BR122020000152B1 - RECOMBINANT DNA MOLECULE AND METHOD FOR PRODUCING A TRANSGENIC CORN PLANT - Google Patents

Abstract

The invention concerns recombinant DNA molecules and constructs and their nucleotide sequences, useful for modulating gene expression in plants. The invention also provides transgenic plants, plant cells, plant parts and seeds comprising a recombinant DNA molecule comprising a DNA molecule operatively linked to a DNA molecule capable of heterologous transcription, as well as methods for their use.

Description

[001] Patent application for division of BR112015023189-6, filed on 03/12/2014.

[002] The listing sequence that is contained in the file called "MONS331WO.txt", which is 54.4 kilobytes (as measured in Microsoft Windows®) and was created on March 12, 2014, is deposited together by electronic submission and is incorporated by reference herein.

FIELD OF INVENTION

[003] The invention relates to the field of plant molecular biology, plant genetic engineering, and DNA molecules useful for modulating gene expression in plants.

FUNDAMENTALS

[004] Regulatory elements are genetic elements that regulate gene activity by modulating the transcription of a DNA molecule that can be transcribed operationally linked. These elements include promoters, leaders, introns, and 3' untranslated regions, and are useful in the field of plant molecular biology and plant genetic engineering.

SUMMARY OF THE INVENTION

[005] The invention provides new regulatory elements for use in plants and constructs comprising regulatory elements. The invention also provides transgenic plants, plant cells, plant parts and seeds comprising regulatory elements. In one embodiment, the invention provides the regulatory elements disclosed herein operatively linked to a DNA molecule that can be transcribed. In certain embodiments, the DNA molecule that can be transcribed is heterologous to a regulatory sequence provided herein. Also provided herein are methods for preparing and using the regulatory elements disclosed herein, including constructs comprising the regulatory elements, and transgenic plants, plant cells, plant parts and seeds comprising the regulatory elements operatively linked to a DNA molecule that can be transcribed. which is heterologous in relation to the regulatory element.

[006] Thus, in one aspect, the invention provides a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: a) a DNA sequence with at least 85 percent sequence identity to any of SEQ ID NOs: 1-20; (b) a DNA sequence comprising any one of SEQ ID NOs: 1-20; and (c) a fragment of any of SEQ ID NOs: 1-20, wherein the fragment has gene regulatory activity; wherein the DNA sequence is operably linked to a DNA molecule that can be heterologously transcribed. "Heterologous transcribeable DNA molecule," means that the transcribed DNA molecule is heterologous with respect to the DNA sequence. In specific embodiments, the recombinant DNA molecule comprises a DNA sequence having at least about 85 percent, at least about 86 percent, at least about 87 percent, at least about 88 percent, at least about of 89 percent, at least about 90 percent, at least about 91 percent, at least about 92 percent, at least about 93 percent, at least about 94 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, or at least about 99 percent sequence identity to the DNA sequence of any one of SEQ ID NOS: 1-20. In particular embodiments, the heterologous DNA molecule that can be transcribed comprises a gene of agronomic interest, such as a gene capable of providing herbicide resistance or pest resistance in plants. In still other embodiments, the invention provides a construct comprising a recombinant DNA molecule, as provided herein.

[007] In another aspect, provided herein are transgenic plant cells comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a DNA sequence with at least about 85 percent identity of sequence for any of SEQ ID NOs: 1-20; (b) a DNA sequence comprising any one of SEQ ID NOs: 1-20; and (c) a fragment of any one of SEQ ID NOs: 1-20, wherein the fragment has gene regulatory activity; wherein the DNA sequence is operably linked to a DNA molecule that can be heterologously transcribed. In certain embodiments, the transgenic plant cell is a monocot plant cell. In other embodiments, the transgenic plant cell is a dicotyledonous plant cell.

[008] In yet another aspect, further provided herein is a transgenic plant, or part thereof, comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a DNA sequence of at least about 85 percent sequence identity to any of SEQ ID NOs: 1-20; (b) a DNA sequence comprising any one of SEQ ID NOs: 1-20; and (c) a fragment of any of SEQ ID NOs: 1-20, wherein the fragment has gene regulatory activity; wherein the DNA sequence is operably linked to a DNA molecule that can be heterologously transcribed. In specific embodiments, the transgenic plant is a progeny plant of any generation relative to an initial transgenic plant and comprises the recombinant DNA molecule. A transgenic seed comprising the recombinant DNA molecule that produces such a transgenic plant when grown is also provided by the invention.

[009] In another aspect, the invention provides a method for producing a commodity from a transgenic plant containing the recombinant DNA molecule of the invention. The commodity products of the invention contain a detectable amount of SEQ ID NOs: 1-20. As used herein, a "commodity product" refers to any composition or product that is composed of material derived from a transgenic plant, plant part, plant cell or seed containing the recombinant DNA molecule of the invention. Commodity products include, but are not limited to, processed seeds, grains, plant parts, and meals. Transgenic plants containing the recombinant DNA molecule of the invention can be used to manufacture any commodity product normally purchased from a plant. A commodity product of the invention will contain a detectable amount of DNA corresponding to the recombinant DNA molecule of the invention. Detection of one or more of this recombinant DNA molecule in a sample can be used to determine the content or source of the commodity product. Any standard detection method for DNA molecules can be used, including methods for detection disclosed herein.

[0010] In yet another aspect, the invention provides a method for expressing a DNA molecule that can be transcribed, as a gene of agronomic interest, in a transgenic plant by obtaining a transgenic plant containing a recombinant DNA molecule of the invention and cultivating the plant.

[0011] Also provided here is a method for providing a transgenic plant by transforming a plant cell with a recombinant DNA molecule of the invention to produce a transformed plant cell, and regenerating the transformed plant cell to produce a transgenic plant.

[0012] Also provided by the invention is a codon-redesigned Escherichia coli (E. coli) β-glucuronidase (GUS) coding sequence; wherein said codon-redesigned GUS coding sequence manifests higher expression in a transgenic plant than the native E. coli GUS coding sequence. In one embodiment, the codon-redesigned GUS coding sequence may be selected from the group consisting of SEQ ID NOs: 29 and 30. The transgenic plant may be a monocot plant. In one embodiment, the monocot plant is selected from the group consisting of corn (Zea mays), rice (Oryza sativa), wheat (Triticum), barley (Hordeum vulgare), sorghum (Sorghum spp.), millet, millet (Pennisetum glaucum) , Giant crow's foot grass (Eleusine coracana), Millet (Panicum miliaceum), Foxtail millet (Setaria italica), Oats (Avena sativa), Triticale, Rye (Secale cereale), Fonio (Digitaria), Onion (Allium spp.), Pineapple (Ananas spp.), Grass, Sugarcane (Saccharum spp.), Palm (Arecaceae), Bamboo (Bambuseae), Banana (Musaceae), Ginger family (Zingiberaceae), Lily ( Lilium), Narcissus (Narcissus), Iris (Iris), Amaryllis, Orchid (Orchidaceae), Cannas, Hyacinth (Hyacinthoides) and Tulips (Tulipa). The transgenic plant can also be a dicotyledonous plant. In one embodiment, the dicotyledonous plant is selected from the group consisting of Soybean (Glycine max), Wild soybean (Glycine longa), Cotton (Gossypium), Tomato (Solanum lycopersicum), Pepper (Piper), Pumpkin (Cucurbita), Pea (Pisum sativum), Alfalfa (Medicago sativa), Medicago truncatula, Beans (Phaseolus), Chickpea (Cicer arietinum), Sunflower (Helianthus annuus), Potato (Solanum tuberosum), Peanut (Arachis hypogaea), Quinoa, Buckwheat ( Fagopyrum esculentum), Carob (onia siliqua), Beetroot (Beta vulgaris), Spinach (Spinacia oleracea) and Cucumber (Cucumis sativus).

BRIEF DESCRIPTION OF FIGURES

[0013] Figures 1a-1c show an alignment between the E. coli β-glucuronidase (GUS) coding sequence (CR-Ec.uidA-1:1:4, SEQ ID NO: 31) and the coding sequence of E. coli GUS codon redesigned (CR-Ec.uidA_nno-1:1:1, SEQ ID NO:30). Identical nucleotides in the alignment are indicated by an asterisk.

BRIEF DESCRIPTION OF SEQUENCES

[0014] SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 23 are promoter sequences.

[0015] SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 are leader sequences.

[0016] SEQ ID NOs: 25 to 28 are amplification primer sequences.

[0017] SEQ ID NOs: 29 and 30 are GUS coding sequence redesigned into codons. SEQ ID NO: 29 comprises a processable intron, while SEQ ID NO: 30 is a contiguous coding sequence.

[0018] SEQ ID NO: 31 is the native Escherichia coli β-glucuronidase coding sequence.

[0019] SEQ ID NO: 32 is a GUS coding sequence with a processable intron based on native E. coli β-glucuronidase of SEQ ID NO: 31.

[0020] SEQ ID NOs: 33, 39 and 40 are 3' UTR sequences.

[0021] SEQ ID NOs: 34-37, 41 and 44 are transcriptional regulatory expression element group sequences (EXPs) comprising a promoter sequence operably linked 5' to a leader sequence that is operably linked 5' to an intron sequence , or in the case of SEQ ID 44, a promoter sequence operably linked 5' to a leader sequence.

[0022] SEQ ID NO: 38 is an intron sequence.

[0023] SEQ ID NOs: 42 and 44 are coding sequences for luciferase proteins derived from Photinus pyralis and Renilla reniformis, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The invention provides DNA molecules having gene regulatory activity in plants. The nucleotide sequences of these DNA molecules are provided as SEQ ID NOs: 1-20. These DNA molecules are, for example, capable of affecting the expression of a transcribed operably linked DNA molecule in plant tissues, and therefore regulating gene expression of an operably linked transgene in transgenic plants. The invention also provides methods for modifying, producing and using the same. The invention also provides compositions including transgenic plant cells, plants, plant parts, and seeds containing recombinant DNA molecules of the invention, and methods for preparing and using the same.

[0025] The following definitions and methods are provided to better define the present invention and guide those skilled in the art in practicing the invention. Unless otherwise indicated, terms should be understood in accordance with conventional usage by those skilled in the relevant art.

DNA molecules

[0026] As used herein, the term "DNA", "DNA molecule" refers to a double-stranded DNA molecule of genomic or synthetic origin, that is, a polymer of deoxyribonucleotide bases. As used herein, the term "DNA sequence" refers to the nucleotide sequence of a DNA molecule. The nomenclature used here corresponds to that in Title 37 of the United States Code of Federal Regulations § 1.822, and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

[0027] As used herein, a "recombinant DNA molecule" is a DNA molecule comprising a combination of DNA molecules that would not naturally occur together without human intervention. For example, a recombinant DNA molecule may be a DNA molecule that is composed of at least two DNA molecules that are heterologous with respect to each other, a DNA molecule that comprises a DNA sequence that departs from the DNA sequences that exist in nature, or a DNA molecule that has been incorporated into the DNA of a host cell by genetic transformation.

[0028] As used herein, the term "sequence identity" refers to the extent to which two ideally aligned polynucleotide sequences or two ideally aligned polypeptide sequences are identical. An ideal sequence alignment is created by manually aligning two sequences, for example, a reference sequence and another DNA sequence, to maximize the number of nucleotide matches in the sequence alignment with appropriate insertions, deletions, or internal nucleotide gaps. As used herein, the term "reference sequence" refers to a DNA sequence provided as SEQ ID NOs: 1-20.

[0029] As used herein, the term "percent sequence identity" or "percent identity" or "% identity" is the fraction of identity multiplied by 100. The "fraction of identity" for an ideally aligned sequence with a reference sequence is the number of nucleotide matches in the ideal alignment, divided by the total number of nucleotides in the reference sequence, for example, the total number of nucleotides in the entire length of the entire reference sequence. Thus, one embodiment of the invention provides a DNA molecule comprising a sequence that, when ideally aligned to a reference sequence, provided herein as SEQ ID NOs: 1-20, has at least about 85 percent identity, at least about of 86 percent identity, at least about 87 percent identity, at least about 88 percent identity at least about 89 percent identity, at least about 90 percent identity, at least about 91 percent identity, at least about 92 percent identity, at least about 93 percent identity, at least about 94 percent identity, at least about 95 percent identity, at least about 96 percent identity, at least about 97 percent identity, at least about 98 percent identity, at least about 99 percent identity, or at least about 100 percent identity for the sequence of reference.

Regulatory Elements

[0030] Regulatory elements such as promoters, leaders, enhancers, introns and transcription termination regions (or 3' UTRs) play an essential role in the general expression of genes in living cells. The term "regulatory element", as used herein, refers to a DNA molecule with gene regulatory activity. The term "gene regulatory activity", as used herein, refers to the ability to affect the expression of an operationally linked transcribeable DNA molecule, e.g., by affecting the transcription and/or translation of the operably linked DNA molecule. operationally linked transcript. Regulatory elements such as promoters, leaders, enhancers, introns and 3' UTRs that function in plants are therefore useful for modifying plant phenotypes through genetic engineering.

[0031] As used herein, a "regulatory expression element group" or "EXP" sequence may refer to a group of operationally linked regulatory elements, such as enhancers, promoters, leaders and introns. Thus, a regulatory expression element group may be composed, for example, of a promoter operably linked 5' to a leader sequence, which in turn operably linked 5' to an intron sequence.

[0032] Regulatory elements can be characterized by their gene expression patterns, for example, positive and/or negative effects such as constitutive expression or temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle response expression , and/or chemistry, and any combination thereof, as well as quantitative or qualitative indications. As used herein, a "gene expression pattern" is any transcription pattern of a DNA molecule operably linked into a transcribed RNA molecule. The transcribed RNA molecule may be translated to produce a protein molecule or may provide a regulatory or antisense or other RNA molecule, such as a double-stranded RNA (dsRNA), a transfer RNA (tRNA), a ribosomal RNA (rRNA). ), a microRNA (miRNA), and the like.

[0033] As used herein, the term "protein expression" is any pattern of translation of a transcribed RNA molecule into a protein molecule. Protein expression can be characterized by its temporal, spatial, developmental, or morphological qualities, as well as by quantitative or qualitative indications.

[0034] A promoter is useful as a regulatory element to modulate the expression of a DNA molecule that can be operationally linked transcription. As used herein, the term "promoter" generally refers to a DNA molecule that is involved in recognizing and binding RNA polymerase II and other proteins, such as transcription factors acting in trans, to initiate transcription. A promoter can originate from the 5' untranslated region (5' UTR) of a gene. Alternately, promoters can be synthetically produced or engineered DNA molecules. Promoters can also be chimeric. Chimeric promoters are produced through the fusion of two or more heterologous DNA molecules. Promoters useful in the practice of the present invention include SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, including fragments or variants thereof. In specific embodiments of the invention, these DNA molecules, and any variants or derivatives thereof as described herein, are also defined as comprising promoter activity, that is, they are capable of acting as a promoter in a host cell, such as in a plant. transgenic. In still other specific embodiments, a fragment may be defined as exhibiting the promoter activity possessed by the initial promoter molecule from which it is derived, or a fragment may comprise a "minimal promoter" that provides a basal level of transcription and is composed of a TATA box or equivalent DNA sequence for recognition and binding of the RNA polymerase II complex for transcription initiation.

[0035] In one embodiment, fragments of a promoter sequence disclosed herein are provided. Promoter fragments may comprise promoter activity as described above and may be useful alone or in combination with other promoters and promoter fragments, such as in the construction of chimeric promoters. In specific embodiments, fragments of a promoter are provided comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about of 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700 , at least about 750, at least about 800, at least about 900, or at least about 1000 contiguous nucleotides, or more, of a polynucleotide molecule having promoter activity as disclosed herein. Methods for producing such fragments of an initial promoter molecule are well known in the art.

[0036] Compositions derived from any of the promoters presented as SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19, as internal or 5' deletions, for example, can be produced using methods known in the art for improving or altering expression, including removing elements that have positive or negative effects on expression, duplicating elements that have positive or negative effects on expression, and/or duplicating or removing elements that have specific effects in the tissue or cell in expression. Compositions derived from any of the promoters presented as SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 3' deletion compounds in which the TATA box element or the equivalent sequence thereof and downstream sequence is removed can be used, for example, to prepare enhancer elements. Other deletions can be prepared to remove any elements that have positive or negative effects; tissue specific; cell specific; or time-specific (such as, among others, circadian rhythms) in expression. Any of the promoters set forth as SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19 and fragments or enhancers derived therefrom can be used to prepare chimeric transcriptional regulatory element compositions composed of any one of the promoters shown as SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 and the fragments or enhancers derived therefrom operably linked to other enhancers and promoters.

[0037] In accordance with the invention, a promoter or promoter fragment can be analyzed for the presence of known promoter elements, i.e., DNA sequence features such as a TATA box and other known transcription factor binding site motifs. . Identification of these known promoter elements can be used by one skilled in the art to design promoter variants having a similar expression pattern to the original promoter.

[0038] As used herein, the term "leader" refers to a DNA molecule from the 5' untranslated region (5' UTR) of a gene and generally defined as a segment of DNA between the transcription start site (TSS ) and the start site of the protein coding sequence. Alternately, leaders can be synthetically produced or engineered DNA elements. A leader can be used as a 5' regulatory element to modulate the expression of an operationally linked transcribed DNA molecule. Leader molecules can be used with a heterologous promoter or with their native promoter. Promoter molecules of the invention, therefore, can be operably linked to their native leader or can be operably linked to a heterologous leader. Useful leaders in the practice of the invention include SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 or fragments or variants thereof. In specific embodiments, these DNA sequences can be defined as being capable of acting as a leader in a host cell, including, for example, a transgenic plant cell. In one embodiment, these DNA sequences can be decoded as comprising leader activity.

[0039] The leader sequences (5' UTR) presented as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 may be composed of regulatory elements or may adopt secondary structures that may have an effect on the transcription or translation of an operably linked DNA molecule. The leader sequences presented as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 can be used in accordance with the invention to prepare chimeric regulatory elements that affect the transcription or translation of a molecule of operably linked DNA. Furthermore, the leader sequences presented as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 can be used to prepare chimeric leader sequences that affect the transcription or translation of a DNA molecule. operationally connected.

[0040] As used herein, the term "intron" refers to a DNA molecule that can be identified from a gene and can be defined generally as a region that leaves by recombination during messenger RNA (mRNA) processing before of the translation. Alternately, an intron can be synthetically produced or engineered DNA element. An intron may contain enhancer elements that affect the transcription of operably linked genes. An intron can be used as a regulatory element to modulate the expression of an operationally linked transcribed DNA molecule. A construct may comprise an intron, and the intron may or may not be heterologous to the DNA molecule that can be transcribed. Examples of introns in the art include the rice actin intron and the maize HSP70 intron.

[0041] In plants, the inclusion of some introns in constructs leads to greater accumulation of mRNA and protein compared to constructs lacking the intron. This effect is called "intron-mediated potentiation" (IME) of gene expression (Mascarenhas et al., Plant Mol. Biol. 15:913-920, 1990). Introns known to stimulate expression in plants have been identified in maize genes (e.g., tubA1, Adh1, Sh1, and Ubi1), in rice genes (e.g., tpi), and in the genes of dicotyledonous plants such as those of petunia (e.g. , rbcS), potato (e.g., st-ls1) and from Arabidopsis thaliana (e.g., ubq3 and pat1). Deletions or mutations within the recombination sites of an intron have been shown to reduce gene expression, indicating that processing may be necessary for IME. However, this processing itself is not necessary, since IME in dicotyledonous plants demonstrated point mutations within the recombination sites of the A. thaliana pat1 gene. Multiple uses of the same intron in a plant have been shown to have disadvantages. In these cases, it is necessary to have a collection of basic control elements for the construction of appropriate recombinant DNA elements.

[0042] As used herein, the term "3' transcription termination molecule", "3' untranslated region" or "3' UTR" herein refers to a DNA molecule that is used during transcription for the untranslated region. translated from the 3' portion of an mRNA molecule. The 3' untranslated region of an mRNA molecule can be generated by specific cleavage and 3' polyadenylation, also known as a polyA tail. A 3' UTR can be operably linked to and located downstream of a DNA molecule that can be transcribed and can include a polyadenylation signal and other regulatory signals capable of affecting transcription, mRNA processing, or gene expression. PolyA tails function in mRNA stability and translation initiation. Examples of 3' transcription termination molecules in the art are the 3' region of nopaline synthase; 3' region of wheat hsp17, 3' region of rubisco pea small subunit, 3' E6 region of cotton and coixin 3' UTR.

[0043] 3' UTRs typically find beneficial use for the recombinant expression of specific DNA molecules. A weak 3' UTR has the potential to generate readthrough, which can affect the expression of the DNA molecule located in neighboring expression cassettes. Appropriate control of transcription termination can prevent read-through into DNA sequences (e.g., other expression cassettes) located downstream and can still allow efficient recycling of RNA polymerase to improve gene expression. Efficient transcription termination (release of RNA polymerase II from DNA) is a prerequisite for transcription restart and thus directly affects the overall transcription level. After completion of transcription, the mature mRNA is released from the site of synthesis and the template is transported to the cytoplasm. Eukaryotic mRNAs are accumulated as poly(A) forms in vivo, making it difficult to detect transcription termination sites by conventional methods. Furthermore, the prediction of functional and efficient 3' UTRs by bioinformatics methods is difficult, as there are no conserved DNA sequences that allow easy prediction of an effective 3' UTR.

[0044] From a practical point of view, it is generally beneficial for a 3' UTR used in an expression cassette to have the following characteristics. The 3' UTR must be able to efficiently and effectively terminate transcription of the DNA molecule that can be transcribed and prevent the reading of the transcript in any neighboring DNA sequence, which can be comprised of another expression cassette, as in the case of multiple expression cassettes that reside on a transfer DNA (T-DNA), or the neighboring chromosomal DNA, into which the T-DNA has been inserted. The 3' UTR should not cause a reduction in the transcriptional activity conveyed by the promoter, leader, enhancers, and introns that are used to drive expression of the DNA molecule. In plant biotechnology, the 3' UTR is often used for initiation of amplification reactions of reverse transcribed RNA extracted from the transformed plant and used to: (1) evaluate the transcriptional activity or expression of the expression cassette once integrated into the plant chromosome ; (2) evaluate the copy number of insertions within the DNA plant; and (3) evaluate the zygosity of the resulting seed after reproduction. The 3' UTR is also used in amplification reactions of DNA extracted from the transformed plant to characterize the integrity of the inserted cassette.

[0045] As used herein, the term "enhancer" or "enhancer element" refers to a cis-acting regulatory element, also known as a cis element that confers an aspect of the overall pattern of expression, but is generally insufficient alone to drive transcription of an operably linked DNA sequence. Unlike promoters, enhancer elements generally do not include a transcription start site (TSS) or TATA box or equivalent DNA sequence. A promoter or promoter fragment may naturally comprise one or more enhancer elements that affect transcription of an operably linked DNA sequence. An enhancer element can also be fused to a promoter to produce a chimeric promoter cis element that confers an aspect of general modulation of gene expression.

[0046] Many promoter enhancer elements are believed to bind to DNA-binding proteins and/or affect DNA topology, producing local conformations that selectively allow or restrict RNA polymerase access to the DNA template or that facilitate the selective opening of the double helix at the transcription start site. An enhancer element may function to bind transcription factors that regulate transcription. Some enhancer elements bind to more than one transcription factor, and transcription factors can interact with different affinities with more than one enhancer domain. Enhancer elements can be identified by various techniques, including deletion analysis, i.e., deleting one or more nucleotides from the 5' end or internal to a promoter; analysis of DNA binding proteins using DNase I footprinting, methylation interference, electrophoretic mobility-shift assays, in vivo genomic footprinting by ligation-mediated polymerase chain reaction (PCR) and other conventional assays; or by DNA sequence similarity analysis using known cis element motifs or enhancer elements as a target sequence or target motif with conventional DNA sequence comparison methods such as BLAST. The fine structure of an enhancer domain can further be studied by mutagenesis (or replacement) of one or more nucleotides or by other conventional methods known in the art. Enhancer elements can be obtained by chemical synthesis or by isolation of regulatory elements that include these elements, and can be synthesized with additional flanking nucleotides that contain useful restriction enzyme sites to facilitate subsequence manipulation. Thus, the design, construction, and use of enhancer elements, in accordance with the methods disclosed herein for modulating the expression of the operably linked transcribeable DNA molecule, are included by the invention.

[0047] As used herein, the term "chimeric" refers to a single DNA molecule produced by the fusion of a first DNA molecule to a second DNA molecule, where neither the first nor the second DNA molecule would normally be found. in this configuration, that is, fused with each other. The chimeric DNA molecule is therefore a new DNA molecule not normally contained in nature. As used herein, the term "chimeric promoter" refers to a promoter produced through such manipulation of DNA molecules. A chimeric promoter can combine two or more DNA fragments, for example, fusing a promoter to an enhancer element. Thus, the design, construction, and use of chimeric promoters, in accordance with the methods disclosed herein for modulating the expression of operably linked transcribeable polynucleotide molecules, are included by the invention.

[0048] As used herein, the term "variant" refers to a second DNA molecule, such as a regulatory element, that is similar in composition to, but not identical to, a first DNA molecule, and in which the second DNA molecule is DNA still maintains the general functionality, that is, the same or similar expression pattern, as the first DNA molecule. A variant may be a shortened or truncated version of the first DNA molecule and/or an altered version of the DNA sequence of the first DNA molecule, such as one with different restriction enzyme sites and/or internal deletions, substitutions and/or insertions . Regulatory element "variants" also include variants arising from mutations that occur naturally in the transformation of bacterial and plant cells. In the invention, a DNA sequence provided as SEQ ID NOs: 1-20 can be used to create variants that are similar in composition, but not identical to the DNA sequence of the original regulatory element, while still maintaining general functionality, i.e., the same or similar expression pattern of the original regulatory element. The production of these variants of the invention is well within the skill of the art in light of the disclosure and is included within the scope of the invention.

[0049] Chimeric regulatory elements can be designed to comprise various constituent elements that can be operably linked by various methods known in the art, such as restriction enzyme digestion and ligation, ligation-independent cloning, modular assembly of PCR products during amplification, or direct chemical synthesis of the regulatory element, as well as other methods known in the art. The resulting various chimeric regulatory elements may be comprised of the same, or variants of, the same constituent elements, but differ in the DNA sequence or DNA sequences that comprise the linker DNA sequence or sequences that allow the constituent elements to be operably linked. In the invention, a DNA sequence provided as SEQ ID NOs: 1-20 may provide a regulatory element reference sequence, wherein the constituent elements comprising the reference sequence may be joined by methods known in the art and may comprise substitutions, deletions and/or insertions of one or more nucleotides or mutations that occur naturally in the transformation of bacterial and plant cells.

[0050] The effectiveness of the modifications, duplications or deletions described herein in the desired expression aspects of a particular transgene can be tested empirically in stable and transient plant assays, such as those described in the working examples herein, in order to validate the results, which can vary depending on the changes made and the purpose of the change in the initial DNA molecule.

Constructs

[0051] As used herein, the term "construct" means any recombinant DNA molecule such as a plasmid, cosmid, virus, phage or linear or circular DNA or RNA molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a DNA molecule wherein at least one DNA molecule has been linked to the other DNA molecule in a functionally operative manner, that is, operably linked. As used herein, the term "vector" means any construct that can be used for transformation purposes, that is, the introduction of heterologous DNA or RNA into a host cell. A construct typically includes one or more expression cassettes. As used herein, an "expression cassette" refers to a DNA molecule comprising at least one transcribeable DNA molecule operably linked to one or more regulatory elements, generally at least one promoter and one 3' UTR.

[0052] As used herein, the term "operationally linked" refers to a first DNA molecule joined to a second DNA molecule, wherein the first and second DNA molecules are then arranged in which the first DNA molecule affects the function of the second DNA molecule. The two DNA molecules may or may not be part of a single contiguous DNA molecule and may or may not be adjacent. For example, a promoter is operably linked to a transcribed DNA molecule if the promoter modulates the transcription of the transcribed DNA molecule of interest in a cell. A leader, for example, is operationally linked to the DNA sequence when it is capable of affecting the transcription or translation of the DNA sequence.

[0053] The constructs of the invention can be provided, in one embodiment, as double tumor-inducing plasmid (Ti) border constructs that have right border (RB or AGRtu.RB) and left border (LB or AGRtu. LB) of the Ti plasmid isolated from Agrobacterium tumefaciens, comprising a T-DNA, which, together with transfer molecules provided by A. tumefaciens cells, allow integration of the T-DNA into the genome in a plant cell (see e.g. , US Patent 6,603,061). The constructs may also contain the plasmid backbone DNA segments that provide replication and antibiotic selection function in bacterial cells, e.g., an Escherichia coli origin of replication such as ori322, a broad host range replication origin such as oriV or oriRi, and a coding region for a selectable marker such as Spec/Strp encoding aminoglycoside adenyltransferase Tn7 (aadA) conferring resistance to spectinomycin or streptomycin, or a gentamicin selectable marker gene (Gm, Gent). For plant transformation, the host bacterial strain is often A. tumefaciens ABI, C58, or LBA4404; however, other strains known to those skilled in the art of plant transformation may work in the invention.

[0054] Methods are known in the art of assembling and introducing constructs into a cell so that the DNA molecule that can be transcribed is transcribed into a functional mRNA molecule that is translated and expressed as a protein. For practicing the invention, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art. Typical vectors useful for expressing nucleic acids in higher plants are well known in the art and include vectors derived from the Agrobacterium tumefaciens Ti plasmid and the transfer control vector pCaMVCN.

[0055] Various regulatory elements can be included in a construct, including any of those provided here. Any of these regulatory elements may be provided in combination with other regulatory elements. These combinations can be engineered or modified to produce desirable regulatory characteristics. In one embodiment, constructs of the invention comprise at least one regulatory element operably linked to a transcribeable DNA molecule operably linked to a 3' UTR.

[0056] Constructs of the invention may include any promoter or leader provided herein or known in the art. For example, a promoter of the invention can be operably linked to a heterologous 5' untranslated leader such as a derivative of a heat shock protein gene. Alternatively, a leader of the invention can be operably linked to a heterologous promoter such as Cauliflower Mosaic Virus 35S transcription promoter.

[0057] Expression cassettes may also include a transit peptide coding sequence that encodes a peptide that is useful for subcellular targeting of an operably linked protein, particularly to a chloroplast, leucoplast or other plastid organelle; mitochondria; peroxisome; vacuole; or an extracellular location. Many chloroplast-localized proteins are expressed from nuclear genes as precursors and are targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, among others, those associated with the small subunit (SSU) of ribulose-1,5,-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, the light harvesting complex protein I and II, thioredoxin F and enolpyruvyl shikimate phosphate synthase (EPSPS). Chloroplast transit peptides are described, for example, in US Patent 7,193,133. It has been demonstrated that non-chloroplast proteins can be targeted to the chloroplast by expression of a heterologous CTP operably linked to the transgene encoding a non-chloroplast protein.

DNA molecules that can be transcribed

[0058] As used herein, the term "transcriptionable DNA molecule" refers to any DNA molecule capable of being transcribed into an RNA molecule, including, but not limited to, those that have protein coding sequences and those producing RNA molecules having sequences useful for gene suppression. The type of DNA molecule may include, among others, a DNA molecule from the same plant, a DNA molecule from another plant, a DNA molecule from a different organism, or a synthetic DNA molecule, such as a DNA molecule containing an antisense message from a gene, or a DNA molecule that encodes an artificial, synthetic, or otherwise modified version of a transgene. Exemplary transcribeable DNA molecules for incorporation into constructs of the invention include, for example, DNA molecules or genes from a species other than the species in which the DNA molecule is incorporated or genes that originate from, or are present in, , the same species, but are incorporated into recipient cells by genetic engineering methods rather than classical breeding techniques.

[0059] A "transgene" refers to a DNA molecule that can be transcribed heterologously to a host cell at least with respect to its location in the genome of the host cell and/or a DNA molecule that can be transcribed artificially incorporated in the genome of a host cell in the current or any previous generation of the cell.

[0060] A regulatory element, such as a promoter of the invention, can be operably linked to a transcribeable DNA molecule that is heterologous to the regulatory element. As used herein, the term "heterologous" refers to the combination of two or more DNA molecules when such a combination is not normally found in nature. For example, the two DNA molecules may be derived from different species and/or the two DNA molecules may be derived from different genes, e.g., different genes from the same species or the same genes from different species. A regulatory element is thus heterologous to a DNA molecule that can be transcribed operationally linked if this combination is not normally found in nature, i.e., the DNA molecule that can be transcribed does not occur naturally operationally linked to the regulatory element.

[0061] The DNA molecule that can be transcribed generally can be any DNA molecule so that expression of a transcript is desired. This expression of a transcript can result in translation of the resulting mRNA molecule and therefore expression of the protein. Alternatively, for example, a DNA molecule that can be transcribed could be engineered to ultimately cause decreased expression of a specific gene or protein. In one embodiment, this can be done using a transcribeable DNA molecule that is oriented in the antisense direction. One skilled in the art is familiar with the use of this antisense technology. Any gene can be downregulated in this manner, and, in one embodiment, a DNA molecule that can be transcribed can be designed to suppress a specific gene through the expression of a dsRNA, siRNA, or miRNA molecule.

[0062] Thus, one embodiment of the invention is a recombinant DNA molecule comprising a regulatory element of the invention, such as those provided as SEQ ID NOs: 1-20, operably linked to a DNA molecule that can be heterologously transcribed in order to modulate the transcription of the DNA molecule that can be transcribed at a desired level or in a desired pattern when the construct is integrated into the genome of a transgenic plant cell. In one embodiment, the transcribeable DNA molecule comprises a protein coding region of a gene, and in another embodiment, the transcribeable DNA molecule comprises an antisense region of a gene.

Genes of Agricultural Interest

[0063] A DNA molecule that can be transcribed can be a gene of agronomic interest. As used herein, the term "gene of agronomic interest" refers to a transcribed DNA molecule that, when expressed in a particular plant tissue, cell, or cell type, confers a desirable characteristic. The product of a gene of agronomic interest may act within the plant to cause an effect on morphology, physiology, growth, development, yield, grain composition, nutritional profile, disease or pest resistance and/or environmental or chemical tolerance of the plant or may act as a pesticidal agent in the diet of a pest that feeds on the plant. In one embodiment of the invention, a regulatory element of the invention is incorporated into a construct such that the regulatory element is operably linked to a transcribeable DNA molecule that is a gene of agronomic interest. In a transgenic plant containing this construct, expression of the gene of agronomic interest can confer a beneficial agronomic trait. A beneficial agronomic trait may include, for example, but not limited to, herbicide tolerance, insect control, modified yield, disease resistance, pathogen resistance, modified plant growth and development, modified starch content, modified oil content, modified fatty acid content, modified protein content, modified fruit ripening, enhanced human and animal nutrition, biopolymer productions, resistance to environmental stress, pharmaceutical peptides, improved processing qualities, improved flavor, hybrid seed production utility, improved production fiber, and desirable biofuel production.

[0064] Examples of genes of agronomic interest known in the art include those for herbicide resistance (US Patents 6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633 .435; and 5,463,175), increased yield (Usre38, 446; 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211; 6,235,971; 6,222,098; and 5,716,837); 93. 293; ; 6,248,536; 6,221,649; 42,664; 5,942,658, 5,880. 275; 5,763,245; and 5,763,241), resistance to fungal diseases (US Patents 6,653,280; 6,573,361; 6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,12 1,436 ; 6,608,241; 6,015,940; 6,013,864; 5,850,023; and 5,304,730), nematode resistance (US Patent 6,228,992), bacterial disease resistance (US Patent 5,516,671), plant growth and development (US Patents 6,723,897 and 6,518,488), starch production ( US Patents 6,538,181; 6,538,179; 6,538,178; 6,476,295), high oil production (Patents US 6,495,739; 5,608,149; 6,483,008; and 6,476,295), modified fatty acid content (US Patents 6,828,849; 6,596,53 8 6,589,767; 6,537,750; 6,489,461; 6,723,837; 6,653,530; 6,541,259; and 6,171,640), biopolymers (US Patents USRE37,543; 6,228,623; and 5,958,745; US Patent 6,072,103), pharmaceutical peptides and secretable peptides (US Patents 6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processing characteristics (US Patent 6,476,295), improved digestibility (US Patent 6,531,648), low raffinose (US Patent 6,166,292), industrial enzyme production (US Patent 5,543,576), flavor improved (US Patent 6,011,199), nitrogen fixation (US Patent 5,229,114), hybrid seed production (US Patent 5,689,041), fiber production (US Patents 6,576,818; 6,271,443; 5,981,834; and 5,869,720) and production of biofuels (US Patent 5,998,700).

[0065] Alternatively, a gene of agronomic interest may affect the characteristics or phenotypes of the aforementioned plant by encoding an RNA molecule that causes modulation of gene expression of an endogenous gene, for example, by antisense (see, for example, US Patent 5,107,065); Inhibitory RNA ("RNAi," including modulation of gene expression by mechanisms mediated by miRNA, siRNA, trans-acting siRNA, and phased sRNA, for example, as described in published applications US 2006/0200878 and US 2008/0066206 and in application US patent 11/974,469); or mechanisms mediated by cosuppression. The RNA could also be a catalytic RNA molecule (e.g., a ribozyme or a riboswitch; see, e.g., US 2006/0200878) designed to cleave a desired endogenous mRNA product. Methods are known in the art for constructing and introducing constructs into a cell so that the DNA molecule that can be transcribed is transcribed into a molecule that is capable of causing suppression of the gene.

[0066] Expression of a DNA molecule that can be transcribed in a plant cell can also be used to suppress plant pests feeding on the plant cell, for example, compositions isolated from coleoteric pests and compositions isolated from nematode pests . Plant pests include, but are not limited to, arthropod pests, nematode pests, and fungal or microbial pests.

Selectable Markers

[0067] Selectable marker transgenes can also be used with the regulatory elements of the invention. As used herein, the term "selectable marker transgene" refers to any transcribed DNA molecule whose expression in a transgenic plant, tissue or cell, or lack thereof, can be screened for or scored in some way. Selectable marker genes, and their associated selection and screening techniques, for use in the practice of the invention are known and include, among others, DNA molecules that can be transcribed encoding β-glucuronidase (GUS), green fluorescent protein (GFP), proteins that confer antibiotic resistance and proteins that confer herbicide tolerance.

β-Glucuronidase

[0068] The β-glucuronidase (GUS) gene isolated from Escherichia coli K-12 is one of the genes reported to be most widely used in the area of plant biotechnology. The E. coli GUS gene, uidA, is part of the GUS operon on the bacterial chromosome. It is induced by a wide variety of β-D-glucuronides. The GUS enzyme is an exohydrolase that catalyzes the hydrolysis of β-D-glucuronide to D-glucuronic acid and the aglycone. E. coli lives in the digestive tract of vertebrates, including humans. Vertebrates use the glucuronidation pathway to decontaminate xenobiotics and endogenous waste compounds such as steroids, aliphatic alcohols, phenol, carboxylic acids, sugars, and several other metabolites. Glucuronidation involves conjugation with D-glucuronic acid. This occurs mainly in the liver, but also occurs in other tissues and organs such as the kidneys, adrenal glands, and the digestive system. Glucuronic acid can be used by E. coli as the main source of carbon and energy. The E. coli GUS protein therefore provides a means by which the bacteria can degrade the products of the glucuronidation pathway in the vertebrate digestive tract to produce glucuronic acid as a source of carbon and energy. The aglycones that are also released by the GUS enzyme are generally not degraded by the bacteria, but used as a transport service for D-glucuronic acid (Gilissen et al., Transgenic Research, 7: 157-163, 1998).

[0069] The use of the E. coli β-glucuronidase gene as a reporter was first described by Jefferson et al. (Proc. Natl. Acad. Sci., 83: 8447-8451, 1986) and was used in the same way as the first described since its introduction. The GUS gene is used to monitor plant gene expression and is often employed to characterize promoters or other expression elements. However, some plant promoters express at very low levels and may not be detected using a GUS-based assay. These lower expression promoters may be valuable for developing transgenic crops with desirable phenotypes such as improved yield.

[0070] From the beginning in the development of transgenic plant crops, promoters that provided high constitutive expression were most desired. These highly constitutive promoters, derived from plant viral genomes such as cauliflower mosaic virus and Figwort mosaic virus, were used to drive transgenes that conferred herbicide tolerance or insect resistance. As the field of plant biotechnology increases in complexity, new transgenic traits are being developed that require more specific patterns of expression or lower levels of expression. Overexpression or expression in the wrong tissues of the plant can lead to unwanted effects in the transformed plant. For example, ectopic expression (expression of a gene in an abnormal location in an organism) of enzyme genes in plants can result in a reduction in the desired end product due to a shortage of precursor at the branch point in a metabolic pathway (Iwase et al. al., Plant Biotech. 26: 29-38, 2009).

[0071] Because transcription factors (TFs) naturally act as master regulators of cellular processes, they are expected to be excellent candidates for modifying complex traits in crop plants, and TF-based technologies are likely to be a prominent part of the next generation of successful biotechnology crops. TF technologies often require optimization to reduce unwanted side effects such as growth retardation or to enhance the desired trait to the level at which it is of commercial value. Optimization is often approached by modifying TF transgene expression. Tissue-specific, developmental, or inducible promoters, rather than the usual constitutive promoters, can be used to limit transgene expression to appropriate tissues or environmental conditions (Century et al., Plant Physiology, 147: 20-29, 2008 ).

[0072] Due in part to these developments, there is a need for a more sensitive assay for expression element characterization to identify expression elements that provide a desired level and pattern of expression. The present invention provides an improved codon-reengineered GUS coding sequence that, when operably linked to a promoter, expresses better than the native E. coli GUS coding sequence commonly used in the art. This improved codon-redesigned GUS coding sequence can be used to provide greater assay sensitivity, both quantitatively and qualitatively, and allows for characterization of promoters and other expression elements that might otherwise not be possible with the E coding sequence. native . coli GUS. The improved codon-redesigned GUS coding sequence can be used to characterize expression elements in monocot and dicot plants. Monocotyledonous plants useful in the practice of the invention include, among others, Corn (Zea mays), Rice (Oryza sativa), Wheat (Triticum), Barley (Hordeum vulgare), Sorghum (Sorghum spp.), Millet, Millet (Pennisetum glaucum), Giant crow's foot grass (Eleusine coracana), Millet (Panicum miliaceum), Foxtail millet (Setaria italica), Oats (Avena sativa), Triticale, Rye (Secale cereale), Fonio (Digitaria), Onion ( Allium spp.), Pineapple (Ananas spp.), Grass, Sugarcane (Saccharum spp.), Palm (Arecaceae), Bamboo (Bambuseae), Banana (Musaceae), Ginger family (Zingiberaceae), Lily (Lilium ), Narcissus (Narcissus), Iris (Iris), Amaryllis, Orchid (Orchidaceae), Cannas, Hyacinth (Hyacinthoides) and Tulips (Tulipa). Dicotyledonous plants useful in the practice of the invention include, among others, Soybean (Glycine max), Wild soybean (Glycine Soya), Cotton (Gossypium), Tomato (Solanum lycopersicum), Pepper (Piper), Pumpkin (Cucurbita), Pea (Pisum sativum) , Alfalfa (Medicago sativa), Medicago truncatula, Beans (Phaseolus), Chickpea (Cicer arietinum), Sunflower (Helianthus annuus), Potato (Solanum tuberosum), Peanut (Arachis hypogaea), Quinoa, Buckwheat (Fagopyrum esculentum ), Carob (onia siliqua), Beetroot (Beta vulgaris), Spinach (Spinacia oleracea) and Cucumber (Cucumis sativus).

Cell Transformation

[0073] The invention is also directed to a method of producing transformed cells and plants that comprise one or more regulatory elements operably linked to a DNA molecule that can be transcribed.

[0074] The term "transformation" refers to the introduction of a DNA molecule into a recipient host. As used herein, the term "host" refers to bacteria, fungi or plants, including any cells, tissues, organs or progeny of bacteria, fungi or plants. Plant tissues and cells of particular interest include protoplasts, callus, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.

[0075] As used here, the term "transformed" refers to a cell, tissue, organ or organism into which a foreign DNA molecule, such as a construct, has been introduced. The introduced DNA molecule is integrated into the genomic DNA of the recipient cell, tissue, organ or organism such that the introduced DNA molecule is inherited by subsequent progeny. A "transgenic" or "transformed" cell or organism may also include progeny of the cell or organism and progeny produced from a breeding program employing a transgenic organism as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a molecule of foreign DNA. The introduced DNA molecule can also be introduced into the recipient cell in such a way that the introduced DNA molecule is not inherited by subsequent progeny. The term "transgenic" refers to a bacteria, fungus or plant that contains one or more heterologous DNA molecules.

[0076] There are several methods known to those skilled in the art for introducing DNA molecules into plant cells. The process generally comprises the steps of selecting a suitable host cell, transforming the host cell with a vector, and obtaining the transformed host cell. Methods and materials for transforming plant cells by introducing a construct into a plant genome in the practice of this invention may include any of the known and demonstrated methods. Suitable methods include, but are not limited to, bacterial infection (e.g., Agrobacterium), binary BAC vectors, direct DNA release (e.g., by PEG-mediated transformation, desiccation/inhibition-mediated DNA uptake, electroporation, agitation with carbide fibers silicon, and acceleration of coated DNA particles), among others.

[0077] The host cells can be any cells or organisms, such as a plant cell, algae cell, algae, fungal cell, fungi, bacterial cell or insect cell. In specific embodiments, the host cells and transformed cells may include cultured plant cells.

[0078] A transgenic plant can subsequently be regenerated from a transgenic plant cell of the invention. Using conventional breeding techniques or self-pollination, seeds can be produced from this transgenic plant. These seeds, and the progeny plant resulting from that seed, will contain the recombinant DNA molecule of the invention and will therefore be transgenic.

[0079] Transgenic plants of the invention can be autogamous to provide seeds for homozygous transgenic plants of the invention (homozygous for the recombinant DNA molecule) or crossed with non-transgenic plants or different transgenic plants to provide seeds for heterozygous transgenic plants of the invention (heterozygous for the recombinant DNA molecule). Both homozygous and heterozygous transgenic plants are referred to here as "progeny plants". Progeny plants are transgenic plants from the progeny of the original transgenic plant that contain the recombinant DNA molecule of the invention. Seeds produced using a transgenic plant of the invention can be harvested and used to grow generations of transgenic plants, i.e., progeny plants of the invention, comprising the construct of this invention and expressing a gene of agronomic interest. Descriptions of breeding methods that are commonly used for different crops can be found in one of several reference books, see, for example, Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U. of CA, Davis, CA , 50-98 (1960); Simmonds, Principles of Crop Improvement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen, Plant breeding Perspectives, Wageningen (ed), Center for Agricultural Publishing and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses, 2nd Edition, Monograph, 16:249 (1987); Fehr, Principles of Variety Development, Theory and Technique, (Vol. 1) and Crop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987).

[0080] Transformed plants can be analyzed for the presence of the gene or genes of interest and the level of expression and/or profile conferred by the regulatory elements of the invention. Those skilled in the art are aware of the numerous methods available for analyzing transformed plants. For example, methods for plant analysis include, but are not limited to, Southern blots or northern blots, PCR-based approaches, biochemical analyses, phenotypic screening methods, field evaluations, and immunodiagnostic assays. Expression of a transcribed DNA molecule can be measured using TaqMan® reagents and methods (Applied Biosystems, Foster City, CA) as described by the manufacturer and PCR cycle number determined using TaqMan® Testing Matrix. Alternatively, Invader® reagents and methods (Third Wave Technologies, Madison, WI) as described by the manufacturer can be used to evaluate transgene expression.

[0081] The invention also provides parts of a plant of the invention. Plant parts include, among others, leaves, stems, roots, tubers, seeds, endosperm, ovule and pollen. The plant parts of the invention may be viable, non-viable, regenerable, and/or non-regenerable. The invention also includes and provides transformed plant cells comprising a DNA molecule of the invention. The transformed or transgenic plant cells of the invention include regenerable and/or non-regenerable plant cells.

[0082] The invention can be more easily understood by reference to the following examples, which are provided by way of illustration, and are not intended to be limiting of the invention unless specified. It should be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have found to work well in the practice of the invention. However, those skilled in the art should, in light of the present disclosure, appreciate that many changes can be made to these specific embodiments that are disclosed and still obtain a similar result without departing from the spirit and scope of the invention, therefore the entire matter established or shown in the attached drawings should be interpreted as illustrative and not in a limiting sense.

EXAMPLES Example 1 Identification and Cloning of Regulatory Elements

[0083] New RCc3 promoters and leaders were identified and cloned from the genomic DNA of the monocot species Coix (Coix lacryma-jobi), Hairy crabgrass (Digitaria sanguinalis (L.) Scop.), Maiden grass (Miscanthus sinensis f. gracillimus) , Gamma grass (Tripsacum dactyloides) and Sugarcane (Saccharum officinarum). The RCc3 protein belongs to the prolamin superfamily, which derives its name from the proline- and glutamine-rich storage proteins soluble in grain alcohol. The prolamin superfamily (also called the seed storage protease inhibitor/lipid transfer protein/2S albumin family; Pfam ID: PF00234) represents one of the most widespread protein superfamilies in the plant genome. Members of the prolamin superfamily are abundant in the fruits, nuts, seeds, and vegetables of a variety of plants. They are known to exhibit diverse function, including seed storage and protection, lipid binding or transfer, and enzyme inhibition. Lipid transfer proteins (LTPs) belong to the prolamin superfamily and are expressed in a variety of plant tissues. The rice RCc3 protein is an LTP that is expressed in rice roots, although not all LTPS proteins are root specific.

[0084] DNA amplification primers (presented as SEQ ID NOs: 25 to 28) were designed using the coding sequences of the twenty-four (24) LTP proteins from Zea mays, Oryza sativa, Sorghum bicolor and Brachypoium distachyon. Amplification primers were used with GenomeWalkerTM libraries (Clontech Laboratories, Inc, Mountain View, CA) constructed following the manufacturer's protocol to clone the 5' region of the corresponding genomic DNA sequence.

[0085] Bioinformatics analysis was performed to identify regulatory elements within the amplified DNA. Using the results of this analysis, regulatory elements were defined within the DNA sequences and primers were designed to amplify the regulatory elements. The corresponding DNA molecule for each regulatory element was amplified using standard polymerase chain reaction (PCR) conditions with primers containing unique restriction enzyme sites and genomic DNA isolated from C. lacryma-jobi, .D. sanguinalis (L.) Scop., M. sinensis f. gracillimus, T. dactyloides, and S. officinarum. The resulting DNA fragments were ligated into vectors and sequenced.

[0086] The DNA sequences of the identified RCc3 promoters and leaders are listed in Table 1. Promoter sequences are provided here as SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. Leader sequences are provided here as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20. Table 1. RCc3 promoters and leaders isolated from various grass species.

Example 2 Analysis of Regulatory Elements Driving GUS in Transgenic Corn

[0087] Corn plants were transformed with vectors, specifically binary plasmid constructs, comprising an RCc3 promoter operably linked to its native RCc3 leader driving expression of the β-glucuronidase (GUS) transgene. The resulting transformed plants were analyzed for GUS protein expression.

[0088] The vectors used in these experiments were constructed using cloning methods known in the art. The resulting vectors comprised a region of the right edge of A. tumefaciens; a first transgene expression cassette to evaluate the RCc3 promoter/leader sequence operably linked to a codon-redesigned GUS coding sequence that possessed a processable intron GOI-Ec.uidA+St.LS1.nno:3 (SEQ ID NO: 29) operably linked 5' to the 3' UTR of foxtail millet gene S-adenosylmethionine synthetase 1 (T-SETit.Ams1-1:1:1, SEQ ID NO:159); a second transgene expression cassette used for the selection of transformed plant cells that confer resistance to the herbicide glyphosate (driven by the rice actin 1 promoter); and a region on the left edge of A. tumefaciens. The resulting plasmids were used to transform corn plants using methods known in the art. GUS expression conferred by the novel RCc3 promoters and leaders was compared to expression driven by the homologous RCc3 promoters and leaders from foxtail millet and rice. Table 2 provides the plasmid constructs, RCc3 promoter and leader sequences, and SEQ ID NOs. Table 2. Binary plant transformation plasmids and associated RCc3 promoter/leader sequences.

[0089] In certain cases, plants were transformed using Agrobacterium-mediated transformation methods known in the art and as described in U.S. Patent Application Publication 2009/0138985.

[0090] GUS histochemistry analyzes were used for qualitative expression analysis of transformed plants. Whole tissue sections were incubated with GUS X-Glyc (5-bromo-4-chloro-3-indolyl-b-glucuronide) staining solution (1 mg/ml) for an appropriate period of time, washed, and visually inspected. for the blue color. GUS activity was qualitatively determined by direct visual inspection or inspection under a microscope using selected plant tissues and organs. R0 plants are inspected for expression in roots and leaves, as well as anther, silk, and developing seed and embryo, 21 days after pollination (21 DAP).

[0091] For quantitative analysis, total protein was extracted from selected tissues of transformed corn plants. One microgram of total protein was used with the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) in a total reaction volume of 50 microliters. The reaction product, 4-methylumbelliferone (4-MU), is maximally fluorescent at high pH, where the hydroxyl group is ionized. The addition of a basic sodium carbonate solution simultaneously stops the assay and adjusts the pH to quantify the fluorescent product. Fluorescence was measured with excitation at 365 nm, emission at 445 nm, using a Fluoromax-3 (Horiba; Kyoto, Japan) with Micromax Reader, with a slit width defined at 2 nm excitation and 3 nm emission. Mean expression values were given as pmol 4MU/μg protein/hour.

[0092] The average GUS R0 expression observed for each transformation was recorded and a mean expression level and standard error determined based on measurements taken from samples derived from various transformation events.

Example 3 Enhancers Derived from Regulatory Elements

[0093] Enhancers are derived from the promoter elements provided herein, such as those presented as SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19. These enhancer elements can be composed of a or more cis regulatory elements that, when operably linked 5' or 3' to a promoter element, or operably linked 5' or 3' to additional enhancer elements that are operably linked to a promoter, can potentiate or modulate the expression of a molecule of DNA that can be transcribed, or provides the expression of a DNA molecule that can be transcribed in a specific cell type or plant organ, or at a particular point of time in development or circadian rhythm. Enhancers are prepared by removing the TATA box or functionally similar elements and any DNA sequence downstream of the promoters that allow transcription to be initiated from the promoters provided herein as described above, including fragments thereof, wherein the TATA box or functionally similar elements and DNA sequence downstream of the TATA box are removed.

[0094] Enhancer elements may be derived from the promoter elements provided herein and cloned using methods known in the art to be operably linked 5' or 3' to a promoter element, or operably linked 5' or 3' to additional enhancer elements that are operably linked to a promoter. Enhancer elements can be cloned to be operably linked 5' or 3' to a promoter element derived from an organism of a different genus, or operably linked 5' or 3' to additional enhancer elements derived from organisms of other genera or organism of the same genus are operationally linked to a promoter derived from organisms of the same or different genera, resulting in a chimeric regulatory element. A GUS expression vector can be constructed using methods known in the art similar to the constructs described previously. Examples where the resulting plant expression vectors contain a right border region of A. tumefaciens; a first transgene cassette for testing a regulatory or chimeric regulatory element comprised of a regulatory or chimeric regulatory element operably linked to an intron derived from the Z. mays heat shock protein HSP70 (I-Zm.DnaK-1:1:1, SEQ ID NO: 38) or any of the introns presented here or any other intron, operably linked to a coding sequence for GUS that has a processable intron (GUS-2 SEQ ID NO: 32) or no intron (CR-Ec. uidA-1:1:4 (GUS.nat), SEQ ID NO: 31) operably linked to the Nopaline synthase 3' UTR of A. tumefaciens (T-AGRtu.nos-1:1:13, SEQ ID NO: 39) or the 3' UTR of the rice lipid transfer protein gene (T-Os.LTP-1:1:1, SEQ ID NO: 40); a second transgene selection cassette used for selecting transformed plant cells that confer resistance to the herbicide glyphosate (driven by the rice Actin 1 promoter), or, alternatively, the antibiotic kanamycin (driven by the rice Actin 1 promoter); and a region on the left edge of A. tumefaciens. The resulting plasmids can be used to transform corn plants or plants of another genus by the methods described above or by other methods known in the art. Alternatively, protoplast cells derived from corn or plants of another genus are transformed using methods known in the art to perform transient assays.

[0095] GUS expression driven by regulatory elements comprising one or more enhancers can be evaluated in stable or transient plant assays to determine the effects of the enhancer element on the expression of a transgene. Modifications to one or more enhancer elements or duplication of one or more enhancer elements can be performed based on empirical experimentation and the resulting gene expression regulation that is observed using each regulatory element composition. Changing the relative position of one or more enhancers in the resulting regulatory element or chimeric regulator can affect the transcriptional activity or specificity of the regulatory element or chimeric regulator and is determined empirically to identify the best enhancers for the desired transgene expression profile within the plant. of corn or plants of other genera.

Example 4 Higher Sensitivity Assay with a Codon Redesigned β-glucuronidase (GUS)

[0096] Plant promoters often express at levels that are below the normal detection limit of many quantitative assays, however, their expression characteristics can be very valuable for the expression of certain transgenes. In earlier plant biotechnology, promoters that drove highly constitutive expression were desirable and were used to drive transcribeable DNA molecules that produced a specific phenotype that necessitated high constitutive expression, such as herbicide tolerance or insect resistance. These highly constitutive promoters were often derived from plant virus genomes rather than plant genomes, for example the 35S promoters derived from cauliflower mosaic virus and Figwort mosaic virus. Notably, in certain cases, highly constitutive expression of certain transcribeable DNA molecules can lead to negative consequences such as transgene silencing, inactive phenotypes, or yield entrainment. For example, high expression of the GUS gene in transgenic sugarcane plants using two different sugarcane-derived ubiquitin promoters as well as a maize ubiquitin promoter resulted in post-transcriptional gene silencing of the GUS gene. (Wei et al., J. Plant Physiol. 160: 12411251, 2003).

[0097] Furthermore, recently, there is demand for promoters that demonstrate specific patterns of expression or express more highly in specific plant tissues. For example, ectopic expression of enzyme genes in plants can result in a reduction in the desired end product due to a shortage of precursor at the branch point in a metabolic pathway (Iwase et al., Plant Biotech. 26:29-38, 2009). In these cases, it is advisable to use a promoter that expresses the operationally linked DNA molecule that can be transcribed in the correct tissue or cell types, or in a particular window of development. Plant genome-derived promoters can often demonstrate desirable tissue, cell, and developmental expression characteristics. Due to the lower expression levels of these plant promoters, expression assays often require the use of enhancers to boost the expression level to allow detection in a quantitative assay. However, the use of these enhancers often alters the overall expression pattern of the plant promoter.

[0098] Improving the expression of the reporter gene used in the assay eliminates the need for potentiation of the plant-derived promoter and therefore provides a more accurate assessment of the expression pattern conferred by a promoter. This Example demonstrates the use of a codon-reengineered GUS coding sequence to improve quantitative assay sensitivity characterizing several different EXPs composed of a promoter sequence, operably linked 5' to a leader sequence, operably linked 5' to a leader sequence. intron.

[0099] Corn plants were transformed with plant expression vectors containing EXP sequences driving expression of the native Escherichia coli β-glucuronidase GUS transgene or codon-redesigned β-glucuronidase transgene (GUS.nno), and the resulting plants were analyzed for GUS protein expression. The EXP and GUS coding sequences were cloned into binary plasmid constructs using methods known in the art.

[00100] The resulting plant expression constructs contain a right border region from A. tumefaciens; a first transgene cassette demonstrating assay sensitivity of the two GUS coding sequences, composed of an EXP operably linked to the native E. coli GUS coding sequence (CR-Ec.uidA-1:1:4 (GUS.nat) , SEQ ID NO: 31) or a codon-redesigned GUS coding sequence (CR- Ec.uidA_nno-1:1:1 (GUS.nno), SEQ ID NO: 30) operably linked 5' to the 3' UTR of rice lipid transfer protein gene (T-Os.LTP-1:1:1 SEQ ID NO: 40); a second transgene selection cassette used for the selection of transformed plant cells that confer resistance to the herbicide glyphosate (driven by the rice Actin 1 promoter); and a left border region of A. tumefaciens. Figs 1a to 1c show an alignment between the native GUS coding sequence (CR-Ec.uidA-1:1:4) and the codon-redesigned GUS coding sequence (CR-Ec.uidA_nno-1:1:1) . Identical nucleotides in the alignment are indicated by an asterisk. The codon-redesigned GUS sequence is 77.9% identical to the native GUS coding sequence and was designed to be better expressed in the plant.

[00101] Three (3) different classes of EXP were used, each conferring a specific expression pattern. The EXPs EXP- SETit.Cab3+Zm.DnaK:1:1 (SEQ ID NO: 34) and EXP- SETit.Cab3+Zm.DnaK:1:2 (SEQ ID NO: 35) confer a leaf expression profile in maize and are essentially identical, with the exception of a five-nucleotide insertion of 5'-CCGGA-3' at nucleotide positions 1408 to 1412 of EXP-SETit.Cab3+Zm.DnaK:1:2. The sequence EXP EXP-CaMV.35S- enh+Os.Rcc3+Zm.DnaK:1:5 (SEQ ID NO: 36) provides an enhanced root expression profile in maize. The sequence EXP EXP-Zm.UbqM1:1:2 (SEQ ID NO: 37) provides a high constitutive expression profile in maize. The resulting plasmids were used to transform corn plants using methods known in the art. Table 3 lists the plasmid construct designations, and the corresponding EXP and GUS sequences. Table 3. Plasmid constructs, EXP sequences, and expression patterns used to compare native GUS vs. codon-redesigned GUS coding sequences.

[00102] In certain cases, plants were transformed using Agrobacterium-mediated transformation methods known in the art and as described in U.S. Patent Application Publication 2009/0138985.

[00103] GUS histochemical analyzes were used for qualitative expression analysis of transformed plants. Whole tissue sections were incubated with GUS X-Glyc (5-bromo-4-chloro-3-indolyl-b-glucuronide) staining solution (1 mg/ml) for an appropriate period of time, washed, and visually inspected. for the blue color. GUS activity was qualitatively determined by direct visual inspection or inspection under a microscope using selected plant tissues and organs. R0 plants are inspected for expression in roots and leaves, as well as anther, silk, and developing seed and embryo, 21 days after pollination (21 DAP).

[00104] For quantitative analysis, total protein was extracted from selected tissues of transformed corn plants. One microgram of total protein was used with the fluorogenic substrate 4-methylumbelliferyl-β-D-glucuronide (MUG) in a total reaction volume of 50 μl. The reaction product, 4-methylumbelliferone (4-MU), is maximally fluorescent at high pH, where the hydroxyl group is ionized. The addition of a basic sodium carbonate solution simultaneously stops the assay and adjusts the pH to quantify the fluorescent product. Fluorescence was measured with excitation at 365 nm, emission at 445 nm, using a Fluoromax-3 (Horiba; Kyoto, Japan) with Micromax Reader, with a slit width defined at excitation 2 nm and emission 3 nm.

[00105] Average GUS expression values for R0 generation transformants are provided in Tables 4, 5 and 6. Table 4. Average R0 generation GUS expression of a native and codon-redesigned GUS coding sequence using an EXP with a leafy expression profile. Table 5. GUS expression in the average R0 generation of a native and codon-redesigned GUS coding sequence using an EXP with a boosted root expression profile. Table 6. GUS expression in the average R0 generation of a native and codon-redesigned GUS coding sequence using an EXP with a constitutive expression profile.

[00106] As can be seen in Tables 4 to 6, there is greater sensitivity in quantitative assays using the codon-redesigned GUS coding sequence when compared to the native GUS coding sequence. Some variability between the GUS.nno and GUS.nat populations is expected, as expression can be affected by T-DNA insertion sites; however, the overall sensitivity trend demonstrates much higher sensitivity using GUS.nno. GUS conducted by EXP- SETit.Cab3+Zm.DnaK:1:1 (SEQ ID NO: 34) and EXP- SETit.Cab3+Zm.DnaK:1:2 (SEQ ID NO: 35) demonstrated a sensitivity of 2.26 to 8.1 times higher using GUS.nno when compared to GUS.nat. Similarly, the potentiated root profile provided by EXP-CaMV.35S-enh+Os.Rcc3+Zm.DnaK:1:5 (SEQ ID NO: 36) was 16.06 to 19.06 times higher using GUS. nno than GUS.nat, making this codon-redesigned GUS coding sequence ideal for screening root promoters, especially those promoters that express at low levels, and can demonstrate GUS levels at or below background levels when using the native GUS coding sequence. The high constitutive expression profile conferred by EXP-Zm.UbqM1:1:2 (SEQ ID NO: 37) demonstrated 1.42 to 4.09 times greater quantitative sensitivity when using GUS.nno compared to GUS.nat.

[00107] Qualitatively, GUS staining was more sensitive and consistently observed in tissue samples using the codon-redesigned GUS coding sequence. Generally, qualitative staining observations tended to be less sensitive than quantitative assays. Use of the codon-redesigned GUS coding sequence provides better and more consistent microscopic inspections of stained tissues. For example, in root tissues where GUS was driven by EXP-CaMV.35S-enh+Os.Rcc3+Zm.DnaK:1:5 (SEQ ID NO: 36), histochemical staining of tissues transformed with the sequence of codon-redesigned GUS encoding was more pronounced and visible in all V7 root samples from the cortex, epidermis, endodermis, root hair, and secondary root tip. In contrast, GUS staining was not observed qualitatively in the corresponding V7 root tissues when the native GUS coding sequence was driven by EXP-CaMV.35S-enh+Os.Rcc3+Zm.DnaK:1:5. The improved codon-redesigned GUS coding sequence (CR-Ec.uidA_nno-1:1:1, SEQ ID NO: 30), provided the greatest sensitivity of the assay and was particularly valuable in measuring expression from promoters that express at levels lows.

Example 5 Analysis of Regulatory Elements Driving GUS in Corn Leaf and Root Protoplasts

[00108] Corn leaf and root protoplasts were transformed with vectors comprising an RCc3 promoter operably linked to their native RCc3 leader driving expression of the β-glucuronidase (GUS) transgene, and the resulting transformed protoplasts were analyzed for GUS protein expression . The RCc3 promoter and leader sequences were cloned into binary plasmid constructs using methods known in the art and as previously described in Example 2.

[00109] Two plasmid constructs for use in cotransformation and data normalization were also constructed using methods known in the art. Each of these plasmid constructs contained a specific luciferase coding sequence that was driven by a constitutive EXP. The pMON19437 vector comprised an expression cassette with a constitutive promoter operably linked 5' to an intron, (EXP-CaMV.35S-enh+Zm.DnaK:1, SEQ ID NO: 41), operably linked 5' to a sequence of encoding firefly (Photinus pyralis) luciferase (LUCIFERASE: 1:3, SEQ ID NO: 42), operably linked 5' to a 3' UTR of the Agrobacterium tumefaciens nopaline synthase gene (T-AGRtu.nos-1: 1:13, SEQ ID NO: 39). The pMON63934 vector comprised an expression cassette with a constitutive EXP sequence (EXP-CaMV.35S-enh-Lhcb1, SEQ ID NO: 44) operably linked 5' to a Renilla reniformis luciferase coding sequence (CR-Ren.hRenilla Lucife-0:0:1, SEQ ID NO: 43), operably linked 5' to a 3' UTR of the Agrobacterium tumefaciens nopaline synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 39) .

[00110] Corn root and leaf protoplasts were transformed using a transformation method based on polyethylene glycol (PEG), which is well known in the art. Protoplast cells were transformed with pMON19437, pMON63934, and one of the plasmids shown in Table 7. After transformation, the transformed protoplasts were incubated overnight in complete darkness. Next, GUS and luciferase measurements were performed by placing aliquots of a lysate preparation of transformed cells as above into two different small well trays. One tray was used for GUS measurements, and a second tray was used to perform a dual luciferase assay using the Dual Luciferase Reporter Assay System (Promega Corp., Madison, WI; see, for example, Promega Notes Magazine, No: 57, 1996, p.02).

[00111] Four transformations for each EXP or promoter + leader + intron sequence were performed. Average expression values for each EXP or promoter + leader + intron sequence were determined from multiple samples of each transformation. Sample measurements were made using four replicates of each EXP construct transformation or promoter + leader + intron sequence plasmid. Background GUS expression was determined using a negative control plasmid construct devoid of the GUS transgene. Average GUS and luciferase expression levels are provided in Tables 7 (leaf) and 8 (root). In these tables, firefly luciferase values (e.g., from pMON19437 expression) are given in the column labeled "FLUC" and Renilla reniformis luciferase values (e.g., from pMON63934 expression) are given as in the column labeled "RLUC". Also provided in Tables 7 and 8 are average GUS/FLUC and GUS/RLUC ratios that provide a relative measure of the strength of expression in the protoplast assays. Table 7. Average GUS, FLUC and RLUC values derived from transformed corn leaf protoplasts. Table 8. Mean values of GUS, FLUC and RLUC derived from transformed corn root protoplasts.

[00112] As demonstrated in Table 7, all RCc3 homologous promoters demonstrated the ability to drive transgene expression in corn leaf protoplasts. Some of the RCc3 homologous promoters drove higher expression than others in this assay based on GUS/FLUC and GUS/RLUC ratios. Furthermore, as demonstrated in Table 8 above, all RCc3 homologous promoters demonstrated the ability to drive transgene expression in maize root protoplasts to varying degrees.

Example 6 Analysis of Regulatory Elements Driving GUS in Transgenic Corn.

[00113] Corn plants were transformed with vectors comprising an RCc3 promoter operably linked to their native RCc3 leader driving expression of the β-glucuronidase (GUS) transgene. The resulting transformed plants were analyzed for GUS protein expression.

[00114] The RCc3 promoter and leader sequences were cloned into binary plasmid constructs using methods known in the art, and as previously described in Example 2. The resulting binary plasmid constructs were pMON264146, pMON264148, pMON264088, pMON264107, pMON264186, pMON264187, pMON264049, pMON264050, pMON264147 and pMON264166. Maize plants were also stably transformed with pMON264108 and pMON264206. Qualitative and quantitative GUS expression analysis was performed as described in Example 2. Plants were evaluated at V4, V7 and VT developmental stages. Sampling on R1 and R3 is shown. Table 9 shows the average quantitative GUS expression for stably transformed maize plants. Table 9. Mean quantitative GUS expression for stably transformed maize plants.

[00115] As demonstrated in Table 9, all RCc3 promoter homologs were able to drive expression of the GUS transgene in stably transformed corn plants. Furthermore, each promoter had an expression pattern that was unique to the specific promoter. For example, expression in the VT flower/anther differed between RCc3 promoter homologs. Expression driven by P-Td.RCc3_3:1 (SEQ ID NO: 17) was the highest expression observed for all promoters, while expression driven by P-MISgr.RCc3-2:2 (SEQ ID NO: 11) was the lowest. Regarding the expression of R1 Cob/silk, P- Td.RCc3_3:1 (SEQ ID NO: 17) demonstrated the highest expression in these tissues and P-MISgr.RCc3-2:2 (SEQ ID NO: 11) expressed the least. Expression driven by P-Td.RCc3_3:1 (SEQ ID NO: 17) increased in late developmental tissues. Expression increased in the root from the V4 to VT phase and was even higher in the VT flowers/anthers, R1 cob/silk and R3 21DAP embryo and endosperm. Expression driven by P-Td.RCc3_3:1 was highest among RCc3 promoter homologues in VT flowers/anthers, R1 cob/silk and R3 21DAP embryo and endosperm.

[00116] With regard to leaf and root expression, some of the RCc3 promoter homologues demonstrated higher expression in the root compared to the leaf. Table 10 shows root-to-leaf expression ratios for all RCc3 promoters analyzed. Table 10. Root-to-leaf expression ratios for stably transformed maize plants.

[00117] As demonstrated in Table 10, each RCc3 promoter homolog demonstrated different root-to-leaf expression ratios and different V4 to VT phase patterns. For example, P- Cl.RCc3:3 (SEQ ID NO: 1) maintained a similar ratio of expression from V4 to VT with a slight decline occurring in the V7 phase. Expression in the root as seen in Table 9 fell slightly from V4 to V7 and then increased through the VT phase. The P-Ds.RCc3_3:1 promoter (SEQ ID NO: 7) demonstrated a change in expression ratios from the V4 to VT phase with higher expression in the root relative to the leaf in the V4 and V7 phase and then a shift approaching equal expression in the leaves in relation to the root in the VT phase (1.25). With this promoter, the average expression shown in Table 9 demonstrates an increase in expression in the leaf from the V4 to VT phase while root expression decreased from the V7 to VT phase. The P-So.RCc3:2 promoter (SEQ ID NO: 19) maintained a root-to-leaf expression ratio of 6.33 in the V4 phase and 6.79 in V7, but then dropped to 2.99 in the VT. However, expression with this promoter increased 3.69- and 3.96-fold in the leaf and root, respectively, from the V4 to V7 phase and then decreased to 2.33 and 1.10 from the V4 phase in VT.

[00118] Notably, not all promoters had a higher root-to-leaf ratio. For example, the promoters P-Ds.RCc3_1:1 (SEQ ID NO: 3) and P-Td.RCc3_1:1 (SEQ ID NO: 13) had root/leaf ratios less than one in the V4 phase. However, expression driven by P-Td.RCc3_1:1 was 6.6-fold higher than P-Ds.RCc3_1:1 in the V4 root. The highest root/leaf ratio in the V4 phase was achieved using P-Td.RCc3_2:1 (SEQ ID NO: 15). The root/leaf expression ratio driven by P-Ds.RCc3_1:1 increased from V4 (0.76) to V7 (2.95) and then returned to a ratio similar to that in V4 (0.79).

[00119] The P-MISgr.RCc3-2:2 promoter (SEQ ID NO: 11) demonstrated an increase in expression in the leaf and root from the V4 to VT phase. This promoter had a root-to-leaf ratio greater than 6.9 throughout all three phases, but the ratio went from 7.46 in the V4 phase to 6.92 in the V7 phase and then rose to 11.44 in the V4 phase. VT. Expression driven by P-MISgr.RCc3-2:2 increased in leaf and root from V4 to VT stage.

[00120] Each of the RCc3 homologous promoters demonstrated expression patterns in stably transformed corn that could not necessarily be predicted by virtue of being derived from homologous genes, especially when used to transform a heterologous species such as corn. Most promoters demonstrated higher expression in the root compared to the leaf at some point in the V4, V7 or VT phase or in all phases previously evaluated. Notably, the magnitude of expression differed widely between promoters. The unique expression properties of each of the RCc3 promoter homologues make some more suitable than others for expression of certain types of transcribeable DNA molecules. For example, the expression of a DNA molecule that can be transcribed may be fundamental for the assimilation of a nutrient in the soil and that is best expressed at a later stage in development, when the plant is about to begin reproduction and production of seed, may better benefit from a promoter such as P-MISgr.RCc3-2:2 (SEQ ID NO: 11) that increases expression in the root close to the VT phase.

[00121] Having illustrated and described the principles of the invention, it should be evident to those skilled in the art that the invention can be modified in arrangement and details without departing from these principles. We claim all modifications that are within the spirit and scope of the claims. All published publications and patent documents cited herein are incorporated herein by reference to the same extent as if each individual publication or patent application were individually and specifically designated to be incorporated herein by reference.

Claims (3)

1. Recombinant DNA molecule, characterized by the fact that it consists of a polynucleotide sequence comprising SEQ ID NO: 7 operably linked 5' to SEQ ID NO: 8, said sequence being operably linked to a heterologous polynucleotide molecule capable of to be transcribed. 2. Recombinant DNA molecule, according to claim 1, characterized by the fact that the heterologous DNA molecule capable of being transcribed comprises a gene of agronomic interest, and the gene of agronomic interest confers herbicide tolerance or tolerance to pest on plants. 3. Method for producing a transgenic corn plant, characterized by the fact that it comprises: (a) transforming a plant cell with the recombinant DNA molecule, as defined in claim 1, to produce a transformed plant cell; and (b) regenerating a transgenic plant from the transformed plant cell.