OA16777A - Plant regulatory elements and uses thereof. - Google Patents

Abstract

The invention provides DNA molecules and constructs, including their nucleotide sequences, useful for modulating gene expression in plants and plant cells. Transgenic plants, plant cells, plant parts, seeds, and commodity products comprising the DNA molecules operably linked to heterologous transcribable polynucleotides are also provided, as are methods of their use.

Description

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

BACKGROUND [04] Regulatory éléments are genetic éléments that regulate gene activity by modulating the transcription of an operably linked transcribable polynucleotide molécule. Such éléments 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 [05] The présent invention provides novel gene regulatory éléments such as promoters, leaders and introns derived from Cticumis melo, a plant species commonly referred to as muskmelon, for use in plants. The présent invention also provides DNA constructs, transgenic plant cells, plants, and sceds comprising the regulatory éléments. The sequences may be provided operably linked to a transcribable polynucleotide molécule which may be heterologous with respect to a regulatory sequence provided herein. The présent invention also provides methods of making and using the regulatory éléments, the DNA constructs comprising the regulatory éléments, and i*/

I the transgenic plant cells, plants, and seeds comprising the regulatory éléments operably linked to a transcribable polynucleotide molécule.

[06] Thus, in one aspect, the présent invention provides a DNA molécule, such as a transcriptional regulatory expression element group, or promoter, or leader, or intron, comprising a polynucleotide sequence selected from the group consisting of: a) a sequence with at Ieast 85 percent sequence identity to any of SEQ ID NOs: 1-199, 211 and 212; b) a sequence comprising any of SEQ ID NOs: 1-199, 211 and 212; and c) a fragment of any of SEQ ID NOs: 1-199, 211 and 212 exhibiting gene-regulatory activity, wherein said DNA molécule is operably linked to a heterologous transcribable polynucleotide molécule. In spécifie embodiments, a transcriptional regulatory expression element group, or promoter, or leader, or intron is at Ieast 90 percent, at Ieast 95 percent, at Ieast 98 percent, or at Ieast 99 percent identical to any of SEQ ID NOs: l-199, 211 and 212. In particular embodiments, the heterologous transcribable polynucleotide molécule comprises a gene of agronomie interest, a gene capable of providing herbicide résistance in plants, or a gene capable of providing plant pest résistance in plants.

[07] The invention also provides a transgenic plant cell containing a DNA molécule such as a transcriptional regulatory expression element group, or promoter, or leader, or intron, comprising a polynucleotide sequence selected from the group consisting of: a) a sequence with at Ieast 85 percent sequence identity to any of SEQ ID NOs: 1-199, 211 and 212; b) a sequence comprising any of SEQ ID NOs: 1-199, 211 and 212; and c) a fragment of any of SEQ ID NOs: 1-199, 211 and 212 exhibiting gene-regulatory activity, wherein said DNA molécule is operably linked to a heterologous transcribable polynucleotide molécule. Further, the transcriptional regulatory expression element group, or promoter, or leader, or intron régulâtes the expression of a gene. The transgenic plant cell can be a monocotyledonous or dicotyledonous plant cell.

[08] Further provided by the invention is a transgenic plant, or part of the transgenic plant containing a DNA molécule such as a transcriptional regulatory expression element group, or promoter, or leader, or intron, comprising a polynucleotide sequence selected from the group consisting of: a) a sequence with at Ieast 85 percent sequence identity to any of SEQ ID NOs: l199, 211 and 2I2; b) a sequence comprising any of SEQ ID NOs: l-199, 211 and 212; and c) a fragment of any of SEQ ID NOs: 1-199, 2ll and 212 exhibiting gene-regulatory activity, wherein said DNA molécule is operably linked to a heterologous transcribable polynucleotide molécule. In spécifie embodiments, the transgenic plant may be a progeny plant of any génération that contains the transcriptional regulatory expression element group, or promoter, or leader, or intron.

[09] Still further provided is a transgenic seed containing a DNA molécule such as a transcriptional regulatory expression element group, or promoter, or leader, or intron, comprising a polynucleotide sequence selected from the group consisting of: a) a sequence with at least 85 percent sequence identity to any of SEQ ID NOs: l -199, 211 and 212; b) a sequence comprising any of SEQ ID NOs: 1-199, 211 and 212; and c) a fragment of any of SEQ ID NOs: 1-199, 211 and 212 exhibiting gene-regulatory activity, wherein said DNA molécule is operably linked to a heterologous transcribabie polynucleotide molécule.

[010] In yet another aspect, the invention provides a method of producing a commodity product from the transgenic plant, transgenic plant part or transgenic seed which contains a DNA molécule such as a transcriptional regulatory expression element group, or promoter, or leader, or intron, comprising a polynucleotide sequence selected from the group consisting of: a) a sequence with at least 85 percent sequence identity to any of SEQ ID NOs: 1-199, 211 and 212; b) a sequence comprising any of SEQ ID NOs: 1-199, 211 and 212; and c) a fragment of any of SEQ ID NOs: 1-199, 211 and 212 exhibiting gene-regulatory activity, wherein said DNA molécule is operably linked to a heterologous transcribabie polynucleotide molécule. In one embodiment, the commodity product is protein concentrate, protein isolate, grain, starch, seeds, meal, flour, biomass, or seed oil.

[011] In another aspect, the invention provides a commodity product comprising a DNA molécule such as a transcriptional regulatory expression element group, or promoter, or leader, or intron, comprising a polynucleotide sequence selected from the group consisting of: a) a sequence with at least 85 percent sequence identity to any of SEQ ID NOs: 1-199, 211 and 212; b) a sequence comprising any of SEQ ID NOs: 1-199, 211 and 212; and c) a fragment of any of SEQ ID NOs: 1-199, 211 and 212 exhibiting gene-regulatory activity, wherein said DNA molécule is operably linked to a heterologous transcribabie polynucleotide molécule.

[012] In still yet another aspect, the invention provides a method of expressing a transcribabie polynucleotide molécule in a transgenic plant using a DNA molécule such as a transcriptional regulatory expression element group, or promoter, or leader, or intron which has a DNA — sequence which is at least 85 percent identical to that of any of SEQ ID NOs: 1-199, 211 and 212, or contains any of SEQ ID NOs: l-199, 211 and 212, or consists of a fragment of any of SEQ ID NOs: 1-199, 211 and 212; and cultivating the transgenic plant.

BRIEF DESCRIPTION OF THE SEQUENCES [013] SEQ ID NOs: l, 5, 7, 9, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 159, 162, 167, 168, 172, 175, 176, 177, 178, 181, 182, 183, 184, 185, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 211 and 212 are Ciicumis transcriptional regulatory expression element groups or EXP sequences which are comprised of either a promoter element, operably linked to a leader element; or a promoter element, operably linked to a leader element and an intron element, or a promoter element, operably linked to a leader element, operably linked to an intron element, operably linked to a leader element.

[014] SEQ ID NOs: 2, 6, 8, 10, 12, 163 and 169 are promoter éléments.

[015] SEQ IDNOs: 3, 164, 166 and 170 are leader sequences.

[016] SEQ ID NOs: 4, 165 and 171 are intron sequences.

[017] SEQ ID NOs: 157, 160, 173, 179 and 186 are sequences wherein a promoter is operably linked to a leader element.

[018] SEQ ID NOs: 158, 161, 174, 180 and 187 are sequences wherein an intron is operably linked to a leader element.

BRIEF DESCRIPTION OF THE DRAWINGS [019] FIGS, la- lf depict alignment of promoter variant segments corresponding to promoter éléments isolated from the Cticumis melo. In particular, Figs, la-If show alignment of the 2068 bp promoter sequence P-CUCme.Ubql-1:1:15 (SEQ ID NO: 2), found in the transcriptional regulatory expression element group EXP-CUCme.Ubql: 1:1 (SEQ ID NO: 1), vs. promoter sequences derived via 5’ délétions of the promoter, P-CUCme.Ubql-l:l:l5. Délétion, for instance of the 5’ end of P-CUCme.Ubql-l:l:l5, produced the promoters, P-CUCme.Ubqll:l:16 (SEQ ID NO: 6) a 1459 bp promoter which is found within EXP-CUCme.Ubql:l:2 (SEQ ID NO: 5); P-CUCme.Ubql-l:l:l7 (SEQ ID NO: 8), a 964 bp sequence comprised within EXPCUCme.Ubql:l:3 (SEQ ID NO: 7); P-CUCme.Ubql-l:l:l8 (SEQ ID NO: 10), a 479 bp sequence comprised within EXP-CUCme.Ubql:l:4 (SEQ ID NO: 9); and P-CUCme.Ubqll: 1:19 (SEQ ID NO: 12), a 173 bp sequence comprised within EXP-CUCme.Ubql:l:5 (SEQ ID NO: 11).

DETAILED DESCRIPTION OF THE INVENTION [020] The invention disclosed herein provides polynucleotide molécules obtained from Cucumis melo having bénéficiai gene regulatory activity. The design, construction, and use of these polynucleotide molécules are described. The nucléotide sequences of these polynucleotide molécules are provided among SEQ ID NOs: 1-199, 2ll and 212. These polynucleotide molécules are, for instance, capable of affecting the expression of an operably linked transcribable polynucleotide molécule in plant tissues, and therefore selectively regulating gene expression, or activity of an encoded gene product, in transgenîc plants. The présent invention also provides methods of modifying, producing, and using the same. The invention also provides compositions, transformed host cells, transgenîc plants, and seeds containing the promoters and/or other disclosed nucléotide sequences, and methods for preparing and using the same, [021] The following définitions and methods are provided to better define the présent 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 conventîonal usage by those of ordinary skill in the relevant art.

DNA Molécules [022] 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 or a polynucleotide molécule, read from the 5’ (upstream) end to the 3’ (downstream) end. As used herein, the term “DNA sequence” refers to the nucléotide sequence of a DNA molécule, — [023] As used herein, the term “isolated DNA molécule” refers to a DNA molécule at least partially separated from other molécules normally associated with it in its native or natural state. In one embodiment, the term “isolated” refers to a DNA molécule that is at least partially separated from some of the nucleic acids which normally flank the DNA molécule in its native or natural state. Thus, DNA molécules fused to regulatory or coding sequences with which they are not normally associated, for example as the resuit of recombinant techniques, are considered isolated herein. Such molécules are considered isolated when integrated into the chromosome of a host cell or présent in a nucleic acid solution with other DNA molécules, in that they are not in their native state.

[024] Any number of methods are known in the to isolate and manipulate a DNA molécule, or fragment thereof, disclosed in the présent invention. For example, PCR (polymerase chain reaction) technology can be used to amplify a particular starting DNA molécule and/or to produce variants of the original molécule. DNA molécules, or fragment thereof, can also be obtained by other techniques such as by directly synthesizing the fragment by chemical means, as is commonly practiced by using an automated oligonucleotide synthesizer.

[025] As used herein, the term “sequence identity” refers to the extent to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. An optimal sequence alignment is created by manually alignîng two sequences, e.g. a reference sequence and another sequence, to maximize the number of nucléotide matches in the sequence alignment with appropriate internai nucieotide insertions, délétions, or gaps. As used herein, the term “reference sequence” refers to a sequence provided as the polynucleotide sequences of SEQ IDNOs: 1-199,211 and 212.

[026] As used herein, the term “percent sequence identity” or “percent identity” or “% identity” is the identity fraction times 100. The “identity fraction” for a sequence optimally aligned with a reference sequence is the number of nucieotide 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 is a DNA molécule comprising a sequence that when optimally aligned to a reference sequence, provided herein as SEQ ID NOs: 1-199, 211 and 212, has at least about 85 percent identity at least about 90 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, or at least about 99 percent identity to the reference sequence. In particular embodiments such sequences may be defined as having gene-regulatory activity or encoding a peptide that functions to localize an operably linked polypeptide within a cell.

Regulatory Eléments [027] A regulatory element is a DNA molécule having gene regulatory activity, i.e. one that has the ability to affect the transcription and/or translation of an operably linked transcribable polynucleotide molécule. The term “gene regulatory activity” thus refers to the ability to affect the expression pattern of an operably linked transcribable polynucleotide molécule by affecting the transcription and/or translation of that operably linked transcribable polynucleotide molécule. As used herein, a transcriptional regulatory expression element group (EXP) may be comprised of expression éléments, such as enhancers, promoters, leaders and introns, operably linked. Thus a transcriptional regulatory expression element group may be comprised, for instance, of a promoter operably linked 5’ to a leader sequence, which is in turn operably linked 5’ to an intron sequence. The intron sequence may be comprised of a sequence beginning at the point of the first intron/exon splice junction of the native sequence and further may be comprised of a small leader fragment comprising the second intron/exon splice junction so as to provide for proper intron/exon processing to facilitate transcription and proper processing of the resulting transcript. Leaders and introns may positively affect transcription of an operably linked transcribable polynucleotide molécule as well as translation of the resulting transcribed RNA. The preprocessed RNA molécule comprises leaders and introns, which may affect the posttranscriptional processing of the transcribed RNA and/or the export of the transcribed RNA molécule from the cell nucléus into the cytoplasm. Following post-transcriptional processing of the transcribed RNA molécule, the leader sequence may be retained as part of the final messenger RNA and may positively affect the translation of the messenger RNA molécule.

[028] Regulatory éléments such as promoters, leaders, introns, and transcription termination régions are DNA molécules that hâve gene regulatory activity and play an integra! part in the overall expression of genes in living cells. The term “regulatory element” refers to a DNA molécule having gene regulatory activity, i.e. one that has the ability to affect the transcription and/or translation of an operably linked transcribable polynucleotide molécule. Isolated regulatory éléments, such as promoters and leaders that function in plants are therefore useful for modifying plant phenotypes through the methods of genetic engineering.

[029] Regulatory éléments may be characterized by their expression pattern effects (qualitatively and/or quantitatively), e.g. positive or négative effects and/or constitutive or other effects such as by their temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and/or chemically responsive expression pattern, and any combination thereof, as well as by quantitative or qualitative indications. A promoter is useful as a regulatory element for modulating the expression of an operably linked transcribable polynucleotide molécule.

[030] 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 antîsense or other regulatory RNA molécule, such as a dsRNA, a tRNA, an rRNA, a miRNA, and the like.

[031] 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.

[032] 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 (trans-acting transcription factors) to initiate transcription. A promoter may be initially isolated from the 5’ untranslated région (5’ UTR) of a genomic copy of a gene. Altemately, promoters may be synthetically produced or manipulated DNA molécules. Promoters may also be chimeric, that is a promoter produced through the fusion of two or more heterologous DNA molécules. Promoters useful in practicing the présent invention include any of SEQ ID NOs: 2, 6, 8, 10, 12, 163 and 169, or the promoter éléments comprised within any of SEQ ID NOs: 13 through 199, 2ll and 212, or fragments or variants thereof. In spécifie embodiments of the invention, such 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 sequence for récognition and binding of the RNA polymerase II complex for intiation of transcription.

[033] In one embodiment, fragments of a promoter molécule are provided. Promoter fragments provide promoter activity, as described above, and may be useful alone or in combination with other promoters and promoter fragments, such as in constructing chimeric promoters. In spécifie embodiments, fragments of a promoter are provided comprising at least about 50, 95, 150, 250, 500, 750, or at least about 1000 contiguous nucléotides, or longer, of a polynucleotide molécule having promoter activity disclosed herein.

[034] Compositions derived from any of the promoters presented as SEQ ID NOs: 2, 6, 8, 10, 12, I63 and 169, or the promoter éléments comprised within SEQ ID NOs: 13 through 199, 211 and 212, such as internai or 5’ délétions, for example, can be produced 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 dupiicating or removing éléments that hâve tissue or cell spécifie effects on expression. Compositions derived from any of the promoters presented as SEQ ID NOs: 2, 6, 8, 10, 12, 163 and 169, or the promoter éléments comprised within SEQ ID NOs: 13 through 199, 211 and 212 comprised of 3’ délétions in which the TATA box element or équivalent sequence thereof and downstream sequence is removed can be used, for cxample, 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 rhythms) effects on expression. Any of the promoters presented as SEQ ID NOs: 2, 6, 8, 10, 12, 163 and 169, or the promoter éléments comprised within SEQ ID NOs: 13 through 199, 2ll and 212, and fragments or enhancers derived there from can be used to make chimeric transcriptional regulatory element compositions comprised of any of the promoters presented as SEQ ID NOs: 2, 6, 8, 10, 12, 163 and 169, or the promoter éléments comprised within SEQ ID NOs: 13 through 199, 211 and 2I2, and the fragments or enhancers derived therefrom operably linked to other enhancers and promoters. The efficacy of the modifications, duplications or délétions described herein on the desired expression aspects of a particular transgene 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 molécule.

[035] As used herein, the term “leader” refers to a DNA molécule isolated from the untranslated 5’ région (5* UTR) of a genomic copy 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 poiynucleotide molécule. Leader molécules may be used with a heterologous promoter or with their native promoter. Promoter molécules of the présent invention may thus be operably linked to their native leader or may be operably linked to a heterologous leader. Leaders useful in practicing the présent invention include SEQ ID NOs: 3, 164, 166 and 170, or the leader element comprised within SEQ ID NOs: I3 through 199, 211 and 2I2, or fragments or variants thereof. In spécifie embodiments, such sequences may be provided defined as being capable of acting as a leader in a host cell, including, for example, a transgenic plant cell. In one embodiment such sequences are decoded as comprising leader activity.

[036] The leader sequences (5’ UTR) presented as SEQ ID NOs: 3, 164, 166 and 170, or the leader element comprised within any of SEQ ID NOs: 13 through 199, 2Il and 212 may be comprised of regulatory éléments or may adopt secondary structures that can hâve an effect on transcription or translation of a transgene. The leader sequences presented as SEQ ID NOs: 3, 164, 166 and 170, or the leader element comprised within SEQ ID NOs: 13 through 199, 211 and 212 can be used in accordance with the invention to make chimeric regulatory éléments that affect transcription or translation of a transgene. In addition, the leader sequences presented as SEQ ID NOs: 3, 164, 166 and 170, or the leader element comprised within any of SEQ ID NOs: 13 through 199, 2ll and 212 can be used to make chimeric leader sequences that affect transcription or translation of a transgene.

[037] The introduction of a foreign gene into a new plant host does not always resuit in a high expression of the incoming gene. Furthermore, if dealing with complex traits, it is sometimes necessary to modulate several genes with spatially or temporarily different expression pattern. Introns can principally provide such modulation. However, multiple use of the same intron in one transgenic plant has shown 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. As the available collection of introns known in the art with expression enhancing properties is limited, alternatives are needed.

[038] Compositions derived from any of the introns presented as SEQ ID NOs: 4, 165 and I7l or the intron element comprised within SEQ ID NOs: I3 through 199, 2ll and 212 can be tv— comprised of internai délétions or duplications of cis regulatory éléments; and/or alterations of the 5’ and 3’ sequences comprising the intron/exon splice junctions can be used to improve expression or specificity of expression when operably linked to a promoter + leader or chimeric promoter + leader and coding sequence. 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.

[039] 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 TATAbox 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 having a similar expression pattern to the original promoter.

[040] As used herein, the term “enhancer” or “enhancer element” refers to a cis-acting transcriptional regulatory element, a.k.a. cis-element, which confers an aspect of the overall expression pattern, but is usually insufficient alone to drive transcription, of an operably linked polynucleotide sequence. Unlike promoters, enhancer éléments do not usually include a transcription start site (TSS) or TATA box or équivalent sequence. A promoter may naturally comprise one or more enhancer éléments that affect the transcription of an operably linked polynucleotide sequence. An isolated enhancer element may also be fused to a promoter to produce a chimeric promoter.cis-element, which confers an aspect of the overall modulation of gene expression. A promoter or promoter fragment may comprise one or more enhancer éléments that effect the transcription of operably linked genes. Many promoter enhancer éléments are believed to bind DNA-binding proteins and/or affect DNA topology, producing local conformations that selectively aliow 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 identifîed 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

II using DNase I footprinting, méthylation interférence, electrophoresis mobility-shift assays, in vivo genomic footprinting by ligation-mediated PCR, and other conventional assays; or by DNA sequence similarity analysis using known cis-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. 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 polynucleotide molécules are encompassed by the présent invention.

[041] 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 (Mascarenhas et al., (1990) Plant Mol. Biol. I5;9l 3-920). Introns known to stimulate expression in plants hâve been identified in maize genes (e.g. tubAl, Adhl, Shl, Ubil (Jeon et al. (2000) Plant Physiol. 123:1005-1014; Callis et al. (1987) Genes Dev. 1:1183-1200; Vasil et al. (1989) Plant Physiol. 91:1575-1579; Christiansen et al. (1992) Plant Mol. Biol, 18:675-689) and in rice genes (e.g. sait, tpi: McElroy et al., Plant Cell 2:163-171 (1990); Xu et al., Plant Physiol. 106:459-467 (1994)). Similarly, introns from dicotyledonous plant genes like those from pétunia (e.g, rbcS), potato (e.g. st-lsl) and from Arabiclopsis thaliana (e.g. ubq3 and patl) hâve been found to elevate gene expression rates (Dean et al. (1989) Plant Cell 1:201-208; Leon et al. (1991) Plant Physiol. 95:968-972; Norris et al. (1993) Plant Mol Biol 21:895-906; Rose and Last (1997) Plant J Λ 1:455-464). 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 (Mascarenhas et al. (1990) Plant Mol Biol. 15:913-920; Clancy and Hannah (2002) Plant Physiol. 130:918-929). However, that splicing per se is not required for a certain IME in dicotyledonous plants has been shown by point mutations within the splice sites of the patl gene from A. thaliana (Rose and Beliakoff (2000) Plant Physiol. 122:535-542).

[042] Enhancement of gene expression by introns is not a general phenomenon because some intron insertions into recombinant expression cassettes fait to enhance expression (e.g. introns from dicot genes (rbcS gene from pea, phaseolin gene from bean and the stls-l gene from Solarium tttberosum) and introns from maize genes (adhl gene the ninth intron, hsp8I gene the first intron)) (Chee et al. (1986) Gene 41:47-57; Kuhlemeier et al. (1988) Mol Gen Genet 212:405-411; Mascarenhas et al. (1990) Plant Mol. Biol. 15:913-920; Sinibaldi and Mettler (1992) In WE Cohn, K Moldave, eds, Progress in Nucleic Acid Research and Molecular Biology, Vol 42. Academie Press, New York, pp 229-257; Vancanneyt et al. 1990 Mol. Gen. Genet. 220:245-250). Therefore, not each intron can be employed in order to manipulate the gene expression level of non-endogenous genes or endogenous genes in transgenic plants. What characteristics or spécifie sequence features must be présent in an intron sequence in order to enhance the expression rate of a given gene is not known in the prior art and therefore from the prior art it is not possible to predict whether a given plant intron, when used heterologously, will cause IME.

[043] 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 first nor 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; an example would be 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 polynucleotide molécules are encompassed by the présent invention.

[044] As used herein, the term “variant” refers to a second DNA molécule that is in composition similar, but not identical to, a first DNA molécule and yet the second DNA molécule still maintains the general functionality, i.e. same or similar expression pattern, 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 ofthe 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. The regulatory element “variants” may also encompass variants arisîng from mutations that naturally occur in bacterial and plant cell transformation. In the présent invention, a polynucleotide sequence provided as SEQ ID NOs: 1-199, 211 and 212 may be used to create variants similar in composition, but not îdentical to, the polynucleotide sequence of the original regulatory element, while still maintaining the general functionality of, i.e. same or similar expression pattern, the original regulatory element. Production of such variants of the présent invention is well within the ordinary skill of the art in light of the disclosure and is encompassed within the scope of the présent invention. “Varients” of chimeric regulatory element comprise the same constituent éléments as a reference chimeric regulatory element sequence but the constituent éléments comprising the chimeric regulatory element may be opcratively 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 chimeric regulatory element as well as other methods known in the art. The resulting “variant” chimeric regulatory element is comprised of the same, or variants of the same, constituent éléments as the reference sequence but difîer in the sequence or sequences that are used to operably link the constituent éléments. In the présent invention, the polynucleotide sequences provided as SEQ ID NOs: 1-199, 2ll and 212 each provide a reference sequence wherein the constituent éléments of the reference sequence may be joined by methods known in the art and may consist of substitutions, délétions and/or insertions of one or more nucléotides or mutations that naturally occur in bacterial and plant cell transformation.

Constructs [045] As used herein, the term “construct” means any recombinant polynucleotide molécule such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molécule, phage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molécule, derived from any source, capable of genomic intégration or autonomous réplication, comprising a polynucleotide molécule where one or more polynucleotide molécule has been linked in a functionally operative manner, i.e. operably linked. As used herein, the term “vector” means any recombinant polynucleotide construct that may be used for the purpose of transformation, i.e. the w~ introduction of heterologous DNA into a host cell. The term includes an expression cassette isolated from any of the aforementioned molécules.

[046] As used herein, the term “operably linked” refers to a first molécule joined to a second molécule, wherein the molécules are so arranged that the first molécule affects the function of the second molécule. The two molécules may or may not be part of a single contiguous molécule and may or may not be adjacent. For example, a promoter is operably linked to a transcribable polynucleotide molécule if the promoter modulâtes transcription of the transcribable polynucleotide molécule of interest in a cell. A leader, for example, is operably linked to coding sequence when it is capable of serving as a leader for the polypeptide encoded by the coding sequence.

[047] The constructs of the présent invention may be provided, in one embodiment, as double Ti plasmid border DNA 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, for example, US Patent 6,603,061). The constructs may also contain the plasmid backbone DNA segments that provide réplication function and antibiotic sélection in bacterial cells, for example, an Escherichia coli origin of réplication such as or/322, 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 in the art of plant transformation can function in the présent invention.

[048] Methods are available for assembling and introducing constructs into a cell in such a manner that the transcribable polynucleotide molécule is transcribed into a functional mRNA molécule that is translated and expressed as a protein product. For the practice of the présent invention, conventional compositions and methods for preparing and using constructs and host cells can be found in, for example, Molecular Cloning: A Laboratory Manual, 3^rd édition Volumes 1, 2, and 3 (2000) J.F. Sambrook, D.W. Russell, and N. Irwin, Cold Spring Harbor Laboratory Press. Methods for making recombinant vectors particularly suited to plant transformation include, without limitation, those described in U.S. Patent No. 4,971,908;

4,940,835; 4,769,061; and 4,757,011 in their entirety. These types of vectors have also been reviewed în the scientific literature (see, for example, Rodriguez, et al., Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston, (1988) and Glick, et al., Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton, FL. (1993)). Typical vectors useful for expression of nucleic acids in higher plants are well known in the art and include vectors derived from the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens (Rogers, et al., Methods in Enzymology 153: 253-277 (1987)). Other recombinant vectors useful for plant transformation, including the pCaMVCN transfer control vector, have also been described in the scientific literature (see, for example, Fromm, et al., Proc. Natl. Acad. Sci. USA 82: 5824-5828(1985)).

[049] 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 présent invention comprise at Ieast one regulatory element operably linked to a transcribable polynucleotide molécule operably linked to a 3’ transcription termination molécule.

[050] Constructs of the présent invention may include any promoter or leader provided herein or known in the art. For example, a promoter of the présent invention may be operably linked to a heterologous non-translated 5’ leader such as one derived from a heat shock protein gene (see, for example, U.S. Patent No. 5,659,122 and 5,362,865). Alternatively, a leader of the présent invention may be operably linked to a heterologous promoter such as the Cauliflower Mosaic Virus 35S transcript promoter (see, U.S. Patent No. 5,352,605). The expression properties imparted by such opérable linkages of heterologous éléments is not necessarily addîtîve of the elucidated properties of each promoter and leader, but rather is determined through empirical analysis of expression driven by the operably linked heterologous promoter and leader.

[051] As used herein, the term “intron” refers to a DNA molécule that may be isolated or identified from the genomic copy of a gene and may be defined generally as a région spliced out during 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 polynucleotide molécule. A DNA \jcS~ construct may comprise an intron, and the intron may or may not be heterologous with respect to the transcribable polynucleotide molécule sequence. Examples of introns in the art include the rice actin intron (U.S. Patent No. 5,641,876) and the corn HSP70 intron (U.S. Patent No. 5,859,347). Introns useful in practicing the present invention include SEQ ID NOs: 4, 165 and 171 or the intron element comprised within any of SEQ ID NOs: 13 through 199, 211 and 212.

[052] As used herein, the term “3’ transcription termination molécule” or “3’ UTR” refers to a DNA molécule that is used during transcription to produce the 3’ untranslated région (3’ UTR) of an mRNA molécule. The 3’ untranslated région of an mRNA molécule may be generated by spécifie cleavage and 3’ polyadenylation, a.k.a. polyA tail. A 3’ UTR may be operably linked to and located downstream of a transcribable polynucleotide molécule and may include polynucleotides that provide 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 are the nopaline synthase 3' région (see, Fraley, et al., Proc. Natl. Acad. Sci. USA, 80: 4803-4807 (1983)); wheat hspl7 3' région; pea rubisco small subunit 3' région; cotton E6 3' région (U.S. Patent 6,096,950); 3' régions disclosed in WO0011200A2; and the coixin 3’ UTR (U.S. Patent No. 6,635,806).

[053] 3’ UTRs typically find bénéficiai use for the recombinant expression of spécifie genes. In animal Systems, a machinery of 3’ UTRs has been well defined (e.g. Zhao et al., Microbiol Mol Biol Rev 63:405-445 (1999); Proudfoot, Nature 322:562-565 (1986); Kim et al., Biotechnology Progress 19:1620-1622 (2003); Yonaha and Proudfoot, EMBO J. 19:3770-3777 (2000); Cramer et al., FEBS Letters 498:179-182 (2001); Kuerstem and Goodwin, Nature Reviews Genetics 4:626-637 (2003)). Effective termination of RNA transcription is required to prevent unwanted transcription of trait- unrelated (downstream) sequences, which may interféré with trait performance. Arrangement of multiple gene expression cassettes in local proximity to one another (e.g. within one T- DNA) may cause suppression of gene expression of one or more genes in said construct in comparison to independent insertions (Padidam and Cao, BioTechniques 31:328-334 (2001). This may interfère with achieving adéquate levels of expression, for instance in cases were strong gene expression from ail cassettes is desired.

[054] In plants, clearly defined polyadenylation signal sequences are not known. Hasegawa et al.. Plant J. 33:1063-1072, (2003)) were not able to identify conserved polyadenylation signal sequences in both in vitro and in vivo Systems in Nicotiana sylvestris and to détermine the actual length of the primary (non-polyadenylated) transcript. A weak 3’ UTR has the potential to generate read-through, which may affect the expression of the genes located in the neighboring expression cassettes (Padidam and Cao, BioTechniques 31:328-334 (2001)), Appropriate control of transcription termination can prevent read-through into 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 pre-requisite 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 to the cytoplasm. Eukaryotic mRNAs are accumulated as poly(A) forms in vivo, so that it is difficult to detect transcriptional termination sites by conventional methods. However, prédiction of functional and efficient 3’ UTRs by bioînformatics methods is difficult in that there are no conserved sequences which would allow easy prédiction of an effective 3’ UTR.

[055] From a practical standpoint, it is typically bénéficiai that a 3’ UTR used in a transgene cassette possesses the following characteristics. The 3’ UTR should be able to effîciently 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 transgene cassette as in the case of multiple cassettes residing in one T-DNA, or the neighboring chromosomal DNA into which the T-DNA has inserted. The 3’ UTR should not cause a réduction in the transcriptional activity împarted by the promoter, leader and introns that are used to drive expression of the transgene. 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 (1) assess the transcriptional activity or expression of the transgene 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.

[056] 3’ UTRs useful in providing expression of a transgene in plants may be identified based upon the expression of expressed sequence tags (ESTs) in cDNA libraries made from messenger RNA isolated from seed, flower and other tissues derived from Foxtail millet (Setaria italica (L.) v—

Beauv). Libraries of cDNA are made from tissues isolated from selected plant species using flower tissue, seed, leaf and root. The resulting cDNAs are sequenced using various sequencing methods. The resulting ESTs are assembled into clusters using bioinformatics software such as clc_ref_assemble_complete version 2.01.37139 (CLC bio USA, Cambridge, Massachusetts 02142). Transcript abundance of each cluster is determined by counting the number of cDNA reads for each cluster. The identified 3’ UTRs may be comprised of sequence derived from cDNA sequence as well as sequence derived from genomic DNA. The cDNA sequence is used to design primers, which are then used with GenomeWalker^IM (Clontech Laboratories, Inc, Mountain View, CA) libraries constructed following the manufacturées protocol to clone the 3’ région of the corresponding genomic DNA sequence to provide a longer termination sequence. Analysis of relative transcript abundance either by direct counts or normalized counts of observed sequence reads for each tissue library can be used to infer properties about patters of expression. For example, some 3’ UTRs may be found in transcripts seen in higher abundance in root tissue as opposed to leaf, This is suggestive that the transcript is highly expressed in root and that the properties of root expression may be attributable to the transcriptional régulation of the promoter, the lead, the introns or the 3’ UTR. Empirical testing of 3’ UTRs identified by the properties of expression within spécifie organs, tissues or cell types can resuit in the identification of 3’ UTRs that enhance expression in those spécifie organs, tissues or cell types.

[057] Constructs and vectors may also include a transit peptide coding sequence that expresses a linked peptide that is useful for targeting of a protein product, particularly to a chloroplast, leucoplast, or other plastid organelle; mitochondria; peroxisome; vacuole; or an extracellular location. For descriptions of the use of chloroplast transit peptides, see U.S. Patent No. 5,188,642 and U.S. Patent No. 5,728,925. 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 assocîated with the small subunit (SSU) of ribulose-l,5,-bisphosphate carboxylase, ferredoxîn, ferredoxin oxidoreductase, the light-harvesting complex protein I and protein II, thioredoxin F, enolpyruvyl shikimate phosphate synthase (EPSPS), and transit peptides described in U.S. Patent No. 7,193,133. It has been demonstrated in vivo and in vitro that non-chloroplasl proteins may be targeted to the chloroplast by use of protein fusions with a heterologous CTP and that the CTP is sufficient to target a protein to the chloroplast. Incorporation of a suitable chloroplast transit peptide such as the Arabidopsis thaliana EPSPS CTP (CTP2) (See, Klee et al., Mol. Gen. Genet. 210:437-442 (1987)) or the Pétunia hybrida EPSPS CTP (CTP4) (See. dellaCioppa et al., Proc. Natl. Acad. Sci. USA 83:6873-6877 (1986)) has been show to target heterologous EPSPS protein sequences to chloroplasts in transgenic plants (See. U.S. Patent Nos. 5,627,061; 5,633,435; and 5,312,910 and EP 0218571; EP 189707; EP 508909; and EP 924299). Transcribabie polynucleotidc molécules [058] As used herein, the term “transcribabie polynucleotide 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. A “transgene refers to a transcribabie polynucleotide molécule heterologous to a host cell at least with respect to its location in the genome and/or a transcribabie polynucleotide molécule artificially incorporated into a host cell’s genome in the current or any prior génération of the cell.

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

[060] The transcribabie polynucleotide molécule may generally be any DNA molécule for which expression of a RNA transcrïpt is desired. Such expression of an RNA transcript may resuit in translation of the resulting mRNA molécule and thus protein expression. Alternatively, for example, a transcribabie polynucleotide 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 transcribabie polynucleotide molécule that is oriented in the antisense direction. Briefly, as the antisense transcribabie polynucleotide molécule is transcribed, the RNA product hybridizes to and sequesters a complimentary RNA molécule inside the cell. This duplex RNA molécule cannot be translated into a protein by the cell’s translational machinery and is degraded in the cell. Any gene may be negatively regulated in this manner.

[061] Thus, one embodiment of the invention is a regulatory element of the présent invention, such as those provided as SEQ ID NOs: 1-I99, 2ll and 212, operably linked to a transcribable polynucleotide molécule so as to modulate transcription of the transcribable polynucleotide molécule at a desired level or in a desired pattern when the construct is integrated in the genome of a plant cell. In one embodiment, the transcribable polynucleotide molécule comprises a protein-coding région of a gene, and the promoter affects the transcription of an RNA molécule that is translated and expressed as a protein product. In another embodiment, the transcribable polynucleotide molécule comprises an antisense région of a gene, and the promoter affects the transcription of an antisense RNA molécule, double stranded RNA or other similar inhibitory RNA molécule in order to inhibit expression of a spécifie RNA molécule of interest in a target host cell.

Genes of Agronomie Interest [062] Transcribable polynucleotide molécules may be genes of agronomie interest. As used herein, the term “gene of agronomie interest” refers to a transcribable polynucleotide molécule that when expressed in a particular plant tissue, cell, or cell type confers a désirable characteristic, such as associated with plant morphology, physiology, growth, development, yield, product, nutritional profile, disease or pest résistance, and/or envïronmental or chemical tolérance. Genes of agronomie interest include, but are not limited to, those encoding a yield protein, a stress résistance protein, a developmental control protein, a tissue différentiation protein, a meristem protein, an environmentally responsive protein, a senescence protein, a hormone responsive protein, an abscission protein, a source protein, a sink protein, a (lower control protein, a seed protein, an herbicide résistance protein, a disease résistance protein, a fatty acid biosynthetic enzyme, a tocopherol biosynthetic enzyme, an amino acid biosynthetic enzyme, a pesticidal protein, or any other agent such as an antisense or RNAi molécule targeting a particular gene for suppression. The product of a gene of agronomie interest may act within the plant in order to cause an effect upon the plant physiology or metaboiism or may be act as a pesticidal agent in the diet of a pest that feeds on the plant.

[063] In one embodiment of the invention, a promoter of the présent invention is incorporated into a construct such that the promoter is operably linked to a transcribable polynucleotide molécule that is a gene of agronomie interest. The expression of the gene of agronomie interest is désirable in order to confer an agronomically bénéficiai trait. A bénéficiai agronomie trait may be, for example, but is not limited to, herbicide tolérance, insect control, modified yield, fungal disease résistance, virus résistance, nematode résistance, bacterial disease résistance, plant growth and development, starch production, modified oils production, high oil production, modified fatty acid content, high protein production, fruit ripening, enhanced animal and human nutrition, biopolymers, environmental stress résistance, pharmaceutical peptides and secretable peptides, improved processing traits, improved digestibility, enzyme production, flavor, nitrogen fixation, hybrid seed production, fiber production, and biofuel production. Examples of genes of agronomie interest include those for herbicide résistance (U.S. Patent No. 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 oil 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).

[064] Altematively, a gene of agronomie interest can affect the above mentloned plant characteristic or phenotype by encoding a RNA molecuie that causes the targeted modulation of gene expression of an endogenous gene, for example via antisense (see e.g. US Patent 5,107,065); inhibitory RNA (“RNAi”, including modulation of gene expression via miRNA-, siRNA-, trans-acting siRNA-, and phased sRNA-mediated mechanisms, e.g. as described in published applications US 2006/0200878 and US 2008/0066206, and in US patent application 11/974,469); or cosuppression-mediated mechanisms. The RNA could also be a catalytic RNA moiecule (e.g. a ribozyme or a riboswitch; see e.g. US 2006/0200878) engineered to cleave a desired endogenous mRNA product. Thus, any transcribable polynucleotide moiecule that encodes a transcrîbed RNA molecuie that affects an agronomically important phenotype or morphology change of interest may be useful for the practice of the présent invention. Methods are known in the art for constructing and introducing constructs into a cell in such a manner that the transcribable polynucleotide moiecule is transcrîbed into a moiecule that is capable of causing gene suppression. For example, posttranscriptional gene suppression using a construct with an anti-sense oriented transcribable polynucleotide molecuie to regulate gene expression in plant cells is disclosed in U.S. Patent Nos. 5,107,065 and 5,759,829, and posttranscriptional gene suppression using a construct with a sense-oriented transcribable polynucleotide molecuie to regulate gene expression in plants is disclosed in U.S. Patent Nos. 5,283,184 and 5,231,020. Expression of a transcribable polynucleotide in a plant cell can also be used to suppress plant pests feeding on the plant cell, for example, compositions isolated from coleopteran pests (U.S. Patent Publication No. US20070124836) and compositions isolated from nematode pests (U.S. Patent Publication No. US20070250947). Plant pests include, but are not limited to arthropod pests, nematode pests, and fungal or microbial pests. Exemplary transcribable polynucleotide molécules for incorporation into constructs of the présent invention include, for example, DNA molécules or genes from a species other than the target species or genes that originate with or are présent in the same species, but are incorporated into récipient cells by genetic engineering methods rather than classical reproduction or breeding techniques. The type of polynucleotide molécule can include, but is not limited to, a polynucleotide molécule that is already présent in the plant cell, a polynucleotide molécule from another plant, a polynucleotide molécule from a different organism, or a polynucleotide molécule generated extemally, such as a polynucleotide molécule containing an antisense message of a gene, or a polynucleotide molécule encoding an artificial, synthetic, or otherwise modified version of a transgene.

Selectable Markers [065] As used herein the term “marker” refers to any transcribable polynucleotide molécule whose expression, or lack thereof, can be screened for or scored in some way. Marker genes for use in the practice of the présent invention include, but are not limited to transcribable polynucleotide molécules encoding β-glucuronidase (GUS described in U.S. Patent No. 5,599,670), green fluorescent protein and variants thereof (GFP described in U.S. Patent No. 5,491,084 and 6,146,826), proteins that confer antibiotic résistance, or proteins that confer herbicide tolérance. Useful antibiotic résistance markers include those encoding proteins conferring résistance to kanamycin (nptH), hygromycin B (aph IV), streptomycin or spectinomycin (aad, spec/strep) and gentamycin (aac3 and aacC4), Herbicides for which transgenic plant tolérance has been demonstrated and the method of the présent invention can be applied, include, but are not limited to: amino-methyl-phosphonic acid, glyphosate, glufosinate, sulfonylureas, imidazolinones, bromoxynil, delapon, dicamba, cyclohezanedione, protoporphyrinogen oxidase inhibitors, and isoxasflutole herbicides. Transcribable polynucleotide molécules encoding proteins involved in herbicide tolérance include, but are not limited to, a transcribable polynucleotide molécule encoding 5-enolpyruvylshikimate-3phosphate synthase (EPSPS for glyphosate tolérance described in U.S, Patent No. 5,627,061; 5,633,435; 6,040,497; and 5,094,945); a transcribable polynucleotide molécule encoding a glyphosate oxidoreductase and a glyphosate-N-acetyl transferase (GOX described in U.S, Patent No. 5,463,175; GAT described in U.S. Patent publication No. 20030083480, and dicamba monooxygenase U.S. Patent publication No. 20030135879); a transcribable polynucleotide molécule encoding bromoxynil nitrilase (Bxti for Bromoxynil tolérance described in U.S. Patent No. 4,810,648); a transcribable polynucleotide molécule encoding phytoene desaturase (crtl) described in Misawa, et al., Plant Journal 4:833-840 (1993) and Misawa, et al., Plant Journal 6:481-489 (1994) for norflurazon tolérance; a transcribable polynucleotide molécule encoding acetohydroxyacid synthase (AHAS, aka ALS) described in Sathasiivan, et al., Nucl. Acids Res. 18:2188-2193 (1990) for tolérance to sulfonylurea herbicides; and the bar gene described in DeBlock, et al., EMBO Journal 6:2513-2519 (1987) for glufosinate and bialaphos tolérance. The promoter molécules of the présent invention can express linked transcribable polynucleotide molécules that encode for phosphinothricin acetyltransferase, glyphosate résistant EPSPS, aminoglycoside phosphotransferase, hydroxyphenyl pyruvate dehydrogenase, hygromycin phosphotransferase, neomycin phosphotransferase, dalapon dehalogenase, bromoxynil résistant nitrilase, anthranilate synthase, aryloxyalkanoate dioxygenases, acetyl CoA carboxylase, glyphosate oxidoreductase, and glyphosate-N-acetyl transferase.

[066] Included within the term “selectable markers” are also genes which encode a secretable marker whose sécrétion can be detected as a means of identifying or selecting for transformed cells. Examples include markers that encode a secretable antigen that can be identified by antibody interaction, or even secretable enzymes which can be detected catalytically. Selectable secreted marker proteins fall into a number of classes, induding small, diffusible proteins which are détectable, (e.g. by ELISA), small active enzymes which are détectable in extracellular solution (e.g, alpha-amylase, beta-lactamase, phosphinothricin transferase), or proteins which are inserted or trapped in the cell wall (such as proteins which include a leader sequence such as that found in the expression unit of extension or tobacco pathogenesis related proteins also known as tobacco PR-S). Other possible selectable marker genes will be apparent to those of skill in the art and are encompassed by the présent invention.

Cell Transformation [067] The invention is also directed to a method of producing transformed cells and plants which comprise a promoter operably linked to a transcribable polynucleotide molécule.

[068] The term “transformation” refers to the introduction of nucleic acid into a récipient host. As used herein, the term “host” refers to bacteria, fungi, or plant, induding any cells, tissue, organs, or progeny of the bacteria, fungi, or plant. Plant tissues and cells of particular interest include protoplasts, calli, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.

[069] As used herein, the term “transformed” refers to a cell, tissue, organ, or organism into which a foreign polynucleotide molécule, such as a construct, has been introduced. The introduced polynucleotide molécule may be integrated into the genomic DNA of the récipient cell, tissue, organ, or organism such that the introduced polynucleotide molécule is inherited by subséquent progeny. A “transgenic” or “transformed” cell or organism 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 polynucleotide molécule. The term “transgenic” refers to a bacteria, fungi, or plant containing one or more heterologous polynucleic acid molécules.

[070] There are many methods for introducing polynucleic acid molécules into plant cells. The method generally comprises the steps of selecting a suitable host cell, transforming the host cell with a recombinant vector, and obtaining the transformed host cell. Suitable methods include bacterial infection (e.g. Agrobacterium), binary bacterial artificial chromosome vectors, direct delivery of DNA (e.g. via PEG-mcdiated transformation, desiccation/inhibition-mediated DNA uptake, electroporation, agitation with silicon carbide fibers, and accélération of DNA coated particles, etc. (reviewed in Potrykus, et al., Ann. Rev. Plant Physiol. Plant Mol. Biol. 42: 205 (I99l)).

[071] Any transformation methods may be utilized to transform a host cell with one or more promoters and/or constructs of the présent invention. Host cells may be any cell or organism such as a plant cell, algae cell, algae, fungal cell, fungi, bacterial cell, or insect cell. Preferred hosts and transformed cells include cells from: plants, Aspergillus, yeasts, insects, bacteria and algae.

[072] Regenerated transgenic plants can be self-pollinated to provide homozygous transgenic plants. Alternatively, pollen obtained from the regenerated transgenic plants may be crossed with non-transgenîc plants, preferably inbred lines of agronomically important species. Descriptions of breeding methods that are commonly used for different traits and crops can be found in one of several reference books, see, for example, Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U. of CA, Davis, CA, 50-98 (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, (Vol. 1) and Crop Species rc·'

Soybean (Vol 2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987). Conversely, pollen from non-transgenic plants may be used to pollinate the regenerated transgenic plants.

[073] The transformed plants may be analyzed for the presence of the genes of interest and the expression level and/or profile conferred by the regulatory éléments of the présent 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 transcribabie polynucleotide 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. Alternatively, the Invader® (Third Wave Technologies, Madison, WI) reagents and methods as described by the manufacturer can be used transgene expression.

[074] The seeds of the plants of this invention can be harvested from fertile transgenic plants and be used to grow progeny générations of transformed plants of this invention including hybrîd plant lines comprising the construct of this invention and expressing a gene of agronomie interest.

[075] The présent invention also provides for parts of the plants of the présent invention. Plant parts, without limitation, include leaves, stems, roots, tubers, seeds, endosperm, ovule, and pollen. The invention also includes and provides transformed plant cells which comprise a nucleic acid molécule of the présent invention.

[076] The transgenic plant may pass along the transgenic polynucleotide molécule to its progeny. Progeny includes any regenerable plant part or seed comprising the transgene derived from an ancestor plant. The transgenic plant is preferably homozygous for the transformed polynucleotide molécule and transmîts that sequence to ail offspring as a resuit of sexual reproduction. Progeny may be grown from seeds produced by the transgenic plant. These additional plants may then be self-pollinated to generate a true breeding line of plants. The progeny from these plants are evaluated, among other things, for gene expression. The gene expression may be detected by several common methods such as western blotting, northem blotting, immuno-precîpitation, and ELISA.

[077] Having now generally described the invention, the same will 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 présent 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

Examplc 1: Identification and Cloning of Regulatory Eléments [078] Novel transcriptional regulatory éléments, or transcriptional regulatory expression element group (EXP) sequences were identified and isolated from genomic DNA of the dicot species Cucttmis melo WSH-39-1070AN.

[079] Transcriptional regulatory éléments were selected based upon proprietary and public microarray data derived from transcriptional profiling experiments conducted in soybean (Glycine ntax) and Arabidopsis as well as homology based searches using known dicot sequences as query against proprietary Cuciimis melo sequences.

[080] Using the identified sequences, a bioinformatic analysis was conducted to identify regulatory éléments within the amplified DNA, followed by identification of the transcriptional start site (TSS) and any bi-directionality, introns, or upstream coding sequence présent in the sequence. Using the results of this analysis, regulatory éléments were defined within the DNA sequences and primers designed to amplify the regulatory éléments. The corresponding DNA molécule for each regulatory element was amplified using standard polymerase chain reaction conditions with primers containing unique restriction enzyme sites and genomic DNA isolated from Cucnmis melo. The resulting DNA fragments were ligated into base plant expression v28 vectors using standard restriction enzyme digestion of compatible restriction sites and DNA ligation methods.

[081] Analysis of the regulatory element TSS and intron/exon splice junctions can be performed using transformed plant protoplasts. Briefly, the protoplasts are transformed with the plant expression vectors comprising the cloned DNA fragments operably linked to a heterologous transcribable polynucleotide molécule and the 5' RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Invtrogen, Carlsbad, California 92008) is used to confirm the regulatory element TSS and intron/exon splice junctions by analyzing the sequence of the mRNA transcripts produced thereby.

[082] Sequences encoding ubiquitin l transcriptional regulatory expression element groups (EXP) were analyzed as described above and each transcriptional regulatory expression element groups (“EXP’s”) was also broken down into the corresponding promoters, leaders and introns comprising each transcriptional regulatory expression element group. Sequences of the identified ubiquitin l transcriptional regulatory expression element groups (“EXP’s”) are provided herein as SEQ ID NOs: l, 5, 7, 9 and 11 and is listed in Table l below. The corresponding ubiquitin l promoters are provided herein as SEQ ID NOs: 2, 6, 8, 10 and 12. The ubiquitin l leader and intron are herein provided as SEQ ID NOs: 3 and 4, respectively.

[083] Sequences encoding other Cucumis transcriptional regulatory expression element groups or EXP sequences which are comprised of either a promoter element, operably linked to a leader element; or a promoter element, operably linked to a leader element and an intron element, or a promoter element, operably linked to a leader element, operably linked to an intron element, operably linked to a leader element are provided as SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, ΙΟΙ, 102, 103, 104, 105, 106, 107, 108, I09, HO, lll, H2, ll3, H4, H5, H6, H7, H8, ll9, 120, I2l, 122, 123, 124, 125, 126, I27, 128, I29, 130, I3l, 132, 133, I34, 135, 136, 137, 138, 139, 140, I4l, 142, I43, I44, 145, 146, 147, 148, 149, 150, I5l, 152, 153, 154, 155, 156, 159, 162, 167, 168, 172, I75, 176, 177, 178, I8l, 182, 183, 184, 185, 188, 189, 190, I9l, I92, I93, 194, 195, 196, 197, 198, 199, 2ll and 212 and are also listed in Table l below. Additional promoter éléments are provided as SEQ ID NOs: 163 and 169. Additional leader éléments are provided as SEQ ID NOs: 164, 166 and I70. Additional intron éléments are provided as SEQ ID NOs: 165 and I7l. Eléments wherein a promoter is operably linked to a leader element are provided as SEQ ID NOs: 157, 160, 173, 179 and 186. Eléments wherein an intron is operably linked to a leader element are provided as SEQ ID NOs: 158, I6l, 174, 180 and 187. With respect to the subset of sequences provided as SEQ ID NOs: I3 through 199, 211 and 212, these sequences were selected and cloned based upon the résulte of experiments such as transcript profiling or expression driven by promoters from homologous genes of a different species suggesting désirable patterns of expression such as constitutive expression, root expression, above ground expression or seed expression. The actual activity imparted by the

Cuctimis sequences is determined empirically and is not necessarily the same as that of a regulatory element derived from a homologous gene from a species other than Citcttmis melo when used in a transformed plant host cell and whole transgenic plant. —

Table 1. Transcriptional regulatory expression element groups, promoters, leaders and introns isolated from Cucumis melo.

Annotation	SEQ ID NO:	Description	Composition Type	Size (bp)	Composition	Coordinates of Eléments within EXP
EXP-CUCme.Ubql:l:l	1	Ubiquitïn 1	EXP	2611	Promoter;Leader;Intron	l-2068;2069- 2150;2151-2608
P-CUCme.Ubql-1:1:15	2	Ubiquitin 1	P	2068	Promoter
L-CUCme.Ubql-1:1:1	3	Ubiquitïn 1	L	82	Leader
I-CUCme.Ubql-l:l:l	4	Ubiquitin 1	I	461	Intron
EXP-CUCme.Ubql:l:2	5	Ubiquitin 1	EXP	2002	Promoter ;Leader; Intron	1-1459; 1460- 1541;1542-1999
P-CUCme.Ubq 1-1:1:16	6	Ubiquitin 1	P	1459	Promoter
EXP-CUCme.Ubql:l:3	7	Ubiquitin 1	EXP	1507	Promoter;Leader; Intron	l-964;9651046; 1047-1504
P-CUCme.Ubql-l:l:17	8	Ubiquitin 1	P	964	Promoter
EXP-CUCme.Ubq 1:1:4	9	Ubiquitin 1	EXP	1022	Promoter;Leader;Intron	l-479;480561;562-1019
P-CUCme.Ubq 1-1:1:18	10	Ubiquitin 1	P	479	Promoter
EXP-CUCme.Ubql:l:5	11	Ubiquitin 1	EXP	716	Promoter; Leader; Intron	1-173;174- 255;256-713
P-CUCme.Ubq 1-1:1:19	12	Ubiquitin 1	P	173	Promoter
P-CUCme.l-l:l:l	13	Phosphatase 2A	EXP	2000	Promoter;Leader; Intron; Leader	Reverse compliment; see SEQ ED NO: 155
P-CUCme.2-l:l:l	14	Actin 1	EXP	2000	Promoter;Leader;Intron; Leader	l-964;965- 1028; 1029- 1991; 1992-2003
P-CUCme.3-l:l:3	15	Actin 2	EXP	1990	Promoter; Leader; Intron ; Leader	1-1243;1244- 1319;13201982;1983-1990
P-CUCme.4-l:l:2	16	Ubiquitin 2	EXP	2005	Promoter;Leader; Intron; Leader	1-1646; 1647- 1704; 1705-

Annotation	SEQ ID NO:	Description	Composition Type	Size (bp)	Composition	Coordinates of Eléments within EXP
						2005;2006-2008
P-CUCme.5-l:l:2	17	Ubiquitin 3	EXP	2004	Promoter ;Leader; Intron	l-748;749819;820-2004
P-CUCme.6-l:l:l	18	Tubulin beta chain	EXP	1935	Promoter;Leader;Intron; Leader	1-1436;1437- 1482;1483- 1919;1920-1935
P-CUCme.8-l:l:2	19	Tubulin beta chain	EXP	1606	Promoter;Leader	l-1527;1528-1606
P-CUCme.9-l:l:2	20	Tubulin beta chain	EXP	1487	Promoter; Leader	1-1384;1385-1487
P-CUCme.lO-l:l:l	21	Tubulin beta chain	EXP	1448	Promoter;Leader	1-1363;1364-1448
P-CUCme. 11-1:1:2	22	Elongation Factor 1 alpha	EXP	1235	Promoter ;Leader; Intron	1-617;618- 677;678- 1213;1214-1235
P-CUCme. 15-1:1:2	23	Elongation Factor 1 alpha	EXP	2003	Promoter;Leader; Intron; Leader	l-1330;1331- 1435; 1430- 1975; 1976-2002
P-CUCme. 16a-l: 1:2	24	Ubiquitin 7	EXP	2015	Promoter;Leader
P-CUCme.l6b-l:l:l	25	Ubiquitin 6	EXP	2006	Promoter; Leader
P-CUCme. 17-1:1:2	26	ubiquitin-40S ribosomal protein S27a	EXP	2017	Promoter;Leader	1-1969; 1970-2017
P-CUCme. 18-1:1:2	27	ubiquitin-40S ribosomal protein S27a	EXP	1353	Promoter;Leader	l-1308;1309-1353
P-CUCme. 19-1:1:2	28	Chloropyll a/b binding protein	EXP	2005	Promoter;Leader	l-1960;1961-2005
P-CUCme.20-l:l:2	29	Chloropyll a/b binding protein	EXP	1445	Promoter;Leader	l-1390;1391-1445
P-CUCme.21-1:1:1	30	Chloropyll a/b binding protein	EXP	1282	Promoter; Leader	1-1233;1234-1282
P-CUCme.22-1:1:3	31	Elongation Factor 4 alpha	EXP	2002
P-CUCme.24-l:l:2	32	S-Adenosylmethionine	EXP	2003	Promoter;Leader;Intron;	l-1067;1068-

Annotation	SEQ ID NO:	Description	Composition Type	Size (bp)	Composition	Coordinates of Eléments within EXP
		Synthetase			Leader	1165;1166- 2001;2002-2003
P-CUCme.26-l:l:2	33	Stress responsive protein	EXP	1372	Promoter;Leader;Intron; Leader	l-577;578- 654;655- 1366; 1367-1372
P-CUCme.28-l:l:2	34	Ribosomal protein S5a	EXP	1122
P-CUCme.29-l:l:2	35	Ribosomal protein S5a	EXP	2017	Promoter;Leader;Intron; Leader	l-490;491- 571;5722012;2013-2017
CumMe WSM SF14398 1.G5150	36	LHCB6 (LIGHT HARVESTING COMPLEX PSII SUBUNIT 6)	EXP	2000
CumMe WSM SF14483 9.G5080	37	EIF2 GAMMA translation initiation factor	EXP	1760
CumMe WSM SF14604 O.G5O5O	38	EIF2 translation initiation factor	EXP	1767
CumMe WSM SF16408 .G5350	39	élongation factor Tu	EXP	2000
CumMe WSM SF16429 .G5670	40	unknown protein	EXP	2000
CumMe WSM SF16444 .G5140	41	histone H4	EXP	2000	Promoter;Leader	1-1947; 1948-2000
CumMe WSM SF16530 .G6000	42	HMGB2 (HIGH MOBILITY GROUP B 2) transcription factor	EXP	2000
CumMe WSM SF16553 .G5090	43	PBG1; threonine-type endopeptidase	EXP	1115

Annotation	SEQ ID NO:	Description	Composition Type	Size (bp)	Composition	Coordinates of Eléments within EXP
CumMe WSM SF16563 .G5560	44	ATARFB1A (ADPribosylation factor B1 A)	EXP	2000	Promoter; Leader; Intron; Leader	l-1329;1330- 1427;1428- 1988; 1989-2000
CumMe WSM SF16675 .G5720	45	chromatin protein family	EXP	2000
CumMe WSM SF16920 .G5650	46	CSD1 (COPPER/ZINC SUPEROXIDE DISMUTASE 1)	EXP	2000
CumMe WSM SF16953 .G5180	47	SCE1 (SUMO CONJUGATION ENZYME 1); SUMO ligase	EXP	2000
CumMe WSM SF17051 .G5470	48	60S ribosomal protein L9 (RPL90D)	EXP	2000
CumMe WSM SF171H .G5790	49	ubiquinol-cytochrome C reductase complex ubiquinone-binding protein	EXP	2000	Promoter;Leader	1-1895;1896-2000
CumMe WSM SF17142 .G5920	50	peptidyl-prolyl cis-trans isomerase, chloroplast	EXP	2000
CumMe WSM SF17190 .G6200	51	PRK (PHOSPHORIBULOKI NASE)	EXP	2000
CumMe WSM SF17250 .G5910	52	LHCB5 (LIGHT HARVESTING COMPLEX OF PHOTOSYSTEM II 5)	EXP	2000
CumMe WSM SF17252 .G7330	53	nascent polypeptideassociated complex (NAC) domain-	EXP	2000	Promoter;Leader; Intron	1-1195;1196- 1297; 1298-2000

Annotation	SEQ ID NO:	Description	Composition Type	Size (bp)	Composition	Coordinates of Eléments within EXP
		containing protein
CumMe WSM SFI7253 .G5150	54	RPS9 (RIBOSOMAL PROTEIN S9)	EXP	1547
CumMe WSM SFI7322 .G5110	55	60S ribosomal protein L22 (RPL22A)	EXP	2000
CumMe WSM SFI7349 .G5770	56	PGRL1B (PGR5-Like B)	EXP	2000
CumMe WSM SFI7357 .G5630	57	40S ribosomal protein S10(RPS10B)	EXP	2000
CumMe WSM SFI7494 .G5140	58	MEE34 (maternai effect embryo arrest 34)	EXP	1591
CumMe WSM SFI7524 .G6410	59	SUS2 (ABNORMAL SUSPENSOR 2)	EXP	2000
CumMe WSM SFI 7672 .G5610	60	PSAK (photosystem I subunit K)	EXP	2000
CumMe WSM SFI7773 .G6620	61	aconîtase C-terminal domain-containing protein	EXP	2000
CumMe WSM SFI7866 .G6050	62	ATPDIL5-1 (PDI-like 5l)	EXP	2000
CumMe WSM SFI 8004 .G6600	63	hydroxyproline-rich glycoprotein family protein	EXP	2000
CumMe WSM SFI8045 .G6670	64		EXP	2000
CumMe WSM SFI8053 .G5410	65	endomembrane protein 70	EXP	2000
CumMe WSM SFI8287 .G5380	66	CP12-1	EXP	2000

Annotation	SEQ ID NO:	Description	Composition Type	Size (bP)	Composition	Coordinates of Eléments within EXP
CumMe WSM SF18488 .G5340	67	caffeoyl-CoA 3-0methyltransferase	EXP	2000	Promoter;Leader	1-1923; 1924-2000
CumMe WSM SF18504 .G5090	68	vacuolar ATP synthase subunit H family protein	EXP	2000
CumMe WSM SF1853O .G5750	69	GUN5 (GENOMES UNCOUPLED 5); magnésium chelatase	EXP	2000
CumMe WSM SF18536 .G6480	70	MBF1A (MULTIPROTEIN BRIDGING FACTOR IA) transcription coactivator	EXP	2000
CumMe WSM SF18575 .G6410	71	unknown protein	EXP	2000
CumMe WSM SF 18634 .G5190	72	60S ribosomal protein L23 (RPL23A)	EXP	2000	Promoter;Leader	1-1971; 1972-2000
CumMe WSM SF 18645 .G5380	73	GS2 (GLUTAMINE SYNTHETASE 2)	EXP	2000
CumMe WSM SF18716 .G5860	74	40S ribosomal protein SI2 (RPS12A); reverse compliment: Auxininduced protein xlOAlike	EXP	2000	Promoter;Leader	Reverse compliment; see SEQIDNO: 184
CumMe WSM SF 18801 .G5040	75		EXP	2000
CumMe WSM SF 18806 .G6220	76	unknown protein	EXP	2000
CumMe WSM SF 18850 .G5630	77	PAC1; threonine-type endopeptidase	EXP	2000

Annotation	SEQ ID NO:	Description	Composition Type	Size (bp)	Composition	Coordinates of Eléments within EXP
CumMe WSM SF18863 .G7550	78	ATP synthase gamma chain, mitochondrial (ATPC)	EXP	2000
CumMe WSM SF18986 .G6110	79	GER1 (GERMIN-LIKE PROTEIN 1 ); oxalate oxidase	EXP	2000
CumMe WSM SF19064 .G5690	80	histone H3.2	EXP	2000	Promoter;Leader;Intron	1-1581;1582- 1670; 1671-2000
CumMe WSM SF19323 .G5120	81	chloroplast outer envelope GTP-binding protein, putative	EXP	2000
CumMe WSM SF19452 .G5090	82	glucan phosphorylase, putative	EXP	1072
CumMe WSM SF19631 .G5170	83	RuBisCO activase, putative	EXP	1730
CumMe WSM SF19647 .G5760	84	6-phosphogluconate dehydrogenase family protein	EXP	2000	Promoter;Leader;Intron; Leader	1 -936;937- 1021;1022- 1992; 1993-2000
CumMe WSM SF19839 .G5090	85	ATPDX1.1 (pyridoxine biosynthesis 1.1)	EXP	1020	Promoter;Leader	l-928;929-1020
CumMe WSM SF19850 .G5130	86	HMGB2 (HIGH MOBILITY GROUP B 2) transcription factor	EXP	2000
CumMe WSM SF19902 .G5260	87	universal stress protein (USP) family protein / early nodulîn ENOD18 family protein	EXP	2000
CumMe WSM SF 19992 .G6100	88	unknown protein	EXP	2000
CumMe WSM SF20132	89	peroxidase 21	EXP	2000	Promoter;Leader	1-1962;1963-2000

'S

Annotation	SEQ ID NO:	Description	Composition Type	Size (bp)	Composition	Coordinates of Eléments within EXP
.G5560
CumMe WSM SF20147 .G7910	90	CSD1 (COPPER/ZINC SUPEROXIDE DISMUTASE 1)	EXP	2000
CumMe WSM SF20355 .G5130	91	ATP synthase family	EXP	2000
CumMe WSM SF20359 .G5870	92	NADH-ubiquinone oxidoreductase 20 kDa subunit, mitochondrial	EXP	2000
CumMe_WSM_SF20368 .G5700	93	PGR5 (proton gradient régulation 5)	EXP	2000
CumMe WSM SF20409 .G5240	94	élongation factor IB alpha-subunît 1 (eEFIBalphal)	EXP	2000
CumMe WSM SF20431 .G6340	95	DHS2 (3-deoxy-darabino-heptulosonate 7phosphate synthase)	EXP	2000
CumMe WSM SF2O5O5 .G5440	96	THIC (ThiaminC); ADPribose pyrophosphohydrolase	EXP	1373
CumMe WSM SF20509 .G5920	97	Y14; RNA binding ! protein binding	EXP	2000
CumMe WSM SF20645 8.G5970	98	FAD2 (FATTY ACID DESATURASE 2)	EXP	2000	Promoter	1-2000
CumMe WSM SF2O653 4.G5200	99	unknown protein	EXP	2000
CumMe WSM SF20997 .G6990	100	ALD1 (AGD2-LIKE DEFENSE RESPONSE PROTEIN 1)	EXP	2000
CumMe WSM SF21O35	101	sodium/calcium	EXP	1078

Annotation	SEQ ID NO:	Description	Composition Type	Size (bp)	Composition	Coordinates of Eléments within EXP
.G5090		exchanger famîly protein
CumMe WSM SF21117 .G5370	102	3 OS ribosomal protein, putative	EXP	2000
CumMe WSM SF21141 .G5630	103	40S ribosomal protein S24 (RPS24A)	EXP	2000
CumMe WSM SF21198 .G5180	104		EXP	1974
CumMe WSM SF21366 .G5980	105	GRF12 (GENERAL REGULATORY FACTOR 12)	EXP	2000
CumMe WSM SF21828 .G5150	106	cpHsc70-l (chloroplast heat shock protein 70-1 )	EXP	1643
CumMe WSM SF21886 .G5080	107	NPQ4 (NONPHOTOCHEMIC AL QUENCHING)	EXP	2000
CumMe WSM SF22008 .G5670	108	NAP1;2 (NUCLEOSOME ASSEMBLY PROTEIN i;2)	EXP	2000
CumMe WSM SF22070 .G5280	109	fructose-bisphosphate aldolase, putative	EXP	2000
CumMe WSM SF22097 .G5540	110	APX3 (ASCORBATE PEROXIDASE 3)	EXP	2000
CumMe WSM SF22254 .G5760	111	40S ribosomal protein S7 (RPS7B)	EXP	2000
CumMe WSM SF22275 .G5780	112	ribosomal protein L17 family protein	EXP	1027
CumMe WSM SF22355 .G5310	113		EXP	2000

Annotation	SEQ ID NO:	Description	Composition Type	Size w	Composition	Coordinates of Eléments within EXP
CumMe WSM SF22531 .G5120	114	eukaryotic translation initiation factor IA, putative	EXP	2000	Promoter;Leader;Intron; Leader	l-759;760- 858;859- 1979; 1980-2000
CumMe WSM SF22870 .G5370	115	ATSARAIA (ARABIDOPSIS THALIANA SECRETIONASSOCIATED RAS SUPER FAMILY 1)	EXP	2000
CumMe WSM SF22934 .G5290	116	T-complex protein 1 epsilon subunit, putative	EXP	2000
CumMe WSM SF23181 .G5100	117	CEV1 (CONSTITUTIVE EXPRESSION OF VSP D	EXP	1025
CumMe WSM SF23186 .G6160	118	ubiquinol-cytochrome C reductase complex 14 kDa protein, putative	EXP	2000
CumMe WSM SF23397 .G5210	119	RPL27 (RIBOSOMAL PROTEIN LARGE SUBUNIT 27)	EXP	2000
CumMe WSM SF23760 .G5200	120	NDPK1; ATP binding/ nucleoside diphosphate kinase	EXP	2000	Promoter; Leader	1-1901;1902-2000
CumMe WSM SF23906 .G6180	121	PSBX (photosystem II subunit X)	EXP	2000
CumMe WSM SF24040 .G5450	122	RPS17 (RIBOSOMAL PROTEIN S17)	EXP	2000
CumMe WSM SF24045 .G5400	123	EXL3 (EXORDIUM LIKE 3)	EXP	2000
CumMe WSM SF24H7	124	6 OS ribosomal protein	EXP	2000

Annotation	SEQ ID NO:	Description	Composition Type	Size (Ï>P)	Composition	Coordinates of Eléments within EXP
.G5600		L26 (RPL26A)
CumMe WSM SF25084 .G5580	125		EXP	2000
CumMe WSM SF25141 .G5160	126	isocitrate dehydrogenase, putative	EXP	1397	Promoter; Leader	1-1322; 1323-1397
CumMe WSM SF25355 .G5000	127	LOS1; copper ion binding translation élongation factor	EXP	2000	Promoter;Leader;Intron; Leader;CDS	l-734;735- 811;812- 1340;1341- 1360; 1361-2000
CumMe WSM SF25370 .G5000	128	PSBP-1 (PHOTOSYSTEM II SUBUNIT P-l)	EXP	1657
CumMe WSM SF25455 .G5370	129	GLY3 (GLYOXALASE Π 3)	EXP	2000
CumMe WSM SF25936 .G5450	130	mitochondrial substrate carrier family protein	EXP	2000	Promoter;Leader	1-1878; 1879-2000
CumMe WSM SF27080 .G5510	131	LIP1 (LIPOIC ACID SYNTHASE 1)	EXP	2000
CumMe WSM SF27222 .G5150	132	DRT 112; copper ion binding / électron carrier	EXP	2000
CumMe WSM SF27957 .G5450	133	SMAP1 (SMALL ACIDIC PROTEIN 1)	EXP	2000
CumMe WSM SF28729 .G5340	134	RNA-binding protein cp29, putative	EXP	1696
CumMe WSM SF288O5 .G6200	135	unknown protein	EXP	2000
CumMe WSM SF31264 | .G5380	136	ATPH1 (ARABIDOPSIS THALIANA PLECKSTRIN HOMOLOGUE 1)	EXP	2000

4l

Annotation	SEQ ID NO:	Description	Composition . ..Type	Size ΦΡ)	Composition	Coordinates of Eléments within EXP
CumMe WSM SF35856 .G5150	137	TIP4;1 (tonoplast intrinsic protein 4;1)	EXP	1575
CumMe WSM SF4O859 .G5250	138	SMT2 (STEROL METHYLTRANSFERA SE 2)	EXP	2000
CumMe WSM SF41124 .G5080	139	40S ribosomal protein S2 (RPS2C)	EXP	1006	Promoter; Leader	l-883;884-1006
CumMe WSM SF41128 .G5410	140	CRY2 (CRYPTOCHROME 2)	EXP	2000
CumMe WSM SF41254 .G5160	141	GDP-D-glucose phosphorylase	EXP	1556
CumMe WSM SF4I588 .G5470	142	PRPL11 (PLASTID RIBOSOMAL PROTEIN Lll)	EXP	2000
CumMe_WSM_SF41644 .G6400	143	SHD (SHEPHERD)	EXP	2000
CumMe WSM SF41983 .G5000	144	catalytic/ coenzyme binding	EXP	1337
CumMe WSM SF42075 .G5100	145	CPN60B (CHAPERONIN 60 BETA)	EXP	2000
CumMe WSM SF42141 .G5110	146	cathepsin B-like cysteine protease, putative	EXP	1212
CumMe WSM SF44933 .G5290	147	EBF1 (EIN3-BINDING F BOX PROTEIN 1) ubiquitin-protein ligase	EXP	2000
CumMe WSM SF44977 .G5000	148	PAP26 (PURPLE ACID PHOSPHATASE 26)	EXP	1254

Annotation	SEQ ID NO:	Description	Composition Type	Size (bp)	Composition	Coordinates of Eléments within EXP
CumMe WSM SF45441 .G5510	149	GAPA-2 (GLYCERALDEHYDE 3-PHOSPHATE DEHYDROGENASE A SUBUNIT 2)	EXP	2000
CumMe WSM SF45882 .G5120	150	fructose-1,6bisphosphatase, putative	EXP	1680
CumMe WSM SF47806 .G5070	151	ATP synthase epsilon chain, mitochondrial	EXP	1524
CumMe WSM SF53106 .G5190	152	CPN60A (CHAPERONIN60ALPHA)	EXP	1851
CumMe WSM SF65588 .G5230	153	vacuolar calcium-binding protein-related	EXP	2000
CumMe WSM SF9060. G5120	154	APE2 (ACCLIMATION OF PHOTOSYNTHESIS TO ENVIRONMENT 2)	EXP	1288
P-CUCme.l-l:l:lrc	155	Phosphatase 2A	EXP	2000	Promoter;Leader; Intron; Leader	1-1135;1136- 1249; 1250- 1990; 1991-2000
EXP-CUCme.4:l:l	156	Ubiquitin 2	EXP	2011	Promoter;Leader;Intron; Leader	1-1646; 1647- 1704; 1705- 2005;2006-2008
P-CUCme.4-l:l:4	157	Ubiquitin 2	P;L	1698	Promoter;Leader
I-CUCme.4-l:l:l	158	Ubiquitin 2	I;L	313	Intron;Leader
EXP-CUCme.5:l:l	159	Ubiquitin 3	EXP	2010	Promoter; Leader; Intron; Leader	l-748;749- 819;8202004;2005-2007
P-CUCme.5-l:l:3	160	Ubiquitin 3	P;L	1107	Promoter; Leader
I-CUCme.5-l:l:l	161	Ubiquitin 3	I;L	903	Intron; Leader

Annotation	SEQ ID NO:	Description	Composition Type	Size ΦΡ)	Composition	Coordinates of Eléments within EXP
EXP-CUCme.eEFla:l:l	162	Elongation Factor 1 alpha	EXP	1235	Promoter; Leader; Intron; Leader	1-617;618- 677;678- 1213;1214-1235
P-CUCme.eEFla-l:l:l	163	Elongation Factor 1 alpha	P	617	Promoter
L-CUCme.eEFla-l:l:l	164	Elongation Factor 1 alpha	L	54	Leader
I-CUCme.eEFla-l:l:l	165	Elongation Factor 1 alpha	I	545	Intron
L-CUCme.eEFla-l:l:2	166	Elongation Factor 1 alpha	L	19	Leader
P-CUCme. 19-1:1:3	167	Chloropyll a/b binding protein	EXP	2003	Promoter;Leader	l-1958;1959-2003
EXP- CUCme.SAMS2:1:1	168	S-Adenosylmethionine Synthetase	EXP	2004	Promoter;Leader;Intron	l-1067;1068- 1165; 1166-2003
P-CUCme.SAMS2-l:l:l	169	S-Adenosylmethionine Synthetase	P	1067	Promoter
L-CUCme.SAMS2-l:l:l	170	S-Adenosylmethionine Synthetase	L	92	Leader
I-CUCme.SAMS2-l:l:l	171	S - Adenosylmethionine Synthetase	I	845	Intron
EXP-CUCme.29:l:l	172	Ribosomal protein S5a	EXP	2018	Promoter;Leader; Intron ; Leader	l-490;491- 571;572- 2012;2013-2018
P-CUCme.29-I:l:4	173	Ribosomal protein S5a	P;L	565	Promoter;Leader
I-CUCme.29-l:l:l	174	Ribosomal protein S5a	i;L	1453	Intron;Leader
P- CUCme.CumMe WSM SF16444.G5140-l:I:l	175	histone H4	EXP	1999	Promoter; Leader; Intron	1-1946;947-1999

Annotation	SEQ ID NO:	Description	Composition Type	Size (bp)	Composition	Coordinates of Eléments within EXP
Ρ- CUCme.CumMe WSM SF16563.G5560-l:l:l	176	ATARFB1A (ADPribosylation factor B1 A)	EXP	2004	Promoter; Leader; Intron ; Leader	1-1331;1332- 1429;14301992; 1993-2004
P- CUCme.CumMe WSM SF17111.05790-1:1:1	177	ubiquinol-cytochrome C reductase complex ubiquinone-binding protein	EXP	2005	Promoter;Leader	1-1901; 1902-2005
EXP- CumMe.WSM SF17252. G7330:l:l	178	nascent polypeptideassociated complex (NAC) domaincontaining protein	EXP	1978	Promoter; Leader; Intron ; Leader	1-1167;1168- 1269;1270- 1972;1973-1975
P- CUCme.WSM SF 17252. 07330-1:1:1	179	nascent polypeptideassociated complex (NAC) domaincontaining protein	P;L	1263	Promoter;Leader
I- CUCme.WSM SF17252. G7330-l:l:l	180	nascent polypeptideassociated complex (NAC) domaincontaining protein	I;L	715	lntron;Leader
P- CUCme.CumMe WSM SF18488.G5340-l:l:l	181	caffeoyl-CoA 3-Omethyltransferase	EXP	2000	Promoter; Leader	l-923;1924-2000
P- CUCme.CumMe WSM SF18536.G648O-1:1:1	182	MBF1A (MULTIPROTEIN BRIDGING FACTOR IA) transcription coactivator	EXP	2000	Promoter;Leader;Intron
P- CUCme.CumMe WSM SF18634.G5190-1:1:1	183	60S ribosomal protein L23 (RPL23A)	EXP	1989	Promoter;Leader	l-1960;1961-1989

’ί

Annotation	SEQ ID NO:	Description	Composition Type	Size (bp)	Composition	Coordinates of Eléments within EXP
P- CUCme.CumMe WSM SF18716.G5860-l:l:l	184	Auxin-induced prtoein X10A-like	EXP	1463	Promoter;Leader	1-1392;1393-1463
EXP- CUCme.WSM SF19064. G5690:l:l	185	histone H3.2	EXP	2006	Promoter;Leader;Intron; Leader	1-1581;15821670;16712000;2001-2003
P- CUCme.WSM SF19064. G5690-l:l:l	186	histone H3.2	P;L	1664	Promoter;Leader
I- CUCme.WSM SF 19064. G5690-l:l:l	187	histone H3.2	I;L	342	Intron; Leader
P- CUCme.CumMe WSM SF19647.G5760-l:l:l	188	6-phosphogluconate dehydrogenase family protein	EXP	2003	Promoter; Leader; Intron; Leader	l-939;940- 1024; 1025- 1995; 1996-2003
P- CUCme.CumMe WSM SF19839.G5O9O-1:1:1	189	ATPDX1.1 (pyridoxine biosynthesis 1.1)	EXP	1024	Promoter;Leader	l-904;905-1024
P- CUCme.CumMe WSM SF20132.G5560-l:l:l	190	peroxîdase 21	EXP	2001	Promoter;Leader	1-1962; 1963-2001
P- CUCme.CumMe WSM SF206458.G5970-l:l:l	191	FAD2 (FATTY ACID DESATURASE 2)	EXP	4175	Promoter ;Leader; Intron; Leader	1-2171;2172- 2325;23264155;4156-4175
P- CUCme.CumMe WSM SF22531.G5120-1:1:1	192	eukaryotîc translation initiation factor IA, putative	EXP	1999	Promoter; Leader; Intron; Leader	l-759;760- 858;8591978; 1979-1999
P- CUCme.CumMe WSM SF23760.G5200-l:l:l	193	NDPK.1; ATPbinding/ nucleoside diphosphate kinase	EXP	2000	Promoter; Leader	1-1901; 1902-2000

Annotation	SEQ ID NO:	Description	Composition Type	Size (bp)	Composition	Coordinates of Eléments within EXP
P- CUCme.CumMe WSM SF23906.G6180-l:l:l	194	PSBX (photosystem II subunit X)	EXP	2000	Promoter;Leader
P- CUCme.CumMe WSM SF25141.G5160-l:l:2	195	isocitrate dehydrogenase, putative	EXP	1400	Promoter; Leader	l-1325;1326-1400
P- CUCme.CumMe WSM SF25355.G5000-l:l:l	196	LOS1; copper ion binding translation élongation factor	EXP	2019	Promoter; Leader;Intron; LeaderjCDS	l-734;735- 811;8121340;1341- 1360; 1361-2019
P- CUCme.CumMe WSM SF25936.G5450-l:l:l	197	mitochondrial substrate carrier family protein	EXP	1999	Promoter;Leader	1-1877;1878-1999
P- CUCme.CumMe WSM SF35856.G5l50-l:l:l	198	TIP4;1 (tonoplast intrinsic protein 4; 1 )	EXP	1578
P- CUCme.CumMe WSM SF41124.G5080-l:l:l	199	40S ribosomal protein S2 (RPS2C)	EXP	1023	Promoter; Leader	l-945;946-1023
P-CUCme.20-l:3	211	Chloropyll a/b binding protein	EXP	1446	Promoter; Leader	1-1390; 1391-1446
EXP-CUCme.29: l :2	212	Ribosomal protein S5a	EXP	2018	Promoter; Leader; Intron; Leader	l-490;491- 57l;5722011;2013-2018

[084] As shown in Table l, for example, the transcriptional regulatory expression element group (EXP) designated EXP-CUCme.Ubql:l:l (SEQ ID NO: l), with components isolated from C. melo, comprises a 2068 base pair sized (bp) promoter element, P-CUCme.Ubql-l:l:l5 (SEQ ID NO: 2), operably linked 5’ to a leader element, L-CUCme.Ubql-l:l:i (SEQ ID NO: 3), operably linked 5’ to an intron element, I-CUCme.Ubql-l:l:l (SEQ ID NO: 4). The transcriptional regulatory expression element group (EXP) designated EXP-CUCme.Ubql:l:2 (SEQ ID NO: 5), with components isolated from C. melo, comprises a 1459 bp promoter element, P-CUCme.Ubql-l:l:l6 (SEQ ID NO: 6), operably linked 5’ to a leader element, LCUCme.Ubql-l:l:l (SEQ ID NO: 3), operably linked 5’ to an intron element, I-CUCme.Ubqll:l:l (SEQ ID NO: 4). The transcriptional regulatory expression element group (EXP) designated EXP-CUCme.Ubql:l:3 (SEQ ID NO: 7), with components isolated from C. melo, comprises a 964 bp promoter element, P-CUCme.(Jbql-l:l:l7 (SEQ ID NO: 8), operably linked 5’ to a leader element, L-CUCme.Ubql-I:l:l (SEQ ID NO: 3), operably linked 5’ to an intron element, l-CUCme.Ubql-l:l:l (SEQ ID NO: 4). The transcriptional regulatory expression element group (EXP) designated EXP-CUCme.(Jbql:l:4 (SEQ ID NO: 9), with components isolated from C. melo, comprises a 479 bp promoter element, P-CUCme.Ubql-l:l:l8 (SEQ ID NO: 10), operably linked 5’ to a leader element, L-CUCme.Ubq 1-1:1:1 (SEQ ID NO: 3), operably linked 5’ to an intron element, I-CUCme.Ubql-l:l:l (SEQ ID NO: 4). The transcriptional regulatory expression element group (EXP) designated EXP-CUCme.Ubql:l:5 (SEQ ID NO: 11), with components isolated from C. melo, comprises a 173 bp promoter element, P-CUCme.Ubql-l:l:19 (SEQ ID NO: 12), operably linked 5’ to a leader element, LCUCme.Ubql-l:l:l (SEQ ID NO: 3), operably linked 5’ to an intron element, I-CUCme.Ubql1:1:1 (SEQ IDNO:4).

[085] An alignment of the ubiquitin 1 promoter sequences is provided in Figs, la-lf. The promoter éléments, P-CUCme.Ubql-l:l:16 (SEQ ID NO: 6), P-CUCme.Ubql-1:1:17 (SEQ ID

NO: 8), P-CUCme.Ubql-l:l:18 (SEQ ID NO: 10) and P-CUCme.Ubql-1:1:19 (SEQ IDNO: 12) were built by introducing varyîng lengths of délétions from the 5’ end of the promoter, PCUCme.Ubql-1:1:15 (SEQ IDNO: 2). y48

Example 2: Analysis of Regulatory Eléments Driving GUS in Soy Cotylédon Protoplasts [086] Soybean cotylédon protoplasts were transformed with plant expression vectors containing a test transcriptional regulatory expression element group driving expression of the Bglucuronidase (GUS) transgene and compared to GUS expression in leaf protoplasts in which expression of GUS is driven by known constitutive promoters.

[087] Expression of a transgene driven by EXP-CUCme.Ubql:l:l (SEQ ID NO: I), EXPCUCme.Ubql:l:2 (SEQ ID NO: 5), EXP-CUCme.Ubql:l:3 (SEQ ID NO: 7), EXPCUCme.Ubql:l:4 (SEQ ID NO: 9) and EXP-CUCme.Ubql:l:5 (SEQ ID NO: 11) was compared with expression from known constitutive promoters. Each plant expression vector was comprised of a right border région from Agrobacterium tumefaciens, a first transgene cassette comprised of an EXP sequence or known constitutive promoter operably linked 5’ to a coding sequence for β-glucuronidase (GUS, SEQ ID NO: 206) containing a processable intron derived from the potato light-inducible tissue-specific ST-LSl gene (Genbank Accession: X04753), operably linked 5’ to a 3’ termination région from the Gossypium barbadense E6 gene (TGb.E6-3b:l:l, SEQ ID NO: 204), the Pisum sativtim RbcS2-E9 gene (T-Ps.RbcS2-E9-l:l:6, SEQ ID NO: 203), or the Gossypium barbadense FbLate-2 gene (T-Gb.FbL2-l:l : l, SEQ ID NO: 205); a second transgene sélection cassette used for sélection of transformed plant cells that either confers résistance to the herbicide glyphosate (driven by the Arabidopsis Actin 7 promoter) or the antibiotic, kanamycin and a left border région from A. tumefaciens. A promoterless control plant expression vector (pMON 124912) served as a négative control for expression. The foregoing test and constitutive expression element groups were cloned into plant expression vectors as shown in Table 2 below.

Table 2. Plant expression vectors and corresponding expression element group and 3* UTR.

Expression Vector	Regulatory Element	SEQ ID NO:	3’ UTR
pMON80585	EXP-At.Atnttl:l:2	200	T-Ps.RbcS2-E9- 1:1:6
pMON 109584	EXP-CaMV.35Senh+Ph.DnaK:l:3	201	T-Gb.E6-3b:l:l
pMON 118756	EXP-At.Act7:l:l 1	202	T-Gb.E6-3b:l:l
pMON 124912	No promoter		T-Gb.FbL2-1:1:1

Expression Vector	Regulatory Elément	SEQ ID NO:	3* UTR
pMONl 38776	EXP-CUCme.Ubql:l:l	1	T-Gb.FbL2-l	1:1
pMON 138777	EXP-CUCme.Ubq 1:1:2	5	T-Gb.FbL2-l	1:1
pMON 138778	EXP-CUCme.Ubql:l:3	7	T-Gb.FbL2-l	1:1
pMON 138779	EXP-CUCme.Ubq 1:1.:4	9	T-Gb.FbL2-l	1:1
pMON 138780	EXP-CUCme.Ubq 1:1:5	11	T-Gb.FbL2-l	1:1

[088] Two plasmids, for use in co-transformation and normalization of data, were also constructed. One transformation control plasmid was comprised of a constitutive promoter, driving the expression of the firefly (Photinus pyralis) luciferase coding sequence (FLuc, SEQ ID NO: 207), operably linked 5’ to a 3’ termination région from the Agrobacterium lumefaciens nopaline synthase gene (T-AGRtu.nos-l:l:l3, SEQ ID NO: 209). The other transformation control plasmid was comprised of a constitutive promoter, driving the expression of the sea pansy (Renilla reniformis) luciferase coding sequence (RLuc, SEQ ID NO: 208), operably linked 5’ to a 3’ termination région from the Agrobacterium lumefaciens nopaline synthase gene.

[089] The plant expression vectors, pMON80585, pMON 109584, pMONl 18756, pMON124912, pMON138776, pMON138777, pMON138778, pMON138779 and pMON138780 were used to transform soybean cotylédon protoplast cells using PEG transformation methods. Protoplast cells were transformed with equimolar amounts of each of the two transformation control plasmids and a test plant expression vector. GUS and luciferase activity was assayed. Measurements of both GUS and luciferase were conducted by placing aliquots of a lysed préparation of cells transformed as above into two different small-well trays. One tray was used for GUS measurements, and a second tray was used to perform a dual luciferase assay using the dual luciferase reporter assay system (Promega Corp., Madison, WI; see for example, Promega Notes Magazine, No: 57, 1996, p.02). Sample measurements were made using 3 or 4 replicates per transformation. The average GUS and luciferase values are presented in Table 3 below. uA—

Table 3. Average GUS and luciferase expression values and GUS/lucifcrase ratios.

Construct	Regulatory Element	SEQ ID NO:	Average GUS	Average FLuc	Average RLuc	GUS/ FLuc	GUS/ RLuc
pMON80585	EXP-At.Atnttl:l:2	200	55173	6498	30503	8.49	1.81
pMON 109584	EXP-CaMV.35Senh+Ph.DnaK:l:3	200	24940	5050.75	35495	4.94	0.70
pMON 118756	EXP-At.Act7:l:l 1	201	9871	6880	40850	1.43	0.24
pMON 124912	No promoter		2000	11670	73187	0.17	0.03
pMONl 38776	EXP- CUCme.Ubq 1:1:1	1	26972	6467.25	37200	4.17	0.73
pMON 138777	EXP- CUCme.Ubql:l:2	5	41307	5902.5	24396	7.00	1.69
pMON 138778	EXP- CUCme.Ubq 1:1:3	7	90140	10710.5	60983	8.42	1.48
pMON 138779	EXP- CUCme.Ubq 1:1:4	9	35526	5590	28001	6.36	1.27
pMON 138780	EXP- CUCme.Ubql:l:5	11	23298	4483.25	19075	5.20	1.22

[090] To compare the relative activity of each promoter in soybean cotylédon protoplasts, GUS values were expressed as a ratio of GUS to luciferase activity and normalized with respect to the 5 expression levels observed for the constitutive expression element groups, EXP-At.Act7:l:l l and EXP-CaMV.35S-enh+Ph.DnaK:l:3. Table 4 below shows the GUS to firefly luciferase (FLuc) ratios normalized with respect to EXP-At.Act7:l:l l and EXP-CaMV.35Senh+Ph.DnaK: l :3. Table 5 below shows the GUS to renilla luciferase (RLuc) ratios normalized with respect to EXP-At.Act7:l:l l and EXP-CaMV.35S-enh+Ph.DnaK:l:3.

Table 4. GUS to firefly luciferase (FLuc) ratios normalized with respect to EXPAt.Act7:l:ll and EXP-CaMV.35S-enh+Ph.DnaK:l:3.

Construct	Regulatory Element	SEQID NO:	GUS/FLuc normalized with respect to EXPAt.Act7:l:ll	GUS/FLuc normalized with respect to EXPCaMV.35Senh+Ph.DnaK:l:3
ΡΜΟΝ80585	EXP-At.Atnttl:l:2	200	5.92	1.72
pMON 109584	EXP-CaMV.35Senh+Ph.DnaK: 1:3	201	3.44	1.00
pMON 118756	EXP-At.Act7:l:ll	202	1.00	0.29

pMON 124912	No promoter		0.12	0.03
pMON 138776	EXP-CUCme.Ubql:l:l	1	2.91	0.84
pMON 138777	EXP-CUCme.Ubql:l:2	5	4.88	1.42
pMON 138778	EXP-CUCme.Ubq 1:1:3	7	5.87	1.70
pMON 138779	EXP-CUCme.Ubql:l:4	9	4.43	1.29
pMON 138780	EXP-CUCme.Ubql:l:5	11	3.62	1.05

Table 5. GUS to renilla lucifcrase (RLuc) ratios normalized with respect to EXPAt.Act7:l:ll and EXP-CaMV.35S-enh+Ph.DnaK:l:3.

Construct	Regulatory Element	SEQ ID NO:	GUS/RLuc normalized with respect to EXPAt.Act7:l:ll	GUS/RLuc normalized with respect to EXPCaMV.35Senh+Ph.DnaK: 1:3
ΡΜΟΝ80585	EXP-At.Atnttl:l:2	200	7.49	2.57
pMON 109584	EXP-CaMV.35Senh+Ph.DnaK:l:3	201	2.91	1.00
pMON 118756	EXP-At.Act7:l:ll	202	1.00	0.34
pMON 124912	No promoter		0.11	0.04
pMON 138776	EXP-CUCme.Ubq 1:1:1	1	3.00	1.03
pMON 138777	EXP-CUCme.Ubq 1:1:2	5	7.01	2.41
pMON 138778	EXP-CUCme.Ubql:l:3	7	6.12	2.10
pMON 138779	EXP-CUCme.Ubql:l:4	9	5.25	1.81
pMON 13 8780	EXP-CUCme.Ubql:l:5	11	5.05	1.74

[091] As can be seen in Tables 4 and 5 above, each of the expression element groups EXPCUCme.Ubql:l:l (SEQ ID NO: l), EXP-CUCme.Ubql:l:2 (SEQ ID NO: 5), EXPCUCme.Ubql:l:3 (SEQ ID NO: 7), EXP-CUCme.Ubql:l:4 (SEQ ID NO: 9) and EXPCUCme.Ubq l:l:5 (SEQ ID NO: 11) demonstrated the ability of driving transgene expression in 10 soybean cotylédon protoplasts. Expression levels were greater than that of EXP-At.Act7:l:l 1 and was 2.9 to 5.8 (FLuc) or 3 to 7 (RLuc) fold higher than EXP-At.Act7:l:l 1 in this assay.

Expression was équivalent or higher than expression observed for EXP-CaMV.35Senh+Ph.DnaK:l:3. Expression levels were 0.8 to 1.7 (FLuc) or 1 to 2.4 (RLuc) fold higher than expression observed for EXP-CaMV.35S-enh+Ph.DnaK: 1:3. v'-''

Example 3: Analysis of Regulatory Eléments Driving GUS in Bombarded Soybean Leaves and Roots.

[092] Soybean leaves and roots were transformed with plant expression vectors containing a test transcriptional regulatory expression element group driving expression of the βglucuronidase (GUS) transgene and compared to GUS expression in roots and leaves in which expression of GUS is driven by known constitutive promoters.

[093] Expression of a transgene driven by EXP-CUCme.Ubql:l:l (SEQ ID NO: l), EXPCUCme.Ubql:l:2 (SEQ ID NO: 5), EXP-CUCme.Ubql:I:3 (SEQ ID NO: 7), EXPCUCme.Ubq I : l :4 (SEQ ID NO: 9) and EXP-CUCme.Ubq l : l :5 (SEQ ID NO: 11 ) was compared with expression from known constitutive promoters in particle bombarded soybean leaves and roots. The plant expression vectors used for transformation of leaves and roots was the same as those presented in Table 2 of Example 2 above.

[094] The plant expression vectors, pMON80585, pMON 109584, pMONl 18756, pMON 124912, pMON 138776, pMON 13 8777, pMON 13 8778, pMON 138779 and pMONl38780 were used to transform soybean leaves and roots using particle bombardment transformat ionmethods.

[095] Briefly, A3244 soybean seeds were surface sterilized and allowed to germinate in trays with a photoperiod of 16 hours light and 8 hours of darkness. After approximately 13 days, leaf and root tissue was harvested under stérile conditions from the seedlings and used for bombardment. The tissue samples were randomly placed on a pétri dish containing plant culture medium. Ten micrograms of plasmid DNA was used to coat 0.6 micron gold particles (Catalog #165-2262 Bio-Rad, Hercules, CA) for bombardment. Macro-carriers were loaded with the DNA-coated gold particles (Catalog #165-2335 Bio-Rad, Hercules CA). A PDS 1000/He biolistic gun was used for transformation (Catalog #165-2257 Bio-Rad, Hercules CA). The bombarded root and leaf tissues were allowed to incubate in the dark for 24 hours at 26 degrees Celsius. Following this overnight incubation, the tissues were stained in solution for GUS expression overnight at 37 degrees Celsius. After staining overnight, the tissues were soaked in 70% éthanol overnight to remove chlorophyll and reveal the GUS staining. The tissues were then photographed and a rating scale of “0”,“+” to “H·I t H” reflecting the level of GUS expression is assigned to each construct (0- no expression, + to -h-h-h- - low to high, respectively). v~— [096] Expression of the GUS transgene demonstrated in each tissue is used to infer the relative potential level and specificity of each element’s capacity to drive transgene expression in stably transformed corn plants. Average GUS expression ratings are provided in Table 6 below.

Table 6. GUS expression ratings for particle bombarded leaf and root.

Construct	Regulatory Element	SEQID NO:	Leaf Expression Rating	Root Expression Rating
pMON80585	EXP-At.Atnttl:l:2	200	4*4“1*4*	4-4-
pMON 109584	EXP-CaMV.35Senh+Ph.DnaK:l:3	201	1 1 1 ! 1	4-4-+
pMON 118756	EXP-At.Act7:l:l 1	202	-H-H-	++
pMON 124912	No promoter		0	0
pMON 13 8776	EXP- CUCme.Ubql: 1:1	1	-H-H-	+++
pMON 13 8777	EXP- CUCme.Ubql: 1:2	5	+++	-H-
pMON 13 8778	EXP- CUCme.Ubql: 1:3	7	+++	4-4-
pMON138779	EXP- CUCme.Ubql: 1:4	9	+++	++
ΡΜΟΝ138780	EXP- CUCme.Ubql: 1:5	11	4—1-	+

[097] As can be seen in Table 6 above, each of the expression element groups EXPCUCme.Ubql: l:l (SEQ ID NO: l), EXP-CUCme.Ubql:l:2 (SEQ ID NO: 5), EXPCUCme.Ubql: 1:3 (SEQ ID NO: 7), EXP-CUCme.Ubql :l:4 (SEQ ID NO: 9) and EXPCUCme.Ubql: 1:5 (SEQ ID NO: 11) demonstrated the ability of driving transgene expression in 10 particle bombarded transformed leaf and root tissues.

Exaniple 4: Analysis of Rcgulatory Eléments Driving GUS in Soy Cotylédon Protoplasts [098] Soybean cotylédon protoplasts were transformed with plant expression vectors containing a test transcriptional regulatory expression element group driving expression of the B15 glucuronidase (GUS) transgene and compared to GUS expression in leaf protoplasts in which expression of GUS is driven by known constitutive promoters.

[099] Expression of a transgene driven by P-CUCme.l-l:l:lrc (SEQ ID NO: 155), PCUCme.2-l:l:l (SEQ ID NO: 14), P-CUCme.3-l:l:3 (SEQ ID NO: 15), EXP-CUCme.4:1:1 (SEQ ID NO: 156), EXP-CUCme.5:l:l (SEQ ID NO: 159), P-CUCme.6-l:l:l (SEQ ID NO:^

18), P-CUCme.8-l:l:2 (SEQ ID NO: 19), P-CUCme.9-l:l:2 (SEQ ID NO: 20), P-CUCme. ÎOl : l : l (SEQ ID NO: 21 ), EXP-CUCme.eEF l a: l : l (SEQ ID NO: 162), P-CUCme. 15-1 : l :2 (SEQ ID NO: 23), P-CUCme.I6a-l:l:2 (SEQ ID NO: 24), P-CUCme.17-1:1:2 (SEQ ID NO: 26), PCUCme, 18-1:1:2 (SEQ ID NO: 27), P-CUCme. 19-1:1:3 (SEQ ID NO: 167), P-CUCme.20-l:3 (SEQ ID NO: 211), P-CUCme.21-l:l:l (SEQ ID NO: 30), P-CUCme.22-l:l:3 (SEQ ID NO: 31), EXP-CUCme.SAMS2:1:1 (SEQ ID NO: 168), P-CUCme.26-1:1:2 (SEQ ID NO: 33), PCUCme.28-l:l:2 (SEQ ID NO: 34) and EXP-CUCme.29:l:2 (SEQ ID NO: 212) was compared with expression from known constitutive expression element groups. Each plant expression vector was comprised of a right border région from Agrobacterium tumefaciens, a first transgene cassette comprised of a test promoter or known constitutive promoter operably linked 5’ to a coding sequence for β-glucuronîdase (GUS, SEQ ID NO: 206) containing a processable intron derived from the potato light-inducïble tissue-specific ST-LS1 gene (Genbank Accession: X04753), operably linked 5’ to a 3’ termination région from the Gossypium barbadense E6 gene (T-Gb.E6-3b:l:l, SEQ ID NO: 204), the Pisum sativum RbcS2-E9 gene (T-Ps.RbcS2-E9-I:l:6, SEQ ID NO: 203), or the Gossypium barbadense FbLate-2 gene (T-Gb.FbL2-l:l:l, SEQ ID NO: 205); a second transgene sélection cassette used for sélection of transformed plant cells that either confers résistance to the herbicide glyphosate (driven by the Arabidopsis Actin 7 promoter) or the antibiotic, kanamycin and a left border région from A. tumefaciens. A promoterless control plant expression vector (pMON124912) served as a négative control for expression. The foregoing test and constitutive expression element groups were cloned into plant expression vectors as shown in Table 7 below.

Table 7. Plant expression vectors and corresponding expression element group and 3’ UTR.

Construct	Regulatory Element	SEQ ID NO:	3’ UTR
pMON80585	EXP-At.Atnttl:l:2	200	T-Ps.RbcS2-E9-l:l:6
pMON 109584	EXP-CaMV.35Senh+Ph.DnaK:l:3	201	T-Gb.E6-3b:I:l
pMON118756	EXP-At.Act7:1:11	202	T-Gb.E6-3b:l:l
PMON124912	Promoterless		T-Gb.FbL2-l:l:l
PMON140818	P-CUCme. 1 -1:1:1 rc	155	T-Gb.FbL2-l:l:l
PMON140819	P-CUCme.2-l:l:l	14	T-Gb.FbL2-l:l:l

Construct	Regulatory Element	SEQ ID NO:	3’ UTR
pMON 140820	P-CUCme.3-l:l:3	15	T-Gb.FbL2-l	I	1
pMON 140821	EXP-CUCme.4:l:l	156	T-Gb.FbL2-l	1	1
pMON 140822	EXP-CUCme.5:l:l	159	T-Gb.FbL2-l	1	1
pMON 140823	P-CUCme.6-l:l:l	18	T-Gb.FbL2-l	1	l
pMON 140824	P-CUCme.8-l:l:2	19	T-Gb.FbL2-l	1	1
pMON 140825	P-CUCme.9-l:l:2	20	T-Gb.FbL2-l	1	1
pMON 140826	P-CUCme.l0-l:l:l	21	T-Gb.FbL2-l	1	1
pMON 140827	EXP-CUCme.eEFla:l:l	162	T-Gb.FbL2-l	1	1
pMON 140828	P-CUCme. 15-1:1:2	23	T-Gb.FbL2-l	1	1
pMON 140829	P-CUCme. 16a-l: 1:2	24	T-Gb.FbL2-l	1	1
pMON 140830	P-CUCme. 17-1:1:2	26	T-Gb.FbL2-l	1	1
pMON 140831	P-CUCme.l8-l:l:2	27	T-Gb.FbL2-l	1	1
pMON 140832	P-CUCme.l9-l:l:3	167	T-Gb.FbL2-l	1	1
pMON 140833	P-CUCme.20-l:3	211	T-Gb.FbL2-l	1	1
pMON 140834	P-CUCme.21-l:l:l	30	T-Gb.FbL2-l	1	1
pMON 140835	P-CUCme.22-l:l:3	31	T-Gb.FbL2-l	1	1
pMON 140836	EXP-CUCme.SAMS2:l : 1	168	T-Gb.FbL2-l	1	1
pMON 140837	P-CUCme.26-l:l:2	33	T-Gb.FbL2-l	1	1
pMON 140838	P-CUCme.28-l:l:2	34	T-Gb.FbL2-l	1	1
pMON 140839	EXP-CUCme.29:l:2	212	T-Gb.FbL2-l	1	1

[ΟΙ00] Two plasmids, for use in co-transformation and normalization of data, were also constructed. One transformation control plasmid was comprised of a constitutive promoter, driving the expression of the firefly (Photinus pyralis) luciferase coding sequence (FLuc, SEQ 5 ID NO: 207), operably linked 5’ to a 3’ termination région from the Agrobacterium tumefaciens nopaline synthase gene (T-AGRtu.nos-l:l:l3, SEQ ID NO: 209). The other transformation control plasmid was comprised of a constitutive promoter, driving the expression of the sea pansy (Renifla reniformis) luciferase coding sequence (RLuc, SEQ ID NO: 208), operably linked 5’ to a 3’ termination région from the Agrobacterium tumefaciens nopaline synthase gene.

[0101] The plant expression vectors, pMON80585, pMON109584, pMONl 18756, pMON124912, pMON140818, pMONI40819, pMON140820, pMON140821, pMONI40822, pMON 140823, pMONl40824, pMON 140825, pMON 140826, pMON 140827, pMON 140828, pMON140829, pMON140830, pMON140831, pMON140832, pMON140833, pMON 140834, pMONI40835, pMONI40836, pMONI40837, pMONI40838 and pMONI40839 were used to transform soybean cotylédon protoplast cells using PEG transformation methods. Protoplast cells were transformed with equimolar amounts of each of the two transformation control plasmids and a test plant expression vector. GUS and luciferase activity was assayed.

Measurements of both GUS and luciferase were conducted by placing aliquots of a lysed préparation of cells transformed as above into two different small-well trays. One tray was used for GUS measurements, and a second tray was used to perform a dual luciferase assay using the dual luciferase reporter assay system (Promega Corp., Madison, WI; see for example, Promega Notes Magazine, No: 57, 1996, p.02). Sample measurements were made using 3 or 4 replicates per transformation. The average GUS and luciferase values are presented in Table 8 below.

Table 8. Average GUS and luciferase expression values and GUS/lucifcrase ratios.

Construct	Regulatory Element	SEQ ID NO:	Average GUS	Average FLuc	Average RLuc	GUS/ FLuc	GUS/ RLuc
PMON80585	EXP-At.Atnttl:l:2	200	586	5220.7	8323	0.1100	0.0700
pMON 109584	EXP-CaMV.35Senh+Ph.DnaK:l:3	201	5768	4275	15098	1.3500	0.3800
pMON 118756	EXP-At.Act7:1:11	202	773	7722	10545	0.1000	0.0700
pMON 124912	Promoterless		48	9746.5	13905	0.0000	0.0000
pMON 140818	P-CUCme. 1-1:1:1 rc	155	194	4772	6363	0.0400	0.0300
pMON 140819	P-CUCme.2-l:l:l	14	171	6855	10123	0.0200	0.0200
pMON 140820	P-CUCme.3-l:l:3	15	37	7089.3	9593	0.0100	0.0000
pMON 140821	EXP-CUCme.4:l:l	156	4211	7626.8	13935	0.5500	0.3000
pMON 140822	EXP-CUCme.5:l:l	159	626	15609.3	21140	0.0400	0.0300
pMON 140823	P-CUCme.6-l:l:l	18	331	15178.5	22818	0,0200	0.0100
pMON 140824	P-CUCme.8-l:l:2	19	238	17514.5	28429	0.0100	0.0100
pMON 140825	P-CUCme.9-l:l:2	20	510	13208	19567	0.0400	0.0300
pMON 140826	P-CUCme.l0-l:l:l	21	352	14805.3	22200	0.0200	0.0200
pMON 140827	EXP- CUCme.eEFIa: 1:1	162	724	9326.8	14476	0.0800	0.0500
pMON 140828	P-CUCme. 15-1:1:2	23	304	11798	17486	0.0300	0.0200
pMON 140829	P-CUCme. 16a1:1:2	24	88	5429	9596	0.0200	0.0100
pMON 140830	P-CUCme.l7-l:l:2	26	180	10477.8	15291	0.0200	0.0100
pMON 140831	P-CUCme.l8-l:l:2	27	111	5059.3	6778	0.0200	0.0200
pMON 140832	P-CUCme.l9-l:l:3	167	121	3765	6032	0.0300	0.0200
pMON 140833	P-CUCme.20-1:3	211	155	10458.8	14748	0.0100	0.0100

Construct	Regulatory Element	SEQ ID NO:	Average GUS	Average FLuc	Average RLuc	GUS/ FLuc	GUS/ RLuc
pMON 140834	P-CUCme.21-l:l:l	30	582	7760	11440	0.0800	0.0500
pMON 140835	P-CUCme.22-l:l:3	31	400	11393.8	18654	0.0400	0.0200
pMON140836	EXP- CUCme.SAMS2:l: 1	168	568	9466.3	13962	0.0600	0.0400
pMON 140837	P-CUCme.26-l:l:2	33	87	6683	8494	0.0100	0.0100
pMON 140838	P-CUCme.28-l:l:2	34	171	19104.8	29619	0.0100	0.0100
pMON 140839	EXP- CUCme.29:l:2	212	90	11247.3	15919	0.0100	0.0057

[0102] To compare the relative activity of each promoter in soybean cotylédon protoplasts, GUS values were expressed as a ratio of GUS to luciferase activity and normalized with respect to the expression levels observed for the constitutive expression element groups, EXP-At.Act7:l:l 1 and EXP-CaMV.35S-enh+Ph.DnaK:l:3. Table 9 below shows the GUS to firefly luciferase (FLuc) ratios normalized with respect to EXP-At.Act7:l:l 1 and EXP-CaMV.35Senh+Ph.DnaK:l:3. Table 10 below shows the GUS to renilla luciferase (RLuc) ratios normalized with respect to EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+Ph.DnaK,: 1:3.

Table 9. GUS to firefly luciferase (FLuc) ratios normalized with respect to EXP10 At.Act7:l:ll and EXP-CaMV.35S-enh+Ph.DnaK:l:3.________________________________

Construct	Regulatory Element	SEQ ID NO:	GUS/FLuc normalized with respect to EXPAt.Act7:l:l 1	GUS/FLuc normalized with respect to EXPCaMV.35Senh+Ph.DnaK: 1:3
PMON80585	EXP-At.Atnttl:l:2	200	1.12	0.08
pMON 109584	EXP-CaMV.35Senh+Ph.DnaK: 1:3	201	13.48	1.00
pMON 118756	EXP-At.Act7:l:l 1	202	1.00	0.07
PMON124912	Promoterless		0.05	0.00
pMON 140818	P-CUCme.l-l:l:lrc	155	0.41	0.03
pMON140819	P-CUCme.2-l:I:l	14	0.25	0.02
PMON140820	P-CUCme.3-l:l:3	15	0.05	0.00
pMON140821	EXP-CUCme.4:l:l	156	5.52	0.41
pMON 140822	EXP-CUCme.5:l:l	159	0.40	0.03
pMON 140823	P-CUCme.6-l:l:l	18	0.22	0.02

Construct	Regulatory Element	SEQ ID NO:	GUS/FLuc normalized with respect to EXPAt.Act7:l:ll	GUS/FLuc normalized with respect to EXPCaMV.35Senh+Ph.DnaK:l:3
pMON 140824	P-CUCme.8-l:l:2	19	0.14	0.01
pMON 140825	P-CUCme.9-1:1:2	20	0.39	0.03
pMON 140826	P-CUCme.l0-l:l:l	21	0.24	0.02
pMON 140827	EXP-CUCme.eEFIa:I:l	162	0.78	0.06
pMON 140828	P-CUCme. 15-1:1:2	23	0.26	0.02
pMON 140829	P-CUCme. 16a-l: 1:2	24	0.16	0.01
pMON 140830	P-CUCme.l7-l:l:2	26	0.17	0.01
pMON 140831	P-CUCme. 18-1:1:2	27	0.22	0.02
pMON 140832	P-CUCme. 19-1:1:3	167	0.32	0.02
pMON 140833	P-CUCme.20-l:3	211	0.15	0.01
pMON 140834	P-CUCme.21-l:l:l	30	0.75	0.06
pMON 140835	P-CUCme.22-l:l:3	31	0.35	0.03
pMON 140836	EXP- CUCme.SAMS2:l:l	168	0.60	0.04
pMON 14083 7	P-CUCme.26-l:l:2	33	0.13	0.01
pMON 140838	P-CUCme.28-l:l:2	34	0.09	0.01
pMON 140839	EXP-CUCme.29:l:2	212	0.08	0.01

Table 10. GUS to renilla luciferasc (RLuc) ratios normalized with respect to EXPAt.Act7:l:ll and EXP-CaMV.35S-enh+Ph.DnaK:l:3.

Construct	Regulatory Element	SEQ ID NO:	GUS/RLuc normalized with respect to EXPAt.Act7:l:ll	GUS/RLuc normalized with respect to EXPCaMV.35Senh+Ph.DnaK:l:3
PMON80585	EXP-At.Atnttl:l:2	200	0.96	0.18
pMON 109584	EXP-CaMV.35Senh+Ph.DnaK:l:3	201	5.21	1.00
pMON 118756	EXP-At.Act7:l:l 1	202	1.00	0.19
pMON 124912	Promoterless		0.05	0.01
pMON 140818	P-CUCme. 1-1:1 : lrc	155	0.42	0.08
pMON 140819	P-CUCme.2-l:l:l	14	0.23	0.04
pMON 140820	P-CUCme.3-l:l:3	15	0.05	0.01
pMON 140821	EXP-CUCme.4:1:1	156	4.12	0.79
pMON 140822	EXP-CUCme.5:l:l	159	0.40	0.08

Construct	Regulatory Element	SEQ ID NO:	GUS/RLuc normalized with respect to EXPAt.Act7:l:ll	GUS/RLuc normalized with respect to EXPCaMV.35Senh+Ph.DnaK:l:3
pMON 140823	P-CUCme.6-l:l:l	18	0.20	0.04
pMON 140824	P-CUCme.8-l:l:2	19	0,11	0.02
pMON 140825	P-CUCme.9-l:l:2	20	0.36	0.07
pMON 140826	P-CUCme.l0-l:l:l	21	0.22	0.04
pMON 140827	EXP- CUCme.eEFla:l:l	162	0.68	0.13
pMON 140828	P-CUCme. 15-1:1:2	23	0.24	0.05
pMON 140829	P-CUCme. 16a-l: 1:2	24	0.13	0.02
pMON 140830	P-CUCme. 17-1:1:2	26	0.16	0.03
pMON 140831	P-CUCme.l8-l:l:2	27	0.22	0.04
pMON140832	P-CUCme. 19-1:1:3	167	0.27	0.05
pMON 140833	P-CUCme.20-l:3	211	0.14	0.03
pMON 140834	P-CUCme.21-1:1:1	30	0.69	0.13
pMON 140835	P-CUCme.22-l:l:3	31	0.29	0.06
pMON 140836	EXP- CUCme.SAMS2:l:l	168	0.55	0.11
pMON 140837	P-CUCme.26-1:1:2	33	0.14	0.03
pMON 140838	P-CUCme.28-l:l:2	34	0.08	0.02
pMON 140839	EXP-CUCme.29:l:2	212	0.08	0.01

[0103] As can be seen in Tables 9 and 10, most of the expression element groups tested, demonstrated the ability to drive transgene expression in soybean cotylédon protoplast cells. One expression element group, EXP-CUCme.4:l:l (SEQ ID NO: 156) demonstrated levels of 5 transgene expression higher than that of EXP-At.Act7:1:11 in this assay.

Example 5: Analysis of Regulatory Eléments Driving GUS in Bombarded Soybean Leaves and Roots.

[0104] Soybean leaves and roots were transformed with plant expression vectors containing a 10 test transcriptional regulatory expression element group driving expression of the Bglucuronidase (GUS) transgene and compared to GUS expression in roots and leaves in which expression of GUS is driven by known constitutive promoters.

[0105] Expression of a transgene driven by P-CUCme. l-l:l:lrc (SEQ ID NO: 155), PCUCme.2-l:l:l (SEQ ID NO: 14), P-CUCme.3-l:l:3 (SEQ ID NO: 15), EXP-CUCme.4: l : I (SEQ ID NO: 156), EXP-CUCme.5:l:I (SEQ ID NO: 159), P-CUCme.6-l:l:l (SEQ ID NO: 18), P-CUCme.8-l:l:2 (SEQ ID NO: 19), P-CUCme.9-l:l:2 (SEQ ID NO: 20), P-CUCme. ÎOl:l:l (SEQ IDNO: 21), EXP-CUCme.eEFla:l:l (SEQ ID NO: 162), P-CUCme. 15-1:1:2 (SEQ ID NO: 23), P-CUCme.l6a-l:I:2 (SEQ ID NO: 24), P-CUCme. 17-1:1:2 (SEQ ID NO: 26), PCUCme.l8-l:l:2 (SEQ ID NO: 27), P-CUCme. 19-1:1:3 (SEQ ID NO: 167), P-CUCme.2O-l:3 (SEQ ID NO: 211), P-CUCme.21-l:l:l (SEQ ID NO: 30), P-CUCme.22-l:l:3 (SEQ ID NO: 31), EXP-CUCme.SAMS2:l:l (SEQ ID NO: 168), P-CUCme.26-l:l:2 (SEQ ID NO; 33), PCUCme.28-l:l:2 (SEQ ID NO: 34) and EXP-CUCme.29: i :2 (SEQ ID NO: 212) was compared with expression from known constitutive expression element groups in particle bombarded soybean leaves and roots. The plant expression vectors used for transformation of leaves and roots was the same as those presented in Table 7 of Example 4 above.

[0106] The plant expression vectors, pMON80585, pMON109584, pMONl 18756, pMON124912, pMON140818, pMON140819, pMON 140820, pMON140821, pMON140822, pMON140823, pMON140824, pMON140825, pMON140826, pMON140827, pMON140828, pMON140829, pMON140830, pMON140831, pMON140832, _PMON140833, pMON140834, pMON140835, pMON140836, pMON140837, pMON140838 and pMON140839 were used to transform soybean leaves and roots using particle bombardment transformation methods.

[0107] Briefly, A3244 soybean seeds were surface sterilized and allowed to germinate in trays with a photoperiod of 16 hours light and 8 hours of darkness. After approximately 13 days, leaf and root tissue was harvested under stérile conditions from the seedlings and used for bombardment. The tissue samples were randomly placed on a pétri dish containing plant culture medium. Ten micrograms of plasmid DNA was used to coat 0.6 micron gold particles (Catalog #165-2262 Bio-Rad, Hercules, CA) for bombardment. Macro-carriers were loaded with the DNA-coated gold particles (Catalog #165-2335 Bio-Rad, Hercules CA). A PDS 1000/He biolistîc gun was used for transformation (Catalog #165-2257 Bio-Rad, Hercules CA). The bombarded root and leaf tissues were allowed to incubate in the dark for 24 hours at 26 degrees Celsius. Following this overnight incubation, the tissues were stained in solution for GUS expression overnight at 37 degrees Celsius. After staining overnight, the tissues were soaked in 70% éthanol overnight to remove chlorophyll and reveal the GUS staining. The tissues were _/tZ' then photographed and a rating scale of “0”, to “++++++” reflecting the level of GUS expression is assigned to each construct (0- no expression, + to l l l l l ·>· - low to high, respectively).

[0l 08] Expression of the GUS transgene demonstrated in each tissue is used to infer the relative 5 potential level and specificity of each element’s capacity to drive transgene expression in stably transformed corn plants. Average GUS expression ratings are provided in Table 11 below.

Table 11. GUS expression ratings for particle bombarded leaf and root.

Construct	Regulatory Element	SEQ IDNO:	Leaf Expression	Root Expression
pMON80585	EXP-At.Atnttl:l:2	200	-f-H-	+++
pMON 109584	EXP-CaMV.35Senh+Ph.DnaK: 1:3	201	1 H -1· 1·	-H-
pMON 118756	EXP-At.Act7:l:l 1	202	1 1 1- !	4-H-
pMON 124912	Promoteriess		0	0
pMON 140818	P-CUCme. 1-1: l:lrc	155	+++	4-
pMON 140819	P-CUCme.2-l:l:l	14	4-4-	+
pMON 140820	P-CUCme.3-l:l:3	15	0	0
pMON 140821	EXP-CUCme.4:1:1	156	1 1 1 H 1	+++
pMON 140822	EXP-CUCme.5:l:l	159	++	4-
pMON 140823	P-CUCme.6-l:l:l	18	-H-	4-
pMON 140824	P-CUCme.8-l:l:2	19	+	+
pMON 140825	P-CUCme.9-l:l:2	20	-H-	4-
pMON 140826	P-CUCme.l0-l:l:l	21	ί Ή1	4-H-
pMON 140827	EXP-CUCme.eEFla:l:l	162	++++	4-H-
pMON 140828	P-CUCme. 15-1:1:2	23	+	Ψ
pMON 140829	P-CUCme. 16a-l: 1:2	24	+	-
pMON 140830	P-CUCme. 17-1:1:2	26		+
pMON 140831	P-CUCme. 18-1:1:2	27	4-H-	4-
pMON 140832	P-CUCme. 19-1:1:3	167	+	4-
pMON 14083 3	P-CUCme.20-l:3	211	+	4-
pMON 140834	P-CUCme.2l-l:l:l	30	+	4-
pMON 140835	P-CUCme.22-l:l:3	31	-H-H-	4-
pMON 140836	EXP-CUCme.SAMS2:l:l	168	1 1 1 1-1-	4-4-4-
pMON 140837	P-CUCme.26-l:l:2	33	+	4-
pMON 14083 8	P-CUCme.28-l:l:2	34	4-	4-
pMON 140839	EXP-CUCme.29:1:2	212		4-

[0109] As can be seen in Table 11 above, ali but one of the expression element groups demonstrated the abilîty to drive transgene expression in particle bombarded soybean leaf and root tissue. Two expression element groups, P-CUCme.28-l:l:2 (SEQ ID NO; 34) and EXPCUCme.4:l:l (SEQ ID NO: 156) demonstrated similar or higher levels of expression relative to expression driven by EXP-CaMV.35S-enh4-Ph.DnaK: l:3 in this assay.

Example 6: Analysis of Regulatory Eléments Driving GUS in Soy Cotylédon Protoplast using Transgene Cassette Amplicons [Ol 10] Soybean cotylédon protoplasts were transformed with transgene cassette amplicons containing a transcriptional regulatory expression element group driving expression of the βglucuronidase (GUS) transgene and compared to GUS expression in leaf protoplasts in which expression of GUS is driven by known constitutive promoters. The transgene cassette amplicons were comprised of an EXP sequence, operably linked to a GUS coding sequence (GUS, SEQ ID NO: 206), operably linked to a 3’ UTR (T-Gb.FbL2-l:l:l, SEQ ID NO: 205). Average GUS expression was compared to the control EXP éléments, P-CaMV.35S-enh-l:l:lO2/L-CaMV.35Sl:l:2 (SEQ ID NO: 210) and EXP-At.Atnttl:l:2 (SEQ ID NO: 200).

[OUI] A plasmid, for use in co-trans format ion and normalization of data was also used in a similar manner as that described above in Example 2. The transformation control plasmid was comprised of a constitutive promoter, driving the expression of the firefly (Photinus pyralis) luciferase coding sequence (FLuc, SEQ ID NO: 205), operably linked 5’ to a 3’ termination région from the Agrobacterium tumefaciens nopaline synthase gene (T-AGRtu.nos-l:l:l3, SEQ ID NO: 209).

[0112] Table 12 below shows the mean GUS expression values conferred by each transgene amplicon. Table 13 below shows the GUS to firefly luciferase (FLuc) ratios normalized with respect to EXP-At.Atnttl: 1:2 and P-CaMV.35S-enh-l:l:l02/L-CaMV.35S-l:l:2

Table 12. Average GUS and luciferase expression values and GUS/Iuciferase ratios.

Amplicon ID	Regulatory Elément	SEQ ID NO:	Mean GUS	Mean Fluc	GUS/Fluc
No DNA			0.00	0.00	0.00
pMON124912	No promoter		54.67	34905.00	0.00
pMON33449	P-CaMV.35S-enh-l : 1:1O2/L-CaMV.35S-l : 1:2	210	107064.67	21757.67	4.92
pMON80585	EXP-At.Atnttl:l:2	200	4962.33	40778.67	0.12
56969	CumMe WSM SF16429.G5670	40	283.67	53452.00	0.01
56877	P-CUCme.CumMe WSM SF16444.G5140-l:l:l	175	5297.67	46576.67	0.11
56749	P-CUCme.CumMe WSM SF16563.G5560-l:l:l	176	280.67	41958.33	0.01
56918	CumMe WSM SF17051.G5470	48	1088.00	36321.00	0.03
56849	P-CUCme.CumMe WSM SF17111 .G5790-1 :1:1	177	196.00	48128.00	0.00
56754	P-CUCme. WSM SF 17252.G7330-1:1:1	179	175.67	45427.00	0.00
56892	CumMe WSM SF17349.G5770	56	34.00	38016.00	0.00
56477	CumMe WSM SF17866.G6050	62	862.00	52203.33	0.02
56842	P-CUCme.CumMe WSM SF18488.G5340-1:1:1	181	2892.67	49144.33	0.06
56852	P-CUCme.CumMe WSM SF18536.G6480-l:l:l	182	3462.67	46549.33	0.07
56497	CumMe WSM SF18575.G6410	71	92.67	47628.33	0.00
56847	P-CUCme.CumMeWSMSF 18634.G5190-1:1:1	183	122.33	36815.33	0.00
56746	P-CUCme.CumMe WSM SF18716.G5860-1:1:1	184	14.33	62483.33	0.00
56883	CumMe WSM SF18986.G6110	79	863.33	54379.33	0.02
56734	EXP-CUCme. WSM SF19064.G5690:1:1	185	142.00	46962.67	0.00
56912	P-CUCme.CumMe WSM SF19647.G5760-l:l:l	188	7659.00	46935.67	0.16
56482	P-CUCme.CumMe WSMSF19839.G5090-1:1:1	189	3279.00	37070.67	0.09
56963	CumMe WSM SF19902.G5260	87	1629.00	55649.00	0.03
56747	P-CUCme.CumMe WSM SF20132.G5560-1:1:1	190	340.33	40577.00	0.01

Amplicon ID	Regulatory Element	SEQ ID NO:	Mean GUS	Mean Flue	GUS/FIuc
56479	CumMe WSM SF20359.G5870	92	192.00	61341.67	0.00
56744	CumMe WSM SF206458.G5970	98	154.67	33139.33	0.00
56948	CumMe WSM SF206534.G5200	99	62.00	52118.00	0.00
56896	CumMe WSM SF22008.G5670	108	1585.00	53540.00	0.03
56919	CumMe WSM SF22275.G5780	112	8.33	48546.33	0.00
56967	CumMe WSM SF22355.G5310	113	74.33	36202.67	0.00
56837	P-CUCme.CumMe WSM SF22531.G5120-1:1:1	192	1526.67	52799.33	0.03
56940	CumMe WSM SF22870.G5370	115	14.67	53663.33	0.00
56495	P-CUCme.CumMe WSM SF23760.G5200-1:1:1	193	196.33	49870.67	0.00
56868	P-CUCme.CumMe WSM SF23906.G6180-l:l:l	194	1584.33	42532.33	0.04
56998	CumMe WSM SF24045.G5400	123	80.67	47553.00	0.00
56976	P-CUCme.CumMe WSM SF25141 .G5160-1:1:2	195	4506.00	57213.00	0.08
56742	P-CUCme.CumMe WSM SF25355.G5000-l:l:l	196	4.00	41114.33	0.00
56915	P-CUCme.CumMe WSM SF25936.G5450-l:l:l	197	965.33	34494.67	0.03
56854	CumMe WSM SF28729.G5340	134	208.33	53956.00	0.00
56936	CumMe WSM SF31264.G5380	136	292.67	42320.67	0.01
56863	P-CUCme.CumMe WSM SF35856.G5150-1:1:1	198	125.00	48705.33	0.00
56751	P-CUCme.CumMe WSM SF41124.G5080-l:l:l	199	31.33	53595.00	0.00
56921	CumMe WSM SF41254.G5160	141	11.67	52643.67	0.00
56884	CumMe WSM SF42141.G5110	146	48.33	40556.67	0.00

Table 13. GUS to firefly luciferase (FLuc) ratios normalized with respect to EXP-At.Atnttl:l:2 and P-CaMV.35S-enh1:1:102/L-CaMV.35S-1:1:2.

Amplicon ID	Regulatory Elément	SEQ ID NO:	GUS/Fluc normalized with respect to EXPAt.Atnttl:l:2	GUS/Fluc normalized with respect to PCaMV.35S-enhl:l:102/LCaMV.35S-l:l:2
No DNA			0.00	0.00
pMON124912	No promoter		0.01	0.00
pMON33449	P-CaMV.35S-enh-1:1:102/L-CaMV.35S-1:1:2	210	40.44	1.00
pMON80585	EXP-At.Atnttl:l:2	200	1.00	0.02
56969	CumMe WSM SF16429.G5670	40	0.04	0.00
56877	P-CUCme.CumMe WSM SF16444.G5140-1:1:1	175	0.93	0.02
56749	P-CUCme.CumMe WSM SF16563.G5560-l:l:l	176	0.05	0.00
56918	CumMe WSM SF17051.G5470	48	0.25	0.01
56849	P-CUCme.CumMe WSM SF17111 .G5790-1:1:1	177	0.03	0.00
56754	P-CUCme.WSM SF17252.G7330-l:l:l	179	0.03	0.00
56892	CumMe WSM SF17349.G5770	56	0.01	0.00
56477	CumMeWSMSF 17866.G6050	62	0.14	0.00
56842	P-CUCme.CumMe WSM SF18488.G5340-l:l:l	181	0.48	0.01
56852	P-CUCme.CumMe WSM SF 18536.G6480-1:1:1	182	0.61	0.02
56497	CumMe WSM SF18575.G6410	71	0.02	0.00
56847	P-CUCme.CumMe WSM SF18634.G5190-l:l:l	183	0.03	0.00
56746	P-CUCme.CumMe WSM SF18716.G5860-l:l:l	184	0.00	0.00
56883	CumMe WSM SF18986.G6110	79	0.13	0.00
56734	EXP-CUCme.WSM SF19064.G5690:l:l	185	0.02	0.00
56912	P-CUCme.CumMe WSM SF19647.G5760-l: 1:1	188	1.34	0.03
56482	P-CUCme.CumMe WSM SF 19839.G5090-1:1:1	189	0.73	0.02
56963	CumMe WSM SF19902.G5260	87	0.24	0.01

Amplicon ID	Regulatory Elément	SEQ ID NO:	GUS/Fluc normalized with respect to EXPAt.Atnttl:l:2	GUS/Fluc normalized with respect to PCaMV.35S-enhl:l:102/LCaMV.35S-l:l:2
56747	P-CUCme.CumMe WSM SF20132.G5560-l:l:l	190	0.07	0.00
56479	CumMe WSM SF20359.G5870	92	0.03	0.00
56744	CumMe WSM SF206458.G5970	98	0.04	0.00
56948	CumMe WSM SF206534.G5200	99	0.01	0.00
56896	CumMe WSM SF22008.G5670	108	0.24	0.01
56919	CumMe WSM SF22275.G5780	112	0.00	0.00
56967	CumMe WSM SF22355.G5310	113	0.02	0.00
56837	P-CUCme.CumMe WSM SF22531 ,G5120-1:1:1	192	0.24	0.01
56940	CumMe WSM SF22870.G5370	115	0.00	0.00
56495	P-CUCme.CumMe WSM SF23760.G5200-l:l:l	193	0.03	0.00
56868	P-CUCme.CumMe WSM SF23906.G6180-1:1:1	194	0.31	0.01
56998	CumMe WSM SF24045.G5400	123	0.01	0.00
56976	P-CUCme.CumMe WSM SF25141.G5160-l:l:2	195	0.65	0.02
56742	P-CUCme.CumMe WSM SF25355.G5000-l:l:l	196	0.00	0.00
56915	P-CUCme.CumMe WSM SF25936.G5450-l:l:l	197	0.23	0.01
56854	CumMe WSM SF28729.G5340	134	0.03	0.00
56936	CumMe WSM SF31264.G5380	136	0.06	0.00
56863	P-CUCme.CumMe WSM SF35856.G5150-l:l:l	198	0.02	0.00
56751	P-CUCme.CumMe WSM SF41124.G5080-1:1:1	199	0.00	0.00
56921	CumMe WSM.SF41254.G5160	141	0.00	0.00
56884	CumMe WSM SF42141.G5110	146	0.01	0.00

[0l 13] As can be seen in Table 12 above, not ail EXP sequences demonstrated the ability to drive transgene expression when compared to the promoterless control. However, the EXP sequences, CumMe_WSM_SFl6429.G5670 (SEQ ID NO: 40), PCUCme.CumMe_WSM_SFl6444.G5l40-l:l:l (SEQ ID NO: I75), PCUCme.CumMe_WSM_SF16563.G5560-l:l:l (SEQ ID NO: 176), CumMe_WSM_SFl705l.G5470 (SEQ ID NO: 48), PCUCme.CumMe_WSM_SFl7l l l.G5790-l:l:l (SEQIDNO: 177), PCUCme.WSM_SFl7252.G7330-l:l:l (SEQ ID NO: 179), CumMe_WSM_SFl7866.G6050 (SEQ ID NO: 62), P-CUCme.CumMe_WSM_SF 18488.G5340-1 : l : l (SEQ ID NO: 181 ), PCUCme.CumMe_WSM_SFI8536.G6480-l:l:l (SEQIDNO: 182), CumMe_WSM_SFl8575.G64l0 (SEQ IDNO: 7l),PCUCme.CumMe_WSM_SFl8634.G5l90-l:l:l (SEQIDNO: I83),

CumMe_WSM_SFl 8986.G61 10 (SEQ IDNO: 79), EXP-CUCmc.WSM SFl9064.G5690:l:l (SEQ IDNO: 185), P-CUCme.CumMe_WSM_SFl9647.G5760-I:l:l (SEQ IDNO: 188), PCUCme.CumMe_WSM_SFl9839.G5090-l:l:l (SEQIDNO: 189), CumMe_WSM_SFl9902.G5260 (SEQ ID NO: 87), PCUCme.CumMe_WSM_SF20132.G5560-1 : l : l (SEQ ID NO: 190), CumMe_WSM_SF20359.G5870 (SEQ ID NO: 92), CumMe_WSM_SF206458.G5970 (SEQ ID NO: 98), CumMe_WSM_SF206534.G5200 (SEQ ID NO: 99),

CumMe_WSM_SF22008.G5670 (SEQIDNO: 108), CumMe_WSMSF22355.G5310 (SEQ IDNO: ll3), P-CUCme.CumMe_WSM_SF2253l.G5l20-l:l:I (SEQIDNO: 192),EXPCUCme.WSM_SFI9064.G5690:l:l (SEQIDNO: 193), PCUCme.CumMe_WSM_SF23906.G6l80-l:l:l (SEQIDNO: 194), CumMe_WSM_SF24045.G5400 (SEQ ID NO: 123), PCUCme.CumMe_WSM_SF25141.G5160-1:1:2 (SEQIDNO: 195), PCUCme.CumMe_WSM_SF25936.G5450-1:1:1 (SEQ ID NO: 197), CumMe_WSM_SF28729.G5340 (SEQIDNO: 134), CumMe_WSM_SF31264.G5380 (SEQ IDNO: 136) and P-CUCme.CumMe_WSM_SF35856.G5150-1:1:1 (SEQIDNO: 198) demonstrated the ability to drive trangene expression in soybean cotylédon protoplasts at a level similar or greater than EXP-At.Atnttl : 1:2. As shown in Table 13 above, the EXP sequence P68

CUCme.CumMe_WSM_SFl9647.G5760-l:l:l (SEQ ID NO: 188) demonstrated the ability to drive transgene expression in this assay at a level greater than EXP-At.Atnttl : l :2.

Example 7: Analysis of Regulatory Eléments Driving GUS in Cotton Leaf Protoplasts [0l 14] Cotton leaf protoplasts were transformed with plant expression vectors containing a test transcriptional regulatory expression element group driving expression of the B-glucuronidase (GUS) transgene and compared to GUS expression in leaf protoplasts in which expression of GUS îs driven by known constitutive promoters.

[Oll 5] Expression of a transgene driven by P-CUCme. I -1 : l : l rc (SEQ ID NO: 155), PCUCme.2-l:l:l (SEQ ID NO: 14), P-CUCme.3-l:l:3 (SEQ ID NO: 15), EXP-CUCme.4:l:l (SEQ ID NO: 156), P-CUCme.6-l:l:l (SEQ ID NO: 18), P-CUCme.8-l:l:2 (SEQ ID NO: 19), P-CUCme.9-l:l:2 (SEQ ID NO: 20), P-CUCme. 10-1 : l : l (SEQ ID NO: 2I), EXPCUCme.eEFla:l:i (SEQ ID NO: 162), P-CUCme. 15-1:1:2 (SEQ ID NO: 23), P-CUCme.l6a1:1:2 (SEQ ID NO: 24), P-CUCme. 17-1:1:2 (SEQ ID NO: 26), P-CUCme. 18-1:1:2 (SEQ ID NO: 27), P-CUCme. 19-1:1:3 (SEQ ID NO: 167), P-CUCme.20-l:3 (SEQ ID NO: 211), PCUCme.21-l:l:l (SEQ ID NO: 30), P-CUCme.22-l:l:3 (SEQ ID NO: 31), EXPCUCme.SAMS2:l:l (SEQ ID NO: 168), P-CUCme.26-1:1:2 (SEQ ID NO: 33), P-CUCme.281:1:2 (SEQ ID NO: 34) and EXP-CUCme.29:l:2 (SEQ ID NO: 212) was compared with expression from known constitutive expression element groups. Each plant expression vector was comprised of a right border région from Agrobacterium tumefaciens, a first transgene cassette comprised of a test promoter or known constitutive promoter operably linked 5’ to a coding sequence for B-glucuronidase (GUS, SEQ ID NO: 206) containing a processable intron derived from the potato light-inducible tissue-specific ST-LS1 gene (Genbank Accession: X04753), operably linked 5’ to a 3’ termination région from the Gossypium barbadense E6 gene (T-Gb.E6-3b:l:l, SEQ ID NO: 204), the Piston sativum RbcS2-E9 gene (T-Ps.RbcS2-E9-l:l:6, SEQ ID NO: 203), or the Gossypium barbadense FbLate-2 gene (T-Gb.FbL2-1:1:1, SEQ ID NO: 205); a second transgene sélection cassette used for sélection of transformed plant cells that either confers résistance to the herbicide glyphosate (driven by the Arabidopsis Actin 7 promoter) or the antibiotic, kanamycin and a left border région from A. tumefaciens. A promoterless control plant expression vector (pMON124912) served as a négative control for expression. The foregoing test and constitutive expression element groups were cloned into plant expression vectors as shown in Table 14 below.

Table 14. Plant expression vectors and corresponding expression element group and 3’

UTR.

Construct	Regulatory Element	SEQ ID NO:	3’ UTR
pMON 109584	EXP-CaMV.35S-enh+Ph.DnaK: 1:3	201	T-Gb.E6-3b:l	:1
pMON 118756	EXP-At.Act7:l:l 1	202	T-Gb.E6-3b: 1	:1
pMON 124912	Promoterless		T-Gb.FbL2-l	1:1
pMON 140818	P-CUCme. 1-1: klrc	155	T-Gb.FbL2-l	1:1
pMON 140819	P-CUCme.2-kl:l	14	T-Gb.FbL2-l	1:1
pMON 140820	P-CUCme.3-1:1:3	15	T-Gb.FbL2-l	1:1
pMON 140821	EXP-CUCme.4:1:1	156	T-Gb.FbL2-l	1:1
pMON 140823	P-CUCme.6-l:l:l	18	T-Gb.FbL2-l	1:1
pMON 140824	P-CUCme.8-1:1:2	19	T-Gb.FbL2-l	1:1
pMON 140825	P-CUCme.9-1:1:2	20	T-Gb.FbL2-l	kl
pMON 140826	P-CUCme.I0-l:l:l	21	T-Gb.FbL2-l	1:1
pMON 140827	EXP-CUCme.eEFla:l:l	162	T-Gb.FbL2-l	kl
pMON 140828	P-CUCme. 15-1:1:2	23	T-Gb.FbL2-l	kl
pMON 140829	P-CUCme. 16a-1:1:2	24	T-Gb.FbL2-l	kl
pMON 140830	P-CUCme. 17-1:1:2	26	T-Gb.FbL2-l	kl
pMON 140831	P-CUCme. 18-1:1:2	27	T-Gb.FbL2-l	kl
pMON 140832	P-CUCme. 19-1:1:3	167	T-Gb.FbL2-l	kl
pMON 140833	P-CUCme.20-l:3	211	T-Gb.FbL2-l	kl
pMON 140834	P-CUCme.21-l:l:l	30	T-Gb.FbL2-l	kl
pMON 140835	P-CUCme.22-1:1:3	31	T-Gb.FbL2-l	kl
pMON 140836	EXP-CUCme.SAMS2:l:l	168	T-Gb.FbL2-l	kl
pMON 140837	P-CUCme.26-1:1:2	33	T-Gb.FbL2-l	kl
pMON 140838	P-CUCme.28-l:l:2	34	T-Gb.FbL2-l	kl
pMON 140839	EXP-CUCme.29;l:2	212	T-Gb.FbL2-l	kl

[0116] Two plasmids, for use in co-transformation and normalization of data, were also constructed. One transformation control plasmid was comprised of a constitutive promoter, driving the expression of the firefly (Photinus pyralis) luciferase coding sequence (FLuc, SEQ ID NO: 205), operably linked 5’ to a 3’ termination région from the Agrobacterium tumefaciens nopaline synthase gene (T-AGRtu.nos-l:l:13, SEQ ID NO: 209). The other transformation,/— control plasmid was comprised of a constitutive promoter, driving the expression of the sea pansy (Renilla reniformis) luciferase coding sequence (RLuc, SEQ ID NO: 206), operably linked 5’ to a 3’ termination région from the Agrobacterium tumefaciens nopaline synthase gene. [0117] The plant expression vectors, pMON80585, pMONl09584, pMONH8756, pMONl249l2, pMONI408I8, pMONl408l9, pMON!40820, pMON!4082l, pMONl40823, pMONl40824, pMONl40825, pMONl40826, pMONl40827, pMONl40828, _PMONl40829, pMON 140830, pMON 140831, pMON 140832, pMON 140833, pMON 140834, pMON 140835, pMON 140836, pMON 140837, pMON 140838 and pMON 140839 were used to transform cotton leaf protoplast cells using PEG transformation methods. Protoplast cells were transformed with equimolar amounts of each of the two transformation control plasmids and a test plant expression vector. GUS and luciferase activity was assayed. Measurements of both GUS and luciferase were conducted by placing aliquots of a lysed préparation of cells transformed as above into two different small-well trays. One tray was used for GUS measurements, and a second tray was used to perform a dual luciferase assay using the dual luciferase reporter assay system (Promega

Corp., Madison, WI; see for example, Promega Notes Magazine, No: 57, 1996, p.02). Sample measurements were made using 4 replicates per transformation. The average GUS and luciferase values are presented in Table 15 below.

Table 15. Average GUS and luciferase expression values and GUS/iucifcrase ratios.

Construct	Regulatory Elément	SEQ ID NO:	Average GUS	Average FLuc	Average RLuc	GUS/ FLuc	GUS/ RLuc
pMON 109584	EXP-CaMV.35Senh+Ph.DnaK:l:3	201	5322.8	14842.8	27990.5	0.3586	0.1902
pMON 11 8756	EXP-At.Act7:l:l 1	202	1006.3	19746.8	25582.3	0.0510	0.0393
pMON 124912	Promoter less		21	19248.5	25012	0.0011	0.0008
pMON 140818	P-CUCme.ll:l:lrc	155	170.3	17796.8	22026.3	0.0096	0.0077
pMON 140819	P-CUCme.2-l:l:l	14	34.8	16326.3	21407.5	0.0021	0.0016
pMON 140820	P-CUCme.3-l:l:3	15	51.5	17356.8	21523.8	0.0030	0.0024
pMON 140821	EXP- CUCme.4:l:l	156	3497.8	18745.3	26065.3	0.1866	0.1342
pMON 140823	P-CUCme.6-1:1:1	18	40.8	19533.8	26361.5	0.0021	0.0015
pMON 140824	P-CUCme.8-1:1:2	19	22	19701	26278	0.0011	0.0008
pMON 140825	P-CUCme.9-1:1:2	20	372.5	21972.3	28755	0.0170	0.0130
pMON 140826	P-CUCme. 101:1:1	21	198	21362.8	28902	0.0093	0.0069

Construct	Regulatory Element	SEQ ID NO:	Average GUS	Average FLuc	Average RLuc	GUS/ FLuc	GUS/ RLuc
pMON 140827	EXP- CUCme.eEFla:l:l	162	725	21589	27635.3	0.0336	0.0262
pMON 140828	P-CUCme. 151:1:2	23	55.3	17706	28846	0.0031	0.0019
pMON 140829	P-CUCme. 16a1:1:2	24	14	23289.5	30190	0.0006	0.0005
pMON 140830	P-CUCme. 171:1:2	26	155.5	23178.3	31602.8	0.0067	0.0049
pMON 140831	P-CUCme. 181:1:2	27	86.8	19085.8	22396.5	0.0045	0.0039
pMON 140832	P-CUCme. 191:1:3	167	130	21520.3	27270.5	0.0060	0.0048
pMON 140833	P-CUCme.20-l:3	211	88.5	22223.8	30786	0.0040	0.0029
pMON 140834	P-CUCme.21- 1:1:1	30	98.5	18579	20506.3	0.0053	0.0048
pMON 14083 5	P-CUCme.22- 1:1:3	31	363	21780.3	28816.3	0.0167	0.0126
pMON 140836	EXP- CUCme.SAMS2:1 :1	168	515	17906	23031	0.0288	0.0224
pMON 140837	P-CUCme.26- 1:1:2	33	125	15529.3	15169.3	0.0080	0.0082
pMON 140838	P-CUCme.281:1:2	34	115.8	17013.5	22236.5	0.0068	0.0052
pMON 140839	EXP- CUCme.29:l:2	212	15.5	16370.3	20409	0.0009	0.0008

[Ol I8] To compare the relative activity of each promoter in cotton leaf protoplasts, GUS values were expressed as a ratio of GUS to luciferase activity and normalized with respect to the expression levels observed for the constitutive expression element groups, EXP-At.Act7:l:l l 5 and EXP-CaMV.35S-enh+Ph.DnaK:l:3. Table 16 below shows the GUS to firefly luciferase (FLuc) ratios normalized with respect to EXP-At.Act7:l:l l and EXP-CaMV.35Senh+Ph.DnaK:l:3. Table 17 below shows the GUS to renilla luciferase (RLuc) ratios normalized with respect to EXP-At.Act7:1:11 and EXP-CaMV.35S-enh+Ph.DnaK: l :3.

Table 16. GUS to firefly luciferase (FLuc) ratios normalized with respect to EXPAt.Act7:l:ll and EXP-CaMV.35S-enh+Ph.DnaK:l:3.

Construct	Regulatory Element	SEQ ID NO:	GUS/FLuc normalized with respect to EXPAt.Act7:l:ll	GUS/FLuc normalized with respect to EXPCaMV.35Senh+Ph.DnaK: 1:3
pMON 109584	EXP-CaMV.35Senh+Ph.DnaK:l:3	201	7.037	1.000
pMONl 18756	EXP-At.Act7:l:l 1	202	1.000	0.142
pMON 124912	Promoterless		0.021	0.003
pMON 140818	P-CUCme.l-l:l:lrc	155	0.188	0.027
pMON 140819	P-CUCme.2-l:l:l	14	0.042	0.006
pMON 140820	P-CUCme.3-l:l:3	15	0.058	0.008
pMON 140821	EXP-CUCme.4:1:1	156	3.662	0.520
pMON 140823	P-CUCme.6-l:l:l	18	0.041	0.006
pMON 140824	P-CUCme.8-l:l:2	19	0.022	0.003
pMON 140825	P-CUCme.9-l:l:2	20	0.333	0.047
pMON 140826	P-CUCme. 1 0-1:1:1	21	0.182	0.026
pMON 140827	EXP-CUCme.eEFla:l:l	162	0.659	0.094
pMON 140828	P-CUCme. 15-1:1:2	23	0.061	0.009
pMON 140829	P-CUCme. I6a-l:l:2	24	0.012	0.002
pMON 140830	P-CUCme. 17-1:1:2	26	0.132	0.019
pMON 140831	P-CUCme. 18-1:1:2	27	0.089	0.013
pMON 140832	P-CUCme. 19-1:1:3	167	0.119	0.017
pMON 140833	P-CUCme.20-l:3	211	0.078	0.011
pMON 140834	P-CUCme.21-1:1:1	30	0.104	0.015
pMON 140835	P-CUCme.22-l:l:3	31	0.327	0.046
pMON 140836	EXP-CUCme.SAMS2:l:l	168	0.564	0.080
pMON 140837	P-CUCme.26-l:l:2	33	0.158	0.022
pMON 140838	P-CUCme.28-l:l:2	34	0.134	0.019
pMON 140839	EXP-CUCme.29:l:2	212	0.019	0.003

Table 17. GUS to renilla lucifcrase (RLuc) ratios normalized with respect to EXPAt.Act7:l:ll and EXP-CaMV.35S-enh+Ph.DnaK:l:3.

Construct	Regulatory Element	SEQ ID NO:	GUS/RLuc normalized with respect to EXPAt.Act7:l:ll	GUS/RLuc normalized with respect to EXPCaMV.35Senh+Ph.DnaK: 1:3
pMON 109584	EXP-CaMV.35Senh+Ph.DnaK:l:3	201	4.83	1.00
pMON 118756	EXP-At.Act7:l:l 1	202	1.00	0.21
pMON 124912	Promoterless		0.02	0.00
pMON 140818	P-CUCme.l-l:l:lrc	155	0.20	0.04
pMON 140819	P-CUCme.2-l:l:l	14	0.04	0.01
pMON 140820	P-CUCme.3-l:l:3	15	0.06	0.01
pMON 140821	EXP-CUCme.4:l:l	156	3.41	0.71
pMON 140823	P-CUCme.6-I:l:l	18	0.04	0.01
pMON 140824	P-CUCme.8-l:l:2	19	0.02	0.00
pMON 140825	P-CUCme.9-l:l:2	20	0.33	0.07
pMON 140826	P-CUCme. 10-1:1:1	21	0.17	0.04
pMON 140827	EXP- CUCme.eEF la: 1:1	162	0.67	0.14
pMON 140828	P-CUCme. 15-1:1:2	23	0.05	0.01
pMON 140829	P-CUCme. 16a-l: 1:2	24	0.01	0.00
pMON 140830	P-CUCme. 17-1:1:2	26	0.13	0.03
pMON 140831	P-CUCme.l8-l:l:2	27	0.10	0.02
pMON 140832	P-CUCme. 19-1:1:3	167	0.12	0.03
pMON 140833	P-CUCme.20-l:3	211	0.07	0.02
pMON 140834	P-CUCme.21-1:1:1	30	0.12	0.03
pMON 140835	P-CUCme.22-l:l:3	31	0.32	0.07
pMON 140836	EXP- CUCme.SAMS2:1:1	168	0.57	0.12
pMON 140837	P-CUCme.26-l:l:2	33	0.21	0.04
pMON 140838	P-CUCme.28-l:l:2	34	0.13	0.03
pMON 140839	EXP-CUCme.29:1:2	212	0.02	0.00

[0119] As can be seen in Tables 16 and 17, most of the expression element groups tested, demonstrated the ability to drive transgene expression in cotton leaf protoplast cells. One expression element group, EXP-CUCme.4:l:l (SEQ ID NO: 156) demonstrated levels of transgene expression higher than that of EXP-At.Act7:1:11 in this assay.

Example 8: Analysis of Regulatory Eléments Driving GUS in Cotton Leaf Protoplasts using Transgene Cassette Amplicons [0120] Cotton leaf protoplasts were transformed with transgene cassette amplicons containing a transcription al regulatory expression element group driving expression of the B-glucuronidase (GUS) transgene and compared to GUS expression in leaf protoplasts in which expression of GUS is driven by known constitutive promoters. The transgene cassette amplicons were comprised of an EXP sequence, operably linked to a GUS coding sequence (GUS, SEQ ID NO: 206), operably linked to a 3’ UTR (T-Gb.FbL2-l:l:l, SEQ ID NO: 205). Average GUS expression was compared to the control EXP éléments, P-CaMV.35S-enh-l:l:102/L-CaMV.35S1:1:2 (SEQ ID NO: 210) and EXP-At.Atnttl:l:2 (SEQ ID NO: 200).

[0121] A plasmid, for use in co-transformation and normalization of data was also used in a similar manner as that described above in Example 2. The transformation control plasmid was comprised of a constitutive promoter, driving the expression of the firefly (Photimis pyralis) luciferase coding sequence (FLuc, SEQ ID NO: 205), operably linked 5’ to a 3’ termination région from the Agrobacterium lumefaciens nopaline synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 209).

[0122] Table 18 below shows the mean GUS expression values conferred by each transgene amplicon. Table 19 below shows the GUS to firefly luciferase (FLuc) ratios normalized with respect to EXP-At. Atnttl : 1:2 and P-CaMV.35S-enh-1:1:102/L-CaMV.35S-1:1:2.

Table 18. Average GUS and luciferase expression values and GUS/luciferase ratios.

Amplicon ID	Regulatory Elément	SEQ ID NO:	Mean GUS	Mean Flue	GUS/Fluc
Empty Vector	No DNA		32.8	14087.5	0.002
pMON124912	No promoter		12	20486.3	0.001
ΡΜΟΝ80585	EXP-At. Atntt 1:1:2	200	55.5	18811	0.003
PMON33449	P-CaMV.35S-enh-l : 1:102/L-CaMV.35S1:1:2	210	12472.5	19126.3	0.652
56741	CumMe WSM SF143981.G5150	36	5.8	17449.5	0.000

Amplicon ID	Regulatory Elément	SEQ ID NO:	Mean GUS	Mean Flue	GUS/Fiuc
56492	CumMe WSM SF144839.G5080	37	27.5	16674	0.002
56877	P- CUCme.CumMe WSM SF16444.G5140l:l:l	175	96.3	17237.8	0.006
56485	CumMe WSM SFI6530.G6000’	42	27.3	17858.5	0.002
56844	CumMe WSM SFI6953.G5180	47	22.3	19398.5	0.001
56500	CumMe WSM SF17250.G5910	52	12.3	23980.3	0.001
56754	P-CUCme.WSM SFI7252.G7330-1: l: I	179	16	13848.8	0.001
56740	CumMe WSM SF17672.G5610	60	12	16646.8	0.001
56870	CumMe WSM SF18287.G5380	66	39.3	13930.5	0.003
56478	CumMe WSMSF 18504.G5090	68	ll.8	15830.5	0.001
56481	CumMe WSM SF18530.G5750	69	6.5	152H.3	0.000
56498	CumMe WSM SF18645.G5380	73	36	I4569.8	0.002
56746	P- CUCme.CumMe WSM SF18716.G5860l:l:l	184	11	18054.5	0.001
56490	CumMe WSM SFI880I.G5040	75	21.5	14147.3	0.002
56488	CumMe WSM SFI9323.G5120	81	15.3	Il 985.3	0.001
56499	CumMe WSM SFI9631.G5170	83	12.5	20140.5	0.001
56482	P- CUCme.CumMe WSM SF19839.G5090l:l:l	189	75	18690.5	0.004
56489	CumMe WSM SF19850.G5130	86	38.3	19756.5	0.002
56476	CumMe WSM SF20355.G5130	91	10.5	27901.8	0.000
56895	CumMe WSM SF20431 .G6340	95	34.8	16283.8	0.002
56744	CumMe WSM SF206458.G5970	98	11	19659	0.001
56480	CumMe WSM SF21366.G5980	105	10.8	17367	0.001
56930	CumMe WSM SF22070.G5280	109	25.3	14210.5	0.002
56484	CumMe WSM SF23181 .G5100	H7	20.3	I3506	0.002
56495	P- CUCme.CumMe WSM SF23760.G5200l:l:l	193	7.8	15138.5	0.001
56971	CumMe WSM SF25O84.G558O	125	16	16135.3	0.001
56742	P- CUCme.CumMe WSM SF25355.G5000l : l:l	196	18	13782.8	0.001
56494	CumMe WSM SF25455.G5370	129	10.5	16089.8	0.001
56751	P-	199	24.3	17884.3	0.001

V—

Amplicon ID	Regulatory Elément	SEQ ID NO:	Mcan GUS	Mean Fine	GUS/Fluc
	CUCme.CumMe WSM SF41124.G50801:1:1
56483	CumMe WSM SF41644.G6400	143	14,5	13130.5	0.001
56904	CumMe WSM SF44933.G5290	147	33	13369	0.002
56743	CumMe WSM SF9060.G5120	154	11.3	15230.8	0.001

Table 19. GUS to firefly luciferase (FLuc) ratios normalized with respect to EXPAt.Atnttl:l:2 and P-CaMV.35S-enli-l:l:102/L-CaMV.35S-l:l:2.

Amplicon ID	Regulatory Element	SEQ ID NO:	GUS/Fluc normalized with respect to EXPAt.Atnttl:l:2	GUS/Fluc normalized with respect to PCaMV.35Senhl:l:102/LCaMV.35S1:1:2
Empty Vector	No DNA
pMON 124912	No promoter
PMON80585	EXP-At.Atnttl:l:2	200	1.000	0.005
PMON33449	P-CaMV.35S-enh-1:1:102/L-CaMV.35S1:1:2	210	221.025	1.000
56741	CumMe WSM SFI43981.G5150	36	0.113	0.001
56492	CumMe WSM SF144839.G5080	37	0.559	0.003
56877	P- CUCme.CumMe WSM SF16444.G51401:1:1	175	1.893	0.009
56485	CumMe WSM SF16530.G6000	42	0.518	0.002
56844	CumMe WSM SF16953.G5180	47	0.390	0.002
56500	CumMe WSM SF17250.G5910	52	0.174	0.001
56754	P-CUCme.WSM SF 17252.G7330-1:1:1	179	0.392	0.002
56740	CumMe WSM SF 17672.G5610	60	0.244	0.001
56870	CumMe WSM SF18287.G5380	66	0.956	0.004
56478	CumMe WSM SF18504.G5090	68	0.253	0.001
56481	CumMe WSM SF18530.G5750	69	0.145	0.001
56498	CumMe WSM SF18645.G5380	73	0.837	0.004
56746	P- CUCme.CumMe WSM SF18716.G5860-	184	0.207	0.001

Amplicon ID	Regulatory Element	SEQ ID NO:	GUS/Fluc normalized with respect to EXPAt.Atnttl :1:2	GUS/Fluc normalized with respect to PCaMV.35Senhl:l:102/LCaMV.35S1:1:2
	I: l: l
56490	CumMe WSM SF18801.G5040	75	0.515	0.002
56488	CumMe WSM SF19323.G5120	81	0.433	0.002
56499	CumMe WSM SF1963I.G5170	83	0.210	0.001
56482	P- CUCme.CumMe WSM SFl 9839.G5090l:l:l	189	1.360	0.006
56489	CumMe WSM SF19850.G5130	86	0.657	0.003
56476	CumMe WSM SF20355.G5I30	91	0.128	0.001
56895	CumMe WSM SF20431.G6340	95	0.724	0.003
56744	CumMe WSM SF206458.G5970	98	0.190	0.001
56480	CumMe WSM SF21366.G5980	105	0.2 11	0.001
56930	CumMe WSM SF22070.G5280	109	0.603	0.003
56484	CumMe WSM SF23181 .G5100	ll7	0.509	0.002
56495	P- CUCme.CumMe WSM SF23760.G5200l:l:l	193	0.175	0.001
56971	CumMe WSM SF25084.G5580	125	0.336	0.002
56742	P- CUCme.CumMe WSM SF25355.G5000l:l:I	196	0.443	0.002
56494	CumMe WSM SF25455.G5370	129	0.221	0.001
56751	P- CUCme.CumMe WSM SF4l 124.G5080l:l :l	199	0.461	0.002
56483	CumMe WSM SF4I644.G6400	143	0.374	0.002
56904	CumMe WSM SF44933.G5290	147	0.837	0.004
56743	CumMe WSM SF9060.G5120	154	0.251	0.001

[0123] As can be seen in Table 18 above, not ail EXP sequences demonstrated the ability to drive transgene expression when compared to the promoterless control. However, the EXP sequences, P-CUCme.CumMe_WSM_SFI6444.G5l40-l:l:l (SEQ ID NO: 175) and P5 CUCme.CumMe_WSM_SFl9839.G5090-l:l:l (SEQ ID NO: 189) demonstrated the ability to drive trangene expression in soybean cotylédon protoplasts at a level similar or greater than

EXP-At.Atnttl:l:2. As shown in Table 19 above, the EXP sequence, PCUCme.CumMe_WSM_SFl9839.G5090-l:l:l (SEQ ID NO: 189) demonstrated the ability to drive transgene expression in this assay at a level greater than EXP-At. Atntt l : l :2.

Example 9: Analysis of Regulatory Eléments Driving GUS in Stably Transformed Soybean [0124] Soybean plants were transformed with plant expression vectors containing an EXP sequence driving expression of the B-glucuronidase (GUS) transgene.

[0125] Expression ofthe GUS transgene driven by EXP-CUCme.Ubql:l:l (SEQ ID NO: l), EXP-CUCme.Ubql:l:3 (SEQ ID NO: 7), P-CUCme.l-l:l:Ire (SEQ IDNO: 155), P-CUCme.2l : l : l (SEQ ID NO: 14), P-CUCme.3-1 : l :3 (SEQ ID NO: 15), EXP-CUCme.4: l : l (SEQ ID NO: 156), EXP-CUCme.5:l:l (SEQ IDNO: 159), P-CUCme.6-1:l:l (SEQ IDNO: 18), P-CUCme.8l : l :2 (SEQ ID NO: 19), P-CUCme.9-1 : l :2 (SEQ ID NO: 20), P-CUCme. 10-1:1 : l (SEQ ID NO: 21), EXP-CUCme.eEFla:l:l (SEQ IDNO: 162), P-CUCme. 15-1:1:2 (SEQ IDNO: 23), PCUCme. 17-1 : l :2 (SEQ ID NO: 26), P-CUCme. 18-1 : l :2 (SEQ ID NO: 27), P-CUCme. 19-1 : l :3 (SEQ ID NO: 167), P-CUCme.20-1:3 (SEQ ID NO: 211 ), P-CUCme.21 -1 : l : l (SEQ ID NO: 30), EXP-CUCme.SAMS2:l:I (SEQ IDNO: 168), P-CUCme.26-l:l:2 (SEQ ID NO: 33), EXPCUCme.29:l:2 (SEQ ID NO: 212), P-CUCme.CumMe_WSM_SF25355.G5000-l:l:l (SEQ ID NO: 196), P-CUCme.CumMe_WSM_SFI7l l l.G5790-l:l:l (SEQ ID NO: 177), PCUCme.CumMe_WSM_SF2253l.G5l20-l:l:l (SEQ IDNO: 192),PCUCme.CumMe_WSM_SFl8488.G5340-l:l:l (SEQ ID NO: I8I), PCUCme.CumMe_WSM_SF23760.G5200-l:l:l (SEQ ID NO: 193), EXPCUCme.WSM_SFl9064.G5690:l:l (SEQ IDNO: 185), P-CUCme. WSM_SFl7252.G7330l:l:l (SEQ IDNO: 179), P-CUCme.CumMe_WSM_SFI8634.G5190-1:1:1 (SEQ IDNO: 183), P-CUCme.CumMe_WSM_SF19647.G5760-l:l:l (SEQ IDNO: 188), PCUCme.CumMe_WSM_SF25936.G5450-1:1:1 (SEQ IDNO: 197), PCUCme.CumMe_WSM_SF19839.G5090-l:l:l (SEQ ID NO: 189), CumMe_WSM_SF206458.G5970 (SEQ ID NO: 98) and PCUCme.CumMe_WSM_SF 18716.G5860-1:1:1 (SEQ ID NO: 184) assayed both qualitatively through inspection of stained tissue sections and quantitatively. Each plant expression vector was comprised of a right border région from Agrobacteritim tumefaciens, a first transgene cassette comprised of an EXP sequence operabîy linked 5’ to a coding sequence for Bglucuronidase (GUS, SEQ ID NO: 206) containing a processable intron derived from the potato light-inducible tissue-specific ST-LSl gene (Genbank Accession: X04753), operabîy linked 5’ to a 3’ termination région from the the Gossypium barbadense FbLate-2 gene (T-Gb.FbL2-l:l:l, SEQ ID NO: 205); a second transgene sélection cassette used for sélection of transformed plant cells that confered résistance to the herbicide glyphosate (driven by the Arabidopsis Actin 7 promoter) and a left border région from A. tumefaciens.

[0126] The foregoing EXP sequences were cloned into plant expression constructgs as shown in Tables 20 through 23 below and used to transform soybean plants using an agrobacterium mediated transformation method. Expression of GUS was assayed qualitatively using histological sections of selected tissues and quantitatively.

[0127] Histochemical GUS analysis was used for qualitative expression analysis of transformed plants. Whole tissue sections were incubated with GUS staining solution X-Gluc (5-bromo-4chloro-3-indolyl-b-glucuronide) (l milligram/milliliter) 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, RI Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, RI Petiole, Yellow Pod Embryo, Yellow Pod Cotylédon, R3 Immature Seed, R3 Pod, R5 Cotylédon and RI Flower.

[0128] For quantitative analysis, total protein was extracted from selected tissues of transformed corn plants. One microgram of total protein was used with the fluorogenic substrate 4methyleumbelliferyl-P-D-glucuronide (MUG) in a total reaction volume of 50 microliters. 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 and émission 3nm.

[0129] Tables 20 and 21 below show the mean quantitative expression levels measured in the R₍₎ génération plant tissues. Those tissued not assayed are shown as blank cells in both tables.

Table 20. Mean GUS expression in Vn5 Root, RI Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf and RI Petiole of Ro génération transformed soybean plants

Construct	Regulatory Element	SEQ ID NO:	Vn5_ Root	RI Root	Vn5_Sink Leaf	Vn5_Source Leaf	Rl_Source Leaf	RI Petiole
pMON 138776	EXP-CUCme.Ubq 1:1:1	1	4				4	4
pMON 138778	EXP-CUCme.Ubql:l:3	7	16		1	2	13	23
pMON 140818	P-CUCme. l-l:l:lrc	155	48.21		22.35	20.24	33.01	78.17
pMON 140819	P-CUCme.2-1:1:1	14
pMON 140820	P-CUCme.3-l:l:3	15
pMON140821	EXP-CUCme.4:1:1	156	96.82		28.32	39.17	322.98	280.03
pMON 140822	EXP-CUCme.5:l:l	159	28.88				41.11
pMON 140823	P-CUCme.6-l:l:l	18	23.94				32.14	30.22
pMON 140824	P-CUCme.8-l:l:2	19
pMON 140825	P-CUCme.9-l:l:2	20	22.06				21.22	23.08
pMON 140826	P-CUCme. 10-1:1:1	21
pMON 140827	EXP-CUCme.eEFla:l:l	162	189.24	153.52	59.6	37.44	103.01	130.6
pMON 140828	P-CUCme. 15-1:1:2	23	30.53
pMON 140830	P-CUCme. 17-1:1:2	26	51.62		30.07	31.08	30.49	60.14
pMON 140831	P-CUCme. 18-1:1:2	27	57.38					30.03
pMON 140832	P-CUCme. 19-1:1:3	167	23.07		50.21	59.73	65.58	137.42
pMON 140833	P-CUCme.20-l:3	211	23.15		61.6	118.76	502.55	119.46
pMON 140834	P-CUCme.21-l:l:l	30					25.49
pMON 140836	EXP-CUCme.SAMS2:1:1	168	230.89	184.88	65.44	53.36	118.82	351.49
pMON 140837	P-CUCme.26-1:1:2	33	56.21		26.81	45.07	51.61	47.42
pMON 140839	EXP-CUCme.29:1:2	212	82.17		45.2	28.27	64.96	109.9
pMON 144926	LL·	196	28.53

Construct	Regulatory Elément	SEQ ID NO:	Vn5_ Root	RI Root	Vn5_Sink Leaf	Vn5_Source Leaf	Rl_Source Leaf	RI Petiole
	CUCme.CumMe WSM S F25355.G5000-1:1:1
pMON 144927	P- CUCme.CumMe WSM S F1711 l.G5790-l:l:l	177	23.62
pMON144928	P- CUCme.CumMe WSM S F22531.G5120-l:l:l	192	75.62		23	20.46	21.78	39.77
pMON144931	P- CUCme.CumMe WSM S F18488.G5340-1:1:1	181	43.2					52.55
pMON 144933	P- CUCme.CumMe WSM S F23760.G5200-l:l:l	193	25.61		20.45	0	0	28.69
pMON 146941	EXP- CUCme.WSM SF19064.G 5690:1:1	185	33.5		0	0	24.27	47.82
pMON144932	P- CUCme.WSM SF17252.G 7330-1:1:1	179	32.54		23.76	21.5	0	22.21
pMON146940	P- CUCme.CumMe WSM S F18634.G5190-I:l:l	183	0		0	0	0	0
pMON 147340	P- CUCme.CumMe WSM S F19647.G5760-l:l:l	188	28.9		0	0	29.77	25.82
pMON 147342	P- CUCme.CumMe WSM S F25936.G5450-l:l:l	197	50.15		24.26	0	29.38	29.91
pMON 147343	P-	189	36.05		25.7	27.54	22.85	37.15

Construct	Regulatory Element	SEQ ID NO:	Vn5_ Root	RI Root	VnSJSink Leaf	Vn5_Source Leaf	Rl_Source Leaf	RI Petiole
	CUCme.CumMe WSM S F19839.G5090-l:l:l
pMON 144929	CumMe_WSM_SF206458. G5970	98
pMON 147304	P- CUCme.CumMe WSM S Fl 8716.G5860-1:1:1	184	35.01		21.17	21.23	22	44.57

Table 21. Mean GUS expression in Yellow Pod Embryo, Yellow Pod Cotylédon, R3 Immature Seed, R3 Pod, R5 Cotylédon and RI Flower of Rn génération transfornied soybean plants

Construct	Regulatory Element	SEQ ID NO:	YeIlow_Pod_ Embryo	YellowPod Cotylédon	R3_ Immature Seed	R3 Po d	R5_ Cotylédon	RI Flower
pMON 138776	EXP-CUCme.Ubq 1:1:1	1	12	9	13	11	10	7
pMON 138778	EXP-CUCme.Ubql:l:3	7	3	1	13	9	13	27
pMON 140818	P-CUCme.l-l:l:lrc	155	100.79	117.5	38.31	84.72	132.27	66.8
pMON 140819	P-CUCme.2-1:1:1	14					20.35	36.18
pMON 140820	P-CUCme.3-l:l:3	15
pMON 140821	EXP-CUCme.4:l:l	156	86.68	225.53	105.62	342.07	119.08	184.92
pMON 140822	EXP-CUCme.5:l:l	159	21.48	32.27	21.47	21.66		36.88
pMON 140823	P-CUCme.6-l:l:l	18	38.75		23.03		25.32	58.7
pMON 140824	P-CUCme.8-l:l:2	19					90.33	25.77
pMON 140825	P-CUCme.9-I:l:2	20	132.04			20.56	34.78
pMON 140826	P-CUCme.l0-l:l:l	21					22.34
pMON 140827	EXP-CUCme.eEFla:l:l	162	200.28	291.26	58.21	131.17	114.29	130.38
pMON 140828	P-CUCme. 15-1:1:2	23			142.24	26.2
pMON 140830	P-CUCme.l7-l:l:2	26	343.34	302.94	65.55	80.94	137.02	62.7

Construct	Regulatory Elément	SEQ ID NO:	Yellow_Pod_ Embryo	Yellow_Pod Cotylédon	R3_ Immature Seed	R3 Po d	R5_ Cotylédon	Rl_ Flower
pMON140831	P-CUCme. 18-1:1:2	27	103.17	135.97	30	34.62	88.14	23.73
pMON 140832	P-CUCme. 19-1:1:3	167	30.96	64.46		316.66		53.46
pMON 140833	P-CUCme.20-l:3	211	174.62	524.88		222.04	59.43	124.68
pMON140834	P-CUCme.21-1:1:1	30			28.15	20.52	23.89
pMON 140836	EXP-CUCme.SAMS2:l:l	168	110.23	159.43	61.99	248.96	49.17	224.24
pMON140837	P-CUCme.26-l:l:2	33	56.73	50.06	70	143.05	25.06	49.92
pMON140839	EXP-CUCme.29:1:2	212	251.76	237.2	49.16	89.28	114.92	57.84
pMON 144926	P- CUCme.CumMe WSM S F25355.G5000-1:1:1	196			21.41		22.23
pMON144927	P- CUCme.CumMe WSM S F1711 l.G5790-l:l:l	177	58.84	28.94			20.97
pMON 144928	P- CUCme.CumMe WSM S F22531.G5120-l:l:l	192	135.62	152.48	30.45	51.71	129.72	42.2
pMON 144931	P- CUCme.CumMe WSM S F18488.G5340-l:l:l	181	866.94		23.26	21.49
pMON 144933	P- CUCme.CumMe WSM S F23760.G5200-1:1:1	193			29.03	34.9	69.63	24.42
pMON 146941	EXP- CUCme.WSM SF19064.G 5690:1:1	185			36.69	83.08	89.81	33.99
pMON 144932	P- CUCme.WSM SF17252.G 7330-1:1:1	179			34.29	39.89	113.83	0
pMON 146940	P-	183			30.25	0	0	0

Construct

Regulatory Element

CUCme.CumMe_WSM_S

F18634.G5190-l:l:l

PCUCme.CumMe_WSM_S pMON 147340 F19647.G5760-1:1:1

PCUCme.CumMeWSMS pMON 147342 F25936.G5450-l:l:l

PpMON 147343 pMON 144929 pMON 147304

SEQ ID

NO:

188

197

Yellow

Embryo

YellowPod Cotylédon

R3_ Immature

Seed

25.73

R5 Cotylédon

28.28 24.04

104.02 80.27 31.06

RI

Flower

23.35

26.8

CUCme.CumMe_WSM_S

F19839.G5090-l:l:l CumMe_WSM_SF206458. G5970_______________

P189

24.42

25.33

29.09

CUCme.CumMe_WSM_S

F18716.G5860-l:l:l

184

283.49

61.43 [ΟΙ30] As can be seen in Tables 20 and 21, the EXP sequences, EXP-CUCme.Ubql:l : l (SEQ ID NO: l ), EXP-CUCme.Ubq l : 1:3 (SEQ ID NO: 7), P-CUCme. I -1 : l : I rc (SEQ ID NO: 155), PCUCme.2-l:I:l (SEQIDNO: 14), EXP-CUCme.4:l:l (SEQ IDNO: I56), EXP-CUCme.5:l:l (SEQIDNO: 159), P-CUCme.6-l:l:l (SEQIDNO: 18), P-CUCme.8-l:l:2 (SEQ IDNO: I9), P-CUCme.9-1:1:2 (SEQ ID NO: 20), P-CUCme. 10-1:1 : l (SEQ ID NO: 21 ), EXPCUCme.eEFl a: I : l (SEQ ID NO: 162), P-CUCme. 15-1 : l :2 (SEQ ID NO: 23), P-CUCme. 17l : l :2 (SEQ ID NO: 26), P-CUCme. 18-1 : l :2 (SEQ ID NO: 27), P-CUCme. 19-1 : l :3 (SEQ ID NO: 167), P-CUCme.20-1:3 (SEQ ID NO: 211 ), P-CUCme.21 -1 : l : l (SEQ ID NO: 30), EXPCUCme.SAMS2:l:l (SEQ ID NO: 168), P-CUCme.26-l:l:2 (SEQ ID NO: 33), EXPCUCme.29:l:2 (SEQ ID NO: 212), P-CUCme.CumMe_WSM_SF25355.G5000-l:l:l (SEQ ID NO: I96), P-CUCme.CumMe_WSM_SFl 71 11.G5790-1:l : l (SEQ ID NO: 177), PCUCme.CumMe WSM_SF2253l.G5l20-l:l:l (SEQIDNO: 192), PCUCme.CumMe_WSM_SFl8488.G5340-l:l:l (SEQ IDNO: I8l), PCUCme.CumMe_WSM_SF23760.G5200-l:l:l (SEQIDNO: 193), EXPCUCme.WSM_SFl9064.G5690:l:l (SEQ IDNO: 185), P-CUCme.WSM SFl7252.G7330l:l:l (SEQ IDNO: 179), P-CUCme.CumMe_WSM_SFl8634.G5l90-l:l:I (SEQIDNO: 183), P-CUCme.CumMe_WSM_SFl9647.G5760-l:l:l (SEQ IDNO: 188), PCUCme.CumMe_WSM_SF25936.G5450-1 : l : l (SEQ ID NO: 197), PCUCme.CumMe_WSM_SFl9839.G5O9O-l:l:l (SEQ IDNO: 189), CumMe_WSM_SF206458.G5970 (SEQ ID NO: 98) and PCUCme.CumMe_WSM_SFl87l6.G5860-l:l:I (SEQ IDNO: 184) demonstrated quantitatively the capacity to drive transgene expression in some or ail tissues assayed, depending upon the EXP sequence used to drive expression.

[0131] Histological analysis of selected tissue sections provided further evidence of expression for many of the EXP sequences. EXP-CUCme.Ubql:l:l (SEQ ID NO: l) and EXPCUCme.Ubql:l:3 (SEQ ID NO: 7) demonstrated a constitutive expression pattern with staining observed in ail tissues, even though quantitative analysis showed fairly low levels of expression. This type of expression pattern can be most adventitîous to driving expression of transgcnes that require a low level of constitutive expression. Expression driven by P-CUCme.l-l:l:lrc (SEQ ID NO: 155) demonstrated expression in sink and source leaf vascular bundles and xylem and in the root cortex, phloem, xylem, endodermis, stele and tip. Expression driven by EXP86

CUCme.4:l:l (SEQ ID NO: 156) was observed in ail tissues with the highest expression observed in the reproductive phase of the plant. Expression driven by P-CUCme.lO-l:l:l (SEQ ID NO: 21) was observed only in in V5 Sink Leaf and RI Flower anthers. Expression driven by EXP-CUCme.eEFla:l:l (SEQ ID NO: 162) demonstrated a constitutive expression pattern with highest expression being observed in yellow pod embryo and cotylédon. The yellow pod embryo activity was 5fold higher in the Rlgeneration than in the RO génération (see Table 23 below). Expression driven by P-CUCme. 15-1:1:2 (SEQ ID NO: 23), P-CUCme.l 7-1:1:2 (SEQ ID NO: 26) and P-CUCme. 18-1:1:2 (SEQ ID NO: 27) demonstrated a constitutive level of expression histologically. Expression driven by P-CUCme. 19-1:1:3 (SEQ ID NO: 167) demonstrated a constitutive pattern of expression histologically with the exception of the V5 root and RI petiole. R3 pod showed the highest expression.

[0132] Expression driven by P-CUCme.20-l:3 (SEQ ID NO: 211) demonstrated a constitutive expression pattern histologically with the exception of expression in V5 root. Expression was highest in the R8 stage cotylédon. Expression driven by EXP-CUCme.SAMS2:l:l (SEQ ID NO: 168) demonstrated a constitutive pattern of expression with expression observed histologically in ail tissues. GUS expression was observed to increase in the RI génération (see Tables 22 and 23 below). The RI stage flowers and pétioles demonstrated the highest levels of expression in soybean. Expression driven by P-CUCme.CumMe_WSM_SF22531 .G5120-1:1:1 (SEQ ID NO: 192) demonstrated a constitutive pattern of expression histologically with highest expression in the R8 stage cotylédon and embryo. Expression driven by PCUCme.CumMe_WSM_SF18488.G5340-l:l:l (SEQ ID NO: 181) demonstrated a constitutive level of expression while quantitatively high expression was observed in the yellow pod embryo. [0133] Ro génération plants transformed with the plasmid constructs comprising EXPCUCme.eEFla:l:l (SEQ ID NO: 162) and EXP-CUCme.SAMS2:i:l (SEQ ID NO: 168) were allowed to set seed and the Ri génération plants analyzed for GUS expression. The R| génération plants were analyzed for expression in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, RI Petiole Yellow Pod Embryo, Yellow Pod Cotylédon, R3 Immature Seed, R3 Pod, R5 Cotylédon and RI Flower. Tables 22 and 23 show the mean GUS expression measured in each tissue of the Ri génération transformed plants.

✓L-'

Table 22. Mean GUS expression in Vn5 Root, Vn5 Sink Leaf, Vn5 Source Leaf, RI Source Leaf, RI Petiole of Ri génération transformed soybean plants

Construct	Regulatory Element	SEQ ID NO:	Vn5_ Root	Vn5_ Sink Leaf	Vn5_ Source Leaf	Rl_ Source Leaf	RI Petiole
pMON140827	EXP- CUCme.eEFla:l:l	162	145.84	50.24	43.73	107.98	357.67
pMON 140836	EXP- CUCme.SAMS2:l:l	168	260.41	65.52	51.12	129.86	623.42

Table 23. Mean GUS expression in Yellow Pod Embryo, Yellow Pod Cotylédon, R3 Immature Seed, R3 Pod, R5 Cotylédon, 5 RI Flower of R] génération transformed soybean plants ________________________________________________________

Construct	Regulatory Element	SEQ ID NO:	Yellow_ Pod Embryo	Yellow Pod_ Cotylédon	R3_ Immature Seed	R3 Pod	R5 Cotylédon	RI Flower
pMON 140827	EXP- CUCme.eEFla:l:l	162	1098.51	764.83	288.77	214.6	459.62	394.77
pMON 14083 6	EXP- CUCme.SAMS2:l:l	168	219.04	291.58	241.48	382.73	397.91	653.23

[0134] As can be seen in Tables 22 and 23 above expression driven in Ri génération by EXP-CUCme.eEFla:l:l (SEQ ID NO: 162) and EXP-CUCme.SAMS2:l:l (SEQ ID NO: 168) shows a constitutive level of expression with increase in expression observed in 10 many tissues at Rj génération relative to Ro génération.

******** [0135] Having illustrated and described the principles of the présent 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. Ail modifications that are within the spirit and scope of the claims are intended to be includcd within the scope of the présent invention. Ail publications and published patent documents cîted herein are hercby 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.

Claims (3)

1. Wliat is claimed is:

   1. A DNA molécule exhibiting a gene regulatory functional activity comprising a polynucleotide sequence selected from the group consisting of;

   a) a sequence with at Ieast 85 percent sequence identity to any of SEQ ID NOs: 1-199, 211 and2l2;

   b) a sequence comprising any of SEQ ID NOs: l-199, 211 and 212; and

   c) a fragment of any of SEQ ID NOs: 1-199, 211 and 212 exhibiting generegulatory activity;

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

   2. The DNA molécule of claim 1, wherein said polynucleotide sequence has at Ieast 90 percent sequence identity to the polynucleotide sequence as set forth in any of SEQ ID NOs: l199,211 and 212.

   3. The DNA molécule of claim 1, wherein said polynucleotide sequence has at Ieast 95 percent sequence identity to the polynucleotide sequence as set forth in any of SEQ ID NOs: 1199,211 and 212.

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

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

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

   7. A transgenic plant cell comprising a heterologous DNA molécule exhibiting a gene regulatory functional activity comprising a polynucleotide sequence selected from the group consisting of:

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

   l -199, 211 and 212;

   b) a sequence comprising any ofSEQ ID NOs: 1-I99, 2ll and 212; and

   c) a fragment of any of SEQ ID NOs: I-199, 211 and 212 exhibiting generegulatory activity;

   wherein said DNA molécule is operably linked to a heterologous transcribable polynucleotide 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 DNA molécule exhibiting a gene regulatory functional activity comprising a polynucleotide sequence selected from the group consisting of:

   a) a sequence with at least 85 percent sequence identity to any of SEQ ID NOs: 1-199, 2ll and2l2;

   b) a sequence comprising any of SEQ ID NOs: 1-199, 2U and2l2;and

   c) a fragment of any of SEQ ID NOs: 1-199, 2ll and 212 exhibiting generegulatory activity;

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

   11. A progeny plant of the transgenïc plant of claim 10, or part thereof, wherein the progeny plant or part thereof comprises said DNA molécule exhibiting a gene regulatory functional activity.

   12. A transgenic seed comprising a DNA molécule exhibiting a gene regulatory functional activity comprising a polynucleotide sequence selected from the group consisting of:

   a) a sequence with at least 85 percent sequence identitÿ to any of SEQ ID NOs: 1-199,211 and 212;

   b) asequencecomprisinganyof SEQ IDNOs: 1-199,211 and212;and

   c) a fragment of any of SEQ ID NOs: 1-199, 211 and 212 exhibiting generegulatory activity;

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

   13. A method of producing a commodity product comprising:

   a) obtaining a transgenic plant or part thereof comprising a DNA molécule exhibiting a gene regulatory functional activity comprising a polynucleotide sequence selected from the group consisting of:

   1) a sequence with at least 85 percent sequence identitÿ to any of SEQ ID NOs: 1-199,211 and 212;
2. 2) a sequence comprising any of SEQ ID NOs: 1-199, 211 and 212; and
3. 3) a fragment of any of SEQ ID NOs: 1-199,211 and 212 exhibiting gene-regulatory activity;

   wherein said DNA molécule is operably linked to a heterologous transcribable polynucleotide molécule; and

   b) 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.

   15. A commodity product comprising a DNA molécule exhibiting a gene regulatory functional activity comprising a polynucleotide sequence selected from the group consisting of:

   a) a sequence with at least 85 percent sequence identity to any of SEQ ID NOs: 1-199,211 and 212;

   b) a sequence comprising any of SEQ ID NOs: 1 -199, 211 and 212; and

   c) a fragment of any of SEQ ID NOs: 1-199, 211 and 212 exhibiting generegulatory activity;

   wherein said DNA molécule is operabîy linked to a heterologous transcribable polynucleotide molécule.

   16. A method of expressing a transcribable polynucleotide molécule comprising:

   a) obtaining a transgenic plant comprising a DNA molécule exhibiting a gene regulatory functional activity comprising a polynucleotide sequence selected from the group consisting of:

   1) a sequence with at least 85 percent sequence identity to any of SEQ ID NOs: 1-199,211 and 212;

   2) a sequence comprising any of SEQ ID NOs: 1-199, 211 and 212; and

   3) a fragment of any of SEQ ID NOs: 1 -199, 211 and 212 exhibiting gene-regulatory activity;

   wherein said DNA molécule is operabîy linked to a heterologous transcribable polynucleotide molécule; and

   b) cultivating said transgenic plant, wherein the transcribable polynucleotide is expressed. xaA~