# Patent

## Title
Plant regulatory elements and uses thereof

## Abstract

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

## Background
REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. provisional application Ser. No. 61/785,245, filed Mar. 14, 2013, which is incorporated by reference herein in its entirety.


INCORPORATION OF SEQUENCE LISTING
The sequence listing that is contained in the file named “MONS331US.txt,” which is 54.4 kilobytes (as measured in Microsoft Windows®) and was created on Mar. 12, 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, 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 gene regulatory elements for use in plants and constructs comprising the regulatory elements. The invention also provides transgenic plants, plant cells, 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 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: 1-20; (b) a DNA sequence comprising any of SEQ ID NOs: 1-20; and (c) a fragment of any of SEQ ID NOs:1-20, wherein the fragment has gene-regulatory activity; wherein the DNA sequence is operably linked to a 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 about 85 percent, at least about 86 percent at least about 87 percent, at least about 88 percent, at least about 89 percent, at least about 90 percent, at least about 91 percent, at least about 92 percent, at least about 93 percent, at least about 94 percent, at least about 95 percent, at least about 96 percent, at least about 97 percent, at least about 98 percent, or at least about 99 percent sequence identity to the DNA sequence of any of SEQ ID NOs: 1-20. In particular embodiments, the heterologous transcribable DNA molecule comprises a gene of agronomic interest, such as a gene capable of providing herbicide resistance or pest resistance in plants. In still other embodiments, the invention provides a construct comprising a recombinant DNA molecule as provided herein.
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: 1-20; (b) a DNA sequence comprising any of SEQ ID NOs: 1-20; and (c) a fragment of any of SEQ ID NOs:1-20, wherein the fragment has gene-regulatory activity; wherein the DNA sequence is operably linked to a 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: 1-20; (b) a DNA sequence comprising any of SEQ ID NOs: 1-20; and (c) a fragment of any of SEQ ID NOs: 1-20, wherein the fragment has gene-regulatory activity; wherein the DNA sequence is operably linked to a 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 another aspect, the invention provides a method of producing a commodity product from a transgenic plant containing the recombinant DNA molecule of the invention. Commodity products of the invention contain a detectable amount of SEQ ID NOs: 1-20. As used herein, a “commodity product” refers to any composition or product which is comprised of material derived from a transgenic plant, plant part, plant cell, or seed containing the recombinant DNA molecule of the invention. Commodity products include, but are not limited to, processed seeds, grains, plant parts, and meal. Transgenic plants containing the recombinant DNA molecule of the invention can be used to manufacture any commodity product typically acquired from a plant. A commodity product of the invention will contain a detectable amount of DNA corresponding to the recombinant DNA molecule of the invention. Detection of one or more of this recombinant DNA molecule in a sample may be used for determining the content or the source of the commodity product. Any standard method of detection for DNA molecules may be used, including methods of detection disclosed herein.
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.
Also provided by the invention is a codon redesigned Escherichia coli (E. coli) β-glucuronidase (GUS) coding sequence; wherein said codon redesigned GUS coding sequence demonstrates higher expression in a transgenic plant than the native E. coli GUS coding sequence. In one embodiment, the codon redesigned GUS coding sequence can be can be selected from the group consisting of SEQ ID NOs: 29 and 30. The transgenic plant may be a monocotyledonous plant. In one embodiment, the monocotyledonous plant is selected from the group consisting of Maize (Zea mays), Rice (Oryza sativa), Wheat (Triticum), Barley (Hordeum vulgare), Sorghum (Sorghum spp.), Millet, Pearl Millet (Pennisetum glaucum), Finger Millet (Eleusine coracana), Proso Millet (Panicum miliaceum), Foxtail Millet (Setaria italica), Oats (Avena sativa), Triticale, Rye (Secale cereale), Fonio (Digitaria), Onions (Allium spp.), Pineapple (Ananas spp.), Turfgrass, Sugarcane (Saccharum spp.), Palm (Arecaceae), Bamboo (Bambuseae), Banana (Musaceae), Ginger family (Zingiberaceae), Lilies (Lilium), Daffodils (Narcissus), Irises (Iris), Amaryllis, Orchids (Orchidaceae), Cannas, Bluebells (Hyacinthoides), and Tulips (Tulipa). The transgenic plant may also be a dicotyledonous plant. In one embodiment, the dicotyledonous plant is selected from the group consisting of Soybean (Glycine max), Wild Soybean (Glycine soja), Cotton (Gossypium), Tomato (Solanum lycopersicum), Pepper (Piper), Squash (Cucurbita), Pea (Pisum sativum), Alfalfa (Medicago sativa), Medicago truncatula, Beans (Phaseolus), Chick pea (Cicer arietinum), Sunflower (Helianthus annuus), Potato (Solanum tuberosum), Peanut (Arachis hypagaea), Quinoa, Buckwheat (Fagopyrum esculentum), Carob (onia siliqua), Beet (Beta vulgaris), Spinach (Spinacia oleracea), and Cucumber (Cucumis sativus).



BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1a-1c show an alignment between the native E. coli β-glucuronidase (GUS) coding sequence (CR-Ec.uidA-1:1:4, SEQ ID NO: 31) and the codon-redesigned E. coli GUS coding sequence (CR-Ec.uidA_nno-1:1:1, SEQ ID NO:30). The identical nucleotides in the alignment are indicated by an asterisk.



BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, and 23 are promoter sequences.
SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 are leader sequences.
SEQ ID NOs: 25-28 are amplification primer sequences.
SEQ ID NOs: 29 and 30 are codon redesigned GUS coding sequences. SEQ ID NO: 29 comprises a processable intron, while SEQ ID NO: 30 is a contiguous coding sequence.
SEQ ID NO: 31 is the native Escherichia coli β-glucuronidase coding sequence.
SEQ ID NO: 32 is a GUS coding sequence with a processable intron based upon the native E. coli β-glucuronidase of SEQ ID NO: 31.
SEQ ID NOs: 33, 39 and 40 are 3′ UTR sequences.
SEQ ID NOs: 34-37, 41 and 44 are sequences of transcriptional regulatory expression element groups (EXPs) comprising either a promoter sequence operably linked 5′ to a leader sequence which is operably linked 5′ to a to an intron sequence, or in the case of SEQ ID 44, a promoter sequence operably linked 5′ to a leader sequence.
SEQ ID NO: 38 is an intron sequence.
SEQ ID NOs: 42 and 44 are coding sequences for luciferase proteins derived from Photinus pyralis and Renilla reniformis, respectively.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides DNA molecules having gene-regulatory activity in plants. The nucleotide sequences of these DNA molecules are provided as SEQ ID NOs: 1-20. These DNA molecules are, for 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 present 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 genomic or synthetic origin, i.e., a polymer of deoxyribonucleotide bases. As used herein, the term “DNA sequence” refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein corresponds to that of 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 polynucleotide sequences or two optimally aligned polypeptide sequences are identical. An optimal sequence alignment is created by manually aligning two sequences, e.g., a reference sequence and another DNA sequence, to maximize the number of 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: 1-20.
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 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 sequence that when optimally aligned to a reference sequence, provided herein as SEQ ID NOs: 1-20, 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, introns and 3′ UTRs 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 present invention include SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19, including fragments or variants thereof. In specific embodiments of the invention, such DNA molecules and any variants or derivatives thereof as described herein, may be 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” that 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 of a promoter sequence disclosed herein are provided. Promoter fragments may comprise promoter activity, as described above, and may be useful alone or in combination with other promoters and promoter fragments, such as in 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 polynucleotide molecule having promoter activity 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: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19, such as internal or 5′ deletions, for example, can be produced using methods known 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: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 comprised of 3′ deletions in which the TATA box element or equivalent 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 rhythm) effects on expression. Any of the promoters presented as SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 and fragments or enhancers derived therefrom can be used to make chimeric transcriptional regulatory element compositions comprised of any of the promoters presented as SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, and 19 and the fragments or enhancers derived therefrom operably linked to other enhancers and promoters.
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 nucleotide 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 present 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: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 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 are decoded as comprising leader activity.
The leader sequences (5′ UTR) presented as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 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: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 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: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 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 identified from 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 gene constructs leads to increased mRNA and protein accumulation relative to constructs lacking the intron. This effect has been termed “intron mediated enhancement” (IME) of gene expression (Mascarenhas et al., Plant Mol. Biol. 15:913-920, 1990). Introns known to stimulate expression in plants have been identified in maize genes (e.g., tubA1, Adh1, Sh1, and Ubi1), in rice genes (e.g., tpi) and in 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, splicing per se might not be 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.
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. Further, 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 transcribable DNA molecule 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 the Basic Alignment Search Tool (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 found 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 polynucleotide 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., the same or similar expression pattern, 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 naturally occur in bacterial and plant cell transformation. In the invention, a DNA sequence provided as SEQ ID NOs: 1-20 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: 1-20 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 ABI, 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 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: 1-20, 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, 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. No. RE38,446; U.S. Pat. Nos. 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. No. RE37,543; U.S. Pat. Nos. 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.
β-Glucuronidase
The β-glucuronidase (GUS) gene isolated from Escherichia coli K-12 is one of the most widely used report genes in plant biotechnology. The E. coli GUS gene, uidA, is part of the GUS operon on the bacterial chromosome. It is induced by a wide variety of β-D-glucuronides. The GUS enzyme is an exohydrolase that catalyses the hydrolysis of β-D-glucuronides into D-glucuronic acid and the aglycone. E. coli lives in the digestive tract of vertebrates, including man. Vertebrates utilize the glucuronidation pathway to detoxify xenobiotics and endogenous waste compounds such as steroids, aliphatic alcohols, phenol, carboxylic acids, sugars, and various other metabolites. Glucuronidation involves conjugation with D-glucuronic acid. This occurs mainly in the liver, but also occurs in other tissues and organs such as the kidney, the adrenal glands, and the alimentary tract. The glucuronic acid can be utilized by E. coli as a main source for carbon and energy. The E. coli GUS protein therefore provides a means by which the bacterium can degrade the products of the glucuronidation pathway in the alimentary tract of vertebrates to yield glucuronic acid as a carbon and energy source. The aglycones that are also liberated by the GUS enzyme are generally not degraded by the bacterium, but utilized as a shuttle for D-glucuronic acid (Gilissen et al., Transgenic Research, 7: 157-163, 1998).
The use of the E. coli β-glucuronidase gene as a reporter was first described by Jefferson et al. (Proc. Natl. Acad. Sci., 83: 8447-8451, 1986) and has been used in much the same manner as first described since its introduction. The GUS gene is used to monitor plant gene expression and is frequently employed to characterize promoters or other expression elements. However, some plant promoters express at very low levels and may be undetectable using a GUS-based assay. These lower expressing promoters may be valuable to the development of transgenic crops with desirable phenotypes such as improved yield.
Early on in the development of transgenic crop plants, promoters that provided high constitutive expression were most desired. These high constitutive promoters, derived from plant viral genomes such as Cauliflower mosaic virus and Figwort mosaic virus, were used to drive transgenes that conferred herbicide tolerance or insect resistance. As the field of plant biotechnology increases in complexity, newer transgenic traits are being developed that require more specific patterns of expression or lower levels of expression. Overexpression or expression in the wrong plant tissues can lead to unwanted effects in the transformed plant. For example, ectopic expression (expression of a gene in an abnormal place in an organism) of enzyme genes in plants can result in a reduction in the desired end product due to a shortage of precursor at the branching point in a metabolic pathway (Iwase et al., Plant Biotech. 26: 29-38, 2009).
Because transcription factors (TFs) naturally act as master regulators of cellular processes, they are expected to be excellent candidates for modifying complex traits in crop plants, and TF-based technologies are likely to be a prominent part of the next generation of successful biotechnology crops. TF technologies often require optimization, either to reduce unwanted side effects such as growth retardation or to enhance the desired trait to the level at which it is of commercial value. Optimization is frequently approached by modifying expression of the TF transgene. Tissue-specific, developmental, or inducible promoters, rather than the usual constitutive promoters, can be utilized to limit expression of the transgene to the appropriate tissues or environmental conditions (Century et al., Plant Physiology, 147: 20-29, 2008).
Due in part to these developments, there is a need for a more sensitive assay for expression element characterization to identify expression elements that provide a desired level and pattern of expression. The present invention provides an improved, codon redesigned GUS coding sequence which, when operably linked to a promoter, expresses better than the native E. coli GUS coding sequence used commonly in the art. This improved, codon redesigned GUS coding sequence can be used to provide greater assay sensitivity, both quantitatively and qualitatively, and allows for the characterization of promoters and other expression elements that might otherwise not be possible with the native E. coli GUS coding sequence. The improved, codon redesigned GUS coding sequence can be used to characterize expression elements in monocot and dicot plants. Monocot plants useful in practicing the invention include, but are not limited to, Maize (Zea mays), Rice (Oryza sativa), Wheat (Triticum), Barley (Hordeum vulgare), Sorghum (Sorghum spp.), Millet, Pearl Millet (Pennisetum glaucum), Finger Millet (Eleusine coracana), Proso Millet (Panicum miliaceum), Foxtail Millet (Setaria italica), Oats (Avena sativa), Triticale, Rye (Secale cereale), Fonio (Digitaria), Onions (Allium spp.), Pineapple (Ananas spp.), Turfgrass, Sugarcane (Saccharum spp.), Palm (Arecaceae), Bamboo (Bambuseae), Banana (Musaceae), Ginger family (Zingiberaceae), Lilies (Lilium), Daffodils (Narcissus), Irises (Iris), Amaryllis, Orchids (Orchidaceae), Cannas, Bluebells (Hyacinthoides) and Tulips (Tulipa). Dicot plants useful in practicing the invention include, but are not limited to, Soybean (Glycine max), Wild Soybean (Glycine soja), Cotton (Gossypium), Tomato (Solanum lycopersicum), Pepper (Piper), Squash (Cucurbita), Pea (Pisum sativum), Alfalfa (Medicago sativa), Medicago truncatula, Beans (Phaseolus), Chick pea (Cicer arietinum), Sunflower (Helianthus annuus), Potato (Solanum tuberosum), Peanut (Arachis hypagaea), Quinoa, Buckwheat (Fagopyrum esculentum), Carob (onia siliqua), Beet (Beta vulgaris), Spinach (Spinacia oleracea), and Cucumber (Cucumis sativus).
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 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 which contain the recombinant DNA molecule of the invention. Seeds produced using a transgenic plant of the invention can be harvested and used to grow generations of transgenic plants, i.e., progeny plants, of the invention, comprising the construct of this invention and expressing a gene of agronomic interest. Descriptions of breeding methods that are commonly used for different crops can be found in one of several reference books, see, 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 RCc3 promoters and leaders were identified and cloned from genomic DNA of the monocot species Coix (Coix lacryma-jobi), Hairy crabgrass (Digitaria sanguinalis (L.) Scop.), Maiden grass (Miscanthus sinensis f. gracillimus), Gama grass (Tripsacum dactyloides) and Sugarcane (Saccharum officinarum). The RCc3 protein belongs to the prolamin superfamily, which derives its name from the alcohol-soluble proline and glutamine rich storage proteins of cereals. The prolamin superfamily (also called protease inhibitor/lipid-transfer protein/seed storage 2S albumin family; Pfam ID: PF00234) represents one of the most wide-spread protein superfamilies in the plant genome. The members of the prolamin superfamily are abundant in the fruits, nuts, seeds, and vegetables of a variety of plants. They are known to exhibit diverse function including seed storage and protection, lipid binding or transfer, and enzyme inhibition. Lipid transfer proteins (LTPs) belong to the prolamin superfamily and are expressed in a variety of plant tissues. The rice RCc3 protein is an LTP that is expressed in the roots of rice, although not all LTPs proteins are root specific.
DNA amplification primers (presented as SEQ ID NOs: 25-28) were designed using the coding sequences of twenty four (24) LTP proteins from Zea mays, Oryza sativa, Sorghum bicolor and Brachypoium distachyon. The amplification 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.
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 were designed to amplify the regulatory elements. The corresponding DNA molecule for each regulatory element was amplified using standard polymerase chain reaction (PCR) conditions with primers containing unique restriction enzyme sites and genomic DNA isolated from C. lacryma-jobi, D. sanguinalis (L.) Scop., M. sinensis f. gracillimus, T. dactyloides, and S. officinarum. The resulting DNA fragments were ligated into vectors and sequenced.
The DNA sequences of the identified RCc3 promoters and leaders are listed in Table 1. Promoter sequences are provided herein as SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19. Leader sequences are provided herein as SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20.







TABLE 1







RCc3 promoters and leaders isolated from various grass species.









Sequence
SEQ ID



Description
NO:
Genus/species












P-C1.RCc3:3
1
Coix lacryma-jobi


L-C1.RCc3:2
2
Coix lacryma-jobi


P-Ds.RCc3_1:1
3
Digitaria sanguinalis (L.) Scop.


L-Ds.RCc3_1:1
4
Digitaria sanguinalis (L.) Scop.


P-Ds.RCc3_2:1
5
Digitaria sanguinalis (L.) Scop.


L-Ds.RCc3_2:1
6
Digitaria sanguinalis (L.) Scop.


P-Ds.RCc3_3:1
7
Digitaria sanguinalis (L.) Scop.


L-Ds.RCc3_3:1
8
Digitaria sanguinalis (L.) Scop.


P-MISgr.RCc3_1:1
9
Miscanthus sinensis f. gracillimus


L-MISgr.RCc3_1:1
10
Miscanthus sinensis f. gracillimus


P-MISgr.RCc3-2:2
11
Miscanthus sinensis f. gracillimus


L-MISgr.RCc3-2:1
12
Miscanthus sinensis f. gracillimus


P-Td.RCc3_1:1
13
Tripsacum dactyloides


L-Td.RCc3_1:1
14
Tripsacum dactyloides


P-Td.RCc3_2:1
15
Tripsacum dactyloides


L-Td.RCc3_2:1
16
Tripsacum dactyloides


P-Td.RCc3_3:1
17
Tripsacum dactyloides


L-Td.RCc3_3:1
18
Tripsacum dactyloides


P-So.RCc3:2
19
Saccharum officinarum


L-So.RCc3:2
20
Saccharum officinarum









Example 2
Analysis of Regulatory Elements Driving GUS in Transgenic Corn
Corn plants were transformed with vectors, specifically binary plasmid constructs, comprising an RCc3 promoter operably linked to its native RCc3 leader driving expression of the β-glucuronidase (GUS) transgene. The resulting transformed plants were analyzed for GUS protein expression.
The vectors utilized in these experiments were built using cloning methods known in the art. The resulting vectors comprised a right border region from A. tumefaciens; a first transgene expression cassette to assay the RCc3 promoter/leader sequence operably linked to a codon redesigned coding sequence for GUS that possessed a processable intron GOI-Ec.uidA+St.LS1.nno:3 (SEQ ID NO:29) operably linked 5′ to the 3′ UTR from the foxtail millet S-adenosylmethionine synthetase 1 gene (T-SETit.Ams1-1:1:1, SEQ ID NO:159); a second transgene expression 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 using methods known in the art. Expression of GUS conferred by the novel RCc3 promoters and leaders was compared to expression driven by the foxtail millet and rice RCc3 homolog promoters and leaders. Table 2 provides the plasmid constructs, the RCc3 promoter and leader sequences, and the SEQ ID NOs.







TABLE 2







Binary plant transformation plasmids and the associated RCc3 


promoter/leader sequences.













SEQ 

SEQ 


Plasmid
Promoter Sequence
ID
Leader Sequence
ID


Construct
Description
NO:
Description
NO:














pMON264146
P-C1.RCc3:3
1
L-C1.RCc3:2
2


pMON264148
P-Ds.RCc3_1:1
3
L-Ds.RCc3_1:1
4


pMON264088
P-Ds.RCc3_2:1
5
L-Ds.RCc3_2:1
6


pMON264107
P-Ds.RCc3_3:1
7
L-Ds.RCc3_3:1
8


pMON264186
P-MISgr.RCc3_1:1
9
L-MISgr.RCc3_1:1
10


pMON264187
P-MISgr.RCc3-2:2
11
L-MISgr.RCc3-2:1
12


pMON264049
P-Td.RCc3_1:1
13
L-Td.RCc3_1:1
14


pMON264050
P-Td.RCc3_2:1
15
L-Td.RCc3_2:1
16


pMON264147
P-Td.RCc3_3:1
17
L-Td.RCc3_3:1
18


pMON264166
P-So.RCc3:2
19
L-So.RCc3:2
20


pMON264108
P-SETit.Rcc3-1:1:10
21
L-SETit.Rcc3-1:1:2
22


pMON264206
P-Os.Rcc3-1:1:24
23
L-Os.Rcc3-1:1:2
24









In certain instances, plants were transformed using Agrobacterium-mediated transformation methods known in the art and as described in U.S. Patent Application Publication 2009/0138985.
Histochemical GUS analysis was used for qualitative expression analysis of the transformed plants. Whole-tissue sections were incubated with GUS staining solution X-Gluc (5-bromo-4-chloro-3-indolyl-b-glucuronide) (1 mg/me 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 were 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 the 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 microliters. The reaction product, 4-methlyumbelliferone (4-MU), is maximally fluorescent at high pH, where the hydroxyl group is ionized. Addition of a basic solution of sodium carbonate simultaneously stops the assay and adjusts the pH for quantifying the fluorescent product. Fluorescence was measured with excitation at 365 nm, emission at 445 nm, using a Fluoromax-3 (Horiba; Kyoto, Japan) with Micromax Reader, with slit width set at excitation 2 nm, emission 3 nm. The average expression values were provided as pmol 4 MU/μg protein/hour.
The average R0 GUS expression observed for each transformation was recorded and an average expression level and standard error determined based upon the measurements taken of samples derived from multiple transformation events.
Example 3
Enhancers Derived from the Regulatory Elements
Enhancers may be derived from the promoter elements provided herein, such as those presented as SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19. These enhancer elements 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 transcribable DNA molecule, or provide expression of a transcribable DNA molecule 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. Enhancer elements can be cloned to be operably linked 5′ or 3′ to a promoter element derived from a different genus organism or operably linked 5′ or 3′ to additional enhancer 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 organisms, resulting in a chimeric regulatory element. A GUS expression vector may be 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 transgene 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: 38) 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: 32) or no intron (CR-Ec.uidA-1:1:4 (GUS.nat), SEQ ID NO: 31) operably linked to the Nopaline synthase 3′ UTR from A. tumefaciens (T-AGRtu.nos-1:1:13, SEQ ID NO: 39) or the 3′ UTR from the rice lipid transfer protein gene (T-Os.LTP-1:1:1, SEQ ID NO: 40); 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 may be used to transform corn plants or other genus plants by the methods described above or by other methods known in the art. Alternatively, protoplast cells derived from corn or other genus plants may be transformed using methods known in the art to perform transient assays.
GUS expression driven by regulatory elements comprising one or more enhancers may be 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 may be 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 4
Greater Assay Sensitivity with a Codon-Redesigned β-Glucuronidase (GUS)
Plant promoters often express at levels that are below the normal detection threshold of many quantitative assays, yet their expression characteristics may be highly valuable for the expression of certain transgenes. In earlier plant biotechnology, promoters that drove high constititutive expression were desirable and were used to drive transcribable DNA molecules that produced a specific phenotype requiring high constitutive expression, such as herbicide tolerance or insect resistance. These high constitutive promoters were often derived from the genomes of plant viruses rather than plant genomes, for example the 35S promoters derived from Cauliflower mosaic virus and Figwort mosaic virus. Notably, in certain instances, high constitutive expression of certain transcribable DNA molecules may lead to negative consequences such as transgene silencing, off-phenotypes, or yield drag. For example, high expression of the GUS gene in transgenic sugarcane plants using two different sugarcane-derived ubiquitin promoters as well as a maize ubiquitin promoter resulted in post transcriptional gene silencing of the GUS gene (Wei et al., J. Plant Physiol. 160: 1241-1251, 2003).
Further, recently there is demand for promoters that demonstrate specific patterns of expression or express more highly in specific tissues of the plant. For example, ectopic expression of enzyme genes in plants can result in reduction of the desired end product due to a shortage of precursor at the branching point in a metabolic pathway (Iwase et al., Plant Biotech. 26:29-38, 2009). In these instances, it is desirable to use a promoter that expresses the operably linked transcribable DNA molecule in the correct tissue or cell types, or at a particular window of development. Plant genome-derived promoters can often demonstrate desirable tissue, cell, or developmental expression characteristics. Due to the lower expression levels of these plant promoters, expression assays often require the use of enhancers to boost the level of expression to permit detection in a quantitative assay. However, the use of such enhancers often changes the overall expression pattern of the plant promoter.
Improving the expression of the reporter gene used in the assay eliminates the need for enhancement of the plant-derived promoter and, thus, provides a more accurate assessment of the expression pattern conferred by a promoter. This Example demonstrates the use of a codon redesigned GUS coding sequence to improve the quantitative assay sensitivity in characterizing several different EXPs comprised of a promoter sequence, operably linked 5′ to a leader sequence, operably linked 5′ to a to an intron sequence.
Corn plants were transformed with plant expression vectors containing EXP sequences driving expression of either a native Escherichia coli β-glucuronidase (GUS) transgene or codon-redesigned β-glucuronidase (GUS.nno) transgene, and the resulting plants were analyzed for GUS protein expression. The EXP and GUS coding sequences were cloned into binary plasmid constructs using methods known in the art.
The resulting plant expression constructs contain a right border region from A. tumefaciens; a first transgene cassette that demonstrates the assay sensitivity of the two GUS coding sequences, comprised of an EXP operably linked to either a native E. coli GUS coding sequence (CR-Ec.uidA-1:1:4 (GUS.nat), SEQ ID NO: 31) or a codon-redesigned GUS coding sequence (CR-Ec.uidA_nno-1:1:1 (GUS.nno), SEQ ID NO: 30) operably linked 5′ to the 3′ UTR from the rice lipid transfer protein gene (T-Os.LTP-1:1:1, SEQ ID NO: 40); 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. FIGS. 1a through 1c show an alignment between the native GUS coding sequence (CR-Ec.uidA-1:1:4) and the codon redesigned GUS coding sequence (CR-Ec.uidA_nno-1:1:1). The identical nucleotides in the alignment are indicated by an asterisk. The codon redesigned GUS sequence is 77.9% identical to the native GUS coding sequence and has been designed to express better in the plant.
Three (3) different EXP classes were used, each conferring a specific expression pattern. The EXPs EXP-SETit.Cab3+Zm.DnaK:1:1 (SEQ ID NO: 34) and EXP-SETit.Cab3+Zm.DnaK:1:2 (SEQ ID NO: 35) confer a leaf expression profile in corn and are essentially identical, with the exception of a five-nucleotide insertion of 5′-CCGGA-3′ in nucleotide positions 1408 through 1412 of EXP-SETit.Cab3+Zm.DnaK:1:2. The EXP sequence EXP-CaMV.35S-enh+Os.Rcc3+Zm.DnaK:1:5 (SEQ ID NO: 36) provides an enhanced root expression profile in corn. The EXP sequence EXP-Zm.UbqM1:1:2 (SEQ ID NO: 37) provides a high constitutive expression profile in corn. The resulting plasmids were used to transform corn plants using methods known in the art. Table 3 lists the plasmid construct designations, and the corresponding EXP and GUS sequences.







TABLE 3







Plasmid constructs, EXP sequences and expression patterns used to compare


native GUS vs. codon-redesigned GUS coding sequences.















SEQ

SEQ


Plasmid

Expression
ID

ID


Construct
EXP Description
Pattern
NO:
GUS
NO:





pMON122599
EXP-
Leaf
35
CR-Ec.uidA-
31



SETit.Cab3 + Zm.DnaK:1:2


1:1:4



pMON122595
EXP-
Leaf
34
CR-
30



SETit.Cab3 + Zm.DnaK:1:1


Ec.uidA_nno-







1:1:1



pMON144050
EXP-CaMV.35S-
Enhanced
36
CR-Ec.uidA-
31



enh + Os.Rcc3 + Zm.DnaK:1:5 
Root

1:1:4



pMON122597
EXP-CaMV.35S-
Enhanced
36
CR-




enh + Os.Rcc3 + Zm.DnaK:1:5
Root

Ec.uidA_nno-
30






1:1:1



pMON144051
EXP-Zm.UbqM1:1:2
Constitutive
37
CR-Ec.uidA-
31






1:1:4



pMON122598
EXP-Zm.UbqM1:1:2
Constitutive
37
CR-
30






Ec.uidA_nno-







1:1:1









In certain instances, plants were transformed using Agrobacterium-mediated transformation methods known in the art and as described in U.S. Patent Application Publication 2009/0138985.
Histochemical GUS analysis was used for qualitative expression analysis of the transformed plants. Whole tissue sections were incubated with GUS staining solution X-Gluc (5-bromo-4-chloro-3-indolyl-b-glucuronide) (1 mg/ml) for an appropriate length of time, rinsed, and visually inspected for blue coloration. GUS activity was qualitatively determined by direct visual inspection or inspection under a microscope using selected plant organs and tissues. The R0 plants were 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 the 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, emission 3 nm.
The average GUS expression values for the R0 generation transformants are provided in Tables 4, 5, and 6.







TABLE 4







Average R0 generation GUS expression of a native and codon-redesigned 


GUS coding sequence using an EXP with a leaf expression profile.










pMON122599
pMON122595



EXP-
EXP-



SETit.Cab3 + Zm.DnaK:1:2/
SETit.Cab3 + Zm.DnaK:1:1/


Tissue
CR-Ec.uidA-1:1:4
CR-Ec.uidA_nno-1:1:1












V4 Leaf
798
1807


V7 Leaf
230
1863


VT Leaf
508
2097


V4 Root
0
0


V7 Root
0
0


VT Root
14
0


Anther
95
1056


Silk
154
1590


21DAP 
24
31


Embryo




21 DAP 
18
61


Endosperm
















TABLE 5







Average R0 generation GUS expression of a native 


and codon-redesigned GUS coding sequence 


using an EXP with an enhanced root expression profile.












pMON144050
pMON122597




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




enh + Os.Rcc3 + 
enh + Os.Rcc3 + 




Zm.DnaK:1:5/
Zm.DnaK:1:5/



Tissue
CR-Ec.uidA-1:1:4
CR-Ec.uidA_nno-1:1:1















V4 Leaf
0
50



V7 Leaf
0
51



VT Leaf
0
82



V4 Root
26
486



V7 Root
16
257



VT Root
18
343



Anther
19
67



Silk
0
12



21DAP 
14
125



Embryo





21 DAP 
17
45



Endosperm

















TABLE 6







Average R0 generation GUS expression of a 


native and codon-redesigned GUS coding sequence 


using an EXP with a constitutive expression profile.










pMON144051
pMON122598



EXP-
EXP-



Zm.UbqM1:1:2/CR-
Zm.UbqM1:1:2/CR-


Tissue
Ec.uidA-1:1:4
Ec.uidA_nno-1:1:1












V4 Leaf
988
3327


V7 Leaf
963
2771


VT Leaf
1777
3787


V4 Root
693
2149


V7 Root
402
1443


VT Root
776
3170


Anther
2247
3190


Silk
975
3324


21DAP Embryo
511
894


21 DAP Endosperm
791
2298









As can be seen in Tables 4 through 6, there is greater sensitivity in the quantitative assays using the codon-redesigned GUS coding sequence when compared with the native GUS coding sequence. Some variability between the GUS.nno and GUS.nat populations is to be expected, since expression may be affected by insertion sites of the T-DNA; however the overall trend in sensitivity demonstrates much greater sensitivity using GUS.nno. GUS driven by EXP-SETit.Cab3+Zm.DnaK:1:1 (SEQ ID NO: 34) and EXP-SETit.Cab3+Zm.DnaK:1:2 (SEQ ID NO: 35) demonstrated a 2.26- to 8.1-fold greater sensitivity using GUS.nno when compared with GUS.nat. Likewise, the enhanced root profile provided by EXP-CaMV.35S-enh+Os.Rcc3+Zm.DnaK:1:5 (SEQ ID NO: 36) was 16.06- to 19.06-fold greater using GUS.nno than GUS.nat, making this codon-redesigned GUS coding sequence ideal for screening for root promoters, especially those promoters that express at low levels, and may demonstrate GUS levels at or below background levels when using the native GUS coding sequence. The high constitutive expression profile conferred by EXP-Zm.UbqM1:1:2 (SEQ ID NO: 37) demonstrated a 1.42- to 4.09-fold greater quantitative sensitivity when using GUS.nno compared with GUS.nat.
Qualitatively, GUS staining was more sensitive and consistently observed in tissue samples using the codon-redesigned GUS coding sequence. Generally, qualitative staining observations tend to be less sensitive than quantitative assays. The use of the codon-redesigned GUS coding sequence provides better and more consistent microscopic inspections of stained tissues. For example, in root tissues where GUS was driven by EXP-CaMV.35S-enh+Os.Rcc3+Zm.DnaK:1:5 (SEQ ID NO: 36), histochemical staining of the tissues transformed with the codon-redesigned GUS coding-sequence was more pronounced and visible in all V7 root samples of the cortex, epidermis, endodermis, root hair and secondary root tip. In contrast, GUS staining was not observed qualitatively in the corresponding V7 root tissues when the native GUS coding sequence was driven by EXP-CaMV.35S-enh+Os.Rcc3+Zm.DnaK:1:5. The improved codon-redesigned GUS coding sequence, (CR-Ec.uidA_nno-1:1:1, SEQ ID NO: 30), provided greater assay sensitivity and was particularly valuable in measuring expression of promoters that express at low levels.
Example 5
Analysis of Regulatory Elements Driving GUS in Corn Leaf and Root Protoplasts
Corn leaf and root protoplasts were transformed with vectors comprising an RCc3 promoter operably linked to its native RCc3 leader driving expression of the β-glucuronidase (GUS) transgene, and the resulting transformed protoplasts were analyzed for GUS protein expression. The RCc3 promoter and leader sequences were cloned into binary plasmid constructs using methods known in the art and as previously described in Example 2.
Two plasmid constructs for use in co-transformation and normalization of data were also constructed using methods known in the art. Each of these plasmid constructs contained a specific luciferase coding sequence that was driven by a constitutive EXP. The vector pMON19437 comprised a expression cassette with a constitutive promoter operably linked 5′ to an intron, (EXP-CaMV.35S-enh+Zm.DnaK:1, SEQ ID NO: 41), operably linked 5′ to a firefly (Photinus pyralis) luciferase coding sequence (LUCIFERASE:1:3, SEQ ID NO: 42), operably linked 5′ to a 3′ UTR from the Agrobacterium tumefaciens nopaline synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 39). The vector pMON63934 comprised an expression cassette with a constitutive EXP sequence (EXP-CaMV.35S-enh-Lhcb1, SEQ ID NO: 44) operably linked 5′ to a sea pansy (Renilla reniformis) luciferase coding sequence (CR-Ren.hRenilla Lucife-0:0:1, SEQ ID NO: 43), operably linked 5′ to a 3′ UTR from the Agrobacterium tumefaciens nopaline synthase gene (T-AGRtu.nos-1:1:13, SEQ ID NO: 39).
Corn root and leaf protoplasts were transformed using a polyethylene glycol (PEG)-based transformation method, which is well known in the art. Protoplast cells were transformed with pMON19437, pMON63934, and one of the plasmids presented in Table 7. After transformation, the transformed protoplasts were incubated overnight in total darkness. Next, measurement of both GUS and luciferase were conducted by placing aliquots of a lysed preparation of cells transformed as noted 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 e.g., Promega Notes Magazine, No: 57, 1996, p. 02).
Four transformations for each EXP or promoter+leader+intron sequence were performed. The mean expression values for each EXP or promoter+leader+intron sequence were determined from several samples from each transformation. Sample measurements were made using four replicates of each EXP or promoter+leader+intron sequence plasmid construct transformation. Background GUS expression was determined using a negative control plasmid construct which lacked the GUS transgene. The average GUS and luciferase expression levels are provided in Tables 7 (leaf) and 8 (root). In these tables, the firefly luciferase values (e.g., from expression of pMON19437) are provided in the column labeled “FLUC” and the sea pansy luciferase values (e.g., from expression of pMON63934) are provided as in the column labeled “RLUC.” Also provided in Tables 7 and 8 are the average GUS/FLUC and GUS/RLUC ratios which provide a relative measure of expression strength in the protoplast assays.







TABLE 7







Average GUS, FLUC and RLUC values derived from transformed corn leaf protoplasts.
















SEQ







Plasmid
Promoter
ID
Average
Average
Average
Average
Average


Construct
Leader
NO:
GUS
FLUC
RLUC
GUS/FLUC
GUS/RLUC

















pMON264146
P-Cl.RCc3:3
1
5328064.75
105434
253107.5
50.73
21.15



L-Cl.RCc3:2
2


pMON264148
P-Ds.RCc3_1:1
3
773613
147918
338149.5
5.23
2.28



L-Ds.RCc3_1:1
4


pMON264088
P-Ds.RCc3_2:1
5
2883555.75
129947.5
309268.5
22.33
9.45



L-Ds.RCc3_2:1
6


pMON264107
P-Ds.RCc3_3:1
7
1093785
124864.75
306178.75
8.70
3.55



L-Ds.RCc3_3:1
8


pMON264186
P-MISgr.RCc3_1:1
9
2613839.75
128887.25
301412.75
20.45
8.83



L-MISgr.RCc3_1:1
10


pMON264187
P-MISgr.RCc3-2:2
11
2370706.25
149383.5
370443.75
15.95
6.53



L-MISgr.RCc3-2:1
12


pMON264049
P-Td.RCc3_1:1
13
7506585.75
150939.25
368035.5
50.15
20.88



L-Td.RCc3_1:1
14


pMON264050
P-Td.RCc3_2:1
15
4447254.25
155356.25
364604.5
28.78
12.40



L-Td.RCc3_2:1
16


pMON264147
P-Td.RCc3_3:1
17
1100118.75
153451
316691.5
7.13
3.48



L-Td.RCc3_3:1
18


pMON264166
P-So.RCc3:2
19
3062045
143684.5
332394.5
21.55
9.45



L-So.RCc3:2
20


pMON264108
P-SETit.Rcc3-1:1:10
21
147483
129834.25
300917.25
1.15
0.50



L-SETit.Rcc3-1:1:2
22


pMON264206
P-Os.Rcc3-1:1:24
23
184905.25
171440.75
386387.25
1.08
0.50



L-Os.Rcc3-1:1:2
24
















TABLE 8







Average GUS, FLUC and RLUC values derived from transformed corn root protoplasts.
















SEQ







Plasmid
Promoter
ID
Average
Average
Average
Average
Average


Construct
Leader
NO:
GUS
FLUC
RLUC
GUS/FLUC
GUS/RLUC

















pMON264146
P-Cl.RCc3:3
1
185142.3
18310
34502.5
10.18
5.43



L-Cl.RCc3:2
2


pMON264148
P-Ds.RCc3_1:1
3
16306.5
17008
31233
0.98
0.53



L-Ds.RCc3_1:1
4


pMON264088
P-Ds.RCc3_2:1
5
101603.8
19201.25
43298
5.23
2.33



L-Ds.RCc3_2:1
6


pMON264107
P-Ds.RCc3_3:1
7
29196
14483.5
34700.75
2.03
0.88



L-Ds.RCc3_3:1
8


pMON264186
P-MISgr.RCc3_1:1
9
87232
18411.75
44755.75
4.80
1.95



L-MISgr.RCc3_1:1
10


pMON264187
P-MISgr.RCc3-2:2
11
510761.5
19093.75
41948.5
26.98
12.30



L-MISgr.RCc3-2:1
12


pMON264049
P-Td.RCc3_1:1
13
884517.8
23881.75
55790
37.23
16.18



L-Td.RCc3_1:1
14


pMON264050
P-Td.RCc3_2:1
15
91634.5
18385
43509.5
5.03
2.18



L-Td.RCc3_2:1
16


pMON264147
P-Td.RCc3_3:1
17
50257.25
18716.75
34489
2.65
1.45



L-Td.RCc3_3:1
18


pMON264166
P-So.RCc3:2
19
508345.3
22335.25
51655.75
22.98
10.13



L-So.RCc3:2
20


pMON264108
P-SETit.Rcc3-1:1:10
21
8123
17750.75
37872.25
0.45
0.23



L-SETit.Rcc3-1:1:2
22


pMON264206
P-Os.Rcc3-1:1:24
23
336095.3
17709.5
40179.5
19.65
8.63



L-Os.Rcc3-1:1:2
24









As demonstrated in Table 7, all of the RCc3 homolog promoters demonstrated the ability to drive transgene expression in corn leaf protoplasts. Some of the RCc3 homolog promoters drove expression higher than others in this assay based upon the GUS/FLUC and GUS/RLUC ratios. Further, as demonstrated in Table 8 above, all of the RCc3 homolog promoters demonstrated the ability to drive transgene expression in corn root protoplasts to varying degrees.
Example 6
Analysis of Regulatory Elements Driving GUS in Transgenic Corn
Corn plants were transformed with vectors comprising an RCc3 promoter operably linked to its native RCc3 leader driving expression of the β-glucuronidase (GUS) transgene. The resulting transformed plants were analyzed for GUS protein expression.
The RCc3 promoter and leader sequences were cloned into binary plasmid constructs using methods known in the art, such as those described in Example 2. The resulting binary plasmid constructs were pMON264146, pMON264148, pMON264088, pMON264107, pMON264186, pMON264187, pMON264049, pMON264050, pMON264147 and pMON264166. The corn plants were also stably transformed with pMON264108 and pMON264206. Qualitative and quantitative GUS expression analysis was performed as described in Example 2. The plants were assayed at V4, V7 and VT stage of development. Sampling at R1 and R3 is shown. Table 9 shows the average quantitative GUS expression for stably transformed corn plants.







TABLE 9







Average quantitative GUS expression in stably transformed corn plants.




























VT

R3



Plasmid
Promoter
SEQ ID
V4
V4
V7
V7
VT
VT
Flower/
R1
21DAP
R3 21DAP


Construct
Leader
NO:
Leaf
Root
Leaf
Root
Leaf
Root
anthers
Cob/silk
Embryo
Endosperm






















pMON264146
P-Cl.RCc3:3
1
25.15
61.31
20.71
42.64
35.96
95.19
298
125.12
21.97
186.52



L-Cl.RCc3:2
2


pMON264148
P-Ds.RCc3_1:1
3
48.34
36.81
42.49
125.25
69.76
55.44
277.93
58
67.08
115.71



L-Ds.RCc3_1:1
4


pMON264088
P-Ds.RCc3_2:1
5
28.31
51.18
59.2
149.2
70.93
158.32
214.47
120.72
141.85
164.68



L-Ds.RCc3_2:1
6


pMON264107
P-Ds.RCc3_3:1
7
67.1
327.44
85.02
365.51
161.65
202.17
787.25
103.63



L-Ds.RCc3_3:1
8


pMON264186
P-MISgr.RCc3_1:1
9
38.66
40.25
39.7
139.98
105.24
308.24
406.38
239.35
118.54
196.48



L-MISgr.RCc3_1:1
10


pMON264187
P-MISgr.RCc3-2:2
11
25.9
193.25
42.13
291.5
48.02
549.37
87.89
41.83



L-MISgr.RCc3-2:1
12


pMON264049
P-Td.RCc3_1:1
13
283.86
238.31



L-Td.RCc3_1:1
14


pMON264050
P-Td.RCc3_2:1
15
51.82
653.38



L-Td.RCc3_2:1
16


pMON264147
P-Td.RCc3_3:1
17
42.49
55.87
41.49
197.51
117.77
282.63
1182.96
938.3
815.36
1240.92



L-Td.RCc3_3:1
18


pMON264166
P-So.RCc3:2
19
34.11
215.86
125.91
855.23
79.33
237.25
347.99
177.13



L-So.RCc3:2
20









As demonstrated in Table 9, all of the RCc3 promoter homologs were able to drive GUS transgene expression in stably transformed corn plants. Further, each promoter had a pattern of expression that was unique to the specific promoter. For example, expression in VT flower/anther differed amongst the RCc3 promoter homologs. Expression driven by P-Td.RCc3_3:1 (SEQ ID NO: 17) was the highest expression observed for all the promoters, while expression driven by P-MISgr.RCc3-2:2 (SEQ ID NO: 11) was the lowest. With respect to R1 Cob/silk expression, P-Td.RCc3_3:1 (SEQ ID NO: 17) demonstrated the highest expression in these tissues and P-MISgr.RCc3-2:2 (SEQ ID NO: 11) expressed the least. Expression driven by P-Td.RCc3_3:1 (SEQ ID NO: 17) increased in later developing tissues. Expression increased in the root from V4 to VT stage and was even higher in VT flowers/anthers, R1 Cob/silk and R3 21DAP embryo and endosperm. Expression driven by P-Td.RCc3_3:1 was highest amongst the RCc3 promoter homologs in VT flowers/anthers, R1 Cob/silk, and R3 21DAP embryo and endosperm.
With respect to leaf and root expression, some of the RCc3 promoter homologs demonstrated higher expression in the root relative to the leaf. Table 10 shows the root-to-leaf expression ratios for all of assayed RCc3 promoters.







TABLE 10







Root/Leaf expression ratios for stably transformed corn plants.














SEQ













Plasmid
Promoter
ID
Average Root/Leaf












Construct
Leader
NO:
V4
V7
VT















pMON264146
P-C1.RCc3:3
1
2.44
2.06
2.65



L-C1.RCc3:2
2





pMON264148
P-Ds.RCc3_1:1
3
0.76
2.95
0.79



L-Ds.RCc3_1:1
4





pMON264088
P-Ds.RCc3_2:1
5
1.81
2.52
2.23



L-Ds.RCc3_2:1
6





pMON264107
P-Ds.RCc3_3:1
7
4.88
4.30
1.25



L-Ds.RCc3_3:1
8





pMON264186
P-MISgr.RCc3_1:1
9
1.04
3.53
2.93



L-MISgr.RCc3_1:1
10





pMON264187
P-MISgr.RCc3-2:2
11
7.46
6.92
11.44



L-MISgr.RCc3-2:1
12





pMON264049
P-Td.RCc3_1:1
13
0.84





L-Td.RCc3_1:1
14





pMON264050 
P-Td.RCc3_2:1
15
12.61





L-Td.RCc3_2:1 
16





pMON264147 
P-Td.RCc3_3:1
17
1.31
4.76
2.40



L-Td.RCc3_3:1 
18





pMON264166
P-So.RCc3:2
19
6.33
6.79
2.99



L-So.RCc3:2
20









As demonstrated in Table 10, each RCc3 promoter homolog demonstrated different ratios of root-to-leaf expression and different patters from V4 to VT stage. For example, P-C1.RCc3:3 (SEQ ID NO: 1) maintained a similar ratio of expression from V4 through VT with a slight decline occurring at V7 stage. Expression in the root as seen in Table 9 dropped slightly from V4 to V7 and then increased by VT stage. The promoter P-Ds.RCc3_3:1 (SEQ ID NO: 7) demonstrated a change in expression ratios from V4 through VT stage with higher expression in the root relative to the leaf in V4 and V7 stage and then a shift approximating equal expression in the leaf relative to the root at VT stage (1.25). With this promoter the average expression shown in Table 9 demonstrates an increase in expression in the leaf from V4 to VT stage while expression in the root declined from V7 to VT stage. The promoter P-So.RCc3:2 (SEQ ID NO: 19) maintained a ratio of root-to-leaf expression of 6.33 at V4 and 6.79 at V7 stage, but then dropped to 2.99 at VT stage. However, expression with this promoter increased 3.69 and 3.96 fold in the leaf and root, respectively, from V4 to V7 stage and then decreased to 2.33 and 1.10 relative to V4 at VT stage.
Notably, not all promoters had a higher root-to-leaf ratio. For example, the promoters P-Ds.RCc3_1:1 (SEQ ID NO: 3) and P-Td.RCc3_1:1 (SEQ ID NO: 13) had root/leaf ratios less than one at V4 stage. However, expression driven by P-Td.RCc3_1:1 was 6.6 fold greater than P-Ds.RCc3_1:1 in V4 root. The highest ratio of root/leaf at V4 stage was achieved using P-Td.RCc3_2:1 (SEQ ID NO: 15). The ratio of root/leaf expression driven by P-Ds.RCc3_1:1 increased from V4 (0.76) to V7 (2.95) and then returned to a ratio similar to that at V4 (0.79).
The promoter P-MISgr.RCc3-2:2 (SEQ ID NO: 11) demonstrated an increase in expression in both leaf and root from V4 to VT stage. This promoter had a root-to-leaf ratio greater than 6.9 throughout all three stages, but the ratio went from 7.46 at V4 stage to 6.92 at V7 stage and then climbed to 11.44 at VT stage. Expression driven by P-MISgr.RCc3-2:2 increased in the leaf and root from V4 to VT stage.
Each of the RCc3 homolog promoters demonstrated patterns of expression in stably transformed corn that could not necessarily be predicted by virtue of being derived from homologous genes, especially when used to transform a heterologous species such as corn. Most of the promoters demonstrated higher expression in the root with respect to the leaf at some point either in V4, V7 or VT stage or in all stages assayed. Notably, the magnitude of expression differed extensively between the promoters. The unique expression properties of each of the RCc3 promoter homologs make some more suitable than others for certain types of transcribable DNA molecule expression. For example, expression of a transcribable DNA molecule that may be critical to the assimilation of a nutrient in the soil and which is best expressed at a later stage of development when the plant is about to begin reproduction and produce seed, may benefit best from a promoter such as P-MISgr.RCc3-2:2 (SEQ ID NO: 11) which increases expression in the root around VT stage.
* * * * * * *
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 with at least 99 percent sequence identity to the full length of SEQ ID NO: 13, wherein the sequence has gene-regulatory activity;
b) a DNA sequence comprising SEQ ID NO: 13; and
c) a fragment comprising at least 600 contiguous nucleotides of SEQ ID NO: 13, wherein the fragment has gene-regulatory activity;
wherein said sequence is operably linked to a heterologous transcribable polynucleotide molecule.

2. The recombinant DNA molecule of claim 1, wherein said DNA sequence has the DNA sequence of SEQ ID NO: 13.

3. The recombinant DNA molecule of claim 1, wherein the heterologous transcribable polynucleotide molecule comprises a gene of agronomic interest.

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

5. The recombinant DNA molecule of claim 3, wherein the gene of agronomic interest confers pest resistance in plants.

6. A transgenic plant cell comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of:
a) a DNA sequence with at least 99 percent sequence identity to the full length of SEQ ID NO: 13, wherein the sequence has gene-regulatory activity;
b) a DNA sequence comprising SEQ ID NO: 13; and
c) a fragment comprising at least 600 contiguous nucleotides of SEQ ID NO: 13, wherein the fragment has gene-regulatory activity;
wherein said sequence is operably linked to a heterologous transcribable polynucleotide molecule.

7. The transgenic plant cell of claim 6, wherein said transgenic plant cell is a monocotyledonous plant cell.

8. The transgenic plant cell of claim 6, wherein said transgenic plant cell is a dicotyledonous plant cell.

9. 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 99 percent sequence identity to the full length of SEQ ID NO: 13, wherein the sequence has gene-regulatory activity;
b) a DNA sequence comprising SEQ ID NO: 13; and
c) a fragment comprising at least 600 contiguous nucleotides of SEQ ID NO: 13, wherein the fragment has gene-regulatory activity;
wherein said sequence is operably linked to a heterologous transcribable polynucleotide molecule.

10. A progeny plant of the transgenic plant of claim 9, or a part thereof, wherein the progeny plant or part thereof comprises said recombinant DNA molecule.

11. A transgenic seed of the transgenic plant of claim 9, wherein the seed comprises the recombinant DNA molecule.

12. A method of producing a commodity product comprising obtaining a transgenic plant or part thereof according to claim 9 and producing the commodity product therefrom.

13. The method of claim 12, wherein the commodity product is processed seeds, grains, plant parts, or meal.

14. 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.