# Patent

## Title
Plant regulatory elements and uses thereof

## Abstract

The invention provides novel recombinant DNA molecules and constructs useful for modulating gene expression in plants, plant cells, seeds, and progeny plants. The invention also provides transgenic plants, plant cells, plant parts, seeds, and progeny plants comprising the recombinant DNA molecules of the invention, along with methods of their use.

## Background
REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. provisional application Ser. No. 61/739,720, filed Dec. 19, 2012, which is incorporated by reference herein in its entirety.


INCORPORATION OF SEQUENCE LISTING
The sequence listing that is contained in the file named “MONS323USseq-corrected.txt”, which is 352,256 bytes (as measured in Microsoft Windows®) and was created on Mar. 6, 2014, is filed herewith by electronic submission and is incorporated by reference herein.
FIELD OF THE INVENTION
The invention relates to the field of plant molecular biology, plant genetic engineering, and DNA molecules useful for modulating gene expression in plants.
BACKGROUND
Regulatory elements are genetic elements that regulate gene activity by modulating the transcription of an operably linked transcribable DNA molecule. Such elements include promoters, leaders, enhancers, introns, and 3′ untranslated regions, and are useful in the field of plant molecular biology and plant genetic engineering.
SUMMARY OF THE INVENTION
The invention provides novel regulatory elements for use in plants and constructs comprising the regulatory elements. The invention also provides transgenic plant cells, plants, plant parts, and seeds comprising the regulatory elements. In one embodiment, the invention provides the regulatory elements disclosed herein operably linked to a transcribable DNA molecule. In certain embodiments, the transcribable DNA molecule is heterologous with respect to a regulatory element sequence provided herein. Also provided herein are methods for making and using the regulatory elements disclosed herein, including constructs comprising the regulatory elements, and transgenic plants, plant cells, plant parts, and seeds comprising the regulatory elements operably linked to a transcribable DNA molecule that is heterologous with respect to the regulatory element.
Thus, in one aspect, the invention provides a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: a) a DNA sequence with at least about 85 percent sequence identity to any of SEQ ID NOs: 1-98 and 168-171; b) a DNA sequence comprising any of SEQ ID NOs: 1-98 and 168-171; and c) a fragment of any of SEQ ID NOs: 1-98 and 168-171, wherein the fragment has gene-regulatory activity; wherein the DNA sequence is operably linked to a heterologous transcribable DNA molecule. By “heterologous transcribable DNA molecule,” it is meant that the transcribable DNA molecule is heterologous with respect to the DNA sequence. In specific embodiments, the recombinant DNA molecule comprises a DNA sequence having at least 90 percent, at least 91 percent, at least 92 percent, at least 93 percent, at least 94 percent, at least about 95 percent, at least 96 percent, at least 97 percent, at least 98 percent, or at least 99 percent sequence identity to the DNA sequence of any of SEQ ID NOs: 1-98 and 168-171. In particular embodiments, the heterologous transcribable DNA molecule comprises a gene of agronomic interest, such as a gene capable of conferring herbicide resistance or pest resistance in plants. In still other embodiments, the invention provides a construct comprising a recombinant DNA molecule as provided herein.
In another aspect, provided herein are transgenic plant cells comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: a) a DNA sequence with at least about 85 percent sequence identity to any of SEQ ID NOs: 1-98 and 168-171; b) a DNA sequence comprising any of SEQ ID NOs: 1-98 and 168-171; and c) a fragment of any of SEQ ID NOs: 1-98 and 168-171, wherein the fragment has gene-regulatory activity; wherein the DNA sequence is operably linked to a heterologous transcribable DNA molecule. In certain embodiments, the transgenic plant cell is a monocotyledonous plant cell. In other embodiments, the transgenic plant cell is a dicotyledonous plant cell.
In still yet another aspect, further provided herein is a transgenic plant, or part thereof, comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: a) a DNA sequence with at least about 85 percent sequence identity to any of SEQ ID NOs: 1-98 and 168-171; b) a DNA sequence comprising any of SEQ ID NOs: 1-98 and 168-171; and c) a fragment of any of SEQ ID NOs: 1-98 and 168-171, wherein the fragment has gene-regulatory activity; wherein the DNA sequence is operably linked to a heterologous transcribable DNA molecule. In specific embodiments, the transgenic plant is a progeny plant of any generation relative to a starting transgenic plant and comprises the recombinant DNA molecule. A transgenic seed comprising the recombinant DNA molecule that produces such a transgenic plant when grown is also provided by the invention.
In still yet another aspect, the invention provides a method of expressing a transcribable DNA molecule, such as a gene of agronomic interest, in a transgenic plant by obtaining a transgenic plant containing a recombinant DNA molecule of the invention and cultivating the plant.
Also provided herein is a method of providing a transgenic plant by transforming a plant cell with a recombinant DNA molecule of the invention to produce a transformed plant cell, and regenerating the transformed plant cell to produce a transgenic plant.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1: Shows an alignment of multiple promoter variants of various sizes corresponding to promoter elements from Agrostis nebulosa. In particular, FIG. 1 shows an alignment of a 2005 base pair (bp) promoter P-AGRne.Ubq1-1:1:5 (SEQ ID NO: 2), contained in the regulatory expression element group (EXP) EXP-AGRne.Ubq1:1:7 (SEQ ID NO: 1), with promoter variants of P-AGRne.Ubq1-1:1:5. Deletion, for instance of the 5′ end of P-AGRne.Ubq1-1:1:5, produced the promoter P-AGRne.Ubq1-1:1:4 (SEQ ID NO: 6), a 999 bp sequence that is contained in EXP-AGRne.Ubq1:1:8 (SEQ ID NO: 5). Another promoter variant shown in FIG. 1 is P-AGRne.Ubq1-1:1:6 (SEQ ID NO: 8), a 762 bp sequence contained in EXP-AGRne.Ubq1:1:9 (SEQ ID NO: 7).
FIG. 2: Shows an alignment of multiple promoter variants of various sizes corresponding to promoter elements from Arundo donax. In particular, FIG. 2 shows an alignment of a 4114 bp promoter P-ARUdo.Ubq1-1:1:4 (SEQ ID NO: 10), contained in the regulatory expression element group EXP-ARUdo.Ubq1:1:4 (SEQ ID NO: 9), with promoter variants of P-ARUdo.Ubq1-1:1:4. Included in the alignment are a 2012 bp promoter P-ARUdo.Ubq1-1:1:5 (SEQ ID NO: 14); a 1000 bp promoter P-ARUdo.Ubq1-1:1:6 (SEQ ID NO: 17); and a 755 bp promoter P-ARUdo.Ubq1-1:1:8 (SEQ ID NO: 22).
FIG. 3: Shows an alignment of multiple promoter variants of various sizes corresponding to promoter elements from Arundo donax. In particular, FIG. 3 shows an alignment of a 2033 bp promoter P-ARUdo.Ubq2-1:1:4 (SEQ ID NO: 24) with promoter variants of P-ARUdo.Ubq2-1:1:4. Included in the alignment are a 2004 bp promoter P-ARUdo.Ubq2-1:1:6 (SEQ ID NO: 28); a 1001 bp promoter P-ARUdo.Ubq2-1:1:5 (SEQ ID NO: 31); and a 696 bp promoter P-ARUdo.Ubq2-1:1:7 (SEQ ID NO: 33).
FIG. 4: Shows an alignment of multiple promoter variants of various sizes corresponding to promoter elements from Bouteloua gracilis. In particular, FIG. 4 shows an alignment of a 2371 bp promoter P-BOUgr.Ubq1-1:1:2 (SEQ ID NO: 35) with promoter variants of the 5′ end of P-BOUgr.Ubq1-1:1:2. Included in the alignment are a 1999 bp promoter P-BOUgr.Ubq1-1:1:3 (SEQ ID NO: 39); a 1022 bp promoter P-BOUgr.Ubq1-1:1:5 (SEQ ID NO: 42); and a 760 bp promoter P-BOUgr.Ubq1-1:1:6 (SEQ ID NO: 44).
FIG. 5: Shows an alignment of multiple promoter variants of various sizes corresponding to promoter elements from Bouteloua gracilis. In particular, FIG. 5 shows alignment of a 2100 bp promoter element, P-BOUgr.Ubq2-1:1:4 (SEQ ID NO: 46) with promoter variants of P-BOUgr.Ubq2-1:1:4. Included in the alignment are a 2043 bp promoter P-BOUgr.Ubq2-1:1:7 (SEQ ID NO: 50); a 2002 bp promoter P-BOUgr.Ubq2-1:1:5 (SEQ ID NO: 53); a 1024 bp promoter P-BOUgr.Ubq2-1:1:6 (SEQ ID NO: 56); and a 749 bp promoter P-BOUgr.Ubq2-1:1:8 (SEQ ID NO: 61).
FIG. 6: Shows an alignment of multiple promoter variants of various sizes corresponding to promoter elements from Miscanthus sinesis. In particular, FIG. 6 shows an alignment of a 5359 bp promoter element, P-MISsi.Ubq1-1:1:2 (SEQ ID NO: 63) with promoter variants of P-MISsi.Ubq1-1:1:2. Included in the alignment are a 2423 bp promoter P-MISsi.Ubq1-1:1:11 (SEQ ID NO: 67); a 1447 bp promoter P-MISsi.Ubq1-1:1:10 (SEQ ID NO: 71); a 899 bp promoter P-MISsi.Ubq1-1:1:13 (SEQ ID NO: 73); a 691 bp promoter P-MISsi.Ubq1-1:1:14 (SEQ ID NO: 75); and a 506 bp promoter P-MISsi.Ubq1-1:1:9 (SEQ ID NO: 77).
FIG. 7: Shows an alignment of multiple promoter variants of various sizes corresponding to promoter elements from Schizachyium scoparium. In particular, FIG. 7 shows an alignment of a 2831 bp promoter element, P-SCHsc.Ubq1-1:1:12 (SEQ ID NO: 79) with promoter variants of P-SCHsc.Ubq1-1:1:12. Included in the alignment are a 2033 bp promoter P-SCHsc.Ubq1-1:1:11 (SEQ ID NO: 83); a 1046 bp promoter P-SCHsc.Ubq1-1:1:10 (SEQ ID NO: 85); and a 547 bp promoter P-SCHsc.Ubq1-1:1:14 (SEQ ID NO: 87).
FIG. 8: Shows an alignment of multiple promoter variants of various sizes corresponding to promoter elements from Sorghastrum nutans. In particular, FIG. 8 shows an alignment of a 2218 bp promoter element, P-SORnu.Ubq1-1:1:4 (SEQ ID NO: 89) with promoter variants of P-SORnu.Ubq1-1:1:4. Included in the alignment are a 1964 bp promoter P-SORnu.Ubq1-1:1:5 (SEQ ID NO: 93); a 1023 bp promoter P-SORnu.Ubq1-1:1:6 (SEQ ID NO: 96); and a 724 bp promoter P—SORnu.Ubq1-1:1:7 (SEQ ID NO: 98).
FIG. 9: Shows expression cassette configurations of the invention.



BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NOs: 1, 5, 7, 9, 13, 16, 18, 19, 21, 23, 27, 30, 32, 34, 38, 41, 43, 45, 49, 52, 55, 58, 60, 62, 66, 70, 72, 74, 76, 78, 82, 84, 86, 88, 92, 95, 97, 99, 103, 106, 108, 110, 114, 116, 118, 120, 122, 126, 128, 132, 134, 138, 140, 144, 148, 150 and 168 are DNA sequences of regulatory expression element groups (EXPs) comprising a promoter sequence operably linked 5′ to a leader sequence which is operably linked 5′ to an intron sequence.
SEQ ID NOs: 2, 6, 8, 10, 14, 17, 22, 24, 28, 31, 33, 35, 39, 42, 44, 46, 50, 53, 56, 61, 63, 67, 71, 73, 75, 77, 79, 83, 85, 87, 89, 93, 96, 98, 100, 104, 107, 109, 111, 117, 119, 121, 123, 129, 135, 141, 145, 151 and 169 are promoter sequences.
SEQ ID NOs: 3, 11, 25, 36, 47, 64, 68, 80, 90, 101, 112, 124, 130, 136, 142, 146, 152 and 170 are leader sequences.
SEQ ID NOs: 4, 12, 15, 20, 26, 29, 37, 40, 48, 51, 54, 57, 59, 65, 69, 81, 91, 94, 102, 105, 113, 115, 125, 127, 131, 133, 137, 139, 143, 147, 149, 153 and 171 are intron sequences.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides DNA molecules having gene-regulatory activity in plants. The nucleotide sequences of these DNA molecules are provided as SEQ ID NOs: 1-98 and 168-171. These DNA molecules are, for instance, capable of affecting the expression of an operably linked transcribable DNA molecule in plant tissues, and therefore regulating gene expression of an operably linked transgene in transgenic plants. The invention also provides methods of modifying, producing, and using the same. The invention also provides compositions that include transgenic plant cells, plants, plant parts, and seeds containing recombinant DNA molecules of the invention, and methods for preparing and using the same.
The following definitions and methods are provided to better define the invention and to guide those of ordinary skill in the art in the practice of the invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.
DNA Molecules
As used herein, the term “DNA” or “DNA molecule” refers to a double-stranded DNA molecule of cellular or synthetic origin, i.e., a polymer of deoxyribonucleotide bases. As used herein, the term “DNA sequence” refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein corresponds to that of by Title 37 of the United States Code of Federal Regulations §1.822, and set forth in the tables in WIPO Standard ST.25 (1998), Appendix 2, Tables 1 and 3.
As used herein, a “recombinant DNA molecule” is a DNA molecule comprising a combination of DNA molecules that would not naturally occur together without human intervention. For instance, a recombinant DNA molecule may be a DNA molecule that is comprised of at least two DNA molecules heterologous with respect to each other, a DNA molecule that comprises a DNA sequence that deviates from DNA sequences that exist in nature, or a DNA molecule that has been incorporated into a host cell's DNA by genetic transformation.
As used herein, the term “sequence identity” refers to the extent to which two optimally aligned DNA sequences are identical. An optimal sequence alignment is created by manually aligning two DNA sequences, e.g., a reference sequence and another DNA sequence, to maximize the number of nucleotide matches in the sequence alignment with appropriate internal nucleotide insertions, deletions, or gaps. As used herein, the term “reference sequence” refers to a DNA sequence provided as SEQ ID NOs: 1-98 and 168-171.
As used herein, the term “percent sequence identity” or “percent identity” or “% identity” is the identity fraction multiplied by 100. The “identity fraction” for a DNA sequence optimally aligned with a reference sequence is the number of nucleotide matches in the optimal alignment, divided by the total number of nucleotides in the reference sequence, e.g., the total number of nucleotides in the full length of the entire reference sequence. Thus, one embodiment of the invention provides a DNA molecule comprising a DNA sequence that when optimally aligned to a reference sequence, provided herein as SEQ ID NOs: 1-98 and 168-171, has at least about 85 percent identity, at least about 86 percent identity, at least about 87 percent identity, at least about 88 percent identity, at least about 89 percent identity, at least about 90 percent identity, at least about 91 percent identity, at least about 92 percent identity, at least about 93 percent identity, at least about 94 percent identity, at least about 95 percent identity, at least about 96 percent identity, at least about 97 percent identity, at least about 98 percent identity, at least about 99 percent identity, or at least about 100 percent identity to the reference sequence.
Regulatory Elements
Regulatory elements such as promoters, leaders, enhancers, introns, and transcription termination regions (or 3′ UTRs) play an integral part in the overall expression of genes in living cells. The term “regulatory element,” as used herein, refers to a DNA molecule having gene-regulatory activity. The term “gene-regulatory activity,” as used herein, refers to the ability to affect the expression of an operably linked transcribable DNA molecule, for instance by affecting the transcription and/or translation of the operably linked transcribable DNA molecule. Regulatory elements, such as promoters, leaders, enhancers, and introns that function in plants are therefore useful for modifying plant phenotypes through genetic engineering.
As used herein, a “regulatory expression element group” or “EXP” sequence may refer to a group of operably linked regulatory elements, such as enhancers, promoters, leaders, and introns. Thus, a regulatory expression element group may be comprised, for instance, of a promoter operably linked 5′ to a leader sequence, which is in turn operably linked 5′ to an intron sequence.
Regulatory elements may be characterized by their gene expression pattern, e.g., positive and/or negative effects such as constitutive expression or temporal, spatial, developmental, tissue, environmental, physiological, pathological, cell cycle, and/or chemically responsive expression, and any combination thereof, as well as by quantitative or qualitative indications. As used herein, a “gene expression pattern” is any pattern of transcription of an operably linked DNA molecule into a transcribed RNA molecule. The transcribed RNA molecule may be translated to produce a protein molecule or may provide an antisense or other regulatory RNA molecule, such as a double-stranded RNA (dsRNA), a transfer RNA (tRNA), a ribosomal RNA (rRNA), a microRNA (miRNA), and the like.
As used herein, the term “protein expression” is any pattern of translation of a transcribed RNA molecule into a protein molecule. Protein expression may be characterized by its temporal, spatial, developmental, or morphological qualities, 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 DNA molecule. As used herein, the term “promoter” refers generally to a DNA molecule that is involved in recognition and binding of RNA polymerase II and other proteins, such as trans-acting transcription factors, to initiate transcription. A promoter may originate from the 5′ untranslated region (5′ UTR) of a gene. Alternately, promoters may be synthetically produced or manipulated DNA molecules. Promoters may also be chimeric. Chimeric promoters are produced through the fusion of two or more heterologous DNA molecules. Promoters useful in practicing the invention include SEQ ID NOs: 2, 6, 8, 10, 14, 17, 22, 24, 28, 31, 33, 35, 39, 42, 44, 46, 50, 53, 56, 61, 63, 67, 71, 73, 75, 77, 79, 83, 85, 87, 89, 93, 96, 98 and 169, including fragments or variants thereof. In specific embodiments of the invention, such DNA molecules and any variants or derivatives 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 specific embodiments, a fragment may be defined as exhibiting promoter activity possessed by the starting promoter molecule 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 equivalent DNA sequence for recognition and binding of the RNA polymerase II complex for initiation of transcription.
In one embodiment, fragments are provided of a promoter sequence disclosed herein. Promoter fragments may comprise promoter activity, as described above, and may be useful alone or in combination with other promoters and promoter fragments, such as in constructing chimeric promoters. In specific embodiments, fragments of a promoter are provided comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, or at least about 1000 contiguous nucleotides, or longer, of a DNA molecule having promoter activity as disclosed herein. Methods for producing such fragments from a starting promoter molecule are well known in the art.
Compositions derived from any of the promoters presented as SEQ ID NOs: 2, 6, 8, 10, 14, 17, 22, 24, 28, 31, 33, 35, 39, 42, 44, 46, 50, 53, 56, 61, 63, 67, 71, 73, 75, 77, 79, 83, 85, 87, 89, 93, 96, 98 and 169, such as internal or 5′ deletions, for example, can be produced using well known methods in the art to improve or alter expression, including by removing elements that have either positive or negative effects on expression; duplicating elements that have positive or negative effects on expression; and/or duplicating or removing elements that have tissue- or cell-specific effects on expression. Compositions derived from any of the promoters presented as SEQ ID NOs: 2, 6, 8, 10, 14, 17, 22, 24, 28, 31, 33, 35, 39, 42, 44, 46, 50, 53, 56, 61, 63, 67, 71, 73, 75, 77, 79, 83, 85, 87, 89, 93, 96, 98 and 169 comprised of 3′ deletions in which the TATA box element or equivalent DNA sequence thereof and downstream sequence is removed can be used, for example, to make enhancer elements. Further deletions can be made to remove any elements that have positive or negative; tissue specific; cell specific; or timing specific (such as, but not limited to, circadian rhythms) effects on expression. Any of the promoters presented as SEQ ID NOs: 2, 6, 8, 10, 14, 17, 22, 24, 28, 31, 33, 35, 39, 42, 44, 46, 50, 53, 56, 61, 63, 67, 71, 73, 75, 77, 79, 83, 85, 87, 89, 93, 96, 98 and 169 and fragments or enhancers derived therefrom can be used to make chimeric regulatory element compositions comprised of any of the promoters presented as SEQ ID NOs: 2, 6, 8, 10, 14, 17, 22, 24, 28, 31, 33, 35, 39, 42, 44, 46, 50, 53, 56, 61, 63, 67, 71, 73, 75, 77, 79, 83, 85, 87, 89, 93, 96, 98 and 169 and the fragments or enhancers derived therefrom operably linked to other enhancers and promoters.
In accordance with the invention, a promoter or promoter fragment may be analyzed for the presence of known promoter elements, i.e., DNA sequence characteristics, such as a TATA box and other known transcription factor binding site motifs. Identification of such known promoter elements may be used by one of skill in the art to design variants of the promoter having a similar expression pattern to the original promoter.
As used herein, the term “leader” refers to a DNA molecule from the untranslated 5′ region (5′ UTR) of a gene and defined generally as a DNA segment between the transcription start site (TSS) and the protein coding sequence start site. Alternately, leaders may be synthetically produced or manipulated DNA elements. A leader can be used as a 5′ regulatory element for modulating expression of an operably linked transcribable DNA molecule. Leader molecules may be used with a heterologous promoter or with their native promoter. Promoter molecules of the invention may thus be operably linked to their native leader or may be operably linked to a heterologous leader. Leaders useful in practicing the invention include SEQ ID NOs: 3, 11, 25, 36, 47, 64, 68, 80, 90 and 170 or fragments or variants thereof. In specific embodiments, such DNA sequences may be defined as being capable of acting as a leader in a host cell, including, for example, a transgenic plant cell. In one embodiment, such DNA sequences may be decoded as comprising leader activity.
The leader sequences (5′ UTR) presented as SEQ ID NOs: 3, 11, 25, 36, 47, 64, 68, 80, 90 and 170 may be comprised of regulatory elements or may adopt secondary structures that can have an effect on transcription or translation of an operably linked DNA molecule. The leader sequences presented as SEQ ID NOs: 3, 11, 25, 36, 47, 64, 68, 80, 90 and 170 can be used in accordance with the invention to make chimeric regulatory elements that affect transcription or translation of an operably linked DNA molecule. In addition, the leader sequences presented as SEQ ID NOs: 3, 11, 25, 36, 47, 64, 68, 80, 90 and 170 can be used to make chimeric leader sequences that affect transcription or translation of an operably linked DNA molecule.
As used herein, the term “intron” refers to a DNA molecule that may be isolated or identified from the genomic copy of a gene and may be defined generally as a region spliced out during messenger RNA (mRNA) processing prior to translation. Alternately, an intron may be a synthetically produced or manipulated DNA element. An intron may contain enhancer elements that effect the transcription of operably linked genes. An intron may be used as a regulatory element for modulating expression of an operably linked transcribable DNA molecule. A construct may comprise an intron, and the intron may or may not be heterologous with respect to the transcribable DNA molecule. Examples of introns in the art include the rice actin intron and the corn HSP70 intron.
In plants, the inclusion of some introns in constructs leads to increased mRNA and protein accumulation relative to constructs lacking the intron. This effect has been termed “intron mediated enhancement” (IME) of gene expression. Introns known to stimulate expression in plants have been identified in maize genes (e.g., tubA1, Adh1, Sh1, and Ubi1), in rice genes (e.g., tpi) and in dicotyledonous plant genes like those from petunia (e.g., rbcS), potato (e.g., st-ls1) and from Arabidopsis thaliana (e.g., ubq3 and pat1). It has been shown that deletions or mutations within the splice sites of an intron reduce gene expression, indicating that splicing might be needed for IME. However, that splicing per se is not required, as IME in dicotyledonous plants has been shown by point mutations within the splice sites of the pat1 gene from A. thaliana. Multiple uses of the same intron in one plant has been shown to exhibit disadvantages. In those cases, it is necessary to have a collection of basic control elements for the construction of appropriate recombinant DNA elements.
Introns useful in practicing the invention include SEQ ID NOs: 4, 12, 15, 20, 26, 29, 37, 40, 48, 51, 54, 57, 59, 65, 69, 81, 91, 94 and 171. Compositions derived from any of the introns presented as SEQ ID NOs: 4, 12, 15, 20, 26, 29, 37, 40, 48, 51, 54, 57, 59, 65, 69, 81, 91, 94 and 171 can be comprised of internal deletions or duplications of cis regulatory elements; and/or alterations of the 5′ and 3′ DNA 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. When modifying intron/exon boundary sequences, it may be beneficial to avoid using the nucleotide sequence AT or the nucleotide A just prior to the 5′ end of the splice site (GT) and the nucleotide G or the nucleotide sequence TG, respectively just after 3′ end of the splice site (AG) to eliminate the potential of unwanted start codons from being formed during processing of the messenger RNA into the final transcript. The DNA sequence around the 5′ or 3′ end splice junction sites of the intron can thus be modified in this manner. Introns and intron variants altered as described herein and through methods known in the art, can be tested empirically as described in the working examples to determine an intron's effect on expression of an operably linked DNA molecule.
As used herein, the term “3′ transcription termination molecule,” “3′ untranslated region” or “3′ UTR” herein refers to a DNA molecule that is used during transcription to the untranslated region of the 3′ portion of an mRNA molecule. The 3′ untranslated region of an mRNA molecule may be generated by specific cleavage and 3′ polyadenylation, also known as a polyA tail. A 3′ UTR may be operably linked to and located downstream of a transcribable DNA molecule and may include a polyadenylation signal and other regulatory signals 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 molecules in the art are the nopaline synthase 3′ region; wheat hsp17 3′ region, pea rubisco small subunit 3′ region, cotton E6 3′ region, and the coixin 3′ UTR.
3′ UTRs typically find beneficial use for the recombinant expression of specific DNA molecules. A weak 3′ UTR has the potential to generate read-through, which may affect the expression of the DNA molecule located in the neighboring expression cassettes. Appropriate control of transcription termination can prevent read-through into DNA sequences (e.g., other expression cassettes) localized downstream and can further allow efficient recycling of RNA polymerase to improve gene expression. Efficient termination of transcription (release of RNA Polymerase II from the DNA) is prerequisite for re-initiation of transcription and thereby directly affects the overall transcript level. Subsequent to transcription termination, the mature mRNA is released from the site of synthesis and template transported to the cytoplasm. Eukaryotic mRNAs are accumulated as poly(A) forms in vivo, making it difficult to detect transcriptional termination sites by conventional methods. However, prediction of functional and efficient 3′ UTRs by bioinformatics methods is difficult in that there are no conserved DNA sequences that would allow easy prediction of an effective 3′ UTR.
From a practical standpoint, it is typically beneficial that a 3′ UTR used in an expression cassette possesses the following characteristics. The 3′ UTR should be able to efficiently and effectively terminate transcription of the transgene and prevent read-through of the transcript into any neighboring DNA sequence, which can be comprised of another expression cassette as in the case of multiple expression cassettes residing in one transfer DNA (T-DNA), or the neighboring chromosomal DNA into which the T-DNA has inserted. The 3′ UTR should not cause a reduction in the transcriptional activity imparted by the promoter, leader, enhancers, and introns that are used to drive expression of the DNA molecule. In plant biotechnology, the 3′ UTR is often used for 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 expression cassette once integrated into the plant chromosome; (2) assess the copy number of insertions within the plant DNA; and (3) assess zygosity of the resulting seed after breeding. The 3′ UTR is also used in amplification reactions of DNA extracted from the transformed plant to characterize the intactness of the inserted cassette.
As used herein, the term “enhancer” or “enhancer element” refers to a cis-acting 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 DNA sequence. Unlike promoters, enhancer elements do not usually include a transcription start site (TSS) or TATA box or equivalent DNA sequence. A promoter or promoter fragment may naturally comprise one or more enhancer elements that affect the transcription of an operably linked DNA sequence. An 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.
Many promoter enhancer elements are believed to bind DNA-binding proteins and/or affect DNA topology, producing local conformations that selectively allow or restrict access of RNA polymerase to the DNA template or that facilitate selective opening of the double helix at the site of transcriptional initiation. An enhancer element may function to bind transcription factors that regulate transcription. Some enhancer elements bind more than one transcription factor, and transcription factors may interact with different affinities with more than one enhancer domain. Enhancer elements can be identified by a number of techniques, including deletion analysis, i.e., deleting one or more nucleotides from the 5′ end or internal to a promoter; DNA binding protein analysis using DNase I footprinting, methylation interference, electrophoresis mobility-shift assays, in vivo genomic footprinting by ligation-mediated polymerase chain reaction (PCR), and other conventional assays; or by DNA sequence similarity analysis using known cis-element motifs or enhancer elements as a target sequence or target motif with conventional DNA sequence comparison methods, such as BLAST. The fine structure of an enhancer domain can be further studied by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods known in the art. Enhancer elements can be obtained by chemical synthesis or by isolation from regulatory elements that include such elements, and they can be synthesized with additional flanking nucleotides that contain useful restriction enzyme sites to facilitate subsequence manipulation. Thus, the design, construction, and use of enhancer elements according to the methods disclosed herein for modulating the expression of operably linked transcribable DNA molecules are encompassed by the invention.
As used herein, the term “chimeric” refers to a single DNA molecule produced by fusing a first DNA molecule to a second DNA molecule, where neither the first nor the second DNA molecule would normally be contained in that configuration, i.e., fused to the other. The chimeric DNA molecule is thus a new DNA molecule not otherwise normally contained in nature. As used herein, the term “chimeric promoter” refers to a promoter produced through such manipulation of DNA molecules. A chimeric promoter may combine two or more DNA fragments, for example, the fusion of a promoter to an enhancer element. Thus, the design, construction, and use of chimeric promoters according to the methods disclosed herein for modulating the expression of operably linked transcribable DNA molecules are encompassed by the invention.
As used herein, the term “variant” refers to a second DNA molecule, such as a regulatory element, that is similar in composition, but not identical to, a first DNA molecule, and wherein the second DNA molecule still maintains the general functionality, i.e., same or similar expression pattern, for instance through more or less or equivalent transcriptional or translational activity, of the first DNA molecule. A variant may be a shortened or truncated version of the first DNA molecule and/or an altered version of the DNA sequence of the first DNA molecule, such as one with different restriction enzyme sites and/or internal deletions, substitutions, and/or insertions. Regulatory element “variants” also encompass variants arising from mutations that occur during or as a result of bacterial and plant cell transformation. In the invention, a DNA sequence provided as SEQ ID NOs: 1-98 and 168-171 may be used to create variants that are similar in composition, but not identical to, the DNA sequence of the original regulatory element, while still maintaining the general functionality, i.e., the same or similar expression pattern, of the original regulatory element. Production of such variants of the invention is well within the ordinary skill of the art in light of the disclosure and is encompassed within the scope of the invention.
Chimeric regulatory elements can be designed to comprise various constituent elements which may be operatively linked by various methods known in the art, such as restriction enzyme digestion and ligation, ligation independent cloning, modular assembly of PCR products during amplification, or direct chemical synthesis of the regulatory element, as well as other methods known in the art. The resulting various chimeric regulatory elements can be comprised of the same, or variants of the same, constituent elements but differ in the DNA sequence or DNA sequences that comprise the linking DNA sequence or sequences that allow the constituent parts to be operatively linked. In the invention, a DNA sequence provided as SEQ ID NOs: 1-98 and 168-171 may provide a regulatory element reference sequence, wherein the constituent elements that comprise the reference sequence may be joined by methods known in the art and may comprise substitutions, deletions, and/or insertions of one or more nucleotides or mutations that naturally occur in bacterial and plant cell transformation.
The efficacy of the modifications, duplications, or deletions 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 DNA molecule.
Constructs
As used herein, the term “construct” means any recombinant DNA molecule such as a plasmid, cosmid, virus, phage, or linear or circular DNA or RNA molecule, derived from any source, capable of genomic integration or autonomous replication, comprising a DNA molecule where at least one DNA molecule has been linked to another DNA molecule in a functionally operative manner, i.e. operably linked. As used herein, the term “vector” means any construct that may be used for the purpose of transformation, i.e., the introduction of heterologous DNA or RNA into a host cell. A construct typically includes one or more expression cassettes. As used herein, an “expression cassette” refers to a DNA molecule comprising at least a transcribable DNA molecule operably linked to one or more regulatory elements, typically at least a promoter and a 3′ UTR.
As used herein, the term “operably linked” refers to a first DNA molecule joined to a second DNA molecule, wherein the first and second DNA molecules are so arranged that the first DNA molecule affects the function of the second DNA molecule. The two DNA molecules may or may not be part of a single contiguous DNA molecule and may or may not be adjacent. For example, a promoter is operably linked to a transcribable DNA molecule if the promoter modulates transcription of the transcribable DNA molecule of interest in a cell. A leader, for example, is operably linked to DNA sequence when it is capable of affecting the transcription or translation of the DNA sequence.
The constructs of the invention may be provided, in one embodiment, as double tumor-inducing (Ti) plasmid border constructs that have the right border (RB or AGRtu.RB) and left border (LB or AGRtu.LB) regions of the Ti plasmid isolated from Agrobacterium tumefaciens comprising a T-DNA that, along with transfer molecules provided by the A. tumefaciens cells, permit the integration of the T-DNA into the genome of a plant cell (see, e.g., U.S. Pat. No. 6,603,061). The constructs may also contain the plasmid backbone DNA segments that provide replication function and antibiotic selection in bacterial cells, e.g., an Escherichia coli origin of replication such as ori322, a broad host range origin of replication such as oriV or oriRi, and a coding region for a selectable marker such as Spec/Strp that encodes for Tn7 aminoglycoside adenyltransferase (aadA) conferring resistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectable marker gene. For plant transformation, the host bacterial strain is often A. tumefaciens AB1, C58, or LBA4404; however, other strains known to those skilled in the art of plant transformation can function in the invention.
Methods are known in the art for assembling and introducing constructs into a cell in such a manner that the transcribable DNA molecule is transcribed into a functional mRNA molecule that is translated and expressed as a protein. For the practice of the invention, conventional compositions and methods for preparing and using constructs and host cells are well known to one skilled in the art. Typical vectors useful for expression of nucleic acids in higher plants are well known in the art and include vectors derived from the Ti plasmid of Agrobacterium tumefaciens and the pCaMVCN transfer control vector.
Various regulatory elements may be included in a construct, including any of those provided herein. Any such regulatory elements may be provided in combination with other regulatory elements. Such combinations can be designed or modified to produce desirable regulatory features. In one embodiment, constructs of the invention comprise at least one regulatory element operably linked to a transcribable DNA molecule operably linked to a 3′ UTR.
Constructs of the invention may include any promoter or leader provided herein or known in the art. For example, a promoter of the invention may be operably linked to a heterologous non-translated 5′ leader such as one derived from a heat shock protein gene. Alternatively, a leader of the invention may be operably linked to a heterologous promoter such as the Cauliflower Mosaic Virus 35S transcript promoter.
Expression cassettes may also include a transit peptide coding sequence that encodes a peptide that is useful for sub-cellular targeting of an operably linked protein, particularly to a chloroplast, leucoplast, or other plastid organelle; mitochondria; peroxisome; vacuole; or an extracellular location. Many chloroplast-localized proteins are expressed from nuclear genes as precursors and are targeted to the chloroplast by a chloroplast transit peptide (CTP). Examples of such isolated chloroplast proteins include, but are not limited to, those associated with the small subunit (SSU) of ribulose-1,5,-bisphosphate carboxylase, ferredoxin, ferredoxin oxidoreductase, the light-harvesting complex protein I and protein II, thioredoxin F, and enolpyruvyl shikimate phosphate synthase (EPSPS). Chloroplast transit peptides are described, for example, in U.S. Pat. No. 7,193,133. It has been demonstrated that non-chloroplast proteins may be targeted to the chloroplast by the expression of a heterologous CTP operably linked to the transgene encoding a non-chloroplast proteins.
Transcribable DNA Molecules
As used herein, the term “transcribable DNA molecule” refers to any DNA molecule capable of being transcribed into a RNA molecule, including, but not limited to, those having protein coding sequences and those producing RNA molecules having sequences useful for gene suppression. The type of DNA molecule can include, but is not limited to, a DNA molecule from the same plant, a DNA molecule from another plant, a DNA molecule from a different organism, or a synthetic DNA molecule, such as a DNA molecule containing an antisense message of a gene, or a DNA molecule encoding an artificial, synthetic, or otherwise modified version of a transgene. Exemplary transcribable DNA molecules for incorporation into constructs of the invention include, e.g., DNA molecules or genes from a species other than the species into which the DNA molecule is incorporated or genes that originate from, or are present in, the same species, but are incorporated into recipient cells by genetic engineering methods rather than classical breeding techniques.
A “transgene” refers to a transcribable DNA molecule heterologous to a host cell at least with respect to its location in the host cell genome and/or a transcribable DNA molecule artificially incorporated into a host cell's genome in the current or any prior generation of the cell.
A regulatory element, such as a promoter of the invention, may be operably linked to a transcribable DNA molecule that is heterologous with respect to the regulatory element. As used herein, the term “heterologous” refers to the combination of two or more DNA molecules when such a combination is not normally found in nature. For example, the two DNA molecules may be derived from different species and/or the two DNA molecules may be derived from different genes, e.g., different genes from the same species or the same genes from different species. A regulatory element is thus heterologous with respect to an operably linked transcribable DNA molecule if such a combination is not normally found in nature, i.e., the transcribable DNA molecule does not naturally occur operably linked to the regulatory element.
The transcribable DNA molecule may generally be any DNA molecule for which expression of a transcript is desired. Such expression of a transcript may result in translation of the resulting mRNA molecule, and thus protein expression. Alternatively, for example, a transcribable DNA molecule may be designed to ultimately cause decreased expression of a specific gene or protein. In one embodiment, this may be accomplished by using a transcribable DNA molecule that is oriented in the antisense direction. One of ordinary skill in the art is familiar with using such antisense technology. Any gene may be negatively regulated in this manner, and, in one embodiment, a transcribable DNA molecule may be designed for suppression of a specific gene through expression of a dsRNA, siRNA or miRNA molecule.
Thus, one embodiment of the invention is a recombinant DNA molecule comprising a regulatory element of the invention, such as those provided as SEQ ID NOs: 1-98 and 168-171, operably linked to a heterologous transcribable DNA molecule so as to modulate transcription of the transcribable DNA molecule at a desired level or in a desired pattern when the construct is integrated in the genome of a transgenic plant cell. In one embodiment, the transcribable DNA molecule comprises a protein-coding region of a gene and in another embodiment the transcribable DNA molecule comprises an antisense region of a gene.
Genes of Agronomic Interest
A transcribable DNA molecule may be a gene of agronomic interest. As used herein, the term “gene of agronomic interest” refers to a transcribable DNA molecule that, when expressed in a particular plant tissue, cell, or cell type, confers a desirable characteristic. The product of a gene of agronomic interest may act within the plant in order to cause an effect upon the plant morphology, physiology, growth, development, yield, grain composition, nutritional profile, disease or pest resistance, and/or environmental or chemical tolerance or may act as a pesticidal agent in the diet of a pest that feeds on the plant. In one embodiment of the invention, a regulatory element of the invention is incorporated into a construct such that the regulatory element is operably linked to a transcribable DNA molecule that is a gene of agronomic interest. In a transgenic plant containing such a construct, the expression of the gene of agronomic interest can confer a beneficial agronomic trait. A beneficial agronomic trait may include, for example, but is not limited to, herbicide tolerance, insect control, modified yield, disease resistance, pathogen resistance, modified plant growth and development, modified starch content, modified oil content, modified fatty acid content, modified protein content, modified fruit ripening, enhanced animal and human nutrition, biopolymer productions, environmental stress resistance, pharmaceutical peptides, improved processing qualities, improved flavor, hybrid seed production utility, improved fiber production, and desirable biofuel production.
Examples of genes of agronomic interest known in the art include those for herbicide resistance (U.S. Pat. Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633,435; and 5,463,175), increased yield (U.S. Pat. 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. Pat. 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 resistance (U.S. Pat. Nos. 6,653,280; 6,573,361; 6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,121,436; 6,316,407; and 6,506,962), virus resistance (U.S. Pat. Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; and 5,304,730), nematode resistance (U.S. Pat. No. 6,228,992), bacterial disease resistance (U.S. Pat. No. 5,516,671), plant growth and development (U.S. Pat. Nos. 6,723,897 and 6,518,488), starch production (U.S. Pat. Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), modified oils production (U.S. Pat. Nos. 6,444,876; 6,426,447; and 6,380,462), high oil production (U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008; and 6,476,295), modified fatty acid content (U.S. Pat. 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. Pat. No. 6,380,466), fruit ripening (U.S. Pat. No. 5,512,466), enhanced animal and human nutrition (U.S. Pat. Nos. 6,723,837; 6,653,530; 6,5412,59; 5,985,605; and 6,171,640), biopolymers (U.S. Pat. Nos. USRE37,543; 6,228,623; and 5,958,745, and 6,946,588), environmental stress resistance (U.S. Pat. No. 6,072,103), pharmaceutical peptides and secretable peptides (U.S. Pat. Nos. 6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processing traits (U.S. Pat. No. 6,476,295), improved digestibility (U.S. Pat. No. 6,531,648) low raffinose (U.S. Pat. No. 6,166,292), industrial enzyme production (U.S. Pat. No. 5,543,576), improved flavor (U.S. Pat. No. 6,011,199), nitrogen fixation (U.S. Pat. No. 5,229,114), hybrid seed production (U.S. Pat. No. 5,689,041), fiber production (U.S. Pat. Nos. 6,576,818; 6,271,443; 5,981,834; and 5,869,720) and biofuel production (U.S. Pat. No. 5,998,700).
Alternatively, a gene of agronomic interest can affect the above mentioned plant characteristics or phenotypes by encoding a RNA molecule that causes the targeted modulation of gene expression of an endogenous gene, for example by antisense (see, e.g. U.S. Pat. No. 5,107,065); inhibitory RNA (“RNAi,” including modulation of gene expression by miRNA-, siRNA-, trans-acting siRNA-, and phased sRNA-mediated mechanisms, e.g., as described in published applications U.S. 2006/0200878 and U.S. 2008/0066206, and in U.S. patent application Ser. No. 11/974,469); or cosuppression-mediated mechanisms. The RNA could also be a catalytic RNA molecule (e.g., a ribozyme or a riboswitch; see, e.g., U.S. 2006/0200878) engineered to cleave a desired endogenous mRNA product. Methods are known in the art for constructing and introducing constructs into a cell in such a manner that the transcribable DNA molecule is transcribed into a molecule that is capable of causing gene suppression.
Expression of a transcribable DNA molecule in a plant cell can also be used to suppress plant pests feeding on the plant cell, for example, compositions isolated from coleopteran pests and compositions isolated from nematode pests. Plant pests include, but are not limited to, arthropod pests, nematode pests, and fungal or microbial pests.
Selectable Markers
Selectable marker transgenes may also be used with the regulatory elements of the invention. As used herein the term “selectable marker transgene” refers to any transcribable DNA molecule whose expression in a transgenic plant, tissue or cell, or lack thereof, can be screened for or scored in some way. Selectable marker genes, and their associated selection and screening techniques, for use in the practice of the invention are known in the art and include, but are not limited to, transcribable DNA molecules encoding β-glucuronidase (GUS), green fluorescent protein (GFP), proteins that confer antibiotic resistance, and proteins that confer herbicide tolerance.
Cell Transformation
The invention is also directed to a method of producing transformed cells and plants that comprise one or more regulatory elements operably linked to a transcribable DNA molecule.
The term “transformation” refers to the introduction of a DNA molecule into a recipient host. As used herein, the term “host” refers to bacteria, fungi, or plants, including any cells, tissues, organs, or progeny of the bacteria, fungi, or plants. Plant tissues and cells of particular interest include protoplasts, calli, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.
As used herein, the term “transformed” refers to a cell, tissue, organ, or organism into which a foreign DNA molecule, such as a construct, has been introduced. The introduced DNA molecule may be integrated into the genomic DNA of the recipient cell, tissue, organ, or organism such that the introduced DNA molecule is inherited by subsequent progeny. A “transgenic” or “transformed” cell or organism may also includes progeny of the cell or organism and progeny produced from a breeding program employing such a transgenic organism as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a foreign DNA molecule. The introduced DNA molecule may also be transiently introduced into the recipient cell such that the introduced DNA molecule is not inherited by subsequent progeny. The term “transgenic” refers to a bacterium, fungus, or plant containing one or more heterologous DNA molecules.
There are many methods well known to those of skill in the art for introducing DNA molecules into plant cells. The process generally comprises the steps of selecting a suitable host cell, transforming the host cell with a vector, and obtaining the transformed host cell. Methods and materials for transforming plant cells by introducing a plant construct into a plant genome in the practice of this invention can include any of the well-known and demonstrated methods. Suitable methods include, but are not limited to, bacterial infection (e.g., Agrobacterium), binary BAC vectors, direct delivery of DNA (e.g., by PEG-mediated transformation, desiccation/inhibition-mediated DNA uptake, electroporation, agitation with silicon carbide fibers, and acceleration of DNA coated particles), among others.
Host cells may be any cell or organism, such as a plant cell, algal cell, algae, fungal cell, fungi, bacterial cell, or insect cell. In specific embodiments, the host cells and transformed cells may include cells from crop plants.
A transgenic plant subsequently may be regenerated from a transgenic plant cell of the invention. Using conventional breeding techniques or self-pollination, seed may be produced from this transgenic plant. Such seed, and the resulting progeny plant grown from such seed, will contain the recombinant DNA molecule of the invention, and therefore will be transgenic.
Transgenic plants of the invention can be self-pollinated to provide seed for homozygous transgenic plants of the invention (homozygous for the recombinant DNA molecule) or crossed with non-transgenic plants or different transgenic plants to provide seed for heterozygous transgenic plants of the invention (heterozygous for the recombinant DNA molecule). Both such homozygous and heterozygous transgenic plants are referred to herein as “progeny plants.” Progeny plants are transgenic plants descended from the original transgenic plant and containing the recombinant DNA molecule of the invention. Seeds produced using a transgenic plant of the invention can be harvested and used to grow generations of transgenic plants, i.e., progeny plants, of the invention, comprising the construct of this invention and expressing a gene of agronomic interest. Descriptions of breeding methods that are commonly used for different crops can be found in one of several reference books, see, e.g., Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U. of CA, Davis, Calif., 50-98 (1960); Simmonds, Principles of Crop Improvement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen, Plant breeding Perspectives, Wageningen (ed), Center for Agricultural Publishing and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses, 2nd Edition, Monograph, 16:249 (1987); Fehr, Principles of Variety Development, Theory and Technique, (Vol. 1) and Crop Species Soybean (Vol. 2), Iowa State Univ., Macmillan Pub. Co., NY, 360-376 (1987).
The transformed plants may be analyzed for the presence of the gene or genes of interest and the expression level and/or profile conferred by the regulatory elements of the invention. Those of skill in the art are aware of the numerous methods available for the analysis of transformed plants. For example, methods for plant analysis include, but are not limited to, Southern blots or northern blots, PCR-based approaches, biochemical analyses, phenotypic screening methods, field evaluations, and immunodiagnostic assays. The expression of a transcribable DNA molecule can be measured using TaqMan® (Applied Biosystems, Foster City, Calif.) 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, Wis.) reagents and methods as described by the manufacturer can be used to evaluate transgene expression.
The invention also provides for parts of a plant of the invention. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovule, and pollen. Plant parts of the invention may be viable, nonviable, regenerable, and/or non-regenerable. The invention also includes and provides transformed plant cells comprising a DNA molecule of the invention. The transformed or transgenic plant cells of the invention include regenerable and/or non-regenerable plant cells.
The invention may be more readily understood through reference to the following examples, which are provided by way of illustration, and are not intended to be limiting of the invention, unless specified. It should be appreciated by those of skill in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention, therefore all matter set forth or shown in the accompanying drawings is to be interpreted as illustrative and not in a limiting sense.
EXAMPLES
Example 1
Identification and Cloning of Regulatory Elements
Novel ubiquitin regulatory elements, or regulatory expression element group (EXP) sequences, were identified and isolated from genomic DNA of the monocot Cloud grass (Agrostis nebulosa), giant reed (Arundo donax), Blue grama (Bouteloua gracilis), Chinese silvergrass (Miscanthus sinesis), Little bluestem (Schizachyium scoparium), Yellow Indiangrass (Sorghastrum nutans) and Coix (Coix lacryma-jobi).
Ubiquitin 1 transcript sequences were identified from each of the above species. The 5′ untranslated region (5′ UTR) of each of the Ubiquitin 1 transcripts was used to design primers to amplify the corresponding regulatory elements for the identified Ubiquitin gene, which comprises a promoter, leader (5′ UTR), and first intron operably linked. The primers were used with GenomeWalkerTM (Clontech Laboratories, Inc, Mountain View, Calif.) libraries constructed following the manufacturer's protocol to clone the 5′ region of the corresponding genomic DNA sequence. Ubiquitin regulatory elements were also isolated from the monocots Setaria italica, Setaria viridis, and Zea mays subsp. Mexicana (Teosinte) using GenomeWalkerTM libraries as described above. In addition, ubiquitin regulatory elements were isolated from the monocot Sorghum bicolor using public sequences that are homologs to the Ubiquitin 4, 6, and 7 genes.
Using the identified sequences, a bioinformatic analysis was conducted to identify regulatory elements within the amplified DNA. Using the results of this analysis, regulatory elements were defined within the DNA sequences and primers designed to amplify the regulatory elements. The corresponding DNA molecule for each regulatory element was amplified using standard polymerase chain reaction (PCR) conditions with primers containing unique restriction enzyme sites and genomic DNA isolated from A. nebulosa, A donax, B. gracilis, M. sinesis, S. scoparium, S. nutans, and C. lacryma-jobi. The resulting DNA fragments were ligated into base plant expression vectors and sequenced. An analysis of the regulatory element transcription start site (TSS) and intron/exon splice junctions was then done using transformed plant protoplasts. Briefly, the protoplasts were transformed with the plant expression vectors comprising the cloned DNA fragments operably linked to a heterologous transcribable DNA molecule and the 5′ RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Invitrogen, Carlsbad, Calif. 92008) was used to confirm the regulatory element TSS and intron/exon splice junctions by analyzing the sequence of the messenger RNA (mRNA) transcripts produced thereby.
DNA sequences of the identified EXPs are provided herein as SEQ ID NOs: 1, 5, 7, 9, 13, 16, 18, 19, 21, 23, 27, 30, 32, 34, 38, 41, 43, 45, 49, 52, 55, 58, 60, 62, 66, 70, 72, 74, 76, 78, 82, 84, 86, 88, 92, 95, 97, 99, 103, 106, 108, 110, 114, 116, 118, 120, 122, 126, 128, 132, 134, 138, 140, 144, 148, 150 and 168, as listed in Table 1 below. Promoter sequences are provided herein as SEQ ID NOs: 2, 6, 8, 10, 14, 17, 22, 24, 28, 31, 33, 35, 39, 42, 44, 46, 50, 53, 56, 61, 63, 67, 71, 73, 75, 77, 79, 83, 85, 87, 89, 93, 96, 98, 100, 104, 107, 109, 111, 117, 119, 121, 123, 129, 135, 141, 145, 151 and 169. Leader sequences are provided herein as SEQ ID NOs: 3, 11, 25, 36, 47, 64, 68, 80, 90, 101, 112, 124, 130, 136, 142, 146, 152 and 170. Intron sequences are provided herein as SEQ ID NOs: 4, 12, 15, 20, 26, 29, 37, 40, 48, 51, 54, 57, 59, 65, 69, 81, 91, 94, 102, 105, 113, 115, 125, 127, 131, 133, 137, 139, 143, 147, 149, 153 and 171.







TABLE 1







Regulatory expression element groups (“EXPs”), promoters, enhancers,


leaders and introns isolated from various grass species.












SEQ


Description and/or regulatory elements of EXP linked in 5′ → 3′


Description
ID NO:
Size (bp)
Genus/Species
direction (SEQ ID NOs):














EXP-AGRne.Ubq1:1:7
1
3143
A. nebulosa
EXP: P-AGRne.Ubq1-1:1:5 (SEQ ID NO: 2); L-AGRne.Ubq1-1:1:1






(SEQ ID NO: 3); I-AGRne.Ubq1-1:1:3 (SEQ ID NO: 4)


P-AGRne.Ubq1-1:1:5
2
2005
A. nebulosa
Promoter


L-AGRne.Ubq1-1:1:1
3
85
A. nebulosa
Leader


I-AGRne.Ubq1-1:1:3
4
1053
A. nebulosa
Intron


EXP-AGRne.Ubq1:1:8
5
2137
A. nebulosa
EXP: P-AGRne.Ubq1-1:1:4 (SEQ ID NO: 6); L-AGRne.Ubq1-1:1:1






(SEQ ID NO: 3); I-AGRne.Ubq1-1:1:3 (SEQ ID NO: 4)


P-AGRne.Ubq1-1:1:4
6
999
A. nebulosa
Promoter


EXP-AGRne.Ubq1:1:9
7
1900
A. nebulosa
EXP: P-AGRne.Ubq1-1:1:6 (SEQ ID NO: 8); L-AGRne.Ubq1-1:1:1






(SEQ ID NO: 3); I-AGRne.Ubq1-1:1:3 (SEQ ID NO: 4)


P-AGRne.Ubq1-1:1:6
8
762
A. nebulosa
Promoter


EXP-ARUdo.Ubq1:1:4
9
5068
A. donax
EXP: P-ARUdo.Ubq1-1:1:4 (SEQ ID NO: 10); L-ARUdo.Ubq1-1:1:1






(SEQ ID NO: 11); I-ARUdo.Ubq1-1:1:2 (SEQ ID NO: 12)


P-ARUdo.Ubq1-1:1:4
10
4114
A. donax
Promoter


L-ARUdo.Ubq1-1:1:1
11
85
A. donax
Leader


I-ARUdo.Ubq1-1:1:2
12
869
A. donax
Intron


EXP-ARUdo.Ubq1:1:8
13
2969
A. donax
EXP: P-ARUdo.Ubq1-1:1:5 (SEQ ID NO: 14); L-ARUdo.Ubq1-1:1:1






(SEQ ID NO: 11); I-ARUdo.Ubq1-1:1:3 (SEQ ID NO: 15)


P-ARUdo.Ubq1-1:1:5
14
2012
A. donax
Promoter


I-ARUdo.Ubq1-1:1:3
15
872
A. donax
Intron


EXP-ARUdo.Ubq1:1:6
16
1954
A. donax
EXP: P-ARUdo.Ubq1-1:1:6 (SEQ ID NO: 17); L-ARUdo.Ubq1-1:1:1






(SEQ ID NO: 11); I-ARUdo.Ubq1-1:1:2 (SEQ ID NO: 12)


P-ARUdo.Ubq1-1:1:6
17
1000
A. donax
Promoter


EXP-ARUdo.Ubq1:1:9
18
1957
A. donax
EXP: P-ARUdo.Ubq1-1:1:6 (SEQ ID NO: 17); L-ARUdo.Ubq1-1:1:1






(SEQ ID NO: 11); I-ARUdo.Ubq1-1:1:3 (SEQ ID NO: 15)


EXP-ARUdo.Ubq1:1:12
19
1957
A. donax
EXP: P-ARUdo.Ubq1-1:1:6 (SEQ ID NO: 17); L-ARUdo.Ubq1-1:1:1






(SEQ ID NO: 11); I-ARUdo.Ubq1-1:1:4 (SEQ ID NO: 20)


I-ARUdo.Ubq1-1:1:4
20
872
A. donax
Intron


EXP-ARUdo.Ubq1:1:11
21
1712
A. donax
EXP: P-ARUdo.Ubq1-1:1:8 (SEQ ID NO: 22); L-ARUdo.Ubq1-1:1:1






(SEQ ID NO: 11); I-ARUdo.Ubq1-1:1:3 (SEQ ID NO: 15)


P-ARUdo.Ubq1-1:1:8
22
755
A. donax
Promoter


EXP-ARUdo.Ubq2:1:4
23
3276
A. donax
EXP: P-ARUdo.Ubq2-1:1:4 (SEQ ID NO: 24); L-ARUdo.Ubq2-1:1:1






(SEQ ID NO: 25); I-ARUdo.Ubq2-1:1:1 (SEQ ID NO: 26)


P-ARUdo.Ubq2-1:1:4
24
2033
A. donax
Promoter


L-ARUdo.Ubq2-1:1:1
25
88
A. donax
Leader


I-ARUdo.Ubq2-1:1:1
26
1155
A. donax
Intron


EXP-ARUdo.Ubq2:1:8
27
3250
A. donax
EXP: P-ARUdo.Ubq2-1:1:6 (SEQ ID NO: 28); L-ARUdo.Ubq2-1:1:1






(SEQ ID NO: 25); I-ARUdo.Ubq2-1:1:2 (SEQ ID NO: 29)


P-ARUdo.Ubq2-1:1:6
28
2004
A. donax
Promoter


I-ARUdo.Ubq2-1:1:2
29
1158
A. donax
Intron


EXP-ARUdo.Ubq2:1:9
30
2247
A. donax
EXP: P-ARUdo.Ubq2-1:1:5 (SEQ ID NO: 31); L-ARUdo.Ubq2-1:1:1






(SEQ ID NO: 25); I-ARUdo.Ubq2-1:1:2 (SEQ ID NO: 29)


P-ARUdo.Ubq2-1:1:5
31
1001
A. donax
Promoter


EXP-ARUdo.Ubq2:1:10
32
1942
A. donax
EXP: P-ARUdo.Ubq2-1:1:7 (SEQ ID NO: 33); L-ARUdo.Ubq2-1:1:1






(SEQ ID NO: 25); I-ARUdo.Ubq2-1:1:2 (SEQ ID NO: 29)


P-ARUdo.Ubq2-1:1:7
33
696
A. donax
Promoter


EXP-BOUgr.Ubq1:1:1
34
3511
B. gracilis
EXP: P-BOUgr.Ubq1-1:1:2 (SEQ ID NO: 35); L-BOUgr.Ubq1-1:1:1






(SEQ ID NO: 36); I-BOUgr.Ubq1-1:1:2 (SEQ ID NO: 37)


P-BOUgr.Ubq1-1:1:2
35
2371
B. gracilis
Promoter


L-BOUgr.Ubq1-1:1:1
36
86
B. gracilis
Leader


I-BOUgr.Ubq1-1:1:2
37
1054
B. gracilis
Intron


EXP-BOUgr.Ubq1:1:6
38
3142
B. gracilis
EXP: P-BOUgr.Ubq1-1:1:3 (SEQ ID NO: 39); L-BOUgr.Ubq1-1:1:1






(SEQ ID NO: 36); I-BOUgr.Ubq1-1:1:3 (SEQ ID NO: 40)


P-BOUgr.Ubq1-1:1:3
39
1999
B. gracilis
Promoter


I-BOUgr.Ubq1-1:1:3
40
1057
B. gracilis
Intron


EXP-BOUgr.Ubq1:1:7
41
2165
B. gracilis
EXP: P-BOUgr.Ubq1-1:1:5 (SEQ ID NO: 42); L-BOUgr.Ubq1-1:1:1






(SEQ ID NO: 36); I-BOUgr.Ubq1-1:1:3 (SEQ ID NO: 40)


P-BOUgr.Ubq1-1:1:5
42
1022
B. gracilis
Promoter


EXP-BOUgr.Ubq1:1:8
43
1903
B. gracilis
EXP: P-BOUgr.Ubq1-1:1:6 (SEQ ID NO: 44); L-BOUgr.Ubq1-1:1:1






(SEQ ID NO: 36); I-BOUgr.Ubq1-1:1:3 (SEQ ID NO: 40)


P-BOUgr.Ubq1-1:1:6
44
760
B. gracilis
Promoter


EXP-BOUgr.Ubq2:1:11
45
3234
B. gracilis
EXP: P-BOUgr.Ubq2-1:1:4 (SEQ ID NO: 46); L-BOUgr.Ubq2-1:1:1






(SEQ ID NO: 47); I-BOUgr.Ubq2-1:1:3 (SEQ ID NO: 48)


P-BOUgr.Ubq2-1:1:4
46
2100
B. gracilis
Promoter


L-BOUgr.Ubq2-1:1:1
47
91
B. gracilis
Leader


I-BOUgr.Ubq2-1:1:3
48
1043
B. gracilis
Intron


EXP-BOUgr.Ubq2:1:7
49
3176
B. gracilis
EXP: P-BOUgr.Ubq2-1:1:7 (SEQ ID NO: 50); L-BOUgr.Ubq2-1:1:1






(SEQ ID NO: 47); I-BOUgr.Ubq2-1:1:1 (SEQ ID NO: 51)


P-BOUgr.Ubq2-1:1:7
50
2043
B. gracilis
Promoter


I-BOUgr.Ubq2-1:1:1
51
1042
B. gracilis
Intron


EXP-BOUgr.Ubq2:1:14
52
3139
B. gracilis
EXP: P-BOUgr.Ubq2-1:1:5 (SEQ ID NO: 53); L-BOUgr.Ubq2-1:1:1






(SEQ ID NO: 47); I-BOUgr.Ubq2-1:1:4 (SEQ ID NO: 54)


P-BOUgr.Ubq2-1:1:5
53
2002
B. gracilis
Promoter


I-BOUgr.Ubq2-1:1:4
54
1046
B. gracilis
Intron


EXP-BOUgr.Ubq2:1:15
55
2160
B. gracilis
EXP: P-BOUgr.Ubq2-1:1:6 (SEQ ID NO: 56); L-BOUgr.Ubq2-1:1:1






(SEQ ID NO: 47); I-BOUgr.Ubq2-1:1:5 (SEQ ID NO: 57)


P-BOUgr.Ubq2-1:1:6
56
1024
B. gracilis
Promoter


I-BOUgr.Ubq2-1:1:5
57
1045
B. gracilis
Intron


EXP-BOUgr.Ubq2:1:16
58
2160
B. gracilis
EXP: P-BOUgr.Ubq2-1:1:6 (SEQ ID NO: 56); L-BOUgr.Ubq2-1:1:1






(SEQ ID NO: 47); I-BOUgr.Ubq2-1:1:6 (SEQ ID NO: 59)


I-BOUgr.Ubq2-1:1:6
59
1045
B. gracilis
Intron


EXP-BOUgr.Ubq2:1:17
60
1885
B. gracilis
EXP: P-BOUgr.Ubq2-1:1:8 (SEQ ID NO: 61); L-BOUgr.Ubq2-1:1:1






(SEQ ID NO: 47); I-BOUgr.Ubq2-1:1:6 (SEQ ID NO: 59)


P-BOUgr.Ubq2-1:1:8
61
749
B. gracilis
Promoter


EXP-MISsi.Ubq1:1:2
62
6813
M. sinesis
EXP: P-MISsi.Ubq1-1:1:2 (SEQ ID NO: 63); L-MISsi.Ubq1-1:1:1






(SEQ ID NO: 64); I-MISsi.Ubq1-1:1:1 (SEQ ID NO: 65)


P-MISsi.Ubq1-1:1:2
63
5359
M. sinesis
Promoter


L-MISsi.Ubq1-1:1:1
64
63
M. sinesis
Leader


I-MISsi.Ubq1-1:1:1
65
1391
M. sinesis
Intron


EXP-MISsi.Ubq1:1:9
66
4402
M. sinesis
EXP: P-MISsi.Ubq1-1:1:11 (SEQ ID NO: 67); L-MISsi.Ubq1-1:1:2






(SEQ ID NO: 68); I-MISsi.Ubq1-1:1:3 (SEQ ID NO: 69)


P-MISsi.Ubq1-1:1:11
67
2423
M. sinesis
Promoter


L-MISsi.Ubq1-1:1:2
68
55
M. sinesis
Leader


I-MISsi.Ubq1-1:1:3
69
1924
M. sinesis
Intron


EXP-MISsi.Ubq1:1:8
70
3426
M. sinesis
EXP: P-MISsi.Ubq1-1:1:10 (SEQ ID NO: 71); L-MISsi.Ubq1-1:1:2






(SEQ ID NO: 68); I-MISsi.Ubq1-1:1:3 (SEQ ID NO: 69)


P-MISsi.Ubq1-1:1:10
71
1447
M. sinesis
Promoter


EXP-MISsi.Ubq1:1:10
72
2878
M. sinesis
EXP: P-MISsi.Ubq1-1:1:13 (SEQ ID NO: 73); L-MISsi.Ubq1-1:1:2






(SEQ ID NO: 68); I-MISsi.Ubq1-1:1:3 (SEQ ID NO: 69)


P-MISsi.Ubq1-1:1:13
73
899
M. sinesis
Promoter


EXP-MISsi.Ubq1:1:11
74
2670
M. sinesis
EXP: P-MISsi.Ubq1-1:1:14 (SEQ ID NO: 75); L-MISsi.Ubq1-1:1:2






(SEQ ID NO: 68); I-MISsi.Ubq1-1:1:3 (SEQ ID NO: 69)


P-MISsi.Ubq1-1:1:14
75
691
M. sinesis
Promoter


EXP-MISsi.Ubq1:1:7
76
2485
M. sinesis
EXP: P-MISsi.Ubq1-1:1:9 (SEQ ID NO: 77); L-MISsi.Ubq1-1:1:2






(SEQ ID NO: 68); I-MISsi.Ubq1-1:1:3 (SEQ ID NO: 69)


P-MISsi.Ubq1-1:1:9
77
506
M. sinesis
Promoter


EXP-SCHsc.Ubq1:1:9
78
4079
S. scoparium
EXP: P-SCHsc.Ubq1-1:1:12 (SEQ ID NO: 79); L-SCHsc.Ubq1-1:1:3






(SEQ ID NO: 80); I-SCHsc.Ubq1-1:1:2 (SEQ ID NO: 81)


P-SCHsc.Ubq1-1:1:12
79
2831
S. scoparium
Promoter


L-SCHsc.Ubq1-1:1:3
80
95
S. scoparium
Leader


I-SCHsc.Ubq1-1:1:2
81
1153
S. scoparium
Intron


EXP-SCHsc.Ubq1:1:8
82
3281
S. scoparium
EXP: P-SCHsc.Ubq1-1:1:11 (SEQ ID NO: 83); L-SCHsc.Ubq1-1:1:3






(SEQ ID NO: 80); I-SCHsc.Ubq1-1:1:2 (SEQ ID NO: 81)


P-SCHsc.Ubq1-1:1:11
83
2033
S. scoparium
Promoter


EXP-SCHsc.Ubq1:1:7
84
2294
S. scoparium
EXP: P-SCHsc.Ubq1-1:1:10 (SEQ ID NO: 85); L-SCHsc.Ubq1-1:1:3






(SEQ ID NO: 80); I-SCHsc.Ubq1-1:1:2 (SEQ ID NO: 81)


P-SCHsc.Ubq1-1:1:10
85
1046
S. scoparium
Promoter


EXP-SCHsc.Ubq1:1:10
86
1795
S. scoparium
EXP: P-SCHsc.Ubq1-1:1:14 (SEQ ID NO: 87); L-SCHsc.Ubq1-1:1:3






(SEQ ID NO: 80); I-SCHsc.Ubq1-1:1:2 (SEQ ID NO: 81)


P-SCHsc.Ubq1-1:1:14
87
547
S. scoparium
Promoter


EXP-SORnu.Ubq1:1:2
88
3357
S. nutans
EXP: P-SORnu.Ubq1-1:1:4 (SEQ ID NO: 89); L-SORnu.Ubq1-1:1:1






(SEQ ID NO: 90); I-SORnu.Ubq1-1:1:1 (SEQ ID NO: 91)


P-SORnu.Ubq1-1:1:4
89
2218
S. nutans
Promoter


L-SORnu.Ubq1-1:1:1
90
86
S. nutans
Leader


I-SORnu.Ubq1-1:1:1
91
1053
S. nutans
Intron


EXP-SORnu.Ubq1:1:6
92
3106
S. nutans
EXP: P-SORnu.Ubq1-1:1:5 (SEQ ID NO: 93); L-SORnu.Ubq1-1:1:1






(SEQ ID NO: 90); I-SORnu.Ubq1-1:1:2 (SEQ ID NO: 94)


P-SORnu.Ubq1-1:1:5
93
1964
S. nutans
Promoter


I-SORnu.Ubq1-1:1:2
94
1056
S. nutans
Intron


EXP-SORnu.Ubq1:1:7
95
2165
S. nutans
EXP: P-SORnu.Ubq1-1:1:6 (SEQ ID NO: 96); L-SORnu.Ubq1-1:1:1






(SEQ ID NO: 90); I-SORnu.Ubq1-1:1:2 (SEQ ID NO: 94)


P-SORnu.Ubq1-1:1:6
96
1023
S. nutans
Promoter


EXP-SORnu.Ubq1:1:8
97
1866
S. nutans
EXP: P-SORnu.Ubq1-1:1:7 (SEQ ID NO: 98); L-SORnu.Ubq1-1:1:1






(SEQ ID NO: 90); I-SORnu.Ubq1-1:1:2 (SEQ ID NO: 94)


P-SORnu.Ubq1-1:1:7
98
724
S. nutans
Promoter


EXP-SETit.Ubq1:1:10
99
2625
S. italica
EXP: P-SETit.Ubq1-1:1:4 (SEQ ID NO: 100); L-SETit.Ubq1-1:1:1






(SEQ ID NO: 101); I-SETit.Ubq1-1:1:3 (SEQ ID NO: 102)


P-SETit.Ubq1-1:1:4
100
1492
S. italica
Promoter


L-SETit.Ubq1-1:1:1
101
127
S. italica
Leader


I-SETit.Ubq1-1:1:3
102
1006
S. italica
Intron


EXP-SETit.Ubq1:1:5
103
2625
S. italica
EXP: P-SETit.Ubq1-1:1:1 (SEQ ID NO: 104); L-SETit.Ubq1-1:1:1






(SEQ ID NO: 101); I-SETit.Ubq1-1:1:2 (SEQ ID NO: 105)


P-SETit.Ubq1-1:1:1
104
1492
S. italica
Promoter


I-SETit.Ubq1-1:1:2
105
1006
S. italica
Intron


EXP-SETit.Ubq1:1:7
106
2167
S. italica
EXP: P-SETit.Ubq1-1:1:2 (SEQ ID NO: 107); L-SETit.Ubq1-1:1:1






(SEQ ID NO: 101); I-SETit.Ubq1-1:1:2 (SEQ ID NO: 105)


P-SETit.Ubq1-1:1:2
107
1034
S. italica
Promoter


EXP-SETit.Ubq1:1:6
108
1813
S. italica
EXP: P-SETit.Ubq1-1:1:3 (SEQ ID NO: 109); L-SETit.Ubq1-1:1:1






(SEQ ID NO: 101); I-SETit.Ubq1-1:1:2 (SEQ ID NO: 105)


P-SETit.Ubq1-1:1:3
109
680
S. italica
Promoter


EXP-Sv.Ubq1:1:7
110
2634
S. viridis
EXP: P-Sv.Ubq1-1:1:1 (SEQ ID NO: 111); L-Sv.Ubq1-1:1:2 (SEQ ID






NO: 112); I-Sv.Ubq1-1:1:2 (SEQ ID NO: 113)


P-Sv.Ubq1-1:1:1
111
1493
S. viridis
Promoter


L-Sv.Ubq1-1:1:2
112
127
S. viridis
Leader


I-Sv.Ubq1-1:1:2
113
1014
S. viridis
Intron


EXP-Sv.Ubq1:1:11
114
2634
S. viridis
EXP: P-Sv.Ubq1-1:1:1 (SEQ ID NO: 111); L-Sv.Ubq1-1:1:2 (SEQ ID






NO: 112); I-Sv.Ubq1-1:1:3 (SEQ ID NO: 115)


I-Sv.Ubq1-1:1:3
115
1014
S. viridis
Intron


EXP-Sv.Ubq1:1:8
116
2176
S. viridis
EXP: P-Sv.Ubq1-1:1:2 (SEQ ID NO: 117); L-Sv.Ubq1-1:1:2 (SEQ ID






NO: 112); I-Sv.Ubq1-1:1:2 (SEQ ID NO: 113)


P-Sv.Ubq1-1:1:2
117
1035
S. viridis
Promoter


EXP-Sv.Ubq1:1:10
118
1822
S. viridis
EXP: P-Sv.Ubq1-1:1:4 (SEQ ID NO: 119); L-Sv.Ubq1-1:1:2 (SEQ ID






NO: 112); I-Sv.Ubq1-1:1:2 (SEQ ID NO: 113)


P-Sv.Ubq1-1:1:4
119
681
S. viridis
Promoter


EXP-Sv.Ubq1:1:12
120
1822
S. viridis
EXP: P-Sv.Ubq1-1:1:3 (SEQ ID NO: 121); L-Sv.Ubq1-1:1:2 (SEQ ID






NO: 112); I-Sv.Ubq1-1:1:3 (SEQ ID NO: 115)


P-Sv.Ubq1-1:1:3
121
681
S. viridis
Promoter


EXP-Zm.UbqM1:1:6
122
1925
Z. mays subsp. Mexicana
EXP: P-Zm.UbqM1-1:1:1 (SEQ ID NO: 123); L-Zm.UbqM1-1:1:1


(Allele-1)



(SEQ ID NO: 124); I-Zm.UbqM1-1:1:13 (SEQ ID NO: 125)


P-Zm.UbqM1-1:1:1 (Allele-
123
850
Z. mays subsp. Mexicana
Promoter


1)


L-Zm.UbqM1-1:1:1 (Allele-
124
78
Z. mays subsp. Mexicana
Leader


1)


I-Zm.UbqM1-1:1:13 (Allele-
125
997
Z. mays subsp. Mexicana
Intron


1)


EXP-Zm.UbqM1:1:10
126
1925
Z. mays subsp. Mexicana
EXP: P-Zm.UbqM1-1:1:1 (SEQ ID NO: 123); L-Zm.UbqM1-1:1:1


(Allele-1)



(SEQ ID NO: 124); I-Zm.UbqM1-1:1:17 (SEQ ID NO: 127)


I-Zm.UbqM1-1:1:17 (Allele-
127
997
Z. mays subsp. Mexicana
Intron


1)


EXP-Zm.UbqM1:1:7
128
1974
Z. mays subsp. Mexicana
EXP: P-Zm.UbqM1-1:1:4 (SEQ ID NO: 129); L-Zm.UbqM1-1:1:5


(Allele-2)



(SEQ ID NO: 130); I-Zm.UbqM1-1:1:14 (SEQ ID NO: 131)


P-Zm.UbqM1-1:1:4 (Allele-
129
887
Z. mays subsp. Mexicana
Promoter


2)


L-Zm.UbqM1-1:1:5 (Allele-
130
77
Z. mays subsp. Mexicana
Leader


2)


I-Zm.UbqM1-1:1:14 (Allele-
131
1010
Z. mays subsp. Mexicana
Intron


2)


EXP-Zm.UbqM1:1:12
132
1974
Z. mays subsp. Mexicana
EXP: P-Zm.UbqM1-1:1:4 (SEQ ID NO: 129); L-Zm.UbqM1-1:1:5


(Allele-2)



(SEQ ID NO: 130); I-Zm.UbqM1-1:1:19 (SEQ ID NO: 133)


I-Zm.UbqM1-1:1:19 (Allele-
133
1010
Z. mays subsp. Mexicana
Intron


2)


EXP-Zm.UbqM1:1:8
134
2008
Z. mays subsp. Mexicana
EXP: P-Zm.UbqM1-1:1:5 (SEQ ID NO: 135); L-Zm.UbqM1-1:1:4


(Allele-2)



(SEQ ID NO: 136); I-Zm.UbqM1-1:1:15 (SEQ ID NO: 137)


P-Zm.UbqM1-1:1:5 (Allele-
135
877
Z. mays subsp. Mexicana
Promoter


2)


L-Zm.UbqM1-1:1:4 (Allele-
136
78
Z. mays subsp. Mexicana
Leader


2)


I-Zm.UbqM1-1:1:15 (Allele-
137
1053
Z. mays subsp. Mexicana
Intron


2)


EXP-Zm.UbqM1:1:11
138
2008
Z. mays subsp. Mexicana
EXP: P-Zm.UbqM1-1:1:5 (SEQ ID NO: 135); L-Zm.UbqM1-1:1:4


(Allele-2)



(SEQ ID NO: 136); I-Zm.UbqM1-1:1:18 (SEQ ID NO: 139)


I-Zm.UbqM1-1:1:18 (Allele-
139
1053
Z. mays subsp. Mexicana
Intron


2)


EXP-Sb.Ubq4:1:2
140
1635
S. bicolor
EXP: P-Sb.Ubq4-1:1:1 (SEQ ID NO: 141); L-Sb.Ubq4-1:1:1 (SEQ ID






NO: 142); I-Sb.Ubq4-1:1:2 (SEQ ID NO: 143)


P-Sb.Ubq4-1:1:1
141
401
S. bicolor
Promoter


L-Sb.Ubq4-1:1:1
142
154
S. bicolor
Leader


I-Sb.Ubq4-1:1:2
143
1080
S. bicolor
Intron


EXP-Sb.Ubq6:1:2
144
2067
S. bicolor
EXP: P-Sb.Ubq6-1:1:1 (SEQ ID NO: 145); L-Sb.Ubq6-1:1:1 (SEQ ID






NO: 146); I-Sb.Ubq6-1:1:2 (SEQ ID NO: 147)


P-Sb.Ubq6-1:1:1
145
855
S. bicolor
Promoter


L-Sb.Ubq6-1:1:1
146
136
S. bicolor
Leader


I-Sb.Ubq6-1:1:2
147
1076
S. bicolor
Intron


EXP-Sb.Ubq6:1:3
148
2067
S. bicolor
EXP: P-Sb.Ubq6-1:1:1 (SEQ ID NO: 145); L-Sb.Ubq6-1:1:1 (SEQ ID






NO: 146); I-Sb.Ubq6-1:1:3 (SEQ ID NO: 149)


I-Sb.Ubq6-1:1:3
149
1076
S. bicolor
Intron


EXP-Sb.Ubq7:1:2
150
2003
S. bicolor
EXP: P-Sb.Ubq7-1:1:1 (SEQ ID NO: 151); L-Sb.Ubq7-1:1:1 (SEQ ID






NO: 152); I-Sb.Ubq7-1:1:2 (SEQ ID NO: 153)


P-Sb.Ubq7-1:1:1
151
565
S. bicolor
Promoter


L-Sb.Ubq7-1:1:1
152
77
S. bicolor
Leader


I-Sb.Ubq7-1:1:2
153
1361
S. bicolor
Intron


EXP-Cl.Ubq10
168
1790
C. lacryma-jobi
EXP: P-Cl.UBQ10 (SEQ ID NO: 169); L-Cl.UBQ10 (SEQ ID NO:






170); I-Cl.UBQ10 (SEQ ID NO: 171)


P-Cl.Ubq10
169
481
C. lacryma-jobi
Promoter


L-Cl.Ubq10
170
93
C. lacryma-jobi
Leader


I-Cl.Ubq10
171
1216
C. lacryma-jobi
Intron









As shown in Table 1, for example, the regulatory EXP sequence designated EXP-AGRne.Ubq1:1:7 (SEQ ID NO: 1), with components isolated from A. nebulosa, comprises a promoter element, P-AGRne.Ubq1-1:1:5 (SEQ ID NO: 2), operably linked 5′ to a leader element, L-AGRne.Ubq1-1:1:1 (SEQ ID NO: 3), operably linked 5′ to an intron element, I-AGRne.Ubq1-1:1:3 (SEQ ID NO: 4). Other EXP sequences are linked similarly, as outlined in Table 1.
As shown in Table 1, the sequence listing, and FIGS. 1-8, variants of promoter sequences from A. nebulosa, A donax, B. gracilis, M. sinesis, S. scoparium, and S. nutans were engineered, which comprise shorter promoter fragments of, for instance, P-AGRne.Ubq1-1:1:5 (SEQ ID NO:2), P-ARUdo.Ubq1-1:1:4 (SEQ ID NO:10), or other respective promoters from other species, and for instance resulting in P-AGRne.Ubq1-1:1:4 (SEQ ID NO: 6) and P-ARUdo.Ubq1-1:1:5 (SEQ ID NO: 14), as well as other promoter fragments.
Also listed in Table 1 are three allelic variants isolated using the same primer sets designed for amplification of genomic DNA from Z. mays subsp. mexicana. Allelic variants of the Z. mays subsp. mexicana EXP sequences are comprised of DNA sequences that share some identity within various regions of other DNA sequences, but insertions, deletions, and nucleotide mismatches may also be apparent within each promoter, leader and/or intron of each of the EXP sequences. The EXP sequences designated EXP-Zm.UbqM1:1:6 (SEQ ID NO: 122) and EXP-Zm.UbqM1:1:10 (SEQ ID NO: 126) represent a first allele (Allele-1) of the Z. mays subsp. mexicana Ubq1 gene regulatory expression element group, with the only difference between the two EXP sequences occurring in the last 3′ nucleotides of each respective intron following the sequence 5′-AG-3′ of the 3′ intron splice junction. The EXP sequences designated EXP-Zm.UbqM1:1:7 (SEQ ID NO: 128) and EXP-Zm.UbqM1:1:12 (SEQ ID NO: 132) represent a second allele (Allele-2) of the Z. mays subsp. mexicana Ubq1 gene regulatory expression element group, with the only difference between the two EXP sequences occurring in the last 3′ nucleotides of each respective intron following the sequence 5′′-AG-3′ of the 3′ intron splice junction. The EXP sequences EXP-Zm.UbqM1:1:8 (SEQ ID NO: 134) and EXP-Zm.UbqM1:1:11 (SEQ ID NO: 138) represent a third allele (Allele-3) of the Z. mays subsp. mexicana Ubq1 gene regulatory expression element group, with the only difference between the two EXP sequences occurring in the last 3′ nucleotides of each respective intron following the sequence 5′-AG-3′ of the 3′ intron splice junction.
Example 2
Analysis of Regulatory Elements Driving GUS in Corn Protoplasts Using GUS Expression Cassette Amplicons
Corn leaf protoplasts were transformed with DNA amplicons derived from plant expression vectors containing an EXP sequence, driving expression of the β-glucuronidase transgene (GUS), and compared to leaf protoplasts in which expression of GUS is driven by known constitutive promoters in a series of experiments presented below.
In a first set of experiments, corn protoplast cells derived from leaf tissue were transformed as above with amplicons produced from amplification of GUS expression cassettes comprising plant expression vectors to compare expression of a transgene (GUS) driven by one of EXP-AGRne.Ubq1:1:7 (SEQ ID NO: 1), EXP-AGRne.Ubq1:1:8 (SEQ ID NO: 5), EXP-AGRne.Ubq1:1:9 (SEQ ID NO: 7), EXP-ARUdo.Ubq1:1:8 (SEQ ID NO: 13), EXP-ARUdo.Ubq1:1:9 (SEQ ID NO: 18), EXP-ARUdo.Ubq1:1:11 (SEQ ID NO: 20), EXP-ARUdo.Ubq2:1:8 (SEQ ID NO: 26), EXP-ARUdo.Ubq2:1:9 (SEQ ID NO: 29), EXP-ARUdo.Ubq2:1:10 (SEQ ID NO: 31), EXP-BOUgr.Ubq1:1:6 (SEQ ID NO: 37), EXP-BOUgr.Ubq1:1:7 (SEQ ID NO: 40), EXP-BOUgr.Ubq1:1:8 (SEQ ID NO: 42), EXP-BOUgr.Ubq2:1:14 (SEQ ID NO: 51), EXP-BOUgr.Ubq2:1:16 (SEQ ID NO: 57), EXP-BOUgr.Ubq2:1:17 (SEQ ID NO: 59), EXP-MISsi.Ubq1:1:8 (SEQ ID NO: 69), EXP-MISsi.Ubq1:1:10 (SEQ ID NO: 71), EXP-MISsi.Ubq1:1:11 (SEQ ID NO: 73), EXP-MISsi.Ubq1:1:7 (SEQ ID NO: 75), EXP-SCHsc.Ubq1:1:9 (SEQ ID NO: 77), EXP-SCHsc.Ubq1:1:7 (SEQ ID NO: 83), EXP-SCHsc.Ubq1:1:10 (SEQ ID NO: 85), EXP-SORnu.Ubq1:1:6 (SEQ ID NO: 91), EXP-SORnu.Ubq1:1:7 (SEQ ID NO: 94), EXP-SORnu.Ubq1:1:8 (SEQ ID NO: 96), EXP-SETit.Ubq1:1:5 (SEQ ID NO: 102), EXP-SETit.Ubq1:1:7 (SEQ ID NO: 105), EXP-SETit.Ubq1:1:6 (SEQ ID NO: 107), EXP-Sv.Ubq1:1:7 (SEQ ID NO: 109), EXP-Sv.Ubq1:1:8 (SEQ ID NO: 115), EXP-Sv.Ubq1:1:10 (SEQ ID NO: 117), EXP-Zm.UbqM1:1:6 (SEQ ID NO: 121), EXP-Zm.UbqM1:1:7 (SEQ ID NO: 127), EXP-Zm.UbqM1:1:8 (SEQ ID NO: 133), Exp-Sb.Ubq4:1:2 (SEQ ID NO: 139), and Exp-Sb.Ubq6:1:2 (SEQ ID NO: 143) with that of known constitutive promoters. Each EXP sequence comprising the amplification template from which the expression cassette amplicon is produced was cloned using methods known in the art into a plant expression vector shown in Table 2 below under the heading of “Amplicon Template.” The resulting plant expression vectors comprise a expression cassette comprised of a EXP sequence, operably linked 5′ to a coding sequence for GUS that either contains a processable intron (“GUS-2”, SEQ ID NO: 154), or a contiguous GUS coding sequence (“GUS-1”, SEQ ID NO: 153), operably linked 5′ to a 3′ UTR T-AGRtu.nos-1:1:13 (SEQ ID NO: 157) or T-Ta.Hsp17-1:1:1 (SEQ ID NO: 158). Amplicons were produced using methods known to those skilled in the art using the plasmid construct templates presented in Table 2 below. Briefly, a 5′ oligonucleotide primer was designed to anneal to the promoter sequence and a 3′ oligonucleotide primer, which anneals at the 3′ end of the 3′ UTR, was used for amplification of each expression cassette. Successive 5′ deletions were introduced into the promoter sequences comprising the expression cassettes, giving rise to different EXP sequences, by the use of different oligonucleotide primers which were designed to anneal at different positions within the promoter sequence comprising each amplicon template.







TABLE 2







GUS plant expression amplicons and corresponding plasmid construct amplicon


templates, EXP sequence, GUS coding sequence and 3′ UTR used


for transformation of corn leaf protoplasts.















SEQ
GUS



Amplicon
Amplicon

ID
Coding


ID
Template
EXP sequence
NO:
Sequence
3′ UTR















PCR0145942
pMON25455
EXP-Os.Act1:1:9
162
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145943
pMON65328
EXP-CaMV.35S-
161
GUS-2
T-




enh + Ta.Lhcb1 + Os.Act1:1:1


Ta.Hsp17-







1:1:1


PCR0145935
pMON140890
EXP-AGRne.Ubq1:1:7
1
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145827
pMON140890
EXP-AGRne.Ubq1:1:8
5
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145828
pMON140890
EXP-AGRne.Ubq1:1:9
7
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145939
pMON140894
EXP-ARUdo.Ubq1:1:8
13
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145837
pMON140894
EXP-ARUdo.Ubq1:1:9
18
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145838
pMON140894
EXP-ARUdo.Ubq1:1:11
21
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145940
pMON140895
EXP-ARUdo.Ubq2:1:8
27
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145841
pMON140895
EXP-ARUdo.Ubq2:1:9
30
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145842
pMON140895
EXP-ARUdo.Ubq2:1:10
32
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145936
pMON140891
EXP-BOUgr.Ubq1:1:6
38
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145829
pMON140891
EXP-BOUgr.Ubq1:1:7
41
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145831
pMON140891
EXP-BOUgr.Ubq1:1:8
43
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145937
pMON140892
EXP-BOUgr.Ubq2:1:14
52
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145833
pMON140892
EXP-BOUgr.Ubq2:1:16
58
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145836
pMON140892
EXP-BOUgr.Ubq2:1:17
60
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145898
pMON136265
EXP-MISsi.Ubq1:1:8
70
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145823
pMON136265
EXP-MISsi.Ubq1:1:10
72
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145824
pMON136265
EXP-MISsi.Ubq1:1:11
74
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145899
pMON136260
EXP-MISsi.Ubq1:1:7
76
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145894
pMON136262
EXP-SCHsc.Ubq1:1:9
78
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145895
pMON136257
EXP-SCHsc.Ubq1:1:7
84
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145813
pMON136257
EXP-SCHsc.Ubq1:1:10
86
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145938
pMON140893
EXP-SORnu.Ubq1:1:6
92
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145839
pMON140893
EXP-SORnu.Ubq1:1:7
95
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145840
pMON140893
EXP-SORnu.Ubq1:1:8
97
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145900
pMON140877
EXP-SETit.Ubq1:1:5
103
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145928
pMON140877
EXP-SETit.Ubq1:1:7
106
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145905
pMON140877
EXP-SETit.Ubq1:1:6
108
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145909
pMON140878
EXP-Sv.Ubq1:1:7
110
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145929
pMON140878
EXP-Sv.Ubq1:1:8
116
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145911
pMON140878
EXP-Sv.Ubq1:1:10
118
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145914
pMON140881
EXP-Zm.UbqM1:1:6
122
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145916
pMON140883
EXP-Zm.UbqM1:1:7
128
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145915
pMON140882
EXP-Zm.UbqM1:1:8
134
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145921
pMON140887
Exp-Sb.Ubq4:1:2
140
GUS-1
T-







AGRtu.nos-







1:1:13


PCR0145920
pMON140886
Exp-Sb.Ubq6:1:2
144
GUS-1
T-







AGRtu.nos-







1:1:13









Plasmid constructs listed as amplicon templates in Table 2 served as templates for amplification of transgene expression cassettes comprising the listed EXP sequences of Table 2. Control plasmids used to generate GUS transgene amplicons for comparison were constructed as previously described with the constitutive EXP sequences EXP-Os.Act1:1:9 (SEQ ID NO: 162) and EXP-CaMV.35S-enh+Ta.Lhcb1+Os.Act1:1:1 (SEQ ID NO: 161). An empty vector not designed for transgene expression was used as a negative control to assess background GUS and luciferase expression.
Two plasmids, for use in co-transformation and normalization of data, were also constructed using methods known in the art. Each plasmid contained a specific luciferase coding sequence that was driven by a constitutive EXP sequence. The plant vector pMON19437 comprises a expression cassette with a constitutive promoter operably linked 5′ to an intron, (EXP-CaMV.35S-enh+Zm.DnaK:1:1, SEQ ID NO: 163), operably linked 5′ to a firefly (Photinus pyralis) luciferase coding sequence (LUCIFERASE:1:3, SEQ ID NO: 156), operably linked 5′ to a 3′ UTR from the Agrobacterium tumefaciens nopaline synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 158). The plant vector pMON63934 comprises a expression cassette with a constitutive EXP sequence (EXP-CaMV.35S-enh-Lhcb1, SEQ ID NO: 164), operably linked 5′ to a sea pansy (Renilla reniformis) luciferase coding sequence (CR-Ren.hRenilla Lucife-0:0:1, SEQ ID NO: 157), operably linked 5′ to a 3′ UTR from the Agrobacterium tumefaciens nopaline synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 158).
Corn leaf protoplasts were transformed using a PEG-based transformation method, which is well known in the art. Protoplast cells were transformed with pMON19437 plasmid DNA, pMON63934 plasmid DNA, and the amplicons presented in Table 2, and were incubated overnight in total darkness. Measurements of both GUS and luciferase were conducted by placing aliquots of a lysed preparation 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, Wis.; see for example, Promega Notes Magazine, No: 57, 1996, p. 02). One or two transformations for each EXP sequence were performed and the mean expression values for each EXP sequence determined from several samples from each transformation experiment. Sample measurements were made using four replicates of each EXP sequence construct transformation, or alternatively, three replicates of each EXP sequence amplicon per one of two transformation experiments. The mean GUS and luciferase expression levels are provided in Table 3. In this table, the firefly luciferase values (e.g., from expression of pMON19437) are provided in the column labeled “FLuc” and the Renilla luciferase values are provided as in the column labeled “RLuc.”







TABLE 3







Mean GUS and Luciferase activity in transformed


corn leaf protoplast cells.












SEQ






ID


EXP Sequence
NO:
GUS
FLuc
RLuc














EMPTY

5
7840.58
205661


EXP-Os.Act1:1:9
162
1540.25
2671.83
105417


EXP-CaMV.35S-
161
12530.8
3067.08
137723


enh + Ta.Lhcb1 + Os.Act1:1:1


EXP-AGRne.Ubq1:1:7
1
39665
3645.83
137384


EXP-AGRne.Ubq1:1:8
5
22805.5
4183.58
140991


EXP-AGRne.Ubq1:1:9
7
5861.5
887.08
34034.3


EXP-ARUdo.Ubq1:1:8
13
26965.5
1052.33
37774.8


EXP-ARUdo.Ubq1:1:9
18
66126
3251.08
114622


EXP-ARUdo.Ubq1:1:11
21
136163

453851


EXP-ARUdo.Ubq2:1:8
27
13222.3
2203.58
72339.1


EXP-ARUdo.Ubq2:1:9
30
30095
6538.58
229201


EXP-ARUdo.Ubq2:1:10
32
16448.5
1842.58
65325.1


EXP-BOUgr.Ubq1:1:6
38
32544.3
2765.08
80330.8


EXP-BOUgr.Ubq1:1:7
41
3826.33
697.11
20709


EXP-BOUgr.Ubq1:1:8
43
9935.5
3372.58
110965


EXP-BOUgr.Ubq2:1:14
52
17828
1575.83
62286.8


EXP-BOUgr.Ubq2:1:16
58
54970.3
3389.08
117616


EXP-BOUgr.Ubq2:1:17
60
48601.3
7139.08
245785


EXP-MISsi.Ubq1:1:8
70
11788.3
3264.58
87751.6


EXP-MISsi.Ubq1:1:10
72
33329.5
2388.58
81000.6


EXP-MISsi.Ubq1:1:11
74
4723.75
3135.33
98059.1


EXP-MISsi.Ubq1:1:7
76
4499
3073.58
84015.1


EXP-SCHsc.Ubq1:1:9
78
5972
1703.33
62310.6


EXP-SCHsc.Ubq1:1:7
84
24173.5
5306.08
155122


EXP-SCHsc.Ubq1:1:10
86
7260
1171.08
38698.1


EXP-SORnu.Ubq1:1:6
92
3966.5
4175.08
129365


EXP-SORnu.Ubq1:1:7
95
23375.5
616.83
25125.3


EXP-SORnu.Ubq1:1:8
97
8431.75
1630.08
55095.6


EXP-SETit.Ubq1:1:5
103
20496.5
2358.83
88695.8


EXP-SETit.Ubq1:1:7
106
75728.5
4723.08
185224


EXP-SETit.Ubq1:1:6
108
44148.3
4962.08
161216


EXP-Sv.Ubq1:1:7
110
15043.8
1888.33
74670.6


EXP-Sv.Ubq1:1:8
116
31997.8
3219.83
113787


EXP-Sv.Ubq1:1:10
118
38952.8
7011.33
220209


EXP-Zm.UbqM1:1:6
122
30528.3
2453.58
90113.1


EXP-Zm.UbqM1:1:8
134
34986.3
2553.78
105725


Exp-Sb.Ubq4:1:2
140
9982.25
2171.58
72593.8


Exp-Sb.Ubq6:1:2
144
33689
3879.58
114710









To compare the relative activity of each EXP sequence, GUS values were expressed as a ratio of GUS to luciferase activity and normalized with respect to the expression levels observed for EXP-Os.Act1:1:1 and EXP-CaMV.35S-enh+Ta.Lhcb1+Os.Act1:1:1. Table 4 below shows the GUS/RLuc ratios of expression normalized with respect to EXP-Os.Act1:1:1 and EXP-CaMV.35S-enh+Ta.Lhcb1+Os.Act1:1:1 driven expression in corn protoplasts. Table 5 below shows the GUS/FLuc ratios of expression normalized with respect to EXP-Os.Act1:1:1 and EXP-CaMV.35S-enh+Ta.Lhcb1+Os.Act1:1:1 driven expression in corn protoplasts.







TABLE 4







GUS/RLuc and GUS/FLuc ratios of expression normalized with


respect to EXP-CaMV.35S-enh + Ta.Lhcb1 + Os.Act1:1:1


(SEQ ID NO: 161) in corn protoplasts.












GUS/FLuc
GUS/RLuc




Relative to
Relative to



SEQ
EXP-CaMV.35S-
EXP-CaMV.35S-



ID
enh + Ta.Lhcb1 +
enh + Ta.Lhcb1 +


EXP Sequence
NO:
Os.Act1:1:1
Os.Act1:1:1













EXP-Os.Act1:1:9
162
0.14
0.16


EXP-CaMV.35S-
161
1
1


enh + Ta.Lhcb1 +


Os.Act1:1:1


EXP-AGRne.Ubq1:1:7
1
2.66
3.17


EXP-AGRne.Ubq1:1:8
5
1.33
1.78


EXP-AGRne.Ubq1:1:9
7
1.62
1.89


EXP-ARUdo.Ubq1:1:8
13
6.27
7.85


EXP-ARUdo.Ubq1:1:9
18
4.98
6.34


EXP-ARUdo.Ubq1:1:11
21

3.3


EXP-ARUdo.Ubq2:1:8
27
1.47
2.01


EXP-ARUdo.Ubq2:1:9
30
1.13
1.44


EXP-ARUdo.Ubq2:1:10
32
2.18
2.77


EXP-BOUgr.Ubq1:1:6
38
2.88
4.45


EXP-BOUgr.Ubq1:1:7
41
1.34
2.03


EXP-BOUgr.Ubq1:1:8
43
0.72
0.98


EXP-BOUgr.Ubq2:1:14
52
2.77
3.15


EXP-BOUgr.Ubq2:1:16
58
3.97
5.14


EXP-BOUgr.Ubq2:1:17
60
1.67
2.17


EXP-MISsi.Ubq1:1:8
70
0.88
1.48


EXP-MISsi.Ubq1:1:10
72
3.42
4.52


EXP-MISsi.Ubq1:1:11
74
0.37
0.53


EXP-MISsi.Ubq1:1:7
76
0.36
0.59


EXP-SCHsc.Ubq1:1:9
78
0.86
1.05


EXP-SCHsc.Ubq1:1:7
84
1.12
1.71


EXP-SCHsc.Ubq1:1:10
86
1.52
2.06


EXP-SORnu.Ubq1:1:6
92
0.23
0.34


EXP-SORnu.Ubq1:1:7
95
9.28
10.23


EXP-SORnu.Ubq1:1:8
97
1.27
1.68


EXP-SETit.Ubq1:1:5
103
2.13
2.54


EXP-SETit.Ubq1:1:7
106
3.92
4.49


EXP-SETit.Ubq1:1:6
108
2.18
3.01


EXP-Sv.Ubq1:1:7
110
1.95
2.21


EXP-Sv.Ubq1:1:8
116
2.43
3.09


EXP-Sv.Ubq1:1:10
118
1.36
1.94


EXP-Zm.UbqM1:1:6
122
3.05
3.72


EXP-Zm.UbqM1:1:8
134
3.35
3.64


Exp-Sb.Ubq4:1:2
140
1.13
1.51


Exp-Sb.Ubq6:1:2
144
2.13
3.23
















TABLE 5







GUS/RLuc and GUS/FLuc ratios of expression normalized with respect


to EXP-Os.Act1:1:9 (SEQ ID NO: 162) in corn leaf protoplasts.












GUS/FLuc
GUS/RLuc



SEQ
Relative to
Relative to



ID
EXP-
EXP-


EXP Sequence
NO:
Os.Act1:1:9
Os.Act1:1:9













EXP-Os.Act1:1:9
162
1
1


EXP-CaMV.35S-
161
7.09
6.23


enh + Ta.Lhcb1 + Os.Act1:1:1


EXP-AGRne.Ubq1:1:7
1
18.87
19.76


EXP-AGRne.Ubq1:1:8
5
9.46
11.07


EXP-AGRne.Ubq1:1:9
7
11.46
11.79


EXP-ARUdo.Ubq1:1:8
13
44.45
48.86


EXP-ARUdo.Ubq1:1:9
18
35.28
39.48


EXP-ARUdo.Ubq1:1:11
21

20.53


EXP-ARUdo.Ubq2:1:8
27
10.41
12.51


EXP-ARUdo.Ubq2:1:9
30
7.98
8.99


EXP-ARUdo.Ubq2:1:10
32
15.49
17.23


EXP-BOUgr.Ubq1:1:6
38
20.42
27.73


EXP-BOUgr.Ubq1:1:7
41
9.52
12.65


EXP-BOUgr.Ubq1:1:8
43
5.11
6.13


EXP-BOUgr.Ubq2:1:14
52
19.63
19.59


EXP-BOUgr.Ubq2:1:16
58
28.14
31.99


EXP-BOUgr.Ubq2:1:17
60
11.81
13.53


EXP-MISsi.Ubq1:1:8
70
6.26
9.19


EXP-MISsi.Ubq1:1:10
72
24.21
28.16


EXP-MISsi.Ubq1:1:11
74
2.61
3.3


EXP-MISsi.Ubq1:1:7
76
2.54
3.67


EXP-SCHsc.Ubq1:1:9
78
6.08
6.56


EXP-SCHsc.Ubq1:1:7
84
7.9
10.67


EXP-SCHsc.Ubq1:1:10
86
10.75
12.84


EXP-SORnu.Ubq1:1:6
92
1.65
2.1


EXP-SORnu.Ubq1:1:7
95
65.74
63.67


EXP-SORnu.Ubq1:1:8
97
8.97
10.47


EXP-SETit.Ubq1:1:5
103
15.07
15.82


EXP-SETit.Ubq1:1:7
106
27.81
27.98


EXP-SETit.Ubq1:1:6
108
15.43
18.74


EXP-Sv.Ubq1:1:7
110
13.82
13.79


EXP-Sv.Ubq1:1:8
116
17.24
19.25


EXP-Sv.Ubq1:1:10
118
9.64
12.11


EXP-Zm.UbqM1:1:6
122
21.58
23.19


EXP-Zm.UbqM1:1:8
134
23.76
22.65


Exp-Sb.Ubq4:1:2
140
7.97
9.41


Exp-Sb.Ubq6:1:2
144
15.06
20.1









As can be seen in Tables 9 and 10, all of the EXP sequences were capable of driving GUS transgene expression in corn cells. Average GUS expression was higher for all of the EXP sequences relative to EXP-Os.Act1:1:9. The EXP sequences, EXP-AGRne.Ubq1:1:7 (SEQ ID NO: 1), EXP-AGRne.Ubq1:1:8 (SEQ ID NO: 5), EXP-AGRne.Ubq1:1:9 (SEQ ID NO: 7), EXP-ARUdo.Ubq1:1:8 (SEQ ID NO: 13), EXP-ARUdo.Ubq1:1:9 (SEQ ID NO: 18), EXP-ARUdo.Ubq1:1:11 (SEQ ID NO: 21), EXP-ARUdo.Ubq2:1:8 (SEQ ID NO: 27), EXP-ARUdo.Ubq2:1:9 (SEQ ID NO: 30), EXP-ARUdo.Ubq2:1:10 (SEQ ID NO: 32), EXP-BOUgr.Ubq1:1:6 (SEQ ID NO: 38), EXP-BOUgr.Ubq1:1:7 (SEQ ID NO: 41), EXP-BOUgr.Ubq2:1:14 (SEQ ID NO: 52), EXP-BOUgr.Ubq2:1:16 (SEQ ID NO: 58), EXP-BOUgr.Ubq2:1:17 (SEQ ID NO: 60), EXP-MISsi.Ubq1:1:10 (SEQ ID NO: 72), EXP-SCHsc.Ubq1:1:7 (SEQ ID NO: 84), EXP-SCHsc.Ubq1:1:10 (SEQ ID NO: 86), EXP-SORnu.Ubq1:1:7 (SEQ ID NO: 95), EXP-SORnu.Ubq1:1:8 (SEQ ID NO: 97), EXP-SETit.Ubq1:1:5 (SEQ ID NO: 103), EXP-SETit.Ubq1:1:7 (SEQ ID NO: 106), EXP-SETit.Ubq1:1:6 (SEQ ID NO: 108), EXP-Sv.Ubq1:1:7 (SEQ ID NO: 110), EXP-Sv.Ubq1:1:8 (SEQ ID NO: 116), EXP-Sv.Ubq1:1:10 (SEQ ID NO: 118), EXP-Zm.UbqM1:1:6 (SEQ ID NO: 122), EXP-Zm.UbqM1:1:8 (SEQ ID NO: 134), EXP-Sb.Ubq4:1:2 (SEQ ID NO: 140), and EXP-Sb.Ubq6:1:2 (SEQ ID NO: 144) demonstrated GUS expression levels above that of EXP-CaMV.35S-enh+Ta.Lhcb1+Os.Act1:1:1.
In a second set of experiments, a GUS expression cassette amplicon comprising the EXP sequence EXP-Zm.UbqM1:1:7 (SEQ ID NO: 128) was compared to the control amplicons, PCR0145942 (EXP-Os.Act1:1:9, SEQ ID NO: 162) and PCR0145944 (EXP-CaMV.35S-enh+Zm.DnaK:1:1, SEQ ID NO: 161) with respect to GUS expression. GUS expression driven by the EXP sequence EXP-Zm.UbqM1:1:7 was higher than that of the two controls. Table 6 below shows the mean GUS and luciferase values determined for each amplicon. Table 7 below shows the GUS/RLuc and GUS/FLuc ratios of expression normalized with respect to EXP-Os.Act1:1:9 and EXP-CaMV.35S-enh+Zm.DnaK:1:1 driven expression in corn protoplasts.







TABLE 6







Mean GUS and Luciferase activity in


transformed corn leaf protoplast cells.











EXP Sequence
SEQ ID NO:
GUS
FLuc
RLuc














EXP-Os.Act1:1:9
162
1512.25
11333.75
190461.00


EXP-CaMV.35S-
161
41176.50
13885.75
330837.25


enh + Ta.Lhcb1 +


Os.Act1:1:1


EXP-Zm.UbqM1:1:7
128
79581.50
15262.50
330755.75
















TABLE 7







GUS/RLuc and GUS/FLuc ratios of expression normalized with respect


to EXP-Os.Act1:1:9 (SEQ ID NO: 161) and EXP-CaMV.35S-enh +


Zm.DnaK:1:1 (SEQ ID NO: 160) in corn leaf protoplasts.











EXP Sequence
SEQ ID NO:
GUS
FLuc
RLuc














EXP-Os.Act1:1:9
162
1512.25
11333.75
190461.00


EXP-CaMV.35S-
161
41176.50
13885.75
330837.25


enh + Ta.Lhcb1 +


Os.Act1:1:1


EXP-Zm.UbqM1:1:7
128
79581.50
15262.50
330755.75









The efficacy of regulatory elements driving GUS expression from amplicons can be similarly studied in sugarcane leaf protoplasts. For instance, sugarcane protoplasts may be transformed with DNA amplicons derived from plant expression vectors containing an EXP sequence, driving expression of the GUS transgene, and compared to leaf protoplast in which expression of GUS is driven by known constitutive promoters.
Example 3
Analysis of Regulatory Elements Driving GUS in Wheat Protoplasts Using GUS Expression Cassette Amplicons
Wheat leaf protoplasts were transformed with DNA amplicons derived from plant expression vectors containing an EXP sequence, driving expression of the GUS transgene, and compared to leaf protoplast in which expression of GUS was driven by known constitutive promoters.
Wheat protoplast cells derived from leaf tissue were transformed using methods known in the art with amplicons produced from amplification of GUS expression cassettes comprising plant expression vectors to compare expression of a transgene (GUS) driven by the EXP sequences listed in Table 3 with that of known constitutive promoters with methodology as described in a previous example (Example 2), using the same GUS expression cassette amplicons as that used for assay in corn in Example 2 above. Control GUS expression cassette amplicons and Luciferase plasmids used for wheat protoplast transformation were also the same as those presented in the previous example and provided in Table 3 above in Example 2. Likewise, negative controls were used for the determination of GUS and Luciferase background, as described above. Wheat leaf protoplasts were transformed using a PEG-based transformation method, as described in Example 2 above. Table 8 lists mean GUS and LUC activity seen in transformed wheat leaf protoplast cells, and Table 9 and 10 shows normalized GUS/FLuc and GUS/RLuc ratios of expression in wheat protoplasts relative to the constitutive EXP controls.







TABLE 8







Mean GUS and Luciferase activity in


transformed wheat leaf protoplast cells.












SEQ ID





EXP Sequence
NO:
GUS
FLuc
RLuc














EMPTY

262.56
1109.78
61422.1


EXP-Os.Act1:1:9
162
2976.33
730.11
53334.8


EXP-CaMV.35S-
161
29299.3
741.78
50717.4


enh + Ta.Lhcb1 + Os.Act1:1:1


EXP-AGRne.Ubq1:1:7
1
27078.3
754.44
44235.8


EXP-AGRne.Ubq1:1:8
5
22082.7
958.11
55774.8


EXP-AGRne.Ubq1:1:9
7
13882.7
699.78
49273.4


EXP-ARUdo.Ubq1:1:8
13
65628
791.44
56358.8


EXP-ARUdo.Ubq1:1:9
18
87615
801.44
53246.4


EXP-ARUdo.Ubq1:1:11
21
19224.3
143.44
14104.1


EXP-ARUdo.Ubq2:1:8
27
25453.3
835.11
57679.4


EXP-ARUdo.Ubq2:1:9
30
26720.7
702.44
47455.4


EXP-ARUdo.Ubq2:1:10
32
37089.3
859.11
57814.4


EXP-BOUgr.Ubq1:1:6
38
35146
995.44
64418.8


EXP-BOUgr.Ubq1:1:7
41
18077
857.78
55793.4


EXP-BOUgr.Ubq1:1:8
43
11723.7
938.44
59362.1


EXP-BOUgr.Ubq2:1:14
52
38109.3
875.11
58048.1


EXP-BOUgr.Ubq2:1:16
58
37384
860.44
52447.8


EXP-BOUgr.Ubq2:1:17
60
24090.7
968.78
53057.8


EXP-MISsi.Ubq1:1:8
70
16456.7
1021.78
61684.1


EXP-MISsi.Ubq1:1:10
72
42816.7
839.78
46688.1


EXP-MISsi.Ubq1:1:11
74
20625.7
987.78
61842.1


EXP-MISsi.Ubq1:1:7
76
4913.67
764.78
64720.1


EXP-SCHsc.Ubq1:1:9
78
9726
937.11
54725.4


EXP-SCHsc.Ubq1:1:7
84
13374.7
1112.44
73815.4


EXP-SCHsc.Ubq1:1:10
86
13650
936.78
62242.1


EXP-SORnu.Ubq1:1:6
92
8188.17
753.83
50572.5


EXP-SORnu.Ubq1:1:7
95
83233.7
854.44
54410.1


EXP-SORnu.Ubq1:1:8
97
21904.7
1011.83
60852


EXP-SETit.Ubq1:1:5
103
39427.7
908.78
57463.1


EXP-SETit.Ubq1:1:7
106
108091
809.44
49330.4


EXP-SETit.Ubq1:1:6
108
58703
809.11
46110.1


EXP-Sv.Ubq1:1:7
110
29330
684.11
43367.1


EXP-Sv.Ubq1:1:8
116
53359
698.11
40076.4


EXP-Sv.Ubq1:1:10
118
49122.7
901.44
53180.8


EXP-Zm.UbqM1:1:6
122
37268
945.78
54088.1


EXP-Zm.UbqM1:1:8
134
51408
677.78
47297.4


Exp-Sb.Ubq4:1:2
140
35660.3
1114.11
62591.1


Exp-Sb.Ubq6:1:2
144
27543
915.11
57826.4
















TABLE 9







GUS/RLuc and GUS/FLuc ratios of expression normalized with


respect to EXP-CaMV.35S-enh + Ta.Lhcb1 +


Os.Act1:1:1 (SEQ ID NO: 161) in wheat protoplasts.












SEQ ID





EXP Sequence
NO:
GUS
FLuc
RLuc














EMPTY

262.56
1109.78
61422.1


EXP-Os.Act1:1:9
162
2976.33
730.11
53334.8


EXP-CaMV.35S-
161
29299.3
741.78
50717.4


enh + Ta.Lhcb1 + Os.Act1:1:1


EXP-AGRne.Ubq1:1:7
1
27078.3
754.44
44235.8


EXP-AGRne.Ubq1:1:8
5
22082.7
958.11
55774.8


EXP-AGRne.Ubq1:1:9
7
13882.7
699.78
49273.4


EXP-ARUdo.Ubq1:1:8
13
65628
791.44
56358.8


EXP-ARUdo.Ubq1:1:9
18
87615
801.44
53246.4


EXP-ARUdo.Ubq1:1:11
21
19224.3
143.44
14104.1


EXP-ARUdo.Ubq2:1:8
27
25453.3
835.11
57679.4


EXP-ARUdo.Ubq2:1:9
30
26720.7
702.44
47455.4


EXP-ARUdo.Ubq2:1:10
32
37089.3
859.11
57814.4


EXP-BOUgr.Ubq1:1:6
38
35146
995.44
64418.8


EXP-BOUgr.Ubq1:1:7
41
18077
857.78
55793.4


EXP-BOUgr.Ubq1:1:8
43
11723.7
938.44
59362.1


EXP-BOUgr.Ubq2:1:14
52
38109.3
875.11
58048.1


EXP-BOUgr.Ubq2:1:16
58
37384
860.44
52447.8


EXP-BOUgr.Ubq2:1:17
60
24090.7
968.78
53057.8


EXP-MISsi.Ubq1:1:8
70
16456.7
1021.78
61684.1


EXP-MISsi.Ubq1:1:10
72
42816.7
839.78
46688.1


EXP-MISsi.Ubq1:1:11
74
20625.7
987.78
61842.1


EXP-MISsi.Ubq1:1:7
76
4913.67
764.78
64720.1


EXP-SCHsc.Ubq1:1:9
78
9726
937.11
54725.4


EXP-SCHsc.Ubq1:1:7
84
13374.7
1112.44
73815.4


EXP-SCHsc.Ubq1:1:10
86
13650
936.78
62242.1


EXP-SORnu.Ubq1:1:6
92
8188.17
753.83
50572.5


EXP-SORnu.Ubq1:1:7
95
83233.7
854.44
54410.1


EXP-SORnu.Ubq1:1:8
97
21904.7
1011.83
60852


EXP-SETit.Ubq1:1:5
103
39427.7
908.78
57463.1


EXP-SETit.Ubq1:1:7
106
108091
809.44
49330.4


EXP-SETit.Ubq1:1:6
108
58703
809.11
46110.1


EXP-Sv.Ubq1:1:7
110
29330
684.11
43367.1


EXP-Sv.Ubq1:1:8
116
53359
698.11
40076.4


EXP-Sv.Ubq1:1:10
118
49122.7
901.44
53180.8


EXP-Zm.UbqM1:1:6
122
37268
945.78
54088.1


EXP-Zm.UbqM1:1:8
134
51408
677.78
47297.4


Exp-Sb.Ubq4:1:2
140
35660.3
1114.11
62591.1


Exp-Sb.Ubq6:1:2
144
27543
915.11
57826.4
















TABLE 10







GUS/RLuc and GUS/FLuc ratios of expression normalized with respect


to EXP-Os.Act1:1:9 (SEQ ID NO: 162) in corn leaf protoplasts.












GUS/FLuc




SEQ
Relative
GUS/RLuc



ID
to EXP-
Relative to


EXP Sequence
NO:
Os.Act1:1:9
EXP-Os.Act1:1:9













EXP-Os.Act1:1:9
162
1
1


EXP-CaMV.35S-
161
9.69
10.35


enh + Ta.Lhcb1 + Os.Act1:1:1


EXP-AGRne.Ubq1:1:7
1
8.8
10.97


EXP-AGRne.Ubq1:1:8
5
5.65
7.09


EXP-AGRne.Ubq1:1:9
7
4.87
5.05


EXP-ARUdo.Ubq1:1:8
13
20.34
20.87


EXP-ARUdo.Ubq1:1:9
18
26.82
29.49


EXP-ARUdo.Ubq1:1:11
21
32.88
24.43


EXP-ARUdo.Ubq2:1:8
27
7.48
7.91


EXP-ARUdo.Ubq2:1:9
30
9.33
10.09


EXP-ARUdo.Ubq2:1:10
32
10.59
11.5


EXP-BOUgr.Ubq1:1:6
38
8.66
9.78


EXP-BOUgr.Ubq1:1:7
41
5.17
5.81


EXP-BOUgr.Ubq1:1:8
43
3.06
3.54


EXP-BOUgr.Ubq2:1:14
52
10.68
11.76


EXP-BOUgr.Ubq2:1:16
58
10.66
12.77


EXP-BOUgr.Ubq2:1:17
60
6.1
8.14


EXP-MISsi.Ubq1:1:8
70
3.95
4.78


EXP-MISsi.Ubq1:1:10
72
12.51
16.43


EXP-MISsi.Ubq1:1:11
74
5.12
5.98


EXP-MISsi.Ubq1:1:7
76
1.58
1.36


EXP-SCHsc.Ubq1:1:9
78
2.55
3.18


EXP-SCHsc.Ubq1:1:7
84
2.95
3.25


EXP-SCHsc.Ubq1:1:10
86
3.57
3.93


EXP-SORnu.Ubq1:1:6
92
2.66
2.9


EXP-SORnu.Ubq1:1:7
95
23.9
27.41


EXP-SORnu.Ubq1:1:8
97
5.31
6.45


EXP-SETit.Ubq1:1:5
103
10.64
12.3


EXP-SETit.Ubq1:1:7
106
32.76
39.26


EXP-SETit.Ubq1:1:6
108
17.8
22.81


EXP-Sv.Ubq1:1:7
110
10.52
12.12


EXP-Sv.Ubq1:1:8
116
18.75
23.86


EXP-Sv.Ubq1:1:10
118
13.37
16.55


EXP-Zm.UbqM1:1:6
122
9.67
12.35


EXP-Zm.UbqM1:1:8
134
18.61
19.48


Exp-Sb.Ubq4:1:2
140
7.85
10.21


Exp-Sb.Ubq6:1:2
144
7.38
8.54









As can be seen in Tables 9 and 10 above, all of the EXP sequences were capable of driving GUS transgene expression in wheat cells. All of the EXP sequences drove GUS expression at levels higher than that of EXP-Os.Act1:1:9 in wheat cells. The EXP sequences EXP-ARUdo.Ubq1:1:8 (SEQ ID NO: 13), EXP-ARUdo.Ubq1:1:9 (SEQ ID NO: 18), EXP-ARUdo.Ubq1:1:11 (SEQ ID NO: 21), EXP-ARUdo.Ubq2:1:10 (SEQ ID NO: 32), EXP-BOUgr.Ubq2:1:14 (SEQ ID NO: 52), EXP-BOUgr.Ubq2:1:16 (SEQ ID NO: 58), EXP-BOUgr.Ubq2:1:17 (SEQ ID NO: 60), EXP-MISsi.Ubq1:1:10 (SEQ ID NO: 72), EXP-SORnu.Ubq1:1:7 (SEQ ID NO: 95), EXP-SETit.Ubq1:1:5 (SEQ ID NO: 103), EXP-SETit.Ubq1:1:7 (SEQ ID NO: 106), EXP-SETit.Ubq1:1:6 (SEQ ID NO: 108), EXP-Sv.Ubq1:1:7 (SEQ ID NO: 110), EXP-Sv.Ubq1:1:8 (SEQ ID NO: 116), EXP-Sv.Ubq1:1:10 (SEQ ID NO: 118), EXP-Zm.UbqM1:1:6 (SEQ ID NO: 122), and EXP-Zm.UbqM1:1:8 (SEQ ID NO: 134) demonstrated levels of GUS expression equal to or greater than GUS expression driven by EXP-CaMV.35S-enh+Ta.Lhcb1+Os.Act1:1:1 in wheat cells.
In a second set of experiments, the amplicon GUS expression cassette comprising EXP-ARUdo.Ubq1:1:11 (SEQ ID NO: 21) was compared to the controls EXP-Os.Act1:1:9 (SEQ ID NO: 162) and EXP-CaMV.35S-enh-FZm.DnaK:1:1 (SEQ ID NO: 161). Table 11 below shows the mean GUS and luciferase values determined for each amplicon. Table 12 below shows the GUS/RLuc ratios of expression normalized with respect to EXP-Os.Act1:1:9 and EXP-CaMV.35S-enh+Zm.DnaK:1:1 driven expression in wheat protoplasts.







TABLE 11







Mean GUS and Luciferase activity in


transformed wheat leaf protoplast cells.











SEQ ID




EXP Sequence
NO:
GUS
RLuc













EMPTY

20.75
187112.50


EXP-Os.Act1:1:9
162
1234.00
176970.50


EXP-CaMV.35S-
161
12883.50
119439.00


enh + Ta.Lhcb1 + Os.Act1:1:1


EXP-ARUdo.Ubq1:1:11
21
30571.50
135037.50
















TABLE 12







GUS/RLuc and GUS/FLuc ratios of expression normalized with respect


to EXP-Os.Act1:1:9 (SEQ ID NO: 161) and EXP-CaMV.35S-enh +


Zm.DnaK:1:1 (SEQ ID NO: 160) in wheat leaf protoplasts.













GUS/RLuc




GUS/RLuc
Relative to



SEQ
Relative to
EXP-CaMV.35S-



ID
EXP-
enh + Ta.Lhcb1 +


EXP Sequence
NO:
Os.Act1:1:9
Os.Act1:1:1













EXP-Os.Act1:1:9
162
1.00
0.06


EXP-CaMV.35S-
161
15.47
1.00


enh + Ta.Lhcb1 + Os.Act1:1:1


EXP-ARUdo.Ubq1:1:11
21
32.47
2.10









As can be seen in Table 12 above, GUS expression driven by EXP-ARUdo.Ubq1:1:11 (SEQ ID NO: 21) was higher than both constitutive controls, EXP-Os.Act1:1:9 and EXP-CaMV.35S-enh+Zm.DnaK:1:1.
Example 4
Analysis of Regulatory Elements Driving GUS in Corn and Wheat Protoplasts
Corn and Wheat leaf protoplasts were transformed with plant expression vectors containing an EXP sequence driving expression of the β-glucuronidase (GUS) transgene and compared to GUS expression in leaf protoplasts in which expression of GUS is driven by known constitutive promoters.
Expression of a transgene driven by EXP-Cl.Ubq10 (SEQ ID NO: 168) was compared with expression from known constitutive promoters. The foregoing EXP sequences were cloned into plant expression vectors as shown in Table 13 below to yield vectors in which an EXP sequence is operably linked 5′ to a GUS reporter that contained a processable intron (referred to as GUS-2, SEQ ID NO: 160) derived from the potato light-inducible tissue-specific ST-LS1 gene (GenBank Accession: X04753) or a contiguous GUS coding sequence (GUS-1, SEQ ID NO: 159), which was operably linked 5′ to a 3′ UTR derived from the A. tumefaciens Nopaline synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 161) or the wheat Hsp17 gene (T-Ta.Hsp17-1:1:1, SEQ ID NO: 162).







TABLE 13







GUS plant expression plasmid construct and corresponding EXP sequence,


GUS coding sequence and 3′ UTR used for transformation of corn leaf


protoplasts. “SEQ ID NO:” refers to given EXP sequence.













GUS/RLuc




GUS/RLuc
Relative to



SEQ
Relative to
EXP-CaMV.35S-



ID
EXP-
enh + Ta.Lhcb1 +


EXP Sequence
NO:
Os.Act1:1:9
Os.Act1:1:1













EXP-Os.Act1:1:9
162
1.00
0.06


EXP-CaMV.35S-
161
15.47
1.00


enh + Ta.Lhcb1 + Os.Act1:1:1


EXP-ARUdo.Ubq1:1:11
21
32.47
2.10









Two plasmids, for use in co-transformation and normalization of data, were also constructed using methods known in the art. Each plasmid contained a specific luciferase coding sequence that was driven by a constitutive EXP sequence. The plant vector pMON19437 comprises an expression cassette with a constitutive promoter operably linked 5′ to an intron, (EXP-CaMV.35S-enh+Zm.DnaK:1:1, SEQ ID NO: 163), operably linked 5′ to a firefly (Photinus pyralis) luciferase coding sequence (LUCIFERASE:1:3, SEQ ID NO: 156), operably linked 5′ to a 3′ UTR from the Agrobacterium tumefaciens nopaline synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 158). The plant vector pMON63934 comprises an expression cassette with a constitutive EXP sequence (EXP-CaMV.35S-enh-Lhcb1, SEQ ID NO: 164), operably linked 5′ to a sea pansy (Renilla reniformis) luciferase coding sequence (CR-Ren.hRenilla Lucife-0:0:1, SEQ ID NO: 157), operably linked 5′ to a 3′ UTR from the Agrobacterium tumefaciens nopaline synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 158).
Corn leaf protoplasts were transformed using a PEG-based transformation method, which is well known in the art. Protoplast cells were transformed with pMON19437 plasmid DNA, pMON63934 plasmid DNA, and the plasmids presented in Table 13 and incubated overnight in total darkness. Measurements of both GUS and luciferase were conducted in a similar manner as that described in Example 2 above. One or two transformations for each EXP sequence were performed and the mean expression values for each EXP sequence determined from several samples from each transformation experiment. Sample measurements were made using four replicates of each EXP sequence construct transformation, or alternatively, three replicates of each EXP sequence construct per one of two transformation experiments. The mean GUS and luciferase expression levels are provided in Table 14. In this table, the firefly luciferase values (e.g., from expression of pMON19437) are provided in the column labeled “FLuc” and the Renilla luciferase values are provided as in the column labeled “RLuc.”







TABLE 14







Mean GUS and Luciferase activity in


transformed corn leaf protoplast cells.












SEQ ID





EXP Sequence
NO:
GUS
FLuc
RLuc














EXP-Os.Act1:1:9
162
83997.3
80983
61619


EXP-CaMV.35S-
161
248832
83589.8
72064.3


enh + Ta.Lhcb1 + Os.Act1:1:1


EXP-Cl.Ubq10
168
30790.8
65807.5
34846.3









Table 15 below shows the GUS/FLuc and GUS/RLuc ratios of expression normalized with respect to EXP-Os.Act1:1:9 and EXP-CaMV.35S-enh+Zm.DnaK:1:1 driven expression in corn protoplasts.







TABLE 15







GUS/FLuc and GUS/RLuc ratios of expression normalized with respect to EXP-


Os.Act1:1:9 (SEQ ID NO: 161) and EXP-CaMV.35S-enh + Zm.DnaK:1:1 (SEQ ID NO: 160)


in wheat leaf protoplasts.














GUS/Fluc
GUS/Rluc
GUS/Fluc
GUS/Rluc




normalized
normalized
normalized with
normalized with




with
with
respect to EXP-
respect to EXP-



SEQ
respect to
respect to
CaMV.35S-
CaMV.35S-



ID
EXP-
EXP-
enh + Ta.Lhcb1 +
enh + Ta.Lhcb1 + Os.


EXP Sequence
NO:
Os.Act1:1:9
Os.Act1:1:9
Os.Act1:1:1
Act1:1:1





EXP-Os.Act1:1:9
162
1.00
1.00
0.35
0.39


EXP-CaMV.35S-
161
2.87
2.53
1.00
1.00


enh + Ta.Lhcb1 + Os.Act1:1:1


EXP-Cl.Ubq10
168
0.45
0.65
0.16
0.26









As can be seen in Table 15 above, EXP-Cl.Ubq10 (SEQ ID NO: 168) was able to drive expression of GUS, but was at a level lower than that of both constitutive controls.
The plasmids listed in Table 13 above were also used to transform wheat leaf protoplast cells in a similar manner as that for corn leaf protoplasts described above. Mean GUS and luciferase values are shown in Table 16 below. Table 17 below shows the GUS/FLuc and GUS/RLuc ratios of expression normalized with respect to EXP-CaMV.35S-enh+Zm.DnaK:1:1 driven expression in corn protoplasts.







TABLE 16







Mean GUS and Luciferase activity in


transformed corn leaf protoplast cells.












SEQ ID





EXP Sequence
NO:
GUS
FLuc
RLuc














EXP-CaMV.35S-
161
134145
1076.67
6858.67


enh + Ta.Lhcb1 + Os.Act1:1:1


EXP-Cl.Ubq10
168
104669
888.67
4516
















TABLE 17







GUS/FLuc and GUS/RLuc ratios of expression normalized with respect to EXP-


CaMV.35S-enh + Zm.DnaK:1:1 (SEQ ID NO: 160) in wheat leaf protoplasts.











SEQ
GUS/Fluc normalized with
GUS/Rluc normalized with



ID
respect to EXP-CaMV.35S-
respect to EXP-CaMV.35S-


EXP Sequence
NO:
enh + Ta.Lhcb1 + Os.Act1:1:1
enh + Ta.Lhcb1 + Os.Act1:1:1





EXP-CaMV.35S-
161
1.00
1.00


enh + Ta.Lhcb1 + Os.Act1:1:1


EXP-Cl.Ubq10
168
0.95
1.19









As can be seen in Table 17 above, EXP-Cl.Ubq10 (SEQ ID NO: 168) expressed GUS at a similar level as that of EXP-CaMV.35S-enh+Zm.DnaK:1:1 (SEQ ID NO: 160) in wheat protoplast cells.
Example 5
Analysis of Regulatory Elements Driving GUS in Transgenic Corn
Corn plants were transformed with plant expression vectors containing a EXP sequences driving expression of the GUS transgene, and the resulting plants were analyzed for GUS protein expression. The ubiquitin EXP sequences were cloned into plant binary transformation plasmid constructs using methods known in the art.
The resulting plant expression vectors contain a right border region from A. tumefaciens, a first expression cassette to assay the EXP sequence operably linked to a coding sequence for GUS that possesses the processable intron GUS-2, described above, operably linked 5′ to the 3′ UTR from the rice lipid transfer protein gene (T-Os.LTP-1:1:1, SEQ ID NO: 159); a second transgene selection cassette used for selection of transformed plant cells that confers resistance to the herbicide glyphosate (driven by the rice Actin 1 promoter), and a left border region from A. tumefaciens. The resulting plasmids were used to transform corn plants. Table 18 lists the plasmid designations, the EXP sequences and the SEQ ID NOs, which are also described in Table 1.







TABLE 18







Binary plant transformation plasmids and the associated EXP sequences.











Plasmid

SEQ ID



Construct
EXP sequence
NO:















pMON140869
EXP-AGRne.Ubq1:1:7
1



pMON140870
EXP-AGRne.Ubq1:1:8
5



pMON142650
EXP-ARUdo.Ubq1:1:8
13



pMON142651
EXP-ARUdo.Ubq1:1:9
18



pMON142652
EXP-ARUdo.Ubq2:1:8
27



pMON142653
EXP-ARUdo.Ubq2:1:9
30



pMON140871
EXP-BOUgr.Ubq1:1:6
38



pMON140872
EXP-BOUgr.Ubq1:1:7
41



pMON140873
EXP-BOUgr.Ubq2:1:14
52



pMON140874
EXP-BOUgr.Ubq2:1:15
55



pMON142887
EXP-MISsi.Ubq1:1:7
76



pMON140875
EXP-SORnu.Ubq1:1:6
92



pMON140876
EXP-SORnu.Ubq1:1:7
95



pMON132037
EXP-SETit.Ubq1:1:10
99



pMON131958
EXP-Sv.Ubq1:1:11
114



pMON131959
EXP-Sv.Ubq1:1:12
120



pMON131961
EXP-Zm.UbqM1:1:10
126



pMON131963
EXP-Zm.UbqM1:1:12
132



pMON131962
EXP-Zm.UbqM1:1:11
138



pMON132932
EXP-Sb.Ubq4:1:2
140



pMON132931
EXP-Sb.Ubq6:1:3
148



pMON132974
EXP-Sb.Ubq7:1:2
150



pMON142738
EXP-Cl.Ubq10
168










Plants were transformed using Agrobacterium-mediated transformations, for instance as described in U.S. Patent Application Publication 2009/0138985.
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-4-chloro-3-indolyl-b-glucuronide) (1 mg/ml) for an appropriate length of time, rinsed, and visually inspected for blue coloration. GUS activity was qualitatively determined by direct visual inspection or inspection under a microscope using selected plant organs and tissues. The R0 plants are inspected for expression in the roots and leaves, as well as the anther, silk, and developing seed and embryo, 21 days after pollination (21 DAP).
For quantitative analysis, total protein was extracted from selected tissues of transformed corn plants. One microgram of total protein was used with the fluorogenic substrate 4-methyleumbelliferyl-β-D-glucuronide (MUG) in a total reaction volume of 50 μl. 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, emission at 445 nm using a Fluoromax-3 (Horiba; Kyoto, Japan) with Micromax Reader, with slit width set at excitation 2 nm and emission 3 nm.
The average R0 GUS expression observed for each transformation is presented in Tables 19 and 20 below.







TABLE 19







Average R0 GUS expression in root and leaf tissue.

















SEQ











ID
V3
V4
V7
VT
V3
V4
V7
VT


EXP sequence
NO:
Root
Root
Root
Root
Leaf
Leaf
Leaf
Leaf



















EXP-AGRne.Ubq1:1:7
1
16

25
14
49

60
48


EXP-AGRne.Ubq1:1:8
5
13

20
22
38

38
52


EXP-ARUdo.Ubq1:1:8
13
18

34
89
117

48
106


EXP-ARUdo.Ubq1:1:9
18
19

20
68
105

33
69


EXP-ARUdo.Ubq2:1:8
27
14

19
27
58

57
47


EXP-ARUdo.Ubq2:1:9
30
14

15
25
40

38
40


EXP-BOUgr.Ubq1:1:6
38
12

28
16
43

46
27


EXP-BOUgr.Ubq1:1:7
41
14

24
114
51

48
48


EXP-BOUgr.Ubq2:1:14
52
17

13
28
46

33
41


EXP-BOUgr.Ubq2:1:15
55
11

67
36
86

72
36


EXP-MISsi.Ubq1:1:7
76
17

28
13
18

12
18


EXP-SORnu.Ubq1:1:6
92
14

45
33
44

64
55


EXP-SORnu.Ubq1:1:7
95
11

18
20
31

36
48


EXP-SETit.Ubq1:1:10
99
0

29
57
58

37
46


EXP-Sv.Ubq1:1:11
114
nd

nd
9
20

55
29


EXP-Sv.Ubq1:1:12
120
63

0
28
184

27
16


EXP-Zm.UbqM1:1:10
126
0

237
18
221

272
272


EXP-Zm.UbqM1:1:12
132
0

21
43
234

231
196


EXP-Zm.UbqM1:1:11
138
124

103
112
311

369
297


EXP-Sb.Ubq4:1:2
140
125

0
95
233

150
88


EXP-Sb.Ubq6:1:3
148
154

13
128
53

39
55


EXP-Sb.Ubq7:1:2
150
37

22
18
165

89
177


EXP-Cl.Ubq10
168

61
67
32

111
58
115
















TABLE 20







Average R0 GUS expression in corn reproductive organs (anther, silk) and


developing seed (embryo and endosperm).













SEQ

VT/





ID
VT
R1
21 DAP
21 DAP


EXP sequence
NO:
Anther
Silk
Embryo
Endosperm















EXP-AGRne.Ubq1:1:7
1
149
36
59
59


EXP-AGRne.Ubq1:1:8
5
73
66
33
58


EXP-ARUdo.Ubq1:1:8
13
321
253
177
355


EXP-ARUdo.Ubq1:1:9
18
242
268
97
266


EXP-ARUdo.Ubq2:1:8
27
104
99
79
157


EXP-ARUdo.Ubq2:1:9
30
78
71
82
139


EXP-BOUgr.Ubq1:1:6
38
58
250
43
63


EXP-BOUgr.Ubq1:1:7
41
58
77
40
49


EXP-BOUgr.Ubq2:1:14
52
236
377
48
137


EXP-BOUgr.Ubq2:1:15
55
203
134
47
180


EXP-MISsi.Ubq1:1:7
76
24
16
29
32


EXP-SORnu.Ubq1:1:6
92
361
80
37
94


EXP-SORnu.Ubq1:1:7
95
195
114
20
55


EXP-SETit.Ubq1:1:10
99
132
85
50
63


EXP-Sv.Ubq1:1:11
114
217
3
45
92


EXP-Sv.Ubq1:1:12
120
120
21
49
112


EXP-Zm.UbqM1:1:10
126
261
506
403
376


EXP-Zm.UbqM1:1:12
132
775
362
253
247


EXP-Zm.UbqM1:1:11
138
551
452
234
302


EXP-Sb.Ubq4:1:2
140
213
0
25
79


EXP-Sb.Ubq6:1:3
148
295
87
51
61


EXP-Sb.Ubq7:1:2
150
423
229
274
90


EXP-Cl.Ubq10
168
237
82
91
210









In R0 corn plants, GUS expression levels in the leaf and root differed amongst the ubiquitin EXP sequences. While all of the EXP sequences demonstrated the ability to drive GUS transgene expression in stably transformed plants, each EXP sequence demonstrated a unique pattern of expression relative to the others. For example, the EXP sequences, EXP-AGRne.Ubq1:1:7 (SEQ ID NO: 1), EXP-AGRne.Ubq1:1:8 (SEQ ID NO: 5), EXP-ARUdo.Ubq1:1:8 (SEQ ID NO: 13), EXP-ARUdo.Ubq1:1:9 (SEQ ID NO: 18), EXP-ARUdo.Ubq2:1:8 (SEQ ID NO: 27), EXP-ARUdo.Ubq2:1:9 (SEQ ID NO: 30), EXP-BOUgr.Ubq1:1:6 (SEQ ID NO: 38), EXP-BOUgr.Ubq1:1:7 (SEQ ID NO: 41), EXP-BOUgr.Ubq2:1:14 (SEQ ID NO: 52), EXP-BOUgr.Ubq2:1:15 (SEQ ID NO: 55), EXP-MISsi.Ubq1:1:7 (SEQ ID NO: 76), EXP-SORnu.Ubq1:1:6 (SEQ ID NO: 92), EXP-SORnu.Ubq1:1:7 (SEQ ID NO: 95), EXP-SETit.Ubq1:1:10 (SEQ ID NO: 99), EXP-Sv.Ubq1:1:11 (SEQ ID NO: 114), EXP-Zm.UbqM1:1:12 (SEQ ID NO: 132), and EXP-Sb.Ubq7:1:2 (SEQ ID NO: 150) demonstrated lower levels of GUS expression in the root at V3 and V7 stages of development relative to EXP-Sv.Ubq1:1:12 (SEQ ID NO: 120), EXP-Zm.UbqM1:1:10 (SEQ ID NO: 126), EXP-Zm.UbqM1:1:11 (SEQ ID NO: 138), EXP-Sb.Ubq4:1:2 (SEQ ID NO: 140), and EXP-Sb.Ubq6:1:3 (SEQ ID NO: 148). Higher levels of GUS expression were observed in later stages of root development (VT) for EXP-ARUdo.Ubq1:1:8 (SEQ ID NO: 13), EXP-ARUdo.Ubq1:1:9 (SEQ ID NO: 18), EXP-BOUgr.Ubq1:1:7 (SEQ ID NO: 41), EXP-Zm.UbqM1:1:11 (SEQ ID NO: 138), and EXP-Sb.Ubq6:1:3 (SEQ ID NO: 148). Root expression driven by EXP-Zm.UbqM1:1:10 (SEQ ID NO: 140) demonstrated no expression at V3 but was high at V7 and then dropped by VT stage. Root expression driven by EXP-Zm.UbqM1:1:11 (SEQ ID NO: 150) was maintained to a similar level throughout development from stages V3, and V7 through VT. Expression of GUS driven by EXP-Cl.Ubq10 (SEQ ID NO: 168) was relatively steady from V4 to V7 stage but dropped to approximately half that of V4 and V7 at VT stage.
GUS expression levels showed dramatic differences in leaf tissue as well. The EXP sequences, EXP-Zm.UbqM1:1:10 (SEQ ID NO: 126), EXP-Zm.UbqM1:1:12 (SEQ ID NO: 132) and EXP-Zm.UbqM1:1:11 (SEQ ID NO: 138) demonstrated the highest level of GUS expression observed across all three stages of development (V3, V7 and VT). The EXP sequence, EXP-Sb.Ubq4:1:2 (SEQ ID NO: 140), showed a decline in expression from V3 to VT stages of development. The EXP sequences, EXP-ARUdo.Ubq1:1:8 (SEQ ID NO: 13) and EXP-Sb.Ubq7:1:2 (SEQ ID NO: 150) demonstrated higher levels of GUS expression in V3 and VT stage of development with a lower level of expression in the middle of growth at V7 stage. The EXP sequence, EXP-ARUdo.Ubq2:1:9 (SEQ ID NO: 30), EXP-BOUgr.Ubq1:1:7 (SEQ ID NO: 41), and EXP-MISsi.Ubq1:1:7 (SEQ ID NO: 76) maintained GUS expression over all three stages, while EXP-ARUdo.Ubq2:1:8 (SEQ ID NO: 27), EXP-BOUgr.Ubq1:1:6 (SEQ ID NO: 38), and EXP-BOUgr.Ubq2:1:15 (SEQ ID NO: 55) showed a slight decrease in expression at VT stage. Expression driven by EXP-Cl.Ubq10 (SEQ ID NO: 168) was similar at V4 and VT stage but dropped to about half the level of V4 and VT at V7 stage.
Likewise, with respect to reproductive tissue (anther and silk) different patterns of expression were observed unique to each EXP sequence. For example, high levels of expression were observed in anther and silk for the EXP sequences EXP-ARUdo.Ubq1:1:8 (SEQ ID NO: 13), EXP-ARUdo.Ubq1:1:9 (SEQ ID NO: 18), EXP-BOUgr.Ubq2:1:14 (SEQ ID NO: 52), EXP-BOUgr.Ubq2:1:15 (SEQ ID NO: 55), EXP-SORnu.Ubq1:1:7 (SEQ ID NO: 95), EXP-Zm.UbqM1:1:10 (SEQ ID NO: 126), EXP-Zm.UbqM1:1:12 (SEQ ID NO: 132), EXP-Zm.UbqM1:1:11 (SEQ ID NO: 138), and EXP-Sb.Ubq7:1:2 (SEQ ID NO: 150). Expression driven by the EXP sequences EXP-AGRne.Ubq1:1:7 (SEQ ID NO: 1), EXP-SORnu.Ubq1:1:6 (SEQ ID NO: 92), EXP-Sv.Ubq1:1:11 (SEQ ID NO: 114), EXP-Sv.Ubq1:1:12 (SEQ ID NO: 120), EXP-Sb.Ubq4:1:2 (SEQ ID NO: 140), EXP-Sb.Ubq6:1:3 (SEQ ID NO: 148), and EXP-Cl.Ubq10 (SEQ ID NO: 168) was high in the anther but lower in the silk relative to each EXP sequence, while expression driven by EXP-BOUgr.Ubq1:1:6 (SEQ ID NO: 38) was higher in the silk in comparison to expression in the anther.
Expression in the developing seed (21 DAP embryo and endosperm) was different among the EXP sequences. The EXP sequences, EXP-Zm.UbqM1:1:10 (SEQ ID NO: 126), EXP-Zm.UbqM1:1:12 (SEQ ID NO: 132), and EXP-Zm.UbqM1:1:11 (SEQ ID NO: 138) drove high expression of GUS in the developing seed embryo and endosperm tissue. Levels of expression in the endosperm were about two-fold or more higher than in the embryo when GUS was driven by the EXP sequences, EXP-ARUdo.Ubq1:1:8 (SEQ ID NO: 13), EXP-ARUdo.Ubq1:1:9 (SEQ ID NO: 18), EXP-ARUdo.Ubq2:1:8 (SEQ ID NO: 27), EXP-BOUgr.Ubq2:1:14 (SEQ ID NO: 52), EXP-BOUgr.Ubq2:1:15 (SEQ ID NO: 55), EXP-SORnu.Ubq1:1:6 (SEQ ID NO: 92), EXP-SORnu.Ubq1:1:7 (SEQ ID NO: 95), EXP-Sv.Ubq1:1:12 (SEQ ID NO: 120), EXP-Sb.Ubq4:1:2 (SEQ ID NO: 140), and EXP-Cl.Ubq10 (SEQ ID NO: 168). Expression of GUS was three-fold higher in the embryo than in the endosperm when driven by EXP-Sb.Ubq7:1:2 (SEQ ID NO: 150). GUS expression levels were relatively equivalent in the embryo and endosperm when driven by the EXP sequences EXP-AGRne.Ubq1:1:7 (SEQ ID NO: 1), EXP-AGRne.Ubq1:1:8 (SEQ ID NO: 5), EXP-BOUgr.Ubq1:1:6 (SEQ ID NO: 38), EXP-BOUgr.Ubq1:1:7 (SEQ ID NO: 41), EXP-MISsi.Ubq1:1:7 (SEQ ID NO: 76), EXP-SETit.Ubq1:1:10 (SEQ ID NO: 99), and EXP-Sb.Ubq6:1:3 (SEQ ID NO: 148).
Each EXP sequence demonstrated the ability to drive transgene expression in stably transformed corn plants. However, each EXP sequence had a pattern of expression for each tissue that was unique and offers an opportunity to select the EXP sequence which will best provide expression of a specific transgene depending upon the tissue expression strategy needed to achieve the desired results. This example demonstrates that EXP sequences isolated from homologous genes do not necessarily behave equivalently in the transformed plant and that expression can only be determined through empirical investigation of the properties for each EXP sequence and cannot be predicted based upon the gene homology from which the promoter was derived.
Example 6
Enhancers Derived from the Regulatory Elements
Enhancers are derived from the promoter elements provided herein, such as those presented as SEQ ID NOs: 2, 6, 8, 10, 14, 17, 22, 24, 28, 31, 33, 35, 39, 42, 44, 46, 50, 53, 56, 61, 63, 67, 71, 73, 75, 77, 79, 83, 85, 87, 89, 93, 96, 98 and 169. The enhancer element may be comprised of one or more cis regulatory elements that, when operably linked 5′ or 3′ to a promoter element, or operably linked 5′ or 3′ to additional enhancer elements that are operably linked to a promoter, can enhance or modulate expression of a transgene, or provide expression of a transgene in a specific cell type or plant organ or at a particular time point in development or circadian rhythm. Enhancers are made by removing the TATA box or functionally similar elements and any downstream DNA sequence from the promoters that allow transcription to be initiated from the promoters provided herein as described above, including fragments thereof, in which the TATA box or functionally similar elements and DNA sequence downstream of the TATA box are removed.
Enhancer elements may be derived from the promoter elements provided herein and cloned using methods known in the art to be operably linked 5′ or 3′ to a promoter element, or operably linked 5′ or 3′ to additional enhancer elements that are operably linked to a promoter. Alternatively, enhancer elements are cloned, using methods known in the art, to be operably linked to one or more copies of the enhancer element which are operably linked 5′ or 3′ to a promoter element, or operably linked 5′ or 3′ to additional enhancer elements that are operably linked to a promoter. Enhancer elements can also be cloned to be operably linked 5′ or 3′ to a promoter element derived from a different genus organism, or operably linked 5′ or 3′ to additional enhancer elements derived from other genus organisms or the same genus organism that are operably linked to a promoter derived from either the same or different genus organism, resulting in a chimeric regulatory element. A GUS expression plant transformation vector is constructed using methods known in the art similar to the constructs described in the previous examples in which the resulting plant expression vectors contain a right border region from A. tumefaciens, a first expression cassette to test the regulatory or a chimeric regulatory element comprised of, a regulatory or chimeric regulatory element, operably linked to an intron derived from the HSP70 heat shock protein of Z. mays (I-Zm.DnaK-1:1:1 SEQ ID NO: 165) or any of the introns presented herein or any other intron, operably linked to a coding sequence for GUS that either possesses a processable intron (GUS-2, SEQ ID NO: 155) or no intron (GUS-1, SEQ ID NO: 154), operably linked to the Nopaline synthase 3′ UTR from A. tumefaciens (T-AGRtu.nos-1:1:13, SEQ ID NO: 158) or the 3′ UTR from the rice lipid transfer protein gene (T-Os.LTP-1:1:1, SEQ ID NO: 160); a second transgene selection cassette used for selection of transformed plant cells that confers resistance to the herbicide glyphosate (driven by the rice Actin 1 promoter), or alternatively, the antibiotic kanamycin (driven by the rice Actin 1 promoter) and a left border region from A. tumefaciens. The resulting plasmids are used to transform corn plants or other genus plants by the methods described above or by other Agrobacterium-mediated or particle bombardment methods known in the art. Alternatively, protoplast cells derived from corn or other genus plants are transformed using methods known in the art to perform transient assays
GUS expression driven by the regulatory element comprising one or more enhancers is evaluated in stable or transient plant assays to determine the effects of the enhancer element on expression of a transgene. Modifications to one or more enhancer elements or duplication of one or more enhancer elements is performed based upon empirical experimentation and the resulting gene expression regulation that is observed using each regulatory element composition. Altering the relative positions of one or more enhancers in the resulting regulatory or chimeric regulatory element may affect the transcriptional activity or specificity of the regulatory or chimeric regulatory element and is determined empirically to identify the best enhancers for the desired transgene expression profile within the corn plant or other genus plant.
Example 7
Analysis of Intron Enhancement of GUS Activity Using Plant Derived Protoplasts
An intron is selected based upon experimentation and comparison with an intronless expression vector control to empirically select an intron and configuration within the vector transfer DNA (T-DNA) element arrangement for optimal expression of a transgene. For example, in the expression of an herbicide resistance gene, such as CP4, which confers tolerance to glyphosate, it is desirable to have transgene expression within the reproductive tissues as well as the vegetative tissues, to prevent the loss of yield when applying the herbicide. An intron in this instance would be selected upon its ability, when operably linked to a constitutive promoter, to enhance expression of the herbicide resistance conferring transgene, particularly within the reproductive cells and tissues of the transgenic plant and thus providing both vegetative and reproductive tolerance to the transgenic plant when sprayed with the herbicide. In most ubiquitin genes, the 5′ UTR is comprised of a leader, which has an intron sequence embedded within it. The regulatory elements derived from such genes are therefore assayed using the entire 5′ UTR comprising the promoter, leader, and intron. To achieve different expression profiles or to modulate the level of transgene expression, the intron from such a regulatory element may be removed or substituted with a heterologous intron.
Introns presented herein as SEQ ID NOs: 4, 12, 15, 20, 26, 29, 37, 40, 48, 51, 54, 57, 59, 65, 69, 81, 91, 94 and 171 are identified using genomic DNA contigs in comparison to expressed sequence tag clusters or cDNA contigs to identify exon and intron sequences within the genomic DNA. In addition, 5′ UTR or leader sequences are also used to define the intron/exon splice junction of one or more introns under conditions when the gene sequence encodes a leader sequence that is interrupted by one or more introns. Introns are cloned using methods known in the art into a plant transformation vector to be operably linked 3′ to a regulatory element and leader fragment and operably linked 5′ to either a second leader fragment or to coding sequences, for instance as depicted in the expression cassettes presented in FIG. 9.
Thus, for instance, a first possible expression cassette (Expression Cassette Configuration 1 in FIG. 9) is comprised of a promoter or chimeric promoter element [A], operably linked 5′ to a leader element [B], operably linked 5′ to a test intron element [C], operably linked to a coding region [D], which is operably linked to a 3′ UTR element [E]. Alternatively, a second possible expression cassette (Expression Cassette Configuration 2 in FIG. 9) is comprised of a promoter or chimeric promoter element [F], operably linked 5′ to a first leader element or first leader element fragment [G], operably linked 5′ to a test intron element [H], operably linked 5′ to a second leader element or first leader element second fragment [I], operably linked to a coding region [J], which is operably linked to a 3′ UTR element [K]. Further, a third possible expression cassette (Expression Cassette Configuration 3 in FIG. 9) is comprised of a promoter or chimeric promoter element [L], operably linked 5′ to a leader element [M], operably linked 5′ to a first fragment of the coding sequence element [N], operably linked 5′ to an intron element [O] element, operably linked 5′ to a second fragment of the coding sequence element [P], which is operably linked to a 3′ UTR element [Q]. Expression Cassette Configuration 3 is designed to allow splicing of the intron in such a manner as to produce a complete open reading frame without a frame shift between the first and second fragment of the coding sequence.
As discussed above, it may be preferable to avoid using the nucleotide sequence AT or the nucleotide A just prior to the 5′ end of the splice site (GT) and the nucleotide G or the nucleotide sequence TG, respectively just after 3′ end of the splice site (AG) to eliminate the potential of unwanted start codons from being formed during processing of the messenger RNA into the final transcript. The DNA sequence around the 5′ or 3′ end splice junction sites of the intron can thus be modified.
The introns are assayed for an enhancement effect through the ability to enhance expression in transient assay or stable plant assay. For transient assay of intron enhancement, a base plant vector is constructed using methods known in the art. The intron is cloned into a base plant vector which comprises an expression cassette comprised of a constitutive promoter such as the Cauliflower mosaic virus promoter, P-CaMV.35S-enh-1:1:9 (SEQ ID NO: 166), operably linked 5′ to a leader element, L-CaMV.35S-1:1:15 (SEQ ID NO: 167), operably linked 5′ to a test intron element (e.g. one of SEQ ID NOs: 4, 12, 15, 20, 26, 29, 37, 40, 48, 51, 54, 57, 59, 65, 69, 81, 91, 94 and 171), operably linked to a coding sequence for GUS that either possesses a processable intron (GUS-2, SEQ ID NO: 155) or no intron (GUS-1, SEQ ID NO: 154), operably linked to the Nopaline synthase 3′ UTR from A. tumefaciens (T-AGRtu.nos-1:1:13, SEQ ID NO: 158). Protoplast cells derived from corn or other genus plant tissue are transformed with the base plant vector and Luciferase control vectors as described previously in Example 2 above, and assayed for activity. To compare the relative ability of the intron to enhance expression, GUS values are expressed as a ratio of GUS to Luciferase activity and compared with those levels imparted by a construct comprising the constitutive promoter operably linked to a known intron standard such as that as the intron derived from the HSP70 heat shock protein of Zea mays, I-Zm.DnaK-1:1:1 (SEQ ID NO: 165), as well as a construct comprising the constitutive promoter, but without an intron operably linked to the promoter.
For stable plant assay of the introns presented as SEQ ID NOs: 4, 12, 15, 20, 26, 29, 37, 40, 48, 51, 54, 57, 59, 65, 69, 81, 91, 94 and 171, a GUS expression plant transformation vector is constructed similar to the constructs described in the previous examples in which the resulting plant expression vectors contains a right border region from A. tumefaciens, a first expression cassette to test the intron comprised of a constitutive promoter such as the Cauliflower mosaic virus promoter, P-CaMV.35S-enh-1:1:9 (SEQ ID NO: 166), operably linked 5′ to a leader element, L-CaMV.35S-1:1:15 (SEQ ID NO: 167), operably linked 5′ to a test intron element provided herein, operably linked to a coding sequence for GUS that either possesses a processable intron (GUS-2, SEQ ID NO: 155) or no intron (GUS-1, SEQ ID NO: 154), operably linked to the Nopaline synthase 3′ UTR from A. tumefaciens (T-AGRtu.nos-1:1:13, SEQ ID NO: 158); a second transgene selection cassette used for selection of transformed plant cells that confers resistance to glyphosate (driven by the rice Actin 1 promoter), or alternatively, the antibiotic kanamycin (driven by the rice Actin 1 promoter) and a left border region from A. tumefaciens. The resulting plasmids are used to transform corn plants or other genus plants by the methods described above or by Agrobacterium-mediated methods known in the art. Single-copy or low copy number transformants are selected for comparison to single-copy or low copy number transformed plants, transformed with a plant transformation vector identical to the test vector but without the test intron to determine if the test intron provides an intron mediated enhancement effect.
Any of the introns presented as SEQ ID NOs: 4, 12, 15, 20, 26, 29, 37, 40, 48, 51, 54, 57, 59, 65, 69, 81, 91, 94 and 171 can be modified in a number of ways, such as deleting fragments within the intron sequence, which may reduce expression or duplication of fragments with the intron that may enhance expression. In addition, DNA sequences within the intron that may affect the specificity of expression to either particular cells types or tissues and organs can be duplicated or altered or deleted to affect expression and patterns of expression of the transgene. In addition, the introns provided herein can be modified to remove any potential start codons (ATG) that may cause unintentional transcripts from being expressed from improperly spliced introns as different, longer or truncated proteins. Once the intron has been empirically tested, or it has been altered based upon experimentation, the intron is used to enhance expression of a transgene in stably transformed plants that can be of any genus monocot or dicot plant, so long as the intron provides enhancement of the transgene. The intron can also be used to enhance expression in other organisms, such as algae, fungi, or animal cells, so long as the intron provides enhancement or attenuation or specificity of expression of the transgene to which it is operably linked.
Having illustrated and described the principles of the invention, it should be apparent to persons skilled in the art that the invention can be modified in arrangement and detail without departing from such principles. We claim all modifications that are within the spirit and scope of the claims. All publications and published patent documents cited herein are hereby incorporated by reference to the same extent as if each individual publication or patent application is specifically and individually indicated to be incorporated by reference.

## Claims

1. A recombinant DNA molecule comprising a DNA sequence selected from the group consisting of:
a) a DNA sequence comprising SEQ ID NO: 17 and
b) a fragment comprising at least 50 contiguous nucleotides of SEQ ID NO: 17, wherein the fragment has gene-regulatory activity;

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

2. The recombinant DNA molecule of claim 1, wherein said DNA sequence comprises said fragment and has at least 90 percent sequence identity to the DNA sequence of SEQ ID NO:16 or 18 and has gene-regulatory activity.

3. The recombinant DNA molecule of claim 1, wherein said DNA sequence comprises said fragment and has at least 95 percent sequence identity to the DNA sequence of SEQ ID NO:16 or 18 and has gene-regulatory activity.

4. The DNA molecule of claim 1, wherein the heterologous transcribable DNA molecule is a gene of agronomic interest.

5. The recombinant DNA molecule of claim 4, wherein the gene of agronomic interest confers herbicide tolerance in a plant.

6. The recombinant DNA molecule of claim 4, wherein the gene of agronomic interest confers pest resistance in a plant.

7. A construct comprising the recombinant DNA molecule of claim 1.

8. A transgenic plant cell comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of:
a) a DNA sequence comprising SEQ ID NO: 17; and
b) a fragment comprising at least 50 contiguous nucleotides of SEQ ID NO: 17, wherein the fragment has gene-regulatory activity;

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

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

10. The transgenic plant cell of claim 8, wherein said transgenic plant cell is a dicotyledonous plant cell.

11. A transgenic plant, or part thereof, comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of:
a) a DNA sequence comprising SEQ ID NO: 17; and
b) a fragment comprising at least 50 contiguous nucleotides of SEQ ID NO: 17, wherein the fragment has gene-regulatory activity;

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

12. A progeny plant of the transgenic plant of claim 11, wherein the progeny plant comprises said recombinant DNA molecule.

13. A transgenic seed of the transgenic plant of claim 11, wherein the seed comprises said recombinant DNA molecule.

14. A method of expressing a transcribable DNA molecule comprising obtaining a transgenic plant according to claim 11 and cultivating said plant, wherein the transcribable DNA molecule is expressed.

15. A method of producing a transgenic plant comprising:
a) transforming a plant cell with the recombinant DNA molecule of claim 1 to produce a transformed plant cell; and
b) regenerating a transgenic plant from the transformed plant cell.

16. The recombinant DNA molecule of claim 1, wherein said DNA sequence is selected from the group consisting of SEQ ID NOs: 16 and 18.

17. The transgenic plant cell of claim 8, wherein said DNA sequence is selected from the group consisting of SEQ ID NOs: 16 and 18.

18. The transgenic plant, or part thereof, of claim 11, wherein said DNA sequence is selected from the group consisting of SEQ ID NOs: 16 and 18.