MXPA98006589A - Nucleic acid that codifies the gai gene of the arabidopsis thali - Google Patents

Abstract

The GAI gene of Arabidopsis thaliana has been cloned, together with the mutant and homologous genetic sequences. The expression of such genes in plants affects the characteristics of plants that include their growth. The expression of the GAI gene inhibits the growth of plants, whose inhibition is antagonized by gibberelin (GA). The expression of the gai mutants confers a dwarf phenotype that is insensitive to GA. Manipulation of GAI expression and gai genes in plants results in tall or dwarf plants. The dwarf plants are particularly useful for the reduction in crop losses resulting from the fall due to the effect of wind or rain.

Description

NUCLEIC ACID THAT CODIFIES THE GAJ GEN OF Arabidqpsis thallana This invention relates to the genetic control of growth and / or development of plants and the cloning and expression of genes involved therein. More particularly, the invention relates to the cloning and expression of the GAI gene of Arabidopsis thaliana, and homologues of other species, and the use of genes in plants. An understanding of the genetic mechanisms which influence the growth and development of plants, includes flowering, providing a means for altering the characteristics of a target plant. The species for which the manipulation of growth and / or development characteristics can be advantageous include all crops, important examples are cereals, rice and maize, probably the most important agronomically in cold climatic zones, and wheat, barley, oats and rye plus acclimated at other temperatures. Important crops for seed production are rapeseed oil or turnip and sugarcane, sugar beet, corn, sunflower, soybean and sorghum. Many crops which are harvested by their roots are, of course, annual growth for seed and the production of seed of any species is very dependent on the ability of the plant to flower to be pollinated and to place seed. In horticulture, the control of the time of growth and development, including flowering, is important. Horticultural plants whose flowering can be controlled include lettuce, endives, and vegetable greens, including cabbage, broccoli, and cauliflower, and carnations and geraniums. Dwarf plants of other shapes and larger sizes, from the workshop plants may be otherwise advantageous and desirable in various agricultural and horticultural contexts. Arabidopsis thaliana is one of the favorite plants of geneticists as a model organism. Because it has a well characterized, small genome, which is relatively easy to transform and regenerate and has a rapid growth cycle, Arabidopsis is an ideal plant model in which growth and development and its control are studied. Many processes of plant development and growth are regulated by specific members of a family of tetracyclic diterpenoid growth factors known as gibberellins (GA) 1. The GAT mutation of Arabidopsis confers a dwarf phenotype and a dramatic reduction in sensitivity.29 9 The molecular cloning of GAI via Ds transfer mutagenesis has been reported.

The phenotype conferred by the insertion of the Ds alleles confirm that the GAI is a gain-of-function mutation, and that the wild-type allele (GAJ) is indispensable5,6. The GAJ encodes a new polypeptide (GAJ) of 532 amino acid residues, of which the 17 amino acid domain is lost in the mutant GAJ polypeptide. This result is consistent with the GAJ that acts as a repressor of plant growth whose activity is antagonized by GA. Although not bound by any particular theory, GAJ can constitutively repress growth because it lacks the domain that interacts with the GA signal. In addition, in accordance with this model the GA regulates the growth of the plant by de-repression. GAI is a mutation of the gain of the dominant function, which confers a brown-green, dwarf phenotype and interferes with the GA reception or subsequent transduction signal.2"9 Dominant mutations that confer similar phenotypes are known in other species of plants, including corn10-12 and wheat.13 The latter are especially important because they are the basis of the high yield of semi-dwarf wheat varieties of the green revolution.14 The increase in the yield of these varieties is due to an increase in production of the grain per ear, and the superior vigor of the straw Short, strong straw, greatly reduces the results of weakening of the plantation, of wheat plants that are knocked down or planted from those that remain standing by rain or wind. Cloning of gai from Arabidopsis is exposed because of its importance for the understanding of the transduction signal GA, and due to the potential use of GA insensitivity in the development of wheat and other crops such as rape seed oil or turnip and rice which can show improvement as great as that which has been seen since in wheat. In accordance with a first aspect of the present invention there is provided a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with a GAJ function. The term "GAJ function" indicates the ability to influence the phenotype of a plant similar to the GAJ gene of Arabidopsis thaliana. The "GAJ function" can be observed phenotypically in a plant as inhibition, suppression, repression or reduction of plant growth, in which the inhibition, suppression, repression or reduction is antagonized by GA. The expression GAJ tends to confer a dwarf phenotype on a plant which is antagonized by GA. Overexpression in a plant from a nucleotide sequence that encodes a polypeptide with GAJ function can be used to confer a dwarf phenotype in a plant which is correctable by treatment with GA. Also, in accordance with one aspect of the present invention, there is provided a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with the ability to confer a gai mutant phenotype on expression. The GAJ mutant plants are dwarf compared to the wild type, the dwarfism is GA insensitive. For gibberellins or GA ^ a diterpenoid molecule with the basic carbon ring structure shown in Figure 1 is suggested and has more biological activity, ie, it refers to biologically active gibberellins. The biological activity can be defined by one or more stimulations of the elongation of the cell, leaf senescence or by the extraction of the a-amylase response of the aleurone of the cereal. There are many standard tests suitable in the art, a positive result in either one or more of the signals proves a gibberel to be biologically active, 9, 30. Convenient assays in the art include the hypocotyl assay of the lettuce, the cucumber hypocotyl test, and the first oat leaf test, all were determined for biological activity based on the ability of a gibberellin applied to cause the elongation of the respective tissue. Preferred assays are those in which the test compositions are applied to a gibberellin deficient plant. Thus, the preferred assays include GA deficient Arabidopsis treatment to determine growth, in the dwarf peas test the elongation of the internodes was determined, in the Tanginbozu dwarf rice test, the elongation of the pod was determined. the leaf, and in the d-5 corn test, the elongation of the leaf sheath was also determined. The elongation bioassay measures the effects of cell elongation in general on the respective organs, and is not restricted to particular cell types. Convenient tests also include the dock (Rumex) test of leaf senescence (Rumex) and the a-amylase assay of aleurone from the cereal. The aleurone cells which round off the endosperm in the grain secrete a-amylase in the germination, which digests the starch to produce sugars that are then used for the growth of the plant. The production of enzyme is controlled by GA. Isolated aleurone cells give GA biologically active secreting α-amylase whose activity can then be assayed, for example by measurement of starch degradation. Structural factors important for high biological activity (exhibited by GA], GA2, GA4 and GA) are a C-6 carboxyl group of the B-ring; C-19, C-10 lactone; and ß-hydroxylation at C3. The β-hydroxylation at C-2 causes inactivity (exhibited by GAg, GA29, GA34, and GA51). GAJ mutants do not respond to GA treatment, for example, treatment with GA ?, GA3 or GA4. Treatment with GA is preferably by spraying with aqueous solution, for example spraying with 10"4M GA3, or GA4 in aqueous solution, perhaps once a week or more frequently, and may be by droplet placement in the plants in Once the spray is applied, the GA can be applied dissolved in an organic solvent such as ethanol or acetone, because it is more soluble in these than in water, but this is not more preferred because these solvents have a tendency to damage the If an organic solvent is used, suitable formulations include 24 μl of 0.6, 0.4 or 300 mM GA3 or GA4, dissolved in 80% ethanol.The plants, for example, Arabidopsis, can grow in a medium containing GA , such as tissue culture media (GM) solidified with agar and containing supplemental GA.

The nucleic acid according to the present invention may have the sequence of a wild-type GAJ gene from Arabidopsis thaliana, or be a mutant, derivative, variant or allele of the provided sequence. The preferred mutants, derivatives, variants and alleles are those which encode a protein which retains a functional characteristic of the protein encoded by the wild-type gene, especially the ability to inhibit the growth of the plant, in which the inhibition is antagonized by GA , or the ability to confer a plant one or more of the other characteristics of response to the GA treatment of the plant. Other preferred mutants, derivatives, variants and alleles that encode a protein confer a gai mutant phenotype, which is said to reduce the growth of the plant in which the reduction is insensitive to GA, ie, not overcome by GA treatment. Changes to a sequence, to produce a mutant, variant or derivative, can be by one or more additions, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one. or more amino acids in the encoded polypeptide. Of course, the changes in the nucleic acid which do not make a difference in the encoded amino acid sequence are included.

A preferred nucleotide sequence for the GAJ gene is one, which encodes the amino acid sequence shown in Figure 4, especially a sequence code shown in Figure 3. A preferred GAJ mutant lacks part or all of the sequence of 17 amino acids underlined in Figure 4. The present invention also provides a construct nucleic acid or vector which comprises nucleic acid with any of the sequences provided, preferably a builder or vector which encodes the polypeptide for the nucleic acid sequence can be expressed. The builder or vector is preferably convenient for transformation into a plant cell. The invention also accompanies a host cell transformed with such a construct or vector, especially a plant cell. In addition, a host cell, such as a plant cell, comprising nucleic acid according to the present invention is provided. Within the cell, nucleic acid can be incorporated into chromosomes. They can be more than one heterologous sequence per haploid genome. This, for example, allows for increased expression of the gene product compared to endogenous levels, as discussed above. A construct or vector comprising nucleic acid according to the present invention need not include a promoter or other regulatory sequence, particularly if the vector is used to introduce the nucleic acid into the cells for recombination within the genome. However, in one aspect of the present invention there is provided a nucleic acid construct comprising a GAJ or gai coding sequence (which includes homologs other than Arabidopsis thaliana), associated for a regulatory sequence for the control of expression, the sequence regulatory is distinct from that naturally fused to the sequence code and preferably from or derived from other genes. The nucleic acid molecules and vectors according to the present invention may be as isolated, provided isolation from their natural environment, in substantially pure or homogeneous form, or free or substantially free of nucleic acid or genes of the species of interest or originating from others other than the sequences encoding a polypeptide capable of influencing growth and / or development, which may include in flowering, for example in the nucleic acid of Arabidopsis thaliana instead of the coding sequence GAJ. The term "isolated nucleic acid" completely or partially accompanies the synthetic nucleic acid.

The nucleic acid can of course be of double or -simple-strand, cDNA or genomic DNA, DNA, full or partially synthetic RNA as appropriate. Of course, when the nucleic acid according to the invention includes RNA, the reference to the sequence shows that it should be constructed with reference to the equivalent RNA, with U substituted for T. The present invention also accompanies the expression product of any of the nucleic acid sequences described and methods of making the expression product by expression from the nucleic acid which therefore encodes convenient conditions in suitable host cells. Those skilled in the art are very capable of constructing vectors and designing protocols for the expression and recovery of recombinant expression products of the gene. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminal fragments, polyadenylation sequences, increased sequences, marker genes and other sequences as appropriate. For additional details see, for example, Molecular Cloning: a Labora tory Manual: 2nd. Edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press. Transformation procedures depend on the host used, but they are well known. Many known techniques and protocols for nucleic acid manipulation, for example, in the preparation of nucleic acid constructs, mutagenesis, sequences, introduction of DNA into cells and expression of the gene, and protein analysis, are described in detail in Protocols in Molecular Biology, Second Edition, Ausbel et al. eds., John Wiley & amp;; Sons, 1992. The specific procedures and vectors previously used with great success on plants are described by Bevan, Nucí. Acid Res. (1984) 12, 8711-8721), and Guerineau and Mullineaux, (1993) Transformation of plant and expression vectors. In: Plant Molecular Biology The fax (Croy RRD of) Oxford, BIOS Scientific Publishers, pp 121-148. The description of Sambrook et al. and Ausubel et al. and all other documents mentioned herein, are incorporated herein by reference. Since the amino acid sequence GAJ of Arabidopsis shown in Figure 4 includes 5 consecutive histidines close to their N-termini, substantial purification of GAJ or gai can be carried out using convenient Ni-NTA resins available from QIAGEN Inc.

(USA) and DIAGEN Gmbh (Germany). See Jacknecht et al31 and EP-A-0253303 and EP-A-0282042. The Ni-NTA resin has high affinity for proteins with consecutive histidines near the N-O C-terminus of the protein and so they can be used to purify GAJ or gai proteins from plants. The parts of plants or extracts or of recombinant organisms such as yeasts or bacteria, for example. E. Coli, express the protein. The purified GAJ protein, for example recombinantly produced by the expression of nucleic acid encoding therefore, can be used to excite antibodies employing techniques which are standard in the art. Antibodies and polypeptides comprise anti-antibody binding fragments that can be used in identification of homologs of other species as discussed above. Methods for producing antibodies include immunizing a mammal (eg, human, rat, mouse, rabbit, horse, goat, sheep or monkey) with the protein or a fragment thereof. Antibodies can be obtained from animals immunized using any of the varieties of techniques known in the art, and could be protected, preferably using antibody binders for antigenes of interest. For example, Western staining or immunoprecipitation techniques can be used (Armitage et al, 1992, Nature 357: 80-82). The antibodies can be polyclonal or monoclonal.

As an alternative or supplement to immunize a mammal, antibodies with appropriate specific bonds can be obtained from a recombinantly produced library. Immunoglobulin expressed in variable domain, for example using lambda bacteriophages or filamentous bacteriophages which exhibit functional immunoglobulin binding domain on their surfaces; for example, see WO92 / 01047. Antibodies raised to a GAJ or gai polypeptide can be used in the identification and / or isolation of homologous polypeptides, and then the coding genes. In addition, the present invention provides a method of identifying or isolating a polypeptide with GAJ function or ability to confer a gai mutant phenotype, which comprises protecting a candidate polypeptide with a polypeptide comprising the antibody binding domain of an antibody (e.g. a whole antibody or a fragment thereof) which is capable of binding an Arabidopsis GAI or gai polypeptide, or preferably has link specificity for such polypeptides such as that having the amino acid sequence shown in Figure 4. The candidate polypeptides for being protected may for example be the products of an expression created in the cabinet using nucleic acid derivatives of a plant of interest, or they may be the product of a purification process from a natural source. A polypeptide that finds binding to the antibody can be isolated and then can be subject to the amino acid sequence. Any convenient technique can be used for the polypeptide sequence either completely or partially (for example a fragment of the polypeptide can be sequenced). The information of the amino acid sequences can be used in the obtaining of the nucleic acid encoding the polypeptide, for example by the designs of one or more of the oligonucleotides (for example a degenerate combination of oligonucleotides) to be used as caladores or first in hybridization for nucleic acid candidates, as will be discussed below. A further aspect of the present invention provides a method of identification and cloning of GAJ homologs of plant species other than Arabidopsis thaliana in which a nucleotide sequence derived from that shown in Figure 3 is employed. Sequences derived from these may be they themselves be used in the identification and cloning of other sequences. The information of the nucleotide sequence provided here, or any part of it, can be used in a database search to find homologous sequences, expression products of which can be tested by the GAJ function. Alternatively, those of cabinet nucleic acids can be protected using techniques well known to those skilled in the art and the homologous sequences thereby identified and then tested. For example, the present invention also provides a method of identifying and / or isolating a GAJ or gai homologous gene, comprising spotting the candidate (or "target") of the nucleic acid with nucleic acid encoding a polypeptide with a GAJ function or a fragment or mutant, derivative or allele thereof. The candidate nucleic acid (which may be, for example, cDNA or genomic DNA) may be derived from any cell or organism which may contain or is suspected to contain nucleic acid encoding such a homologue. In a preferred embodiment of this aspect of the present invention, the nucleic acid used for setting the nucleic acid candidate encodes an amino acid sequence shown in Figure 4, a sequence complementary to a sequence code, or a fragment of either they, more preferably comprise a nucleotide sequence shown in Figure 3. Alternatively, as discussed, a draft can be designed using an amino acid sequence information obtained by the sequence of a polypeptide identified as being capable of binding by an antigen domain. binder of an antibody which is capable of binding a GAJ or gai polypeptide such as one to the amino acid sequence shown in Figure 4. Preferred staging or test conditions are those which are quite longitudinal to be a simple pattern with a small number of hybridizations identified as positive, which can be further investigated onally. It is well known in the art to increase the stringency of hybridization gradually to only a few remnants of positive clones. As a further alternative, although still employing nucleic acid hybridization, oligonucleotide designs for amplifying DNA sequences from GAJ genes can be used in PCR or other methods involving nucleic acid amplification, using routine procedures. See for example "PCR protocols; A Guide for Methods and Applications ", Eds. Innis er al, 1990, Academic Press, New York Convenient preferred amino acid sequences for use in the design of openings or early PCRs are conserved sequences (completely, substantially or partially) between genes GAJ.

One of the bases of the information of the amino acid sequences, oligonucleotide cleavage or digest can be designed, taking into account the degeneracy of the genetic code, and, where appropriate, use the organism derived codon from the nucleic acid candidate. The present invention also extends to the nucleic acid encoding a GAJ homologue obtained using a nucleotide sequence derived from that shown in Figure 3. Also included within the scope of the present invention are the nucleic acid molecules which encode the sequences of amino acids which are homologous to the polypeptides encoded by the Arabidopsis thaliana GAJ. A homolog may be of a different species than Arabidopsis thaliana. The homology can be to the nucleotide sequence and / or amino acid sequence level. Preferably, the nucleic acid and / or the homology distributed in the amino acid sequence with the sequence encoding the nucleotide sequence of Figure 3, preferably at least about 50% or 60% or 70% or 80% homology, more preferably at least 90% or 95% homology. The nucleic acid encoding such a polypeptide can preferably be distributed with the GAI gene of Arabidopsis thaliana which has the ability to confer a particular phenotype of expression in a plant, preferably a phenotype which is a GA response (i.e., there is a change in a characteristic of the plant in the treatment with GA), such as the ability to inhibit the growth of the plant where the inhibition is antagonized by the GA. As noted, the expression GAI in a plant can affect one or more of the other characteristics of the plant. A preferred feature that can be distributed with the GAI gene of Arabidopsis thaliana is the ability to complement a null GAJ mutant phenotype in a plant such as Arabidopsis thaliana, such a phenotype is resistant to the dwarfing effect of paclobutrazol. Some preferred embodiments of the polypeptides according to the present invention (encoded by the nucleic acid modalities according to the present invention) include the sequence of 17 amino acids which are underlined in Figure 4 or a contiguous sequence of amino acid residues with at least about 10 residues with similarity or identity to the respective corresponding residue (in terms of position) in 17 amino acids which are underlined in Figure 4, more preferably, 11, 12, 13, 14, 15, 16, or 17 residues ).

As is well understood, the homology at the amino acid level is generally in terms of similarity or identity of the amino acid. The similarity allows "conservative variation", i.e., substitution of a hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine. The similarity can be defined and determined by the TBLASTN program of Altschul et al. (1990) J. Mol. Biol. 215: 403-10, which is in use as a standard of the art. The homology may be on the full length of the GAJ sequence of Figure 4, or it may be more preferably on a continuous sequence of 17 amino acids, compared to the 17 amino acids underlined in Figure 4, or a long sequence, eg, about 20, 25, 30, 40, 50 or more amino acids compared to the amino acid sequence of Figure 4 and preferably including the 17 underlined amino acids. At the level of the nucleic acid, the homology may be on full length or more preferably by comparison with the sequence encoding nucleotide 51 within the sequence of Figure 3 and encoding the amino acid sequence 18 underlined in Figure 4, or a long sequence, for example, about 60, 70, 80, 90, 100, 120, 150 or more nucleotides and preferably including nucleotide 51 of Figure 3 which encodes the sequence of 17 underlined amino acids of Figure 4. homologs for GAJ mutants are also provided by the present invention. These can be mutants where the wild type includes 17 underlined amino acids in Figure 4, or a contiguous sequence of 17 amino acids with at least about 10 (most preferably 11, 12, 13, 14, 15, 16, or 17) which have similarity or identity to the corresponding residue in the 17 amino acid sequence underlined in Figure 4, but the mutant does not. The nucleic acid encoding such mutant polypeptides can, in expression in a plant, confer a phenotype which is insensitive or unresponsive to the treatment of the plant with GA, which is a mutant phenotype which does not overcome or is not reversible to the type phenotype. wild in the treatment of the plant with GA (although it may only be a response in the plant in GA provision or reduction). A further aspect of the present invention provides an isolated nucleic acid having a nucleotide sequence encoding a polypeptide which includes an amino acid sequence which is a mutant, allele or derivative or variant sequence of the amino acid sequence GAJ of the Arabidopsis species thaliana shown in Figure 4, or is a homolog of other species, or a mutant, allele, derivative or variant thereof, wherein said mutant, allele, derivative, variant or homologous differs from the amino acid sequence shown in Figure 4 by means of the insertion, elimination, addition and / or substitution of one or more amino acids, obtained to produce transgenic plants by the transformation of plants which have a null GAJ mutant phenotype, in which the phenotype is resistant to the effect of dwarfing of paclobutrazol, with nucleic acid testing, causing or allowing expression of nucleic acid tests within plants t ransgenic, transgenic plants are protected to exhibit complementation of the null GAJ mutant phenotype to identify nucleic acid tests capable of complementing the null GAJ mutant, by suppressing the nucleic acid thus identified by being able to complement the null GAJ mutant to a nucleotide sequence encoding the sequence of 17 amino acids underlined in Figure 4 or a contiguous sequence of 17 amino acids in which at least 10 residues have similarity or identity to the respective amino acid at the corresponding position in the sequence of 17 amino acids underlined in Figure 4, more preferably 11, 12, 13, 14, 15, 16 or 17.

The GAJ and gai gene homologues can be identified from economically important monocotyledonous plant cultures such as wheat, rice and corn. Although the genes encoding the same protein in monocotyledonous and dicotyledonous plants show relatively little homology at the nucleotide level, the amino acid sequences are conserved. In the public sequence database, several EST sequences were recently identified that were obtained in random sequence programs and that provided homology with GAJ. Table 2 gives details, showing that the homologous sequences that were found in several species, include Zea mayz (Maize), O. sativa (rice), and Brassica napus (turnip). The sequence is obtained by studying expression models and examining the effect of the alteration of its expression, the homologous GAJ gene, which carries out a similar function in other plants. Of course, new uses and mutants, derivatives and alleles of these sequences are included within the scope of the various aspects of the present invention in the same terms as discussed above for the Arabidopsis thaliana gene. A nucleic acid-containing cell of the present invention represents a further aspect of the invention, particularly a plant cell or a bacterial cell. The cell can comprise the nucleic acid encoding the enzyme by virtue of introduction into the cell or an ancestor thereof of the nucleic acid, for example, by transformation using any convenient technique available to those skilled in the art. Also in accordance with the invention, a plant cell having nucleic acid incorporated within its genome is provided as described. The present invention also provides a plant comprising such a plant cell. Also in accordance with the invention, there is provided a plant cell having incorporated into its genome a nucleotide sequence as provided by the present invention, under the operative control of a regulatory sequence for the control of expression. A further aspect of the present invention provides a method of making such a plant cell involving the introduction of a vector comprising the nucleotide sequence within a plant cell and causing or allowing recombination between the vector and the cell genome. of the plant to introduce the sequence of the nucleotides within the genome.

A plant according to the present invention may be one, which does not actually multiply in one or more properties. Plant varieties, particularly varieties of plants registered in accordance with Plant Multiplication Rights, may be excluded. It is noted that a plant does not need to be considered as a "plant variety" simply because it contains within its genome a stable transgene, introduced into a plant cell or an ancestor thereof. In addition to a plant, the present invention provides any clone of such plant, seed, or hybrid progeny and their own descendants; and any part of any of them, such as cutting seeds. The invention provides any plant propagule, which is any part which can be used in sexual or asexual reproduction or propagation, including cuts or seeds. Also accompanied by the invention is a plant which is a fruit propagated sexually or asexually, a clone or descendant of such plant, or any part or propagule of said plant, fruit, clone or descendant. The invention further provides a method for influencing the characteristics of a plant comprising the expression of a sequence of GAJ gene or heterogeneous gai (or mutant, allele, derivative or homologue thereof, as discussed), within the cells of the plant . The term "heterologous" indicates that the gene / nucleotide sequence in question has been introduced into said cells of the plant, or an ancestor thereof, using genetic engineering, which is known to be by human intervention, which may comprise transformation . The gene can be in an extra genomic or incorporated vector, preferably stable within the genome. The heterologous gene can be replaced by an endogenous equivalent gene, i.e. one which normally performs the same or a similar function in the control of growth and / or development, or the inserted sequence can be additional to an endogenous gene. An advantage of the introduction of a heterologous gene is the ability to place the expression of the gene under the control of a selection promoter, in order to be able to influence the expression of the gene, and thereby the growth and / or development of the gene. plant in accordance with the preference. In addition, mutants and derivatives of the wild-type gene can be used in place of the endogenous gene. The inserted gene may be endogenous or exogenous to the host cell, for example, from other plant species. The main characteristic which can be altered using the present invention is growth.

In accordance with the GAJ gene model as a growth repressor, low expression of the gene can be used to promote growth, at least in plants which have only one endogenous gene conferring GAJ function (not for example Arabidopsis which has endogenous homologs which could compensate). This may involve the use of anti-sensor or sensory regulation. The workshop plants can be made by beating the GAJ or the relevant homologous gene in the plant of interest. Plants can be made with resistance to compounds which inhibit GA biosynthesis, such as paclobutrazol, for example to allow the use of a GA biosynthesis inhibitor to maintain dwarf grasses to allow the growth of stems in plant crops. Overexpression of the GAJ gene can lead to a dwarf plant to be corrected by GA treatment, as predicted by the GAJ repression model. Since gai mutant genes are dominant in phenotype, they can be used for the production of GA-insensitive dwarf plants. This can be applied for example to any transformable plant crop, trees or fruit tree species. It can provide high yields / reduced plantings similar to Rth wheat. In rice this can provide GA-insensitive rice resistance to Bakana disease, which is a problem in Japan and elsewhere. Dwarf ornamentals can be of value for the markets of horticulture and flower cutting. The manipulation of the sequence can provide several degrees of severity of dwarfism, GA-insensitive phenotype, allowing to adapt the degree of severity to the needs of each plant culture or wishes of the manipulator. The over expression of the GAJ mutant sequences is potentially the most used. A second characteristic that can be altered is the development of the plant, for example flowering. In some plants, and under certain environmental conditions, a GA signal is required for floral induction. For example, mutant Arabidopsis plants with poor GA that grow under short day conditions will not flower unless they are treated with GA: these plants flower normally when they grow under long day conditions. The Arabidopsis gai mutant plants show suppression of flowering under short-day conditions: several mutants may not flower all. In addition, for example, by the expression of GAJ or gai gene or expression, plants can be produced that allow vegetative residues to give the GA treatment to induce flowering. This can be used in horticultural contexts or in spinach, lettuce and other crops where the suppression of the blow is desirable. The nucleic acid according to the invention can be placed under the control of an externally inducible promoter gene to place the GAJ or gai code sequence under user control. The term "inducible" as applied to a promoter is well understood to those skilled in the art. In essence, expression under the control of an inducible promoter is "connected in" or increased in response to an applied stimulus. The nature of the stimulus varies among the promoters. Some inducible promoters cause small or undetectable levels of expression (or non-expression) in the absence of the appropriate stimulus. Other inducible promoters cause detectable constitutive expression in the absence of the stimulus. Whatever the level of expression is in the absence of the stimulus, the expression of any inducible promoter is increased in the presence of the correct stimulus. The preferable situation is when the level of expression is increased over the application of the relevant stimulus by an effective amount by altering the phenotypic characteristics. In addition an inducible (or connected) promoter that can be used causes a basic level of expression in the absence of the stimulus in which the level is also low to bring about a desired phenotype (and they can in fact be zero). Upon the application of the stimulus, the expression increases (or connects in) at a level which brings approximately the desired phenotype. Suitable promoters include the promoter of the 35S virus gene of the Cauliflower Mosaic (CaMV) which is expressed at a high level in virtually all plant tissues (Benfey et al, 1990a, and 1990b); the promoter of the isoform II gene of corn glutanione-s-transferase (GST-II-27) is activated in response to an application of exogenous (WO93 / 02194, ICI Ltd); the meri 5 promoter of the cauliflower is expressed in the vegetative apical meristem as well as in the positions located in the body of the plant, for example internal phloem, floral primordium, points of the branches, in the root and stem (Medford, 1992 Medford et al, 1991) and the LEAFY promoter of Arabidopsis thaliana that is expressed very close in the development of flowering (Weigel et al, 1992). The GST-II-27 gene promoter has been shown to induce certain chemical compounds which can be applied to growing plants. The promoter is functional in both monocotyledonous and dicotyledonous. One can therefore use the control of gene expression in a variety of genetically modified plants, including crop fields such as cañola, sunflower flower, tobacco, sugar beet, cotton; cereals such as wheat, oats, rice, corn, sorghum, fruits such as tomatoes, mangoes, peaches, apples, pears, strawberries, bananas and melons; and vegetables such as carrots, lettuce, cabbage and onions. The GST-II-27 promoter is also suitable for use in a variety of tissues, including roots, leaves, stems and reproductive tissues. In accordance with the present invention there is provided in a further aspect a gene construct comprising an inducible promoter operably linked to a nucleotide sequence provided by the present invention, such as the GAJ gene of Arabidopsis thaliana, a homolog of other plant species or any mutant, derivative or allele thereof. This control allows the expression of the gene. The invention also provides plants transformed with said building genes into a plant cell and / or induction of the expression of a builder within a plant cell, by the application of a convenient stimulus, an effective exogenous inducer. The promoter may be the promoter of the GST-II-27 gene or any other inducible plant promoter. When a gene constructed within a cell is chosen in the introduction, certain considerations must be taken into account, well known to those skilled in the art. The nucleic acid to be inserted should be assembled within a constructor which contains effective regulatory elements which will handle the transcription. A method of transporting the constructor within the cell could be convenient. Once the constructor is inside the cell membrane, integration within the endogenous microsomal material will occur or not occur. Finally as much as possible, the concerned plants of the target cell type may be such that the cells can be regenerated within the whole plants. The selective genetic markers that can be used consist of chimeric genes conferring selective phenotypes such as resistance to antibiotics such as kana icin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, genta icin, spectinomycin, imidazolinone > and glyphosate. One aspect of the present invention is the use of nucleic acid according to the invention in the production of a transgenic plant. A further aspect provides a method that includes introducing the nucleic acid into a plant cell and causing or allowing the incorporation of the nucleic acid into the genome of the cell.

Any suitable method of transforming the plant can be used to generate plant cells comprising nucleic acid according to the present invention. Following the transformation, plants can be regenerated from cells and tissues of transformed plants. The cells and / or plants transformed successively, that is, with the built-in builder within their genome, can be selected following the introduction of the nucleic acid into the cells of the plants, optionally followed by regeneration inside a plant, for example , using one or more gene markers such as those resistant to antibiotics (see above). Plants transformed with the DNA segment containing the sequence can be produced by standard techniques which are widely known for the genetic manipulation of plants. The DNA can be transformed into plant cells using any convenient technology, such as a disarmed Ti-plasmid vector made by Asgrobacterium exploiting its ability to transfer the natural gene (EP-A-270355, EP-A-0116718, NAR 12 (22) 87-11-87215 1984), bombardment of particles or microprojectiles (US 5100792, EP-A-444882, EP-a-434616) microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966 , Green et al. (1987 Plant Tissue and Cell Culture, Academic Press), electroporation (EP 290395, WO 8706614 Gelvin Debeyser - see annexes) other forms of direct DNA comprehension (DE 4005152, WO 9012096, US 4684611), liposome mediated comprehension DNA (eg Freeman et al., Plant Cell Physiol., 29: 1353 (1984)), or the vortex method (eg, Kindle, PNAS USA 87: 1228 (190d).) Physical methods for transforming Plant cells are reviewed in Oard, 1991, Biotech, Adv. 9: 1-11 The transformation of agrobac Terium is widely used by those skilled in the art to transform dicotyledonous species. Recently, there has been substantial progress towards the routine production of transgenic, fertile, stable plants in at least all economically relevant onocotyledonous plants (Toriyama, et al. (1988) Bio / Technology 6, 1072-1074; Zhang, et al. 1988) Plant Cell Rep. 1, 379-384; Zhang, et al. (1988) Theor Appl Genet 76, 835-840; Schimamoto, et al. (1989) Nature 338, 274-276; Datta, et al. 1990) Bio / Technology 8, 736-740; Christou, et al. (1991) Bio / Technology 9, 957-962; Peng, et al. (1991) International Rice Research Institute Manila, Philippines 563-574; Cao, et. al. (1992) Plant Cell Rep. 11, 585-591; Li, et al. (1993) Plant Cell Rep. 12, 250-255; Rathore, et. to the. (1993) Plant Molecular Biology 21, 871-884; Froinm et al. (1990) Bio / tecnology 8, 833-839; Gordon-Kamm et al. (1990) Plant Cell 2, 603-618; D'Haullin, et al. (1992) Plant Cell 4, 1495-1505; Walters et al (1992) Plant Molecular Biology 18, 189-200; Koziel, et al (1993) Biotechnology 11, 194-200; Vasil, I.K. (1994) Plant Molecular Biology 25, 925-937; Weeks et al (1993) Plant Physiology 102, 1077-1084; Somers, et al. (1992) Bio / Tecnology 10, 1589-1594; WO 92/14828). In particular, the average transformation of Agrobacterium is now also emerging as a highly efficient transformation method in monocots (Hiei et al. (1994) The Plant Journal 6, 271-282). Generation of fertile transgenic plants have been carried out in rice cereals, corn wheat, oats and barley (reviewed in Shimamoto K. (1994) Current Opinion in Biotechnology 5, 158-162; Vasil et al. (1992) Bio / Technology 10, 667-674, Vain et al., 1995, Biotechnology Advances 13 (4): 653-671, Vasil, 1996, Nature Biotechnology 14 page 702). The bombardment of microprojectiles, electroporation and direct DNA of understanding are preferred where the Agrobacterium is inefficient or ineffective.

Alternatively, in combination of different techniques can be used to achieve the efficiency of the transformation of the process, for example the bombardment with Agrobacterium coated with microparticles (EP-A-486234) or bombardment of myocroprojectiles to induce the lesion followed by co-cultivation with Agrobacterium (EP-A-486233). The transformation of Brassica napus is described in Moloney et al. (1989) Plant Cell Reports 8: 238-242. Following the transformation, a plant can be regenerated, for example, by individual cells, callus tissues or leaf discs, as in standards in the art. Any plant can be completely regenerated from cells, tissues and organs of the plant. Convenient techniques are reviewed by Vasil et al., Cell Cul ture and Soma tic Cell Genetics of Plants, Vol. I, II and III, Labora tory Procedures and Their Applications, Academic Press, 1984, and Weissbach and Weissbach, Methods for plant Molecular Bilology, Academic Press, 1989. The particular selection of a transformation technology will be determined by its efficiency - to transform certain species of plants as well as the experience and preference of the practicing person of the invention with a particular chosen methodology. It will be apparent to the person skilled in the art that the particular selection of a transformation system for introducing the nucleic acid into the plant cells is not essential for a limitation of the invention, or it is the selection of a technique for the regeneration of plants. In the present invention, overexpression can be carried out by introducing the nucleotide sequence in a sense of orientation. In addition, the present invention provides a method for influencing a characteristic of a plant, the method comprising causing or allowing the expression of the nucleic acid according to the invention from the nucleic acid within the cells of the plant. The low expression of the polypeptide product of the gene can be done using anti-sensing technology or "sensory regulation". The use of anti-sensor genes or partial gene sequences for the low expression of the regulated gene is now well established. The DNA is placed under the control of a promoter so that the transcription of the "anti sensor" DNA strand obtains RNA which is complementary to the normal mRNA transcribed from the "sensor" strand of the target gene. For double-stranded DNA this is achieved by placing a code sequence or fragment thereof in a "reverse orientation" under the control of a promoter. The complementary anti-sensory RNA sequence is then linked through an mRNA to form a duplo, inhibiting the translation of the endogenous mRNA from the target gene into the protein. Either way or not, this current model of action is still uncertain. However, the fact of the work of the technique is established. See for example, Rothstein et al, 1987; Smith et al (1988) Na ture 334, 724-726; Zhang et al, (1992); The plant Cell 4, 1575-1588, English et al. , (1996) The Plant Cell 8, 179-188. Antisensing technology is also reviewed in the review in Bourque, (1995), Plant Science 105, 125-149, and Flavell, (1994) PNAS USA 91, 3490-3496. The complete sequence corresponding to the sequence of code in the orientation does not need to be used. For example, fragments of sufficient lengths can be used. It is a routine matter for the person skilled in the art to protect fragments of various sizes or of various parts of the code sequence to optimize the level of anti-sensor inhibition. It may be advantageous to include the initial methionine ATG codon, and perhaps one or more nucleotides contrary to the initial codon. An additional possibility of an objective is a regulatory sequence of a gene, for example, a sequence that is characteristic of one or more genes in one or more pathogens against which resistance is desired. A convenient fragment may have at least about 14-23 nucleotides, for example about 15, 16 or 17, or more, at least about 25, at least about 30, at least about 40, at least about 50, or more. Such fragments in the sensory orientation can be used in co-suppression (see below). The total complement of the sequence is not essential, although it may be preferred. One or more nucleotides may differ in the anti-insensitive construction of the target gene. It may be preferred to be sufficiently homologous to the anti-sensor and sensor of respective RNA molecules, to hybridize particularly under the conditions existing in a plant cell. In addition, the present invention also provides a method for influencing a characteristic of a plant, the method comprising causing or allowing anti-sensing transcription of the nucleic acid according to the invention within the cells of plants. When additional copies of the target gene are inserted into the sensor that is the same, the orientation with the target gene, a range of phenotypes occurs in which individuals are included where over-expression occurs and some where over-expression occurs. the protein of the target gene. When the inserted gene is only part of the endogenous gene, the number of over individual expressions in the transgenic population is increased. The mechanism by which sensor regulation occurs, particularly down regulation, is not well understood. However, this technique is also well reported in scientific and patent literature and is routinely used for gene control. See, for example, van der Krol et al. , (1990) The Plant Cell 2, 291-299; Napoli et al. , (1990) The plant Cell 2, 279-289; Zhang et al. , (1992) The Plant Cell 4, 1575-1588, and US-A-5, 231, 020. In addition, the present invention also provides a method for influencing the characteristics of a plant, the method comprising causing or allowing expression of the nucleic acid according to the invention within the cells of the plant. This can be used to influence growth. Aspects and modalities of the present invention will now be illustrated by means of the example, with reference to the accompanying figures. Additional aspects and modalities will be apparent to those skilled in the art. All the documents mentioned in this text are incorporated here by reference.

The following figures are included here: Figure 1: the basic structure of the carbon ring of gibberellins.

Figure 2: The gai-t6 line containing a transfer Ds which interrupts a transcribed gene. Figure 2a: Plants showing (left to right) ho ocigotos for GAJ, gai and gai-tß. The GAJ and gai-tß of the plants are indistinguishable. Figure 2b: Hybridization of gel-labeled DNA using a Ds test. The DNA in the GAJ line lacks Ds. The gai course contains DNA from plants homozygous for gai and for T-DNA A2645, which contains Ds (EcoRI fragment 18.0 kb). The gai-t6 course contains DNA from plants homozygous for A264 and for a transfer Ds (fragment 15.5 kb). Figure 2c: hybridization of gel-labeled DNA using a radiomaraked GAJ cDNA test. The DNA hybrids with a Bcll 5.1 kb fragment in DNA from GAJ and gai are replaced in gai-tβ by the 6.4 and 2.8 kb fragments. Since the Bcll cuts once inside Ds, the Ds insert is flanked on either side by the gene (GAJ) encoding the cDNA. Hybridization weakened to 1.7 kb is one of several views at long exposures and identifies a sequence reported for GAJ. Figure 3: A nucleotide sequence of a GAJ gene encoding a polypeptide with a GAJ function. Figure 4: Primary structure of GAJ and gai proteins. The expected amino acid sequence is shown from the genomic sequence of GAJ. The segment of the 17 amino acids deleted in GAJ is shown in bold face and double underscore. Figure 5: repression model for the regulation of plant growth by GA. Figure 6: nucleotides and amino acid sequences encoding the derived GAJ alleles Figure 6a: nucleotide sequence of gai-dl. Figure 6b: amino acid sequence of gai-dl Figure 6c: nucleotide sequence of gai-d2 Figure 6d: amino acid sequence of gai-d2 Figure 6e: nucleotide sequence of gai-d5 Figure 6f: amino acid sequence of gai-d5 Figure 6g: nucleotide sequence of gai-dl Figure 6h: amino acid sequence of gai-d7 EXAMPLE 1 Cloning and characterization of GAI and gai genes The GAJ maps for chromosome 12 of Arabidopsis, approximately 11 cM of a T-DNA insert bearing a Transfer DS5'15. Genetic analyzes suggest that a loss of allele functions confer a high phenotype indistinguishable from that conferred by the allele (GAJ) 5'6 wild type. The clone GAJ is insured via insertional mutagenesis, exploiting the tendency of Ds to preferentially transcribe for linked sites16,17. Plants with homozygous lines were constructed for A264 and gai, which contain a transgene (? Nael-sAc (GUS) -l-) expressed transcription Ac. Plants homozygous for a putative Ds allele insert, in which gai-t6 were designated, were isolated from this material as follows5. The material was accumulated, by self pollination, for several generations. During this accumulation, the search was made for plants which have a leaf system more elongated than expected for a homozygous gai. The seeds obtained from the self-pollination of such leaves were planted for close examination. The progeny of such a leaf of segregated plants, at a frequency of about a quarter, exhibit a high phenotype indistinguishable from that conferred by GAJ (Figure 2a). These plants were homozygous for a new gai allele, which was designated gai-tß. The gel-labeled DNA experiments revealed that the gai-t6 contains transfer Ds (Figure 2b), inserted within a region (approximately 200 kb) of chromosome 1 known to contain GAJ (data not shown). The DNA genomic preparation and the gel-labeled hybridizations were performed as described5. The digested EcoRIs were hybridized with the Ds test (radiolabelled 3.4 kb Xhol-BamHI subfragment of Ac). The gai-tß has been lost (? Nael-sAc (GUS) -l-) via genetic segregation. Additional experiments showed that the transfer Ds interrupts the transcribed region of a gene (GAJ), and that the Arabidopsis genome contains at least one proportional significant sequence of the additional gene homologous with GAJ (Figure 2c). A radiolabeled IPCR fragment containing genomic DNA adjacent to the 3 'end of the transfer Ds in gai-t6 was isolated as previously described24. It is necessary to be careful in the use of this test since they are potentially contaminated with sequences derived from the 3 'T-DNA of Ds in A264 (which is still present in the gai-t6 line). However, the fact that the test hybridized with DNA from plants lacking any T-DNA insertion indicates that the region of the genomic DNA within the transfer Ds in the gai-t6 was used for the purposes of cloning. inserted This test showed to hybridize the genomic DNA of previously identified cosmid clones linked by containing GAI by cloning based on the map. One of these cosmids was used to identify, by hybridization, clones from cabinet cDNA made from mRNA isolated from aerial parts of the plant. { Arabidopsis). These cDNAs were classified according to their hybridization to the genomic DNa of GAJ, gai and gai-tβ. Some of these clones were hybridized once a week to fragments containing GAJ (as defined by the alteration in the size of fragments caused by the Ds insertion in gai-tß), but more strongly to other sequences reported. These cDNAs are presumably derived from mRNAs transcribed from genes reported in the sequence for GAJ, but not for GAJ itself, and were set aside for future research. A cDNA, pPCl, strongly hybridized to GA, less strong to the fragments containing sequences reported for GAJ. The DNA sequence of part of this cDNA was identical with approximately 150 bp of genomic DNA flanking the insertion Ds in gai-t6. The reversal analysis showed that the excision of Ds from gai-tβ was associated with the restoration of a dominant dwarf phenotype. The DNA sequence for two overlapping GAJ cDNAs revealed an open reading structure (ORF) encoding a protein (GAJ; of 532 amino acid residues. DNA fragments containing this ORF were amplified from GAJ and genomic DNA gai.

The first oligonucleotides derived from the DNA sequences of overlapping pPCI and pPC2 cDNAs were used to amplify, via PCR, 1.7 kb fragments of GAI and gai genomic DNA. The sequences of the first ones used were: Primer Nß: 5'TAG AAG TGG TAG TGG3 ': First ATI: 5'ACC ATG AGA CCA GCC G3'. The sequence of the first ATI differs by a base from the sequence of the genomic and c-DNA clones. The first was synthesized very clearly in the projected sequence, before the end of the corrected version the sequence was obtained. The DNA sequences of fragments for duplicate amplifications were determined, in addition, errors introduced by PCR were avoided. The GAJ genomic sequence was at least identical with that of the overlapping cDNAs. Three nucleotide substitutions could be made due to the difference between species ecotypes and those which do not alter the expected amino acid sequence of GAJ. The sequences of these genomic fragments revealed that the ORF is not interrupted by the introns (Figure 3). The Ds insertion in gai-tβ is located between codons Glu182 and Asn183 (Figure 4). The expected secondary structure of GAI showed few salient features. GAJ is a broadly hydrophilic protein with a polyhistidine tract of unknown significance close to the amino terminus, and a weak hydrophobic domain rounding a possible glycosylation site to Asn183. Computer analysis indicated a relatively low probability that this region is a dominant transmembrane. The search of the protein and DNA sequence database did not reveal a domain of obvious functional significance within GAJ. The gai contains a deletion of 51 bp within the GAJ ORF. This results in the suppression of the stretch in the absence, in gai, of a segment of 17 amino acid residues located near the amino terminus of the expected GAJ protein (Figure 4). Laurenzio et al45, reported after the priority date of the present invention a sequence for the SCR gene (SCARECROW) of Arabidopsis, a mutation in which the results in roots are lost in the cell layer. The SCR sequence described has little homology with the Arabidopsis GAI sequence of the present invention, but lacks the 17 amino acid motif discussed. A previous publication describes isolation, followed by mutagenesis? -irradiation, alleles5 gai derivatives. These alleles, when they are homozygous, confer a high phenotype indistinguishable from that conferred by GAJ5. The sequence of amplified fragments from several of the derived alleles (gai-dl, gai-d2, gai-d5 and gai-d7) showed that each contains the 51 bd deletion characteristic of gai. The nucleotide and the amino acid sequence encoding these alleles are shown in Figure ß. They also contain mutations additions that can confer a non-functional gene product (Table 1). The fact of the loss of the gai mutant phenotype is correlated with each of these mutations, together with the reversion data (see above), confirming that the GAJ has been cloned. Additionally, these results are consistent with predictions that the gai-d alleles could be annular alleles5,6. The cloning of gai via insertional mutagenesis is possible due to a gain mutation function. Such mutations can have dominant effects for several reasons, including ectopic or increased expression of the normal gene product, or altered function of a mutant gene product. Here it has been found that the gai mutation is associated with an altered product. The suppression of a 17 amino acid residue of gai domain results in a mutant protein (gai) which, in a genetically dominant fashion, causes dwarfism. This strongly suggests that GAJ is a growth repressor and that GA de-repression of growth antagonizes the action of GAJ. The loss of domain in the GAJ mutant protein may be responsible for interacting with the GA signal or with the same GA. The gai could then constitutively repress growth because it does not antagonize GA. A model of repression for the regulation of the growth of the plant by GA mediator is also elaborated in figure 5, but it should be noted that it is proposed and should not be taken as a limit of the scope of the present invention. Recognizing the current GAJ and gai action model, i.e., the work, is not a prerequisite for operation of the present invention, which is found in cloning of wild-type and mutant versions of the GAJ gene. Mutations to the SPINDLY (SPY) sites of Arabidopsis confer increased resistance to inhibitors of GA biosynthesis and a reduced dependence on GA for growth regulation18, the phenotype characteristics of the inappropriate mutants previously described in other plant species19-23 . Recent experiments have shown that the dwarf phenotype conferred by GAJ can be partially suppressed by mutations to SPY at the other sites6'9. It is again proposed without limiting the scope of the present invention that, SPY together with the proteins encoding these other sites, is involved with the down-transduction of the growth-repressor signal that originates with GAJ (FIG.

). In accordance with the model shown in Figure 5, the growth of the de-repression GA plant due to this (or a GA signaling component) antagonizes gai activity, a current protein represses growth. The growth repression signal is transmitted via SPY6'18, GAR26, GAS2 (J.P. and N.P.H., unpublished) and other proteins. Normal plants (GAJ) grow high because the level of endogenous GA is sufficiently high to substantially antagonize the activity of the GAJ repressor. GA deficient plants contain insufficient GA to antagonize GAJ repression to the same degree, and they are also dwarfed.25"27 GAJ mutant plants are dwarf2 because the gai mutant protein is not anatagonized by GA, and represses growth in a The spy, gar2, and gas2 mutations partially suppress the gai phenotype, and confer resistance to the 6'18 inhibitors of GA biosynthesis.The pairwise combinations of these three mutations confer more extreme gai suppression and resistance to inhibition of biosynthesis GA that is conferred by any of the spy, gar2 or gas2 alone.In addition, these genes are proposed to encode downstream components that are responsible for the transmission of the growth repression signal from GAJ. It is possible that the gai mutation is a functional homologous of insensitive GA mutations in corn10"12 and wheat13. In addition, this model can be used to provide a general explanation for the regulation of plant growth by GA. Independent studies of insensitive GA dwarf mutants in corn11'12 and weak GA independent mutants in peas and barley19, 23 have previously implicated the involvement of a repressor function in the transduction signal GA.The indications from the work described here are all the likelihood that Arabidoppsis GAJ is such a repressor.An important implication of this is that the GA then regulates the growth of plants not via activation but by de-repression.

EXAMPLE 2 Cloning of GAI homologs from wheat, rice and Brassica spp. The DNA containing homologous potential is isolated from wheat, rice and Brassica by the reduced stromal cDNA cabinet test or genomic DNA containing DNA from these species. The hybridizing clones are then purified using standard techniques. Alternatively, potential GAI homologs are identified by the EST database projection for cDNA and other sequences showing statistically significant homology with the GAJ sequence. The clones are then obtained by the requirements from the relevant distribution scales. Table 2 gives in detail the search results in public sequence database containing EST sequences that are obtained in random sequence programs, showed that the homologous sequences have been found in several species, including Zea maiz (Maize), O. sativa (rice) and Brasica napus (turnip). In the case of wheat and corn, it is important to know if these homologous sequences correspond to the previously characterized Rht and to the genetic site D8. This is determined as follows. The cDNA or gemomic DNA of rice, wheat or corn is delineated within the genomic tracing of wheat, in addition, it is determined if the position of the DNA trace corresponds to the position of the trace of the Rht site in wheat. In addition, in the case of maize, the potential of the DS transfer insertion alleles exists, and they are used to provide DS cloning in the same manner as gai cloning from Arabidopsis was provided. By sequencing these various cDNAs and genomic DNA clones, by studying their expression patterns and examining the effect of altering their expression, we obtain that the genes perform a similar function for GAJ in the regulation of plant growth. The mutants, variant derivatives and alleles of these sequences are made and identified as appropriate.

EXAMPLE 3 Expression of GAI and gai proteins in E. coli DNA fragments containing the GAJ or full gai of open reading structures were amplified using PCR from genomic DNA clones (not introduced into genes) containing the GAJ genes and gai. The amplifications were made using charges which are converted into the initial ATG translation codon at a BamH1 restriction endonuclease site. The fragments have a PstI endonuclease restriction site at the other end (beyond the stop codon). The products were cloned and their DNA sequences determined to allow no errors to be introduced duate the course of the PCR. Correct fragments were cloned into the PQE30 expression vector assimilated by BamHI / PstI (Qiaexpressionist Kit of the Company Quiagen), resulting in builders with the potential to express GAJ and gai proteins in E. Coli. Expression in this vector is regulated by an IPTG-inducible promoter, and the resulting proteins carry an N-terminal polyhistidine terminus which can be used to purify it from cell extracts. Introduction with IPTG results in expression of high levels of GAJ and gai proteins in E. coli.

EXAMPLE 4 Builders of expression and transformation of plants. (a) Normal expression levels, using endogenous promoters. The GAJ and gai genes were isolated as EcoRI / EcoRV 5kb fragments (containing approximately 1.5 kb of non-coding sequences flanking the code sequence) by subcloning their appropriate genomic clones. These fragments were cloned into the Bluescript vector, isolating again as EcoRI / Xbal fragments, and ligated into binary vectors for mobilization in Agrobacterium tumefaciens C58C1, with the T-DNA being introduced into Arabidopsis plants and tobacco as described by Valvekens et al. .32 or by the latest 33 method of vacuum filtration, and in Brassica napus using the high efficiency transformation technique for Agrobacterium. as described in Moloney et al.34. (b) Overexpression using an exogenous promoter Constructs have been constructed using DNA from pJITβO vectors, which contain a 35S double promoter and pJIT62, a pJITβ modifier containing a single 35S promoter. Promoters from these vectors were fused to a non-coding sequence of approximately 5 'IOOp, followed by an ATG and the complete GAJ or gai open reading structures, followed by a translational stop codon, followed by an approximately non-coding sequence. 20 bp 3 ', followed by a polyadenylation signal: all these were carried out in a SstI / XhoI fragment. This fragment has been ligated into binary vectors for introduction into transgenic plants, either by the use of Agrobacterium tumefaciens or as pure DNA, as described earlier.

EXAMPLE 4 Modification of GAJ and gai sequences A short segment of the open reading structure of GAJ rounding off the gai deletion is amplified from GAJ and gai by the use of an appropriate first oligonucleotide PCR designed on the basis of the sequence of information provided here. The amplified segment is then subjected to one or more vectors of various forms of mutagenesis (see for example Sambrook et al.) Resulting in a series of overlapping deletion mutants, or if desired, substitutions of individual nucleotides in this region. The amplified mutant segment is then replaced by the equivalent segment in GAJ, via restriction endonuclease assimilation and a subsequent ligation reaction. This new variant is then expressed in transgenic plants either at normal levels or via overexpression as described above. The builders are studied to test their effects on the regulation of the growth of plants in models (for example Arabidopsis and tobacco) and crop species (for example, wheat, rice and corn). The different constructors confer different degrees of dwarfism and can be individually located especially for the modifications and improvement of species of particular crops for growth of crops in particular environments.

EXAMPLE 5 Null GAJ alleles that confer increased resistance to paclobutrazol: Paclocutrazol is a triazole derivative that specifically inhibits GA biosynthesis in the reaction36'37 of kaurene oxidase, in addition, reduces endogenous GA levels and confers a dwarfing phenotype in exposed plants to that. Weak mutants of pea and barley are resistant to the effects of dwarfism of paclobutrazol38"42, with the constitutive Arabidopsis mutant response GA spy43'44.In addition, in these mutants the elongation is stopped at least partially not coupled from the characteristic GA mediator control of normal plants Interestingly, the gai-tß mutant also exhibits resistance to paclobutrazol.When growing in a medium containing paclobutrazol, the gai-t6 mutants exhibit greater rapid flowering arrests than with GAJ control plants. This result suggests that the loss of GAJ function causes a reduction in AG dependency on the arrest of elongation or elongation, but otherwise, a GAJ null mutant appears to require less endogenous GA to reach a certain degree of growth than a normal plant.The GA dependency is not completely abolished by gai-tß possibly because the gene products reported n the sequence for GAJ (see above) can substantially but not completely compensate for the loss of the GAJ function. These observations are significant, because they demonstrate that the wild-type gene product, GAJ, is a component of the GA transduction signal.

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TABLE 1 Mutations in alleles GAJ Alleles Nature Position in consequence of that of the mutation mutation * sequence of codes gai-dl CAG to TAG Glu 239 Codon stopped, truncated polypeptide gai-d2 GAT a GA Asp 274 Change structure, a base of addition of two new amino acid deletion, truncated polypeptide gai-d5 1 base of following Change structure, deletion Leu281 addition of 18 new also C to amino acids, G truncated polypeptide gai-dl GTT to GT, Val 156 Change structure, an addition base of 27 new amino acid deletions, truncated polypeptides * the stitched lines denote substitution of nucleotides in each of the alleles. The alleles were isolated following mutagenesis of? -radiation of homozygotes5 gai. The 1.7 kb fragments were amplified from genomic DNA of each of the alleles and sequenced as described above. Each of the alleles contains the 51 bp deletion characteristic of gai, confirming that they are all genuine gai derivatives and are not contaminants.

Search in Database on 11/01/96 Table 2 ESTs with homology for the GAJ c-DNA 1.- HOMOLOGY TO THE FIRST 200 AMINO CIDOS Clone ID Species probability Blast Poisson EM_EST1: ATTS3217 A. Thaliana 4.8 • e "32 EM_EST1: AT7823 A. Thaliana 4.8 • e" 24 EM_EST1: AT7938 A. Thaliana 7.2 • e "22 EM_EST3: OSSO803A O. Sativa (rice) 7.8 • e "11 EM_EST1: AT5178 A. Thaliana 0.014 EM EST1: AT9456 A. Thaliana 0.026 2. - HOMOLOGY FOR AMINO ACIDS 200-400, Clone ID Species Probability of Blast Poisson EM_EST1: ATTS4818 A. Thaliana 1.5 • e "21 EM_EST3: ZM3101 Zea Mayz (corn) 9.1 • e" 14 EM_EST1: ATTS1110 A. Thaliana 7.9 • e "10 EM_EST1: ATTS3935 A. Thaliana 1.7 • e "9 EM_STS: ZM7862 Zea Maiz (maize) 4.5 • e" 7 EM_EST1: AT7938 A. Thaliana 0.00011 EM EST3: 0SS3989A 0. Sativa (rice) 0.00050 3. -HOMOLOGY AT THE LATEST 132 AMINO ACIDS Clone ID Species Probability of Poisson Blast EM_EST1: AT2057 A. Thaliana 3.1 • e -52 EM_EST1: ATTS3359 A. Thaliana 3.2 • e "42 EM_EST3: OS0713A 0. Sativa (rice) 2.8 • e" 10 EM_EST1: BN6691 B. Napus (turnip) 3.0 • e "5 EM_EST1: ATTS3934 A. Thaliana 0.00034 EM_EST1: ATTS4819 A Thaliana 0.00059 EM_EST1: AT4893 A. Thaliana 0.00060 EM_EST1: ATTS1327 A. Thaliana 0.00073 EM-EST1: AT1868 A. Thaliana 0.0054 EM_EST1: AT79316 A. Thaliana 0.092 EM EST1: AT7747 A. Thaliana 0.35 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the manufacture of the objects to which it refers. Having described the invention as above, the content of the following is claimed as property.

Claims (48)

1. An isolated nucleic acid having a nucleotide sequence of codes for a polypeptide which includes the amino acid sequence shown in Figure 4.

2. A nucleic acid according to claim 1, characterized in that the nucleotide sequence of codes includes the nucleotide sequence of codes shown in Figure 3.

3. Nucleic acid according to claim 1, characterized in that the nucleotide sequence of codes includes a mutant, allele, derivative or variant, by means of addition, substitution, insertion and / or deletion of one or more nucleotides, of the nucleotide sequence of codes shown in Figure 34.

An isolated nucleic acid having a code nucleotide sequence for a polypeptide which includes an amino acid sequence which is a mutant, allele, derivative or variant sequence of the GAJ amino acid sequence of the Arabidopsis thaliana species shown in Figure 4, or is a homolog of other species or a mutant, allele, derivative or variant thereof, wherein said mutant, allele, derivative, variant or homolog differs from the amino acid sequence shown in Figure 4 by means of insertion, deletion, addition and / or substitution of one or more amino acids, wherein the expression of said nucleic acid in a plant results in an inhibition of the growth of the plant, the inhibition is antagonized by gibberellin (GA).

5. Nucleic acid according to claim 4, characterized in that the over expression of said nucleic acid in a plant confers a dwarfing phenotype in the plant, in which the dwarfing phenotype is corrected by treatment with GA.

6. Nucleic acid according to claim 4 or 5 ', * characterized in that said polypeptide includes the sequence of 17 amino acids underlined in Figure 4.

7. Nucleic acid according to claim 4 or 5, characterized in that said polypeptide includes a contiguous sequence of 17 amino acid residues in which at least 10 residues have similarity to a residue at the corresponding position in the sequence of 17 amino acids underlined in the Figure Four.

8. An isolated nucleic acid having a code nucleotide sequence for a polypeptide characterized in that it includes an amino acid sequence which is a mutant, allele, derivative or variant sequence of the GAJ amino acid sequence of the Arabidopsis thaliana species shown in Figure 4 or is a homolog of another species or mutant, allele, derivative or variant thereof, wherein said mutant, allele, variant derivative or homolog differs from the amino acid sequence shown in Figure 4 by means of insertion, deletion, addition and / or substitution one or more amino acids, wherein the expression of said nucleic acid complements a null GAJ mutant phenotype in a plant, such a phenotype is resistant to the dwarfing effect of paclobutrazol.

9. Nucleic acid according to any of claims 4 to 8, characterized in that said plant is Arabidopsis thaliana.

10. Isolated nucleic acid having a nucleotide sequence code for a polypeptide characterized in that it includes the amino acid sequence encoding the nucleic acid according to claim 8 except for the deletion of the sequence of 17 amino acids underlined in Figure 4 or a sequence contiguous of 17 amino acids in which at least 10 residues have similarity to a residue in the corresponding position in the sequence of 17 amino acids underlined in Figure 4.

11. An isolated nucleic acid having a nucleotide sequence of codes for a polypeptide characterized in that it includes an amino acid sequence which is a mutant, allele, derivative or variant sequence, by means of the insertion, deletion, addition and / or substitution of one or more amino acids, for the GAJ amino acid sequence of the Arabidopsis thaliana species shown in Figure 4 or a homolog of other species, where the expression of the nucleic acid in a plant confers a phenotype on the plant in which the dwarfism is not a response or replica of gibberellin.

12. Nucleic acid according to claim 11, characterized in that the polypeptide includes the amino acid sequence shown in Figure 4 with the 17 underlined deleted amino acids.

13. Nucleic acid according to claim 12, characterized in that the nucleotide sequence of codes includes the nucleotide sequence of codes shown in Figure 3 but with the nucleotides which encode the amino acids underlined in Figure 4 deleted.

14. Nucleic acid according to claim 12, characterized in that the nucleotide sequence of codes includes a nucleotide sequence which are a mutant, allele, derivative or variant sequence, by means of the insertion, deletion, addition and / or substitution of one or more nucleotides, of the nucleotide sequence shown in Figure 3 but with the nucleotides which encode the underlined amino acids in Figure 4 deleted.

15. Nucleic acid according to claim 11, characterized in that the polypeptide has an amino acid sequence which is a mutant, allele or derivative or variant sequence of the amino acid sequence shown in Figure 4 by means of deletion of the 17 underlined amino acids in Figure 4 and the addition, insertion, substitution and / or deletion of one or more amino acids.

16. Nucleic acid according to any of claims 11 to 15, characterized in that said plant is Arabidopsis thaliana.

17. Nucleic acid having a nucleotide sequence of codes for a polypeptide characterized in that it includes an amino acid sequence which is a mutant, allele, derivative or variant sequence, by means of the insertion, deletion, addition and / or substitution of one or more amino acids , of the GAJ amino acid sequence of the Arabidopsis thaliana species shown in Figure 4, wherein the poplipeptide has the amino acid sequence shown in Figure 6b, Figure 6d, Figure 6f or Figure 6h.

18. Nucleic acid according to claim 17, characterized in that the code nucleotide sequence is that shown in Figure 6a, Figure 6c, Figure 6e or Figure 6g.

19. Nucleic acid according to any of claims 1 to 18, characterized in that it also includes a regulatory sequence for the expression of said nucleotide sequence code.

20. Nucleic acid according to claim 19, characterized in that the regulatory sequence includes an inducible promoter.

21. An isolated nucleic acid having a nucleotide sequence complementary to a sequence of at least 14 contiguous nucleotides of the sequence or sequence code complementary to the nucleic acid sequence code according to any one of claims 1 to 15 suitable for use in anti-regulation -sensor or sensor ("co-suppression") of the expression of said sequence code.

22. Nucleic acid according to claim 21, characterized in that it is DNA and wherein said complementary nucleotide sequence is under control of the regulatory sequence for anti-sensorial transcription.

23. Nucleic acid according to claim 22, characterized in that the regulatory sequence includes an inducible promoter.

24. A nucleic acid vector suitable for the transformation of a plant cell and including nucleic acid according to any of the preceding claims.

25. A host cell containing heterologous nucleic acid according to any of the preceding claims.

26. A host cell according to claim 25, characterized in that it is microbial.

27. A host cell according to claim 25 characterized in that it is a plant cell.

28. A plant cell according to claim 27, which heterologous said nucleic acid within its genome.

29. A plant cell according to claim 28, characterized in that it has more than one nucleotide sequence per haploid genome.

30. A plant cell according to any of claims 27 to 29 characterized by porous is comprised in a plant, a plant part or a plant propagule, or an extract or derivative of a plant.

31. A method of producing a cell according to any of claims 25 to 30, characterized in that the method includes the incorporation of said nucleic acid into the cell by means of transformation.

32. A method according to claim 31 characterized in that it includes the recombination of the nucleic acid with the nucleic acid of the genome of the cell, so that it is stably incorporated therein.

33. A method according to claim 31 or claim 32, characterized in that it includes the regeneration of a plant from one or more transformed cells.

34. A plant comprising a plant cell according to any of claims 27 to 29.

35. A plant which is a fruit propagated sexually or asexually, clone or descendant of a plant, according to claim 31, or any part or propagule of said plant, clone or descending fruit.

36. A part or propagule, or extract or derivative of a plant according to claim 35.

37. A method of producing a plant, the method includes the incorporation of nucleic acid according to any of claims 1 to 24 in a plant cell and regenerating a plant from said plant cell.

38. A method according to claim 37, characterized in that it includes sexual or asexually propagation or growth of the fruit or a descendant of the regenerated plant from said plant cell.

39. A method of influencing a characteristic of a plant, characterized in that the method includes causing or allowing expression of heterologous nucleic acids according to any of claims 1 to 3 within the cells of the plant.

40. A method of influencing a characteristic of a plant, characterized in that the method includes causing or allowing the expression of heterologous nucleic acid according to any of claims 4 to 7 within the cells of the plant.

41. A method of influencing a characteristic of a plant, characterized in that the method includes causing or allowing the expression of the heterologous nucleic acid according to claim 8 or 9 within the cells of the plant.

42. A method of influencing a characteristic of a plant, characterized in that the method includes causing or allowing the expression of the heterologous muclic acid according to claims 10 to 16 within the cells of the plant.

43. A method of influencing a characteristic of a plant, characterized in that the method includes causing or allowing the transcription of the heterologous muclic acid according to any of claims 21 to 23 within the cells of the plant.

44. Use of nucleic acid according to any of claims 1 to 3 in the production of a transgenic plant.

45. Use of nucleic acid according to any of claims 4 to 7 in the production of a transgenic plant.

46. Use of nucleic acid according to claim 8 or claim 9, in the production of a transgenic plant.

47. Use of nucleic acid according to any of claims 10 to 16 in the production of a transgenic plant.

48. Use of nucleic acid according to any of claims 21 to 23 in the production of a transgenic plant.