EP1025250A2 - Modification of the tocopherol content of transgenic plants - Google Patents

Abstract

The invention relates to novel nucleic acid sequences which provide the coding for a geranyl geranyl reductase, to a method for producing novel plants which contain a novel nucleic acid sequence and whose tocopherol and/or chlorophyl content is modified in relation to wild type plants, to these plants and to parts, products and plant cells thereof and to the use of the nucleic acid sequences for influencing the tocopherol, chlorophyl and/or vitamin K1 content of transgenic plants and parts, products and plant cells thereof.

Description

 Influencing the tocopherol content in transgenic plants

The present invention relates to new nucleic acid sequences. which code for a geranylgeranyl reductase, a method for producing new plants which contain a new nucleic acid sequence and whose content of tocopherol and / or chlorophyll has changed compared to wild type plants, these new plants, their parts and products and plant cells and the use of Nucleic acid sequences for influencing the tocopherol, chlorophyll and / or vitamin K, content in transgenic plants, their parts and products and

Plant cells.

The diterpene geranylgeranyl pyrophosphate (GGPP) is produced as a 20 C intermediate in the plant isoprenoid metabolism. It results from the addition of one unit of isopentenyl pyrophosphate (IPP) to farnesyl pyrophosphate, a 15-C-

Sesquiterpene. GGPP flows into various synthetic routes of plant secondary metabolism. For example, two molecules of GGPP "tail to tail" can be joined together to form 40-C bodies, the tetraterpenes, which are generally referred to as carotenoids and which include, for example, ß-carotene. By adding further molecules IPP, GGPP also flows into the biosynthesis of polyterpenes, such as rubber and gutta-percha.

GGPP can also be used in other diterpenes, e.g. Phytyl pyrophosphate (PPP). The 20 C body phytol is a mandatory

Intermediate in the biosynthesis of tocopherols (Soll and Schulz (1981) Biochem. Biophys. Res. Commun. 99, 907-912) and the synthesis of chlorophylls (Beale and Weinstein (1990) In: Biosynthesis of Heme and Chlorophyll (Dailey HA, ed.) McGraw Hill. NY, 287-391). While the basic structure of all chlorophylls (Chlorophyll a, b, c, etc.) is a porphyrin system made up of 4 pyrrole rings, to which the phytol is bound like an ester via the pyrrole ring IV, the tocopherols are characterized by a structure consisting of homogenate and a phytol tail.

The group of tocopherols, commonly referred to as vitamin E, includes a number of structurally closely related fat-soluble vitamins, namely α-, β-, γ-, δ- and £ -tocopherol, of which α-tocopherol is the most biologically important. The tocopherols are found in many vegetable oils, the seed oils of soy, wheat, corn, rice, cotton, are particularly rich in tocopherols.

Alfalfa and nuts. Fruits and vegetables, e.g. Raspberries, beans, peas, fennel, peppers, etc. contain tocopherols. As far as is known, tocopherols are only synthesized in plants or photo-synthetically active organisms.

Due to their redox potential, tocopherols help to prevent the oxidation of unsaturated fatty acids by atmospheric oxygen; in humans - tocopherol is the most important fat-soluble antioxidant. It is believed that the tocopherols, through their function as antioxidants, contribute to the stabilization of biological membranes, since the membrane fluidity is maintained by protecting the unsaturated fatty acids of the membrane lipids. According to recent findings, the formation of arteriosclerosis can also be countered with the regular intake of relatively high doses of tocopherol. A further beneficial physiological property of tocopherols has been described as delaying late-stage damage caused by diabetes, reducing the risk of cataract formation, reducing oxidative stress in smokers, anticarcinogenic effects, protective effect against skin damage such as erythrems and skin aging, etc. - ->

- Because of their antioxidant properties, the tocopherols are not only used in terms of food technology, but also in paints based on natural oils, in deodorants and other cosmetics, e.g. sunscreens, skin care products, lipsticks etc. Tocopherol compounds such as tocopheryl acetate and succinate are the usual forms of application for use as vitamin E, in blood circulation-promoting and lipid-lowering agents and in veterinary medicine as a feed additive.

In the biosythesis of tocopherols, especially α-tocopherol, seems

Phytyl pyrophosphate to be a limiting factor. Previous investigations indicate that PPP arises from sequential hydrogenation of the isoprenoid group from GGPP, with dihydro-GGPP and tetrahydro-GGPP being formed as intermediates (GGPP -> dihydro-GGPP -> tetrahydro-GGPP -> PPP; cf., for example. Bollivar et al. (1994) Biochemistry 33, 12763-12768).

The gradual hydrogenation of GGPP to PPP is currently believed to be catalyzed by the enzyme geranylgeranyl reductase (GGPP reductase, also known as geranylgeranyl pyrophosphate hydrogenase or GGPP hydrogenase), which is encoded in plants in the Chl P gene . The enzyme

Ger.anylgeranyl reductase is part of the isoprenoid metabolism and works for two metabolic pathways: tocopherol synthesis and chlorophyll synthesis.

The essential importance of this enzyme has been shown for the first time in chlorophyll biosynthesis (Benz et al. (1980) Plant Sei. Lett. 19, 225-230; Soll and Schultz (1981) Biochem. Biophys. Res. Commun. 99, 907 -912; Schoch et al. (1977) Z. Plant Physiol. 83, 427-436). The final step in chlorophyll biosynthesis is the esterification of chlorophyllide. which can be done with both phytyl pyrophosphate and geranylgeranyl pyrophosphate. Systematic _ Studies of Rhodobacter capsulatus mutants have shown that bacteriochlorophyllid is first esterified with GGPP and then esterified chlorophyll-GG is hydrogenated (Katz et al. (1972) J. Am. Chem. Soc. 94, 7938-7939). Phytyl chlorophyll (chlorophyll-P) is mostly detected in higher plants (Rüdiger and Schoch (1991) in chlorophylls

(Scheer, H., Ed.) Pp. 451-464, CRC Press, Boca Raton, Florida, USA). It has not yet been clarified with which substrates the reductase reaction takes place in plants. It is currently assumed that the plant geranylgeranyl reductase is able to convert both chlorophyll-GG to chlorophyll-P (Schoch et al. (1978) Z. Vegetable Physiol. 83, 427-436) and to hydrogenate GGPP to PPP, which is then subsequently combined with chlorophyllide (Soll et αl. (1983) Plant Physiol. 71, 849-854).

GGPP serves as a substrate for the synthetic pathways of tocopherol and phylloquinone in chloroplast envelope membranes and for chlorophyll synthesis in the thylakoid membranes. The reduction from GGPP to PPP was first described in 1983 by Soll et αl. (1983, Plant. Physiol. 71, 849-854). So far, however, the isolation and characterization of nucleic acid sequences which code for the plant enzyme and can be used for influencing the tocopherol content in transgenic plants has not been successful.

The essential role of geranylgeranyl reductase in tocopherol and chlorophyll metabolism makes this enzyme a particularly valuable tool for molecular biotechnology. Using molecular biological techniques such as the transfer of DNA sequences that are essential for geranylgeranyl

Encoding reductase, changes in tocopherol and / or chlorophyll biosynthesis performance could be achieved in plants. In this way it would be possible, for example, to produce transgenic plants with an increased or decreased tocopherol content. Such transgenic plants or their parts, cells and / or Products could then be used as food and feed or more generally as a production facility for tocopherol, which is used in the chemical, pharmaceutical and cosmetic industries.

In addition, it can be assumed that plants which have a higher antioxidative tocopherol content than wild type plants also have an increased resistance to stress conditions, in particular to oxidative stress.

It is therefore an object of the invention to provide new nucleic acid sequences by means of which the tocopherol content in plants, plant cells, parts and / or products can be influenced.

Furthermore, it is an important object of the invention to provide transgenic plants, cells, products and parts with a tocopherol content which is different from that of wild type plants.

Another object of the invention is to demonstrate possibilities of using the DNA sequences according to the invention, their gene products and the transgenic plants according to the invention for plant breeding practice.

Further objects of the invention will become apparent from the following description. These tasks are covered by the subject matter of the independent claims, in particular based on the provision of the DNA

Sequences, the gene products of which are directly involved in tocopherol biosynthesis, and the transfer of these DNA sequences to plants resulting in an altered tocopherol content, were solved. The present invention thus relates to DNA sequences which code for proteins with the biological activity of a geranylgeranyl reductase (also called geranylgeranyl pyrophosphate hydrogenase) or a biologically active fragment thereof. In the context of this invention, biologically active fragment means that the mediated biological activity has an effect on the

Tocopherol content is sufficient. In particular, the invention relates to plant DNA sequences which code for proteins with the biological activity of a geranylgeranyl reductase or a biologically active fragment thereof; the invention particularly preferably relates to those in SEQTD NO. 1 specified DNA sequence (see also Fig. 1).

Furthermore, the invention relates to alleles and derivatives of the DNA sequences according to the invention which code for a protein with the biological activity of a geranylgeranyl reductase, in particular nucleic acid molecules, the sequences of which differ from the DNA sequences according to the invention due to the degeneration of the genetic code which code for a protein or a fragment thereof which has the biological activity of a geranylgeranyl reductase.

The invention further relates to nucleic acid molecules which contain the DNA sequences according to the invention or which have arisen from, or have been derived from, naturally occurring or by genetic engineering or chemical processes and synthesis processes. This can be, for example, DNA or RNA molecules, cDNA, genomic DNA, mRNA etc.

The invention also relates to those nucleic acid molecules in which the DNA sequences according to the invention are linked to regulatory elements which ensure transcription and, if desired, translation in the plant cell. The DNA sequences according to the invention can be expressed in plant cells, for example, under the control of constitutive, but also inducible or tissue- or development-specific regulatory elements, in particular promoters. While, for example, the use of an inducible promoter is the specifically triggered expression of the DNA sequences according to the invention in

Enables plant cells, e.g. the use of tissue-specific, for example seed-specific, promoters the possibility of changing the tocopherol content in certain tissues, for example in seed tissue. Thus, the DNA sequences according to the invention are present in a preferred embodiment in combination with tissue-specific promoters, in particular seed-specific promoters.

The invention further relates to proteins with the biological activity of a geranylgeranyl reductase or active fragments thereof, which are encoded by a DNA sequence according to the invention or a nucleic acid molecule according to the invention. It is preferably a plant geranylgeranyl reductase, preferably from Nicotiana tabacum, particularly preferably a protein with the number shown in SEQ: ID No. 2 (see also Fig. 2) amino acid sequence shown or an active fragment thereof.

Another object of the invention is to provide vectors and microorganisms, the use of which enables the production of new plants in which an altered tocopherol content can be achieved. This object is achieved by the provision of the vectors and microorganisms according to the invention which contain nucleic acid sequences coding for enzymes with the activity of a geranylgeranyl reductase.

The present invention thus also relates to vectors, in particular plasmids, cosmids, viruses, bacteriophages and other vectors which are common in genetic engineering, which contain the nucleic acid molecules according to the invention described above and can optionally be used for the transfer of the nucleic acid molecules according to the invention to plants or plant cells.

The invention also relates to transformed microorganisms, such as bacteria,

Viruses, fungi, yeasts etc. which contain the nucleic acid sequences according to the invention.

In a preferred embodiment, the nucleic acid molecules contained in the vectors are linked to regulatory elements that form the

Ensure transcription and, if necessary, translation in prokaryotic and eukaryotic cells.

Optionally, the nucleic acid sequences of the invention can be supplemented by enhancer sequences or other regulatory sequences. These regulatory sequences include e.g. also signal sequences that ensure that the gene product is transported to a specific compartment.

It is also an object of the invention to provide new plants, plant cells, parts or products which are distinguished by an altered tocopherol content which, if appropriate, can be coupled with a chlorophyll biosynthesis performance which is altered in comparison with wild-type plants.

These tasks are solved by the transfer of the nucleic acid molecules according to the invention and their expression in plants. By providing the nucleic acid molecules according to the invention, it is now possible to use genetic engineering methods to modify plant cells to the effect that they produce a new or modified geranylgeranyl compared to wild-type cells. Have reductase activity and, as a result, there is an altered tocopherol biosynthesis performance and an altered tocopherol content.

In a preferred embodiment, plants or their

Cells and parts in which the tocopherol content is increased compared to wild type plants due to the presence and expression of the nucleic acid molecules according to the invention.

However, the invention also relates to plants in which the transfer of the nucleic acid molecules according to the invention leads to a reduction in the tocopherol and / or chlorophyll content. A reduced tocopherol and / or chlorophyll biosynthesis performance can be achieved, for example, by the transfer of antisense constructs or other suppression mechanisms, such as, for example, cosuppression.

The invention further relates to transgenic plant cells or plants comprising such plant cells and their parts and products in which the new nucleic acid molecules are integrated into the plant genome. The invention also relates to plants in whose cells the nucleic acid sequence according to the invention is present in self-replicating form, i.e. the plant cell contains the foreign DNA on an independent nucleic acid molecule.

The plants which are transformed with the nucleic acid molecules according to the invention and in which a modified amount of tocopherol and / or chlorophyll is synthesized due to the introduction of such a molecule can in principle be any plant. It is preferably a monocot or dicot crop. Examples of monocot plants are the plants belonging to the genera Avena (oats), Triticum (wheat), Seeale (rye), Hordeum (barley), Oryza (rice), Panicum, Pennisetum, Setaria, Sorghum (millet), Zea (corn). The dicotyledonous useful plants include legumes, such as legumes and in particular alfalfa, soybean, rapeseed, tomato, sugar beet, potatoes, ornamental plants, trees. Other useful plants can be, for example, fruit (in particular apples, pears, cherries, grapes, citrus, pineapple and bananas), oil palms, tea, cocoa and coffee bushes, tobacco, sisal, cotton, flax, sunflower and medicinal plants and pasture grasses and fodder plants. Cereals, wheat, rye, oats, barley, rice, corn and millet, feed grain, sugar beet, rapeseed, soya,

Tomato, Potato, Sweet Grasses, Forage Grasses, and Clover. It is self-evident that the invention relates in particular to conventional food or fodder plants. In addition to the plants already mentioned, peanut, lentil, broad bean, black beet, buckwheat, carrot, sunflower, Jerusalem artichoke, turnip, white mustard, turnip and stubble are worth mentioning.

The invention furthermore relates to propagation material from plants according to the invention, for example seeds, fruits, cuttings, tubers, rhizomes etc., this propagation material optionally containing transgenic plant cells described above, and parts of these plants such as

Protoplasts, plant cells and calli.

The invention also relates to plant cells which, owing to the presence and possibly expression of the nucleic acid molecules according to the invention, changed one compared to plant cells which do not contain the nucleic acid molecules

Vitamin K content. The fat-soluble vitamin K, which is particularly contained in plants, has an important function in the formation of coagulation factors, lack of vitamin K, leads to a reduction in blood clotting, which is why it is also used as an antihemoragic or coagulation vitamin referred to as. Since the expression of the nucleic acid molecules according to the invention results in an altered geranylgeranyl reductase activity and consequently an altered PPP synthesis performance and since the phylloquinone referred to as vitamin K, like the tocopherols, comprises a unit of phytol, such plant cells or plants are also the subject of the invention. that changed one

Vitamin K, content alone or in combination with an altered tocopherol content.

In a preferred embodiment, these are transgenic plant cells or plants and their parts and products which, owing to the presence and, where appropriate, expression of a DNA sequence coding for a geranylgeranyl reductase from plants, have a tocopherol content which is changed compared to non-transformed cells . The DNA sequence contained in the plant cells is preferably a sequence coding for geranylgeranyl reductase, which comes from tobacco. These are particularly preferably those in SEQ: ID NO. 1 DNA sequence shown (see also Fig. 1). In a particularly preferred embodiment, the DNA sequences according to the invention code for a geranylgeranyl reductase precursor enzyme which comprises a transit sequence for translocation in plastids.

The invention further relates to plants in which, in addition to the chl P gene, a gene for hydroxyphenylpyruvate dioxygenase (HPD) is also expressed. The enzyme HPD catalyzes the conversion of 4-hydroxyphenyl pyruvate into homogentisate, which, as mentioned above, is the second building block of tocopherols alongside phytol. The enzyme HPD and its position in the plant

Isoprenoid metabolism is more alia in Norris et al. (1995) The Plant Cell 7, 2139-2149. By the joint expression, preferably overexpression, of sequences which code for geranylgeranyl reductase and HPD, the tocopherol content in transgenic plants can be additionally increased compared to those plants which only contain the sequences for CHL P coding according to the invention.

In a further embodiment, the invention relates to host cells, in particular prokaryotic and eukaryotic cells that have been transformed or infected with a nucleic acid molecule or a vector described above, and cells that are derived from such host cells and contain the described nucleic acid molecules or vectors. The host cells can be bacteria, algae, yeast and fungal cells as well as plant or animal cells. The invention also relates to such host cells which, in addition to the nucleic acid molecules according to the invention, contain one or more nucleic acid molecules which are transmitted by genetic engineering or natural means and which carry the genetic information for enzymes involved in tocopherol, chlorophyll and / or vitamin K, biosynthesis .

The present invention is also based on the object of methods for producing plant cells and plants which are changed by a

Label tocopherol content to provide.

This object is achieved by methods with the aid of which the production of new plant cells and plants which have changed as a result of the transfer of nucleic acid molecules coding for geranylgeranyl reductase

Have tocopherol content is possible.

Furthermore, this object is achieved by methods with the aid of which it is possible to produce new plant cells and plants which, owing to the ^" joint transmission of nucleic acid molecules coding for geranylgeranyl reductase and for nucleic acid molecules coding for HPD or the transmission of nucleic acid molecules coding for geranylgeranyl reductase and for HPD have a tocopherol content that is different from that of wild type plants.

Various methods are available for producing such new plant cells and plants. On the one hand, plants or plant cells can be modified using conventional genetic engineering transformation methods in such a way that the new nucleic acid molecules are integrated into the plant genome, i.e. that stable transformants are generated. On the other hand, a nucleic acid molecule according to the invention, the presence and optionally expression of which in the plant cell causes an altered tocopherol biosynthesis, can be contained in the plant cell or the plant as a self-replicating system. For example, the nucleic acid molecules of the invention can e.g. contained in a virus with which the plant or plant cell comes into contact.

According to the invention, plant cells which have an altered tocopherol content due to the expression of a nucleic acid sequence according to the invention are produced by a method which comprises the following steps:

a) Production of an expression cassette which comprises the following DNA sequences: a promoter which ensures transcription in plant cells; at least one nucleic acid sequence according to the invention which codes for a protein or a fragment with the enzymatic activity of a geranylgeranyl reductase, the nucleic acid sequence being in the sense orientation at the 3 'end of the Promoter is coupled; and optionally a termination signal for the termination of the

Transcription and the addition of a poly-A tail to the corresponding one

Transcript that is at the 3 'end of the coding

Region is coupled.

b) Transformation of plant cells with the expression cassette produced in step a).

c) Regeneration of transgenic plants and, if appropriate, the multiplication of the plants.

Alternatively, one or more nucleic acid sequences according to the invention can be introduced into the plant cell or plant as a self-replicating system.

As a further alternative, step a) of the above method can be modified such that the at least one nucleic acid sequence according to the invention, which codes for a protein or a fragment with the enzymatic activity of a geranylgeranyl reductase, in antisense orientation at the 3 'end of the

Promoter is coupled.

Another object of the invention is to show uses of the nucleic acid sequences according to the invention and of the nucleic acid molecules they contain.

This object is achieved by the uses according to the invention of the new DNA molecules for the production of plant cells and plants which are characterized by a tocopherol content that is modified, preferably increased, compared to wild type cells or plants, solved.

Furthermore, the invention relates to the use of the nucleic acid sequences according to the invention for producing plants which have changed

Have chlorophyll content.

The invention further relates to the use of the nucleic acid sequences according to the invention for the production of plants which are distinguished by an altered, preferably increased, vitamin K content.

Another object of the invention is to record the possibilities of using the plants according to the invention or their cells, parts and products.

The invention relates in particular to the use of the plants according to the invention as a forage and / or food plant. Depending on the achieved increase in the content of vitamin E and / or vitamin K, in the transgenic useful plant or its products and parts, an otherwise common and often also necessary admixture of the corresponding vitamins, in particular vitamin E, can be limited in quantity or become completely superfluous. Irrespective of this, the invention relates generally to increasing the nutritional value of useful plants by increasing the tocopherol and / or phylloquinone content.

Furthermore, the invention relates to the use of the plant cells according to the invention, plants, their parts and products, as production sites for vitamin E and / or vitamin K. In addition to their use due to their vitamin character, for example in dietetic and pharmaceutical products, Cosmetics, skin care products, generally for vitamin E supplementation, etc. tocopherols are also used as antioxidants in chemical products such as fats and oils. The plants according to the invention thus represent an important source for the production of tocopherols and / or vitamin K, for a broad spectrum of commercial purposes.

The invention also relates to the use of the nucleic acid sequences according to the invention in combination with seed-specific promoters for the production of plants in which the seed tissue in particular is distinguished by a changed, preferably increased, tocopherol content. In a preferred embodiment of the invention, the nucleic acid sequences according to the invention are used in combination with the USP (Bäumlein et al. (1991) Mol. Gen. Genet. 225, 459-467) or Hordein promoter (Brandt et al. (1985) Carlsberg Res. Commun. 50, 333-345).

These promoters mentioned, in particular the seed-specific promoters, are also particularly suitable for the targeted reduction of the tocopherol or chlorophyll content in transgenic seeds using the DNA sequences according to the invention in connection with the antisense technique.

The invention further relates to the use of a geranylgeranyl reductase gene for producing an altered tocopherol content in plants.

The invention further relates to the use of a protein with the enzymatic activity of a geranylgeranyl reductase in order to achieve an altered tocopherol content in plants.

Furthermore, the invention relates to the use of the nucleic acid molecules according to the invention, the proteins according to the invention with geranylgeranyl Reductase activity and / or the transgenic plants or host cells according to the invention with new or modified geranylgeranyl reductase activity for identifying new herbicidal active compounds for crop protection. Thanks to the key position of geranylgeranyl reductase in chlorophyll and tocopherol biosynthesis, the DNA sequences according to the invention and the proteins encoded by them represent an extremely valuable target for herbicide research. For example, the proteins according to the invention with enzymatic geranylgeranyl reductase activity can be used for X-ray structure analysis , NMR spectroscopy, molecular modeling, and drug design are used to identify or synthesize inhibitors and / or effectors of geranylgeranyl reductase and thus potential herbicides based on the knowledge gained from these methods.

The invention further relates to the use of the nucleic acid sequences according to the invention for the production of herbicide-tolerant plants. So for

Sequences encoding geranylgeranyl reductase can be changed using standard methods or expanded to include new sequence elements and then transferred to plant cells. The introduction of sequences derived from the sequences according to the invention can e.g. be used to change the properties of plants in such a way that in the transgenic

Plant more or less functionally active geranylgeranyl reductase or a variant of geranylgeranyl reductase with modified properties is formed or that the level of expression of the chl P gene present in the transgenic plant is reduced. For example, an increase in the tolerance to herbicides which inhibit chlorophyll biosynthesis can be achieved by increasing the CHL P activity. For example, the expression of modified geranylgeranyl reductase genes in transgenic plant cells can also be associated with an increase in herbicide tolerance. The invention further relates to the use of the nucleic acid sequences according to the invention or a protein encoded by them for the production of antibodies.

The present invention thus encompasses every possible form of use of the nucleic acid molecules according to the invention, the presence and expression of which, if appropriate, in plants causes a change in the tocopherol content and / or chlorophyll content, and the use of the proteins or fragments thereof, the enzymatic activity of which brings about such a change.

In principle, any promoter which is functional in the plants selected for the transformation and which fulfills the condition that the expression regulated by it leads to an altered tocopherol synthesis performance is suitable for the promoter mentioned. With regard to the use of the transgenic plants as food or fodder plants, especially those appear for this

Promoters that ensure seed-specific expression make sense. Examples of such promoters are the USP promoter, hordein promoter and napin promoter.

If such promoters are not known or are not available, the concept for isolating such promoters is in any case known to the person skilled in the art. In a first step, the poly (A) ⁺ RNA is isolated from seed tissue and a cDNA library is created. In a second step, cDNA clones based on poly (A) ⁺ RNA molecules from a non-seed tissue are used to identify those clones from the first bank by hybridization whose corresponding poly (A) ⁺ RNA Molecules are only expressed in the seed tissue. Promoters are then isolated with the aid of these cDNAs identified in this way and are then used for the expression of the coding nucleic acid sequences described here can be. Analogously, other tissue-specific or development-specific or inducible by abiotic stimulators promoters can be isolated and used according to the invention.

Alternatively, it may be desirable that the plant has a changed, preferably increased tocopherol content in many areas due to the expression of the nucleic acid molecules according to the invention. In this case it is advisable to use a constitutive promoter, for example the 35S RNA promoter from Cauliflower Mosaic Virus.

The invention also relates to nucleic acid molecules which code for proteins with the biological activity of a geranylgeranyl reductase or biologically active fragments thereof and which hybridize with one of the nucleic acid molecules described above. The term biologically active fragments refers to fragments that can change the tocopherol content. 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) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Nucleic acid molecules that hybridize with the molecules of the invention can e.g. can be isolated from genomic or from cDNA libraries.

Such nucleic acid molecules can be identified and isolated using the nucleic acid molecules according to the invention or parts of these molecules or the reverse complements of these molecules, for example by means of hybridization using standard methods (see for example Sambrook et al., Supra). To identify and isolate such nucleic acid molecules, sequence sequences derived from the DNA sequences according to the invention, for example degenerate oligonucleotide primers, can be used.

The invention thus also relates to the use of a DNA sequence according to the invention or parts thereof for the identification and isolation of homologous ones

Sequences from plants or other organisms.

As a hybridization probe, e.g. Nucleic acid molecules are used that have the nucleotide sequences listed above or parts of these sequences exactly or essentially. In the used as a hybridization probe

Fragments can also be synthetic fragments that have been produced with the aid of common synthetic techniques and whose sequence essentially corresponds to that of a nucleic acid molecule according to the invention. If genes have been identified and isolated which hybridize with the nucleic acid sequences according to the invention, the sequence must be determined and the analysis carried out

Properties of the proteins encoded by this sequence are required. For this purpose, a number of standard molecular biological, biochemical and biotechnological methods are available to the person skilled in the art.

Those hybridizing with the nucleic acid molecules according to the invention

Molecules also include fragments, derivatives, and allelic variants of the DNA molecules described above, which encode a geranylgeranyl reductase or a biological, i.e. enzymatically active fragment thereof. Fragments are understood to mean parts of the nucleic acid molecules that are long enough to contain a polypeptide or protein with the enzymatic activity of a geranylgeranyl

To encode reductase or a comparable enzymatic activity that causes an altered tocopherol content. The term derivative in this context means that the sequences of these molecules differ from the sequences of the nucleic acid molecules described above on one or differentiate multiple positions and have a high degree of homology to these sequences. Homology means a sequence identity of at least 40%, in particular an identity of at least 60%, preferably over 80% and particularly preferably over 90%. The deviations from the nucleic acid molecules described above can be caused by deletion. Addition,

Substitution, insertion or recombination have arisen.

Homology also means that there is functional and / or structural equivalence between the nucleic acid molecules in question or the proteins encoded by them. The nucleic acid molecules which are homologous to the molecules described above and which are derivatives of these molecules are generally variations of these molecules which are modifications which have the same biological function. This can involve both naturally occurring variations, for example sequences from other organisms, or mutations, it being possible for these modifications to have occurred naturally or to have been introduced by targeted mutagenesis. Furthermore, the variations can be synthetically produced sequences. The allelic variants can be both naturally occurring and synthetically produced variants or those produced by recombinant DNA techniques.

The proteins encoded by the different variants and derivatives of the nucleic acid molecules according to the invention usually have certain common characteristics. For this, e.g. Enzyme activity, molecular weight, immunological reactivity, conformation etc. belong. More common

Characteristics can include physical properties such as e.g. B. the running behavior in gel electrophoresis, chromatographic behavior, sedimentation coefficient, solubility, spectroscopic properties, stability, pH optimum, temperature Represent optimum etc. Furthermore, the products of the reactions catalyzed by the proteins can of course have common or similar features.

To prepare the introduction of foreign genes into higher plants, 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 used for the transformation of E. co / cells. Transformed E. coli cells are grown in a suitable medium and then harvested and lysed. The plasmid is recovered. Restriction analyzes, gel electrophoresis and other biochemical-molecular biological methods are generally used as the analysis method for characterizing the plasmid DNA obtained. After each manipulation, the plasmid DNA can be cleaved and DNA fragments obtained can be linked to other DNA sequences. Each plasmid DNA sequence can be cloned into the same or different plasmids.

A variety of known techniques are available for introducing DNA into a plant host cell, and the person skilled in the art can determine the appropriate method without difficulty. 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 of isolated DNA into protoplasts, the microinjection and electroporation of DNA, the introduction of DNA by means of biolistic method and other possibilities. J -

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 are used. However, if whole plants are to be regenerated from such transformed cells, the presence of a selectable one is usually the case

Marker gene necessary. 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 introduction method, in addition to the desired gene or genes

Presence of additional DNA sequences may be required. E.g. If 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 and Ri plasmid, must be connected as a flank region to the genes to be introduced .

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 vir region necessary for the transfer of the T-DNA. Intermediate vectors cannot replicate in agrobacteria. Using a helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors 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 (Holsters et al. (1978) Molecular and General Genetics 163, 181- 187). The agrobacterium serving as the host cell is said to contain a plasmid which carries a vir region. The vir region is necessary for the transfer of the T-DNA 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 is sufficient in EP 120 515; Hoekema in: The Binary Plant Vector System, Offsetdrokkerij Kanters B.V., Alblasserdam (1985) Chapter V; Fraley et al. (1993) Crit. Rev. Plant. Sci., 4, 1-46 and An et al. (1985) EMBO J.

4, 277-287.

For the transfer of the DNA into the plant cell, plant explants can expediently be cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenes. From the infected plant material (e.g. leaves,

Leaf pieces, stem segments, roots, but also protoplasts or suspension-cultivated plant cells) can then regenerate whole plants again in a suitable medium, which can contain antibiotics or biocides for the selection of transformed cells. The plants are regenerated using conventional regeneration methods using known nutrient media.

The plants thus obtained can then be examined for the presence of the introduced DNA. Other possibilities of introducing foreign DNA using the biolistic method or by means of protoplast transformation are known (cf. e.g. Wilmitzer L. (1993) Transgenic Plants, in: Biotechnology, A Multi-Volume Comprehensive Treatise (H. Rehm, G. Reed, A. Pühler, P.

Stadler, eds.) Vol. 2, 627-659, VCH Weinheim - New York - Basel - Cambridge). While the transformation of dicotyledonous plants via Ti plasmid vector systems with the help of Agrobacterium tumefaciens is well established, recent work indicates that monocotyledonous plants can also be transformed using 4grobαcter w / n-based vectors (Chan et al. (1993) Plant Mol. Biol. 22, 491-506; Hiei et al. (1994) Plant J. 6, 271-282; Deng et al. (1990) Science in China 33, 28-34; Wilmink et al. (1992) Plant Cell Reports 11, 76-80; May et al. (1995) Bio / Technology 13, 486-492; Conner and Domiss (1992) Int. J. Plant Sei. 153, 550-555; Ritchie et al (1993) Transgenic Res. 2, 252-265).

Alternative systems for the transformation of monocotyledonous plants are transformations using the biolistic approach (Wan and Lemaux (1994) Plant Physiol. 104, 37-48; Vasil et al. (1993) Bio / Technology 11, 1553-1558; Ritala et al. (1994) Plant Mol. Biol. 24, 317-325; Spencer et al. (1990) Theor. Appl. Genet. 79, 625-631; Altpeter et al. (1996) Plant Cell Reports 16, 12-17), the

Protoplast transformation, the electroporation of partially permeabilized cells, the introduction of DNA using glass fibers.

The transformation of maize is described in various ways in the literature (cf. e.g. WO 95/06128, EP 0 513 849; EP 0 465 875; Fromm et al.

(1990) Biotechnology 8, 833-844; Gordon-Kamm et al. (1990) Plant Cell 2, 603-618; Koziel et al. (1993) Biotechnology 11, 194-200). EP 292 435 describes a process by means of which fertile plants can be obtained starting from a slimy, soft, granular corn callus. Shillito et al. ((1989) Bio / Technology 7, 581) have observed in this connection that it is also necessary for the regenerability to fertile plants to start from callus suspension cultures, from which one divides Protoplast culture with the ability to regenerate plants can be produced. After an in vitro cultivation time of seven to eight months, Shillito et al. Plants with viable offspring.

Prioli and Sondahl ((1989) Bio / Technology 7, 589) describe the regeneration and extraction of fertile plants from maize protoplasts, the Cateto maize inbred line Cat 100-1. The authors suspect that protoplast regeneration to fertile plants can be caused by a number of different factors, e.g. depends on the genotype, the physiological state of the donor cells and the cultivation conditions.

The successful transformation of other cereals has also been described, e.g. for barley (Wan and Lemaux, supra; Ritala et al., supra) and for wheat (Nehra et al. (1994) Plant J. 5, 285-297; Altpeter et al, supra).

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 normally contains a selection marker which shows the transformed plant cells resistance to a biocide or an antibiotic such as kanamycin, G418, bleomycin, hygromycin, methotrexate,

Glyphosate, streptomycin, sulfonyl urea, gentamycin or phosphinotricin and others taught. The individually selected marker should therefore allow the selection of transformed cells from cells that lack the inserted DNA.

The transformed cells grow within the plant in the usual way

(see also McCormick et al. (1986) Plant Cell Reports 5, 81-84). The resulting plants can be grown normally and crossed with plants that have the same transformed genetic makeup or other genetic makeup become. The resulting hybrid individuals have the corresponding phenotypic properties. Seeds can be obtained from the plant cells.

Two or more generations should be grown to ensure that the phenotypic trait is stably maintained and inherited. Seeds should also be harvested to ensure that the appropriate phenotype or other characteristics have been preserved.

Likewise, transgenic lines can be determined by customary methods, which are homozygous for the new nucleic acid molecules and whose phenotypic behavior with regard to an altered tocopherol content is examined and compared with that of hemizygotic lines.

The expression of the proteins according to the invention with geranylgeranyl reductase

Activity can be accomplished using conventional molecular biological and biochemical methods. These techniques are known to the person skilled in the art and he is easily able to select a suitable detection method, for example a Northern blot analysis for detecting geranylgeranyl reductase-specific RNA or for determining the level of accumulation of geranylgeranyl reductase-specific RNA, a Southern blot analysis for identification of DNA sequences coding for geranylgeranyl reductase or a Western blot analysis for detection of the protein encoded by the DNA sequences according to the invention, preferably CHL P. Detection of the enzymatic activity of geranylgeranyl reductase can, for example, with that of

Soll and Schultz (1981) in Biochem. Biophys. Res. Commun. 99, 907-912 described enzyme assay on the formation of chlorophyll-phytyl. The invention is based on the successful isolation of a cDNA clone coding for a geranylgeranyl reductase from a cDNA library from Nicotiana tabacum cv. Petit Havana SRI. The sequence of this cDNA clone, which comprises a complete open reading frame, is in SEQ: ID NO. 1 shown. Using the sequence according to SEQ: ID No. 1 succeeded in producing transgenic plants that have a different tocopherol content compared to wild type plants.

The DNA sequence according to SEQ: ID NO. 1 containing cDNA clone was transformed into Escherichia coli and the corresponding E. coli strain on

16.10.1997 deposited with the German Collection for Microorganisms and Cell Cultures GmbH (DSMZ), Mascheroder Weg lb, D-38124 Braunschweig, under the deposit number DSM 11816 according to the Budapest contract.

The following examples serve to explain the invention.

EXAMPLES

Example 1 :

Cloning of a tobacco cDNA encoding geranylgeranyl reductase (CHL P)

To identify the geranylgeranyl reductase cDNA from tobacco, a Lambda ZAP II cDNA library (Nicotiana tabacum SRI, Stratagene, USA) was screened using an EST from Arabidopsis thaliana coding for the locus 4D9T7P. The EST sequence used is similar to the known bch P / chl P sequences from Rhodobacter capsulatus (Young et al. (1989) Mol. Gen. Genet. 218, 1-12; Bollivar et al. (1994) J. Mol. Biol. 237, 622-640; Bollivar et al. (1994) Biochemistry 33, 12763-12768) and Synechocystis PCC6803 (Addlesee et al. (1996) FEBS Lett. 389, 126-130).

The hybridization probe used encompasses the region of the EST sequence from base 1 to base 364 shown in 4D9T7P (Accession No. T04791). The probe was obtained as a NotI / Sall restriction fragment from the PRL2 library from A. thaliana (vector: λZipLox) ( Newman et al. (1994) Plant Physiol. 106: 1241-1255) isolated and radioactively labeled with [α- ³² P] dCTP using nick translation (Life Technologies, Eggenstein).

The hybridization was carried out according to the following protocol.

2 h pre-hybridization at 55 ° C with hybridization solution following

Composition: 5 x SSC, 0.1% SDS, 5 x Denhard reagent, 100 μg / ml denatured Salmon sperm DNA; - 12 h main hybridization at 55 ° C with fresh hybridization solution of the above composition plus radiolabelled probe;

Wash: 2 x 10 min. at 55 ° C with 2 x SSC and 0.1% SDS, and

1 x 5 min. at 55 ° C with 1 x SSC and 0.1% SDS.

The plasmid DNA isolated after cDNA bank screening was sequenced. The identified and in SEQ: ID NO. 1 shown chI P cDNA sequence comprises 1510 nucleotides (without polyA tail), of which nucleotides 1 to 1392 for a 52 kDa protein of 464 amino acids (including the start methionine, and without the stop codon (nucleotides 1393 to 1395 ) calculated) code. The deduced amino acid sequence of the CHL P is in SEQ: ID NO. 2 shown. In the

SEQ: ID NO. 1 nucleotide sequence shown comprises a 3 ^' untranslated region from nucleotide 1396 to 1510. For DNA-RNA isolation, sequence analysis, restriction, cloning, gel electrophoresis, radioactive labeling, Southern, standards and Western blot analyzes, hybridization and the like, common methods have been used as described in relevant laboratory manuals, such as Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

Example 2: Transformation of tobacco plants and regeneration of intact plants

For the production of tr.ansgenic plants which overexpress CHL P and therefore have a higher tocopherol content than non-transformed plants, the DNA sequence according to SEQ: ID NO. 1 by means of the restriction enzymes BamHI and Sall in the multiple cloning interface of the pBluescript vector cut out of the vector and in sense orientation in the binary vector BinAR-TX (Höfgen and Willmitzer (1990) Plant Science 66, 221-230), one pBIB derivative (Becker (1990) Nucleic Acid Res. 18, 203), which was digested with the same restriction endonucleases, ligated behind the CaMV 35S promoter. For clarification, a restriction map of the vector BinAR-TX is attached as Figure 3.

Instead of the binary vector BinAR-TX mentioned, any vector suitable for plant transformation can be used to produce a chimeric gene consisting of a fusion of the CaMV 35S promoter or another

Promotors, which ensures the transcription and translation in plant cells, and DNA sequences coding for CHL P are used. The recombinant vector pCHLPbin was then transformed into Agrobacterium tumefaciens (strain GV2260; Horsch et al. (1985) Science 227, 1229-1231) and used to transform tobacco plants (SNN) using the leaf disc transformation technique (Horsch et al., Supra) .

For this purpose, an overnight culture of the corresponding Agrobacterium tumefaciens clone was centrifuged for 10 minutes at 5000 rpm, and the bacteria were resuspended in 2YT medium. Young tobacco leaves from a sterile culture (Nicotiana tabacum cv. Samsun NN) were cut into small pieces measuring approx. 1 cm ² and briefly placed in the bacterial suspension. The leaf pieces were then placed on MS medium (Murashige and Skoog (1962) Physiol. Plant. 15, 473; 0.7% agar) and incubated for two days in the dark. The leaf pieces were then sprouted on MS medium (0.7% agar) with 1.6% glucose, 1 mg / 1 6-benzylaminopurine, 0.2 mg / 1 naphthyl acetic acid, 500 mg / 1 claforan (cefotaxime, Hoechst, Frankfut) and 50 mg / 1

Kanamycin laid. The medium was changed every seven to ten days. When shoots had developed, the leaf pieces were transferred to glass jars containing the same medium. Resulting shoots were cut off and placed on MS medium with 2% sucrose and 250 mg / 1 Claforan and regenerated to whole plants.

Example 3:

Analysis of transgenic tobacco plants that contain the recombinant vector pCHLPbin

Transgenic tobacco plants were transformed, selected and regenerated as described above. After rooting in sterile culture, about 100 independent transformants were transferred to soil in the greenhouse. The tobacco plants were in Greenhouse at 60% humidity and 20-25 ° C for 16 hours in the light and 18-20 ° C for 8 hours in the dark.

The transformants with normal or increased tocopherol or chlorophyll contents showed neither a changed appearance with regard to their phenotype nor a different growth rate compared to control plants.

Some of the primary transformants showed a tocopherol content that was up to four to four times higher than that of wild type plants. This increase in tocopherol content could be seen in offspring of the TI and T2 generation and in homozygotes

Daughter plants obtained by customary self-pollination and subsequent determination of the split pattern of the seeds on medium containing kanamycin are additionally increased.

It was also observed that the tocopherol levels in transgenic

Plants under stress conditions, e.g. Cultivation under low and elevated temperatures or strong light, and in senescent leaves were additionally increased compared to control plants.

The tocopherol content was determined according to the following protocol:

Leaf disks were homogenized in liquid nitrogen and extracted three times in methanol. The extracts were collected and eluted on a LiCrospher 100 HPLC RP-18 column (Merck, Darmstadt, Germany) at a flow of 1 ml / min with the following gradient: 94% solvent B (100% methanol) / 6% solvent A ( 30% methanol, 10% 0.1 M ammonium acetate, pH 5.1) for 7 min., For a further 17 min. 99% solvent B / 1% solvent A, then another 26 min. 94% solvent B / 6% solvent A. Alternatively, the collected extracts were analyzed by HPLC in an isocratic gradient (gradient consists of 2% solution A [10% methanol and 10% acetic acid] and 98% methanol (solution B); flow rate 1 ml / min.). A Waters LC module system with Shimadzu RF 551 fluorescence detector (295 nmex, 325 nm) was used.

The result of a tocopherol assay is shown in Figure 4 in the form of a bar chart. The comparison of leaves 6, 9, 12 (counted from the plant tip) of transformants 28 and 30 with the corresponding leaves of the control plant (SNN) shows an up to 6-fold increased tocopherol content in the transgenic lines.

Regardless of their ability to grow on kanamycin-containing medium, the transgenic tobacco plants were also analyzed in a Southern blot. This resulted in hybridization with a labeled cDNA fragment for CHL

P additional radio-labeled bands of the genomic DNA of the transformants digested with restriction enzymes in comparison to control plants

Northern blot analysis revealed an increased amount of specific RNA in the transformants compared to the CHL P-RNA contents of the control plants.

An increased geranylgeranyl reductase expression in the transgenic plants could also be detected in the Western blot. The transformants showed an increased amount of CHL P protein compared to control plants.

In addition, plastid import experiments (carried out according to Grimm et al. (1989) Plant Mol. Biol. 13, 583-593) were able to confirm that the sequence according to SEQ: ID NO. 1 encoded CHL P precursor protein was imported into the plastids after in vitro transcription and translation. Example 4:

Production of CHL P antisense constructs and transfer to tobacco

While the transgenic plants produced and analyzed in Examples 2 and 3 had an increased tocopherol content due to the overexpression of the DNA sequences according to the invention, the following antisense construct was generated and produced for the production of transgenic tobacco plants which have a reduced activity of CHL P Transfer tobacco.

The cDNA sequence according to SEQ: ID NO. 1 was cut out of the vector using the restriction enzymes Kpnl and Xbal in the multiple cloning interface of the pBluescript vector and in the antisense orientation in the binary vector BinAR-TX (see Example 2), which was digested with the same restriction enzymes, with the Cauliflower Mosaic Virus 35S promoter fused. The resulting recombinant vector pCHLPASbin was transferred to tobacco as described in Example 2 using Agrobacterium tumefaciens-mediated leaf disc transformation. Subsequently, transgenic plants were regenerated. Approximately 100 independent transgenic lines were regenerated and copies of the transgene were inserted using standard techniques (e.g. Southern

Blot hybridization).

The transformants showed a reduced growth compared to control plants, a bleached phenotype, reduced RNA and protein contents for CHL P, a high content of geranylgeranyl chlorophyll (up to 50% of the

Total chlorophyll content compared to 100% phytyl chlorophyll in wild type plants) and a reduced chlorophyll and tocopherol content. Example 5:

Overexpression of active geranylgeranyl reductase in Escherichia coli

For the production of expression clones that overexpress the recombinant CHL P in E. coli, the open reading frame was assumed to be a mature one

(Processed) protein using the oligonucleotide primers

CSYN 1 5'-cgc cat ggg ccg caa tct tcg tgt tgc ggt-3 'and

CSYN 2 5'-gca gat ctg tcc att tcc ctt ctt agt gca-3 '

from the DNA sequence according to SEQ: ID No. 1 amplified by PCR (1 min. 94

° C; 2 min. 60 ° C; 3 min. 72 ° C for 25 cycles). The amplified PCR fragment was purified and cut with the restriction enzymes Ncol and BglII and ligated into the expression vector pQE60 (Qiagen, Hilden) digested with the same enzymes. After the initiation codon ATG (part of the recognition sequence for Ncol) was followed by the coding Chl P sequence, starting with nucleotide No. 148 of the open reading frame of the CHL P cDNA sequence. After methionine, this results in the incorporation of a glycine (amino acid No. 50 of the protein translated from the cDNA sequence).

The E. co // strains XL 1 were used for the expression of the plant CHL P

Blue (Stratagene, LaJolla, CA, USA) or SG 13009 (Gottesman et al. (1981) J. Bacteriol. 148, 265-273) were transformed with the recombinant vector. After induction of the transcription of the recombinant gene by means of IPTG, a protein with a molecular weight of approx. 47 kDa was expressed in the E. co / z ^' strains. The protein was detected in the pelletable fraction of the bacterial extract and purified from the total extract under denaturing conditions using a nickel affinity column in accordance with the supplier's instructions (Qiagen, Hilden). The purified protein was injected into rabbits for antibody production.

The protein from the total extract has a geranylgeranyl reductase activity in a combined enzyme assay with bacterial bacteriochlorophyll synthase using chlorophyllide and GGPP. The

Enzyme assay was carried out according to the protocol in Oster et al. (1997) J. Biol. Chem. 272, 9671-9676.

Chlorophyll-GG and chlorophyll-phytol were separated on an HPLC with the RP 18 column using the solvent mixture solution

A (60% acetone) and solution B (100% acetone). The gradient of solvents was used: t ₀ 75% solution A and 25% solution B, 2 min .; within t ₂ . ₄ to 45% solution A and 55% solution B; within t ₄ . ₁₃ to 30% solution A and 70% solution B; within t _13.17 to 100% solution B; t ₁₇ . ₂₁ 100% solution B isocratic; then within 5 min. on 75% solution A and 25% solution B; then another 5 min. 75% solution A and 25% solution B isocratic. A fluorescence detector was used to detect the tetrapyrroles (λ _ex 425 nm, λ _em 665 nm).

Example 6:

Co-expression of the CHL P gene and the HPD gene in Nicotiana tabacum

Using the oligonucleotide primers hpdolil 5'-tta ggt acc atg ggc cac caa aac gcc gcc gtt tca g-3 'and hpdoli2 5'-tga gtc gac cac aat cct tta gtt ggt tct tct tct tg-3'

the sequence of the HPD cDNA (Accession No .: AF 000228) between nucleotide 37 and 1404 was amplified from an Arabidopsis tbα / zαttα cDNA library, cloned and sequenced. The fragment was cut with the restriction endonucleases Kpnl and Sall and ligated into the Kpnl / Sall-cut binary vector Bin-Hyg-TX. The vector Bin-Hyg-TX is (as in the vector BinAR-TX used in Example 2) a pBIB descendant (Becker, supra) which expresses the one in the multiple

Enables the cloning site ligated coding region under control of the 35S RNA promoter from Cauliflower Mosaic Virus. In contrast to the BinAR-TX vector used for the expression of the CHL P gene, which enables selection for kanamycin in plant cells, the binary vector Bin-Hyg-TX carries a hygromycin resistance gene as a selection marker for plant cells. For clarification, a restriction map of the vector Bin-Hyg-TX is attached as Figure 5.

Instead of the binary vector Bin-Hyg-TX mentioned, any vector suitable for plant transformation can be used to produce a chimeric gene, consisting of a fusion of the CaMV 35S promoter or another promoter which ensures transcription and translation in plant cells and DNA- Sequences encoding HPD are used.

The recombinant vector pBinHygHPD obtained was then transferred to tobacco SNN by means of Agrobacterium tumefaciens as described in Example 2 above, transgenic tobacco sprouts being selected on hygromycin-containing medium. The plants obtained after regeneration served as control plants.

In addition, transformants 28 and 30 described in Example 2 and optionally other transformants that control the CHL P gene under control of the 35S Overexpress RNA promoters, transformed with the recombinant vector pBinHygHPD using leaf disc transformation and whole plants regenerated with selection for kanamycin and hygromycin.

In all transgenic plants, the successful transmission and

Expression of the chimeric HPD gene was confirmed by suitable Southern and Northern blot experiments with the above-mentioned PCR fragment for HPD as a probe.

A comparison of the tocopherol contents (analysis as in Example 3) in leaves of plants which only express the CHL P gene (see Example 3, transformants

28 and 30), and those plants which co-express the HPD gene and the CHL P gene (transformants 28 + HPD and 30 + HPD) showed that the tocopherol content was additionally increased by the simultaneous expression of the hydroxyphenylpyruvate dioxygenase gene could be.

Alternatively, transgenic plants that express both the CHL P gene and the HPD gene can also be obtained by crossing homozygous "CHL P lines" with homozygous "HPD lines".

An additional increase in tocopherol content in double transformants (or

Double crossings) can be expected under stress conditions (e.g. increased temperature, light stress, etc.).

In addition to the double transformants described above, which express HPD and CHL P, transgenic HPD lines were also examined for their tocopherol content and the influence of stress conditions on the tocopherol content. It could be shown that overexpression of HPD in tobacco leaves is a 2- to 3-fold increase in tocopherol content and that, in particular, the values increase under stress conditions compared to non-transgenic control plants.

Figure 6 shows tocopherol levels in leaves 4, 7 and 10 of 12-week-old transgenic takak lines (# 2, 6 and 33) that overexpress the Arabidopsis enzyme hydroxyphenyl pyruvate dioxygenase (HPD) compared to control plants (SNN). The plants were removed either at 38 ° C. or at 10 ° C. and a light intensity of approx. 200 μmol photons / m ² / s. Tocopherol is enriched in the plants with increasing leaf age. In the older leaves in particular, the tocopherol content in the plants grown below 38 ° C rises much more steeply and reaches twice the amount compared to the control plants. In contrast, the tocopherol levels in the transformants in the cold are only slightly increased compared to those of the wild type plants.

The results indicate that HPD particularly favors the regeneration of these antioxidants in transgenic plants when there is an increased need for tocopherols, in particular under stress conditions.

Example 7:

Increased resistance of the transgenic plants to oxidative stress

Transgenic plants produced according to Examples 2 and 3 were in a

Leaf disc incubation experiment examined for the influence of inhibitory and oxidative substances. 15 seedlings were incubated in 20 mM potassium phosphate buffer (pH 7.1) for 10 h in the light. In addition to the control batches (water), the plants were batched with 3.3 μM (low concentration = NK) or 33 μM (high concentration = HK) Acifluorfen (BASF, Ludwigshafen, Germany), an inhibitor of protoporphyrinogen oxidase, and in other approaches with 1.7 μM (low concentration = NK) or 17 μM (high concentration = HK) rose bengal ( 4,5,6,7-tetrachloro-2 ', 4', 5 ', 7'-tetraiodofluorescein, Sigma-Aldrich, Deisenhofen, Germany), a producer of reactive oxygen species. The tocopherol content in the examined transgenic lines (28, 30) was increased by 2 to 3 times both in the buffer control batches and under oxidative stress conditions compared to wild type plants (SNN). The results are illustrated in Figure 7.

The results indicate that the plants according to the invention are notable for increased resistance to oxidative stress, owing to their higher tocopherol content compared to wild type plants. An increased antioxidative protection of the cellular membranes against reactive oxygen species in the plants according to the invention is therefore to be expected.

Example 8:

Tocopherol content in seeds of transgenic plants

As described above, tocopherol was extracted from seeds from transgenic plants which, owing to the expression of CHL P under the control of the CaMV 35S promoter (cf. Example 2), are characterized by an increased tocopherol content in leaves in comparison to wild type plants. The results of the tocopherol quantification performed by HPLC are compared to

Tobacco control plants shown in Figure 8.

In addition to α-tocopherol, γ-tocopherol was also quantified. The latter is generally present in larger quantities in tobacco seeds; the ratio of γ-tocopherol to α-tocopherol in tobacco seeds is approx. 10: 1. The levels of both forms of tocopherols, especially α-tocopherol, are around 2 to 3 times higher than the control plants in the transgenic plants with increased geranylgeranyl reductase expression. times increased.

These results, which reflect the effects of constitutive CHL P expression under the control of the 35S promoter on the tocopherol content in the seed, clearly show that when a seed-specific promoter (or a seed inducible promoter) is used, the expression of geranylgeranyl reductase in the Seeds and thereby the tocopherol content in seeds of transgenic plants can be additionally increased.

If molecular biological work has not been adequately described in any way, this was done using standard methods, as described by Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition,

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. With regard to the transformation of plants, reference is made to generally known review articles and to the publications mentioned above.

DESCRIPTION OF THE PICTURES

Figure 1: SEQ: ID NO. 1 shows the nucleotide sequence of a chl P cDNA of geranylgeranyl reductase (CHL P) from Nicotiana tabacum.

Figure 2: SEQ: ID NO. 2 shows an amino acid sequence of the enzyme CHL P from N tabacum, derived from the SEQ shown in FIG. 1: ID Ν0.1.

Figure 3: Restriction map of the binary vector BinAR used for plant transformation. BinAR (Höfgen and Willmitzer (1990) Plant Science 66, 221) is a Bin19 derivative (Bevan (1984) Nucl. Acids Res. 12, 8711) which contains an expression cassette for the constitutive expression of chimeric genes in plants, the Expression cassette via the EcoRI and Hindlll

Restriction interfaces of Binl9 is cloned. The cassette comprises a 770 bp EcoRI / HindIII fragment which contains the CaMV 35S promoter, a partial pUC18 polylinker and the termination signal of the octopine synthase gene (OCS). The unique interfaces of the pUC18 polylinker, namely Kpnl, Smal, BamHI, Xbal and Sall, are particularly suitable for the insertion of coding sequences. The binary vector BinAR carries a kanamycin resistance gene as a plant selection marker.

Figure 4: Bar chart showing the tocopherol content in leaves 6, 9, 12 (counted from the plant tip) of transgenic tobacco plants (lines 28 and 30) versus the corresponding leaves of control plants (SNN). Figure 5: Restriction map of the binary vector Bin-Hyg-TX used for plant transformation, which is a pBIB derivative (Becker, supra; Bevan, supra) that contains an expression cassette for the constitutive expression of chimeric genes in plants . The unique interfaces of the pUC18 polylinker, namely Hpal, Kpnl, Smal, Xbal and Sall, are particularly suitable for the insertion of coding sequences. The binary vector Bin-Hyg-TX carries a hygromycin resistance gene as a plant selection marker.

Figure 6: Bar graph of tocopherol levels in leaves 4, 7 and 10 in 12-week-old transgenic lines (# 2, 6, 33) that overexpress the Arabidopsis enzyme HPD and in control plants (SNN).

Figure 7: Tocopherol content in 12-day-old seedlings which were exposed to acifluoride at 3.3 μM (NK) or 33 μM (HK) or with 1 during a 10-hour exposure in 20 mM potassium phosphate buffer (pH 7.1, control = water) , 7 μM (NK) and 17 μM (HK) Rose Bengal were incubated (SNN = wild-type control plants, 28 and 30 = transgenic lines).

Figure 8: Relative values of α-tocopherol and γ-tocopherol in seeds from transgenic tobacco plants (# 7, 39, 67, 96) and wild-type tobacco plants (SNN). 100% α-tocopherol corresponds to 6 ng / mg seeds; 100% γ-tocopherol corresponds to 62.8 ng / mg seeds.

Claims

EXPECTATIONS 1. nucleic acid sequence, characterized in that it codes for a protein with the activity of a geranylgeranyl reductase or an active fragment thereof. 2. Nucleic acid sequence according to claim 1, characterized in that it codes for a vegetable protein. 3. Nucleic acid sequence according to claim 1 or 2, characterized in that it codes for a protein from tobacco. 4. Nucleic acid sequence according to SEQ: ID No. 1. 5. Nucleic acid sequence according to claim 1, characterized in that it is contained in the clone DSM 11816. 6. Alleles and derivatives of the nucleic acid sequence according to one of the preceding claims, characterized in that they code for a protein with the activity of a geranylgeranyl reductase or an active fragment thereof. 7. Nucleic acid molecule, characterized in that it comprises a nucleic acid sequence according to one of the preceding claims. 8. Nucleic acid molecule according to claim 7, characterized in that it comprises a nucleic acid sequence according to one of claims 1-6 in combination with regulatory elements which ensure the transcription and translation in prokaryotic or eukaryotic cells. 9. Nucleic acid molecule according to claim 7 or 8, characterized in that it comprises a nucleic acid sequence according to any one of claims 1-6 in combination with promoters which ensure transcription and translation in plant cells. 10. Nucleic acid molecule according to claim 9, characterized in that the promoter is a constitutive, inducible, tissue-specific or development-specific promoter. 11. Nucleic acid molecule according to claim 10, characterized in that the promoter is a seed-specific promoter. 12. Nucleic acid molecule according to one of claims 7-11, characterized in that it additionally comprises enhancer sequences, sequences coding for signal peptides or other regulatory sequences. 13. Nucleic acid molecule according to one of claims 7-12, in which the coding nucleic acid sequence is in the sense orientation. 14. Nucleic acid molecule according to one of claims 7-12, in which the coding nucleic acid sequence is present in the antisense orientation. 15. Protein with the biological activity of a geranylgeranyl reductase or an active fragment thereof, characterized in that it is characterized by a DNA sequence or a nucleic acid molecule according to one of the preceding Claims is encoded. 16. Protein with the biological activity of a geranylgeranyl reductase or an active fragment thereof, characterized in that it has the in SEQ: ID NO. 2 amino acid sequence shown or regions thereof. 17. Microorganism, characterized in that it has a Contains nucleic acid sequence or a nucleic acid molecule according to any one of claims 1-14. 18. Transgenic plants which have a nucleic acid sequence, a nucleic acid sequence derived therefrom or a nucleic acid molecule according to one of the Claims 1-14 contain, as well as parts of these plants and their nutrients, such as protoplasts, plant cells, calli, seeds, tubers or cuttings, etc., and the progeny of these plants. 19. Plants according to claim 18, which have a modified tocopherol content compared to wild-type plants. 20. Plants according to claim 19, which have an increased tocopherol content compared to wild type plants. 21. Plants according to claim 18, which have a modified vitamin K content compared to wild type plants. 22. Plants according to claim 21, which have an increased vitamin K content compared to wild type plants. 23. Plants according to claim 18, which have a different chlorophyll content compared to wild type plants. 24. Plants according to claim 23, which have an increased chlorophyll content compared to wild type plants. 25. Dicotyledonous plants according to one of claims 18-24. 26. Plants according to claim 25, which are useful plants, food and / or fodder plants, in particular rape, soybean, tomato, potato, sugar beet, clover. 27. Monocot plants according to one of claims 18-24. 28. Plants according to claim 27, which are useful plants, food and / or fodder plants, in particular cereals, such as wheat, barley, corn, oats, rye, rice, sweet and pasture grasses. 29. Plants according to one of claims 18-28, in which a nucleic acid sequence according to one of claims 1-6 is integrated in the genome of the plant, and parts of these plants and their propagation material, such as protoplasts, plant cells, calli, seeds, tubers or cuttings, etc., as well as the descendants of these plants. 30. Transgenic plant cells, including protoplasts, which contain a nucleic acid sequence, a nucleic acid sequence derived therefrom or a nucleic acid molecule according to any one of claims 1-14. 31. Plant cells according to claim 30, including protoplasts which have a tocopherol, vitamin K, and / or chlorophyll content which is modified, in particular increased, compared to non-transformed plant cells. 32. Plants or plant cells according to any one of claims 18-31, which additionally contain a nucleic acid sequence coding for hydroxyphenylpyruvate dioxygenase. 33. A method for producing plants or plant cells according to any one of claims 18-32, comprising the following steps: a) Preparation of a nucleic acid sequence consisting of the following Components that are strung together in the 5 '-3' orientation: - a functional, especially seed-specific, Promoter, at least one nucleic acid sequence which codes for a protein with the activity of a geranylgeranyl reductase or an active fragment thereof and - optionally a termination signal for the termination of the Transcription and the addition of a poly-A tail to the corresponding transcript, and, if appropriate, DNA sequences derived therefrom; b) transfer of the nucleic acid sequence from a) to plant cells and, if appropriate, integration of the nucleic acid sequence into the plant genome, c) if desired, regeneration of completely transformed plants and, if appropriate, propagation of these plants. 34. Use of a plant according to one of claims 18-29 and 32 as a food or forage plant. 35. Use of a plant according to one of claims 18-29 and 32 as a production site for tocopherols and / or vitamin K ,. 36. Use of a nucleic acid sequence or a nucleic acid molecule according to any one of claims 1-14 for identification, Isolation or amplification of a nucleic acid sequence coding for a protein with the activity of a geranylgeranyl reductase or an active fragment thereof. 37. Use of a nucleic acid sequence, a nucleic acid molecule or a protein according to any one of claims 1-16 for the production of antibodies. 38. Use of a nucleic acid sequence or a nucleic acid molecule according to any one of claims 1-14 for the production of transgenic plants or plant cells. 39. Use according to claim 38, wherein the plants or plant cells have an altered tocopherol content. 40. Use according to claim 38, wherein the plants or plant cells have an altered chlorophyll content. 41. Use according to claim 38, wherein the plants or plant cells have an altered vitamin K, content. 42. Use according to claim 38, wherein the plants or plant cells have an increased herbicide tolerance compared to wild type plants. 43. Use according to any one of claims 38-42, wherein the plants or plant cells additionally contain a nucleic acid sequence coding for hydroxyphenylpyruvate dioxygenase. 44. Use of a nucleic acid sequence, a nucleic acid molecule, a protein or a microorganism according to any one of claims 1-17 for finding effectors of plant geranylgeranyl reductase.