WO2000050598A1

WO2000050598A1 - Dna sequence of a protein that is similar to fkbp

Info

Publication number: WO2000050598A1
Application number: PCT/DE2000/000506
Authority: WO
Inventors: Burkhard Schulz
Original assignee: Sympore Gmbh
Priority date: 1999-02-22
Filing date: 2000-02-18
Publication date: 2000-08-31
Also published as: AU3654900A; CA2363816A1; DE19907598A1; EP1159425A1

Abstract

The present invention relates to a nucleic acid sequence according to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:7 or the fragment or derivative thereof or a nucleic acid sequence which is hybridised with the nucleic acid sequence according to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:7 and which is provided with the biological activity of the nucleic acid sequence according to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:7. The invention also relates to transgenic plants and the seeds thereof, whereby said plants comprise a recombinant inventive nucleic acid sequence.

Description

DNA sequence of a protein similar to FKBP

The present invention relates to a nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ LD NO: 5 or SEQ LD NO: 7 or its fragment or derivative or a nucleic acid sequence which corresponds to the nucleic acid sequence according to SEQ LD NO: 1, SEQ LD NO: 2, SEQ LD NO: 5 or SEQ LD NO: 7 hybridizes and has the biological activity of the nucleic acid sequence according to SEQ LD NO: 1, SEQ LD NO: 2, SEQ LD NO: 5 or SEQ LD NO: 7. The invention further relates to transgenic plants and their seeds comprising a recombinant nucleic acid sequence according to the invention.

The sessile way of life of plants demands a high adaptability to the environmental conditions of the location. The endogenous growth and development programs must be tailored to exogenous factors. This requires the perception of exogenous factors that are vital for the plant. Since the site of perception is usually different from the location of the stimulus response, inter- and intracellular signal transduction must take place. Although stimuli are perceived in plants and animals via different receptors and lead to different stimulus responses, they often use the same principles to convey signals. G proteins, calcium or calmodulin, protein kinases and protein phosphatases are elements of signal transduction chains that occur in plants and animals. The general mechanism of signaling is conserved in many cases.

Immunophilins are a large family of conserved proteins, the functions of which are still poorly known in signaling (Schreiber, 1991, Science 251: 283-287). The immunophilins are a superfamily whose members can be found in bacteria, yeasts, plants and animals. They are located in different cell compartments and involved in the most diverse processes for signal transmission. They have been identified as intracellular receptors for immunosuppressive substances in mammalian cells (Handschumacher et al., 1984, Science 266: 544-547). The immunophilins can be divided structurally and by their affinity for immunosuppressants into three classes: Cyclophiline, the CyclosporinA bind, FK506 binding proteins that bind FK506 or rapamvcin and parvulins that have no affinity for immunosuppressive substances. CyclosporinA, FK506 and rapamycin are substances that are synthesized by soil-dwelling fungi. Their effect in mammals is to suppress the immune response, which is used in transplant medicine to reduce the rejection reaction against a foreign organ.

The FK506-binding proteins (FKBPs) are classified according to their size. The smallest FKBP in eukaryotes, FKBP 12, is a relatively well-studied immunophilin. It provides different responses depending on the bound immunosuppressive substance (Bram et al., 1993, Mol. Cell. Biol. 13: 4760-4769; Brown et al., 1994, Nature 369: 756-758; Liu et al, 1991 , Cell 66: 807-815). The binding of FK506 to FKBP 12 leads to complex formation with the calcium / calmodulin-dependent protein phosphatase calzineurin.

Calzineurin is involved in numerous signal transduction and its inhibition by the

FKBP12-FK506 complex mediates, among other things, the suppression of T cell activation. The suppression of the immune response by FK506 is mediated, in contrast to rapamycin, by the inactivation of calcineurin (Schreiber and Crabtree, 1992, Immunology Today 13: 136-142). In complex with rapamycin, FKBP 12 interacts with the protein mTOR (mammalian target of rapamycin). One domain of the protein mTOR has sequence homology to the catalytic domain of phosphatidylinositol kinases (Sabatini et al., 1994, Cell 78: 35-43). The response that FKBP 12 mediates with rapamycin results in interleukin-2 stimulated T cells to be locked in the Gl phase of the cell cycle. In the absence of immunosuppressive substances, FKBP12 interacts with elements of other signal transduction, for example with the receptor for the transforming growth factor-b (TGF-b receptor) and modulates its function in cell cycle control (Wang et al, 1994, Science 265: 674-676) . FKBP 12 is also involved in the regulation of two intracellular calcium channels, namely the inositol-1,4,5-triphosphate receptor and the ryanodine receptor (Brillantes et al, 1994; Cameron et al, 1995, Cell 83: 463-472). FK506 or rapamycin led to the dissociation of FKBP 12 and calcineurin from the calcium channel complexes and above that to an increased calcium efflux through these channels. The regulation of the calcium channels by FKBP 12 could be based on investigations of a transgenic mouse mutant that did not functional FKBP12 exposed, confirmed (Shou et al, 1998, Nature 391 489-492) FKBP12-deficient mice died before or shortly after birth of a cardiac muscle weakness, which was also observed in patients who were treated with FK506. The calcium conductivity the ryanodine receptors in skeletal muscles of this mouse resembled those of a purified receptor without bound FKBP 12

FKBP59 from mammals was identified as an essential component of steroid receptor complexes not bound to ligands (Sanchez et al, 1990, Biochemistry 29 5145-5152). In this multiprotein complex there are also two heat shock proteins. Hsp70 and Hsp90, have been identified. The binding of FKBP59 to the steroid receptor takes place indirectly via Hsp90 (Peattie et al, 1992). The interaction of FKBP59 and Hsp90 is mediated by a conserved protein-protein interaction motif, the so-called tetramine peptide repeats (TPR) TPR motif is a 34 amino acid sequence that was originally found in proteins which are involved in cell cycle regulation, transcription regulation, protein transport and in the heat shock response (Goebl and Yanagida, 1991, TIBS 16 173-177). The TPR domains of Type III consists of the repetition of the TPR motif three times, two of the repetitions directly following one another. The distance to the first TPR motif is conserved and is 10-16 amino acids. The sequence motifs form amphipatic α-heights, which act as a "knob-hole" structure have been referred to and can mediate a specific protein-protein interaction

The binding of a steroid hormone to the receptor complex leads to the dissociation of FKBP59 and Hsp90. The hand-bound steroid receptor can now get into the nucleus and is bound to DNA in the construction of a transcription complex. It is discussed that FKBP59 and the Hsp proteins do not preserve the conformation of the Ligand-bound steroid receptor are required (Pratt and Welsh, 1994, Sem Cell Biol 5 83-93)

A few years ago, immunophilins were isolated from plant extracts from Vicia faba via their affinity for FK506 and CyclosporinA (Luan et al, 1994, Proc Natl Acad Sei USA 91 984-988). An FKBP12 was also isolated, which has high sequence homology to FKBP12 from yeast and In animals, however (between 47% -51% amino acid sequence identity), this FKBP12 from Vicia faba showed only slight affinity for calcineurin, and when exposed to yeast, it did not mediate the effects of FK506 and rapamycin (Xu et al., 1998, Plant J 15 511 - 519). In Vicia faba, injected FK506 could only inhibit calcium-dependent regulation of potassium channels in guard cells if human FKBP 12 was also administered exogenously (Luan et al, 1993, Proc. Natl. Acad Sei USA 90: 2202-2206), which is a The presence of an FKBP12-FK506 signal transduction chain in plant cells is indicated without an endogenous receptor for FK506.

Plant breeding has been trying for a long time to improve the desired properties of useful and ornamental plants. So far, these improvements have been achieved through very long-term and costly methods of conventional breeding. However, the development of new plant varieties and products often takes 10 to 15 years. An alternative strategy then consists in providing plants with an improvement in the desired properties for useful and ornamental plants by using genetic information such as "marker-supported breeding" and genetic engineering changes. A desired aspect is to increase the yield by increasing the number or the Volume of harvestable seeds (1000 grain weight), which are the yield-determining organs in many crops, but the aim is not only to increase the number and volume parameters, but also to avoid seed loss due to seed fall before harvesting and to reduce the number of seeds Loss of threshing during harvest: During seed ripening, after filling the seeds with storage substances, the phase of seed rest is initiated.This development phase is characterized by the drying of the seed-bearing organs, eg pods or other fruit r phase along the seams of the organ to ensure the spreading of the seeds From a production point of view, this process, which is important for the spreading of the generative organs of a plant, is not desired. Seeds that are released from the fruit by weather influences such as precipitation or wind before the start of the harvest seeds, as well as seeds that fall to the ground due to mechanical manipulations during the harvesting process, must be considered as loss of harvest.

The change in the overall architecture of a plant with the aim of reducing shoot growth has long been a declared breeding goal for crops whose yield is determined by harvestable reproductive organs. On the one hand, there is the possibility of shifting the ratios of the biomass from vegetative areas of the plant that are not relevant to yield into yield-determining organs. Second, through the shortening of shoot areas increases the strength of the plant against weather influences. These aspects are particularly important when growing cereal crops, as a relevant part of the loss of yield due to stalks (lodging) is made up before the harvest. Grain yields have increased significantly over the past 50 years, with dwarfism-mediating mutations such as Rhtl, Rht2, Rht3 in wheat or D8 and D9 in maize, which have been crossed into commercially used varieties, making a significant contribution to this increase in yield . The result of this breeding work was lines that reacted with the addition of artificial fertilizer not with lengthening the stalk but with increasing the seed yield (Silverstone and Sun, Trends in Plant Science 5: 1-2 (2000).

Reduced growth is also often desirable for ornamental plants. Here, particular attention is paid to the production of bonsai plants as well as reduced versions of many ornamental plants and cut flowers e.g. Sunflowers pointed out. In this context, the twisted growth can also be of interest, since shrub and tree plants with twisted growth e.g. Corkscrew willows or ficus can be found in the range of ornamental plants. In the production of timber, twisted growth of the shoot and branches can produce desirable properties. In trees, on the one hand, wood with modified strength properties and modified yields can be obtained by forming so-called "compression wood". Plants with strongly lignified shoot organs produce increased amounts of compression wood when the given direction of growth changes to relieve mechanical stress. This property can be used in the production of wood for paper production. Wood that is used as construction timber or for the production of furniture can be produced by twisted growth of the harvestable shoot areas with changed strength, since this changes the pressure and tensile strength properties of the shoot areas. Due to the twisted growth, fiber-producing plants can produce plant fibers with new, desired processing properties and physical properties (strength, etc.).

The tasks mentioned here are solved by the subject matter of the claims.

The invention is explained in more detail by the following figures: FIG. 1 shows the genomic sequence of the twisted dwarf gene from Arabidopsis thahana, okotype Wassilewskija, including the promoter area. The start and stop codon are shown underlined. Exon sequences are shown in bold, intron sequences are printed in italics. The numbered positions are indicated at the beginning of the nucleotide sequence the lines below the nucleotide sequence each indicate the amino acid sequence of the open reading frame. Amino acid positions are numbered at the end of the line

FIG. 2 shows a representation of the amino acid comparison of the twd gene from Arabidopsis thahana (TWD) and Lvcopersicon esculentum (TTP). Identical amino acids are connected by a vertical stitch, similar amino acids are connected by two points

FIG. 3 shows a representation of the amino acid comparison of the twd gene from Arabidopsis thahana (TWD) and Zea maγs (ZmTWD). Identical amino acids are connected by a vertical stitch, similar amino acids are connected by two points

The term "vector" used here denotes naturally occurring or artificially produced constructs for the uptake, multiplication, expression or transfer of nucleic acids, for example plasmids, phagemids, cosmids, artificial chromosomes. Bacteriophages, viruses, retroviruses

The term “derivatives” used here denotes nuclear acid sequences or amino acid sequences which have one or more modifications such as deletions, substitutions, additions, insertions and / or inversions

The term "fragments" used here denotes nuclear acid sequences or amino acid sequences which comprise a partial area of the nuclear acid sequences or amino acid sequences according to the invention

The term "transformed plant cell" used here denotes plant cells and plants or plant organs resulting therefrom which are genetically generated by transmission of nucleic acids, for example plasmids, phagemids, cosmids, artificial chromosomes, bacterophages, viruses, retroviruses or nucleic acid sequences which were not included in vector constructs have been changed.

The term "control elements" used here denotes nuclear acid sequences which serve for the regulation of the expression of a gene. These nucleic acid sequences contain both promoter regions of a gene and also regulatory regions within the translated and untranslated regions of a gene.

The term "hybridization or" hybridize "as used herein means stringent and less stringent conditions, see Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory (1989), ISBN 0-87969-309-6. An example of stringent hybridization conditions is : Hybridization in 4 x SSC at 65 ° C (alternatively in 50% formamide and 4 X SSC at 42 ° C), followed by several washing steps in 0.1 x SSC at 65 ° C for a total of one hour - an example of less stringent Hybridization conditions are hybridization in 4 x SSC at 37 ° C, followed by several washing steps in 1 x SSC at room temperature.

The term "homologous sequence" or "homologous" used here denotes a nucleic acid or protein sequence which the activity of the nucleic acid or protein sequences according to SEQ LD NO: 1, SEQ LD NO: 2, SEQ FD NO: 5 or SEQ LD NO: 7 have. Homologous sequences also include nucleic acid sequences which hybridize with the sequence according to SEQ LD NO: 1, SEQ LD NO: 2, SEQ LD NO: 5 or SEQ LD NO: 7 or parts of these sequences under stringent or less stringent conditions. Furthermore, homologous sequences are to be considered nucleic acid or protein sequences or parts thereof which, with the aid of the similarity algorithm BLAST (Basic Local Alignment Search Tool, Altschul et al., Journal of Molecular Biology 215, 403-410 (1990) (Matrix: Blosum 62 , Gap existence cost: 11, Per residue gap cost: l) have a significant similarity to the nucleic acid and amino acid sequences of the present invention, and sequences used as significantly similar, as used here, are those which, for example, use standard parameters in the blast service of the NCBI have a probability level of P <le ^"30 if they are compared with the sequences according to SEQ D NO: 1 or SEQ ID NO: 2 or parts thereof.

The term "marker-supported breeding" used here denotes the selection of Plants using genetic information and molecular markers derived from them such as AFLP, RFLP, SNP etc. in breeding programs. The molecular markers mentioned represent all types of nuclear acid sequence changes which can be detected by means of diagnostic DNA analyzes such as PCR, restriction analysis or hybridization and used to screen plant populations

From a population of Arabidopsis thahana varieties that were mutagemated by T-DNA insertion, a mutant could be isolated that is characterized by a drastic change in its phenotype. The twisted dwarf mutant (hereinafter referred to as twd) has a pleiotropic phenotype, the Expression manifested in plant architecture and physiology The n mutant is greatly reduced in its overall size, at the time of senescence it only reaches a third of the wild type overall size (approx. 25 cm) The mutant, like other Arabidopsis dwarf mutants, is darker green and appears compact due to the shortened inflorescences The growth of the rosette leaves is characterized by extremely epinastic curvature and an irregular surface. Spread rosette leaves show that the ratio of leaf length to leaf blade is smaller than that of wild-type rosette leaves.The greatly shortened shoot axis of inflorescence has oneLarger diameter than wild-type plants The disoriented growth of the shoot axis gives the mutant an unusual appearance for Arabidopsis thahana, which is reminiscent of a tendril plant. The disoriented growth of the plant organs can also be observed in the stamens and in the ovary

The DNA sequences of the mutated gene were isolated from the mutant using the plasmid rescue method in E. coli. The fact that the T-DNA used for the mutagenesis contains two sequence regions which enable replication and selection in E coh cells is used By cleavage of genomic DNA of the twd mutant with suitable residual endonucleases (here EcoRI), DNA fragments were generated which were introduced into transformation-competent E cσ / z cells after self-healing. The selection of plasmid-bearing clones was carried out by resistance to the antibiotic ampicilhn on solid Nutrient media From these clones, DNA of the transformed plasmids was isolated and identified by cleavage with residual function endonucleases and subsequent hybridization with hybridization samples of the T-DNA clones used which, in addition to T-DNA sequences, also contained DNA sequences of the mutated twd locus. These DNA sequences were isolated and subcloned into the vector pBluescript (SK-) ® (Stratagene, USA). The inserted DNA sequences were sequenced using the Sanger chain termination method. For the subsequent isolation of genomic and cDNA clones of the twd gene from gene libraries, the cloned DNA sequences were used as hybridization probes.

The present invention relates to a nucleic acid sequence according to SEQ ID NO: 1, SEQ LD

NO: 2, SEQ LD NO: 5 or SEQ LD NO: 7 or their fragment or derivative or one

Nucleic acid sequence that corresponds to the nucleic acid sequence according to SEQ LD NO: 1, SEQ LD NO: 2,

^~ SEQ LD NO: 5 or SEQ LD NO: 7 hybridized and has the biological activity of the nucleic acid sequence according to SEQ LD NO: 1, SEQ LD NO: 2, SEQ LD NO: 5 or SEQ LD NO: 7. The invention further relates to a nucleic acid sequence which hybridizes with the nucleic acid sequence according to SEQ LD NO: 1, SEQ LD NO: 2, SEQ LD NO: 5 or SEQ ID NO: 7 under stringent conditions. The nucleic acid sequence according to SEQ LD NO: 1 represents the genomic DNA sequence, the nucleic acid sequence according to SEQ LD NO: 2 the cDNA sequence of the twisted dwarf gene from Arabidopsis thaliana, SEQ ID NO: 5 a fragment of the cDNA sequence of the homologous twisted dwarf gene from Lvcopersicon esculentum, SEQ LD NO: 7 represents a fragment of the cDNA sequence of the homologous twisted dwarf gene from Zea mays.

The invention further relates to a polypeptide comprising an amino acid sequence according to SEQ ID NO: 3, SEQ LD NO: 4 or SEQ LD NO: 8. The amino acid sequence can be modified so that the amino acid sequence has amino acid additions, deletions or insertions at one or more positions. The amino acid sequence according to SEQ LD NO: 3 represents the amino acid sequence of the twisted dwarf protein from Arabidopsis thaliana, SEQ LD NO: 6 the amino acid sequence of the homologue twisted dwarf protein from Lvcopersicon esculentum, SEQ LD NO: 8 the amino acid sequence of the homologue twisted dwarf protein from Zea mays.

The present invention relates to nucleic acid sequences from a plant genome, particularly preferably from Arabidopsis thaliana, Zea mays or Lvcopersicon esculentum, which contain the coding region of a FKBP-like (FK506 binding protein) gene (twisted dwarf), the activity of which particularly affects the expression of the overall architecture of the plant Cell growth, growth orientation, degree of branching, etc. controlled. The loss of this activity, for example due to mutation or deletion in a plant genome, leads to a change in the overall architecture of the plant by reducing cell growth, disorientation of the Growth of all above-ground and underground organs, reduction of the degree of branching of the shoot, changes in the response to brassinosteroids and their precursors and derivatives, and change in the response of the root to gravitropic stimuli by changing the ethylene production and ethylene-induced signal transmission. The nucleic acid sequence according to the invention can be inserted into a vector which additionally comprises one or more control elements which control the transcription and / or translation of the nucleic acid sequence according to the invention. The invention further relates to vectors, for example plasmids, and host cells, for example yeasts and bacteria, comprising the nucleic acid sequence according to the invention.

The invention further relates to the use of the nucleic acid sequence according to the invention for the identification and isolation of homologous or related FKBP-like genes and the amino acid sequence derived therefrom from other dicotyledonous and monocotyledonous plants by database comparison, hybridization or by PCR techniques, all of which are known to the person skilled in the art.

To find homologous or related FKBP-like genes from other plants by means of a database comparison, the nucleic acid sequences according to the invention or the polypeptide sequences according to the invention derived therefrom can be compared in databases using the BLAST similarity algorithm (Basic Local Alignment Search Tool, Altschul et al., Journal of Molecular Biology 215, 403 -410 (1990), blastn for comparisons with nucleic acid sequences, tblastn for comparisons with polypeptide sequences) using standard parameters in the blast service of the NCBI. Here, gene sequences with a significance level of P <le ^{* 30} , which also have a similar domain structure to the nucleic acid sequences according to the invention or the polypeptide sequences derived therefrom, are referred to as homologous or related to the twd gene.

The present invention further relates to a method for producing plants, comprising the stable integration of at least one nucleic acid sequence according to the invention into the genome of plant cells or plant tissues and regeneration of the plant cells or plant tissues obtained to plants. The invention preferably relates to a method, the integrated nucleic acid sequence further comprising one or more control elements which ensure the transcription and / or translation of the nucleic acid sequence. Especially the invention preferably relates to a method in which the integrated nucleic acid sequence is expressed in an antisense orientation. The invention further particularly preferably relates to a method in which the integrated nucleic acid sequence has the activity of a ribozyme which suppresses the biological activity of the endogenous nucleic acid sequence which codes for a protein similar to FKBP. The nucleic acid sequence according to the invention and optionally its control elements can be integrated into the genomic DNA of the target cells via homologous recombination. The homologous recombination can also be carried out in such a way that the nucleic acid sequence according to the invention is integrated into the genomic region of the endogenous gene which encodes a protein similar to FKBP.

The method according to the invention is not limited to a specific type of plant, but can be used in all plants. Preferred plants are e.g. Useful or ornamental plants e.g. Cereals such as wheat, corn, rice, rye or barley, legumes such as peas, beans, chickpeas, lentils or soybeans, brassicaceae such as rape or mustard, fiber plants such as flax, hemp or cotton, trees such as spruce, poplar, beech, oak or walnut, Ornamental shrubs, or nightshade plants such as tomatoes or potatoes.

The invention further relates to transformed plant cells or transformed plant tissue, comprising a nucleic acid sequence according to the invention which is stably integrated into the genome of the plant cell or plant tissue. Transformed plant cells or transformed plant tissue that can be regenerated into a seed-producing plant are preferred. The invention further relates to transgenic plants and their seeds comprising a recombinant nucleic acid sequence according to the invention.

The invention further relates to mutants, for example in Arabidopsis thaliana, Zea mays or Lycopersicon esculentum, in which the endogenous twisted dwarf DNA sequence (FKBP-like (FK506 binding protein) gene), for example by T-DNA insertion or by deletion or insertion of DNA of different sizes -Areas changed and show the phenotypic changes mentioned. The invention further relates to transgenic plants in which mutated DNA sequences of the gene sequence indicated have been phenotypically restored to the wild type by introducing intact gene copies. The nucleic acid sequence according to the invention or the method according to the invention can be used to produce transgenic plants with an altered, ie disoriented, growth. The disoriented growth manifests itself in a change in the structure of cell walls and intracellular support and form elements (e.g. cytoskeleton). Such changes can be used to make plants that are used to produce fiber and other materials that have new, altered material properties. Due to the twisted growth of lignified support organs, for example in trees, wood with changed strength properties and possibly changed yields can be obtained on the one hand by forming so-called "compression wood". Due to the twisted growth, fiber-producing plants can produce plant fibers with new, desired processing properties and physical properties (strength, etc.).

Another aspect of the twisted growth concerns the twisting of the growth direction about the longitudinal axis of opening fruits e.g. Pods of crops. This growth results in a reduced spontaneous bursting of the opening fruits at the time of seed ripening. At the time of seed ripening, after filling the seeds with storage substances, the phase of seed rest is initiated, which is characterized by drying and bursting of the opening fruits to spread the seeds. A twisted growth of the opening fruits e.g. Pods prevent the pod from opening completely and thus lead to reduced seed fall. This reduces the loss of threshing during mechanical manipulation during the harvesting process and the loss of harvesting due to unintentional early fall. These properties are particularly important for all crops with harvestable opening crops, e.g. Soy, rapeseed, mustard or legumes of all kinds are useful.

It was also possible to show that the nucleic acid sequence according to the invention is not only responsible for the direction of growth but also for the size of the plant itself. The present invention thus also relates to transgenic plants which have a reduced habit compared to their wild-type plants. On the one hand, this applies to all cereals such as wheat, maize, rice, rye, barley etc., which, due to the shortening of the stalk, on the one hand provide greater stability during growth and harvesting and thus reduce stalk breakage, e.g. in the event of precipitation and wind, as well as higher production of Show biomass in the harvestable organs. Furthermore, reduced growth is often desirable even in ornamental plants. Here is particular on the production of bonsai plants as well as reduced versions of many Ornamental plants and cut flowers such as sunflowers pointed out. In this context, the twisted growth can also be of interest, since shrub and tree plants with twisted growth, for example, corkscrew willows or ficus can be found in the range of ornamental plants.

The pod, like the flower, is reduced in its overall length less than the other organs, but the flower style is greatly shortened. The seeds of the twisted dwarf mutant strikingly do not show the large size reduction of the other plant organs. In comparison to wild-type seeds, there is even a larger volume in the seeds of twisted dwarf mutants. Furthermore, the present invention thus relates to an increase in the total harvest weight of seed-bearing plants. It could be shown that the average number of seeds per pod in twd mutants of Arabidopsis thaliana (20) is reduced to approximately one third of the number of a corresponding wild-type plant (57). However, the number of pods on a plant was significantly increased in twd mutants (417) compared to the wild type (136). Therefore, the average total number of seeds harvested per plant for twd mutants results in an approximately 10% increase in seed yield. The dimensions of the seeds also show considerable differences in twd mutants and corresponding wild-type plants. Estimates of the seed volumes according to Leon-Klosterziel et al., Plant Cell 6: 385-392 (1994) show a volume increased by approximately 50% for seeds of twd plants. These properties are particularly important for all crops with harvestable pods e.g. Soy, rapeseed. Mustard or legumes of all kinds useful.

Irrespective of the differently pronounced size reduction of individual plant organs, the irregular and disoriented growth has been observed in all plant organs, including the root of Arabidopsis thaliana. In addition to changes in growth, the twisted dwarf mutation also slows down the development of the plant. This can be seen in an extended life cycle of the twisted dwarf mutant compared to the wild type. After about 6 weeks under long day conditions (light phase at least 16 hours), senescence begins in the wild type. The life cycle of the twisted dwarf mutant is extended by about one week under long day conditions, and about three weeks under short day conditions (light phase maximum 9 hours). Due to the defect in the twd gene an extension of the vegetative life phase by 5 days (approx. 20% longer vegetative phase) of the mutants can be observed. This fact can be exploited for the production of plants, where a delay in entering the generative phase and a delayed entry of senescence is desired, for example in ornamental plants.

The reduced overall size of the twisted dwarf mutant is caused by a shortening of the cells. Epidermal cells from primary inflorescences of the wild type and the twisted dwarf mutant, which were detached with tweezers and then stained with safranin red, show a shortening of the cells from the twisted dwarf mutant to approx. 33%. Spray tests of twisted dwarf mutants growing on earth with 10 ^"7 M of the brassinosteroid brassinolide showed increased elongation growth compared to control experiments. However, double mutants of twisted dwarf and the campesterol reductase mutant det2, which is a dwarf mutant of Arabidopsis thaliana, caused by exogenous Application of brassinosteroids can be complemented to the wild type, sprayed with 10 ^"7 M brassinolide, no reaction to the administration of brassinolide was found in these plants, which have extreme dwarfism. This result therefore shows that the twisted dwarf mutant is a plant which is involved in the reception or signal transmission of the brassinosteroid response in plants. On the one hand, by producing such mutants, plants with reduced growth as ornamental plants or other useful plants can be produced in a targeted manner. On the other hand, such plants can be used as models for studying the

Steroid hormone effects can be used.

The apparent involvement in the brassinosteroid signal reception and signal processing of the TWD gene product enables plants to be produced which, by altering the TWD gene product itself or the amount of TWD gene product, via the reaction to the plant growth agent brassinosteroid and its derivatives in habitus , Life cycle, yield etc. can be influenced. This also makes it possible to create model systems for studying the mode of action of brassinosteroid and its derivatives in useful plants, which lead to the development of specific growth substances and effectors.

The disoriented growth of the twisted dwarf mutant raised the question of whether the mutant is capable of directed, asymmetric growth in response to a unidirectional stimulus (tropism). Like the wild type, the aerial plant organs reacted with positive phototropism and with negative gravitropism. The root gravitropism of the mutant twisted dwarf and the wild type was increased to vertical by growing seedlings positioned agar plates. The vertical reorientation _. of the plates after 7 days by 90 ° led to a change in the direction of growth, the angle of which was measured after a further 5 days. A curvature angle between 80 ° - 100 ° was defined as gravitropic (Yamamoto and Yamamoto, 1998, Plant Cell Physiol. 39: 660-664). Only 27% of the roots of the twisted dwarf mutant showed gravitropic growth. The remaining 73% showed agravitropic growth. The roots of wild-type seedlings all changed the growth direction by approximately 90 °, which corresponds to positive-gravitropic growth. Agravitropic root growth was also observed in the mutants eirl (Luschnig et al., 1998, Genes Dev. 12: 2175-2187) and auxl (Mower and Martindale, 1980, Biochem. Genet. 18: 1041-1053). These mutants are insensitive to exogenous applications of ethylene. In order to investigate the ethylene sensitivity of the twisted dwarf mutant, seedlings were incubated under the same conditions as described for the control, only 10 ppm of ethylene were added to the air. First, the twisted dwarf mutant was examined for its phenotype. The increased ethylene concentration caused phenotypic changes, which included a shortening of the root, an increase in the circumference of the hypocotyl and a decrease in the leaf blade. These changes were observed in the wild type and in the twisted dwarf mutant alike. It was striking that the roots of twisted dwarf mutants, which had grown under increased ethylene concentration, all showed gravitropic growth which corresponded to that of the wild type. However, the increased ethylene concentration could not reverse any other properties of the twisted dwarf phenotype. The measurement of the angles of curvature showed that all the roots of the twisted dwarf mutants which had been grown under 10 ppm ethylene grew gravitropically, but only 27% of the twisted dwarf roots grown in air showed normal gravitropism. The roots of wild-type seedlings grew gravitropically under both conditions.

In order to check whether the root gravitropism of the twisted dwarf mutant was corrected by the action of the phytohormone ethylene, the influence of inhibitors of ethylene biosynthesis and of inhibitors of the ethylene action on the root gravitropism of twisted dwarf and wild-type seedlings was investigated. The same experiment as described above was carried out, but with the addition of silver nitrate in the Arabidopsis medium, an inhibitor of the ethylene action. A concentration of 1 μM silver nitrate in the growth medium and 10 ppm ethylene in the growth chamber led to agravitropic growth at the roots of the twisted dwarf mutant. This effect has been Plants not found. Aminoethoxyvinylglycine (AVG), which inhibits endogenous ethylene biosynthesis, resulted in a concentration of 1 μM that only 12% of the roots of twisted dwarf mutants grew gravitropically. In this experiment, 35% of the roots of twisted dwarf mutants had grown gravitropically under control conditions (air). With the addition of 1 μM AVG, however, 41% of the roots of wild-type plants also showed agravitropic growth. Ethylene is important for root gravitropism, since the inhibition of endogenous ethylene synthesis also leads to agravitropic behavior in wild-type roots. Mutants of twisted dwarf in Arabidopsis thaliana or Lycopersicon esculentum and other plants can be used to produce plants and plant organs that produce or accumulate a reduced amount of ethylene. This effect can be used in a targeted manner to influence the ripening of fruit and seeds, and to extend and control the flowering phase of ornamental and useful plants, since these processes are controlled by the amount of ethylene in the corresponding organs or plants.

Since the gravitropism of the root in twd mutants is greatly reduced under normal growth conditions and is only reconstituted when the gaseous phytohormone ethylene is added exogenously, this characteristic can be used to easily regulate the expression of the root gravitropism. This affects the anchoring of the root in the soil, which has an impact on the strength of soil-covering plants, and generally the anchoring of useful plants in the substrate. The induction of root gravitropism can be initiated by ethylene at any point in the development. This fact can also be exploited to regulate other plant development processes using ethylene.

Since twd is a mutant in a FK506 binding protein, these plants can be used as non-animal models for studies on appropriate immunosuppressants (especially FK506 (tacrolimus), rapamycin, cyclosporin A and other substances with a similar effect) and processes of Signaling the effect of immunosuppressive agents, used in pharmaceutical research. Here, new, plant-specific cellular interactions and mechanisms of action can be studied that are not present in the form in animal systems. Among other things, models depleted of FKBPs can be developed in which the effects of not only immunosuppressive substances but also genetically modified ligands for these substances can be tested. The comparison of the derived armnosa sequence of the open reading frame of the twisted dwarf cDNA in the current sequence databases shows a sequence identity of 30-33% and a sequence similarity of 43-53% to FKBPs from humans, animals and other plants (PILEUP, Genetic Computer Group, Wisconsin Package Version 9 1-Unιx, Sept. 1997 (gap creation penalty- 5; gap extension pealty: 1)) The derived twisted dwarf peptide has identical and in four other cases conserved ammosaic exchanges in the four identified amino acid positions for an FK506 interaction . A triple repetition of a TPR motif can be found in the C-terminal region of the peptide. For these motifs, human systems have been shown to interact with Hsp90 with FKBPs (Callebaut et al, 1992, Proc. Natl Acad. Be USA 89 6270-6274). Plant mutants of this class of protein genes can be developed as non-Tien model systems for the study of the effects and signal transmission of immunophiles

The following exemplary embodiments serve to explain the invention and are not to be interpreted as restrictive.

I. General methods

1. Clomeration process

The lambda ZipLox phage vector and the resulting plasmid PZL-1 (Newman et al, 1994, Plant Phys. 106 1241-1255) and the phagemid pBluescnpt (pBS) (Short et al, 1988, Nucl. Acids Res 16 7583 -7600) is used. The expression vector pET3-Hιs (Novagen) was used to express m E. coh. The vectors pAS1 and pACT2 (Clontech, Matchmaker 2-Hybnd System) and pRS314 (Sikorski and Hieter, 1989, Genetics 122 19-27) were used for the transformation of yeast. For the plant transformation, the gene constructions m pRT-W Notl (Überlacker and Wen, 1996, Mol. Breedmg 2. 293-295) and m the binary vector pGPTV-Bar (Becker et al, 1992, Plant Mol Biol. 20: 1195- 1197).

2. Bacterial and yeast strains For the pBluescnpt KS (pBS) vector, the plasmid pZL-1 and for pASl, pACT2 and pGPTV constructs, the E.cob strain DH5α (Hanahan et al., 1983, J. Mol. Biol. 166: 557-580). The twisted dwarf protein was expressed in the E coh strain B121 (Studier and Moffat, 1986). The transformation of the pGPTV constructs into Arabidopsis plants was undertaken using the Agrobacterium tumefaciens strain GV3101: pMP90 (Koncz and Schell, 1990, Mol. Gen. Genet. 204: 383-396). The transformation of 2 hybrid constructs was carried out in the yeast strain Y190.

3. Transformation of Agrobacterium tumefaciens

The transfer of the DNA into agrobacteria was carried out by direct transformation with naked DNA according to Höfgen and Willmitzer (1988, Nucl. Acids Res. 16: 9877). The plasmid DNA of transformed agrobacteria was isolated by the method of Birnboim and Doly (1979, Nucl. Acids Res. 7: 1513-1523) and, after a suitable restriction cleavage, was checked by gel electrophoresis for the correctness and orientation of the inserted DNA.

4. Plant transformation

150 ml of antibiotic-containing YEB medium were inoculated with a positive colony and 2

Shaken at 28 ° C for days. 500 ml of antibiotics-containing YEB medium were inoculated with 10-15 ml of this preculture. This culture was incubated overnight at 28 ° C. on a shaker and centrifuged the next day at 4000 rpm for 15 min. The sedimented bacteria were taken up in infiltration medium. The concentration of the suspension was determined by turbidity measurement and adjusted to an OD600 (optical density) between 0.8 and 1.2. 400 ml plastic beakers filled with agrobacterial suspension were placed in a vacuum desiccator. Pots of Arabidopsis plants were placed upside down on the beakers so that the plant's fluorescence dipped into the agrobacterial suspension. A vacuum of 10-30 mbar was applied for 15 min and then the vacuum desiccant was quickly ventilated. A bacterial suspension was used for up to four consecutive infiltrations. The plants were then kept under long-day conditions (16 hours light / 8 hours dark) until the pods ripened. The 10 plants in a pot were put in two bags (2 pools) of 5 plants each to collect the seeds when the oldest pods of the plants were ripe. The well-dried seeds could be sown directly on soil for selection with BASTA ^® (Aventis CropScience, SA, Lyon France).

Sowing was carried out separately in large bowls according to pools. The seedlings were sprayed with BASTA ^® solution for the first time when the cotyledons were fully developed. The seedlings were sprayed 1-2 more times with BASTA ^® in the following 6 days. Not BASTA ^® - resistant seedlings faded at the cotyledon stage and did not develop further. The resistant seedlings were allowed to continue growing until seed maturity and the seeds of these plants were harvested individually.

II. Embodiments

Embodiment 1: Isolation of the FKBP-like twisted dwarf gene from T-DNA-tagged insertion lines of Arabidopsis thaliana by means of plasmid rescue and isolation of full-length cDNA and genomic clones from gene libraries.

An Arabidopsis thaliana line transformed by T-DNA (Feldmann, 1991, Plant J. 1: 71-82; Forsthoefel et al., 1992, Aust. J. Plant Physiol. 19: 353-366) was transformed into plasmid rescue (Schulz et al, 1995, Plant Mol. Biol. Manual, pp 1-17) isolated a 200 bp long DNA sequence of the twisted dwarf gene flanking the T-DNA insertion. Radiolabelled samples prepared from the plasmids obtained from plasmid rescue (clone pBUB 52) were used to screen the CD4-7 1PRL-2 cDNA library (Newman et al., 1994, Plant Phys. 106: 1241- 1255) and the CD4-11 genomic cosmid library (Schulz et al, 1995, Plant Mol. Biol. Manual, pp 1-17). Approximately 200,000 clones from the cDNA library were screened with these hybridization samples. A plasmid was isolated from a positively reacting 1 clone by tvzvo excision and by determining the DNA sequence (dideoxy method: Sanger et al., 1977, Proc. Natl. Acad. Sci. USA 74: 5463-5467) analyzed. The primary structure of the twisted dwarf protein was derived from this DNA sequence (clone pBUB 65). The cDNA was then used to isolate a genomic clone from the cosmid library.

Embodiment 2: Expression of twisted dwarf peptides in E. coli, purification of the proteins and extraction of antisera against the protein in rabbits

Partial cDNA sequences coding for amino acid positions 1-324 and 1-187 were amplified by PCR and, after cleavage with BamHI and XhoII, ligated into the reading frame of the His tag sequence of the vector pET3 linearized with BamHI. Competent B121 cells were transformed with the ligations and the expression of the peptides after induction with LPTG in crude extracts on Laemmli gels was demonstrated. The fusion peptides with His-Tag were Purified via Ni-NTA agarose (Novagen). The apparent molecular weights were determined after comparison with large markers as 33 kDa for the peptide spanning the region from position 1-187 and as 44 kDa for the peptide in the region from position 1-324 for immunization The purified peptide (pos. 1-187) from rabbits was purified via a preparative SDS-PAGE gel. The protein band was identified by staining the gel with Cu2 + ions, cut out and crushed. The comminuted gel was taken up in buffer and used to immunize rabbits from from BioGenes (Berlin) used After the first immunization, two further booster immunizations were carried out before antisera against the twisted dwarf protein were obtained by bleeding the animals. The detection of the twisted dwarf protein by the antiserum was tested in immunoblot experiments

Exemplary embodiment 3 Transformation of mutated Arabidopsis thahana plants with a construction for overexpression of the code region of the twisted dwarf protein

To complement twisted dwarf mutants (twisted dwarfl-1, twisted dwarfl-3, twisted dwarfl-4), the open reading frame of the twisted dwarf gene was amplified using PCR from plasmid BUB65 and after BamHI / Bglll cleavage the BamHI- Interface of the vector pRT-Ω Notl clomers The pRT-Ω Notl has a CaMV 35S promoter and an Ω sequence from the tobacco mosaic virus in front of the BamHI residual sequence, which can increase the translation of different reporter genes in plants 2-10 times ( Galhe et al, 1989, Plant Cell 1 301-31 1) A polyadenyherization signal from the cauliflower mosaic virus was behind the BamHI residual sequence. Sequencing of the insert showed that the cloned twisted dwarf cDNA sequence had no sequence changes. The cassette was loaded with the residual functional enzyme Ascl is split out of the pRT-Ω Notl and after filling the overhanging ends by Klenow polymerase into the HindLU residual site of the binary, which is also filled Plant vector pGPTV-BAR hgiert The uidA reading frame had previously been deleted from the pGPTV-BAR by a Smal / EcoRI cleavage

The transformation of twisted dwarf mutants based on vacuum infiltration of bleeds was carried out with the binary vectors using the agrobacter strain GV3101 pMP90 Transgenic Arabidopsis thahana-? finances were selected for herbicide resistance and phenotypically analyzed as described. The presence and structure of the transformed constructs in the transgenic plants were determined by DNA gel blot analysis. All mutant plants transformed with the gene sequence of the intact twisted dwarf gene showed a reversion of the phenotype to the wild type.

Exemplary embodiment 4 Sequence analysis of different mutated twisted dwarf alleles in Arabidopsis thahana mutants

Other Arabidopsis thahana mutants that had the twisted dwarf mutant phenotype were isolated from various mutagenized populations. A cross analysis with the mutant generated by T-DNA insertion showed that the different mutants represent alleles of the same gene. A residual functional fragment length polymorphism (RFLP) was demonstrated for two of the mutants by means of a DNA gel blot expansion.

For a more precise analysis of the twisted dwarf mutants, PCR products of the mutated alleles of the gene were sequenced and compared with the wild-type sequence. The T-DNA insertion in the twisted dwarf allele 1-1 is in the fifth exon at position +1484. A deletion of 593 bp from position -122 to +471 led to the loss of part of the promoter, the transcription start, in the mutant twisted dwarfl-3 as well as the first 35 bp of the open reading frame. The shortening of an EcoRI fragment of approximately 600 bp had already been observed in a DNA gel blot expansion. A nucleotide insertion in the dntten exon at position +823 and a nucleotide exchange from Adenm to Guanm at position +829 have been identified in the mutant twisted dwarfl-4. The nucleotide shift causes a reading frame shift and thus leads to a translation stop after 85 amino acids. All twisted dwarf alleles are so-called null alleles that can no longer produce a functional gene product. All examined twisted dwarf mutants show the same expression of the twisted dwarf phenotype described above.

Exemplary embodiment 5- Identification of homologs of the twd gene from other plant species.

1. Identification of the twd homolog from Lvcopersicon esculentum

For the amplification and subsequent identification of homologs of the n gene from others

The ohgonucleotides twd-S and twd-A were used as plants from the gene sequence of the plants 9?

derived twd gene. Using these oligonucleotides, sequences of the twrf homologue from tomato were isolated in a PCR under the following conditions on DNA from a cDNA library of tomato (Lycopersicon esculentum):

1 x 94 ° C 2 min then 35 cycles: 94 ° C 1 min 58 ° C 1 min 72 ° C 2 min then 4 ° C until it is removed from the PCR machine.

Sequence of the primer twd-S:

5 '-CT (C / T) (G / T) TG C (A / T) T GT (G / T) (G / T) GC TGG GAA TTA G-3' Sequence of the primer twd-A: 5 ' -CCA TCC ATT TT (C / T) CTT CT (A / G) T (G / C) T GCT GC-3 '

The resulting PCR product (SEQ LD NO: 4) was cloned into the vector pGEM-T easy ^® (Promega) and sequenced by chain termination method according to Sanger. With the help of the sequences of the EST clones AW038756, AI895686, AW441601, AW222544 from tomato (Lycopersicon esculentum) (GenBank online, release <115), which with the help of the similarity algorithm BLAST (Basic Local Alignment Search Tool, Altschul et al, Journal of Molecular Biology 215, 403-410 (1990) (tblastn, cutoff for P value: 6e ^{~ 26} , Matrix: Blosum 62, Gap existence cost: 11, Per residue gap cost: l) with the amino acid sequence of the Arabidopsis TWD protein, It was possible to assemble a cDNA contig over a total of 1142 base pairs (TomTWDContig; SEQ ID NO: 5). The area of the sequence overlaps of the EST clones with the clone TomTWD spans nucleotide positions 1 to 95 of TomTWD with AW441601 and 121 to 140 of TomTWD with AW222544 The translation of the longest open reading frame of the nucleotide sequence of TomTWDContig into amino acids results in a continuous peptide (TTP) with a length of 320 amino acids (SEQ LD NO: 6) .The identity to the TWD protein from Arabidopsis is 74.1%, the similarity is 85.3% to amino acid positions 1 to 316 of the TWD protein from Arabidopsis. 2. Identification of the Zea mays twd homolog

With the help of the sequences of the EST clones AW216068 and AW171820 from Zea mays (GenBank online, release <115), which with the help of the similarity algorithm BLAST (Basic Local Alignment Search Tool, Altschul et al., Journal of Molecular Biology 215, 403-410 (1990), tblastn, cutoff for P value <le ^"31 , Matrix: Blosum 62, Gap existence cost: 11, Per residue gap cost: l) with the amino acid sequence of the Arabidopsis TWD protein in the non-redundant database of GenBank EST Division / Subdivision Zea mays was found, a cDNA contig with a total of 776 base pairs could be put together (ZmTWDContig, SEQ LD NO: 7). The translation of the longest open reading frame of the nucleotide sequence of ZmTWDContig into amino acids results in a continuous peptide (ZmTWD, SEQ ID NO : 8) with a length of 168 amino acids, the identity to the TWD protein from Arabidopsis is 68.5%, the similarity 79.8%> to the amino acid positions 196 to 365 of the TWD protein from Arabidopsis.

Claims

claims

1. Nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ HD NO: 5 or SEQ ID NO: 7 or its fragment or derivative or a nucleic acid sequence which is identical to the nucleic acid sequence according to SEQ ID NO: 1, SEQ LD NO : 2, SEQ LD NO: 5 or SEQ LD NO: 7 and has the biological activity of the nucleic acid sequence according to SEQ LD NO: 1, SEQ LD NO: 2, SEQ LD NO: 5 or SEQ LD NO: 7.

2. Nucleic acid sequence according to claim 1, wherein the hybridizing nucleic acid sequence under stringent conditions with the nucleic acid sequence according to SEQ LD NO: 1, SEQ HD

NO: 2, SEQ CD NO: 5 or SEQ ID NO: 7 hybridized.

3. Polypeptide comprising an amino acid sequence according to SEQ ID NO: 3, SEQ ID NO: 6 or SEQ ID NO: 8.

4. Vector comprising a nucleic acid sequence according to claim 1 or 2.

5. The vector according to claim 4, further comprising one or more control elements which ensure the transcription and / or translation of the nucleic acid sequence according to claim 1 or 2.

6. A process for the production of plants, comprising the stable integration of at least one nucleic acid sequence according to claim 1 or 2 into the genome of plant cells or plant tissues and regeneration of the plant cells or plant tissues obtained to plants.

7. The method according to claim 6, wherein the integrated nucleic acid sequence further comprises one or more control elements which ensure the transcription and / or translation of the nucleic acid sequence.

8. The method according to claim 6 or 7, wherein the integrated nucleic acid sequence is expressed in antisense orientation.

9. The method according to claim 6 or 7, wherein the integrated nucleic acid sequence

Has activity of a ribozyme which suppresses the biological activity of the endogenous nucleic acid sequence according to claim 1 or 2.

10. The method according to claim 6, wherein the nucleic acid sequence is integrated into the genomic region of the homologous endogenous gene by homologous recombination.

11. Transformed plant cell or transformed plant tissue, comprising a nucleic acid sequence stably integrated into the genome of the plant cell or plant tissue according to claim 1 or 2.

12. Plant cell or plant tissue according to claim 11, regenerable to a seed-producing plant.

13. The transgenic plant and its seeds comprising a recombinant nucleic acid sequence according to claim 1 or 2.