MXPA96006552A - Plant genes that affect the biosynthesis of giberel acid - Google Patents

Abstract

Genes that control the biosynthesis of gibbetic acid in genetic engineering are used to alter the development of the plant. Alterations in the nature of the quantity of products of the genes affect the development of the plant. A family of An genes in monocotyledons encodes a cyclase involved in the first steps of the biosynthesis of gibberelic acid (GA). The members of the family are identified in wheat, barley, sorghum and corn. In maize, two members of the family were identified, the An1 and An2 genes. The An1 gene is cloned and the function of the gene is characterized. An2 is isolated and identified by homology with An1. The levels of gibberellic acid are manipulated using recombinant genetic technology. Changes in the levels of gibbetic acid alter the phenotypes of the monocotyledonous plant, for example, by increasing or decreasing height and fertility.

Description

PLANT GENES AFFECTING THE BIOSYNTHESIS OF GIBBEL ACID BACKGROUND OF THE INVENTION The present invention relates to genes that encode regulators of gibberellic acid biosynthesis in plants. The development of the plant is affected by alterations in the nature or quantity of expression products of these genes. A family of An genes, found in monocotyledonous plants (monocots), codes for an essential composition for the conversion of GGPP to ei2t-aurene involved in the first steps of the biosynthesis of gibberellic acid (GA, for its acronym in English ). Illustrative members of the family, the Anther earl (Anl) and An ther ear2 (An2) genes are identified in maize cloning and the functional attributes of the Anl and An2 genes are described. Ap genes are also identified in barley, sorghum and wheat because of their homology with the Anl gene of corn. The fact that gibberellic acid is important in the development of the plant is illustrated by the correlation between increased vigor in hybrid corn and higher levels of gibberellic acid compared to mother levels, and the best response of the inbreeding (compared to hybrids) to the exogenously applied gibberellic acid content (Rood et al., 1988). In addition, the RFLP analysis points to the known biosynthetic sites of gibberellic acid as the sites of quantitative traits (LRCs) for height in maize hybrids (Beavis et al., 1991), suggesting a role for gibberellic acid in heterosis. The importance of gibberellic acid in the development of the plant is more evident in the phenotype of maize gibberellic acid-deficient mutants, which include: reduced stature of the plant due to shorter internode lengths; shorter shorter leaves; minor branch of the spikelets; and the development of anthers on the usually pistillated ear, resulting in perfect flowers (Emerson and Emerson, 1922). In corn and probably other plant species, reduced stature is primarily the result of a decrease in the final length of germinating cells. A reduction in the number of cells per patient is also a factor. Although deficiency of gibberellic acid affects maize germination and mesocotyl cell length, the coleoptile cell lengths are not affected, suggesting that the extension of the coleoptile cell is independent of gibberellic acid. The reduced plant height of deficient / responsive gibberellic corn mutants is a common feature of the deficient / responsive gibberellic acid mutants of many plant species including Arabidopsis, tomato, rice, pea, and barley. Interestingly, it appears that the reduced height phenotype is more responsive to gibberellic acid levels than the development of anthers on the cob. This is true because, regardless of the semi-dwarf or non-dwarf stature of the Anl mutants, they remain with anthers on the ear. The levels of gibberellic acid also affect fertility in plants. For example, can I spray acid? gibberellic directly in plants to affect fertility. The nature of the effect is specific to the species, that is, in some species the excess of gibberellic acid increases fertility; while, in other species, gibberellic acid reduces fertility. The effect depends on the reproductive mechanics of the species, and on the structure or function that affects the gibberellic acid. In corn, a monoecious plant with diclinas flowers, the staminiferous flowers are formed in the spikelet, while the pistillate flowers are formed in the ear. The ears of corn emerge from axillary buds. They develop protuberances in a gradient acropétalo in the ear that bifurcates becoming double lobed. However, the diclina nature of the mature flowers contradicts the fact that all the flowers on the spikelet and the cob are initially perfect. Very early during its development, the differentiation of the pistillate and staminiferous structures stops at the spikelet and the ear, respectively (Cheng et al., 1983). The flowers, known as floret in corn, are in pairs on the cob. Each pair emerges from the bifurcation of a spike, with one floret close to the axis of the ear and the other distal. The development of the staminiferous structures in the cob stops in both florets as the development of the pistillate structure in the proximal floret. Therefore, the distal flocculus ovum contains the only mature gametophyte found on the ear, and when fertilized the locked ovum and polar nucleus develop into a grain. The florets in the anther arise in a similar manner, with the development of the pistillate structures of both florets arrested very early, while stamens develop in both florets. The reduced levels of gibberellic acid affect the development of the pistils and stamens in the corn by stopping the development of the stamens in both florets of the ear. This results in a staminiferous flower in the proximal floret and a perfect mature flower in the distal floret. The development of the pistils and stamens in the spikelet of mutants deficient in gibberellic acid is delayed, but otherwise it is not affected. Therefore, gibberellic acid is required for the normal arrested development of the stamens that are observed in both florets of the cob. The nearby anthers on the ears of the deficient / responsive mutants of gibberellic acid produce mature pollen that accumulates starch and possesses a germ pore; these are indications of a functional gametophyte. The sexual determination of the spikelet florets in these mutants appears to be normal, with both florets developing fertile anthers, while the pistillate structures do not develop. The effect of these ».. mutations in spikelets appear to be limited to the reduction of branching and to causing a poor spread of pollen, apparently because the glumes do not open. In corn, spikelets and sprouts have served as sources for the identification of many intermediaries biosynthetics of gibberellic acid (Suzuki et al., 1992; Hedden et al., 1982). In addition to being present in shoots, gibberellic acids have been shown to be present at the tips of the Pisum root (Coolbaugh, 1985) and in immature seeds of the Pharbi tis (Barendse et al., 1983). Gibberellic acids are synthesized from isoprenoid GGPP, beginning with the cyclizations of GGPP to CPP, after CPP to ent-kaurene, catalyzed by syntans A and B of kaurene (formerly synthetases A and 5 B of kaurene), respectively (Duncan et al., 1981). It is believed that higher plants are like corn because, in corn, ent-kaurene is oxidized in steps to 7-hydroxy-kaurenóic acid, which is converted to the first true gibberellin; GA12-aldehyde (Suzuki et al., 1992). The latter compound is then further oxidized to an active gibberellic acid by one of three parallel paths. In corn, the dominant trajectory seems to be the first 13-hydroxyl trajectory (Hedden et al., 1982), with GA1 being the penultimate active product, typically present in amounts of less than 1 μg / 100 gfwt (for weight of fresh growth tissue) (Fujioka et al., 1988). The biosynthetic block has been predicted in four of the five maize-deficient gibberellic acid mutants documented, by measuring the accumulation of endogenous gibberellic acid biosynthetic intermediates, and measuring the growth responses to, and determining the fate of, intermediaries (Fujioka et al., 1988). So far, the precise biosynthetic role of the fifth place, Anl. Mutations in Anl result in a deficient phenotype of gibberellic acid, which can be cured with ent-kaurene applied, suggesting that the Anl gene product functions in the synthesis of ent-kaurene. However, the An genes have not been cloned, isolated or sequenced. Therefore, genetic engineering methods for the manipulation of An genes to control plants are not available in the art. The availability of genetic engineering for levels of gibberellic acid would accelerate and improve the previously available classical breeding programs. Maize genes have been cloned using the Mutator (Mu) transferable element family to generate labeled mutants of the gene. Among the genes thus cloned are (O'Reilly et al., 1985); bz2 (McLaughlin et al., 1987); hcfl Oß (Marteinssen et al, 1989); hml (Johal et al., 1992); ioj ap (Han et al., 1992); vpl (McCarty et al., 1989) and yl (Buckner et al., 1990). However, the use of the Mu system for cloning does not have predictable success. COMPENDIUM OF THE INVENTION The control of the levels of gibberellic acid (GA) in plants by means of genetic engineering techniques requires the identification and isolation of the genes whose expression affects the operation of the biosynthetic path that leads to the gibberellic acids. The control of the levels of gibberellic acid is a means to control the development of the plant. One aspect of the present invention is to identify, isolate and characterize a family of genes in monocots, which is capable of encoding a product that functions to convert GGPP to ent-kaurene in the biosynthesis of gibberellic acid. Monocotyledons include sorghum, wheat, corn, barley. The gene family is defined by an ability to hybridize, under conditions of high stringency, with the Anl gene of corn, and is therefore designated "An". The genes of this family encode products that are necessary for the conversion of GGPP to ent-kaurene in the biosynthesis of gibberellic acid. Without being bound theoretically, it is believed that the product is an isoprenoid cyclase. A representative member of the family is the Anther earl (Anl) gene from Zea mays, which has been isolated, cloned, sequenced and characterized. The Anl gene is required for the accumulation of normal levels of gibberellic acid in corn, and it is understood that it encodes ent-kaurene synthase A, the enzyme involved in the first step made of the biosynthesis of gibberellic acid. Defective mutations in this gene cause plants to be dwarf, with anthers on the ear and late blooming. Other members of the family of the An genes of the present invention were located in barley, wheat and sorghum, through the ability of a candidate gene to hybridize with an oligonucleotide probe of a nucleotide sequence of the Anl gene. corn of the present invention. As a probe part of an Anl clone was used. Genomic DNA was extracted from barley, sorghum, and wheat plants. Each gender was analyzed separately. The genomic DNA was digested and separated by gel electrophoresis. The DNA was stained separately. An Anl DNA probe was used to search for the homologous nucleotide sequences in barley, sorghum and wheat. In addition, an An2 gene of corn is detected in corn. The products of An2 mutant genes decrease the levels of gibberellic acid, although to a lesser degree than that effected by the Anl gene product. A doubly imitative plant can be characterized, this is a plant with a mutation in both Anl and An2 by a more severe phenotype than any single mutant, that is, a phenotype with severe dwarfism. DNA and RNA gel spot analyzes show that Anl is a single-copy gene. Sequence analysis of an 2.8 kb Anl-cDNA clone shows homology with plant cyclase genes and a polyrenyl pyrophosphate binding domain. The initial steps in the biosynthetic trajectory of gibberellic acid involve the fixation of a geranylgeranyl pyrophosphate of polyprenylpyrophosphorylated substrate, which is converted to kaurene by cyclization, steps for which the Anl plants are defective. Northern analysis of the Anl transcript indicates that it accumulates in buds, roots, spikelets, buds, pollen and grains. In shoots of seedlings the induction of light has been demonstrated by the Anl transcripts. The cloning of the biosynthetic genes of gibberellic acid provides recombinant genetic tools that lead to a better understanding of the role of gibberellic acid in the growth and development of corn. In addition, control over the levels of gibberellic acid can be used to manipulate the development of the plant using recombinant DNA technology for specific purposes. The Anl is one of the five genes identified in the -is corn that are involved in the biosynthesis of gibberellic acid. The mutants of the five genes (Anl, di, d2, d3 and d5) have anthers on the cob, but the Anl is different from the others because its stature is invariably semi-dwarf rather than dwarf. It seems that the semi-dwarf stature is the result of a redundancy in the maize genome for the Anl function. Evidence for this redundancy comes from anl -, - bz2 - 6923, a deletion mutant that lacks the Anl gene and still accumulates ent-kaurene, a downstream product of Anl activity. From the Southern analysis of low stringency of the DNA anl-bz2-6923 comes more support for redundancy, which shows the presence of sequences with some homology with Anl. One of these sequences is identified as the An2 gene, the existence of which was not suspected of the classic reproduction experiments, which identified the other biosynthetic corn genes of gibberellic acid.

The Anl gene product is involved in the synthesis of kaurene, early in the biosynthetic pathway of gibberellic acid (GA). Therefore, the loss of the Anl function results in a deficient phenotype of gibberellic acid, which causes an altered development, including a reduced height of the plant and the development of perfect flowers in normally pistillated ears. An Anl allele was generated by Mutator-induced mutagenesis, and the gene was cloned using a DNA fragment that is common to both MuI and Mu2 as a mutant gene probe. The Anl gene of the corn was cloned using a mutant fragment as a gene probe. In an ann - 891339 isolated from labeled Anl, Mu2 was inserted into the coding region of the Anl gene. This results in a deficient phenotype of gibberellic acid. The identity of the Anl clone was confirmed by a comparison of the predicted amino acid sequence with that of a GA1 gene from Arabidopsis (See PCT patent application WO / 9316096). The two genes are 47 percent identical and 68 percent similar (GCG package, Genetics Computer, Inc., University of Wisconsin) at the amino acid level, suggesting that they have a common function. Anl contains a polyprenyl pyrophosphorylase binding domain and shares homology in this region with other plant cyclase genes. Southern analysis of a deletion mutant, anl-bz2-6923, showed that the Apl coding region is completely within the suppression. But the deletion mutant accumulates kaurene, indicating that the Anl function is partially supplemented by additional activity. In fact, the low-stringency Southern analysis of the DNA of the deletion mutant demonstrates the presence of DNA sequences homologous to Apl, for example, the Ap2 gene, which was isolated by the RT-PCR method. Therefore, it is likely that the semi-dwarf stature of the Apl mutants, as opposed to the dwarf stature of the other gibberellic acid-deficient mutants in corn, is based on the redundancy in this step of the biosynthetic path of the acid gibberellic A double mutant, with deficiency in the levels of gibberellic acid effected by more than one gene, may show a more severe phenotype than a single mutant. Antibodies have been prepared for the Apl gene product. Antibodies coupled with in vivo and in vitro assays of kaurene synthase A and B activity from a cloned Apl construct within E. coli expression vectors allow the Apl gene product to be tested for the activity of synthase A and B of kaurene. Complexes were formed with kain-synthase A and the gene product of clone Apl. The identity of a second gene product that catalyzes the first step performed in the synthesis of the gibberellic acid of the plant hormone has been determined through the use of oligonucleotide primers derived from the Apl sequence. Oligonucleotides homologous to the nucleotide sequence Apl were generated, and were used to synthesize a 485 bp RT-PCR fragment that is highly homologous to, but distinct from, Apl, as evidenced by a restriction site analysis of a corresponding nucleotide stretch in Apl. This fragment has been designated Ap2. The resulting 485 bp RT-PCR product is used to derive specific primers from Ap2. These primers are used to isolate full length An 2 cDNAs and to determine their mRNA sequence. In the past, changes have been made in the activity and development performance of the plant, through conventional breeding, which requires that a complete genome be recombined, rather than a single gene or selected set of genes, and which is limited to natural genetic variability, rather than being susceptible to genetic engineering. The family of genes provided by the present invention allows for the engineering placement of such genes in a uniform medium, for better control of aspects of plant development, such as height and fertility, and the manipulation of genes per se for achieve specific plant reproduction objectives. For example, add An genes to the plant to increase the levels of gibberellic acid, or add an anti-sense molecule to lower the levels of gibberellic acid. Definitions In the following description, many terms are used extensively. The following definitions are provided to facilitate understanding of the present invention. In eukaryotes, RNA polymerase II catalyzes the transcription of a structural gene to produce mRNA. It can be designated that a DNA molecule contains an RNA polymerase II template, in which the RNA transcript has a sequence that is complementary to that of a specific mRNA. The RNA transcript is called an anti-sense RNA and a DNA sequence that encodes anti-sense RNA is called an anti-sense gene. The anti-sense RNA molecules are able to bind to the mRNA molecules, resulting in an inhibition of the translation of mRNA. A cloning vector is a DNA molecule, such as a plasmid, cosmid, or bacteriophage that has the ability to replicate autonomously in a host cell. Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites, in which foreign DNA sequences can be inserted in a determinable manner without loss of an essential biological function of the vector, as well as a marker gene which is suitable for use in the identification and selection of cells transformed with the cloning vector. Marker genes typically include genes that provide resistance to tetracycline or resistance to ampicillin. Exocene denotes an article that is foreign to its surroundings, and particularly applies in the present to a genetic construction class that is not found in the normal genetic complement of the host plant. Accordingly, in the present invention an exogenous construct that is used to produce a plant by transformation, includes an operable promoter and an isolated DNA molecule having a nucleotide sequence of a member of the gene family of the present invention. An expression vector is a DNA molecule that comprises a gene that is expressed in a host cell. Typically, the expression of the gene is placed under the control of certain regulatory elements, including constitutive or capable promoters, regulatory elements specific to a tissue and enhancers. It is said that such a gene is "operably linked to" the regulatory elements. Heterologist is a modifier that indicates a source that is different. For example, a heterologous promoter that is used with a structural gene of the present invention is a promoter that is different from that of the structural gene. An isolated DNA molecule is a fragment of DNA that is not integrated into the genomic DNA of an organism. For example, the nucleotide sequence of the Anl gene is a DNA fragment that has been separated from the genomic DNA of a maize plant. Another example of an isolated DNA molecule is a chemically synthesized DNA molecule that is not integrated into the genomic DNA of an organism. The isolated ones are mutant plants derived from independent sources. A recombinant host may be a prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the gene (s) cloned in the chromosome or genome of the host cell. RT-PCR is a method known to those skilled in the art. The components that are used herein for RT-PCR were obtained with GIBCO-BRL, Garthersburg, Md. The manufacturer's instructions were followed. Two nucleic acid molecules are considered to have a substantial sequence similarity. if their nucleotide sequences share a similarity of at least 50 percent. The sequence similarity determinations can be carried out, for example, using the FASTA program (Genetics Computer Group; Madison, WI). Alternatively, sequence similarity determinations can be carried out using BLASTP (Basic Local Alignment Search Tool) of the Experimental GENIFO (R) BLAST Network Service. See Altschul et al., J. Mol. Biol. 215: 403 (1990). Also see Pasternak and collaborators, "Searches of i? ' Sequence Similarity, Multiple Sequence Alignments, and Molecular Tree Construction "(" Sequence Similarity Searches, Multiple Sequence Alignments, and Molecular Tree Building ") in Methods in Plant Molecular Biology and Biotechnology (Methods in Plant Molecular Biology and Biotechnology) Glick et al. (Eds.), Pages 251-267 (CRC Press, 1993). A suitable promoter is a promoter that controls the expression of the gene in cells that are going to be altered experimentally by manipulating genes or controlling the biosynthesis of gibberellic acid. A transgenic plant is a plant that has one or more plant cells that contain an expression vector. BRIEF DESCRIPTION OF THE DRAWINGS FIGURE ÍA is a schematic representation of the steps of the biosynthesis of gibberellic acid and FIGURE IB is concentrated in the steps catalyzed by the synthase A and B of kaurene. FIGURE 2 is a sequence comparison of amino acids between gene products of the corn Apl gene (top) and an Arabidopsis gene, GA1 (bottom). FIGURE 3 A and B are the cDNA sequence of the gene Apl isolated from maize (access number of Gen Bank L37750). FIGURE 4 illustrates the role of gibberellic acid in maize maturity by reference to a comparison of the days required for maturation for anl-bz2-6923 and its wild type siblings. GDUSHD is the units of heat to the dispersion of the pollen, 25 units during 1 day. FIGURE 5 is a map of the DP6464 plasmid. FIGURE 6 is a cDNA sequence of an An2 gene isolated from maize, aligned with a fragment of a corresponding segment of the nucleotide sequence of the Anl gene illustrated in FIGURE 3. FIGURE 7 is a map of the restriction site of the genes. corresponding nucleotide sequences of Anl and An2, in accordance with FIGURE 6. FIGURE 8 A and B is a nucleotide sequence of the promoter of the nucleotide sequence of An2 of the FIGURE 3. Position 7 in Figure 8A is the start of the promoter; position 2075 in Figure 8B is the promoter and position 2076 is the starting site of Apl transcription. DETAILED DESCRIPTION OF THE PREFERRED MODALITY Gibberellic acid (GA) levels are important factors in the development of the plant. The control of the levels of gibberellic acid by means of genetic engineering technology allows the alteration of the phenotypes of the plant such as fertility and size. For this purpose, the identification and isolation of genes that control the biosynthesis of gibberellic acid are required. A gene family has been identified that is capable of coding a product that is necessary for the conversion of GGPP to ent-kaurene in the biosynthesis of gibberellic acid. The product is consistent in structure with a cyclase. The members of this family of genes hybridize with the Apl gene under conditions of high stringency. These genes also encode products that are the functional equivalents of the sequence in FIGURE 2 inside the box. FIGURE 2 shows the correlation between the predicted amino acid sequence of Apl (upper) and that of GA1 (lower). The steps catalysed by the kauran synthase are as follows: Two rings are closed in the conversion of GGPP to CPP by the kaurene synthase A. The third ring is closed, the pyrophosphate group is split, and a carbon-carbon bond is broken and reformed at a nearby site as the CPP is converted to ent-kaurene by the kaurene synthase B (FIGURE IB). In addition, as noted, Anl is one of five genes identified in maize that are involved in the biosynthesis of gibberellic acid. The mutants Apl, di, d2, d3, and d5 of corn make up a class of recessive mutants that are gibberellic deficient and responsive to gibberellic acid. It seems that all of them are defective in some step of the biosynthetic trajectory of gibberellic acid, and these share many phenotypes, including the reduced stature and the development of anthers on the normally pistillated ear. Within this class of mutants there are two distinct groups in relation to height. The alleles of di, d2, d3, and d5 typically have severe dwarfism, exhibiting a reduction of 80 percent or greater in the final height of the plant. By contrast, Apl alleles have less severe dwarfism, typically semi-dwarfism, and in some cases there is no reduction in their final height. The severity of reduction in height of the shoot for both groups is also reflected in the degree of reduction in leaf lengths. For the entire class, the reduction in height in lar can be recorded. growth seedlings in both light and shade. In seedlings of Apl growth in the shade of six days, the base of the reduced height lies in the mesocotyl cells. In Apl seedlings the number of coleoptile cells is slightly reduced, while the average cell length of coleoptile cells is the same as that found in wild-type seedlings (Table 1). This is in contrast to mesocotyl, where the number of cells is reduced by half and the average length of the cell is reduced to a quarter of that observed in wild-type seedlings. Consequently, the reduced stature in the shade growth seedlings is mainly due to the greatly reduced final cell lengths.

Table 1. Comparison of Cell Length and Cell Numbers in Shaded Growth Seedling Buds. L Loonnggiittuudd ((mmmm)) N Number of Average Length Cell Cells (mm) Brother High Coleoptile 18 228 0.08 M Meessooccoottiilloo 7 700 2 29944 0.18 Total 88 522 Dwarfism (Anl) Coleoptile 14 171 0.08 Mesocotile _6 130 0.05 Total 20 301 The seedlings were cultured for six days in total darkness. The Apl gene was cloned using transposon labeling. A key convenience for labeling genes with the mutator is 50-fold or more increase in mutation frequency compared to spontaneous rates. See Walbot, 1992 for a review. Transposon labeling involves the use of any of a number of naturally occurring plant transposons - Mu, Ac, Spm and the like - to create a "molecular label" to recover the mutated gene. Although it has been used before, the proposed labeling with transposon for the recovery of a gene of interest is unpredictable, is plagued by a low frequency of mutation, and technically it is very difficult. First, genetic materials have to be phenotypically traced to look for mutants of interest. There is no way to direct the transposon to a particular gene or to produce a particular phenotype. After a mutant phenotype of interest is found, it is also necessary to determine if the mutant was actually caused by the insertion of a transposon, because not all mutations are caused by transferable elements. A gene can be isolated by transposon labeling only if a particular transposon has been inserted into the gene. Each transposon system has major conveniences and inconveniences. The Ac and the Spm, for example, occur in fewer copies per genome than the Mu and therefore, promote a lower frequency of mutations. Because these two elements are excised from the germ line at a higher frequency than the Mu, however, it is possible to use the powerful genetic tool to look for a reversal of the mutant phenotype as a result of the removal of the element from the germline. germline This provides very strong evidence that a particular mutant was caused by insertion of the transposon. The Mu has the advantage of having a high copy number, in such a way that the frequency of causing mutations is higher (up to 10-100X higher than the previous mutation index). Because the frequency of removal of the germ line is very low (~ 1 in 10,000), however, standard tests for reversion are not practical. Other labor-intensive elements need to be used to verify that the gene was labeled by the transposon. These methods are molecular detection methods that involve isolating the DNA from the mutant plants of interest, and probing the DNA to look for the presence of a Mu element which is co-segregated with the mutant phenotype. With Mu this is particularly difficult, because there are many copies of Mu per genome - in fact, some genomes have more than 200 copies (Walbot and Arren, 1988). For the present invention, the co-segregation of an anl-891339 phenotype and the Mu2 containing restriction fragments were demonstrated by Southern blot analysis. The DNA of individual homozygous dwarf ani-891339 dwarves F2 was analyzed to determine the link between the mutation and the Mu element. The DNA was restricted with Sstl, and the blot was probed with an internal Mu2-DNA fragment. A Mu2 containing a restriction fragment of 5.7 kb was identified, common to all tested individuals. This Mu2 was cloned containing a restriction fragment within a lamd vector. A DNA gel spot analysis of a restriction digestive of the clone was performed. Double digestives P, of the cloned fragment were in Row 2 (Sstl and HindIII) and Row 3 (Sstl and Xbal). Flanking sequence DNA was identified, and a 2.6 kb flanking sequence fragment (g2.6Xba) was subcloned and used as a probe. Southern blot analysis of the deletion mutant (anl-bz2-6923) was performed as follows: Southern blots of digested Sstl genomic DNA from the deletion mutant and the wild type sibling were analyzed. A spot probed with the genomic flanking frequency sub-clone g2.6Xba showed that the deletion mutant 0 plants lacked DNA homologous to g2.6Xba. Using the g2.6Xba as a probe, a 2.8-kb cDNA clone was recovered from a corn cDNA library. It appears that this cDNA represents the full-length mRNA based on RNA gel spot analysis: the primary product is a transcribed homolog of a 2.8-kb. The cDNA contains an open reading frame of 2.5 kb or 823 amino acids, as illustrated in FIGURE 3. A comparison of the sequences of the maize Apl and GAl of Arabidopsis showed that the amino acid sequences predicted complete of Apl and GAl are similar. Its global identity of 47 percent (68 percent similarity) is surprising, but it is even stronger in an internal segment of 300 amino acids that is 68 percent identical (94 percent similar). As for the pyrophosphate binding domain of P, putative polyprenyl within this segment, Apl and GAl share a 100 percent similarity. Other sequenced plant genes that used polyprenylpyrrophosphorylated substrates (geranyl pyrophosphate, farnisyl, and geranylgeranyl) also share significant homology with Apl in this domain (Facchini et al., 1992), but much less global homology with Anl (20 to 25 percent of identity). These sequence homologies clearly indicate that the Apl encodes a cyclase that functions in the conversion of GGPP to ent-kaurene. 0 Although it is highly homologous to GAl, it is important to note that Apl is different from GAl in its amino terms (only 11 percent identical for the first 100 amino acids) and carboxyl (only 18 percent identical for the last 283 amino acids). In addition, the amino terminus 5 of Apl has the expected characteristics of a chloroplast targeting sequence, including a net positive charge (12 of 43 amino acids are basic, while only two are acidic). In addition, the amino terminus of Apl also has a similarity greater than 50 percent with the amino-5-terminus of a rice aspartate aminotransferase cDNA clone (Gene Bank Source D16340). Aspartate aminotransferase has many isoforms, of which at least one is located in the chloroplast (Matthews et al., 1993). This suggests that the amino terminus of Anl serves as a chloroplast targeting sequence. The support for a chloroplastic localization of the kaurene synthesis comes from the demonstration that purified chloroplast cell-free assays synthesize kaurene (Simcox et al., 1975). If the Apl and the GAl code for the In the same chloroplast direction activity, their direction sequences are different. The low homology between Anl and GAl at its carboxyl terminus can be functionally important. Although many plant cyclase activities share a conserved polyrenyl pyrophosphate binding domain, these act on different substrates and cyclize by different mechanisms. The basis for these differences is not obvious from an examination of the primary sequences of amino acids. A Southern blot analysis was performed using high and low stringency. Southern blots of DNA from anl-bz2-6923 and its wild-type homozygous suppression brother were compared from high stringency (at a temperature of 65 ° C) and low (at a temperature of 25 ° C) washes. Genomic DNAs were digested with BamHI. The probe was 5 Apl-cDNA. Therefore, at high stringency, the probe DNA hybridizes only to wheat DNA and its tall brother, while, at low stringency, hybridization occurs with the mutant deletion maize. A related sequence in wheat is likely. i. Northern blot analysis shows accumulation of Apl transcripts. Northern blots of the total RNA preparations were probed with Apl-cDNA. The tissues analyzed were: (A) shoots and roots of light and shade seedlings, - and 15 (B) reproductive structures. The blot revealed accumulation of Anl transcripts in all tissues, and increased accumulation in shoots of growth to light. Since gibberellic acid plays roles important development, its control is a useful avenue to alter development with specific purposes. The anl-bz2- ß 923 allele of Anl is consistent with a robust plant that shows little or no reduction in plant height or leaf length compared to its siblings. wild type. In spite of its simi larity in growth, the first day of average pollen spread was delayed in this mutant, in the example shown this delay is 5 days (FIGURE 4). This shows that the decrease in the levels of gibberellic acid reduces the time for maturation 5 in corn, possibly by means of reducing the time required between germination and flower initiation. In FIGURE 4 is a comparison of the days required for the maturity of anl-bz2-6923 and its wild-type brothers, as a spot of the height of? < r brothers of wild type and mutants anl-bz2 -6923 against GDUSHD (heat units for pollen spread, 25 units «1 day). Although there is no difference in the final heights, there is an average of 200 GDUSHDs of delay for the mutant plants. The shortened time of maturity is an advantage in some growing areas (climates), - while, the increased maturity time is advantageous in other growing areas. Therefore, the ability to manipulate the levels of gibberellic acid by recombination techniques is advantageous for the development of commercial monocotyledons.

The isolation of genes such as Anl provides some of the tools needed in this endeavor. The Anl gene will also be useful for probing other homologous genes in other species. A gene homologous to the Apl was isolated by RT-PCR. As shown by Bensen and colleagues (1995), the construction of The primers used to generate the 485 bp RT-PCR product were completely dependent on the previously determined Anl cDNA sequence. Additional oligonucleotide primers were generated from the 485 bp RT-PCR product. Specific primers for An2 are used in a reverse genetic screening. A collection of maize families is used that has a high frequency and, possibly, many mutations. A large number of families are screened in games of approximately 50 by gene mutations in areas of interest. The PCR primers are defined for the elements of the mutator.

The primers of the Ap2 fragment are matched to those of the families to detect specific families having a Mu inserted near the tested primer product of interest. These families are then used for different breeding crosses. The families of plants selected by this screening have Mu insertions in the An2 gene. The seeds of the F2 progeny of the plant families are grown. A dwarfing phenotype is not likely for these families, because the Ap2 mutants only have a reduction in the level of gibberellic acid of 20%. However, the crosses between these families and Anl mutant plants produce double mutants which have severe dwarfism, because both 20% and 80% reduction are combined. Alternatively, if Apl and Ap2 are different, a complementation occurs.

The present invention is illustrated in more detail in the following examples. These examples are included for the purpose of explanation and should not be considered as limiting the present invention. EXAMPLE 1 Cloning of the Apl Gene Reports in the literature suggest that gibberellic acid levels may be a partial cause of heterosis. To develop the transgenic tools to improve the performance of crop plants using genes that affect the synthesis of ag, one of the goals was to clone genes that encode enzymes of the ag biosynthetic path. Several maize mutants with gibberellic acid deficiency (di, d2, d3, d5, Apl) have been described which were associated with a dwarf stature and the andromonic flowering (perfect flowers on the cob). If these mutations actually occurred in IC genes encoding directly for the ag biosynthetic enzymes, it was difficult to perceive how to identify and isolate the genes without having to purify the enzymes still uncharacterized in the path of ag. One possibility was to use transposon labeling, which has been used with great success in some cases to label and isolate genes (Albot, 1992). But dwarfs are very rare and, in addition, transposon-induced alleles known to any dwarf mutant have not previously been reported. Patrick Schnable (Iowa State Univerisity) obtained an ear mutation with anther (Apl) segregating on a corn track containing Mu, and experiments were carried out to determine if a transferable element associated with the mutant gene could be found. The possibility of this was questionable, however, because they had never before identified such dwarf mutants labeled by transposon. The method used for the detection of mutants included isolating the DNA from mutant plants of interest and then probing the DNA to look for the presence of a Mu element which is co-segregated with the mutant phenotype. This was particularly difficult because there are many copies of Mu per genome; in fact, some genomes have more than 200 copies (Walbot and Warren, 1988). In order to reduce the immense amount of Mu hybridizing bands, it was first necessary to carry out repeated crosses to plants that inactivated and diluted most of the Mu elements. It was also necessary that the search for the mutant Apl gene would use Southern blots for probe the genomic DNA separately with a DNA fragment that is unique to each of the nine different Mu families. Even so, the number of copies per Mu family is around 25, making it very difficult to identify a hybridizing band in the stain that co-segregates with the Mu element used as a probe. In doing this DNA screening for the Apl, it was necessary to prepare DNA from 50 different individual plants and to probe each of these samples in a Southern blot with each of the specific Mu probes, Muí, Mu2 and Mu3, which are characteristic of the sub-family. After a Mu labeling, a co-segregating restriction fragment was found, the fragment was isolated by cloning and sequenced to identify the location of the Mu insert. The flanking regions were also sequenced to locate the structural gene of interest. For a gene such as Anl, not identified or previously isolated and, therefore, of unknown sequence, it can be very difficult to determine the exact limits of the gene and even prove that the clone contains the mutant gene of interest. As indicated by Walbot in his strategy report for mutagenesis and gene cloning using 1992 transposon labeling, the identification of a co-segregating side is not direct. In addition, the identification of such a band is not proof that the band in question defines the gene of interest. It was observed that a family with a phenotype characteristic of gibberellic acid deficiency segregates as a simple characteristic recessive trait in an active Mu track. The mutation was shown to be allelic with Apl, and identified as anl-891339. Southern analysis of the Sstl genomic DNA of the anl-891339 deletion mutant and its wild-type siblings identified a restriction fragment containing Mu2, "Approximately 5.4 kb, which was co-segregated with the mutation. This fragment was aired from an agarose gel preparation, cloned into a lamdol shaker vector and purified by foil using an internal Mu2 fragment as a probe. Analysis of the cloned fragment, by restriction with Xbal or with HindIII, identified flanking sequence DNA fragments. A 2.6kb flanking sequence fragment Xbal (g2.6Xba) was subcloned into a plasmid and used as a probe for the Southerns and screening for cDNA libraries of the maize. Southern analysis of maize genomic DNA showed that g2.6Xba was a single copy DNA. Different cDNA clones from maize cDNA libraries were selected using g2.6Xba as a probe, thus demonstrating that g2.6Xba is located in a transcribed region of the genome. The frequency of positive clones in each of the two amplified libraries was 8 per 360,000 sheets. It was sub-cloned into a plasmid and the longest of the cDNAs, 2.8 kb, was sequenced. It appears that this cDNA represents the full-length mRNA. The comparison of cDNA and the genomic DNA sequence of Apl identifies many exons. The comparison also shows that the Mu2 element that causes the mutation is inserted in or on the edge of an intron, at 1.6 kbp from the carboxy terminal of the transcript and at 900 bp from the amino terminus. It was necessary to take many approaches to confirm the identity of the putative clone of the Anl gene. It was necessary to establish a strong link between the clone and the gene by test, to show that the clone did not hybridize to DNA from a known genetic suppression mutant of Apl. This evidence placed the clone in a few map units (4 centimorgans) of the genetic site for Anl., based on the resolution of this mapping experiment. This distance corresponds to ~ 8.4 Mb x 106 bp, so it is possible that the clone could have been located as far as 8.4 mb from the genetic place for Apl. The next step was to isolate and sequence a cDNA clone. To do this, it was necessary to determine where the putative Apl gene was expressed, in such a way that a cDNA library could be created that probably contained the gene. Because the size of the mRNA was known to be quite large, (~ 3 kb), the recovery of a full-length clone was very difficult. The first clone was only 2.5 kb in size, so it was necessary to track a second library to recover a longer clone of 2.8 kb. The cDNA sequence showed a similarity of ~ 40 percent in only one region of the clone to a type of isoprenoid cyclase binding region, based on other known cyclase-like genes. 5 It is known that the biochemical function of the Apl for the accumulation of kaurene and is similar to the cyclase that converts the GGPP to CPP. It is known that this is the first step made in the biosynthesis of gibberellic acid (kaurene synthase A). o Homology with other cyclase was consistent with one of the possible functions for the gene product Apl. The homology that was seen was very limited and much lower than the global homology that is typically among the cyclase, so that only tentative conclusions could be drawn regarding the identity of the isolated gene. Therefore, additional evidence had to be obtained from others '_ technical approaches. Peptides corresponding to predicted antigenic domains were synthesized from the protein that was encoded by the clone. Antibodies arose against many peptides. Only 2 of the 4 antibody preparations could be used. It was shown that some of the antibodies precipitate the cyclase activity of GGPP-a-CPP from cucurbitic endosperm extracts, providing additional evidence to support the possibility that the isolated gene was Apl. Finally, an amino acid sequence comparison between our clone and an AGI clone of the Arabidopsis revealed significant homology along the length of the protein. It has been shown that AGI encodes the cyclase of GGPP-a-CPP (Tai-Ping Sun et al., Personal communication). These data provide a convincing case that the Apl was cloned, but clearly, the process was difficult and uncertain. Although the transposon labeling made cloning of the Apl gene possible, the success was far from predicted. The efficiency to obtain an insertion mutant depends on a variety of factors, including the activity phase of the autonomous element (s), the number of moving elements, the location of the elements and the susceptibility of the target site (Walbot, 1992). As Walbot states in his review, "Although 'it is not reported frequently, some directional mutagenesis traces fail completely, despite the reasonable sizes of the progeny." Table 2 in its review indicates many examples where failed attempts to direct epothilic icoty genes by transposon insertion. Based on the previous failure to identify any dwarf mutants that were labeled with transposon, it was not unreasonable to assume that the target site for the genes in the path of gibberellic acid might not be susceptible to labeling. Therefore, it was very uncertain that the Apl mutant of the Mu genetic materials was in fact labeled by Mu. However, as shown herein, the Apl gene has been cloned. EXAMPLE 2 Bases for Nature I-Dwarfed Plant Apl As described above, the Apl is different from the other mutants deficient / responsive to corn gibberellic acid in that it is a semi-dwarf. This is true of the four Apl isolates examined. Apl plants respond to the application of several ag biosynthetic intermediates, including ent-kaurene. Since the biosynthesis of gibberellic acid is initiated by the conversion of GGPP to CPP, followed by the conversion of CPP to ent-kaurene, it appears that the Apl is deficient in the conversion of GGPP to ent-kaurene. The DNA probe of anl-bz2-6923 in a Southern blot with either g2.6Xba or full-length Apl-cDNA resulted in the absence of detectable probe hybridization. Similar results were observed in Northern blots of the deletion mimic RNA. Ethyl indicates that the Anl gene transcript lies completely within the deletion and, therefore, is not present in the anl-bz2-6923 plants. Therefore, it would be expected that this mutant would be absolutely defective in the synthesis of ent-kaurene. All in all, the anl-bz2 ~ 6923 seedlings of light growth accumulate ent-kaurene in vivo albeit at a very low rate (20 percent) compared to their wild-type siblings (Table 2). This activity was attributed to the Ap2 gene product, an activity that is not Apl that complements the ent-kaurene Apl production. It is thought that the complementary activity is not unique to corn. It is also expected that a deletion mutant of Arabidopsis, GAl -3, is free of exit-kaurene, since the GAl coding region is completely suppressed (Tai-Ping Sun et al., 1992).

However, GAl-3 plants convert GGPP to CPP and CPP to ent-kaurene in cell-free extracts from the siliques. Notably, there are many GAI isolates that demonstrate a uniform but variable reduction in plant height, similar to that observed for isolates in the Apl in corn. However, there is no accumulation of ent-kaurene in the d5 mutants. The d5 mutant is believed to be defective in kaurene B synthase as is the GA2 mutant of Arabidopsis, which has' A, but not B activity in free cell extracts from immature siliques. When Southern stringency is reduced for restricted DNA spots of anl-bz2-6923, by altering temperatures, bands that share homology with Apl can be identified, suggesting that homologous sequences provide functional equivalents Ap.

Consequently, the "cracked" or consistent semi-dwarf phenotype observed for all Anl mutants documented in maize is probably the result of a redundancy for the Apl function. This redundancy does not exist, or is of little importance, for the B synthase of kaurene that encodes the d5 genes of maize and GA2 of the Arabidopsis, since its block in the synthesis of kaurene seems complete. EXAMPLE 3 Distribution and Expression of the Anl Transcript The transcription of the Apl gene in corn occurs in many tissues, as demonstrated by Northern blots. The vegetative parts of the plant, shoots and roots, contain mRNA of Apl. All reproductive tissues including spikelets, developing ears, buds and embryos contain Apl mRNA. It is interesting to note that the bleached bud tissue seems to have very little, if any, of ANl mRNA compared to shoots grown to light. This presence of message in the roots separates this transcription induced by light from the dependence in the development of the chloroplast. Both qualitative and quantitative measurements of the Apl transcript were made, using specific Apl-derived primers from the cDNA sequence of the Apl. The primers used were: 5 '-TTGCCAA- GCTCTGCATCAGCTTGAGTGT-3' as a forward primer, and 5'- GGAAACATGTCTATCGATCATATGTTGTGGGGA-3 'as a reverse primer. Through the reverse transcriptase polymerase chain reaction (RT-PCR), using these primers, the distribution of the Anl transcript in corn was determined to include: shoots, roots, buds, pollen, and spikelets. With the quantitative (Q-) RT-PCR using a competitive template (a cDNA sub-clone of Apl with a lamda insert of 120 bp), it was determined that the Apl transcripts accumulate io after exposure to light in the outbreaks of corn. Therefore, the accumulation of the transcript Apl is induced by light. Using the same Q-RT-PCR approach, it was shown that the accumulation of the Apl transcript was repressed by the treatment of plant gibberellic acid. EXAMPLE 4 Cloning of Ap2 by RT-PCR A deletion mutant in maize, designated as anl-bz2-6923, produces 20 percent of the wild-type amount of the biosynthetic product of the gene • Anl. This Production occurs despite the fact that the deletion mutant is totally lacking the Apl transcript and that there is no evidence of genomic DNA apr. Therefore, it was believed that the Apl gene has a functional homologue that catalyzes the production of 20 percent of the residual activity. A priori this functional homologue element could be, but is not necessarily homologous to Anl. To localize the structural homologue element to Apl, a large number of primers were generated and tested for Anl by RT-PCR to see if any of them produced a PCR product using RNA isolated from the deletion mutant. Based on the absence of DNA from the Apl in the deletion mutant, RT-PCR products derived in this manner were used. A primer pair yielded an RT-PCR product. That primer pair was 5'CTTCGAGATCGCCTTCCTTCTCTCA-3 '(5266) as the forward primer, and 5 '-TAGCCCAGCAAATCCCATCTTCAGTCCA-3 * (5267) as the reverse primer. This primer pair produced a 485 bp product that was sub-cloned and sequenced. The nucleotide sequence was 82 percent identical to the Apl, as it is aligned in FIGURE 6. The predicted amino acid level was 82 percent identical and 91 percent similar to that of the Apl. This large percentage of homology suggested that An2 is a functional duplicate of Apl. EXAMPLE 5 Distinguishing Anl from Ap2 In the 485 bp region of interest, each Anl and An2 have unique PstI sites which allows the two genes to be distinguished when analyzing PCR products, cDNA libraries, or when selecting a colony. The PstI polymorphism was used to track libraries searching for the presence of Ap2. The presence of both genes in a corn seedling library resulted in the PstI digested pattern of the four bands shown in FIGURE 7. The upper and lower bands can be attributed to the Apl, and the two bands in half can be attributed to the Apl. attribute to Ap2. The original primers, # 5266 and # 5267, were put in pairs with the homolog of the primers to "anchor" sequences located at the 5 'and 3' ends of a cDNA library of a seedling which has been shown to contain Anl and An2, and whole Anl and An2 cDNAs were generated by PCR in two fragments, size 1.2 and 2.1 kb. The sub-cloning and transformation of these fragments into competent cells was followed by an analysis of plasmid preparations from individual clones. The PstI digestion of the plasmid preparations revealed cDNA clones of Ap2 for the two fragments 1.2 and 2.1. EXAMPLE 6 Use of Recombinant Genetic Methods to Affect the Development of a Plant Recombinant genetic methods made use of an isolated DNA molecule that encoded a gene product which is necessary to convert GGPP to ent-kaurene in the ag biosynthesis. The isolated DNA molecule is incorporated into a plasmid, such as that shown in FIGURE 5, and transferred to a host plant. The expression of DNA in the host will generally increase the endogenous levels of ag. The effect will depend on the species and the increase in ag levels. As shown herein, mutations can still affect maturity time. Generally a strong constitutive promoter is preferred to regulate a gene of the present invention in a host cell. Examples of suitable promoters are ubiquitin and 35S. The reduction of endogenous gibberellic acid levels is achieved by introducing an antisense molecule to a gene product of the present invention. The knowledge of the sequence of the binding domain (FIGURE 2) allows such anti-sense molecules to be constructed. The direct mutation is useful for changing a phenotypic gene of the present invention in such a way that ag levels are reduced. The effects of reduced levels of ag have been described above. Knowledge of a sequence of a corn Apl and a partial sequence of a corn Ap2 gene will facilitate site-specific mutations directed not only in corn, but also in other monocots, which, as described herein, have homologs to Anl of corn.

Table 1. Accumulation of Kaureno in Buds of Growing Maize Seedlings to Light. Content of £ nt-Kaurene (pmoles / gfwt) Length of the Leaf (mm) Leaf Without Treatment 48 h in 2a. Sheet 3a. Plant Tetcyclacis anl -bz2-6923 High 120 1330 42 83 Dwarf 33 209 30 58 Anl - 891339 High 61 710 Dwarf 54 216 d5 Dwarf not detected not detected B73 94 1093 The seedlings were grown in continuous light for six days, at which time tetcyclacis mM (an inhibitor of the kaurene mechanism) was applied directly to the shoots. Forty-eight hours later, the outbreaks of treated and untreated plants were analyzed to see their ent-kaurene content. EXAMPLE 7 Constructs and Fusion Expression of the Apl Promoter-GUS Two thousand bases have been cloned and sequenced immediately after 5 'at the start of the transcription Apl (ie, the Apl promoter). In FIGURES 8A and B the sequence is shown. This 2 kb promoter region was merged into GUS. Transient expression assays in germination seedlings demonstrated that the Apl promoter is sufficient for the expression of GUS fusion protein in roots and shoots. METHODS Plant Material A labeled label of Mu2 from anl - 891339 from the maize family of lines with active Mu elements (lines originating from Pat Schnable, Iowa State University) was selected. 5 Additional Apl alleles that were used in this study include; anlbm2 (110D, Maize Genetics Cooperation Stock Center, U. Illinois). The idd * -2286A was mutated both in the indeterminate place (id) and in the place Anl (d), but it seems to be a deletion mutant, as progeny of themselves from __ of this segregated material for id and Anl. Conversely, it appears that the anl-bz2-6923 is a deletion mutant. The degree of suppression was not defined although Id (two map units close to Apl) and Ad (two map units away from Bz2) are not affected by the deletion. 15 Southern Analysis Total DNA was extracted from the leaf tissue by the urea extraction method (Dellaporta et al., 1983). Southern blots were performed as described previously (Johal, 1992) using Duralose-UV membranes (Stratagene). Mu2 probes were synthesized by random priming (Amersham) a Levigated internal Aval-BstEII muI fragment isolated from pA / B5 (Chandler, 1986). This internal MuI fragment contains regions of homology with Mu2, thus allowing the hybridization of the sequences of both Mui and Mu2.

Cloning Protocol The restriction fragment of genomic DNA containing the Mu2 element that is determined to cause the anl-891339 mutation was electro-leaked following the agarose gel electrophoresis of the digested total dialysis of Sstl DNA, and concentrated by precipitation of ethanol. The precipitated fragments were previously hardened to restricted Sstl arms of the bacteriophage vector lamda sep6 / lac5 (Meyerowitz, de _ ^ Marteinssen, CSH) and they were packed using Gigapack Gold (Stratagene). This library was screened for the phage containing the Mu2, with the Sstl insert of a clone containing the purified Mu2 from plaque, then transferred to the bacteriophage vector Lamda-ZapII (Stratagene). This insert and others clones that were used for probing or sequencing were subcloned into the Bluescript SK + plasmid and maintained '• in SURE cells (Stratagene). Tracking of cDNA library Two cDNA libraries were prepared, which served as sources for the Anl cDNAs, from the buds of B73 seedlings of growth in the light of 14 days, a gift from A. Barkan, University of Oregon (Barkan, 1991) and from whole grains (30 DAP) of W22, a gift from Karen Cone, University of Missouri. Loftstrand Labs Limited generated data Sequence 25 of an Apl cDNA; 2.8 kb.

Preparation of RNA and Northern Analysis Total DNA was prepared as described above (Chomczynski et al., 1987). It enriched the PolyA + RNA using the PolyA-Tract System III (Promega) following the manufacturer's protocol. The Northerns were run, stained and probed as described above (Johal, 1992) using sub-clones of 1.5 kb and 1.1 kb of Apl to generate random primed probes. Analysis of the Synthetase Activity of ept-Kaureno and Kaureno ^ .- < ? An analysis of the in vivo accumulation of ent-kaurene in maize seedlings of light growth was performed. Cell-free assays of the kaurene synthetase A and B activities were performed using immature silicas from Arabidopsis seedlings. (Bensen, 1995). Production of a Transgenic Plant A transgenic plant containing a construction having a gene of the present invention can be regenerated from a culture transformed with that same construction, as long as the plant species involved is susceptible to regeneration. The "culture" in that context comprises an aggregate of cells, a callus, or derivatives thereof which are suitable for culture. A plant is regenerated from a cell or transformed culture, or from an explantation, by methods described herein that are known to those skilled in the art. The methods vary according to the species of the plant. The seed is obtained from the regenerated plant or from a cross between the regenerated plant and a suitable plant of the same species, using reproduction methods known to those skilled in the art. Example of Corn Transformation Methods (Can be Modified for Specific Promoters and Structural Genes) Specific Promoter of Corn Mats: Experimental Protocols of Stable Transformations. Repetition 1, 2, and 5, - Target: Recover colonies, plants and transgenic progeny of maize, resistant to Basta / Bialophos and expressing GUS guided by the specific promoter SGB6gl mat. Genotype: 54-68-5 Bl-1 (Repetition 1) or 54-68-5 161F3 (Repetition 3) 54-68-5 161F4 (Repetition 5). Medium: liquid suspension medium 237 for corn 115, callus maintenance medium for corn 115E, selection medium Basis of 5mg / L of callus 115B, selection medium Bialaphos of 3mg / L of callus Treatment of the tissue - Screen the cells through a mesh one day after subculture - Re-suspend in PEG at 237 + 3 percent at a plate density of 50 g / ml - Incubate in 3 percent PEG overnight - Put cells on plate, 0.5 ml / plate on 934 -AH glass filters on top of a Whatman filter - moistened with one milliliter of PEG medium at 237 + 3 percent - Transfer the cells on the glass filter to medium 115 following the bombardment Bombardment of Particle Gun DuPont Helium Gun (Repetitions 1 and 5) 650 PSI Burst Discs (Repetitions 1 and 5) DuPont PDS-1000 Gun (Repetition 2) 0.230 '' Arrest Plates, Acetyl Macroprojectiles (Repetition 2) A Bombardment per sample (Repetici 1 and 5) Two bombings per sample (Repetition 2). Modified tungsten DNA precursor protocols, specific for each gun. DNA: DP687 + DP610 DP460 + DP610 DP1952 + DP610 DP2125 + DP610. Treatment / Test after bombardment - Search for an R gene expression 24 to 48 hours after bombardment - Transfer samples to 115E (Repetition 1) 48 hours after bombardment. Transfer the samples to 115B (Repetitions 2 and 5) 7 days after the bombardment - Transfer the cells out of the filters 2 weeks after the transfer for selection. - Practice PCR colonies for reporter gene before regeneration of the plant - Keep the samples at 28 ° C in the dark. Method of transformation of corn to recover stable transgenic plants Day-l The cells were placed in the liquid medium and screened (710um), 100-200 milligrams of cells were collected in a 5.5-centimeter glass fiber filter over an area of 3.5 centimeters The cells were transferred to the medium and medium of incubation overnight. Day 0 The filter and the cells of the medium were removed, they were dried and bombed. The filter and the cells were again placed in the medium. Day 5 The cells in the filter were transferred to the selection medium (Bialophos of 3 milligrams). Day 12 The cells in the filter were transferred to the fresh selection medium. Day 19 The filter cells were scraped and dispersed in 5 milliliters of selection medium containing wild-type agarose with low melting point at 0.6 percent. The cells and medium were spread on the surface of two plates of 100 millimeters by 15 millimeters, containing 20 milliliters of gel-rite solidified medium. Day 40 Putative transformers were collected from the plate. Day 61 Plates were checked for new colonies. REFERENCES The references listed below are incorporated herein by reference to the extent to which they complement, explain, provide a background for or teach methodology, techniques or compositions that are employed herein. Altschul et al. (1990). J. Mol. Biol. 215: 403. Barendse, G. W. M., Dijkstra, A. and Moore, T. C. (1983). The biosynthesis of the ept-kaurene gibberellin precursor in cell-free extracts, and the endogenous gibberellins of the Japanese bluebell vines in relation to the development of the seed (The biosynthesis of the gibberellin precursor ent-kaurene in cell-free extracts and the endogenous gibberellins of Japanese morning glory in relation to seed development). J. Plant Growth Regul. 2, 165-175. Barkan, A. and Marteinssen, R. A. (1991). Proc. Nati Acad. Sci. USA 88, 3502. Beavis, W. D., Grant, D., Albertsen, M. and Fincher, R. (1991) Theor. Appl. Genet 83: 141-145. Bensen, R. J. et al. (1995) Cloning and characterization of the Anl gene (Cloning and characterization of the An gene), The Plant Cell 7: 75-84. Buckner, B., Kelson, T. L. and Robertson, D. S. (1990). The Plant Cell 2: 867-876. Chandler, V. L. and Walbot, V. 1986. 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Proc. Nati Acad. Sci. USA 83: 1767. Chang, P. C, Greyson, R. I. and Walden, D. B. (1983). Organ initiation and the development of unisexual flowers in the spikelet and ear of zea mays (organ initiation and the development of unisexual flowers in the tassel and ear of zea mays). Amer. J. Bot. 70, 450-462. Chomczynski, P. and Sacchi,? (1987). Anal Biochem. 162, 156. Coolbaugh, R. C. (1985). Sites of gibberellin biosynthesis in pea seedlings (Sites of gibberelline biosynthesis in pea seedlings). Plant Physiol. 78, 655-657. Dellaporta, S.L., Wood, J.B. and Hicks, J.B. (1983). Plant Mol. Biol. Rep. 1, 18 Duncan, J. D. and West, C. A. (1981). Properties of the jahren synthetase of Marah endosperm inacrocarpus -. evidence for the participation of separate but interacting enzymes (Properties of kaurene synthetase from Marah macrocarpus endosperm: evidence for the participation of separated but interacting enzymes). Plant Physiol. 68, 1128-1134. Emerson, R. A. and Emerson, S. E. (1922). Genetic interrelations of two maize andromonotic types (Genetic interrelations of two andromonecious types of maize). Geneti cs 7, 203-227. Facchini, P. and Chappell, J. (1992). Gene family for a sesquiterpene cyclase induced by an extractor in tobacco (Gene family for an elicitor-induced sesquiterpene cyclase in tobáceo). Proc. Nati Acad. Sci. USA 89, 11088-11092. Fujioka, S., Yamane, H., Spray, CR, Gaskin, P., MacMillain, J., Phinney, BO yTakahashi, N. (1988) Qualitative and quantitative analysis of gibberellins in plant shoots of normal, dwarf seedlings - 1 , dwarf -2, dwarf -3, and dwarf -5 of Zea mays (Qualitative and quantitative analysis of gibberellins in vegetative shoots of normal, dwarf-1, dwarf -2, dwarf-3, and dwarf -5 seedlings of Zea mays) . L. Plant Physiol. 88: 1367-1372. Han, C. D., Coe, Jr. , E. H. and Marteinssen, R. A. (1992). EMBO 11: 4037-4046. Hedden, P., Phinney, B. O., Heupel, R., Fujii, D. Cohen, H., Gaskin, P., MacMillian, J. and Graebe, J. E. (1982). Hormones of young spikelets of Zea mays. Phytochemistry 21: 391-393. Johal, G. S. and Briggs, S. P. (1992). Reductase activity encoded by the HM1 disease resistance gene in maize (Reductase activity encoded by the HM1 disease resistance gene in maize, Science 258: 985-987, McCarty, D.R., Carlson, C.B., Stinard, P.S.

Robertson, D. S. (1989). The Plant Cell 1: 523-532. McLaughlin, M. and Walbot, V. (1987). Geneti cs 117: 771-776. Marteinssen, R. A., Barkan, A., Freeling, M. and Taylor, W. C. (1989). EMBOJ 8: 1633-1639. Matthews, B. F., Wadsworth, G., Gebhardt, J. S. and Wilson, B. (1993). Cloning and expression of genes encoding aspartate aminotransferase in soybean (Cloning and expression of genes encoding aspartate aminotransferase in soybean). I proved Crop and Plan t Products Through Biotechnology, Abs. Xl-324, pp. 105. Metzger, J. D. and Zeevart, J. A. D. (1980). Effect of photoperiod on endogenous gibberellin levels in spinach, as measured by ion current monitoring selected by combined gas chromatography (Effect of photoperiod on the levéis of endogenous gibberellins in spinach as measured by combined gas chromatography-selected ion current monitoring). Plant Physiol. 66, 844-846. Metzger, J. D. and Zeevart, J. A. D. (1982). Photoperiodic control of the metabolism of gibberellin in spinach (Photoperiodic control of gibberellin metabolism in spinach). Plant Physiol. 69, 287-291. O'Reilly, C. 0., Shepherd, N. S., Pereira. A., Schwartz-Summer, Z. , Bertram, I., Robertson, D. S., Petersson, P. A. and Saedler, H. (1985). EMBOJ 4: 877-882. Pasternak et al. (1993). Searches of Sequence Similarity, Multiple Sequence Alignments, and Molecular Tree Construction (Sequence Similarity Searches, Multiple Sequence Alignment, and Molecular Tree Building). Methods in Plant Molecular Biology and Biotechnology, Glick et al. (Eds.), (CRC Press), pp. 251-267. Rood, S.B., Buzzell, R.I., Mauder, L. N., Pearce, D. and Pharis, R. P. (1988). Science 241.- 1216-1218. • Simcox, P. D., Dennis, D. T. and West, C. A. (1975).

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Claims (14)

1. An isolated DNA molecule capable of hybridizing with a low monocot nucleotide sequence 5 conditions of high stringency, said molecule encoding a product that affects the conversion of GGPP to ent-kaurene in the biosynthesis of gibberellic acid, and the molecule not comprising the GAl gene.

2. The DNA molecule of claim 1, characterized in that it is capable of hybridizing to a nucleotide sequence in accordance with the Anl sequence of FIGURE 3, under conditions of high stringency.

3. The isolated DNA molecule of claim 1, characterized in that it has the nucleotide sequence of FIGURE 3. The isolated DNA molecule of claim 1, characterized in that it has a partial sequence of FIGURE 6. The isolated DNA molecule of claim 20, characterized in that it has a mutation that alters the product which affects the conversion of GGPP to ent-kaurene. 6. A cloned gene Apl of corn. 7. A gene Ap2 cloned from corn. 8. An expression vector comprising the DNA molecule of claim 1 and a promoter controlling the expression of the molecule. 9. The expression vector of claim 8, characterized in that the promoter is according to FIGURE 8A and B. 10. A polypeptide encoded by the expression vector of claim 8. 11. The polypeptide of claim 10, characterized because it has an amino acid sequence of Apl, according to FIGURE 2. 12. A method for altering the level of endogenous gibberellic acid in a plant of a first species, comprising the transfer of an isolated DNA molecule of claim 1, capable of coding a product that is used for the conversion of GGPP to ent-kaurene to a host cell, from which the plant is regenerated. The method of claim 12, characterized in that the transferred DNA molecule affects the level of endogenous gibberellic acid, in such a way that the time to mature of said plant is altered in relation to the norm for said first species. A method for altering the level of endogenous gibberellic acid in a plant of a first monocotyledonous species, said method comprising the construction of an anti-sense molecule for the isolated DNA molecule of claim 1, and sending the anti-sense molecule. sense to the plant in sufficient quantities and at appropriate times in the development to decrease the levels of gibberellic acid.