MXPA06007152A - Maize metallothionein promoter - Google Patents

Abstract

The present invention provides compositions and methods for regulating expression of heterologous nucleotide sequences in a plant. Compositions include a novel nucleotide sequence for a promoter for the gene encoding metallothionein. A method for expressing a heterologous nucleotide sequence in a plant using the promoter sequences disclosed herein is provided. The method comprises transforming a plant or plant cell with a nucleotide sequence operably linked to one of the promoters of the present invention.

Description

METALOTIONEIN DE MAIZE PROMOTER 'FIELD OF THE INVENTION The present invention relates to the field of plant molecular biology, more particularly to the regulation of gene expression in plants. BACKGROUND OF THE INVENTION Expression of heterologous DNA sequences in a plant host is dependent on the presence of an operably linked promoter that is functional within the plant host. The choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed. Where expression in specific tissues or organs is desired, preferred tissue promoters can be used. Where gene expression is desired in response to a stimulus, inducible promoters are the regulatory element of choice. In contrast, where continuous expression is desired by all cells of a plant, constitutive promoters are used. Additional regulatory sequences upstream and / or downstream of the core promoter sequence can be included in the expression constructs of transformation vectors to give rise to varying levels of expression of heterologous nucleotide sequences in a transgenic plant. It is often desirable to express a sequence of DNA in particular tissues or organs of a plant. For example, the increased resistance of a plant to infection by pathogens carried by the soil and by air could be accomplished by genetic manipulation of the plant genome to comprise a preferred tissue promoter operably linked to a pathogen resistance gene. heterologous such that the pathogen resistance proteins are produced in the desired plant tissue. Alternatively, it may be desirable to inhibit the expression of a native DNA sequence within the tissues of a plant to achieve a desired phenotype. In this case, such inhibition could be performed with the transformation of the plant to comprise a preferred promoter of tissue operably linked to an antisense nucleotide sequence, such that the expression of the antisense sequence produces an RNA transcript that interferes with the translation of the mRNA of the native DNA sequence. Until now, the regulation of gene expression in plant roots has not been adequately studied despite the importance of the root for the development of the plant. To some degree this is attributable to a lack of readily available biochemical specific root functions whose genes can be cloned, studied and manipulated. The genetic alteration of the plants through the use of genetic engineering techniques and in this way the Production of a plant with useful attributes requires the availability of a variety of promoters. An accumulation of promoters would allow researchers to design recombinant DNA molecules that are capable of being expressed at desired cellular local levels. Therefore, a collection of preferred tissue promoters would allow a new attribute to be expressed in the desired tissue. Several genes have been described by Ta ahashi et al. (1991) Plant J. 1: 327-332; Takahashi et al. (1990) Proc. Nati Sci. USA 87: 8013-8016; Hertig et al. (1991) Plant Mol Biol. 16: 171-174; Xu et al. (1995) Plant Mol Biol 27: 237-248; Capone et al. (1994) Plant Mol Biol 25: 681-691; Masuda et al. (1999) Plant Cell Physiol. 40 (11): 1177-81; Luschnig et al. (1998) Genes Dev. 12 (14): 2175-87; Goddemeier et al (1998) 36 (5): 799-802; and Yamamoto et al. (1991) Plant Cell. 3 (4): 371-82 to express preferentially in plant root tissues. Metallothioneins (MT's) are proteins or polypeptides that link and sequester ionic forms of certain metals in plant and animal tissues. Examples of such metals include copper, zinc, cadmium, mercury, gold, silver, cobalt, nickel and bismuth. The specific metals sequestered by MTs vary given the structurally distinct proteins / polypeptides that occur in different organisms. The plants are presented that contain a diversity of MTs that binds the metal with the potential to perform different functions in the metabolism of different metal ions. In plants, MTs suggest that they have functions in metal accumulation, metal poisoning and embryogenesis (Thomas et al. (2003) Biotechnol. Prog. 19: 273-280; Dunstan (1996) Planta 199: 459-466; Kawashima et al. (1992) Eur. J. Biochem. 209: 971-976) Typically, MTs and MT-like proteins are Cys rich proteins that are characterized by the presence of portions of Cys-Xaa-Cys suggesting the ability to bind metal ions. Additional categories of MT-like proteins have been proposed in the base of the predicted locations of the Cys residues and types 1 and 2 have been designated. In type 1 there are exclusively Cys-Xaa-Cys portions, whereas in type 2 there is a pair of Cys-Cys and a Cys-Xaa-Xaa-Cys within the N-terminal domain. The type 1 portion has been implicated in copper binding and sequestration (Murphy et al. (1997) Plant Physiol 113: 1293-1301 and Carr et al. (2002) J. Biol. Chem. 277: 31237-31242). Several plant genes similar to metallothionein have been isolated, including those of pea, corn, barley, Mimulus (monacata flower), soybean, castor and Arabidopsis.

See Robinson et al. (1993) Biochem J. "295: 1-10 The sequences expressed in roots have been reported to be isolated from peas, as described in Evans et al. (1990) FEBS Lett 262: 29-32. A corn root MT gene has been described in U.S. Patent No. 5,466,785, although this sequence is also expressed in the leaves, marrow and seed, as described by Framond (1991) FEBS Lett 290: 103-106. the isolation and characterization of tissue-preferred promoters, particularly preferred at the root, which can serve as regulatory regions for the expression of the heterologous nucleotide sequences of interest in a preferred tissue manner are necessary for the genetic manipulation of plants. BRIEF DESCRIPTION OF THE INVENTION Compositions and methods are provided for regulating the expression of a heterologous nucleotide sequence of interest in a plant or plant cell. novel nucleotide sequences for promoters that initiate transcription. The embodiments of the invention comprise the nucleotide sequence set forth in the SEQ ID NO: a complement thereto, the nucleotide sequence comprising the promoter sequence of the deposited plasmid plant, as Patent Deposit No. NRRL B-30792 or a complement to it; a nucleotide sequence comprising at least 20 contiguous nucleotides of the SEQ ID NO: 1, wherein the sequence initiates transcription in a plant cell; and, a nucleotide sequence comprising a sequence having at least 85% sequence identity to the sequence set forth in SEQ ID NO: 1, wherein the sequence initiates transcription in the plant cell. A method is provided for expressing a heterologous nucleotide sequence in a plant or plant cell. The method comprises introducing into a plant or a plant cell, an expression cassette comprising a heterologous nucleotide sequence of interest operably linked to one of the promoters of the present invention. In this way, the promoter sequences are useful for controlling the expression of the operably linked heterologous nucleotide sequence. In specific methods, the heterologous nucleotide sequence of interest is expressed in a preferred root manner. In addition, a method for expressing a nucleotide sequence of interest in a preferred root manner in a plant is provided. The method comprises introducing into a plant cell an expression cassette comprising a promoter of the invention operably linked to a heterologous nucleotide sequence of interest. The expression of the nucleotide sequence of interest can provide modification of the plant phenotype. Such modification includes the modulation of the production of an endogenous product, in terms of the amount, relative distribution or the like, or the production of an exogenous expression product to provide a novel function or product in the plant. In specific methods and compositions, the heterologous nucleotide sequence of interest comprises a gene product that contains herbicide resistance, pathogen resistance, insect resistance and / or altered tolerance to salt, cold or drought. Expression cassettes comprising the promoter sequences of the invention operably linked to a heterologous nucleotide sequence of interest are provided. Additionally, plant cells, plant tissues, seeds and transformed plants are provided. DETAILED DESCRIPTION OF THE INVENTION The invention relates to compositions and methods related to plant promoters and methods of their use. The compositions comprise nucleotide sequences for the promoter region of the metallothionein (MT) gene. The compositions further comprise DNA constructs comprising a nucleotide sequence for the promoter region of the metallothionein 1 (MT1) gene operably linked to a heterologous nucleotide sequence of interest. In particular, the present invention provides isolated nucleic acid molecules comprising the nucleotide sequence set forth in SEQ ID NO: 1, and the plant promoter sequence deposited in bacterial hosts as Patent Deposit No. NRRL B-30792, on December 1, 2004, and fragments, variants and complements of it. Plasmids containing the plant promoter nucleotide sequence of the invention were deposited on December 1, 2004 with the Patent Depositary of the Agricultural Research Services Growing Collection of the National Center for the Investigation of the Utilization of Agriculture, at 1815 N. University Street, Peoria, IL. 61604, and Patent Deposit No. NRRL B-30792 was assigned. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience to those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112. The deposit irrevocably and without restriction or condition will be available to the public in the issuance of a patent.

However, it is going to be understood that the availability of a 'deposit does not constitute a license to practice the present invention in derogation of patent rights granted by the government's action. The MTl promoter sequences of the present invention include nucleotide constructs that allow the initiation of transcription in a plant. In specific embodiments, the MTl promoter sequence allows the initiation of transcription in a preferred manner of tissue, more particularly a preferred root form. Such constructs of the invention comprise regulated transcription initiation regions associated with the regulation of plant development. Thus, the compositions of the present invention comprise novel plant promoter nucleotide sequences, particularly preferred root promoter sequences for the MT gene, more particularly a sequence of the corn MTl promoter. The sequence for the corn MTl promoter region is set forth in the SEO ID N0: 1. The compositions of the invention include the nucleotide sequences for the native MTl promoter and fragments and variants thereof. The promoter sequences of the invention are useful for expressing sequences. In specific embodiments, the promoter sequences of the invention are useful for expressing sequences of interest in a preferred tissue manner, particularly in a preferred root manner. The sequences of the invention also find use in the construction of expression vectors for the subsequent expression of a heterologous nucleotide sequence in a plant of interest or as probes for the isolation of these MTl-like promoters. The related metalotion promoter sequences are disclosed in U.S. Application No. 09 / 520,268 and in U.S. Provisional Application No. 60 / 532,180 filed on December 23, 2003, the descriptions of which are incorporated herein by reference. In particular, the present invention provides isolated DNA constructs comprising the promoter sequence set forth in SEQ ID NO: 1 operably linked to a nucleotide sequence of interest. The invention comprises isolated or substantially purified nucleic acid compositions. An "isolated" or "purified" nucleic acid molecule or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemical substances. when they are synthesized chemically. An "isolated" nucleic acid is free of sequences (optimally protein coding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5 'and 3' ends of the nucleic acid) in the genomic DNA of the human body. which nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule may contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or Ol kb of nucleotide sequences that naturally flank the acid molecule nucleic acid in the genomic DNA of the cell from which the nucleic acid is derived. The MTl promoter sequences of the invention can be isolated from the 5 'untranslated region flanking their respective transcription initiation sites. The fragments and variants of the promoter sequences disclosed are also encompassed by the present invention. In particular, fragments and variants of the MTl promoter sequence of SEQ ID NO: 1 can be used in the DNA construct of the invention. As used herein, the term "fragment" means a portion of the nucleic acid sequence. Fragments of a MTl promoter sequence can retain the activity of initiating transcription. Alternatively, fragments of a nucleotide sequence that is useful as hybridization probes can not necessarily retain biological activity. Fragments of a nucleotide sequence for the promoter region of the MTl gene can vary from at least about 20 nucleotides, about 50 nucleotides, about 100 nucleotides and up to full-length nucleotide sequence of the invention for the promoter region of the gene. A biologically active portion of a MTl promoter can be prepared by isolating a portion of the MTl promoter sequence of the invention, and by estimating the promoter activity of the fragment. Nucleic acid molecules that are fragments of a nucleotide sequence of the MTl promoter comprise at least about 16, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 , 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800 nucleotides or up to the number of nucleotides present in a full length MTl promoter sequence disclosed herein (eg, 1815 nucleotides for the SEO ID NO: l). As used herein, the term '"variants" means substantially similar sequences. For nucleotide sequences, naturally occurring variants can be identified with the use of well-known molecular biology techniques, such as, for example, with the polymerase chain reaction (PCR) and hybridization techniques as summarized herein. . For nucleotide sequences, a variant comprises a deletion and / or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and / or a substitution of one or more nucleotides in one or more sites in the native polynucleotide. As used herein, a "native" nucleotide sequence comprises a naturally occurring nucleotide sequence. For nucleotide sequences, naturally occurring variants can be identified with the use of well-known molecular biology techniques, such as, for example, with the polymerase chain reaction (PCR) and hybridization techniques as summarized below. The nucleotide sequences of variants also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis. Generally, variants of a particular nucleotide sequence of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular nucleotide sequence, as determined - by the sequence alignment programs and the parameters described elsewhere in the present. A biologically active variant of a nucleotide sequence of the invention may differ from those sequences by as few as 1-15 nucleic acid residues, as few as 1-10, such as 6-10, as few as 5, so few as 4, 3, 2 or even 1 nucleic acid residue. The variant nucleotide sequences also comprise sequences derived from a mutagenic or recombinogenic process such as DNA intermixing. With such a procedure, one or more different MTl nucleotide sequences for the promoter can be manipulated to create a new MTl promoter. In this manner, libraries of recombinant polynucleotides are generated from a population of related sequence polynucleotides comprising regions of sequence that have substantial sequence identity and can be homologously recombined in vi tro or in vivo. Strategies for such intermingling of DNA are known in the art. See, for example, Stemmer (1994) Proc. Nati Acad. USA: 10747-10751; Stemmer (1994) Nature 370: 389-391; Cra eri (1997) Nature Biotech. 15: 436-438; Moore et al. (1997) J. Mol. 'Biol. 272: 336-347; Zhang et al. (1997) Proc. Nati Acad. USA 94: 4504-4509; Crameri et al (1998) Nature 391: 288-291; and U.S. Patent Nos. 5,605,793 and 5,837,458. The nucleotide sequences of the invention can be used to isolate corresponding sequences from other organisms, particularly other plants, more particularly other monocots. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology or the sequences displayed in the I presented. Isolated sequences based on their sequence identity in the complete MTl sequences set forth herein or to fragments thereof are encompassed by the present invention. In a PCR procedure, the oligonucleotide primers can be designed for use in PCR reactions to amplify the corresponding DNA sequences from a genomic DNA extracted from any plant of interest. Methods for designing PCR primers and PCR cloning are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York), then in the present Sambrook. See also Innis et al., Eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York) and; Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using peer primers, spliced primers, individual specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially-mismatched primers and the like. In hybridization techniques, all or part of a The known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of genomic DNA fragments cloned from a selected organism. The probes Hybridization can be labeled with a detectable group such as 32P, or any other detectable label. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the MTl promoter sequence of the invention. Methods for the preparation of probes for hybridization and for the construction of genomic libraries are generally known in the art and are disclosed in Sambrook. For example, the entire MTl promoter sequence disclosed herein, or one or more portions thereof, can be used as a probe capable of specifically hybridizing sequences of corresponding MTl promoter and messenger RNAs. To achieve specific hybridization under a variety of conditions, such probes include sequences that are unique among the MTl promoter sequences and are at least about 10 nucleotides in length or at least about 20 nucleotides in length. Such probes can be used to amplify corresponding MTl promoter sequences from a selected plant by PCR. This technique can be used to isolate additional coding sequences from an organism desired or as a diagnostic assay to determine the presence of coding sequences in an organism. Hybridization techniques include the hybridization classification of DNA libraries placed on plates (either plates or colonies; see, for example, Sambrook et al. (1989) Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Plainview , New York.) Hybridization of such sequences can be carried out under severe conditions The terms "severe conditions" and "conditions of severe hybridization" are proposed to imply conditions under which a probe will hybridize its target sequence to a degree detectably larger than other sequences (for example, at least 2 times over in background) .Strict conditions are dependent on the sequences will be different in different circumstances.When controlling the severity of hybridization and / or washing conditions, the target sequences that are 100% complementary to the probe can be identified (homologous probe). Severity can be adjusted to allow some sequence mismatches so that lower degrees of similarity are detected (heterologous polling). Generally, a probe is less than about 1000 nucleotides in length or less than 500 nucleotides in length.

Typically, severe conditions will be those in which the salt concentration is less than about 1.5 M NA ion, typically about 0.1 to 1.0 M Na (or other salts) concentration at pH 7.0 to 8.3 and the temperature is at least about 30 ° C for short probes (for example, 10 to 50 nucleotides) and at least about 60 ° C for long probes (for example, greater than 50 nucleotides). Severe conditions can also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization or a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37 ° C and a wash at IX to 2X SSC (20X SSC = 3.0 M NaCl / trisodium citrate 3 M) at 50 to 55 ° C. Exemplary moderate severity conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37 ° C, and a 0.5X to IX SSC wash at 55 to 60 ° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37 ° C and a final wash in 0.1 X SSC at 60 to 65 ° C for a duration of at least 30 minutes . The duration of the hybridization is generally less than about 24 hours, usually from about 4 to about 12 hours. The duration of the type of washing will be at least a sufficient length of time to reach the equilibrium. The specificity is typically the function of the post-hybridization washes, the critical factors that are of ionic strength and the temperature of the final wash solution. For DNA-DNA hybrids, the Tm (thermal melting point) can be approximated from the Meinkoth and Wahl equation (1984) Anal. Biochem. 138: 267-284: Tm = 81.5 ° C + 16.6 (log M) + 0. 41 (% GC) - 61 (% form) -500 / L; where M is the molarity of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in DNA,% of 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 (defined low ionic strength and pH) in which 50% of a complementary hybrid target sequence is a perfectly matched probe. Tm is reduced by approximately 1 ° C for every 1% of unequalization; thus, Tm, hybridization and / or wash conditions can be adjusted to hybridize the sequences of the desired identity. For example, if sequences with 90% identity are searched, the Tm can be decreased to 10 ° C. Generally, severe conditions are selected to be about 5 ° C lower than the Tm for the specific sequence is a complement at a defined ionic strength and pH. However, severely severe conditions may utilize hybridization and / or washing at 1, 2, 3 or 4 ° C less than Tm; the conditions moderately severe they can use a hybridization and / or washing at 6, 7, 8, 9 or 10 ° C lower than the Tm; the conditions of low severity can use a hybridization and / or washing at 11, 12, 13, 14, 15 or 20 ° C lower than the Tm. Using the equation, the hybridization and washing compositions, and the desired Tm, those of ordinary skill will understand that variations in the hybridization severity and / or washing solutions are inherently described. If the desired degree of unequalization results in a Tm of less than 45 ° C (aqueous solution) or 32 ° C (formamide solution), it is preferred to increase the SSC concentration so that it can be used at a higher temperature. An extensive guide for nucleic acid hybridization is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridisation wi th Nucleic Acid Probes, Part. I, Chapter 2 (Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See also Sambrook. Thus, isolated sequences having preferred root promoter activity and hybridizing under severe conditions to the MTl promoter sequences disclosed herein, or fragments thereof are encompassed by the present invention. The following terms are used to describe sequence relationships between two or more nucleic acids or polynucleotides: (a) "reference sequence", (b) "comparison window", (c) "sequence identity", (d) "percent sequence identity "and (e)" substantial identity ". (a) As used herein, "reference sequence" is a defined sequence used as a basis for the sequence comparison. A reference sequence can be a subset in the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. (b) As used in the present "comparison window" it refers to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window can comprise additions and deletions (ie, spaces) compared to the reference sequence (which does not include additions or deletions) for the optimal alignment of the two sequences. Generally, the comparison window is at least 20 nucleotides contiguous in length, and optionally may be 30, 40, 50, 100 or longer. Those skilled in the art understand that to avoid a high similarity of a reference sequence due to the inclusion of spaces in the polynucleotide sequence aSpace sanction is typically introduced and is subtracted from the number of equalizations. Methods of sequence alignment for comparison are well known in the art. Thus, the determination of the percent sequence identity between any of the two sequences can be performed using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (1988) CABIOS 4: 11-17; the local alignment algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the global alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453; the local search alignment method of Pearson and Lipman (1988) Proc. Nati Acad. 85: 2444-2448; the algorithm of Karlin and Altschul (1990) Proc. Nati Acad. USA 872264, as modified by Karlin and Altschul (1993) Proc. Nati USA 90: 5873-5877. Computer implementations of these mathematical algorithms can be used for sequence comparison to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC / Gene program (available from Intelligenetics, Mountain View, California); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics GCG Software Package, Version 10 (available from Accelrys Inc., 9685 Scranton Road, San Diego, California, USA). Alignments using these programs can be performed using the error parameters. The CLUSTAL program is well known by Higgins (1988) Gene 73: 237-244 (1988); Higgins et al. (1989) CABIOS 5: 151-153; Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang et al. (1992) CABIOS 8: 155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24: 307-331. The ALIGN program is based on the algorithm of Myers and Miller (1988) supra. A weighted residue table PAM120, a space length penalty of 12 and a space penalty of 4 can be used with the ALIGN program when comparing amino acid sequences. The BLAST program of Altschul et al. (1990) J. Mol. Biol. 215: 403 are based on the algorithm of Karlin and Altschul (1990) supra. BLAST nucleotide searches can be performed with the BLASTN program, log = 100, word length = 12, to obtain nucleotide sequences homologous to a nucleotide sequence encoding a protein of the invention. BLAST protein searches can be performed in the TLASTX program, register = 50, word length = 3, to obtain amino acid sequences homologous to a protein or polypeptide of the invention. To obtain spaced alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When using BLAST, Gapped BLAST and PSI-BLAST, the error parameters of the respective programs (for example, BLASTN for nucleotide sequences, BLASTX for proteins) can be used. See the National Center for Biotechnology Information on the website of the network on the world network in nebí .nim.nih.gob. The alignment can also be done manually by inspection. Unless stated otherwise, the sequence identity / similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters:% identity and% similarity for a nucleotide sequence using the Weight GAP of 50 and the length weight of 3 and the registration matrix nwsgapdna. emp; or any equivalent program of it. By "equivalent program" it is proposed to imply any sequence comparison program that, for either of two sequences in question, generates an alignment that has identical nucleotide or amino acid residue matches and an identical sequence identity percent when compared to the corresponding alignment generated by GAP Version 10. GAP uses the algorithm of Needleman and Wunsch (1970) supra, to find the alignment of two complete sequences that maximizes the number of equalizations and minimizes the number of spaces. GAP considers all possible alignments and space positions and creates the alignment with the largest number of equalized bases and the smallest spaces. This allows the provision of a space creation sanction and a space extension penalty in units of equal bases. GAP must make use of the space creation sanction number of the matches for each space it inserts. If a space extension penalty greater than zero is selected, GAP must also make use of each space inserted in the length of space times of the space extension penalty. The error space creation sanction values and Version 10 space extension sanction settings of the Wisconsin Genetics GCG Software package for protein sequences are 8 and 2, respectively. For nucleotide sequences the penalty space creation penalty is 50 while the error space extension penalty is 3. Space creation space creation penalties may be expressed as a whole number selected from the group of numbers integers consisting of 0 to 200, so, for example, The space extension space creation sanctions can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or larger. GAP presents a member of the family of best alignments. There may be many members of this family, but no other member has a better quality. GAP exhibits four figures of merit for the alignments: Quality, Relation, Identity and Similarity. Quality is the maximized metric in order to align the sequences. The relationship is the quality divided by the number of bases in the shortest segment. The percent identity is the percent of the symbols that actually match. The percent of similarity is the percent of the symbols that are similar. The symbols that are through the spaces are ignored. A similarity is recorded when the matrix value is recorded for a pair of symbols is greater than or equal to 0.50, the similarity threshold. The registration matrix used in Version 10 of the Wisconsin Genetics Software Package GCG is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Nati. Acad. Sci. USA 89: 10915). (c) As used herein, "sequence identity" or "identity" in the context of 'two polypeptide nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence over a comparison window specified When the percentage of sequence identity is used in reference to proteins it is recognized that residue positions that are not identical frequently differ by conservative amino acid substitutions, where the amino acid residues are replaced by other amino acid residues with similar chemical properties (for example, loading or hydrophobicity) and therefore do not change the functional properties of the molecule. When the sequences differ in conservative substitutions, the percent sequence identity can be adjusted upward to correct the conservative nature of the substitution. The sequences that differ with such conservative substitutions are said to have "sequence similarity" or "similarity". Means for making this adjustment are well known to those skilled in the art. Typically, this involves registering a conservative substitution as a partial unequalization before it completes, in order to increase the percentage of sequence identity. Thus, for example, where an identical amino acid is given a record of 1 and a non-conservative substitution is given a record of zero, a conservative substitution is given a record between zero and 1. The record of conservative substitutions is calculated, for example, how it is implemented in the PC / GENE program (Intelligenetics, Mountain View, California). (d) As used herein, "percent sequence identity" means the value determined by comparing two optimally aligned sequences on a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (ie, spaces) as compared to the reference sequence (which does not comprise additions or deletions) for the optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to produce the number of equalized positions, by dividing the number of equalized positions by the total number of positions in the comparison window, and multiplying the result by 100 to produce the percentage of sequence identity. (e) The term "substantial identity" of the polynucleotide sequence means that a polynucleotide comprises a sequence having at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and much more preferably at least 95%, compared to a reference sequence using one of the alignment programs described using standard parameters. Another indication of which nucleotide sequences are substantially identical is if two molecules hybridize to each other under severe conditions. Generally, severe conditions are selected to be approximately 5 ° C lower than Tra for the specific sequence at a defined ionic strength and pH. However, severe conditions comprise temperatures in the range of about 1 ° C to about 20 ° C lower than the Tm depending on the desired degree of severity as otherwise qualified herein. As used herein, the term "plant" includes plant cells, plant protoplasts, tissue cultures of plant cells from which plants, plant corns, plant groups, and plant cells that are intact can be regenerated. in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, cores, spikes, corncobs, farfollas, stems, roots, root tips, anthers and the like. The grain is proposed to imply mature seed produced by commercial growers for purposes other than growth in the reproduction of the species. Progeny, variants and mutants of regenerated plants are also included within the scope of the invention, with the proviso that these parts comprise the introduced polynucleotide. The present invention can be used for transformation of any species of plant, including, but not limited to, monocots and dicotyledons. Examples of plant species include, but are not limited to, maize (Zea mays), Brassica sp. (for example, B. napus, rapa, B. j uence), particularly those Brassica species useful as a source of seed oil, alfalfa . { Medicago sativa), rice (Oryza sativa), rye (Sécale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (for example, pearl millet (Pennisetum glaucum), millet proso (Panícum miliaceum), millet of foxtail ( Setaria i tálica), extended millet (Eleusine coracana), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Tri ticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hipogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), casava (Manihot esculenta), coffee (Coffea spp.), Coco (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), Cacao (Theobroma cacaco), tea (Camellia sinensis), banana (Musa spp.), Avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew nut (Anacardium occidentale), macadamia (Macadamia integrifolia), almonds (Prunus amygdalus), beets (Beta vulgaris), sugar cane (Saccharum spp.), Oats, barley, vegetables, ornamental plants and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (for example, Lactuca sativa), green beans (Phaseolus vulgaris), beans (Phaseolus limensis), peas (Lathyrus spp.), And members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantal upensis), and melon (C. meló). Ornamental plants include azalea (Rhododendron spp.), Hydrangea (Macrophylia hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), Tulips (Tulipa spp.), Daffodils (Narcissus spp.), Petunias (Petunia hybrida), carnation (Dianthus caryophyllus), red shepherdess (Euphorbia pulcherrima), and chrysanthemums. The conifers that may be employed in the practice of the present invention include, for example, pine trees such as incense pine (Pinus taeda), felling pine (Pinus ellíotii), ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta). ), and Monterey pine (Pinus radiata); Douglas fir (Pseudotsuga menziesil); western pinabete (Tsuga canadensis); Sitka fir (Picea glauca); red wood (Sequoia sempérvirens); typical firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as western red cedar (Thuja plicata) and yellow Alaskan cedar (Chamaecyparis nootkatensis). In specific embodiments, the plants of the present invention are crop plants (e.g., corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanuts, sorghum, wheat, millet, tobacco, etc.). In other modalities, corn and soybean plants are optimal, and in other modalities, corn plants are still optimal. Other plants of interest include grain plants that provide seeds of interest, oilseed plants and leguminous plants. The seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil seed plants include cotton, soybean, safflower, sunflower, Brasíca, corn, alfalfa, palm, coconut, etc. Legume plants include beans and peas. The beans include guar, carob, fenegreco, soybeans, garden beans, cowpeas, mung beans, beans, fava beans, lentils, chickpeas, etc. The heterologous coding sequences expressed by the MTl promoters of the invention can be used to vary the phenotype of a plant. Several changes in the phenotype are of interest including the modification of the expression of a gene in a plant root, the alteration of a defense mechanism to the pathogens or insects of the plant, the increase of the tolerance of the herbicide plants in a plant, the alteration of the development of the root when responding to environmental stress, the modulation of the response of the plant to salt, temperature (hot and cold), drought, and the like. These results can be achieved by expressing a sequence of heterologous nucleotides of interest comprising an appropriate gene product. In specific embodiments, the heterologous nucleotide sequence of interest is an endogenous plant sequence whose level of expression is increased in the plant or part of the plant. Alternatively, the results can be achieved by providing a reduction of expression of one or more endogenous gene products, particularly enzymes, transporters or cofactors, or by affecting the uptake of nutrients in the plant. These changes result in a change in the phenotype of the transformed plant. General categories of nucleotide sequences of interest for the present invention include, for example, those genes involved in the information, such as zinc extensions, those involved in communication, such as kinases, and those involved in maintenance., such as heat shock proteins. More specific categories of transgenes, for example, include genes that encode important attributes for agronomy, insect resistance, disease resistance, herbicide resistance, and resistance to environmental stress (altered tolerance to cold, salt, drought, etc.). It is recognized that any gene of interest can be operably linked to the promoter of the invention expressed in the plant. The insect resistance genes can encode the resistance to pests that have great performance impairment such as rootworm, cutworm, European corn borer and the like. Such genes include, for example, the toxic protein genes of Bacillus thuringiensis (U.S. Patent Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48: 109); and the similar ones. Genes encoding disease resistance attributes include detoxification genes, such as those that detoxify fumonisin (U.S. Patent No. 5,792,931); avirulence (avr) and disease resistance genes (R) (Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432 and Mindrinos et al. (1994) Cell 78: 1089); and the similar ones. Attributes of herbicide resistance may include genes that code for resistance to herbicides that act to inhibit the action of acetolactate synthase (ALS) and in particular sulfonylurea-type herbicides (eg, the acetolactate synthase (ALS) gene that contains mutations that lead to to such resistance, in particular mutations S4 and / or Hra) genes encoding for herbicide resistance that act to inhibit the action of glutamine synthase, such as phosphinothricin or coarse (eg, the bar gene), glyphosate (eg, the EPSPS gene and the gat gene; see, for example, the U.S. Publication No. 20040082770 and WO 03/092360) and other such genes known in the art. The geb bar codes for resistance to the coarse herbicide, the nptll gene codes for resistance to antibiotics and kanamycin and geneticin, and ALS gene mutants code for resistance to the herbicide chlorsulfuron. Resistance to glyphosate is imparted by the mutant 5-enolpiruvl-3-fosfiquimato (EPSP) and aroA genes. See, for example, U.S. Patent No. 4,940,835 to Shah et al., Which discloses the nucleotide sequence of an EPSPS form that can confer resistance to glyphosate. U.S. Patent No. 5,627,061 to Barry et al also discloses genes encoding EPSPS enzymes. See also U.S. Patent Nos. 6,248,876 Bl; 6,040,497; 5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114 Bl; 6,130,366; 5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E; and 5,491,288; and the international publications WO 97/04103; WO 97/04114; WO 00/66746; WO 01/66704; WO 00/66747 and WO 00/66748, which are incorporated herein by reference for this purpose. Resistance to glyphosate is also imparted in plants that express a gene encoding a glyphosate oxide-reductase enzyme as more fully described in U.S. Patent Nos. 5,776,760 and 5,463,175, which are incorporated herein by reference for this purpose. In addition to glyphosate resistance, it can be imparted to plants by overexpression of genes encoding glyphosate N-acetyltransferase. See, for example, U.S. patent application serial numbers 10 / 004,357 and 10 / 427,692. Exogenous products include enzymes from plants and products as well as those from other sources including prokaryotes and other eukaryotes. Such products include enzymes, cofactors, hormones and the like. Examples of other applicable genes and their associated phenotype include the gene encoding the viral coat protein and / or RNA, or other viral or plant genes that contain viral resistance; with genes that confer fungal resistance; genes that promote improvement in performance and genes that provide resistance to stress, such as cold, dehydration that results from drought, heat and salinity, toxic metal or minor elements or the like. As mentioned, the heterologous nucleotide sequence operably linked to the MTl promoters disclosed herein may be an antisense sequence for a targeted gene. Thus the promoter sequences disclosed herein can be operably linked to the antisense DNA sequences to reduce or inhibit the expression of a native protein in the root of the plant. "RNAi" refers to a series of related techniques for reducing the expression of genes (See, for example, U.S. Patent No. 6,506,559). More previous techniques referred to by other names are now thought to depend on the same mechanism, but are given different names in the literature. These include "antisense inhibition", the production of antisense RNA transgene capable of suppressing the expression of the target protein and "co-suppresion" or "sense suppression" which refers to the production of sense transgene RNA capable of suppressing the expression of identical substantially identical foreign or endogenous genes (U.S. Patent No. 5,231,020, incorporated herein by reference). Such techniques depend on the use of constructs that result in the accumulation of double-stranded RNA with a strand complementary to the target gene that is silenced. The MT2 promoters of the modalities can be used to induce the expression of constructs that will result in RNA interference including microRNAs and siRNAs. The term "promoter" or "transcriptional initiation region" is intended to mean a regulatory region of DNA usually comprising a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the transcription initiation site appropriate for a particular coding sequence. A promoter may additionally comprise other recognition sequences generally positioned upstream or 5 'to the TATA box, referred to as upstream promoter elements, which influence the rate of transcription initiation. It is recognized that having identified the nucleotide sequences for the promoter regions disclosed herein, it is within the art to isolate and identify additional regulatory elements in the 5 'untranslated region upstream of the particular promoter regions identified in Additionally, chimeric promoters may be provided, such chimeras include portions of the promoter sequence fused to fragments and / or variants of heterologous transcriptional regulatory regions, Thus, the promoter regions disclosed herein may comprise upstream regulatory elements such as, those responsible for the tissue and temporal expression of the coding sequence, enhancers and the like In the same way, the promoter elements, which allow expression in the desired tissue, such as the root, can be identified, isolated and used with other promoters of n cleo to confer root-preferred expression. In this aspect of the invention, a "core promoter" is proposed to mean a promoter without promoter element.

In the context of this description, the term "regulatory element" also refers to a DNA sequence, usually, but not always, upstream (5 ') to the coding sequence of a structural gene, which includes sequences that control the expression of the coding region by providing recognition for RNA polymerase and / or other factors required for transcription to start at a particular site. An example of a regulatory element that provides recognition for RNA polymerase or other transcription factors to ensure initiation at a particular site is a promoter element. A promoter element comprises a core promoter element, responsible for the initiation of transcription, as well as other regulatory elements (as discussed elsewhere in this application) that modify the expression of the gene. It is to be understood that the sequence of nucleotides, located within introns or 3 'of the coding region sequence may also contribute to the regulation of the expression of a coding region of interest. Examples of suitable introns include, but are not limited to, the corn IVS6 intron, or the corn actin intron. A regulatory element may also include elements located downstream (3 ') to the transcription initiation site, or within the transcribed regions, or both. In the context of the present invention a post-transcriptional regulatory element may include elements that are active after the initiation of transcription, for example translational and transcriptional enhancers, translational and transcriptional repressors, and mRNA stability determinants. The regulatory elements, or variants or fragments thereof, of the present invention can be operatively associated with heterologous regulatory elements or promoters in order to modulate the activity of the heterologous regulatory element. Such modulation includes increasing or repressing the transcriptional activity of the heterologous regulatory element, modulating the posttranscription events, or either increasing or repressing the transcriptional activity of the heterologous regulatory element, and modulating the post-transcription events. For example, one or more regulatory elements, or fragments thereof, of the present invention may be operatively associated with constitutive, inducible or tissue-specific promoters or fragments thereof, to modulate the activity of such promoters within desired tissues. in plant cells. The regulatory sequences of the present invention, or variants or fragments thereof, when operably linked to heterologous nucleotide sequences of interest can induce the preferred expression root of the heterologous nucleotide sequence in the root (or part of the root) of the plant expressing this construction. The term "preferred root" is intended to imply that the expression of the heterologous nucleotide sequence is more abundant in the root or part of the root, including, for example, the root layer, apical meristem, protoderma , meristema of the earth, procambio, endodermis, cortex, vascular cortex, epidermis and the like. While some level of expression of the heterologous nucleotide sequence may occur in other types of plant tissue, expression occurs more abundantly in the root or part of the root including the primary, lateral and adventitious roots. A "heterologous nucleotide sequence" is proposed to imply a sequence that is not occurring naturally with the promoter sequence of the invention. While this nucleotide sequence is heterologous to the promoter sequence, it may be homologous, either native or heterologous, or foreign to the plant host. The isolated promoter sequences of the present invention can be modified to provide a range of expression levels of the heterologous nucleotide sequence. Thus, less than the complete promoter regions can be used and the ability to induce expression of the nucleic acid sequence of arrested interest. It is recognized that the levels of mRNA expression can be altered in different ways by deletions of portions of the promoter sequence. The levels of mRNA expression can be decreased or alternatively, expression can be increased as a result of promoter deletions if, for example, there is a native regulatory element (for a repressor) that is removed during the truncation process. Generally, at least about 20 nucleotides of an isolated promoter sequence has been used to induce the expression of a nucleotide sequence. It is recognized that in order to increase the levels of transcription, the enhancers can be used in combination with the promoter regions of the invention. Enhancers are nucleotide sequences that act to increase the expression of a promoter region. Enhancers are known in the art and include the SV40 enhancer region, the enhancer element 35S, and the like. Some enhancers are also known to alter normal promoter expression patterns, for example, by causing a promoter to be expressed constitutively when the enhancer, the same promoter is expressed only in a specific tissue or a few specific tissues. Modifications of promoter sequences isolated from the present invention can provide a range of expression of the heterologous nucleotide sequence. Thus, it can be modified to be weak promoters or strong promoters. Generally, a "weak promoter" is proposed to imply a promoter that induces the expression of a coding sequence at a low level. A "low level" of expression is proposed to imply the expression and levels of approximately 1/10, 000 transcripts to approximately 1 / 100,000 transcripts to approximately .1 / 500,000 transcripts. Conversely, a strong promoter induces the expression of a coding sequence at a high level, or from about 1/10 transcripts to about 1/100 transcripts to about 1 / 1,000 transcripts. It is recognized that the promoters of the invention can be used with their native MT coding sequences to increase or decrease expression, in order to thereby result in a change in the phenotype of the transformed plant. This phenotypic change could also affect an increase in the gene in the tissue metal gene levels of the transformed plant. The nucleotide sequences disclosed in the present invention, as well as variants and fragments thereof, are useful in the genetic manipulation of any plant. The MTl promoter sequences are useful in this aspect when they are operably linked to a sequence of heterologous nucleotides whose expression is to be controlled to achieve a desired phenotypic response. The term "operably linked" is intended to imply that the transcription or translation of the heterologous nucleotide sequence is under the influence of the promoter sequence. In this manner, the nucleotide sequences for the promoters of the invention can be provided in expression cassettes together with heterologous nucleotide sequences of interest for expression in the plant of interest, more particularly in the root of the plant. Such expression cassettes will comprise a transcriptional initiation region comprising one of the promoter nucleotide sequences of the present invention, or variants or fragments thereof operably linked to the heterologous nucleotide sequence. Such an expression cassette can be provided with a plurality of restriction sites for the insertion of the nucleotide sequence that is under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain selectable marker genes as well as 3 'termination regions. The expression cassette may include, in the 5 '-3' direction of transcription, a transcriptional initiation region (i.e., a promoter, or variant or fragment thereof, of the invention), a translational initiation region, a heterologous nucleotide sequence of interest, a translational termination region and optionally, a transcriptional termination region, functional in the host organism. The regulatory regions (ie, promoters, transcriptional regulatory regions and translational termination regions) and / or polynucleotides of the modalities may be native / analogous to the host cell or to each other. Alternatively, the regulatory regions and / or polynucleotides of the embodiments may be heterologous to the host cell or to each other. As used herein, "heterologous" in reference to a sequence is a sequence that originates from a foreign species, or, if it is from the same species, is substantially modified from its native form in composition and / or genomic site by the deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide is a species different from the species from which the polynucleotide was derived, or if it is from the same / analogous species, one or both are substantially modified from their original form and / or site genomic, or the promoter is not the native promoter for the operably linked polynucleotide. While it may be preferable to express a heterologous nucleotide sequence using the promoters of the invention, the native sequences may be expressed. Such constructs would change the expression levels of the MT protein in the plant or plant cell. Thus, the phenotype of the plant or plant cells is altered. The termination region may be native to the transcriptional initiation region, may be native to the operably linked DNA sequence of interest, may be native to the plant host, or may be derived from another source (i.e., foreign or heterologous). to the promoter, the sequence of 'DNA that is expressed, the plant host, or any combination thereof). Suitable termination regions are available from the Ti plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon and collaborators (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al (1990) Gene 91: 151-158; Bailas et al. (1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al. (1987) Nucleic Acid Res. 15: 962 '-9639. The expression cassette comprising the sequences of the present invention may also contain at least one additional nucleotide sequence for a gene that is cotransformed in the organism. Alternatively, the (s) Additional sequence (s) can be provided in another expression cassette. Where appropriate, nucleotide sequences whose expression will be under the control of the preferred root promoter sequence of the present invention and any additional nucleotide sequence (s) can be optimized for increased expression in the transformed plant. That is, these nucleotide sequences can be synthesized using preferred plant codons for improved expression. See, for example, Campbell and Gowi (1990) Plant Physiol. 92: 1-11 for a discussion of the use of the host's preferred codon. Methods are available in the art to synthesize preferred plant genes. See, for example, U.S. Patent Nos. 5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17: 477-498, incorporated herein by reference. Additional sequence modifications are known to increase gene expression in a cell host. These include the elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats and other such well-characterized sequences that may be detrimental to gene expression. The G-C content of the heterologous nucleotide sequence can be adjusted to average levels for a given cell host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid the predicted secondary hairpin mRNA structures. The expression cassettes can additionally contain 5 'leader sequences. Such guide sequences can act to increase translation. Translation guides are known in the art and include: picornavirus guides, eg, EMCV guide (5 'non-coding region of Encephalomyocarditis) (Elroy-Stein et al. (1989) Proc. Nat. Acad. Sci. USA 86 : 6126-6130), potyvirus guides, for example, VTE guide (Tobacco Etch Virus) (Allison et al. (1986) Virology 154: 9-29)); MDMV guide (dwarf corn mosaic virus), a protein that binds the human immunoglobulin heavy chain (BiP) (Macejak et al. (1991) Nature 353: 90-94); untranslated guidance of the mRNA coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625), tobacco mosaic virus (TMV) guide (Gallie et al. 1989) Molecular Biology of RNA, pages 237-256); and corn chlorotic mottled virus guide (MCMV) (Lommel et al. (1991) Virology 81: 382-385). See also Della Cioppa et al. (1987) Plant Physiology 84: 965-968. Methods Known for increasing the stability of mRNA can also be used, for example, introns, such as the corn Ubicuitin intron (Christensen and Quail (1996) Transgenic Res, 5: 213-218; Christensen et al. (1992) Plant Molecular Biology 18 : 675-689) or the Adhl intron of corn (Kyozuka et al. (1991) Mol. Gen. Genet. 228: 40-48; Kyozuka et al. (1990) Maydica 35: 353-357) and the like. In the preparation of the expression cassette, the various DNA fragments can be manipulated, to provide the DNA sequences in the proper orientation and, as appropriate, in the appropriate reading structure. Towards this end, adapters or linkers can be used to join DNA fragments or other manipulations can be involved to provide convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, re- substitutions, for example, transitions or transversions, may be involved. Reporter genes or selectable marker genes can be included in the expression cassettes. Examples of suitable reporter genes known in the art can be found in, for example Jefferson et al. (1991) in Plant Molecular Biology Manual, ed.

Gelvin et al. (Kluwer Academic Publishers), pp. 1-33; DeWet et al. (1987) Mol. Cell. Biol. 7: 725-737; Goff et al. (1990) EMBO J. 9: 2517-2522; Kain et al. (1995) BioTechniques 19: 650-655; and Chiu et al. (1996) Current Biology 6: 325-330. Selectable marker genes for the selection of transformed cells or tissues may include genes that confer antibiotic resistance or herbicide resistance. Examples of suitable selectable marker genes include, but are not limited to, genes encoding chloramphenicol resistance (Herrera Estrella et al. (1983) EMBO J. 2: 987-992); metrotexate (Herrera Estrella et al. (1983) EMBO J. 2: 987-992; methotrexate (Herrera Estrella et al. (1983) Nature 303: 209-213; Meijer et al. (1991) Plant Mol. Biol. 16: 807-820 ), hygromycin (Waldron et al. (1985) Plant Mol. Biol. 5: 103-108; Zhijian et al. (1995) Plant Science 108: 219-227); streptomycin (Jones et al. (1987) Mol. Gen. Genet. 210: 86-91); spectinomycin (Bretagne-Sagnard et al. (1996); Transgenic Res. 5: 131-137); bleomycin (Hille et al. (1990) Plant Mol. Biol 7: 171-176); sulfonamide (Guerineau et al. (1990) Plant Mol. Biol. 15: 127-136); Bromoxynil (Stalker et al. (1988) Science 242: 419-423); glyphosate (Shaw et al. (1986) Science 233: 478- 481); phosphinothricin (DeBlock et al. (1987) EMBO J. 6: 2513-2518). Other genes that could be useful in the recovery of transgenic events but could not be required in the final product would include, but are not limited to, examples such as GUS (beta-glucuronidase; Jefferson (1987) Plant Mol. Bi ol. Rep. 5: 387), GFP (green fluorescence protein, Chalfie et al. (1994) Sci en 263: 802), luciferase (Riggs et al. (1987) Nucleic Acids Res. 15 (19): 8115 and Luehrsen et al. (1992). ) Methods Enzymol 216: 397-414) and the maize genes coding for the production of anthocyanin (Ludwig et al. (1990) Sci en 247: 449). The expression cassette comprising the MTl promoter of the present invention, operably linked to a nucleotide sequence of interest, can be used to transform any plant. In this way, genetically modified plants, plant cells, plant tissue, seed, root and the like can be obtained. The methods of the invention involve introducing a nucleotide sequence into a plant. "Introduction" is intended to imply the presentation to the plant of the nucleotide sequence in such a way that the sequence gains access to the interior of a plant cell. The methods of the invention do not depend on a particular method to introduce a sequence of a plant, only that the nucleotide sequence gains access to the interior of at least one cell of the plant. Methods for introducing nucleotide sequences into plants are known in the art including, but not limited to, stable transformation methods, transient transformation methods and virus mediated methods. "Stable transformation" is proposed to imply that the construction of nucleotides introduced into a plant is integrated into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" is proposed to imply that a nucleotide sequence is introduced into the plant and is not integrated into the genome of the plant or a polypeptide is introduced into a plant. Transformation protocols as well as protocols for introducing nucleotide sequences in plants can vary depending on the type of plant or plant cell, i.e., monocot and dicot, directed for transformation. Suitable methods for introducing nucleotide sequences into plant cells and subsequent insertion into the genome of the plant include microinjection (Crossway et al. (1986) Biotechniques 4: 320-334), electroporation (Riggs et al. (1986) Proc. Nati. Acad. Sci. USA 83: 5602-5606, transformation mediated by Agrobacterium (U.S. Patent Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3: 2717-2722), and acceleration of ballistic particles (see, for example, US Pat. 4,945,050; 5,879,918; 5.88'6.244; 5,932,782; Tomes et al. (1995) in: Plant Cell, Tissue, and Organ Culture Fundamental Methods, ed. Gamborg and Philips (Springer-Verlag Berlin); McCabe et al. (1988) Biotechnology 6: 923-926); and the transformation of Lecl (WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22: 421-477; Sanford et al. (1987) Particulate Science and Technology 5: 27-37 (onion); Christou et al. (1988) Plant Physiol. 87: 671-674 (soybean); McCabe et al. (1988) Bio / Technology 6: 923-926 (soybean); Finer and McMullen (1991 In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96: 319-324 (soybean); Datta et al. (1990) Biotechnology 8 : 736-740 (rice), Klein et al (1988) Proc. Nati, Acad. Sci. USA 85: 4305-4309 (corn), Klein et al. (1988) Biotechnology 6: 559-563 (corn); North American patents ,240,855; 5,322,783 and 5,324,646; Klein and collaborators (1988) Plant Physiol. 91: 440-444 (corn); Fromm et al (1990) Biotechnology 8: 833-839 (corn); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311: 763-764; U.S. Patent No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Nati Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman and collaborators (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9: 415-418 and Kaeppler et al. (1992) Theor. Appl. Genet 84: 560-566 (transformation mediated by whisker); D'Halluin et al. (1992) Plant Cell 4: 1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12: 250-255 and Christou and Ford (1995) Annals of Botany 75: 407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14: 745-750 (maize via Agrobacterium tumefaciens); all of which are incorporated herein by reference. In specific embodiments, DNA constructs comprising the promoter sequences of the invention can be provided to a plant using a variety of transient transformation methods. Such transient transformation methods include, but are not limited to, viral vector systems and precipitation of the polynucleotide in a manner that prevents subsequent release of the DNA. Thus, transcription of DNA bound to particles can occur, but the frequency with which it is released to become integrated into the genome is greatly reduced. Such methods include the use of particles coated with polyethyleneimine (PEI; Sigma # P3143). In other embodiments, the polynucleotide of the invention can be introduced into plants by contacting plants with a virus or viral nucleic acids. Generally, such methods involve incorporating a nucleotide construct of the invention into a viral DNA or RNA molecule. Methods for introducing polynucleotides into plants and expressing a protein encoded therein, involve viral DNA or RNA molecules, are known in the art. See, for example, U.S. Patent Nos. 5,889,191, 5,889,190, 5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) Molecular Biotechnology 5: 209-221; incorporated herein by reference. The methods are known in the art for the targeted insertion of a nucleotide sequence at a specific location in the genome of the plant. In one embodiment, the insertion of the DNA construct comprising the heterologous nucleotide sequence of interest into a desired genomic location is achieved using a site-specific recombination system. See, for example, W099 / 25821, W099 / 25854, W099 / 25840, W099 / 25855 and W099 / 25853, all of which are incorporated herein. or reference, Briefly, the promoter of the invention can be contained in a transfer cassette flanked by the two non-recombinogenic recombination sites. The transfer cassette is interested in a plant that has stably incorporated its genome into a target site that is flanked by two non-recombinogenic recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated into the target site. The DNA construct comprising the heterologous nucleotide sequence of interest is thus integrated into a specific chromosomal position in the genome of the plant. The cells that have been transformed can be grown in plants according to conventional manners. See, for example, McCormick et al. (1986) Plant Cell Reports 5: 81-84. These plants can then be cultured, either pollinated with the same transformed strain or different strains, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations can be cultured to ensure that the expression of the desired phenotypic characteristic is stably maintained and inherited and then the seeds recirculated to ensure the expression of the desired phenotypic characteristic that has been achieved. In this way, the present invention provides seed transformed (also referred to as "transgenic seed") having a nucleotide construct of the invention, for example, an expression cassette of the invention, stably incorporated in its genome. The article "a" and "an" is used in the present to refer to one or more than one (ie, at least one) of the grammatical object of the article. By way of example, "an element" means one or more elements. Throughout the specification the word "comprising" or variations such as "is understood" or "comprising" will be understood to imply the inclusion of an established element, integer or stage, or groups of elements, integers or steps, but not the exclusion of any other element, integer or stage, or group of elements, integers or stages. The following examples are offered by way of illustration and not by way of limitation. EXPERIMENTAL Example 1: Amplification and Reconstruction of the 1.8 kb Metalotionin (MTl) Promoter The 1.8 kb promoter sequence upstream of the root metallothionein (MTl) gene was obtained by walking the genome using the GenomeWalker ™ equipment (BD Biosciences Clontech, Palo Alto, CA) following the manufacturer's protocol. The 1.8 kb MTl promoter sequence is generated by performing three stages of genome passage upstream of a TM promoter of 747 base pairs (bp) smaller (SEO ID NO: 2). Two specific gene spliced primers were designed using SEQ ID NO: 2 as a template. The first stage, two gene-specific primers spliced named rootmet5 (SEO ID NO: 3) and rootmet7 (SEQ ID NO: 4) were used to amplify a 683 bp fragment via the polymerase chain reaction (PCR). which was cloned into the vector pCR®2.1-TOPO® (Invitrogen, Carlsbad, CA). This clone was named pTOPO®-RM22. In the second stage, two different spliced gene-specific primers designed from the pTOPO-RM22 sequence, named rootmetl (SEQ ID NO: 5) and rootmet 2 (SEQ ID NO: 6) were used to amplify a 300 bp fragment by PCR. . This PCR product was cloned into the vector pCR®2.1-TOPO® to generate a clone named pTOPO®-Rootmetl2. The insert in pTOPO®-Rootmetl2 was then sequenced. In the final stage, a third set of spliced primers named rootmet3KRW3 (SEQ ID NO: 7) and rootmet3KRW4 (SEQ ID NO: 8) was used to perform an additional genome step step upstream of the insert sequence pTOPO @ - Rootmetl2. This final step generated an 871 bp PCR product that was cloned into pCR®2.1-TOPO® and then sequenced. This final clone was named pTOPO®-FRAG53.

For each step of the genome, the following PCR conditions were used: 94 ° C for 1 min followed by 20 cycles of: 94 ° C for 15 sec. and 65 ° C for 4 min. (reduced by 0.5 ° C per cycle) and 15 cycles of: 94 ° C for 15 sec. and 55 ° C for 4 min. The full length MTl promoter was then assembled from a subset of the cloned genome passage fragments using the splice by overlay extension (SOE) as described below. pOPO®-Rootmetl2 was not used in the SOE process since the SOE primers that were designed to bridge the sequence of pTOPO®-RM22 and pTOPO®-FRAG53 included the sequence of pTOPO®-Rootmetl2 not present in any of the clones previous SOE was performed due to the difficulties encountered during the PCR amplification of the 1.8 kb promoter fragment directly from the maize genomic DNA. SOE - Round 1 In the first stage of SOE, round 1, the PCR primers were used to amplify promoter fragments using pTOPO®-RM22 and pTOPO®-FRAG53 individually as templates. An 894 bp promoter fragment was amplified by PCR from pTOPO®-FRAG53 using primers named RMSOE1 (SEQ ID NO: 9) and RMSOE4 (SEO ID NO: 10). A BamHI site was added to the 5 'end of RMSOE1 to facilitate subcloning of the entire promoter fragment.

A 672 bp promoter fragment was amplified by PCR of pTOPO®-RM22 using the primers named RMSOE3 (SEQ ID NO: ll) and RMSOE2 (SEQ ID NO: 12). RMSOE3 and RMSOE4 were designed such that the 36 nucleotides at the 3 'end of each primer were complemented with each other in order to facilitate the annealing of the two PCR products separated in the second stage of SOE. The PCR conditions for the first stage of SOE, round 1, were as follows: 94 ° C for 30 sec. followed by 30 cycles of: 94 ° C for 30 sec, 42 ° C for 1 min, and 72 ° C for 30 sec. followed by 72 ° C for 5 min. The PCR products were gel purified and used as templates for the second stage of SOE. In the second stage of SOE, the PCR products of 894 and 672 bp purified from stage 1 were used as templates for PCR with the primers named RMSOE1 (SEQ ID NO: 9) and RMSOE2 (SEQ ID NO: 12). The PCR conditions for the second stage of SOE, round 1, were as follows: 94 ° C for 5 min. followed by 30 cycles of: 94 ° C for 30 sec, 50 ° C for 1 min., and 72 ° C for 30 sec, followed by 72 ° C for 7 min. The resulting 1.52 kb fragment was cloned into pCR®2.1-TOPO® and the sequence was confirmed. This clone was named pTOPO®-SOEl. SOE - Round 2 An additional round of SOE, round 2, was performed to add the remainder of the sequence to the 1.8 kb MTl promoter. In the first step two primers named Bamrootmetprod (SEQ ID NO: 13) and pSOEB (SEQ ID NO: 14) were used to PCR amplify a 1.2 kb promoter fragment of the clone named pTOPO®-SOEl. Then, the primers named pSOEA (SEQ ID NO: 15) and Xhorootmetlb (SEQ ID NO: 16) were used to PCR amplify a 656 bp product of a plasmid containing the original MT 747 bp promoter (SEQ ID NO: 2) . An Xhol site was incorporated into the Xhorootmetlb primer to facilitate future subcloning. pSOEA and pSOEB. they contained 20 nucleotides of complementary sequence at their 5 'ends so that the two PCR products in the second SOE stage would be easily annealed. Each PCR reaction for the first stage of round 2 used the following conditions: 94 ° C for 5 min. followed by 30 cycles of: 94 ° C for 30 sec, 50 ° C for 1 min., and 72 ° C for 30 sec, followed by 72 ° C for 7 min. The 1.2 kb and 0.66 kb PCR products from step one of round 2 were gel purified and used as templates for the second stage (round 2) of SOE. The primers used in the second step were BAMrootmetprod (SEQ ID NO: 13) and Xhorootmetlb (SEQ ID NO: 16). The PCR conditions for the second stage of SOE, round 2, were as follows: 94 ° C for 30 sec. followed by 30 cycles of: 94 ° C for 30 sec., 50 ° C for 1 min., and 72 ° C for 30 sec., followed by 72 ° C for 5 min. The resulting 1.8 kb PCR fragment (i.e., the 1.8 kb MTl promoter) was cloned into pCR®2.1-TOPO® and the sequence was confirmed. This clone was named RM2-2. Example 2 Transformation and Regeneration of Transgenic Plants Embryos of immature maize from greenhouse donor plants were bombarded with a plasmid containing a gene of interest operably linked to an MTl promoter of the invention, plus a plasmid containing the selectable marker gene PAT ( Wohlleben et al. (1988) Gene 70: 25-37) which confers resistance to the herbicide Bialaphos. The transformation is done as follows. The recipe for the media is shown right away. To prepare the target tissue, the ears were sterilized on the surface in 30% Clorox bleach plus 0.5% Micro detergent for 20 minutes, and rinsed twice with sterile water. The immature embryos were removed and placed with the axis of the embryo side down (scutellum side up), 25 embryos per plate and the 560Y medium for 4 hours and then aligned within the target area 2.5 cm in the preparation for the bombing.

To prepare DNA, a plasmid vector comprising a gene of interest operably linked to an MTl promoter of the invention is made. This plasmid DNA plus plasmid DNA containing a selectable PAT marker is precipitated on tungsten pellets of 1.1 μm (average diameter) using a CaCl 2 precipitation procedure as follows: 100 μL of tungsten particles prepared in water; 10 μL (1 μg) of DNA in TrisEDTA buffer (1 μg total); 100 μL of 2.5 M CaCl; 10 μL of 0.1 M spermidine. Each reagent is sequentially added to the suspension of tungsten particles, while remaining in the multi-tube vortex apparatus. The final mixture is briefly sonicated and allowed to incubate under constant vortex formation for 10 minutes. After the precipitation period, the tubes are centrifuged briefly, the liquid is removed, washed with 500 mL of 100% ethanol and centrifuged for 30 seconds. Again the liquid is removed and 105 μL of 100% ethanol is added to the final tungsten particle pellet. For particle bombardment, the tungsten / DNA particles are briefly sonicated and 10 μL is stained on the center of each macrocarrier and allowed to dry approximately 2 minutes before bombardment. The sample plates are bombarded at the levels # 4 in the particle gun # HE34-1 or # HE34-2. All samples receive a single shot at 650 PSI, with a total of 10 aliquots taken from each tube of prepared particles / DNA. After bombardment, the embryos are conserved in 560Y medium for 2 days, then transferred to the 560R selection medium containing 3 mg / liter of Bialaphos, and subcultured every 2 weeks. After about 10 weeks of selection, callus clones resistant to selection are transferred to selection medium 288J to initiate regeneration of the plant. After maturation of the somatic embryo (2-4 weeks), well-developed somatic embryos are transferred into the medium for germination and transferred to the lit culture room. Approximately 7-10 days later, the developing seedlings are transferred to the 272V hormone-free medium in tubes for 7-10 days until the seedlings are well established. The plants are then transferred to inserts in seed boxes (equivalent to a 2.5"pot) containing potted earth and grown for a week in a growth chamber, subsequently 1-2 additional weeks are grown in a greenhouse, then transferred to 600 classic pots (1.6 gallons) and grown to maturity.The plants are monitored and recorded for the preferred root activity of the gene of interest or for the levels of altered metal ion. The means of bombardment and cultivation employed is as follows. Bombardment medium (560Y) comprises 4.0 g / L of base N6 salts (SIGMA C-1416), 1.0 mL / L of Eriksson Vitamin Mix (1000X SIGMA-1511), 0.5 mg / L of thiamine HCl, 120.0 g / L of sucrose, 1.0 mg / L of 2,4-D, and 2.88 g / L of L-proline (brought to a volume with H20 DI after adjustment to pH 5.8 with KOH); 2.0 g / l Gelrite (added later to bring the volume with H20 D-I); and 8.5 mg / L of silver nitrate (added after sterilizing the medium and cooling to room temperature). The means of selection (560R) comprises 4.0 g / L of base salts N6 (SIGMA C-1416), 1. 0 mL / L of Eriksson Vitamin Mixture (1000X Sigma-1511), 0.5 mg / L of thiamine HCl, 30.0 g / L of sucrose, and 2.0 mg / L of 2,4-D (brought to volume with H20 of DI after adjustment to pH 5.8 with KOH); 3.0 g / L of Gelrite (added after bringing to volume with H20 D-I); and 0.85 mg / L of silver nitrate and 3.0 mg / L of bialaphos (both added after sterilizing the medium and cooling to room temperature). The plant regeneration medium (288J) comprises 4.3 g / L of MS salts (GIBCO 11117-074), 5.0 mL / L of MS vitamins extract solution (0.100 g of nicotinic acid, 0.02 g / L of thiamine HCl , 0.10 g / l of pyridoxine HCl and 0.40 g / L of glycine brought to volume with H20 D- I purified) (Murashige and Skoog (1962) Physiol. Plant 15: 473), 100 mg / L of myo-inositol, 0.5 mg / L of zeatin, 60 g / L of sucrose and 1.0 mL / L of abscisic acid 0.1 mM (brought to volume with H20 DI purified after adjustment to pH 5.6); 3.0 g / L of Gelrite (added after bringing to volume with H20 D-I); and 1.0 mg / L of indoleacetic acid and 3.0 mg / L of bialaphos (were added after sterilizing the medium and cooling to 60 ° C). The hormone-free medium (272 V) comprises 4.3 g / L of MS salts (GIBCO 11117-074), 5.0 mL / L of MS vitamins extract solution (0.100 g / L of nicotinic acid, 0.02 g / L of HCl of thiamin, 0.10 g / L of pyridoxine HCL and 0.40 g / L of glycine carried to volume with purified H20 DI) and 0.1 g / L of myo inositol, and 40.0 g / L of sucrose (brought to volume with purified H20 DI after adjusting the pH to 5.6); and 6 g / L of bacto-agar (added after bringing to volume with purified H20 D-I), sterilized and cooled to 60 ° C. Example 3: Expression Data Using the Promoter Sequences of the Invention The B73 seeds were placed along one edge of the growth paper soaked in a 7% sucrose solution. An additional piece of growth paper identical in size to the first was also soaked in 7% sucrose and overlaid on the seed. The interleaved set of paper-growth-seed-paper of The growth was subsequently coiled solidly with the edge of the seed at the top of the roll. The roll was placed directionally in a glass of 7% sucrose solution with the seeds on top to allow the growth of the straight root. The seeds were allowed to germinate and developed for 2-3 days in the dark at 27-28 ° C. Prior to bombardment, the external film layer of the cotyledon was removed and the seedlings were placed in a sterile petri dish (60 mm) on a layer of Whatman # 1 filter paper moistened with 1 mL of H20. Two seedlings per plate were arranged in opposite orientations and fixed to the filter paper with a 0.5% agarose solution. Root tip sections of 2-3 cm were also excised from the seedlings and the plates were arranged longitudinally for bombardment. The DNA / gold particle mixtures were prepared for bombardment in the following method. Sixty mg of 0.6-1.0 micron gold particles were prewashed with ethanol, rinsed with sterile distilled H20 and resuspended in a total of 1 mL of sterile H20. Aliquots of 50 μL of the suspension of gold particles were stored in siliconized Eppendorf tubes at room temperature. The DNA was precipitated on the surface of the gold particles by combining, in order, the aliquot of 50 μL of pre-washed 0.6 μM gold particles, 5-10 μg of test DNA, 50 μL of CaCl2 of 2.5 M and 25 μL of 0.1 M spermidine. The solution was immediately vortexed for 3 minutes and centrifuged briefly to pellet the DNA / gold particles. The DNA / gold was washed once with 500 μL of 100% ethanol and suspended in a final volume of 50 μL of 100% ethanol. The DNA / gold solution was incubated at -20 ° C for at least 60 minutes before aliquoting 6 μL of the DNA / gold mixture on each Mylar ™ macrocarrier. The seedlings prepared as indicated above and the root tips excised were bombarded twice using the PDS-1000 / He gun at 1000 psi under 27-28 inches Hg vacuum. The distance between the macrocarrier and the detention screen was between 6-8 cm. Plates were incubated in sealed containers for 24 hours in the dark at 27-28 ° C after bombardment. After 18-24 hours of incubation the bombarded seedlings and root tips were analyzed for transient GUS expression. Seedlings and roots excised were immersed in 10-15 mL of assay buffer containing 100 mM NaH2P04-H20 (pH 7.0), 10 mM EDTA, 0.5 mM K4Fe (CN) 6-3H20 (CN), Triton X- 100 0.1% and 2 mM 5-bromo-4-chloro-3-indoyl glucuronide. The tissues were incubated in the dark for 24 hours at 37 ° C. Replacement of the GUS stain solution with 100% ethanol stopped testing. The GUS / spotting expression was displayed under the microscope. Table 1 shows the results of the transient bombardment for the 1.8 kb root met promoter: GUS construct, as well as a ubiquitin control promoter: GUS construction in the leaf, in the excised root and the seedling tissue. The GUS expression induced by the root met promoter was observed in the roots and seedlings but not in the tissue of the leaf. The expression GUS-induced construction control of ubiquitin was observed in all tissues. Table 1: Expression of the Rootmetl Promoter in Bombed Tissues Record: - no expression + weak expression levels compared to Ubi: control GUS ++ mean expression levels compared to Ubi: control GUS +++ strong expression levels compared to Ubi: GUS control ++++ Very strong expression levels compared to Ubi: GUS control Example 4: Transformation and Regeneration of Transgenic Plants using the Agroba ct eri um-mediated transformation For the Agrobacterium-mediated transformation of maize with a MTl promoter sequence of the embodiments, the Zhao method is used (U.S. Patent No. 5,981,840 (hereinafter the "840 Patent") and the PCT Patent Publication W098 / 32326; the contents of which are incorporated herein by reference). Agrobacterium were grown in a 800 medium plate and cultured at 28 ° C in the dark for 3 days, and then stored at 4 ° C for up to one month. Agrobacterium um work plates were plated with medium 810 and incubated in the dark at 28 ° C for one to two days. Briefly, the embryos were dissected in fresh sterilized corncobs and stored in 561Q medium until all the required embryos were harvested. The embryos were then contacted with an Agrobacterium suspension prepared from the work plate, in which the Agrobacterium contained a plasmid comprising the promoter sequence of the modalities. The embryos were co-cultured with the Agrobacterium in 562P plates, with the Embryos placed with the shaft down on the plates, according to the protocol of the? 840 patent. After one week in 562P medium, the embryos were transferred to medium 5630. The embryos were subcultured in the fresh 5630 medium at 2 week intervals and the incubation was continued under the same conditions. Callus events began to appear after 6 to 8 weeks in the selection. After what the calluses have reached the appropriate size, the calluses were cultured in the regeneration medium (288W) and kept in the dark for 2-3 weeks to initiate the regeneration of the plant. After the maturation of the somatic embryo, well developed somatic embryos were transferred to the medium for germination (272V) and transferred to a lighted culture room. Approximately 7-10 days later, the developing seedlings were transferred to the 272V hormone-free medium in tubes for 7-10 days until the seedlings were well established. The plants were then transferred to inserts in seed boxes (equivalent to 2.5"pot) containing potted earth and were grown for 1 week in a growth chamber, subsequently 1-2 additional weeks were grown in the greenhouse, then transferred. to 600 classic pots (1.6 gallons) and were grown to maturity.

Means used in Agrobacterium-mediated transformation and regeneration of transgenic maize plants: Medium 561Q comprises 4.0 g / L of N6 basal salts (SIGMA C-1416), 1.0 mL / L of Eriksson Vitamin Mixture (lOOOx SIGMA- 1511), 0.5 mg / L of thiamine HCl, 68.5 g / L of sucrose, 36.0 g / L of glucose, 1.5 mg / L of 2,4-D and 0.69 g / L of L-proline (brought to volume with H20 after adjusting to pH 5.2 with KOH); 2.0 g / L of Gelrite ™ (added after bringing to volume with H20 di); and 8.5 mg / L of silver nitrate (added after sterilizing the medium and cooling to room temperature). Medium 800 comprises 50.0 mL / 1 of extract solution A and 850 mL of H20 di, and brought to volume minus 100 mL / L with H20 di, after which 9.0 g of phytagar are added. After sterilization and cooling, 50.0 mL / L of extract B solution is added, along with 5.0 g of glucose and 2.0 mL of a 50 mg / mL extract solution of spectinomycin. The extract A solution comprises 60.0 g of dibasic -K2HP04 and 20.0 g of monobasic sodium phosphate, dissolved in 950 mL of water, adjusted to pH 7.0 with KOH, and brought to a volume of 1.0 L with H20 di. The solution of extract B comprises 20.0 g of NH4C1, 6.0 g of MgSO4β7H20, 3.0 g of potassium chloride, 0.2 g of CaCl2 and 0.05 g of FeS04 »7 H20, all brought to volume with H20 di sterilized and cooled.

Medium 810 comprises 5.0 g of yeast extract (Difco), 10.0 of peptone (Difco), 5.0 g of NaCl, dissolved in H20 di brought to volume after adjusting the pH to 6.8. 15.0 g of bacto-agar are then added, the solution is sterilized and cooled and 1.0 mL of a 50 mg / mL extract solution of spectinomycin is added. The 562P medium comprises 4.0 g / l of N6 basal salts (SIGMA C-1416), 1.0 mL / L of Eriksson's Vitamin Mixture (lOOOx SIGMA-1511, 0.5 mg / L of thiamine HCl, 30.0 g / L of sucrose and 2.0 mg / L of 2,4-D (brought to volume with H20 di after adjustment to pH 5.8 with KOH), 3.0 g / L of Gelrite ™ (was added after bringing to volume with H20 di) and 0.85 mg / 1 silver nitrate and 1.0 mL of a 100 mM extract of acetosyringone (both were added after sterilizing the medium and cooling to room temperature) Medium 5630 comprises 4.0 g / L of N6 base salts (SIGMA C-1416 ), 1.0 mL / 1 Eriksson Vitamin Mix (1000 x SIGMA-1511), 0.5 mg / 1 thiamin HCl, 30.0 g / l sucrose, 1.0 mg / L 2,4-D, 0.69 g L-proline and 0.5 g of MES buffer (brought to volume with H20 di after adjustment to pH 5.8 with KOH) Then, 6.0 g / L of Ultrapure ™ agar (EM Science) is added and the medium is sterilized and subsequently cooled 0.85 mg / L of nitrate a, 3.0 mL of an extract of 1 mg / mL of Bialaphos and 2.0 mL of an extract of 50 mg / mL of carbenicillin are added. 288 W comprises 4.3 g / L of MS salts (GIBCO 11117-074), 5.0 mL / L of MS vitamins extract solution (0.100 g of nicotinic acid, 0.02 g / l of thiamine HCl, 0.10 g / L of HCl of pyridoxine and 0.40 g / L of glycine brought to volume with H20 di purified) (Murashige and Skoog (1962) Physiol. Plant 15: 473), 100 mg / L of myo-inositol, 0.5 mg / L of zeatin and 60 g / L sucrose, which is then brought to volume with H20 di purified after adjusting to pH 5.6. Then, 6.0 g / L of Ultrapure ™ agar-agr (EM Science) is added and the medium is sterilized and cooled. Subsequently, 1.0 mL / L of 0.1 mM abscisic acid; 1.0 mg / L of indoleacetic acid and 3.0 mg / L of Bialaphos are added, together with 2.0 mL of a 50 mg / mL extract of carbenicillin. A recipe for 272V is provided in Example 3. Example 5: Expression Pattern of MTl in Transgenic Plants Stable transformed plants were created using the Agrobacterium transformation protocols according to Example 4, to allow a more detailed characterization of the activity of the promoter . To begin, the leaf and root tissue of regenerated plants grown on nutrient agar stably transformed with an expression cassette containing the 1815 bp MTl promoter (SEQ ID NO: 1) operably linked to a GUS gene (abbreviated as MTl: GUS). ) was sampled to test the 'presence of the GUS activity. Histochemical analysis showed that GUS was expressed in approximately 95% of the events generated (21 out of 22 events). In the group of expression plants, approximately 1/3 or 7 plants had their expression only in the roots. The 2/3 or 14 remaining plants had expression in both leaves and roots. To further characterize the MTl promoter, 17 transgenic plants were taken to the greenhouse where they were evaluated under normal growth conditions. Four of the 17 plants sent had expression in the roots only. The other 13 plants had expression in both the leaves and the root. Leaf and root tissue were sampled from 16 plants (one plant died by reducing the total number of plants in the greenhouse to 16) at the developmental stage, V5 (5 collar leaves). All 16 plants had GUS expression at the nodal roots, as determined by histochemical staining. Fifteen of these plants were classified as having a level of machado that was comparable to plants that express ubi: GUS. Identical results were obtained for lateral roots. The ubicuitin promoter is considered to be a strong promoter (Christensen et al. (1989) Plant Mol. 'Biol. 12: 619-632 and Christensen et al. (1992) Plant Mol. Biol. 18: 675-689) and plants expressing ubi: GUS were generated as a positive control for the evaluation of MTl events.

GUS expression was observed in the leaves. The abaxial sections of leaf V5 were stained histochemically and 15 of the 16 plants had spotted GUS observable. Nine of the 15 plants had a level of spotting comparable to the spotting level of the leaves of the Ubi-GUS plants. Interestingly, of the 4 plants that had only the root expression while growing on the nutrient agar, 3 now had the GUS expression on the leaves. The GUS expression was also detected in spikelets. Fourteen of every 14 plants stained for GUS. The silky parts or hairs, on the other hand, did not have much GUS expression and only 3 of 13 plants had expression in the silky parts. No spotting was observed on the pollen of any of the plants. In addition to the lack of expression in the pollen, the spotting observed, particularly in the leaves and spikelets, does not correlate well with the MPSS data for the rootmetl gene, which indicated the expression of the gene that was preferred by the root. MPSS or Massively Parallel Signature Sequencing (see Brenner S. et al. (2000) Nature Biotechnology 18: 630-634, Brenner S et al. (2000) Proc Nati Acad Sci USA 97: 1665-1670) is a method that can be used for determine the expression pattern of a particular native gene and its level of expression in different tissues. The inconsistency between the expression pattern The native and the expression pattern and the transgenic plants could be due to the presence of the 35S enhancer of the test vector. The enhancer altered the expression pattern of a related promoter, MT2 (North American provisional application No. 60 / 531,793 filed on December 22, 2003, incorporated herein by reference). Alternatively, it is possible that the elements necessary for the preferred root expression are not present in the MTl promoter fragment. However, the transient bombardment results of Example 3 showed preferred root expression. Despite this, the expression data show that the MTl promoter is a functional genetic element. It is capable of directing transgene expression in corn plants and could be useful in cases where low or no expression is desired in pollen and silky parts. The MTl promoter would also be particularly useful in cases when expression in pollen is not specifically desired. All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are incorporated herein by reference to the same degree as if each individual publication or patent application was specifically or individually indicated to be incorporated by reference. Although the above invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims (24)

1. CLAIMS 1. An isolated nucleic acid molecule, characterized in that it comprises a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 1 or a complement thereof; b) a nucleotide sequence comprising the promoter sequences of the plasmids deposited as Patent Deposit No. NRRL B-30792 or a complement thereof; c) a nucleotide sequence comprising at least 20 contiguous nucleotides of the sequence set forth in SEQ ID NO: 1, wherein the sequence initiates transcription in a plant cell; and, d) a nucleotide sequence comprising a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 1, wherein the sequence initiates transcription in the plant cell.
2. 2. An expression cassette, characterized in that it comprises a nucleotide sequence of claim 1 operably linked to a heterologous nucleotide sequence of interest.
3. 3. A vector, characterized in that it comprises the expression cassette of claim 2.
4. 4. A plant cell, characterized in that it comprises the expression cassette of claim 2.
5. 5. The plant cell according to claim 4, characterized in that the expression cassette is stably incorporated into the genome of the plant cell.
6. 6. The plant cell according to claim 4 or 5, characterized in that the plant cell is a monocot.
7. 7. The plant cell according to claim 6, characterized in that the monocot is corn.
8. 8. The plant cell according to claim 4 or 5, characterized in that the plant cell is of a dicotyledon.
9. 9. A plant, characterized in that it comprises the expression cassette of claim 2.
10. 10. The plant in accordance with the claim 9, characterized because the plant is a monocot.
11. 11. The plant in accordance with the claim 10, characterized because the monocotyledon is corn.
12. 12. The plant according to claim 9, characterized in that the plant is a dicot.
13. 13. The plant according to claim 9, characterized in that the expression cassette is Stably incorporated into the genome of the plant.
14. 14. A transgenic seed of the plant of claim 13, characterized in that the seed comprises the expression cassette.
15. 15. The plant in accordance with the claim 9, 10, 11, 12 or 13, characterized in that the heterologous nucleotide sequence of interest comprises a gene product that confers resistance to herbicide, salt, cold, drought, pathogens or insects.
16. 16. A method for expressing a nucleotide sequence in a plant, the method characterized in that it comprises introducing into the plant an expression cassette, the expression cassette comprising a promoter operably linked to a heterologous nucleotide sequence of interest, wherein the promoter comprises a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 1; b) a nucleotide sequence comprising the plant promoter sequences of the plasmids designated as Patent Deposit No. NRRL B-30792; c) a nucleotide sequence comprising at least 20 contiguous nucleotides of the sequence set forth in SEQ ID NO: 1, wherein the nucleotide sequence initiates transcription in the plant; Y, d) a nucleotide sequence comprising a sequence having at least 95% sequence identity in the sequence set forth in SEQ ID NO: 1, wherein the nucleotide sequence initiates transcription in a plant cell.
17. 17. The method according to claim 16, characterized in that the heterologous nucleotide sequence of interest is expressed in a preferred root manner.
18. 18. A method for expressing a nucleotide sequence in a plant cell, the method characterized in that it comprises introducing into the plant cell an expression cassette comprising a promoter operably linked to a heterologous nucleotide sequence of interest, wherein the promoter comprises a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence comprising the sequence set forth in SEQ ID NO: l; b) a nucleotide sequence comprising the plant promoter sequences of the plasmids designated as Patent Deposit No. NRRL B-30792; c) a nucleotide sequence comprising at least 20 contiguous nucleotides of the sequence set forth in SEQ ID NO: 1, wherein the nucleotide sequence initiates transcription in a plant cell; Y, d) a nucleotide sequence comprising a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 1, wherein the nucleotide sequence initiates transcription in the plant cell.
19. 19. A method for expressing a nucleotide sequence of interest in a preferred root manner in a plant, the method characterized in that it comprises introducing into an plant cell an expression cassette and regenerating a plant from the plant cell, the plant that has stably incorporated into its genome the expression cassette, the expression cassette comprising a promoter operably linked to. the heterologous nucleotide sequence of interest, wherein the promoter comprises a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence comprising the sequence set forth in SEQ ID NO: 1; - b) a nucleotide sequence comprising the plant promoter sequences of the plasmids deposited as Patent Deposit No. NRRL B-30792; c) a nucleotide sequence comprising at least 20 contiguous nucleotides in the sequence set forth in SEQ ID NO: 1, wherein the sequence initiates transcription in a plant root cell; Y, d) a nucleotide sequence comprising a sequence having at least 95% sequence identity to the sequence set forth in SEQ ID NO: 1, wherein the sequence initiates transcription in a plant root cell. The method according to claim 19, characterized in that the expression of the heterologous nucleotide sequence of interest alters the phenotype of the plant. 21. The method according to any of claims 16, 17, 18, 19 or 20, characterized in that the plant or plant cell is monocotyledonous. 22. The method according to claim 21, characterized in that the monocot plant or plant cell is corn. 23. The method according to any of claims 16, 17, 18, 19 or 20, characterized in that the plant or plant cell is dicotyledonous. 24. The method according to any of claims 16, 17, 18, 19 or 20, characterized in that the heterologous nucleotide sequence of interest comprises a gene product that confers resistance to herbicide, salt, cold, drought, pathogens or insects. .