OA17484A - Plant regulatory elements and uses thereof. - Google Patents

Abstract

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

Description

PLANT REGULATORY ELEMENTS AND USES THEREOF REFERENCE TO RELATED APPLICATIONS [001] This application daims the benefit of United States provisional application Ser.

No. 61/785,268, filed March 14, 2013, which is incorporated by reference herein in its entirety.

INCORPORATION OF SEQUENCE LISTING [002] The sequence listing contained in the file named “MONS332WO.txt”, which is 52.7 kilobytes (size as measured in Microsoft Windows®) and was created on March 11, 2014, .orated by reference hereinis filed herewith by electronic submission and is incorp

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

BACKGROUND [004] Regulatory éléments are genetic éléments that regulate gene activity by modulating the transcription of an operably linked transcribable DNA molécule. Such éléments may include promoters, leaders, introns, and 3 ' untranslated régions and are useful in the field of plant molecular biology and plant genetic engineering.

SUMMARY OF THE INVENTION [005] The invention provides novel regulatory éléments for use in plants, and constructs comprising the regulatory éléments. The invention also provides transgenic plant cells, plants, and seeds comprising the regulatory éléments. In one embodiment disclosed herein, the regulatory éléments are operably linked to a transcribable DNA molécule. In certain embodiments, the transcribable DNA molécule is heterologous with respect to the regulatory sequence. Also provided herein are methods for making and using the regulatory éléments disclosed herein, including constructs comprising the regulatory éléments, and the transgenic plant cells, plants, and seeds comprising the regulatory éléments operably linked to a transcribable DNA molécule that is heterologous with respect to the regulatory element.

[006] Thus, in one aspect, the invention provides a recombinant DNA molécule comprising a DNA sequence selected from the group consisting of: (a) a DNA sequence with at least about 85 percent sequence identity to any of SEQ ID NOs: 1-37; (b) a DNA sequence comprising any of SEQ ID NOs: 1-37; and (c) a fragment of any of SEQ ID NOs: 1-37, wherein the fragment has gene-regulatory activity; wherein the DNA sequence is operably linked to a heterologous transcribable DNA molécule. By “heterologous transcribable DNA molécule,” it is meant that the transcribable DNA molécule is heterologous with respect to the DNA sequence to which it is operably linked. In spécifie embodiments, the recombinant DNA molécule comprises a DNA sequence having at least 90 percent, at least 91 percent, at least 92 percent, at least 93 percent, at least 94 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, or at least 99 percent sequence identity to the DNA sequence of any of SEQ ID NOs: 137. In particular embodiments, the heterologous transcribable DNA molécule comprises a gene of agronomie interest, such as a gene capable of providing herbicide résistance or pest résistance in plants. In still other embodiments, the invention provides a construct comprising a recombinant DNA molécule as provided herein.

[007] In another aspect, provided herein are transgenic plant cells comprising a recombinant DNA molécule comprising a DNA sequence selected from the group consisting of: (a) a DNA sequence with at least about 85 percent sequence identity to any of SEQ ID NOs: 137; (b) a DNA sequence comprising any of SEQ ID NOs: 1-37; and (c) a fragment of any of SEQ ID NOs: 1-37, wherein the fragment has gene-regulatory activity; wherein the DNA sequence is operably linked to a heterologous transcribable DNA molécule. In certain embodiments, the transgenic plant cell is a monocotyledonous plant cell. In other embodiments, the transgenic plant cell is a dicotyledonous plant cell.

[008] In still yet another aspect, further provided herein is a transgenic plant, or part thereof, comprising a recombinant DNA molécule comprising a DNA sequence selected from the group consisting of: a) a DNA sequence with at least 85 percent sequence identity to any of SEQ ID NOs: 1-37; b) a DNA sequence comprising any of SEQ ID NOs: 1-37; and c) a fragment of any of SEQ ID NOs: 1-37, wherein the fragment has gene-regulatory activity; wherein the DNA sequence is operably linked to a heterologous transcribable DNA molécule. In spécifie embodiments, the transgenic plant is a progeny plant of any génération relative to a starting transgenic plant and comprises the recombinant DNA molécule. A transgenic seed comprising the recombinant DNA molécule that produces such a transgenic plant when grown is also provided herein.

[009] In another aspect, the invention provides a method of producing a commodity product comprising obtaining a transgenic plant or part thereof containing a recombinant DNA molécule of the invention and producing the commodity product therefrom. In one embodiment, the commodity product is processed seeds, grains, plant parts, and meal.

[0010] In still yet another aspect, the invention provides a method of producing a transgenic plant comprising a recombinant DNA molécule of the invention comprising transforming a plant cell with the recombinant DNA molécule of the invention to produce a transformed plant cell and regenerating a transgenic plant from the transformed plant cell.

BRIEF DESCRIPTION OF THE FIGURES [0011] FIG. 1: Shows expression cassette configurations of the invention.

BRIEF DESCRIPTION OF THE SEQUENCES [0012] SEQIDNOs: 1-30, 38-41, 49 and 56 are 3'UTR sequences.

[0013] SEQ ID NOs: 31, 35, 42, 47, 48, 50, 51, 52, 53, 54 and 55 are DNA sequences of regulatory expression element groups (EXPs) comprising a promoter sequence operably linked 5' to a leader sequence, which is operably linked 5' to an intron sequence; or a promoter sequence operably linked 5' to a leader sequence.

[0014] SEQ ID NOs: 32, 36, and 43 are promoter sequences.

[0015] SEQ ID NOs: 33 and 37 are leader sequences.

[0016] SEQ ID NO: 34 is an intron sequence.

[0017] SEQ ID NO: 44 is a coding sequence for B-glucuronidase (GUS) that possesses a processable intron.

[0018] SEQ ID NOs: 45 and 46 are luciferase coding sequences.

DETAILED DESCRIPTION OF THE INVENTION [0019] The invention provides DNA molécules having gene-regulatory activity in plants.

The nucléotide sequences of these DNA molécules are provided as SEQ ID NOs: 1-37. These DNA molécules are capable of affecting the expression of an operably linked transcribable DNA molécule in plant tissues, and therefore regulating gene expression of an operably linked transgene in transgenic plants. The invention also provides methods of modifying, producing, and using the same. The invention also provides compositions that include transgenic plant cells, plants, plant parts, and seeds containing the recombinant DNA molécules of the invention, and methods for preparing and using the same.

[0020] The following définitions and methods are provided to better define the invention and to guide those of ordinary skill in the art in the practice of the présent invention. Unless otherwise noted, ternis are to be understood according to conventional usage by those of ordinary skill in the relevant art.

DNA Molécules [0021] As used herein, the term “DNA” or “DNA molécule” refers to a double-stranded DNA molécule of genomic or synthetic origin, i.e., a polymer of deoxyribonucleotide bases. As used herein, the term “DNA sequence” to the nucléotide sequence of a DNA molécule. The nomenclature used herein corresponds to that of Title 37 of the United States Code of Fédéral Régulations § 1.822, and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.

[0022] As used herein, a “recombinant DNA molécule” is a DNA molécule comprising a combination of DNA molécules that would not naturally occur together without human intervention. For instance, a recombinant DNA molécule may be a DNA molécule that is comprised of at least two DNA molécules heterologous with respect to each other, a DNA molécule that comprises a DNA sequence that deviates from DNA sequences that exist in nature, or a DNA molécule that has been incorporated into a host cell’s DNA by genetic transformation.

[0023] As used herein, the term “sequence identity” refers to the extent to which two optimally aligned DNA sequences are identical. An optimal sequence alignment is created by manually aligning two sequences, e.g., a reference sequence and another DNA sequence, to maximize the number of nucléotide matches in the sequence alignment with appropriate internai nucléotide insertions, délétions, or gaps. As used herein, the term “reference sequence” refers to a DNA sequence provided as SEQ ID NOs: 1-37.

[0024] As used herein, the term “percent sequence identity” or “percent identity” or “% identity” is the identity fraction multiplied by 100. The “identity fraction” for a DNA sequence optimally aligned with a reference sequence is the number of nucléotide matches in the optimal alignment, divided by the total number of nucléotides in the reference sequence, e.g., the total number of nucléotides in the full length of the entire reference sequence. Thus, one embodiment of the invention provides a DNA molécule comprising a DNA sequence that, when optimally aligned to a reference sequence, provided herein as SEQ ID NOs: 1-37, has at least about 85 percent identity, at least about 86 percent identity, at least about 87 percent identity, at least about 88 percent identity, at least about 89 percent identity, at least about 90 percent identity, at least about 91 percent identity, at least about 92 percent identity, at least about 93 percent identity, at least about 94 percent identity, at least about 95 percent identity, at least about 96 percent identity, at least about 97 percent identity, at least about 98 percent identity, at least about 99 percent identity, or at least about 100 percent identity to the reference sequence.

Regulatory Eléments [0025] Regulatory éléments such as promoters, leaders, enhancers, introns, and transcription termination régions (or 3 ' UTRs) play an intégral part in the overall expression of genes in living cells. The term “regulatory element,” as used herein, refers to a DNA molécule having gene-regulatory activity. The term “gene-regulatory activity,” as used herein, refers to the ability to affect the expression of an operably linked transcribable DNA molécule, for instance by affecting the transcription and/or translation of the operably linked transcribable DNA molécule. Regulatory éléments, such as promoters, leaders, enhancers, introns and 3’ UTRs that function in plants are therefore useful for modifying plant phenotypes through genetic engineering.

[0026] As used herein, a “regulatory expression element group” or “EXP” sequence may refer to a group of operably linked regulatory éléments, such as enhancers, promoters, leaders, and introns. Thus, a regulatory expression element group may be comprised, for instance, of a promoter operably linked 5 ' to a leader sequence, which is in tum operably linked 5 ' to an intron sequence.

[0027] Regulatory éléments may be characterized by their gene expression pattern, e.g., positive and/or négative effects such as constitutive, temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and/or chemically responsive expression, and any combination thereof, as well as by quantitative or qualitative indications. As used herein, a “gene expression pattern” is any pattern of transcription of an operably linked DNA molécule into a transcribed RNA molécule. The transcribed RNA molécule may be translated to produce a protein molécule or may provide an antisense or other regulatory RNA molécule, such as double-stranded RNA (dsRNA), a transfer RNA (tRNA), a ribosomal RNA (rRNA), a microRNA (miRNA), and the like.

[0028] As used herein, the term “protein expression” is any pattern of translation of a transcribed RNA molécule into a protein molécule. Protein expression may be characterized by its temporal, spatial, developmental, or morphological qualities, as well as by quantitative or qualitative indications, [0029] A promoter is usefiil as a regulatory element for modulating the expression of an operably linked transcribable DNA molécule. As used herein, the term “promoter” refers generally to a DNA molécule that is involved in récognition and binding of RNA polymerase II and other proteins, such as trans-acting transcription factors, to initiate transcription. A promoter may be initially identified from the 5' untranslated région (5' UTR) of a gene. Altemately, promoters may be synthetically produced or manipulated DNA molécules. Promoters may also be chimeric. Chimeric promoters are produced through the fusion of two or more heterologous DNA molécules. Promoters usefiil in practicing the invention include SEQ ID NOs: 32 and 36, including fragments or variants thereof. In spécifie embodiments of the invention, the claimed DNA molécules and any variants or dérivatives thereof as described herein, are further defined as comprising promoter activity, i.e., are capable of acting as a promoter in a host cell, such as in a transgenic plant. In still further spécifie embodiments, a fragment may be defined as exhibiting promoter activity possessed by the starting promoter molécule from which it is derived, or a fragment may comprise a “minimal promoter” which provides a basal level of transcription and is comprised of a TATA box or équivalent DNA sequence for récognition and binding of the RNA polymerase II complex for initiation of transcription.

[0030] In one embodiment, fragments of a promoter sequence disclosed herein are provided. Promoter fragments may comprise promoter activity, as described above, and may be usefiil alone or in combination with other promoters and promoter fragments, such as in constructing chimeric promoters, or in combination with other EXPs and EXP fragments. In spécifie embodiments, fragments of a promoter are provided comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, or at least about 1000 contiguous nucléotides, or longer, of a DNA molécule having promoter activity as disclosed herein. Methods for producing such fragments from a starting promoter molécule are well known in the art.

[0031] Compositions derived from any of the promoters presented as SEQ ID NOs: 32 and 36, such as internai or 5' délétions, for example, can be produced using methods well known in the art to improve or alter expression, including by removing éléments that hâve either positive or négative effects on expression; duplicating éléments that hâve positive or négative effects on expression; and/or duplicating or removing éléments that hâve tissue- or cell-specific effects on expression. Compositions derived from any of the promoters presented as SEQ ID NOs: 32 and 36 comprised of 3' délétions in which the TATA box element or équivalent sequence thereof and downstream sequence is removed can be used, for example, to make enhancer éléments. Further délétions can be made to remove any éléments that hâve positive or négative; tissue spécifie; cell spécifie; or timing spécifie (such as, but not limited to, circadian rhythm) effects on expression. Any of the promoters presented as SEQ ID NOs: 32 and 36 and fragments or enhancers derived therefrom can be used to make chimeric transcriptional regulatory element compositions.

[0032] In accordance with the invention, a promoter or promoter fragment may be analyzed for the presence of known promoter éléments, i.e., DNA sequence characteristics, such as a TATA box and other known transcription factor binding site motifs. Identification of such known promoter éléments may be used by one of skill in the art to design variants of the promoter having a similar expression pattern to the original promoter.

[0033] As used herein, the term “leader” refers to a DNA molécule identified from the untranslated 5' région (5' UTR) of a gene and defined generally as a nucléotide segment between the transcription start site (TSS) and the protein coding sequence start site. Altemately, leaders may be synthetically produced or manipulated DNA éléments. A leader can be used as a 5' regulatory element for modulating expression of an operably linked transcribable DNA molécule. Leader molécules may be used with a heterologous promoter or with their native promoter. Leaders useful in practicing the invention include SEQ ID NOs: 33 and 37 or fragments or variants thereof. In spécifie embodiments, such DNA sequences may be defined as being capable of acting as a leader in a host cell, including, for example, a transgenic plant cell. In one embodiment, such DNA sequences may be decoded as comprising leader activity.

[0034] The leader sequences presented as SEQ ID NOs: 33 and 37 may be comprised of regulatory éléments, or may adopt secondary structures that can hâve an effect on transcription or translation of an operably linked transcribable DNA molécule. The leader sequences presented as SEQ ID NOs: 33 and 37 can be used in accordance with the invention to make chimeric regulatory éléments that affect transcription or translation of an operably linked DNA molécule.

[0035] As used herein, the term “intron” refers to a DNA molécule that may be identified from a gene and may be defîned generally as a région spliced out during messenger RNA (mRNA) processing prior to translation. Altemately, an intron may be a synthetically produced or manipulated DNA element. An intron may contain enhancer éléments that effect the transcription of operably linked genes. An intron may be used as a regulatory element for modulating expression of an operably linked transcribable DNA molécule. A construct may comprise an intron, and the intron may or may not be heterologous with respect to the transcribable DNA molécule. Examples of introns in the art include the rice actin intron and the corn HSP70 intron.

[0036] In plants, the inclusion of some introns in gene constructs leads to increased mRNA and protein accumulation relative to constructs lacking the intron. This effect has been termed “intron mediated enhancement” (IME) of gene expression. Introns known to stimulate expression in plants hâve been identified in maize genes (e.g., tubAl, Adhl, Shl, and Ubil), in rice genes (e.g., tpi) and in dicotyledonous plant genes like those from pétunia (e.g., rbcS), potato (e.g., st-lsl) and from Arabidopsis thaliana (e.g., ubq3 and patl). It has been shown that délétions or mutations within the splice sites of an intron reduce gene expression, indicating that splicing might be needed for IME. However, IME in dicotyledonous plants has been shown by point mutations within the splice sites of the patl gene from A. thaliana. Multiple uses of the same intron in one plant has been shown, in certain circumstances, to exhibit disadvantages. In those cases, it is necessary to hâve a collection of basic control éléments for the construction of appropriate recombinant DNA éléments.

[0037] Introns useful in practicing the invention include SEQ IN NO: 34. Compositions derived from the intron presented as SEQ ID NO: 34 can be comprised of internai délétions or duplications of czs-regulatory éléments; and/or alterations of the 5' and 3' DNA sequences comprising the intron/exon splice junctions can be used to improve expression or specifïcity of expression when operably linked to a promoter + leader or chimeric promoter + leader and coding sequence. When modifying intron/exon boundary sequences, it may be bénéficiai to avoid using the nucléotide sequence AT or the nucléotide A just prior to the 5’ end of the splice site (GT) and the nucléotide G or the nucléotide sequence TG, respectively, just after the 3’ end of the splice site (AG) to eliminate the potential of unwanted start codons from being formed during processing of the messenger RNA into the final transcript. The DNA sequence around the 5’ or 3’ end splice junction sites of the intron can thus be modified in this manner. Intron and intron variants altered as described herein and through methods known in the art can be tested empirically as described in the working examples to détermine the intron’s effect on expression of an operably linked DNA molécule. Alterations of the 5' and 3' régions comprising the intron/exon splice junction can also be made to reduce the potential for introduction of false start and stop codons being produced in the resulting transcript after processing and splicing of the messenger RNA. The introns can be tested empirically as described in the working examples to détermine the intron’s effect on expression of a transgene.

[0038] As used herein, the terms “3' transcription termination molécule,” “3' untranslated région” or “3' UTR” refer to a DNA molécule that is used during transcription to the untranslated région of the 3 ' portion of an mRNA molécule. The 3 ' untranslated région of an mRNA molécule may be generated by spécifie cleavage and 3 ' polyadenylation, also known as a polyA tail. A 3' UTR may be operably linked to and located downstream of a transcribable DNA molécule and may include a polyadenylation signal and other regulatory signais capable of affecting transcription, mRNA processing, or gene expression. PolyA tails are thought to function in mRNA stability and in initiation of translation. Examples of 3' transcription termination molécules in the art are the nopaline synthase 3 ' région; wheat hsp 17 3' région, pea rubisco small subunit 3 ' région, cotton E6 3 ' région, and the coixin 3 ' UTR.

[0039] 3' UTRs typically find bénéficiai use for the recombinant expression of spécifie

DNA molécules. A weak 3' UTR has the potential to generate read-through, which may affect the expression of the DNA molécule located in the neighboring expression cassettes. Appropriate control of transcription termination can prevent read-through into DNA sequences (e.g., other expression cassettes) localized downstream and can further allow efficient recycling of RNA polymerase to improve gene expression. Efficient termination of transcription (release of RNA polymerase II from the DNA) is prerequisite for re-initiation of transcription and thereby directly affects the overall transcript level. Subséquent to transcription termination, the mature mRNA is released from the site of synthesis and template transported to the cytoplasm. Eukaryotic mRNAs are accumulated as poly(A) forms in vivo, making it difficult to detect transcriptional termination sites by conventional methods. However, prédiction of functional and efficient 3' UTRs by bioinformatics methods can be difficult in that there are few conserved DNA sequences that would allow for easy prédiction of an effective 3 ' UTR. — [0040] From a practical standpoint, it is typically bénéficiai that a 3' UTR used in an expression cassette possesses the following characteristics. The 3' UTR should be able to efficiently and effectively terminate transcription of the transgene and prevent read-through of the transcript into any neighboring DNA sequence, which can be comprised of another expression cassette as in the case of multiple expression cassettes residing in one transfer DNA (T-DNA), or the neighboring chromosomal DNA into which the T-DNA has inserted. In plant biotechnology, the 3' UTR is often used for priming of amplification reactions of reverse transcribed RNA extracted from the transformed plant and used to: (l) assess the transcriptional activity or expression of the expression cassette once integrated into the plant chromosome; (2) assess the copy number of insertions within the plant DNA; and (3) assess zygosity of the resulting seed after breeding. The 3' UTR is also used in amplification reactions of DNA extracted from the transformed plant to characterize the intactness of the inserted cassette.

[0041 ] As used herein, the term “enhancer” or “enhancer element” refers to a czs-acting regulatory element, a.k.a. czs-element, which confers an aspect of the overall expression pattern, but is usually insufficient alone to drive transcription, of an operably linked transcribable DNA molécule. Unlike promoters, enhancer éléments do not usually include a transcription start site (TSS) or TATA box or équivalent DNA sequence. A promoter or promoter fragment may naturally comprise one or more enhancer éléments that affect the transcription of an operably linked transcribable DNA molécule. An enhancer element may also be fused to a promoter to produce a chimeric promoter czs-element, which confers an aspect of the overall modulation of gene expression.

[0042] Many promoter enhancer éléments are believed to bind DNA-binding proteins and/or affect DNA topology, producing local conformations that selectively allow or restrict access of RNA polymerase to the DNA template or that facilitate sélective opening of the double hélix at the site of transcriptional initiation. An enhancer element may function to bind transcription factors that regulate transcription. Some enhancer éléments bind more than one transcription factor, and transcription factors may interact with different affinities with more than one enhancer domain. Enhancer éléments can be identified by a number of techniques, including délétion analysis, i.e., deleting one or more nucléotides from the 5' end or internai to a promoter; DNA binding protein analysis using DNase I footprinting, méthylation interférence, electrophoresis mobility-shift assays, in vivo genomic footprinting by ligation-mediated polymerase chain reaction (PCR), and other conventional assays; or by DNA sequence similarity analysis using known cA-element motifs or enhancer éléments as a target sequence or target motif with conventional DNA sequence comparison methods, such as BLAST. The fine structure of an enhancer domain can be further studied by mutagenesis (or substitution) of one or more nucléotides or by other conventional methods known in the art. Enhancer éléments can be obtained by chemical synthesis or by isolation from regulatory éléments that include such éléments, and they can be synthesized with additional flanking nucléotides that contain useful restriction enzyme sites to facilitate subsequence manipulation. Thus, the design, construction, and use of enhancer éléments according to the methods disclosed herein for modulating the expression of operably linked transcribable DNA molécules are encompassed by the invention.

[0043] As used herein, the term “chimeric” refers to a single DNA molécule produced by fusing a first DNA molécule to a second DNA molécule, where neither the first nor the second DNA molécule would normally be found in that configuration, i.e., fused to the other. The chimeric DNA molécule is thus a new DNA molécule not otherwise normally found in nature. As used herein, the term “chimeric promoter” refers to a promoter produced through such manipulation of DNA molécules. A chimeric promoter may combine two or more DNA fragments, for example, the fusion of a promoter to an enhancer element. Thus, the design, construction, and use of chimeric promoters according to the methods disclosed herein for modulating the expression of operably linked transcribable DNA molécules are encompassed by the présent invention.

[0044] As used herein, the term “variant” refers to a second DNA molécule, such as a regulatory element, that is similar in composition, but not identical to, a first DNA molécule, and wherein the second DNA molécule still maintains the general functionality, i.e., same or similar expression pattern, for instance through more or less or équivalent transcriptional or translational activity, of the first DNA molécule. A variant may be a shorter or truncated version of the first DNA molécule and/or an altered version of the sequence of the first DNA molécule, such as one with different restriction enzyme sites and/or internai délétions, substitutions, and/or insertions. A “variant” can also encompass a regulatory element having a nucléotide sequence comprising a substitution, délétion, and/or insertion of one or more nucléotides of a reference sequence, wherein the dérivative regulatory element has more or less or équivalent transcriptional or translational activity than the corresponding parent regulatory molécule. Regulatory element “variants” also encompass variants arising from mutations that occur during or as a resuit of bacterial and plant cell transformation. In the invention, a DNA sequence provided as SEQ ID NOs: l-37 may be used to create variants that are in similar in composition, but not identical to, the DNA sequence of the original regulatory element, while still maintaining the general functionality, i.e., the same or similar expression pattern, of the original regulatory element. Production of such variants of the invention is well within the ordinary skill of the art in light of the disclosure and is encompassed within the scope of the invention.

[0045] Chimeric regulatory éléments can be designed to comprise various constituent éléments which may be operatively linked by various methods known in the art, such as restriction enzyme digestion and ligation, ligation independent cloning, modular assembly of PCR products during amplification, or direct chemical synthesis of the regulatory element, as well as other methods known in the art. The resulting various chimeric regulatory éléments can be comprised of the same, or variants of the same, constituent éléments but differ in the DNA sequence or DNA sequences that comprise the linking DNA sequence or sequences that allow the constituent parts to be operatively linked. In the invention, a DNA sequence provided as SEQ ID NOs: 1-30 or 31-37 may provide a regulatory element reference sequence, wherein the constituent éléments that comprise the reference sequence may be joined by methods known in the art and may comprise substitutions, délétions, and/or insertions of one or more nucléotides or mutations that naturally occur in bacterial and plant cell transformation.

[0046] The efficacy of the modifications, duplications, or délétions described herein on the desired expression aspects of a particular transcribable DNA molécule may be tested empirically in stable and transient plant assays, such as those described in the working examples herein, so as to validate the results, which may vary depending upon the changes made and the goal of the change in the starting DNA molécule.

Constructs [0047] As used herein, the term “construct” means any recombinant DNA molécule such as a plasmid, cosmid, virus, phage, or linear or circular DNA or RNA molécule, derived from any source, capable of genomic intégration or autonomous réplication, comprising a DNA molécule where at least one DNA molécule has been linked to another DNA molécule in a functionally operative manner, i.e., operably linked. As used herein, the term “vector” means any construct that may be used for the purpose of transformation, i.e., the introduction of vV heterologous DNA or RNA into a host cell. A construct typically includes one or more expression cassettes. As used herein, an “expression cassette” refers to a DNA molécule comprising at least a transcribable DNA molécule operably linked to one or more regulatory éléments, typically at least a promoter and a 3’ UTR.

[0048] As used herein, the term “operably linked” refers to a first DNA molécule joined to a second DNA molécule, wherein the first and second DNA molécules are arranged so that the first DNA molécule affects the function of the second DNA molécule. The two DNA molécules may or may not be part of a single contiguous DNA molécule and may or may not be adjacent. For example, a promoter is operably linked to a transcribable DNA molécule if the promoter is capable of affecting the transcription or translation of the transcribable DNA molécule.

[0049] The constructs of the invention may be provided, in one embodiment, as double tumor-inducing (Ti) plasmid border constructs that hâve the right border (RB or AGRtu.RB) and left border (LB or AGRtu.LB) régions of the Ti plasmid isolated from Agrobacterium tumefaciens comprising a T-DNA that, along with transfer molécules provided by the A. tumefaciens cells, permit the intégration of the T-DNA into the genome of a plant cell (see, e.g., U.S. Patent 6,603,061). The constructs may also contain the plasmid backbone DNA segments that provide réplication function and antibiotic sélection in bacterial cells, e.g., an Escherichia coli origin of réplication such as ori322, a broad host range origin of réplication such as oriV or oriRi, and a coding région for a selectable marker such as Spec/Strp that encodes for Tn7 aminoglycoside adenyltransferase (aadA) conferring résistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectable marker gene. For plant transformation, the host bacterial strain is often A. tumefaciens ABI, C58, or LBA4404; however, other strains known to those skilled in the art of plant transformation can function in the invention.

[0050] Methods are known in the art for assembling and introducing constructs into a cell in such a manner that the transcribable DNA molécule is transcribed into a functional mRNA molécule that is translated and expressed as a protein. For the practice of the invention, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art. For example, typical vectors useful for expression of nucleic acids in higher plants are well known in the art and include vectors derived from the Ti plasmid of Agrobacterium tumefaciens and the pCaMVCN transfer control vector.

[0051] Various regulatory éléments may be included in a construct, including any of those provided herein. Any such regulatory éléments may be provided in combination with other regulatory éléments. Such combinations can be designed or modified to produce désirable regulatory features. In one embodiment, constructs of the invention comprise at least one regulatory element operably linked to a transcribable DNA molécule operably linked to a 3' UTR.

[0052] Constructs of the invention may include any promoter or leader provided herein or known in the art. For example, a promoter of the invention may be operably linked to a heterologous non-translated 5' leader such as one derived from a heat shock protein gene. Altematively, a leader of the invention may be operably linked to a heterologous promoter such as the Cauliflower Mosaic Virus 35S transcript promoter.

[0053] Expression cassettes may also include a transit peptide coding sequence that encodes a peptide that is useful for sub-cellular targeting of an operably linked protein, particularly to a chloroplast, leucoplast, or other plastid organelle; mitochondria; peroxisome; vacuole; or an extracellular location. Many chloroplast-localized proteins are expressed from nuclear genes as precursors and are targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, those associated with the small subunit (SSU) of ribulose-l,5,-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, the light-harvesting complex protein I and protein II, thioredoxin F, and enolpyruvyl shikimate phosphate synthase (EPSPS). Chloroplast transit peptides are described, for example, in U.S. Patent No. 7,193,133. It has been demonstrated that nonchloroplast proteins may be targeted to the chloroplast by the expression of a heterologous CTP operably linked to the transcribable DNA molécule encoding non-chloroplast proteins.

Transcribable DNA molécules [0054] As used herein, the terni “transcribable DNA molécule” refers to any DNA molécule capable of being transcribed into a RNA molécule, including, but not limited to, those having protein coding sequences and those producing RNA molécules having sequences useful for gene suppression. The type of DNA molécule can include, but is not limited to, a DNA molécule from the same plant, a DNA molécule from another plant, a DNA molécule from a different organism, or a synthetic DNA molécule, such as a DNA molécule containing an antisense message of a gene, or a DNA molécule encoding an artifïcial, synthetic, or otherwise — modified version of a transgene. Exemplary transcribable DNA molécules for incorporation into constructs of the invention include, e.g., DNA molécules or genes from a species other than the species into which the DNA molécule is incorporated or genes that originate from, or are présent in, the same species, but are incorporated into récipient cells by genetic engineering methods rather than classical breeding techniques.

[0055] A “transgene” refers to a transcribable DNA molécule heterologous to a host cell at least with respect to its location in the host cell genome and/or a transcribable DNA molécule artificially incorporated into a host cell’s genome in the current or any prior génération of the cell.

[0056] A regulatory element, such as a promoter of the invention, may be operably linked to a transcribable DNA molécule that is heterologous with respect to the regulatory element. As used herein, the term “heterologous” refers to the combination of two or more DNA molécules when such a combination is not normally found in nature. For example, the two DNA molécules may be derived from different species and/or the two DNA molécules may be derived from different genes, e.g., different genes from the same species or the same genes from different species. A regulatory element is thus heterologous with respect to an operably linked transcribable DNA molécule if such a combination is not normally found in nature, i.e., the transcribable DNA molécule does not naturally occur operably linked to the regulatory element.

[0057] The transcribable DNA molécule may generally be any DNA molécule for which expression of a transcript is desired. Such expression of a transcript may resuit in translation of the resulting mRNA molécule, and thus protein expression. Altematively, for example, a transcribable DNA molécule may be designed to ultimately cause decreased expression of a spécifie gene or protein. In one embodiment, this may be accomplished by using a transcribable DNA molécule that is oriented in the antisense direction. One of ordinary skill in the art is familiar with using such antisense technology. Any gene may be negatively regulated in this manner, and, in one embodiment, a transcribable DNA molécule may be designed for suppression of a spécifie gene through expression of a dsRNA, siRNA or miRNA molécule.

[0058] Thus, one embodiment of the invention is a recombinant DNA molécule comprising a regulatory element of the invention, such as those provided as SEQ ID NOs: l-37, operably linked to a heterologous transcribable DNA molécule so as to modulate transcription of the transcribable DNA molécule at a desired level or in a desired pattern when the construct is integrated in the genome of a transgenic plant cell. In one embodiment, the transcribable DNA molécule comprises a protein-coding région of a gene and in another embodiment the transcribable DNA molécule comprises an antisense région of a gene.

Genes of Agronomie Interest [0059] A transcribable DNA molécule may be a gene of agronomie interest. As used herein, the term “gene of agronomie interest” refers to a transcribable DNA molécule that, when expressed in a particular plant tissue, cell, or cell type, confers a désirable characteristic. The product of a gene of agronomie interest may act within the plant in order to cause an effect upon the plant morphology, physiology, growth, development, yield, grain composition, nutritional profile, disease or pest résistance, and/or environmental or chemical tolérance or may act as a pesticidal agent in the diet of a pest that feeds on the plant. In one embodiment of the invention, a regulatory element of the invention is incorporated into a construct such that the regulatory element is operably linked to a transcribable DNA molécule that is a gene of agronomie interest. In a transgenic plant containing such a construct, the expression of the gene of agronomie interest can confer a bénéficiai agronomie trait. A bénéficiai agronomie trait may include, for example, but is not limited to, herbicide tolérance, insect control, modified yield, disease résistance, pathogen résistance, modified plant growth and development, modified starch content, modified oil content, modified fatty acid content, modified protein content, modified fruit ripening, enhanced animal and human nutrition, biopolymer productions, environmental stress résistance, pharmaceutical peptides, improved processing qualities, improved flavor, hybrid seed production utility, improved fiber production, and désirable biofuel production.

[0060] Examples of genes of agronomie interest known in the art include those for herbicide résistance (U.S. 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), increased yield (U.S. Patent Nos. USRE38,446; 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211; 6,235,971; 6,222,098; and 5,716,837), insect control (U.S. 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 disease résistance (U.S. 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 résistance (U.S. Patent Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; and 5,304,730), nematode résistance (U.S. Patent No. 6,228,992), bacterial disease résistance (U.S. Patent No. 5,516,671), plant growth and development (U.S. Patent Nos. 6,723,897 and 6,518,488), starch production (U.S. Patent Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), modified oils production (U.S. Patent Nos. 6,444,876; 6,426,447; and 6,380,462), high oïl production (U.S. Patent Nos. 6,495,739; 5,608,149; 6,483,008; and 6,476,295), modified fatty acid content (U.S. 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; 6,489,461; and 6,459,018), high protein production (U.S. Patent No. 6,380,466), fruit ripening (U.S. Patent No. 5,512,466), enhanced animal and human nutrition (U.S. Patent Nos. 6,723,837; 6,653,530; 6,5412,59; 5,985,605; and 6,171,640), biopolymers (U.S. Patent Nos. USRE37,543; 6,228,623; and 5,958,745, and 6,946,588), environmental stress résistance (U.S. Patent No. 6,072,103), pharmaceutical peptides and secretable peptides (U.S. Patent Nos. 6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processing traits (U.S. Patent No. 6,476,295), improved digestibility (U.S. Patent No. 6,531,648) low raffinose (U.S. Patent No. 6,166,292), industrial enzyme production (U.S. Patent No. 5,543,576), improved flavor (U.S. Patent No. 6,011,199), nitrogen fixation (U.S. Patent No. 5,229,114), hybrid seed production (U.S. Patent No. 5,689,041), fiber production (U.S. Patent Nos. 6,576,818; 6,271,443; 5,981,834; and 5,869,720) and biofuel production (U.S. Patent No. 5,998,700).

[0061] Altematively, a gene of agronomie interest can affect the above mentioned plant characteristics or phenotypes by encoding a RNA molécule that causes the targeted modulation of gene expression of an endogenous gene, for example by antisense (see, e.g. U.S. Patent 5,107,065); inhibitory RNA (“RNAi,” including modulation of gene expression by miRNA-, siRNA-, trans-acting siRNA-, and phased sRNA-mediated mechanisms, e.g., as described in published applications U.S. 2006/0200878 and U.S. 2008/0066206, and in U.S. Patent application 11/974,469); or cosuppression-mediated mechanisms. The RNA could also be a catalytic RNA molécule (e.g., a ribozyme or a riboswitch; see, e.g., U.S. 2006/0200878) engineered to cleave a desired endogenous mRNA product. Methods are known in the art for constructing and introducing constructs into a cell in such a manner that the transcribable DNA molécule is transcribed into a molécule that is capable of causing gene suppression.

Selectable Markers [0062] Selectable marker transgenes may also be used with the regulatory éléments of the invention. As used herein the term “selectable marker transgene” refers to any transcribable DNA molécule whose expression in a transgenic plant, tissue or cell, or lack thereof, can be screened for or scored in some way. Selectable marker genes, and their associated sélection and screening techniques, for use in the practice of the invention are known in the art and include, but are not limited to, transcribable DNA molécules encoding β-glucuronidase (GUS), luciferase, green fluorescent protein (GFP), proteins that confer antibiotic résistance, and proteins that confer herbicide tolérance.

Cell Transformation [0063] The invention is also directed to a method of producing transformed cells and plants that comprise one or more regulatory éléments operably linked to a transcribable DNA molécule.

[0064] The term “transformation” refers to the introduction of a DNA molécule into a récipient host. As used herein, the term “host” refers to bacteria, fiingi, or plants, including any cells, tissues, organs, or progeny of the bacteria, fungi, or plants. Plant tissues and cells of particular interest include protoplasts, calli, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.

[0065] As used herein, the term “transformed” refers to a cell, tissue, organ, or organism into which a foreign DNA molécule, such as a construct, has been introduced. The introduced DNA molécule may be integrated into the genomic DNA of the récipient cell, tissue, organ, or organism such that the introduced DNA molécule is inherited by subséquent progeny. A “transgenic” or “transformed” cell or organism may also includes progeny of the cell or organism and progeny produced from a breeding program employing such a transgenic organism as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a foreign DNA molécule. The introduced DNA molécule may also be transiently introduced into the récipient cell such that the introduced DNA molécule is not inherited by subséquent progeny. The term “transgenic” refers to a bacterium, fungus, or plant containing one or more heterologous DNA molécules.

[0066] There are many methods well known to those of skill in the art for introducing

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

[0067] Host cells may be any cell or organism, such as a plant cell, algal cell, algae, fungal cell, fungi, bacterial cell, or insect cell. In spécifie embodiments, the host cells and transformed cells may include cells from crop plants.

[0068] A transgenic plant subsequently may be regenerated from a transgenic plant cell of the invention. Using conventional breeding techniques or self-pollination, seed may be produced from this transgenic plant. Such seed, and the resulting progeny plant grown from such seed, will contain the recombinant DNA molécule of the invention, and therefore will be transgenic.

[0069] Transgenic plants of the invention can be self-pollinated to provide seed for homozygous transgenic plants of the invention (homozygous for the recombinant DNA molécule) or crossed with non-transgenic plants or different transgenic plants to provide seed for heterozygous transgenic plants of the invention (heterozygous for the recombinant DNA molécule). Both such homozygous and heterozygous transgenic plants are referred to herein as “progeny plants.” Progeny plants are transgenic plants descended from the original transgenic plant and containing the recombinant DNA molécule of the invention. Seeds produced using a transgenic plant of the invention can be harvested and used to grow générations of transgenic plants, i.e., progeny plants of the invention, comprising the construct of this invention and expressing a gene of agronomie interest. Descriptions of breeding methods that are commonly used for different crops can be found in one of several reference books, see, e.g., Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U. of CA, Davis, CA, 50-98 (I960); Simmonds, Principles of Crop Improvement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen, Plant breeding Perspectives, Wageningen (ed), Center for Agricultural Publishing and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses, 2nd Edition, Monograph, 16:249 (1987); Fehr, Principles of Variety Development, Theory and Technique,

M/' (Vol. I) and Crop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY, 360376(1987).

[0070] The transformed plants may be analyzed for the presence of the gene or genes of interest and the expression level and/or profile conferred by the regulatory éléments of the invention. Those of skill in the art are aware of the numerous methods available for the analysis of transformed plants. For example, methods for plant analysis include, but are not limited to, Southem blots or northem blots, PCR-based approaches, biochemical analyses, phenotypic screening methods, field évaluations, and immunodiagnostic assays. The expression of a transcribable DNA molécule can be measured using TaqMan® (Applied Biosystems, Foster City, CA) reagents and methods as described by the manufacturer and PCR cycle times determined using the TaqMan® Testing Matrix. Altematively, the Invader® (Third Wave Technologies, Madison, WI) reagents and methods as described by the manufacturer can be used to evaluate transgene expression.

[0071] The invention also provides for parts of a plant of the invention. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovule, and pollen. Plant parts of the invention may be viable, non viable, regenerable, and/or non-regenerable. The invention also includes and provides transformed plant cells comprising a DNA molécule of the invention. The transformed or transgenic plant cells of the invention include regenerable and/or non-regenerable plant cells.

[0072] The invention also provides a commodity product that is produced from a transgenic plant or part thereof containing the recombinant DNA molécule of the invention. Commodity products of the invention contain a détectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NO: 1-37. As used herein, a “commodity product” refers to any composition or product which is comprised of material derived from a transgenic plant, seed, plant cell, or plant part containing the recombinant DNA molécule of the invention. Commodity products include but are not limited to processed seeds, grains, plant parts, and meal. A commodity product of the invention will contain a détectable amount of DNA corresponding to the recombinant DNA molécule of the invention. Détection of one or more of this DNA in a sample may be used for determining the content or the source of the commodity product. Any standard method of détection for DNA molécules may be used, including methods of détection disclosed herein. _y<_r~ [0073] The invention may be more readily understood through reference to the following examples, which are provided by way of illustration, and are not intended to be limiting of the invention, unless specified. It should be appreciated by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the invention. However, those of skill in the art should, in light of the présent disclosure, appreciate that many changes can be made in the spécifie embodiments that are disclosed and still obtain a like or similar resuit without departing from the spirit and scope of the invention, therefore ail matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.

EXAMPLES

Example 1 Identification and Cloning of Regulatory Eléments [0074] Regulatory expression element groups (EXPs) and transcription termination régions (3' UTRs) were identified and cloned from the genomic DNA of the dicot species Medicago tnincatula (Barrel Medic). The sélection of the Medicago tnincatula 3’ UTRs was, in part, based on expression patterns observed in homologous soybean genes.

[0075] The identification and cloning of Medicago tnincatula 3' UTRs began with the sélection of soybean genes of interest based upon the soybean genes’ expression pattern in soy tissue surveys and proprietary transcript profiling experiments. The selected soybean genes were then used to find homologous genes in Medicago tnincatula using publicly available DNA sequences. Next, tissue samples derived from Medicago tnincatula were isolated from plants grown under different environmental conditions. Then, messenger RNA (mRNA) was isolated from the Medicago tissues and used in real time polymerase chain reaction (PCR) experiments to détermine the expression pattern of the Medicago genes. From these experiments, a subset of the Medicago tnincatula genome was selected for cloning and characterization.

[0076] Using public Medicago tnincatula sequence data, a bioinformatic analysis was performed to identify regulatory éléments within the selected Medicago gene loci. For example, bioinformatic analysis was performed to identify 3' UTR sequences that comprise the polyadenylation and termination régions of the mRNA and sequences extending further to the end of the identified gene locus. 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 comprising a promoter, leader and intron, and DNA fragments comprising a promoter and leader. The resulting DNA fragments were ligated into base plant expression vectors and sequenced.

[0077] For applicable DNA fragments, an analysis of the regulatory element transcription start site (TSS) and intron/exon splice junctions was then performed using transformed plant protoplasts. In this analysis, the protoplasts were transformed with the plant expression vectors comprising the cloned DNA fragments operably linked to a heterologous transcribable DNA molécule. Next, 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 splice junctions by analyzing the DNA sequence of the produced mRNA transcripts.

[0078] The DNA sequences of the identified 3' UTRs are provided herein as SEQ ID NOs: l-30. In addition, identified promoter DNA sequences are provided herein as SEQ ID NOs: 32 and 36; identified leader DNA sequences are provided herein as SEQ ID NOs: 33 and 37; and an identified intron DNA sequence is provided as SEQ ID NO: 34. Further, the DNA sequences of the identified EXPs are provided herein as SEQ ID NOs: 31 and 35. The regulatory expression element group EXP-Mt.Ubq2:1:2 (SEQ ID NO: 31) comprises a promoter element, PMt.Ubq2-l:l:l (SEQ IDNO: 32), operably linked 5' to a leader element, L-Mt.Ubq2-l:l:l (SEQ ID NO: 33), operably linked 5' to an intron element, I-Mt.Ubq2-l:l:2 (SEQ ID NO: 34) and the regulatory expression element group EXP-Mt.AC145767v28:l:l (SEQ ID NO: 35) comprises a promoter element, P-Mt.AC145767v28-l:2:l (SEQ ID NO: 36), operably linked 5' to a leader element, L-Mt.AC145767v28-l:l:2 (SEQ ID NO: 37). Each of the DNA sequences identified and cloned from Medicago truncatula are listed in Table 1.

Table 1. 3' UTRs, Regulatory expression element groups, promoters, leaders, and introns cloned from Medicago truncatula.

Description	SEQ ID NO:	Annotation
T-Mt.AC145767v28-l:l:2	1	AC 145767.28
T-Mt.AC140914v20-l:2:l	2	AC 140914.20
T-Mt.AC 139600v 16-1:2:1	3	AC 139600.16

Description	SEQ ID NO:	Annotation
T-Mt.ACl53l25V10-l:2:l	4	AC153125.10
T-Mt.Apx-l:l:2	5	cytosolic ascorbate peroxidase
T-Mt.EFla-l:l:2	6	élongation factor 1 alpha
T-Mt.Exprl-1:2:1	7	putative oxidoreductase
T-Mt.FBA-l:l:5	8	fructose biphasphate aldolase, cytoplasmic isozyme 2
T-Mt.FBA-l:2:l	9	fructose biphasphate aldolase, cytoplasmic isozyme 2
T-Mt.Gapdh-l:2:l	10	glyceraldehyde-3-phosphate dehydrogenase
T-Mt.Gpi-1:2:1	11	GPI-anchored protein
T-Mt.Hsp20-l:2:l	12	heat shock protein 20
T-Mt.Lhcb2-l:2:l	13	chlorophyll a/b binding protein type II precursor
T-Mt.Lox-l-l:2:l	14	lipoxygenase
T-Mt.Methm-1:2:1	15	5-methyltetrahydropteroyltriglutamate-homocysteine S-methyltransferase
T-MLMP21-1:2:1	16	seed maturation protein
T-Mt.Oxr-l:2:l	17	putative oxidoreductase
T-Mt.Pip 1-1:2:1	18	plasma membrane intégral protein
T-Mt.Prx-l:l:l	19	peroxidase
T-Mt.PSII-T_A-l:2:l	20	photosystem II 5 kDa protein, chloroplast precursor
T-Mt.PSII-T_B-l:2:l	21	photosystem II 5 kDa protein, chloroplast precursor
T-Mt.Ptl-1:2:2	22	phosphate Transporter
T-Mt.Pt2-1:2:2	23	phosphate Transporter
T-Mt.RD22-1:2:1	24	dehydration-responsive protein
T-Mt.RpL3-l:2:l	25	ribosomal protein L3
T-Mt.Sali3-2-1:2:1	26	aluminum-induced Sali3-2 protein
T-Mt.Scp-l:2:l	27	serine carboxypeptidase-related protein
T-Mt.SeqID#21-1:2:1	28	peroxidase
T-Mt.Suil-l:l:2	29	SUI1 translation initiation factor

Description	SEQ ID NO:	Annotation
T-Mt.Zfp-1:2:1	30	CCCH-type zinc finger protein
EXP-Mt.Ubq2:l:2	31	Ubiquitin 2
P-Mt.Ubq2-l:l:l	32	Ubiquitin 2
L-Mt.Ubq2-l:l:l	33	Ubiquitin 2
I-Mt.Ubq2-l:l:2	34	Ubiquitin 2
EXP-Mt.AC145767v28:l:l	35	AC 145767.28
P-Mt.AC145767v28-1:2:1	36	AC145767.28
L-Mt.AC145767v28-l:l:2	37	AC145767.28

Example 2

Analysis of the Effect of 3' UTRs on Constitutive GUS Expression in Soybean Leaf Protoplasts [0079] Soybean leaf protoplasts were transformed with vectors, specifïcally plasmid constructs, to assess the effect of selected Medicago truncatula 3' UTRs on expression. Soybean leaf protoplasts were transformed with DNA vectors containing a constitutive EXP sequence driving expression of the β-glucuronidase (GUS) transgene operably linked to a Medicago 3' UTR. These Medicago 3 ' UTR-transformed soybean leaf protoplasts were compared to soybean leaf protoplast in which expression of the GUS transgene was driven by a constitutive promoter, and the GUS transgene was operably linked to a 3' UTR derived from Gossypium hirsutum or Gossypium barbadense.

[0080] The plant vectors utilized in these experiments were built using cloning methods known in the art. The resulting vectors comprised a left border région from A. tumefaciens', a first transgene expression cassette for sélection of transformed plant cells that confers résistance to either the herbicide glyphosate or the antibiotic spectinomycin (both driven by the Arabidopsis Actin 7 promoter); a second transgene expression cassette used to assess the activity of the 3' UTR, which comprised an EXP or promoter sequence operably linked 5 ' to a DNA sequence for GUS that possesses a processable intron (GUS-2, SEQ ID NO: 44), which is operably linked 5' to 3 ' UTR derived from Medicago truncatula, Gossypium hirsutum, or Gossypium barbadense', and a right border région from A. tumefaciens. The vectors that comprised a 3 ' UTR derived from Medicago (i.e., pMON109593, pMONl 16803, pMONl 16812, pMONl 16813, pMONl 16815, pMONl 16826, pMONl 16827, pMONl 16830, pMONl22852, pMONl22853, pMON122854, pMON122855, pMON122856, pMON122857, pMON122858, pMON122859, 5 pMON122862, pMON122864, pMON122865, pMON122866, pMON122867, and pMON122868) used the constitutive regulatory expression element group EXP-CaMV.35Senh+Ph.DnaK:l:3 (SEQ ID NO: 42) to drive GUS. The vectors that comprised a 3’ UTR derived from Gossypium hirsutum or Gossypium barbadense (i.e., pMON81345, pMON81347, and pMON83002) used the constitutive promoter P-CaMV.35S-enh-l:l:l 1 (SEQ ID NO: 43) to 10 drive GUS.

[0081] Table 2 provides the plasmid constructs with the corresponding 3' UTR and SEQ ID NO used to transform the soybean protoplasts in experiments presented in this Example.

Table 2. Plasmid constructs used to transform soybean leaf protoplasts and 3' UTR descriptions.

Plasmid Construct	3’ UTR Description	SEQ ID NO:
pMON81345	T-Gb.FbL2-l:l:l	41
pMON81347	T-Gh.E6-4A-0:2:l	38
pMON83002	T-Gb.H6-1:2:1	39
pMONl 09593	T-Mt.Pt 1-1:2:2	22
pMONl 16803	T-Mt.AC140914v20-l:2:l	2
pMONl 16812	T-Mt.Lhcb2-l:2:l	13
pMONl 16813	T-Mt.PSII-T_B-l:2:l	21
pMONl 16815	T-Mt.AC145767v28-l:l:2	1
pMONl 16826	T-Mt.Lox-l-l:2:l	14
pMONl 16827	T-Mt.Gpi-l:2:l	11
pMONl 16830	T-Mt.Scp-l:2:l	27
pMONl 22852	T-Mt.Methm-1:2:1	15
pMONl 22853	T-Mt.Prx-l:l:l	19

Plasmid Construct	3’ UTR Description	SEQ ID NO:
pMON 122854	T-Mt.Gapdh-l:2:l	10
pMON 122855	T-Mt.FBA-l:l:5	8
pMON 122856	T-Mt.Zfp-1:2:1	30
pMON 122857	T-Mt.AC139600vl6-l:2:l	3
pMON 122858	T-Mt.MP21-1:2:1	16
pMON 122859	T-Mt.Oxr-l:2:l	17
pMON 122862	T-Mt.Suil-l:l:2	29
pMON 122864	T-Mt.Pip 1-1:2:1	18
pMON 122865	T-Mt.AC153125V10-l:2:l	4
pMON 122866	T-Mt.Sali3-2-l:2:l	26
pMON 122867	T-Mt.Hsp20-1:2:1	12
pMON 122868	T-Mt.Exprl-l:2:l	7

[0082] Two plant vectors, specifically plasmid constructs, for use in co-transformation and normalization of data were also built using cloning methods known in the art. Each of these plasmid constructs contained a spécifie luciferase coding sequence that was driven by a 5 constitutive EXP. The plant vector pMONl9437 comprised an expression cassette with a constitutive EXP comprising a promoter operably linked 5' to a leader sequence which is operably linked 5’ to an intron (EXP-CaMV.35S-enh+Zm.DnaK:l:l, SEQ ID NO: 47), operably linked 5' to a firefly (Photinus pyralis) luciferase coding sequence (LUCIFERASE: 1:3, SEQ ID NO: 45), operably linked 5' to a 3' UTR from the Agrobacterium tumefaciens nopaline synthase 10 gene (T-AGRtu.nos-l:l:13, SEQ ID NO: 49). The plant vector pMON63934 comprised an expression cassette with a constitutive EXP sequence comprising a promoter operably linked 5’ to a leader sequence (EXP-CaMV.35S-enh-Lhcbl, SEQ ID NO: 48), operably linked 5' to a sea pansy (Renilla reniformis) luciferase coding sequence (CR-Ren.hRenilla Lucife-0:0:l, SEQ ID NO: 46), operably linked 5' to a 3' UTR from the Agrobacterium tumefaciens nopaline synthase 15 gene (T-AGRtu.nos-l:l:13, SEQ ID NO: 49).

[0083] The soybean leaf protoplasts were transformée! using a polyethylene glycol (PEG)-based transformation method, as is well known in the art. Each protoplast cell was transformed with the pMON 19437 plasmid construct, the pMON63934 plasmid construct, and one of the plasmid constructs presented in Table 2. After transformation, the transformed 5 soybean leaf protoplasts were incubated ovemight in total darkness. Next, measurement of GUS and luciferase was conducted by placing aliquots of a lysed préparation of transformed cells into two different small-well trays. One tray was used for GUS measurements, and a second tray was used to perforai a dual luciferase assay using the dual luciferase reporter assay System (Promega Corp., Madison, WI; see, e.g., Promega Notes Magazine, NO: 57, 1996, p.02).

[0084] One or two transformations were performed for each plasmid construct presented in Table 2. The mean expression values for each 3' UTR were determined from several samples from each transformation. Sample measurements were made using four replicates of each plasmid construct transformation, or altematively, three replicates of each plasmid construct per one of two transformation experiments. The mean GUS and luciferase expression levels are provided in Table 3. In this Table, the firefly luciferase values (e.g., from expression of pMON19437) are provided in the column labeled “FLuc” and the sea pansy luciferase values (e.g., from expression of pMON63934) are provided in the column labeled “RLuc.”

Table 3. Mean GUS and Luciferase assay values in transformed soybean leaf protoplasts.

Plasmid Construct	3’ UTR Description	SEQ ID NO:	GUS	FLuc	RLuc
pMON81345	T-Gb.FbL2-l:l:l	41	795	2332.5	3701
pMON81347	T-Gh.E6-4A-0:2:l	38	73	584.3	802
pMON83002	T-Gb.H6-1:2:1	39	91	1142.8	1995
pMON 109593	T-Mt.Ptl-1:2:2	22	4783	3619	12341
pMON 116803	T-Mt.AC140914v20-l:2:l	2	15053	4801.7	15876
pMONl 16812	T-Mt.Lhcb2-1:2:1	13	9771	4202.3	10976
pMONl 16813	T-Mt.PSII-T_B-l:2:l	21	7482	3347.3	8395
pMONl 16815	T-Mt.AC145767v28-l:l:2	1	30469	6428	17764
pMONl 16826	T-Mt.Lox-l-l:2:l	14	22330	3580.5	9984

Plasmid Construct	3’ UTR Description	SEQ ID NO:	GUS	FLuc	RLuc
pMON 116827	T-Mt.Gpi-l:2:l	11	269	343.7	478
pMON 116830	T-Mt.Scp-1:2:1	27	3909	4683.7	10180
pMON 122852	T-Mt.Methm-1:2:1	15	33403	11049	28226
pMON122853	T-Mt.Prx-l:l:l	19	12833	11198	22722
pMON 122854	T-Mt.Gapdh-1:2:1	10	14811	8775.5	25229
pMON 122855	T-Mt.FBA-l:l:5	8	40383	17826	50299
pMON 122856	T-Mt.Zfp-1:2:1	30	21870	16141.3	56362
pMON 122857	T-Mt.AC139600vl6-l:2:l	3	24386	6782.7	15024
pMON122858	T-Mt.MP21-1:2:1	16	30753	12929.8	40571
pMON 122859	T-Mt.Oxr-1:2:1	17	14499	5586.7	15222
pMON 122862	T-Mt.Suil-l:l:2	29	27768	14680	35263
pMON 122864	T-Mt.Pipl-l:2:l	18	40579	15837.7	36515
pMON 122865	T-Mt.AC 153125V10-1:2:1	4	34867	17285.5	52519
pMON 122866	T-Mt.Sali3-2-1:2:1	26	33664	11923	27663
pMON 122867	T-Mt.Hsp20-1:2:1	12	7088	9885.3	19590
pMON 122868	T-Mt.Exprl-1:2:1	7	14539	7563.5	22320

[0085] Further, to compare the relative activity of each 3' UTR, GUS values were expressed as a ratio of GUS to luciferase activity and normalized to the best expressing nonMedicago 3' UTR, i.e., T-Gb.FbL2-l:l: l (SEQ ID NO: 41). Table 4 shows the GUS/Luciferase ratios and the normalized ratios. Again, in this Table, the firefly luciferase values are labeled 5 “FLuc” and the sea pansy luciferase values are labeled “RLuc.”

Table 4. GUS/FLuc and GUS/RLuc ratios of expression normalized with respect to TGb.FbL2-l:l:l (SEQ ID NO: 41) in transformed soybean leaf protoplasts.

3’ UTR Description	SEQ ID NO:	GUS/FLuc	GUS/RLuc	GUS/FLuc Normalized toT- Gb.FbL2- 1:1:1	GUS/RLuc Normalized to TGb.FbL21:1:1
T-Gb.FbL2-l:l:l	41	0.34	0.21	1.00	1.00
T-Gh.E6-4A-0:2:l	38	0.12	0.09	0.37	0.42
T-Gb.H6-1:2:1	39	0.08	0.05	0.23	0.21
T-Mt.Ptl-l:2:2	22	1.32	0.39	3.88	1.80
T-Mt.ACl 40914v20-1:2:1	2	3.13	0.95	9.20	4.41
T-Mt.Lhcb2-l:2:l	13	2.33	0.89	6.82	4.14
T-Mt.PSII-T_B-l:2:l	21	2.24	0.89	6.56	4.15
T-Mt.AC145767v28-l:l:2	1	4.74	1.72	13.91	7.98
T-Mt.Lox-1 -1:2:1	14	6.24	2.24	18.30	10.41
T-Mt.Gpi-l:2:l	11	0.78	0.56	2.30	2.62
T-Mt.Scp-l:2:l	27	0.83	0.38	2.45	1.79
T-Mt.Methm-1:2:1	15	3.02	1.18	8.87	5.51
T-Mt.Prx-l:l:l	19	1.15	0.56	3.36	2.63
T-Mt.Gapdh-l:2:l	10	1.69	0.59	4.95	2.73
T-Mt.FBA-l:l:5	8	2.27	0.80	6.65	3.74
T-Mt.Zfp-l:2:l	30	1.35	0.39	3.98	1.81
T-Mt.AC139600vl6-l:2:l	3	3.60	1.62	10.55	7.56
T-Mt.MP21-1:2:1	16	2.38	0.76	6.98	3.53
T-Mt.Oxr-1:2:1	17	2.60	0.95	7.61	4.43
T-Mt.Suil-l:l:2	29	1.89	0.79	5.55	3.67
T-Mt.Pipl-l:2:l	18	2.56	1.11	7.52	5.17
T-Mt.AC153125V10-l:2:l	4	2.02	0.66	5.92	3.09
T-Mt.Sali3-2-1:2:1	26	2.82	1.22	8.28	5.67
T-Mt.Hsp20-1:2:1	12	0.72	0.36	2.10	1.68
T-Mt.Exprl-l:2:l	7	1.92	0.65	5.64	3.03

[0086] As demonstrated in Table 4, GUS expression was enhanced using ail of the selected Medicago 3' UTRs compared to the 3' UTRs derived from Gossypium hirsutum or Gossypium barbadense. For example, expression of GUS was 2.1- to 18.3-fold higher using a Medicago-derived 3' UTR based upon the GUS/FLuc ratios normalized with respect to TGb.FbL2-l:l:l, the best expressing 3' UTR of those derived from Gossypium hirsutum or Gossypium barbadense. Similarly, expression of GUS was 1.61- to 10.48-fold higher using a Medicago-derived 3' UTR based upon the GUS/RLuc ratios normalized with respect to TGb.FbL2-l:l:l.

Example 3

Analysis of the Effect of 3' UTRs on Constitutive GUS Expression in Stably Transformed Soybean Plants [0087] Soybean plants were transformed with vectors, specifically plasmid constructs, to assess the effect of selected Medicago truncatula 3 ' UTRs on expression. Specifically, soybean plants were transformed with vectors containing a constitutive EXP sequence driving expression of the β-glucuronidase (GUS) transgene operably linked to a Medicago 3' UTR. These Medicago 3 ' UTR-transformed soybean plants were compared to transformed soybean plants in which expression of the GUS transgene was driven by a constitutive promoter, and the GUS transgene was operably linked to a 3 ' UTR derived from Gossypium barbadense.

[0088] The plant vectors utilized in these experiments were built using cloning methods known in the art. The resulting vectors comprised a left border région from A. tumefaciens-, a first transgene expression cassette for sélection of transformed plant cells that confers résistance to the antibiotic spectinomycin (driven by the Arabidopsis Actin 7 promoter); a second transgene expression cassette used to assess the activity of the 3' UTR, which comprised the regulatory expression element group EXP-CaMV.35S-enh+Ph.DnaK:l:3 (SEQ ID NO: 42) operably linked 5' to a coding sequence for GUS that possesses a processable intron (GUS-2, SEQ ID NO: 44), which is operably linked 5' to a 3' UTR derived from Medicago truncatula or Gossypium barbadense-, and a right border région from A. tumefaciens. The vectors that comprised a 3 ' UTR derived from Medicago were pMON109593, pMONl 16803, pMONl 16812, pMONl 16813, pMONl 16815, pMONl 16826, pMONl 16827, pMONl 16830, pMONl22850, pMON122851, pMON122852, pMON122853, pMON122854, pMON122855, pMON122856, pMON122857, pMON122858, pMON122859, pMON122861, pMON122862, pMON122863, pMONl22864, pMON122865, pMONl22866, pMON122867, and pMON122868. The vector that comprised a 3’ UTR from Gossypium barbadense was pMON102167.

[0089] Table 5 provides the plasmid constructs with the corresponding 3' UTR and SEQ ID NO used to transform the soybean plants in experiments presented in this Example.

Table 5. Plasmid constructs used to transform soybean plants and the 3' UTR descriptions.

Plasmid Construct	3’ UTR Description	SEQ ID NO:
pMON 102167	T-Gb.E6-3b:l:l	40
pMON 109593	T-Mt.Ptl-l:2:2	22
pMON 116803	T-Mt.AC140914v20-l:2:l	2
pMON 116812	T-Mt.Lhcb2-1:2:1	13
pMONl 16813	T-Mt.PSII-T_B-l:2:l	21
pMONl 16815	T-Mt.AC145767v28-l:l:2	1
pMONl 16826	T-Mt.Lox-l-l:2:l	14
pMONl 16827	T-Mt.Gpi-l:2:l	11
pMONl 16830	T-Mt.Scp-1:2:1	27
pMON 122850	T-Mt.RpL3-1:2:1	25
pMON 122851	T-Mt.RD22-l:2:l	24
pMON 122852	T-Mt.Methm-1:2:1	15
pMON 122853	T-Mt.Prx-l:l:l	19
pMON 122854	T-Mt.Gapdh-1:2:1	10
pMON 122855	T-Mt.FBA-l:l:5	8
pMON 122856	T-Mt.Zfp-l:2:l	30
pMON 122857	T-Mt.AC139600vl6-l:2:l	3
pMON 122858	T-MLMP21-1:2:1	16
pMON122859	T-Mt.Oxr-1:2:1	17
pMON 122861	T-Mt.Apx-l:l:2	5
pMON 122862	T-Mt.Suil-l:l:2	29

pMON 122863	T-Mt.EFla-l:l:2	6
pMON 122864	T-Mt.Pipl-l:2:l	18
pMON 122865	T-Mt.AC153125V10-l:2:l	4
pMON 122866	T-Mt.Sali3-2-l:2:l	26
pMON 122867	T-Mt.Hsp20-1:2:1	12
pMON 122868	T-Mt.Exprl-l:2:l	7

[0090] The soybean plants were transformer! using Agrobacteriiim-mediated transformation methods known in the art. Expression of GUS was assayed qualitatively using histological sections of selected tissues. For the histochemical GUS analysis, whole tissue sections were incubated with the GUS staining solution X-Gluc (5-bromo-4-chloro-3-indolyl-bglucuronide) (1 mg/ml) for an appropriate length of time, rinsed, and visually inspected for blue coloration. GUS activity was qualitatively determined by direct visual inspection or inspection under a microscope using selected plant organs and tissues. The Ro génération plants were inspected for expression in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, RI Petiole, RI Flower, Yellow Pod Embryo (approximately R8 development stage), Yellow Pod Cotylédon (approximately R8 development stage), R3 Immature Seed, R3 Pod, and R5 Cotylédon.

[0091] The quantitative changes of GUS expression relative to expression imparted by pMON102167, which comprised the 3' UTR derived from Gossypium barbadense, was also analyzed, as demonstrated in Tables 6-13. For this quantitative analysis, total protein was extracted from selected tissues of transformed plants. One microgram of total protein was used with the fluorogenic substrate 4-methyleumbelliferyl-P-D-glucuronide (MUG) in a total reaction volume of 50 μΐ. The reaction product, 4-methlyumbelliferone (4-MU), is maximally fluorescent at high pH, where the hydroxyl group is ionized. Addition of a basic solution of sodium carbonate simultaneously stops the assay and adjusts the pH for quantifying the fluorescent product. Fluorescence was measured with excitation at 365 nm, émission at 445 nm using a Fluoromax-3 (Horiba; Kyoto, Japan) with Micromax Reader, with slit width set at excitation 2 nm, émission 3 nm.

[0092] Tables 6 and 7 show the mean quantitative expression levels measured in the Ro génération plant tissues. Those tissues not assayed are shown as blank cells in both tables.

Table 6. Mean GUS expression in Ro génération plants in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, RI Petiole, and RI Flower.

RI Flower

412.30

215.37

243.11

294.38

179.31

876.08

1980.93

235.01

41.73

202.68

407.35

184.02

220.29

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94.21

115.33

421.81

308.11

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RI Flower	117.70	219.67	281.30	355.00	32.62	45.26	OO uS CN	157.45
RI Petiole	103.70	182.58	323.10	369.00	104.64	753.02	668.83	87.40
RI Source Leaf	263.00	342.22	171.40	401.00	239.01	976.91	675.68	235.62
Vn5 Source Leaf	510.50	604.88	398.30	240.00	298.16	1490.54	395.30	692.82
Vn5 Sink Leaf	433.60	710.62	281.00	203.00	160.51	1176.10	544.73	608.21
Vn5 Root	974.70	1131.24	667.00	448.00	385.42	2274.70	753.94	1151.60
SEQ ID NO:		29		OO		26	CN
3’ UTR Description	CN 1 X CL H	CN t ’5 C/3 H	T-Mt.EFla-l:l:2	CN 1 CL £ H	T-Mt.AC153125V10-l:2:l	CN 1 CN m « (Z) 4-» S H	[ T-Mt.Hsp20-1:2:1	T-Mt.Exprl-l:2:l
Plasmid Construct	pMON122861	pMON122862	pMON122863	pMON122864	pMON122865	pMONl 22866	pMONl 22867	pMONl 22868

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R5 Cotylédon	101.34	71.91	190.51	407.40	50.92	566.93	341.60	27.80
R3 Pod	433.54	406.13	467.72	526.08	282.34	2309.72	2868.17	37.22
R3 Immature Seed	67.45	28.63	209.92	163.76	78.12	989.47	231.15	10.96
Yellow Pod Cotylédon	49.45	170.11	181.62	120.30	279.84	1192.69	577.87	127.74
1 Yellow Pod Embryo	47.86	18.56	100.42	74.53	127.65	358.03	280.48	81'811
SEQ ID NO:	40	CN CN	CN	m	CN	-		«
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Plasmid Construct	pMONl 02167	pMON109593	pMONl 16803	pMONl 16812	pMONl 16813	pMONl 16815	pMONl 16826	pMONl 16827

R5 Cotylédon

98.36

264.40

72.23

518.51

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245.85

306.13

57.17

452.09

169.16

43.69

174.50

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LD m [0093] As demonstrated in Tables 6 and 7, expression driven by the same EXP was distinct in tissues of stably transformed soybean plants comprising different Medicago 3 ' UTRs when compared to the Gossypium barbadense-derived 3 ' UTR.

[0094] Tables 8 and 9 show the fold expression différences in the tissues of stably transformed soybean plants comprising different Medicago 3' UTRs when compared to the Gossypium barbadense-derived 3’ UTR. \}S

Table 8. Fold expression in Ro génération transformed soybean plants in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, RI Petiole, and RI Flower.

RI

Flower

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R5 Cotylédon	1.00	0.71	00 00	4.02	O un o	5.59	Fen en	FCM ό	0.97	2.61
R3 Pod	1.00	0.94	1.08	CM	0.65	5.33	6.62	60Ό	0.63	1.12
R3 Immature Seed	1.00	0.42	en	2.43	Ό	14.67	3.43	9Γ0	0.36	0.85
Yellow Pod Cotylédon	1.00	3.44	3.67	2.43	5.66	24.12	11.69	2.58	1.46	06'6
Yellow Pod Embryo	1.00 1 _____________________________________________________________________________________________________________________________________________________________________________________________1	0.39	2.10	1.56	2.67	00 d- F·*	5.86	2.47	Cri	5.54
m e g (Λ Z	40	22	CM	m	CM	-	d	-	27	un CM
3’ UTR Description	Js m 1 W xi O 1 H	CM CM 1 E â H	T-Mt.AC140914v20-l:2:l	T-Mt.Lhcb2-l:2:l	T-Mt.PSII-T B-l:2:l	T-Mt.AC145767v28-l:l:2	CM* 1 t X O U H	CM 1 Έ O 4>J S H	CM 1 CL Q un	CM en J CL 4-J S H
Plasmid Construct	F kO CM O Z O 2 CL	m o\ un Cri o § s CL	pMONl 16803	pMONl 16812	pMONl 16813	pMONl 16815	pMONl 16826	pMONl 16827	pMONl 16830	pMONl 22850

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σ» m [0095] As demonstrated in Tables 8 and 9, expression in the tissues of transformed soybean plants comprising different Medicago 3 ' UTRs was distinct when compared to that of soybean plants transformed with pMONl02l67, which comprised a 3' UTR derived from Gossypium barbadense. For example, two Medicago 3' UTRs, T-Mt.AC145767v28-l:l:2 (SEQ ID NO: 1) and T-Mt.Lox-1-1:2:1 (SEQ ID NO: 14) caused enhanced expression of the constitutive EXP, EXP-CaMV.35S-enh+Ph.DnaK:l:3 (SEQ ID NO: 42), across ail tissues. Other Medicago 3' UTRs provided enhanced expression of the constitutive EXP in some tissues, while reducing expression in others. For example, the 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 the Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, Yellow Pod Embryo, and Yellow Pod Cotylédon, while reducing expression in the RI Flower and R5 Cotylédon. Further, the 3' UTR T-Mt.ACl40914v20-1:2:1 (SEQ ID NO: 2) provided a 1.88- to 4.12-fold increase in expression in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, Yellow Pod Embryo, Yellow Pod Cotylédon, R3 Immature Seed, and R5 Cotylédon, while reducing expression in the RI Source Leaf, RI Flower, and keeping expression relatively the same in the R3 Pod. In addition, the 3' UTR T-Mt.Oxr-l:2:l (SEQ ID NO: 17) provided a 2.19-to 10.90-fold increase in expression in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, Yellow Pod Embryo, Yellow Pod Cotylédon, and R3 Pod, while reducing expression in the RI Flower and R5 Cotylédon, and keeping expression relatively the same in R3 Immature Seed.

[0096] Some of the transformed soybean plants comprising different Medicago 3 ' UTRs were taken to the Ri génération. Tables 10 and 11 show the mean GUS expression values of the assayed tissues. Tables 12 and 13 show the fold différence in expression relative to the 3’UTR derived from Gossypium barbadense..

Table 10. Mean GUS expression in Ri génération transformed soybean plants in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, RI Petiole, and RI Flower.

RI Flower	19Ύ09	506.70	2584.82	1041.40	919.57
RI Petiole	570.64	MD OO	2825.41	99Ό9Ι1	1087.69
RI Source Leaf	856.01	1159.28	2654.13	2666.68	2012.63
Vn5 Source Leaf	1210.30	1495.65	4447.42	3862.55	2510.49
Vn5 Sink Leaf	992.31	1169.79	5146.48	3090.41	2856.04
Vn5 Root	934.22	1462.92	5555.77	3726.08	3438.35
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3’ UTR Description	JS m 1 md W jS O 1 H	T-Mt.PSII-T B-l:2:l	1 T-Mt.AC145767v28-l:l:2	CM tL X O H	CM 1 CM 1 ë5 S H
Plasmid Construct	pMON102167	pMONl 16813	pMONl 16815	pMONl 22859	pMONl 22866

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R3 Pod	OO un	976.84	un 3- MD O un ττ	1947.45	1068.10
R3 Immature Seed	298.03	ΟΟΊΖ.Ι	o en Œ m O	245.45	488.62
Yellow Pod Cotylédon	174.11	537.77	rO\ en CM	1098.62	988.27
Yellow Pod Embryo	85.27	468.66	Tf N; ’T en	730.81	686.08
SEQ ID NO:	40	CM	-	r-	26
3’ UTR Description	F* JS en W JD O 1 H	T-Mt.PSII-T B-l:2:l	CM 1 OO CM > r- run Tj- O < H	T-Mt.Oxr-1:2:1	CM CM en cq
Plasmid Construct	pMONl 02167	pMONl 16813	un OO MD Z o 2 CL	Os un OO CM CM § CL	pMON122866

[0097] As demonstrated in Tables 10 and 11, expression driven by the same EXP was distinct in tissues of stably transformed soybean plants comprising different Medicago 3' UTRs when compared to the Gossypium barbadense-derived 3' UTR. Tables 12 and 13 show the fold expression différences in the tissues of stably transformed soybean plants comprising different Medicago 3'

to εύ

H

RI Flower	00T	0.84	4.28	1.73	1.52
RI Petiole	1.00		4.95	2.03
RI Source Leaf	1.00	1.35	3.10	3.12	2.35
Vn5 Source Leaf	00'l	1.24	3.67	1 3.19	2.07
m e .g S > ΪΛ hJ	1.00	OO	5.19	511	2.88
Vn5 Root	1.00	1.57	5.95	3.99	3.68
SEQID NO:	O tJ-	CN	-	Γ-	KD CN
3’ UTR Description	x> KO W X O 1 H	T-Mt.PSII-T B-1:2:1	T-Mt.AC145767v28- 1:1:2	T-Mt.Oxr-1:2:1	CN CN 1 C/2 H
Plasmid Construct	pMONl 02167	pMONl 16813	pMONl 16815	pMONl 22859	pMON122866

CN

Table 13. Fold expression différences in Ri génération transformed soybean plants in Yellow Pod Embryo, Yellow Pod Cotylédon, R3 Immature Seed, R3 Pod and R5 Cotylédon.

Plasmid Construct	3’ UTR Description	SEQ ID NO:	Yellow Pod Embryo	Yellow Pod Cotylédon	R3 Immature Seed	R3 Pod	R5 Cotylédon
pMON102167	T-Gb.E6-3b:l:l	40	1.00	1.00	1.00	1.00	1.00
pMONl 16813	T-Mt.PSII-T B-l:2:l	21	5.50	3.09	0.57	1.72	4.02
pMON116815	T-Mt.AC145767v28-l:l:2	1	15.42	12.26	3.49	7.94	21.65
pMON122859	T-Mt.Oxr-1:2:1	17	8.57	6.31	0.82	3.43	4.97
pMON122866	T-Mt.Sali3-2-l:2:l	26	8.05	5.68	1.64	1.88	8.90

[0098] As demonstrated in Tables 12 and 13, several of the Medicago 3' UTRs enhanced expression of the constitutive EXP element, EXP-CaMV.35S-enh+Ph.DnaK:l:3 (SEQ ID NO: 42), relative to plants transformed with pMON 102167, which comprised a 3' UTR derived from Gossypium barbadense in the Ri génération. For example, the 3' UTR T10 Mt.AC145767v28-l:l:2 (SEQ ID NO: 1) provided a 3.10- to 21.65-fold enhancement of GUS expression in ail of the tissues assayed. The 3' UTR T-Mt.Sali3-2-l:2:l (SEQ ID NO: 26) provided a 1.52- to 8.90-fold enhancement of GUS expression in ail of the tissues assayed. The 3' UTR T-Mt.Oxr-l:2:l (SEQ ID NO: 17) provided enhancement in most tissues, but reduced expression in the R3 Immature Seed relative to plants transformed with T-Gb.E6-3b: 1:1 (SEQ ID 15 NO: 40).

[0099] The forgoing experiments demonstrate that the Medicago truncatula derived 3' UTR éléments affected expression of the constitutive EXP element EXP-CaMV.35Senh+Ph.DnaK:l:3 (SEQ ID NO: 42) in different ways depending upon the spécifie 3' UTR selected. In many cases, there was an enhancement of expression in certain tissues of plants 20 transformed with plant expression vectors comprising a Medicago 3' UTRs relative to plants transformed with pMON 102167, which comprised a 3' UTR derived from Gossypium barbadense. However, the enhancement effect was not seen in ail plant tissues and, in many cases, expression was attenuated in some tissues and enhanced in others using a Medicago 3' UTR. Thus, the use of selected Medicago 3 ' UTRs allows for one to “fine tune” the expression profile of a particular transgene and can be used in combination with different expression éléments, such as promoters, leaders and introns, in opérable linkage with a transcribable DNA molécule to provide optimal expression in spécifie tissues, while reducing expression in tissues that are less désirable for a spécifie transcribable DNA molécule.

Example 4

Analysis of the Effect of 3' UTRs on Seed Preferred GUS Expression in Stably Transformed Soybean Plants [00100] Soybean plants were transformed with vectors, specifically plasmid constructs, to assess the effect of selected Medicago 3 ' UTRs on expression. Specifically, soybean plants were transformed with DNA vectors containing a seed expressing EXP sequence driving expression of the β-glucuronidase (GUS) transgene operably linked to a Medicago 3' UTR. These Medicago 3' UTR-transformed soybean plants were compared to transformed soybean plants in which expression of the GUS transgene was driven by a seed expressing EXP sequence and the GUS transgene was operably linked to 3 ' UTR derived from Gossypium barbadense.

[00101] The plant vectors utilized in these experiments were built using cloning methods known in the art. The resulting vectors comprised a left border région from A. tumefaciens·, a first transgene expression cassette for sélection of transformed plant cells that confers résistance to the antibiotic spectinomycin (driven by the Arabidopsis Actin 7 promoter); a second transgene expression cassette used to assess the activity of the 3 ' UTR, which comprised the EXP element, EXP-Gm.Sphasl:l:l (SEQ ID NO: 50), which provides seed preferred expression, operably linked 5' to a coding sequence for GUS that possesses a processable intron (GUS-2, SEQ ID NO: 44), which is operably linked 5' to a 3' UTR derived from Medicago truncatula or Gossypium barbadense', and a right border région from A. tumefaciens. The plant expression vectors that comprised a 3' UTR derived from Medicago were pMON 116832, pMON 116834, pMONl 16835, pMONl 16841, pMON122869, pMON122870, pMON122871, pMON122872, pMON 122873, pMON 122874, pMON 122875, pMON 122876, pMON 122878, pMON 122879, pMON122880, pMON122881, pMON122882, pMON122883, pMON122885, pMON122887, pMON122888, and pMON126122. The vector that comprised a 3’ UTR from Gossypium ôarZwi/ense was pMON83028. »>/ [00102] Table 14 provides the plasmid constructs with the corresponding 3' UTR, SEQ ID

NO, and génération for which quantitative assay data is provided.

Table 14. Plasmid constructs used to transform soybean plants and corresponding 3' UTR.

Plasmid Construct	3’ UTR Description	SEQ ID NO:	Génération For which Data is Provided
pMON83028	T-Gb.E6-3b:l:l	40	R.
pMONl 16832	T-Mt.AC140914v20-l:2:l	2	Ro
pMONl 16834	T-Mt.PSII-T_A-l:2:l	20	Ro
pMONl 16835	T-Mt.AC145767v28-l:l:2	1	Ro
pMONl 16841	T-Mt.PSII-T_B-l:2:l	21	Ro
pMON122869	T-Mt.RpL3-l:2:l	25	Ro
pMON122870	T-Mt.RD22-l:2:l	24	Ro
pMONl 22871	T-Mt.Methm-l:2:l	15	Ro
pMONl 22872	T-Mt.Prx-l:l:l	19	Ro
pMON122873	T-Mt.Gapdh-l:2:l	10	Ro
pMONl 22874	T-Mt.FBA-l:2:l	9	Ro
pMON122875	T-Mt.Zfp-1:2:1	30	Ro and Ri
pMON122876	T-Mt.AC139600vl6-l:2:l	3	Ro
pMONl 22878	T-Mt.Oxr-l:2:l	17	Ro
pMONl 22879	T-Mt.Apx-l:l:2	5	Ro and Ri
pMONl 22880	T-Mt.Suil-l:l:2	29	Ro and R,
pMON122881	T-Mt.EFla-l:l:2	6	Ro and Ri
pMON122882	T-Mt.Pipl-l:2:l	18	Ro
pMONl 22883	T-Mt.AC153125V10-l:2:l	4	Ro
pMONl 22885	T-Mt.Exprl-l:2:l	7	Ro
pMONl 22887	T-Mt.Ptl-l:2:2	22	Ro
pMONl 22888	T-Mt.Pt2-l:2:2	23	Ro
pMON126122	T-Mt.Exprl-l:2:l	7	Ro

[00103] The soybean plants were transformed and GUS assayed as described in Example

3. Tables 15 and 16 provide the quantitative mean GUS values for the Ro génération of stably transformed soybean plants. vJ

Table 15. Mean GUS expression in Ro génération of transformed soybean plants in Yellow Pod Embryo, Yellow Pod Cotylédon, R3 Immature Seed, R3 Pod, and R5 Cotylédon.

3’UTR Description	SEQID NO:	Yellow Pod Embryo	Yellow Pod Cotylédon	R3 Immature Seed	R3 Pod	R5 Cotylédon
T-Mt.AC140914v20-l:2:l	2	572	1045	9	6	8
T-Mt.PSII-T_A-l:2:l	20	210	371	7	6	61
T-Mt.AC145767v28-l:l:2	1	1445	4264	11	8	47
T-Mt.PSII-T_B-l:2:l	21	218	774	15	16	60
T-Mt.RpL3-l:2:l	25	683	1087
T-Mt.RD22-l:2:l	24	3164	6809	30	15	24
T-Mt.Methm-l:2:l	15	459	2136	7	6	74
T-Mt.Prx-l:l:l	19	109	794	9	6	42
T-Mt.Gapdh-l:2:l	10	241	745	6	5
T-Mt.FBA-l:2:l	9	622	772	10	6	100
T-Mt.Zfp-l:2:l	30	192	193	2	2	31
T-Mt.AC139600vl6-l:2:l	3	319	2150	8	6	157
T-Mt.Oxr-1:2:1	17	995	3220	5	4	235
T-Mt.Apx-l:l:2	5	41	272	10	9	10
T-Mt.Suil-l:l:2	29	120	546	15	116	16
T-Mt.EFla-l:l:2	6			10	9	17
T-Mt.Pipl-l:2:l	18	670	614	8	9	5
T-Mt.AC153125V10-l:2:l	4	2079	4192	8	6	62
T-Mt.Exprl-1:2:1	7	385	1092	11	5	299
T-Mt.Ptl-l:2:2	22	142	630	14	14	426
T-Mt.Pt2-1:2:2	23	440	513	2	1	10
T-Mt.Exprl-1:2:1	7	527	1122	15	6	154

Table 16. Mean GUS expression in Ro génération transformed soybean plants in Vn5 Root, Vn5 SinkLeaf, Vn5 Source Leaf, RI Source Leaf, RI Petiole, and RI Flower.

3’ UTR Description	SEQ ID NO:	Vn5 Root	Vn5 Sink Leaf	Vn5 Source Leaf	RI Source Leaf	RI Petiole	RI Flower
T-Mt.AC140914v20-l:2:l	2	23	4	6	4	4	4
T-Mt.PSII-T_A-l:2:l	20	29	5	8	6	3	3
T-Mt.AC145767v28-l:l:2	1	10	3	4	0	0	0
T-Mt.PSH-T_B-l:2:l	21	8	5	5	5	5	6
T-Mt.RpL3-1:2:1	25	60	26	22	7	8	9
T-Mt.RD22-l:2:l	24	21	2	3	12	11	11
T-Mt.Methm-l:2:l	15	8	4	4	0	0	0
T-Mt.Prx-l:l:l	19	5	5	5	0	0	0
T-Mt.Gapdh-l:2:l	10	20	8	6	8	6	8
T-Mt.FBA-l:2:l	9	9	3	3	18	15	17
T-Mt.Zfp-l:2:l	30	41	13	14	7	5	6
T-Mt.AC139600vl6-l:2:l	3	7	5	5	0	0	0
T-Mt.Oxr-l:2:l	17	7	3	8	0	0	0
T-Mt.Apx-l:l:2	5	31	16	19	1173	294	357
T-Mt.Suil-l:l:2	29	29	20	19	10	5	4
T-Mt.EFla-l:l:2	6	8	3	3	16	19	19
T-Mt.Pipl-l:2:l	18	15	7	6	8	4	3
T-Mt.AC153125V10-l:2:l	4	16	5	3	0	0	0
T-Mt.Exprl-l:2:l	7	22	8	10	6	3	3
T-Mt.Ptl-l:2:2	22	8	6	5	5	6	6
T-Mt.Pt2-1:2:2	23	34	11	11	6	6	6
T-Mt.Exprl-l:2:l	7	15	6	8	5	4	4

[00104] As can be seen in Tables 15 and 16, most of the Medicago 3' UTRs affected expression of the seed preferred EXP element, EXP-Gm.Sphasl:l:l (SEQ ID NO: 50), in only seed-derived tissues, with the exception of T-Mt.Apx-l:l:2 (SEQ ID NO: 5), which enhanced expression of GUS in the RI Source Leaf, RI Petiole, and RI Flower. Several Medicago 3' UTRs provided high expression in the Yellow Pod Embryo and Yellow Pod Cotylédon, such as

T-Mt.AC145767v28-l:l:2 (SEQ ID NO: 1), T-Mt.RD22-1:2:1 (SEQ ID NO: 24), and T

Mt.ACl53l25V10-l:2:l (SEQ ID NO: 4). Thus, these 3' UTRs may be idéal to enhance expression of a seed promoter during the later stages of seed development. The 3' UTR TMt.Exprl-l:2:l (SEQ ID NO: 7) provided high expression in both R5 Cotylédon and Yellow Pod Cotylédon relative to many of the other 3 ' UTRs, and thus may be useful in providing high cotylédon expression for a wider window of seed development. In some cases, the 3' UTR provided a more uniform level of seed expression both in the Yellow Pod Embryo and Yellow Pod Cotylédon, such as when T-Mt.FBA-l:2:l (SEQ ID NO: 9), T-Mt.Zfp-l:2:l (SEQ ID NO: 30), T-Mt.Pip 1-1:2:1 (SEQ ID NO: 18), and T-Mt.Pt2-1:2:2 (SEQ ID NO: 23) were used.

[00105] The Ro génération plants comprising T-Mt.Zfp-1:2:1 (SEQ ID NO: 30), TMt.Apx-l:l:2 (SEQ ID NO: 5), T-Mt.Suil-l:l:2 (SEQ ID NO: 29), and T-Mt.EFla-l:l:2 (SEQ ID NO: 6) were allowed to set seed and were planted for Ri génération studies. Table 17 shows a comparison of the mean quantitative assay data for events comprising these Ri génération plants comprising Medicago 3' UTRs and plants transformed with pMON83028, which comprised the 3' UTR T-Gb.E6-3b:l:l (SEQ ID NO: 40) derived from Gossypium barbadense.

Table 17. Mean GUS expression in Ri génération transformed soybean plants in Yellow Pod Embryo, Yellow Pod Cotylédon, and R5 Cotylédon.

Plasmid Construct	3’ UTR Description	SEQ ID NO:	Yellow Pod Embryo	Yellow Pod Cotylédon	R5 Cotylédon
pMON83028	T-Gb.E6-3b:l:l	40	102	362	7
pMON 122875	T-Mt.Zfp-1:2:1	30	56	153	498
pMON 122879	T-Mt.Apx-l:l:2	5	205	645	777
pMON 122880	T-Mt.Suil-l:l:2	29	462	1241	355
pMON 122881	T-Mt.EFla-l:l:2	6	415	1059	11

[00106] As can be seen in Table 17, the Medicago 3' UTRs affected expression differently than T-Gb.E6-3b:l:l in the embryo and cotylédon tissues. For example, T-Mt.Apx-l:l:2 (SEQ ID NO: 5) and T-Mt.Suil-l:l:2 (SEQ ID NO: 29) enhanced expression of the seed-preferred EXP element in the Yellow Pod Embryo, Yellow Pod Cotylédon, and R5 Cotylédon relative to T-Gb.E6-3b:l:l. T-Mt.EFla-l:l:2 (SEQ ID NO: 6) enhanced expression in the Yellow Pod

Embryo and Yellow Pod Cotylédon, but not in the R5 Cotylédon. T-Mt.Zfp-1:2:1 (SEQ ID NO: 30) reduced expression in the later developing Yellow Pod Embryo and Yellow Pod Cotylédon, but enhanced expression in the R5 Cotylédon.

[00107] Thus, each of the different Medicago 3' UTRs affect expression differentially in the developing seed when in opérable linkage with a seed preferred promoter. These différences in the effect on expression can be utilized to provide a more refined and tailored approach to seed expression and may be ideally suited for “fine tuning” the expression profile of spécifie transcribable DNA molécules where seed expression is desired.

Example 5

Analysis of the Effect of 3' UTRs on Constitutive GUS Expression in Stably Transformed Soybean Plants.

[00108] Soybean plants were transformed with vectors, specifically plasmid constructs, to assess the effect of selected Medicago truncatula 3' UTRs on expression. Specifically, soybean plants were transformed with vectors containing two different EXP éléments that exhibit a constitutive expression profile driving expression of the β-glucuronidase (GUS) transgene operably linked to a Medicago 3' UTR. These Medicago 3' UTR-transformed plants were compared to transformed soybean plants in which expression of the GUS transgene was operably linked to a 3 ’ UTR derived from Gossypium barbadense.

[00109] The plant vectors utilized in these experiments were built using cloning methods known in the art. The resulting vectors comprised a left border région from A. tumefaciens', a first transgene expression cassette for sélection of transformed plant cells that confers résistance to the antibiotic spectinomycin (driven by the Arabidopsis Actin 7 promoter); a second transgene expression cassette used to assess the activity of the 3 ' UTR, which comprised the EXP éléments EXP-CaMV.35S-enh+Ph.DnaK:l:3 (SEQ ID NO: 42) or EXP-DaMV.FLT:l:2 (SEQ ID NO: 51) operably linked 5' to a coding sequence for GUS that possesses a processable intron (GUS-2, SEQ ID NO: 44), which is operably linked 5' to a 3' UTR derived from Medicago truncatula or Gossypium barbadense', and a right border région from A. tumefaciens. The vectors that comprised a 3' UTR derived from Medicago were pMON 118768, pMON 153701 and pMONl 16803. The vectors that comprised a 3’ UTR from Gossypium barbadense were pMON 121042 and pMON 102167.

[00110] Table 18 provides the plasmid constructs with the corresponding EXP element, 3' UTR and SEQ ID NO used to transform the soybean plants presented in this Example.

Table 18. Plasmid constructs used to transform soybean plants and the corresponding EXP element and 3 ' UTR.

Plasmid Construct	EXP Description	EXP SEQ ID NO:	3' UTR Description	3’ UTR SEQ ID NO:
pMONl 21042	EXP-DaMV.FLT:l:2	51	T-Gb.E6-3b:l:l	40
pMONl 18768	EXP-DaMV.FLT:l:2	51	T-Mt.Sali3-2-l:2:l	26
pMONl 53701	EXP-DaMV.FLT: 1:2	51	T-Mt.AC140914v20-l:2:l	2
pMONl 02167	EXP-CaMV.35S-enh+Ph.DnaK: 1:3	42	T-Gb.E6-3b:l:l	40
pMON122866	EXP-CaMV.35S-enh+Ph.DnaK: 1:3	42	T-Mt.Sali3-2-l:2:l	26
pMONl 16803	EXP-CaMV.35S-enh+Ph.DnaK: 1:3	42	T-Mt.AC140914v20-l:2:l	2

[00111] Plants were transformed and GUS assayed as described in Example 3. Tables 19 and 20 provide the quantitative mean GUS values for the Ro génération of stably transformed 15 soybean plants,

Table 19. Mean GUS expression in Ro génération transformed soybean plants in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, RI Petiole, and RI Flower.

RI Flower	467.94	1398.80	739.97	412.30	45.26	243.11
		Έ te	69	S©	.28		02	47
		ω Ph	Os Γ-	un CN	CN Os		en un	O 00
			m		Γ—		r-	'd-
		Si
	o		CN	Un	1.99	en		00
	Q U	«	o	un	Os	Ov	Γ-;
Si	fi		o	en	en	O	\©	Tf
O	J	CN	's©	un	Γ-	’Φ
	CO		m	en		en	Ch	en
Vn5	Source	Leaf	509.35	1208.48	6'9011	605.29	1490.54	2187.61
un	44	U-	en Os	.59	00 en	s©	O	.95
fi >	e CO	<υ J	688.	1009	725.	551.	1176	2269
	m	w	OS	.43	.66	O Ch	.70	.76
	fi			CN	CN		'T	s©
	>	si	00 o	00 r-	Ch C'en	o	rCN CN	o en
		fi
		o			CN			CN
		•c						• ·
		Q.			|			i
					o			O
		u 5Λ © o	-	-1:2:1	CN > Os	-	-1:2:1	CN > Tt Ch
		Si	-D	CN 1	o	X)	CN 1	O
		H	en	en		en	en
		5	s©		o	s©		o
		*	w	CO	<	tu	CO	<
			_D	M-i		Λ	«J	4J
			o	s	s	o	s	s
			H	H	H	H	H	H
						en	en	en
		fi				c	c	c
						Q	Q	Q
		fi.				JC	Jfi	-C
						PU	PU	PU
		Q	CN			+	+	+
		(A Φ Q	CN	CN	<D	Q	ê <υ
		&	H	H 1—1	H >-1	CO un	CO un	CO un
		w	U-i	M-t		en	en	en
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			s	s	s	s	s	s
			fi	fi	fi	fi
			Q	Q	ü	U	U	0
			pu	PU	eu	pu	PU	CP
			X	X	X	X	X	X
			ω	w	tu	PU	PU	PU

un

R5 Cotylédon	64.18	247.75	s© 00	101.34	59.33	190.51
		o ©		00	en un	54		72
		R3 P	859.	1157	un un tjCN	433.		467.
	<x>
en	fi w		.02	00 00	un un	45		.92
si	Ë Ë	0) CO	0 Tt en	84.	s© un Tt	67.		209
	Yellow Pod	otyledon	9Γ91Ι	832.99	CN 00 0 un 0	49.45	334.27	181.62
	U
		©	00	un			en	CN
©	-0	U	un		(V>	un
Yell	© Pu	Λ g W	104.	CN 00 un	s© Ch	47.1	126.	Ό01
		fi			CN			CN
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					CN			CN
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		Q		1	Os	V—«	1	Os
				CN	0		CN	O
		si	JP en	en	Th	jp en	1 en	Tt
		E	S©		0	1 s©		U
		pu	PU	CO	<	PU	CO	<
		en	-O	t-»	1-i	X)	*-*
		0		s	□	s	s
			H	H	H	H	H	H
						en	en	en
								·—H
		fi				C	C	c
		_©				Q	Q	Q
						JP	J=	JP
						PU	PU	PU
		U				+	+	+
		(Λ ©	CN	CN	CN	nh	nh	nh
		a &				<L>	D	<υ
		H >-l	H J	H >-U	1 CO un	1 CO un	1 CO un
		X		bu	tu	en	en	en
		M	>	>	>	>	>	>
				s	s		s
			fi	fi	fi			0
			ü	Q	Q	0	0	0
			PU	eu	pu	pu	ώ	PU
			X	X	X	X	X	X
			PU	PU	PU	w	w	PU

[00112] As demonstrated in Tables 19 and 20, the Medicago 3' UTRs T-Mt.Sali3-2-l:2:l (SEQ ID NO: 26) and T-Mt.AC 140914v20-1:2:1 (SEQ ID NO: 2) affected expression of the constitutive EXP element EXP-DaMV.FLT: 1:2 (SEQ ID NO: 51) differently than the Gossypium barbadense-denved 3' UTR T-Gb.E6-3b:l:l (SEQ ID NO: 40). In many of the sampled tissues, there was an enhancement of expression using the Medicago 3 ' UTRs. With respect to the 3 ' UTR T-Mt.AC140914v20-l:2:l, enhancement was seen in most tissues in plants also comprising the EXP element EXP-CaMV.35S-enh+Ph.DnaK:l:3 (SEQ ID NO: 42). Tables 21 and 22 show the fold différences of the quantitative GUS expression relative to the expression imparted by pMON121042 (T-Gb.E6-3b: 1:1 (SEQ ID NO: 40)), which comprises a 3’ UTR derived from Gossypium barbadense. yS

Table 21. Fold expression différences in Ri génération transformed soybean plants in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, RI Petiole and RI Flower.

	RI	Flower	00T	2.99	00	00’l	O	0.59
		OJ			2.62
	S	etiol	1.00	SO en
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R5 Cotylédon	1.00	3.86	cs C<î	1 00'ï	0.59	1.88
R3 Pod	1.00	1.35	KO oo ri	1.00		801
R3 Immature Seed	1.00	0.25	Tien	1.00 1		en
Yellow Pod Cotylédon	1.00	7.23	en Os	1 00T	6.76	3.67
Yellow Pod Embryo	1.00	15.13	Os O	1.00 1	2.64	2.10
3' UTR Description	JD en KO W JD (0 1 H	CS 1 CS en 13 <z H	ri 1 O CS > tJ- Os O Tj- O < S H	JD m KD W xi O l H	CS t cs 1 en *cS (Z) H	T-Mt.AC140914v20-l:2:l
EXP Description	EXP-DaMV.FLT:l:2	EXP-DaMV.FLT:l:2	cs H tu > S ce Q S W	EXP-CaMV.35S-enh+Ph.DnaK:l:3	EXP-CaMV.35S-enh+Ph.DnaK: 1:3	EXP-CaMV.35S-enh+Ph.DnaK: 1:3

[00113] The forgoing experiments demonstrate that each of the Medicago 3' UTRs has different effects upon the level of expression of each of the constitutive EXP éléments relative to pMONl21042 (T-Gb.E6-3b:l:l (SEQ ID NO: 40)), which comprises the 3’ UTR derived from Gossypium barbadense. For example, expression of EXP-DaMV.FLT:l:2 was enhanced 1.14to 15.13-fold in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, RI Petiole, RI Flower, Yellow Pod Embryo, Yellow Pod Cotylédon, R3 Pod, and R5 Cotylédon, but reduced in the R3 Immature Seed using T-Mt.Sali3-2-l:2:l. This same EXP element, when combined with T-Mt.AC140914v20-l:2:1, resulted in a 1.34- to 13.42-fold enhancement in Vn5 Root, Vn5 Source Leaf, RI Source Leaf, RI Petiole, RI Flower, Yellow Pod Embryo, Yellow Pod Cotylédon, R3 Immature Seed, R3 Pod, and R5 Cotylédon, but remained about the same as TGb.E6-3b:l:l (SEQ ID NO: 40) in the V5 Sink Leaf. Expression in Yellow Pod Embryo was about twice that of Yellow Pod Cotylédon using T-Mt.Sali3-2-l:2:l (15.13- vs. 7.23-fold enhancement), while expression in these two tissues was relatively the same when using TMt.AC140914v20-l:2:l (9.19- vs. 9.13-fold enhancement). With respect to the EXP element EXP-CaMV.35S-enh+Ph.DnaK:l:3, combination with T-Mt.AC140914v20-l:2:l produced less enhancement in many of the sampled tissues than when this same 3' UTR was combined with EXP-DaMV.FLT:l:2. In RI Flower, there was a réduction of expression relative to T-Gb.E63b: 1:1 when EXP-CaMV.35S-enh+Ph.DnaK:l:3 was combined with T-Mt.AC140914v20-l:2:l. The combination of EXP-CaMV.35S-enh+Ph.DnaK:l:3 with T-Mt.Sali3-2-l:2:l provided enhancement in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, Yellow Pod Embryo, and Yellow Pod Cotylédon, but reduced expression in the RI Flower and R5 Cotylédon relative to T-Gb.E6-3b: 1:1 (SEQ ID NO: 40).

[00114] Each of the two Medicago 3' UTRs, T-Mt.Sali3-2-l:2:l and T-Mt.AC 140914v201:2:1, affected the expression of the two different constitutive EXP éléments, EXPDaMV.FLT:l:2 and EXP-CaMV.35S-enh+Ph.DnaK:l:3, differently. In many tissues, there was an enhancement of expression relative to T-Gb.E6-3b:l:l (SEQ ID NO: 40), but in some tissues, a réduction of expression occurred. Thus, by using different Medicago 3' UTRs, one may be able to more precisely control expression in the plant and better “fine tune” the expression of spécifie transcribable DNA molécules to provide optimal expression where the expression of the transcribable DNA molécule is required, while reducing expression in tissues that might negatively affect the plant.

Example 6

The Medicago truncatula 3' UTR T-Mt.AC145767v28-l:l:2 Causes Enhancement of GUS Expression When Combined with Many Different EXP Eléments in Stably Transformed Soybean Plants [00115] Soybean plants were transformed with vectors, specifically plasmid constructs, to assess the effect of the Medicago 3' UTR T-Mt.AC145767v28-l:l:2 (SEQ ID NO: 1) on expression. Specifically, the soybean plants were transformed with vectors containing several different EXPs with a constitutive expression profile driving expression of the β-glucuronidase (GUS) transgene operably linked to the Medicago 3' UTR T-Mt.AC145767v28-l:l:2 (SEQ ID NO: 1). These Medicago 3' UTR- transformed soybean plants were compared to transformed soybean plants in which the GUS transgene was operably linked to a 3' UTR derived from Gossypium barbadense.

[00116] The vectors utilized in these experiments were built using cloning methods known in the art. The resulting vectors comprised a left border région from A. tumefaciens; a first transgene expression cassette for sélection of transformed plant cells that confers résistance to the antibiotic spectinomycin (driven by the Arabidopsis Actin 7 promoter); a second transgene expression cassette used to assess the activity of the 3' UTR T-Mt.AC145767v28-l:l:2 (SEQ ID NO: 1) which comprises the EXP éléments, EXP-Mt.AC145767v28:l:l (SEQ ID NO: 35), EXPCaMV.35S-enh+Ph.DnaK:l: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:l:2 (SEQ ID NO: 51), EXPCUCme.eEFla:l:l (SEQ ID NO: 54), or EXP-Mt.Ubq2:l:2 (SEQ ID NO: 31) operably linked 5' to a coding sequence for GUS that possesses a processable intron (GUS-2, SEQ ID NO: 44) which is operably linked 5' to the 3' UTR T-Mt.AC145767v28-l:l:2 (SEQ ID NO: 1) derived from Medicago truncatula, or to the 3’UTRs T-Gb.E6-3b:l:l (SEQ ID NO: 40) or T-Gb.FbL21:1:1 (SEQ ID NO: 41) derived from Gossypium barbadense', and a right border région from A. tumefaciens. The vectors that comprised T-Mt.AC145767v28-l:l:2 (SEQ ID NO: 1) were pMON 118798, pMON 116815, pMON118769, pMON 153709, pMON 118771, pMON 153707, and pMON 155502. Notably, vector pMONl 18798 comprised the native EXPMt.AC145767v28:l:l which is comprised of a promoter element operably linked to a leader^ element cloned from the same gene locus as the 3' UTR T-Mt.AC145767v28-l:l:2 (SEQ ID NO: l). The vectors that comprised the 3’UTR from Gossypium barbadense were pMONl02167, pMONl 13874, pMON121030, pMON121042, pMON140827, and pMON 125841.

[00117] Table 23 provides the plasmid constructs with the corresponding EXP element, 3'

UTR, and SEQ ID NO used to transform the soybean plants presented in this Example.

Table 23. Plasmid constructs used to transform soybean plants and the corresponding EXP element and 3' UTR.

Plasmid Construct	EXP Description	EXP SEQ ID NO:	3' UTR Description	3 'UTR SEQ ID NO:
pMONl 18798	EXP-Mt.AC 145767v28:1:1	35	T-Mt.AC145767v28-l:l:2	1
pMON102167	EXP-CaMV.35S-enh+Ph.DnaK: 1:3	42	T-Gb.E6-3b:l:l	40
pMONl 16815	EXP-CaMV.35S-enh+Ph.DnaK: 1:3	42	T-Mt.AC145767v28-l:l:2	1
pMONl 13874	EXP-BSAcVNV.FLT: 1:2	52	T-Gb.E6-3b:l:l	40
pMON 118769	EXP-BS Ac VNV.FLT: 1:2	52	T-Mt.AC145767v28-l:l:2	1
pMONl 21030	EXP-CERV.FLT:1:2	53	T-Gb.E6-3b:l:l	40
pMON153709	EXP-CERV.FLT:1:2	53	T-Mt.AC145767v28-l:l:2	1
pMON121042	EXP-DaMV.FLT:l:2	51	T-Gb.E6-3b:l:l	40
pMONl 18771	EXP-DaMV.FLT:l:2	51	T-Mt.AC145767v28-l:l:2	1
pMON140827	EXP-CUCme.eEFla:l:l	54	T-Gb.FbL2-l:l:l	41
pMONl 53707	EXP-CUCme.eEFla:l:l	54	T-Mt.AC145767v28-l:l:2	1
pMON125841	EXP-Mt.Ubq2:l:2	31	T-Gb.FbL2-l:l:l	41
pMONl 55502	EXP-Mt.Ubq2:l:2	31	T-Mt.AC145767v28-l:l:2	1

[00118] The soybean plants were transformed and GUS assayed as described in Example

3. Tables 24 and 25 provide the quantitative mean GUS values for the Ro génération of stably transformed soybean plants. Table cells marked as “bdl” indicate tissues that were quantitatively analyzed but in which expression was below the level of détection. Tables 26 and 27 provide the 15 fold changes in expression of each EXP element operably linked to T-Mt.AC145767v28-l:l:2 relative to T-Gb.E6-3b: 1:1(SEQ ID NO: 40).

Table 24. Mean GUS expression in Ro génération transformed soybean plants in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, RI Petiole and RI Flower.

RI

Flower

bdl

412.30

876.08

64.38

581.97

130.87

3608.75

467.94

2131.16

130.38

405.97 1

482.13

398.10

RI

Petiole

23.00

1001.37

88.14

1380.90

285.15 1

5071.90

379.69

3610.84

130.60

1628.65

1195.97

875.67

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17.47

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Vn5

Source

Leaf

34.00

605.29

3250.38

19.46

419.52

480.25

3262.73

509.35

3923.47

37.44

216.21

275.48

1118.76

c >

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Leaf

32.00

551.61

1939.40

96'61

477.98

344.15

1618.72

688.93

5870.15

59.60

160.99

202.73

293.68

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8.03

81.72

55.79

13.52

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Table 25. Mean GUS expression in Ro génération transformed soybean plants in Yellow Pod Embryo, Yellow Pod Cotylédon, R3 Immature Seed, R3 Pod and R5 Cotylédon.

R5

Cotylédon

26.00

101.34

566.93

11.35

65.67

34.43

900.82

64.18

401.66

114.29

521.64

72.66

352.81

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67.45

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128.15

64.42

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340.02

518.90

58.21

209.77

400.15

247.18

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15.16

14.87

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83.48

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358.03

28.31

547.47

68.57

1474.35

104.58

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200.28

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Table 26. Fold expression différences in R] génération transformed soybean plants in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, RI Petiole and RI Flower.

RI Flower

00’l

2.12

1.00

9.04

1.00

27.57

1.00

4.55

1.00

en

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Table 27. Fold expression différences in Ri génération transformed soybean plants in Yellow Pod Embryo, Yellow Pod Cotylédon, R3 Immature Seed, R3 Pod and R5 Cotylédon.

X

Cotylédon

OOT

5.59

1.00

5.78

1.00

26.16

1.00

6.26

1.00

4.56

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1 4.86

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[00119] As demonstrated in Tables 24 and 25, the Medicago 3' UTR TMt.AC145767v28-l:l:2 (SEQ ID NO: 1) boosted expression of the six constitutive EXP éléments relative to T-Gb.E6-3b:l:l(SEQ ID NO: 40), but in different ways depending upon the spécifie EXP element and tissue. The EXP element, EXP-Mt.AC145767v28:l:l, when used to drive GUS and operably linked to its native 3' UTR T-Mt.AC145767v28-l:l:2 expressed very low in ail of the tissues assayed and was undetectable in R3 Immature Seed, R3 Pod, and RI Flower. Some tissues of plants comprising the EXP element EXP-Mt.Ubq2:l:2 and the 3' UTR T-Mt.AC145767v28-l:l:2 demonstrated reduced expression relative to the combination of EXPMt.Ubq2:l:2 and T-Gb.FbL2-l:l:l. This reduced expression was seen in R3 Immature Seed, RI Flower, and RI Petiole while, in contrast, Vn5 Sink Leaf and R5 Cotylédon expression was enhanced greater than four-fold. There was no change in root expression (Vn5 Root) with EXPMt.Ubq2:1:2 and either 3 ' UTR.

[00120] The regulatory expression element groups EXP-CERV.FLT:1:2 and EXPDaMV.FLT:l:2 provided the highest levels of expression. As demonstrated in Tables 26 and 27, these two EXPs were enhanced in ail tissues with T-Mt.AC145767v28-l:l:2 relative to the same EXPs combined with T-Gb.E6-3b:l:l(SEQ ID NO: 40). The regulatory expression element group EXP-CERV.FLT:1:2 was enhanced 60.50-fold in the developing Yellow Pod Cotylédon and less so in the Yellow Pod Embryo (21.50-fold), while the regulatory expression element group EXP-DaMV.FLT:l:2 was enhanced to a greater degree in the Yellow Pod Embryo than in the Yellow Pod Cotylédon (26.80- vs. 15.76-fold enhancement, respectively). These expression and enhancement différences offer an opportunity to provide tailored expression of a transgene in the later stage developing seed. The regulatory expression element group EXPBSAcVNV.FLT:l:2 expressed highest in the R3 Pod and Vn5 Root when combined with TGb.E6-3b:l:l (see Tables 25 and 26). The expression of EXP-BSAcVNV.FLT:l:2 in these two tissues was enhanced dramatically when combined with T-Mt.AC145767v28-l:l:2, particularly in Vn5 Root. Further, the expression of EXP-BSAcVNV.FLT:l:2 was boosted 58.63-fold when combined with T-Mt.AC145767v28-l:l:2 relative to this same EXP combined with T-Gb.E63b: 1:19 (SEQ ID NO: 40) [00121] In sum, the Medicago truncatula 3' UTR T-Mt.AC145767v28-l:l:2 (SEQ ID NO: 1) enhanced expression of six different constitutive EXP éléments which were derived from both plant and plant viral genomic DNA. In addition, this 3 ' UTR enhanced expression of the seed-preferred EXP element EXP-Gm.Sphasl:l:l (SEQ ID NO: 54) relative to most of the other Medicago-àedNQà 3' UTRs. Accordingly, this 3' UTR is suited for providing enhanced expression of a promoter or combination of operably linked expression éléments in a construct.

Example 7

Analysis of EXP-Mt.Ubq2:l:2 (SEQ ID NO: 31) in Stably Transformed Soybean Plants [00122] Soybean plants were transformed with vectors, specifically plasmid constructs, comprising the constitutive regulatory expression element group EXP-Mt.Ubq2:l:2 (SEQ ID NO: 31) operably linked to a GUS coding sequence. These transformed plants were then assayed for GUS expression in stably transformed soybean plants.

[00123] The plant vectors utilized in these experiments were built using cloning methods known in the art. The resulting vectors comprised a left border région from A. tumefaciens', a first transgene expression cassette for sélection of transformed plant cells that confers résistance to the antibiotic spectinomycin (driven by the Arabidopsis Actin 7 promoter); a second transgene expression cassette used to assess the activity of EXP-Mt.Ubq2:l:2 (SEQ ID NO: 31) which comprised EXP-Mt.Ubq2:l:2 operably linked 5' to a coding sequence for B-glucuronidase (GUS) that possesses a processable intron (GUS-2, SEQ ID NO: 44) operably linked 5' to the 3' UTR T-Mt.AC145767v28-l:l:2 (SEQ ID NO: 1) derived from Medicago truncatula, or the 3’ UTRs T-Gb.E6-3b:l:l (SEQ ID NO: 40) or T-Gb.FbL2-l:l:l (SEQ ID NO: 41) derived from Gossypium barbadense’, and a right border région from A. tumefaciens.

[00124] The resulting vectors were used to transform soybean plants as described in

Example 3. Tables 28 and 29 show the average quantitative GUS expression values assayed in various tissues and developmental time points for the stably transformed soybean plants.

Table 28. Average GUS expression in leaf, root and flower for stably transformed soybean plants comprising EXP-Mt.Ubq2:l:2 (SEQ ID NO: 31).

Plasmid Construct	3 ’ UTR Description	Vn5 Root	Vn5 Sink Leaf	Vn5 Source Leaf	RI Source Leaf	RI Petiole	RI Flower
pMON125840	T-Gb.E6-3b:l:l	252.58	126.69	86.01	49.05	108.41	83.23
pMONl 25841	T-Gb.FbL2-l:l:l	800.93	202.73	275.48	143.6	1195.97	482.13
pMONl 55502	T-Mt.AC145767v28-l:l:2	855	293.68	1118.76	254.25	875.67	398.1

Table 29. Average GUS expression in pod and seed tissues for stably transformed soybean plants comprising EXP-Mt.Ubq2:l:2 (SEQ ID NO: 31).

Plasmid Construct	3' UTR Description	R3 Immature Seed	R3 Pod	R5 Cotylédon	Yellow Pod Embryo	Yellow Pod Cotylédon
pMON125840	T-Gb.E6-3b:l:l	2.22	111.19	3.21	24.31	50.98
pMON125841	T-Gb.FbL2-l:l:l	400.15	875.75	72.66	129.84	83.45
pMONl 55502	T-Mt.AC145767v28-l:l:2	247.18	1324.98	352.81

[00125] As demonstrated in Tables 28 and 29, EXP-Mt.Ubq2:1:2 (SEQ ID NO: 31) is able to drive constitutive expression of a transcribable DNA molécule in stably transformed soybean plants. Further, different 3’ UTRs affect the degree of expression in each tissue. For example, combining EXP-Mt.Ubq2:1:2 with T-Gb.E6-3b:l:l resulted in lower expression in ail of the tissues assayed than the other two 3' UTRs, T-Gb.FbL2-l:l:l and T-Mt.AC145767v28-l:l:2. However, regardless of which 3' UTR was applied, EXP-Mt.Ubq2:l:2 provides medium-to-high constitutive expression, the degree of which can be modulated by a sélection of which 3' UTR is operably linked to the EXP.

Example 8

Enhancers Derived from the Regulatory Eléments [00126] Enhancers may be derived from the promoter éléments provided herein, such as

SEQ ID NOs: 32 and 36. An enhancer element may be comprised of one or more czs-regulatory éléments that, when operably linked 5' or 3' to a promoter element, or operably linked 5' or 3' to additional enhancer éléments that are operably linked to a promoter, can enhance or modulate expression of a transcribable DNA molécule, or provide expression of a transcribable DNA molécule in a spécifie cell type or plant organ or at a particular time point in development or circadian rhythm. Enhancers are made by removing the TATA box or functionally similar éléments and any downstream sequence that allow transcription to be initiated from the promoters or promoter fragments.

[00127] Enhancer éléments may be derived from the promoter éléments provided herein and cloned using methods known in the art to be operably linked 5' or 3' to a promoter element, or operably linked 5' or 3' to additional enhancer éléments that are operably linked to a promoter. Altematively, enhancer éléments maybe cloned, using methods known in the art, to be operably linked to one or more copies of the enhancer element which are operably linked 5' or 3 ' to a promoter element, or operably linked 5' or 3' to additional enhancer éléments that are operably linked to a promoter. Further, enhancer éléments can be cloned to be operably linked 5' or 3' to a promoter element derived from a different genus organism, or operably linked 5' or 3' to additional enhancer éléments derived from other genus organisme or the same genus organism that are operably linked to a promoter derived from either the same or different genus organism, resulting in a chimeric regulatory element. A GUS expression plant transformation vector maybe constructed using methods known in the art similar to the constructs described in the previous Examples in which the resulting plant expression vectors contain a lefit border région from A. tumefaciens; a first transgene sélection cassette that confers résistance to an antibiotic or herbicide and is utilized for sélection of transformed plant cells; and a second transgene cassette in which an enhancer element is operably linked to a promoter forming a chimeric promoter element, which is operably linked 5' to a leader element, which is operably linked 5' to a coding sequence for GUS that possesses a processable intron (GUS-2, SEQ ID NO: 44), operably linked to a 3' UTR such as T-Gb.E6-3b:l:l or any of those described above from Medicago truncatula; and a right border région from A. tumefaciens.

[00128] GUS expression driven by a regulatory element comprising one or more enhancers maybe evaluated in stable or transient plant assays as described herein to détermine the effects of the enhancer element on expression of a transcribable DNA molécule. Modifications to one or more enhancer éléments or duplication of one or more enhancer éléments maybe performed based upon empirical expérimentation, and the resulting gene expression régulation that is observed using each regulatory element composition. Altering the relative positions of one or more enhancers in the resulting regulatory or chimeric regulatory éléments may affect the transcriptional activity or specifïcity of the regulatory or chimeric regulatory element and is determined empirically to identify the best enhancers for the desired transgene expression profile within a plant.

Example 9

Analysis of the Effect of 3' UTRs on Constitutive GUS Expression in Stably Transformed Corn Plants [00129] Corn plants were transformed with binary plasmid constructs to assess the effect of the Medicago 3' UTR T-Mt.Oxr-1:2:1 (SEQ ID NO: 17) on expression relative to two 3' UTRs used frequently in corn plants. Specifically, the corn plants were transformed with vectors containing an EXP that exhibited a constitutive expression profile driving expression of the βglucuronidase (GUS) transgene, which was operably linked to the Medicago 3' UTR T-Mt.Oxr1:2:1 (SEQ ID NO: 17). These transformed corn plants were compared to transformed corn plants in which GUS was operably linked to either the 3' UTR T-AGRtu.nos-l:l:13 (SEQ ID NO: 49) or the 3’ UTR T-Os.LTP:l (SEQ ID NO: 56).

[00130] The binary plasmid constructs utilized in these experiments were built using cloning methods known in the art. The resulting vectors contained a right border région from A. tumefaciens\ a first expression cassette to assay the 3' UTR sequence wherein a constitutive regulatory expression element group EXP-FMV.35S-enh+Ta.Lhcbl+Zm.DnaK:l:2 (SEQ ID NO: 56) is operably linked 5’ to a coding sequence for GUS that possesses a processable intron (GUS-2, SEQ ID NO: 44), which is operably linked 5' to one of the following three 3' UTRs: TMt.Oxr-1:2:1 (SEQ ID NO: 17), T-AGRtu.nos-l:l:13 (SEQ ID NO: 49) or T-Os.LTP:l (SEQ ID NO: 56); a second transgene expression cassette used for sélection of transformed plant cells that confers résistance to the herbicide glyphosate (driven by the rice Actin 1 promoter); and a left border région from A. tumefaciens. The resulting plasmids were used to transform corn plants.

[00131] Histochemical GUS analysis was used for qualitative expression analysis of the transformed plants. Whole tissue sections were incubated with GUS staining solution X-Gluc (5-bromo-4-chloro-3-indolyl-b-glucuronide) (1 mg/ml) for an appropriate length of time, rinsed, and visually inspected for blue coloration. GUS activity was qualitatively determined by direct visual inspection or inspection under a microscope using selected plant organs and tissues. The Ro plants were inspected for expression in the roots and leaves, as well as the anther, silk, and developing seed and embryo, 21 days afiter pollination (21 DAP).

[00132] Quantitative analysis for the transformed corn plants was also performed. For the quantitative analysis, total protein was extracted from selected tissues of the transformed corn plants. One microgram of total protein was used with the fluorogenic substrate 4methyleumbelliferyl-P-D-glucuronide (MUG) in a total reaction volume of 50 μΐ. The reaction 5 product, 4-methlyumbelliferone (4-MU), is maximally fluorescent at high pH, where the hydroxyl group is ionized. Addition of a basic solution of sodium carbonate simultaneously stops the assay and adjusts the pH for quantifying the fluorescent product. Fluorescence is measured with excitation at 365 nm, émission at 445 nm using a Fluoromax-3 (Horiba; Kyoto, Japan) with Micromax Reader, with slit width set at excitation 2 nm and émission 3nm.

[00133] Table 30 shows the average quantitative GUS expression measured demonstrating different effects of each 3' UTR on the same constitutive expressing EXP.

Table 30. Average GUS expression in corn plants transformed with different 3' UTRs.

	Plasmid Construct
Develop mental Stage	Tissue	pMON128881 T-Mt.Oxr-l:2:l (SEQ ID NO: 17)	pMONl 19693 T-Os.LTP:l (SEQ ID NO: 56)	pMON132035 T-AGRtu.nos:13 (SEQ ID NO: 49)
V4	Leaf	205	232	222
Root	126	134	44
V7	Leaf	277	534	293
Root	nd	135	nd
VT	Leaf	314	429	194
Root	198	1043	291
Flower/Anther	527	486	308
RI	Cob/Silk	169	1258	319
R3	Embryo 21DAP	179	72	101
Endosperm 21DAP	516	207	243

[00134] As can be seen in Table 30, each 3' UTR had a different effect on constitutive expression driven by EXP-FMV.35S-enh+Ta.Lhcbl+Zm.DnaK:l:2 (SEQ ID NO: 56). For example, the 3' UTR T-Os.LTP:l (SEQ ID NO: 56) appeared to enhance expression in the VT Root and RI Cob/Silk relative to the other two 3' UTRs. The 3' UTR T-Mt.Oxr-1:2:1 (SEQ ID NO: 17) appeared to enhance expression in the R3 seed, both in the 21DAP endosperm and 21DAP Embryo relative to T-AGRtu.nos-1:1:13 (SEQ ID NO: 49) and T-Os.LTP:l (SEQ ID NO: 56). Expression in the Flower/Anther was also higher using T-Mt.Oxr-1:2:1 (SEQ ID NO: 17) relative to the other two 3' UTRs. The différences in expression observed for each of the 3' UTRs demonstrates the usefulness of each 3' UTR in modulating expression. Thus, these experiments demonstrate that the sélection of a 3 ' UTR can be used in transgene cassettes to fine tune expression of a particular transcribable DNA molécule. This experiment also demonstrates the ability of a dicot-derived 3' UTR, such as T-Mt.Oxr-l:2:l, to affect transcription in a monocot species such as corn.

Example 10

Analysis of Intron Enhancement of GUS Activity Using Plant Derived Protoplasts [00135] Generally, an intron is selected based upon expérimentation and comparison with an intronless vector control to empirically select an intron and configuration within the vector transfer DNA (T-DNA) element arrangement for optimal expression of a transgene. For example, in the expression of an herbicide résistance gene, such as CP4 (US RE39247), which confers tolérance to glyphosate, it is désirable to hâve transgene expression within the reproductive tissues as well as the végétative tissues in order to prevent the loss of yield when applying the herbicide. An intron in this instance would be selected upon its ability, when operably linked to a constitutive promoter, to enhance expression of the herbicide résistance conferring transgene, particularly within the reproductive cells and tissues of the transgenic plant, and thus providing both végétative and reproductive tolérance to the transgenic plant when sprayed with the herbicide. In most ubiquitin genes, the 5' UTR is comprised of a leader, which has an intron sequence embedded within it. The regulatory éléments derived from such genes are therefore assayed using the entire 5' UTR comprising the promoter, leader, and intron. To achieve different expression profiles or to modulate the level of transgene expression, the intron from such a regulatory element may be removed or substituted with a heterologous intron.

[00136] The intron presented herein as SEQ ID NO: 34 was identified using genomic DNA contigs in comparison to expressed sequence tag clusters, or cDNA contigs, to identify exon and intron sequences within the genomic DNA. In addition, 5' UTR or leader sequences were also used to defme the intron/exon splice junction of one or more introns under conditions when the gene sequence encodes a leader sequence that is interrupted by one or more introns. Introns were cloned using methods known in the art into a plant transformation vector to be operably linked 3' to a regulatory element and leader fragment and operably linked 5' to either a second leader fragment or to coding sequences, such as the expression cassettes presented in FIG. 1.

[00137] Thus, for example, a first possible expression cassette, such as Expression

Cassette Configuration 1 in FIG. 1, is comprised of a promoter or chimeric promoter element [A], operably linked 5' to a leader element [B], operably linked 5' to a test intron element [C], operably linked to a coding région [D], which is operably linked to a 3' UTR element [E]. Altematively, a second possible expression cassette, such as Expression Cassette Configuration 2 in FIG. 1, is comprised of a promoter or chimeric promoter element [F], operably linked 5' to a first leader element or first leader element fragment [G], operably linked 5' to a test intron element [H], operably linked 5' to a second leader element or first leader element second fragment [I], operably linked to a coding région [J], which is operably linked to a 3' UTR element [K]. Further, a third possible expression cassette, such as Expression Cassette Configuration 3 in FIG. 1, is comprised of a promoter or chimeric promoter element [L], operably linked 5' to a leader element [M], operably linked 5' to a first fragment of the coding sequence element [N], operably linked 5' to an intron element [O] element, operably linked 5' to a second fragment of the coding sequence element [P], which is operably linked to a 3' UTR element [Q]. Notably, Expression Cassette Configuration 3 is designed to allow splicing of the intron in such a manner as to produce a complété open reading frame without a frame shift between the first and second fragment of the coding sequence.

[00138] As discussed herein, it may be préférable to avoid using the nucléotide sequence

AT or the nucléotide A just prior to the 5’ end of the splice site (GT) and the nucléotide G or the nucléotide sequence TG, respectively just after 3 ' end of the splice site (AG) to eliminate the potential of unwanted start codons from being formed during processing of the messenger RNA w~ into the final transcript. The DNA sequence around the 5' or 3' end splice junction sites of the intron can thus be modified.

[00139] Introns may be assayed for an enhancement effect through the ability to enhance expression in transient assay or stable plant assay. For transient assay of intron enhancement, a base plant vector is constructed using methods known in the art. The intron is cloned into a base plant vector which comprises an expression cassette comprised of a constitutive EXP comprised of a promoter and leader such as EXP-CaMV.35S-enh+Ph.DnaK:l:3 (SEQ ID NO: 42), operably linked 5' to a test intron element (e.g. one SEQ ID NO: 34), operably linked to a coding sequence for GUS that possesses a processable intron (GUS-2, SEQ ID NO: 44), operably linked to the 3' UTR from (T-Gb.E6-3b:l:l, SEQ ID NO: 40). Protoplast cells derived from soybean or other genus plant tissue can be transformed with the base plant vector and Luciferase control vectors as described previously in Example 2 above, and assayed for activity. To compare the relative ability of the intron to enhance expression, GUS values are expressed as a ratio of GUS to Luciferase activity and compared with those levels imparted by a construct comprising the constitutive promoter operably linked to a known intron standard such as that as the intron derived from the Nicotiana tabacum élongation factor 4A10 gene, I-Nt.eIF4A10-1:1:1 (SEQ ID NO: 57), as well as a construct comprising the constitutive promoter, but without an intron operably linked to the promoter.

[00140] For stable plant assay of the intron presented as SEQ ID NO: 34, a GUS expression plant transformation vector can be constructed similar to the constructs described in the previous examples in which the resulting plant expression vectors contains a right border région from A. tumefaciens; a first expression cassette comprised of a constitutive EXP comprised of a promoter and leader such as EXP-CaMV.35S-enh+Ph.DnaK:l:3 (SEQ ID NO: 42), operably linked 5' to a test intron element (e.g., SEQ ID NO: 34), operably linked to a coding sequence for GUS that possesses a processable intron (GUS-2, SEQ ID NO: 44), operably linked to the 3' UTR from Gossypium barbadense (T-Gb.E6-3b:l:l, SEQ ID NO: 40). Protoplast cells derived from corn or other genus plant tissue may be transformed with the base plant vector and luciferase control vectors, as described previously in Example 2 above, and assayed for activity. To compare the relative ability of the intron to enhance expression, GUS values are expressed as a ratio of GUS to luciferase activity and compared with those levels imparted by a construct comprising the constitutive promoter operably linked to a known intron jy^- standard such as that as the intron derived from the Nicotiana tabacum élongation factor 4A10 gene, I-Nt.eIF4A10-l:l:l (SEQ ID NO: 57), as well as a construct comprising the constitutive promoter, but without an intron operably linked to the promoter.

[00141]

It should be noted that the intron presented as SEQ ID NO: 34 can be modified in a number of ways, such as deleting fragments within the intron sequence, which may reduce expression or duplication of fragments with the intron that may enhance expression. In addition, DNA sequences within the intron that may affect the specificity of expression to either particular cells types or tissues and organs can be duplicated or altered or deleted to affect expression and patterns of expression of the transgene. In addition, the intron provided herein can be modified to remove any potential start codons (ATG) that may cause unintentional transcripts from being expressed from improperly spliced introns as different, longer or truncated proteins. Once the intron has been empirically tested, or it has been altered based upon expérimentation, the intron may be used to enhance expression of a transgene in stably transformed plants that can be of any genus monocot or dicot plant, so long as the intron provides enhancement of the transgene. The intron can also be used to enhance expression in other organisms, such as algae, fungi, or animal cells, so long as the intron provides enhancement or atténuation or specificity of expression of the transgene to which it is operably linked.

[00142]

Having illustrated and described the principles of the invention, it should be apparent to persons skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. We claim ail modifications that are within the spirit and scope of the daims. Ail publications and published patent documents cited herein are hereby incorporated by reference to the same extent as if each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

4süwBe^cÂmeroun

Claims (5)

WHAT IS CLAIMED IS:

1. A recombinant DNA molécule comprising a DNA sequence selected from the group consisting of:

a) a DNA sequence with at least 85 percent sequence identity to any of SEQ ID NOs: 137;

b) a DNA sequence comprising any of SEQ ID NOs: 1-37; and

c) a fragment of any of SEQ ID NOs: 1-37, wherein the fragment has gene-regulatory activity;

wherein said DNA sequence is operably linked to a heterologous transcribable DNA molécule.

2. The recombinant DNA molécule of claim 1, wherein said DNA sequence has at least 90 percent sequence identity to the DNA sequence of any of SEQ ID NOs: 1-37.

3. The recombinant DNA molécule of claim 1, wherein said DNA sequence has at least 95 percent sequence identity to the DNA sequence of any of SEQ ID NOs: 1-37.

4. The recombinant DNA molécule of claim 1, wherein the heterologous transcribable polynucleotide molécule comprises a gene of agronomie interest.

5. The recombinant DNA molécule of claim 4, wherein the gene of agronomie interest confers herbicide tolérance in plants.

6. The recombinant DNA molécule of claim 4, wherein the gene of agronomie interest confers pest résistance in plants.

7. A transgenic plant cell comprising a recombinant DNA molécule comprising a DNA sequence selected from the group consisting of:

a) a DNA sequence with at least 85 percent sequence identity to any of SEQ ID NOs: 137; Z

b) a DNA sequence comprising any of SEQ ID NOs: 1-37; and

c) a fragment of any of SEQ ID NOs: 1-37, wherein the fragment has gene-regulatory activity;

wherein said DNA sequence is operably linked to a heterologous transcribable DNA molécule.

8. The transgenic plant cell of claim 7, wherein said transgenic plant cell is a monocotyledonous plant cell.

9. The transgenic plant cell of claim 7, wherein said transgenic plant cell is a dicotyledonous plant cell.

10. A transgenic plant, or part thereof, comprising a recombinant DNA molécule comprising a DNA sequence selected from the group consisting of:

a) a DNA sequence with at least 85 percent sequence identity to any of SEQ ID NOs: 137;

b) a DNA sequence comprising any of SEQ ID NOs: 1-37; and

c) a fragment of any of SEQ ID NOs: 1-37, wherein the fragment has gene-regulatory activity;

wherein said DNA sequence is operably linked to a heterologous transcribable DNA molécule

11. A progeny plant of the transgenic plant of claim 10, or a part thereof, wherein the progeny plant or part thereof comprises said recombinant DNA molécule.

12. A transgenic seed of the transgenic plant of claim 10 wherein the transgenic seed comprises the recombinant DNA molécule.

13. A method of producing a commodity product comprising obtaining a transgenic plant or part thereof according to claim 10 and producing the commodity product therefrom.

14. The method of claim 13, wherein the commodity product is protein concentrate, protein isolate, grain, starch, seeds, meal, flour, biomass, or seed oil.

5 15. A method of producing a transgenic plant comprising:

a) transforming a plant cell with the recombinant DNA molécule of claim 1 to produce a transformed plant cell; and

b) regenerating a transgenic plant from the transformed plant cell. \jJ