RU2145636C1 - Method of inducing plant flowering - Google Patents

Abstract

FIELD: plant growing. SUBSTANCE: DNA sequences encoding protein manifesting citrate-synthase activity is smoothly incorporated into plant cell genom. Whole plant is then regenerated from cell. Owing to strengthened production of citrate- synthase in plant cells, accelerated and extended flowering is induced in these cells. EFFECT: increased productivity of plant. 25 cl, 3 tbl, 10 ex

Description

 The present invention relates to methods of inhibiting the formation of flowers and to methods of inducing the formation of flowers in plants, as well as to methods of improving the storage ability of storage organs of useful plants and to methods of reducing tuber germination in tuberous plants. The present invention also relates to DNA sequences encoding plant citrate synthase, and to new plasmids containing these DNA sequences that, as a result of integration with the plant genome, alter the activity of citrate synthase in a plant, as well as transgenic plants in which changes in citrate activity synthases are obtained by introducing the indicated DNA sequences.

 Due to the continuous increase in food demand, resulting from the constant growth of the world's population, one of the tasks of biotechnological research is the desire to increase the yield of useful plants. One of the possibilities to achieve this is, for example, changing the nature of the flowering of beneficial agricultural plants. An increase in the number of flowers is, for example, desirable for plants whose flowers, fruits or seeds are of agricultural importance. Earlier flower formation leads to a shortening of the period between sowing and flowering, which, thus, allows you to cultivate plants in climatic zones with shorter vegetation periods or to use two crops during one vegetation period. Inhibition of flower formation may be appropriate for plants that propagate predominantly by the vegetative method, and can lead to increased accumulation of stored substances in storage organs. One example of such a useful agricultural plant is potato.

 However, a directed change in the nature of flowering in plants has not yet been possible, since the process of inducing the formation of flowers in plants has still not been sufficiently clear. Various substances are considered as flower inducers, such as, for example, carbohydrates, cytokinins, auxin, polyamines and calcium. However, it was generally recognized that flowering is a complex process resulting from the interaction of several factors that have not yet been sufficiently studied (Bernier et al., (1993), Plant Cell, 5: 1147-1155).

 Currently, to change the nature of flowering, as a rule, chemicals are used. For example, it is known that inhibition of flower formation in sugarcane, which leads to a significant increase in sugar production, can be achieved by exogenously introducing various synthetic growth regulators (monuron, diuron, diquat). The use of such synthetic substances, however, is usually associated with significant costs and environmental hazards, which are difficult to assess.

 In this regard, it is desirable to develop methods that allow for a directed change in the nature of flowering, in particular, inhibition or induction of flower formation of various useful plants, while eliminating the use of synthetic substances.

 Thus, it is an object of the present invention to provide methods for producing plants with a modified flowering characteristic, in particular plants that inhibit flower formation, or plants with an earlier flower formation and an increased amount thereof.

 The present invention describes genetic engineering methods that lead to a change in the flowering characteristics of plants as a result of changes in the activity of the enzyme involved in the respiration processes in cells.

 It was unexpectedly found that a strong inhibition of the activity of citrate synthase in the cells of potato plants leads to a complete inhibition of the formation of flowers in these plants and that an increase in the activity of citrate synthase in the cells of transformed potato plants also leads to a change in the nature of flowering plants, in particular to an earlier formation flowers and to increase their number.

 To obtain plants with reduced activity of citrate synthase, DNA sequences encoding enzymes with the enzymatic activity of citrate synthase were isolated from various plant species. These sequences are DNA sequences from plants of the nightshade family (Solanaceae), in particular from Solanum tuberosum and Nicotiana tabacum, and sequences from plants of the creeper family (Chenopodiacae), in particular from sugar beet (Beta vulgaris).

 Therefore, the subject of the present invention is DNA sequences from plants of the nightshade family, in particular from the species Solanum tuberosum and Nicotiana tabacum, and from plants of the cinnamon family, in particular from the species Beta vulgaris, encoding enzymes having the enzymatic activity of citrate synthase, and which, after integration with the plant genome, it is possible to obtain transcripts in which the activity of endogenous citrate synthase can be suppressed, or to obtain transcripts in the cells of which citrate synthase activity can be increased. In particular, the invention relates to DNA sequences encoding a protein having one of the amino acid sequences shown in Seq ID N 1, Seq ID N 2 or Seq ID N 3, or a protein having an almost identical amino acid sequence, and to DNA sequences having one of the nucleotide sequences shown in Seq ID N 1, Seq ID N 2 or Seq ID N 3, or a substantially identical nucleotide sequence. The invention also relates to derivatives of the sequences shown in Seq ID N 1-3, which can be obtained from them by insertion, deletion or substitution of one or more nucleotides or by recombination, and which encode for proteins having the enzymatic activity of citrate synthase .

 Recombinant DNA molecules, for example, plasmids, and bacteria containing these DNA sequences or their parts, or their derivatives, are also the subject of the present invention.

 The term “substantially identical” with respect to DNA and amino acid sequences means that the sequences in question have a high degree of homology and that there is functional and / or structural equivalence between the DNA sequences or amino acid sequences in question. A high degree of homology is understood to mean that the sequence is at least 40% identical, preferably more than 60% identical, and particularly preferably more than 80% identical. Sequences that are homologous to the sequences of the invention and differ from the DNA sequence or amino acid sequence of the invention in one or more positions, are usually variations or derivatives of this sequence and are modifications that perform the same function. In addition, they can also be variations of natural origin, for example sequences from other organisms, or mutations, moreover, these mutations can be caused naturally or be introduced as a result of directed mutagenesis. These variations may also be artificial sequences.

 Proteins encoding various variants of the DNA sequence of the invention have certain common characteristics. The latter may include, for example, enzymatic activity, immunological reactivity, conformation, etc., and physical properties, such as, for example, gel electrophoresis mobility, chromatographic behavior, sedimentation coefficients, solubility, spectroscopic characteristics, stability, etc. .

 It was found that inhibition of flower formation occurs in transformed plants if the DNA sequences encoding citrate synthase are introduced into plant cells and expressed in the antisense orientation, which leads to a decrease in citrate synthase activity in cells.

 In the context of the present invention, inhibition of flower formation means that flowers in transformed plants either no longer develop at all or fewer flowers form than non-transformed plants, or some flowers may form, but do not turn into flowers with normal functions. Inhibition of flower formation also means that the plants still develop flowers, but they are sterile and do not lead to the formation of seeds or fruits, or they are able to function only to a limited extent and lead to the formation of fewer seeds compared to wild-type plants. In particular, inhibition of flower formation means that male sterile flowers or flowers are formed, in the male reproductive organs of which only a small amount of fertile pollen is formed. The term also means that flowers form in plants in which the female reproductive organs are absent, do not have normal functions or their functions are reduced compared to wild-type plants.

 Inhibition of flower formation also means that transformed plants, if they have the ability to bloom, bloom later than non-transformed plants, usually later for several days, preferably later for one or several weeks, most preferably later for 2-4 weeks.

 Therefore, the subject of the present invention is the use of DNA sequences encoding citrate synthase for inhibiting flower formation in plants and the use of such sequences for the expression of untranslated mRNA that inhibits the synthesis of endogenous citrate synthases in cells.

 The present invention also relates to a method of inhibiting the formation of flowers in plants, characterized in that the activity of citrate synthase is reduced in plant cells, moreover, this decrease is obtained preferably by inhibiting the expression of DNA sequences encoding citrate synthase.

 Most preferred are methods in which inhibition of flower formation is achieved by inhibiting the expression of endogenous citrate synthase genes using antisense RNA.

The present invention relates, in particular, to methods of inhibiting the formation of flowers in plants, characterized in that
a) DNA that is complementary to the citrate synthase gene present in the cell is stably integrated into the genome of the plant cell,
b) the specified DNA is expressed in its own way or it is induced by combination with the corresponding elements that control transcription,
c) the expression of endogenous citrate synthase genes is inhibited as a result of the antisense effect and
d) plants regenerate from transgenic cells.

The expression of DNA complementary to the citrate synthase gene present in the cell is usually obtained by integrating a recombinant double-stranded DNA molecule in the plant genome, including a polygenic expression cluster having the following components and expressing them:
A) a promoter that functions in plants,
B) a DNA sequence encoding a citrate synthase, which is fused with the promoter in an antisense orientation so that a non-coding chain is transcribed and, if necessary
C) a signal functioning in plants to terminate the transcription and polyadenylation of an RNA molecule.

 Such DNA molecules are also the subject of the invention. The present invention relates to such DNA molecules that contain the described polygenic expression clusters in the form of pKS-CSa plasmid (DSM 8880) containing the coding region for potato citrate synthase and the TCSAS plasmid (DSM 9359) containing the coding region for tobacco citrate synthase , the structure of which is described in examples 3 and 8, respectively.

 In principle, any promoter active in plants can be used as a promoter. The promoter must ensure that the selected gene is expressed in the plant. It is possible to use such promoters that guarantee significant expression in all plant tissues, such as, for example, the 35S promoter of the cauliflower mosaic virus, and such promoters that guarantee expression only in a certain tissue, at a certain stage of plant development or at a time external influences. The promoter may be homologous or heterologous to the transformable plant.

 The use of tissue-specific promoters is a preferred subject of the invention.

 A DNA sequence encoding a protein having the enzymatic activity of citrate synthase can, in principle, be from any selected organism, preferably from plants. Preferably, the sequence used is derived from those plant species that are used for transformation, or from closely related plant species.

 A preferred embodiment of the above method is that a DNA sequence derived from a plant of the Solanaceae family or the Chenopodiaceae family, in particular Solanum tuberosum, Nicotiana tabacum or Beta vulgaris, is used as the DNA sequence encoding the citrate synthase. Most preferred embodiments of the invention include the use of a DNA sequence encoding a protein having one of the amino acid sequences shown in Seq ID N 1, Seq ID N 2 or Seq ID N 3, or a substantially identical amino acid sequence, in particular a DNA sequence that is identical or almost identical to one of the DNA sequences presented in Seq ID N 1, Seq ID N 2 or Seq ID N 3.

 In addition, using standard methods and the already known DNA sequences encoding citrate synthase, can be isolated from any organisms, preferably from plants, other DNA sequences that encode proteins having the enzymatic activity of citrate synthase. These sequences can also be used in the methods of the present invention.

 The antisense orientation with respect to the promoter of the coding DNA sequence indicated in paragraph B) leads to the formation of untranslated mRNA in transformed plant cells, which prevents the synthesis of endogenous citrate synthase.

 Instead of the complete DNA sequences shown in Seq ID N 1, Seq ID N 2 and Seq ID N 3, partial sequences can also be used for antisense inhibition in accordance with the present invention. Sequences with a minimum length of 15 base pairs may be used. However, an inhibitory effect is not excluded when shorter sequences are used. Longer sequences of 100-500 base pairs are preferably used; in particular, sequences having more than 500 base pairs are preferred for effective antisense inhibition. Typically, sequences having less than 5,000 base pairs are used, preferably sequences that have less than 2,500 base pairs.

 In the method of the present invention, it is also possible to use DNA sequences having a high degree of homology with the DNA sequences of the present invention, but which are not completely identical. Minimum homology should be higher than approximately 65%. It is preferable to use sequences homologous to 95-100%.

 DNA sequences derived from the sequences shown in Seq ID N 1, Seq ID N 2 or Seq ID N 3 can also be used by insertion, deletion or substitution so that the inhibitory effect of the antisense sequence is not disturbed.

 DNA fragments used to construct antisense constructs can also be synthetic DNA fragments obtained using modern DNA synthesis methods.

Plants obtained using the described method and which are characterized by the fact that they exhibit reduced citrate synthase activity in cells as a result of the expression of antisense RNA that is complementary to DNA sequences encoding a protein having the enzymatic activity of citrate synthase are also the subject of the invention. Such plants are also characterized in that they contain a polygenic expression cluster stably integrated into the genome containing the following sequences:
A) a promoter that functions in plants,
B) a DNA sequence encoding a citrate synthase, which is fused with the promoter in an antisense orientation so that a non-coding chain is transcribed and, if necessary
C) a signal functioning in plants to terminate the transcription and polyadenylation of an RNA molecule.

 Plants are preferably the above plants.

 As described in the exemplary embodiments of the invention, when using potato as an example, in potato plants, inhibition of flower formation in transformed plants occurs due to a decrease in citrate synthase activity as a result of the antisense effect. In particular, transformed potato plants exhibit more or less pronounced phenotypes depending on the degree of reduction of citrate synthase activity. A significant decrease in the activity of citrate synthase leads to a complete inhibition of flower formation. Plants with less pronounced inhibition can form buds, from which nevertheless flowers with normal function do not develop. Plants on which flowers develop, but whose female reproductive organs do not have normal function, can also be obtained.

 Similar effects are observed in transgenic tobacco plants, which have reduced activity of citrate synthase. In this case, flowers also develop, the female reproductive organs of which are greatly reduced in size.

 Inhibition of flower formation by reducing the activity of citrate synthase is of interest, however, not only for potatoes or tobacco, but has a broader significance for plant breeding and agriculture. For example, it may be noted that chronologically defined induction or inhibition of flowering can be achieved by combining the DNA sequences of the present invention with exogenously controlled controls. This may play a role in increasing frost resistance.

 The methods of the present invention can be used both in relation to dicotyledonous and in relation to monocotyledonous plants. Plants of particular interest are useful plants, such as some types of crops (e.g. rye, wheat, corn, oats, barley, maize, rice, etc.), some types of fruit crops (e.g. apricot, peach (apple, plum, etc.), certain types of vegetable crops (e.g. tomato, broccoli, asparagus, etc.), ornamental plants or other types of plants of economic importance (e.g. potato, tobacco, rape, soybean , sunflower, sugarcane, etc.).

 The use of the present invention, in particular with respect to sugar beets, is of particular interest, since in this case, “stem” can be prevented by inhibiting the formation of flowers. Since stem temperatures are induced by low temperatures, the seeds are planted relatively late (in April / May) to prevent stalking. Reducing the stalk of sugar beets can be achieved by inhibiting citrate synthase. This allows you to plant sugar beet seeds earlier, which subsequently leads to an increase in yield due to an increase in the growing season.

 In addition to inhibiting the formation of flowers in transformed potato plants, which have a reduced activity of citrate synthase in the cells, a decrease in tuber germination and a decrease in respiration in tuber cells were observed compared to untransformed plants. This leads to a decrease in storage loss and an increase in shelf life of tubers. Therefore, the method of the present invention is also suitable for producing plants with improved storage stability of storage organs, and increased storage stability, as understood in the context of the present invention, means that stored storage organs of transformed plants have less loss of raw tissue mass and mass in dry state after a storage period compared to that for untransformed plants. Under storage organs are understood as ordinary plant organs harvested, such as seeds, fruits, tubers and root crops.

 In particular, the method is suitable for producing transgenic potato plants, the tubers of which are characterized by increased storage stability, reduced storage losses and reduced tuber germination ability compared to wild-type plants. The reduced tuber germination ability of the tubers means that sprouts are formed on the tubers of the transformed plants, which have a lower mass of raw tissue and a dry mass compared to the sprouts of non-transformed plants. The economic benefits of this are obvious.

 Therefore, the invention also provides methods for enhancing storage stability of plant storage organs, characterized in that the activity of citrate synthase in the cells of these plants is reduced, and the decrease in activity is preferably achieved by inhibiting the expression of DNA sequences encoding citrate synthase.

 Most preferred are methods in which the activity of citrate synthase is reduced by inhibiting the expression of endogenous citrate synthase genes using antisense RNA.

The present invention relates, in particular, to methods for increasing storage stability of storage organs of plants, characterized in that
a) DNA that is complementary to the citrate synthase gene present in the cell is stably integrated into the genome of the plant cell,
b) the specified DNA is expressed in its own way or it is induced by combination with the corresponding elements that control transcription,
c) the expression of endogenous citrate synthase genes is inhibited as a result of the antisense effect and
d) plants regenerate from transgenic cells.

 Such methods can be used for all types of plants that develop storage organs, preferably for beneficial agricultural plants, and most preferably for certain types of crops (e.g. rye, barley, wheat, corn, rice, etc.) , certain types of fruit crops, vegetables, plants in which tubers are formed, such as, for example, potatoes or cassava, and plants in which root crops are formed as storage organs, in particular sugar beets.

 The invention also relates to methods for producing transgenic tuberous plants, the tubers of which have reduced germination, characterized in that the activity of citrate synthase in the cells of these plants is reduced, and a decrease in activity is preferably achieved by inhibiting the expression of DNA sequences encoding citrate synthase.

 Most preferred are methods in which the activity of citrate synthase is reduced by inhibiting the expression of endogenous citrate synthase genes using antisense RNA.

The present invention relates, in particular, to methods for producing transgenic tuberous plants, the tubers of which have reduced germination, characterized in that
a) DNA that is complementary to the citrate synthase gene present in the cell is stably integrated into the genome of the plant cell,
b) the specified DNA is expressed in its own way or it is induced by combination with the corresponding elements that control transcription,
c) the expression of endogenous citrate synthase genes is inhibited as a result of the antisense effect and
d) plants regenerate from transgenic cells.

 Such methods can preferably be used to produce transgenic potato and cassava plants.

 The above in relation to the method of inhibiting the formation of flowers is also applicable to various variants of the implementation of these methods, in particular with respect to the choice and length of the used DNA sequence that encodes citrate synthase and the choice of promoter.

 As an alternative to reducing the activity of citrate synthase in plant cells using an antisense effect, this reduction can also be achieved by introducing a DNA sequence encoding a ribozyme that specifically cleaves endogenous citrate synthase gene transcripts in an endonucleotide manner. Ribozymes are catalytically active RNA molecules that have the ability to cleave RNA molecules in specific target sequences. Using methods of genetic engineering, it is possible to change the specificity of the ribozyme. There are various classes of ribozyme. In practical applications, for the targeted cleavage of the transcript of a particular gene, representatives of two different ribozyme groups are preferably used. The first group includes ribozymes, which can be assigned to the introns of group I ribozymes. The second group includes ribozymes, which have the main characteristic structural feature - the sequence-motive, the so-called "hammer head". The specific recognition of the target RNA molecule can be modified by changing the sequences adjacent to the motif sequence. By pairing the bases of the sequences of the target molecule, these sequences determine the site at which the catalytic reaction occurs and, therefore, the cleavage of the target molecule. Since the sequence requirements for efficient cleavage are extremely low, it is in principle possible to obtain specific ribozymes for almost any RNA molecule.

Genetically modified plants in which the activity of citrate synthase is significantly reduced, therefore, can also be obtained by introducing into the plants and expression of a recombinant double-stranded DNA molecule containing:
a) a promoter that functions in plants,
b) a DNA sequence encoding the catalytic region of the ribozyme and "adjacent" to which are located DNA sequences homologous to the sequences of the target molecule and, if necessary
c) a signal functioning in plants to terminate the transcription and polyadenylation of an RNA molecule.

 The sequences defined in paragraph b) include, for example, the catalytic region of the satellite DNA of the SCMo virus (Davies et al., 1990, Virology, 177: 216-2224) or that of the satellite DNA of the TobR virus (Steinecke et al., 1992, EMBO J., 11: 1525-1530; Haseloff and Gerlach, 1988, Nature 334: 585-591).

 DNA sequences adjacent to the catalytic region are formed from DNA sequences that are homologous to endogenous citrate synthase gene sequences.

 The above for the construction of antisense structures is also applicable to the sequences indicated in clauses a) and c).

 Another subject of the present invention is the expression in the sense orientation of DNA sequences encoding proteins having enzymatic activity of citrate synthase in plant cells in order to increase the activity of this citrate synthase. For this, the DNA sequence encoding citrate synthase is fused with the promoter in a sense orientation, i.e. The 3 ′ end of the promoter is linked to the 5 ′ end of the DNA coding sequence. This leads to the expression of mRNA encoding citrate synthase, and, consequently, to an increase in the synthesis of this enzyme.

It was unexpectedly found that as a result of an increase in the activity of citrate synthase in the cells of transformed plants, a change in the nature of flowering occurs compared with non-transformed plants. In particular, flower formation is induced. By this in the context of the present invention is meant the following:
a) earlier flower formation (in this case, this means that the transformed plants bloom earlier than non-transformed plants, usually several days earlier, preferably one or several weeks earlier), and / or
b) an increase in the formation of flowers (in this case, this means that the transformed plants form more flowers, preferably at least 10% more flowers compared to untransformed plants).

 This effect is desirable for a number of cultivated and useful plants, such as some types of vegetables, for example, tomatoes, peppers, pumpkin, melon, Antilles cucumber, zucchini, canola, some types of crops, corn or cotton, and various types of ornamental plants.

 Another object of the present invention is, therefore, the use of DNA sequences encoding proteins having enzymatic activity of citrate synthase for inducing flower formation in plants and methods for inducing flower formation in plants, characterized in that the activity of citrate synthase in the cells of these plants is increased . The activity of citrate synthase is preferably increased by introducing into the plant cells a recombinant DNA molecule containing a coding region for citrate synthase and leading to the expression of citrate synthase in transformed cells.

Such methods preferably include the following steps:
a) stable integration into the genome of a plant cell DNA, which is homologous or heterologous to that inherent in a given organism, and which encodes a protein having citrate synthase activity,
b) the expression of this DNA in its own way or as a result of induction by combining with the appropriate elements that control transcription,
c) an increase due to this activity of citrate synthase in cells and
d) plant regeneration from transgenic cells.

The expression of DNA, which encodes a protein with the enzymatic activity of citrate synthase, is usually achieved by integration into the plant genome of a recombinant double-stranded DNA molecule, including a polygenic expression cluster, having the following components and expressing them:
A) a promoter that functions in plants,
B) a DNA sequence encoding a citrate synthase, which is fused with the promoter in a sense orientation, and if necessary
C) a signal functioning in plants to terminate the transcription and polyadenylation of an RNA molecule.

 Such DNA molecules are also the subject of the invention. The present invention relates to such DNA molecules that contain the indicated polygenic expression clusters in the form of the plasmid pHS-mCS, which contains the coding region for citrate synthase from S. cerevisiae, and the plasmid pEC-mCS, which contains the coding region for citrate synthase from E .coli.

 The DNA sequences encoding citrate synthase shown in step a) of this method can be either homologous or native, or heterologous or of foreign origin to the host plant to be transformed. They can be of both pro- and eukaryotic origin. For example, DNA sequences encoding citrate synthase are known from the following organisms: Bacillus subtilis (U05256 and U05257), E. coli (V01501), R.prowazekii (M17149). P.aeruginosa (M29728), A. anitratum (M33037) (see Schendel et al. (1992) Appl. Environ. Microbiol. 58: 335-345 and references cited therein), Haloferax volcanii (James et al. ( 1992) Biochem. Soc. Trans. 20:12), Arabidopsis thaliana (Z17455) (Unger et al. (1989) Plant Mol. Biol. 13: 411-418), B. coagulans (M74818), C. burnetii (M36338 ) (Heinzen et al. (1991) Gene 109: 63-69), M.smegmatis (X60513), T. acidophilum (X55282), T.thermophila (D90117), pig (M21197) (Bloxham et al. (1981) Proc. Natl. Acad. Sci. 78: 5381-5385), N. crassa (M84187) (Ferea et al. (1994), Mol. Gen. Genet. 242: 105-110) and S. cerevisiae (Z11113, Z23259 , M14686, M54982, X00782) (Suissa et al. (1984) EMBO J. 3: 1773-1781). The numbers in parentheses in each case represent the registration numbers under which these sequences are registered in the GenEMBL database. Sequences can be isolated from these organisms using modern molecular biology methods or can be obtained artificially.

 A preferred embodiment of the method of the present invention includes the use of DNA sequences encoding citrate synthases, which are deregulated or unregulated compared to citrate synthases commonly found in plants, i.e. their enzymatic activity is not regulated by regulatory mechanisms that affect the activity of citrate synthase in plant cells. Deregulation means, in particular, that these enzymes are not equally inhibited by inhibitors or activated by activators that normally inhibit or activate plant citrate synthases. In the context of the present description, unregulated citrate synthases are understood to mean citrate synthases that are not regulated by inhibitors or activators in plant cells.

 Preferred are prokaryotic, in particular bacterial, DNA sequences that encode citrate synthases, since they have the advantage that the proteins encoded by these sequences are unregulated or poorly regulated in plant cells. Therefore, it is possible that an increase in citrate synthase activity occurs due to the expression of additional citrate synthase in plant cells.

 In a preferred embodiment of the method, DNA sequences from E. coli are used that encode a protein with citrate synthase activity, in particular the glt A gene (Sarbjit et al., 1983, Biochemistry 22: 5243-5249).

 Another preferred example of the method of the present invention relates to the use of DNA sequences from Saccharomyces cerevisiae that encode citrate synthase, in particular to the use of DNA sequences described by Suissa et al. (1984, EMBO J. 3: 1773-1781).

 In the case where DNA sequences of plant origin are used, DNA sequences encoding a protein having one of the amino acid sequences shown in Seq ID N 1 or Seq ID N 2 or Seq ID N 3 or a substantially identical amino acid sequence are preferably used. Shorter DNA sequences encoding only portions of the amino acid sequences set forth in Seq ID N 1 or Seq ID N 2 or Seq ID N 3 can also be used, provided that the resulting protein guarantees the enzymatic activity of citrate synthase.

 The most preferred embodiment relates to a method in which the DNA sequence encoding the activity of citrate synthase comprises the nucleotide sequence shown in Seq ID N 1 or Seq ID N 2 or Seq ID N 3, or a substantially identical nucleotide sequence or part thereof and this part is long enough to encode a protein exhibiting citrate synthase activity.

 In addition, using standard methods using already known DNA sequences encoding citrate synthase, other DNA sequences that encode proteins with citrate synthase activity can be isolated from any organism, preferably from plants and prokaryotic organisms. These sequences can also be used in the methods of the present invention.

 Using the methods of the present invention, it is possible to increase citrate synthase activity in principle in each compartment of the transformed cell. Preferably, an increase in activity should occur in mitochondria, glyoxysomes, or a cytosol. In order to guarantee the localization of citrate synthase in a particular compartment of transformed cells, the coding sequence must be linked to the sequences necessary for localization in the corresponding compartment. Such sequences are known. To localize citrate synthase, for example, in mitochondria, it is necessary for the expressed protein to have a target mitochondrial sequence (signal sequence) at the N-terminus that guarantees the transport of the protein expressed in the cytosol to the mitochondria. If the gene used does not already include the sequence encoding the signal peptide, then such a sequence should be introduced using genetic engineering methods. The sequence that encodes the mitochondrial target sequence is known, for example, from Braun et al., (1992, EMBO J. 11: 3219-3227). This sequence should be linked to the coding region so that the polypeptide encoded by the target sequence is in the same reading frame as the subsequent DNA sequence encoding citrate synthase.

 If bacterial DNA sequences encoding citrate synthase are used, then all 5'-untranslated regions are preferably deleted. If the bacterial enzyme has signal sequences, they are preferably replaced with plant signal sequences.

 The above with respect to the methods of the present invention for inhibiting flower formation is equally applicable to the selection of appropriate transcriptional regulatory sequences, in particular promoters for the expression of a DNA sequence encoding citrate synthase and signals for terminating transcription.

 The described method can be applied to both dicotyledonous and monocotyledonous plants. Plants of particular interest are useful plants such as some types of crops (e.g. rye, wheat, corn, barley, maize, etc.), some types of fruit crops (e.g. apricot, peach, apple, plum etc.), certain types of vegetable crops (for example, tomato, pepper, pumpkin, melon, Antilles cucumber, zucchini, broccoli, asparagus, etc.), ornamental plants or other types of plants of economic importance (for example, tobacco, rape, soy, cotton, sunflower, etc.).

The subject of the present invention is also plants obtained using the described method, characterized in that they exhibit increased activity of citrate synthase in cells as a result of additional expression of a DNA sequence encoding a protein having the enzymatic activity of citrate synthase. Such plants are also characterized in that they contain a polygenic expression cluster stably integrated into the genome containing the following sequences:
A) a promoter that functions in plants,
B) a DNA sequence encoding a citrate synthase, which is fused with the promoter in a sense orientation, and if necessary
C) a signal functioning in plants to terminate the transcription and polyadenylation of an RNA molecule.

 Plants are preferably the above plants.

 When combining the DNA sequences of the invention with exogenously regulated transcriptional control elements, for example, temperature-induced promoters, using the described methods to inhibit or induce flower formation, there may also be the possibility of inducing chronologically defined flowering or inhibition of flowering depending on whether the sequence is drained DNA with a promoter in sense or in antisense orientation. Thus, among others, promoters are known for specific expression in flower buds (Huisjer et al., 1992, EMBO J. 11: 1239-1249) or in photosynthetic-active tissues, for example, the ST-LS1 promoter (Stockhaus et al., 1989 EMBO J. 8: 2445-2451). To prevent the germination of potato tubers and to reduce storage losses due to metabolism of stored substances, acceptable promoters are those that guarantee activation of transcription in storage organs. For potatoes, promoters are known that guarantee specific expression in the tuber, for example, class I patatin gene promoters. An example is the promoter of the patatin gene B33 Solanum tuberosum (Rocha-Sosa et al., 1989, EMBO J. 8: 23-29). By combining with exogenously regulated control elements, for example, damage-induced promoters or temperature-controlled promoters, the problem of vegetative propagation can be solved in the case of potato plants whose tubers do not germinate due to inhibition of citrate synthase. In the case of sugar beets in a similar way, using a beet-specific promoter, one can reduce respiration and, as a result, reduce crop losses due to decomposition of sugar in the root crop.

 A large number of cloning vectors that contain a replication signal for E. coli and a marker gene for selection of transformed bacterial cells are acceptable for introducing a foreign gene into higher plants. Examples of such vectors are pBR322, pUC series, M13mp series, pACYC184, etc. The desired sequence can be introduced into the vector at the corresponding restriction cleavage site. The resulting plasmid is used to transform E. coli cells. Transformed E. coli cells are grown in an appropriate medium, then harvested and lysed. The plasmid is regenerated. Analytical methods used to characterize the obtained plasmid DNA, usually include restriction analysis, gel electrophoresis and other methods of biochemistry and molecular biology. After each operation, the DNA plasmid can be cleaved and combined with other DNA sequences. Each plasmid DNA sequence can be cloned in the same or in other plasmids. Numerous techniques are suitable for introducing DNA into a plant host cell. These techniques include transforming plant cells using T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as transformation agents, protoplast fusion, injection, DNA electroporation, DNA injection using the bio-ballistic method and other features.

 When injecting and electroporating DNA into plant cells, there are no special requirements for the plasmids used. Simple plasmids, such as, for example, pUC derivatives, can be used. However, if whole plants need to be regenerated from cells transformed in this way, a selectable marker gene is necessary. In accordance with the method of introducing the required genes into the plant cell, other DNA sequences may be necessary. If, for example, a Ti- or Ri plasmid is used to transform plant cells, then at least the right border region of the T-DNA of the Ti or Ri plasmid, although often the right and left border regions, should be attached as flanking (adjacent ) areas to introduced genes.

 If agrobacteria are used for transformation, the introduced DNA must be cloned in special plasmids, either in an intermediate vector or in a binary vector. Intermediate vectors can be integrated into the Ti- or Ri plasmid of an agrobacterium by homologous recombination, since their sequences are homologous to T-DNA sequences. They also contain the vir region necessary for T-DNA transfer. Intermediate vectors cannot be replicated in agrobacteria. Using the helper plasmid, the intermediate vector can be transferred to Agrobacterium tumefaciens (conjugation). Binary vectors can replicate in both E. coli and agrobacteria. They contain a selection marker gene and a linker (ligation agent) or polylinker, which are limited to the right and left border regions of T-DNA. They can be transformed directly in agrobacteria (Holsters et al. (1978) Mol.Gen.Genet. 163: 181-187). An agrobacterium that serves as a host cell may contain a plasmid carrying the vir region. The vir region is necessary for the transfer of T-DNA into the plant cell. Additional T-DNA may be present. Agrobacterium transformed in this way is used to transform plant cells.

 The use of T-DNA for the transformation of plant cells has been extensively studied and duly described in EP 120516; Hoekema, in The Binary Plant Vector System Offsetdrukkerij Kanters B.V., Alblasserdam (1985), part V; Fraley et al., Crit. Rev. Plant Sci., 4: 1-46 and An et al. (1985) EMBO J. 4: 277-287.

 To transfer DNA into a plant cell, it may be appropriate to jointly cultivate plant explants and Agrobacterium tumefaciens or Agrobacterium rhizogenes. Entire plants can then be regenerated from infected plant material (e.g., leaf pieces, stem segments, roots or also protoplasts or suspension-cultured plant cells) in a suitable medium for this purpose, which may contain antibiotics or biocides for breeding transformed cells. The plants thus obtained can then be examined for the presence of introduced DNA.

 If the introduced DNA is integrated into the genome of a plant cell, then as a rule this integration is stable and remains even in the next generations of the cell, which was originally transformed. It usually contains a selection marker that makes the transformed plant cell resistant to a biocide or antibiotic, such as kanamycin, G 418, bleomycin, hygromycin or phosphinotricin, etc. Thus, an individually selected marker should distinguish transformed cells from cells that lack introduced DNA.

 Transformed cells develop 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 in a normal way and crossed with plants that have the same transformed genetic code or other genetic codes. The resulting hybrid individuals have the corresponding phenotypic properties.

 Two or more generations must be grown to ensure that the phenotypic trait is stably preserved and inherited. A seed crop must also be harvested to ensure that the appropriate phenotype or other characteristics are maintained.

 In addition to the applications already indicated, the DNA sequences of the present invention can also be introduced into plasmids, which allows mutagenesis or sequence modification by insertion, deletion or recombination of DNA sequences in prokaryotic or eukaryotic systems. The sequences can also be provided with control elements for expression in pro- and eukaryotic cells and can be introduced into the corresponding cells.

 The DNA sequences of the invention can also be used to isolate various types of homologous sequences also encoding citrate synthase from the plant genome. In this context, homology means a sequence that is at least 60% identical, preferably more than 80%, and particularly preferably more than 95%. The identification and isolation of such sequences is carried out in accordance with standard methods (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). In turn, based on these sequences, constructs for transforming plants or microorganisms can be obtained.

Escrow
The plasmids obtained and used within the scope of the present invention were deposited at the German Collection of Microorganisms (DSM) in Brunswick, Federal Republic of Germany, recognized as an international depository in accordance with the requirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the purposes of the patenting procedure . The following plasmids were deposited at the German Microorganism Collection (DSM) on December 28, 1993 under deposit numbers:
Plasmid pPCS - (DSM 8879)
Plasmid pKS-CSa - (DSM 8880)
The following plasmids were deposited at the German Microorganism Collection (DSM) on 08/10/1994 under deposit numbers:
Plasmid pTCS - (DSM 9357)
Plasmid pSBCS - (DSM 9358)
Plasmid TCSAS - (DSM 9359)
Abbreviations Used
BSA - Bovine Serum Albumin
EDTA - (ethylene dinitrile) tetraacetic acid
50x Denhart solution - 5 g of ficol (type 400, Pharmacia), 5 g of polyvinylpyrrolidone, 5 g of bovine serum albumin (fraction V, Sigma), water up to 500 ml
FADN ₂ - reduced flavin adenine dinucleotide
MOPS - 3- (N-morpholino) propanesulfonic acid
NADH - b-nicotinamide adenine dinucleotide, reduced
PCR - polymerase chain synthesis reaction (polymerase chain reaction)
PMSF - phenylmethylsulfonyl fluoride
SCMo virus - "underground clover mosaic virus"
SDS - sodium dodecyl sulfate
20xSSC 175.3 g NaCl, 88.2 g sodium citrate, up to 1000 ml water, pH 7.0 with 10N NaOH
TobR - "tobacco ring spot virus"
Trizin - N-Tris (hydroxymethyl) methylglycine
Description of drawings
In FIG. 1 is a schematic of the pPCS plasmid (DSM 8879). The closed line corresponds to the pBluescript KS sequence. The bold line corresponds to cDNA encoding a citrate synthase from Solanum tuberosum. The restriction cleavage sites of the insert segment are shown.

 In FIG. 2 shows a schematic of the plasmid pKS-CSa (DSM 8880).

The structure of the plasmid:
A corresponds to fragment A: CaMV 35S promoter, nucleotides 6909-7437 (Frank et al., (1980) Cell 21: 285-294);
B corresponds to fragment B: cDNA from Solanum tuberosum encoding citrate synthase; BamHI / SalI fragment from pPCS, approximately 1900 base pairs. Orientation with respect to the promoter: antisense;
C corresponds to fragment C: nucleotides 11748-11939 from the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., (1984) EMBO J. 3: 835-846).

 In FIG. 3 shows the scheme of the plasmid pSBCS (DSM 9358). The closed line corresponds to the sequence pBluescript SK. The bold line corresponds to cDNA encoding citrate synthase from Beta vulgaris L. The restriction cleavage sites of the insert segment are shown.

 In FIG. 4 shows the scheme of the plasmid pTCS (DSM 9357). The closed line corresponds to the sequence pBluescript SK. The bold line corresponds to the cDNA encoding citrate synthase from Nicotiana tabacum. The restriction cleavage sites of the insert segment are shown.

 In FIG. 5 is a schematic representation of the plasmid TCSAS (DSM 9359).

The structure of the plasmid:
A corresponds to fragment A: CaMV 35S promoter, nucleotides 6909-7437 (Frank et al., (1980) Cell 21: 285-294);
B corresponds to fragment B: cDNA from Nicotiana tabacum encoding citrate synthase; BamHI / SalI fragment from pTCS, approximately 1800 base pairs. Orientation with respect to the promoter: antisense;
C corresponds to fragment C: nucleotides 11748-11939 from the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., (1984) EMBO J. 3: 835-846).

In FIG. Figure 6 shows the experimental results obtained by Northern blotting. In each case, 2 μg of poly (A ⁺ ) mRNA from various transgenic potato plants (bands 4-8) and three non-transformed potato plants (bands 1-3) were used for analysis.

 Lanes 1, 2 and 3 correspond to the wild type Solanum tuberosum of the Desiree variety.

 Lane 4 corresponds to potatoes of the T6 transgenic line.

 Lane 5 corresponds to potatoes of the transgenic line T21.

 Lane 6 corresponds to the potato transgenic line T29.

 Lane 7 corresponds to potatoes of the T50 transgenic line.

 Lane 8 corresponds to potatoes of the T55 transgenic line.

 For hybridization, radioactively labeled potato citrate synthase cDNA was used.

In FIG. 7 shows transgenic potato plants of the T6 line (NN 3 and 4) and T29 (NN 5 and 6), which were transformed with pKS-CSa plasmid, in comparison with wild-type plants (NN 1 and 2). Plants were grown in a greenhouse at a relative humidity of 60%, at 22 ^° C. for a 16-hour light period, and at 15 ^° C. for an 8-hour dark period.

 In FIG. Figure 8 shows in a diagram the number of flowers formed on potato plants that were transformed with the plasmid pKS-CSa, in comparison with wild-type plants. The number of plants with fully developed open flowers during the flowering period is shown. Five transgenic lines (T6, T21, T29, T50 and T55) were compared with wild-type plants. The T21 transgenic line is a transgenic line in which there is no inhibition of citrate synthase (100% citrate synthase activity). During the study period, no flowers on any plant of the T29 line developed, and plants of the T6 and T50 line began to bloom only about 3 weeks later than wild-type plants. Day 1 indicates the first day on which well-defined buds were found on the plants.

 "wt" refers to the wild type.

 t6, t21, t29, t50, t55 denote transgenic lines T6, T21, T29, T50 and T55.

 In FIG. 9 shows longitudinal sections of flower buds of wild-type plants and for comparison of transgenic plants of the T29 line.

A: Flower bud of a wild-type plant
B: Larger view of the structure of the ovary of the kidney from A
C: Flower bud of a transgenic T29 line plant
D: Larger view of the structure of the ovary of the kidney from C
an: anthers
ov: ovary
re: petals
se: sepals
The damage to the tissue of the ovaries of transgenic plants is clearly visible.

 In FIG. 10 shows the germination process of tubers of T6 line potato plants (left), which were transformed with pKS-CSa plasmid, in comparison with wild-type tubers (right). The tubers were stored for 9 months in the dark at room temperature.

 In FIG. Figure 11 shows a flower of a tobacco plant that was transformed with the plasmid TCSAS (left), compared with a flower of an untransformed tobacco plant (right). The flower pistil of the transformed plant is much shorter than the flower pistil of the wild-type plant.

 In FIG. 12 is a schematic diagram of the plasmid pHS-mCS.

The structure of the plasmid:
A corresponds to fragment A: CaMV 35S promoter, nucleotides 6909-7437 (Frank et al., (1980) Cell 21: 285-294);
B corresponds to fragment B: a 99 bp DNA fragment encoding a target mitochondrial matrix sequence of a processing peptidase (MPP) (Braun et al., 1992, EMBO J. 11: 3219-3227);
C corresponds to fragment C: a DNA sequence from Saccharomyces cerevisiae encoding citrate synthase (nucleotides 376-1818; Suissa et al., 1984, EMBO J. 3: 1773-1781); orientation with respect to the promoter: semantic;
D corresponds to fragment D: nucleotides 11748-11939 from the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., (1984) EMBO J. 3: 835-846).

 In FIG. 13 shows two transgenic potato plants from two independent lines that were transformed with the plasmid pHS-mCS (in the middle and on the right), in comparison with the wild-type plant (on the left). Plants are grown in a greenhouse and their age is approximately 6 weeks. While wild-type plants have not yet formed inflorescences, each of the two transgenic potato plants already has fully developed inflorescences.

 In FIG. 14 is a schematic diagram of the pEC-mCS plasmid.

The structure of the plasmid:
A corresponds to fragment A: CaMV 35S promoter, nucleotides 6909-7437 (Frank et al., (1980) Cell 21: 285-294);
B corresponds to fragment B: a DNA fragment with a length of 99 base pairs encoding a mitochondrial target sequence of a matrix processing peptidase (MPP) (Braun et al., 1992, EMBO J. 11: 3219-3227);
C corresponds to fragment C: a DNA sequence from E. coli encoding citrate synthase (nucleotides 306-1589; Sarbjit et al., 1983, Biochemistry 22: 5244-5249); orientation with respect to the promoter: semantic;
D corresponds to fragment D: nucleotides 11748-11939 from the T-DNA of the Ti plasmid pTiACH5 (Gielen et al., (1984) EMBO J. 3: 835-846).

 To illustrate the examples, the most important techniques used are presented below.

1. The cloning procedure
For cloning in E. coli, the pBluescript KS vector and the pBluescript SK vector (Stratagene, USA) were used.

 To transform plants, gene constructs were cloned into the pBinAR binary vector.

2. Bacterial strains
For pBluescript vectors and pBinAR vectors, E. coli strain DH5α (Bethesda Research Laboratories, Gaithersburg, USA) was used. For in vivo excision, E. coli strain XL1-Blue was used.

 Plasmid transformation in potato and tobacco plants was carried out using the Agrobacterium tumefaciens strain C58C1 (Rocha-Sosa et al., (1989) EMBO J. 8: 23-29).

3. Transformation of Agrobacterium tumefaciens
DNA was transferred by direct transformation in accordance with the methods of Hofgen & Willmitzer (1988, Nucleic Acids Res. 16: 9877).

 The plasmid DNA of the transformed agrobacteria was isolated according to the Birnboim & Doly method (1979, Nucleic Acids Res. 7: 1513-1523) and analyzed after appropriate restriction digestion using gel electrophoresis.

4. Potato Transformation
Ten scalpel-cut small leaves of a sterile potato culture (Solanum tuberosum L., Desiree variety) were placed in 10 ml of MS medium (Murashige & Skood (1962) Physiol. Plant. 15: 473) with 2% sucrose containing 50 μl grown in overnight with selection of Agrobacterium tumefaciens culture. After careful shaking for 3-5 minutes, the resulting system was further incubated in the dark for 2 days. Thereafter, for callus induction, the leaves were transferred to MS medium containing 1.6% glucose, 5 mg / L naphthylacetic acid, 0.2 mg / L benzylaminopurine, 250 mg / L claforan, 50 mg / L kanamycin and 0.80% Bactoagar . After incubation for 1 week at 25 ^o C and illumination of 3000 lux for the induction of shoot formation, the leaves were transferred to MS medium containing 1.6% glucose, 1.4 mg / l zeatinribose, 20 mg / l naphthyloacetic acid, 20 mg / l gibberillic acids, 250 mg / l of claforan, 50 mg / l of kanamycin and 0.80% bactoagar.

5. Tobacco transformation
An overnight culture of the corresponding Agrobacterium tumefaciens clone was separated by centrifugation (6500 rpm; 3 min) and the bacteria were resuspended in YEB medium. Leaves of tobacco sterile tobacco culture (Nicotiana tabacum, cultivar Samsun NN) were cut into small pieces of approximately 1 cm ² and immersed in a bacterial suspension. Pieces of leaves were then added to MS medium (0.7% agar) and incubated for 2 days in the dark. Then, leaf pieces for the induction of shoot formation were transferred to MS medium (0.7% agar) containing 1.6% glucose, 1 mg / L benzylaminopurine, 0.2 mg / L naphthylacetic acid, 500 mg / L claforan and 50 mg / L kanamycin. The environment was changed every 7-10 days. If shoots developed, pieces of leaves were transferred to glass vessels containing the same medium. The formed shoots were cut off and placed on MS medium with the addition of 2% sucrose and 250 mg / L claforan and whole plants were regenerated from them.

6. Determination of citrate synthase activity in tissues of transgenic potato and tobacco plants and untransformed potato and tobacco plants
To determine citrate synthase activity, crude extracts were obtained from tubers, leaves, and flowers, and mitochondria were isolated from potato tubers. To obtain crude extracts, the test material was frozen in liquid nitrogen, homogenized in extraction buffer (Neuhaus and Stitt 1 (1990) Planta 182: 445-454), centrifuged, and a liquid supernatant was used for subsequent activity assessment. To isolate mitochondria from potato tubers, 100-200 g of freshly picked tubers were purified and homogenized in 100 ml of “grinding buffer” (0.4 M mannitol, 1 mM EDTA, 25 mM MOPS, 0.1% BSA, 10 mM b-mercaptoethanol, 0 , 05 mM PMSF, pH 7.8). The homogenate was filtered through 4 layers of cotton gauze and centrifuged for 4 min at 3500g. The supernatant was filtered through 2 layers: Miracloth (Calbiochem) and centrifuged again for 30 minutes at 18000g. Debris was resuspended using a soft brush in 2 ml of resuspending buffer (0.4 M mannitol, 20 mM Trisin, 2 mM EDTA, pH 7.2). After two homogenization in a potter homogenizer, the extract was applied to percoll with a variable gradient and centrifuged for 1 hour at 72000g. Mitochondria were removed from the interphase 28% / 45%, washed and centrifuged twice for 15 min at 14500g in “washing buffer” (0.4 M mannitol, 5 mM MOPS, 0.1% BSA, 0.2 mM PMSF, pH 7 ,5). Next, mitochondria were resuspended in 100 μl of resuspended buffer. To determine citrate synthase activity, 5 μl of a suspension of mitochondria was extracted into 100 μl of extraction buffer (Neuhaus and Stitt (1990) Planta 182: 445-454).

The activity of citrate synthase was determined by spectrophotometry at 412 nm and 30 ^o C in accordance with the Srere method (1967, Methods in Enzymology 13: 3-22).

7. RNA Extraction and Experiments Using Northern Blotting
RNA was isolated from frozen plant material as described by Logemann et al. (1987, Anal. Biochem. 163: 21-26). RNA was denatured in 40% formamide. Then, RNA was separated by gel electrophoresis on formaldehyde / agarose gels and, after passage of the gel, they were blotted on a nylon membrane (Hybond N; Amersham, UK). Hybridization with a radioactively labeled DNA sample was carried out in accordance with standard methods.

8. Cultivation of plants
Potato plants (Solanum tuberosum) were grown in a greenhouse at a relative humidity of 60% and at a temperature of 22 ^° C. for a 16-hour light period and at a temperature of 15 ^° C. for an 8-hour dark period. Tobacco plants (Nicotiana tabacum) were grown in a greenhouse at a relative humidity of 60% and at a temperature of 25 ^o C for a 14-hour light period and at a temperature of 20 ^o C for a 10-hour dark period.

Examples
Example 1
Potato citrate synthase cDNA cloning
To identify cDNA from potato encoding citrate synthase, a DNA fragment was first amplified from the already known citrate synthase cDNA from Arabidopsis thaliana (Tal cutter) (Unger et al. (1989) Plant Mol.Biol. 13: 411-418). For this, whole DNA was extracted from the green (chlorophyll-containing) plant tissue of Arabidopsis thaliana plants and poly (A ⁺ ) mRNA was obtained from it. Then it was used to obtain cDNA. Using oligonucleotides
5'-AAGTGGATCCATGGTGTTTTTTCCGCAGCGTAT-3 '(Seq ID N 4) and
5'-CATAGGATCCTTAAGCAGATGAAGCTTTCTTA-3 '(Seq ID N 5),
which are complementary to the 5 ′ or 3 ′ end of the coding region of a citrate synthase cDNA from Arabidopsis thaliana (Unger et al. (1989) Plant Mol. Biol. 13: 411-418), a 1438 bp DNA fragment encoding citrate synthase from Arabidopsis thaliana, cDNA was isolated from this preparation using a "polymerase chain reaction" (PCR). BamHI cleavage sites were additionally introduced into the nucleotides used at both ends of the amplified DNA fragment. The resulting PCR DNA fragment was digested with BamHI and ligated with plasmid pUC9.2 digested with BamHI. The insertion cDNA of this plasmid was further used as a heterologous sample to identify cDNA encoding potato citrate synthase.

To obtain a cDNA library, poly (A ⁺ ) mRNA was isolated from the leaves of potato plants. Starting with poly (A ⁺ ) mRNA, cDNA equipped with EcoRI / NotI linkers was prepared and the cDNA library was placed with them in the Lambda ZAP II vector (Strategene, USA) (Kossmann et al. (1992) Planta 188: 7-12 ) Using a heterologous sample from Arabidopsis thaliana, 250,000 plaques from this cDNA library were examined for the presence of DNA sequences homologous to this sample. For this, plaques were transferred onto nitrocellulose filters and denatured by treatment with NaOH. Then the filters were neutralized and DNA was fixed on the filters using heat treatment. The filters were pre-hybridized for 2 hours at 42 ^° C in a composition containing 25% formamide, 0.5% BSA, 1% SDS, 5xSSC, 5x Denhart solution, 40 mM sodium phosphate buffer with a pH of 7.2 and 100 μg / ml salmon sperm DNA. Then the filters, after adding P ^32- labeled cDNA encoding citrate synthase from Arabidopsis thaliana, were hybridized overnight at 42 ^° C in a composition containing 25% formamide, 0.5% BSA, 1% SDS, 5xSSC, 5x Denhart solution, 40 mM sodium phosphate buffer with a pH of 7.2 and 100 μg / ml salmon sperm DNA. The filters were washed for 30 min in 5xSSC and 0.5% SDS at 42 ^o C. and 20 minutes in 3xSSC and 0.5% SDS at 42 ^o C.

 Phage clones from the cDNA library that underwent hybridization using cDNA from Arabidopsis thaliana were further purified using standard techniques. Using in vivo excision method, E. coli clones were obtained from positive phage clones that contain the double-stranded plasmid pBluescript with the corresponding cDNA insert at the EcoRI cleavage site of the polylinker. After controlling the size and type of restriction of the inserts, the corresponding clone was subjected to sequencing.

Example 2
Sequencing of cDNA insert of plasmid pPCS (DSM 8879)
Plasmid pPCS (Fig. 1) was isolated from the E. coli clone obtained in accordance with Example 1, and its cDNA insert was determined by standard methods using the dideoxy method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467). The insertion length is 1891 base pairs. The nucleotide sequence corresponds to the following sequence (Seq ID N 1).

Example 3
Construction of the plasmid pKS-CSa (DSM 8880) and transfer of the plasmid to potato plants
A DNA fragment of approximately 1.9 kbp length having the following sequence (Seq ID N 1) and containing a cloning region for potato citrate synthase was isolated from the pPCS plasmid by digestion with BamHI / SalI. This DNA fragment was cloned into the pBinAR vector (Hofgen and Willmitzer (1990) Plant Sci. 66: 221-230) digested with BamHI / SalI. The pBinAR vector is derived from the binary vector Binl9 (Bevan (1984) Nucleic Acids Res. 12: 8711-8721).

 The resulting plasmid, designated pKS-CSa, is shown in FIG. 2.

 By inserting a cDNA fragment, a polygenic expression cluster is obtained, which is constructed as follows from fragments A, B and C (Fig. 2).

 Fragment A (529 base pairs) contains the 35S promoter of cauliflower mosaic virus (CaMV). The fragment includes CaMV nucleotides 6909 to 7437 (Franck et al. (1980) Cell 21: 285-294).

 Fragment B contains the protein coding region of potato citrate synthase. It was isolated as described above as a BamHI / Sall fragment from pPCS and fused in the antisense orientation with a promoter in the pBinAR vector.

 Fragment C (192 base pairs) contains the polyadenylation signal of the T-DNA gene 3 of the Ti-plasmid pTiACH5 (Gielen et al. (1984) EMBO J. 3: 835-864). The size of the pKS-CSa plasmid is approximately 12.9 kb.

The pKS-CSa vector was transferred to potato plants using the Agrobacterium tumefaciens-pipelined transformation. Intact plants were regenerated from transformed cells. As a result of this transformation, transgenic potato plants showed varying degrees of reduction in mRNA encoding citrate synthase (see FIG. 6). In experiments using Northern blotting, 2 μg of poly (A ⁺ ) mRNA was hybridized with a potato citrate synthase probe. The transcript encoding citrate synthase and characteristic of wild-type plants (bands 1 to 3) is shorter than the antisense transcript of the polygenic expression cluster (see, for example, band 6), which shows that the decrease in endogenous transcripts occurring in different transgenic plants, different.

 The citrate synthase activity of various tissues of transgenic potato plants was studied, which showed a decrease in mRNA encoding citrate synthase. The results of these studies of leaves, tubers and mitochondria isolated from tubers are given in table. 1.

 A decrease in the activity of citrate synthase has a significant effect on the formation of flowers in transgenic plants, the severity of which depends on the degree of inhibition of the activity of citrate synthase.

 In transformed potato plants, in which the activity of citrate synthase is greatly reduced (see Table 1), flower formation is inhibited to a large extent or completely (see Figure 7).

 Plants in which the activity of citrate synthase is reduced only to a relatively small extent exhibit slow flower formation, and they form fewer flowers or develop only flower buds, which later die, without turning into flowers with normal functions. This can be seen in FIG. 8. It shows the number of plants with fully developed open flowers during one flowering period. Five transgenic lines (T6, T21, T29, T50, and T55) were compared with wild-type plants. The T21 transgenic line is a transgenic line that lacks citrate synthase inhibition (100% citrate synthase activity). During the experiment, no plants of the T29 line developed flowers, and in plants of the T6 and T50 lines, flower formation began only about 3 weeks later than in wild-type plants.

 In other plants, flowers develop, but do not become functional, as female reproductive organs (ovaries) are seriously damaged. In these plants, the ovaries disintegrate during formation. This is shown in FIG. 9. This figure shows a comparison of longitudinal sections through the flower buds of wild-type plants and transgenic T29 plants. The tissues of the ovaries of transgenic plants are seriously damaged compared to wild-type plants.

 Therefore, using the present invention, it is also possible, in accordance with the method of the invention, to obtain plants in which the activity of citrate synthase is inhibited to various degrees, so that transgenic plants could be selected with the desired phenotype, for example, with complete inhibition of flower formation, or those in which the onset of flower formation is delayed compared to non-transformed plants, or those that develop buds that do not turn into flowers with normal functions.

 A decrease in citrate synthase activity can also have a significant effect on various properties of tubers of transformed potato plants. For example, tubers of transformed potato plants exhibit less storage loss after relatively long storage periods compared to tubers of non-transformed plants. This is expressed in a smaller reduction in the mass of raw tissue and dry mass during storage. The following table shows the wet weight and dry weight of tubers of transformed potato plants (line T6) and wild-type plants of the Desiree variety. The tubers were stored for 9 months at room temperature. The masses of tubers are given as a percentage relative to the masses of raw tuber tissue at the beginning of storage. The indicated values are given as mean values obtained from 3-12 measurements, with standard deviations. The values of the mass in the dry state and the mass of raw tissue of tubers of wild-type plants after 9 months of storage are taken as 100% (Table 2).

 In tubers of transformed potato plants, a change in the characteristics of germination was also found. The sprouts of these tubers are significantly smaller compared to those of wild-type tubers and have a significantly lower mass of raw tissue and dry mass. The following table presents the values of the mass of raw tissue and the mass in the dry state of sprouts of tubers of transformed potato plants (line T6) and wild-type plants of the Desiree variety. Sprouts were taken from tubers stored in the dark for 9 months at room temperature. The masses of sprouts for each case are given in grams. The indicated values are given as average values obtained from 3-12 measurements, with standard deviations (Table 3).

 Altered germination characteristics are also shown in FIG. 10. In each case, 3 tubers of transformed T6 line potato plants and 3 tubers of the wild-type Desiree plant are shown. The tubers were stored in the dark for 9 months at room temperature. On the tubers of transformed plants (left), the sprouts are significantly smaller and shorter than those on wild-type tubers (on the right).

Example 4
Cloning of cDNA encoding tobacco citrate synthase (Nicotiana tabacum)
To identify cDNA from Nicotiana tabacum encoding citrate synthase, a tobacco leaf cDNA bank was created as described in Example 1 for potatoes. Using a radioactive DNA probe for sequences encoding citrate synthase, 250,000 plaques from this cDNA bank were screened. The coding citrate synthase cDNA from Solanum tuberosum (1.4 kb, NruI / HindII fragment from pPCS plasmid; see examples 1 and 2 and Seq ID N 1) was used as a probe. The identification and isolation of phage clones hybridized with the used radioactive DNA probe was carried out similarly to that described in Example 1, with the difference that the plaques were transferred to nylon membranes and the following buffer was used for prehybridization and hybridization: 0.25 M sodium phosphate buffer with pH 7.2 , 10 mM EDTA, 7% SDS, 10 mg BSA.

 Using in vivo excision method, E. coli clones that contain the double-stranded pBluescript plasmid with the corresponding cDNA insert were obtained from positive phage clones. After controlling the size and type of restriction of the inserts, the corresponding clone was subjected to sequencing.

Example 5
Sequencing of cDNA insert of plasmid pTCS (DSM 9357)
Plasmid pTCS (Fig. 4) was isolated from the E. coli clone obtained in accordance with Example 4 and its cDNA insert was determined by standard methods using the dideoxy method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467). The insertion length is 1747 base pairs. The nucleotide sequence corresponds to the following sequence (Seq ID N 3).

Example 6
Cloning of cDNA encoding sugar beet citrate synthase (Beta vulgaris L.)
To identify cDNA from sugar beet encoding citrate synthase, a sugar beet leaf tissue cDNA bank (Beta vulgaris L. cultured line 5S0026) was created by isolating poly (A ⁺ ) mRNA from leaf tissue and using it for cDNA synthesis using commercially available kits (Pharmacia LKB, Stratagen, USA) in accordance with the method of Gubler and Hoffmann (1983, Gene 25: 263-269). Using radioactive DNA probes for sequences encoding citrate synthase, we screened 250,000 plaques from this cDNA bank as described in Example 4. As a probe, a mixture consisting of citrate synthase encoding radioactively labeled cDNA from Solanum tuberosum was used (see examples 1, 2 and 4 and Seq ID N 1) and a radiolabeled cDNA coding citrate synthase from Nicotiana tabacum (see Examples 4 and 5 and Seq ID N 3). Identification and isolation of phage clones hybridized with the used radioactive DNA sample was carried out similarly to that described in Example 1. Using in vivo excision method, E. coli clones containing the double-stranded pBluescript plasmid with the corresponding cDNA insert were obtained from positive phage clones. After controlling the size and type of restriction of the inserts, the corresponding clone was subjected to sequencing.

Example 7
Sequencing of cDNA insert of plasmid pSBCS (DSM 9358)
Plasmid pSBCS (Fig. 3) was isolated from the E. coli clone obtained in accordance with Example 6, and its cDNA insert was determined by standard methods using the dideoxy method (Sanger et al. (1977) Proc. Natl. Acad. Sci. USA 74: 5463-5467). The insertion length is 1,551 base pairs. The nucleotide sequence corresponds to the following sequence (Seq ID N 2).

Example 8
Construction of the plasmid TCSAS (DSM 9359) and transfer of the plasmid to tobacco plants
A DNA fragment of approximately 1,800 kbp length, having the following sequence (Seq ID N 3) and containing the coding region for Nicotiana tabacum citrate synthase, was isolated from the pTCS plasmid by digestion with BamHI / SaII. This DNA fragment was cloned into a pBinAR vector digested with BamHI / Sall (Hofgen and Willmitzer (1990) Plant Sci. 66: 221-230). The pBinAR vector is derived from the binary vector Bin19 (Bevan (1984) Nucleic Acids Res. 12: 8711-8721). The resulting plasmid, designated TCSAS, is shown in FIG. 5.

 By inserting a cDNA fragment, a polygenic expression cluster is obtained, which is constructed as follows from fragments A, B and C (Fig. 5).

 Fragment A (529 base pairs) contains the 35S promoter of cauliflower mosaic virus (CaMV). The fragment includes CaMV nucleotides 6909 to 7437 (Franck et al. (1980) Cell 21: 285-294).

 Fragment B, in addition to flanking regions, contains the protein coding region of citrate synthase from Nicotiana tabacum. It was isolated as described above as a BamHI / SalI fragment from pTCS and fused in the antisense orientation with a promoter in the pBinAR vector.

 Fragment C (192 base pairs) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al. (1984) EMBO J. 3: 835-864).

 The size of the plasmid TCSAS is approximately 12.75 TPN

 The plasmid was transferred to tobacco plants using the conveyor transformation of agrobacteria, as described above. Entire plants were regenerated from transformed cells.

 The success of plant genetic modification was evaluated by analyzing the whole RNA for the absence of endogenous mRNA encoding citrate synthase. The activity of citrate synthase in various tissues of transgenic tobacco plants was determined. The results of these studies showed that using this method can be obtained tobacco plants, citrate synthase activity of which is reduced to varying degrees.

 Therefore, as in the case of potato plants, various tobacco lines can be obtained that differ in the degree of reduction of citrate synthase activity.

 Transformed plants with altered flowering characteristics have also been found in tobacco. Of particular interest is the fact that lines can be obtained in which flowers form with strongly shortened pestles compared to those of flowers of untransformed plants. This can be seen in FIG. 11, which shows flowers of transformed and non-transformed tobacco plants. This means that the inhibition of flower formation as a result of a decrease in the activity of citrate synthase in both tobacco and potato primarily affects the female organs of the flowers. Another phenotypic manifestation of these lines is that they form significantly fewer seeds compared to wild-type plants, and the number of seeds is determined in relation to the total mass of the formed seeds.

Example 9
Construction of the plasmid pHS-mCS and transfer of the plasmid to potato plants
To construct the plasmid, the pHS-mCS was first integrated into the pUC18 vector the DNA sequence encoding the mitochondrial target sequence of the matrix processing peptidase (MPP). This sequence was isolated by polymerase chain synthesis (PCR) reaction from the pBluescript plasmid, which contains the MPP cDNA sequence (Braun et al., 1992, EMBO J. 11: 3219-3227) using the following oligonucleotides:
oligonucleotide a): 5'-GATC GGT ACC ATG TAG AGA TGC GCA TCG TCT-3 '(Seq ID N 6) and
oligonucleotide b): 5'-GTAC GGA TCC CTT GGT TGC AAC AGC AGC TGA-3 '(Seq ID N 7).

 The resulting DNA fragment contained nucleotides 299 to 397 of the sequence shown by Braun et al. (1992, EMBO J. 11: 3219-3227) encoding matrix processing peptidase. At the 5'-end of the oligonucleotide sequence a), an Asp718 cleavage site was inserted. BamHI cleavage site was inserted at the 3'-end of oligonucleotide sequence b).

 The DNA fragment obtained by PCR was digested with Asp718 and BamHI and cloned in pUC18 vector digested with Asp718 and BamHI. The resulting vector was designated as pMTP.

A citrate synthase coding DNA sequence from Saccharomyces cerevisiae was cloned into the pMPT plasmid behind the mitochondrial target sequence in the same reading frame. For this, genomic DNA was obtained from yeast by modern methods and a fragment of a length of 1443 base pairs containing the coding region of yeast citrate synthase using PCR was isolated using oligonucleotides:
oligonucleotide c): 5'-CTAG GGA TCC ATG TCA GCG ATA TTA TCA ACA ACT AGC AAA AGT-3 '(Seq ID N 8) and
oligonucleotide g): 5'-GATT GGA TCC TTA GTT CTT ACT TTC GAT TTT CTT TAC CAA CTC-3 '(Seq ID N 9).

 In particular, the sequence includes nucleotides 376-1818 of the sequence shown by Suissa et al. (1984, EMBO J. 3: 1773-1781). The oligonucleotides used introduce the BamHI cleavage site at both ends of the amplified DNA sequence. The resulting DNA fragment was digested with BamHI restriction endonuclease and then ligated with the BamHI digested pMPT vector and transformed in E. coli cells. By determining the type of restriction, a clone was selected in which the insertion of the PCR fragment occurred in such a way that the coding region was attached to the target mitochondrial sequence in a sense orientation, i.e. so that the 5'-end of the coding region is connected to the 3'-end of the target sequence. The resulting plasmid was designated as pMTP-YCS.

 Using restriction endonucleases Asp718 and Xbal, a fragment of approximately 1,550 base pairs containing the mitochondrial target sequence and the coding region of yeast citrate synthase was isolated from the pMTP-YCS vector. This fragment was ligated with the binary vector pBinAR (Hofgen and Willmitzer, 1990, Plant Sci. 66: 221-230) digested with Asp718 and XbaI. The resulting plasmid pHS-mCS is shown in FIG. 12. The binary vector pBinAR is derived from the binary vector Bin19. The vector contains a 35S promoter and a transcription termination signal, between which there is a polylinker that can be used to insert various DNA sequences.

 By inserting a cDNA fragment encoding a yeast citrate synthase with a target mitochondrial sequence at the N-terminus, a polygenic expression cluster is obtained which is constructed as follows from fragments A, B, C and D (Fig. 12).

 Fragment A (529 base pairs) contains the 35S promoter of cauliflower mosaic virus (CaMV). The fragment includes CaMV nucleotides 6909 to 7437 (Franck et al. (1980) Cell 21: 285-294).

 Fragment B contains a 99 bp DNA fragment encoding the mitochondrial target sequence of the matrix processing peptidase (nucleotides 299-397 of the sequence shown by Braun et al. (1992, EMBO J. 11: 3219-3227).

 Fragment C contains the coding region of citrate synthase from Saccharomyces cerevisiae (nucleotides 376-1818 of the sequence shown by Suissa et al. (1984, EMBO J. 3: 1773-1781) fused in the sense orientation with the 3'-end of the target sequence and in the same reading frame as the target sequence.

 Fragment D (192 base pairs) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al. (1984) EMBO J. 3: 835-846).

 The size of the pHS-mCS plasmid is approximately 12.5 kb.

 A transcript encoding yeast citrate synthase and containing at its N-terminus an amino acid sequence guaranteeing protein transport to mitochondria is transcribed from this polygenic expression cluster using the 35S promoter.

 The plasmid was transferred to potato plants using the conveyor transformation of agrobacteria, as described above. Whole plants were regenerated from the transformed cells.

 As a result of transformation, transgenic potato plants were capable of expressing yeast citrate synthase in their cells. This has been demonstrated by Western blotting using polyclonal antibodies that specifically recognize yeast citrate synthase.

 Transformed potato plants that have a high ability to express yeast citrate synthase have altered flowering characteristics compared to untransformed potato plants. On the one hand, it was found that transformed plants began to form flowers much earlier (in greenhouse conditions, on average, 2-4 weeks) and formed more flowers compared to non-transformed plants.

 An earlier flower formation in transgenic potato plants is shown in FIG. 13. It shows two transgenic potato plants that have been transformed with the plasmid pHS-mCS, in comparison with the wild-type Desiree plant.

 Transgenic plants also formed significantly more flowers.

 In particular, after the first inflorescences wither, transgenic plants, as a rule, developed second inflorescences, and in some cases even third inflorescences. In contrast, wild-type plants bloom only once and die after these inflorescences fade.

Example 10
Construction of the plasmid pEC-mCS and transfer of the plasmid to potato plants
To obtain the pEC-mCS plasmid, the DNA sequence from E. coli encoding citrate synthase was cloned into the pMPP plasmid described in Example 9, behind the mitochondrial target sequence in the same reading frame. For this, genomic DNA was obtained from E. coli DH5α strain by modern methods and a fragment of approximately 1280 base pairs containing the coding region of citrate synthase from E. coli was isolated using oligonucleotides using PCR
oligonucleotide d): 5'-GTAGGGATCC ATGGCTGATA CAAAAGCAA-3 '(Seq ID N 10) and
oligonucleotide e): 5'-GATTGGATCCTTAACGCTTGATATCGCTT-3 '(Seq ID N 11).

 The sequence, in particular, contains nucleotides 306-1589 of the sequence given by Sarbjit et al. (1983, Biochemistry, 22: 5243-5249). The oligonucleotides used introduce the BamHI cleavage site at both ends of the amplified DNA sequence. The resulting DNA fragment was digested with BamHI restriction endonuclease and then ligated with the BamHI digested pMPT vector, and introduced into E. coli cells during transformation. By determining the type of restriction, a clone was selected in which the insertion of the PCR fragment occurred in such a way that the coding region was attached to the target mitochondrial sequence in sense orientation, i.e., in such a way that the 5'-end of the coding region was connected to the 3'-end target sequences. The resulting plasmid was designated as pMTP-ECCS. Using restriction endonucleases Asp718 and XbaI, a fragment containing the target mitochondrial sequence and the coding region of E. coli citrate synthase was isolated from this vector. This fragment was ligated with the pBinAR binary vector digested with Asp718 and XbaI (Hofgen and Willmitzer, 1990, Plant Sci. 66: 221-230). The resulting plasmid pEC-mCS is shown in FIG. fourteen.

 By inserting a DNA fragment encoding E. coli citrate synthase with a target mitochondrial sequence at the N-terminus, a polygenic expression cluster is obtained which is constructed as follows from fragments A, B, C and D (Fig. 14).

 Fragment A (529 base pairs) contains the 35S promoter of cauliflower mosaic virus (CaMV). The fragment includes the nucleotides CaMV 6909-7437 (Franck et al. (1980) Cell 21: 285-294).

 Fragment B contains a 99 bp DNA fragment encoding the mitochondrial target sequence of the matrix processing peptidase (nucleotides 299-397 of the sequence shown by Braun et al. (1992, EMBO J. 11: 3219-3227).

 Fragment C contains the coding region of citrate synthase from E. coli (nucleotides 306-1589 of the sequence shown by Sarbjit et al., 1983, Biochemistry, 22: 5243-5249), fused in the sense orientation with the 3'-end of the target sequence and in the same reading frame as the target sequence.

 Fragment D (192 base pairs) contains the polyadenylation signal of gene 3 of the T-DNA of the Ti plasmid pTiACH5 (Gielen et al. (1984) EMBO J. 3: 835-846).

 The size of the pEC-mCS plasmid is approximately 12.4 kb.

 A transcript encoding E. coli citrate synthase and containing an amino acid sequence guaranteeing protein transport to mitochondria is transcribed from this polygenic expression cluster using the 35S promoter.

 The plasmid was transferred to potato plants using the conveyor transformation of agrobacteria, as described above. Whole plants were regenerated from the transformed cells and analyzed for citrate synthase activity.

 A sequence listing is provided at the end of the description.

Claims (9)

 1. A method of inducing flower formation in plants, in which the activity of citrate synthase is increased in plant cells.  2. The method according to claim 1, in which an increase in citrate synthase activity is achieved by expression of a recombinant DNA molecule that is stably integrated into the genome of plant cells and which contains the coding region of citrate synthase and leads to the expression of citrate synthase in transformed cells.  3. The method according to claim 2, in which a) a DNA that is homologous or heterologous in origin and which encodes a protein having citrate synthase activity is stably integrated into the genome of the plant cell; b) the specified DNA is expressed in its own way or by induction by combination with appropriate elements that control transcription; c) as a result of this expression, the activity of citrate synthase in transgenic cells is increased, and d) plants are regenerated from transgenic cells.  4. The method according to claim 2 or 3, in which the DNA sequence contains a nucleotide sequence encoding a protein having the amino acid sequence represented in SegID 1, or SegID 2, or SegID 3, or almost identical amino acid sequence or part of these sequences, where the protein encoded by a DNA sequence or part thereof exhibits citrate synthase activity.  5. The method according to claim 2 or 3, in which the DNA sequence contains the nucleotide sequence represented in SegID 1, or SegID 2, or SegID 3, or a substantially identical nucleotide sequence or part thereof, where the protein encoded by the DNA sequence or part thereof, exhibits citrate synthase activity.  6. The method according to claim 2 or 3, where the DNA sequence encodes a deregulated or unregulated citrate synthase.  7. The method according to claim 3, where the DNA sequence is derived from Saccharomyces cerevisiae.  8. The method of claim 3, wherein the DNA sequence is from a prokaryotic organism.  9. The method of claim 8, wherein the prokaryotic organism is E. coli. Priority on points and signs:
03.09.94 PP 1 to 3, as well as an attribute relating to the sequence of SEQID 1 in paragraphs 4 and 5;
09/22/94 by claims 7 to 9, as well as features related to the sequences of SEQID 2 and SEQID 3 in claims 4 to 5;
03/07/95 according to claim 6.

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