MX2007003048A - Promoter molecules for use in plants - Google Patents

Abstract

The present invention relates to polynucleotide molecules for regulating gene expression in plants. In particular, the invention relates to promoters isolated fromGlycine maxandArabidopsis thalianathat are useful for regulating gene expression of heterologous polynucleotide molecules in plants. The invention also relates to expression constructs and transgenic plants containing the heterologous polynucleotide molecules.

Description

PROMOTING MOLECULES FOR USE IN PLANTS BACKGROUND PE THE INVENTION This application claims the priority of the provisional application for E.U.A. No. 60 / 609,770, filed September 14, 2004, the description of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION The invention relates to the field of plant molecular biology and more specifically relates to polynucleotide molecules useful for the expression of transgenes in plants. The invention relates to the promoters P-Gm.701202739 and P-Gm.701209813 isolated from Glicine max and to the promoter P-At.TT2 isolated from Arabidopsis thaliana. The promoters are useful for the expression of transgenes of importance in agronomy in plants for harvest.

BACKGROUND OF THE INVENTION One of the objectives of plant genetic engineering is to produce plants with desirable characteristics or traits in agronomy. The Advances in genetic engineering have provided the tools required to transform plants to contain and express foreign genes. Technological advances in the transformation and regeneration of plants have allowed researchers to take an exogenous polynucleotide molecule, such as a gene from a heterologous or native source, and incorporate that polynucleotide molecule into a plant genome. The gene can then be expressed in a plant cell to exhibit the added feature or trait. In one method, the expression of a gene in a plant cell or in a plant tissue that does not normally express said gene may confer a desirable phenotypic effect. In another method, transcription of a gene or part of a gene in an antisense orientation can produce a desirable effect by preventing or inhibiting the expression of an endogenous gene. Promoters are polynucleotide molecules that comprise the 5 'regulatory elements, which play an integral part in the general expression of genes in living cells. Isolated promoters that work in plants are useful for the modification of plant phenotypes through genetic engineering methods. The first step in the process for producing a transgenic plant includes the assembly of various genetic elements within a polynucleotide construct. The construct includes a transcribable polynucleotide molecule (gene of interest) that confers a desirable phenotype when expressed (transcribed) in plant cells by a promoter that is operatively associated with the gene of interest. A developer in a construction it can be homologous or heterogeneous to the gene of interest also contained in it. The construction is then introduced into a plant cell by various plant transformation methods to produce a transformed plant cell and the transformed plant cell is regenerated into a transgenic plant. The promoter controls the expression of the gene of interest to which the promoter is operatively associated and therefore affects the characteristic or trait conferred by the expression of the transgene in plants. For the production of transgenic plants with various desired characteristics, it may be advantageous to have a variety of promoters to provide expression of the gene such that a gene is transcribed efficiently in the amount necessary to produce the desired effect. The commercial development of genetically improved geasm has also advanced to the stage of introducing multiple traits into crop plants, often referred to as a gene stacking method. In this method, multiple genes can be introduced that confer different characteristics of interest within a plant. It is often desired, when multiple genes are introduced into a plant, that each gene is modeled or controlled for optimal expression, leading to a requirement for various regulatory elements. In light of these and other considerations, it is evident that optimal control of gene expression and diversity of the regulatory element are important in plant biotechnology.

A variety of different types or classes of promoters can be used for genetic engineering of plants. Promoters can be classified based on characteristics such as time interval or developmental interval, transgene expression levels, or tissue specificity. For example, promoters referred to as constitutive promoters are able to transcribe efficiently associated genes efficiently and express those genes in multiple tissues. Different types of promoters can be obtained by isolating the upstream regulatory regions upstream of the genes that are transcribed and expressed in the desired manner, eg, constitutive, tissue enhanced, or developmentally induced. In the literature, numerous promoters have been described, which are active in plant cells. These include the nopaline synthase (nos) promoter and the octopine synthase (oes) promoters that are carried on Agrobacterium tumefaciens tumor-inducing plasmids and the caulimovirus promoters such as the 19S or 35S promoter of the mosaic virus of the Cauliflower (CaMV) (US Pat. No. 5,352,605), CaMV 35S promoter with a duplicated enhancer (US Patents 5,164,316, 5,196,525, 5,322,938, 5,359,142, and 5,424,200), and the 35S promoter of the figwort mosaic virus (FMV) ( U.S. Patent 5,378,619). These promoters and many others have been used in the creation of constructs for the expression of the transgene in plants. Other useful promoters are described, for example, in the patents of E.U.A. 5,391, 725; 5,428,147; ,447,858; 5,608,144; 5,614,399; 5,633,441; 6,232,526; and 5,633,435, all of which are incorporated in the present invention as references. Promoters are also necessary for the expression of genes in seeds for the production of vegetable oils and other traits. The cover of the seed is a tissue of maternal origin. Once the fertilization has been carried out, the cover of the seed is responsible for maintaining the integrity of the fertilized life inside the seed, both for the protection of the embryo and for providing adequate nutrition, so far in that the conditions for growth are presented. The present invention describes promoters that have been found to be expressed in the seed coat. Although previous work has provided numerous useful promoters to direct transcription in transgenic plants, there is still a need for novel promoters with beneficial expression characteristics. In particular, there is a need for promoters that are capable of directing the expression of exogenous genes, for oil production, in seeds. Many previously identified promoters can not provide the standards or expression levels required to fully obtain the benefits of expression of the oil-associated genes improved in seeds in transgenic plants. Therefore, there is a need in the plant genetic engineering technique of novel promoters for use in oily seeds.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to promoters that are expressed in the seed coat. The seed coat promoters are useful for the production of transgenic plants with desired seed traits. These include, but are not limited to, altered nutrient intake, storage, metabolism and carbon transport, carbon / nitrogen biosynthesis, phenylpropanoid biosynthesis, seed composition or size, oil content, protein quality, or quality of micronutrients. In one embodiment, the present invention provides a promoter comprising a polynucleotide sequence substantially homologous to a polynucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 5, and fragments of they are capable of regulating the transcription of operably associated polynucleotide molecules. Polynucleotide sequences comprising at least about 70% sequence identity with respect to any of these sequences are provided by the invention in particular embodiments., including sequences with approximately 75%, 80%, 83%, 85%, 88%, 90%, 92%, 94%, 95%, 96%, 98%, 99% or more of sequence identity with respect to any one or more of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5 or a fragment thereof capable of regulating the transcription of polynucleotide molecules operatively associated, for example, that have promoter activity. In particular embodiments, a fragment of a sequence provided in the present invention is defined as comprising at least about 30, 40, 50, 75, 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 600, 750, 800, or more contiguous nucleotides of any of the promoter sequences described in the present invention such as, for example, SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 5. In another embodiment, the invention provides a plant expression construct comprising a polynucleotide sequence substantially homologous to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 5, or any fragments or regions thereof, wherein said promoter is operatively associated with respect to a transcriptional polynucleotide molecule and may also be operatively associated with a polynucleotide molecule 3 'from the transcription terminus. In still another embodiment, the invention provides a transient plant stably transformed with a plant expression construct comprising a promoter including a polynucleotide sequence substantially homologous to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 5, or any fragments thereof, wherein said promoter is operatively associated with a transcribable polynucleotide molecule, which it may be operatively associated with a polynucleotide molecule 3 'from the end of the transcription. In another embodiment, the invention provides a method for making a vegetable oil, comprising the steps of incorporating into the genome of an oil plant a promoter of the present invention operatively associated with a transcribable polynucleotide molecule that confers altered oil content and / or protein, growth of the oily plant to produce oily seeds, and extract the oil and / or protein from the oily seed. The foregoing and other aspects of the invention will be more apparent from the following detailed description and accompanying drawings.

Brief description of the sequences SEQ ID NO: 1 discloses a polynucleotide sequence of a P-Gm.701202739 promoter from Glicine max. SEQ ID NO: 2 discloses a polynucleotide sequence of a P-Gm.701209813 promoter from Glicine max. SEQ ID NOs: 3-4 expose sequence of the primers. SEQ ID NO: 5 discloses a polynucleotide sequence of a P-At.TT2 promoter from Arabidopsis thaliana.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates pMON65410. Figure 2 illustrates pMON65409. Figure 3 illustrates pMON65427. Figure 4 illustrates pMON65431. Figure 5 illustrates pMON65432.

DETAILED DESCRIPTION OF THE INVENTION The following definitions and methods are provided to better define the present invention and to guide those skilled in the art in the practice of the present invention. Unless otherwise mentioned, the terms should be understood in accordance with conventional use by those skilled in the related art. As used in the present invention, the phrase "polynucleotide molecule" refers to single or double-stranded DNA or RNA of genomic or synthetic origin, for example, a polymer of deoxyribonucleotide or riboucleotide bases, respectively, read from the terminus. 5 '(upstream) to the 3' end (downstream). As used in the present invention, the phrase "polynucleotide sequence" refers to the sequence of a polynucleotide molecule. HE uses the nomenclature of DNA bases as set forth in 37 CFR §1.822.

Promoters As used in the present invention, the term "promoter" refers to a polynucleotide molecule which in its native state is located upstream or 5 'to a codon for the start of translation of a open reading frame (or coding region of the protein) and that participates in the recognition and binding of RNA polymerase II and other proteins (transcription factors that act in tans) to initiate transcription. A "plant promoter" is a native or non-native promoter that is functional in plant cells. Constitutive promoters in plants are functional in most or all tissues of a plant throughout plant development. Any plant promoter can be used as a 5 'regulatory element for the modulation of the expression of a particular gene or genes operatively associated therewith. When it is operatively associated with a transcribable polynucleotide molecule, typically a promoter causes the transcribable polynucleotide molecule to be transcribed in a manner similar to that with which the promoter is normally associated. Plant promoters may include promoters produced through the manipulation of known promoters to produce artificial, chimeric, or hybrid promoters. These promoters can also combine elements in cis from one or more promoters with their own partial or complete regulatory elements. Therefore, the design, construction, and use of chimeric or hybrid promoters comprising at least one cis-element of SEQ ID NO: 1, 2, or 5 for the modulation of the expression of operably associated polynucleotide sequences is included by the present invention. As used in the present invention, the term "cis-element" refers to a cis-acting transcription regulatory element that confers an aspect of general control of gene expression. An element in cis may function to bind to other transcription factors, trans-acting protein factors that regulate transcription. Some elements in cis bind to more than one transcription factor, and transcription factors can interact with different affinities with more than one element in cis. Promoters of the present invention desirably contain cis-elements that can confer or modulate gene expression. The cis elements can be identified by numerous techniques, including reelection analysis, for example, by deleting one or more nucleotides from the 5 'end or internal to a promoter; analysis of DNA binding protein using footprinting technique by DNAse I, methylation interference, mobility change assays in electrophoresis, genomic footprinting in vivo by means of PCR-mediated ligation, and other conventional assays; or by DNA sequence similarity analysis with known cis-element motifs by conventional methods of comparison of DNA sequence. The fine structure of a cis element can be further studied by mutagenesis (or inclusion) of one or more nucleotides or by other conventional methods. The cis-elements can be obtained by chemical synthesis or by isolation from promoters including said elements, and can be synthesized with additional flanking nucleotides containing sites useful for restriction enzyme to facilitate subsequent manipulation. In one embodiment, the promoters of the present invention comprise multiple elements in cis each of which confers a different aspect to the general control of gene expression. In a preferred embodiment, cis-elements from the polynucleotide molecules of SEQ ID NO: 1, 2, or 5 are identified using computer programs specifically designed to identify cis-elements, domains, or motifs within the sequences. The elements in cis can regulate the expression of the gene either positively or negatively, depending on the conditions. Therefore, the present invention comprises cis-elements of the described promoters. As used in the present invention, the phrase "substantially homologous" refers to polynucleotide molecules that generally demonstrate a substantial percent sequence identity with the promoters provided in the present invention. Of particular interest are polynucleotide molecules where the polynucleotide molecules function in plants to direct transcription and have at least about 70% sequence identity, at least about 80% sequence identity, at least about 90% sequence identity, or even greater sequence identity, such as 98% or 99% sequence identity with respect to the polynucleotide sequences of the promoters described in the present invention. Polynucleotide molecules that are capable of regulating the transcription of operably associated transcribable polynucleotide molecules and that are substantially homologous to the polynucleotide sequences of the promoters provided in the present invention are included within the scope of this invention. As used in the present invention, the phrase "percent sequence identity" refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference polynucleotide molecule (or its complementary strand) as compared to a polynucleotide test molecule (or its complementary string) when the two sequences are aligned optimally (with insertionsdeletions, or appropriate nucleotide spaces totaling less than 20% of the reference sequence in the comparison window). The optimal alignment of the sequences for the alignment of a comparison window is well known to those skilled in the art and can be carried out by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, Pearson's similarity search method and Lipman, and preferably through computerized implementations of these algorithms such as GAP, BESTFIT, FAST, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA). An "identity fraction" for aligned segments of a test sequence and a reference sequence is the number of identical components that are shared by the two aligned sequences divided by the total number of components in the segment of the reference sequence, ie , the total reference sequence or a smaller defined part of the reference sequence. The percent sequence identity is represented as 100 times the identity of the fraction. The comparison of one or more polynucleotide sequences can be with respect to a polynucleotide sequence of full length or with respect to a portion thereof, or to a longer polynucleotide sequence. As used in the present invention, the term "homology" refers to the level of similarity or percentage identity between polynucleotide sequences in terms of percent identity of the nucleotide position, i.e., sequence similarity or identity. As used in the present invention, the term homology also refers to the concept of similar functional properties between different polynucleotide molecules, for example, promoters having similar functions may have elements in cis homologues. Polynucleotide molecules are homologous when, under certain circumstances, they hybridize specifically to form a duplex molecule. Under these conditions, referred to as conditions of severity, a polynucleotide molecule can be used as a probe or primer to identify other polynucleotide molecules that share homology. The phrase "stringent conditions" is defined functionally with respect to the hybridization of a nucleic acid probe with respect to a target nucleic acid (i.e., with respect to a particular nucleic acid sequence of interest) by the specific hybridization method discussed in Molecular Cloning: A Laboratory Manual, 3rd edition, Volumes 1, 2, and 3. JF Sambrook, D.W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press, 2000 (referred to in the present invention as Sambrook, et al.). Accordingly, the nucleotide sequences of the invention can be used for their ability to selectively form duplex molecules with complementary extensions of fragments of the polynucleotide molecule. Depending on the application considered one may wish to employ variable hybridization conditions to achieve varying degrees of selectivity of the probe with respect to the target sequence. For applications requiring high selectivity, one will typically want to employ relatively high severity conditions to form the hybrids, for example, one will select relatively low salt conditions and / or high temperature, such as those provided by about 0.02 M to about 0.15. M NaCl at temperatures from about 50 ° C to about 70 ° C. For example, a condition of high severity is to wash the hybridization filter at least twice with a pH regulator for high severity wash (0.2X SSC, 0.1% SDS, 65%). Appropriate moderate severity conditions that promote DNA hybridization, for example, 6.0 x sodium chloride / sodium citrate (SSC) at about 45 ° C, followed by a wash of 2.0 x SSC at 50 ° C, are known by those skilled in the art. Additionally, the salt concentration in the wash step can be selected from a low severity of about 2.0 x SSC at 50 ° C to a high severity of about 0.2 x SSC at 50 ° C. Additionally, the temperature in the wash step can be increased from low stringency conditions at room temperature, from about 22 ° C, to high stringency conditions at about 65 ° C. Both the temperature and the salt can vary, or either the temperature or the salt concentration can be kept constant while the other variable changes. These selection conditions tolerate small inconsistencies between the probe and the mold or the white chain. The detection of polynucleotide molecules via hybridization is well known to those skilled in the art, and the teachings of the Patents of E.U.A. 4,965,188 and 5,176,995 are exemplary of the methods of analysis by hybridization. Homology can also be determined by computer programs that align polynucleotide sequences and estimate the ability of polynucleotide molecules to form duplex molecules under certain conditions of severity. The polynucleotide molecules from from different sources that share a high degree of homology are referred to as "homologs". Methods well known to a person skilled in the art may be used to identify promoters of interest having similar activity with respect to the promoters described in the present invention. For example, cDNA libraries can be constructed using cells or tissues of interest and selected to identify genes that have a similar expression pattern with respect to that of the promoters described in the present invention. The cDNA sequence for the identified gene can then be used to isolate the gene promoter for further characterization. See, for example, US Patents. 6,096,950; 5,589,583; and 5,898,096, incorporated in the present invention as references. Alternatively, transcriptional or northern electronic profiling techniques may be used to identify genes that have a similar expression pattern with respect to that of the promoters described in the present invention. Once these genes have been identified, their promoters can be isolated for further characterization. See, for example, US Patents. 6,506,565 and 6,448,387, incorporated in the present invention as references. The northern electronic technique refers to a computer-based sequence analysis that allows comparing sequences from multiple cDNA libraries electronically based on the parameters that the researcher identifies including abundance in EST populations in multiple cDNA libraries, or exclusively in the ESTs established from one or combinations of libraries. The transcriptional profiling technique is a high resolution method used for the systematic monitoring of gene expression profiles for thousands of genes. This technology based on DNA fragments has thousands of cDNA sequences on the surface of a support. These arrangements are hybridized simultaneously with a population of labeled cDNA probes prepared from RNA samples from different cells or tissue types, allowing a direct expression comparison analysis. This method can be used for the isolation of regulatory sequences such as promoters associated with those genes. In another embodiment, the promoter described in the present invention can be modified. Those skilled in the art can create promoters that have variations in the polynucleotide sequence. The polynucleotide sequences of the promoters of the present invention as shown in SEQ ID NO: 1, 2, or 5 can be modified or altered to improve their control characteristics. A preferred method of altering a polynucleotide sequence is to use PCR to modify the selected nucleotides or regions of the sequences. These methods are well known to those skilled in the art. The sequences can be modified, for example by insertion, deletion, or replacement of template sequences in a PCR-based DNA modification method. A "variant" is a promoter that contains changes in which one or more nucleotides of an original promoter is deleted, added, and / or substituted, preferably at the same time that the function of the promoter is substantially maintained. For example, one or more base pairs can be deleted from the 5 'or 3' end of a promoter to produce a "truncated" promoter. One or more base pairs can also be inserted, deleted, or substituted internally in a promoter. In the case of a promoter fragment, the variants of the promoters may include changes that affect the transcription of a minimal promoter to which it is operatively associated. A minimal or basal promoter is a polynucleotide molecule that is capable of recruiting and binding the machinery of basal transcription. An example of a basal transcription machinery in eukaryotic cells is the complex of RNA polymerase II and its accessory proteins. Promoter variants can be produced, for example, by standard techniques of DNA mutagenesis or by chemical synthesis of the promoter variant or a portion thereof. Novel chimeric promoters can be engineered or engineered by numerous methods. Many promoters contain elements in cis that activate, improve, or define the strength and / or specificity of the promoter. For example, promoters may contain "TATA" boxes that define the transcription initiation site and other elements in cis located upstream of the transcription initiation site that modulate transcription levels. For example, a chimeric promoter can be produced by fusing a first promoter fragment containing the cis-activating element from a promoter with respect to a second promoter fragment containing the cis-activating element. from another promoter; the resulting chimeric promoter can cause an increase in the expression of an operably associated transcriptionally polynucleotide molecule. The promoters can be constructed in such a way that fragments or elements of the promoter are operatively associated, for example, by placing said fragment upstream of a minimal promoter. The cis elements and fragments of the present invention can be used for the construction of said chimeric promoters. Methods for the construction of chimeric promoters and variants of the present invention include, but are not limited to, combined control elements from different promoters or duplicating portions or regions of a promoter (see, e.g., U.S. Patent Nos. 4,990,607; 5,110,732; 5,097,025, all of which are incorporated herein by reference). Those skilled in the art are familiar with standard resource materials describing specific conditions and procedures for the construction, manipulation, and isolation of macromolecules (e.g., polynucleotide molecules, plasmids, etc.), as well as the generation of recombinant organisms and the selection and isolation of polynucleotide molecules. In another embodiment, a promoter comprising the polynucleotide sequence shown in SEQ ID NO: 1, 2, or 5 includes any length of said polynucleotide sequence that is capable of regulating an operably associated transcribable polynucleotide molecule. For example, promoters as described in SEQ ID NO: 1, 2, or 5 may be truncated or portions may be deleted as long as it retains the ability to regulate the transcription of an operably associated polynucleotide molecule. In a related embodiment, a cis-element of the described promoters can confer a particular specificity such as by conferring improved expression of polynucleotide molecules operatively associated in certain tissues and therefore also capable of regulating the transcription of operably associated polynucleotide molecules. Accordingly, any fragments, portions, or regions of the promoters comprising the polynucleotide sequences shown in SEQ ID NO: 1, 2, or 5 can be used as regulatory polynucleotide molecules, including but not limited to cis elements or motifs. the polynucleotide molecules described. The substitutions, deletions, insertions, or any combination thereof may be combined to produce a final construction.

Polynucleotide Constructs As used in the present invention, the term "construct" refers to any recombinant polynucleotide molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or single or single-stranded DNA or RNA polynucleotide molecule. of double chain, linear or circular derived from any source, capable of carrying out integration to the genome or autonomous replication, comprising a polynucleotide molecule where one or more Polynucleotide molecules have been associated in a functionally operative manner. As used in the present invention, the phrase "operatively associated" refers to a first polynucleotide molecule, such as a promoter, connected to a second transcribable polynucleotide molecule, such as a gene of interest, wherein the polynucleotide molecules are disposed of so that the first polynucleotide molecule affects the function of the second polynucleotide molecule. Preferably, the two polynucleotide molecules are part of a particular contiguous polynucleotide molecule and more preferably are adjacent. For example, a promoter is operatively associated with a gene of interest if the promoter regulates or mediates the transcription of the gene of interest in a cell. As used in the present invention, the phrase "transcribable polynucleotide molecule" refers to any polynucleotide molecule capable of being transcribed to an RNA molecule. Methods for introducing constructs into a cell are known in such a way that the transcribable polynucleotide molecule is transcribed into a functional mRNA molecule that is translated and therefore expressed as a protein product. The constructs can also be constructed to be capable of expressing antisense RNA molecules, in order to inhibit the translation of an RNA molecule of specific interest. For the practice of the present invention, they are well known to an expert in the technique conventional compositions and methods for the preparation and use of constructs and host cells (see, for example, Sambrook, et al.) - The constructions of the present invention could typically contain a promoter operatively associated with a transcribable polynucleotide molecule operatively associated with a polynucleotide molecule towards 3 'of transcription termination. In addition constructs may include but are not limited to additional regulatory polynucleotide molecules from the untranslated region to 3 '(3' UTR) of the plant genes (eg, a 3 'UTR to increase the stability of the mRNA, such as the Pl-ll termination region of the potato or the 3 'termination regions of octopine or nopaline synthase). Constructs may include but are not limited to the untranslated regions towards 5 '(5' UTR) of a polynucleotide mRNA molecule which may play an important role in the initiation of translation and may also be a genetic component in a construct of vegetal expression. For example, it has been shown that the 5'-untranslated leader polynucleotide molecules derived from the heat shock protein genes improve gene expression in plants (see, for example, U.S. Patent Nos. 5,659,122 and 5,362,865; US Published No. 2002/0192812, incorporated herein by reference). These additional upstream and cascading downstream regulatory polynucleotide molecules can be derived from a source that is native or heterologous with respect to the other elements present in the construction of the promoter. Therefore, the constructs of the present invention comprise promoters such as those provided in SEQ ID NO: 1, 2 or 5 or modified as described above, operatively associated with a transcribable polynucleotide molecule so as to direct the transcription of said polynucleotide molecule transcribable at a desired level or in a desired tissue or in a developmentally dependent pattern after the introduction of said construction into a plant cell. In some cases, the transcribable polynucleotide molecule comprises a region of a gene that is encoding the protein, and the promoter is provided for the transcription of a functional mRNA molecule that is translated and expressed as a protein product. Constructs can also be constructed for the transcription of antisense RNA molecules or other similar inhibitory RNAs in order to inhibit the expression of a specific RNA molecule of interest in a white host cell. Exemplary transcriptable polynucleotide molecules for incorporation into constructs of the present invention include, for example, DNA molecules or genes from a species other than the target gene species, or even genes that originate with or are present in the same species, but that are incorporated into recipient cells by genetic engineering methods rather than by the classic reproduction or crossing techniques. It is intended that the exogenous gene or genetic element refers to any gene or DNA molecule that is introduced into a recipient cell. The type of DNA included in the exogenous DNA may include DNA "that is already present in the plant cell, DNA from another plant, DNA from a different organism, or DNA that is generated externally, such as a DNA molecule that contains an antisense messenger of a gene, or a DNA molecule that encodes an artificial or modified version of a gene The promoters of the present invention can be incorporated into a construct using marker genes as described and tested in transient analyzes that provide an indication of gene expression in stable plant systems As used in the present invention, the phrase "marker gene" refers to any transcribable polynucleotide molecule whose expression can be selected or evaluated in some way. Methods for the evaluation of marker gene expression in transient assays are known to those skilled in the art. Transient expression of marker genes has been reported using a variety of plants, tissues, and DNA delivery systems. For example, types of transient assays may include, but are not limited to, direct administration of the gene via electroporation or bombardment of tissues by particles in any transient plant assay utilizing a plant species of interest. Such transient systems could include, but are not limited to, electroporation of protoplasts from a variety of sources of tissue or bombardment of specific interest tissues by particles. The present invention includes the use of any transient expression system for evaluating the promoters or promoter fragments operatively associated with any "transcribable" polynucleotide molecules, including but not limited to selected reporter genes, marker genes, or genes of interest in agriculture. of plant tissues considered to be evaluated in transients via an adequate administration system could include, but are not limited to, tissues of the leaf base, callus, cotyledons, roots, endosperm, embryos, floral tissue, pollen, and epidermal tissue. Any selectable or selectable marker gene can be used in a transient assay Preferred marker genes for transient analyzes of the promoters or fragments of the promoters of the present invention include a GUS gene (US Patent 5,599,670, incorporated herein by reference) ) or a GFP gene (US Patent 5 , 491, 084, incorporated herein by reference). Constructs containing the promoters or promoter fragments operatively associated with a marker gene are administered to the tissues and the tissues are analyzed by the appropriate mechanism, depending on the marker. Quantitative or qualitative analyzes are used as a tool to evaluate the potential expression profile of promoters or promoter fragments when they are operatively associated with genes of interest in agronomy in stable plants.

Therefore, in a preferred embodiment, a polynucleotide molecule of the present invention is incorporated as shown in SEQ ID NO: 1 to 4 or fragments, variants, or derivatives thereof within a construct such that a promoter of the present invention is operatively associated with a transcribable polynucleotide molecule that is provided for a selectable, classifiable, or assessable marker. Markers for use in the practice of the present invention include, but are not limited to, transcribable polynucleotide molecules encoding β-glucuronidase (GUS), green fluorescent protein (GFP), luciferase (LUC), proteins encoding resistance to antibiotics, or proteins that confer tolerance to herbicide. Useful markers for resistance to antibiotics, including those proteins that encode conferring resistance to kanamycin (nptll), hygromycin B (aph IV), streptomycin or spectinomycin (aad, spec / strep), and gentamicin (aac3 or aaC4) are known in the technique. The herbicides from which tolerance has been demonstrated by the transgenic plants and the methods of the present invention can be applied, including but not limited to: glyphosate, glufosinate, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrionic inhibitors synthase, and isoxasflutol type herbicides. Polynucleotide molecules that encode proteins involved in herbicide tolerance are well known in the art, and include, but are not limited to, a polynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) described in U.S. Pat. 5,627,061; 5,633,435; Y 6,040,497; and AroA described in the US patent. 5,094,945 for glyphosate tolerance; a polynucleotide molecule encoding Bromoxynil Nitrilasse (Bxn) described in the U.S. Patent. 4,810,648 for tolerance to Bromoxinil; a polynucleotide molecule encoding phytoene desaturase (crtl) described in Misawa et al., Plant J., 4: 833-840 (1993) and Misawa et al., Plant J., 6: 481-489 (1994) for tolerance to norflurazon; a polynucleotide molecule encoding acetohydroxy synthase acid (AHAS, aka ALS) described in Sathasiivan et al., Nucí. Acids Res., 18: 2188-2193 (1990) for tolerance to sulfonylurea herbicides; and the bar gene described in DeBlock, et al., EMBO J., 6: 2513-2519 (987) for tolerance to glufosinate and bialaphos. In a preferred embodiment, a polynucleotide molecule of the present invention as shown in SEQ ID NO: 1, 2, or 5, or fragments, variables, or derivatives thereof are incorporated into a construct so that a promoter of the present invention is operatively associated with a transcribable polynucleotide molecule that is a gene of interest in agronomy. As used in the present invention, the phrase "gene of interest in agronomy" refers to a transcribable polynucleotide molecule that includes, but is not limited to, a gene that provides a desirable characteristic associated with plant morphology, physiology, growth and development, performance, nutritional improvement, resistance to disease or pest, or environmental or chemical tolerance. The expression of a gene of interest in agronomy is desirable in order to achieve an important feature in agronomy. A gene of interest in agronomy that provides a beneficial agronomic trait to harvest plants may be, for example, including, but not limited to, genetic elements comprising herbicide resistance (US Patents 5,633,435 and 5,463,175), increased yield (US Patent 5,716,837), insect control (Patents US 6,063,597, 6,063,756, 6,093,695, 5,942,664, and 6,110,464), resistance to fungal diseases (US Patents 5,516,671, 5,773,696, 6,121, 436, 6,316,407, and 6,506,962), virus resistance (US Patents 5,304,730 and 6,013,864), resistance to nematodes (US Patent 6,228,992), resistance to bacterial diseases (US Patent 5,516,671), starch production (US Patents 5,750,876 and 6,476,295), modified oil production (US Patent 6,444,876), high oil production (US Patents) 5,608,149 and 6,476,295), modified fatty acid content (US Patent 6,537,750), high protein production (US Patent 6,380,466), fruit ripening (U.S. Patent 5,512,466), improved animal and human nutrition (U.S. Patents 5,985,605 and 6,171, 640), biopolymers (U.S. Pat. 5,958,745 and Published Application of E.U.A. No. 2003/0028917), resistance to environmental stress (US Patent 6,072,103), pharmaceutical peptides (US Patent 6,080,560), improved processing features (US Patent 6,476,295), improved digestion (US Patent 6,531, 648), low raffinose production (US Patent 6,166,292), industrial enzyme production (US Patent 5,543,576), improved flavor (US Patent 6,011, 199), nitrogen fixation (US Patent 5,229,114), seed production hybrid (U.S. Patent 5,689,041), and biofuel production (U.S. Patent 5,998,700), the genetic elements and transgenes described in the patents listed above are incorporated herein by reference. Alternatively, a transcribable polynucleotide molecule can affect the aforementioned phenotypes by encoding a non-translatable RNA molecule that causes targeted inhibition of the expression of an endogenous gene, for example via antisense, RNAi, or mechanisms mediated by cosuppression. The RNA could also be a catalytic RNA molecule (e.g., a ribozyme) designed to cleave a desired product of endogenous mRNA. Therefore, any polynucleotide molecule that encodes a protein or mRNA that expresses a change in phenotype or morphology of interest is useful for practicing the present invention. Constructs of the present invention may comprise DNA constructs with double Ti-plasmid border having the right border regions (RB or AGRtu.RB) and the left border (LB or AGRtu.LB) of the Ti plasmid isolated from Agrobacterium tumefaciens which comprises a T-DNA, which together with the transfer molecules provided by the Agrobacterium cells, allow the integration of the T-DNA into the genome of a plant cell. The constructs can also contain the DNA segments with the basic structure of the plasmid that provide replication of the function and selection of antibiotic in bacterial cells, by example, an E. coli with origin of replication such as or2322, a host with a wide range of origin of replication such as oriV or oriRi, and a coding region for a selection marker such as Spec / Strep coding for the Tn7 aminoglycoside adenyltransferase (aadA) that confers resistance to spectinomycin or streptomycin, or a selection marker gene for gentamicin (Gm, Gent). For plant transformation, the host bacterial strain is generally Agrobacterium tumefaciens ABI, C58, or LBA4404, however, other strains known to those skilled in the plant transformation art may function in the present invention.

Plants and transformed plant cells As used in the present invention, the term "transformed" refers to a cell, tissue, organ or organism into which a foreign polynucleotide molecule has been introduced, such as a construct. Preferably, the introduced polynucleotide molecule is integrated into the genomic DNA of the recipient cell, tissue, organ, or organism such that the introduced polynucleotide molecule is inherited by the subsequent progeny. A "transgenic" or "transformed" cell or organism also includes progeny of the cell or organism produced from a cross program that employs said transgenic plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a foreign polynucleotide molecule.

A plant transformation construct containing a promoter of the present invention can be introduced into plants by any method for plant transformation. Methods and materials for transforming plants by "introducing a plant expression construct into a plant genome in the practice of this invention can include any of the well-known and demonstrated methods including electroporation as illustrated in U.S. Patent 5,384,253; bombardment of microprojectiles as illustrated in U.S. Patents 5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865, mediated transformation by Agrobacterium as illustrated in U.S. Patents 5,635,055, 5,824,877, 5,591, 616, 5,981, 840, and 6,384,301; and protoplast transformation as illustrated in U.S. Patent 5,508,184, all of which are incorporated herein by reference .. Methods for specifically transforming dicots are well known to those skilled in the art. these methods has been described for numerous sews chas including, but not limited to, cotton (Gossypium hirsutum), soybean (Glycine max), peanut (Arachis hypogaea), members of the genus Brassica; and alfalfa (Medicago sativa) and members of the genus Brassica. It is evident to those skilled in the art that numerous transformation methodologies can be used and modified for the production of plants stable transgenic for any number of dicotyledonous white crops that are of interest. Methods for transforming specifically monocotyledons are well known to those skilled in the art Plant transformation and regeneration using these methods has been described for numerous crops including, but not limited to, barley (Hordeum vulgarae); corn (Zea mays); oats (Avena sativa), orchid grass (Dactylis glomerata), rice (Oryza sativa, including Indica and Japanese varieties), sorghum (Sorghum bicolor), cane sugar (Saccarum sp.), tall cane (Festuca arundinacea); (Agrostis) and wheat (Tritticum aestivum) It is evident to those skilled in the art that numerous transformation and modification methodologies can be used for the production of stable transgenic plants from any number of monocotyledonous white crops that are of interest Many seeds, nuts, and grains contain oil that can be extracted and used for cooking, as an ingredient in other foods, or a nutritional supplement, such as raw material for making soap, body and hair oils, detergents, paints, as well as replacements for certain lubricants and petroleum-based fuels. As used in the present invention, these seeds, nuts, and grains are collectively referred to as "oilseeds" (National Sustainable Agriculture Information Service (ATT RA), Fayetteville, AR). Table 1 lists examples of seeds, nuts, and grains commonly classified as oily seeds.

TABLE 1 Seeds, nuts, grains containing oil In another embodiment, the invention provides a method for making a vegetable oil, comprising the steps of incorporating within the genome of an oil plant a promoter of the present invention operatively associated with a transcribable polynucleotide molecule that confers altered oil content and / or protein, plant growth oil to produce oily seeds, and extraction of the oil and / or protein from the oily seed. Transformed plants are analyzed for the presence of genes of interest and the level of expression and / or profile conferred by the promoters of the present invention. Those skilled in the art are aware of the numerous methods available for the analysis of transformed plants. For example, methods for the analysis of plants include, but are not limited to Southern blot or northern blot, methods based on PCR, biochemical analysis, phenotypic selection methods, field evaluations, and immunodiagnostic assays. The seeds of this invention can be harvested from fertile transgenic plants and can be used to grow generations of progeny of transformed plants of this invention including hybrid plant lines comprising the construction of this invention and expressing a gene of interest in agronomy. The promoter of the present invention is provided for differential expression in plant tissues, preferably in at least one tissue of plant seed that includes seed coating, embryo, aleurone, and endosperm. The promoters are referred to in the present invention as "improved seed promoters". The phrase "micronutrient content" means the amount of micronutrients, for example, vitamins A, E, K, tocopherols, or carotenoids within a seed expressed on a weight basis.

The phrase "oil content" means level of oil, which can be determined, for example, by low resolution 1H nuclear magnetic resonance (NMR) (Tiwari et al., JAOCS, 51: 104-109 (1974) or Rubel). , JAOCS, 71: 1057-1062 (1994)) or near infrared transmittance spectroscopy (NIT) (Orman, et al., JAOCS, 69 (10) 1036-1038 (1992); Patrick, et al., JAOCS, 74 (3): 273-276 (1997)). The phrase "protein quality" means the level of one or more essential amino acids, whether free or incorporated into a protein namely histidine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan, and valine . As used in the present invention, the phrase "oil composition" means the ratio of different fatty acid or oil components within a sample. Said sample may be a plant or plant part, such as a seed. Said sample can also be a collection of plant parts. As used in the present invention, the phrase "percent content" in a preferred embodiment means the percentage of total weight of a particular component, relative to another similar component of related components. As used in the present invention, the phrase "improved oil" or "oil improvement" includes an increased oil yield or an altered performance in the oil composition.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques described in the examples below represent techniques discovered by the inventors to function well in the practice of the invention. However, those skilled in the art should appreciate, in light of the present disclosure, that many changes can be made in the specific embodiments that are described and still obtain a similar or similar result without departing from the spirit and scope of the invention. , therefore what is established or shown in the attached drawings must be interpreted as illustrative and not in a limiting sense.

EXAMPLES EXAMPLE 1 Isolation of the promoters P-Gm.701202739 v P-Gm.701209813 The improved ESTs for expression of the seed coat were identified to the library subtraction and the electronic Northems were obtained using the PhytoSeq SOYMON035 database. The DNA from the soybean cultivar A3244 was extracted and used to construct a GenomeWalker library (BD Biosciences, Palo Alto, CA). The Two promoters from the previously identified ESTs were isolated by PCR amplification from the GenomeWalker libraries: 701202739H1 (0.76 kb) and 701209813H1 (0.78 kb). The upstream 5 'cascade regulatory sequences for the putative promoters from the soy bean seed coat were cloned and the sequences verified: P.701202739H1 (SEQ ID NO: 1) (pMON57315) and 701209813H1 (SEQ ID NO: 2 ) (pMON57314).

EXAMPLE 2 Isolation of the P-ALTT2 promoter The gene encoding a protein of the R2R3 MYB domain of Arabidopsis thaliana TT2 includes an accumulation of proanthocyanidin in the seed coat under development. A promoter sequence was first identified by BLASTing of the coding sequence (AJ299452) against a database of Arabidopsis thaliana sequence Monsanto ArabGDNA which contained all of the Arabidopsis genomic sequences from all sources. Clone P1 MOK9 (AB015477) contained the TT2 coding sequence (base pairs 64541-67240). The primers were designed to amplify the sequences upstream of the TT2 coding region (corresponding to base pairs 64658-65620 of P1 MOK9) from the Arabidopsis genomic DNA (strain ID CS3176). The predicted ATG of TT2 was converted to an Ncol site and a Srfl site was added to the 'end of the second primer to facilitate cloning. The primer sequences were SEQ ID NO: 3 and SEQ ID NO: 4. The promoter sequence (SEQ ID NO: 5) was cloned into PCR2.1 Mole (Invitrogen, Carlsbad, CA) to prodpMON65418.

EXAMPLE 3 Constructions for the transformation of Arabidopsis pMON65410 (P-Gm.701202739-0: 3: 1 :: GUS) A 698 bp fragment containing P-Gm.701202739-0: 3: 1 was removed from pMON57315 by digestion with PstI and Ncol. The fragment was ligated into pMON69802, which had been digested with Sse8387l and Ncol. The resulting plasmid, which contained P-Gm.701202739-0: 3: 1 which directs the E. coli uidA gene and with the napin 3 'UTR was designated pMON65410. The nucleic acid sequence was determined using known methodology and the integrity of the cloned linkages was confirmed. This vector was used in the subsequent transformation of Arabidopsis. pMON65409 (P-Gm.701209813-0: 3: 1 :: GUS) A 704 bp fragment containing P-Gm.701209813-0: 3: 1 was removed from pMON57314 by digestion with PstI and Ncol. The fragment was ligated into pMON69802, which had been digested with Sse8387l and Ncol. The resulting plasmid, which contained P-Gm.70 209813- 0: 3: 11 which directs the E. coli uidA gene and with the naprin 3 'UTR was named pMON65409. The nucleic acid sequence was determined using known methodology and the integrity of the cloning linkages was confirmed. This vector was used in the subsequent transformation of Arabidopsis. The transgenic Arabidopsis thaliana plants were obtained as described by Bent et al., Science, 265: 856-860 (1994) or Bechtold et al., C.R. Acad. Sci, Life Sciences, 316: 194-99 (1993). Cultures of Agrobacterium tumefaciens strain ABI containing any of the transformation vectors pMON65410 op ON65409 were grown overnight in LB (10% bacto-tryptone, 5% yeast extract, and 10% NaCl with kanamycin (75 mg / L), chloramphenicol (25 mg / L), and spectinomycin (100 mg / L)). The bacterial cultures were centrifuged and resuspended in 5% sucrose + .05% solution of Silwet-77. The aerial portions of the total Arabidopsis thaliana plants (at approximately 5-7 weeks of age) were immersed in the resulting solution for 2-3 seconds. The excess solution was removed by blotting the plants on paper towels. The submerged plants were placed laterally on a coated plate and transferred to a growth chamber at 19 ° C. After 16 to 24 hours the dome was removed and the plants were placed vertically. When the plants had reached maturity, the water was retained for 2-7 days before the seed was harvested. The harvested seed was passed through a stainless steel sieve screen (40 holes / 2.5 cm) to remove the debris. The seed harvested was stored in a paper cover at room temperature until analysis. The Arabidopsis seeds were sterilized on the surface using a steam phase sterilization protocol. An open seed container was placed in a desiccator with a beaker containing 100 ml of household bleach. Immediately before sealing the desiccator, 3 ml of concentrated HCl was added to the bleach. The desiccator was sealed and vacuum applied to allow sterilization by chlorine vapors. The seeds were incubated for several hours. The sterilized seeds were spread on the medium for germination of Arabidopsis (MS salts (1X), sucrose (1%), myo-lnositol (100 mg / L), Tiamine-HCI (1 mg / L), pyridoxine-HCI (500 mg / L), nicotinic acid (500 mg / L), MES pH 5.7 (0.05%), and Phytagar (0.7%) supplemented with 50 mg / L of glyphosate.

EXAMPLE 4 Characterization of the promoter in transgenic plants of Arabidopsis The expression of ß-glucuronidase (the product of the E. coli uidA gene) was analyzed in Arabidopsis thaliana plants transformed with pMON65409 or pMON65410 using chemical staining. Up to 16 glyphosate-resistant seedlings were transplanted into 5.6 cm pots, one seedling per pot, which contained MetroMix 200 and were grown under the conditions described above until the initial siliques that had formed they started to stand out. Tissue (rosette leaf, stem leaf, stem, flowers, flower buds, and growing siliques) was removed from each T1 plant for subsequent histochemical staining. The tissue (rosette leaf, stem leaf, stem, flowers, flower buds, and growing siliques) were removed from each T1 plant for subsequent histochemical staining. The tissues, prepared as described above, were incubated for approximately 24 hours at 37 ° C in a solution containing 50 mM NaP04 (pH 7.2); 100 OM of potassium ferricyanide; 100 DM of potassium ferrocyanide, 0.03% of Triton X-100; 20% methanol and 2.5 mg / ml 5-bromo-4-chloro-3-indoyl glucuronic acid (X-gluc). The tissue was lacking in chlorophyll by overnight incubation in 70% ethanol / 30% H20 at 37 ° C. The stained fabrics were photographed immediately or transferred to a solution of 70% ethanol / 30% glycerol (v / v) and stored at 4 ° C until they were photographed. For pMON65409, 5 of 5 plants that were selected had detectable levels of activity in the seed from at least one time point. For pMON65410, 5 of 5 selected plants had detectable levels of activity in the seed from at least one time point.

EXAMPLE 5 Constructions for the transformation of Cañóla PMON65431 (P-Gm.701202739-0: 3: 1 :: GUS) A 745 bp fragment containing P-Gm.701202739-0: 3: 1 was removed from PMON57315 by digestion with Notl and Ncol. The fragment was ligated into pMON65424, which had been digested with Notl and Ncol. The resulting plasmid, which contained P-Gm.701202739-0: 3: 11 which directs the E. coli uidA gene and with the napin 3 'UTR, was designated p ON65431. The nucleic acid sequence was determined using known methodology and the integrity of the clone junctions was confirmed. This vector was used in the subsequent transformation of Cañóla.

PMON65432 (P-Gm.701209813-0: 3: 1 :: GUS) A 746 bp fragment containing P-Gm.701209813-0: 3: 1 was removed from pMON57314 by digestion with Notl and Ncol. The fragment was ligated into pMON65424, which had been digested with Notl and Ncol. The resulting plasmid, which contained P-Gm.701209813-0: 3: 11 which directs the E. coli uidA gene and with the naptin 3 'UTR, was designated pMON65432. The nucleic acid sequence was determined using known methodology and the integrity of the clone junctions was confirmed. This vector was used in the subsequent transformation of Cañóla. pMON65427 (P-At.TT2-0: 3: 2 :: GUS) A 966 bp fragment containing P-At.TT2-0: 3: 2 was removed from pMON65418 by digestion with Smal and Ncol. The fragment was ligated into pMON65424. which had been digested with Pmel and Ncol. The resulting plasmid G, which contained P-At.TT2-0: 3: 21 which directs the E. coli uidA gene and with the napin 3 'UTR, was designated pMON65427. The nucleic acid sequence was determined using known methodology and the integrity of the clone junctions was confirmed. This vector was used in the subsequent transformation of Cañóla. The vectors pMON65422, 65431 and pMON65432 were introduced into A. tumefaciens strain ABI for transformation into Brassica napus. Canola plants were transformed using the protocol described by Moloney and Radke in the U.S. Patent. 5,720,871. Briefly, B. napus cv Ebony seeds were planted in 5 cm pots containing Metro Mix 350 (The Scotts Company, Columbus, OH). The plants were grown in a growth chamber at 24 ° C, and at a photoperiod of 6/8 hours, with light intensity of 400 uEm "2 sec" 1 (HID lamps). After 2-1 / 2 weeks, the plants were transplanted into 15 cm pots and grown in a growth chamber at 15/0 ° C day / night temperature, 16/8 hours photoperiod, light intensity 800 uEm "2 sec" 1 (HID lamps). Four terminal internments from plants just before the flower formation process or in the flower formation process but before of the flowering were removed and the surface sterilized in 70% v / v ethanol for 1 minute, 2% w / v sodium hypochlorite for 20 minutes and rinsing 3 times with sterile deionized water. Five to seven segments of the stem were cut into 5 mm discs, maintaining the orientation of the basal end. The Agrobacterium crops used to transform Cañola was grown overnight in an agitator with rotation at 24 ° C in 2 mis of Luria Broth, LB (10% bacto-tryptone, 5% yeast extract, and 10% NaCl) containing 50 mg / l of kanamycin, 24 mg / l of chloramphenicol, and 100 mg / l of spectinomycin. A 1: 10 dilution was made in MS medium (Murashige and Skoog, Physiol. Plant., 15: 473-497, 1962) yielding approximately 9x108 cells per ml. The stem discs (explants) were inoculated with 1.0 ml of Agrobacterium and the excess was aspirated from the explants. The explants were placed with the basal side down in Petri dishes containing the medium comprising 1/10 MS salts, vitamins B5 (1% inositol, 0.1% thiamine HCI, 0.01% nicotinic acid, 0.01% pyridoxine-HCl), 3% sucrose, 0.8% agar, pH 5.7, 1.0 mg / l of 6-benzyladenine (BA). The plates were stacked with 1.5 ml of medium containing MS salts, vitamins B5, 3% sucrose, pH 5.7, 4.0 mg / l of p-chlorophenoxyacetic acid, 0.005 mg / l of kinetin and covered with sterile filter paper. After a co-culture for 2 to 3 days, the explants were transferred to plates of deeper Petri dishes containing MS salts, vitamins B5, 3% sucrose, 0.8% agar, pH 5.7, 1 mg / l of BA, 500 mg / l of carbenicillin, 50 mg / l of cefotaxime, 200 mg / l of kanamycin, or 175 mg / l of gentamicin for selection. Seven explants were placed in each plate. After 3 weeks, 5 explants per plate were transferred to fresh medium. The explants were grown in a room for growth at 25 ° C, with continuous light (Cool White). The transformed plants were grown in a growth chamber at 22 ° C in a light-dark cycle of 16-8 hours with light intensity of 220 uEm "2s" 1 for several weeks before transferring to the greenhouse. The plants were kept in a greenhouse under standard conditions. The developing seed was harvested at several stages after pollination and stored at minus 70 ° C. The mature seed was collected and stored under controlled conditions that consisted of approximately 17 ° C and 30% humidity.

EXAMPLE 6 Characterization of the promoter in transgenic plants of Cañóla Up to 5 siliques were harvested from the individual R0 plants transformed with pMON65427, pMON65431, or pMON65432 at various time points after pollination. The siliques were evaluated with an 18 gauge needle to allow the staining solution to come into contact with the developing seed. The siliques were incubated for approximately 24 hours at 37 ° C in a solution containing 50 mM of NaP04 (pH 7.2); 100 uM of potassium ferricyanide; 100 uM of potassium ferrocyanide, 0.03% of Triton X-100; 20% methanol and 2.5 mg / ml 5-bromo-4-chloro-3-indoyl glucuronic acid (X-gluc). The chlorophyll was removed from the stained fabric by incubation overnight in 70% ethanol / 30% H2O at 37 ° C. The stained fabrics were photographed immediately or transferred to a solution of 70% ethanol / 30% glycerol (v / v) and stored at 4 ° C until they were photographed. The samples were classified as positive (+) or negative (-) for the blue color. For pMON65431, 6 of 10 selected plants had detectable levels of activity in the seed from at least one time point. The time course data for the individual transformations are presented in tables 2 and 3.

TABLE 2 P-Gm.701202739-0: 3: 1 seed expression of canola in development TABLE 3 P-Gm.701209813-0: 3: 1 expression in seed of canola in development For pMON65427, 10 of 10 selected plants had detectable levels of activity in the seed for at least one point of time; the time course data for the individual transformants are presented in table 4 TABLE 4 P-At.TT2-0: 3: 2 Expression in seeds of canola in development Having illustrated and described the principles of the present invention, it should be apparent to those skilled in the art that the invention can be modified in accordance and detail without departing from said principles. The inventors claim all modifications that are within the spirit and scope of the appended claims. All publications and published patent documents cited in this specification are incorporated herein by reference to the same extent, as if each individual publication or patent publication was specifically and individually indicated to be incorporated by reference.

Claims (17)

1. NOVELTY OF THE INVENTION CLAIMS 1. - A promoter comprising a polynucleotide sequence selected from the group consisting of (a) a polynucleotide sequence comprising the nucleic acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 5; (b) a polynucleotide sequence comprising a fragment of the polynucleotide sequence of (a) capable of regulating the transcription of an operably associated transcribable polynucleotide molecule; and (c) a polynucleotide sequence comprising at least 70% sequence identity with respect to the polynucleotide sequence of (a) or (b) capable of regulating the transcription of an operably associated transcribable polynucleotide molecule. 2. - The promoter according to claim 1, further characterized in that said promoter comprises a polynucleotide sequence with from about 90% identity to about 99% sequence identity with respect to the polynucleotide sequence of (a) or fragment of ( b) 3. - The promoter according to claim 1, further characterized in that said promoter comprises a polynucleotide sequence with approximately 80% identity to approximately 89% sequence identity with respect to the polynucleotide sequence of (a) or fragment of (b). 4. - The promoter according to claim 1, further characterized in that said promoter comprises a polynucleotide sequence with from about 70% identity to about 79% sequence identity with respect to the polynucleotide sequence of (a) or fragment of ( b) 5. A construct comprising the promoter according to claim 1, operatively associated with a transcribable polynucleotide molecule. 6. The construction according to claim 5, further characterized in that said transcribable polynucleotide molecule is a gene of interest in agronomy. 7. The construction according to claim 5, further characterized in that said transcribable polynucleotide molecule is a marker gene. 8. - A transgenic plant or part of it transformed in a stable manner with the construction according to claim 5. 9. - The transgenic plant according to claim 8, further characterized in that said plant is a dicotyledonous plant selected from of the group consisting of tobacco, tomato, potato, soybeans, cotton, sugarcane, sunflower, and alfalfa. 10. - The transgenic plant according to claim 8, further characterized in that said transcribable polynucleotide molecule confers altered oil content in the seed of said transgenic plant. 11.- The transgenic plant in accordance with the claim 8, further characterized in that said transcribable polynucleotide molecule confers altered protein quality in the seed of said transgenic plant. 12. - The transgenic plant according to claim 8, further characterized in that said transcribable polynucleotide molecule confers altered content of micronutrients to said transgenic plant. 13. - A transgenic seed transformed with the construction according to claim 5. 14. - A transgenic cell transformed with the construction according to claim 5. 15. - The oil from the transgenic seed in accordance with the claim 13, further characterized in that the oil comprises a detectable nucleic acid comprising the promoter according to claim 1. 16.- The flour from the transgenic seed according to claim 13, further characterized in that the flour comprises a nucleic acid. detectable comprising the promoter according to claim 1. 17. - A method for preparing a vegetable oil, comprising the steps of: a) obtaining the seed according to claim 13; and b) extract the oil from the seed. 18. - A method for making a flour, comprising the steps of: a) obtaining the plant or part thereof according to claim 8; and b) prepare flour from the plant or part of it. 19. - A method for the preparation of food or fodder comprising the steps of: a) obtaining the plant or part thereof according to claim 8; and b) prepare food or fodder from the plant or part of it.