EP1190077A2 - Genetic sequence which codes for the flavon synthase ii enzyme and use of the same - Google Patents

Abstract

The invention relates to genetic sequences which code for the enzyme of the flavonoid metabolism, in particular, for flavon synthase II (FNS II) or derivates thereof and to their use for specifically modifying the colour of flowers, for modifying the flavon content or expression in leaves, flowers and other plant or organic tissue. The use also covers expression systems for synthesising natural, functional flavons for medical or similar applications, for example, for treating cancer or for improving the human immune defence.

Description

 GENETIC SEQUENCE ENCODING FLAVON SYNTHESIS II ENZYMES AND THEIR USE

The present invention relates to genetic sequences which code for enzymes of the flavonoid metabolism, especially for the flavone synthase II (FNS II) or derivatives thereof and their use for the targeted change in the flower color, for changing the flavon content or pattern in leaves, flowers and other tissues of plants and other organisms and moreover the use in expression systems for the synthesis of natural, functional flavones for medical or similar applications, for example for cancer therapy or improvement of the human immune system.

Flavonoids and their function in the plant

Flavonoids are the most important and most widespread plant pigments that are found in various tissues such as Flowers, leaves or roots have been detected. In addition, they are among the best characterized secondary metabolites in plants. More than 3000 different flavonoids have been characterized so far. They have been divided into different subclasses (e.g. flavones, flavonols or anthocyanins) based on the degree of oxidation of the central C-ring. Each type can also be modified by hydroxylation, acylation and glycosylation (Heller and Forkmann, 1994). Due to the different physicochemical properties of the molecules, the subclasses sometimes have very different biological functions.

The accumulation of certain flavonoids in a plant cell depends on the availability of the respective enzymes, the availability of the enzymes ultimately depending on the expression of the corresponding gene. The regulation of the expression of flavonoid biosynthesis genes is essentially determined by the plant species, the stage of development and the environmental conditions.

Flavonoids play an important role both inside and outside the plant. For example, it has been shown that certain flavonols are required for pollen tube growth. If the accumulation of flavonols is suppressed by blocking the biosynthetic pathway, sterile pollen is obtained (Taylor and Jorgensen, 1992). Both biotic and abiotic signals can be generated during plant interaction lead to flavonoid accumulation with their environment. For example, UV radiation leads to an accumulation of flavonols and flavones. This is achieved by inducing the transcription of the respective flavonoid biosynthesis genes in different types (Kubasek et.al., 1992). However, other stress factors such as wounding, extreme temperature fluctuations and water stress can also induce flavonoid accumulation and / or gene expression in various ways (Hrazdina, 1982). When plants interact with other organisms, the flavonoids are assigned a dual function. On the one hand, the flavonoids as phenolic compounds have an effect as phytoalexin against various pathogens and as a deterrent against predators (Harborne and Grayer, 1994), and on the other hand they are responsible for the communication between plants from the legume family and certain microorganisms. Flavonoids serve as signaling substances for nitrogen-fixing bacteria, in which genes are then expressed that are required for symbiosis with the plant (Redmond et.al., 1986). In flowers, leaves and fruits, flavonoids, especially the colored anthocyanins, but also chalcones, aurones, flavones and flavonols, are responsible for the coloring and the patterns of the various secondary ingredients. The latter, together with other characteristics, such as, for example, the scent, is important for recognition by various animals, but also for the person who uses the plant as jewelry or for food (Harborne and Grayer, 1994). In addition, certain flavonoids, such as fiavon apigenin and flavonol quercetin, influence auxin transport within the plant (Jacobs and Rubery, 1988).

The Flavonoid Biosynthesis Weq (Fig. 1A)

The structure of the flavonoids consists of two aromatic rings (A and B) and a central heterocycle (C) (Fig. 1 B). They are formed in the plant starting from L-phenylalanine via the phenylpropanoid pathway through the enzymatic reaction of phenylalanine ammonia lyase (PAL) and cinnamic acid 4-hydroxylase (4CL). The resulting 4-coumaroyl-CoA together with 3 molecules of malonyl-CoA results in tetrahydroxy chalcone. This reaction is synthesized by chalcon synthase (CHS), the key enzyme in flavonoid biosynthesis (Fig. 1A). The tetrahydroxy chalcone (THC) is usually quickly isomerized to naringenin (NAR) by the enzyme chalconisomerase (CHI). The anthocyanins are formed in various other reactions. Flavones are formed on a side path through FNS I or FNS II, a cytochrome P450 enzyme. This class of enzymes is widespread and natural Various genes for cytochrome P450 enzymes have been isolated and sequenced from vertebrates, insects, yeasts, fungi, bacteria and plants.

The flavone synthase uses various flavanones, e.g. NAR or eriodictyol (ERI) as a substrate to synthesize the corresponding flavones apigenin (Ap) and luteolin (Lu). As shown in Figure 1 B, a double bond is inserted between the C2 and C3 positions. Flavones can be present in the plant in a glycosylated or methylated form.

In vitro, the formation of flavones from flavanones was first observed with enzyme preparations from UV-irradiated Petroselium crispt-zm cell suspension cultures. The corresponding enzyme is a soluble 2-oxoglutarate-dependent dioxygenase called flavone synthase I (FNS I). In the flowers of various flavon-producing plants, e.g. Sinningia cardinalis, Antirrhinum majus, Verbena hybrida, Columnea hybrida, Chrysanthemum morifolium, Gerbera hybrids and osmotically induced Glycine max cell suspension cultures, however, this reaction is catalyzed by the FNS II mentioned above, a NADPH-dependent, the microsomal enzyme, the Cytochrome P450 belongs (Heller and Forkmann, 1994). The locus Fns controls the formation of flavones in the flowers of gerbera hybrids. A synthesis of flavones can only be detected in lines with a dominant allele; no FNS II activity is detectable in homozygous recessive lines (Fig. 2; Martens and Forkmann, 1998).

Three types of pigments are responsible for the colors in flowers: betalaine, carotenoids and flavonoids. Betalaine only come in a few families

Centrospermae, where they are responsible for yellow, orange, red and violet colors.

Carotenoids produce orange or yellow tones and are the main pigments in most orange and yellow flowers. Flavonoids are the main and most common

Flower or plant pigments. This group includes the coloring anthocyanins, which are glycosylated and often acylated in the vacuole. Different anthocyanins can produce different colors. The flower color is also determined by the pH of the vacuole, the complexation with metals and the

Glycosylation or acylation patterns influenced (Forkmann, 1991). Another important factor for the development of different flower colors is the copigmentation of anthocyanins with colorless flavonoids, e.g. Flavones or

Flavonols or tannins (Scott-Moncrieff, 1936). Anthocyanins made with flavones copigmented can take on different colors, depending on the basic structure of the anthocyanin, and can vary between purple and blue (Äsen and Horowitz, 1974; Goto and Kondo, 1991). Various flavones, such as isoetin, have also been identified as yellow flower pigments (Harborne, 1978).

An important goal in horticultural plant breeding is the development of new varieties for flowering ornamental plants. Classic breeding methods have been successfully used in the past to establish a number of different colors in many economically important ornamental plants. However, the gene pool of the individual species naturally limits the possibilities for such approaches. For this reason, there are only a few species that have the entire color spectrum. In addition, no precise change can be achieved with the classic breeding methods. Since the aesthetic value of a flower depends on various factors, e.g. Shape, fragrance and color are determined and a change in one of these factors by crossing can usually only be achieved at the expense of similar, other visible characteristics or can only be achieved with a great deal of time and money, a more effective way to new varieties must be used . The ability to specifically change the flower colors of plants brings clear advantages over other methods. This is particularly important in an area that has a high product turnover and where novelty is an important market factor. For example, the development of blue-flowering varieties for the main cut flower types such as roses, chrysanthemums, carnations, lilies, tulips and gerberas would bring a significant market advantage in the cut flower market, but also in the potted plant market. The possibility of controlling the synthesis of copigments, e.g. Flavone, in plants, is a useful application for the targeted change of flower colors. In addition to the flowers, this application can also be used on fruits and other useful plants, e.g. on fruit and vegetable plants and on leaves, e.g. for ornamental plants.

In addition to their contribution to the color of the flowers, the flavonoids, especially the flavones, have other biological properties and effects. For example, they were found in some plants as feeding stimulants for monophage and oligophage insects (Harborne and Grayer, 1994). In most cases, the glycosides show a far greater effect than the corresponding Aglyka, which is probably due to the better solubility of the glycosides. In addition, the insects can differentiate between different sugar residues, thereby further differentiating the active Components is given. The basic structure of the Aglyka can also lead to different effects. In comparison to many other phytonutrients, the flavonoids or flavones obviously do not have a very toxic effect on insects. Nevertheless, there are some flavones that act as a deterrent to feeding insects even at low concentrations or can significantly inhibit the growth of the animals. An influence of the type of glycosylation could not be shown here, but that of the hydroxylation or methoxylation of the flavone (Harborne and Grayer, 1994).

Various flavones also stimulate butterflies to lay eggs on certain plants. The animals were shown to lay their eggs only after the stimulus was recognized. Such stimulating substances include e.g. the Flavone Vicenin-2 and various luteolin derivatives. If the synthesis of these substances in the respective host plants is prevented, the butterflies' oviposition and thus damage by eating can be prevented by their caterpillars.

By exploiting the plants' natural chemical defense mechanisms, problems associated with the use of synthetic insecticides, such as the environmental impact caused by residues of the substances used in the fruit and soil is avoided. In addition, resistance formation, as is observed with most synthetic pesticides, is avoided or at least delayed and the sometimes considerable costs for agents and application are saved.

Another important biological property of the flavonoids concerns the activation of the nodulation genes in different Rhizobium species. These bacteria infect legumes and form nitrogen-fixing root nodules. The flavonoids produced by the host plant act as a "signaling substance", whereby the bacteria initiate the infection process. These plant-specific, active compounds also include various flavones, such as, for example, apigenin, luteolin and 7,4'-dihydroxyflavone (Firmin et.al., 1986; Redmond et al., 1986) If the flavone production and its release are changed by the root or the flavon pattern in this tissue, an improvement in nitrogen fixation or possibly an establishment of this mechanism can be achieved in non-leguminous plants. By using these natural symbiotic mechanisms, nitrogen fertilization and thus the pollution of the environment by leaching out the nutrients can be reduced. In addition, costs for fertilizers and their application are saved.

Various naturally occurring flavonoids affect the plant, e.g. the Fiavon apigenin or the flavonols Kaempferol and Quercetin, the auxin transport in different plant tissues and transport systems. They behave similarly to synthetic transport inhibitors. Auxins, as plant-specific growth regulators, influence cell extension, cell division, apika dominance, new root and shoot formation as well as Parthenokaφie. An induced, changed flavonoid concentration (endogenous change and / or exogenous application) can thus significantly influence the growth of plants via the interaction with auxins. This can replace synthetic growth inhibitors.

As bioactive substances, flavonoids also have a significant role in human and animal nutrition. They occur in fruits, vegetables, nuts, seeds, stems, but also in tea and wine. Anti-allergic, anti-inflammatory, anti-viral, anti-proliferation and anti-carcinogenic properties have long been attributed to flavonoids, including various flavones. An influence on human and animal metabolism and the highly complex immune system is also described. In this context, the flavonoids or flavones influence a large number of different enzymes (e.g. hyaluronidase or aldose reductase), they have important enzyme-inducing and antioxidative properties, can scavenge free radicals, chelate different metal cations and also influence cellular protein phosphorylation (Middleton and Kandaswami, 1994).

The improvement of useful plants, which are important for the nutrition of humans and animals due to their content of health-promoting flavonoids, through the targeted change in the content or pattern of the corresponding compounds, makes a significant contribution to the healthy nutrition of humans and animals.

It was therefore an object of the present invention to provide means and methods to specifically change and control the flavonoid biosynthesis or the formation of flavones in plants, for example to change the flower color of a plant or to improve the resistance properties and symbiotic ability of a plant. A further object of the present invention was to provide means and methods which can be used for the targeted synthesis of defined flavones. Flavones obtained in this way can be used, among other things, in cancer therapy or can contribute to the health of humans and animals in the form of drugs.

This object is achieved by the claims.

The invention therefore relates to a nucleic acid sequence which codes for a flavone synthase II (FNS II). In one embodiment, the invention relates to a nucleic acid sequence shown in SEQ ID NO: 1 or a part thereof. In another embodiment, the invention relates to a nucleic acid sequence with the SEQ ID NO: Nucleic acid sequence or 1 shown a portion hybridizes thereof and / or at least a 40%, better at least 45% ιge ^', more preferably at least 55% or, best of all, at least 65-70% homology, or most preferably greater than 85% at the level of the nucleic acid or amino acid sequence to at least one or more regions (preferably over the entire region) of the sequence shown in SEQ ID NO: 1 . The nucleic acid sequence preferably encodes a protein or polypeptide with the biological activity of a flavone synthase. In a further embodiment, the invention relates to a nucleic acid sequence which is degenerate with respect to a nucleic acid sequence according to the preceding embodiments. In a preferred embodiment, the nucleic acid sequence according to the invention is DNA or RNA and is derived from a plant containing flavones, such as, for example, from gerbera (Gerbera hybrids), aster (Callistephus chinensis), snapdragons (Antirrhinum majus), chrysanthemum (Chrysanthemum indicum), dahlia (Dahlia hybrids) ), Gloxinia (Sinningia hybrids), verbena (Verbena hybrids) and streptocarpus (S. hybrids). In a further preferred embodiment, the nucleic acid sequence according to the invention is a recombinant nucleic acid sequence. The invention further relates to a nucleic acid sequence which is complementary to the sequence coding for flavone synthase II (FNS II).

Accordingly, the present invention provides an isolated nucleic acid encoding one for flavone synthase II (FNS II) or for a functional derivative of this enzyme

Nucleic acid or a complementary nucleic acid. The term "FNS II enzyme" means enzymes of the flavonoid biosynthetic pathway that flavanones such as narine Use genin and eriodictyol or other compounds from this class as a substrate for the synthesis of the corresponding flavones.

Preferred is a nucleic acid according to the invention which has been isolated from the natural environment or chemically synthesized. Particularly preferred are nucleic acid molecules that are formed or obtained in vitro, including genomic DNA fragments, recombinant and synthetic molecules and nucleic acids in combination with heterologous nucleic acids. This also includes genomic DNA or cDNA or parts thereof which encode the FNS II or parts thereof in reverse orientation to the corresponding or another promoter. Naturally occurring, closely related nucleic acid sequences are also included.

The term "nucleic acid sequence encoding a flavone synthase II" is used here in its most general form and encompasses each successive series of nucleotide bases which, directly or via a complementary series of bases, determines an amino acid sequence of an FNS II.

A polypeptide with part or all of the amino acid sequence of flavone synthase II means a full (full length) FNS II or an active, incomplete form thereof.

In a further embodiment, the invention relates to oligonucleotides which can be used as genetic samples or as "antisense" molecules for regulating the expression of the corresponding gene in plants or other organisms. An "antisense molecule" as described herein also includes a gene construct that consists of a structural, genomic, or a cDNA gene or a portion thereof in reverse orientation with respect to his or another promoter.

In a further embodiment, the nucleic acid sequence which codes for the FNS II or for various functional derivatives thereof is used to reduce the activity of endogenous FNS II, or alternatively a nucleic acid sequence which codes for this enzyme or various derivatives or parts thereof is used in antisense Orientation used to reduce the activity of FNS II. In addition, however, it is also possible for an antisense transcript of the FNS II or a fragment or part of the FNS II (for example an oligonucleotide molecule) to undergo a duplex with all or part of the naturally occurring mRNA which the enzyme specifies such an accumulation of or prevents translation of the mRNA into the active enzyme. Another possibility is the use of ribozymes to inactivate special nucleic acid sequences.

Changes in FNS II activity mentioned here relate to an increase or reduction in activity of up to 30% or better from 30 to 50% or even better 50 to 75% or best of all 75% or even higher or lower than normal, endogenous or existing value of the activity. The level of activity can easily be tested using the method described by Martens and Forkmann (1998) (see Example 3).

The nucleic acid described in this invention can also be a ribonucleic acid or a deoxyribonucleic acid, which can be present as a single-stranded or double-stranded and linear or covalently closed molecule. The nucleic acid molecule is usually present as a cDNA. The present invention also encompasses other nucleic acid molecules which hybridize under low, better under medium and best under high-stringent conditions with nucleic acid molecules according to the invention or specifically with the sequence shown in SEQ ID NO: 1 or a part or a part thereof. A particularly preferred embodiment relates to a nucleic acid molecule comprising the nucleic acid sequence shown in SEQ ID NO: 1 or a molecule which contains at least a 40%, better an at least 45%, even better an at least 55% or best at least a 65- 70% or, best of all, a similarity higher than 85% at the level of the nucleic acid sequence or amino acid sequence to at least one or more regions (preferably over the entire region) of the sequence shown in SEQ ID NO: 1 and wherein the nucleic acid is coding or complementary to is a sequence encoding an enzyme that has FNS II activity.

The present invention further comprises nucleic acid molecules in the form of oligonucleotide primers or competent samples for hybridization with a part of the nucleic acid molecules considered above and especially with the one shown in SEQ ID NO: 1. The hybridization can take place under low, better under medium and best under highly stringent conditions. The part preferably corresponds to the 5 'or 3' end of the gene. For convenience, the 5 'end is defined here as the region mainly between the start codon of the structural, genetic sequence and the middle region of the gene. The 3 'end is considered here as the region that defines the structural genetic sequence between the central region of the gene and the stop codon. It is therefore clear that oligonucleotides or samples have the 5 'end or the 3 'end or with one region together to hybridize to both the 5' or 3 'end. The present invention includes all such samples. Preferred oligonucleotides are shown in Example 4.

The nucleic acid or its complementary form can encode the complete enzyme or a part or a derivative. By "derivative" is meant a single or multiple amino acid substitution, deletion and / or addition in relation to the naturally occurring enzyme, the flavone synthase II activity preferably being retained. In this context, the nucleic acid according to the invention comprises the naturally occurring nucleotide sequence which codes for the FNS II and single or multiple nucleotide substitutions, deletions and / or additions. The nucleic acid of the present invention or its complementary form can also encode part of the FNS II, either active or inactive. Such a nucleic acid molecule can be used as an oligonucleotide sample, as a primer for the polymerase chain reaction, in various mutagenic techniques or for the preparation of antisense molecules.

The invention further relates to a recombinant DNA molecule containing a nucleic acid sequence according to the invention. In a preferred embodiment, the recombinant DNA molecule is a vector or a vector with a promoter. In a particularly preferred embodiment, the promoter is capable of expressing the nucleic acid according to the invention. The nucleic acid molecules according to the invention can be present in one of the two orientations in combination with a vector molecule, for example an expression vector. The term "vector molecule" is used in its most general meaning to include any intermediate vehicles for the nucleic acid molecule which, for example, make it possible to transfer the nucleic acid into cells, in particular plant cells, and / or to integrate it into a genome. These vector molecules or parts thereof are preferably used for integration into a plant genome. Such vector molecules can be replicated and / or expressed in prokaryotic cells and / or in eukaryotic cells. An intermediate vehicle can be adapted, for example, for use in electroporation, in microprojectile bombardment, in transfer with the aid of agrobacteria or in the insertion via DNA or RNA viruses. The intermediate vehicle and / or the nucleic acid molecule contained therein can be stably integrated into the plant genome. The nucleic acid molecule can additionally also contain a promoter sequence which is used to initiate expression of the nucleic acid molecule in a cell, in particular one Plant cell, is suitable. The nucleic acid molecule and promoter can also be introduced into the cell by various methods (see above).

The invention also relates to host cells which contain these DNA molecules according to the invention. The host cells can be prokaryotic or eukaryotic cells, in particular yeast cells, insect cells, plant cells and mammalian cells.

The invention further relates to a polypeptide which is encoded by a nucleic acid according to the invention. In a preferred embodiment, the invention relates to a polypeptide with the amino acid sequence shown in SEQ ID NO: 2 or a part thereof or derivatives thereof. In a further preferred embodiment the polypeptide is derived from a plant containing flavon, e.g. from Gerbera (Gerbera hybrids), Aster (Callistephus chinensis), Snapdragons (Antirrhinum majus), Chrysanthemum (Chrysanthemum indicum), Dahlia (Dahlia hybrids), Gloxinie (Sinningia hybrids), Verbena (Verbena hybrids) and Streptocarpus. In a particularly preferred embodiment, the polypeptide according to the invention has flavone synthase II activity.

Derivatives in the sense of this invention are amino acid insertion derivatives, deletion derivatives and / or substitution amino acid variants of the amino acid sequence from SEQ ID NO: 2.

Amino acid insertion derivatives of the FNS II according to the invention include both amino and carboxylene mergers as well as insertions of single or more amino acids within the sequence. Insertion amino acid sequence variants are those in which one or more amino acid residues have been inserted into the protein at a predetermined position, although a random insertion with an appropriate screening of the product is also possible. Deletion variants are characterized by the removal of one or more amino acids from the sequence. Substitution amino acid variants are those in which at least one residue in the sequence has been removed and another residue has been inserted at this point. Typical substitutions are shown in Table 1. TABLE 1 Suitable residues for amino acid substitution

Original amino acid Exemplary substitution by

Aln Ser

Arg Lys

Asn Gin; His

Asp Glu

Cys Ser

Gin Asn

Glu Asp

Gly Pro

His Asn; gin

Ue Leu; Nal

Leu Ile; Nal

Lys Arg; Gin; Glu

Met Leu; Ile

Phe Met; Leu; Tyr

Ser Thr

Thr Ser

Trp Tyr

Tyr Trp; Phe

Val Ile; Leu

In general, amino acids are replaced by amino acids with similar properties, such as hydrophobicity, hydrophilicity, electronegativity, very extensive side chains and the like. Substitutions of amino acids usually affect only one residue, whereas insertions usually affect a range of 1 to 10 amino acid residues and deletions affect a range of 1 to 20 residues. Deletions and insertions are preferably carried out on adjacent pairs, e.g. a two residue deletion or an insertion of two residues.

The amino acid variants of derivatives according to the invention described above can easily be produced with the aid of known peptide synthesis techniques such as, for example, by solid phase synthesis and similar processes or by recombinant DΝA manipulations. Techniques for making substitution mutations at predetermined locations in DNA that have a known or partially known sequence are well known and include, for example, M13 mutagenesis. The manipulation of DNA sequences for the production of proteins with substitutions, insertions or deletions is described, for example, in Sambrook et.al. (1989). Other examples of recombinant or synthetic mutants and derivatives of the FNS II according to the invention include single or multiple substitutions, deletions and / or additions of any molecules associated with the enzyme, such as carbohydrates, lipids and / or proteins or polypeptides.

The terms "analog" and "derivative" also extend to all functional chemical equivalents of FNS II and also to all amino acid derivatives which have already been described above.

Another aspect of the present invention concerns recombinant forms of the FNS II. The recombinant forms of the enzyme provide a way to more active for example enzymes or systems for various in ^'tto production flavones for use in various fields, such as to develop human medicine. The latter system can be used, among other things, in cancer research.

In a further embodiment, the invention relates to transgenic plants containing a nucleic acid sequence according to the invention. In a preferred embodiment, the nucleic acid sequence is suitable for expression and can optionally be regulated or is regulated in the plant depending on development. In a further preferred embodiment, the transgenic plant is selected from the group of plants containing flavon, e.g. from Gerbera (Gerbera hybrids), Aster (Callistephus chinensis), Snapdragons (Antirrhinum majus), Chrysanthemum (Chrysanthemum indicum), Dahlia (Dahlia hybrids), Gloxinie (Sinningia hybrids), Verbena (Verbena hybrids) and Streptocarpus, S. streptocarpus carries an endogenous FNS II and also contains a non-endogenous FNS II nucleic acid sequence according to the invention.

According to the present invention, a nucleic acid sequence coding for an FNS II or a derivative or a part thereof can be introduced into and expressed in a plant in one of two possible orientations and thereby offer the possibility of either naringenin (NAR) and / or other suitable substrates when synthesized in the plant cell to convert, which ultimately leads to the formation of different flavones. In addition, the formation of these metabolites can be prevented by reducing or eliminating endogenous or existing FNS II activity. The synthesis of flavones leads to a change in the color of the flowers in gerberas. Using the example of the locus Fns heterozygotes, Flavone-containing, orange variety "Th 58" (fns * fns) could show a variation in color from dark red (flavone-free; genotype: fns fns) to various orange-red tones (containing flavone; genotype: fns * fns) to a yellow-orange (strongly flavonic; genotype) : fns * fns *) are shown. This experiment can be arbitrarily transferred to other varieties heterozygous for the Locus Fns (see also Example 2). The expression of the nucleic acid sequence in one of two possible orientations in the plant can be constitutive, inducible or development-related and also tissue-specific. The word "expression" is used in its most general meaning to include the production of RNA or both, RNA and protein. It is also extended to partial expression of nucleic acid molecules.

The invention relates to a process for the production of transgenic plants which are capable of synthesizing FNS II or active mutants or derivatives. This method comprises the stable transformation of a cell of a suitable plant with a nucleic acid molecule which comprises a nucleotide sequence which codes for said FNS II under conditions which induce the possible expression of said nucleic acid molecule, the regeneration of a transgenic plant from the cell and the Growth of this transgenic plant for a certain time and under conditions suitable for inducing the expression of the nucleic acid. The transgenic plant may show higher values of FNS II activity compared to the value measured in comparable, non-transgenic plants or the values may be lower compared to those of comparable, non-transgenic plants.

One aspect of the present invention relates to a method for producing a transgenic plant with reduced, endogenous or existing FNS II activity. This method involves the stable transformation of a cell of a suitable plant with a nucleic acid molecule comprising a nucleotide sequence encoding a sequence or a complementary sequence of the FNS II, the regeneration of a transgenic plant from the cell and, if necessary, the cultivation of this transgenic plant under suitable conditions to achieve expression of nucleic acids.

Another aspect of the invention relates to a method for producing a genetically modified plant with reduced endogenous or existing FNS II activity. This procedure involves the modification of the FNS Il gene by a Modification of the endogenous sequence via homologous recombination, starting from a correspondingly modified gene of an FNS II or a derivative or a part thereof. The gene is introduced into the plant cell and a genetically modified plant is regenerated from the cell.

Another aspect of the present invention relates to a method for producing a transgenic, flowering plant with altered flower properties. This method comprises introducing the nucleic acid sequence according to the invention into a cell of a suitable flavon-free plant, regenerating a transgenic plant from the cell and cultivating a transgenic plant at a time and under conditions in order to achieve the expression of the introduced nucleic acid sequence according to the invention. The transgenic plant can be selected, for example, from the group of plants containing flavones, consisting of Euphorbia (E. pulchem ^' ma), Cyclamen (Cyclamen persicum), Rose (Rosa Hybrida), Pelargonium (P. spec), Begonia (ß. Spec) , Carnation (Dianthus caryophyllus) and tulip (Tulipa hybrids). In a further preferred embodiment, the transgenic plant is capable of expressing an endogenous flavone synthase II. Such a transgenic plant can be selected, for example, from the group of plants consisting of gerbera (Gerbera hybrids), aster (Callistephus chinensis), snapdragons (Antirrhinum majus), chrysanthemum (Chrysanthemum indicum), dahlia (Dahlia hybrids), gloxinia (Sinningia hybrids) , Verbena (Ve-n ena hybrids) and streptocarpus (S. hybrids). In a further preferred embodiment, this endogenous flavone synthase II is co-expressed when the introduced nucleic acid according to the invention is expressed.

In one embodiment, the endogenously present flavone synthase II activity is reduced by introducing the nucleic acid sequence. This method comprises the stable transformation of a cell of a suitable plant with a nucleic acid sequence according to the invention or a sequence complementary thereto, the regeneration of a transgenic plant from the cell and the cultivation of this transgenic plant at a time and under suitable conditions to the level of activity of the endogenous or to change existing FNS II. The changed value is preferably less than the endogenous or existing level of FNS II activity in a comparable, non-transgenic plant. If necessary, expression of the introduced nucleic acid sequence or its complementary sequence is necessary for the reduction of the endogenous FNS II activity. Expression of the introduced genetic sequence or its complementary analogue may be necessary to achieve the desired effect. This essentially means a flowering plant with changed flower properties.

In this connection, the present invention relates to a method for producing a flowering plant which exhibits different flowering properties. This method involves changes in the FNS II gene by modification of the endogenous sequences via homologous recombination of a correspondingly modified gene of an FNS II or a derivative or a part thereof, the introduction into the plant cell and the regeneration of the genetically modified plant from the cell.

The nucleic acid molecule according to the invention can also be regulated depending on development. As a rule, a changed inflorescence includes the production of weakly colored flowers or other colors, depending on the physiological conditions of the recipient plant. By "recipient plant" is meant a plant that produces a measurable amount of substrate for the FNS II enzyme or the FNS II itself and has the corresponding physiological properties and genotype that are necessary for the development of the desired colors. This includes, but is not limited to, the following plants: Gloxinia (Sinningia hybrids), Snapdragons (Antirrhinum majus), Columnea (Columnea hybrids), Dahlia (Dahlia variabilis), Gloxinia (Sinningia cardinalis), Streptocarpus (Streptocarpus Hybridus), Verbena (Verbena hybrida), chrysanthemum (Chrysanthemum indicum), single leaf (Spathiphyllum wallisii), petunia (Petunia hybrida), cyclamen (Cyclamen persicum), rose (pink hybrids) and geranium (P. spec).

Accordingly, the present invention encompasses a method for producing a transgenic plant that measurably expresses a recombinant gene encoding FNS II or a portion thereof or that carries a nucleic acid sequence that is substantially complementary to all or a portion of the mRNA molecule , which, if necessary, can be easily transcribed to effect the regulation of FNS II. This method comprises the stable transformation of a cell of a suitable plant with the isolated nucleic acid, comprising a nucleotide sequence coding for an FNS II or a derivative or a part thereof, or a sequence complementary to the coding nucleotide sequence, if necessary under conditions which a Allow expression of said isolated nucleic acid, and by regeneration of a transgenic plant from a cell. Those skilled in the art immediately notice the possibilities of use in the present invention, such as increasing or reducing the expression of the enzymes that are naturally present in a target plant. This leads to different new flower hues, such as different orange or dark red tones.

The present invention therefore relates to all transgenic plants which contain all or part of the nucleic acid sequence according to the invention and / or which have a homologous or related form thereof or an antisense form of any of the described, and in particular those transgenic plants which differ Show flower properties. The transgenic plants can contain introduced nucleic acid molecules that comprise a nucleotide sequence encoding an FNS II or a complementary sequence. In general, the nucleic acid is stably introduced into the plant genome, although the present invention also introduces an FNS II nucleotide sequence within an autonomously replicating nucleic acid sequence such as e.g. Includes DNA or RNA viruses that are capable of replicating in a plant cell. The invention also includes seeds of the transgenic plant, especially if they contain flavon.

With the help of the FNS II gene according to the invention, the flavonoid biosynthesis or the formation of flavones in plants can be specifically changed in various ways.

In the field of plants, two different starting points must be taken into account. On the one hand, plants are available that naturally form flavones or their glycosides, e.g. Antirrhinum and Verbena in flowers, Clerodendron and Citrus in leaves, Althaea and Sophora (Leguminosae) in roots and Prunus and Pinus spec. in hardwood (Wollenweber, 1994; Williams and Harborne, 1994). In addition to this very large group, there are also some plants that do not synthesize flavones in certain tissues. This is e.g. this is the case with some flowers of important target plants such as Pelargonium, Cyclamen and Petunia and in apple leaves.

Accordingly, the present invention relates to an altered FNS II activity in plants and other organisms, which can be achieved both by increasing and reducing the naturally occurring FNS II activity by means of the introduction of the sequence from the present invention. A reduction in the level of FNS II activity can also be described as a down-regulation. In plants containing flavones, the synthesis of flavones can be specifically up-regulated or down-regulated with the help of suitable methods, or switched off entirely. This has various consequences for the plant. Basically, such a change in the biosynthetic pathway affects the entire flavonoid biosynthesis, since there is constant competition from several enzymes (FNS II, FHT, F3'H, F3 ', 5'H) for the substrate (flavanones, eg naringenin). Specifically, this means that, for example, an upregulation of FNS II reduces the synthesis of flavonoids in the later biosynthetic pathway (Fig. 1A) and thus, for example, paler flower colors can be achieved. In addition, this targeted increase in flavon, which can only affect certain tissues, can significantly improve the resistance and symbiosis of the plant. In contrast, down regulation leads to an increase in the synthesis of other flavonoids that may be more useful for the plant. This can affect the flavonols important for UV protection as well as the coloring anthocyanins or resistance-inducing proanthocyanidins and phytoalexins. Complete suppression of flavone formation increases the effects of down-regulation and can remove possible stimulants for insects and thus contribute to the development of resistance.

A completely new synthesis of flavones can be achieved by the targeted introduction of FNS II into plants without natural activity. Depending on the existing substrates, the flavon content and the flavon pattern can be regulated. By establishing this step, flower colors, resistance properties and the ability to symbiosis with nitrogen-fixing bacteria can be specifically changed. In addition, the opening of a new biosynthetic pathway can reduce the formation of less useful flavonoids for the plant (e.g. feeding stimulants) or also establish the synthesis of biflavones that arise from flavones.

With the help of these approaches, important flavon-containing or flavon-free useful plants in their flavonoid pattern and content, including the flavones, can be changed in such a way that their positive properties with regard to the biology of humans and animals are optimized.

The present invention further relates to methods for specifically changing the flake content or pattern in various plant tissues (flowers, roots, leaves, etc.) and other organisms, and thus generally changing the Flavonoid composition, especially the change of flower colors through copigmentation, resistance properties and the ability of nodulation in legumes.

In a further embodiment, the invention relates to the use of a polypeptide according to the invention for the synthesis of flavones. In suitable expression systems, the enzyme itself and ultimately natural flavones can be synthesized, starting from suitable substrates. One possibility is the use of suitable expression systems for obtaining health-promoting, natural flavones. These expression systems can be plant-based or consist of cell or yeast cultures. The expression of the FNS II in plants or in cell cultures can be used for the direct extraction of the corresponding flavones, it being possible for the yeast expression system to be preferred for the extraction of the intact enzyme. This enzyme can then be used to convert chemical or natural precursors (flavanones) to the corresponding fiavon. The flavones synthesized in this way can be used, among other things, in cancer therapy or can contribute to the health of humans and animals in the form of drugs.

The present invention is described in detail by the following figures and examples. The present invention is based on a nucleic acid sequence

Gerbera hybrids was derived, illustrated. It is now obvious that similar

Sequences easily from various other sources such as other plants or special microorganisms. Examples of other flavone-producing plants include gloxinia (Sinningia hybrids), snapdragons (Antirrhinum majus), Columnea (columnea hybrids), dahlia (Dahlia variabilis), gloxinia

(Sinningia ca dinalis), Streptocaφus (Streptocarpus Hybridus), Verbena (Verbena

Hybrida), Chrysanthemum (Chrysanthemum indicum), Einblatt (Spathiphyllum wallisii), but are not limited to these. All of these nucleic acid sequences that directly or indirectly encode an enzyme of the flavonoid biosynthetic pathway and in particular the FNS II, regardless of their source, are included in the present invention.

FIGURE 1A-C shows a schematic representation of the general flavonoid biosynthetic pathway and chemical structure of various flavonoids.

The enzymes involved are abbreviated as follows in Fig. 1A and 1C: CHS = chalcone synthase; CHI = chalconisomerase; FHT = flavanone 3-hydroxylase; DFR = dihydroflavonol 4- Reductase; ANS = anthocyanidine synthase; FGT = flavonoid 3-glucosyltransferase; FNS II = flavone synthase II; FLS = flavonol synthase; F3Η = flavonoid 3'-hydroxylase; F3 ', 5'H = flavonoid 3', 5'-hydroxylase. The level of flavon formation is particularly marked (++++) in Figure 1A. The top part of Figure 1B shows the FNS II reaction in the presence of NADPH with some common flavanones. Other important flavonoids are shown in the lower part. Figure 1C describes the flavonoid biosynthetic pathway as it occurs in gerbera hybrids. The following abbreviations have been used for the different flavonoids: THC = tetrahydroxy chalcone; PHC = Pentahydroxychalkon; NAR = naringenin; ERI = eriodictyol; Ap = apigenin; Lu = luteolin; DHK = dihydrokaempferol; DHQ = dihydroquercetin; Km = kaempferol; Qu = quercetin; LPg = leucopelargonidine; LCy = leucocyanidin; Pg = pelargonidine; Cy = cyanidine.

FIGURE 2 shows the activity or the lack of activity of FNS II in enzyme extract from petals of different gerbera lines: "Th 58" (genotype fns ⁺ fns), "147-150" (fns ⁺ .) And "147-146" ( fns fns). The lines "147-150" and "147-146" are self-progeny from "Th 58". The activity of FNS II was measured by the conversion of ¹ C-labeled naringenin to the corresponding fiavon of apigenin.

FIGURE 3 shows the FNS II activity (■) and the accumulation of flavones (D) in the line "Th 58" (fns ⁺ fns) over various stages of flowering. The different flower stages are defined in Example 2. The flavon content was determined by extraction with ethyl acetate and HPLC analysis, as described in Martens and Forkmann (1998).

FIGURE 4A + B (after Schopfer and Ebel, 1998) shows the known structure of the cytochrome P450 sequences with areas of high sequence conservation. The proline-rich, the oxygen binding and the heme binding region are shown. Fig. 4B shows the heme binding region in detail and also the primers derived from it for the DD-RT-PCR. A set of eight non-degenerate 5 'primers was created according to the possible nucleotide sequence.

FIGURE 5 shows a schematic representation of the various generated cytochrome P450 DNA fragments. All clones contain the labeled heme binding site. pDDd7a: a 358 bp fragment could be PCR by means of the Oligo's "Decamer d7" and "Oligo A" are generated with a DNA template recovered from a differentially expressed band.

pTABATA: a 1519 bp fragment was started from cDNA from Gerbera "Th58" using a PCR-based RACE method with the oligos "GSP7", "GSP8", "GSP9" and "AAP" (GIBCO-BRL) or " backrace "isolated.

pCYPFNSI: a 1589 bp fragment with an open reading frame was isolated by PCR using the oligo's "CypFNSI H" and "CypFNSI R". CDNA from Gerbera "Th58" was used as template.

FIGURES 6 and 7 are representations of the nucleic acid and the amino acid sequence of the complete clone derived therefrom. The start codon and the various stop codons are marked separately.

FIGURE 8 shows a diagram of the existing restriction site for standard restriction enzymes.

FIGURE 9 shows an FNS II test with yeast microsomes. [ ¹⁴ C] Naringenin was used as substrate. Microsomes were prepared from transformed (INVSd - CypFNSI) and untransformed yeast (INVSc 1). The autoradiogram shows the conversion of [ ¹ C] naringenin to the corresponding fiavon, the [ ¹ C] apigenin, with an extract of the transformed yeast (INVSc 1 - CypFNSI). No activity was measured in the control experiment (INVSc 1). The product was identified in four different eluents by co-chromatography with authentic apigenin.

FIGURE 10 shows an autoradiography of an RNA gel blot that was hybridized with a ³² P-labeled cDNA of the insert CypFNSI. Each lane contains 20 μg total RNA, which was applied as follows: {1} Simm (genotype fns ⁺ .), {2} Delphi (fns \), {3} T3 (fns fns), {4} 147-150 ( fns ⁺ .), {5} Clivia (fns fns), {6} 147-146 (fns fns), {7} Regina (fns ⁺ .), {8} Gerbera leaves (fns fns), {9} 10s Pool (fns ⁺ .), {10} pool of 10 (fns fns). EXAMPLE 1

materials

Chemicals, enzymes and radiochemicals

Naringenin, eriodictyol, apigenin and luteolin were obtained from Carl Roth (Karlsruhe, Germany). [ ¹⁴ C] Naringenin was derived from [ ¹ C] malonyl-CoA (ARC, St. Louis, USA) and p-coumaroyl-CoA (Dr. Werner Heller, GSF, Neuherberg, Germany) according to the method described in Britsch et.al. (1981) described method with partially purified chalcone synthase (CHS) and chalcone isomerase from parsley suspension culture. All other enzymes were obtained from commercial suppliers and used according to their description.

Bacterial and yeast strains

The following Escherichia coli strains were used: TOP10F 'and TOP10, both from Invitrogen (Groningen, The Netherlands). The following yeast strain was also used: INVSd (Invitrogen).

The cloning vectors pCR2.1 and pYES2 were obtained from Invitrogen.

The insert and vector pCR2.1 were ligated and the bacteria were transformed according to the manufacturer's instructions.

Plant material

There were chemically defined Ge-nbera clone varieties (Tyrach and Hörn, 1997) and

Progeny progeny of the line "Th58" are available. In addition, the current cut gerbera varieties "Regina" from Terra Nigra (DeKwakel, Niederiande) and "Delphi" from Florist (DeKwakel, Netherlands) were included. A detailed one

A description of the plant material can be found in Table 2.

TABLE 2

Plant material used

Gerbera line genotype origin

Th58 fhs ⁺ fhs Tyrach and Hörn, 1997

Delphi fhs ⁺ fhs florist, Tyrach and Hörn, 1997

Simm fhs ⁺ fhs Tyrach and Hörn, 1997

T3 fhs fhs Tyrach and Hörn, 1997

147-150 fhs ⁺ fns Th 58 x S

Clivia fhs fiis Tyrach and Hörn, 1997

147-146 fhs fhs Th 58 x S

Regina fhs ⁺ . Terra Nigra

Laub Regina fhs fhs Terra Nigra

Pool of 10 (147-. ••) fhs ⁺ . Th 58 x S

Pool of 10 (147- ...) fhs fhs Th 58 x S

EXAMPLE 2

Cultivation of plants, methods of cross and flower stages

Plants of gerbera hybrids were cultivated in a greenhouse under normal conditions. The day length was at least 14 hours with a light intensity of 10,000 lux and at 22 ° C.

Self-cultivations of the variety "Th58" (genotype fns ^* fns) were carried out within an inflorescence or between the inflorescences of the same plant. This self-experiment can be performed with any variety or line heterozygous for the Fns locus. The corresponding chemogenetic and biochemical methods are described in detail in Tyrach and Hörn (1997) and Martens and Forkmann (1998). Controlled blooming was achieved with glassine bags drawn over the flowers. The pollinations were ideally repeated up to four times a day. Further information on pollination methods and flowering in gerberas can be found in Maurer (1967).

Gerbera flowers were harvested at various stages of development, which were defined as follows (according to Martens and Forkmann, 1998):

Stage 1 bud closed, petals smaller than 5 mm. Stage 2 ray flowers visible, 5 - 10 mm long. Stage 3 ray flowers 10-15 mm long. Stage 4: beginning of pigmentation, length 15-23 mm.

Stage 5: Ligules of the ray flowers pigmented, 23 - 26 mm long.

Stage 6: ray flowers 26 - 35 mm long.

Stage 7: inflorescence half open, 35 - 40 mm long. Stage 8: inflorescence fully open, 40 - 50 mm long.

Stage 9: ray flowers 50 - 55 mm long.

Stage 10: ray flowers 55 - 60 mm long.

Stage 11: senescent inflorescence, 55 - 60 mm long.

EXAMPLE 3

Biochemical and environmental characterization of the progeny of self

Known standard methods were used for the extraction and identification of flavonoids (Marbry et.al., 1970; Harborne, 1967). Flavones were also detected under UV light (243 nm) before and after vaporization with ammonia. Flavanones were identified by reduction with sodium borohydride and subsequent treatment with hydrochloric acid vapors (Eigen et al., 1957). Thin layer chromatography was carried out on precoated cellulose plates G1440 from Schleicher & Schüll (Dassel, Germany). The following solvents were used: (1) chloroform - acetic acid - water (10: 9: 1); (2) 30% acetic acid; (3) acetic acid - hydrochloric acid - water (30: 3: 10) and (4) tert-butanol - acetic acid - water (3: 1: 1). The flavon content of buds and flowers during development was determined by extracting the pigments with ethyl acetate from petals of various stages. The tissue to extractant ratio was 1:40 (g / ml) and the extraction time was 48 hours at 4 ° C. in the dark. Characterization and quantification was carried out using HPLC. 10 μl of 75% methanol extract were applied to a Spherisorb ODS II column (particle size 5 μm, 250 x 4.6 mm, Bischoff, Leonberg, Germany) and separated (Lange et.al., 1994). Detection was carried out using a Model 168 diode array detector (Beckmann, Munich, Germany).

Enzyme processing and FNS II tests were carried out as described in Martens and Forkmann (1998). The mixture was worked up with 6.0 ml of Tris-HCl buffer (pH 7.5), which received 28 mmol / l of 2-mercaptoethanol and 10 mmol / l of sodium ascorbate, at 4 ° C with 1.0 g of petals, 0.5 g of Dowex (equilibrated with Tris-HCl buffer , pH 7.5) and 0.5 g sea sand. To "-.

After homogenizing in a pre-cooled mortar, the homogenate was transferred to Eppendorf tubes and twice for 5 min. Centrifuged at 10,000 xg. The now clear supernatant served as a crude extract or was used to precipitate microsomes with MgCl ₂ according to Diesperger et.al. (1974) used. The protein content of the workup was determined using the Bradford (1976) method.

The standard test for the FNS II contained in a total volume of 200 μl: 175 μl Tris-HCl buffer (pH 7.5), 0.3 nmol radioactive labeled substrate (83 Bq; Naringenin), 2.0 nmol unlabeled substrate, 10 μl 20 mmol / l NADPH and 15 μl crude extract or microsome preparation. After an incubation of 20 min. at 25 ° C the reaction was stopped by the addition of 20 ul methanol containing a mixture of the corresponding flavonoids. The reaction mixture was extracted twice with 100 or 50 μl of ethyl acetate. The upper phase was chromatographed on cellulose thin-layer plates in solvent 1 (see above). The radioactivity was localized and quantified using a Fuji BAS 1000 bio-imaging analyzer (Fuji Photo Film Co., Tokyo, Japan) and the TINA software package (Raytest, Straubenhardt, Germany).

Fig. 2 shows an example of the results of the enzyme tests with the Gerbera variety "Th 58" and the lines "147-150" and "147-146". The corresponding genotypes can be found in Table 2.

EXAMPLE 4

Synthesis of oligonucleotides

Oligonucleotides were synthesized by Metabion (Martinsried, Germany). The following oligonucleotides were used (5 '- 3'):

Oligo A: 5'- 1 I I I I I I I I I T (A, C, G) A-3 '(SEQ ID NO: 3)

Oligo C: 5'- 1 I I I I I I I I I T (A, C, G) C-3 '(SEQ ID NO: 4)

Oligo G: 5'- 1 IIIIIIIIIT (A, C, G) G-3 '(SEQ ID NO: 5) decamer 1 5'-CGCCATTTGG-3' (SEQ ID NO: 6) decamer 2 5'-CGCCATTCGG-3 '(SEQ ID NO: 7) Decamer 3 5'-CGCCCTTTGG-3' (SEQ ID NO: 8) Decamer 4 S'-CGCCCTTCGG-S '(SEQ ID NO: 9) Decamer 5 5'-CGCCGTTTGG-3' ( SEQ ID NO: 10) Decamer 6 5'-CGCCGTTCGG-3 '(SEQ ID NO: 11) Decamer 7 5'-CGCCTTTTGG-3' (SEQ ID NO: 12) Decamer 8: 5'-CGCCTTTCGG-3 '(SEQ ID NO: 13) GSP7: S'-ATCπC AGTGTTTCCTCGTTCC-S' (SEQ ID NO: 14) GSP8: 5'-AATGGAACACACACAAAATCTACC-3 '(SEQ ID NO: 15) GSP9: 5'-TCACCACTGAGAGTTCTCTCATGG-3 '(SEQ ID NO: 16) AAP: 5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3' (SEQ ID NO: 17) Backrace: 5'-GCCACGCGTCGACTAGTACQFN '(C) : δ'-CAAAGGATCCCAACACCATGAATACACTCC-S '(SEQ ID NO: 19) CypFNSI R: 5'-AGATAGACCGACTGCCATCAAGAAAGC-3' (SEQ ID NO: 20) The three oligo's and eight decamers were synthesized according to Schopfer and Ebel (1998).

EXAMPLE 5

Cloning of a P450 fragment from Gerbera hybrids

Isolation of total RNA from gerbera petals

Total RNA was obtained from gerbera petals of various defined genotypes (fns ⁺ . Or fns fns; Tab. 2) of stages 2-4, in which the FNS II activity increased (Fig. 3), according to a study by Giuliano et.al. (1993) isolated method. 1.2 g of plant material frozen in liquid nitrogen was ground into fine powder in a pre-cooled Moser and transferred to a likewise pre-cooled Corex tube. The tissue was placed in 3 ml of extraction buffer consisting of 4 M guanidium thiocyanate, 0.15 M sodium acetate (pH 5.3), 0.2% sodium sacrosinate and 0.7% ß-mercaptoethanol, and 2.4 ml of water-equilibrated phenol (saturated with 0.1 M citrate buffer , pH 4.3, Sigma, Deisenhofen, Germany) homogenized by vigorous vortexing. After the addition of 0.6 ml of chloroform, the well-mixed homogenate was left on ice for 20 min and then centrifuged at 15,000 xg (Sorvall RC-5B plus; SS34). The removed top phase was mixed with 1 volume of isopropanol and incubated again on ice for 60 min. After centrifugation at 15,000 xg (Sorvall see above) for 30 min, the upper phase was discarded and the pellet was carefully resuspended in sterile H ₂ O. To remove the polysaccharides, 100% ethanol (20% (v / v) final concentration) was added to the solution, incubated on ice for 20 min and centrifuged at 10,000 xg and 4 ° C. for 10 min. The supernatant containing the nucleic acids was mixed with 1/3 vol. 8 M lithium chloride. After 30 min incubation on ice, centrifugation was carried out at 15,000 xg for 20 min and the supernatant was discarded. The precipitated RNA was washed twice with 1 ml each of 80% ethanol and then resuspended in 50 μl H ₂ O and stored at -70 ° C. Determination of the RNA concentration was carried out spectrophotometrically at a wavelength of 260 nm (Pharmacia Biochrome 4060).

If necessary, poly (A) ⁺ RNA was also isolated from the total RNA by two cycles of oligo (dT) cellulose chromatography (Sambrook et.al., 1989).

Reverse transcription of RNA

5 μg total RNA or 500 ng poly (A) ⁺ RNA, isolated from the different genotypes or flower stages described above, was in a 25 μl batch with one of the three oligo (dT) primers using reverse transcriptase (SuperScript ™ II, GIBCO BRL, Paisley, UK) rewritten to cDNA. 1 μl of the respective oligo (dT) primer (1 μM final) and water up to a volume of 17 μl were added to the RNA. This approach was then at 70 ° C for 10 min. denatured and then put on ice. After the addition of 4 μl 5 x SuperScript First Strand Buffer, 2 μl 100 mM dithiothreitol, 1 μl 10 mM dNTP Mix, the mixture was stirred at 42 ° C. for 2 min. pre-incubated. Then 1 μl (200 U) SuperScript ™ reverse transcriptase was pipetted in directly. The reaction was at 42 ° C for 60 min. and then for 15 min. incubated at 70 ^C C. The following cDNAs were thus available: fns ⁺ A, C or G and correspondingly for the recessive line fns ^" A, C or G. These cDNAs were used directly as templates for the PCR approach. PCR amplification with P450-specific primers

The various cDNAs were amplified by means of PCR, a second, non-degenerate, P450-specific primer (decamer 1-8) being used in addition to the oligo (dT) anchor primer. The PCR mixture contained the following components in a total volume of 20 μl: 6.2 μl water, 1 μl 20 × polymerase buffer, 2.5 mM MgCl ₂ , 0.2 μM dNTP mix, 1 μl ³⁵ S-ATP [1000 Ci / mmol] (ICN Pharmaceutics, Irvine, USA), 0.5 μM one of the eight decamer primers, 1 μM of the oligo (dT) primer corresponding to the cDNA 4 μl from the cDNA approach described above and 0.2 μl 5 U / μl replitherm polymerase (Epicenter, Madison, USA). The following PCR parameters were used: a denaturation step at 94 ° C. for 10 minutes, then 40 cycles at 94 ° C. for 30 seconds, 40 ° C. (annealing) for 2 minutes. and 72 ° C (extension) for 30 sec. and finally a final extension at 72 ° C for 7 min. 4 μl of the PCR product were now mixed with 2 μl formamide loading buffer {80% formamide; 10 mM EDTA (pH 8.0), 1 mg / ml Xylencyanol FF and 1 mg / ml bromophenol blue} provided, mixed, for 2 min. denatured at 95 ° C and then placed directly on ice. The samples prepared in this way were loaded onto a 5% denaturing polyacrylamide gel and separated for 3 hours at a maximum of 40 W. The gel on Whatman paper was then peeled off the glass plate and in fixed in a gel dryer. The radioactivity or differential bands were localized using a Fuji BAS 1000 bio-imaging analyzer (Fuji) and the TINA software package (Raytest). Differently expressed bands in the order of 300 to 500 bp were cut out of the fixed gel with the Whatman paper using a sharp scalpel, transferred to an Eppendorf tube and placed in 100 μl water for 10 min. rehydrated at room temperature. The tube was then at 100 ° C for 10 min. incubated and 2 min. centrifuged at fill speed in the table centrifuge to separate gel residues and paper. The supernatant was transferred to a new Eppendorf tube. After the addition of 10 ul 3 M sodium acetate, 5 ul 10 mg / ml glycogen as carrier and 400 ul 100% ethanol, the DNA was for 60 min. precipitated at -70 ° C. The precipitated DNA was then pelleted by centrifugation at 4 ° C and 14,000 φm, washed twice with 85% ice-cold ethanol, air dried and resuspended in 10 ul water. The cDNA obtained in this way was reamplified in 50 μl PCR batches which contained the following components: 27.8 μl water, 2 μl 20 × polymerase buffer, 4 μl 25 mM MgCl ₂ , 3.2 μl 500 μM dNTP mix, 4 μl 5 μM of the corresponding Decamer primer, 4 μl 25 μM of the corresponding oligo (dT) primer, 4 μl from the eluted DNA described above and 0.5 μl 5 U / μl Replitherm polymerase (Epicenter). The PCR parameters correspond to those of the first amplification. The PCR products were separated using a 1.5% agarose gel and the corresponding amplificates were cloned into the TOPO pCR2.1 vector and then transformed into TOPO 10F 'one-shot competent cells (Invitrogen) according to the manufacturer's instructions. Plasmid isolation from transformed bacteria identified via blue-white screening was carried out using the plasmid Miniprep - Quantum Prep - Kit (Bio-Rad, Munich, Germany) according to the manufacturer's instructions. The inserts with a length between 300 and 500 bp identified after digestion with suitable restriction enzymes (eg Eco Rl; Boehringer Mannheim, Germany) and separation on a 1.5% agarose gel were analyzed with the QIAEX II Gelelutions KIT (QIAGEN, Hilden, Germany) eluted from the gel. These DNA fragments were ³² P-labeled with the Rediprime Labeling Kit (Amersham, Braunschweig, Germany) and used for Northern blot analyzes. DNA sequencing of these and other clones was essentially carried out according to the method described by Sanger et al (1977) with the aid of the Sequenase enzyme version 2.1 (Amersham, Braunschweig, Germany). EXAMPLE 6

Northern blot analysis

Total RNA was isolated as described in Example 5. 10 μg of different total RNA samples were electrophoresed on a 2.2 M formaldehyde / 1.2% (w / v) agarose gel. The running buffer contained 20 mM MOPS (pH 7.0), 5 mM sodium acetate and 1 mM EDTA (pH 7.0). The RNA was transferred to Hybond-NX membrane (Amersham) according to the manufacturer's instructions and hybridized with a ³² P-labeled Eco Rl-Eco Rl pDDd7a cDNA fragment. Prehybridization (1 to 3 hours at 42 ° C) and hybridization (16 to 24 hours at 42 ° C) was carried out in 50% deionized formamide, 5 x SSPE, 5 x Denhardt's, 0.5% SDS. Denatured herring sperm DNA (100 μg / ml) was added to the hybridization step together with the ³² P-labeled sample. The filters were washed twice for 15 minutes. washed at 42 ° C with 2 x SSPE, 1% SDS (w / v) and then once or twice at 65 ° C with 1 x SSPE, 1% SDS (w / v). The radioactivity was localized and quantified using a Fuji BAS 1000 bio-imaging analyzer (Fuji) and the TINA software package (Raytest).

Northern blot analyzes showed that the gene which corresponds to the cDNA clone pDDd7a only in lines with the genotype fns ⁺ . is expressed. No lines of hybridization could be detected in lines with the recessive genotype fns fns (Fig. 10). In addition, the expression pattern across the various flower stages runs parallel to the enzyme activity or fiavon accumulation measured in the flowers (Martens and Forkmann, 1998).

EXAMPLE 7

Isolation of the full-length clone of pDDd7a

The cDNA clone pDDd7a does not correspond to a full-length clone, but only the area from the heme binding site to the poly (A ⁺ ) end (3 'end) of the sequence, including several stop codons. To get the full clone of pDDd7a, one was

PCR-based RACE method according to Frohman et.al. (1988) using the 5'-RACE system,

Version 2.0 (GibcoBRL) used.

First, various gene-specific 5'-RACE primers (GSP7-9) were constructed on the basis of pDDd7a and additionally the nested amplification primer "Backrace".

With the help of GSP7, total RNA was cDNA according to the method in Example 5 rewritten. The First Strand product was then cleaned of excess nucleotides and GSP7 using the High Pure PCR Product Purification Kit (Boehringer Mannheim) according to the manufacturer's instructions. The purified cDNA was provided with an oligo-dC tail by the terminal transferase (TdT). The tailing approach was as follows: 6.5 ul water, 5.0 ul 5 x tailing buffer, 2.5 ul 2 mM dCTP and 10 ul cDNA. This mix was for 3 min. denatured at 94 ° C and then for 1 min. put on ice. The reaction was started by adding 1 μl of TdT and then for 10 min. incubated at 37 ° C. The enzyme was inactivated by incubation at 65 ° C. for 10 min. The PCR amplification of the dC-detailed cDNA was carried out in 0.5 ml thin-walled PCR tubes according to the following protocol: single denaturation at 94 ° C. for 2 min., Then 35 cycles consisting of 94 ° C. for 1 min., 57 ° C. for 1 min. and 72 ° C for 2 min. Finally, a final extension step of 7 min. carried out. The PCR approach consisted of the following components: 31.5 μl water, 5.0 μl 10 × PCR buffer, 3.0 μl 25 mM MgCl ₂ , 1.0 μl 10 mM dNTP, 2.0 μl 10 μM GSP8, 2.0 μl 10μM AAP, 5.0 μl dC -tailed cDNA and 0.5 μl 5 U / μl Taqf DNA polymerase (Promega, Madison, USA). 15 μl of the δ'-RACE product were analyzed on a 1.5% agarose gel using suitable length standards. Specific individual bands in the range of approximately 1.5 kb were cloned in and verified by Northern blot or sequence analysis. With the help of the 5'-RACE a 1.5 kb long specific fragment (pTABATA) was amplified, which only contains total RNA from Gerbera lines with the genotype fns *. hybridized, not with RNA from recessive genotypes. In the range from the gene-specific primer (GSP8) to the heme binding site, this new fragment is homologous to the pDDd7a fragment. The total clone (1698 bp) has an open reading frame and, at the amino acid level, shows 58% homology to the cytochrome P450 clone CYP93B1 (Akashi et al., 1998).

EXAMPLE 8

Expression of pCYPFNSI in yeast

Construction of pYeCYPFNSI

A 1.5 kb Barn HI-Eco R1 fragment, corresponding to pCYPFNSI, was ligated into the yeast expression vector pYES2 opened with Barn HI-Eco R1. The resulting plasmid, designated pYeCYPFNSI, contained the pCYPFNSI cDNA fragment in a sense orientation as an insert. Yeast transformation

The yeast strain INVSc 1 was plasmid pYeCYPFNSI in accordance with the Gietz et.al. (1992). Transformed yeast cells were selected using a complementation marker.

Preparation of yeast microsomes for flavone synthase II tests

Individual colonies of INVSc 1 / pYeCYPFNS1 and INVSc 1 on selection medium

SGI {20 g glucose (w / v), 1 g peptone (Fluka), 6.7 g Yeast Nitrogen Base without amino acids (Difco) and 20 mg L-tryptophan (Fluka) per liter} were then used in 5 x 5 ml SGI-broth inoculated and incubated at 200 φ and 30 ° C for 24 hours. With an OD ₆₀ o of 0.2-0.4 of the 1:10 diluted preculture, complete inoculation was carried out in 250 ml of YPGE {5 g of glucose, 10 g of peptone, 10 g of yeast extract (Fluka) and 3% by volume of ethanol per liter}. The main culture was incubated at 30 ° C and 120 φm. With an OD ₆₀ o of 0.8 to 1.2, induction was carried out by adding 27 ml of 200 g / l sterile galactose solution.

After 12 to 15 hours, with an OD ₆₀ o of about 0.6 to 1.2 of the main culture diluted 1:10, the yeast cells were harvested by centrifugation, once with TEK {50 mM Tris-HCl pH 7.4, 1 mM EDTA and 0.1 M KCI} washed and resuspended in TES-B ^* {50mM Tris-HCl pH 7.4, 1mM EDTA, 0.6mM sorbitol and 2mM DTT}. The yeasts were digested at 4 ° C. with 15 g of glass beads (Sigma) per batch. The glass beads were then washed three times with 5 ml of TES-B ^* and the combined supernatant was adjusted to a final concentration of 0.15 M with 4 M NaCl. The microsomes were precipitated by the addition of 2.5 g PEG-4000 (Fluka) and after a washing step with 2 ml TES-B * in a Potter in 2.5 ml TEG * {50 mM Tris-HCl pH 7.4, 1 mM EDTA, 2 mM DTT} homogenized. The homogenate obtained served as a microsomal enzyme source for the FNS II tests.

FNS II activity was measured using the method of Martens and Forkmann (1998). The standard test for flavone synthase II contained in a total volume of 200 μl: 140 μl Tris-HCl buffer (pH 7.5), 0.3 nmol radioactive labeled substrate (83 Bq; [ ^14C ] naringenin), 10 μl 20 mmol / l NADPH and 50 μl the yeast microsome preparation. After an incubation of 20 min. at 25 ° C the reaction was stopped by the addition of 20 ul methanol containing a mixture of naringenin and apigenin (product). The reaction mixture was extracted twice with 100 or 50 μl of ethyl acetate. The upper phase was carried out on cellulose thin-layer plates in solvent 1 to 4 (see above) chromatographed. The radioactivity was localized and quantified using a Fuji BAS 1000 bio-imaging analyzer (Fuji) and the TINA software package (Raytest). The enzyme extract, which was prepared from INVSc 1 / pYeCYPFNS1, shows a clear FNS II activity, whereas the corresponding fraction from untransformed yeasts showed no activity (Fig. 9). The results of the yeast expression confirm that the cDNA insert pCYPFNSI codes for an FNS II enzyme. Furthermore, the result shows that the expression of the enzyme which is encoded by the Gerbera cDNA clone in yeast is sufficient to achieve the direct formation of flavones. This suggests that only a single enzyme is required for the introduction of the double bond between C2 and C3 (Fig. 1B) and that the cDNA clone, which was described by Akashi et al. (1998), does not represent FNS II, but rather represents a flavanone 2-hydroxylase.

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Claims

PATENT CLAIMS

1. A nucleic acid sequence encoding a flavone synthase II (FNS II), selected from the group consisting of:

(a) the nucleic acid sequence of SEQ ID NO: 1 or a part thereof,

(b) a nucleic acid sequence which hybridizes to the nucleic acid sequence (a) and / or has at least 40% homology to it and which encodes a protein or polypeptide with the biological activity of a flavone synthase II,

(c) a nucleic acid sequence that is degenerate, based on a nucleic acid sequence according to (a) or (b).

2. Nucleic acid sequence which is complementary to the nucleic acid sequence from claim 1.

3. A nucleic acid sequence according to claim 1 or 2, wherein the nucleic acid sequence is DNA or RNA.

4. A nucleic acid sequence according to claim 1, wherein the nucleic acid sequence is derived from a plant from the group of plants consisting of gerberas (Gerbera hybrids), asters (Callistephus chinensis), snapdragons (Antirrhinum majus), chrysanthemums (Chrysanthemum indicum), dahlia ( Dahlia hybrids), gloxinia (Sinningia hybrids), verbena (Verbena hybrids) and streptocarpus (S. hybrids).

5. A recombinant DNA molecule containing a nucleic acid sequence according to one of claims 1 to 4.

6. Recombinant DNA molecule according to claim 5, wherein the recombinant DNA

Molecule is a vector or a vector with a promoter.

7. A host cell containing a DNA molecule according to claim 6.

8. A host cell according to claim 7 which is a bacterial cell, a yeast cell, an insect cell, a plant cell or a mammalian cell.

9. polypeptide encoded by a nucleic acid sequence according to any one of claims 1 to 4.

10. Polypeptide with part or all of the amino acid sequence from SEQ ID NO: 2 or derivatives thereof.

11. The polypeptide according to claim 10, wherein the polypeptide has flavone synthase II activity.

12. The polypeptide according to claim 11, wherein the polypeptide is derived from a plant from the group of plants consisting of gerbera (Genbera hybrids), aster (Callistephus chinensis), snapdragons (Antirrhinum majus), chrysanthemum (Chrysanthemum indicum), dahlia (Dahlia Hybrids), gloxinia (Sinningia hybrids), verbena (Verbena hybrids) and streptocarpus (S. hybrids).

13. Transgenic plant containing a nucleic acid sequence according to one of claims 1 to 4.

14. The transgenic plant according to claim 13, wherein the nucleic acid sequence is suitable for expression and this expression is optionally regulatable or regulated by development.

15. Transgenic plant according to one of claims 13 or 14, selected from the group of plants consisting of gerbera (Gerbera hybrids), aster (Callistephus chinensis), snapdragons (Antirrhinum majus), chrysanthemum (Chrysanthemum indicum), dahlia (Dahlia hybrids), Gloxinie (Sinningia hybrids), Verbena (Ve ena hybrids) and Streptocaφus (S. hybrids).

16. A method for producing a transgenic plant with a changed flower color, comprising introducing a nucleic acid sequence according to one of claims 1 to 4 into a cell of a suitable plant and regenerating a transgenic plant from this cell and cultivating this transgenic plant over a suitable period of time and under conditions suitable for the expression of the introduced nucleic acid sequence.

17. The method according to claim 16, wherein the introduced nucleic acid sequence is expressed in the plant.

18. The method according to claim 16 or 17, wherein the transgenic plant is selected from the group of plants consisting of Euphorbia (E. pulcherrima), cyclamen (Cyclamen persicum), rose (Rosa hybrida), pelargonium (P. spec), begonia ( ß. spec), carnation (Dianthus caryophyllus) and tulip (Tulipa hybrids).

19. The method according to claim 16 or 17, wherein the plant is capable of expressing an endogenous flavone synthase II (FNS II) and this is co-expressed upon expression of the introduced nucleic acid sequence.

20. The method according to claim 19, wherein the transgenic plant is selected from the group of plants consisting of gerbera (Gerbera hybrids), aster (Callistephus chinensis), snapdragons (Antirrhinum majus), chrysanthemum (Chrysanthemum indicum), dahlia (Dahlia hybrids), Gloxinia (Sinningia hybrids), Verbena (Verbena hybrids) and Streptocarpus (S. hybrids).

21. The method according to claim 19 or 20, wherein the endogenously present flavone synthase II (FNS II) activity is reduced by introducing the nucleic acid sequence.

22. Use of a polypeptide according to one of claims 9 to 12 for the synthesis of flavones.

23. Use according to claim 22, wherein the flavones are used as a medicament.

24. Use according to claim 23, wherein the flavones are used in cancer therapy.

25. Use according to claim 22, wherein the flavones are used as bioactive substances.