Plant regulatory elements and uses thereof

Abstract

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

Description

[0001] This application is a divisional application of the invention patent application entitled "Plant Regulatory Elements and Their Uses" filed on March 11, 2014, with application number 201480022713.6 (application number 201810544674.6, filed on May 30, 2018, with invention title "Plant Regulatory Elements and Their Uses").

[0002] Citation of relevant applications

[0003] This application claims the benefit of U.S. Provisional Serial No. 61 / 785,268, filed March 14, 2013, which is incorporated herein by reference in its entirety.

[0004] Merging of sequence lists

[0005] The sequence list contained in the file named "MONS332WO.txt" is submitted electronically with this document and incorporated herein by reference. The sequence list is 52.7 kilobytes in size (as defined in Microsoft...). (Measured size) and created on March 11, 2014. Invention Field

[0006] This invention relates to the fields of plant molecular biology, plant genetic engineering, and DNA molecules suitable for regulating gene expression in plants. Background Technology

[0007] Regulatory elements are genetic elements that regulate gene activity by modulating transcription through operable links to transcribed DNA molecules. These elements can include promoters, leader regions, introns, and 3′ untranslated regions and are applicable to the fields of plant molecular biology and plant genetic engineering. Invention Overview

[0008] This invention provides novel regulatory elements for use in plants and constructs containing regulatory elements. The invention also provides transgenic plant cells, plants, and seeds containing regulatory elements. In one embodiment disclosed herein, the regulatory element is operatively linked to a transcribed DNA molecule. In some embodiments, the transcribed DNA molecule is heterologous relative to the regulatory sequence. This invention also provides methods for manufacturing and using the disclosed regulatory elements, including constructs containing regulatory elements, and transgenic plant cells, plants, and seeds containing regulatory elements operatively linked to transcribed DNA molecules heterologous to the regulatory element.

[0009] Therefore, in one aspect, the present invention provides a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a DNA sequence having at least about 85% sequence identity with any one of SEQ ID NO: 1-37; (b) a DNA sequence comprising any one of SEQ ID NO: 1-37; and (c) a fragment of any one of SEQ ID NO: 1-37, wherein the fragment has gene regulatory activity; wherein the DNA sequence is operatively ligated to a heterologous transcribed DNA molecule. A “heterologous transcribed DNA molecule” means that the transcribed DNA molecule is heterologous to the DNA sequence operatively ligated thereto. In a specific embodiment, the recombinant DNA molecule comprises a DNA sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 1-37. In a specific embodiment, the heterologous transcribed DNA molecule comprises a gene of agronomical importance, such as a gene capable of providing herbicide resistance or pest resistance in plants. In other embodiments, the present invention provides constructs comprising recombinant DNA molecules as provided herein.

[0010] On the other hand, this document provides a transgenic plant cell comprising a recombinant DNA molecule containing a DNA sequence selected from the group consisting of: (a) a DNA sequence having at least about 85% sequence identity with any one of SEQ ID NO: 1-37; (b) a DNA sequence comprising any one of SEQ ID NO: 1-37; and (c) a fragment of any one of SEQ ID NO: 1-37, wherein said fragment has gene regulatory activity; wherein said DNA sequence is operatively ligated to a heterologous transcribed DNA molecule. In some embodiments, the transgenic plant cell is a monocotyledonous plant cell. In other embodiments, the transgenic plant cell is a dicotyledonous plant cell.

[0011] In another aspect, this document further provides a transgenic plant or a portion thereof comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a DNA sequence having at least 85% sequence identity with any one of SEQ ID NO: 1-37; (b) a DNA sequence comprising any one of SEQ ID NO: 1-37; and (c) a fragment of any one of SEQ ID NO: 1-37, wherein said fragment has gene regulatory activity; wherein said DNA sequence is operatively ligated to a heterologous transcribed DNA molecule. In a specific embodiment, the transgenic plant is a progeny plant of any generation relative to a starting transgenic plant and comprises a recombinant DNA molecule. This document also provides transgenic seeds comprising a recombinant DNA molecule that produces such transgenic plants upon growth.

[0012] In another aspect, the present invention provides a method for producing a commercial product, comprising obtaining a transgenic plant or a portion thereof containing the recombinant DNA molecule of the present invention and producing the commercial product therefrom. In one embodiment, the commercial product is processed seeds, granules, plant parts, and powder.

[0013] In another aspect, the present invention provides a method for producing transgenic plants comprising the recombinant DNA molecules of the present invention, comprising transforming plant cells with the recombinant DNA molecules of the present invention to produce transformed plant cells and regenerating transgenic plants from said transformed plant cells. Brief description of the attached diagram

[0014] Figure 1 : Demonstrates the expression box configuration of the present invention.

[0015] Sequence Summary

[0016] SEQ ID NO:1-30, 38-41, 49 and 56 are 3′UTR sequences.

[0017] SEQ ID NO:31, 35, 42, 47, 48, 50, 51, 52, 53, 54 and 55 are DNA sequences of a regulatory expression element set (EXP) containing a promoter sequence 5′ operably linked to a leader sequence, the leader sequence being 5′ operably linked to an intron sequence; or a promoter sequence 5′ operably linked to a leader sequence.

[0018] SEQ ID NO:32, 36 and 43 are promoter sequences.

[0019] SEQ ID NO:33 and 37 are leader sequences.

[0020] SEQ ID NO:34 is an intron sequence.

[0021] SEQ ID NO:44 is the coding sequence for β-glucuronidase (GUS) with processable introns.

[0022] SEQ ID NO:45 and 46 are luciferase coding sequences. Invention Details

[0023] This invention provides DNA molecules with gene regulatory activity in plants. The nucleotide sequences of these DNA molecules are provided as SEQ ID NO:1-37. These DNA molecules are capable of influencing the expression of operably linked, transcribed DNA molecules in plant tissues, and thus regulating the expression of operably linked transgenic genes in transgenic plants. This invention also provides methods for modifying, producing, and using these molecules. This invention also provides compositions comprising transgenic plant cells, plants, plant parts, and seeds containing the recombinant DNA molecules of this invention, and methods for preparing and using said compositions.

[0024] The following definitions and methods are provided to better define the present invention and to guide those skilled in the art in practicing it. Unless otherwise indicated, the terminology should be understood in accordance with its conventional usage by those skilled in the art.

[0025] DNA molecules

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

[0027] As used herein, a “recombinant DNA molecule” is a DNA molecule that contains a combination of DNA molecules that would not naturally occur without human intervention. For example, a recombinant DNA molecule may be a DNA molecule that contains at least two DNA molecules that are heterologous to each other, a DNA molecule that contains a DNA sequence that deviates from the DNA sequence that exists in nature, or a DNA molecule that has been incorporated into the DNA of a host cell through genetic transformation.

[0028] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned DNA sequences are identical. Optimal sequence alignment is established by manually aligning two sequences, such as a reference sequence and another DNA sequence, to maximize the number of nucleotide matches in sequence alignments with appropriate internal nucleotide insertions, deletions, or gaps. As used herein, the term "reference sequence" refers to the DNA sequence provided as SEQ ID NO:1-37.

[0029] As used herein, the term “% sequence identity” or “% identity” is the identity score multiplied by 100. The “identity score” of a DNA sequence that is best aligned to a reference sequence is the number of nucleotide matches in the best 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 reference sequence. Therefore, one embodiment of the present invention provides a DNA molecule comprising a DNA sequence that, when best aligned with a reference sequence provided herein as SEQ ID NO:1-37, has at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or at least about 100% identity.

[0030] Control element

[0031] Regulatory elements such as promoters, leader regions, enhancers, introns, and transcription termination regions (or 3′UTRs) play a holistic role in overall gene expression in living cells. As used herein, the term "regulatory element" refers to a DNA molecule with gene regulatory activity. As used herein, the term "gene regulatory activity" refers to the ability to influence the expression of operatively linked transcribed DNA molecules, for example, by affecting the transcription and / or translation of operatively linked transcribed DNA molecules. Therefore, regulatory elements functioning in plants, such as promoters, leader regions, enhancers, introns, and 3′UTRs, are suitable for modifying plant phenotypes via genetic engineering.

[0032] As used herein, a “regulatory expression element set” or “EXP” sequence may refer to a group of regulatory elements, such as enhancers, promoters, leader regions, and introns, that are operatively linked. Thus, a regulatory expression element set may include, for example, a promoter operatively linked at 5′ to a leader sequence, which in turn is operatively linked at 5′ to an intron sequence.

[0033] Regulatory elements can be characterized by their gene expression patterns, such as positive and / or negative effects like constitutive, temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and / or chemical responses, and any combination thereof, as well as quantitative or qualitative indications. As used herein, a “gene expression pattern” is any pattern that can be operatively linked to the transcription of DNA molecules into transcribed RNA molecules. Transcribed RNA molecules can be translated to produce protein molecules or provide antisense or other regulatory RNA molecules, such as double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small RNA (miRNA), etc.

[0034] As used herein, the term "protein expression" refers to any pattern of translation from transcribed RNA molecules to protein molecules. Protein expression can be characterized by its temporal, spatial, developmental, or morphological properties, as well as by quantitative or qualitative indicators.

[0035] Promoters are suitable as regulatory elements that regulate the expression of operatively linked, transcribed DNA molecules. As used herein, the term "promoter" generally refers to a DNA molecule involved in recognizing and binding RNA polymerase II and other proteins, such as trans-acting transcription factors, to initiate transcription. Promoters may initially be recognized from the 5′ untranslated region (5′UTR) of a gene. Alternatively, promoters may be synthetically generated or manipulated DNA molecules. Promoters may also be chimeric. Chimeric promoters are generated via the fusion of two or more heterologous DNA molecules. Promoters suitable for carrying out the invention include SEQ ID NO:32 and 36, comprising fragments or variants thereof. In specific embodiments of the invention, the claimed DNA molecules and any variants or derivatives thereof as described herein are further defined as containing promoter activity, i.e., capable of acting as a promoter host cell, such as in transgenic plants. In a more specific implementation, a fragment may be defined as exhibiting promoter activity of the initiator promoter molecule from which it originates, or the fragment may contain a “minimal promoter” that provides basal-level transcription and consists of a TATA box or equivalent DNA sequence for recognizing and binding the RNA polymerase II complex to initiate transcription.

[0036] In one embodiment, a fragment of the promoter sequence disclosed herein is provided. The promoter fragment may include promoter activity as described above and may be used alone or in combination with other promoters and promoter fragments, such as in the construction of a chimeric promoter, or in combination with other EXPs and EXP fragments. In a specific embodiment, a fragment of a promoter is 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 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 adjacent nucleotides, or longer, of a DNA molecule with promoter activity disclosed herein. Methods for generating such fragments from a starter promoter molecule are well known in the art.

[0037] Compositions derived from any promoter represented by SEQ ID NO:32 and 36, such as internal or 5′ deletions, can be generated, for example, using methods well known in the art to improve or alter expression, including removing elements that have a positive or negative effect on expression; replicating elements that have a positive or negative effect on expression; and / or replicating or removing elements that have a tissue or cell-specific effect on expression. Compositions derived from any promoter represented by SEQ ID NO:32 and 36 containing a 3′ deletion in which a TATA box element or its equivalent sequence and downstream sequence are removed can be used, for example, to prepare enhancer elements. Further deletions can be generated to remove any elements that have a positive or negative effect on expression; tissue-specific; cell-specific; or timing-specific (such as, but not limited to, circadian rhythm) effect. Any promoter represented by SEQ ID NO:32 and 36 and fragments or enhancers derived therefrom can be used to prepare chimeric transcriptional regulatory element compositions.

[0038] According to the present invention, the promoter or promoter fragment can be analyzed for known promoter elements, i.e., DNA sequence characteristics, such as the presence of TATA boxes and other known transcription factor binding site motifs. The identification of such known promoter elements can be used by those skilled in the art to design promoter variants having an expression pattern similar to the original promoter.

[0039] As used herein, the term "leader region" refers to a DNA molecule recognized from the untranslated 5′ region (5′UTR) of a gene and is generally defined as a nucleotide segment between the transcription start site (TSS) and the protein-coding sequence start site. Alternatively, a leader region may be a synthetically generated or manipulated DNA element. A leader region can serve as a 5′ regulatory element regulating the expression of a operatively linked, transcribed DNA molecule. Leader region molecules can be used with a heterologous promoter or with its native promoter. Leader regions suitable for carrying out the present invention include SEQ ID NO: 33 and 37 or fragments thereof. In specific embodiments, such DNA sequences can be defined as capable of functioning as leader regions in host cells, including, for example, transgenic plant cells. In one embodiment, such DNA sequences can be decoded to contain leader region activity.

[0040] The leader sequences represented by SEQ ID NO:33 and 37 may contain regulatory elements or may employ secondary structures that can have an effect on the transcription or translation of operably linked transcribed DNA molecules. The leader sequences represented by SEQ ID NO:33 and 37 may be used according to the present invention to obtain chimeric regulatory elements that influence the transcription or translation of operably linked DNA molecules.

[0041] As used herein, the term "intron" refers to a region of a DNA molecule that is recognizable from a gene and can be generally defined as a region spliced ​​during pre-translational messenger RNA (mRNA) processing. Alternatively, an intron can be a synthetically generated or manipulated DNA element. Introns may contain enhancer elements that enable transcription of operably linked genes. Introns can serve as regulatory elements that regulate the expression of operably linked transcribed DNA molecules. Constructs may include introns, and introns may or may not be heterologous relative to transcribed DNA molecules. Examples of introns in the art include the rice actin intron and the maize HSP70 intron.

[0042] In plants, the presence of certain introns in a gene construct leads to increased mRNA and protein accumulation compared to constructs lacking introns. This effect is known as "intron-mediated enhancement" (IME) of gene expression. Introns known to stimulate expression in plants have been identified in maize genes (e.g., tubA1, Adh1, Sh1, and Ubi1), rice genes (e.g., tpi), and dicotyledonous genes such as those from petunia (e.g., rbcS), potato (e.g., st-ls1), and Arabidopsis (e.g., ubq3 and pat1). Deletion or mutation within the splice site of an intron has been shown to reduce gene expression, indicating that splicing may be required for IME. However, IME in dicotyledons has been demonstrated through point mutations within the splice site of the pat1 gene from Arabidopsis. The repeated use of the same intron in a single plant has proven detrimental in some cases. In those cases, a set of basic control elements is required to construct appropriate recombinant DNA elements.

[0043] Introns suitable for carrying out the present invention include SEQ ID NO:34. Compositions derived from introns represented by SEQ ID NO:34 may contain internal deletions or duplications of cis-regulatory elements; and / or alterations to the 5′ and 3′ DNA sequences containing the intron / exon splice junction can be used to improve expression or expression specificity when operatively linked to a promoter + leader region or a chimeric promoter + leader region and coding sequence. When modifying the intron / exon boundary sequence, avoiding the use of the nucleotide sequence AT or nucleotide A just before the 5′ end of the splice site (GT) and the corresponding nucleotide G or nucleotide sequence TG just after the 3′ end of the splice site (AG) can help eliminate the possibility of unwanted start codons formed during the processing of messenger RNA into the final transcript. Thus, the DNA sequence surrounding the 5′ or 3′ splice junction of the intron can be modified in this manner. Introns and intron variants modified as described herein and via methods known in the art can be empirically tested to determine the effect of the intron on the expression of operatively linked DNA molecules, as described in the working examples. Alterations can also be made in the 5′ and 3′ regions containing intron / exon splicing junctions to reduce the likelihood of introducing incorrect start and stop codons into the transcript obtained after processing and splicing messenger RNA. Introns can be empirically tested to determine their effect on transgene expression, as described in the working examples.

[0044] As used herein, the terms “3′ transcription termination molecule,” “3′ untranslated region,” or “3′UTR” refer to a DNA molecule used during transcription of the untranslated region of 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 polyadenylated tail. The 3′UTR can be operatively linked to and located downstream of a transcribed DNA molecule and can include polyadenylation signals and other regulatory signals capable of influencing transcription, mRNA processing, or gene expression. The polyadenylated tail is thought to play a role in mRNA stability and the initiation of translation. Examples of 3′ transcription termination molecules in the art are the 3′ region of carmine synthase; the 3′ region of wheat hsp17; the 3′ region of the small subunit of pea rubiscoase; the 3′ region of cotton E6; and the 3′UTR of coixol.

[0045] 3′UTRs are often advantageous for the recombinant expression of specific DNA molecules. Weak 3′UTRs have the potential to generate readthroughs, which can influence the expression of DNA molecules located in adjacent expression cassettes. Proper control of transcription termination can prevent readthroughs to downstream DNA sequences (e.g., other expression cassettes) and can further allow efficient recycling of RNA polymerase to improve gene expression. Efficient termination of transcription (release of RNA polymerase II from DNA) is a prerequisite for restarting transcription, thus directly affecting the overall transcriptional level. After transcription termination, mature mRNA is released from the site of synthesis and the template is transported to the cytoplasm. Eukaryotic mRNA accumulates in vivo in the form of aggregates (A), making it difficult to detect transcription termination sites using conventional methods. However, predicting functional and effective 3′UTRs using bioinformatics methods can be challenging because few conserved DNA sequences allow for easy prediction of effective 3′UTRs.

[0046] From a practical standpoint, 3′UTRs used in expression cassettes generally advantageously possess the following characteristics: The 3′UTR should be able to efficiently and effectively terminate transgene transcription and prevent transcript readthrough into any adjacent DNA sequence, which may contain another expression cassette, such as in the case where multiple expression cassettes reside in a single transfer DNA (T-DNA), or adjacent chromosomal DNA into which the T-DNA is inserted. In plant biotechnology, 3′UTRs are frequently used to induce amplification reactions of reverse-transcribed RNA extracted from transformed plants and for: (1) assessing the transcriptional activity or expression of expression cassettes once integrated into the plant chromosome; (2) assessing the copy number within the inserted plant DNA; and (3) assessing the conjugation of resulting seeds after breeding. 3′UTRs are also used to characterize the integrity of insert cassettes in amplification reactions of DNA extracted from transformed plants.

[0047] As used herein, the term "enhancer" or "enhancer element" refers to a cis-acting regulatory element, also known as a cis-element, that confers aspects of the overall expression pattern but is generally insufficient to independently drive transcription that operatively links a transcribed DNA molecule. Unlike a promoter, an enhancer element typically does not include a transcription start site (TSS) or TATA box or equivalent DNA sequence. A promoter or promoter fragment may naturally include one or more enhancer elements that influence transcription that operatively links a transcribed DNA molecule. Enhancer elements may also be fused to a promoter to produce a chimeric promoter cis-element, which confers aspects of the overall regulation of gene expression.

[0048] Many promoter enhancer elements are thought to bind DNA-binding proteins and / or influence DNA layout, creating local conformations of the double helix that selectively allow or restrict RNA polymerase access to the DNA template or promote selective opening of transcription start sites. Enhancer elements can function to bind transcription factors that regulate transcription. Some enhancer elements bind more than one transcription factor, and transcription factors can interact with more than one enhancer domain with varying affinities. Enhancer elements can be identified using a variety of techniques, including deletion analysis (i.e., deletion of one or more nucleotides at the 5′ end or within the promoter); DNA-binding protein analysis using DNase I footprinting, methylation interference, electrophoretic mobility shift assays, in vivo genomic footprinting via ligation-mediated polymerase chain reaction (PCR), and other conventional assays; or DNA sequence similarity analysis using known cis-element motifs or enhancer elements as target sequences or target motifs using conventional DNA sequence comparison methods such as BLAST. The fine structure of enhancer domains can be further investigated by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods known in the art. Enhancer elements can be obtained through chemical synthesis or by isolation from regulatory elements containing such elements, and they can be synthesized with additional side nucleotides containing suitable restriction enzyme sites to facilitate subsequence manipulation. Therefore, this invention covers the design, construction, and use of enhancer elements according to the methods disclosed herein for regulating the expression of operably linked transcribed DNA molecules.

[0049] As used herein, the term "chimerism" refers to a single DNA molecule produced by fusing a first DNA molecule to a second DNA molecule, wherein neither the first nor the second DNA molecule is typically present in the configuration, i.e., they are fused together. Therefore, a chimeric DNA molecule is a novel DNA molecule that is not typically found elsewhere in nature. As used herein, the term "chimeric promoter" refers to a promoter produced via such manipulation of a DNA molecule. Chimeric promoters can combine two or more DNA fragments, for example, by fusing a promoter to an enhancer element. Therefore, this invention covers the design, construction, and use of enhancer elements according to the methods disclosed herein for regulating the expression of operably linked transcribed DNA molecules.

[0050] As used herein, the term "variant" refers to a second DNA molecule, such as a regulatory element, that is compositionally similar to but not identical to a first DNA molecule, and wherein the second DNA molecule retains the general functionality of the first DNA molecule, i.e., the same or similar expression pattern, for example, via more or less or equivalent transcriptional or translational activity. A variant can be a shortened or truncated form of a first DNA molecule and / or a modified form of a first DNA molecule, such as a DNA molecule with different restriction enzyme sites and / or internal deletions, substitutions, and / or insertions. "Variant" may also encompass a regulatory element having a nucleotide sequence with substitutions, deletions, and / or insertions comprising one or more nucleotides of a reference sequence, wherein the derived regulatory element has more or less or equivalent transcriptional or translational activity compared to the corresponding parental regulatory molecule. Regulatory element "variants" also include variants resulting from mutations that occur during or as a result of bacterial and plant cell transformation. In this invention, the DNA sequences provided as SEQ ID NO:1-37 can be used to generate variants that are compositionally similar to but not identical to the DNA sequence of the original regulatory element, but still retain the general functionality of the original regulatory element, i.e., the same or similar expression pattern. In view of this disclosure, such variations of the invention are entirely within the scope of ordinary skill in the art and are covered by the scope of the invention.

[0051] Chimeric regulatory elements can be designed to comprise a variety of constituent elements that can be operatively linked by a variety of 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 comprise the same constituent elements or variations thereof, but differ in that they constitute one or more ligation DNA sequences that allow the constituent elements to be operatively linked. In this invention, DNA sequences provided as SEQ ID NO: 1-30 or 31-37 provide a reference sequence for the regulatory element, wherein the constituent elements constituting the reference sequence can be linked by methods known in the art and may include substitutions, deletions, and / or insertions of one or more nucleotides or mutations that occur naturally during bacterial and plant cell transformations.

[0052] The efficacy of the modifications, duplications, or deletions described herein for the desired expression of specific transcribed DNA molecules can be empirically tested in stable and transient plant assays, such as those described in the working examples herein, to validate the results, which may vary depending on the changes produced and the target of the changes in the starting DNA molecule.

[0053] Buildings

[0054] As used herein, the term "construct" means any recombinant DNA molecule, such as plasmids, granules, viruses, bacteriophages, or linear or circular DNA or RNA molecules, derived from any source and capable of genome integration or autonomous replication, including at least one DNA molecule functionally linked to another DNA molecule, i.e., an operably linked DNA molecule. As used herein, the term "vector" means any construct that can be used for transformation purposes, i.e., to introduce heterologous DNA or RNA into host cells. Constructs typically include one or more expression cassettes. As used herein, an "expression cassette" is a DNA molecule containing at least one transcribed DNA molecule operablely linked to one or more regulatory elements, typically at least a promoter and a 3′ UTR.

[0055] As used herein, the term "operably linked" means that a first DNA molecule is linked to a second DNA molecule, wherein the first and second DNA molecules are arranged such that 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 adjacent DNA molecule and may or may not be adjacent. For example, if a promoter affects the transcription or expression of a DNA molecule, the promoter is operably linked to the DNA molecule.

[0056] In one embodiment, the construct of the present invention may be provided as a dual tumor-inducing (Ti) plasmid boundary construct having a right boundary (RB or AGRtu.RB) and a left boundary (LB or AGRtu.LB) region of a Ti plasmid isolated from *Agrobacterium tumefaciens* containing T-DNA, which, together with a delivery molecule provided by *Agrobacterium tumefaciens* cells, allows T-DNA integration into the genome of the plant cell (see, for example, U.S. Patent 6,603,061). The construct may also contain plasmid backbone DNA segments that provide replication function and antibiotic selection in bacterial cells, such as *E. coli* origin of replication like ori322, broad host-wide origin of replication like oriV or oriRi, and coding regions for selectable markers such as Spec / Strp encoding Tn7 aminoglycoside adenosyltransferase (aadA) conferring resistance to spectinomycin or streptomycin, or gentamicin (Gm, Gent) selectable marker genes. For plant transformation, the host strain is often Agrobacterium tumefaciens ABI, C58 or LBA4404; however, other strains known to those skilled in the art of plant transformation may function in this invention.

[0057] Methods for assembling and introducing constructs into cells in a manner that causes transcribed DNA molecules to be transcribed into functional mRNA molecules that are translated and expressed as proteins are known in the art. Conventional compositions and methods for preparing and using constructs and host cells are well known to those skilled in the art in practice. For example, typical vectors suitable for expressing nucleic acids in taller plants are well known in the art and include vectors derived from the Ti plasmid of Agrobacterium tumefaciens and the pCaMVCN delivery control vector.

[0058] Various regulatory elements may be included in the construct, including any of those elements provided herein. Any such regulatory element may be combined with other regulatory elements to provide a construct. Such combinations may be designed or modified to produce desired regulatory characteristics. In one embodiment, the construct of the present invention includes at least one regulatory element operatively linked to a transcribed DNA molecule operatively linked to the 3′UTR.

[0059] The constructs of this invention may include any promoter or leader region provided herein or known in the art. For example, the promoter of this invention may be operatively linked to a heterologous untranslated 5′ leader region, such as a leader region from a heat shock protein gene. Alternatively, the leader region of this invention may be operatively linked to a heterologous promoter, such as the cauliflower mosaic virus 35S transcript promoter.

[0060] Expression cassettes may also include transport peptide-coding sequences that encode peptides operatively linked to proteins suitable for subcellular targeted delivery, particularly to chloroplasts, leucoplasts, or other plastid organelles; mitochondria; peroxisomes; vacuoles; or extracellular locations. Many chloroplast-localized proteins are expressed from nuclear genes as precursors and are targeted to chloroplasts via chloroplast transport peptides (CTPs). Examples of such isolated chloroplast proteins include, but are not limited to, those associated with small subunit (SSU) ribulose-1,5,2-bisphosphate carboxylase, feroxin, feroxin oxidoreductase, light-gathering complex I and II, thioredoxin F, and enolpyruvate shikimate phosphate synthase (EPSPS). Chloroplast transport peptides are described, for example, in U.S. Patent No. 7,193,133. It has been demonstrated that nonchloroplast proteins can be targeted to chloroplasts via expression of heterologous CTPs operatively linked to transcribed DNA molecules encoding nonchloroplast proteins.

[0061] Transcribed DNA molecules

[0062] As used herein, the term "transcribed DNA molecule" means any DNA molecule capable of being transcribed into an RNA molecule, including, but not limited to, those molecules having protein-coding sequences and those molecules that produce RNA molecules with sequences suitable for gene repression. Types of DNA molecules may include, but are not limited to, DNA molecules from the same plant, DNA molecules from another plant, DNA molecules from different organisms, or synthetic DNA molecules, such as DNA molecules containing antisense information of a gene, or DNA molecules encoding artificial, synthetic, or otherwise modified transgenic forms. Exemplary transcribed DNA molecules incorporated into the constructs of this invention include, for example, DNA molecules or genes from species other than the species in which the DNA molecule is incorporated, or genes derived from or present in the same species but incorporated into recipient cells by genetic engineering methods rather than classical breeding techniques.

[0063] "Transgenic" refers to a transmissible DNA molecule that is at least heterologous to the host cell genome and / or artificially incorporated into the host cell genome in the current or any previous generation of cells, at least relative to its position in the host cell genome.

[0064] Regulatory elements, such as the promoters of this invention, can be operatively linked to a transcribed DNA molecule that is heterologous to the regulatory element. As used herein, the term "heterologous" refers to a combination of two or more DNA molecules that is not normally found in nature. For example, two DNA molecules may come from different species and / or two DNA molecules may come from different genes, for example, different genes from the same species or the same gene from different species. Thus, a regulatory element is heterologous to the operatively linked transcribed DNA molecule if such a combination is not normally found in nature, i.e., the transcribed DNA molecule is not naturally and operatively linked to the regulatory element.

[0065] Transcriptable DNA molecules can generally be any DNA molecule that requires the expression of a transcript. Such expression of a transcript can lead to the translation of the resulting mRNA molecule, and thereby protein expression. Alternatively, for example, a transcriptable DNA molecule can be programmed to ultimately result in reduced expression of a specific gene or protein. In one embodiment, this can be accomplished using a transcriptable DNA molecule oriented in an antisense direction. Those skilled in the art are familiar with the use of such antisense techniques. Any gene can be negatively regulated in this manner, and in one embodiment, the transcriptable DNA molecule can be programmed to repress a specific gene via the expression of dsRNA, siRNA, or miRNA molecules.

[0066] Therefore, one embodiment of the present invention is a recombinant DNA molecule comprising the regulatory elements of the present invention, such as those provided as SEQ ID NO:1-37, said regulatory elements being operatively linked to a heterologous transcribed DNA molecule to regulate the transcription of the transcribed DNA molecule at a desired level or in a desired mode when the construct is integrated into the genome of a transgenic plant cell. In one embodiment, the transcribed DNA molecule comprises a protein-coding region of a gene, and in another embodiment, the transcribed DNA molecule comprises an antisense region of a gene.

[0067] Genes of agricultural importance

[0068] Transcribed DNA molecules can be genes of agronomic importance. As used herein, the term "gene of agronomic importance" refers to a transcribed DNA molecule expressed in a specific plant tissue, cell, or cell type that confers desired characteristics. The product of agronomically important genes can function in plants to result in effects on plant morphology, physiology, growth, development, yield, grain composition, nutritional profile, disease or pest resistance, and / or environmental or chemical tolerance, or can act as an insecticide in the diet of plant-feeding pests. In one embodiment of the invention, the regulatory element of the invention is incorporated into a construct such that the regulatory element is operatively linked to a transcribed DNA molecule that is a gene of agronomic importance. In transgenic plants containing such constructs, expression of agronomically important genes can confer favorable agronomic traits. Beneficial agronomic traits may include, for example but not limited to, herbicide tolerance, insect control, improved yield, disease resistance, pathogen resistance, improved plant growth and development, modified starch content, improved oil content, improved fatty acid content, improved protein content, improved fruit ripening, enhanced animal and human nutrition, biopolymer production, environmental stress resistance, pharmaceutical peptides, improved processing quality, improved flavor, hybrid seed propagation efficiency, improved fiber production, and desired biofuel production.

[0069] Examples of genes known in the art to be of agronomic importance include those for traits such as herbicide resistance (US Patent Nos. 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), and increased yield (US Patent Nos. USRE 38,446; 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211; 6,235,971; 6,222,0). 98; and 5,716,837), Insect Control (US Patent Nos. 6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030; 6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109; 6,537,756; 6,521,442; 6,501,009; 6,468,523; 6,326,351; 6,313,378; 6,284,949; 6,281,016; 6,248,536; 6,242,241; 6,221,649; 6, 177,615; 6,156,573; 6,153,814; 6,110,464; 6,093,695; 6,063,756; 6,063,597; 6,023,013; 5,959,091; 5,942,664; 5,942,658; 5,880,275; 5,763,245; and 5,763,241), fungal resistance (US Patent Nos. 6,653,280; 6,573,361; 6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,121,436; 6,316,407; and 6,506 ,962), virus resistance (US Patent Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; and 5,304,730), nematode resistance (US Patent No. 6,228,992), bacterial disease resistance (US Patent No. 5,516,671), plant growth and development (US Patent Nos. 6,723,897 and 6,518,488), starch production (US Patent Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), improved oil production (US Patent Nos. 6,444,876; 6,426,447);Higher oil production (US Patent Nos. 6,495,739; 5,608,149; 6,483,008; and 6,476,295), improved fatty acid content (US Patent Nos. 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538; 6,589,767; 6,537,750; 6,48 9,461; and 6,459,018), higher protein production (US Patent No. 6,380,466), fruit ripening (US Patent No. 5,512,466), enhanced animal and human nutrition (US Patent Nos. 6,723,837; 6,653,530; 6,5412,59; 5,985,605; and 6,171,640), biopolymers (US Patent Nos. USRE37,543; 6,228,623; and 6,461,640). 5,958,745, and 6,946,588), environmental stress resistance (US Patent No. 6,072,103), pharmaceutical peptides and secretory peptides (US Patent Nos. 6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processing properties (US Patent No. 6,476,295), improved digestibility (US Patent No. 6,531,648), low raffinose (US Patent No. 6,166,295). 2) Industrial enzyme production (US Patent No. 5,543,576), flavor improvement (US Patent No. 6,011,199), nitrogen fixation (US Patent No. 5,229,114), hybrid seed breeding (US Patent No. 5,689,041), fiber production (US Patent Nos. 6,576,818; 6,271,443; 5,981,834; and 5,869,720), and biofuel production (US Patent No. 5,998,700).

[0070] Alternatively, agronomically important genes can influence the aforementioned plant characteristics or phenotypes by encoding RNA molecules that target the regulation of gene expression leading to endogenous genes, for example, through antisense (see, for example, U.S. Patent 5,107,065); repressive RNAs (“RNAi”, including regulation of gene expression through miRNA-, siRNA-, trans-acting siRNA-, and staged sRNA-mediated mechanisms, as described, for example, in published applications US2006 / 0200878 and US2008 / 0066206, and U.S. Patent Application 11 / 974,469); or co-repressive mediating mechanisms. The RNA can also be a catalytic RNA molecule engineered to cleave the desired endogenous mRNA product (e.g., a ribozyme or riboswitch; see, for example, US2006 / 0200878). Methods for constructing and introducing constructs into cells in a manner that causes transcribed DNA molecules to be transcribed into molecules capable of causing gene repression are known in the art.

[0071] Selective marking

[0072] Selective marker transgenes can also be used in conjunction with the regulatory elements of this invention. As used herein, the term “selective marker transgene” refers to any transcribed DNA molecule whose expression, or absence, in a transgenic plant, tissue, or cell is screenable or can be recorded in some way. Selective marker genes used in the practice of this invention, and their associated selection and screening techniques, are known in the art and include, but are not limited to, transcribed DNA molecules encoding β-glucuronidase (GUS), luciferase, green fluorescent protein (GFP), proteins conferring antibiotic resistance, and proteins conferring herbicide resistance.

[0073] Cell transformation

[0074] The present invention also relates to a method for producing transformed cells and plants comprising one or more regulatory elements operatively linked to a transcribed DNA molecule.

[0075] The term “transformation” refers to the introduction of DNA molecules into a recipient host. As used herein, the term “host” refers to bacteria, fungi, or plants, including any cell, tissue, organ, 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.

[0076] As used herein, the term "transformation" refers to a cell, tissue, organ, or organism into which an external polynucleotide molecule (such as a construct) has been introduced. Preferably, the introduced polynucleotide molecule is integrated into the genomic DNA of the recipient cell, tissue, organ, or organism so that the introduced polynucleotide molecule is inherited by subsequent offspring. "Transgenic" or "transformed" cells or organisms also include the offspring of the cells or organisms and offspring produced in breeding programs that use such transgenic plants as parents in hybridization and exhibit altered phenotypes caused by the presence of the external polynucleotide molecule. The introduced DNA molecule may also be transiently introduced into the recipient cell so that the introduced DNA molecule is not inherited by subsequent offspring. The term "transgenic" refers to a bacterium, fungus, or plant containing one or more heterologous DNA molecules.

[0077] Numerous methods exist well known to those skilled in the art for introducing DNA molecules into plant cells. This process generally involves selecting suitable host cells, transforming the host cells with a vector, and obtaining transformed host cells. In the practice of this invention, methods and materials for transforming plant cells by introducing constructs into the plant genome can include any well-known and proven methods. Suitable methods include, in particular, bacterial infection (e.g., Agrobacterium), binary BAC vectors, direct DNA delivery (e.g., PEG-mediated transformation, drying / inhibition-mediated DNA uptake, electroporation, agitation using silicon carbide fibers, and acceleration of DNA-coated particles).

[0078] The host cell can be any cell or organism, such as plant cells, algal cells, fungal cells, bacterial cells, or insect cells. In a specific embodiment, the host cell and transformed cell may include cells derived from crop plants.

[0079] The transgenic plant can then be regenerated from the cell of the transgenic plant of this invention. Seeds can be produced from this transgenic plant using conventional breeding techniques or self-pollination. These seeds, and the offspring plants obtained from these seeds, contain the recombinant DNA molecules of this invention and are therefore transgenic.

[0080] The transgenic plants of this invention can self-pollinate to provide seeds of homozygous transgenic plants (homozygous for the recombinant DNA molecule) of this invention, or hybridize with non-transgenic plants or different transgenic plants to provide seeds of heterozygous transgenic plants (heterozygous for the recombinant DNA molecule) of this invention. These homozygous and heterozygous transgenic plants are referred to herein as "progeny plants." Progeny plants are transgenic plants originating from the original transgenic plant and containing the recombinant DNA molecule of this invention. Seeds produced using the transgenic plants of this invention can be harvested and used to grow several generations of transgenic plants of this invention, i.e., progeny plants, which contain the constructs of this invention and express genes of agronomical importance. Descriptions of breeding methods 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 ed., Monograph, 16:249 (1987); Fehr, Principles of Variety Development, Theory and Technique, (Vol. 1) and Crop Species Soybean (Vol. 2), Iowa State University, Macmillan Pub.Co.,NY, 360-376 (1987).

[0081] Transformed plants can be analyzed for the presence of one or more genes of interest and their expression levels and / or profiles conferred by the regulatory elements of this invention. Those skilled in the art will recognize many methods that can be used to analyze transformed plants. For example, plant analysis methods include, but are not limited to, DNA blotting or RNA blotting, PCR-based methods, biochemical analysis, phenotypic screening methods, field assessment, and immunodiagnostic assays. Expression of transcribed DNA molecules can be performed using methods as described by the manufacturer. (Applied Biosystems, Foster City, CA) Reagents and methods for measuring and PCR cycle time were used. The Testing Matrix can be used to determine this. Alternatively, it can be as described by the manufacturer. (Third Wave Technologies, Madison, WI) reagents and methods can be used to evaluate transgenic expression.

[0082] This invention also provides plant parts of the invention. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen. Plant parts of the invention can be viable, non-viable, regenerable, and / or non-regenerable. This invention also includes and provides transformed plant cells comprising the DNA molecules of the invention. Transformed or transgenic plant cells of the invention comprise regenerable and / or non-regenerable plant cells.

[0083] This invention also provides commercial products derived from transgenic plants or portions thereof containing the recombinant DNA molecule of this invention. The commercial products of this invention contain a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NO: 1-37. As used herein, "commercial product" means any composition or product comprising material derived from transgenic plants, seeds, plant cells, or plant parts containing the recombinant DNA molecule of this invention. Commercial products include, but are not limited to, processed seeds, grains, plant parts, and flour. The commercial products of this invention contain a detectable amount of DNA corresponding to the recombinant DNA molecule of this invention. Detection of one or more of this DNA in a sample can be used to determine the contents or source of the commercial product. Any standard method for detecting DNA molecules, including the detection methods disclosed herein, can be used.

[0084] This invention can be more readily understood by referring to the following embodiments, which are provided for illustration and are not intended to limit the invention unless otherwise specified. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to work well in practice. However, given this disclosure, those skilled in the art will understand that many variations can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention. Therefore, all matters set forth or illustrated in the drawings should be understood as illustrative and not limiting. Example

[0085] Example 1

[0086] Identification and cloning of regulatory elements

[0087] The regulatory expression element set (EXP) and transcription termination region (3′UTR) were identified and cloned from the genomic DNA of the dicotyledonous species *Barrel Medic*. The selection of the *Barrel Medic* 3′UTR was based in part on the expression pattern observed in homologous soybean genes.

[0088] The identification and cloning of the *Alfalfa trichomoniasis* 3′UTR began with the selection of soybean genes of interest based on soybean gene expression patterns observed in soybean tissue surveys and proprietary transcript analysis experiments. These soybean genes were then selected for the discovery of homologous genes in *Alfalfa trichomoniasis* using publicly available DNA sequences. Subsequently, tissue samples from *Alfalfa trichomoniasis* were isolated from plants grown under different environmental conditions. Messenger RNA (mRNA) was then isolated from alfalfa tissues and used in real-time polymerase chain reaction (PCR) experiments to determine alfalfa gene expression patterns. From these experiments, a subset of the *Alfalfa trichomoniasis* genome was selected for cloning and characterization.

[0089] Using publicly available *Alfalfa* sequence data, bioinformatics analyses were performed to identify regulatory elements within selected alfalfa loci. For example, bioinformatics analyses were performed to identify 3′UTR sequences containing polyadenylation and termination regions of mRNA, and sequences extending further to the ends of the identified loci. Amplification primers were then designed and used to amplify each of the identified regulatory element DNA fragments, such as 3′UTR DNA fragments, DNA fragments containing promoters, leader regions, and introns, and DNA fragments containing promoters and leader regions. The resulting DNA fragments were ligated into a basic plant expression vector and sequenced.

[0090] For applicable DNA fragments, transformed plant protoplasts were then used to perform analyses of regulatory element transcription start sites (TSS) and intron / exon splicing junctions. In this analysis, protoplasts were transformed with plant expression vectors containing clonal DNA fragments operatively linked to heterologous transcribed DNA molecules. Subsequently, the 5′RACE System for Rapid Amplification of cDNA Ends, version 2.0 (Invitrogen, Carlsbad, California 92008) was used to confirm the regulatory element TSS and intron / exon splicing junctions by analyzing the DNA sequence of the resulting mRNA transcripts.

[0091] The identified 3′UTR DNA sequences are provided herein as SEQ ID NO:1-30. Additionally, the identified promoter DNA sequences are provided herein as SEQ ID NO:32 and 36; the identified leader region DNA sequences are provided herein as SEQ ID NO:33 and 37; and the identified intron DNA sequences are provided herein as SEQ ID NO:34. Furthermore, the identified EXP DNA sequences are provided herein as SEQ ID NO:31 and 35. The regulatory expression element group EXP-Mt.Ubq2:1:2 (SEQ ID NO:31) includes a promoter element, P-Mt.Ubq2-1:1:1 (SEQ ID NO:32), wherein the promoter element 5′ is operatively connected to a leader element, L-Mt.Ubq2-1:1:1 (SEQ ID NO:33), wherein the 5′ is operatively connected to an infiltrator element, I-Mt.Ubq2-1:1:2 (SEQ ID NO:34). Furthermore, the regulatory expression element group EXP-Mt.AC145767v28:1:1 (SEQ ID NO:35) includes a promoter element, P-Mt.AC145767v28-1:2:1 (SEQ ID NO:36), wherein the promoter element 5′ is operatively connected to a leader element, L-Mt.AC145767v28-1:1:2 (SEQ ID NO:37). Each of the DNA sequences identified and cloned from clover is listed in Table 1.

[0092] Table 1. 3′UTR, regulatory expression element set, promoter, leader region and introns from alfalfa clones.

[0093]

[0094]

[0095] Example 2

[0096] Analysis of the effect of 3′UTR on constitutive GUS expression in soybean leaf protoplasts

[0097] Soybean leaf protoplasts were transformed with vectors, particularly plasmid constructs, to evaluate the effect of selected alfalfa 3′UTRs on expression. Soybean leaf protoplasts were transformed with DNA vectors containing constitutive EXP sequences driving the expression of a β-glucuronidase (GUS) transgene operably linked to the alfalfa 3′UTR. These alfalfa 3′UTR-transformed soybean leaf protoplasts were compared with soybean leaf protoplasts in which GUS transgene expression was constitutively driven by a promoter and the GUS transgene was operably linked to a 3′UTR from upland or sea island cotton.

[0098] The plant vectors used in these experiments were constructed using cloning methods known in the art. The resulting vectors comprised a left-bound region from *Agrobacterium tumefaciens*; a first transgenic expression cassette selecting for resistance to the herbicide glyphosate or the antibiotic spectinomycin (both driven by the Arabidopsis thaliana myofibrillar 7 promoter); a second transgenic expression cassette for assessing the activity of a 3′UTR, the 3′UTR comprising an EXP or promoter sequence 5′ operably linked to a DNA sequence of a GUS having a processable intron (GUS-2, SEQ ID NO:44), the processable intron 5′ operably linked to a 3′UTR from *Alfalfa tumefaciens*, upland cotton, or sea island cotton; and a right-bound region from *Agrobacterium tumefaciens*. Vectors containing 3′UTRs from alfalfa (i.e., pMON109593, pMON116803, pMON116812, pMON116813, pMON116815, pMON116826, pMON116827, pMON116830, pMON122852, pMON122853, pMON122854, pMON122855, pMON122856, pMON122857, pMON122858, pMON122859, pMON122862, pMON122864, pMON122865, pMON122866, pMON122867, and pMON122868) were used with the constitutive regulatory expression element set EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID). GUS is driven by the constitutive promoter P-CaMV.35S-enh-1∶1∶11 (SEQ ID NO:43). Vectors containing 3'UTRs (i.e., pMON81345, pMON81347, and pMON83002) from upland or sea island cotton are driven by the constitutive promoter P-CaMV.35S-enh-1∶1∶11 (SEQ ID NO:43).

[0099] Table 2 provides plasmid constructs with the corresponding 3′UTR and SEQ ID NO for transforming soybean protoplasts in the experiments presented in this embodiment.

[0100] Table 2. Plasmid constructs and 3′UTR descriptions used for transforming soybean leaf protoplasts.

[0101] plasmid constructs 3'UTR description SEQ ID NO: pMON81345 T-Gb.FbL2-1∶1∶1 41 pMON81347 T-Gh.E6-4A-0∶2∶1 38 pMON83002 T-Gb.H6-1∶2∶1 39 pMON109593 T-Mt.Pt1-1∶2∶2 22 pMON116803 T-Mt.AC140914v20-1∶2∶1 2 pMON116812 T-Mt.Lhcb2-1∶2∶1 13 pMON116813 T-Mt.PSII-T_B-1∶2∶1 21 pMON116815 T-Mt.AC145767v28-1∶1∶2 1 pMON116826 T-Mt.Lox-1-1∶2∶1 14 pMON116827 T-Mt.Gpi-1∶2∶1 11 pMON116830 T-Mt.Scp-1∶2∶1 27 pMON122852 T-Mt.Methm-1:2:1 15 pMON122853 T-Mt.Prx-1∶1∶1 19 pMON122854 T-Mt.Gapdh-1∶2∶1 10 pMON122855 T-Mt.FBA-1∶1∶5 8 pMON122856 T-Mt.Zfp-1∶2∶1 30 pMON122857 T-Mt.AC139600v16-1∶2∶1 3 pMON122858 T-Mt.MP21-1∶2∶1 16 pMON122859 T-Mt.Oxr-1∶2∶1 17 pMON122862 T-Mt.Sui1-1∶1∶2 29 pMON122864 T-Mt.Pip1-1∶2∶1 18 pMON122865 T-Mt.AC153125V10-1∶2∶1 4 pMON122866 T-Mt.Sali3-2-1∶2∶1 26 pMON122867 T-Mt.Hsp20-1∶2∶1 12 pMON122868 T-Mt.Expr1-1∶2∶1 7

[0102] Two plasmid constructs were used for co-transformation and data normalization, and in particular, the plasmid constructs were constructed using methods known in the art. Each of these plasmid constructs contains a specific luciferase-coding sequence driven by a constitutive EXP. The plant vector pMON19437 contains an expression cassette with a constitutive EXP containing a promoter operatively linked to a leader sequence 5′ operatively linked to an intron (EXP-CaMV.35S-enh+Zm.DnaK:1∶1, SEQ ID NO:47), the intron 5′ operatively linked to a firefly (Photinus pyralis) luciferase-coding sequence (LUCIFERASE:1:3, SEQ ID NO:45), and the coding sequence 5′ operatively linked to a 3′ UTR (T-AGRtu.nos-1:1:13, SEQ ID NO:49) from the Agrobacterium tumefaciens carmine synthase gene. The plant vector pMON63934 contains an expression cassette with a constitutive EXP sequence comprising a 5′ promoter operatively linked to a leader sequence (EXP-CaMV.35S-enh-Lhcb1, SEQ ID NO:48), the leader sequence being operatively linked to a Renilla reniformis luciferase coding sequence (CR-Ren.hRenilla Lucife-0:0:1, SEQ ID NO:46), and the coding sequence being operatively linked to a 3′ UTR of the Agrobacterium tumefaciens carmine synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO:49).

[0103] Soybean leaf protoplasts were transformed using a polyethylene glycol (PEG)-based transformation method well-known in the art. Each protoplast cell was transformed with one of the pMON19437 plasmid constructs, the pMON63934 plasmid construct, and one of the plasmid constructs presented in Table 2. After transformation, the transformed soybean leaf protoplasts were incubated overnight in complete darkness. Subsequently, GUS and luciferase measurements were performed by placing aliquots of the lysate preparation of the transformed cells in two separate well trays. One tray was used for GUS measurements, and the second tray was used to perform dual luciferase assays using a dual luciferase reporter gene assay system (Promega Corp., Madison, WI; see, for example, Promega Notes Magazine, Vol. 57, 1996, p. 02).

[0104] One or two transformations were performed for each plasmid construct presented in Table 2. The mean expression value for each 3′ UTR was determined from multiple samples from each transformation. Sample measurements were performed using four replicates of each plasmid construct transformation, or alternatively, three replicates of each plasmid construct for each of the two transformation experiments. Mean GUS and luciferase expression levels are provided in Table 3. In this table, firefly luciferase values ​​(e.g., expression from pMON19437) are provided in the column labeled “FLuc” and Renida luciferase values ​​(e.g., expression from pMON63934) are provided in the column labeled “RLuc”.

[0105] Table 3. Average GUS and luciferase levels in transformed soybean leaf protoplasts.

[0106]

[0107] Furthermore, to compare the relative activity of each 3′UTR, the GUS value is expressed as the ratio of GUS to luciferase activity and normalized relative to the optimal expression non-alfalfa 3′UTR, i.e., T-Gb.FbL2-1∶1∶1 (SEQ ID NO: 41). Table 4 shows the GUS / luciferase ratio and normalized ratio. Additionally, in this table, firefly luciferase values ​​are labeled “FLuc” and kidney luciferase values ​​are labeled “RLuc”.

[0108] Table 4. GUS / FLuc and GUS / RLuc expression ratios normalized to T-Gb.FbL2-1∶1∶1 (SEQ ID NO: 41) in transformed soybean leaf protoplasts.

[0109]

[0110]

[0111] As shown in Table 4, GUS expression was enhanced using all selected alfalfa 3′UTRs compared to 3′UTRs from upland or Sea Island cotton. For example, based on a GUS / FLuc ratio normalized to T-Gb.FbL2-1∶1∶1, using alfalfa-derived 3′UTRs resulted in 2.1 to 18.3-fold higher GUS expression, with the best-expressing 3′UTRs from upland or Sea Island cotton. Similarly, based on a GUS / RLuc ratio normalized to T-Gb.FbL2-1∶1∶1, using alfalfa-derived 3′UTRs resulted in 1.61 to 10.48-fold higher GUS expression.

[0112] Example 3

[0113] Analysis of the effect of 3′UTR on the stable transformation of constitutive GUS expression in soybean plants

[0114] Soybean plants were transformed using vectors, particularly plasmid constructs, to evaluate the effect of selected alfalfa 3′UTRs on expression. Specifically, soybean plants were transformed with vectors containing constitutive EXP sequences that drive the expression of a β-glucuronidase (GUS) transgene operably linked to the alfalfa 3′UTR. These alfalfa 3′UTR-transformed soybean plants were compared with transformed soybean plants in which GUS transgene expression was constitutively driven by a promoter and the GUS transgene was operably linked to a 3′UTR from sea island cotton.

[0115] The plant vectors used in these experiments were constructed using cloning methods known in the art. The resulting vectors comprised a left-bound region from *Agrobacterium tumefaciens*; a first transgenic expression cassette for selecting transformed plant cells conferring resistance to the antibiotic spectinomycin (driven by the Arabidopsis thaliana myofibrillarin 7 promoter); a second transgenic expression cassette for assessing the activity of a 3′UTR containing a set of regulatory expression elements EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO:42) operatively linked to a coding sequence of GUS having a processable intron (GUS-2, SEQ ID NO:44), the processable intron 5′ being operatively linked to a 3′UTR from *Alfalfa tumefaciens* or *Alfalfa tumefaciens*; and a right-bound region from *Agrobacterium tumefaciens*. Vectors containing 3′UTRs from alfalfa are pMON109593, pMON116803, pMON116812, pMON116813, pMON116815, pMON116826, pMON116827, pMON116830, pMON122850, pMON122851, pMON122852, pMON122853, and pMON109593. N122854, pMON122855, pMON122856, pMON122857, pMON122858, pMON122859, pMON122861, pMON122862, pMON122863, pMON122864, pMON122865, pMON122866, pMON122867, and pMON122868. The vector containing the 3' UTR from Sea Island cotton is pMON102167.

[0116] Table 5 provides plasmid constructs with the corresponding 3′UTR and SEQ ID NO for transforming soybean plants in the experiments presented in this embodiment.

[0117] Table 5. Plasmid constructs and 3′UTR descriptions used for transforming soybean leaf plants.

[0118]

[0119]

[0120] Soybean plants were transformed using Agrobacterium-mediated transformation methods known in the art. GUS expression was qualitatively determined using histological sections of selected tissues. For histochemical GUS analysis, whole tissue sections were incubated with the GUS staining solution X-Gluc (5-bromo-4-chloro-3-indolyl-β-glucuronide) (1 mg / ml) for an appropriate duration, rinsed, and visually examined for blue staining. GUS activity was qualitatively determined using selected plant organs and tissues by direct visual inspection or microscopic examination. R0 generation plants were examined for expression in Vn5 roots, Vn5 sapodilla leaves, Vn5 progenitor leaves, R1 progenitor leaves, R1 petioles, R1 flowers, yellow round pod embryos (approximately R8 developmental stage), yellow round pod cotyledons (approximately R8 developmental stage), R3 immature seeds, R3 round pods, and R5 cotyledons.

[0121] Quantitative changes in GUS expression were also analyzed relative to expression conferred by pMON102167 containing the 3′UTR from sea island cotton, as shown in Table 6-13. For this quantitative analysis, total protein was extracted from selected tissues of the transformed plant. One microgram of total protein was used with the fluorescent substrate 4-methylumbelliferone-β-D-glucuronide (MUG) in a total reaction volume of 50 μl. The reaction product, 4-methylumbelliferone (4-MU), fluoresced maximally at a higher pH where the hydroxyl groups were ionized. An alkaline solution of sodium carbonate was added to stop the assay and adjust the pH for quantification of the fluorescent product. Fluorescence was measured using a Fluoromax-3 (Horiba; Kyoto, Japan) with a Micromax reader, excitation at 365 nm and emission at 445 nm, the reader having a slit width set to 2 nm for excitation and 3 nm for emission.

[0122] Tables 6 and 7 show the mean quantitative expression levels measured in R0 generation plant tissues. Tissues that were not measured are shown as blank cells in both tables.

[0123]

[0124]

[0125]

[0126] As shown in Tables 6 and 7, the expression driven by the same EXP differs in tissues of stable transformed soybean plants containing different alfalfa 3′UTRs compared to sea island cotton-derived 3′UTRs.

[0127] Tables 8 and 9 show the fold change in expression in tissues of stable transformed soybean plants containing different alfalfa 3′UTRs compared to sea island cotton-derived 3′UTRs.

[0128]

[0129]

[0130]

[0131] As shown in Tables 8 and 9, expression in transformed soybean plants containing different alfalfa 3′UTRs differed compared to soybean plants transformed with pMON102167 containing 3′UTRs from Sea Island cotton. For example, two alfalfa 3′UTRs, T-Mt.AC145767v28-1:1:2 (SEQ ID NO:1) and T-Mt.Lox-1-1:2:1 (SEQ ID NO:14), resulted in enhanced expression of the constitutive EXP, EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO:42), in all tissues. Other alfalfa 3′UTRs provided enhanced expression of the constitutive EXP in some tissues and reduced expression in others. For example, 3′UTR T-Mt.Sali3-2-1:2:1(SEQ ID NO:26) provided a 2.19 to 8.05-fold increase in expression in Vn5 roots, Vn5 follicles, Vn5 source leaves, R1 source leaves, yellow round pod embryos, and yellow round pod cotyledons, while expression was reduced in R1 flowers and R5 cotyledons. Furthermore, 3′UTR T-Mt.AC140914v20-1:2:1(SEQ ID NO:2) provided a 1.88 to 4.12-fold increase in expression in Vn5 roots, Vn5 follicles, Vn5 source leaves, yellow round pod embryos, yellow round pod cotyledons, R3 immature seeds, and R5 cotyledons, while expression was reduced in R1 source leaves and R1 flowers, and remained relatively constant in R3 round pods. In addition, 3′UTR T-Mt.Oxr-1:2:1 (SEQ ID NO:17) provided a 2.19 to 10.90-fold increase in expression in Vn5 roots, Vn5 follicles, Vn5 source leaves, R1 source leaves, yellow round pod embryos, yellow round pod cotyledons, and R3 round pods, while expression was reduced in R1 flowers and R5 cotyledons, and remained relatively the same in R3 immature seeds.

[0132] Several transformed soybean plants containing different alfalfa 3′UTRs were brought to the R1 generation. Tables 10 and 11 show the mean GUS expression values ​​of the measured tissues. Tables 12 and 13 show the fold change in expression relative to the 3′UTR from sea island cotton.

[0133]

[0134] As shown in Tables 10 and 11, expression driven by the same EXP differs in tissues of stable transformed soybean plants containing different alfalfa 3′UTRs compared to Sea Island cotton-derived 3′UTRs. Tables 12 and 13 show the fold-change expression differences in tissues of stable transformed soybean plants containing different alfalfa 3′UTRs relative to tissues transformed with pMON102167 containing 3′UTRs from Sea Island cotton.

[0135]

[0136] Table 13. Differences in fold expression in yellow round pod embryos, yellow round pod cotyledons, R3 immature seeds, R3 round pods, and R5 cotyledons of R1 generation transformed soybean plants.

[0137]

[0138] As shown in Tables 12 and 13, several alfalfa 3′UTRs enhanced the expression of constitutive EXP elements, EXP-CaMV.35S-enh+Ph.DnaK:1∶3 (SEQ ID NO: 42), relative to plants transformed with pMON102167 containing a 3′UTR from sea island cotton in the R1 generation. For example, 3′UTR T-Mt.AC145767v28-1∶1∶2 (SEQ ID NO: 1) provided a 3.10 to 21.65-fold enhancement of GUS expression in all tested tissues. 3′UTR T-Mt.Sali3-2-1∶2∶1 (SEQ ID NO: 26) provided a 1.52 to 8.90-fold enhancement of GUS expression in all tested tissues. 3′UTR T-Mt.Oxr-1∶2∶1 (SEQ ID NO: 17) provides enhancement in most tissues, but reduces R3 expression in immature seeds compared to plants transformed with T-Gb.E6-3b∶1∶1 (SEQ ID NO: 40).

[0139] The aforementioned experiments demonstrate that, depending on the specific 3′UTR selected, alfalfa-derived 3′UTR elements affect the expression of the constitutive EXP element EXP-CaMV.35S-enh+Ph.DnaK:1∶3 (SEQ ID NO: 42) in different ways. In many cases, enhanced expression exists in certain tissues of plants transformed with plant expression vectors containing alfalfa 3′UTRs, relative to plants transformed with pMON102167 containing 3′UTRs from sea island cotton. However, the enhancement effect is not observed in all plant tissues, and in many cases, expression is attenuated in some tissues and enhanced in others using alfalfa 3′UTRs. Therefore, the use of selected alfalfa 3′UTRs allows for “fine-tuning” the expression profile of specific transgenes and can be combined with different expression elements, such as promoters, leader regions, and introns, in operatively linked to transcribed DNA molecules to provide optimal expression in specific tissues while reducing expression in tissues less suitable for specific transcribed DNA molecules.

[0140] Example 4

[0141] Analysis of the effect of 3′UTR on stable transformation of seed-optimized GUS expression in soybean plants

[0142] Soybean plants were transformed using vectors, particularly plasmid constructs, to evaluate the effect of selected alfalfa 3′UTRs on expression. Specifically, soybean plants were transformed with a seed-expressing EXP sequence containing a β-glucuronidase (GUS) transgene expression that drives operatively linked to the alfalfa 3′UTR. These alfalfa 3′UTR-transformed soybean plants were compared with transformed soybean plants in which GUS transgene expression was driven by the seed-expressing EXP sequence and the GUS transgene was operatively linked to a 3′UTR from sea island cotton.

[0143] The plant vectors used in these experiments were constructed using cloning methods known in the art. The resulting vectors comprised a left-bound region from *Agrobacterium tumefaciens*; a first transgenic expression cassette for selecting transformed plant cells to confer resistance to the antibiotic spectinomycin (driven by the Arabidopsis thaliana myofibrillar 7 promoter); a second transgenic expression cassette for evaluating the activity of a 3′UTR containing a seed-preferred expression EXP element EXP-Gm.Sphas1∶1∶1 (SEQ ID NO: 50) operatively linked to a coding sequence of GUS having a processable intron (GUS-2, SEQ ID NO: 44), the processable intron 5′ being operatively linked to a 3′UTR from *Alfalfa tumefaciens* or *Cotton Island*; and a right-bound region from *Agrobacterium tumefaciens*. Plant expression vectors containing 3′UTRs from alfalfa are pMON116832, pMON116834, pMON116835, pMON116841, pMON122869, pMON122870, pMON122871, pMON122872, pMON122873, pMON122874, pMON122875, pMON122876, pMON122878, pMON122879, pMON122880, pMON122881, pMON122882, pMON122883, pMON122885, pMON122887, pMON122888, and pMON126122. The vector containing 3′UTRs from sea island cotton is pMON83028.

[0144] Table 14 provides plasmid constructs with corresponding 3′UTR, SEQ ID NO, and generations that provide targeted quantitative assay data.

[0145] Table 14. Plasmid constructs and corresponding 3′UTRs used for transforming soybean plants.

[0146] plasmid constructs 3'UTR description SEQ ID NO: The generation that provides data pMON83028 T-Gb.E6-3b∶1∶1 40 <![CDATA[R1]]> pMON116832 T-Mt.AC140914v20-1∶2∶1 2 <![CDATA[R0]]> pMON116834 T-Mt.PSII-T_A-1∶2∶1 20 <![CDATA[R0]]> pMON116835 T-Mt.AC 145767v28-1∶1∶2 1 <![CDATA[R0]]> pMON116841 T-Mt.PSII-T_B-1∶2∶1 21 <![CDATA[R0]]> pMON122869 T-Mt.RpL3-1∶2∶1 25 <![CDATA[R0]]> pMON122870 T-Mt.RD22-1∶2∶1 24 <![CDATA[R0]]> pMON122871 T-Mt.Methm-1∶2∶1 15 <![CDATA[R0]]> pMON122872 T-Mt.Prx-1∶1∶1 19 <![CDATA[R0]]> pMON122873 T-Mt.Gapdh-1∶2∶1 10 <![CDATA[R0]]> pMON122874 T-Mt.FBA-1∶2∶1 9 <![CDATA[R0]]> pMON122875 T-Mt.Zfp-1∶2∶1 30 <![CDATA[R0 and R1]]> pMON122876 T-Mt.AC139600v16-1∶2∶1 3 <![CDATA[R0]]> pMON122878 T-Mt.Oxr-1∶2∶1 17 <![CDATA[R0]]> pMON122879 T-Mt.Apx-1∶1∶2 5 <![CDATA[R0 and R1]]> pMON122880 T-Mt.Sui1-1∶1∶2 29 <![CDATA[R0 and R1]]> pMON122881 T-Mt.EF1a-1∶1∶2 6 <![CDATA[R0 and R1]]> pMON122882 T-Mt.Pip1-1∶2∶1 18 <![CDATA[R0]]> pMON122883 T-Mt.AC153125V10-1∶2∶1 4 <![CDATA[R0]]> pMON122885 T-Mt.Expr1-1∶2∶1 7 <![CDATA[R0]]> pMON122887 T-Mt.Pt1-1∶2∶2 22 <![CDATA[R0]]> pMON122888 T-Mt.Pt2-1∶2∶2 23 <![CDATA[R0]]> pMON126122 T-Mt.Expr1-1∶2∶1 7 <![CDATA[R0]]>

[0147] Soybean plants were transformed, and the GUS values ​​were measured as described in Example 3. Tables 15 and 16 provide the quantitative average GUS values ​​of stable R0 generation transformed soybean plants.

[0148] Table 15. Mean GUS expression in yellow round pod embryos, yellow round pod cotyledons, R3 immature seeds, R3 round pods, and R5 cotyledons of R0 generation transformed soybean plants.

[0149]

[0150]

[0151] Table 16. Mean GUS expression in Vn5 roots, Vn5 saponins, Vn5 source leaves, R1 source leaves, R1 petioles and R1 flowers of R0 generation transformed soybean plants.

[0152]

[0153] As can be seen in Tables 15 and 16, most alfalfa 3′UTRs affect the expression of the seed-preferred EXP element, EXP-Gm.Sphas 1∶1∶1 (SEQ ID NO: 50), only in seed-derived tissues, except for T-Mt.Apx-1∶1∶2 (SEQ ID NO: 5), which enhances GUS expression in R1 source leaves, R1 petioles, and R1 flowers. Several alfalfa 3′UTRs provide higher expression in yellow round pod plumules and yellow round pod cotyledons, such as T-Mt.AC145767v28-1∶1∶2 (SEQ ID NO: 1), T-Mt.RD22-1∶2∶1 (SEQ ID NO: 24), and T-Mt.AC153125V10-1∶2∶1 (SEQ ID NO: 4). Therefore, these 3′UTRs can ideally enhance seed promoter expression during later stages of seed development. Compared to many other 3′UTRs, 3′UTRT-Mt.Expr1-1∶2∶1 (SEQ ID NO: 7) provides higher expression in both R5 cotyledons and yellow round pod cotyledons, thus making it suitable for providing higher cotyledon expression over a wider window of seed development. In some cases, the 3′UTR provides more uniform levels of seed expression in both yellow round pod plumules and yellow round pod cotyledons, as seen when using T-Mt.FBA-1∶2∶1 (SEQ ID NO: 9), T-Mt.Zfp-1∶2∶1 (SEQ ID NO: 30), T-Mt.Pip1-1∶2∶1 (SEQ ID NO: 18), and T-Mt.Pt2-1∶2∶2 (SEQ ID NO: 23).

[0154] Seeds were produced from R0 generation plants containing T-Mt.Zfp-1∶2∶1 (SEQ ID NO: 30), T-Mt.Apx-1∶1∶2 (SEQ ID NO: 5), T-Mt.Sui1-1∶1∶2 (SEQ ID NO: 29), and T-Mt.EF1a-1∶1∶2 (SEQ ID NO: 6) and planted for R1 generation studies. Table 17 shows a comparison of mean quantitative data for events including these R1 generation plants containing alfalfa 3′UTR and plants transformed with pMON83028 containing 3′UTR T-Gb.E6-3b∶1∶1 (SEQ ID NO: 40) from sea island cotton.

[0155] Table 17. Mean GUS expression in yellow round pod embryos, yellow round pod cotyledons, and R5 cotyledons of R1 generation transformed soybean plants.

[0156]

[0157] As can be seen in Table 17, compared with T-Gb.E6-3b∶1∶1, the alfalfa 3′UTR differently affects expression in embryonic and cotyledonary tissues. For example, compared with T-Gb.E6-3b∶1∶1, T-Mt.Apx-1∶1∶2 (SEQ ID NO: 5) and T-Mt.Sui1-1∶1∶2 (SEQ ID NO: 29) enhance the expression of seed-preferred EXP elements in the yellow round pod embryo, yellow round pod cotyledon, and R5 cotyledon. T-Mt.EF1a-1∶1∶2 (SEQ ID NO: 6) enhances expression in the yellow round pod embryo and yellow round pod cotyledon, but does not enhance expression in the R5 cotyledon. T-Mt.Zfp-1∶2∶1 (SEQ ID NO: 30) reduces expression in later-developing yellow round pod embryo and yellow round pod cotyledon, but enhances expression in the R5 cotyledon.

[0158] Therefore, when operatively linked to a seed-preferred promoter, each different alfalfa 3′UTR differently affects expression in developing seeds. These differences in the effects on expression can be used to provide more refined and customized pathways for seed expression and are ideally suited for “fine-tuning” the expression profile of specific transcribed DNA molecules required for seed expression.

[0159] Example 5

[0160] The effect of 3′UTR on the stable transformation of constitutive GUS expression in soybean plants was analyzed.

[0161] Soybean plants were transformed using vectors, particularly plasmid constructs, to evaluate the effect of selected alfalfa 3′UTRs on expression. Specifically, soybean plants were transformed with vectors containing two distinct EXP sequences exhibiting constitutive expression profiles that drove the expression of a β-glucuronidase (GUS) transgene operably linked to the alfalfa 3′UTR. These alfalfa 3′UTR-transformed plants were compared with soybean plants in which the expression of the GUS transgene was operably linked to a 3′UTR from sea island cotton.

[0162] The plant vectors used in these experiments were constructed using cloning methods known in the art. The resulting vectors comprised a left-bound region from *Agrobacterium tumefaciens*; a first transgenic expression cassette selecting transformed plant cells to confer resistance to the antibiotic spectinomycin (driven by the Arabidopsis thaliana myofibrillar 7 promoter); a second transgenic expression cassette for evaluating the activity of a 3′UTR containing an EXP element, EXP-CaMV.35S-enh+Ph.DnaK∶1∶3 (SEQ ID NO: 42) or EXP-DaMV.FLT∶1∶2 (SEQ ID NO: 51), operably linked to a 5′ processing intron (GUS-2, SEQ ID NO: 44) coding sequence of GUS, said processing intron 5′ operably linked to a 3′UTR from *Alfalfa tumefaciens* or *Cotton Island*; and a right-bound region from *Agrobacterium tumefaciens*. Vectors containing 3′UTR from alfalfa are pMON118768, pMON153701, and pMON116803. Vectors containing 3′UTR from Sea Island cotton are pMON121042 and pMON102167.

[0163] Table 18 provides plasmid constructs having the corresponding EXP element, 3′UTR, and SEQ ID NO for transforming the soybean plants presented in this embodiment.

[0164] Table 18. Plasmid constructs and corresponding EXP elements and 3′UTRs used for transforming soybean plants.

[0165]

[0166] The plants were transformed and the GUS values ​​were measured as described in Example 3. Tables 19 and 20 provide the quantitative average GUS values ​​of stable R0 generation transformed soybean plants.

[0167]

[0168] As shown in Tables 19 and 20, alfalfa 3′UTRs T-Mt.Sali3-2-1:2:1 (SEQ ID NO:26) and T-Mt.AC140914v20-1:2:1 (SEQ ID NO:2) differently affect the expression of the constitutive EXP element EXP-DaMV.FLT:1:2 (SEQ ID NO:51) compared to sea island cotton derived 3′UTRT-Gb.E6-3b:1:1 (SEQ ID NO:40). Enhanced expression using alfalfa 3′UTR was observed in many sampled tissues. Enhancement was also found in most tissues of plants that also contain the EXP element EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO:42) relative to 3′UTRT-Mt.AC140914v20-1:2:1. Tables 21 and 22 show the fold differences in quantitative GUS expression relative to the expression conferred by pMON121042 (T-Gb.E6-3b:1:1(SEQ ID NO:40)) containing the 3'UTR from Sea Island cotton.

[0169]

[0170] The aforementioned experiments showed that, relative to pMON121042 (T-Gb.E6-3b:1:1 (SEQ ID NO:40)) containing a 3'UTR from Sea Island cotton, each alfalfa 3'UTR had a different effect on the expression level of each constitutive EXP element. For example, the expression of EXP-DaMV.FLT:1:2 was enhanced by 1.14 to 15.13 times in Vn5 roots, Vn5 follicles, Vn5 source leaves, R1 source leaves, R1 petioles, R1 flowers, yellow round pod embryos, yellow round pod cotyledons, R3 round pods, and R5 cotyledons, but expression was reduced in R3 immature seeds when using T-Mt.Sali3-2-1:2:1. This same EXP element, when combined with T-Mt.AC140914v20-1:2:1, resulted in a 1.34 to 13.42-fold increase in expression in Vn5 roots, Vn5 primary leaves, R1 primary leaves, R1 petioles, R1 flowers, yellow round pod embryos, yellow round pod cotyledons, R3 immature seeds, R3 round pods, and R5 cotyledons, but remained approximately the same in V5 cotyledons as with T-Gb.E6-3b:1:1 (SEQ ID NO:40). Expression in yellow round pod embryos was approximately twice that in yellow round pod cotyledons using T-Mt.Sali3-2-1:2:1 (15.13-fold increase compared to 7.23-fold increase), while expression in both tissues was relatively similar when using T-Mt.AC140914v20-1:2:1 (9.19-fold increase compared to 9.13-fold increase). Compared to the EXP element EXP-CaMV.35S-enh+Ph.DnaK:1:3, the same 3′ UTR combination with EXP-DaMV.FLT:1:2 produced less enhancement in many sampled tissues compared to the combination with T-Mt.AC140914v20-1:2:1. In R1 flowers, reduced expression was observed relative to T-Gb.E6-3b:1:1 in the EXP-CaMV.35S-enh+Ph.DnaK:1:3 combination with T-Mt.AC140914v20-1:2:1. The combination of EXP-CaMV.35S-enh+Ph.DnaK:1:3 and T-Mt.Sali3-2-1:2:1 provided enhancement in Vn5 roots, Vn5 follicles, Vn5 source leaves, R1 source leaves, yellow round pod embryos, and yellow round pod cotyledons, but reduced expression in R1 flowers and R5 cotyledons compared to T-Gb.E6-3b:1:1 (SEQ ID NO:40).

[0171] Each of the two alfalfa 3′UTRs, T-Mt.Sali3-2-1:2:1 and T-Mt.AC140914v20-1:2:1, differently affects the expression of two constitutive EXP elements, EXP-DaMV.FLT:1:2 and EXP-CaMV.35S-enh+Ph.DnaK:1:3. Enhanced expression relative to T-Gb.E6-3b:1:1 (SEQ ID NO:40) was observed in many tissues, but decreased expression occurred in some. Therefore, by using different alfalfa 3′UTRs, it is possible to more precisely control expression in plants and better “fine-tune” the expression of specific transcribed DNA molecules to provide optimal expression when needed, while reducing expression in tissues that might negatively impact plant growth.

[0172] Example 6

[0173] The combination of *Alfalfa trichomoniasis* 3′UTR T-Mt.AC145767v28-1∶1∶2 with many different EXP molecules in stably transformed soybean plants resulted in enhanced GUS expression.

[0174] Soybean plants were transformed with vectors, particularly plasmid constructs, to evaluate the effect of alfalfa 3′UTR T-Mt.AC145767v28-1∶1∶2 (SEQ ID NO: 1) on expression. Specifically, soybean plants were transformed with vectors containing constitutive expression profiles of β-glucuronidase (GUS) transgenes operatively linked to alfalfa 3′UTR T-Mt.AC145767v28-1∶1∶2 (SEQ ID NO: 1). These alfalfa 3′UTR-transformed soybean plants were compared with soybean plants operatively linked to a 3′UTR from sea island cotton.

[0175] The vectors used in these experiments were created using cloning methods known in the art. The resulting vector contains a left-bound region from *Agrobacterium tumefaciens*; a first transgenic expression cassette for selecting transformed plant cells conferring resistance to the antibiotic spectinomycin (driven by the Arabidopsis thaliana myofibrillar 7 promoter); and a second transgenic expression cassette for evaluating the activity of 3′UTR T-Mt.AC145767v28-1∶1∶2 (SEQ ID NO: 1) containing EXP elements, namely EXP-Mt.AC145767v28∶1∶1 (SEQ ID NO: 35), EXP-CaMV.35S-enh+Ph.DnaK∶1∶3 (SEQ ID NO: 42), EXP-BSAcVNV.FLT∶1∶2 (SEQ ID NO: 52), EXP-CERV.FLT∶1∶2 (SEQ ID NO: 53), EXP-DaMV.FLT∶1∶2 (SEQ ID NO: 51), and EXP-CUCme.eEFla∶1∶1 (SEQ ID NO: 52). NO: 54) or EXP-Mt.Ubq2∶1∶2 (SEQ ID NO: 31) 5′ is operatively linked to the coding sequence of GUS having a processable intron (GUS-2, SEQ ID NO: 44), said processable intron 5′ being operatively linked to 3′UTR T-Mt.AC145767v28-1∶1∶2 (SEQ ID NO: 1) from alfalfa, or 3′UTR T-Gb.E6-3b∶1∶1 (SEQ ID NO: 40) or T-Gb.FbL2-1∶1∶1 (SEQ ID NO: 41) from sea island cotton; and the right boundary region from Agrobacterium tumefaciens. Vectors containing T-Mt.AC145767v28-1∶1∶2 (SEQ ID NO: 1) are pMON118798, pMON116815, pMON118769, pMON153709, pMON118771, pMON153707, and pMON155502. Notably, vector pMON118798 contains native EXP-Mt.AC145767v28∶1∶1, which includes a promoter element operatively linked to a leader region element cloned from the same gene locus as 3′UTR T-Mt.AC145767v28-1∶1∶2 (SEQ ID NO: 1). The carriers containing 3'UTR from Sea Island cotton are pMON102167, pMON113874, pMON121030, pMON121042, pMON140827 and pMON125841.

[0176] Table 23 provides plasmid constructs having the corresponding EXP element, 3′UTR, and SEQ ID NO for transforming the soybean plants presented in this embodiment.

[0177] Table 23. Plasmid constructs and corresponding EXP elements and 3′UTRs used for transforming soybean plants.

[0178]

[0179]

[0180] Soybean plants were transformed, and the measured GUS values ​​were as described in Example 3. Tables 24 and 25 provide the quantitative mean GUS values ​​of stable R0 generation transformed soybean plants. Table cells labeled “bdl” indicate tissues that were quantitatively analyzed but expressed below the detection level. Tables 26 and 27 provide the fold change in expression relative to each EXP element operatively linked to T-Mt.AC145767v28-1:1:2 of T-Gb.E6-3b:1:1 (SEQ ID NO:40).

[0181]

[0182]

[0183]

[0184] As shown in Tables 24 and 25, alfalfa 3′UTR T-Mt.AC145767v28-1:1:2 (SEQ ID NO:1) enhanced the expression of six constitutive EXP elements relative to T-Gb.E6-3b:1:1 (SEQ ID NO:40), but in different ways depending on the specific EXP element and tissue. The EXP element, EXP-Mt.AC145767v28:1:1, was expressed very low in all assayed tissues and was undetectable in R3 immature seeds, R3 round pods, and R1 flowers when used to drive GUS and operatively linked to its native 3′UTR T-Mt.AC145767v28-1:1:2. Compared to the combination of EXP-Mt.Ubq2:1:2 and T-Gb.FbL2-1:1:1, some tissues of plants containing the EXP element EXP-Mt.Ubq2:1:2 and the 3′UTR T-Mt.AC145767v28-1:1:2 showed reduced expression. This reduced expression was found in R3 immature seeds, R1 flowers, and R1 petioles, while Vn5 cotyledon and R5 cotyledon expression was increased by more than four-fold in contrast. Root expression (Vn5 roots) remained unchanged when using EXP-Mt.Ubq2:1:2 and the 3′UTR.

[0185] The regulatory expression element sets EXP-CERV.FLT:1:2 and EXP-DaMV.FLT:1:2 provided the highest levels of expression. As shown in Tables 26 and 27, these two EXPs were enhanced in all tissues with T-Mt.AC145767v28-1:1:2 compared to the same EXP combined with T-Gb.E6-3b:1:1 (SEQ ID NO:40). The regulatory expression element set EXP-CERV.FLT:1:2 was enhanced 60.50-fold in developing yellow round pod cotyledons and less enhanced in yellow round pod plumules (21.50-fold), while the regulatory expression element set EXP-DaMV.FLT:1:2 was enhanced to a greater extent in yellow round pod plumules (26.80-fold vs. 15.76-fold enhancement, respectively) compared to yellow round pod cotyledons. These differences in expression and enhancement provide opportunities for customized expression of transgenes in late-developing seeds. When combined with T-Gb.E6-3b:1:1, the expression modulator EXP-BSAcVNV.FLT:1:2 was most highly expressed in the R3 pod and Vn5 root (see Tables 25 and 26). When combined with T-Mt.AC145767v28-1:1:2, the expression of EXP-BSAcVNV.FLT:1:2 was significantly enhanced in both tissues, especially in the Vn5 root. Furthermore, compared to the same EXP combined with T-Gb.E6-3b:1:19 (SEQ ID NO:40), the expression of EXP-BSAcVNV.FLT:1:2 was enhanced by 58.63-fold when combined with T-Mt.AC145767v28-1:1:2.

[0186] In summary, the *Alfalfa trichomoniasis* 3′UTR T-Mt.AC145767v28-1:1:2 (SEQ ID NO:1) enhances the expression of six distinct constitutive EXP elements derived from plant and plant virus genomic DNA. Furthermore, this 3′UTR enhances the expression of the seed-preferred EXP element EXP-Gm.Sphas1∶1∶1 (SEQ ID NO:54) compared to most other alfalfa-derived 3′UTRs. Therefore, this 3′UTR is suitable for providing enhanced expression of combinations of operatively linked expression elements in promoters or constructs.

[0187] Example 7

[0188] Analysis of EXP-Mt.Ubq2∶1∶2 (SEQ ID NO: 31) in stably transformed soybean plants

[0189] Soybean plants were transformed with vectors, particularly plasmid constructs, containing a constitutive regulatory expression element set EXP-Mt.Ubq2∶1∶2 (SEQ ID NO: 31) operably linked to a GUS coding sequence. GUS expression in these transformed plants was then determined in stably transformed soybean plants.

[0190] The plant vectors used in these experiments were constructed using cloning methods known in the art. The resulting vectors contained a left-bound region from *Agrobacterium tumefaciens*; a first transgenic expression cassette selecting transformed plant cells to confer resistance to the antibiotic spectinomycin (driven by the Arabidopsis thaliana myofibrillarin 7 promoter); and a second transgenic expression cassette for evaluating the activity of EXP-Mt.Ubq2∶1∶2 (SEQ ID NO: 31), wherein the 5′ of EXP-Mt.Ubq2∶1∶2 is operatively linked to a coding sequence for β-glucuronidase (GUS) having a processable intron (GUS-2, SEQ ID NO: 44), wherein the 5′ of the processable intron is operatively linked to a 3′UTRT-Mt.AC145767v28-1∶1∶2 (SEQ ID NO: 1) from *Alfalfa truncatula*, or a 3′UTRT-Gb.E6-3b∶1∶1 (SEQ ID NO: 44) from *Cotton Island*. NO: 40) or T-Gb.FbL2-1∶1∶1 (SEQ ID NO: 41); and the right boundary region from Agrobacterium tumefaciens.

[0191] The resulting vector was used to transform soybean plants as described in Example 3. Tables 28 and 29 show the mean quantitative GUS expression values ​​determined in various tissues and the developmental time points of the stably transformed soybean plants.

[0192] Table 28. Mean GUS expression in leaves, roots and flowers of stable transformed soybean plants containing EXP-Mt.Ubq2∶1∶2 (SEQ ID NO: 31).

[0193]

[0194] Table 29. Mean GUS expression in round pods and seed tissues of stable transformed soybean plants containing EXP-Mt.Ubq2∶1∶2 (SEQ ID NO: 31).

[0195]

[0196] As shown in Tables 28 and 29, EXP-Mt.Ubq2∶1∶2 (SEQ ID NO: 31) can drive constitutive expression of transcribed DNA molecules in stable transformed soybean plants. Furthermore, different 3'UTRs affect the expression levels in each tissue. For example, combining EXP-Mt.Ubq2∶1∶2 with T-Gb.E6-3b∶1∶1 resulted in lower expression in all tested tissues compared to the other two 3'UTRs, T-Gb.FbL2-1∶1∶1 and T-Mt.AC145767v28-1∶1∶2. However, regardless of which 3'UTR was applied, EXP-Mt.Ubq2∶1∶2 provided intermediate to high constitutive expression, the extent of which could be modulated by selecting which 3'UTR was operatively linked to EXP.

[0197] Example 8

[0198] enhancer from control element

[0199] Enhancers may be derived from promoter elements provided herein, such as SEQ ID NO: 32 and 36. An enhancer element may comprise one or more cis-regulatory elements that, when operably linked at 5′ or 3′ to a promoter element or operably linked at 5′ or 3′ to an additional enhancer element operably linked to the promoter, can enhance or regulate the expression of a transcribed DNA molecule, or provide transcribed DNA molecule expression specific to a cell type or plant organ, or at a specific time point in development or diurnal rhythm. Enhancers are generated by removing a TATA box or a functionally similar element and allowing transcription to initiate from the promoter or any downstream sequence of the promoter fragment.

[0200] The enhancer element may be derived from the promoter element provided herein and cloned using methods known in the art to be 5′ or 3′ operably connected to the promoter element or to an additional enhancer element 5′ or 3′ operably connected to the promoter. Alternatively, the enhancer element may be cloned using methods known in the art to operably connect to one or more copies of the enhancer element, 5′ or 3′ operably connected to the promoter element, or 5′ or 3′ operably connected to an additional enhancer element operably connected to the promoter. Furthermore, enhancer elements may be cloned to be 5′ or 3′ operably connected to promoter elements from different genera or organisms or to additional enhancer elements from other genera or organisms of the same genera, said additional enhancer elements operably connected to promoters from the same or different genera, thereby producing chimeric regulatory elements. GUS expression plant transformation vectors can be constructed using methods known in the art similar to those described in previous examples, wherein the resulting plant expression vector contains a left boundary region from *Agrobacterium tumefaciens*; a first transgenic selection cassette conferring resistance to antibiotics or herbicides and for selecting transformed plant cells; and a second transgenic cassette wherein an enhancer element is operatively linked to a promoter to form a chimeric promoter element 5′ operatively linked to a leader element 5′ operatively linked to the coding sequence of GUS having a processable intron (GUS-2, SEQ ID NO:44), the processable intron being operatively linked to a 3′ UTR such as T-Gb.E6-3b:1:1 or any 3′ UTR from *Alfalfa tumefaciens* as described above; and a right boundary region from *Agrobacterium tumefaciens*.

[0201] GUS expression driven by regulatory elements containing one or more enhancers can be assessed in stable or transient plant assays as described herein to determine the effect of enhancer elements on the expression of transcribed DNA molecules. Modification of one or more enhancer elements or duplication of one or more enhancer elements can be performed based on empirical experiments and the resulting gene expression regulation observed using each regulatory element combination. Altering the relative positions of one or more enhancers within the resulting regulatory or chimeric regulatory element can affect transcriptional activity or the specificity of the regulatory or chimeric regulatory element and empirically determine the optimal enhancer for identifying the desired transgenic expression profile within the plant.

[0202] Example 9

[0203] Analysis of the effect of 3′UTR on the stable transformation of constitutive GUS expression in maize plants

[0204] Maize plants were transformed with binary plasmid constructs to evaluate the effect of alfalfa 3′UTR T-Mt.Oxr-1∶2∶1 (SEQ ID NO: 17) on expression relative to two 3′UTRs commonly used in maize plants. Specifically, maize plants were transformed with vectors containing an EXP that exhibits a constitutive expression profile of the expression of a β-glucuronidase (GUS) transgene operatively linked to alfalfa 3′UTR T-Mt.Oxr-1∶2∶1 (SEQ ID NO: 17). These transformed maize plants were compared with transformed maize plants in which GUS is operatively linked to 3′UTR T-AGRtu.nos-1∶1∶13 (SEQ ID NO: 49) or 3′UTR T-Os.LTP:1 (SEQ ID NO: 56).

[0205] The binary plasmid constructs used in these experiments were created using cloning methods known in the art. The resulting vector contains a right-boundary region from *Agrobacterium tumefaciens*; a first expression cassette with a 3′ UTR sequence in which the constitutive regulatory expression element group EXP-FMV.35S-enh+Ta.Lhcb1+Zm.DnaK:1:2 (SEQ ID NO: 56) 5′ is operatively linked to the coding sequence of GUS having a processable intron (GUS-2, SEQ ID NO: 44), the processable intron 5′ being operatively linked to one of the following three 3′ UTRs: T-Mt.Oxr-1∶2∶1 (SEQ ID NO: 17), T-AGRtu.nos-1∶1∶13 (SEQ ID NO: 49), or T-Os.LTP:1 (SEQ ID NO: 56); a second transgenic expression cassette for selecting transformed plant cells conferred resistance to the herbicide glyphosate (driven by the rice myofibrillar 1 promoter); and a left-boundary region from *Agrobacterium tumefaciens*. The resulting plasmid was used to transform maize plants.

[0206] Histochemical GUS analysis was used for qualitative expression analysis in transformed plants. Whole tissue sections were incubated with GUS staining solution X-Gluc (5-bromo-4-chloro-3-indolyl-β-glucuronide) (1 mg / ml) for an appropriate period of time, rinsed, and visually inspected for blue staining. GUS activity was qualitatively determined using selected plant organs and tissues by direct visual inspection or microscopic examination. Expression in R0 plants was examined in roots and leaves, as well as pollen sacs, filaments, and developing seeds and embryos 21 days post-pollination (21 DAP).

[0207] Quantitative analysis of the transformed maize plants was also performed. For quantitative analysis, total protein was extracted from selected tissues of the transformed maize plants. One microgram of total protein was used with the fluorescent substrate 4-methylumbelliferone-β-D-glucuronide (MUG) in a total reaction volume of 50 μl. The reaction product, 4-methylumbelliferone (4-MU), fluoresces maximally at a higher pH where the hydroxyl groups are ionized. An alkaline solution of sodium carbonate was added to simultaneously stop the assay and adjust the pH for quantification of the fluorescent product. Fluorescence was measured using a Fluoromax-3 (Horiba; Kyoto, Japan) with a Micromax reader, excitation at 365 nm and emission at 445 nm, the reader having a slit width set to 2 nm for excitation and 3 nm for emission.

[0208] Table 30 shows the measured average quantitative GUS expression, demonstrating the different effects of each 3′UTR on the same constitutive expression EXP.

[0209] Table 30. Mean GUS expression in maize plants transformed with different 3′UTRs.

[0210]

[0211] As can be seen in Table 30, each 3′UTR has a different effect on constitutive expression driven by EXP-FMV.35S-enh+Ta.Lhcb1+Zm.DnaK∶1∶2 (SEQ ID NO: 56). For example, 3′UTR T-Os.LTP:1 (SEQ ID NO: 56) appears to have enhanced expression in VT roots and R1 rachis / filaments relative to the other two 3′UTRs. 3′UTR T-Mt.Oxr-1∶2∶1 (SEQ ID NO: 17) appears to have enhanced expression in R3 seeds in both 21DAP endosperm and 21DAP plumules relative to T-AGRtu.nos-1∶1∶13 (SEQ ID NO: 49) and T-Os.LTP:1 (SEQ ID NO: 56). Compared to the other two 3′UTRs, the expression in flowers / pollen sacs was also higher using T-Mt.Oxr-1∶2∶1 (SEQ ID NO: 17). The observed expression differences for each 3′UTR demonstrate the usefulness of each 3′UTR in regulating expression. Therefore, these experiments show that the selection of 3′UTRs can be used in transgenic cassettes to fine-tune the expression of specific transcribed DNA molecules. This experiment also demonstrates that dicotyledonous derived 3′UTRs, such as T-Mt.Oxr-1∶2∶1, affect the ability of transcription in monocotyledonous species such as maize.

[0212] Example 10

[0213] Analysis of intron enhancement of GUS activity using plant-derived protoplasts

[0214] In general, introns are selected based on experiments and comparisons using intronless vector controls to empirically choose the optimal vector for transgene expression, including the introns and their configuration within the delivery DNA (T-DNA) element arrangement. For example, in the expression of herbicide resistance genes conferring glyphosate tolerance, such as CP4 (US RE39247), transgene expression in both reproductive and vegetative tissues is required to prevent yield loss upon herbicide application. In this example, introns are selected based on their ability to enhance herbicide resistance conferring transgene expression, particularly in the reproductive cells and tissues of the transgenic plant, when operatively linked to a constitutive promoter, thus providing nutritional and reproductive tolerance to the transgenic plant upon herbicide application. In most ubiquitin genes, the 5′UTR contains a leader region with embedded intron sequences. Therefore, regulatory elements from these genes are determined using the entire 5′UTR, including the promoter, leader region, and introns. To obtain different expression profiles or regulate transgene expression levels, introns from these regulatory elements can be removed or replaced with heterologous introns.

[0215] This document describes the intron, SEQ ID NO: 34, as being identified using genomic DNA contigs, or cDNA contigs, compared to expressed sequence tag clusters, to identify exon and intron sequences within the genomic DNA. Additionally, in cases where the gene sequence encodes a leader sequence interrupted by one or more introns, the 5′ UTR or leader sequence is also used to define intron / exon splicing of one or more introns. The intron is cloned into a plant transformation vector using methods known in the art, operatively ligated at 3′ to a regulatory element and a leader region fragment, and operatively ligated at 5′ to a second leader region fragment or coding sequence, as presented. Figure 1 The expression box in the middle.

[0216] Therefore, for example, the first possible expression box, such as Figure 1 The expression box configuration 1 includes a promoter or chimeric promoter element [A], whose 5′ is operatively connected to a preamble element [B], the preamble element 5′ being operatively connected to a test intron element [C], the test intron element being operatively connected to a coding region [D], and the coding region being operatively connected to a 3′ UTR element [E]. Alternatively, a second possible expression box, such as... Figure 1The expression box configuration 2 includes a promoter or chimeric promoter element [F], whose 5′ is operatively connected to a first preamble element or a segment of a first preamble element [G]. The first preamble element or the segment of the first preamble element 5′ is operatively connected to a test integrator element [H]. The test integrator element 5′ is operatively connected to a second preamble element or a second segment of the first preamble element [I]. The second preamble element or the second segment of the first preamble element is operatively connected to a coding region [J]. The coding region is operatively connected to a 3′ UTR element [K]. Furthermore, a third possible expression box, such as... Figure 1 The expression box configuration 3 includes a promoter or chimeric promoter element [L], whose 5′ is operatively connected to a preamble element [M]. The preamble element 5′ is operatively connected to a first segment of a coding sequence element [N]. The first segment 5′ is operatively connected to an intron element [O]. The element 5′ is operatively connected to a second segment of a coding sequence element [P]. The second segment is operatively connected to a 3′ UTR element [Q]. Notably, the expression box configuration 3 is designed to allow intron splicing in a manner that produces a fully open interpretable code group without frame changes between the first and second segment coding sequences.

[0217] As discussed herein, it is preferable to avoid using the nucleotide sequence AT or nucleotide A immediately before the 5' end of the splice site (GT) and the corresponding nucleotide G or nucleotide sequence TG immediately after the 3' end of the splice site (AG) to eliminate the possibility of unnecessary start codons being formed during the processing of messenger RNA into the final transcript. Therefore, the DNA sequence surrounding the 5' or 3' splice junction of the intron can be modified.

[0218] Introns can be measured for enhancement effects via their ability to enhance expression in transient or stable plant assays. For transient assays of intron enhancement, the basic plant vector is constructed using methods known in the art. Introns are cloned into a basic plant vector containing an expression cassette containing a constitutive EXP containing a promoter and a leader region such as EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO:42), the promoter and leader region 5′ being operatively linked to a test intron element (e.g., a SEQ ID NO:34), the test intron element being operatively linked to a coding sequence of a GUS having a processable intron (GUS-2, SEQ ID NO:44), the processable intron being operatively linked to a 3′ UTR (T-Gb.E6-3b:1:1, SEQ ID NO:40). Protoplast cells from soybean or other plant genera can be transformed using a basic plant vector and a luciferase control vector as previously described in Example 2 above, and activity can be measured. To compare the relative ability of intron-enhanced expression, GUS values ​​are expressed as the ratio of GUS to luciferase activity and compared to those levels conferred by constructs containing constitutive promoters operatively linked to known intron criteria, such as introns from the tobacco elongation factor 4A10 gene, I-Nt.eIF4A10-1:1:1 (SEQ ID NO:57), and constructs containing constitutive promoters but without operatively linked introns.

[0219] For stable plant assays of the intron presented as SEQ ID NO:34, the GUS expression plant transformation vector can be constructed similarly to the constructs described in previous examples, wherein the resulting plant expression vector contains a right-boundary region from Agrobacterium tumefaciens; a first expression cassette comprising a constitutive EXP including a promoter and a leader region such as EXP-CaMV.35S-enh+Ph.DnaK:1:3 (SEQ ID NO:42), the promoter and leader region 5′ being operatively linked to a test intron element (e.g., SEQ ID NO:34), the test intron element being operatively linked to a coding sequence of GUS having a processable intron (GUS-2, SEQ ID NO:44), the processable intron being operatively linked to a 3′ UTR from Sea Island cotton (T-Gb.E6-3b:1:1, SEQ ID NO:40). Protoplast cells from maize or other plant tissues can be transformed using the basic plant vector and a luciferase control vector as previously described in Example 2 above, and activity can be measured. To compare the relative ability of introns to enhance expression, GUS values ​​are expressed as the ratio of GUS to luciferase activity and compared with those levels conferred by constructs containing constitutive promoters operatively linked to known intron criteria, such as introns from the tobacco elongation factor 4A10 gene, I-Nt.eIF4A10-1∶1∶1 (SEQ ID NO: 57), and constructs containing constitutive promoters but without introns operatively linked to promoters.

[0220] It should be noted that the intron presented as SEQ ID NO:34 can be modified in several ways, such as by deleting segments within the intron sequence, which may reduce the expression or replication of segments for introns that can enhance expression. Additionally, DNA sequences within the intron that can affect expression specificity for specific cell types or tissues and organs can be replicated, altered, or deleted to affect transgene expression and expression patterns. Furthermore, the introns provided herein can be modified to remove any potential start codon (ATG) that could cause unintentional transcript expression of the intron from improper splicing, resulting in different, longer, or truncated protein forms. Once an intron has been empirically tested or experimentally modified, it can be used to enhance transgene expression in stable transformed plants of any genus of monocotyledonous or dicotyledonous plants, provided the intron provides enhancement of the transgene. Introns can also be used to enhance expression in other organisms, such as algae, fungi, or animal cells, provided the intron provides enhancement or attenuation or specificity of the expression of the transgene it operatively links.

[0221] *******

[0222] The principles of the invention have been shown and described, and it will be apparent to those skilled in the art that modifications in arrangement and detail may be made without departing from these principles. We claim all modifications within the spirit and scope of the claims. All publications herein are incorporated by reference as if expressly and individually indicated that individual publications or patent applications are incorporated herein by reference.

Claims

1. A recombinant DNA molecule comprising the DNA sequence of SEQ ID NO: 12; wherein the DNA sequence is operatively ligated to a heterologous transcribed DNA molecule.

2. The recombinant DNA molecule of claim 1, wherein the heterotranscribed DNA molecule comprises a gene of agronomic importance.

3. The recombinant DNA molecule of claim 2, wherein the gene of agronomic importance confers herbicide tolerance in plants.

4. The recombinant DNA molecule of claim 2, wherein the agronomically important gene confers pest resistance in the plant.

5. A method of producing a commercial product, comprising obtaining a transgenic plant or a portion thereof comprising the recombinant DNA molecule according to claim 1 and producing the commercial product therefrom.

6. The method of claim 5, wherein the commodity product is biomass.

7. The method of claim 5, wherein the product is selected from protein concentrates, protein isolates, seeds, or crude powder.

8. The method of claim 5, wherein the product is starch.

9. The method of claim 5, wherein the commodity product is grain flour.

10. The method of claim 5, wherein the commodity product is grain or seed oil.

11. A method for producing transgenic plants, comprising: a) Transforming plant cells with the recombinant DNA molecule as described in claim 1 to produce transformed plant cells; and b) To regenerate the transgenic plant from the transformed plant cells.

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