EP1250352A1

EP1250352A1 - Receptor-like protein kinases from nicotiana tabacum

Info

Publication number: EP1250352A1
Application number: EP00983273A
Authority: EP
Inventors: Thomas SCHMÜLLING; Silke Schäfer
Original assignee: Bayer AG; Bayer CropScience AG
Current assignee: Bayer CropScience AG
Priority date: 1999-12-20
Filing date: 2000-12-11
Publication date: 2002-10-23
Also published as: DE19961519A1; US20030041345A1; AU2007501A; JP2003529342A; WO2001046233A1

Abstract

The invention relates to nucleic acids which code for plant polypeptides with the biological activity of receptor-like protein kinases and the corresponding polypeptides per se.

Description

RECIPE-LIKE PROTEIN KINASES FROM NICOTIANA TABACUM

The invention relates to nucleic acids which code for plant polypeptides with the biological activity of receptor-like protein kinases, and the corresponding polypeptides per se

Receptor-like kinases usually span the cell membrane and thus have an extracytoplasmic and a cytoplasmic part. They act in organisms as mediators of signals that are passed on from the outside into the cell interior (e.g. van der Geer at al, 1994) by binding a ligand the extracellular domain is activated and the cytoplasmic protein kinase domains are activated. This is done by autophosphoryeration of either the Senn and / or Threonm residues, the tyrosine or histidine residues (e.g. Fantl et al, 1993). Animal receptor kinases autophosphoryheres mainly tyrosine residues, in contrast, seem almost exclusively in plants Seπn- / Threomn-Kιnasen occur (Becraft, 1998)

Seπn- / Threonιn-Kιnases catalyze the reversible transfer of the γ-phosphate residue from ATP to amino acids of a recipient protein. The presence of 1 1 conserved domains essentially determines the enzymatic function of protemkinases (Hanks et al, 1988). These domains contain a total of 9 Amino acids that are invariable in all previously identified protein kinases. They are involved in ATP binding and presumably in the recognition of the amino acid to be phosphorylated, such as Senn Threonm or tyrosine

The subject of the present invention is nucleic acids which encode plant poh peptides with the biological activity of a protemkinhe protemkinase which encompasses the amino acid sequence according to SEQ I D \ 0 2. In particular, encode the inventive sequences for icceptor-like proteins.

Threonm kinases The nucleic acids according to the invention are in particular single-stranded or double-stranded deoxyribonucleic acids (DNA) or ribonucleic acids (RNA). Preferred embodiments are fragments of genomic DNA, which may contain introns, and cDNAs.

The nucleic acids according to the invention are preferably DNA fragments which correspond to genomic DNA from tobacco plants.

The nucleic acids according to the invention particularly preferably comprise a sequence selected from

a) the sequence according to SEQ ID NO: 1,

b) sequences which code for a polypeptide which comprises the amino acid sequence according to SEQ ID NO: 2,

c) at least 14 base pairs long partial sequences of the sequences defined under a) or b),

d) sequences which hybridize to the sequences defined under a) or b),

e) sequences which have at least 60%, preferably at least 80%, particularly preferably at least 90% identity with the sequences defined under a) or b),

0 sequences which are at least 60%, preferably at least one

80%, particularly preferably at least 90% identity with the N-terminal receptor domain defined under a) or b)

Have sequences g) sequences which are complementary to the sequences defined under a) or b), and

h) sequences which, owing to the degeneracy of the genetic

Coding codes for the same amino acid sequence as the sequences defined under a) to f)

A very particularly preferred embodiment of the nucleic acids according to the invention is a cDNA molecule with the sequence according to SEQ ID NO 1

The term "hybridize" as used herein describes the process in which a single-stranded nucleic acid molecule with a complementary strand undergoes base pairing. In this way, starting from the sequence information disclosed herein, for example, DNA fragments from others

Plants are isolated as tobacco plants which code for receptor-like protein kinases which have the same or similar properties as the kinase with the amino acid sequence according to SEQ ID NO 2

Hybπdisierungsbedingungen are approximately calculated according to the following Fonuel

The melting temperature Tm = 81 5 ° C + 16 6 log {c (Na ⁺ )] + 0 41 (% G + Q) - 500 / n (Lottspeicher and Zorbas, 1998)

Here, c is the concentration and n the length of the hybridizing sequence section in base pairs and a sequence> 100 bp, the expression 500 / n is omitted. With maximum rigidity, at a temperature case, 5-15 ° C below Tm and an ion peak of 15 mM Well (corresponds to 0 1 \ SSC) if a RNA sample ends too early, the melting point is 10-1 ^ C higher Preferred hybridization conditions are given below

Hybridization solution 6X SSC / 5X Denhardt's solution / 50% formamide, hybridization temperature 36 ° C, preferably 42 ° C, 1 washing step 2X SSC, 30 min at room temperature,

2 washing step IX SSC, 30 min at 50 ° C, preferably 0.5X SSC, 30 min at 65 ° C, particularly preferably 0.2X SSC, 30 min at 65 ° C

The term “N-terminal receptor domains” as used in the present context refers to a peptide region which functionally corresponds to the peptide region with an amino acid sequence from position 1 to position 394 of the sequence according to SEQ ID NO 2

The degree of identity of the nucleic acids is preferably determined with the aid of the NCBI BLASTN version 2 0 4 program (Altschul et al, 1997)

The present invention also relates to the regulatory regions which naturally control the transcription of the nucleic acids according to the invention in plant cells, in particular in tobacco plants

The term "regulatory regions", as used herein, refers to untranslated regions of the gene in question, such as promoters, enhancers, repressor or activator binding sites or termination sequences that interact with cellular proteins, thereby controlling transcription

The present invention furthermore relates to DNA constructs which comprise a nucleic acid according to the invention and a heterologous promoter

Dei \ usdi uck "heteiologci Promotoi" as ei hieπn v erw ends w. Refers to a Piomotoi, which has other properties than its own Piomotoi, which in the

Original organism controls the expression of the gene in question The choice of heterologous promoters depends on whether pro- or eukaryotic cells or cell-free systems are used for expression. Examples of heterologous promoters are the 35S promoter of the cauliflower mosaic virus for plant cells, the promoter of alcohol dehydrogenase for yeast cells, the T3,

T7 or SP6 promoters for prokaryotic cells or cell-free systems

The present invention furthermore relates to vectors which contain a nucleic acid according to the invention, a regulatory region according to the invention or a DNA construct according to the invention. All phages, plasmids, phagmids, phasmids, phasmids, cosmids, YACs used in molecular-biological laboratories can be used as vectors. BACs, artificial chromosomes or particles that are suitable for particle bombardment can be used

Preferred vectors are pBIN (Bevan, 1984) and its derivatives for vegetable

Cells, pFL61 (Minet et al, 1992) for yeast cells, pBLUESCRIPT vectors for bacterial cells, lamdaZAP (Stratagene) for phages

The present invention also relates to host cells which contain a nucleic acid according to the invention, a DNA construct according to the invention or a vector according to the invention

The term “host cell” as used in the present context refers to cells which do not naturally contain the nucleic acids according to the invention

Both prokaryotic cells, preferably E coh, are suitable as host cells. as well as eukaryotic cells, ie cells from Saccharomyces cerevisiae, Pichia pastons, insects, plants. Fioschooocytes and cell nuts from nipples

The present invention further relates to poly peptides with the biological activity of receptor-like protemkinases which are derived from the Measured nucleic acids are encoded in particular polypeptides that represent Seπn- / Threonm-Kιnasen according to the invention

The term "polypeptides" as used herein refers to both short amino acid chains, commonly referred to as peptides, oligopeptides, or oligomers, and longer amino acid chains, commonly referred to as proteins. It includes amino acid chains that are either natural Processes, such as post-translational processing, or can be modified by chemical processes which are state of the art. Such modifications can occur at various locations and multiple times in a polypeptide, for example on the peptide backbone, on the amino acid side chain, on the amino and / or at the carboxy terminus They include, for example, acetylation, acylation, ADP ribosylation, amidation, covalent linkage with flavins, ham moieties, nucleotides or nucleotide derivatives, lipids or lipid derivatives or phosphatyhnositol, cyclization, disulfide bridging,

Demethyers, cystine formations, formers, gamma-carboxyers, glycosylers, hydroxylations, iodinations, methylers, mythersters, oxidations, proteolytic processing, phosphorylers, selenoyls and tRNA-mediated additions of amino acids

The polypeptides according to the invention can be in the form of "mature" proteins or as parts of larger proteins, for example as fusion proteins. Furthermore, they can secrete or "leader" sequences, pro-sequences, sequences which enable easy purification, such as multiple histidine Have residues or additional stabilizing amino acids

The polypeptides according to the invention do not have to represent complete receptor-like protemkinases, but can also be nui fragments thereof as long as they still have at least one biological activity of the complete protemkinases exercise are still considered to be in accordance with the invention. The polypeptides according to the invention need not be derivable from receptor-like protein kinases from tobacco. Polypeptides which correspond to receptor-like protein kinases of the following plants, for example, or fragments thereof which can still exert the biological activity thereof are also considered to be according to the invention: corn, wheat, barley, oats, rice, rye, tomatoes, legumes, potato plants, Lactuca sativa, brassicaceae , Wood plants, Physcomitrella patens.

The polypeptides according to the invention can have deletions or amino acid substitutions in comparison to the corresponding region of naturally occurring receptor-like protein kinases, as long as they still exert at least a biological activity of the complete receptors. Conservative substitutions are preferred. Such conservative substitutions include variations in which one amino acid is replaced by another amino acid from the following group:

1. Small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro and Gly;

2. Polar, negatively charged residues and their amides: Asp, Asn, Glu and Gin;

3. Polar, positively charged residues: His, Arg and Lys;

4. Large aliphatic, non-polar residues: Met, Leu, Ile, Val and Cys; and

5. Aromatic residues: Phe, Tyr and Trp.

The following list shows preferred conservative substitutions:

Original remainder substitution

Ala Gly, Ser

Arg Lys

Asn Gin, His

Asp Glu

Cys | Ser

The present invention thus also relates to polypeptides which exercise at least one biological activity of a receptor-like protein kinase and comprise an amino acid sequence which have an at least 60% identity, preferably an at least 80% identity, particularly preferably an at least 90% identity, very particularly preferably 97-99% identity, with the sequence according to SEQ ID NO 2 over its entire length

The degree of identity of the amino acid sequences is preferably determined with

Help of the BLASTP + BEAUTY version 2 0 4 (Altschul et al 1997)

A preferred embodiment of the polypeptides according to the invention is the cvto kinmregulieite receptor-like protein kinase (CRK 1) with the amino acid sequences / according to SEQ ID NO 2 In the CRK1 amino acid sequence, the 1 1 kinase domains could be determined in which all amino acids defined according to Hanks et al., 1988 are present. It is therefore a serine / threonine kinase. The CRKl protein has a transmembrane domain in the region of amino acids 390 and 410. The first 28 amino acids with a positively charged amino acid residue and a hydrophobic one

A region of 15 amino acids at the N-terminus represents a signal sequence. The cleavage site is probably located between amino acids 28 and 29. This signal sequence ensures translocation of the newly synthesized CRK1 protein through the membrane of the endoplasmic reticulum and further transport into it plasma membrane. The N-terminal domain of

CRKl between amino acids 29 and 390 is located extracytoplasmic. This extracellular domain comprises four regions which have> 70% homology to ATP / GTP binding sites or to binding sites for cyclic nucleotides. In the case of CRK1, cytokinins are possible ligands. As adenine derivatives, cytokinins are structurally similar to purines.

Cytokinins belong to the group of plant hormones and have the ability to induce cell division. In the meantime, more than 40 naturally occurring cytokinins have been isolated, all of the known cytokinins being adenine derivatives. The activity is determined by the nature of the substituents, especially the N ⁶ group (overview in Shaw, 1994 and Kaminek, 1992).

There is a diverse range of effects associated with cytokinins. In addition to inducing cell division in combination with aux, cytokinins also influence differentiation processes. A high cytokinin to auxin ratio leads to shoot formation in vitro in callus, a high auxm to cytokinin ratio to root formation (Skoog and Miller, 1957). Cytokinins cause a reduction in apical dominance, which results in the budding of the side buds hat (Wickson and Thimann, 1958). The cytokinins also play an important role in delaying leaf senescence

Cytokinins cause inactivation of proteolytic enzymes, among others Lipases and Lipoxykmasen, which are responsible for the degradation processes. To further classic effects of the cytokinins the demanding influence on the chloroplast development and the inhibition of the root length growth pays

Cytokinins are necessary for normal plant development. An oversupply of this hormone leads to extremely compact, sterile plants

Since the amount of CRKl transcript is regulated by cytokinins, it can be a component of the cytokinin signal transduction pathway. The amount of transcript in CRKl is transiently regulated by cytokinins. The CRK1 -

The gene is then subject to early regulation, 30 minutes after the addition of 5 x 10 ⁷ M BAP there is an almost complete reduction in the amount of transcript. Depending on the cytokinin concentration, the CRK1 transcripts accumulate again after 9-24 h of cytokinin treatment, so that one can assume a transient regulation This regulation could represent negative feedback in order to ensure that a cell is competent for a signal, but this sensitivity can be changed by reducing the amount of protein in signal transduction components. For the CRK1 receptor, it is postulated that the cell after the signal has been recorded is unable to respond to further cytokine signals for a period of time

When determining the dose-dependency of the regulation, a very positive result has been found

1 7 rriinnggee CCyyttookkiinniinnkkoonnzzeenntenntraattiioonn vvoon 5 x 10 M BAP as sufficient to completely reduce the amount of transcripts

Other plant hormones, such as gibberelhnic acid, ACC, brassinosteroids and jasmonic acid, as well as the structural analogue adenine do not lead to a change in the CRKl -transformabundance in comparable concentration The term "biological activity of a receptor-like protein kinase" as used herein means a change in cell activity and plant growth after ligand binding

The present invention furthermore relates to antibodies which bind specifically to the polypeptides according to the invention. Such antibodies are produced in the customary manner. These antibodies can be used, for example, to identify expression clones, for example a gene bank, which carry the nucleic acids according to the invention

The term "antibody" as used herein also extends to parts of complete antibodies, such as Fa-, F (ab) ₂ - or Fv fragments, which still have the ability to bind to the epitopes of the polypeptides according to the invention

The present invention furthermore also relates to processes for producing the nucleic acids according to the invention. The nucleic acids according to the invention can be prepared in the customary manner. For example, the nucleic acid molecules can be synthesized completely chemically. Short pieces of the nucleic acids according to the invention can also be synthesized chemically and such ohgonucleotides can be labeled radioactively or with a fluorescent dye

The labeled ohgonucleotides can also be used to search cDNA banks produced from plant mRNA. Clones to which the labeled ohogonucleotides hybridize are selected for the isolation of the relevant DNA fragments. After the characterization of the isolated DNA, the nucleic acids according to the invention

The nucleic acids according to the invention can also be produced by means of PCR methods with the use of chemically synthesized ohgonucleotides The term "oligonucleotide (s)" as used herein means DNA molecules consisting of 10 to 50 nucleotides, preferably 15 to 30 nucleotides. They are chemically synthesized and can be used as probes.

The present invention furthermore relates to processes for producing the polypeptides according to the invention. To produce the polypeptides which are encoded by the nucleic acids according to the invention, host cells which contain nucleic acids according to the invention can be cultivated under suitable conditions. The desired polypeptides can then be isolated from the cells or the culture medium in a conventional manner. The polypeptides can also be used in in vitro

Systems are manufactured.

A rapid method for isolating the polypeptides according to the invention, which are synthesized by host cells using a nucleic acid according to the invention, begins with the expression of a fusion protein, whereby the fusion partner can be easily affinity-purified. The fusion partner can be, for example, glutathione S-transferase. The fusion protein can then be purified on a glutathione affinity column. The fusion partner can be separated by partial proteolytic cleavage, for example on linkers between the fusion partner and the polypeptide according to the invention to be purified. The linker can be designed to include target amino acids, such as arginine and lysine residues, which define sites for trypsin cleavage. Standard cloning methods using oligonucleotides can be used to generate such linkers.

Other possible cleaning methods are based on preparative electrophoresis. FPLC, HPLC (eg using gel filtration, reverse phase or slightly hydrophobic columns). Gel filtration, differential precipitation, ion exchange chromatography and affinity chromatography. Since receptor-like protein kinases are membrane proteins, detergent extractions are preferably carried out in the cleaning processes, for example using detergents which have little or no effect on the secondary and tertiary structures of the polypeptides, such as nonionic detergents

The purification of the polypeptides according to the invention can include the isolation of membranes from host cells which expand the nucleic acids according to the invention. Preferably, such cells expand the polypeptides according to the invention in a sufficient number of copies so that the amount of the polypeptides in a membrane fraction is at least 10 times higher than that which is found in comparable membranes of cells which naturally expand the CRK1 gene, the amount is particularly preferably at least 100 times, very particularly preferably at least 1000 times higher

The terms "isolation or purification" as used herein mean that the polypeptides according to the invention are separated from other proteins or other macromolecules of the cell or the tissue. A composition containing the polypeptides according to the invention is preferred in terms of protein content compared to a preparation from the Host cells enriched at least 10 times and particularly preferably at least 100 times

The polypeptides according to the invention can also be affinity-purified without a fusion partner with the aid of antibodies which bind to the polypeptides

The present invention also relates to processes for finding chemical compounds which bind to the polypeptides according to the invention and change their properties. Due to the diverse functions of the receptor-like protein kinases according to the invention, modulators which influence the activity can represent new growth-regulating or herbicidal active compounds The term "agonist" as used in the present context refers to a molecule which accelerates or amplifies the signal transduction of the receptor-like protein kinases according to the invention, ie the autophosphorylation of the serine and / or threonine residues.

The term "antagonist" as used herein refers to a molecule that mediates signal transduction of the receptor-like protein kinases of the invention, i.e. the autophosphorylation of the serine and / or threonine residues is slowed down or prevented.

The term “modulator” as used herein represents the generic term for agonist or antagonist. Modulators can be small organic chemical molecules, peptides or antibodies that bind to the polypeptides according to the invention. Furthermore, modulators can be small organic chemical molecules, peptides or antibodies which bind to a molecule which in turn binds to the polypeptides according to the invention and thereby influences their biological activity. Modulators can be natural substrates and ligands or structural or functional mimetics thereof. However, the term "modulator" does not include cytokinins.

The modulators are preferably small organic chemical compounds.

The binding of the modulators to the receptor-like protein kinases according to the invention can change the cellular processes in a way which leads to the death of the plants treated with them.

Furthermore, the present invention comprises methods for finding chemical compounds which change the expression of the polypeptides according to the invention. Such "expression modulators" can also represent new growth-regulating or herbicidal active ingredients. Expression modulators can small organic msch-chemical molecules, peptides or antibodies which bind to the regulatory regions of the nucleic acids coding for the polypeptides according to the invention. Furthermore, expression modulators can be small organic-chemical molecules, peptides or antibodies which bind to a molecule which in turn binds to regulatory regions of the encoding the polypeptides of the invention

Nucleic acids bind and their expression influences expression modulators can also be antisense molecules

The present invention therefore also extends to the use of modulators of the polypeptides according to the invention or of expression modulators as

Plant growth regulators or herbicides

The methods according to the invention include high throughput screening (HTS). Both host cells and cell-free preparations containing the nucleic acids according to the invention and / or the polypeptides according to the invention can be used for this

In order to find modulators, a synthetic reaction mix (eg products of the in vz / ro transcription) or a cellular component, such as a membrane or any other preparation which contains the polypeptides according to the invention, together with a labeled substrate or ligand of the polypeptides in the presence and the absence of a candidate molecule, which may be an agonist or antagonist. The ability of the candidate molecule to increase or to inhibit the activity of the polypeptides according to the invention is evident from an increased or decreased binding of the labeled ligand or from an increased or decreased conversion of the labeled Substrate molecules that bind well and lead to increased activity of the polypeptides according to the invention are agonists. Molecules that bind well but do not trigger the biological activity of the polypeptides according to the invention are probably good antagonists. The detection of biological Activity of the polypeptides according to the invention can be improved by a so-called reporter system. include, but are not limited to, colorimetrically labeled substrates that are converted to a product or a reporter gene that responds to changes in the activity or expression of the polypeptides of the invention, or other known binding assays.

Another example of a method with which modulators of the polypeptides according to the invention can be found is a displacement test in which, under suitable conditions, the polypeptides according to the invention and a potential modulator with a molecule which is known to bind to the polypeptides according to the invention, such as a natural substrate or ligand or a substrate or ligand mimetic. The polypeptides of the invention themselves can be labeled, e.g. radioactive or colorimetric so that the number of polypeptides that are bound to a ligand or that have undergone a reaction can be determined exactly. In this way, the effectiveness of an agonist or antagonist can be measured.

The invention furthermore relates to the use of a nucleic acid according to the invention, a DNA construct according to the invention or a vector according to the invention for producing transgenic plants, and the corresponding transgenic plants as such or their parts or propagation material.

Transgenic plants, parts of plants, protoplasts, plant tissue or plant propagation materials in which, after introduction of a nucleic acid according to the invention, a DNA construct according to the invention or a vector according to the invention, the intracellular concentration of the receptor-like protein kinases in the

The present invention also relates to an increase or decrease in comparison to the corresponding wild-type forms.

The term "plant parts" as used herein means all above-ground and underground parts and organs of the plants, such as shoot, leaf, flower and

Root, as well as protoplasts and tissue cultures made from it. The term "propagation material" as used herein means vegetative and generative propagation material such as cuttings, tubers, rhizomes, cuttings and seeds

The invention also relates to plants, parts of plants, protoplasts, plant tissue or plant propagation materials in which changes to the sequence coding for endogenous receptor kinases are made and selected, which lead to the production of a receptor kinase according to the invention or in which mutagenesis increases or decreases the endogenous receptor kinases. activity is achieved

For example, endogenous receptor kinase genes already in the plant can be modified by mutagenesis, for example using ethyl methanosulfonate (EMS). After the mutagenesis, individuals can be specifically selected on the basis of the. Sequence analysis, ligand binding studies or analysis of a biochemical reaction caused by the ligand binding Mutagenesis according to the invention produce receptor kinases with increased or reduced activity. In the same way, the expression of a receptor kinase gene according to the invention which is already in the plant can be changed by mutagenesis of the regulatory sequences in such a way that plants with a reduced or increased ligand sensitivity are produced Plants can be identified by well-known methods of gene expression analysis such as Northern blot or Western blot

Since the receptor-like protein kinases according to the invention, in particular the CRK1 receptor, play a role in signal perception, one obtains in transgenic "sense" plants or in plants which are linked to an increased amount or activity of corresponding endogenous receptor kinases or in signaling induction Elements were specifically selected, increased ligand sensitivity

On the one hand, such changes can result in lower ligands Concentrations are recorded and forwarded as a signal. It can also be achieved by switching on the signal transmission pathway ligand action in the absence of a ligand. On the other hand, in the case of constitutive expression, no negative feedback can occur, so that the cells have constant competence for the signal. A reaction to this situation is increased ligand

Effects. Transgenic "antisense" plants or plants with reduced receptor kinase activity have a reduced ligand sensitivity. An increase or decrease in receptor kinase activity can lead to plants that have a changed development, changed physiology or a changed mophology.

Examples

example 1

Isolation of the Nucleotide Sequence CRK1 Described:

Using the Representational Difference Analysis (RDA) method (Hubank and Schatz, 1994), a 400 bp fragment of CRKl from cell suspension cultures was obtained from Nicotiana tabacum L Kultivar Wisconsin 38 (Skoog and Miller, 1957) ISO-

Representational Difference Analysis (RDA) is a method for isolating differentially exposed genes. The two samples to be compared were formed by a tobacco cell culture, which was cultivated without cytokine addition, and a cell culture which after 5 days of subculturing for 45 min 10 ⁷ M BAP (Benzylaminopu- πn) was added

The fragments obtained were clomerized into the vector pUC19. The fragment of CRKl was further used as a probe for Northern and Southern blots and in a cDNA library screening to isolate the complete cDNA from

CRKl used as a probe

Isolation of the complete cDNA sequence from CRKl

(modified according to Stratagene)

A cDNA library was produced by the company SCInet in the λ-ZAP-Express Vector according to the manufacturer Stratagene. 400 μg total RNA of the tobacco suspension culture W 38 was added to the Fπ ma It was previously ensured that the searched mR \ \ is expressed in the culture after receipt of the finished cDNA library is called and it is called up in a five-step process Fig. Screen searched for the complete CRKl cDNA The titer of the cDNA library was 1 x 10 ⁹ pfu / ml

200 μl of E coh XLl-Blue MRF 'cells were incubated for 20 min at RT with 100,000 pfu of the corresponding cDNA bank, 7 ml of top agar were added and the mixture was preheated (37 ° C.) to NZY aga plates (diameter of 145 mm) Poured out After incubation UN at 37 ° C., phages were identified which carry an insertion homologous to the probe used

Pπmar screen

The phage plates were cooled for 1 h at 4 ° C., nylon filters placed for 1 mm, with the DNA side up for 2 min on Whatman 3M filter paper soaked in denaturation solution and 5 min on Whatman 3M filter paper soaked in re-solution solution. The filters were briefly incubated dipped in a 2 x SSC solution, dried and irradiated with UV light for DNA fixation (UV-Transil-

2 luminator, 0, 12 J / cm)

After identifying positive plaques, they were cut out generously (approx. 1 cm) in the Pπmar screen with a scalpel and shaken for at least one hour with 3-5 ml of SM buffer. 1 μl and

1 μl of a 1 10 dilution used Positive plaques in the secondary screen were excised with sterile Pasteuφipettes, dissolved in 500 μl SM buffer and 30 μl of a 10 ² dilution used for a tertiary screen. The same dilution was used for a further round of screening and partial plaques could be obtained. These positive single plaques became a fifth

Round in which all the individual plaques obtained on the plate had to be positive in order for the corresponding clone to be processed further. From these individual plaques, the cDNA inserts were converted into plasmid DNA by means of an excision reaction in an E coh XLOLR strain and isolated was experienced according to the manufacturer Stiatagene v 573-RACE

To isolate the 5 'and 3' ends missing in the identified cDNA clone, the 573 'RACE method was used

The 5'-RACE is based on the specific amplification of the 5 end of a gene from mRNA. With the aid of a sequence-specific polymer and the AMV reverse transceptase, the first strand of cDNA is synthesized. A poly (A) tail is attached to the product so that an ohgo dT anchor primer and a nested sequence-specific primer can be used in the subsequent PCR. In a second PCR, another nested primer can be used to ensure the specificity

In the 3 'RACE, the cDNA first strand synthesis takes place with an ohgo dT polymer and the subsequent PCR reactions with sequence-specific polymers. The products were cloned with the "TA cloning kit" from Pharmacia

Example 2

Northern blot analyzes were carried out to determine the differential expression of CRK1 in response to cytokinin

Total RNA isolation from tobacco suspension cultures

(Puissant and Houdebine, 1990)

To obtain total RNA from suspension cell cultures, the cells were briefly dried over a Nutsche which was connected to a vacuum pump

The material was frozen in liquid nitrogen and stored at -80 ° C until the start of RNA isolation

1 g of plant material is homogenized at 4 ° C. with 5 ml of cooled RNA isothermal buffer using mortars. The homogenized plant material is transferred to Greiner tubes. 0.5 ml of 2 M sodium acetate (pH 40) is added and on a vortex vibration mixer Treated After adding 5 ml of phenol, the solution is again mixed on the vibration mixer. For better phase separation, 1 ml of chloroform is added and centrifuged for 10 min at 4000 rpm. The water phase is removed and transferred to a fresh Greiner tube

The RNA is precipitated with 0.7 parts by volume of isopropanol and centrifugation at 4000 rpm for 10 mm. The collected pellet is resuspended in 2 ml of 4 M lithium chloride for the purification of polysaccharides and again centrifuged for 10 min at 4000 rpm. The pellet is dissolved in 2 ml of TES. Buffer resuspended and mixed with 2 ml chloroform for the purification of proteins and phenol residues and centrifuged for 10 min at 4000 rpm to separate the phases 0.3 M sodium acetate (pH 5.2) filled in. A centrifugation is carried out for 30 min at 4000 rpm. Then, salts from the RN A pellet are washed with 70% alcohol Centrifuged at 4000 rpm for 10 min. After removing the alcohol, the pellet is dried in vacuo and dissolved in 17 μl TE.

Isolation of polyA-mRNA from total RNA preparations (according to the Dynal protocol)

The principle of purification is based on the separation of polyadenylated mRNA by o / z ^' go-öT-coated Dynabeads according to the manufacturer's instructions. The yield of mRNA was approximately 1-2% of the total amount of RNA used.

The application was carried out according to the manufacturer.

The agarose is with a final concentration of 1.5% agarose in H ₂ O dest. Set on and boiled. After cooling to 60 ° C, 1/10 part by volume of 10 x MOPS buffer and 1/6 part by volume of formaldehyde (37%) are added. EtBr is added to determine identical quantities. After curing, the gel is set to 19 V for 10 min. 50 μg of the nucleic acid samples and 3 μl of the length standard (RNA: 0.24-9.5 kb RNA ladder from Gibco BRL Lifetechnologies) are mixed with 2 parts by volume of sample buffer and denatured for 10 min at 65 ° C. in the heating block. The RNA samples are electrophoresed at 45 V for about 5 h.

Transfer of the RNA to a nylon membrane

Those in the denaturing agarose formaldehyde gel electrophoretically separated

Nucleic acid samples are blotted on a nylon membrane. For this purpose, 3M Whatman absorbent paper is impregnated with 10 x SSC buffer on a glass pane and attached over two containers so that there are no air bubbles so that its ends are immersed in the containers. These are also filled with 10 x SSC buffers. The gel is placed, topside down, on the 3M Whatman absorbent paper without air bubbles and with the

Nvlon membrane covered. The membrane should not be larger than the gel and Lie on the gel without air bubbles. Two layers of 3M Whatman absorbent paper are then placed on top of the nylon membrane. The agarose gel is now sealed with film so that no liquid can be sucked past it. About 3 cm of absorbent paper, a larger glass pane and then on top of the 3M Whatman absorbent paper a weight of about half a kilogram is placed. Blotting is carried out for at least 6 hours. The RNA is fixed by \ J / -cι os shnkιng with a Fluo Link UV lamp. The dose is 0.12 J / cm

Radioactive labeling by "in vitro transcription"

The fragment to be labeled must be clomerized in a vector which has a T3 and / or a T7 promoter, eg pBluescπpt. The plasmid is digested with a restriction enzyme which is downstream of the sequence to be transcribed to transcribe the entire plasmid prevent after one

Proteinase K digestion, the DNA is subjected to a phenol / chloroform extraction and precipitated

The sequence of the 15 clone was cut out of the pUC19 vector via Eco RI and Xba I sections and clomerized into the pBluescπpt SK vector using the same restriction sections. Using the T3 promoter, an antisense RNA can be used with the T3 polymerase Synthesized 1 μg DNA was used for labeling. The Stratagene '7/7 vüi o transcπptιon "-Kιt was used and the manufacturer's instructions followed. After the" In viti o Transcnption ", a DNase digestion of 15 min was carried out The RNA was

Phenol / chloroform shaken out and precipitated. The incorporation rate averaged 5 10 cpnVμg DNA. After a denaturation, the tagged probe was added to ori-modified Membi Separation of the non-incorporated nucleotides

A Sephadex G50 column ("Nick Columns", Pharmacia) is washed with 10 ml of water. The labeling mixture and 350 μl of water are pipetted on, the eluate is discarded. 400 μl of water are added to the column. The eluate contains the marked sample of 1 μl of the Eluates are taken to determine the specific activity (cpm / μg DNA). This should be at least 10 ⁸ cpm / μg of DNA used. The remaining eluate is denatured for 10 min at 95 ° C. and, after briefly cooling on ice, is added to the hybridization tubes

Hybridization with "P-labeled nucleic acid fragments

The nylon membrane with the fixed nucleic acid is placed in a hybridization tube. To block unspecific binding sites on the membrane, a prehybination is carried out first with sperm semen. For this purpose, 10 ml of hybridization solution per 10 cm ² membrane are added to the hybridization tubes and with 1 ml of freshly denatured herring sperm solution μg / ml) each 10 ml hybridization solution added. The pre-hybridization takes place, like the hybridization, at 68 ° C. in the hybridization oven. The pre-hybridization should take at least half an hour, after which the labeled and freshly denatured DNA sample is added. The hybridization is carried out overnight

The next day, non-hybridized DNA sample and non-specific bindings are washed off the membrane

Room temperature with 2 x SSC, 0.1% SDS solution and then washed for 30 mm at Hvbπdisierung temperature with 0.2 x SSC, 0.1% SDS solution

The membrane is sealed in foil and an X-ray film is placed on the detection literature

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J.Z .; Miller W. and Lipman, D.J. 1997. Gapped BLAST and PSI-BLAST generafion of protein database search programs. Nucleic Acids Res. 25: 3389-3402.

Becraft, P.W. 1998. Receptor kinases in plant development. TIPS 3: 384-388.

Bevan M. 1984. Binary Agrobacterium vectors for plant transformation. Nucleic

Acids Res 12 (22): 871 1 -8721.

Fantl, W.J., Johnson, D.E. and Williams, L.T. 1993. Signaling by receptor tyrosine kinases Ann. Rev. Biochem. 62: 453-481.

Hanks, S.K., Quinn, A.M. and Hunter, T. 1988. The protein kinase familiy: Conserved features and deduced phylogeny of the catalytic domains. Science 241: 42-52.

Hubank, M. and Schatz, D.G. 1994. Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucl. Acids Res. 22: 5640-5648.

Kaminek, M. 1992. Progress in cytokinin research. TIBTECH 10, 159-164.

Lottsspeich, F., Zorbas H. (ed.). 1998. Bioanalytics. Spectrum of academic

Publishing house, Heidelberg, Berlin.

Minet, M., Dufour. M.-E. and Lacroute, F. 1992. Complementation of Saccharomyces cerevisiae au otrophic mutants by Arabidopsis thaliana cDNAs. Plant J. 2: 417-422. Puissant, C. and Houdebine, L.-M. 1990. An improvement in the single-step method of the RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. BioTechniques 8: 148-149.

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Cytokimns, CRC Press, Boca Raton: 15-34.

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Claims

claims

1. Nucleic acids which code for plant polypeptides with the biological activity of a receptor-like protein kinase which comprises the amino acid sequence according to SEQ ID NO: 2.

2. Nucleic acids according to claim 1, characterized in that they code for polypeptides with the activity of serine / threonine kinases.

3. Nucleic acids according to claim 1 or 2, characterized in that it is single-stranded or double-stranded DNA or RNA.

4. Nucleic acids according to claim 3, characterized in that they are fragments of genomic DNA or cDNA.

5. Nucleic acids according to one of claims 1 to 4, characterized in that they come from tobacco plants.

6. Nucleic acids according to one of claims 1 to 5, comprising a sequence selected from

(a) the sequence according to SEQ ID NO: 1,

(b) sequences which code for a polypeptide which comprises the amino acid sequence according to SEQ ID NO: 2,

(c) partial sequences of at least 14 base pairs long of the sequences defined under (a) or (b),

(d) sequences which hybridize to the sequences defined under (a) or (b), (e) sequences which have at least 60% identity with the sequences defined under (a) or (b),

(f) sequences which have at least 60% identity with the N-terminal receptor domain of the sequences defined under a) or b),

(g) sequences which are complementary to the sequences defined under a) or b), and

(h) sequences which, owing to the degeneracy of the genetic code, code for the same amino acid sequence as the sequences defined under (a) to (e)

Regulatory region, which naturally transcribes a

Nucleic acid according to one of claims 1 to 6 in plant cells, in particular in tobacco plants, controlled

DNA construct comprising a nucleic acid according to any one of claims 1 to 6 and a heterologous promoter

Vector comprising a nucleic acid according to any one of claims 1 to 6, a regulatory region according to claim 7 or a DNA construct according to claim 8

Vector according to claim 9, characterized in that the nucleic acid is functionally linked to regulatory sequences which guarantee the exposition of the small-molecule strain in pi o or the eucarvotic cells

1 1. Host cell containing a nucleic acid according to one of claims 1 to 6, a DNA construct according to claim 8 or a vector according to claim 9 or 10.

12. Host cell according to claim 1 1, characterized in that it is a prokaryotic cell, in particular E. coli.

13. Host cell according to claim 1 1, characterized in that it is a eukaryotic cell, in particular a yeast, insect, mammal or plant cell.

14. A polypeptide with the biological activity of a receptor-like kinase which is encoded by a nucleic acid according to one of claims 1 to 6.

15. Polypeptide with the biological activity of a receptor-like kinase, which comprises an amino acid sequence which has at least 60% identity with the sequence according to SEQ ID NO: 2.

16. Antibody which binds specifically to a polypeptide according to claim 14 or 15.

17. A method for producing a nucleic acid according to any one of claims 1 to 6, comprising the following steps:

(a) Complete chemical synthesis in a manner known per se or

(b) chemical synthesis of oligonucleotides, labeling of the ohgonucleotides, hybridization of the ohgonucleotides to DNA of a genomic or cDNA library which was produced from plant cells on the basis of genomic DNA or mRNA, selecting positive cloning and isolating the hybridizing DNA from positive clones or

(c) chemical synthesis of oligonucleotides and amplification of the target DNA by means of PCR.

18. A method of making a polypeptide according to claim 14 comprising

(a) cultivating a host cell according to any one of claims 1 1 to

13 under conditions which ensure the expression of the nucleic acid according to one of claims 1 to 6, or

(b) expressing a nucleic acid according to any one of claims 1 to 6 in an in tro system, and

(b) the recovery of the polypeptide from the cell, the culture medium or the in vitro system.

19. A method for finding a chemical compound that binds to a polypeptide according to claim 14 or 15, comprising the following steps:

(a) contacting a host cell according to any one of claims 1 to 13 or a polypeptide according to claim 14 or 15 with a chemical compound or a mixture of chemical compounds under conditions which allow the interaction of a chemical compound with the polypeptide, and

(b) Determine the chemical compound that specifically binds to the polypeptide. A method of finding a compound which changes the expression of polypeptides according to claim 14, comprising the following steps

(a) contacting a host cell according to any one of claims 1 1 to 13 with a chemical compound or a mixture of chemical compounds,

(b) determining the polypeptide concentration, and

(c) Determining the compound that specifically affects expression of the polypeptide

Use of a nucleic acid according to one of claims 1 to 6, a DNA construct according to claim 8, a vector according to claim 9 or

10, a host cell according to any one of claims 1 1 to 13, a polypeptide according to claim 14 or 15, or an Antikoφers according to claim 16

Finding new herbicidal agents

Use of a modulator of a polypeptide according to claim 14 or 15 as a plant growth regulator or herbicide

Use of a nucleic acid according to one of claims 1 to 6, a DNA construct according to claim 8, a vector according to claim 9 for the production of transgenic plants

Transgenic plants, plant parts, piotoplasts, plant tissues or planters are used in propagation material, characterized in that after inserting a nucleic acid according to one of Claims 1 to 6 of a D \ A construct according to Claim 8 or a vector according to Claim 9, the intracellular concentration of a polypeptide according to claim 14 is increased or decreased compared to the corresponding wild-type cells.

25. Plants, parts of plants, protoplasts, plant tissue or plant propagation materials, characterized in that they are a polypeptide according

Claim 15 contain, whose biological activity or expression pattern is changed compared to the corresponding endogenous polypeptides.

26. A method for producing plants, plant parts, protoplasts, plant tissues or plant propagation materials according to claim 25, characterized in that a nucleic acid according to one of claims 1 to 6 or a regulatory region according to claim 7 is changed by endogenous mutagenesis.