Plant regulatory elements and uses thereof

Abstract

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

Description

[0001] Citation of relevant applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 016,904, filed April 28, 2020, the entire contents of which are incorporated herein by reference.

[0003] Merging of sequence lists

[0004] The file named "MONS481WO-sequence_listing.txt" contains a sequence list of 13,550 bytes (as shown in Microsoft...). This article (as measured in China) was created on April 22, 2021, submitted electronically, and is incorporated into this paper by reference. Technical Field

[0005] This invention relates to the fields of plant molecular biology and plant genetic engineering. More specifically, this invention relates to DNA molecules that can be used to regulate gene expression in plants. Background Technology

[0006] Regulatory elements are genetic elements that regulate gene activity by modulating the transcription of operably linked, transcribed DNA molecules. These elements can include promoters, leader sequences, introns, and 3′ untranslated regions, and are used in plant molecular biology and plant genetic engineering. Summary of the Invention

[0007] This invention provides gene regulatory elements for plants. It also provides recombinant DNA molecules comprising regulatory elements. Furthermore, it provides transgenic plant cells, plants, and seeds comprising regulatory elements. In one embodiment, the regulatory element is operatively linked to a transcribed DNA molecule. In some embodiments, the transcribed DNA molecule may be heterologous relative to the regulatory sequence. Therefore, in specific embodiments, the regulatory element sequence provided by this invention can be defined as operatively linked to a heterologous transcribed DNA molecule. This invention also provides methods for using regulatory elements and for preparing and using recombinant DNA molecules comprising regulatory elements, as well as transgenic plant cells, plants, and seeds comprising regulatory elements operatively linked to transcribed DNA molecules.

[0008] Therefore, in one aspect, the present invention provides a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a sequence having at least about 85% sequence identity with any of SEQ ID NO:1-5; (b) a sequence comprising any of SEQ ID NO:1-5; and (c) a fragment of any of SEQ ID NO:1-5, wherein the fragment has gene regulatory activity; wherein the sequence is operatively linked to a heterologous transcribed DNA molecule. The term "heterologous transcribed DNA molecule" means that the transcribed DNA molecule is heterologous relative to the polynucleotide sequence operatively linked thereto. In a specific embodiment, the recombinant DNA molecule comprises a DNA sequence having at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the DNA sequences in SEQ ID NO:1-5.

[0009] On the other hand, this document provides transgenic plant cells comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: (a) a sequence having at least about 85% sequence identity with any of SEQ ID NO:1-5; (b) a sequence comprising any of SEQ ID NO:1-5; and (c) a fragment of any of SEQ ID NO:1-5, wherein the fragment has gene regulatory activity; wherein the DNA sequence is operatively ligated to a heterologous transcribed DNA molecule. In some embodiments, the transgenic plant cell is a monocotyledonous plant cell. In other embodiments, the transgenic plant cell is a dicotyledonous plant cell.

[0010] In another aspect, this document also provides a transgenic plant or a portion thereof comprising a recombinant DNA molecule comprising a DNA sequence selected from the group consisting of: a) a sequence having at least 85% sequence identity with any of SEQ ID NO:1-5; b) a sequence comprising any of SEQ ID NO:1-5; and c) a fragment of any of SEQ ID NO:1-5, wherein the fragment has gene regulatory activity; wherein the sequence is operatively linked to a heterologous transcribed DNA molecule. In a specific embodiment, the transgenic plant is a progeny plant of any generation comprising the recombinant DNA molecule. This document also provides transgenic seeds comprising a recombinant DNA molecule that produces such transgenic plants upon growth.

[0011] In another aspect, the present invention provides a method for producing a commercial product, comprising obtaining a transgenic plant or a portion thereof containing the recombinant DNA molecule of the present invention and producing the commercial product therefrom. In one embodiment, the commercial product is seeds, processed seeds, protein concentrates, protein isolates, starch, grains, plant parts, seed oils, biomass, flour, and coarse flour.

[0012] In another aspect, the present invention provides a method for producing a transgenic plant comprising the recombinant DNA molecule of the present invention, the method comprising transforming plant cells with the recombinant DNA molecule of the present invention to produce transformed plant cells and regenerating the transgenic plant from the transformed plant cells.

[0013] Sequence Summary

[0014] SEQ ID NO:1 is a DNA sequence of the regulatory expression element group or EXP, namely EXP-ANDge.Ubq:5, which contains a promoter (P-ANDge.Ubq:7) operatively linked to the 5′ of a leader sequence (L-ANDge.Ubq:2) operatively linked to the 5′ of an intron (I-ANDge.Ubq:2), derived from Andropogongerardii.

[0015] SEQ ID NO:2 is the DNA sequence of the promoter, namely P-ANDge.Ubq:7, which is derived from *Miscanthus sinensis*.

[0016] SEQ ID NO:3 is the leader sequence or 5′UTR, i.e., L-ANDge.Ubq:2, of DNA, derived from *Miscanthus sinensis*.

[0017] SEQ ID NO:4 is the DNA sequence of the intron I-ANDge.Ubq:2, which is derived from *Miscanthus sinensis*.

[0018] SEQ ID NO:5 is the DNA sequence of the 3′UTR, namely T-ARUdo.TubA:1, which is derived from Arundo donax.

[0019] SEQ ID NO:6 is the DNA sequence of EXP, namely EXP-CaMV.35S+Zm.DnaK:12.

[0020] SEQ ID NO:7 is a 3′UTR DNA sequence, namely T-Sb.Nltp4-1:1:2, derived from dicolor sorghum.

[0021] SEQ ID NO:8 is a synthetic coding sequence (GUS, GOI-Ec.uidA+St.LS1.nno:1) optimized for plant expression of β-glucuronidase, which has a processable intron derived from the potato light-induced tissue-specific St-LS1 gene (Genbank accession number: X04753). Detailed Implementation

[0022] This invention provides regulatory elements with gene-regulating activity in plants. The nucleotide sequences of these regulatory elements are provided as SEQ ID NO:1-5. These regulatory elements are capable of influencing the expression of operably linked, transcribed DNA molecules in plant tissues, and thus regulating gene expression of operably linked transgenes in transgenic plants. This invention also provides methods for modifying, producing, and using recombinant DNA molecules containing the provided regulatory elements. This invention further provides compositions comprising transgenic plant cells, plants, plant parts, and seeds containing the recombinant DNA molecules of this invention, and methods for their preparation and use.

[0023] The following definitions and methods are provided to better define the present invention and to guide those skilled in the art in practicing it. Unless otherwise stated, the terminology should be understood in accordance with its conventional usage by those skilled in the art.

[0024] DNA molecules

[0025] As used herein, the term “DNA” or “DNA molecule” refers to a double-stranded DNA molecule of genomic or synthetic origin, that is, a polymer of deoxyribonucleotide bases or a DNA molecule read from the 5′ (upstream) end to the 3′ (downstream) end. As used herein, the term “DNA sequence” refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein corresponds to Title 37 of § 1.822 of the United States Federal Code and is set forth in the tables of WIPO Standard ST.25 (1998), Annex 2, Tables 1 and 3.

[0026] As used herein, a “recombinant DNA molecule” is a DNA molecule that comprises a combination of DNA molecules that would not naturally exist together without human intervention. For example, a recombinant DNA molecule can be a DNA molecule composed of at least two DNA molecules that are heterologous to each other, a DNA molecule containing a DNA sequence that deviates from the DNA sequence that exists in nature, a DNA molecule containing a synthetic DNA sequence, or a DNA molecule that has been incorporated into the host cell through genetic transformation or gene editing.

[0027] In this application, references to "isolated DNA molecule" or equivalent terms or phrases are intended to refer to a DNA molecule that exists alone or in combination with other compositions but is not present in its natural environment. For example, nucleic acid elements (such as coding sequences, intron sequences, nontranslation leader sequences, promoter sequences, transcription termination sequences, etc.) naturally present in the DNA of an organism's genome are not considered "isolated" as long as the element is located within the organism's genome and at its naturally present location within the genome. However, each of these elements and sub-parts of these elements will be considered "isolated" within the scope of this disclosure as long as the element is not within the organism's genome and at its naturally present location within the genome. Similarly, the nucleotide sequence encoding an insecticidal protein or any naturally present insecticidal variant of that protein will be an isolated nucleotide sequence, provided that the nucleotide sequence is not present in the DNA of the bacteria encoding the protein sequence. For the purposes of this disclosure, synthetic nucleotide sequences encoding the amino acid sequence of a naturally present insecticidal protein will be considered isolated. For the purposes of this disclosure, any transgenic nucleotide sequence (i.e., a nucleotide sequence of DNA inserted into the genome of a plant or bacterial cell or present in an extrachromosomal vector) will be considered an isolated nucleotide sequence, whether or not such nucleotide sequence is present in a plasmid or similar structure used to transform the cell, in the genome of a plant or bacteria, or in a detectable amount in a tissue, progeny, biological sample, or commercial product derived from a plant or bacteria.

[0028] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide sequences or two optimally aligned polypeptide sequences are identical. Optimal sequence alignments are achieved by manually aligning two sequences (e.g., a reference sequence and another sequence) to maximize the number of nucleotide matches in the alignment through appropriate internal nucleotide insertions, deletions, or gaps. As used herein, the term "reference sequence" refers to the DNA sequences provided as SEQ ID NO:1-5.

[0029] As used herein, the term “% sequence identity” or “% identity” is an identity score multiplied by 100. The “identity score” of the sequence best aligned to the reference sequence is the number of nucleotide matches in the best alignment divided by the total number of nucleotides in the reference sequence, for example, the total number of nucleotides in the entire length of the reference sequence. Therefore, one embodiment of the present invention provides a DNA molecule comprising a sequence that, when best aligned with a reference sequence provided herein as SEQ ID NO:1-5, has at least about 85% identity, at least about 86% identity, at least about 87% identity, at least about 88% identity, at least about 89% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, or at least about 100% identity.

[0030] Control element

[0031] Regulatory elements such as promoters, leader sequences (also known as 5'UTRs), enhancers, introns, and transcription termination regions (or 3'UTRs) play a holistic role in overall gene expression in living cells. As used herein, the term "regulatory element" refers to a DNA molecule that has gene regulatory activity. As used herein, the term "gene regulatory activity" refers to the ability to influence the expression of operably linked transcribed DNA molecules, for example, by affecting the transcription and / or translation of operably linked transcribed DNA molecules. Regulatory elements that function in plants, such as promoters, leader sequences, enhancers, introns, and 3'UTRs, are suitable for modifying plant phenotypes via genetic engineering.

[0032] As used herein, a “regulatory expression element set” or “EXP” sequence can refer to a group of operatively linked regulatory elements, such as enhancers, promoters, leader sequences, and introns. For example, a regulatory expression element set may include, for example, a promoter operatively linked at 5′ to a leader sequence and operatively linked at 5′ to an intron sequence. An EXP that can be used to practice this invention is represented as SEQ ID NO:1.

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

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

[0035] Promoters can serve as regulatory elements for controlling the expression of operably linked, transcribed DNA molecules. As used herein, the term "promoter" generally refers to a DNA molecule involved in recognizing and binding RNA polymerase II and other proteins, such as trans-acting transcription factors, to initiate transcription. Promoters can initially be isolated from the 5′ untranslated region (5′UTR) of a genomic copy of a gene, or, for the purposes of this disclosure, the promoters provided herein consist of a promoter operably linked to a leader sequence at the 5′ end. Alternatively, promoters can be DNA molecules generated or manipulated synthetically. Promoters can also be chimeric. Chimeric promoters are generated via the fusion of two or more heterologous DNA molecules. Promoters that can be used in the practice of the present invention are represented as SEQ ID NO:2, or fragments or variants thereof. In specific embodiments of the invention, the claimed DNA molecule and any variants or derivatives thereof as described herein are further defined as containing promoter activity, i.e., capable of acting as a promoter in a host cell, such as in transgenic plants. In another specific implementation, a fragment may be defined as exhibiting the promoter activity of the initiator promoter molecule from which it originates, or the fragment may contain a “minimal promoter” that provides a basic level of transcription and consists of a TATA box or equivalent DNA sequence for recognizing and binding the RNA polymerase II complex to initiate transcription.

[0036] In one embodiment, a fragment of the promoter sequence disclosed herein is provided. The promoter fragment may contain promoter activity, as described above, and may be used alone or in combination with other promoters and promoter fragments, such as for constructing chimeric promoters, or in combination with other expression elements and expression element fragments. In a specific embodiment, a promoter fragment is provided comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, or at least about 1000 consecutive nucleotides or longer of a DNA molecule having promoter activity as disclosed herein. Methods for generating such fragments from a starter promoter molecule are well known in the art.

[0037] Compositions derived from promoter elements (such as internal or 5′ deletions) of, for example, SEQ ID NO:2 can be produced using methods known in the art to improve or alter expression, including by deleting elements that have a positive or negative effect on expression; replicating elements that have a positive or negative effect on expression; and / or replicating or deleting elements that have a tissue or cell-specific effect on expression. Compositions derived from promoter elements of SEQ ID NO:2 (including those containing a 3′ deletion of a TATA box element or its equivalent sequence and a downstream sequence) can be used, for example, to prepare enhancer elements. Further deletions can be made to remove any elements that have a positive or negative, tissue-specific, cell-specific, or time-specific effect on expression (such as, but not limited to, circadian rhythms). Promoter elements as provided in SEQ ID NO:2 and their derived fragments or enhancers can be used to prepare chimeric transcriptional regulatory element compositions.

[0038] According to the present invention, the presence of known promoter elements, i.e., DNA sequence features such as TATA boxes and other known transcription factor binding site motifs, can be analyzed in the promoter or promoter fragment. Those skilled in the art can use the identification of such known promoter elements to design promoter variants with expression patterns similar to the original promoter.

[0039] As used herein, the term "leader sequence" refers to a DNA molecule isolated from the untranslated 5′ region (5′UTR) of a gene and is generally defined as a nucleotide segment between the transcription start site (TSS) and the protein-coding sequence start site. Alternatively, the leader sequence can be a synthetically generated or manipulated DNA element. The leader sequence can be used as a 5′ regulatory element to regulate the expression of operatively linked transcribed DNA molecules. The leader sequence molecule can be used with a heterologous promoter or with its native promoter. A leader sequence that can be used in the practice of the present invention is represented as SEQ ID NO:3, or a fragment or variant thereof. In a specific embodiment, such a DNA sequence can be defined as capable of functioning as a leader sequence in a host cell, including, for example, transgenic plant cells. In one embodiment, such a sequence is decoded to contain leader sequence activity.

[0040] The leader sequence (also known as the 5′UTR) of SEQ ID NO:3 may contain a regulatory element or may employ a secondary structure that can effect the transcription or translation of operably linked transcribed DNA molecules. The leader sequence of SEQ ID NO:3 may be used according to the present invention to prepare a chimeric regulatory element that influences the transcription or translation of operably linked transcribed DNA molecules.

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

[0042] In plants, the inclusion of certain introns in a gene construct leads to increased mRNA and protein accumulation compared to constructs lacking introns. This effect is known as "intron-mediated enhancement" (IME) of gene expression. Introns known to stimulate expression in plants have been identified in maize genes (e.g., tubA1, Adh1, Sh1, and Ubi1), rice genes (e.g., tpi), and dicotyledonous genes such as those from petunia (e.g., rbcS), potato (e.g., st-ls1), and from Arabidopsis thaliana (e.g., ubq3 and pat1). Deletion or mutation within the splice site of an intron has been shown to decrease gene expression, suggesting that IME may require splicing. However, IME in dicotyledons has been demonstrated through point mutations within the splice site of the pat1 gene in Arabidopsis. Repeated use of the same intron in a single plant has been shown to be disadvantageous. In these cases, it is necessary to collect basic control elements to construct appropriate recombinant DNA elements. An exemplary intron that can be used to practice this invention is represented by SEQ ID NO:4.

[0043] As used herein, the terms “3′ transcription termination molecule,” “3′ untranslated region,” or “3′UTR” refer to the DNA molecule used during transcription of the untranslated region of the 3′ portion of an mRNA molecule. The 3′ untranslated region of an mRNA molecule can be generated through specific cleavage and 3′ polyadenylation, also known as a polyadenylation tail. The 3′UTR can be operatively linked to and located downstream of a transcribed DNA molecule and may contain polyadenylation signals and other regulatory signals capable of influencing transcription, mRNA processing, or gene expression. The polyadenylation tail is thought to play a role in mRNA stability and the initiation of translation. Examples of 3′ transcription termination molecules in the art are the 3′ region of carmine synthase, the 3′ region of wheat hsp17, the 3′ region of the pea rubisco small subunit, the 3′ region of cotton E6, and the 3′UTR of Job's tears.

[0044] 3′UTRs are commonly used for recombinant expression of specific DNA molecules. Weak 3′UTRs can lead to readthroughs, which can affect the expression of DNA molecules located in adjacent expression cassettes. Proper control of transcription termination can prevent readthroughs to downstream DNA sequences (e.g., other expression cassettes) and can further allow efficient recycling of RNA polymerase to improve gene expression. Efficient termination of transcription (release of RNA polymerase II from DNA) is a prerequisite for restarting transcription, thus directly affecting the overall level of transcription. After transcription termination, mature mRNA is released from the site of synthesis, and the template is transported to the cytoplasm. Eukaryotic mRNA accumulates in vivo as polyadenylates, making it difficult to detect transcription termination sites using conventional methods. However, predicting functional and efficient 3′UTRs using bioinformatics methods is challenging because there are no conserved DNA sequences that allow for easy prediction of efficient 3′UTRs.

[0045] From a practical perspective, the following characteristics are generally beneficial for the 3′UTR used in expression cassettes. First, the 3′UTR should be able to efficiently and effectively terminate the transcription of the transgene and prevent the transcript from reading through to any adjacent DNA sequences, which may consist of another expression cassette (as in the case of multiple expression cassettes in a single transfer DNA (T-DNA)) or adjacent chromosomal DNA inserted into the T-DNA. Second, the 3′UTR should not result in a reduction in the transcriptional activity conferred by the promoter, leader sequence, enhancer, and introns that drive the expression of the DNA molecule. Finally, in plant biotechnology, the 3′UTR is commonly used to initiate the amplification reaction of reverse transcribed RNA extracted from transformed plants and is used for: (1) assessing the transcriptional activity or expression of the expression cassette integrated into the plant chromosome; (2) assessing the copy number of the insertion within the plant DNA; and (3) assessing the conjugation of seeds obtained after breeding. The 3′UTR is also used for the amplification reaction of DNA extracted from transformed plants to characterize the integrity of the insertion cassette. The 3′UTR that can be used to practice the present invention is represented by SEQ ID NO:5.

[0046] As used herein, the term "enhancer" or "enhancer element" refers to a cis-acting regulatory element, also known as a cis-element, that confers aspects of the overall expression pattern but is generally insufficient to drive transcription alone, operatively linking transcribed DNA molecules. Unlike promoters, enhancer elements typically do not contain a transcription start site (TSS) or TATA box or equivalent DNA sequence. Promoters or promoter fragments may naturally contain one or more enhancer elements that influence transcription of operatively linked DNA sequences. Enhancer elements may also be fused to promoters to produce chimeric promoter cis-elements, which confer aspects of the overall regulation of gene expression.

[0047] Many promoter enhancer elements are believed to bind DNA-binding proteins and / or influence DNA topology, producing local conformations that selectively allow or restrict RNA polymerase access to the DNA template or promote selective opening of the double helix at the transcription start site. Enhancer elements can also bind transcription factors that regulate transcription. Some enhancer elements bind more than one transcription factor, and transcription factors can interact with more than one enhancer domain with varying affinities. Enhancer elements can be identified using a variety of techniques, including deletion analysis (i.e., deletion of one or more nucleotides from the 5′ end or within the promoter); DNA-binding protein analysis using DNase I footprinting analysis, methylation interference, electrophoretic mobility shift analysis, in vivo genomic footprinting analysis via ligation-mediated polymerase chain reaction (PCR), and other conventional analyses; or DNA sequence similarity analysis using conventional DNA sequence comparison methods such as BLAST with known cis-element motifs or enhancer elements as target sequences or target motifs. The fine structure of enhancer domains can be further investigated by mutagenesis (or substitution) of one or more nucleotides or by other conventional methods known in the art. Enhancer elements can be obtained by chemical synthesis or by isolation from regulatory elements containing such elements, and they can be synthesized with additional flanking nucleotides containing available restriction enzyme sites to facilitate subsequence manipulation. Therefore, the design, construction, and use of enhancer elements according to the methods disclosed herein for regulating the expression of operably linked transcribed DNA molecules are covered in this invention. Enhancers can be derived from the promoter represented by SEQ ID NO:2.

[0048] As used herein, the term "chimerism" refers to a single DNA molecule produced by fusing a first DNA molecule to a second DNA molecule, wherein neither the first nor the second DNA molecule would normally exist in this configuration (i.e., fused together). Therefore, chimeric DNA molecules are novel DNA molecules that do not normally exist in nature. As used herein, the term "chimeric promoter" refers to a promoter produced by such manipulation of a DNA molecule. Chimeric promoters can bind two or more DNA fragments, for example, through the fusion of a promoter with an enhancer element. Therefore, the design, construction, and use of chimeric promoters according to the methods disclosed herein for regulating the expression of operatively linked, transcribed DNA molecules are covered within this invention.

[0049] Chimeric regulatory elements can be designed to comprise a variety of constituent elements 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 chimeric regulatory elements may consist of the same constituent elements or variants of the same constituent elements, but differ in that they include one or more ligation DNA sequences that allow for operative linking of the constituent elements. In this invention, the DNA sequences provided as SEQ ID NO: 1-5 can provide reference sequences for the regulatory element, wherein the constituent elements constituting the reference sequence can be linked by methods known in the art and may contain substitutions, deletions, and / or insertions of one or more nucleotides, or mutations that occur naturally during bacterial and plant cell transformations.

[0050] As used herein, the term "variant" refers to a second DNA molecule, such as a regulatory element, whose composition is similar to but not identical to that of a first DNA molecule, and wherein the second DNA molecule retains the general functionality of the first DNA molecule, i.e., the same or similar expression pattern, for example, through more or less equivalent transcriptional activity. A variant can be a shortened or truncated form of the first DNA molecule or a modified form of the first DNA molecule, such as a DNA molecule with different restriction enzyme sites and / or internal deletions, substitutions, or insertions. "Variant" can also encompass regulatory elements having a nucleotide sequence with substitutions, deletions, or insertions comprising one or more nucleotides of a reference sequence, wherein the derived regulatory element has more or less or equivalent transcriptional or translational activity than the corresponding parental regulatory molecule. Regulatory element "variants" will also encompass variants resulting from mutations that occur naturally during bacterial and plant cell transformations. In this invention, the polynucleotide sequences provided as SEQ ID NO:1-5 can be used to generate variants that are compositionally similar to but not identical to the DNA sequence of the original regulatory element, while retaining the general function of the original regulatory element, i.e., the same or similar expression pattern. In view of this disclosure, the generation of such variants of the invention is entirely within the scope of ordinary skill in the art and is covered by the scope of this invention.

[0051] The efficacy of the modifications, duplications, or deletions described herein in the desired expression of a particular transgene can be empirically tested in stable transient plant assays, such as those described in the working examples herein, to verify the results, which may vary depending on the changes made and the target of the changes in the starting DNA molecule.

[0052] Construct

[0053] As used herein, the term "construct" means any recombinant DNA molecule, such as plasmids, granules, viruses, bacteriophages, or linear or circular DNA or RNA molecules, derived from any source and capable of genome integration or autonomous replication, including DNA molecules in which at least one DNA molecule is functionally linked (i.e., operably linked) to another DNA molecule. As used herein, the term "vector" means any construct that can be used for transformation purposes, i.e., to introduce heterologous DNA or RNA into host cells. Constructs typically include one or more expression cassettes. As used herein, an "expression cassette" refers to a recombinant DNA molecule that includes at least a transcribed DNA molecule operably linked to one or more regulatory elements, typically at least a promoter and a 3′ UTR.

[0054] As used herein, the term "operably linked" refers to a first DNA molecule linked to a second DNA molecule, wherein the first and second DNA molecules are arranged such that the first DNA molecule influences the function of the second DNA molecule. These two DNA molecules may or may not be part of a single adjacent DNA molecule, and may be adjacent or not. For example, if a promoter regulates the transcription of a transcribed DNA molecule of interest in a cell, then the promoter is operably linked to that transcribed DNA molecule. For example, a leader sequence is operably linked to a DNA molecule when it can influence the transcription or translation of a DNA sequence.

[0055] In one embodiment, the construct of the present invention can be provided as a double tumor-inducing (Ti) plasmid boundary construct having a right boundary (RB or AGRtu.RB) region and a left boundary (LB or AGRtu.LB) region of a Ti plasmid isolated from *Agrobacterium tumefaciens* containing T-DNA, the Ti plasmid, together with transfer molecules provided by *Agrobacterium tumefaciens* cells, allowing T-DNA integration into the genome of plant cells (see, for example, U.S. Patent 6,603,061). The construct may also include plasmid backbone DNA fragments that provide replication function and antibiotic selectivity in bacterial cells, such as *Escherichia coli* replication origins such as ori322, broadly host-wide replication origins such as oriV or oriRi, and coding regions for selective markers such as Spec / Strp encoding Tn7 aminoglycoside adenosyltransferase (aadA) conferring resistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent) selective marker gene. For plant transformation, the host bacterial strains are typically Agrobacterium tumefaciens ABI, C58, or LBA4404; however, other strains known to those skilled in the art of plant transformation may also function in this invention.

[0056] Methods for assembling and introducing constructs into cells in a manner that causes transcribed DNA molecules to be transcribed into functional mRNA molecules that are translated and expressed as proteins are known in the art. Conventional compositions and methods for preparing and using constructs and host cells are well known to those skilled in the art in practice. Typical vectors that can be used to express 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.

[0057] Various regulatory elements may be included in the construct, including any of those elements provided herein. Any such regulatory element may be provided in combination with other regulatory elements. Such combinations may be designed or modified to produce desired regulatory characteristics. In one embodiment, the construct of the present invention includes at least one regulatory element operatively linked to a transcribed DNA molecule operatively linked to the 3′UTR.

[0058] The constructs of this invention may contain any promoter or leader sequence provided herein or known in the art. For example, the promoters of this invention may be operatively linked to a heterologous untranslated 5′ leader sequence, such as a leader sequence from a heat shock protein gene. Alternatively, the leader sequence of this invention may be operatively linked to a heterologous promoter, such as the cauliflower mosaic virus 35S transcript promoter.

[0059] The expression cassette may also contain a transport peptide encoding a subcellular targeting peptide that can be operatively linked to a protein, specifically targeting chloroplasts, leucoplasts, or other plastid organelles; mitochondria; peroxisomes; vacuoles; or extracellular locations. Many chloroplast-localizing proteins are expressed as precursors from nuclear genes and target chloroplasts via chloroplast transport peptides (CTPs). Examples of such isolated chloroplast proteins include, but are not limited to, those associated with ribulose-1,5-bisphosphate carboxylase, ferrugin, ferrugin oxidoreductase, light-harvesting complex I and II, thioredoxin F, and the small subunit (SSU) of enolpyruvylshikimate synthase (EPSPS). Chloroplast transport peptides are described, for example, in U.S. Patent No. 7,193,133. It has been demonstrated that non-chloroplast proteins can target chloroplasts by expressing heterologous CTPs operatively linked to transgenes encoding non-chloroplast proteins.

[0060] Transcribed DNA molecules

[0061] As used herein, the term "transcribed DNA molecule" refers to any DNA molecule capable of being transcribed into an RNA molecule, including but not limited to those having protein-coding sequences, those encoding guide RNA, and those producing RNA molecules with sequences that can be used for gene repression. The types of DNA molecules may include, but are not limited to, DNA molecules from the same plant, DNA molecules from another plant, DNA molecules from different organisms, or synthetic DNA molecules, such as DNA molecules containing antisense information of a gene, or transgenic DNA molecules encoding artificial, synthetic, or otherwise modified forms. Exemplary transcribed DNA molecules incorporated into the constructs of this invention include, for example, DNA molecules or genes from species other than the species in which the DNA molecule is incorporated, or genes derived from or present in the same species but incorporated into recipient cells through genetic engineering methods rather than classical breeding techniques.

[0062] "Transgenic" refers to a transmissible DNA molecule that is at least heterologous to the host cell genome and / or artificially incorporated into the host cell genome in the current or any previous generation of cells, at least relative to its position in the host cell genome.

[0063] Regulatory elements, such as the promoters of this invention, can be operatively linked to a heterologous transcribed DNA molecule relative to the regulatory element. As used herein, the term "heterologous" refers to a combination of two or more DNA molecules that is not normally found in nature. For example, two DNA molecules may originate from different species and / or two DNA molecules may originate from different genes, for example, different genes from the same species or the same gene from different species. Thus, a regulatory element is heterologous relative to an operatively linked transcribed DNA molecule if such a combination is not normally found in nature, i.e., the transcribed DNA molecule is not naturally and operatively linked to the regulatory element.

[0064] Transcriptable DNA molecules can generally be any DNA molecule that requires the expression of a transcript. Such expression of the transcript leads to the translation of the resulting mRNA molecule, and thus to protein expression. Alternatively, for example, a transcriptable DNA molecule can be programmed to ultimately result in reduced expression of a specific gene or protein. In one embodiment, this can be accomplished by using a transcriptable DNA molecule oriented in an antisense direction. Those skilled in the art are familiar with the use of such antisense techniques. Any gene can be negatively regulated in this manner, and in one embodiment, the transcriptable DNA molecule can be programmed to repress a specific gene via the expression of dsRNA, siRNA, or miRNA molecules.

[0065] Therefore, one embodiment of the present invention is a recombinant DNA molecule comprising the regulatory element of the present invention, such as those provided as SEQ ID NO:1-5, which is operatively linked to a heterologous transcribed DNA molecule to regulate the transcription of the transcribed DNA molecule at a desired level or in a desired mode when the construct is integrated into the genome of a transgenic plant cell. In one embodiment, the transcribed DNA molecule comprises a protein-coding region of a gene, and in another embodiment, the transcribed DNA molecule comprises an antisense region of a gene.

[0066] Genes with agronomic significance

[0067] Transcribed DNA molecules can be genes of agronomic significance. As used herein, the term "gene of agronomic significance" refers to a transcribed DNA molecule that, when expressed in a particular plant tissue, cell, or cell type, confers a desired characteristic. The product of agronomically significant genes can function within plants, thereby influencing plant morphology, physiology, growth, development, yield, cereal composition, nutritional status, disease or pest resistance, and / or environmental or chemical tolerance, or can be used as an insecticide in the diet of plant-feeding pests. In one embodiment of the invention, the regulatory element of the invention is incorporated into a construct such that the regulatory element is operatively linked to a transcribed DNA molecule that is a gene of agronomic significance. In transgenic plants containing such a construct, expression of agronomically significant genes can confer beneficial agronomic traits. Beneficial agronomic traits may include, but are not limited to, herbicide tolerance, insect control, improved yield, disease resistance, pathogen resistance, improved plant growth and development, improved starch content, improved oil content, improved fatty acid content, improved protein content, improved fruit maturity, enhanced animal and human nutrition, biopolymer production, environmental stress resistance, pharmaceutical peptides, improved processing quality, improved flavor, practicality in hybrid seed production, improved fiber production, enhanced carbon sequestration, and desired biofuel production.

[0068] Examples of agronomically significant genes known in the art include those for: herbicide resistance (US Patent Nos. 6,803,501, 6,448,476, 6,248,876, 6,225,114, 6,107,549, 5,866,775, 5,804,425, 5,633,435, and 5,463,175), and increased yield (US Patent Nos. USRE 38,446, 6,716,474, 6,663,906, 6,476,295, 6,441,277, 6,423,828, 6,399,330, 6,372,211, 6,235,971, 6,222,098, and 5,716,8). 37) Insect control (US Patent Nos. 6,809,078, 6,713,063, 6,686,452, 6,657,046, 6,645,497, 6,642,030, 6,639,054, 6,620,988, 6,593,293, 6,555,655, 6,538,109, 6,537,756, 6,521,442, 6,501,009, 6,468,523, 6,326,351, 6,313,378, 6,284,949, 6,281,016, 6,248,536, 6,242,241, 6,221,649, 6,177,615, 6,156, 573, 6,153,814, 6,110,464, 6,093,695, 6,063,756, 6,063,597, 6,023,013, 5,959,091, 5,942,664, 5,942,658, 5,880,275, 5,763,245 and 5,763,241), fungal resistance (US Patent Nos. 6,653,280, 6,573,361, 6,506,962, 6,316,407, 6,215,048, 5,516,671, 5,773,696, 6,121,436, 6,316,407 and 6,506,962), viral resistance (US Patent Nos. 6,6 17,496, 6,608,241, 6,015,940, 6,013,864, 5,850,023 and 5,304,730), nematode resistance (US Patent No. 6,228,992), bacterial resistance (US Patent No. 5,516,671), plant growth and development (US Patent Nos. 6,723,897 and 6,518,488), starch production (US Patent Nos. 6,538,181, 6,538,179, 6,538,178, 5,750,876, 6,476,295), improved oil production (US Patent Nos. 6,444,876, 6,426,447 and 6,380,462), high-oil production (US Patent No. 6,495,739, 5,608,149, 6,483,008 and 6,476,295), improved fatty acid content (US Patent Nos. 6,828,475, 6,822,141, 6,770,465, 6,706,950, 6,660,849, 6,596,538, 6,589,767, 6,537,750, 6,489,461 and 6,459,018), high protein Protein production (US Patent No. 6,380,466), fruit ripening (US Patent No. 5,512,466), enhanced animal and human nutrition (US Patent Nos. 6,723,837, 6,653,530, 6,5412,59, 5,985,605, and 6,171,640), biopolymers (US Patent Nos. USRE 37,543, 6,228,623, and 5,958,745, and 6,9...). 46,588), environmental stress resistance (US Patent No. 6,072,103), pharmaceutical peptides and secretory peptides (US Patent Nos. 6,812,379, 6,774,283, 6,140,075 and 6,080,560), improved processing properties (US Patent No. 6,476,295), improved digestibility (US Patent No. 6,531,648), low raffinose (US Patent No. 6,166,292), industrial enzyme production Production (US Patent No. 5,543,576), flavor improvement (US Patent No. 6,011,199), nitrogen fixation (US Patent No. 5,229,114), hybrid seed production (US Patent No. 5,689,041), fiber production (US Patent Nos. 6,576,818, 6,271,443, 5,981,834, and 5,869,720), and biofuel production (US Patent No. 5,998,700).

[0069] Alternatively, agronomically significant genes can influence the aforementioned plant traits or phenotypes by encoding RNA molecules that target and regulate the expression of endogenous genes, for example, through antisense (see, for example, U.S. Patent 5,107,065); repressive RNAs (“RNAi”, including mechanisms that regulate gene expression through miRNA, siRNA, trans-acting siRNA, and phased sRNA, for example, as described in published applications US2006 / 0200878 and US2008 / 0066206, and U.S. Patent Application 11 / 974,469); or co-repressive mechanisms. The RNA can also be a catalytic RNA molecule engineered to cleave the desired endogenous mRNA product (e.g., a ribozyme or riboswitch; see, for example, US2006 / 0200878). Methods for constructing and introducing constructs into cells in a manner that causes transcribed DNA molecules to be transcribed into molecules capable of inducing gene repression are known in the art.

[0070] Selective marking

[0071] Selectively marked transgenes can also be used in conjunction with the regulatory elements of this invention. As used herein, the term "selectively marked transgene" refers to any transcribed DNA molecule whose expression or absence in a transgenic plant, tissue, or cell can be screened or scored in some way. Selectively marked genes used in the practice of this invention, and their associated selection and screening techniques, are known in the art and include, but are not limited to, transcribed DNA molecules encoding β-glucuronidase (GUS), green fluorescent protein (GFP), proteins conferring antibiotic resistance, and proteins conferring herbicide resistance. An example of a selectively marked transgene is provided in SEQ ID NO:8.

[0072] Genome editing

[0073] Several embodiments relate to a recombinant DNA construct comprising an expression cassette containing a sequence having at least about 85% sequence identity with any of SEQ ID NO:1-5 or fragments thereof operatively linked to a heterologous DNA sequence encoding a site-specific genome-modifying enzyme and / or any associated protein for genome modification. These nuclease expression cassettes may be present in the same molecule or vector as a donor template for template editing (cis-), or on a separate molecule or vector (trans-). Several methods for editing are known in the art involving different sequence-specific genome-modifying enzymes (or complexes of proteins and / or guide RNA) for modifying genomic DNA. In some embodiments, the site-specific genome-modifying enzyme modifies the genome by inducing double-strand breaks (DSBs) or nicks at desired genomic sites or loci. In some embodiments, during the repair of DSBs or nicks introduced by the genome-modifying enzyme, donor template DNA may be integrated into the genome at the site of the DSB or nick. In some embodiments, during the repair of DSBs or nicks introduced by the genome-modifying enzyme, insertion or deletion mutations (indels) may be introduced into the genome. In some embodiments, the site-specific genome-modifying enzyme comprises cytidine deaminase. In some embodiments, the site-specific genome-modifying enzyme comprises adenine deaminase. In this disclosure, the site-specific genome-modifying enzyme includes endonucleases, recombinases, transposases, deaminases, helicases, reverse transcriptases, and any combination thereof.

[0074] Several embodiments relate to a gene regulatory element as described herein, operatively linked to a heterologous transcribed DNA molecule encoding one or more components of a genome editing system. The genome editing system can be used to introduce one or more insertions, deletions, substitutions, base modifications, translocations, or inversions into the genome of a host cell. In some embodiments, the gene regulatory element as described herein is operatively linked to a heterologous transcribed DNA molecule encoding a sequence-specific DNA-binding domain, such as a CRISPR-Cas effector protein, zinc finger protein, or transcription activator (TAL) protein. In some embodiments, the sequence-specific DNA-binding domain may be a fusion protein. In some embodiments, the gene regulatory element as described herein is operatively linked to a heterologous transcribed DNA molecule encoding a CRISPR-Cas effector protein. In some embodiments, the CRISPR-Cas effector protein is selected from type I, type II, type III, type IV, type V, or type VI CRISPR-Cas systems. In some embodiments, the gene regulatory element, as described herein, is operatively linked to a heterologous transcribed DNA molecule encoding a guide RNA. As used herein, "guide RNA" or "gRNA" refers to RNA that recognizes a target DNA sequence and guides or "directs" a CRISPR effector protein to the target DNA sequence. The guide RNA consists of a region complementary to the target DNA (called crRNA) and a region binding to the CRISPR effector protein (called tracrRNA). The guide RNA can be a single RNA molecule (sgRNA) or two separate RNA molecules (2-part gRNA). In some embodiments, the gRNA may also contain an RNA template (pegRNA) for reverse transcriptase.

[0075] Several embodiments involve a gene regulatory element as described herein, operatively linked to a heterologous transcribed DNA molecule encoding one or more components of a CRISPR-Cas genome editing system comprising CRISPR-Cas effector proteins and guide RNA. Examples of CRISPR-Cas effector proteins include, but are not limited to, Cas9, C2c1, C2c3, C2c4, C2c5, C2c8, C2c9, C2c10, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas12h, Cas12i, Cas12g, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3'', Cas4, Cas... 5. Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csf1, Csf2, Csf3, Csf4(dinG), Csf5, Cas14a, Cas14b, and Cas14c effector proteins. In some embodiments, the gene regulatory element as described herein is operatively linked to a CRISPR-Cas effector protein containing a mutation at its nuclease active site (e.g., RuvC, HNH, e.g., the RuvC site of the Cas12a nuclease domain; e.g., the RuvC site of the Cas9 nuclease domain and / or the HNH site). CRISPR-Cas effector proteins with a mutation at their nuclease active site and therefore no longer containing nuclease activity are often referred to as “dead,” e.g., dCas. In some embodiments, the domains or peptides of a CRISPR-Cas effector protein with a mutation at its nuclease active site may have impaired or reduced activity compared to the same CRISPR-Cas effector protein without the mutation. In some embodiments, the gene regulatory element as described herein is operatively linked to a CRISPR-Cas effector protein with a mutation at its nuclease active site to produce nicking enzyme activity operatively linked to a reverse transcriptase.

[0076] Cell transformation

[0077] The present invention also relates to a method for producing transformed cells and plants comprising one or more regulatory elements operatively linked to a transcribed DNA molecule.

[0078] The term "transformation" refers to the introduction of DNA molecules into a recipient host. As used herein, the term "host" refers to bacteria, fungi, or plants, including any cell, tissue, organ, or progeny of bacteria, fungi, or plants. Plant tissues and cells of particular interest include protoplasts, callus, roots, tubers, seeds, stems, leaves, seedlings, embryos, and pollen.

[0079] As used herein, the term "transformation" refers to a cell, tissue, organ, or organism into which a foreign DNA molecule, such as a construct, has been introduced. Preferably, the introduced DNA molecule is integrated into the genomic DNA of the recipient cell, tissue, organ, or organism so that the introduced DNA molecule is inherited by subsequent offspring. "Transgenic" or "transformed" cells or organisms may also include offspring of the cell or organism, as well as offspring produced by breeding programs using such transgenic organisms as hybridization parents and exhibiting altered phenotypes due to the presence of the foreign DNA molecule. The introduced DNA molecule may also be introduced transiently into the recipient cell so that the introduced DNA molecule is not inherited by subsequent offspring. The term "transgenic" refers to a bacterium, fungus, or plant containing one or more heterologous DNA molecules.

[0080] Numerous methods exist well-known to those skilled in the art for introducing DNA molecules into plant cells. This process typically involves selecting suitable host cells, transforming the host cells with a vector, and obtaining transformed host cells. In the practice of this invention, methods and materials for transforming plant cells by introducing plant constructs into the plant genome can include any well-known and proven methods. Suitable methods include, but are not limited to, bacterial infection (e.g., Agrobacterium), binary BAC vectors, direct delivery of DNA (e.g., transformation via PEG, drying / inhibition-mediated DNA uptake, electroporation, stirring with silicon carbide fibers, and acceleration of DNA-coated particles), gene editing (e.g., CRISPR-Cas systems), etc.

[0081] The host cell can be any cell or organism, such as plant cells, algal cells, fungal cells, bacterial cells, or insect cells. In a specific embodiment, the host cell and transformed cell may include cells derived from crop plants.

[0082] The transgenic plant can then be regenerated from the transgenic plant cells of the present invention. Seeds can be produced from the transgenic plant using conventional breeding techniques or self-pollination. Such seeds, and the offspring plants obtained from such seeds, will contain the recombinant DNA molecules of the present invention, and will therefore be transgenic.

[0083] The transgenic plants of this invention can be self-pollinated to provide seeds of homozygous transgenic plants (homozygous for the recombinant DNA molecule) of this invention, or hybridized with non-transgenic plants or different transgenic plants to provide seeds of heterozygous transgenic plants (heterozygous for the recombinant DNA molecule) of this invention. Such homozygous and heterozygous transgenic plants are referred to herein as "progeny plants." Progeny plants are transgenic plants originating from the original transgenic plant and containing the recombinant DNA molecule of this invention. Seeds produced using the transgenic plants of this invention can be harvested and used to cultivate generations of transgenic plants, i.e., progeny plants of this invention, which contain the recombinant DNA molecule of this invention and express genes of agronomical significance. Descriptions of breeding methods commonly used for different crops can be found in several reference books, such as Allard, Principles of Plant Breeding, John Wiley & Sons, NY, U. of CA, Davis, CA, 50-98 (1960); Simmonds, Principles of Crop Improvement, Longman, Inc., NY, 369-399 (1979); Sneep and Hendriksen, Plant Breeding Perspectives, Wageningen (ed.), Center for Agricultural Publishing and Documentation (1979); Fehr, Soybeans: Improvement, Production and Uses, 2nd ed., Monograph, 16:249 (1987); Fehr, Principles of Variety Development, Theory and Technique, (Vol. 1) and Crop Species Soybean (Vol. 2), Iowa State University, Macmillan. Pub.Co.,NY, 360-376 (1987).

[0084] This method can analyze the presence of one or more genes of interest in transformed plants, as well as the expression levels and / or status conferred by the regulatory elements of this invention. Those skilled in the art are aware of a variety of methods that can be used to analyze transformed plants. For example, plant analysis methods include, but are not limited to, DNA blotting or RNA blotting, PCR-based methods, biochemical analysis, phenotypic screening methods, field assessment, and immunodiagnostic assays. Expression of transcribed DNA molecules can be performed using methods as described by the manufacturer. (Applied Biosystems, Foster City, CA) Reagents and methods and their use The PCR cycle time is determined by the Testing Matrix. Alternatively, as described by the manufacturer. (Third Wave Technologies, Madison, WI) reagents and methods can be used to evaluate transgenic expression.

[0085] This invention also provides plant parts of the invention. Plant parts include, but are not limited to, leaves, stems, roots, tubers, seeds, endosperm, ovules, and pollen. Plant parts of the invention can be viable, non-viable, regenerable, and / or non-regenerable. This invention also includes and provides transformed plant cells containing the DNA molecules of the invention. Transformed or transgenic plant cells of the invention include regenerable and / or non-regenerable plant cells.

[0086] This invention also provides commercial products derived from transgenic plants or portions thereof containing the recombinant DNA molecule of this invention. The commercial products of this invention contain a detectable amount of DNA comprising a DNA sequence selected from the group consisting of SEQ ID NO: 1-5. As used herein, “commercial product” means any composition or product comprising material derived from transgenic plants, seeds, plant cells, or plant parts containing the recombinant DNA molecule of this invention. Commercial products include, but are not limited to, processed seeds, cereals, plant parts, and flours. The commercial products of this invention will contain a detectable amount of DNA corresponding to the recombinant DNA molecule of this invention. One or more detections of this DNA in a sample can be used to determine the content or origin of the commercial product. Any standard DNA molecule detection method can be used, including the detection methods disclosed herein.

[0087] The invention can be more readily understood by referring to the following embodiments, which are provided by way of example only and are not intended to limit the invention unless otherwise specified. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to be effective in practicing the invention. However, given this disclosure, those skilled in the art will understand that many variations can be made to the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the invention. Therefore, all matters set forth or illustrated in the drawings should be understood as illustrative and not limiting.

[0088] Example

[0089] Example 1

[0090] Determination and cloning of control elements

[0091] This embodiment describes the identification, synthesis, and cloning of regulatory expression elements derived from *Miscanthus sinensis* (large blue stem) and *Phragmites australis* (giant reed).

[0092] A novel ubiquitin regulatory element was identified and isolated from the genomic DNA of the monocotyledonous species *Miscanthus sinensis* (large blue stem). Primers were designed using the 5' untranslated region (5′UTR) of each ubiquitin 1 transcript to amplify the corresponding regulatory element of the identified ubiquitin gene, which contains an operatively linked promoter, leader sequence (5′UTR), and first intron. The primers were used with GenomeWalker primers constructed according to the manufacturer's protocol. TM The library (Clontech Laboratories, Inc., MountainView, CA) was used together to clone the 5′ region of the corresponding genomic DNA sequence.

[0093] Furthermore, novel 3′UTRs were identified and isolated from the genomic DNA of the monocotyledonous species *Phragmites australis* (giant reed). Table 1 presents the identified EXPs and their corresponding promoters, leader sequences, and introns from *Miscanthus sinensis*, as well as the 3′UTRs from *Phragmites australis*.

[0094] Table 1. Regulatory expression element set, promoter, leader sequence, intron and 3′UTR.

[0095]

[0096] In the expression cassette for driving β-glucuronidase (GUS) expression, the identified EXP and 3′UTR were synthesized using methods known in the art and cloned into a binary plant transformation vector construct to evaluate their activity in stably transformed maize plants, as described in Examples 2 and 3.

[0097] Example 2

[0098] Analysis of EXP-ANDge.Ubq:5 driving GUS expression in stably transformed maize plants

[0099] Maize plants were transformed with the plant expression vector EXP-ANDge.Ubq:5, specifically containing the EXP that drives the expression of the β-glucuronidase (GUS) transgene. GUS protein expression in the resulting plants was analyzed to assess the effect of the regulatory element set (EXP) on expression and compared with the standard control EXP.

[0100] Maize plants were transformed using a plant GUS expression construct. EXP, specifically EXP-ANDge.Ubq:5 (SEQ ID NO:1), was cloned into a basic plant expression vector using standard methods known in the art. The resulting plant expression vector contained a left border region (B-AGRtu.left border) from Agrobacterium tumefaciens, a first transgenic selection cassette for selecting transformed plant cells conferring resistance to the herbicide glyphosate, a second transgenic cassette for evaluating the activity of EXP-ANDge.Ubq:5, and a right border region (B-AGRtu.right border) from Agrobacterium tumefaciens. The second transgenic cassette contained EXP-ANDge.Ubq:5 (SEQ ID NO:1), operatively linked to the 5′ of the GUS coding region, which contained a processable intron (SEQ ID NO:8) operatively linked to the 3′ UTR (T-Sb.Nltp4-1:1:2, SEQ ID NO:7). GUS expression driven by EXP-ANDge.Ubq:5 was compared with the expression of a similar control construct containing a GUS transgenic expression cassette containing EXP, namely EXP-CaMV.35S+Zm.DnaK:12 (SEQ ID NO:6), which is operatively linked to the 5' of the GUS coding region, which is operatively linked to the 3' UTR, namely T-Sb.Nltp4-1:1:2.

[0101] As is well known in the art, maize plant cells were transformed using the aforementioned binary transformation vector construct via Agrobacterium-mediated transformation. The resulting transformed plant cells were induced to form complete maize plants.

[0102] Qualitative and quantitative GUS analysis was used to assess the activity of expression elements in selected plant organs and tissues of transformed plants. For qualitative analysis of GUS expression by histochemical staining, whole or sectioned tissues were incubated with a GUS staining solution containing 1 mg / mL X-Gluc (5-bromo-4-chloro-3-indolyl-β-glucuronide) at 37°C for 5 hours and destained with 35% EtOH and 50% acetic acid. GUS expression was qualitatively determined by visually examining the blue color of selected plant organs or tissues under a dissecting microscope or a combination microscope.

[0103] To quantify GUS expression by enzymatic assay, total protein was extracted from selected tissues of transformed maize plants. One to two micrograms of total protein were incubated with the fluorescent substrate 4-methylumbelliferone-β-D-glucuronide (MUG) at a concentration of 1 mM in a total reaction volume of 50 μL. After incubation at 37 °C for 1 hour, the reaction was stopped by adding 350 μL of 200 mM sodium bicarbonate solution. The reaction product 4-methylumbelliferone (4-MU) exhibited maximum fluorescence at high pH when the hydroxyl groups were ionized. The assay was stopped by adding alkaline sodium carbonate solution, and the pH was adjusted to quantify the fluorescent product 4-MU. The amount of 4-MU formed was estimated by measuring its fluorescence using a FLUOstar Omega Microplate Reader (BMG LABTECH) (excited at 355 nm, emitted at 460 nm). GUS activity values ​​were provided as nanomoles of 4-MU / hour / mg total protein.

[0104] The following tissues were sampled to determine GUS expression in the R0 generation: leaves and roots at the V4 stage; leaves and roots at the V7 stage; leaves, flowers / anthers, and pollen at the VT stage; rachis / filaments at the R1 stage; and seed embryos and seed endosperm at the R3 stage 21 days post-pollination (DAP). Table 2 shows the mean quantitative GUS expression in the sampled tissues.

[0105] Table 2. Mean GUS expression in stably transformed maize plants.

[0106]

[0107] As can be seen in Table 2, EXP-ANDge.Ubq:5 (SEQ ID NO:1) provides a distinctly different expression profile compared to the control EXP-CaMV.35S+Zm.DnaK:12 (SEQ ID NO:6). For example, EXP-ANDge.Ubq:5-driven expression was higher than EXP-CaMV.35S+Zm.DnaK:12-driven expression in VT anthers and pollen. While EXP-ANDge.Ubq:5-driven expression in leaves appeared to increase from V4 to V7 and then decrease to VT, EXP-ANDge.Ubq:5-driven expression remained consistent across all three stages. EXP-ANDge.Ubq:5-driven expression was higher in R1 rachis / filaments compared to the control. EXP-ANDge.Ubq:5 (SEQ ID NO:1) exhibits unique expression characteristics and provides a novel tool for expressing selected transgenes.

[0108] Example 3

[0109] 3′UTR, or T-ARUdo.TubA:1, drives GUS expression in stably transformed maize plants.

[0110] Maize plants were transformed with a plant expression vector, specifically the T-ARUdo.TubA:1 vector containing the 3′UTR that regulates β-glucuronidase (GUS) transgene expression. GUS protein expression in the resulting plants was analyzed to assess the effect of the 3′UTR regulatory element on expression.

[0111] Maize plants were transformed with a plant GUS expression construct and a control construct. The constructs were similar to those described above in Example 2. Both constructs contained an EXP, namely EXP-CaMV.35S+Zm.DnaK:12 (SEQ ID NO:6), which was operatively linked to the 5′ of a GUS coding sequence containing a processable intron (SEQ ID NO:8). The control construct was as described above in Example 2, wherein the GUS coding sequence was operatively linked to a 3′ UTR, namely T-Sb.Nltp4-1:1:2. Another construct used a 3′ UTR, namely T-ARUdo.TubA:1 (SEQ ID NO:5), to terminate the transcription of the GUS transgene.

[0112] Maize plants were transformed and GUS expression was determined as described in Example 2. Tissues were sampled for GUS expression in the R0 generation: leaves and roots at the V4 stage; leaves and roots at the V7 stage; leaves, flowers / anthers, and pollen at the VT stage; cob / filament at the R1 stage; and seed embryos and seed endosperm at the R3 stage 21 days post-pollination (DAP). Table 3 shows the mean quantitative GUS expression in the sampled tissues and the fold increase in GUS provided by T-ARUdo.TubA:1.

[0113] Table 3. Mean GUS expression in stably transformed maize plants.

[0114]

[0115]

[0116] As can be seen in Table 3, the 3′UTR, T-ARUdo.TubA:1 (SEQ ID NO:5), enhanced GUS transgene expression in each tissue tested compared to the 3′UTR, T-Sb.Nltp4-1:1:2. Transcriptional analysis showed appropriate termination and polyadenylation of GUS mRNA. In some cases, the enhancement of transgene expression was quite significant. For example, expression in VT leaves was 176.8-fold higher than the control. Expression in V7 roots was 93.7-fold higher than the control. Therefore, the 3′UTR, T-ARUdo.TubA:1 (SEQ ID NO:5), provided enhanced transgene expression relative to the control.

[0117] The principles of the invention have been shown and described, and it will be apparent to those skilled in the art that modifications may be made in arrangement and detail without departing from these principles. We claim protection for all modifications within the spirit and scope of the claims. All publications herein are incorporated by reference as if expressly and individually indicated that individual publications or patent applications are incorporated herein by reference.

Claims

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

2. The recombinant DNA molecule of claim 1, wherein the DNA sequence contains gene regulatory activity.

3. The recombinant DNA molecule of claim 1, wherein the heterologous transcribed DNA molecule comprises a gene of agronomic significance.

4. The recombinant DNA molecule of claim 3, wherein the agronomically significant gene confers herbicide tolerance to the plant.

5. The recombinant DNA molecule of claim 3, wherein the agronomically significant gene confers pest resistance to the plant.

6. The recombinant DNA molecule of claim 1, wherein the heterotranscribed DNA molecule encodes dsRNA, miRNA, or siRNA.

7. A non-renewable transgenic plant cell comprising a recombinant DNA molecule containing the sequence of SEQ ID NO:1; wherein the sequence is operatively linked to a heterologous transcribed DNA molecule.

8. The non-renewable transgenic plant cell as described in claim 7, wherein the transgenic plant cell is a monocotyledonous plant cell.

9. The non-renewable transgenic plant cell of claim 7, wherein the transgenic plant cell is a dicotyledonous plant cell.

10. A method of producing a commodity product, the method comprising obtaining a transgenic plant or a portion thereof comprising the recombinant DNA molecule of claim 1 and producing the commodity product therefrom; wherein the commodity product is selected from the group consisting of: seeds, processed seeds, protein concentrates, protein isolates, starch, cereals, plant parts, seed oils, biomass, flour, and coarse flour.

11. A method for expressing a transcribed DNA molecule, the method comprising obtaining a transgenic plant comprising the recombinant DNA molecule of claim 1 and culturing the plant, wherein the transcribed DNA is expressed.

12. A transgenic plant or a portion thereof, comprising the recombinant DNA molecule as described in claim 1.

13. A progeny plant or a portion thereof of the transgenic plant as claimed in claim 12, wherein the progeny plant or a portion thereof comprises the recombinant DNA molecule.

14. A transgenic seed, wherein the seed comprises the recombinant DNA molecule as claimed in claim 1.

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