MXPA01006841A - Maize alternative oxidase genes and uses thereof - Google Patents

Abstract

The invention provides isolated alternative oxidase nucleic acids and their encoded proteins. The present invention provides methods and compositions relating to altering alternative oxidase levels in plants. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions.

Description

ALTERNATIVE CORN OXIDASE GENES AND USES OF THE SAME TECHNICAL FIELD The present invention relates in general to the molecular biology of plants. More specifically, it is related to nucleic acids and to methods for modulating their expression in plants. BACKGROUND OF THE INVENTION There are several obstacles to the development of crop varieties. These challenges include the development of varieties with a greater tolerance to cold; the development of varieties with greater resistance to pathogens; the achievement of male sterility for hybrid crops with new methods; the possibility of overcoming the limitations of the selectable markers for the transformation; and the manipulation by genetic engineering of the targeting of proteins to the mitochondria. An important determinant of the performance of hybrid corn seeds in Nordic climates is the vigor of the seedlings in the cold soil or in the cold weather conditions of spring. Cold soils and weather also affect the performance of other crops such as wheat, soy, rice, barley, oats, sunflower and rye. Improving the vigor of the seedlings under cold conditions will increase the number of standing plants, extend the effective growing season for a given cold climate zone and increase overall yield. Moreover, it will allow the emergence of new opportunities for the use of genetically modified and higher yielding varieties in these regions.

Cold conditions inhibit normal respiration in plants. Cellular respiration is vital for aerobic life and occurs in three main steps: a) carbohydrates, fatty acids and some amino acids are oxidized into 2-carbon chemical subunits and present in the form of acetyl-CoA; b) these acetyl groups enter the citric acid cycle, obtaining carbon dioxide and protons and electrons from hydrogen; and c) these high energy electrons cascade down the respiratory chain whereupon ATP is produced and finally oxygen is reduced to form water. The terminal oxidase of the normal cyanide-sensitive airway is cytochrome oxidase. This pathway is also known simply as the cytochrome pathway. The cytochrome is labile to cold and poor behavior in cold conditions. When normal breathing is inhibited, such as under cold conditions, the movement of carbohydrates is delayed through the citric acid cycle, whereby cellular respiration is depressed and an accumulation of various intermediates, such as citric acid, occurs. which can be stressful or toxic to the cells. In addition, the inhibition of normal cellular respiration can result in an accumulation of free radicals, reactive oxygen species such as superoxide, hydrogen peroxide or hydroxyl radicals. These radicals can cause cell damage due to their high chemical reactivity. They can also dramatically change genetic expression, including the activation of defense systems, which can be stressful or toxic to the plant. An alternative airway has been observed in bacteria, plants and some fungi. This pathway involves an enzyme known as alternative oxidase. A higher expression of the alternative oxidase can cause elevated temperatures in the plant organs. This increase is more dramatic in thermogenic plants that produce heat during flowering in volatilized odorant compounds that attract pollinating insects. These thermogenic plants include Sauromautum guttatum, Symplocarpus foetidus and Arum maculatum. In these plants there is a massive increase in the mRNA and the alternative oxidase protein that is regulated by salicylic acid. It has also been observed that plant tissue infected by pathogens has high levels of alternative oxidase and higher organ temperatures. The increase in the levels of the alternative oxidase protein correlates with a high level of activity in the alternative route (Vanlerberghe, 1992a). The cold causes a high expression of the alternative oxidase and the activation of the alternative oxidase pathway in plants. It has been observed that those species and varieties that have a better tolerance to cold have a greater expression of the alternative oxidase pathway. For example, winter wheat has higher expression levels than spring wheat. (McCaig et al., 1977) In cultivated tobacco - with cold, as much as 45% of respiration is done by the alternative route. (Vanlerberghe, 1992a) Cold-grown corn seedlings also show increased activity in the alternative oxidase pathway (Stewart et al., 1990a). There are two alternative rice oxidase genes whose expression is induced at the mRNA level under cold conditions. (Ito et al., 1997) The alternative oxidase pathway adapts to cold conditions because it allows the advance of the citric acid cycle and respiration, relieves stress caused by chemical species and generates heat. The increase in the resistance of the crop species to attack by pathogens is another area of concern for the development of new crop varieties. Every year large portions of crop are lost due to susceptibility to various pathogens. An increase in resistance to these pathogens would reduce these losses. There is a growing information that relates the alternative oxidase with the responses of the plants to an attack by pathogens. This relationship focuses on reactive chemical species, such as reactive oxygen species (ROS) such as superoxide, hydrogen peroxide and hydroxyl radicals. There are various conditions that produce ROS, such as an inhibition of respiration via the cytochrome, saturation of the respiratory cytochrome or normal responses of the plant to pathogens, which result in an activation of the expression of the alternative oxidase. The regulation of the expression of the alternative oxidase is sensitive to the oxidation-reduction. There are reports on the transcriptional activation of alternative plant oxidase genes and the proteins themselves are activated after * transcription due to ROS stress. There are additional data that compromise alternative oxidase in the responses of plants to pathogens. It has been known for some time that salicylic acid is a chemical inducer of the expression of alternative oxidase in thermogenic flowers. It is also known that salicylic acid is an inducer of the expression of proteins related to pathogenesis, of inducible resistance to pathogens and of systemic resistance acquired to pathogens. More recently it has been shown that the treatment of tobacco leaves with salicyclic acid causes an increase in the expression of alternative oxidase and of the flow through the alternative respiratory route. It should be noted that SHAM (salicylic hydroxamic acid), which blocks the activity of alternative oxidase, also blocks the induced and systemic resistance acquired to pathogens (such as the tobacco mosaic virus in tobacco). Less clear is the role it can play in resistance to bacteria and fungi (Chivasa, et al., 1997). Apparently, salicylate is not a determinant of alternative oxidase levels in non-induced stationary states, but actually determines levels of induced expression (Lennon, et al., 1997). Other factors, such as ROS, can also fulfill some function. In summary, the expression of the alternative oxidase contributes to resistance to pathogens by some unknown mechanism. Another challenge for the development of new crop varieties is constituted by the special requirements involved in the development of hybrid seeds. The production of hybrid seeds for crop plants involves the crossing of two parent varieties to obtain a more desirable hybrid, usually higher yielding progeny. * The production of these hybrid seeds is costly both in terms of time and money. The production of hybrid corn seeds often involves a manual or mechanical emasculation of the parent that functions as a female plant and the donation of pollen to it by another parent. Male sterility lines are convenient in that they greatly simplify the production of hybrid seeds by eliminating the need for physical emasculation. Several progenitors of male sterility have been proposed and a few were implemented, with variable results. The need for more diverse and more effective approaches to achieve male sterility is clear. There is evidence about the commitment of mitochondria to male fertility. In fact, certain male sterility is inherited via the cytoplasm by virtue of genetic abnormalities in the mitochondria. The CMS (Cytoplasmic Male Sterility) of Texas is a famous example. Some plants with male sterility comprise abnormally low levels of expression and / or activity of the alternative oxidase (Connett and Hanson, 1990; Musgrave et al., 1986). The manipulation of the alternative oxidase pathway has been used to generate male sterility in tobacco (International Patent Application WO 96/31113). A partial sequence for an alternative oxidase corresponding to ZmAOX2 has been published in this application (Polidoros, A.N. GenBank, Direct Deposit 30-DIC-1997, Accession No. AF040566, bases 1 to 447). Genetic manipulation of crop plants has been limited both in terms of variety and volume of possible genetic manipulation events due to limitations of selectable markers during transformation. There are relatively few selectable markers for plant transformation protocols. This causes various problems.

One of them is that some of the markers are not clearly selectable. Another is that with so few markers, the stacking of multiple genetic manipulated characteristics brings disadvantages since the plant to be transformed with a new gene may already have a transgenic construction with the same selectable marker.

There are indications that alternative oxidase could be used as a selectable marker. For example, it has been shown that tobacco cells treated with inhibitors for the cytochrome airway, such as potassium cyanide, can survive thanks to the respiration of the alternative oxidase pathway, but their growth is slow. Said cyanide-treated tobacco cells grew faster when they were transformed with the alternative oxidase gene under the direction of the 35S promoter which causes a high (higher than normal) expression of the alternative oxidase gene (Vanlerberghe et al., 1997a ), presumably due to increased respiration as a consequence of the alternative route. The targeting of proteins to mitochondria is another area where advances in art are also needed. The alternative oxidase genes are encoded by the nucleus, but the protein is located in the mitochondria. The importation of proteins in the mitochondria depends on transit peptides located at the N-terminal end of the primary peptide transcripts. These transit peptides are separated by cleavage after entering the mitochondria. The direct sequencing of the N-terminal end of the mature AOX peptide of Sauromatum guttatum allowed to locate the starting point of the mature peptide (Rhoads and Mclntosh, 1993). This region of processing sites is highly conserved in other alternative plant oxidases (Whelan, et al., 1995). The mature peptide usually begins with "XST", and the transit peptide has a residue (R) arginine at the amino acid position minus 2, which is necessary for import (the mutagenesis of this arginine inhibits importation).

In art the need remains for the alternative oxidase sequences necessary to provide a means to use the alternative oxidase pathway to improve the vigor of the seedlings under cold conditions. In addition, the need persists in the art for means to increase resistance to attack by pathogens, means to generate male sterility, means to improve the efficiency of plant transformation, new selectable markers and means to manipulate the targeting of proteins to mitochondria. The present invention provides these and other advantages. COMPENDIUM OF THE INVENTION. In general, one of the objectives of the present invention is to provide nucleic acids and proteins related to the alternative oxidase 1 of Zea mays, the alternative oxidase 2 of Zea mays and the alternative oxidase 3 of Zea mays, called ZmAOXI, ZmAOX2 and ZmAOX3 , respectively, in this document. Another objective of the present invention is to provide transgenic plants comprising the nucleic acids of the present invention and methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention. Therefore, in one aspect the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide having a specific sequence identity with a polynucleotide encoding a polypeptide of the present invention; (b) 'a polynucleotide that is complementary to the polynucleotide of (a); and (c) a polynucleotide comprising a specific amount of contiguous nucleotides of a polynucleotide of (a) or (b). The isolated nucleic acid can be DNA.

In other aspects the present invention relates to: 1) recombinant expression cassettes, comprising a nucleic acid of the present invention operatively linked to a promoter, 2) a host cell into which the recombinant expression cassette has been introduced and 3) a transgenic plant comprising said recombinant expression cassette. The host cell and plant are optionally a corn cell or a corn plant, respectively. Definitions Units, prefixes and symbols can be expressed in their accepted Sl form. Unless otherwise indicated, nucleic acids are written from left to right, with 5 'to 3' orientation; the amino acid sequences are written from left to right, with amine to carboxyl orientation, respectively. The numerical ranges indicated herein include the numbers that define the range and include each integer within the defined range. In this document, amino acids can be named with the well-known symbols of three letters or with the symbol of a letter recommended by the IUPAC-IUB Biochemical Nomenclature Commission. In the same way, nucleotides can be named by their commonly accepted single-letter codes. Unless otherwise indicated, the terms of computing, electricity and electronics used herein are defined according to The New IEEE Standard Dictionary of Electrical and Electronics Terms (5th Edition, 1993). The terms defined below will be better understood with reference to the specification as a whole. The term "amplified" means the construction of multiple copies of a nucleic acid sequence or multiple complementary copies of a nucleic acid sequence using at least one of the nucleic acid sequences as a template. Amplification systems include the polymerase chain reaction (PCR) system, the ligase chain reaction (LCR) system, amplification based on a nucleic acid sequence (NASBA, Cangene, Mississauga, Ontario ), Q-Beta replicase systems, transcription based amplification (TAS) systems and chain shift amplification (SDA). See, for example, Diagnostic Molecular Microbiology: Principles and Applications, D, H. Persing et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of the amplification is called an amplicon. As used herein, the term "antisense orientation" includes reference to a double polynucleotide sequence that is operably linked to a promoter in the orientation in which the antisense strand is transcribed. The antisense strand is sufficiently complementary to an endogenous transcription product, such that translation of said endogenous transcription product is often inhibited. The terms "coding" or "coding", with respect to a specific nucleic acid, means that it contains the information necessary for translation into a specific protein. The nucleic acid encoding a protein can comprise untranslated sequences (e.g., introns) 'within the translated regions of the nucleic acid or may lack such intervening untranslated sequences (eg, as in the cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid by the "universal" genetic code. However, variants of the universal code can be used, as is the case in some mitochondria of plants, animals and fungi, the bacterium Mycoplasma capricolum or the ciliate Macronucleus, when the nucleic acid is expressed in them. When the nucleic acid is synthetically prepared or altered, the codon preferences of the intended host where the nucleic acid will be expressed can be exploited. For example, although the nucleic acid sequences of the present invention can be expressed in both monocotyledonous and dicotyledonous species, the sequences can be modified to respond to the preferences of specific codops and the GC content preferences in monocots and dicots. since it has been shown that these preferences differ (Murray et al., Nucí Acids Res. 17: 477-498 (1989)). Therefore, the preferred corn codons for a particular amino acid can be derived from known maize gene sequences. The use of corn codons for 28 genes of maize plants is listed in Table 4 of Murray et al., Supra. As used herein, a "full length sequence", relative to a specific polynucleotide or the protein encoded by it, refers to that which possesses an entire amino acid sequence in a native (non-synthetic) manner, endogenous, biologically 'active of the specified protein. Methods for determining whether a sequence is full length are well known in the art including examples of techniques such as Northern or Western blots, primer extension, Sl protection and ribonuclease protection. See, for example, Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Comparison with known full-length homologous sequences (orthologs and / or paralogs) can also be used to identify the full-length sequences of the present invention.

In addition, the consensus sequences typically present in the 5 'and 3' untranslated regions of the mRNA aid in the identification of a polynucleotide as one of full length. For example, an ANNNNAUGG consensus sequence, where the underlined codon represents N-terminal methionine, aids in the determination of whether the polynucleotide has a complete 5 'end; Consensus sequences at the 3 'end, such as polyadenylation sequences, aid in the determination of whether the polynucleotide has a complete 3' end. The term "genetic activity" refers to one or more steps involved in gene expression, including transcription, translation and functioning of the protein encoded by the gene. As used herein, the term "heterologous" refers to a nucleic acid that originates from a foreign species or, if it comes from the same species, is substantially modified with respect to its native form in the composition and / or genomic locus by deliberate human intervention. For example, a promoter operatively linked to a heterologous structural gene is from a different species from which the gene is derived.

Structural or, if it is of the same spice, one or both are modified substantially with respect to their original form. The heterologous protein may originate from a foreign species or, if it is from the same species, is substantially modified with respect to its original form by deliberate human intervention. A "host cell" means a cell that contains a vector and supports the replication and / or expression of said vector. The host cells can be prokaryotic cells, such as E. coli, or eukaryotic cells, such as yeast, insect, amphibian or mammalian cells. It is preferred that the host cells are monocotyledonous or dicotyledonous plant cells. A particularly preferred monocot host cell is a maize host cell. The term "introduced" in the context of insertion of a nucleic acid into a cell means "transfection" or "transformation" or "transduction" and includes any reference to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell, where the Nucleic acid can be incorporated into the genome of the cell (eg, chromosomal, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon or transiently expressed (eg, transfected mRNA). The term "isolated" refers to a material, such as a nucleic acid or a protein, that is: (1) substantially or essentially free of the components that usually accompany or interact with it as it is found in its natural environment. The isolated material optionally comprises material that is not usually found with the material in its natural environment; or (2) if the material is in its natural environment, has been synthetically altered '(not naturally) by deliberate human intervention in a composition and / or has been placed in a locus in the cell (e.g., in the genome or in a subcellular organelle) not native to a material found in that environment. The alteration to obtain the synthetic material can be carried out on the material in its natural state or, removed from it. For example, a natural nucleic acid becomes an isolated nucleic acid if it is altered or produced by non-natural synthesis methods, or if it is transcribed from DNA that has been altered by non-natural, synthetic methods. See, for example, Compounds and Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting in Eukaryotic Cells; Zarling et al., PCT / US93 / 03868. The isolated nucleic acid can also be produced by the synthetic rearrangement (intermixed) of one or several parts of one or more allelic forms of the gene of interest. In the same way, a natural nucleic acid (eg, a promoter) becomes isolated if it is introduced by an unnatural medium into the locus of a non-native genome for said nucleic acid. Nucleic acids that are "isolated" as defined in this documentThey are also called "heterologous" nucleic acids. Unless otherwise indicated, the term "ZmAOXI, ZmAOX2 or ZmAOX3 nucleic acid" means a nucleic acid of the present invention and means a nucleic acid comprising a polynucleotide of the present invention (a polynucleotide "ZmAOXI, ZmAOX2 or ZmAOX3" ) encoder of a ZmAOXI, ZmAOX2 or ZmAOX3 polypeptide. A "gene ZmAOXI, ZmAOX2 or ZmAOX3"is a gene of the present invention and refers to a full-length ZmAAOXI, ZmAOX2 or ZmAOX3 polynucleotide As used herein, the term" nucleic acid "refers to a deoxyribonucleotide or ribonucleotide polymer , or chimeras thereof, either in the form of a single or double chain and, unless otherwise limited, encompasses known analogs possessing the essential nature of natural nucleotides in that they hybridize with single-stranded nucleic acids similar to the way natural nucleotides do (eg, peptide nucleic acids) A "nucleic acid library" means a collection of isolated DNA or RNA molecules that comprises and substantially represents the entire transcribed fraction of an organism's genome The construction of examples of nucleic acid libraries, such as genomic and cDNA libraries, is explained in standard molecular biology references, such as Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology, Vol. 152, Academic Press, Inc., San Diego, CA (Berger); Sambrook et al., Molecular Cloning - A Laboratory Manual, 2nd Ed., Vol. 1-3 (1989); and Current Protocols in Molecular Biology, F.M. Ausubel et al., Eds., Current Protocols, a joint work between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994). As used herein, the term "operably linked" refers to a functional link between a promoter and a second sequence, wherein a promoter sequence initiates and mediates the transcription of a DNA sequence corresponding to said second sequence. In general, "operably linked" means that the nucleic acid sequences that bind are contiguous and, when necessary, join two protein coding regions, contiguous and in the same "reading frame." As used herein, the term "plant" refers to whole plants, plant organs (eg, leaves, stems, roots, etc.), seeds and plant cells and the progeny thereof A plant cell, as used herein, includes, without limitations, seed suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores Plant cells can also include modified cells, such as protoplasts, obtained from the tissues mentioned previously The class of plants that can be used in the methods of the invention is, in general, as broad as the class of higher plants that can be subjected to a to transformation techniques, including both monocotyledonous and dicotyledonous plants. A particularly preferred plant is Zea mays. As used herein, a "polynucleotide" refers to a deoxyribopolinucleotide, ribopolynucleotide or analogs thereof that possess the same essential nature of a natural ribonucleotide, in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence that the natural nucleotides and / or allow the translation in the same amino acids or the natural nucleotides. A polynucleotide can be full-length or a subsequence of a structural or regulatory gene, native or heterologous. Unless indicated otherwise, the term refers to a specific sequence as well as a complementary sequence thereof. Accordingly, the DNA or RNA whose main structures were modified for greater stability or for other reasons are "polynucleotides" according to the meaning intended in this document. Moreover, DNA or RNA comprising uncommon bases, such as inosine, or modified bases, such as tritylated bases, to name but two examples, are polynucleotides as used herein. It will be understood that large modifications have been made to DNA and RNA that serve many useful purposes known to those skilled in the art. The term polynucleotide, as used herein, encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, among others, simple and complex cells. The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms are applied to amino acid polymers in which one or more amino acid residues constitute artificial chemical analogues of the corresponding natural amino acids, as well as to natural amino acid polymers. The essential nature of said natural amino acid analogues is that, when incorporated into a protein, said protein is specifically reactive to antibodies generated against the same protein, but consists of natural amino acids in their entirety. The terms "polypeptide", "peptide" and "protein" also include modifications including, but not limited to, glycosylation, lipid binding, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. In addition, this invention contemplates the use of both methionine-containing variants and the amino-terminal variants without methionine of the protein of the invention. As used herein, the term "promoter" refers to a region of DNA toward the 5 'end of transcription initiation and which is involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells, whether or not it is a plant cell, Examples of plant promoters include, but are not limited to, those obtained from plants, plant viruses and bacteria that contain genes that are expressed in plant cells such as Agrobacterium or Rhizobium Examples of promoters that are under development control include promoters that initiate transcription preferentially in certain tissues, such as leaves, roots, or seeds. known as "with preference for tissues." Promoters that only initiate transcription in certain tissues are called "tissue-specific." A specific promoter of a "cell type" directs primarily expression in certain cell types in one or more organs, For example, vascular cells in roots or leaves An "inducible" or "repressible" promoter is a promoter that It is under environmental control. Examples of environmental conditions that can cause transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue-specific promoters, preferably tissue-specific, cell-type and inducible, constitute the class * of "non-constitutive" promoters. A "constitutive" promoter is a promoter that is active under most environmental conditions. The term "ZmAOXI, ZmAOX2 or ZmAOX3 polypeptide" is a polypeptide of the present invention and refers to one or more amino acid sequences, in their glycosylated or non-glycosylated form. The term also includes fragments, variants, homologs, alleles or precursors (e.g., preproproteins or proproteins) thereof. A "ZmAOXI, ZmAOX2 or ZmAOX3 protein" is a protein of the present invention and comprises a ZmAOXI, ZmAOX2 or ZmAOX3 polypeptide. As used herein, the term "recombinant" refers to a cell or vector that has been modified by the introduction of a heterologous nucleic acid or from which a cell so modified is derived. Thus, for example, recombinant cells express genes that are not found in an identical form within the native (non-recombinant) form of the cell or express native genes that are expressed, abnormally under-expressed otherwise or are not expressed as a result of the deliberate human intervention. The term "recombinant", as used herein, does not cover alteration of the cell or vector by natural events (eg, spontaneous mutation, transformation / transduction / natural transposition) such as those that occur without deliberate human intervention. As used herein, a "recombinant expression cassette" is a recombinant or synthetic generated nucleic acid construct, with a series of specific nucleic acid elements that allow the transcription of a particular nucleic acid in a host cell . The recombinant expression cassette can be incorporated "into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid fragment Typically, the cassette portion of recombinant expression of an expression vector includes, among other sequences, an acid Nucleic to be transcribed and a promoter The terms "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide or peptide (collectively, "protein") The amino acid may be a natural amino acid and, unless otherwise limited, may encompass the unnatural analogs of natural amino acids that can function in a manner similar to natural amino acids s The term "selectively hybridizes" refers to to a hybridization, under stringent hybridization conditions, of a nucleic acid sequence with a white acid sequence nucleic acids specific to a greater degree of detection (for example, at least 2 times with respect to the basal level) than the same hybridization with a non-target or nucleic acid sequence and with the substantial exclusion of non-white nucleic acids. Selective hybridization sequences typically have at least about 80% sequence identity, preferably 90% sequence identity and more preferably 100% sequence identity (i.e., complementarity) with each other. 5 The terms "stringent conditions" or "stringent hybridization conditions" refer to conditions under which a probe will hybridize to its target sequence, up to a greater degree of detection than other sequences (e.g., at least 2-fold with respect to at the basal level). Strict conditions depend on a sequence and will be different under different circumstances. By controlling the severity of the hybridization and / or washing conditions, white sequences can be identified that are 100% complementary to the probe (tests with "homologous" probes) Alternatively, the severity conditions can be adjusted to allow a certain lack of coincidence in the sequences in order to detect lower degrees of similarity (tests with heterologous probes) .In general, a probe is less than 1000 nucleotides in length, optionally less than 500 nucleotides in length.Typically, the conditions of severity are those in which the salt concentration is less than about 1.5 M Na ion, typically 0.01 to 1.0 M Na (or other salts) ion concentration at a pH of 7.0 at 8.3 and the temperature is at least about 30 ° C for short probes (for example, 10 to 50 nucleotides) and at least 60 ° C for long probes (for example, more than 50 nucleotides) The conditions of seve can also be achieved with the addition of destabilizing agents, such as formamide. Examples of low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulfate) at 37 ° C and 1X to 2X SSC wash (SSC 20X = 3 , 0 M NaCl / 0.3 M trisodium citrate) at 50-55 ° C. Examples of conditions of moderate severity include hybridization in formamide 40 to 45%, NaCl 1 M, SDS 1% at 37 ° C and a wash in SSC 0.5X a 1X at 55 - 60 ° C. Examples of conditions of high severity include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37 ° C and a wash in 0.1X SSC at 60-65 ° C. The specificity is typically a function of the post-hybridization washes, the critical factors being the ionic strength and the temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be calculated from the equation of Meinkoth and Wahl, Anal. Biochem., 138: 267-284 (1984): Ym = 81, 5 ° C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% form) - 500 / L, where M is the molarity of the monovalent cations,% GC is the percentage of the guanosine and cytosine nucleotides in the DNA,% form is the percentage of formamide in the hybridization solution and L is the length of the hybrid in base pairs. The Tm is the temperature (under a defined ionic strength and pH) at which 50% of a complementary white sequence is hybridized with a perfectly matching probe. The Tm is reduced by approximately 1 ° C for every 1% of mismatch; therefore, the Tm, the hybridization and / or washing conditions can be adjusted to hybridize with sequences of the desired identity. For example, if you search for sequences with > 90% identity, Tm can be decreased by 10 ° C. In general, the severity conditions are selected such that they are 5 ° C lower than the thermal melting point (Tm) of a specific sequence and its complement at a defined ionic strength and pH. However, conditions of great severity may employ hybridization and / or washing at 1, 2, 3 or 4 ° C below the thermal melting point (Tm); the conditions of moderate severity can employ hybridization and / or washing at 6, 7, 8, 9 or 10 ° C below the thermal melting point (Tm); Low stringency conditions can employ hybridization and / or washing at 11, 12, 13, 14, 15 or 20 ° C below the thermal melting point (Tm). Using the equation, the hybridization and washing compositions and the desired Tm, the specialists will understand that the variations in the severity of the hybridization and / or washing solutions are inherently described. If the degree of mismatch desired results in a Tm less than 45 ° C (aqueous solution) or 32 ° C (formamide solution) it is preferred to increase the concentration of SSC in such a way that a higher temperature can be employed. An extensive guide on nucleic acid hybridization can be found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acids Probes, Part 1, Chapter 2"Overview of Principles of Hybridization and the Strategy of Nucleic Acids Probes Assays", Elsevier, New York (1993); and Current Protocols in Molecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995). As used herein, a "transgenic plant" refers to a plant that contains a heterologous polynucleotide in its genome. In general, the heterologous polynucleotide is stably integrated into the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide can be integrated into the genome alone or as part of a cassette of recombinant expression. The term "transgenic" is used herein for any cell, cell line, callus, tissue, part of a plant or a plant, whose genotype was altered by the presence of the heterologous nucleic acid, including the transgenics so altered initially, as those created by sexual crossings or asexual propagation from the initial transgenic. The term "transgenic" as used herein does not cover alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by natural events, such as random cross-fertilization, non-recombinant viral infection, transformation non-recombinant bacterial, non-recombinant transposition or spontaneous mutation. As used herein, a "vector" refers to a nucleic acid that is used in the transfection of a host cell and into which a polynucleotide can be inserted. Vectors are often replicons. Expression vectors allow the transcription of a nucleic acid inserted therein. The following terms are used to describe the sequence relationships between two or more nucleic acid or polynucleotide sequences: (a) "reference sequence", (b) "comparison window", (c) "sequence identity" and ( d) "percentage of sequence identity". (a) As used herein, a "reference sequence" is a defined sequence that is used as a basis for comparing sequences with a polynucleotide / polypeptide of the present invention. A reference sequence can be a subset or the whole of a specific sequence; for example, as a segment of a full-length gene or cDNA sequence or a complete sequence of the gene or cDNA. (b) As used herein, a "comparison window" includes a contiguous and specific segment of a polynucleotide / polypeptide sequence, wherein the polynucleotide / polypeptide sequence can be compared to a reference sequence and where the of a polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) that are compared to a reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. In general, the comparison window is at least 20 contiguous nucleotides in length and, optionally, may be 30, 40, 50, 100 nucleotides or longer. Those skilled in the art will understand that to avoid a great similarity to a reference sequence due to the inclusion of slits in a polynucleotide sequence, a slit mismatch is typically introduced and subtracted from the number of matches. Methods of sequence alignment for comparison are well known in the art. An optimal sequence alignment for comparison can be carried out with the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2: 482 (1981); with the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48: 443 (1970); with the similarity search method of Pearson and Lipman, Proc. Nati Acad. Sci. 85. 2444 (1988); with computerized implementations of these algorithms, including but not limited to: CLUSTAL in the Intelligenetics PC / Gene program, Mountain View, California, GAP, BESTFIT, BLAST, FASTA and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wisconsin, USA; the CLUSTAL program is very well described by Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al, Computer Applications in the Biosciences 8: 155-65 (1992) and Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994). The BLAST family of programs that can be used for database similarity searches includes: BLASTN for queries of nucleotide sequences against nucleotide sequence databases; BLASTX for queries of nucleotide sequences against protein sequence databases; BLASTP for queries of 'protein sequences against protein sequence databases; TBLASTN for queries of sequences of proteins against databases of nucleotide sequences and TBLASTX for queries of nucleotide sequences against databases of nucleotide sequences. See, Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995). Unless indicated otherwise, the identity / sequence similarity values provided refer to the value obtained using the BLAST 2.0 program set using the predetermined parameters. Altschut et al., J. Mol. Biol., 215: 403-410 (1990); Altschut et al. in Nucleic Acids Res. 25: 3389-3402 (1997). The software to perform BLAST analyzes are public knowledge, eg. through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high-qualification sequence pairs (HSP) by identifying short words of length W in the query sequence, which either match or satisfy some positively rated T-limit scores when they are aligned with a word of the same length in a sequence of the database. T is defined as the qualification limit of a neighboring word. These initial hits of neighboring words act as seeds to initiate searches in order to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for all that is possible to increase the cumulative alignment qualification. Cumulative ratings are calculated using nucleotide sequences, M parameters (score compensated for a pair of matching residues, always> 0) and N (mismatch score for non-matching residues, always <0) . For the amino acid sequences, a rating matrix is used to calculate the accumulated score. The extension of word hits in each direction stops when: the cumulative alignment score is set aside in the amount X of the maximum value achieved; the cumulative rating reaches zero or less due to the accumulation of one or more alignments of negative rating residues; or the end of any of the sequences is reached. The W, T and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, a hope (E) of 10, a cutoff value of 100, M = 5, N = -4 and a comparison of both chains. For the amino acid sequences, the BLASTP program uses a word length (W) of 3, a hope (E) of 10 and the qualification matrix BLOSUM62 as default values (see Henikoff &Henikoff (1989) Proc. Nati. Acad. Sci. USA, 89: 10915). In addition to calculating the percent identity of the sequence, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & amp;; Altschul, Proc. Nat'l. Acad. 'Sci. USA 90: 5873-5787 (1993)). One measure of the similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which indicates the probability that a match between two nucleotide or amino acid sequences will occur randomly. Searches for BLAST assume that proteins can be modeled as random sequences. However, many real proteins comprise regions of non-random sequences which may be homopolymeric pathways, short-period repeats or regions enriched in one or more amino acids. Said regions of low complexity can be aligned between unrelated proteins even when other regions of the protein are totally dissimilar. A number of low complexity filter programs can be employed to reduce such low complexity alignments. For example, the low-complexity SEC filters can be used (Wooten and Federhen, Comput, Chem., 17: 149-163 (1993)) and XNU (Claverie and States, Comput, Chem., 17: 191-201 (1993)). )) alone or in combination. GAP can also be used to compare a polynucleotide or a polypeptide of the present invention with a reference sequence. GAP employs the algorithm of Needleman and Wunsch (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of mismatches. GAP takes into account all possible alignments and positions of matches and creates an alignment with the most matching bases and the least number of mismatches. Allows the creation of a mismatch and an extension of mismatch "in matching base units, GAP must get a gain of the number of matches for each mismatch it inserts. that zero, GAP must, in addition, achieve a profit for each inserted mismatch that consists of the 'length of the mismatch multiplied by the extent of the mismatch. The default values for the creation of mismatches and the mismatch extension in Version 10 of the Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively. For nucleotide sequences, the penalty for creating a mismatch is 50, while the mismatch extension is 3. The penalties for creating a mismatch and an extension of mismatch can be expressed as a whole number selected from the group of integers consisting of 0 to 1000. Thus, for example, the creation of mismatch and the extension of mismatch may each be independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60 or greater. GAP shows a member of the family of best alignments. This family can consist of many members, but no other member is of better quality. GAP shows four values of merit for the alignments: Quality, Relation, Identity and Similarity. Quality is the maximized metric to align the sequences. The relationship is quality divided by the number of bases in the shortest segment. The percentage of identity is the percentage of symbols that really coincide. The percentage of similarity is the percentage of symbols that are similar. The symbols passing through the mismatches are not taken into account. A similarity is scored when the value of the qualification matrix for a pair of symbols is greater than or equal to 0.50, such as the similarity threshold. The scoring matrix used in Version 10 of the Wisconsin Genetics Software Package is BLOSUM62 (see Henikoff &Henikoff (1989) Proc Nati Acad Sci USA, 89: 10915). (c) As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences refers to the residues in the two sequences that are the same when aligned by correspondence maximum in a specific comparison window. When the percentage of sequence identity is used with reference to proteins it will be understood that the positions of the residues that are not identical often differ by conservative substitutions of amino acids, where the amino acid residues are replaced by other amino acid residues with similar chemical properties ( example, loading or hydrophobicity) and therefore does not change the functional properties of the molecule. When the sequences differ by conservative substitutions, the percentage of sequence identity can be adjusted upward in order to correct the conservative nature of the substitution. It is said that the sequences that differ by said conservative substitutions possess "sequence similarity" or "similarity". The means for effecting this adjustment are well known to those skilled in the art. Typically, it involves qualifying a conservative substitution as a partial mismatch rather than a complete one, thereby increasing the percentage of sequence identity. Thus, for example, when an identical amino acid receives a rating of 1 and a non-conservative substitution receives a rating of zero, the conservative substitution receives a rating between zero and 1. The qualification of conservative substitutions is calculated, for example, in accordance with the algorithm of Meyers and Miller, Computer Applic. Biol. Sci, 4: 11-17 (1988), for example, as implemented in the PC / GENE program (Intelligenetics, Mountain View. 'California, USA). (d) As used herein, the "percent sequence identity" means the value determined by comparison of two sequences aligned optimally in a comparison window, where the portion of a polynucleotide sequence in the window of comparison may include additions or deletions (ie, slits) compared to a reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in which the acid base or amino acid residue appears in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage of sequence identity. DETAILED DESCRIPTION OF THE INVENTION Introduction The present invention provides, among others, compositions and methods for modulating (i.e., increasing or decreasing) the level of the polynucleotides and polypeptides of the present invention in plants. In particular, the polynucleotides and polypeptides of the present invention can be expressed temporally or spatially, for example, at developmental stages, in tissues and / or in amounts, which are not characteristic of plants that were not manipulated recombinantly. Accordingly, the present invention is useful in examples of applications such as the improvement of cold tolerance in plants, the improvement in disease resistance in plants, the achievement of male sterility in plants, the development of selectable genetic markers for the production of transgenic plants and the manipulation of the targeting of proteins towards the mitochondria.

Cold Tolerance The present invention can be used to improve cold tolerance in plants. The coding regions of any of the three alternative corn oxidase genes will be expressed in the transgenic plant, such as corn, under the direction of promoters with preference for tissues and development that cause a high level of expression in seedlings. Two of these promoters are those of the alpha-amylase genes regulated by GA and of a cysteine protease, which causes a high level of seed expression (scutellum and aleurone). A thpromoter, of the betaglucosidase gene (Glu1), causes a high level of expression in the mesocotil, coleoptile and young leaves of the seedlings. Those skilled in the art will understand that it is also possible to use other promoters. After germination, these plants have high levels of alternative oxidase and a better tolerance to cold, which is manifested as a better and more complete establishment of the plants when the germination was in cold lands or under cold climatic conditions.

Alternatively, a modified coding region of any of the alternative oxidase genes can be used to produce an alternative oxidase with a higher intrinsic activity in the same process as previously described. This is carried out using a targeted protein manipulation strategy comprising the alteration of one or both cysteine residues at amino acid positions 120 and 170 in ZmAOXI and 102 and 152 in ZmAOX2 with an amino acid not containing sulfhydryl, such as Serine These cysteine residues are responsible for the dimerization of the alternative oxidase. The dimeric form is less active than the monomeric form and is also less susceptible to pyruvate stimulation. After re-introducing the modified alternative oxidase gene, increased activity of the alternative oxidase and greater tolerance to cold will be observed. Since the enzyme will be more intrinsically active, other preferred / seed-specific promoters may be used that do not necessarily exhibit such high levels of expression. Another option to modify any of the alternative oxidase genes to produce an alternative oxidase with higher intrinsic activity is the use of a targeted protein manipulation strategy comprising the intermixing of the alternative corn oxidase sequences together with coding regions or synthetic oligonucleotides of other alternative plant and non-plant oxidases. The highest activity can be evaluated first by means of complementary microbes, such as E. coli or yeast, whose own alternative oxidases are mutated, with the modified alternative oxidases. It is expected that those who were supplemented with more active alternative oxidases will grow more actively in vitro. The recovered clones are then introduced into the plant. Again, since the enzyme will be more active, other preferred / seed-specific promoters may be employed that do not necessarily exhibit such high levels of expression. Improvement of disease resistance The present invention can also be used to improve resistance to diseases in plants. You can use any of the three 'alternative corn oxidase sequences to improve resistance to diseases in plants caused by various pathogens. An example of a plant is corn. Examples of pathogens include, but are not limited to, viral pathogens. In preferred embodiments, this invention improves inducible resistance to pathogens, but it can also be used in constitutive resistance mechanisms. The inducible resistance mechanisms comprise an improvement in the level and synchronization of the alternative oxidase expression after an attack by pathogens in order to increase the expression and, therefore, the resistance. The present invention can be used in any of several ways to achieve it. A. Native ZnlAOX The coding regions of any of the alternative corn oxidase genes can be expressed in a transgenic plant under the direction of pathogen-inducible promoters that cause high expression (when possible, very high expression) after the attack by pathogens. Corn is a particularly preferred transgenic plant. Examples of implantable promoters include the inducible maize promoters that are described in U.S. Pat. N °: 09 / 257,583 with the title "Inducible Maize Promoters", presented on February 25 *, 1999. The specialists will understand that it is possible to use other promoters, including promoters who have a certain preference for tissues or development for their expression, that They will focus their induction capacity on tissues or stages particularly susceptible to the pathogen of interest.

'After the attack by pathogens, these transgenic plants have high levels of alternative oxidase and a greater resistance to diseases.

B. Site-specific genetically engineered ZmAOX Alternatively, the coding region of any of the alternative oxidase genes can be modified to produce an alternative oxidase with a higher intrinsic activity. This is carried out by a targeted protein targeting strategy of, for example, one or both cysteine residues at amino acid positions 120 and 170 in ZnlAOX 1 and 102 and 152 in ZmAOX2, with an amino acid not containing sulfhydryl, such as serine. These cysteine residues help in the dimerization of the alternative oxidase. The dimeric form is less active than the monomeric form and is also less susceptible to stimulation with pyruvate.

After the re-introduction, the alternative oxidase gene modified in the manner described, greater activity of the alternative oxidase and greater resistance to diseases are observed. As the aforementioned enzyme is more active, other preferred / seed-specific promoters may be employed which do not necessarily exhibit such high expression levels. C. ZmAOX of intermixed sequence Alternatively, the coding region "of any of the alternative oxidase genes can be modified to produce an alternative oxidase with a higher intrinsic activity.This is carried out by means of a targeted manipulation strategy. of proteins comprising the intermixing of the sequences of these two alternative corn oxidase 'together with coding regions or synthetic oligonucleotides of other alternative plant and non-vegatable oxidases. The highest activity can be evaluated first by means of complementary microbes, such as E. coli or yeast, whose own alternative oxidases are mutated, with the modified alternative oxidases. Those that were supplemented with alternative oxidases will grow more actively in vitro. The recovered clones are then introduced into the plant as previously described. The enzyme will be more active, therefore, other preferred / specific promoters of seedlings can be used that do not necessarily have such high expression levels. The use of the coding regions of the native versions, manipulated in a site-specific manner or of interspersed sequences of the alternative corn oxidases, allows non-inducible resistance to be achieved by directing its expression with a promoter that ensures the appropriate expression levels of oxidase. alternative in the tissue and / or the desired stages of development. Male sterility The present invention provides methods for the creation of transgenic plants of male sterility in order to obtain hybrid seeds.

The coding region of one of the genes of the alternative corn oxidase described herein is used. A. Mutagenesis It can be mutated to any of the alternative corn oxidase genes so that it is not functional. One of the methods that can be used to achieve this is an insertion mutagenesis of transposons. Those skilled in the art will understand that other known methods can be employed. When one or more of the alternative corn oxidase genes are normally expressed in the spindle, mutagenesis can result in male sterility. This can be useful in the creation of hybrids; the male parent will donate a functional copy of the gene so that the resulting hybrid plant is fertile. B. Antisense Any of these alternative corn oxidase genes can be expressed in an antisense configuration under the direction of a spike-specific promoter. Male sterility is achieved when the expression of the alternative oxidase in the spout is sufficiently reduced. This strategy requires a restorer gene in the hybrid plant to counteract the suppression of antisense expression. Selectable genetic markers for the production of transgenic plants Alternative oxidase can be used as a selectable marker. For example, it has been shown that tobacco cells treated with cytochrome airway inhibitors, such as potassium cyanide, can survive thanks to the respiration of the alternative oxidase pathway, but with a slower growth rate. Said cyanide treated tobacco cells grew faster when they were transformed with the alternative oxidase gene under the direction of the 35S promoter which causes a high (higher than normal) expression of the alternative oxidase gene (Vanlerberghe et al., 1997a), due to an increase in breathing carried out by the alternative route. The alternative oxidase genes, in particular the maize genes described herein, can be used as selectable markers. In this invention, one or more of the ZmAOX genes can be cotransformed with the transformation gene of interest. The ZmAOX gene will be under the direction of a very active promoter. The cells, tissue or calluses of faster growth will then be subcultured, since they represent the tissue transformed with success. Ideally, the promoter will be preferentially active during the early stages of development or cultivation and will have a minor impact later in development or even in successive generations. There are several known inhibitors of alternative oxidase, among which can be mentioned SHAM (salicilhidroxamic acid) and disulfuram. It is possible to create a variant of alternative corn oxidase genes that is resistant to these inhibitors. This variant can be created in one of the following ways: a) directed manipulation of proteins to modify specific sites or structures involved in the susceptibility to these inhibitors; b) intermixing sequences to create such resistant versions, followed by selection to achieve resistance in E. coli or yeast mutants lacking the function of the alternative oxidase; or c) with more conventional mutagenesis, such as by EMS, etc., followed by selection to achieve resistance in E. coli or yeast mutants lacking the function of the alternative oxidase. The resistant variant, under the direction of a moderately strong constitutive promoter, although the native ZmAOX promoter may be sufficient and its use later in development, is reintroduced into the plant during a cotransformation process with the 'transformative gene of interest. The selection medium will contain an inhibitor (like SHAM or disulfuram) and the cells that grow are those that were transformed with the gene of interest and linked to the construction with the alternative oxidase selectable marker. This selection is even more effective when SHAM or disulfuram is added to inhibit the alternative pathway plus an inhibitor of the cytochrome pathway, such as cyanide or antimycin A. Under these conditions, respiration only occurs through the alternative oxidase resistant to inhibitor and only those cells that were transformed with it will survive. In addition to its function as a potential selectable marker, the alternative oxidase transformation strategies previously described are also useful for improving the frequency of transformation. The alternative oxidase pathway is more active after various types of stress. Bombardment of particles, a commonly used transformation technique, is undoubtedly stressful for tissue. The alternative route is more active in bombed tissues. This greater activity is adaptive in that it contributes to the reduction of reactive oxygen species that accumulate after different types of stress, such as bombardment, and causes cell de and decreases transformation efficiency. An increase in the alternative oxidase pathway after transformation, as by one of the described methods or by a high transient expression of the coding region of the gene by inclusion thereof in the form of DNA or RNA on the transforming particles, then improves the rates of cell survival, and consequently the frequency of transformation. Manipulation of protein targeting to mitochondria The present invention provides transit peptides for the alternative oxidase genes. The transit peptides direct these and other proteins towards the mitochondria. As transit peptides are an important determinant to direct proteins towards mitochondria, the coding region of the transit peptides can be used for any of the genes of the present invention to genetically manipulate the targeting of other proteins towards the mitochondria. This is achieved by fusing the coding region of the transit peptide to the N-terminal end of the mitochondria-bound protein. After translation, the chimeric protein is directed to the mitochondria and after entering them, said transit peptide is proteolytically separated by the proteases present in the mitochondria and then the released protein resides and functions in the mitochondria. According to the protein involved, said genetic manipulation of the protein targeting to the mitochondria is useful in various areas including, but not limited to, resistance to herbicides, selectable markers of transformation, production of metabolites, male sterility (and restoration of fertility). ), cell cycle control and apoptosis. With respect to the use of selectable markers, it should be borne in mind that many antimicrobial / antipyretic compounds / drugs are known. Many of them affect translation in prokaryotes. The mitochondria and plant chloroplasts are relatively similar to prokaryotes in terms of their translation apparatus, since they were apparently incorporated by a primitive eukaryotic cell by an endosymbiosis. The mitochondrial transit peptide of the oxidase genes * Alternative can be used to direct the genes that encode resistance to these drugs to the mitochondria. They can be used as such as selectable markers in plant transformation.

It should be taken into account that although the transit peptide alone can direct the proteins towards the mitochondria, no single transit peptide will direct all the proteins towards the mitochondria. There are other factors, such as the structure of the mature peptide region, which will cause the 5 transit peptides to have less or greater capacity to direct the protein towards the mitochondria. Moreover, a comparison between closely related AOX proteins, as are these alternative oxidases of various plant species, indicates that divergent transit peptide sequences exist, both within a species and between species. The present invention also provides isolated nucleic acids containing polynucleotides of sufficient length and complementarity with a gene of the present invention to allow their use as probes or amplification primers in the detection, quantification or isolation of gene transcripts. For example, nucleic acids isolated from the i- '> present invention can be used as probes for the detection of deficiencies at the level of mRNA in the search of the desired transgenic plants, for the detection of mutations in the gene (for example, substitutions, deletions or additions), for the monitoring of "the supersensitization of expression or changes in enzymatic activity in compound search assays, for the detection of any number of allelic variants (polymorphisms), orthologs or gene paralogs or for site-directed mutagenesis in eukaryotic cells (see, for example, 'U.S. Patent No.: 5,565,350). The isolated nucleic acids of the present invention can also be used for the recombinant expression of the polypeptides encoded by them or for their use as immunogens in the preparation and / or the search for antibodies. The isolated nucleic acids of the present invention can also be used for the deletion in the sense of the reading or antisense framework of one or several genes of the present invention. The binding of chemical agents that bind, intercalate, clone and / or crosslink with the isolated nucleic acids of the present invention can also be used to modulate transcription or translation. The present invention also provides isolated proteins comprising a polypeptide of the present invention (e.g., preproenzymes, proenzymes or enzymes). The present invention also provides proteins comprising at least one epitope of a polypeptide of the present invention. The proteins of the present invention can be used in assays for enzymatic agonists or antagonists of enzyme function or for use as immunogens or antigens in order to obtain antibodies specifically immunoreactive with the protein of the present invention.

Said antibodies can be used in the assays for expression levels, to identify and / or isolate the nucleic acids of the present invention from the expression libraries for the identification of homologous polypeptides from other species or for the purification of polypeptides of the present invention. invention. The isolated nucleic acids and polypeptides of the present invention can be used with a wide range of plant types, in particular with 'monocotyledons such as the species of the family Gramineae which include Hordeum, Sécale, Triticum, Sorghum (for example, S. bicolor) and Zea (for example Z. mays). The nucleic acid and the isolated proteins of the present invention can also be used with species of the genera: Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Triganella, Vigna, Citrus, Linum, Geranium, Manihot , Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyosyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis- Pelargoniun7, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cuczanis, Browallia: Glvchie, Pisum, Phaseolus, Lolium, Oryza and Oats. Nucleic Acids The present invention provides, inter alia, nucleic acids isolated from RNA, DNA and analogues and / or chimeras thereof, comprising a polynucleotide of the present invention. A polynucleotide of the present invention includes: (a) a polynucleotide encoding a polypeptide of SEQ ID NOS: 2, 3, 5, 6, 8 including the examples of polynucleotides of SEQ ID NOS: 1, 4, 7; (b) a polynucleotide that is the product of the amplification of a Zea mays nucleic acid library using primer pairs that selectively hybridize under stringent conditions with loci within a polynucleotide selected from the group consisting of SEQ ID NO: 1 , 4, 7; (c) a polynucleotide that selectively hybridizes with a polynucleotide of (a) or (b); (d) a polynucleotide having a specific sequence identity with the polynucleotides of (a), (b) or (c); (e) a polynucleotide that encodes a protein containing a specific amount of contiguous amino acids of a prototype polypeptide, wherein the protein is recognized specifically by antisera generated by presentation of the protein and where the protein does not exhibit a detectable immunoreactivity with antiserum that was completely immunosorbed with the protein; (f) polynucleotide sequences complementary to (a), (b), (c), (d) or (and); (g) polynucleotides comprising the sequences obtained from the clones deposited in a bacterial host in the American Type Culture Collection (ATCC) on January 14, 2000, and which received Accession No. PTA-1209; and (h) a polynucleotide comprising at least a specific amount of contiguous nucleotides of a polynucleotide of (a), (b), (c), (d), (e), (0 or (g). Polynucleotides that encode a polypeptide of the present invention As indicated in (a), previously, the present invention provides isolated nucleic acids comprising a polynucleotide of the present invention, wherein the polynucleotide encodes a polypeptide of the present invention. each nucleic acid sequence encoding a polypeptide also describes, by reference the genetic code, each possible silent variation of the nucleic acid.The skilled person will understand that it is possible to modify each codon in a nucleic acid (except AUG, which is usually the only codon for methionine and UGG, which is usually the only codon for tryptophan) to obtain a functionally identical molecule, therefore, each silent variation of a nucleic acid and encodes a polypeptide of the present invention is implicit in each described polypeptide sequence and is within the scope of the present invention. Therefore, the present invention includes the polynucleotides of SEQ ID NOS: 1, 4 or 7 and the polynucleotides encoding the polypeptides of SEQ ID N °: 2, 3, 5, 6 or 8. ß. Amplified Polynucleotides from a Zea mays Nucleic Acid Library As indicated in (b), previously, the present invention provides an isolated nucleic acid comprising a polynucleotide of the present invention, wherein the polynucleotides are amplified from a library of nucleic acids of Zea mays. The lines B73, PHRE1, A632, BMS-P2 # 10, W23 and Mo17 of Zea mays are known and available to the public. Other corn lines known to the public and available can be obtained from Maize Genetics Cooperation (Urbana, IL). The nucleic acid library can be a cDNA library, a genomic library or a library constructed in general from nuclear transcripts at any stage of introns processing. The cDNA libraries are normalized to increase the representation of relatively rare cDNAs. In optional embodiments, the DNA library is constructed using a full-length cDNA synthesis method. Examples of such methods include "Oligo Capping" (Maruyama, K. and Sugano, S. Gene 138: 171-174, 1994), biotinylated CAP sequestrant (Caminci, P., Kvan, C, et al.

Genomics 37: 327-336, 1996) and CAP retention procedure (Edery, E., * Chu, L. L., et al. Molecular and Cellular Biology 15: 3363-3371, 1995). The synthesis of cDNA is catalyzed, preferably, at 50-55 ° C to prevent the formation of the secondary structure of the RNA. Examples of reverse transcriptases that are relatively stable at these temperatures are: SuDerScript II reverse transcriptase (Life Technologies, Inc.), AMV reverse transcriptase (Boehringer Mannheim) and RetroAmp reverse transcriptase (Epicenter). Preferably, rapidly growing tissues or rapidly dividing cells are used as sources of mRNA. A preferred source of tissues from which it is possible to isolate alternative oxidase mRNAs are corn seedlings with cold stress (for example, seedlings in step V3 treated for a maximum of 24 hours at 10 ° C). Another preferred tissue source is maize leaves infected with pathogens (for example, leaves of V6 plants 48 hours after inoculation with conidia of Cochliobolus heterostrophus). Another preferred source of tissues are corn spikelets in the early stages of pollen release. The present invention also provides subsequences of the polynucleotides of the present invention. Various subsequences can be obtained using primers that hybridize selectively, under stringent conditions, with at least two sites within the polynucleotides of the present invention or with two sites within the flanking nucleic acid and containing a polynucleotide of the present invention or with a site within a polynucleotide of the present invention and a site within the nucleic acid containing it. The primers are chosen to hybridize selectively, under stringent hybridization conditions, with a polynucleotide of the present invention. In general, the primers are complementary to 'a sub-sequence of the nucleic acid of interest that they amplify, but may have a sequence identity ranging from about 85% to 99% relative to the sequence of polynucleotides for which they were designed to be aligned. As will be understood by those skilled in the art, the sites with which the primer pairs are to selectively hybridize are chosen such that a unique contiguous nucleic acid can be formed under the desired amplification conditions. In optional embodiments, the primers are to be constructed in such a way that they selectively hybridize under stringent conditions with a sequence (or with the complement thereof) within the nucleic acid of interest that contains the codon encoding the carboxy or amino acid amino acid residue. terminal (i.e., the 3 'terminal coding region and the 5' terminal coding region, respectively) of the polynucleotides of the present invention. Optionally, in these embodiments, the primers will be constructed to be selectively hybridized completely within the coding region of the interest polynucleotide of the present invention, such that the amplification product of a blank cDNA will consist of the coding region of said cDNA. The length of the primer expressed in nucleotides is selected from the group of integers consisting of at least 15 to 50. Thus, the primers can be at least 15, 18, 20, 25, 30, 40 or 50 nucleotides of length. Those skilled in the art will understand that an elongated primer sequence can be employed to increase the binding specificity (i.e., alignment) with a target sequence. A sequence that does not align at the 5 'end of the primer (a "tail") can be added, for example, to introduce a cloning site at the terminal ends of the amplicon. The amplification products can be translated using expression systems well known to those skilled in the art and as will be discussed, infra. The resulting translation products can be confirmed as polypeptides of the present invention by, for example, evaluation of the appropriate catalytic activity (e.g., specific activity and / or substrate specificity) or by verification of the presence of one or more linear epitopes that are specific for a polypeptide of the present invention. Methods for the synthesis of proteins from hardened derivatives of a PCR are known in the art and are commercially available. See, for example, Amersham Life Sciences, Inc., Catalog '97, p. 354. Methods for obtaining 5 'and / or 3' ends of the insert of a vector are well known in the art. See, for example, RACE (Rapid Amplification of Complementary Ends) described in Frohman, M.A., in PCR Protocols: A Guide to Methods and Applications, M.A. Innis, D, H. Gelfand, J.J. Sninsky, T.J. White, Eds. (Academic Press, Inc., San Diego, 1990), p. 28-38.); see also, U.S. Pat. No. 5,470,722 and Current Protocols in Molecular Biology, Unit 15.6, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995) Frohman and Martin, Techniques 1: 165 (1989). C. Polynucleotides that selectively hybridize with a polynucleotide of (A) or (B) As previously indicated in (c), the present invention provides isolated nucleic acids comprising the polynucleotides of the present invention, wherein the polynucleotides are selectively hybridized, low 'Selective hybridization conditions, with one of the polynucleotides of paragraphs (A) or (B) described above. Accordingly, the polynucleotides of this embodiment can be used to isolate, detect and / or quantify nucleic acids comprising the polynucleotides of (A) or (B). For example, the polynucleotides of the present invention can be used to identify, isolate or amplify partial or full length clones in a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated or complementary in some other way from a dicotyledonous or monocotyledonous nucleic acid library. Examples of dicotyledonous and monocotyledonous species include, but are not limited to, corn, barley, soybeans, cotton, wheat, sorghum, sunflower, alfalfa, oats, sugar cane, millet, barley and rice. Optionally, the cDNA library comprises at least 30% to 95% of full length sequences (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of full-length sequences.CND libraries can be normalized to increase the representation of rare sequences.Hybridization conditions of low severity are typically, but not exclusively, used with sequences that have a reduced sequence identity relative to the complementary sequences, moderate and high severity conditions can be used optionally for the sequences of greater identity.The low severity conditions allow a selective hybridization of the sequences that possess approximately 70% to 80% of sequence identity and that can be used to identify orthologous sequences or paralogs D. Polynucleotides that have a specific sequence identity with the polynucleotides of (A), (B) or (C) As indicated or previously in (d), the present invention provides isolated nucleic acids comprising the polynucleotides of the present invention, wherein the polynucleotides possess a specific identity at the nucleotide level with one of the polynucleotides described above in paragraphs (A), (B) ) or (C). The identity can be calculated using, for example, the BLAST or GAP algorithms with the default values. The percent identity with a reference sequence is at least 60% and, rounded to the nearest whole number, can be expressed as an integer selected from the group of integers consisting of 60 to 99. Thus, for example, the percent identity with a reference sequence can be at least 70%, 75%, 80%, 85%, 90% or 95%. Optionally, the polynucleotides of this embodiment will encode a polypeptide that shares an epitope with a polypeptide encoded by the polynucleotides of (A), (B) or (C). Accordingly, these polynucleotides encode a first polypeptide that allows to produce antisera that contain antibodies that are specifically reactive with the second polypeptide encoded by a polynucleotide of (A), (B) or (C). However, the first polypeptide does not bind antisera generated against itself when said antisera have been completely immunoabsorbed with the first polypeptide. Therefore, the polynucleotides of this embodiment can be used to generate antibodies that can be used, for example, in the search in libraries of expression of the nucleic acids comprising the polynucleotides of (A), (B) or (C) or for the purification of, or in immunoassays for the polypeptides encoded by the polynucleotides of (A), (B) or (C). The polynucleotides of this embodiment encompass nucleic acid sequences that can be employed for selective hybridization to a polynucleotide encoding a polypeptide of the present invention. The search for polypeptides for specific binding to antisera can be conveniently carried out using peptide expression libraries. This method comprises the search in large collections of peptides of individual members having the desired function or structure. The search for antibodies in peptide expression libraries is well known in the art. The expression sequences of the peptides can be from 3 to 5000 or more amino acids in length, often 5-100 amino acids in length and often from 8 to 15 amino acids in length approximately. In addition to direct chemical synthesis methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves the expression of a peptide sequence on the surface of a bacteriophage or a cell. Each bacteriophage or cell contains the nucleotide sequence that encodes the particular expressed peptide sequence. These methods are described in the Publications of PCT Patent No.: 91/172711, 91/18980, 91/19818 and 93/08278. Other systems for generating peptide libraries contain aspects of both in vitro chemical synthesis and recombinant methods. See, PCT Patent Publications No.:92/05258, 92/14843 and 96/20078. See also, U.S. Pat. No. 5,658,754; and 5,643,768. Peptide expression libraries, vectors and sets of elements for searching are commercially available from suppliers such as Invitrogen '(Carlsbad, CA).

E. Polynucleotides that encode a protein containing a subsequence of a polypeptide prototype and that cross-react with the polypeptide prototype. As previously indicated in (e), the present invention provides isolated nucleic acids comprising the polynucleotides of the present invention. , wherein the polynucleotides encode a protein having a subsequence of contiguous amino acids of a prototype polypeptide of the present invention, such as those previously provided in (a). The length of contiguous amino acids of the polypeptide prototype is selected from the group of integers consisting of at least 10 up to the number of amino acids of the sequence prototype. Thus, for example, the polynucleotide can encode a polypeptide having a subsequence that has at least 10, 15, 20, 25, 30, 35, 40, 45 or 50 contiguous amino acids of the polypeptide prototype. In addition, the amount of said subsequences encoded by a polynucleotide of this embodiment can be any integer selected from the group consisting of 1 to 20, such as 2, 3, 4 or 5. The subsequences can be separated by any integer number of nucleotides of 1 up to the amount of nucleotides present in the sequence, such as at least 5, 10, 15, 25, 50, 100 or 200 nucleotides. The proteins encoded by the polynucleotides of this embodiment, when presented as an immunogen, cause the production of polyclonal antibodies that specifically bind to a prototype of 'polypeptide, such as, but not limited to, a polypeptide encoded by the polynucleotide of (a) or (b), above. In general, however, a protein encoded by a polynucleotide of this embodiment does not bind with antisera raised against the polypeptide prototype when said antisera have been completely immunoabsorbed with the polypeptide prototype. Methods for making and evaluating the specificity / binding affinity of antibodies are well known in the art. Examples of immunoassay formats include ELISA, competitive immunoassays, radioimmunoassays, Western blots, indirect immunofluorescence assays and the like. In a preferred evaluation method, fully immunoabsorbed and pooled antisera, which were generated against the prototype polypeptide, can be used in a competitive binding assay to evaluate the protein. The concentration of polypeptide prototype required to inhibit 50% of the binding of the antisera to the polypeptide prototype is determined. If the amount of protein needed to inhibit the binding is less than twice the amount of protein prototype, then the protein is said to bind specifically with the antisera raised against the immunogen. Accordingly, the proteins of the present invention encompass allelic variants, conservatively modified variants and minor recombinant modifications of the polypeptide prototype. The polynucleotide of the present invention optionally encodes a protein having a molecular weight of non-glycosylated protein within the % of the molecular weight of the full length non-glycosylated polypeptide of the present invention. The molecular weight can be easily determined by 'SDS-PAGE under reducing conditions. Optionally, the molecular weight is within 15% of that of a full-length polypeptide of the present invention, more preferably within 10% or 5% and more preferably even within 3%, 2% or 1% of the a full-length polypeptide of the present invention. Optionally, the polynucleotides of this embodiment can encode a protein having a specific enzymatic activity of at least 50%, 60%, 80% or 90% of a cell extract comprising the native, endogenous, full-length polypeptide of the present invention. In addition, the proteins encoded by the polynucleotides of this embodiment may optionally have an affinity constant (Km) and / or a catalytic activity (i.e., the microscopic rate constant (kcat) substantially similar to those of the native endogenous protein of It will be understood by those skilled in the art that the kcat / Km value determines the specificity for competitive substrates and is often referred to as the specificity constant.The proteins of this embodiment may have a value of kcat / Km which represents minus 10% of the value of the full length polypeptide of the present invention determined using the endogenous substrate of that polypeptide Optionally, the value of kcat / Km will represent at least 20%, 30%, 40%, 50% and, more preferably, at least 60%, 70%, 80%, 90% or 95% of the k ^ / Km value of the full-length polypeptide of the present invention. The values of kcat, Km and kcat / Km can be made by any of the means well known to those skilled in the art. For example, the initial velocities (that is, the first 5% or less of the reaction) can be determined using techniques from 'rapid sampling and mixing (for example, continuous flow techniques, stopped flow or rapid stop), instantaneous photolysis or relaxation methods (for example, temperature jumps) together with examples of measurement methods such as spectrophotometry, spectrofluorometry, magnetic resonance nuclear or radioactive procedures. The kinetic values are conveniently obtained using a Lineweaver-Burk or Eadie-Hofstee graphic. F. Polynucleotides complementary to the polynucleotides of (A) - (E) s As previously indicated in (f), the present invention provides isolated nucleic acids comprising polynucleotides complementary to the polynucleotides of paragraphs A-E, above. It will be understood by those skilled in the art that the complementary sequences are matched at their bases along the length of their length with the polynucleotides of paragraphs (A) - (E) (ie they possess 100% sequence identity throughout. the length).

The complementary bases are associated by means of hydrogen bonds in double-stranded nucleic acids. For example, the following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil. G. Polynucleotides that are subsequences of the polynucleotides of 5 (A) - (F) As previously indicated in (g), the present invention provides isolated nucleic acids comprising polynucleotides comprising at least 15 contiguous bases of the polynucleotides the paragraphs (A) a * (F) mentioned above. The length of the polynucleotide is expressed as an integer selected from the group consisting of at least 15 up to the length of the nucleic acid sequence of which the polynucleotide is a subsequence. Thus, for example, the polynucleotides of the present invention 'include polynucleotides comprising at least 15, 20, 25, 30, 40, 50, 60, 75 or 100 contiguous nucleotides in length of the polynucleotides of (A) - (F). Optionally, the amount of said subsequences encoded by a polynucleotide of this embodiment can be any integer selected from group 1 to 20, such as 2, 3, 4 or 5. The subsequences can be separated from each other by any integer number of nucleotides, 1 up to the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100 or 200 nucleotides. The subsequences of the present invention may comprise structural features of the sequence from which they are derived. Alternatively, the subsequences may lack certain structural features of the longer sequence from which they are derived such as the poly (A) tail. Optionally, the subsequence of a polynucleotide encoding a polypeptide that possesses at least one linear epitope in common with a prototype polypeptide set forth above in (a), can encode an epitope in common with the prototype sequence. Alternatively, it may be that the subsequence does not encode an epitope in common with the prototype sequence, but it can be used to isolate the larger sequence, for example, by hybridization of the nucleic acid with the sequence from which it is derived. The subsequences can be used to modulate or detect the expression of genes by introducing into the subsequences compounds that bind, intercalate, clivan and / or crosslink with the nucleic acids. Examples of compounds include conjugates of acridine, psoralen, phenanthroline, naphthoquinone, daunomycin or chloroetlaminaryl. Nucleic acid construction The isolated nucleic acids of the present invention can be made using (a) standard recombinant methods, (b) synthetic techniques or combinations thereof. In some embodiments, the polynucleotides of the present invention can be cloned, amplified or otherwise constructed from a monocot. In preferred embodiments, the monocot is Zea mays. The nucleic acids may conveniently comprise other sequences in addition to a polynucleotide of the present invention. For example, a multiple cloning site, comprising one or more restriction sites of endonucleases in the nucleic acid can be inserted to aid in the isolation of the polynucleotide. Translatable sequences can also be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a marker sequence of hexa-histidine provides a convenient means for purifying the proteins of the present invention. A polynucleotide of the present invention can be attached to a vector, adapter or linker for the cloning and / or expression of a polynucleotide of the present invention. Additional sequences can be added to said cloning and / or expression sequences to optimize their function in cloning and / or expression, to aid in the isolation of the polynucleotide or to improve the introduction of the polynucleotide into a cell. Typically, the length of the nucleic acid of the present invention less than the "length" of the corresponding polynucleotide of the present invention is less than 20 kllobase pairs, often less than 15 kb and often less than 10 kb.The use of cloning vectors , expression vectors, adapters and linkers is well known and is widely described in the art. 'of the various nucleic acids see, for example, Stratagene Cloning Systems, Catalogs of 1995, 1996, 1997 (La Jolla, CA); and Amersham Life Sciences, Inc., Catalog * 97 (Arlington Heights, IL).

A. Recombinant methods for the construction of nucleic acids The isolated nucleic acid compositions of this invention, such as RNA, cDNA, genomic DNA or a hybrid thereof, can be obtained from biological sources using any number of cloning methodologies known to the art. specialists in art. In some embodiments, oligonucleotide probes that hybridize selectively, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library. The isolation of RNA and the construction of cDNA and genomic libraries are well known to those skilled in the art. See, for example, Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); and Current Protocols in Molecular Biology, Ausubel, et. al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995). A number of protocols for cDNA synthesis have been described that provide substantially pure full-length cDNA libraries.

The substantially pure full-length cDNA libraries are constructed such that they comprise at least 90% and, more preferably, at least 93% or 95% of full length inserts between the clones containing the inserts. The length of the insert in said libraries can be from 0 to 8, 9, 10, 11, 12, 13 or more kilobase pairs. Vectors for accommodating inserts of these sizes are known in the art and are commercially available. See, for example, lambda ZAP 'Stratagene Express (cDNA cloning vector with 0 to 12 kb cloning capacity). An example of a method that can be used to construct a full length cDNA library with more than 95% purity was described by Caminci et al., Genomics, 37: 327-336 (1996). Other methods for producing full-length libraries are known in the art. See, for example, Edery et al., Mol. Cell Biol., 15 (6): 3363-3371 (1995); and PCT Application WO 96/34981. A1. Normalized or subtracted cDNA libraries A non-normalized cDNA library represents the mRNA population of the tissue from which it was obtained. Given that the unique clones are in numerical inferiority with respect to the clones derived from genes of great expression, their isolation can be difficult. Normalization of a cDNA library is the process by which a library is created in which each clone is represented more equally. The construction of standardized libraries is described in Ko, Nucí. Acids Res., 18 (19): 5705-5711 (1990); Patanjali et al., Proc. Nati Acad, USA, 88: 1943-1947 (1991); U.S. Patent Nos. No. 5,482,685 and 5,637,685. In an example of a method described by Soares et al., Normalization resulted in the reduction of the abundance of clones from a range of four orders of magnitude to a narrow range of only 1 order of magnitude. Proc. Nati Acad. Sci. EE. UU., 91: 9228-9232 (1994). Subtracted cDNA libraries provide another means to increase the proportion of the less abundant cDNA species. In this procedure, the sequences present in a second pool of mRNA are removed from the cDNA prepared from a mRNA pool.

'Hybridization. The cDNA.RNAn hybrids are removed and the remaining unhybridized cDNA pool is enriched with unique sequences for that pool. See, Foote et al, in, Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Kho and Zarbl, Technique, 3 (2): 58-63 (1991); Sive and St. John, Nucí. Acids Res., 16 (22): 10937 (1988); Current Protocols in Molecular Biology, Ausubel, et al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995); and Swaroop et al., Nucí. Acids Res., 19) 8): 1954 (1991). The sets of elements for subtraction of cDNA are commercially available. See, for example, PCR-Select (Clontech, Palo Alto, California). In order to construct genomic libraries, large segments of genomic DNA are generated by fragmentation, for example using restriction endonucleases, and ligated with the DNA of a vector to form concatemers that can then be packaged into the appropriate vector. The methodologies for doing so and the sequencing methods for verifying the sequence of the nucleic acids are well known in the art. Examples of appropriate molecular biology techniques and the instructions necessary to direct the specialist in the various construction, cloning and search methodologies are described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory , Vols. 1-3 (1989), Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, Berger and Kirmnel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocols in Molecular Biology, Ausubel, et al. ., Eds., Greene Publishing and Wiley-lnterscience, New York (1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). The games of elements 'for the construction of genomic libraries are also commercially available. The cDNA or genomic library can be examined using a probe based on the sequence of a polynucleotide of the present invention, such as those described herein. The probes can be used for hybridization with genomic DNA or cDNA sequences to isolate homologous genes from the same plant species or from a different species. Those skilled in the art will understand that various degrees of hybridization severity can be employed in the assay; and that any of the hybridization or washing media can be stringent. The nucleic acids of interest can also be amplified from nucleic acid samples using amplification techniques. For example, polymerase chain reaction (PCR) technology can be used to amplify the polynucleotide sequences of the present invention and related genes, directly from genomic or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences encoding the proteins to be expressed, to make nucleic acids that can be used as probes to detect the presence of mRNA in samples, for the sequencing of nucleic acids or for other purposes. The 32 protein of the T4 gene (Boehringer Mannheim) can be used to improve the performance of long PCR products. PCR-based search methods were also described.

Wilfinger et al. describe a PCR-based method in which the longest cDNA is identified in the passage of the primer, such that 'eliminate incomplete clones from the study. BioTechniques, 22 (3): 481-486 (1997). Such methods are particularly effective in combination with the previous full-length cDNA construction methodology.

H.H. Synthesis Methods for the Construction of Nucleic Acids The isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis with methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 68: 90-99 (1979); the phosphodiester method of Brcwn et al., Meth. Enzymol. 68: 109-151 (1979); the diethylphosphoramidite method of Beaucage et al., Tetra. Lett. 22: 1859-1862 (1981); the solid phase phosphoramidite triester method described by Beaucage and Caruthers, Tetra. Letts. 22 (20): 1859-1862 (1981), for example, using an automated synthesizer, for example, as described by Needham-VanDevanter et al., Nucleic Acids Res., 12: 6159-6168 (1984); and the solid support method of US Pat. No. 4,458,066. In general, chemical synthesis produces a single-stranded oligonucleotide. This can be converted to double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as an annealing. The artisan will understand that as long as the chemical synthesis of DNA has better results for sequences of about 100 bases or less, longer sequences can be obtained by means of ligation of shorter sequences. Recombinant expression cassettes The present invention also provides recombinant expression cassettes comprising a nucleic acid of the present invention. The nucleic acid sequence encoding the desired polypeptide of the The present invention, for example a cDNA or genomic sequence, which encodes a full-length polypeptide of the present invention, can be used to construct a recombinant expression cassette that can be introduced into the desired host cell. The recombinant expression cassette will typically comprise a polynucleotide of the present invention operably linked to regulatory sequences of the start of transcription., which will direct the transcription of the polynucleotide in the intended host cell, such as the tissue of a transformed plant. For example, plant expression vectors can include (1) a cloned plant gene under the control of transcription of 5 'and 3' regulatory sequences and (2) a dominant selection marker. Said plant expression vectors may also contain, if convenient, a promoter regulatory region (e.g., a region that confers inducible or constitutive expression, regulated by the environment or by the development or selective / specific of cells or tissues), a start site of the start of transcription, a ribosome binding site, an RNA processing signal, a transcription termination site and / or a polyadenylation signal. A fragment of a plant promoter which will direct expression of a polynucleotide of the present invention in all tissues of the regenerated plant may be employed. In this document, said promoters are called "constitutive" promoters and are active under most environmental conditions and states of cell development or differentiation. Examples of constitutive promoters include the initiation region of the 35S transcript of the cauliflower mosaic virus (CaMV), the 1 '- or 2'-promoter derived from the Agrobacterium tumefaciens T-DNA, the ubiquitin I promoter, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Patent No. 5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter, the GRP1-8 promoter, and other initiation regions of the transcription of several plant genes known to specialists. Alternatively, the plant promoter can direct the expression of a polynucleotide of the present invention in a specific tissue or can be otherwise found under more precise environmental or developmental control. In this document, said promoters are called "inducible" promoters. Environmental conditions that can affect transcription by inducible promoters include attack by pathogens, anaerobic conditions or the presence of light. Examples of inducible promoters are the Adh1 promoter which is inducible by cold stress or hypoxia, the Hsp70 promoter which is inducible by heat stress and the PPDK promoter which is inducible by light. Examples of promoters that are under development control include promoters that initiate transcription only or, preferably, in certain tissues, such as leaves, roots, fruits, seeds or flowers. Examples of promoters include the anther-specific promoter 5126 (U.S. Patent Nos. 5,689,049 and 5,689,051), the glob-1 promoter and the gamma-zein promoter. The function of a promoter can also vary according to its location in the genome. Accordingly, an inducible promoter can become fully or partially constitutive in certain locations. Both heterologous and non-heterologous (ie, endogenous) promoters can be used to direct the expression of the nucleic acids of the present invention. These promoters can also be used, for example, in recombinant expression cassettes to direct the expression of antisense nucleic acids to reduce, increase or alter the concentration and / or composition of the proteins of the present invention in a desired tissue. Therefore, in some embodiments, the nucleic acid construct will comprise a functional promoter in a plant cell, such as in Zea mays, operably linked to a polynucleotide of the present invention. Promoters used in these embodiments include endogenous promoters that direct the expression of a polypeptide of the present invention. In some embodiments, isolated nucleic acids that serve as promoter or enhancer elements in the appropriate position (generally towards the 5 'end) of a non-heterologous form of a polynucleotide of the present invention can be introduced., so as to increase or decrease the expression of a polynucleotide of the present invention. For example, endogenous promoters can be altered in vivo by mutation, deletion and / or substitution (see, Kmiec, U.S. Patent No. 5,565,350).; Zarling et al., PCT / US93 / 03868) or isolated promoters can be introduced into a plant cell in the appropriate orientation and distance of one of the genes of the present invention in order to control the expression of the gene. Gene expression can be modulated under conditions suitable for plant growth in order to alter the total concentration and / or composition of the polypeptides of the present invention in the plant cell. Therefore, the present invention provides compositions and methods for making heterologous enhancers and / or promoters operably linked to a native, endogenous (ie, non-heterologous) form of a polynucleotide of the present invention. If expression of the polypeptide is desired, it is generally convenient to include a polyadenylation region towards the 3 'end of the coding region of the polynucleotide. L? The polyadenylation region can be derived from a natural gene, from a variety of other plant genes or from T-DNA. The sequence of the 3 'end to be added may be derived from, for example, the genes of nopaline synthetase or octopine synthetase, or, alternatively, from other plant genes or, less preferably, from any other eukaryotic gene. An intron sequence can be added to the 5 'untranslated region or to the coding sequence of the partial coding sequence to increase the amount of mature messenger that accumulates in the cytosol. It has been demonstrated that the inclusion of a processable intron in the transcription unit of both plant and animal constructions increases the gene expression, both at the mRNA and protein levels, up to 1000 times, Buchman and Berg, Mol. Cell Biol, 8: 4395-4405 (1988); Callis et al., Genes Dev. 1: 1183-1200 (1987). Said enhancement with introns of gene expression is typically greater when placed near the 5 'end of the transcription unit. Using the introns of corn, intron Adh1-S 1, 2 and 6, the Bronze-1 intron is known in the art. See, generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, New York (1994). The vector comprising the sequences of a polynucleotide of the present invention typically comprises a marker gene that confers a selectable phenotype on plant cells. Vectors that are useful for the expression of genes in higher plants are well known in the art and include vectors derived from the tumor-inducing plasmid (Ti) of Agrobacterium tumefacíens described by Rogers et al., Meth. in Enzymol., 153: 253-277 (1987). A polynucleotide of the present invention can be expressed in either the sense orientation of the reading frame or antisense as desired. It will be understood that the control of gene expression in any of the two orientations can have a direct impact on the observable characteristics of the plant. Antisense technology can be conveniently employed to inhibit gene expression in plants. For this, a segment of nucleic acids of the desired gene is cloned and is operatively linked to a promoter in such a way that the antisense RNA chain is transcribed. Then the construction is transformed into plants and the antisense chain of RNA It has been shown in plant cells, that antisense RNA inhibits gene expression because it prevents the accumulation of mRNA encoding the enzyme of interest, see, for example, Sheeby et al, Proc. Nat'l. Acad. Sci. (USA) 85: 8805-8809 (1988); and Hiatt et al., U.S. Pat. N °: 4,801,340. Another method of suppression is the suppression in the sense of the reading frame. It has been shown that the introduction of nucleic acids configured with orientation in the sense of the reading frame is an effective means by which the transcription of the target genes is blocked. For an example of the use of this method to modulate the expression of endogenous genes see, Napoli et al., The Plant Cell 2: 279-289 (1990) and U.S. Pat. N °: 5,034,323.

Catalytic RNA molecules or ribozymes can also be used 'to inhibit the expression of plant genes. It is possible to design ribozymes that specifically pair with virtually any white RNA and clive the phosphodiester structure in a specific location, thereby rendering the target RNA functionally inactive. By carrying out this cleavage, the ribozyme itself is not altered and in this way it can be recycled and other molecules cleaved, becoming a true enzyme. The inclusion of ribozyme sequences within the antisense RNA confers on them the RNA cleavage activity, thereby increasing the activity of the constructions. The design and use of specific ribozymes of the target RNA are described in Haseloff et al., Nature 334: 585-591 (1988). A variety of crosslinking agents, alkylating agents and radical generating species can be used as pendant groups on the polynucleotides of the present invention in order to bind, label, detect and / or cleave the nucleic acids. For example, Vlassov, V.V., et al !, Nucleic Acids Res (1986) 14: 4065-4076, describe the covalent attachment of a single-stranded DNA fragment with alkylating derivatives of nucleotides complementary to the target sequences. A report of a similar work by the same research group is that of Knorre, D. Q., et al., Biochimie (1985) 67: 785-789. Iverson and Dervan also showed a specific cleavage of the single-stranded DNA sequence mediated by the incorporation of a modified nucleotide that was capable of activating cleavage (J Am Chem 'Soc (1987) 109: 1241-1243). Meyer, R.B., et al., J Am Chem Soc (1989) 111: 8517-8519, performed a covalent crosslinking with a white nucleotide using an alkylating agent complementary to the single chain nucleotide sequence of interest. Photoactivated cross-linking with single chain oligonucleotides mediated by psoralen was described by Lee, B. L., et al., Biochemistry. (1988) 27: 3197-3203. The use of crosslinking in triple helix forming probes was described by Home, et al., J Am Chem Soc (1990) 112: 2435-2437. The use of N4, N4-ethancytosine as alkylating agent to cross-link single-stranded oligonucleotides was described by Webb and Matteucci, J Am Chem Soc (1986) 108: 2764-2765; Nucleic Acids Res (1986) 14: 7661-7674; Feteritz el al "J. Am. Chem. Soc. 113: 4000 (1991). In the art, several compounds are known for binding, detecting, labeling and / or cleaving nucleic acids. See, for example, U.S. Pat. No. 5,543,507; 5,672,593; 5,484,908; 5,256,648; and 5,681,941. Proteins The isolated proteins of the present invention comprise an i) polypeptide having at least 10 amino acids encoded by any of the polynucleotides of the present invention as already discussed above, or polypeptides that are conservatively modified variants thereof. The proteins of the present invention or the variants thereof may comprise any amount of waste contiguous amino acids of a polypeptide of the present invention, wherein said amount is selected from the group of integers consisting of up to the amount of residues of a full-length polypeptide of the present invention. Optionally, this subsequence of contiguous amino acids is at least 15, 20, 25, 30, 35 or 40 amino acids in length, or often at least 50, 60, 70, 80 or 90 amino acids in length. In addition, the amount of said subsequences can be any integer selected from the group consisting of 1 to 20, such as 2, 3, 4 or 5. The present invention further provides a protein comprising a polypeptide having a sequence identity determined with a polypeptide of the present invention. The percentage of sequence identity is an integer selected from the group between 50 and 99. Examples of sequence identity values include 60%, 65%, 70%, 75%, 80% 85%, 90% and 95% . The sequence identity can be determined using, for example, the GAP or BLAST algorithms. Those skilled in the art will understand that the present invention includes the catalytically active polypeptides of the present invention (ie, enzymes). The catalytically active polypeptides have a specific activity of at least 20%, 30% or 40%, and preferably at least 50%, 60% or 70% and more preferably at least 80%, 90% or more. 95% of the endogenous, native (non-synthetic) polypeptide activity. In addition, and optionally, the substrate specificity (kcat / Km) is substantially similar to that of the endogenous, native (non-synthetic) polypeptide. Typically, the Km will be at least 30%, 40% or 50% of that of the endogenous, native (non-synthetic) polypeptide; and more preferably at least 60%, 70%, 80% or 90%. Methods for evaluating and quantifying the measurements of enzyme activity and substrate specificity (kcat / Km) are well known to those skilled in the art. In general, the proteins of the present invention will cause, to be presented as an immunogen, the production of an antibody specifically reactive with a polypeptide of the present invention. In addition, the proteins of the present invention will not bind to antisera raised against a polypeptide of the present invention that has been completely immunoabsorbed with the same polypeptide. Immunoassays to determine binding are well known to those skilled in the art. A preferred immunoassay is a competitive immunoassay as previously described. Therefore, the proteins of the present invention can be employed as immunogens for the construction of immunoreactive antibodies with a protein of the present invention for use in, for example, techniques such as immunoassays or protein purification techniques. Expression of proteins in host cells The use of the nucleic acids of the present invention allows the expression of a protein of the present invention in recombinantly manipulated cells, such as cells of bacteria, yeast, insects, mammals or, preferably, plant cells. The cells produce the protein in a non-natural condition (for example, in terms of quantity, composition, location and / or time), because they have been genetically altered by human intervention. It is believed that those skilled in the art are aware of the numerous expression systems available for the expression of a nucleic acid encoding a protein of the present invention. The various known methods for the expression of proteins in prokaryotes or eukaryotes will not be described in detail. In summary, the expression of the isolated nucleic acids encoding a protein of the present invention is typically achieved by operably linking, for example, the DNA or cDNA to a promoter (either constitutive or regulatable), followed by its incorporation into a vector expression. The vectors may be suitable for replication and integration in prokaryotes or eukaryotes. Typical expression vectors contain transcription and translation terminators, initiation sequences and promoters that are useful in regulating the expression of DNA encoding a protein of the present invention. In order to obtain a high level of expression of a cloned gene, it is convenient to construct expression vectors containing at least one strong promoter to direct transcription, a ribosome binding site for the start of translation and a terminator of transcription / translation. The skilled artisan will understand that modifications to the protein of the present invention can be made without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression or incorporation of the molecule of interest in a fusion protein. Such modifications are well known to those skilled in the art and include, for example, the addition of a methionine in the terminal amine to provide a starting site or additional amino acids (eg, poly His) located at any of the terminations for create purification sequences in a convenient location. It is also possible to introduce restriction sites or termination codons. Transfection / Cell Transformation The transformation / transfection method is not critical to the present invention; currently there are several methods of transformation or transfection. As newer methods are available to transform cultures or other host cells they can be applied directly. Accordingly, a wide variety of methods have been developed to insert a DNA sequence into the genome of a cell 'host in order to obtain the transcription and / or translation of the sequence that causes the phenotypic changes in the organism. Therefore, any method that provides efficient transformation / transfection can be employed. A. Transformation of plants The DNA sequence encoding the desired polypeptide of the present invention, for example a cDNA or genomic sequence, encoding a full-length protein will be employed to construct a recombinant expression cassette that can be introduced into the desired plant. The preferred method of plant transformation for the present invention is by bombardment of immature corn embryo particles. See, for example, Tomes, et al., Direct DNA Transfer into Intact Plant Cells Via Microprojectile Bombardment. pp.197-213 in Plant Cell, Tissue and Organ Culture, Fundamental Methods. eds. O. L. Gamborg and G.C. Phillips. Springer-Verlag Berlin Heidelberg New York, 1995, and Songstad, D.D., B.M. Hairston and C.L. Armstrong 1993. Stable Transformation of Maize by Microprojectile Bombardment of Embryos. Agronomy Abstrais p. 183. The isolated nucleic acids of the present invention can be introduced into plants according to techniques known in the art. In general, expression cassettes are prepared as described above and are suitable for the transformation of plant cells. The techniques for transforming a wide variety of higher plant species are well known and are described in the technical, scientific and patent literature. See, for example, Weising et al., Ann. Rev. Genet. 22: 421-477 (1988). For example, the DNA construct can be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation, polyethylene glycol (PEG) poration, particle bombardment, distribution with silicone fibers or microinjection in protoplasts or embryogenic cell calluses. vegetables. See, for example, Tomes et al., Direct DNA Transfer into Intact Plant Cells via Microprojectile Bombardment, p. 197-213 in Plant Cell, Tissue and Organ Culture, Fundamental Methods. Eds. O.L. Gamborg and G.C. Phillips. Springer-Verlag, Berlin, Heidelberg, New York, 1995. Alternatively, the DNA constructs can be combined with suitable T-DNA flanking regions and then can be introduced into a conventional host vector of Agrobacterium tumefaciens. The virulence functions of the host Agrobacterium tumefaciens will direct the insertion of the construct and the adjacent marker into the DNA of the plant cell when the cell is infected by the bacterium. See U.S. Pat. N °: . 591,616. The introduction of DNA constructs using polyethylene glycol (PEG) precipitation was described by Paszkowski et al., Embo J. 3: 27 \ -2722 (1984). Electroporation techniques were described by Fromm et al., Proc. Nati Acad. Sci. 82: 5824 (1985). Ballistic transformation techniques were described by Klein et al., Nature 327: 70-73. (1987) Transformation techniques mediated by Agrobacterium tumefaciens are well described in the scientific literature. See, for example, Horseh et al., Science 233: 496-498 (1984), and Fraley et al., Proc. Nati Acad. Sci. 80: 4803 (1983). Although Agrobacterium is primarily useful in dicots, it is possible to transform some monocotyledons by Agrobacterium. For example, transformation of corn with Agrobacterium is described in US Pat. N °: 5.550.318. Other methods of transfection or transformation include (1) transformation mediated by Agrobacterium rhizogenes (see, for example, Lichtenstein and Fuller in: Genetic Engineering, vol 6, PWJ Rigby, Ed., London, Academic Press, 1987, and Lichtenstein, CP , and Draper, J. In: DNA Cloning, Vol. II, DM Glover, Ed., Oxford, IRI Press, 1985); in Patent Application PCT / US87 / 02512 (WO 88/02405, published April 7, 1988) describes the use of strain A4 of A. rhizogenes and its plasmid Ri together with vectors pARC8 or pARC16 of A. Tumefaciens; (2) liposome-mediated DNA uptake (see, for example, Freeman et al., Plant Cell Physiol. 25: 1353, 1984), (3) the vortex method (see, for example, Kindle, Proc. Nati. Acad. Sci., USA, 87: 1228, (1990) .DNA can also be introduced into plants by direct transfer of DNA in pollen as described by Zhou et al., Methods in Enzymology, 101: 433 (1983); D. Hess, Intern. Rev. Cytol., 107: 367 (1987); Luo et al., Plant Mol. Biol. Repórter, 6: 165 (1988). The expression of genes encoding the polypeptide can be obtained by injection of the DNA into the reproductive organs of a plant as described by Pena et al., Nature, 325: 274 (1987). The DNA can also be injected directly into the cells of immature embryos and with subsequent rehydration of dried embryos, as described by Neuhaus et al., Theor. Appl. Genet, 75: 30 (1987); and Benbrook et al., in Proceedings-Bio Expo 1986, Butterworth, Stoneharn, Mass., pgs. 27-54 (1986). A variety of plant viruses can be employed as vectors that are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, brominated mosaic virus and tobacco mosaic virus. S. Transfection of prokaryotic, lower eukaryotic and animal cells Animal and lower eukaryotic host cells (e.g., yeast) are competent or competent for transfection by various means. There are several well-known methods for introducing DNA into animal cells. These methods include: calcium phosphate precipitation, fusion of recipient cells with bacterial protoplasts containing DNA, treatment of recipient cells with liposomes containing DNA, DEAE dextran, electroporation, bioiistics and micro-injection of DNA directly into the cells. The transfected cells are cultured by means well known in the art. Kuchler, R.J., Biochemical Methods in Cell Culture and Virology, Dowden, Hutchinson and Ross, Inc. (1977). Synthesis of Proteins The proteins of the present invention can be constructed using synthetic non-cellular methods. The solid phase synthesis of proteins of less than about 50 amino acids in length can be carried out by ligating the C-terminal amino acid of the sequence is an insoluble support followed by the sequential addition of the amino acids remaining in the sequence. The techniques for solid-phase synthesis were described by Barany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, Part A .; Merrifield, et al, J. Am. Chem. Soc, 85: 2149-2156 (1963), and Stewart et al., Solid-Phase Peptide Synthesis, 2nd Ed .. Pierce Chem. Co., Rockford, lll. (1984). Longer proteins can be synthesized by condensation of the amino and carboxyl endings of more fragments 'shorts. Methods for forming peptide bonds by activation of a terminal carboxyl terminus (for example, with the use of the coupling agent N, N'-dicyclohexylcarbodiimide) are known to those skilled in the art.

Purification of Proteins The proteins of the present invention can be purified by standard techniques well known to those skilled in the art. The recombinantly produced proteins of the present invention can be expressed directly or can be expressed as a fusion protein. The recombinant protein is purified by the combination of cell lysis (eg, sonication, pressing) and affinity chromatography. For the fusion products, the subsequent digestion of the fusion protein with an appropriate proteolytic enzyme releases the desired recombinant protein. Lae proteins of this invention, recombinant or synthetic, can be purified to a substantial purity by standard techniques well known in the art, including methods of solubilization with detergents, selective precipitation with substances such as ammonium sulfate, column chromatography, immunopurification and others. . See, for example, R. Scopes, Protein Purification: Principles and Practice, Springer-Verlag: New York (1982); Deutscher, Guide to Protein Purification, Academic Press (1990). For example, antibodies against the proteins can be generated as described herein. Purification from E. coli can be achieved by the methods described in U.S. Pat. N °: 4,511,503. The protein can then be isolated from the cells expressing the protein and further purified by standard protein chemistry techniques as described herein. The detection of 'Expressed protein is achieved by methods known in the art and includes, for example, radioimmunoassays, Western blotting techniques or immunoprecipitation.

Regeneration of transgenic plants Transformed plant cells derived by means of any of the transformation techniques mentioned can be cultured to regenerate an entire plant containing the transformed genotype. Such regeneration techniques are often based on the manipulation of certain phytohormones in a tissue culture growth medium. For the transformation and regeneration of corn see Gordon-Kamm et al. The Plant Cel !, 2: 603-618 (1990). Plant cells transformed with a plant expression vector can be regenerated, for example, from individual cells, callus tissue or leaf discs, by means of standard techniques of plant tissue culture. It is a well-known fact in the art that several cells, tissues and organs can be successfully cultured from almost any plant in order to regenerate an entire plant. The regeneration of plants from cultured protoplasts is described in Evans et al., Protoplasts Isolatíon and Culture, Handbook of Plant Cell Culture, Macmillan Publishing Company, New York, pgs. 124-176 (1983); and Binding, Regeneration of Plants, Plant Pr? toplasts, CRC Press, Boca Raton, p. 21-73 (1985). Regeneration of plants containing the foreign gene introduced by Agrobacterium from leaf explants can be achieved as described by Horsch et al., Science, 227: 1229-1231 (1985). In this procedure, the transformants are cultured in the presence of an agent of Selection and in a medium that induces the regeneration of shoots in the plant species that is being transformed, as described by Fraley et al., Proc. Nati Acad. Sci. USA, 80: 4803 (1983). This procedure typically allows shoots to be obtained within two to four weeks and these transformants are then transferred to an appropriate root inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. The transgenic plants of the present invention can be fertile or sterile. Regeneration can also be achieved from calluses, explants and plant organs or parts thereof. Such regeneration techniques are generally described in Klee et al., Ann. Rev. of Plant Phys. 38: 467-486 (1987). The regeneration of plants from individual plant protoplasts or various explants is well known in the art. See, for example, Methods for Plant Molecular Biology, A. Weissbach and H. Weissbach, Eds., Academic Press, Inc., San Diego, Calif. (1988). This process of regeneration and growth includes the steps of selection of transforming cells and shoots, the rooting of the transforming shoots and the cultivation of seedlings on land. For the cultivation and regeneration of corn cells, see generally, The Maize Handbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn and Corn Improvement, 3rd edition, Sprague and Dudley Eds., American Society of Agronomy, Madison, Wisconsin (1988). The artisan will understand that after the recombinant expression cassette was stably incorporated into the transgenic plants and its functionality confirmed, it can be introduced into other plants by sexual cross-linking. You can use any of the standard breeding techniques, depending on the species to be crossed. In vegetatively propagated crops, mature transgenic plants can be propagated by taking segments or by tissue culture techniques in order to produce multiple identical plants. The selection of the desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use. In seed-propagated crops, mature transgenic plants can self-cross to produce a homozygous inbred plant. The inbred plant produces seeds that contain the recently introduced heterologous nucleic acid. These seeds can be grown to produce plants that in turn will produce the selected phenotype. The parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruits and the like, are included in the invention, provided that these parts comprise cells containing an isolated nucleic acid of the present invention. Progeny and variants and mutants of regenerated plants are also included in the scope of the invention, provided that these parts contain the introduced nucleic acid sequences. Transgenic plants expressing in selection marker can be examined by the transmission of the nucleic acid of the present invention by, for example, standard immunoblot and DNA detection techniques. The transgenic lines are also typically evaluated by the levels at which the heterologous nucleic acid is expressed. The expression at the RNA level can be determined initially to identify and quantify positive expression plants. Standard RNA analysis techniques can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the RNA tempers 'heterologous and hybridization assays in solution using as probe the specific heterologous nucleic acid. The positive RNA plants can then be analyzed for their protein expression by western blot analysis using the specifically reactive antibodies of the present invention. In addition, in situ hybridization and immunocytochemistry can be performed according to standard protocols using polynucleotide probes of the specific heterologous nucleic acid and antibodies, respectively, to localize the expression sites within the transgenic tissue. In general, a number of transgenic lines are examined by the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles. A preferred embodiment is a transgenic plant that is homozygous for the added heterologous nucleic acid; that is, a transgenic plant that contains two added nucleic acid sequences, a gene at the same locus on each chromosome of a pair of chromosomes. A homozygous transgenic plant can be obtained by sexual mating (autocrossing) of a heterozygous transgenic plant containing a single added heterologous nucleic acid, germination of some of the seeds produced and analysis of the resulting plants produced for the altered expression of a polynucleotide of the present invention in relation to a control (i.e., native, non-transgenic) plant. Backcrossing with a progenitor plant and crossing with a non-transgenic plant is also contemplated. Modulation of polypeptide levels and / or composition The present invention further provides a method for modulating (i.e., increasing or decreasing) the concentration or ratio of the polypeptides of the present invention in a plant or a part thereof. The modulation can be carried out by increasing or decreasing the concentration and / or the ratio of the polypeptides of the present invention in a plant. The method comprises introducing into a plant cell a cassette of recombinant expression comprising a polynucleotide of the present invention as described above to obtain a transformed plant cell, cultivating said transformed plant cell under conditions favorable to the growth of the plant cell and inducing or repressing the expression of a polynucleotide of the present invention in the plant for a time sufficient to modulate the concentration and / or the ratios of the polypeptides in the plant or part thereof. In some embodiments, the concentration and / or ratios of the polypeptides of the present invention can be modulated in a plant by altering, in vivo or in vitro, the promoter of a gene to increase or decrease the expression of the gene. In some embodiments, the coding regions of the native genes of the present invention can be altered by means of a substitution, addition, insertion or deletion in order to decrease the activity of the encoded enzyme. See, for example, Kmiec, U.S. Pat. N °: . 565,350; Zarling et al., PCT / US93 / 03868. And in some embodiments, an isolated nucleic acid (e.g., a vector) is transfected which contains a promoter sequence in a plant cell. Next, the plant cell containing the promoter operably linked to a polynucleotide of the present invention is selected by means known to those skilled in the art, such as, but not limited to, Southern blotting, DNA sequencing or PCR analysis using primers. specific 'for the promoter and the gene and detection of amplicons produced therefrom. The plant or part of the altered or modified plant is cultivated with the aforementioned embodiments under planting conditions for a time sufficient to modulate the concentration and / or ratios of the polypeptides of the present invention in the plant. The conditions of plant formation are well known in the art and discussed briefly, supra. In general, the concentration and / or the ratios of the polypeptides is increased or decreased by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90 % in relation to a plant, part of a plant or native control cell that does not contain the cassette of recombinant expression mentioned above. In the present invention the modulation may take place during and / or after the cultivation of the plant until the desired development stage. The modulation of nucleic acid expression either temporarily and / or in particular tissues can be controlled by employing an appropriate promoter operably linked to a polynucleotide of the present invention, for example, with orientation in the sense of the reading frame or antisense as discussed in more detail, supra. The induction of the expression of a polynucleotide of the present invention can also be controlled by exogenous administration of an effective amount of an inducing compound. Inducible promoters and inducing compounds that activate expression from these promoters are well known in the art. In preferred embodiments, the polypeptides of the present invention are modulated in monocots, in particular in maize, Codon Preference and UTR In general, it has been found that the efficiency of the translation is regulated by specific sequence elements in the non-coding region. 'or untranslated (5' UTR) of the RNA. Positive sequence motifs include consensus start translation sequences (Kozak, Nucleic Acids Res., 15: 8125 (1987)) and the protective structure of 7-methylguanosine (Drummond et al., Nucleic Acids Res. 13: 7375 ( 1985)). Negative elements include stable intramolecular 5 'UTR loop type structures (Muesing et al., Cell 48: 691 (1987)) and AUG sequences or short open reading frames preceded by an appropriate AUG in the 5' UTR (Kozak, supra). , Rao et al., Mol. And Cell, Biol. 8: 284 (1988)). Accordingly, the present invention provides 5 'and / or 3' untranslated regions for the modulation of the translation of heterologous coding sequences. In addition, the polypeptide coding segments of the polynucleotides of the present invention can be modified to alter the codon usage. An altered codon usage can be employed to alter the efficiency of the translation and / or to optimize the coding sequence for expression in a desired host or to optimize codon usage in a heterologous sequence for expression in maize.

The use of codons in the coding regions of the polynucleotides of the present invention can be analyzed statistically using commercially available software packages, such as "Codon Preference" available from the Genetics Computer Group of the University of Wisconsin (see Devereaux et al. , Nucleic Acids Res. 12: 387-395 (1984)) or MacVector 4.1 (Eastman Kodak Co., New Haven, Conn.). Therefore, the present invention provides a frequency utilization characteristic of codons of the coding region of at least one of the polynucleotides of the present invention. The amount of polynucleotides that can be used to determine the frequency of codon usage can be any integer from 1 to the amount of polynucleotides of the present invention described herein. Optionally, the polynucleotides are full length sequences. An example of the number of sequences for statistical analysis may be at least 1, 5, 10, 20, 50 or 100. Intermixing or combination of sequences The present invention provides methods for combining sequences using the polynucleotides of the present invention. invention and the compositions resulting therefrom. The combination of sequences is described in PCT Patent Publication WO 97/20078. See also, Zhang, J.-H., et al. Proc. Nati Acad. Sci. USA 94: 4504-4509 (1997). In general, the combination or intermixing of sequences provides a means to generate libraries of polynucleotides that possess a desired characteristic that can be selected or tracked. Recombinant polynucleotide libraries are generated from a population of related polynucleotide sequences that contain said regions of sequences that exhibit substantial sequence identity and that can be homologously recombined in vitro or in vivo. The population of polynucleotides recombined with sequences comprises a subpopulation of polynucleotides which possesses the desired or advantageous characteristics and which can be selected with an appropriate selection or search method. The characteristics can be any property or attribute capable of being selected or detected in a search system and can include 'properties of an encoded protein, of an element of transcription, of a sequence that controls transcription, RNA processing, stability of RNA, conformation of chromatin, translation or other property of the expression of a gene or transgene, an element of replication, a protein binding element or the like, as well as any characteristic that confers a selectable or detectable property. In some embodiments, the selected feature will be a lower Km and / or a greater Kcat with respect to the wild type protein, as shown herein. In other embodiments, the protein or polynucleotide generated from the intermixing or the combination of sequences will have a higher ligand binding affinity than the non-combined wild-type polynucleotide. The increase in said properties can be at least 110%, 120%, 130%, 140% or at least 150% with respect to the wild-type value.

Generic and Consensus Sequences The polynucleotides and polypeptides of the present invention further include those that possess: (a) a generic sequence of at least two homologous polynucleotides or polypeptides, respectively, of the present invention; and (b) a consensus sequence of at least three homologous polynucleotides or polypeptides, respectively, of the present invention. The generic sequence of the present invention comprises each of the polypeptide or polynucleotide species comprised of "a generic polypeptide or polynucleotide sequence, respectively." The individual species comprised of a polynucleotide that possesses a consensus amino acid or nucleic acid sequence can be used to generate antibodies or produce nucleic acid probes or primers for 'Conduct homologous searches on other species, genera, families, orders, classes, queues or kingdoms. For example, a polynucleotide having a consensus sequence from a family of Zea mays genes can be used to generate antibodies or probes or nucleic acid primers for other Gramíneae species, such as wheat, rice or sorghum. Alternatively, a polynucleotide having a consensus sequence generated from orthologous genes can be used to identify or isolate orthologs from another taxon. Typically, a polynucleotide having a consensus sequence will be at least 9, 10, 15, 20, 25, 30 or 40 amino acids in length or 20, 30, 40, 50, 100 or 150 nucleotides in length. As will be understood by those skilled in the art, a conservative amino acid substitution that differs from the aligned sequence but that comes from the same conservative substitution group, as described above, can be used. Optionally, no more than 1 or 2 conservative amino acids are substituted for each 10 amino acids in length of the consensus sequence. Similar sequences used for the generation of a consensus or generic sequence include any amount and combination of allelic variants of the same gene, orthologous sequences or paralogs provided herein. Optionally, similar sequences used in the generation of a consensus or generic sequence are identified using the lowest sum probability (P (N)) of the BLAST algorithm. The various providers of sequence analysis software are listed in Chapter 7 of Current Protocols in Molecular Biology, F. M. Ausubel et al., , Current Protocols, a joint work between Greene Publishing Associates, Inc. and John 'Wiley & Sons, Inc. (Supplement 30). A polynucleotide sequence is considered similar to a reference sequence if the likelihood of a minor sum in the comparison of a test nucleic acid with the reference nucleic acid is less than about 0.1, preferably less than 0.01 or 0.001. approximately and more preferably less than about 0.0001 or 0.00001. Similar polynucleotides can be aligned and a consensus or generic sequence can be generated using multiple sequence alignment software available from numerous commercial providers such as the PILEUP software from Genetics Computer Group (Madison, Wl), ALIGNX from Vector NTI (North Bethesda , MD) or SEQUENCHER of Genecode (Ann Arbor, Ml). The predetermined parameters of said software can be conveniently used to generate the consensus or generic sequences. Computer Applications The present invention provides equipment, data structures and processes for modeling or analyzing the polynucleotides and polypeptides of the present invention. A. Data structures and equipment The present invention provides an equipment that has a memory that contains the data representing a sequence of a polynucleotide or polypeptide of the present invention. The equipment of the present invention is typically a digital computer. The memory of said equipment includes, but is not limited to, ROM or RAM or computer reading means such as, but not limited to, magnetic media such as floppy disks or hard drives or media such as CD-ROM. Accordingly, the present invention also 'provides a data structure comprising the sequence of a polynucleotide of the present invention incorporated into a computer reading medium. Those skilled in the art will understand that the shape of the computer memory of the present invention or the particular embodiment of the computer reading means is not a critical element of the invention and can be of various forms, ß. Homology Searches The present invention provides a process for identifying a homologous candidate (ie, an ortholog or paralog) of a polynucleotide or polypeptide of the present invention. The homologous candidate has a statistically significant probability of having the same biological function (for example, catalyze the same reaction, bind to homologous proteins / nucleic acids) than the reference sequence with which it is compared. Accordingly, the polynucleotides and polypeptides of the present invention are useful in the identification of homologs in animals or other plant species, in particular those belonging to the Gramineae family such as, but not limited to, sorghum, wheat or rice. The process of the present invention comprises obtaining data that represent a sequence of test polynucleotides or polypeptides. The test sequences are generally at least 25 amino acids in length or at least 50 nucleotides in length. Optionally, "the test sequence can be at least 50, 100, 150, 200, 250, 300 or 400 amino acids in length.The test polynucleotide can be at least 50, 100, 200, 300, 400 or 500 nucleotides in length, often the test sequence will be a full-length sequence.

The test can be obtained from a nucleic acid of an animal or a plant. Optionally, the test sequence is obtained from a plant species other than corn whose function is uncertain but which will be compared with the test sequence in order to determine sequence similarity or sequence identity; for example, said plant species may belong to the Gramíneae family, such as wheat, rice or sorghum. The data of the test sequence is entered into the equipment, typically a computer, whose memory contains the data representing a reference sequence. The reference sequence may be the sequence of a polypeptide or a polynucleotide of the present invention and is often at least 25 amino acids or 100 nucleotides in length. As will be understood by those skilled in the art, the greater the identity / sequence similarity between the reference sequence of known function and a test sequence, the greater is the probability that said test sequence will have the same or a similar function as the of the reference sequence. The kit further comprises a means for comparing sequences in order to determine the identity or sequence similarity between the test sequence and the reference sequence. Examples of sequence comparison means were mentioned for the sequence analysis software described above. Optionally, a sequence comparison can be made using the BLAST suite of programs. You can display the results of the comparison between the test and reference sequences. In general, a value of the smallest sum probability (P (N)) less than 0.1 or, alternatively, less than 0.01, 0.001, 0.0001 or 0.00001 using the set of BLAST 2.0 algorithms with the predetermined parameters allows to identify a test sequence as a homologous candidate (i.e., an allele, an ortholog or a paralog) of the reference sequence. A nucleic acid comprising a polynucleotide possessing the sequence of the candidate homolog can be constructed using well-known in vitro (eg, phosphoramidite), library isolation, cloning or chemistry synthesis techniques, such as those described herein. . In further embodiments, a nucleic acid comprising a polynucleotide having a sequence represented by the candidate homolog in a plant is introduced; typically, these polynucleotides are operably linked to a promoter. Confirmation of the function of the candidate homolog can be carried out by operably linking the homologous nucleic acid candidate to, for example, an inducible promoter or by the expression of an antisense transcript and analyzing in the plant the phenotype changes consistent with the presumed function of the homologous candidate. Optionally, the plant in which these nucleic acids are introduced is a monocot, for example from the Gramineae family. Examples of plants include corn, sorghum, wheat, rice, barley, alfalfa, cotton and soybeans. C. Computer Modeling The present invention provides a process for modeling / analyzing data representative of the sequence a polynucleotide or polypeptide of the present invention. The process comprises entering the sequence data of * a polynucleotide or polypeptide of the present invention in a kit, manipulating the data to model or analyze the structure or activity of the polynucleotide or polypeptide and visualizing the results of the modeling or analysis. There are various modeling and analytical tools well known in the art and available from commercial sources, such as Genetics Computer Group (Version 9, Madison, Wl). The modeling / analysis tools include methods for: 1) recognizing overlapping sequences (eg, from a sequencing project) with a polynucleotide of the present invention and creating an alignment called "contig"; 2) identifying the restriction enzyme sites of a polynucleotide of the present invention; 3) identify the products of a T1 ribonuclease digestion of a polynucleotide of the present invention; 4) identify PCR primers with minimal self-complementarity; 5) compare two sequences of proteins or nucleic acids and identify points of similarity or dissimilarity between them; 6) calculate the paired distances between the sequences in an alignment, reconstruct phylogenetic trees using the distance methods and calculate the degree of divergence of two protein coding regions; 7) identify patterns such as coding regions, terminators, repeats and other consensus patterns in the polynucleotides of the present invention; 8) identify the RNA secondary structure; 9) identify sequence motifs, isoelectric points, secondary structure, hydrophobicity and antigenicity in the polypeptides of the present invention; and 10) translating the polynucleotides of the present invention and backtracking the polypeptides of the present invention. Nucleic Acid Detection The present invention further provides methods for detecting a polynucleotide of the present invention in a nucleic acid sample, suspected of containing a polynucleotide of the present invention, such as a lysate of plant cells, in particular a corn lysate. In some embodiments, a gene of the present invention or a portion thereof can be amplified before the step of placing the nucleic acid sample in contact with a polynucleotide of the present invention. The nucleic acid sample is contacted with the polynucleotide to form a hybridization complex. The polynucleotide is hybridized under stringent conditions to a gene encoding a polypeptide of the present invention. The hybridization complex formation is used to detect a gene encoding a polypeptide of the present invention in the nucleic acid sample. The skilled artisan will understand that an isolated nucleic acid containing a polynucleotide of the present invention should not contain cross hybridization sequences in common with genes that are not of interest, which will lead to a false positive result. The detection of the hybridization complex can be achieved using any of the well-known methods. For example, the nucleic acid sample, or a portion thereof, can be evaluated with hybridization formats including, without limitation, hybridization assays in solution phase, solid phase, mixed phase or in situ. Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Marks that are useful in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent dyes, radioactive labels, enzymes and colorimetric labels. Other brands include ligands that bind antibodies labeled with fluorophores, chemiluminescent agents, and enzymes. The labeling of the nucleic acids of the present invention can be achieved easily by the use of labeled PCR primers. Example 1 In this example, the construction of the cDNA libraries is described.

Isolation of total RNA Total RNA was isolated from corn tissues with the TRIzol ™ reagent (Life Technology Inc., Rockville, MD) using a modification of the guanidine isothiocyanate / acid-phenol process described by Chomezynski and Sacchi (Chomezynski, P ., and Sacchi, N. Anal. Biochem. 162, 156 (1987)). Briefly, samples of plant tissue were sprayed in liquid nitrogen before the addition of the TRIzol reagent and then further homogenized in mortar. The addition of chloroform followed by centrifugation was performed to separate the aqueous phase from the organic phase. The total RNA was recovered by precipitation with isopropyl alcohol from the aqueous phase.

Isolation of Poly (A) RNA + Selection of poly (A) + RNA from total RNA was performed using the PoIyATract® system (Promega Corporation, Madison, Wl). Briefly, biotinylated oligo (dT) primers were used for hybridization with the poly (A) 3 'tails in the mRNA. The hybrids were captured using streptavidin assembled to paramagnetic particles and a magnetic separation device. The mRNA was washed under conditions of great severity and eluted with RNAse-free deionized water. Construction of the cDNA library cDNA was synthesized and unidirectional cDNA libraries were constructed using the SuperScript ™ plasmid system (Life Technology Inc. Rockville, MD). The first strand of the cDNA was synthesized in 'a test with primers with the oligo primer (dT) containing a Not I site.

The reaction was catalyzed by the SuperScript ™ II reverse transcriptase at 45 ° C. The second strand of cDNA was labeled with alpha-32P-dCTP and a portion of the reaction was analyzed by electrophoresis on an agarose gel to determine the sizes of the cDNA. The cDNA molecules less than 500 base pairs and the unbound adapters were removed by chromatography with Sephacryl-S400. The selected cDNA molecules were ligated into the pSPORTI vector between the Not I and Sal I sites. Example 2 In this example, cDNA sequencing and library subtraction are described. Preparation of sequencing tempering Individual colonies were excised and DNA was prepared either by PCR with the forward primers M13 and the reverse primers M13 or by plasmid isolation. All cDNA clones were sequenced using the reverse primers M13. Subtraction procedure Q-bot The cDNA libraries subjected to the subtraction procedure were plated on a 22 x 22 cm2 agar plate at a density of about 3,000 colonies per plate. The plates were incubated in an incubator at 37 ° C for 12-24 hours. The colonies were plated in 384 well plates by an automatic colony collimator, Q-bot (GENETIX Limited). These plates were incubated overnight at 37 ° C.

Once a sufficient number of colonies had been replazed, they were applied to 22 x 22 cm2 nylon membranes using Q-bot. Each membrane contained '9,216 colonies or 36,864 colonies. These membranes were placed on an agar plate with the appropriate antibiotic. The plates were incubated at 37 ° C overnight. After recovering the colonies on the second day, these filters were placed on filter paper pre-wetted with a denaturing solution for four minutes, then incubated on a boiling water bath for an additional four minutes. The filters were placed on pre-wet filter paper with a neutralizing solution for four minutes. After removing the excess solution by placing the filters on dry filter papers for one minute, the filters were placed, on the side of the colonies, in a Proteinase K solution and incubated at 37 ° C for 40-50 minutes. The filters were placed on dry filter papers and allowed to dry overnight. The DNA was then crosslinked with nylon membranes by UV light treatment.

Hybridization of the colonies was carried out as described by Sambrook, J., Fritsch, E. F. and Maniatis, T., (in Molecular Cloning: A Laboratory Manual, 2nd Edition). The following probes were used in the hybridization of the colonies: 1. cDNA from the first chain of the same tissue as that used to obtain the library in order to eliminate the most redundant clones. 2. 48-192 more redundant cDNA clones from the same library based on the previous sequencing data. 3. 192 clones of the most redundant cDNA in the entire maize sequence database. 4. Oligonucleotide Sal-A20: TCG ACC CAC GCG TCC GAA AAA AAA AAA AAA AAA AAA, which removes clones containing a poly A tail 'but not the cDNA. 5. cDNA clones derived from rRNA. The autoradiography image was scanned and incorporated into the computer and the signal intensity and locations of cold colonies in each plate of colonies were analyzed. The reordering of cold colonies from 384 cavities plates to 96-well plates was carried out using Q-bot. Example 3 This example describes the identification of the gene from a computer homology search. The identities of the genes were determined with searches of similarity with BLAST (Basic Local Alignment Search Tool; Altschul, SF, et al., (1993) J. Mol. Biol. 215: 403-410, see also www.ncbi.nlm .nih.gov / BLAST /) using the default parameters, with sequences contained in the BLAST database "nr" (which includes all the non-redundant CDS translations of GenBank, the sequences derived from the three-dimensional structure in the Brookhaven Protein Data Bank , the last important release of the SWISS-PROT protein sequence database and the EMBL and DDBJ databases). The similarity of the cDNA sequences was analyzed with all the DNA sequences available to the public contained in the "nr" database using the BLASTN algorithm. The DNA sequences were translated in all the reading frames and compared for their similarity with all the sequences of proteins available to the public contained in the database "nr" using the algorithm BLASTX (Gish, W. and States, D. J. Nature Genetics 3: 266-272 (1993)) provided by NCBI. In some cases, sequencing data from two or more clones containing overlapping DNA segments were used to construct contiguous DNA sequences.

Example 4 In this example an analysis of the transit peptides of the present invention is provided in comparison with other alternative plant oxidase genes. Table 1 Alignment of multiple sequences CLUSTAL W (1.74) of selected alternative plant oxidase genes. ZmAOXI Zea mays, Oxidase alternative 1, in this document. ZmAOX2 Zea mays, Oxidase alternative 2, in this document. OsAOXIa Oryza sativa, Oxidase alternative 1a, Acc. AB004864 OSAOX1 b Oryza sativa, Oxidase alternative 1 b, Acc. AB004865 SgAOXI Sauromatum guttatum, Oxidase alternative 1, Acc. NtAOXI Nicotiana tabacum, Oxidase alternative 1, Acc. X79768 NtAOX Nicotiana tabacum, Oxidase alternative, Acc. S71335 CrAOX Catharanthus roseus, Alternative Oxidase, Acc. AB009395 AtAOXYa Arabidopsis thaliana, Alternative Oxidase 1a, Acc. D89875 AtAOXIb Arabidopsis thaliana, Alternative Oxidase 1b, Acc. D89875 AtAOXIc Arabidopsis thaliana, Alternative Oxidase 1c, Acc. AB003175 GmAOXI Glycine max, Oxidase alternative 1, Acc. X68702 GmAOX2 Glycine max, Oxidase alternative 2, Acc. U87906 GmAOX3 Glycine max, Oxidase alternative 3, Acc. U87907 * ZmAOXl_ MSTRA AGSALLRHLGPRVFG PVFSPAVAPPRPLLAL OsAOXlb_ MSSRM AGATLLRHL-GPRLFA EPVYSGLAASARGVMPA ZmAOX2 - MMSSR AGSI RHAG-SRLFT AAAISP AAASRPL OsAOXla_ MSSRM AGSAI RHVGGVR FT ASATSPAAAAAAAARPF SgA0Xl_ - -MISSRL AGTALCRQLSHVPVPQY LPA RPTADTASSLLHR N AOXl_ NtAOX_ MMTRGATRMTRTVLGHMGPRYFSTAIFR-NDAGTGVMS-GAAVFMHGVPANPSEKA CrA0X_ MMSRGATRISRSLICQISPRYFSSAAVRGHEPSLGILTSGGTTTFLHGNPGNGSERT AtAOXlb_ - -MMMSRR- -YGAK METAVTH SHLLNPRVPL AtA0Xlc_ MITTLLRRSLLDASKQATSIN GILFHQLAP- AtA0Xla_ - -MMITRGGAKAAKSLLVAAGPRLFSTVRTV SSHEALSASHILKPGVTS GmA0Xl_ MMMMMSRS GANRVANTAM F-VAKGLSG GmAOX2_ - -MKLTALNSTVRRALLNGRNQN GNRLGSAALMPYAAAETRLL GmAOX3 - -MKNV VRSAAR-ALLGG GGRSYYRQLSTAATVEQRHQ ZmAOX1_ AGGGERGGALVWVRVRL LST-SAAEAKEEVAASKGNSGS-TAAAKAEAVEAAKEGDGKRD OsAOXlb_ AAR IFPARM ASTSSAGADVKEGAAEKLPEPAATAAAAAT DPQNK- - ZmA0X2_ LAGGNGVP- VM-LRL MSTSSPAAP TEAKD- -EAAKASKVGGD KKA OsAOXla_ LAGGEAVP VWGLRL MSTSSVAS TEA AAKAEAKKADA EKE SgAOXl_ CSAAAPAQRAGLWPPSWFSPPRH I ASTLSARAQDGGKEKAAGTAGKVPPG EDGGAEK NtAOXl_ - -MWVRH-FPVMGPRS ASTVALND-KQHDKKVENGG- -AAASGG . GDGGDE NtA0X_ WTWVRH-FPVMGSRS AMSMALND-KQHDKKAENGS- - -AAATGG GDGGDE CrAOX_ ALTWIK-LPMMRARS ASTVATVDQKDKDEKREDKN- - -GVADG ENGN- AtAOXlb_ VTENIRV-PAMGWRV FSKMTFEKKKTTEEK-GSS - - - -GGKA DQGNKGE AtA0Xlc_ -AKYFRV-PAVGGLRD DFSKMTFEKKKTSEEEEGSGD GVKV-N DQGNKGE tAOXla_? AWIWTRA-PTIGGMRF ASTITLGEKTPMKEEDANQKKTENESTGGDA-A GGNNKGD GmAOXl_ EVGGLRA- YGGG RS ESTLALSEKEKIEKKVGLSS AGGNKEE GmAOX2 CAGGANGW-FFYWKRT MVSPAEAKVPEKEKEKEK AKAEKS GmA0X3_ HGGGAFG SFHLRR "MSTLP-EVKDQHSEEKKNE- -VNGTSN (Cleavage site of the transit peptide) ZmA0Xl_ KWSSYWGVA-PS -KLMNKDGAEWRWSCFRPWEAYKPDTTIDLNRHHEPKVLLDKIAYWT OsAOXlb_ KAWSYWGIQ-PP-KLVKEDGTEWKWLSFRPWDTYTSDTSIDVTKHHEPKGLPDKLAYWT ZmA0X2_ WINSYWGIE -QNNKLARDDGTEWKWTCFRPWETYTADTSIDLTRHHEPKTLMDKVAYWT 0sA0Xla_ WVNSYWGIE-QSKKLVREDGTEWKWSCFRPWETYTADTSIDLTKHHVPKTLLDKIAYWT SgA0Xl_ EAWSYWAVP-PS -KVSKEDGSEWRWTCFRPWETYQADLSIDLHKHHVPTTILDKLALRT Nt AOX 1_ KS WS WGVP - PS - KVTKEDGTEWKWNCFRPWETYKADLS IDLTKHHAPTTFLDKFAYWT Nt A0X_ KS WS YWGVQ - PS - KVTKEDGTEWKWNCFRPWETYKADLS IDLTKHHAPTTFLDKFAYWT CrA0X_ KAWSYWGVE - AP - KLTKEDGTVWRWTCFRPWETYKPDTDIELKKHHVPVTLLDKVAFFT AtA0Xlb_ QLIVSYWGVK-PM- KITKEDGTEWKWSCFRPWETYKSDtTIDLKKHHVPSTLPDKLAYWT AtA0XlC_ QLIVSYWGVK- PM-KITKEDGTEWKWSCFRPWETYKADLTIDLKKHHVPSTLPDKIAYWM AtA0Xla_ KGIASYWGVE- PN-KITKEDGSEWKWNCFRPWETYKADITIDLKKHHVPTTFLDRIAYWT GmAOXl_ KVIVSYWGIQ- PS - KITKKDGTEWKWNCFSPWGTYKADLSIDLEKHMPPTTFLDYMAFWT GmA0X2_ WESSYWGIS - -RPKWREDGTEWPWNCF PWESYRSNVSIDLTKHHVPKNVLDKVAYRT GmA0X3 A TSYWGIT- -RPKVRREDGTEWPWNCFMPWDSYHSDVSIDVTKHHTPKSLTDKVAFRA **. * * * **. * * * *. * ZmA0Xl_ VKLLRVPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHLRSLRRFEHSGGWIRALLEEA 0sA0Xlb_ VRSLAVPRDLFFQRRHASHALLLETVAGVPGMVGGMLLHLRSLRRFEQSGGWIRALLEEA ZmA0X2_ VKSLRFPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHLRSLRRFEQSGGWIRALLEEA 0sA0Xla_ VKSLRFPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHLRSLRRFEQSGGWIRTLLEEA SgAOXl_ VKALRWPTDIFFQRRYACRAMMLETVAAVPGMVGGVLLHLKSLRRFEHSGGWIRALLEEA NtA0Xl_ VKALRYPTDIFFQRRYGCRAMMLETVAAVPGMVGGMLLHCKSLRRFEQSGGWIKALLEEA NtAOX_ VKSLRYPTDIFFQRRYGCRAMMATVAAVPGMVGGMLLHCKSLRRFEQSGGWIKTLLDEA CrAOX_ VKALRWPTDLFFQRRYGCRAMMLETVAAVPGMVGGMLLHCKSLRRFEHSGGWIKALLEEA AtAOXlb_ VKSLRWPTDLFFQRRYGCRAMMLETVAAVPGMVGGMLVHCKSLRRFEQSGGWIKALLEEA AtAOXlc_ VKSLRWPTDLFFQRRYGCRAIMLETVAAVPGMVGGMLMHFKSLRRFEQSGGWIKALLEEA AtAOXla_ VKSLRWPTDLFFQRRYGCRAMMLETVAAVPGMVGGMLLHCKSLRRFEQSGGWIKALLEEA GMA0X1_ VKVLRYPTDVFFQRRYGCRAMMLETVAAVPGMVAGMLLHCKSLRRFEHSGGWFKALLEEA GmA0X2_ VKLLRIPTDLFFKRRYGCRAMMLETVAAVPGMVGGMLLHLRSLRKFQQSGGWIKALLEEA GmAOX3 VKFLRVLSDIYFKERYGCHAMMLETIAAVPGMVGGMLLHLKSLRKFQHSGGWIKALLEEA * .. *. *,. * .. ***. * ***** *. *. *. *. *. * .. **** ... **. ** ZmAOXl_ ENERMHLMTFMEVAKPKWYERALVLAVQGVFFNAYFLGYLISPKFAHRWGYLEEEAIHS OsAOXlb_ ENERMHLMTFLEVMQPRWWERALVLAAQGVFFNAYFVGYLVSPKFAHRFVGYLEEEAVSS ZmAOX2_ ENERMHLMTFMEVAKPRWYERALVITVQGVFFNAYFLGYLLSPKFAHLWGYLEEEAIHS OsAOXla_ ENERMHLMTFMEVANPKWYERALVITVQGVFFNAYFLGYLLSPKFAHRWGYLEEEAIHS SgAOXl_ ENERMHLMTFMEVAQPRWYERALVLAVQGVFFNAYFLGYLLSPKFAHRWGYLEEEAIHS NtAOXl_ ENERMHLMTFMEVAKPNWYERALVFAVQGVFINAYFVTYLLSPKLAXRIVGYLEEEAIHS NtAOX_ ENERMHLMTFMEVAKPNWYERALVFAVQGVFFNAYFVTYLLSPKLAHRIVGYLEEEAIHS CrAOX_ ENERMHLMTFMEVSKPRWYERALVFAVQGVFFNAYFLTYLASPKLAHRIVGYLEEEAIHS AtAOXlb_ ENERMHLMTFMEVAKPNWYERALVIAVQGIFFNAYFLGYLISPKFAHRMVGYLEEEAIHS AtAOX1c_ ENERMHLMTFMEVAKPKWYERALVISVQGVFFNAY IGYIISPKFAHRMVGYLEEEAIHS AtAOXla_ ENERMHLMTFMEVAKPKWYERALVITVQGVFFNAYFLGYLISPKFAHRMVGYLEEEAIHS GmAOXl_ ENERMHLMTFMEVAKPKWYERALVITVQGVFFNAYFLGYLLSPKFAHRMFGYLEEFAIHS GmAOX2_ ENERMHLMTMVELVKPKWYERLLVLAVQGVFFNAFFVLYILSPKVAIiRIVGYLEEEAIHS GmAOX3- ENERMHLMTMVELVKPSWHERLLIFTAQGVFFNAFFVFYLLSPKAAHRFVGYLEEEAVIS ********* .. * .. ***** .. **. *. * .... *. **. * *******. * ZmAOXl_ YTEYLKDLEAGKIEPPIPAPAIAIDYWQLPADATLKDWWRSDEAHHRDVNHFASDIHF OsAOXlb_ YTEYLKDLEAGKIENTPAPAIAIDYWRLPADATLKDWTVIRADEAKHRDLHHFASDIQQ ZmA0X2_ YTEYLKDLEAGKIENVPAPAIAIDYWRLPANATLKDWTWRADEAHHRDVNHFASDIHC OsAOXla_ YTEFLKDLEAGKIDNVPAPAIAIDYWRLPANATLKDWTWRADEAHHRDVNHFASDIHY SgAOXl_ YTEFLKDIDNGAIQDCPAPAIALDYWRLPQGSTLRDWTWRADEAHHRDVNHFASDVHY NtA0Xl_ YTEFLKELDKGNIENVPAPAIAIDYWRLPKDSTLRDWLWRADEAHHRDVNHFAPDIHY NtAOX_ YTEFLKELDKGNIENVPAPAIAIDYCRLPKDSTLLDWLWRADEAHHRDVNHFASDIHY CrAOX_ YSEFLNFLDKGNIENVPAPAIAIDYWQMPPDSTLRDVVMWRADEALHRDVNHYASDIHY AtA0Xlb_ YTEFLKELDNGNIENVPAPAIAIDYWRLEADATLRDWMWRADEAHHRDVNHYASDIHY AtA0Xlc_ YTEFLKELDNGNIENVPAPAIAVDYWRLEADATLRDWMWRADEAHHRDVNHYASDIHY AtAOX1a_ YTEFLKELDKGNIENVPAPAIAIDYWRLPADATLRDWMWRP-DEAHHRDVNHFASDIHY GmAOXl_ YTEFLKELDKGNIENVPAPAIAIDYWQLPPGSTLRDWMWRADEAHHRDVNHFASDIHY GmA0X2_ YTEYLKDLESGAIENVPAPAIAIDYWRLPKDARLKDVITVIRADEAHHRDVNHFASDIHF GmAOX3 YTQHLNAIESGKVENVPAPAIAIDYWRLPKDATLKDWTVIRADEAHHRDVNHFASDIHH *****. **. . ***. *. *. ******. ** ZmAOX1_ QGMQLKETPAPIEYH OsAOXlb_ QGMKLKDTPAPIGYH ZmAOX2_ QGMQLKQSPAPIGYH OsAOXla_ QGMELKQTPAPIGYH SgAOXl_ QDLELKTTPAPLGYH NtAOXl_ QGQQLKDSPAPIGYH NtAOX_ QGQQLKDSPAPIGYH CrAOX KGLELKEAAAPLDYH AtAOXlb_ QGRELKEAPAPIGYH AtA0XlC_ QGHELKEAPAPIGYH AtAOXla_ QGRELKEAPAPIGYH GmAOXl_ QGRELREAAAPIGYH GMA0X2 QGKELREAPAPIGYH GMAOX3 QGKELKEAPAPIGYH **. ** The known or predicted transit peptide cleavage sites are indicated with a vertical line (I). The first three amino acids of the mature peptides are underlined. The penultimate amino acid of the transit peptide, Arginine conserved, is highlighted. It should be taken into account that the transit peptides are considerably divergent in size and sequence, while most of the coding region of the protein is highly conserved. The alignment of the maize sequences with these other plant AOX gene sequences clearly reveals the likely cleavage site of the transit peptide, even though the transit peptides, with the possible exception of those of the rice AOX clones, are quite different. Example 5 In this example the determination of the mRNA profile of the ZmAOX3 in suspension cultures of GS3 cells of Zea mays after exposure to spores of the fungus Fusarium monilíforme or to quito-oligosaccharides.

The maize GS3 (HYII) cells were cultured as a suspension until the mid-log phase, at which time they were treated with 1 ml of water (control), 1 ml of Fusarium monilíforme spores to obtain a final concentration of 100,000 spores. ml or 1 ml of quito-oligosaccharide mixture to obtain a final concentration of 100 μg / ml. The quito-oligosaccharide mixture was a partial hydrolyzate of crab carapace chitin from CarboMer, Inc. (Westborough, MA). (See Yalpani, M. and D. Pantaleone (1994) Carbohydrate Research 256: 159-.175 for details on the preparation of quito-oligosaccharides.) Cells were harvested 2 hours and 6 hours after treatment and immediately frozen in liquid nitrogen and kept at -80 ° C until RNA extraction. RNA extraction and isolation of pol 'A + RNA were carried out as described in Example 1. The double-stranded cDNA was synthesized using the SuperScript ™ plasmid system (Life Technologies, Inc., Rockville, MD) . The in vitro transcription of cRNA with ribonucleotides conjugated with biotin was carried out with the MEGAscnpt ™ T7 element set (Ambion, Inc., Austin, TX), followed by the QIAGEN mini-protocol, Inc. (Valencia, CA) RNeasy ® for the purification of RNA. The resulting cRNA was fragmented and hybridized for 16 hours with a custom order Gene Chip of Zea rriays oligonucleotides, then washed and stained with streptavidin, R-phycoerythrin conjugate, using the Affymetrix GeneChip Fluidics Station. The Hewlett-Packard G2500A Gene Array scanner and the Affymetrix GeneChip Analysis software suite were used to analyze the results. The data for ZmAOX3 showed the following changes in the level of expression in response to the described treatment: 2 hours of exposure to monosodium Fusarium spores: increase of 4.8 times 6 hours of exposure to Fusarium moniliforrne spores: increase of 5 , 0 times 2 hours of exposure to Fusarium moniliforrne spores: increase of 9.5 times 6 hours of exposure to Fusarium moniliforrne spores: increase of 8.9 times These data support the conclusion that the ZmAOX genes are sensitized in defense situations and illustrates its potential usefulness in the manipulation of cold tolerance and resistance to plant diseases. References Chivasa, S. et al. (1997) Plant Cell 9, 547-557. Connett, M.B. and Hanson, M.R. (1990) Plant Physiol. 93, 1634-1640. Ito, Y., et al. (1997) Gene 203 (2), 121-129. Lennon, A.M. et al. (1997) Plant Physiol. 115, 783-791. McCGig, T.N. et al. (1977) Can. J. Bot. 55: 549-555. Musgrave, M.E. et al. (1986) Plant Sci. 33, 7-11. Polidoros, A.N. et al. (1997) GenBank Direct Submission. Accession AF040566. Submitted (30-DEC-1997). Rhoades, D.M. et al. (1993) Plant Mol. Bio. Int. J. Mol. Biol. Biochem. Genet Eng. 21, 615-624. Stewart, C.R. et al. (1990a) Plant Physiol. 92, 755-760. Stewart, C.R. et al. (1990b) Plant Physiol. 92, 761-766. Vanlerberghe, G.C. et al- (1992a) Plant Physiol. 100, 115-119. Vanlerberghe, G.C. et al. (1992b) Plant Physiol. 100, 1846-1851. Vanlerberghe, G.C. et al. (1997a) Plant Physiol. 113, 657-661. Vanlerberghe, G.C. et al. (1997b) Ann Rev. Plant. Physiol. Plant Mol. Bio. 48, 703-734. Whelan, J. et al. (1995) Plant Mol. Bio. 27, 769-778.

The above examples are provided for the purpose of illustrating the invention but not to limit its scope. Other variants of the invention will be apparent to those skilled in the art and are encompassed by the appended claims. All publications, patents, patent applications and computer programs referred to in this document are incorporated herein by reference. The polynucleotides of SEQ ID N °: 1, 4 and 7 are contained in a deposit made at the American Type Culture Collection (ATCC) on January 14, 2000, and received Accession No. PTA-1209. American Type Culture Collection is located at 10801 University Blvd., Manassas, VA 20110-2209.

The ATCC deposit is maintained under the terms of the Budapest Treaty on the International Recognition of Deposits of Microorganisms for Patent Procedure. The deposit is provided according to need to those skilled in the art and said deposit is not to be understood as an acknowledgment of any obligation under the art.

U.S.C. Article 112. The deposited sequences, as well as the polypeptides encoded by the sequences, are incorporated herein by way of reference and control in the case of any conflict, such as a sequencing error, with the description in this application.

LIST OF SEQUENCES < 110 > Pioneer Hi-Bred International, Inc. < 120 > Alternative maize oxidase genes and uses thereof < 130 > 0963-PCT < 150 > US 60 / 117,776 < 151 > 1999-01-29 < 160 > 9 < 170 > FastSEQ for Windows Version 3.0 < 210 > 1 < 211 > 1520 < 212 > DNA < 213 > Zea mays 0 < 220 > < 221 > CDS < 222 > (124) ... (1164) < 400 > 1 caggcaaaca ctgaaacact agtaccacag tagcagcgag gagcaaacag caaagcacgc 60 tctggagaaa tccctcgtct accttcacca gttgaccgca ctagtggccg tcgtccgacc 120 acg atg age acc cgc gc gca gga gcc tcc etc etc cgc falls ctg ggt 168 Met Ser Thr Arg Ala Ala Gly Ser Ala Leu Leu Arg His Leu Gly 5 1 5 10 15 ccg cgc gtc gtc gtc gtc gtc gtc ct gcc gcg gcc ccg gcc gcg gcc gcg gcg pro ggc ggc ggg cg ggc ggg ggg cg ggc ggg gcg etc gtg 264 Pro Leu Leu Wing Leu Wing Gly Gly Gly Glu Arg Gly Gly Wing Leu Val 35 40 45 tgg gtg cgg gtg cgg ct ctg tcc ccc tcc acc gcc gac gcg aag gag 312 Trp Val Arg Val Arg Leu Leu Ser Thr Be Wing Wing Glu Wing Lys Glu 50 55 60 gag gtg gcg gcg tcc aag ggg aac tea gga age acc gcg gcg gcg aag 360 Qlu Val Wing Wing Ser Lys Gly Asn Ser Gly Ser Thr Wing Wing Ala Lys 65 70 75 gcg gag gcg gtg gag gcc gag aag gag ggt gac gga aag aga gac aaa 408 Wing Glu 'Wing Val Glu Wing Wing Lys Glu Gly Asp Gly Lys Arg Asp Lys 80 85 90 95 gtg g tg age age tac tgg ggc gtc gcg ccg tcg aag ctg atg aac aag 456 Val Val Ser Ser Tyr Trp Gly Val Ala Pro Ser Lys Leu Met Asn Lys 100 105 110 gac ggc gcc gag tgg agg tgg tct tgc ttc agg cea tgg gag gcg tac 504 Asp Gly Wing Glu Trp Arg Trp Ser Cys Phe Arg Pro Trp Glu Wing Tyr 115 120 125 aag ccg gac acc acg gat gat etc aac aga falls falls gaa ccc aag gtg 552 Lys Pro Asp Thr Thr He Asp Leu Asn Arg His His Glu Pro Lys Val 130 135 140 ctg etc gac aag ate gcc tat tgg acc gtc aaa tta ctg cgc gtg ccc 600 Leu Leu Asp Lys He Ala Tyr Trp Thr Val Lys Leu Leu Arg Val Pro 145 150 155 acc gac ata ttc ttc cag agg agg tac ggc tgc cgt gct atg atg ctg 648 Thr Asp He Phe Phe Gln Arg Arg Tyr Gly Cys Arg Ala Met Met Leu 160 165 170 175 gaa here gtg gcg gcg gtg ccg ggg atg gtg ggc ggc atg ctg ctt cae 696 Glu Thr Val Wing Wing Val Pro Gly Met Val Gly Gly Met Leu Leu His 180 185 190 ctc cgc tcg etc cgc cgc ttc gag fall age ggc ggc tgg ate cgg gcg 744 Leu Arg Ser Leu Arg Arg Phe Glu His Ser Gly Gly Trp He Arg Ala 195 200 205 ctg ctg gag gag gcg gag aat gaa cgc atg falls etc atg acc ttc atg 792 Leu Leu Glu Glu Wing Glu Asn Glu Arg Met His Leu Met Thr Phe Met 210 215 220 gag gtg gcc aag ccc aag tgg tac gag cgc gcg ctt gtc etc gcc gtg 840 Glu Val Wing Lys Pro Lys Trp Tyr Glu Arg Ala Leu Val Leu Ala Val 225 230 235 cag ggc gtc ttc ttc aac gcc tac ttc etc ggc tac etc tcc tcc ccc 888 Gln Gly Val Phe Phe Asn Wing Tyr Phe Leu Gly Tyr Leu He Ser Pro 240 245 250 255 aag ttc gcg falls cgt gtc gtt ggg tac etc gag gag gag gcc tees falls 936 Lys Phe Wing His Arg Val Val Gly Tyr Leu Glu Glu Glu Wing He His 260 265 270 tea tat acc gaa tac etc aag gac etc gag gcc ggc aag ate gag aac 984 Ser Tyr Thr Glu Tyr Leu Lys Asp Leu Glu Wing Gly Lys He Glu Asn 275 280 285 gtc ccc gcg ccg gcc att gcc ate gac tac tgg cag etc cea gct gat 1032 Val Pro Wing Pro Wing Wing He Asp Tyr Trp Gln Leu Pro Wing Asp 290 295 300 gcg acg etc aag gat gtg gtt gtc gtg gtg cgc tcc gac gag gcg falls 1080 Wing Thr Leu Lys Asp Val Val Val Val Val Serg Arg Asp Glu Ala His 305 '310 315 cgc gac gtc aat falls falls tcc gcg tcg gac ata cat ttc cag ggt atg 1128 his arg asp val asn his phe wing be asp he his phe phe gln gly met 320 325 330 335 cag etc aag gag here ect gca ccg att gag tac cat tgaacaatcg 1174 Gln leu Lys Glu Thr Pro Ala Pro He Glu Tyr His 340 345 gggtcctgtg acgcttgaga gttccagttc attttggcta gctgtaggta getaaagatg 1234 cttgagaaat aaaaagaaaa tgcctggctg ctatgagtag caaagatetc gtgggttgat 1294 cctaaaatct tttacgtgag tgtttgtaag gacattgata tagagataca cgtgcactga 1354 gaacatacca atcttgtcca tcgacttggt tggttccaca gagaaaactt tatatcegat 1414 tgtaacagag tttttttttc ctgaaaagga gcaacgaagg agccgcgcgt cgtcgatttt 1474 aaaaaaaaaa ctaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 1520 <; 210 > 2 < 211 > 347 < 212 > PRT < 213 > Zea mays < 400 > 2 Met Ser Thr Arg Ala Ala Gly Be Ala Leu Leu Arg His Leu Gly Pro 1 5 10 15 Arg Val Phe Gly Pro Val Phe Ser Pro Ala Val Ala Pro Pro Pro Arg Pro 20 25 30 Leu Leu Ala Leu Ala Gly Gly Gly Glu Arg Gly Gly Ala Leu Val Trp 35 40 45 Val Arg Val Arg Leu Leu Ser Thr Ser Ala Ala Glu Ala Lys Glu Glu 50 55 60 Val Ala Ala Ser Lys Gly Asn Ser Gly Ser Thr Ala Ala Ala Lys Ala 65 70 75 80 Glu Ala Val Glu Ala Ala Lys Glu Gly Asp Gly Lys Arg Asp Lys Val 85 90 95 Val Ser Ser Tyr Trp Gly Val Ala Pro Ser Lys Leu Met Asn Lys Asp 100 105 110 Gly Wing Glu Trp Arg Trp Ser Cys Phe Arg Pro Trp Glu Wing Tyr Lys 115 120 125 Pro Asp Thr Thr He Asp Leu Asn Arg His His Glu Pro Lys Val Leu 130 135 140 Leu Asp Lys He Wing Tyr Trp Thr Val Lys Leu Leu Arg Val Pro Thr 145 150 155 160 Asp He Phe Phe Gln Arg Arg Tyr Gly Cys Arg Wing Met Met Leu Glu 165 170 175 Thr Val Wing Wing Val Pro Gly Met Val Gly Gly Met Leu Leu His Leu 180 185 190 Arg Ser Leu Arg Arg Phe Glu His Ser Gly Gly Trp He Arg Ala Leu 195 200 205 L eu Glu Glu Ala Glu Asn Glu Arg Met His Leu Met Thr Phe Met Glu 210 215 220 Val Wing Lys Pro Lys Trp Tyr Glu Arg Wing Leu Val Leu Wing Val Gln 225 230 235 240 Gly Val Phe Phe Aen Wing Tyr Phe Leu Gly Tyr Leu He Ser Pro Lys 245 250 255 Phe Ala His Arg Val Val Gly Tyr Leu Glu Glu Glu Ala He His Ser 260 265 270 Tyr Thr 'Glu Tyr Leu Lys Asp Leu Glu Ala Gly Lys He Glu Asn Val 275 280 285 Pro Ala Pro Wing He Wing He Asp Tyr Trp Gln Leu Pro Wing Asp Wing 290 295 300 Thr Leu Lys Asp Val Val Val Val Arg Ser Asp Glu Wing His His 305 310 315 320 Arg Asp Val Asn His Phe Wing Being Asp He His Phe Gln Gly Met Gln 325 330 335 Leu Lys Glu Thr Pro Ala Pro He Glu Tyr His 340 345 < 210 > 3 < 211 > 52 < 212 > PRT < 13 > Zea mays < 400 > 3 Met Ser Thr Arg Ala Ala Gly Ser Ala Leu Leu Arg His Leu Gly Pro 1 5 10 15 Arg Val Phe Gly Pro Val Phe Ser Pro Ala Val Ala Pro Pro Arg Pro 20 25 30 Leu Leu Ala Leu Ala Gly Gly Gly Glu Arg Gly Gly Ala Leu Val Trp 35 40 45 Val Arg Val Arg 50 < 210 > 4 < 211 > 1457 < 212 > DNA, < 213 > Zea mays < 220 > < 221 > CDS < 222 > (108) ... (1094) < 400 > 4 cgaaaaccca cgtctaaaac aggtggecca ccaacgattc acttccccga tcccaggggg 60 cggcgatcgg aattcgcaac tcctcccacg cggcgaacac ggcagag atg atg age 116 Met Met Ser 1 tcc cgg gcc gga tcc ate etc etc cgc falls gcc ggc tcc cgt etc ttc 164 Ser Arg Ala Gly Ser He Leu Leu Arg His Ala Gly Be Arg Leu Phe 5 10 15 acc gca gcg gcg ate tct ccg gcg gcg gcc tcg agg cea ctg etc gcc 212 Thr Wing Wing Wing Be Pro Pro Wing Wing Wing Being Arg Pro Leu Leu Wing 20 25 30 35 ggc ggc aat ggt gtt ccg gca gtc atg cta cgg ctt atg tcc acg tcc 260 Gly Gly Asn Gly Val Pro Wing Val Met Leu Arg Leu Met Ser Thr Ser 40 45 50 tcc ccc gcc gcc ccc acg gac gcg aag gac gac gca gcc aag gcc tcc 308 Ser Pro Ala Wing Pro Thr Glu Wing Lys Aßp Qlu Wing Wing Lyß Wing Wing 55 60 65 aag gtg 'gga gga gac aag aag gcg gtg gtg ate aac age tac tgg ggg 356 Lys Val Gly Gly Asp Lys Lys Wing Val Val He Asn Ser Tyr Trp Gly 70 75 80 ate gag "caac aac aac aag cta gcg cgg gac gac ggc acc gag tgg aag 404 He Glu Gln Asn Asn Lys Leu Wing Arg Asp Asp Gly Thr Glu Trp Lys 85 90 95 tgg act tgc ttt agg cea tgg gag acg tac acg gcg gac acg tcc att 452 Trp Thr Cys Phe Arg Pro Trp Glu Thr Tyr Thr Wing Asp Thr Ser He 100 105 110 115 gac etc acc aga falls cat gag ccc aag acg ctg atg gat aag gtc gca 500 Asp Leu Thr Arg His His Glu Pro Lys Thr Leu Met Asp Lye Val Wing 120 125 130 tac tgg acc gtc aag tcg ctg cgc tc ccc acc gac ate ttc tcc cag 548 Tyr Trp Thr Val Lys Ser Leu Arg Phe Pro Thr Asp He Phe Phe Gln 135 140 145 agg cgg tat ggc tgc cgg gcg atg atg ctg gaa acg gtg gct gcg gtg 596 Arg Arg Tyr Gly Cys Arg Ala Met Met Leu Glu Thr Val Ala Wing Val 150 155 160 ect ggg atg gtg ggc ggc atg ctg etc falls ctg cgc tea etc cgc cgc 644 Pro Gly Met Val Gly Gly Met Leu Leu His Leu Arg Ser Leu Arg Arg 165 170 175 tgc gag cag age ggc ggc tgg ate cgc gct ttg ctg gag gag gcc gag 692 Phe Glu Gln Gly Gly Trp He Arg Wing Leu Leu Glu Glu Wing Glu 180 185 190 195 aac gag cgc atg drops etc atg acc ttc atg gag gtg gcg aag ccg agg 740 Asn Glu Arg Met His Leu Met Thr Phe Met Glu Val Wing Lys Pro Arg 200 205 210 tgg tac gag cgc gcg etc gtt ate acc gtc cag ggc gtc ttc ttc aac 788 Trp Tyr Glu Arg Ala Leu Val He Thr Val Gln Gly Val Phe Phe Asn 215 220 225 ca tac ttc etc ggc tac etc ttg tcc ccg aag ttc gcg drops etc gtc 836 Wing Tyr Phe Leu Gly Tyr Leu Leu Ser Pro Lys Phe Wing His Leu Val 230 235 240 gtc ggc tac cg gag gag gag gcc tcg falls tac acc gag tac etc 884 Val Gly Tyr Leu Glu Glu Glu Wing His Ser Tyr Thr Glu Tyr Leu 245 250 255 aag gat ctg gag gcc ggc aag ate gag aac gtc ccc gcc ccg gcc ate 932 Lys Asp Leu Glu Wing Gly Lys He Glu Asn Val Pro Ala Pro Ala He 260 265 270 275 gcc ate gac tac tgg cgc etc ccc gct aac gcc acg etc aag gac gta 980 Wing He Asp Tyr Trp Arg Leu Pro Wing Asn Wing Thr Leu Lys Asp Val 280 285 290 gtc acc gtc gtc cgc gcc gac gag gct falls falls cgc gac gtc aac falls 1028 Val Thr 'Val Val Arg Wing Asp Glu Wing His His Arg Asp Val Asn His 295 300 305 ttt gca tcg gac ate cat tgc cag gga atg cag ctg aag cag tcc ect 1076 phe Ala Ser Asp He His Cys Gln Gly Met Gln Leu Lys Gln Ser Pro 310 315 320 gcg ccg ate gga tac falls tgaggatgtt tgtgctcttc ttaattttgc 1124 Ala Pro He Gly Tyr His 325 atcgctaata agcaattgtc tttaagggaa ggaaaggatg cttattgagt taegagtact 1184 gctacggcga ttaggatatt ttccaaccag ttgtttgaga gtgaaaccta ttatatgtac 1244 gcatgttaca tgtacatatc tctaagtgcg agagatgett ttctggcgtt tatcacttct 1304 tcctggagtt cctttgttct tcatgtcagc tgaattgggc tegetaatat cgaatgtaca 1364 aaaaaaaaaa atttttgcat aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1424 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaa 1457 < 210 > 5 < 211 > 329 < 212 > PRT < 13 > Zea mays < 400 > 5 Met Met Ser Ser Arg Ala Gly Ser He Leu Leu Arg His Wing Gly Ser 1 5 10 15 Arg Leu Phe Thr Wing Wing Wing He Pro Wing Wing Wing Wing Arg Pro 20 25 30 Leu Leu Wing Gly Gly Asn Gly Val Pro Wing Val Met Leu Arg Leu Met 35 40 45 Ser Thr Ser Ser Pro Wing Wing Pro Thr Glu Wing Lys Asp Glu Wing Wing 50 55 60 Lys Wing Ser Lys Val Gly Gly Asp Lys Lys Wing Val Val He Asn Ser 65 70 75 80 Tyr Trp Gly He Glu Gln Asn Asn Lys Leu Wing Arg Asp Asp Gly Thr 85 90 95 Glu Trp Lys Trp Thr Cys Phe Arg Pro Trp Glu Thr Tyr Thr Wing Asp 100 105 110 Thr Ser He Asp Leu Thr Arg His His Glu Pro Lys Thr Leu Met Asp 115 120 125 Lys Val Wing Tyr Trp Thr Val Lys Ser Leu Arg Phe Pro Thr Asp He 130 135 140 Phe Phe Gln Arg Arg Tyr Gly Cys Arg Ala Met Met Leu Glu Thr Val 145 150 155 160 Ala Ala Val Pro Gly Met Val Gly Gly Met Leu Leu His Leu Arg Ser 165 170 175 Leu Arg Arg Phe Glu Gln Ser Gly Gly Trp He Arg Wing Leu Leu Glu 180 185 190 Glu Wing Glu Asn Glu Arg Met His Leu Met Thr Phe Met Glu Val Wing 195 200 205 L ys Pro Arg Trp Tyr Glu Arg Ala Leu Val He Thr Val Gln Gly Val 210 215 220 Phe Phe Asn Wing Tyr Phe Leu Gly Tyr Leu Leu Ser Pro Lys Phe Wing 225 230 235 240 His Leu Val Val Gly Tyr Leu Glu Glu Glu Wing He His Ser Tyr Thr 245 250 255 Glu Tyr Leu Lys Asp Leu Glu Wing Gly Lys He Glu Asn Val Pro Wing 260 265 270 Pro Wing Wing He Asp Tyr Trp Arg Leu Pro Wing Asn Wing Thr Leu 275 280 285 Lys Asp Val Val Thr Val Val Arg Wing Asp Glu Wing His His Arg Asp 290 295 300 Val Asn His Phe Wing Being Asp He His Cys Gln Gly Met Gln Leu Lys 305 310 315 320 Gln Ser Pro Wing Pro He Gly Tyr His 325 < 210 > 6 < 211 > 47 < 212 > PRT < 213 > Zea mays < 400 > 6 Met Met Being Ser Arg Ala Gly Being He Leu Leu Arg His Wing Gly Being 1 5 10 15 Arg Leu Phe Thr Wing Wing Wing Being Pro Wing Wing Wing Being Arg Pro 20 25 30 Leu Leu Wing Gly Gly Asn Gly Val Pro Wing Val Met Leu Arg Leu 35 40 45 < 210 > 7 < 211 > 420 < 212 > DNA < 13 > Zea mays < 220 > < 221 > CDS < 222 > (2) ... (420) < 400 > 7 a ccc acg cgt ccg ccc acg cgt ccg ccc acg cgt ccg gca gtc gtc age 49 Pro Thr Arg Pro Pro Thr Arg Pro Pro Thr Arg Pro Wing Val Val Ser 1 5 10 15 tac tgg ggc ate gac acg ccc aag etc gtg aag gaa gac ggc acg gag 97 Tyr Trp Gly He Asp Thr Pro Lys Leu Val Lys Glu Asp Gly Thr Glu 20 25 30 tgg aag tgg acc age ttc cgg ccg tgg gac gcg tac acg tcg gac acg 145 Trp Lys Trp Thr Ser Phe Arg Pro Trp Asp Ala Tyr Thr Ser Asp Thr 35 40 45 tcc ate gac ata ggg aag falls falls gcg ccg acg acg ctg ccc gac aag 193 Ser He Asp He Gly Lys His His Wing Pro Thr Thr Leu Pro Asp Lys 50 55 60 gcg gcg tac ctg ate gtc aag tcg ctg cgc gtg ccc atg gac etc ttc 241 Ala Ala Tyr Leu He Val Lys Ser Leu Arg Val Pro Met Asp Leu Phe 65 70 75 80 ttc cag cgc cgg drops gcc age drops gcg ctg ctg etc gag acg gtg gcg 289 Phe Gln Arg Arg His Ala Ser His Wing Leu Leu Leu Glu Thr Val Wing 85 90 95 gcc gtg 'ccg ggc atg gtg ggc ggc atg etc etc falls ctg cgc tcc etc 337 Wing Val Pro Gly Met Val Gly Gly Met Leu Leu His Leu Arg Ser Leu 100 105 110 cgc cgc ttc gag falls age ggc ggc tgg ate cgc gcg ctg etc gag gag 385 Arg Arg Phe Glu His Ser Gly Gly Trp He Arg Wing Leu Leu Glu Glu 115 120 125 gcc gag aac gag cgc atg caa etc atg acg ttc te 420 Wing Glu Asn Glu Arg Met Gln Leu Met Thr Phe 130 135 < 210 > 8 < 211 > 140 < 212 > PRT < 213 > Zea mays < 400 > 8 Pro Thr Arg Pro Pro Thr Arg Pro Pro Thr Arg Pro Wing Val Val Ser 1 5 10 15 Tyr Trp Gly He Asp Thr Pro Lys Leu Val Lys Glu Asp Gly Thr Glu 20 25 30 Trp Lys Trp Thr Ser Phe Arg Pro Trp Asp Wing Tyr Thr Ser Asp Thr 35 40 45 Ser He Asp He Gly Lys His His Wing Pro Thr Thr Leu Pro Asp Lys 50 55 60 Wing Wing Tyr Leu He Val Lys Ser Leu Arg Val Pro Met Asp Leu Phe 65 70 75 80 Phe Gln Arg Arg His Ala Ser His Wing Leu Leu Leu Leu Glu Thr Val Wing 85 90 95 Wing Val Pro Gly Met Val Gly Gly Met Leu Leu His Leu Arg Ser Leu 100 105 110 Arg Arg Phe Glu His Ser Gly Gly Trp He Arg Ala Leu Leu Glu Glu 115 120 125 Wing Glu Asn Glu Arg Met Gln Leu Met Thr Phe Ser 130 135 140 < 210 > 9 < 211 > 36 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Oligonucleotide designed on the basis of the adapter sequence and poly T to remove clones that have a poly A tail but not the cDNA < 400 > 9 tcgacccacg cgtccgaaaa aaaaaaaaaa aaaaaa 36 1

Claims (13)

1. CLAIMS: 1. An isolated nucleic acid comprising a member selected from the group consisting of: (a) a polynucleotide having at least 80% sequence identity, determined with the BLAST 2.0 algorithm with the predetermined parameters, with a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOS: 2, 5 and 8; (b) a polynucleotide encoding a polypeptide selected from the group consisting of SEQ ID NOS: 2, 5 and 8; (c) a polynucleotide amplified from a Zea mays nucleic acid library using primers that selectively hybridize, under stringent hybridization conditions, to loci within a polynucleotide selected from the group consisting of SEQ ID NO: 1, 4 and 7; (d) a polynucleotide that hybridizes selectively, under stringent hybridization conditions and a 2X SSC wash at 50 ° C, with a polynucleotide selected from the group consisting of SEQ ID NOS: 1, 4 and 7; (e) a polynucleotide selected from the group consisting of SEQ ID N °: 1, 4 and 7; (f) a polynucleotide that is complementary to a polynucleotide of (a), (b), (c), (d) or (e); and (g) a polynucleotide comprising at least 25 contiguous nucleotides of a polynucleotide of (a), (b), (c), (d), (e) or (f).
2. 2. A cassette of recombinant expression, comprising a member of clause 1 operatively linked, with orientation in the sense of the reading frame or antisense, to a promoter.
3. 3. A host cell comprising the recombinant expression cassette of clause 2.
4. 4. A transgenic plant comprising the recombinant expression cassette of clause 2.
5. 5. The transgenic plant of clause 4, wherein said plant is a monocot.
6. 6. The transgenic plant of clause 4, where said plant is selected from the group consisting of: corn, soybean, sunflower, sorghum, cañola, wheat, alfalfa, cotton, rice, barley and millet.
7. 7. A transgenic seed of the transgenic plant of clause 4.
8. 8. A method for modulating the level of activity of the ZmAOX 1, ZmAOX2 or ZmAOX3 genes in a plant, comprising: (a) Introducing a cassette into a plant cell of recombinant expression comprising a ZmAOXI, ZmAOX2 or ZmAOX3 polynucleotide of clause 1, operatively linked to a promoter; (b) cultivating the plant cell under growth conditions suitable for said plant cell; e (c) inducing the expression of said polynucleotide for a sufficient time to modulate the activity level of the ZmAOXI, ZmAOX2 or ZmAOX3 genes in said plant.
9. 9. The method of clause 8, where the plant is corn.
10. 10. An isolated protein comprising a member selected from the group consisting of: a) a polypeptide of at least 20 contiguous amino acids of a polypeptide selected from the group consisting of SEQ ID NOS: 2, 5 and 8; (b) a polypeptide selected from the group consisting of SEQ ID NOS: 2, 5 and 8; (c) a polypeptide having at least 80% sequence identity, and having at least one linear epitope in common, with a polypeptide selected from the group consisting of SEQ ID NOS: 2, 5 and 8, wherein said sequence identity is determined using BLAST 2.0 with the predetermined parameters; and (d) at least one polypeptide encoded by a member of clause 1.
11. 11. A process for cloning a candidate homologue of the ZmAOXI genes, ZmAOX2 or ZmAOX3, comprising the steps of: (a) collecting data representing a sequence of test polynucleotides or polypeptides; (b) entering the data of step (a) in a device having a memory containing (i) the data representing a reference sequence selected from the group consisting of SEQ ID N °: 2, 5, 8 or a subsequence of at least 25 amino acids thereof and SEQ ID NO: 1, 4, 7 or a subsequence of at least 50 nucleotides thereof and (i) a means for comparing the sequences; (c) comparing said test sequence with said reference sequence using a means to determine the sequence identity or similarity; (d) expressing the results of said comparison, where a comparison that made it possible to obtain a sum probability value less than about 0.1 determined with BLAST 2.0 using the predetermined parameters, identifies said test sequence as a candidate homologue of said sequence of reference; and (e) cloning or synthesizing a nucleic acid comprising a polynucleotide having the sequence of said candidate homologue.
12. 12. A plant cell of the Gramineae family comprising a heterologous polynucleotide having the sequence of the candidate homologue of clause 11.
13. 13. A plant transformed with a polynucleotide of SEQ ID No. 1, 4 or 7, wherein said plant exhibits selected characteristics of the group consisting of: a greater tolerance to cold; greater resistance to diseases; male sterility; and an altered protein expression directed to the mitochondria.