MXPA98008583A - Novedoso steroid of planta 5alfa reductasa, d - Google Patents

Abstract

A novel 5-reductase plant steroid, DET2, as well as polynucleotides encoding DET2 is provided. DET2 or the mammalian steroid 5alpha-reductase is useful to promote increased yield of the plant and / or increased biomass of the plant. Genetically modified plants are also provided, characterized by having increased yield, and methods to produce such plants

Description

NOVEDOSO ESTEROIDE DE PLANTA 5a REDUCTASA, DET2 Field of the Invention The present invention relates generally to plant genetic engineering, and specifically to the performance of genetically engineered plants, characterized as having an increased yield yield phenotype. BACKGROUND OF THE INVENTION The growth and development of the plant are governed by complex interactions between environmental signals and internal factors. Light regulates many development processes throughout the life cycle of the plant, from seed germination to flower induction (Chory, J. Trends Genet., 9: 161, 1993; McNellis and Deng, Plant Cell, 7: 1749, 1995), and causes profound morphological changes in young seedlings. In the presence of light, the growth of hypocotile is inhibited, cotyledons expand, leaves develop, chloroplasts differentiate, chlorophylls are produced, and many genes that can be induced by light are expressed in a coordinated manner. It has been suggested that plant hormones, which are known to affect the division, elongation, and differentiation of cells, are directly involved in the response of plants to light signals (PJ Davies, Plant Hormones: Physiology, Biochemistry and Molecular Biology, pages 1-836, 1995; Greef and Freddericq, Photomorphogenesis, pages 401-427, 1983). The interactions between the trajectories of phototransduction and the plant hormones, however, are not well understood. Brassinosteroids are a unique class of biologically active natural products, which possess highly specific activity and plant steroidal hormone activity. Their low effective concentrations for use in crops make them environmentally safe, and those brassinosteroids that are used on a large scale are generally non-toxic. At the physiological level, brassinosteroids produce many changes and may represent a new class of hormones in plants. The economic aspects of brassinosteroids can have global effects. The brassinosteroids can be used as protectors of the plant both against pesticides and against environmental adversity. In addition, it seems that brassinosteroids are important for insect control. In addition, brassinosteroids can regulate some stage of the reproductive cycle in plants, and other species, providing by means of the same the means to increase or decrease the reproductive process. For example, in certain horticultural crops it may be desirable to eliminate the flowering process to ensure the continued performance of other tissues such as leaves, bulbs, and other storage organs. This modulation of the reproductive process can be important in the control of certain weeds that contain seeds, where the suspension of the flowering cycle eliminates the following generations. It seems that brassinosteroids also stimulate the growth of the root, and external application does not cause any deformity of the plants. Brassinosteroids qualify for classification as biochemical pesticides. These pesticides are generally distinguished from conventional chemical pesticides by their unique modes of action, low effective concentration, target species, and specificity. Historically, brassinosteroids have not been used in real agricultural applications due to the expense involved in producing them, as well as the difficulty in their purification. Compendium of the Invention Although steroid hormones are important for animal development, the physiological role of plant steroids is largely unknown. The present invention is based on the discovery of the DET2 gene, which encodes a protein that shares significant sequence identity with the mammalian spheroid 5? F-reductase, and is involved in the biosynthetic path of brasinolide. A mutation of glutamate 204, which is required for human spheroid reductase activity, suppresses the in vivo activity of DET2, and leads to defects in light-regulated development. These defects can be improved by the application of the plant spheroid, brasinolide. In a first embodiment, the invention provides the DET2 polypeptide and the polynucleotide sequences encoding DET2. In another embodiment, the invention provides a method for producing a genetically modified plant, characterized in that it has an increased yield compared to a wild-type plant. The method includes transferring at least one copy of a polynucleotide encoding DET2, or a polynucleotide encoding another spheroid 5 -reductase (e.g., mammalian) operably associated with a promoter to a plant cell, to obtain a transformed plant cell, and produce a plant from the transformed plant cell. These genetically modified plants may exhibit increased yield of crop or increased biomass, for example. In still another embodiment of the invention, there is provided a method for producing a plant characterized in that it has an increased yield by contacting the plant having a native DET2 gene operably linked to its native promoter, with an amount that induces the promoter of an agent that induces the expression of the DET2 gene, wherein the induction of the expression of the DET2 gene results in the performance of a plant having an increased yield compared to a plant that does not come into contact with the agent of induction. Accordingly, transcription factors or chemical agents can be used to increase the expression of DET2 in a plant, in order to provide increased yield. Brief Description of the Drawings Figure 1 is a schematic illustration of the cloning and sequence analysis of the DET2 gene. Figure 1A shows a compendium of positional cloning. Three classes of cDNA from an Arabidopsis cDNA library with cosmid 217-61 were identified as a probe, and their relative positions and transcriptional directions (5 '- * 3') are indicated. Figure IB shows a map of the genetic structure of DET2 and mutations in the DET2 gene. The thick lines indicate the exons, and the open box denotes an intron. The positions of the mutations are relative to the initiation codon. Z, stop codon. Figure 1C shows the nucleotide and deduced amino acid sequence of DET2 (SEQ ID NO: 1 and 2, respectively). Figure 2 shows a photo of seedlings that grew by light, 12 days old (Figure 2A), and seedlings that grew in the dark of 10 days of age (Figure 2B), after complementation of det2 by the DET2 gene of wild type. (From left to right in each panel) Col-0 wild type, det2-l, and transgenic det2-l containing cosmid 217-61. Figure 3 shows a comparison of sequences of DET2 with mammalian spheroidal 5a-reductases. Figure 3A is the deduced amino acid sequence of the DET2 gene aligned with rat spheroidal 5a-reductases (rS5R1 and rS5R2) and human (hS5R1 and hS5R2). The dashes indicate introduced separations to maximize alignment, and the residues conserved in at least two of the five sequences are shaded. The arrow indicates the mutated glutamate in the alleles det2-l and det2-6. (The abbreviation of a single letter for amino acid residues are as follows: A, High; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, The; V, Val; , Trp; and Y, Tyr). Figure 3B shows a phylogenetic analysis of the relationship between the DET2 protein and the mammalian spheroidal 5a-reductases. The scale measures the relative distance between the sequences. Figure 4A shows the proposed function of the DET2 protein in the biosynthetic trajectory of brasinolide. The asterisk (*) indicates six intermediate steps (Fujicka, and collaborators, supra). Figure 4B shows Arabadopsis seedlings that grew in the dark, 10 days old, and Figure 4C shows seedlings that grew in light, 12 days old. From left to right, wild-type, det2-l, and det2-l plants treated with brasinolide are shown in each panel. Figure 4D shows an elongation of the hypocotyle induced by the brasinolide of the seedlings that grew in the dark and wild type plants that grew in light. The data represent the mean term ± SE obtained from triplicate determinations, each with an average sample size of 12 seedlings. Figure 5 shows the chemical reactions catalyzed by 5-mammalian spheroidalreductases (panel A) and the DET2 of Arabidopsis (panel B). Figure 6 shows that Arabidopsis DET2 has spheroidal 5-reductase activity. Thin-layer chromatography analysis of the steroids produced by incubating 64 nM of [14 C] progesterone with human 293 embryonic cells transfected with a pCMV5 expression plasmid containing human spheroidal 5a-reductase type 1 cDNA ( lane 1), no insert (lane 2), DET2 wild type (lane 3), or det2-l (lane 4). Figure 7 shows the biochemical characterization of spheroidal 5a-reductase activity of DET2. Figure 7A is a Lineweaver-Burk diagram for the [14C] testosterone. Figure 7B is a Lineweaver-Burk diagram for [14C] progesterone. Figure 7C shows the competitive inhibition of the enzyme activity of DET2 by 4-MA. Tests were performed on the activity of spheroidal 5α-reductase in the presence of the indicated concentration of 4-MA, and either 0.34 mM or 0.68 mM of [14] progesterone. The intersection of the two lines defines the K ± (Dixon, M. and Webb, E.C., (1979) Enzymes Academic, New York). Figure 7D shows a pH profile of the spheroidal 5c¿-reductase activity of DET2 using [14C] testosterone as a substrate. Tests were performed at the indicated pHs in the presence of 5 mg of cell lysis product protein, 1 J [M] of [14 C] testosterone, and 2.0 mM of NADPH for 20 minutes, at 37 ° C. Figure 8 shows that human spheroidal 5-reductase can complement the mutation det2-l. Figure 8A shows the schematic representations of the pMD-hS5R expression plasmids used to transform the det2-l mutants. Two constructs were made for each type of human spheroidal 5-reductase cDNA: a shorter one containing a truncated region not translated in 3 ', and a longer one containing full-length human cDNA. The indicated DNA fragments are human spheroidal 5a-reductase (hS5R), nopaline synthase promoter (NosPro) and transcriptional terminator (Nos-ter), neomycin phosphotransferase (NPTII) gene, 35S promoter of cauliflower mosaic virus ( 35Spro), and multiple cloning sites (MCS). Figure 8B-D shows the complementation of the det2-l mutation by the human spheroidal 5a-reductase cDNA. Figure 8B shows seedlings that grew in the dark, 7 days old. Figure 8C shows seedlings that grew in the light, 7 days old. Figure 8D shows seedlings that grew in light, 3 weeks old. (From left to right in panel B, C, and D) Col-0 wild-type, transgenic det2-l containing the full-length human spheroidal 5-crescent type 1 cDNA • ta, and transgenic det2-l containing full length human spheroidal 5o-reductase type 2 cDNA. Figure 9 shows the effect of 4-MA on the hypocotyl lengths of plants of det2-l, wild type and transgenic det2-l. The data represent the mean term ± SE obtained from 25 seedlings of each genotype. Figure 10 shows that the level of expression of human spheroidal 5a-reductase l ^ L cDNA is correlated with the phenotype in transgenic plants of det2-l. Figure 10A shows the length of the hypocotileo of seedlings that grew in the dark of wild-type Col-0, det-2 and the segregation progeny of primary transgenic det-2-l plants. The data represent the mean term ± SE obtained from a population with an average sample size of 60 seedlings. Figure 10B shows a Northern blot analysis of transgene expression. Each lane contained 20 milligrams of RNA or total plant. Figure 10C shows the morphology of 14-day-old seedlings that grew in light. (From left to right in each panel), 1, det2-l; 2, hS5Rl-1 .3kb (04); 3, hS5Rl- 1.3kb (08); 4, hS5Rl-2. lkb (04); 5, hS5Rl-2. lkb (17); 6, wild type 5; 7, hS5R2-0.8kb (01); 8, hS5R2-0.8kb (12); 9, hS5R2-0.8kb (15); 10, hS5R2-2.4kb (04); 11, hS5R2-2.4kb (05); 12, hS5R2- 2.4kb (12). DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides a novel spheroid 5 -reductase, DET2, which is involved in the synthesis of spheroidal plant hormone, brasinolide. Overexpression of the DET2 reductase or mammalian spheroid 5 -reductase (eg, human, monkey, rat, mouse) in transgenic plants causes these plants to become significantly longer and more robust than their wild-type counterparts, increasing this way the productions of the plants. As used herein, the term "yield" or "plant yield" refers to the increased growth of the plant, the increased growth of the crop, and / or the increased production of biomass. In a first embodiment, the present invention provides a substantially pure DET2 polypeptide. The DET2 polypeptide is exemplified by the amino acid sequence shown in Figure 1C and SEQ ID NO: 2. The DET2 polypeptide is characterized as having a predicted molecular weight of 31 kDa, as determined by polyacrylamide gel electrophoresis. of sodium dodecyl sulfate, which has spheroidal 5a-reductase activity, and functioning in the biosynthetic path of brasinolide. The deduced amino acid sequence of the DET2 gene is similar to that of the 5-spheroid mammalian reductase, with 38 to 42 percent sequence identity. The sequence similarity is increased to 54 to 60 percent when conservative substitutions are taken into account. Two isozymes (types 1 and 2) of spheroidal 5a-reductase have been isolated in rats and humans (Wilson, et al., Endocr. Rev., 1.4: 577, 1993, Russell and Wilson, Annu., Rev. Biochem., 63. : 25, 1994). Phylogenetic analysis shows that the DET2 is at least as closely related to the type 2 enzymes, as the type 2 enzymes are related to the type 1 enzymes. The term "substantially pure" as used herein, refers to the DET2 polypeptide that is substantially free of other proteins, lipids, carbohydrates, or other materials with which it is naturally associated, one skilled in the art can purify DET2 using standard techniques for protein purification. The substantially pure polypeptide will yield a single band greater than about 31kD in a denaturing polyacrylamide gel. The purity of the DET2 polypeptide can also be determined by amino acid sequence analysis with amino terminal. The invention includes the functional DET2 polypeptide, and functional fragments thereof. As used herein, the term "functional polypeptide" refers to a polypeptide that possesses biological function or activity that is identified through a defined functional assay, and that is associated with a particular biological, morphological, or phenotypic alteration in the cell. The term "functional fragments of the DET2 polypeptide", refers to all fragments of the DET2 that retain the activity of the DET2, for example, spheroidal 5-reductase 5 activity. Biologically functional fragments, for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule, to a large polypeptide capable of participating in the induction or programming characteristic of phenotypic changes within a cell. The spheroidal 5a-reductase activity of DET2, and its role in the biosynthetic path of brasinolide, can be used in bioassays to identify biologically active fragments of the DET2 polypeptide or related polypeptides. For example, DET2 can catalyze the conversion of campesterol to campestanol, therefore, an assay can be performed to detect the enzymatic activity of DET2. DET2 inhibitors can be used to cause loss of DET2 function, resulting in, for example, sterile male or female plants, reduced stature, and so on. For example, the inhibition of DET2 is useful in horticulture to create dwarf varieties. Minor modifications of the primary amino acid sequence of DET2 can result in proteins having activity substantially equivalent to the DET2 polypeptide described herein in SEQ ID NO: 2 (Figure 1C). These modifications can be deliberate, such as through site-directed mutagenesis, or they can be spontaneous. All polypeptides produced by these modifications are included herein as long as the biological activity of the DET2 is present, for example, the activity of spheroidal 5α-reductase is present to promote the increased yield and / or biomass of the plant or crop . In addition, the deletion of one or more amino acids can also result in a modification of the structure of the resulting molecule, without significantly altering its activity. This may lead to the development of a smaller active molecule that may have wider utility. For example, it may be possible to remove amino acids with amino or carboxy termini that are required for DET2 activity. The DET2 polypeptide includes amino acid sequences substantially the same as the sequence set forth in SEQ ID NO: 2. The term "substantially equal" refers to amino acid sequences that retain the activity of DET2 as described herein, example, spheroidal 5a-reductase activity. The DET2 polypeptides of the invention include conservative variations of the polypeptide sequence. The term "conservative variation" as used herein, denotes the replacement of an amino acid residue with another, biologically similar residue. Examples of conservative variations include the substitution of a hydrophobic residue such as isoieucine, valine, leucine, or methionine, on the other, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted mother amino acid, with the proviso that antibodies raised for the substituted polypeptide also immunoreact with the unsubstituted polypeptide. The proteins of the invention can be analyzed by standard sodium dodecylsulfate polyacrylamide gel electrophoresis and / or immunoprecipitation analysis and / or Western blot analysis, for example. In addition, the in vi tro synthesized protein assay (IVS), as described in the present examples, can be used to analyze the product of the DET2 protein. The invention also provides an isolated polynucleotide sequence encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The DET2 gene has been mapped to a range of 150-kb on chromosome 2 of Arabidopsis. The DET2 transcript contains a single long open reading frame that encodes a protein of 262 amino acids. The term "isolated" as used herein, includes polynucleotides substantially free of other nucleic acids, proteins, lipids, carbohydrates or other materials with which they are naturally associated. The polynucleotide sequences of the invention include DNA, cDNA and RNA sequences encoding DET2. It is understood that polynucleotides that encode all the variable portions or portions of DET2 are included herein, as long as they encode a polypeptide with DET2 activity. These polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides, as well as splice variants. For example, portions of the mRNA sequence may be altered due to alternative RNA splicing patterns, or to the use of alternating promoters for RNA transcription. On the other hand, the DET2 polynucleotides of the invention include polynucleotides having alterations in the nucleic acid sequence that still encode functional DET2. Alterations in the nucleic acid of DET2 include, but are not limited to, intragenetic mutations (eg, dot mutation, nonsense (stop), anti-sense, splice site, and frame change) and heteroclot deletions or homocs. Detection of such alterations can be made by standard methods known to those of skill in the art, including sequence analysis, Southern blot analysis, polymerase chain reaction based assays (e.g., multiple polymerase chain reaction , labeled sites of sequence (STSs)) and in situ hybridization. The polynucleotide sequences of the invention also include anti-sense sequences. The polynucleotides of the invention include sequences that degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences are included in the invention, as long as the amino acid sequence of the DET2 polypeptide encoded by those nucleotide sequences retains the spheroidal 5α-F-reductase activity of DET2. A "functional polynucleotide" denotes a polynucleotide that encodes a functional polypeptide as described herein. In addition, the invention also includes a polynucleotide that encodes a polypeptide having the biological activity of an amino acid sequence of SEQ ID NO: 2, and having at least one epitope for an antibody immunoreactive with the DET2 polypeptide. As used herein, the terms polynucleotide and nucleic acid sequences of the invention refer to DNA, RNA and cDNA sequences. The polynucleotide encoding DET2 includes the nucleotide sequence in Figure 1C (SEQ ID NO: 1), as well as the complementary nucleic acid sequences for that sequence. A complementary sequence may include an anti-sense nucleotide. When the sequence is RNA, the deoxyribonucleotides A, G, C and T of Figure 1C are replaced by ribonucleotides A, G, C and U, respectively. Also included in the invention are fragments ("probes") of the nucleic acid sequences described above that are at least 15 bases in length, which is sufficient to allow the probe to selectively hybridize to DNA encoding the protein of Figure 1C (SEQ ID NO: 2). "Selective hybridization" as used herein refers to hybridization under moderately stringent or highly stringent physiological conditions. (See, for example, the techniques described in Maniatis et al., 1989 Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., incorporated herein by reference), which distinguishes the nucleotide sequences of related DET2 from the unrelated ones. In nucleic acid hybridization reactions, the conditions that are used to achieve a particular level of stringency will vary depending on the nature of the nucleic acids being hybridized. For example, the length, the degree of complementarity, the composition of the nucleotide sequence (e.g., GC v. AT content), and the type of nucleic acid (e.g., RNA and DNA) of the regions can be considered. of hybridization of nucleic acids, when selecting the hybridization conditions. A further consideration is whether one of the nucleic acids is immobilized, for example, in a filter. An example of progressively higher stringency conditions is as follows: 2 x SSC / 0.1% SDS at about room temperature (hybridization conditions); 0.2 x SSC / 0.1% SDS at approximately room temperature (low stringency conditions); 0.2 x SSC / 0.1% SDS at approximately 42 ° C (moderate stringency conditions); and 0.1 x SSC at approximately 68 ° C (high stringency conditions). Washing can be performed using only one of these conditions, for example, high stringency conditions, or each of the conditions can be used, for example, for 10-15 minutes each, in the order listed above, repeating either or all steps listed. However, as mentioned above, optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically. A cDNA sequence for DET2 is specifically described herein. Figure 1C shows the complete and deduced cDNA protein sequences (SEQ ID NO: 1 and 2, respectively). The DNA sequences of the invention can be obtained by many methods. For example, DNA can be isolated using hybridization or computer-based techniques that are well known in the art. Such techniques include, but are not limited to: 1) hybridization of genomic or cDNA libraries with probes to detect homologous nucleotide sequences; 2) Antibody classification of the expression libraries to detect cloned DNA fragments with shared structural characteristics; 3) polymerase chain reaction (PCR) in genomic DNA or cDNA, using primers capable of annealing to the DNA sequence of interest; 4) computer searches of the sequence databases for similar sequences; and 5) differential classification of a subtracted DNA library. 5 Classification procedures that depend on nucleic acid hybridization make it possible to isolate any gene sequence from any organism, provided that the appropriate probe is available. Oligonucleotide probes, which correspond to a part of the DET2 lft sequence encoding the protein in question, can be chemically synthesized. This requires that short elongations of the oligopeptide of the amino acid sequence be known. The DNA sequence that encodes the protein can be deduced from the genetic code, however, it must be taken into account the degeneracy of the code. When the sequence is degenerate it is possible to perform a mixed addition reaction. This includes a heterologous mixture of denatured double-stranded DNA. For this classification, hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. Hybridization is particularly useful in the detection of derived cDNA clones from sources where an extremely low amount of mRNA sequences related to the polypeptide of interest is present. In other words, by using stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow autoradiographic visualization of a specific cDNA clone by hybridizing the target DNA to that individual probe in the mixture that is its complete complement (Wallace, et al, Nucí, Acid Res., 9.:879, 1981). Alternatively, a library that tends to decrease, as illustrated herein, is useful for the removal of nonspecific cDNA clones. Among standard procedures for isolating cDNA sequences of interest is the formation of cDNA libraries containing plasmid or phage, which are derived from the reverse transcription of mRNA that is abundant in donor cells that have a high level of gene expression . When used in combination with polymerase chain reaction technology, up to rare expression products can be cloned. In those cases where significant portions of the amino acid sequence of the polypeptide are known, the production of labeled single or double-stranded DNA or RNA probe sequences, which duplicate a putatively present sequence in the target cDNA, can be employed. , in DNA / DNA hybridization procedures performed on cloned copies of the cDNA that has been denatured to a single chain form (Jay, et al., Nucí, Acid Res., 1.1: 2325, 1983). A cDNA expression library, such as lambda gtll, can be indirectly classified for the DET2 peptides, using antibodies specific for DET2. These antibodies can be derived either polyclonally or monoclonally, and used to detect the expression product that indicates the presence of DET2 cDNA. DNA sequences encoding DET2, or other 5α-5 reductases, can be expressed in vitro by transferring DNA to a suitable host cell. "Host cells" are cells in which a vector and its expressed DNA can be propagated. The term also includes any progeny or grafting material, for example, of the subject host cell. 1f0 It is understood that all the progeny may not be identical to the stem cell, since there may be mutations that occur during the replication. However, that progeny is included when the term "host cell" is used. Stable transfer methods are known in the art, meaning that foreign DNA is continuously maintained in the host. In the present invention, the DET2 or other polynucleotide (e.g., spheroidal 5a-reductase of ^ mammalian) sequences, within a recombinant expression vector. The terms "recombinant expression vector" or "vector "Expression vectors" refer to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the genetic sequence of DET2 These expression vectors contain a promoter sequence that facilitates efficient transcription of the sequence of DET2 inserted. The expression vector typically contains a replication origin, a promoter, as well as specific genes that allow phenotypic selection of the transformed cells. Methods that are well known to those skilled in the art can be used to construct expression vectors containing the coding sequence of DET2., or for example, for the mammalian spheroidal 5a-reductase coding sequence, and appropriate transcriptional / translational control signals. These methods include recombinant DNA techniques in vitro, synthetic techniques, and recombinant / genetic techniques in vivo. A variety of host expression vector systems can be used to express the coding sequence of DET2. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage DNA expression vectors, plasmid DNA or cosmid DNA, which contain the coding sequence of D? T2; yeast transformed with recombinant yeast expression vectors containing the coding sequence of DET2; plant cell systems infected with recombinant virus expression vectors (eg, cauliflower mosaic virus, cauliflower mosaic virus, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (eg, plasmid) Ti) containing the coding sequence of DET2; insect cell systems infected with recombinant virus expression vectors (eg, baculovirus) that contain the coding sequence of DET2; or animal cell systems infected with recombinant virus expression vectors (e.g., retroviruses, adenoviruses, vacciniaviruses) that contain the DET2 coding sequence, or animal cell systems designed for stable expression. Depending on the host / vector system used, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc., may be used in the expression vector (see , for example, Bitter et al., 1987, Methods in Enzymology 153: 516-544). For example, when cloning into bacterial systems, inducible promoters such as pL of the bacteriophage α, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used. When cloning into mammalian cell systems, promoters derived from the genome of mammalian cells (eg, metallothionein promoter) or from mammalian virus (eg, the long terminal repeat of the retrovirus; adenovirus; the 7.5K vacciniavirus promoter). Promoters produced by recombinant DNA or synthetic techniques can also be used to allow transcription of the inserted DET2 coding sequence. The isolation and purification of the recombinantly expressed polypeptide, or fragments thereof, provided by the invention, can be carried out by conventional means including preparation chromatography and immunological separations involving monoclonal or polyclonal antibodies. The invention also includes immunoreactive antibodies to the DET2 polypeptide or antigenic fragments thereof. The antibody is provided consisting essentially of clustered monoclonal antibodies with different epitopic specificities, as well as different monoclonal antibody preparations. Monoclonal antibodies are made from antigen containing fragments of the protein, by methods well known to those skilled in the art.

(Kohler, et al., Na ture, 256: 495, 1975). The term "antibody" as used in this invention includes intact molecules as well as fragments thereof, such as Fab, F (ab ') 2, and Fv which are capable of binding to an epitope determinant present in the DET2 polypeptide. These antibody fragments retain some ability to selectively bind with their antigen or receptor. The methods for making these fragments are known in the art. (See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988), incorporated herein by reference). As used in this invention, the term "epitope" refers to an antigenic determinant in an antigen to which the paratope of an antibody is fixed. Epitope determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Antibodies that bind to the DET2 polypeptide of the invention can be prepared using an intact polypeptide or fragments containing small peptides of interest as the immunization antigen. For example, it may be desirable to produce antibodies that specifically bind to the N or C terminal domains of DET2. The polypeptide or peptide that is used to immunize an animal, which is derived from translated or chemically synthesized cDNA that can be conjugated to a carrier protein, if desired. These commonly used carriers that chemically attach to the immunization peptide include orifice limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. The polyclonal or monoclonal antibodies of the invention can be further purified, for example, by binding to, and leaching from, a matrix to which the polypeptide or a peptide for which the antibodies arose is attached. Those of skill in the art will know different techniques common in the immunology art for the purifi cation and / or concentration of polyclonal antibodies, as well as monoclonal antibodies (See for example, Coligan, et al., Unit 9, Current Protocols in Immunology. , Wiley Interscien-ce, 1994, incorporated as reference). It is also possible to use anti-idiotype technology to produce the monoclonal antibodies of the invention that mimic an epitope. For example, an anti-idiotypic monoclonal antibody made for a first monoclonal antibody will have a binding domain in the hypervariable region which is the "image" of the epitope fixed by the first monoclonal antibody. In another embodiment, the invention provides a method for producing a genetically modified plant, characterized as having an increased yield as compared to a plant that has not been genetically modified (eg, a wild-type plant). The term "yield" has been previously defined herein. The method includes the steps of contacting a plant cell with at least one vector that contains at least one nucleic acid sequence encoding a spheroid 5 -reductase, e.g., DET2, or a mammalian enzyme (e.g. spheroid 5a-reductase type 1 or human type 2), wherein the nucleic acid sequence is operably associated with a promoter, to obtain a transformed plant cell; produce a plant from the transformed plant cell; and after that, select a plant that exhibits increased yield.

The term "genetic modification" as used herein, refers to the introduction of one or more heterologous nucleic acid sequences, for example, the sequence encoding DET2 or 5 -reductase type 1 or human type 2, within of one or more plant cells, which can generate viable, complete, sexually competent plants. The term "genetically modified" as used herein, refers to a plant that has been generated through the process mentioned above. The genetically modified plants of the invention are capable of self-pollination or cross-pollination with other plants of the same species, in such a way that the foreign gene, contained in the germline, can be inserted or reproduced within agriculturally useful plant varieties. The term "plant cell" as used herein, refers to protoplasts, gamete producing cells, and cells that regenerate into whole plants. In accordance with the foregoing, the definition of "plant cells" includes a seed comprising multiple plant cells capable of regeneration in a whole plant. As used herein, the term "plant" refers to either a whole plant, a part of the plant, a plant cell, or a group of plant cells, such as plant tissue, for example. Plantlets are also included within the meaning of "plant". The plants included in the invention are any plants willing to accept transformation techniques, including angiosperms, gymnosperms, monocotyledons and dicots. Examples of monocotyledonous plants include, but are not limited to, asparagus, forage and sweet corn, barley, wheat, rice, sorghum, onion, pearl millet, rye and oats. Examples of dicotyledonous plants include, but are not limited to, tomato, tobacco, cotton, rapeseed, field beans, soybeans, peppers, lettuce, peas, alfalfa, clover, cabbage crops or Brassica oleracea (for example, repo). -lio, broccoli, cauliflower, Brussels sprouts), radish, carrot, beets, eggplant, spinach, cucumber, chayote, melons, cantaloupe, sunflowers and different ornamental plants. Wood species include poplar, pine, redwood, cedar, oak, and so on. The term "heterologous nucleic acid sequence" as used herein, refers to a foreign nucleic acid for the host host plant, or native to the host if the native nucleic acid is substantially modified from its original form. For example, the term includes a nucleic acid that originates from the host species, wherein the sequence is operably linked to a promoter that differs from the wild-type or natural promoter. In the broad method of the invention, at least one nucleic acid sequence encoding DET2 or another spheroid 5a-reductase (e.g., mammalian enzyme) is operably linked to a promoter. It may be desirable to introduce more than one copy of DET2 or another spheroid 5a-reductase polynucleotide into a plant, for enhanced expression of the corresponding polypeptide. For example, multiple copies of the DET2 gene would have the effect of increasing the production of DET2 in the plant. It should be understood that the nucleic acid sequences encoding the spheroid 5-reductase refers to human type 2 or type 2 DET2, 5a-reductase, and other spheroidal 5α-reductases. The method of the invention also includes any combination of these enzymes. The genetically modified plants of the present invention are produced by contacting a plant cell with a vector that includes at least one nucleic acid sequence encoding DET2. To be effective once introduced into the plant cells, the nucleic acid sequence of DET2 must be operably associated with a promoter that is effective in the plant cells, to cause the transcription of DET2. Additionally, a polyadenylation sequence or transcription control sequence, also recognized in plant cells, can also be employed. It is preferred that the vector harboring the nucleic acid sequence to be inserted also contains one or more selectable marker genes., such that transformed cells can be selected from untransformed cells in culture, as described herein.

The term "operably associated" refers to the functional link between a promoter sequence and the nucleic acid sequence of DET2 regulated by the promoter. The operably linked promoter controls the expression of the nucleic acid sequence of DET2. The expression of the structural genes used in the present invention can be promoted by many promoters. Although the endogenous promoter, or native of a structural gene of interest for the transcriptional regulation of the gene, can be used, preferably, the promoter is a foreign regulatory sequence. For plant expression vectors, suitable viral promoters include the 19S RNA and 19S RNA promoters of cauliflower mosaic virus (Brisson, et al., Na ture, 310: 511, 1984; Odell, et al., Na ture, 313: 810, 1985); the full-length transcript promoter from Scrofular Mosaic Virus (FMV) (Gowda, et al., J. Cell Biochem., 13D: 301, 1989) and the shell protein promoter for TMV (Takamatsu, et al., EMBO J. 6: 307, 1987). Alternatively, plant promoters such as the light-inducible promoter can be used from the small subunit of ribulose bisphosphate carboxylase (ssRUBISCO) (Coruz-zi, et al., EMBO J., 3: 1671, 1984; Broglie, et al., Science, 224: 838, 1984); Manopine synthase promoter (Velten, et al., EMBO J., 3.:2723, 1984); nopaline synthase (NOS) and octopine synthase (OCS) promoters (contained in Agrobacterium tumefaciens tumor induction plasmids) or heat shock promoters, eg, soybean hspl7.5-E or hspl7.3-B ( Gurley, et al., Mol. Cell, Biol., 6: 559, 1986; Severin, et al., Plant Mol. Biol., 1_5: 827, 1990). Promoters useful in the invention include both constitutive and inducible natural promoters, as well as designed promoters. Cauliflower mosaic virus promoters are examples of constitutive promoters. To be more useful, an inducible promoter must 1) provide low expression I (j ^ in the absence of the inducer; 2) provide high expression in the presence of the inducer; 3) use an induction scheme that does not interfere with the normal physiology of the plant; 4) have no effect on the expression of other genes. Examples of inducible promoters useful in plants include those induced by chemical means, such as the promoter of yeast halothionein which is activated by copper ions (Mett, et al., Proc. Nati. Acad. Sci., USA, .90.: 4567, 1993); regulatory sequences In2-1 and In2-2 which are activated by substituted benzenesulfonamides, for example, herbicidal insurers (Hershey, et al., Plant Mol. Biol., 17: 679, 1991); and the GRE regulatory sequences that are induced by glucocorticoids (Schena, et al., Proc. Nati, Acad. Sci., U. S.A., 8: 10421, 1991). Other promoters, both constitutive 5 and inducible, will be known to those of skill in the art.

The particular promoter selected must be capable of causing sufficient expression to result in the production of an effective amount of structural genetic product, e.g., DET2, to cause increased yield and / or increased biomass. If desired, the promoters that are used in the constructions of the vector of the present invention can be modified to affect their control characteristics. Tissue-specific promoters can also be used in the present invention. An example of a tissue-specific promoter is the active promoter in firing meristems (Atanassova, et al., Plant J., 2: 291, 1992). For those of skill in the art other tissue-specific promoters useful in transgenic plants will be known, including the cdc2a promoter and the cyc07 promoter. (See, for example, Ito, et al., Plant Mol. Biol., 24: 863, 1994; Martínez, and collaborators, Proc. Nati Acad. Sci. USA, 89: 7360, 1992; Medford, et al., Plant Cell, 3: 359, 1991; Terada, et al., Plant Journal, 3: 241, 1993; Wissenbach, et al., Plant Journal, 4: 411, 1993). Optionally, a selectable marker may be associated with the nucleic acid sequence to be inserted. As used herein, the term "marker" refers to a gene that encodes a trait or phenotype that allows the selection of, or classification for, a plant or plant cell that contains the marker. Preferably, the marker gene is an antibiotic resistance gene by which the appropriate antibiotic can be used to select for the transformed cells from among the cells that are not transformed. Examples of suitable selectable markers include adenosine aminase, dihydro-folate reductase, hygromycin B-phosphotransferase, thymidine kinase, xanthine-guanine phospho-ribosyltransferase, and aminoglycoside 3'-O-phosphotransferase-II (kanamycin, neomycin and resistance G418). Other suitable markers will be known to those skilled in the art. Although the following descriptions describe DET2, it is understood that this is an exemplary 5a-reductase, and mammalian 5a-reductases or others are also included in the following descriptions. The vector (s) employed in the present invention for the transformation of a plant cell includes (n) a nucleic acid sequence encoding DET2, operably associated with a promoter. To begin a transformation process in accordance with the present invention, it is first necessary to construct a suitable vector and properly introduce it into the plant cell. Those of the construction vectors used herein are known to those skilled in the art of plant genetic engineering. The nucleic acid sequences of DET2 used in the present invention can be introduced into the plant cells using Ti plasmids from Agrobacterium tumefaciens, root induction plasmids (Ri), and plant virus vectors. (For reviews of these techniques see, for example, Weissbach and Weissbach, 1988, Methods for Plant Molecular Biology, Academic 5 Press, NY, Section VIII, pages 421-463, and Grierson and Corey, 1988, Plant Molecular Biology, Second Edition , Blackie, London, Chapters 7-9, and Horsch, et al., Science, 227: 1229, 1985, incorporated herein by reference). In addition to the transformation vectors of derived plants from HL the Ti or root induction (Ri) plasmids of Agrobacterium, alternative methods may involve, for example, the use of liposomes, electroporation, chemicals that increase the uptake of free DNA, transformation using viruses or pollen, and the use of microprojection One of ordinary skill in the art will be able to select an appropriate vector to introduce the encoding nucleic acid sequence DET2 in a relatively intact condition. This ^^ way, any vector that produces a plant that carries the introduced DNA sequence should be sufficient, one would expect that even the use of a naked piece of DNA conferred properties of this invention, typically guided by the selected transformation method. The transformation of plants according to the invention and essentially any of the different ways known to those skilled in the art of plant molecular biology. (See, for example, Methods of Enzymology, volume 153, 1987, Wu and Grossman, editors, Academic Press, incorporated herein by reference). As used herein, the term "transformation" means the alteration of the genotype of a host plant by the introduction of the nucleic acid sequence DET2. For example, a DET2 nucleic acid sequence can be introduced into a plant cell using the Agrobacterium tumefaciens containing the Ti plasmid, as briefly mentioned above. When using a culture of A. tumefaciens as a transformation vehicle, it is most advantageous to use a non-oncogenic strain of Agrobacterium as the carrier of the vector so that the non-oncogenic differentiation of the transformed tissues is possible. It is also preferred that the Agrobacterium harbor a binary system of the Ti plasmid. This binary system comprises 1) a first Ti plasmid having a virulence region essential for the introduction of the transfer DNA (T-DNA) into the plants, and 2) a chimeric plasmid. The latter contains at least one boundary region of the T-DNA region of a wild-type Ti plasmid flanking the nucleic acid to be transferred. The binary systems of the Ti plasmid have been shown to be efficient in transforming plant cells (De Framond, Biotechno-J-ogy, JL: 262, 1983, Hockema et al., Nature, 303: 179, 1983). This binary system is preferred because it does not require integration into the Ti plasmid of the Agrobacterium, which is an older methodology. Methods that include the use of Agrobacterium in the transformation in accordance with the present invention, include, but are not limited to: 1) Agrobacterium co-culture with isolated cultured protoplasts; 2) transformation of plant cells or tissues with the Agrobacterium; or 3) transformation of the seeds, apices or meristems with the Agrobacterium. In addition, gene transfer can be achieved by in-plant transformation by Agrobacterium, as described by Bechtold et al. (C. R. Acad. Sci. Paris, 316: 1194, 1993) and as exemplified in the Examples in the present. This approach is based on the vacuum infiltration of a suspension of Agrobacterium cells. The preferred method for introducing DTE2 coding nucleic acid into plant cells is to infect these plant cells, an explant, a meristem or a seed, with the transformed Agrobacterium tumefaciens, as described above. Under the appropriate conditions known in the art, the transformed cells of the plant are grown to form suckers, roots, and further developed within the plant. Alternatively, DTE2 coding nucleic acid sequences can be introduced into a plant cell using mechanical or chemical means. For example, nucleic acid can be mechanically transferred into the cell of the plant by microinjection, using a micropipette. Alternatively, the nucleic acid can be transferred into the cell of the plant using polyethylene glycol which forms a precipitation complex with genetic material that is taken up by the cell. Sequences of DTE2 encoding nucleic acid can also be introduced into a plant cell by electroporation (Fromm et al, Proc.Nat.Acid.Sci., EUA, 82.:5824, 1985, which is incorporated herein by reference). reference). In this technique, the protoplasts of the plant are electroporated in the presence of the nucleic acids containing the relevant nucleic acid sequences. The electric impulses of high field resistance, reversibly permebeali-z membranes, allowing the introduction of nucleic acids. The protoplasts of the plant that were passed through electroporation reform the cell wall, divide and form a plant callus. The selection of the transformed cells of the plant with the transformed gene can be achieved using phenotypic markers, as described herein. Another method for introducing the DET2 nucleic acid into a plant cell is the high speed ballistic penetration by small particles with the nucleic acid to be introduced content either within the matrix of these particles, or on the surface of the same ones (Klein et al., Nature, 327: 70, 1987). Methods of bombardment transformation are also described in Sandford et al. (Techniques 3: 3-16, 1991) and in Klein et al. (Bio / Techniques, 1_0: 286, 1992). Although, typically only a single introduction of a new nucleic acid sequence is required, this method provides in particular for multiple introductions. Coli-flower mosaic virus (CaMV) can also be used as a vector for introducing nucleic acid into the cells of the plant (U.S. Pat. No. 4,407,956). The viral DNA genome of the cauliflower mosaic virus is inserted into a parent bacterial plasmid, creating a recombinant DNA molecule which can be propagated in the bacteria. After cloning, the recombinant plasmid can be cloned again and further modified by introducing the desired nucleic acid sequence. The modified viral portion of the recombinant plasmid of the parent bacterial plasmid is then excised and used to inoculate the cells of the plant or plants. As used herein, the term "contacting" refers to any means of introducing the DET2 into a plant cell, including chemical and physical means, as described above. Preferably, contacting refers to the introduction of the nucleic acid or vector into the cells of the plant (including an explantation, a meristem or a seed), by means of the Agrobacterium tumefaciens transformed with the nucleic acid encoding DET2, as described previously. 5 Normally, a plant cell is regenerated to obtain a complete plant from the transformation process. The immediate product of the transformation is referred to as a "transgenote". The term "cultivar" or "regeneration" as used herein, means growing a plant H completes from a plant cell, a group of plant cells, a plant part (including the seeds), or a piece of plant (for example, from a part of protoplast, callus, or tissue ). The regeneration from the protoplast varies from species to species of plants, but generally a suspension of protoplasts is first made. In some species, the formation of the embryo can be induced, from the suspension of the protoplast, to the stage of maturation and germination as in natural embryos. The culture medium will contain generally different amino acids and hormones, necessary for growth and regeneration. Examples of the hormones that are used include auxins and cytokinins. Sometimes it is advantageous to add glutamic acid and proline to the medium, especially for plant species such as corn and alfalfa.

Efficient regeneration will depend on the medium, the genotype, and the history of the crop. If these variables are controlled, regeneration can be reproduced. Regeneration also occurs from the callus, explantations, organs or parts of the plant. The transformation can be carried out in the context of the regeneration of the organ or part of the plant. (See Methods in Enzymology, volume 118 and Klee et al., Annual Review of Plant Physiology, 38: 467, 1987). When using the leaf-transformation-regeneration disc method of Horsch et al., Science, 227: 1229, 1985, the discs are cultured in a select medium, followed by shoot formation in about 2-4 weeks. The shoots that develop from the calluses are removed and transplanted to an appropriate root inducer selected. The seedlings are transplanted to the ground as soon as possible after the root appears. The seedlings can be potted again as needed until they reach maturity. In crops that are vegetatively propagated, mature transgenic plants are propagated by using cuts or tissue culture techniques to produce multiple identical plants. The selection of desirable transgenotes is made and new varieties are obtained and propagated vegetatively for commercial use. In crops that are propagated by seed, transgenic plants can cross themselves to produce a homozygous inbred plant. The resulting inbred plant produces seed that contains the newly introduced gene (s). These seeds can be grown to produce plants that would produce the selected phenotype, for example, increased production. The parts that are obtained from the regenerated plant, such as flowers, seeds, leaves, branches, roots, fruits, and the like are included in the invention, as long as these parts comprise the introduced nucleic acid sequences. Plants that exhibit production or biomass compared to wild-type plants can be selected by visual observation. The invention includes plants produced by the method of the invention, as well as plant tissue and seeds. In still another embodiment, the invention provides a method for producing a genetically modified plant cell, such that a plant that is produced from this cell produces increased yield compared to a wild-type plant. The method includes contacting the plant cell with a DET2 nucleic acid sequence to obtain a transformed plant cell.; the cultivation of the transformed cell of plant under conditions of plant formation to obtain a plant that has increased production. Conditions such as environmental and promoter inducing conditions vary from species to species, but should be the same within a species. In another embodiment, the invention provides a method for producing a plant characterized in that it has increased production by contacting a susceptible plant with a DET2 promoter-inducing amount of an agent that induces expression of the DET2 gene, wherein the induction compared to a plant that has not contacted the agent. A "susceptible plant" refers to a plant that can be induced to use its endogenous D? T2 gene to achieve increased production. The term "promoter-inducing amount" refers to the amount of an agent, necessary to elevate the expression of the DET2 gene above the expression of DET2 in a plant cell that has not been contacted with the agent. For example, a transcription factor or a chemical agent can be used to elevate the expression of the gene from the innate DET2 promoter, thereby inducing the promoter and expression of the DET2 gene. In yet another aspect of the invention, it is contemplated that increased expression of DET2 or other 5α-reductase steroids (eg, mammal) in a plant cell or in a plant increases the resistance of that cell / plant to the pests of the plant or the pathogens of the plant. For example, field studies have shown that brasinolides are effective as pesticides, therefore, increased expression of DET2 or other 5o-reductases would result in increased amounts of brasinolide in the plant. In addition, increased expression of DET2 or other 5-α-reductases may also cause resistance to pesticides (insurers). Therefore, the DET2 or other 5a-reductases, protect plants against pests, as well as against pesticides. The above description generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples, which are provided for purposes of illustration only and are not intended to limit the scope of the invention.

Examples Previously, Arabidopsis mutants having the characteristics of light culture plants still grown in the dark have been isolated. At least 11 of these gene sites have been identified, known as det, cop and fus (Bowler and Chua, Plant Cell, 6: 1529, 1994). Analyzes of double mutants with different photoreceptor mutants suggest that DET1, COP1, and COP9 act on a signal transduction path, whereas DET2 acts on a different trajectory (Bowler and Chua, supra). DETl, COPl, and C0P9 encode the localized nuclear proteins the way of acting of which is still not understood (Bowler and Chua, supra). The loss-of-function mutations in the DET2 have pleiotropic effects (Chory et al., Ibid, 3 .: 445, 1991). In the dark, det2 mutants are short, have thick hypocotyls, accumulate anthocyanins, have open, expanded cotyledons, and develop primary leaf buds. These morphological changes are accompanied by a depression of 10 to 20 parts of different genes responsive to light. In light, the det2 mutants are smaller and darker green than the wild type, show reduced cell size in the tissues examined (hypocotyl, cotyledons, and leaves), and have reduced apical predominance and male fertility. Det2 mutations also affect photoperiodic responses and cause a delay in flowering, a shortening of the circa-target period of CAB gene expression (chlorophyll a / b binding proteins), inappropriate regulation of day and night of expression of the gene, and a delay in leaf and chloroplast senescence. (Chory et al., Supra; chory and collaborators, Plant physiol., 104: 339, 1994; Millar et al., Science, 267: 1163, 1995). These phenotype differences show that DET2 plays an important role throughout the development of Arabidopsis. Example 1 The DET2 gene was mapped at a 150-kb interval on an Arabidopsis chromosome 2 (Figure 1A). Figure 1 is a schematic illustration of the cloning and sequence analysis of the DE 2 gene. Figure 2A shows a summary of positional cloning. The mutant of the homozygous det2-l (Col-0) was crossed with either the non-0 or the wild-type La-er (isolated geographical designations). DNA from the F2 det2 plantings was prepared (Deilaporta et al., Plant Mol. Biol. Rep., .1: 19, 1983) for single sequence length polymorphisms (SSLPs) (Bell and Ecker, Genomic, 19: 137, 1994) and the amplified polymorphic adhered sequences (CAPS) (Konieczny and Ausubel, Plant J., 4.:403, 1993). The overlapping clones of the yeast artificial chromosome (YAC) were isolated from three separate libraries of yeast artificial chromosome from Arabidopsis (Ward and Jen, Plant Mol. Biol., 14: 561, 1990; JR Ecker, Methods, 1: 186, 1990; Grill &Somerville, Mol. Gen. Genet., 226: 484, 1991). A fine analysis of restriction fragment length polymorphism was performed with F2 det2 plants with recombination breakpoints, either in the m323-ZE2'2 region (68 recombinants, two mapping populations), or in the DET2 interval -ng & 168 (31 recombinants). The nga 168 molecular marker was used at the starting point to identify 8 overlapping clones of the yeast artificial chromosome that covered ~ 800 kb of the Arabidopsis genomic DNA. The novel markers of the amplified polymorphic sequences adhered directly from the ends of the yeast artificial chromosome inserts or derived from the phage clones of a genomic library of the Arabidopsis isolated with end probes of the artificial chromosome of yeast. Restriction fragment length polymorphism analysis delimited the DET2 site to a region of 150 ~ kb between the left end of yUP2C12 and yUPSElO chromosome 2 of the Arabidopsis. A contiguous cosmid was assembled within this region from the cosmid and phage clones isolated from the two genomic libraries of Arabidopsis (Olszewski et al., Nucleic Acids Res. , 1_6: 10765, 1988) by hybridization with yUP2C12, and UPSElO and the end probes of the artificial chromosome of yeast, or probes derived from cosmid. The cosmid DNAs were transformed into det2-l plants by a modified method of vacuum infiltration (Bechtold et al., Acad. Sci. Paris, 316: 1194, 1993; Bent et al., Science, 265: 1856, 1994) to identify the cosmids that contain the DET2 gene. Three cosmids, 2C12-19, 2C12-21, and 217-61, det2 mutant phenotypes rescued. We identified a 20-kb genomic fragment that can rescue the phenotypes of det2. Figure 2 shows a photograph of 12-day-old seedlings cultured in light (Figure 2A) and 10-day-old seedlings cultured in the dark (Figure 2B) after complementation of det2 by the wild-type DET2 gene. (From left to right in each panel) Col-0 wild-type, det2, and transgenic det2-l containing cosmid 217-61. Fragments of cosmid 217-61 labeled Eco Rl were used as probes to classify clones of ~2 x 106 from a supplementary DNA library (cDNA) of Arabidopsis constructed in lambda ZAPII (Kieber, et al., Cell, 72.:427, 1993 ). Positive clones were converted to plasmids by in vivo excision according to the manufacturer's protocol (Stratagene) and sequenced with gene-specific primers. The 20 kb fragment gives rise to at least three transcripts (Figure 1A), one of which is altered in all the analyzed alleles of det2 and derived from the DET2 gene (Figure IB). The DET2 transcript contains a single, long-gap reading frame that encodes an amino acid protein 262. The corresponding genomic sequences were determined from 8 alleles of det2, all of which have similar mutant phenotypes. The transcribed region of the DET2 gene was amplified by polymerase chain reaction (PCR) from the genomic DNAs of wild-type Col-0 and eight alleles of the det2, subcloned into the vector pGEM-t (Promega ), and they were sequenced. To minimize errors of the polymerase chain reaction, at least four different clones were pooled for sequencing from two independently amplified fragments. Four alleles contain frame change deletions, and two other mutations cause premature termination of the DET2 protein. The two remaining alleles have a non-conservative lysine substitution for glutamate at position 204 (Figure IB). Figure IB shows a map of the gene structure of DET2 and mutations in the DET2 gene. Thick lines indicate exons, and the open box denotes an intron. The positions of the mutations are relative to the initiation codon. Z, interruption codon. Figure 1C shows the nucleotide and deduced amino acid sequence of DET2 (SEQ ID N0: 1 and 2, respectively). Figure 3 shows a sequence comparison of DET2 with mammalian spheroid 5a-reductases. Figure 3A shows the deduced amino acid sequence of the DET2 gene aligned with the 5 -reductases of spheroid from rat (rS5R1 and rS5R2) and human (hS5R1 and hS5R2). The dashes indicate entered free spaces to maximize the alignment, and the conserved residues are shaded in at least two of the five sequences. The arrow indicates the mutated glutamate in the alleles of det2-1 and det2. (The one-letter abbreviations for the amino acid residues are as follows: A, High; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, The; V, Val; W, Trp; and Y, Tyr). Figure 3B shows a phylogenetic analysis of the relationship between the protein of DET2 and the 5 -reductases of mammalian spheroid. The scale measures the relative distance between the sequences. The deduced amino acid sequence of the DET2 gene is similar to that of mammalian spheroid 5a-reductases, with 38 to 42 percent sequence identity. (Database research was conducted at the US National Center for Biotechnology Information with the BLAST program (Altschul et al., J. Mol. Biol., 215: 403, 1990.) Alignment of the sequence and Phylogenetic analysis was performed with the Megalign program (DNAStar) by the method of J. Hein (Higgins and Sharp, Comput.Appl. Biosci., 5_: 151, 1989). The similarity of the sequence increases to 54 to 60 percent when conservative substitutions are taken into account. Two isozymes (types 1 and 2) of the spheroidal 5-reductase have been isolated in rats and humans (Wilson et al., Endocr. Rev., 14: 577, 1993; Russel and Wilson, Annu., Rev. Biochem., 63.:25, 1994). Phylogenetic analysis shows that DET2 is at least as closely related to type 2 enzymes, as type 2 enzymes are to type 1 enzymes (Figure 2). 3B). Eighty percent of the residues conserved in an absolute manner in mammalian enzymes are found in the protein of DET2 predicted. The mammalian spheroid 5-reductase catalyzes the dinucleotide phosphate of nicotinamide adenine (reduced) (NADPH) -dependent conversion of testosterone to dihydrotestosterone, which is a key step in the metabolism of the spheroid and is essential for the development ' embryonic of male external genitalia and prostate (Wilson et al., Supra). The importance of this reaction is evident from certain hereditary forms of male pseudohermaphroditism in humans, caused by the deficiency of spheroid 5a-reductase. The analysis of the sequences of the type 2 gene of spheroid 5-reductase from affected families identifies a mutation of the wrong sense that causes a conservative substitution of aspartate for Glu197 (Wilson et al., Supra), which corresponds to Glu204 in the DET2. in the alleles of det2-l and det2-6, this glutamate is changed to lysine, indicating that this glutamate has a similar critical function as in human 5a-reductase. Because a 1 ^ conservative substitution in this position causes the deactivation of the human enzyme (Wilson et al., Supra), it is predicted that the non-conservative change of glutamate-a-lysine completely eliminates the activity of the DET2. This could explain the severe phenotypes of the two alleles of the wrong direction.

Taken together, the data suggest that the enzyme of the DET2 can catalyze a biochemical reaction similar to the reaction that catalyzes the human enzyme. f ^^ Example 2 Many steroids have been identified in plants 20 (JMC Geuns, Phytochemistry, 17: 1, 1978), but only brassinosteroids (BRs) have a wide distribution throughout the kingdom plant and biological activity unique in the growth of plants when applied exogenously (NB Mandava, Annu., Rev. Plant 25 Physiol. Plant Mol. Biol., 39:23, 1988; RN Arteca, (2), 206- 213). Recently, a trajectory for the biosynthesis of brasinolide, the most active brasinosteroid, has been proposed, based on evidence from cell suspension cultures and complete sowing experiments (Fujioka and 5 collaborators, Biol. Sci. Biotechnical. ., 5_9: 1973, 1995). Although the biosynthesis of brasinolide includes many oxidation steps, only two steps involve reduction. One occurs early on the path where a double union in the campesterol is reduced to form campestanol. This reaction is H similar to that which is catalyzed by mammalian spheroid 5o-reductases, suggesting that DET2 could catalyze the conversion of campesterol to campestenol. Because the Arabidopsis genome does not contain any other sequence that is closely related to DET2, one possibility is that the The phenotype of det2 is due to the reduction or elimination of biosynthesis of the brasinosteroid. To test this hypothesis, the seedlings were treated ^^ of det2 with exogenous brasinolide. Figure 4A shows the proposed function of the DET2 protein in the biosynthetic path of brasinolido. The asterisk (*) indicates intermediate steps (Fujioka et al., Supra). Figure 4B shows the 10-day-old seedlings grown in the dark and the Figure 4C shows the 12-day-old seedlings cultured in light. Wild type plants, det2-l, and those treated with brasinolide det2-l, are described from left to right in each panel. Seeds were germinated in wet Whatman papers placed in MS medium (0.5xMS salts (Gibco), lx Vitamins B5 of Gamborg (Sigma), phytagar at 8 percent, and sucrose at 1 percent pH 5.7) for 2 days, and were transferred to fresh plates supplemented with different concentrations of auxin (IAA, 0 to 10- 5 M), brasinolide (0 to 10 ~ 6 M), and gibberellins (GA1 and GA4, 0 to 10"5 M) The hormones were sterile filtered into cooling medium MS For seedlings grown in the dark, the seeds were exposed to a treatment of two hours of light before their plates were wrapped with three layers of aluminum foil and the seedlings were transferred under a green safety light. The hypocotyl lengths of 10-day-old bleached seedlings and wild-type plants grown in the light were measured. Figure 4D shows a response to the dose of elongation of the hypocotyl induced by brasinolide of the seedlings grown in the dark and of the wild type cultivated in the light. The data F ^^ represent the mean SD that was obtained from the triplicate determinations, each with an average sample size of 12 seedlings. Although the addition of the 10"6 M brasinolide to the culture medium had no effect on the wild-type seedlings in the dark, the short hypocotyl phenotype of the cultured det2 seedlings was rescued in the dark (Figures 4, B and D). ).

Similarly, when added to 10"7 M, brasinolide had no effect on the petioles and leaves of wild-type seedlings, but completely suppressed the dwarf phenotypes of these organs in det2 plants grown in light (Figure 4C) In contrast, none of the applied gibberellins 5 (GA1 or GA4, 10 ~ 8 to 10"5 M), nor the auxins (1A, A 10 ~ 6 and 10 ~ 5 M) rescued det2 defects . The treatment of brasinolide reversed the inhibition of hypocotyl elongation caused either by detl mutation or by light (Figure 4D), but did not complement the mutant phenotypes of any of the seedlings HL cultured in the dark or in the light of betl, supporting the previous genetic studies that the DETL and the DET2 act on separate trajectories that control the processes regulated by light. Example 3 Mutants of Arabidopsis det2 are small dark green dwarfs that display pleiotropic defects in the light-regulated development during the multiple stages of the cycle. ^^ plant life. The DET2 gene encodes a protein that shares sequence identity of ~ 40 percent with the 5 -reductases of mammalian spheroid and that is involved in the synthesis of a class of plant steroids, the brasinosteroids. The following example shows that the protein of DET2, when expressed in human embryonic kidney cells 293, catalyzes the reduction of different substrates of animal spheroid and has kinetic properties similar to those of the enzymes of the 5? -reductase of mammalian spheroid. On the other hand, the 5α-reductases of human spheroid that are expressed in mutant plants of det 2 can substitute the DET2 in the biosynthesis of the brasinosteroid. These data indicate that the DET2 is an ortholog of the 5 -reductases of mammalian spheroid and provide additional evidence that the brassinosteroids play an essential role in the development of plant regulated by light. Figure 5 shows the chemical reactions catalyzed by the mammalian spheroid 5a-reductases (A) and the DET2 (B) of the Arabidopsis. 1. Plant materials and culture conditions: The standard wild-type genotype that was used was the Arabidopsis thaliana Columbia (Col-0). The mutant of det2-l that was used in this study has been described (Chory, J. Nagpal, P. &Peto, C.A. (1991) Plant Cell 3, 445-459). The sterilized seeds were worked on the surface by washing for 1 minute in 95 percent ethanol, followed by 12 minutes in a 1: 3 solution of bleach (Clorox) containing 0.02 percent Tween-20 (v / v). . The seeds were then washed 3 times with sterilized distilled water, and resuspended in 0.08 percent phytate (Gibco BRL). After treatment for 2 days at 4 ° C to induce germination, the seeds were seeded in Petri dishes containing medium (pH 5.7) of 0.5X MS (Murashige, T. &Skoog, F. (1962) Physiol. Plant 15, 473-497), supplemented with 1 percent sucrose, phytagar at 0.8 percent, and IX vitamins B5 from Gamborg (Sigma) and kept in culture chambers at 21 ° C with a photoperiod of 16 hours. The plants were grown in the dark by wrapping the plates with three layers of aluminum foil. For all experiments, det2 and all wild-type Col-0 plants were grown side by side under the same light and moisture conditions. 2. Expression of DET2 cDNA in human embryonic kidney cells 293: A 948-bp fragment was ligated from a clone of DET2 cDNA containing 18 bp of 5 'untranslated sequence, an open reading frame of 786 bp and 154 bp of the 3' untranslated sequence, within the expression vector of pCMV5 (Anderson, S. Davis, DL, Dahlback , H., Jornvall, H. and Russell, DW (1989), J. Biol. Chem., 264, 8222-8229). As a control, a mutant det2 protein was also expressed in the pCMV5 vector (pCMV5-det2). This construct, which contains a simple nucleotide (GA) mutation that changes Glu204 in Lys204, was made by replacing the wild type sequence DET2 with the same restriction fragment that was derived from the amplified det2-1 genomic DNA. by polymerase chain reaction. The resulting plasmid was then digested with EcoRI / BglII and cloned into the expression vector of pCMV5. Five milligrams of the expression plasmid containing either the wild-type DET2, or the mutated cDNA of the det2-l, were transfected into the human embryonic kidney cells 293 (ATCC CRL1573) by a calcium phosphate precipitation method, as HE ^ B described above (Normington, K. and Russel, D.W. (1992) J. Biol. Chem. 267, 19548-19554). Sixteen hours after the After transfection, the activity of spheroid 5a-reductase was assayed in any of the intact cells or cell lysates, as described (Anderson, S., Bishop, RW and Russell, DW (1989), J. Biol. Chem. ., 264, 16249-16255; Thigpen, AE and Russell, DW (1992), J. Biol. Chem., 267, 8577-8583). 3. Transformation of the det2-l mutants: A human cDNA encoding spheroid 5a-reductase, either type 1 or type 2, was cloned into a vector pMDl, a derivative of pBI121 (Clontech), which contains the promoter of the 35S mosaic virus of cauliflower (CaMV) and a transcriptional terminator of the nopaline synthase (Noster), to generate pMDl-hS5R plasmids (human spheroid 5a-reductase) (Figure 8A). The Agrobacterium strain GV3101, transformed with a pMDl-hS5R construct, was used to transform the det2-l mutants by the vacuum infiltration method, as described above (Bechtold, N., Ellis, J. and Pelletier, G. (1993) C. R. Acad. Sci. Paris 316, 1188-1193). Transformants (TI) were selected on medium 0.5X MS (H 5.7) (supra), and 25 milligrams / milliliters kanamycin. The kanamycin-resistant seedlings were transferred to the soil, maintained at 23 ° under a cycle of 16 hours of light and 8 hours of darkness, allowing it to pollinate itself, and T2 seeds were collected. 4. Treatment of bleached seedlings with 4-MA. Seeds were germinated on medium (pH 7.5) of 0.5X MS (supra) supplemented with 1 percent sucrose, vitamins B5 of IX Gamborg, phytagar at 0.8 percent, and variable concentrations of 4-MA (17b- (N, N-diethyl) carbamoyl-4-methyl-4-aza-5a-androstan-3-one, a gift from Merck Sharp &Dohme Research Laboratories). After a 2 hour light treatment, the plates were wrapped with three layers of aluminum foil and kept at 21 ° in a culture chamber. The hypocotyl lengths of bleached seedlings of 10 days of age were measured. 5. Analysis of DNA and RNA. The Arabidopsis DNA was isolated as described previously (li, J. and Chory, J., (1996) in Methods in Molecular Biology: Arabidopsis Protocols, editors Martinez-Zapater, JM and Salinas, J. (Humana, •• , NJ), in press). For the amplification of a genomic DET2 fragment, 1 milliliter (10-20 ng) of plant DNA was used as a template in a 50 milliliter reaction mixture containing 5 milliliters of polymerase pH regulator of 10X Taq ( Stratagene), 200 milliliters mM of deoxynucleoside triphosphates (dNTPs), 125 ng of forward and reverse primers, and 2.5 units of Taq polymerase. The amplification reactions were conducted in a thermal cycler (ERICOMP) by denaturing the DNA template for 10 minutes at 95 ° C, followed by 40 cycles of denaturation at 94 ° C for 45 seconds, annealing at 50 ° C for 45 seconds, extension at 72 ° C for 90 seconds, and a final extension period of 10 minutes at 72 ° C. For the cDNAs of human spheroid 5a-reductase in the transgenic det2-l plants, 10 percent DMSO was added in the polymerase chain reaction (to overcome the problem that caused the high GC content of the cDNA's ), together with the primers that are derived from the CaMV 35S promoter and each of the cDNA's. Total RNA was isolated from 2-week-old seedlings by the Napoli et al. Method (Napoli, C, Le-mieux, C, and Jorgensen, R. (1990) Plant Cell 2, 279-289) and performed gel blot hybridizations of RNA as described previously (Chory, J., Nagpal, P. and Peto, CA (1991) Plant Cell 3, 445-459). To determine whether the DET2 site of Arabidopsis encodes a functional spheroid 5o-reductase, a full-length DET2 cDNA was cloned into a mammalian expression vector, pCMV5 (supra) and introduced into kidney cells. human embryos 293 by a transfection protocol mediated by CaP04 (supra). Sixteen hours after the transfection, the radio labeled steroids were added to the cell medium and their conversion to 5a-reductase forms was verified by thin layer chromatography (Normington, K. and Russell, D.W. (1992) J. Biol. Chem. 267, 19548-19554. As shown in Figure 6, cells transfected with an expression vector pCMV5 lacking a cDNA insert, did not visually show spheroid 5a-reductase activity that could be measured, while the introduction of a vector of pCMV5 expression containing spheroid 5a-reductase cDNA either human type 1, or Arabidopsis DET2, resulted in the reduction of radiolabelled progesterone to 4-5-dihydroprogesterone. In consistency with our previous prediction (Li, J., Nagpal, P., Vitart, V., McMorris, CT and Chory, J. (1996) Science 272, 398-401), the Glu204Lys mutation of det2-q, completely deactivated the activity of spheroid 5a-reductase of DET2, since 293 cells that were transfected with the DNA of det2-l could not convert progesterone labeled with radio to its 5a-reduced form (Figure 6). Like the 5 -reductasas of mammalian spheroid (Russell, DW and Wilson, JD 1994 Annu. Rev. Biochem., 63, 25-f ^^ 61), Arabidopsis DET2 can catalyze the 5a-reduction of many steroids with a structure of 3-oxo-D4.5 , including testosterone and androstenedione. Since the hypothetical substrates of Arabidopsis DET2 in biosynthesis of brasinosteroid is campesterol or its analogs (sitosterol or stigmasterol), which contains a structure of 3b-hydroxyl-D5,6, we measured the activity of 5a-reductase of DET2 towards different steroids 25 labeled with radium including this structure cholesterol, pregnenolone, and dehydroepiandrosterone. In line with the results obtained with the 5 -reductases of mammalian spheroid (Hsia, SL and Voigt, W. (1974) J. Invest. Derma tol., 62, 224-227), the 293 cells that were transfected with a cDNA of spheroid 5a-reductase either human type 1, or of DET2 could not 5a-reduce these substrates (data not shown), implying that plants require the presence of an additional enzyme to convert campesterol to 3-oxo-D4,5 -campesterol, before 5a-reduction by the enzyme DET2. Kinetic properties of the expressed protein of the DET2. An in vitro assay (supra) was used to study the enzyme kinetics of the DET2 protein expressed by renal cell 293. It was determined that the apparent Km value for testosterone was 2.5 mM with a Vmax of 0.2 nmol / ( min-1 * mg-1) (Figure 7A), while the apparent Km value for progesterone was 0.4 mM with a Vmax of 0.5 nmol / (min-1 * mg-1) (Figure 7B). These values compare favorably with those calculated for the human isozyme type 1 (Km = 1.7 mM for testosterone and Km = 1.3 mM for progesterone). Like mammalian enzymes, the 5a-reductase activity of DET2 requires NADPH rather than NADH, as a cofactor and the catalyzed reaction was irreversible. The 4-azasteroids are a class of selective and potent inhibitors of the mammalian spheroid 5-reductase isozymes. To determine whether the enzyme of the DET2 was also inhibited by these drugs, cell lysates containing the recombinant DET2 were incubated, with the progesterone as the substrate and different amounts of the 4-azasteroid, 4-MA. Increasing the concentration of 4-MA in the reaction mixture revealed a competitive mode of inhibition with calculated apparent Ki of 300 nM. In the experiments that are not shown, the other two inhibitors of the 5-reductase isozymes, finasteride (a 4-azasteroid) and LY191704 (a non-spheroidal benzoquinolinone), did not affect the activity of the recombinant DET2. The isozymes type 1 and type 2 of the 5a: -reductase of mammal spheroid are distinguished by their optimal pH. In all the species that have been examined so far, the isozyme type 1 has an optimum of alkaline pH (Vmax at approximately a pH of 8.0), and the isozyme type 2 has an acidic optimum pH (Vmax at a pH of 5.0-5.5) (supra). The pH optimum of the plant enzyme was measured to determine which mammalian isozyme most resembled the enzyme of the DET2, with respect to this biochemical parameter. The data of Figure 7D show that the pH against the activity curve of the enzyme that was obtained with the cell lysates containing the recombinant DET2 protein, was symmetric with an optimum at a pH of 6.8, which is between the optimum of the pH of the two mammalian isozymes. The human 2-spheroid 5-reductase cDNA complements the mutation of the plant det2-l. If the DET2 is a functional homolog of the 5a-reductases of human spheroid, then the expression of one or other of the human enzymes should complement the mutation of det2 in the plants, and rescue the mutant phenotypes. To test this hypothesis, we stably introduced cDNAs encoding human spheroid 5o-reductase, either type or type 2, into det2-l mutants using the Agrobacterium-mediated transformation (supra). In these experiments, two constructs were prepared with either a full length cDNA or a cDNA with a 3 'untranslated truncated region for each type of human spheroid 5a-reductase cDNA. These were cloned into a binary vector, pMDl, which contains a cauliflower 35S mosaic virus promoter and a 3 'nopaline synthase sequence not translated (Figure 8A). In a set of transformation experiments, 64 transgenic plants were obtained. Among these, 57 visually displayed wild type phenotypes and the remaining 7 lines visually displayed the intermediate phenotypes between det2-l and wild-type plants (Table 1). As shown in Figures 8B-8D, the presence of any human spheroid 5a-reductase cDNA in the middle of the det2-l mutant, rescued both the dark and light phenotypes of the mutation. All 64 transgenic lines were tested for the insertion of a human cDNA into their genomes by the polymerase chain reaction using oligonucleotide primers derived from the CaMV 35S promoter and a 5a-reductase cDNA. The products of the polymerase chain reaction of the expected size were amplified for any type of human spheroid 5a-reductase cDNA, from the genomic DNAs of all the transgenic plants (summarized in Table 1), whereas the controls untransformed wild type or heterozygous det2-l and homozygous plants, did not produce positive amplification signals. Since the mutation of the det2-l causes a GA transition, leading to the removal of an Mnll restriction site, there is a restriction fragment length polymorphism (RFLP) of Mnll between the det2-ly mutants the wild type plants. A restriction fragment length polymorphism analysis based on polymerase chain reaction was used to confirm that each of the 64 transgenic lines still contained the det2-l mutation (Table 1), thereby eliminating the possibility that the phenotypic normalization observed was due to an accidental introduction of a wild-type DET2 gene into their genomes or to the inversion of the mutated site.

Table 1 Summary of transsénic det2-l plants carrying cDNAs of human spheroid 5-reductase Number of Plants Constructions Showing resis- Containing Bearing mutation Deploying phenotency to the canami- cDNA of hS5R tion of the det2-l type of type silvescina tre hS5Rl-1.3kb 8 8 8 8 hS5Rl-2.1kb 18 18 18 18 hhSS55RR22--00..88kkbb 1 188 1 188 18 16 hS5R2-2.4kb 20 20 20 15 The transgenic plants of each construction were obtained from two independent transformation experiments. Observe the above description and Figure 8A for a description of the constructions. To prove that the phenotypic normalization that was observed was caused by the expression of a human spheroid 5Oi-reductase cDNA in the transgenic plants, the germinated seeds were harvested from two representative transgenic lines, containing human cDNA, either of type 1 or type 2, on a synthetic culture medium with different concentrations of 4-MA, an inhibitor of the 5 -reductases of mammalian spheroid. Figure 9 shows the effect of increasing concentrations of 4-MA on the elongation of the hypocotyl of plants grown in the dark. While the increasing concentrations of 4-MA had little effect on the elongation of the hypocotyl of the seedlings, either wild-type or det2-l, the drug caused a significant decrease in the growth of the hypocotyl in the two transgenic lines. The differential effects of 4-MA on the det2-l, the wild type and the two transgenic lines that were observed in these experiments, were consistent with the biochemical properties of human spheroid 5a-reductases and DET2. The Ki for the 4-MA of human spheroid 5a-reductase type 1 (8.0 nM), is twice that of the type 2 enzyme (4.0 nM) (supra), while the Ki for 4-MA of the DET2 is at least 30 parts higher (300 nM) than those of the human isozymes (Figure 7C). The second line of evidence that the expression of the human spheroid 5o-reductase cDNAs rescued the mutant phenotypes of det2-l, comes from observations that the progeny of some of the individual transformants looked more like the mutants of the det2-l than to the wild-type plants, although their parental lines exhibited a complete wild-type phenotype. We suspect that this phenomenon was due to the loss of transgene expression in the generation of T2, which had previously been observed in different transgenic lines with the binary vector of pMDl. Therefore, the expression levels of human cDNAs in different transgenic lines were examined by RNA staining. The stable state level of human spheroid 5a-reductase mRNA that was expressed in the transgenic det2-l plants (Figure 10B), was correlated with the degree of mutant rescue, as indicated by both the length of the hypocotyl of the seedlings cultured in the dark (Figure 10A), as the total morphology of the plants grown in the light (Figure 10C). The plants that showed the highest level of human transcripts among all the lines examined, exhibited full stature of wild type in the light and their hypocotyls were even longer than those of the wild type controls in the dark. In contrast, the lines that showed the lowest levels of human mRNAs, looked more like the mutants of the det2-l than the wild-type plants in the light and had intermediate lengths of hypocotyl in the dark. Based on these results, we conclude that human cDNAs can rescue det2-l defects and that the expression of human spheroid 5a-reductase cDNAs in transgenic det2-l plants correlates with the degree of rescue of their phenotypes mutants Example 4 The rice was transformed with two constructions, one of which allowed overexpression and the other the subexpression (antisense) of the DET2. Standard Agrobacterium expression vectors were used, in which the coding sequence of the DET2 was expressed in reverse orientation from the promoter of the 35S mosaic virus of the cauliflower (Metzlaff et al., Cell 8.:845, 1997). The antisense plants had phenotypes similar to those of the det2 mutants, that is, they were small with reduced male fertility and reduced apical predominance. The lines with moderate overexpression were longer than the wild type and based on a few lines, they had an increase in production of approximately 15 percent (the seed mass is 15 percent larger than the wild type seeds) . High overexpositories are not healthy, which is consistent with the idea that too much hormone is harmful. Although the invention has been described with reference to the modalities that are currently preferred, it should be understood that various modifications can be made without departing from the spirit of the invention. In accordance with the above, the invention is limited only by the following claims.

ISTADQ OF SEQUENCES NUMBER OF SEQUENCES: 7 (2) INFORMATION FOR THE SEQUENCE ID NO: l: (i) CHARACTERISTICS OF THE SEQUENCE: 5 (A) LENGTH: 974 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: l: (AATTCCATAA CCCGAAAAAT GGAAGAAATC GCCGATAAAA CCTTCTTCCG ATACTGTCTC 60 CTCACTCTTA TTTTCGCCGG CCCACCAACC GCCGTCCTTC TGAAATTCCT CCAAGCTCCT 120 TACGGTAAAC ACAACCGTAC CGGATGGGGT CCCACCGTAT CTCCACCGAT TGCTTGGTTC 180 GTCATGGAGA GCCCAACCTT GTGGCTCACT CTCCTCCTCT TCCCCTTTGG TCGTCACGCT 240 CTCAACCCTA AATCTCTACT TCTATTCTCT CCTTATCTCA TTCATTACTT CCACCGCACC 300 5 ATCATTTACC CTCTTCGCCT CTTCCGCAGC TCCTTCCCCG CCGGTAAAAA CGGATTTCCG 360 ATCACCATCG CCGCCTTGGC TTTCACCTTT AATCTCCTCA ATGGTTATAT CCAGGCGAGG 420 TGGGTTTCGC ATTACAAGGA TGACTACGAA GACGGAAACT GGTTCTGGTG GCGGTTTGTT 480 ^ ATCGGTATGG TGGTTTTCAT AACCGGCATG TATATAAATA TCACGTCGGA CCGCACTTTG 540 GTACGATTGA AGAAAGAGAA CCGGGGAGGT TATGTGATAC CGAGAGGAGG CTGGTTCGAG 600 0 TTGGTAAGCC GTCCGAATTA TTTTGGAGAG GCGATTGAGT GGTTGGGCTG GGCTGTTATG 660 ACTTGGTCTT GGGCCGGTAT TGGATTTTTT CTGTACACGT GTTCCAATTT GTTTCCGCGT 720 GCACGTGCGA GTCACAAGTG GTACATTGCC AAGTTCAAGG AAGAGTATCC CAAGACTCGT 780 AAAGCTGTTA TTCCTTTTGT GTACTGAGAA TTGAGAAAGT TGAAAACTAG TTTATCATAT 840 GTTATGTGTC AATTTGTTTC CAAACTACCT TTGTCAAAAT TTCCAGTAAC CGGTTTAATT 900 CCAACACGGT TTAGATCTTA TGTTGGTATC TTCAACAATG CACAACAAAC TGTGTATTCT TTAGACAAAT TTTA 960 974 (2) INFORMATION FOR THE SEQUENCE ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 262 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: irrelevant (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: u Thr Leu lie Phe Wing Gly Pro Pro Thr Wing Val Leu Leu Lys Phe Leu Gln 20 25 30 Wing Pro Tyr Gly Lys His Asn Arg Thr Gly Trp Gly Pro Thr Val Ser 15 35 40 45 Pro Pro lie Wing Trp Phe Val Met Glu Ser Pro Thr Leu Trp Leu Thr 50 55 60 ^^ Leu Leu Leu Phe Pro Phe Gly Arg His Wing Leu Asn Pro Lys Ser Leu 65 70 75 80 Leu Leu Phe Ser Pro Tyr Leu lie His Tyr Phe His Arg Thr lie lie 85 90 95 Tyr Pro Leu Arg Leu Phe Arg Ser Ser Phe Pro Ala Gly Lys Asn Gly 100 105 110 Phe Pro lie Thr lie Ala Ala Leu Ala Phe Thr Phe Asn Leu Leu Asn 25 115 120 125 Gly Tyr lie Gln Wing Arg Trp Val Ser His Tyr Lys Asp Asp Tyr Glu 130 135 140 Asp Gly Asn Trp Phe Trp Trp Arg Phe Val lie Gly Met Val Val Phe 145 150 155 160 lie Thr Gly Met Tyr lie Asn lie Thr Ser Asp Arg Thr Leu Val Arg 165 170 175 Leu Lys Lys Glu Asn Arg Gly Gly Tyr Val lie Pro Arg Gly Gly Trp 180 185 190 Phe Glu Leu Val Ser Arg Pro Asn Tyr Phe Gly Glu Ala Glu Trp Gly Phe Phe 210 215 220 Leu Tyr Thr Cys Ser Asn Leu Phe Pro Arg Ala Arg Ala Ser His Lys 225 230 235 240 Trp Tyr He Wing Lys Phe Lys Glu Glu Tyr Pro Lys Thr Arg Lys Wing 245 250 255 Val He Pro Phe Val Tyr 260 (2) INFORMATION FOR SEQUENCE ID NO: 3: 0 (i) SEQUENCE CHARACTERISTICS: (A ) LENGTH: 246 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: irrelevant (D) TOPOLOGY: linear 5 (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: Leu He Phe Ala Gly Pro Pro Thr Ala Val Leu Leu Lys Phe Leu Gln 1 5 10 15 Wing Pro Tyr Gly Lys His Asn Arg Thr Gly Trp Gly Pro Thr Val Ser 20 25 30 Pro Pro He Wing Trp Phe Val Met Glu Ser Pro Thr Leu Trp Leu Thr 35 40 45 Leu Leu Leu Leu Pro Phe Gly Arg His Wing Leu Asn Pro Lys Ser Leu 50 55 60 Leu Leu Phe Ser Pro Tyr Leu He His Tyr Phe Leu Arg Thr He He • 65 70 75 80 Tyr Pro Leu Arg Leu Phe Arg Be Ser Phe Pro Wing Gly Lys Asn Gly 85 90 95 Phe Pro He Thr He Wing Wing Leu Wing Phe Thr Phe Asn Leu Leu Asn 15 100 105 110 Gly Tyr He Gln Wing Arg Trp Val Ser His Tyr Lys Asp Asp Tyr Glu 115 120 125 ^^ Asp Gly Asn Trp Phe Trp Trp Arg Phe Val He Gly Met Val Val Phe 130 135 140 20 He Thr Gly Met Tyr He Asn He Thr Ser Asp Arg Thr Leu Val Arg 145 150 155 160 Leu Lys Lys Glu Asn Arg Gly Gly Tyr Val He Pro Arg Gly Gly Trp 165 170 175 Phe Glu Leu Val Ser Arg Pro Asn Tyr Phe Gly Glu Wing He Glu Trp 25 180 185 190 Leu Gly Trp Wing Val Met Thr Trp Ser Trp Wing Gly He Gly Phe Phe 195 200 205 Leu Tyr Thr Cys Ser Asn Leu Phe Pro Arg Ala Arg Ala Ser His Lys 210 215 220 Trp Tyr He Ala Lys Phe Lys Glu Glu Tyr Pro Lys Thr Arg Lys Ala 225 230 235 240 Val He Pro Glu Val Tyr 245 (2) INFORMATION FOR THE SEQUENCE ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 239 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: irrelevant (D) ) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: Gly Phe Met Wing Phe Val Ser He Val Gly Leu Arg Ser Val Gly Ser 1 5 10 15 Pro Tyr Gly Arg Tyr Ser Pro Gln Trp Pro Gly He Arg Val Pro Wing 20 25 30 Arg Pro Wing Trp Phe He Gln Glu Leu Pro Ser Met Wing Trp Pro Leu 40 45 Tyr Glu Tyr He Arg Pro Wing Wing Wing Arg Leu Gly Asn Leu Pro Asn 50 55 60 Arg Val Leu Leu Wing Met Phe Leu He His Tyr Val Gln Arg Thr Leu 65 70 75 80 Val Phe Pro Val Leu He Arg Gly Gly Lys Pro Thr Leu Leu Val Thr 85 90 95 Phe Val Leu Wing Phe Leu Phe Cys Thr Phe Asn Gly Tyr Val Gln Ser 100 105 110 Arg Tyr Leu Ser Gln Phe Wing Val Tyr Wing Glu Asp Trp Val Thr His 115 120 125 Pro Cys Phe Leu Thr Gly Phe Ala Leu Trp Leu Val Gly Met Val lie 130 135 140 Asn He His As Asp His He Leu Arg Asn Leu Arg Lys Pro Gly Glu 145 150 155 160 Thr Gly Tyr Lys He Pro Arg Gly Glu Leu Phe Glu Tyr Val Ser Wing 165 170 175 Wing Asn Tyr Phe Gly Glu Leu Val Glu Trp Cys Gly Phe Ala Leu Wing 180 185 190 Ser Trp Ser Leu Gln Val Val Phe Ala Leu Phe Thr Leu Ser Thr 195 200 205 Leu Leu Thr Arg Ala Lys Gln His His Gln Trp Tyr His Glu Lys Phe 210 215 220 Glu Asp Tyr Pro Lys Ser Arg Lys He Leu He Pro Phe Val Leu 225 230 235 (2) INFORMATION FOR SEQUENCE ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 243 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: irrelevant (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Ala Tyr Leu Gln Cys Ala Val Gly Cys Ala Val Phe Ala Arg Asn Arg 1 5 10 15 Gln Thr Asn Ser Val Tyr Gly Arg His Ala Leu Pro Ser His Arg Leu 20 25 30 u Pro Ser Leu Ala Leu Pro Leu Tyr Gln Tyr Ala Ser Glu Ser Ala Pro Arg Leu Arg 50 55 60 Ser Ala Pro Asn Cys He Leu Leu Ala Met Phe Leu Val His Tyr Gly 65 70 75 80 His Arg Cys Leu He Tyr Pro Phe Leu Met Arg Gly Gly Lys Pro Met 85 90 95 ^^ Pro Leu Leu Wing Cys Thr Met Wing He Met Phe Cys Thr Cys Asn Gly 100 105 110 20 Tyr Leu Gln Ser Arg Tyr Leu Ser His Cys Wing Val Tyr Wing Asp Asp 115 120 125 Trp Val Thr Asp Pro Arg Phe Leu He Gly Phe Gly Leu Trp Leu Thr 130 135 140 Gly Met Leu He Asn He His As Asp His He Leu Arg Asn Leu Arg 145 150 155 160 Lys Pro Gly Asp Thr Gly Tyr Lys He Pro Arg Gly Gly Leu Phe Glu 165 170 175 Tyr Val Thr Ala Ala Asn Tyr Phe Gly Glu He Met Glu Trp Cys Gly 180 185 190 Tyr Ala Leu Ala Ser Trp Ser Val Gln Gly Ala Ala Phe Ala Phe Phe 195 200 205 Thr Phe Gly Phe Leu Ser Gly Arg Ala Lys Glu His His Glu Trp Tyr 210 215 220 Leu Arg Lys Phe Glu Glu Tyr Pro Lys Phe Arg Lys He He He Pro 225 230 235 240 Phe Leu Phe (2) INFORMATION FOR THE SEQUENCE ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 238 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: irrelevant (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Leu Ala Thr Met Gly Thr Leu He Leu Cys Leu Gly Lys Pro Ala Ser 1 5 10 15 Tyr Gly Lys His Thr Glu Ser Val Ser Ser Gly Val Pro Phe Leu Pro 20 25 30 Wing Arg He Wing Trp Phe Leu Gln Glu Leu Pro Ser Phe Val Val Ser 35 40 45 Val Gly Met Leu Wing Trp Gln Pro Arg Ser Leu Phe Gly Pro Pro Gly 50 55 60 Asn Val Leu Leu Ala Leu Phe Ser Wing His Tyr Phe His Arg Thr Phe 65 70 75 80 He Tyr Ser Leu Leu Thr Arg Gly Arg Pro Phe Pro Wing Val Leu Phe 85 90 95 Leu Arg Wing Thr Wing Phe Cys He Gly Asn Gly Leu Leu Gln Wing Tyr 100 105 110 Tyr Leu Val Tyr Cys Wing Glu Tyr Pro Glu Glu Trp Tyr Thr Asp Val 115 120 125 Arg Phe Ser Phe Gly Val Phe Leu Phe He Leu Gly Met Gly He Asn 130 135 140 He His As Asp Tyr Thr Leu Arg Gln Leu Arg Lys Pro Gly Glu Val 145 150 155 160 He Tyr Arg He Pro Arg Gly Gly Leu Phe Thr Tyr Val Ser Gly Wing 165 170 175 Asn Phe Leu Gly Glu He He Glu Trp He Gly Tyr Wing Leu Wing Thr 180 185 190 Trp Ser Val Pro Wing Phe Wing Phe Wing Phe Phe Thr Leu Cys Phe Leu. 195 200 205 Gly Met Gln Wing Phe Tyr His His Arg Phe Tyr Leu Lys Met Phe Lys 210 215 220 Asp Tyr Pro Lys Ser Arg Lys Ala Leu He Pro Phe He Phe 225 230 235 (2) INFORMATION FOR THE SEQUENCE ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 241 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: irrelevant (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: Leu Val Ala Leu Gly Ala Leu Ala Leu Tyr Val Ala Lys Pro Ser Ser 1 5 10 15 Being Ser Gly Tyr Gly Lys His Thr Glu Being Leu Lys Pro Wing Ala Thr 25 30 Arg Leu Pro Ala Arg Ala Ala Trp Phe Leu Gln Glu Leu Pro Ser Phe 40 45 Wing Val Pro Wing Gly He Leu Wing Arg Gln Pro Leu Ser Leu Phe Gly 50 55 60 Pro Pro Gly Thr Val Leu Leu Gly Leu Phe Cys Val His Tyr Phe His 65 70 75 80 Arg Thr Phe Val Tyr Ser Leu Leu Asn Arg Gly Arg Pro Tyr Pro Wing 85 90 95 He Leu He Leu Arg Gly Thr Wing Phe Cys Thr Gly Asn Gly Val Leu 100 105 110 Gln Gly Tyr Tyr Leu He. Tyr Cys Wing Glu Tyr Pro Asp Gly Trp Tyr 115 120 125 Thr Asp He Arg Phe Ser Leu Gly Val Phe Leu Phe He Leu Gly Met 130 135 140 Gly He Asn He His As Asp Tyr He Leu Arg Gln Leu Arg Lys Pro 145 150 155 160 Gly Glu Be Ser Tyr Arg He Pro Gln Gly Gly Leu Phe Thr Tyr Val 165 170 175 Ser Gly Wing Asn Phe Leu Gly Glu He He Glu Trp He Gly Tyr Wing 180 185 190 Leu Ala Thr Trp Ser Leu Pro Ala Leu Ala Phe Ala Phe Phe Ser Leu 195 200 205 Cys Phe Leu Gly Leu Arg Ala Phe His His His Arg Phe Tyr Leu Lys 210 215 220 Met Phe Glu Asp Tyr Pro Lys Ser Arg Lys Ala Leu He Pro Phe He 225 230 235 240 Phe

Claims (30)

1. SUBJECTS 1. A substantially purified DET2 polypeptide.
2. 2. The polypeptide according to claim 1, wherein DET2 is characterized as: a) having a molecular weight of about 31 KD, as determined by SDS-PAGE; b) having spheroidal 5a-reductase activity; and c) functioning in the biosynthetic trajectory of brasinolide.
3. 3. The polypeptide according to claim 1, wherein the amino acid sequence of said protein is substantially the same amino acid sequence set forth in SEQ ID NO: 2 (Figure 1C).
4. 4. The polypeptide according to claim 1, wherein the amino acid sequence of said protein is substantially the same amino acid sequence set forth in SEQ ID NO: 2 (Figure 1C).
5. 5. An isolated polynucleotide encoding the DET2 polypeptide of claim 1.
6. 6. An isolated polynucleotide according to claim 5, having a nucleotide sequence as set forth in SEQ ID NO: 1 (Figure 1C), or variations of they encode the same amino acid sequence, but employ different codons for some of the amino acids, or sequences of nucleotides variably of them.
7. 7. A recombinant expression vector containing a polynucleotide sequence according to claim 5.
8. 8. A host cell containing the vector of claim 7.
9. 9. An antibody that binds the protein of claim 1, or antigenic fragments of said protein.
10. 10. A method of producing a genetically modified plant, characterized by having an increased yield compared to a wild type plant, said method comprising: contacting a plant cell with at least one nucleic acid sequence encoding a 5a-reductase spheroid, said nucleic acid sequence operatively associated with a promoter, to obtain a transformed plant cell; producing a plant from said transformed plant cell; and selecting a plant that exhibits said increased yield.
11. 11. The method of claim 10, wherein the spheroidal 5o-reductase is a plant enzyme. The method of claim 11, wherein the 5α-reductase spheroid is DET2. The method of claim 10, wherein the spheroidal 5a-reductase is spheroidal 5a-reductase is a mammalian enzyme. The method of claim 10, wherein the 5α-reductase spheroid is selected from the group consisting of human 5-reductase spheroid, rat, mouse and monkey type 1 or type 2. 15. The method of claim 10, wherein the contacting is by physical means. 16. The method of claim 10, wherein the contacting is by chemical means. The method of claim 10, wherein the plant cell is selected from the group consisting of protoplasts, gamete producing cells, and cells that are regenerated in whole plants. 18. The method of claim 10, wherein the promoter is selected from the group consisting of a constitutive promoter and an inducible promoter. 19. A plant produced by the method of claim 10. 20. Plant tissue derived from a plant produced by the method of claim 10. 21. A seed derived from a plant produced by the method of claim 10. 22. A method for genetically modifying a plant cell such that a plant, produced from said cell, produces an increased yield as compared to a wild-type plant, said method comprising: contacting said plant cell with the polynucleotide of the plant; claim 5 or a polynucleotide encoding 5 a mammalian spheroid 5a-reductase to obtain a transformed plant cell; and culturing the transformed plant cell under plant formation conditions to obtain a plant that has increased yield. 23. The method of claim 22, where to induce an increased growth is achieved by inducing the expression of J 5a-reductase spheroid in the plant. The method of claim 22, wherein the 5α-reductase spheroid is a plant enzyme. 25. The method of claim 24, wherein the spheroid 5a-15 reductase is DET2. 26. The method of claim 22, wherein the 5α-reductase spheroid is a mammalian enzyme. / 27. The method of claim 26, wherein the spheroidal 5α-reductase is selected from the group consisting of human 5α-reductase, spheroid, rat, mouse and mono type 1 or type 2. 28. A method of producing a plant characterized by having increased yield, said method comprising: contacting a susceptible plant with an inducing amount of the DET2 promoter of an agent necessary to elevate the expression of the DET2 gene on the expression of DET2 in a plant not contacted with the agent. J ~ 29. The method of claim 28, wherein the agent is a transcription factor. 30. The method of claim 28, wherein the agent is a chemical agent.