or cotton, agricultural auxiliary plants, trees and shrubs, and ornamental plants.

For the production of the transgenic plants according to the invention, Plant parts and seeds come not only from wild-type plants, also plants that are already in relation to other Pa parameters modified genetically or by classic mutation were considered. These other mutation changes can among other things, changes in the metabolism of the plant meet, for example, such changes that lead to a ver reduced or increased synthesis of gaseous substances or of their precursors. Reduced perspiration (Water release in the form of water vapor) a resulting  transgenic plant can e.g. B. by combining a reducer stomatal opening with a reduced water transport capacity ability to be achieved through the xylem. For an enhanced Water delivery would allow an increased stomatal opening coupled with improved xylem water transport ability. A increased drought tolerance of plants could be caused by a ver wrestled stomatal opening with deeper down into the ground roots. Modified Xylem or Wur Individual parameters could be the result of such a further gene technical modification.

Various techniques are used to produce transgenic plants ken and methods for cloning the corresponding DNA construct te, for the construction of suitable transformation vectors and for Introduction of the genetic information contained in the vector tion in a plant cell known to those skilled in the art. The techniques for introducing genetic information into a plant cells include direct DNA transfer (e.g. in protoplasts by means of electroporation or by using an osmotic Using high molecular weight as well as biolistic methods, where DNA coated particles in plant tissue be shot) as well as the use of natural Host / vector systems (e.g. from agrobacteria or plants viruses). For permanent extrachromosomal localization and Maintenance of the DNA constructs can be speci fish viruses, such as the tobacco mosaic virus (TMV) or potato Virus X, in whose genome the DNA constructs in this case be used.

The invention further includes a kit especially for ver Application in one of the above-described inventive Method, which is characterized in that he invented DNA construct according to the invention or a vector according to the invention or a plasmid, cosmid, bacmid, virus or Phage genome includes. The kit can also contain suitable buffers  and other reagents and enzymes for manipulating the con strukts or the vector or other type of carrier system, such as also, in the case of a DNA construct without the coding already inserted Sequence, reagents for cloning a user to be include tuning gene. In addition, the kit can be added a carrier system, such as a vector and the other systems described above, for an integra tion of the DNA construct and possibly also host microorganisms Recording and possible transfer of the carrier system with the DNA Include construct. The Ti plasmid and Agrobacterium tumefaciens host organisms are called can be used to transform plants. This additional reagents and organisms can be separately or also in the form of a combination of certain components in the kit will see.

Furthermore, the identification of the for a guard cell-specific expression of essential sequence motifs TAAAG or AAAAG or complementary sequence motifs as Characteristic of such a cell-specific expression another new method of identification Decoration of previously unknown, in lock cells from Pflan zen expressed genes. This procedure is already in use Available or future genomic DNA Sequences 5'-regulatory areas back and analyzed the se for the presence of identified, for lock cell specific cis-elements characteristic sequence motifs TAAAG or AAAAG and / or the sequence motifs with the specified nu small acid sequences complementary nucleic acid sequence. Dement speaking, the invention further comprises a method for identi detection of genes expressed in plant guard cells, in which by nucleic acid sequencing of 5'- regulatory areas of gene-derived nucleic acid sequence data on the presence of at least one of the for regulatory, a lock cell-specific gene expression ver  average cis-active element characteristic nucleic acid sequence motifs TAAAG and / or AAAAG and / or each of them complementary nucleic acid sequence motifs checked. The review fung can be done computer aided by looking at programs served for sequence homology comparisons, or can also be personal Lich carried out by a processor who by Nu Sequences of small acid obtained on the above Looks through nucleic acid sequence motifs.

The check for the two or more fa is preferably carried out che presence of the nucleic acid indicated above reference motifs within a 5 'regulatory area.

After identifying a gene as a potential candi data for a lock cell-specific expression should be Promoter range then based on functionality and cell specificity Fusion of the promoter area with a reporter gene and detection a lock cell-specific or additive expression of the Re portergens after stable or transient transformation of Plants or guard cells tested with the fusion construct become. The method according to the invention can thus be used for preselection tion of promoters or genes active in guard cells be set.

The invention will now be described with reference to the following Figures and examples that merely illustrate the invention Chen and should not limit, further described.

Examples

One for the lock cell-specific expression in plants essential area and two contained therein, for the lock cell-specific expression essential nucleic acid quenz motifs that form the basis of the processes and methods claimed here Could be formed by a deletion analysis of the kstl promoter from potato (Solanum tuberosum) identified become.  

After determining an area which is essential for the lock cell-specific expression in potato and which has approximately 83 nucleotides, as in SEQ. ID. NO. 1, included, but it is not to be said that this region is only essential in its entirety for lock cell-specific expression, three overlapping, double-stranded oligonucleotides representing this region (AB (nucleotides 1-40), CD (nucleotides 41-83) and EF (nucleotides 23-62); SEQ. ID. NO. 2, 3 and 4; Fig. 2). These were tetramerized and fused to a mini-promoter (Δ35S), which was not active even in guard cells, and which was linked to a reporter (GUS) within the construct. The starting region comprising 83 nucleotides was also fused in a corresponding manner in tetrameric form (“83 nt”). With these constructs, plants were stably transformed using agrobacteria and the transgenic plants were examined for reporter gene activity (GUS activity). For the tetramer of the 83 nucleotide output region ("83 nt") as for the tetramer AB, a strong expression for the tetramers CD and EF could be demonstrated in lock cells ( FIG. 1).

The non-overlapping functional sequences AB and CD both have the sequence motif TAAAG, the sequence CD also has a further element with the sequence AAAAG ( FIG. 2). The functional significance of the TAAAG motif was demonstrated by the introduction of a block mutation (CGCGA) at this point in the context of the construct AB × 4 (= AB tetramer). This change led in two independent experiments (ABmut × 4 and ABmutN × 4) to a drastic decrease in the lock cell-specific expression ( FIG. 3). As a further comparison, a tetra mer of the 83 nucleotides starting area (“90 × 4”) was taken in FIG .

The functionality of the motifs TAAAG and AAAAG was also demonstrated in the context of the original promoter shortened to 240 nucleotides (Kst240), the nucleotide sequence of which is shown in FIG. 5 and in SEQ. ID. NO. 5 is specified. As can be seen from Fig. 5 ent, it includes near the 5 'end in SEQ. ID. NO. 1 specified range of 83 nucleotides, which he had shown to be essential for the lock cell specificity of the starting promoter. In addition, it essentially only comprises the putative CCAAT box, the putative TATA box and the transcription initiation site of the original or original pro motor kstl from Solanum tuberosum. In FIG. 4, of the Kst240 promoter, only the relevant region from nucleotide 14 to nucleotide 60 from the one shown in SEQ. ID. NO. 1 indicated section of 83 nucleotides shown. The sequential mutation of the motifs TAAAG and AAAAG to the sequence CGCGA (Kst240mut1, Kst240mut2 and Kst240mut3) in turn leads to a significant decrease in the lock cell-specific activity ( FIG. 4).

example 1 Cloning of the promoter region of the potato kst1 gene

A genomic library of the nuclear DNA of a monohaploid Line of Solanum tuberosum L. (Liu et al., 1991) was established with ei a Sacl fragment of the kstlcDNA (Müller-Röber et al., 1995) and the stringent conditions. By restrictionana lysis and stringent hybridizations using Tei len of the kst1 cDNA as a probe could be shown that the clone L # 7.5 approx. 4.5 kb sequence upstream from the kst1 start codon held, which then as a SalI / SacI fragment in the same ge cut vector pBluescriptSKII (Stratagene) was subcloned de. The sequence of the kst1 promoter region was up to position 1909 upstream of the start codon of the kst1cDNA by sequencing determined (Ehrhardt, 1998).  

Example 2 Identification of the for the lock cell-specific expression of the kst1 gene relevant promoter region

To analyze the regulatory region of the kstl gene, two differently long promoter fragments were transcriptionally fused with the β-glucuronidase reading frame from E. coli (Jefferson et al., 1987). At the position of the kstl start codon, an SmaI interface was introduced by means of PCR with the primer KstPSmaI ( 5 '-TTCCCCCGGGTATTATATATTGCTGCTTC) (construct PVP2, Ehrhardt, 1998). For the transcriptional fusion to the β-glucuronidase reading frame, a 3 kb BamHI / SmaI and a 1.5 kb EcoRV / SmaI promoter region were cloned into the vector pBin 101.1 (Clontech) cut with BamH / SmaI or SmaI and the correct orientation checked by test verdau. The constructs obtained in this way were designated pDKP1 (3 kb promoter) or pDKP3 (1.5 kb promoter). The constructs were transferred to the Agrobacterium strain GV 2260 (Deblaere et al., 1985), which was then used for gene transfer to potatoes. Transgenic potato plants were obtained by selective regeneration on kanamycin-containing medium. Expression of the reporter gene could be detected for both constructs in approximately 1/3 of the cases by detection of the β-glucuronidase activity (conversion of the colorless substrate X-Gluc to a blue precipitate) in guard cells. Accordingly, the 1.5 kb regulatory sequence of the kst1 gene contained in the construct pDKP3 are sufficient for the generation of lock cell-specific expression (Ehrhardt, 1998). Reporter gene activity was evident in all lock cells on the top and underside of sink and source leaves as well as on the stems through blue staining. A comparable lock cell specificity for tobacco and Arabidopsis could be demonstrated for this construct.

Example 3 Narrowing down the regulations relevant for the specificity of the guard cell toric areas by a promoter deletion analysis

By means of a step-by-step deletion analysis using existing restriction interfaces, the region relevant for the closing cell specificity could be limited within the 1.5 kb regulatory sequences ( FIG. 1). The construction of the constructs pDKPl and pDKP3 has already been described above. For the construct KstBclI900, an approximately 900 bp long BclI / Smal fragment was isolated from the construct PVP2 and cloned into the vector pBI101.1 cut with BamHI / SmaI. Correspondingly, the constructs KstEcoRI650 and KstBg1II300 were prepared by cutting the PVP2 with EcoRI / SmaI (EcoRI interface subsequently "blunted") or BglII / SmaI and cloning into the vector pBI101.1 cut with Smal or BamHI / SmaI. The constructs were, as described above, transferred to potato plants using Agrobacterium. The reporter gene activity was detected by incubation with the synthetic substrate X-Gluc and the number of plants with strong, medium and weak expression was determined. The further shortening of the regulatory range in the constructs Kst240 (position -239, relative to the start codon of the kstl gene) and Kst200 (position -193) was carried out using the primers Kst240dw (5'-GCAGATCTGCAAGTAGCAATGTCACG-3 ') and KstPSmaI or Kst200dw (5'-CGGGATCCGAATCACAATAAAGAAACAC-3 ') and KstPSmaI using the clone p7-Ssub7 (Ehrhardt, 1998) as a template. The fragments cut with BglII / SmaI or BamHI / SmaI were cloned into the vector pBI101.1 cut with BamHI / SmaI. While the deletion Kst240 retained the lock cell activity, the further shortening to position -193 (construct Kst200) led to a drastic reduction in reporter gene activity. Accordingly, there are relevant sequence motifs for the lock cell expression in the described 239 bp long regulatory area and at least partially localized in the area between positions -239 and -193 (Figure 1).

Example 4 Description of short regulatory areas for the Mediation of lock cell-specific activity sufficient are

In order to define short regulatory areas in the kst1 promoter that are sufficient for mediating lock cell-specific activity, relevant parts of the kstl promoter were tetramerized in the form of PCR products or double-stranded oligonucleotides for effect enhancement (Gosh et al., 1993) and fused to a -56 shortened version of the 35S promoter (Froh berg et al., 1991) as a minimal control unit (minimal promoter). For the cloning of the tetramer 90 × 4, the 83 bp long region between positions -239 and -153 was amplified with the primers Kst240dw and Kst150 (5'-GGGTGATACACGGGTCAAG-3 ') and inserted into the SmaI interface of the vector pKSII (Strata gene) cloned. The clone of correct orientation obtained (Kst240dw primer facing the PstI interface) was cut with BamHI / ScaI or BglII / ScaI and the fragments each containing the PCR product were ligated together. A tetramer was produced from the dimer thus obtained using the same principle. This was isolated as a HindIII / SpeI fragment and together with an approximately 2 kb-sized SpeI / SacI fragment (355 minimal promoter plus the section coding for the β-glucuronidase) from the vector pDoAHpaGus (Frohberg et al., 1991) in the cloned with HindIIII / SacI cut vector pUCGusA (van de Löcht et al., 1990). The entire cassette of tetramer, mini malpromotor and reporter gene was then obtained from this construct as a HindIII / SacI fragment and cloned into the corresponding interfaces of the vector pBI101.1. In this way it could be demonstrated that an 83 bp region between positions -239 and -153 is sufficient for the mediation of lock cell-specific activity. This area was further broken down into three overlapping fragments of approx. 40-45 bp and examined separately. To this end, the oligonucleotides were KStA (5'-CCGGGCAAGTAGCAATGTCACGTTCTTAAAGCTAAATGCTTTTT-'3 ') and kstb (5'-CCGGAAAAAGCATTTAGCTTTAAGAACGTGACATTGCTACTTGC-3') or KSTC (5'-CCGGAAAAGAATCACAATAAAGAAACACTTGACCCGTGTATCAC-3 ') and KSTD (5'-CCGGGTGATACACGGGTCAAGTGTTTCTTTATTGTGATTCTTTT-3') or KstE (5'-CCGGAAAGCTAAATGCTTTTTCAAAAGAATCACAATAAAGAAA-3 ') and KstF (5'-CCGGTTTCTTTATTGTGATTCTTTTGAAAAAGCATTTAGCTTT-3') phosphorylated, hybridized with each other and ligated to multimers. These were then trimmed with NaeI / MroI (AB), SmaI / MroI (CD) and PinAI / MroI in order to obtain only fusions of the correct orientation. Subsequently, fragments were isolated according to the tetramer size and ligated into the XmaI (corresponds to the SmaI interface, only cut with another enzyme) interface of the vector pKSII. Clones of the desired orientation and correct sequence were used for the production of the fusion with the minimal promoter and the reporter gene according to the strategy given above. Since different cell strengths were found for all three tested tetramers, at least two independent elements can be assumed in this area for the cell expression. ( Fig. 2)

Example 5 Identification of a short sequence motif for the activi must be present in lock cells

In the two active and non-overlapping sequences of approx. 45 base pairs (AB and CD, see Fig. 2), the nucleotide sequence 5'-TAAAG-3 'could be identified as a conserved and thus possibly functional element. A change in this sequence to the nucleotide sequence 5'-CGCGA-3 'in the context of the active tetramer AB × 4 by appropriately modified versions of the oligonucleotides KstA or KstB led to a drastic reduction in the lock cell-specific activity ( Fig. 3). The cloning and functional analysis were carried out as already described above.

It can be concluded that the sequence sequence described 5'- TAAAG-3 'a central function for the switching of closing has cell-specific activity.

Example 6 Confirmation of the functionality of the TAAAG motif in the context of the shortened kst1 promoter (-239)

The shortened to 239 bp, still active version of the kstl promoter has in the characterized region up to position -153 two TAAAG nucleotide sequences and another similar, possibly also active AAAAG motif. In order to demonstrate the functionality of these elements in the context of the shortened kstl promoter, they were gradually converted into the non-functional sequence sequence 5'CGCGA-3 'by means of PCR. The primers St240mut1 (5'-GCAGATCTGCAAGTAGCAATGTCACGTTCTCGCGACTAAATGC-3 ') and KstPSmaI were used for the construct Kst240mut1. A "megaprimer" strategy was used for the other constructs, in which a PCR product is used as a primer in a subsequent PCR. For the construct Kst240mut2, the primers St240mut1 and Kst240mutrev (5'-TTGTGATTTCGCGGAAAAAGCATTTAGC-3 ') were used and the product obtained was used as a primer for amplification with the primer KstPSmaI. For the construct Kst240mut3, the primer Kst240mut2rev (5'-CAAGTGTTTTCGCGTTGTGATTTCGCGGAAAAAGCATTTAGC-3 ') replaced the primer Kst240mutrev. The original genomic clone p7-Ssub7 served as the template. The products were each cut with BglII / SmaI, cloned into the vector pUCGusA cut with BamHI / SmaI and sequenced. The entire cassettes were in turn cloned as HindIII / SacI fragments in the binary vector pBI101.1 and the construct thus obtained was used for the transformation of potato plants. The introduction of these block mutations led to a gradual reduction in the lock cell-specific activity. For the construct Kst240mut3, in which all three motifs were changed, almost no closing cell activity could be detected ( FIG. 4).

Description of the figures

Fig. 1: Deletion analysis of the kstl promoter from potatoes and identification of a total of three short, overlapping sequence elements of 40, 43 and 39 base pairs (AB, CD and EF) which, after fusion with a self-inactive minimal starter region (35S minimal promoter), are specific to the closing cell Arrange activity. The promoter-reporter gene fusions shown were cloned into a binary vector (pBI101.1, Clontech), and potato cells were transformed by means of gene transfer mediated by agrobacteria. Reporter gene expression in guard cells was determined by enzymatic detection of reporter gene activity (GUS activity).

Fig. 2: sequence able to convey the non-overlapping fragments AB and CD from the KSTL promoter, the cell-specific closing by fusion to the 35S minimal promoter gene expression. The sequence motif TAAAG occurring in both elements is highlighted ben.

Fig. 3: Evidence of the importance of the motif TAAAG for the lock cell-specific expression in the context of minimal promoter fusions. The lock cell-specific activity of various promoter constructs is shown as a percentage of plants with reporter gene activity in lock cells. This shows a drastic reduction in activity if the TAAAG element is replaced by a block mutation (CGCGA) (construct ABmut × 4 or by an independent repetition of the experiment) in the context of the tetramer AB × 4 (tetramer of AB) Production of the construct ABmut × 4, construct ABmutN × 4).

Fig. 4: Evidence of the meaning of the motifs TAAAG and AAAAG in the context of the shortened original promoter Kst240. The successive replacement of the motifs TAAAG and AAAAG by the block mutation CGCGA in the constructs Kst240mut1, Kst240mut2 and Kst240mut3 leads to a drastic reduction in the lock cell-specific activity of the promoter.

Fig. 5: Nucleotide sequence of the original promoter Kst240. The position of the sequence elements AB, CD and EF, the putative TATA box and the start codon is given within the nucleotide sequence.

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Claims (27)

1. Regulatory cis-active element that at least one of the Nucleic acid sequences TAAAG and / or AAAAG and / or at least a Se which is complementary to the nucleic acid sequences mentioned quenz includes and in the context of a promoter a lock cell spec gene expression mediated. 2. Regulatory cis-active element according to claim 1, characterized characterized in that there is essentially at least one of the in SEQ. ID. NO. 1, 2, 3 or 4 specified nucleotide sequences sums up. 3. Regulatory cis-active element according to claim 1, characterized characterized in that it comprises a nucleic acid sequence derived from the nucleic acid sequence of the regulato defined in claim 2 Rische cis-active element by mutation, especially Dele tion, substitution, addition and / or insertion of individual Nucleotides or smaller group derived from nucleotides is. 4. Use of the regulatory cis-active element according to ei nem of claims 1 to 3 for the identification of transcripts ons factors that bind to this cis-active element. 5. Process for the production of a recombinant promoter for a lock cell-specific or lock cell additive gene expression sion, characterized in that in an output promoter, the no lock cell-specific or lock cell additive gene express  sion causes, or components of such a starting promo tors at least one regulatory, one lock cell-specific Gene expression mediating cis-active element comprising the Nucleic acid sequence (s) TAAAG and / or AAAAG and / or at least a Se which is complementary to the nucleic acid sequences mentioned quenz, added or generated by mutation. 6. The method according to claim 5, characterized in that for Production of the recombinant promoter two to twenty, esp special two to eight cis-active elements, comprising the nu Small acid sequence (s) TAAAG and / or AAAAG and / or at least a Se which is complementary to the nucleic acid sequences mentioned quenz, can be used. 7. The method according to claim 5 or 6, characterized in that that the cis-active element is essentially one of those in SEQ. ID. NO. 1, 2, 3 or 4 specified nucleotide sequences. 8. Method for producing a recombinant promoter with one modified compared to an output promoter, in particular increased activity in guard cells, characterized in that in an output promoter that is a lock cell specific or Closure cell additive gene expression causes at least one regu latent cis-active element comprising at least one sequence mo tiv TAAAG and / or AAAAG and / or at least one of them nucleic acid sequences complementary sequence added or is generated by mutation. 9. The method according to claim 8, characterized in that the cis-active element essentially one of those in SEQ. ID. NO. 1, 2, 3 or 4 specified nucleotide sequences. 10. Method for producing a recombinant promoter with one modified compared to an output promoter, in particular reduced activity in guard cells, characterized in  that in the output promoter, which is a lock cell specific or closing cell additive gene expression causes at least one with the sequence motifs TAAAG and AAAAG or the sequence motifs with the specified nucleic acid sequences of complementary sequence de leased or by block substitution or by deletion, insertion tion and / or substitution of one or more nucleotides is changed. 11. Method for producing a recombinant promoter with an activity changed compared to an output promoter in Locking cells, characterized in that in an output pro motor, which is a lock cell-specific or lock cell additive Gene expression causes the location of at least one sequence motif TAAAG and / or AAAAG and / or at least one sequence motif with a Se which is complementary to the nucleic acid sequences mentioned sequence based on at least one further element of the promoter is changed. 12. Recombinant promoter, obtainable by the method according to one of claims 5 to 11. 13. DNA construct, in particular expression cassette, comprising the recombinant promoter according to claim 12. 14. DNA construct according to claim 13, which also a a nucleic acid segment encoding a gene product, which is operative or is functionally linked to the recombinant promoter, includes. 15. DNA construct according to claim 13, which also has a Nucleic acid section, the transcription of which is a transcription product, e.g. B. a ribozyme or an "antisense" DNA, and the operatively or functionally with the recombinant promoter ties is included.   16. Use of a DNA construct according to claim 14 for expressing sion of a homologous or heterologous gene in guard cells transformed plants. 17. Use of a DNA construct according to claim 14, in which the coding nucleic acid section a reporter protein ko diert, in a process for the production of lock cell specififi promoters with one versus that in the construct maintained promoter changed activity in guard cells Mutation. 18. Use of a DNA construct according to claim 14, in which the coding nucleic acid section is a reporter protein encoded, in a procedure for the detection of the presence of Mu aunts in the signaling pathway for guard cell specificity in one Locking cell after a mutation of an output cell that the DNA construct comprises, or a plant or a plant in part, the cells of which comprise the DNA construct, and, if necessary, to identify one for a change Responsibility for the signaling pathway for guard cell specificity literal mutation. 19. Use of a DNA construct according to claim 14, in which the coding nucleic acid section is a reporter protein encoded in a process for the identification of chemical Connections that define the signaling pathway for lock cell spec influence in a locking cell. 20. Use of a DNA construct according to claim 14, in which the coding nucleic acid section a reporter protein ko that allows the status of cellular concentrations to determine certain ions or metabolites in one process to detect the presence of mutants in the signaling Pathway for the generation and / or regulation of intrazel lular concentration of these ions or metabolites in one  Locking cell after a mutation of an output cell that the DNA construct comprises, or a plant or a plant partly, whose or whose cells comprise the DNA construct, and if necessary to identify one for a change mutation responsible for this signaling pathway. 21. Use of a DNA construct according to claim 14, in which the coding nucleic acid section a reporter protein ko that allows the status of cellular concentrations to determine certain ions or metabolites in one process for the identification of chemical substances that cellular concentrations of these ions or metabolites change. 22. Use of a DNA construct according to claim 14, in which the coding nucleic acid section a reporter protein ko that allows the status of cellular concentrations to determine certain ions or metabolites in one process to investigate an influence on the status of cellular Concentrations of these ions or metabolites by Um world influences. 23. vector, plasmid, cosmid, bacmid, virus or phage genome, comprising a DNA construct according to any one of claims 13 to 15. 24. Transformed microorganism, characterized in that it contains a DNA construct of one of claims 13 to 15. 25. Transgenic plant, transgenic plant part, transgenic plant cell or seed, characterized in that she or he a Contains DNA construct according to any one of claims 13 to 15. 26. Kit especially for use in one of the methods according to one of claims 5 to 11, characterized in that it is a DNA construct according to one of claims 13 to 15 or one  Vector, a plasmid, cosmid, bacmid, virus or phage genome according to claim 23. 27. Method of identifying in lock cells of Plants expressed genes, characterized in that one by nucleic acid sequencing of 5 'regulatory areas nucleic acid sequence data obtained from genes for presence be at least one of those for a regulatory, a closing cell-specific gene expression-mediating cis-active element characteristic nucleic acid sequence motifs TAAAG and / or AAAAG and / or the sequence motifs with the specified nucleus acid sequences of complementary nucleic acid sequence checked.