MX2007003050A - 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 that 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 OF THE INVENTION This application claims the priority of the provisional application for E.U.A. No. 60 / 609,535, filed September 13, 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 in particular refers to the promoters P-Dgat1 and P-Dgat2 isolated from Arabidopsis thaliana and useful for the expression of transgenes of importance in agronomy in seeds of 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. 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 to prevent or inhibit 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 promoter in a construct can be homologous or heterogeneous to the gene of interest also contained therein. 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 germplasm 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 interval in development, 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 written, 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 (Ca V) (US Patent 5,352,605), CaMV 35S promoter with a duplicate enhancer (US Patent 5,164,316; 5,196,525; 5,322,938; 5,359,142; and 5,424,200), and the 35S promoter of the escrofularia 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; 5,447,858; 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. Diacylglycerol acyltransferase (hereinafter referred to as Dgat) is an integral membrane protein that catalyzes the final enzymatic step in the production of triacylglycerols in plants, fungi, and mammals (Harwood, Biochem Biophysics, Acta, 13017-13056 (1996); Daum et al., Yeast, 16: 1471-1510 (1998); and Coleman et al., Annu. Tev. Nutr., 20: 77-103 (2000) .Dgat is responsible for the transfer of an acyl group from from acyl-coenzyme-A to 1, 2-diacylglycerol (Dag) to form triacylglycerol (TAG) Almost all commercially important fats and oils of vegetable origin consist of triacylglycerols Triacylglycerols are hereinafter referred to as "oils" or vegetable oils. "In plants, particularly oilseeds, Dgat is associated with membrane and body lipid fractions, and play a key role in oil synthesis (Kennedy et al., J. Biol. Chem., 222 : 193 (1956); Finnlayson et al., Arch. Biochem. Biophys., 199 : 179-185 (1980)). Two different families of Dgat proteins have been identified. The first Dgat protein family (hereinafter referred to as Dgatl) is related to acyl-coenzyme A: cholesterol acyltransferase (ACAT) and has been described in the literature (see, for example, U.S. Patents 6,100, 077 and 6,344,548). A second family of Dgat proteins (hereinafter referred to as Dgat2), unrelated to the first identified family of Dgatl proteins, is described in the Published Application of E.U.A. US 20030028923. The present invention describes promoters associated with these Dgat families. 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 provides genetic tools that respond to the need both for the alteration of the composition of oils produced in plants as well as the percentage content thereof in relation to other components of a seed, including, for example, the flour content thereof. . The present invention includes promoters of diacylglycerol acyltransferase (Dgat), which must be used to direct the transcription of genes involved in the production in seeds of important traits in agriculture such as increased oil, starch and protein. 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 and SEQ ID NO: 4, and fragments thereof which are capable of regulate the transcription of operably associated polynucleotide molecules. Also provided by the invention are polynucleotide sequences comprising at least about 70% sequence identity with respect to any of these sequences, including sequences with about 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 or SEQ ID NO: 4 or a fragment thereof capable of regulate the transcription of operably associated polynucleotide molecules, 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, 900, 1000, or more contiguous nucleotides of any of the promoter sequences described in the present invention, including, for example, SEQ ID NO: 1 and SEQ ID NO: 4. In another embodiment, the invention provides an expression construct in plant comprising a polynucleotide sequence substantially homologous to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1 and 4 or any fragments thereof, wherein said promoter is operatively associated with respect to a polynucleotide molecule transcribed and may also be operatively associated with a polynucleotide molecule 3 'of the transcription terminus. In still another embodiment, the invention provides a transgene plant stably transformed with a plant expression construct provided by the invention. In one embodiment, the construct comprises a promoter comprising a polynucleotide sequence substantially homologous to a polynucleotide sequence selected from the group consisting of SEQ ID NOs: 1 and 4 or any fragments or regions thereof, wherein said promoter is operatively associated with a transcribable polynucleotide molecule, and optionally, is operatively associated with a polynucleotide molecule upstream of the transcription terminus. In another embodiment, the invention provides a method for making a vegetable oil, comprising obtaining a transgenic plant provided by the invention, for example, by incorporating an oily plant of a promoter of the present invention operatively associated within the genome. with a transcribable polynucleotide molecule that confers an altered content of oil and / or protein, and the extraction of oil and / or protein from seeds and / or other plant parts. In one aspect, the plant can be defined as an oily plant. The method can comprise the growth of the plant to produce protein and / or oil. The invention also provides methods for the production of food and fodder from a plant provided in the present invention. In one aspect, this includes obtaining a plant or part thereof provided by the invention and preparing food or fodder therefrom. In one aspect, the invention provides a method for making a vegetable oil comprising the incorporation into the genome of an oil plant of a promoter of the present invention operatively associated with a transcribable polynucleotide molecule that encodes a gene that improves the oil, conferring altered oil content, for example, Diacylglycerol Acyltransferase (Dgat, EC 2.3.1.20, US Patent 6,444,876), Phosphatidic Acid Phosphatase (Pap, EC 3.1.3.4, US Patents 6,495,739 and 6,406,294), and leucoantocyanidin dioxygenase (Dox , EC 1.14.11, WO 04/046336). The method may comprise growing the oily plant to produce oily seeds, and extracting the oil and / or protein from the oily seed. In another embodiment, a food or fodder prepared by the invention is a food product or fodder product, such as a vegetable starch. The invention thus provides a method comprising the incorporation into the genome of an oil plant of a promoter of the present invention (SEQ ID NO: 1 and 4) operatively associated with a transcribable polynucleotide molecule that encodes a gene that improves starch, which it confers an increased content of starch, comprising sucrose phosphorylase (Sm. gtfA, EC 2.4.1.7, US Patent 6,235,971), by growing the oil plant to produce seeds enriched in starch, and extracting the starch and / or protein from the oil seed. In another embodiment, the invention provides a method for altering cell proliferation, comprising the steps of incorporating within the genome of an oil plant a promoter of the present invention (SEQ ID NO: 1 and 4) operatively associated with a transcribed polynucleotide molecule. which encodes a cell proliferation gene. 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-Dgat1 promoter. SEQ ID NO: 2 discloses a primer sequence of P-Dgat1. SEQ ID NO: 3 discloses a primer sequence of P-Dgat1. SEQ ID NO: 4 discloses a polynucleotide sequence of a P-Dgat2 promoter. SEQ ID NO: 5 exhibits a primer sequence of P-Dgat2. SEQ ID NO: 6 exhibits a sequence of the initiator of P-Dgat2.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 illustrates pMON65429. Figure 2 illustrates pMON65430.

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. The nomenclature of DNA bases is used 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 translation start of an open reading frame (or region). encoding the protein) and involved in the recognition and binding of RNA polymerase II and other proteins (transcription factors that act on 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 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. Said promoters may 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 and 4 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. Cis elements can be identified by numerous techniques, including reelection analysis, for example, deleting one or more nucleotides from the 5 'end or internal to a promoter.; DNA-binding protein analysis using the footprinting technique by DNase I, interference from methylation, mobility change assays in electrophoresis, live genomic footprinting by PCR-mediated ligation, and other conventional assays; or by DNA sequence similarity analysis with known cis element motifs by conventional DNA sequence comparison methods. 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 and 4 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 substantial percent sequence identity with the promoters provided in the present invention. Of particular interest are polynucleotide molecules wherein the polynucleotide molecules function in plants to direct transcription and have at least about 70% sequence identity, at least about 75% sequence identity, at least about 80% sequence identity , at least about 85% sequence identity, and at least about 90% or more sequence identity, including at least about 92%, 95%, 96%, 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 transcriptionally operable polynucleotide molecules 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 strand) when the two sequences are aligned optimally (with insertions, deletions, 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, the Pearson and Lipman similarity search method, 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, AC). 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 "severe 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 procedure discussed in Molecular Cloning: A Laboratory Manual, 3rd edition, Volumes 1, 2, and 3. J.F. 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. Polynucleotide molecules from different sources that share a high degree of homology are referred to as "homologs." Methods well known to one skilled in the art may be used to identify promoters of interest having similar activity with respect to the promoters described in 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 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 and 4 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 while substantially maintaining the function of the promoter. 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 a variant of the promoter 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 may cause an increase in the expression of an operably associated transcribable 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 and 4 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 and 4 may be truncated or portions may be deleted as long as they retain 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. Consequently, any fragments, portions, or regions of the promoters comprising the polynucleotide sequences shown in SEQ ID NO: 1 and 4 can be used as regulatory polynucleotide molecules, including but not limited to elements in cis or motifs of the polynucleotide molecules described. The substitutions, deletions, insertions, or any combination thereof may be combined to produce a final construction.

Polynucleotide constructions As used in the present invention, the term "construction" refers to any recombinant polynucleotide molecule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, phage, or DNA or RNA polynucleotide molecule, single-chain or double-stranded, linear or circular derived from any source, capable of carrying out integration to the genome or autonomous replication, comprising a polynucleotide molecule in which 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, conventional compositions and methods for the preparation and use of host cell constructs and cells are well known to one skilled in the art (see, for example, Sambrook, et al.). The constructs of the present invention may contain a promoter operatively associated with a transcribable polynucleotide molecule associated with a polynucleotide molecule up to 3 'of transcription termination. In addition, constructs may include but are not limited to additional regulatory polynucleotide molecules from the 3 'untranslated region (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 regulatory polynucleotide molecules upstream and downstream 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 and 4 or modified as described above, operatively associated with a transcribable polynucleotide molecule so as to direct the transcription of said transcribable polynucleotide molecule to 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 which are incorporated into recipient cells by genetic engineering methods rather than by classical breeding or cross breeding techniques. It is intended that the exogenous gene or the 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 can include DNA that is already present in the plant cell, DNA from another plant, DNA from a different organism, or a DNA that is generated externally, such as a molecule of DNA that contains an antisense messenger of a gene, or a DNA molecule that encodes an artificial or modified version of a gene. 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 may 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 tissue sources or bombardment of tissue of specific interest by particles. The present invention includes the use of any transient expression system for evaluating the promoters or promoter fragments operatively associated with any transcriptional polynucleotide molecules, including but not limited to selected reporter genes, marker genes, or genes of interest in agriculture. Examples of plant tissues considered to be evaluated in transients via a suitable delivery system could include, but are not limited to, tissues of the leaf base, callus, cotyledons, roots, endosperm, embryos, floral tissue, pollen, and tissue epidermal. Any marker gene that can be evaluated or selected can be used in a transient trial. 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) in the present invention as 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. Thus, 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 it is operatively associated with a transcribable polynucleotide molecule that is provided for a selectable, classifiable, or evaluable 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; and 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 (1987) for tolerance to glufosinate and bialaphos. In a preferred embodiment, a polynucleotide molecule of the present invention as shown in SEQ ID NO: 1 and 4 or fragments, variables, or derivatives thereof are incorporated within a construct such 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 crop 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 (Patent of US Pat. US 5,716,837), insect control (US Patents 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 (Patents US 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 production of oils (US Patent 6,444,876) , high oil production (U.S. Patents 5,608,149 and 6,476,295), modified fatty acid content (US Patent 6,537,750), high production protein ion (Patent of US Pat. No. 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 taste (US Patent 6,011, 199), nitrogen fixation (US Patent 5,229.1 14), hybrid seed production (US Pat. U.S. Patent 5,689,041), and biofuel production (U.S. Patent 5,998,700), the genetic elements and transgenes described in the above-listed patents 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 (for example, 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. The constructions of the present invention may comprise DNA constructions with double Ti-plasmid border having the right border isolated from Agrobacterium tumefaciens comprising a T-DNA, which together with the transfer molecules provided by the Agrobacterium cells, allow the integration of T-DNA into the genome of a plant cell. The constructs may 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, for example, an E. coli with origin of replication such as OR322, 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 encoding the Tn7 aminoglycoside adenylyltransferase (aadA) that confers resistance to spectinomycin or streptomycin, or a marker gene of selection 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 US Pat. 5,384,253; bombardment of microprojectiles as illustrated in U.S. Patent 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. Patent 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 transforming specifically dicotyledons are well known to those skilled in the art. Plant transformation and regeneration using these methods has been described for numerous crops including, not limited to, cotton (Gossypium hirsutum), soybean (Glycine max), peanut (Arachis hypogaea), members of the genus Brassica; and alfalfa (Medicago sativa). 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, not limited to, barley (Hordeum vulgarae); corn (Zea mays); avenas (Avena sativa); orchid grass (Dactylis glomerata); rice (Oryza sativa, including Indica and Japanese varieties); sorghum (Sorghum bicolor); cane sugar (Saccarum sp.); high cañuela (Festuca arundinacea); turf (Agrostis); wheat (Tritticum aestivum), millet, and rye. 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 white crops of interest. Many seeds, nuts, and grains contain oil that can be extracted and used for cooking, as an ingredient in other foods, as a nutritional supplement, as an unprocessed 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 (AURA), 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 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 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 analyzing plants include, but are not limited to Southern blot or northern blot, PCR-based methods, 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 terms "seeds" and "grains" are understood as equivalent in their meaning. The term grain is often used in the description of the seed of a corn plant or a rice plant. In all plants the seed is the mature ovule consisting of a seed coat, embryo, aleuron, and an endosperm. 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 present invention relates to the alteration of the cell cycle in plants through the manipulation of the expression of the genes for cell proliferation in the seed to alter the number of cells and the size of the specific organs of the seed such as the embryo and / or the aleurone. The larger specific organs of the seed result in higher content of oil, micronutrients, protein, or starch produced by the seed. Various strategies for modifying the cell cycle are considered by the present invention. The phrase "cell proliferation" refers to cells that undergo mitotic cell divisions, such as in rapidly growing tissues. Cell proliferation can be enhanced by the expression of genes involved in the cell proliferation process, including but not restricted to, CYCD2; 1 (CYCD2) and CYCD; 1 (CYCD3) (accesses GenBank AJ 294533, Richard et al., Plant Cell, Tissue and Organ Culture, 69: 167-176 (2002)), AINTEGUMENTA (access GenBank U411339, Elliot et al. ., Plant Cell, 8 (2): 155-168 (1996)). Similarly, cell proliferation can be interrupted or inhibited by expression of genes involved in cell proliferation including but not restricted to, KRP1 and KRP2 (accesses GenBank U94772 and AJ251851, De Veylder et al., The Plant Cell, 13 : 1653-1667 (2001)) and AtWEEI (access GenBank CAD28679, Sorrell et al., Planta, 215 (3): 518-522 (2002)). 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)). 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. As used in the present invention, the phrase "sucrose phosphorylase" means an enzyme, which catalyzes a reversible conversation of sucrose and inorganic phosphate to alpha-D-glucose-1-phosphate and D-fructose. It can be isolated from many microbial sources, including Streptococcus mutans, Clostridium pasteurianum, Pseudomonas saccharophila, Pseudomonas putrifaciens, Pullularia pullulans, Acetobacter xylinum, Agrobacterium sp., And Leuconoston mesenteroides (Patent of US Pat. No. 6,235,971). As used in the present invention, the phrase "starch improvement" refers to genes or combinations of genes that result in increased levels of polysaccharides, for example, starch. As used in the present invention, the term "starch" refers to a carbohydrate polymer that occurs in a granular form in certain plant species notably in cereals, tubers and legumes such as corn, wheat, rice, potato and soybeans. The polymer consists of anhydro-a-D-glucose associated units. This may have to be a mainly linear structure (amylase) or a branched structure (amylopectin). The phrase "protein quality" refers to the level of one or more essential amino acids either free or incorporated in the protein, namely histidine, isoleucine, leucine, lysine, methionine, cysteine, phenylalanine, tyrosine, threonine, tryptophan, and Valina 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 P-Dqat1 and P-Dgat2 promoters A genomic DNA library was constructed using genomic DNA isolated from Arabidopsis thaliana using a modified genomic DNA isolation protocol described below (Dellaporta et al., (1983) Plant Molecular Biology Reporter, 1: 19-21). The Arabidopsis seedlings were grown in soil or in Petri dishes, harvested, and kept cold in liquid nitrogen until extraction. The tissue was milled to a fine powder using a mortar and pestle while keeping the tissue frozen with liquid nitrogen. The tissue powder was transferred to a Waring blender containing 200 ml of pH buffer for cold DNA extraction (0 ° C) (350 mM sorbitol).; 100 mM Tris; 5 mM EDTA; pH 7.5 with HCI; Sodium bisulfite (3.8 mg / ml) was adjusted just before use), and homogenized at a high speed for 30-60 seconds. The homogenate was filtered through a cheesecloth layer and collected in a bottle for centrifugation. The samples were centrifuged at 2500 xg for 20 minutes. The supernatant and any loose green material were discarded. The concentrate was then resuspended in 1.25 mL of pH regulator for extraction and transferred to a 50 ml polypropylene tube. Then the pH regulator for nuclear lysis was added (1.75 mL containing 200 mM Tris, 50 mM EDTA, 2 mM NaCl, 2.0% (w / v) of C , pH adjusted to 7.5 with HCI), followed by the addition of 0.6 mL of sarcosil at 5% (w / v). The tubes were mixed gently, and the samples were incubated at 65 ° C for 20 minutes. An equal volume of c! Oroform: isoamyl alcohol (24: 1) was added and the tubes mixed gently. The tubes were then centrifuged at 2500 xg for 15 minutes, and the resulting supernatant was transferred to a clean tube. An equal volume of ice-cold sopropanol was poured onto the sample, and the sample was inverted several times until a precipitate formed. The precipitate was removed from the solution using a glass pipette and the residual alcohol was removed by allowing the precipitate to air dry for 2-5 minutes. The precipitate was resuspended in 400 uL of pH regulator TE (10 mM Tris-HCl, 1 mM EDTA, pH adjusted to 8.0).

Promoter P-Dgat1 The P-Dgat1 promoter (SEQ ID NO: 1) was isolated by the use of PCR (polymerase chain reaction). The reaction conditions of the PCR reaction followed the manufacturer's protocol (PE Applied Biosystems, Foster City, CA). Approximately 00 ng of Arabidopsis genomic DNA, prepared as described above, was amplified using 30 nmol of each of the forward primers (SEQ ID NO: 2) and of the reverse primer (SEQ ID NO: 3) and 10 micromoles of each of dATP, dCTP, dGTP, and TTP, 2.5 units of Taq TaKaRaLa in pH II regulator for 1X LA PCR (Takara Bio INC, Shiga, Japan). After the initial incubation at 94 ° C for 1 minute, 35 cycles of PCR were carried out at 94 ° C for 45 seconds, followed by fixation at 60 ° C for 45 seconds, 72 ° C for 1 minute 15 seconds, followed for 1 cycle of 72 ° C for 7 minutes. The construction of the reporter gene p-Dgat1: glucuronidase from Escherichia coli (uidA) was performed by the isolation of a 1250 bp fragment containing the P-Dgat1 promoter by PCR, followed by a digestion reaction with the restriction enzymes Pstl and Spel. The resulting fragment was ligated into pMON63925, which had also been digested with PstI and Spel. The resulting plasmid was designated pMON63922. A 1496 bp fragment containing the P-Dgat1 and P-CaMV-70 promoters was removed from pMON63922 by digestion with the restriction enzymes Ncol and Notl. The resulting fragment was ligated into pMON65424, which had been digested with Ncol and Notl. The resulting plasmid, containing the P-Dgat1 promoter, in operative association with the reporter gene of E. coli glucuronidase (uidA) and the 3 'UTR of napin, was subsequently named pMON65430 (FIG. 2). Glyphosate was used as the selection marker (U.S. Patent 5,633,435). The nucleic acid sequence was determined using standard methodology as set out in PE Applied Biosystems BigDye terminator v3.0 (PE Applied Biosystems, Foster City, CA) and the integrity of the cloning junctions was confirmed. The vector pMON65430 was used in the subsequent transformation of Arabidopsis and Cañóla as described in Example 2, below.

Promoter P-Dqat2 The P-Dgat2 promoter (SEQ ID NO: 4) was isolated by the use of PCR (polymerase chain reaction). The reaction conditions of the PCR reaction followed the manufacturer's protocol (PE Applied Biosystems, Foster City, CA). Approximately 100 ng of Arabidopsis genomic DNA, prepared as described above, were amplified using 30 nmol of each of the forward primers (SEQ ID NO: 5) and of the reverse primer (SEQ ID NO: 6) and 10 micromoles of each of dATP, dCTP, dGTP, and TTP, 2.5 units of AmpliTaq Gold in pH regulator 3 Opti-Prime ™ 1X (Stratagene, La Jolla, California, USA). After the initial incubation at 95 ° C for 0 minutes, 15 PCR cycles were carried out at 92 ° C for 15 seconds, followed by 15 cycles starting at 62 ° C and decreasing 0.7 ° C per cycle for 20 seconds, 72 ° C for 2 minutes, followed by 20 cycles at 92 ° C for 15 seconds, 52 ° C for 20 seconds, and 72 ° C for 2 minutes, followed by 1 cycle of 72 ° C for 7 minutes. The product of the PCR reaction was purified using the QIAquick PCR purification kit (Qiagen Inc. Valencia, CA, USA) in accordance with the manufacturer's instructions and cloned into pCR2.1 Topo (Invitrogen Corp. Carlsbad, CA , USA) in accordance with the manufacturer's instructions. The resulting plasmid was named pMON65421. The entire sequence of this clone was determined using standard sequencing methodologies as established by PE Applied Biosystems (Perkin-Elmer Applied Biosystems Inc., Foster City, USA). The construction of the reporter gene p-Dgat2: glucuronidase from Escherichia coli (uidA) was carried out by isolating a 1082 bp fragment containing the P-Dgat2 promoter by restriction digestion with the restriction enzymes Pmel and Ncol. The resulting fragment was ligated into pMON65424, which had also been digested with Pmel and Ncol. The resulting plasmid, containing P-Dgat2, in operative association with the E. coli glucuronidase reporter gene (uidA) and the 3 'UTR of napin, was subsequently designated pMON65429 (FIG. 1). Glyphosate was used as the selection marker (U.S. Patent 5,633,435). The nucleic acid sequence was determined, and the integrity of the cloning junctions was confirmed, using the methodology established by the PE Applied Biosystems BigDye terminator v.3.0 protocol (PE Applied Biosystems, Foster City, CA). This vector was used in the subsequent transformation of Arabidopsis and Cañóla as described in example 2.

EXAMPLE 2 Transformation of Arabidopsis and canola containing constructs of the P-Dgat promoter The promoters described in Example 1 were operatively associated with the marker gene, uidA from Escherichia coli, in a construct to demonstrate expression in Arabidopsis and canola. Table 2 lists the promoter-specific constructions.

TABLE 2 Constructions for transformation of Arabidopsis and canola The Arabidopsis plants were grown by sowing the seeds in 10 cm pots containing water in reverse osmosis (ROW) stuck with MetroMix 200 (The Scotts Company, Columbus, OH, USA). The plants were vernalized by placing the pots on a flat plate covered in a chamber for growth at 4-7 ° C, 8 hours light / day, for 4-7 days. The plates were transferred to a growth chamber at 22 ° C, with 55% relative humidity, and 16 hours of light / day at an average intensity of 160-200 uEinstein / s / m2. The cover was lifted and slid 2.5 cm after germination, and then removed when the true leaves were formed. The plants were watered from below as necessary with ROW until 2-3 weeks after germination. The plants were then watered from below as necessary with a Plantex 15-15-18 solution (Plantex Corporation, Ottawa, Canada) at 50 ppm N2. The pots were culled so that 1 plant remained per pot 2-3 weeks after germination. Once the plants began to form the flower, the primary fluorescence was able to encourage the growth of axillary buds. Transgenic plants of Arabidopsis thaliana were obtained as described by Bent et al., Science, 265: 1856-1860 (1994) or Bechtold et al., C.R. Acad. Sci. Life Sciences, 300 16: 1194-1199 (1993). Cultures of Agrobacterium tumefaciens strain ABI containing any of the transformation vectors pMON65429 or pMON65430 were grown overnight in LB (10% bactotriptone, 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 a 5% solution of sucrose + 0.05% of Silwet-77. The aerial portions of the total Arabidopsis thaliana plants (approximately 5-7 weeks old) were immersed in the resulting solution for 2-3 seconds. The excess solution was removed by placing the plants on paper towels. The submerged plants were placed on their side in a flat covered plate and transferred to a growth chamber at 19 ° C.

After 16 to 24 hours the dome was removed and the plants were placed upwards. When the plants had reached maturity, the water retained for 2-7 days before harvesting the seeds. The harvested seeds were passed through a stainless steel mesh screen (40 holes / 2.5 cm) to promote waste. The harvested seeds were stored in paper wraps at room temperature until analysis. Arabidopsis seeds were sterilized on the surface using a steam phase sterilization protocol. An open container of seeds was placed in a desiccator with a beaker containing 00 ml of homemade 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 smoke. The seeds incubated by several works. The sterilized seeds were splashed on Arabidopsis germination medium containing MS salts (1X); sucrose (1%); myo-inositol (100 mg / L); Thiamine-HCl (1 mg / L), Pyridoxine-HCl (500 mg / L); nicotinic acid (500 mg / L); MES pH 5.7 (0.05%); and Phytagar (0.7%) supplemented with 50 mg / L glyphosate. Up to 16 glyphosate-resistant seedlings were transferred to 8 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 were started to dry. The tissue (of rosette leaves, cauline leaf, stem, flowers, flower buds, and developing siliques) were removed from each T1 plant for subsequent histochemical staining. The expression of β-glucuronidase was analyzed in Arabidopsis thaliana plants transformed with pMON65430 using histochemical staining. Tissues from the transformed and control plants were incubated for approximately 24 hours at 37 ° C in a solution containing 50 mM NaPO 4 (pH 7.2); potassium ferricyanide 100 uM; potassium ferrocyanide 100 uM, Triton X- 00 0.03%; 20% methanol; and 2.5 mg / ml of 5-bromo-3-indol glucuronic acid (X-gluc). The chlorophyll was removed from the stained tissue 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. For pMOC65430, 14 of 15 events that had detectable levels of GUS activity in developing seeds were evaluated.

EXAMPLE 3 Transformation of canopy containing p-Dqat constructions Vectors pMON65429 and pMON65430 were introduced into Agrobacterium 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, Brassica napus cv ebony seeds were planted in 10 cm pots containing Metro Mix 350 (The Scotts Company, Columbus, OH, USA). The plants were grown in a growth chamber at 24 ° C, and a photoperiod of 16/8 hours, with light intensity of 400 mEm-2 sec-1 (HID lamps). After 2-1 / 2 weeks, the plants were transplanted into 15.2 cm pots and grown in a growth chamber at 15/10 ° C day / night temperature, 6/8 hour photoperiod, 800 light intensity mEm-2 sec-1 (HID lamps). Four terminal internodos were removed from the plants just before the flower formation or in the flower formation process but before flowering and the surface was sterilized with 70% ethanol (v / v) for 1 minute, 2 % sodium hypochlorite (w / v) for 20 minutes and rinsed three times with sterile deionized water. Six to seven segments of the stem were cut into 5 mm discs, maintaining the orientation of the basal end. The culture of Agrobacterium used to transform barley was grown overnight on a rotary shaker at 24 ° C in 2 mLs of Luria broth, LB (10% bactotriptone, 5% yeast extract, and 10% NaCl) contained 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 5: 473-497, (1962)) which produced approximately 9 x 108 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 medium comprising MS 1/10 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 covered with 1.5 mL of medium containing MS salts, vitamins B5, 3% sucrose, pH 5.7, 4.0 mg / L p-chlorophenoxyacetic acid, 0.005 mg / L decinetine and covered with sterile filter paper. After 2 to 3 days of co-culture, the explants were transferred to deep Petri dishes containing MS salts, B5 vitamins, 3% sucrose, 0.8% agar, pH 5.7, 1 mg / L BA, 500 mg / l of carbenicillin, 50 mg / L of cefotaxime, and 25 mg / L of glyphosate for selection. Seven explants were placed in each plate. After three weeks, five explants per plate were transferred to fresh medium. The explants were grown in a room for growth at 25 ° C under continuous light conditions (Cool White). The transformed plants were grown in a growth chamber at 22 ° C, 16/8 hours of light-dark cycle with a light intensity of mEm "1 sec" 1 for several hours before transferring to the greenhouse. The plants were kept in a greenhouse until they were harvested. The growing siliques were harvested at various stages after politicization and stored at -70 ° C. The stems, flowers, and leaves were also collected; and stained (as described below) shortly before harvesting without prior freezing. The mature seeds were harvested and stored under controlled conditions that consisted of approximately 17 ° C and 30% humidity. Up to 5 siliques were harvested from individual R0 plants at various time points after politicization. The siliques were evaluated with an 18-gauge needle to allow the staining solution to come in contact with the developing seed. The siliques and the seeds were incubated for approximately 24 hours at 37 ° C in a solution containing 50 mM NaPO4 (pH 7.2); 100 mM potassium ferricyanide; potassium ferrocyanide 100 mM, Triton X 100 0.03%; 20% methanol and 2.5 mg / ml 5-bromo-4-chloro-3-indoyl gluconic 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 immediately photographed or transferred to a solution of 70% ethanol / 30% glycerol (v / v) and stored at 4 ° C until they were photographed. The samples were evaluated as positive (+) with negative (-) for the blue color. For plants transformed with pMON65429, 8 of 10 evaluated plants had detectable levels of GUS activity in the seed from at least one time point (Table 3). For plants transformed with pMON65430, 7 of 10 evaluated plants had detectable levels of GUS activity in the seed from at least one time point (Table 4).

TABLE 3 Expression of p ON65429 in seed of canola in development TABLE 4 Expression of pMON65430 in seed 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 (9)

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 or SEQ ID NO: 4; (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 (b) .

3. - The promoter according to claim 1, further characterized in that said promoter comprises a polynucleotide sequence with from about 80% identity to about 89% sequence identity with respect to the polynucleotide sequence of (a) or (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 (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 6, further characterized in that the gene of interest in agronomy is a gene that improves the oil content selected from the group consisting of diacylglycerol acyltransferase, phosphatidic acid phosphatase, and leucoantocianidin dioxygenase.

8. The construction according to claim 6, further characterized in that the gene of interest in agronomy is a gene that improves the amount of starch comprising sucrose phosphorylase.

9. The construction according to claim 5, further characterized in that said transcribable polynucleotide molecule is a marker gene. 0. - A transgenic plant or part of it transformed in a stable manner with the construction according to claim 5. 11. - The transgenic plant or part thereof according to claim 10, further characterized in that said plant is a dicotyledonous plant selected from the group consisting of tobacco, tomato, potato, soybean, cotton, canola, sunflower, and alfalfa. 12. The transgenic plant or part thereof according to claim 10, further characterized in that said transcribable polynucleotide molecule confers altered cell proliferation in the embryo, aleurone, or both to said transgenic plant. 13. - The transgenic plant or part thereof according to claim 10, further characterized in that said transcribable polynucleotide molecule confers altered oil content in the embryo, aleurone, or both to said transgenic plant. 14. - The transgenic plant or part thereof according to claim 10, further characterized in that said transcribable polynucleotide molecule confers altered protein quality in the embryo, aleurone, or both to said transgenic plant. 5. - The transgenic plant or part thereof according to claim 10, further characterized in that said transcribable polynucleotide molecule confers altered content of micronutrients to said transgenic plant. 16. A seed transformed with the construction according to claim 5. 17. A cell transformed with the construction according to claim 5. 18. The oil of the transgenic plant according to claim 10, further characterized in that the oil comprises a detectable nucleic acid comprising the promoter according to claim 1. 19. The flour of the transgenic plant according to claim 10, further characterized in that the flour comprises a detectable nucleic acid comprising the promoter according to claim 1. 20. A method for preparing a vegetable oil, comprising the steps of: a) obtaining the transgenic seed according to claim 16; and b) extract the oil from the seed. 21. - A method for the preparation of a vegetable protein, comprising the steps of: a) obtaining the transgenic seed according to claim 16; and b) extracting the protein from the seed. 22. - The method according to claim 20, further characterized in that the promoter is operatively associated with a transcribibie polynucleotide molecule that confers altered protein content. 23. A method for preparing food or fodder comprising: a) obtaining the plant or part thereof in accordance with claim 10; and b) prepare food or fodder from the plant or part of it.